WO2000004903A1

WO2000004903A1 - The use of cd4-binding small molecules to inhibit hiv infection

Info

Publication number: WO2000004903A1
Application number: PCT/US1999/016358
Authority: WO
Inventors: Ziwei Huang; James Hall; Xiaobing Han; Xueyou Hu
Original assignee: Thomas Jefferson University
Current assignee: Thomas Jefferson University
Priority date: 1998-07-21
Filing date: 1999-07-20
Publication date: 2000-02-03
Anticipated expiration: 2001-01-21
Also published as: EP1100500A1; CA2338329A1

Abstract

A method of inhibiting HIV-1 infection is provided that comprises administering to a subject an effective amount of an active compound having a molecular weight of between 200 daltons and 650 daltons. At a concentration of not more than 100 νM, compounds useful in the practice of the invention inhibit greater than 50 % of the binding of gp120 to CD4. Preferred compounds are further characterized in that they inhibit less than 10 % of the binding of human CD4-expressing, CD4-transfected COS cells to Raji cells.

Description

THE USE OF CD4-BINDING SMALL MOLECULES TO INHIBIT HIV INFECTION

Cross-Reference to Related Applications

This application claims priority from provisional applications Ser. No. 60/128,105, filed April 7, 1999, and 60/093,562, filed July 21, 1998, the entire disclosures of which are incorporated herein by reference.

Field of the Invention

The present invention generally relates to the fields of immunology and infectious diseases, and more particularly to compounds that selectively bind to a functional surface pocket of the Dl domain of the CD4 protein, thereby interfering with the interaction of CD4 and the gp 120 envelope glycoproteins of the human immunodeficiency virus.

Background of the Invention

CD4 plays an important role in immune response and infection of human immunodeficiency virus type 1 (HIV- 1 ) . The entry of HIV- 1 into the target cell is mediated by CD4 as the primary receptor (Dalgleish et al., Nature 312:763- 766 (1984); Klatzmann et al, Nature 312:767-768 (1984)), as well as chemokine receptors such as CCR5, CXCR4, CCR3 and CCR2b as necessary coreceptors (Feng et al, Science 272:872-877 (1996); Dragic et al, Nature 381:667-673 (1996); Dengeta/. Natwre381:661-666(1996);Alkhatibetfl/.,₁S'c ence 272:1955-8 (1996)). Macrophage-tropic (M-tropic) strains are those HIV-1 isolates that are capable of using CCR5. T-tropic strains utilize CXCR4 for entry, while dual -tropic strains utilize CXCR4 or CCR5 (Simmons et al, J. Virol. 70:8355-60 (1996)). While these HIV-1 strains adopt different chemokine coreceptors, they all require the CD4 protein for their entry into the target cell. CD4 is a glycoprotein expressed on the surface of helper T cells that consists of four immunoglobulin-like extracellular domains (D1-D4) (White et al, J. Exp. Med. 148:664-673 (1978)). The HIN envelope glycoprotein gp 120, anchored to the viral membrane by its non- covalent association with the gp41 transmembrane glycoprotein, forms the spike on the surface of the virion (Kowalski etal, Science 237:1351-1355 (1987); Lu et al, Nature Struct, biol 2:1075-1082 (1995)). The gpl20 glycoprotein binds to the Dl domain of CD4, and mutagenesis indicates that the second complementarity- determining region (CDR2) of CD4 D 1 is a critical binding site for gp 120 (Moebius et al. , Journal of Experimental Medicine 176:507-517 (1992); Sweet et al. , Curr. Opin. Biotechnol. 2:622-33 (1991)).

A recent advance in the understanding of the biology of the HIV fusion process includes (1) resolution of the crystal structure of an HIV gpl20 glycoprotein bound to the D1D2 fragment of CD4 and to the antigen-binding fragment of a neutralizing antibody 17b (Kwong et al, Nature 393:648-659 (1998)). HIV-1 fusion may involve the initial binding of HIV-1 gpl20 to its primary receptor CD4 which results in conformational changes in gpl20 and probably also CD4 (Gershoni et al, FASEB Journal 7:1185-7 (1993); Clements et al. , AIDS Research & Human Retroviruses 7:3-16 (1991); Sattentau et al , Journal of Virology 67:7383-93 (1993)). The gpl20-CD4 complex interacts with the chemokine coreceptor to form a heterotrimeric complex of gpl 20-CD4-coreceptor (Lapham etal, Science 274:602-5 (1996); Wu etal, Nature 6605:179-83 (1996); Trkola et al, Nature 384:184-7 (1996)). This triggers further conformational changes that allow the gp41 fusion peptide to spring into the target membrane and initiate fusion.

In addition to serving as the primary receptor for HIV gpl 20, the CD4 molecule is known to mediate critical functions in the immune system. CD4 binds to a non-polymorphic region of the β-chain of the major histocompatibility complex (MHC) class II molecule on the antigen-presenting cell, thereby increasing the avidity of the T-cell receptor (TCR) for its ligand (Doyle et al. , Nature 330:256- 259 (1987); Gay etal, Nature 328:626-629 (1987); Konig etal, Nature 356:796- 798 (1992)). Thus, the CD4-MHC class II interaction is critical for the activation of T cells during immune response. The presence of CD4 can potentiate the T cell response as much as 300 fold ( Janeway, Semin. Immunol. 3:153-160 (1991)). CD4 is also capable of functioning as a signal transduction molecule as studies have indicated that the cytoplasmic tail of CD4 is associated with the tyrosine kinase p56^lck (Veillette et al. , Cell 55:301 -308 (1988); Barber et al. , Proc. Natl Acad. Sci. USA 86:3277-3281 (1989); Turner etal, Cell 60:755-765 (1990)). The binding of antigen in association with MHC class II to the TCR facilitates interaction of the CD4 molecule with TCR/CD3 (Saizawa et al. , Nature 328:260-263 (1987); Rivas et al, J. Immunol. 140:2912-2918 (1988) and CD45 (Dianzani et al, Eur. J. Immunol. 20:2249-2257 (1990)). The assembled complex brings about the apposition of the CD4 tyrosine kinase p56^lck, the TCR tyrosine kinase p59^fyn, and the CD45 tyrosine phosphatase (Veillette et al. , Cell 55:301 -308 (1988); Barber et al, Proc. Natl. Acad. Sci. USA 86:3277-3281 (1989)). This may then result in the dephosphorylation and activation of the kinases, leading to a cascade of signals that activate the T cell.

