SHAIR MATTHEW D
TAN DEREK S
FOLEY MICHAEL A
STOCKWELL BRENT R
STERNSON SCOTT M
WO1998016830A2 | 1998-04-23 |
TAN, DEREK S. ET AL: "Stereoselective Synthesis of over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assays" J. AM. CHEM. SOC. (1998), 120(33), 8565-8566, XP002136146
1. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: providing one or more template structures; synthesizing one or more diversifiable scaffold structures containing reactive moieties and at least one stereocenter in one or more synthetic steps from said one or more template structures; and diversifying said one or more scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
2. | The method of claim 1, wherein said one or more isolated complex compounds comprises a library of isolated complex compounds containing at least 1,000,000 library members. |
3. | The method of claim 1, wherein said one or more isolated complex compounds comprises a library of isolated complex compounds containing at least 2,000,000 library members. |
4. | The method of claim 1, wherein said one or more diversifiable scaffold structures each contain at least four stereocenters and at least four diversifiable functionalities. |
5. | The method of claim 1, wherein providing said one or more template structures comprises synthesizing said one or more template structures in four steps or fewer and wherein said one or more diversifiable scaffold structures contain at least four stereocenters and at least four diversifiable functionalities. |
6. | The method of claim 1, further comprising attaching said one or more template structures to a solid support unit prior to the step of synthesizing said one or more diversifiable scaffold structures. |
7. | The method of claim 1, wherein providing said one or more template structures comprises synthesizing each of said one or more template structures directly on solid support units. |
8. | A method for generating a novel orthonitrobenzyl photolabile linker comprising: (a) providing an imine having the following structure: wherein R is a protecting group; and (b) forming an amino ester by the addition of the imine to the lithium enolate of methyl isobutyrate to generate a novel orthonitrobenzyl photolabile linker having the following structure: wherein R2 is selected from the group consisting of protecting group, spacer, isolated complex compound reminiscent of natural products, biomolecule, polymer and hydrogen; and R3 is a solid support unit. |
9. | A method for generating a novel orthonitrobenzyl photolabile linker comprising: (a) providing an imine having the following structure: wherein R is a protecting group; (b) forming an amino ester by addition of the imine to the lithium enolate of methyl isobutyrate to generate a compound having the following structure: wherein R2 is selected from the group consisting of protecting group, spacer, complex compound reminiscent of natural products, biomolecule, polymer and hydrogen; R3 is a solid support unit; and (c) saponifying the methyl ester to generate an acid which is subsequently reacted with an amine or amine containing moiety to generate a novel orthonitrobenzyl photolabile linker having the following structure: wherein R2 is selected from the group consisting of protecting group, spacer, complex compound reminiscent of natural products, biomolecule, polymer and hydrogen; and X is a solid support unit. |
10. | The method of claim 9, further comprising reaction of said amino ester with a solid support to generate a novel solid support bound orthonitrobenzyl photolabile linker. |
11. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein RlR9 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
12. | The method of claim 11, wherein synthesizing said epoxyol template comprises: providing ()shikimic acid; reaction of shikimic acid with DEAD and triphenylphosphine to yield an epoxide; reaction of said epoxide with benzoic acid, triphenylphosphine and DEAD to yield the benzoate ester; reaction of said benzoate ester with lithium hydroxide to yield one enantiomer of a carboxylic acid epoxyol template. |
13. | The method of claim 11, wherein synthesizing said epoxyol template comprises: providing ()shikimic acid; reaction of shikimic acid with acetoxybutyrylbromide; epoxidation with NaOCH3 and subsequent Payne rearrangement; reaction of said epoxide with lithium hydroxide to yield one enantiomer of a carboxylic acid epoxyol template. |
14. | The method of claim 11, further comprising attachment of said epoxyol template to the solid support unit. |
15. | A method for generating a library of isolated complex compounds reminiscent of natural products comprising: (a) providing a collection of solid supports; (b) reacting said collection of solid supports with one or more spacers and one or more skip codons to generate distinct solid support units; (c) reacting each of said distinct solid support units with enantiomers of an epoxyol template to generate distinct solid support bound epoxyol templates; (d) reacting each of said solid support bound epoxyol templates with one or more nitrones to generate tetracyclic templates; (e) reacting each of said tetracyclic templates with one or more of a particular class of reagents and optionally one or more skip codons; and (f) repeating step (e) for different classes of reagents until a desired library of natural productlike compounds is obtained. |
16. | The method of claim 15, wherein each of said tetracyclic templates is reacted sequentially with one or more terminal alkynes and optionally one or more skip codons; one or more amines and optionally one or more skip codons; and one or more acids and optionally one or more skip codons to generate a library of natural productlike compounds. |
17. | The method of claim 15 or 16, wherein each of said terminal alkynes is selected from the group consisting of acetaldehyde ethyl propargyl acetal, tertbutyl lmethyl2propynyl ether, 4 (tertbutyl) phenylacetylene, tertbutyldimethylsilyl acetylene, 2 (3butynloxy) tetrahydro2H pyran, lchloro4ethynylbenzene, 1,4decadiyne (50% in hexane), 1,5decadiyne, 3 dibutylamino1propyne, mdiethynylbenzene, 3,3dimethyl1butyne, 1dimethylamino2 propyne, 1dodecyne, ethyl ethynyl ether (50% in hexanes), ethynyl ptolyl sulfone, 1ethynyl4 fluorobenzene, 1ethynylcyclohexene, ethynylestradiol 3methyl ether, 2ethynylpyridine, 4 ethynyltoluene, 1,5hexadiyne (50% in hexane), 1hexyne, 5hexynenitrile, methyl propargyl ether, 2methyl1buten3yne, methylNpropargylbenzylamine, 1,8nonadiyne, 1pentyne, 4 phenyl1butyne, 3phenyl1propyne, phenylacetylene, propargyl ether, propargynlH benzotriazole, N (propargyloxy) phthalimide, Npropargylphthalimide, propargyltriphenylphosphonium bromide, proiolaldehyde diethyl acetal, tetrahydro2 (2 propynyloxy)2Hpyran, triethylsilylacetylene, tripropargylamine, 2 (3butynloxy) tetrahydro 2Hpyran, 3,5dimethyl1hexyn3ol, 1, 1diphenyl2propyn1ol, 1ethynyllcyclohexanol, 1 ethynyl4fluorobenzene, 9ethynyl9fluorenol, 1ethynylcyclopentanol, 1heptyne, 3methyl1 pentyn3ol, 2phenyl3butyn2ol, and propiolaldehyde diethyl acetal. |
18. | The method of claim 15 or 16, wherein each of said amines is selected from the group consisting of allylamine, 2amino1propene1, 1, 3tricarbonitrile, 3amino1 Hisoindole hydrochloride, 3amino5methylisoxazole, aminoacetaldehyde diethyl acetal, aminoacetaldehyde dimethyl acetal, aminoacetonitrile bisulfate, 4 (2 aminoethyl) benzenesulfonamide, 4 (2aminoethyl) morpholine, 2 (2aminomethyl) pyridine, 1 (2 aminoethyl) pyrrolidine, 2aminoindan hydroxchloride, (R)()laminoindan, (S)(+)1 aminoindan, 2 (aminomethyl)15crown5, 4 (aminomethyl) benzenesulfonamide hydrochloride, (aminomethyl) cyclopropane, 2pyrenemethylamine hydrochloride, 3 (aminomethyl) pyridine, 4 (aminomethyl) pyridine, 3aminopropionitrile fumarate, 1 (3aminopropyl)2pyrrolidinone, 1 (3 aminopropyl) imidazole, 3aminopropyltrimethoxysilane, (R) (+)3aminoquinuclidine dihydrochloride, (S) ()3aminoquinuclidine dihydrochloride, ammonia (0.5 M in dioxane), benzylamine, Sbenzylcysteamine hydrochloride, (R) (+)bomylamine, butylamine, cyclobutylamine, cyclohexanemethylamine, cyclohexylamine, cyclopentylamine, cyclopropylamine, (R) (+)cycloserine, 3 (diethoxymethylsilyl) propylamine, 3,4 dimethoxyphenethylamine, 4 (dimethylamino) benzylamine dihydrochloride, 3 dimethylaminopropylamine, N, Ndimethylethylenediamine, ethylamine (2.0 M in THF), 1 ethylpropylamine, 2fluoroethylamine hydrochloride, 4fluorophenethylamine, furfurylamine, geranylamine, 3fluorobenzylamine, (1R, 2R, 3R, 5S) ()isopinocampheylamine, (IS, 2S, 3S, 5R) (+)isopinocampheylamine, isopropylamine, 2methoxybenzylamine, 4 methoxybenzylamine, 2methoxyethylamine, 2methoxyphenethylamine, 3 methoxyphenethylamine, 4methoxyphenethylamine, 3methoxypropylamine, methylamine (2.0M in THF), ()cismyrtanylamine, 1napthylenemethylamine, 3nitrobenzylamine hydrochloride, 4nitrophenethylamine hydrochloride, octylamine, phenethylamine, trans 2phenylcyclopropylamine hydrochloride, 2phenylglycinonitrile hydrochloride, piperonylamine, propargyl amine, (R) ()tetrahydrofurfurylamine, (S) (+)tetrahydrofurfurylamine, N, N, 2,2 tetramethyl1, 3propanediamine, 2thiopheneethylamine, 2,2,2trifluoroethylamine, tryptamine, veratrylamine, 2 (2aminoethyl) pyridine, 3 (aminomethyl) pyridine, (R) ()secbutylamine, (S) (+)secbutylamine, (R) ()1cyclohexylethylamine, (S) (+)1cyclohexylethylamine, isoamylamine, (R) (+)amethylbenzylamine, (S) ()l (lnapthyl) ethylamine, 4 (trifluoromethyoxy) benzylamine, and 3 (trifluoromethyl) benzylamine. |
19. | The method of claim 15 or 16, wherein each of said sixty two acids is selected from the group consisting of acetic acid, 4acetoxybenzoic acid, acetylsalicyclic acid, acrylic acid, m anisic acid, oanisic acid, panisic acid, benzoic acid, 2butynoic acid, (3 carboxypropyl) trimethylammonium chloride, 3chloropropionic acid, crotonic acid, cyanoacetic acid, 3cyanobenzoic acid, 4cyanobenzoic acid, cyclohexanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentylacetic acid, cyclopropanecarboxylic acid, 3,4dihydro 2,2dimethyl4oxy2Hpyran6carboxylic acid, 1, 4dihydro2methylbenzoic acid, 3 dimethylaminobenzoic acid, 4dimethylaminobenzoic acid, N, Ndimethylglycine, ferroceneacetic acid, formic acid, trans3furanacrylic acid, 2furoic acid, 3furoic acid, furylacrylic acid, 2,4hexadienoic acid (Sorbic acid), isobutyric acid, isonicotinic acid, isovaleric acid, levulinic acid, linolenic acid, (+)menthoxyacetic acid, ()menthoxyacetic acid, methacrylic acid, methoxyacetic acid, (R) ()amethoxyphenylacetic acid, (S) (+)a methoxyphenylacetic acid, 2methoxyphenylacetic acid, 3methoxyphenylacetic acid, 4 methoxyphenylacetic acid, 1methyl (IS, 2R) (+)cis1, 2,3,6tetrahydrophthalate, monomethyl glutarate, monomethyl phthalate, monomethyl terephthalate, [lR (la, 2b, 3a)] (+)3methyl2 (nitromethyl)5oxocyclopentaneacetic acid, 4 (3methyl5oxo2pyrazolin1yl) benzoic acid, 6 methylchromone2carboxylic acid, 3,4 (methylenedioxy) phenylacetic acid, 1methylindole2 carboxylic acid, nicotinic acid, 5nitro2furoic acid, 4nitrobenzoic acid, 4nitrophenylacetic acid, 3nitropropionic acid, 2norbornaneacetic acid, orotic acid monohydrate, (S) (+)2oxo4 phenyl3oxazolidineacetic acid, anti3oxotricyclo [2.2.1.0 (2,6)] heptane7carboxylic acid, phenylacetic acid, phenylpropiolic acid, phthalylsulfathiazole, picolinic acid, propionic acid, 2 pyrazinecarboxylic acid, 2pyridylacetic acid hydrochloride, 3pyridylacetic acid hydrochloride, 4pyridylacetic acid hydrochloride, (2pyrimidylthio) acetic acid, pyruvic acid, tetrahydro2 furoic acid, tetrahydro3furoic acid, thioctic acid, 2thiopheneacetic acid, 3thiopheneacetic acid, 2thiophenecarboxylic acid, 3thiophenecarboxylic acid, 2thiopheneglyoxylic acid, (a, a, a trifluoroptolyl) acetic acid, vinylacetic acid, acetoxyacetic acid, 2benzofurancarboxylic acid, cinnoline4carboxylic acid, 3,5diido4pyridone1acetic acid, 3,3dimethylacrylic acid, ferrocenecarboxylic acid, 5methoxy1indanone3acetic acid, 1methyl2pyrrolecarboxylic acid, 3oxo1indancarboxylic acid, trans3 (3pyridyl) acrylic acid, 3 (2thienyl) acrylic acid, a, a, atrifluoromtoluic acid, a, a, atrifluorootoluic acid, and a, a, atrifluoroptoluic acid. |
20. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: (a) synthesizing one or more epoxyol templates having the following structure: wherein R,R7 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more ortho acetates with said one or more epoxyol templates to yield one or more diversifiable scaffolds having the following structure: wherein R,R8 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. (c) diversifying said one or more scaffold structures at said one or more reactive moieties with one or more reagents or a skip codon to generate one or more isolated complex compounds reminiscent of natural products. |
21. | The method of claim 20, further comprising reaction with one or more palladium allylation catalysts and one or more nucleophiles after reaction with said one or more ortho acetates to yield one or more diversifiable scaffolds having the following structure: wherein Rupu is selected from the group consisting of hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X is selected from the group consisting of, any of the above, a solid support, a biomolecule and polymer; and Y is a nucleophiles selected from the group consisting of amine, phenol, maleonate, thiol, carboxylic acid, and azide. |
22. | The method of claim 21, further comprising reaction with one or more nitrones after reaction with said one or more palladium catalysts to generate one or more diversifiable scaffolds having the following structure: wherein RiRi 1 is selected from the group consisting of, hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle, wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X is any of the above, a solid support unit, a biomolecule or polymer. |
23. | The method of claim 20,21, or 22, wherein synthesizing said epoxyol template comprises: providing ()shikimic acid; reacting shikimic acid with DEAD and triphenylphosphine to yield an epoxide; reacting said epoxide with benzoic acid, triphenylphosphine and DEAD to yield the benzoate ester; reaction of said benzoate ester with lithium hydroxide to yield a carboxylic acid epoxyol template. |
24. | The method of claim 20,21, or 22, further comprising attachment of said one or more epoxyol templates to a solid support prior to the step of synthesizing said one or more diversifiable scaffold structures. |
25. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: (a) synthesizing one or more epoxyol templates having the following structure: wherein RlR8 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting said one or more epoxyol templates with one or more acylating agents, tosyl azide, and a catalyst capable of effecting cyclopropanation to yield one or more scaffolds having the following structure: wherein RlR8 independently comprises any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocyclyl wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and wherein X is any of the above, a solid support, or any biomolecule or polymer. (c) diversifying said one or more solid support bound scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
26. | The method of claim 25, wherein synthesizing said epoxyol template comprises: providing ()shikimic acid; reacting shikimic acid with DEAD and triphenylphosphine to yield an epoxide; reaction of said epoxide with benzoic acid, triphenylphosphine and DEAD to yield the benzoate ester; reaction of said benzoate ester with lithium hydroxide to yield a carboxylic acid epoxyol template. |
27. | The method of claim 25, further comprising attachment of each of said one or more epoxyol templates to one or more solid support units prior to the step of synthesizing said one or more diversifiable scaffold structures. |
28. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: (a) providing one or more isonicotinamide templates; (b) reacting said one or more isonicotinamide templates with one or more nucleophilic acylation reagents, dienophiles and amines to yield one or more diversifiable isoquinuclidine scaffolds having the following structure: wherein R,R7 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is NR, wherein R includes, but is not limited to any substituted or unsubstituted alkyl or aryl moiety, CH2, 0 or S; Y is hydrogen, solid support unit, a polymer or a biomolecule, and Z is hydrogen or indole. (c) diversifying said one or more scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
29. | The method of claim 28, further comprising reacting said one or more isoquinuclidine scaffolds with one or more nitrones to generate one or more diversifiable polycyclic alkaloid scaffold structures having the following structure: wherein RloR, 3 is hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle, wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X includes, but is not limited to NR, wherein R includes, but is not limited to, any substituted or unsubstituted alkyl or aryl moiety, CH2, O, or S; and Y includes, but is not limited to, hydrogen, a solid support unit, a polymer or biomolecule. |
30. | The method of claim 28 or 29, further comprising attachment of each of said one or more template structures to a solid support unit prior to the step of synthesizing said one or more diversifiable scaffold structures. |
31. | The method of claim 28 or 29, wherein synthesizing said one or more isonicotinamide template structures comprises synthesizing said one or more template structures directly on a solid support unit. |
32. | The method of claim 31, wherein synthesis of said isonicotinamide template structure directly on a solid support unit comprises: providing nitrobenzylsulfonyl chloride; reacting said sulfonyl chloride with a solid support unit to generate a solid support bound sulfonamide ; reacting said solid support bound sulfonamide with a substituted alcohol, triphenylphosphine or tributylphosphine and DEAD or TMAD (N, N', N", N"' tetramethylazodicarboxamide) to generate a solid support bound sulfonamide containing a diversity position; reacting said solid support bound sulfonamide with thiophenylate, wherein the counterion is selected from the group consisting of sodium, potassium, cesium, and amine bases, and wherein said amine base is selected from the group consisting of DBU, MTBD, DIPEA and triethylamine; reacting said diversifiable support bound moiety with isonicotinoyl chloride to yield an isonicotinamide derivative containing a diversity position. |
33. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: (a) providing one or more isonicotinamide templates; (b) reacting said one or more isonicotinamide templates with one or more nucleophilic acylation reagents, dienophiles and amines to yield one or more diversifiable isoquinuclidine scaffolds having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein Y is a hydrogen, solid support unit, a polymer or a biomolecule; and Z is a hydrogen or indole; and (c) diversifying said one or more scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
34. | The method of claim 33, further comprising reacting said one or more isoquinuclidine scaffolds with one or more nitrones to generate one or more diversifiable polycyclic alkaloid scaffold structures having the following structure:. |
35. | The method of claim 33 or 34, further comprising attachment of each of said one or more template structures to a solid support unit prior to the step of synthesizing said one or more diversifiable scaffold structures. 3<a. |
36. | The method of claim 33 or 34, wherein synthesizing an isonicotinamide template structure comprises synthesizing said template structure directly on a solid support unit. |
37. | The method of claim 36, wherein synthesis of said isonicotinamide template structure directly on a solid support unit comprises: providing nitrobenzylsulfonyl chloride; reacting said sulfonyl chloride with a solid support unit to generate a solid support bound sulfonamide ; reacting said solid support bound sulfonamide with a substituted alcohol, triphenylphosphine or tributylphosphine and DEAD or TMAD to generate a solid support bound sulfonamide containing a diversity position; reacting said solid support bound sulfonamide with thiophenylate; reacting said diversifiable support bound moiety with isonicotinoyl chloride to yield an isonicotinamide derivative containing a diversity position. |
38. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: providing one or more isonicotinamide templates; reacting said one or more isonicotinamide templates with bromoacetophenone, triethylamine, and a double bond containing electron withdrawing group to yield one or more diversifiable piperidine scaffolds having the following structure: wherein RIRI, each independently comprise hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle, wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X is any of the above, a solid support, a biomolecule or a polymer; and Z is NR, wherein R is any substituted or unsubstituted alkyl or aryl moiety, CH2, 0 or S. diversifying said one or more piperdine scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
39. | The method of claim 38, further comprising attachment of each of said one or more template structures to a solid support unit prior to the step of synthesizing said one or more diversifiable scaffold structures. |
40. | The method of claim 38, wherein synthesizing one or more isonicotinamide template structures comprises synthesizing said one or more template structures directly on a solid support unit. |
41. | The method of claim 38, wherein said method of synthesizing an isonicotinamide template directly on a solid support unit comprises: providing nitrobenzenesulfonylchloride ; reacting said sulfonylchloride with a solid support unit to generate a solid support bound sulfonamide; reacting said solid support bound sulfonamide with a substituted alcohol, triphenylphosphine or tributylphosphine, and DEAD or TMAD to generate a solid support bound sulfonamide containing a diversity position; reacting said solid support bound sulfonamide with thiophenylate; reacting said diversifiable support bound moiety with isonicotinoyl chloride to yield an isonicotinamide derivative containing a diversity position. |
42. | A method for generating one or more isolated complex compounds reminiscent of natural products comprising: providing one or more isonicotinamide templates; reacting said one or more isonicotinamide templates with bromoacetophenone, triethylamine, and a double bond containing electron withdrawing group to yield one or more diversifiable piperidine scaffolds having the following structure: wherein RlRl, each independently include hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X is any of the above, a solid support, a biomolecule or polymer; and diversifying said one or more piperdine scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
43. | The method of claim 42, further comprising attachment of each of said one or more template structures to a solid support unit prior to the step of synthesizing said one or more diversifiable scaffold structures. |
44. | The method of claim 42, wherein providing said one or more isonicotinamide templates comprises synthesizing said template structure directly on a solid support unit. |
45. | The method of claim 44, wherein said method of providing a solid support bound isonicotinamide template comprises: providing nitrobenzenesulfonylchloride ; reacting said sulfonylchloride with a solid support unit to generate a solid support bound sulfonamide ; reacting said solid support bound sulfonamide with a substituted alcohol, triphenylphosphine or tributylphosphine, and DEAD or TMAD to generate a solid support bound sulfonamide containing a diversity position; reacting said solid support bound sulfonamide with thiophenoxide, wherein the counterion is selected from the group consisting of sodium, potassium, cesium, and amine bases, and wherein said amine bases are selected from the group consisting of DBU, MTBD, DIPEA, and triethylamine; and reacting said diversifiable support bound moiety with isonicotinoyl chloride to yield an isonicotinamide derivative containing a diversity position. |
46. | A library of templates for use in the development of complex compounds reminiscent of natural products comprising the structure: wherein RlRv each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
47. | A library of isolated complex compounds reminiscent of natural products comprising the following structure: wherein RlRg each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
48. | The library of claim 47 produced by the method comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein RlRg independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
49. | An isolated natural productlike compound comprising the following structure: wherein RlRg each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo,. hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
50. | The composition of claim 49, wherein X comprises a solid support unit. |
51. | A library of isolated complex compounds reminiscent of natural products comprising the following structure: wherein RIR, 4 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
52. | The library of claim 51 produced by the process comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein RlR, each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein RlRg independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
53. | A natural productlike compound comprising the following structure: wherein RiR) 4 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
54. | The compound of claim 53, wherein X is a solid support unit. |
55. | A library of isolated complex compounds reminiscent of natural products comprising the following structure: wherein RlRs 1 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
56. | The library of claim 55 produced by the process comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein RfRy each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein RlRg independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
57. | A natural productlike compound comprising the following structure: wherein RiRi 1 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
58. | The compound of claim 57, wherein X comprises a solid support unit. |
59. | A library of isolated complex compounds reminiscent of natural products comprising the following structure: wherein RlRl, each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
60. | The library of claim 59 produced by the process comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein RlRg independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
61. | A natural productlike compound comprising the following structure: wherein Rureach independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
62. | The compound of claim 61, wherein X comprises a solid support unit. |
63. | A library isolated complex compounds reminiscent of natural products comprising the following structure: wherein RiRn each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
64. | The library of claim 63 produced by the process comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein RlRg independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
65. | A natural productlike compound comprising the following structure: wherein RlRs I each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
66. | The compound of claim 65, wherein X comprises a solid support unit. |
67. | A library of isolated complex compounds reminiscent of natural products comprising the following structure: wherein RlR, 3 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
68. | The library of claim 67 produced by the process comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein RlRg independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
69. | A natural productlike compound comprising the following structure: wherein R,R13 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
70. | The compound of claim 69, wherein X comprises a solid support unit. |
71. | A library of isolated complex compounds reminiscent of natural products comprising the following structure: wherein RlRs3 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
72. | The library of claim 71 produced by the process comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein RlR7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocyce is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein Ruru independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
73. | A natural productlike compound comprising the following structure: wherein RlRt3 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
74. | The compound of claim 73, wherein X comprises a solid support unit. |
75. | A library of natural productlike compounds having the following structure: wherein RlRz3 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
76. | The library of claim 75 produced by the process comprising: (a) synthesizing one or more expoxyol templates having the following structure: wherein R,R each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocyce is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more nitrone carboxylic acids with said one or more expoxyol templates to yield one or more diversifiable tetracyclic scaffolds having the following structure: wherein Riru independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (c) diversifying said one or more tetracyclic scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more complex compounds reminiscent of natural products. |
77. | A natural productlike compound comprising the following structure: wherein RIRI3 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
78. | The compound of claim 77, wherein X comprises a solid support unit. |
79. | A library isolated complex compounds reminiscent of natural products having the following structure: wherein RlR7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycly is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein X is NR, wherein R inlcudes but is not limited to any substituted or unsubstitued alkyl or aryl moiety, CH2, O, or S; Y is hydrogen, a solid support unit, a polymer or biomolecule; and Z is hydrogen or indole. |
80. | The library of claim 79 produced by the process comprising: (a) providing one or more isonicotinamide templates; (b) reacting said one or more isonicotinamide templates with one or more nucleophilic acylation reagents, dienophiles and amines to yield one or more diversifiable isoquinuclidine scaffolds having the following structure: wherein RlR7 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is NR, wherein R includes, but is not limited to any substituted or unsubstituted alkyl or aryl moiety, CH2, 0 or S; Y is hydrogen, solid support unit, a polymer or a biomolecule, and Z is hydrogen or indole. (c) diversifying said one or more scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
81. | A natural productlike compound comprising the following structure: wherein RP ? each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycly is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein X is NR, wherein R inlcudes but is not limited to any substituted or unsubstitued alkyl or aryl moiety, CH2, O, or S; Y is hydrogen, a solid support unit, a polymer or biomolecule; and Z is hydrogen or indole. |
82. | The compound of claim 81, wherein X comprises a solid support unit. |
83. | A library of isolated complex compounds reminiscent of natural products having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycly is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein Y is hydrogen, a solid support unit, a polymer or biomolecule; and Z is hydrogen or indole. |
84. | The library of claim 83 produced by the process comprising: (a) providing one or more isonicotinamide templates; (b) reacting said one or more isonicotinamide templates with one or more nucleophilic acylation reagents, dienophiles and amines to yield one or more diversifiable isoquinuclidine scaffolds having the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein Y is a hydrogen, solid support unit, a polymer or a biomolecule; and Z is a hydrogen or indole; and (c) diversifying said one or more scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
85. | A natural productlike compound comprising the following structure: wherein R,R7 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycly is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein Y is hydrogen, a solid support unit, a polymer or biomolecule; and Z is hydrogen or indole. |
86. | The compound of claim 85, wherein X comprises a solid support unit. |
87. | An isolated library of complex compounds reminiscent of natural products having the following structure: wherein RlRz3 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein X is NR, wherein R is any substituted or unsubstituted alkyl or aryl moiety, CH2, S or O ; and Y is a solid support unit or hydrogen. |
88. | The library of claim 87 produced by the process comprising: (a) providing one or more isonicotinamide templates; (b) reacting said one or more isonicotinamide templates with one or more nucleophilic acylation reagents, dienophiles and amines to yield one or more diversifiable isoquinuclidine scaffolds having the following structure: wherein RlR7 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is NR, wherein R includes, but is not limited to any substituted or unsubstituted alkyl or aryl moiety, CH2, 0 or S; Y is hydrogen, solid support unit, a polymer or a biomolecule, and Z is hydrogen or indole; (c) reacting said one or more isoquinuclidine scaffolds with one or more nitrones to generate one or more diversifiable polycyclic alkaloid scaffold structures having the following structure: (d) diversifying said one or more scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
89. | A natural productlike compound comprising the following structure: wherein RIRI3 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; wherein X is NR, wherein R is any substituted or unsubstituted alkyl or aryl moiety, CH2, S or O ; and Y is a solid support unit or hydrogen. |
90. | The compound of claim 90, wherein X comprises a solid support unit. |
91. | A library of isolated complex compounds reminiscent of natural products having the following structure: wherein RlR8 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
92. | The library of claim 91 produced by the process comprising: (a) synthesizing one or more epoxyol templates having the following structure: wherein R,R7independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more ortho acetates with said one or more epoxyol templates to yield one or more diversifiable scaffolds having the following structure: wherein RlR8 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. (c) diversifying said one or more scaffold structures at said one or more reactive moieties with one or more reagents or a skip codon to generate one or more isolated complex compounds reminiscent of natural products. |
93. | An isolated natural productlike compound comprising the following structure: wherein RlR8 each independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer. |
94. | The compound of claim 93, wherein X comprises a solid support unit. |
95. | An isolated library of complex compounds reminiscent of natural products comprising the following structure: wherein RlRg each independently comprises hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted heterocycle, wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X is any of the above, a solid support, a biomolecule or polymer; and Y is a nucleophile selected from the group consisting of amine, phenol, maleonate, thiol, carboxylic acid, and azide. |
96. | The library of claim 95 produced by the process comprising: (a) synthesizing one or more epoxyol templates having the following structure: wherein RlR7 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more ortho acetates with said one or more epoxyol templates to yield one or more diversifiable scaffolds having the following structure: wherein R,R8 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; and (c) reaction with one or more palladium allylation catalysts and one or more nucleophiles after reaction with said one or more ortho acetates to yield one or more diversifiable scaffolds having the following structure: (d) diversifying said one or more scaffold structures at said one or more reactive moieties with one or more reagents or a skip codon to generate one or more isolated complex compounds reminiscent of natural products. |
97. | An isolated natural productlike compound comprising the following structure: wherein RIR8 each independently comprises hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted heterocycle, wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X is any of the above, a solid support, a biomolecule or polymer; and Y is a nucleophile selected from the group consisting of amine, phenol, maleonate, thiol, carboxylic acid, and azide. |
98. | The compound of claim 97, wherein X comprises a solid support unit. |
99. | An library of isolated complex compounds reminiscent of natural products having the following structure: wherein R,R"each independently comprise hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstitued heterocycle wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X is any of the above, a solid support unit, biomolecule or polymer. |
100. | The library of claim 99 produced by the process comprising: (a) synthesizing one or more epoxyol templates having the following structure: wherein R,R7 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting one or more ortho acetates with said one or more epoxyol templates to yield one or more diversifiable scaffolds having the following structure: wherein RlR8 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; and (c) reaction with one or more palladium allylation catalysts and one or more nucleophiles after reaction with said one or more ortho acetates to yield one or more diversifiable scaffolds having the following structure: (d) reaction with one or more nitrones after reaction with said one or more palladium catalysts to generate one or more diversifiable scaffolds having the following structure: wherein RjRn is selected from the group consisting of, hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle, wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X is any of the above, a solid support unit, a biomolecule or polymer; and (e) diversifying said one or more scaffold structures at said one or more reactive moieties with one or more reagents or a skip codon to generate one or more isolated complex compounds reminiscent of natural products. |
101. | An isolated natural productlike compound comprising the following structure: wherein RlRI I each independently comprise hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstitued heterocycle wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X is any of the above, a solid support unit, biomolecule or polymer. |
102. | The compound of claim 101, wherein X comprises a solid support unit. |
103. | A library of isolated complex compounds reminiscent of natural products having the following structure: wherein RlR8 independently comprises any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylallkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and whrein X is any of the above, a solid support, or any biomolecule or polymer. |
104. | The library of claim 103 produced by the process comprising: (a) synthesizing one or more epoxyol templates having the following structure: wherein RlR8 independently comprises any linear or branched, substituted or unsubstituted alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and wherein X is any of the above, hydrogen, a solid support unit, a biomolecule or a polymer; (b) reacting said one or more epoxyol templates with one or more acylating agents, tosyl azide, and a catalyst capable of effecting cyclopropanation to yield one or more scaffolds having the following structure: wherein RlR8 independently comprises any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocyclyl wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and whrein X is any of the above, a solid support, or any biomolecule or polymer; and (c) diversifying said one or more solid support bound scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
105. | An isolated natural productlike compound comprising the following structure: wherein RlR8 independently comprises any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylallkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and whrein X is any of the above, a solid support, or any biomolecule or polymer. |
106. | The compound of claim 105, wherein X comprises a solid support unit. |
107. | A library of isolated complex compounds reminiscent of natural products having the following structure: wherein RlRt I are any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylallkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and X is a any of the above, a solid support, or any biomolecule or polymer; and Z is NR, wherein R is any substitued or unsubstituted alkyl or aryl moiety, CH2, O, or S. |
108. | The library of claim 107 produced by the process comprising: providing one or more isonicotinamide templates; reacting said one or more isonicotinamide templates with bromoacetophenone, triethylamine, and a double bond containing electron withdrawing group to yield one or more diversifiable piperidine scaffolds having the following structure: wherein RiRn 1 each independently comprise hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle, wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X is any of the above, a solid support, a biomolecule or a polymer; and Z is NR, wherein R is any substituted or unsubstituted alkyl or aryl moiety, CH2, 0 or S. diversifying said one or more piperdine scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
109. | An isolated natural productlike compound comprising the following structure: wherein RlRi I are any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylallkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and X is a any of the above, a solid support, or any biomolecule or polymer; and Z is NR, wherein R is any substitued or unsubstituted alkyl or aryl moiety, CH2, O, or S. |
110. | The compound of claim 109, wherein X comprises a solid support unit. |
111. | A library of isolated complex compounds reminiscent of natural products having the following structure: wherein R,R, are independently any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylallkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and X is a any of the above, a solid support, or any biomolecule or polymer. |
112. | The library of claim 111 produced by the process comprising: providing one or more isonicotinamide templates; reacting said one or more isonicotinamide templates with bromoacetophenone, triethylamine, and a double bond containing electron withdrawing group to yield one or more diversifiable piperidine scaffolds having the following structure: wherein RlRz I each independently include hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstitued heterocycle wherein said substituted heterocycle is preferably substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X is any of the above, a solid support, a biomolecule or polymer; and diversifying said one or more piperdine scaffold structures at one or more of said reactive moieties with one or more reagents or a skip codon, to generate one or more isolated complex compounds reminiscent of natural products. |
113. | An isolated natural productlike compound comprising the following structure: wherein RlRI are independently any linear or branched alkyl, alkenyl, linear or branched aminoalkyl, linear or branched acylamino, linear or branched acyloxy, linear or branched alkoxycarbonyl, linear or branched alkoxy, linear or branched alkylaryl, linear or branched hydroxyalkyl, linear or branched thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylallkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, and substituted or unsubstituted heterocycle wherein said heterocycle is substituted with 15 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy, and any derivative incorporating phosphorous; and X is a any of the above, a solid support, or any biomolecule or polymer. |
114. | The compound of claim 113, wherein X comprises a solid support unit. |
115. | A method for determining one or more biological activities of members of a library of compounds comprising : subjecting a library to a biological target, wherein said library is an isolated complex library of compounds reminiscent of natural products; determining a statistically significant change in a biochemical activity relative to the level of biochemical activity in the absence of the library of compounds; and identification of the library members producing said statistically significant change. |
116. | A kit for determining one or more biological activities of a library member comprising: providing a binding reagent; and providing a library of compounds, wherein said library of compounds is an isolated complex library of compounds reminiscent of natural products. |
Background of the Invention The identification of small organic molecules that affect specific biological functions is an endeavor that impacts both biology and medicine. Such molecules are useful as therapeutic agents and as probes of biological function. For example, progress in whole genome sequencing (see, for example, Collins, F. S.; Patrinos, A.; Jordan, E.; Chakravarti, A.; Gesteland, R.; Walters, L.; and the members of the DOE and NIH planning groups Science 1998,282,682) has facilitated a related method of exploring biological systems. The sequencing of, for example, the estimated 80,000 to 100,000 genes in the human genome is uncovering a myriad of novel genes with unknown functions. In the"reverse genetic"approach, a deletion, or"knockout"mutation is targeted to a known gene of unknown function. This is followed by a broad search for all resulting biological effects, allowing the function of the gene to be inferred. In but one example from the emerging field of chemical genetics, in which small molecules can be used to alter the function of biological molecules to which they bind, these molecules have been effective at elucidating signal transduction pathways by acting as chemical protein knockouts, thereby causing a loss of protein function. (Schreiber et al. J. Am. Chem. Soc. 1990,112,5583; Mitchison, Chez. and Biol. 1994, l, 3) Additionally, due to the interaction of these small molecules with particular biological targets and their ability to affect specific biological functions, they may also serve as candidates for the development of therapeutics.
Because it is difficult to predict which small molecules will interact with a biological target, intense efforts have been directed towards the generation of large numbers, or"libraries", of small organic compounds. These libraries can then be linked to sensitive screens to identify the active molecules. In many cases, researchers have developed"biased"libraries, in which all members share a particular characteristic, such as an ability to interact with a particular target
ligand, or a characteristic structural feature designed to mimic a particular aspect of a class of natural compounds. For example, a number of libraries have been designed to mimic one or more features of natural peptides. Such"peptidomimetic"libraries include phthalimido libraries (WO 97/22594), thiophene libraries (WO 97/40034), benzodiazopene libraries (US 5,288,514), libraries formed by the sequential reaction of dienes (WO 96/03424), thiazolidinone libraries, libraries of metathiazanones and their derivatives (US 5,549,974), and azatide libraries (WO 97/35199) (for review of peptidomimetic technologies, see Gante, J., Angew. Chem. Int. Ed.
Engl. 1994,33,1699-1720 and references cited therein).
Each of these libraries has provided solid phase synthetic strategies for compounds possessing specific core functionalities, but none achieves the complexity of structure found in natural products, or in other lead compounds prepared through traditional chemical synthetic routes. Complex natural products commonly contain several different functionalities and often are rich in stereochemical complexity. Such diversity and complexity are difficult to achieve if the synthesis is restricted to a specific class of compounds.
Recognizing the need for development of synthetic strategies that produce large numbers of complex molecules, Boger et al. (EP 0774 464) have recently developed a solution-phase synthetic strategy for producing a library of compounds based on a functionalizable template core, to which various reagents can be added.
However, there remains a need for development of solid-phase strategies, where the more rapid production methods such as split-and-pool strategies can be employed to generate larger (> 1, 000,000), more complex, preferably natural product-like, libraries. Additional solution-phase strategies would, of course, also be valuable.
Summary of the Invention The present invention provides methods for the production of compounds and libraries of complex compounds reminiscent of natural products from diversifiable scaffold structures. In particular, the present invention provides synthetic strategies that allow production of complex compounds and preferably large collections of complex compounds that are reminiscent of natural products in that they contain one or more stereocenters, and a high density and diversity of functionality. In preferred embodiments, the compounds of the present inventive libraries are structurally related to a natural product. Alternatively or additionally, the compounds of the inventive libraries possess the capability of acting as a ligand in a biological system to produce a desired inhibitory or promoter effect, and thus may also be functionally reminiscent of natural products.
According to the present invention, the inventive compounds and combinatorial libraries are synthesized from diversifiable solid support bound scaffolds, which are synthesized from readily available or easily synthesizable template structures. In certain embodiments, the inventive compounds and libraries are generated from diversifiable scaffolds synthesized from a shikimic acid based epoxyol template. In other embodiments, the inventive compounds and libraries are generated from diversifiable scaffolds synthesized from the pyridine-based template isonicotinamide.
In addition to providing complex compounds reminiscent of natural products, combinatorial libraries thereof, and methods of their production, the present invention also provides a novel ortho-nitrobenzyl photolinker, and a method for its synthesis, that can be used in the preparation of solid suppport bound compounds and combinatorial libraries.
The present invention further provides a method for determining one or more biological activities of a library member. In a preferred embodiment, the method for determining one or more biological activities of the inventive compounds comprises contacting the inventive compounds with a biological target, such as a binding target or transcription based assay, and determining a statistically significant change in a biochemical activity relative to the level of biochemical activity in the absence of the compound.
The present invention further provides a kit comprising a library of compounds and reagents for determining one or more biological activities of the library member. To give but one example, the biological activity can be determined by providing a kit containing a binding reagent, such as a direct reagent (binding target) or an indirect reagent (transcription based assay) and a library of compounds.
The present invention additionally provides pharmaceutical compositions containing one or more library members. In a preferred embodiment, the pharmaceutical composition preferably comprises one or more of the inventive compounds and a pharmaceutically acceptable carrier.
Definitions "Combinatorial library" : As used herein, a"combinatorial library"is a plurality of complex compounds reminiscent of natural products synthesized from diversifiable scaffold structures by employing different reactants, or monomers, at each stage of the diversification of the scaffold structures. The combinatorial libraries of the present invention may be prepared in solution or on the solid phase.
"Diversifiable scaffold structures" : As used herein, a"diversifiable scaffold structure"is a compound synthesized from a template structure, which contains unique latent or active
functionalities capable of being further reacted with synthetic reagents to generate at least one new functionality, but, particularly in the case of a latent functionality, may generate more than one. As used herein, a"latent functionality"is one that is present, but is temporarily inactive.
Upon release with an activator or reagent, the latent functionality becomes active, and is thus available for further diversification. For example, a diversifiable scaffold structure may contain an epoxide moiety, which, upon reaction with a nucleophile releases a latent alcohol functionality and generates an additional functionality at the site of nucleophilic attack.
Furthermore, the alcohol functionality can be subseqently diversified using electrophiles to yield other functionalities including, but not limited to, ether, ester, carbamate and thioester.
"Complex compounds reminiscent of natural products" : As used herein, a complex compound reminiscent of a natural product is a compound that, similarly to complex natural products which nature has selected through evolution, contains more than one stereocenter, a high density and diversity of functionality, and a diverse range of atoms within one structure.
This term can also, for the purposes of the present invention, be used interchangeably with the term"natural product-like"compound. In this context, diversity of functionality can be defined as varying the topology, charge, size, hydrophilicity, hydrophobicity, and reactivity, to name a few, of the functional groups present in the compounds. The term,"high density of functionality", as used herein, can preferably be used to define any molecule that contains at least four latent or active diversifiable functional moieties. These structural characteristics may additionally render the inventive compounds functionally reminiscent of complex natural products, in that they may interact specifically with a particular biological receptor, and thus may also be functionally natural product-like.
"Small Molecule" : As used herein, the term"small molecule"refers to an organic compound either synthesized in the laboratory or found in nature. Typically, a small molecule is characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 1500, although this characterization is not intended to be limiting for the purposes of the present invention. Examples of"small molecules"that occur in nature include, but are not limited to, taxol, dynemicin, and rapamycin. Examples of"small molecules"that are synthesized in the laboratory include, but are not limited to, the inventive compounds incorporated herein.
"Linker" : The term"linker", as used herein, refers to a molecule or group of molecules connecting a solid support and a combinatorial library member. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and the library member by a specific distance.
"Radially Arrayed" : The term"radially arrayed"as used herein, refers to a spatial arrangement of functionality that projects outwardly in all directions, from the synthesized scaffold structure.
"Protecting Group" : The term"protecting group"as used herein, refers to a chemical group that reacts selectively with a desired fuctionality in good yield to give a derivative that is stable to further reactions for which protection is desired, can be selectively removed from the particular functionality that it protects to yield the desired fuctionality, and is removable in good yield by reagents compatible with the other functional group (s) generated during the reactions.
"Support" : The term"support", as used herein interchangeably as beads, solid surfaces, substrates, particles, supports, etc. These terms are intended to include 1) solid supports such as beads, pellets, disks, capillaries, pore-glass beads, silica gels, polystyrene beads optionally cross- linked with divinylbenzene, grafted co-poly beads, poly-acrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N, N'-bis acryloyl ethylene diamine, glass particles coated with a hydrophobic polymer, or any other material having a rigid or semi- rigid surface; and 2) soluble supports such as low molecular weight non-cross-linked polystyrene. These materials also contain functionalities such that identifiers and/or templates, scaffolds, and inventive compounds can be attached to them. It is particularly preferred for the purposes of the present invention that the solid support Tentagel is used.
"Identifier Tag" : The term"identifier tag"as used herein, refers to a means for recording a step in a series of reactions used in the synthesis of a chemical library. For the purposes of this application, the terms encoded chemical library and tagged chemical library both refer to libraries containing a means for recording each step in the reaction sequence for the synthesis of the chemical library.
Description of the Drawing Figure 1 depicts several examples of natural product-like compounds.
Figure 2 depicts the diverse reaction products of one embodiment of the inventive method.
Figure 3 depicts the use of a small molecule to bind the Human Growth Hormone receptor.
Figure 4 depicts the inventive method for the shikimic acid based combinatorial library.
Figure 5 depicts the synthesis of different enantiomers of the epoxyol templates.
Figure 6 depicts the synthesis of an isonicotinamide template.
Figure 7 depicts the use of a preferred Tentagel amino resin.
Figure 8 depicts the use of a photocleavable linker to attach the solid phase resin to the desired template structure.
Figure 9 depicts the synthesis of a novel ortho-nitrobenzyl photolabile linker.
Figure 10 depicts alternative ortho-nitrobenzyl photolinkers.
Figure 11 depicts a dithiane-protected benzoin photolinker.
Figure 12 depicts addition of a diversity position via Fukuyama sulfonamide alkylation.
Figure 13 depicts the synthesis and tandem reaction of the nitrone portion.
Figure 14 depicts the synthesis of iodophenyl nitrones.
Figure 15 depicts the synthesis of alternative scaffold structures.
Figure 16 depicts acetoacetate as a synthetic intermediate.
Figure 17 depicts the solid phase synthesis of rigid polycyclic core structures.
Figure 18 depicts the synthesis of isoquinuclidine scaffolds.
Figure 19 depicts the asymmetric synthesis of 1,2-dihydropyridines.
Figure 20 depicts the use of a sugar based chiral auxiliary.
Figure 21 depicts a novel rearrangement from photolytic cleavage.
Figure 22 depicts examples of solid phase cycloaddition chemistry.
Figure 23 depicts further reactions of isoquinuclidine scaffolds.
Figure 24 depicts solution phase lactone aminolysis.
Figure 25 depicts aminolysis of the tetracycle with n-butylamine.
Figure 26 depicts 2-hydroxypyridine-catalyzed butyrolactone aminolysis.
Figure 27 depicts acylation of the unmasked hydroxyamide.
Figure 28 depicts epoxide ring opening reactions.
Figure 29 depicts additional epoxide ring opening reactions.
Figure 30 depicts chemoselective solvolysis with AcSH and AcOH.
Figure 31 depicts epoxide thiolysis.
Figure 32 depicts solid phase palladium chemistry.
Figure 33 depicts examples of palladium cross-coupling reactions at the aryl iodide.
Figure 34 depicts rhodium-catalyzed hydroacylation and azide cycloaddition at the aryl alkyne.
Figure 35 depicts nitrone and nitrile oxide, alkyne cycloadditions.
Figure 36 depicts representative potential nucleation points of the isoquinuclidine scaffold.
Figure 37 depicts the efficient synthesis of N-arylimide derivatives.
Figure 38 depicts representative diversity sites for the cup-like pentacyclic scaffold.
Figure 39 depicts a synthetic plan for the geneation of 46.5 million complex molecules.
Figure 40 depicts a synthetic plan for the generation of 30 million complex molecules.
Figure 41 depicts a test library synthesis library quality control.
Figure 42 depicts monomer screening.
Figure 43 depicts library quality control for a small test library.
Figure 44 depicts demonstration compounds.
Figure 45 depicts the synthesis of a test library of isoquinuclidine-based compounds.
Figure 46 depicts the use of photorelease of the inventive compounds into nanodroplets.
Figure 47 depicts the ability of the shikimic acid test library to activate the 3TP promoter.
Figure 48 depicts the antagonism of TGF-p-induced reporter gene activity.
Figure 49 depicts the inhibition of mink lung cell growth by the test library.
Figure 50 depicts the ability of KC233 to arrest mink lung cells in the S-phase of the cell cycle.
Figure 51 depicts fully elaborated products 42a-f.
Figure 52 depicts testing of potential building blocks for the shikimic acid-based library.
Figure 53 depicts alkyne building blocks.
Figure 54 depicts amine building blocks.
Figure 55 depicts carboxylic acid building blocks.
Figure 56 depicts representative LC-MS data for testing of building blocks.
Figure 57 depicts tetracycle and building blocks used in the test library.
Figure 58 depicts alkyne and amine building block masses and the resulting 64 unique y- hydroxyamide product masses.
Figure 59 depicts respresentative LC-MS data for test library pool 43 {X, X, 4} acylated with Acid 4.
Figure 60 depicts the coupling of Still's polyhaloaromatic EC-GC tags directly to the polystyrene backbone of beads using mild carbene insertion chemistry.
Figure 61 depicts results for a mink lung cell proliferation assay.
Figure 62 depicts activators of the TGF--responsive reporter gene.
Figure 63 depicts results for TGF-p-responsive reporter gene assay.
Figure 64 depicts results for TGF-p-reponsive reporter gene assay.
Figure 65 depicts numbered acid building blocks tested.
Figure 66 depicts numbered amine building blocks tested.
Figure 67 depicts numbered alkyne building blocks tested.
Figure 68 depicts representative EC-GC trace for binary encoding tag analysis.
Description of Certain Preferred Embodiments As described herein, the present invention provides complex radially arrayed compounds and libraries of compounds, and methods for making such libraries. In general, the present invention provides synthetic strategies that allow production of compounds and large collections of compounds that are reminiscent of complex natural products in that they contain at least one stereocenter, a high density and diversity of functionality displayed in a radial array, and a diverse range of atoms within one structure. In this context, diversity of functionality can be defined as varying a specific characteristic or set of characteristics of the functional groups present in the molecule including, but not limited to, topology, size, charge, hydrophilicity, hydrophobicity, and reactivity. Examples of ways in which functional groups may differ from one another include, but are not limited to, variations in either the shape or chain length of a particular collection of atoms or variations in the particular atoms present in the functional groups. Additionally, functional groups may also differ from one another by variations in both the shape or chain length and variations in the particular atoms present in the functional groups.
In the context of the present invention, a high density of functionality can be defined as a large number of chemical moieties present in an inventive compound or library member. In preferred embodiments the inventive compounds and library members contain at least four chemical moieties. For example, in a preferred embodiment, an inventive compound or library member may contain substituted aryl, epoxide, amine and ester functionalities, and will contain at least one stereocenter. Figure 1 depicts examples of inventive compounds containing stereochemical complexity and a high density and diversity of functionality, qualities that render them reminiscent of natural products (examples include, but are not limited to, trapoxin, Taxo (+)- discodermolide, or rapamycin) or"natural product-like". Figure 2 depicts examples of some of the inventive compounds. Furthermore, as discussed previously, the functionality is displayed in a radial array, which, unlike many polymers or chains of peptides or other molecules, enables diversification in all directions, thus adding to the complexity of the inventive compounds and providing them with a greater likelihood of interacting with biological molecules. In certain embodiments, this complexity is achieved by designing the inventive compounds and libraries of compounds based on an existing natural product, such as ibogamine or catharanthine, or based on a receptor for a particular protein, such as the"hot spot"of human growth hormone (Figure 3). In other embodiments, the present invention also provides compounds and libraries of compounds that, although not based on an existing natual product, are reminiscent of natural products because of their stereochemical and functional complexity and diversity, and thus may be thought of as"non-natural"natural products. Whether the compounds are"non-natural"or
are based on an existing natural product, the compounds and libraries of compounds are expected to be useful as therapeutics and biological probes because of their ability to interact with biomolecules, such as proteins, carbohydrates, and nucleic acids.
In particular, the inventive method involves the synthesis of combinatorial libraries from solution phase or solid support bound scaffolds, which are synthesized from readily available or easily synthesizable template structures. The synthesis of the scaffolds and combinatorial libraries from solid support bound templates is particularly preferred because of the ease with which large numbers (> 1,000,000) of compounds can be synthesized. The template structures are preferably selected for the inventive method because they are easily synthesizable or readily available, they contain multiple reactive sites where individual combinatorial units can be added to generate scaffold structures in preferably four steps or fewer, and possess the potential for stereochemical diversity. The resulting scaffold structures are characterized by their rigidity, stereochemical and functional group complexity, high density and diversity of functionality radially arrayed (e. g., at least four functionalizable sites) from which to generate highly diversified libraries, and by the minimal need to employ protecting groups (e. g., no more than one functionality in the molecule contains a protecting group, or in the case of certain scaffold structures, no protecting groups need be employed) during the synthesis of the scaffold structures and combinatorial libraries. Preferred template and scaffold structures also include those that are capable of reacting with reagents without the need for a catalyst. Importantly, the diversity of these highly complex compounds and libraries of compounds reminiscent of natural products, as discussed above, results both from the ability to diversify the templates and combinatorializable units used to synthesize the scaffold structures, and from the diversity generated upon reaction with the latent and non-latent functionalities in the scaffold structure. This diversity, as discussed above, results from the changing of the shape, size, hydrophilicity, hydrophobicity, charge and reactivity to name a few, when introducing new functionality. In the method of the presently claimed invention, solution phase or solid phase techniques may be employed to generate combinatorial libraries containing as many as or more than one million members of complex radially arrayed compounds reminiscent of natural products, and more preferably libraries containing as many as or more than two million members of complex compounds reminiscent of natural products.
Particularly preferred embodiments of the present invention include the synthesis of compounds and libraries of compounds starting from a shikimic acid based epoxyol template and the synthesis of compounds and libraries of compounds starting from a pyridine based template, isonicotinamide. Figure 4 depicts the inventive method for the shikimic acid based
combinatorial library, in which the boxed regions depict the potential diversity nucleation points.
Each chemical step thus performed in the inventive method will deliver a new monomer while concurrently generating a new position for functionality.
Various characteristics of the templates and resulting scaffolds and reactions utilized in certain preferred embodiments of the present invention are discussed in more detail below; certain examples of inventive reactions and compounds are also presented.
Synthesis of Template Structures In one particularly preferred embodiment, the present invention provides a method for the synthesis of complex compounds and combinatorial libraries generated from scaffold structures that are synthesized from shikimic acid based epoxyol templates. In another particularly preferred embodiment, the present invention provides a method for the synthesis of complex compounds and combinatorial libraries generated from scaffold structures synthesized from a readily available isonicotinamide template. These epoxyol and isonicotinamide templates are subjected to different reaction conditions to yield different highly complex diversifiable scaffold structures from which the complex compounds and libraries of the present invention are generated.
As discussed above, the epoxyol and isonicotinamide templates are selected for the inventive method because they are easily synthesizable or readily available, contain multiple reactive sites from which to synthesize complex diversifiable structures in a minimal number of steps, preferably four steps or fewer, and possess the potential for stereochemical diversity. As will be appreciated by one of ordinary skill in the art, the method of the present invention is intended to encompass all possible stereoisomers and diastereomers for each of the reaction conditions employed.
In one particularly preferred embodiment, the synthesis of desired epoxyol templates is achieved from the natural product (-)-shikimic acid (McGowan et al. J. Org. Chem. 1981,46, 2381; Wood et al. J. Am. Chem. Soc. 1990,112,8907; Mitsunobu, O. Synthesis 1981, 1-28).
Additionally, employing different reaction conditions in the presence of methyl shikimate enables the synthesis of enantiomers of the desired epoxyol templates as shown in Figure 5. For example, reaction under Berchtold reaction conditions, subsequent reaction with DEAD (diethylazo dicarboxylate), triphenylphosphine and benzoic acid, and reaction with LiOH yields the R, S, S acid. The other enantiomer is readily synthesized using acetoxyisobutyryl bromide, subsequent epoxidation with NaOCH3 and Payne rearrangement, and finally reaction with LiOH
to yield the S, R, R acid. These epoxyol templates can be utilized for further reaction in solution, or may subsequently be attached to a solid support.
In another particularly preferred embodiment, an isonicotinamide template is easily synthesized from the commercially available reagent isonicotinoyl chloride and an amine. The use of isonicotinoyl chloride as a starting material is preferred because it provides a handle for solid phase attachment, if desired, and also because it blocks the 4-position in a tandem reaction as shown in Figure 6 (Yamaguchi et al. J. Org. Chem. 1985,50,287; Yamaguchi et al. J. Org.
