WO1997003055A1

WO1997003055A1 - Compounds, compositions, and methods for inhibiting replication of retroviruses and for inhibiting tumor promoter initiated transcription

Info

Publication number: WO1997003055A1
Application number: PCT/US1996/011699
Authority: WO
Inventors: Hans J. Prochaska
Original assignee: Memorial Sloan Kettering Cancer Center
Current assignee: Memorial Sloan Kettering Cancer Center
Priority date: 1995-07-13
Filing date: 1996-07-12
Publication date: 1997-01-30
Anticipated expiration: 1998-01-13
Also published as: AU6676796A

Abstract

The subject invention provides a pharmaceutical composition for inhibiting replication of a retrovirus which comprises a compound having structure (I), wherein Z1 is either C-R5 or N; wherein Z2 is N; wherein X1 and X2 are the same or different and O, S, or N-R4; and wherein R1, R2, R3, R4, and R5 are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons. The invention further comprises a pharmaceutical composition for inhibiting replication of a retrovirus which comprises a first compound which inhibits replication of the retrovirus and a second compound having the above-defined structure. Also provided are methods of inhibiting retroviral replication in a subject using the aforementioned compositions. This invention also provides a pharmaceutical composition for inhibiting tumor-promoter initiated transcription which comprises a compound having the above-defined structure. Also provided is a method for preventing the formation of tumors in a subject which comprises administering the aforementioned composition for inhibiting tumor-promoter initiated transcription.

Description

COMPOUNDS. COMPOSITIONS, AND METHODS FOR INHIBITING REPLICATION OF RETROVIRUSES AND FOR INHIBITING TUMOR PROMOTER INITIATED TRANSCRIPTION

This application claims the benefit of U.S. Provisional Application No. 60/001,110, filed July 13, 1995, the contents of which are hereby incorporated by reference into the present application.

Background of the Invention The invention disclosed herein was made with Government support under National Cancer Institute Cancer Center Support Grant NCI-P30-CA08748. Accordingly, the U.S. Government has certain rights in this invention.

Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of this application. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Oltipraz (5-pyrazinyl-4-methyl-l, 2-dithiole-3-thione) , which was developed and tested in humans as an antischistosomal agent, is a highly effective inhibitor of chemical carcinogenesis (1-3) . The effectiveness of oltipraz in preventing neoplasia in a wide variety of experimental models has prompted a clinical evaluation of oltipraz as a potential human anticarcinogen (4-8) . Oltipraz is also an inhibitor of HIV-1 replication (9) . RT (reverse transcriptase) is a potential target, because oltipraz irreversibly inhibits RT in the template/primer binding domain (9) . However, because oltipraz also inhibits viral replication in a chronic infection model (10) , oltipraz appears to possess an additional antiviral mechanism. The potency of the drug in Ul cells is markedly potentiated by preincubation of the cells with oltipraz before PMA stimulation (10) . This invention involves the novel notion that metabolites of oltipraz may also play a role in the inhibition of viral replication.

The metabolic fate of oltipraz is complex (7, 8, 11, 12) . Less than 1% of the administered dose is excreted unaltered in the urine. The major metabolic pathway for oltipraz is likely to occur via a scenario involving opening of the 1, 2-dithiole- 3-thione ring by a nucleophile (perhaps glutathione) , followed by a two-electron reduction of the adduct, elimination of the nucleophile, and recyclization (13) . The resulting species is then methylated to form 7-methyl-6-8-bis (methylthio) pyrrolo [1,2-a] pyrazine. 7-Methyl-6, 8-bis (methylthio) pyrrolo [1,2- a] pyrazine, termed "metabolite II" by Bieder et al. (11) , is the common precursor for 10 other, closely related metabolites (which differ primarily in the oxidation state of the sulfur atoms and the pyrazine ring) . The amounts of rearranged metabolites isolated from human serum or urine were found to be much larger than the amount of oltipraz (11) . Until this invention, there were no known biological activities of metabolite III and related congeners.

There is evidence that oltipraz exerts its antiviral activity by more than one mechanism (10) . To address the question of whether metabolites of oltipraz possess antiviral activity, oltipraz and metabolite III were tested as described in the "Experimental Details" Section below in H9, Ul, ACH-2, and CEM cell lines, as well as in PBMCs. As noted above, the antiviral effects of oltipraz have been demonstrated in H9 (9) and PMA-stimulated Ul (10) cell lines, as models of acute and chronic HIV-1 replication, respectively. Because Ul cells are representative of a monocytoid lineage, ACH-2 cells were examined in the experiments described below as a model of chronic T cell infection. ACH-2 cells produce low levels of infectious virus under basal conditions but HIV-1 replication can be markedly induced with PMA (14) . CEM cells were used in the experiments described below not only because they represent another widely utilized model for acute viral replication in T cells but also because they are progenitors of A3.01 cells, from which the ACH-2 cell line was derived. Thus, it would be expected that differences in metabolism and uptake of oltipraz would be minimized between CEM and ACH-2 cells, rendering more valid a comparison of oltipraz and metabolite III in models of acute versus chronic viral replication. PBMCs were tested in the experiments described below because they are the closest model of in vivo conditions. This invention involves the discovery, established by the experiments described below, that metabolite III possesses antiviral activity at a step distal to viral integration and is synergistic with the parent compound (oltipraz) .

Oltipraz, which was developed clinically as an antischistosomal agent, has received intensive scrutiny since it is a highly effective inhibitor of experimental carcinogenesis. The anticarcinogenic activity of oltipraz was predicted by the ability of the drug to induce Phase II detoxication enzymes (i.e., GSH transferases) and enzymes concerned with the synthesis and maintenance of GSH pool (1, 25) . The induction of Phase II enzymes appears to be relevant to its ability to prevent cancer in a number of experimental models. In particular, oltipraz is a highly effective inhibitor of aflatoxin Bj-induced hepatocarcinogenesis in the F344 Fischer rat (2, 22), and these results have prompted a Phase II clinical trial in Qidong, China to ascertain if the drug can reduce urinary levels of DNA-aflatoxin adducts (26) . Although the chemopreventive activity of oltipraz can be attributed to the induction of Phase II enzymes under many circumstances, the mechanism for protection is less clear in others. In an incidental finding, Roebuck et al. (2) reported that 0.075% oltipraz in the diet at five to nine weeks of age significantly reduced the incidence of hematopoietic neoplasms from 53 to 30% in two-year old Fischer rats as compared to controls (p < 0.05) . Although both groups had also received ten does of 25 μg aflatoxin B_x in weeks 6-8, aflatoxin is not recognized to be carcinogenic to hematopoietic cells. Microscopic examination of several of these tumors have shown that they are large granulocytic leukemias (27) , which develops spontaneously in elderly Fischer F344 rats (28, 29) . Reddy and colleagues (24) have also observed that oltipraz is as effective as an inhibitor of tumor promotion as it is in the initiation phase of azoxymethane (AOM) -induced colon carcinogenesis. Although these investigators have shown that dietary oltipraz has modest inhibitory effects of tyrosine kinase activity in the liver and colon shortly after AOM exposure (30) , the relevance and mechanism for this observation has not been clarified further.

