IMMUNOMODULATORY COMPOUNDS AND METHODS OF USE THEREOF BACKGROUND OF THE INVENTION Vaccines have proven to be successful, highly acceptable methods for the prevention of infectious diseases. They are cost effective, and do not induce antibiotic resistance to the target pathogen or affect normal flora present in the host. In many cases, such as when inducing anti-viral immunity, vaccines can prevent a disease for which there are no viable curative or ameliorative treatments available.

Vaccines function by triggering the immune system to mount a response to an agent, or antigen, typically an infectious organism or a portion thereof that is introduced into the body in a non-infectious or non-pathogenic form. Once the immune system has been"primed"or sensitized to the organism, later exposure of the immune system to this organism as an infectious pathogen results in a rapid and robust immune response that destroys the pathogen before it can multiply and infect enough cells in the host organism to cause disease symptoms.

The agent, or antigen, used to prime the immune system can be the entire organism in a less infectious state, known as an attenuated organism, or in some cases, components of the organism such as carbohydrates, proteins or peptides representing various structural components of the organism.

In many cases, it is necessary to enhance the immune response to the antigens present in a vaccine in order to stimulate the immune system to a sufficient extent to make a vaccine effective, i. e. , to confer immunity. Many protein and most peptide and carbohydrate antigens, administered alone, do not elicit a sufficient antibody response to confer immunity. Such antigens need to be presented to the immune system in such a way that they will be recognized as foreign and will elicit an immune response. To this end, additives (adjuvants) have been devised which immobilize antigens and stimulate the immune response.

The best known adjuvant, Freund's complete adjuvant, consists of a mixture of mycobacteria in an oil/water emulsion. Freund's adjuvant works in two ways: first, by enhancing cell and humoral-mediated immunity, and second, by blocking rapid dispersal of the antigen challenge (the"depot effect"). However, due to frequent toxic physiological and immunological reactions to this material, Freund's adjuvant cannot be used in humans.

Another molecule that has been shown to have immunostimulatory or adjuvant activity is endotoxin, also known as lipopolysaccharide (LPS). LPS stimulates the immune system by triggering an"innate"immune response-a response that has evolved to enable an organism to recognize endotoxin (and the invading bacteria of which it is a component) without the need for the organism to have been previously exposed. While LPS is too toxic to be a viable adjuvant, molecules that are structurally related to endotoxin, such as monophosphoryl lipid A ("MPL") are being tested as adjuvants in clinical trials. Currently, however, the only FDA-approved adjuvant for use in humans is aluminum salts (Alum) which are used to"depot" antigens by precipitation of the antigens. Alum also stimulates the immune response to antigens.

Thus, there is a recognized need in the art for compounds which can be co- administered with antigens in order to stimulate the immune system to generate a more robust antibody response to the antigen than would be seen if the antigen were injected alone or with Alum.

SUMMARY OF THE INVENTION In one aspect, the present invention is directed to novel compounds that function as immunological adjuvants when co-administered with antigens such as vaccines for bacterial and viral diseases.

In a second aspect, the present invention is directed to novel adjuvant formulations which comprise at least one of the adjuvant compounds of the invention.

In a third aspect, the invention is directed to novel immunostimulatory compositions which comprise an antigen and at least one of the adjuvant compounds of the invention.

In another aspect, the present invention is directed to methods for the immunization of an animal by co-administration of a compound of the invention with an antigen against which the animal is to be immunized.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph that shows the results of an in vitro assay for induction of TNF-alpha cytokine release by compounds 100,184 or 186 of the invention.

Figure 2 is a graph that shows stimulation of alkaline phosphatase expression from an inducible reporter construct with the TNF promoter (TNF-PLAP) in THP-1 cells by compounds 106 and 126 in the absence and presence of 10 % serum.

Figure 3 is a graph showing stimulation of IL-10 release from normal mouse splenocytes by compounds 104,106, 124,126, 160, and 162 of the invention.

Figure 4 is a graph showing stimulation of interferon-gamma release from normal mouse splenocytes by compounds 104,106, 124,126, 160, and 162 of the invention.

Figure 5 is a graph illustrating the results of serum titration analysis for determining the amounts of antibody that are produced in response to keyhole limpet hemocyanin in the absence and presence of compounds 100,124, and 126 of the invention.

Figure 6 is a graph illustrating the results of serum titration analysis for determining amounts of antibody produced in response to tetanus toxoid in the absence and presence of compounds 100,116, 126,160 and 184 of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed in part to the novel compounds of the formula I, II, and in ; immunological adjuvant for mutations comprising a compound of formula I, II, or in ; and at least one additional component; methods of using the compounds of the formula I, II or III ; and methods of using the immunological formulations comprising compounds of the formula I, II or III and at least one additional component.

Wherein for each of formula I, II, or m : R1 is selected from the group consisting of (a) C (O); (b) C (O)-Cl-14 alkyl-C (O), wherein said Cl l4 alkyl is optionally substituted with hydroxy, C1-5 alkoxy, C15 alkylenedioxy, C1-5 alkylamino, or C1-5- alkyl-aryl, wherein said aryl moiety of said Cl-is-alkyl-aryl is optionally substituted with C1-5 alkoxy, C1-5 alkyl amino, C1-5 alkoxy-amino, C1-5 alkylamino-Cl-5 alkoxy, - O-Cl s alkylamino-Cl s alkoxy,-O-Cl s alkylamino-C (O)-Cl s alkyl C (O) OH,-0-Cl-5 alkylamino-C (O)-C1-5 alkyl-C(O)-C1-5 alkyl ; (c) C2 to C15 straight or branched chain alkyl optionally substituted with hydroxy or alkoxy; and (d) -C (O)-C6-12 arylene-C (O)- wherein said arylene is optionally substituted with hydroxy, halogen, nitro or amino; a and b are independently 0,1, 2,3 or 4; d, d', d', e, e'and ev are independently an integer from 0 to 4; XI, X2, Y1 and Y2 are independently selected from the group consisting of null, oxygen, NH and N (C (O) C1-4 alkyl), and N (CI-4 alkyl) 2; Wl and w2 are independently selected from the group consisting of carbonyl, methylene, sulfone and sulfoxide;

R and R5 are independently selected from the group consisting of: (a) C2 to C20 straight chain or branched chain alkyl which is optionally substituted with oxo, hydroxy or alkoxy, (b) C2 to CZn straight chain or branched chain alkenyl or dialkenyl which is optionally substituted with oxo, hydroxy or alkoxy; (c) C2 to C20 straight chain or branched chain alkoxy which is optionally substituted with oxo, hydroxy or alkoxy; (d)-NH-C2 to C20 straight chain or branched chain alkyl, wherein said alkyl group is optionally substituted with oxo, hydroxy or alkoxy; and wherein Z is selected from the group consisting of O and NH, and M and N are independently selected from the group consisting of C2 to C20 straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino; R1 and R6 are independently selected from the group consisting of C2 to C20 straight chain or branched chain alkyl or alkenyl optionally substituted with oxo or fluoro; R4 and R are independently selected from the group consisting of C (O) C2 to C20 straight chain or branched chain alkyl or alkenyl; C2 to C20 straight chain or branched chain alkyl ; C2 to C20 straight chain or branched chain alkoxy; C2 to C20 straight chain or branched chain alkenyl; wherein said alkyl, alkenyl or alkoxy groups can be independently and optionally substituted with hydroxy, fluoro or Ci to C5 alkoxy; G1, G2, G3 and G4 are independently selected from the group consisting of oxygen, methylene, amino, thiol,-NHC (O) -,-C (O) NH-, -C (O) O-,-OC (O)- and - N (C (O) C14 alkyl)-, or G2R4 or G4R7 may together be a hydrogen atom or hydroxyl; or a pharmaceutically acceptable salt thereof; and wherein for Formula II : a'and b'are independently 2,3, 4,5, 6,7, or 8, preferably 2;

Z1 is selected from the group consisting of-OP (O) (OH) 2,-P (O) (OH) 2,- OP (O) (OR8) (OH) where Rs is a C1-C4 alkyl chain, -OS (O) 2OH,-S (O) 2OH-,-CO2H,- OB (OH) 2,-OH,-CH3,-NH2, NR93 where R9 is a C1-C4 alkyl chain; 2 is-OP (O) (OH) 2,-P (O) (OH)2', -OP(O)(OR10) (OH) where Rl°is a C1-C4 alkyl chain, -OS (O) 20H,-S (O) 2OH, C02H,-OB (OH) 2,-OH, CH3, -NH2, -NR11, where Rll is a Cl-C4 alkyl chain; and wherein for Formula 3: R12 is selected from H and a C1-C4 alkyl chain; or a pharmaceutical salt thereof, with the proviso that the compounds of formula I, II, or III are not In preferred compounds of the invention, one or more of the following is present: each of a and b is 2; each of Xl and Y1 is NH; Rl is C (O) or C (O)-C1-4 alkyl- C (O) ; each of d'and e'is 1; each of d-and C is 1; X is O or NH, more preferably NH; and W is C (O) ; or each of d'and e'are 2.

In further preferred embodiments, R'is a C (O) C1-14 alkyl-C (O), wherein said Cl l4 alkyl is substituted, for example with a C1-5 alkoxy group; In a most preferred embodiment, the invention is directed to compounds ER 803022, ER 803058, ER 803732, ER 804053, ER 804057, ER 804058, ER 804059, ER 804442, ER 804680 and ER 804764, and compositions containing these compounds.

The invention is also directed to novel immunostimulatory compositions which include an antigen and an immunological adjuvant formulation of the invention as disclosed above.

Also provided are methods for the immunization of an animal by co- administration of a compound of the invention or an adjuvant formulation of the invention with an antigen against which the animal is to be immunized.

Definitions Carbonyl, as used herein, is a (C=O) moiety.

Dicarbonyl, as used herein, is a moiety with the structure (C=O)-alkyl- (C=O) or (C=O)-aryl- (C=O), which is bonded to a molecule through the carbon atoms of both of the terminal carbonyl moieties.

Oxo, as used herein, is a =O group.

Alkyl ester, as used herein, is a moiety with the structure 0- (C=O)-alkyl, which is bonded to a molecule through the non-double bonded oxygen of the ester group.

Alkenyl ester, as used herein, is a moiety with the structure 0- (C=O)-carbon chain, where the carbon chain contains a carbon-to-carbon double bond, which is bonded to a molecule through the non-double bonded oxygen of the ester group.

The term"alkylene"means a bivalent straight chain or branched alkyl hydrocarbon group.

The term"alkenylene"means a bivalent straight chain or branched hydrocarbon group having a single carbon to carbon double bond.

The term"dialkenylene"means a bivalent unsaturated straight chain or branched chain hydrocarbon group having two carbon to carbon double bonds.

The term"arylene"refers to a bivalent aromatic group.

The abbreviation"Boc"as used herein means t-butyloxycarbonyl.

As used herein with reference to compounds and compositions of the invention, the term"type 1"refers to those compounds of the invention corresponding to formula I above where the values of a and b are the same; the values of d and e are the same; the values of d'and e'are the same; the values of d-and e-are the same; Xl and Y'are the same; X2 and y2 are the same; Wl and W2 are the same; R and R5 are the same; G1 and G3 are the same; R3 and R6 are the same; G and G4 are the same; and R4 and R7 are the same. "Type 2", as used herein, refers to compounds or

compositions corresponding to formula I where any one or more of the following applies: the values of a and b are different, the values of d and e are the same, the values of d'and e'are different; the values of dÆ and e"are the same; Xl and Y'are different; X2 and Y2 are different; Wl and W2 are different; R2 and R5 are different ; gel and G3 are different; R3 and R6 are different; G2 and G4 are different; or R4 and R7 are different.

All patents, patent applications, and publications referred to herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

General Synthetic Methods 1. Synthesis of Diamide Compounds In general, a 2-amino-1, 3-dihydroxypropane or () serinol is transformed into the 2-azido compound by reaction with trifluoromethanesulfonyl azide followed by protection as the per-acetate for easy manipulation. The resulting compound is deacetylated, followed by reaction with an appropriately activated primary alcohol of a diol moiety. The primary alcohol moiety of the product of this reaction is then protected, e. g. , by using TBDPSCL, followed by reaction with phosgene and then allyl alcohol, to yield a fully protected diol. The protected diol is then treated to cleave the protecting group from the primary alcohol. The unprotected alcohol is reacted with a properly functionalized phosphorylating reagent with formula (11) as indicated in the Examples, to form a phosphate ester compound. The azido moiety of the product is reduced, and then reacted with an activated acyl acid to form an amide.

The protected terminal amine on the functionalized phosphate is deprotected, and subsequently reacted with a phosgene or a dicarboxylic acid in the presence of a dehydrating agent, such as EDC. The phosphate groups of the resulting compound are then deprotected, yielding a racemic amide.

2. Synthesis of Chiral Diamide Compounds of Type 1 In general, a chiral amino acid ester with the desired structure is protected with a benzimidate ester. The protected compound is reacted with a reducing agent, e. g., DIBAL or the like, to reduce the acid moiety of the amino acid to an alcohol. The resulting alcohol compound is reacted with an appropriately activated primary alcohol of a diol moiety, followed by cleavage of the benzimidate protecting group, yielding

an amino-diol. The diol is then reacted with an appropriate acid chloride to yield a diol-amide.

The diol-amide is then reacted with a properly functionalized phosphorylating reagent at the free primary hydroxyl group. The resulting compound is esterified at the secondary alcohol group with an appropriate acyl moiety. The N-BOC group is then cleaved from the amino group introduced by phosphorylating reagent (11), yielding a phosphate ester compound with a free primary amine. This product is then reacted with phosgene or a dicarboxylic acid in the presence of a dehydrating agent, to yield a diamide product. The protected phosphate groups of the diamide product are then deprotected, typically with palladium (0) and phenylsilane.

3. Synthesis of Chiral Diamide Compounds of Type 2 Chiral diamide compounds of Type 2 are synthesized essentially as described for chiral diamide compounds of Type 1, up to the point just after cleavage of the protecting group from the primary amine group of the phosphate ester compound. At this point, a dicarboxylic acid which has one of the acid moieties protected is reacted with the primary amine group, to yield a monoamide. The protecting group on the other carboxylic acid is then cleaved, providing a free carboxylic acid which can then be reacted with a primary amine from an alternative, appropriately substituted phosphate system, in the presence of a dehydrating agent to yield a diamide of type 2, which can then be treated to deprotect the phosphate group or groups to yield a desired compound of the invention.

In the special case of chiral urea compounds of type 2 of the invention, the primary amino group of the N-BOC amino group of the phosphate ester is deprotected and then reacted with trichloromethyl chloroformate or the like, in order to form an isocyanate compound. The isocyanate is then reacted with a primary amine from an alternative, appropriately substituted phosphate system to yield a urea product of type 2. This product can then be treated to deprotect the phosphate group or groups.

4. Glycerol Diamide Analogs These compounds of the invention have an ester moiety attached to the carbon which is beta to the phosphate group, instead of an amide moiety.

In general, these compounds are prepared by the etherification of a protected chiral glycerol with an activated primary alcohol of a diol moiety, followed by esterification of the secondary alcohol moiety and subsequent deprotection of the glycerol moiety, to yield a new diol. The primary hydroxyl group of the diol is then

protected, and the secondary hydroxyl group is condensed with an acyl moiety to yield a diester. The primary hydroxyl is deprotected, followed by esterification with a phosphorylating agent, of which compound (11), below is exemplary. Following deprotection of the amine group introduced by the phosphorylating agent, the product is reacted with phosgene or a dicarboxylic acid using a dehydrating agent such as EDC. Subsequent deprotection of the phosphate groups yield compounds of the invention.

In the synthesis described generally above, the substituent at R of the compounds of the invention can easily be varied by utilizing different dicarboxylic acid compounds. Such acids can be coupled to the amine group of the phosphate ester intermediate of the reaction scheme outlined above, either using a dehydrating agent such as EDC, or by activating the dicarboxylic acid by synthesizing, e. g. , the corresponding diacid chloride.

The substituents represented by variables R2 and Rs in formula I above can easily be varied by utilizing an appropriate activated acid or acid chloride in the amidation or esterification reaction of the heteroatom represented by X or Y in formula I.