Chem. 1988,53,3507). In yet another particulary preferred embodiment, an alternative isonicotinamide template is synthesized via Fukuyama sulfonamide alkylation, in which a diversifiable amide functionality is created by alkylation of the nitrogen under Mitsunobu conditions. Nitrobenzenesulfonylchloride is reacted with a solid support to generate a solid support-bound sulfonamide. Subsequent reaction with triphenylphosphine or tributylphosphine and DEAD or TMAD generates a solid support bound sulfonamide containing a diversity position. Subsequent cleavage of the sulfonamide with thiophenylate, or more generally a thiophenoxide, wherein the counterion includes, but is not limited to, sodium, potassium, cesium or amine bases, wherein said amine bases include, but are not limited to, DBU, MTBD, DIPEA, or triethylamine, yields a functionalized moiety available for further reaction with isonicotinoyl chloride to yield the functionalized isonicotinamide template. In preferred embodiments, the diversifiable functionality present on the nitrogen includes but is not limited to branched or unbranched, substituted or unsubstituted alkyl, aryl, and arylalkyl moieties.
Once the synthesis of either a desired solution phase or solid support bound template has been completed, the template is then available for further reaction to yield the desired solution phase or solid support bound scaffold structure. The use of solid support bound templates is particularly preferred because it enables the use of more rapid split and pool techniques to generate libraries containing as many as or more than 1,000,000 members.
A solid support, for the purposes of this invention, is defined as an insoluble material to which compounds are attached during a synthesis sequence. The use of a solid support is advantageous for the synthesis of libraries because the isolation of support-bound reaction products can be accomplished simply by washing away reagents from the support-bound material and therefore the reaction can be driven to completion by the use of excess reagents.
Additionally, the use of a solid support also enables the use of specific encoding techniques to "track"the identity of the inventive compounds in the library. A solid support can be any material which is an insoluble matrix and can have a rigid or semi-rigid surface. Exemplary solid supports include but are not limited to pellets, disks, capillaries, hollow fibers, needles,
pins, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, poly-acyrlamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N-N'-bis-acryloylethylenediamine, and glass particles coated with a hydrophobic polymer. One of ordinary skill in the art will realize that the choice of a particular solid support will be limited by the compatibility of the support with the reaction chemistry being utilized. In one particularly preferred embodiment, a Tentagel (see, Rapp Polymere Home Page. http://www. rapp-polymere. com (accessed June 1999) amino resin, a composite of 1) a polystyrene bead crosslinked with a divinylbenzene and 2) PEG (polyethylene glycol), is employed for use in the present invention, as shown in Figure 7.
Tentagel is a particulary useful solid support because it provides a versatile support for use in on- bead or off-bead assays, and it also undergoes excellent swelling in solvents ranging from toluene to water.
The compounds of the present invention may be attached directly to the solid support or may be attached to the solid support through a linking reagent, as shown in Figure 7. Direct attachment to the solid support may be useful if it is desired not to detach the library member from the solid support. For example, for direct on-bead analysis of biological activity or analysis of the compound structure, a stronger interaction between the library member and the solid support may be desirable. Alternatively, the use of a linking reagent may be useful if more facile cleavage of the inventive library members from the solid support is desired.
Furthermore, any linking reagent used in the present invention may comprise a single linking molecule, or alternatively may comprise a linking molecule and one or more spacer molecules, as depicted in Figure 7. A spacer molecule is particularly useful when the particular reaction conditions require that the linking molecule be separated from the library member, or if additional distance between the solid support/linking unit and the library member is desired. In one particularly preferred embodiment, photocleavable linkers are employed to attach the solid phase resin to the desired template structure, as shown in Figure 8. Photocleavable linkers are particularly advantageous for the presently claimed invention because of the ability to use these linkers in in vivo screening strategies. Once the template is released from the solid support via photocleavage, the complex small molecule is able to enter the cell.
In addition to providing for the synthesis of scaffold structures, compounds and libraries of compounds, in another aspect, the present invention provides a novel ortho-nitrobenzyl photolabile linker (3-amino-3- (2'-nitrophenyl)-2, 2-dimethylpropionic acid (I) and a method for the synthesis of the photolabile linker, as shown in Figure 9. As shown in Figure 9, the imine (1) is synthesized in two steps from commercially available 2-nitrobenzaldehyde by modification of
a published procedure. (Kanazawa, A. M. et al., J. Org. Chem. 1994,59,1238) The amino ester (2) is then formed by the addition of a pre-cooled solution of (1) to the lithium enolate of methyl isobutyrate. Subsequent recrystallization from 40: 60 ether/petroleum ether, hydrolysis of the synthesized ester with lithium hydroxide (LiOH), and coupling toTentagel S NH, using HATU yields the support bound linker (4). Importantly, this linker is incapable of P-elimination, a common decomposition pathway for photolinkers, and is stable to acid, base, and Lewis acid/arnine conditions.
Referring to (1), R, includes, but is not limited to a protecting group, a complex compound reminiscent of a natural product, a spacer, a biomolecule, or a polymer; and X is a solid support unit.
In other particulary preferred embodiments, alternative ortho-Nitrobenzyl photolinkers are employed, such as the Rich Linker (Nba), Geysen Linker (Anp) (see, Brown et al. Mol. Div.
1995, l, 4), Linker (A), and Affymax Linkers (Hep, Hmp, Aep) as shown in Figure 10.
Additionally, a dithiane-protected benzoin photolinker, as shown in Figure 11 may be employed.
One of ordinary skill in the art will also realize that any of these photolinkers as well as other photolinkers can be employed with the limitation that they will not degrade in the presence of the complex reaction steps employed in the synthesis of the compounds and combinatorial libraries.
Furthermore, the method of the present invention is not limited to the use of photocleavable linkers; rather other linkers may be employed, preferably those that are capable of delivering the desired compounds in vivo.
Furthermore, as mentioned above, it may also be desirable, or even necessary, to utilize a spacer unit, to ensure that the photolinker is sufficiently distanced from the desired compound.
Representative spacer units include but are not limited to aminocaproic acid (Aca), glycine, and any amino acid that does not contain a functionality capable of being acylated.
In certain embodiments, the completed template may be attached to the solid phase, through a linking unit, or directly, and subsequently used in the synthesis of desired scaffold structures. In particularly preferred embodiments, attachment of the completed templates of the present invention to the solid phase is achieved by reaction under standard amide coupling conditions. In one example, Figure 5 depicts the attachment of completed epoxyol templates to the solid phase by reaction with PyBOP, Hunig's Base and NMP, to yield a support bound epoxyol template. One of ordinary skill in the art will realize that attachment of templates to the solid phase may also be effected through alternative means, such as, but not limited to, ether linkages. This choice of linkage will depend upon the reactivity of the functionalities available in the compounds and the solid support units (including any combination of a solid support, and linking reagent) and the stability of these linkages.
In other embodiments, one of the reagents used in the synthesis of the desired template may be attached to the solid support and the template synthesis completed while on the solid support. For example, as shown in Figure 6, attachment of isonicotinoyl chloride to the solid phase to yield a support bound isonicotinamide, is achieved by reaction with Anp-Tgl and DIPEA. Furthermore, as shown in Figure 12, alkylation of the nitrogen via Fukuyama sulfonamide alkylation, wherein nitrobenzenesulfonylchloride is reacted with a solid support to generate a solid support-bound sulfonamide, and subsequent reaction with triphenylphosphine or tributylphosphine and DEAD or TMA, generates a solid support bound sulfonamide containing a diversity position (see, Fukuyama et al. Tet. Lett. 1995,36,6373). Subsequent cleavage of the sulfonamide with thiophenylate, or more generally thiophenoxide, wherein the counterion includes, but is not limited to, sodium, potassium, cesium or amine bases, and wherein said amine bases include, but are not limited to, DBU, MTBD, DIPEA, or triethylamine, yields the alkylated support bound moiety available for further reaction with isonicotinoyl chloride to yield an alkylated isonicotinamide derivative. In preferred embodiments, the diversifiable functionality, Ro, includes but is not limited to, branched or unbranched, substituted or unsubstituted alkyl, aryl, and arylalkyl moieties.
Each of the templates synthesized according to the method of the present invention, whether in the solution phase or attached to a solid support, can then be subsequently used in the synthesis of desired scaffold structures.
Shikimic acid based scaffold structures The above-described epoxyol templates provide useful starting materials for the synthesis of diversifiable scaffold structures. In one particularly preferred embodiment, the synthesis of a
tetracyclic scaffold is achieved by reaction of the epoxyol bound template with a nitrone under transesterification conditions to yield a tetracycle as shown in Figure 13. Tamura and co- workers have described the synthesis of a tricyclic compound by tandem transesterification- cycloaddition reaction of a nitrone methyl ester and a cyclohexen-3-ol. A modified sequence for this tricyclic structure was used to yield the core tetracyclic template (see, Tamura et al.
Tetrahedron 1995,51,107; Tamura et al. Tetrahedron 1995,51,119). One of ordinary skill in the art will realize that any commonly used transesterifiction reagent may be employed to yield the desired tetracycle structure, such as the Otera catalyst, (SCNBu2Sn) 2O. Moreover, the nitrone employed in the reaction can also be varied to yield different derivatives of the tetracyclic scaffold. As shown in Figure 13, a benzyl nitrone is synthesized from a benzaldehyde precursor.
In other embodiments, other aldehydes, such as any aromatic or aliphatic aldehyde, can be substituted to yield different nitrones. Alternatively, Figure 14 depicts the synthesis of different iodophenyl nitrones from the nitrophenyliodides. These nitrophenyliodides are reduced, preferably with Zn/NH4Cl, to the N-iodophenylhydroxylamine, followed by condensation with glyoxylic acid monohydrate to form the N-iodophenylnitrones. Any of the abovementioned nitrones, or derivatives thereof can be subsequently reacted with the epoxyol template to yield a desired tetracycle, such as the tetracycle (as shown in Figure 13) and shown in (II) below. Referring to (11), R,-Rg each independently includes, but is not limited to hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and any substituted or unsubstituted heterocycle wherein said substituted
heterocycle is preferably substituted with 1-5 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, benzyloxy; and X includes, but is not limited, to any of the above, a solid support, a biomolecule or polymer. Furthermore, each of the above functionalities may be unsubstituted or substituted with appropriate chemical moieties. In a particularly preferred embodiment, R2-Rg are each hydrogen, R, is an substituted or unsubstitued alkyl, aryl, or alkylaryl, and X is a solid support unit or hydrogen.
In another particularly preferred embodiment, alternative scaffold structures can be obtained in which the epoxyol bound template is treated with an orthoacetate, such as trimethylorthoacetate to undergo a Johnson ortho-ester-like Claisen rearrangement to yield the ester (1), as shown in Figure 15 and in (III) below.
Referring to (III), R-Rs each independently includes, but is not limited to, hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 1-5 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X includes, but is not limited to, any of the above, a solid support, a biomolecule or polymer. Furthermore, each of the above functionalities may be unsubstituted or substituted with appropriate chemical moieties. In a particularly preferred embodiment, R2-R8 are each hydrogen and R, is a lower alkyl group, such as methyl, and X is a hydrogen or a solid support unit.
Reaction of this scaffold structure with other reagents also yields alternative diversifiable scaffold structures, as shown in Figure 15. For example, reaction with a palladium allylation catalyst such as Pd (dba) 2 and a nucleophile (Y), yields an alternative epoxide opened structure (2), as shown in Figure 15 and (IV) below.
Referring to (IV), R,-Rg each independently includes, but is not limited to hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 1-5 substitutents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X includes, but is not limited to, any of the above, a solid support, a biomolecule or polymer; and Y includes, but is not limited to nucleophiles selected from the group consisting of amine, phenol, maleonate, thiol, carboxylic acid, and azide. Furthermore, each of the above functionalities may be unsubstituted or substituted with appropriate chemical moieties. In a particulary preferred embodiment, R2-R8 are each hydrogen and R, is a lower alkyl group, such as methyl, X is a hydrogen or a solid support unit, and Y is an amine, phenol, maleonate, thiol, carboxylic acid, or azide.
Subsequent reaction with a nitrone, under standard conditions, yields an alternative diversifiable scaffold structure (3), as shown in Figure 15 and (V) below, where the addition of reagents, such as but not limited to, amines or boronic acid, yields diversified structures, as shown in Figure 15.
Referring to (V), R,-R"each independently includes, but is not limited to, hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 1-5 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X includes, but is not limited to, any of the above, a solid support unit, a biomolecule or polymer. Furthermore, each of the above functionalities may be unsubstituted or substituted with appropriate chemical moieties. In a particularly preferred embodiment, R-R5 and R7-R, are each hydrogen, R6 is a substituted or unsubstituted aryl, alkyl, arylalkyl ; and X is hydrogen or a solid support unit.
Additionally, in another particularly preferred embodiment, a different scaffold can be constructed whereby the inventive epoxyol template is treated with an acylating agent including, but not limited to a diketene, to yield the diketone, as shown in Figure 16. Subsequent reaction with tosyl azide yields the diazo p-keto ester (2), as shown in Figure 16. Finally, cyclopropanation with a rhodium or copper catalyst yields the cyclic scaffold structure (3), as shown in Figure 16 and (VI) below, which contains several radially diversifiable moieties.
Referring to (VI), R,-R8 each independently includes, but is not limited to, hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 1-5 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; and X includes, but is not limited to any of the above, a solid support unit, a biomolecule or polymer. Furthermore, each of the above functionalities may be unsubstituted or substituted with appropriate chemical moieties. In particularly preferred embodiments, R,-R6 and R8 are each hydrogen, R7 is a lower alkyl, such as methyl, and X is a hydrogen or a solid support unit.
One of ordinary skill in the art will appreciate that the particular functional groups available at any site in the template structures must be compatible with the particular reaction chemistry being utilized in the synthesis of the scaffold structures. Additionally, the compounds described herein contain one or more centers of asymmetry and may thus give rise to enantiomers, diastereomers and other stereoisomeric forms. The present invention is meant to include all such possible stereoisomers as well as their racemic and optically pure forms.
Optically active (R) and (S) isomers may be prepared using chiral synthesis, chiral reagents, or resolved using conventional techniques. When the compounds disclosed herein contain olefinic double bonds, it is intended to include both E and Z geometric isomers. Furthermore, the examples and scaffolds, and the functional groups contained therein, presented above are not
intended to be exclusive; rather all equivalents thereof are intended to be within the scope of the present invention.
Synthesis of Pyridine Based Scaffold Structures The present invention also provides a method for the synthesis of compounds and complex combinatorial libraries based on isonicotinamide templates in the solution phase or on the solid support, as discussed previously. In preferred embodiments, the synthesis of polycyclic alkaloids is achieved from 1,2-dihydropyridines. As shown in Figures 17 and 18, each of the resulting pentacycles share isonicotinamide as a starting material and feature a 1,2- dihydropyridine synthetic intermediate. Cycloadditions are used in each synthesis to build up structural complexity and functional group manipulations are used to elaborate the rigid core structures.
In particularly preferred embodiments, the solid support bound isonicotinamide can be first converted into an azomethine ylide in the synthesis of diversifiable scaffold structures. For example, in one particularly preferred embodiment, the cup-like pentacyclic piperidine scaffold (1), as shown in Figure 17, can be obtained by reaction of the template with bromoacetopheone, triethylamine and N-methylmaleimide to yield the azomethine ylide. Subsequently, reaction with N-methylmalimide under reflux conditions yields the desired pentacycle, as shown in (VIIA) below, wherein Z is N-R, and wherein R is preferably a substituted or unsubstituted alky or aryl mioety and which contains several sites of latent functionality for diversification. One of ordinary skill in the art will realize that the synthesis of the scaffold is not limited to the pentacyclic structure and may also be diversified by employing any double substituted or unsubstituted bond containing an electron withdrawing group, to yield alternative piperidine structures for (VIIA), in which Z is CH2, O or S, or structures as shown in (VIIB).
Referring to (VIIA and VIIB), R,-Rl, each independently includes hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 1-5 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X is a any of the above, a solid support, a biomolecule or polymer; and Z is NR, wherein R includes but is not limited to any substituted or unsubstituted alkyl or aryl mioety, CH2, O, or S. In particularly preferred embodiments, R, is hydrogen or any aliphatic group, R2- R6 and R8-R"are each hydrogen, R7 is a benzoyl moiety, X is a hydrogen, or a solid support unit; and in the case of Figure 7a, Z is NR, wherein R includes but is not limited to any substituted or unsubstituted alkyl or aryl mioety.
In another particularly preferred embodiment, the resin bound isonicotinamide template is converted to the allyl derivative, from which isoquinuclidine scaffolds are synthesized, as shown in Figure 18. First, the resin bound template is treated with allyltributyltin to yield the allyl intermediate. One of ordinary skill in the art will realize that this reaction may also be effected stereoselectively to yield stereochemically pure scaffold structures. For example, in one particularly preferred embodiment, the synthesis of an enantiomerically pure compound may be effected by the asymmetric synthesis of 1,2 dihydropyridine as shown in Figure 19, which can
then be used in the synthesis of enantiomerically pure scaffold structures and combinatorial libraries. Figure 20 also depicts a method for the stereoselective synthesis of 1,2- dihydropyridines utilizing a sugar based chiral auxiliary. Alkylation of the pyridine with glucosyl bromide yields the pyridinium salt which is then capable of directing the addition of nucleophiles stereoselectively. In addition to providing stereochemically pure compounds, the inventive method also provides a novel rearrangement of the allyl intermediate as shown in Figure 21. Upon exposure to light, the allyl intermediate undergoes a rearrangement to yield a new intermediate which can subsequently be utilized in the synthesis of the scaffold, thus providing further diversity. The intermediate, as shown in Figures 17 or 18, or any of the intermediates discussed above, may be subsequently reacted with dienophiles, including, but not limited to maleic anhydride, aza-dicarboximide, and dimethylacetylenedicarboxylate, in a Diels- Alder reaction to yield various tricyclic intermediates, as shown in Figure 22 and more generally in (VIIIA and VIIIB), shown below. Subsequent reaction of the imide intermediate with a primary amine, and removal of the protecting group yields alternative isoquinuclidine scaffolds, as shown in Figure 18, and more generally in (VIIIB) Referring to (VIIIA and VIIIB), R,-R7 each independently includes, but is not limited to hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, hydrogen, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any
functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 1-5 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X includes, but is not limited to NR, wherein R includes but is not limited to any substituted or unsubstituted alkyl or aryl mioety, CH2, O or S; Y includes, but is not limited to hydrogen, a solid support unit, a polymer or biomolecule; and Z includes, but is not limited to, hydrogen or indole. Furthermore, each of the above functionalities may be unsubstituted or substituted with appropriate chemical moieties. In particulary preferred embodiments, RI-R. are each hydrogen, X is NR, wherein R includes but is not limited to any substituted or unsubstituted alkyl or aryl moiety, Y is a solid support unit, and Z is an indole to generate an ibogamine-like compound, as shown in Figure 18. Furthermore, as shown in Figure 23, an indole substituted allyl scaffold (1) is also capable of undergoing palladium insertion to yield the cyclic structure (2). Reaction with dimethyl sulfate and DBU yields an alternative structure (3) depicted in Figure 23.
In yet another particularly preferred embodiment, the tandem acylation and [ (3 + 2] cyclization employed in the shikimic acid based combinatorial library discussed above can also be utilized to generate a polycyclic alkaloid from the deprotected isoquinuclidine scaffold as shown in Figure 18 and (IXA and IXB) below.
Referring to IXA and IXB above, Rl-Rl3 each independently includes, but is not limited to hydrogen, any linear or branched alkyl, alkenyl, aminoalkyl, acylamino, acyloxy, alkoxycarbonyl, alkoxy, alkylaryl, hydroxyalkyl, thioalkyl, acyl, amino, hydroxy, thio, aryloxy, arylalkoxy, alkynyl, halogen, cyano, sulfhydryl, carbamoyl, nitro, trifluoromethyl, any functionality incorporating phosphorous, and substituted or unsubstituted heterocycle wherein said substituted heterocycle is preferably substituted with 1-5 substituents selected from the group consisting of lower alkyl, halo, hydroxy, amino, thio, lower alkoxy, lower alkylthio, lower alkylamino, nitro, phenoxy, and benzyloxy; X includes, but is not limited to, NR, wherein R includes but is not limited to any substituted or unsubstituted alkyl or aryl moiety, CH2, O or S; andY includes, but is not limited to, hydrogen, a solid support unit, a polymer or biomolecule.
Furthermore, each of the above functionalities may be unsubstituted or substituted with appropriate chemical moieties. In particularly preferred embodiments, Ri is a benzyl, and R2-Rl3 are each hydrogen, X is NR, wherein R includes but is not limited to any substituted or unsubstituted alkyl or aryl moiety, and Y is a solid support unit.
One of ordinary skill in the art will appreciate that the particular functional groups available at any site in the isonicotinamide-based template structures must be compatible with the particular reaction chemistry being utilized in the synthesis of the scaffold structures.
Additionally, the compounds described herein contain one or more centers of asymmetry and may thus give rise to enantiomers, diastereomers and other stereoisomeric forms. The present invention is meant to include all such possible stereoisomers as well as their racemic and optically pure forms. Optically active (R) and (S) isomers may be prepared using chiral synthesis, chiral reagents or resolved using conventional techniques. When the compounds disclosed herein contain olefinic double bonds, it is intended to include both E and Z geometric isomers. Furthermore, the templates and scaffolds, and the functional groups contained therein and the reagents utilized, presented above are not intended to be exclusive; rather all equivalents thereof are intended to be within the scope of the presently claimed invention.
Reactions at latent functionality in the inventive scaffolds Once the inventive scaffolds have been synthesized as discussed above, diversification reactions may be employed at each of the different latent functionality sites present in the scaffold. One of ordinary skill in the art will appreciate that the reactivity of a particular functionality must be considered when selecting a reagent for diversification.
In one particularly preferred embodiment, diversification reactions are employed on the shikimic acid based tetracyclic scaffold. Examples of specific reactions to which some or all of the shikimic acid based tetracyclic systems can be subjected in solution or on the solid support include i) addition of nucleophiles (primary and secondary amines) to the y-lactone function as shown in Figures 24,25 and 26; ii) functionalization of the free hydroxyl with electrophiles (for example, isocyanates, anhydrides, or acid chlorides as depicted in Figure 27); iii) opening of the epoxide with nucleophiles, such as amines, under ytterbium catalysis (see, for example, Ryan et al. Tetrahedron 1973,29,3649; Lindsay Smith et al. J. Chem. Soc., Perkin Trans. 1 1975, 1200) as shown in Figures 28 and 29, or thiols or hydroxyls as shown in Figures 30 and 31) ; iv) cleavage of the N-O bond of tetrahydroisoxazole to release a 1,3 amino alcohol that can be functionalized with various electrophiles such as acid chlorides, sulfonyl chlorides, or isocyanates; and v) functionalization at the iodide in the aromatic ring. For example, functionalization of the iodide in the aromatic ring can be effected by conversion to such structures as amines, amides, aromatic rings, alkenes, alkynes, and heterocycles using palladium- catalyzed chemistry, as shown in Figure 32 which depicts various diversification reactions that can be employed on an iodoaromatic ring, such as Buchwald-Hartwig aminations, Heck (see, Heck, R. F. In Comprehensive Organic Synthesis ; Trost, B. M.; Fleming, I., Eds.; Permagon Press: Oxford, 1991; Vol. 4, pp. 833-863; Hiroshige et al. Tetrahedron Lett. 1995,36,4567) and Stille couplings (see, Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986,25,508; Despande, M. S.
Tetrahedron Lett. 1994,35,5613) Sonogashira/Castro-Stephens couplings (see, Sonogashira, et al. Tetrahedron Lett. 1975,4467; Stephens, R. D.; Castro, C. E. J. Org. Chem. 1963,28,3313; Young et al. J. Am. Chem. Soc. 1994,116,10841; Collini et al. Tetrahedron Lett. 1997,38, 7963; Odingo, J.; Sharpe, B. A.; Oare, D. Presented at the 213th National Meeting of the American Chemical Society, San Francisco, CA, April 1997; ORGN 574), Suzuki and Stille couplings, and carbonylations. More specifically, Figure 33 depicts palladium cross-coupling reactions at the aryl iodide using the Sonogashira-Castro-Stephens, Suzuki and Stille reactions.
Furthermore, resulting aryl alkynes can undergo rhodium-catalyzed hydroacylation and azide cycloaddition as shown in Figure 34, and nitrone and nitrile oxide cycloaddition as shown in Figure 35.
In another particularly preferred embodiment, the isoquinuclidine core as shown in Figure 36, can be diversified by reaction at potential diversity dites such as the amine, the bridge carbon and the amide functionality. For example, the amide may be functionalized using a Mitsunobu reaction to generate alcohols such as straight chain, branched, and cyclic alcohols. In
particularly preferred embodiments, the alcohol should not have an unprotected site that could be acylated, such as an amine, or thiol. The bridge amine can be subjected to reaction to yield chloroformates, by reacting alcohols with phosgene, and anything that can acylate or alkylate an amine, such as alkyl bromides, mesylates, and aldehydes to name a few. The bridge carbon may also be functionalized to yield an allyl and any allyl derivative of allyltributyltin, thiazole or indole, but is not limited to these functionalities. Furthermore, the carboximide may be functionalized by reaction with reagents including, but limited to, amines, amino acids, and alcohols. Figure 37 also depicts the use of amino acids to generate more diversity. Additionally, Figure 38 depicts the potential diversity sites for the cup-like pentacyclic scaffold structure.
One of ordinary skill in the art will realize that the above examples are representative of the reactions that can be used to diversify the templates, scaffolds, compounds, and libraries of compounds of the presently claimed invention and are not intended to be exclusive. Rather, all equivalents thereof are intended to be within the scope of the presently claimed invention. A skilled artisan will be able to readily identify those reagents capable of reacting to create further diversity at selected sites in the inventive scaffold structures to generate compounds and libraries of compounds reminiscent of natural products.
Combinatorial Methods for the Synthesis of Complex Natural Product-Like Libraries According to the method of the present invention, the synthesis of libraries from the above-described scaffold structures can be performed using established combinatorial methods for solution phase, solid phase, or a combination of solution phase and solid phase synthesis techniques. The synthesis of combinatorial libraries is well known in the art and has been reviewed (see, e. g.,"Combinatorial Chemistry", Chemical and Engineering News, Feb. 24,1997, p. 43; Thompson, L. A., Ellman, J. A., Chem. Rev. 1996,96,555.) One of ordinary skill in the art will realize that the choice of method will depend upon the specific number of compounds to be synthesized, the specific reaction chemistry, and the availability of specific instrumentation, such as robotic instrumentation for the preparation and analysis of the inventive libraries. In particularly preferred embodiments, the reactions to be performed on the inventive scaffolds to generate the libraries are selected for their ability to proceed in high yield, and in a stereoselective fashion, if applicable.
In one embodiment of the present invention, the inventive libraries are generated using a solution phase technique. Traditional advantages of solution phase techniques for the synthesis of combinatorial libraries include the availability of a much wider range of organic reactions, and
the relative ease with which products can be characterized. Notable disadvantages of solution phase techniques includes the inability to easily synthesize libraries of compounds containing very large numbers, such as one million or more library members, because one reaction vessel must be provided for each library member, and the inability to use excess reagents without time- consuming purification steps, such as chromatography. Recently, however, advances have been made in solution. phase synthesis techniques such as the use of a"covalent scavenger"which selectively removes from solution via covalent bond formation. The"covalent scavenger"is essentially a solid phase bound nucleophile or electrophile that reacts with these excess reagents.
(Kaldor, Eli Lilly, Frechet et al., Tetrahedron Lett., 21,617 (1980)). In a preferred embodiment, for the generation of a solution phase combinatorial library, a parallel synthesis technique is utilized, in which all of the products are assembled separately in their own reaction vessels. In a particularly preferred parallel synthesis procedure, a microtitre plate containing n rows and m columns of tiny wells which are capable of holding a few milliliters of the solvent in which the reaction will occur, is utilized. It is possible to then use n variants of reactant A, such as a carboxylic acid, and m variants of reactant B, such as an amide to obtain n x m variants, in n x m wells. One of ordinary skill in the art will realize that this particular procedure is most useful when smaller libraries are desired, and the specific wells can provide a ready means to identify the library members in a particular well.
In another more particularly preferred embodiment of the present invention, a solid phase synthesis technique is utilized, in which the desired scaffold structures are attached to the solid phase directly or though a linking unit, as discussed above. Advantages of solid phase techniques include the ability to more easily conduct multi-step reactions and the ability to drive reactions to completion because excess reagents can be utilized and the unreacted reagent washed away. Perhaps one of the most significant advantages of solid phase synthesis is the ability to use a technique called"split and pool", in addition to the parallel synthesis technique, develped by Furka. (Furka et al., Abstr. 14th Int. Congr. Biochem., Prague, Czechoslovakia, 1988,5,47; Furka et al., Int. J. Pept. Protein Res. 1991, 37, 487; Sebestyen et al., Bioorg. Med.
Chem. Lett., 1993,3,413.) In this technique, a mixture of related compounds can be made in the same reaction vessel, thus substantially reducing the number of containers required for the synthesis of very large libraries, such as those containing as many as or more than one million library members. As an example, the solid support scaffolds can be divided into n vessels, where n represents the number species of reagent A to be reacted with the scaffold structures. After reaction, the contents from n vessels are combined and then split into m vessels, where m
represents the number of species of reagent B to be reacted with the scaffold structures. This procedure is repeated until the desired number of reagents is reacted with the scaffold structures to yield the inventive library.
The use of solid phase techniques in the present invention may also include the use of a specific encoding technique. Specific encoding techniques have been reviewed by Czarnik.
(Czarnik, A. W., Current Opinion in Chemical Biology, 1997,1,60.) As used in the present invention, an encoding technique involves the use of a particular"identifiying agent"attached to the solid support, which enables the determination of the structure of a specific library member without reference to its spatial coordinates. One of ordinary skill in the art will also realize that if smaller solid phase libraries are generated in specific reaction wells, such as 96 well plates, or on plastic pins, the reaction history of these library members may also be identified by their spatial coordinates in the particular plate, and thus are spatially encoded. It is most preferred, however for large combinatorial libraries, to use an alternative encoding technique to record the specific reaction history.
Examples of particulary preferred alternative encoding techniques that can be utilized in the present invention include, but are not limited to, spatial encoding techniques, graphical encoding techniques, including the"tea bag"method, chemical encoding methods, and spectrophotometric encoding methods. Spatial encoding refers to recording a reaction's history based on its location. Graphical encoding techniques involve the coding of each synthesis platform to permit the generation of a relational database. Examples of preferred spectrophotometic encoding methods include the use of mass spectroscopy, fluorescence emission, and nuclear magnetic resonance spectroscopy. In a most preferred embodiment, chemical encoding methods are utilized, which uses the structure of the reaction product to code for its identity. Decoding using this method can be performed on the solid phase or off of the solid phase. One of ordinary skill in the art will realize that the particular encoding method to be used in the present invention must be selected based upon the number of library members desired, and the reaction chemistry employed.
In an exemplary embodiment of the method of the present invention, more than 2,000,000 members of a shikimic acid based library can be generated. The preferred method of the invention begins with the attachment of one or more spacers to the linking reagent, preferably a photolinker. Subsequently, the resin can be pooled, divided into two portions, and one enantiomer of epoxycyclohexenol carboxylic acid coupled to each pool. After pooling and division into three portions, iodobenzyl nitrone acids can be coupled resulting in a total of 18
tetracyclic scaffolds. The stereoselective synthesis of the library of complex compounds reminiscent of natural products can be completed by reaction with 30 terminal alkynes, 62 primary amines, and finally 62 carboxylic acids, employing a split and pool technique at each step. Each of the reagents utilized are preferably selected for their ability to generate diversity and for their ability to react in high yield. As one of ordinary skill in the art will realize, the use also of a skip codon (Combs et al. J. Am. Chem. Soc. 1996,118,287), or"blank", at each step yields further diversity. Furthermore, in particulary preferred embodiments, after each reaction step, the beads are"tagged"to encode the particular reaction choice employed. Preferred alkynes for use in the presently claimed invention include, but are not limited to acetaldehyde ethyl propargyl acetal, tert-butyl 1-methyl-2-propynyl ether, 4- (tert-butyl) phenylacetylene, tert- butyldimethylsilyl acetylene, 2- (3-butynloxy) tetrahydro-2H-pyran, 1-chloro-4-ethynylbenzene, 1,4-decadiyne (50% in hexane), 1,5-decadiyne, 3-dibutylamino-1-propyne, m-diethynylbenzene, 3,3-dimethyl-1-butyne, 1-dimethylamino-2-propyne, 1-dodecyne, ethyl ethynyl ether (50% in hexanes), ethynyl p-tolyl sulfone, 1-ethynyl-4-fluorobenzene, 1-ethynylcyclohexene, ethynylestradiol 3-methyl ether, 2-ethynylpyridine, 4-ethynyltoluene, 1,5-hexadiyne (50% in hexane), 1-hexyne, 5-hexynenitrile, methyl propargyl ether, 2-methyl-1-buten-3-yne, methyl-N- propargylbenzylamine, 1,8-nonadiyne, 1-pentyne, 4-phenyl-1-butyne, 3-phenyl-1-propyne, phenylacetylene, propargyl ether, propargyn-lH-benzotriazole, N- (propargyloxy) phthalimide, N- propargylphthalimide, propargyltriphenylphosphonium bromide, proiolaldehyde diethyl acetal, tetrahydro-2- (2-propynyloxy)-2H-pyran, triethylsilylacetylene, tripropargylamine, 2- (3- burynloxy) tetrahydro-2H-pyran, 3,5-dimethyl-1-hexyn-3-ol, 1, 1-diphenyl-2-propyn-1-ol, 1- ethynyl-1-cyclohexanol, 1-ethynyl-4-fluorobenzene, 9-ethynyl-9-fluorenol, 1- ethynylcyclopentanol, 1-heptyne, 3-methyl-1-pentyn-3-ol, 2-phenyl-3-butyn-2-ol, and propiolaldehyde diethyl acetal. Preferred primary amines include, but are not limited to, allylamine, 2-amino-1-propene-1, 1, 3-tricarbonitrile, 3-amino-1 H-isoindole hydrochloride, 3- amino-5-methylisoxazole, aminoacetaldehyde diethyl acetal, aminoacetaldehyde dimethyl acetal, aminoacetonitrile bisulfate, 4-(2-aminoethyl) benzenesulfonamide, 4-(2-aminoethyl) morpholine, 2- (2-aminomethyl) pyridine, 1- (2-aminoethyl) pyrrolidine, 2-aminoindan hydroxchloride, (R)- (-)- 1-aminoindan, (S)- (+)-l-aminoindan, 2- (aminomethyl)-15-crown-5, 4- (aminomethyl) benzenesulfonamide hydrochloride, (aminomethyl) cyclopropane, 2- pyrenemethylamine hydrochloride, 3- (aminomethyl) pyridine, 4- (aminomethyl) pyridine, 3- aminopropionitrile fumarate, 1- (3-aminopropyl)-2-pyrrolidinone, 1- (3-aminopropyl) imidazole, 3-aminopropyltrimethoxysilane, (R)- (+)-3-aminoqauinuclidine dihydrochloride, (S)- (-)-3-
aminoquinuclidine dihydrochloride, ammonia (0.5 M in dioxane), benzylamine, S- benzylcysteamine hydrochloride, (R)- (+)-bomylamine, butylamine, cyclobutylamine, cyclohexanemethylamine, cyclohexylamine, cyclopentylamine, cyclopropylamine, (R)- (+)- cycloserine, 3- (diethoxymethylsilyl) propylamine, 3,4-dimethoxyphenethylamine, 4- (dimethylamino) benzylamine dihydrochloride, 3-dimethylaminopropylamine, N, N- dimethylethylenediamine, ethylamine (2.0 M in THF), 1-ethylpropylamine, 2-fluoroethylamine hydrochloride, 4-fluorophenethylamine, furfurylamine, geranylamine, 3-fluorobenzylamine, (1R, 2R, 3R, 5S)- (-)-isopinocampheylamine, (1S, 2S, 3S, 5R)- (+)-isopinocampheylamine, isopropylamine, 2-methoxybenzylamine, 4-methoxybenzylamine, 2-methoxyethylamine, 2- methoxyphenethylamine, 3-methoxyphenethylamine, 4-methoxyphenethylamine, 3- methoxypropylamine, methylamine (2.0M in THF), (-)-cis-myrtanylamine, 1- napthylenemethylamine, 3-nitrobenzylamine hydrochloride, 4-nitrophenethylamine hydrochloride, octylamine, phenethylamine, trans-2phenylcyclopropylamine hydrochloride, 2- phenylglycinonitrile hydrochloride, piperonylamine, propargyl amine, (R)- (-)- tetrahydrofurfurylamine, (S)- (+)-tetrahydrofurfurylamine, N, N, 2,2-tetramethyl-1,3- propanediamine, 2-thiopheneetthylamine, 2,2,2-trifluoroethylamine, tryptamine, veratrylamine, 2- (2-aminoethyl) pyridine, 3- (aminomethyl) pyridine, (R)- (-)-sec-butylamine, (S)- (+)-sec- butylamine, (R)- (-)-1-cyclohexylethylamine, (S)- (+)-1-cyclohexylethylamine, isoamylamine, (R)- (+)-a-methylbenzylamine, (S)- (-)-1- (l-napthyl) ethylamine, 4- (trifluoromethyoxy) benzylamine, and 3- (trifluoromethyl) benzylamine. Preferred carboxylic acids include, but are not limited to, acetic acid, 4-acetoxybenzoic acid, acetylsalicyclic acid, acrylic acid, m-anisic acid, o-anisic acid, p-anisic acid, benzoic acid, 2-butynoic acid, (3- carboxypropyl) trimethylammonium chloride, 3-chloropropionic acid, crotonic acid, cyanoacetic acid, 3-cyanobenzoic acid, 4-cyanobenzoic acid, cyclohexanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentylacetic acid, cyclopropanecarboxylic acid, 3,4-dihydro- 2,2-dimethyl-4-oxy-2H-pyran-6-carboxylic acid, 1, 4-dihydro-2-methylbenzoic acid, 3- dimethylaminobenzoic acid, 4-dimethylaminobenzoic acid, N, N-dimethylglycine, ferroceneacetic acid, formic acid, trans-3-furanacrylic acid, 2-furoic acid, 3-furoic acid, furylacrylic acid, 2,4-hexadienoic acid (Sorbic acid), isobutyric acid, isonicotinic acid, isovaleric acid, levulinic acid, linolenic acid, (+)-menthoxyacetic acid, (-)-menthoxyacetic acid, methacrylic acid, methoxyacetic acid, (R)- (-)-a-methoxyphenylacetic acid, (S)- (+)-a- methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3-methoxyphenylacetic acid, 4- methoxyphenylacetic acid, 1-methyl (1S, 2R)- (+)-cis-1, 2,3,6-tetrahydrophthalate, mono-methyl
glutarate, mono-methyl phthalate, mono-methyl terephthalate, [lR- (l-a, 2b, 3a)]- (+)-3-methyl-2- (nitromethyl)-5-oxocyclopentaneacetic acid, 4- (3-methyl-5-oxo-2-pyrazolin-1-yl) benzoic acid, 6- methylchromone-2-carboxylic acid, 3,4- (methylenedioxy) phenylacetic acid, 1-methylindole-2- carboxylic acid, nicotinic acid, 5-nitro-2-furoic acid, 4-nitrobenzoic acid, 4-nitrophenylacetic acid, 3-nitropropionic acid, 2-norbornaneacetic acid, orotic acid monohydrate, (S)- (+)-2-oxo-4- phenyl-3-oxazolidineacetic acid, anti-3-oxotricyclo [2.2.1.0 (2,6)] heptane-7-carboxylic acid, phenylacetic acid, phenylpropiolic acid, phthalylsulfathiazole, picolinic acid, propionic acid, 2- pyrazinecarboxylic acid, 2-pyridylacetic acid hydrochloride, 3-pyridylacetic acid hydrochloride, 4-pyridylacetic acid hydrochloride, (2-pyrimidylthio) acetic acid, pyruvic acid, tetrahydro-2- furoic acid, tetrahydro-3-furoic acid, thioctic acid, 2-thiopheneacetic acid, 3-thiopheneacetic acid, 2-thiophenecarboxylic acid, 3-thiophenecarboxylic acid, 2-thiopheneglyoxylic acid, (a, a, a- trifluoro-p-tolyl) acetic acid, vinylacetic acid, acetoxyacetic acid, 2-benzofurancarboxylic acid, cinnoline-4-carboxylic acid, 3,5-diido-4-pyridone-1-acetic acid, 3,3-dimethylacrylic acid, ferrocenecarboxylic acid, 5-methoxy-1-indanone-3-acetic acid, 1-methyl-2-pyrrolecarboxylic acid, 3-oxo-1-indancarboxylic acid, trans-3- (3-pyridyl) acrylic acid, 3- (2-thienyl) acrylic acid, a, a, a-trifluoro-m-toluic acid, a, a, a-trifluoro-o-toluic acid, and a, a, a-trifluoro-p-toluic acid.
Additionally, Figure 39 depicts a plan for the synthesis of over 46.5 million complex molecules.
In another exemplary embodiment, the present invention provides a method for synthesizing over 30,000,000 members of an isoquinuclidine library as depicted in Figure 40.
First, 63 derivatized isonicotinamide templates are provided and reacted with allyltributyltin and TeocCl to yield a racemic mixture, thus providing 126 compounds. Subsequent reaction with maleic anhydride, 63 amino acids, and 63 amines, yields 500,094 compounds. Further reaction with 3 nitrone isomers, and 20 arylboronic acids yields over 30,000,000 complex compounds reminiscent of natural products.
Subsequent characterization of the library members can be performed using standard analytical techniques, such as mass spectrometry, Nuclear Magnetic Resonance Spectroscopy, and gas chromatrograpy. One of ordinary skill in the art will realize that the selection of a particular analytical technique will depend upon whether the inventive library members are in the solution phase or on the solid phase. As but one example, Figures 41 though 44 more particularly depict the synthesis and analysis of a test library of shikimic acid-based compounds; these examples are not intended to limit the scope of the present invention, however. In yet another example, Figure 45 depicts the synthesis of a test library of isoquinuclidine-based compounds, as also described in more detail in the Examples.
Uses The methods, compounds and libraries of the present invention can be utilized in various disciplines. For example, one aspect of the present invention concerns a method for identifying natural product-like small molecules from the inventive libraries of compounds, which modulate the biological activity of a biological target, such as a protein, nucleic acid, lipid or combination thereof. In one preferred embodiment, the compounds of the present invention are utilized in chemical genetics assays to alter, i. e. inhibit or initiate, the action of such biological molecules.
Alternatively or additionally, the compounds may be used in in vitro assays, or any other system that allows detection of a chemical or biological function.
In a particularly preferred embodiment of the invention, one or more inventive compounds is contacted with a biological target having a detectable biochemical activity. Such biological targets include, for example, enzymes, receptors, subunits involved in the formation of multimeric complexes. Such multimeric complex subunits may be characterized by catalytic capabilities (such as, for example, an ability to catalyze substrate conversion), or may alternatively be primarily active in binding to one or more other molecule. The biological target can be provided in the form of a purified or semi-purified composition, a cell lysate, a whole cell or tissue, or even a whole organism. The level of biochemical activity is detected in the presence of the compound, and a statistically significant change in the biochemical activity, relative to the level of biochemical activity in the absence of the compound, identifies the compound as a modulator, e. g. inhibitor or potentiator of the biological activity of the target protein. In some cases, particularly where assays are done on whole cells or organisms, the effect of the chemical compound may be to alter the amount, in addition to or instead of the activity, of the particular biological target."Modulators", therefore, are chemical compounds that alter the level or activity of a particular target molecule.
In one particularly preferred embodiment of the present invention, multiple compounds are assayed simultaneously in a high-throughput format, preferably allowing simultaneous analysis of at least 500,000 compounds, preferably at least 1,000,000 compounds, and most preferably at least or more than 2,000,000 compounds. One such format, referred to herein as "nanodroplet format"is described in US patent application 08/951,930, entitled"Droplet Assay System", which is incorporated herein by reference. In brief, the format involves ordered or stochastic arrays of small volume (preferably about 50-200 nL, most preferably about 100 nL) droplets into which chemical compounds to be assayed are distributed. Those of ordinary skill in the art will readily appreciate that this nanodroplet format can be employed for any of a large
variety of assays. Any assay whose result may be observed in the context of a discrete liquid droplet is appropriate for use with the present invention. Preferred read-out assays for use in accordance with the present invention analyze chemical or biological activities of test compounds. Read-out assays can be designed to test in vitro or in vivo activities. Example 1 describes the preferred droplet assay procedure, and Examples 2-4 describe particularly preferred assays for analysis of the inventive chemical compounds.
As discussed above, once a specific desired effect on a biological target has been associated with a particular compound of the inventive library, the compounds of the present invention may be utilized as a therapeutic agent for a particular medical condition. A therapeutic agent for use in the present invention may include any pharmacologically active substances that produce a local or systemic effect in animals, preferably mammals, or humans. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human. The therapeutic agent may be administered orally, topically or via injection by itself, or additionally may be provided as a pharmaceutical composition comprising the therapeutic agent and a biologically acceptable carrier. The inventive compositions can be, but are not limited to an aqueous solutions, emulsions, creams, ointments, suspensions, gels, and liposomal suspensions. Particularly preferred biologically acceptable carriers include but are not limited to water, saline, Ringer's solution, dextrose solution and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol, and vegetable oils. It is also possible to include suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents, for example including but not limited to BHA, BHT, citric acid, ascorbic acid, and tetracycline. The therapeutic agents of the presently claimed invention may also be incorporated or encapsulated in a suitable polymer matrix or membrane, thus providing a sustained-release delivery device suitable for implantation near the site to be treated locally.
As one of ordinary skill in the art will realize, the amount of the therapeutic agent required to treat any particular disorder will of course vary depending upon the nature and severity of the disorder, the age and condition of the subject, and other factors readily determined by one or ordinary skill in the art.
In alternative embodiments, the compounds and libraries of the present invention may also be used for the development of cosmetics, food additives, pesticides, and lubricants to name a few. Furthermore, the compounds and libraries of the present invention may also be used for
the development of novel catalysts and materials. For example, the inventive compounds may be useful as ligands for transition metal catalysts and the inventive libraries may be useful for the rapid identification of novel ligands. These compounds and libraries of compounds may also function by acting in concert with a particular transition metal catalyst to effect a particular desired chemical reaction. Additionally, the inventive compounds and libraries of compounds are also useful in the area of materials science. Because of the reactive moieties present in these compounds, molecules such as lipids and other polymeric materials may be attached and thus generate potentially important biomaterials.
One of ordinary skill in the art will realize that the present invention is not intended to be limited to the abovementioned uses, but rather may be employed in many contexts and disciplines.
Furthermore, the specific examples presented below, and also the specific examples presented in the Appendix (for the more detailed experimentals for the synthesis of compounds and libraries of compounds, the characterization of said compounds and libraries of compounds, and the testing of the biological activity of said compounds and libraries of compounds) are intended to more particulary describe the present invention, but are not intended to limit the scope of the presently claimed invention.
Examples Example 1 : Nanodroplet assay : The ability of the preferred procedure utilized for the library synthesis to controllably release compounds from the individual 90 i diameter beads into nanodroplet containing engineered wells enables the use of these miniaturized cell-based assays to detect specific characteristics of library members. In a particularly preferred embodiment of the invention, the compounds in an inventive encoded combinatorial library are attached to beads through a photocleavable linker. Each bead is labeled with a tag that identifies the bound compound.
Additionally, the concentration of the test compound released in the droplet can be controlled by controlling the time of exposure to UV radiation. The amount of compound released in any particular experiment, of course, will depend on the efficiency of bead loading and the extent of bead functionalization. Figure 46 depicts the photorelease of an inventive compound.
In particular, the present invention specifically contemplates the screening of the inventive compounds, especially libraries of these compounds in assays designed to detect their
protein-binding properties (e. g., small molecule inactivation of protein targets or small molecule activation of protein targets).
Example 2: Assay to detect activation of gene expression: The inventive compounds and libraries of compounds synthesized by the inventive method are tested for activation of a luciferase reporter gene with pathway specific promoters such as a TGF-P responsive promoter/enhancer. The luciferase gene is a particularly preferred reporter gene because the determination of the expressed luciferase enzyme is rapid, easy to perform and detection is extremely sensitive. Furthermore, luciferase is a monomeric protein that does not require any post-translational processing and can thus be measured as a genetic reporter immediately upon translation. As shown in Figure 47,8 different pools, each containing 64 different isolated compounds selected from the shikimic acid test library as described in Appendix A, were tested for the ability to induce luciferase activity and all were found to activate the reporter gene to various extents. Interestingly, KC233, an isolated compound selected from the inventive isoquinuclidine library, does not activate the reporter gene and furthermore also prohibits TGF-ß from activating the reporter gene. Figure 48 depicts this in greater detail.
These results suggest that compounds 43 {5, 8,1} and 43 {6, 8,1}, as described in the library synthesis below, are useful for the activation of a signaling pathway that results in activation of the 3TP promoter, and that KC233, a member of the isoquinuclidine library is effective in preventing TGF-ß-induced activation of the 3TP promoter/enhancer. One of ordinary skill in the art will realize that other reporter genes can be utilized to test the ability of the inventive compounds and libraries of compounds to promote different cellular responses. Exemplary reporter genes include, but are not limited to secreted alkaline phosphatase (seap), P-lactamase, chloramphenicol transferase (cat), and green fluorescent protein.
Example 3 : Cell Proliferation Studies In another illustrative embodiment, the inventive compounds and libraries of compounds were tested for their ability to inhibit cell proliferation in mink lung cells. Figure 49 depicts the ability of each of the specific pools of 64 compounds (1, uM per compound) selected from the shikimic acid test library to inhibit cell proliferation. These results suggest that the inventive compounds and libraries of compounds are useful as inhibitors of cell proliferation, and thus may also be useful as potential therapeutics for cancer or other conditions such as autoimmune diseases in which the inhibition of cell proliferation, specifically tumor cell proliferation or hematopoietic cell growth is important. Furthermore, Figure 50 depicts the ability of KC233, a member of the inventive isoquinuclidine library (KC233 shown in Figure 48), to arrest mink
lung cells in the S-phase of the cell cycle. After treatment of mink lung cells with 10 uM KC233 for 40 hours, the DNA content corresponding to the G1, G2 and M phases decreases, and the corresponding DNA content associated with the S phase increases. Thus, these results suggest that KC233 is useful as a therapeutic for arresting lung cell cancers. Additionally, the ability of KC233 to act as a general cell cycle arresting agent suggests its ability to function analogously to other cell-cycle arresting drugs. For example, hydroxyurea, the currently cytotoxic agent of choice for treatment of chronic myelocytic leukemia, also arrests cells in the S-phase. Another example of a cell-cycle arresting drug in which the cell cycle is arrested in mitosis (M-phase) is the well-known anticancer drug paclitaxel (Taxol), currently approved for ovarian cancer and head and neck cancer. One of ordinary skill in the art will realize that these represent only a few examples of cell-cycle arresting drugs, and that the inventive compounds and libraries of compounds may function as analogues of other cell-cycle arresting drugs.
General Materials and Methods for Assays: Cell Culture: MvlLu mink lung epithelial cells were obtained form the American Type Culture Collection (catalog # CCL64). Clone 6f is a stably transfected derivative of MvlLu cells containing the p3TPLux reporter plasmid as well as the construct MFp, 3Tl [D] (see Stockwell, B. R.; Schreiber, S. L. Curr. Biol. 1998,8,761). MvlLu and 6f cells were cultured in DMEM with 10% FBS, 100 units/mL penicillin G sodium, 100 pg/mL streptomycin sulfate and 100 uM each of the amino acids Ala, Asp, Glu, Gly, Asn, and Pro.
Luciferase Assay: 2.0 x 105 6f cells were seeded in each 35 mm well of a six well dish in 10% FBS. After 20 hours, the cells were washed once and incubated in DMEM containing 0.2 % FBS and the non-essential amino acids (NEAA) and the reagent of interest (e. g., library pool, KC233, or TGF-P) for 25 to 30 hours. Cells were incubated on ice for 15 minutes, washed three times with HBSS and lysed in extraction buffer (25 mM glycylglycine, pH 7.8,15 mM MgSO4, 4 mM egta, 1 Triton X, 1 mM DTT, 1 mM PMSF) by shaking gently at 4°C for 30 minutes. The lysates were centrifuged for 5 minutes at 10,000 g at 4°C and stored on ice. 100 pL of lysate was added to 150 u. L of assay mixture (25 mM glycylglycine pH 7.8,15 mM MgSO4, 4 mM egta, 15 mM K2HPO4 pH 7.8,1 mM DTT, 4 mM ATP) and 150 uL ofluciferin buffer (25 mM glycylglycine pH 7.8,15 mM MgSO4, 4 mM egta, 10 mM DTT, 167 uM D- luciferin). This mixture was placed in a 500 uL microfuge tube inside a glass scintillation vial, and luminescence was detected by counting in single photon mode (SPM) on a Beckman LS 6500 liquid scintillation counter for 15 seconds. The error bars reported represent plus or minus one standard deviation. All experiments were performed multiple times in triplicate.
Growth Inhibition Assay: MvlLu cells were seeded in 6 well clusters (20,000 cells per well) and allowed to attach overnight in 10% serum. Media was changed to 1% FBS with or without the test compound. After four days the cells were washed, trypsinized and counted. The cell number reported represent live cells, since dead cells detach and are washed away by this protocol.
Example 4: Testing the inventive libraries for the ability to act as a ligand for the receptor of human growth hormone: Another interesting application for the complex radially arrayed combinatorial libraries of the presently claimed invention is as a ligand for the receptor for human growth hormone, which induces homodimerization of the receptor and initiates the intracellular growth hormone signalling pathway, as depicted in Figure 3. The"hot spot", which is a small patch of residues identified as being responsible for the majority of the binding energy between hGH and its receptor is an excellent target for the library.
Example 5: Test Library Synthesis for ibogamine-like compounds (as shown in Figure 45) : With the viability of the synthetic route proven, rigorous quality control experiments required for the synthesis of large collections of polycyclic alkaloid natural product-like molecules have been undertaken. Polystyrene resin (400-450 um) loaded with a photo-cleavable linker was chosen for the building block screening studies. The resin was chosen from a screen of solid supports with a photo-cleavable linker because it provided the best balance between loading and reaction kinetics.
As shown in Figure 4 5 the building block studies began with the coupling of eleven Fmoc-amino acids onto polystyrene resin (400-450 um) loaded with a photocleavable linker.
After removal of the Fmoc protecting group and acylation with isonicotinoyl chloride, a portion of each sample was photolyzed and analyzed by TLC and LCMS. Ten of the eleven building blocks were converted in >90% purity to the desired product (see Chart A below).
In the second step, each of the resulting isonicotinamides was treated with allyltributyltin and TeocCl to yield N-acyl-1, 2-dihydropyridines. A portion of each sample was photolyzed and analyzed by TLC and LCMS. All samples were converted in >90% purity to the desired product (See Chart B below).
In the third step, each of the ten N-acyl-1, 2-dihydropyridines was then reacted with maleic anhydride. Each sample was photolyzed and analyzed by TLC and LCMS to ensure that all of the building blocks from the first step would perform equally well in two batch steps (a-
allylation of N-acylpyridinium salt and Diels-Alder reaction). All samples were converted in >90% purity to the desired product (see Chart C below).
Assured that all of the building blocks selected in the first step could withstand the two batch steps, a single isoquinuclidine was scaled up for the next step. In the fourth step, an isoquinuclidine with glycine in the first building block position was scaled up for testing in the imide forming reaction. Of 20 amines tested in the 2-pyridone mediated imide forming reaction 18 were converted in > 90% purity to the desired product (See Chart D below).
In the final building block testing step, a single isoquinuclidinium salt was scaled up for building block testing in the nitrogen alkylation/acylation reaction (see Chart E below).