The understanding in the structure and biology of HIV viral entry machinery has inspired new therapeutic strategies. For example, several inhibitors targeting the chemokine coreceptor CXCR4 can block the entry of certain HIV-1 strains (Murakami etal. . Exp. Med. 186:1389-1393 (1997); Schools etal,J Exp. Med. 186:1383-1388 (1997); Donzella et al, Nature Medicine 4:72-77 (1998); Doranz et al, J. Exp. Med. 186:1395-1400 (1997)). In the development of new anti-HIV drugs based on the viral entry mechanism, the interaction between gp 120 and CD4 is a particularly important target since such an interaction is the critical initial step in the HIN fusion process, and CD4 is the primary receptor required by various virus strains.

The current treatments available for HIV infection in humans target the proteins required for viral replication and processing. Used in combination therapy, the reverse transcriptase (RT) and protease inhibitors dramatically decrease the levels of HIV RNA measured in infected individuals. However, prolonged usage of these treatments may cause mutations in RT and HIV protease, thus diminishing the effectiveness of these drugs.

Mutational studies have shown that the regions of CD4 involved in MHC class II interactions overlap with each other. The primary binding site for gpl 20 is the CDR2 region with mutations at residues 43 and 47 abrogating gpl 20 binding to CD4. Other evidence implicates the CDR3 region on CD4 as another important interaction site between CD4 and gpl 20.

In the development of new anti-HIV drugs based on the viral entry mechanism, the interaction between gpl 20 and CD4 is a particularly important target since such an interaction is the critical initial step in the HIV fusion process, and CD4 is the primary receptor required by various virus strains. For CD4 as a drug target, the challenge is to design selective inhibitors that block CD4 interaction with HIV gpl 20 but do not interfere with the CD4-MHC class II interaction which is important for normal immune response.

Such compounds would advantageously comprise small molecules. In contrast to large protein-based therapeutics such as monoclonal antibodies, small molecules are advantageous since they are more likely to be non-immunogenic, orally administrable, and amenable to chemical synthesis.

Summary of the Invention

A method of inhibiting HIV-1 infection comprises administering to a subject an effective amount of an active compound having a molecular weight of between 200 daltons and 650 daltons, which compound, at a concentration of not more than lOO M inhibits greater than 50% of the binding of gpl 20 to CD4.

The compounds may be selected based upon a DOCK3.5 complementarity score with the GFCC'C" pocket of CD4. The compounds have a DOCK3.5 complementarity score of at least 150. According to one preferred embodiment, the active compound inhibits less than 10% of the binding of human CD4-expressing, CD4-transfected COS cells to Raji cells.

According to one embodiment, compounds having the desired DOCK3.5 complementarity score are selected from: (i) TJU101, 5-(4- chlorobenzylthio)-3 - { [(4-chlorophenyl)-2-thiazolyl] methylthiomethyl } -4-methyl- 1 ,2,4-triazole; (ii) TJU1021, 4-(4-methoxy-ρhenyl)-l-[imidazol(2,l- B)benzothiazole; (iii) TJU103, N-(3-indomethylene)isonicotinic hydrazone; and (iv) TJU104, N-(2,4-dichlorophenyl)-3-(l ,2,4-triazol- 1 -ylmethyl)- 1 ,2,4-triazole-5- carboxamide, the structures of which are as follows:

According to another embodiment, the active compound used in the method of the invention has the formula:

wherein R is

or

Y is C or N;

R¹ is selected from the group consisting of C_rC₅ alkyl, phenyl, pyridyl, cyclohexyl substituted with one, two, or three halogen atoms, phenoxy substituted with one or two halogen atoms, phenyl, pyridyl, cyclohexyl, cyclopentyl, cyclopentyl substituted with one or two NH₂ OH, OCH₃, CF₃, or COOH, a five-member or six-member heterocyclic ring substituted with halogen, NH₂ OCH₃, CF₃ or COOH, and NHCOA, NHCONHA, NHCSNHA, COA, and CONHA, where A is selected from the group consisting of phenyl, mono-, di-, or tri-halogen substituted phenyl, and phenyl having mono-, or di-substituted NH₂, OH, OCH₃, CF₃, COOH, and COOCH₃;

R², R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H, F, CI, Br, CH₃, CF₃, SCH₃, OCH₃, NH₂, OH, COOH, NO₂, COOCH₃, and CONH₂ provided at least two of R², R\ R⁴, R⁵, and R⁶ are H;

Z, is selected from the group consisting of CH₂, NH, O, and S;

Z₂ and Z₃ are independently CH or N provided Z₂ and Z₃ are CH when Z_x is S; only one ofZ₂ and Z₃ is N when Z, is O;

R⁷ is selected from the group consisting of H, F, CI, Br, CF₃, SCH₃, OCH₃, C_rC₃ alkyl, C,-C₃ alkoxy, NH₂, OH, COOH, NO2, COOCH₃, phenoxy, pyridyl, phenyl, halogen-substituted phenyl, phenylamino, or hydroxyphenyl;

X is selected from the group consisting of NH, O, CO, NHCO, NHCONH, NHNHCH₂, NHNHCONH, NHCS, NHCSNH, NHNHCSNH, CH₂CO, CH₂COCH₂, CH₂CH₂CO, OCH₂, and OCH₂CH₂;

or pharmaceutically acceptable salts thereof.

Preferably, the active compound has the above formula wherein Z, is selected from the group consisting of CH₂, NH, and O; and R⁷ is selected from the group consisting of H, C,-C₃ alkyl, and C_rC₃ alkoxy. Another preferred embodiment of the active compound has the formula X is NH and R¹ is selected from the group consisting of NHCOA, NHCONHA, NHCSNHA, COA, and CONHA, where A is selected from the group consisting of phenyl, mono-, di-, or tri-halogen substituted phenyl, and phenyl having mono-, or di-substituted NH₂, OH, OCH₃, CF₃, COOH, and COOCH₃

Another preferred embodiment of the active compound has the formula

wherein

R⁸ is selected from the group consisting of dichlorophenyl and trifluoromethyl-phenyl; and

R⁹ is selected from the group consisting of trifluoromethyl-phenyl, mono- or di-halogenated phenyl, and halogenated methylphenyl; B, is S or O; or pharmaceutically acceptable salts thereof.

