WO2006088824A2 - Compounds useful for treating hiv - Google Patents

Abstract

Compounds, e.g., of the Formulae (Ia), (Ib) and (Ic) are useful for treating HIV infection.

Description

COMPOUNDS USEFUL FOR TREATING HIV

This application claims the benefit of the filing date of U.S. Provisional Application Serial No. 60/652,742 filed February 15, 2005, which is incorporated by reference herein.

Tremendous progress has been made over the years in developing antiretroviral therapy in order to reduce morbidity and mortality in HIV-1 infected individuals. However, emergence of viral resistance to protease as well as reverse transcriptase inhibitors and more recently to fusion inhibitors has posed new challenges to develop new classes of potent and safe antiretroviral drugs to help achieve sustained suppression of viral replication.

SUMMARY OF THE INVENTION

The present invention relates to the treatment of HIV (e.g., for inhibition of HIV-1 RNase H) comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formulae:

or

wherein R¹ is

OH, =O, -O-CO-CH2-C-(CH₃)₂COOH, -CO-CH₂-C-(CH₃)₂COOH,

R² is

Z is H, a basic cation forming a pharmaceutically acceptable salt, C-ι-₆ alkyl, C₂-₆ alkenyl, C₁-6 alkoxyalkyl or -CO2C1-6 alkyl and the like ester groups.

V, X and Y are identical or different and each is H, OH₁ C1-6 alkyl, Ci_-6 alkoxy, Ci-6 alkoxyalkyl, C₂-e alkenyi, halogen (F, Cl, Br, I), COOH, -CO₂(Ci_-6 alkyl), -CF₃, CF₃O- , CH₃C(O)-, CN; CH₃SO₂-, CF₃SO₂-, -NH₂, -NH(Ci_-6 alkyl); -NO₂, -NH-C(=O)-CH₃, -NH-C(=O)-Ci_-6 alkyl, -NHSO₂(C_1-6 alkyl), -CH=NO(Ci_-6 alkyl), -NH-COCF₃, -NHCO₂(Ci_-6 alkyl), -OCF₃, -CO(C_1-6 alkyl), -CHF₂, -CH₂F, -N-OH, -S(Ci_-6 alkyl), -SO(Ci_-6 alkyl), -SO₂(Ci_-6 alkyl), -SO₂NH₂, -SO₂NH(Ci_-6), -O(Ci_-6alkyl), -NHC(=O)CH=CHC(=O)ONa, -NHC(=O)CH=CHC(=O)OH, -OSO₂CF₃ , or together two of V, X and Y form together with the C atoms to which they are attached, a five or six membered ring having 1 or 2 O, N or S (preferably O) atoms therein.

Preferred R² groups in formulae (Ia), (Ib) and (Ic) include:

Preferred Z groups of formulae (Ia), (Ib) or (Ic) include H or Na. Preferred V, X and Y groups of formulae (Ia), (Ib) or (Ic) include OH, H, OCH₃, -NH-C(=O)-CH=CH-CO₂Na, -CO₂H, -NHC(=O)CH₃, F₁ Cl, Br, Or -NHCH₃.

The preferred R¹ group of formulae (Ia)₁ (Ib) or (Ic) is OH.

The invention also relates to pharmaceutical compositions containing compounds of formulae (Ia), (Ib) and/or (Ic). These can contain only one or more compounds of the invention. They can also be administered as a single dosage form or simultaneously or sequentially as separate dosage forms, typically with one or more, preferably one to eight, other anti-viral agents useful in anti-HIV-1 therapy. The antiviral agents contemplated for use in combination with the compounds of the present invention comprise nucleoside and nucleotide reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors of HIV-1 and other antiviral drugs for treating HIV-1 not falling within these classifications.

The present invention also relates to the treatment of HIV comprising administering to a human in need thereof a therapeutically effective amount of an RNase H inhibitor represented by formulae (Ia), (Ib) or (Ic), and/or a compound of the following chemical series: di and tricaffeolyquinic acids and derivatives, caffeic glucoside esters and derivatives, rosmarinic acid derivatives, triterpenes, lignans and coumarins the structures of which are presented below.

The following Table lists compounds of the present invention. The biochemical and cellular assay's are described in example III. . Table 1. Compounds of the present invention

The detail of semi-synthesis of compounds 32, 36-38 is described below.

The compounds of the present invention are especially useful for treating HIV infection. They are preferably administered in substantially pure form. By "substantially pure" is meant that the compound is either synthesized or purified from a naturally occurring state such that it has only trace quantities of materials that occur with it in the natural state (e.g., amounts whereby no effect on a patient can be observed). By "isolated" is meant that the compound is in a state other than the natural state. If desired, synthesized, isolated or purified compounds may be present along with a pharmaceutical carrier as described below.

The term "alkyl" is used herein at all occurrences (as a group per se or a part of a group e.g., alkoxy etc.) to mean straight or branched chain alkyl groups of 1 to 6 carbon atoms, unless the chain length is otherwise specified or limited, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and the like.

Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, sec.-butoxy, isobutoxy, tert.-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, 2,2-dimethylpropoxy, heptoxy. and the like.

Alkenyl represents C₂-C₆ carbon chains having one or two unsaturated bonds, provided that two unsaturated bonds are not adjacent to each other.

The term "halogen" herein means chlorine, fluorine, iodine or bromine.

Many of the compounds of the present invention can be isolated from natural sources such as, for example, Melianthus comosus, Rhoiptelea chiliantha, Myrica cerifera, Tecoma stans, Melissa officinalis, Antennaria canadensis and Ochroma pyramidale. Several suitable methods for extracting the compounds of the present invention are known in the art. A highly preferred extraction method typically comprises crushing of the plant material (e.g., plant cell culture), blending the crushed material with a polar, preferably aqueous, solvent, filtering the slurry of crushed material and polar solvent and purifying the filtrate. Several purification methods like one-step or multi-step liquid-liquid extraction, solid phase extraction, chromatography and the like are known from the state of the art.

Compounds according to the invention can also be synthesized. The reaction schemes disclosed herein illustrate such syntheses. The other compounds of the invention can be made analogously, including using other conventional chemical reactions.

The compounds according to the invention can also be semi-synthesized. For example, the semi-synthetic synthesis of the myricerol analogs described in Schemes 9-14 represents a significant improvement in the development of these and other compounds of this class. For example, the conventional complete chemical synthesis of compound 39 requires 10 synthetic steps from oleanolic acid (see J. Org. Chem 1997, 62: 960-966). However, according to the present invention, synthesis of the compounds of the invention can be done in far fewer steps. For example, isolation of compounds from plant cell culture technology allows production of compound 39 in a single saponification step from compound 31. See for example, Scheme 8. This represents a major breakthrough in the availability of starting materials for further semi-synthetic steps for the production of the compounds of the present invention.