CHART A
CHART B
Chart C
CHART D
CHARTE
APPENDIX A: Methods and Experimentals for the Synthesis and Evaluation of a Library of Shikimic Acid Based Library of Polycyclic Small Molecules I. General Experimental Details: General. Solution phase reactions were performed in oven-or flame-dried glassware under positive N2 pressure. Small-scale solid phase reactions (5-10 mg resin) were performed in 500 JL polypropylene Eppendorf tubes (VWR Scientific Products; 20170-310) with mixing provided by a Vortex Genie-2 vortexer (VWR 58815-178, setting V2-V3) fitted with a 60 microtube insert. Medium-scale solid phase reactions (20-500 mg resin) were performed in 2 mL fritted polypropylene Bio-SpinW chromatography columns (Bio-Rad Laboratories, Hercules, CA; 732-6008) or 10 mL fritted polypropylene PD-10 columns (Pharmacia Biotech, Piscataway, NJ; 17-0435-01) with 360° rotation on a Barnstead-Thermolyne LabquakeTM Shaker (VWR 56264-306). Large-scale solid phase reactions (>500 mg resin) were performed in silanized 50 or 100 mL fritted glass tubes equipped for vacuum filtration and N2 bubbling. The tubes were silanized by treatment with 20% dichlorodimethylsilane/CH2Cl2 for 15 min, MeOH for 15 min, followed by oven heating at 120 °C for at least 2 h.
After small-scale reactions, resin samples were transferred to 2 mL BioSpin columns via vacuum cannula. Resin samples in polypropylene columns were washed on a Vac-Mant) Laboratory Vacuum Manifold (Promega, Madison, WI; A7231) fitted with nylon 3-way stopcocks (Biorad 732-8107). Resin samples in glass tubes were washed in the reaction vessels with alternating periods of N2 bubbling and vacuum draining. The following standard wash procedure was used: 3 x THF, 3 x DMF (Method A) or NMP (Method B), 3 x iPrOH, 3 x DMF/NMP, 3 x CH2C12, 3 x DMF/NMP, 3 x CH3CN, 3 x THF, 3 x CH2C12.
Resin samples were then transferred via spatula to 500 pL Eppendorf tubes and suspended in Ar-degassed HPLC grade CH3CN. The tubes were wrapped with parafilm and fixed with rubber bands to a 2"x 3"piece of cardboard that had been wrapped with aluminum foil. The tubes were then placed on a vortexer (setting S1-S2) under a UVP High Intensity Longwave UV Lamp (Fisher Scientific, Pittsburgh, PA; 11-984-79) at a distance of 3 inches ("21. 7 mW/cm2). Photocleavage products were recovered by filtration and evaporation or by sampling of the supernatant.
Atom numbers shown in structures below refer only to NMR peak assignments and not to CAS or trivial nomenclature. Compound numbers followed by R represent molecules still attached to the solid support.
Sources. Reagents were obtained from Advanced Chemtech (Louisville, KY), Aldrich Chemical (Milwaukee, WI), Eastman Chemicals (Rochester, NY), Fluka (Milwaukee, WI), GFS Chemicals (Powell, OH), Novabiochem (San Diego, CA), Pierce (Rockford, IL), or Strem Chemicals (Newburyport, MA) and used without further purification. Tentagel S NH2 was obtained from Rapp Polymere (Germany). Solvents were obtained from Mallinckrodt or E.
Merck. Wash solvents were used as received. Reaction solvents were distilled under N2 as follows: Tetrahydrofuran (THF), diethyl ether (Et20), and dimethoxyethane (DME) from sodium/benzophenone ketyl; methylene chloride (CH2C12), ethyl acetate (EtOAc), benzene, toluene, pyridine, 2,6-lutidine, and N, N-diisopropylethylamine (DIPEA) from calcium hydride; methanol (MeOH) from magnesium methoxide. Anhydrous NN-dimethylformamide (DMF), N, N-dimethylacetamide (DMA), 1-methyl-2-pyrrolidinone (NMP), and trimethyl orthoformate (TMOF) were obtained from Aldrich in SureSeal bottles. Water (H20) was double distilled.
Purification and Analysis. Flash chromatography was performed on E. Merck 60 230- 400 mesh silica gel. TLC was performed on 0.25 mm E. Merck silica gel 60 F254 plates and visualized by UV (254 nm) and cerium ammonium molybdate (CAM). HPLC was performed on a Nest Group (Southborough, MA) Hypersil C 18 100 A 3 u 4.6 mm x 6 cm column using a flow rate of 3 mL/min and a 4 min gradient of 0-99.9% CH3CN in H20/0. 1% TFA, constant 0.1% MeOH with diode array LTV detection. Melting point determinations were performed on a Laboratory Devices (Cambridge, MA) Mel-temp apparatus and are uncorrected (benzoic acid, lit.
122-123 °C, found 119.0-121.5 °C). Optical rotations were measured on a Perkin-Elmer 241 Polarimeter. IR spectra were recorded on a Nicolet 5PC FT-IR Spectrometer with peaks reported in cm-1. NMR spectra were recorded on Varian Inova 600 and Bruker DMX500, AM500, and AM400 instruments. Chemical shifts are expressed in ppm relative to TMS (0.00 ppm) or residual solvent signals (CDC13 7.26 ppm/77.0 ppm; CD3CN 1.93 ppm/1.3 ppm, CD30D 3.30 ppm/49.0 ppm). Peak assignments were made based on extensive homonuclear decoupling and/or two-dimensional DQF-COSY, TOCSY, and NOESY experiments. Mass spectra were obtained on JEOL AX-505H or SX-102A mass spectrometers by electron impact ionization (EI), chemical ionization (CI) with ammonia (NH3), or fast atom bombardment ionization (FAB) with glycerol or 3-nitrobenzyl alcohol/sodium iodide (NBA/NaI) matrices. Time-of-flight electrospray ionization (TOF-ESI) data were obtained on a Micromass LCT mass spectrometer.
Tandem high pressure liquid chromatography-mass spectrometry (LC-MS) data were obtained on a Micromass Platform II mass spectrometer in atmospheric pressure chemical ionization (AP- CI) mode attached to a Hewlett-Packard Series 1050 HPLC system. LC-MS chromatography was performed on a Hewlett-Packard ODS Hypersil 5, 2.1 mm x 10 cm column using a flow
rate of 0.4 mL/min and a 5 min gradient of 30-90% CH3CN in H20, constant 0.1% formic acid with detection at 214 nm.
Atom numbers shown in structures below refer only to NMR peak assignments and not to CAS or trivial nomenclature. Compound numbers followed by R represent molecules still attached to the solid support.
II. General Description of Experimental Plan: Library Validation Protocols. Split-pool synthesis provides the theoretical means to synthesize the full matrix of every combination of building blocks in a multi-step synthesis.
Such large numbers of molecules will likely be required for successful outcomes in chemical genetic screens. However, these syntheses present enormous analytical challenges. We have developed a four-stage validation protocol in order to provide maximum confidence that a complex, split-pool synthesis of encoded molecules yields the anticipated products in high purity and efficiency. The first three protocols are concerned with the synthetic molecules and the fourth with the encoding step.
First, the suitability of the reaction sequence for library synthesis was demonstrated by execution of the entire reaction sequence six times, each time using different building blocks.
The fully elaborated final products, 42a-f, as shown in Figure 51, were recovered in 80-90% purity following photolysis. These products, as well as the 20 intermediates preceding them (38a, 38d, 39a-f, 40a-f, 41a-f), were fully characterized by multidimensional 1H-NMR, HR- FAB-MS, TLC, and HPLC. This experiment showed that the reaction sequence could be used to synthesize library members in satisfactory purity.
Second, potential building blocks were tested by reaction with a selected substrate at each step (Figure 52). While it is impossible to test the complete matrix of building block combinations, this experiment indicated which building blocks are compatible with the coupling reactions. Thus, 50 alkynes (Figure 53) were tested in reactions with iodobenzyltetracycle 39d, 87 amines (Figure 54) in reactions with alkynylbenzyltetracycle 40d, and 98 acids (Figure 55) in reactions with y-hydroxyamide 41d. Nearly every commercially available terminal alkyne was tested, along with a variety of amines and acids representing different steric and electronic functional groups. Photocleavage products were analyzed by HPLC and LC-MS (certain acid- sensitive products were also analyzed by TLC and FAB-MS) and their purities and percent conversions were estimated from these data (Figure 56). There were no obvious trends among the alkynes that were unsuitable for the Sonogashira/Castro-Stephens reaction, however, in the aminolysis and esterification reactions, electron poor amines and electron rich or enolizable acids generally did not react with suitable efficiency. In addition, several of the acids were insoluble under the reaction conditions. Of the building blocks tested, 23 alkynes, 54 amines, and 44 acids
reacted with greater than or equal to 90% conversion and purity. These building blocks, along with a limited number of less optimal candidates (generally reacting with greater than or equal to 70% conversion and purity), were selected for inclusion in library synthesis.
Third, a small test library was generated from iodobenzyltetracycle 39b (Figure 57) in order to investigate whether any unforeseen complications, such as interactions between building blocks coupled at different sites, might arise during synthesis in a split-pool format. The building blocks were carefully selected such that every product within each final acylated pool would have a unique mass (Figure 58), allowing analysis by LC-MS. Thus, the tetracycle- containing resin was divided into eight portions and the seven alkynes were coupled to the first seven portions. The eighth portion was left as the parent aryl iodide, representing a"skip codon". (Combs et al., J. Am. Chem. Soc. 1996,118,287). After pooling and splitting, the seven amines were coupled and the eighth portion of resin was left as the lactone-closed skip codon.
Finally, after a third round of pooling and splitting, the seven acids were coupled and the eighth portion was left as the free C-6 hydroxyl skip codon. Because all eight final pools, designated 43 {X, X, 1} through 43 {X, X, 8}, (Test library compounds are designated as 43 {R,, Rb, Rc} where Ra signifies the alkyne building block, Rb signifies the amine building block, and Rc signifies the acid building block. Pools of compounds are signified by Rn=X where X represents all eight building blocks at a given position). contained the same eight y-butyrolactone compounds corresponding to the aminolysis skip codon, a total of 456 compounds was generated.
Each pool was photocleaved to yield a mixture of 64 compounds that were analyzed by LC-MS (Figure 59). Of the 456 expected masses, all 456 (100%) were detected at some level, 418 (92%) were detected at greater than or equal to 10% of the average intensity for the given pool, and 400 (88%) were detected at greater than or equal to 20% of the average intensity for the given pool. All of the weak signals resulted from compounds having one of two building blocks at the amine position. 1 (-3-Aminopropyl)-2-pyrrolidinone (Amine 6) is known to cyclize to DBN with loss of H20. Since strong bases had been found to be incompatible with our linker- support combination, this building block was excluded from full-scale library synthesis. The skip codon (Amine 1) left lactone-closed tetracycles that were partially hydrolyzed during the final acylation step. As a result, during full-scale library synthesis, the aminolysis skip codon pool was set aside before the final pooling, splitting, and acylation steps.
Binary Encoding. Assuming an ideal, efficient split-pool synthesis, each support carries a single compound. Several solutions to the problem of compound identification have been developed, falling into two general categories: recursive deconvolution and encoding (Czarnik, A. W. Curr. Opin. Cliem. Biol. 1997, l, 60). Since recursive deconvolution requires several rounds of resynthesis, we chose the particularly powerful binary encoding strategy, having used
this method successfully in previous work (Combs et al., J. Am. Chem. Soc. 1996,118,287; Czarnik, A. W. Curr. Opin. Chem. Biol. 1997,1,60; Kapoor et al. J. Am. Chem. Soc. 1998,120, 23). Still's polyhaloaromatic EC-GC tags, 44, were selected since they are relatively unreactive and can be coupled directly to the polystyrene backbone of beads using mild carbene insertion chemistry (Figure 60) (Ohlmeyer et al. Proc. Natl Acad. Sci. USA 1993,90,10922; Nestler et al.
J. Org. Chem. 1994,59,4723). Unfortunately, the published procedures gave inconsistent and unsatisfactory results in our hands. Referring to Figure 60 in the discussion below, substitution of the reported rhodium bis (trifluoroacetate) catalyst with a bulkier rhodium bis (triphenylacetate) catalyst (Callot et al. Tetrahedron 1985,41,4495) suppressed solution-phase diazoketone dimerization and substantially improved the efficiency of tag-bead coupling to form cycloheptatrienes 45. We also found that, after reaction with an initial set of tags, attachment of subsequent tags to the same beads required multiple couplings. It is possible that the initial reactions occurred at the most accessible sites in the polymer, making subsequent reactions more difficult. Finally, the reaction conditions for oxidative cleavage of the tags from 45 with ceric ammonium nitrate (CAN) were optimized, reducing the required cleavage time to 10 min from the reported 4 h. This improved the yields of the polyhaloaromatic alcohol products, 46, and allowed rapid and consistent analysis by EC-GC.
Full-Scale Library Planning and Synthesis. Completion of the validation protocols above set the stage for full-scale encoded library synthesis. First, building blocks were selected for each step of the synthesis (Figure 51). (o-Aminocaproic acid and glycine were selected as spacer elements with the"no spacer"skip codon providing a third structure for 37. Use of both enantiomers of epoxycyclohexenol 7 resulted in six structures for 38. Inclusion of all three iodobenzyl nitrone carboxylic acids lib-d led to 18 iodobenzyltetracycle structures for 39.
The three remaining diversity positions, corresponding to the Sonogashira/Castro- Stephens, lactone aminolysis, and C-6 acylation reactions, allowed the use of substantially larger numbers of building blocks. Optimal use of the binary encoding tags dictates that 2n-2 building blocks should be used at a given position. This accounts for one skip codon (the"all one"code) and allows for exclusion of the undesirable"all null"code that cannot be differentiated from a failed tagging reaction. As a practical matter, coupling of up to 26-2 = 62 building blocks at a given step was deemed feasible.
Only 25-2 = 30 building blocks were selected for the Sonogashira/Castro-Stephens reaction because of the relatively small number of available terminal alkynes. Most of the alkynes that reacted efficiently during building block screening (Figure 53) were selected.
Several racemic alkynes were also included, although diastereomeric products would likely result. Furthermore, several alkynols were included, despite their potential reactivity in the final
acylation reaction. Control experiments indicated that these hydroxyl groups were efficiently acylated by a variety of alkyl and aromatic acids under the DIPC-mediated coupling conditions.
Coupling of 30 different alkynes with exclusion of a 31st skip codon portion would result in 558 structures for 40.
Wider selections of building blocks were available at the amine and acid positions. For each reaction, 62 building blocks were selected, representing a range of sizes and functional groups (Figures 54 and 55). Coupling of 62 different amines with exclusion of a 63rd aminolysis skip codon portion would result in 35,154 structures for 41. As discussed above, the 558 lactone-closed compounds corresponding to the aminolysis skip codon would not react at the final acylation step. Therefore, the total number of final library compounds, 42, resulting from acylation with 62 acids and exclusion of a 63rd skip codon portion is calculated as follows: [ (62 x 558 = 34,596) x 63 = 2,179,548] + 558 = 2,180,106 compounds.
Synthesis of three copies of the library was planned, based upon a calculation that indicates that three copies should be screened to ensure 95% confidence that every compound has been sampled (Nolan, G. P., FACS Screening Web Page. http ://www. stanford. edu/group/nolan/FACSscrn. html (accessed Jun 1999)). Although this calculation does not address the number of copies required to ensure that every possible compound has been synthesized, we recognized that, if necessary, the library could be resynthesized on larger scale in the future.
Library synthesis began with coupling of Fmoc-protected Geysen linker to 90 4m TentaGel S NH2. After deprotection, the resin, 36, was split into three portions by weight and labeled with the tags corresponding to the spacer position. For the fourth validation protocol, after each tagging step in the synthesis, several beads were removed from every portion of the resin and the tags were cleaved and analyzed to ensure adequate incorporation levels. Fmoc- Aca-OH and Fmoc-Gly-OH were then coupled to two of the portions and deprotected under standard conditions. The resin, 37, was pooled, mixed, and split into two equal portions. After tagging, one enantiomer of epoxycyclohexenol 7 was coupled to each portion. Resin 38 was then pooled, mixed, and split into three equal portions for tagging and reactions with iodobenzyl nitrones llb-d to yield resin 39. These tetracycle-containing resins were pooled, mixed, and split into 31 equal portions. Each was tagged and the appropriate 30 terminal alkynes were coupled using the Sonogashira/Castro-Stephens reaction. The 31 st portion of resin was set aside as the skip codon. Resin 40 was pooled, mixed, and split into 63 portions. In this case, the 63rd portion of resin, corresponding to the aminolysis skip codon, was 1/63rd the size of the other 62 portions. This modification was required to avoid overrepresentation of lactone-closed compounds in the completed library. The 62 large portions of resin were tagged and reacted with
the appropriate amines to yield resin 41. The 63rd skip codon portion was set aside for the remainder of the synthesis to avoid hydrolysis of the lactone-closed compounds during the final acylation step. The remaining 62 portions of resin were pooled, mixed, and split into 63 equal portions. After tagging, the appropriate 62 acids were coupled and the 63rd portion of resin was left as the unreacted C-6 hydroxy compounds. Finally, the 63 acylation portions and the lactone aminolysis skip codon portion were pooled and mixed to yield the final library 42, calculated to contain three copies of 2,180,106 compounds. The entire process was completed by two of us (D. S. T. and M. A. F.) working over a period of three weeks. The bulk of this time was spent verifying that the encoding tags had successfully coupled to every portion of the resin during each step of the synthesis.
Cell Permeation and Pathway Modulation. It seemed worthwhile to begin an analysis of these compounds by screening the 456-compound test library (Figure 57) in cellular assays even before the full-scale library was completed. Although our compounds were designed to contain structural features common to natural products, we had no general sense of their ability to either permeate cells or alter cellular pathways.
These initial studies relied upon traditional non-miniaturized assays, requiring more material than was contained on a single synthesis bead. Thus, the test library was screened as mixtures of compounds cleaved in bulk. The eight final pools, 43 {X, X, 1} through 43 {X, X, 8}, each contained 64 compounds. The compounds in each pool were photolyzed from the resin, recovered, and dissolved in DMSO at an estimated concentration of 1 mM per compound (64 mM overall) (Based upon previous results a 50% yield was assumed). The eight pools, assayed at concentrations up to 10 M per compound, showed no suppression of rapamycin-based growth inhibition in S. cerevisiae (The rapamycin concentration was 100nM). In addition, none of the pools, assayed at concentrations up to 12.5 J. M per compound, showed inhibitory activity in a Xenopus laevis oocyte extract assay that indicates modulation of the cyclin B degradation pathway.
However, all eight pools showed a significant inhibitory effect on mink lung cell proliferation when assayed at a concentration of 1 uM per compound (Figure 61). Moreover, when the library was assayed at a concentration of 250 nM per compound, pool 43 {X, X, 8} (Figure 62), was found to activate a TGF--responsive reporter gene (Carcamo et al. J. Mol.
Cell. Biol. 1995,15,1573) in a stably transfected mink lung cell line (Figure 63) (Stockwell et al., Curr. Biol. 1998,8,761) Since this library is not encoded with chemical tags, a recursive deconvolution strategy was used to investigate this activity further.
The 64 compounds in 43 {X, X, 8} were resynthesized as eight pools, designated 43 {X, 1,8} through 43 {X, 8,8}, each containing eight different compounds. Each pool was aminolyzed with
a different amine and all pools were acylated with Acid 8. As a negative control, pool 43 {X, X, 3} was also resynthesized as eight pools, 43 {X, 1,3} through 43 {X, 8,3}. Surprisingly, pool 43 {X, 8,3}, assayed at a concentration of 1 tM per compound, was a stronger activator of the TGF-p-responsive reporter gene than any of the other 15 eight-compound pools or either of the parent 64-compound pools (data not shown).
A final round of resynthesis deconvoluted pool 43 {X, 8,3}, yielding single compounds 43 {1, 8,3} through 43 {8, 8,3}. These eight compounds and all of the 16 intermediates preceding them were recovered in high purity as determined by 1H-NMR and HR-TOF-ESI-MS analysis.
All 24 compounds were screened and compounds 43 {5,8,3} and 43 {6, 8,3} were found to activate the TGF-p-responsive reporter gene (Figure 64). However, the alkynylbenzyltetracycle (43 {5, 1,1} and 43 {6, 1,1}) and y-hydroxyamide (43 {5, 8,1} and 43 {6,8,1}) precursors to these compounds were even more active. The six active compounds were purified by silica gel chromatography and reassayed, verifying that the desired major product is responsible for the activity in each case. The ECso for the strongest activator, 43 {6, 1,1}, is approximately 50 uM.
Discovery of these compounds, while somewhat fortuitous, gave us a measure of confidence in the cell permeating and pathway modulating properties of members of the library.
These results also highlight the shortcoming of screening mixtures of compounds. Active compounds may be masked by competing cytotoxic, cytostatic, or antagonistic compounds in the same pool. Alternatively, activity may arise from synergistic effects between two or more compounds, making deconvolution to a single structure impossible. For these reasons, we generally avoid screening mixtures in high-throughput chemical genetic screens.
Chemical Genetic Screens Using Compounds Released from Single Beads. Solid phase synthesis was originally developed to facilitate the purification of peptides from reaction mixtures. Much of the more recent solid phase organic synthesis has been used for a similar purpose, but involves non-oligomeric small molecules. Split-pool synthesis provides a new and arguably more powerful incentive for performing solid phase organic synthesis: It yields large numbers of spatially segregated small molecules. To take full advantage of this feature, assay formats must be developed that use compounds derived from single beads. In our early studies of assay formats using single beads, two such systems were used. Binding-based assays were performed by detecting soluble proteins that had been recruited to individual beads via their interactions with a tethered small molecule (see, Combs et al. J. Am. Chem. Soc. 1996,118,287; Kapoor et al. J Am. Chem. Soc. 1998,120,23; Morken et al. J. Am. Chem. Soc. 1998,120,30).
Phenotype-based assays were performed using cells and synthesis beads contained in small volumes of cell culture. These"nanodroplets"were generated either stochastically (see,
Borchardt et al. Chem. Biol. 1997,4,961) or on a molded, polydimethylsiloxane (PDMS) grid (You et al. Chem. Biol. 1997,4,969). Although these assays have yielded useful information, they suffer from several shortcomings, including the fact that the synthesis beads are not easily used more than once. Moreover, the synthesis described above was performed on 90 um TentaGel beads, having a loading capacity of only approximately 80-100 picomolar equivalents per bead. In mink lung cell cytoblot assays searching for small molecule suppressors of trapoxin's and rapamycin's actions and using a subset of approximately 77,000 beads, these shortcomings manifested themselves in several ways, including the need for resynthesis of compounds corresponding to apparent positives. On the other hand, our early experience in screening these synthesis beads has revealed the ability to recover positive beads, cleave their EC-GC tags, and decode them successfully.
To address the problem noted above, colleagues at the Harvard Institute of Chemistry and Cell Biology have developed instrumentation and robotics that provide efficient arraying of synthesis beads into high density wells, releasing and transferring of compounds into high density stock solutions, and transferring of these solutions into high density PDMS assay plates.
These assay plates are the preferred format for cytoblot assays (see, Stockwell et al. Chem. Biol.
1999,6,71), and the high density stock solutions are also well-suited for small molecule printing (with reference to provisional application number 60/133,595 entitled"Small Molecule Printing" filed May 11, 1999, the entire contents of which are incorporated herein by reference) To allow efficient release of compounds into storage wells without the need for removal of toxic byproducts, we have explored various linkers. To provide sufficient amounts of released compounds to allow their use in large numbers of assays, we have explored beads with higher loading capacities.
III. Specific Synthetic Procedures and Demonstration Compound Synthesis: Shikimic acid, methyl ester (4) (Fischer et al. Helv. Chim. Acta (-)- Shikimic acid, 3, (Aldrich or Fluka, 7.0 g, 40.2 mmol, 1. 0 equiv) and Amberlite IR-120 (plus) resin (12.0 g, 22.8 mmol, 0.57 equiv) were combined in 210 mL MeOH. The mixture was refluxed with stirring for 36 h, cooled to rt, and filtered. The MeOH was evaporated to yield methyl ester 4 as a white solid (7.56 g, 100%) that was used without further purification. TLC: Rf0. 25 (9: 1 CH2Cl2/MeOH). 1H-NMR (400 MHz, CD30D) : 6 6.78 (m, 1H), 8 4.36 (m, 1H), 8 3.98 (dt, 1H, J= 6.9,5.3), 8 3.73 (s, 3H), 6 3.68 (dd, 1H, J= 7.1,4.2), 8 2.68 (app ddt, 1H, J= 18.2,4.9), 8 2.19 (app ddt, 1H, J= 18.2,5.3). CI-MS (NH3) (rel int): 206 ( [M+NH4J+, 100). HRMS (NH3) m/z calcd for C8Hl6NO5 206.1029; found 206.1036.
(1S, 5R, 6S)-5-Hydroxy-7-oxabicyclol4. 1.0] hept-3-ene-3-carboxylic acid, methyl ester (5). Epoxycyclohexenol 5 was prepared by a modified version of the literature procedure (McGowan et al. J. Org. Chem. 1981,46,2381) as follows: Shikimic acid, methyl ester, 4, (4.31 g, 22.9 mmol, 1. 0 equiv) and triphenylphosphine (6.61 g, 25.2 mmol, 1. 1 equiv) were dissolved in 100 mL THF and cooled to 0 °C in an ice bath. Diethylazodicarboxylate (3.97 mL, 25.2 mmol, 1. 1 equiv) was dissolved in 10 mL THF and added dropwise with stirring via addition funnel. The mixture was stirred for 30 min at 0 °C, then warmed to rt and stirred for 1 h. The THF was evaporated and the residue was refluxed in 125 mL toluene for 90 min. The toluene was evaporated and the crude mixture was taken up in 100 mL hot Et20, cooled to rt, and filtered. This process was repeated with 100 mL Et20 then 75 mL Et20. The crude product (8.22 g) was recovered as a brown residue that was determined by 1H-NMR to consist of 53% desired epoxide 5,26% triphenylphosphine oxide, and 21% bis (carboethoxy) hydrazine. The crude product (96% calculated yield) could be used without further purification or purified by silica flash chromatography (1 : 1 hexanes/EtOAc) to yield the pure product having analytical data consistent with the literature. TLC: Rf 0. 16 (1 : 1 hexanes/EtOAc).
(lS, 5S, 6S)-5-Benzoyloxy-7-oxabicyclo [4.1.0] hept-3-ene-3-carboxylic acid, methyl ester (Benzoyl epoxycyclohexenol, methyl ester; 6). Epoxycyclohexenol 5 (4.75 g, 27.9 mmol, 1.0 equiv) was dissolved in 125 mL THF. Triphenylphosphine (13.18 g, 50.3 mmol, 1.8 equiv) and benzoic acid (6.14 g, 50.3 mmol, 1.8 equiv) were added and the solution was cooled to 0 °C in an ice bath. Diethylazodicarboxylate (7.9 mL, 50.3 mmol, 1.8 equiv) was added via syringe and the reaction was allowed to warm slowly to rt. After stirring overnight, the THF was evaporated and the crude mixture was taken up in 150 mL Et20 and filtered twice to remove the triphenylphosphine oxide and bis (carboethoxy) hydrazine byproducts. The solvent was evaporated and the crude mixture was taken up in 100 mL Et20 and again filtered twice. The crude product (17.8 g) was purified by silica gel flash chromatography (17: 3 hexanes/EtOAc) to yield the pure benzoyl ester 6 as a clear, colorless oil (6.77 g, 88%). TLC: Rf 0. 35 (3: 1 hexanes/EtOAc). IR (film): 1718,1669,1246. lH-NMR (400 MHz, CDC13) : 6 8. 06 (dd, 2H, J = 6.3,3.3, Cl 1-H, C15-H), 7.69 (t, 2H, J= 7.7, C12-H, C14-H), 7.59 (tt, 1H, J= 7.4,1.4, C13-
H), 6.86 (ddd, 1H, J=7. 7,2.9,1.7, C3-H), 5.92 (app dt, 1H, J=4. 6,2.0,0.9, C4-H), 3.77 (s, 3H, C8-H3), 3.51 (dd, 1H, J= 3. 7,2.8, C5-H), 3.41 (ddd, 1H, J= 4. 5,2.7,1.7, C6-H), 3.05 (ddd, 1H, J = 20.0,2.7,1.3, C7-Ha), 2.76 (ddd, 1H, J = 20.0,4.8,2.7, C7-Hb). 13C-NMR (100 MHz, CDC13) : 8 166.2,165.3,133.2,129.7,129.5,129.4,128.8,128.2,64.9,51.8,50 .4,50.1,24.1.
CI-MS (NH3) mlz (rel int): 292 ( [M+NH4] +, 100), 275 ( [M+H] +, 58). HRMS (NH3) mlz calcd for C15Hl8NO5 292.1185; found 292.1179.
(+)- (lS, 5S, 6S)-5-Hydroxy-7-oxabicyclo [4. 1.0] hept-3-ene-3-carboxylic acid ( (+)- Epoxycyclohexenol carboxylic acid, (+)-7). Benzoyl epoxycyclohexenol methyl ester 6 (1.05 g, 3.84 mmol, 1.0 equiv) was dissolved in 40 mL THF and 10 mL H20 and cooled to 0°C in an ice bath. Lithium hydroxide monohydrate (483 mg, 11.52 mmol, 3.0 equiv) was dissolved in 10 mL H20 and added dropwise via addition funnel to the stirring reaction mixture. When the reaction was complete by TLC, the solution was acidified at 0 °C to pH 5 with Amberlite IR- 120 (plus) resin, filtered, and evaporated to yield the crude product as an off-white solid. NMR analysis indicated approximately 25% Payne rearrangement. Purification on silica gel (25: 75: 1 hexanes/EtOAc/AcOH, dry loaded from THF) afforded epoxycyclohexenol carboxylic acid (+)-7 as a white solid (352 mg, 59%). TLC: Rf 0. 24 (25: 75: 1 hexanes/EtOAc/AcOH); Rf 0. 49 (85: 15: 1 CH2Cl2/MeOH/AcOH). mp: 115.5-116.5°C. [a'_ +57. 6 (c 1.0, MeOH). IR (KBr pellet): 3700-2800,1713,1661,1248.1H-NMR (500 MHz, CD3CN): 8 6.67 (m, 1H, C3-H), 4.46 (m, 1H, C4-H), 3.36 (m, 1H, C6-H), 3.14 (m, 1H, C5-H), 2.76 (app dq, 1H, J = 19.8,1.4, C7-Ha), 2.57 (app dq, 1H, J= 19.8,2.4, C7-Hp). 1H-NMR (400 MHz, CD30D) : 6 6.73 (m, 1H, C3-H), 4.47 (m, 1H, C4-H), 3.41 (m, 1H, C6-H), 3.19 (m, 1H, C5-H), 2.81 (app dq, 1H, J= 19.8,1.3, C7-Ha), 2.60 (app dq, 1H, J= 19.8,2.5, C7-Hp). 13C-NMR (125 MHz, CD3CN) : 8 168.6 (C1), 135.9 (C3), 127.0 (C2), 63.4 (C4), 53.6 (C5), 51.3 (C6), 25.1 (C7). 13C-NMR (125 MHz, CD30D): 6 170.0 (C1), 135.4 (C3), 127.8 (C2), 63.7 (C4), 54.1 (C5), 51.9 (C6), 25.4 (C7). CI- MS (NH3) mlz (rel int): 174 ([M+NH4] +, 66). HRMS (NH3) mlz calcd for C7H12NO4 174.0766; found 174.0762.
(-)- (lR, 5R, 6R)-5-Hydroxy-7-oxabicyclo [4.1.0] hept-3-ene-3-carboxylic acid ( (-)- Epoxycyclohexenol carboxylic acid, (-)-7). Epoxycyclohexenol, methyl ester 9 was prepared essentially as previously described (Wood et al. J. Am. Chem. Soc. 1990,112,8907) and recovered as a 1.6: 1 mixture with the Payne rearranged isomer in 51% combined yield. This mixture (1.21 g, 7.15 mmol, 1.0 equiv) was dissolved in 14 mL 1: 1 THF/H20 and cooled to 0 °C in an ice bath. Lithium hydroxide (330 mg, 7.87 mmol, 1. 1 equiv) in 3.3 mL H20 was added dropwise over 10 min. The reaction was stirred at 0 °C until the starting material was consumed (approx 2 h, TLC: 25: 75: 1 hexanes/EtOAc/AcOH). The solution was acidified at 0 °C to pH 5 with Amberlyte IR-120 (plus) resin, filtered, and evaporated to yield the crude product as a clear oil. Purification on silica gel (0-5% MeOH in CH2C12 gradient) afforded epoxycyclohexenol carboxylic acid (-)-7 as a white solid (477 mg, 43% based on mixture). TLC and 1H-NMR identical to (+)-7 above. [ccf =-50. 6 (c 1.0, MeOH). CI-MS (NH3) m/z (rel int): 174 ( [M+NH4] +, 75). HRMS (NH3) m/z calcd for C7H12NO4 174.0766; found 174.0770.
General Procedure for Synthesis of [l (Iodophenyl) methylloxidoiminolacetic acids (Iodobenzyl Nitrone Acids) (Keirs, D.; Overton, K. Heterocycles 1989,28,841). The appropriate N (iodobenzyl) hydroxylamine (see Supporting Information, 1.0 equiv) and glyoxylic acid monohydrate (1.05 equiv) were dissolved in CH2C12 and stirred at rt until the reaction was complete by NMR (24 h). The reaction mixture was washed with 2 x H20 and 1 x brine, dried (MgS04), filtered, and evaporated to yield the crude nitrone. The crude product was slurried in THF, then Et20 was added with vigorous stirring. After trituration overnight, the pure nitrone carboxylic acid 11 was recovered by vacuum filtration in 47-67% yield.
[ [ (2-lodophenvl) methyl] oxidoiminol acetic acid (2-Iodobenzyl nitrone acid, llb). N- (2-Iodobenzyl) hydroxylamine (9.46 g, 38.0 mmol) was reacted in 250 mL CH2C12. The crude product was recovered as a slightly yellow solid (10.9 g) and slurried in 10 mL THF then 250 mL Et20. The pure product was recovered as white flakes (6.22 g, 54%). mp: 82 °C (dec). IR (film) : 1715,1470,1414. IH-NMR (400 MHz, CDC13) : 6 7.96 (dd, 1H, J= 8.0,1.0), 7.51 (dd, 1H, J= 7.6,2.0), 7.48 (td, 1H, J= 7.4,1.1), 7.44 (br s, 1H), 7.22 (s, 1H), 7.19 (ddd, 1H, J= 7.9, 7.2,2.0), 5.21 (s, 2H). 13C-NMR (125 MHz, CDC13) : 160.6,140.5,132.4,132.3,132.1,130.5,
129.4,101.0,74.2. CI-MS (NH3) (rel int): 340 ([M+2NH3+H] +, 7), 323 ( [M+NH4] +, 100), 306 ( [M+H] +, 10).
[ [ (3-lodophenyl) methyll oxidoimino] acetic acid (3-Iodobenzyl nitrone acid, llc). N- (3-Iodobenzyl) hydroxylamine (7.96 g, 32.0 mmol) was reacted in 250 mL CH2C12. After dilution with 250 mL CH2Cl2 and washing, the crude product was recovered as a slightly yellow solid (9.1 g) and slurried in 10 mL THF then 350 mL Et20. The pure product was recovered as white flakes (6.58 g, 68%). mp: 109.0-109.5°C (dec). IR (film): 1715,1470,1412.1H-NMR (400 MHz, CDC13) : 8 7.84 (dt, 1H, J= 8.0,1.3), 7.80 (t, 1H, J= 1.7), 7.63 (br s, 1H), 7.42 (dt, 1H, J = 7.7), 7.29 (s, 1H), 7.23 (t, 1H, J = 7.8), 5.00 (s, 2H). 13C-NMR (125 MHz, CDC13) : 160.5,139.4,138.6,131.8,131.0,130.0,128.9,94.9,69.8. CI-MS (NH3) m/z (rel int): 340 ( [M+2NH3+H] +, 14), 323 ( [M+NH4] +, 100), 306 ( [M+H] +, 4).
[ [ (4-lodophenyl) methyl] oxidoiminol acetic acid (4-Iodobenzyl nitrone acid, lld). N- (4-Iodobenzyl) hydroxylamine (11.8 g, 47.4 mmol) was reacted in 300 mL CH2C12. The crude product was recovered as a white powder (10.2 g) and slurried in 15 mL THF then 300 mL Et20.
The pure product was recovered as a white powder (6.89 g, 48%). mp: 124.0 °C (dec, peach), 156-173 °C (dec, brown oil). IR (film): 1711,1466,1447,1424,1402.1H-NMR (400 MHz, CDC13) : 6 7. 82 (d, 2H, J= 8. 4), 7.27 (s, 1H), 7.17 (d, 2H, J= 8. 3), 4.99 (s, 2H). 13C-NMR (100 MHz, CDC13) : 160.5,138.7,131.4,129.8,129.2,96.8,70.2. EI-MS m/z (rel int) : 305 (M+, 2), 261 ( [M-C021+, 6), 217 ( [M-HOOC-CH-NO] +, 100). FAB-MS (NBA/NaI) m/z (rel int): 328 ( [M+Na] +, 42), 306 ( [M+H] +, 25).
4-Iodobenzyl alcohol. (Acheson, R. M.; Lee, G. C. M. J. Chem. Soc. Perkin Trans. I 1987,2321-2332.) To a stirred suspension of sodium borohydride (5.68 g, 150 mmol, 2.0 equiv) in 50 mL dioxane at 0 °C was added dropwise a solution of 4-iodobenzoyl chloride (19.99 g, 75 mmol, 1.0 equiv) in 50 mL dioxane over 25 min. The resulting mixture was heated to 100 °C for 90 min under a reflux condenser then cooled to 0 °C. 50 mL H20 was added cautiously under a flowing stream of nitrogen. [CAUTION: Evolves gas!] The mixture was extracted 3 x 125 mL CH2C12 and the combined organic extracts were washed with 2 x H20, 2 x 0. 1N HC1, 2 x IN NaOH, H20, and brine, dried (MgS04), filtered, and evaporated to yield 16.9 g of crude 4- iodobenzyl alcohol as a white solid, determined by NMR to contain 78% desired product with
the remainder residual starting material and 4-iodobenzoic acid. The crude product was used without further purification. mp: 61.0-66.5°C. TLC Rf 0. 27 (3: 1 hexanes/EtOAc). IR (film): 3306,1005,791. IH-NMR (400 MHz, CDCl3) : 8 7.69 (d, 2H, J= 8.3, C4-H, C6-H), 7.12 (d, 2H, J= 8.5, C3-H, C7-H), 4.66 (br d, 2H, J= 4.1, C1-H2), 1.67 (br t, 1H, C1-OH). 13C-NMR (125 MHz, CDC13) : 8 140. 4,137.6,128.8,93.0,64.6. EI-MS m/z (rel int) : 234 (M+, 100).
General Procedure for Synthesis of Iodobenzaldehydes. (Acheson, R. M.; Lee, G. C.
M. J. Chem. Soc. Perkin Trans. I 1987, 2321-2332.) To a stirred suspension of pyridinium dichromate (1.5 equiv) in CH2C12 was added the appropriate iodobenzyl alcohol (1.0 equiv) at rt.
The mixture was stirred vigorously for 20-40 h until the reaction was complete by TLC. Et20 was added and the mixture was filtered through a column of 2"celite over 2"silica gel. Elution of the product with additional Et20 and evaporation of solvents yielded the crude iodobenzaldehyde which was approximately 95% pure by tH-NMR and used without further purification.
2-Iodobenzaldehyde. Commercially available 2-iodobenzyl alcohol (10.0 g, 42.7 mmol) was dissolved in 200 mL CH2C12. Upon completion, the reaction was diluted with 250 mL Et20. The product was recovered as a brown liquid (10.3 g, 104%). TLC: Rf 0. 57 (3: 1 hexanes/EtOAc). IR (neat): 3061,2853,2745,1696,1580,1561. tH-NMR (400 MHz, CDCl3) : 6 10.07 (s, 1H, Cl-H), 7.95 (dd, 1H, J= 7.9,1.0, C4-H), 7.88 (dd, 1H, J= 7.7,1.8, C7-H), 7.47 (td, 1H, J= 7.5 0.8, C6-H), 7.29 (td, 1H, J= 7.6,1.8, C5-H). 13C-NMR (100 MHz, CDCl3) : 6 195.1,140.2,135.1,134.7,129.9,128.4,100.5. EI-MS m/z (rel int): 232 (M+, 100), 231 ( [M- H] +, 40), 203 ( [M-CHO] +, 15), 105 ( [M-I] +, 3).
3-Iodobenzaldehyde. Commercially available 3-iodobenzyl alcohol (5.32 mL, 41.9 mmol) was dissolved in 200 mL CH2Cl2 Upon completion, the reaction was diluted with 250 mL Et20. The product was recovered as off-white crystals (8.1 g, 83.4%) mp: 48.0-55.0°C.
TLC: Rf0. 54 (3: 1 hexanes/EtOAc). IR (neat): 3058,2824,2728,1698,1586,1566.1H-NMR (400 MHz, CDCl3) : 5 9.93 (s, 1H, Cl-H), 8.22 (t, 1H, J= 1.6, C3-H), 7.96 (dt, 1H, J= 7.7,1.4, C5-H), 7.85 (dt, 1H, J = 7.7,1.3, C7-H), 7.29 (t, 1H, J= 7.7, C6-H). 13C-NMR (125 MHz, CDCl3) : 5 190.6,143.1,138.4,138.0,130.7,128.8,94.6. EI-MS m/z (rel int): 232 (M+, 100), 231 ( [M-H] +, 25), 203 ( [M-CHO] +, 14), 104 ([M-HI] +, 38).
4-Iodobenzaldehyde. 4-Iodobenzyl alcohol prepared above (16.9 g, 72.2 mmol) was dissolved in 350 mL CH2C12. Upon completion, the reaction was diluted with 250 mL Et20.
The product was recovered as a white solid (13.7 g, 81.8%) mp: 71.0-73.5°C. TLC: Rf 0. 50 (3: 1 hexanes/EtOAc). IR (film): 2820,2726,1690,1584,1564,804.1H-NMR (400 MHz, CDCl3) : 8 9.93 (s, 1H, C1-H), 7.92 (d, 2H, J = 8.5, C4-H, C6-H), 7.59 (d, 2H, J= 8.1, C3-H, C7-H). 13C-NMR (100 MHz, CDC13): 8 191.4,138.4,135.6,130.8,102.8. EI-MS m/z (rel int): 232 (M+, 100), 203 ( [M-CHO] +, 24).
General Procedure for Synthesis of N-(Iodobenzyl) hydroxylamines. (Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem. Soc. 1971,93,2897-2904.) To a stirred solution of the appropriate iodobenzaldehyde (1.0 equiv) in a mixture of MeOH and THF was added a trace of Methyl Orange at rt. Hydroxylamine hydrochloride (1.25 equiv) was dissolved in H20 and added to the iodobenzaldehyde solution. The pH was raised to 9 with 6N KOH and additional THF, MeOH, and/or H20 were added to form a homogeneous solution. Solid sodium cyanoborohydride (1.0 equiv) was added and 2N HCl in aq MeOH was added via addition funnel until the solution was ruby red. CAUTION : Evolves gas!] Additional acid was added as necessary to maintain the color during the reaction. After the reaction was complete by NMR (15-20 h), the bulk of the MeOH and THF were evaporated. The remaining aq solution was adjusted to pH 12 with 6N KOH and extracted with 4 x CH2C12. The combined organic extracts were washed with H20 and brine, dried (MgS04), filtered, and evaporated to yield the crude N- (iodobenzyl) hydroxylamine which was determined by NMR to contain 90-94% of the desired product with the remainder N, N-bis (iodobenzyl) hydroxylamine. The crude product was used without further purification.
N- (2-Iodobenzyl) hydroxylamine. 2-Iodobenzaldehyde prepared above (10.3 g, 44.4 mmol) was dissolved in 50 mL MeOH and 10 mL THF. After addition of H2NOH-HCl (10 mL H20) and 6N KOH, an additional 50 mL THF, 30 mL H20, and 30 mL MeOH were added. The product was recovered as a cloudy orange oil (9.46 g, 85.6%, 90% desired product). IR (film) : 3256,3057,2872,1564,1466,1435,1013,748.1H-NMR (400 MHz, CDC13): 6 7.92 (dd, 1H, J = 7.8,1.2, C4-H), 7.58 (dd, 1H, J= 7.4,1.8, C7-H), 7.34 (td, 1H, J= 7.4,1.2, C6-H), 7.00 (td, 1H, J= 7.6,1.8, C5-H), 5.5 (br s, 1H, Cl-NHOH), 5.0 (br s, 1H, C1-NHOH), 4.13 (s, 2H, Cl- H2) 13C-NMR (100 MHz, CDC13) : 8 139.5,139.1,131.0,129.4,128. 3,100.1,62.0. EI-MS mlz (rel int): 249 (M+, 36), 217 ([M-NHOH] +, 100), 122 ( [M-I] +, 30). CI-MS (NH3) mlz (rel int): 284 ( [M+2NH3+H] +, 30), 267 ([M+NH4] +, 100), 250 ( [M+H] +, 27).
N- (3-Iodobenzyl) hydroxylamine. 3-Iodobenzaldehyde prepared above (8.1 g, 34.9 mmol) was dissolved in 50 mL MeOH and 20 mL THF. After addition of H2NOH*HCl (10 mL H20) and 6N KOH, an additional 20 mL H20, 10 mL THF, and 10 mL MeOH were added. The product was recovered as a white solid (7.96 g, 91.6%, 92% desired product). mp: 63.0-70.0°C.
IR (film): 3256,3056,2857,1591,1564,1470,1420,1063,995,777.1H-NMR (400 MHz, CDC13) : 8 7.73 (t, 1H, J= 1.6, C6-H), 7.63 (dt, 1H, J= 7.9,1.3, C5-H), 7.31 (dt, 1H, J= 7.6, C7-H), 7.09 (t, 1H, J= 7.8, C6-H), 5.5 (br s, 1H, C1-NHOH), 5.1 (br s, 1H, C1-NHOH), 3.98 (s, 2H, C1-H2). 13C-NMR (100 MHz, CDC13) : 6 139.9,137.9,136.6,130.2,128.2,94.4,57.4. EI- MS m/z (rel int): 249 (M+, 65), 217 ( [M-NHOH] +, 100). CI-MS (NH3) mlz (rel int): 284 ( [M+2NH3+H] +, 28), 267 ([M+NH4] +, 100), 250 ( [M+H] +, 45).
N- (4-Iodobenzyl) hydroxylamine. 4-Iodobenzaldehyde prepared above (13.7 g, 59.0 mmol) was dissolved in 80 mL MeOH and 60 mL THF. After addition of H2NOHHCl (10 mL H20) and 6N KOH, an additional 30 mL H20 was added. The product was recovered as a white solid (11.8 g, 80.3%, 94% desired product). mp: 89.0-95.0°C. IR (film): 3245,3173,2916, 2847,1483,1007,787.1H-NMR (400 MHz, CDC13) : 67. 66 (d, 2H, J= 8. 3, C4-H, C6-H), 7.06 (d, 2H, J= 8.3, C3-H, C7-H), 5.4-4.7 (br s, 2H, Cl-NHOH), 3.90 (s, 2H, C1-H2). 13C-NMR (100 MHz, CDC13) : 137.6,137.4,131.0,93.2,57.4. EI-MS m/z (rel int): 249 (M+, 29), 217
( [M-NHOH] +, 100). CI-MS (NH3) m/z (rel int): 267 ([M+NH4] +, 40), 250 ( [M+H] +, 100), 234 ([M-NHOH+NH3] +, 52), 217 ( [M-NHOH] +, 63).
Demonstration Compounds-General. For demonstration compound photocleavage reactions, 50 mg of resin was divided between two 500 uL Eppendorf tubes, suspended in 450 uL CHsCN each, and photolyzed for 2 h. Trace impurities resulting only from the photocleavage reaction were identified by photolysis of underivatized 3-amino-3-o- nitrophenylpropionic acid (Anp)-loaded resin (see below) and discounted in purity calculations.
Identification of Photolysis Byproducts. 50 mg of underivatized H2N-Anp-Tentagel resin was photolyzed and analyzed by TLC, HPLC, 1H-NMR and FAB-MS as follows: TLC (trace amounts, detectable by UV only): Rf 0. 55 (9: 1 CH2Cl2/MeOH) ; Rf 0. 71,0.82 (1 : 1 CH2C12/THF) ; Rf 0. 47,0.71 (4: 1 CH2Cl2/THF) ; Rf 0. 18,0.63 (1 : 1 CH2Cl2/EtOAc). HPLC (trace amounts): tR = 2.073 min, Amas = 217,244,303 nm ; tR = 2.462 min, = 242,301 nm; tR = 2.980 min, Ama = 243 nm ; tR = 3.141 min, Amax = 239 nm. 1H-NMR (500 MHz, CD3CN, trace amounts except for PEG): 6 7. 70 (dd, J= 5. 6,3.3), 7.60 (obs md, J= 5. 9,2.5), 7.57 (td, J= 7.8,1.3), 7.52 (d, J= 7.4), 7.09 (td, J= 7.5,0.8), 6.95 (d, J= 7.9), 4.44 (br s), 4.30 (q, J= 7.1), 4.22 (m), 3.55 (s, PEG), 2.85-2.50 (br), 1.31 (t, J= 7.1), 1.26 (br s). FAB-MS (glycerol) m/z (rel int): 503 ([M+H] +, 2). FAB-MS (NBA/NaI) m/z (rel int): 569 ( [M+Na] +, 100), 547 ( [M+H] +, 13), 553 ( [M+Na] +, 58), 531 ( [M+H] +, 25). Dibutylphthalate was occasionally detected by HPLC and LC-MS (HPLC: tR = 3.30 min; LC-MS: tR = 5.0 min, [M+H] + = 279). HPLC analysis also showed varying amounts of a secondary peak which trailed each product by 0.3-0.4 min and was highly UV active at 254 and 280 nm. This impurity could not be identified by LC- MS but might result form product cleavage at polyethyleneglycol rather than at the Anp linker.
Adventitious oxidation of polyethyleneglycol to labile peroxides or esters has been discussed in the literature (Rapp Polymere Home Page. http ://www. rap-polymere. com (accessed June 1999).
3-Amino-3- (2-nitrophenyl) propionic acid (H-Anp-OH). The photolinker was synthesized as the free amino acid by a modified version of the literature procedure (Brown et al.
Mol. Div. 1995,1,4) as follows: 2-Nitrobenzaldehyde (20.0 g, 132 mmol, 1.0 equiv) and malonic acid (17.8 g, 171 mmol, 1.3 equiv) were slurried in 20 mL glacial acetic acid (AcOH) and warmed to 45 °C with stirring. Solid ammonium acetate (25.0 g, 324 mmol, 2.5 equiv) was added in one portion and the mixture heated to 60 °C to form a brown solution. After 15 min, a brown solid precipitated and was broken up with a spatula. 15 mL AcOH was added and the mixture stirred for an additional 45 min at 60 °C. Another 15 mL AcOH was added and the mixture was heated to 98-100 °C and stirred for 3 h, eventually forming a deep red solution. 70
mL concd HCl was added and the solution was stirred for another 1 h at 98-100 °C. The solution was cooled to rt, diluted with 150 mL H20, and washed with 200 mL Et20. The aqueous layer was adjusted to pH 4.5, resulting in formation of a precipitate. The solids were collected by filtration and washed with Et20 to yield H-Anp-OH as a yellow solid (18.3 g, 66%) exhibiting satisfactory analytical data.
N- (9-Fluorenylmethyloxycarbonyl)-3-Amino-3- (2-nitrophenyl) propionic acid (Fmoc-Anp-OH). The Fmoc-protected photolinker was synthesized by a modified version of the literature procedure (Brown et al. Mol. Div. 1995,1,4) as follows: H-Anp-OH (5 g, 23.8 mmol, 1.0 equiv), N- (9-fluorenylmethyloxycarbonyloxy) succinimide (Fmoc-OSu, 8.8 g, 26.1 mmol, 1.1 equiv) and 0.85M aq sodium carbonate (100 mL, 85 mmol, 3.5 equiv) were combined in 150 mL THF and stirred for 90 min. The reaction mixture was washed with 2 x 100 mL hexanes, acidified to pH 6, and extracted with 3 x 150 mL EtOAc. The combined organic layers were washed with 2 x IN HC1, 1 x H20, 1 x brine, dried (MgS04), filtered, and evaporated to yield the crude product as a light brown solid. The solid was taken up in 250 mL hot EtOAc and filtered hot. Hexanes were added until precipitate began to form and the mixture was allowed to cool to rt overnight. The desired product (5.5 g) was recovered by filtration as an off-white solid having analytical data consistent with the literature. A second crop of product (0.85 g) was recovered for a combined yield of 62%.
H2N-Anp-TentaGel Resin (36R, 37a-cR). TentaGel S NH2 (10.0 g, 0.29 meq/g, 2.9 mmol, 1.0 equiv) was placed in a 100 mL fritted glass tube and swollen in distd THF with N2 bubbling for 2 min. The vessel was drained and the resin was swollen in distd CH2Cl2 for another 2 min. The vessel was drained and Fmoc-Anp-OH (1.881 g, 4.35 mmol, 1.5 equiv), HATU (1.654 g, 4.35 mmol, 1.5 equiv), NMP (50 mL), and DIPEA (1.52 mL, 8.70 mmol, 3.0 equiv) were added in sequence. The reaction was allowed to proceed for 5 h. The resin was washed with 4 x NMP and 4 x CH2Cl2 to yield Fmoc-Anp-TentaGel which was negative to Kaiser ninhydrin test. The Fmoc group was removed by 2 x 15 min treatments with 50 mL of freshly prepared 20% piperidine in DMF. The resin was washed as above to yield H2N-Anp- TentaGel resin, 36R, 37a-cR (10.6 g, 100% by mass) which turned brown after heating for 2 min under Kaiser conditions.
H2N-Aca-Anp-TentaGel Resin (37d-fR). H2N-Anp-TentaGel resin, 36R, (3.18 g, 0.27 meq/g, 0.873 mmol, 1.0 equiv) was placed in a 50 mL fritted glass tube and swollen in distd CH2C12 for 2 min. The vessel was drained and N-Fmoc-oz-Aminocaproic acid (Fmoc-Aca-OH, 925.6 mg, 2.619 mmol, 3.0 equiv), PyBOP (1.363 g, 2.619 mmol, 3.0 equiv), 30 mL NMP, and DIPEA (0.760 mL, 4.365 mmol, 5. 0 equiv) were added in sequence. After 1 h, the resin was washed as above to yield Fmoc-Aca-Anp-TentaGel resin which was negative to Kaiser test.
Fmoc deprotection as above yielded H2N-Aca-Anp-TentaGel resin, 37d-fR (3.24 g, 99% by mass) which was positive to Kaiser test.
(lS, 5S, 6S)-5-Hydroxy-7-oxabicyclo [4.1.0] hept-3-ene-3-carboxamide (Epoxycyclohexenol carboxamide, 38a-c). H2N-Anp-TentaGel resin, 37a-cR (1.59 g, 0.27 meq/g, 0.436 mmol, 1.0 equiv) was placed in a 50 mL fritted glass tube. Epoxycyclohexenol carboxylic acid (+)-7 (74.9 mg, 0.480 mmol, 1.1 equiv), PyBOP (249.8 mg, 0.480 mmol, 1.1 equiv), 20 mL NMP, and DIPEA (228 pL, 1.309 mmol, 3.0 equiv) were added in sequence.
After 2 h, the resin was washed as above to yield Epoxycyclohexenol-Anp-TentaGel resin 38a- cR (1.6343 g, 99% by mass) which was negative to Kaiser test. Photolysis of the resin yielded the crude epoxycyclohexenol carboxamide, 38a-c, as a yellow oil. TLC: Rf 0.18 (9: 1 CH2Cl2/MeOH) ; Rf0. 11 (l : l CH2Cl2/THF). HPLC: tR = 0.306 min, Amax = 215 nm. 1H-NMR (400 MHz, CD3CN) : 8 6.28 (m, 1H, C3-H), 4.42 (m, 1H, C4-H), 3.35 (m, 1H, C5-H), 3.13 (m, 1H, C6-H), 2.73 (ddq, 1H, J= 4.0,1.3, C7-Ha), 2.59 (app dq, 1H, J= 7.3,2.5, C7-Hb). CI-MS (NH3) mlz (rel int): 173 ([M+NH4] +, 90), 156 ( [M+H] +, 33). HRMS (NH3) mlz calcd for C7H13N203 173.0926; found 173.0929 (lS, 5S, 65)-N-(6-amino-6-oxoheXyl)-5-hydroxy-7-oxabicyclo [4. 1.0] hept-3-ene-3- carboxamide (Epoxycyclohexenol co-amino caproic carboxamide, 38d-f).
