GB2319026A - HIV integrase inhibitors - Google Patents

Abstract

Natural products such as certain ermophilane sesquiterpenoids and derivatives thereof are described. These compounds are useful in the inhibition of HIV integrase, the prevention or treatment of infection by HIV and the treatment of AIDS, either as compounds, pharmaceutically acceptable salts, pharmaceutical composition ingredients, whether or not in combination with other antivirals, immunomodulators, antibiotics or vaccines. Methods of treating AIDS and methods of preventing or treating infection by HIV are also described. The novel fungal culture MF6254, Xylaria sp. (ATCC 74397) is also described and disclosed from which the compounds of the invention are isolated.

Description

TITLE OF THE INVENTION HIV INTEGRASE INHIBITORS BACKGROUND OF THE INVENTION A retrovirus designated human immunodeficiency virus (HIV) i8 the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is the insertion by virally-encoded integrase of proviral DNA into the host cell genome, a required step in HIV replication in human T-lymphoid cells.

Integration is believed to occur in three stages: cleavage of two nucleotides from the 3' termini of the linear proviral DNA; covalent joining of the recessed 3' OH termini of the proviral DNA at a staggered cut made at the host target site; repair synthesis by host enzymes.

Nucleotide sequencing of HIV shows the presence of a pol gene in one open reading frame tRatner, L. et al., Nature, 313, 227 (1985. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an integrase and an HIV protease [Toh, H. et al., EMBO J. 4, 1267 (1985). Power, M. D. et al., Science, 231, 1567 (1986); Pearl, L.H. et al., Nature 329, 351 (1987)].

It is known that some antiviral compounds act as inhibitors of HIV and are effective agents in the treatment of HIV and similar diseases, e.g., azidothymidine or AZT. Applicants demonstrate that the compounds of this invention are inhibitors of HIV integrase, by inhibiting strand transfer and cleavage activity. The particular advantage of the present invention is specific inhibition of HIV integrase.

Applicants have discovered that certain ermophilane sesquiterpenoid esters are potent inhibitors of HIV integrase. These compounds are useful for the treatment of AIDS or HIV infections.

BRIEF DESCRIPTION OF THE INVENTION Compounds of formula I, as herein defined, are disclosed.

These compounds are useful in the inhibition of HIV integrase, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment ofAIDS and/or ARC, either as compounds, pharmaceutically acceptable salts or hydrates (when appropriate), pharmaceutical composition ingredients, whether or not in combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV are also disclosed.

The compounds of formula I are obtained from the fermentation of the novel fungal culture MF6254, Xylaria sp. (ATCC 74397), which is also described and disclosed.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS This invention is concerned with compounds of formula I, combinations thereof, or pharmaceutically acceptable salts thereof, in the inhibition of HIV integrase, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS). Compounds of formula I are defined as follows:

wherein X is H, C1-4 alkyl, C1-4 alkyl substituted with a substituent selected from (a) phenyl, and (b) phenyl substituted with methyl, methoxy, halogen, or hydroxy; or a pharmaceutically acceptable salt thereof.

One embodiment of the present invention is directed to compound (A):

or a pharmaceutically acceptable salt thereof.

Also covered by the present invention are pharmaceutical compositions useful for inhibiting HIV integrase, comprising an effective amount of a compound of this invention. Pharmaceutical compositions useful for treating infection by HIV, or for treating AIDS or ARC, are also encompassed by the present invention, as well as a method of inhibiting HIV integrase, and a method of treating infection by HIV, or of treating AIDS or ARC. This invention also discloses the culture MF 6254, Xylaria sp. (ATCC 74397).

The present invention also relates to the preparation of compounds of structural formula I comprising: (a) fermenting a culture of MF 6254, Xylaria sp. (ATCC 74397) to produce a fermentation broth, (b) extracting the fermentation broth with an organic solvent, (c) purifying the organic extract to obtain the compounds of structural formula (1).

When any variable (e.g., X, etc.) occurs more than one time in any constituent or in formula I, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

The compounds of the present inventions are useful in the inhibition of HIV integrase, the prevention or treatment of infection by human immunodeficiency virus (HIV) and the treatment of consequent pathological conditions such as AIDS. Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection: AIDS, ARC (AIDS related complex), both symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the compounds of this invention are useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, exchange of body fluids, bites, accidental needle stick, or exposure to patient blood during surgery.

The compounds of this invention are useful in the preparation and execution of screening assays for antiviral compounds.

For example, the compounds of this invention are useful for isolating enzyme mutants, which are excellent screening tools for more powerful antiviral compounds. Furthermore, the compounds of this invention are useful in establishing or determining the binding site of other antivirals to HIV integrase, e.g., by competitive inhibition. Thus, the compounds of this invention are commercial products to be sold for these purposes.

