AU667900B2 - Lipid-A analogs: New monosaccharide and disaccharide intermediates for eliciting therapeutic antibodies and for antitumor and antiviral activities - Google Patents

Description

OPI DATE 08/11/93 APPLN. ID AOJP DATE 13/01/94 PCT NUMBER 39390/93 PCT/US93/02903 Slll Iii 1111lll AUllll i lll lll933 19111IIII AU9339390 (51) International Patent Classification 5 A61K 31/70, 31/715, 37/02 C07H 15/06, C12N 9/00 (21) International Application Number: (22) International Filing Date: 26 h Priority data: 861,362 27 March 19! 871,229 17 April 199; (11) International Publication Number: Al (43) International Publication Date: WO 93/19761 14 October 1993 (14.10.93)

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PCT/US93/02903 larch 1993 (26.03,93) 92 (27.03.92) 2 (17,04.92) -7 •P t iurninMi 1 Nmaret f-9J5 I (71)Applicant: IGEN, INC. [US/US]; 1530 East Jefferson Street, Rockville, MD 20852 (US).

(72) Inventors: KAMIREDDY, Balreddy 1001 Rockville Pike, #1006, Rockville, MD 20852 DARSLEY, Michael, James 5905 Halsey Road, Rockville, MD 20851 (US).

SIMPSON, David, Michael 8306 Rosette Lane, Adelphi, MD 20783 MASSEY, Richard, J. 5 Valerian Court, Rockville, MD 20852 (US).

(74) Agents: EVANS, Barry et al.; Curtis, Morris Safford, 530 Fifth Avenue, New York, NY 10036 (US).

(81) Designated States: AU, CA, JP, KR, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

Published With international search report, 66909t (54)Title: LIPID-A ANALOGS: NEW MONOSACCHARIDE AND DISACCHARIDE INTERMEDIATES FOR ELICIT- ING THERAPEUTIC ANTIBODIES AND FOR ANTITUMOR AND ANTIVIRAL ACTIVITIES (57) Abstract Compounds are disclosed that are useful for eliciting antibodies which bind to Lipid-A, induce protective immunity against gram-negative bacterial infection, protect against viral infection, control tumor growth, and bind to receptors in competition with Lipid-A. Compositions containing these compounds are also disclosed. Further, antibodies elicited by the compounds are disclosed, as well as uses for such antibodies, for instance in treating septicemia.

L-~

WO 93/19761 PCT/US93/02903 1 LIPID-A ANALOGS: NEW MONOSACCHARIDE AND DISACCHARIDE INTERMEDIATES FOR ELICITING THERAPEUTIC ANTIBODIES AND FOR ANTITUMOR AND ANTIVIRAL ACTIVITIES CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of copending application Serial No. 871,229, filed April 17, 1992, which in turn is a continuation-in-part of copending application Serial No. 861,362, filed March 27, 1992, each of which are hereby incorporated herein by reference. This application is also a continuation-inpart of U.S. Application Serial No. 761,868, having an international filing date of May 4, 1989 under 35 U.S.C.

§363, and a date of September 3, 1991 under 35 U.S.C.

§§102(e) and 371(c). This application is also a continuation-in-part of PCT/US89/01950 designating the U.S. and filed May 4, 1989. Said application Serial No.

761,868 and said PCT/US89/01950 being hereby incorporated herein by reference. Reference is also made to copending U.S. application Serial No. 07/700,210 having an international filing date of May 4, 1989 under 35 U.S.C.

§363, and a date of June 12, 1991 under 35 U.S.C.

§§102(e) and 371(c), said application Serial No.

07/700,210 being hereby incorporated herein by reference.

Reference is further made to copending U.S. application Serial No. 07/190,271, filed May 4, 1988, also incorporated herein by reference.

FIELD OF THE INVENTION This invention relates to Lipid-A analogs and methods of using such analogs such as for eliciting catalytic antibodies for detoxification of LPS and Lipid- A, which bind to Lipid-A and LPS for the treatment of septic shock, for inducing protective immunity against the harmful effects of gram-negative bacterial infection, for protecting against viral infection, for treating and controlling tumor growth, and for binding to receptors in competition with Lipid-A. The invention also relates to Lipid-A analogs as transition state analogs for eliciting -rt SIIC. ~-i WO 93/19761 PCT/US93/02903 2 catalytic antibodies for the detoxification of LPS and Lipid-A, compositions which are useful for protective activity against gram-negative bacterial infection, as an antiviral composition, as an antitumor composition and for binding to receptors in competition with Lipid-A.

The invention further relates to catalytic antibodies which cleave the ester bonds and glycosidic bond of Lipid-A for the detoxification of LPS and Lipid-A, and an antibody which binds to LPS, Lipid-A and the Lipid-A analogs and to methods of using such an antibody; for instance, in treating septicemia.

This invention further relates to a novel method for generating antibodies of both the IgG and the IgM isotype against Lipid-A and its analogs and amphipathic molecules of similar structures.

Various documents are cited parenthetically throughout the text of this disclosure, with full citation to these documents appearing as a list immediately preceding the claims. These documents pertain to the field of this invention; and, each of these documents is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION Bacteremia is a severe infection of microorganisms in the bloodstream. The organisms cause a variety of symptoms including fever, shock, transient leukopenia and thrombocytopenia. Septic shock results when cardiac output cannot maintain the blood pressure due to a loss of intravascular volume. Septic shock can be caused by Gram-negative and Gram-positive bacteria, fungi, and although infrequently, by rickettsias or viruses. Escherichia coli account for a major portion of the cases of Gram-negative bacteremia.

The incidence of nosocomial bacteremia due to Gram-negative bacilli has been increasing since the 1950's despite the advent of antibiotics. It is now estimated to affect 260,000 to 300,000 patients per year WO 93/19761 PCT/US93/02903 3 in the United States with a mortality rate of 20 to even when treated under optimal situations. When the bacteremia is further complicated by renal or respiratory failure, the mortality approaches 90 100% Sepsis and septic shock may result from many causes, such as wound contamination in a traumatized patient, postoperative surgical complications, dissemination of a localized infection, or invasion of microorganisms through or around invasive instruments.

Sepsis can be further complicated by immune suppression, as often occurs during critical illnesses. Most patientdie from progressive multi-organ failure, which is thought to be due to the release of toxic substances from the infecting organisms endotoxin), the release of secondary endogenous mediators, and altered metabolic status, resulting in progressive tissue ischemia.

Many of the symptoms and effects of Gramnegative bacteremia are consistent with the premise that endotoxin, or the lipopolysaccharide (LPS) component of the bacterial outer membrane, is the causative agent of the bacteria-induced shock. The Lipid-A portion of LPS is structurally conserved amongst the Enterobacteriaceae and is responsible for most of the biological effects attributed to endotoxin. Although the length, number, saturation, and position of the acyl and acyloxyacyl chains are heterogeneous in the Lipid-A structure, the basic structure is common to all the Lipid-A molecules.

Lipid-A molecules from some organisms such as E. coli are not as variable. LPS stimulates various cell types to release mediators, hormones or other factors, in particular tumor necrosis factor (TNF), interleukin-1 and interleukin-6, which in turn act on other organs or target tissues Current therapeutic intervention includes antibiotics and fluids to increase intravascular volume, though these treatments are often unsuccessful at halting the cascading effects triggered by LPS.

i WO 93/19761 PCT/US93/02903 4 Antibiotics may reduce the bacteremia, but may also increase the amount of LPS shed into the bloodstream As secondary complications occur, the physician must use other therapies to compensate for or to save the affected target organs. Experimental treatments employing murine and human monoclonal antibodies specific for Lipid-A are yielding encouraging results 7).

It has also been proposed to employ antibodies specific for the Lipid-A moiety of LPS as treatment for septicemia or septic shock. Such antibodies have been IgM antibodies which are relatively large molecules and do not easily penetrate tissues.

Most monoclonal antibodies generated against the Lipid-A region of LPS have been produced by immunizing with killed cells of R-mutant gram-negative bacteria, or with such cells coated with additional Lipid-A or analogs. This approach has the disadvantage that the immune system is presented with natural Lipid-A structures at the same time as it is presented with the analog so that it is not certain exactly what the eliciting antigen for a given Mab might have been. In order to generate novel antibodies against Lipid-A with improved therapeutic properties including catalytic activity, it is desired to specifically stimulate the immune system with defined analogs.

Liposomes have been used widely and successfully as the basis for immunogens and vaccines to generate antibody responses to otherwise poorly immunogenic proteins or to obviate the need for harmful adjuvants It has also long been known that liposomes incorporating Lipid-A could induce antibodies capable of reacting with purified Lipid-A Recently liposomes incorporating Lipid-A have been used to raise antibodies against short synthetic peptides which react with the native protein from which the peptide sequence was derived (11).

Recently it has been shown that monoclonal 1 r,

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WO93/19761 PCT/US93/02903 antibody fragments can be isolated by methods other than the conventional process of fusing specific B-cells with myeloma cells to generate hybridomas which secrete MAbs.

The new method involves the isolation of the gene fragments encoding antibody molecules by their amplification, by the polymerase chain reaction (PCR), and cloning followed by expression as functional antigen binding molecules on the surface of filamentous phage particles (12, 13), or into the periplasmic space of bacteria infected with recombinant lambda-phage (14).

The starting point for the PCR amplification of antibodyencoding gene fragments can be any of the following: splenocytes isolated from a mouse or other animal immunized with an antigen, eg. a transition state analog; splenocytes isolated from an unimmunized animal; peripheral blood lymphocytes isolated from a human donor.

Throughout this disclosure wherever reference is made to antibodies (or catalytic antibodies) or fragments thereof it is recognized that this applies both to antibodies derived by hybridoma technology and to antibodies isolated using the bacteriophage technologies outlined above. A fuller description of this technology is given in the copending application Serial No. 07/841,648, filed February 24, 1992, and incorporated herein by reference.

The manner in which catalytic antibodies carry out chemical reactions on substrates (or antigens) is essentially governed by the same theoretical principles that describe how enzymes carry out chemical reactions.

For most chemical transformations to occur, substantial activation energy is required to overcome the energy A barrier that exists between reactant and product.

Enzymes catalyze chemical reactions by lowering the activation energy required to form the short-lived unstable chemical species found at the top of the energy barrier, known as the transition state (15, 16).

Four basic mechanisms are e..ployed in enzymatic catalysis to lower the free energy of the rate limiting

A

WO 93/ 9761 PCT/US93/02903 6 transition state, thereby accelerating the rate of a chemical reaction. Firstly, the active site of an enzyme is complementary in atomic and electronic structure to the transition state, such that the energy of the transition state is lower when bound to the enzyme than when free in solution. Secondly, general acid and base residues are often found optimally positioned for participation in catalysis within catalytic active sites causing the reaction to proceed via alternative and lower energy transition states. A third mechanism involves the formation of covalent enzyme-substrate intermediates.

Fourth, model systems have shown that binding reactants in the proper orientation for reaction can increase the "effective concentration" of reactants by at least seven orders of magnitude (17).

Drawing upon this understanding of enzymatic catalysis, several antibodies with catalytic activity have been designed v<i isolated Antibodies are elicited to compounds that resemble the transition state of a desired reaction transition state analogs).

Several laboratories have studied the breakdown and detoxification of LPS ahd Lipid-A analogs (19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33). Their studies suggest that synthet' analogs of Lipid-A coli) having one or more acyloxyacyl groups removed were less toxic than Lipid-A itself. The potential for detoxification of LPS is also demonstrated by the hydrolysis of the ester bonds of Lipid-A with the enzyme acyloxyacyl hydrolase, which removes the 3-hydroxytetradecanoyl chains of LPS, leaving hydroxyl groups (34, 35, 36, 37). Another study suggests that synthetic 0-deacylated Lipid-A compounds are non-toxic (38, 39).

Based on these obser ations it is desired to develop transition state analogs of Lipid-A and LPS to elicit catalytic antibodies which can cleave the ester bonds of Lipid-A and LPS to detoxify the endotoxins. The I WO 93/19761 PCT/US93/02903 7 target bonds for the catalytic antibodies are shown with arrows in Fig. 40 wherein the targets for ester bond hydrolysis are designated Immu-1, Immu-2, and Immu-3. It has also been shown that the synthetic monosaccnaride analogs of Lipid-A and LPS are non-toxic Based on this fact, it is also desired to design transition state analogs which will elicit catalytic antibodies which cleave the glycosidic bond of LPS and Lipid-A to the monosaccharide derivative (site Immu-4 in Fig. It is desired to achieve this by eliciting catalytic antibodies to amidin transition state analogs of Lipid-

A.

It is desired to employ catalytic antibodies to treat septicemia or septic shock. Catalytic antibodies are more efficacious than ordinary antibodies as a single molecule can inactivate many molecules of LPS. Catalytic antibodies can have better penetration of tissues as catalytic antibodies can be IgG-type antibodies or catalytically active fragments thereof Fab, Fv, SCAb (single-chain antibodies) which are smaller in size). If these are conventional (non-catalytic) LPS binding antibodies of smaller size than IgM IgG or fragments) they are unlikely to be effective in treatment or are less effective because they lack the effector functions associated with therapeutic action. On the contrary, the effector function of catalytic antibodies, namely the chemical detoxification of LPS, is mediated by the binding site such that any fragment which contains that site, however small, will be equally active. It is also desired to have new IgM-type antibodies which are I also useful for treating septicemia or septic shock.

Further, it is desired to be able to employ a cocktail of antibodies of either various catalytic antibodies or of a mix of catalytic and binding antibodies (and of either IgG, IgM, antibody fragments, or a mix of IgG and IgM and antibody fragments) to treat septicemia or septic shock. Thus, it is highly desired to obtain Lipid-A

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8 i 1 WO 93/19761 PCT/US93/02903 8 analogs which are capable of eliciting the desired catalytic and binding antibodies.

Further, therapeutic catalytic antibodies are desired for the treatment of sepsis and septic shock by means of cleavage of the glycosidic bond between the two monosaccharides for the detoxification of Lipid-A and LPS. In addition, antibodies generated against the analogs which bind to the Lipid-A moiety of LPS can have therapeutic efficacy in treatment of sepsis and are, therefore, also desired. Catalytic antibodies for reducing the toxicity of other compounds, e.g., glycosidic bond containing drugs or for activating compounds, glycosidic prodrugs, are also desired.

It is also desired to be able to elicit such antibodies as an immunological response, for instance, to vaccinate against toxic substances. Thus, it is highly desired to obtain Lipid-A analogs which elicit these desired antibodies and which can be administered.

Lipid-A, which is responsible for endotoxic activity also exhibits beneficial antitumor (TNF inducing) and antiviral (IFN inducing) activities.

Lipid-A analogs having decreased toxicity have been prepared and their biological activities studied (39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 87, 88, 89, 90, 91). Monophosphorylated Lipid-A analogs exhibited Limulus, mitogenic, PBA, TNF (tumor necrosis factor) -inducing and IFN (interferon)inducing activities, as well as protective activity against gram-negative bacteria, antiviral activity and antitumor activity.

It is also desired to prepare Lipid-A analogs having decreased toxicity but equivalent or increased beneficial antitumor and antiviral activities. The

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WO93/19761 PCT/US93/02903 9 above-referenced studies demonstrate that both monosaccharide derivatives of Lipid-A having acyl chains and monophosphoryl Lipid-A analogs are non-toxic but retain some of their beneficial activities. The activity of these molecules may be dependent upon the composition of the lipid chains the presence and position of functional groups and individual lipid chains); but, Applicants do not wish to necessarily be bound by any one particular theory. For instance, the presence of both a free hydroxyl group hydroxy tetradecanoic) on the lipid chain attached to the 3-hydroxy group and a lauroyloxy tetradecanoic acid group attached to the 2amino group of the monosaccharide was shown to produce a compound having good TNF inducing activity but only moderate IFN inducing activity. In contrast, reversing the position of these two groups, a lauroyloxy tetradecanoic acid group attached to the 3-hydroxy group and a hydroxy tetradecanoic acid group attached to the 2amino position resulted in good IFN inducing activity but only moderate TNF inducing activity. These observations show that the presence and position of functional groups in the hydrophobic lipid region of Lipid-A and LPS can play an important functional role in the expression of activity.

It is desired to design new and novel compounds for use as therapeutic agents. For instance, it is desired to design new compounds having a hydroxy functionality in the lipid region without changing the hydrophobicity by eliminating an acyl chain.

It is especially desired to synthesize analogs having the pentavalent phosphorus in place of carbonyl carbon at the ester bond, allowing both a hydroxyl group and a lipid chain at the same position. The structureactivity relationship noted above indicates that such compounds have unique, improved and desired biological activities.

It is desired to obtain Lipid-A analogs which WO 93/19761 PCT/US93/02903 exhibit the beneficial activities of Lipid-A or of previous Lipid-A analogs with reduced toxicity or even without toxicity. It is also desired to obtain Lipid-A analogs having conformational rigidity (for instance so that the analog's activities are closer to the activities of Lipid-A), as well as a means for introducing nucleophilic functionality, such as hydroxyl, without decreasing lipophilicity (for instance by a pentavalent phosphorous in the lipophilic region). It is further desired to have a Lipid-A analog which allows direct attachment of pentavalent phosphorous to a sugar backbone. More particularly, it is desired to have both ester and hydroxy moieties at either the Immu-1 or Immu-2 positions (Fig. 40) so that the resultant Lipid-A analog exhibits superior IFN and TNF inducing activities.

When one further considers the four positions labeled in the accompanying structure of Lipid-A, e.g., Immu-l, Immu-2, Immu-3 and Immu-4 (Fig. 40), these would be sites for cleavage of Lipid-A by a catalytic antibody so as to detoxify Lipid-A. However, heretofore, no such catalytic antibody or compounds to elicit such have been described. It is thus desired to provide compounds which mimic the transition state (transition state analogs) of hydrolytic reactions at these positions in Lipid-A so as to elicit superior catalytic antibodies which can detoxify Lipid-A, by cleavage of ester bonds and/or glycosidic bonds of Lipid-A or LPS.

SUMMARY OF THE INVENTION The invention disclosed herein entails the production of catalytic antibodies which detoxify the endotoxin activity by hydrolyzing the LPS or Lipid-A to products which are inactive or have reduced toxicity.

Clinical trials have demonstrated some success in treating sepsis with conventional antibodies of the IgM class, i.e. the pharmacokinetics for antibody therapy are favorable. Addition of catalytic degradation by the present invention offers the advantage of reduced doses WO 93/19761 PCr/US93/02903 11 with higher efficacy and the ability to use smaller antibody fragments which better penetrate the tissues and detoxify the LPS before it reaches the bloodstream.

The present invention provides a novel method of generating high titre IgG and IgM immune responses to Lipid-A, its analogs and other similarly amphipathic molecules. It employs liposomes as a vehicle and specific stimulatory peptides to enlist the involvement of T-Cells in the response. The generation of high titre IgM responses by this method means that such formulations are most effective as vaccines to induce protective immunity against the harmful effects of endotoxemia and gramnegative sepsis. The ability to generate a T-dependent immune response to Lipid-A and its analogs with the resulting high affinity, highly diversified IgG component greatly increases the ability to isolate catalytic monoclonal antibodies capable of detoxifying LPS which are therefore effective in treating endotoxemia and gramnegative sepsis.

A novel feature of this invention in using liposomes incorporating Lipid-A to raise antibodies is the use of a peptide sequence which is known to be the major T-Cell stimulatory region in Balb/c (H-2d) mice immunized with Hen Egg Lysozyme (HEL) (92) in the liposome formulation to recruit T-cells into, and so dramatically increase, the immune response to Lipid-A and its analogs.

This invention encompasses the development of therapeutic catalytic antibodies for the treatment of Gram-negative bacteremia and septic shock. Four sites of the Lipid-A and LPS molecule are targeted for cleavage by the catalytic antibodies: 1) the ester bond of the acyloxyacyl chain which is linked to the 2'-N glucosamine position (designated Immu-1 1R Fig. 40) the ester bond of the acyloxyacyl chain of the 3'-0 acyl group (designated Immu-2 in Fig. 40) the ester bond of the O-acyl chain at the 3'-0 position of the glucosamine 1h L 1 i WO 93/19761 PCT/US93/02903 12 (designated Immu-3 in Fig. 40); and, 4) the bond between beta 1-6 linked glucosamine residues (Immu-4 in Fig. The present invention provides Lipid-A analogs having a hydroxy functionality in the lipid region without changing the hydrophobicity (without eliminating an acyl chain) by substituting pentavalent phosphorus for tetravalent carbon at specific locations in the lipid region. The transition state analogs of the present invention contain hydroxy and ester functionalities at the same position and hence elicit superior antibodies.

It is therefore and object of the invention to provide Lipid-A analogs which elicit the desired therapeutic antibodies either binding or catalytic antibodies, or both. It is a further object of this invention to provide novel Lipid-A analogs which exhibit the beneficial activities of Lipid-A or previous Lipid-A analogs, but have reduced or no toxicity. It is a further object of the invention to provide Lipid-A analogs having any or all of: conformational rigidity; means for introducing nucleophilic functionality, such as hydroxyl, without decreasing lipophilicity pentavalent phosphorous in lipophilic region); and means for direct attachment of pentavalent phosphorous to a sugar backbone.

Thus, the present invention provides a compound of formula 0

A

0 3 020 0 0 30 0=Y 2 X-0- I (I) i 2 wherein: each of R 1

R

2 and R 2 independent of each

I!(

ii WO 93/19761 PCr/US93/O2903 13 other is a substituted or unsubstituted, branched or linear C1- 1 2 alkyl, alkene or alkyne group, R 3 is OH,

OCH

3

CH

2 COOH or 0 *1 H 0- H HON H wherein each R 2 1 R 2 1' of R 2 and

R

2 ,11, independent of each other, is a substituted or unsubstituted, branched or linear C 1

-CI.

2 alkyl, alkene or alkyne group, and: A NH 2 X Y =Z B (if present) OCH, or A OH, X Y =Z B (if present)- OCH, or A =OCO (CH 2 nNH 2 X P (OH) Y Z C, B (if present) OCH 3 wherein n 1-10, or A OH, X P(OH), Y =Z C, B (if present)

O(CH

2 )COH, wherein n 1-10, or A OH, X P(OH), Y Z B (if present)-

(CH

2 )nCO 2 H, wherein n =1-10, or A NH 2 X Z C, P (OH) B (if present)=

OCH

3 or A =OH, X Y =Z B (if present) OCH, or A OCO (CH 2 nNH 2 X =Z Y P B (if present) =OCH, wherein n 1-10, or A OH, X Z C, Y P (OH) B 0 O(CH 2 nC0 2

H,

&wherein n 1-10, or A =OH, X Z Y=P(OH), B (if present)-

(CH

2

)CO

2 -I wherein n =1-11, or

A=NH

2 X Y Z B (if present)

OCH

3 or A =OH, X Y Z B (if present)-

OCH

3 or WO 93/19761 PCT/US93/02903 14 A OCO(CH 2 )nNH 2 X Y C, Z P(OH), B (if present) OCH 3 wherein n 1-10, or A OH, X Y C, Z P(OH), B (if present)

O(CH

2 )nCO 2 H, wherein n 1-10, or A OH, X Y C, Z P(OH), B (if present)

(CH

2 )nCO 2 H and n=l-ll.

The substituents which may be on the branched or linear C_1- 1 alkyl, alkene or alkyne groups of any or all of R 1

R

1

R

2

R

2 1

R

2 and R 2 can be any substituents which permit antibodies to be elicited to the formula compound and preferably which do not significantly reduce the therapeutic utilities of the formula compound. These substituents can be one or more substituents selected from the group consisting of -OH, alkyl, chloro, fluoro, bromo, iodo, -S0 3 aryl, -SH, O 0 -C-OH, ester groups, ether groups, alkenyl, alkynyl 0 -N2, cyano, epoxide groups, heterocyclic groups, and

-N(R

4 2 wherein R 4 is H or a substituent listed above (so as to include substituted and unsubstituted amines).

The present invention also provides a method for eliciting catalytic antibodies to cleave the ester bonds or glycosidic bond of Lipid-A and LPS, and a method for eliciting antibodies comprising administering to an animal, as an immunogen, a compound of formula or formula (II) as part of a composition designed to maximize the antibody response.

The present invention further provides a composition for protective activity against the effects of gram-negative bacterial infection comprising a suitable carrier and a compound of formula or formula (II) Likewise, the present invention includes a method WO 93/19761 PCr/US93/02903 for inducing protective activity against the effects of gram-negative bacterial infection in an animal in need of such protection comprising administering the composition.

Further, the invention provides an antitumor composition comprising a suitable carrier and a formula or formula (II) compound; and, a method for treating or controlling tumor growth comprising administering the antitumor composition.

The invention also contemplates an antibody which binds to both Lipid-A and a compound of formula (I) or formula as well as an antibody which binds to Lipid-A and a formula or formula (II) compound, and has been made by a process comprising: immunizing an animal with a composition comprising the compound of formula I, removing antibody-producing lymphocytes from said animal, and fusing the lymphocytes with myeloma cells and thereby producing hybridoma cells producing the antibody.

The invention also comprehends this process.

The invention further comprehends a method for producing an antibcdy which binds to Lipid A and a compound of formula or (II) comprising immunizing an animal with a composition comprising the compound, isolating spleen cells from the animal, amplifying at least one gene fragment encoding all or part of both heavy and light chains of at least one antibody from said spleen cells, inserting said gene fragment in a recombinatoral fashion into a viral vector, producing a library of viable virus particles which express a protein derived from the gene fragment, and screening said library for binding and/or catalytic activity by expressed antigen binding protein of the gene fragment preferably with respect to Lipid A WO 93/19761 PCT/US93/02903 16 or the compound.

The invention also encompasses an antibody produced by this method, including a catalytic antibody.

In the foregoing antibody producing methods involving immunization of an animal, in preferred embodiments the immunizing comprises immunizing the animal with the compound complexed with a suitable immunogenic carrier, immunizing the animal with the compound, Lipid A, and a T-cell stimulatory peptide such as HEL[105-120].

In this disclosure an "antibody-encoding gene" includes fragments of such a gene which encode and express the antibody or a functional (by binding or catalytic activity) antibody fragments thereof.

The antibodies of the invention can be catalytic antibodies. These antibodies are useful for treating septicemia by administering them to a patient in need of such treatment; and thus, the invention includes a method for treating septicemia comprising administering a composition comprising such an antibody and a suitable carrier.

The invention also includes a method for binding to receptors in competition with Lipid-A comprising administering a composition comprising a suitable carrier and a formula compound.

With respect to formula in preferred embodiments

R

3 is

HC,-

S---0 n HO

HO

RP

2 1 and each of R 1

R

1

R

2 R, R 2 and R 2 is a C 1 2 linear alkyl group, such as C 12

H

24 and: Y C, Z C, X P(OH), B=OCH 3 and A OH; or, Y C, Z C, X P(OH), B=OCH 3 and A NH 2 or, Y C, Z C, X P(OH), B=OCH 3 and A Vi -a I I 1 WO 93/19761 PCT/US93/02903

OCO(CH

2 )nNH 2 wherein n 1-10; or, Y P(OH), Z C, X C, B=OCH 3 and A OH; or, Y P(OH), X C, Z C, B=OCH 3 and A NH 2 or, Y P(OH), X C, Z C, B=OCH 3 A

OCO(CH

2 )nNH 2 wherein n 1-10; or, Y C, X C, Z P(OH), B=OCH 3 and A OH; or, Y C, X C, Z P(OH), B=OCH 3 and A NH 2 or, Y C, X C, Z P(OH), B=OCH 3 and A

OCO(CH

2 )nNH 2 wherein n 1-10.

The invention further relates to novel amidine compounds of formula (II) which are also transition state analogs and which are therefore useful in eliciting antibodies, especially catalytic antibodies, for binding to and cleavage of compounds containing glycosidic bonds.

Like the formula compounds, the amidine compounds and antibodies elicited thereto of the invention are useful for several applications. The antibodies elicited to the amidine compounds bind to Lipid-A and LPS; the catalytic antibodies elicited to the amidine compounds catalyze cleavage of glycosidic bonds (to render LPS and Lipid-A less or non toxic). The novel amidine compounds can be used as receptor antagonists of Lipid-A/LPS, as well as antitumor agents, antiviral agents, and as compounds which can provide protection against bacterial infection.

