AU2008202882B2 - Immunological adjuvant compound - Google Patents

Description

Australian Patents Act 1990 - Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Immunological adjuvant compound" The following statement is a full description of this invention, including the best method of performing it known to us:- IMMUNOLOGICAL ADJUVANT COMPOUND This application is a divisional of Australian Patent Application No. 2004201147, the entire content of which is incorporated herein by reference. BACKGROUND OF THE INVENTION 5 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. 10 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 15 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. 20 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 25 as foreign and will elicit an immune response. To this end, additives (adjuvants) have been devised which immobilize antigens and stimulate the immune response.

-2 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 5 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 10 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. 15 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 20 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 25 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.

-3 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph that shows the results of an in vitro assay for induction of 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 5 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 10 splenocytes by compounds 104, 106, 124, 126, 160, and 162 of the invention. Figure 5 is a graph illustrating the results of serum tritration 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 15. 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 novel compounds of formula I -4 X'-R' Y

(CH

2 )a

(CH

2 )b 0 0 HO-P=0 HO-P~- 0 (CH2)d

(CH

2 )c x 2

Y

2 W\ (CH1 2 )d' (CH 2 ). W2

R

2 G' G3 R5

(CH

2 )d-

(CH

2

)

G2 G4 4 R R7 R 6 wherein: R' is selected from the group consisting of (a) C(O); (b) C(O)-C,.

4 alkyl-C(O), wherein said C,., alkyl is optionally substituted 5 with hydroxy, C,., alkoxy, C,., alkylenedioxy, C,., alkylamino., or C,.5-alkyl-aryl, wherein said aryl moiety of said C,.5-alkyl-aryl is optionally substituted with C,., alkoxy, C,., alkyl amino, C,., alkoxy-amino, C,., alkylamino-C,., alkoxy, -0-C,., alkylamino-C,., alkoxy, -0-C,., alkylamino-C(O)-C,., alkyl C(O)OH, -0-C,., alkylamino-C(O)-C,., alkyl-C(O)-C,., alkyl; 10 (c) C 2 to C,, straight or branched chain alkyl optionally substituted with hydroxy or alkoxy; and -5 (d) -C(O)-C..

2 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 e" are independently an integer from I to 4; 5 X', X 2 , Y' and Y 2 are independently selected from the group consisting of null, oxygen, NH and N(C(O)C,.

4 alkyl), and N(C,.

4 alkyl),; W' and W 2 are independently selected from the group consisting of carbonyl, methylene, sulfone and sulfoxide;

R

2 and R' are independently selected from the group consisting of: 10 (a) C, to C, 0 straight chain or branched chain alkyl which is optionally substituted with oxo, hydroxy or alkoxy, (b) C 2 to C, 0 straight chain or branched chain alkenyl or dialkenyl which is optionally substituted with oxo, hydroxy or alkoxy; (c) C2 to C, 0 straight chain or branched chain alkoxy which is optionally 15 substituted with oxo, hydroxy or alkoxy; (d) -NH-C 2 to C 10 straight chain or branched chain alkyl, wherein said alkyl group is optionally substituted with oxo, hydroxy or alkoxy; and (e) 0 Z M N wherein Z is selected from the group consisting of 0 and NH, and M and N are 20 independently selected from the group consisting of C, to CO straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylamino; RI and R' are independently selected from the group consisting of C, to CO straight chain or branched chain alkyl or alkenyl optionally substituted with oxo or fluoro; -6

R

4 and R' are independently selected from the group consisting of C(O)C, to Co straight chain or branched chain alkyl or alkenyl; C, to C 2 0 straight chain or branched chain alkyl; C 2 to C 20 straight chain or branched chain alkoxy; C 2 to CO straight chain or branched chain alkenyl; wherein said alkyl, alkenyl or alkoxy groups can be independently 5 and optionally substituted with hydroxy, fluoro or C, to Cs alkoxy; G', G 2 , G 3 and G4 are independently selected from the group consisting of oxygen, methylene, amino, thiol, -NHC(O)-, and -N(C(O)CI. alkyl)-; or G 2

R

4 or G'R' may together be a hydrogen atom or hydroxyl; or a pharmaceutically acceptable salt thereof; 10 with the proviso that the compounds are not 0 0 h0 Or CH 0 or 0 0 0 cc ,~ 0 0 }a 40(4)cac o' o 0

O

.7. The present invention also encompasses immunological adjuvant formulations comprising a compound of formula 1 X'-R'-Y

(CH

2 )a

(CH

2 ), O 0 HO-P~O HO -P--- 0 O O (CH2)d

(CH

2 )e x2 __ _ 2 W

(CH

2 )d'

(CH

2 )e 2 R G G3 R5 (CH2)d- (CH2)e" G4 R ' Gi7 -\ R 6 R wherein: R' is selected from the group consisting of 5 (a) C(O); (b) C(O)-C 1 .1, alkyl-C(O), wherein said C.

4 alkyl.is optionally substituted with hydroxy, C 1

.

5 .alkoxy, C,.

5 alkylenedioxy, C,1.alkylamino, or C,.

5 -alkyl-aryl, wherein said aryl moiety of said C, 5 -alkyl-aryl is optionally substituted with C 1

.

5 alkoxy, C,.5 alkyl-amino, CI.

5 alkoxy-amino, C,.

5 alkylamino-C,., alkoxy, -0-C,.s alkylamino-C,.

5 alkoxy, 10 -0-C,., alkylamino-C(O)-C,.

5 alkyl C(O)OH, -0-C,., alkylamino-C(O)-C,., alkyl-C(O)-C,.

5 alkyl; -8 (c) C2 to C,, straight or branched chain alkyl optionally substituted with hydroxy or alkoxy; and (d) -C(O)-C 6 , arylene-C(O)- wherein said arylene is optionally substituted with hydroxy, halogen, nitro or amino; 5 a and b are independently 0, 1, 2, 3 or 4; d, d', d'', e, e' and e" are independently an integer from I to 4;

X

1 , X 2 , Y' and Y 2 are independently selected from the group consisting of null, oxygen, NH and N(C(O)C,.

4 alkyl), and N(C. alkyl) 2 ; W' and W 2 are independently selected from the group consisting of carbonyl, 10 methylene, sulfone and sulfoxide;

R

2 and RI are independently selected from the group consisting of: (c) C 2 to CID straight chain or branched chain alkyl which is optionally substituted with oxo, hydroxy or alkoxy, (d) C, to CO straight chain or branched chain alkenyl or dialkenyl which is 15 optionally substituted with oxo, hydroxy or alkoxy; (c) C, to Co straight chain or branched chain alkoxy which is optionally substituted with oxo. hydroxy or alkoxy; (d) -NH-C, to Co straight chain or branched chain alkyl, wherein said alkyl group is optionally substituted with oxo, hydroxy or alkoxy; and 20 (e) 0 Z M N wherein Z is selected from the group consisting of 0 and NH, and M and N are independently selected from the group consisting of C, to C 20 straight chain or branched chain alkyl, alkenyl, alkoxy, acyloxy, alkylamino, and acylanino; R1 and R' are P.\0PER~ %ma~nats\244 544 rnpon- do. 130003 -9 independently selected from the group consisting of C 2 to C 20 straight chain or branched chain alkyl or alkenyl optionally substituted with oxo or fluoro;

R

4 and R 7 are independently selected from the group consisting of C(O)C 2 to C 20 straight chain or branched chain alkyl or alkenyl; C 2 to C 2 0 straight chain or 5 branched chain alkyl; C 2 to C 20 straight chain or branched chain alkoxy; C 2 to C 20 straight chain or branched chain alkenyl; wherein said alkyl, alkenyl or alkoxy groups can be independently and optionally substituted with hydroxy, fluoro or C, to Cs alkoxy; G', G 2 , G3 and G 4 are independently selected from the group consisting of oxygen, methylene, amino, thiol, -NEC(O)-, and -N(C(O)CI.

4 alkyl) 10 or G 2

R

4 or G 4

R

7 may together be a hydrogen atom or hydroxyl; or a pharmaceutically acceptable salt thereof; and at least one additional component. In preferred compounds of the invention, one or more of the following is present: each of a and b is 2; each of X1 and Y' is NH; R' is C(O) or C(0)-C.

14 alkyl-C(O); each of 15 d' and e' is 1; each of d" and e" is 1; X is 0 or NH, more preferably NH; and W is C(0); or each of d' and e' are 2. In further preferred embodiments, R' is a C(0)C 1

.

1 4 alkyl-C(0), wherein said CI.

1 4 alkyl is substituted, for example with a C 1 .5 alkoxy group; In a most preferred embodiment, the invention is directed to compounds ER 20 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. 25 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. Also provided is the use of a compound of the invention or an adjuvant formulation of the invention with an antigen against which the animal is to be immunized for the 30 manufacture of a medicament for stimulating an immune response to an antigen. Definitions Carbonyl, as used herein is a (C=O) moiety.

