HIV integrase inhibitors

BACKGROUND OF THE INVENTION

A retrovirus designated human immunodeficiency virus (HIV) is 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 and monocytoid cells. Integration is believed to be mediated by integrase in three steps: assembly of a stable nucleoprotein complex with viral DNA sequences; 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. The fourth step in the process, repair synthesis of the resultant gap, may be accomplished by cellular enzymes.

Nucleotide sequencing of HIV shows the presence of a pol gene in one open reading frame [Ratner, L. et al., Nature, 313, 277(1985)]. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, 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)]. All three enzymes have been shown to be essential for the replication of HIV.

It is known that some antiviral compounds which act as inhibitors of HIV replication are effective agents in the treatment of AIDS and similar diseases, e.g., azidothymidine or AZT. Applicants demonstrate that the compounds of this invention are inhibitors of HIV integrase. The applicants additionally demonstrate that inhibition of integrase in vitro is a direct result of inhibiting the strand transfer reaction catalyzed by the recombinant integrase in vitro. The particular advantage of the present invention is highly specific inhibition of HIV integrase and HIV replication. The compounds of the present invention inhibit integrases of closely related lentiviruses such as HIV 2 and SIV, but not integrases from more distantly related retroviruses, for example RSV. These compounds do not inhibit binding or catalysis of other nucleic acid binding proteins, including enzymatic reactions such as those catalyzed by HIV reverse transcriptase, HIV Rnase H, Influenza transcriptase, Hepatitis C polymerase, Yeast DNA polymerase, DNase I, Eco RI endonuclease, or mammalian polymerase II.

Zhao et al., (J. Med Chem. vol. 40, pp. 937-941 and 1186-1194 (1997)) describe hydrazide and arylamide HIV integrase inhibitors. Bis-catechols useful for inhibiting HIV integrase are described in LaFemina et al. (Antimicrobial Agents & Chemotherapy, vol. 39, no. 2, pp. 320-324, February 1995).

Applicants have discovered that certain tetracyclic aromatic ketones 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 of AIDS 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. Further, the cultures Sterile Fungus MF6388 (ATCC 74478) and Ascochyta sp. MF6591 (ATCC 74477) are also disclosed, as well as processes for making compounds of the present invention employing the cultures.

DETAILED DESCRIPTION OF THE INVENTION

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:

R is selected from:

(a) —CH

2

OH, and

(b) —CH(═O);

or a pharmaceutically acceptable salt thereof.

Particular compounds of structural Formula I include:

Also included within the present invention are pharmaceutical compositions useful for inhibiting HIV integrase, comprising an effective amount of a compound of this invention, and a pharmaceutically acceptable carrier. 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. Additionally, the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention 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.

The compounds of the present invention may have asymmetric centers and may occur, except when specifically noted, as mixtures of stereoisomers or as individual diastereomers, or enantiomers, with all isomeric forms being included in the present invention.

This invention also discloses the culture MP6388 (ATCC 74478) identified as Sterile Fungus. A culture of MF6388 (ATCC 74478) is defined as substantially free of its natural soil contaminants and capable of forming a compound of Formula (I) in a recoverable amount. The culture should be free from viable contaminating microorganisms deleterious to the production of a compound of Formula (I). A biologically pure culture of MF6388 (ATCC 74478) may also be employed. In one embodiment, the present invention includes a culture of MF6388 (optionally biologically pure), or a mutant thereof, capable of producing in a recoverable amount a compound of Formula (I).

Suitable mutant strains of MF6388 can be obtained by chemically induced mutagenesis using mutagens such as nitrosoguanidine, 1-methyl-3-nitro-1-nitrosoguanidine, ethyl methane sulfonate, 2-aminopurine, and the like. Mutant strains can also be obtained by radiation-induced mutagenesis, such as by irradiation with ultraviolet light (e.g., using a germicidal lamp), X-rays, or gamma rays (e.g., using a cobalt-60 source). Recombinant DNA techniques such as protoplast fusion, plasmid incorporation, gene transfer and the like may also be employed. Further description of mutagenic technqiues can be found in Vinci and Bing, “Strain Improvement by Nonrecombinant Methods”, in

Manual of Industrial Microbiology and Biotechnology

1999, 2d edition, edited by Demain et al., ASM Press, 103-113; and in Carlton and Brown, “Gene Mutation”, Chapter 13 in

Manual of Methods for General Bacteriology

1985, edited by Gerhardt et al., ASM Press, 222-229.

In addition, compounds of the present invention may be prepared by fermentation of the culture MF6388, ATCC 74478.

The present invention also relates to the preparation of compounds of structural Formula I comprising:

(a) fermenting a culture of MF6388 (ATCC 74478), Sterile Fungus or a mutant thereof to produce a fermentation broth,

(b) extracting the fermentation broth with an organic solvent,

(c) isolating the compounds of structural Formula I.

The compounds of structural Formula I are preferably isolated by partitioning the fermentation extract between the organic solvent and water, followed by size exclusion chromatography and normal or reverse-phase chromatography.

This invention also discloses the culture MF6591 (ATCC 74477) identified as Ascochyta sp. A culture of MF6591 (ATCC 74477) is defined as substantially free of its natural soil contaminants and capable of forming a compound of Formula (I) in a recoverable amount. The culture should be free from viable contaminating microorganisms deleterious to the production of a compound of Formula (I). A biologically pure culture of MF6591 (ATCC 74477) may also be employed. In one embodiment, the present invention includes a culture of MF6591 (optionally biologically pure), or a mutant thereof, capable of producing in a recoverable amount a compound of Formula (I). Suitable mutant strains of MF6591 can be obtained using the methods disclosed above for obtaining mutants of MF6388.

