GB2271566A - HIV integrase inhibitors - Google Patents

Abstract

Compounds of the general formula: <IMAGE> wherein n is 0-3; p is 1-2; q is 1-2; X is CH2, O or N-R<1>, and R<1> is H, C1-4alkyl or C3-5cycloalkyl; R is (a) C1-6 alkyl; (b) C1-6alkoxy; (c) hydroxyl; (d) halogen; (e) CN; (f) NO2; (g) NHSO2CHÏ; or (h) COOH; G is H or R, and salts or hydrates thereof have HIV integrase inhibiting activity.

Description

TITLE OF THE INVENTION DIBENZYLHETEROCYCLIC OR DIBENZYLCYCLOALKYL DERIVATIVES AS INHIBITORS OF HIV INTEGRASE 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 celi 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 (Ratner, L. et al., Nature, 313, 277(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 AIDS and similar diseases, e.g., azidothymidine or AZT. Applicants demonstrate that the compounds of this invention are inhibitors of HIV integrase, probably by inhibiting its endonucleolytic cleavage activity rather than its binding function.

The particular advantage of the present invention is highly specific inhibition of HIV integrase. The compounds of the present do not inhibit a.variety of other protein-nucleic acid interactions, including enzymatic reactions involving HIV reverse transcriptase, mammalian topoisomerase I, mammalian topoisomenase II, Eco RI endonuclease, or mammalian polymerase II, as well as other related interactions, e.g., involving HIV TAT protein.

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.

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 n is 0-3; p is 1-2; q is 1-2; X is CH2, 0 or N-R1, and Rl is H, C14alkyl or C3~5cycloakyl; R is (a) C16 alkyl; (b) C1-6 alkoxy; (c) hydroxyl; (d) halogen; (e) CN; (f) NO2; (g) NHSO2CH3; or (h) COOH; G is H or R, or pharmaceutically acceptable salt or hydrate thereof.

A preferred compound of the present invention is Compound A as follows: A:

L-619,323, 7,8-Dihydroxy-1-(3,4-dihydroxyphenyl)-3-methyl-2,3,4,5- tetrahydro-lH-3-benzazepine, or pharmaceutically acceptable salt or hydrate thereof.

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

When any variable (e.g., G, R, 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.

As used herein except where noted, "alkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.

"Halogen" or "halo" as used herein, means fluoro, chloro, bromo and iodo.

The compounds of the present invention can be synthesized by the following methods.

SCHEME I

Deprotect

One method of synthesizing the compounds of the present invention is found in Scheme I. The phenyl aldehyde reactant is treated with an alkylene Grignard reagent to give 2. Protection of the hydroxyl, followed by ozonolysis in the presence of a reducing agent affords S. The Wittig approach can then be used, e.g. the aldehyde 1 is reacted with 4, which is a benzyl substituted with alkylene triphenyl phosphorane.

The resulting condensation product 5 yields an unsaturated alkylene bridge, which is then hydrogenated, deprotected, then subjected to acid-catalyzed cyclodehydration, to yield i. Further deprotection of R or G groups may or may not be needed, as the case may be. For example, OH groups protected with methyl substituents may be dealkylated by reaction with pyridine hydrochloride. It will be understood that the appropriate protecting groups will be added to prevent undesired side reactions with G or R substituents.

When X=NR1 cyclodehydration is conducted with phosphorus oxychloride in the Bischler-Napieralski reaction instead of polyphosphoric acid (PPA). See Examples 1-6, as well as Grethe, b. (ed.) Isoquinolines Wiley New York 1981, pp. 142-161.

SCHEME II

Scheme II provides a method for synthesizing compounds of Formula I when X=CH2. In Scheme II, formation of a Grignard reagent is performed by reaction of 7 with magnesium turnings. -After reaction with oxirane, conversion of the resulting alcohol into the corresponding bromide 8 occurs by reaction with phosphorus tribromide. Preparation of a second Grignard reagent followed by reaction with the appropriate aromatic aldehyde, e.g. parabenzylvanillinaldehyde, gives the alcohol intermediate e.

Formation of a good leaving group for cyclization is accomplished by, for example, benzylation with NaH as base, to afford 10. Intramoleular Friedel-Crafts cyclization in the presence of BF3^Et20 followed by hydrogenation provides compounds of the present invention 11. For extensive discussion of synthetic routes related to Scheme II, see, for example, Boissin, P. et al., Tetrahedron 48, 687 (1992).