-10 Dicarbonyl, as used herein, is a moiety with the structure (C=0)-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 =0 group. 5 Alkyl ester, as used herein, is a moiety with the structure 0-(C=0)-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. 10 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. 15 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 I" 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 20 same; the values of d" and e" are the same; X' and Y' are the same; X 2 and Y 2 are the same; W' and W 2 are the same; R 2 and R' are the same; G' and G' are the same; R' and R' are the same;

G

2 and G 4 are the same; and R' and R' 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 different, the values of d' and 25 e' are different; the values of d" and e" are different; X' and Y' are different;

X

2 and Y 2 are different; W' and W 2 are different;

R

2 and R' are different; G' and G 3 are different;

R

3 and R6 are different;

G

2 and G 4 are different; or R 4 and R 7 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 30 is controlling.

General Synthetic Methods 1. Synthesis of Diamide Compounds In general, a 2-amino-1,3-dihydroxypropane or (t) serinol is transformed into the 2 azido compound by reaction with trifluoromethanesulfonyl azide followed by protection as 5 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 TBDPSCI 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. 10 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 aide. 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 15 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 bcnzimidate ester. The protected compound is reacted with a reducing agent, e.g., DIBAL or 20 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 25 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 (II), 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 30 protected phosphate groups of the diamide product are then deprotected, typically with palladium(0) and phenylsilane.

-12 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 pointjust after cleavage of the protecting group from the primary amine group of the phosphate ester compound. At this point, a 5 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 10 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, 15 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. 20 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 25 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. 30 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 -13 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 R 2 and R' in formula I above can easily be 5 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 R' and R" 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 (OSOCH 3 , OSO 2

CF

3 , 10 OSOCHC 6

H

4 -p-CH 3 ) which can be reacted with the azido diol, amino alcohol, or glycerol starting materials. The substituents represented by variables R' and R' 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. 15 The values of a and b in compounds of formula I can be varied by using the appropriate N-BOC-protected amino alcohol when synthesizing the phosphorylating reagent, exemplified by 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. 20 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. 25 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 30 some applications to employ an antigen covalently linked to an amino, carboxyl, hydroxyl -14 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. 5 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: 10 (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; 15 (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 20 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 25 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 30 vaccine antigens, for example through an amino, carbonyl, hydroxyl or phosphate moiety. The compounds of the invention may be bonde, Methods of linking the adjuvant -15 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. Hoffman 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., Int. J. Peptide 5 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. Chen. 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. 10 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 15 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 (ID), intraperitoneal 20 (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 expemrientation, by use of conventional antibody titer determination techniques and 25 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 30 and/or other biologically active substances.

-16 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 5 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 10 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

NH

2

N

3 1. NaN 3 trifluoromethanesulfonic anhydride DMAP -.... __ __ _ __ __ __'__ _ __ _ AcO OAc HO OH 2. acetic anhydride, pyridine 1 2 To a solution of sodium azide (107.67 g) in 250 mL of water was added 300 mL of 15 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 -17 of (±)-2-amino-l,3-dihydroxypropane (1) (9.89 g) in methanol (200 mL) and 4-NN 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 5 (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 N 3 Na metal AcO OAc N HO OH Dowex 50-8 2 3 The diacetate (2) (16 g) was dissolved in methanol (150 mL) and sodium metal (2.0 g) 10 was slowly added. The mixture was stirred for 90 minutes and Dowex* 50-8 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).

N

3 TsO 7

H

1 5 NaH HO N 3 HC1is HO + - P. O O C 7 ~ HO~k~HOH DMFTHF OH 3 4 5 To a suspension of sodium hydride (1.24 g of a 60% oil dispersion washed three times 15 with hexanes and dried under nitrogen) in dimethylfornamide (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-1-0-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* 125 H' for 25 minutes and concentrated to dryness. Chromatography gave 4.37 g of (5).

-18 N3 TBDPSCI

N

3 HO o y15 TBADP, TBDPSO,, O,,,Z 1Hi OH OH 5 6 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 (TBDPSCI, 5 mL) and the mixture was stirred overnight. The mixture was worked up as usual. 5 Chromatography gave 3.6 g of secondary alcohol (6) as an oil. TBDPSo J..O...> cH i Allyl alcohol , TBDPSO.. .<O c7H 15 OH Pnosgene. Pyridine 0 0 O O or OAOC 0 6 7 To a solution of the secondary alcohol (6) (2.95 g) in toluene (30 mL) was added pyridine (1.8 rnL) 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 10 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. N3PO~J.~..N.~I HF N3 TB DPSO,, O,,,, 7H is CH3CN I- HO O7His5 OAOC OAOC 7 8 To a solution of protected alcohol (7) (1.29 g) in methylene chloride (3 mL) was added hydrofluoric acid (IF, 4 mL) in acetonitrile (12 mL). The mixture was stirred overnight and 1 5 worked up in the usual way. Chromatography gave 150 mg of the alcohol (8) as an oil.

-19 BocHN-N\ O 'P-N N3 N3 HO _O -7H1s IO OAOC Tetrazole BocHN '.O'.' c?H s 2. Oxone 0 OADC 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 5 3 hours, the reaction was worked up in the usual way. Chromatography gave 242 mg of (9) as an oil, BocHN :ieOH BocHN-N- __\ / - O2 T e tra z o le 0O 10 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 10 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 15 by the suspension and filtering of the residue with hexanes (200 mL). The resulting hexanes filtrate was concentrated to dry and azeotroped with 2, 1 0-mL portions of toluene to provide the crude product (11) (21.54 g) as an oil.

-20 (PhS)2Sn BocHN PhSH, TEA BocHN O,.Dy7H15 0 OAOC 0 OAOC 9 12 To a suspension of dithiophenol tin (1.3 g) in methylene chloride (7.8 mL) was added thiophenol (400 pL) followed by TEA (543 pL). The reaction mixture was stirred at room 5 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. 0 0 cMN HaEDC - SH OAOC 8 OADC 12 13 10 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. 0 .0 TFA, TES BocHN H -H2 A H~ll0...- Al, 8 C 8O3C 15 13 14 -21 To a solution of the protected phosphate (13) (232 mg) in methylene chloride (1 mL) was added triethylsilane (TES, 120 pL) 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 5 up in the usual manner to give 174 mg of free amine (14) as an oil. 0 o0 HN CH H Succinic Acid OAC

H

2 N O EDC 0 OAOC 0 HN HN 7H5 0 OAOC 14 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 I hour, the reaction was worked up in the usual manner. Chromatography gave 143.1 mg of blocked diphosphate (15) as an oil. 0 0 OHcH5 Na HN c HN H s 1.' Pd(PPH3)4. PhSi3 0 0 o 0AO ooc 3 2. DEAE OAOC 0 OADC 0O 10 15 16 To a solution of blocked diphosphate (15) (177.9 mg) in degassed chloroform (1.7 mL) in a dry box was first added phenylsilane (PhSiH,, 50 pL) and then tetrakis-triphcnylphosphine palladium (0) (Pd(PPH 3

)

4 , 70 mg). After I hour, the reaction was removed from the box and chloroform: methanol: water, 2:3:1, was added and the mixture stirred for I hour. It was poured 15 onto a diethylamino-ethylcellulose (DEAE)chromatography column. Elution of the column with -22 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 pL) followed by lyopholization gave 136.2 mg of (16) as a white solid. 5 Example 2: Chiral Malonate-Type I C I OC 2

H

5 K2CO3 . OC 2

H

5 NH HCI NH 17 18 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 10 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 0 1,2-Dichloroethane 0 OCH 3 HO OCH 3 + O C 2 H5 N

NH

2 - HCI H 19 18 21 To a solution of L-serine methyl ester hydrochloride (19) (41.6 g) in 1,2-dichloroethane 15 (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.

-23 0 OCH 3 OIBAL O -,OH 21 22 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 I M solution in hexane). The mixture was allowed to warm to room temperature overnight and then carefully poured onto an aqueous 5 solution of Rochelle's salt (500 g in 1.0 L water). The mixture stirred for I hour and worked up in the usual manner. Chromatography gave 25.5 g of alcohol (22) as a white solid. OH O j-OH 0 "N--;CIH Is -N TsO C 7

H

15 NaH I -N OH DMF/THF 22 4 24 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 T-IF. After 30 minutes, the tosylate (4) was 10 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. OH OH O O- C 7 His 1. 4 N HCI HO O C7H 1s N 2. 1NNaOH NH 2 24 25 -24 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. 0 OH C rKC1 3

H

2 7 OH HO O 7H1i Na HCO ' OO37 15

NH

2 3 HN C 13

H

27 0 5 25 26 The diol (25)(2.88 g) wasdissolved 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 10 give 3.07 g of alcohol (26) as an oil. BocHN -\,\. OH P N OH HO'-f-'-'" 'C7His5 BocHNWO O 'C7H 15 HN r 13H27 Tetrazole HN 13

H

27 0 2. oxone O 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 15 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.