In addition, compounds of the present invention may be prepared by fermentation of the culture MF6591, ATCC 74477.

The present invention also relates to the preparation of compounds of structural Formula I comprising:

(a) fermenting a culture of MF6591 (ATCC 74477), Ascochyta sp. or a mutant thereof to produce a fermentation broth,

(b) extracting the fermentation broth with an organic solvent,

(c) isolating the compounds of structural Formula I.

The compounds of structural Formula I are preferably isolated by partitioning the fermentation extract between the organic solvent and water, followed by size exclusion chromatography and normal or reverse-phase chromatography.

When any variable (e.g., R

1

, R

2

, 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.

The present invention also provides for the use of a compound of structural Formula (I) to make a pharmaceutical composition useful for inhibiting HIV integrase and in the treatment of AIDS or ARC.

Applicants have discovered that compounds of structural Formula (I), are useful for inhibiting HIV integrase.

ATCC Deposit of MF6388 (ATCC 74478), identified as Sterile Fungus.

Before the U.S. filing date of the present application, a sample of MF6388 (ATCC 74478), Sterile Fungus, was deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, United States of America, under the terms of the Budapest Treaty. The culture access designation is 74478. 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.

ATCC Deposit of MF6591 (ATCC 74477), identified as Ascochyta sp.

Before the U.S. filing date of the present application, a samnple of MF6388 (ATCC 74477), Ascochyta sp., was deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, United States of America, under the terms of the Budapest Treaty. The culture access designation is 74477. 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 Characteristics and Descriptions of MF6388 (ATCC 74478), Sterile Fungus and MF6591 (ATCC 74477), Ascochyta sp.

In the following descriptions, MF6388 and MF6591 were edge inoculated with a 5 mm diameter plug on 2,100 mm petri dishes for each the following growth media. All cultures were incubated for 20 days at 25° C. and 67% relative humidity in 12 hr photoperiod in fluorescent light unless otherwise indicated. In addition, all capitalized color names are from Ridgway, Color Standards and Nomenclature, (Published by author, Washington D.C., 1912) 43p. +53.

Sterile Fungus (MF6388)

MF6388 was isolated from herbivore dung collected in New Mexico.

On oatmeal agar (Difco) culture mat attaining a diameter of 40 mm. Culture mat woolly from inoculation point to midway through the colony then thickly cottony until margin, grey-green (Pale Olive-Gray, Olive Gray), margin entire, white. Exudate forming as large, clear to light brown droplets near inoculation point and smaller, clear droplets closer to margin area. Reverse dark brown (Drab, Hair Brown). Soluble pigment absent.

On potato-dextrose agar (Difco), culture mat attaining a diameter of 64 mm. Culture mat densely cottony, more dense at the inoculation point than closer to the margin, white aerial mycelium over green (Sage Green, Slate Olive) mycelium, margin entire, white. Exudate forming as sparse, clear droplets. Soluble pigment dark red brown (Dark Indian Red, Indian Red).

On cornmeal agar (Difco), culture mat attaining a diameter of 75 mm (plate covered). Colony mat appressed, uncolored to slightly white. Margin entire, hyaline. Exudate, reverse and soluble pigment absent.

On YME agar (malt extract, 10.0 g; yeast extract, 4.0 g; dextrose, 4.0 g; agar, 20.0 g, distilled water, 1L) culture mat attaining a diameter of 66 mm. Culture mat woolly, forming dense tufts, tufts becoming smaller towards margin area, white throughout, margin entire, white. Exudate forming as medium sized, light brown to sparse (at margin), clear droplets. Soluble pigment dark red brown (Dark Indian Red, Indian Red). No growth at 37° C., in the dark and no humidity control.

Microscopic: Hyphae hyaline, branched, thin-walled, usually 2-4 μm wide. No sporulation observed.

Ascochyta sp. (MF6591)

MF6591 was isolated from

Urtica urens

collected in Ontigola, Aranjuez, Spain.

On oatmeal agar (Difco) colony mat attaining a diameter of 40 mm. Culture mat cottony, dissected, light gray (Pale Neutral Gray, Gull Gray), abundant black fruiting bodies oozing light pink near inoculation point. Margin entire, light brown. Reverse dark gray (Deep Purplish Gray, Purplish Gray). Soluble pigment and exudate absent.

On potato-dextrose agar (Difco), colony mat attaining a diameter of 33 mm. Culture mat cottony, consistent throughout, gray-green (Olive Gray, Light Olive-Gray, Deep Olive-Gray), sparse white aerial mycelium near inoculation point. Soluble pigment deep red-brown (Indian Red, Deep Indian Red). Reverse and exudate absent.

On cornmeal agar (Difco), colony mat attaining a diameter of 45 mm. Culture mat appressed, uncolored. Fruiting bodies black, clustered around inoculation plug in contact with agar, immature fruiting bodies scattered throughout culture mat, abundant near inoculation point. Reverse, soluble pigment and exudate absent.

