KLEIDERNIGG OLIVER PAUL (AT)
KLEIDERNIGG OLIVER PAUL (AT)
O.P. KLIEDERNIGG, ET AL.: "Synthesis and application of a new isothiocyanate as a chiral agent for the indirect resolution of chiral amino alcohols and amines", JOURNAL OF CHROMATOGRAPHY A, vol. 729, no. 1-2, January 1996 (1996-01-01), AMSTERDAM, NL, pages 33 - 42, XP000604369
T. TOYO'OKA, ET AL.: "Development of optically active fluorescent Edman-type reagents", ANALYST, vol. 120, no. 2, February 1995 (1995-02-01), LETCHWORTH, GB, pages 385 - 390, XP000603667
| 1. | Compound of the general formula (I) wherein the groups R are the same or different but preferentially the same and are aliphatic dio groups or optionally substituted aromatic groups or that both groups R together with the C atoms marked with *form a carbocyclic or heterocyclic or anellated ring system (with 310, preferably 58 ring members) of the formula (II) (CR'R")n (H) wherein R' and R" are the same or different and are hydrogen atoms, aliphatic Cl10 groups, optionally substituted aromatic groups or optionally substituted hetero aromatic groups or anellated groups, and n is an integer from 116, preferably from 110 and RΪ is a group of the general formula (III): wherein t are aliphatic CMS groups or optionally substituted aromatic groups or optionally substituted hetero aromatic groups or aralkyl groups or Ri is a group of the general Formula IV wherein R5 is C1C10 aliphatic group or optionally substituted aralkyl groups selected from groups consisting of or Ri is a group of the general formula V wherein ; is an optionally substituted aromatic group an optionally substituted heteroaromatic group like e.g. the dansyl residue . |
| 2. | Compound according to claim 1, wherein the groups R are the same or different and are selected from the group consisting of R= wherein m is an integer from 16, wherein R' and R" in the group of formula (II) are the same or different, are hydrogen atoms, aliphatic Cl10 groups, optionally substituted aromatic groups or optionally substituted hetero aromatic groups or anellated groups and n is an integer from 16, and wherein Rt is selected from the group consisting of — C(CH3)3 (CH2)8— CH=CH2 wherein R3 ins an aliphatic CMO group or an optionally substituted methylaryl, or an optionally substituted benzyl group. |
| 3. | Compound according to claim 2 wherein R3 is a group selected from the formulae: CH3 } — H5 Compounds according to any of the claims 13, which are optically pure. |
| 4. | Compounds according to any one of claims 14, namely (1R,2R) and (l'S,2S) N[(2 isothiocyanato)cyclohexyl](3,5)dinitrobenzoyl amide. |
| 5. | Compounds according to any one of claims 14, namely (1R,2R) and (1S,2S) N[(2 isothiocyanato)cyclohexyl]pivalinyl amide. |
| 6. | Compounds according to any one of claims 14, namely (1R,2R) and (lS,2S)N[(2 isothiocyanato)cyclohexyl]6methoxy4quinolinylamide. |
| 7. | Compounds according to any one of claims 14, namely (1R,2R) and (1S,2S) N[(2 isothiocyanato)cyclohexyl]6methoxyNmethyl4chinolinylamide. |
| 8. | Compounds according to any one of claims 14, namely (1R.2R) and (1S,2S)N [(2isothiocyanato)cyclohexyl]undecenylamide. |
| 9. | Compounds according to any one of claims 14, namely (1R,2R) and (lS,2S)N[(2 isothiocyanato)cyclohexyl]6methoxyNallyl4chinolinylamide. |
| 10. | Compounds according to any one of claims 14, namely (1R,2R) and (lS,2S)l[3 trimethoxysilylpropyl]N[(cyclohexyl)2(3,5)dinitrober_zoyl amide]thiourea. |
| 11. | Compounds according to any one of claims 14, namely (1R2R) and (lS,2S)N[(2 isothiocyanato) 1 ,2diphenylethyl]6methoxy4chinolinylamide. |
| 12. | Compounds according to any one of claims 14, namely (1R,2R) and (lS,2S)N[(2 isothiocyanato)cyclohexyl]4nitrobenzyloxy amide. |
| 13. | Method for the preparation of a compound according to any one of claims 14, wherein a compound of the general formula (VI) which may be optically pure and wherein R and Ri are as defined in claims 14, is subjected to a ring opening process. |
| 14. | Method according to claim 14, wherein the ring opening process is a thermal ring opening process. |
| 15. | Method according to claims 14 or 15 using an aprotic or protic solvent for the ring opening process. |
| 16. | Use of a compound according to any one of claims 113 for the specific tagging of compounds which contain functional groups which react with isothiocyanates. |
| 17. | Use of a compound according to any one of claims 113 for the tagging and separation of chiral compounds. |
| 18. | Use according to claims 17 or 18, wherein the chiral compounds are selected from the group consisting of chiral thiols, chiral amines and mixtures thereof each of which may have one or more of their respective functionalities. |
| 19. | Use according to claim 19 wherein the chiral amines are selected from the group consisting of primary and secondary amines, amino alcohols and mixtures thereof. |
| 20. | Use according to claim 17 or 18 wherein the chiral compounds are selected from the group consisting of amino acids, peptides and proteins. |
| 21. | Use according to claim 21 wherein the chiral amines are selected from amino acids whereby the resulting chiral thiourea derivatives may be converted to the corresponding chiral thiohytantoins and/or the corresponding openchained products. |
| 22. | Use according to claim 19 wherein the chiral thiols are selected from thiol group containing acids whereby the resulting chiral dithiocarbamate derivatives may be converted to the corresponding chiral thiazolidines. |
| 23. | Use of a compound according to any one of claims 113 for the tagging of amino groups in peptides or proteins and subsequent selective cleavage of the Nterminal peptide bond resulting in the corresponding chiral thiohydantoins and/or openchained products of the tagged Nterminal amino acid moieties. |
| 24. | Use of compound according to any one of claims 113 as building block for the synthesis of chiral selectors or chiral stationary phases (CSPs) to be used for the liquid phase separations of chiral compounds. |