-25 0 OH H BocHN 9,-- ,, 7H1 HO H2 BocH 7H 15 HN C 1 3 H7 EDC. DMAP 0 HN Qc3H2 27 28 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 stir-red 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 5 usual manner and chromatographed to give 2.07 g of N-BOC-protected amine (28). 0 9)I0 2 9'"CII H23 9 TFA BocHN 7H15 TF H2N 7H15-OPI' HN C 1 3

H

2 ? TES HN C 1 3

H

7 7 0\ 0 28 29 To a solution of the N-BOC-protected amine (28)(187.3 mg) in methylene chloride (1.5 mL) was added TES (240 pL) and TFA (300 pL) and the mixture stirred for 45 minutes. Toluene was added and the solvent removed under reduced pressure. The residue was dissolved 10 in methylene chloride and worked up in the usual manner to give 154 mg of amine (29) as an oil. O H N 13H27 H 0 0 CAIH 23 0 o eH1 Malonic acid O1 H2N EOC O

HN),<C

1 3 H27 H O 0 CH 3 HN., C13H27 29 30 -26 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 I 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). o 0 HN /HN 13HV HN OH HNN 13H 0 0 1 Pd(PPHi3) PhSiH 3 N 0 2. DEAE 0 H 0 0- IH23 0 1 Ha HN i H2 OH HN .

1 3HV 100 5 30 31 To a solution of the dimer (30) (54 mg) in degassed chloroform (2mL) was added PhSiH, (14 pL) and Pd(PPh 3 ), (18 mg) followed by removal of the cooling bath. After 1 hour, the reaction was quenched with chloroforn:methanol:water (2:3:1) and poured onto a DEAE 10 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 PL) was added and the mixture. lyophilized to yield-white solid (31) (25 mg). Example 3: Chiral Malonate-Type 2 0 1.2-Dichloroethane 0 OCH 3 HO OCH + OC 2 H - N

NH

2 - HCI H 32 20 33 -27 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. O 0 OCH 3 OIBAL O "OH N N 5 33 36 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 I hour and worked up. Chromatography gave 18.6 g of 10 alcohol (36) as a white solid. Os 1H ~ TsO y C7H1s NaH OOCCH 1 s OH DMFITHF 36 23 37 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 m-L). The mixture was stirred 15 for I 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.

-28 OH OH Oi 1. 4N HCI N 2. 1 N NaOH NH 2 37 39 The alcohol (37) (1.03 g) 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 5 with I 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). 0 OH OH

NH

2 NaHCO 3

HNX

13

H

27 0 39 41 To a solution of amino-diol (39) (553 mg) in THIF (3 mL) was added saturated aqueous 10 sodium bicarbonate (6 mL) followed by myristoyl chloride (628 PL). After 50 minutes, the reaction was worked up in the usual way. Chromatography gave 389 mg of amide-diol (41) as an oil.

-29 BocHN-\ OH N 0OH HOOB'cH 1 1 I HO7His P--BcH W 'O7 is HN f,,C13H27 Tetrazole HN 13H27 0 2. Oxone 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 5 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. 0 O Hc H H O ' H pOH - - - - -O -eK- , H 0o OH 11H23 HN Ci3CH27 EDc, DMAP HN 1CI3H27 0\ 0 10 42 43 To a solution of the phosphate alcohol (42) (3.9 g) in methylene chloride (126 mL) was added EDC (10.8 g), DM AP (66 mg) and dodecyl acid (1.62 g). The reaction mixture was stirred oyemight. Additional acid (1.6 g), EDC (I 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 15 amine (43).

-30 o 0 BocHN 9

C

1

H

2 3 TFA

H

2 N C 0HNC 3 H TES HN C 3

H

7 7 43 44 To a solution of the protected amine (43) (194 mg) in methylene chloride (1.5 mL) was added TES (240 pL), and TFA (300 pL). The mixture was stirred for 20 minutes and additional TES (50 pL) and TFA (50 pL) was added. After I hour, toluene was added and the solvent 5 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. H2N 9 O nH2 HO U O> 9 0 nH2

HNC

3

H

2 7 EDC 0 0 HN (C 1 H2 7 29 45 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 p L), EDC (12.4 mg) and a trace of DMAP. The ice bath 10 was removed and after 2 hours, the reaction was worked up in the usual manner. Chromatography gave 44 mg of amide (45). H O tHD 1FA H 9 O N TES , H - N 0 0 HN CH2 0 0 HN..<jHr 4 O 45 46 -31 To a solution of the amide (45) (44 mg) in methylene chloride (0.5 mL) was added TES (90 iL) and TFA (100 pL). 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). 0 H o 0~ ~ H 0 N H15C1H7 EDc HN O O O cyH15 o 0 O1 o%1IH2 HN O ~' I I H234 HN rC 1 3

H

27 HN...C 1 3

H

27 5 44+46 47 To an ice cold solution of the free acid (46)(41 mg) and the free amine (44) (26.3 mg) in methylene chloride (500 pL) 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 I hour, the reaction was worked up in the usual manner. Chromatography gave 32.7 mg of coupled compound 47 10 as an oil. 0 O 0)UH23 HN HN 13 H2 - OHis o O HN OH HN 13H27 /2 1. Pd(PPH3), PhSiH 3 O o H N k2. DEAE O rH2 HN 7 0 15 N 13H Z 6K.OH HNC13HO 0 0 47 48 To a solution of the coupled compound (47) (32.7 mg) in degassed chloroform (1.5 mL) in a dry box was added PhSiH 3 (8.5 pL) and Pd(PPH 3

)

4 ( 1I mg). The mixture was removed from -32 the box and cooled in ice. After 5 minutes, the ice bath was removed and after I 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 5 treatment followed by lyophilization. Example 4: Chiral Urea-Type I - O 7 His 'I y uCH3~ Phosgene HN HN C 13

H

27 H2N O= 7Hs' O C H N 7 1 H N /0 0 H& )(C1 3 11 27 lot9 9 'k 1 jH3 HN 1C 3 Hz 7 O 44 49 To a solution of amine (44) (46.1 mg) in toluene was added saturated sodium bicarbonate (0.5 mL) followed by phosgene (15 pL of a 1.93 M solution in toluene). After 30 minutes, 10 additional phosgene (10 pL) was added. After 2 hours, additional phosgene (5 pL) 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. SOO 0 ~ 0kCjjH23 0 o kC:1 H?3 HN r - HN -C1H27 O-jP Na HN c13H27 o 1. Pd(PPH 3

)

4 . PhSiH3 HN H o 2. DEAE HN o O CnH2 HN03Hv 0 Na HN SYCi 3 Hr 0 . 49 50 -33 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 PhSiH, (8 pL). The reaction vessel was removed from the dry box and placed on ice. Pd(PPh) 4 (10 mg) was added and after 5 minutes the ice bath removed. After I hour, the reaction was quenched by addition of 5 chloroforrn: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 01 H OUtH ntrororneiyi ch~oroformle 9.. 1 ~ H2 H .8 ic(ormethyam no on e O' IN H1Nyrzi3H7 HN,.CH~y O 0 29 SI 10 To an ice-cold solution of trichloromethyl chloroformate (2.6 p L) in methylene chloride (200 .iL) was added a solution of free amine (29) (35 mg) and 1,8-bis-(dimethylamino) naphthalene in methylene chloride (200 pL). After S 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. 0 101~ ~ enLClH230 o=c=N,, 0 0o-c H H23 HNy 13H27 y1

CH

2 cI 2 HN HN C1 3

H

27 + 0 HN 0 0 O CIH3 6] H23 H2N _,- 0 ,,O r~ H c,3Hyr H 5 H H 15 51 +44 61 -34 To a solution of the isocyanate (5 1) (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. 0 0 9 l ClIH23 0 9l CIIH2 3 H /-0 HN,, 1l3H27 1. Pd(PPh3) 4 . PhSiH 3 O Na HN CH2 9H O O 2. DEAE Chromatography MNO 0 OH HN O C,,H2 HN O CIIHZ 0-FfI-O '""CHi O50- Po Hi . HN c, 3 H 0 Na HN C 13

H

27 0 0 5 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 PhSiH, (6.6 4L) and Pd(PPH 3

)

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 I hour, chloroform:methanol:water (2:3:1) was added and the mixture stirred for 15 minutes and stored 10 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 I H H OH OH TsO 7H1 5 NaH/DMF OH THF 63 4 65 15 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) (0.4 i mL) in 8 mL of THF dropwise -35 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). H H 1. C IH 23

CO

2 H. EDC. DMAP H O C 1

H

2 3 Q O C7H15 .HO7O 15 7

H

1 5 A 2. HOAc-H 2 0 HO 5 65 66 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 10 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 0.77 g of semi-solid diol (66). 0 0 H O ClH 23 TBDPSCI H 0 C 1 H23 HO 'O_'- C 7

H

15 TEA TBDPSO "O '-_'C 7

H

15 HO DMAP H6 66 67 A solution of the diol (66) (0.22 g), DMAP (6.3 mg), TEA (100 pL) and TBDPSCl (164 15 pL) 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.