The formula (II) compounds of the present invention have the structure: WO 93/19761 PCT/US93/02903 18

OH

0

SNH+

NH

H 0 HO SIL/"

HO

d R Rf wherein each of Ra, Rb, Rc, Rd, Re and Rf, independent of each other is a substituted or unsubstituted, branched or linear Cl_ 11 alkyl, alkene or alkyne group; the substituents can be those for R 1

R

1

R

2

R

2 R2,. and

R

2 provided above, preferably each of Ra, Rb, Rc, Rd and Rf is a linear alkyl group, most preferably each is CllH 23 and, E is NH or O.

The invention also comprehends pharmaceutically acceptable salts and pharmaceutically acceptable derivatives of formulae and (II) compounds. Further, the invention encompasses the novel intermediates of formulae and (II) compounds, as well as pharmaceutically acceptable salts and pharmaceutically acceptable derivatives thereof; and, the novel methods for preparing compounds of formulae and the intermediates thereof, and the pharmaceutically acceptable salts and derivatives thereof.

BRIEF DESCRIPTION OF DRAWINGS The following Detailed Description will be better understood by reference to the accompanying Figures, incorporated herein by reference, wherein: Figs. 1-39 and 48-52 show reaction Schemes 1 to (Figs. 1-10), reaction Scheme 10a (Fig. 11), reaction u WO 93/19761 PCT/US93/02903 19 Scheme 11 to 19 (Figs. 12-20), reaction Scheme 19a (Fig.

21), reaction Schemes 20 to 37 (Figs. 22-39), reaction Schemes 41 to 42 (Figs. 48, 49), reaction Scheme 43a (Fig. 50), reaction Scheme 43b (Fig. 51) and reaction Scheme 44 (Fig. 52) for the synthesis of formula (I) compounds and intermediates thereof; these figures are discussed in the Detailed Description by reference to the particular "Scheme"; Fig. 40 shows the structure of Lipid-A with positions Immu-1, Immu-2, and Immu-3 and Immu-4 labelled; as the targets for catalytic action by a catalytic antibody for detoxification of Lipid-A and LPS; Fig. 41 shows the structure of the amidine TS analog (79) of the present invention; Figs. 42-44 show reaction Schemes 38 to 40 for the synthesis of formula (II) compounds and intermediates thereof; Figs. 45-47 show reaction Schemes for the synthesis of formula (II) compounds and intermediates thereof; and Fig. 53 shows compounds of formula of the invention.

DETAILED DESCRIPTION The synthuJis of compounds of formulae and (II) can be according to the schemes which appear as Figures accompanying this description. The compounds of formulae and (II) and of the amidine TS analogs can be used as Lipid-A and previous Lipid-A analogs aro used in the literature.

It should be noted that, whereas some synthetic methods and compounds are specific as to a particular stereochemistry, this is not meant in any way to exclude methods and compounds of different stereochemistry or of a mixture of stereochemistries (or stereoisomers). The preferred embodiments of this invention entail methods and compounds having the same stereochemistry as naturally occuring Lipid-A and LPS.

L -I WO 93/19761 PCT/US93/02903 The compounds of formula allow for both hydroxy and ester groups at the Immu-1 and Immu-2 positions of Lipid-A (Fig. 40) to provide compounds which exhibit superior IFN and TNF inducing activities. IFN activity is an antiviral activity; TNF activity is antitumor activity. Furthermore, the compounds of formulae and (II) are transition state analogs of Lipid-A, and therefore are not only useful for eliciting antibodies, but are useful for eliciting catalytic antibodies. As to methods of eliciting catalytic antibodies, reference is made to Schochetman et al., U.S.

Patent No. 4,888,281, incorporated herein by reference.

These antibodies have therapeutic uses (see also the applications referenced and incorporated herein above in the Cross-Reference to Related Applications). These therapeutic uses include reducing the toxicity of compounds such as the treatment of septicemia or septic shock which is caused by lipopolysaccharide or LPS. As to formula compounds, three sites of the Lipid-A molecule are targeted by the catalytic antibodies elicited thereto: the ester bond of the acyloxyacyl chain which is linked to the 2'-N glucosamine position; the ester linked o-acyi chain at the 3'-0 position of the glucosamine; and the bond between the beta 1-6 linked glucosamine residues. The catalytic antibodies elicited to formula (II) compounds target glycosidic bonds, including glycosidic bonds of Lipid-A. Monoclonal antibodies produced against the formulae and (II) of the transition states for each of the hydrolysis reactions are screened for binding to and cleavage (catalytic activity) of Lipid-A (and LPS) to yield detoxified Lipid-A (and LPS) structures. The catalytic antibodies, alone or in combination, (for instance as a "cocktail") are expected to have therapeutic efficacy in several animal models of lethal bacteremia and septic shock and in treating human disease. Thus, the novel Lipid-A transition state analogs target one or more bonds 1i t i WO 93/19761 PCT/US93/02903 21 of Lipid-A (see Fig. Other utilities include causing conditions such as the activation of prodrugs such as glycosidic prodrugs. Further, formulae and (II) compounds are also useful to elicit antibodies as an immunological response; for instance, to vaccinate against toxic substances. Likewise, the formulae and (II) compounds are useful for treating those in need of treatment for endotoxemia or for septicemia or septic 10 shock because the formulae and (II) compounds can compete for binding sites with LPS or Lipid-A (receptor antagonists), or stimulate an immune response thereto.

The compounds of formulae and or antibodies elicited thereto (to either or both of formula and (II) are administered in typical doses to a patient, by the skilled artisan, physician or veterinarian, taking into consideration such typical factors as the condition being treated viral, bacterial, tumor, LPS, etc.), and the age, weight, sex and general health of the patient.

In general, with reference to Figs. 1-39, the synthesis and uses of c.pounds of formula are as follows.

Synthesis of immunogen BK-1 is accomplished as shown in the retro-synthetic analysis (Scheme i) (Fig.

Synthesis is achieved by first preparing the key intermediate 1, which is then transformed to BK-1 via appropriate chemical reactions. The intermediate 1 in turn is synthesized starting from three key intermediates 2, 3 and 4. Aicordingly comp 'i.nds 2, 3 and 4 are S* prepared as follows.

Intermediate 2 is prepared starting from commercially available tri-O-acetyl-D-glucal.

Azidonitration of tri-O-acetyl- 7local using ceric ammonium nitrate in acetonitrile followed by hydrolysis using aqueous sodium hitrate in dioxane affords the mixture of gluco and manno derivatives 5 a and 5 b WO 93/19761 PCT/US93/02903 22 (Scheme 2) (Fig. 2) The mixture is subjected to a silylation reaction using t-butyldimethylsilyl chloride in DMF in the presence of imidazole to afford the mixture of /-gluco 6 a and e-manno 6 b derivatives in the ratio of 2.4:1. P-Gluco compound 6 a is separated and treated with sodium methoxide in methanol to give the corresponding triol, which on treatment with 2,2-dimethoxypropane in methylene chloride in the presence of ptoluenesulphonic acid affords the azidosugar compound 2.

Other key intermediates, acid 3 and acid 4 are prepared by the following sequence of reactions. Both the intermediates 3 and 4 are prepared from the common intermediate 10, which is obtained from the allyl alcohol 7 (Scheme 3) (Fig. The alkyl alcohol 7 can be substituted or unsubstituted, branched or linear.

Synthesis of acid 3 is accomplished starting from dodecyl aldehyde (Scheme 4) (Fig. Wittig Sreaction of the aldehyde with methyl (triphenylphosphoralenyledene) acetate in methylene chloride affords the corresponding E-a, 3 unsaturated ester which on reduction with DIBAL-H in i methylene chloride at -78 C affords the allyl alcohol 7 The allyl alcohol on enantioselective epoxidation (96, 97) using diethyl tartrate, titanium tetraisopropoxide and t-butyl hydroperoxide in methylene chloride at -20 0 C affords the epoxide 8. The i epoxide is then reduced with Red-Al (9P) to afford the 1,3 diol 9 along with 1,2 diol in the ratio of 10:1. The ratio of the regioisomers is determined by NMR of their o 30 acetate derivatives. The stereochemistry and optical purity of the 1,3 diol is determined by converting the diol to 3-hydroxytetradecanoic acid (Scheme 4) (Fig. 4) and is comparable with the literature reported values (99).

The diol 9 is a key intermediate for the preparation of both acids 3 and 4. The primary alcoholic functionality of the diol 9 is silylated selectively as R WO 93/19761 PCT/US93/02903 23 the t-butyldimethylsilyl ether to afford the compound (Scheme 5) (Fig. Compound 10 is coupled with lauric acid in the presence of DCC and DMAP to afford the coupling compound 11. Desilylation of compound 11 is achieved by treating with PTSA in methanol at 0°C to afford the corresponding primary alcoholic compound which after Jones oxidation affords the acid 3.

Preparation of the other acid 4, is achieved by the following sequence of reactions (Scheme 6) (Fig. 6).

The phosphochloridate 12, a necessary component for the synthesis of acid 4, is prepared starting from dibenzyl phosphite. Dibenzyl phosphite is alkylated with lauryl bromide in dry DMF in the presence of potassium tbutoxide to afford the alkylated phosphate 14. The phosphate compound 14 is treated with phosphorus pentachloride in dry chlorofo-m to afford the phosphochloridate 12. The phosphochloridate 12 is coupled with the hydroxy silyl compound 10 in methylene chloride in the presence of DMAP to give the corresponding coupled compound 13. The coupled compound 13 is then converted to the acid 4 by the same sequence of reactions used for the preparation of acid 3 from compound 11.

Having obtained the three intermediates, azido sugar 2, acid 3 and acid 4, BK-I is prepared by the following sequence of reactions (Scheme 7) (Fig. The azido compound 2 is coupled with acid 3 in methylene chloride in the presence of DCC and DMAP to afford the corresponding coupled compound 15. The acetonide of compound 15 is deprotected by using trifluoroacetic acid in methylene chloride to afford the corresponding diol, which is subjected to selective silylation with tbutyldimethyl silyl chloride to afford the monosilylated compound. The obtained monosilylated compound is then hydrogenated using Pd-C in methanol to afford the corresponding amino compound 16. The amino compound 16 is coupled with acid 4 in methylene chloride in the WO 93/19761 PCT/US93/02903 24 presence of EDC and HOBT to afford the compound 1.

Compound 1 is phosphitylated at the 4 position with N,Ndiisopropylamino dibenzyl phosphite in methylene chloride in the presence of tetrazole, followed by oxidation of the obtained phosphite with m-CPBA to afford the corresponding phosphate 17. Hydrogenation of the compound 17 using Pd-C in methanol, followed by desilylation using HF-pyridine, affords the immunogen BK- 1.

Synthesis of immunogen, BK-3 is accomplished as shown in Scheme 8 (Fig. Accordingly a key intermediate 20 is prepared from three intermediates; acid 3, amino compound 18 and phosphochloridate 19.

The intermediate phosphochloridate 19 is prepared starting from commercially available dimethyl methylphosphonate (Scheme 9) (Fig. Dimethyl methylphosphonate is deprotonated using n-butyllithium at -78 0 C and methyl laurate is added to afford the keto phosphonate 21. The keto phosphonate is reduced with sodium borohydride in methanol to give the corresponding hydroxy compound 22. Compound 22 is coupled with lauric acid in the presence of DCC to afford the corresponding ester 23. Demethylation of compound 23 with TMSI in dichloromethane affords the dihydroxy compound 24.

Monobenzylation of compound 24 is achieved using benzyl alcohol and trichloroacetonitrile in pyriaine and chloroform to afford the monobenzylated compound Compound 25, after treatment with phosphorus pentachloride in chloroform, affords the phosphochloridate 19.

S"The immunogen BK-3 is then prepared by the following sequence of reactions. The amino sugar 18 (see discussion of BK-1 synthesis, above) is coupled with acid 3 (see discussion of BK-1 synthesis, above) under standard EDC reaction conditions to afford the acylated sugar 26 (Scheme 10) (Fig. 10). The hydroxy compound 26 is coupled with phosphochloridate 19 to afford the i I- ii WO 93/19761 PC/US9/02903 diacylated sugar 27. Deprotection of the acetonide of compound 27 is achieved using trifluoroacetic acid in dichloromethane to afford the corresponding diol 28 (Scheme lOa) (Fig. 11). Compound 28 is subjected to hydrogenolysis using Pd-C in ethyl acetate to give the corresponding debenzylated compound, which after desilylation using HF-pyridine affords the immunogen BK- 3.

Synthesis of immunogen 3M is accomplished as shown in the retro-synthetic analysis (Scheme 11) (Fig.

12). Immunogen 3M is prepared by making use of the intermediate 29, phosphochloridate 19 and acid 3 via suitable chemical reactions to afford compound 29, which is subsequently converted to immunogen 3M.

One of the intermediates for the synthesis of immunogen 3M, amino compound 30, is prepared starting from commercially available D-glucosamine hydrochloride (Scheme 12) (Fig. 13). Accordingly, glucosamine hydrochloride is treated with Cbz-Cl in aqueous sodium bicarbonate at room temperature to afford the aminoprotected compound, which is then treated with methanolic HC1 to afford compound 31. Compound 31 is then treated with 2,2-dimethoxypropane in dichloromethane in the presence of PTSA at room temperature to afford protected compound 32. Compound 32 is deprotected by hydrogenation using Pd-C in ethyl acetate to afford amino compound The other intermediates phosphochloridate 19 and acid 3 are prepared by following the same procedures used for BK-1 and BK-3.

The amino compound 30 is converted to immunogen 3M via the following sequence of reactions (Scheme 13) (Fig. 14). Amino compound 30 is reacted with acid 3 in dichloromethane in the presence of EDC and HOBT to afford the acylated compound 33. Compound 33 is treated with t-butyldimethylsilyl chloride in DMF in the presence of imidazole to afford the silylated compound 34. The WO 93/19761 PCT/ US93/02903 26 acetonide functionality of compound 34 is removed by treating compound 34 with trifluoroacetic acid in dichloromethane to give diol 35. The primary alcohol functionality of compound 35 is converted to the tosylate by treating it with tosyl chloride in the presence of DMAP, and the tosylate intermediate (Scheme 14) (Fig. is reacted with sodium azide in DMF to afford the azido compound 36. Phosphorylation of compound 36 is accomplished by treatment with N,N-diisopropylamino dibenzyl phosphite in dichloromethane in the presence of tetrazole, followed by oxidation with m-CPBA to afford the corresponding phosphorylated compound 29.

Desilylation of the phosphorylated compound 29 is achieved using PTSA in methanol and dichloromethane to give the corresponding hydroxy compound 37 (See structure in Scheme 15 (Fig. The hydroxy compound 37 is coupled with phosphochloridate 19 in dichloromethane in the presence of DMAP and triethylamine to afford the coupled compound 38. Compound 38 is deprotected by hydrogenolysis using Pd-C in ethyl acetate to afford immunogen 3M (Scheme 14) (Fig. i The synthesis of immunogen 2M is achieved as shown in Scheme 15 (Fig. 16). Intermediate 37 is prepared according to the procedure described above for immunogen 3M. In Scheme 16 (Fig. 17) compound 37 is coupled with acid 4 using DCC in dichloromethane to afford compound 39. Compound 39 after hydrogenation j affords immunogen 2M.

Synthesis of disaccharide immunogen 3D is j 30 accomplished as shown in Scheme 17 (Fig. 18). A key step Sinvolved in the synthesis of the disaccharide immunogen is the coupling reaction between the imidate 40 and diol 41 to afford the corresponding coupled compound and subsequent suitable chemical transformations to lead to immunogen-3D.

Accordingly, the synthesis of intermediate is started from compound 6a (Schemes 18 and 19) (Figs. 19 1 -i 1 WO 93/19761 PCT/US93/02903 27 and 20). Compound 6a is prepared by the same procedure as described for the synthesis of BK-1. Compound 6a is hydrogenated using Pd-C as a catalyst in ethyl acetate to afford the corresponding amino compound, which after treatment with Troc-Cl in pyridine and dichloromethane, affords the trichloroethyl carbamate-protected compound 42. Compound 42 is treated with sodium methoxide in methanol to give the corresponding triol, which after treatment with 2,2-dimethoxypropane in dichloromethane in the presence of PTSA, affords the corresponding acetonide 43. Compound 43 is coupled with phosphochloridate 19 (the synthesis of phosphochloridate 19 is described above with respect to the synthesis of 3M) in the presence of DMAP in dichloromethane to afford the coupled compound 44. Then compound 44 is treated with tetrabutylammonium fluoride in THF and dichloromethane and subsequently with trichloroacetonitrile to afford a key intermediate, imidate 40, as an anomeric mixture.

The other intermediate diol 41, is prepared by coupling compound 30 (the preparation of compound 30 is described with respect to the synthesis of 3M) and compound 47 and subsequent deprotection Scheme 19a (Fig.

21).

Compound 47 is prepared from compound 10 (the preparation of compound 10 is described with respect to the synthesis of BK-1). Compound 10 is first benzylated using benzyl 2,2,22-trichloro-acetimidate in dichloromethane in the presence of triflic acid to afford I compound 45. Compound 45 is desilylated using PTSA in methanol and dichloromethane to afford the corresponding hydroxy compound 46. Compound 46 is subjected to Jones oxidation to afford compound 47. Compound 47 is coupled with compound 30 (Scheme 19a) (Fig. 21) to afford the coupled compound 48. Compound 48, after treatment with trifluoroacetic acid in dichloromethane, affords the diol compound 41.

Having thus obtained the glycosyl donor 40 and WO 93/19761 PCT/US93/02903 28 the acceptor 41, the coupling reaction is performed using Lewis acids such as boron trifluoride etherate and/or TMS triflate. Treating compound 41 and compound 40 in dichloromethane with boron trifluoride etherate and/or TMS triflate affords the coupled compound 49 (Scheme (Fig. 22). The coupled compound 49 is treated with tbutyldimethylsilyl chloride in DMF to afford the silylated compound. The acetonide functionality of the silylated compound is removed using trifluoroacetic acid in dichloromethane to afford the diol The primary alcohol functionality of the diol is selectively protected as the t-butyldimethyl silyl ether using silylating reaction conditions (Scheme 21) (Fig. 23) to afford compound 51. Compound 51 is deprotected using zinc in acetic acid and THF to afford the diastereomeric mixture of amino compound 52. The amino compound 52 is coupled with acid 3 to afford the coupled compound 53. Phosphorylation of compound 53 at the 4'-position is accomplished by treating compound 53 with N,N-diisopropylamino dibenzyl phosphite in dichloromethane in the presence of tetrazole to afford the corresponding phosphite, which after oxidation with m-CPBA affords the phosphorylated compound 54 (Scheme 22) (Fig. 24). Compound 54 is debenzylated by hydrogenation over Pd-C in ethyl acetate to afford compound Compound 55 is treated with HF-pyridine to afford immunogen 3D.

Synthesis of immunogen 1D is achieved starting from the intermediates 56 and compound 41 (Scheme 23) 30 (Fig. SPreparation of intermediate 56 is accomplished by following the sequence of reactions outlined in Scheme 24 (Fig. 26). Accordingly, the compound 43 after coupling with acid 3 in dichloromethane in the presence of DCC and DMAP, affords the coupled product 57.

Compound 57, after treatment with tetrabutylammonium fluoride in THF followed by addition of Jl -e 1 WO93/19761 PCT/US93/02903 29 trichloroacetonitrile, affords an anomeric mixture of imidates 56.

The imidate compound 56 and diol compound 41 in dichloromethane in the presence of Lewis acid (either boron trifluoride etherate or TMS-triflate)(Scheme (Fig. 27), affords the coupled compound 58. The hydroxyl functionality of compound 58 is protected as the tbutyldimethylsilyl ether using t-butyldimethylsilyl chloride in DMF in the presence of imidazole, and the resulting silylated compound, after treatment with trifluoroacetic acid, affords the corresponding diol compound 59. The primary hydroxyl functionality of the diol compound 59 is selectively protected as the TBDMS ether (Scheme 26) (Fig. 28) by using t-butyldimethylsilyl chloride (1.1 eq) in DMF in the presence of imidazole (2.2 eq) and the resulting compound after treatment with zinc in acetic acid and THF affords the amino compound The amino compound 60 on coupling with acid 4 in dichloromethane in the presence of EDC and HOBT affords the coupled compound 61.

The C-4' hydroxyl position of compound 61 is phosphorylated using the same method as used for the synthesis of immunogen 3D. The phosphorylated compound 62 is converted to immunogen ID by hydrogenation followed by desilylation using HF- pyridine (Scheme 27) (Fig. 29).

Synthesis of immunogen 2D is achieved by coupling the imidate 62a and compound 41 to afford the coupling compound, which on suitable chemical transformations affords the immunogen 2D (Scheme 28) (Fig. The hydroxy compound 43 is treated with 4methoxybenzyl chloride in THF in the presence of sodium hydride to afford compound 63. Compound 63 after treatment with tetrabutyl-ammonium fluoride in THF followed by treatment with 62a trichloroacetonitrile, yields imidate 62a as an anomeric mixture (Scheme 29) (Fig. 31). The imidate is coupled with compound 41 in WO 93/19761 PCT/US93/02903 the presence of a Lewis acid (either boron trifluoride etherate or TMS-triflate) to afford the coupled compound 64. Compound 64 on treatment with t-butyldimethylsilyl chloride in DMF in the presence of imidazole, affords the corresponding silylated compound, which after treatment with trifluoroacetic acid, gives the diol (Scheme (Fig. 32). The primary hydroxyl functionality of the diol compound is selectively protected as the t-butyldimethylsilyl ether by treatment with t-butyldimethylsilyl chloride (1.1 eq) in DMF in the presence of imidazole (2.2 eq) to afford the compound 65. Compound 65, after treatment with zinc in acetic acid and THF, gives the corresponding amino compound, which after treatment with acid 3 in dichloromethane in the presence of EDC and HOBT, affords the coupled compound 66.

The C-4' hydroxyl of compound 66 is phosphorylated using the method utilized for the other immunogens to afford the corresponding phosphorylated compound, which after treatment with DDQ affords the corresponding deprotected compound 67 (Scheme 31) (Fig.

33). Compound 67, after treatment with acid 4 in dichloromethane in the presence of DCC and DMAP, affords the coupled compound 68. Compound 68 is converted to immunogen 2D by hydrogenation followed by desilylation (HF-pyridine).

Synthesis of immunogen 3D-A is achieved starting from the intermediate 54 (Scheme 32) (Fig. 34).

Accordingly, compound 54 on treatment with PTSA in methanol affords the corresponding hydroxy compound. The hydroxy compound on treatment with MsCl gives the mesylate, which on treatment with sodium azide gives the corresponding azide compound. The azido compound on hydrogenation with Pd-C in ethyl acetate and followed by treatment with HF-pyridine affords the immunogen 3D-A.

Synthesis of other immunogens 2D-A and 1D-A is also accomplished by the similar sequence of reactions starting from 68 (Scheme 33) (Fig. 35) and 62 (Scheme 34)

I

WO 93/19761 PCT/US93/02903 31 (Fig. 36) respectively.

Synthesis of immunogen 3D-AL is achieved starting from the intermediate 54 (Scheme 35) (Fig. 37).

AC>or-dingly compound 54 on treatment with PTSA in me.h ;gol affords the corresponding hydroxy compound. The hydrc-ty compound on treatment with the amino protected acid in dichloromethane in the presence of DCC gives the corresponding coupled compound. The coupled compound after hydrogenation with Pd-C in ethyl acetate and followed by treatment with HF-pyridine affords the immunogen 3D-AL.

Synthesis of other immunogens 2D-AL and 1D-AL is accomplished starting from intermediates 68 (Scheme 36) (Fig. 38) and 62 (Scheme 37) (Fig. 39) respectively by the similar sequence of reaction adopted for 3D-AL from 54.

With reference to Schemes 38 to 45, (Figs. 41- 47) synthesis and uses of the formula amidine TS analogs of Lipid-A of the present invention, particularly with respect to a preferred embodiment thereof, is as follows: Synthesis of amidine TS analog 79 (Fig. 41) is performed as follows. In Fig. 41, compound 79 is shown as having R C 11

H

23 however, in formula each of Ra, Rb, Rc, Rd and Re independent of each other can be a branched or linear, substituted or unsubstituted C 1

-C

11 alkyl, alkene or alkyne. From the herein described synthesis method for analog 79 the skilled artisan, without undue experimentation, can modify the procedure to obtain the compounds encompassed by the herein definition of formula The synthesis of analog 79 is performed by first preparing two intermediate compounds 71 and 77 and coupling them by appropriate chemical reactions and then converting the coupled compound to the amidine compound.

Synthesis of intermediate 71 (synthesis see immunogen 3D) is started from the diol compound 41 (Fig.

WO 93/19761 PCT/US93/02903 32 42). Compound 41 is treated with about 2.4 eq TBDMS-Cl in DMF in the presence of imidazole to afford the disilylated compound. The disilylated compound on treatment with a catalytic amount PTSA in methanol at 0 C affords the primarysilyl cleaved compound 69. Hydroxy compound 69 is converted to mesylate by treatment with Ms-Cl, which on treatment with sodium azide affords the corresponding azide compound 70. Compound 70 on hydrogenation affords the amino compound 71.

The other intermediate 77 is prepared from the commercially available lactone 72 (Fig. 43). Compound 72 on treatment with 2,2 dimethoxy propane affords the acetonide which, on treatment with about 2.4 eq TBDMS-C1 affords the compound 73. Compound 73, on treatment with benzyl alcohol at about reflux temperature, affords the hydroxy benzylester compound. The hydroxy ester compound on treatment with MS-C1 gives the mesylate compound, which on treatment with sodium iodide gives the corresponding iodo compound. The iodo compound on treatment with sodium azide affords the azido compound 74. Compound 74 on hydrogenation gives the lactam, which on treatment with tetrabutylammonium fluoride affords the hydroxy lactam 75. Compound 75 on treatment with about 2.2 eq acid 3 (synthesis of acid 3: see BK-1) in the presence of DCC and DMAP affords the coupled compound.

The coupled compound, on treatment with trifluoroacetic acid affords the diol, which on reacting with about 1.1 eq TBDMS-C1 affords selectively the primary hydroxy protected compound 76. The phosphorylation at the 4position is accomplished by treatment of compound 76 with N,N-diisopropylamino dibenzylphosphite in the presence of tetrazole. This results in forming the corresponding phosphite, which on oxidation with m-CPBA affords the corresponding phosphate compound 77 (Fig. 44). Compound 77, on treatment with Meerwein reagent followed by addition of compound 71 affords the coupled compound 78.

Compound 78 is converted to compound 79 by hydrogenation WO 93/1

I

;1 d lii-----rr a~r 9761 PCT/US93/02903

I~

followed by treatment with HF-pyridine. Alternatively, compound 78 is also converted to 79 by treatment with TMS-I followed by acidification.

Amidine TS analog 89 is prepared from compound 71, the synthesis of which has been described above, and compound 87 (Fig. 47). Compound 87 is prepared in the following way. The mixture of compounds 5a and 5b (for the synthesis, see Scheme 2) is oxidized using Jones reagent to give compound 80 (Fig. 45). The hydroxyl protecting groups of compound *0 are replaced following standard chemical procedures to give lactone 81. The lactone ring of compound 81 is solvolyzed using benzyl alcohol to give an intermediate 6-hydroxy ester. Then, the hydroxyl group is substituted by an azido group using double inversion of chirality to obtain compound 82, which has the correct stereochemistry. Hydrogenation of compound 82 reveals an a,6-diamino acid, which undergoes lactamization selectively to form the 6-lactam, compound 83. The amino group of compound 83 is protected with one 4-nitrobenzyl group to give compound 84. The silylprotected hydroxyl group of compound 84 is deprotected to give compound 85 (Fig. 46), and then compound 85 is condensed with two equivalents of acid 3 (for the synthesis, see Scheme 5) to give compound 86. Hydrolysis of the isopropylidene protecting group of compound 86 produces a diol, the primary hydroxyl group of which is selectively reprotected with a TBDMS group.

