WO2000053577A1

WO2000053577A1 - Hydrazide inhibitors of hiv-1 integrase

Info

Publication number: WO2000053577A1
Application number: PCT/US2000/006361
Authority: WO
Inventors: Nouri Neamati; Yves Pommier; Zhaiwei Lin; Terrence Burke
Original assignee: The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, The National Institutes Of Health
Priority date: 1999-03-12
Filing date: 2000-03-10
Publication date: 2000-09-14
Also published as: AU3875400A; WO2000053577A9

Abstract

The design, synthesis and antiviral activity of novel mercaptosalicylhydrazides are disclosed, which inhibit human immunodeficiency virus type-1 integrase (HIV-1 IN), an essential enzyme for effective viral replication. Examples of the compounds are N,N'-bis(2-mercaptobenzoyl)hydrazide, N,N'-bis(2,2'-dithiosalicyl)hydrazide, and N,N'-bis(2-mercaptobenzyl)-2,2'-dithiosalicylhydrazide. These compounds are effective against the integrase catalytic core domain, inhibit integrase binding to HIV LTR DNA, and inhibit integrase in preassmbled integrase-DNA complexes. The disclosed mercaptosalicylhydrazides are 300 fold less cytotoxic than other known salicylhydrazides, and exhibit antiviral activity. They are also active in Mg+2-based assays, while integrase inhibition by salicylhydrazides is strictly Mn+2-dependent. The mercaptosalicylhydrazides have no detectable effect on other retroviral targets, including reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerase I at concentrations that effectively inhibited integrase. The mercaptosalicylhydrazides of the invention are therefore selective inhibitors of HIV-1 integrase, are useful in therapeutic compositions for the treatment of HIV disease, and can also be used to find related antiviral drugs.

Description

HYDRAZIDE INHIBITORS OF HIV-1 INTEGRASE

FIELD OF THE INVENTION

The present invention concerns anti-retroviral drugs, and particularly prophylactic and therapeutic treatments for infections with the human immunodeficiency virus (HIV).

BACKGROUND OF THE INVENTION

HIV is a retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS). HIV disease is characterized by progressive functional deterioration of the immune system. The treatment of HIV disease has been significantly advanced by the recognition that combining different drugs with specific activities against different biochemical functions of the virus can help reduce the rapid development of drug resistant viruses that were seen in response to single drug treatment. However, even with combined treatments, multi-drug resistant strains of the virus have emerged. There is therefore a continuing need for the development of new anti-retroviral drugs that act specifically at different steps of the viral infection and replication cycle. The integrase (IN) enzyme is an example of such a specific target.

This enzyme catalyzes the insertion by virally-encoded integrase of proviral DNA into the host cell genome, which is the mechanism by which HIV and other retroviruses are introduced into human T-lymphoid cells. For HIV-1 , this process is mediated by a 32 kD virally encoded integrase, having conserved sequences in the HIV long terminal repeats (LTR) (1). Following reverse-transcription in the cytoplasm of infected cells, integrase cleaves two nucleotides from each of the viral DNA ends which contain a highly conserved CA motif. The cleaved DNA migrates to the nucleus as a part of a large nucleoprotein complex, where the integrase catalyzes the insertion of viral DNA into a host chromosome by a direct transesterification reaction. In vitro assays have previously been developed to identify integrase inhibitors (2,3), and have permitted the discovery of diverse classes of drugs that inhibit integrase (4,5). However, the drugs discovered by these assays have not been highly selective and potent inhibitors of the integrase enzyme. Many of these drugs have additionally been non-selective inhibitors of reverse transcriptase or HIV protease, which limits their usefulness in combination therapy directed to different specific steps of the retroviral life cycle.

One class of reported integrase inhibitors is catechol-containing hydroxylated aromatics, which are non-selective integrase inhibitors that can also cross-link proteins (6) and chelate metals (7). Non-catechol containing compounds, however, have been found to be cytotoxic, perhaps because they are unable to form reactive quinones. Such generalized cytotoxicity is a disadvantage, because it can affect host cells without being selective for retroviral eradication or inhibition. Some hydrazides have been reported to be novel noncatechol-containing inhibitors of integrase (8-10). Structure-activity relationship studies among these inhibitors have indicated that the salicyl moiety is required for activity (9,10). Moreover, substitution of a mercapto group for one of the hydroxyls in the salicyl moiety has been found to decrease the anti-integrase potency of the salicyl hydrizide (4).

SUMMARY OF THE INVENTION

It has now surprisingly been found that salicylhydrazides can be substituted at both hydroxyls with a mercapto group, to produce novel anti- integrase mercaptosalicylhydrazides, and analogs and derivatives thereof. In vitro assays can be used to screen for other anti-integrase inhibitors that have the same specific activity. The novel inhibitors are active in the presence of both Mn⁺² and Mg⁺², unlike prior integrase inhibitors which were relatively inactivated by Mg⁺² (and would be expected to be relatively inactive in the presence of physiological concentrations of Mg⁺²). In addition, the disclosed mercaptosalicylhydrazides are selective integrase inhibitors that do not appear to have a substantial effect on other steps of the retroviral life cycle, such as reverse transcription or protease activity.

In one embodiment, the compounds are o o

II II

A— C — NH — Y — NH — C — A

where A is a 2-mercapto aryl or 2-mercapto heterocyclic group, and Y is a substituted or unsubstituted lower alkyl. In particular embodiments, A is an aromatic ring system such as benzene, pyridine, pyrazidine, pyrimidine, pyrazine, naphthalene, or quinoline, which is substituted at the 2 position (ortho to the carbonyl) with a sulfur, and wherein a nitrogen of the heterocycle (if present) is at the 3, 4, 5, or 6 position on the ring. Particular examples of the aromatic rings are 2-mercapto pyridine or 2-mercapto quinoline, which may have one or more additional non- interfering ring substitutions. Examples of A are therefore:

where any of the aromatic rings may be substituted with halogen, lower alkyl, lower alkoxy, or nitro, or with a nitrogen at any of the 3, 4, 5 or 6 position on the ring. Specific disclosed embodiments of the invention include a compound of the formula (or a pharmaceutically acceptable salt of):

wherein

X is one or more H, halogen, lower alkyl, lower alkoxy, or nitro; Y is substituted or unsubstituted lower alkyl;

Z is C or N (and the N can alternatively be at the 3, 4, 5 or 6 ring position); and R, is H, COR₃ , or O₃H (so that the resulting SO₃H is a sulfonic acid); wherein R₃ is lower alkyl, hydroxy, or alkoxy. In particular disclosed embodiments:

Y is lower alkyl or (CH₂)_n where n= 1-2; X is halogen, methyl, methoxy or nitro; and R_j is H, COCH₃ (acetyl) or O₃H;

Y is (CH₂)_n and n=0-5, or Y is CHR₂, wherein R₂ is a carboxy (so that Y is, for example, CHC(O)OH);

Z is C, n=0, and R is H, COCH₃ or O₃H.

In many embodiments, X is hydrogen.