-36 0 00 C IH 2 3 0 t-CIH3C13H27CO2H1. )Hn2 H C) CH23 ,TBDPSO0' "' 7H15 TBDPSO O' 0 "' C7H15 EDC. MAP H27 HO 0 67 68 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 5 usual way and chromatographed to give 0.4 g of silyl protected ether product (68). O O H 0 CIIH 23 H C 11 H23 TBDPSO O -';'C7H15 HF HO OCi o C13H 27 0 C 13

H

27 0 0 68 69 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).

-37 OoBocHNcO H OUIHz3 HP- O n~H23j HO 7H 15 BocHNO 7H o C13H 27 Teirazole O CH 27 0 2. Oxone 0 69 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. Afier four hours, the mixture was cooled to 0 *C followed by the addition of oxone (82 mg) in THF (0.5 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). 0 -O O 715 0 3P.

1 127V O ~HN13y 0 1 H Malonic acid 0 BocHN AO7HO5 E0c 27 HN O o Ak Hz 0 O ffZjH27 70 71 To a solution of the protected phosphate (70) (72 mg) in methylene chloride (I mL) was 10 added TES (120 p L) and tri fluoroacetic acid (0.6 mL) followed by stirring for I 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.

-38 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). o 0 0 H O't 1 1Hz 3 O H O I H23 0 0 CH 15 }-O O -- C 7

H

1 5 HN O 3H27HN 0 Na O C 13 H27 1. Pd(PPht) 4 . PhSiH3 a O O O O H e2 H 0 H 0 CnIH23 0 0 C7His 2 DEAE Chromatography - - e7Hs O C0H27 Na O C13H 27 0 0 71 72 5 The protected dimer (71) (32.5 mg) from the preceding reaction was dissolved in degassed chloroform (2 mL) and PhSiH 3 (8.6 pL) was added in a dry box. The mixture was removed from the dry box and Pd(PPh 3

)

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 IN sodium hydroxide (34.2 pL) followed by lyophilization.

39 Preparation of ER-804253 OH OMs 5 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 10 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. N O OMs N 3 15 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 20 over silica gel using a 4:1 ratio of hexanes to ethyl acetate and the dried product fractions give 420 mg. NN N3 NH2 25 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.

40 Pt N N O

NH

2 NH.11H3 0 The amine (930 mg) was dissolved in THF (10 mL) and sat. sodium bicarbonate (22 5 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. HO N O H 2 N NH C 11

H

23 NH CIIH 2 3 0 0 10 The aide (1.11 g) was dissolved in methanol (14 mL) and 4 N hydrochloric acid (8 mL) added. The mixture was stirred for I hour 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 15 washed with water, dried and the solvent removed to give 930 mg. HO NH C 11

H

23 H 2 N O 'KHO HN NH...lC 1

H

23 HN1 The amino alcohol was dissolved in THF (6 mL) and saturated sodium bicarbonate 20 added (13 mL). After 5 minutes the mixture was cooled to 0 * C and myristoyl 41 chloride ( 300 pL) added. After 30 minutes, the mixture was worked up in the usual way to give 430 mg. NHBoc0 NH) r 1

,H

23 gHNHKCI

H

23 51 OH'-r O C7H H23 ,1=0071 HN C13H28 C N 132 00 The alcohol (101.7 mg) was dissolved in ice cold methylene chloride and phosphorylating reagent 11 (90 pL) 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 10 and chromatographed to give 174 mg (not purified) NHBoc N H 2 0 NH CIIH 2 3 NH) 'CiH2 O=P-O 'y'Th 7

H

15 7

H

1 5 I A HN C 13

H

28 HN C 13

H

2 8 O 00 The protected amine from the above reaction was dissolved in ice cold methylene 15 chloride (1 mL), trifluoroacetic acid (1 mL) was added and the mixture stirred for I hour. The TFA was removed and the mixture purified to give 106.7 mg.

42 0 HN .. H1 2 H28 H2 NH C 1 H23 HN~ O0Ii 12 O= -O O )" 7

H

15 HN0 O ~ HN <C1 3

H

28 NH" 11 H23 7

H

15 SHN C 13

H

28 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 5 and worked up to give 1 12.3 mg. O= 0 . HN 13H28- CjH 0 O'0 7H155 f -' N~yil11H 23 HN0 O O ER-804253 HN O

NH..C

1 H2 3 0 HN. 13

H

28 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 10 mg) and the mixture stirred for 1 hour. The mixture was chromatographeci on a DEAE column to give 27.7 mg of ER-804253. H N -- CC 1 H 2I 0I 43 Preparation of ER-804130 HN 0 _ 5 CH, 7 C1 C 8

H

17 Ph Ph To a solution of the amide (3 g) in THF (65 mL) at -78 0 C was added an equivalent 10 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. 1 5 H O MSO, ACI 0

H

2 1 To a solution of the alcohol (5 g) in ice cold methylene chloride (100 mL) was added 20 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. MsO ,-z&,C 1

OH

21 . BK,-N.C 1

OH

21 25 The a 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. 30 44 CH N Br C10H 2 1 C 7 H 15 N O

C

10

H

21 Ph Ph 5 A solution of the aide (2.8 g) in THF (15 mL) was added to a -78 *C solution of sodium bis-trimethylsilylamide in THF (15 mL). After I hour, the bromide was added and the mixture allowed to warm to room temperature and worked up in the 10 usual manner to give 1.02 g. 00 0 0 15H 1

C

10

H

21 C Ci H 2 O P h Ph To a solution of the olefin (1.02 g) in EtOAc was added palladium on carbon (126 20 mg) and the mixture placed under hydrogen. After 4 hours, the mixture was worked up in the usual manner to give 1.0 g. 0 0 0 25 C 7 H 15 N-'-0 C7 1 5 O

C

10

H

21 CoH 21 Ph To a solution of the amide (1.0 g) in ice cold THF (20 mL) was added water, 30 hydrogen peroxide and lithium hydroxide. The next day, the mixture was worked up in the usual manner to give 590 mg of acid.

45

C

7 His OH C H C 7C1H2 C0H21 5 O lH1:D 5 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 10 material was dissolved in ice cold ether and LAH solution (2 mL of IM) added. After 5 minutes, the mixture was worked up in the usual manner to give 556 mg of the alcohol. 1

H

C 7H OH

C

7

H

ClOH 21 Cl 0

H

2 1, : 20 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 (I 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. 25 CH Ph 3

P*CH

2 0MeBr-

C

7

H

1 OMe 3C1H21 30 46 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 5 worked up in the usual manner to give 342 mg.

C

7

H

15 ,z.OMe

C

7

H

1

C

1 0

H

2 CO C 10

H

21 C 10 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 15 manner to give 325 mg.

C

7

H

15

C

7

H

15 O C1 0

H

2 1 H2C OH 20 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 25 to give 303 mg of the alcohol. 0 7 H 15 OH

C

7 H 1 5 OMS C 1 0 H 21 C 10

H

2 C 30 47 To an ice cold solution of the alcohol (303 mg) in methylene chloride (10 mL) was added triethylamine (150 pL) and mesyl chloride (76 pL). After 4 hours, the reaction was worked up in the usual manner to give 352 mg.

C

7

H

15 OMs OH O N 0 CClH 21 " C 10

H

2 1 To a solution of the oxazoline (I mL) in ice cold THF (5 mL) was added potassium t butoxide solution (2.2 mL of IM). After 30 minutes, the mesylate was added in TIHF 10 (5 mL) and the mixture stirred for 8 hours. The usual work-up gave 318.5 mg. P

NH

2 HO C10 H21 C02 15 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. 20 48 NH2 9 Cr I.uiH 27 HNJ C 13

H

27 HO~ 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 5 chloride was added. After 30 minutes, the reaction was worked up in the usual manner to give 146 mg. 0 O HN C 1 3

H

27 O I r2O HN C 13

H

27 HO OCHsBocHN BocHN~r O0OO 7i HO1 0,-'-C 7

H

1 5 Hod-IN: O 7H C12H25 OC1 2

H

2 5 10 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 ( I mL) and THF (2 mL) was added. After 90 minutes, thiosulfate was used to quench the reaction. Standard work up gave 140 mg. 15 o MY C 13

H

27 HN C 13

H

27 BocHN OC

H

2 N POC7HI O NC12H25 0C 12

H

25 To a solution of the substrate in ice cold methylene chloride was added triethylsilane 20 (370 pL) and trifluoroacetic acid (110 pL). After 5 minutes, the volatiles were removed to give 148 mg.