A method of inhibiting HIV- 1 viral entry into a human T-cell is also provided comprising administering to a subject an effective amount of an active compound having a molecular weight of between 200 daltons and 650 daltons, which compound, at a concentration of not more than 1 OOμM inhibits greater than 50% of the binding of gpl20 to CD4. Preferably, the compound inhibits less than 10% of the binding of human CD4-expressing, CD4-transfected COS cells to Raji cells. The preferred active compounds are as described above in connection with the method for inhibiting HIV infection. Brief Description of the Drawings

Figure 1 is a graph showing the inhibition of Leu3a binding to sCD4 a function of HBO 1 concentration.

Figures 2A-2F are graphs showing the results of a Leu3a-FITC FACS analysis of human peripheral blood lymphocytes.

Figure 3 is a graph showing the inhibition of CD4-gpl20 binding by HB01 as a function of HBO 1 concentration.

Figure 4 is a graph showing the inhibition of CD4-MHC class II interaction by HB01 and HBOl-1 at lOOμM.

Figure 5 is a graph showing the inhibition of gp 120-CD4-mediated cell fusion by selected compounds: CD4-, CD4 negative cells were fusion target cells; Control, no inhibitor; Leu3a, TJU103 , TJU104 and # 19: inhibitor was Leu3a antibody, compound TJU103, compound TJU104, or α-(l-benzyl-4-hydroxy-4- piperidyl)-γ-butyrolactone (control) .

Detailed Description of the Invention

Methods of inhibiting FUN- 1 infection are provided by administering an effective amount of a small molecule inhibitor of HIN-1 binding to CD4. According to one preferred embodiment, the compounds used in the practice of the invention are selective for inhibiting CD4-gp 120 binding. That is, they block CD4 interaction with HIV gp 120, but not the CD4-MHC class II interaction. The latter interaction is important for normal immune response.

As shown below, representative compounds useful in the practice of the invention have been demonstrated experimentally to bind to a pocket on CD4 which lies below CD4 amino acid Phe 43. The pocket is important for gpl 20 recognition. The compounds useful in the practice of the invention significantly block CD4-gpl20 interaction in a concentration-dependent manner, inhibiting HIV- 1 viral entry in a cell-cell fusion assay. Most significantly, preferred compounds do not inhibit CD4-MHC class II interaction at concentrations which inhibit CD4- gpl20 binding.

A. Methods of Identifying CD4-gpf 20 Inhibitors

1. Computer-assisted Structure-based Screening

A computer-assisted structure-based screening process was undertaken to identify small organic molecules that bind to CD4 at the location where gpl 20 binds to CD4. The crystal structure of the CD4-gp 120 complex reveals a deep pocket on gpl 20, that when optimally positioned just above the CDR2 loop of the CD4 protein, docks with the aromatic side chain of Phe 43 at the CD4 CDR2 loop.

Specifically, a synthetic peptide mapping approach was employed to identify potential surface binding epitopes of the CD4 protein. As described in detail in Satoh, T., et al, 1996, Biochem. Biophys. Res. Com. 224:438-443, which is hereby incorporated by reference, this strategy consisted of two steps. In the first step, theoretical analyses using surface binding site searching algorithms of APROPOS, Peters, K.P., et al, 1996, J. Mol Biol 256:201, and DOCK, Meng, E.C., et al, 1992, J. Comput. Chem. 13:505, and solvent-accessible surface area calculations, Lee, B. & Richards, F.M., 1971, J. Mol. Biol. 55:379, were carried out for the CD4 Dl domain to identify surface structural features that might mediate protein-protein interactions. Other computational methods such as Delphi, Gilson, M.K., et al, 1988, J. Comput. Chem. 9:327, were also used to analyze the electrostatic properties of CD4 surface structures. Informed by these calculations, an area was selected bounded by the three loops termed: FG, residues 86-89; CC, residues 29-35; and C'C", residues 40-43. In our model, the amino acids that form the binding site and are available to interact with an inhibitory ligand include: Gin²⁵, His²⁷, Glu⁸⁷, Asp⁸⁸, Gin⁸⁹, and Lys⁹⁰. DOCK3.5 is an automatic algorithm to screen small-molecule databases for ligands that can bind a given receptor. Meng et al., J. Comp. Chem. 15:505 (1992)(incorporated herein by reference). DOCK3.5 was used to screen a database of small molecules for complementqrity between ligand and receptor in shape and electrostatic interactions. The small molecule database employed was The Available Chemicals Directory (Molecular Design Limited, San Leandro, C A), incorporated herein by reference. This database currently contains about 200,000 commercially available compounds. The structures of the molecules were generated using a heuristic algorithm, CONCORD, developed by R. Pearlman at the University of Texas.

DOCK3.5 characterizes the surface of the active site to be filled with sets of overlapping spheres. The generated sphere centers constitute an irregular grid that is matched to the atomic centers of the potential ligands. The quality of the fit of the ligand to the site is judged by either the shape complementarity or by a simplified estimated electrostatic interaction energy that takes into account van der Waals forces or coulombic interactions.

The 1000 molecules with the best shape complementarity scores and the 1000 molecules with the best force field scores were selected from the DOCK3.5 screening. The compounds used in the method of the invention preferably have a DOCK 3.5 complementarity score of at least 150 for the GFCC'C" pocket of the CD4 molecule. This surface pocket in the Dl domain of CD4 is formed by the GFCC'C" sheet and the FG, CC\ and C'C" loops. The GFCC C" pocket is defined by residues 26-46 and 80-97 of human CD4, according to the atomic coordinates as published by Wang et al. , 1990, Nature 348, 411-418 (incorporated herein by reference).

The resulting 2000 compounds were then visually screened thrice independently in the context of the CD4 Dl domain surface binding pocket using the molecular display software Insight II (Biosym, Inc., San Diego, CA). A set of diverse compounds that possessed distinctive chemical structures, receptor binding modes, and electrostatic and shape complementarity were selected for further testing. The control parameters for DOCK3.5 that were used are given in Table 2 of WO 98/25469 (1998); the entire disclosure of WO 98/25469 is incorporated herein by reference.