Secondly, the synthetic methodology developed and described in Schemes 9-15 also represents a marked improvement, from 16 steps (see Organic Process Research and Development 1999, 3: 347-351) to 9 steps herein, from the previously described synthesis of compound 36. This improvement is important both in its synthetic simplicity and specificity to achieve the desired compounds. These syntheses allow an even wider variety of semi-synthetic compounds to be produced. Overall, the semi-synthetic routes described herein represent a technological advancement for the development of these and other RNase inhibitors.

As a matter of convenience, the following description of the preparation of several preferred compounds of the invention is shown below:

Organic Transformation of some Active Compounds

Compound 31 is treated with 5 % of sodium hydroxide in methanol to furnish compound 39 (scheme 1).

Schemel

Jones oxidation of compound 31 gave compound 34 that when treated with sodium hydroxide afforded compound 40 (scheme 2).

Scheme 2

Synthesis of compounds 41-44 (scheme 3)

Scheme 3 Synthesis of compounds 26-28 (scheme 4)

Scheme 4

Semi-Synthesis of Compounds 32, 36-38 of the general formula (scheme 5)

Scheme 5

A library of compound with the general formula of scheme 5 are prepared with the appropriate cinnamic acid (scheme 6).

Scheme 6

General synthetic scheme for 27-O-trans-cinnamoyl analogs (scheme 7):

Scheme 7

Synthesis of 3-β-hydroxy-27-O-trans-(3-hydroxycaffeoyl) myriceroi (scheme 8)

In scheme 2, 31 is first saponified with LAH to myriceroi which is then esterified with cinnamic acid analogs on both C-3 and C-27 alcohol functions to afford the corresponding dimer, a selective saponification of the C-3 cinnamic ester lead to the 27-O-trans-cinnamoyl analogs. For example, this allow the synthesis of 3-β- hydroxy-27-O-trans-(3-hydroxycaffeoyl) myriceroi 37 .

Preparation of myriceroi 39 (scheme 8)

Myriceroi 39

Scheme 8

Preparation of 3-β-27-di-O-trans-(3-acetoxy caffeoyl)-myricerol. (scheme 9)

Scheme 9

Preparation of 3-β-27-O-trans-(3-hydroxy-caffeoyl) myricerol 37 (scheme 10)

Scheme 10

Synthesis of 3- β-27-O-trans-(4-hydroxy-caffeoyl) myricerol 38 (scheme11)

This method is amendable to a variety of other compounds. For example, compound 38 is prepared according to the same above experimental conditions.

The synthesis of the compounds of the general formula (Scheme 11)

Scheme 11

A library of compounds with the above general formula of scheme 11 are prepared as below (scheme 12).

In scheme 12, 31 is first saponified with LAH to get myricerol, diacetylation of myricerol followed by selective hydrolysis of diacetyl myricerol gave 27-0- acetylmyricerol, which is oxidized with Jones reagent followed by removal of the acetyl to afford myricerone which is converted to the corresponding phosphonate, a Horner-Wadsworth-Emmons (HWE) olefination of the phosphonate in the presence of a library of aldehydes afforded a variety of α, β- unsaturated cinnamic ester (Ref 31 : J. Org. Chem. 1997, 62, 960-966 ; Organic Process Researchδc Development 1999, 3, 347-351). When Z is methyl ester, the cinnamic ester underwent a selective methyl ester saponification with LiOH to afford the corresponding acids, which are converted to double salt upon treatment with 2 equiv of aqueous NaOH.

Scheme 12

The above methodology can be used to synthesize a variety of compounds as determined in the following.

Synthesis of (+)-Disodium 27-((E)-3-(2-((E)-3-carboxypropenoylamino)-5- hydroxyphenyl)propenoyloxy)3-oxoolean-12-ene-28-oate; (+)-Disodium 27-((E)- 3-(2-((E)-3-carboxypropenoylamino)-5-hydroxyphenyl)propenoyloxy)3-oxoolean- 12-ene-28-oate 36 ;

This following sequence of synthesis is made from the phosphonate of myricerone according to the experimental conditions of ref 31 (scheme13)

Scheme13

Scheme 14

This compound could be prepared in the same manner as (+)-Disodium 27-((E)- 3-(2-((E)-3-carboxypropenoylamino)-5-hydroxyphenyI)propenoyloxy)3-oxoolean- 12-ene-28-oate starting from 4, 5 dihydroxy-2-(3-methoxycarbonylacryIoylamino) benzaldehyde.

Preparation of 4,5 dihydroxy-2(3-methoxycarbonyIacryloylamino) benzaldehyde.

The 4,5-dihydroxy-2-nitrobenzaldehyde is synthesized from 6-nitropiperonal (Ref 32: JOC 1980, 45, 2750) which is then subjected to the reduction of the nitro function to the corresponding amino which upon addition of trans-β- methoxycarbonyl acryloyl chloride afforded the desired 4, 5 dihydroxy-2(3- methoxycarbonylacryloylamino) benzaldehyde (scheme 15).

Scheme 15

One of ordinary skill in the art will recognize that some of the compounds of the present invention can exist in different geometrical isomeric forms. In addition, some of the compounds of the present invention possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers, as well as in the form of racemic or nonracemic mixtures thereof, and in the form of diastereomers and diastereomeric mixtures inter alia. All of these compounds, including cis isomers, trans isomers, diastereomic mixtures, racemates, nonracemic mixtures of enantiomers, and substantially pure and pure enantiomers, are within the scope of the present invention. Substantially pure enantiomers contain no more than 5% w/w of the corresponding opposite enantiomer, preferably no more than 2%, most preferably no more than 1%.

The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example, by the formation of diastereoisomeric salts using an optically active acid or base or formation of covalent diastereomers. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. Mixtures of diastereoisomers can be separated into their individual diastereomers on the basis of their physical and/or chemical differences by methods known to those skilled in the art, for example, by chromatography or fractional crystallization. The optically active bases or acids are then liberated from the separated diastereomeric salts. A different process for separation of optical isomers involves the use of chiral chromatography (e.g., chiral HPLC columns), with or without conventional derivation, optimally chosen to maximize the separation of the enantiomers. Suitable chiral HPLC columns are manufactured by Diacel, e.g., Chiracel OD and Chiracel OJ among many others, all routinely selectable. Enzymatic separations, with or without derivitization, are also useful. The optically active compounds of the present invention, particularly those of Formula (Ia), (Ib) or (Ic) can likewise be obtained by chiral syntheses utilizing optically active starting materials.