The substituents represented by variables R3 and R6 of formula I can be varied by using an intermediate containing the desired number of carbon atoms which also contains an activated carbon functionality, e. g. , a halogen or sulfonate (OS02CH3, OS02CF3, OSO2CH2C6H4-p-CH3) which can be reacted with the azido diol, amino alcohol, or glycerol starting materials.

The substituents represented by variables R4 and R7 in formula I above can be varied by using an appropriate activated acid or acid chloride in the esterification of the secondary hydroxyl group used in the reaction schemes outlined above.

The values of a and b in compounds of formula I can be varied by using the appropriate compound (11) below. The values of variables d and e in compounds of formula I can be modified by using the appropriate 2-aminodiol or 2-hydroxydiol starting materials Adjuvant and Vaccine Formulation and Administration The present invention is also directed to adjuvant formulations comprising adjuvant compounds of the invention, as well as vaccine and other immunostimulatory formulations which comprise the adjuvant compounds of the invention. Methods for

the stimulation of an immune response to a particular antigen are also within the scope of the invention.

The host animals to which the adjuvant and adjuvant-containing vaccine formulations of the present invention are usefully administered include human as well as non-human mammals, fish, reptiles, etc.

Typically, an antigen is employed in mixture with the adjuvant compounds of the invention. In other formulations of the adjuvant of the present invention, it may be useful in some applications to employ an antigen covalently linked to an amino, carboxyl, hydroxyl and/or phosphate moiety of the adjuvant compounds of the invention. The specific formulation of therapeutically effective compositions of the present invention may thus be carried out in any suitable manner which will render the adjuvant bioavailable, safe and effective in the subject to whom the formulation is administered.

The invention broadly contemplates therapeutic adjuvant formulations, which may for example comprise (i) at least one therapeutically effective antigen or vaccine; and (ii) at least one adjuvant compound according to the invention.

Such therapeutic composition may for example comprise at least one antigenic agent selected from the group consisting of: (A) live, heat killed, or chemically attenuated viruses, bacteria, mycoplasmas, fungi, and protozoa; (B) fragments, extracts, subunits, metabolites and recombinant constructs of (A); (C) fragments, subunits, metabolites and recombinant constructs of mammalian proteins and glycoproteins; (D) tumor-specific antigens; and (E) nucleic acid vaccines.

The therapeutic composition may therefore utilize any suitable antigen or vaccine component in combination with an adjuvant compound of the invention, e. g., an antigenic agent selected from the group consisting of antigens from pathogenic and non-pathogenic organisms, viruses, and fungi, in combination with an adjuvant compound of the invention.

As a further example, such therapeutic compositions may suitably comprise proteins, peptides, antigens and vaccines which are pharmacologically active for disease states and conditions such as smallpox, yellow fever, distemper, cholera, fowl

pox, scarlet fever, diphtheria, tetanus, whooping cough, influenza, rabies, mumps, measles, foot and mouth disease, and poliomyelitis. In the resulting vaccine formulation, comprising (i) an antigen, and (ii) at least one adjuvant compound of the invention the antigen and adjuvant compound are each present in an amount effective to elicit an immune response when the formulation is administered to a host animal, embryo, or ovum vaccinated therewith.

In further embodiments, the compounds of the invention may be covalently bonded to vaccine antigens, for example through an amino, carbonyl, hydroxyl or phosphate moiety. Methods of linking the adjuvant compositions of the invention to vaccine antigens are understood by persons of ordinary skill in the art in view of this disclosure. The adjuvant compositions may be linked to vaccines by any of the methods described in P. Hoffinan et al., Biol. Chem. Hoppe-Sayler, 1989,370 : 575- 582 ; K. -H. Wiesmuller et al., Vaccine, 1989,7 : 29-33; K.-H Wiesmuller et al., lilt. J.

Peptide Protein Res., 1992,40 : 255-260; J. -P. Defourt et al. , Proc. Natl. Acad. Sci.

1992, 89 : 3879-3883 ; T. Tohokuni et al. , J. Am. Chem. Soc., 1994,116 : 395-396; F.

Reichel, Chem. Commun., 1997, 2087-2088 ; H. Kamitakahara, Angew. Chem. Int. Ed.

1998, 37: 1524-1528 ; W. Dullenkopf et al. , Chem. Eur. J., 1999,5 : 2432-2438; all of which are hereby incorporated by reference.

The resulting vaccine formulations, including (i) an antigen, and (ii) an adjuvant compound, are usefully employed to induce an immunological response in an animal, by administering to such animal the vaccine formulation, in an amount sufficient to produce an antibody response in such animal.

The modes of administration may comprise the use of any suitable means and/or methods for delivering the adjuvant, adjuvant-containing vaccine, or adjuvant and/or antigen to one or more corporeal loci of the host animal where the adjuvant and associated antigens are immumostimulatively effective. Delivery modes may include, without limitation, parenteral administration methods, such as subcutaneous (SC) injection, transcutaneous, intranasal (IN), ophthalmic, transdermal, intramuscular (IM), intradermal (nid), intraperitoneal (IP), intravaginal, pulmonary, and rectal administration, as well as non-parenteral, e. g. , oral, administration.

The dose rate and suitable dosage forms for the adjuvant and vaccine compositions of the present invention may be readily determined by those of ordinary skill in the art without undue experimentation, by use of conventional antibody titer determination techniques and conventional bioefficacy/biocompatibility protocols, and

depending on the particular antigen or therapeutic agent employed with the adjuvant, the desired therapeutic effect, and the desired time span of bioactivity.

The adjuvant of the present invention may be usefully administered to the host animal with any other suitable pharmacologically or physiologically active agents, e. g. , antigenic and/or other biologically active substances.

Formulations of the invention can include additional components such as saline, oil, squalene, oil-water dispersions, liposomes, and other adjuvants such as QS- 21, muramyl peptides, Freunds's incomplete adjuvant, and the like.

Synthetic Examples All reaction products in the synthetic methods described below gave satisfactory NMR spectra and thin layer chromatography profiles on silica gel. All chromatography was performed on silica gel and the elution monitored by thin layer chromatography. All completed reactions were determined by thin layer chromatographic analysis. All reactions were run under nitrogen at room temperature unless otherwise specified. All reaction solvents were anhydrous unless otherwise noted. The typical work-up for the chemical reactions described below includes aqueous washings, drying over anhydrous sodium sulfate and removal of solvent under reduced pressure.

Example 1: Succinate-1 1. NaN3, NH2 Trifluromethanesulfonic anhydride N3 HOOH DMAP AcOOAc 2. Acetic Anhydride, Pyridine 2 To a solution of sodium azide (107.67 g) in 250 mL of water was added 300 mL of methylene chloride. The mixture was cooled to 0°C and trifluoromethanesulfonic anhydride (57 mL) was added dropwise at a 0. 32 mL/minute rate. The mixture was stirred for an additional 6 hours at 0°C and stored at-20°C for 72 hours. The mixture was warmed to 10°C followed by extraction with methylene chloride in a Teflon° separatory funnel. The combined organic layers were dried (magnesium sulfate). The above suspension was slowly filtered into a stirred solution of ()-2-amino-1, 3-dihydroxypropane (1) (9. 89 g) in methanol (200 mL) and 4-N, N

dimethylaminopyridine (DMAP, 54 g) at 10°C. The resultant reaction mixture was stirred for 17 hours at room temperature.

The solvent was removed under reduced pressure and the residue dissolved in pyridine (200mL) and cooled to 0°C. Acetic anhydride (50 mL) was added dropwise and the mixture stirred for 20 hours at room temperature. Additional acetic anhydride (20 mL) was added and after 4 hours, the mixture was poured onto ice and worked up in the usual manner. Chromatography gave 16 g of diacetate (2) as an oil. N3 1. Na metal N3 AcO9Xz 2. Dowex50-8 XOH -'" 2. Dowex 50-8 -' 2 3 The diacetate (2) (16 g) was dissolved in methanol (150 mL) and sodium metal (2.0 g) was slowly added. The mixture was stirred for 90 minutes and Dowexs 50-S resin was added until the pH was less than or equal to 7. The mixture was filtered followed by concentration of the filtrate and chromatography to give 6.73 g of the diol (3).

To a suspension of sodium hydride (1.24 g of a 60% oil dispersion washed three times with hexanes and dried under nitrogen) in dimethylformamide (DMF, 200 mL) was added dropwise the azido-diol (3) (6.73 g) in THF (100 mL), followed by the dropwise addition of 3-R-hydroxy-1-O-tosyl-1-decanol (4) (tosylate, 9.44 g) in THF (100 mL). The mixture was stirred for 16 hours, diluted with methanol (200 mL), stirred with Amberlite@ 25 H for 25 minutes and concentrated to dryness.

Chromatography gave 4.37 g of (5).

To a solution of the diol (5) (5.32 g) in methylene chloride (30 mL) was added triethylamine (TEA, 6 mL) and DMAP (trace), followed by t-butyldiphenylsilyl chloride (TBDPSC1,5 mL) and the mixture was stirred overnight. The mixture was worked up as usual. Chromatography gave 3.6 g of secondary alcohol (6) as an oil. N3 Na TBDPSO<O oC7H1s Aìlyl aìcohol TBDPSOgtzO, C7H15 6 OH Phosgene, Pyridine 7 0 O

To a solution of the secondary alcohol (6) (2.95 g) in toluene (30 mL) was added pyridine (1.8 mL) followed by a slow addition of phosgene (4.5 mL of a 1.93 M solution in toluene) at 0°C. After stirring at 0°C for 20 minutes, allyl alcohol (3.1 mL) was added dropwise. After an additional stirring for 60 minutes at room temperature, the reaction was worked up in the usual way. Chromatography gave 3.24 g of protected alcohol (7) as an oil.

To a solution of protected alcohol (7) (1.29 g) in methylene chloride (3 mL) was added hydrofluoric acid (HF, 4 mL) in acetonitrile (12 mL). The mixture was stirred overnight and worked up in the usual way. Chromatography gave 150 mg of the alcohol (8) as an oil. N3 1. 11, Tetrazole N3 HOJO <oC7H1s 2. Oxone BocHN~Ospl, OJO oC7H1s OAOC Õ OAOC 8 9 To a solution of alcohol (8) (150 mg) in methylene chloride (0.6 mL) was added tetrazole (74 mg) and the phosphorylating reagent (11) (175 mg). After 30 minutes, oxone (323 mg) in a cooled THF (0.5 mL)-water (0.5 mL) solution was added to the cooled reaction mixture. After 3 hours, the reaction was worked up in the usual way. Chromatography gave 242 mg of (9) as an oil. BocHNH O I O, P N O_P, N/ Tetrazole r r-i 10 \/2 BocHN 11 To make phosphorylating reagent 11, to a solution of distilled diisopropylamine (9.0 mL) in methylene chloride was added tetrazole (4.51 g) at room temperature followed by stirring for 1.5 hours. Allyl phosphorodiamidite (10) (20.5

mL) was added dropwise at a 6.5 mL/hour rate followed by stirring for an additional 3 hours. N-Boc-2-aminoethanol (10.36 g) in methylene chloride (50 mL) was added to the above reaction mixture dropwise at a 8.4 mL/hour rate followed by stirring for an additional 18 hours. The white suspension was filtered through Celite 545 with two 20 mL washings with methylene chloride. The filtrate was concentrated followed by the suspension and filtering of the residue with hexanes (200 mL). The resulting hexanes filtrate was concentrated to dry and azeotroped with 2, 10-mL portions of toluene to provide the crude product (11) (21.54 g) as an oil.

To a suspension of dithiophenol tin (1.3 g) in methylene chloride (7.8 mL) was added thiophenol (400 pL) followed by TEA (543 I1L). The reaction mixture was stirred at room temperature for 15 minutes followed by stopping the stirring and allowing the residue to settle to the bottom of the flask. 1.0 mL of the above solution was added to a solution of the azide (9) (242 mg) in methylene chloride (0.5 mL) and allowed to stir for 30 minutes. Quenching with 0.1 N NaOH followed by the usual work-up afforded 193.1 mg of the amine (12) as an oil.

To a dried solution of the amine (12) (193 mg) and acyl acid (which can be made according to Christ et al. , U. S. Pat. No. 5,530, 113) (132 mg) in methylene chloride was added 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 93 mg). After stirring at room temperature for 90 minutes the reaction was quenched and processed in the usual way to provide 232 mg of protected phosphate (13) as an oil.

To a solution of the protected phosphate (13) (232 mg) in methylene chloride (1 mL) was added triethylsilane (TES, 120 u. L) and trifluoroacetic acid (TFA, 1.2 mL) followed by stirring for 30 minutes. The TFA was removed under reduced pressure followed by azeotroping with 3,5-mL portions of toluene. 20 mL of methylene chloride was added and the mixture was worked up in the usual manner to give 174 mg of free amine (14) as an oil. 0 9''sHia 0 O O O""CgHig HN 'CHis HNPOOCHs HN CHS Succlnc Acid p O OAOC 0 OAOC 0 c6H13 14 OAOC \=O [C6Hl3 14 HN oJ HN4C7H1s 11 0--^Y 0 OAOC 15

To a dried solution of the free amine (14) (174 mg) in methylene chloride (0.5 mL) was added succinic acid (12.1 mg) and EDC (59 mg). After 1 hour, the reaction was worked up in the usual manner. Chromatography gave 143.1 mg of blocked diphosphate (15) as an oil.

To a solution of blocked diphosphate (15) (177.9 mg) in degassed chloroform (1.7 mL) in a dry box was first added phenylsilane (PhSiH3, 50 uL) and then tetrakis- triphenylphosphine palladium (0) (Pd (PPH3) 4,70 mg). After 1 hour, the reaction was removed from the box and chloroform: methanol: water, 2: 3: 1, was added and the mixture stirred for 1 hour. It was poured onto a diethylamino-ethylcellulose (DEAE) chromatography column. Elution of the column with a linear gradient of 0.0 M to 0.1 M ammonium acetate in chloroform: methanol: water, 2: 3: 1, extraction of the desired fractions with an equal volume of chloroform, concentration to dryness and the addition of 0.1 N NaOH (175 uL) followed by lyopholization gave 136.2 mg of (16) as a white solid.

Example 2: Chiral Malonate-Type 1

To a cooled solution of potassium carbonate (165 g) in water (575 mL) was added methylene chloride (200 mL) followed by ethyl benzimidate hydrochloride (17) (100 g) after which time the mixture was stirred for 8 minutes. The layers were separated and the aqueous layer extracted with methylene chloride. The organic layers were combined, dried and the solvent removed under reduced pressure to give 83 g of (18). 0 1, 2-Dichloroethane OOCH3 HO<OCH3 + (/OC2H5 --N NH2 HCI NH 19 18 < 21

To a solution of L-serine methyl ester hydrochloride (19) (41.6 g) in 1,2- dichloroethane (450 mL) was added ethyl benzimidate (18) (36 g). The mixture was heated to reflux for 20 hours, cooled, filtered through diatomaceous earth, and concentrated to dryness to give 56 g of ethyl ester (21) as a white solid.

To an ice cold solution of ethyl ester (21) (56 g) in THF (500 mL) was added dropwise diisobutylaluminum hydride (DIBAL, 545 mL of a 1. M solution in hexane).

The mixture was allowed to warm to room temperature overnight and then carefully poured onto an aqueous solution of Rochelle's salt (500 g in 1.0 L water). The mixture stirred for 1 hour and worked up in the usual manner. Chromatography gave 25.5 g of alcohol (22) as a white solid.

To a suspension of washed sodium hydride (5.3 g of a 60% oil dispersion) in DMF (200 mL) was added the alcohol (22) (24 g) in 250 mL of THF After 30 minutes, the tosylate (4) was added in 250 mL of THF over 2.5 hours and stirred overnight. The mixture was cooled in ice, methanol was added, the solvent removed under reduced pressure and chromatographed to give 4.32 g of alcohol (24) as an oil.

The alcohol (24) (4.3 g) was dissolved in 4 N aqueous hydrochloric acid and heated to reflux for 20 hours. The mixture was cooled, filtered, extracted with ether, made basic with sodium hydroxide and extracted twice with chloroform. The combined chloroform layers were dried and the solvent removed to give 2.88 g of diol (25) as an oil.