Phosphitylation of the secondary hydroxyl group and subsequent oxidation of the trialkylphosphite to the phosphate gives compound 87.

The coupling of compounds 87 and 71 to form compound 88 (see Fig. 47) is carried out using the same methodology as used for the preparation of compound 78 (Fig. 44). Subsequent deprotecton of compound 88 gives the amidine TS analog, compound 89.

Preparation of substituted or alkene or alkyne compounds within the definition of formulae and (II) k- j WO 93/19

I--

T

761 PCT/US93/02903

I

is as provided above, except that the appropriate precursor compound is substituted or has the alkene or alkyne group on it.

In addition to being useful for eliciting antibodies (catalytic and binding), the compounds of formulae and (II) are useful as antiviral, antitumor and antibacterial agents. At a dose of about 10 Ag/kg in Japanese white rabbits, the formula and (II) compounds do not show pyrogenic activity, whereas natural Lipid-A exhibits marked pyrogenicity at a dose of 0.001 ig/kg. While Lipid-A at a dose of 1 jg/mouse exhibits protective activity against gram-negative bacteria, at ,arious doses including about 1-100, preferably about 1and most preferably about 10 Ag/mouse, formula (I) and (II) compounds exhibit protective activity against gram-negative bacteria aeruginosa). In these tests the mice are inoculated and thereafter challenged. As to antiviral activity, mice are intravenously inoculated with various doses such as about 0.1-100, preferably about 1-10 pg of formula or (II) and thereafter challenged with a suitable quantity, about 104 pfu of vaccinia virus. The formulae and (II) compounds perform similar to Lipid-A. With respect to antitumor activity, 1 or 2 X 105 Meth A fibrosarcoma cells, or about 105 melanoma cells, or 106 Pro b cells are administered into mice; Lipid-A or formula or (II) compounds are administered at a dose of 250, 3,00 or Ag/mouse Meth A fibrosarcoma) or up to 10 ig/kg body weight, either intravenously or intratumorly after transplantation or administration of the tumor cells.

Tumor growth is retarded or prevented in r,~ce receiving Lipid-A or formula or (II) compounds. From these results, the skilled artisan can, without undue experimentation, determine the proper dosage for administering formulae and (II) compounds and antibodies thereto to any suitable animal or human patient, taking into consideration such typical factors r WO 93/19761 PCT/US93/01903 as the nature of the patient, the condition being treated, and the age, weight, sex and general health of the patient.

The formulae and (II) compounds and antibodies thereto can be administered in any suitable form which is effective, for instance, orally, intravenously, subcutaneously, intradermally, intratumorally, and the like. The formulae and (II) compounds and antibodies thereto can be administered in any suitable carrier or adjuvant, such as saline. These formulations can be coadministered with other treatments; for instance, formula compounds and/or formula (II) compounds can be administered with other antineoplastic agents, or the antibodies can be administered with antibiotics. Furthermore, the antibodies to a formula or formula (II) compound can be administered as a mixture with other antibodies, for instance to antibodies other formula compounds, to other formula (II) compounds and/or with antibodies to other Lipid-A analogs and/or with antibodies to Lipid-A, the antibodies of the present invention can be administered as a "cocktail". This cocktail can include both IgG and IgM antibodies as well as both binding and catalytic antibodies. And, by "antibodies" the invention includes either binding or catalytically active fragments of antibodies.

In addition, the invention contemplates that the formula and (II) compounds and antibodies thereto can be dispensed in concentrated form or lyophilized form for dilution by the ultimate user. These preparations can be in kit form. The kit ftrm can also include suitable instructions for administration in accordance with this invention.

The following non-limiting Examples are given by way of illustration only and are not to be considered a limitation of this invention, ma,y apparent variations of which are possible without departing from the spirit WO093/19761 PCT/US93/02903 3 G or scope thereof.

EXAMPLES

With res~pect to the Examples, thR following are general remarks: Melting pcoints were recorded c: Haake Buqhier melting poinit apparatus, with electric f.oil1 heating were uncorreuted. E. Merck precoated silica gel with F 254 (0.25 mm thickness) thin-lay chromatograph (TLC) plates were used for monitoring the reactions. S. Merck silica gel 60 (70-230) mesh) wc~s used for columii chromatography.

organic layers were dried with anhydrous MgSO 4 Infrared (IR) aboorption spectra were recorded on a Perkin-elmer (1710) spectrophotome'ter as neat unless otherwise mentioned. Proton nuclear magnetic resonance (HNM-) spectra and Carbon nuclear magnetic resonance 1 3C NMR) were measured on General Electric QE-300 (300 Mhz) spectrometer with CDCl 3 as a solvent unless otherwise mentioned, using tetramethylsilane as internal standard.

In this disclosure and in particular in the following Examples. the following abbreviations (as defined) are used: AHB Acid Hydroly~sed Bacteria Cbz-Cl: Benzylchlorol onnate CFA Complete Freund's Adjuyant DCC: 1,3 Dicyclohexylcarbodijimide DET: Diethyl tartrate DIPT: Diisopropyl tart, tte DMAP: 4-Dimethylaminopyridine DMF: N, N-Dimethylformamiide EDC: 1-Ethyl 3 (3 dimethylaminopropyl) carbodimide ELISA Enzyms-Labelled ImmunoSorbant Assay HCl: Hydrochloric acid HEL Hen Egg-white Lysozyme i ____IILI qll WO 93/19761 PCT/US93/02903 HEL[105-1203

HOBT:

IFA

IFN

IL-1 IL-6 LDs 5

LPS

m-CPBA: MAb

PBS

PPBE

PTSA:

SCAb

SRBC

TBDMS-Cl:

TFA:

THF:

A peptide having an amino acid sequence corresponding to positions 105-120 of HEL preferably with an additional Cterminal cysteine residue, or a homologous peptide, or a peptide having an amino acid sequence which has substantial homology with positions 105-120 of HEL (preferably with the additional C-terminal cysteine residue) and preferably the peptide is a synthetic peptide l-Hydroxybenzotriozole Incomplete Freund's Adjuvant Interferon (alpha, beta or gamma) Interleukin-1 Interleukin-6 The dose of a toxic moiety sufficient to kill 50% of animals to which it is administered Lipopolysaccharide 3-Chloroperoxybenzoic acid Monoclonal Antibody Phosphate Buffered Saline Na phosphate, 0.15M NaC1, pH 7.2) Proteose Peptone Beef Extract (Bacterial culture medium) p-Toluenesulfonic acid Single Chain Antibody Sheep Red Blood Cells ter-Butyldimethylsilyl chloride Trifluoroacetic acid Tetrahydrofu.'un WO 93/19761 PCT/US93/02903 38 TMS-I: Trimethylsilyl iodide TMS-Tf: Trimethylsilylmethyl trifluoromethanesulfonic acid TNF Tumor Necrosis Factor alpha Troc-Cl: 2,2,2-Trichloroethyl chloroformate Example 1 Preparation of 2-Azido-2-deoxy-3,4,6-tri- 0-acetyl P-D-glucopyranose (Sa) and 2azido-2-deoxy-3,4,6 tri-O-acetyl a-D-mannopyranose To a stirred solution of tri-O-acetyl-D-glucal (27.24 g, 0.1 m) in dry acetonitrile (600 ml) at -25 0

C,

was added a mixture of sodium azide (7.2 g, 0.11 mol) and ceric ammonium nitrate (219.20 g, 0.4 mol) and the suspension was stirred at -25 0 C for 16 h (TLC analysis showed the completion of the reaction). Then the cold ether (500 mL) and water (200 mL) were added to the reaction mixture and was separated the organic layer, dried (MgS0 4 filtered and concentrated to give the oily compound (Rf 0.72, silica, ethyl acetate:petroleum ether 1:1, 29 g, 77 The .'rude compound obtained from the above procedure was subjected to the next reaction without purification. A mixture of the above compound (29 g, 77 mmol) in dioxane (500 mL), and an aqueous solution of sodium nitrite (27.6 g, 0.4 mol in 200 mL of water) was heated at 80 0 C for 8 h. Then the organic layer was separated and the aqueous layer was extracted with ethyl acetate (300 mL). The combined organic layers were dried (MgSO 4 and concentrated to give a pale yellow oil as an inseparable mixture of gluco and manno pyranoses 5a and 5b (24 g, 94%).

Example 2 Preparation of t-Butyldimethylsilyl-2azido-2-deoxy-3,4,6 tri-O-acetyl b-Dglucopyranose (6 a) and tbutyldimethylsilyl-2-azido-2-deoxy 3,4,6 tri-O-acetyl a-D- Mannopyranose (6 b) To a solution of hydroxy compounds (obtained above as a mixture, 23 g, 69 mmol) in N,N- WO 93/19761 PCT/US93/02903 39 dimethylformamide at 0 C, was sequentially added imidazole (11.26 g, 166 mmol, 2.4 eq) and tbutyldimethylsilyl chloride (12.5 g, 83 mmol 1.2 eq), and the mixture was stirred for 4 h. After the completion of the reaction (TLC) the mixture was diluted with ethyl acetate (300 mL) and washed with water (3 x 150 mL). The organic layer was dried and concentrated to give a mixture of two compounds which were separated by flash chromatography (silica) using 8% ethyl acetate in petroleum ether as eluent to afford 6a and 6b. Compound- 6a (Rf 0.60, silica, ethyl acetate:petroleum ether, 1:4, 16.8 g. 55%) was found to be p gluco derivative H NMR (CDC1 3 4.915 2H), 4.604 J=7.8 Hz, 1H, C- 4.110 2 3.633 1H), 3.366 IH), 2.020 3H, OAc), 2.006 3H, OAc), 1.958 3H, OAc), 0.881 9H, t-bu) and 0.08 6H, Me 2 Si).

13C NMR: 170.324, 169.778, 169.487, 97.002, 72.128, 71.757, 68.788, 65.788, 65.914, 62.251, 25.443, 20.556, 20.459, 17.857, -4.557, -5.319. IR: 3484, 2958, 2859, 2114, 1752, 1659, 1435, 1240, 1107, 1007, 940, 843, 785 and 676.

The slower moving compound 6b (Rf 0.51, silica, ethyl acetate:petroleum ether, 1:4, 7 g, 23%) was found to be the a manno derivative.

1 H NMR (CDC13): 5.12 1H), 4.96 2H), 4.0-4.19 (m, 3H), 3.61 1H), 2.06 3H, OAc) 2.02 3H, OAc) 1.94 3H, OAc), 0.91 9H, t-bu), 0.15 3H, MeSi) and 0.1 3H, MeSi).

Example 3 Preparation of t-Butyldimethylsilyl-2azido-2-deoxy-3-hydroxy-4,6-0isopropyledene-P-D-glucopyranose (2) A mixture Of compound 6a (19 g, 42 mmol), sodium methoxide (3 ml, 1 M) in methanol (120 mL) was stirred at room temperature for 2 h. After completion of the reaction (TLC), methanol was removed in vacuo and the resulting material was filtered through a small pad of silica gel using ethyl acetate as eluent to afford the corresponding triol as an oil (Rf 0.21, silica, WO 93/19761 PCT/US93/02903 petroleum ether:ethyl acetate, 1:1, 12.7 g, IH NMR (CDC1 3 4.60 J=7.2 Hz, 1H, anomeric), 3.841 2H), 3.60 1H), 3.32 3H), 0.91 9H, t-bu), 0.1 6H, Me 2 Si).

13 C NMR: 97.167, 75.362, 74.351, 70.027, 68.314, 61.751, 25.564, 17.920, -4.311, and -5.232.

IR: 3369, 2956, 2860, 2114, 1566, 1464, 1364, 1218, 1173, 1079, 957, 843, 760 and 691.

The triol compound obtained above (12 g, 37 mmol), was treated with 2,2-dimethoxy propane (7.70 g, 74 mmol) in methylene chloride (150 mL) in the presence of p-toluenesulfonic acid (0.3 g) and the mixture was stirred at room temperature for 4 h. Then the reaction mixture was diluted with methylene chloride (200 mL), and washed with water (200 mL), sodium bicarbonate solution 100 mL) and water (150 mL). The organic layer was separated, dried, and concentrated to give a thick mass, which after flash chromatography afforded compound 2 as an oil (Rf 0.32, silica, ethyl acetate:petroleum ether 1:4, 11.4 g, 86%).

1 H NMR (CDC1 3 4.601 J=7.5 Hz, 1H, anomeric), 3.84 2H), 3.589 9.3 Hz, 1H), 3.470 J=9.3 Hz, 1H), 3.30 1H), 3.0 (bs, 1H, exchanged with D 2 2.232 (n, 2H), 1.58 3H, other CH 3 1.46 3H, CH 3 of isopropylidene), 0.94 9H, t-bu), 0.12 3H, CH 3 0.1 3H, CH3).

13C NMR: 99.930, 97.503, 73.497, 72.072, 69.151, 67.930, 61.909, 28.952, 25.519, 19.049, -4.397, -5.238.

IR: 3453, 2932, 2114, 1643, 1382, 1279, 1040, 866, and 680.

Example 4 Preparation of Tetradodec-ene-l-ol v (7) A mixture of dodecanal (18.4 g, 100 mmol), methyl (triphenylphosphoralenyledene) acetate (40.3 g, 120 mmol), in chloroform (400 mL), was stirred at room temperature for 4 h. After completion of the reaction (TLC), the solvent was removed and the resulting material was dissolved in ether (100 mL). The precipitated Lh .i WO 93/19761 PCT/US93/02903 triphenylphosphine oxide was removed by filtration. The filtrate was concentrated in vacuo and subjected to the flash chromatography to afford a mixture of the E and Z isomers of a, a-unsaturated esters as an oil (Rf 0.62, silica, ethyl acetate:hexane 1:7, 20 g, 86%).

H NMR (CDC1 3 6.98 1H, olefinic), 5.82 J=7.2 Hz, olefinic), 3.76 3H, OCH 3 2.1 2H, allylic methylene), 1.20 (bs, 18H, 9 x CH 2 and 0.86 3H, terminal CH 3 To a solution of the above obtained ester compound (18 g, 75 mmol) in methylene chloride (250 mL), at -78 0 C, was added a solution of DIBAL-H (160 mL, 1 M), and the reaction mixture was stitred at that temperature for 1 h. After completion of the reaction (TLC), the excess DIBAL-H was quenched with methanol (15 mL) and transferred to a conical flask containing ethyl acetate (750 mL) and rochelle salt solution (200 mL) and stirred until two clear phases separated (approximately 1 h).

Then the organic layer was separated, dried, and concentrated to give a colorless oil, which was purified by flash chromatography to afford the allyl alcohol 7 as a colorless oil (Rf 0.25, silica, ethyl acetate:hexane, 1:4, 14.5 g, 92%).

IH NM (CDC1 3 5.60 2H, olefinic), 4.06 2H), 2.02 2H, allylic CH 2 1.20 (bs, 18H, 9 x CH 2 and 0.9 (t, 3H, CH 3 Example 5 Preparation of (2S, 3R)-2,3-Epoxy tetradecan-1-ol (8) To a solution of methylene chloride (150 mL) containing powdered activated molecular sieves (4 A, 8 g) and titaniumtetra-isopropoxide (1.41 g, 5 mmol, 10% by weight), at -200C sequentially added diethyl tartrate (1.23 g, 6 mmol, allyl alcohol 7 (10.50 g, 50 mmol) in methylene chloride (50 mL) and t-butyl hydroperoxide in methylene chloride (15 ml, 5.19 M, 1.5 eq) with 5 min between each addition. The mixture was stirred for 2 h at that temperature and allowed to stand at -200C overnight (at which time reaction was completed, TLC).

n:- WO 9319761 PCr/US93/02903 42 The molecular sieves were removed by filtration and the filtrate was treated with a saturated solution of sodium sulfate (5 ml) for 2 h. The precipitated inorganic salts were removed by filtration through celite, the solvent was removed, and the product was purified by flash chromatography to afford the epoxide 8 as a colorless solid (Rf 0.54, silica, ethyl acetate:hexane 1:5, 9.10 g, m.p 85-86 0

C.

1 H NMR (CDC1 3 3.92 1H, diastereotopic proton of C- 2.98 2H), 3.60 1H), 1.61 2H), 1.20 (bs, 18H, 9 x CH 2 and 0.9 3H, CH 3 13C NMR: 63.211, 60.124, 57.397, 33.232, 32.899, 30.983, 30.863, 30.720, 30.662, 27.262, 23.995, 15.394.

IR: 3573, 2918, 2848, 1584, 1549, 1480, 1430, 1377, 1252, 1154.

Example 6 Preparation of (R)-3-Hydroxy tetradecan-.od (9) To a solution of epoxide 8 (9.00 g, 40 mmol) in tetrahydrofuran (80 mL) at -200C was added a solution of Red-Al (40 mL, 3M) and the reaction temperature was slowly raised to 0°C and subsequently to room temperature and stirred for 12 h. After completion of the reaction (TLC), the excess of Red-Al was quenched with methanol mL) and poured in to a conical flask containing ethyl acetate (400 mL) and rochelle salt solution (100 mL) and stirred for 2 h until clear separation of two layers occurred. The organic layer was separated, dried and concentrated to give the diol 9 as a colorless solid (8.1 g, m.p. 59-60 0

C.

3-H NMR: 3.783 3H) 1.652 2H) 1.236 0.855 J=6.6 Hz, 3H).

13C NMR: 73.033, 62.528,. 39.697, 39.122, 33.268, 31.019, 30.709, 26.978, 24.024 and 15.429.

IR: 3310, 2917, 2849, 1565, 1431, 1108, 1029, 980, 860 and 721.

The diol was characterized as its acetate derivative. The acetate was prepared by the standard procedure using acetic anhydride, DMAP in methylene i;P*1 WO 93/19761 PCT/US93/02903 i It i? chloride.

1H NMR (CDC1 3 4.98 1H, 4.04 2H, 2.04 (S 6H, 2 x OAc), 1.84 (4 1.30 (bs, 18H, 9 x CH 2 0.9 3H, CH 3 Example 7 Preparation of Compound 9a To a solution of diol 9 (920 mg, 4 mmol) and DMAP (610 mg, 5 mmol) in dichloromethane (20 mL) at 0°C, a solution of pivoloyl chloride (600 mg, 5 mmol) in dichloromethane (1 mL) was added and the mixture was stirred at 0 C for 2 h. After completion of the reaction excess chloride was neutralized with methanol and solvents were removed in vacuo to afford an oil, which after purification by flash chromatography afforded the pivoloyl protected compound as an oil (Rf 0.62, silica, 8% ethyl acetate in hexane, 1.04 g, 83%).

1H NMR: 4.12 2H), 3.46 1H), 1.56 4H), 1.24 (bs, 18H), 1.16 9H), 0.86 3H).

To the above-obtained compound (520 mg, 1.65 mmol) in DMF (8 mL) was sequentially added imidazole (272 mg, 4 mmol) and t-butyldimethylsilyl chloride (308 mg, 2 mmol) and the mixture was stirred at room temperature for 4 h. After completion of the reaction, mixture was diluted with ethyl acetate (10 mL) and washed with water (3 x 5 mL). The organic phase was separated, dried, concentrated to afford an oil, which after purification by flash chromatography afforded compound 9a as an oil (Rf 0.61, 5% ethyl acetate in hexane, 0.592 g, 84%).

1 H NMR: 4.16 2H), 3.46 1H), 1.64 4H), 1.26 (bs, 18H), 1.16 9H), 0.842 12H), 0.03 6H).

Example 8 Preparation of R 3-Hydroxy 14tetradecanoic acid To a solution of compound 9a (690 mg, 1.6 mmol) in dichloromethane (8 mL) at -78 0 C, a solution of DIBAL-H mL, 4 mmol, 1.6M in toluene) was added under an argon atmosphere and mixture was stirred at that temperature for 1 h. After completion of the reaction, excess DIBAL-H was neutralized with methanol (2 mL) and mixture was transferred to a conical flask containing

L

WO 93/19761 PCT/US93/02903 44 ethyl acetate (30 mL) and saturated solution of rochelle's salt (10 mL) and stirred for 1 h. The organic phase was separated, dried, concentrated and purification by flash chromatography afforded the hydroxy compound as thick oil (Rf 0.24, 8% ethyl acetate in hexane, 430 mg, 78%).

1 H NMR: 3.48 3H), 1.58 4H), 1.26 (bs, 18H), 0.86 12H) 0.03 6H).

The above-obtained alcohol (0.342, 1 mmol) was dissolved in acetone (10 mL) and cooled to 0 C and added Jones reagent until orange color persisted. After completion of the reaction, solvent was removed in vacuo, the resulting material was dissolved in ethyl acetate mL) and washed with water (3 x 5 mL), separated the organic phase, dry, concentrate and purified by flash chromatography to afford the corresponding acid as an oil (Rf 0.18, 10% ethyl acetate in hexane, 195 mg, 54%).

IH NMR: 10.02 (bs, 1H), 3.48 1H), 2.24 2H), 1.64 2H), 1.24 (bs, 18H), 0.84 12H) 0.03 6H).

The above-obtained silyl acid (180 mg, mmol) was dissolved in THF (2 mL) in a plastic container and cooled to -20 0 C and HF-pyridine (1 mL) was added and the mixture was stirred for 1 h. After completion of the reaction excess acid was neutralized with sodium bicarbonate solution 2 mL) and extracted with dichloromethane (5 mL). The organic phase was separated, dried, concentrated and purified by flash chromatography to afford the hydroxy acid as colorless solid (Rf 0.16, ethyl acetate in hexane, 85 mg, m.p. 74 0

C

(a)D 21 16.44 (C 0.46, CHC1 3 Literature m.p. 73 0 C, (a) 2 5 D 16.20 (C 2, CHC1 3 IH NMR: 10.12 1H), 3.46 1H), 2.36 2H), 1.26 (bs, 20H), 0.846 3H).

I; WO 93/19761 PCT/US93/02903 Example 9 Preparation of Butyldimethylsilyloxy- 3-hydroxytetradecane To a solution of diol compound 9 (2.28 g, mmol) in N,N-dimethyl formamide (50 mL) at 0°C was added sequentially imidazole (1.63 g, 24 mmol), and tbutyldimethylsilyl chloride (1.65 g, 11 mmol), and the reaction mixture was stirred for 2 h. After completion of the reaction (TLC). The mixture was diluted with ethyl acetate (200 mL), washed with water (2 x 50 mL), dried and concentrated to give a colorless oil, which after purification by flash chromatography, afforded the pure monosilyl compound 10 as an oil (Rf 0.71, silica, ethyl acetate and hexane, 1:10, 2.80 g, 84%).

The alcohol was characterized as its acetate derivative.

H NMR (CDC1 3 5.0 1H, C-3) 3.62 2H, 1.80 2H), 1.56 2H), 0.92 12H, CH 3 and t bu), 0.1 6H, Me 2 Si).

Example 10 Preparation of Butyldimethylsiloxy-3dodecanoyloxy tetradecane (11) A mixture of compound 10 (1.02 g, 3 mmol) and lauric acid (720 mg, 3.6 mmol), DCC (680 mg, 3.3 mmol) and DMAP (400 mg, 3.3 mmol) in methylene chloride (12 mL) was stirred at room temperature for 4 h. After completion of the reaction the insoluble material was removed by filtration, the solvent was removed, and the product was isolated by flash chromatography to afford coupling compound 11 as an oil (Rf 0.64, silica, ethyl acetate:hexane 1:9, 1.26 g, 81%).

1H NMR (CDC1 3 4.96 1H), 3.75 J=6.6 Hz, 2H), 2.228 J=7.5 Hz, 2H), 1.691 4H), 1.512 2H), 1.228 (bs, 36H), 0.91 15H), 0.002 6H, methyls of silyl).

13C NMR: 174.469, 72.660, 72.623, 60.903, 38.585, 35.936, 35.738, 33.261, 30.970, 30.917, 30.828, 30.686, 30.532, 27.244, 27.184, 27.059, 26.534, 26.463, 24.008, 19.511, WO 93/19761 PCT/US93/02903 46 15.392.

IR: 2926, 1738, 1585, 1494, 1415, 1255, 1176, 1097, 940, 837, 723.

Example 11 Preparation of (R)-3-Dodecanoyioxy tetradecanoic acid (3) To a solution of compound 11 (2.63 g, 5 mmol) in methanol and methylene chloride (20 mL, 1:1 mixture) at 0 0 C, was added PTSA (200 mg) and the resulting mixture was stirred at 0 0 C for 2 h. After completion of the reaction, acid was neutralized with triethylamine (1 mL) and the solvents were removed in vacuo and the product was purified by flash chromatography to give the corresponding alcohol as an oil. (Rf 0.24, silica, ethyl acetate:hexane, 1:1, 1.44 g, IH NMR: 4.984 IH), 3.501 2H), 2.775 (bs, 1H, OH), 2.329 J= 7.2 Hz, 2H), 1.571 6H), 1.226 (bs, 36H), 0.864 (t, J=6.6 Hz, 6H).

3C NMR: 175.488, 72.550, 69.959, 59.824, 38.913, 38.864, 37.732, 35.997, 35.891, 35.676, 33.251, 30.963, 30.875, 30.682, 30.613, 30.513, 26.979, 26.780, 26.462, 26.324, fi 24.012, 15.408.

IR: 3535, 2924, 1739, 1692, 1565, 1494, 1427, 1379, 1171, 1059, 936.

The alcohol obtained-above (1.49 g, 3.61 mmol) was dissolved in acetone (40 mL), and Jones reagent was added slowly until the orange color persisted. After completion of the reaction, the solvent was removed in vacuo and the resulting material was dissolved in ethyl acetate (50 mL) and washed with water (3 x 30 mL) until ~,organic phase was almost colorless. The organic layer was separated, dried, and concentrated to give an oil, which was purified by flash chromatography to afford acid 3 as a colorless solid (1.32 g, 86%).

IH NMR: 9.86 (bs, 1H, OH of COOH), 5.186 2H), 2.579 J=6.6 Hz, 6H), 2.44 J=7.5 Hz, 2H), 1.592 (bs, 4H), 1.231 (bs, 36 0.847 J=6.6 Hz, 6H, terminal WO 93/19761 PCT/US93/02903 47 methyl).

13C NMR: 177.659, 174.650, 71.390, 40.277, 35.774, 35.292, 33.247, 30.969, 30.880, 30.636, 30.617, 30.445, 26.326, 24.010, 15.383.

IR: 3573, 2921, 2675, 1746, 1713, 1675, 1549, 1513, 1466, 1379, 1283, 1206, 1164, 1114, 1070, 1012, 935, 723.

Example 12 Preparation of Dibenzyl dodecanephosphonate (14) To a solution of potassium t-butoxide (6.16 g, 55 mmol), in dry DMF (50 mL), under an argon atmosphere was added dropwise via syringe a solution of dibenzyl phosphite (13.1 g, 50 mmol) in dry DMF (5 mL), and the reaction mixture was stirred at room temperature for min. Then a solution of lauryl bromide (18.6 g, 75 mmol) in dry THF (15 mL), was added dropwise under argon atmosphere, and the mixture was stirred at room temperature for an additional 1 h. After completion of the reaction, the mixture was neutralized with dilute HC1 mL) and extracted with ethyl acetate (200 mL), the organic phase was dried, concentrated, and purified by flash chromatography to afford the pure dibenzyl phosphate 14 as a colorless oil (Rf 0.46, silica, ethyl acetate and hexane, 1:2, 10.5 g, 1 H NMR (CDC1 3 7.36 10H, aromatic) 5.02 4H benzylic) 1.52 2H), 1.24 (bs, 18H), 0.9 3H).

Example 13 Preparation of Phosphochloridate (12) A mixture of compound 14 (6 g, 14 mmol) and phosphorus pentachloride (2.9 g, 14 mmol), in chloroform mL), was heated at 60°C for 2 h. After completion of the reaction (progress of the reaction was monitored by NMR) solvent was removed in vacuo to afford the phosphochloridate 12 as an oil.

IH NMR (CDC1 3 7.36 5H, aromatic) 5.24 2H, benzylic), 1.26 /bs, 18H), 0.92 3H).