Epoxycyclohexenol-Aca-Anp-TentaGel resin 38d-fR was synthesized essentially as above from H2N-Aca-Anp-TentaGel resin, 37d-fR (97% yield by mass). Photolysis of the resin yielded the crude epoxycyclohexenol carboxamide, 38d-f, as a yellow oil. TLC: Rf 0.09 (9: 1 CH2Cl2/MeOH) ; Rf0. 03 (1: 1 CH2Cl2/THF). HPLC: tR = 1.717 min, Amac= 203,211 nm. 1H- NMR (500 MHz, CD3CN) : 8 6.60 (br s, 1H, C7-NH), 6.20 (m, 1H, C9-H), 6.04 (br s, 1H, Cl- NHa), 5.51 (br s, 1H, Cl-NHb), 4.41 (m, 1H, C10-H), 3.35 (m, 1H, Cll-H), 3.17 (q, 2H, J= 6. 8, C6-H2), 3.14 (m, 1H, C12-H), 2.72 (app ddd, 1H, J= 19.8,2.7,1.3, C13-Ha), 2.58 (app dq, 1H, J = 19.6,2.4, C13-Hb), 2.11 (t, 2H, J = 7.5, C2-H2), 1.54 (quint, 2H, J = 7.6, C5-H2), 1.47 (quint, 2H, J = 7.3, C3-H2), 1.29 (m, 2H, C4-H2). FAB-MS (NBA/NaI) m/z (rel int): 291 ([M+Nal+,
100), 269 ( [M+H] +, 22). HRMS (NBA/NaI) m/z calcd for C13H2oN204Na 291.1321; found 291.1320.
General Procedure for Tandem Acylation-1, 3-Dipolar Cycloaddition Reaction. In a PD-10 column were placed the appropriate epoxycyclohexenol resin, 38R (533 mg, 0.26 meq/g, 133.9 umol, 1.0 equiv), PyBroP (127.6 mg, 273.8 llmol, 2.0 equiv), and the appropriate iodobenzyl nitrone acid, 11 (83.5 mg, 273.8 pmol, 2.0 equiv). CH2C12 (5.3 mL) was added and the tube was flushed with Ar, capped, vortexed briefly, and immediately cooled to 0 °C in an ice bath. DIPEA (95.4 pL, 547.5 (J. mol, 4.0 equiv) was added and the tube was vortexed briefly and returned to 0 °C. DMAP (18.4 mg, 150.6 pmol, 1.1 equiv) was added as 97.4 LL of a CH2C12 stock solution and the tube was vortexed briefly and returned to 0 °C for 10 min. The tube was then wrapped with parafilm, wrapped in foil, and transferred to a Labquake in a 4 °C cold cabinet. After mixing overnight, the resin was washed (Method B) and exposed to the coupling conditions twice more to yield the iodobenzyl tetracycle resin, 39R. Photolysis of the resin yielded the crude iodobenzyl tetracycle, 39, as a yellow oil.
[2aS- (2aa, 4aa, Sap, 6ap, 6ba, 6ca)]-Hexahydro-3- [ (2-iodophenyl) methyl]-2-oxo-2H- furo [4,3,2-cdl oxireno [fl [1,2] benzisoxazole-4a (3H)-carboxamide (2-Iodobenzyl tetracycle carboxamide, 39a). TLC: Rif 0. 38 (4: 1 CH2C12/THF) ; Rf0. 27 (1: 1 CH2Cl2/EtOAc). HPLC: tR = 2.573 min, amas = 202,230 nm. 1H-NMR (400 MHz, CD3CN): 8 7.87 (dd, 1H, J= 7.9,1.2, C13-H), 7.52 (dd, 1H, J= 7.8,1.7, C16-H), 7.38 (td, 1H, J= 7.5,1.2, C15-H), 7.04 (td, 1H, J= 7.5,1.8, C14-H), 6.06 (br s, 1H, C1-NHa), 5. 51 (br s, 1H, C1-NHb), 5.12 (dd, 1H, J= 7.2,2.7, C6-H), 4.38 (d, 1 H, J = 8.2, C9-H), 4.35 (d, 1H, J= 14.6, C 10-Ha), 4.09 (d, 1H, J= 14.6, C10- Hb), 3.87 (t, 1H, J = 7.5, C7-H), 3.51 (dd, 1H, J = 3.6,2.7, C5-H), 3.30 (dd, 1H, J = 6. 2,2.4, C4- H), 2.34 (dd, 1H, J= 16.8,1.7, C3-Ha), 2.25 (dd, 1H, J= 16.8,2.7, C3-Hb). FAB-MS (glycerol) mlz (rel int): 443 ( [M+H] +, 42). HRMS (glycerol) mlz calcd for C16Hl6lN205 443.0104; found 443.0110.
[2aS-(2aa, 4aa, 5aß, 6aß, 6ba, 6ca)]-Hexahydro-3-[(3-iodophenyl) methyl]-2-oxo-2H- furo [4,3,2-cdloxireno [t [1, 2] benzisoxazole-4a (3H)-carboxamide (3-Iodobenzyl tetracycle carboxamide, 39b). TLC: Rf 0.38 (4:1 CH2Cl2/THF) ; Rf 0.30 (1: 1 CH2Cl2/EtOAc). HPLC : tR = 2.893 min, AM,, = 203,229 nm. 1H-NMR (400 MHz, CD3CN) : 8 7.77 (app d, 1H, J= 1.6, C12-H), 7.65 (dd, 1H, J= 7.8,1.4, C14-H), 7.37 (dd, 1H, J= 7.7,1.0, C16-H), 7.12 (t, 1H, J= 7.8, C15-H), 6.09 (br s, 1H, C1-NHa), 5.70 (br s, 1H, C1-NHb), 5.09 (dd, 1H, J= 7.2,2.6, C6- H), 4.32 (d, 1 H, J = 8.2, C9-H), 4.26 (d, 1 H, J = 14.2, C 10-Ha), 3.95 (d, 1H, J= 14.2, C10-Hb), 3.86 (t, 1H, J= 7.5, C7-H), 3.49 (dd, 1H, J= 3.6,2.7, C5-H), 3.28 (dd, 1H, J= 6.2,2.3, C4-H), 2.34 (dd, 1H, J= 16.8,1.7, C3-Ha), 2.25 (dd, 1H, J= 16.8,2.7, C3-Hb). FAB-MS (glycerol) mlz (rel int): 443 ( [M+H] +, 38). HRMS (glycerol) m/z calcd for C16Hl6IN205 443.0104; found 443.0105.
[2aS- (2aa, 4aa, 5ap, 6a (3, 6ba, 6ca)]-Hexahydro-3- [ (4-iodophenyl) methyl]-2-oxo-2H- furo [4, 3,2-edloxirenolt [1,2] benzisoxazole-4a (3H)-carboxamide (4-Iodobenzyl tetracycle carboxamide, 39e). TLC: Rf 0. 38 (4: 1 CH2Cl2/THF) ; Rf 0.27(1:1 CH2Cl2/EtOAc). HPLC: tR = 2.898 min, #max = 202,234 nm. 1H-NMR (400 MHz, CD3CN) : 8 7.69 (d, 2H, J = 8.3, C13-H, C15-H), 7.17 (d, 2H, J = 8.3, C12-H, C16-H), 6.06 (br s, 1H, Cl-NHa), 5.65 (br s, 1H, C1-NHb), 5.09 (dd, 1H, J= 7.3,2.6, C6-H), 4.32 (d, 1H, J= 8.2, C9-H), 4.24 (d, 1H, J= 14.1, C10-Ha), 3.95 (d, 1H, J = 14.1, C10-Hb), 3.85 (t, 1H, J= 7.5, C7-H), 3.49 (dd, 1H, J = 3.6,2.7, C5-H), 3.28 (dd, 1H, J= 6.2,2.4, C4-H), 2.34 (dd, 1H, J= 16.8,1.7, C3-Ha), 2.23 (dd, 1H, J= 16.8,2.7, C3-Hb). FAB-MS (glycerol) mlz (rel int): 443 ( [M+H] +, 28). HRMS (glycerol) mlz calcd for C16H16IN205 443.0104; found 443.0102.
[2aS-(2aα, 4aa, 5ap, 6ap, 6ba, 6ea)]-N-(6-Amino-6-oxohexyl) hexahydro-3-[(2- iodophenyl) methyl]-2-oxo-2H-furo [4,3,2-cd] oxireno [f] [1,2] benzisoxazole-4a (3H)- carboxamide (2-Iodobenzyl tetracycle (o-aminocaproic carboxamide, 39d). TLC: Rf 0. 42 (9: 1 CH2C12/MeOH) ; Rf0. 14 (1 : 1 CH2C12/THF). HPLC: tR = 2.894 min, #max = 202,229 nm.
¹H-NMR (400 MHz, CD3CN) : 87. 89 (dd, 1H, J= 7. 9,1.2, C19-H), 7.48 (dd, 1H, J= 8. 3,2.3, C22-H), 7.40 (td, 1H, J= 7. 5,1.2, C21-H), 7.05 (td, 1H, J= 7. 6,1.8, C20-H), 6.11 (br s, 1H, C7- NH), 6.02 (br s, 1H, Cl-NHa), 5.50 (br s, 1H, Cl-NHb), 5.11 (dd, 1 H, J = 7.2,2.7, C12-H), 4.39 (d, 1H, J= 8.2, C15-H), 4.33 (d, 1H, J= 14.7, C16-Ha), 4.07 (d, 1H, J = 14.6, C16-Hb), 3.86 (t, 1H, J= 7.7, C13-H), 3.50 (dd, 1H, J= 3.6,2.7, Cll-H), 3.29 (dd, 1H, J= 6.3,2.5, C10-H), 3.02 (m, 1H, C6-Ha), 2.85 (m, 1H, C6-Hb), 2.31 (dd, 1H, J= 16.6,1.9, C9-Ha), 2.21 (dd, 1H, J= 16.8,2.7, C9-Hb), 2.07 (t, 2H, J= 7.5, C2-H2), 1.46 (m, 2H, C3-H2), 1.26 (m, 2H, C5-H2), 1.14 (m, 2H, C4-H2). FAB-MS (glycerol) m/z (rel int): 556 ([M+H] +, 33). HRMS (glycerol) mlz calcd for C22H27IN306 556.0945; found 556.0957.
[2aS-(2aα, 4aa, Sap, 6ap, 6ba, 6ca)]-N (6-Amino-6-oxohexyl) hexahydro-3- [ (3- iodophenyl) methyll-2-oxo-2H-furo [4,3,2-cdl oxireno U1 [1, 2] benzisoxazole-4a (3H)- carboxamide (3-Iodobenzyl tetracycle m-ammoeaproic carboxamide, 39e). TLC: Rf 0. 38 (9: 1 CH2Cl2/MeOH) ; Rf0. 15 (1: 1 CH2Cl2/THF). HPLC: tR = 2.962 min, #max = 207,229 nm.
1H-NMR (400 MHz, CD3CN) : 5 7.75 (app d, 1H, J = 1.4, C18-H), 7.67 (dd, 1H J = 7.9,1.2, C20-H), 7.37 (dd, 1H, V= 7.7,1.0, C22-H), 7.14 (t, 1H, J= 7.8, C21-H), 6.25 (br s, 1H, C7-NH), 6.02 (br s, 1H, Cl-NHa), 5.50 (br s, 1H, Cl-NHb), 5.09 (dd, 1H, J = 7.3,2.6, C12-H), 4.34 (d, 1H, J= 8.2, C15-H), 4.24 (d, 1H, J= 14.2, C16-Ha), 3.94 (d,1H, J = 14.2, C16-Hb), 3.84 (t, 1H, J = 7.7, C13-H), 3.49 (dd, lH, J=3. 6,2.7, C 11-H), 3.28 (dd, 1H, J = 6.0,2.6, C10-H), 3.07 (m, 1H, C6-Ha), 2.96 (m, 1H, C6-Hb), 2.28 (dd, 1H, J= 16.8,1.9, C9-Ha), 2.21 (dd, 1H, J= 16.8, 2.8, C9-Hb), 2.08 (t, 2H, J = 7.5, C2-H2), 1.50 (m, 2H, C3-H2), 1.26 (m, 2H, C5-H2), 1.18 (m, 2H, C4-H2). FAB-MS (glycerol) m/z (rel int) : 556 ( [M+H] +, 100). HRMS (glycerol) m/z calcd for C22H27IN306 556.0945; found 556.0953.
[2aS-(2aa, 4aa, 5ap, 6ap, 6ba, 6ca)]-N-(6-Amino-6-oxohexyl) hexahydro-3-[(4- iodophenyl) methyl]-2-oxo-2H-furo [4,3,2-cdl oxireno [fl [1,2] benzisoxazole-4a (3H)- carboxamide (4-Iodobenzyl tetracycle eo-aminocaproic carboxamide, 39f). TLC: Rf 0.32 (9: 1 CH2Cl2/MeOH) ; Rf0. 15 (1: 1 THF/CH2C12). HPLC : tR = 2.974 min, Amax = 204,234 nm.
1H-NMR (400 MHz, CD3CN) : 8 7.71 (d, 2H, J = 8.3, C19-H, C21-H), 7.15 (d, 2H, J= 8.3, C18-H, C22-H), 6.21 (br s, 1H, C7-NH), 6.02 (br s, 1H, C1-NHa, 5.51 (br s, 1H, Cl-NHb), 5.08 (dd, 1H, J= 7.2,2.6, C12-H), 4.32 (d, 1H, J= 8.2, C15-H), 4. 22 (d, 1H, J= 14.1, C 16-Ha), 3.94 (d, 1H, J= 14.1, C16-Hb), 3.83 (t, 1 H, J = 7.7, C13-H), 3.49 (dd, 1H, J= 3.6,2.7, C11-H), 3.27 (dd, 1H, J= 6.2,2.4, C10-H), 3.03 (m, 1H, C6-Ha), 2.92 (m, 1H, C6-Hb), 2.28 (m, 1H, C9-Ha), 2.20 (m, 1H, C9-Hb), 2.09 (t, 2H, J= 7.7, C2-H2), 1.5 (m, 2H, C3-H2), 1.3-1.1 (m, 4H, C5-H2, C4-H2). FAB-MS (glycerol) mlz (rel int): 556 ( [M+H] +, 100). HRMS (glycerol) mlz calcd for C22H27IN306 556.0945; found 556.0947.
General Procedure for Sonogashira/Castro-Stephens Alkyne Coupling Reaction. To 50 mg (10.5 jj. mol) of the appropriate iodobenzyl tetracycle resin, 39R, in a 2 mL Bio-Spin column was added copper (I) iodide (4.4 mg, 23.1 mol, 2.2 equiv) and bis (triphenylphosphine) palladium (II) chloride (8.1 mg, 11.55 umol, 1.1 equiv). DMF (500 pL) was added and the tube was flushed with Ar, capped, and shaken to dissolve the reagents.
DIPEA (54.9 L, 315 mol, 30 equiv) and the appropriate alkyne (20 equiv) were added and the tube was capped, shaken, wrapped with parafilm, and wrapped in foil. After mixing at rt (para : 15 min, meta : 30 min, ortho : 45 min), the resin was washed (Method A) and dried under vacuum. Photolysis of the resin, 40R, yielded the crude alkynylbenzyl tetracycle, 40, as a yellow oil.
General Procedure for Sonogashira/Castro-Stephens Alkyne Coupling Reaction with Bis (Terminal Alkynes). The same procedure was used as above except (Ph3P) 2PdCl2 was replaced with tetrakis (triphenylphosphine) palladium (0) (prepared as previously described) (Coulson, D. L. Inorg. Synth. 1972,13,121) and 70 equiv of DIPEA and 50 equiv of alkyne were used.
[2aS- (2aa, 4aa, 5aß, 6aß, 6ba, 6ca)]-Hexahydro-3-[[2-(3-phenyl-1- propynyl) phenyl] methyl]-2-oxo-2H-furo [4,3,2-cdloxireno [f] [1,2] benzisoxazole-4a (3H)- carboxamide (o- (3-Phenyl-1-propynyl) benzyl Tetracycle Carboxamide, 40a). TLC: Rf 0.44 (4: 1 CH2C12/THF) ; Rf 0. 30 (1: 1 CH2Cl2/EtOAc). HPLC: tR = 3.114 min, = (203), 208, 246 nm. 1H-NMR (500 MHz, CD3CN) : 8 7.50 (d, 1H, J= 7.4, C13-H), 7.44 (d, 1H, J= 7.4, C16-H), 7.43 (obs d, 2H, C21-H, C25-H), 7.36 (t, 2H, J= 7.7, C22-H, C24-H), 7.33 (td, 1H, J= 7.5,1.4, C14-H), 7.28 (td, 1H, J= 7.5,1.3, C15-H), 7.26 (t, 1H, J= 7.2, C23-H), 6.04 (br s, 1H, C1-NHa), 5.58 (br s, 1 H, C 1-NHb), 5.05 (dd, 1H, J= 7.1,2.7, C6-H), 4.41 (d, 1 H, J = 14.0, C10- Ha), 4.24 (d, 1H, J= 13.9, C10-Hb), 4.24 (d, 1H, J= 8.2, C9-H), 3.88 (s, 2H, C19-H2), 3.79 (t, 1H, J = 7.6, C7-H), 3.49 (t, 1H, J = 3. 3, C5-H), 3.28 (app dd, 1H, J= 6.2,2.5, C4-H), 2.39 (d, 1H, J= 16. 5, C3-Ha), 2.20 (dd, 1H, J= 16. 7,2.6, C3-Hb). FAB-MS (glycerol) m/z (rel int): 431 ( [M+H] +, 33). FAB-MS (NBA/NaI) m/z (rel int): 453 ( [M+Na] +, 40), 431 ( [M+H] +, 5). HRMS (NBA/NaI) m/z calcd for C25H22N205Na 453.1426; found 453.1432.
[2aS-(2aα, 4aα, 5aß, 6aß, 6bα, 6cα)]-Hexahydro-3-[[3-(3-methyl-3-buten-1- ynyl) phenyll methyl]-2-oxo-2H-furol4, 3,2-cdl oxireno [fl [1,2] benzisoxazole-4a (3H)- carboxamide (m- (3-Methyl-3-buten-1-ynyl) benzyl tetracycle carboxamide, 40b). TLC: Rf
0.41 (4: 1 CH2C12/THF) ; Rf 0.33 (1: 1 CH2Cl2/EtOAc). HPLC: tR = 3.182 min, 4. = (203), 212,270, (282) nm. 1H-NMR (500 MHz, CD3CN) : 6 7. 57 (s, 1H, C12-H), 7.37 (m, 2H, C14-H, C16-H), 7.33 (t, 1H, J= 7.4, C15-H), 6.09 (br s, 1H, C1-NHa), 5.64 (br s, 1H, C1-NHb), 5.38 (app q, 1H, J= 1.0, C20-Hz), 5.36 (app q, 1H, J= 1.7, C20-HE), 5.09 (dd, 1H, J= 7.2,2.6, C6- H), 4.32 (d, 1H, J= 8.2, C9-H), 4.29 (d, 1H, J=13.9, C10-Ha), 4.00 (d, 1H, J=13. 9, C10-Hb), 3.86 (t, 1H, J = 7.7, C7-H), 3.50 (t, 1H, J = 3.2, C5-H), 3.29 (dt, 1H, J= 3.7,2.5, C4-H), 2.36 (dd, 1H, J =16. 9,1.6, C3-Ha), 2.26 (dd, 1H, J =16. 8,2.7, C3-Hb), 1.97 (dd, 3H, J= 2.5,1.4, C21-H3). FAB-MS (glycerol) mlz (rel int): 381 ( [M+H] +, 100). FAB-MS (NBA/NaI) mlz (rel int): 403 ( [M+Na] +, 27). HRMS (glycerol) m/z calcd for C2lH2lN205 381.1450; found 381.1442.
[2aS- (2aa, 4aa, 5ap, 6ars, 6ba, 6cα)]-3-[[4-(4- Chlorophenyl) ethynyl] phenyl] methyl] hexahydro-2-oxo-2H-furo [4,3,2- cdloxireno [f] [1,2] benzisoxazole-4a (3H)-carboxamide (p- (4-Chlorophenylethynyl) benzyl tetracycle carboxamide, 40c). TLC: Rif 0. 40 (4: 1 CH2C12/THF) ; Rf0. 30 (1: 1 CH2C12/EtOAc).
HPLC: tR = 3.618 min, Amas = 202, (222), (276), 289,303 nm. 1H-NMR (400 MHz, CD3CN) : 8 7.51 (app d, 4H, J= 8.4, C13-H, C15-H, C20-H, C24-H), 7.41 (obs d, 2H, J= 8.7, C12-H, C16-H), 7.41 (obs d, 2H, J= 8.2, C21-H, C23-H), 6.06 (br s, 1H, Cl-NHa), 5.66 (br s, 1H, Cl- NHb), 5.10 (dd, 1H, J= 7.2,2.7, C6-H), 4.35 (d, 1H, J= 8.2, C9-H), 4.32 (d, 1H, J= 14.2, C10- Ha), 4.03 (d, 1H, J= 14.2, C10-Hb), 3.87 (t, 1H, J= 7.7, C7-H), 3.50 (dd, 1H, J= 3.5,2.9, C5- H), 3.29 (dt, 1H, J= 3. 8,2.5, C4-H), 2.35 (dd, 1H, J= 16.8,1.5, C3-Ha), 2.25 (dd, 1H, J= 16.8, 2.8, C3-Hb). FAB-MS (glycerol) mlz (rel int) : 451 ( [M+H] +, 46). HRMS (glycerol) mlz calcd for C24H2oclN2os 451.1061; found 451.1060.
[2aS-(2aa, 4aa, 5aß, 6aß, 6ba, 6ca)]-N-(6-Amino-6-oxohexyl)-3-[[2-(3, 3-dimethyl-1- butynyl) phenyl] methyl] hexahydro-2-oxo-2H-furo [4,3,2-cdl oxireno [fl [1,2] benzisoxazole- 4a (3H)-carboxamide (o-(3,3-Dimethyl-1-butynyl)benzyl tetracycle w-aminocaproic carboxamide, 40d). TLC: Rf 0.43 (9:1 CH2Cl2/MeOH) ; Rf 0.20 (1:1 CH2Cl2/THF). HPLC: tR = 3.257 min, #max = 209,247 nm. ¹H-NMR(500 MHz, CD3CN) : 5 7.44 (d, 1H, J= 7.5, C19- H), 7.35 (dd, 1H, J = 7.7,1.2, C22-H), 7.32 (td, 1H, J = 7.7,1.4, C20-H), 7.26 (td, 1H, J = 7.4, 1.3, C21-H), 6.14 (br s, 1H, C7-NH), 6.00 (br s, 1H, Cl-NHa), 5.50 (br s, 1H, Cl-NHb), 5.10 (dd, 1H, J= 7.2,2.6, C12-H), 4.37 (d, 1H, J= 8.2, C15-H), 4.35 (d, 1H, J= 16.6, C16-Ha), 4.18 (d, 1H, J= 14.4, C16-Hb), 3.85 (t, 1 H, J = 7.6, C13-H), 3.50 (t, 1 H, J = 3.2, C11-H), 3.28 (dd, 1H, J= 6.0,2.6, C10-H), 3.01 (m, 1H, C6-Ha), 2.88 (m, 1H, C6-Hb), 2.31 (d, 1H, J= 16.8, C9- Ha), 2.19 (dd, 1H, J= 16.7,2.8, C9-Hb), 2.06 (t, 2H, J= 7.5, C2-H2), 1.46 (m, 2H, C3-H2), 1.31 (obs m, 2H, C5-H2), 1.30 (s, 9H, C26-H3, C27-H3, C28-H3), 1.13 (m, 2H, C4-H2). FAB-MS (glycerol) m/z (rel int): 510( [M+H]+, 100). HRMS (glycerol) mlz calcd for C28H36N306 510.2604; found 510.2612.
[2aS-(2aa, 4aa, 5aß, 6aß, 6ba, 6cα)]-N-(6-Amino-6-oxohexyl)-3-[[3-(3,3-diethoxy-1- propynyl) phenyl] methyl] hexahydro-2-oxo-2H-furo [4,3,2-cdloxireno [f] [1,2] benzisoxazole- 4a (3H)-carboxamide (m- (3, 3-Diethoxy-l-propynyl) benzyl tetracycle co-aminocaproic carboxamide, 40e). TLC: Rf0. 39 (9: 1 CH2Cl2/MeOH) ; Rf0. 17 (1 : 1 CH2C12/THF). HPLC: tR
= 3.105 min, #max = 206, 243, 246 nm. ¹H-NMR (500 MHz, CD3CN): â 7. 51 (s, 1H, C18-H), 7.43-7.34 (m, 3H, C20-H, C21-H, C22-H), 6.22 (br s, 1H, C7-NH), 6.00 (br s, 1H, C1-NHa), 5.46 (br s, 1H, Cl-NHb), 5.45 (s, 1H, C25-H), 5.09 (dd, 1H, J= 7.3,2.5, C12-H), 4.34 (d, 1H, J = 8.1, C15-H), 4.28 (d, 1H, J= 14.1, C16-Ha), 3.98 (d, 1H, J= 14.2, C16-Hb), 3.85 (t, 1H, J= 7.7, C13-H), 3.74 (m, 2H, C26-Ha, C28-Ha), 3.60 (m, 2H, C26-Hb, C28-Hb), 3.50 (app t, 1H, J= 3.5,2.8, C 11-H), 3.28 (app q, 1H, V= 5. 8,2.7, C10-H), 3.06 (m, lH, J=13. 2,6.4, C6-Ha), 2.94 (m, 1H, J= 13.2,5.6, C6-Hb), 2.28 (dd, 1H, J= 16.9,1.4, C9-Ha), 2.21 (dd, 1 H, J = 16.8,2.8, C9-Hb), 2.09 (t, 2H, J= 7.4, C2-H2), 1.49 (quint, 2H, J= 7.5, C3-H2), 1.24 (obs m, 2H, C5-H2), 1.20 (t, 6H, J= 7.1, C27-H3, C29-H3), 1.16 (obs m, 2H, C4-H2). FAB-MS (glycerol) m/z (rel int): 510 ( [M-OEt] +, 100), 556( [M+H]+,7). FAB-MS (NBA/NaI) m/z (rel int): 578 ( [M+Na] +, 100), 510 ( [M-OEt] +, 11). HRMS (NBA/NaI) m/z calcd for C29H37N30gNa 578.2478; found 578.2475.
[2aS-(2aa, 4aa, 5aß, 6aß, 6ba, 6ca)]-N-(6-Amino-6-oxohexyl) hexahydro-2-oxo-3-[[4- (1-pentynyl)phenyl]methyl]-2H-furo[4,3,2-cd]oxireno [f][1,2]benzisoxazole-4a(3H)- carboxamide (p-(l-Pentynyl) benzyl tetracycle e-aminocaproic carboxamide, 40f). TLC: Rf 0.31 (9: 1 CH2Cl2/MeOH); Rf 0.18(1:1 CH2C12/THF). HPLC: tR = 3.25 5 min, = 203,248, 251 nm. 1H-NMR (500 MHz, CD3CN) : 5 7.35 (d, 2H, J= 8.3, C19-H, C21-H), 7.30 (d, 2H, J= 8.1, C18-H, C22-H), 6.21 (br s, 1H, C7-NH), 6.01 (br s, 1H, Cl-NHa), 5.50 (br s, 1H, Cl-NHb), 5.08 (dd, 1H, J= 7. 2, 2.6, C12-H), 4.32 (d, 1H, J= 8.1, C15-H), 4.26 (d, 1 H, J = 14.1, C16-Ha), 3.98 (d, 1H, J= 14.1, C16-Hb), 3.83 (t, 1H, J = 7.7, C13-H), 3.49 (app t, 1 H, J = 3.2, C 11-H), 3.28 (app dd, 1H, J= 5.9,2.7, C10-H), 3.02 (m, 1H, C6-Ha), 2.91 (m, 1H, C6-Hb), 2.37 (t, 2H, J = 7.0, C25-H2), 2.27 (br d, 1H, J= 16.8, C9-Ha), 2.20 (dd, 1H, J = 16.8,2.9, C9-Hb), 2.08 (t, 2H, J= 7.7, C2-H2), 1.59 (sxt, 2H, J= 7. 2, C26-H2), 1.49 (quint, 2H, J = 7.4, C3-H2), 1.22 (m, 2H, C5-H2), 1.17 (m, 2H, C4-H2), 1.02 (t, 3H, J = 7.3, C27-H3). FAB-MS (glycerol) mlz (rel int): 496 ( [M+H] +, 100). FAB-MS (NBA/NaI) m/z (rel int): 518 ( [M+Na] +, 100), 496 ( [M+H] +, 13).
HRMS (glycerol) m/z calcd for C27H34N306 496.2448; found 496.2463.
General Procedure for Lactone Aminolysis. To 50 mg (10.5 J. mol) of the appropriate alkynylbenzyl tetracycle resin, 40R, in a 2 mL Bio-Spin8 column was added 2-hydroxypyridine (5.0 mg, 52.5 mol, 5 equiv). THF (500 J. L) was added and the tube was flushed briefly with Ar, capped, and shaken until the 2-hydroxypyridine was dissolved. The appropriate amine (25 equiv) was added and the tube was immediately capped, shaken, wrapped with parafilm, and wrapped in foil. After mixing 12-16 h at rt, the resin was washed (Method A) and dried under vacuum. Photolysis of the resin, 41R, yielded the crude alkynylbenzyl y-hydroxyamido tricycle, 41, as a yellow oil.
General Procedure for Lactone Aminolysis with a-Branched Amines. The same procedure was used as above except 10 equiv 2-hydroxypyridine and 50 equiv amine were used.
General Procedure for Lactone Aminolysis with Amine Hydrochlorides. To 50 mg (10.5 pmol) of the appropriate alkynylbenzyl tetracycle resin, 40R, in a 2 mL Bio-Spin column was added 2-hydroxypyridine (5.0 mg, 52.5 p. mol, 5 equiv) and the amine hydrochloride (25 equiv). CH2C12 (300 J. L) and DMF (200 1L) were added and the tube was flushed briefly with N2. DIPEA (91.5 J. L, 525 pmol, 50 equiv) was added and the tube was immediately capped, shaken, wrapped with parafilm, and wrapped in foil. After mixing 12-16 h at rt, the resin was washed (Method A + 3 x 20% DIPEA/CH2C12) and dried under vacuum. Photolysis of the resin, 41R, yielded the crude alkynylbenzyl y-hydroxyamido tricycle, 41, as a yellow oil.
[35-(3a, 3aß, 4a, 4aa, 5aa, 6aD)]-(N3-cyclobutyl) hexahydro-4-hydroxy-2-[[2-(3- phenyl-1-propynyl) phenyl] methyl]-oxireno [fl-1, 2-benzisoxazole-3, 6a (2H)-dicarboxamide (o- (3-Phenyl-l-propynyl) benzyl cyclobutylamido hydroxy tricycle carboxamide, 41 a).
TLC: Rf0. 14 (4: 1 CH2C12/THF) ; Rf0. 06 (1 : 1CH2Cl2/EtOAc). HPLC: tR = 3. 141 min, = = (203), 209,247 nm. 1H-NMR (500 MHz, CD3CN): 6 7.56 (br s, 1H, C9-NH), 7.50 (dd, 1H, J= 7.3,1.4, C13-H), 7.48 (obs d, 2H, C21-H, C25-H), 7.43 (dd, 1H, J= 7.5,1.3, C16-H), 7.38 (t, 2H, J = 7. 7, C22-H, C24-H), 7.32 (td, 1 H, J = 7. 5,1.5, C14-H), 7.27 (td, 3 H, J = 7. 5,1.4, C15-H, C23-H), 6.54 (brs, 1H, Cl-NHa), 5.89 (brs, 1H, Cl-NHb), 5.11 (d, 1H, J= 9. 7, C6-OH), 4.43 (d, 1H, J=12. 8, C10-Ha), 4.11 (obs sxt, 1H, J= 8. 1, C26-H), 4.11 (d, 1H, J=12. 8, C10-Hb),
3.91 (s, 2H, C19-H2), 3.90 (obs m, 1H, C6-H), 3.86 (d, 1H, J = 8.4, C9-H), 3.62 (app ddd, 1H, J = 8.4,5.4,1.4, C7-H), 3.12 (td, 1H, J= 4.0,2.7, C4-H), 3.09 (dd, 1H, J= 4.0,3.1, C5-H), 2.26 (dd, 1H, J= 16.4,3.7, C3-Ha), 2.10 (obs m, 1H, C27-Ha), 2.05 (obs m, 1H, C29-Ha), 1.99 (dt, 1H, J= 16.4,2.1, C3-Hb), 1.75 (app sxt, 2H, J= 10.2, C27-Hb, C29-Hb), 1.60 (m, 2H, C28-H2).
FAB-MS (glycerol) mlz (rel int): 502 ( [M+H] +, 62). FAB-MS (NBA/NaI) mlz (rel int): 524 ( [M+Na] +, 70), 502 ( [M+H] +, 13). HRMS (glycerol) m/z calcd for C29H32N305 502.2342; found 502.2336.
[3S- (3a, 3ap, 4a, 4aa, 5aa, 6a (3)]-Hexahydro-4-hydroxy-2- [ [3- (3-methyl-3-buten-1- ynyl) phenyl] methyl]-N3- (2-propenyl) oxireno [fl-1, 2-benzisoxazole-3, 6a (2H)-dicarboxamide (m- (3-Methyl-3-buten-1-ynyl) benzyl allylamido hydroxy tricycle carboxamide, 41b). TLC: Rf0. 12 (4: 1 CH2C12/THF) ; Rif 0. 07 (1: 1 CH2Cl2/EtOAc). HPLC: tR = 3.034 min, #max = (203), 213,270, (283) nm. 1H-NMR (400 MHz, CD3CN) : 8 7.64 (br t, 1H, C8-NH), 7.51 (s, 1H, C12- H), 7.38 (dt, 1H, J= 7. 0,1.7, C14-H), 7.35 (dd, 1H,. J = 5.7,1.7, C16-H), 7.32 (t, 1H, J= 7. 5, C15-H), 6.42 (s, 1H, C1-NHa), 5.93 (s, 1H, C1-NHb), 5.79 (ddt, 1H, J=17. 2,10.5,5.3, C23-H), 5.37 (obs m, 1H, C20-Hz), 5.36 (obs m, 1H, C20-HE), 5.11 (dq, 1H, J=17. 4,1.6, C24-Hz), 5.06 (dq, 1H, J =10. 3,1.5, C24-HE), 5.02 (d, 1H, J = 9.4, C6-OH), 4.17 (d, 1H, J =13. 7, C10-Ha), 3.93 (obs m, 1H, C6-H), 3.92 (obs d, 1H, J= 8.1, C9-H), 3.91 (obs d, 1H, J =13. 9, C10-Hb), 3.78 (tt, 2H, J = 5.8,1.4, C22-H2), 3.64 (dd, 1H, J= 8.3,5.1, C7-H), 3.14 (obs m, 1H, C5-H), 3.13 (obs m, 1H, C4-H), 2.26 (dd, 1H, J=16. 3,3.4, C3-Ha), 1.97 (t, 3H, J= 1.3, C21-H3), 1.91 (obs dd, 1H, J= 1.9, C3-Hb). FAB-MS (glycerol) m/z (rel int): 438 ( [M+H] +, 60). FAB-MS (NBA/NaI) mlz (rel int) : 438 ( [M+H] +, 78), 460 ( [M+Na] +, 43). HRMS (NBA/NaI) mlz calcd for C24H27N305Na 460.1848; found 460.1853.
[3S-(3α, 3aß, 4α, 4aα, 5aα, 6aß)]-2-[[4-[2-(4-Chlorophenyl)-1- ethynyl] phenyl] methyl] hexahydro-4-hydroxy-N3- [ (2-methoxyphenyl) methyl] oxireno [f]-1, 2- benzisoxazole-3, 6a (2H)-dicarboxamide (p- (4-Chlorophenylethynyl) benzyl 2- methoxybenzylamido hydroxy tricycle carboxamide, 41c). TLC: Rf0.13 (4: 1 CH2Cl2/THF) ; Rf 0.06 (1:1 CH2Cl2/EtOAc). HPLC: tR = 3.778 min, 201, 222, (276), 289,303 nm. 1H- NMR (500 MHz, CD3CN) : 6 7.97 (br t, 1H, C8-NH), 7.52 (d, 1H, J= 8.5, C20-H, C24-H), 7.49 (d, 1H, J= 8. 1, C13-H, C15-H), 7.42 (obs d, 1H, J= 8. 1, C21-H, C23-H), 7.42 (obs d, 1H, C12- H, C16-H), 7.27 (td, 1H, J= 7.9,1.4, C29-H), 7.13 (dd, 1H, J= 7.3, C31-H), 6.97 (d, 1H, J= 8.1, C28-H), 6.89 (td, 1H, J= 7.4, C30-H), 6.37 (br s, 1H, C1-NHa), 5.88 (br s, 1H, C1-NHb), 5.13 (d, 1H, J = 10.0, C6-OH), 4.37 (dd, 1H, J=15. 0,6.2, C25-Ha), 4.33 (dd, 1H, J=14. 8,6.1, C25-Hb), 4.21 (d, 1H, J=13. 8, C 10-Ha), 3.95 (d, 1H, J= 8.6, C9-H), 3.93 (d, 1H, J = 13.9, C10- Hb), 3.85 (s, 3H, C32-H), 3.83 (obs m, 1H, C6-H), 3.67 (dd, 1H, J= 8.4,5.5, C7-H), 3.01 (t, 1H, J= 3.7, C5-H), 2.78 (m, 1H, C4-H), 2.15 (dd, 1H, J=16. 4,4.1, C3-Ha), 1.82 (dd, 1H,. J=16. 4, 2.6, C3-Hb). FAB-MS (glycerol) m/z (rel int): 588/590 ( [M+H] +, 52/28). FAB-MS (NBA/NaI) mlz (rel int): 610/612 ( [M+Na] +, 22/10). HRMS (glycerol) mlz calcd for C32H31ClN306 588.1901; found 588.1896.
[3S- (3a, 3ap, 4a, 4aa, 5aa, 6aD)]-N6a-(6-Amino-6-oxohexyl)-2-[[2-(3, 3-dimethyl-1- butynyl)phenyl]methyl]hexahydro-4-hydroxy-N³- [(4-methoxyphenyl)methyl]oxireno[f]-1,2- benzisoxazole-3, 6a (2H)-dicarboxamide (o-(3,3-Dimethyl-1-butynyl)benzyl 4- methoxybenzvlamido hydroxy tricycle co-aminocaproic carboxamide, 41d). TLC: Rf 0. 43
(9: 1 CH2Cl2/MeOH) ; Rif 0. 20 (1 : 1 CH2C12/THF). HPLC: tR = 3.385 min, #max = 202,230,245, (275) nm. 1H-NMR (500 MHz, CD3CN) : 6 8.09 (br t, 1H J= 5.8, C14-NH), 7.58 (d, 1H J= 7.5, C19-H), 7.33 (dd, 1H, J= 7.4,1.1, C22-H), 7.31 (td, 1H, J= 7.3,1.3, C20-H), 7.25 (td, 1H, J = 7.4,1.3, C21-H), 7.11 (d, 2H, J = 8.7, C31-H, C35-H), 6.82 (d, 2h J = 8.6, C32-H, C34-H), 6.67 (br t, 1H, J= 5.7, C7-NH), 5. 99 (br s, 1H, C1-NHa), 5.46 (br s, 1H, Cl-NHb), 5.38 (d, 1 H, J = 10.8, C12-OH), 4.26 (dd, 1 H, J = 14.6,6.5, C29-Ha), 4.25 (d, 1H, J = 13.1, C 16-Ha), 4.18 (dd, 1H, J = 14.6,6.1, C29-Hb), 4.08 (d, 1H, J= 13.1, C16-Hb), 3.97 (d, 1 H, JI= 8. 8, C 15-H), 3.84 (dd, 1H, J= 9.0,5.3, C13-H), 3.74 (s, 3H, C36-H3), 3.73 (obs m, 1H, C12-H), 3.11 (m, 2H, C6- H2), 2.97 (dd, 1H, J= 4.2,3.3, C 11-H), 2.83 (td, 1H J = 5.4,4.4, C10-H), 2.17 (obs m, 1H, C9- Ha), 2.06 (t, 2H, J= 7.4, C2-H2), 1.62 (dd, lh J= 16.4,3.2, C9-Hb), 1.48 (m, 2H, C3-H2), 1.38 (m, 2H, C5-H2), 1.22 (m, 2H, C4-H2), 1.30 (s, 9H, C26-H3, C27-H3, C28-H3). FAB-MS (glycerol) mlz (rel int) : 647 ( [M+H] +, 100). HRMS (glycerol) mlz calcd for C36H47N407 647.3445; found 647.3463 [3S-(3α, 3aß, 4a, 4aa, 5aα, 6aß)]-N6a- (6-Amino-6-oxohexyl)-2- [ [3- (3, 3-diethoxy-l- propynyl) phenyl] methyl]-N3-(2, 2-dimethoxyethyl) hexahydro-4-hydroxyoxireno [fl-1, 2- benzisoxazole-3, 6a (2H)-dicarboxamide (m- (3, 3-Diethoxy-l-propynyl) benzyl 2,2- dimethoxyethylamido hydroxy tricycle m-aminocaproic carboxamide, 41e). TLC: Rf 0. 33 (9: 1 CH2Cl2/MeOH) ; Rf0.09 (1:1 CH2CI2/THF). HPLC: tR = 2.995 min, = 204,243,246 nm. 1H-NMR (500 MHz, CD3CN) : 6 7.78 (br t, 1H, J= 5.5, C14-NH), 7.51 (s, 1H, C18-H), 7.40 (m, 2H, C20-H, C22-H), 7.35 (t, 1H, J= 7.5, C21-H), 6.68 (br t, 1H, J= 5.7, C7-NH), 6.07 (br s, 1H, C1-NHa), 5.61 (br s, 1H, C1-NHb), 5.46 (s, 1H, C25-H), 4.87 (d, 1H, J= 8.4, C12- OH), 4.38 (t, 1H, J= 5.0, C31-H), 4.12 (d, 1H, J= 13.9, C16-Ha), 4.01 (ddd, 1H, J= 8.4,4.6, 3.7, C12-H), 3.89 (d, 1H, J= 13.9, C16-Hb), 3.86 (d, 1H, J= 8.0, C15-H), 3.74 (dq, 2H, J= 9.5, 7.1, C26-Ha, C28-Ha), 3.60 (dq, 2H, J = 9.4,7.1, C26-Hb, C28-Hb), 3.53 (obs dd, 1H, J = 8.2, 4.9, C13-H), 3.37 (m, 1H, C30-Ha), 3.32 (s, 3H, C32-H3), 3.32 (s, 3H, C33-H3), 3.22 (obs m,
1H, C30-Hb), 3.19 (obs m, 3H, C6-H2, C11-H), 3.14 (obs m, 1H, C10-H), 2.24 (dd, 1H, J= 16.3, 3.4, C9-Ha), 2.12 (obs t, 2H, J = 7.3, C2-H2), 1.93 (obs m, 1H, C9-Hb), 1.53 (m, 2H, C3-H2), 1.45 (m, 2H, C5-H2), 1.27 (m, 2H, C4-H2), 1.20 (t, 6H, J = 7.1, C27-H3, C29-H3). FAB-MS (glycerol) mlz (rel int): 661 ( [M+H] +, 55), 615 ( [M-OEt] +, 18). HRMS (glycerol) mlz calcd for C33H49N4010 661.3449; found 661.3464.
[3- (3a, 3ap, 4a, 4aa, 5aa, 6ap)]-/V- (6-Amiao-6-oxohexyt) hexahydro-4-hydroxy- N³-[2-(4-methoxyphenyl)ethyl]-2- [[4-(1-pentynyl)phenyl]methyl]oxireno [f]-1,2- benzisoxazole-3, 6a (2H)-dicarboxamide (p- (1-Pentynyl) benzyl 4-methoxyphenethylamido hydroxy tricycle c3-aminocaproic carboxamide, 41f). TLC: Rif 0. 23 (9: 1 CH2Cl2/MeOH) ; Rf 0.09 (1: 1 CH2Cl2/THF). HPLC: tR = 3.428 min, Amas = 201,232,251, (279) nm. 1H NMR (500 MHz, CD3CN) : 8 7.60 (br t, 1H, C14-NH), 7.32 (d, 2H, J= 8.1, C19-H, C21-H), 7.23 (d, 2H, J= 8.1, C18-H, C22-H), 7.11 (d, 2H, J= 8.6, C31-H, C35-H), 6.82 (d, 2H, J= 8.7, C32-H, C34-H), 6.63 (br t, 1H, J= 6.0, C7-NH), 6.05 (br s, 1 H, C 1-NHa), 5.52 (br s, 1H, Cl-NHb), 5.05 (d, 1H, J= 8.8, C 12-OH), 4.06 (d, 1 H, J = 13.9, C16-Hb), 3.79 (m, 3H, C12-H, C15-H, C16-Ha), 3.73 (s, 3H, C36-H3), 3.52 (obs m, 1H, C13-H), 3.42 (sxt, 1H, J= 6.3, C28-Ha), 3.36 (sxt, 1H, J = 6.2, C28-Hb), 3.12 (t, 2H, J= 6.6, C6-H2), 3.02 (app dd, 1H, J= 7.0,4.0, C10-H), 2.68 (td, 2H, J= 6.9,2.7, C29-H2), 2.37 (t, 2H, J= 7.0, C25-H2), 2.16 (obs d, 1H, C9-Ha), 2.09 (t, 2H, J= 7.4, C2-H2), 1.82 (dd, 1H, J= 16.2,2.9, C9-Hb), 1.59 (sxt, 2H, J = 7.3, C26-H2), 1.51 (m, 2H, C5- H2), 1.41 (m, 2H, C3-H2), 1. 25 (m, 2H, C4-H2), 1.02 (t, 3H, J= 7.4, C27-H3). FAB-MS (glycerol) mlz (rel int): 647 ( [M+H] +, 85). FAB-MS (NBA/NaI) mlz (rel int): 669 ( [M+Na] +, 100), 647 ( [M+H] +, 60). HRMS (NBA/NaI) m/z calcd for C24H27N305Na 669.3264; found 669.3252.
General Procedure for Alcohol Esterification. To 50 mg (10.5 umol) of the appropriate alkynylbenzyl y-hydroxyamido tricycle resin, 41R, in a 2 mL Bio-Spin column was added 200 µL CH2Cl2. The tube was flushed with Ar and cooled to 0 °C in an ice bath. The appropriate carboxylic acid (50 equiv) was dissolved or suspended in 400 tL CH2CI2 in an oven-dried 2 mL Wheaton vial and activated with DIPC (41.1 1, 262.5 Hmol, 25 equiv). After
stirring at rt for 2 min, DIPEA (91.5 pL, 525 pL, 50 equiv) was added and stirring continued for another 3 min. The activated acid solution was then added to the resin via pipette with manual agitation followed by DMAP (6.4 mg, 52.5 µmol, 5 equiv) in 50 iL CH2CI2. After standing 15 min at 0 °C, the tube was warmed to rt, wrapped with parafilm, wrapped in foil, and mixed at rt for 12-16 h. After washing (Method A + 3 x 20% DIPEA/CH2Cl2), photolysis of the resin, 42R, yielded the crude alkynylbenzyl amido acyl tricycle, 42, as a yellow oil.
[3S- (3a, 3a (3, 4a, 4aa, 5aa, 6ap)]-4-Methoxyphenylacetic acid, 6a- (aminocarbonyl)-<BR> 2-[[2-(3-phenyl-1-propynyl) phenyl] methyl] oetahydro-3- [ (cyclobutylamino) carbonyl] oxireno [fl-1, 2-benzisoxazol-4-yl ester (o- (3-Phenyl-1- propynyl) benzyl cyclobutylamido 4-methoxyphenylacetyl tricycle carboxamide, 42a). TLC: Rf0. 25 (4: 1 CH2C12/THF) ; Rf0.10 (1:1 CH2Cl2/EtOAc). HPLC: tR = 3.532 min, = 202, (209), 235, (250), (274) nm. 1H-NMR (500 MHz, CD3CN) : 6 7. 49 (d, 3H, J= 7. 4, C13-H, C21- H, C25-H), 7.46 (d, 1H, J= 7.7, C16-H), 7. 36 (t, 2H, J= 7.7, C22-H, C24-H), 7.33 (td, 1H, J= 7.5,1.6, C14-H), 7.29 (td, lH, J=7. 6,1.4, C15-H), 7.25 (t,1H, J = 7.5, C23-H), 7.17 (d, 2H, J= 8.7, C33-H, C37-H), 6.87 (d, 1H, J= 8.7, C8-NH), 6.84 (d, 2H, J= 8.7, C34-H, C36-H), 6.64 (br s, 1H, C1-NHa), 5.91 (br s, 1H, C1-NHb), 5.31 (t, 1H, J= 3.9, C6-H), 4.61 (d, 1H, J= 12.3, C10- Ha), 4.17 (d, 1H, J= 12.3, C10-Hb), 3.92 (app d, 2H, J = 2.7, C19-H2), 3.81 (d, 1H, J= 8.6, C9- H), 3.80 (obs m, 1H, C26-H), 3.75 (s, 3H, C38-H3), 3.69 (dd, 1H J = 8.5,3.8, C7-H), 3.48 (d, 1H, J = 15.5, C31-Ha), 3.37 (d, 1H, J = 15.5, C31-Hb), 3.31 (t, 1H, J = 4.1, C5-H), 3.10 (ddd, 1H, J= 4. 2,3.0,1.9, C4-H), 2.40 (dd, 1H, J= 16. 2,3.0, C3-Ha), 2.34 (dd, 1H, J= 16. 1,1.8, C3- Hb), 1.94 (obs m, 1H, C27-Ha), 1.84 (obs m, 1H, C29-Ha), 1.63 (quint, 1H, J= 9.8, C27-Hb), 1.50 (m, 2H, C28-H2), 1.33 (quint, 1H, J= 9.9, C29-Hb). FAB-MS (glycerol) m/z (rel int): 650 ( [M+H] +, 65). FAB-MS (NBA/NaI) m/z (rel int): 672 ( [M+Na] +, 35), 524 ( [M+H] +, 9). HRMS (glycerol) m/z calcd for C38H4oN307 650.2866; found 650.2836.
[3S-(3a, 3aß, 4a, 4aa, 5aa, 6aß)]-Benzoic acid, 6a- (aminocarbonyl)-2- [ (3- (3- methylbut-3-en-1-ynyl) phenyl] methyl] octahydro-3-[[(2- propenyl) amino] carbonyl] oxireno [fl-1, 2-benzisoxazol-4-yl ester (m- (3-Methyl-3-buten-1- ynyl) benzyl allylamido benzoyl tricycle carboxamide, 42b). TLC: Rf 0.22 (4: 1 CH2Cl2/THF) ; Rf 0.13 (1:1 CH2Cl2/EtOAc). HPLC: tR = 3.601 min, = 201,220, (236), 270, (282) nm. ¹H-NMR (500 MHz, CD3CN) : 6 8.15 (dd, 2H, J= 8.2,1.2, C27-H, C31-H), 7.64 (tt, 1H, J= 7. 4,1.5, C29-H), 7.56 (s, 1H, C12-H), 7.55 (obs t, 2H, J= 7. 6, C28-H, C30-H), 7.37 (m, 3H, C14-H, C15-H, C16-H), 6.71 (br t, 1H, C8-NH), 6.53 (br s, 1H, Cl-NHa), 5.99 (br s, 1H, C1-NHb), 5.63 (t, 1H, J= 3.9, C6-H), 5.40 (m, 2H, C20-H2), 5.22 (ddt, 1H, J= 16.8,10.3, 6.1, C23-H), 4.70 (app dq, 1H, J= 10.2,1.3, C24-HE), 4.64 (app dq, 1H, J= 17.1,1.5, C24-Hz), 4.26 (d, 1H, J= 14.1, C10-Ha), 3.96 (d, 1H, J= 14.1, C10-Hb), 3.86 (d, 1H, J= 8.0, C9-H), 3.77 (dd, 1H, J= 8.0,3.6, C7-H), 3.54 (obs m, 1H, C5-H), 3.53 (obs m, 1H, C22-Ha), 3.17 (app quint, 1H, J= 2.1, C4-H), 3.01 (dddt, 1H, J= 15.2,6.2,4.9, C22-Hb), 2.44 (dd, 1H, J= 16.6,2.5, C3- Ha), 2.40 (dd, 1H, J= 16.5,1.8, C3-Hb), 1.99 (app t, 3H, J= 1.2, C21-H3). FAB-MS (glycerol) mlz (rel int): 542 ( [M+H] +, 100). FAB-MS (NBA/NaI) m/z (rel int): 564 ( [M+Na] +, 100), 542 ( [M+H] +, 18). HRMS (NBA/NaI) m/z calcd for C31H3lN306Na 564. 2111; found 564.2100.
[3S-(3α, 3aß, 4α, 4aα,5aα, 6aß)]-2-Methylpropanoic acid, 6a-(aminocarbonyl)-2- [[4- [2- (4-chlorophenyl)-1-ethynyl] phenyl] methyl]-3- [ [ [ (2- methoxyphenyl) methyl] amino] carbonyl] octahydrooxirenoUl-1, 2-benzisoxazol-4-yl ester (p- (4-Chlorophenylethynyl) benzyl 2-methoxybenzylamido isobutyryl tricycle carboxamide,
42c). TLC: Rf0. 19 (4: 1 CH2CI2/THF) ; Rf0. 12 (1: 1 CH2Cl2/EtOAc). HPLC: tR = 4.072 min, . = 202,222, (270), 291,304 nm. 1H-NMR (500 MHz, CD3CN) : 8 7.52 (d, 2H, J = 8.5, C20-H, C24-H), 7.45 (d, 2H, J= 8.1, C13-H, C15-H), 7.42 (d, 2H, J = 8.5, C21-H, C23-H), 7.39 (obs m, 1H, C8-NH), 7.35 (d, 2H, J= 8.1, C12-H, C16-H), 7.25 (td, 1H, J= 7.8,1.7, C29-H), 7.02 (dd, 1H, J = 7.4,1.3, C31-H), 6.94 (d, 1H, J = 8.2, C28-H), 6.87 (t, 1H, J= 7.5, C30-H), 6.48 (br s, 1H, C1-NHa), 5.92 (br s, 1H, C1-NHb), 5.36 (t, 1H, J= 4.0, C6-H), 4.39 (dd, 1H, J =14. 6,7.3, C25-Ha), 4.18 (d, 1H, J=14. 1, C10-Ha), 4.06 (dd, 1H, J=14. 7,4.8, C25-Hb), 3.87 (d, 1H, J=14. 2, C10-Hb), 3.78 (obs d, 1H, J= 8.6, C9-H), 3.76 (s, 3H, C32-H3), 3.67 (dd, 1H, J= 8.4,3.9, C7-H), 3. 37 (t, 1H, J= 4.2, C5-H), 3.15 (dt, 1H, J= 4.1,2.6, C4-H), 2.40 (sept, 1H, J= 7.0, C34-H), 2.35 (dd, 1H, J= 16.4,2.9, C3-Ha), 2.29 (dd, 1H, J= 16.2,2.0, C3-Hb), 1.13 (d, 3H, J= 7. 1, C35-H3), 1.09 (d, 3H, J= 7. 0, C36-H3). FAB-MS (glycerol) m/z (rel int) : 658/660 ( [M+H] +, 6/3). FAB-MS (NBA/NaI) m/z (rel int): 680/682 ( [M+Na] +, 20/10), 658/660 ( [M+H] +, 12/6). HRMS (NBA/NaI) m/z calcd for C36H36ClN307Na 680.2139; found 680.2147.