2. Biological Assays The capacity of a compound to bind to the CD4 pocket can be determined by competition binding experiments using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA). The candidate inhibitor is tested in this manner for ability to compete with CD4 pocket-specific antibodies for binding to the pocket. The candidate inhibitor is tested in an ELISA at concentrations ranging from lμM to 100 μM for its ability to compete with the CD4 binding of mAb Leu3a antibody, which is known to specifically recognize the CD4 pocket (Sutlor et al, J. Immunol. 149:1452-1461; 1992). A useful small molecule inhibitor binds to CD4 and blocks Leu3a from binding to the CD4. Satisfactory results are obtained when the inhibitor molecule inhibits 50% of the target binding sites at a concentration (IC₅₀) of less than 1 OOμM.

The binding of candidate small molecule inhibitors to CD4 may also be confirmed using fluorescence-activated cell sorter (FACS) analysis. Fluorescein-conjugated Leu3a antibody (Leu3a-FITC) is used to label CD4⁺ cells present in a population of human peripheral blood lymphocytes. Dilutions of the candidate small molecule inhibitor are added to the cells. The extent to which the candidate inhibitor blocks Leu3a-FITC binding to the CD4⁺ cells is determined. Satisfactory results are obtained when the sample containing the inhibitor molecule produces a result substantially similar to that of the positive control, at the same concentration. The inhibitory effect and specificity of a candidate small organic molecule for inhibition of CD4 binding to gpl 20 can also be determined by a competition assay using recombinant gpl 20 ELISA. For example, recombinant HIV- 1 gp 120-L AV,πfβ competes with the prospective organic inhibitor, at various dilutions, to bind with recombinant soluble CD4. Bound gpl20 may be measured with a primary antibody that detects a region of gpl 20 not involved in CD4 binding. A rabbit anti-mouse IgG horseradish peroxidase conjugate may be used as a secondary antibody. The extent to which the organic inhibitor blocks gpl 20 binding to immobilized CD4 can be determined by absorbance measurement. Satisfactory results are obtained when the inhibitor molecule exhibits an IC₅₀ less than lOOμM.

A cell adhesion assay may be conducted to determine the effect of the prospective organic inhibitors on CD4-MHC class II binding. The capacity of a compound to inhibit the interaction between CD4 and class II MHC gene products can be determined by a cell rosetting assay. This cell-cell adhesion assay has also been used by other laboratories and shown to accurately reflect specific functional CD4-MHC class II binding. Doyle et al, Nature, 330:256-259 (1987); Fleury et al, Cell, 66:1037-1049 (1991). A cell line, such as Cos-7, Cos-1 or the like, can be transiently transfected with a plasmid bearing a human CD4 cDNA operably linked to a promoter. In an alternative embodiment, a COS-1 cell line may be stably transformed with a human CD4 expression plasmid. The human CD4 expressing cells and a human class II MHC expressing cell are mixed so that cellular rosettes are formed.

Specific blockage of CD4-MHC class II binding by a compound is evidenced by a significant reduction in the number of rosettes when the compound is present. According to the present invention, a useful inhibitor of CD4-gpl20 binding should cause no more than about 10% reduction in the number of rosettes when the inhibitor is present in the rosetting medium at a concentration of at most 100 μM.

The invention encompasses the use of any compound that is susceptible to computational screening by programs such as DOCK3.5, and that inhibits the binding of HIV-1 gpl20 to CD4. Preferably, the compound does not appreciably inhibit CD4-MHC class II interactions. The molecular weight of such compounds is between about 250 daltons and about 650 daltons, and preferably between about 430 daltons and about 550 daltons. C. Therapeutic Administration

The effective amount of compound needed to treat a subject may be routinely determined through procedures well known to those skilled in the art which address such parameters as biological half-life, bioavailability, andtoxicity. Such determination is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

The present invention provides pharmaceutical compositions that comprise the compounds of the invention and pharmaceutically acceptable carriers or diluents. For parenteral administration, the compounds of the invention can be, for example, formulated as a solution, suspension, or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer' s solution, dextrose solution, and 5% human serum albumin. The vehicle or lyophilized powder may contain additives that maintain isotonicity {e.g. , sodium chloride, mannitol) and chemical stability {e.g. , buffers and preservatives). For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution. The formulation can be sterilized by any commonly used technique. The pharmaceutical compositions according to the invention may be administered as a single dose or in multiple doses. The pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with each other or with other therapeutic agents. The treatments of the present invention may be combined with conventional therapies, which may be administered sequentially or simultaneously.

The pharmaceutical compositions of the present invention may be administered by any means that enables the active agent to reach the targeted cells.

Because compounds of the invention may be subject to being digested when administered orally, parenteral administration, i.e., intravenous, subcutaneous or intramuscular, would ordinarily be used to optimize absorption. Intravenous administration may be accomplished with the aid of an infusion pump. Alternatively, the compounds of the invention can be formulated as aerosol medicaments for intranasal inhalation or topical administration.

The dosage administered varies depending upon factors such as: pharmacodynamic characteristics; its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment; and frequency of treatment. Usually, the dosage of the compound of the invention can be about 1 to 3000 milligrams per 50 kilograms of body weight; preferably 10 to 1000 milligrams per 50 kilograms of body weight; more preferably 25 to 80 milligrams per 50 kilograms of body weight. Ordinarily, 8 to 800 milligrams are administered to an individual per day in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.

D. Compounds of the Invention

The compounds for use in the method of the invention have a molecular weight of between about 200 daltons and about 650 daltons, preferably between about 350 daltons and about 550 daltons. At concentrations of not more than 100 μM, the compounds for use in the method of the invention inhibit greater than 50% of gpl 20 binding to CD4 as measured by an ELISA assay, and preferably inhibit less than 10% of the binding of human CD4-expressing, CD4-transfected COS cells to Raji cells. A preferred compound utilized in the method of the invention is N- l-(4-bromo-3-methylρhenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4-tetraazol-2- yl]acetyl}hydrazine-l-carbothioamide (referred to herein as "HB01", and having a molecular weight of 515.222 daltons). The following is a synthesis scheme for HBO 1. The scheme may be extended to analogs of HBO 1 :

The preferred analogs of HBO 1 include the following compounds identified by structure (Table 1), systematic chemical name, internal designation (HB01-_) and molecular weight(daltons):

N-l-(3,4-dichlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide {HBOl-1; 491.189);

N-l-(3,4-dichlorophenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydr.azine-l-carbothioamide {HB01-2; 491.189);

N-l-(4-bromo-3-chlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H- l,2,3,4-tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide (HB07-i; 535.64);

N-l-(2-fluorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide {HB01-4; 440.289);

N-l -(4-chlorophenyl)-2- { 2- [5 -(4-trifluoromethylphenyl)-2H- l,2,3,4-tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide (H507-5, - 440.836); N-l-(4-trifluoromethylphenyl)-2-{2-[5-(5-trifluoromethylphenyl)- 2H-l,2,3,4-tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide(H507-d 459.^0J ; and

N-l -(4-trifluoromethylphenyl)-2- {2-[5-(2,3-dichlorophenyl)-2H- l,2,3,4-tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide {HBO 1-7; 480.298).