The present invention also relates to useful forms of the compounds as disclosed herein, such as pharmaceutically acceptable salts and prodrugs of all the compounds of the present invention. Pharmaceutically acceptable salts include those obtained by reacting the main compound, functioning as a base, with an inorganic or organic acid to form a salt, for example, salts of hydrochloric acid, sulfuric acid, phosphoric acid, methane sulfonic acid, camphor sulfonic acid, oxalic acid, maleic acid, succinic acid and citric acid. Pharmaceutically acceptable salts also include those in which the main compound functions as an acid and is reacted with an appropriate base to form, e.g., sodium, potassium, calcium, mangnesium, ammonium, and choline salts. Those skilled in the art will further recognize that acid addition salts of the claimed compounds may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts are prepared by reacting the compounds of the invention with the appropriate base via a variety of known methods. The following are further examples of acid salts that can be obtained by reaction with inorganic or organic acids: acetates, adipates, alginates, citrates, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, camphorates, digluconates, cyclopentanepropionates, dodecylsulfates, ethanesulfonates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, fumarates, hydrobromides, hydroiodides, 2-hydroxy-ethanesulfonates, lactates, maleates, methanesulfonates, nicotinates, 2-naphthalenesulfonates, oxalates, palmoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, succinates, tartrates, thiocyanates, tosylates, mesylates and undecanoates.

Preferably, the salts formed are pharmaceutically acceptable for administration to mammals. However, pharmaceutically unacceptable salts of the compounds are suitable as intermediates, for example, for isolating the compound as a salt and then converting the salt back to the free base compound by treatment with an alkaline reagent. The free base can then, if desired, be converted to a pharmaceutically acceptable acid addition salt.

The compounds of the present invention, particularly those of formulae (Ia), (Ib) or (Ic) are useful for treating HIV infection alone or with co- infections e.g., opportunistic infections such as viral, bacterial or parasitic co-infection. Thus another aspect of the invention is the administration of the compounds of the present invention, particularly those of formulae (Ia), (Ib) and/or (Ic) along with an effective amount of an agent to treat the co-infection. Typical opportunistic infections ("Ol's") and other disorders commonly present with HIV disease are bacterial and mycobacterial infections such as, for example, Mycobacterium Avium Complex (MAC, MAI), Salmonellosis, Syphilis, Neuroshyphilis, Turberculosis (TB) and Bacillary angiomatosis (cat scratch disease); fungal infections such as, for example, Aspergillosis, Candidiasis (thrush, yeast infection), Coccidioidomycosis, Cryptococcal Meningitis and Histoplasmosis; malignancies such as, for example, Kaposi's Sarcoma, Lymphoma, Systemic Non-Hodgkin's Lymphoma (NHL) and Primary CNS Lymphoma; protozoal infections such as, for example, Cryptosporidiosis, Isosporiasis, Microsporidiosis, Pneumocystis Carinii Pneumonia (PCP) and Toxoplasmosis; viral infections such as, for example, Cytomegalovirus (CMV), Hepatitis, Herpes Simplex (HSV, genital herpes), Herpes Zoster (HZV, shingles), Human Papiloma Virus (HPV, genital warts, cervical cancer), Molluscum Contagiosum .Oral Hairy Leukoplakia (OHL) and Progressive Multifocal Leukoencephalopathy (PML); neurological conditions such as, for example, AIDS Dementia Complex (ADC) and Peripheral Neuropathy. Other conditions and complications of HIV infection include, for example, Apthous Ulcers, malabsorption, depression, diarrhea, thrombocytopenia, wasting syndrome, idiopathic thrombocytopenic purpura, Listeriosis, pelvic inflammatory disease, Burkitt's lymphoma and immunoblastic lymphoma. Treatment for these opportunistic infections and other disorders are conventionally known. Suitable agents which may be co-administered or used in combination therapy with the compounds of the present invention can be found, for example, at http://www.aegis.com/topics/oi/.

The activity of the compounds of the present invention can be determined by any one of a number of known assays available to one skilled in the art . The activity of the compounds according to the invention can also be determined by assays such as those described in the Examples.

The compounds of the invention can be administered alone, but preferably as an active ingredient of a formulation. Thus, the present invention also includes pharmaceutical compositions containing a compounds of the present invention, particularly a compound of formula(la), (Ib) or (Ic), for example, and one or more pharmaceutically acceptable carriers, excipients etc.

Numerous standard references are available that describe procedures for preparing various formulations suitable for administering the compounds according to the invention. Examples of potential formulations and preparations are contained, for example, in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (current edition); Pharmaceutical Dosage Forms: Tablets (Lieberman, Lachman and Schwartz, editors) current edition, published by Marcel Dekker, Inc., as well as Remington's Pharmaceutical Sciences (Arthur Osol, editor), 1553-1593 (current edition).

In view of their high degree efficacy in reducing HIV viral load, the compounds of the present invention can be administered to anyone requiring or desiring reduction in HIV viral load, including patients having HIV with co-infection. Administration may be accomplished according to patient needs, for example, orally, nasally, parenterally (subcutaneously, intraveneously, intramuscularly, intrasternally and by infusion), by inhalation, rectally, vaginally, topically, locally, transdermal^, and by ocular administration.

Various solid oral dosage forms can be used for administering compounds of the invention including such solid forms as tablets, gelcaps, capsules, caplets, granules, lozenges and bulk powders. The compounds of the present invention can be administered alone or combined with various pharmaceutically acceptable carriers, diluents (such as sucrose, mannitol, lactose, starches) and excipients known in the art, including but not limited to suspending agents, solubilizers, buffering agents, binders, disintegrants, preservatives, colorants, flavorants, lubricants and the like. Time release capsules, tablets and gels are also advantageous in administering the compounds of the present invention.

Various liquid oral dosage forms can also be used for administering compounds of the invention, including aqueous and non-aqueous solutions, emulsions, suspensions, syrups, and elixirs. Such dosage forms can also contain suitable inert diluents known in the art such as water and suitable excipients known in the art such as preservatives, wetting agents, sweeteners, flavorants, as well as agents for emulsifying and/or suspending the compounds of the invention. The compounds of the present invention may be injected, for example, intravenously, in the form of an isotonic sterile solution. Other preparations are also possible.

Suppositories for rectal administration of the compounds of the present invention can be prepared by mixing the compound with a suitable excipient such as cocoa butter, salicylates and polyethylene glycols. Formulations for vaginal administration can be in the form of a pessary, tampon, cream, gel, paste, foam, or spray formula containing, in addition to the active ingredient, such suitable carriers as are known in the art.

For topical administration the pharmaceutical composition can be in the form of creams, ointments, liniments, lotions, emulsions, suspensions, gels, solutions, pastes, powders, sprays, and drops suitable for administration to the skin, eye, ear or nose. Topical administration may also involve transdermal administration via means such as transdermal patches.

Aerosol formulations suitable for administering via inhalation also can be made. For example, the compounds according to the invention can be administered by inhalation in the form of a powder (e.g., micronized) or in the form of atomized solutions or suspensions. The aerosol formulation can be placed into a pressurized acceptable propellant.