[3S-(3α, 3aß, 4α, 4aα, 5aα, 6aß)]-3-Methylbutanoic acid, 6a-[[(6-amino-6- oxohexyl) amino] carbonyl]-2-[[2-(3,3-dimethyl-1-butynyl)phenyl]methyl]-3-[[[ (4- methoxyphenyl) methyl] amino] carbonyl] octahydrooxireno [f]-1,2-benzisoxazol-4-yl ester (o- (3,3-Dimethyl-1-butynyl) benzyl 4-methoxybenzylamido isovaleryl tricycle co-aminocaproic carboxamide, 42d). TLC: Rf0. 49 (9: 1 CH2Cl2/MeOH) ; Rf0.29 (1:1 CH2Cl2/THF). HPLC: tR = 3. 742 min, (203), (215), 232,247, (275) nm. 1H-NMR (500 MHz, CD3CN) : 6 7.46 (d, 1H, J = 8.0, C19-H), 7.27 (obs dd, 1H, J = 7.6,1.2, C22-H), 7.25 (obs td, 1H, J = 7.5,1.6, C20- H), 7.20 (td, 1H, J = 7.4,1.2, C21-H), 7.18 (obs br t, 1H, C14-NH), 6.88 (d, 2H, J= 8.5, C31-H, C35-H), 6.83 (br t, 1H, C7-NH), 6.77 (d, 2H, J= 8.7, C32-H, C34-H), 5.97 (br s, 1H, C1-NHa), 5.45 (br s, 1H, C1-NHb), 5.31 (t, 1H, J= 4.1, C12-H), 4.38 (d, 1 H, J = 13.1, C16-Ha), 4.05 (d, 1H, J= 13.2, C16-Hb), 3.99 (dd, 1H, J= 14.6,6.3, C29-Ha), 3.91 (dd, 1 H, J = 14.6,5.9, C29- Hb), 3.87 (d, 1H, J= 8.9, C15-H), 3.77 (dd, 1H, J= 8.8,4.1, C13-H), 3.74 (s, 3H, C36-H3), 3.40 (t, 1 H, J = 4.2, C11-H), 3.18 (q, 2H, J= 6.7, C6-H2), 3.15 (obs m, 1H, C10-H), 2.29 (app s, 2H,
C9-H2), 2.09 (obs m, 5H, C2-H2, C38-H2, C39-H), 1.52 (obs m, 2H, C3-H2), 1.47 (obs m, 2H, C5-H2), 1.31 (s, 9H, C26-H3, C27-H3, C28-H3), 1.27 (obs m, 2H, C4-H2), 0.89 (t, 6H, J= 5.9, C40-H3, C41-H3). FAB-MS (glycerol) m/z (rel int): 731 ( [M+H] +, 13). FAB-MS (NBA/NaI) mlz (rel int): 753 ( [M+Na] +, 100), 731 ([M+H]-, 22). HRMS (NBA/NaI) mlz calcd for C41H54N408Na 753.3839; found 753.3842.
[35-(3a, 3aß, 4a (E), 4aa, 5aa, 6aß)]-2-Butenoic acid, 6a- [ [ (6-amino-6- oxohexyl)amino]carbonyl]-2-[[3-(3,3-diethoxy-1-propynyl)phen yl]methyl]-3-[[(2,2- dimethoxyethyl) amino] carbonyl] octahydrooxireno [fl-1, 2-benzisoxazol-4-yl ester (m- (3, 3- Diethoxy-1-propynyl) benzyl 2,2-dimethoxyethylamido crotonyl tricycle m-amiaocaproic carboxamide, 42e). TLC: Rf0. 40 (9: 1 CH2Cl2/MeOH) ; Rf0. 16 (1 : 1 CH2C12/THF). HPLC: tR = 3. 285 min, #max = 207, 241,246 nm. 1H-NMR (500 MHz, CD3CN) : 67. 53 (s, 1H, C18-H), 7.40 (m, 3H, C20-H, C21-H, C22-H), 7.09 (obs br t, 1H, C14-NH), 7.05 (dq, 1H, J= 15.4,6.9, C36-H), 6.78 (br t, 1H, J= 6. 5, C7-NH), 6.04 (s, 1H, C1-NHa), 5.83 (dq, 1H, J = 15.4, 1.7, C35- H), 5.55 (s, 1H, C1-NHb), 5.46 (s, 1H, C25-H), 5.34 (t, 1H, J= 4.0, C12-H), 4.25 (dd, 1H, J= 5.8,4.5, C31-H), 4.04 (d, 1H, J= 14.3, C16-Ha), 3.89 (d, 1H, J= 14.3, C16-Hb), 3.74 (obs m, 2H, C26-Ha, C28-Ha), 3.73 (obs m, 1H, C15-H), 3.64 (dd, 1H, J= 8.0,4.0, C13-H), 3.60 (app dq, 2H, J = 9.5,7.1, C26-Hb, C28-Hb), 3.43 (dd, 1H, J = 7.8,4.5, C30-Ha), 3.40 (obs m, 1H, C11-H), 3.29 (s, 3H, C32-H3), 3.27 (s, 3H, C33-H3), 3.24 (m, 2H, C6-H2), 3.15 (m, 1H, C10-H), 2.86 (ddd, 1H, J=13. 7,5.8,3.8, C30-Hb), 2.27 (app d, 2H, J = 2.4, C9-H2), 2.12 (m, 2H, C2- H2), 1.89 (dd, 3H, J = 6.9,1.7, C37-H3), 1.57 (m, 2H, C3-H2), 1.53 (m, 2H, C5-H2), 1.31 (m, 2H, C4-H2), 1.20 (t, 6H, J= 7.1, C27-H3, C29-H3). FAB-MS (glycerol) m/z (rel int): 729 ( [M+H] +, 13). FAB-MS (NBA/NaI) m/z (rel int): 751 ( [M+Na] +, 100), 729 ( [M+H] +, 6).
HRMS (NBA/NaI) m/z calcd for C37H52N4011Na 751.3530; found 751.3536.
[3- (3a, 3ap, 4a, 4aa, 5aa, 6ap)]-Propanoic acid, 6a- [ [ (6-amino-6- oxohexyl) amino] carbonyl]-3-[[[2-(4-methoxyphenyl) ethyl] amino] carbonyl] octahydro-2-[[4- (1-pentynyl) phenyl] methyl] oxireno [fl-1, 2-benzisoxazol-4-yl ester (p- (1-Pentynyl) benzyl 4- methoxyphenethylamido propionyl tricycle co-aminocaproic carboxamide, 42f). TLC: Rf 0.33 (9: 1 CH2Cl2/MeOH) ; Rif 0. 23 (1 : 1 CH2Cl2/THF). HPLC: tR = 3.602 min, #max = 202,231, 252, (280) nm. 1H-NMR (500 MHz, CD3CN) : 6 7. 33 (d, 2H, J= 8. 1, C19-H, C21-H), 7.20 (d, 2H, J= 8. 1, C18-H, C22-H), 7.04 (d, 2H, J= 8. 4, C31-H, C35-H), 6.90 (br t, 1H, J = 5.5, C14- NH), 6.74 (d, 2H, J= 8.7, C32-H, C34-H), 6.72 (br t, 1H, J= 5.5, C7-NH), 6.03 (br s, 1H, C1- NHa), 5.55 (br s, 1H, C1-NHb), 5.25 (t, 1H, J= 4.0, C12-H), 3.98 (d, 1H, J= 14.2, C16-Ha), 3.74 (d, 1H, J= 14.4, C16-Hb), 3.67 (s, 3H, C36-H3), 3.63 (d, 1H, J= 8.4, C15-H), 3.60 (dd, 1H, J= 8.4,4.1, C13-H), 3.50 (obs m, 1H, C28-Ha), 3.32 (app t, 1H, J= 4.2, C11-H), 3.20 (obs m, 1H, C6-Ha), 3.16 (obs m, 1H, C6-Hb), 3.14 (obs m, 1H, C10-H), 3.02 (m, 1H, C28-Hb), 2.64-2.50 (m, 2H, C29-H2), 2.38 (t, 2H, J= 7.0, C25-H2), 2.28-2.16 (m, 4H, C9-H2, C38-H2), 2.10 (t, 2H, J = 7.3, C2-H2), 1.59 (sxt, 2H, J 7. 2, C26-H2), 1.53 (quint, 2H, J= 7.7, C3-H2), 1.47 (m, 2H, C5-H2), 1.27 (m, 2H, C4-H2), 1.03 (t, 3H, J = 7.5, C39-H3), 1.02 (t, 3H, J= 7. 3, C27-H3). FAB- MS (glycerol) mlz (rel int): 703 ( [M+H] +, 18). FAB-MS (NBA/NaI) mlz (rel int): 725 ( [M+Na] +, 100), 703 ( [M+H] +, 17). HRMS (NBA/NaI) m/z calcd for C39H5oN408Na 725.3526; found 725.3527.
IV. Building Block Testing: Building Block Testing-General. All solids were measured to within 10%. All liquids were dispensed via Gilson automatic pipettemen with polypropylene tips. Reactions were performed using the small-scale solid phase reaction procedures described above. Resin samples were photolyzed in sets of 11 for 1 h. After photolysis, the samples were centrifuged briefly and 10 u. L of the supernatant was submitted for HPLC analysis. An additional 5 I1L was diluted to 50 µL with CH3CN and submitted for LC-MS analysis (10 tL injection). Where necessary, additional samples were removed for TLC and FAB-MS analysis.
To ensure that all of the building blocks included in the library synthesis were viable coupling partners, a total of 235 building blocks were tested in the Sonogashira/Castro-Stephens, lactone aminolyis, and esterification reactions (Figures 65-67). A representative sample of the LC-MS data is shown in Figure 56 of the Manuscript. Complete HPLC (52 pages) and LCMS data (235 pages) have also been obtained. These data are summarized in tabular format below (Tables A-C).
Building Block Testing-Alkynes. 2-Iodobenzyl tetracycle-aminocaproic-Anp- TentaGel resin 39dR (10 mg, 0.24 meq/g, 2.39 umol, 1.0 equiv), copper (I) iodide (1.0 mg, 5.26 ) J. mol, 2.2 equiv), and bis (triphenylphosphine) palladium (II) chloride (1.84 mg, 2.63 pmol, 1.1 equiv) or tetrakis (triphenylphosphine) palladium (0) for polyynes (3.04 mg, 2.63 umol, 1. 1 equiv) were combined and 100 L DMF was added, followed by DIPEA (12.5 L, 71.76 pmol, 30 equiv for monoynes; 29.17 uL, 167.43 u. mol, 70 equiv for diynes; or 43.75 L, 251. 1 pmol, 105 equiv for triynes). The tube was vortexed vigorously, centrifuged briefly, then the appropriate alkyne (47.84 µmol, 20 equiv for monoynes; 119.6, umol, 50 equiv for diynes; or 179.4 pmol, 75 equiv for triynes) was added as a neat liquid or solid. The tube was again vortexed vigorously, centrifuged briefly, wrapped with parafilm, and finally vortexed gently for 1 h. After washing, the resin was photolyzed in 125 u. L CH3CN.
Building Block Testing-Amines. o- (3, 3-Dimethyl-l-butynyl) benzyl tetracycle o- aminocaproic-Anp-TentaGel resin 40dR (5 mg, 0.24 meq/g, 1.21 µmol, 1.0 equiv) and solid amines where appropriate (30.23 pmol, 25 equiv for non-a-branched amines; 60.49 jj. mol, 50 equiv for a-branched amines) were combined, then 2-hydroxypyridine (0.575 mg, 6.05 jj. mol, 5 equiv for non-a-branched amines; 1.150 mg, 12.09 J. mol, 10 equiv for a-branched amines) was added as a 50 L stock solution in THF (free amines) or 2: 1 CH2Cl2/DMF (amine hydrochlorides). Neat liquid amines (30.23, umol, 25 equiv for non-a-branched amines; 60.49 , umol, 50 equiv for a-branched amines) were added where appropriate. DIPEA was added as necessary to neutralize amine hydrochlorides (10.53 µL, 60.46 pmol, 50 equiv for non-a- branched monohydrochlorides; 21.06 uL, 120.92 J. mol, 100 equiv for non-a-branched dihydrochlorides and a-branched monohydrochlorides ; 42.12 p. L, 241.84 µmol. 200 equiv for a- branched dihydrochlorides). The tubes were wrapped with teflon tape and parafilm and vortexed gently for 13 h. After washing, the resin was photolyzed in 60 iL CH3CN.
Building Block Testing-Acids. In 2 mL oven-dried Wheaton vials fitted with teflon septum caps and stir bars were placed the appropriate carboxylic acids (292.6 jn. mol, 250 equiv) and 182.62 iL CH2CI2. DIPC (22.90 L, 146.3 J. mol, 125 equiv) was added to each vial and the
mixtures stirred for 2 min. DIPEA (50.95 iL, 292.6 pmol, 250 equiv; 101.9 pL, 585.2 llmol, 500 equiv for amino acid hydrochlorides) was added to each vial and the mixtures stirred for another 5 min. Approximately 1/Sth of each preactivation mixture (60 pL normally; 70 pL for hydrochlorides; 25 equiv activated acid) was added to o- (3, 3-dimethyl-l-butynyl) benzyl 4- methoxybenzylamido hydroxy tricycle co-aminocaproic-Anp-TentaGel resin 41dR (5 mg, 0.23 meq/g, 1.17 pmol, 1.0 equiv). DMAP (0.715 mg, 5.85 pLmol, S equiv) was added to each tube as a 10 tL stock solution in CH2C12 and the tubes were wrapped with teflon tape and parafilm then vortexed gently for 14 h. The resin was exposed to the standard wash procedure with an additional 20% DIPEA in CH2Cl2 wash inserted between the CH2Cl2 and DMF wash steps.
Finally, the resin was photolyzed in 60 pL CH3CN.
V. Test Library Synthesis and Deconvolution : Test Library Synthesis-General. All solids were measured to within 10%. All liquids were dispensed via Gilson automatic pipettemen with polypropylene tips. Reactions were performed in tared 2 mL BioSpin columns. Resin was distributed to each column as 1 mL of an 8 mL isopicnic slurry in DMSO/CH2C12 with a P1000 pipetteman fitted with a P1000 polypropylene tip trimmed by approximately 2 mm. The resin was washed with distd THF and distd CH2Cl2, dried, and weighed. This method of resin distribution proved consistent to within 5% (data not shown). After reaction according to the medium-scale solid phase procedures described above, the resin portions were washed using the standard wash procedure (Method A) and a sample was photolyzed for 2 h followed by HPLC and LC-MS analysis of the supernatant.
The remaining resins were pooled in a PD-10 column via vacuum cannula transfer from the reaction vessels and mixed thoroughly by repeated washing with CH2Cl2.
A representative sample of the LC-MS data is shown in Figure 56. Complete HPLC (14 pages) and LC-MS data (168 pages) have been obtained. These data are summarized in tabular format below (Tables D-F).
Test Library Synthesis-3-Alkynylbenzyl tetracycle-Anp-TentaGel resins (43R {X, 1,1}). To each of seven aliquots of 3-iodobenzyl tetracycle-Anp-TentaGel resin 39bR (31.25 mg, 0.25 meq/g, 7.68 Hmol, 1.0 equiv) was added in sequence copper (I) iodide (3. 2 mg, 16.90 mol, 2.2 equiv), bis (triphenylphosphine) palladium (II) chloride (5.9 mg, 8.45 pLmol, 1. 1 equiv), and 300 L DMF. The tubes were flushed with Ar and vortexed briefly. DIPEA (40.15 L, 230.5 mol, 30 equiv) was added to each tube followed by the appropriate alkyne (153.65 pmol, 20 equiv). The tubes were wrapped with parafilm and mixed for 2 h. After washing, approx 1 mg of resin was removed from each tube and photolyzed in 30 L CH3CN. 10 L was
submitted for HPLC analysis and an additional 4 p. L was diluted to 30 I1L and submitted for LC- MS analysis (10 pL injection). The remaining resin was pooled to yield a mixture of eight 3- alkynylbenzyl tetracycle-Anp-TentaGel resins 43R {X, 1,1}.
Test Library Synthesis-3-Alkynylbenzyl y-hydroxyamido tricycle-Anp-TentaGel resins (43R {X, X, 1}). To each of seven aliquots of 3-alkynylbenzyl tetracycle-Anp-TentaGel resin 43R {X, 1,1} (30 mg, 0.25 meq/g avg 7.43 jmol avg, 1.0 equiv) was added 2- hydroxypyridine (3.53 mg, 37.14 jj. mol, 5 equiv) as a 300 pL stock solution in THF. An additional 5 equiv of 2-hydroxypyridine was added as a solid to Pool #4 (a-branched amine).
The tubes were flushed with Ar, and the appropriate amine (185.7 mol, 25 equiv; 371.4 pmol, 50 equiv for Pool #4) was added to each. The tubes were wrapped with teflon tape and parafilm and mixed for 15 h. After washing, approx 2 mg of resin was removed from each tube and photolyzed in 40 je. L CH3CN. 10 L was submitted for HPLC analysis and an additional 10 L was submitted without dilution for LC-MS analysis. The remaining resin was pooled to yield a mixture of sixty-four 3-alkynylbenzyl y-hydroxyamido tricycle-Anp-TentaGel resins 43R {X, X, 1}.
Test Library Synthesis-3-Alkynylbenzyl amido acyl tricycle-Anp-TentaGel resins (43R {X, X, 1} through 43R {X, X, 8}). In each of seven 2 mL oven-dried Wheaton vials fitted with septum caps and stir bars were placed the appropriate carboxylic acids (326.5 pmol, 50 equiv) and 300 pL CH2C12. DIPC (25.60 uL, 163.25 llmol, 25 equiv) was added to each vial and the mixtures were stirred for 2 min. DIPEA (56.9 L, 326.5 pLmol, 50 equiv) was added to each vial and the mixtures were stirred for another 5 min. Seven aliquots of 3-alkynylbenzyl y- hydroxyamido tricycle-Anp-TentaGel resin 43R {X, X, 1} (27 mg, 0. 24 meq/g avg, 6.53 mol avg, 1.0 equiv) were each swollen with 100 J. L CH2C12, flushed with Ar, and cooled to 0°C in an ice bath. The appropriate preactivated acid was then added to each tube followed by DMAP (3.99 mg, 32.65 pmol, 5 equiv) as a 50 jj. L stock solution in CH2C12. Each tube was vortexed briefly and allowed to stand at 0°C for 15 min. The tubes were then warmed to rt, wrapped with teflon tape and parafilm and mixed for 10 h. A 20% DIPEA/CH2C12 wash was added between the CH2C12 and DMF steps of the standard wash procedure. After drying, 12 mg of each 3- alkynylbenzyl amido acyl tricycle-Anp-TentaGel resin 43R {X, X, 1} through 43R {X, X, 8} was photolyzed in 120 L CH3CN. The supernatant from each tube was filtered through a BioSpin column into a new Eppendorf tube and the photolysis tubes and resin rinsed with an additional 50 pL CH3CN. The eight samples were concentrated for 15 min on a Savant AES 1000 SpeedVac at Low Drying Rate, redissolved in 11 I1L CH3CN, and transferred to an HPLC
autosampler vial. Each tube was rinsed with an additional 11, uL CH3CN transferred to the same vials. 10 pu of each sample was submitted for HPLC analysis and 10 pL was submitted for LC- MS analysis.
Test Library Deconvolution Syntheses. TGF-p-responsive reporter gene assay activity was detected in pool 43 {X, X, 8}, which contains 64 compounds. To deconvolute this activity, the eight-compound subpools 43 {X, 1,8} through 43 {X, 8,8} were synthesized essentially as described above from 3-iodobenzyl tetracycle-Anp-TentaGel resin 39bR (12.5 mg, 0.25 meq/g, 3.07 pmol, 1.0 equiv) except that the resin portions were not repooled after lactone aminolysis. All eight portions were acylated separately with Acid 8 (monomethyl terephthalic acid). The presence of all eight expected compounds in each pool was verified by LC-MS analysis (data not shown).
Pool 43 {X, X, 3} showed lower activity in the TGF-p-responsive reporter gene assay and was deconvoluted as a negative control. The eight-compound subpools 43 {X, 1,3} through 43 {X, 8,3} were synthesized as above and acylated with Acid 3 (methoxyacetic acid).
Of the 16 eight-compound subpools, 43 {X, 8,3} showed the highest activity in the TGF-ß- responsive reporter gene assay. To deconvolute this activity, the eight individual compounds comprising the subpool, 43 {1, 8,3} through 43 {8, 8,3}, were synthesized in parallel essentially as described for Demonstration Compounds in the Manuscript from 3-iodobenzyl tetracycle-Anp- TentaGel resin 39bR (150 mg, 0.25 meq/g, 36.88 umol, 1.0 equiv). The final acylated products, as well as the 3-alkynylbenzyl tetracycle intermediates, 43 {1, 1,1} through 43 {8, 1,1}, and the y-hydroxyamido tricycle intermediates, 43 {1,8,1} through 43 {8, 8,1}, were analyzed by IH-NMR, TOF-ESI-MS, and HR-TOF-ESI-MS. All compounds exhibited satisfactory 1H-NMR data and were recovered in approximately 80-90% purity.
43 {1, 1, 1} HPLC: tR = 3.022 min. TOF-ESI-MS m/z (rel int) : 381 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C21H2lN205 381.1450; found 381.1449.
43 {2, 1,1} HPLC: tR = 2.588 min. TOF-ESI-MS m/z (rel int) : 385 ([M+H] +, 100). HR- TOF-ESI-MS m calcd calcd for C2oH21N206 385.1400; found 385.1388.
43 {3, 1,1} HPLC: tR = 3. 184 min. TOF-ESI-MS rnlz (rel int) : 397 ([M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C22H25N205 397.1763; found 397.1781.
43 {4, 1,1} HPLC: tR = 2.696 min. TOF-ESI-MS m/z (rel int): 408 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C22H22N305 408.1559; found 408.1539.
43 {5, 1,1} HPLC : tR = 3.190 min. TOF-ESI-MS m/z (rel int) : 417 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C24H21N205 417.1450; found 417.1429.
43 {6, 1,1} HPLC: tR = 3.214 min. TOF-ESI-MS m/z (rel int): 431 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C25H23N205 431.1607; found 431. 1584.
43 {7, 1,1} HPLC: tR = 2. 741 min. TOF-ESI-MS m/z (rel int): 443 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C16Hl6lN205 443.0104; found 443.0107.
43 {8,1,1} HPLC: tR = 3.467 min. TOF-ESI-MS m/z (rel int): 449 ( [M+H] +, 38). HR- TOF-ESI-MS m/z calcd for C26H29N205 449.2076; found 449.2055.
43 {1, 8,1} HPLC: tR = 3.023 min. TOF-ESI-MS m/z (rel int): 548 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C3oH34N307 548.2397; found 548.2369.
43 {2, 8,1} HPLC: tR = 2. 664 min. TOF-ESI-MS (rel int): 552 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C29H34N3Og 552.2346; found 552.2320.
43 {3, 8,1} HPLC: tR = 3. 147 min. TOF-ESI-MS mlz (rel int): 564 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C31H3gN307 564.2710; found 564.2730.
43 {4, 8,1} HPLC: tR = 2. 726 min. TOF-ESI-MS mlz (rel int): 575 ( [M+H] +, 100). HR- TOF-ESI-MS calcd for C3lH35N407 575.2506; found 575. 2524.
43 {5, 8,1} HPLC: tR = 3. 174 min. TOF-ESI-MS m/z (rel int): 584 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C33H34N307 584.2397; found 584.2416.
43 {6, 8,1} HPLC: tR = 3. 172 min. TOF-ESI-MS m/z (rel int): 598 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd calcd for C34H36N307 598.2553; found 598.2546.
43 {7, 8,1} HPLC: tR = 2. 768 min. TOF-ESI-MS mlz (rel int): 610 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C25H29IN307 610.1050; found 610.1064.
43 {8, 8,1} HPLC: tR = 3. 450 min. TOF-ESI-MS m/z (rel int): 616 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C35H42N307 616.3023; found 616.3010.
43 {1, 8,3} HPLC: tR = 3. 074 min. TOF-ESI-MS mlz (rel int): 620 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C33H38N309 620.2608; found 620.2621.
43 {2, 8,3} HPLC: tR = 2. 764 min. TOF-ESI-MS m/z (rel int): 624 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C32H38N301o 624.2557; found 624.2554.
43 {3, 8,3} HPLC: tR = 3. 235 min. TOF-ESI-MS m/z (rel int): 636 ( [M+H] +, 100). HR- TOF-ESI-MS wiz calcd for C31H42N3Og 636.2921; found 636.2899.
43 {4, 8,3} HPLC: tR = 2. 812 min. TOF-ESI-MS m/z (rel int): 647 ( [M+H] +, 100). HR- TOF-ESI-MS Hl/Z calcd for C34H39N409 647.2717; found 647.2700.
43 {5, 8,3} HPLC: tR = 3. 241 min. TOF-ESI-MS m/z (rel int): 656 ( [M+H] +, 100). HR- TOF-ESI-MS w/z calcd for C36H36N309 656.2608; found 656.2615.
43 {6, 8,3} HPLC: tR = 3. 249 min. TOF-ESI-MS mlz (rel int) : 670 ([M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C37H4oN309 670.2765; found 670.2777.
43 {7, 8,3} HPLC: tR = 2.857 min. TOF-ESI-MS mlz (rel int) : 682 ([M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C28H33IN309 682.1262; found 682.1285.
43 {8, 8,3} HPLC: tR = 3.488 min. TOF-ESI-MS (relint) : 688 ( [M+H] +, 100). HR- TOF-ESI-MS m/z calcd for C38H46N309 688.3234; found 688.3226.
Test Library Photolysis for Biological Assays. 3-Alkynylbenzyl amido acyl tricycle- Anp-TentaGel resins 43R {X, X, 1} through 43R {X, X, 8} prepared above (10 mg, 0.24 meq/g avg, 2.37 mol avg) were placed in Eppendorf tubes, swollen in 120 ilL CH3CN, and photolyzed for 90 min. The resins were filtered through BioSpin columns and rinsed with an additional 2 x 60 } je CH3CN. The filtrates were concentrated for 30 min on a SpeedVac at Low Drying Rate. The samples were redissolved in 18.5 L DMSO at an estimated average concentration of 1 mM per compound, assuming 50% photocleavage yield. These stock solutions were used in assays for suppression of rapamycin-based growth inhibition in S. cerevisiae, modulation of the cyclin B degradation pathway in a Xenopus laevis oocyte extract assay, inhibition of mink lung cell proliferation (see Experimentals herein), and activation of a TGF-p-responsive reporter gene (see Experimentals herein).
3-Alkynylbenzyl amido 2-methoxyacetyl tricycle-Anp-TentaGel resins 43R {X, 1,3} through 43R {X, 8,3} and 3-alkynylbenzyl amido methylterephthaloyl tricycle-Anp-TentaGel resins 43R {X, 1,8} through 43R {X, 8,8} prepared above (3-4 mg, 0.23-0.25 meq/g, 0.7-2.0 pmol) were weighed into Eppendorf tubes and swollen with 50 L CH3CN. After 2 h photolysis, the resins were filtered through BioSpin columns and rinsed with an additional 2 x 100 L CH3CN. The filtrates were concentrated as above and redissolved in 43-62 JL DMSO at an estimated concentration of 1 mM per compound, assuming 50% photocleavage yield. These stock solutions were used in the TGF-p-responsive reporter gene assay.
3-Alkynylbenzyl tetracycle-Anp-TentaGel resins 43R {1, 1,1} through 43R {8, 1,1} (50 mg, 0.25 meq/g avg, 12.38 mol avg), 3-alkynylbenzyl veratrylamido hydroxy tricycle-Anp- TentaGel resins 43R {1, 8,1} through 43R {8, 8,1} (50 mg, 0.24 meq/g avg, 11.89 mol avg), and 3-alkynylbenzyl veratrylamido 2-methoxyacetyl tricycle-Anp-TentaGel resins 43R {1, 8,3} through 43R {8, 8,3} prepared above (41-47 mg, 0.23 meq/g avg, 10.23 umol avg) were placed in two Eppendorf tubes per sample. The resin in each tube was swollen with 400 J. L CH3CN and photolyzed for 2 h. 20 L of each sample was removed for HPLC and LC-MS analysis. The remainder of each sample was filtered through a BioSpin't) column and rinsed with CH3CN. The
filtrates were concentrated by rotary evaporation in tared 4 mL glass vials. The residue was redissolved in 888.9 J. L CD3CN. 800 L was used for NMR analysis and set aside. The remaining 88.9 L of each sample was concentrated for 15 min on a SpeedVac at Low Drying Rate and redissolved in 61.9,59.4, or 51.1 I1L DMSO at an estimated average concentration of 10 mM per compound, assuming 50% photocleavage yield. These stock solutions were used in the TGF-p-responsive reporter gene assay.
After initial screening, the six active compounds were recovered from the NMR samples and purified by silica gel chromatography (CH2C12/THF) to yield the pure products 43 {5, 1,1}, 43 {6, 1,1}, 43 {5, 8,1}, 43 {6, 8,1}, 43 {5, 8,3}, and 43 {6, 8,3} (0.5-0.8 mg, 7-12%) as clear residues.
The purified products were dissolved in DMSO at a concentration of 20 mM and retested in the TGF-ß-responsive reporter gene assay.
VI. Full-Scale Library Synthesis and Tagging Full-Scale Library-General. Spacer, epoxycyclohexenol, iodobenzyl nitrone carboxylic acid, alkyne, amine, and acid building blocks included in full-scale library synthesis are listed below (Tables G-J). Pooling steps were performed by rinsing all resin portions into a silanized 50 mL fritted glass tube followed by thorough mixing by N2 bubbling in CH2C12. The resin was then slurried in CH2C12 and transferred via Gilson P5000 pipetteman to a PD-10 column (placed under high vacuum for 30 min then tared) with drainage provided by a VacMan manifold. The resin was washed several times with distd CH2C12, allowed to dry several minutes by drawing air through the tube, then washed down with additional distd CH2Cl2. The entire tube was then placed under high vacuum for 30 min and reweighed. Splitting steps were accomplished by weighing aliquots of the pooled resin into the appropriate vessels. The last portion of resin was removed from the pooling tube via vacuum cannula transfer to the appropriate vessel.
Tagging reactions were performed before each building block coupling step. Beads from every portion of the library were analyzed to verify tag coupling. The binary tagging code is shown below (Table K). A representative sample of the EC-GC data is shown below (Figure 68). Complete EC-GC data (165 pages) have been obtained.
Alkyne, amine, and acid coupling reactions were performed in sets of seven to facilitate washing after the reactions. PD-10 and BioSpinR columns were capped at both ends and sealed with teflon tape and parafilm.
Full-Scale Library-Spacer resins (37R). In each of six PD-10 columns was placed H2N-Anp-TentaGel 36R (416.7 mg, 114.4 p. mol, 1.0 equiv). After tagging, to two each of the six resin portions was added Fmoc-Gly-OH (102 mg, 343.2 mol, 3.0 equiv) or Fmoc-Aca-OH
(121 mg, 343.2 pmol, 3.0 equiv). The remaining two portions were set aside as the R1 skip codon. PyBOP (179 mg, 343.2 pmol, 3.0 equiv) was added to each of the four tubes being coupled. NMP (5 mL) and DIPEA (99.6 L, 572.0 Hmol, 5.0 equiv) were then added to each tube with brief vortexing between each addition. After mixing for 80 min, the resin portions were washed with 5 x NMP, 5 x CH2Cl2 and a small sample of each treated with the Kaiser ninhydrin test to verify complete coupling. The portions were then treated with 20% piperidine in DMF for 2 x 15 min and washed as above. The deprotection reaction was verified by Kaiser ninhydrin test.
Full-Scale Library-Epoxycyclohexenol resins (38R). After pooling, the spacer- containing resins, 37R, were split into two equal portions in silanized 50 mL fritted glass tubes and tagged. Both of the resin portions (1.25 g, 0.27 meq/g avg, 337.5 pmol, 1.0 equiv) were then washed with 1 x 20% DIPEA in CH2C12, 3 x CH2C12, and 1 x anhyd NMP. The resin was bubbled in minimal distd CH2Cl2 and the appropriate epoxycyclohexenol carboxylic acid, 7 (58 mg, 371.3 umol, 1. 1 equiv) and PyBOP (193.2 mg, 371.3 mol, 1.1 equiv) were added to each vessel, followed by NMP (25 mL). DIPEA (176.4 pL, 1.01 mmol, 3.0 equiv) was added to each tube and the reactions were allowed to proceed with N2 bubbling for 9 h. The resins were washed with 5 x NMP and 5 x CH2C12 and complete conversion was verified by Kaiser ninhydrin test.
Full-Scale Library-Iodobenzyl tetracycle resins (39R). After pooling, the epoxycyclohexenol-containing resins, 38R, were split into six equal portions in PD-10 columns and tagged. To two each of the six tagged resin portions (429 mg, 0.26 meq/g avg, 111.8 pmol, 1.0 equiv, dried under high vacuum) were added the appropriate nitrone carboxylic acid, 11 (68.2 mg, 223.6 umol, 2.0 equiv) and PyBroP (104.2 mg, 223.6 pmol, 2.0 equiv). The tubes were flushed with Ar and cooled to 0 °C in an ice bath. CH2C12 (4 mL), DIPEA (77.9 tL, 447.2 llmol, 4.0 equiv), and solid DMAP (15.0 mg, 123.0 mol, 1. 1 equiv) were added in sequence with immediate vortexing and recooling to 0 °C between each addition. The tubes were transferred to a Labquake in a 4 °C cold cabinet for 2 h, then mixed at rt for 2-10 h. After the standard wash (Method B), approx 1 mg of resin was removed from each tube and photolyzed in 30 L CH3CN for 2 h. Percent conversion was analyzed by TLC (17: 3 CH2Cl2/MeOH and 1: 1 CH2CI2/THF). The process was repeated until no epoxycyclohexenol carboxamides, 38, could be detected. LC-MS analysis of photocleaved samples from each of the six pools indicated the presence of all three of the expected tetracycles, 39, in each pool.
Full-Scale Library-Alkynylbenzyl tetracycle resins (40R). After pooling, the iodobenzyl tetracycle-containing resins, 39R, were split into 31 equal portions in 2 mL BioSpin columns and tagged. To each tagged resin portion (86 mg, 0.24 meq/g, 20.85 llmol, 1.0 equiv) was added copper (I) iodide (8.7 mg, 45.87 mol, 2.2 equiv) and bis (triphenylphosphine) palladium (II) chloride (16.1 mg, 22.94 llmol, 1.1 equiv) or tetrakis (triphenylphosphine) palladium (0) (26.5 mg, 22.94 umol, 1.1 equiv). DMF (860 pL) was added and the tubes were flushed with Ar and vortexed briefly. DIPEA (monoynes: 109 uL, 625.5 pmol, 30 equiv; diynes: 254.3 uL, 1.460 mmol, 70 equiv) was added followed immediately by the appropriate alkyne (monoynes: 417 umol, 20 equiv; diynes 1.043 mmol, 50 equiv). The tubes were vortexed briefly and mixed for 2 h followed by the standard wash procedure (Method B).
Full-Scale Library-Alkynylbenzyl amido hydroxy tricycle resins (41R). After pooling, the alkynylbenzyl tetracycle-containing resins, 40R, were split into 63 portions in 2 mL BioSpinX columns such that the 63rd (aminolysis skip codon) portion was 1/63rd the weight of the other equal 62 portions. Following tag coupling, the 63rd portion was set aside and to each of the remaining resin portions (40.45 mg, 0.24 meq/g, 9.82 J. mol, 1.0 equiv) was added 2- hydroxypyridine (non-a-branched amines: 4.67 mg, 49.09 pmol, 5 equiv; a-branched amines: 9.34 mg, 98.17, umol, 10 equiv) as a 404.5 L stock solution in THF (free amines) or 3: 2 CH2C12/DMF (amine hydrochloride salts). The tubes were flushed with Ar and the appropriate amine (non-a-branched amines: 245.43 umol, 25 equiv; a-branched amines 490.86 pmol, 50 equiv) was added to each tube followed by DIPEA (85.5 L, 490.86 J. mol, 50 equiv) where appropriate. The tubes were vortexed briefly and mixed for 15 h followed by the standard wash procedure (Method A).
Full-Scale Library-Alkynylbenzyl amido acyl tricycle resins (42R). The first 62 alkynylbenzyl y-hydroxyamido tricycle-containing resin portions, 41R, above were pooled and split into 63 equal portions in 2 mL BioSpin columns and tagged. The 63rd (aminolysis skip codon) portion above was set aside. After tagging, to each of the resin portions (37.14 mg, 0.235 meq/g, 8.72 Hmol, 1.0 equiv) was added 150) LiL CH2C12. The tubes were flushed with Ar and cooled to 0 °C in an ice bath. The appropriate acids (871.8 mol, 100 equiv) were placed in oven-dried 8 mL teflon-capped vials and dissolved in 532 L CH2Cl2. DIPC (68.5 aL, 435.9 mol, 50 equiv) was added and the mixture was stirred for 2 min. DIPEA (75.9 pL, 435.9 llmol, 50 equiv) was added and the mixture was stirred another 3 min. Half of each preactivated acid mixture was added to the appropriate BioSpin't column. Each tube was vortexed briefly and
returned to 0 °C. DMAP (5.325 mg, 43.58 mol, 5 equiv) was added to each tube as a 50, uL stock solution in CH2C12, and the tubes were wrapped with parafilm, vortexed briefly, and returned to 0 °C for 30 min. The tubes were warmed to rt and mixed for 11 h followed by the standard wash procedure (Method A) with an additional 3 x 20% DIPEA in CH2C12 wash. The 63 acylated resins and the lactone aminolysis skip codon resin were then combined to yield the completed full-scale library, 42R, as a brown resin.
VII. Encoding Methods and Biological Testing: Binary Encoding-General. HPLC grade CH3CN, spectrophotometric grade DMF, and 99+% decane (Aldrich) were used in bead picking and tag cleavage procedures. DMF and decane were stored over activated 4A MS during use. N, O-Bistrimethylsilylacetamide (BSA, Pierce, Rockford, IL; 38836) was obtained in ampules and stored as stocks at-20 °C. Solvent and BSA aliquots were prepared fresh daily. Ammonium cerium nitrate (CAN, Aldrich, 136 mg) was dissolved in 0.5 mL distd THF and 0.5 mL ddH20 and used within 2 h of preparation.
Sonication was performed in an Ultrasonic Cleaner water bath (Cole-Parmer, Vernon Hills, IL; 8892). Centrifugation was performed at 2000 x g with a National Labnet C-1200 Mini Centrifuge (VWR 20668-212). EC-GC analysis was performed on a Hewlett-Packard 5890E Series II Plus gas chromatograph equipped with an Ultra-1 crosslinked methyl siloxane 25 m x 0.2 mm x 0.33 J. m film thickness capillary column (HP 19091A-102) and a 63Ni electron capture detector (HP 19233-69576).
Binary Encoding-Tag Coupling. The resin to be tagged was washed with 5 x distd CH2C12. Resins containing free amine functionalities were washed further with 5 x 0.2% TFA in distd CH2C12. Rhodium triphenylacetate prepared as previously described (Callot et al.
Tetrahedron 1985,41,4495) (180 nmol per 100 mg resin) was dissolved in distd EtOAc (1 mL per 100 mg resin) by sonication for 20 sec and added to the resin. The mixture was agitated for 10 min by N2 bubbling, 360° rotation, or gentle vortexing as appropriate for the reaction vessel.
The diazoketone tags synthesized as previously described (Ohlmeyer et al. Proc. Natl. Acad. Sci.
USA 1993,90,10922; Nestler et al. J. Org. Cliem. 1994,59,4723) were dissolved in EtOAc at a concentration of approximately 24 mM. The appropriate stock solutions (500 p. L per 100 mg resin) were combined to generate the binary code for each building block (see Supporting Information). The combined stock solution was added to the resin in four equal portions at 30 min intervals. 2 h after the final addition, the resin was drained and the procedure repeated. The second coupling reaction was allowed to proceed overnight, then the resin was washed with 5 x CH2C12 and 5 x CH3CN.
Binary Encoding-Tag Cleavage and Analysis. Several beads were removed from each reaction tube with the aid of a flame-pulled capillary tube and placed on a glass 25 x 75 mm microscope slide (VWR 48300-025). CH3CN was added to the plate and a"Microliter 705"50 1L syringe (Hamilton, Reno, NV; 80530) with a 22s gauge removeable needle (Hamilton 80464) was used to pick single beads with the aid of an Olympus CK2 microscope. The beads were transferred to 1.1-1.2 I. D. x 100 mm glass capillary tubes (Corning, Corning, NY; 9530-2) which had been cut to approximately 3 cm. The tubes were centrifuged briefly, the CH3CN was removed with a"Microliter 701"10 L syringe (Hamilton 80330) with a stainless steel taper needle for 320 um columns (HP 5182-0831), and the tubes were centrifuged again. 2 tL CAN solution then 3 ptL decane were added with centrifugation after each addition. The tubes were allowed to stand for 10 min, sonicated for 1 min, then centrifuged. The 10 L syringe was rinsed with 3 x CH3CN, 3 x DMF, 3 x decane, and 2 L neat BSA. The syringe barrel was coated with the BSA plug, which was then ejected. The top decane layer from the capillary tube was drawn into the syringe and the sample plug drawn up and down in the BSA-coated portion of the barrel.
The sample was allowed to stand for 1 min inside the syringe, then analyzed by EC-GC using the published method (Ohlmeyer et al. Proc. Natl. Acad. Sci. USA 1993,90,10922) EC-GC analysis of single bead cleavage samples from all tagged resin portions indicated satisfactory tag incorporation with clearly defined peaks.
Cell Proliferation Assay. 10,000 MvlLu mink lung epithelial cells were seeded in each well of a 12-well dish in 1 mL Dubelco's Modified Eagle Medium (DMEM, GibcoBRL, Gaithersburg, MD; 11995-040) containing 10% fetal bovine serum (FBS, GibcoBRL 10438- 026), 100 units/mL penicillin G sodium (GibcoBRL 15140-122), 100 g/mL streptomycin sulfate (GibcoBRL 15140-122), and 100 g/mL each of Ala, Asp, Glu, Gly, Asn, Pro (Sigma, St.
Louis, MO or ICN Biomedicals, Aurora, OH). After 24 h, 1 J. L of DMSO was added to the DMSO control wells, and 1 aL of 1 mM 43 {X, X, 1} through 43 {X, X, 8} in DMSO was added to the assay wells. After 4 days, no cell death was observed. The cells were washed with Hanks Balanced Salt Solution (HBSS, GibcoBRL 24020-117), trypsinized, and counted. Experiments were performed in triplicate.
TGF-ß-Responsive Reporter Gene Assay. Transforming growth factor beta (TGF-, Sigma T-1654) was stored in 20 aL aliquots at-80 °C as 40 nM stock solutions (100-1000X) in 0.2 llm-filtered 4 mM HCl with 1 mg/mL bovine serum albumin (Sigma A2153). The plasmid p3TPLux, which contains three copies of the phorbol myristate acetate response element from the collagenase gene as well as a fragment of the plasminogen activator inhibitor type 1 (PAI-1) promoter, was obtained from Joan Massague (Carcamo et al. J. Mol. Cell. Biol. 1995,15,1573)
MvlLu mink lung epithelial cells were obtained from the American Type Culture Collection (Manassas, VA; CCL64). 6F mink lung cells, a stably-transfected clone containing p3TPLux as well as another plasmid, are derived from MvlLu cells. The generation of this clone was described previously (Stockwell et al. Curr. Biol. 1998,8,761). Both MvlLu and 6F cells were cultured in 10% mink medium, which consists of DMEM with 10% FBS, 100 units/mL penicillin G sodium, 100 pg/mL streptomycin sulfate and 100 aM each of Ala, Asp, Glu, Gly, Asn, Pro. 700 llg/mL G418 sulfate (GibcoBRL 11811-031) was added to cultures of 6F cells.