Applicants have discovered that certain ermophilance sesquiterpenoids, recovered from a culture of MF6254, identified as Xylaria sp. (ATCC 74397) are useful for inhibiting HIV integrase. The compounds of formula (I) are prepared by an aerobic fermentation procedure employing a novel fungal culture MF6254 (ATCC 74397), identified as Xylaria sp., or a mutant thereof. A mutant refers to an organism in which some gene on the genome is modified, leaving the gene or genes responsible for the organism's ability to produce the compounds of formula (I) in recoverable amounts functional and heritable.

ATCC Deposit MF 6245. Xvlaria. sn.

Before the U.S. filing date of the present application, on October 16, 1996, a sample of MF 6254, Xylaria sp. had been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852. The culture access designation is ATCC 74397.

This deposit will be maintained in the ATCC for at least 30 years and will be made available to the public upon the grant of a patent disclosing it. It should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

General Charactenstics of ATCC 74397 Because of the characteristic stromata and general colony morphology, this endophytic fungus is easily placed in the genus Xylaria (Pyrenomycetes, Ascomycotina). The isolate produces no teleomorph (sexual state) or anamorph (asexual state) in culture making species identification particularly difficult. A synoptic key (Callan, B.E. and J.D. Rogers. 1992. A synoptic key to Xylaria species from continental United States and Canada based on cultural and anamorphic features.

Mycotaxon 46:141-154) to twenty-three species of Xylaria based on cultural characters was employed. However, the characteristics of MF6254 do not fit well into any of the described species. Some key characters of this isolate, such as growth rate or stroma morphology, may correspond to one of the species, but the combination of the characters together do not fit well into any of the reported species.

Therefore this isolate is designated simply as Xylaria sp.

MF6254 was isolated as an endophyte by the method of Bills and Polishook, 1991, as JP3770 from twigs of Aristolochia potheri (Aristolochiaceae)(156-88033) collected in Thailand. See, Bills, G. F. and J.D. Polishook. 1991. Microfungi from Carpinus caroliniana. Can. J.

Bot. 69(7): 1477-1482.

In the following description, all capitalized color names are from Ridgway, R. 1912. Color standards and color nomenclature.

Publ. by the author, Washington, D.C. 43 p. + 53 pl.

On oatmeal agar (Difco) colony, with center inoculation, covering a 100 mm petri dish after 21 days at 25 C and 67% relative humidity in 12 hr photoperiod in fluorescent light. Colony mat white, floccose with a scalloped appearance; stroma abundant, forming loose concentric rings, stout, 4 - 5 mm tall x 1 - 2 mm wide, velvety, when mature olivaceous (Deep Slate-Olive, Olivaceous Black); margin hyaline, indistinct; reverse light brown (Clay Color); exudate abundant, clear; soluble pigment absent.

On PDYA (potato-dextrose agar (Difco) wl 5 g(L yeastextract) covering the petri dish after 21d under the same inoculation and incubation conditions. Colony mat cottony, white, with 5 mm wide bands of black to olivaceous green (Deep Slate-Olive) mycelium; stroma rare, limited to inoculation point, 3 - 4 large, 5 x 3 mm, black; reverse brown (Clay Color, Tawny-Olive); exudate rare, small, clear droplets on mycelial tufts; soluble pigment absent.

On MEA (2% malt extract, Difco) covering the petri dish after 21d under the same inoculation and incubation conditions. Colony mat white, floccose to velvety, underlying a colony center dark green (Deep Slate-Olive), heavily dissected, an appearance of finger-like projections; margin, white, entire, with rare white tufts; reverse, exudate and soluble pigment absent.

No sporulation or conidiation observed. Hyphae thin walled, hyaline to dark brown, septate, 3-4 Rm wide.

In general, MF 6254, Xylaria sp. (ATCC 74397) is cultured on a solid medium, or in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen. For example, the cultures can be grown under submerged aerobic conditions (e.g., shaking culture, submerged culture, etc.) The aqueous medium is preferably maintained at a pH of about 6-8 at the initiation and termination (harvest) of the fermentation process. The desired pH may be maintained by the use of a buffer such as morpholinoethane-sulfonic acid (MES), morpholinopropanesulfonic acid (MOPS), and the like, or by choice of nutrient materials which inherently possess buffering properties.

The preferred source of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, sucrose, dextrin, and the like. Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, sodium succinate, and the like.

The preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cottonseed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates, and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distiller's solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g., ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use.

When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like. If necessary, especially when the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.