49 O HNI C 13

H

27 0 0 O ,' 'C7His5 0 HN I C13H27 C12H25 H2N O -O O7H15 CH2H25 1 0 CI2H25 P..z/ 0 HN yC 13H27 0 To a solution.of the amine (66.9 mg) in ice cold methylene chloride (0.8 mL) was 5 added saturated aqueous sodium bicarbonate (0.8 mL) and phosgene solution ( 20 pL of 1.93 M). After one hour, additional phosgene (10 .L) was added. After 30 minutes, the usual work-up gave 47.7 mg. 0 HN

C

13

H

2 7

C

12

H

2 5 HN O ' ER-804121 HN d 0 0 C3HH RN yC 13

H

2 , O 20. 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.

50 After 5 minutes, the mixture was applied to a DEAE column and chromatographed to give 31 mg of ER-804130. Preparation of ER-804558 5 0 CIOH21

C

10

H

21 TBDPSiO -OC63 TBDPSiO O C63 NH2 NH S H 23 0-"0 To a solution of the amine (325 mg) in methylene chloride was added triethylarnine 10 (321 pL) and 1-dodecanesulfonyl chloride. After 3 hours, the'usual work up gave 384 mg. 0 0 CioH 21 CI0H21 TBDPSiO C6H3 " HO O C6HI3 NH S CIIH23 NH S CIH23 -1/ \ 0// \ 15 To a solution of the protected alcohol (384 mg) in THF (4 mL) was added tetrabutylamnnonium fluoride (123 mg) and acetic acid (29 FL). After 2 hours, the usual work up gave 180 mg. 20 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.

51 Preparation of ER-804442 The diol amine was mono-protected as its t-butyl-diphenylsilylether outlined above. 5 NHW2 PhPh TPSiO" ' ''03TD~ O H 0 T D P1O OC BH 13 OH The amine (2.6 g) and benzophenone imine (1.1 mL) were mixed and heated to 40 0 C for 4 days to give after chromatography 3.3 g. 10 NPh R Ph TBNP - b- TB TBD PS iO O ,C6H 13 - TBPS iO" 6HrC 13 OH 11

H

23 0 To a solution of the imine (3.3 g) was in ice cold methylene chloride was added lauric 15 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. Pt Ph ,

NH

2 TBDPSiO 0 H13- T 11 H 23 O 0 To a solution of the imine (3.14 g) in ether was added I N aqueous hydrochloric acid. The next day, the reaction was worked up in the usual manner to give 2.81 g. 20 52

NH

2 O TBDPSiO O is NH )-NH-C12H25

C

12

H

25

NH

2 ---. C 12

H

25 NCO -- H1K1123 13 OrCIH23 0 To a solution of trichloromethylchloroformate (12 jiL) in ice cold methylene chloride (250 pL) was added dodecylamine (18 pL) and diisopropylethylamine (27 gL). After 5 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 p±L). After 2 hours, the usual work up gave, after chromatography, 60.9 mg. 10 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 I5 00

C

1

H

2 3<kI C 11

H

2 3 N OH 0 20 To an ice cold solution of glycine (8.26 g) in aqueous sodium hydroxide (4.4 g in 60 rnL) 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. 25 OH OH HO O -'~ C 6 Hi 3

TFN

3 HOO

NH

2

N

3 53 : 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 5 worked up in the usual manner to give after chromatography 1.14 g. OH OH HO OC 6

H

1 3 ' TBDPSiO O' 6

H

1 3

N

3

N

3 10 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. 0 OHO OH OA CIOH21 T TBDPSiO" O6Hi

N

3

N

3 15 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. 20 0 0 Q..,kCIOH 2 ] 0

-KCIOH

2 1 TBDPSiO O C 6

H

13 ' HO O 13

N

3

N

3 54 To a solution of the azide (778 mg) in THF was added acetic acid (77 LL) and TBAF (323 mg). The next day, the usual work up gave, after chromatography, 428 mg. .5 )L-C10H21 O A CI OH21 0 HO - O 6

H

13 - BOCNH - -- O6

N

3 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 10 mg in 3 mL). The reaction was quenched with thiosulfate. Usual work up, after chromatography, gave 392 mg. O O

)Q..CIOH

21 ) CIOH21 0 OC

H

2 N O-P-0 0>OC683 BOCNH O-P -OO6H3 0 N 3 O

N

3 15 The protected amine (460 mg) was dissolved in methylene chloride and trifluoroacetic acid (394 kL) and triethylsilane (308 pL). After 1.5 hour, the usual work up gave, 392 mg. 20 55 N 30 O-- -O OC6H3O 1 3 o N 3 NH2 - OIOH21 HN O OO NO C 3 O N112,A~

C

6

H

1 0 CoH2 10 0 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 5 toluene). After 15 minutes, the usual work up, after chromatography gave 342 mg. 0 9 C io H 21 0

)Q...CIOH

2 1 0 0 O -ON.O - C6H3 0 O N 3 O NH 2 HN HN O O HN HN O i O- ON C6His O O613 0 0 10 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. 15 56 0 0 0 ) CIOH 2 1 0

,COH

2 1 O- 0 C6H3 0 -

C

0 6

H

1 3 0 NH 2 O HN O HN HN 0 H C H 23 HN HN 0 F Osp - 2 p HN 23 0 0NKC -O O -- C6His -- 06 O5 Ct H2 CioH21 To a ice cold solution of the urea (55 mg) in methylene chloride was added the acid 5 (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. 10 ER-804222 was prepared in a similar manner except that the condensation product between lauryl chloride and glycine, 15-methylrnyristic-acid was used. 15 Preparation of ER-804281 P Ph \N N OPM a OH 20 To a ice cold solution of the protected alcohol (8.3 g) in acetonitrile: water was added CAN (41.4 g). After I hour, the usual work up gave 5.7 g.

57 2 H O O OHOH A solution of the alcohol (5.63 g) in 4 N HCl solution was heated to reflux for I hour, 5 cooled, neutralized with sodium hydroxide and worked up in the usual manner to give 2.1 g. bH 2

NH

2 HO, XO - TBD PSi O OH OH 10 To an ice cold solution of the alcohol (2.2 g) in methylene 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. 15 Ph

NH

2 TBDPSiO O C 7

H

1 5 N Ph OH 1 OH TBDPSiO,_ ,,O 7H 15 OH To a solution of the alcohol (1.93 g) in methylene chloride (40 mL) was added benzophenone imine (0.8 mL). After I day, the mixture was heated to reflux 20 ovemight. The usual work up gave 1.67 g.

58 Ph PhPh t Ph TBDPSiO O C 7

H

1 5 TBD PSiO O-. HC7H i5 O CH23 OH 0 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 5 work up gave 74% yield.

NH

2 TBDPSiOh TBD PSiO ' O , C7H 15 i7H 15O

C

11

H

23 O C 11

H

23 0 10 To an ice cold solution of the imine (2.9 g) in ether (50 mL) was added I N HCI (50 mL). The next day, the usual work up gave 2.09 g.

NH

2 HNL14H 29 TBDPSiO O C 7 H15 TBDPSiO AHis O IH23 O C 1 1

H

23 0 0 15 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.

59 HN14H29 AOC., A 14

H

2 9 TBDPSiO .O < C 7 H1 5 TBD PSi O C7H15 0 11H 23 0 IH 23 0 O To a ice cold solution of the amine (221 mg) in dioxane was added allyl chloroformate (40 mg) and 308 pL of I N NaOH solution. After 2 hours, the usual 5 work up gave 200 mg. AOC N H29 AOC, AH29 TBDPSiO , <O C 7 H15 HOO ' O C 7 H15

IIH

2 3 n H 23 0 0 10 To an ice cold solution of the protected alcohol (365 mg) in THF was added TBAF (1924 pL) and acetic acid (122 pL). 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 15 above to give ER-804281. Preparation of ER-804339 ER-804281 - ER-804339 20 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 pL). After 4 days, the usual work-up gave 1.1 mg.

60 Preparation of ER-804674 ER-804281

-

ER-804674 5 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 10 additional 3 days, additional bicarbonate (28 mg) and Mel (16 pL). After an additional 6 days, the mixture was worked up to give 9.1 mg of product. Preparation of ER-804596 15

N

3 N HO O C 7

H

15 iP--O OPMB OPMB To a solution of the alcohol (393 mg) in methylene chloride (2 mL) was added diisopropylamine (210 iL), tetrazole (105 mg) and phosphorylating reagent (as 20 described above) (488 mg). After 2 1/5 hours, the usual work up gave the desired product.