The initial pools 43 {X, X, 1} through 43 {X, X, 8} were assayed using a previously described scintillation counter method (Stockwell et al. Chem. Biol. 1998,5,385). Deconvoluted pools 43 {X, 1,3} through 43 {X, 8,3} and 43 {X, 1,8} through 43 {X, 8,8}, and individual compounds 43 {1, 1,1} through 43 {8, 1,1}, 43 {1, 8,1} through 43 {8, 8,1}, and 43 {1, 8,3} through 43 {8, 8,3} were assayed in 384-well plates as follows: 20,000 6F cells were seeded in 50 L of 10% mink medium in each well of a white 384-well plate (Nalge Nunc International, Naperville, IL; 164610) using a Multidrop 384 liquid dispenser (Lab Systems, Helsinki, Finland). After 16 hours, medium was removed using a 24 channel wand (V&P Scientific, San Diego, CA; VP186L), the cells were washed with 75 L of 0.2% mink medium (containing 0.2% FBS), and reagents were added in 40 J. L of 0.2% medium. For the primary screen, reagents were added by pin transfer using 384 polypropylene pin arrays (Matrix Technologies, Hudson, NH). After 24 hours, the cells were cooled on ice and washed twice with 75 IlL HBSS. Then 20 uL lysis buffer (25 mM glycylglycine (Sigma G7278) pH 7.8,15 mM MgS04 (Sigma M5921), 4 mM EGTA (Sigma E0396), 1% Triton X-100 (Sigma T9284), 1 mM dithiothreitol (DTT, Sigma D5545), 1 mM phenylmethylsulfonyl fluoride (Sigma P7626)) was added to each well with a Multidrop dispenser. After incubating the cells for five minutes on ice, 20 gel of ATP/luciferin solution was added (25 mM glycylglycine pH 7.8,15 mM MgS04,4 mM EGTA, 6.25 mM K2HP04 (Sigma P5504) pH 7.8,5 mM DTT, 75 aM D-luciferin (Sigma L9504), 2 mM ATP (Sigma A7699)). Light output was immediately measured with an LJL Analyst 384-well platereader, with 0.5 s counting time per well. Table A. Alk ne building blocks tested. mono terminal alkynes 47. 84 umol alkyne (20 eq) bis terminal alkynes (italicized) 119.60 umol alkyne (50 eq) Test mg or uL d = 290% conversion & urit # Vendor Catalog # ChemicaI Name 2 ! te MW d HPLC Mass LCMS TLC 1 Aldrich 33, 482-0 Acetaidehyde ethyl propargyl acetal y 128. 17 0. 898 50% 555 60% 2 Aldrich 38, 425-9 But I 1-meth I-2-ro n I ether, ter-- 3GFS115730Buty)) pheny) acety ! ene, 4- (tert-158. 000. 88980% 585 If 4 Aldrich 39, 926-4 Butyldimethylsilyl) acetvlene, (tert-fi 140. 30 0. 751 50% 567 50% + 5 Aldrich 30, 586-3 Butynloxv) tetrahyd ro-2H-pyran, 2-(3-ß 154. 21 0. 984 NR 581 NR 50% c 6 Aldrich 20, 647-4 Chloro-4-ethynylbenzene, i-it 136. 58 1. 000 4 564 q 7GFB126504 Decadiyne (50% in hexane), 1,4-134. 220. 50020% 56120% N 8 C 126706 Decadi ne, 1 5-134. 22 1. 000 1 561 129103 Dibutylamino-1-propyne, 3-y 111. 19 0. 804 NR 538 NR 10 1 30 1 00 Diethynylbenzene, m-> 126. 15 1. 000 60% 553 80% 11 Aldrich 24, 439-2 Dimethyl-1-butyne,3,3- 82. 15 0. 667 4 509 4 r-- 1 2 Aldrich 14, 306-5 Dimethylamino-2-propyne, 1-. 83. 13 0. 772 50% 510 10°/a 80%c 13 Aldrich 24, 440-6 Dodesvne, 1-0 | llw 166. 31 0. 778 80% 5 93 90% 1 4 Aldrich 27, 136-5 Ethyl ethynyl ether (50% in hexanes) 70. 090. 500NPt497NR 1 5 Aldrich 41, 986-9 Ethvnyl p-tolyl sulfone | 1 180. 23 1. 000 iS 607 NR 1 6 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-120. 13 1. 048 n. d. 547 n. d. 1 7 Aldrich 31, 657-1 Ethynylcyclohexene, 1-| 106. 17 0. 903 ßl 533 ; 18 Aldrich 85, 587-1 Ethynylestradiol 3-methyl ether l 310. 44 1. 000 ; 737 ; 19GFS143907 Ethynytpyridino, 2-103. 120. 940W53050% 70% c 2 0 Aldrich 20, 650-4 Ethynyltoluene, 4-l 116. 16 0. 916 4 543 ; 21 Aldrich 40, 729-1 Hexadiyne (50% in hexane, 1, 5- 78. 110.500 30% 505 40% |W 2 2 Aldrich 24, 442-2 Hexyne, 1-82. 150. 715509 23Aldrich 27, 134-9 Hexynenitrile, 5-93. 13 0. 889 4 520 4 1 24 Aldrich 17, 719-9 Methyl propargyl ether | 70. 09 0. 830 70% 497 80% 2 5 Aldrich M3 280-1 Methvl-1-buten-3-yne, 2-| 4 66. 10 0. 695 4 493 ; 2 6 Aldrich M7 425-3 Methyl-N-propargylbenzylamine, N-| > 159. 23 0. 944 ; 586 ßi 90°/Oc 2 7 Aldrich 16, 130-6 Nonadiyne, 1, 8-l 120. 20 0. 799 nuked 547 nuked baseline 28 Aldrich 25 656-0 Pentyne. 1-68. 120. 691495 Table A. Alk ne buildin blocks l _ Y g I monoterminal alkynes 47. 84 umol alk) bis terminal alkynes (italicized) 119.60 umol alk ne 50 e Test m or uL = Z90% conversion & puritv # Vendor Catalo # Chemical Name al a MW d HPLC Mass LCMS TLC 11 Aldrich 33, 482-0 Acetaldeh de eth I ro ar I acetal GV'' : 128. 17 0. 898 50% 555 60% +2 Aldrich 38, 425-9 Butyl 1-methyl-2-propynyl ether, tert-126. 20 0. 795 553 3GRS115730Buty !) pheny) acety ! ene, 4- (tert-158. 000. 88980%; 4Aldrich 39, 926-4 But Idimeth Isil I acet lene, tert-'140. 30 0. 751 50% 567 50% +5 Aldrich 30, 586-3 Butynloxy) tetrahydro-2H-pyran, 2-(3-M 154. 21 0. 984 NiR 581 NR 50% c 6Aldrich 20, 647-4 Chloro-4-ethynyibenzene, 1-e E 136. 58 1. 000 71 564 ; 7 126504 Decadiyne (50% in hexane), 1,4-134. 22 0. 500 20% 561 20% 8 126706 Decadiyne, 1, 5-134. 22 1. 000 > 5 61 ; 9GFB129103 D ! buty) am ! no-1-propyne, 3-111. 190. 804N3538NF ! 10 GIs 130100 Diethynylbenzene, m-126. 15 1. 000 60% 553 80% 11 Aldrich 24, 439-2 Dimethy !-1-butyne, 3. 3-82. 150. 667509 12 Aldrich 14, 306-5 Dimethvlamino-2-propyne, 1-5 y 83. 13 0. 772 50% 510 10% 80% c 1 3 Aldrich 24 440-6 Dodecyne, 1-W 166. 31 0. 778 80% 593 90% 1 4 Aldrich 27, 136-5 Eth I eth nyl ether (50% in hexanes) E 70. 09 0. 500 NR 497 NR 15Aldrich 41, 986-9 Ethynyl p-tolyl sulfone n 180. 23 1. 000 NR 607 iM 1 6 Aldrich 40, 433-0 Ethynv1-4-fluorobenzene, 1-m 120. 13 1. 048 n. d. 547 n. d. 17 Aldrich 31, 657-1 Ethynylcyclohexene, 1-n 106. 17 0. 903 > 533 ; 18 Aldrich 85, 587-1 Ethynylestradiol 3-methyl ether n 310. 44 1. 000 ; 737 ; 19GR143907Ethynyipyridine, 2-103. 120. 94053050% 70% c 20 Aldrich 20, 650-4 Ethynyltoluene, 4-n 116. 16 0. 916 ; 543 ßI 21 Aldrich 40, 729-1 Hexadiyne (50% in hexane2, 1, 5- 78. 11 0. 500 30% 505 40% y 22 Aldrich 24, 442-2 Hexyne, 1-82. 150. 715509 23 Aldrich 27 134-9 Hexynenitrile, 5: Qvt 93. 13 0. 889 I 520 2 4 Aldrich 17, 719-9 Methyl propargyl ether 1 70. 09 0. 830 70% 497 80% 25 Aldrich M3, 280-1 Methyl-l-buten-3-yne, 2-66. 10 0. 695 493 2 6 Aldrich M7, 425-3 Methyl-N-propargylbenzylamine, N-y 159. 23 0. 944/5 86 ; 90% c 2 7 Aldrich 16, 130-6 Nonadiyne, 1, 8-y 120. 20 0. 799 nuked 547 nuked baseline 28 Aldrich 25 656-0 Pent ne 1-2 68. 12 0. 691 495 Table A. Alkyne bui ding blocks tested. mono terminal alkynes 47. 84 umol alkyne (20 eq) bis terminal alkynes (italicized) 119.60 umol alk ne 50 e Test mg or uL->90% conversion & purity # Vendor Cataloq # Chemical Name alkvne MW HPLC Mass LCMS TLC_ , 1 Aldrich 33, 482-o Acetaldehyde ethvl propargyl acetal g 128. 17 0. 898 50% 555 60% t2 Aldrich 38, 425-9 ButyI 1-methyl-2-propynyl ether, tert-126. 20 0. 795 4 553 4 3 GFS 115730 Butyl) phenylacetylene, 4- (tert- 158. 00 0. 889 80%q 4 Aldrich 39, 926-4 Butyidimethvisilyi) acetylene, (tert-X 140. 30 0. 751 50% 567 50% 5Aidrich30, 586-3 Butyntoxy) tetrahydro-2H-pyran, 2- (3-154. 210. 984NR581 ! 50% c 6 Aldrich 20, 647-4 Chloro-4-ethynylbenzene, 1-136. 58 1. 000 I 564 7GPS126504Decadiyne (50% inhexane), 1. 4-134. 220. 50020% 56120% 8GR5126706 Decadiyne, 1, 5-134. 221. 000561 9 G 129103 Dibutylamino-1-propyne, 3-4 | | 111. 19 0. 804 bR 538 i\R 10 GI-S 130100 Diethynylbenzene, m--Li 126. 15 1. 000 60% 5 1 1 Aldrich 24, 439-2 Dimethy1-1-butyne, 3, 3-| |, 82. 15 0. 667 4 509 51 1 2 Aldrich 14, 306-5 Dimethylamino-2-propyne, 1-83. 13 0. 772 50% 510 10% 80% c 1 3 Aldrich 24, 440-6 Dodecyne, 1-| m 166. 31 0. 778 80% 593 90% 14 Aldrich 27, 136-5 Ethyl ethynyl ether (50% ! n hexanes) 70. 090. 500ft497R 1 5 Aldrich 41, 986-9 Ethynyl p-tolyl sulfone i1 E 1 180. 23 1. 000 ihR 607 M 16 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-120. 13 1. 048 n. d. 547 n. d. 17 Aldrich 31, 657-1 Ethynylcyclohexene, 1-i i 106. 17 0. 903 4 533 51 18 Aldrich 85, 587-1 Ethvn lestradiol 3-methyl ether £ 310. 44 1. 000 4 7737 19GF5143907 Ethyny ! pyndine, 2-103. 120. 94053050% 70% c 2 0 Aldrich 20, 650-4 Ethynyltoluene, 4-i S 116. 16 0. 916 4 543 » 21 Aldrich 40, 729-1 Hexadiyne (50% in hexane, 1, 5-'8. 78. 11 0. 500 40% 22 Aldrich 24, 442-2 Hexyne, 1-| ll 82. 15 0. 715 w 509 2 3 | Aldrich 27, 134-9 Hexynenitrile, 5-S m 93. 13 0. 889 4 520 71 24 Aldrich 17, 719-9 090. 83070% 49780% 25 Aldrich M3, 280-1 Methyl-1-buten-3-yne, 2-M l m 66. 10 0. 695 ; 493 q 26 Aldrich M7, 425-3 Methy !-N-propargy ! benzytam ! ne, N-159. 230. 94458690% c 27 | Aidrich 16, 130-6 Nonadivne, 1, 8-|. | 120. 20 0. 799 nuked 547 nuked baseline 28 Aldrich 25 656-0 Pent ne 1-. :,-2 68. 12 0. 691 495 Table A. Alkyne building blocks tested. mono terminal alkynes 47. 84 umol alkyne (20 eq) bis terminal alkynes (italicized) 119.6C umol alkyn L (50 eq) Test m or uL = 290% conversion & urit # Vendor Catalo # Chemical Name al ne MW d HPLC Mass LCMS TLC 1 Aldrich 33, 482-0 Acetaldehyde ethyl propargyl acetal 128. 17 2 Aldrich 38, 425-9 Butvl 1-methyl-2-propynyl ether, tert-126. 20 0.795 4 553 4 3 115730 Butyl) phenylacetylene, 4-(tert-| 158.00 0.889 80% 51 4 Aldrich 39, 926-4 Butyidimethylsilyl) acetylene, (tert-140. 30 0.751 50% 567 50% t 5 Aldrich _ 30, 586-3 Butynloxy) tetrahydro-2H-pyran, 2-(3-M ß 154. 21 0.984 w 6 Aldrich 20, 647-4 Chloro-4-ethynylbenzene, 1-ß M 136. 58 1.000 4 564 4 7GFS126504 Decad ! yne (50% ! n hexane), 1. 4-134. 220.500 20% 561 20% 7 8GFS126706 Decadiyne, 1, 5-134. 22 1. 000561 9GF3129103Dibuty ! amino-1-propyne, 3-111. 19 0.804 w 538 m 10 G : s 130100 Diethynylbenzene, m-126. 5 1. 000 60% 553 80% 11 Aldrich 24, 439-2 Dimethyl-1-butvne, 3, 3-n l 82. 15 0.667 4 509 71 12 Aldrich 14, 306-5 Dimethylamino-2-propyne, 1-y 83. 13 0.772 50% 510 10% 80% c 1 3 Aldrich 24, 440-6 Dodecyne, 1-166. 31 0.778 80% 593 90% 1 4 Aldrich 27, 136-5 Ethyl ethynyl ether (50% in hexanes) 70.09 0.500NFt497NR 1 5 Aldrich 41, 986-9 Ethynyl p-tolyI sulfone | m 180. 23 1.000 607 inR 1 6 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-| | 120. 13 1.048 n. d. 547 n. d. 17 Aldrich 31, 657-1 Ethynylcyclohexene, 1-106. 17 0.903 533 1 8 Aldrich 85, 587-1 Ethynylestradiol 3-methyl ether 310. 44 1.000 737 19GFS143907Ethyny ! pyridine, 2-103. 12 0.940 w 530 50% 1 20 Aldrich 20, 650-4 Ethynyltoluene, 4-116. 16 0.916 4 543 21 Aldrich 40, 729-1 Hexadivne (50% in hexane), 1, 5-| | 78. 11 0. 500 30% 505 40% 2 2 Aldrich 24, 442-2 Hexvne, 1-l | 82. 15 0. 715 q 509 71 2 3 Aldrich 27, 134-9 Hexynenitrile, 5-3R1 R 93. 13 0.889 520 2 4 Aldrich 17, 719-9 Methvi propargvl ether El 70.0.0. 830 70% 497 80% 25 Aldrich M3, 280-1 Methyl-1-buten-3-yne, 2-66. 10 0.695 493 4 2 6-ildrich M7, 425-3 Methyl-N-proparqylbenzylamine, N-159. 23 0.944 586 4 90% c 2 7 Aldrich 16, 130-6 Nonadiyne, 1, 8-y S 120. 20 0.799 nuked 547 nuked baseline 28 Aldrich 25 656-0 Pentvne, 1- 68. 12 0.691 71 495 w Table A. Alkyne building blocks ß mono terminal alkynes 47. 84 umol alkyne (20 eq) bis terminal alkynes (italicized) 119.60 umol alk ne 50 e Test mg or uL->90% conversion & punfy # Vendor 4 Cataloa # Chemical Name ! t MW d HPLC Mass LCMS TLC 1Atdrich33, 482-0 Aceta ! dehyde ethy ! propargyi acetai128. 170. 89850% 55560% +2 Aldrich 38, 425-9 Butyl 1-methyl-2-propynyl ether, tert-126. 200. 795553 3GR115730Butyt) pheny ! acety ! ene, 4- (tert-158. 000. 88980% 585 4 Aldrich 39, 926-4 Butyidimethylsilyl) acetylene, (tert-B fi 140. 30 0. 751 50% 567 50% 5 Aldrich 30, 586-3 Butynioxy) tetrahydro-2H-pyran, 2-(3-m-154. 21 0. 984 SR 58t I\R 50% c 6 Aldrich 20, 647-4 Chloro-4-ethynelbenzene, 1-B 136. 58 1. 00C q 564 ßl 7 126504 Decadiyne (50% in hexane), 1, 4-S 134. 22 0. 50C 20% 561 20% 8 C5 126706 Decadi ne,1,5 1. 000 561 9GFS129103 Dibuty) amino-1-propyne, 3-111. 190. 804rft538hf ! 10 (X 130100 Diethynvibenzene, m-M 126. 15 1. 000 60% 553 80% 1 1 Aldrich 24, 439-2 Dimethyl-1-butyne, 3,3- 82. 15 0. 667 4 5-09 4 1 2 Aldrich 14 306-5 Dimethviamino-2-propyne, 1-83. 13 0. 772 50% 510 10% 80% c 1 3 Aldrich 24, 440-6 Dodecyne, 1-2 166. 31 0. 77E 80% 593 90% 14 Aldrich 27, 136-5 Ethyl ethynyl ether (50% in hexanes) 70. 09 0. 500 m 497 15--ldrich Ethynyl p-tolyl sulfone 180. 23 1. 000 NR 607 t\R 1 6 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-n 120. 13 1. 048 n. d. 547 n. d. 17Aldrich 31, 657-1 Eth n ic clohexene 1- : 6 : 106. 17 0. 903 I 533 1 8 Aldrich 85, 587-1 Ethynylestradiol 3-methyl ether n 310. 44 1. 00C » 737 4 1 ! pyrid ! ne, 2-103. 120. 940NR53050% 70% c 2 0 Aldrich 20, 650-4 Ethynyltoluene, 4-W 116. 16 0. 916 4 543 51 21 Aldrich 40 729-1 Hexadivne (50% in hexanq), 1, 5- 78. 11 0, 500 30% 505 40% 2 2 Aldrich 24, 442-2 Hexyne, 1-1 82. 15 0. 715 1 509 4 23 Aldrich 27, 134-9 Hexynenitrile, 5-V 93. 13 0. 889 4 520 4 24Aldrich 17, 719-9 Methyl propargyl ether 70. 09 0. 830 70% 497 80% 25 Aldrich M3, 280-1 Methyt-l-buten-3-yne, 2-66. 100. 695493 26Aldrich M7, 425-3 Methyl-N-propargylbenzylamine, N-y 159. 23 0. 944 4 586 51 90% c 2 7 Aldrich 16, 130-6 Nonadiyne, 1, 8-W 120. 20 0. 799 nuked 547 nuked baseline 2 8 Aldrich 25 656-0 Pent ne 1-68. 12 0. 691 l 495 I Table A. Alkyne building blocks l mono terminal alkynes 47. 84 umol alkyne (20 eq) bis terminal alkynes (italicized) 119.60 umol alk ne 50 e Testm or uL.. = 290% conversion & urit # Vendor Cataloq # Chemical Name li ryne MW d HPLC Mass LCMS TLC + 1 Aldrich 33, 482-0 Acetatdehyde ethy ! propargy ! aceta ! 128. 170. 89850% 55560% t2 Aldrich 38, 425-9 But I 1-meth i-2-ro n I ether, tert-' ; : 126. 20 0. 795 553 3GR115730Buty)) phenyiacetytene. 4- (tert-158. 000. 88980% 585 4 Aldrich 39, 926-4 Butyidimethylsilyl) acetylene, (tert-X 3 140. 30 0. 751 50% 567 50% t5 Aldrich 30, 586-3 Butynioxy) tetrahydro-2H-pyran. 2- (3-154. 210. 984N=t581NR 50% c 6 Aldrich 20, 7GFS126504Decadiyne (50% inhexane),1,4-134. 220. 50020% 56120% _, | 0 | 8GFS126706 Decadiyne,1,5-134. 221. 000561 9GR129103Dibutyiamino-1-propyne, 3-111. 190. 804M=) 538W or 10 1 30 1 00 Diethynvibenzene, rn-2 126. 15 1. 000 60% 553 80% 11 Aldrich 24, 439-2 Dimethyl-l-butyne, 3, 3- 82. 15 0. 667 4 509 4 1 2 Aldrich 14, 306-5 Dimethylamino-2-propyne, 1-| | 83. 13 0. 772 50% 51 0 10% 80% c 13Aldrich 24, 440-6 Dodecyne, 1-| B 166. 31 0. 778 80% 593 90% 14 Aldrich Ethyl ethynyl ether (50% in hexanes) 70. 09 0. 500 m 497 N : l 4 15 Aldrich 41, 986-9 EthynyI p-tolyI sulfone | E 180. 23 1. 000 bR 607 ioR 1 6 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-| S 120. 13 1. 048 n. d. 547 n. d. 1 7 Aldrich 31, 657-1 Ethynylcyclohexene, 1-g IS 106. 17 0. 903 A 533 4 1 8 Aldrich 85, 587-1 Ethynylestradiol 3-methyl ether | | 310. 44 1. 000 q 737 4 19GFS143907Ethyny) pyridine, 2-103. 120. 940tt53050% 70% c 20 Aldrich 20, 650-4 Ethynyltóluene, 4-| | 116. 16 0. 916 ; 543 ; 21 Aldrich 40, 729-1 Hexadiyne L50% in hexane), 1, 5- 78. 11 0. 500 30% 505 40% I 22 Aldrich 24, 442-2 Hexyne, 1-| E 82. 15 0. 715 ; 509 23 Aldrich 27, 134-9 He nenitrile, 5-93. 13 0. 889 I 520 24 Aldrich 17, 719-9 Meth i ro ar I ether 70. 09 0. 830 70% 497 80°0 25 Aldrich M3, 280-1 Methyl-1-buten-3-vne, 2-| l | 66. 10 0. 695 ; 493 nj 26 Aldrich M7, 425-3 Methyl-N-propargylbenzvlamine, N-| l 159. 23 0. 944 ; 586 i | 90% c 27 Aldrich 16, 130-6 Nonadiyne,1, 8- 8g 120. 20 0. 799 nuked 547 nuked baseline 28 Pentyne,-68. 12 0. 691 4 495 4 Table A. Alk ne buildin blocks mono terminal alkynes 47. 84 umol alkyne (20 eq) bis terminal alkynes (italicized) 119.60 umol alkyne 50 eq) Test mg or uL, l = 290% conversion &purit # Vendor Catalo # Chemical Name a ! ne MW d HPLC Mass LCMS TLC I 1 Aldrich 33, 482-0 Acetatdehyde ethyi propargyi aceta ! 128. 170. 89850% 55560% +2 Aldrich 38, 425-9 Butyl 1-methvl-2-proPynyl ether, tert-t ß 126. 20 0. 795 51 553 51 3 GE 115730 Butyl) phenylacetylene, 4-(tert-G ß 158. 00 0. 889 80%4 4 Aldrich 39, 926-4 Butyidimethylsilyl) acetylene, (tert-_ 8 140. 30 0. 751 50% 567 50% +5 Aldrich 30, 586-3 Butynloxy) tetrahydro-2H-pyran, 2-(3-ß 154. 21 0. 984 NR 581 NR 50% c 6 Aldrich 20, 647-4 Chloro-4-ethynylbenzene, 1-136. 58 1. 000 4 564 q 7GFS126504Decadiyne (50% inhexane).1,4-134. 22 0. 500 20% 561 20% 8 126706 Decadiyne,1,5- 134. 22 1. 000 561 9GR129103 Dibuty ! amino-1-propyne. 3-111. 190. 804W538bF) 10 130 1 00 Diethynylbenzene, m-| i 126. 15 1. 000 60% 553 80% 11 Aldrich 24, 439-2 Dimethvl-1-butyne, 3,3- 82. 15 0. 667 4 509 4 1 12 Aldrich 14, 306-5 Dimethy) amino-2-propyne, 1-83. 130. 77250% 51010% 80% c 1 3 Aldrich 24, 440-6 Dodeovne, 1-< | l % 166. 31 0. 778 80% 593 90% 1 4 Aldrich 27, 136-5 Ethyl ethynyl ether (50% in hexanes) l | 70. 09 0. 500 NR 497 NR 1 5 Aldrich 41, 986-9 EthynyI p-tolyI sulfone i n 180. 23 1. 000 NR 607 NR 16 Aldrich 40, 433-0 Ethyny1-4-fluorobenzene, 1-| | 120. 13 1. 048 n. d. 547 n. d. 17 Aldrich 31, 657-1 Ethynylcyclohexene, 1-106. 17 0. 903 I 533 18 Aldrich 85, 587-1 Ethynylestradiol 3-methyl ether'310. 44 1. 000 I 737 19 GFS 143907 Ethynylpyridine, 2-103. 12 0. 940 N3 530 50% 70% c 20 Aldrich 20, 650-4 Ethyny ! to) uene. 4-116. 160. 916543 21 Aldrich 40, 729-1 Hexadiyne (50% in hexang), 1, 5- 78. 11 0. 500 30% 505 40% 2 S 22 Aldrich 24, 442-2 Hexyne. 1-82. 150. 715509 23Aldrich 27, 134-9 Hexynenitrile, 5- 93. 13 0. 889 4 520 4 24 Aldrich 17, 719-9 Methyl propargyl ether | > 70. 09 0. 830 70% 497 80% 25 Aldrich M3, 280-1 Methyl-1-buten-3-yne, 2-g g 66. 10 0. 695 nl 493 v 26 Aldrich M7, 425-3 Methyl-N-propargylbenzvlamine, N-t3. 159. 23 0. 944 q 586 4 90% c 27 Aldrich 16, 130-6 Nonadiyne, 1, 8- 120. 20 0 799 nuked 547 nuked baseline 28 Aldrich 25 656-0 Pent ne 1-", Tv2 68. 12 0. 691 495 Table A. Alkyne bui cks Test m o uL l = z90% conversion & urit es Vendor Catalog # Chemical Name ai MW d H9LC Mass LCMS TLC 29GFB184701 PhenyM-butyne, 4-S 130. 190. 926557 30 Aldrich 37, 684-1 Phenyl-1-propVne, 3-X W 116. 16 0. 934 4 543 l 31 Aldrich 11, 770-6 Phenylacetylene 102. 14 0. 930 529 32 Aldrich 41, 696-7 Proparqyl ether S g 94. 11 0. 9 14 AR 521 30% 33Aldrich 44, 694-7 Propargyl-1H-benzotriazole, 1-| 157. 18 1. 000 30% 584 10% 34 Aldrich P5 133-8 Pro ar lox hthalimide, N-62 201. 18 1. 000 N3 628 10% 3 5 187530 Proparqylphthalimide N-0 0 « i 185. 18 1. 000 40% 61 2 30% ., | S 36 Aldrich 22, 648-3 Propargyltriphenylphosponium bromide i | 381. 26 1. 000 NR 808 NR 37 Aldrich 30, 360-7 Propiolaldehyde diethyl acetal 128. 17 0. 894 NR 555 NR 3 8 Aldrich 30, 081-0 Tetrahydro-2-(2-propynyloxy)-2H-pvran M 140. 18 0. 997 40% 5 67 40% 39 Aldrich 34, 697-7 Triethylsilyl) acetylene, (140. 30 0.783 70% 567 80% 40GPS193080 Trimethy) s ! -1. 4-pentad ! yne, 1-136. 271. 000W56320% 41Aldrich 36, 005-8 ilyl) acetylene, 284. 44 1. 000 40% 711 40% 42 Aldrich T8, 496-4 Tripropargvlamine ; 131. 18 0. 927 PR 558 30% h 43 Aldrich 30, 586-3 ahydro-2H-pyran, 2- (3- 154. 21 0. 984 8 1- 44 M 133502 Dimethvl-1-hexvn-3-ol, 3, 5-11 126. 20 1. 000 4 553 >1 4 5 136101 Diphenv1-2-propyn-1-ol 1, 1-| | 208. 26 1. 000 w 635 4 46 Aldrich E5, 140-6 Ethenyl-1-cyclohexanolt 1-| | 124. 18 0. 967 4/NR ? 551 80% p 47 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-120. 13 1. 048- 4 547 48GFS143705 Ethynyt-9-ftuoreno !, 9-206. 251. 000633 49 Aldrich 13, 086-9 Ethyny ! cyc ! opentanoL 1- 110. 160. 962/NR ? 537 80% p _ q q 5 0 Aldrich 24, 441-4 Heptene, 1-1 » 96. 17 0. 733 70% c 523 60% c 51 Aldrich 13, 756-1 Methyl-1-pentyn-3-ol, 3-y g 98. 15 0. 866 80% p 525 80% p 5 2 184903 Phenyl-3-butyn-2-ol, 2-E 146. 19 1. 000 4 573 q 53Aldrich 30, 360-7 Propiolaldehyde diethvl acetal w 128. 17 0. 894 40% c 555 40% c Table A. Alk ne buildin blocks l l Y g l l I Test mg or uL = 290% conversion & urit # Vendor Catalog # Chemical Name aih nQ MW d HPLC Mass LCMS TLC 2 9 GS 184701 PhenVl-1-butyne, 4-§ § 130. 19 0. 926/557 q 30! drich37. 684-1PhenyM-propyne. 3-116. 160. 934543, 31Aldrich 11, 770-6 Phenylacetylene k 102. 14 0. 930 q 529 4 32 Aldrich 41, 696-7 Propargyl ether W 94. 1 1 0.914 NR 521 30% 33 Aldrich 44, 694-7 Propargyl-1H-benzotriazole, 1-S 157. 18 1. 000 30% 584 10% 34 Aldrich P5, 133-8 Propargyloxy) phthalimide, N-(M 201. 18 1. 000 NR 628 10% 35GFB187530 Propargyfphthafimide. N-185. 181. 00040% 61230% g 36 Aldrich 22, 648-3 Propargyltriphenylphosponium bromide E 381. 26 1. 000 NR 808 NR 37 Aldrich hyde diethyl acetal 128. 17 0. 894 m 555 m + 38 Aldrich 30, 081-0 Tetrahydro-2- (2-propynytoxy)-2H-pyran140. 180. 99740% 56740% 39Aldrich 34, 697-7 Triethvlsilvi) acetylene, (140. 30 0. 7831 70% 567 80% 40 (SS 193080 Trimethylsilyl-1, 4-pentadiyne, 1-it B 136. 27 1. 000 NR 563 20% 41 Aldrich 36, 005-8 Triphenylsilyl acetylene, 284. 44 1. 000 40% 711 40% 4 2 Aldrich T8, 496-4 Tripropargvlamine ; 131. 18 0. 92 7 558 30% 43 Aldrich 30, 586-3 Butynloxy) tetrahydro-2H-pyran, 2-(3-154. 21 0. 984 1/581 4 44 C 133502 Dimeth I-1-hex n-3-oi,3,5-S B 126. 20 1. 000 4 553 4 4S GB 136101 Diphenyl-2-propyn-1-ol,1,1-208. 261. 000635 4 6 Aldrich E5, 140-6 Ethynyl-1-cyclohexanol, 1-t B 124. 18 0. 967 4/N R ? 551 80% p 47 Aldrich 40, 433-0 Ethvnyl-4-fluorobenzene, 1- » 120. 13 1. 048 w 547 q 4 8 GFS 143705 Ethynyl-9-fluorenol, 9-t 206. 25 1. 000/633 v 49 Aldrich 13, 086-9 Ethynylcyclopentanol, 1-| g 110. 16 0. 962 FI/NR ? 537 80% p _ llN w 50. Aldrich 24, 441-4 Heptyne, I-96. 17 0. 733 70% c 523 60% c 51 Aldrich 13, 756-1 Methyl-l-pentyn-3-ol, 3-98. 15 0. 866 80% p 525 80% p 52GFS184903Pheny !-3-butyn-2-o), 2-146. 191. 000V573 53 Aldrich 30, 360-7 Propiolaidehyde diethyl acetal 128. 17 0. 8941 40% c 555 40% c Table A. Alkyne building blocks tested. Test mg or uL d = z90% conversion & urit # Vendor Catalog # Chemical Name ~ > MW d HPLC Mass LCMS TLC 57 30 Aldrich 37, 684-1 Phen i-1-ro ne, 3-. . 116. 934543 31 Aldrich 11, 770-6 Phenylacetylene tX S 102. 14 0. 930 4 529 q 3 2 Aldrich 4 >, 696-7 Pro ar I ether 3'94. 11 0. 9 i 4 lR 5 21 30 % 33 Aidrich 44, 694-7 Pro ar I-1H-benzotriazole, 1-5v 157. 18 1. 000 30% 584 10% 34Aldrich P5, 133-Propargyloxy) phthalimide, N-(ffi 201. 18 1. 000 NR 628 10% 35GFS187530 Propargytphthatimide. N-185. 181. 00040% 61230% 3 6 Aldrich 22, 648-3 ProPargyltriphenvlphosponium bromide g 381. 26 1. 000 NR 808 NR 37-30, 360-7 Propiolaldehyde diethyl acetal 128. 17 0. 894 m 555 m s38 Aldrich 30, 081-0 Tetrahydro-2- (2-propyny ! oxy)-2H-pyran140. 180. 99740% 56740% 399 Aldrich 34, 697-Triethvlsilyl) acetylene, (æ æ 140. 30 0. 783 70% 567 80% 4 0 193080 Trimethylsilyl-1, 4-nentadiyne, 1-t B 136. 27 1. 000 NR 5 63 20% 41 Aldrich 36, 005-8 Triphenylsilyl) acetylene, 284. 44 1. 000 40% 711 40% 4 2 Aldrich T8, 496-4 Tripropargylamine 131. 18 0. 927 AR 558 30 % 43 Aldrich 30, 586-3) tetrahydro-2H-pyran, 2- (3- 154. 21 0. 984 4 581 4 44 _ 133502 Dimethyl-1-hexyn-3-ol, 3, 5-126. 20 1. 000 4 553 51 4 5 136101 Diphenyl-2-propvn-1-ol, 1, 1-s 208. 26 1. 000 4 635 q 46 Aldrich E5, 140-6 Ethvnyl-1-cyclohexanol, 1-| 124. 18 0. 967 4/NR ? 551 80% p 47 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-120. 13 1. 048 4 547 4 4 8 143705 Ethynyl-9-fluorenol, 9-Q 206. 25 1. 000 4 633 q 49 Aldrich 13, 086-9 Ethynyicyclopentanol, 1-=8. 110. 16 0. 962 l/NR ? 537 80% p 5 0 Aldrich 24, 441-4 Heptyne, 1-| 96. 17 0. 733 70% c 523 60% c 51 Aldrich 13, 756-1 Methyl-1-pentyn-3-ol, 3-fi 98. 15 0. 866 80% p 525 80% p 5 2 M 184903 Phenyl-3-butyn-2-ol, 2-X 146. 19 1. 000 4 573 w 5 3 Aldrich 30, 360-7 Propiolaldehyde diethyl acetal M 128. 17 0. 894 40% c 555 40% c Table A. _ ding blocks _ Test mg or uL d = 290% conversion & urit # Vendor Catalog # Chemical Name MW d HPLC Mass LCMS TLC 2g CC3 184701 Phen i-1-but ne, 4-'6 :.. : 130. 19 0. 926 I 557 xzzt 30A ! drich37, 684-1Pheny)-1-propyne, 3-116. 160. 934543 31 Aldrich 11, 770-6 Phenylacetylene 32 Aldrich 41, 696-7 Propargy/efher94. H0. 94M ? 52130% 33 Aldrich 44, 694-7 PropargI-iH-benzotriazole, 1-157. 18 1. 000 30% 584 10% 34 Aldrich P5, 133-8 Pronargyloxy) Dhthalimide, N-(0 u 201. 18 1. 000 NR 628 10% 3 5 187530 Pronar I hthålimide N-0 i 185. 18 1. 000 40% 61 2 30% 36 Aldrich 22, 648-3 Propargvitriphenylphosponium bromide | 381. 26 1. 000 NR 808 NR 37 Aldrich 30 360-7 Propiolaldehyde diethyl acetal 8 8 128. 17 0. 894 NR 555 NR s38 Aldrich 30, 081-0 Tetrahvdro-2-(2-propynyloxy)-2H-pyran B N 140. 18 0. 997 40% 567 40% 39 Aldrich 34, 697-7 Triethy ! si ! yt) acety ! ene, ( ! 140. 300. 78370% 56780% 4 0 193080 Trimethvlsilyl-1, 4-pentadiyne, 1-M 136. 27 1. 000 NR 5 63 20% 41 Aldrich 36, 005-8 Triphenylsilyl) acetylene,. l 284. 44 1. 000 40% 711 40% 4 2 Aldrich T8, 496-4 Triproparqylamine 131. 18 0. 927 N9 558 30% 43Aldrich 30, 586-3 Butynloxy) tetrahvdro-2H-pyran, 2-(3-N 154. 21 0. 984 581 44 GT 133502 Dimethyl-1-hexyn-3-ol, 3, 5-. M 126. 20 1. 000 » 553 ; 45GfB136101 Dipheny)-2-propyn-1-o),1,1- 208. 26 1. 000 4 635 46 Aldrich E5, 140-6 Ethynyl-l-cyclohexanol, 1-124. 18 0. 967 4/NR ? 551 80% p 47 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-120. 13 1. 048 4 547 4 8 ; 143705 Ethynyl-9-fluorenol, 9-s fi 206. 25 1. 000 í 633 4 49 Aldrich 13, 086-9 Ethynylcyclopentanol, 1-110. 16 0. 962 4/NR ? 537 80% p . F o 50 Aldrich 24, 441-4 Heptyne, 1-S 96. 17 0. 733 70% c 523 60% c _ t°'. a' 98. 15 0. 866 80% 525 80°/a 51 Aldrich 13, 756-1 Methyl-1-pentyn-3-ol, 3- 52GFS184903 Pheny !-3-butyn-2-o !, 2-146. 191. 000573 53 Aldrich 30, 360-7 Propioialdehvde diethyl acetal i d 128. 17 0. 894 40% c 555 40% c Table A. Alkyne building blocks tested. Test m or uL = z90% conversion & urit # Vendor Catalog # Chemical Name al no mw d HPLC Mass LCMS TLC 2g ('S 184701 PhenyI-1-butyne, 4-130. 19 0. 926 I 557 30 Aldrich 37, 684-1 Phen I-1-ro ne, 3-5 : x'y 116. 16 0. 934 ! 543 31 Aldrich 11, 770-6 Pheny) acety) ene102. 140. 930529 32 Aldrich 41, 696-7 ProparQyl ether g 94. 1 1 0. 914 PR 521 30% 33Aldrich 44, 694-7 Proparnyl-1H-benzotriazole, 1-. O 157. 18 1. 000 30% 584 10% 3 4 Aldrich P5, 133-8 ProParqvloxy) phthalimide, N-(, 201. 18 1. 000 NR 628 10% 35GF5187530 Propargytphtha ! imide, N-185. 181. 00040% 61230% 36Aldrich 22, 648-3 PropargyttriphenyIphosponium bromide | g 381. 26 1. 000 NR 808 NR 37 Aldrich 30 360-7 Propiota ! dehydediethytaceta ! 128. 170. 894W555F) 38A ! drich30, 081-0 Tetrahydro-2- (2-propyny) oxy)-2H-pyran140. 180. 99740% 56740% 39A ! drich34. 697-7 Tr ! ethytsity)) acetyiene, (140. 300. 78370% 56780% 40 193080 Trimethylsilyl-1, 4-pentadiyne, 1-y 136. 27 1. 000 w 563 20% 41 Aldrich 36, 005-8 Triphenylsilyl) acetvlene, (0 284. 44 1. 000 40% 711 40% 4 2 Aldrich T8, 496-4 Tripropargvlamine | | 1 31. 18 0. 927 M 558 30% 43 Aldrich 30, 586-3 But nlox tetrah dro-2H-ran, 2-3-Q 154. 21 0. 984 581 44 133502 Dimethyl-1-hexyn-3-ol, 3, 5-126. 2C 1. 000 4 553 71 4 5 136101 Diphenyl-2-propyn-1-ol, 1, 1-208. 26 1. 000 4 635 4 46 Aldrich E5, 140-6 Ethynyl-1-cyclohexanol, 1-n 124. 13 0. 967 4/N R ? 551 80% p 47 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-120. 13 1. 048 4 547 7 48 143705 Ethynyl-9-fluorenol, 9-0 | B 206. 25 1. 000 » 633 q 49 Aldrich 13, 086-9 EthynyIcyclopentanol, 1-110. 16 0. 962 4/NR ? 537 80% 50 Aldrich 24, 441-4 Heptyne, 1-96. 17 0. 733 70% c 523 60% c 51Aldrich 13, 756-1 Methyl-1-pentyn-3-ol, 3-| | 98. 15 0. 866 80% p 525 80% p 52GR184903 Pheny)-3-butyn-2-o), 2-146. 191. 000573 53Aldrich 30, 360-7 Propiolaldehvde diethvl acetal _ Wè 128. 17 0. 894 40% c 555 40% c I Table A. Alkyne building blocks l m or uL = z90% conversion & urit Test # Vendor Catalo # Chemical Name a ! ne MW d HPLC Mass LCMS TLC Catalo # 30dr ! ch37. 684-1PhenyM-propyne. 3-116. 160. 934543 37, 684-1 32 Aldrich 41, 696-7 Pro ar I ether ; 94. 11 0. 9 Ivfi 521 30% 33Aldrich 44, 694-7 Propargyl-lH-benzotiazole, 1-157. 18 1. 000 30% 584 10% 34A ! drichP5, 133-8 Propargytoxy) phthatim ! de, N- (201. 181. 000tt62810% 3 5 187530 Propargvlphthalimide, N-| | 185. 18 1. 000 40% 612 30% 36 Aldrich 22, 648-3 Propargvltriphenvlphosponium bromide | 381. 26 1. 000 NR 8 0 8 NR 37 Aldrich 30, 360-7 Propiolaldehyde diethyl acetal 128. 17 0. 894 NR 555 NR 38Atdrich30, 081-0 Tetrahydro-2- (2-propyny ! oxy)-2H-pyran140. 180. 99740% 56740% 39 Aldrich 34, 697-7 Triethylsilyl) acetylene, 140. 30 0. 783 70% 567 80% 4 0 (E 193080 Trimethvisilyl-1, 4-pentadiyne, 1-2 92 136. 27 1. 000 NR 563 20% 41005-8 Triphenyisilyl) acetylene, 284. 44 1. 000 40% 711 40% ldrich 131. 18-0. 927 AR-558 30% 43 Aldrich 30, 586-3 Butynloxv) tetrahydro-2H-pyran, 2-(3-| | 154. 21 0. 984 581 44GF3133502 Dimethy !-1-hexyn-3-o !, 3, 5-126. 201. 000553 45 136101 Diphenyl-2-proDyn-1-ol, 1, 1-, I 208. 26 1. 000 4 635 4 4 6 Aldrich E5, 1 40-6 Ethynv1-1-cvolohexanol, 1-X 124. 18 0. 967 FI/N R ? 551 80% p 47 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-120. 13 1. 048 547 48 GFS 143705 Ethynyl-9-fluorenol, 9-206. 25 1. 000 633 4 9 Aldrich 13, 086-9 Ethynylcyclopentanol, 1-| 1 10. 16 0. 9 62 4/N R ? 537 80% 50 Aldrich 24, 441-4 Heptyne, 1-y 96. 17 0. 733 70% c 523 60% c 51 Aldrich 13, 756-1 Methv1-1-pentyn-3-ol, 3-y 98. 15 0. 866 80% p 525 80% p 52GF5184903Pheny)-3-butyn-2-ot, 2-146. 191. 000573 53Aldrich 30, 360-7 Propiolaldehvde diethyl acetal I B 128. 17 0. 894 40% c 555 40% c Table A. Alkyne building blocks tested. Test m or uL = z90% conversion & purity es Vendor Catalog # Chemical Name _ _ MW d HPLC Mass LCMS TLC 29GFS184701 PhenyM-butyne, 4-130. 190. 926557 300 Aldrich 37, 684-1 Phenvl-1-propyne, 3-| M 116. 16 0. 934 4 543 q 31Aldrich 11, 770-6 Phenylacetylene 102. 14 0. 930 529 32 Aldrich 41, 696-7 Pro ar I ether 94. 11 0. 914 lW 521 30% 33 Aldrich 44 694-7 Propargyl-lH-benzotriazole, 1-157. 18 1. 000 30% 584 10% 34 Aldrich P5, 133-8 Propargloxy) phthalimide, N-(201. 18 1. 000 NR 628 10% 3 5 187530 Propargyiphthalimide, N-185. 18 1. 000 40% 61 2 30% 3 6 Aldrich 22, 648-3 Propargyltriphenylphosponium bromide ! 381. 26 1. OOC NA 808 NR 37Aldrich 30, 360-7 Propiolaldehvde diethVl acetal 3 128. 17 0. 894 ioR 555 NR : t38 Aldrich 30, 081-0 Tetrahvdro-2- (2-propynyloxy)-2H-pyran 140. 18 0. 997 40% 567 40% 39 Aldrich 34, 697-7 Triethy ! si ! y !) acetytene. (140. 300. 78370% 56780% 40M 193080 Trimethylsilyl-1, 4-pentadivne, 1-136. 27 1. 00C NR 563 20% 41 Aldrich 36, 005-8 Triphenylsilvl) acetylene, (284. 44 1. 000 40% 711 40% 4 2 Aldrich T8, 496-4 Tripropargvlamine 1 31. 18 0. 92 7 55 8 30 % 43 Aldrich 30, 586-3 Butynloxy) tetrahydro-2H-pyran, 2- (3-154. 21 0. 984 581 44 G"S 133502 Dimethyl-l-hexyn-3-ol, 3, 5- 126. 20 1. 000 553 45 C5 136101 Di hen I-2-ro n-1-ol, 1, 1- 208. 26 1. 000 I 635 46 Aldrich E5, 140-6 Ethynyl-1-cyclohexanol, 1-n | 124. 18 0. 967 4/NR ? 551 80% p 47 Aldrich 40, 433-0 Ethynyl-4-fluorobenzene, 1-1. 048 4 547 48 M 143705 Ethynyl-9-fluorenol, 9- 206. 25 1. 000 4 633 > 49 Aldrich 13, 086-9 Ethynylcyclopentanol, 1-E 110. 16 0. 962 4/N R ? 537 80% p 50 Aldrich 24, 441-4 He t ne 1-96. 17 0. 733 70%c 523 60%c 51Aldrich 13, 756-1 Methyl-1-pentyn-3-ol, 3- 98. 15 0. 866 80% p 525 80% p 52 C5 184903 Phen I-3-but n-2-ol, 2-146. 19 1. 000 I 573 53 Aldrich 30, 360-7 Propiolaldehvde diethyl acetal 128. 17 0. 894 40% c 555 40% c Table B. Amine building blocks tested. l I beta-branched or greater (mult = 1) 30.23 umol amine (25 eq) alpha-branched (mult = 2) 60.49 umol amine (50 eg) 2-hydroxypyridine (2-pyr) stock solutions 1. 0 6. 05 umol (5 eq) in THF 1. X 6.05 umol (5 eq) in 3: 2 CH2CI2/DMF 2. 0 12.09 umol (10 eq) in THF 2. X 12.09 umol 10 e in 3: 2 CH2CI2/DMF ->90% conversion FAB Test Aldrich m or u uL acid and urit rel int # Catalog # Chemical Name 2-r amfne DIPEA MW d mult salt HPLC Mass LCMS TLC Pdt/SM 1 24,107-5 Alivlamine l S7. 10 0.761 1 q 5661 4 2 10, 741-7 Amino-1-propene-1, 1, 3-tricarbonitrile, 2-0 132. 13 1. 000 2 NR ? 641 NR NR 3 41, 592-8 Amino-1H-isoindole hydrochloride,3-168. 63 1. 000 2 1 NR? 642 NR 30% ? 5/100 4 23, 227-0 Amino-5-methylisoxazole, 3-98. 10 1.000 2 NR ? 607 tR N 5 A 720-0 Aminoacetaidehyde diethyl acetal 0 ; 0 133. 19 0.916 1 4 642 4 6 12,196-7 Aminoacetatdeh de dimeth I acetal 0 105. 14 0. 965 1 I 614 7 13, 052-4 Aminoacetonitrile bisulfate 92. 53 1. 000 1 1 NR ? 565 NR NR 5/100 8 27, 524-7 Aminoethyl benzenesulfonamide, 4-2-6= 5 ; 200. 26 1.000 1 ; 709 70% 9 A5, 500-4 Amin oethyl) morrhol i ne, 4- (2- 130. 191 0. 9921 10'A6, 540-9 Aminomethyl) pyridine 2-2- 2-$ 108. 141.062 1 611 rR 90% 100/2 1 1 A5, 535-7 Aminoethyl) pvrrolidine, 1-(2-8 8 l 114. 19 0. 901 1 _ 50% 623 NR 70% 100/- 12 A5, 952-2 Aminoindan hydrochloride 2-26 169. 66 1.000 2 1 NR ? 643 20% 40% _ 13 44,534-7 Aminoindan, R---1-133. 19 1.038 2 _ 4 642 4 1 4 44, 535-5 Aminoindan, S-+-1-133. 19 1.038 2 642 80% s15 38,841-6 Aminomethyl)-15-crown-5, 2-249. 31 1. 134 1 d 758 70°/a 16 A6, 180-2 Aminomethyl) benzenesulfonamide hydrochloride, 4-222. 69 1. 000 000 1 40% 695 40% 40% 17 35, 952-1. Aminomethyl cyclopropane, (71. 12 0.820 1 NR ? 4 18'40, 163-3 Pyrenemethylamine hvdrochloride, 2-l E | E 267. 76 1. 000 1 1 4 740 4 ? 1 9 A6, 540-9 AminomethVl) pyridine, 3-(E l E 108. 14 1.062 1 NR ? 617 NR W 20 A6, 560-3 Aminomethyl) pyridine, 4- (108. 14 1. 065 1 4 617 nuked 80% 21 A7, 642-7 Amino ro ionitrile fumarafe, 3-8 0 128. i3 1.000 1 1 NR? 579 NR 2 2 1 3, 656-5 Amino ro I-2-rrolidinone, 1-3-142. 20 1. 014 1. I 651 v 23 27, 226-4 Amino ropy)) imidazole, 1- (3-125. 18 1.049 1 4 634 60% q ? 100/0 24 28, 177-8 Amino ro ltrimethox silane 3-0 19. 29 1.027 1 4 688 20% 4 ? 100/2 25 41,571-5 Aminoquinuclidine R-+-3-04 4 199. 14 1.000 2 2 NR ? 636 NR Ru 26 41, 572-3 Amino uinuclidine dih drochloride, S---3-199. 14 1. 000 2 2 NR ? 6 3 6 fW tR 27 40, 766-6 Ammonia (0. 5M in dioxane) 2000 1. 000 1 4 526 526 M M-/100 28 18, 570-1 Benzylamine 0. 9 8 1 1 NR ? 616 Table B. Amine building blocks = = = = == = l = V = Z90% conversion FAB Test Aldrich m or u uL acid and urit rel int # Cataìog # Chemloal Name VIW d mult salt HPLC Mass LCMS TLC Pdt/SM 29 40,817-4 Benzylcysteamine hydrochloride, S-203. 74 1. 000 1 1 60% 677 30 35,993-9 Bornvlamine, (R)-(+)-153. 27 1. 000 2/662 70% 31 23,991-7 Butylamine 73.14 0.740 1 1 582 q 32 25,518-5 C clobut lamine 71.12 0.833 2 V ? 580 4 33 10, 184-2 C clohexanemeth lamine 113. 20 0. 870 1 4. 622 34 24,064-8 C clohe lamine 99.18 0.867 2 4 1608 4 35 Cl 1, 500-2 mine 3 6 12, 550-4 Cvolopropylamine 57. 10 0.824 2 90% 566 70% 37 85, 857-9 Cycloserine, ()- (+)- 102. 09 1. 000 2 NM 611 M NR 38 37, 189-0 Diethoxymethylsi ! yl) propylamine, 3- (191. 35 0. 916 1 700. 80% 4 100/0 39 D13, 620-4 Dimethox heneth lamine 34-181. 24 1.074 1 6901 4 40 28,563-3 Dimethvlamino) benzvamine dihvdrochloride, 4-(223. 15 1. 000 1 2 NR ? 659 NR NR-/100 41 24. 005-2 Dimethylaminopropylamine, 3-102. 18 0.812 1 _ 50% 611 N1 50% 100/2 42 D15, 780-5 Dimethylethvlenediamine, N, N-88. 15 0.828 1ho NR 50% 0/27 i 43 39 507-2 Eth tamine 2. OM in THF 500. 00 1. 000 1 I 554 44 19, 019-5 Eth I ro lamine 1-87. 17 0. 748 2 596 80% 45 42,905-8 Fluoroethvlamine hedrochloride, 2-99. 54 1. 000 1 1 80% 573 50% 70% 46 36, 182-8 Fluoro heneth lamine 4-139. 17 1. 061 1 I 648 l 47F2, 000-9 Furfu lamine 97. 12 1. 099 1 30% 606 30% 10% 48 41,264-3 Geranvlamine 153. 27 0. 829 1 d 662 d 49'12, 689-6 Fluorobenz lamine 3-125. tb 1. 097 1 634 5 0 39,165-4 Isopinocampheylamirle, (lR, 2R, 3R, 5S)- (-)- 153. 27 0. 909 2--662 51 39, 166-2 Isopinocamphevlamine, (1S, 2S, 3S, 5R)-(+)-153. 27 0. 909 2 4 662 ; 52 10,906-1 Isopropyiamine 59. 11 0. 694 2 ßi ? 568 ; 53 15, 988-3 Methoxybenzylamine, 2-137. 18 1. 051 1 ; 646 « 54 M1 110-3 Methox benz lamine 4-137. 18 1. 050 1 646 5524, 106-7 Methoxyethvlamine, 2-75. 11 0. 864 1 l 584 V _ 5637,359-1 Methoxyphenethylamine, 2-151. 21 1. 033 1 d 660 J 57 27, 022-9 Methox heneth lamine 3-151. 21 1. 038 1 d 660 58 18, 730-5 Methox heneth lamine 4-151. 2f 1. 033 1 6 6 0 _ 59 M2, 500-7 Methoxypropylamine, 3-89. 14 0.874 1 598 60 39, 505-6 Methvlamine (2. 0M in THF) 5ûO. 00 1. 000 1 540 4 6 T 18 080-7 M rtan lamine--cis-153. 27 0.915 1 ; 662 ; 62 12 703-5 Na th lenemeth lamine 1-157. 22 1. 073 1 666 70% 63 19, 166-3 Nitrobenzvlamine hvdrochloride, 3-188. 62 1. 000 1 1 60% 662 50% 60% Table B. Amine building blocks tested. ? 90% conversion FAB Test Aldrich m or u ul acid and urif rel int # Catalog # Chemical Name 2-r amine DIPEA MW d mult salt HPLC Mass LCMS TLC Pdt/SM 64 18, 480-2 Nitrophenethylamine hVdrochloride, 4-202. 64 1. 000 1 1 NP ? 676 10% 10% 1 65 0-580-2 Octylamine 129. 25 0.782 1 ; q 6 6 4 û, 726-7 Phenethylamine 121.18 0.965 1 6 3 0 +67 P2, 237-0 PhenylcVclopropylamine hydroehloride ! trans-2-169. 66 1. 000 2 1 40% 642 40% NR ? 100/68 68 P2, 555-8 Phenylglycinonitrile hydrochloride, 2-168. 63 1.000 21 W 641 10% NR? 10/96 6 9 P4, 950-3 Piparonylamine 151. 17 1.214 1 660 70 P5, 090-0 Propargvi amine | | 55. 08 0.803 1 80% 564 80% 71 | 41, 293-7 Tetrahydrofurfu lamine, (R- (-- 101. 15 0.980 1 610 80% 72 41, 294-5 Tetrahydrofurfu lamine, (S)- (+)- 101. 15 0. 980 1 4 610 4 73 22, 741-2 Tetramethyl-1, 3-propanediamine, N, N, 2, 2-IFÉ M | 130 24 0. 818 1 70% 639 10% 70% 100/- 74 42, 327-0 Thiopheneethylamine, 2-lE a 127. 21 1.087 1 4 636 4 75 26, 904-2 TritiuoroethYlamine, 2, 2, 2-iW X 99. 06 1.245 1 NR? 608 10% Ni 5/100 76 19, 374-7 Tryptamine 160. 22 1. 000 1 70% 669 1 77 V130-9 Verat iamine 167. 21 1. 109 1 NR ? 676 80% 509 78 AS 530-6 Aminoeth I ridine 2-2-122. 17 1. 021 1 1 » 631 nuked 79 | A6, 540-9 Aminometh I ridine, 3- 0 8 i 08. 14 1.062 1/617 80% c 80 29, 664-3 But lamine, R---sec-d 6 0 73. 14 0. 731 2 i 582 p 81 29, 665-1 But lamine, S-+-sec-0 6 0 73. 14 0. 731 2 5 82 80% 82 33, 650-5 C clohex leth lamine R---1-$ 127. 33 0.856 2 636 80% p 83 33, 651-3 C clohex leth lamine S-+-1-127. 33 0. 856 2 I 636 I 127. 33 0. 856 2 509 8 5 42 193-6 Methylbenzvlamine, (R- (+)-a-121. 