| 25. | Chiral selector type products obtainable by reacting of a compound according to any of claims 113 with a chiral or nonchiral anchoring group to be immobilized to silica or any other adsorbens. |
| 26. | Use of a compound according to any of claims 14, wherein at least one of the compounds of the general formula III, IV, V, or VI reacts in situ with compounds containing functional groups, which react with isothiocyanates. |
| 27. | Process for the production of diastereomeric derivatives from mixtures of chiral compounds which comprises reacting a mixture of chiral compounds with at least one compound according to any one of claims 113. |
| 28. | Process according to claim 28, wherein the mixture of chiral compounds is comprised of thiols, primary and secondary amines, amino alcohols, peptides and/or amino acids. |
| 29. | Process for the separation of the products obtainable according to any one of claims 28 or 29 which comprises subjecting the products to liquid phase separation systems. |
| 30. | Process according to claim 30, wherein the liquid phase separation systems are normal or reversed LC or any type of electrochromatography. |
| 31. | Process according to claim 30, wherein the separation system is an electrophoretic process. |
| 32. | Process for the specific detection of the products according to claims 113 and / or 17, optionally separated by a process according to any one of claims 3032, which comprises subjecting the products to an electrochemical detection UV/VIS detection fluorescence detection chemiluminescence detection conductivity detection. |
| 33. | Process according to to claim 33 wherein the detection method is specifically selected for the product to be detected. |
The present invention relates to new chiral isothiocyanates their preparation and their application as chiral derivatizing agents (CD A), which may also be termed „reactive chiral selector", for the indirect analytical and preparative liquid chromatographic resolution of chiral amino compounds such as amines, amino acids, amino alcohols, peptides and thiol compounds as diastereomeric derivatives. Similarly, such diastereomers may also be separated by capillary electrochromatographic and capillary electrophoretic methods. The new chiral isothiocyanates may also serve as building blocks for chiral selectors and chiral stationary phases (CSPs), to be used for the liquid phase resolution of chiral compounds.
In general terms, the new CD As comprise three parts: 1) The reactive part (isothiocyanate moiety) for the chemically selective derivatization of primary and secondary amino- compounds and thiols.
2) The chiral template realized by trans- (R,R)- or (S,S)-l,2-diaminocyclohexane (DACH), trans- (R.R)- or (S,S)- 1,2-diphenylethanediamine (DPEDA) or similar 1,2- trans diamines. Similar chiral templates may also be introduced by taking a meso (R,S)- or (S,R)- 1,2 - cis diamine and substituting it asymmetrically.
3) The detectoφhore for the selective and sensitive detection of above mentioned diastereomeric compounds at trace levels (pM-aM range). This feature is realized by the introduction of e.g. a strong fluorophore (e.g. methoxy-quinolinic acid) or a potent chromophore like 3,5-dinitro benzoic acid. The detectoφhoric group may also be considered as rationally designed substituent of the chiral selector to introduce specific intra- and intermoleCular interactions with substituents stemming from the analyte. Accordingly, the detectoφhoric group may also be considered as selectoφhoric group in the case of chiral selectors or CSPs derived from the new CD As. Other demands considered to be important for a useful CDA were the chemical and slereochemical stability, its enantiomeric purity (> 99.9 ee) and high diastereoselectivity.
The concept of using CDAs in stereoselective analysis of chiral amino and thiol compounds is well established and various CDAs with different functional groups are known [W. Lindner, C. Pettersson in Liquid Chromatography in Pharmaceutical Development, I. Wainer, Ed., Part 1, Aster, Springfield 1985]. Focusing only on isothiocyanate based CDAs the following examples should be mentioned:
In the category of carbohydrate based chiral isothiocyanates the most important representatives are 2,3,4,6-tetra-O- acetyl-β- D- glucopyranosyl isothiocyanate (GITC) [ T. Kinoshita et.al., J. Chromatogr., 210 (1981) 77.], 2,3,4-tri- O-acetyl-α- D-arabinopyranosyl isothiocyanate (AITC)
[ T. Kinoshita et.al., J. Chromatogr., 210 (1981) 77.] and 2,3,4,6-tetra-O- benzoyl-β- D- glucopyranosyl isothiocyanate (BGIT) [ M. Lobell et.al., J. Chromatogr., 633 (1993) 287.]. These CDAs were and are used quite often for the indirect resolution of chiral amino compounds [ J. Gal, J. Chromatogr., 307 (1984) 220.; j. Gal, J. Chromatogr., 331 (1985) 349.; J. Gal, J. Liquid Chromatogr., 9 (1986) 673.; M. Fujimaki et.al., J. Pharm. Sci., 79 (1990) 568.; I. S. Lurie, J. Chromatogr. A, 605 (1992) 269.] and thiols [ S. Ito et.al., J. Chromatogr., 626 (1992) 187.]. One disadvantage of these sugar based isothiocyanates and of the corresponding thioureas is their relative low chemical stability in basic aqueous media due to the easily hydrolizable ester functionalities, leading to a loss in diastereoselectivity and/or unclear mixture of reaction and decomposition compounds. This is, however, a major drawback in the field including bioanalysis, amino acid- and peptide- analysis etc.. Another disadvantage is the relatively low stereoselectivity, making optical trace analysis and applications, where high selectivity is a must, almost impossible. The enantiomeric forms of these CDAs are also not available due to the lack of the corresponding carbohydrate moieties. Last but not least the detectability - only depending on the thiocarbonyl absoφtion in UV - is very limited, because of absence of a potent chromophore (with the exception of BGIT). Therefore trace analysis in the pM-aM range may not be managed.