The compounds of the present invention, particularly a compound of formulae (Ia), (Ib) or (Ic) can be administered as the sole active agent or in combination with one or more, preferably one to eight, anti-viral agents useful in anti-HIV-1 therapy e.g., protease inhibitors, reverse transcriptase inhibitors, fusion inhibitors ("FI¹¹S) or other antiviral drugs such as ribavirin. Suitable antiviral agents which may be co-administered or used in combination therapy include, for example, suitable nucleoside and nucleotide reverse transcriptase inhibitors (¹¹NRTI" s) including, for example, zidovudine (AZT) available under the RETROVIR tradename from Glaxo-Welicome Inc., Research Triangle, N. C. 27709; didanosine (ddl) available under the VIDEX tradename from Bristol-Myers Squibb Co., Princeton, N.J. 08543; zalcitabine (ddC) available under the HIVID tradename from Roche Pharmaceuticals, Nutley, NJ. 07110; stavudine (d4T) available under the ZERIT trademark from Bristol-Myers Squibb Co., Princeton, NJ. 08543; lamivudine (3TC) available under the EPIVIR tradename from Glaxo- Wellcome Research Triangle, N.C. 27709; abacavir (1592U89) disclosed in WO96/30025 and available under the ZIAGEN trademark from Glaxo-Wellcome Research Triangle, N.C. 27709; adefovir dipivoxil [bis(POM)-PMEA] available under the PREVON tradename from Gilead Sciences, Foster City, Calif. 94404; lobucavir (BMS-180194), a nucleoside reverse transcriptase inhibitor disclosed in EP-0358154 and EP-0736533 and under development by Bristol-Myers Squibb, Princeton, NJ. 08543; BCH-10652, a reverse transcriptase inhibitor (in the form of a racemic mixture of BCH-10618 and BCH-10619) under development by Biochem Pharma, Laval, Quebec H₇ V, 4A7, Canada; emitricitabine [(-)-FTC] licensed from Emory University under Emory Univ. U.S. Pat. No. 5,814,639 and under development by Triangle Pharmaceuticals, Durham, N.C. 27707; beta-L- FD4 (also called beta-L-D4C and named beta-L-2',3'-dicleoxy-5-fluoro-cytidene) licensed by Yale University to Vion Pharmaceuticals, New Haven Conn. 06511 ; DAPD, the purine nucleoside, (-)-beta-D-2,6,-diamino-purine dioxolane disclosed in EP 0656778 and licensed by Emory University and the University of Georgia to Triangle Pharmaceuticals, Durham, N.C. 27707; and lodenosine (FddA), 9-(2,3- dideoxy-2-fluoro-b-D-threo-pentofuranosyl)adenine, an acid stable purine-based reverse transcriptase inhibitor discovered by the NIH and under development by U.S. Bioscience Inc., West Conshohoken, Pa. 19428.

Typical suitable non-nucleoside reverse transcriptase inhibitors ("NNRTI¹¹S) include nevirapine (BI-RG-587) available under the VIRAMUNE tradename from Boehringer Ingelheim, the manufacturer for Roxane Laboratories, Columbus, Ohio 43216; delaviradine (BHAP, U-90152) available under the RESCRIPTOR tradename from Pharmacia & Upjohn Co., Bridgewater NJ. 08807; efavirenz (DMP-266) a benzoxazin-2-one disclosed in WO94/03440 and available under the SUSTIVA tradename from DuPont Pharmaceutical Co., Wilmington, Del. 19880-0723; PNU-142721 , a furopyridine-thio-pyrimide under development by Pharmacia and Upjohn, Bridgewater N.J. 08807; AG-1549 (formerly Shionogi #S- 1153); 5-(3,5-dichlorophenyl)-thio-4-isopropyl-1-(4-pyridyl)methyl-1H-imidazol-2- ylmethyl carbonate disclosed in WO 96/10019 and under clinical development by Agouron Pharmaceuticals, Inc., LaJoIIa Calif. 92037-1020; MKC-442 (1-(ethoxy- methyO-δ-CI-methylethyO-θ-CphenylmethyO^^CIH.SHJ-pyrimidinedione) discovered by Mitsubishi Chemical Co. and under development by Triangle Pharmaceuticals, Durham, N.C. 27707; and (+)-calanolide A (NSC-675451) and B, coumarin derivatives disclosed in NIH U.S. Pat. No. 5,489,697 licensed to Med Chem Research, which is co-developing (+) calanolide A with Vita-Invest as an orally administrable product.

Protease inhibitors ("Pl") include compounds having a peptidomimetic structure, high molecular weight (7600 daltons) and substantial peptide character, e.g. CRIXIVAN(available from Merck) as well as nonpeptide protease inhibitors e.g., VIRACEPT (available from Agouron).

Typical suitable PIs include, for example, saquinavir (Ro 31-8959) available in hard gel capsules under the INVIRASE tradename and as soft gel capsules under the FORTOVASE tradename from Roche Pharmaceuticals, Nutley, N.J. 07110-1199; ritonavir (ABT-538) available under the NORVIR tradename from Abbott Laboratories, Abbott Park, III. 60064; indinavir (MK-639) available under the CRIXIVAN tradename from Merck & Co., Inc., West Point, Pa. 19486-0004; nelfnavir (AG-1343) available under the VIRACEPT tradename from Agouron Pharmaceuticals, Inc., LaJoIIa Calif. 92037-1020; amprenavir (141W94), tradename AGENERASE, a non-peptide protease inhibitor under development by Vertex Pharmaceuticals, Inc., Cambridge, Mass. 02139-4211 and available from Glaxo-Wellcome, Research Triangle, N.C. under an expanded access program; lasinavir (BMS-234475) available from Bristol-Myers Squibb, Princeton, N.J. 08543 (originally discovered by Novartis, Basel, Switzerland (CGP-61755); DMP-450, a cyclic urea discovered by Dupont and under development by Triangle Pharmaceuticals; BMS-2322623, an azapeptide under development by Bristol-Myers Squibb, Princeton, N.J. 08543, as a 2nd-generation HIV-1 Pl; ABT- 378 under development by Abbott, Abbott Park, III. 60064; and AG-1549 an orally active imidazole carbamate discovered by Shionogi (Shionogi #S-1153) and under development by Agouron Pharmaceuticals, Inc., LaJoIIa Calif. 92037-1020.