As to the conditions for the production of cells in massive amounts, submerged aerobic cultural conditions is one method of culturing the cells. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative forms of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism produced in a "slant" and culturing said inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, in which the inoculum is produced, is generally autoclaved to sterilize the medium prior to inoculation. The pH of the medium is generally adjusted to about 6-7 to the autoclaving step.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment, or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.

The fermentation is usually conducted at a temperature between about 200C and 300C, preferably 22-25"C, for a period of about 1$ 30 days, which may be varied according to fermentation conditions and scales.

Preferred culturing/production media for carrying out the fermentation include the media as set forth in the Examples.

After growth is completed, either the whole broth or the cells, harvested by conventional methods, e.g., centrifugation and filtration, are extracted with the appropriate solvent, e.g., methylethylketone.

The product of the present invention can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known substances. The substances produced may be found in either or both the cultured mycelium and broth filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, such as methylene chloride or methanol and the like, pH adjustment, treatment with a conventional resin (e.g., anion or cation exchange resin, nonionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like. A preferred method is extraction of cultured whole broth with methylethylketone, followed by filtration of the extract through filtering aid such as diatomaceous earth. The methylethylketone layer of the filtrate was separated and concentrated to dryness initially by evaporating under reduced pressure followed by lyophilization. The compounds were finally isolated either by solvent partitioning and crystallization or by preparative HPLC on reversed phase systems.

Compounds of formula (I) may be isolated from the aerobic fermentation of a culture of MF 6254, Xylaria sp. (ATCC 74397). A culture of MF 6254, Xylaria sp (ATCC 74397) is defined as substantially free of its natural soil contaminants and capable of forming compounds of structural formula (I) in recoverable amounts. The culture employed in the present invention should be free from viable contaminating microorganisms deleterious to the production of the compound of structural formula (I). A biologically pure culture of MF 6254, Xylaria sp (ATCC 74397) may also be employed.

The pharmaceutically acceptable salts of the compounds of this invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chl oropro caine , diethanolamine, procaine, N-benzylphenethylamine, di ethyl amine, piperazine, tris(hydroxymethyl )aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, e.g. by reacting the free acid with a suitable organic or inorganic base.

For these purposes, the compounds of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles.

The terms "administration of' and or "administering a" compound should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to the individual in need of treatment.

Thus, in accordance with the present invention there is firther provided a method of treating and a pharmaceutical composition for treating HIV infection and AIDS. The treatment involves administering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceutical carrier and a therapeutically- effective amount of a compound of the present invention.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

These pharmaceutical compositions may be in the form of orally-aflministrable suspensions or tablets, nasal sprays, sterile injectible preparations, for example, as sterile injectible aqueous or oleagenous suspensions or suppositories.

When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

The injectible solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterallyacceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable no nitiating =ipirrt, such as cocoa butter, synthetic glycerine esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

The compounds of this invention can be administered orally to humans in a dosage range of 0.1 to 1000 mgllog body weight in divided doses. One preferred dosage range is 0.1 to 200 mg/kg body weight orally in divided doses. Another preferred dosage range is 0.5 to 100 mg/kg body weight orally in divided doses. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0, 75.0, 100.0, 160.0, 150.0,200.0,250.0, 300.0,400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specified compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

The present invention is also directed to combinations of the HIV integrase inhibitor compounds with one or more agents useful in the treatment of AIDS. For example, the compounds of this invention may be effectively administered, whether at periods of preexposure and/or post-exposure, in combination with effective amounts of the AIDS antivirals, imunomodulators, antiinfectives, or vaccines, such as those in the following table.

ANTIVIRALS i:)rug Name Indication Awl 721 Ethigen ARC, PGL (Los Angeles, CA) HIV positive, AIDS Recombinant Human Triton Biosciences AIDS, Kaposi's Interferon Beta (Almeda, CA) sarcoma, ARC Acemannan Carrington Labs ARC (Irving, TX) (See also immunomodulators) Cytovene Syntex sight Ganciclovir (Palo Alto, CA) threatening CMV peripheral CMV retinitis d4T Bristol-Myers AIDS, ARC Didehydrodeoxy- (New York, NY) thymidine ddI Bristol-Myers AIDS, ARC Dideoxyinosine (New York, NY) EL10 Elan Corp, PLC HIV infection (Gainesville, GA) (See also immunomodulators) Trisodium Astra Pharm. CMV retinitis, HIV Phosphonoformate Products, Inc infection, other CMV (Westborough, MA) infections Dideoxycytidine; Hoffman-La Roche AIDS, ARC ddC (Nutley, NJ) Novapren Novaferon Labs, Inc. HIV inhibitor (Akron, OH) Diapren, Inc.