Another group of optically active and fluorescence active isothiocyanates are the benzoxadiazole derivatives namely 4-(3- isothiocyanato-2-pyrrolidin - 1-yl) -7-nitro-2,l,3- benzoxadiazole (NBD-PyNCS) [ T. Toyooka et.al., Analyst, 120 (1995) 385.] and 4-(3- isothiocyanato- pyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)- 2,1,3- benzoxadiazole (DBD-PyNCS) [ T. Toyooka et.al., Analyst, 120 (1995) 385.]. Like the carbohydrate based isothiocyanates latter CDAs react selectively with primary and secondary amines forming the corresponding diastereomeric thiourea derivatives. These diastereomers then can be separated into their optically isomeric forms, preferably by liquid chromatographic [ T. Toyooka et.al., Analyst, 120 (1995) 385.] or electrochromatographic [ I. S. Lurie, J. Chromatogr. A, 605 (1992) 269.] methods. However, CDAs with more then one stereogenic center preferably integrated in a conformational rigid ring system seem to be superior in terms of stereoselectivity of the resulting derivatives. Compared to the isothiocyanates based on proline and developed by Toyooka the new class of chiral CDAs fullfill this element of improvement.
Besides chiral isothiocyanates also other classes of CDAs and methods to be applied for the derivatization of amines have been reported in literature: N-(5-fluoro-2,4-dinitrophenyl)-L- alanine amide (Marfey's reagent) [ H. Bruckner et.al., J. Chromatogr., 555 (1991) 81.], (+) and (-)-l-(9-fluorenyl)-ethyl chloroformate ((+)- and (-)-FLEC) [ S. Einarsson et.al., Anal Chem.,
59 (1987) 1191.] and the achiral ortho-phthaldialdehyde (OP A) [ A. L. L. Duchateau et.al., J.
Chromatogr., 623 (1992) 237.] together with chiral thiols which provide the chiral information.
All these CDAs react more or less selectively with chiral primary and / or secondary amino functions forming the corresponding diastereomers. The only exception of the above mentioned
CDAs is the OPA reaction, which only can be applied to primary amines.
One inherent disadvantage of the approach in general and most of the chiral derivatizing agents
(CDAs) used to date may be their relatively low chemical and chiral stability in basic (pH>8.5) aqueous media. Furthermore the diastereoselectivity of the above cited CDAs is also not always satisfying.
A new class of chiral isothiocyanates has now been discovered which can function as particularly efficient chiral derivatizing agents.
1.) The object underlying the present invention is to provide chiral isothiocyanate type compounds and chiral derivatizing agents as defined in claims 1-5. Preferred compounds have the general formula
and are recovered from symmetric or unsymmetric but preferentially symmetric (R,R)-, (S,S)-, (S,R)- and (R,S)-l,2-diamines wherein
by cyclization of the above mentioned 1,2- diamines with CS 2 in accordance with Davies et al. [ S. G. Davies et.al., Tetrahedron, 49 (1993) 4419, S.G. Davies, European Patent Application (03.05.1991) EP 0 457 469 Al] forming the corresponding imidazolidine-2-thiones of the general formula
and subsequent selective monoacylation of the above mentioned imidazolidine thiones with acid chlorides. These cyclic thioureas undergo monoacylation suφrisingly followed by a thermal ring opening and proton migration to the desired isothiocyanates under neutral and acidic conditions, preferably in protic and aprotic media in a temperature range from 35 - 180° C depending on the choice of the solvents. The above mentioned ring opening of the cyclic thioureas was carried out in accordance to Ulrich et al. [Ulrich, H. et.al., J. Org. Chem. 43 (8) 1978] describing a thermal ring opening of macrocyclic ureas to isocyanates, but not thioureas to isothiocyanates, and, definitely excluding, however, the ring opening of 5- and 6- membered ring systems. Along this line Davies et al. [ S. G. Davies et.al., Tetrahedron, 49 (1993) 4419, S.G. Davies, European
Patent Application (03.05.1991) EP 0 457 469 Al] described the bisacylation of imidazolidine thiones and their application as chiral auxiliaries, however, also in these studies ring opening reaction has not been reported.
Furthermore, the present invention comprises the synthesis of subtypes of CDAs which can be ionized and their application to electrophoretic separation of the resulting diastereomeric reaction products with chiral amines and thiols.
2.) The tagging of primary and secondary chiral amines, amino alcohols, amino acids, thiol compounds and their combinations - even with the simultaneous presence of more than one of the above mentioned functionalities - with the new chiral isothiocyanates, mentioned under point 1.), forming the corresponding diastereomeric thioureas, Edman chemistry corresponding anilinothiazolinones (ATZ), thiohydantoines, dithiocarbamates as well as thiazolidines. However, this principal application of chiral isothiocyanates is in accordance with citations in literature related to amino alcohols [ M. Fujimaki et.al., J. Pharm. Sci., 79 (1990) 568.], amino acids [ M. Lobell et.al.,, J. Chromatogr., 633 (1993) 287.] and thiols [ S. Ito et.al., J. Chromatogr., 626 (1992) 187.].