HB01 may be commercially obtained. A source of HB01 is Maybridge Chemical Company Ltd. (MDL Cat. No. MFCDOOl 04396). The analogs of HBO 1 may be similarly obtained from Maybridge Chemical Company: HB01-1 (MDL Cat. No. MFCDOOl 04400); HBOl-2 (MFCD00104397); HBOl-3 (MFCDOOl 04402); HBOl-4 (MFCD00104399); HB01-5 (MFCD00207592); HB01-6 (MFCDOOl 05250); and HB01-7 (MFCD00104398).

Table 1

HB01 may be synthesized, as shown above. 5-(2,3-dichlorophenyl)- tetraazole can be prepared by treating 2,3-dichlorobenzonitrile with trimethylsilyl azide in the presence of di-n-butylin oxide. Alkylation of the aryl substituted tetraazole with 2-bromo acetyl acetate gives ethyl [5-(2,3-dichlorophenyl)-2H- l,2,3,4-tetraazol-2-yl] acetate as a major product, which can be coupled with an aryl substituted thiosemicarbazide to give the final product. With such coupling, the ester group can be transformed to its acid form, and then reacted with a thiosemicarbazide.

Analogs of HBO 1 useful in the practice of the invention may be synthesized by modifying the 4-bromo-5-methylphenyl carbothioamide moiety of HB01. For example, N-l-(benzyl)-2-{2-[5(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl} hydrazine ("S-8") may be prepared by the reaction of 2- substituted acetic hydrazide with a benzyl chloride in the presence of the base, trimethylamineprepared as follows:

S-8

N- 1 -(benzyl)-2- {2-[5-(2,3-dichlorophenyl)-2H- 1 ,2,3,4-tetraazol-2- yl] acetyl} hydrazine-carboamide ("S-9") may be prepared by treating phenyl urea with hydrazine to form phenylsemicarbazide, which in turn can be reacted with an acid to give the compound S-9:

S-9

The practice of the invention is illustrated by the following non- limiting examples.

Example 1 HBOl Binding to Immobilized CD4 Competition binding experiments using an ELISA were conducted to confirm the binding of HBOl to the CD4 Dl domain surface binding pocket. Purified recombinant soluble CD4 (supplied by SmithKline Beecham Pharmaceuticals, PA) was coated on the surface of plastic Nunc Polysorp 96-well microtiter plates. HBOl in 0.25% DMSO and PBS at concentrations ranging from lμM to lOOμM was added to the coated plates and incubated at room temperature for 30 minutes. mAb Leu3a antibody (Becton Dickinson, San Jose, CA) was then added to the wells and incubated for 30 minutes at room temperature. Leu3a is a known CD4 binder. A secondary rabbit anti-mouse horseradish peroxidase conjugate (Sigma, St. Louis, MO) was added to the wells and incubated for one hour at room temperature. To develop a signal in the wells, 100 μL/well of ortho- phenylenediamine (OPD) substrate was added. The OPD substrate was prepared by dissolving a urea H₂O₂ tablet in 20 ml distilled water, then dissolving the OPD tablet in the urea H₂O₂ buffer. Color development ceased upon the addition of 25 μL/well 3M H₂SO₄. The absorbance (Abs) of each well at 490 nm was measured using a Bio-Rad Model 3550 Microplate reader (Bio-Rad Laboratories, Hercules, CA). Percent inhibition was calculated by using the following formula:

Percent Inhibition = [(Abs of Leu3a alone - Abs of organic inhibitor)/ Abs of Leu3a alone] x 100

The results of an assay are shown in Figure 1. HBO 1 strongly bound CD4 and blocked the binding of Leu3 a to CD4. A control compound, α-( 1 -benzyl-

4-hydroxy-4-piperidyl)-γ-butyrolactone, did not exhibit any CD4 binding activity.

Example 2 HBOl Binding to CD4 on Human Lymphocytes The binding of HBOl to CD4 on the cell surface was further confirmed via independent experiments using fluorescence-activated cell sorter (FACS) analysis. Human peripheral blood lymphocytes were isolated from a blood sample for use in a FACS assay using Lymphoprep™ solution (Gibco BRL, Gaithersburg, MD). Dilutions of HBOl were prepared from 50 mM DMSO stock solutions into phosphate buffered solution (PBS), then added to the cells for one hour. The assay was conducted at 4 °C. Each sample contained about 2 x 10⁵ cells. Fluorescein-conjugated Leu3a antibody (Becton-Dickinson, San Jose, CA) was added to label CD4⁺ cells present in the isolated population for one hour. Each sample was washed three times with PBS and fixed with cold 2% formaldehyde/PBS. Each sample was then analyzed by flow cytometry. The results of this experiment are presented in Figures 2A-F. In Fig.

2A, the cells were incubated with an anti-mouse IgG FITC antibody as a negative control. Fig. 2B shows the Leu3a-FITC antibody binding to CD4⁺ cells. Soluble CD4 added to the mixture prevented the Leu3a-FITC antibody from attaching to CD4⁺ cells leading to the decrease in the signal observed in Fig. 2C. HBOl tested at a concentration of lOOμM (Fig. 2D) also blocked the Leu3a-FITC binding to CD4⁺ cells. Figs. 2E and 2F show that two negative control organic molecules (α- ( 1 -benzyl-4-hydroxy-4-piperidyl)-γ-butyrolactone and "18A11", 2-[5 -(phenyl)- l,2,3,4-tetraazol-2-yl]-ethanohydrazide)did not interfere with the Leu3a-FITC and CD4⁺ cell interaction. Thus, HBOl binds to CD4.