The diol (25) (2.88 g) was dissolved in saturated aqueous sodium bicarbonate (45 mL) and THF (25 mL) and allowed to stir for five minutes. Myristoyl chloride (3.4 mL) was added dropwise over a 25-minute period after which time the reaction mixture was allowed to stir for an additional hour. The reaction was worked up in the usual manner and chromatographed to give 3.07 g of alcohol (26) as an oil. BocHN \ ou OH 1. P N O OH HOtOC7H15 BocHN O'IPoAf OC7Hr5 HNG1sH27 Tetrazo, e) HN C13H27 O 2. Oxone 26 27

To a solution of the alcohol (26) (1.78 g) in methylene chloride (140 mL) was added tetrazole (683 mg), followed by the phosphorylating reagent (11) (1.6 mL).

After 30 minutes, the mixture was cooled in ice, and THF (105 mL) was added followed by an oxone solution (3 g in 90 mL of water). After 5 minutes, the ice bath was removed and the mixture stirred for 30 minutes. The reaction was worked up in the usual manner and chromatographed to give 2.99 g of alcohol (27) as an oil.

To a solution of the alcohol (27) (3.9 g) in methylene chloride (126 mL) was added EDC (10.8 g), DMAP (66 mg) and dodecyl acid (1.62 g) and stirred overnight.

Additional acid (1.6 g), EDC (1 g) and DMAP (0.5 g) was added. After 3 hours, the reaction was worked up in the usual manner and chromatographed to give 2.07 g of N- BOC-protected amine (28).

To a solution of the N-BOC-protected amine (28) (187.3 mg) in methylene chloride (1.5 mL) was added TES (240, ut) and TFA (300 fiL) and the mixture stirred for 45 minutes. Toluene was added and the solvent removed under reduced pressure.

The residue was dissolved in methylene chloride and worked up in the usual manner to give 154 mg of amine (29) as an oil. 0 0 6 CHsg 11 p-p-ooc 9/P HN C 8-° G11H23 Ma, Onjc acid) =/> H2N OC fi HN II 11 Malonic acid Etc HNC13H2 FiN O _ T 0 23 P-OC H 29 HNUCI3Hz s ici /@ O 30 To an ice cold solution of the amine (29) (75.6 mg) and malonic acid (4. 8 mg) in methylene chloride (0.5 mL) was added EDC (27 mg), followed by removal of the cooling bath. After 1 hour, a trace of DMAP was added. After 2.5 hours, the mixture was directly chromatographed to give 54.1 mg of dimer product (30).

To a solution of the dimer (30) (54 mg) in degassed chloroform (2mL) was added PhSiH3 (14, uL) and Pd (PPh3) 4 (18 mg) followed by removal of the cooling bath. After 1 hour, the reaction was quenched with chloroform: methanol: water (2: 3: 1) and poured onto a DEAE cellulose chromatography column and chromatographed to give a semi-solid. The solid was dissolved in sterile water and 0.1 N aqueous sodium hydroxide (306 KL) was added and the mixture lyophilized to yield white solid (31) (25 mg).

Example 3: Chiral Malonate-Type 2 0 ° 1, 2-Dichloroethane O. 4OCH3 HOt OCH3 + AOC2H5') =N NH2 HCI NH 32 20 O 33

To a solution of D-serine methyl ester hydrochloride (32) (25 g) in dichloroethane (270 mL) was added ethyl benzimidate (20) (24 g). After 20 hours at reflux, the mixture was cooled to room temperature, filtered through diatomaceous earth and the solvent removed under reduced pressure to give 34 g of methyl ester (33) as a white solid.

To a ice cold solution of the methyl ester (33) (34 g) in THF (300 mL) was added dropwise a solution of DIBAL in hexane (1.0 M, 322 mL). The mixture was allowed to warm to room temperature overnight and then carefully poured into an aqueous solution of Rochelle's salt. The mixture was then stirred for 1 hour and worked up. Chromatography gave 18.6 g of alcohol (36) as a white solid.

To a suspension of washed sodium hydride (4 g of a 60% oil suspension) in DMF (100 mL) was added a solution of the alcohol (36) (8.6 g) in THF (40 mL). The mixture was stirred for 1 hour and a solution of the tosylate (23) (17.5 g) in THF (40 mL) was added. The mixture was stirred overnight and then additional tosylate (23) was added (5g) and stirred for another 4 hours. Methanol was added to the cooled reaction mixture, the solvent was removed under reduced pressure, and the residue was chromatographed to give 1.03 g of alcohol (37) as a solid.

The alcohol (37) (1.03) was dissolved in 4 N aqueous hydrochloric acid (25 mL) and the mixture was heated to 100 °C for 20 hours. Additional hydrochloric acid (5 mL) was added and the reflux continued for 6 hours. The mixture was cooled, washed with ether, made basic with 1 N aqueous sodium hydroxide, and extracted (3x) with chloroform. The combined chloroform layers were dried and the solvent removed under reduced pressure to give 553 mg of amino-diol (39).

To a solution of amino-diol (39) (553 mg) in THF (3 mL) was added saturated aqueous sodium bicarbonate (6 mL) followed by myristoyl chloride (628 uL). After 50 minutes, the reaction was worked up in the usual way. Chromatography gave 389 mg of amide-diol (41) as an oil.

BocHN \ 0)- -,-., 0, P, OH 1 PN BocHN Oz IPsozoz H O HIVUC13H27 Tetrazole HN'C13H27 T 2. Oxone 0 41 42 To a solution of the amide-diol (41) (531 mg) in methylene chloride (40 mL) was added tetrazole (203 mg) and the mixture was stirred for 5 minutes. Then phosphorylating reagent (11) (482 mg) was added. After 20 minutes, additional phosphorylating reagent (11) (100 mg) was added and after an additional 20 minutes, 100 mg more was added. After an additional 20 minutes, 50 mg more was added.

After 20 minutes, the mixture was poured into a cold solution of THF (30 mL)/oxone (1.07 g) /water (30 mL). The mixture was stirred at 0°C for 5 minutes, and then 20 minutes at room temperature after which time the reaction was worked up in the usual manner. Chromatography gave 852 mg of phosphate alcohol (42) as an oil.

To a solution of the phosphate alcohol (42) (3.9 g) in methylene chloride (126 mL) was added EDC (10.8 g), DMAP (66 mg) and dodecyl acid (1.62 g). The reaction mixture was stirred overnight. Additional acid (1.6 g), EDC (1 g) and DMAP (0.5 g) was added. After 3 hours, the reaction was worked up in the usual manner and chromatographed to give 2.07 g of protected amine (43).

To a solution of the protected amine (43) (194 mg) in methylene chloride (1.5 mL) was added TES (240 fiL), and TFA (300, uL). The mixture was stirred for 20 minutes and additional TES (50 pL) and TFA (50, ut) was added. After 1 hour, toluene was added and the solvent removed under reduced pressure and then the mixture was worked up in the usual manner. The crude free amine product (44) was used immediately in the next reaction.

To an ice cold solution of the free amine (29) (43.5 mg) in methylene chloride (250 pL) was added mono-t-butyl malonate (8. 3, uL), EDC (12.4 mg) and a trace of DMAP. The ice bath was removed and after 2 hours, the reaction was worked up in the usual manner. Chromatography gave 44 mg of amide (45).

To a solution of the amide (45) (44 mg) in methylene chloride (0.5 mL) was added TES (90 pL) and TFA (100 uL). After 2 hours, toluene was added and the solvent removed under reduced pressure. The mixture was worked up in the usual manner to give 44.2 mg of free acid (46).

To an ice cold solution of the free acid (46) (41 mg) and the free amine (44) (26.3 mg) in methylene chloride (500 uL) was added EDC (13 mg) and the mixture was stirred for 30 minutes. Additional EDC (5 mg) and DMAP (2 mg) was added and after 1 hour, the reaction was worked up in the usual manner. Chromatography gave 32.7 mg of coupled compound 47 as an oil. 0 0 O-kCliH23 0) CIIH23 -P-0 0'-"'C7H P- HN 0 HN y 27 HN 0 Na HN y C13H27 ho il 1. Pd (PPh3) 4, PhSiH3 fi 0 So !) 2. DEAEChromatography \-0 HN C11H23 HN O OCljl23 'I-P-0 15 y Na 0 Na HNC13H27 /o !) % 47 O 40

To a solution of the coupled compound (47) (32.7 mg) in degassed chloroform (1.5 mL) in a dry box was added PhSiH3 (8.5 KL) and Pd (PPH3) 4 (11 mg). The mixture was removed from the box and cooled in ice. After 5 minutes, the ice bath was removed and after 1 hour, chloroform: methanol : water (2: 3: 1) was added and the mixture stirred for 15 minutes and stored in the freezer overnight. The mixture was then poured onto a DEAE chromatography column. Chromatography gave 13.9 mg of a compound (48) as a white powder after 0.1 N NaOH treatment followed by lyophilization.

Example 4: Chiral Urea-Type 1 0 O ° C11H23 O o-p-o. M°C7H15 O HNUCI3Hz A..,,/- q HN, CigH27 O O C11H23 Phosgene HN O'I 0 HN H N )) CllH23 O o HÑrCt3H27 0 HNy C13H27 z 49 49

To a solution of amine (44) (46.1 mg) in toluene was added saturated sodium bicarbonate (0.5 mL) followed by phosgene (15 I1L of a 1.93 M solution in toluene).

After 30 minutes, additional phosgene (10 uL) was added. After 2 hours, additional phosgene (5 uL) was added. The reaction was quenched with aqueous sodium bicarbonate and worked up in the usual manner to give 29.6 mg of urea (49) with protected phosphates. 0 0 o o O O C11H23 o °C11H23 O-P'O'C7H /-'6 HÑ Ci3Hs7 1. Pd (PPh3h, PhSiH3//o NaHÑ C13H27 ) 0 2. DEAE Chromatography HN) ==o 0 J HN % OXC11H23, ° C11H23 _P_7Hls 0 HNC13H27 0 Na HN C13H27 in il 0 49 O 50

To a solution of the urea with protected phosphates (49) (29.6 mg) in degassed chloroform (1.5 mL) in a dry box was added PhSiH3 (8 pL). The reaction vessel was removed from the dry box and placed on ice. Pd (PPh3) 4 (10 mg) was added and after 5 minutes the ice bath removed. After 1 hour, the reaction was quenched by addition of chloroform: methanol : water. The mixture was stirred for 15 minutes and stored overnight in the freezer. It was chromatographed on DEAE to give 24.1 mg of (50) as a white powder after 0.1 N NaOH treatment followed by lyophilization.

Example 5: Chiral Urea-Type 2 0 0 0 ll H N p CH23 Trichloromethylchloroformate p=C-N p C » H23 0 1 0'-'r-0----C, H15-0 101^o---, -c7H, 5 1 /HNCH27/HN. CH27 0 0 29 51

To an ice-cold solution of trichloromethyl chloroformate (2.6 pL) in methylene chloride (200 pu) was added a solution of free amine (29) (35 mg) and 1,8-bis- (dimethylamino)-naphthalene in methylene chloride (200 u, L). After 5 minutes, the ice bath was removed. After 15 minutes, additional methylene chloride was added and the mixture worked up in the usual manner. Chromatography gave 9.4 mg of isocyanate (51) as an oil.

To a solution of the isocyanate (51) (9.4 mg) in methylene chloride (0.2 mL) was added a solution of the amine (44) (10.3 mg) in methylene chloride. After 15 minutes, the reaction was worked up in the usual manner. Chromatography gave 5.5 mg of coupled compound (61) as an oil. 1 o o crc3 o oc3 8 o cl 1 H23, °, o) 4cl 1 H23 HN// ;) HNC13H27 1. Pd (PPh3) 4 HN'Na 0 HN C13H27 HN 3 HN u 13 27 HN 0 HN 0 p O p O Jk 2. DEAE HN JX 0 CIH itography V--\ 0 0 ClIH23 O-pu °o C7Hls ° tPoz O C H /HNUC13H2 HIVC13H2 o 0 /> 0 0 61 62

To a solution of the coupled compound (61) (25.2 mg) in degassed chloroform (0.5 mL) in a dry box was added PhSiH3 (6. 6 uL) and Pd (PPH3) 4 (8. 8 mg). The mixture was removed from the box and cooled in ice. After 5 minutes, the ice bath was removed and after 1 hour, chloroform: methanol: water (2: 3: 1) was added and the mixture stirred for 15 minutes and stored in the freezer overnight. The mixture was then poured onto a DEAE chromatography column. Chromatography gave 7.5 mg of (62) as a white powder after 0.1 N NaOH treatment followed by lyophilization.

Example 6: Chiral Glycerol Analogue of Type 1

To a stirred suspension of sodium hydride (145.5 mg of 60% oil dispersion washed with hexanes) in DMF (12 mL) was added (S)- (+)-alcohol (63) (0.41 mL) in 8 mL of THF dropwise over a 1 hour period. The mixture was stirred for an additional 30 minutes followed by a dropwise addition of the tosylate (4) (0.789 g) in 10 mL of THF over a 10-minute period. The resulting reaction mixture was stirred overnight.

The usual work up gave 0.56 mg of the desired adduct (65).

A solution of lauric acid (1.40 g), EDC (1.35 g), DMAP (0.04 g) and the alcohol adduct (65) (0.564 g) in 4 mL of methylene chloride was stirred for 15 hours at room temperature. Brine and saturated aqueous sodium bicarbonate (1: 1) were added and the mixture extracted with methylene chloride. The mixture was worked up in the usual manner and chromatographed. The desired fraction was dissolved in 20 mL of 4: 1 acetic acid: water and stirred for 15 hours. The solvent was removed under reduced pressure and the residue chromatographed to give 0.77g of semi-solid diol (66).

A solution of the diol (66) (0.22 g), DMAP (6.3 mg), TEA (100 pL) and TBDPSCI (164 1L) was stirred for 24 hours at room temperature. Methanol (2 mL) and a trace of aqueous hydrochloric acid was added followed by extraction with methylene chloride. The mixture was worked up in the usual way. The residue was chromatographed to give 0.3 g of alcohol (67) as an oil.

A solution of the alcohol (67) (0.3 g), DMAP (5.5 mg), EDC (258 mg), myristic acid (308 mg) in methylene chloride (4 mL) was stirred for 18 hours at room temperature followed by the addition of brine and saturated sodium bicarbonate. The mixture was worked up in the usual way and chromatographed to give 0.4 g of silyl protected ether product (68).

To a solution of the silyl protected ether (68) (195 mg) in acetonitrile (2.7 mL) was added 48% hydrofluoric acid (0.756 ml). After 30 hours, saturated sodium bicarbonate was added and the mixture worked up in the usual way. Chromatography gave 94.7 mg of free alcohol (69). BocHN 0 N -0 II J - O C H 11 23 BocHN \ O CH BocHN p HO-OC7Hts BocHN o OO~\o C7H15 13 27 O 69 2. Oxone,/@ o 70

To a solution of the free alcohol (69) (57 mg) in methylene chloride (0.5 mL) was added tetrazole (15.6 mg) and phosphorylating reagent (11) (40 mg) at room temperature. After four hours, the mixture was cooled to 0°C followed by the addition of oxone (82 mg) in THF (0.5 mL): water (0.6 mL). The mixture was warmed to room temperature and stirred for 80 minutes. The final reaction mixture was worked up in the usual manner. Chromatography gave 72 mg of protected phosphate (70). o u 0 OCHzg ~/1 o+ozC7Hr5 , HN {° Õ C13H27 n A."\ OCisH 11 H llH23 1. TFA, TES BocHN P / 0 C13H27 0 0'0 C13H 7 15 0 0 2. Malonic acid, EDC I I H ak -0----C. H. (orC, 3H27 z 71 To a solution of the protected phosphate (70) (72 mg) in methylene chloride (1 mL) was added TES (120, uL) and trifluoroacetic acid (0.6 mL) followed by stirring for 1 hour. The TFA was removed under reduced pressure followed by azeotroping

with 10 mL of toluene. 20 mL of methylene chloride was added and the mixture was worked up in the usual manner to give 0.52 g of an oil.