N

3 HO 9 H O OP7i Na- 0 OPMB OPMB OPMB N3 61 To a solution of the diol (73 mg) in acetonitrile was added tetrazole (1.75 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. 5

N

3

N

3 OJ O' C7H15 O > C 7 His OPMB OH 6 O OPMB 6 O OH 0~0 0

N

3 N3C 7 H1 5 OC 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 I hour, the usual work up provided 10 68.5 mg. N3

N

3 O O C7H 1 O 1

H

23 OH )6 0 0 4H O C15 0 C 11 H 23

N

3 O P-0 O"' C 7 H15

N

3 15 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.

62 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. 5 Preparation of ER-804732 -P-P N OH N OMs 10 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 15 chloride (3.69 mL) was added dropwise after which time the reaction was stirred at room temperature for I hour. The usual work up gave 5.551 g. 20 N OMs N N The mesylated (1.114 g) was dissolved in DMF (30 mL) followed by sodium azide 25 (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. 30 NO H 2 P0N 4 \ HO

N

3 63 The protected aminoalcohol (0.466 g) was hydrolyzed using 4 N HCJ (15 mL) at 107 *C for 3 hours. After cooling to room temperature, the reaction mixture was filtered 5 and extracted with ethyl ether, dried, concentrated and used in the next reaction. blH2

HO-K.N

3 HN C 1 3

H

27 10 HO The crude aminoalcohol was dissolved in THF (5 mL) with saturated sodium 15 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. 20 0 0 Hbdl C 13

H

27 HN 13H7 HO N3 TBDPSO,,, N 3 25 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. 30 64 O O HLA'1 3

H

27 HVAJ C 1 3

H

2 7 TBDPSO.,_ N 3 TBDPSO A NH 2 5 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. 10 OPMB H OPMB

HO'-C

7

H

15 O 7

H

1 5 15 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 20 *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. OPMB O 0 ~ ~ C 7

H

1 5 HN KC 1 3

H

27 Hl>'C 13 H2 7 H TBDPSO NH 2 TBDPSO. N C 7 Hi 5 OPMB 25 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 65 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. H 1C3H27 HL1 3 H27 H TBDPSO N C7H15 TBDPSO,, .zN< N,7H 15 OPMB OPMB 5 The secondary amine was dissolved in 1,4-dioxane (15 mL) and cooled to 0 *C followed by the slow addition of I M sodium hydroxide (3.0 mL). After stirring for 10 minutes allyl chloroformate (0.236 mL) was added dropwise after which time the 10 reaction was warmed to room temperature and stirred for 16 hours. Work up in the usual manner gave 0.613 g. HN C 1 3

H

27 HtC 1 3

H

27 TBDPSON C7H15 TBDPSOO C7H OPMB OH 15 The para-methoxybenzyl ether (0.613 g) was dissolved in a 4 to I 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. 20 66 H t C 1 3

H

27 HN'L '13H27 STBDPSO - N 7H15 TBDPSO,,,; y 7H 15 AOC O OH 0 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 5 in the usual manner gave 0.436 g. O O HN C 1 3

H

27 Ht C 1 3

H

27 TBDPSO.,,- N 7

H

1 5 . HO-,,A-_ IN 7Hs AM CO Or 11 H23 Or 11H 23 0 0 10 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. 15 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.

67 Preparation of ER-804680 0 OPMB 5 PMB 5 C7H15 HO C 7

H

15 The aldehyde (1.54 g) was dissolved in THF (28 mL) and cooled to 0 "C after which 10 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 15 1.55 g. 0 OPMB HO' - C 7

H

15 Ht4 13H27

HNA

1 kC 3

H

27 H TBDPSO-

NH

2 H27 TBDPSO .CN C7H15 0 OPMB 20 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. O HIM C 13

H

27 H 1 3

H

27 H H TBOPSON C7H1N 7H15 OPMB ~0H 68 The methoxybenzyl ether (0.567 g) was dissolved in a I to I ratio of acetonitrile to water (16 mL) with methylene chloride (8 mL) and cooled to 0 *C. CAN (1.53 g) was 5 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. 0 Ht4J 1

K

13

H

27 HtJ C 13

H

27 H HTH TBDPSO NY 7H15 TBDPSO -,- Ny- 7H 1s -y 7j 0 OH 0 0 10 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 15 work up procedures provided 0.622 g. Ht% 13H 27 HN C13H27 H H TBDPSO 7H1 HO15 7Hs O - O 1 CH 23 0 CiH23 o 0 20 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.

69 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. 5 Preparation of ER-804679 HA C 13

H

27 HtA 13

H

27 TBDPSO_, 4 .. . C7His 1 TBDPSO N CHis O 11lH23 0 11H23 0 0 10 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 1 5 chloride, washed with water, dried, concentrated, and chromatographed to give 0.068 g. H0 13H27 H C 13 H27 TBDP SO N j-zN C7H 1 HO, C7H 15 0 0 IZH 23 0 11CH23 o 0 20 The silyl ether was deprotected in THF (5 mL) with acetic acid (0.025 mL) with the addition of tert-butylammonuium 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.

70 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. 5 Preparation of ER-804764 HWJl C 13

H

27 HJ C 13

H

27 HO O -O O IH23 H O C1H 2 3 0 0 10 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 15 "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. H MeOCH 2 P*Ph 3 BH 3 11 H23 11H23 0 0 20 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.

71 HN*'%1 3 H27 H H 13

H

27 MeO-" O O n H 23

O

1

C

11

H

23 0 0 5 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. 10 H HN C 13

H

27 Hl C 13

H

27 Q CH23 I C H23 0 0 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 15 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 20 above to give ER-804764.

72 Preparation of ER-804772 Me M MeO HO ~ ~ ''~ C0H 8 H 1 7 10 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 15 high vacuum. Silica gel chromatography of the resultant crude product using 10% ethyl acetate in hexanes gave 2.084 g. M OPMB 20 i ) b , . HO HOH1 OH S1C

H

17 25 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 30 tartarate. The normal work up gave 2.1 g.

73 PMB PMB HO C 8

H

17 17 5 The alcohol (1.286 g) was mixed with triethylamine (0.883 g) in methylene chloride (15 mL) and cooled to O *C. Methanesulfonyl chloride (0.575 g) was added dropwise followed by stirring for 20 minutes at 0 *C and room temperature for 2 hours. The normal work up gave 1.496 g. 10 N H 0 PM B P- OPMB 15 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 0 *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. 20 As with examples provided above, the para-methoxybenzyl protecting group was hydrolyzed with CAN, the protected amino alcohol hydrolyzed using aqueous HCI then KOH, acylation of the amine with tetradecanoyl chloride, silylation of the primary alcohol with TBDPS, acylation of the secondary alcohol with dodecanoyl 25 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.

74 Preparation of ER-804947 0 5 pH-Ph OBn N H -N 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. 10 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.

NH

2 15 P N OBn HOOBn The protected aminoalcohol (0.189 g) was hydrolyzed using 4 N hydrochloric acid 20 (2.5 mL) followed by 40% sodium hydroxide (2.5 mL) as described previously to provide 0.121 g. 0 bH 2 H J H 25 HO M OBn H C Hy HO,, K .,,OBn The arninoalcohol (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 30 mL) was added dropwise. After continued stirring for 2 hours the mixture was worked.up in the usual manner and gave 0.181 g.

75 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 5 stirring for 16 hours at room temperature. The usual work up gave 0.3 10 g. HN C 13

H

2 7 HN C 13

H

27 BnON)K O%->A7H 15 BnO ) O -1C7H 15 10 The para-methoxybenzyl ether (0.305 g) was dissolved in acetonitrile (8 mL) with water (2 mL) and cooled to O *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 15 gave crude alcohol. 0 ~HN C13H27 HN C13H27 BnO.-l, O Y 7H15 J O OH O CH 23 0 20 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.

76 0N4C 2 HN 13

H

27 B nOOC7HH15HO 21 C7H 15 B nOLj-.<, CHl Y .'<Y 1 H O O nH3 0 O nZH23 0 OyJ ZiH3 0 0 00 The benzyl ether (0.355 g) was dissolved in ethyl acetate (50 mL) with palladium 5 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 10 the allyl protecting groups removed with phenylsilane and palladium as described abovetogive ER-804947.