On YME agar (malt extract, 10.0 g; yeast extract, 4.0 g; dextrose, 4.0 g; agar, 20.0 g, distilled water, 1L) attaining a diameter of 33 mm. Culture mat cottony to thick velvety, light orange to pink (Vinaceous Pink, Shell Pink), sections near inoculation point collapsed, blackish. Margin region very faint yellow-brown (Light Buff, Pale Ochraceous Buff), very edge of margin entire, uncolored, submerged. Soluble pigment dark red-brown to light red-brown (Chestnut Brown, Cinnamon-Rufous). Exudate and reverse absent. At 37° C., in the dark and no humidity control, no growth.

Microscopic:

Hyphae hyaline

to light brown, thin-walled, branched, septate, 4-5 μm wide.

Conidiomata pycnidial,

thin-walled, black, sphaerical, mostly immersed in agar, 150-200 μm, mature conidia oozes as a pink mass from ostiole. Conidiogenous cells phialidic, 1-celled, hyaline, 5-10×4-5 μm. Conidia, hyaline, cylindrical, 1 medially septate, thin-walled, 10-15×3-4 μm.

In general, MF6388 (ATCC 74478) is a strain cultured on a solid medium, or in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen. For example, the cultures may be grown under submerged aerobic conditions (e.g., shaking culture, submerged culture, etc.)

In general, MF6591 (ATCC 74477) is strain that may be cultured on a solid medium, or in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen. For example, the cultures may be grown under submerged aerobic conditions (e.g., shaking culture, submerged culture, etc.)

The sources 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 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.

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 20° C. and 30° C., preferably 22-25° C., for a period of about 14-21 days, which may be varied according to fermentation conditions and scales.

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

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. The use of fermentors (tanks) is preferred for the generation of large quantities of materials. Fermentors can be sterilized with the production medium or can be sterilized empty and the medium sent through a continuous sterilizer, which is preferred for very large fermentations (20,000 gallons or larger). Preferably, the pH of the medium is adjusted to about 6-7, generally using acid or base additions, preferably made automatically with a pH electrode and a controller. The parameters for fermenter operation include agitation, aeration, temperature and pressure. Agitation is preferably carried out by mixing the medium with a number of impellers mounted on a rotating agitator shaft located in the midst of the tank. Aeration may be carried out by a variety of means, preferably by bubbling sterile air into the medium (subsurface sparging). The tank is preferably maintained under positive pressure. Temperature is preferably maintained at between about 20° C. and 30° C.

When the growth is carried out in large tanks, vegetative forms of the organism for inoculation in the production tanks may be employed in order to avoid growth lag in the process of production. This requires production of 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 transferring the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, in which the inoculum is produced, is generally sterilized prior to inoculation. The pH of the medium is generally adjusted to about 6-7 prior to the autoclaving step, generally using acid or base additions, preferably made automatically with a pH electrode and a controller.

Preferred culturing/production media for carrying out the fermentation are those set forth in the Examples.

After growth is completed, the cells harvested by adding the appropriate solvent, e.g., methylethylketone, to the entire culture medium and cells. If the culture is grown in a liquid fermentation, the growth could be harvested by other conventional methods, e.g., centrifugation and filtration, and then 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, non-ionic 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 is separated and concentrated to dryness initially by evaporating under reduced pressure followed by lyophilization. The compounds are finally isolated either by solvent partitioning, crystallization, gel filtration or by preparative HPLC on reversed phase systems.

Compounds of Formula (I) may be isolated from the aerobic fermentation of a culture of ME6388 (ATCC 74478). A culture of MF6388 (ATCC 74478) 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 MF6388 (ATCC 74478) may also be employed.

Compounds of Formula (I) may be isolated from the aerobic fermentation of a culture of MF6591 (ATCC 74477). A culture of MF6591 (ATCC 74477) 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 MF6591 (ATCC 74477) may also be employed.

The compounds of the present invention may be administered in the form of pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” is intended to include all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, isothionate, triethiodide, lactate, panoate, valerate, and the like which can be used as a dosage form for modifying the solubility or hydrolysis characteristics or can be used in sustained release or pro-drug formulations. Depending on the particular functionality of the compound of the present invention, 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, omithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, e.g. by reacting a free acid with a suitable organic or inorganic base. Where a basic group is present, such as amino, an acidic salt, i.e. hydrochloride, hydrobromide, acetate, pamoate, and the like, can be used as the dosage form.

Also, in the case of an acid (—COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed, e.g. acetate, maleate, pivaloyloxymethyl, and the like, and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.

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 pharmaceutically-acceptable 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 further 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.

By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

These pharmaceutical compositions may be in the form of orally-administrable 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, parenterally-acceptable 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 non-initiating excipient, such as cocoa butter, synthetic glyceride 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 1 to 1000 mg/kg 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, 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 specific 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 pre-exposure and/or post-exposure, in combination with effective amounts of the AIDS antivirals, imunomodulators, antiinfectives, or vaccines, such as those in the following table.