As mentioned above, oltipraz has also been determined to inhibit HIV-1 replication in vi tro (9) . Oltipraz may act as an antiretroviral by irreversibly inhibiting HIV-1 RT in acute infection models, however the finding that the drug possesses antiretroviral activity in chronically infected Ul promonocytic leukemia cells led to the conclusion that oltipraz exerts another antiviral mechanism of action (10) .

As indicated above, the metabolism of oltipraz is complex with the generation of series of rearranged metabolites that are formed from 7-methyl-6, 8-bis (methylthio)pyrrolo- [1, 2-a] - pyrazine (metabolite III;) (11, 12) . Most of the rearranged metabolites differ from metabolite III primarily in the oxidation state of the pyrazine ring or sulfur atoms.

Summary of the Invention

This invention provides a pharmaceutical composition for inhibiting replication of a retrovirus in a subject infected with the retrovirus which comprises a pharmaceutically acceptable carrier and a compound having the structure:

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are O, S, or N-R₄; and wherein Ri , R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

This invention also provides a pharmaceutical composition for inhibiting replication of a retrovirus in a subject infected with the retrovirus which comprises a pharmaceutically acceptable carrier, a first compound which inhibits replication of the retrovirus and a second compound having the structure:

wherein Zj is either C-R₅ or N; wherein Z₂ is N; wherein X_{ and X₂ are the same or different and are 0, S, or N-R₄; and wherein R,, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

The subject invention also provides a method of inhibiting replication of a retrovirus in a subject infected with the retrovirus which comprises administering to the subject in an amount effective to inhibit retroviral replication the composition described above which comprises a pharmaceutically acceptable carrier and a compound having the structure:

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein Xj and X₂ are the same or different and are O, S, or N-R₄; and wherein Rj, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

This invention further provides a method of inhibiting replication of a retrovirus in subject infected with the retrovirus which comprises administering to the subject in an amount effective to inhibit retroviral replication the above- described composition which comprises a pharmaceutically acceptable carrier, a first compound which inhibits replication of the retrovirus and a second compound having the structure:

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are 0, S, or N-R₄; and wherein R_{l f} R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

This invention further provides a pharmaceutical composition for inhibiting tumor-promoter initiated transcription which comprises a pharmaceutically acceptable carrier and a compound having the structure:

wherein Z is either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are 0, S, or N-R₄; and wherein R,, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

This invention also provides a method for preventing the formation of tumors in a subject which comprises administering to the subject in an amount effective to inhibit tumor formation the above described composition which comprises a pharmaceutically acceptable carrier and a compound having the structure:

This invention further describes a compound having the structure:

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein X_! and X₂ are the same or different and are 0, S, or N-R₄; and wherein Rj, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons; with the proviso that when Zj and Z₂ are both N, X_j and X₂ are both S, and Rj and R₂ are both methyl, R₃ is not methyl.

Brief Description of the Figures

Fig. 1. Proposed routes for the biotransformation of oltipraz to metabolite III. The scheme was adapted from the work of Fleury et al . (13) . "Nuc" is nucleophile.

Fig. 2. Inhibition of p24 antigen release into culture supernatants of H9 (upper) and CEM (lower) cell lines as a function of the concentration of metabolite III or oltipraz. The inhibition of p24 antigen release was determined by normalizing p24 antigen levels to those of matched, HIV-1-infected (drug-free) , control cells. Culture conditions and assays were as described in Experimental Procedures. Symbols, mean + standard error of the percentage inhibition of p24 antigen release for seven and five independent experiments for the H9 and CEM cell lines, respectively. The IC₅₀ values for zidovudine were determined to be 0.050 and 0.010 μM for the H9 and CEM cell lines, respectively.

Fig. 3. Effects of oltipraz and metabolite III on RT activity as a function of concentration (upper) and the effect of metabolite III as an irreversible inhibitor of RT (lower) . Upper, 10-μl aliquots of PBS containing RT, 1 mg/ml bovine serum albumin, 3.5% Triton X-100, and 0-525 μM metabolite III were added to 25 μl of RT reaction buffer (50 mM Tris- HCL, 75mM KCI, 5 mM MgCl₂, 1 mM dithiothreitol, 1 mM

EGTA, 0.1% Nonidet P-40, 0.1% Triton X-100, 10 μg/ml poly(A)⁺ RNA, 2.5 μg/ml oligo(dT) , 4 μM dTTP, 20 μCi/ml [α-³²P]dTTP, pH 7,8] in well of 96-well microtiter plate. After the mixtures were incubated for 0.5 hour at 37^°, the remainder of the isotopic assay was performed as described by Chavan and Prochaska (16) . Symbols , mean ± standard error for the enzyme assay. Lower, RT dissolved in PBS containing 1 mg/ml bovine serum albumin was preincubated with 56 (x) , 28 (Δ) , 14(D) , or 0(O) μM metabolite III for the times indicated. After incubation, 10-μl aliquots (in triplicate) were removed and assayed for RT activity as described above.

Fig. 4. Inhibition of p24 antigen release into culture supernatants from PMA-stimulated ACH-2 cells by metabolite III (M III) and interferon-α (IFN) but not oltipraz (OLT) . ACH-2 cells (50,000 cells/microtiter well, 200-μl final volume) were incubated with various concentrations of oltipraz or metabolite III for 24 hours before the addition of 2.5 nM PMA, in triplicate sets of wells. The cells were grown for an additional 48 hours, and p24 antigen release into culture supernatants was determined. Shown are the means (■) , standard errors (D) , and p values (numbers on the right) ; p values of <0.05 were considered significantly different from PMA-stimulated controls.