-77 BIOLOGICAL EXAMPLES Example 7: Induction of Cytokines (in vitro) There are no validated in vitro assays that can be used as an indicator of adjuvant activity. However, the inability of a molecule to trigger any stimulatory response in macrophages is a 5 strong indication that it is not likely to be an immunoadjuvant. For this reason, the ability of compounds to stimulate release of TNF-alpha and other cytokines from immune cells may be indicative of their ability to stimulate an immune response which may result in adjuvant activity. A. Assays in human whole blood The most readily available human system to test compound activity on 10 monocytes/macrophages is in whole blood. Various concentrations of compounds of the invention were added as lOx stocks in 50 pl of Ca", Mg"- free Hank's balanced salt solution (HBSS) followed by 50 pl of HBSS into 400 pl of heparinized 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 pl/well (final concentration of whole blood was 80%). After a 3-hour 15 incubation with gentle shaking at 37*C in a 5% CO 2 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 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-bome cells 20 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 pM) ranges from inactive (such as compound 110) to compounds demonstrating greater activity than the LPS standard. B. Cultured human cell lines 25 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, -78 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 5 as the presence of serum. Table 1. Stimulation of cytokine release by compounds in vitro ER - Compound Whole Blood THP-1 ccli Stimulation (% of (% of LPS. at compound 100 at 10 pM)(' OJM) +scrum - serum MPL Standard 29 112022 131 * 10.2 (n= * 6) 111230 49 10 111231 17 111232 158 155 225 This is important to determine if serum components such as lipopolysaccharide binding protein are necessary for drug activity.

-79 1 11233 141 a W 112043 0 112047 0 112047 0 9 5 112048 0 24 112049 0 112063 Jil -go 112064 50 H12066 162 330 112071 0 0 .0w H 0 S 112072 0 112091 0 10 11209 0 112092 0 -81 112093 0 112098 0 112100 0 5 112859 00 112860 0 -82 112861 0 113634 0 113635 0 113643 0 5 113644 0 113651 133 4.4 (n=4) 215 254 r-J 113665 -83 113666 18023 63 019772 69 1 18989 159 5 118999 105 0*07 119000 60 119001 113 1 18949 1138 -84 119327 1653 n-3) 119328 180: i4 2 (n=3) 119329 2 x2 (n=2) 11952) 103 5 119522 129 .Z-yw 119523 -~176 803022 0 _______ 164 803045 65 -85 803056 151 *42 803058 149 * 37 (n=2) 803059 2 803592 15 *-1 5 "' Response in each assay was compared to 10 pM compound 100 internal standard which typically induced 2.3 fold increase in TNF-alpha PLAP expression over basal.

'

2 Tested at @5.8 pM. C. Murine splenocytes The ability of compounds to stimulate cytokine release from splenocytes can be 10 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, I mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin and 50 pM beta-mercaptoethanol, various concentrations of test compound for 20-24 hours, after which the cell culture supernatant is tested for the presence of cytokines. 15 Spleen cells harvested from mice were cultured for 24 hours 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, 126, 160, and 162.

WO 00/44758 -86 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 5 same media. It is possible that this cytokine "milieu" is responsible for part of this robust immune responses. Example 8: In vivo 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 10 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 gpl 20 protein. The P18 21aa peptide, corresponding to amino acids 308-322 15 of the V3 loop of HIV 1IIB gp120 protein, has been reported to be immunogenic. This peptide with glycine/alaninc/glycine spacer residues plus an amino terminal cysteine residue was synthesized by Genosys (Woodlands TX). The peptide sequence is as follows: CGAGIRIORGPGRAFVTIGKG with the underlined amino acids representing the native sequence. The peptide was isolated to >80% purity using HPLC by the supplier. This peptide was 20 coupled via the cysteine residue to bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) using maleimide activated conjugation (Pierce Immunochemical; cat #77107). The KLH conjugated peptide was used as the immunogen and the BSA conjugate as the screening target antigen for P18 specific antibodies. The indicated amount of KLH-P18 conjugate, was routinely used along with 300 pg of test compound, Alum or PBS was injected at 2 or 3 week intervals (as 25 indicated), into male Balb/c mice (Charles River Laboratories) approximately 6-8 weeks old (1 8 25g). All injections were subcutaneous at the back of the neck with 200pl 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 2"' and 3 'd injections. Sample bleeds are designated as to when taken (i.e. secondary bleed is one week after 30 the second protein injection or two weeks after the second polysaccharide injections, tertiary -87 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 serum separator tubes. Serum was separated from the red cells by microcentrifugation and tested by ELISA for antigen specific IgG levels. 5 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 (PI8-BSA) and coated onto an ELISA plate. As shown in figure 5 and Tables 2 and 3, mice injected with the various compounds along 10 with KLH-P18 antigen demonstrated greater response (higher levels of antibody) than those injected with the P1 8-KLH peptide conjugate alone. Table 2. Stimulation of antibody generation to P18 peptide by compounds ER # Compound Average Serum anti-P 18 IgG Concentration' (fold increase over no adjuvant) 112022 6.7 15 112065 19.4 112066 39.2 'Concentration of IgG assayed at the tertiary bleed. Average IgG for serum from 5 mice that were injected with 300 pg compound and 5 pg KLH-P18 conjugate antigen as described in the Methods section. Antigen-specific ELISAs were performed as described in the Methods Section 20 with Costar EIA/RIA plates coated with 50 pl of 5 pg/ml BSA-P18 conjugate in PBS.

-88 Table 3. Stimulation of antibody generation to P18 peptide by compounds Compound # Compound Average Serum anti-P18 IgG Concentration' (fold increase over no adjuvant) 111232 7.6 112066 13.4* 5 113651 10.S* * Y 118989 4.35* -Co 119327 J 16.5* 119328 26.8* 'Concentration of IgG assayed at the tertiary bleed. Average IgG for serum from 5 mice that were injected with 10 300 pg compound and antigen (below) as described in the Methods section. Antigen-specific ELISAs were performed as described in the Methods Section with Costar EIA/RJA plates coated with 50 pl of 5 pg/ml BSA PI8 conjugate in PBS. As comparison, addition of Alum increased IgG levels 7.4- fold over PBS/antigen alone. Antigen used: primary :1 pg KLH-conjugated PI 8 peptide. 2* and 30 boosts: 0.5 pg KLH-conjugated PI 8 peptide *p<0.05 by Student's two-Eailed t-test (unequal variance) compared to the PBS + antigen group.

-89 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-3 1 antigen. Increases in response are also seen when tetanus toxoid (Figure 6) and menningococcal C polysaccharide 5 (Table 5) are used as challenge antigens. In the assays, I pg of meningococcal C PS or 1.5 pg tetanus toxoid or 5 pg 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 antibody generation to Influenza X31 by compounds 10 ER # Compound Average Serum anti-Influenza X-31 IgG Concentration' (fold increase over no adjuvant) 112022 1.7 112048 5.4 112066 2.3 113651 -90 119327 7.85 * 1 19328 26.8 'Concentration of IgG assayed at the tertiary bleed. Average IgG for serum from 5 mice that were injected with 300 pg compound and 5 pg antigen as described in the 5 Methods section. Antigen-specific ELISAs were performed as described in the Methods Section with Costar EIA/RJA plates coated with 50 pl of 10 pg/mi 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 Menningococcal polysaccharide by compounds ER # Compound Average Serum anti-Menningococcal PS IgG Concentration' (fold increase over no adjuvant) 10 112032 8.3 112048 1.8 112066 ?12.9 113651 18.3 -91 119327 15.8 'Concentration of IgG assayed at the secondary bleed. Average IgG for serum from 5 mice that were injected with 300 pg compound and I pg antigen as described in the Methods section. Antigen-specific ELISAs were performed as described in the Methods Section 5 with Costar EIA/RJA plates coated with 50 pl of5 pg/ml meningococcal PS in PBS plus methylated human serum albumin as described Gheesling et al. 1. Clin Microbiol.32; 1475-82 (1994). In comparison, addition of Alum increased lgG 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. 10 Menningococcal C polysaccharide (PS) was supplied by Pasteur Merrieur 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/mi with phosphate buffered saline (PBS; cat # P-3813; Sigma Chemical 15 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. lmjectR 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, 20 were mixed with the compounds, MPL, or Alum such that the final concentration of the compound or MPL was 300 pg (unless otherwise noted) in the 200 pl 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 25 coated onto 96 well Costar EIA/RIA plates. Plates were coated with 50pl/ well 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 200pl / well of 0.5% gelatin in PBS for I hr at room temperature (RT) and washed 3x with PBS-t. Mouse sera was diluted in PBS-t plus 0.3% BSA and 100pl of various dilutions were added, in duplicate to the antigen 30 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., -92 Birmingham AL, cat #1031-08) was diluted 1:5000 in PBS-t and I 00p] / 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 5 TMB substrate (Kirkegaard and Perry Labs) for 5 minutes. Color development was stopped with the addition of an equal volume of I M 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 10 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-P 18 conjugate. The anti-FAb reagent orients the purified IgG in a mainer 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 15 antigen-specific capture of antibodies. The same reagent solutions used for detection of the antibodies bound to BSA-P18 conjugate, namely biotinylated anti-IgG (Fe-specific) followed with HRP-strepiavidin, 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 20 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).