The compounds of the present inventions are useful in the inhibition of HIV integrase the prevention of 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 EIV 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 HIVby e.g., blood transfusion, exchange of body fluids, bites, accidental needle stick, or exposure to patient blood during surgery.

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.

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.

These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable preparations, for example, as sterile injectable 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 injectable 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-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify 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 100 mg/kg body weight orally in divided doses. Another preferred dosage range is 0.1 to 200 mg/kg body weight orally in divided doses. 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, immunomodulators, antiinfectives, or vaccines, such as those in the following table.

TABLE ANT IV IRALS Drug Name Manufacturer Indication AL-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 threatening CMV Ganciclovir (Palo Alto, CA) 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) Drug Name Manufacturer Indication 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 (Blmont, CA) Sequence Zidovudine; AZT Burroughs Wellcome AIDS, a dv, ARC (Rsch. Triangle Park, pediatric AIDS, NC) Kaposi's sarcoma, asymptomatic HIV infection, less severe HIV disease, neurological involvement, in combination with other therapies.

Drug Name Manufacturer Indication Ansamycin LM 427 - Adria Laboratories ARC (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, HIV (Rsch. Triangle in combination Park, NC) w/Retrovir Acyclovir Burroughs Wellcome AIDS, ARC, asymptomatic HIV positive, in combination with AZT.

Antibody which Advanced Biotherapy AIDS, ARC neutralizes pH Concepts labile alpha aber- (Rockville, MD) rant Interferon in an immunoadsorption column Drug Name Manufacturer Indication L-697,661 Merck AIDS, ARC, (Rahway, NJ) asymptomatic HIV positive, also in combination with AZT.

L-696,229 Merck AIDS, ARC, (Rahway, NJ) asymptomatic HIV positive, also in combination with AZT.

L-735,524 Merck Aids, ARC, (Rahway, NJ) asymptomatic HIV positive, also in combination with AZT.

IMMUNO-MODULATORS Drug Name Manufacturer Indication AS-101 Wyeth-Ayerst Labs. AIDS (Philadelphia, PA) Bropirimine Upjohn advanced AIDS (Kalamazoo, MI) Acemannan Carrington Labs, Inc. AIDS, ARC (Irving, TX) (See also 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, w/TNF (tumor necrosis CA) factor) Granulocyte Genetics Institute AIDS Macrophage Colony (Cambridge, MA) Stimulating Sandoz Factor (East Hanover, NJ) Drug Name Manufacturer Indication Granulocyte Hoeschst-Roussel AIDS Macrophage Colony (Somerville, NJ) Stimulating Immunex Factor (Seattle, WA) Granulocyte Schering-Plough AIDS Macrophage Colony (Madison, NJ) Stimulating Factor AIDS, in combination 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 Immunex w/AZT Immune Globulin Cutter Biological pediatric AIDS, in Intravenous (Berkeley, CA) combination (human) w/AZT IMREG-1 Imreg AIDS, Kaposi's (New Orleans; LA) sarcoma, ARC, PGL IMREG-2 Imreg AIDS, Kaposi's (New Orleans, LA) sarcoma, ARC, PGL DrUp Name Manufacturer Indication 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- (Summit, NJ) Tripeptide Granulocyte Amgen AIDS, in combination Colony (Thousand Oaks, CA) w/AZT Stimulating Factor rCD4 Genentech AIDS, ARC Recombinant (S. San Francisco, Soluble Human CD4 CA) 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 Druz Name Manufacturer Indication SK & 106528 Smith, Kline & French HIV infection Soluble T4 Laboratories (Philadelphia, PA) Thymopentin Immunobiology HIV infection Research Institute (Annandale, NJ) Tumor Necrosis Genentech ARC, in combina Factor; TNF (S.San Francisco, tion w/gamma CA) Interferon ANTI-INFECTIVES Drug Name Manufacturer Indication 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 (Rosemont, IL) & IV) Drug Name Manufacturer Indication Piritrexim - Burroughs Wellcome 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. histoplasmosis; 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.

w/AIDS Total Enteral Norwich Eaten 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-infectives 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.