The crude amine was dissolved in methylene chloride (0.7 mL) followed by the addition of malonic acid (4.5 mg) and EDC (25.6 mg). The mixture was stirred overnight and worked up in the usual way to give 32.5 mg of the dimer product (71). 0 0 0 9) CllH23 i 7H15 O P DppHis HN {° o>gcr3H27 HN O Na O C13H27 1. Pd (PPh3) a PhSiH3 O II O » 0 0 b (O HN C11H2a HN p H CttHza 0 1-0-+-'-O---C7HIS 2. DEAE Chromatography 0 9 õC13H27 o Na 0.., Ci3Hz7 II II 71 0 0 72

The protected dimer (71) (32.5 mg) from the preceding reaction was dissolved in degassed chloroform (2 mL) and PhSiH3 (8.6 uL) was added in a dry box. The mixture was removed from the dry box and Pd (PPh3) 4 (22.6 mg), previously weighed in the dry box, was added. After 2 hours, the mixture was chromatographed on DEAE to give 27.9 mg of white solid (72) after the addition of 1N sodium hydroxide (34.2 pL) followed by lyophilization.

Preparation of ER-804253

The alcohol (558 mg) was dissolved in methylene chloride (5 mL) cooled to 0°C and triethylamine (0.466 mL) was added under a nitrogen atmosphere. After stirring for 5 minutes methanesulfonyl chloride (0.142 mL) was added dropwise. The mixture was stirred for an additional 5 minutes at 0°C and then warmed to room temperature.

After stirring for an additional hour, the mixture was worked up with sat. sodium bicarbonate, extracted with ethyl acetate and the extract washed with water, dilute aqueous hydrochloric acid, water, brine, dried and the solvent removed to give 630 mg.

The mesylate (630 mg) and sodium azide (299 mg) were dissolved in DMSO (6 mL) and heated to 60°C for 90 minutes. After cooling to room temperature the reaction mixture was diluted with methylene chloride, washed with water and brine. After extracting the aqueous washes the combined organic was dried, concentrated, purified over silica gel using a 4: 1 ratio of hexanes to ethyl acetate and the dried product fractions give 420 mg.

The azide (295 mg) was dissolved in ethanol (5 mL) and Lindlar catalyst (200 mg) was added. After stirring under an atmosphere of hydrogen gas at atmospheric pressure, the filtered solution was dried to give 274 mg.

The amine (930 mg) was dissolved in THF (10 mL) and sat. sodium bicarbonate (22 mL). After stirring for 5 minutes, lauroyl chloride (0.712 mL) was added dropwise over 20 minutes. The final mixture was extracted with chloroform, dried to give 1.45 g.

The protected amine from the above reaction was dissolved in ice cold methylene chloride (1 mL), trifluoroacetic acid (1 mL) was added and the mixture stirred for 1 hour. The TFA was removed and the mixture purified to give 106.7 mg.

The amine was dissolved in methylene chloride (3 mL) and saturated. sodium bicarbonate solution (3 mL) was added. The mixture was cooled in ice and phosgene in toluene (0.55 equiv. ) was added dropwise. The mixture was stirred for 20 minutes and worked up to give 112.3 mg.

To a solution of the blocked phosphate (40.5 mg) in ice cold chloroform (2.6 mL) was added phenylsilane (10.7 mg) and tetrakis (triphenylphosphine) palladium [0] (28.7 mg) and the mixture stirred for 1 hour. The mixture was chromatographed on a DEAE column to give 27.7 mg of ER-804253.

Preparation of ER-804130

The amide (1.11 g) was dissolved in methanol (14 mL) and 4 N hydrochloric acid (8 mL) added. The mixture was stirred for 1 our at 50°C and then concentrated.

Methanol (16 mL) and 40% sodium hydroxide (8 mL) was added and the mixture refluxed for 1 hour. It was cooled, extracted with methylene chloride and the extract washed with water, dried and the solvent removed to give 930 mg.

The amino alcohol was dissolved in THF (6 mL) and saturated sodium bicarbonate added (13 mL). After 5 minutes the mixture was cooled to 0 ° C and myristoyl chloride (300 u, L) added. After 30 minutes, the mixture was worked up in the usual way to give 430 mg.

The alcohol (101.7 mg) was dissolved in ice cold methylene chloride and phosphorylating reagent 11 (90 pal) was added and the mixture stirred for 30 minutes.

Ice cold oxone (166.3 mg) was added and the mixture stirred for 30 minutes. The reaction was quenched with thiosulfate. The mixture was worked up the usual way and chromatographed to give 174 mg (not purified)

To a solution of the amide (3 g) in THF (65 mL) at-78 ° C was added an equivalent of butyllithium, followed by a solution of nonanoyl chloride in THF (6 mL). Aqueous ammonium chloride was added and the mixture worked up in the usual manner to give 5.35 g.

To a solution of the alcohol (5 g) in ice cold methylene chloride (100 mL) was added triethylamine (4.1 mL) and mesyl chloride (2.1 mL). The mixture was stirred for 4 hours and worked up in the usual manner to give 6.99 g.

To the solution of the mesylate (6.99 g) in ice cold DMF (100 mL) was added potassium bromide. The mixture was allowed to warm to room temperature and stirred for five hours. It was worked up in the usual manner to give 4.63 g of clear oil.

A solution of the amide (2. 8 g) in THF (15 mL) was added to a-78 °C solution of sodium bis-trimethylsilylamide in THF (15 mL). After 1 hour, the bromide was added and the mixture allowed to warm to room temperature and worked up in the usual manner to give 1.02 g.

To a solution of the olefin (1.02 g) in EtOAc was added palladium on carbon (126 mg) and the mixture placed under hydrogen. After 4 hours, the mixture was worked up in the usual manner to give 1.0 g. To a solution of the amide (1.0 g) in ice cold THF (20 mL) was added water, hydrogen peroxide and lithium hydroxide. The next day, the mixture was worked up in the usual manner to give 590 mg of acid.

To a solution of the acid in ice cold THF (10 mL) was added diborane: THF complex and the mixture allowed to warm slowly. After seven hours, dilute hydrochloric acid was added carefully and the mixture worked up in the usual manner. The crude material was dissolved in ice cold ether and LAH solution (2 mL, of 1M) added.

After 5 minutes, the mixture was worked up in the usual manner to give 556 mg of the alcohol.

To a-78 °C solution of oxalyl chloride (2.2 mL) in methylene chloride (10 mL) was added DMSO (1. 1 mL) and after 2 minutes the alcohol (556 mg) was added in methylene chloride (5 mL). After 20 minutes, triethylamine (1 mL) was added and the mixture warmed to 0° C. The mixture was diluted with ether and worked up in the usual manner to give 567 mg. The Wittig reagent (679 mg) was suspended in THF (10 mL) and KHMDS solution (4 mL of 0.5 M) added. After 20 minutes, the mixture was cooled to-78 °C and the aldehyde (567 mg) in THF (5 mL) was added. After 15 minutes, the mixture was worked up in the usual manner to give 342 mg.

To a solution of the enol ether (342 mg) in acetonitrile (3.5 mL) and water (0.15 mL) was added hydroiodic acid. After 4 hours, the mixture was worked up in the usual manner to give 325 mg.

To a solution of the aldehyde (325 mg) in methanol (10 mL) was added sodium borohydride (38 mg). After 3 hours, the reaction was worked up in the usual manner to give 303 mg of the alcohol.

To an ice cold solution of the alcohol (303 mg) in methylene chloride (10 mL) was added triethylamine (150 pL) and mesyl chloride (76 iL). After 4 hours, the reaction was worked up in the usual manner to give 352 mg.

To a solution of the oxazoline (1 mL) in ice cold THF (5 mL) was added potassium t- butoxide solution (2.2 mL of 1M). After 30 minutes, the mesylate was added in THF (5 mL) and the mixture stirred for 8 hours. The usual work-up gave 318.5 mg.

A solution of the oxazoline in methanol (8 mL) and hydrochloric acid (4 mL of 4M) was warmed to 50 ° C for 90 minutes. Additional methanol was added and the solvent removed. The residue was dissolved in methanol (8 mL) and sodium hydroxide solution (4 mL) and briefly warmed to 50 ° C. The mixture was cooled and extracted with chloroform. The usual work-up gave 114 mg. To a solution of the amine (114 mg) in THF (2 mL) and saturated aqueous sodium bicarbonate (2 mL) was added the acid chloride. After 30 minutes additional acid chloride was added. After 30 minutes, the reaction was worked up in the usual manner to give 146 mg.

To a solution of tetrazole (48 mg), the phosphorylating reagent (122 mg) in ice cold methylene chloride (2 mL) was added the alcohol (146 mg). Oxone (230 mg) in water (1 mL) and THF (2 mL) was added. After 90 minutes, thiosulfate was used to quench the reaction. Standard work up gave 140 mg. To a solution of the substrate in ice cold methylene chloride was added triethylsilane (370, uL) and trifluoroacetic acid (110 pL). After 5 minutes, the volatiles were removed to give 148 mg.

To a solution of the amine (66.9 mg) in ice cold methylene chloride (0.8 mL) was added saturated aqueous sodium bicarbonate (0.8 mL) and phosgene solution (20 uL of 1. 93 M). After one hour, additional phosgene (10 uL) was added. After 30 minutes, the usual work-up gave 47.7 mg.

To a solution of phenylsilane (15 uL), tetrakis (triphenylphosphine) palladium [0] (24. 8 mg) in ice cold chloroform under an inert atmosphere was added the substrate.

After 5 minutes, the mixture was applied to a DEAE column and chromatographed to give 31 mg of ER-804130.

Preparation of ER-804558

To a solution of the amine (325 mg) in methylene chloride was added triethylamine (321 pL) and 1-dodecanesulfonyl chloride. After 3 hours, the usual work up gave 384 mg.

To a solution of the protected alcohol (384 mg) in THF (4 mL) was added tetrabutylammonium fluoride (123 mg) and acetic acid (29 uL). After 2 hours, the usual work up gave 180 mg.

The remainder of the synthesis was completed as outlined above for other compounds of the present invention, i. e. phosphorylating, deblocking, coupling with phosgene, and deprotecting with phenylsilane and palladium.

Preparation of ER-804442 The diol amine was mono-protected as its t-butyl-diphenylsilylether outlined above.

The amine (2.6 g) and benzophenone imine (1.1 mL) were mixed and heated to 40 ° C for 4 days to give after chromatography 3.3 g.

To a solution of the imine (3.3 g) was in ice cold methylene chloride was added lauric acid (1.5 g), EDC (1.7 g) and DMAP (155 mg). The next day, the reaction was worked up in the usual manner to give 3.15 g. To a solution of the imine (3.14 g) in ether was added 1 N aqueous hydrochloric acid.

The next day, the reaction was worked up in the usual manner to give 2.81 g.

To a solution of trichloromethylchloroformate (12 N. L) in ice cold methylene chloride (250 pL) was added dodecylamine (18 pL) and diisopropylethylamine (27 p1L). After 30 minutes, the solvent was removed. The residue was dissolved in ice cold methylene chloride, to which was added the amine (55.6 mg) and additional diisopropylethylamine (13 suL). After 2 hours, the usual work up gave, after chromatography, 60.9 mg.

This product was de-protected with fluoride, phosphorylated, de-protected with TFA, dimerized with phosgene, and un-blocked with phenylsilane and palladium as outlined above to give ER-804442.

Preparation of ER-804221 To an ice cold solution of glycine (8.26 g) in aqueous sodium hydroxide (4.4 g in 60 mL) was added lauroyl chloride (21.8 g). After 1 hour, acid was added and the mixture worked up in the usual way. Recrystallization from ethyl acetate gave 9.7 g. To an ice cold solution of the amine (1.4 g) in methanol was added triflic azide (20 mg). The next day, additional azide was added. After 2 hours, the reaction was worked up in the usual manner to give after chromatography 1.14 g.

To a solution of the alcohol (1.14 g) in methylene chloride was added t-butyl- diphenylsilyl chloride (1.09 mL), triethylamine (1.8 mL) and DMAP (50 mg). After 3 hours, the usual work up gave 1.4 g.

To a solution of the alcohol (1.4 g) in ice cold methylene chloride was added lauric acid (826 mg), EDC (1.05 g) and DMAP (33 mg). The next day, the usual work up gave after chromatography 778 mg. To a solution of the azide (778 mg) in THF was added acetic acid (77 fiL) and TBAF (323 mg). The next day, the usual work up gave, after chromatography, 428 mg.

To a solution of the azide (460 mg) in methylene chloride was added tetrazole (165 mg), the phosphorylating reagent (390 mg), and after 30 minutes, oxone in water (722 mg in 3 mL). The reaction was quenched with thiosulfate. Usual work up, after chromatography, gave 392 mg.

The protected amine (460 mg) was dissolved in methylene chloride and trifluoroacetic acid (394 ZL) and triethylsilane (308 pL). After 1.5 hour, the usual work up gave, 392 mg.

To an ice cold solution of the amine in methylene chloride (5.5 mL) was added saturated sodium bicarbonate (5.5 mL), and phosgene (164 pL of a 1.93 M solution in toluene). After 15 minutes, the usual work up, after chromatography gave 342 mg.

To a ice cold solution of the azide (187 mg) in methylene chloride was added the tin reagent (1.5 mL) which was prepared as outlined in U. S. 5,756, 718 incorporated herein by reference. After 30 minutes, the mixture was chromatographed to give 187 mg.

To a ice cold solution of the urea (55 mg) in methylene chloride was added the acid (59 mg) (prepared as above) and EDC (44 mg). The next day, additional EDC (5 mg) and acid (5 mg) was added. After 2 hours, the normal work up provided 45.7 mg.

Normal removal of the protecting groups with phenylsilane and palladium gave ER- 804221.

ER-804222 was prepared in a similar manner except that the condensation product between lauryl chloride and glycine, 15-methylmyristic acid was used.

Preparation of ER-804281 To a ice cold solution of the protected alcohol (8.3 g) in acetonitrile: water was added CAN (41.4 g). After 1 hour, the usual work up gave 5.7 g.

A solution of the alcohol (5.63 g) in 4 N HCl solution was heated to reflux for 1 hour, cooled, neutralized with sodium hydroxide and worked up in the usual manner to give 2.1 g.

To an ice cold solution of the alcohol (2.2 g) in rmiethylene chloride was added imidazole (0.7 g), t-butyl-diphenylsilyl chloride in 15 mL of methylene chloride. The next day, the usual work up gave 1.54 g. To a solution of the alcohol (1.93 g) in methylene chloride (40 mL) was added benzophenone imine (0.8 mL). After 1 day, the mixture was heated to reflux overnight. The usual work up gave 1.67 g.

To an ice cold solution of the alcohol (1.67 g) in methylene chloride was added DMAP (159 mg), EDC (0.99 g) and lauric acid (1.04 g). After one day, the usual work up gave 74% yield.

To an ice cold solution of the imine (2.9 g) in ether (50 mL) was added 1 N HCl (50 mL). The next day, the usual work up gave 2.09 g.

To a solution of the amine (1.24 g) in dichloroethane was added sodium cyanoborohydride (178 mg) and tetradecanal (411 mg). The next day, the usual work up gave 1.5 g.

To a ice cold solution of the amine (221 mg) in dioxane was added allyl chloroformate (40 mg) and 308 FLL of 1 N NaOH solution. After 2 hours, the usual work up gave 200 mg.

To an ice cold solution of the protected alcohol (365 mg) in THF was added TBAF (1924 iL) and acetic acid (122 1L). The next day, the usual work up gave 271 mg.

This material was phosphorylated, deblocked with TFA, dimerized with phosgene and the allyl protecting groups removed with phenylsilane and palladium as described above to give ER-804281.

Preparation of ER-804339 ER-804281 ER-804339 To a ice cold solution of ER-804281 (7 mg) in methylene chloride was added triethylamine (5 pL), DMAP (0.6 mg) and acetyl chloride (1. 8 µL). After 4 days, the usual work-up gave 1.1 mg.

Preparation of ER-804674 ER-804281-3 ER-804674 To a solution of ER-804281 (12.7 mg) in THF (1.0 mL) was added methyl iodide (9.2 mg) and sodium bicarbonate (6.8 mg). The mixture was stirred for 5 days and sodium bicarbonate (14 mg) and additional methyl iodide (8 mL) was added. After an additional 3 days, additional bicarbonate (28 mg) and Mei (16 uL). After an additional 6 days, the mixture was worked up to give 9.1 mg of product.

Preparation of ER-804596

To a solution of the alcohol (393 mg) in methylene chloride (2 mL) was added diisopropylamine (210 vuL), tetrazole (105 mg) and phosphorylating reagent (as described above) (488 mg). After 2 1/5 hours, the usual work up gave the desired product. To a solution of the diol (73 mg) in acetonitrile was added tetrazole (175 mg), the azide (1 equivalent). After 3 hours, the mixture was cooled and ozone (1229 mg) added. The next day, usual work up gave the desired product.