-93 Table 6. WB ED 5 , vs. % of LPS at IOng/ml ER# Structure WB ED, vs LPS@ lOng/ml SePL >> 10 pm Standard r 1 p 5 112022 - 0.696 pm 111230 111231 0.29 pm 111232 111233 -94 112043 * j O 112044 112047 112048 >> l0 pM 5 112049 112063 112064 -95 112065 0.25 pM 0.04 pM 112066 - 112071 112072 5 112091 112092 112093 -96 112098 o 112099 112100 112859 5 112860 112861 113634 -97 113635 113643 113644 113651 0.70 pM 5 113665 113666 118023 019772 1y -98 118989 - 0.1 PM 118999 119000 119001 -. 23 pM 5 118949 119327 . 0.015 pM 119328 >> 10 pM 119329- -99 119521 119522 -Y 119523 -'-. Y 803022 0.06 pM 5 803028 803045 803056 803058 -0.022 pM -100 803059 0.89 pM 803592 803596 803597 5 803598 803599 803613 ~ 803731 > 10 pM -10 803732 0.85 M 803733 0.70 pM 803751 803783 5 803784 803789 0.10 PM 804053 1.34 pM C:\" thCCX " 2 I-LDOC-2004d2ol I O-Na+ HN (CH 2

)

1 0

CH

3 O-P-O,-K,- O ,- (CH 2

)

6

CH

3 HN Oy(CH 2

)

0

CH

3 804057 /=o0 0 00.008 4M HN 0 HN (CH 2

)I

0

CH-

3 0 y~ (CH 2

)I

0

CH

3 0 -102 804058 0.03 4M 804059 > 10 pM 804061 2.5 M 804097 - 0.3 pM 5 CM:R~cntbNCCRXSU624 I I.DOC-IOMI/2O II - 102X 0 OMe 804121 HN 0.46 jiM FIN OMe 15 20 C:WRPonbliDCCX560824I-I DOC-20/42011 - 1025 804130 0.66 piM 804221 2.2 p M 804222 0.008 pM 804252 400 nM (576-021) + EtOH 804253 > 10 PM C:WRPonbl\DCC\RX5\3602411DOC-1 Il1u201 I -101 O-0 I- -- o HN I-/O O 804281 HN O HN 0.45 pM OMe 0 0 OH 0HN 804313 OH 0 0.014 M O- -o0 HN ON 0 O -P-O' OH HN OH 0 OMe 0 C \V"w bflDCCMRXS60824l ID.M ZOO42O I I -103A 804372 -,. , .0.4 pM 804442 0.007 itM 804503* 0.3 p -104 804558 L.6p 804596 > 10 M 804674 1.2 pM 804678 0.018 pM 5 804679 0.53 pM 804680 0.015 pM 804732 <0.001 pM 804764 0.015 pM -105 804772 ~Y 0.008 pM >>10 PM 804947 Table 7 below contains the compound number as referenced herein to the corresponding ER number. 5 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 10 50 803022 158 113644 62 803789 160 113651 72 803592 164 113665 100 112022 166 113666 102 111230 168 118023 15 104 111231 170 019772 106 111232 172 118989 108 111233 176 118999 110 112043 178 119000 112 112047 180 119001 20 114 112047 182 118949 116 112048 184 119327 118 112049 186 119328 120 112063 188 119329 122 112064 190 119521 25 124 112065 192 119522 126 112066 194 119523 128 112071 196 803022 130 112072 198 803045 132 112091 200 803056 PAOPERWALO2010 I 2P. dM.29MZfl~I -106 134 112092 202 803058 136 112093 204 803059 138 112098 206 803592 140 112099 142 112100 146 112859 148 112860 150 112861 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group 5 of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an 10 acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 15

Claims (16)

1. 2. The compound of claim 1, wherein R1 is -C(0)-.
2. 3. The compound of claim 2, having formula (a), (b) or (c) , O O HN c~oooa HNHN O HN OH (0 P.OPERVAAL\2DOO\357baI Claim for DV %26 14 AU3 DOC-6/30/2008 - 108 0 0 O HN O-A-o0 0 /jOH HN 0 C0 - 00 o HN 0 HN OH 0 (b) ,or 0 0 O HN HN OH C=o 0 0 HN 0-P-0~~ OH 0- o 0 (c) 0 or a pharmaceutically acceptable salt thereof.
3. 4. The compound of claim 3, having formula (a).
4. 5. The compound of claim 4 is in the form of a sodium salt.
5. 6. A compound of Formula (II): P\OPER\MAL\208\30571%l Carms for DV %26 14 AU3 DOC4/30/20M - 109 0 0 o HN""l""**[""""" o---o 0 OH HN OH R 0 0 R OORN HN 0 HN OH (II) wherein: R' is selected from the group consisting of -C(O)- and -C(O)(CH 2 ) 2 -C(O)-; or a pharmaceutically acceptable salt thereof.
6. 7. A compound of Formula (1II): 0 OH IN\ 00 0 HN O 0 O-P -O OH HN 0 wherein: R 1 is selected from the group consisting of -C(O)- and -C(O)(CH 2 )-C(O)-; or a pharmaceutically acceptable salt thereof.
7. 8. The compound of claim 7, wherein R' is -C(O)-. P3OPER\MAL\2008\30571901 CLms ro DV %26 14 AU3 DOC.6/30/2X)s - 110
8. 9. The compound of claim 7 or 8, having formula (d), (e) or (f) 0 0 OH 0- I-OO HNO HN HN~ 0 0 /R O HNH H 0 O-P 0O II 0 (d) 0 OH O- -O O' HN O H /R 0 O N 0 0 (e) or P :OPER\MAL\20030571901 Claim for DV %26 14 AU3 DOC.63/2005 0 OH o-O O HN /R0 0 HN O 0 R O OH HN 0 or a pharmaceutically acceptable salt thereof.
9. 10. A compound of Formula (IV): OHO O- 0 0 00 0 O O 0 6H 0-0 (IV) wherein: R' is -C(O)(CH 2 )-C(O) or -C(O)-; or a pharmaceutically acceptable salt thereof. I1. A compound of Formula (V): P:kOPER\MAL\2 10371 'A)I Ct m (oDV 9626 14 AU3 DOC.4/30/I - 112 O HNY" 0- o 0 o / [O H HNO HN 0 0 0- o o OH 000 (V) wherein: R' is selected from the group consisting of -C(O)- , -C(0)(CH2)2 -C(O)-, -C(O)(CH2)4 -C(O)-, -C(0)(CHi2)6 -C(O)-, -C(O)(CH2)s-C(0)-, and -C(O)(CH(R 2))-C(O)-; R 2 is selected from a group of formula (g) (CH2) ORH 0 R3 is selected from the group consisting of -H and -(CH2)2NHBoc; or a pharmaceutically acceptable salt thereof. P3OPER\MALA23057190l Claim for DV %26 14 AU3 DOC.6/3/2(0 - 113 12. A compound of Formula (VI): 0 OH 00 HN O HN 0-p -O 0 OH 0 0 (VI) wherein: R' is selected from the group consisting of -C(O)- , -C(O)(CH 2 )-C(O) and -C(O)(CH(R 2 ))-C(O)-; (CH 2 ) OR 3 R 2 is -(CH 2 ) 4 NHBoc or is selected from a group of formula (h) (h) R 3 is selected from the group consisting of -(CH 2 ) 2 NHBoc, (CH 2 ) 2 NH 2 , -(CH 2 ) 2 NH(CO)(CH 2 ) 4 C(O)OH; or a pharmaceutically acceptable salt thereof. P:OPER\MAL\2008\30511901 Clmms for DV 9626 14 AU3 DOC.6/3/2008 -114
10. 13. A compound of Formula (VI): 0 OH O H 0 (VTI) wherein: R' is selected from a group of formula (i) 0 0 o00 CC () or a pharmaceutically acceptable salt thereof.
11. 14. A compound having formula P:OPER\ALU20\30571%I Claim for DV%26 14 AU3 DOC6/30/2008 - 115 0 OH O HN HN O /)-==o HN 0 O 07 6HHN 0 or a pharmaceutically acceptable salt thereof.
12. 15. A compound having formula 0 0' 0 O-P- O-' 6HH HN 0 0 0 0 HNo 0 OH HN 0 or a pharmaceutically acceptable salt thereof.
13. 16. A compound having formula PAOPER\MAL\200\3057191 Clms tor DV %26 14 AU3.DOC-6/302(XIS - 116 0 0 0 O- P-OO HN OH HN HN 0 0 OH HN 0 or a pharmaceutically acceptable salt thereof. PMOPER\MAL\20\30571901 CLams for DV %26 14 AU3 DOC46/30/200 - 117
14. 17. A compound having formula O 0 O HN 0-p0 O-P-0 OHO N O HN 0- 0 0 OH 0 or a pharmaceutically acceptable salt thereof.
15. 18. The compound of any one of claims 1, 2, 6, 7. 8, 11 or 12, wherein the compound is in the form of a chiral isomer or a racemic mixture.
16. 19. The compound of any preceding claim, wherein the compound is in the form of a sodium salt.