In more particular embodiments, R_{ is H, COCH₃ or O₃H. In other embodiments, in which Y is CHR₂, then R₂ is carboxy. Alternatively, when X is hydrogen, R is hydrogen, COCH₃ or O₃H.

In some embodiments the compound is

wherein Z is C or N;

Y is lower alkyl, or (CH₂)_n where n=0; and

R is H, COCH₃ or O₃H.

In particular the compound may be

wherein R, is H, COCH₃, or O₃H. Alternatively, the compound may be selected from the group of:

2

These compounds are useful in the inhibition of a retroviral integrase, such as HIV integrase, the treatment or prevention of infection by HIV, and in the treatment of AIDS, either as compounds, pharmaceutically acceptable salts or hydrates, and pharmaceutical composition ingredients.

The invention also includes a method of inhibiting HIV integrase, by administering to a mammal an effective amount of one or more of the specific anti-integrase compounds, such as bis(thiosalicyl)hydrazide, or its prodrugs such as a dimeric bisthiosalicylhydrazide. The method also includes treating or preventing HIV infection in a mammal by administering to the mammal an effective amount of one or more of the anti-integrase compounds. The compound or compounds can also be administered in combination with an other compound for the treatment of prevention of HIV infection, such as an HIV reverse transcriptase or protease inhibitor, or another drug that is not an integrase inhibitor. The other drug may be an HIV antiviral agent, an HIV anti-infective agent, and/or an immunomodulator.

Also included in the invention is a process for making the novel dimeric bisthiosalicylhydrazide anti-integrase inhibitors, by providing a homodimeric disulfide, in which an internal disulfide bond acts as a thiol-blocking group, and reacting the homodimeric disulfide with a thiosalicylhydrazide to form a dimeric bisthiosalicylhydrazide which is subsequently reduced (for example with triethylphosphine) to a dimeric bisthiosalicylhydrazide anti-integrase inhibitor. In particular embodiments, the homodimeric disulfide is

the thiosalicyl hydrazide is

and the dimeric bis-thiosalicylhydrazide is

wherein Rj is H. The compounds of the present invention are superior integrase inhibitors to

N ,N ' -bis-salicylhydrazide :

The invention also includes a method of screening for an HIV integrase inhibiting drug, by using an assay of HIV integrase inhibition to screen for analogs or derivatives of any of the disclosed compounds, particularly those that inhibit HIV integrase activity in the presence of Mg⁺². The analogs or derivatives can, for example, be screened in the disclosed assays to obtain a salicylhydrazide compound that inhibits human immunodeficiency virus type-1 integrase, including the integrase core domain, in both Mg⁺²-based assays and Mn⁺² based assays; inhibits integrase binding to HIV LTR DNA; inhibits integrase in preassembled integrase-DNA complexes; is at least 100 times (e.g. 300 times) less cytotoxic than salicylhydrazide 1; and exhibits specific anti-integrase activity in cell culture. In particular, the drugs have no detectable effect on reverse transcriptase, protease, virus attachment, and human topoisomerase I at concentrations that effectively inhibit integrase. In particular embodiments, the analogs and derivatives are mercaptosalicylhydrazides. The disclosed compounds can be used to screen for analogs, derivatives and mimetics of the compounds (particularly mercaptosalicylhydrazides, including substituted mercaptosalicylhydrazides), which inhibit HIV integrase activity in the presence of Mg⁺², for example in one of the assays provided in this specification. In particular embodiments, the compounds inhibit human immunodeficiency virus type-1 integrase (HIV-1 IN), including the integrase core domain, in both Mg⁺²- based assays and Mn⁺² based assays; inhibit integrase binding to HIV LTR DNA; inhibit integrase in preassembled integrase-DNA complexes; are at least 100 times less cytotoxic than salicylhydrazide 1; and exhibit HIV antiviral activity. Particular compounds may also have no detectable effect on reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerase I at concentrations that effectively inhibit integrase.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description of a several embodiments which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structural formulas illustrating a synthetic route to mercaptosalicylhydrazides .

FIG. 2 A is a schematic diagram illustrating an assay for integrase mediated integration of HIV DNA into the host cell genome. A 21-mer blunt-end oligonucleotide corresponding to the U5 end of the HIV-1 LTR, 5' end-labeled with 32p_{5 1S} reacted with purified integrase. The enzyme causes nucleolytic cleavage of two bases from the 3 '-end (3' processing), resulting in a 19-mer oligonucleotide. Subsequently, 3' ends are covalently joined to another identical oligonucleotide that serves as the target DNA (strand transfer reaction).

FIGS. 2B and 2C are graphs illustrating concentration dependent inhibition of HIV-1 integrase by hydrazides 1-4 using Mn⁺² (B) or Mg⁺² (C) as cofactor. Drug concentrations in μM are indicated above each lane. (D) Quantitation of the results of panel C.

FIG. 3. Inhibition of IN-DNA preassembled complexes. Concentration-dependent inhibition of IN assembled product by hydrazides 1- 4 in the presence of and Mn⁺² (A) or Mg⁺² (B). DNA and IN were assembled on ice for 15 min prior to the addition of drugs. Drug concentrations in μM are indicated above each lane. (C) Quantitation of the results of panel B.

FIG. 4. Inhibition of Ca⁺²-mediated IN-DNA preassembled complexes. (A) Concentration-dependent inhibition of Mn⁺²-induced strand transfer. (B) Inhibition of Mg⁺²-induced strand transfer. Drugs and concentrations in μM are indicated above each lane. (C) Quantitation of the results of panel B.

FIG. 5. Inhibition of the IN core domain disintegration activity by salicylhydrazides. (A) Schematic representation of the disintegration assay. The substrate oligonucleotide mimics a strand transfer product, i.e. a Y oligonucleotide. The 15-mer oligonucleotide is 5' end-labeled with 3 p Disintegration generates a 30-mer oligonucleotide. (B) Concentration- dependent inhibition of IN50-212r i85jQ__me(jj_atecι disintegration by compounds 1-4. (C) Quantitation of the results shown in panel B. FIG. 6. Global inhibition of nucleophilic attack in the 3'- processing reaction by salicylhydrazides. (A) Schematic for the nucleophilic reactions. Nucleophilic substitutions by water, glycerol, or the 3'-hydroxyl group of the viral DNA terminus yield a linear trinucleotide with a 5'- phosphate (L), a linear trinucleotide with a glycerol esterified to the 5'- phosphate (G), and a cyclic trinucleotide (C). (B). Concentration-dependent inhibition of nucleophilic substitutions by compounds 1-4. Drug concentrations in μM are indicated above each lane.

FIG. 7. Metal-specific inhibition of IN binding to LTR DNA by salicylhydrazides. (A) The assay uses IN-DNA crosslinking by formation of a Schiff base between IN and a duplex oligonucleotide containing an abasic site. Effect of salicylhydrazides 1-4 on DNA binding of IN in the presence of Mn⁺² (B ) or Mg (C). Phosphorlmager picture showing the inhibition of the 39 kDa product corresponding to the IN-DNA covalent complex in the presence of indicated concentrations of drug.