Drug Name

Manufacturer

Indication

ANTIVIRALS

097

Hoechst/Bayer

HIV infection, AIDS,

ARC

(non-nucleoside

reverse transcriptase

inhibitor)

Amprenavir

Glaxo Wellcome

HIV infection, AIDS,

141 W94

ARC

GW 141

(protease inhibitor)

Abacavir

Glaxo Welcome

HIV infection, AIDS,

GW 1592

ARC

1592U89

(reverse transcriptase

inhibitor)

Acemannan

Carrington Labs

ARC

(Irving, TX)

Acyclovir

Burroughs Wellcome

HIV infection, AIDS,

ARC, in

combination with

AZT

AD-439

Tanox Biosystems

HIV infection, AIDS,

ARC

AD-519

Tanox Biosystems

HIV infection, AIDS,

ARC

Adefovir dipivoxil

Gilead Sciences

HIV infection

AL-721

Ethigen

ARC, PGL

(Los Angeles, CA)

HIV positive, AIDS

Alpha Interferon

Glaxo Wellcome

Kaposi's sarcoma, HIV in

combination w/Retrovir

Ansamycin

Adria Laboratories

ARC

LM 427

(Dublin, OH)

Erbamont

(Stamford, CT)

Antibody which

Advanced Biotherapy

AIDS, ARC

neutralizes pH

Concepts

labile alpha aberrant

(Rockville, MD)

Interferon

AR177

Aronex Pharm

HIV infection, AIDS,

ARC

beta-fluoro-ddA

Nat'l Cancer Institute

AIDS-associated diseases

BMS-232623

Bristol-Myers Squibb/

HIV infection, AIDS,

(CGP-73547)

Novartis

ARC

(protease inhibitor)

BMS-234475

Bristol-Myers Squibb/

HIV infection, AIDS,

(CGP-61755)

Novartis

ARC

(protease inhibitor)

CI-1012

Warner-Lambert

HIV-1 infection

Cidofovir

Gilead Science

CMV retinitis, herpes,

papillomavirus

Curdlan sulfate

AJI Pharma USA

HIV infection

Cytomegalovirus

MedImmune

CMV retinitis

immune globin

Cytovene

Syntex

sight threatening CMV

Ganciclovir

peripheral CMV

retinitis

Delaviridine

Pharmacia-Upjohn

HIV infection, AIDS,

ARC

(protease inhibitor)

Dextran Sulfate

Ueno Fine Chem.

AIDS, ARC, HIV

Ind. Ltd.

positive asymptomatic

(Osaka, Japan)

ddC

Hoffman-La Roche

HIV infection, AIDS,

Dideoxycytidine

ARC

ddI

Bristol-Myers Squibb

HIV infection, AIDS,

Dideoxyinosine

ARC; combination with

AZT/d4T

DMP-450

AVID

HIV infection, AIDS,

(Camden, NJ)

ARC

(protease inhibitor)

EL10

Elan Corp, PLC

HIV infection

(Gainesville, GA)

Efavirenz

DuPont

HIV infection, AIDS,

(DMP 266)

(SUSTIVA ®),

ARC

(−)6-Chloro-4(S)-

Merck (STOCRIN ®)

(non-nucleoside RT

cyclopropylethynyl-

inhibitor)

4(S)-trifluoro-

methyl-1,4-

dihydro-2H-3,1-

benzoxazin-2-one,

Famciclovir

Smith Kline

herpes zoster, herpes

simplex

FTC

Emory University

HIV infection, AIDS,

ARC

(reverse transcriptase

inhibitor)

GS 840

Gilead

HIV infection, AIDS,

ARC

(reverse transcriptase

inhibitor)

HBY097

Hoechst Marion

HIV infection, AIDS,

Roussel

ARC

(non-nucleoside reverse

transcriptase inhibitor)

Hypericin

VIMRx Pharm.

HIV infection, AIDS,

ARC

Recombinant

Triton Biosciences

AIDS, Kaposi's

Human

(Almeda, CA)

sarcoma, ARC

Interferon Beta

Interferon alfa-n3

Interferon Sciences

ARC, AIDS

Indinavir

Merck

HIV infection, AIDS,

ARC, asymptomatic HIV

positive, also in

combination with

AZT/ddI/ddC

Compound Q

Merck

HIV infection, AIDS,

ARC, asymptomatic

HIV positive

ISIS 2922

ISIS Pharmaceuticals

CMV retinitis

KNI-272

Nat'l Cancer Institute

HIV-assoc. diseases

Lamivudine, 3TC

Glaxo Wellcome

HIV infection, AIDS,

ARC (reverse

transcriptase

inhibitor); also with

AZT

Lobucavir

Bristol-Myers Squibb

CMV infection

Nelfinavir

Agouron

HIV infection, AIDS,

Pharmaceuticals

ARC

(protease inhibitor)

Nevirapine

Boeheringer Ingleheim

HIV infection, AIDS,

ARC

(protease inhibitor)

Novapren

Novaferon Labs, Inc.

HIV inhibitor

(Akron, OH)

Peptide T

Peninsula Labs

AIDS

Octapeptide

(Belmont, CA)

Sequence

Trisodium

Astra Pharm.

CMV retinitis, HIV

Phosphonoformate

Products, Inc

infection, other CMV

infections

PNU-140690

Pharmacia Upjohn

HIV infection, AIDS,

ARC

(protease inhibitor)

Probucol

Vyrex

HIV infection, AIDS

RBC-CD4

Sheffield Med. Tech

HIV infection, AIDS,

(Houston TX)

ARC

Ritonavir

Abbott

HIV infection, AIDS,

ARC

(protease inhibitor)

Saquinavir

Hoffmann-LaRoche

HIV infection, AIDS,

ARC

(protease inhibitor)

Stavudine; d4T

Bristol-Myers Squibb

HIV infection, AIDS,

Didehydrodeoxy-

ARC

thymidine

Valaciclovir

Glaxo Wellcome

genital HSV & CMV

infections

Virazole

Viratek/ICN

asymptomatic HIV

Ribavirin

(Costa Mesa, CA)

positive, LAS, ARC

VX-478

Vertex

HIV infection, AIDS,

ARC

Zalcitabine

Hoffmann-La Roche

HIV infection, AIDS,

ARC, with AZT

Zidovudine; AZT

Glaxo Wellcome

HIV infection, AIDS,

ARC, Kaposi's sarcoma, in

combination with other

therapies

ABT-378

Abbott

HIV infection, AIDS,

ARC (protease inhibitor)

JE2147/AG1776

Agouron

HIV infection, AIDS,

ARC (protease inhibitor)

T-20

Trimeris

HIV infection, AIDS,

T-1249

ARC (fusion inhibitor)

BMS 232632

Bristol-Myers-Squibb

HIV infection, AIDS,

ARC (protease inhibitor)

IMMUNO-MODULATORS

AS-101

Wyeth-Ayerst

AIDS

Bropirimine

Pharmacia Upjohn

advanced AIDS

Acemannan

Carrington Labs, Inc.