(Roseville, MN, marketer) Peptide T Peninsula Labs AIDS Octapeptide (Belmont, CA) Sequence Zidovudine; AZT Burroughs Wellcome AIDS, adv, ARC (Rsch. Triangle Park, pediatric AIDS, NC) Kaposi's sarcoma, asymptomatic HIV infection, less severe HIV disease, neurological involvement, in combination with other therapies.

Ansamycin Adria Laboratories ARC LM 427 (Dublin, OH) Erbamont (Stamford, CT) Dextran Sulfate Ueno Fine Chem. AIDS, ARC, HIV Ind. Ltd. positive asymptomatic (Osaka, Japan) Virazole Viratek/ICN asymptomatic HIV Ribavirin (Costa Mesa, CA) positive, LAS, ARC Alpha Interferon Burroughs Wellcome Kaposi's sarcoma, (Rsch. Triangle HIV in combination Park, NC) wiRetrovir Acyclovir Burroughs Wellcome AIDS, ARC, asymptomatic HIV positive, in combination with AZT.

Antibody which Advanced Biotherapy AIDS, ARC neutralizes pH Concepts labile alpha aberrant (Rockville, MD) Interferon in an immuno- adsorption column Indinavir Merck (Rahway, NJ) AIDS, ARC, asymptomatic HIV positive, also in combination with AZT.

Nevirapine Boeheringer AIDS, ARC Ingleheim (protease inhibitor) Delaviridine Pharmacia-Upjohn AIDS, ARC (protease inhibitor) Ritonavir Abbott AIDS, ARC (protease inhibitor) Saquinavir Hoffmann- AIDS, ARC LaRoche (protease inhibitor) Nelfinavir Agouron AIDS, ARC Pharmaceuticals (protease inhibitor) 141 W94 Glaxo-Wellcome AIDS, ARC (protease inhibitor) DMP-266 DuPont-Merck AIDS, ARC Pharmaceuticals (non-nucleoside reverse transcriptase inhibitor) IMMUNO-MODULATORS Drug Name Manufacturer Indication AS-lOl Wyeth-Ayerst Labs. AIDS (Philadelphia, PA) Bropirimine Upjohn advanced AIDS (Kalamazoo, MI) Acemannan Carrington Labs, Inc. AIDS, ARC (See also (Irving, TX) anti-virals) CL246,738 American Cyanamid AIDS, Kaposi's (Pearl River, NY) sarcoma Lederle Labs (Wayne, NJ) EL10 Elan Corp, PLC HIV infection (Gainesville, GA) (See also anti virals) Gamma Interferon Genentech ARC, in combination (S. San Francisco, CA) w/TNF (tumor necrosis factor) Granulocyte Genetics Institute AIDS Macrophage Colony (Cambridge, MA) Stimulating Sandoz Factor (East Hanover, NJ) Granulocyte Hoeschst-Roussel AIDS Macrophage Colony (Sommerville, NJ) Stimulating Immunex Factor (Seattle, WA) Granulocyte Schering-Plough AIDS Macrophage Colony (Madison, NJ) AIDS, in combination Stimulating Factor w/AZT HIV Core Particle Rorer seropositive HIV Immunostimulant (Ft. Washington, PA) IL-2 Cetus AIDS, in combination Interleukin-2 (Emeryville, CA) w/AZT IL-2 Hoffman-La Roche AIDS, ARC, HIV, in Interleukin-2 (Nutley, NJ) combination w/AZT Immunex Immune Globulin Cutter Biological pediatric AIDS, in Intravenous (Berkeley, CA) combination w/AZT (human) IMREG-1 Imreg AIDS, Kaposi's (New Orleans, LA) sarcoma, ARC, PGL IMREG-2 Imreg AIDS, Kaposi's (New Orleans, LA) sarcoma, ARC, PGL Imuthiol Diethyl Merieux Institute AIDS, ARC Dithio Carbamate (Miami, FL) Alpha-2 Schering Plough Kaposi's sarcoma Interferon (Madison, NJ) w/AZT: AIDS Methionine- TNI Pharmaceutical AIDS, ARC Enkephalin (Chicago, IL) MTP-PE Ciba-Geigy Corp. Kaposi's sarcoma Muramyl-Tripeptide (Summit, NJ) Granulocyte Am g en AIDS, in combination Colony Stimulating (Thousand Oaks, CA) w/AZT Factor rCD4 Genentech AIDS, ARC Recombinant (S. San Francisco,CA) Soluble Human CD4 rCD4-IgG AIDS, ARC hybrids Recombinant Biogen AIDS, ARC Soluble Human CD4 (Cambridge, MA) Interferon Hoffman-La Roche Kaposi's sarcoma Alfa 2a (Nutley, NJ) AIDS, ARC, in combination w/AZT SK & F106528 Smith, Kline & French HIV infection Soluble T4 Laboratories (Philadelphia, PA) Thymopentin Immunobiology HIV infection Research Institute (Annandale, NJ) Tumor Necrosis Genentech ARC, in combination Factor; TNF (S. San Francisco, CA) wlgamma Interferon ANTI-INFECTIVE S Drug Name Manufacturer Clindamycin with Upjohn PCP Primaquine (Kalamazoo, MI) Fluconazole Pfizer cryptococcal (New York, NY) meningitis, candidiasis Pastille Squibb Corp. prevention of Nystatin Pastille (Princeton, NJ) oral candidiasis Ornidyl Merrell Dow PCP Eflornithine (Cincinnati, OH) Pentamidine LyphoMed PCP treatment Isethionate (IM & IV) (Rosemont, IL) Trimethoprim antibacterial Trimethoprim/sulfa antibacterial Piritrexim Burroughs Well come PCP treatment (Rsch. Triangle Park, NC) Pentamidine Fisons Corporation PCP prophylaxis isethionate for (Bedford, MA) inhalation Spiramycin Rhone-Poulenc cryptosporidial Pharmaceuticals diarrhea (Princeton, NJ) Intraconazole- Janssen Pharm. histoplasmo Si s; R51211 (Piscataway, NJ) cryptococcal meningitis Trimetrexate Warner-Lambert PCP OTHER Drug Name Manufacturer Indication Recombinant Human Ortho Pharm. Corp. severe anemia Erythropoietin (Raritan, NJ) assoc. with AZT therapy Megestrol Acetate Bristol-Myers treatment of (New York, NY) anorexia assoc.