Other antiviral agents include, for example, hydroxyurea, ribavirin, IL-2, IL-12, pentafuside and Yissum Project No.11607. Hydroyurea (Droxia), a ribonucleoside triphosphate reductase inhibitor, the enzyme involved in the activation of T-cells, was discovered at the NCI and is under development by Bristol-Myers Squibb; in preclinical studies, it was shown to have a synergistic effect on the activity of didanosine and has been studied with stavudine. IL-2 is disclosed in Ajinomoto EP-0142268, Takeda EP-0176299, and Chiron U.S. Pat. Nos. RE 33,653, 4,530,787, 4,569,790, 4,604,377, 4,748,234, 4,752,585, and 4,949,314, and is available under the PROLEUKIN (aldesleukin) tradename from Chiron Corp., Emeryville, Calif. 94608-2997 as a lyophilized powder for IV infusion or sc administration upon reconstitution and dilution with water; a dose of about 1 to about 20 million IU/day, sc is preferred; a dose of about 15 million IU/day, sc is more preferred. IL-12 is disclosed in WO96/25171 and is available from Roche Pharmaceuticals, Nutley, NJ. 07110-1199 and American Home Products, Madison, N.J. 07940; a dose of about 0.5 microgram/kg/day to about 10 microgram/kg/day, sc is preferred. Pentafuside (DP-178, T-20) a 36-amino acid synthetic peptide, disclosed in U.S. Pat. No. 5,464,933 licensed from Duke University to Trimeris which is developing pentafuside in collaboration with Duke University; pentafuside acts by inhibiting fusion of HIV-1 to target membranes. Pentafuside (3-100 mg/day) is given as a continuous sc infusion or injection together with efavirenz and 2 Pi's to HIV-1 positive patients refractory to a triple combination therapy; use of 100 mg/day is preferred. Yissum Project No. 11607, a synthetic protein based on the HIV-1 Vif protein, is under preclinical development by Yissum Research Development Co., Jerusalem 91042, Israel. Ribavirin, 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamicle, is available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif; its manufacture and formulation are described in U.S. Pat. No. 4,211,771.

The term "anti-HIV-1 therapy" as used herein means any anti-HIV-1 drug found useful for treating HIV-1 infections in man alone, or as part of multidrug combination therapies, especially the HAART triple and quadruple combination therapies. Typical suitable known anti-HIV-1 therapies include, but are not limited to multidrug combination therapies such as (i) at least three anti-HIV-1 drugs selected from two NRTIs, one Pl, a second Pl, and one NNRTI; and (ii) at least two anti-HIV-1 drugs selected from NNRTIs and PIs. Typical suitable HAART- multidrug combination therapies include:

• (a) triple combination therapies such as two NRTIs and one Pl; or (b) two NRTIs and one NNRTI; and (c) quadruple combination therapies such as two NRTIs, one Pl and a second Pl or one NNRTI. In treatment of naive patients, it is preferred to start anti-HIV-1 treatment with the triple combination therapy; the use of two NRTIs and one Pl is prefered unless there is intolerance to PIs. Drug compliance is essential. The CD4+ and HIV-1 -RNA plasma levels should be monitored every 3-6 months. Should viral load plateau, a fourth drug, e.g., one Pl or one NNRTI could be added. Typical therapy schemes are described below:

ANTI-HIV-1 MULTI DRUG COMBINATION THERAPY SCHEMES

A. Triple Combination Therapies

• 1. Two NRTIs +one Pl

• 2. Two NRTIs +one NNRTI

B. Quadruple Combination Therapies

• Two NRTIs+one Pl+a second Pl or one NNRTI

C. ALTERNATIVES:

• Two NRTI • One NRTI +one Pl

. Two Pl's±one NRTI or NNRTI

• One Pl +one NRTI +one NNRTI

• One Pl +one NRTI +one NNRTI + one Fl

In such combinations, each active ingredient can be administered either in accordance with their usual dosage range or a dose below its usual dosage range.

The dosages of the compounds of the present invention depend upon a variety of factors including the particular syndrome to be treated, the severity of the symptoms, the route of administration, the frequency of the dosage interval, the particular compound utilized, the efficacy, toxicology profile, pharmacokinetic profile of the compound, and the presence of any deleterious side-effects, among other considerations.

The compounds of the invention are typically administered at therapeutically effective dosage levels and in a mammal an amount customary for HIV viral load reduction such as those known compounds mentioned above. For example, the compounds can be administered, in single or multiple doses, by oral administration at a dosage level of, for example, 0.01-100 mg/kg/day, preferably 0.1-70 mg/kg/day, especially 0.5-10 mg/kg/day. Unit dosage forms can contain, for example, 0.1-50 mg of active compound. For intravenous administration, the compounds can be administered, in single or multiple dosages, at a dosage level of, for example, 0.001-50 mg/kg/day, preferably 0.001-10 mg/kg/day, especially 0.01-1 mg/kg/day. Unit dosage forms can contain, for example, 0.1-10 mg of active compound. A therapeutically effective amount is an amount sufficient to lower HIV-1 RNA plasma levels. In carrying out the procedures of the present invention it is of course to be understood that reference to particular buffers, media, reagents, cells, culture conditions and the like are not intended to be limiting, but are to be read so as to include all related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another and still achieve similar, if not identical, results. Those of skill in the art will have sufficient knowledge of such systems and methodologies so as to be able, without undue experimentation, to make such substitutions as will optimally serve their purposes in using the methods and procedures disclosed herein.

The present invention will now be further described by way of the following non- limiting examples. In applying the disclosure of these examples, it should be kept clearly in mind that other and different embodiments of the methods disclosed according to the present invention will no doubt suggest themselves to those of skill in the relevant art.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications, cited above and below, are hereby incorporated by reference.

EXAMPLE I Cell line generation

For all cell lines discussed below biological material (Erigeron Canadensis, Tecoma stans, Melissa officinalis, Antennaria canadensis and Ochroma pyramidale) is obtained from commercial seed or plant sources, the Jardin de Botanique de Montreal or naturally collected. All culture protocols are based on methods traditionally used for the culture of plant cells. See for example R.A. Dixon Ed: Plant Cell Culture a practical approach.1995 IRL Press Limited, Oxford.

Biological materials (plant material, seeds, etc.) are sterilized with 5% sodium hypoclorite containing 1% surfactant (e.g. Triton X-100). Sterilized materials are placed on typical media solidified with agar (0.8%) or phytagel (0.4%). Medium consists of the inorganic salts proposed by Gamborg et a/. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158 or Mmurashige and Skoog (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497, 3% sucrose and various combinations of plant growth regulators (including 2,4- diphenoxyacetic acid, naphthaleneacetic acid, benzyladenine, kinetin, etc.) to select for suitable cell lines.

Cell lines (and medium) are selected from these plates based solely on the growth of the resulting calli, generated from the sterilized material, and are subsequently transferred (every two months) onto the selected medium. Once the cell lines are stabilized (6-8 transfers) they are initiated into liquid cultures using the identical medium as for the solid transfer without the solidifying agent. Once fully established (3 months or more) cells lines are then scaled up by serial transfer (every fourteen days) into larger Erlenmeyer flasks. Cell lines are prepared for biosynthesis of metabolites by culturing into 1 L flasks for use as inocula.

Example Il Biosynthesis

Compound biosynthesis is conducted in 4 L sterile glass bioreactors equipped with an impeller, dissolved oxygen probe, inoculating and sampling ports. These bioreactors are connected to peristaltic pumps for feeding nutrients, two mass flow controllers supply and control the oxygen and dissolved carbon dioxide concentrations and finally the gas exhaust is connected to a carbon dioxide monitor. All instrumentation is connected to a real time control system to perform advanced control algorithms and data monitoring.