Example 3 Inhibitory Effect and Specificity of HBOl on CD4-gpl20 Binding

The inhibitory effect and specificity of HBO 1 on CD4-gp 120 binding was demonstrated by ELISA. In this assay, 50 μL of 5 μg/ml recombinant soluble CD4 (sCD4) in 0.1 M sodium bicarbonate (pH=9.5) was immobilized onto Nunc Polysorp 96 well plates overnight at 4°C. A 2% BS A/PBS solution was used to block the wells and the plate was then washed with PBS. Dilutions of HBOl (ranging from lμM to lOOμM) were prepared from 10 mM stock solution into PBS, then added to the plate.

50μL of purified recombinant HIV-1 gpl20-LAV, IIIB (Protein Sciences Corporation, Meridian, CT) was used at 500 ng/ml to compete with the candidate inhibitor for binding to immobilized sCD4. The primary antibody for detecting the CD4-bound gpl 20 (DuPont NEN, Wilmington, DE) detects a sequence specific region of gpl 20 not involved in CD4 binding, permitting measurement of bound gpl 20. A rabbit anti-mouse Igg horseradish peroxidase conjugate (Sigma, St. Louis, MO) was used as the secondary antibody. In between each step, the plate was washed with PBS/0.05% Tween 20. Peroxidase substrate was added to each well and color development was then stopped by addition of IN H₂SO₄. Absorbance was measured at 490 nm on a Bio-Rad Model 3550 Microplate reader. Percent inhibition of CD4-gpl20 binding was calculated according to the formula:

Percent Inhibition = [(Absorbance of gpl 20 alone - Absorbance of HBOl)/Absorbance of gpl20 alone] x 100. α-(l-Benzyl-4-hydroxy-4-piperidyl)-γ-butyrolactone was used as a negative control compound (bar marked "control" in Fig. 3). The positive control was the absence of HBOl (bar marked "sCD4" in Fig. 3).

The results of this assay are shown in Figure 3. HBOl blocked gpl 20 binding to CD4 in a concentration dependent manner. The negative control compound did not show any effect. At an IC₅₀ of less than 10 μM, HBOl selectively blocked CD4-gpl20 interaction.

Examples 4-10 The inhibitory effect of compounds HBOl-1 through HBO 1-7 on CD4-gpl20 binding at 100 μM was demonstrated by the ELISA described in Example 2. The results are shown in Table 2.

Table 2. Inhibitory effect of HBOl analogs on CD4-gpl20 binding 000 uM).

Example 11 Effect of Compounds on gpl20-CD4-Mediated Cell Fusion Assay

Following a modified procedure published by others, a gene reporter fusion assay was used to determine the inhibitory effect of small organic molecules on binding of CD4 and gpl 20. HIV-1 Env proteins and T7 RNA polymerase were introduced into effector 293 cells by infection with recombinant vaccinia virus at a multiplicity of infection of 10 for 2 hours. Infected cells were then trypsinized, washed with PBS, resuspended in medium and incubated overnight at 32°C in the presence of rifampicin (100 mg/ml). QT6 target cells were co-transfected in 6-well plates with plasmids encoding CD4 and luciferase under control of T7 promoter, using the calcium phosphate precipitation method. CD4^" QT6 cells were also prepared, for use as controls. Four to six hours after transfection cells were lifted, washed with PBS, seeded in 24-well plates and incubated at 37 °C overnight. Organic inhibitor solutions were prepared from 10 mM stock solution into fusion medium. The medium on the target cells was aspirated and then organic inhibitor solutions or control substances were added (500 μM TJU103, TJU104 and compound #19; 2 μg/ml, Leu3a). To initiate fusion, 105 effector cells were added to each well and incubated at 37 °C in the presence of ara-C and rifampicin. After 5 hours of fusion, cells were lysed in 150 ml of reporter lysis buffer (Promega) and assayed for luciferase activity by using commercially available reagents (Promega).

The results are shown in Fig. 5 : Control: no inhibitor; CD4-: CD4 negative cells were the target cells; Leu3a: inhibitor was Leu3a, an antibody against CD4; TJU103: inhibitor was compound TJU103; TJU104: inhibitor was compound TJU104; #19: inhibitor was α-(l-benzyl-4-hydroxy-4-piperidyl)-γ-butyrolactone, a randomly-selected compound used as a control. Compounds TJU103 and TJU104 effectively blocked gpl20-CD4-mediated cell fusion.

Example 12 Effect of Compounds on CD4-MHC Class II Binding A cell adhesion assay was conducted to determine the effect of compounds of the invention on CD4-MHC class II binding.

The sequence of human CD4 cDNA is given in Maddon, P.J. et al, 1985, Cell 42:93-104, which sequence is hereby incorporated by reference in its entirety. Maddon et al. refers to human CD4 as "T4." The plasmid T4-pMV7, containing the human CD4 cDNA is available from the AIDS Research and Reference Reagent

Program, Division of AIDS, NIAID, NIH (McKesson BioServices, Rockville, MD).

The 3.0 Kb Eco Rl fragment of T4-pMV7 contains the 1.5 Kb CD4 cDNA.

For transfection of COS-7 cells, a CD4-expression plasmid T4-pcDNA3, was constructed by subcloning the 3.0 Kb Eco Rl fragment of T4-pMV7 into the

EcoRI site of the mammalian expression vector pcDNA3 (INVITROGEN).

Transfection was accomplished by DOSPER liposomal transfection reagent

(BOEHRINGER MANNHEIM), by the following modification of the manufacturer's protocol. 1. In a six-well or 35mm tissue culture plate, ~5xl 0⁴ cells were seeded per well in 2 ml DEME containing 10% FCS (fetal calf sera, GIBCO) and nonessential amino acids.

2. The cells were incubated at 37 °C, 5% CO₂ in a cell culture incubate until the cells were 70-80% confluent. This usually takes 18-24 h. 3. A DOSPER/DNA mixture was prepared:

- Solution A: 2 μg T4 -pcDNA3 recombinant plasmid DNA diluted with 20mM HBS (Hepes-buffered saline, GIBCO) to a final volume of 50μl.

- Solution B: 6 μl DOSPER diluted with 20 mM HBS to a final volume of 50μl. Solutions A and B were combined, gently mixed, and incubated at room temperature for 15~30min to allow the DOSPER/DNA complex to form.

4. On the day of transfection, the culture medium was replaced shortly before adding the DOSPER/DNA mixture with 1ml serum-free DMEM.

5. Without removing the culture medium previously added, 100 μl of the DOSPER DNA complex was added dropwise to the cultures. It was essential to add the DOSPER/DNA complex dropwise. The culture plate was gently rocked to ensure uniform distribution.