The compound L-697,661 is an inhibitor of HIV reverse transcriptase and is 3-CF4,7-dichloro-l,3-benzo- xazol-2-yl)methyl]amino)-5-ethyl-6-methyl-pyridin2(1H)-one or pharmaceutically acceptable salt thereof.' The compound L-696,229 is an inhibitor of HIV reverse transcriptase and is 3-E2-Cl,3-benzoxazol- 2-yl)ethyl]-5-ethyl-6-methyl-pyridin-2(lH)-one or pharmaceutically acceptable salt thereof. The compound L-735,524 is an inhibitor of HIV protease and is N-(2(R)-hydroxy-l(S)-indanyl)-2(R)-phenylmethyl-4-(S) hydroxy-5-(1-(4-(3-pyridyl-methyl)-2(S)-N'-(t-butylcar- boxamido)-piperazinyl))-pentaneamide, or pharmaceutically acceptable salt thereof.

EXAMPLE 1 1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-3,4-dihydro i soguinol ine A stirred solution of N-(3,4-dimethoxyphenylethyl)-3,4-dimethoxyphenylacetamide (5g, prepared according to Weisbach, J.A. et al J. Med. Chem. II, 752 (1968) and Wiegrebe, W., Archiv. Pharm. 297,362 (1964)), POC13 (2;5my) in toluene (25 ml) was ref fluxed with stirring for 3 hours. The exotherm was cooled to room temperature, and diluted with petroleum ether (100 ml).

The supernatant was discarded, and the gummy residue washed with petroleum ether (2 x 50 ml). A volume of 100 ml ice water was added to the washed residue, the resulting mixture basified with conc. NH40H, and extracted with CH2C12 (4-x 50 ml). The combined CH2C12 extracts were washed with H20 (50 ml), sat. NaCl solution (50 ml) and dried (Na2S04). After 1 hour the Na2SO4 was filtered off, the ether solvent removed, affording a viscous, amber oil (4.6 g).

EXAMPLE 2 1-(3,4-dimethoxybenzoyl)-6,7-dimethoxy-3,4-dihydro- i soguinoline The product of Example 1 (4.6 g) was dissolved in ethanol (15 ml) and bubbled with 'air while stirring overnight. A pale yellow precipitate formed, which was filtered. The filtrate was washed (2 ml ethanol), dried and the residue chromatographed on silica gel (CHC13:MeOH, 9:1). Yield: 0.9g, mp = 180-187"C. The filtrate was suspended in ethanol (15 ml) and 02 bubbled with stirring overnight. About 0.65 g more of the pale yellow solid precipitated out, and was worked up as above. Total yield: 1.55 g.

EXAMPLE 3 1-(3,4-dimethoxybenzoyl)-6,7-dimethoxy-2-methyl-3,4- dihvdroi-soguinolinium iodide The ketone product of Example 2 (1.55 g, 4.4 mmol) was suspended in ethanol (25 ml), and heated to near reflux. Methylating agent CH3I (1.25 g, 0.55 ml, 8.8 mmol) was added and the mixture refluxed with stirring for 5 hours. An additional 0.25 ml of CH3I was added to drive the reaction to completion and ref fluxing continued overnight. At 20 hours, the mixture was cooled to room temperature and filtered.

The filtrate was a yellow solid, 1.5 g, mp = 178-180"C. An analytical TLC on silica gel (CHC13:MeOH, 9:1) confirmed nearly quantitative yield.

EXAMPLE 4 1-(3,4-dimethoxy-alpha-hydroxybenzyl)-6,7-dimethoxy 2-methyl-i .2.3 .4-tetrahydroisoauinoline hvdrochloride To the ketone iodide product of Example 3 (i.4 g), suspended in MeOH (25 ml, not completely soluble), was added stepwise NaBH4 (1 g). The mixture had vigorous gas evolution. It was refluxed with stirring in a steam bath for 2 hours, cooled to room temperature and the solvent removed. The residue was taken up in 35 ml water, extracted with CH2CH2 (4 x 50 ml). The CH2CH2 extracts were combined, washed with 50 ml of saturated NaCl solution and dried (Na2S04).