FIG. 8. Mercaptosalicylhydrazides (2-4), but not salicylhydrazide (1) protect HIV-1 infected CEM cells. Antiviral activities were performed in CEM-SS cells using the standard NCI XTT cytoprotection assay.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations IN: Integrase

Definitions

A "hydrazine" refers to a compound containing H₂NNH₂, while a "hydrazide" is a class of compounds resulting from replacement of at least one of the hydrazine hydrogens with an acid group. A "salicylhydrazide" is a hydrazide in which at least one of the hydrogens of the hydrazine is replaced with a salicyl group, such as:

A mercapto group is -SH, and a 2-mercaptosalicylhydrazide is a compound containing the functional group:

A bis-mercaptosalicylhydrazide is a hydrazide in which the mercaptosalicylhydrazide is repeated twice.

The term "alkyl" refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, and unless otherwise mentioned contains one to twelve carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.

The term "lower alkyl" refers to a cyclic, branched or straight chain monovalent alkyl radical of one to five carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl, and n-amyl. Lower alkyl groups can also be unsubstituted or substituted, where a specific example of a substituted alkyl is 1,1 -dimethyl propyl. "Hydroxyl" refers to -OH.

"Alcohol" refers to R-OH, wherein R is alkyl, especially lower alkyl (for example in methyl, ethyl or propyl alcohol). An alcohol may be either linear or branched, such as isopropyl alcohol. "Carboxyl" refers to the radical -COOH, and substituted carboxyl refers to -COR where R is alkyl, lower alkyl or a carboxylic acid or ester.

The term "aryl" refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl or anthryl), which can optionally be unsubstituted or substituted with, e.g. , halogen, alkyl, alkoxy, mercapto (-SH), alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.

The term "alkoxy" refers to a substituted or unsubstituted alkoxy, where an alkoxy has the structure -O-R, where R is substituted or unsubstituted alkyl. In an unsubstituted alkoxy, the R is an unsubstituted alkyl. The term "substituted alkoxy" refers to a group having the structure -O-R, where R is alkyl which is substituted with a non-interfering substituent.

The term "heterocycle" refers to a monovalent saturated, unsaturated, or aromatic carbocyclic group having a single ring (e.g. benzyl, morpholino, pyridyl or furyl) or multiple condensed rings (e.g. naphthyl, quinolinyl, indolizinyl or benzo[b]thienyl) and having at least one heteroatom, defined as N, O, P, or S, within the ring, which can optionally be unsubstituted or substituted with, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.

The term "halogen" refers to fluoro, bromo, chloro and iodo substituents. The term "amino" refers to a chemical functionality -NR1R2 where

Rland R2are independently hydrogen, alkyl, or aryl. A "pharmaceutical agent" or "drug" refers to a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.

All chemical compounds include both the (+) and (-) stereoisomers, as well as either the (+) or (-) stereoisomer.

An analog is a molecule, that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, or a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington: The Science and Practice of Pharmacology , 19^th Edition (1995), chapter 28. A derivative is a biologically active molecule derived from the base structure. A mimetic is a biomolecule that mimics the activity of another biologically active molecule. Biologically active molecules can include both chemical structures and peptides of protein entities that mimic the biological activities of the mercaptosalicylhydrazides of the present invention.

Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (1985) and The Condensed Chemical Dictionary (1981).

A "mammal" includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

An animal is a living multicellular vertebrate organism, a category which includes, for example, mammals and birds. "HIV disease" refers to a well recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, as determined by antibody or western blot studies. Laboratory findings associated with this disease are a progressive decline in T- helper cells. Materials and Methods

Melting points were determined on either a Gallenkamp or a Mel Temp II melting point apparatus and are uncorrected. Elemental analysis were obtained from Atlantic Microlab Inc. , Norcross, GA and are within 0.4% of the theoretical except where indicated. Fast atom bombardment mass spectra (FABMS) were acquired with a VG Analytical 7070E mass spectrometer under the control of a VG data system. Η NMR data were obtained on a Bruker AC250 spectrometer (250 MHz) and are reported in ppm relative to TMS and referenced to the solvent in which they were run. Anhydrous solvents were obtained commercially and used without further drying. Flash column chromatography was performed using E. Merck silica gel 60 (particle size, 230-400 Mesh). Thiosalicylhydrazide (TSH) was prepared from methylthiosalicylate (MTS) and 2,2'-Dithiosalicylic acid (DSA) was purchased from Sigma-Aldrich Co.

EXAMPLE 1 Synthesis of N, N-Bis(2-mercaptobenzoyl)hydrazide In initial attempts to prepare N,N'-bis(2-mercaptobenzoyl)hydrazide 2, salicylmonohydrazides bearing protected thiol groups were reacted with thiosalicyl chloride. These reactions either produced intractable dark-colored mixtures, underwent premature deprotection, or presented difficulties in removing thiol protection. Alternatively, refluxing methyl thiosalicylate in pure, excess hydrazine yielded only the corresponding monohydrazide. In light of these problems, an alternative "internal protection" scheme was employed which relied on initial formation of homodimeric disulfide DSA (Fig. 1). In this approach, an internal disulfide bond served as a temporary thiol-blocking group, which could be removed once the bisthiosalicylhydrazide had been formed.

Therefore, starting from the commercially available 2,2'-dithiosalicylic acid, dimeric acid chloride DSC was prepared by treatment with thionyl chloride. After reaction with thiosalicylhydrazide TSH, dimeric bisthiosalicylhydrazide 3 resulted. Triethylphosphine-mediated reduction of 3 gave the desired final product 2 (Fig. 1). Compounds 2-4 provided analytical data consistent with their assigned structures. (Compound 4 gave combustion analysis which deviated 0.62% from theoretical N value).

Preparation of 2,2'-dithiosalicyl chloride (DSC): To a suspension of

2,2'-dithiosalicylic acid (DSA) (5.0g, 16.3 mmole) in benzene (100 mL) was added thionyl chloride (18.5g, 155.6 mmole). The mixture was refluxed for three hours, then solvent was removed in vacuo, to provide crude DSC as brown solid in sufficient purity for further use. Mp 142-147 °C. 'H NMR (CDC1₃) δ 8.47 (dd, J- = 8.1 Hz, J₂ = 1.5 Hz, 2H, C₆H₄-), 7.84 (dd, J. = 8.1 Hz, J₂ = 1.2 Hz, 2H, C₆H₄-), 7.84 (td, J_t = 8.1 Hz, J₂ = 1.5 Hz, 2H, C₆H₄-), 7.47 (td, J, = 8.1 Hz, J₂ = 1.2 Hz, 2H, C₆H₄-).

N,N'-Bis(2-mercaptobenzoyl)-2,2'-dithiosalicylhydrazide 3: To a solution of thiosalicylhydrazide (1.77g, 10.6 mmole) in pyridine (20 mL) under argon was added a solution of 2,2'-dithiosalicyl chloride (DSC) (2.0g, 5.3 mmole) in toluene (40 mL) and DMF (20 mL), then the mixture was stirred at room temperature for 36 hours, and solvent was removed in vacuo to produce a brown residue. The residue was treated with H₂O (40 mL) to yield crude 3 as a white precipitate. Recrystallization (methanol/diethyl ether) afforded pure product as an off-white solid (2.50 g, 78% yield). Mp 255 °C (dec). Η NMR (d₆-DMSO) δ 10.82 (d, J = 22.9 Hz, 2H, 2NH), 10.59 (d, J = 21.7 Hz, 2H, 2NH), 7.90-7.24 (m, 16H, 4C₆H₄-), 5.40 (br s, 2H, 2SH). FABMS m/z 607 (M+H), Anal. C₂₈H₂₂N₄O₄S₄: C, H, N.