6. The culture plate was incubated for 6 h at 37°C, 5% CO₂ in a cell culture incubator. 7. Following incubation, 1 ml DMEM with 20% FCS was added without removing the transfection mixture. 8. The medium containing DOSPER/DNA mixture was replaced 24 h after transfection with 2ml fresh DEME with 10% FCS.

9. The CD4 expression levels were determined as a measure of transfection efficiency by flow cytometry analysis. Usually between 30% and 40% of transfected COS-7 cells expressed human CD4 as defined by immunofluorescence binding assay for the interaction between CD4 and class II MHC proteins in the presence of organic chemicals by rosette formation: Raji B cells 10⁷ in 1 ml of RPMI medium with 10% FCS and 200 mM glutamine were added to each well 48 h post-transfection and incubated with transfected COS-7 cells in the presence of the test compound (individually 200 μM) at 37° C for 1 h. Following incubation, wells were washed five or six times by dropping RPMI medium with FCS into wells. Rosette formation between Raji cells and transfected COS-7 cells was scored microscopically at 100-fold magnification. The number of rosette containing more than five Raji cells was counted in 10 random optical fields in each individual well; 300-400 rosettes per well were counted as the positive control for rosette formation without any inhibition in the absence of any chemicals. The inhibition activity for rosette formation for each chemical was determined by the ratio of the number of rosettes in the positive control. COS-7 cells transfected with pcDN A3 vector alone served as negative controls for rosette formation. No rosettes should be observed in the negative control wells.

TJU103 (N-(3-indoylmethylene)-isonicotinic hydrazone) was used as a positive control. TJU103 inhibits CD4-MHC class II interaction. α-(l-Benzyl-4- hydroxy-4-piperidyl)-γ-butyrolactone was employed as a negative control.

The results of the assay are shown in Figure 5. HBO 1 and its analog HBO 1 - 1 (N-l-(4-(3-chloro-4-bromophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2yl] acetyl} hydrazine- 1 -carbothioamide), both at concentrations of lOOμM, did not inhibit the CD4-MHC class II interaction as demonstrated by this cell adhesion assay. Both compounds significantly inhibited CD4-gpl20 binding at the same concentration (lOOμM). Both compounds also failed to exhibit any inhibitory activity with respect to CD4-MHC class II interaction at a concentration of 200μM (data not shown). All references cited herein are incorporated herein by reference.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention.

Claims

We claim:

1. A method of inhibiting HIV- 1 infection comprising administering to a subject an effective amount of an active compound having a molecular weight of between 200 daltons and 650 daltons, which compound, at a concentration of not more than lOOμM inhibits greater than 50%) of the binding of gpl 20 to CD4.

2. The method of claim 1, wherein the DOCK3.5 complementarity score for the active compound and the GFCC'C" pocket of the CD4 molecule is at least 150.

3. The method of claim 1 wherein the active compound, at a concentration of not more than lOOμM, inhibits less than 10% of the binding of human CD4-expressing, CD4-transfected COS cells to Raji cells.

The method of claim 3, wherein the active compound has the formula:

wherein:

R is

Y is C or N;

R is selected from the group consisting of C_rC₅ alkyl, phenyl, pyridyl, cyclohexyl substituted with one, two, or three halogen atoms, phenoxy substituted with one or two halogen atoms, phenyl, pyridyl, cyclohexyl, cyclopentyl, cyclopentyl substituted with one or two NH₂ OH, OCH₃, CF₃, or COOH, a five- member or six-member heterocyclic ring substituted with halogen, NH₂ OCH₃, CF₃ or COOH, and NHCOA, NHCONHA, NHCSNHA, COA, and CONHA, where A is selected from the group consisting of phenyl, mono-, di-, or tri-halogen substituted phenyl, and phenyl having mono-, or di-substituted NH₂, OH, OCH₃, CF₃, COOH, and COOCH₃;

Z, is selected from the group consisting of CH₂, NH, O, and S;

Z₂ and Z₃ are independently CH or N provided

Z₂ and Z₃ are CH when Z_λ is S; only one of Z₂ and Z₃ is N when Z_λ is O;

R⁷ is selected from the group consisting of H, F, CI, Br, CF₃, SCH₃, OCH₃, C,-C₃ alkyl, C_rC₃ alkoxy, NH₂, OH, COOH, NO2, COOCH₃, phenoxy, pyridyl, phenyl, halogen-substituted phenyl, phenylamino, or hydroxyphenyl;

or pharmaceutically acceptable salts thereof.

5. The method of claim 4, wherein Z_λ is selected from the group consisting of CH₂, NH, and O; and R⁷ is selected from the group consisting of H, -C_j alkyl, and C_rC₃ alkoxy.

6. The method of claim 4, wherein X is NH and R¹ is selected from the group consisting of NHCOA, NHCONHA, NHCSNHA, COA, and CONHA, where A is selected from the group consisting of phenyl, mono-, di-, or tri-halogen substituted phenyl, and phenyl having mono-, or di-substituted NH₂, OH, OCH₃, CF₃, COOH, and COOCH₃

7. The method of claim 4, wherein the compound has the formula

wherein

R⁸ is selected from the group consisting of dichlorophenyl and trifluoromethyl-phenyl;

R⁹ is selected from the group consisting of trifluoromethyl-phenyl, mono- or di-halogenated phenyl, and halogenated methylphenyl; and B^s S or O; or pharmaceutically acceptable salts thereof.

8. The method of claim 7, wherein the compound is selected from the group consisting of

N-l-(4-bromo-3-methylphenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl } hydrazine- 1 -carbothioamide ;

N-l-(3,4-dichlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl} hydrazine- 1 -carbothioamide; N-l-(3,4-dichlorophenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide;

N-l-(4-bromo-3-chlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl} hydrazine- 1 -carbothioamide; N-l-(2-fluorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4-tetraazol-2- yl] acetyl } hydrazine- 1 -carbothioamide ;

N-l-(4-chlorophenyl)-2-{2-[5-(4-trifluoromethylphenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide;

N-l-(4-trifluoromethylphenyl)-2-{2-[5-(5-trifluoromethylphenyl)-2H- l,2,3,4-tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide; and

N-l-(4-trifluoromethylphenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl} hydrazine- 1 -carbothioamide; and pharmaceutically acceptable salts thereof.