After filtering off Na2S04, and removing solvent, a viscous, glassy residue was obtained (0.9 g). The residue was dissolved in ethanol/HCl, stirred, scratched to induce crystallization, and placed in an ice water bath. The resulting white solid was filtered and dried, to afford the title compound. mp = 218-221"C (dec.) EXAMPLE 5 7,8-Dimethoxy-1-(3,4-dimethoxyphenyl)-3-methyl 2.3.4,5-tetrahvdro-lH-3-benzBzePine HC1 A mixture of the product of Example 4 (14.3 g) in proprionic acid (190 ml) was warmed until the mixture turned yellow. The mixture was heated to reflux with vigorous stirring, then Zn dust (26.4 g) was added gradually over about 10 minutes. The reflux continued with vigorous stirring for 7 hours. A resulting white solid was filtered and washed with proprionic acid.The filtrate and washings were combined and dried, taken up in water (200 ml) and combined with a 50 ml H20 wash of the white solid, to give a combined liquid wash. The combined liquid wash was basified with 10% NaOH, extracted with ether (4 x 250 ml). The combined ether extracts were washed with water (200 ml); sat. NaCl solution (200 ml) and dried (Na2S04), to give a yellow oil (11.6 g). A white solid crystallized in EtOH/HCl, mp = 196"-198"C.

Anal Calcd. for C22H27N04 C, 64.03; H, 7.16; N, 3.56.

Found C, 63.77; H, 7.01; N, 3.37.

EXAMPLE 6 7,8-Dihydroxy-1-(3,4-dihydroxyphenyl)-3-methyl-2,3,4,5- tetrahydro-lH-3-benzazepine hydrobromide sesquihydrate, a hvdrate of Compound A The methoxy product of Example 5 (2 g, 5.1 mmol) was dissolved in CH2C12 (200 ml) and cooled in a dry ice-acetone bath. Boron tribromide (2.4 ml in 50 ml CH2C12) was added dropwise over 5 minutes, and the resulting mixture stirred in the bath with gradually rising temperature to about 15 C. After 4 hours of stirring, methanol was added dropwise until gas evolution had ceased, giving a white precipitate. The precipitate was washed with CH2C12, filtered and dried under vacuum. The precipitate was purified by successive steps of dissolution and drying using ethanol, acetone, and finally water. mp = 192 -194.5 dec.Mass spectrometry showed a molecular ion at 301 without detectable boron.

Anal Calcd. for C17H19N04 + IIBr + 1.5 H20 C, 49.89; H, 5.66; N, 3.42.

Found C, 50.37; H, 5.36; N, 3.19.

EXAMPLE 7 Annealing and Klenow Labeling of Integrase Substrate Oligonucleotide 1. HIV integrase substrate oligonucleotide (SEQ ID No:l) has been gel purified and is at a concentration of 0.65 OD/ml (21.45 Fg/ml), or 1 g = 46.6 l 2. Lyophilize 1 g of substrate oligo (SEQ. ID NO:1).

3. Resuspend in 48, > 1 dH20, add 1 > 1 1M Tris-HCl pH 7.5 and 1 l 5M NaCl.

4. Heat at 85 C for 15 min, take heating block out of.

heating unit and allow to slow cool on the bench top to room temperature, usually for 1.5 to 2 hours, affording annealed oligonucleotide (SEQ.ID No. 1), which forms a hairpin loop.

5. Divide into 5 l aliquots and store at -20 C.

6. Prepare fresh 10X Klenow buffer: 500 l 1M Tris-HCl pH 7.2 100 l 1M MgC12 50 l 5M NaCl 1 l 1M DTT 349 l dH2O 1000 l 7. Klenow reaction mix: 4 pl dH20 2 l 10X Klenow buffer 5 l annealed oligonucleotide 5 l 32p-dGTP 2 l lmM TTP 2 l Klenow enzvme (5 Units/ l) 20 pl Incubate at room temperature in a plexiglass shielded box for 2 hours.

8. Add 30 ul 20mM Tris-HCl pH 7.5, 100mM NaCl, lOmM EDTA buffer to the mix to stop the reaction.

9. Extract with 50 l of Phenol/chloroform (1:1).

10. Extract with 50 pl of Chloroform/isoamyl alcohol (24:1).

11. The reaction mix is now ready for-molecular sieve chromatography. It is stored at -20 C until chromatography can be done.

12. TCA precipitate aliquots coming off the column, then count both pellet and supernatant. Pool and save fractions having low counts in supernatant and high counts in the pellet, which contain labeled integrase substrate oligonucleotide.

HIV INTEGRASE ASSAY: TCA PRECIPITATION 1. On day of assay, pipet 5 1 of sample or 10% DMSO for controls into tubes kept on ice.

2. Prepare fresh reaction buffer on day of assay, and dispense eight (8) aliquots into tubes on ice for dispensing with multipipettor. Pipet 11 1 per tube.