N, N'-Bis(2,2'-dithiosalicyl)hydrazide 4: Iodine (0.21 g, 0.82 mmole) was added to a solution of N,N'-bis(2-mercaptobenzoyl)-2,2'- dithiosalicylhydrazide 3 (0.5 g, 0.82 mmole) in DMF (3 mL) with methanol (3 mL), and the mixture was stirred at room temperature for 15 hours. Then H₂O (10 mL) was added to the mixture, whereupon crude product came out of solution as a brown solid. Recrystallization (DMF/H₂O) afforded pure 4 as white powder. (0.46 g, 90%). Mp 148 °C (dec). Η NMR (d₆-DMSO): δ 11.75 (br s, 4H, 2NHNH), 8.07-7.48 (m, 16H, 4C₆H₄). FABMS m/z 605 (M+H). Anal. C₂₈H₂₀N₄O₄S₄: C, H, N.

N, N'-Bis(2-mercaptobenzoyl)hydrazide 2: Triethylphosphine (0.19g, 1.6 mmole) containing H₂O (4 mL) was slowly added to a suspension of N,N'- bis(2-mercaptobenzoyl)-2,2'-dithiosalicylhydrazide 3 (0.50 g, 0.82 mmole) in tetrahydrofuran (40 mL was added slowly and the mixture was stirred under argon (0.5 h). Solvents were removed in vacuo, yielding a brown solid, which was purified by silica gel flash chromatography (gradient of chloroform and acetic acid) to afford pure product as beige solid (0.40 g, 80%). Mp 170 °C (dec). 'H NMR (CDClj) δ 10.55 (s, 2H, NHNH), δ 7.71 (d, J = 7.6 Hz, 2H, C₆H₄-), 7.53 (d, J = 7.8 Hz, 2H, C₆H₄-), 7.42 (t, J = 7.8 Hz, 2H, C₆H₄-), 7.28 (t, J = 7.6 Hz, 2H, C₆H₄-), 5.40 (br s, 2H, 2SH). FABMS m/z 303 (M-H). Anal. C₁₄H₁₂N₂O₂S₂0.2H₂O: C, H, N.

All compounds were dissolved in DMSO and the stock solutions were stored at -20° C.

EXAMPLE 2 Integrase Assays The HPLC purified oligonucleotides AE 117 (5 ' - ACTGCT AGAGATTTTCC AC AC-3 ' ) ; AE118 (5'-GTGTGGAAAATCTCTAGCAGT-3'); AE157 (5 -GAAAGCGACCGCGCC-3');

AE 146 (5 ' -GGACGCC ATAGCCCCGGCGCGGTCGCTTTC-3 ' ) ; AE156 (5 -GTGTGGAAAATCTCTAGCAGGGGCTATGGCGTCC-3 '); RM22M (5 '-T ACTGCT AGAGATTTTCCACAC-3'); and RMAB2 (5'-GTGTGGAAAATCTCTAGCUGT-3') were purchased from Midland Certified Reagent Company (Midland, TX). An expression system for the wild-type integrase and the IN50-212 (F185K) were obtained from the Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD. To analyze the extent of 3 '-processing and strand transfer using 5'- end labeled substrates, AE118 was 5 '-end labeled using T4 polynucleotide kinase (Gibco BRL) and γ[³²P]-ATP (Dupont-NEN). To determine the extent of 30-mer target strand generation during disintegration, AE157 was 5 '-end labeled and annealed to AE156, AE146, and AE117. The kinase was heat- inactivated and AE117 was added to the same final concentration. The mixture was heated at 95 °C, allowed to cool slowly to room temperature, and run through a G-25 Sephadex quick spin column (Boehringer Mannheim, Indianapolis, IN) to separate annealed double-stranded oligonucleotide from unincorporated label. To analyze the extent of site-specific cleavage of 3 '-end-labeled substrate by integrase, AE118 was 3 '-end-labeled using γ[³²P]-cordycepin triphosphate (Dupont-NEN) and terminal transferase (Boehringer Mannheim). The transferase was heat-inactivated, and RM22M was added to the same final concentration. The mixture was heated at 95°C, allowed to cool slowly to room temperature, and run through a G-25 spin column as before.

To determine the extent of Schiff base formation, RMAB2 was 5 '-end labeled and reacted with AE117 as described above. The uracil was removed from duplex oligonucleotide containing deoxyuridine by incubation of 40 μl of end-labeled DNA (500 nM stock solution) with 1 unit of uracil DNA glycosylase (Life Technologies, Inc.) for 90 minutes at 30^° C. The reaction was then loaded on a G-25 Sephadex quick spin column to remove the unincorporated label and the uracil.

To determine the extent of 3 '-processing and strand transfer, integrase was preincubated at a final concentration of 200 nM with the inhibitor in a reaction buffer (50 mM NaCl, 1 mM HEPES, pH 7.5, 50 μM EDTA, 50 μM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl₂, 0.1 mg/ml bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide, and 25 mM MOPS, pH 7.2) at 30°C for 30 minutes. Then, 20 nM of the 5 '-end ^-^P-labeled linear oligonucleotide substrate was added, and incubation was continued for an additional one hour. Reactions were quenched by the addition of an equal volume (16 μl) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025 % bromophenol blue). An aliquot (5 μl) was electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris-borate pH 8.3, 2 mM EDTA, 20% acrylamide, 8M urea).

Gels were dried, exposed in a Phosphorlmager cassette, and analyzed using a Molecular Dynamics Phosphorlmager (Sunnyvale, CA). Percent inhibition was calculated using the following equation:

%I = 100 X [1 - (D - C)/(N - C)] where C, N, and D are the fractions of 21-mer substrate converted to 19-mer (3 '-processing product) or strand transfer products for DNA alone (C), DNA plus integrase (N), and integrase plus drug (D). All IC50 values were determined by plotting the drug concentration versus percent inhibition, and determining the concentration which produced 50% inhibition. To determine the effects of drugs on the choice of nucleophile in the 3 '-processing, reactions were performed essentially as described above with a 3 '-end labeled oligonucleotide. Disintegration reactions were performed as above with a Y oligonucleotide (i.e. , the branched substrate in which the U5 end was "integrated" into target DNA). Table 1

Metal-dependent Inhibition of Assembled IN-DNA Complexes by

Hydrazides 1-4

Compound Preassembly" Postassembly*

'-Processing Strand transfer 3'- ■Processing Strand transfer

(Mn⁺²) IC₅₀ (ΩM)

1 2.0 ± 0.7 0.7 ± 0.1 12 10

2 5.1 + 1.2 5.0 + 1.0 35 12 3 3.2 ± 1.0 3.7 ± 1.1 8 9 4 2.4 ± 0.9 7.2 ± 2.3 5 5

(Mg⁺

1 > 1000 > 1000 > 1000 > 1000

2 20 11 20 18 3 15 10 20 18 4 11 11 7 5 ^a Integrase was preincubated with metals and drugs for 30 min followed by DNA for 1 hr.