18 0.940 2 630 86 27, 745-0 Na th I eth lamine S---1-1-2 9 171. 25 1. 060 2 80%c 680 80%c 87 34, 098-7 Trifluoromethoxy) benzylamine, 4- 91. 15 1. 252 1 700 88 26. 349-4 Trifluorometh t benz lamine 3-1.. 50 ? 4 175. 16 1. 222 1 684 Table C. Acid building blocks tested. carboxylic acids 58.52 umol (50 eq) amino acid hydrochlorides (italics) neutralized with an additional 50 eg DIPEA Tes Aldrich mg or uL v = 2909 conversi n and pu # Catalo # Chemical Name mw d HPLC Mass LCMS Comment 1 33, 882- Acetic acid 57. 061. 049V « 2 24, 851-7 Acetoxybenzoic acid, 4-180. 16 1. 000 NR/i ? 808 20% p 80% OAc 3 23, 936-1 Acetylsalicvlic acid 180. 16 1. 000 4 808 20% p 80% OAc 4 14,723-0 Ac ! ic acid 72. 06 1. 051 50% 700 50% 5 11,771-4 Anisic acid, m-152. 15 1. 000 4 780 80% p 6 16,997-8 Anisic acid, o-152. 15 1. 000 60% c 780 60% c 7 11,739-0 Anisic acid, p-152. 15 1. 000 70% p 780 20% p 30% c 8 24, 238-1 Benzoic acid 122. 12 1. 000 q 750 80% p 930,366-6 Butynoic acid, 2-84. 07 1 000 70% P 712 40% c 10 40,324-5 Carboxypropyl) trimethylammonium chloride, (3-181. 66 1. 000 NFT ? 774 NW 11 13, 269-1 Chioropropionicacid. 3-108. 521. 00020% p73710% p 12 23. 956-9 Crotonic acid86. 091. 00071470% p 13 C8, 850-5 Cyanoacetic acid 85. 06 1. 000 ; 713 10% c _ 0 1415,716-3Cyanobenzoicacid, 3-147. 131. 00077580% p 15 C8, 980-3 Cyanobenzoic acid, 4-147. 13 1. 000 775 80% p- 16 10. 183-4 Cyciohexanecarboxyiic acid128. 171. 0337S6 17 C11, 200-3 C clo entanecarbox lic acid 114. 14 1. 053 742 1812,549-q Cyclopentylacetic acid 128. 17 1. 022 4 756 4 1 9 C11, 660-2 C cio ro anecarbox lic acid 3 86. 09 1. 088 714 80% 20 19,572-3 Dih dro-22-dimeth I-4-oxo-2H-ran-6-carbox lic acid, methyl-4-oxo-2H 4- 170. 16 1. 000 60% T98 60% 3 isomers 21 30,035-7 Dihydro-2-methylbenzoic acid, 1, 4-138. 17 1. 000 d 766 80% p 22 Dimethylaminobenzoic acid, 3-165. 19 1. 000 40% c ? 23 D13, 945-9 Dimethylaminobenzoic acid, 4 X 165. 19 1. 000 iF 793 NR 24 29, 223-0 Dimethylglycine, N, N- 103. 12 0 R ? 7 31 1 0% c 2533,504-5 Ferroceneacetic acid _ X 244. 08 1. 000 70% c 872 70% c 26 25, 136-4 Formic acid46. 031. 220NR/ ? 674N=) 2733,638 6 Furanacrvilc acid, trans-3-138. 12 1. 000 4 766 4 28 F2, 050-5 Furoicacid, 2-112. 081. 000740 Table C. Acid building blocks tested. r Test Aldrich mg or uL->90% conversion and pu itv #Catalog # Chemical Name acid MW d HPLC Mass LCMS Comment 29 16,339-2 uroicacid, 3-112. 081. 000 740 30 F2, 080-7 Fu lac iic acid 0 138. 12 1. 000 40% p 766 1 0% P 798 ? 31 24, 010-9 Hexadienole acid, 2,4- (Sorbic acid) 112. 13 1. 000 4 740 4 32 24, 016-8 Isobutvric acid _ V 88. 11 0. 950 4 716 i 3 3 i-1, 750-8 isonicotinic acid 12 3. 11 1. 000 ; 7 51 » 34 12, 954-2 Isovaleric acid 102. 13 0. 937 I77 0 35 L200-9 Levulinic acid 116. 12 1. 134 80% p 7 4 4 80% p 36 85, 601-0 Linolenic acid 1 278. 44 0. 914 d 906 938 ? FAB : » 37'44, 869-7 Menthoxvacetic acid, (+)-_ 214 31 1. 020 l 842 11 38 M300-0 Menthoxyacetic acid,-2f 4. 31 1. 020 51 842 v 39 39, 537-4 Methacrylic acid 86. 09 1. 015 70% p 714 70% p 40 19,455-7 Methoxyacetic acid E 90. 08 1. 174 ; 718 » 41 24, 896-7 Methoxyphenylacetic acid, (R)- (-)-a- 166. 18 1. 000 70% P 794 60% p 40% diast 42 24,898-3 ethoxyphenytaceticacid, (S)- (+)-a-166. 181. 000 60% 794 60% p 40% diast 43 18, 065- Met oxyphenylacetic acid, 2-166. 18 1. 000 794 44 M1 900-7 Methoxyphenyiacetic acid, 3-166. 181. 000 80% c79480% c MI, 920-1 Methoxypheny) aceticacid, 4-166. 181. 000 794 46 36, 728-1 Methy) (1S. 2R)- (+)-cis-1, 2. 3, 6-tetrahydrophtha) ate, 1-184. 191. 000 l 812 ß/ 47 M4, 735-3 Methyl cilutarate, mono- 48 31,764-0 Methyl phthalate, mono-180. 16 1. 000 808 49 32,838-3 Methyl terephthalate, mono-180. 16 1. 000 J 808 J 50 29, 29S-8 Methyi-2-(nitremethyl)-5-oxoevclopentaneacetic acid, 215, 21 1. 000 N3 43 1 R-1-a, 2b, 3a-+-3- 51 19, 755-6 Methyl-5-oxo-2-razolin-1-yl) benzoic acid, 4- (3- 218. 21 1. 000 NR? 846 NR 52 41,749-1 Methy) chromone-2-carboxyiicacid, 6-204. 191. 000 10% p83230% p 53 32, 967-3 Meth lenediox hen lacetic acid, 34-1 180. 16 1. 000 80%c 808 54 13,415-5 Methylindole-2-arboxylic acid, 1-175. 19 1. 000 iO3 55 N785-0 Nicotinic acid 1. 000 751 56 15 571-3 Nitro-2-turoic acid 5-d. 9. 6 157. 08 t, 000 50% 785 10% 80% 754 ? Table C. Acid building blocks tested. Test Aldrich mg or uL/= 2905 conversi In and purify # Catalo # Chemical Name acid MW d HPLC Mass LCMS Comment 57 N1, 179-5 Nitrobenzoic acid, 4-, O 167. 12 1 000 60% p 795 60% p 809 ? 58 N2, 020-4 Nitrophenytacetic ac ! d, 4-181. 151. 000NR/ ? 809 NR 59 N2, 290-8 Nitropropionic acid, 3-119. 081. 000NFt747 60 12, 726-4 Norbornaneacetic acid, 2-154. 21 1. 065 4 782 4 61 0-840-2 Orotic acid monohydrate 174. 11 1. 000 N R/4 ? 7 8 4 NR 62 39, 134-4 Oxo-4-phenyl-3-oxazolidineacetic acid, (S)- (+)-2-221. 211. 000849 63 32, 285-7 Oxotric clo 2. 2.1.0 (2, 6)] heptane-7-carboxylic acid, anti-3-152. 15 1. 000 4 780 64 P1, 662-1 Phen lacetic acid 136. 151 1. 081 8 764 90% c 65120-5 Pheny) propio ! ic acid146. 151. 00060% c ? 77410% c 66 21, 183-4 PhthalvIsulfathlazole | t 403. 44 1. 000 NR 1031 NR 67 P4, 280-0 Picolinic acid 123. 111 1. 000 751 6840. 290-7 Propionicacid74. 080. 993702 69P5, 610-0 Pyrazinecarboxytic acid, 2-124. 101. 000752 70 P6, 560-6 Pyridylacetic acid hydrochloride, 2-5Q, $'07173. 60 1. 000 rR 766 Na WR 71 P6, 580-0 Pyridylacetic acid hydrochloride, 3-1. 60 1. 000 NR ? 766 m 72 P6, 585-1 Pyridylacetic acid hvdrochloride, 4-d 173. 60 1. 000 NR ? 766 NR 7327, 553-0 Pyrimidy ! thio) acetic acid, (2-170. 19 1. 000 N R/4 ? 798 NR 74 10, 736-0 P ruvic acid 2U,'4 88. 06 1. 267 NR/ ? 716 rR 7534, 151-7 Tetrahydro-2-furoic acid116. 121. 20960% p74460/40 2diast : t76 33, 995-4 Tetrahvdro-3-furoic acid w 116. 1. 1. 214 ; 744 ; 2 diast 77T2. 860-6 Thiocticacid206. 331. 000nuked8340% p 60%866? 78 19, 594-4 Thiopheneacetic acid, 2-142. 18 1. 000 NR ? 770 NR 79 22, 063-9 Thiopheneacetic acid, 3-142. 181. 00050% c77050% c 80 T3, 260-'Thiophenecarboxylic acid, 2-> 128. 1i 1. 00C « 756 ; 8124, 776-6 Thiophenecarboxytic acid, 3-128. 151. 000756 82 22, 227-5 Thiophenegiyoxytic acid, 2-156. 161. 000R784N=t 83'23, 302-1 Trif luoro-p-tolyl) acetic acid, (a, a, a-204. 151. 000N383220% c 84 1 l- , nylacetic acid Table C. Acid building blocks tested. Questionable starting material quality for 85-98 I Test Aldrich m or ul = 290% conversion and purity # Catalog # Chemical Name acid MW d HPLC Mass LCMS Comment 85 30, 234-1 Acetoxyacetic acid118. 09 1. 000 70% p 746 p 4 4 162. 14 1. 000 30% 790 60% OK 86 30, 727-0 Benzofurancarboxylic acid 2-.. ;, 87 C8, 215-9 Cinno) ine-4-carboyx ! icacid174. 16 1.000 30% p 802 60% p OK 88 D12, 380-3 Diiodo-4-ridone-1-acetic acid, 3,5- 404.93 1.000 10% p 1033 NR 89 D13, 860-6 Dimethvlaerylie aeid, 3, 3-100. 12 1.000 10% p 728 50% c 90 10, 688-7 Ferroeeneearboxylie aeid | 230. 05 1.000 20% p 858 NR 91 22, 528-2 Methoxy-l-indanone-3-acetic acid, 5-220. 23 1. 000 30% p 848 50% p OK 92 15, 314-1 Meth 1-2-rrolecarboxylic acid, 1-125. 13 1.000 20% D 753 m 93 41 077-2 Oxo-1-indancarboxylic acid, 3-176. 17 1.000 40% c 804 ion 94 P6,620-3 Pvridyl) acrylic aeid, trans-3-(3-Z, 149. 15 1.000 /NR ? 777 80% p OK 95 13, 058-3 Th ! eny)) acry) ic acid, 3- (2-154. 19 1. 000 40% p 782 60% p OK 96 18,834-4 Trifluoro-m-toluic acid, à a a-s 190. 12 1.00C 40% p 818 70% p OK 97 19,688-6 Trifluoro-o-toluic acid,. a a a-190. 12 1.000 40% p 818 60% p OK 98 19, 689-4 Trifluoro-p-toluic acid, a, a, a-5069 190. 12 1.000 40% p 818 60% p OK Table D. Alkyne building blocks ttsed in test library synthesis. mono terminal alkyne 153. 65 umol al ne 20 e Testmg or uL BB# # Chemical Name al ne MW d Vendor Catalo # HPLC Mass Int Int 1 25 Methvl-1-buten-3-Yne, 2- 66. 10 0.695 Aldrich M3, 280-1 4 380 0. 0047 0. 12 2 24 Methyl propargyl ether . 70. 09 0.830 Aldrich 17, 719-9 80% c 384 0. 0020 0. 05 3 11 Dimethyl-1-butyne, 3, 3-§9g2 82. 15 0.667 Aldrich 24, 439-2 4 396 0. 0433 1. 12 4 23 Hexynenitrile, 5i6. 9'93. 13 0.889 Aldrich 27, 134-9 4070. 00920. 24 5 31 Phenylacetylene 102. 14 0.930 Aldrich 11, 770-6 4 416 0. 0271 0. 70 6 30 PhenVl-1-propyne, 3- 116. 16 0.934 Aldrich 37, 684-1 v 430 0. 0297 0. 77 7. SIQP CODON 128. 00 1.000 v 442 0. 1160 2. 99 8 8 Decadivne, 1, 5- 134.22 1.000 C 126706 ßl 44 8 0. 0788 2. 03 AVERAGE99.00 413 0. 0388 Table . Amine buildin blocls used in test library synthesis. beta-branched or greater (mult = 1) 185.70 umol amine (25 eq) alphabranched (mult = 2) 371.40 umol amine 50 e 2-hydroxypyridine (2-pyr) 1.0 37.14 stock solutions 2.0 = = Test mg or ul Aldrich LCMS Rel BB# # Chemical Name 2-pyr amine MW d mult Catalog # Mass Int Int 1-SIdPCODON 0. 00-380 0.032 0.57 384 0. 016 0. 28 3960.114 2. 07 407 0.056 1.02 416 0.052 0.94 430 0.055 1.01 442 0.046 0.83 448 0. 0741.34 AVERAGE 413 0. 055 2 60 Methvlamine (2. 0M in THF) v v 500. 00 1.000 1 39, 505-6 411 0.056 10. 39 415 0. 080 0. 55 __ __--_ _____. ___ _. _ _. __. 427 0. 209 1. 45 4380.103 0. 72 d 447 0.140 0.97 = B_ 461 0. 182 1.26 2#. 473 0. 057 0.39 479 0. 328 2. 28 AVER4GE 444 0. 144 3 55 Methoxyethylamine, 2-75. 11 0. 864 1 24106-7 455 0. 109 0. 48 4590. 1420. 62 » 471 0. 410 1. 79 482 0. 162 0.71 4820. 1620. 71 .-_ ; s : : 5170880. 38 5233851. 68 rxar--r _ : -, -'. . '"w- r::, AVERAGE 488 0.229 4 35 Cyclopentylamine 85.15 0.863 2 C11, 500-2 465 0. 080 0. 50 469 0. 116 0.73 481 0. 319 1. 99 _ 4920. 1791. 12 r 5 01 0. 173 1. 08 5150. 1080. 68 527 0. 109 0. 68 533 0.194 1.21 AVERAGE 4980.160 5 33 Cyctohexanemethytamine5 113. 20 0.870 1 10,184-2 493 0. 389 0. 76 497 0. 455 0. 89 5090. 8191. 59 5200. 4010. 78 ... 529 0. 553 1. 08 . 543 0. 614 1. 19 55_. 303 0. 59 sr . 5 61 0.578 1.12 AVERAGE 526 0. 514 Table E. Amine building bloclGs used in test libr synthesis. Test m or ul Aldrich LCMS Rel BB# # Chemical Name 2-pyr amine MW d mult Catalog # Mass Int Int 6 22 Aminopropyl)-2-pyrrolidinone, IW*-. 009 0. 64 1-3-526 0. 013 0. 91 538 0. 022 1. 57 549 0. 0120.84 558 0. 010 0.71 572 0. 010 0. 71 584 0.005 0.35 590 0.029 2.10 AVERAGE 5550.014 7 62 Na th lenemeth lamine 1- 157. 22 1.073 1 12, 703-5 537 0. 144 0. 45 541 0. 324 324 02 553 0. 524 1.65 564 0.369 1.16 573 0. 247 0.78 587 0.279 0.88 e w 599 0. 2870.91 605 0. 365 1.15 AVERAGE 570 0. 317 8 77 Veratrlamine 167.21 1.109 1 V130-9 547 0.168 0.66 551 0. 136 0. 53 mm _ _ 563 0. 532 2. 08 ; 574 0. 227 0. 89 583 0. 229 0.89 597 0.266 1.04 609 0. 184 184 72 615 0. 303 1.18 £ AVERAGE 5800.256 AVERAGE155. 01 Table F. Acid buildin blocks v-, in test library synthesis. carboxylic acids 326.5 umol (50 eq) I Expected Mass Coding Scheme: Acid, Alkyne, Amine) followed by mass calculations Butyrolactone (aminolysis skip codon) coteounds are common to each pool'italicized) Test mg or uL Ret LCMS Rei < 10% < 20% Mult BB# # Chemical Name acid MW d Mass Time Intensity Int Rel Int Rel Int Peaks 1-SlaP CODON 1, 1, 1) 442. 0+-18. 0+18. 0+-128. 0+66. 0+0.0+0.0= 380 5.62 0.054 0.21 (1, 1, 2) 442.0+-18.0+18.0+-128.0+66.0+0.0+31.0= 411 4.24 0.109 _ 1 1 3 442. 0+-18.0+18.0+-128.0+66.0+0.0+75.0= 455 4.58 0.266 1.06 442. 0+-18.0+18.0+-128.0+66.0+0.0+85.0= 465 5.60 0.191 0.76 1 1, 5) 442.0+-18.0+18.0+-128.0+66.0+0.0+113.0= 493 6.45 0.270 1.07 (1, 1, 6) 442.0+-18.0+18.0+-128.0+66.0+0.0+142.0= 522 5.49 0.067 _ (1, 1, 7) 442. 0+-18.0+18.0+-128.0+66.0+0.0+157.0= 537 6.40 0.270 _ (1, 1, 8) 442.0+-18.0+18.0+-128.0+66.0+0.0+167.0= 547 5.33 0.182 0.72 (1, 2, 1) 442. 0+-18. 0+18. 0+-128. 0+70. 0+0.0+0.0= 384 3.97 0.031 0.12 1 (1, 2, 2) 442.0+-18.0+18.0+-128.0+70.0+0.0+31.0= 415 1.62 0.122 0.48 (1, 2, 3) 442.0+-18.0+18.0+-128.0+70.0+0.0+75.0= 459 1.92 0.262 1.04 (1, 2, 4) 442.0+-18.0+18.0+-128.0+70.0+0.0+85.0= 469 4.10 0.241 0.96 1, 2, 5) 442.0+-18.0+18.0+-128.0+70.0+0.0+113.0= 497 5.36 0.414 1.64 (1, 2, 6) 442.0+-18.0+18.0+-128.0+70.0+0.0+142.0= 526 1.44 0.018 0.07 1 1 1, 2, 7) 442.0+-18.0+18.0+-128.0+70.0+0.0+157.0= 541 5.44 0.344 1. 37 (1,2, 8) 442. 0+-18. 0+18.0+-128. 0+70.0+0.0+167. 0= 551 3. 86 0. 219 0.87 1, 3, 1) 442. 0+-18.0+18.0+-128.0+82.0+0.0+0.0= 396 6.05 0.121 0.48 1, 3, 2) 442.0+-18.0+18.0+-128.0+82.0+0.0+31.0= 427 4.90 0.307 1.22 (1, 3, 3)442.0+-18.0+18.0+-128.0+82.0+0.0+75.0= 471 5. 17 0. 647 2.57 ; 1, 3. 4) 442. 0+-18. 0+18. 0+-128. 0+82. 0+0. 0+85. 0= 481 6. 10 0. 422 1. 67 1, 3, 5 442. 0+-18. 0+18. 0+-128. 0+82.0+0.0+113. 0= 509 6. 99 0. 565 2.24 1, 3, 6 4442. 0+-18. 0+18. 0+-128. 0+82. 0+0. 0+142. 0= 538 4. 56 0. 050 0. 20 (1, 3, 7)442.0+-18.0+18.0+-128.0+82.0+0.0+157.0= 553 6. 88 0. 410 1.63 (1, 3, 8) 442.0+-18.0+18.0+-128.0+82.0+0.0+167.0= 563 5. 78 0. 438 1.74 (1, 4, 1) 442.0+-18.0+18.0+-128.0+93.0+0. 0+0.0= 407 4.50 0.146 0.58 (1, 4, 2) 442.0+-18.0+18. 0+-128.0+93.0+0.0+31. 0= 438 2. 02 0. 190 0.75 (1, 4, 3) 442.0+-18.0+18.0+-128.0+93.0+0.0+75.0= 482 2. 50 0. 253 1.00 (1, 4. 4) 442.0+-18.0+18.0+-128.0+93.0+0.0+85.0= 492 4. 53 0. 283 1.12 (1, 4, 5) 442.0+-18.0+18.0+-128.0+93.0+0.0+113.0= 520 5. 49 0. 532 2.11 1 4 6442.0+-18.0+18.0+-128.0+93.0+0.0+142.0= 549 1 : 76 0. 010 0. 04 1 1 (1, 4, 7)442.0+-18.0+18.0+-128.0+93.0+0.0+157.0= 564 5. 57 0. 377 1.50 (1 4 8)442.0+-18. 0+18.0+-128.0+93.0+0.0+167.0= 574 4. 37 0. 291 1.15 1, 5, 1) 442. 0+-18. 0+18. 0+-128. 0+102. 0+0. 0+0.0= 416 5. 97 0. 086 0.34 (1, 5, 2) 442.0+-18.0+18.0+-128.0+102.0+0.0+31. 0= 447 4. 93 0. 201 0. 80 (1, 5, 3)442.0+-18.0+18.0+-128.0+102.0+0.0+75.0= 491 5. 14 0. 483 1.92 (1. 5. 4) 442.0+-18.0+18.0+-128.0+102.0+0.0+85.0= 501 6. 00 0. 303 1.20 (1,5, 5) 442.0+-18.0+18.0+-128.0+102.0+0.0+113.0= S29 6. 77 0. 385 1.53 (1, 5, 6) 442. 0+-18.0+18.0+-128.0+102.0+0.0+142.0= 558 4. 61 0. 020 0. 08 1 1 (1,5, 7) 442.0+-18.0+18.0+-128.0+102.0+0.0+157.0= 573 6. 69 0. 315 1. 25 (1, 5,8) 442.0+-18.0+18.0+-128.0+102.0+0.0+167.0= 583 5.70 0.2911.15 Table F. Acid building blocks used in test library synthesis. Testmg or uLRetLCMSRd<10% < 20% Mult BB# # Chemical Name acid MW d Mass Time Intensit Int Rel Int Rel Int Peaks 1-SKiPCODONl (1, 6, 1) _ (1, 6, 2) 442.0+-18.0+18.0+-128.0+116.0+0.0+31.0= 461 5. 06 0. 242 0.96 (1, 6, 3) 442.0+-18.0+18.0+-128.0+116.0+0.0+75.0= 505 5. 28 0. 516 2.05 (1, 6, 4) 442.0+-18.0+18.0+-128.0+116.0+0.0+85.0= 515 6. 05 0. 279 1.11 (1, 6, 5) 442.0+-18.0+18.0+-128.0+116.0+0.0+113.0= 543 6. 85 0. 324 1.29 (1, 6, 6) 442. 0+-18.0+18.0+-128.0+116.0+0.0+142.0= 572 4. 96 0. 009 0. 04 1 _ (1, 6, 7) 442.0+-18.0+18.0+-128.0+116.0+0.0+157.0= 587 6. 72 0. 311 1.23 (1 6 8) 442. 0+-18.0+18.0+-128.0+116.0+0.0+167.0= 597 5. 78 0. 266 1.06 1, 7, 1) 442.0+-18.0+18.0+-128. 0+128.0+0.0+0. 0= 442 4. 64 0. 114 0. 45 (1, 7, 2) 442._ 1 7 3 442.0+-18.0+18.0+-128.0+128.0+0.0+75.0= 517 2. 56 0. 291 1.15 (1, 7, 4) 442.0+-18.0+18.0+-128.0+128.0+0.0+85.0= 527 4. 77 0. 234 0.93 (1, 7, 5) 442.0+-18.0+18.0+-128.0+128.0+0.0+113.0= 555 5. 84 0. 365 1.45 (1, 7. 6) _ (1, 7, 7) 442. 0+-18.0+18.0+-128.0+128.0+0.0+157.0= 599 5. 84 0. 307 1.22 (1, 7, 8) 442.0+-18.0+18.0+-128.0+128.0+0.0+167.0= 609 4. 45 0. 236 0.94 (1, 8, 1) 442. 0+-18. 0+18. 0+-128. 0+134.0+0.0+0.0= 448 6. 80 0. 095 0. 38 (1, 8, 2) 442.0+-18.0+18.0+-128.0+134.0+0.0+31.0= 479 5. 92 0. 311 1.23 (1, 8, 3) 442.0+-18.0+18.0+-128.0+134.0+0.0+75.0= 523 6. 10 0. 483 1.92 (1, 8,4) 442.0+-18.0+18.0+-128.0+134.0+0.0+85.0= 533 6. 90 0. 254 1.01 (1, 8, 5) 442.0+-18.0+18.0+-128.0+134.0+0.0+113.0= 561 7. 68 0. 377 1.50 (1, 8, 6) 442.0+-18.0+18.0+-128.0+134.0+0.0+142.0= 590 5. 62 0. 028 0. 11 1 (1, 8, 7) 442.0+-18.0+18.0+-128.0+134.0+0.0+157.0= 605 7. 52 0. 299 1.19 (1, 8,8) 442.0+-18.0+18.0+-128.0+134.0+0.0+167.0= 615 6. 53 0. 319 1. 27 AVERAGE 5 0 9 5. 2 0. 252 OTAL 4 6 2 1 Acetic acid 2. 7>S76 57. 06 1. 049 = t 2, 1, 1) 380 5. 57 0. 029 0. 05 1 1 (2, 1, 2) 442.0+-18.0+60.0+-128.0+66.0+0.0+31.0= 453 4. 90 0. 348 0.66 (2, 1, 3) 442.0+-18.0+60.0+-128. 0+66. 0+0. 0+75. 0=4975. 220. 7821. 47 (2, 1, 4) 442.0+-18.0+60.0+-128.0+66.0+0.0+85.0= 507 5. 94 0. 315 0.59 2, 1, 5) 442.0+-18.0+60.0+-128.0+66.0+0.0+113.0= 535 6. 66 0. 610 1.15 (2, 1, 6) 442.0+-18.0+60.0+-128.0+66.0+0.0+142.0= 564 4. 56 0. 020 0.04 1 1 2, 1 7) 442.0+-18.0+60.0+-128.0+66.0+0.0+157.0= 579 6. 64 0. 397 0.75 (2, 1, 8) _ {2, 2, i 384 4. 21 0. 046 0. 09 1 1 (2, 2, 2) 442.0+-18.0+60.0+-128.0+70.0+0.0+31.0= 457 2. 31 0. 299 0.56 (2, 2, 3) 442.0+-18.0+60.0+-128.0+70.0+0.0+75.0= 501 3. 10 0. 389 0.73 (2, 2, 4) 442.0+-18.0+60.0+-128.0+70.0+0.0+85.0= 511 4. 80 0. 795 1.50 (2, 2, 5) _ (2, 2, 6) 442.0+-18.0+60.0+-128.0+70.0+0.0+142.0= 568 1. 89 0. 038 0.07 1 1 (2, 2, 7) 442.0+-18.0+60.0+-128.0+70.0+0.0+157.0= 583 5. 76 0. 406 0.76 (2, 2, 8) 442.0+-18.0+60.0+-128.0+70.0+0.0+167.0= 593 4. 42 0. 541 1.02 (2, 3, 1) I I 396 6. 98 0. 080 0. 15 1 (2, 3, 2) 442.0+-18.0+60.0+-128.0+82.0+0.0+31.0= 469 5. 38 1. 100 2.07 2, 3, 3) 442.0+-18.0+60.0+-128.0+82.0+0.0+75.0= 513 5. 70 0. 831 1.56 (2, 3, 4) 442.0+-18.0+60.0+-128.0+82.0+0.0+85.0= 523 6. 42 1. 050 1.98 2, 3. 5) 442.0+-18.0+60.0+-128.0+82.0+0.0+113.0= 551 7. 17 1. 230 2. 2, 3, 6) 442.0+-18.0+60.0+-128.0+82.0+0.0+142.0= 580 5. 09 0. 900 1.69 (2, 3, 7) 442.0+-18.0+60.0+-128.0+82.0+0.0+157.0= 595 7. 06 1. 100 2. 07 (2, 3,8) 442. 0+-18.0+60.0+-128.0+82.0+0.0+167.0= 605 5. 97 0. 688 1.30 Table F. Acid building blocks need in test library synthesis. l I Test mg or uL Ret LCMS Rel < 10% < 20% Mult BB# # Chemical Name acid MW d Mass Eme Intensity Int Rel Int Rel Int Peaks 2 1 Acetic acid 57. 06 1 1 4, 1). 407 4. 50 0. 116 0. (2, 4, 2) 442.0+-18.0+60.0+-128.0+93.0+0.0+31.0= 480 3. 09 0. 340 0.64 2, 4, 3) 442.0+-18.0+60.0+-128.0+93.0+0.0+75.0= 524 3. 97 0. 594 1.12 2, 4, 4) 442.0+-18.0+60.0+-128.0+93.0+0.0+85.0= 534 5. 01 0. 807 1.52 (2, 4, 5) 442.0+-18.0+60.0+-128.0+93.0+0.0+113.0= 62 5. 73 0. 725 1.37 2, 4, 6) 442.0+-18.0+60.0+-128.0+93.0+0.0+142.0= 591 2. 50 0. 020 0.04 1 1 (2, 4, 7) 442.0+-18.0+60.0+-128.0+93.0+0.0+157.0= 606 5. 81 0. 692 1.30 2, 4, 8) 442.0+-18.0+60.0+-128.0+93.0+0.0+167.0= 616 4. 72 0. 643 1. 21 _ 2, 5, 1) I i 416 6. 02 0. 04S 0. 09 1 1 2, 5, 2) 442. 0+-18.0+60.0+-128.0+102.0+0.0+31.0= 489 5. 38 0. 590 1. 11 2, 5, 3) 442. 0+-18.0+60.0+-128.0+102.0+0.0+75.0= 533 5. 62 0. 815 1.53 (2, 5, 4) 442. 0+-18.0+60.0+-128.0+102.0+0.0+85.0= 543 6. 29 0. 557 1.05 5. 442. 0+-18.0+60.0+-128.0+102.0+0.0+113.0= 571 6. 96 0. 582 1.10 2, 5, 6) 442. 0+-18.0+60.0+-128.0+102.0+0.0+142.0= 600 5. 04 0. 042 0.08 1 1 (2, 5, 7) 442. 0+-18.0+60.0+-128.0+102.0+0.0+157.0= 615 6. 90 0. 639 1.20 2, 5, 8) 442.0+-18.0+60.0+-128.0+102.0+0.0+167.0= 625 5. 92 0. 557 1.05 2 6, 1 430 6. 05 0. 078 O. iS 1 (2, 6,2) 442. 0+-18.0+60.0+-128.0+116.0+0.0+31.0= 503 5. 46 0. 569 1.07 2, 6, 3) 442.0+-18.0+60.0+-128.0+116.0+0.0+75.0= 547 5. 70 0. 492 0.93 2, 6,4) 442.0+-18.0+60.0+-128.0+116.0+0.0+85.0= 557 6. 34 0. 569 1.07 (2, 6, 5) 442.0+-18.0+60.0+-128.0+116.0+0.0+113.0= 585 6. 98 0. 737 1.39 (2, 6, 6) 442.0+-18.0+60.0+-128.0+116.0+0.0+142.0= 614 5. 17 0. 041 0.08 1 1 (2, 6, 7) 442.0+-18.0+60.0+-128.0+116.0+0.0+157.0= 629 6. 93 0. 643 1.21 (2, 6, 8) 442. 0+-18.0+60.0+-128.0+116.0+0.0+167.0= 639 5. 97 0. 504 0. 95 (2, 7, i 442 4. 66 0. 096 0. 18 1 (2, 7, 2) 442.0+-18.0+60. 0+-128.0+128.0+0.0+31. 0= 515 3. 25 0. 208 0.39 (2, 7, 3) 442. 0+-18. 0+60. 0+-128. 0+128. 0+0. 0+75.0= 559 4. 21 0. 688 1.30 (2, 7, 4) 442.0+-18. 0+60. 0+-128. 0+128.0+0.0+85.0= 569 5. 30 0. 606 1.14 (2, 7, 5 442. 0+-18. 0+60. 0+-128. 0+128.0+0.0+113.0= 597 6. 13 0. 795 1.50 (2, 7, 6) 442. 0+-18. 0+60. 0+-128. 0+128. 0+0. 0+142. 0= 626 5. 92 0. 251 0. 47 (2, 7,7) 442. 0+-18. 0+60. 0+-128. 0+128. 0+0. 0+157. 0= 641 6. 16 0. 766 1. 44 (2, 7, 8) 442. 0+-18. 0+60. 0+-128. 0+128. 0+0. 0+167. 0= 651 4. 85 0. 418 0. 79 2, 8, 1) I I 448 6. 82 o 108 0. 20 (2, 8, 2) 442. 0+-18. 0+60. 0+-128. 0+134. 0+0.0+31. 0= 521 6. 26 0. 938 1. 77. (2, 8,3) 442. 0+-18. 0+60. 0+-128. 0+134. 0+0.0+75. 0= 565 6. 50 1. 080 2.03 (2, 8, 4) 442. 0+-18. 0+60. 0+-128. 0+134. 0+0. 0+85.0= 575 7. 17 0. 493 0.93 (2, 8,5) 442. 0+-18. 0+60. 0+-128. 0+134. 0+0. 0+113.0= 603 7. 81 1. 060 2.00 (2, 8, 6) 442. 0+-18. 0+60. 0+-128. 0+134. 0+0.0+142. 0= 632 5. 97 0. 046 0. 09 1 1 (2, 8, 7) 442. 0+-18. _ (2, 8, 8) 442. 0+-18.0+60.0+-128. 0+134. 0+0. 0+167. 0= 657 6. 69 0. 766 1.44 AVERAGE 546 5. 5 0. 531 TOTAL 9 12 l I 3 Methoxyacetic acid-90. 08 1.174 (3, 1, 1) 1380 5. 57 0. 025 0. 04 1 1 (3, 1, 2) 442. 0+-18.0+90.0+-128.0+66.0+0.0+31.0= 483 4. 90 0. 524 0.84 (3, 1, 3) _ (3, 1, 4)442.0+-18.0+90.0+-128.0+66.0+0.0+85.0= 537 5. 97 0. 360 0.58 (3, 1, 5) _ (3, 1,6) 442.0+-18.0+90.0+-128.0+66.0+0.0+142.0= 594 4. 64 0. 030 0.05 1 1 (3, 1,7) 442.0+-18.0+90.0+-128.0+66.0+0.0+157.0= 609 6. 61 0. 582 0. 93 (3, 1,8) 442. 0+-18.0+90.0+-128.0+66.0+0.0+167.0= 619 5. 57 0. 561 0. Table F. Acid building blocks uSed in test library synthesis. Test m or uL Ret LCMS Rel < 10% < 20% Mult BB## Chemical Name acid NIW d Mass Time Intensitv Int Rel Int Rel Int Peaks 3 40 Methoxyacetic acid 90. 08 1.174 (3, 2, 1) 384 4. 24 0. 047 0. 07 1 1 (3, 2, 2) 442.0+-18.0+90.0+-128.0+70.0+0.0+31.0= 487 2. 32 0. 340 0. 54 (3, 2, 3) 442. 0+-18.0+90.0+-128.0+70.0+0.0+75.0= 531 3. 22 0. 442 0.71 (3, 2, 4) 442.0+-18.0+90.0+-128.0+70.0+0.0+85.0= 541 4. 80 0. 635 1.01 (3, 2, 5) 442.0+-18.0+90.0+-128.0+70.0+0.0+113.0= 569 5. 65 0. 725 1.16 (3, 2, 6) 442.0+-18.0+90.0+-128.0+70.0+0.0+142.0= 598 1. 97 0. 042 0.07 1 (3, 2, 7) 442.0+-18.0+90.0+-128.0+70.0+0.0+157.0= 613 5. 73 0. 586 0.94 (3, 2, 8) 442.0+-18.0+90.0+-128.0+70.0+0.0+167.0= 623 4. 45 0. 713 1. 14 (3, 3, 1) 396 6. 98 0. 082 0. 13 1 3 (3, 3, 2) _ (3, 3, 3) 442.0+-18.0+90.0+-128.0+82.0+0.0+75.0= 543 5. 70 1. 050 1.68 (3, 3, 4) 442.0+-18.0+90.0+-128.0+82.0+0.0+85.0= 553 6. 42 1. 310 2.09 (3, 3, 5) 442.0+-18.0+90.0+-128.0+82.0+0.0+113.0= 581 7. 17 1. 510 2.41 3, 3, 6) 442.0+-18.0+90.0+-128.0+82.0+0.0+142.0= 610 5. 14 0. 100 0. 16 (3, 3, 7) 442.0+-18.0+90.0+-128.0+82.0+0.0+157.0= 625 7. 06 1. 440 2.30 (3. 3, 8) 442.0+-18.0+90.0+-128.0+82.0+0.0+167.0= 635 6. 00 1. 080 1.73 (3, 4, 1 407 4. 53 0. 115 0. 18 (3, 4, 2) 442.0+-18.0+90.0+-128.0+93.0+0.0+31.0= 510 3. 20 0. 307 0.49 (3, 4, 3) 442.0+-18.0+90.0+-128.0+93.0+0.0+75.0= 554 3. 97 0. 717 1.15 (3, 4,4) 442.0+-18.0+90.0+-128.0+93.0+0.0+85.0= 564 5. 01 0. 668 1.07 (3, 4, 5) 442.0+-18.0+90.0+-128.0+93.0+0.0+113.0= 592 5. 76 0. 684 1. 09 (3, 4, 6) 1 1 (3, 4, 7) _ (3, 4, 8) 442.0+-18.0+90.0+-128.0+93.0+0.0+167.0= 646 4. 72 0. 864 1. 38 (3, 5, 1) 416 6.00 0.034 0.05 1 1 3, 5,2) 442.0+-18.0+90.0+-128.0+102.0+0.0+31. 0= 519 5. 38 0. 709 1.13 3, 5,3) 442. 0+-18. 0+90. 0+-128. 0+102.0+0.0+75.0= 563 5. 62 0. 897 1. 43 3, 5, 4) 442. 0+-18. 0+90. 0+-128. 0+102. 0+0.0+85. 0= 573 6. 29 0. 676 1. 08 3, 5, 5 442. 0+-18. 0+90. 0+-128. 0+102.0+0.0+113.0= 601 6. 93 0. 848 1.35 3, 5, 6) 442. 0+-18. 0+90. 0+-128. 0+102.0+0. 0+142. 0= 630 5. 12 0. 058 0.09 1 1 3, 5, 7) 442. 0+-18. 0+90. 0+-128. 0+102. 0+0.0+157. 0= 645 6. 90 0. 844 1. 35 (3, 5,8) 442. 0+-18. 0+90. 0+-128. 0+102. 0+0. 0+167. 0= 655 5. 89 0. 860 1. 37 (3, 6, 1) 430 6.02 0.071 0.11 1 (3, 6, 2) 442.0+-18.0+90.0+-128.0+116.0+0.0+31. 0= 533 5. 49 0. 725 1.16 (3, 6,3) 442. 0+-18. 0+90.0+-128. 0+116.0+0.0+75.0= 577 5. 70 0. 602 0.96 (3, 6, 4) 442. 0+-18. 0+90. 0+-128. 0+116. 0+0. 0+85. 0=5876. 320. 7051. 13 3, 6, 5) 442. 0+-18. 0+90. 0+-128. 0+116. 0+0.0+113.0= 615 6. 98 0. 913 1.46 (3, 6, 6) 442. 0+-18. 0+90. 0+-128. 0+116. 0+0.0+142. 0= 644 5. 20 0. 054 0.09 1 1 (3, 6, 7) 442.0+-18.0+90.0+-128.0+116.0+0.0+157.0= 659 6. 93 0. 754 1.20 (3,6, 8) 442.0+-18.0+90.0+-128.0+116.0+0.0+167 0= 669 5. 97 0. 578 0.92 (3, 7, 1). 442 4. 64 0. 092 0. 15 1 3, 7, 2) 442.0+-18. 0+90. 0+-128. 0+128.0+0.0+31. 0= 545 3. 46 0. 231 0.37 3, 7, 3) 442.0+-18.0+90.0+-128.0+128.0+0.0+75.0= 589 4. 24 0. 885 1.41 (3, 7, 4) 442.0+-18.0+90.0+-128.0+128.0+0.0+85.0= 599 5.33 0.508 0.81 (3, 7,5) 442.0+-18.0+90.0+-128.0+128.0+0.0+113.0= 627 6. 13 1. 110 1.77 (3, 7, 6) 442. 0+-18. 0+90. 0+-128. 0+128. 0+0. 0+142. 0= 656 5. 89 0. 311 0. 50 (3. 7, 7) 442. 0+-18. 0+90. 0+-128. 0+128. 0+0.0+157. 0= 671 6. 13 0. 831 1.33 (3, 7,8) 442.0+-18.0+90.0+-128.0+128.0+0.0+167.0= 681 4.88 0.573 0.92 Table F. Acid building blocks used in test library synthesis. Test mg or uL. _ Ret LCMS Rel < 10% < 20% Mult BB# # Chemical Name acid MW d Mass Time IntensitV Int Rel Int Rel Int Peaks 3 40 Metho acetic acid :.-25. 05 90. U8 1.174 * 3 81 1) 448 6. 80 0. 134 0. 21 (3. 8, 2) 442. 0+-18.0+90.0+-128.0+134.0+0.0+31.0= 551 6. 26 0. 987 1.58 (3. 8, 3) 442. _ _ (3 8 4) 442.0+-18.0+90.0+-128.0+134.0+0.0+85.0= 605 7. 14 0. 582 0.93 3 8 5) 442.0+-18.0+90.0+-128.0+134.0+0.0+113.0= 633 7. 81 1. 460 2.33 (3, 8, 6) 442.0+-18.0+90.0+-128.0+134.0+0.0+142.0= 662 5. 97 0. 074 0. 12 1 3 8 7) _ (3, 8, 8) 442.0+-18.0+90.0+-128.0+134.0+0.0+167.0= 687 6. 69 0. 991 1.58 A GE 1572 5. 5 0. 626 ROTAL 8 14 lsovaledc acid 102. 13 0. 937 (4, 1, 1) 380 5. 57 0. 032 0. 04 1 1 (4, 1 2) 442.0+-18.0+102.0+-128.0+66.0+0.0+31.0= 495 6. 00 0. 557 0.74 (4, 1 3) 442.0+-18.0+102.0+-128.0+66.0+0.0+75.0= 539 6. 29 0. 999 1.33 (4, 1 4) 442.0+-18.0+102.0+-128.0+66.0+0.0+85.0= 549 7. 01 0. 598 0.80 (4, 1 5) 442.0+-18.0+102.0+-128.0+66.0+0.0+113.0= 577 7. 68 0. 827 1.10 (4, 1 6) 442.0+-18.0+102.0+-128.0+66.0+0.0+142.0= 606 5. 62 0. 070 0.09 1 1 4, 1, 7) 442.0+-18.0+102.0+-128.0+66.0+0.0+157.0= 621 7. 49 0. 582 0.78 4, 1, 8) 442.0+-18.0+102.0+-128.0+66.0+0.0+167.0= 631 6. 40 0. 639 0. 85 (4, 2, 1 384 3. 89 0. 047 0. 06 1 4, 2,2) 442.0+-18.0+102.0+-128.0+70.0+0.0+31.0= 499 4. 95 1. 100 1.47 (4, 2, 3) 442.0+-18.0+102.0+-128.0+70.0+0.0+75.0= 543 5. 27 1. 560 2. 08 (4, 2, 4) 442.0+-18.0+102.0+-128.0+70.0+0.0+85.0= 553 6. 02 0. 680 0.91 (4, 2, 5) 442.0+-18.0+102.0+-128.0+70.0+0.0+113.0= 581 6. 72 0. 668 0.89 (4, 2, 6) 442.0+-18.0+102.0+-128.0+70.0+0.0+142.0= 610 4. 47 0. 106 0. 14 (4, 2, 7) 442.0+-18.0+102.0+-128.0+70.0+0.0+157.0= 625 6. 61 0. 569 0.76 (4, 2 8) 442.0+-18.0+102.0+-128.0+70.0+0.0+167.0=-o-889 1. 19 (4,3,1) 396 6. 32 0. 081 0. 11 1 4, 3, 2) 442.0+-18.0+102.0+-128.0+82.0+0.0+31. 0= 511 6. 42 1. 520 2.03 (4, 3, 3) 442. 0+-18. 0+102.0+-128. 0+82.0+0.0+75.0= 555 6. 74 1. 150 1.53 (4, 3,4) 442. 0+-18. 0+102. 0+-128. 0+82. 0+0. 0+85. 0=5657. 490. 9011. 20 (4, 3,5) 442. 0+-18. 0+102. 0+-128. 0+82. 0+0. 0+113.0= 593 8. 18 1. 460 1.95 (4, 3, 6) 442. 0+-18. 0+102. 0+-128. 0+82. 0+0.0+142. 0= 622 6. 10 0. 471 0.63 (4, 3, 7) 442. 0+-18. 0+102.0+-128. 0+82.0+0.0+157.0= 637 7. 94 1. 440 1.92 (4, 3, 8) 442. 0+-18. 0+102. 0+-128. 0+82. 0+0. 0+167.0= 647 6. 82 1. 310 1.75 4,4 1) 407 4.47 0.137 0.18 (4, 4, 2) 442. 0+-18. 0+102. 0+-128. 0+93. 0+0. 0+31. 0= 522 5. 11 1. 340 1. 79. _ (4, 4, 3) 442. 0+-18. 0+102.0+-128.0+93.0+0.0+75.0= 566 5.38 1.310 1.75 (4, 4, 4) 442.0+-18. 0+102. 0+-128. 0+93.0+0.0+85. 0= 576 6. 02 0. 696 0. 93 (4, 4, 5) 442.0+-18. 0+102. 0+-128. 0+93. 0+0.0+113.0= 604 6. 61 0. 868 1.16 (4, 4, 6) 442.0+-18. 0+102. 0+-128. 0+93.0+0.0+142.0= 633 4.77 0.117 0.16 1 (4, 4, 7) 442. 0+-18. 0+102. 0+-128. 0+93. 0+0.0+157. 0= 648 6. 53 1. 080 1.44 4 4 8) 442. 0+-18. 0+102.0+-128. 0+93.0+0.0+167. 0= 658 5. 65 1. 010 1.35 (4, 5, 1) 1 1 416 5. 97 0. 040 0. 05 1 1 (4, 5, 2)442.0+-18.0+102.0+-128.0+102.0+0.0+31. 0= 531 6. 32 1. 060 1.41 (4, 5, 3) 442. 0+-18. 0+102. 0+-128. 0+102. 0+0. 0+75.0= 575 6. 58 0. 782 1.04 (4, 5, 4) 442. 0+-18. 0+102. 0+-128. 0+102. 0+0.0+85.0= 585 7. 28 0. 668 0.89 (4, 5. 5) 442. 0+-18. 0+102. 0+-128. 0+102. 0+0.0+113.0= 613 7. 86 0. 958 1.28 (4, 5, 6) 442. 0+-18. 0+102.0+-128. 0+102.0+0.0+142.0= 642 5.94 0.130 0.17 1 (4, 5, 7) 442. 0+-18. 0+102. 0+-128. 0+102.0+0.0+157. 0= 657 7. 73 1. 020 1.36 (4, 5, 8) 442. 0+-18. 0+102. 0+-128. 0+102.0+0. 0+167. 0= 667 6. 66 0. 561 0.75 Table F. Acid building blocks used in test library synthesis. Testmg or uLRetLCMSRet <10% <20% Mutt BB# # Chemical Name acid MW d Mass Time lntensit Int Re ! Int Rel Int Peaks 4 34 Isovaleric acid a, >5 102. 13 0.937 (4 6s 1) 430. 6. 82 0. 031 0. 04 1 1 (4, 6, 2) 442.0+-18.0+102.0+-128.0+116.0+0.0+31.0= 545 6. 34 0. 844 1.13 (4, 6, 3) 442.0+-18.0+102.0+-128.0+116.0+0.0+75.0= 589 6. 61 0. 598 0.80 (4 6, 4) 442.0+-18.0+102.0+-128.0+116.0+0.0+85.0= 599 7. 30 0. 725 0.97 (4, 6, 5) 442. 0+-18.0+102.0+-128.0+116.0+0.0+113.0= 627 7. 92 0. 918 1.22 (4, 6, 6) 442.0+-18.0+102.0+-128.0+116.0+0.0+142.0= 656 6. 02 0. 047 0.06 1 (4, 6, 7) 442.0+-18.0+102.0+-128.0+116.0+0.0+157.0= 671 7. 70 1. 050 1.40 (4, 6, 8) 442.0+-18.0+102.0+-128.0+116.0+0.0+167.0= 681 6. 72 0. 610 0.81 (4, 7, 1) 1 1 442 4. 61 0. 121 0. 16 1 (4, 7, 2) 442.0+-18.0+102.0+-128.0+128.0+0.0+31.0= 557 5. 38 0. 586 0. 78 (4, 7, 3) 442. 0+-18.0+102.0+-128.0+128.0+0.0+75.0= 601 5. 73 1. 180 1.57 (4, 7, 4) 442. 0+-18.0+102.0+-128.0+128.0+0.0+85.0= 611 6. 50 0. 479 0.64 (4, 7, 5) 442.0+-18.0+102.0+-128.0+128.0+0.0+113.0= 639 7. 22 0. 852 1.14 4 7, 6) 442.0+-18.0+102.0+-128.0+128.0+0.0+142.0= 668 5. 03 0. 050 0.07 1 1 4 7, 7) 442.0+-18.0+102.0+-128.0+128.0+0.0+157.0= 683 7. 09 0. 893 1.19 (4, 7, 8) 442.0+-18.0+102.0+-128.0+128.0+0.0+167.0= 693 5. 89 1. 010 1.35 (4 8, 1) 448 6. 85 0. 029 0. 04 1 1 (4, 8, 2) 442. 0+-18.0+102.0+-128.0+134.0+0.0+31.0= 563 7. 12 1. 160 1.55 (4, 8, 3) 442.0+-18.0+102.0+-128.0+134.0+0.0+75.0= 607 7. 41 1. 310 1.75 4, 8,4) 442.0+-18.0+102.0+-128.0+134.0+0.0+85.0= 617 8. 08 0. 963 1.28 4, 8,5) 442.0+-18.0+102.0+-128.0+134.0+0.0+113.0= 645 8. 72 1. 700 2.27 (4, 8,6) 442.0+-18.0+102.0+-128.0+134.0+0.0+142.0= 674 6. 72 0. 084 0. 11 1 4, 8,7) 442.0+-18.0+102.0+-128.0+134.0+0.0+157.0= 689 8. 45 1. 740 2.32 (4, 8,8) 442.0+-18.0+102.0+-128.0+134.0+0.0+167.0= 699 7. 44 0. 979 1.31 AVERAGE 583 6. 5 0. 750 OTAL 8 15 Hexadienoic acid, 2, 4- 112. 13 _ (5,l, 1 380 5. 62 0. 033 0. 09 1 1 5, 1, 2) 442.0+-18.0+112.0+-128.0+66.0+0.0+31.0= 505 6. 05 0. 311 0. 85 2 (5, 1, 3) 442.0+-18.0+112.0+-128.0+66.0+0.0+75.0= 549 6. 23 0. 406 1.11 (5, 1,4) 442.0+-18.0+112.0+-128.0+66.0+0.0+85.0= 559 6. 93 0. 270 0.74 (5, 1, 5) 442.0+-18.0+112.0+-128.0+66.0+0.0+113.0= 587 7. 65 0. 463 1.27 (5, 1, 6) 442.0+-18.0+112.0+-128.0+66.0+0.0+142.0= 616 5. 51 0. 050 0. 14 1 (5, 1, 7) 442.0+-18.0+112.0+-128.0+66.0+0.0+157.0= 631 7. 46 0. 520 1.42 (5, 1, 8) 442.0+-18.0+112.0+-128.0+66.0+0.0+167.0= 641 6. 34 0. 393 1.07 (5,2, 1) l l 384 3. 97 0. 031 0. 08 1 1 (5, 2, 2) 442.0+-18.0+112.0+-128.0+70.0+0.0+31. 0= 509 6. 98 0. 532 1. 45 2 (5, 2, 3) _ (5, 2, 4) 442.0+-18.0+112.0+-128.0+70.0+0.0+85.0= 563 5. 94 0. 262 0.72 (5, 2, 5) 442.0+-18.0+112.0+-128.0+70.0+0.0+113.0= 591 6. 63 0. 401 1.10 (5, 2, 6) 442. 0+-18.0+112.0+-128.0+70.0+0.0+142.0= 620 4. 42 0. 036 0. 10 1 (5, 2, 7) 442.0+-18.0+112.0+-128.0+70.0+0.0+157.0= 635 6. 53 0. 356 0. 97 (5, 2, 8) 442.0+-18.0+112.0+-128.0+70.0+0.0+167.0= 645 5. 49 0. 397 1.08 (5, 3, 1) l l 396 6. 07 0. 073 0. 20 (5. 3,2) 442.0+-18.0+112.0+-128.0+82.0+0.0+31. 0= 521 6. 45 0. 602 1.64 5, 3,3) 442.0+-18.0+112.0+-128.0+82.0+0.0+75.0= 565 6. 66 0. 573 1.57 5, 3, 4) 442. 0+-18. 0+112.0+-128.0+82.0+0.0+85.0= 575 7. 41 0. 324 0.89 5, 3, 5) 442.0+-18.0+112.0+-128.0+82.0+0.0+113.0= 603 8. 16 0. 692 1.89 (5, 3, 6) 442.0+-18.0+112.0+-128.0+82.0+0.0+142.0= 632 5. 91 0. 078 0.21 5, 3, 7) 442.0+-18.0+112.0+-128.0+82.0+0.0+157.0= 647 7. 92 0. 905 2.47 5, 3, 8) 442. 0+-18. 0+112.0+-128.0+82.0+0.0+167.0= 657 6. 74 0. 836 2. 28 Table F. Acid building blocks _d in test library synthesis. Test m or uL Ret LCMS Rel < < < < 20% Mult BBX # Chemical Name acid MW d Mass Eme Intensíty Int Rel Int Rel Int Peaks 5 31 Hexadienoic acid, 2,4- 112. 13 = _ (5,4, 1) 1 407 4. 50 0. 143 0. 39 5 4, 2) 442. 0+-18. 0+112.0+-128.0+93.0+0.0+31.0= 532 5. 22 0. 344 0.94 (5, 4, 3) 442.0+-18.0+112.0+-128.0+93.0+0.0+75.0= 576 5. 33 0. 463 1.27 5 4 4) 442. 0+-18.0+112.0+-128.0+93.0+0.0+85.0= 586 5. 94 0. 266 0.73 442.0+-18.0+112.0+-128.0+93.0+0.0+113.0= 614 6. 53 0. 352 0.96 (5, 4, 6) 442.0+-18.0+112.0+-128.0+93.0+0.0+142.0= 643 4. 71 0. 051 0. 14 1 (5, 4, 7) 442. 0+-18.0+112.0+-128.0+93.0+0.0+157.0= 658 6. 47 0. 455 1. 24 (5, 4, 8) 442. _ {5, 5 1) I i 416 1. 51 0. 040 0. 11 1 (5, 5, 2) 442.0+-18.0+112.0+-128.0+102.0+0.0+31.0= 541 6. 34 0. 430 1.17 (5, 5, 3) 442.0+-18.0+112.0+-128.0+102.0+0.0+75.0= 585 6. 50 0. 385 1.05 (5, 5, 4) 442.0+-18.0+112.0+-128.0+102.0+0.0+85.0= 595 7. 17 0. 251 0.69 (5, 5, 5) 442.0+-18.0+112.0+-128.0+102.0+0.0+113.0= 623 7. 84 0. 508 1.39 (5, 5,6) 442.0+-18.0+112.0+-128.0+102.0+0.0+142.0= 652 5. 14 0. 307 0.84 (5, 5, 7) 442. 0+-18.0+112.0+-128.0+102.0+0.0+157.0= 667 7. 70 0. 737 2.01 (5, 5, 8) 442. 0+-18.0+112.0+-128.0+102.0+0.0+167.0= 677 6. 58 0. 414 1.13 (5,6 1 430 6. 07 0. 057 0. 15 1 (5, 6, 2) 442. 0+-18.0+112.0+-128.0+116.0+0.0+31.0= 555 6. 39 0. 319 0. 87 2 (5, 6,3) 442. 0+-18.0+112.0+-128.0+116.0+0.0+75.0= 599 6. 55 0. 418 1.14 (5, 6,4) 442. 0+-18.0+112.0+-128.0+116.0+0.0+85.0= 609 7. 20 0. 217 0.59 5, 6, 5) 442.0+-18.0+112.0+-128.0+116.0+0.0+113.0= 637 7. 84 0. 430 1.17 (5, 6, 6) 442.0+-18.0+112.0+-128.0+116.0+0.0+142.0= 666 5. 94 0. 034 0.09 1 1 2 (5, 6,7) 442.0+-18.0+112.0+-128.0+116.0+0.0+157.0= 681 7. 68 0. 582 1.59 (5, 6, 8) 442. 0+-18.0+112.0+-128.0+116.0+0.0+167.0= 691 6. 63 0. 410 1. 12 {5, 7, 1 1 442 4. 63 0. 115 0. 31 (5, 7, 2) 442.0+-18.0+112.0+-128. 0+128.0+0.0+31. 0= 567 5. 43 0. 232 0.63 (5, 7,3) 442. 0+-18. 0+112. 0+-128. 0+128. 0+0. 0+75. 0= 611 5. 65 0. 446 1. 22 5, 7, 4) 442. 0+-18. 0+112. 0+-128. 0+128. 0+0. 0+85. 0= 621 6. 42 0. 191 0. 52 (5, 7,5) 442. 0+-18. 0+112. 0+-128. 0+128. 0+0. 0+113. 0= 649 7. 17 0. 426 1. 16 (5, 7, 6) 442. 0+-18. 0+112. 0+-128. 0+128. 0+0. 0+142. 0= 678 4. 77 0. 028 0. 08 1 5, 7,7) 442. 0+-18. 0+112. 0+-128. 0+128. 0+0. 0+157. 0= 693 6. 98 0. 639 1. 75 5, 7. 8) 442. 0+-18. 0+112. 0+-128. 0+128. 0+0. 0+167. 0=7035. 780. 4671. 28 (5,8,1). I I 448 6. 82 0. 067 0. 18 1 5, 8,2) 442.0+-18.0+112.0+-128.0+134.0+0.0+31. 0= 573 7. 12 0. 594 1.62 (5, 8, 3) 442. 0+-18. 0+112.0+-128. 0+134.0+0.0+75.0= 617 7. 33 0. 487 1.33 (5, 8, 4) 442. 0+-18. 0+112. 0+-128. 0+134. 0+0. 0+85. 0= 627 8. 02 0. 426 1. _ 5, 8 5) 442. 0+-18. 0+112. 0+-128. 0+134. 0+0.0+113. 0= 655 8. 69 0. 733 2.00 (5, 8, 6) 442. 0+-18. 0+112. 0+-128. 0+134. 0+0. 0+142. 0= 684 6. 71 0. 035 0. 09 1 (5, 8, 7) 442. 0+-18. 0+112. 0+-128. 0+134.0+0.0+157. 0= 699 8. 45 0. 827 2.26 (5, 8. 8) 442. 0+-18. 0+112. 0+-128. 0+134. 0+0. 0+167. 0=7097. 380. 6961. 90 AVERAGE591 6. 4 0. 366 OTAL 5 11 6 33 Isonicotinic acid 123. 11 1.000 (6, 1, 1) 380 5. 60 0. 044 0. 10 1 (6, 1, 2) 442.0+-18.0+123.0+-128.0+66.0+0.0+31. 0= 516 5. 28 0. 438 0.98 6, 1, 3) 442. 0+-18. 0+123. 0+-128. 0+66. 0+0. 0+75.0= 560 5. 44 0. 512 1.15 6, 1, 4) 442. 0+-18. 0+123.0+-128. 0+66. 0+0. 0+85. 0= 570 6. 10 0. 206 0.46 6, 1,5) 442. 0+-18. 0+123. 0+-128. 0+66. 0+0.0+113. 0= 598 6. 85 0. 459 1.03 6, 1, 6) 442. 0+-18. 0+123. 0+-128. 0+66. 0+0.0+142. 0= 627). 22 0. 015 0.03 1 6, 1, 7) 442.0+-18.0+123.0+-128.0+66. 0+0.0+157.0= 642 6. 77 0. 360 0.81 6, 1, 8) 442. 0+-18. 0+123.0+-128.0+66.0+0.0+167.0=6525. 650. 4420.99 Table F. Acid building blocks ased in test library synthesis. Test mg or uL Ret LW6 Rel < 10% < 20% Mult BB## Chemical Name acid MW d Mass Time Intensit Int Rel Int Rel Int Peaks 6 33sonicotinicacid'0 ? 123. 11 1. _. (6, 2, 1) 384 4. 45 0. 030 0. 07 1 1 2 (6, 2, 2) 442.0+-18.0+123.0+-128.0+70.0+0.0+31. 0= 520 3. 30 0. 319 0.72 (6, 2, 3) 442.0+-18.0+123.0+-128.0+70.0+0.0+75.0= 564 3. 83 0. 565 1.27 ré, 2, 4) _ (6, 2, 5) 442. _ (6, 2, 6) 442.0+-18.0+123.0+-128.0+70.0+0.0+142.0= 631 2. 98 0. 029 0.06 1 1 (6, 2, 7) 442.0+-18.0+123.0+-128.0+70.0+0.0+157.0= 646 5. 73 0. 377 0. 85 (6, 2, 8) 442. 0+-18.0+123.0+-128.0+70.0+0.0+167.0= 656 4. 55 0. 606 1.36 (6,3 1 396 6. 08 0. 053 0. 12 1 3 (6, 3, 2) 442.0+-18.0+123.0+-128.0+82.0+0.0+31.0= 532 5. 73 0. 717 1. 61 6 3, 3) 442.0+-18.0+123.0+-128.0+82.0+0.0+75.0= 576 5. 97 0. 905 2.03 442.0+-18.0+123.0+-128.0+82.0+0.0+85.0= 586 6. 61 0. 844 1. 89 (6, 3, 5) 442.0+-18.0+123.0+-128.0+82.0+0.0+113.0= 614 7. 41 1. 040 2.33 (6, 3, 6) 442. 0+-18.0+123.0+-128.0+82.0+0.0+142.0= 643 5. 76 0. 042 0.09 1 1 (6, 3, 7) 442.0+-18.0+123.0+-128.0+82.0+0.0+157.0= 658 7. 25 0. 799 1.79 6, 3, 8) 442.0+-18.0+123.0+-128.0+82.0+0.0+167.0= 668 6. 10 0. 750 1.68 (6, 4, 1 407 4. 47 0. 108 0. 24 (6, 4, 2) 442.0+-18.0+123.0+-128.0+93.0+0.0+31. 0= 543 2. 37 0. 754 1. 69 g (6, 4, 3) 442.0+-18.0+123.0+-128.0+93.0+0.0+75.0= 587 4. 34 0. 795 1.78 (6, 4, 4) 442.0+-18.0+123.0+-128.0+93.0+0.0+85.0= 597 5. 17 0. 733 1.64 (6, 4, 5) 442. 0+-18.0+123.0+-128.0+93.0+0.0+113.0= 625 5. 81 0. 557 1.25 (6, 4,6) 442.0+-18.0+123.0+-128.0+93.0+0.0+142.0= 654 3. 70 0. 031 0.07 1 (6. 4, 7) 442.0+-18.0+123.0+-128.0+93.0+0.0+157.0= 669 5. 81 0. 520 1.17 (6, 4,8) 442.0+-18.0+123.0+-128.0+93. 0+0. 0+167. 0= 6794. 820. 7661. 72 (6,(6, 5, 1)-I 416 6. 00 0. 047 o. 1t i- 6 5, 2) 442. 0+-18. 0+123. 0+-128. 0+102. 0+0.0+31. 0= 552 5. 73 0. 295 0.66 6, 5,3) 442. 0+-18. 0+123. 0+-128. 0+102.0+0.0+75.0= 596 5. 92 0. 483 1.08 6, 5,4) 442. 0+-18. 0+123. 0+-128. 0+102. 0+0. 0+85. 0= 606 6. 50 0. 516 1. 16 6, 5,5) 442. 0+-18. 0+123. 0+-128. 0+102. 0+0. 0+113.0= 634 7. 17 0. 606 1.36 6, 5,6) 442. 0+-18. 0+123. 0+-128. 0+102. 0+0. 0+142. 0= 663 5. 70 0. 037 0. 08 1 1 2 (6, 5, 7) 442. 0+-18.0+123.0+-128.0+102.0+0.0+157.0= 678 7. 09 0. 532 1.19 6, 5,8) 442.0+-18.0+123.0+-128.0+102.0+0.0+167.0= 688 6. 10 0. 459 1.03 (6,6,1) 430 6.08 0.042 0.09 1 1 (6, 6,2) 442.0+-18.0+123.0+-128.0+116.0+0.0+31.0= 566 5. 84 0. 324 0.73 6, 6, 3) 442.0+-18.0+123.0+-128.0+116.0+0.0+75.0= 610 5. 97 0. 573 1.28 6, 6, 4) 442.0+-18.0+123.0+-128.0+116.0+0.0+85.0= 620 6. 50 0. 455 1.02 (6,6, 5) 442. 0+-18.0+123.0+-128.0+116.0+0.0+113.0= 648 7. 20 0. 627 1.41 (6, 6, 6) 442.0+-18.0+123.0+-128.0+116.0+0.0+142.0= 677 5. 