3.) Taking advantage of the introduced deiectophore within tne new CDAs mentioned under point 1.) with respect to the following detection principles a) electrochemical detection b) UV-VIS detection c) fluorescence detection d) chemiluminescence detection
also in combination with all kinds of liquid phase separation systems.
4.) Application of the new chiral isothiocyanate type CDAs mentioned under point 1.) for the indirect resolution of chiral analytes as diastereomeric derivatives by all liquid phase separation systems.
5.) Application of the new chiral isothiocyanate type CDAs mentioned under point 1.) for the synthesis of chiral selectors or chiral stationary phases (CSPs) to be used for the resolution of chiral compounds by any liquid phase separation technique.
The invention will now be described in more detail with reference to the following examples:
Examples:
Example 1
Synthesis of (1R,2R)- and (1S.2S) N-[(2-isothiocyanato)-cyclohexyl]-(3,5)-dinitrobenzoyl amide ((1R,2R)-DNB-DACH-ITC and (1S,2S)-D B-DACH-ITC) DDITC:
a) Cyclization of trans 1,2 (R,R)- or (S,S)-diaminocy ohexane (DACH) with carbon disulfide , forming trans 4,5- Tetramethyleneimidazolidine -2- thione , was accomplished according to a method described by Davies et. al. [ S. G. Davies et.al., Tetrahedron, 49 (1993) 4419, S.G. Davies, European Patent Application (03.05.1991) EP 0 457 469 Al] . Spectroscopic data, yields and melting points were almost identical as described by Davies. b) For the monoacylation of the cyclic thiourea 6g (38.4 mM) of 4,5- tetramethyleneimidazolidine -2- thione were dissolved in 120 ml dichloroethane in presence of a catalytic amount of 4-(dimethylamino)-pyridine and added to a suspension of 7.97g (34.58 mM) 3,5 - dinitrobenzoylchloride in 30 ml dichloroethane at a temperature of 50°C over a period of 15 minutes. During the reaction all reactants dissolved and the solution was refluxed (90°C) for 2 n . A slightly yellowish crystalline solid (1 st crop) was filtered uncooled, washed and the mother liquor was evaporated to dryness. The residue was redissolved in 60 ml dichloroethane and refluxed for another 16 n to thermally dispropose the 5-membered ring product to the open isothiocyanate. f ,R.-enantiomer : C14H14N4O5S = 350.35, yellowish crystals; m.p.: >250°C; total yield of both fractions 6.9g (51.2%); 1 H- MR (ppm) in DMSO-d6 1.15-2.29 (8H, m, cyclohexyl), 3.90 (1H, m CH-NCS), 4.10 (1H, iό, CH-NH), 9.01 (1H, m, dinitrobenzoyl . 9.09 (1H, m dinitrobenzoyl) 9.30 (1H, d -NH); IR (KBr): 3254 (NH), 2953 (CH), 2055 (-
NCS, strong), 1650 (-CO-), 1540 (-NO2), 1343 cm" 1 (-NO2). [α]546=-133°.c=l in . acetonitrile, Elemental analysis calculated: C (48%), H (4.03%), N (15.99%); found: C (47.76%), H (3.99%), N (15.62%). (S.S , enantiomer : C14H14N4O5S = 350.35; all physicochemical properties except the specific rotation were identical to the (R,R)-enantiomer. [α]546= +135° c=l in acetonitrile; Elemental analysis calculated: C (48%), H (4.03%), N (15.99%); found: C (47.68%), H (4.07%), N (15.92%).
Example 2
Preparative scale derivatization of (R)- and (S)-Propranolol with (R,R)-DDITC:
200 mg (0.77 mM) optically pure (R)- and (S)-propranolol and 240 mg (0.70 mM) of
(R,R)-DDITC were dissolved in 7 ml acetonitrile and kept at 60°C for one hour. The reaction solution was evaporated under reduced pressure and the residue redissolved in 15 ml dichloromethane. The organic phase was acidified with aqueous 0.2 m HC1. This two phase system was stirred intensively in order to extract the excess propranolol into the aqueous phase. After separation of the phases the organic layer was washed with 15 ml water, dried over Na2SO4 and evaporated to dryness. The crude product was purified via flash chromatography (eluent toluene/acetone = 3/1).
_R.R.R.-diastereomer: C30H36N5O7S = 610.31; yellow crystals; mp. 168°C; yield 380 mg (91%); 1H-NMR (ppm) in CDCI3 1.02 (3H, d, -CH3), 1.24 (3H, d, -CH3), 1.31-1.55 (4H, m, cyclohexyl cβ), 1.69-2.39 (4H, m, cyclohexyl Cα), 3.55(2H, m, -CHfc-N), 3.71 (IH, s, -OH), 3.88 (IH, m, -CH-NH-CO-), 4.11 (2H, m, -O-CH2-), 4.30 (IH, m, -CH- OH), 4.82 (IH, m, -CH-NH-CS-), 5.50 (IH, m, N-CH-), 6.73 (IH, d, Aromat), 7.22-7.52 (4H, m, Aromat), 7.77 (IH, m, Aromat), 8.11 (IH, m, Aromat), 8.62 (IH, d, -NH), 8.95 (IH, m, DNB-Aromat), 9.05 (2H, m, DNB-Aromat); IR (KBr) 3255 (-NH), 3074 (Aromat), 2933 (-CH-), 1652 (-CO-), 1580 (-NH), 1539 (-NO2), 1344 cm "1 (-NO2); [α]546=-202°, c=1.03 in CH2CI2.