Integrase was preincubated with metals and DNA on ice for 10 min followed by drugs for 1 hr.

Data obtained from these studies showed that the hydrazides of the present invention inhibited integrase catalytic activities. The integrase catalyzed 3 '-processing and DNA strand transfer were measured by the in vitro assay employing purified integrase, a 21-mer duplex oligonucleotide corresponding to the U5 end of the HIV LTR sequence (Fig. 2A), and divalent metal ion (Mn⁺² or Mg⁺²). Figure 2B shows a representative gel illustrating inhibition of both 3'- processing and strand transfer reactions by the hydrazides of the present invention. All inhibitors exhibited comparable IC₅₀ values (2-5 μM) indicating that mercapto groups (compound 2) can substitute for hydroxyls (compound 1) and that cyclization does not adversely influence potency.

Since integrase assays are routinely carried out in the presence of 10 mM of 2-mercaptoethanol, under these assay conditions it is plausible that 4 could be reduced to 3 and subsequently to 2, a possibility which would be a particular advantage in the design of these compounds. Similarly, under reducing conditions found in vivo, the disulfides could also be reduced to the corresponding mercapto derivatives. In the integrase reaction buffer, which contains mercapto-ethanol, a significant portion of 4 converted to 3 in approximately half an hour. This was corroborated by TLC analysis, where after 30 minutes, solutions of 4 in plain DMF, or in integrase buffer solution, clearly indicated the conversion between 2 and 3 in the presence of mercaptoethanol (data not shown).

The hydrazides were also found to exert a remarkable divalent metal dependency. Although in vitro assays are generally more efficient in the presence of Mn⁺² as a cofactor, it has been proposed that the actual physiological divalent cation is Mg⁺² [(see for example (25)]. For that reason, the extent of 3'- processing and strand transfer in the presence of either Mn⁺² (Fig. 2B) or Mg⁺² (Fig. 2C and D, Table 1) was determined. Although compound 1 was inactive in Mg⁺², the mercapto-containing compounds 2-4 were active with either Mg⁺² or Mn⁺². Hence in contrast to other salicylhydrazides and more generally polyhydroxylated aromatics (7,26,27), the mercaptosalicylhydrazides (compounds 2-4) inhibit integrase in reactions catalyzed with either Mn⁺² or Mg⁺².

To further examine the metal dependency of compound 1 in purified integrase assays, a variety of different conditions were employed. For example, when integrase was allowed to mix without metal and in the presence of all possible combinations of Mn⁺², Mg⁺², or Ca⁺², compound 1 was inhibitory only in Mn⁺²-containing reactions (data not shown). This inhibitory effect of 1 in the presence of Mn⁺² was independent of the order of the addition of DNA (data not shown). This implies that inhibition of integrase by compound 1 is absolutely a Mn⁺² dependent phenomenon. The Mn⁺²-dependent activity of 1 was further established with other assays as described below.

Additionally, assays were performed with different concentrations of the various metals. Integrase can cleave its substrate DNA in the presence of Mn⁺² with concentrations as low as 3 mM and as high as 50 mM, and in the presence of Mg⁺² in the range of 7 mM to 25 mM (data not shown). When compounds 1 and 2 were examined within these concentration ranges similar IC₅₀ values were observed as for 1 at all the tested concentrations of Mn⁺², whereas 1 was slightly more potent at higher concentrations of Mn⁺² (data not shown).

In order to investigate whether mercaptosalicyhydrazides can affect the assembled complex, integrase was allowed to bind to DNA on ice first, before addition of drugs. Figure 3 demonstrates that the salicylhydrazides (1-4) inhibited 3 '-processing and strand transfer in the presence of Mn⁺² within the same range (IC₅₀ values 5-35 μM). However, when similar reactions were performed in the presence of Mg⁺², compound 1 was inactive, while the mercaptosalicylhydrazides 2-4 inhibited both 3 '-processing and strand transfer as efficiently as in Mn⁺² (Fig. 3B and 3C and Table 1). Integrase is also known to be capable of assembling with its DNA substrate in the presence of Ca⁺² without proceeding to enzymatic cleavage of the DNA (28). Using this assay (Fig. 4), it was again found that the mercaptosalicylhydrazides (2-4) were active both in the presence of Mg⁺² and Mn⁺², while the salicylhydrazide 1 was most active in the presence of Mn⁺². The N- and C-terminal regions of integrase are not required for inhibition of disintegration by salicylhydrazides. In order to further examine the mechanism of integrase inhibition, an integrase deletion mutant, IN^0"212 _was employed, which lacks the amino-terminal zinc-binding region and the carboxy 1- terminal DNA-binding domain (30,31). This mutant is capable of catalyzing an apparent reversal of the integration reaction, known as "disintegration" (Fig. 5A) (31). In the disintegration assay, all compounds exhibited comparable inhibition (Fig. 5B and 5C). These results demonstrate that hydrazides can interfere with the activity of the IN core region irrespective of the presence or absence of thiols, and that their inhibitory activity does not require the presence of the zinc binding and C-terminal domains of IN. However, when 1 was assayed for the inhibition of disintegration in the presence of a combination of Mn⁺² and Zn⁺², it was observed that inhibition by 1 was stimulated by the presence of Zn⁺² and that this effect was more pronounced when the full length IN was used instead of the core (data not shown). Inhibition of 3 '-processing by salicylhydrazides was shown to result from inhibition of the integrase-mediated nucleophilic reactions. The 3'- processing reaction involves hydrolysis of a single phosphodiester bond 3 ' of the conserved CA-3' dinucleotide (Fig. 6A). However, in addition to this hydrolysis reaction, retroviral integrases can utilize glycerol or the hydroxyl group of the viral DNA terminus as the nucleophile in the 3 '-processing reaction, yielding respectively, a glycerol esterified to the 5 '-phosphate, or a circular di- or trinucleotide (Fig. 6 A) (32-34). In order to examine the effect of hydrazides 1-4 on the choice of nucleophiles in the 3 '-processing reaction, a substrate DNA labeled at the 3 '-end with 32p._cor(jy_Cepi_{n was} employed (34). All compounds inhibited glycerolysis, hydrolysis, and circular nucleotide formation similarly (Fig. 6B). These results indicate that hydrazides indiscriminately block all IN -catalyzed nucleophilic reactions.