All components of the bioreactor are either glass or stainless steel and are steam sterilized. Cultures are inoculated with a suitable volume of a plant cell suspension. Sampling is done every 48h for measurement of biomass and nutrient concentrations. All off-line and on-line information is collected by an expert control system allowing quality control of the bioprocess. This system also adjusts rate and composition of nutrient feeds to optimize growth and production rate. Once optimal growth is achieved secondary metabolites are induced. After 10 days of production, the culture is harvested by filtration to separate the solid phase (which contains the compounds of interest) from the medium.

The resulting cakes are extracted repeatedly (5 x) using acidified methanol (0.1% v/v acetic acid)). The resulting methanolic extracts are concentrated in vacuo and dried onto C4 stationary phase material. This C4 material is packed into injection columns and then the metabolites are separated using a SEPBOX™ , a two dimension automated preparative chromatographic system (SEPIAtec GmbH, Berlin, Germany). Standard reverse phase separation methods are used at every level. For example: compound 1 is isolated from a SEPBOX™ fraction eluted at 70% methanol/ 30% buffer in the first level of separation and at 25% acetonitrile/ 75% buffer in the second level of separation. This fraction is freed from buffer through automated solid phase extraction and eluted to a fraction collector in an organic mixture of methanol:acetonitrile:acetone (80:10:10).

Example III

Biochemical And Cell based assays

A High-Throughput screen for the HIV-1 RNase H target is carried out according to McLellan et al. (2002) Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format in BioTechniques 33: 424-429, 2002. Briefly a primer/template complex is transferred into wells of a streptavidin pre-coated microplate. In this case the immobilized complex is made up of a DIG-labeled tRNA molecule hybridized to biotinylated oligodeoxynucleotide (ODN) as described. Incubation of this complex with HIV-1 reverse transcriptase leads to degradation of this complex due to the activity of RNase H present in this enzyme and causes a signal reduction to background levels. On the other hand the presence of an inhibitor of HIV-1 RNase H in the incubation mixture leads to an increased signal relative to background levels. 10,000 fractions derived from plant cell cultures present in the library are screened and several fractions are obtained that show inhibition of HIV-1 RNase H activity. These fractions are further purified and the active compounds are identified. Finally the structures of these compounds are elucidated.

Other useful assays for use in this invention include in a cell based assay consisting of Human Peripheral Blood Mononuclear Cells (PBMC) infected with HIV-1. Typically, fresh human PBMCs, seronegative for HIV-1 and HBV, are isolated from screened donors. Cells are pelleted and washed 2-3 times by low speed centrifugation and re-suspended in PBS (Phosphate Buffered Saline) to remove contaminating platelets. The leukophoresed blood is then diluted with Dulbeco's Phosphate Buffered Saline (DPBS) and layered over Lymphocyte Separation Medium (LSM; Cellgro^® by Mediatech, Inc.; density 1.078 +/- 0.002 g/ml; Cat. # 85-072-CL) in a 50 mL tube and then centrifuged. The buffy coat layer is gently aspirated from the resulting interface and subsequently washed with PBS by low speed centrifugation. After the third wash, cells are resuspended in RPMI 1640 supplemented with fetal bovine serum (FBS), L-glutamine, streptomycin, and phytohemagglutinin (PHA, Sigma). The cells are incubated at 37⁰C. After two days of incubation, PBMCs are centrifuged and are re- suspended in RPMI 1640 with FBS, L-glutamine, penicillin, streptomycin, and recombinant human IL-2 (R&D Systems, Inc). IL-2 is included in the culture medium to maintain the cell division initiated by the PHA mitogenic stimulation. For the standard PBMC assay, PHA stimulated cells from at least two normal donors are pooled, diluted in fresh media and plated in the interior wells of a 96 well round bottom microplate. Pooling PBMCs from more than one donor is used to minimize the variability observed between individual donors, resulting from quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 activation of primary lymphocyte populations. Each plate contains PBMC/virus control wells (cells + virus), experimental wells (drug + cells + virus) and compound control wells (drug + media, no cells). Test drug dilutions are prepared in microtiter tubes and each concentration is placed in appropriate wells using a standard format. Following addition of the drug dilutions to the PBMCs, a predetermined dilution of virus stock is then placed in each test well at a final Multiplicity of Infection (MOI) of approximately 0.1. The low passage, lymphotropic clinical isolate HIVJEKI is used in all assays. This clinical isolate is obtained from a pediatric patient . Since HIV-1 is not cytopathic to PBMCs, the same assay plate is used for both antiviral efficacy and cytotoxicity measurements. The PBMC cultures are maintained for seven days following infection at 37⁰C, 5% CO₂. After this period, cell-free supernatant samples are collected for analysis of reverse transcriptase activity and/or HIV p24 content. Following removal of supernatant samples, compound cytotoxicity is measured by addition of MTS to the plates for determination of cell viability. Wells are also examined microscopically and any abnormalities are noted. IC₅₀ (50% inhibition of virus replication), TC₅₀ (50% cytotoxicity) and Therapeutic Index (Tl = TC50/IC50) values are measured.

Example IV Chemistry

General Protocol for Isolation of Active compounds from Plant cell cultures

Compounds 1-4 are isolated from the extract of Erigeron canadensis plant cell cultures. The active fractions identified by HTS are further purified using preparative HPLC (C-18 Luna, ACE and/or Synergy columns) with water/acetonitrile gradients. Active fractions identified from Tecoma stans, Melissa officinalis, Antennaria canadensis and Ochroma pyramidale are treated in the same way as Erigeron canadensis to isolate, respectively, the following compounds: 5-9, 10-17, 18-24 and 31, 33. Structures of the isolated compounds are established according to their 1 D and 2D NMR data.

Example V

Betulinic acid, compound 39 and 40 are treated with 2,2-dimethylsuccinic anhydride in pyridine in the presence of 4-dimethylaminopyridine to furnish the corresponding 3-0 and/or 27-O (3',3-dimethylsuccinyl) derivative compounds 41- 44 (see scheme 3).

A solution of compound 40 (6 mg, 0.013 mmol) with dimethylaminopyridine (1.6 mg, 0.011 mmol) and 2,2-dimethyisuccinic anhydride in anhydrous pyridine (5 ml) is refluxed over night. The reaction mixture is diluted with ice water and extracted with EtOAc. The organic layer is washed with water and dried over MgSO₄, and concentrated under reduced pressure. The residue is chromatographed using a preparative HPLC column (ACE, 150x21.2 mm, 10 urn) with water/acetonitrile gradient to afford compound 41.