(S.R.R.-diastereomer: C30H36N5O7S = 610.31; yellow crystals; mp. 172°C; yield 370 mg (89%); 1H-NMR (ppm) in CDCI3 1.17-1.23 (6H, m, -CH(M_θ2), 1.32-1.59 (4H, m, cyclohexyl cβ), 1.72-2.40 (4H, m, cyclohexyl Cα), 3.55(2H, m, -CH2-N), 3.65 (IH, s, - OH), 3.73 (IH, m, -CH-NH-CO-), 4.16 (2H, m, -O-CH2-), 4.29 (IH, m, -CH-OH), 4.75 (IH, m, -CH-NH-CS-), 5.55 (IH, m, N-CH-), 6.77 (IH, d, Aromat), 7.30-7.40 (4H, m,
Aromat), 7.72 (IH, d, Aromat), 8 11 (IH, d, Aromal), 8.55 '(IH, ά, -NH)/S.94 (IH, m, DNB-Aromat), 8.97 (2H, m, DNB-Aromat), 9.25 (IH, d, -NH); IR (KBr) 3265 (-NH),
3055 (Aromat), 2927 (-CH-), 1650 (-CO-), 1579 (-NH), 1541 (-NO2), 1343 cm" 1 (- NO2); [α]546=-l l*5°, c=1.07 in CH2CI2.
Example 3
Synthesis of (1S,2S) N-[(2-isothiocyanato)-cyclohexyl]-pivalinyl amide ((S,S)-PDITC):
a) Cyclization of trans 1,2 diaminocyclohexane (DACH) with carbon disulfide , forming trans 4,5- Tetramethyleneimidazolidine -2- thione , was accomplished according to a method described by Davies et. al. [ S. G. Davies et.al., Tetrahedron, 49 (1993) 4419, S.G. Davies, European Patent Application (03.05.1991) EP 0 457 469 Al] . b) For the monoacylation of the cyclic thiourea 2g (12.8 mM) of 4,5- tetramethyleneimidazolidine -2- thione were dissolved in 50 ml dichloroethane in presence of a catalytic amount of 4-(dimethylamino)-pyridine and added to a suspension of 2.09 g
(17.33 mM) pivalinic acidchloride in 10 ml dichloroethane at a temperature of 50°C over a period of 15 minutes. During the reaction all reactants dissolved and the solution was refluxed (90°C) for 2 n . The reaction mixture was evaporated to dryness. The residue was redissolved in 60 ml dichloromethane and extracted twice with diluted aqueous HC1. The organic phase was washed with brine, dried over Na 2 SO 4 and evaporated to dryness. The crude product was purified via flash chromatography (eluent toluene/acetone = 9.5/0.5).
C12H20N2OS = 240.36, white fluffy solid; m.p.: 152°C; yield: 1.21 g (39.5%);1H-NMR (ppm) in CDC1 3 1.21 (9H, s, t-butyl) 1.25-2.18 (8H, m, cyclohexyl), 3.61 (IH, m CH- NCS), 3.87 (IH, m, CH-NH), 5.65 (IH, d, -NH-); IR (KBr): 3316 (NH), 2943 (CH), 2085
(-NCS, strong), 1633 (-CO-) cm "1 . [α]546=-123°.c=l in acetonitrile
Example 4
Synthesis of (1R,2R)- and (lS,2S)-N-[(2-isothiocyanato)-cyclohexyl]-6-methoxy-4- quinolinylamide ((1R.2R)- and (1S,2S)-CDITC):
a) Cyclization of trans 1,2 diaminocyclohexane (DACH) with carbon disulfide , forming trans 4,5- Tetramethyleneimidazolidine -2- thione , was accomplished according to a method described by Davies et. al. [ S. G. Davies et.al., Tetrahedron, 49 (1993) 4419, S.G. Davies, European Patent Application (03.05.1991) EP 0 457 469 Al] . b) Synthesis of 6-methoxy-4-quinolinic acid was accomplished according to Skraup et al. [Skraup Monatshefte Chem. 2 (1881) 592 ]. c) For the monoacylation of the cyclic thiourea 2g (12.8 mM) of 4,5- tetramethyleneimidazolidine -2- thione were dissolved in 50 ml dichloroethane in presence of a catalytic amount of 4-(dimethylamino)-pyridine and added to a suspension of 3g (11.7 mM) 6-methoxy-4-quinolinic acidchloride hydrochloride in 30 ml dichloroethane at a temperature of 50°C over a period of 15 minutes. During the reaction all reactants dissolved and the solution was refluxed (90°C) for 2 n . A slightly yellowish crystalline solid
(1 st crop of isothiocyanate) was filtered, washed and the mother liquor was evaporated to dryness (2nd crop, mixture of isothiocyanate, ringclosed product and 4,5- tetramethyleneimidazolidine -2- thione). 1st and 2nd crop were separately dissolved in 50 ml DCM and extracted with 50 ml 6.5 % NaHCO 3 solution. The organic phases were washed with brine, dried over Na 2 SO 4 and evaporated to dryness. The residue of the 2nd crop was redissolved in 60 ml dichloroethane and refluxed for another 20" to thermally dispropose (fig. 1) the 5-membered ring product to the open isothiocyanate. The crude fractions were recrystallized in toluene.