65 0. 023 0.05 1 1 (6, 6, 7) 442.0+-18.0+123.0+-128.0+116.0+0.0+157.0= 692 7. 12 0. 594 1.33 (6, 6, 8) 442.0+-18.0+123.0+-128.0+116.0+0.0+167.0= 702 6. 18 0. 512 1.15 (6, 7, 1) l l 442 4. 66 0. 087 0. 20 (6, 7, 2) 442.0+-18.0+123.0+-128.0+128.0+0.0+31.0= 578 4. 02 0. 242 0.54 (6,7, 3) 442.0+-18.0+123.0+-128.0+128.0+0.0+75.0= 622 4. 42 0. 631 1.41 (6, 7, 4) 442.0+-18.0+123.0+-128.0+128.0+0.0+85.0= 632 5. 73 0. 717 1.61 6, 7, 5) 442.0+-18.0+123.0+-128.0+128.0+0.0+113.0= 660 6. 21 0. 553 1.24 (6, 7, 6) 442.0+-18.0+123.0+-128.0+128.0+0.0+142.0= 689 3. 81 0. 017 0.04 1 1 (6, 7, 7) 442.0+-18.0+123.0+-128.0+128.0+0.0+157.0= 704 6. 16 0. 414 0. 93 (6, 7,8) 442. 0+-18.0+123.0+-128.0+128.0+0.0+167.0= 714 4.88 0.369 0.83 Table F. Acid building blocks _JC I in test library synthesis. Testmg or uLRet LCMS Re) < 10% < 20% Mult BB## Chemical Name acid MW d Mass Time Intensity Int Rel Int Rel Int Peaks 6 33 Isonicotinic acid 4Pw2 ; 0 123.11 1. 000 {6, 8, 1) 448 6.80 0. 069 0. 15 1 (6, 8, 2) 442.0+-18.0+123.0+-128.0+134.0+0.0+31.0= 584 6.64 0. 647 1.45 (6, 8, 3) 442.0+-18.0+123.0+-128.0+134.0+0.0+75.0= 628 6.80 0.770 _. (6, 8, 4) 442.0+-18.0+123.0+-128.0+134.0+0.0+85.0= 638 7.41 0.455 _ _ (ô, 8, 5) 442.0+-18.0+123.0+-128.0+134.0+0.0+113.0= 666 8.10 0.877 1.97 (6, 8, 6) 442.0+-18.0+123.0+-128.0+134.0+0.0+142.0= 695 6.80 0.030 0.07 12 (6, 8, 7) 442.0+-18.0+123.0+-128.0+134.0+0.0+157.0= 710 8.00 1.060 2.38 (6, 8, 8) 442.0+-18.0+123.0+-128.0+134.0+0.0+167. 0=720 6.96 1. 34 AVER4GE 601 5. 8 0. 446 roTAL 10 14 7 45 Methoxvphenylacetic acid, 4-85eM26 166. 18 1.000 7, 1, 1) 380 5. 54 0.035 0.06 1 1 (7, 1, 2) 442.0+-18.0+166.0+-128.0+66.0+0.0+31. 0= 559 6.00 0.430 0.71 (7, 1, 3) 442.0+-18.0+166.0+-128.0+66.0+0.0+75.0= 603 6.21 0.659 1.09 (7, 1 4 442. 0+-18.0+166.0+-128.0+66.0+0.0+85.0= 613 6.87 0.352 0.58 (7, 1, 5) 442.0+-18.0+166.0+-128.0+66.0+0.0+113.0= 641 7.46 0.795 1.31 (7, 1, 6) 442.0+-18.0+166.0+-128.0+66.0+0.0+142.0= 670 5.65 0.033 0.05 1 1 7, 1, 7) 442.0+-18.0+166.0+-128.0+66.0+0.0+157.0= 685 7.38 0.422 0.70 (7, 1, 8) 442. 0+-18.0+166.0+-128.0+66.0+0.0+167.0= 695 6.37 0.467 0.77 (7, 2, 1) _, I I 284 0.70_ 0.068 (7, 2, 2) 442. 0+-18. 0+166. 0+-128. 0+70.0+0.0+31.0= 563 5.06 0.872 1.44 7, 2, 3) 442. 0+-18. 0+166. 0+-128. 0+70. 0+0. 0+75. 0= 607 5. 30 1. 200 1. 98 7 2, 4) 442. 0+-18. 0+166. 0+-128. 0+70.0+0.0+85. 0= 617 5. 97 0. 504 0.83 7, 2,5) 442. 0+-18. 0+166. 0+-128. 0+70. 0+0. 0+113.0= 645 6. 55 0. 659 1.09 7, 2, 6) 442. 0+-18. 0+166. 0+-128. 0+70. 0+0. 0+142. 0= 674 4. 74 0. 050 0. 08 1 1 (7, 2, 7) 442. 0+-18. 0+166. 0+-128. 0+70. 0+0.0+157. 0= 689 6. 53 0. 508 0.84 7, 2, 8 442. 0+-18. 0+166. 0+-128. 0+70. 0+0. 0+167. 0= 699 5. 59 0. 651 1. 07 (7, 3, 1 396 7. 67 0. 397 0. 66 7, 3, 2) 442. 0+-18. 0+166.0+-128.0+82.0+0.0+31. 0= 575 6. 39 1. 050 1.73 7, 3, 3) 442. 0+-18. 0+166. 0+-128. 0+82. 0+0. 0+75.0= 61 9 6. 63 1. 080 1.78 7, 3,4) 442. 0+-18. 0+166. 0+-128. 0+82. 0+0. 0+85. 0= 629 7. 33 0. 659 1. 09 7, 3,5) 442. 0+-18. 0+166. 0+-128. 0+82. 0+0. 0+113.0= 657 7. 94 1. 640 2.71 (7, 3,6) 442. 0+-18. 0+166. 0+-128. 0+82. 0+0. 0+142. 0= 686 6. 05 0. 087 0. 14 1 (7,3, 7) 442.0+-18.0+166.0+-128.0+82.0+0.0+157.0= 701 7.78 1.390 2.29 (7, 3, 8)442.0+-18.0+166.0+-128.0+82.0+0.0+167. 0= 711 6. 74 0. 495 0.82 407 4. 45 0. 161 0. 27 (7, 4, 2) 442.0+-18.0+166.0+-128.0+93.0+0.0+31. 0=5865, 190. 6511.07 (7, 4, 3) 442. 0+-18. 0+166. 0+-128. 0+93. 0+0.0+75.0= 630 5. 41 0. 991 1.64 (7, 4, 4) 442.0+-18.0+166.0+-128.0+93.0+0.0+85.0= 640 6. 00 0. 537 0.89 (7, 4, 5) 442.0+-18.0+166.0+-128.0+93.0+0.0+113.0= 668 6. 47 0. 717 1.18 (7, 4, 6) 442.0+-18.0+166.0+-128.0+93.0+0.0+142.0= 697 4. 90 0. 043 0.07 1 1 (7, 4, 7) 442.0+-18.0+166.0+-128.0+93.0+0.0+157.0= 712 6. 47 0. 647 1.07 (7, 4, 8) 442.0+-18.0+166.0+-128.0+93.0+0.0+167.0= 722 5.65 0.766 1.26 7, 5 1 416 5. 97 0. 050 0. 08 1 1 (7, 5,2) 442.0+-18.0+166.0+-128.0+102.0+0.0+31.0= 5956. 260. 6801. 12 7, 5, 3) 442.0+-18.0+166.0+-128.0+102.0+0.0+75.639 6. 47 0. 713 1. 181 7, 5, 4) 442.0+-18.0+166.0+-128.0+102.0+0.0+85.0= 649 7. 11 0. 590 0. 97 7, 5,5) 442.0+-18.0+166.0+-128.0+102.0+0.0+113.0= 677 7. 65 1. 050 1.73 (7, 5,6) 442.0+-18.0+166.0+-128.0+102.0+0.0+142.0= 706 5. 91 0. 060 0. 10 1 7, 5, 7) 442.0+-18.0+166.0+-128.0+102.0+0.0+157.0= 721 7. 57 0. 664 1.10 (7, 5, 8) 442.0+-18.0+166.0+-128.0+102.0+0.0+167.0= 731 6. 61 0. 520 0.86 Table F. Acid building blocks 6>ed in test library synthesis. Test mg or u Ret LCMS Ret < 10% < 20% Mult BB# # Chemical Name acid MW d Mass Time Intensit Int Rel Int Rel Int Peaks 7 45 Methoxyphenylacetic acid, 4-166. 18 1 (7 430 6. 74 0. 040 0. 07 1 1 (7, 6, 2) 442.0+-18.0+166.0+-128.0+116.0+0.0+31.0= 609 6. 31 0. 610 1.01 (7, 6, 3) 442.0+-18.0+166.0+-128.0+116.0+0.0+75.0= 653 6. 53 0. 647 1.07 (7, 6,4) 442.0+-18.0+166.0+-128.0+116.0+0.0+85.0= 663 7. 14 0. 598 0.99 (7, 6, 5) 442.0+-18.0+166.0+-128.0+116.0+0.0+113.0= 691 7. 67 0. 975 1.61 (7, 6, 6) 442.0+-18.0+166.0+-128.0+116.0+0.0+142.0= 720 6. 00 0. 264 0.44 (7, 6,7) 442. 0+-18.0+166.0+-128.0+116.0+0.0+157.0= 735 7. 57 0. 930 1. 53 (7, 6, 8) 442. 0+-18.0+166.0+-128.0+116.0+0.0+167.0= 745 6. 66 0. 483 0.80 7 7 1 442 4. 58 0. 115 0. 19 1 (7, 7, 2) _ 7 7, 3) 442. 0+-18.0+166.0+-128.0+128.0+0.0+75.0= 665 5. 70 0. 975 1.61 (7, 7, 4) 442.0+-18.0+166.0+-128.0+128.0+0.0+85.0= 675 6. 42 0. 315 0.52 7 7, 5) 442. 0+-18.0+166.0+-128.0+128.0+0.0+113.0= 703 7. 01 0. 844 1.39 (7, 7, 6) 442.0+-18.0+166.0+-128.0+128.0+0.0+142.0= 732 5. 09 0. 020 0.03 1 1 (7, 7, 7) 442. 0+-18.0+166.0+-128.0+128.0+0.0+157.0= 747 6. 93 0. 848 1.40 (7, 7, 8) 442.0+-18.0+166.0+-128.0+128.0+0.0+167.0= 757 5. 89 0. 627 1.03 (7, 8 1) 1 1 1 448 6. 82 0. 049 0. 08 1 1 (7. 8, 2) 442. 0+-18.0+166.0+-128.0+134.0+0.0+31.0= 627 7. 06 1 0. 987 1.63 (7, 8, 3) 442.0+-18.0+166.0+-128.0+134.0+0.0+75.0= 671 7. 27 1. 390 2.29 7, 8 4) 442.0+-18.0+166.0+-128.0+134.0+0.0+85.0= 681 7. 89 0. 786 1.30 (7, 8,5) 442.0+-18.0+166.0+-128.0+134.0+0.0+113.0-709 8. 45 1. 380 2.28 7, 8,6) 442.0+-18.0+166.0+-128.0+134.0+0.0+142.0= 738 6. 71 0. 054 0.09 1 1 (7, 8,7) 442.0+-18.0+166.0+-128.0+134.0+0.0+157.0= 753 8. 26 1. 360 2.24 (7, 8, 8) 442._ I AVERAGE 639 6. 5 0.606 OTAL 9 13 Methyl terephthalate, mono-180. 16 1. 000 (8,1, 1) 380 5.60 0.023 0.04 1 1 8, 1, 2) 442.0+-18.0+180.0+-128.0+66.0+0.0+31.0= 573 6. 00 0. 442 0.78 (8, 1, 3) 442. 0+-18.0+180.0+-128.0+66.0+0.0+75.0= 617 6. 10 0. 676 1.19 8, 1, 4) 442.0+-18.0+180.0+-128.0+66.0+0.0+85.0= 627 6. 74 0. 389 0.69 8, 1,5) 442.0+-18.0+180.0+-128.0+66.0+0.0+113.0= 655 7. 33 0. 688 1.22 8, 1, 6) 442.0+-18.0+180.0+-128.0+66.0+0.0+142.0= 684 5. 57 0. 050 0.09 1 1 (8, 1, 7) 442.0+-18.0+180.0+-128.0+66.0+0.0+157.0= 699 7. 17 0. 492 0.87 (8,1, 8) 442.0+-18.0+180.0+-128.0+66.0+0.0+167.0= 709 6. 18 0. 500 0.88 8 2 1 384 5. 62 0. 063 0. i 1 1 2 (8, 2, 2) 442. 0+-18.0+180.0+-128.0+70.0+0.0+31.0= 577 5. 17 0. 864 1.53 (8, 2, 3) 442.0+-18.0+180.0+-128.0+70.0+0.0+75.0= 621 5. 28 0. 713 1.26 (8, 2, 4) 442.0+-18.0+180.0+-128.0+70.0+0.0+85.0= 631 5. 92 0. 578 1. 02 2 (8,2, 5) 442.0+-18.0+180.0+-128.0+70.0+0.0+113.0= 659 6. 50 0. 573 1.01 (8, 2, 6) 442.0+-18.0+180.0+-128.0+70.0+0.0+142.0= 688 4. 53 0. 069 0. 12 8, 2, 7) 442.0+-18.0+180.0+-128.0+70.0+0.0+157.0= 703 6. 37 0. 442 0.78 8, 2, 8) 442.0+-18.0+180.0+-128.0+70.0+0.0+167.0= 713 5.46 0.5530.98 (8,3,1) i i 396 6. 08 0. 103 0. 18 1 2 8, 3,2) 442.0+-18.0+180.0+-128.0+82.0+0.0+31.0= 589 6. 40 0. 532 0. 94 8, 3,3) 442.0+-18.0+180.0+-128.0+82.0+0.0+75.0= 633 6. 56 0. 926 1.64 (8, 3,4) 442.0+-18.0+180.0+-128.0+82.0+0.0+85.0= 643 7.17 0.930 1.64 (8, 3,5) 442.0+-18.0+180.0+-128.0+82.0+0.0+113.0= 671 7. 78 1. 690 2.99 8, 3, 6) 442.0+-18.0+180.0+-128.0+82.0+0.0+142.0= 700 5. 94 0. 150 0.27 8, 3,7) 442.0+-18.0+180.0+-128.0+82.0+0.0+157.0= 715 7. 60 1. 030 1.82 (8, 3, 8) 442. 0+-18.0+180.0+-128.0+82.0+0.0+167.0= 725 6.58 0.639 1.13 Table F. Acid building blocks u-, d in test library synthesis. Test mg or uL Ret LCMS Rel < 10% < 20% Mult BB# # Chemical Name acid MW d Mass Time Intensit Int Rel Int Rel Int Peaks 8 49 Methyl terephthalate, mono-w 180. 16 1.000 (8, 4, 1) 407 4. 42 0. 164 0. 29 (8, 4, 2) 442.0+-18.0+180.0+-128.0+93.0+0.0+31.0= 600 5. 28 0. 553 0.98 (8, 4, 3) 442.0+-18.0+180.0+-128.0+93.0+0.0+75.0= 644 5. 36 0. 897 1.58 (8, 4, 4) 442.0+-18.0+180.0+-128.0+93.0+0.0+85.0= 654 5. 92 0. 668 1.18 (8, 4, 5) 442.0+-18.0+180.0+-128.0+93.0+0.0+113.0= 682 6. 42 0. 729 1.29 (8, 4, 6) 442.0+-18.0+180.0+-128.0+93.0+0.0+142.0= 711 4. 80 0. 070 0. 12 1 (8, 4, 7) 442.0+-18.0+180.0+-128.0+93.0+0.0+157.0= 726 6. 34 0. 676 1.19 (8, 4, 8) _ (8, 5, 1) l l 416 5. 97 0. 052 0. 09 1 1 (8, 5, 2) 442.0+-18.0+180.0+-128.0+102.0+0.0+31.0= 609 6. 29 0. 532 0.94 (8, 5, 3) 442.0+-18.0+180.0+-128.0+102.0+0.0+75.0= 653 6. 40 0. 430 0.76 (8, 5, 4) 442.0+-18.0+180.0+-128.0+102.0+0.0+85.0= 663 6. 96 0. 668 1.18 (8, 5, 5) 442.0+-18.0+180.0+-128.0+102.0+0.0+113.0= 691 7. 52 1. 010 1.78 (8, 5, 6) 442.0+-18.0+180.0+-128.0+102.0+0.0+142.0= 720 5. 86 0. 088 0. 16 1 (8, 5, 7) 442.0+-18.0+180.0+-128.0+102.0+0.0+157.0= 735 7. 38 0. 733 1.30 (8, 5,8) 442.0+-18.0+180.0+-128.0+102.0+0.0+167.0= 745 6. 45 0. 299 0.53 (8,6,1) l l 430 6. 02 0. 068 0. 12 1 2 (8, 6,2) 442.0+-18.0+180.0+-128.0+116.0+0.0+31.0= 623 6. 40 0. 291 0.51 (8, 6,3) 442.0+-18.0+180.0+-128.0+116.0+0.0+75.0= 667 6. 50 0. 668 1.18 (8, 6,4) 442.0+-18.0+180.0+-128.0+116.0+0.0+85.0= 677 7. 04 0. 786 1.39 8, 6,5) 442.0+-18.0+180.0+-128.0+116.0+0.0+113.0= 705 7. 62 0. 979 1.73 8, 6,6) 442.0+-18.0+180.0+-128.0+116.0+0.0+142.0= 734 6. 00 0. 050 0.09 1 1 (8, 6, 7) 442.0+-18.0+180.0+-128.0+116.0+0.0+157.0= 749 7. 46 0. 795 1.40 (8, 6, 8) 442. 0+-18.0+180.0+-128.0+116.0+0.0+167.0= 759 6. 58 0. 332 0. 59 (8, 7, 1) 442 4.56 0.117 0.21 (8, 7, 2) 442. 0+-18. 0+180. 0+-128. 0+128.0+0.0+31. 0= 635 5.44 0.389 0. 69 8, 7, 3) 442. 0+-18. 0+180. 0+-128. 0+128. 0+0.0+75.0= 679 0.56 1.020 1.80 (8, 7,4) 442. 0+-18. 0+180. 0+-128. 0+128. 0+0. 0+85. 0= 689 6.24-0.459 0.81 (8, 7, 5) 442. 0+-18. 0+180. 0+-128. 0+128. 0+0. 0+113. 0= 717 6.88 0.987 1.74 8, 7, 6) 442. 0+-18. 0+180. 0+-128. 0+128. 0+0. 0+142. 0= 746 4.85 0.030 0.05 1 1 8, 7, 7) 442. 0+-18. 0+180. 0+-128. 0+128. 0+0. 0+157. 0=761 6.69 0.553 0.98 8, 7, 8) 442.0+-18.0+180.0+-128.0+128.0+0.0+167.0= 771 5.68 0.516 0.91 8, 8, 1) 1 1 448 6.77 0. 120 0.21 (8, 8, 2) 442.0+-18.0+180.0+-128.0+134.0+0.0+31.0= 641 7.12 0.938 1.66 (8, 8, 3) 442.0+-18.0+180.0+-128.0+134.0+0.0+75.0=685 7. 251.160 2.05 (8,8, 4) 442.0+-18.0+180.0+-128.0+134.0+0.0+85.0= 695 7.84 0.979 1.73 (8, 8, 5) 442.0+-18.0+180.0+-128.0+134.0+0.0+113.0= 723 8.37 1.290 2.28 (8, 8, 6) 442.0+-18.0+180.0+-128.0+134.0+0.0+142.0= 752 6.72 0.056 0.10 1 2 (8, 8, 7) 442.0+-18.0+180.0+-128.0+134.0+0.0+157.0= 767 8.16 1.180 2.08 (8, 8, 8) 442.0+-18.0+180.0+-128.0+134.0+0.0+167.0= 777 7.28 0.602 1.06 AVERAGE 651 6. 3 0. 557 OTAL 5 12 SUBTOTAL 58 97 less redundant butyrolactone misses-20-41 GRAND TOTAL 38 56 total compounds 456 456 % HITS 91.7 87.7 Table G. Spacer, epoxycyçlohexenol and nitrone building blockb used in library synthesis. BB# Chemical Name Spacer Postion 1 1SKIP CODON 2Glycine 3 6-Aminocaproic acid Epoxycyctohexenot Position 2 1(+)-Epoxycyclohexenol 2(-)-Epoxycyclohexenol Nitrone Position 3 12-lodobenzyl nitrone 23-lodobenzyl nitrone 34-lodobenzyl nitrone Table H. Alkyne building blocks used in library synthesis. mono terminal alkyne bis terminal al ne italicized 1042. 6 umol alkyne BB #23 was tested separately in an NMR scale reaction data not shown Test mg or uL BB#Chemical Name mw d Vendor Cataloci Size 1 81 Acetaldehvde ethvl propargyl acetal 128. 17 0.898 Aldrich 33, 482-0 g 2 t2 Butyl 1-methvl-2-propvnVl ether, tert-w66, ; 126. 20 0.795 Aldrich 38, 425-9 100 mL 3 3 But I hen lacet lene 4-tert-158. 00 0.889 GFS 115730 10 a 4 +5, 43 Butvnloxy) tetrahydro-2H-pvran, 2-(3-=3 154. 21 0.984 Aldrich 30, 586-3 5q 5 6 Chloro-4-ethynylbenzene, I-6. 58 1.000 Aldrich 20, 1 a 6 8 Decadiyne. 1. 5-134. 22 1.000 (Ri 126706 100 7 10 Diethynylbenzene, m-| mi, 126. t5 1. 000 (E 130100 5 9 X X 8 1 1 Dimethyl-1-butvne, 3, 3-n m 82. 15 0.667 Aldrich 24, 439-2 9 9 12 IDimethylamino-2-propyne, I-83. 13 0.772 Aldrich 14, 306-5 5 10 13 Dodecvne, 1-mW 166. 31 0.778 Aldrich 24,440-6 5g 11 46 Ethynvl-1-cyclohexanol, 1-wo (124. 18 0.967 Aldrich E5, 140-6 5 mL J2 47 Ethyny)-4-ftuorobenzene, 1-120. 13 1. 048 Aldrich 40, 433-0 500 mg 13 48 Eth n I-9-fluorenol 9-860. 206. 25 1. 000 GFS 143705 g 14 17 Ethvnylcyclohexene, 1-w !. f 106. 17 0.903 Aldrich 31, 657-1 5g 1 5 4 9 Ethvnvicyclopentanol, 1-= 110. 16 0.962 Aldrich 13,086-9 5 1 6 18 Ethynylestradiol 3-methvl ether 2. 9~. 310. 44 1.000 Aldrich 85, 587-1 5 g 1 7 19 EthvnylpYridine, 2-. 103. 12 0.940 GFS 143907 1 g 18 20 Ethynyltoluene, 4-116. 16 0.916 Aldrich 20, 650-4 5 19 22 Hexyne, 1-82. 15 0.715 Aldrich 24,442-2 25 mL 20 23 Hexvnenitrile, 5-3A. 93. 13 0. 889 Aldrich 27, 134-9 5 g 21 24 Methvl propargvl ether S 70. 09 0.830 Aldrich 17,719-9 10 g 22 25 Methv1-1-buten-3-vne, 2-3v9E7 66. 10 0.695 Aldrich M3, 280-1 5g 23 MethYl-3-butyn-2-ol, 2-wP ». ; 84. 12 0.868 Aldrich 12, 976-3 5 mL 24 26 Methel-N-propargvlbenzvlamine, N-> 159. 23 0.944 Aldrich M7,425-3 50 25 27 Nonadiyne, 1, 8- 120. 20 0.799 Aldrich 16, 130-6 10 q 26 28 Pentvne, 1-S-i 68. 12 0. 691 Aldrich 25, 656-0 5 9 27 29 Phenvl-1-butvne, 4-. 130. 19 0.926 GFS 184701 5 28 30 Phenvl-1-propvne, 3-. r 116. 16 0.934 Aldrich 37, 684-1 5g wEw. 29 31 Phenylacetylene 102.14 0.930 Aldrich 11, 770-6 25 mL 30 37, 53 Propiolaldehyde diethyl acetal, 59 ; 8 128. 17 0.894 Aldrich 30, 360-7 g 31 SIdP CODON 0.., 0 127. 90 AVERAGE 123. 85 Table 1. Amine building blocks used in library synthesis. beta-branched or greater mult=1 245.43 umol amine 25 e alpha-branched (mult = 2) 490.86 umol amine (50 eg) 2-hydroxypyridine (2-pyr) 1.0 49.09 umol 5 e in _ _ stock solutions 1. 1 49.09 umol (5 eq) in 3: 2 CH2CI2/DMF 2. 0 98.17 umol (10 eq) in _ _ Test m or uL uL salt Aldrich BB# # Chemical Name 2- e'd DIPEA MW d mult Catalo # Size 1 1 Allyfamine 57. 10 0.761 1 24. 107-5 50 mL 2 5 laminoacetaidehyde diethyl acetal 133. 19 0.916 1 A3, 720-0 25 mL 3 6 Aminoacetatdehyde dimethy ! acetat 105. 14 0.965 1 12, 196-7 25 mL 4 8 Aminoethyl) benzenesulfonamide, 4-(2-a | 200. 1.0001 1 27,524-7 a 5 9 Aminoethyl) morpholine, 4- (2- 130. 19 0.992 1 A5 500-4 g 6 78 Aminoeth I ridine 2-2-122. 17 1. 021 1 A5 530-6 i0 7 11 Aminoethyl) pyrrolidine, 1-(2-1 ww iw 114. 19 0.901 1 A5 535-7 g 8 13 Aminoindan, (R)-(-)-1-E 133. 19 1.038 2 44, 534-7 9 8 13 Aminoindan. (RH-)-1-133. 19 1.038 2 44, 535-5-La 9 14 Aminoindan, (S)- (+)-1-44.535-51j 1 0 t15 Aminometh I-15-crown-5, 2-. _, U 4t, ; : 249. 31 1. 134 1 38 841-6 i 11 17 Aminomethyl) cyclopropane, 71. 12 0. 820 1 35, 952-1 1 mL 12 10'Aminomethyl) pyridine, 2-(S Q 108.14 1.062 1 A6, 540-9 5 -) 3 19. 79Aminomethy !) pyridine, 3- (108. 14 1.062 1 A6, 540-9 9 14 20 Aminomethyl) pyridine, 4-(108. 14 1.065 1 A6, 560-3 25 g -----x w B 15 23 Aminopropyl) imidazole, 1- (3- 125. 18 1. 049 1 27, 226-4 g 16 24 Aminopropyltrimethoxysilane, 3-29 1. 027 1 28, 177-8 5 mL 17 28 Benzylamine 107. 16 0. 981 1 18,5 9 1 8 30 Bom lamine R-+-'..'c'2.. Q_ 5_2 : = 153. 27 1. 000 2 35, 993-9 500 mg 1931Butytamine73. 140. 740123. 991-7g 20 80 Butylamine, (R)-(-)-sec-g ~. 73. 14 0. 731 2 _ 29,1 9 21 81 Butylamine, (S)-(+)-sec-W §. | S 73. 14 0. 731 2 = 29, 665-1 <1 'a ; ; .,-,. 22 32 Cvclobutvlamine w S 71. 12 0. 833 2 _ 25, 5 i8-5 1 2334Cydohexytamine99. 180. 867224, 064-8 5mL 2482Cyc) ohexy) ethytamine. (R)- (-)-1-127. 330. 856233. 650-5 5g 2583Cyctohexytethytamine. (S)- (+)-1-127. 330. 856233,651-35g 26 35 clo ent lamine 26 35 clopentylamine 85. 15 0. 863 2 Cl 1, 500-2 5 g 27 36 CyclopropYiamine m | | 57. 10 0. 824 2 _ 12, 550-4 10 28 38 Diethoxvmethylsilyl) propelamine, 3-($ = w 191. 35 0. 916 1 _ 37,189-050 mL , N S ; _ _. iv'C'TS^'Y 2939Dimethoxyphenethytamine. 3. 4-181. 241. 0741'"D13, 620-4'25g 30 41 Dimethvlaminopropvlamine, 3-0 W 102. 18 0. 812 1 24, 005-2 50 mL 31 43 Eth famine 2. OM in Tl-I ,. : ; ;,", 500. 00 1. 000 1 39, 507-2 100 mL 32 49'Fluoobenz lamine, 3-p'28., : 0 c-_ 125. 15 1. 097 1 33 46 Fluorophenethylamine, 4-139. 17 1. 061 1 36, 182 10 g 3448Geranytamine153. 270. 829141, 264-3 5 35 50 Iso inocam he amine, U ; 2 ; 8 153. 27 0. 909 2 39 165-4 5 (1R. 2R. 3R. 5SH-)- Table 1. Amine building blocks used in library synthesis. Test mg or uL uL salt Aldrich BB# # Chemical Name 2- req'd DIPEA MW d mult Catato # Size 36 51 lsopinocampheylamine, 153. 27 0. 909 2-39, 166-2 5gi 1S2S3S5R-+- 37 52 IsopropyWamine m 59. 11 0.694 2 10, 906-1 25 mL 38 53 Methoxvbenzvlamine, 2-B 137. 18 1. 051 1 15 988-3 5 39 54 MethoxVbenzelamine 4-m 137. 18 1.050 1 MI, 110--3- g 40 55 Methoxvethylamine, 2-B 75. 11 0.864 1 24, 106-7 50 mL 41 56 Metho heneth amine 2-5. 151. 21 1.033 1 37,359-1 5g 42 57 Metho heneth amine 3-1. 5. 151. 2i 1. 038 1 27, 022-9 g . E ! | | m m _ _ _ 43 58 Metho heneth lamine 4-0 5 9 151. 21 1.033 1 18, 730-5 5 g 44 59 Metho o lamine 3-,. 89. 14 0.874 1 M2, 500-7 25 mL 45 60 Meth amine 2. OM in THF)..... 500. 00 1. 000 1 = 39, 505-6 100 mL 46 85 Methylbenalamine, (R)- (+)-a- 121. 18 0. 940 2 42, 193-6 5 mL 47 61 Myrtanylamine, (-) wis-ç w4six X 153. 27 0.915 1 18, 080-7 9 4886Napthy)) ethytamine. (S)- (-)-1- (l-171. 25 1.060 227. 745-0 5 g 49 62 Na th lenemeth amine 1-6 U 157. 22 1.073 1 12, 703-5 5 50 63 Nitrobenzylamine hydrochloride, 3-188. 62 1. 000 1 1 19,166-3 5 51 65 Octylamine w w S 129. 25 0. 782 1 = 0-580-2 a 52 66 Pheneth lamine". :, y : 8 : 121. 18 0. 965 1 40, 726-7 100 mL 53 69 Piperonybmine151. 17 1. 214 1P4, 950-39 54 70 PropargYl amine ffi w w 55. 08 0.803 1 P5, 090-0 5 55 71 Tetrahydrofurfurylamine, (R)-(-)- » 101. 15 0. 980 1 _ 41, 293-7 101.15 0.980 1 41,293-7 1 56 72 Tetrahydrofurfurylamine, W)-(+)-> 101. 15 0. 980 1 41, 294-5 1 . . _..... ;.,. _ _, ... _. . : 57 73 ITetramethyl-1, 3-propanediamine, 130. 24 0. 818 1 22, 741-2 25 N, N, N, 2, 2- 5874Thiopheneethy) amine, 2-127. 21 1.087 1 42,327-0 5g 59 87 Trifluoromethoxy) benzylamine, 4-(mw 191. 15 1.252 1 34, 1g 60 88 Trifluoromethyl) benzylamine, 3-(R m w 175. 16 1. 222 1 _ 26 349-4 5 61 76 Tryptamine160. 22 1. 000 119, 374-7 10 g 62 77 Veratrylamine 167. 21 1. 109 1 V130-9 5g CODON AVERAGE 139. 95 Table J. Acid building blocks used in library synthesis. = = g rary y carboxylic acid 871.77 umol (2 x 50 eq) Test mg or uL Aldrich SB# # Chemical Name acid NMI d Catalo # Size 1 1 Acetic acid 60. 05 1.049 33, 882-6 25 mL 2 85 Acetoxyacetic acid E = 118. 09 1.000 30, 234-1 5 g 3 5 Anisic acid, m-. 6 152. 15 1.000 1 1, 771-4 25 g 4 86 Benzofurancarboxptic acid, 2-g 162. 14 1.000 30, 727-0 5 5 8 Benzoic acid 122. 12 1.000 24, 238-1 25 g 6 9 Bu noic acid 2-84. 07 1. 000 30, 366-6 5 g 7 11 Chloropropionic acid, 3-B W 108. 52 1.000 13, 269-1 5 g W E 8 8 7 Cinnoline-4-carboyxlic acid E s 174. 16 1.000 C8, 2 15-9 1 g 9 12 Crotonic acid 86. 09 1.000 23, 956-9 50 1014Cyanobenzoicacid. 3-147. 13 1.000 15, 716-3 1 1 1 1 5 Cyanobenzoic acid, 4-147. 13 1.000 C8, 980-3 59 12 16 Cyclohexanecarboxylic acid 128. 17 1.033 10, 183-4 5 9 1317Cyciopentanecarboxytic acid114. 14 1.053 C11, 200-3 5 14 18 Cyclopentylacetic acid : 128. 17 1. 022 12, 549-0 5 g « ' ^ucs 15 19 Cyclopropanecarboxylic acid 86. 09 1. 088 Ci1, 660-2 25 1 6 2 0 Dihydro-2, 2-dimethyl-4-oxo-2H-<8t 170. 16 1.000 19, 572-3 5a pyran-6-carboxylic acid, 3,4- S., 1 7 21 Dihydro-2-methylbenzoic acid, 1, 4- 138. 17 1.000 30,035-7 5 g 1889Dimethytacryiic acid. 3. 3-100. 12 1. 000 013, 860-65g 19 25 Ferroceneacetic acid 244. 08 1.000 33, 504-5500 mg 20 27 Furanacrylic acid, trans-3-138. 12 1.000 33, 638-6 21 28 Furoic acid, 2-2 11 2. 0E 1.000 F2, 050-5 5 2 2 29 Furoic acid, 3->. 7 112 08 1. 000 16, 339-2 5 9 23 31 Hexadienoic acid, 2,4- (Sorbic acid) i. : N 112. 13 2 4 3 2 Isobutyric acid 8p » o. 9 88. 11 0. 950 24, 016-8 50 mL 2 5 3 3 Isonicotinic acid @ 0 ; 7-B. 3 123. 11 1. 000 i-1, 750-8 5 g 2 6 34 Isovaleric acid 102. 13 0. 937 12, 954-2 5 mL 2 7 35 Levulinic acid ,.,. 116. 12 1. 134 L200-9 50 9 2 8 36 Linotenic acid 265* : 6 278. 44 0. 914 85, 601-0 5 9 gS 29 37'Menthoxyacetic acid, 30 38 Menthoxyacetic acid, (-)-. g.. 2 214. 31 1. 020 M300-0 10 9 3 1 39 Methacrytic acid m 86. 09 1. 015 39, 537-4 5 mL 3 2 91 Methoxy-1-indanone-3-acetic acid, 5-92Y2. C 220. 23 1. 000 22, 528-2 1 g 33 40 Methoxyacetic acid i. X6=M ? 90. 08 1. 174 19, 455-7 50 9 3 4 41 Methoxyphenylacetic acid, (R)-(-)-a-gm 166. 18 1. 000 24, 896-7 1 9 35 43 Methox hen lacetic acid, 2- ; 4,.'9 166. 18 1. 000 18 065-3 5 36 44 Methoxyphenylacetic acid, 3-166. 18 1. 000 M1, 900-7 5 g 37 45 Methoxyphenvlacetic acid, 4-3til4Mm9 166. 18 1. 000 M1, 920-1 5 g 3 8 46 Methyl (1S, 2R)-(+)-cis-1,2,3,6- <184. 191. 00036. 728-11 g _ tetrahydrophthalate, 1-S 39 4 7 Methyl glutarate, mono--3 5 40 48 Methyl phthalate, mono-180. 16 1. 000 31, 764-0 25 41 49 Methyl terephthalate, mono-180. 16 1. 000 32, 838-3 5 42 92 Methyl-2-pyrrolecarboxylic acid, 1-i 9 125. 13 1. 000 15, 314-1 5 ql Table J. Acid building blots used in library synthesis. Test Aldrich BB## Chemical Name acid mw d Cataloq Size 43 53 Meth enedio hen lacetic acid, 3 4- 1'180. 16 1. 000 32 967-3 5 44 54 Meth indole-2-carbo lic acid 1-. 2. 175. 19 1.000 13, 415-5 g 45 55 Nicotinic acid 123. 11 1. 000 5 g 4660 ofbomaneaceticacid, 2-154. 21J. 065 12. 726-4 5 47 62 Oxo-4-hen-3-oxazolidineacetic acid S-+-2- 8 221. 21 1.000 39, 134-4 1 48 63 Oxotricyclo [2. 2.1.0 (2, 6) 1heptane-7-carboxylic acid,, 1Ba2 152. 15 1. 000 32, 285-7 1 anti-3- 49 64 Phenylacetic acid S 136. 15 1. 081 P1, 662-1 9 5 0 6 7 Picolinic acid _ W X 123. 1 1 1.000 P4, 280-0 5 51 68 Proplonic acid 74. 08 0.993 40, 290-7 100 mL 5 2 6 9 Pyrazinecarboxviic acid, 2-v iN 124. 10 1. 000 P5, 610-0g 53 94 Pyridyl) acrylic acid, trans-3- (3- 149. 15 1.000 P6, 620-3 5 g 5 4t75 Tetrahydro-2-furoic acid116. 12 1.209 34, 151-7 5 5 5 : t76 Tetrahydro-3-furoic acid B'-. 3S 1 16. 12 1.214 33, 995-4 5 56 95 Thienyl) acrylic acid, 3- (2- 154. 19 1.000 13,058-3 9 5 7 8 0 Thiophenecarboxvlic acid, 2-, % i, XJ 128. 15 1. 000 T3, 260-3 g 5 8 81 Thiophenecarboxylic acid, 3-il1iq~$ 128. 15 1. 000 24, 776-6 5 9 59 96 Trifluoro-m-toluic acid, a, a, a- 190. 12 1. 000 18,834-4 9 60 97 Trifluoro-o-toluic acid,. a, a, a- 190. 12 1.000 19, 688-6 5 61 98 Trifluoro-p-toluic acid, a, a, a-X 190. 12 1. 000 19, 689-4 5 g 1; 657 190.12 1.000 19,689-4 5 62 84 Vinylacetic acid 86. 09 1.013 13,471-6 g 63 SiQPMj AVERAGE123. 32 143. 07 Table K. i Binarv encoding scheme for diazoketone ! I I I-I I I I ! Note that taqs 5B, 6B, and 7B were not i I I I ' Buiiding Spacer Epoxyo ! Nitrone Atkyne ! Block# 1C I 2C 1B 1D 28 ! 2D 3B 3D I 4B ! 4D ! 5D 1 0 1 0 0 0 0 I 0 I 0'x. 20 0 0 0 0 0".. 3 o g 3'1 0 I 0 0 k'1 ,. 1 _5 f. |_i To I o MiW _o S 0 0 0 1 6 0 I 0 '1.''t 0 7 0 0 0 0 ili, 0 . rl 0 I 0 ; :'t, 1 0 0 :.'It ''1 0 i a 1 17 itKi l> ! o i o uLI 1 2 0 t'1. . ' : a 0 112 MIX= a 1 3 =law 0 0 2 2 _ o S. l i o 15 mom 0 2 4 9 X D 30WiS o I o I o 17 o o o 2 6 8 j O Wi 000 2 0, r. 0 : 'l 0 0 20 o, Wl W4 o I o wiz 2 2 w wu 2 3. b'' 0 ''tT v 1 . it, , __ i 2 4,, 1 , le 0 I 0 I 0 2 5 . !'k... ,''1 : I 0' : ti" 2 6 - 1' 0''1', e. 0 2 7'y. , .. : 0 1'=, E1 28 1 : 1 :. a_. 7 0 0 2 s i . : j-: o i 30 0 31't v1. i I I I I i I i I i I I i i I I i I I I I i I i i i i AmineAcid 6D I 7D I 8B 8D I 9B 9D 10B 10D I 7A I 8A (9A I 10A o I o i o ! o o r ° ! ° ! ° I ° o iMXß oIoIoIoIo . ololo o-Io s o! o! o o o o! o t o asS o o o I o I o 1. o o I o 0 o i. o o I o I o 0 0 o I o I o o o o i o | J =o i o S | w 0 00 0 0 1 o o o o 0 Im-o! o ! o o o I o.. 1 , i o o I o, y, ; oi o : a, i"., = : o I o Io o I o 4o I o I o o o t oM o o I o i o o I o I o w w R o o I o i @Si Oo o 0 0 o o 0 I 0 a'_. 0 0 0 0 v 1 0 I 0 o ! o iS ° W ° ° I ° Zi o w o uF :. 1 r s li. YfN. G 0 0 0 0 0 0 SS 0 0 0 0 J3M00 O M O 0 0 0 o I o i w o o ! o w o o M ! o t o o M o o o b o o l o ii o o iX o o o l o o X o ! o 1-S_ o° I ° ! ° iSiS ........ . ,... o R o I o-o o wm o I o O j O j O I O O jM, JMj O W O I O 0.... 1 : : 0 L 1.. 0 0 0 1. o imi o mais o o o iwlli o 0M 0 0 ? ? 0 'fa ! ! o o ! o go o M I 0 ' '1 ; 0 I 0 I''p F 0. p p' :' ° ; ; I o gHie ° i o o _ o o . n rw u. , z i u ! 0 0 o o 0 0 0 0 0 , y 0 0 0 0'1 v. . 0.'1..
Appendix B: Isoquinuclidine Based Synthesis Experimental Section General Methods : All reactions were performed on Tentagel polystyrene resin purchased from Rapp Polymere, Gennany. In addition to standard polyethylene glycol spacers, the resin was charged with a photocleavable linker element. Cleavage of compounds from solid phase at any step of synthesis was carried out by placing resin in a minimal amount of acetonitrile followed by exposure to UV (300 nm) for approximately 1 hour. All reactions were carried out at room temperature unless otherwise noted.
Isonicotinamide. I Tentagel resin (0.24 mmol/g) was placed in a 10 mL reaction barrel and allowed to swell in dry CH2CI'. Isonicotinoyl chloride hydrochloride was added (213.61 mg, 5 eq) and the resulting suspension mixed well. Freshly distilled diisopropylethylamine was slowly added (0. 641 mL, 15 eq) resulting in dissolution of any remaining insoluble acid chloride. The reaction was shaken and allowed to proceed for 15 min., after which the resin was drained of reactants and washed well with CH5CI2, THF and iPrOH 3 times. The resin was given a final wash with trimethylorthoformate (TMOF) followed by anhydrous THF, and dried under a nitrogen stream.'H NMR: 6 8.77 (dd, J = 4.46,1.68 Hz, 2 H), 7.65 (dd, J = 4.42,1.72,2H), 6.08 (d, J = 107 Hz, 2H) ; NMR : 5 167.22,150.73,140.43,121. 07; IR (NaCI plate): 3327.6, 3059.4,1682.1,1622. 3cm-l.
1 g resin (approx. 0.24 mmol ! g) was swelled with dry CHCL in a 10 mL reaction barrel.
Allyltributyltin was added (1.86 mL. 25 eq) and the resulting solution shaken well and cooled to 0°C. Teoc-CI (trimethylsilylethoxycarbonylc chloride) was then added (1.08 mL, 25 eq), the reaction barre) vented, and shaken for 6 hours. warming to room temperature after the first hour.
The reaction vessel was then drained and the resin washed with alternating solutions of anhydrous hexane and CH, Cl,. THF, DMF, MeCN, and iPrOH (3x). The final wash of TMOF followed by THF dried the resin, which was stored under N2. The product generated in this step is vulnerable to Ut-induced photorearrangement ; the data shown below pertain to desired product only.'H NMR: 5 6.90,6.78, (d, J = 15.5 Hz), 6.22 (m), 5. 95-5. 55 (m), 5.09,5.01,4.95 (m), 4.45.4.40 (ni), 2. 35 (dm), 1.05 (m), 0.05. HPLC ret (reverse phase): 2. 488 min. MS: M+ 309.
I g resin (approx. 0.24 mmol/g) was placed dry into a 20 mL screw top glass vial.
Anhydrous toluene (8 mL) was then added, and the solution shaken to disperse the resin uniformly. 3 eq maleic anhydride (70 mg) were dissolved in a minimal amount of anhydrous acetonitrile. and added to the resin. The vial threads were sealed with teflon tape. the reaction heated to SO"C for 12 hr. and the vessel shaken well every 3 hours. The resin was filtered into a fritted reaction vessel and the glass vial washed with CH'CIn to remove any adherent beads. The resin was washed 3x with CH2Cl2, THF, DMF.McCN, and iPrOH. TMOF and THF solutions were used to dry the resin, which was stored under N,. H NMR : (j 7.15,7.05 (d), 6. 31. 5.79 (m). 5.49. 5.37 (t). 5.17-5.05 (m). 4.26 (m). 3.91. 3.31 (dd). 3.21. 3.11. 2.59. 2.49.1.86 (m), 1.69, 1. 39, 1.05 (m). 0.07. HPLC ret. (reverse phase): 2.224 min.MS: M- = 407.
1 g resin (approx. 0.24 mmolig) was placed dry illtO a 20 mL screw top glass vial.
Anhydrous totuene (8 mL) was then added, and the solution shaken to disperse the resin uniformly. 5 eq benzylamine (0.131 mL) was then added to the resin. The vial threads were sealed with teflon tape, the reaction heated to 80°C for 12 hr., and the vessel shaken well every 3 hours. The resin was filtered into a fritted reaction vessel and the glass vial washed with CH2CI2 to remove anv adherent beads. The resin was washed W with CH=Cl_. THF. DNIF MeCNz and iPrOH. TMOF and THF solutions were used to dry the resin, which was stored under C'2. HPLC ret (reverse phase): 2.545 min.MS: M@ + Na = 518.
1 g resin (approx. 0.24 mmoli'g) was swelled with CH2Cl2 in a 10 mL reaction vessel. The resin was drained and CH2Cl2 sufficient to cover the resin added. Approximately 2 mL TFA was added. the reaction vessel shaken. and vented. Shaking was continued for 10 min, after which the solution was drained, and the TFA treatment repeated for 15 min. The resin was drained, fresh CH, Cl-, added. and approximately I mL DIPEA (diisopropylethvlamine) added to neutralize any residual TFA. The resin was washed with CH2CI2 THFs DMF. MeCN, and iPrOH (3 times). TMOF and THF solutions were used to dry the resin, which was stored under N.'HNMR : 6 7.35 (m), 7.13 (d). 6.80 (d), 6.05 (m).5.75 (m), 5.03 (m), 4.7-4.4 (nif.
4.45 (d) * 3.6 (d). 3. 25 (dd). 3. 11 (td), 3.08 (dd), 2.80 (m), 1.90 (m), 1.79 (dm), 1. 37 (m). HPLC ret (reverse phase) : 1.808 min. MS: M+ = 352. resin (approx. 0.24 mmol ! g) was swelled with 7 mL CH2CI2 in a 10 mL reaction vessel. 25 eq. p-iodobenzylnitrone (1.82 g) was added and the vessel shaken to dissolve the solid material. 25 eq. PyBroP (2. 79 g) was then added, and the mixture shaken again. Upon dissolution of all solid reacJents. the vessel was cooled to-20°C for 6 hr. Subsequent washing was performed 3x with CH2CI.. THF. DMF, MeCN, and iPrOH. TMOF and THF solutions were used to dry the resin, which was then stored under N2.'H NMR: 6 7.91 (d), 7.54 (d), 7. 30 (m), 7.02 (t), 6.53 (dd). 5. 75 (dd), 5. 09 (t), 4.60 (t), 4.07 (t), 3.81. 3.76 (m), 3. 65 (d), 3. 4-3. 3 (m), 3. 14, 1.95 (m), 1.78. HPLC ret. (reverse phase): 2.182 min. MS: M = 639.
Methyl (4R)-2- (t-butyl)-3- (1, 3)-oxazolidine-4-carboxylate. (Dieter Seebach and Johannes D. Aebi, Tetrahedr-on Lettens, Vol. 25, No. 24, pp. 2545-2548) To a 100 mL round bottomed flask equipped with a stir bar and Dean Stark trap and purged with N2 was added D- serine methyl ester hydrochloride (6.2 g, 40 mmol 1 eq.) followed by n-pentane (50 mL Pivaldehvde (8.8 mL. 80 mmol. 2 eq) was added to the mixture followed by triethylamine (6.1 mL, 44 mmol. 1.1 eq). The mixture was heated to reflux for 16 h with removal of water. The mixture was cooled to 23"C\ tittered, washed with ether (50 mL) and concentrated to an oil which was used without further purification in the next step.
Methyl (2S, 4R)-2- (t-butyl)-3-chlorocarbonyl- (1, 3)-oxazolidine-4-carboxylate.
Jaques Streith, Arnaud Boiron, Thierry Sifferlen. Christiane Strehler. Theophile Tschamber, Tetrahedron Letters, Vol. 35, No. 23, pp. 3927-3930). To a stirred solution of oxazolidine (7.02 g, 37.5 mmol. 1 eq) in CH, Cl-, (141 ml) at-15°C was added a 1.93 M solution of phosgene in toluene (29 ml, 56 mmol, 1.5 eq) dropwise. Triethyl amine (6.7 mL. 48 mmol, 1.3 eq) was added dropwise and the reaction was allowed to warm to 23°C. After 2 h N2 was bubbled through the reaction mixture in order to remove excess phosgene. The solvents were evaporated and the residue was slurried with AcOEt/cyclohexane (3 : 7) and the mixture was filtered. The filtrate was concentrated and purified by flash chromatography (rf=). Recrystallization from pentane yielded Methyl (2S, 4R)-2- (t-butyl)-3-chlorocarbonyl- (1, 3)-oxazolidine-4-carboxylate (7.9 g, 85 %), m. p. = 78"C. NMR (400 MHz, CDCl3): 5 5. 17 (s, 1H, C2-H), 4.89 (dd, 1H. J = 7. 9,4.8, C4-H), 4.39 (dd, lHJ=8845CS~H) 422 (ddw lH, J=8. 8,8.1, C5-Ha), 3.81 (s, 3H, CO2CH3), 0.97 (s, 9H, C(CH3)3).
Methyl (4S)-2- (t-butyl)-3- (1, 3)-oxazolidine-4-carboxylate. (Dieter Seebach and Johannes D. Aebi, Tetrahedron Letters. Vol. 25, No. 24, pp. 2545-2548). To a 100 mL round- bottomed flask equipped with stir bar and Dean Stark trap and purged with N, was added L- serine methyl ester hydrochloride (6. 2 g, 40 mmol, I eq) followed by n-pentane (50 mL).
Privaldehyde (8.8 mL, 80 mmol. 2 eq) was added to the mixture followed by triethylamine (6.1 mL, 44 mmol, 1.1 eq). The mixture was heated to reflux for 16 h with removal of water. The mixture was cooled to 23°C, filtered, washed with ether (50 mL) and concentrated to an oil which was used without further purification in the next step.
Methyl (2R, 4S)-2- (t-butyl)-3-chlorocarbonyl- (1, 3)-oxazolidine-4-carboxylate.
(Jacques Streith, Amaud Boiron, Thierry Sifferlen, Christiane Strehler, Theophile Tschamber Tetrahedrorz Letters, Vol. 35, No. 23, pp. 3927-3930). To a stirred solution of oxazolidine (7. 02 g, 37. 5 mmol, I eq) in CHCL (141 ml) at-15°C was added a 1.93 M solution of phosgene in toluene (29 mL. 56 mmol, 1.5 eq) dropwise. Triethyl amine (6.7 mL, 48 mmol, 1.3 eq) was added dropwise and the reaction was allowed to warm to 230C. After 2 h N2 was bubbled through the reaction mixture in order to remove excess phosgene. The solvents were evaporated and the residue was slurried with AcOEt/cyclohexane (3 : 7) and the mixture was filtered. The filtrate was concentrated and purified by flash chromatography (rf=). Recrystallization from pentane yielded Methyl (2R, 4S)-2- (t-butyl)-3-chlorocarbonyl- (1, 3)-oxazolidine-4-carboxylate (7.9 g, 85%), m. p. = 78°C. IH NMR (400 MHz, CDC13) : d 5.17 (s, 1H, C2-H), 4.89 (dd, 1H, = 7.9,4.8, C4-H), 4.39 (dd, 1H, J = 8. 8,4.5, C5-Hß), 4.22 (dd, 1H, J = 8.8,8.1, C5-Ha), 3.81 3 H, CO2CHt). 0.97 (s, 9H, C (CH1) 3).
Isonicotinamide. 3-Amino-3-(2'-nitrophenyl)-2, 2-dimethylproponylcarboxamide- Tentagel resin (200 mg, 0.27 meq/g, 54 umol, 1 eq) was placed in a PD-10 column.
Isonicotinoylchloride hydrochloride (48 mg, 270 umol, 5 eq), distilled CH2C12 (2.4 mL), and DIPEA (141 il, 810 Amol, 15 eq) were added in sequence. After 1 h the resin was washed 3 x DMF. 3 x IPA, 3 x DMF, 3 x CH, CI2, 3 x DMF, 3 x CH1CN, 3 x THF, 3 x CH : to yield isonictinoyl-3-Amino-3- (2'-nitrophenyl)-2, 2-dimethylproponylcarboxamide-Tentagel resin which was negative to Kaiser ninhydrin test. Photolysis of the resin yielded the crude isonicotinamide, as a yellow oil. IR (NaCI) 3175, 1684,1554,1506,1412.612 cm-1. ¹H NMR (500 MHz, CD CN) : 6 8.70 (br m. 2H), 7.65 (dd, J = 4.4,1.7,2H). EI-MS (Direct) m/z (rel int): 122 (M), 100). 106 (33).
Methyl (2R, 2'R, 4'S)-3'- [2-allyl-4-carboxamide-1, 2-dihydro-1-pyridinyl]-carbonyl- 2'-t-butyl-(1,3)-oxazoline-4-carboxylate. Isonicotinamide resin (80 mg, 0.27 meq/'g. 21. 6 umol, 1 eq) was placed in a new PD-10 column along with metny, (2R, 4S)-2-(t-butyl)-3-chlorocarbonyl-(1,3)-oxazolidine-4-carboxy late (54 mg, 216 tmol, 10 eq), NaI (65 mg, 432 µmol. 20 eq) and toluene (800 p1). The mixture was agitated by 360 rotation for 5 days during which time the resin changed colors from tan to burgundy, the resin was filtered and washed with toluene 10 x 1 mL, resuspended in toluene (900 µL), cooled to 0°C and treated with allyltributyltin (860 111, 2.8 mmol, 130 eq). The mixture was agitated by 360 rotation for 1 day. The resin washed with hexanes 50 x 1 mL, CH) Cl-, 50 x 1 mL Methyl (2S, 2'S, 4'R)-3'- [2-allyl-4-carboxamide-1, 2-dihvdro-1-pyridinyl]-carbonyl- 2'-t-butyl- (1, 3)-oxazoline-4-carboxytate. Isonicotinamide resin (80 mg, 0. 27 meq/g, 21.6 umol, 1 eq) was placed in a new PD-10 column along with methyl (2S.4R)-2-(t-butyl)-3- chlorocarbonyl- (1, 3)-oxazoline-4-carboxylate (54 mg, 216 µmol, 10 eq), NaI (65 mg, 432 ummol, 20 eq) and toluene (800 ul). The mixture was agitated by 360 rotation for 5 days during which time the resin changed colors from tan to burgundy. The resin was filtered and washed with toluene 10 x 1 mL, resuspended in toluene (900 uL), cooled to 0°C and treated with allyltributyltin (860, ul, 2.8 mmol, 130 eq). The mixture was agitated by 360 rotation for 1 day.
The resin was washed with hexanes 50 x 1 mL, CH2CI2 50 x lmL.
Solution Phase Studies Methyl (2R, 2'R, 4'S)-3'-12-allvl-4-butylcarboxamide-1 ? 2-dihvdro-1-pyridinyl]- carbonyl-2'-t-butyl- (1, 3)-oxazoline-4-carboxylate. To a flame dried 5 mL round-bottomed flask equipped with stir bar and purged with N2 was added N-butyl isonicotinamde (50 mg, 280 , mol, 1 eq), Methyl (2R, 4S)-2-(t-butyl) 3-chlorocarbonyl-(1, 3)-oxazolidine-4-carboxylate (70 mg, 280 mol, 1 eq), NaI (84 mg, 560 mol, 2 eq) and toluene (1.2 mL). The flask was capped with a glass-stopper, sealed with parafilm, and stirred for 5 days. The flask was then fitted with a nitrogen inlet and cooled to 0°C.
Allyltributyltin (86 pilz 308 jmol, 1. 1 eq) was added and the flask was allowed to warm to 23°C with stirring overnite. The mixture was filtered, concentrated and purified by column chromatography (Si02, 10 % MeOH/CHC13) to afford 109 mg, 90% of Methyl (2R, 2'R, 4'S)-3'- [2-allyl-4-butylcarboxamide-1, 2-dihydro-1-pyridinyl] carbonyl-2'-t-butyl- (1, 3)-oxazoline-4- carboxylate. NMR (400 MHz, CDC13) : 6.99 (d, 1H, J = 7. 6, C5-H), 6.17 (d, 1H, J = 6.2, C6- H), 5.8 (m, 1H, Cl"-H), 5.72 (t, 1H, NH), 5.67 (dd, 1H, J = 7. 6, 1.7, C3-H), 5.43 (s, 1H, C2'-H), 5. 05 (m, 1H, C3"-HC), 5.01 (brs, 1H, C3"-H «), 4.74 (dd, 1H, J = 6.3, C4'-H), 4. 36 9d, 1H, J = 8.8, C5'-Ha), 4. 09 (d, 1H, J = 6.03, C5'-Hp), 3.79 (m, 1H, C2-H), 3. 75 (s, 3H, CO2CH3), 3.32 (m, 2H, NHCH2), 2.4-2.3 (m, 2H, Cl"-H), 1.4-1.2 (m, 4H, (CH2) 2), 0.97 (s, 9H, C2'-t-Bu), 0.96 (t, 3H, CH3).