To an ice cold solution of the protected alcohol (92.9 mg) in acetonitrile: water (6mL: 1.5 mL) was added CAN (358 mg). After 1 hour, the usual work up provided 68.5 mg.

To an ice cold solution of the diol (68.5 mg) in methylene chloride was added lauric acid (76.5 mg), DMAP (4.7 mg) and EDC (73 mg). The next day, the usual work up gave 76.5 mg.

The azides were reduced using the tin reagent described above. The diamine was acylated with dodecanoyl chloride, and the protecting groups removed with phenylsilane and palladium as described above to give ER-804596.

Preparation of ER-804732 The alcohol (7.04 g) was dissolved in methylene chloride (300 mL) with triethylamine (11.13 mL) and then cooled to 0°C under a nitrogen atmosphere. Methanesulfonyl chloride (3.69 mL) was added dropwise after which time the reaction was stirred at room temperature for 1 hour. The usual work up gave 5.551 g.

The mesylated (1.114 g) was dissolved in DMF (30 mL) followed by sodium azide (0.9337 g). The reaction mixture was warmed to 57 °C and stirred for 16 hours and then to 104 °C for and additional 3 hours. After cooling to room temperature the mixture was worked up in the usual manner and gave 0.466 g. The protected aminoalcohol (0.466 g) was hydrolyzed using 4 N HC1 (15 mL) at 107 °C for 3 hours. After cooling to room temperature, the reaction mixture was filtered and extracted with ethyl ether, dried, concentrated and used in the next reaction.

The crude aminoalcohol was dissolved in THF (5 mL) with saturated sodium bicarbonate (6 mL) and cooled to 0 °C. Myristoyl chloride (0.79 mL) was added dropwise after which time the reaction was warmed to room temperature and stirred for 2 hours. The reaction mixture was worked up using the usual methods and gave 0.751 g.

The alcohol (0.185 g) was dissolved in DMF (3.0 mL) with imidazole (0.077 g) and tert-butyldiphenylsilyl chloride (0.197 mL). The reaction mixture was stirred at room temperature for 16 hours after which time the usual work up gave 0.320 g.

The azide (0.975 g) was dissolved in methanol (20 mL) with 10 % palladium on carbon (0.180 g). The mixture was stirred under an atmosphere of hydrogen gas under atmospheric pressure for 2 hours after which time the gas was evacuated and the mixture filtered over Celite 545 and concentrated. Purification using the usual methods gave 0.873g.

DMSO (1.5 mL) was added dropwise to oxalyl chloride (0.92 mL) in methylene chloride (30 mL) at 78 °C. After stirring for 15 minutes the alcohol (1.727 g) in methylene chloride (30 mL) was added dropwise and stirred for an additional 30 minutes. Triethylamine (4.90 mL) was added dropwise, the reaction was warmed to 0 °C and quenched using saturated ammonium chloride. Purification of the crude product using silica gel chromatography with 20 % ethyl acetate in hexanes gave 1.653 g.

The primary amine (0.135 g) and aldehyde (0.077 g) were dissolved in 1,2- dichloroethane (5 mL) followed by the addition of sodium cyanoborohydride (0.032 g). The reaction was stirred for 20 hours after which time acetic acid (0.02 mL) was added and the reaction worked up in the usual manner to give 0.103 g.

The secondary amine was dissolved in 1,4-dioxane (15 mL) and cooled to 0 °C followed by the slow addition of 1 M sodium hydroxide (3.0 mL). After stirring for 10 minutes allyl chloroformate (0.236 mL) was added dropwise after which time the reaction was warmed to room temperature and stirred for 16 hours. Work up in the usual manner gave 0.613 g.

The para-methoxybenzyl ether (0.613 g) was dissolved in a 4 to 1 ratio of acetonitrile to water (15 mL), cooled to 0 °C and then CAN (1.525 g) was added. The reaction mixture was stirred at 0 °C for 2 hours and then worked up in the usual manner to give 0.357 g.

The alcohol (0.357 g) was dissolved in methylene chloride (5 mL) with lauric acid (0. 184 g), EDC (0.175 g) and cooled to 0 °C. 4-Dimethylaminopyridine (0.012 g) was

added and the resulting mixture was stirred at room temperature for 2 hours. Work up in the usual manner gave 0.436 g.

The silyl protected alcohol (0.211 g) was dissolved in THF (5 mL) with acetic acid (0. 03 mL). Tetrabutylammonium fluoride (0.115 g) was added in one portion and the reaction mixture was stirred at room temperature for 16 hours. A normal work up gave 0.150 g.

This material was phosphorylated, deblocked with TFA, dimerized with phosgene and the allyl protecting groups removed with phenylsilane and palladium as descried above to give ER-804732.

Preparation of ER-804680 The aldehyde (1.54 g) was dissolved in THF (28 mL) and cooled to 0 °C after which time 2-methyl-2-butene (14 mL) and tert-butyl alcohol (28 mL) was added. A stirred suspension of sodium chlorite (3.70 g) and sodium trihydrogen phosphate (4.09 g) in water (42.7 mL) was added to the above mixture and stirred at 0 °C for 1.5 hours. The completed reaction was diluted with ethyl acetate (100 mL) and washed with 10 % sodium bisulfite, brine, dried, concentrated and silica gel chromatographed to give 1.55 g.

The amine (0.553 g) and acid (0.381 g) were mixed in methylene chloride (8 mL) and cooled to 0 °C after which time EDC (0.230 g) was added and the reaction mixture was stirred at room temperature for 72 hours. The usual work up gave 0.567 g.

The methoxybenzyl ether (0.567 g) was dissolved in a 1 to 1 ratio of acetonitrile to water (16 mL) with methylene chloride (8 mL) and cooled to 0 °C. CAN (1.53 g) was added and the reaction mixture was stirred for 1 hour after which time it was worked up in the usual manner to give the crude alcohol.

The crude alcohol from above was dissolved in methylene chloride (15 mL) with lauric acid (0.280 g) and 4 dimethylaminopyridine (0.017 g). The reaction mixture was cooled to 0 °C and EDC (0.267 g) was added in one portion after which time the reaction mixture was warmed to room temperature and stirred for 16 hours. Normal work up procedures provided 0.622 g.

The silyl ether (0.563 g) was dissolved in THF (10 mL) with acetic acid (0.087 mL).

Tert-butylammonium fluoride (0.330 g) was added and the reaction was stirred at room temperature for 16 hours. Work up in the usual manner gave 0.384 g.

This material was phosphorylated, deblocked with TFA, dimerized with phosgene and the allyl protecting groups removed with phenylsilane and palladium as described above to give ER-804780.

Preparation of ER-804679 The protected secondary amine (0.071 g) was dissolved in degassed chloroform (3 mL) with phenylsilane (0.017 mL) and acetic anhydride (0.014 mL). The reaction mixture was cooled to 0 °C followed by the addition of tetrakistriphenylphosphine palladium (0) (0.002 g). The reaction mixture was warmed to room temperature and allowed to stir for 30 minutes. The completed reaction was diluted with methylene chloride, washed with water, dried, concentrated, and chromatographed to give 0. 068 g- The silyl ether was deprotected in THF (5 mL) with acetic acid (0.025 mL) with the addition of tert-butylammonium fluoride (0.092 g). After stirring at room temperature for 16 hours the reaction was worked up in the usual manner to give 0.120 g.

This material was phosphorylated, deblocked with TFA, dimerized with phosgene and the allyl protecting groups removed with phenylsilane and palladium as described above to give ER-804679.

Preparation of ER-804764

DMSO (0.33 mL) was added dropwise to oxalyl chloride (0.203 mL) in methylene chloride (10 mL) at 78 °C. After stirring for 15 minutes the alcohol (0.993 g) in methylene chloride (3 mL) was added dropwise and stirred for an additional 30 minutes. Triethylamine (1.08 mL) was added dropwise, the reaction was warmed to 0 °C and quenched using saturated ammonium chloride. Purification of the crude product using silica gel chromatography with 20 % ethyl acetate in hexanes gave 0.743 g.

The 1.6 M n-butyl lithium in hexanes (1.5 mL) was added dropwise to the phosphonium salt (0.797 g) in THF (10 mL) at 0 °C. After stirring for 30 minutes the aldehyde (0.734 g) in THF (15 mL) was added dropwise. After stirring at room temperature for one hour the reaction was worked up in the usual manner to give 0.193 g.

The enol ether (0.193 g) was hydrolyzed with 57% hydrogen iodide (0.114 L) in acetonitrile (2 mL). After stirring at room temperature for 2 hours the reaction was quenched with saturated sodium bicarbonate, extracted with methylene chloride, and dried to give 0.211 g crude aldehyde.

The crude aldehyde (0.211 g) was dissolved in methanol (3 mL) and sodium borohydride (0.033 g) was added at 0 °C. After stirring for 30 minutes the reaction

was diluted with water, extracted with methylene chloride, dried, concentrated and purified by silica gel chromatography to give 0.148 g.

This material was phosphorylated, deblocked with TFA, dimerized with phosgene and the allyl protecting groups removed with phenylsilane and palladium as described above to give ER-804764.

Preparation of ER-804772 The commercially available diol (1.486 g) was mixed with the acetal (1.864 g) and para-toluenesulfonic acid (0.195 g) in DMF (10 mL). After stirring for 20 hours at room temperature under a nitrogen atmosphere, the reaction was quenched with sat. sodium bicarbonate, extracted with methylene chloride, dried and concentrated via high vacuum. Silica gel chromatography of the resultant crude product using 10% ethyl acetate in hexanes gave 2.084 g.

The acetal (2.084 g) was cooled to 78 °C under a nitrogen atmosphere in methylene chloride (30 mL) followed by the dropwise addition of 1. 0 M DIBAL in hexanes (14.3 mL). After additional DIBAL (14 mL) was added, the reaction mixture was stirred for 1 hour, warmed to room temperature and quenched with sodium, potassium tartarate. The normal work up gave 2.1 g.

The alcohol (1.286 g) was mixed with triethylamine (0.883 g) in methylene chloride (15 mL) and cooled to 0 °C. Methanesulfonyl chloride (0.575 g) was added dropwise followed by stirring for 20 minutes at O°C and room temperature for 2 hours. The normal work up gave 1.496 g.

The alcohol (1.495 g) in DMF (10 mL) was added dropwise to a stirring suspension of washed 60 % sodium hydride (0.257 g) in DMF (20 mL) at O °C. After stirring for 3 hours the mesylate (0.925 g) in DMF (10 mL) was added dropwise. After stirring for an additional 3 days, the reaction was quenched and worked up in the usual manner gave 0.905 g.

As with examples provided above, the para-methoxybenzyl protecting group was hydrolyzed with CAN, the protected amino alcohol hydrolyzed using aqueous HCl then KOH, acylation of the amine with tetradecanoyl chloride, silylation of the primary alcohol with TBDPS, acylation of the secondary alcohol with dodecanoyl chloride, and hydrolysis of the silyl protecting group using TBAF to give the primary alcohol. This material was phosphorylated, deblocked with TFA, dimerized with phosgene and the allyl protecting groups removed with phenylsilane and palladium as described above to give ER-804772.

Preparation of ER-804947 The alcohol (0.263 g) in THF (5 mL) was added dropwise to washed 60% sodium hydride (0.216 g) in DMF (2.0 mL) at room temperature under a nitrogen atmosphere.

The reaction mixture was stirred for 30 minutes after which time benzyl bromide (0.272 mL) with a catalytic amount (0.05 g) of tetrabutylammonium iodide. The final reaction mixture was stirred for an additional hour after which time the mixture was quenched and worked up in the usual manner to give 0.365 g.

The protected aminoalcohol (0.189 g) was hydrolyzed using 4 N hydrochloric acid (2.5 mL) followed by 40% sodium hydroxide (2.5 mL) as described previously to provide 0.121 g.

The aminoalcohol (0.121 g) was dissolved in methylene chloride (2 mL) with saturated sodium bicarbonate (2 mL). After cooling to 0 °C, myristoyl chloride (0.199 mL) was added dropwise. After continued stirring for 2 hours the mixture was worked up in the usual manner and gave 0.181 g.

The alcohol (0.181 g) was dissolved in methylene chloride (5 mL) with the acid (0.180 g) and 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDC 0.133 g). The mixture was cooled to 0 °C and 4-dimethylaminopyridine was added follow by stirring for 16 hours at room temperature. The usual work up gave 0.310 g.

The para-methoxybenzyl ether (0.305 g) was dissolved in acetonitrile (8 mL) with water (2 mL) and cooled to 0 °C. Cerium ammonium nitrate (1.110 g) was added and the reaction mixture was stirred for 2 hours after which time using the normal work up gave crude alcohol.

The crude alcohol was dissolved in methylene chloride (8 mL) with lauric acid (0.126 g) and 4-dimethylaminopyridine (0. 011 g). After cooling to 0 °C, EDC (0.119 g) was added and the mixture was stirred at room temperature for 16 hours. The usual work up gave 0.355 g.

The benzyl ether (0.355 g) was dissolved in ethyl acetate (50 mL) with palladium hydroxide (0.048 g) and acetic acid (0.25 mL). The reaction mixture was placed under 50 psi of a hydrogen atmosphere and shaken for 10 hours. Work up in the usual manner gave 0.255 g. This material was phosphorylated, deblocked with TFA, dimerized with phosgene and the allyl protecting groups removed with phenylsilane and palladium as described above to give ER-804947.

Preparation of ER 805718 To a stirred solution of diol (6.90 g) in methylene chloride (100 mL) at room temperature was added triethylamine (6.63 mL) and DMAP (0.50 g) followed by the drop wise addition of tert-butyldiphenylchlorosilane (10. 38 mL). After stirring for 18 h the reaction was worked up in the normal fashion followed by silica gel purification to provide 11.70 g of the desired silyl ether.

To a stirred solution of silyl ether (11.70 g) in methylene chloride (60 mL) at room temperature was added lauric acid (8.60 g). After the mixture became a clear solution the reaction mixture was cooled to 0 °C followed by the addition of EDC (8.3 g) and DMAP (0.35 g). After stirring for 16 h the reaction was worked up in the normal fashion and purified using silica gel chromatography to provide 16.43 g of the desired ester.

To a stirred solution of ester (16.40 g) in THF (100 mL) at room temperature was added acetic acid (2.20 mL) followed by the drop wise addition of 1 M TBAF in THF (33 mL). After stirring for 16 h the reaction was worked up in the normal fashion and purified using silica gel chromatography to provide 10.32 g of the desired alcohol. To a stirred solution of alcohol (1.03 g) in THF (12 mL) at 0 °C was added triphenylphosphine (1.50 g) and diphenylphosphoryl azide (1.24 mL) followed by a drop wise addition of DEAD (1.1 mL). After stirring for 1.5 h the reaction was worked up in the normal fashion and purified using silica gel chromatography to provide 0.95 g of the desired azide. To a stirred solution of azide (0.473 g) in methanol (10 mL) at room temperature under an argon atmosphere was added 10 % palladium on carbon (0.20 g) followed by charging the reaction vessel with hydrogen gas. After stirring at atmospheric pressure for 2 h the reaction was worked up in the normal fashion to provide 0.390 g of the desired amine.

To a stirred solution of amine (0.188 g) and protected L-serine (0.150 g) in methylene chloride (2 mL) at 0 °C was added EDC (0.152 g) followed by DMAP (0.006 g).

After stirring for 16 h the reaction was worked up in the normal fashion and purified using silica gel chromatography to provide 0.292 g of the desired amide.

To a stirred solution of amide (0.292 g) in methanol (7 mL) at room temperature was added para-toluenesulfonic acid (0.70 g). After stirring for 4 h the reaction was worked up using sat. sodium bicarbonate followed by extraction and concentration in the normal fashion. To a stirred solution of the crude intermediate in methylene chloride (7 mL) was added at room temperature triethylsilane (1.2 mL) followed by trifluoroacetic acid (2.1 mL). After stirring for 1 h the reaction mixture was worked up in the normal fashion and purified using silica gel chromatography to provide 0.170 g of the desired amino alcohol. To a stirred solution of amino alcohol (0.170 g) in THF (4 mL) at room temperature was added sat. sodium bicarbonate (4 mL) followed by myristoyl chloride (0.114 mL).