. R.-enantiomer: C18H18N3O2S = 341.42, yellowish crystals; m.p.: 190°C; total yield of both fractions 2.9g (65.2%); 1 H-NMR (ppm) in CDC1 3 : 1.25-2.25 (8H, m, cyclohexyl), 3.62 (IH, m CH-NCS), 3.95 (3H, s, -OCHj), 4.22 (IH, m, CH-NH), 6.39 (IH, d, -NH), 7.36-7.41 (2H, m, quinolinic H3 and H7), 7.51 (IH, d, quinolinic H5), 7.94 (IH, d, quinolinic H8), 8.70 (IH, d, quinolinic H2); IR (KBr): 3260 (NH), 2927 (CH), 2100 (-
NCS, strong), 1640 (-CO-) cm" 1 ; [α]546=-H2°.c=0.98 in DCM
(S.S . enantiomer: C18H18N3O2S = 341.42; all physicochemical properties except the specific rotation were identical to the (R,R)-enantiomer. [α]546= +115° c= 1.04 in DCM
Example 5
Synthesis of (lR,2R)-N-[(2-isothiocyanato)-cyclohexyl]-6-methoxy-N-methyl -4- chinolinylamide hydrochloride ((lR,2R)-N-methyl-CDITC):
500 mg (1.46 mM) of (1R,2R)-CDITC were dissolved in 50 ml acetonitrile. This solution was cooled to 5°C, the 5 ml CH3I were added dropwise. The reaction mixture was stirred 2 h at 5°C and then heated to 30°C for 48 h. The solution was evaporated to dryness and stirred in 20 ml Et2O (abs.) to cystallize the product. The residue was filtered in vaccuo, dissolved in 50 ml MeOH and purified via an ion exchanger (Cl ' -form).
C19H22N3O2SCI = 391.91, resulting 380 mg (65%) yellowish crystals; m.p.: 224°C; ] H- NMR (ppm) in DMSO-d 6 :1.21-2.28 (8H, m, cyclohexyl), 3.92 (IH, m CH-NCS), 4.02 (3H, s, -OCHj), 4.15 (IH, m, CH-NH), 4.68 (3H, s, N-CHj), 7.73 (IH, d, quinolinic H3), 8.01 (IH, m, quinolinic H7), 8.13 (IH, d, quinolinic H8), 9.53 (IH, d, quinolinic H2),
9.81 (IH, d, -NH); IR (KBr): 3286 (NH), 2949 (CH), 210S (-NCS, strong), 1640 (-CO-) cm" 1 ; [α]546=-108°,c=0.98 in ACN
Example 6
General derivatization protocol for primary and secondary amines, amino alcohols (e.g. β- blockers) and thiol-compounds
A 0.01 mM amount of the amino-compound, in free base form or as salt (e.g. hydrochloride), was dissolved in 0.5ml of acetonitrile in a test tube. If the compound was in salt form 0.25 ml of 5% Na2CO3 was added. Then 0.5 ml acetonitrile containing 0.05 mM DDITC was added, the test tube tightly capped, vortexed for 1 minute and stored in an oven at 60°C. After a period of 120 minutes (or less, depending on the CDA and analytes) the reaction was stopped and the excess of CDA was quenched by the addition of 0.1 mM (L)-Proline and the reaction mixture put into the oven for another 30 minutes. Then an lOOμl aliquot was taken out, diluted with mobile phase, neutralized with acetic acid and lOμl of this mixture were directly injected onto the HPLC-column.
Indirect resolution of chiral amino alcohols using (R,R)- and (S,S)-DDITC as CDA
Indirect resolution of chiral amino alcohols using (R,R)- and (S,S)-DDITC as CDA in comparison to GITC
Influence of column surface and CDA on the resolution of amino alcohols and amines deriv. conditions k'ι' compound k'2 1 α*
Metoprolol DDITC A 5.33 7.83 1.47 6.67 GITC A 3.81 4.62 1.21 2.58
Normetoprolol DDITC B 4.47 4.69 1.05 0.67 GITC B 2.47 - 1.00 0.00
Carvedilol DDITC A 66..1111 77..3355 11..2200 3.23
GITC A 55..4411 55..9988 11..1111 1.05
General HPLC conditions:
A: Column = Lichrospher 60 RP select B (125 X 4 mm i.d., 5μm); mobile phase composition: CH3CN/NH4AC (20mM) = 55/45, apparent pH of rthe mobile phase
7.30;: flow: lml/min; detection UV 254nm;
B: Column = Hypersil ODS(125 X 4 mm i.d., 5μm : mobile phase composition:
CH3CN/NH4AC (20mM) = 45/55, apparent pH of rthe mobile phase 7.30;: flow: lml/min; detection UV 254nm;
*α=k's/k' R ; ** R s =1.18 X t2-tl/w(0.5)ι+w(0.5)2
Indirect resolution of chiral thiol compounds using (R,R)- and (S,S)-DDITC as CDA
compound mobile phase ki ' k2 ' α s
(R, S)-phenylethylmercaptan A 14.60 20.86 1.43 5.18
(R, S)-phenylethylamine A 14.75 16.23 1.10 1.58
(R,S)-2-mercapto succinc B 1.90 2.51 1.32 1.37 acid
(R,S)- 2-mercapto proprionic B 3.19 4.53 1.42 3.28 acid
*** B 8.46 11.90 1.41 4.78
(R,S)-N-acetyl cystein B 2.04 2.28 1.12 0.57
General HPLC conditions: flow: lml min; detection UV 254nm; mobile phase composition A: CH3CN/NH4AC (20mM) = 45/55, pH= =7.30; B:
CH3OH/NH4AC (20mM) = 55/45, pH=3.38 adjusted with acetic acid;