wIAlDS Total Enteral Norwich Eaton diarrhea and Nutrition Pharmaceuticals malabsorption (Norwich, NY) related to AIDS It will be understood that the scope of combinations of the compounds of this invention with AIDS antivirals, immunomodulators, anti-inEectives or vaccines is not limited to the list in the above Table, but includes in principle any combination with any pharmaceutical composition useful for the treatment of AIDS.

Indinavir is an inhibitor of HIV protease and is the sulfate salt of N-(2(R)-hydroxy- 1(S )-indanyl )-2(RphenylmethylA--(S)-hydroxy-5 - (1-(4-(3 -pyridyl-methyl)-2( S)-N'-(t-butylcarboxamido )-piperazinyl))- pentaneamide ethanolate, and is synthesized according to U.S. 5,413,999.

Indinavir is generally administered at a dosage of 800 mg three times a day.

EXAMPLE1 Fermentation A. Media: Seed medium contained the following in g/L: corn steep liquor, 5g; tomato paste, 40; oat flour, 10; glucose, 10; agar, 4; FeSO4-7H2O, 0.01; MnS04-4H20, 0.01; CuCl2-2H20,0.00025; CaC12, 0.001; H3BO3, 0.00056; (NH4)6Mo7024-4H2O,0.00019; ZnSO4-7H2O, 0.002. The pH was adjusted to 6.8.

Production media contained the following per 250 mL flask: brown rice, 10g; base liquid, 20 mL. Base liquid contained the following in g/L: yeast extract, 1; sodium tartrate, 0.5; KH2PO4, 0.5. The flasks were autoclaved for 15 minutes at 1210C, 15 psi and stored. Prior to innoculation, 15 mL of distilled water were added per flask and the flasks were sterilized for 20 minutes at minutes at 1210 C, 15 psi.

B. Innoculum Preparation Frozen vegetative mycelia (FVM) were prepared by inoculating 50 mL of seed medium in a 250 mL flask and incubating at 250C, 85% relative humidity and at 200 rpm for 3-5 days. Aliquots of the culture were frozen and used as a source of innoculum for future experiments.

C. Seed Culture To 50 mL of seed media in a 250 mL flask, 2.0 mL of FVM was added as inoculum and the flasks were incubated at 250C, 85% relative humidity and at 200 rpm for days 2-3 days.

D. Production of Culture and Extractlon To 35 mL of production media in a 250 mL flask, 1 mL of seed culture was added as innoculum and the flasks were incubated a 25"C, 85% relative humidity and at 200 rpm for days 24 days. Each flask was then extracted with 50 mL of methyl ethyl ketone and the solids were discarded.