AIDS, ARC

(Irving, TX)

CL246,738

American Cyanamid

AIDS, Kaposi's

Lederle Labs

sarcoma

EL10

Elan Corp, PLC

HIV infection

(Gainesville, GA)

FP-21399

Fuki ImmunoPharm

blocks HIV fusion with

CD4 + cells

Gamma Interferon

Genentech

ARC, in combination

w/TNF (tumor necrosis

factor)

Granulocyte

Genetics Institute

AIDS

Macrophage Colony

Sandoz

Stimulating

Factor

Granulocyte

Hoeschst-Roussel

AIDS

Macrophage Colony

Immunex

Stimulating

Factor

Granulocyte

Schering-Plough

AIDS, combination

Macrophage Colony

w/AZT

Stimulating Factor

HIV Core Particle

Rorer

seropositive HIV

Immunostimulant

IL-2

Cetus

AIDS, in combination

Interleukin-2

w/AZT

IL-2

Hoffman-La Roche

AIDS, ARC, HIV, in

Interleukin-2

Immunex

combination w/AZT

IL-2

Chiron

AIDS, increase in CD4

Interleukin-2

cell counts

(aldeslukin)

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

Alpha-2

Schering Plough

Kaposi's sarcoma

Interferon

w/AZT, AIDS

Methionine-

TNI Pharmaceutical

AIDS, ARC

Enkephalin

(Chicago, IL)

MTP-PE

Ciba-Geigy Corp.

Kaposi's sarcoma

Muramyl-Tripeptide

Granulocyte

Amgen

AIDS, in combination

Colony Stimulating

w/AZT

Factor

Remune

Immune Response

immunotherapeutic

rCD4

Corp. Genentech

AIDS, ARC

Recombinant

Soluble Human

CD4

rCD4-IgG

AIDS, ARC

hybrids

Recombinant

Biogen

AIDS, ARC

Soluble Human

CD4

Interferon

Hoffman-La Roche

Kaposi's sarcoma

Alfa 2a

AIDS, ARC, in

combination w/AZT

SK&F106528

Smith Kline

HIV infection

Soluble T4

Thymopentin

Immunobiology

HIV infection

Research

Institute

Tumor Necrosis

Genentech

ARC, in combination

Factor; TNF

w/gamma Interferon

etanercept

Immunex Corp

rheumatoid arthritis

(Enbrel ®)

infliximab

Centocor

rheumatoid arthritis and

(Remicade ®)

Crohn's disease

ANTI-INFECTIVES

Clindamycin with

Pharmacia Upjohn

PCP

Primaquine

Fluconazole

Pfizer

cryptococcal

meningitis, candidiasis

Pastille

Squibb Corp.

prevention of

Nystatin Pastille

oral candidiasis

Ornidyl

Merrell Dow

PCP

Eflornithine

Pentamidine

LyphoMed

PCP treatment

Isethionate

(Rosemont, IL)

(IM & IV)

Trimethoprim

antibacterial

Trimethoprim/sulfa

antibacterial

Piritrexim

Burroughs Wellcome

PCP treatment

Pentamidine

Fisons Corporation

PCP prophylaxis

isethionate for

inhalation

Spiramycin

Rhone-Poulenc

cryptosporidial

diarrhea

Intraconazole-

Janssen Pharm.

histoplasmosis;

R51211

cryptococcal

meningitis

Trimetrexate

Warner-Lambert

PCP

OTHER

Daunorubicin

NeXstar, Sequus

Karposi's sarcoma

Recombinant

Ortho Pharm. Corp.

severe anemia

Human

assoc. with AZT

Erythropoietin

therapy

Recombinant

Serono

AIDS-related wasting,

Human

cachexia

Growth Hormone

Leukotriene B4

—

HIV infection

Receptor

Antagonist

Megestrol Acetate

Bristol-Myers Squibb

treatment of

anorexia assoc. w/AIDS

Soluble CD4 Protein

—

HIV infection

and Derivatives

Testosterone

Alza, Smith Kline

AIDS-related wasting

Total Enteral

Norwich Eaton

diarrhea and

Nutrition

Pharmaceuticals

malabsorption

related to AIDS

It will be understood that the scope of combinations of the compounds of this invention with HIV/AIDS antivirals, immunomodulators, anti-infectives or vaccines is not limited to the list in Table 1 above, but includes in principle any combination with any pharmaceutical composition useful for the treatment of AIDS.