After stirring for 3 h the reaction mixture was worked up in the normal fashion and purified using silica gel chromatography to provide 0.244 g of the desired alcohol.

To a stirred solution of alcohol (0.256 g) in methylene chloride (4 mL) at room temperature was added tetrazole (0.069 g) followed by the phosphorylating reagent (0.163 g). After stirring for 30 minutes a tetrazole (0.065 g) and phosphorylating reagent (0.165 g) were added. The reaction mixture was stirred for an additional 1 h the reaction mixture and then poured over a stirred suspension of oxone (0.601 g) in THF (4 mL) and water (4 mL) at 0 °C. After stirring for an additional 16 hours at room temperature, the reaction was worked up in the normal fashion and purified using silica gel chromatography to provide 0.40 g of the desired protected amino- phosphate.

To a stirred solution of the protected amino-phosphate (0.400 g) in methylene chloride (4 mL) was added at room temperature triethylsilane (0.21 mL) followed by trifluoroacetic acid (0.34 mL). After stirring for 2 h the reaction mixture was worked up in the normal fashion to provide the desired crude amine.

To a stirred solution of the crude amine in THF (3 mL) at room temperature was added sat. sodium bicarbonate (3 mL) followed by a drop wise addition of 20 % phosgene in toluene (0.115 mL). After stirring for 16 h at room temperature, the reaction was worked up in the normal fashion and purified using silica gel chromatography to provide 0.0. 81 g of the desired urea.

To a stirred solution of the urea (0.081 g) in degassed chloroform (2 mL) at 0 °C was added phenylsilane (0.044 mL) followed by tetrakis (triphenylphosphine) palladium (0) (0.115 mL). After stirring for 1 h, the reaction was worked up in the normal fashion and purified using DEAE cellulose ion exchange chromatography, then silica gel chromatography followed by SP Sephadex cation exchange to provide 0.054 g of the ER-805718.

Reaction Scheme for Phosphate Triester Analogues (Formula II) Persons familiar with the art can easily envision the preparation of the phosphate triester analogues depicted in Formula II by altering the phosphorylating reagent 11 that will accommodate the new structures listed. As exemplified in the Scheme below, replacing the allyl-protecting group with the appropriately functionalized substituent provides the desired structure by normal synthetic processes. In general the altered phosphorylating reagent is prepared stepwise by the addition of N-Boc-l- amino-2-ethanol to phosphorus trichloride in the presence of pyridine. After transforming the dichlorophosphorylmonoester to the activated bis (diisoproplyamide), the appropriately functionalized alcohol in the presence of tetrazole is added to provide the desired protected functionality or a precursor thereof. The altered phosphorylating reagent is then used in place of the original phosphorylating reagent 11 described in the general experimental. Instead of deprotection of the allyl group used for the typical synthetic route, the new functionality incorporated into the structure is deprotected by methods available to persons familiar to the art to provide the alternative desired product listed. 0 c. JN J PCI3 + HNHBoc CI P_N HBoc Cl y 4Protected-Z NHBoc. ( Tetrazole Xp o~NHBoc + HO*Protected-Z. N'\ H NHBoc Reaction Scheme for Quaternary Amine Analogues (Formula III) Persons familiar with the art can easily envision the preparation of quaternary amine compounds. As exemplified in the Scheme below, oxidation of an alcohol to an aldehyde, reductive animation with the appropriately functionalized amine,

followed by protection of the ensuing secondary amine with a protecting group such as Fmoc provides the desired protected intermediate. Selective deprotection of the Boc-group on the primary amine followed by condensation with the appropriate linker such as phosgene provides the protected dimer. The final desired product can be produced by the deprotection of the secondary amine followed by dialkylation of the amine in the presence of an simple alkyl halide, such as methyl iodide. The product is purified by cation exchange chromatography using CM-Sephadex using dilute HC1 as the eluting counter ion, followed by silica gel chromatography, and then anion exchange with SP-Sephadex containing the appropriate anionic counter ion using similar elution solvents as described in the previous experimentals. 0 0 o o HNCi3H27 ...,. HN CHz ? HN C13H27 1. Oxidation (Swern) HN) 4C13H27 -O C7H15 > BocHN~ ^ ° C7H15 'u 2. Reduct ! veAnimation H fu OCHss (NaCNBHs) OCHza O t1 23 (NaCNBH3) T O 0 0 OII Fmoc O C11 H23 Hs f HNC13H27 3. Protection (Fmoc20) HN 0 6. Hydrolysis (Peperdine) O 4. Hydrolysis (TFA) HN 0 7. Alkylation (CH31) 5. Coupling (Phosgene) (HN"CigH27 8. Purification (CM-Sephatdex, \NO\ ^/CHS Silica Gel, SP-Sephadex) i 1 Fmoc 0y CliH23 a O o N ° C1 1 H23 0 \ OCHas i c f X'HNyC13H27 HN O ho HN o tx X-HN) C13H27 0y CllH23 OC, iH 0 o BIOLOGICAL EXAMPLES Example 7: Induction of Cytokines (in vitro) A. Assays in human whole blood The most readily available human system to test compound activity on monocytes/macrophages is in whole blood. Various concentrations of compounds of

the invention were added as lOx stocks in 50 ul of Caw, Mg-free Hank's balanced salt solution (HBSS) followed by 50 gl of HBSS into 400 pi ofheparinized whole blood obtained from normal volunteers (18-51 years old; 110-230 lb.) into the wells of plastic assay plates, for a total volume of 500 ul/well (final concentration of whole blood was 80%). After a 3-hour incubation with gentle shaking at 37°C in a 5% C02 atmosphere, the assay plates were centrifuged at 1000 x g for 10 min. at 4° C and plasma was drawn off and frozen at-80° C. Plasma samples were analyzed for TNF- alpha, IL-10, and IL-12 by ELISA (Genzyme Corp. , Cambridge, MA). Each assay point was tested in triplicate.

As shown in Figure 1, compounds such as 100,184 and 186 stimulate blood- borne cells to release TNF-alpha. This stimulatory activity can be compared to that of 10 ng/ml endotoxin (or LPS) present in similar incubations in the same assay. As shown in Table 1, activity of compounds (tested at 10 uM) ranges from inactive (such as compound 110) to compounds demonstrating greater activity than the LPS standard.

B. Cultured human cell lines Similar results can be obtained when compounds of the invention are tested in a cell-culture model. In this assay, compounds of the invention are tested for their ability to stimulate secretion of alkaline phosphatase from THP-1 cells that have been transfected with the gene for secreted alkaline phosphatase under the control of the TNF-alpha promoter, as described in detail in Goto et al., Molecular Pharmacology 49; 860-873 (1996). In this assay, however, the effects of removing serum-a condition that may more-likely mimic a subcutaneous environment-can be evaluated.

As shown in Figure 2 and described in Table 1, results from these assays indicate that compounds of the invention stimulate induction of genes under the control of the TNF-alpha promoter when added to cells in the absence as well as the presence of serum. 1 This is important to determine if serum components such as lipopolysaccharide binding protein are necessary for drug activity.

Table 1. Stimulation of cytokine release by compounds in vitro ER# Compound Whole Blood THP-1 cell Stimulation (% of (% of LPS at compound 100 at 10 uM)'" 10aM) +serum-serum MPL Standard 29 (2) iS 112022 131 + 10. 2 0 (n=6) 0--OOC, H,., =o o o O O i), o 0 (O CJc6H, 3 H O Na H aC,, 14. d b 111230 49 O Q) >C2Hrs 0 ou QsH, s HN 0 N. H) r^y CIIH2 (t O 1 sO p H o p O-t-Oto~C7H O Na HNn CIrHa3 //fi 23 O O 111231 17 0 O OJc6Hst o_p oYo~c, H, s HN O NaHN) Cr1H2s (W O gO o Q C6H" "¢ OOW s O Na HNn CerH2s O O 111232 158 155 225 0 p 9""sHis ., M 0 Na HN, C,, H,, 0 IH13 HX O Na HNn C"H3 Q C6H,, O'uv-CsHna HN-- O o->o~||to~c, Ht, 0 Na HN ^'C, Hz3 1f lf 0 0 111233 141 0 p Q""zsHta -P'OCHns O Né C3H O O --'-p HN Q 112043'0 112043 b Na HNwC » Hz3 O O 112043 0 OH ii 0 Na HNCHzs 0 0 O O o 9" 1 I or'H15 -o Na HN. CjH,, O Na HN) C1H23 o ri 112044 0 go p oh O--O O H 0 Na HNC, H,, O O CH3 0 O pu mom H O Na HN H' 0 °b c6H" 0 112047 0 OH ici O_p OX OH HNs o Na HN) C"H23 0 iry 2 0 ho oh M7Hts o Na HN) C1rH23 0 0 0 0 112048 0 24 p oh wP-o'1^o'c H H ; O eH c6H CBH13 0 0 H0 go His Õ Na HN),-yG7H, s O O) r-,,,-, _=, C6Hi3 ''2 ° 112049 0 OH 112049 UN O OH (X e II 2 O O HN OH b MpHs 0 Na HN) C1sH23 0 0 112063 0 0 p 9''sH, 3 HN 0 Na yy OllH23 () , 0 0 0 O O 2 O HN OH 0-'r'- ^ H 2064 50 fl II O O 112064 50 0 0 0 9 CHsa HNs o Na HN) C"H23 0 0 0 0 HN O-G1yH23 O O H o Na HN) C,, Hs3 0 0 112065 86 o Q c, 1H23 0 0 oc"H , 0 Na HN. . C, (t o HN O o cH33 0 2Q O HN 0 0) CIIH23 I 7H, 5 ) =o 5 5 C o'O Na HN) CH23 0 0 112066 162 330 0 0 CIIH2a o O 0 Na N , » H2 s i-o o or o HN OCnHza o 0 0'C,, H o_p_otOC7H, s /=0 0 0 J ! O Na HAN,, C,, H23 0 0 O O 2071 0 O , pp-OO O'Na 0 zozo O'Na" b P-OO O HN O 112072 0 0 O o (cHzlsCHa (_O Nal < (CH2) sCH O'Na I (cH5cH3 ou D'Na* (CH, CIi3 O-P-O Õ 0-- (CH2) sCHs 0 112091 0 0 O 0J~4H I , H,s -0 Na HNC, H, s CBH13 O O ) S=0 o o) C6H" HN o. 0 0 HOM H 6 Na<Y rGs s Has Tf I o °f CsHs 0 112092 0 0II - 0 0 cH, 3 HN o O c6H" CBH3 2 : 0 0 O OII o M CaHta °PO ° o O Na NNCN23 0 oceH, a o 112093 0 o M I MAHns HN O Na HNCH, S O OCsHs O O HN o Q C6H H C. H, 3 I"^Cihlis O Na HNC,, H2, O 0] =CcH" 0 112098 0 o B" o, M QH Y Y HN Na HNCNs 0 O H,. 4 0 O pH "C° o 0" 0 N. HN,, 0,, H. 112049 0 OH 0 OH 112049 0 0 ou n--ooc, H, s HN-/'0 Na Na HN CHs ) ° °row c6H 0 O OH "C° 0 °" O Na HNCiH23 O Na HN. -OgHa O 112100 0 0 qH o-a-o'1^o^^c, H, s X o oeC6H, 6 O O cH13 0 H O g) c6Hg6 \0 PO<, H, 6 6 Na HN. C,, H 17 23 112859 0 0 ? H O H HN õ Na HNGH, s O O -CSHns . O QH 9H 0 oN \o_p_o4H" 6 Na HNC,, Hs ozzcH3 112860 QH -- 0 Na HN. , C, H, s ""0 SH HN/° <7H" H O N NN ^'CHns 0 o HM ici 0 H 0 PH -b-W H,s 0Na Na HN, C.. Ha3 o 0CH3 0 112861 0 o QH o on Hl ~ Õ Na HN>, q, C7H" O Na HNCHs 0 Ht O QH o S" qu O 'o 9-- I OMCH, s o Na H'fr"YcIIH23 zu -aHta O 113634 0 ou o-P-o sH, 6 Han O Na HNc7H, 6 \/o_ =o o Ofs C6H" - O go Ho 0 O O o 9" 113635 0 O Ne HNC » HZ sHa 0 113635 0 0 9H HNo- Na HN Hs wW s o OOSFlna HNt O 9H O HN , QH 0 0 Na HNC"Hi3 II 1_ _s o Hta 0 113643 0 0 ° vO-r-O<o C7Hts HN Na HNwCHy H = O QH o o pH \~o-OYo~caHrs b Na HNq OsHs O O 113644 0 ap 0 0' Ht O 9H -HOeOC, H i O - ° '"C°0 SH 0 0 0 Na MM. , C,, H :, 0 _COH, 0 113651 133 4. 4 (n=4) 215 254 o Na [- ^ , o-p-o~c7H, S HN Ne Na HNqCH23 H =° P O O 0 ll Q' -CgHn S"I OMCnHns 113665 0 0 113665 OH O H HN O Na r-YC7H, s ) =0 0 HN 9 gH I 6 O Ne HNC » lip3 ovo C6Hr3 0 O 113666 o B" o OH H je Na N3 HNq CsH3 0 Hoo. ° o QH H N u oH o 9JOsH, 3 o 118023na 0 118023 63 0 C, =0 o O -0 Na HN-, C,, H,, w n xa o 0 o o o Ho-o oM H 0 Na HNn tt, in ici O o 019772 69 0p O r-OtO~caH HNF b N3 HNnfsH33 O p p H 0 o y''aHna o-Na'Y s HBoc 0 O 118989 159 o o Q) c3Hr3 o--o-y-o H o 0 J0 o0. ,, Q'v"'cHns Hl-'6 N. HN y I 0 118999 105 o Q ~ c6Hr3 o 118999'KM H O QJc5Hr3 HI-o-Na HN yc"H. C, H, =0 0 v v-CSHna Õ N6 HNwsH73 o 119000 60 0 p cH,. O',^,N C O Q O NaH C6H, H., Qu, Cw-ceHna ONaHN.. CisH3) T O 119001 113 119001 113 p ~o-tH3 119001 ~---113 o QJssHr3 o oh 0fuzz H 0 o Na oH HN o < ; uc7H3 118949 138 O zu p Y""CsHna C ° o 1 s na O O o J [ p sHna o N O Na HNI='psH3 o 119327 16533 (n=3) 0 o 0 CrrH23 O-P-OmOC7H, 5 H ONaHN C,, O HN Q O C » Hp3 O H OMaHNC, T 119328181. 42 (n=3) o 9JC6H, 3 0 Y v6H13 c7HIS HN ON. HNYC, 314N 0 p'I HN__o 0 I"GFlns O Na HNCg3H27 0 11932922 (n=2) 0 o Q) RQ6H2 _q5_p C7Hr2 O Na HNCnsHn O O JO [- _ ») HN\ O 9CsHs6 ,- o H L-NH. 6 N. HN xc O 119521 103 0 p ,''OsHna o-r-oYoc7Ha3 o Na Na HN Cr3Hz7 O O oJvc5H, 3 O o'I HBxH O sHna v- oMC H 5 o Ne C, 3Hi 0 O Nt HN Cz3Hz7 119522 129 0 p Q""'csH, a o--O I OMCnHns b Na HNC3Han O O 6 N. HNC, H,, U HNA O QJW OzHs3 OLx H NH 0 Na H yC, H 0 119523 176 0 0 yCaH) 3 0 0 z O O O B""CeHna NH, H o hH2 _ OM H O2>tC, 2H2, 0 803022 164 0 0 oc"H HAN Na Na HNCr6H27 HN 0 i o-P-Oto~C7Hr6 Õ Na HN Co3H27 0 P " p 9-C O 803045 65 0p o-¢-e~+c7H HN o Na H9) trzhn zozo 0 J0 1 \ H Q p 9 "vsHna ''''pp o N HNC H koH OH 80305615142 0 o oJ (cH3 O-0--T-, 7c HN õ Na HN cf6H27 C =0 6 O O o c H wP-O<OC, Ht 0 Na HN yC13HV 0 803058 149 37 (n=2) o Q cr1H23 0 0 clip23 0--0joc, H, s HN Na yci3HZr z O OII han\ O o) c"H3 HN 0 CIIHM o Na HN yC13H27 0 803059 2 0 0 BCMHN S B 0'U, G"H. (-O O O HN 9) C11H. C A ONa6, CMH :, i5 0 0' p COHza O-P-Oo~c, H o Na õ c, 3Hs . O O o) ßcH3 -P-0--"A'r-7H,, 0 Na 00, 37 0 õ Ns OCr6Hn (Response in each assay was compared to 10 RM compound 100 internal standard which typically induced 2-3 fold increase in TNF-alpha PLAP expression over basal.