EXAMPLE 3

DNA Binding and Topoisomerase Assays

DNA binding assay using Schiff base formation

This assay was previously described in detail (13). Briefly, integrase (200 nM) was preincubated with the inhibitor (at the indicated concentration) for 30 minutes at 30°C. Subsequently, an oligonucleotide containing an abasic site (13) (Fig. 9A) in reaction buffer as described above was added for 2 minutes at room temperature. A freshly prepared solution of sodium borohydride (0.1 M final concentration) was added, and reaction was continued for an additional two minutes. An equal volume (16 μl) of 2X SDS-PAGE buffer (100 mM Tris, pH 6.8, 4% 2-mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was added to each reaction, and the reaction was heated at 95 °C for three minutes prior to loading a 20 μl aliquot on a 12% SDS-polyacrylamide gel. The gel was run at 120 V for 1.5 hr, dried, and exposed in a Phosphorlmager cassette. Gels were analyzed using a Molecular Dynamics Phosphorlmager. The hydrazides were also shown to inhibit integrase-DNA Binding. As previously described, DNA-integrase cross-linking assays can be employed to assess inhibition of DNA binding (13). A 21-mer oligonucleotide having uracil substituted for adenine in the conserved C A dinucleotide on the distal end of the U5 LTR, is treated with uracil DNA glycosylase to generate an abasic site (Fig. 7A). Subsequent incubation with integrase and stabilization with sodium borohydride of the Schiff base formed between free aldehyde on the DNA backbone and the amino group of lysine(s) from integrase, produced a cross-linked product running as a 39 kDa band in SDS-PAGE (Fig. 7, lane 2). The salicylhydrazides 1-4 inhibited the formation of integrase-DNA complexes in the presence of Mn⁺² (Fig. 7A). However, only the mercaptosalicylhydrazides (2-4) were active in the presence of Mg⁺² (Fig. 7B). UV-cross linking experiments provided similar results (data not shown). Assays were performed with different combinations of metals in order to investigate the apparent absolute Mn⁺² requirement of 1. When the Schiff-base assays were performed in the presence of other divalent metals (Ca⁺², Zn⁺², Co⁺², and all possible combinations of these metals) 1 inhibited DNA cross-linked product only in reactions where Mn⁺² was present (data not shown). These results and the dependency on the type of divalent metal are consistent with the inhibition of DNA integration results, and suggest that salicylhydrazides 1-4 inhibit IN activity by blocking IN binding to its DNA. Topoisomerase Reactions

Reactions were performed in 10 μl reaction buffer (0.01 M tris- HC1 pH 7.5, 150 mM KC1, 5 mM MgCl₂, 0.1 mM EDTA, 15 mg/ml bovine serum albumin) with the following duplex oligonucleotide substrate (14) labeled with γ³²P-cordycepin at the 3 '-end of the upper strand (asterisk):

5'-GATCTAAAAGACΓTGGAAAAATTTTTAAAAAA* ATTTTCTGAA-CCTTTTTAAAAATTTTTTCTAG-5'

This oligonucleotide contains a single topoisomerase I cleavage site (caret on the upper strand). Approximately 50 fmoles oligonucleotides per reaction were incubated with 10 units of calf thymus DNA topoisomerase I (Gibco BRL, Gaithersburg, Maryland). Reactions were stopped by adding sodium dodecylsulfate (0.5% as final concentration). Proteolysis was halted by the addition of 36 μl 2.5 X loading buffer (98 % formamide, 0.01 M EDTA, 1 mg/ml xylene cyanol and 1 mg/ml bromophenol blue). An aliquot (5 μl) was electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris-borate pH 8.3, 2 mM EDTA, 20% acrylamide, 8M urea).

In this assay specific for eukaryotic topoisomerase I (14), none of the four salicylhydrazides induced cleavage complex or inhibited the ability of topoisomerase I to generate camptothecin-mediated cleavage complexes at concentrations that effectively inhibited integrase. Hence the tested hydrazide compounds were more selective for integrase, and their inhibitory potency is not due to non-specific binding to the integrase protein.

EXAMPLE 4 Anti-HIV Assays in Cultured Cells

The anti-HIV drug testing was performed at NCI based on a protocol described by Weislow et al. (15). All compounds were dissolved in dimethyl sulfoxide and diluted 1 :100 in cell culture medium. Exponentially growing T4 lymphocytes (CEM cell line) were added at 5000 cells per well. Frozen virus stock solutions were thawed immediately before use, suspended in complete medium to yield the desired multiplicity of infection (m. o. i. « 0.1), and added to the microtiter wells, resulting in a 1 :200 final dilution of the compound. Uninfected cells with the compound served as a toxicity control, and infected and uninfected cells without the compound served as basic controls.

Cultures were incubated at 37 C in a 5 % CO2 atmosphere for 6 days. The tetrazolium salt, XTT was added to all wells, and cultures were incubated to allow formazan color development by viable cells. Individual wells were analyzed spectrophotometrically to quantitate formazan production, and in addition were viewed microscopically for detection of viable cells and confirmation of protective activity.

All positive control compounds for individual assays except AZTTP were obtained from the NCI chemical repository. The reference reagents for the individual assays were as follows: attachment: Farmatalia (NSC 65016) (16) and dextran sulfate (NSC 620255); reverse transcriptase inhibition: rAdT Template/primer-AZTEC (Sierra BioResearch, Tuscon, AZ), rCdG Template/primer-UC38 (17) (NSC 629243); protease inhibition: KNI-272 (18) (NSC 651714).

The mercaptosalicylhydrazides 2-4 protected HIV-1 infected cells with 50% inhibitory concentration (IC₅₀) values ranging from 14 to 34 μM and 50% effective concentration (EC₅₀) values ranging from 4 to 18 μM (Table 2, Fig. 8). In contrast, compound 1 was cytotoxic and exhibited an IC₅₀ value of 0.1 μM without showing protection of the HIV-1 infected cells. There was a 300-fold reduction in cytotoxicity of mercaptosalicylhydrazides relative to 1.

EXAMPLE 5

HIV-1 Cell and Target-based Assays The cell-based p24 attachment assay has been described in detail elsewhere (19). Assays for activity against HIV-1 reverse transcriptase rAdT (template/primer) and rCdG (template/primer) using recombinant HIV-1 reverse transcriptase (from ABL Basic Research NCI-FCRDC, Frederick, MD) have been previously described (20). The substrate cleavage of recombinant HIV-1 protease in the presence of test compounds was quantified using an HPLC -based methodology with the artificial substrate Ala-Ser-Glu- Asn-Try -Pro-He- Val-amide (Multiple Peptide Systems, San Diego, CA) previously described (19,21).

Hydrazides were assayed for inhibition of retroviral targets other than integrase. At concentrations that inhibited integrase, none of these agents exhibited detectable activity on HIV-1 RT, protease, and viral attachment (Table 2).

Table 2 Inhibition of Viral Proteins by Hydrazides

Concentration of DM

NR, not reached due to cytotoxicity

Salicylhydrazide 1 is an inhibitor of integrase (8,9). However, nanomolar cytotoxicity associated with 1 limited further studies in cell-based assays (10). The thiol compounds of the present invention, however, are 300-fold less cytotoxic than the salicylhydrazide compound 1. Moreover, the salicylhydrazide 1, like other hydroxy lated aromatics, has an absolute requirement for a divalent metal ion for the inhibition of integrase. This is a drawback not shared by compounds 2-4. The low inhibitory potency of the mercaptosalicylhydrazides 2-4 against various other viral and nonviral proteins tested, attests to their selectivity for integrase. Therefore, this class of compounds is different from previously described hydroxy lated aromatics, which frequently do not exhibit such selectivity.