¹H NMR (CD₃OD, 500 MHZ) δ : 5.57 (t, 1 H, J=3.5 Hz); 4.30 (d, 1H, J=12.5 Hz); 4.08 (d, 1 H, J=12.5 Hz); 2.93 (dd, 1H, Ji=3.5, J₂=13.5 Hz); 2.60(d, 1H, J=16.0 Hz); 2.50 (d, 1 H, J=16.0 Hz); 1.27 (s, 3H); 1.25 (s, 3H); 1.08 (s, 3H); 1.06 (s, 3H); 1.04 (s, 3H); 0.95 (s, 3H); 0.88 (s, 3H); 0.86 (s, 3H).

Example Vl

Compounds 26-30 are prepared by condensing the corresponding acid and amine in the presence of N-hydroxysuccinimide and dicyclohexylcarbodiimide in dimethylformamide (see scheme 4).

To a solution of caffeic acid (155.5 mg, 0.86 mmol), tyrosine (200.0 mg, 0.86 mmol) and N-hydroxysuccinimide (248.3 mg, 2.2 mmol) in dimethylformamide (DMF, 7 ml) is added dicyclohexylcarbodiimide (DCC, 356.1 mg, 1.7 mmol) in DMF (1 ml), the mixture being stirred for 24 hours at room temperature. The reaction mixture is then poured into water and extracted with EtOAc. The resulting extract is ished with 5% NaHCO₃ and evaporated to give a crude amide, which is purified using preparative HPLC column (Synergy 150x21.2 mm, 4 urn) with water/acetonitrile gradient to afford 27.

¹H NMR (CD₃OD, 500 MHZ) δ : 7.36 (d, 1H, J=15.5 Hz); 7.03 (d, 1 H, J=8.5 Hz); 6.99 (d, 1H, J=1.5 Hz); 6.90 (dd, 1H, J^=2.0, J₂=8.5 Hz); 6.76 (d, 1 H, J=8.5 Hz); 6.70 (d, 1 H, J=8.5 Hz); 6.41 (d, 1H, J=15.5 Hz); 4.71 (m, 1 H); 3.08 (dd, 1H, J_Ϊ=6.0, J₂=13.7 Hz); 2.94 (dd, 1 H, J,=6.0, J₂=I 3.7 Hz).

Example VII

Synthesis of 3-β-hydroxy-27-O-trans-(3-hydroxycaffeoyl) myricerol In scheme 2, 31 is first saponified with LAH to myricerol which is then esterified with cinnamic acid analogs on both C-3 and C-27 alcohol functions to afford the corresponding dimer, a selective saponification of the C-3 cinnamic ester lead to the 27-O-trans-cinnamoyl analogs. For example, this allow the synthesis of 3-β- hydroxy-27-O-trans-(3-hydroxycaffeoyl) myricerol 37 (see Scheme 8).

Example VIII

Preparation of pure myricerol

To a solution of 31 (0.435 g, 0.686 mmoles) in dry ether (435 ml) is added dropwise LAH (1 M in THF, 2.75 ml), the mixture is heated at 48⁰C (4 hours), it is then quenched at 0 ⁰C by the addition of water (10 ml) followed with a solution of sulfuric acid (1 M, 1.5 ml). The two layers are separated and the aqueous layer is ished with ether (2.50 ml), The ether layer is dried with MgSO₄, filtered off, the solvent is evaporated to dryness under vacuum. The obtained residue is purified over Silica gel (gradient : Hexane/EtOAc: 5/1 to 0/1) to afford a white solid which upon recrystallisation in methanol gave pure myricerol 39 (44 % yield) (see scheme 8).

¹H NMR (CD₃OD, 500 MHZ) δ : 5.61 (s, 1 H); 3.73 (d, 1H, J=12 Hz); 3.46 (d, 1 H, J=12.5 Hz); 3.14 (d, 1 H, J=11 Hz); 2.91 (d, 1H, J=10.5 Hz); 1.95-0.83 (m, 22 H); 0.97; 0.96; 0.92; 0.91 ; 0.77 (S, 6*CH3).

Example IX

Preparation of 3-β-27-di-O-trans-(3-acetoxy caffeoyl)-myricerol (see Scheme 9)

To a solution of myricerol 39 (37 mg, 0.078 mmoles) in dry dichloromethane (4 ml) is added 3-acetoxychlorocinnamic acid chloride in 0.48 ml of dichloromethane (52 mg, 0.234 mmoles) followed with DMAP (47 mg, 0.39 mmol) ), the mixture is heated at 50 ⁰C for 13 H, the solvent is then evaporated under vacuum to dryness. The obtained crude material is solubilised in THF (10 ml) and NaOH (0.1 N, 3,7 ml) is added, the mixture is stirred for 10 min, it is then diluted with EtOAc (10 ml), the pH of the solution is adjusted to 3 with a solution of HCMN.

The solution is extracted with EtOAc, the organic layer is dried with MgSO₄, filtered, and the solvent is evaporated to dryness under vaccum. The obtained residue is purified over Silica gel to afford the desired dimer (13 mg).

¹H NMR (CDCI₃, 500 MHZ) δ : 12.18 (s, 1H); 7.61 (d, 1H, J=16.5 Hz); 7.47-7.05 (m, 9 H); 6.41 (d, 1H, J=15.5 Hz); 6.36 (d, 1H, J=16 Hz); 5.64 (s, 1H); 4.59 (m, 1H); 4.36 (d, 1 H, J= 13 Hz); 4.17 (d, 1H, J=12.5 Hz); 2.91 (d, 1H, J=12 Hz); 2.33; 2.30 (s, 2*CH3); 1.98-0.06 (m, 22 H); 0.98; 0.93; 0.90; 0.85; 0.80 (S, 6*CH3).

Example X

Preparation of 3-β-27-O-trans-(3-hydroxy-caffeoyl) myricerol 37 (see scheme 10) and 3- β-27-O-trans-(4-hydroxy-caffeoyl) myricerol

To a solution of 3- β -27-di-O-trans-(3-acetoxy caffeoyl) myricerol (Dimer) (10 mg, 0.01118 mmol) in a solution of KOH (5 % in MeOH, 2.5 ml) is refluxed for 1 hour. The mixture is diluted with EtOAc (10 ml) and the pH of the solution is adjusted to 3 with a solution of HCI (1 N).

The solution is extracted with EtOAc, the organic layer is dried with MgSO₄, filtered off, and the solvent is evaporated to dryness under vacuum. The obtained residue is purified by HPLC to afford the desired compound 37 (3 mg).

¹H NMR (CDCI₃, 500 MHZ) δ : 7.56 (d, 1 H, J=16 Hz); 7.27 (m, 1H); 7.09 (d, 1H, J= 7.5 Hz); 7.0 (s, 1H); 6.88 (d, 1H, J=8 Hz); 6.34 (d, 1H, J=16 Hz); 5.63 (S, 1H); 4.34 (d, 1H, J=13 Hz); 4.16 (d, 1H, J= 12.5 Hz); 3.21-3.18 (m, 1 H); 2.91 (d, 1H, J=13 Hz); 1.98-0.07 (m, 22 H); 0.97; 0.92; 0.84; 0.78; 0.77 (S, 6*CH3).