Fig. 5. Antiviral activity of oltipraz (•) , metabolite III (M III) (X) , and the combination of oltipraz/metabolite III (01T:M III) at a fixed ratio of 2:1 ( ,▼) on acutely infected PBMCs. PHA-

P stimulated lymphocytes were prepared and treated with oltipraz and/or metabolite III and virus as described in the "Experimental Details" Section. Symbols , mean ± standard error for five independent experiments (wherein the combination of oltipraz and metabolite II was tested) . Inset, corresponding combination index as a function of fractional inhibition of p24 antigen release. The median-effect plots [-log [ ( 1/fractional inhibition) -1] as a function of log

(concentration) ; see Ref. 17 for derivitation] yielded the following parameters, which were used to generate the combination index plot shown

(inset); oltipraz, ED₅₀ = 13.0 μM, slope = 1.94, and r = 0.925; metabolite III, ED₅₀ = 27.1 μM, slope =

0.626, and r = 0.994; oltipraz/metabolite III (2:1) , ED₅₀ = 7.31 μM, slope = 1.94, and r = 0.953.

Fig. 6. Structures of oltipraz and metabolite III (7- methyl-6, 8-bis (methylthio)pyrrolo- [1, 2-a] -pyrazine

(metabolite III) . Fig. 7. Inhibition of PMA-stimulated expression of LTR- driven jS-galactosidase in 293,27.2 cells by metabolite III and NAC, but not by oltipraz or 1,2- dithiole-3-thione. 293.27.2 cells were plated at a density of 3000 cells per 0.32 cm² well in 96-well microtiter plates in 200 μl medium as detailed by Roederer et al.¹⁹, and cells were exposed to test compounds 48 (+) , 24 (A) , 2 (•) , and 0 (Δ) h prior to stimulation with PMA in triplicate sets of plates utilizing the methods and plate format described²¹. DMSO (dimethyl sulfoxide) was utilized as the solvent for the test compounds except NAC, and all cells received 0.1% final concentration DMSO. The upper four rows of wells of each plate received 10 ng/ml final concentration PMA 72 h after plating and the plates were allowed to incubate an additional 24 h. /3-galactosidase assays were performed with one set of plates as described¹⁹, except that the rate of formation of the 0- nitrophenol from 0 - nitropheny1 - β -D - g a 1 a c t o py r a n o s i de was determined spectrophotometrically with a microtiter plate scanner at 405 nm. Cell densities were determined by staining the remaining two sets of plates with

^* crystal violet²² or MTT²³. /3-galactosidase specific activities were determined by normalizing the β- galactosidase activity to the MTT staining for all compounds except NAC, where the β-galactosidase activities were normalized to crystal violet staining. The treated to control ratios were determined by normalizing the drug-treated specific activities to the specific activities of PMA- stimulated, vehicle (DMSO) -treated control cells on the same plate. The fold-increase in β- galactosidase by PMA stimulation was made by comparing the fold-increase in β-galactosidase specific activities from DMSO-treated cells from the same plate. Metabolite III does not inhibit the enzymatic activity of 3-galactosidase.

Fig. 8. Potential mechanisms whereby oltipraz and metabolite III may affect differing stages of the viral life-cycle and carcinogenesis. Left, oltipraz inhibits HIV-1 replication in acute T-cell infection models, and oltipraz inactivates HIV-1 RT²'³. This is the likely antiviral mechanism for oltipraz since HIV-1 replication is not blocked by oltipraz treatment in chronically infected ACH-2 T- cell lymphoma cells¹. Metabolite III affects LTR- driven transcription, and can block viral replication in chronically infected cells. Right , oltipraz can inhibit HIV-1 the initiation phase of carcinogenesis by diverting ultimate carcinogens form interacting with DNA (by induction of Phase II enzymes^4,5'⁸ and the inhibition of Phase I enzymatic activities⁶) . Metabolite III is proposed to block the promotion phase of carcinogenesis by blocking cellular proliferations by mitogens and cytokines. This scheme implies that oltipraz may be a more effective antiviral/anticarcinogen than can be appreciated by considering the effects of oltipraz or metabolite III alone.

Detailed Description of The Invention

; wherein _j iε either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are 0, S, or N-R₄; and wherein R. , R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

In one embodiment of the above composition, Zj and Z₂ are both N; X_j and X₂ are both S; and R_1# R₂, and R₃ are each methyl, i.e. the compound of the composition is 7-methyl-6, 8 bis (methylthio)pyrrolo[1, 2-a]pyrazine, which is sometimes referred to as "metabolite III".

The compound of the above composition, and therefore the composition itself, is useful for inhibiting the replication of any retrovirus, including, but not limited to HIV-1 (Human Immunodeficiency Virus Type 1) , HIV-2 (Human Immunodeficiency Virus Type 2) , HTLV-1 (Human T-Cell Leukemia Virus Type 1) , FIV (Feline Immunodeficiency Virus) , or FLV (Feline Leukemia Virus) . The compound appears to be able to block a transcription factor from binding to an LTR (Long Terminal Repeat) sequence of such retroviruses, and thereby inhibits the activation of transcription of the viral genome of these retroviruses. Accordingly, the composition of this invention is also useful for inhibiting the replication of any virus in which this transcription factor plays a role in the virus's life-cycle, i.e. in the metabolic steps which comprise replication of the virus.

In one embodiment, the subject in which the above composition inhibits replication of a retrovirus, the subject is a mammal. The subject may be any mammal. For example, in one embodiment the subject is a human, in which case the composition of the invention particularly inhibits in the subject replication of retroviruses which most commonly infect humans, such as HIV-1 or HIV-2, or HTLV-I. However, should a human be infected with a retrovirus which does not commonly infect humans, the composition would still be useful for inhibiting replication of that retrovirus. In another embodiment, the subject is a cat, in which case the composition of the invention particularly inhibits replication of retroviruses which most commonly infect cats, including, but not limited to, FIV or FLV. However, the composition of the invention also inhibits in cats the replication of retroviruses which do not commonly infect cats.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutically accepted carriers known to those of ordinary skill in the art. Examples of such standard carriers include, but are not limited to, phosphate buffered saline solution, water, emulsions such as oil/water emulsions or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules. A suitable pharmaceutically acceptable carrier may be selected taking into account factors known to those of ordinary skill in the art, for example the mode of administration by which the composition is to be administered to the subject.

Methods for synthesizing compounds having the structure defined above which may be used in the above-described composition may be ascertained by those of ordinary skill in the art using generally-known techniques. Examples of such techniques include the general organic synthesis techniques disclosed in such texts as March, J. Advanced Organic Chemistry, 3rd ed. (Wiley; New York: 1985) , the contents of which are hereby incorporated by reference. Techniques for synthesizing the above-defined compounds are also described in Fleury, M.B., et al . , Studies of the reaction of 1,3-dithiole- 3-thiones with nucleophiles, Tetrahedron 41:3705-3715 (1985), the contents of which are hereby incorporated by reference.

wherein Zj is either C-R₅ or N; wherein Z₂ is N; wherein X_] and X₂ are the same or different and are O, S, or N-R₄; and wherein R_lf R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

In one embodiment of the above-described composition, Z_j and Z₂ of the second compound are both N; Xj and X₂ are both S; and Ri, R₂, and R₃ are each methyl. As discussed above, this compound is sometimes referred to as "metabolite III".