Example 14 Preparation of Compound 13 A mixture of hydroxy compound 10 (1.05 g, 3 mmol) and phosphochloridate compound 12 (1.4a g, 4 mimol), was stirred in methylene chloride (12 mL) in the WO 93/19761 PCT/US93/02903 48 i presence of DMAP (0.41 g, 3.3 mmol) and triethylamine (2 mL) for 3 h. After completion of the reaction, methanol (1 mL) was added, the solvent was removed in vacuo and the product was purified by flash chromatography (Rf 0.46, silica, ethyl acetate and hexane 1:9, 1.36 g, 68%) H NMR (CDC1 3 7.42 5H, aromatic), 5.02 (in, 2H, i benzylic) 4.58 3.76 2H), 1.82 6H), 1.28 (bs, 36H), 0.92 9H), 0.12 3H) and 0.1 3H).

Example 15 Preparation of Acid 4 A mixture of compound 13 (800 mg, 1. 2 mmol), and PTSA (60 mg) in methanol and methylene chloride (6 mL, 1:1 mixture) was stirred at 0°C for 1 h. After completion of the reaction the acid was neutralized with triethylamine (2 mL), the solvents were removed, and the mixture was subjected to flash chromatography to afford the corresponding hydroxy compound as an oil (Rf 0.24, silica, ethyl acetate and hexane, 1:4, 580 mg, 87%).

IH NMR: 7.36 5H, aromatic), 5.124 2H, benzylic), 4.612 (m 1H), 3.728 2H), 3.052 (bs, OH), 1.642 (m, 4H), 1.264 (bs, 40H) 0.846 6H).

The above alcohol compound (550 mg, 1 mmnol) was dissolved in acetone (10 mL) and cooled to 0°C, and Jones reagent was added until a red color persisted and the mixture was stirred at that temperature for 1 h. After completion of the reaction the acetone was removed, and the resulting material was dissolved in ethyl acetate mL) and washed with water (3 x 5 mL) until the organic layer became almost colorless. The organic layer was dried and concentrated to give acid 4 as an oil (520 mg, SJ 81%).

1H NM.: 7.36 5H, aromatic), 5.124 2H), 4.746 (m, 1H), 2.562 2H), 1.462 4H), 1.264 (bs, 40H), and 0.861 6H).

Example 16 Preparation of Compound A mixture of compound 2 (1.79 g, 5 mmol), acid 3 (2.13 g, 5 rmol), DCC (1.24 g, 6 mmol) and DMAP (0.74 WO93/19761 PCT/US93/02903 49 g, 6 mmol) in methylene chloride (20 mL) was stirred at room temperature overnight. The insoluble material was removed by filtration, the solvent was removed, and the product was purified by flash chromatography to afford the coupled compound 15 as an oil (Rf 0.24, silica, ethyl acetate and hexane 1:19, 2.76 g, 72%).

IH NMR (CDC1 3 5.26 1H), 4.98 1H), 4.70 J=8.

6 Hz, 1H, anomeric), 3.80 3H), 3.32 2H), 2.72 (AB quartet, 2H), 2.32 2 1.68 2H), 1.42 3H, isopropylidene Me), 1.30 3H, isopropylidene Me), 1.26 (bs, 36H), 0.92 15H), 0.2 (2 x s, 6H).

Example 17 Preparation of Amino Compound 16 A mixture of compound 15 (2.37 g, 3.1 mmol) and trifluoroacetic acid (8 mL) in methylene chloride (20 mL) was stirred at room temperature. After completion of the reaction the acid was neutralized with triethylamine mL), and the solvents were removed to give a thick oil.

The product was purified by flash chromatography to afford the diol as an oil (Rf 0.36, silica, ethyl acetate and hexane, 1:6, 1.62 g, 72%).

H NMR (CDC1 3 5.20 1H), 4.80 4.70 1H, anomeric), 3.82 (AB quartet, 2H), 3.60 1H), 3.41 (m, 1H), 3.30 (dd, 1H), 3.08 1H), 2.60 2 2.32 (t, 2H), 1.60 2H), 1.28 (bs, 36H), 0.94 9H), 0.90 (t, 6H) and 0.1 6H).

The primary alcohol functionality of the diol obtained above (1.53 g, 2.1 mmol) was protected as tbutyldimethylsilyl ether following the same procedure used for the preparation of ether 8 to give the primary silyl ether (Rf 0.34 silica, ethyl Lctate and hexane, 1:19, 1.54 g, 87%).

1H NMR: 5.20 1H), 4.82 1H), 4.61 1H, anomeric), 3.84 2H), 3.60 1H), 3.40 IH), 3.30 (dd, 1H), 2.61 2H), 2.30 2H), 1.60 2H), 1.26 (bs, 36H), 0.92 24H), 0.14 (2 x s, 6H) 0.1 6H) The monosilylated compound obtained above (1.49 g, 1.77 mmol), was hydrogenated over Pd-C, 200 mg) i WO 93/19761 PCT/US93/02903 in methanol (10 mL) to afford the corresponding amino compound 16 as an oil (Rf 0.24, silica, ethyl acetate:hexane, 1:9, 1.38 g, 96%).

IH NMR (benzene-d 6 5.40 1H), 5.14 1H), 4.51 (d, 1H, anomeric), 3.82 2H), 3.72 1H), 3.30 1H), 2.91 3H, after exchange with D 2 0 integration decreases, amino), 2.60 2H), 2.20 2H), 1.62 (m, 2H), 1.24 (bs, 36H), 0.92 24H), 0.2 (2 x s, 6H) 0.0 6H).

Example 18 Preparation of Compound 1 To a solution of acid 4 (526 mg, 1 mol) in methylene chloride (5 mL), was added EDC (200 mg, 1 mmol) and the reaction mixture was stirred for 10 min. Then the amino compound 16 (815 mg, 1 mmol), and 1-hydroxy benzotriazole (130 mg, 1 mmol) were added and the resulting mixture was stirred under argon atmosphere for 4 h. After completion of the reaction the mixture was diluted with ether (10 mL) and washed with water (2 x mL) dried, concentrated and chromatographed to give the coupled compound 1 as an oil (Rf 0.71, silica, methylene chloride:ether, 3:1, 640 mg, 49%).

1 NR: 7.40 5H, aromatic), 6.32 1H, NH) 4.60- 5.23 5H), 3.82 2H), 3.40 2H), 3.36 2H), 3.30 1H), 2.52 2H) 2.24 21H), 1.62 2H), 1.24 (bs, 36H), 0.91 24H) and 0.06 (bs, 12H).

Example 19 Preparation of compound 17 A mixture of compound 1 (400 mg, 0.302 mmol), N,N-diisopropylamino dibenzyl phosphite (164 mg, mmol), and tetrazole (55 mg, 0.75 mmol) in methylene chloride (2 mL) was stirred at room temperature for min. After completion of the reaction (TLC), the mixtur was cooled to -20 0 C and m-CPBA (140 mg, 0.85 mmol) was added and the reaction mixture was stirred for 30 min.

After completion of the oxidation the mixture was diluted with ether (10 mL) and washed with water (5 mL), and then again washed with sodium bicarbonate solution 5 mL).

The organic layer was dried, concentrated, and purified 1 WO 93/19761 PCT/US93/02903 51 by flash chromatography to afford the phosphorylated compound 17 as an oil (Rf 0.36, silica, ethyl acetate and hexane, 1:3, 435 mg, 91%).

1 E NMR: 7.340 15H, aromatic), 5.012 6H), 4.742 2H), 4.395 1H), 3.676 2H), 3.264 2H), 2.421 8H), 1.521 8H), 1.246 (bs, 72H), 0.862 (m, and 0.08 12H).

Example 20 Preparation of Immunogen BK-1 A suspension of compound 17 (220 mg, 0.014 mmol) in methanol (1 mL) was subjected to hydrogenation using Pd-C 20 mg). After completion of the reaction, catalyst was removed by filtration and the solvent was then removed to afford the debenzylated compound (Rf 0.36, silica, 30% methanol in ethyl acetate, 165 mg, 1 H NMR: 5.102 2H), 4.642 2H), 3.962 2H), 3.24 2H), 2.42 8H), 1.521 8H), 1.241 (bs, 72H), 0.824 30H) and 0.08 12H).

The compound obtained above (131 mg, 0.01 mmol) was treated with HF-pyridine (0.5 mL), in THF (1 mL), in a plastic container at 0°C for 30 min. After completion of the reaction, the mixture was diluted with ethyl acetate (5 mL) and stirred with sodium bicarbonate 2 mL) solution for 20 min. The organic layer was separated and the aqueous solution was washed with chloroform mL), organic phases were dried and the solvents were removed in vacuo to give an oil. The oil was dissolved in a chloroform-methanol mixture 10 mL) and insoluble particles were filtered. The filtrate was dried and concentrated to give a thick oil and the residue was redissolved in water and lyophilized to give BK-1 (Rf 0.21, silica, 50% methanol in chloroform, 55 mg, 51%).

IH NMR: 5.112 2H), 4.462 2H), 3.621 4H), 2.221 8H), 1.562 8H), 1.221 (bs, 72H), and 0.824 12H).

I i I 1 WO 93/19761 PCU/US93/02903 Example 21 Preparation of Compound 18 A suspension of azido compound 2 (3.59 g, mmol) and Pd-C 0.35 g) in ethyl acetate (40 mL) was stirred under hydrogen atmosphere. After completion of the reaction (TLC) the mixture was filtered through a pad of celite to remove catalyst and the solvent was removed in vacuo to give a thick oil. The oil was purified by flash chromatography to afford compound 18 as a colorless solid (Rf 0.26, silica, ethyl acetate, 3.0 g, m.p.

154-155°C H NMR: 4.463 J=7.8 Hz, 1H, 3.785 2H), 3.538 J=9.3 Hz, 1H), 3.415 J=9.3 Hz, 1H), 3.229 1H), 2.682 1H) 1.463 3H), 1.385 3H), 0,869 9H, t-butyl), 0.082 SiMe 2 1 3 C NMR: 99.655, 99.313, 74.049, 73.104, 67.557, 62.130, 59.904, 29.080, 25.682, 19.149, -4.088, -5.190.

IR: 3573, 3391, 2931, 2249, 1666, 1512, 1464, 1383, 1266, 1181, 1141.

Example 22 Preparation of Compound 26 To a solution of amino compound 18 (2.85 g, 8.55 mmol) and acid 3 (3.83 g, 9 mmol) in dichloromethane (18 mL) under an Ar atmosphere, was added sequentially EDC (1.91 g, 10 mmol) and HOBT (1.30 gms, 10 mmol), and the resulting reaction mixture was stirred at room temperature for 4 h. After completion of the reaction the solvent was removed in vacuo and the product was isolated as an oil after flash chromatography (Rf 0.34, silica, ethyl acetate:hexane 3:7, 4.81 g, 76%).

1 H NMR: 6.249 J=6.9 Hz, 1H, NH) 5.133 1H, CHOCO), 4.875 J=8.1 Hz, 1H, 3.833 3H), 3.615 J=9 Hz, 1H), 3.415 1H), 3.280 1H), 2.439 2H) 2.263 J=7.2 Hz, 2H) 1.513 3H, acetonide methyl) 1.427 3H, acetonide methyl), 1.247 (bs, 34H), 0.876 15H, t-butyl and terminal methyls) and 0.08 6H, SiMe 2 13 C NMR: 174.242, 171.041, 99.719, 95.891, 74.284, 71.657, 71.406, 67.280, 62.036, 60.810, 42.508, 34.534, 34.478, A i I WO 93/19761 PCT/US93/02903 31.880, 29.594, 29.513, 29.482, 29.262, 29.167, 29.036, 25.608, 25.259, 24.271, 22.642, 19.035, 17.836, 14.080, -4.135, -5.196.

IR: 3298, 2928, 1733, 1654, 1465, 1306, 1257, 1098, 941, 842, 784, 673.

Example 23 Preparation of Keto Phosphonate 21 To a cooled solution (-78 0 C) of dimethyl methylphosphonate (6.20 g, 50 mmol) in dry THF (250 mL) was added under Ar atmosphere a solution of nbutyllithium (1.6 M, 30 mL, 52.8 mmol) through a syringe and the mixture was stirred at that temperature for 1 h.

Then a solution of methyl laurate (12.8 g, 60 mmol) in THF (30 mL) was added and the reaction mixture was stirred further for 1 h at -78°C and then at 0°C for min. After completion of the reaction, the base was neutralized with ammonium chloride solutic 100 mL) and the organic layer was separated, dried, and concentrated to give an oil. The product, keto phosphonate 21, was purified by flash chromatography to afford a colorless solid (Rf 0.46, ethyl acetate, 9.8 gms. m.p. 43-44 0

C.

1 H NMR: 3.757 3H, OMe), 3.720 3H, OMe), 3.03B (d, J=22.5 Hz, 2H, CH 2 2.559 J=7.5 Hz, 2H), 0.826 (t, J=6.6 Hz, 3H, side-chain terminal methyl).

C NMR: 203.012, 54.122, 54.043, 45.291, 43.231, 41.534, 33.070, 30.777, 30.634, 30.503, 30.562, 30.114, 24.567, 23.849, 15.252.

IR: 2967, 1704, 1588, 1474, 1378, 1237, 1193, 1105, 1057, 895, 826, 794.

Example 24 Preparation of Hydroxy Phosphonate 22 To a solution of keto phosphonate 21 (6.12 g, mmol), in methanol (100 mL) cooled to 0°C was added sodium borohydride (0.95 g, 25 mmol) in four portions, and the mixture was stirred at 0 C for 1 h. After completion of the reaction, the excess hydride reagent was quenched with water (2 mL) and the solvent was removed in vacuo to afford the crude hydroxy phosphonate.

WO 93/19761 PCr/US93/02903 The product was purified by flash chromatography to afford the hydroxy phosphonate 22 as a colorless solid (Rf 0.21, silica, ethyl acetate, 5.80 g, m.p. 72- 73 0

C

H kMR: 4.02 1H, CHOH), 3.7325 J=9.9 Hz, 6H, P

OCH

3 3.410 (bs, 1H, OH), 1.976 2H, CH 2 1.352 (m, 4H, 2 x CH 2 1.206 (bs, 16H 0.826 J=6.6 Hz, 3H, terminal methyl).

1 3 C NMR: 90.241, 67.796, 67.711, 40.124, 39.538, 34.855, 33.211, 33.026, 30.944, 30.919, 30.885, 30.787, 30.636, 26.721, 23.972, 15.381.

IR: 3338, 2956, 2743, 1741, 1471, 1266, 1129, 1030, 988, 854, 727, 572.

Example 25 Preparation of Compound 23 To a solution of hydroxy phosphonate 22 (5.54 g, 18 mmol) and lauric acid (4.0 g, 20 mmol) in dichloromethane (40 mL), was added sequentially DCC (4.12 g, 20 mmol) and DMAP (2.44 g, 20 mmol) and the contents were stirred at room temperature for 4 h under argon atmosphere. After completion of the reaction (4 h, TLC), the insoluble material was removed by filtration and the filtrate was concentrated to give the crude ester 23, which after flash chromatography, afforded the ester 23 as a colorless solid (Rf 0.64, ethyl acetate, 7.23 g, m.p. 74-75 0

C

IH NMR: 5.186 1H, CHCOO), 3.732 J=6.9 Hz, 6H, P

OCH

3 2.346 J=7.5 Hz, 2H), 2.102 2H), 1.614 (m, 4H, 2 x CH 2 1.240 (bs, 32 0.826 J=6.2 Hz, 6H) 13C NMR: 174.005, 69.761, 53.545, 53.460, 36.221, 36.111, 35.587, 33.099, 32.151, 30.804, 30.670, 30.526, 30.430, 30.336, 30.290, 26.257, 26.079, 23.850, 15.222.

IR: 2926, 2855, 1739, 1494, 1251, 1 4 d3, 1035, 844, 756, 666.

Example 26 Preparation of Diacid 24 Under an Ar atmosphere, to a solution of compound 23 (6.86 gms, 14 mmol) in dichloromethane mL) cooled by an ice bath was added trimethylsilyl iodide 4 WO 93/19761 PCT/US93/02903 (7 mL), and the mixture was stirred at room temperature for 1 h. After completion of the reaction, the mixture was diluted with dichloromethane (20 mL) and stirred with sodium thiosulfate solution 20 mL) for 10 min.

Then the organic layer was separated and concentrated to give the crude diacid 24. The crude material was dissolved in THF (50 mL) dilute HCl 20 mL) and the solution was stirred for 1 h. To the solution was added dichloromethane (100 mL), and the organic layer was separated, dried, and concentrated to give compound 24 as a colorless solid (5.30 g, m.p. 78-79 0

C

1H NMR: 5.186 1H, CHOH), 2.324 J=7.2 Hz, 2H), 2.084 2H), 1.632 4H), 1.286 (bs, 34 0.826 (t, J=6.6 Hz, 6H).

13C NMR: 175.488, 70.250, 36.755, 36.610, 35.875, 33.299, 31.058, 31.058, 31.037, 30.947, 30.738, 30.582, 26.392, 26.227, 24.049, 15.436.

IR: 2954, 1740, 1687, 1471, 1277, 1170, 1090, 998, 900, 855, 720, 610, 570.

Example 27 Preparation of Compound A mixture of compound 24 (4.62 g, 10 mmol), benzyl alcohol (3.24 g, 30 mmol), trichloroacetonitrile mL) pyridine (10 mL) and chloroform (20 mL) was heated at 50 0 C overnight. After completion of the reaction, the solvents were removed in vacuo and the I resulting material was dissolved in THF (40 mL) and stirred with HC1 20 mL) for 1 h. Then the organic layer was separated, concentrated, and the product was purified by flash chromatography to afford the monobenzylated compound 25 as a brown thick oil (3.64 g, 64%) H NMR: 11.56 (bs, 1H, OH), 7.363 5H, aromatic), 5.197 (ni, 1H), 5.062 2H), 2.112 4H), 1.652 (m, 4H), 1.264 (bs, 36 0.896 3H).

13C NMR: 174.442, 137.520, 137.615, 129.845, 129.172, 129.367, 69.930, 67.947, 36.553, 36.430, 35.770, 33.581,, 33.302, 31.024, 30.966, 30.738, 30.693, 30.548, 26.393, WO93/19761 PCT/US93/02903 56 26.244, 24.065, 15.476.

IR: 3359, 3063, 2957, 2873, 1869, 1717, 1636, 1499, 1382, 1072, 997.

Example 28 Preparation of Phosphochloridate 19 A solution of compound 25 (1.707 g, 3 mmol) and phosphorus pentachloride (0.615 g, 3 mmol) in dry chloroform (10 mL) was stirred at room temperature in an Ar atmosphere for 3 h. After completion of the reaction (3 h, NMR) the solvent was removed and the resulting product phosphochloridate was used in the next reaction without the need for further purification.

1H NMR: 7.340 5H, aromatic), 5.124 3H, benzylic CHCOO), 2.241 4H), 1.42 4H), 1.24 (bs, 34H), 0.864 6H, terminal methyls).

Example 29 Preparation of Compound 27 To a solution of compound 26 (1.48 g, 2 mmol), DMAP (0.244 g, 2 mmol) and triethylamine (4 mL) in dichloromethane (8 mL) under an Argon atmosphere was added a solution of phosphochloridate 19 (1.76 gms, 3 mmol, crude) in dichloromethane (5 mL) through a syringe over a period of min and the resulting reaction mixture was stirred at room temperature for a further 1 h. After completion of the reaction, the solvents were removed in vacuo and the product was purified by flash chromatography to afford compound 27 as an oil (Rf 0.36, silica, ethyl acetate:hexane 1:5, 1.5 g, 56%).

1 H NMR: 7.340 5H, aromatic) 6.589 1H, NH), 5.033 4H, benzylic and 2 x CHCOO), 4.665 1H, C-l), 4.371 1H), 3.774 3H), 3.349 1H), 2.231 (m, 8H) 1.592 8H), 1.521 3H, acetonide methyl), 1.367 3H, acetonide methyl), 1.258 (bs, 68H), 0.874 21H, t-butyl and side-chain terminal methyls), 0.08 3H, SiMe), 0.07 3H, SiMe).

13C NMR: 174.678, 172.091, 169.665, 128.516, 127.875, 99.558, 97.067, 72.538, 70.366, 68.523, 66.840, 61.811, 56.701, 42.021, 34.448, 31.835, 29.884, 29.002, 25.569, *i 1 1

P.,

WO 93/19761 PCT/US93/02903 57 25.196, 22.642, 19.109, 17.192, 14.054, -4.300 and 5.416.

IR: 3289, 2993, 1744, 1661, 1464, 1306, 1252, 1199, 1098, 942, 842, 732, 698, 525.

Example 30 Preparation of Compound 28 To a solution of compound 27 (0.51 g, 0.4 mmol) in dichloromethane (5 mL) cooled to 0 0 C was added trifluoroacetic acid (3 mL) and the resulting reaction mixture was stirred at 0 0 C for 1 h. After completion of the reaction, the acid was neutralized with triethylamine (4 mL), and the solvents were removed in vacuo to afford the crude diol 28 as an oil, which after purification by flash chromatography afforded the pure diol 28 as an oil (Rf 0.16, ethyl acetate:hexane, 1:4, 0.30 g, 81%).

1H NMR: 7.346 5H, aromatic) 6.211 1H, NH) 5.116 4H), 4.842 J=7.2 Hz, 1H, 4.420 1H), 3.642 5H), 2.228 8H), 1.33 8H), 1.261 (bs, 68H), 0.824 21 H, terminal methyls and t-butyl), 0.08 3H, SiMe), 0.074 3H, SiMe).

13 C NMR: 173.799, 173.287, 169.818, 128.634, 128,347, 95.666, 74.927, 70.810, 68.884, 62.879, 34.524, 34.405, 31.892, 29.775, 29.682, 29.397, 29.332, 29.129, 25.607, 24.974, 24.939, 24.868, 24.848, 22.658, 17.822, 14.083, -4.002, -5.014.

IR: 3286, 2955, 2362, 1736, 1656, 1500, 1416, 1379, 1229, 1081, 939, 784.

Example 31 Preparation of BK-3 A suspension of compound 28 (123 mg, 0.1 mmol) and Pd-C 12 mg) in ethyl acetate (2 mL) was stirred under a hydrogen atmosphere. After completion of the reaction the catalyst was removed by filtration and removal of the solvent afforded the debenzylated compound as an oil (103 mg, 1H NMR: 6.208 1H, NH), 5.012 1H), 4.782 2H), 3.742 3H), 3.124 2H), 2.228 8H), 1.32 (m, 8H), 1.261 (bs, 68H), 0.824 21H), 0.80 6H).

WO 93/19761 PCT/US93/02903 58 The above-obtained compound (100 mg, 0.087 mmnol) was dissolved in THF (2 mL) in a plastic container cooled to 0°C and HF-pyridine (0.2 mL) was added. After completion of the reaction (30 min) the mixture was diluted with dichloromethane (5 mL), and stirred with sodium bicarbonate solution 2 mL). The organic layer was separated, dried, and concentrated to give BK-3 as an oil. The oil was dissolved in water (5 mL) and i lyophilized to give BK-3 as a thick oil. (65 mg, 74%).

1 H NMR: 6.021 IH, NH), 5.034 1H), 4.420 2H), 3.342 5H), 2.228 8H), 1.30 8H), 1.24 (m, 68H), 0.832 12H).

SExample 32 Preparation of Compound 31 J To a solution of glucosamine hydrochloride (21.55 g, 100 mmol) in aqueous sodium bicarbonate (14.4 g, 300 mmol, 400 mL water) cooled to 0°C was added dropwise Cbz-Cl (18.75 g, 110 mmol) and the reaction mixture was stirred at 0°C for 2 h and then at room temperature overnight. The precipitated compound was filtered, washed with cold acetone (40 mL) and dried to give the amino protected compound as a colorless solid (26.90 g, 86%).

The above-obtained amino-protected compound g, 79.8 mmol) was dissolved in methanolic hydrogen chloride solution v/w, 350 ml), and the mixture was heated at reflux J for 4 h. After completion of the reaction, the solvent was removed in vacuo to afford compound 31 as a colorless solid (Rf 0.24, Silica, 10% methanol in dichloromethane, 20.30 g, 78%).

Example 33 Preparation of Compound 32 A solution of compound 31 (22 g, 67.2 mmol), PTSA (0.5g) in acetone (200 mL), dichloromethane (300 mL), and 2,2-dimethoxypropane (28 g, 4 eq) was stirred at room temperature for 4 h. After completion of the reaction, the acid was neutralized with triethylamine WO 93/19761 PCF/US93/02903 mL) and washed with water (50 mL), dried, and concentrated to give a slurry, which after flash chromatography, afforded compound 32 as a colorless solid (Rf 0.52, ethyl acetate:hexane 2:3, 20.71 g, m.p.

64-65 0

C

1 H NMR: 7.313 5H, aromatic), 5.362 J=9Hz, 1H, NH), 5.093 2H, benzylic), 4.683 J=3 Hz, 1H, C-l), 3.709 6H), 3.308 3H, OCH 3 2.432 (bs, 1H, OH), 1.483 3H, acetonide methyl), 1.414 3H, acetonide methyl).

13C NMR: 158.159, 137.547, 129.879, 129.714, 129.546, 101.202, 100.075, 75.902, 71.398, 68.531, 64.723, 63.658, 56.570, 30.435, 20.461.

IR: 3422, 3382, 2995, 1708, 1531, 1374, 1268, 1163, 1022, 991, 895, 752.

Example 34 Preparation of Amino Compound A suspension of compound 32 (19 g, 51.7 rmol) and Pd-C 1 in ethyl acetate (200 mL) was stirred under an atmosphere of hydrogen. After completion of the reaction, the catalyst was removed by filtration through a pad of celite and removal of the solvent afforded amino compound 30 as a colorless solid (Rf 0.46, silica, ethyl acetate, 11.3 g. 92%).

m.p. 152-153 0

C

1 H NMR: 4.637 J=3.6 Hz, 1H, 3.714 2H), 3.483 3H), 2.700 1H), 2.342 (bs, 2H, NH 2 1.481 3H), 1.398 3H).

13 C NMR: 102.532, 100.963, 75.960, 73.320, 64.811, 63.823, 58.070, 58.030, 56.601, 30.512, 20.522.

IR: 3454, 2938, 1580, 1466, 1383, 1273, 1201, 1122, 1041, 994, 898, 752, 738.

Example 35 Preparation of Compound 33 To a solution of amino compound 30 (4.66 g, mmol) and acid 3 (8.52 g, 20 mmol) in dichloromethane (100 mL) was added sequentially EDC (4.18 g, 22 mmol) and HOBT (2.86 g, 22 mmol) and the mixture was stirred at room temperature under an argon atmosphere for 2 h.

k. 61 1

-II

WO 93/19761 PCT/US93/02903 After completion of the reaction, the mixture was transferred to a separation funnel, washed with water mL), dried, and concentrated to give an oil. The product was purified by flash chromatography to afford compound 33 as an oil (Rf 0.41, silica, ethyl acetate:hexane, 9.58 g, 68%).

1 H NMR: 6.134 J=9 Hz, 1H, NH), 5.159 1H, CHOCO), 4.634 J=3. 9Hz, 1H, 4.134 1H), 3.766 (m, 3.352 3H, OCH 3 3.193 (bs, 1H, OH), 2.483 (d, J=6 Hz, 2H), 2.290 J=7.5 Hz, 2H), 1.605 4H), 1.524 3H), 1.431 3H, CH 3 of acetonide), 1.246 (bs, 34H), 0.875 J=6.6 Hz, 3H, terminal CH 3 1 3 C NMR: 174.960, 172.237, 101.191, 100.162, 75.913, 72.549, 72.176, 64.671, 63.618, 56.441, 55.535, 43.149, 35.884, 35.451, 33.237, 30.954, 30.863, 30.837, 30.668, 30.515, 26.633, 26.318, 24.004, 20.423, 15.425.

IR: 3418, 3354, 2994, 1734, 1652, 1467, 1339, 1267, 1197, 1131, 1074, 994, 896, 735.