EXAMPLE 2 Isolation of HIV Integrate Inhibltor Compound A Ten mL methyl ethyl ketone extract was prepared from a ten mL broth of the Xylaria sps prepared according to the fermentation procedures in Example 1, above by shaking the broth with methyl ethyl ketone for 20-30 min followed by filtration through a filtration aid. The filtrate was concentrated to dryness initially at reduced pressure on a rotatory evaporator followed by lyophilization to give a 50 mg of colorless solid. The solid was dissolved in 3 mL methanol and filtered.The filtrate was concentrated to a volume of 1.5 mL and was injected to a ZorbaxR HPLC column (22 x 250 mm). The column was eluted at a flow rate of 8 mL per min with a 50 to 80% linear gradient of acetonitrile in water over 60 min. A 220 nm in-line ultraviolet light detection was used for monitoring the elution profile in HPLC. The compound responsible for HIV integrase activity eluted between 30-35 min which upon concentration at reduced pressure followed by lyophilization gave compound A as a colorless powder.

EXAMPLE 3 Large Scale Isolation of HIV Inteerase Inhibitor Comnound A A 700 mL whole broth of Xylaria species as described in Example 1 above was extracted with 700 mL methyl ethyl ketone by shaking for - 30 minutes at room temperature. The insoluble portion of the broth was removed by filtration through a filter aid such as diatomaceous earth. The filtrate which contained all of the biological activity (as determined by employing the assay described in Examples 14 and 15) was concentrated under reduced pressure to a small volume which upon lyophilization gave a 3.8 g of crude material that contained a significant amount of Compound A.

The crude material was suspended in a 200 mL watermethanol mixture and acidified to pH 2.0 by addition of dilute hydrochloric acid. This mixture was washed with hexane (200 mL) and extracted with ethyl acetate (3 x 300 mL). The latter extract was dried over sodium sulfate and concentrated to dryness under reduced pressure to give a still-crude mixture of compounds weighing 3.5 g containing Compound A. The crude mixture was dissolved in 6 mL methanol and was subdivided into 3 equal portions of 2 mL each. One of the three portions was purified on a PhenomenexR prime sphere (50 x 250 mm) HPLC column using a 60 min linear gradient of 40 to 80% acetonitrile in water (both solvents contained 0.1% trifluoroacetic acid).

The column was eluted at a flow rate of 60 mL per nun. The HIV integrase inhibitor Compound A eluted between 29-33 minutes. This chromatographic process was repeated with the remainding two portions of the crude mixture. The HPLC fractions containing Compound A were combined from the three chromatographic runs and was concentrated on a rotatory evaporator at reduced pressure to remove most of the acetonitrile. Subsequent lyophilization of the fraction gave Compound A as a colorless, amorphous powder.

EXAMPLE 4 Phvsical Properties of Compounds of Structure I.

Compound A: The compound of structural formula I wherein X is hydrogen.

Mass Snectral Data: Mass spectra were recorded on Jeol SX-102A(electron impact, EI, 90eV) and JEOL HX110 (Fast Atom Bombardment, FAB) mass spectrometers. Exact mass measurements were performed at high resolution (HR-EI) using perfluorokerosene (PFK) as an internal standard. The FAB spectrum was run in a matrix of dithiothreitol/ dithioerythritol (20/80).

The molecular weight of Compound A was determined by FAB-MS and EI-MS to be 430. High resolution EI-MS indicated a molecular formula of C25H3406 (found 430.2370, calculated 430.2355).

Critical fragment ions were observed at m/z 153 and 260.

UV 1w (MeOH) hoax: 220 nm; Optical Rotation [ai25D = +38.3 (c, 0.63, MeOH).

13C NMR andlH NMR All of the NMR spectra were recorded on a Varian Unity 400 MHz spectrometers operating at a field strength of 400 MHz for proton NMR, 100 MHz for carbon NMR respectively. The data are summarized in following Table.

Table: NMR Assignment of Compound A in CDCl3 at 25 0C

3C so 3H, mult, J 1 196.85 2 193.13 1 9.54,s

3 177. 1 3 177.87 -- 4 16678 5 158.90 6 149.69 6.54, dq, 10, 1.2 7 147.66 8 136.47 6.35, 5 6.25, 5 9 129.60 6.10, s 10 125.83 ~ 11 72.65 5.25, t, 2.8 12 53.31 2.46, dd, 13.2,3.2 13 43.18 2.26, t, 13.6 2.12, dd, 13.2, 4.0 14 43.08 3.73, dd, 14.8,4.4 15 38.28 -- 16 36.53 1.4, m; 1.3, m 17 33.34 2.50, m 18 29.77 2.15, m; 1.7, m 19 29.66 1.2, m 20 22.78 1.3, m 21 20.12 2.3, m; 1.9, m 22 19.98 1.00, d, 6.6 23 19.53 1.50, s 24 14.08 0.87,t,7.2 25 12.65 1.80, d, 1.6 EXAMPLE 5 Preparation of an Ammomum Salt A 0.1 mmol sample of Compound A, the free acid of the compound of formula (I) is dissolved in 10 mL ethyl acetate. The resulting solution is saturated with gaseous ammonia and the ammonium salt precipitates from solution.