The first compound of the above-described composition may be any compound which is known to inhibit retroviral replication. Some of the resulting compositions would comprise a ^* combination of a first compound and a second compound which would be expected to provide a synergistic inhibition of retroviral replication. Accordingly, the subject invention also includes synergistic compositions according to the above- described composition comprising a first compound which inhibits retroviral replication and a second compound having the above-defined structure.

As discussed above, the compound in the above-described composition appears to be able to block a transcription factor from binding to the LTR (Long Terminal Repeat) sequences of retroviruses, and thereby inhibits activation of transcription of the integrated viral genome of these retroviruses. The compound of the above-described composition therefore inhibits retrovirus transcription by blocking the transcription activation step in the life-cycle of retroviruses. In one embodiment of the above-described composition, the first compound inhibits retroviral replication by interfering with a step in the life-cycle of retroviruses other than the transcription activation step. Such steps in the life-cycle of retroviruses are well known to those of ordinary skill in the art and include, but are not limited to, binding of the retrovirus to a CD4 receptor on a host cell, internalization of the retrovirus by the host cell, uncoating of the retrovirus, reverse transcription of the retroviral genome, DNA integration into the host cell's genome, and, following transcription activation, production of genomic mRNA, translation of genomic mRNA, processing of retroviral proteins, assembly, and release. In another embodiment, the first compound inhibits transcription activation in the retroviral life-cycle. If the first compound may, however, inhibit transcription activation by a mechanism other than by blocking the aforementioned transcription factor from binding to an LTR of the retrovirus.

In one embodiment of the above-described composition, the first compound inhibits protease, an enzyme which processes retroviral proteins during the step of processing retroviral proteins in the retroviral life-cycle.

Any compound which inhibits protease may be used in the above- described composition. Examples of compounds which inhibit protease include, but are not limited to, saquinavir (made by Hoffman LaRoche) and indinavir sulfate (made by Merck) .

In another embodiment, the first compound inhibits reverse transcriptase, the enzyme which reverse transcribes the viral genome into DNA during the reverse transcription step of the retroviral life-cycle.

Any compound which inhibits reverse transcriptase may be used in the above-described composition. Compounds which inhibit reverse transcriptase include, but are not limited to, AZT

(3' -azido-3 ' -deoxythimidine, also known as zidovudine), DDI (2' , 3' -dideoxyinosine, also known as didanosine) , DDC (2', 3' dideoxycytidine, also known as zalcitabine) , D4T (2', 3'- didehydro-3' -deoxythimidine, also known as stavudine) , 3TC (2' -deoxy-3' -thiacytidine, also known as lamivudine) , nevirapine (6, 11-dihydro-11-cyclopropyl-4-methyldipyrido [2,3- jb:2',3'-e] [1,4] diazapene-6-one) , or oltipraz (5-pyrazinyl-4- methyl-1, 2-dithiole-3-thione) .

The above-described composition is useful for inhibiting the replication of any retrovirus, including, but not limited to HIV-1 (Human Immunodeficiency Virus Type 1) , HIV-2 (Human Immunodeficiency Virus Type 2) , HTLV-1 (Human T-Cell Leukemia Virus Type 1) , FIV (Feline Immunodeficiency Virus) , or FLV (Feline Leukemia Virus) .

In one embodiment, the subject in which the above-described composition inhibits replication of a retrovirus, the subject is a mammal. The subject may be any mammal. For example, in one embodiment the subject is a human, in which case the composition of the invention particularly inhibits in the subject replication of retroviruses which most commonly infect humans, such as HIV-1 or HIV-2, or HTLV-I. However, should a human be infected with a retrovirus which does not commonly infect humans, the composition would still be useful for inhibiting replication of that retrovirus. In another embodiment, the subject is a cat, in which case the composition of the invention particularly inhibits replication of retroviruses which most commonly infect cats, including, but not limited to, FIV or FLV. However, the composition of the invention also inhibits in cats the replication of retroviruses which do not commonly infect cats.

The term "pharmaceutically acceptable carrier" in the description of above composition is as defined above.

Methods for synthesizing compounds having the structure defined above for the second compound which may be used in the above-described composition may be ascertained by those of ordinary skill in the art using general organic synthesis techniques as described above.

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein X_x and X₂ are the same or different and are 0, S, or N-R₄; and wherein R_j, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons .

In the above-described method, any suitable means known to those of ordinary skill in the art may be used to administer the pharmaceutical composition to the subject. In one embodiment, administering the pharmaceutical composition to the subject comprises orally administering the composition to the subject. If oral administration is employed, the pharmaceutical composition may be in the form of a capsule, tablet, or solution.

In another embodiment, the pharmaceutical composition is injected into the subject. Injection may be intramuscular, intraperitoneal, intravenous, or subcutaneous. The pharmaceutical composition may be injected into any part of the subject's body.

In a further embodiment, the pharmaceutical composition is topically applied to the subject. If the pharmaceutical composition is topically applied, the pharmaceutical composition may, for example, be in the form of a lotion or cream.

As used herein, the term "amount effective to inhibit retroviral replication" is intended to mean any amount which will inhibit the replication of the retrovirus in the infected subject. This amount will depend on various factors known to those of ordinary skill in the art. Such factors include, but are not limited to, the size, weight and age of the subject and the concentration of the retrovirus in the infected subject.

Retrovirus the replication of which may be inhibited according to the above-described method include, but not limited to HIV- 1, HIV-2, HTLV-1, FIV, and FLV.

In one embodiment of the above-described method, the subject is a mammal, including, but not limited to, humans and cats.

This invention further provides a method of inhibiting replication of a retrovirus in subject infected with the retrovirus which comprises administering to the subject in an amount effective to inhibit retroviral replication the above- described composition which comprises a pharmaceutically acceptable carrier, a first compound which inhibits replication of the retrovirus and a second compound having the structure :

wherein Zj is either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are O, S, or N-R₄; and wherein R_j, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

The term "amount effective to inhibit retroviral replication" in the above-described method is as defined above.

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are O, S, or N-R₄; and wherein R_j, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

In one embodiment of the above composition, Zj and Z₂ are both N; X_j and X₂ are both S; and Rj, R₂, and R₃ are each methyl, i.e. the compound of the composition is 7-methyl-6, 8- bis (methylthio)pyrrolo [1,2-a]pyrazine, which is sometimes referred to as "metabolite III".