Example 36 Preparation of Compound 34 To a solution of compound 33 (7.16 g, 11.17 mmol) in DMF (40 mL) was added sequentially imidazo.le (1.76 g, 26 mmol) and t-butyldimethylsilyl chloride (1.95 g, 13 mmol) and the mixture was stirred at room temperature under an argon atmosphere for 6 h. After completion of the reaction, the mixture was transferred to a separatory funnel, diluted with ethyl acetate (150 mL), and washed with water (2 x 100 mL). The organic layer was separated, dried, and concentrated to give an oil. The product was isolated after flash chromatography i 30 to afford the silylated compound 34 as an oil (Rf 0.62, silica, ethyl acetate:hexane, 6.83 g, 81%).

1H NMR: 5.726 J=9.6 Hz, 1H, NH), 5.124 1H, OCHCO), 4.615 J=3.6 Hz, 1H, 4.190 1H), 3.827-3.537 5H), 3.31 3H, OCH 3 2.462 2H,

CH

2 CO), 2.287 J=7.5 Hz, 2H, CH 2 CO), 1.612 4H), 1.522 3H), 1.432 3H), 1.321 (bs, 34H), 0.894 (m, t-butyl and side-chain terminal CH3), 0.042 6H,

I

WO 93/19761 PCT/US93/02903 61 3iMe 2 1 3 C NXR: 174.419, 170.738, 100.637, 100.736, 76.157, 73.000, 72.947, 72.888, 72.280, 64.977, 63.768, 56.360, 55.388, 43.169. 35.860, 35.765, 33.229, 30.935, 30.865, 30.810, 30.654, 30.616, 30.501, 30.382, 27.112, 27.064, 27.031, 26.473, 26.323, 23.992, 20.271, 19.482, 15.408.

IR: 2995, 1724, 1667, 1516, 1382, 1267, 1164, 1083, 1005, 910, 838, 780, 734.

Example 37 Preparation of Compound To a solution of acetonide compound 34 (4.53 g, 6 mnol) in dichloromethane (10 mL) at 0°C was added trifluoroacetic acid (5 mL) and the mixture was stirred at 0°C for 2 h. After completion of the reaction the acid was neutralized with triethylamine (6 mL) and the mixture was washed with water (10 mL). The organic layer was separated, dried, and concentrated to give the crude diol, which after purification by flash chromatography afforded the diol 35 as an oil (Rf 0.36 silica, ethyl acetate:hexane, 1:1, 3.98 g, 93%).

IH NMR: 5.724 J=9.6 Hz, IH, NH), 5.124 1H, OCHCO), 4.612 J=3.6 Hz, 1H, 3.724-3.401 (m, 3.32 3H, OCH 3 2.416 J=7.6 Hz, 2H), 2.232 J=7.6 Hz, 2H), 1.618 4H), 1.321 (bs, 34H), 0.894 12H, t-butyl and side chain terminal CH 3 0.042 (s, '3H, SiMe), 0.038 3H, SiMe).

13C NMR: 174.519, 171.030, 100.153, 74.732, 73.220, 72.992, 72.611, 63.675, 56.391, 54.923, 43.272, 35.873, 35.790, 33.231, 30.941, 30.872, 30.820, 30.686, 30.652, 30.517, 27.107, 26.107, 26.476, 26.339, 23.992, 19.420, 15.393, -2.602, -3.060.

IR: 3567, 3434, 3300, 2922, 1719, 1654, 1466, 1254, 1190, 1055, 861, 756, 736.

Example 38 Preparation of Ct4Miiad 36 To a solution of compfun4 2 (2.14 g, 3 =mol) and DMAP (0.488 g. 4 mmol) in dichloromethane (10 mL), a solution of tosyl chloride (0.686 g, 3.6 nunmmol) in dichloromethane (3 mL) was added at 0°C slowly through a L WO 93/19761 PCT/US93/02903 62 syringe under an argon atmosphere and the mixture was stirred at 0 C for 4 h. After completion of the reaction, the excess tosyl chloride was reacted with methanol (1 mL), and the mixture was transferred to a separatory funnel, washed with water (10 mL), dried and concentrated to give an oil. The monotosylated compound was purified by flash chromatography to afford an oil (Rf 0.34, silica, ethyl acetate:hexane, 1:4, 2.16 g. 83%).

i 1 NMR: 7.814 J=8.1 Hz, 2H, aromatic), 7.363 (d, J=8.1 Hz, 2H, aromatic), 5.674 J=9.3 Hz, 1H, NH), 5.088 1H, CHOCO), 4.517 J= 3.6 Hz, 1H, C-l), 4.058 3H), 3.665 3H), 3.279 3H, OCH 3 2.458 3H, CH3), 2.276 4H), 1.638 4H), 1.267 (bs, 34H) 0.864 12H, t-butyl terminal side chain methyl), 0.042 3H, SiMe), 0.038 3H, SiMe).

13C NMR: 174.402, 170.848, 146.180, 134.360, 131.103, 129.283, 100.006, 74.633, 72.905, 72.465, 70.911, 70.515, 56.'4724 54.594, 43.192, 35.829, 35.774, 33.205, 30.902, 30.838, 30.790, 30.624, 30.602, 30.482, 27.058, 26.440, 26.310, 23.964, 22.897, 19.371, 15.' 6, -2.688, -3.092.

IR: 3524, 3359, 2914, 2535, 2063, 1992, 1780, 1717, 1652, 1541, 1348, 1292, 1174, 1098, 1065, 1004, 978, 862, 835.

The above-obtained monotosylate compound (1.73 g, 2 mmol) was dissolved in DMF (10 mmol) and heated at 0 C in the presence of sodium azide (0.39 g, 6 mmol) for 6 h. After completion of the reaction, the mixture was transferred to a separatory funnel, diluted with ethyl acetate (20 mL) and washed with water (10 mL) and the organic layer was separated, dried, and concentrated to give an oil, which after purification by flash chromatography, afforded azide 36 as an oil (Rf 0.58, silica, ethyl acetate:hexane, 1:4, 1.15 g, 78%).

H NMR: 5.701 J=9.6 Hz, 1H, NH), 5.093 1H, CHOCO), 4.619 J=3.6 Hz, 1H, 4.138 1H), 3.63 5H), 3.372 3H, OCH 3 2.417 2H), 2.285 (t,

L

WO093/19761 PCT/US93/02903 63 J=7.6 Hz, 2H), 1.620 (in, 4H) 1.264 (bs, 34H) 0.874 (Mn, t-butyl and terminal side chain methyl), 0.056 (s, 3H, SiMe) 0.052 3H, SiMe) 1 3 C NMR: 174.470, 170.885, 100.118, 90.241, 74.834, 73.804, 72.969, 72.263, 56.535, 54.634, 52.959, 43.280, 35.853, 35.791, 33.219, 30.926f 30.855, 30.803f 30.643, 30.618, 30.501, 27.055, 26,457, 26.327, 23.980, 19.971, 15.396, -2.672, -2.993.

IR: 3567, 3434, 2920, 2852, 2252, 2100, 1718, 1654, 1466, 1259, 1125, 1063, 910, 839, 735.

Example 39 Preparation of Phosphorylated Compound 29 Phosphorylation of compound 36 was accomplished by the same procedure used to convert compound 1 to compound 17 (Example 19), to afford the correspond~ing phosphorylated compound 29 as an oil (Rf 0.54, sillica, ethyl acetate:hexane, 1:4, 82%).

1 H I4MR: 7.343 (in, 10 H, aromatic), 5.653 I'd, J=9.6 Hz, 1H, NH) 5. 07 (in, 5H, 2 x CH 2 0 and CHCOO) 4.608 (d, J=3.6 Hz, MH, 4.112 (mn, 2H), 3.802 (mn, 2H), 3.553 (mn, 2H) 3.367 3H, OCH 3 2.419 (mn, 2H), 2.287 (t, AJ=6 i6 Hz, 2H) 1. 63 0 (mn, 4H) 1 1. 261 (bs, 34H), 0.892 (t, J=6.2 Hz, 6H, side chain terminal CH 3 0.841 9H, tbutyl) 0. 084 3H, SiM.) 0. 064 3H, SiMe).

3 C WM: 174.465, 171.200i 136.394, 131.437, 130,980, 130.183, 130.103e 129.991, 129.961, 129.933t 129-795, 99.3()5, 73.067, 72.449, 71.497f 71.041, 66.439, 56.749, I54.7521, 52.616, 43.341, 35.827, 35.880, 33.257, 30.959, 30.889, 30.841, 30.534, 27.150, 26.483, 26.355, 24.030, 19.163, 15.163, 15.447, -2.523, -2.701.

IR: 2927, 2101, 1728, 1679, 1529, 1464, 1$380, 1257, 1135, 1016, 924, 858, 78o, 698, 675, 647.

Exami~le 40 Preparation of compound 37 The above-obtained phosphorylated compound 29 (1.1 g, 1.1. iniol) was dissolved mnethanol (3 inL) and dichloromethane (3 xnL) cooled to 00e. And stirred with a catalytic amount of PTSA (100 ing) for 2 h. After completion of the reaction, the acid was neutralized with WO 93/19761 PCr/US93/O29O3 64 triethylamine (1 mL) and the solvents were removed in vacuc to afford the crude hydroxy compound as an oil, which after purification by flash chromatography afforded the hydroxy compound 37 as an oil (Rf 0.46, silica, ethyl acetate:hexane, 1:1, 0.53 g, 59%).

1 H NMR: 7.343 (in, lOH, aromatic) 6.281 J=8.4 Hiz, 1H, NH), 5.067 (mn, 5H, 2 x OCH 2 Ph, and CHOCO) 4.717 (d, J=3.6 Hz, 1H, 4.185 (in, 2H), 3,851 (in, 2H), 3.361 3H, OCH 3 3.300 (in, 2H), 2.493 (in, 2H), 2.281 J=7.5 Hz, 2H), 1.608 (mn, 4 1. 2 47 (bs, 3 4H) 0. 8 77 J= 6. 6 H z, 6H, side chain _ermninal methyl).

1 3 C kflMR: 174.723, 171.801, 136.977, 136.889, 136.821, 130.046, 129.970, 129.897, 129.694, 129.464, 129.299, 99.374, 79.880, 79.249, 72.526, 72.363, 71.508, 71.431, 71.257, 71.187f 70.726, 70. 627, 69.910, 56.748, 54.912, 52.338, 42.953, 38.520, 35.869, 35.124, 33.246, 30.958, 30.875, 30.844, 30.705, 30.669, 30.533, 27.178, 26.614, 26.331, 24.015, 15.441.

IR: 34119, 3348, 2921, 2100, 1719, 1654, 1641, 1457, 1380, 1283, 1214, 1152, 1121f 998, 887, 698, 597, 486.

Example 41 Preparjtion of Coupled Compound 38 The coupling reaction of the above-obtained hydroxy compound 37 (0.42 g, 0.48 iniol) and phosphochioridate 19 (0.380 g, 0.672 iniol) was achieved by using the same procedure described fo~r the preparation of compound 27 from compound 26 (Example 29) to afford compound 38 (Rf 0.42 silica, ethyl acetate, hexane, 1:4, 0.37 g, 56%).

1 H NM: 7. 3 47 (in, 15H, aromatic) 7. 013 (m 1H, NH) 4.997 (mn, 8H, 3 x OCH 2 Ph and 2 x CHOCO), 4.710 J=3.6 Hz, 4. 169 (mn, 4.235 (in, 1H) 4. 039 (mn, 1H), 3.794 (in, 1H), 3.486 (mn, 1H) 3.352 3H, OCH 3 2.262 (mn, 8H), 1.535 (in, 8H), 1.26 (bs, 68H), 0.870 12H).

EXAMple 42 Preparation of Immunogen 3M A suspension of the above obtained coupled compound 38 (0.24 g, 0.17 iniol) in methanol (4 mL), was WO 93/19761

I:

PCU/US93/02903 hydrogenated using Pd-C 30 mg) for 4 h. After completion of the reaction, the catalyst was removed by filtration through a small pad of celite and the solvents were removed in vacuo to afford an oil. The oil was dissolved in chloroform (5 mL) and acidified with acetic acid (0.5 mL) and filtered through a small pad of celite to remove any solid particles. The solvents were removed in vacuo to give a thick oil. The oil was dissolved in acetonitrile (1 mL), and water (3 mL), sonicated (the mixture was still turbid) and lyophilized to give immunogen 3M as a colorless solid (140 mg, 78%).

1H NMR: 5.206 2H, CHOCO), 4.682 2H), 4.242 (m, 3.801 1H), 3.401 3H, OCH 3 2.842 2H), 2.400 6H), 1.807 8H), 1.26 68H), 0.892 (t, 12H, side chain terminal methyls).

Example 43 Preparation Of Compound 39 To a solution of compound 37 (1 eq), and acid 4 (1.2 eq) in dichlororomethane is added DCC (1.2 eq) and the resulting reaction mixture is stirred for 4 h. After completion of the reaction, by filtration the salts are separated. Removal of the solvent and flash chromatography affords compound 39.

Example 44 Preparation of Immunogen 2M Hydrogenation of compound 39 by a suspension of compound 39 and Pd-C in ethyl acetate, affords the immunogen 2M.

Example 45 Preparation of Compound 42 A suspension of compound 6a (4.45 g, 10 mmol) and Pd-C 0.5 g) in ethyl acetate (50 mL) was stirred under an atmosphere of hydrogen overnight. The progress of the reaction was monitored by TLC and showed the disappearance of the starting material and the formation of a new, more polar, compound. The catalyst was removed by filtration and the solvent was removed in vacuo to afford an oil, which, after flash chromatography, afforded the amine as a colorless crystalline solid (Rf 0.16, ethyl acetate:hexane 1:3, 3.98 g, WO 93/19761 PCT/US93/02903 66 m.p. 63-64-C.

1 H NMR: 4.869 2H, CHOAc), 4.41 J=7.8 Hz, 1H, anomeric), 4.075 2H, CH 2 0Ac), 3.610 1H), 2.74 (m, 1H), 1.96 6H, two acetates), 1.92 3H, acetate), 1.36 (bs, 2H, NH 2 0.824 9H, t-butyl), 0.0 6H, Si (CH 3 2) C NMR: 171.837, 171.767, 171.022, 100.343, 90.241, 76.622, 76.459, 73.197, 70.629, 70.463, 63.964, 58.986, 26.989, 22.040, 21.939, 19.275, -2.914, -3.877.

IR: 2954, 2862, 2777, 2743, 2482, 2127, 1939, 1771, 1717, 1594, 1473, 1386, 1331, 1282, 114D, 1017, 973.

To a solution of the amino compound (2.72 g, mmol) in dichloromethane (20 mL) and pyridine (12 mL) under an Ar atmosphere was added dropwise through a 1.ringe a solution of trichloroethyl chlorformate (1.60 g, 7.6 mmol) in dichloromethane (3 mL), and the mixture was stirred for 3 h. After completion of the reaction (TLC), the mixture was transferred to a separatory funnel, diluted with dichloromethane (40 mL), and washed successively with water (10 mL), sodium bicarbonate mL) and water (10 mL). The organic layer was separated, dried, and concentrated to give the crude product, which after purification by flash chromatography, afforded the carbamate 42 as a colorless solid (Rf 0.51, silica, ethyl acetate:hexane, 1:3, 3.55 g, 92%).

m.p. 170-1710C.

H NMR: 5.541 J= 9.3 Hz, 1H, NH), 5.227 J= 9.9 Hz, 1H, 4.980 J= 9.6 Hz, 1H, 4.776 J= 8.1Hz, 1H, 4.652 (AB, 2H, CH 2 of T, 7t) 4.146 2H, C-6), 3.675 2H, C-2 and 2.J3 3H, Acetate), 2.012 6H, two acetates), 0.839 9H, t-bu), 0.05 3H, FiMe), 0.03 3H, SiMe).

3 C NMR: 172.175, 171.951, 170.830, 155.488, 97.508, 75.921, 73.460, 73.092, 70.654, 63.927, 59.472, 32.982, 26.860, 23.950, 21.943, 21.975, 22.004, 19,242, 15.429, 2.962, -3.934.

i WO 93/19761 PCT/US93/02903 67 IR (KBr): 3291, 3171, 3087, 2959, 2932, 2531, 1752, 1747, 1708, 1473, 1464, 1435, 1370, 1230, 1191, 1141, 1108, 980, 957, 887.

Example 46 Preparation of compound 43 A solution of compound 42 (3.56 g, 6 mmol) in methanol (25 mL) and sodium methoxide in methanol (1M, 0.2 mL) was stirred at room temperature for 1 h. After completion of the reaction, the base was neutralized with acetic acid (1 mL) and the solvents were removed in vacuo. The crude product was purified by flash chromatography to afford the corresponding trihydroxy compound as a colorless crystalline solid (Rf 0.45, silica, ethyl acetate, 2.42 g, m.p. 136-137°C 1 H NMR (CD30D): 4.657 2H, CH 2 of Troc), 4.595 (d, J=7.5 Hz, 1H, 3.790 2H), 3.349 4H), 0.840 9H, t-butyl), 0.031 3H, SiMe), 0.020 3H, SiMe).

13 C NMR (CD 3 0D): 156.828, 97.823, 79.208, 79.107, 77.464, 75.930, 75.430, 72.144, 62.897, 61.030, 26.483, 26.442, 18.954, -3.605, -4.721.

IR (KBr): 3365, 2957, 2887, 2805, 2712, 2045, 1718, 1545, 1362, 1255, 1175, 1083, 949, 785.

A solution of the above-obtained trihydroxy compound (2.33 g, 5 mmol), dichloromethane (20 mL), PTSA (200 mg), and 2,2-dimethoxy propane (1.56 g, 15 mmol) was stirred under an argon atmosphere for 4 h. After completion of the reaction (TLC), the acid was neutralized with triethylamine (1 mL) and the mixture was transferred to a separatory funnel. The mixture was diluted with dichloromethane (40 mL) and washed with water (10 mL). The organic layer was separated, dried (MgS0 4 and the product was purified by flash chromatography to afford compound 43 as an oil. (Rf 0.46, silica, ethyl acetate:hexane, 1:4, 2.12 g, m.p. 90-91°C.

1 H NMR: 5.711 J=7.5 Hz, 1H, NHCO), 4.824 J=6.9 WO093/19761 PCT/US93/02903 68 Hz, 1H, C-i) 4.715, (AB, 2H, CH 2 pf Troc), 3.874 (mn, 2H), 3.771 J=10.2 Hz, 1H), 3.578 J=9.2 Hz, 1H), 3.496 (bs, 1H, OH) 3.22 (mn, 2H) 1.496 3H, CH 3 of acetonide), 1.415 3H, CH 3 of acetonide), 0.861 (s, 9H, t- bu) 0. 086 3H, SiGH 3 0.069 3H, SiCH 3 1CNMR: 155. 904, 110.996, 101.176, 97.499, 96.654, 76.134, 75.670, 72.318, 68.507, 63.400, 62.285, 30.435, 26.958, 20.520, 19.255, -2.870, -3.874.

IR (KBr): 3094, 2996, 2193, 1987, 1718, 1560, 1474, 1382, 1288, 1200, 1166, 1095, 986, 842, 735.

Example 47 Preparation of Compound 44 Under an Ar atmosphere, to a solution of compound 43 (2.02 g, 4 inmol) in dichioromethane (20 inL) containing DMAP (0.488 g, 4 minol) and triethylamine (1.10 g) was added (through a syringe) over a period of min, a solution of phosphochloridate 19 (2.70 g, 5 minol) in dichioroinethane (5 inL) After completion of the reaction (TLC) the contents were transferred to a separatory funnel and washed with water (10 mL). The organic layer was separated, dried and concentrated to give an oil. The product was purified by flash chromatography to afford compound 44 as an oily compound (Rf 0.61 silica, ethyl acetate:hexane 1:3, 2.90 g. 72%).

1 H NMR (CDCl 3 7.299 (mn, 5H, aromatic), 6.979 lH, NH), 5.190 (1H, CHCO), 5.00 (in, 2H, OCH 2 Ph), 4.775 (in, 1H, C-1, anoineric), 4.605 (mn, 1H), 4.431 (AB, Cr1 2 of Troc), 3.916-3.796 (mn, 4H), 3.513 (mn, lH), 2.165 (in, 4H,

CH

2 CO,and CH 2 1.548 3H, CH 3 acetonide), 1.423 (s, 3H, CH 3 of acetonide), 1.26 (in, 28H, CH 2 0.860 (in, side chain terminal Cr1 3 and t-bu Si), 0.003 6H, SiMe).

13 C NMR: 174.00, 155.943, 137.255, 129.898, 129.137, 100.932, 98.674, 96.467, 76.050, 74.144, 69. 842, 67.722, 67.409, 63.252, 60.571, 35.675, 30.984, 26.878, 24.040, 20.498, 20.436, 19.161, 15.469, -2.863, -3.778.

IR: 2926, 1718, 1671, 1520, 1430, 1354, 1214, 1156, 1021, 908, 864, 740.

~C

WO 93/19761 PC/US93/02903 69 Example 48 Preparation of Imidate A solution of compound 44 (1.61 g, 1.6 mmo!) in dichloromethane (3 mL) was stirred with tetrabutylammonium fluoride (1 M solution in THF, 2.6 ml) for 4h. After completion of the reaction (TLC) trichloroacetonitrile (3 mL) was added and the solution stirred for additional 4 h. After the intermediate hydroxy compound was nearly consumed (TLC), the solvents were removed in vacuo and the residue was purified by flash chromatography to afford the imidate as an anomeric mixture.

a Imidate (Rf, 0.52, silica, ethylacetate:hexane 1:3, 0.583 g, 34%) 1H NMR: 8.746 1H, NH of imidate), 7.289 aromatic), 6.489 J=3.9 Hz, 1H, 6.234 J=7.8 Hz, 1H, NH of Troc), 5.064 3H, OCH 2 Ph and CHOCO), 4.

571 3H, CH 2 of Troc and 1H), 4.173 1H), 3.835 (m, 4H), 2.199 4H), 1.422 3H, Methyl of acetonide), 1.391 3H, methyl of acetonide), 1.26 (bs, 34H), 0.824 6H).

p Imidate (Rf 0.49, silica, ethyl acetate:hexane, 1:3, 0.59 g, 34%).

1 H NMR: 8.721 1H, NH of imidate), 7.361 aromatic), 6.501 1H, NH), 6.392 J=7.5 Hz, 1H, C- 5.018 3H, OCH 2 Ph and CHOCO), 4.512 3H), 4.186 1H), 3.993 4H), 2.241 m, 4H), 1.462 3H, methyl of acetonide), 1.392 3H, methyls of acetonide), 1.201 (bs, 34H), 0.910 6H).

Example 49 Preparation of Benzyloxy-l-tertbutyldimethylsiloxytetradecane Trifluoromethanesulphonic acid (0.14 mL, 1.6 mmol) was added to a mixture of alcohol 10 (6.9 g, mmol) and benzyl trichloroacetimidate (12.3 g, 49 mmol) in a 1:1 mixture of dichloromethane and cyclohexane (320 mL) at room temperature. After completion of the reaction (5 h) a saturated solution of sodium bicarbonate mL) was added and the phases were separated. The 55~- I~ 1111)11~- WO93/19761 PCI/US93/02903 aqueous phase was extracted with ethyl acetate (2 x 50 mL) and the organic phases were washed with brine mL), dried (MgS0 4 and concentrated in vacuo. The product was purified by flash chromatography to afford compound 45 as an oil (Rf 0.18, silica, 2% ethyl acetate in hexane, 7.7 g, 88%).

1 H NMR: 7.49-7.23 5H, aromatic), 4.60 (AB, 2H, benzylic), 3.85-3.70 2H), 3.67-3.55 1H), 1.90- 1.77 2H), 1.67-1.55 2H), 1.45-1.35 18H), 0.97 12H), 0.11 6H).

13 C NMR: 139.25, 128.19, 127.64, 127.26, 76.25, 71.02, 59.89, 37.52, 34.21, 31.89, 29.78, 29.60, 29.30, 25.94, 25.27, 22.63, 18.23, 14.00, -5.33.

IR (Neat): 3089, 3066, 3031, 2927, 2855, 1497, 1464, 1388, 1361, 1255, 1224, 1097, 1029, 1006, 939, 836, 776, 733, 697, 682, 666.

Example 50 Preparation of (R)-3-Benzyloxy-ltetradecanol (46) A solution of aqueous HF 12 mL) was added to a mixture of compound 45 (6.3 g, 14.5 mmol) in acetonitrile (120 mL) and the mixture was stirred for min. After completion of the reaction, the acid was neutralized with saturated sodium bicarbonate solution and extracted with ethyl acetate (3 x 150 mL) and the organic phase was separated, dried, concentrated, and the product was purified by flash chromatography to afford compound 46 as colorless oil (Rf 0.42, 25% ethyl acetate in hexane, 3.4 g, 74%).

1H NMR: 7.43-7.25 5H), 4.60 (AB, 25H), 3.90-3.73 (m, 2H), 3.70-3. 63 1H), 1.92-1.47 4H), 1.42-1.23 18H), 0.93 3H).

13C NMR: 138.44, 128.42, 127.81, 127.67, 78.55, 70.92, 60.74, 35.92, 33.44, 31.91, 29.81, 29.60, 29.34, 25.13, 22.67, 14.10.

Example 51 Preparation of Benzyloxytetradecanoic acid (47) Jones reagent was added in portions to a mixture of alcohol 46 (3.23 g, 10.1 mmol) in acetone WO 93/19761 PCT/US93/02903 71 mL) at 0°C until the alcohol was consumed. The mixture was partitioned between 0.1 M HCl (100 mL) and ethyl acetate (400 mL), and the organic phases were washed with brine (100 mL), dried, and concentrated in vacuo. The product was purified by flash chromatography to give compound 47 as an oil (Rf 0.38, silica, 25% ethyl acetate in hexane, 2.09 g, 62%).

H NMR: 7.42-7.26 5H), 4.61 (bs, 2H), 3.91 1H), 2.63 (ddd, 2H), 1.76-1.51 2H), 1.46-1.24 0.93 3H).

13 C NMR: 177.29, 138.16, 128.36, 127.83, 127.67, 75.74, 71.54, 39.56, 34.18, 31.91, 29.63, 29.57, 25.12, 22.69, 14.11.

IR (neat): 3400-2400 (broad), 3065, 3032, 2926, 2855, 1713, 1497, 1377, 1353, 1295, 1240, 1207, 1097, 1070, 1029, 939.

Example 52 Preparation of Compound 48 A solution of amino compound 30 (2.35 g, mmol), compound 47 (3.34 g, 10 mmol), EDC (1.91 g, mmol), and HOBT (1.35 g, 10 mmol) in dry dichloromethane mL) was stirred at room temperature under an argon atmosphere for 4 h. After completion of the reaction the solvents were removed in vacuo, and the resulting material was purified by flash chromatography to afford the coupled compound as an oil (Rf 0.31, silica, ethyl acetate:hexane, 1:1, 4.93 g, 1 H NMR: 7.321 5H, aromatic), 4.512 3H, benzylic and 3.814 7H) 3.194 3H, OCH 3 2.421 2H), 1.546 3H, methyl of acetonide), 1.486 3H, methyl of acetonide), 1.214 (bs, 20H), 0.894 3H, terminal side chain methyl).

To the above-obtained compound (4.40 g, 8 mmol) and compound 47 (3.34 g, 10 mmol) in dichloromethane mL) sequentially added was DCC (2.06 g, 10 mmol) and DMAP (1.22 g, 10 mmol) and the mixture was stirred at room temperature under an argon atmosphere for 4 h. After L i WO 93/19761 PCT/US93/02903 72 completion of the reaction, the insoluble material was removed by filtration and the solvent was evaporated in vacuo to afford an oil. The crude product was purified by flash chromatography to afford compound 48 as an oil (Rf 0.60, silica, ethyl acetate:hexane 3:7, 6.25 g, 1 H NMR: 7.362 10H, aromatic), 6.312 J=7.6 Hz, 1H, NH), 5.200 J=7.6 Hz, 1H, CHCOO), 4.562 6H), 3.812 6H), 3.189 3H, OCH 3 2.741-2.346 4H), 1.456 3H, acetonide methyl), 1.312 acetonide I 10 methyl), 1.216 (bs, 40H), 0.816 J=6.3 Hz, 6H).