This method is amendable to a variety of other compounds. For example, 3- β- 27-O-trans-(4-hydroxy-caffeoyl) myricerol 38 is prepared according to the same above experimental conditions

¹H NMR (CD₃OD, 500 MHZ) δ : 7.57 (d, 1H, J=16 Hz); 7.43 (d, 1H; J=8.5Hz); 6.81 (d, 1H, J= 8.5 Hz); 6.23 (d, 1 H, J=15.5 Hz); 5.60 (s, 1H); 4.41 (d, 1H, J=12 Hz); 4.14 (d, 1H, J= 13 Hz); 3.12-3.08 (m, 1H); 2.94 (d, 1H, J=10.5 Hz); 2.00- 0.80 (m, 22 H); 0.96; 0.95; 0.93; 0.84; 0.83; 0.76 (S, 6*CH3).

Example Xl

Synthesis of (+)-Disodium 27-((E)-3-(2-((E)-3-carboxypropenoylamino)-5~ hydroxyphenyl)propenoyloxy)3-oxoolean-12-ene-28-oate

Preparation of 3,27-diacetate of myricerol

A solution of myricerol 39, previously described, (0.5 g, 1.05 mmoles) in a mixture of pyridine (7 ml) and acetic anhydride (7 ml) is stirred overnight at room temperature, after usual work up the crude is purified on silica gel to afford 0.55 g of 3,27 diacetate of myricerol.

¹H NMR (CD₃OD, 500 MHZ) δ : 5.54 (s, 1H); 4.44 (m, 1 H); 4.29 (d, 1 H, J=12.5 Hz); 4.06 (d, 1 H, J=13 Hz); 2.91 (d, 1H, 13.5 Hz); 2.02; 2.00 (s, 2*CH3); 1.93- 0.80 (m, 22 H); 0.97; 0.95; 0.88; 0.87; 0.80 (S, 6*CH3).

Example XII

Preparation of 27-acetate of myricerol A solution of 3, 27-diacetate of myricerol (0.55 g, 1 mmoles) in 5% potassium hydroxide in MeOH (165 ml) is stirred for 3 H, the pH of the solution is adjusted to 3 with a solution of HCI 1N and the mixture is extracted with EtOAc, the organic layer is dried with MgSO₄, filtered off, the solvent is evaporated to dryness under vaccum, the crude obtained is purified on silica gel to afford 0.27 g of 27-acetate of myricerol.

¹H NMR (CDCI₃, 500 MHZ) δ : 5.59 (s, 1H); 4.19 (d, 1H, J=12 Hz); 4.06 (d, 1H, J=12.5 Hz); 3.25-3.19 (m, 1H); 2.92-2.86 (m, 1H); 2.03 (s, 3H); 1.92-0.70 (m, 22 H); 0.98; 0.94; 0.90; 0.87; 0.77; 0.74; 0.71 (S, 6*CH3).

Example XlII

Preparation of 27-acetate of myricerone

A solution of 27-acetate of myricerol (0.1 g, 0.194 mmoles) in CHCI₃ (2 ml) is added Jones reagent drop wise under ice cooling, the mixture is stirred for 45 minutes then it is neutralized with H₂O and extracted with CHCL₃. The organic layer is dried with MgSO₄, filtered, and the solvent evaporated to dryness under vacuum, the crude obtained is purified on silica gel to afford 0.044 g of 27- acetate of myricerone.

¹H NMR (CDCI₃, 500 MHZ) δ : 5.60 (s, 1H); 4.21 (d, 1H, J=13 Hz); 4.06 (d, 1H, J=12.5 Hz); 2.93-2.80 (m, 1H); 2.60-2.50 (m, 1H); 2.4-2.35 (m, 1 H); 2.02 (s, 3H); 1.98-0.70 (m, 20 H); 1.09; 1.04; 1.02; 0.94; 0.88; 0.79 (S, 6*CH3).

Example XIV

Preparation of myricerone A solution of 27-acetate of myricerone (0.095 g, 0.185 mmoles) in 5% potassium hydroxide in MeOH (4 ml) is stirred overnight at room temperature, it is then diluted with EtOAc, the pH of the solution is adjusted to 3 with with a solution of HCI 1N and the mixture is extracted with EtOAc, the organic layer is dried with MgSO₄, filtered off, the solvent is evaporated to dryness under vacuum, the crude obtained is purified on silica gel to afford 0.084 g of myricerone.

¹H NMR (CDCI₃, 500 MHZ) δ : 5.90 (s, 1H); 3.78 (d, 1H, J=11.5 Hz); 3.24 (d, 1H, J=12.0 Hz); 2.94 (d, 1 H, J=13.5 Hz); 2.60-2.48 (m, 1 H); 2.42-2.36 (m, 1H); 2.10- 0.88 (m, 20H); 1.08; 1.01; 0.96; 0.91; 0.76 (S, 6*CH3).

Example XV

B- Synthesis of (+)-Disodium 27-((E)-3-(2-((E)-3-carboxypropenoylamino)-5- hydroxyphenyl)propenoyloxy)3-oxoolean-12-ene-28-oate 36

The synthesis is made from the phosphonate of myricerone according to the experimental conditions of ref compound 31 (see scheme13)

¹H NMR (D₂O, 500 MHZ) δ : 7.53 (d, 1H, J=16 Hz); 7.15 (s, 1H); 7.11 (d, 1H, J= 8.5 Hz); 6.93 (d, 1H, J=8.5 Hz); 6.86 (d, 1H, J=15.5 Hz); 6.80 (d, 1H, J= 16 Hz); 6.39 (d, 1H, J=16 Hz); 5.54 (s, 1H); 4.40 (d, 1H, J=13 Hz); 4.02 (d, 1H, J= 12.5 Hz); 2.76 (d, 1H, J=11.5 Hz); 2.47-2.42 (m, 1H); 2.25-2.21 (m, 1 H); 1.96-0.7 (m, 20 H); 0.96; 0.92; 0.82; 0.75; 0.69 (s, 6*CH3).

Example XVI

Synthesis of (+)-Disodium 27-((E)-3-(2-((E)-3-carboxypropenoylamino)-4,5- dihydroxyphenyl)propenoyloxy)3-oxoolean-12-ene-28-oate (see scheme 14) Example XVII

Preparation of 4, 5 dihydroxy-2-(3-methoxycarbonylacryloyIamino) benzaldehyde

The 4,5-dihydroxy-2-nitrobenzaldehyde is synthesized from 6-nitropiperonal (Ref 32: JOC 1980, 45, 2750) which is then subjected to the reduction of the nitro function to the corresponding amino which upon addition of trans-β- methoxycarbonyl acryloyl chloride afforded the desired 4, 5 dihydroxy-2(3- methoxycarbonylacryloylamino) benzaldehyde (see scheme 15).

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

We claim:

1. A method of treating HIV comprising administering compound number 15.