One preferred combination is a compound of the present invention and a nucleoside inhibitor of HIV reverse transcriptase such as AZT, 3TC, ddC, or ddI. Another preferred combination is a compound of the present invention and a non-nucleoside inhibitor of HIV reverse transcriptase, such as efavirenz, and optionally a nucleoside inhibitor of HIV reverse transcriptase, such as AZT, 3TC, ddC or ddI. Still another preferred combination is any one of the foregoing combinations further comprising an additional HIV protease inhibitor such as indinavir, Compound Q, nelfinavir, ritonavir, saquinavir, amprenavir, or abacavir. A preferred additional inhibitor of HIV protease is the sulfate salt of indinavir. Other preferred additional protease inhibitors are nelfinavir and ritonavir. Still another preferred additional inhibitor of HIV protease is saquinavir which is administered in a dosage of 600 or 1200 mg tid.

Other preferred combinations include a compound of the present invention with the following (1) efavirenz, optionally with AZT and/or 3TC and/or ddI and/or ddC, and optionally with indinavir; (2) any of AZT and/or ddI and/or ddC and/or 3TC, and optionally with indinavir; (3) d4T and 3TC and/or AZT; (4) AZT and 3TC; and (5) AZT and d4T.

In such combinations the compound of the present invention and other active agents may be administered together or separately. In addition, the administration of one agent may be prior to, concurrent to, or subsequent to the administration of other agent(s). These combinations may have unexpected effects on limiting the spread and degree of infection of HIV.

Efavirenz is (−)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-1,4-dihydro-2H-3,1-benzoxazin-2-one, also known as DMP-266 or SUSTIVA® (DuPont) or STOCRIN® (Merck). Efavirenz and its utility as an HIV reverse transcriptase inhibitor is described in U.S. Pat. No. 5,519,021 and in the corresponding PCT published application, WO 95/20389. Efavirenz can be synthesized by the protocol of U.S. Pat. No. 5,633,405. Additionally, the asymmetric synthesis of an enantiomeric benzoxazinone by a highly enantioselective acetylide addition and cyclization sequence is described in Thompson et al., {i Tetrahedron Letters 1995, 36: 8937-40, as well as in the PCT publication, WO 96/37457.

AZT is 3′-azido-3′-deoxythymidine, is also known as zidovudine, and is available from Burroughs-Wellcome under the tradename RETROVIR®. Stavudine is 2′,3′-didehydro-3′-deoxythymidine, is also known as 2′,3′-dihydro-3′-deoxythymidine and d4T, and is available from Bristol-Myers Squibb under the tradename ZERIT®. 3TC is (2R-cis)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone, is also known as (−)-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine and lamivudine, and is available from Glaxo Wellcome under the tradename EPIVIR®. ddC is 2′,3′-dideoxycytidine, is also known as zalcitabine, and is available from Hoffman LaRoche under the tradename HIVID®. ddI is 2′,3′-dideoxyinosine, is also known as didanosine, and is available from Bristol-Myers-Squibb under the tradename VIDEX®. The preparation of ddC, ddI and AZT are also described in EPO 0,484,071.

Indinavir is N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4-(S)-hydroxy-5-(1-(4-(3-pyridyl-methyl)-2(S)-N′-(t-butylcarboxamido)-piperazinyl))-pentaneamide, and can be prepared as described in U.S. Pat. No. 5,413,999. Indinavir is generally administered as the sulfate salt at a dosage of 800 mg three times a day. Indinavir is available from Merck under the tradename CRIXIVAN®.

Compound Q is N-(2(R)-hydroxy-1(S)-indanyl)-2(R)-phenylmethyl-4(S)-hydroxy-5-(1-(4-(2-benzo[b]furanylmethyl)-2(S)-N′-(t-butylcarboxamido)-piperazinyl))pentaneamide, preferably administered as the sulfate salt. Compound Q can be prepared as described in U.S. Pat. No. 5,646,148.

Ritonavir is [5S-(5R*,8R*,10R*, 11R*)])-10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11 -bis(phenylmethyl)-2, 4, 7, 12-tetraazatridecan-13-oic acid 5-thiazolylmethyl ester, also known as 5-thiazolylmethyl [(aS)-a-[(1S,3S)-1-hydroxy-3-[(2S)-2-[3-[(2-isopropyl-4-thiazolyl)methyl]-3-methylureido]-3-methylbutyramido]-4-phenylbutyl]phenethyl]carbamate. It is available from Abbott under the tradename NORVIR®. Ritonavir can be prepared as described in U.S. Pat. No. 5,484,801.

Nelfinavir is [3S-[2(2S*,3S*),3a,4ab,8ab]]-N-(1,1-dimethylethyl)decahydro-2-[2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-3-isoquinolinecarboxamide monomethanesulfonate, also known as (3S,4aS,8aS)-N-tert-Butyl-2-[(2R,3R)-3-(3,2-crestoamido)-2-hydroxy-4-(phenylthio)butyl]decahydro-3-isoquinolinecarboxamide monomethanesulfonate and VIRACEPT®, which is commerically available from Agouron. Nelfinavir can be prepared as described in U.S. Pat. No. 5,484,926.

Saquinavir is N-tert-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide, also known as INVIRASE®. Saquinavir can be prepared in accordance with procedures disclosed in U.S. Pat. No. 5,196,438. INVIRASE® (saquinavir mesylate) is available from Roche Laboratories. Saquinavir can be prepared as described in U.S. Pat. No. 5,196,438.