Tested at @5.8 µM.

C. Murine splenocytes The ability of compounds to stimulate cytokine release from splenocytes can be assessed in a mouse model. Spleen cells harvested from C57BL/6 mice are cultured for 24 hours in RPMI 1640 cell culture medium containing 5% FBS, 1 mM

sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin and 50 JIM beta-mercaptoethanol, various concentrations of test compound for 20-24 hours, after which the cell culture supernatant is tested for the presence of cytokines.

Spleen cells harvested from mice were cultured for 24 house with test compound and the supernatant was tested for release of cytokines. As shown in Figures 3 and 4, the release of cytokines such as IL-10 and interferon-gamma from splenocytes is stimulated by compounds such as 104,106, 124,160, and 162.

These assays utilized a heterogeneous population of cells derived from the spleen. This makes it possible that cytokine induction can be caused both by direct effects of test compounds on cells and through more indirect stimulation of cytokine "cascades"where the release of a cytokine by one type of cell can induce release of other cytokines in other cells present in the same media. It is possible that this cytokine"milieu"is responsible for part of this robust immune response.

Example 8: Ili 72ZU0 Induction of Antibody Response The most critical test of adjuvant activity is the determination if the addition of a compound to an antigen preparation increases immune response by elevating the level of antibodies generated to that antigen when administered to a living animal.

Initial experiments involved the injection of mice (Balb/c) with compounds of the invention plus a peptide conjugated to a carrier such as keyhole limpet hemocyanin. The peptide chosen for these studies is a peptide (P18) that corresponds to amino acids 308-322 of the V3 loop of HIV IIIB gpl20 protein. The P18 21aa peptide, corresponding to amino acids 308-322 of the V3 loop of HIV IIIB gpl20 protein, has been reported to be immunogenic. This peptide with glycine/alanine/glycine spacer residues plus an amino terminal cysteine residue was synthesized by Genosys (Woodlands TX). The peptide sequence is as follows: CGAG1 : RIQRGPGRAFVTIGKG with the underlined amino acids representing the native sequence. The peptide was isolated to >80% purity using HPLC by the supplier. This peptide was coupled via the cysteine residue to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) using maleimide activated conjugation (Pierce Immunochemical ; cat&num 77107). The KLH conjugated peptide was used as the immunogen and the BSA conjugate as the screening target antigen for PI 1 specific antibodies. The indicated amount of KLH-P18 conjugate, was routinely used along with 300 jug of test compound, Alum or PBS was injected at 2 or 3 week intervals (as

indicated), into male Balb/c mice (Charles River Laboratories) approximately 6-8 weeks old (18-25g). All injections were subcutaneous at the back of the neck with 200go of a mixture of antigen plus adjuvant in PBS administered every two weeks (three weeks for polysaccharides or influenza) for a total of three injections. Mice were bled one-or two weeks post 2nd and 3rd injections. Sample bleeds are designated as to when taken (i. e. secondary bleed is one week after the second protein injection or two weeks after the second polysaccharide injections, tertiary bleed is after the third injection of antigen/adjuvant. Blood was collected after nicking the tail vein and drops collected into Becton Dickinson brand microtainer serun separator tubes.

Serum was separated from the red cells by microcentrifugation and tested by ELISA for antigen specific IgG levels.

Immune response to the peptide can be tested by enzyme-linked immunosorbent assay (ELISA), which can quantitate levels of serum antibody that bind to P18 peptide conjugated to another non-cross-reacting protein such as bovine serum albumin (P18-BSA) and coated onto an ELISA plate.

As shown in figure 5 and Tables 2 and 3, mice injected with the various compounds along with KLH-P18 antigen demonstrated greater response (higher levels of antibody) than those injected with the P18-KLH peptide conjugate alone.

Table 2. Stimulation of antibodv veneration to P18 nentide bv comnounds ç V A A V s ER # Compound Average Serum anti-P18 IgG Concentration' (fold increase over no adjuvant) 112022 112022 6. 7 o 9JGC6H, 6 o -ONaHN. C,, H ; ; 3 j 0 o NaHNnrCrrH2s (t o 4 : 0 0 0 o NaHNwCHsa w QII 0 0'"-C,, H. a "S-0 I OMCiHns o Na HNwC"H 0 0) CIIH23 0 OCH n o O Na H O H Hs HN\ O o za O O 2 O O f' HN OCH O CH 0 Na HN. C,, H O O 0 0

112066 112066 39. 2 o 0 O-C ici )-0 & S ? / 1 23 NN OCnHza O O P OppHs O Na HN. C"H23 if 1f 0 0 'Concentration of IgG assayed at the tertiary bleed.

Average IgG for serum from 5 mice that were injected with 300 gg compound and 5 llg KLH-P18 conjugate antigen as described in the Methods section. Antigen-specific ELISAs were performed as described in the Methods Section with Costar EIA/RIA plates coated with 50 ul of 5 µg/ml BSA-P18 conjugate in PBS.

Table 3. Stimulation of antibody generation to P18 peptide by compounds Compound # Compound Average Serum anti-P18 IgG Concentrationl (fold increase over no adjuvant) 111232 111232 7. 6 0 O =OuC6H,, HN 0 Na HN,,"YC"H n zs 0 Na 6 ° °) c6H, 3 0 9"'sHna O H O Na HNCHza s O O 112066 112066 13. 4* 0 0 9) CIIH23 OH '0 NaHN-CH /=0 0 0 !) un O CiHp3 O OH O Na HN) Cr1H23 0 0 113651 113651 10. 5* 0 o Q 0 HS Nv 0 0 O o Y v"-CsHna 0 Ma HN.. CnHs3 0 0 118989 118989 4. 35* 0 S Y W0 0 Hz 0 Op O Q'vv-CSHna O_P_O,"o Õ N3 HN O, 3H37 O

119327 119327 16. 5* 0 0 ClIH23 oMHts ... ONaHNC, ho) lu O o c, 1Hz3 usW OM H ONaHMC, 3H. <T O 119328 119328 26. 8* 0 p 9""sHna o Q) C6H, 6 O O J ONa H yC, H. , O H \o-0-Q<O c7H 0 'Concentration of IgG assayed at the tertiary bleed. Average IgG for serum from 5 mice that were injected with 300 llg compound and antigen (below) as described in the Methods section. Antigen- specific ELISAs were performed as described in the Methods Section with Costar EIA/RIA plates coated with 50 ul of 5 FLg/ml BSA-P18 conjugate in PBS. As comparison, addition of Alum increased IgG levels 7. 4- fold over PBS/antigen alone. Antigen used: primary: 1 ug KLH-conjugated P18 peptide. 2° and 3° boosts : 0.5 ug KLH-conjugated P18 peptide *p<0. 05 by Student's two-tailed t-test (unequal variance) compared to the PBS + antigen group.

Adjuvant activity has also been obtained with compounds of the invention when tested with other antigens. Compounds such as 100,116, 126,160, 184, and 186 can stimulate antigen-specific antibody production by up to 26.8-fold (Table 4) to influenza X-31 antigen. Increases in response are also seen when tetanus toxoid (Figure 6) and meningococcal C polysaccharide (Table 5) are used as challenge antigens. In the assays, 1 Rg of meningococcal C PS or 1.5 ug tetanus toxoid or 5 ug of influenza X31 (SPAFAS laboratories) were used. Tetanus toxoid from Accurate Chemical (cat #sstettox) was used as a challenge antigen while the purified toxoid from List Biologicals (cat #191) was used as target antigen for the ELISA assay.

Table 4. Stimulation of antibodv generation to Influenza X31 bv compounds ER # Compound Average Serum anti-Influenza X-31 IgG Concentration' (fold increase over no adjuvant) 112022 112022 1. 7 0 o Bc. H,, HN/° N<H6 HN Na HN ry. C"H. cl o (), 0 0 H\-"o 0 0 Na HN,, yCliHm b O 112048 112048 5. 4 0 OH o-¢-otoc7H, HN/'0 N. HN C7HIS OY-cH, s il ; =0 O 0 QH "-P-OpMyHfs o Ne HNwc, H, s O O C11 H23 0 112066 112066 23 0ici 0 9CllH23 0 C H HN O Na HN) C11H23 O O O H O C » H23 0 O_p_OYoC7H, s O Na HN) C11H23 0 0 113651 113651 0 HlX o Na HNyfOsH2a /o-b I"MyHu O Na HNwC"Hz j=o o O H) - CH13 "1-0OMCnHns 0 Na HN,, Y" 0 0 119327 119327 7. 85 0 o Qc"H, HN 0 Na HNY013HZ ? C) o nHza o-P-omOClH1s o Na HN c13H27 0

119328 119328 26. 8 0 O O °JA C6H3 p p-""-cstlns HN ONa HN ClaHz7 O 13 27 O p 9'"sHns vMHns O Nua po 0 o 'Concentration of IgG assayed at the tertiary bleed.

Average IgG for serum from 5 mice that were injected with 300 µg compound and 5 Rg antigen as described in the Methods section. Antigen-specific ELISAs were performed as described in the Methods Section with Costar EIA/RIA plates coated with 50 Ill of 10 µg/ml Influenza X-31 antigen in 0.5 M sodium carbonate buffer pH 9.6. As comparison, addition of Alum increased IgG levels 3.5- fold over PBS/antigen alone.

Table 5. Stimulation of antibody generation to Meningoeoccal polysaccharide by compounds ER # Compound Average Serum anti-Meningococcal PS IgG Concentration' (fold increase over no adjuvant) 112022 112022 8. 3 0 0 C. e 3Hs3 o-D c7Hr6 HNt õ NaHNn CssH# 0 0. o ° csHß3 6 Na HN,,-Yc"m. õ Na HN) CxlH23 d O 112048 112048 1. 8 o gH o B" HN/--/0 Na Him-r, 7H,, CeH, s {) 0 0 °< =z°eHra (l ICI HN O gH ''2 0 Oh I OMH, s Na Na HN) C7Hrs O O) C, Hla 0 112066 112066 12. 9 0 o 0 C11H23 O-P-O<OC7H1s HN O Na HNc1lH2o O =o TT l un Q o C » HZ3 -""C7HIs O Na HN) C11H23 0 0 0 0

113651 113651 18. 3 0 0 O Q) CeHra HM* 0 Ma HA-, C,, H,, H9 coo O O O O H p Y""OsHna HNV_'o 0.. 13 0 Na HNwC » Hz3 0 0 119327 119327 15. 8 0 ) C11H, c 9"c,, Hzi HN a HN O Q c11H23 i ONaHN, CH.,, 0 'Concentration of IgG assayed at the secondary bleed.

Average IgG for serum from 5 mice that were injected with 300 llg compound and 1 g antigen as described in the Methods section. Antigen-specific ELISAs were performed as described in the Methods Section with Costar EIA/RIA plates coated with 50 p1 of 5 llg/ml meningococcal PS in PBS plus methylated human serum albumin as described Gheesling et al. J. Clin Microbiol. 32; 1475-82 (1994). In comparison, addition of Alum increased IgG levels 2. 3- fold over PBS/antigen alone.

Influenza virus X-31 was purchased from SPAFAS (Storrs, CT) and was inactivated and confirmed to be inactive [Payne et al. Vaccine 16; 92-98 (1998) ] by the supplier. Meningococcal C polysaccharide (PS) was supplied by Pasteur Merieux Connaught (Swiftwater PA). Methylated human albumin can be obtained according to the methods described by Gheesling et al. J. Clin Microbiol. 32; 1475-82 (1994).

For the preparation of antigen/adjuvant mixtures, lyophilized test compounds were reconstituted to 2 mg/ml with phosphate buffered saline (PBS; cat # P-3813; Sigma Chemical Co, St Louis MO) and sonicated in a chilled water bath for two minutes. Monophosphoryl Lipid A, MPL, (Ribi Immunochemical) was reconstituted to 2 mg/ml with sterile water for injection, incubated at 50 °C for 15 minutes and then sonicated as above. ImjectR Alum, purchased from Pierce Immunochemical, was used according to manufacturer's guidelines, and comprised approximately 20-30% of the injection volume. Indicated amounts of antigen, diluted in PBS, were mixed with the compounds, MPL, or Alum such that the final concentration of the compound or MPL was 300 u, g (unless otherwise noted) in the 200 fil injection volume. The mixtures were incubated at room temperature for 40 minutes with continuous shaking prior to injection.

Antigen specific IgG levels were monitored by direct ELISA where antigen was passively coated onto 96 well Costar EIA/RIA plates. Plates were coated with 50well of the indicated antigen and incubated overnight (ON) at 4°C and washed 3x with PBS + 0.05% tween 20 (PBS-t) in an automated plate washer. Plates were then blocked with 200, u1/well of 0.5% gelatin in PBS for 1 hr at room temperature (RT) and washed 3x with PBS-t. Mouse sera was diluted in PBS-t plus 0.3% BSA and lOOul of various dilutions were added, in duplicate to the antigen coated wells (or BSA coated wells as a control) and incubated at RT for 1 hr. and again washed 3x with PBS-t. Biotinylated goat anti-mouse IgG (Southern Biotechnology Associates Inc. , Birmingham AL, cat #1031-08) was diluted 1: 5000 in PBS-t and lOOul/well was applied and incubated at RT for Ihr, washed 3x with PBS-t and followed by the addition of 1: 10,000 streptavidin-horseradish peroxidase conjugate (Southern Biotechnology Associates Inc. ) in PBS-t for 30 minutes at RT and again washed 3x with PBS-t. Wells were then incubated in 100 pL TMB substrate (Kirkegaard and Perry Labs) for 5 minutes. Color development was stopped with the addition of an equal volume of 1M phosphoric acid and the absorbance was read at 450nm on a Titertek Multiscan plate reader with Deltasoft software analysis package.

For relative quantitation of antigen-specific IgG levels, curves were compared to one another by determining the dilution necessary to obtain a fixed amount of antibody-generated color. In some cases, a total IgG assay using an anti-FAb-specific reagent to capture known amounts of purified IgG (purchased from Southern Biotech.) as an IgG standard curve was run in conjunction with the direct ELISA on the BSA- P18 conjugate. The anti-FAb reagent orients the purified IgG in a manner similar to how an antibody would bind to an antigen through the FAb region. This allows detection of bound antibody by the same reagents used to measure antigen-specific capture of antibodies. The same reagent solutions used for detection of the antibodies bound to BSA-P18 conjugate, namely biotinylated anti-IgG (Fc-specific) followed with HRP-streptavidin, were simultaneously applied to the anti-FAb total IgG quantitative assay and to the antigen-specific assay. Hence, the signal from the binding of the purified IgG standard curve is equivalent to that generated to equal amounts of IgG bound in the anti-target antigen assay. The amount of antibody in the serum is then interpolated from the purified IgG standard using a 4-parameter curve fit (DeltaSoft 3 software package).