The compound of the above composition, and therefore the composition itself, is useful for inhibiting transcription which is initiated by a tumor promoter.

Tumor promoters are well known to those of ordinary skill in the art and include, but are not limited to, phorbol ester and phenobarbital. The term "tumor promoter" also encompasses complete carcinogens, i.e. compounds which exhibit, inter alia, tumor promoter activity. Complete carcinogens include, but are not limited to, benzpyrene and the polycyclic aromatic hydrocarbons which are found, for example, in cigarette smoke.

The term "pharmaceutically acceptable carrier" for purposes of the above-described composition is as defined above.

Methods for synthesizing compounds having the structure defined in the above-described composition may be ascertained by those of ordinary skill in the art using general organic synthesis techniques as described above.

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are 0, S, or N-R₄; and wherein R_j, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons. In the above-described method, any suitable means known to those of ordinary skill in the art may be used to administer the pharmaceutical composition to the subject. In one embodiment, administering the pharmaceutical composition to the subject comprises orally administering the composition to the subject. If oral administration is employed, the pharmaceutical composition may be in the form of a capsule, tablet, or solution.

As used herein, the term "amount effective to inhibit tumor formation" is intended to mean any amount which will inhibit the formation of tumors in the subject. This amount will depend on various factors known to those of ordinary skill in the art. Such factors include, but are not limited to, the size, weight and age of the subject.

In one embodiment of the above-described method, the subject is a mammal. In one embodiment of the above-described method wherein the subject is a mammal, the subject is a human.

In another embodiment of the above-described method, the subject is likely to be exposed to a tumor promoter, such as any of the tumor promoters, including the complete carcinogens, identified above. For example, a subject who is likely to be exposed to cigarette smoke is a subject likely to be exposed to tumor promoters.

In another embodiment of the above-described method, the subject is predisposed to developing tumors. Subjects predisposed to developing tumors are well known to those of ordinary skill in the art. An example of a subject predisposed to developing tumors is a subject predisposed to having cancer, such as a subject with ulcerative colitis, which subject is therefore predisposed to developing colon cancer.

This invention further describes a compound having the structure:

wherein Z_x is either C-R₅ or N; wherein Z₂ is N; wherein X, and X₂ are the same or different and are O, S, or N-R₄; and wherein R_j, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons; with the proviso that when Zj and Z₂ are both N, X_j and X₂ are both S, and Rj and R₂ are both methyl, R₃ is not methyl.

The above-described compounds are useful for inhibiting the replication of retroviruses and for inhibiting tumor promoter initiated transcription.

This invention will be better understood from the Examples in the "Experimental Details" Section which follows. However, one skilled in the art will readily appreciate that the specific methods and results discussed in the "Experimental Details" Section are merely illustrative of, and are not intended to, and should not be construed to limited the invention as described above.

Experimental Details

Abbreviations: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; PBMC, peripheral blood mononuciear cell; PBS, phosphate-buffered saline; PHA-P, phytohemagglutinin P; IL-2, interleukin-2; DMSO, dimethylsulfoxide; MTT, 3- (4, 5-dimethylthiazo-2-yl) -2, 5- diphenyltetrazolium bromide; PMA, phorbol-12-myristate-13- acetate; EGTA, ethylene glycol bis (/3-aminoethyle ether) - N,N,N' ,N' -tetraacetic acid; NAC, n-acetyl-L-cysteine.

Materials. Oltipraz was synthesized as described (15) . PBMCs were obtained from the New York Blood Center as buffy coats; cell lines were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program

(Rockville, MD) . PHA-P was obtained from Difco Laboratories

(Detroit, MI) , purified human IL-2 was purchased from

Schipparelli Biosystems (Columbia, MD) , interferon-cv was from

Hoffman-LaRoche (Nutley, NJ) , Ficoll was obtained from Organon Technica (Durham, NC) , and 0.2-mm silica gel/aluminum-backed thin layer chromatography plates were purchased from E. Merck (Darmstadt, Germany) . All other materials were obtained from the sources cited (9,10) or were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher (Springfield, NJ) . NMR spectra were recorded with a Bruker AMX400 spectrometer, IR spectra were recorded with a Perkin Elmer 1600 series Fourier transform-IR spectrometer, and mass spectra were recorded with a Delsi Nermag Automass high performance gas chromatograph- mass spectrometer.

Synthesis of metabolite III. Metabolite III was prepared according to the method of Fleury et al. (13) . Briefly, oltipraz (O.llg, 0.50mmol) was dissolved 500ml of absolute ethanol under argon and heated to 35^° , yielding a deep orange- colored solution. While this mixture was being stirred, 20ml of a yellow-brown solution of absolute ethanol containing 5mmol of sodium ethoxide and 0.30g (2.5mmol) of L-cysteine were added via hypodermic syringe over 5 minutes. Although there was no obvious change in the color of the reaction, thin layer chromatography revealed complete disappearance of oltipraz 1 hour after addition of the sodium ethoxide/cysteine. After an additional 1 hour of stirring, 3.1ml (7.1g, 50mmol) of CH₃I were added via hypodermic syringe. There was an immediate change in color from deep orange to lemon yellow. After the reaction was allowed to proceed for an additional 1 hour, the flask was removed from the oil bath, and small portions of crushed dry ice were added to neutralize the reaction mixture. The ethanol was removed by rotary evaporation, and the resulting yellow paste was partitioned between water and ethyl acetate. After the organic fraction was dried over MgSo₄ the ethyl acetate was removed by rotary evaporation. The resulting orange oil was dissolved in toluene, applied to a silica column (2.5 x 10cm) , and eluted with toluene/acetone (98:2) . The pooled fractions were dried by rotary evaporation and yielded 0.10g (91% yield) of straw-colored solid [m.p., 62-63° (uncorrected) ; literature m.p., 66^°; Η NMR (400 MHz, CDCI₃) , δ 2.213 (s 3 H, C₈-SCH₃) , δ 2.274 (s, 3 H, C₆-SCH₃) , δ 2.489 (s, 3 H, C₇-CH₃) , δ 7.6852 (d, J = 4.8 Hz, 1 H, C₃-H) , δ 8.188 (dd, J = 4.8 and 1.4 Hz, 1 H, C₄-H) , and δ 8.962 (d, J = 1.4Hz, 1 H, Cj-H) ; ¹³C NMR (100 MHz, CDCI₃) , δ 10.45 (C₇-CH₃) , δ 17.64 (C₈-SCH₃) , δ 20.31 (C₆-SCH₃) , δ 107.68 (C₈) , δ 115.64 (C₇) , δ 116.07 (C₄) , δ 128.27 (C₃) , δ 131.02(C₈), δ 135.76 (C₆) , and δ 142.86 (Cj) ; IR (KBr pellet) , 2917.9, 1604.2, 1498.5, 1435.4, 1419.7, 1370.6, 1336.7, 1370.6, 1336.7, 1306.7, 1288.7, 1230.8, 1117.5, 968.9, 949.3, 709.7, 721.9, 662.4, 595.8, and 583.2 cm^"1; UV (absolute ethanol) , 250.0, 305.6, 314.8, and 354.0 nm; mass, 225.03 (M+l)] . Treatment of cells. Cell lines were grown in RPMI 1640 medium supplemented with 20% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and lOOμg/ml streptomycin, at 37° in humidified incubators with 5% C0₂. PBMCs, isolated from buffy coats with a ficoll density gradient, were washed three times in PBS and were grown in the aforementioned medium supplemented with lμg/ml PHA-P. After 24 hour exposure to PHA-P, the supernatant was replaced with medium supplemented with 5% IL-2 (without PHA-P) . Oltipraz and metabolite III were dissolved in DMSO and were stored at -70^°, The stabilities of oltipraz and metabolite III in DMSO were assessed by high performance liquid chromatography using a Microsorb C₁₈ column (Ranin, Emeryville, CA) run under isocratic elution conditions (methanol/water, 80:20) , and the antiretroviral activities of old stocks were routinely compared with those of freshly prepared stocks at the time that the old stocks were replaced. There was neither chemical nor biological evidence for degradation of either drug in DMSO stored at -70^° for >1 year (data not shown) . The DMSO stocks were diluted into medium, to 0.2% maximal final concentration, just before treatment of the cells. Because the cells were cultured with 20% fetal bovine serum, no obvious precipitation of oltipraz or metabolite III was observed with final concentrations up to 200 μM.