Amprenavir is 4-amino-N-((2 syn,3S)-2-hydroxy4-phenyl-3-((S)-tetrahydrofuran-3-yloxycarbonylamino)-butyl)-N-isobutyl-benzenesulfonamide, also known as Compound 168 and 141 W94. Amprenavir is an aspartyl protease inhibitor that can be prepared by following the procedures described in U.S. Pat. No. 5,585,397. Amprenavir is available under the tradename AGENERASE® from Glaxo Wellcome. Amprenavir can be prepared as described in U.S. Pat. No. 5,783,701.

Abacavir is (1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, also known as 1592U89. Abacavir can be prepared by following the protocol of EP 0434450. The following examples are provided to further illustrate details for the preparation and use of the compounds of the present invention. The examples are not intended to be limitations on the scope of the instant invention in any way, and they should not be so construed. Furthermore, the compounds described in the following examples are not to be construed as forming the only genus that is considered as the invention, and any combination of the compounds or their moieties may itself form a genus. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are in degrees Celsius unless noted otherwise.

Abbreviations: DMF is dimethyl formamide; ESIMS represents Electron Spray Ionization Mass Spectroscopy; Et represents ethyl; HPLC is high pressure liquid chromatography; HREIMS respresents High Resolution Electron Impact Mass Spectroscopy; IPA is isopropyl alcohol; MEK is methyl ethyl ketone; Me represent methyl; rh represents relative humidity, TFA is trifluoroacetic acid; THF is tetrahydrofuran; TLC is thin layer (SiO

2

) chromatography.

EXAMPLE 1

Fermentation of MF6388 (ATCC 74478) Sterile Fungus

A. Media

SEED MEDIUM:

Component

g/L

Yeast extract

4.0

Malt extract

8.0

Glucose

4.0

Junlon

1.5

The medium was prepared with distilled water, the pH adjusted to 7.0 prior to sterilization, and was dispensed at 50 mL/250 mL unbaffled Erlenmeyer flask. Cotton closures were used. Sterilization was at 121° C. for 20 minutes.

PRODUCTION MEDIUM:

1. Solid portion:

675 cc vermiculite was added to a 2 liter roller bottle which was plugged with latex closure and autoclaved for 60 minutes, plus 30 minutes and dried.

2. Liquid portion

Component

Amount

Glucose

150.0 g/L

Fructose

15.0 g/L

Sucrose

40.0 g/L

Casamino acids

2.0 g/L

Asparagine

2.0 g/L

Yeast extract

1.0 g/L

Na

2

HPO

4

0.5 g/L

MgSO

4

.7H

2

O

1.0 g/L

CaCl

2

0.5 g/L

K-elements

1.0 mL/L

pH to 7.0

The medium was prepared with distilled water, dispensed at 220 mL in 500 mL bottles and sterilized at 121° C. for 20 minutes.

K-elements

Component

Amount

FeCl

3

.6H

2

O

5.8 g/L

MnSO

4

.H

2

O

0.1 g/L

CoCl

2

.6H

2

O

0.02 g/L

CuSO

4

.5H

2

O

0.015 g/L

NaMoO

4

.2H

2

O

0.012 g/L

ZnCl

2

0.02 g/L

SnCl

2

.2H

2

O

0.005 g/L

H

3

BO

3

0.01 g/L

KCl

0.02 g/L

HCl (concentrated)

2.0 mL/L

B. Inoculum Preparation

An agar slant was used to prepare FVMs (frozen vegetative mycelia). A portion of the agar slant was transferred aseptically to seed medium. (The composition of the seed medium is detailed above). The flasks were incubated on a 2-inch throw gyratory shaker, 220 rpm for 7 days at 25° C, 85% relative humidity (rh), to obtain biomass. Portions of the biomass were transferred into sterile vials containing glycerol and frozen (as FVM). These were maintained in a final concentration of 10-15% glycerol at −75° C.

C. Seed Culture

Frozen vials (FVM) were thawed to room temperature and used to inoculate seed cultures, at 1.0 mL per 50 mL seed medium. The cultures were grown on a gyratory shaker (220 rpm) for 4 days at 25° C., 85% rh, until a sufficient amount of biomass was obtained.

D. Production

The composition of the solid substrate fermentation medium is shown above. An aliquot (12 mL) of each grown seed was placed into 220 mL of the liquid portion of the production. This was swirled vigorously to disperse the biomass. The contents were dispensed by pouring into a 2-liter roller culture vessel which contained 675 cubic centimeters of steam-sterilized large-particle vermiculite. The contents of the roller bottle were shaken/mixed to insure homogeneous inoculation and coverage. The roller bottles were incubated horizontially, revolving at approximately 4 rpm on a Wheaton roller apparatus at 22° C., 75% rh for 20 days, to obtain secondary metabolite production in the fermentation medium. The contents of each roller bottle were extracted with 200-250 mnL methyl ethyl ketone and the solids discarded.

EXAMPLE 2

Fermentation of MF6591 (ATCC 74477) Ascochyta sp

A. Media:

Seed medium contained the following in g/L: corn steep liquor, 5 g; tomato paste, 40; oat flour, 10; glucose, 10; agar, 4; FeSO

4

·7H

2

O, 0.01; MnSO

4

·4H

2

O, 0.01; CuCl

2

·2H

2

O, 0.00025; CaCl

2

, 0.001; H

3

BO

3

, 0.00056; (NH

4

)

6

Mo

7

O

24

·4H

2

O, 0.00019; ZnSO

4

·7H

2

O, 0.002. The pH was adjusted to 6.8.

Production media contained the following per 250 mL flask: brown rice, 10 g; base liquid, 20 mL. Base liquid contained the following in g(L: yeast extract, 1; sodium tartrate, 0.5; KH

2

PO

4

, 0.5. The flasks were autoclaved for 15 minutes at 121° C, 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 121° C., 15 psi.