Table 6. WB ED50 vs. % of LPS at 10ng/ml ER# Structure WB ED50 vs LPS@ IQng/ml lOng/ml HO ., offofX 0fof O NH yoYv H O 0 0 p O MPL Standard/ » l0 pm O o o o) C, H,, O O 112022 c), o 0 0. 696 n iY °--aHna o N H O gW NHIX Na HN) clsHz 111230 () c, p "_ slins --oxo o 0 111230 () 6 0 0 fizz o_ vHls 16 111230 0 N Q) CsH w n za 6 CaH p _ _ sHna I"MHns } =0 S E O O 111231 HN/O 0. 29pu /=°) ; p ""aHta _ _p Hs O Nn H C H O O O o c. Hl 111232 "T. HN Na HNwoHns 111232 0 0 J o-r-o+p, 6 I"-"Hns o Na HNe CrgHzo O O OII p eHns HN o Na HN),-YCIIH2. II II 111233 o o o Ht L- CGH, 3 o 0 Na HN, ,, H. O O in Hs HN O NaHN) CrrH2s II II 112043 HN° ° ° OH \ \oi oYoc7H1s 0 Na HN,"CiIH23 0 0 0 OU HN õ XmH6 HN b Na HNCiH, s O O CsHs ) =° 0 O.. C, H,, 112044 S MiH, s 0 Na H CHS O OM-CsHna 0 OU u O C H HNF O Na HN) CraH2s 112047 ( ) (10 HN OH o OH 0 Na HNC » H23 0 O OH M --------- 9 (G O C6H, a il ) =° 0 O-. CeHta 112048 HN O QH » 10 0 -P-OM H O Na HNCH 6 o OSoC6r3 go p oh I M7Hts HN O N6 HNf >, C, H, s t0 ° °ec6H3 O OCSHta 112049 o HN\__\o0 PH HN__ o oH O P Op7Hs Õ N6 HN) C"H33 0 0 o Na C6H, 3 p ""-CSlia -P-oMHns 0 õ Na HN) CH23 O O 112063 (() 2 o OH \o-R-Oto C7H, 6 Na w-HN) c"H23 O O O p oG7 o-P-oCoC7Hg6 HNF O Na HN) cH23 Cl 0 0 112064) 40 0 0 ici HN QC » Nza HN\ O Q) <cH22 O Na HNCiH23 O O 0 Q 0'c,, Hz ! 0 O H HN Na Na HN) C"H23 0 0 112065 Coo 0 0. 25 I o HN 0) O ! HN gC,, o-p-oYoc7Hrõ O Na HNC, H23 It O O 0 if O P OMCFys O Na HNCiyH23 112066 0 0 0 °II OC » Hza O o Na HN) C1aH23 O Na 4 ^/C » H23 O O O s, O C 1'Na 112071 oN o Õ HN, >/ O HN O O oJS (CH2) sCHs 112071 OH 0 Oh 112072 f OH O O'Na* OI 00r" (CH) CH 0 O 0. . y 0 C. H, a O p '"CsHna I MHis HN Õ NaHNf>oC7He6 t0 o °tc6Hr3 112091' ° o HN\ °¢ D) c6He3 o-p-omoS, Hr6 0 Na HNC » Hz3 O gJc6Hra O oit 0 CaHI3 O-P-O M HN Õ Na HNqoc7Hrs t0 o Oec6913 112092 ° ? 0 ici g o o--oYo''c, H, s O Na HN) C"H33 O OCaH3 0 OII p CsHa - MtHs HN/° Na HNCHS O OCsH3 O 112093 0 0 H O Ho0 CGH, A "-P-OpMFis O N6 HN) oCtgH22 O OCgH3 O gH QH IvHs o Na HNCHS g - o Na ? c6H, 3 112098 HN\ o gH O--O. OCyH,, o Na HN) CH22 O OCgFIg 0 ou I"Hts HN/° N 4 t13 '1 O OCaHta 'g O 112099 9H qu -OpMHs b Na HNI, C » Hsa o laH3 O Q OH O- McHu HN/0 Na H'r-yoH, o WsHas 112100 ho -CroM9 Hns HN O o) ~ 0 go Q oh O -opMpHis Õ N6<QF han n 112859 qu 112859'h°, ° J Na HNCHz3 o OCaH3 O R, gH o O Na HV NCHns Hns ah 0 0 bs oH Zou "'o-N H MHns o 0. -csHaa 0 u OH -S M>Hns HN O Na NNCHs OH O 1,2861 NN OH 112861 o "\ o S" O OCgHs O gH 0 Na HNr-yC,, H 0 N HNwC H 0w0 C6Hr6 113634" oI, pH "'S-0^pMys 4 Na o n r 0 go OH HOX g e _C6Hs6 H r o Na HNCNis HOQ 0 HO O 113635 HO o 0 H 0 gii 'OMCH, s 0 Na HN-C,, Hm O OC6H =OU ou o-b-OCHS H HN ° 9H C6H3 O OM-CsHaa 113643 HN OH =s 3 O Na HNCHza O O sHs O p ,, OH o-b-O I"WHns 6° H07HIS ol-CIHII 113644 Hf o I 'ciHns O Ne HN1. CyHb V VcHn O oOsHss QJ C, H" O -O MCHns HN°"Na H C » Hzs 113651 CO O O 0. 70 Ai 0 O Q) ~CßH 0 Q""z% elina 0 O-P-O<OC, H, 6 o a OH HN N. H Y-y-C7H,, OrHis O OC6H3 II 113665 H o OH His 6 Na MN. , C,, HM o N6 HN wCrsHza OH O gH oH D-U-oto~CTHr HN 6 Na HNo7H, 6 Hon, ° 0c6Hgs HOO. II 113666 H°H, O QH 0 t4. H p5 MHs O Na HNbm>CrsH2a O °e C6H" O Oui' p M sHna I MHns HN 6 N2=H6 0 0 118023 gO 0 I HN HN o n ta "-oMCNns b Na HNwc"Hi O o 0II LI) --- C. H, -S I"MHns H0 Na HNn CH6s cl 019772 0 0 00 /H p -"sHaa O-f-omo~czH B-0 yC,, H3 O O O p Q""n ; sHa O Nn HN CMHu na m 118989 CO 0 0. 1 pm 0 ici pu M-oOsHna 5- I VHns HC O QJC3Hr6 o N6 HN OzsHs7 p U""z% sHis MCHas o-P-o~Co~czH, 6 HNs O Na HN C"H" 118999 0 Htk_\o0 b-ir-oto~c, 6 M H O Na HNCoH23 O 0 p ""vsHna O Na HN C H u ns an 119000 C. 0 ^^ HN O'v v'CSHa b--oHu O Na C6H, 6 O O Q _ BH13 O-P-O<OC7Hrs HN/6 Na HN), cH26 119001 to 1. 23 pu 0 fI C H H° o Bc. H,, HN 0 Na-CGHt3 0 OH han0 p o _"sHs HN/° Na HN<, CbH, 3 118949 0 9-1-CIIH23 b-P-o~o~c, H6 0 0 CHsa O C11H23 MHs HN 0 Na HN yC13H27 0 119327 HN o, 0. 015 jDM HN O Q C H23 O O C H ONa HN.. C, 3H27 o o =O) +C6H, 3 0 c6H13 p-'"sH,a HM--OMa HNCH 119328 ° 5 j ? » 10pM H p O H o Na HN Cr3H27 O I 0 O-P-OpGrHis HN Na HNUC3Hz 119329 0 ° o p'"CaHns \ O_P-O M 00 o" IVHBOC O pp Q'"CsHn3 o QC6H, 6 O Na HNC3Hx H0 Na H yClH, 119521 NHBoc N\_ Ht3 "-P COlins o Na HNC,, H22 0 o Q 6Hr3..... 0 9-1---C. H13 O C H 0 Na HNC, aH 119522 o , \O/, H Nt 0 0 L-\IH2'0 N. HN yC13H27 o C6Hts 0 0 p 0''sHa O C H j 0 Na HNCHsy 119523 0 0 0 J _ NNZ _"_ O Q' 'OSFya 0 :' 0 9)] s 07H, s T O p iH2a j] O--OoCyH, : UM-ONa HNCjaH 803022 HN T ? 0. 06 pM M q "Hza 0 Na HN yC13H27 O Na HNUC3H2 T 803028 y u-0 MaHNCisHs ? 80MOy u O Na HNNC, sHz 803028' o 0 fI HN na HN\__, o0 O Na HNCtaHs7 0 oui' p asHna I"MCrHns , » 0 Y 0 0 803045 > t c6H,, N. HNyC13 V b-OH o 0 po O''C » Hy HN 0 a N yC13HU 803056 o =0 J ! UN oC, Hz, n o H 0 Ma HNC), H ;, 7 0 O o, ° C"Ha /-0 Ma HNCH HN 803058 HN ° 0 0. 022 N. M "M o-c,, H 1"MGHis 0 Na HNYC, HV O 0 p o'c"Haa - MOM H HN, o Na o, CuHs7 803059 0 ° O 0. 89 O HN p 9 oHza UN 0 0-c,, Hs ont Na 0, cr3H27 0 O1I p QCmNaa O-P-OMOMCHs O Na OuCgHr, O 803592) 5 P 0 MMHis 0 Na yC13H27 0 0 O C6HI3 0--0" HN Na HNUC3H2 803596 ° 0 J0 _ HN 0 g'v v-CfiHa I MH, s HN 0 C6H13 O OH o Na HNKcr3H27 O_e_owO7Hr6 HN o Na HN yC13H27 =o O 803597 0 HN OH O-P-OoC7H, 6 i 0 Na HN yC, aH27 0 ou O C H HN O Na HÑCr3H27 ) =0 5 803598 HN OH 0-"-0 o c7HI5 0 O Na HÑC13H27 OH 0 9H OH ii O C H HN 0 Na HR yC13H27 803599 zozo 803599 =o ? C11H23 O o-P-oYo~C7H15 O Na HNCiaH27 0 OH C13H27 O C H HN Na HNCHsy I I II 803613 HN 0 ° 0 C11H, 0 O C H ONa HN.-CH Y g- 0 . o 0 9), ClIH23 HN/--"'0 N. HR CisH27 803731 HN ° o >10pM o HN O O C » Hp3 ou 0 Na HNC) 3H27 0 CAH23 p 9etHza u MO H HN 0 Na HN yC13H27 803732 H S g 0. 85 juM 0 O P-MMW His 0 Na HN yCl3H27 T 0 0 0 CHg C11H23 O C H HN o O Na HNUC3HM 803733 0 0. 70 N, M zozo Han O C » H23 0 °~P°oC7H1s ONa HN., CH27 -P-0 0 C. gHl3 O C H o Na HÑC, 3H27 803751 HO 0 HO zozo N 0 p 9'"eHia O C H ONaHN., C,, Hz7 0 O 0--o", -0- HN Na HÑC13H27 O O 803783 HO 0 <_p_o<O'C1SH37 o Na HNC : 3H27 0 0 /°-P-o. o'C1SH37 HN/--/0 ONa HNC, aH27 O O 803784 HN 0 A p o, o-C18H37 o Na HÑC13H27 O ° C1 1 H23 0 0 9 H23 p 'tiH23 O F OMCFIts 803789) =o g P 0. 10 pM 80 78 o un O Cii-Ipg -P-MOMHs o Na HÑC, 3H27 ° 0 0 0 CdH u O C H HNO O Na NNC H 3 s 804053 =o o o 9 1. 34 Ai 0 O CiHzs OrPOOC7H, s O Na HÑ) CH23 in ici o J 0 0 CHa O--OYoC. H 0 CllH23 zo-P-OYOC7Hts "\ o 9c<i p 9 n Hxa u 0 0 0 ____ o 1f 1f 0 0 O_p OYoC7H1s 0 O MG, F s HN/° Na HN) c"H23 804058 0 o o 0. 03 PM HN O C6H, 3 o-P-OYOC7H s O C H 0 0 0 0 CeHig 0 Na HN) C11H23 o o O O ti p 9""sNs u O C H HN 0 Na HN Y--r C, 1 Hp3 804059 oo o 0 0 > 10 M 0 HN 9""CsHis P-o' O Na HÑ) C"H23 Ir n 0 0 0 0 9""CsHm ici O~P~Oz, oC7H, s HN o Na HN) C"H23 I If 804061 ° o 0 0 2. 5 AI zozo HN ^^ w O :"C7H, s UMC7H15 0 0 0 0 0 CsHna 0 pNDCi 1 H23 O C H HNO Na NN ^/Ci H 3 804097 =0 0 0 0 0. 3 HN C17H23 0 H\ gcH, 3 O Na HÑ) C1tH23 0 0 0 0 o C, 2H2s 0 Ci2H25 O P O O'v'Cyits HN ONa HNC, 3H27 II 804121 n °,, 0. 46 pM 0 O P O Hts O Na HNCr3H27 0 0 O C12H25 O-P-O 1 p'Hs HN/--/0 Na Na HNC3H27 0 804130 o 0. 66 AI HN 0 C12H25 p zHzs WO CHiS O Na HNC13H27 0 OII 0 CHs p LJ O ° C1rH23 O-P-O I O Õ C7H1s HN'0 Na HN N C » HZ3 804221/=° ° o 2. 2, uM HN C11H23 O HN) NAC"H23 --\ 0 Y C,, H, 3 o -p-o' ON. 0 M Qr'11H23 HNO O Na HN CHS 804222 =o S j ? I 0. 008 AI ,., p m z Xo-P-OYOC7Hrs O Na HNY 0 U ONa Ct, H23 O-P-O : oC7H1s 804252 HNF c14H29 O EtOH 804252 H p'"°II EtOH OC 1 Hp3 O C14H29 O HNACttH23 14"S9 OII 0 J ! O''C H O-P-O-Y-oCyH HN o Na HNC13H27 804253) =o o A > 10 I NON 0 P O ^ O P O O'v Clis O Na HNUC13H23 T O O Q ° C11H23 O-P-OOC H O Na HN 804281"o °i4"29 0 0. 45 p ; M HN HN O O C1tH23 \-\ 0 O P O OCH5 ONa HN t4"29 0 0-\H23 Hs UM-ONa HN. 804313 HNF AoHCs2H2s o 0. 014, uM UN \ O o Ct1H23 0 7 AH Na HN 0--- --- o QC11H23 O-P-OOMC H 5 804339 HNF of CX4H29 0 1. 06 N, M HN 0) CIIH23 Q C11H23 --'4C7H 0 Na N. QH O' p Hs , OMCHfs O Q C H23 O Y 804372 HN O Q^CH23 0-4 |lM 0 0 Na HNUC13H2 0 O o °C1tH23 il C7HIs HN 0 Na H HN N 804442 T , 0. 007 M Q CilH ONa) H Han-. N HNUN HN yN'Ci2H23 0 t) Q OCs o °Ct1H23 804503 C H O-P-0 0'-'-'-'C7HI5 Ho ou ont je p nH2a HN Y Na ° o. 0 O o °C1tH23 Q P-OpMCNis 0 804558 H o o S o ° HN 0 OC1H23 O-P-O<oC7H, s 0 Na HN, Sx'c12H2s Oxo 0 Ji g'"c,, Hz3 /~ ONa HN Cr3H37 804596) ° o > 10, uM tX o g CarH23 0--0 O Na HNCg3H27 O - _-__ _ o o 0 CrrH23 sO-P-O<OC7Hts 804674" °'o, 0 Na N, HN O_'CaHZa O C14H29 O Na N g P CH OII g ? ttHza O-P-OpMNl5 HN 0 Na HN>Cr3H27 804678 o II NN 9CnHza S T 0 0 0'Q'kCllH23 0-OC » H23 N CHls M1N H 804679 =o 9 0. 53 pM 0 0 0 CiIH23 Lu, 0 O _ O P O NMCFIts O Na HNC3H2 0 oit 0 OC » H23 O-P-0' N' C H HN O NaH ts 04680 804679 O Jl 0. 015 . M O O CiHzs O M ONa t H O-PO I N4C7H : s '-O'rH 0 Na HN yC13H27 0 HNo O Na H is HN C3Hz O 804732 H ° o <0. 001, uM O CiH23 O 0 Na, rH O Na 1 H HNrC13H27 0 0 19 O'fl'CllH23 O-P-O- P 00, 3 hINUC 1i2 o UN"HNC 804764 HN O 0. 015 uM õ C » H23 ONT O C11H23 HNCi3H27 0 0 O M O-P-OoC u-ONa HNC) 3H27 804772) H ? 0. 008 uM HN O WPWO C H o Na HN Cr3H27 Y 0 0 0 0 CIIH23 I I J 11-"1 15 HN ONa HN C13H27 804947 H o $ » 10 . M " 0 P 0 CH <Poto4c7H15 o Na HN Ct3H27 ! ! 0 Table 7 below contains the compound number as referenced herein to the corresponding ER number.

Table 7. Correspondence of Compound Nos. to ER Nos. Compound ER# Compound ER 16 112048 152 113634 31 803058 154 113635 48 803733 156 113643 50 803022 158 113644 62 803789 160 113651 72 803592 164 113665 100 112022 166 113666 102 111230 168 118023 104 111231 170 019772 106 111232 172 118989 108 111233 176 118999 110 112043 178 119000 112 112047 180 119001 114 112047 182 118949 116 112048 184 119327 118 112049 186 119328 120 112063 188 119329 122 112064 190 119521 124 112065 192 119522 126 112066 194 119523 128 112071 196 803022 130 112072 198 803045 132 112091 200 803056 134 112092 202 803058 136 112093 204 803059 138 112098 206 803592 140 112099 142 112100 146 112859 148 112860 150 112861