Infection and assay of HIV-1 replication in H9 and CEM cells. Antiviral activity was assessed in H9 and CEM cells as described (9) . Briefly, H9 and CEM cells were infected with HIV-1 (isolate HTLV-IIIB) at 1000 50% tissue culture infectious doses/10⁶ cells. Virus was allowed to adsorb for 1 hour, after which the unadsorbed virus was removed by centrifugation. After the cells were washed twice with PBS, they were added to 96-well microtiter plates with medium containing up to 0.2% DMSO and various concentrations of oltipraz or metabolite III, at a final density of 500,000 cells/ml (final volume/well, 200 μl) . On day 3 or 4, 50% of each supernatant was discarded and replaced with freshly prepared medium containing the same drug concentration as that in which the cells were plated initially. On day 7, cell densities were estimated by staining with MTT (10) and HIV-1 replication was assessed by measurement of p24 antigen in culture supernatants (coulter, Hialeah, FL) or by indirect immunofluorescence, as described (9) .

Infection and assay of HIV-1 replication in Ul and ACH-2 cells. Ul and ACH-2 cells were treated as described (10) , by incubating the cells (50,000 cells/microtiter well, 200-μl final volume) with various concentrations of oltipraz or metabolite III for 24 hours before the addition of 2.5 nM PMA. The cells were grown for an additional 48 hours, and p24 antigen release was measured in the culture supernatants.

Infection and assay of HIV-1 replication in PBMCs. PBMCs were used for experiments 2-6 days after PHA-P stimulation. Except for two experiments, PBMCs for each experiment were obtained from different donors . The PBMCs were treated as described for H9/CEM cells lines, except that the cells were plated at a density of 10₆ cells/ml in medium supplemented with 5% IL-2.

Cytotoxicity assays. Uninfected PBMCs, H9 cells, and CEM cells were treated as described above for infected cells . On day 7, cell densities were estimated by MTT staining (10) . For Ul and ACH-2 cells, the cytotoxicity of oltipraz and metabolite III was assessed by MTT staining of matched non-

PMA-stimulated cells.

Assay for inhibition of RT. Isotopic RT assays were performed as described (16) , except that 4 μM (final concentration) unlabeled dTTP was added to all incubation mixtures. Assays were performed in duplicate or triplicate.

Statistical treatment of results. All data presented for H9, CEM, and Ul cells and PMBCs are expressed as percentage inhibition of HIV-1 or cell growth, relative to matched controls. For each experiment, the effects of treatment with various drug concentrations were assessed in duplicate sets of wells, whereas two to four wells of cells were used for controls. To adequately present the results of multiple independent experiments without bias, the individual experiments were treated as independent points and the average ± standard error values of the percentage inhibition for all experiments were determined. The ED₅₀ (concentration for 50% inhibition of viral replication) , IC₅₀ (concentration for 50% reduction in cell density) , and synergy estimates from pooled or individual experiments were determined by the median-effect principle, as described (17) . two independent experiments were performed with ACH-2 cells, wherein all drug concentrations and controls were assayed in triplicate sets of wells. The data presented are results from one of these experiments and are representative of the other. Statistical significance was analyzed by unpaired t test; p<0.05 was taken as a statistically significant difference from PMA-stimulated controls.

Assay for the inhibition of long terminal repeat (LTR) -driven transcription

The U293.27.2 cell line is a stably-transfected clone derived from the 293S embryonic human kidney cell line which contains a construct of HIV-1 LTR fused to the bacterial β- galactosidase gene (32) . Cells were plated at a density of 3000 cells per 0.32 cm² well as described (32) in triplicate set of plates utilizing the plate format outlined by Prochaska (33) . Cells were pretreated with test compounds 24 hours after plating and 0-48 hours prior to the addition of 10 ng/ml PMA (upper four rows of each plate) . One set of plates were assayed for 3-galactosidase as described (32) 24 hours after the addition of PMA except that the formation of 0-nitrophenol from 0-nitropheyl-3-D-galactopyranoside was determined spectrophotometrically with a microtiter plate scanner at 405nm. Cell densities were determined by staining the remaining sets of plates with either crystal violet (34) or MTT (35) . β-galactosidase specific activities were determined by normalizing the jS-galactosidase activity to the MTT [3- (4,5- dimethylthiazo-2-yl) -2, 5-diphenyltetrazolium bromide] staining for all compounds tested except NAC, where β-galactosidase activities were normalized to crystal violet staining. The treated-to-control ratios were determined by normalizing the drug-treated specific activities to the specific activities of the PMA-stimulated, vehicle (DMSO) -treated control cells on the same plate. The fold-increase in /3-galactosidase specific activities from DMSO-treated cells from the same plate. Metabolite III does not inhibit the enzymatic activity of β- galactosidase.