9. The method of claim 8, wherein the compound is N-l-(4-bromo-3-methylphenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide;

N- 1 -(3,4-dichlorophenyl)-2- { 2- [5-(2,3 -dichlorophenyl)-2H- 1 ,2,3,4- tetraazol-2-yl] acetyl} hydrazine- 1 -carbothioamide;

N-l-(2-fluorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4-tetraazol-2- yl] acetyl} hydrazine- 1 -carbothioamide; and

N-l-(4-bromo-3-chlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl } hydrazine- 1 -carbothioamide.

10. The method of claim 2, wherein the active compound is selected from the group consisting of 5-(4-chlorobenzylthio)-3-{[(4-chlorophenyl)-2- thiazolyl]methylthiomethyl} -4-methyl- 1 ,2,4-triazole; 4-(4-methoxy-phenyl)- 1 - [imidazol(2,l-B)benzothiazole; N-(3-indomethylene)isonicotinic hydrazone; and N-(2,4-dichlorophenyl)-3-(l,2,4-triazol-l-ylmethyl)-l,2,4-triazole-5-carboxamide.

11. A method of inhibiting HIV-1 viral entry into a human T-cell comprising administering to a subject an effective amount of an active compound having a molecular weight of between 200 daltons and 650 daltons, which compound, at a concentration of not more than lOOμM, inhibits greater than 50%) of the binding of gp 120 to CD4.

12. The method of claim 11, wherein the DOCK3.5 complementarity score for the active compound and the GFCC'C" pocket of the CD4 molecule is at least 150.

13. The method of claim 11 wherein the active compound, at a concentration of not more than lOOμM, inhibits less than 10%) of the binding of human CD4-expressing, CD4-transfected COS cells to Raji cells.

14. The method of claim 13, wherein the active compound has the formula:

wherein R is

Y is C orN;

R, is selected from the group consisting of C_rC₅ alkyl, phenyl, pyridyl, cyclohexyl substituted with one, two, or three halogen atoms, phenoxy substituted with one or two halogen atoms, phenyl, pyridyl, cyclohexyl, cyclopentyl, cyclopentyl substituted with one or two NH₂ OH, OCH₃, CF₃, or COOH, a five- member or six-member heterocyclic ring substituted with halogen, NH₂ OCH₃, CF₃ or COOH, and NHCOA, NHCONHA, NHCSNHA, COA, and CONHA, where A is selected from the group consisting of phenyl, mono-, di-, or tri-halogen substituted phenyl, and phenyl having mono-, or di-substituted NH₂, OH, OCH₃, CF₃, COOH, and COOCH₃.

R², R³, R⁴, R⁵, and R⁶ are independently selected from the group consisting of H, F, CI, Br, CH₃, CF₃, SCH₃, OCH₃, NH₂, OH, COOH, NO₂, COOCH₃, and CONH₂ provided at least two of R², R³, R⁴, R⁵, and R⁶ are H;

Z is selected from the group consisting of CH₂, NH, O, and S;

Z₂ and Z₃ are independently CH or N provided

Z₂ and Z₃ are CH when Z, is S; only one of Z₂ and Z₃ is N when Z_λ is O;

or pharmaceutically acceptable salts thereof.

15. The method of claim 14, wherein Z_λ is selected from the group consisting of CH₂, NH, and O; and R⁷ is selected from the group consisting of H, C_rC₃ alkyl, and C,-C₃ alkoxy.

16. The method of claim 14, wherein X is NH and R¹ is selected from the group consisting of NHCOA, NHCONHA, NHCSNHA, COA, and CONHA, where A is selected from the group consisting of phenyl, mono-, di-, or tri-halogen substituted phenyl, and phenyl having mono-, or di-substituted NH₂, OH, OCH₃, CF₃, COOH, and COOCH₃

17. The method of claim 14, wherein the compound comprises the formula

O

\ H H

_-N — ^___

N -~ _— -—"^N

/ H I

wherein

R⁹ is selected from the group consisting of trifluoromethyl-phenyl, mono- or di-halogenated phenyl, and halogenated methylphenyl; and

B, is S or O; or pharmaceutically acceptable salts thereof.

18. The method of claim 14, wherein the compound is selected from the group consisting of

N-l-(4-bromo-3-methylphenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide; N-l-(3,4-dichlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothio.amide;

N-l-(3,4-dichlorophenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine- 1 -carbothioamide; N-l-(4-bromo-3-chlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl} hydrazine- 1 -carbothioamide;

N-l-(2-fluorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4-tetraazol-2- yl] acetyl } hydrazine- 1 -carbothioamide;

N-l-(4-chlorophenyl)-2-{2-[5-(4-trifluoromethylphenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl} hydrazine- 1 -carbothioamide;

N- 1 -(4-trifluoromethylphenyl)-2- {2-[5-(5-trifluoromethylphenyl)-2H- 1 ,2,3 ,4-tetraazol-2-yl]acetyl } hydrazine- 1 -carbothioamide;

N-l-(4-trifluoromethylphenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl } hydrazine- 1 -carbothioamide ; and pharmaceutically acceptable salts thereof.

19. Themethodof claim 18, wherein the compound is N-l-(4-bromo-3 - methylphenyl)-2- {2-[5-(2,3-dichlorophenyl)-2H- l ,2,3,4-tetraazol-2- yl] acetyl} hydrazine- 1 -carbothioamide;

N-l-(3,4-dichlorophenyl)-2-{2-[5-(2,3-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl]acetyl}hydrazine-l-carbothioamide;

N-l-(2-fluorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4-tetraazol-2- yl]acetyl}hydrazine-l-carbothioamide; and

N-l-(4-bromo-3-chlorophenyl)-2-{2-[5-(3,5-dichlorophenyl)-2H-l,2,3,4- tetraazol-2-yl] acetyl} hydrazine- 1 -carbothioamide.

20. The method of claim 12, wherein the active compound is selected from the group consisting of 5-(4-chlorobenzylthio)-3-{[(4-chlorophenyl)-2- thiazolyljmethylthiomethyl } -4-methyl- 1 ,2,4-triazole; 4-(4-methoxy-phenyl)- 1 - [imidazol(2,l-B)benzothiazole; N-(3-indomethylene)isonicotinic hydrazone; and N-(2,4-dichlorophenyl)-3-(l,2,4-triazol-l-ylmethyl)-l,2,4-triazole-5-carboxamide.