Ij Example 53 Preparation of Compound 41 A cooled solution of compound 48 (4.30 g, i mmol) in dichloromethane (20 mL), and trifluoroacetic acid (6 mL) was stirred at 0°C for 2 h. After completion of the reaction the acid was neutralized with I triethylamine (6 mL), the solvent was evaporated, and the I residue was purified by flash chromatography to afford the diol compound 48 as an oil (Rf 0.24, silica, ethyl iacetate:hexane, 1:1, 3.55 g, 86%).

1 H NMR: 7.360 10H, aromatic), 6.346 J=7.5 Hz, 1H, NH), 5.226 1H), 4.612 J=3 Hz, 1H, 4.502 I 4H), 3.724 5H), 3.191 3H, OCH 3 2.464 (m, 4H), 1.264 (bs, 40H), 0.826 J= 6.6 Hz, 6H).

13C NMR: 172.449, 172.077, 138.329, 137.902, 128.810, 128.313, 127.551, 127.395, 98.050, 97.975, 71.274, 70.412, 69.926, 69.544, 62.426, 54.911, 51.941, 50.813, 41.275, 33.951, 31.873, 29.598, 29.309, 25.407, 25.306, 21.643, 13.911, 13.778.

IR: 3324, 2952, 1728, 1674, 1499, 1168, 965, 834.

Example 54 Preparation of Compound 49 Under an Ar atmosphere, to a cooled solution 78 0 C) of imidate 40 (either a or 8, 0.685 g, 0.63 mmol) and compound 41 (0.570 g, 0.689 mmol) in dichloromethane mL) was added (through a syringe) a solution of boron trifluoride etherate (0.1 mL) in dichloromethane (1 mL).

The reaction mixture was stirred at -78 0 C for 2 h and then at -20 0 C for 12 h. After completion of the i i WO 93/19761 PCT/US93/02903 reaction, triethylamine (0.3 mL), was added, the mixture was transferred to a separatory funnel, diluted with dichloromethane (5mL), and washed with sodium bicarbonate 5 mL). The organic layer was separated, dried, and concentrated to give an oil, which was subjected to flash chromatography to afford compound 49 as an oil (Rf 0.54, silica, ethyl acetate:hexane 1:1, 0.75 g, 68%).

1 H NMR: 7.332 15H, aromatic), 6.521 (bs, 1H, NH of Troc), 6.224 J=6.6 Hz, 1H, NH), 5.082 4H), 4.556 7H), 3.874 12H), 3.242 3H, OCH 3 2.462 (m, 8H), 1.542 3H, methyl of acetonide), 1.464 3H, methyls of acetonide), 1,246 (bs, 80 0.846 12H, terminal methyls).

1C NMR: 174.485, 174.414, 173.967, 172.385, 156.138, 156.171, 139.830, 139.729, 137.065, 136.946, 129.925, 129.701, 129.653, 129.187, 128.894, 109.246, 105.421, 100.724, 96.246, 78.610, 72.889, 72.354, 70.246, 67.424, 63.246, 43.246, 41.208, 35.481, 33.290, 31.012, 30.729, 30.513, 26.585, 26.501, 26.240, 24.061, 21.142, 15.492.

IR: 3276, 2996, 1735, 1670, 1499, 1379, 1178, 1095, 941, 821.

Example 55 Preparation of Compound A solution of compound 49 (0.84 g, 0.48 mmol), imidazole (0.068 g, 1 mmol) and t-butyldimethylsilyl chloride (0.135 g, 0.9 mmol) in dry DMF (3 mL) was stirred at 50 0 C for 12 h. After completion of the reaction, the mixture was diluted with ethyl acetate mL) and washed with water (5 mL). The organic layer was separated, dried and concentrated to give an oil. The oil was purified to give the silyl compound as an oil (Rf 0.42, silica, ethyl acetate:hexane 1:3, 0.69 g, 78%).

1H NMR: 7.312 5H, aromatic), 6.541 (bs, 1H, NH of troc), 6.194 J=6.3 Hz, 1H, NH), 5.014 4H), 4.524 7H), 4.184 2H), 3.746 12H), 3.186 3H,

OCH

3 2.462 8H), 1.542 3H, methyl of acetonide), 1.462 3H, methyls of acetonide), 1.246 (bs, L .I i WO93/19761 PCT/US93/02903 74 0.884 12H, terminal side chain methyls), 0.812 (s, 9H, t-butyl), 0.04 3H, SiMe), 0.032 3H, SiMe).

The above-obtained silyl compound (1.05 gm, 0.57 mmol) was dissolved in dichloromethane (5 mL) and cooled to 0°C and trifluoroacetic acid (1.5 mL) was added. The resulting solution was stirred at that temperature for 3 h. After completion of the reaction (TLC) the acid was neutralized with triethylamine (2 mL), and the solvents were removed in vacuo. The diol compound 50 was purified by flash chromatography (Rf 0.42, silica, ethyl acetate:hexane 1:1, 0.84 g, 81%).

IH NMR: 7.294 15H, aromatic), 6.192 J=6.3 Hz, 1H, NH), 5.120 4H), 4.542 7H), 4.169 2H), 3.642 12H), 3.184 3H, OCH3), 2.824-2.246 8H), 1.246 (bs, 80H), 0.892 32H), 0.812 9H, t-butyl), 0.040 3H, SiMe), 0.036 3H, SiMe).

Example 56 Preparation of Compound 51 To diol 50 (0.82 g, 0.45 mmol) in DMF (4 mL) at 0 C, was added sequentially imidazole (0.068 g, 0.1 mmol) and t-butyldimethylsilyl chloride (75 mg, 0.5 mmol) and the resulting solution was stirred at that temperature for 2 h. After completion of the reaction, the mixture was diluted with ethyl acetate (10 mL), and washed with water (2 x 5 mL). The organic layer was separated, dried, and concentrated to give an oil, which after purification by flash chromatography, afforded compound 51 as an oil (Rf 0.41, ethyl acetate:hexane, 1:3, 0.69 g, I NMR: 7.310 15H, aromatic), 6.146 J=6.3 Hz, 1H, NH), 5.146 4H), 4.746 7H), 4.184 2H), 3.746 12H), 3.178 3H, OCH 3 2.476 8H), 1.246 (bs, 0.876 30H), 0.040 12H).

Example 57 Preparation of Compound 52 A suspension of compound 51 (240 mg, 0.12 mmol) in aqueous THF 2 mL) and zinc dust (300 mg) was stirred at room temperature. Then acetic acid (0.5 mL) was added to the reaction and the mixture was stirred at

L

WO93/19761 PCT/US93/02903 room temperature for 4 h. After completion of the reaction (TLC, silica, ethyl-acetate:hexane, the inorganic salts were removed by filtration and washed with chloroform (10 mL). The organic layer was neutralized with triethylamine (1 mL), diluted with ethyl acetate (5mL), washed with water (5 mL) dried, concentrated, and purified by flash chromatography to afford amino compound 52 (diastereomers were separated).

Product A (Rf 0.40, silica, ethyl acetate:hexane 1:1, 100 mg, 48%): 1 H NMR: 7.324 15H, aromatic), 6.246 J=6. 3 Hz, 1H, NH), 5.124 4H), 4.674 7H), 4.14-3.64 (m, {i 12H), 3.124 3H, OCH 3 2.468-2.124 8H), 1.246 (bs, 80H), 0.892 20H), 0.04 6H).

Product B (Rf 0.14, silica, ethyl acetate:hexane 1:1, 64 mg,

S

1 H NMR: 7.294 5H, aromatic), 6.204 J=6.3 Hz, 1 H, 1 NH), 5.184 4H), 4.584 (in, 5H), 4.241 3H), 3.786 i 11H), 3.126 3H, OCH 3 2.942 1H) 2.642-2.242 i 20 8H), 1.296 (bs, 80H), 0.896 30H), 0.04 12H).

Example 58 Preparation of Compound 53.

A solution of amino compound 52 (340 mg, 0.195 mmol, product A from Example 57), acid 3 (90 mg, 0.211 i mmol), EDC (45 mg, 0.2 mmol), and HOBT (27 mg, 0.2 mmol) in dichloromethane (2 mL) was stirred at room temperature ifor overnight. After completion of the reaction, the solvent was removed in vacuo and purification by flash chromatography afforded compound 53 as an oil (diasteromeric mixture was separated).

i 30 Product 53a (Rf 0.62 silica, ethyl acetate:hexane, 2:3, 105 mg, 1H NMR: 7.346 15H, aromatic), 6.196 2H, NH), 5.196 5H), 4.562 5H), 4.21-3.624 12H), 1.296 (bs, 120H), 0.918 36H), 0.04 12H).

Product 53b (Rf 0.41, silica, ethyl acetate: hexane, 2:3, 140 mg, 33%): 1 H NMR: 7.304 15H, aromatic), 6.346 1H, NH),

LC-FII~Y

WO 93/19761 PCT/US93/02903 76 6.192 1H, NH), 5.210 5H), 4.612 5H), 4.201- 3.562 14H), 3.192 3H, OCH 3 2.746-2.196 (m, 12H), 1.286 (bs, 120H), 0.942 36H), 0.04 12H).

Example 59 Preparation Of Compound 54 To a solution of compound 53 (72 mg, 0.033 mmol) and N,N-diisopropylamino dibenzyl phosphite (17 mg, eq) in dichloromethane (1.0 mL) was added tetrazole (1.4 mg, 2 eq) and the mixture was stirred at room temperature for 2 h. After the alcohol was consumed (TLC), the reaction mixture was cooled to 0°C, m-CPBA (2 eq) was added, and the mixture was stirred for 1 h.

After completion of the reaction, the mixture was diluted with dichloromethane (5 mL) and washed with sodium bicarbonate 5 mL). The organic layer was separated, dried, and concentrated to give an oil, which was purified by flash chromatography to afford compound 54 as an oil (Rf 0.28, silica, ethyl acetate:hexane, 3:7, 55 mg, 69%).

1 H NMR: 7.346 25H, aromatic), 6.246 2H, NH), 5.146 9H), 4.521 5H), 4.124-3.426 14H), 3.102 3H, OCH 3 2.746-2.194 12H), 1.286 (bs, 120H), 0.942 36H), 0.036 12H).

Example 60 Preparation of Compound A suspension of compound 54 (39 mg, 0.016 mmol) and Pd-C 6 mg) in ethyl acetate was stirred under a hydrogen atmosphere at room temperature for 3 h. After completion of the reaction, the catalyst was removed by filtration, and the solvent was evaporated to afford compound 55 as an oil (28 mg, 92%).

1 H NMR (CDC13 TFA 5.246 3H), 4.624 4H), 3,942 11H), 2.412 12H), 1.294 (bs, 120H), 0.896 Example 61 Preparation Of Immunogen 3D To a solution of compound 55 (24 mg, 0. 012 mmol) in THF (1 mL) cooled to -200C was added HF-pyridine (0.2 mL) and the mixture was stirred under argon atmosphere for 1 h at -20 0 C and then at 0 C for 1 h.

w0 i IFII~DLI3~1 i93/19761 PCT/US93/02903 After completion of the reaction, the reaction mixture was diluted with chloroform (5 mL), and stirred with sodium bicarbonate ImL). The organic layer was separated, the aqueous layer was extracted with chloroform, and the combined organic phases were dried and concentrated to give immunogen 3D as an oil. The compound was dissolved in acetonitrile (2 mL) and water (6 mL) and sonicated to give an almost homogeneous solution, which after lyophilization, afforded the immunogen 3D as a colorless powder (18 mg, 86%).

Example 62 Preparation of Compound 57 A solution of hydroxy compound 43 (1 eq), acid 3 (1.1 eq), DCC (1.1 eq) and DMAP (1.1 eq) in dichloromethane (0.2 M) is stirred at room temperature for 4 h. After completion of the reaction, the insoluble material is filtered off, and removal of the solvent and flash chromatography affords compound 57.

Example 63 Preparation Compounds 58 through Immunogen

ID

Preparation of all these compounds is accomplished by following similar procedures to those set forth above to prepare compounds 49 through immunogen 3D (Example 54 to 61), in particular: Preparation Compound 56: To a solution of compound 57 in THF and dichloromethane 0.2 M) under an Ar atmosphere, a solution of tetrabutylammonium fluoride (1 eq) is added and the resulting mixture is stirred at room temperature until the starting material (compound 57) disappears, then trichloroacetonitrile (4 eq) is added and the stirring is continued for additional 4 hr. After completion of the reaction solvents, are removed in vacuo and flash chromatography affords the compound 56 as an anomeric mixture.

Preparation Of Compound 58: To a solution of imidate compound 56 (1 eq) and compound 41 (1.2 eq) in dichloromethane (0.2 M) at -78 0 C under an Ar atmosphere a solution of borontrifluoride etherate (catalytic amount) in dichloromethane is added and the resulting mixture is i i 1

F

I

O 9 *--L*l*ii 3/19761 PCT/US93/02903 stirred initially at -780C, then at -200C for 12 hr.

After completion of the reaction (TLC), the mixture is neutralized with triethylamine, and solvents are removed in vacuo and flash chromatography affords compound 58.

Preparation of Compound 59: To a solution of compound 58 (1 eq) in dry DMF (0.2 M) at room temperature sequentially added are: imidazole (2.4 eq) and ter- butyldimethylsilyl chloride (1.2 eq); and the resulting mixture is stirred at that temperature under an Ar atmosphere until the starting material disappears (TLC). After completion of the reaction the mixture is transferred into separatory funnel and diluted with ethyl acetate and washed with water. The organic phase is separated, dried and concentrated, and flash chromatography affords the silylated compound. To the silyl compound (1 eq) in dichloromethane (0.2 M) a solution of trifluoroacetic acid (2 eq) is added and the resulting mixture is stirred at 0°C until the starting material disappears (TLC). After completion of the reaction acid is neutralized with triethylamine at 0 C and solvents are removed in vacuo and flash chromatography affords the compound 59.

Preparation of Compound 60: To a solution of compound 59 (1 eq) in dry DMF (0.2 M) is added sequentially imidazole (2.4 eq) and ter butyldimethylsilyl chloride (1.2 eq) at 0 °C and the resulting mixture is stirred at that temperature until starting material disappears. After completion of the reaction the mixture is transferred into a separatory funnel, diluted with ethyl acetate and washed with water; the organic phase is separated, dried and solvents are removed in vacuo and flash chromatography affords the monosilylated compound. A suspension of the silylated (1 eq) compound in THF (0.2 acetic acid and zinc powder is stirred at room temperature until starting material disappears. After completion of the reaction, the inorganic salts are filtered off and the solvents are

I

WO 93/19761 PCT/US93/02903 I i removed in vacuo. Purification by flash chromatography affords the compound Preparation of Compound 61: A solution of compound 60 (1 eq), acid 4 (1.2 eq) EDC (1.2 eq) and HOBT (1.2 eq) is stirred in dichloromethane (0.2 M) under an Ar atmosphere until the starting material disappears.

After completion of the reaction, the solution is transferred into a separating funnel and washed with water. The organic phase is separated, dried and concentrated, and flash chromatography affords the compound 61.

Preparation of Compound 62: A solution of compound 61 (1 eq), N,N- diisopropylamino dibenzylphosphite (1.2 eq) in dichloromethane (0.2 M) is stirred under an Ar atmosphere in the presence of tetrazole. After completion of the reaction (TLC), the mixture is cooled to 0°C and m-CPBA (1.4 eq) is added and the mixture is stirred further until the starting material has disappeared (TLC). After completion of the reaction solvents are removed in vacuo and the product 62 is purified by flash chromatography.

Preparation Of Immunogen ID: A suspension of compound 62 (1 eq) in ethyl acetate in the presence of Pd-C is hydrogenated by using a hydrogen balloon.

After completion of the reaction, catalyst is filtered through a pad of celite and removal of the solvent affords the hydrogenated compound. The hydrogenated compound (1 eq) is dissolved in THF in a plastic container, cooled to 0 C and HF-pyridine is added. The resulting mixture is stirred at 0°C for 0.5 hr. After completion of the reaction sodium bicarbonate solution is added and the organic layer is separated, dried and concentrated to affords immunogen ID.

Example 64 Preparation of Compound 63 To a cooled solution of compound 43 (1 eq) in dry THF, sodium hydride (60% suspension in mineral oil, 1.1 eq) is added and the resulting suspension is stirred WO93/19761 PCT/US93/02903 under an argon atmosphere for 1 h. Then 4-methoxybenzyl chloride (1.4 eq) is added to the reaction mixture and the stirring continued for 2 h. After completion of the reaction (TLC), the excess base is neutralized with ammonium chloride solution, and the organic layer is separated, dried, concentrated, and purified by flash chromatography to afford compound 63.

Example 65 Preparation of Imidate Compound 62a To a solution of compound 63 (1 eq) in dichloromethane, tetrabutylammonium fluoride (1 M in THF, 1.1 eq) is added and the reaction mixture is stirred under an argon atmosphere until the starting material (Compound 63) disappears (TLC). Then trichloroacetonitrile (3 eq) is added to the reaction mixture and stirring is continued for an additional 4 h.

After completion of the reaction, the solvents are removed and flash chromatography affords the imidates as an anomeric mixture.

Example 66 Preparation of Compound 64 To a cooled solution (-20 0 C) of imidate compound 62a (1 eq) and diol compound 41 (1 eq) in dichloromethane is added a catalytic amount of a Lewis acid (boron trifluoride etherate or TMS-Triflate) and the mixture is stirred under an argon atmosphere at that temperature overnight. After completion of the reaction, the acid is neutralized with ammonium chloride solution, the organic layer is separated, doied, concentrated, and purified by flash chromatography to afford the coupled compound 64.

Example 67 Preparation of Compound To a solution of compound (4 (1 eq) in dry DMF (0.2M) is added sequentially imidazole (2.4 eq) and tbutyldimethylsilyl chloride (1.2 eq) and the mixture is stirred at room temperature under argon for 6 h. After completion of the reaction, the reaction mixture is diluted with chloroform and washed with water and the organic phase is separated, dried, concentrated and

I

i- 1~ L WO 93/19761 PCT/ US93/02903 81 purified by flash chromatography to afford the silylated compound.

Trifluoroacetic acid is added to a solution of the silylated compound in dichloromethane at 0 C and the mixture is stirred for 2 h. After completion of the reaction, the acid is neutralized with triethylamine, the solvents are removed and the diol is purified by flash chromatography.

To a cold solution (0 C) of the diol (1 eq) in dry DMF under an argon atmosphere is added sequentially imidazole (2.2 eq) and t-butyldimethylsilyl chloride (1.1 eq), and the mixture is stirred at that temperature for 2 h. After completion of the reaction, the mixture is diluted with dichloromethane and washed with water, the organic layer is separated, dried, concentrated and purified by flash chromatography to afford the compound Example 68 Preparation of compound 66 Zinc powder is added to a solution of compound 65 (1 eq) in THF and acetic acid, and the mixture is stirred until the starting material (compound disappears (TLC). After completion of the reaction, the salts are removed by filtration, the filtrate is neutralized with triethylamine and the solvent is removed. The amino compound is purified by flash chromatography. To a solution of the amino compound (1 eq) and acid 3 (1.1 eq) in dichloromethane is added sequentially EDC (1.1 eq) and HOBT (1.1 eq) and the mixture is stirred overnight. After completion of the reaction, the mixture is diluted with dichloromethane, and washed with water, and the organic layer is separated, dried, concentrated, and the product 66 is purified by flash chromatography.

Example 69 Preparation of Compound 67 A mixture of compound 66 (1 eq), N,Ndiisopropylamino dibenzyl phosphite (1.2 eq) and tetrazole (1.4 eq) in dichloromethane is stirred for 2 h L WO 93/19761 PCT/US93/02903 under an argon atmosphere. After completion of the phosphitylation reaction (TLC), the mixture is cooled to 0°C and m-CPBA (1.3 eq) is added and stirring is continued for an additional 1 h. After completion of the oxidation reaction, the mixture is diluted with dichloromethane, and washed with sodium bicarbonate solution The organic layer is separated, dried, and concentrated, and the product is purified by flash chromatography.

To a solution of the phosphorylated compound (1 eq) in acetonitrile, is added DDQ (1.5 eq) and the mixture is stirred for 1 h. After completion of the reaction, an aqueous workup followed by flash chromatography affords the compound 67.

Example 70 Preparation of Compound 68 To a solution of compound 67 (1 eq) and acid 4 (1.2 eq) in dichloromethane is added sequentially DCC (1.2 eq) and DMAP (1.2 eq) and the mixture is stirred for 6 h. After completion of the reaction, the insoluble material is filtered off, the filtrate is concentrated, and the product, compound 68, is purified by flash chromatography.

Example 71 Preparation of Immunogen 2D Conversion of compound 68 to immunogen 2D is accomplished by conditions similar to those set forth above for the conversion of compound 62 to immunogen ID.

In particular, a suspension of compound 68 (1 eq) in ethyl acetate is hydrogenated by using a hydrogen balloon in the presence of Pd-C After completion of the reaction catalyst is filtered through a pad of celite and removal of the solvent affords the hydrogenated compound. The hydrogenated compound (1 eq) is dissolved in dry THF (0.2 M) in a plastic container and HF-pyridine is added and the mixture is stirred under an L WO 93/19761 PCT/US93/02903 83 Ar atmosphere at 0OC. After completion of the reaction a solution of sodium bicarbonate is added and with stirring. The organic layer is separated and dried, and removal of the solvent affords Immunogen 2D.

Example 72 Preparation of Immunogens 3D-A, 2D-A and 1D-A A solution of compound 54 in methanol and a catalytic amount of PTSA is stirred at 0°C until the starting material disappears (TLC). After completion of the reaction, acid is neutralized with triethylamine and removal of the solvents and purification by flash chromatography affords the hydroxy compound.

To a solution of the above hydroxy compound (1 eq) and triethylamine (2eq) in dichloromethane, methanesulphonic chloride is added at 0 C and the mixture is stirred in an argon atmosphere for 1 h. After completion of the reaction, solvents are removed in vacuo and the mesylate reaction product is used in the next reaction without the need for further purification.

A solution of the mesylate compound (reaction product above) (1 eq) and sodium azide (3 eq) in dry DMF is heated at 50 0 C in an argon atmosphere for 4 h. After completion of the reaction the mixture is diluted with dichloromethane and washed with water, the organic phase is separated, dried, concentrated, and purified by flash chromatography to afford the azide.

A suspension of the azide and Pd-C in ethyl acetate is stirred under an hydrogen atmosphere for 4 h. After completion of the reaction, the catalyst is filtered, and removal of the solvent affords the corresponding amino compound, which on treatment with HFpyridine, affords immunogen 3D-A.

Preparation of Immunogens 1D-A: A solution of compound 62 in methanol and a catalytic amount of PTSA is stirred at 0°C until the starting material disappears (TLC). After completion of the reaction, acid is neutralized with triethylamine and removal the solvents and purification by flash chromatography affords the W093/19761 PCT/US93/02903 84 hydroxy compound. To a solution of the hydroxy compound (1 eq) and triethylamine (2 eq) in dichloromethane, a solution of methanesulphonic chloride is added at 0 C and the mixture is stirred in an argon atmosphere for 1 h.

After completion of the reaction, solvents are removed in vacuo and the mesylate compound used for next reaction without purification. A solution of the mesylate compound (1 eq) and sodium azide (3 eq) in dry DMF is heated at 50 0 C in an argon atmosphere for 4 h. After completion of the reaction the mixture is diluted with dichloromethane and washed with water. The organic phase is separated, dried and concentrated, and the azide is purified by flash chromatography. A suspension of the azide and Pd-C in ethyl acetate is stirred under a hydrogen atmosphere for 4 h. After completion of the reaction the catalyst is filtered and removal of the solvent affords the corresponding amino compound, which on treatment with HF-pyridine affords the immunogen ID-A.

Preparation of Immunogens 2D-A: A solution of compound 68 in methanol and a catalytic amount of PTSA is stirred at 0 C until the starting material disappears (TLC). After completion of the reaction, acid is neutralized with triethylamine and removal the solvents and purification by flash chromatography affords the hydroxy compound. To a solution of the hydroxy compound (1 eq) and triethylamine (2 eq) in dichloromethane, a solution of methanesulphonic chloride is added at 0°C and the mixture is stirred in an argon atmosphere for 1 h. After completion of the reaction, solvents are removed in vacuo and the mesylate compound is used for next reaction without purification.

A solution of mesylate compound (1 eq) and sodium azide (3 eq) in dry DMF is heated at 50 0 C in argon atmosphere for 4 h. After completion of the reaction the mixture is diluted with dichloromethane and washed with water. The organic phase is separated, dried and concentrated and the azide is purified by flash chromatography. A WO 93/19761 PCT/US93/02903 suspension of the azide and Pd-C in ethyl acetate is stirred under a hydrogen atmosphere for 4 h. After completion of the reaction the catalyst is filtered and removal of the solvent affords the corresponding amino compound, which on treatment with HF-pyridine affords the i immunogen 2D-A.

Example 73 Preparation of Immunogens 3D-AL, 2D-AL, and 1D-AL To a solution of compound 54 in methanol and a catalytic amount of PTSA at 0 C is stirred until starting material (c~mpound 54) disappears (TLC). After completion of the reaction, acid is neutralized with triethylamine and removal of the solvents and flash chromatography affords the corresponding hydroxy compound.

To a solution of the above hydroxy compound (1 eq) and CBZ-protected amino acid (1.2 eq) in dichloromethane is added sequentially DCC (1.2 eq) and DMAP (1.2 eq); and the mixture is stirred in an argon atmosphere for 4 h. After completion of the reaction, insoluble salts are removed by filtration. The filtrate is concentrated in vacuo and purification of the product by flash chromatography affords the coupled compound.

A suspension of the above coupled compound and Pd-C in ethyl acetate is stirred under an Ar atmosphere 4 h. After completion of the reaction the catalyst is filtered and removal of the solvent affords the amino compound, which on treatment with HF-pyridine, affords immunogen 3D-AL Preparation of Immunogens 2D-AL: A solution of compound 68 in methanol and a catalytic amount of PTSA at 0°C is stirred until the starting material disappears (TLC). After completion of the reaction, acid is neutralized with triethylamine and removal of the solvents and flash chromatography affords the corresponding hydroxy compound. To a solution of the hydroxy compound (1 eq) and CBZ protected amino acid (1.2 eq) in dichloromethane is added sequentially DCC (1.2 eq) WO 93/19761 PCT/'IS93/02903 86 and DMAP (1.2 eq) and the mixture is stirred in argon atmosphere for 4 h. After completion of the reaction, insoluble salts are removed by filtration. The filtrate is concentrated in vacuo and purification of the product by flash chromatography affords the coupled compound. A suspension of the coupled compound and Pd-C in ethyl acetate is stirred under an Ar atmosphere 4 h.

After completion of the reaction the catalyst is filtered and removal of the solvent affords the amino compound, which on treatment with HF-pyridine affords the immunogen 2D-AL.

Preparation of Immunogens ID-AL: A solution of compound 62 in methanol and catalytic amount of PTSA at 0°C is stirred until the starting material disappears (TLC). After completion of the reaction, acid is neutralized with triethylamine and removal of the solvents and flash chromatography affords the corresponding hydroxy compound. To a solution of the hydroxy compound (1 eq) and CBZ protected amino acid (1.2 eq) in dichloromethane is added sequentially DCC (1.2 eq) and DMAP (1.2 eq) and the mixture is stirred in argon atmosphere for 4 h. After completion of the reaction, insoluble salts are removed by filtration. The filtrate is concentrated in vacuo and purification of the product by flash chromatography affords the coupled compound. A suspension of the coupled compound and Pd-C in ethyl acetate is stirred under an Ar atmosphere 4 h.

After completion of the reaction the catalyst is filtered and removal of the solvent affords the amino compound, which on treatment with HF-pyridine affords the immunogen 1D-AL.

Example 74 Immunization with Lipid-A Analogs for the Generation of Monoclonal Antibodies BK-1 Sheep Red Blood Cells (SRBC) were coated with BK-1 as follows (100): i) Formaldehyde fixed, lyophilized SRBC (Sigma

L

1~ _i WO 93/19761 PCT/US93/02903 87 Chemicals) were resuspended in sterile PBS to give a v/v suspension. 0.5 ml of this suspension 'as washed three times with 10 ml PBS by centrifugation and finally resuspended to 0.5 ml. BK-1 was dissolved in 0.05% triethylamine by sonication to a concentration of 1mg/ml.