EXAMPLE 6 Preparation of a Potassium Salt A solution of 0.1 mmol of Compound A, the free acid of a compound of formula (I), in 10 mL methanol is treated with an aqueous or methanolic solution containing 0.1 mmol of potassium hydroxide.

Evaporation of the solvent affords the potassium salt.

In a similar fashion, the sodium and lithium salts can be formed.

EXAMPLE 7 preparation of a Calcium Salt A solution of 0.1 mmol of Compound A, the free acid of a compound of formula (I), in 20 mL 6:4 methanol:water is treated with an aqueous solution of 0.1 mmol of calcium hydroxide. The solvents are evaporated to give the corresponding calcium salt.

EXAMPLE8 Preparation of an Ethvlenediarmne Salt A solution of 0.1 mmol of Compound A, the free acid of a compound of formula (I), in 10 mL of methanol is treated with 0.1 mmol of ethylenediamine. Evaporation of the solvent affords the ethylenediamine salt.

The procedure can also be applied to the preparation of the N,N"-dibenzylethylenediamine salt.

EXAMPLE 9 Preparation of a Tns(hvdroxvviethvl)aminomethane Salt To a solution of 0.1 mmol of Compound A, the free acid of a compound of formula (I) in 10 mL of methanol is added 0.1 mmol of tris(hydroxymethyl)aminomethane dissolved in 10 mL of methanol.

Evaporation of the solvent gives a corresponding salt form.

Similarly prepared are the salts of L-ornithine, L-lysine, and N-methylglutamine.

EXAMPLE 1Q Preparation of an L-armne Salt A solution of 0.1 mmol of Compound A, the free acid of a compound of formula (I), in 20 ml of 6:4 methanol:water is treated with an aqueous solution of 0.1 mmol of L-arginine. Evaporation of the solvent affords the title salt.

Similarly prepared are the salts of Lornithine, 1--lysine, and N-methylglutamine.

EXAMPLE 11 Preparation of the Compound of Structural Formula I wherein X is CH3 (Method 1) Compound A (0.6 mg) is dissolved in 1 mL diethyl ether and stirred at 0 C. Etheral cyanamide is added dropwise until the solution remains yellow. The solution is evaporated under a stream of nitrogen to yield the title compound.

EXAMPLE 12 Preparation of the Compound of Structural Formula I wherein X is CH3 To a cooled (-78 OC) solution of Compound A (0.6 mg) in methylene chloride (0.3 mL) and methanol (0.02 mL) was added an excess solution of tetramethylsilyl diazomethane in hexane. The solution was stirred for 10 min and then quenched with a 2 drops of acetic acid.

The volatile components of the reaction mixture were removed under a stream of nitrogen and the reaction product was purified on a Pasteur pipette filled with silica gel. Elution with 40% ethyl acetate in hexane gave pure methyl ester of Compound A as a gum.

Mass spectral data of methyl ester: High Resolution EI MS (m/z) :444.2502 (M+, 1%, calcd. for C26H3606: 444.2512).

EXAMPLE 13 Preparation of the Compound of Structural Formula I wherein X is IsoDroDvl A solution of 5 mg of Compound A in 0.5 mL of tetrahydrofuran (THF) is treated at room temperature with 1 equivalent of N,N'-diisopropyl-O-benzyl isourea for 18 hours. The reaction mixture is then chilled to -15C, filtered to remove the urea. The filtrate is concentrated under reduced pressure to yield the title compound.

The method of Example 13 is also suitable for the preparation of other ester derivatives such as: 1) methyl and the other lower alkyls, and 2) substituted benzyl esters, using the appropriately substituted isourea.

EXAMPLE 14 HIV Integrate Substrate Cleavage and Integration Assav An assay for trimming of the 3' end of HIV long terminal repeat terminus by HIV-1 integrase was conducted according to LaFemina, R.L. et al., J. Virol 10, 5624 (1991), herein incorporated by reference for these purposes. To assay inhibition of HIV integrase substrate cleavage, the reaction was conducted with inhibitor having various concentrations in the range of 0.1 to 100 FLM. Results follow: Compound QQ A 5-10 pM EXAMPLE 15 Strand Transfer Assav for HIV Integrase A microtiter assay for ligation of processed donor (HIV) DNA to unspecific, nicked host DNA was conducted according to Hazuda, D.J. et al., Nucl. Acids, Res. 22, 1121 (1994), herein incorporated by reference for these purposes. To assay inhibition of such strand transfer by HIV integrase, the reaction was conducted with inhibition having various concentrations in the range of 0.1 to 100 Results follow.