Column: Hypersil ODS (125 X 4 mm i.d., 5μm); *α=k ' 2/k ' l ; ** Rs= =1.18 X t2- t]/wι+w2 *** the corresponding thiazolidines of 2- mercaptoproprionic acid via intramolecular condensation at acidic conditions
Example 7
General derivatization procedure for the derivatization of - and β- amino acids and peptides by using (R,R)- or (S,S)- CDITC as CDA
A portion of 100 μl (0.862mM) aqueous D- or L- Pro solution was transferred into a 2ml vial. The solution was alkalized with 5 μl 5 % aqueous Na2CO3 solution, diluted with 100 μl H2O and 100 μl ACN. Then a 100 μl aliquot of the derivatizing reagent stock solution in ACN (1.724 μM) was added to the reaction vial, which got tightly capped, vortexed and stored at 50°C over a period of 90 minutes. The cooled reaction mixture was diluted and neutralized with 200 μl acidic mobile phase and 10 μl of this solution were directly injected onto the reversed phase column
Indirect resolution of chiral α-amino acids using (R,R)- and (S,S)-CDITC as CDA
Indirect resolution of chiral β-amino acids using (R,R)- and (S,S)-CDITC as CDA
Chromatographic data of (R,R)-CDITC derivatized ] DL - β - amino acids racemic β-amino acids mobile k'ι k' 2 Re e.o. phase β-amino isobutyric acid D 34.35 42.60 1.24 3.20 D<L β-amino butyric acid D 21.88 27.38 1.25 2.88 D<L
2-methyl-taurine D 4.87 6.04 1.24 1.48 D<L
General HPLC conditions: Column: Hvpersil ODS (125 x 4 mm i.d.. 5 μm). Flow: lml/min: Detection Fluorescence λκ > = 325 nm. λ m = 420 nm: Mobile phase compositions: D: MeOH/ ammonium acetate (20 mM) = 40/60 apparent pH = 4.22;
Example 8
General derivatization procedure for the derivatization of primary and secondary amines, amino alcohols, α- and β- amino acids and peptides by using (R,R)- or (S,S)- PDITC as CDA; derivatization protocols see also examples 6 and 7.
Indirect resolution of chiral α-amino acids, amines, amino alcohols and thiols using (S,S)- PDITC as CDA in comparison to (S,S)-CDITC and the well established GITC
Example 9
Micellar Electrochromatographic resolution of (R,S)-Leucine derivative using (R,R)-CDITC as CDA
Figure 1/4
Example 10
Synthesis of (lR,2R)-N-[(2-isothiocyanato)-cyclohexyl]-undecenylamide ((lR,2R)- UDITC):
a) Cyclization of trans 1,2 diaminocyclohexane (DACH) with carbon disulfide , forming trans 4,5- Tetramethyleneimidazolidine -2- thione , was accomplished according to a method described by Davies et. al. [ S. G. Davies et.al., Tetrahedron, 49 (1993) 4419, S.G. Davies, European Patent Application (03.05.1991) EP 0 457 469 Al]. b) For the monoacylation of the cyclic thiourea 0.7 mg (4.5 mM) of 4,5- tetramethyleneimidazolidine -2- thione and 0.68 g (6.7 mM) triethylamine were dissolved in 40 ml dichloroethane in presence of a catalytic amount of 4-(dimethylamino)-pyridine, heated to 90° C and a suspension of 1.34 g (6.64mM) undecenoyl chloride in 4 ml dichloroethane was added at a temperature of 90°C over a period of 15 minutes. The solution was refluxed for 14 n . The residue was extracted twice with diluted aqueous 40 ml 0.12 n HC1. The organic phase was washed with brine, dried over Na 2 SO 4 and evaporated to dryness. The crude product was purified via flash chromatography (eluent toluene).
C18H29 2OS = 322.42, yellow oil; yield: 0.52 g (52%); 1 H-NMR (ppm) in CDC1 3 1.31 (14H, s, -(CH 2 ) 7 -) 1.50-2.00 (8H, m, cyclohexyl), 2.10 (2H, d, -CO-CH 2 -), 3.15 (IH, m
CH-NCS), 3.52 (IH, m, CH-NH), 4.95 (2H, in, CH 2 -CH-), 5.80 (IH, m, CH 2 -CH-); IR (KBr): 3293 (NH), 3076 (double bond), 2076 (-NCS, strong), 1708 (-CO-) cm '1 .
Example 11
Synthesis of ( lR,2R)-N-[(2-isothiocyanato)-cyclohexyl]-6-methoxy-N-allyl-4 - chinolinylamide hydrochloride ((lR,2R)-N-allyl-CDITC):
500 mg (1.46 mM) (1R,2R)-CDITC were dissolved in 50 ml acetonitrile. This solution was cooled to 5°C, the 5 ml allyl bromide were added dropwise. The reaction mixture was stirred 2 h at 5°C and then heated to 30°C for 48 h. The solution was evaporated to dryness and stirred in 20 ml Et2O (abs.) to crystallize the product. The residue was filtered in vaccuo, dissolved in 50 ml MeOH and purified via an ion exchanger (Cl ' -form).