B. Inoculum Preparation

Frozen vegetative mycelia (FVM) were prepared by inoculating 50 mL of seed medium in a 250 mL flask and incubating at 25° C., 85% relative humidity and at 200 rpm for 3-5 days. Aliquots of the culture were frozen and used as a source of inoculum 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 25° C., 85% relative humidity and at 200 rpm for days 2-3 days.

D. Production of Culture and Extraction

To 50 mL of production media in a 250 mL flask, 1 mL of seed culture was added as inoculum and the flasks were incubated at 25° C., 85% relative humidity for 24 days. Each flask was then extracted with 50 mL of methyl ethyl ketone and the solids were discarded.

EXAMPLE 3

Isolation of Compounds A and B from MF6591:

The fungus was grown on a brown rice based liquid medium for 31 days and 1.4 L of the broth was extracted with 1.2 volumes of methyl ethyl ketone (MEK). The layers were separated to give 1.4 L MEK extract. The extract was concentrated under reduced pressure to give an aqueous solution that was diluted by addition of 300 mL of water and 50 mL of methanol. This methanolic aqueous solution was partitioned three times with one volume each of hexane followed by five times with one volume each of ethyl acetate. All of the integrase activity was concentrated in ethyl acetate extract. This extract was concentrated to dryness to give a brown gum. Five grams of this material was chromatographed on a 2.0 L SEPHADEX LH20 column. Elution of the column with methanol eluted the activity in a broad zone from 0.6 L to 1.7 L column volume. The most active fraction contained mostly the mixture of Compounds A and B. An aliquot of this fraction was chromatographed on a reverse phase HPLC using ZORBAX RX C-8 (22×250 mm) column. The column was eluted at 8 mL/min with a gradient of acetonitrile in water containing 0.05% trifluoroacetic acid. The gradient started with 20% acetonitrile to 40% acetonitrile in 50 minutes, held at 40% for 10 minutes and then the acetonitrile concentration was increased to 100% over 20 minutes. Lyophilization of fractions eluting in between 31-35 and 52-59 minutes gave Compounds B and A, respectively, as brown powders. Compound B: UVλ

max

: 210, 230, 315 nm. ESIMS (m/z) 405 (M+H)

+

, 403 (M−H)-; HREIMS (m/z): 404.1110 (M

+

, calcd for C

20

H

20

O

9

: 404.1107), 209.0436 (calcd for C

10

H

9

O

5

: 209.0450), 195.0623 (calcd for C

10

H

11

O

4

: 195.0657); for

1

H and

13

C NMR see Table 1. Compound A: UVλ

max

: 215, 235, 320 nm. ESIMS (m/z) 403 (M+H)

+

, 401 (M—H)-; HREIMS (m/z): 402.0960 (M

+

, calcd for C

20

H

18

O

9

: 402.0950), 209.0452 (calcd for C

10

H

9

O

5

: 209.0450), 194.0576 (calcd for C

10

H

11

O

4

: 194.0579); for

1

H and

13

C NMR see Table 1.

EXAMPLE 4

Isolation of Compound A from MF6388:

Sixty mL methyl ethyl ketone extract of MF6388 grown on vermiculite solid medium was concentrated to dryness to give a brown gum. This material was dissolved in a 1:1 mixture of methylene chloride and methanol and chromatographed on a 1.0 L column of SEPHADEX LH20. The column was eluted with methanol. A single combined active fraction was chromatographed on a ZORBAX RX C-8 reverse phase HPLC column. Gradient elution with 20 to 80% acetonitrile in water containing 0.05% TFA over 40 minutes at a flow rate of 8 mL/min gave after lyophilization Compound A as a brown powder.

TABLE 1

1H NMR and 13C NMR spectral data of Compounds B and A

Compound B; R = CH

2

OH

Compound A; R = CHO

δC

δH

δC

δH

Compound A

Compound A

Compound B

Compound B

CDCl

3

+

CDCl

3

+

Position

CD

3

CN

CD

3

CN

CD

3

CN

CD

3

CN

2

97.8

97.8

3

121.3

120.7

4

140.1*

140.1*

5

140.4

142.2

6

148.6

148.7

7

101.4

7.03, s

101.8

7.06, s

8

121.1

120.4

9

193.8

193.7

10

77.4

77.1

11

114.1

121.3

12

130.5

126.0

13

107.3

6.70, s

105.7

7.14, s

14

147.7

147.8

15

134.4

140.9

16

142.0*

140.8*

18

26.4

2.09, s

26.5

2.15, s

19

22.2

1.76, s

25.8

1.87, s

20

60.9

4.58, d, 12.8

190.6

10.21, s

4.40, d, 12.8

21

56.4

3.75, s

56.6

3.75, s

22

56.6

3.83, s

56.7

3.85, s

EXAMPLE 5

HIV Integrase Assay: Strand Transfer Catalyzed by Recombinant Integrase

Assays for the strand transfer activity of integrase were conducted according to Wolfe, A. L. et al., J. Virol. 70, 1424 (1996), hereby incorporated by reference for these purposes. Representative compounds tested in the integrase assay demonstrated IC50's less than 10 micromolar.

EXAMPLE 6

Oral Composition

As a specific embodiment of an oral composition of a compound of this invention, 50 mg of a compound of the present invention is formatted 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 their equivalents.

HIV integrase inhibitors

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