Compound ICp A 5-10 pM EXAMPLE 16 Assav for Assemblv of HIV Inteerase/donor (HIV) DNA Complexes An assay measuring the binding of HIV Integrase to immobilized donor (HIV) DNA was conducted according to Wolfe, A.L.

et al., J. Virol. 70, 1424 (1996), herein incorporated by reference for these purposes. To assay inhibition of such assembly or binding, the reaction was conducted with inhibitor having various concentrations in the range of 0.1 to 100 pM. Results follow.

Compound Q A 10pM EXAMPLE 17 Oral Composition As a specific embodiment of an oral composition of a compound of this invention, 50 mg of Compound A is formulated with sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adoptions, or modifications, as come within the scope of the following claims and its equivalents.

Claims (17)

WHAT IS CLAIMED:

1. compound of the formula

wherein X is selected from: (1) H, (2) C1-4 alkyl, and (3) C1-4 alkyl substituted with a substituent selected from (a) phenyl, and (b) phenyl substituted with methyl, methoxy, halogen, or hydroxy; or a pharmaceutically acceptable salt thereof.

2. The compound according to Claim 1, of the structure

or a pharmaceutically acceptable salt thereof.

3. A compound of molecular formula C25H3406 and molecular weight 430 characterized by (a) the 13CNMR chemical shifts as measured in CD Cl3 at 25 C at 100 MHz: 196.85, 193.13,177.87, 166.78, 158.90, 149.69, 147.66,

136.47, 129.60, 125.83,72.65, 53.31, 43.18,43.08,38.28,

36.53,33.34, 29.77,29.66,22.78, 20.12, 19.98, 19.53,

14.08, 12.65; (b) 1HNMR chemical shifts as measured in CDCl3 at 25 C at 400 MHz:

9.54(s), 6.54 (dq, 10, J=1.2), 6.35(s), 6.25(s), 6.10 (s),

5.25 (t, J=2.8), 2.46 (dd, J=13.2, 3.2), 2.26 (t, J=13.6),

2.12 (dd, J=13.2, 4.0), 3.73(dd, J=14.8, 4.4), 1.4 (m), 1.3 (m), 2.50(m), 2.15 (m), 1.7 (m), 1.2 (m), 1.3 (m), 2.3 (m), 1.9 (m), 1.00 (d, J=6.6), 1.50 (s), 0.87 (t, 7.2), 1.80 (d, 1.6); (c) an ultraviolet absorption spectrum in methanol which exhibits absorption maximum at 220 nM, and (d) an optical rotation of [α]25D of [a]25D=+38.3.

4. A pharmaceutical composition useful for inhibiting HIV integrase, comprising an effective amount of a compound according to Claim 1 and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of Claim 4, useful for treating infection by HIV, or for treating AIDS or ARC.

6. A pharmaceutical composition comprising a therapeutically effective amount of a compound of Claim 1 in combination with a therapeutically effective amount of an AIDS treatment agent selected from (1) an AIDS antiviral agent, (2) an anti-infective agent, and (3) an immunomodulator.

7. The composition of Claim 6 wherein the antiviral agent is an HIV protease inhibitor.

8. The composition of Claim 7 wherein the HIV protease inhibitor is N-(2(R)-hydroxy- 1-(S)-indanyl)-2(R)-phenylmethyl-4(S)- hydroxy-5-( 1-(4-(3-pyridylmethyl)-2(S)-N'-(t-butylcarboxamido )piperazinyl))-pentaneamide or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition made by combining the compound of Claim 1 and a pharmaceutically acceptable carrier.

10. A process for making a pharmaceutical composition comprising combining a compound of Claim 1 and a pharmaceutically acceptable carrier.

11. A method of inhibiting HIV integrase, comprising the administration to a mammal in need of such treatment a therapeutically effective amount of a compound of Claim 1.

12. A method of treating infection by HIV, or of treating AIDS or ARC, comprising the administration to a mammal in need of such treatment a therapeutically effective amount of a compound of Claim 1.

13. A biologically pure culture of MF 6254, Xylaria sp.

(ATCC 74397).

14. A culture of MF 6254, Xylaria sp. (ATCC 74397).

15. A biologically pure culture of Claim 13 or mutant thereof capable of producing a compound of structure

in recoverable amounts.

16. A culture of Claim 14 or a mutant thereof capable of producing a compound of the structure:

in recoverable amounts.

17. A process of making a compound according to Claim 2 comprising cultivating MF 6254, Xylaria sp. (ATCC 74397) or a mutant thereof, under conditions suitable for the formation of the compound and recovering the compound.