This solution was then neutralized by the addition of 1/10 volume of 1M Tris buffer pH 7. 150 gl of this solution was diluted to 3ml with PBS to give a concentration of 50 Mg/ml and this was added to the washed SRBC to give a total volume of 3.5 ml. This mixture was incubated overnight on a rotary mixer at room temperature. The coated SRBC were then pelleted by centrifugation and washed twice with 4ml of PBS, finally being resuspended to 1.5 ml in PBS to give a 3.3% suspension.

ii) Mice were immunized with these BK-1-coated SRBC, in the absence of adjuvant, multiple times. Following the later bleeds :vrum was obtained from individual mice by bleeding from the retro-orbital sinus and BK-l-specif.c IgM and IgG antibody was measured separately in these sera by ELISA assays. Mice immunized in this way produced only IgM responses. Those individuals giving the highest titer anti-BK-1 responses were selected and hybridomas secreting monoclonal antibodies were generated from heir splenocytes by conventional cell fusion techniques.

BK-3 Acid hydrolysed bacteria (AHB) coated with BK-3 were prepared as follows (101): i) E. coli strain HB101 were grown to late log phase by inoculating 100 ml volumes of PPBE medium with 0.5 ml of stationary phase overnight cultures in the same medium.

Cultures were grown at 37°C with rotary mixing at 250 rpm. for approximately i hrs until reaching an 0.D.

540 of 0.85. Cells were harvested by centrifugation, washed in 1% acetic acid and harvested again by centrifugation. A total of 500 ml of culture yielded 0.92 g wet weight of WO 93/19761 PCT/US93/2903 88 bacteria. These cells were resuspended in 45 ml of 1% acetic acid, hydrolysed for 2 hrs in a boiling water bath at 100 C then kept at 4°C overnight. The cells were washed twice in 1% acetic acid, once in Tiater, once in acetone then dried under vacuum, yielding 130 mg of acid hydrolysed bacteria. These were resuspended in water, aliquotted into 10 mg fractions, dried under vacuum and stored at -20°C until used.

ii) 2mg of AHB were suspended in 4 ml water in a 50 ml round bottomed flask. 80 Al of a vigorously sonicated mg/ml solution (200 of BK-3 in 0.05% triethylamine was added. The mixture was dried on a rotary evaporator, resuspended in 2 ml water by sonication and dried again. These antigen coated AHB weote stored dry at 43C until used for immunization.

Coated AHB were resuspended in 0.9% NaCl to give a concentration of 1 mg/ml AHB (100 Ag/ml BK-3) and mixed with an equal volume of Complete or Incomplete Freund's Adjuvant for injection. Mice were immunized with 100 Al of this suspension either intraperitoneally or subcutaneously, the first immunization used CFA and subsequent immunizations ised IFA.

iii) Mice were immunized as above, multiple times.

Following the later bleeds serum was obtained from individual mice by bleeding from the retro-orbital sinus and BK-3-specific IgM and IgG antibody was measured separately in these sera by ELISA assays. Mice which showed the highest titer anti-BK3 IgG responses were selected and hybridomas secreting monoclonal antibodies were generated from their splenocytes by conventional cell fusion techniques.

Immunogen 3D Immunogen 3D was incorporated into liposomes as follows: i) A stock solution of lipids for the formation of liposomes was prepared by mixing 10 mM solutions of Sdimyristoyl phosphatidyl choline, dicetyl phosphate and WO 93/19761 PCT/US93/02903 89 cholesterol (all from Sigma Chemicals) in the ratio 1.0:0.11:0.75. This stock was aliquotted into 1.0 ml volumes and dried under argon and stored at -20°C until used.

ii) 500 Ag of immunogen 3D dissolved in CHCl 3 was dried into a 50 ml round bottomed flask on a rotary evaporator.

One aliquot of the lipid stock was dissolved in 1 ml of CHC13:MeOH and added to the dried immunogen 3D.

The dissolved lipids were thoroughly mixed then dried together on a rotary evaporator under argon until all solvent had been removed and then for an additional mins. The flask was then placed in a high vacuum dessicator for 2 hrs. 1.0 ml of swelling solution, approximately 0.9% NaCl, was added to the dry lipid film with 0.5 g of glass beads and mixed vigorously by swirling and vortexing. Liposomes were allowed to swell at room temperature overnight. The nominal concentration of immunogen 3D in these liposome preparations was therefor 500 Ag/ml (;0.25 mM), and the concentration of the other lipids was 10 mM.

iii) Optimal conditions for immunization of mice with these liposomes were found in preliminary experiments to be the injection of 0.1 ml (50 Ag immunogen 3D) in the absence of adjuvant. Mice were therefor immunized in this way on day 0, day 21 and day 35 then bled from the retro-orbital sinus on day 42 for the determination of the immunogen 3D response by ELISA.

iv) In a modification of this basic protocol, liposomes were synthesized which incorporated a synthetic peptide known to be a dominant T-cell stimulatory epitope in Balb/c mice. This peptide comprises residues 105-120 of Hen Egg Lysozyme (HEL) and was incorporated into liposomes by dissolving it to a concentration of 25 Ag/ml in the swelling solution. This preparation was used to immunize mice according to the schedule described above.

These mice had been primed 10 days before the first injection of liposomes with 10gg of HEL in CFA.

WO 93/19761 PCT/US93/02903 Example 75 Monoclonal Antibodies Generated Against Lipid-A Analogs BK-1 A total of 14 MAbs which bound specifically to Lipid-A in ELISA were generated from two fusions with BK-l/SRBC. All of these MAbs were of the IgM/kappa isotype. Inhibition ELISAs were performed to determine the relative affinities of these MAbs for Lipid-A analogs S(analogs used were: monophosphoryl Lipid-A, MPL; ReLPS from salmonella minnesota strain R595; BK-1 and BK-3).

The MAbs fell into four specificity groups based on their relative affinities for these ligands: I MPL ReLPS BK1 BK3 II MPL ReLPS BK1 BK3 III MPL BK1 ReLPS BK3 IV MPL ReLPS BK1 BK3 Similar inhibition ELISAs with different LPS chemotypes showed that all of the MAbs reacted with ReLPS, some reacted with low affinity with ReLPS but none of them had i any affinity for fully O-glycosylated E. coli S-LPS.

Five of these MAbs (4 from group II and 1 from group I) were purified from ascites fluid and tested for their ability to release fatty acyl chains from 3 H-Lipid-A and 3 H-ReLPS substrates. None were catalytically active. However, each of these MAbs is useful for binding to LPS and for treating septicemia or septic shock. This also demonstrates that formula (I) compounds can compete for binding sites with Lipid-A and ILPS and are thus useful for treatment in this manner.

BK-3 Five fusions were performed and a total of 24 MAbs selected which showed specific binding to Lipid-A and to BK-3 as measured by inhibition ELISAs. Of these MAbs 23 were IgM/kappa and one was IgG/kappa. Twenty three MAbs demonstrated a higher affinity for Lipid-A than for BK-3, whilst two had a higher affinity for BK-3 WO 93/19761 PCT/US93/02903 91 than for Lipid-A. Eleven of these MAbs were tested for their ability to release fatty acyl chains from 3 H-Lipid- A and 3 H-ReLPS substrates; none were catalytically active. However, each of these MAbs is useful for binding LPS and for treating septicemia or septic shock.

This also demonstrates that formula compounds can compete for binding sites with Lipid-A and LPS and are thus useful for treatment in this manner.

Immunoqen 3D Fusions are performed as set forth above with respect to BK-1 and BK-3. Both IgM and IgG MAbs are generated. The MAbs have high affinity for both immunogen 3D and Lipid-A; none are catalytically active; but all are useful for binding to LPS and are thus useful for treating septicemia or septic shock. This also demonstrates that formula compounds can compete for binding sites with Lipid-A and LPS and are thus useful for treatment in this manner.

EXAMPLE 76 Catalytic Antibodies The procedures of Examples 74 and 75 are performed and the anti-BK-1, anti-BK-3 and anti-immunogen 3D MAbs are tested for their ability to release fatty acyl chains from 3 H-Lipid-A and 3 H-ReLPS; these MAbs release the fatty acyl chains and are thus catalytic antibodies. These catalytic antibodies are useful for treating septicemia or toxic shock, either alone, or in admixture with each other or in admixture with MAbs of Example 75 (as a cocktail), as further described below.

MAbs of any isotype, but preferably IgG, generated against transition state analogs (formula (I) compounds) as described above are screened for their ability to hydrolyse ester bonds, resulting in the liberation of fatty acyl chains from 3 H-labelled ReLPS according to the method described in Munford and Hall (36).

Those MAbs which are catalytically active in this assay (or smaller fragments derived from them, 1 16. WO 93/19761 PCT/US93/02903 92 either by protein chemical techniques or by the expression in any system of the genes encoding the antibodies or fragments of those genes encoding parts of the antibodies, which include the antigen combining site) (herein termed ABZYMEs

T

are then tested for their ability to react in purified form with LPS in such a way as to neutralize its in vitro and in vivo bioactivities which are well known to those skilled in the art, including but not limited to: Reactivity in, or immunoprotection from, challenge in the Dermal Schwartzman Reaction; pyrogenicity; leukopenia; complement activation; cytokine (TNF, IL-1, IL-6) induction; priming of neutrophils for oxygen radical release; and induction of procoagulant activity in cultured epithelial cells. The catalytic MAbs or fragments thereof of the invention suitably react in purified form with LPS in such a way as to neutralize its in vitro and in vivo bioactivities.

Catalytic MAbs are also be tested for their ability to reduce the toxicity of LPS preparations towards both galactosamine sensitized and unsensitized mice in assays which are well known to those skilled in the art: Galactosamine primed mice (62) are injected with increasing doses of LPS treated with individual catalytic antibodies and untreated LPS and the LD 50 in such mice is determined after a period of three days.

The catalytic Mabs or fragments thereof of the present invention reduce the toxicity of LPS preparations.

The ability of the catalytic MAbs to afford protection against lethal gram-negative bacteremia is assessed in the standard mouse toxicity assay (102).

Briefly, female CF1 mice of 6 to 8 months of age are injected with live cultures of E. coli diluted to give a graded response. The prophylactic protection afforded by the catalytic antibodies is determined by injection of the mice with the MAbs 18 hrs prior to infection. The LDSo of mice treated in this way is determined after a WO 93/19761 PCI/US93/02903 93 period of three days. In order to test for the efficacy of the antibodies in treating bacteremia, the MAbs which are effective in the above experiment are administered following bacterial infection. The catalytic Mabs or fragments thereof of the present invention afford protection against gram-negative bacteremia.

Following these pre-clinical animal studies, then there is testing of the MAbs for therapeutic action in humans. Two major studies on the treatment of gramnegative sepsis with MAbs as an adjunct to conventional therapy including antibiotic treatment and intensive supporting care have been carried out these studies define the criteria for enrolling a suitable patient population and the preferred protocols for the administration of antibody based therapeutics in this disease. These protocols are followed in studies using catalytic MAbs or fragments derived therefrom of the present invention ("ABZYME").

Initial dose escalation safety and toxicity studies are carried out, according to methods well known in the art, to establish the maximum tolerable dose of selected "ABZYMEs7". Efficacy testing then involves the administration of the "ABZYMEm" via intravenous, intramuscular or intraperitoneal route to patients suspected of having or being susceptible to a gramnegative bacterial infection. The "ABZYME m is administered in a physiologically acceptable solution such as phosphate buffered saline, which can be supplemented with an excipient such as dextran or human serum albumin, at a dose determined by the body weight of the host. This dose is preferably in the range of about 0.1 mg/kg to about 40 mg/kg and usually in the range of about mg/kg to about 10 mg/kg of host body weight, not to exceed the maximum tolerable dose determined as described above. Treatment is repeated at intervals as necessary until the recovery of the patient from infection is effected. The catalytic Mabs or fragments thereof of the ii' WO 93/19761 PCT/US93/02903 94 present invention are effective.

Example 77 Preparation of Compound 69 To a solution of diol compound 41 (1 eq) in dry DMF (0.2 under an Ar atmosphere the following are sequentially added: imidazole (4.8 eq) and tbutyldimethylsilyl chloride (2.4 eq); and the mixture is stirred at room temperature until the starting material (compound 41) disappears. After completion of the reaction, the mixture is diluted with ethyl acetate (0.1 M) and washed with water. The organic phase is separated and dried and removal of the solvent and flash chromatography affords the corresponding disilylated compound.

The above-obtained compound (1 eq) is dissolved in methanol (0.2 M) cooled to 0°C and PTSA (catalytic amount) is added. The resulting mixture is stirred at that temperature until the starting material disappears (TLC). After completion of the reaction acid is quenched with triethylamine, and removal of the solvent and flash chromatography affords compound 69.

Example 78 Preparation Of Compound To a solution of compound 69 (1 eq) in dry dichloromethane (0.2 M) containing triethylamine (3 eq) at 0°C under an Ar atmosphere is added MsCI (1.2 eq) dropwise through a syringe. The mixture is stirred at that temperature until the hydroxy compound disappears.

After completion of the reaction, a solution of ammonium chloride is added and the organic phases are separated, dried and concentrated. Removal of the solvent and flash chromatography affords the corresponding mesylate compound.

A mixture of the above-obtained mesylate compound (1 eq) and sodium azide (3 eq) in dry DMF (0.2 M) is heated at 50°C under an Ar atmosphere until the starting material disappears. After completion of the reaction, the mixture is diluted with ethyl acetate (0.1 M) and washed with water.

i i 1 .i WO 93/19761 PCT/US93/02903 The organic phase is separated, dried and concentrated and flash chromatography affords the azido compound Example 79 Preparation Of Compound 71.

A suspension of compound 70 (1 eq) in ethyl acetate (0.2 M) in the presence of catalyst (Pd-C, is hydrogenated by using a hydrogen balloon. After completion of the reaction (TLC) the catalyst is filtered through a pad of celite and removal of the solvent affords the amino compound 71.

Example 80 Preparation of Compound 73 A solution of commercially available lactone 72 (1 eq) and 2,2 dimethoxy propane, in dry DMF (0.1 M) in the presence of PTSA (catalytic amount) is stirred at room temperature under an Ar atmosphere. After completion of the reaction (TLC) the solvent is removed in vacuo and fresh dry DMF (0.2 M) is added followed by sequential addition of imidazole (4.8 eq) and tbutyldimethylsilyl chloride (2.4 eq). The mixture is stirred under an Ar atmosphere until the starting material disappears. After completion of the reaction, the mixture is diluted with ethyl acetate (0.1 washed with water; and the organic phase is separated, dried and concentrated, and flash chromatography affords the compound 73.

Example 81 Preparation of compound 74 A solution of compound 73 (1 eq) in benzyl alcohol (10 eq) is heated at reflux, until the starting material disappears. After completion of the reaction benzyl alcohol is removed in vacuo and the resulting mixture on flash chromatography affords the hydroxy ester compound.

To a solution of the above-obtained hydroxy ester compound (1 eq) in dry dichloromethane (0.2 M) in the presence of triethylamine (3 eq), Ms-C1 (1.2 eq) is added through a syringe under an Ar atmosphere and the A WO 93/19761 PCT/US93/02903 96 mixture is stirred at room temperature. After completion of the reaction (TLC), a solution of ammonium chloride is added and the organic layer is separated, dried and concentrated, and flash chromatography affords the corresponding mesylate compound.

A solution of the mesylate compound (1 eq), sodium iodide (2 eq) in dry acetone (0.2 M) is heated at 0 C under an Ar atmosphere. After completion of the reaction (TLC), removal of the solvent and flash chromatography affords the iodo compound.

A solution of the above-obtained iodo compound (1 eq) and sodium azide (3 eq) in dry DMF (0.2M) is heated at 50°C under an Ar atmosphere. After completion of the reaction (TLC) the solution is diluted with ethyl acetate (0.1 and washed with water. The organic phase is separated, dried, and concentrated, and flash chromatography affords the azido compound 74.

Example 82 Preparation of Compound A suspension of compound 74 (1 eq) in ethyl acetate (0.2 M) in the presence of catalyst (Pd-C, is stirred under a hydrogen atmosphere. After completion of the reaction the catalyst is filtered and removal of the solvent affords the silyl lactam.

To a solution of silyl lactam (1 eq) in dry THF (0.2 a solution of n-tetrabutylammonium fluoride (1M solution in THF, 2.4 eq) is added through a syringe under an Ar atmosphere. After completion of the reaction, the solution is diluted with ethyl acetate and washed with a minimum amount of water. The organic phase is separated, dried and concentrated, and flash chromatography affords the compound Example 83 Preparation of Compound 76 A solution of compound 75 (1 eq), DCC (2.4 eq), DMAP (2.4 eq) and acid 3 (2.4 eq) in dry dichloromethane (0.2 M) is stirred under Ar atmosphere at room temperature. After completion of the reaction insoluble particles are separated by filtration and removal of the WO 93/19761 PCT/US93/02903 97 solvent and flash chromatography affords the coupling compound.

The above-obtained compound (1 eq) is stirred in a solution of dichloromethane (0.2 M) and trifluoroacetic acid (3 eq) at room temperature under an Ar atmosphere. After completion of the reaction, the excess acid is neutralized with triethylamine, the solvents are then removed in vacuo, and flash chromatography affords the diol compound.

To a solution of the above-obtained diol (1 eq) in dry DMF (0.2 M) at 0°C under an Ar atmosphere sequentially added are: imidazole (2.2 eq) and tbutyldimethylsilyl chloride (1.1 eq); and the mixture is stirred at that temperature until the starting material disappears. After completion of the reaction, the mixture is diluted with ethyl acetate, washed with water, dried and concentrated and flash chromatography affords the compound 76.

Example 84 Preparation of Compound 77 A solution of compound 76 (1 eq), N,N diisopropylamino dibenzylphosphite, tetrazole in dry dichloromethane (0.2 M) is stirred under an Ar atmosphere at room temperature. After completion of the reaction the mixture is cooled to 0°C and m-CPBA (1.2 eq) is added and the mixture is stirred until completion of the reaction. After completion of the reaction, the solvent is removed and flash chromatography affords the compound 77.

Example 85 Preparation Of Compound 78 To a solution of compound 77 (1 eq) in dry dichloromethane (0.2 a solution of triethyloxonium tetrafluoroborate (1 eq, 1 M solution in dichloromethane, Meerwein reagent) is added and the mixture is stirred for 1 h under an Ar atmosphere at 0°C. Then a solution of amino compound 71 (1.2 eq) in dichloromethane (0.2 M) is added and the mixture is stirred until the completion of the reaction. After completion of the reaction the i Il93196 PCr/US93/02903 solvent is removed and flash chromatography affords the compound 78.

Example 86 Preparation of Compound 79 Method-A: A suspension of compound 78 (1 eq) in ethyl acetate (0.2 M) is stirred under a hydrogen atmosphere in the presence of catalyst (Pd-C, After completion of the reaction the catalyst is filtered and the solvent is removed to afford the hydrogenated compound. A solution of the hydrogenated compound (1 eq) in THF (0.1 M) in a plastic container is stirred with HFpyridine at 0 C. After completion of the reaction, the solution is neutralized with sodium bicarbonate. The organic phase is separated and removal of the solvent affords compound 79.

Method-B: A solution of compound 78 (1 eq), trimethylsilyl iodide (2 eq) in dry dichloromethane (0.2 M) is stirred at 0°C under an Ar atmosphere. After completion of the reaction, the solution is neutralized with dilute HC1 and stirred for 1 h. The organic phase is separated and removal of the solvent affords the compound 79.

EXAMPLE 87 Pyrogenicity of Formula and (II) Compounds Japanese white rabbits are administered 1 and 10 g/kg of BK-1, BK-3 and immunogen 3D, and compound 79 as well as 0.001 Ag/kg Lipid-A. The formula and (II) compounds do not show pyrogenicity whereas Lipid-A does.

This demonstrates that the formula and (II) compounds are less toxic than Lipid-A and that the immunopharmaocological activities, B cell and macrophage activation, Ifn, TNF inducing activities, are separate from toxic activities such as pyrogenicity, lethality and Schwartzman reactivity.

EXAMPLE 88 Nonspecific Protection against Bacterial Infection The ability of compounds BK-1, BK-3, immunogen 3D and compound 79 to enhance the nonspecific resistance to bacterial infection is measured in the Pseudomonas i 1 Ni 1 WO 93/19761 PCT/US93/02903 99 aeruginosa model described by Nakatsuka et al. (103).

Briefly, the compounds of the invention are injected at various doses (1-10 pg/mouse) ip. into ICR mice one day prior to ip. infection with graded doses of viable P.aeruginosa organisms (of a clinically isolated strain such as 5E81-1), between 107 and 2x10 8 CFU/mouse. A number of mice survive for seven days following infection and this is recorded as the final result, control treated mice aeruginosa without prior administration of inventive compound) die within about three days following infection. The compounds of the present invention enhance the nonspecific resistance to bacterial infection.

EXAMPLE 89 Protection from Viral Challenge The ability of compounds BK-1, BK-3, immunogen 3D and compound 79 of the present invention to enhance the nonspecific resistance to viral infection is measured in the vaccinia virus model described by Ikeda et al.

Briefly, groups of 10 20 four week old female ddY mice are injected iv. with the compounds of the present invention one day before iv. infection with 104 pfu of vaccinia virus. Seven days following viral challenge, vaccinia virus lesions on the tails of the mice are counted following visualization by staining wi.h 1% fluorescein-0.5% methylene blue. The anti-viral potency is measured as the reduction in the number of lesions in treated mice relative to untreated controls.

Mice treated with the compounds of the present invention exhibit a reduction in the number of lesions relative to untreated control mice; thus, the compounds of the present invention exhibit antiviral activity.

EXAMPLE 90 Antitumor Agents The antitumor action of the compounds BK-1, BK- 3, immunogen 3D and compound 79 is investigated in the animal models well known to those skilled in the art, for example: The B16 murine melanoma metastasis model, I, A! h i WO 93/19761 PCT/US93/02903 100 described by Nakatsuka et al. (104). Briefly, the compounds of the present invention aze injected iv. into mice in various doses (0.1-10 pg/mouse) and with varying frequency and timing of administration, prior to the iv.

inoculation with 105 B16 melanoma cells. The number of melanoma metastases on the surface of the lungs of mice are counted with the aid of a dissecting microscope at day 21 following transplantation of the tumor cells.

Efficacy of the compounds of the present invention is manifested as a reduction in the number of metastases in mice treated with compounds of the present invention.

The rat colon carcinoma model described by Jeannin et al. (105). Briefly, BDIX rats are inoculated with 106 Pro b cells ip. Fourteen days later treatment with the compounds of the present invention is initiated: compounds are injected at various doses, up to 10mg/kg body weight, and according to various schedules, up to 5 injections made twice a week. The extent of the disease is monitored six weeks after tumor transplantation by inspection of the size :ind number of tumor nodules in the abdominal cavity of sacrificed rats.

Rats treated with compounds of the present invention exhibit less and smaller tumor nodules than control rats.

ThF Meth A fibrosarcoma model as described by Nakatsuka et al. 10 s or 2x10 5 Meth A cells are injected intradermally into the flank of 7 week old Balb/c mice. The compounds of the present invention are injected, at doses of 100g/mouse, either intravenously or directly into the growing tumor mass, on days 7 and 9 following tumor transplantation. The size cl the tumors are measured with calipers at intervals of 2-4 days.

After four weeks mice are sacrificed and the tumors excised and weighed. The number of mice in which the tumors have resolved is also scored. Mice treated with compounds of the present invention exhibit smaller tumors

V

L i i WO 93/19761 PCT/US93/02903 101 or tumors which have resolved. Thus, the compounds of the present invention are useful as antitumor agents.

Example 91 Receptor Antagonists of Lipid-A/LPS The ability of compounds BK-1, BK-3, immunogen 3D and compound 79 to compete for receptor binding and so block the endotoxic effects of Lipid-A or LPS are evaluated in the mouse model described by Quereshi et al.

(106) Briefly, groups of Balb/c mice 8-12 weeks of age are injected with the compound(s) in PBS or PBS alone as control, followed 60 mins later by 1Ag of ReLPS.

After a further 60 mins animals are exsanguinated and serum levels of TNF measured using the L929 fibroblast toxicity assay described by Flick and Gifford (107).

The compounds of the present invention significantly reduce the levels of TNF production in this assay and are further tested for their ability to inhibit TNF production by human monocytes in culture as described by Golenbock et al. (108); the compounds of the present invention inhibit TNF production in human monocytes.

Example 92 Antibodies to Formula (II) Compounds Using the procedures set forth in Examples 74, and 76, binding and catalytic antibodies are elicited against compound 79. These antibodies are useful like those elicited against BK-1, BK-3, and immunogen 3D.

Example 93 Immunization with Liposomes Incorporating T-Cell stimulatory Peptides Mice which were primed with HEL and immunized with Immunogen 3D("Imm-3D") and Lipid-A incorporated separately into liposomes along with the peptide HEL[105- 120] (synthetic, with additional C-terminal cysteine residue) according to the schedule outlined in Example 74 gave enhanced IgG antibody responses to the eliciting antigen. This was best shown in an experiment with Lipid-A: Four groups of five Balb/c mice each were immunized with 50pg of Lipid-A incorporated in liposomes.

Half of the mice were primed with HEL 10 days before the first immunization, half of the mice received liposomes which incorporated the peptide HEL[105-120] (synthetic, WO 93/19761 PCT/US93/02903 102 with additional C-terminal cysteine residue). Seven days after the final immunization, serum samples were taken and tne titres of IgG and IgM anti-Lipid-A antibodies determined separately by ELISA. The results are given in the Table I, below: Table I

I

Li

I'

A

GROUP HEL PRIMED PEPTIDE IN IqM TITRE I G TITRE

LIPOSOMES

A 1:60,000 1:10,000 B 1: 6,000 1: 150 C 1:10,000 1: 1,000 D 1:15,000 1: 100 These data show that the generation of a high-titre IgG response is dependent upon both HEL priming and the inclusion of the peptide in the liposome preparations.

The tenfold enhanced IgG titre of group C over groups B and D is presumably due to the fact that the earlier doses of peptide in the liposomes prime specific T-Cells for the later immunizations, resulting in T-Cell help and the resulting switch from IgM to IgG isotype antibodies.

The combinat ~n of HEL priming and poptide incorporation into the liposomes (group A) also increases the IgM titre by 4-10 fold over groups B-D. This titre of 1:60,000 against Lipid-A is the highest we have seen from any of the protocols tried, including prolonged immunization regimes using killed bacterial cells coated with large amounts of Lipid-A. This shows that formulations of this type are most effective for use as a vaccine to induce an IgM response capable of protecting animals from the harmful effects of endotoxemia or gramnegative sepsis.

In a simultaneous experiment mice were immunized with Imm-3D in ',.1Jsomes plus HEL[105-120} (synthetic, with additional C-terminal cysteine rezir.,e) following priming with HEL. The results are given in Table II below: I- WO 93/19761 PCT/US93/02903 103 Table II ISOTYPE TITRE ON IMM-3D TITRE ON LIPID-A IgM 1: 3,000 1:3,000 IgG 110, 00 1:1,500 This immunization ha; therefore resulted in a i higher titre IgG response than IgM response to Imm-3D.

In addition there is a degree of antigen specificity in the IgG antibodies raised against Imm-3D; the titre on Lipid-A is six-fold lower than on the homologous antigen.

The IgM response, on the other hand, is more crossreactive, producing the same titre on Lpid-A and Imm-3D.

Example 94 Movioclonal Antibody Fragments Isolated from a Bacteriophage 2xpreslsion Library which Bind to BK3 Mice were immunized with BK3-coated E. coli HB101 cells as described in Example 74. Splenocytes from two of these mice were used for the preparation of mRNA i by standard methods. cDNA was synthesized from this mRNA using primers based in the CH1 domains of IgG, IgM and the CK domain. PCR amplification of this initial cDNA was then performed using standard methodologies, known to those skilled in the art, and sets of primers as described in U.S. application Serial No. 07/841,648, filed February 24, 1992 and previously incorporated Sherein by reference (reference is also made to PCT patent publication WO92/01047, published January 23, 1992, entitled "Methods for Producing Members of Specific Binding Pairs", as well as to PCT publications W091/17271 and WO91/19818; and all of these PCT publications are hereby incorporated herein by reference). Two fd-phage expression libraries were generated, one from cDNA synthesized from the IgM specific primer and the other from cDNA synthesized from the IgG specific primer.