Results and Discussion

Metabolite III was synthesized in 91% yield according to the method of Fleury et al. (13) . The synthesis of metabolite III from cysteine demonstrates that oltipraz is an electrophile.

The electrophilicity of oltipraz may be relevant to its biological properties, because oltipraz is thought to inactivate and/or modify proteins that are critical to its antischistosomal (i.e. schistosomal glutathione S- transferases) (18) , anticarcinogenic (i.e., transcription factors) (19,20), and antiviral (i.e., RT) (9) activities.

Metabolite III was tested as an antiviral agent because it is the major metabolic intermediate of oltipraz in vivo and the results with PMA-stimulated Ul cells suggested that oltipraz may be a prodrug in this chronic infection model (10) .

In acutely infected H9 and CEM cells, metabolite III treatment resulted in a concentration-dependent inhibition of p24 antigen release into culture supernatant (Fig.2) . For both cell lines, the ED₅₀ was approximately 25 μM. Oltipraz was approximately 2.5-fold more potent than metabolite III. Oltipraz and metabolite III inhibited viral replication in H9 cells with similar potencies, using indirect immunofluorescence as a surrogate marker for viral replication (data not shown) . Although the assays examining the antiviral activity of metabolite III and oltipraz were not performed above 60 μM, cytotoxicity studies in uninfected cells indicated that metabolite III was relatively nontoxic (Table 1) . The selectivity index (IC₅₀/ED₅o) for metabolite III was 6.6 in H9 cells and 4.4 in CEM cells.

The inactivation of RT by oltipraz is a proposed mechanism for the antiretroviral activity of the drug (9) and, as noted above, it is likely that oltipraz acts as an electrophile by covalently modifying a nucleophilic amino acid residue of HIV- 1 RT. In stark contrast, metabolite III had negligible effects on purified recombinant HIV-1 RT when assayed for its ability to reversibly or irreversibly inhibit the enzyme (Fig. 3) . The inability of metabolite II to inactivate RT is not surprising, because metabolite III is not an electrophile. Moreover, given the hypothesis that the antiparasitic and anticarcinogenic activities of oltipraz are related to the ability of oltipraz to modify target proteins, the ineffectiveness of metabolite III as an antischistosomal agent and as an inducer of phase III enzymes would be expected (20) .

The antiviral effects of metabolite III in the setting of chronic infection were examined because the drug clearly possessed antiviral activity but had no effect on RT.

Determining whether metabolite III could block a point in the viral life cycle that was distal to viral integration was examined so as to perhaps explain the results with Ul cells (10) . The ability of oltipraz and metabolite III to inhibit

PMA-stimulated HIV-1 replication in chronically infected Ul

(promonocytic leukemia) and ACH-2 (T cell lymphoma) cell line was examined. In Ul cells, oltipraz (10) and metabolite III

(40.4 ± 6.5% inhibition of HIV-1 replication of 60 μM, six independent experiments) inhibited PMA-induced HIV-1 replication. In ACH-2 cells, metabolite III was an effective inhibitor of PMA-induced HIV-1 replication, whereas oltipraz was inactive (Fig. 4) . Metabolite III did not exhibit any cytotoxicity in either cell line at up to 60 μM.

The results with ACH-2 cells were particularly useful for narrowing the potential mechanisms by which oltipraz and metabolite III inhibit HIV-1 replication. Although oltipraz was active as an inhibitor of HIV-1 replication in acutely infected H9 and CEM T cell lymphoma models (Fig.2) , oltipraz was devoid of antiviral activity in chronically infected ACH-2

(T cell lymphoma) cells (Fig.4) . Although it is possible that differences in the uptake and metabolism of oltipraz are responsible for the inactivity of oltipraz in ACH-2 cells, that is less likely than an intrinsic difference in the mechanism of action of oltipraz and metabolite III, because ACH-2 cells are ultimately derived from the CEM cell line. The ability to inhibit acute, but not chronic, replication in these T cell models supports our proposal that oltipraz inhibits HIV-1 replication via the inactivation of RT (9) . In stark contrast, metabolite III is an effective inhibitor of HIV-1 replication in ACH-2 cells, suggesting that metabolite III acts to inhibit viral replication at a step distal to the integration of the viral genome. The findings in Ul cells may result from the ability of monocytoid cells to metabolize oltipraz to metabolite III and would account for the markedly increased potency of oltipraz with prolonged incubation before PMA stimulation (10) . These results suggest further investigation as to whether oltipraz is metabolized differently in monocytoid versus T cell lines.

Because metabolite III could inhibit HIV-1 replication in ACH- 2 cells, metabolite III was tested for the ability to inhibit HIV-1 transcription. Thus, it was determined whether metabolite III could inhibit the LTR-driven transcription of /3-galactosidase in stably transfected 293S human embryonic kidney cells (clone 293.27.2) (31) . Cells were pretreated with test compounds 0-48 h prior to the addition 10 ng/ml PMA 72 hours after plating the cells, and were assayed for β- galactosidase 24 hours after PMA stimulation. NAC (N-acetyl- L-cysteine) was included as a positive control, since it is able to inhibit HIV-1 replication and LTR-driven transcription (31, 32) .

The microscopic appearance of unstimulated and PMA-stimulated 293.27.2 cells treated with 60 μM metabolite III appeared indistinguishable from DMSO-treated controls, and cell densities as determined by MTT [3- (4, 5-dimethylthiazo-2- yl) 2, 5-diphenyltetrazolium bromide] staining were only modestly decreased with 24-72 hours exposure to the drug (14- 30%) . Cells treated with 30 _mM NAC showed a greater decrease in cell density (~ 50%) , and the cells treated with this concentration of NAC were more rounded and less adherent. 60 μM oltipraz produced a modest decrease in cell density ( 30%) . With the exception of the NAC-treated plates, results obtained by crystal violet staining were similar to results obtained with MTT (NAC reduces MTT spontaneously to the formazan dye) . It was thus concluded that metabolite III is not cytotoxic for prolonged periods of exposure in 293.27.2 cells at concentrations up to 60 μM.

After 24 hours incubation with 10 ng/ml PMA, the specific activity of -galactosidase was increased 24- to 52- fold in DMSO-treated cells relative to unstimulated cells. Figure 2 (31, 33-35) shows that metabolite III is able to inhibit the LTR-driven transcription of HIV-1 metabolite III in PMA- stimulated cells with an IC₅₀ 15 μM, in good agreement with IC₅₀ values determined for HIV-1 replication in a number of T- cell models. The effects of metabolite III were essentially identical to the results obtained with NAC, except that metabolite III is approximately 1000-fold more potent. The potencies of metabolite III and NAC were not altered with the length of preincubation, indicating that both compounds are able to exert their effects without further biotransformation. In contrast to metabolite III, oltipraz was completely inactive as an inhibitor of PMA-induced /3-galactosidase as was the unsubstituted 1, 2-dithiole-3-thione.