EXAMPLE 9 Methods of Treatment The present invention includes a treatment for HIV disease and associated diseases, in a subject such as an animal, for example a rat or human. The method includes administering the compound of the present invention, or a combination of the compound and one or more other pharmaceutical agents, to the subject in a pharmaceutically compatible carrier and in an amount effective to inhibit the development or progression of HIV disease. Although the treatment can be used prophylactically in any patient in a demographic group at significant risk for such diseases, subjects can also be selected using more specific criteria, such as a definitive diagnosis of the condition.

The vehicle in which the drug is delivered can include pharmaceutically acceptable compositions of the drugs, using methods well known to those with skill in the art. Any of the common carriers, such as sterile saline or glucose solution, can be utilized with the drugs provided by the invention. Routes of administration include but are not limited to oral and parenteral rountes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal.

The drugs may be administered intravenously in any conventional medium for intravenous injection, such as an aqueous saline medium, or in blood plasma medium. The medium may also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like. A more complete explanation of parenteral pharmaceutical carriers can be found in Remington: The Science and Practice of Pharmacy (19^th Edition, 1995) in chapter 95.

Embodiments of other pharmaceutical compositions can be prepared with conventional pharmaceutically acceptable carriers, adjuvants and counterions as would be known to those of skill in the art. The compositions are preferably in the form of a unit dose in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions.

The compounds of the present invention are ideally administered as soon as possible after potential or actual exposure to HIV infection. For example, once HIV infection has been confirmed by laboratory tests, a therapeutically effective amount of the drug is administered. The dose can be given by frequent bolus administration.

Therapeutically effective doses of the compounds of the present invention can be determined by one of skill in the art, with a goal of achieving tissue concentrations that are at least as high as the IC₅₀ of each drug tested in the foregoing examples. The low toxicity of the compound makes it possible to administer high doses, for example 100 mg/kg, although doses of 10 mg/kg, 20 mg/kg, 30 mg/kg or more are contemplated. An example of such a dosage range is 0.1 to 200 mg/kg body weight orally in single or divided doses. Another example of a dosage range is 1.0 to 100 mg/kg body weight orally in single or divided doses. For oral administration, the compositions are, for example, provided in the form of a tablet containing 1.0 to 1000 mg of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 100, 200, 400, 500, 600, and 1000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject being treated.

The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, 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, and severity of the condition of the host undergoing therapy.

The pharmaceutical compositions can be used in the treatment of a variety of retroviral diseases caused by infection with retroviruses that require integrase activity for infection and viral replication. Examples of such diseases include HIV-1 , HIV-2, the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV), HTLV-1 , HTLV-2, spumavirus (human foamy virus) and feline infectious leukemia. EXMAPLE 10 Combination Therapy

The present invention also includes combinations of HIV integrase inhibitor compounds with one or more agents useful in the treatment of HIV disease. For example, the compounds of this invention may be administered, whether before or after exposure to the virus, in combination with effective doses of other anti-virals, immunomodulators, anti-infectives, or vaccines. The term "administration" refers to both concurrent and sequential administration of the active agents. Example of antivirals that can be used in combination with the integrase inhibitors of the invention are: AL-721 (from Ethigen of Los Angeles, CA), recombinant human interferon beta (from Triton Biosciences of Alameda, CA), Acemannan (from Carrington Labs of Irving, TX), gangiclovir (from Syntex of Palo alto, CA), didehydrodeoxythymidine or d4T (from Bristol-Myers-Squibb), EL10 (from Elan Corp. of Gainesville, GA), dideoxycytidine or ddC (from Hoffman-LaRoche), Novapren (from Novaferon labs, Inc. of Akron, OH), zidovudine or AZT (from Burroughs Wellcome), ribaririn (from Viratek of Costa Mesa, CA), alpha interferon and acyclovir (from Burroughs Wellcome), Indinavir (from Merck & Co.), 3TC (from Glaxo Wellcome), Ritonavir (from Abbott), Saquinavir (from Hoffmann-LaRoche), and others.

Examples of immuno-modulators that can be used in combination with the integrase inhibitors of the invention are AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F106528, and TNF (Genentech).

Examples of some anti-infectives with which the integrase inhibitors can be used include clindamycin with primaquine (from Upjohn, for the treatment of pneumocystis pneumonia), fluconazlone (from Pfizer for the treatment of cryptococcal meningitis or candidiasis), nystatin, pentamidine, trimethaprim- sulfamethoxazole, and many others. The combination therapies are of course not limited to the lists provided in these examples, but includes any composition for the treatment of HIV disease (including treatment of AIDS).

In view of the many possible embodiments to which the principles of the invention may be applied, it should be recognized that the illustrated embodiments are only particular examples of the invention and should not be taken as a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

References

1. Brown (1998) Integration. Retroviruses (Coffin, J. M., Hughes, S. H., and Varmus, H. E. , Eds.), Cold Spring Harbor Press, Cold Spring Harbor

2. Mazumder et al. (1998) in Methods in Molecular Medicine (Kinchington and Schinazi, eds), pp. 1998, The Humana Press, Inc. , Totowa, NJ

3. Chow (1997) Methods 12(4), 306-17

4. Neamati et al. (1997) Drug Discovery Today 2, 487-498

5. Pommier and Neamati (1998) Adv Virus Res (in press)

6. Stanwell et al. (1996) Biochem Pharmacol 52(3), 475-80 7. Fesen et al. (1994) Biochem Pharmacol 48(3), 595-608

8. Hong et al. (1997) J Med Chem 40(6), 930-6

9. Zhao et al. (1997) J Med Chem 40(6), 937-41

10. Neamati et al. (1998) J Med Chem 41, 3202-3209

11. Pecoraro et al. (1984) Biochemistry 23(22), 5262-71 12. Sarnovsky et al. (1996) Embo J 15(22), 6348-61

13. Mazumder et al. (1996) J Biol Chem 271(44), 27330-8

14. Pommier et al. (1995) Proc Natl Acad Sci USA 92, 8861-8865

15. Weislow et al. (1989) J Natl Cancer Inst 81 , 577-586

16. Clanton et al. (1995) Antiviral Res 27(4), 335-54 17. Bader et al. (1991) Proc Natl Acad Sci USA 88(15), 6740-4

18. Kageyama et al. (1993) Antimicrob Agents Chemother 37(4), 810-7

19. Rice et al. (1995) Science 270(5239), 1194-7

20. Turpin et al. (1996) J Virol 70(9), 6180-9

21. Riceet al. (1997) Antimicrob Agents Chemother 41(2), 419-26 22. Urpi and Vilarrasa (1986) Tetrahedron Lett 27, 4623-4624

23. Maiti et al. (1988) Synth Commun 18, 575-582

24. Houk and Whitesides (1987) J Amer Chem Soc 109, 6825-6836

25. Cowan (1995) The biological chemistry of magnesium (Cowan, J. A. , Ed.), VCH 26. Hazuda et al. (1997) J Virol 71(9), 7005-11