These libraries contained 1.2 x 106 and 2.8 x 106 independent clones respectively. Phage particles expressing antibody fragments capable of binding to BK3 were selected by a "panning" procedure on a solid surface WO 93/19761 PCT/US93/02903 104 coated with BK3. 35mm diameter petri dishes were coated with BK3 by evaporating a solution containing 15Lg of the compound in CHC13/MeOH (3:1 ratio) to dryness. The dishes were incubated with a 3% solution of dried milk in PBS for lhr to block non-specific binding then phage from the libraries incubated in them for 2-3 hrs at room temperature. After washing with PBS/0.1% Tween 20 and PBS, specific phage were eluted with 1OOmM triethylamine in water and used to infect E coli TG1 cells. The resultant expanded phage population was further selected by repeating this procedure a total of three times.

Phage particles isolated from the third round of panning were expanded again in TG1 cells and then plated out and single clones picked and used to infect individual cultures of bacteria. Supernatants from these cultures were analyzed for binding to BK3 coated plastic wells in a standard ELISA, well known to those skilled in the art (100pl of phage supernatant was reacted with the antigen coated wells, binding was detected with sheep anti-fd serum). Ten clones from a total of 156 which were analyzed from the IgM library demonstrated strong binding to BK3 in this assay; four out of 240 analyzed from the IgG library did likewise. Thus, antibodies from these clones are useful for treating septicemia. Ten of these binders were further characterized.

The genetic diversity of the clones isolated in this way was investigated. The insert encoding the antibody chains was amplified by PCR and the product digested with the restriction enzyme BstNl, which cuts frequently in antibody gene sequences, and the resulting fragments analyzed on a 4% agarose gel. This analysis revealed that ten distinctly different antibody molecules had been selected from the library. Subsequent sequence analysis revealed the usage of three different VK genes, with one employed by 8 antibodies and two others by one antibody each. At least four different VH genes were used by the antibodies. The antibodies expressed by this

K-

WO93/19761 PCT/US93/02903 105 method show binding and catalytic activity and are thus useful in treating septicemia. Screening for catalytic activity can be as described in U.S. application Serial No. 07/841,648, filed February 24, 1992.

Alternatively, splenocytes or peripheral blood lymphocytes from unimmunized animals or humans can be used as the source of immunoglobulin mRNA and libraries constructed as above.

Example 95 Preparation of Compound Compound 5a,b (10 mmol) is dissolved in 50 mL of acetone and cooled to 0 C. Jones reagent is added until a red color persists, and the mixture is stirred at that temperature for 1 h. After completion of the reaction the acetone is evaporated in vacuo, and the residue is dissolved in ethyl acetate (100 mL) and washed with water (3x50 mL) until the organic layer becomes almost colorless. The organic layer is dried over anhydrous MgSO 4 and concentrated in vacuo. Purification by flash chromatography gives compound 80 as an oil.

Example 96 Preparation of Compound 81 1 M Sodium methoxide in methanol (0.8 ml) is added to a mixture of compound 80 (10 mmol) and 30 mL of methanol, and the mixture is stirred at room temperature for 2 h. After completion of the reaction, the volatile components are evaporated in vacuo and the residue is filtered through a small pad of silica gel using ethyl acetate as eluent to afford the triol as an oil.

PTSA (0.1 g) is added to a mixture of the above triol (10 mmol), 2,2-dimethoxypropane (25 mmol), and mL of methylene chloride, and the mixture is stirred at room temperature for 4 h. Then the reaction mixture is diluted with methylene chloride (70 mL) and washed with water (70 mL), 5% sodium bicarbonate (30 mL) and water mL). The organic phase is dried over anhydrous MgSO 4 and concentrated in vacuo. Purification by flash chromatography gives the acetonide as an oil.

A mixture of the above acetonide (10 mmol),

I

wI 1 0 93/19761 PCT/US93/02993 106 imidazole (22 mmol), and TBDMS-Cl (11 mmol) in 10 mL of DMF cooled to 0 0 C is stirred until the starting material is consumed. The mixture is diluted with ethyl acetate (150 mL), washed with water (2x50 mL) and brine (50 mL), dried over anhydrous MgSO 4 and concentrated in vacuo.

Purification by flash chromatography gives compound 81 as a colorless oil.

Example 97 Preparation of Compound 82 A solution of compound 81 (5 mmol) in benzyl alcohol (50 mmol) is heated at reflux until the starting material is consumed. After completion of the reaction, the excess benzyl alcohol is removed in vacuo.

Purification by flash chromatography gives the hydroxy ester.

Triethylamine (5.5 mmol) is added to a mixture of methanesulfonyl chloride (5.5 mmol) and the above hydroxy ester (5.0 mmol) in 50 mL of THF cooled to 0°C.

After the starting material is consumed, the mixture is partitioned between water (75 mL) and ethyl acetate (3 x 50 mL), and the organic phases are washed with brine mL), dried over anhydrous MgSO 4 and concentrated in vacuo. Purification by flash chromatography gives the methanesulfonate as a colorless solid.

A mixture of the above methanesulfonate mmol) and sodium iodide (10 mmol) in acetone (25 mL) is heated at reflux. After the starting material is consumed, the solvent is removed in vacuo, and purification by flash chromatography gives the product as a yellow oil.

A mixture of the above iodide (5 mmol) and sodium azide (15 mmol) in 10 mL of DMF is heated at oC. After completion of the reaction, the mixture is partitioned between water (75 mL) and ethyl acetate (3x50 mL), and the organic phases are washed with brine '2,

'I

WO 93/19761 PCT/US93/02903 107 mL), dried over anhydrous MgS0 4 and concentrated in vacuo. Purification by flash chromatography gives compound 82 as a colorless solid.

Example 98 Preparation of Compound 83 A suspension of compound 82 (5 mmol) and 5% Pd- C (0.5 mmol) in 25 mL of methanol is stirred under a hydrogen atmosphere. After the starting material is consumed, the catalyst is removed by filtration, and evaporation of the solvent in vacuo gives compound 83.

Example 99 Preparation of Compound 84 4-Nitrobenzaldehyde (5 mmol) is added to a solution of compound 83 (5 mmol) and triethylamine mmol) in 25 mL of methylene chloride cooled to 0°C.

After the starting material is consumed, the solvent is evaporated in vacuo, and the residue is partitioned between saturated sodium bicarbonate (30 mL) and ether (4x80 mL), the organic phases are dried over anhydrous potassium carbonate, and the solvent is evaporated in vacuo. Purification by flash chromatography gives compound 84 as an oil.

Example 100 Preparation of Compound Tetra-n-butylammonium fluoride (I M solution in THF, 12 mmol) is added to a solution of compound 84 mmol) in 35 mL of THF at room temperature. After the starting material is consumed, the mixture is washed with saturated sodium bicarbonate (2x10 mL), the aqueous phases are extracted with ether (2 x 20 mL) and the combined organic phases are dried over anhydrous potassium carbonate and concentrated in vacuo.

Purification by flash chromatography gives compound 85 as an oil.

Example 101 Preparation of Compound 86 A solution of compound 85 (1 mmol), EDC (2.2 mmol), DMAP (2.2 mmol) and compound 3 (2.2 mmol) in 20 mL of methylene chloride is stirred at room temperature until the starting materials are consumed. The mixture is poured into water (60 mL) and extracted with ethyl WO 93/1976

I

1 PCT/US93/02903

I

acetate (3x60 mL), and the organic phases are washed with brine (50 mL), dried over anhydrous MgSO 4 and concentrated in vacuo. Purification by flash chromatography gives compound 86 as a colorless oil.

Example 102 Preparation of Compound 87 Trifluoroacetic acid (3 mL) is added to a mixture of compound 86 (1 mmol) and 3 mL of methylene chloride cooled to 0 C. After 30 min, the acid is neutralized by the slow addition of triethylamamine while maintaining the temperature at 0 C. Then the mixture is poured carefully into saturated sodium bicarbonate mL) and extracted with ethyl acetate (4 x 60 mL), and the organic phases are washed with brine (50 mL), dried over anhydrous MgS0 4 and concentrated in vacuo. Purification by flash chromatography gives the diol as a colorless oil.

A mixture of the above diol (1 mmol), imidazole (2.2 mmol), and TBDMS-C1 (1.1 mmol) in 2 mL of DMF cooled to 0°C is stirred until the starting material is consumed. The mixture is diluted with ethyl acetate mL), washed with water (2x10 mL) and brine (10 mL), dried over anhydrous MgSO 4 and concentrated in vacuo.

Purification by flash chromatography gives the silyl ether as a colorless oil.

A solution of the above secondary alcohol (1 mmol), N,N-diisopropylamino dibenzylphosphite (2 mmol), and tetrazole (2 mmol) in 10 mL of methylene chloride is stirred under an argon atmosphere at room temperature.

After the starting material is consumed, the mixture is cooled to 0 C and m-CPBA (2 mmol) is added. After 2 h, the solvent is evaporated in vacuo. Purification by flash chromatography affords compound 87 as a colorless oil.

Example 103 Preparation of Compound 88 A 1 M solution of triethyloxonium tetrafluoroborate in methylene chloride (1 mmol) is added to a solution of compound 87 (1 mmol) in 9 mL of methylene chloride cooled to 0 C. After 1 h, a solution a-- WO 93/19761 PCT/US93/02903 109 of compound 71 (1.2 mmol) in 4 mL of methylene chloride is added. After completion of the reaction, the solvent is evaporated in vacuo, and purification by flash chromatography affords compound 88.

Example 104 Preparation of Compound 89 A suspension of compound 88 (0.1 mmol) and Pd-C (10 weight percent) in 5 mL of ethyl acetate is stirred under a hydrogen atmosphere. After completion of the reaction, the catalyst is removed by filtration, the solvent is evaporated in vacuo, and the residue is taken up in 4.75 mL of acetonitrile in a plastic container.

48% Aqueous hydrofluoric acid (0.25 mL) is then added to the resulting mixture. After 1 h, the volatile components are evaporated in vacuo to give compound 89 as a solid.

Example 105 Synthesis of Compound 94 (Reaction Scheme 41; Fig. 48) Compound 94 was synthesized starting with Dglucosamine hydrochloride 90; amino protection using benzyl chloroformate and sodium bicarbonate followed by acetonation using 2,2-dimethoxypropane and paratoluenesulfonic acid catalyst gave the protected glucosamine 91. Reaction of the aldo form of compound 91 with carbomethoxymethyltriphenylphosphorane in acetonitrile at reflux for 48 hours gave the C-glycoside 92 contaminated with triphenylphosphine oxide after purification by flash chromatography. The Wittig reaction proceeded with a selectivity of 3.5:1 favoring the a isomer. In order to obtain a better separation from the phosphine oxide and to separate the a and p isomers, the hydroxyl of compound 92 was acetylated and gave compound 93. Purification by flash chromatography using 30% ethyl acetate/hexane separated the phosphine oxide contaminant. Separation of the a and p isomers was achieved by flash chromatography using 2% methanol/methylene chloride. Reduction of compound 93 using 4 equivalents of DIBAL-H in methylene chloride at 78 0 C resulted in complete deacetylation, as expected, but I i WO93/19761 PCT/US93/02903 110 resulted in only partial reduction of the methyl ester; a mixture of the aldehyde and the alcohol resulted. The reduction was completed: the mixture was treated with sodium borohydride in methanol for 10 minutes which gave diol 94.

Example 106 Synthesis of Compound 100 (Reaction Scheme 42; Fig. 49) I (R)-3-Benzyloxyhexanoic acid, compound 100, was synthesized starting from trans-2-hexen-l-ol. A Sharpless asymmetric epoxidation with (-)-diisopropyl tartrate was utilized to introduce the chiral center of epoxide 95. Red-Al reduction of the epoxide gave 1,3-hexanediol, compound 96, selectively in good yield.

The primary hydroxyl of diol 96 was selectively silylated under standard conditions to give alcohol 97. The remaining hydroxyl was benzylated using benzyl trichloroacetimidate and catalytic trifluoromethanesulfonic acid which gave compound 98.

The primary hydroxyl was deprotected using acidic methanol which gave alcohol 99, which was oxidized to the acid 100 using Jones reagent.

Example 107 Preparation of Compound 107 (Reaction Schemes 43a, 43b; Figs. 50,51) The primary alcohol functionality of the diol 101 was selectively protected as the t-butyldimethyl silyl ether and the CBZ group was removed by catalytic hydrogenation which afforded the amino compound 102.

Subsequent condensation of 102 with acid 100 using EDC yielded compound 103. Further acylation of 103 using acid 100, EDC and DMAP gave the fully protected compound 104. Removal of the silyl group was performed using tetrabutylammonium fluoride in THF which afforded the hydroxy compound 105. Acylation of 105 with N-CBZ- Glycine using DCC and DMAP gave compound 106. The diol 107 was prepared by the acid hydrolysis of 106 with trifluoroacetic acid.

Example 108 Preparation of Compounds 110 and 112 (Reaction Scheme 44; Fig. 52) WO 93/19761 PCT/US93/02903 111 The syntheses of acids 110 and 112 originated from the same chiral intermediate, alcohol 97.

Butylphosphonic dichloride was reacted with DMAP and excess benzyl alcohol. The resultant dibenzyl ester was reacted with 1.1 equivalents of phosphorus pentachloride in chloroform at reflux which gave monochloridate 108.

Compound 108 was reacted with alcohol 97 and base which gave compound 109. Compound 109 was desilylated using acidic methanol at 0 C and the resultant alcohol was oxidized to the acid 110 using Jones reagent. In a similar manner, alcohol 97 was acylated using butyryl chloride and DMAP which gave compound 111. Careful acidic methanolysis of the TBDMS ether at 0 C was carried out in order to minimize 1,3-acyl migration and, the resultant primary alcohol was oxidized using Jones reagent which afforded acid 112.

Example 109 Preparation of Compound 1D-SC (Fig. 53) The synthesis of 1D-SC (Fig. 53) followed essentially the same chemical steps as the synthesis of ID (Scheme 27). The difference in the two syntheses was the types of reagents used. For compound ID-SC, the acid 112 (Scheme 44) was used instead of compound 3 (Scheme 24), and for disaccharide formation, Compound 113 (shown in Fig. 53) was coupled to 107 rather than 41 (Scheme 25). For the acylation of the amino group of the disaccharide, compound 110 (Scheme 41) was used instead of 4 (Scheme 26). Confirmation of the synthesis was provided by 1H NMR analysis.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the appended claims is not to be limited by particular details set forth in the above-description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

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Claims (54)

1. A compound of the formula: 0 A (HO) 2 -P-0 0 Y 2 X-0- O=Y \2 I R 2 RI R 1 i I i ii (I) wherein: each of RI, RI, R 2 and R 2 independent of each other is a branched or linear, substituted or unsubstituted, C_1 12 alkyl, alkene or alkyne group, R 3 is OH, OCH 3 CH 2 COOH or *St* t* t tt «Ir £r S t e 0 R2 1 I S S wherein each of R2'' and independent of each other is a branched or linear, substituted or unsubstituted cl_ 12 alkyl, alkene or alkyne group and: A NH 2 X P(OH), Y Z C, B, if present, OCH3, or A OH, X P(OH), Y= Z C, B, if present, OCH 3 or A OCO(CH 2 )nNH 2 X P(OH), Y present, OCH 3 wherein n 1-10, or A OH, X P(OH), Y Z C, B, O(CH 2 )nCO 2 H, wherein n 1-10, or A OH, X P(OH), Y Z C, B, C(CH 2 )nCO 2 H, wherein n 1-10, or Z C, B, if if present, if present, I 121 A NH 2 ,X Z= C, Y P(OH), B, if present, OCH 3 or A OH, X Z C, Y P(OH), B, if present, OCH 3 or A OCO(CH 2 )nNH 2 X Z C, Y P(OH), B, if present, OCH 3 wherein n 1-10, or A OH, X Z C, Y P(OH), B, if present, O(CH 2 )nCO 2 H, wherein n 1-11, or A OH, X Z C, Y P(OH), B, if present, (CH 2 )nCO 2 H, wherein n 1-10, or A NH 2 X Y C, Z P(OH), B, if present, OCH 3 or A OH, X Y C, Z P(OH), B, if present, OCH 3 or A OCO(CH 2 nNH 2 X Y C, Z P(OH), B, if present, OCH3, wherein n 1-10, or A OH, X Y C, Z P(OH), B, if present, 0 (CH 2 )nC0 2 H, wherein n 1-10, or A 20 A= OH, X Y C, Z P(OH), B, if present, (CH2)nCO 2 H and n=1-11.

2. The compound of claim 1 wherein R 3 .is o -o HO S0- 25 000: NHa HO-H HO R211 2 1 and each of R 1 R R 2 R 2 R2 and R 2 1 is a C 1 2 linear alkyl group, and wherein Y C, Z C, X P(OH), B=0CH 3 and A OH; or wherein Y C, Z C, X P(OH), B=OCH 3 and A NH2; or wherein Y C, Z C, X P(OH), B=OCH 3 and A OCO(CH 2 )nNH 2 wherein n 1-10; or At I, PY r WO 93/19761 PCT/US93/02903 122 OH; or wherein Y P(OH), Z C, X C, B=OCH 3 and A wherein Y P(OH), X C, Z C, B=OCH 3 and A NH2; or wherein Y P(OH), X C, Z C, OCO(CH 2 )nNH 2 wherein n 1-10; or wherein Y C, X C, Z P(OH), OH; or B=OCH 3 A B=OCH 3 and A wherein Y C, X C, Z P(OH), B=OCH 3 and A NH2; or wherein Y C, X C, Z P(OH), OCO(CH 2 )nNH 2 wherein n 1-10. B=OCH 3 and A

3. A method for eliciting antibodies in an animal which bind to Lipid A or LPS comprising administering to the animal, as an immunogen, a composition comprising a compound as claimed in any one of claims 1 or 2.

4. A composition for protective activity against gram-negative bacterial infection comprising a suitable carrier and a compound as claimed in any one of claims 1 or 2. A method for inducing protective activity against gram-negative bacterial infection in an animal in need of such protection comprising administering to said animal a composition comprising a suitable carrier and a compound as claimed in any one of claims 1 or 2.

6. An antiviral composition comprising a suitable carrier and a compound as claimed in any one of claims 1 or 2.

7. A method for protecting against viral infection in an animal in need of such protection comprising administering to the animal a composition comprising a suitable carrier and a compound as claimed in any one of claims 1 or 2.

8. An antitumor composition comprising a suitable carrier and a compound as claimed in any one of claims 1 or 2. I I I,« __IL WO 93/19761 PCT/US93/02903 123

9. A method for controlling tumor growth in an animal in need of such control comprising administering a composition comprising a suitable carrier and a compound as claimed in any one of claims 1 or 2.

10. The method of claim 3 wherein the animal is a mouse.

11. The method of claim 5 wherein the animal is a human.

12. The method of claim 7 wherein the animal is a human.

13. The method of claim 9 wherein the animal is a human.

14. An antibody which binds to both Lipid-A and a compound as claimed in any one of claims 1 or 2.

15. An antibody elicited using a compound as claimed in any one of claims 1 or 2 which catalyzes the hydrolysis of a chemical bond in the Lipid-A moiety of LPS.

16. An antibody which binds to Lipid-A and a compound as claimed in any one of claims I or 2, said antibody having been made by a process comprising: immunizing an animal with a composition comprising the compound, removing antibody-producing lymphocytes from said animal, and fusing the lymphocytes with a myeloma cells and thereby producing hybridoma cells producing the antibody.

17. A method for treating septicemia comprising administering to a patient in need of such treatment a composition comprising a suitable carrier and an antibody as claimed in claim 14.

18. A method for treating septicemia comprising administering to a patient in need of such treatment a composition comprising a suitable carrier and an antibody as claimed in claim

19. A method for treating septicemia comprising administering to a patient in need of such ~LI 124 treatment a composition comprising a suitable carrier and an antibody as claimed in claim 16. A method for binding to receptors in competition with Lipid A in a patient in need of such binding comprising administering to said patient a composition comprising a suitable carrier and a -compound of formula I as claimed in any one of claims 1 or 2.

21. A compound of the formula OH I I NH+ (HO) 2 -P-O N io o ~NH HO 0 0 0_ 0 "o o 0 0 0( 0 0 NI e d: Re HO t0 I j wherein each of Ra, Rb, Rc, Rd, and Re and Rf, independent of each other, is a branched or linear, substituted or unsubstituted C.-_II alkyl, alkene or alkyne group, and E is NH or O.

22. The compound of claim 21 wherein each of Ra, Rb, Rc, Rd, Re and Rf is CIIH 23

23. The compound of claim 22 wherein E is NH.

24. The compound of claim 22 wherein E is 0.

25. A method for eliciting antibodies in an animal which bind to Lipid A or LPS comprising administering to the animal, as an immunogen a composition comprising a compound as claimed in any one of claims 21-24. .26. A composition for protective activity against gram-negative bacterial infection comprising a A. WO 93/19761 PCT/US93/02903 125 suitable carrier and a compound as claimed in anyone of claims 21-24.

27. A method for inducing protective activity against gram-negative bacterial infection in an animal in need of such protection comprising administering to said animal a composition comprising a suitable carrier and a compound as claimed in any one of claims 21.-24.

28. An antiviral composition comprising a suitable carrier and a compound as claimed in any one of claims 21-24.

29. A method for protecting against viral infection in an animal in need of such protection comprising administering to the animal a composition comprising a suitable carrier and a compound as claimed in any one of claims 21-24. An antitumor composition comprising a suitable carrier and a compound as claimed in any one of claims 21-24.

31. A method for controlling tumor growth in an animal in need of such control comprising administering a composition comprising a suitable carrier and a compound as claimed in any one of claims 21-24.

32. The method of claim 25 wherein the animal is a mouse.

33. The method of claim 27 wherein the animal is a human.

34. The method of claim 29 wherein the animal is a human. The method of claim 31 wherein the animal is a human.

36. An antibody which binds to both Lipid-A or LPS and a compound as claimed in any one of claims 21-24.

37. An antibody elicited using a compound as claimed in any one of claims 21-24 which catalyzes the hydrolysis of a chemical bond in the Lipid-A moiety of LPS. IA WO 93/19761 PC'/US93/02903 126

38. An antibody which binds to Lipid A or LPS and a compound as claimed in any one of claims 21-24, said antibody having been made by a process comprising: immunizing an animal with a composition comprising the compound, removing antibody-producing lymphocytes from said animal, and fusing the lymphocytes with myeloma cells and thereby producing hybridoma cells producing the antibody.

39. A method for treating septicemia comprising administering to a patient in need of such treatment a composition comprising a suitable carrier and an antibody as claimed in claim 36.. A method for treating septicemia comprising administering to a patient in need of such treatment a composition comprising a suitable carrier and an antibody as claimed in claim 3.

41. A method for treating septicemia comprising administering to a patient in need of such treatment a composition comprising a suitable carrier and an antibody as claimed in claim 38.

42. A method for binding to receptors in i competition with Lipid A in a patient in need of such I binding comprising administering to said patient a composition comprising a suitable carrier and a compound as claimed in any one of claims 21-24.

43. A method for producing a antibody which binds to Lipid A and a compound, said compound as claimed in any one of claims 1-2 or 21-24, said method comprising: immunizing an animal with a composition comprising the compound, isolating spleen cells from the animal, amplifying at least one gene fragment encoding all or part of both heavy and light chains of at least one antibody from said spleen cells, _I j WO 93/19761 PCT/US93/02903 127 inserting said gene fragment in a recombinatoral fashion into a viral vector, producing a library of viable virus particles which express a protein derived from the gene fragment, and screening the library for binding and/or catalytic activity by expressed antigen binding protein of the gene fragment.

44. An antibody which binds to Lipid-A and a compound, said compound as claimed in any one of claims 1-2 or 21-24, said antibody having been produced by a process comprising: immunizing an animal with a composition comprising the compound, amplifying at least one gene fragment encoding all or part of both heavy and light chains of at least one antibody from said spleen cells, inserting said gene fragment in a recombinatoral fashion into a viral vector, producing a library of viable virus particles which express a protein derived from the gene fragment, and screening said library for binding and/or catalytic activity by expressed antigen binding protein of the gene fragment.

45. The method of claim 43 wherein the antibody-encoding gene is expressed on the surface of the virus.

46. The antibody of claim 44 wherein the antibody-encoding gene is expressed on the surface of the virus.

47. The antibody of claim 46 wherein the antibody is catalytic.

48. A method for treating septicemia comprising a administering to a patient in need of such treatment a composition comprising a suitable carrier and a antibody as claimed in claim 46. I IWdSLn1I-e I A i i, Galanos, C. and Luderitz, 0. Tetrahedron Lettl983, WO 93/19761 PCT/US93/02903 128

49. A method for producing an antibody which binds to Lipid A and a compound, said compound as claimed in any one of claims 1-2 or 21-24, said method comprising: immunizing an animal with the compound and a T- cell stimulating peptide incorporated into liposomes, removing antibody-producing lymphocytes from the animal, and fusing the lymphocytes with myeloma cells and thereby producing hybridoma cells producing the antibody. The method of claim 49 wherein the T-cell stimulatory peptide comprises HELl105-120].

51. The antibody of claim 16 wherein the immunizing comprises immunizing the animal with the compound and a T-cell stimulating peptide incorporated into liposomes.

52. The antibody of claim 38 wherein the immunizing comprises immunizing the animal with the compound and a T-cell stimulating peptide incorporated into liposomes.

53. The method of claim 43 wherein the Simmunizing comprises immunizing the animal with the compound and a T-cell stimulating peptide incorporated into liposomes.

54. The antibody of claim 44 wherein the immunizing comprises immunizing the animal with the compound and a T-cell stimulating peptide incorporated into liposomes.

55. The method of claim 3 wherein said administering comprises administering the compound and a T-cell stimulating peptide incorporated into a liposome.

56. The method of claim 25 wherein said administering comprises administering the compound and a T-cell stimulating peptide incorporated into a liposomes.

57. An antibody which catalyzes the hydrolysis of a chemical bond in the Lipid-A moiety of LPS. WO 93/19761 PCI/US93/02903 129

58. A method for producing an antibody which binds to Lipid-A and a compound, said compound as claimed in any one of claims 1-2 or 21-24, said method comprising: amplifying at least one gene fragment encoding all or part of both heavy and light chains of at least one antibody from peripheral lymphocytes, inserting said gene fragment in a recombinatoral fashion into a viral vector, producing a library of viable virus particles which express a protein derived from the gene fragment, and screening the library for binding and/or catalytic activity by expressed antigen binding protein of the gene fragment.

59. An antibody which bonds to Lipid-A and a compound, said compound as claimed in any one of claims 1-2 or 21-24, said antibody having been produced by a process comprising: amplifying at least one gene fragment encoding all or part of both heavy and light chains of at least one antibody from peripheral lymphocytes, inserting said gene fragment in a recombinatoral fashion into a viral vector, producing a library of viable virus particles A 25 which express a protein derived from the gene fragment, and screening the library for binding and/or catalytic activity by expressed antigen binding protein I of the gene fragment. The antibody of claim 43 wherein the antibody is catalytic. 1 ,it 130

61. A compound of the formula as set out in claim 1, substantially as hero-,abfore described with reference to any one of Examples 1 to 70, 77 to 86, or 95 to

109. 62. An antibody which binds to both Lipid-A and a compound of the formula (I) as set out in claim 1, said antibody, substantially as hereinbefore described with reference to any one of Examples 71 to 76, or 87 to 94. 63. A process for producing a compound of the formula as set out in claim 1, substantially as hereinbefore described with reference to any one of Examples 1 to 70, 77 to 86, or 95 to 109. 64. A process for producing an antibody which binds to Lipid-A and a compound of the formula as set out in claim 1, said process, substantially as hereinbefore described with reference to any one of Examples 71 to 76, or 87 to 94. Dated 6 February, 1996 Igen, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON I,, S I I I, r [N\LIBU]17860:KBH