C21H24N3O2SCI = 417.91, resulting 415 mg (68%) yellowish crystals; d.p.: 196°C; 1H- NMR (ppm) in DMSO-d 6 : 1.21-2.30 (8H, m, cyclohexyl), 3.15 (IH, m CH-NCS), 3.52 (IH, m, CH-NH), 4.05 (3H, s, -OCH ), 4.58 (3H, s, N-Cϊfc), 4.95 (2H, m, CH-z-CH-), 5.80 (IH, m, CH 2 -CH-); 7.75 (IH, d, quinolinic H3), 8.02 (IH, m, quinolinic H7), 8.14 (IH, d, quinolinic H8), 9.55 (IH, d, quinolinic H2), 9.82 (IH, d, -NH); IR (KBr): 3286 (NH), 3075 (double bond), 2086 (-NCS, strong), 1708 (-CO-) cm- I .[α]546=-H7 θ ,c=0.98 in ACN
Example 12
Synthesis of (lR,2R)-l-[3-trimethoxysilyl-propyl]-N-[(cyclohexyl)-2-(3,5) -dinitrobenzoyl amide]-thiourea and its immobilization onto silica
2 g (5.71 mM) of (R,R)-DDITC was dissolved in 100 ml absolute dioxane, heated up to 40°C, and 1.13 g (6.27 mM) 3-aminopropyl-trimethoxysilane were added. Then the solution was refluxed for 4h. The residue was evaporated to dryness and this crude product purified via flash chromatography (eluent EE/DCM=3/6). C20H3 lNsOsSSi = 529.81, yellow oil; yield 3.0 g (99%); 1 H-NMR (ppm) in CDC1 3 0.58 (2H, t, Si-CH 2 ), 1.5-2.45 (10H, m, 4 x CH 2 cyclohexyl, 1 x CH 2 propyl), 3.30 (2H, m, CH 2 -NH-CS-, 3.52 (9H, s, 3 x -O- CH 3 ), 3.88 (IH, m CH-NH-CS-), 4.65 (IH, m, CH-NH-CO-), 5.96 (IH, d, NH), 6.38 (IH, t, NH), 8.75 (IH, d, -NH-CO-), 9.14 (3H, m, dinitrobenzoyl; IR (KBr): 3311 (NH), 3097 (Ar), 2953 (CH), 1648 (-CO-), 1536 (-NO2), 1345 (-NO2), 1079 (-0 CH 3 ) cm"*.
3g of the chirally modified siloxane and 5g silica (Kromasil, 100 A, 5μm) were refluxed 19 h in absolute toluene in the presence of a catalytic amount of p-toluene sulfonic acid. Elemental analysis of the modified silica: C (10.58%), H (1.62%), N (3.14%); leading to calculated surface coverage of 440 μM/g silica. 6g of the chirally modified silica were suspended in 150 toluene (abs.) and endcapped with 3 ml bistrimethylsilyl-acetamide by refluxing the supension for 3.5h. Elemental analysis: C (11.48%), H (1.90%), N (2.98%). This mentioned CSP was slurry packed in a HPLC stainless steel column (150 x 40 mm i.d.) which served as „chiral column".
Example 13
Application of Example 12 as chiral stationary phase (CSP) in HPLC
Example 14
Capillary electrophoretic migration of (R,R)-CDITC at pH 3
Figure 2/4
Example 15
Synthesis of (lR,2R)-l-acridinoyl-trans-4,5-tetramethylenimidazolidine-2- thione and in situ reaction with (RS)-propranolol to the corresponding diastereomeric thioureas
(R,S)-PropranoIol
Cyclization of trans 1,2 diaminocyclohexane (DACH) with carbon disulfide , forming trans 4,5- Tetramethyleneimidazolidine -2- thione , was accomplished according to a method described by Davies et. al. [ S. G. Davies et.al., Tetrahedron, 49 (1993) 4419; S.G. Davies, European Patent Application (03.05.1991) EP 0 457 469 Al] .
For the monoacylation of the cyclic thiourea 0.76 g (4.9 mM) of 4,5- tetramethyleneimidazolidine -2- thione were dissolved in 20 ml dichloroethane in presence of a catalytic amount of 4-(dimethylamino)-pyridine and added to a suspension of 1.5 g (5.4 mM) acridinic acidchloride hydrochloride in 20 ml dichloroethane at a temperature of
50°C over a period of 15 minutes. During the reaction all reactants dissolved and the solution was refluxed (90°C) for 2 n . A orange-yellowish crystalline solid was filtered containing the 5-membered ringproduct. The residue was dissolved in 50 ml DCM and extracted with 50 ml 6.5 % NaHCO 3 solution. The organic phase was washed with brine, dried over Na 2 SO and evaporated to dryness.
C21H19N3OS = 361.46, orange-yellowish crystals; m.p.: 195°C; yield: 1.07 g
(55.2%); 1 H-NMR (ppm) in CDC1 3 : 1.1525-2.29 (8H, m, cyclohexyl), 3.88 (2H, m, 2 x CH- ), 6.99 (IH, d, -NH), 7.56-8.38 (8H, m, Ar); IR (KBr): 3175 (NH), 2931 (CH), 1634 (-
CO-) cm' 1 ;
In an analytical scale reaction the monoacylated 5-membered ring product was heated with (R,S)-propranolol in o-dichlorobenzol to yield the diastereomeric thioureas. Chromatogram of the indirect resolution shown in figure 3/4.
Example 16
Synthesis of (1R,2R) DADEA mono Quininolinoy amide isothiocyanate and in situ reaction with (R,S)-propranolol to the corresponding diastereomeric thioureas
Syntheses of (R,R)-DADEA was accomplished according to [Brienne, M.-J. et.al., Bull. Soc. Chim. Fr., (1973) 190; Trost, B. M. et. al., J. Am. Chem. Soc. 114 (1992) 9327]. The monoacylation and in situ reaction was performed as described in example 15. The result of the derivatization yielding the diastereomeric thioureas is illustrated in figure 4/4.