27. Neamati et al. (1997) J Med Chem 40(6), 942-51 28. Ellison and Brown (1994) Proc Natl Acad Sci USA 91(15), 7316-20

29. Hazuda et al. (1997) J Virol 71(1), 807-11

30. Bushman et al. (1993) Proc Natl Acad Sci USA 90(8), 3428-32

31. Chow et al. (1992) Science 255(5045), 723-6 32. Katzman and Sudol (1996) J Virol 70(4), 2598-604

33. Vink et al. (1991) Nucleic Acids Res 19(24), 6691-8

34. Mazumder et al. (1996) J Med Chem 39(13), 2472-81

35. Neamati et al. (1997) Antimicrob Agents Chemother 41(2), 385-93

36. Neamati et al. (1997) Antimicrob Agents Chemother 8, 485-495 37. Lubkowski et al. (1998) Proc Natl Acad Sci USA 95(9), 4831-6

Claims

We claim:

1. A compound of the formula

O o

II II

A— C — NH— Y— NH — C — A

wherein A is a 2-mercapto substituted aromatic or aromatic heterocyclic ring system, and Y is substituted or unsubstituted lower alkyl, or pharmaceutically effective salts thereof.

2. The compound of claim 1, wherein the aromatic ring system comprises a system selected from the group consisting of benzyl, naphthyl, pyridyl, pyrazidinyl, pyrimidinyl, pyrazinyl, naphthyl, or quinoline, or a nitrogen containing heterocyclic aromatic ring with a N at any position on the ring except the 2- position.

3. The compound of claim 1, wherein Y is (CH₂)_n wherein n=0-2.

4. The compound of claim 3, wherein n=0.

5. The compound of claim 1 having a formula

wherein X is H, halogen, lower alkyl, lower alkoxy, or nitro:

Y is substituted or unsubstituted lower alkyl;

Z is C or N; and

R[ is H, COR₃, or O₃H, wherein R₃ is hydroxy or lower alkyl or alkoxy; or a pharmaceutically acceptable salt thereof.

6. The compound of claim 5, wherein the compound is

wherein

X is H, halogen, NO₂, lower alkyl, or lower alkoxy; and Rj is H, COR₂, or O₃H, wherein R₂ is lower alkyl.

7. The compound of claim 5, wherein Y is (CH₂)_n where n= 1-2; X is halogen, methyl, methoxy or nitro; and R_j is H, COCH₃ or O₃H.

8. The compound of claim 6, wherein X is halogen, methyl, methoxy or nitro; and R_γ is H, COCH₃ or O₃H.

9. The compound according to claim 5, of the formula:

wherein n=0; and

R. is H, COOCH₃ or O₃H.

10. The compound according to claim 1, of the formula:

wherein Y is lower alkyl.

11. The compound of claim 10, wherein Y is CHR₂, wherein R₂ is carboxy.

12. The compound of claim 1, wherein the compound is selected from the group of:

VI and VII

VIII

13. The compound of claim 12, wherein the compound is

14. The compound of claim 5, wherein Y is (CH2)_n where n=0-5, or CHR₂, wherein R₂ is carboxy;

15. The compound of claim 2, wherein X is hydrogen, and Rj is hydrogen, COCH₃ or O₃H.

16. The compound according to claim 5 of the formula I, wherein X is halogen, CH₃, OCH₃ or NO₂, and R, is H, COCH₃ or O₃H.

17. The compound according to claim 9 of the formula II, wherein Z is C, and R, is H, COCH₃ or O₃H.

18. The compound according to claim 9 of the formula

wherein R, is H, COCH₃, or O₃H.

19. Use of one or more of the compounds of claim 1 , for inhibiting HIV integrase.

20. Use of one or more of the compounds of claims 1 , 5, 6, 7, 8, 9, 10, 11 or 12 for treatment of HIV disease.

21. Use of one or more of the compounds of claims 1 , 5, 6, 7, 8, 9, 10, 11 or 12 for preventing HIV infection, in a mammal subject in need thereof, by administering to the mammal an effective amount of one or more of the compounds of claim 1.

22. Use of one of more of the compounds of claim 21 , in combination with an other compound for the treatment or prevention of HIV infection, wherein the other compound is not an integrase inhibitor.

23. Use of one or more of the compounds of claim 22, wherein the other compound is one or more of an AIDS antiviral agent, and anti-infective agent, and an immunomodulator.

24. A pharmaceutical composition, comprising a therapeutically effective amount of one or more of the compounds of claim 1 , and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 39, wherein the compound is one or more of the compounds of claim 5, 6, 7, 8, 9, 10, 11 or 12.

26. A process for making a medicament, comprising combining a pharmaceutically acceptable carrier with one or more of the compounds of claim

7.

27. A pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds of claim 1 , a pharmaceutically acceptable carrier, and one or more of an AIDS antiviral agent, and anti-infective agent, and an immunomodulator.

28. A process for making a compound of claim 1, comprising providing a homodimeric disulfide, in which an internal disulfide bond acts as a thiol-blocking group.

29. The process of claim 28, further comprising reacting the homodimeric disulfide with a thiosalicylhydrazide to form a dimeric bisthiosalicylhydrazide.

30. The process of claim 29, further comprising reducing the dimeric bisthiosalicylhydrazide .

31. The process of claim 30, wherein reducing the dimeric bisthiosalicylhydrazide comprises reducing with triethylphosphine.

32. A process for making a bisthiosalicylhydrazide, comprising: reacting a homodimeric disulfide with a thionyl chloride to produce a dimeric acid chloride; reacting the dimeric acid chloride with a thiosalicylhydrazide to produce a dimeric bisthiosalicylhydrazide; and reducing the dimeric bisthiosalicylhydrazide with triethylphosphine to produce bisthiosalicylhydrazide.

33. The process of claim 32, wherein the homodimeric disulfide is

and the dimeric acid chloride is

34. The process of claim 32, wherein the dimeric bisthiosalicylhydrazide is

35. A method of screening for an anti-HIV integrase drug, comprising providing an assay of HIV integrase inhibition; and using the assay to screen for drugs that inhibit HIV integrase activity in the presence of Mg+2.

36. The method of claim 35, wherein using the assay to screen for drugs comprises screening for analogs or derivatives of any of the compounds of claim 1.

37. The method of claim 35, wherein using the assay to screen for drugs comprises screening for analogs or derivatives of any of the compounds of claims 2, 8 or 13.

38. The method of claim 35, wherein the assay detects a salicylhydrazide compound that: inhibits human immunodeficiency virus type-1 integrase (HIV-1 IN), including the integrase core domain, in both Mg⁺²-based assays and Mn⁺² based assays; inhibits integrase binding to HIV LTR DNA; inhibits integrase in preassembled integrase-DNA complexes; is at least 100 times less cytotoxic than other known salicylhydrazides; and exhibits HIV antiviral activity.

39. The method of claim 38, further comprising detecting a salicylhydrazide having no detectable effect on reverse transcriptase, protease, and virus attachment, and exhibiting no detectable activity against human topoisomerase I at concentrations that effectively inhibit integrase.

40. The method of claim 35, wherein the compounds that are screened are mercaptosalicylhydrazides.