Reaction buffer: 20 l 1M Tris-HCl pH 7.8 3 > 1 1M MnC12 3.5 1 1.4 M Beta-mercap toethanol (1:10 dilution of commercial stock solution) 5 l 10 mg BSA/ml 518.5 1 dH 550 1, enough for 50 reactions 3. Thaw 1 tube of pooled integrase on ice.

4. Thaw labeled integrase substrate oligonucleotide, allow approx. 10000 cpm per reaction, dilute if necessary with 20mM Tris-HCl pH 7.5, 100-mM NaCl, 0.1 mM EDTA. Pipet 2 > 1 of substrate solution to be used in the assay into 208 l of the dilution buffer, and sample 150 l into one polypropylene tube, place on ice until end of assay (=input counts). Pipet eight aliquots into tubes on ice for dispensing with multipipettor.

5. Pipet 2 l of HIV integrase enzyme (expressed in E. coli BL21(DE3) as described in LaFemina, R. et al., J. Virol. 65, 5624(1991)) into all reaction tubes except No Enzyme Control tubes. No Enzyme Control tubes receive 2 l each HA Buffer B (50mM Tris-HCl pH 7.5, 10% glycerol, lmM DTT, O.lmM EDTA, and 1M NaCl). Immediately add 2 l of substrate to all tubes.

6. Finger tap to mix gently. Place tubes into prewarmed racks in 37 C water bath. Incubate 60 min.

7. Add 5 > 1 of 1 mg tRNA/ml solution to each tube, followed by 185 l of 11X cold TCA. Vortex hard.

Place on ice at least 60 min.

8. Filter through polyvinylidene difluoride microporous filters to trap undesired TCA precipitate.

9. Determine average cpm of No Enzyme-Controls (background). Determine average cpm of Enzyme Controls (100 Z value). Substract background from all samples, divide by Enzyme control value, multiply by 100, and substract from 100% to determine % Inhibition.

HIV Integrase Assay: Gel Cleavage Gel cleavage was conducted according to LaFemina, R. et al., J. Virol. 65, 5624(1991), as follows. A 5'-end-labeled oligonucleotide representing the terminal 20 nucleotides of the U5 LTR plus strand (SEQ ID NO:2) was annealed with its complement (SEQ ID NO:3) and 1 ng of the resulting radiolabelled substrate was incubated with or without inhibitor for 1 h at 37"C with 80 ng (1 > 1) of HIV integrase purified from E.

coli BL21(DE3)/pET3c. The reaction was in 20 1 of 10 mM Tris-HCl (-pH 7.8)-5 mM 2-mercaptoethanol containing 3 mM MnC12. Products were analyzed by electrophoresis on 20% sequencing gels. The positions and sizes of the substrate, 20 nucleotides, and the primary cleavage product, 18 nucleotides, are readily determined. To assay inhibition of HIV integrase, the reaction is conducted with inhibitor having various concentrations in the range of O.l-lOg/ml. Compound A shows detectable inhibition as low as 0.5 Fg/ml.

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, adaptations, or modifications, as come within the scope of the following claims and its equivalents.

Claims (6)

WHAT IS CLAIMED IS:

1. A compound of the formulas:

wherein .n is 0-3; p is 1-2; q is 1-2; X is CH2, 0 or N-R1, and R1 is H, C14 alkyl or C35 cycloalkyl; R is (a) C1-6 alkyl; (b) C1-6 alkoxy; (c) hydroxyl; (d) halogen; (e) CN; (f) N02; (g) NHS02CH3; or (h) COOH; G is H or R, or pharmaceutically acceptable salt or hydrate thereof

2. The compound

or pharmaceutically acceptable salt or hydrate thereof.

3. A method of inhibiting HIV integrase, comprising administering to a mammal an effective amount of a compound of any of Claims 1 or 2.

4. A method of preventing infection of HIV, or of treating infection by HIV or of treating AIDS or ARC, comprising administering to a mammal an effective amount of a compound of any of Claims 1 or 2.

5. A pharmaceutical composition useful for inhibiting HIV integrase, comprising an effective amount of a compound of any of Claims 1 or 2, and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition useful for preventing or treating infection of HIV or for treating AIDS or ARC, comprising an effective amount of a compound of any of Claims 1 or 2, and a pharmaceutically acceptable carrier.