Since the activation of NF-κB plays an important role in the PMA-induced transcription of HIV-1 (36) , the binding activity of NF-KB from nuclear extract of 293,27,2 cells treated with test compounds for 2 hours prior to the addition of 10 ng/ml PMA was determined for four hours prior to harvesting of the cells. Nuclear extracts and EMSA were performed ad described

(31) , and typical results are shown in Fig. 3. Surprisingly,

PMA-inducible NF-κB binding activity was essentially unchanged by treatment with metabolite III, and contrasts sharply with the recognized ability of NAC to abolish NF-KB binding (31, 32) . Oltipraz and 1,2-dithiole-3-thione had modest inhibitory effects on NF-/B binding.

The ability of oltipraz and metabolite III to inhibit viral replication in acutely infected PMBCs (Fig. 5) was assessed. Oltipraz was able to inhibit HIV-1 replication with an ED₅₀ of

II μM, whereas the ED₅₀ for metabolite III was 18 μM (eight independent experiments) . The dose-response curve for inhibition of viral replication by metabolite III was much shallower than that for oltipraz. It was also clear that the inhibitory activity of metabolite III varied more markedly than did that of oltipraz. For example, in three of eight independent experiments the ED₅₀ for metabolite III was _<5 μM. Whether this reflects differences in donor lymphocytes and/or the time at which stimulated PBMCs were exposed to metabolite

III and HIV-1 after PHA-P treatment is at present unknown and is under investigation. It might be expected that agents acting at a site distal to integration of the viral genome would be more susceptible to differences in donor lymphocytes and/or the time of culture. The cytotoxicities of oltipraz and metabolite III in PBMCs are given in Table 1. The selectivity indices for oltipraz and metabolite III in PBMCs were 5- and 3- fold, respectively.

TABLE 1

Cytotoxicity of oltipraz and metabolite III in H9 cells, CEM cell, and PBMCs

Cell densitites were determined by MTT staining in uninfected cells cultured for 7 days in medium containing 0-200 μM oltipraz or metabolite III, as described in Experimental Procedures. The IC₅₀ for each independent experiment was determined by the median-effect principle (17) . The results shown are the mean ± standard error of independent IC₅₀ values.

IC,

Drug

H9 cells CEM cells PBMCs μM

Oltipraz >200 (n=l)^a*^b >200(n=l)^a 55.6 ± 5.6 (n=6)

Metabolite III 166 ± 13(n=3) 109 ± 3(n=3) 54,8 ± 8.7 (n=5)

^a No cytotoxicity at concentrations up to 60 μM in multiple independent experiments. ^b n, number of independent experiments. All experimental points were determined with two to four wells.

Because metabolite III is formed from oltipraz in vivo, determination of whether they were synergistic was sought. The dose-response curves for oltipraz, metabolite III, and oltipraz/metabolite III (2:1) are shown in Fig. 5, and the combination indices calculated from these data are shown in Fig. 5, inset. Oltipraz and metabolite III were synergistic at fractional inhibitions of >15% (a combination index of <1 indicates synergy) . The observation that oltipraz and metabolite III are synergistic inhibitors of HIV-1 replication raises questions regarding the potential in vivo activity of oltipraz, especially because most of the absorbed drug undergoes rearrangement to metabolite III (11,12) . Significantly, rearranged metabolites related to metabolite III represent >75% of ¹⁴C-labeled oltipraz recovered from the serum or urine (11,12) . Moreover, the serum concentration of oltipraz at nontoxic doses in humans and rodents has been documented to be in the low micromolar range (up to 20 μM) (3-8) . Thus, oltipraz and its arranged metabolites are found in serum at concentrations that inhibit HIV-1 replication in vi tro, and the above-described results suggest that oltipraz and its metabolites could act to synergistically inhibit HIV-1 replication in vivo .

In conclusion, the results presented here demonstrate that metabolite III inhibits HIV-1 replication in vi tro. This is the first reported biological activity for this compound. In T cells, metabolite III appears to be an inhibitor of HIV-1 replication at a point in the replicative life cycle that is distal to viral integration. Therefore, just as oltipraz represents a new lead for the design of inhibitors of acute replication (and RT) , metabolite III represents a new lead compound for the prevention of HIV-1 replication in chronically infected cells. Oltipraz and metabolite III inhibit HIV-1 replication via different mechanisms, and the results with PBMCs demonstrate that oltipraz and metabolite III are synergistic. Thus, oltipraz may be a more effective antiretroviral agent in vivo than it is in vi tro . This possibility suggests a clinical trial evaluating the ability of oltipraz to reduce viral load which should include an examination of oltipraz and metabolite III pharmacokinetics. Although many of the known biological effects of oltipraz have been attributed to the parent compound (9, 18, 20) , the above results demonstrate that oltipraz is a pro-drug, albeit affecting very different biological processes. It is not surprising that the biological properties of oltipraz and metabolite III are so distinct since the structures of the two compounds differ significantly. The antischistosomal, anticarcinogenic, and antiviral activities of the parent compound (oltipraz) are thought to be mediated by the ability of the drug to covantly modify target proteins (i.e. schistosomal glutathione S-transferase (18) , transcription factors involved in the regulation of Phase II enzymes (20) , and RT (9) , respectively) . In stark contrast to oltipraz, metabolite III: a) is not an electrophile, b) does not induce Phase II enzymes nor activate EpRE (Electrophile Response Element) -driven transcription) (20) ; and c) does not inhibit nor inactivate HIV-1 RT. The ability of the major metabolic intermediate of oltipraz to interfere with LTR-driven transcription provides a rational mechanism for the observed antiviral activity of metabolite III in chronically infected ACH-2 cells. Since metabolite III and closely-related congeners represent more than 75% of [¹⁴C] -labeled oltipraz recovered from serum or urine (12) , metabolite III may represent a lead compound for the identification of nontoxic inhibitors of mitogenic signal transduction.

The clinical implications for the observations of the above- described experiments are two-fold (Fig. 4 (2, 9, 21-23, 25) ) . The experiments show that oltipraz and metabolite III are synergistic in acutely infected PBMCs, and the action of oltipraz on RT and metabolite III on LTR-driven transcription is likely to account for this effect. In the case of cancer chemoprevention, the above results may rationalize the chemopreventive effects of oltipraz that were considered to be unrelated to the induction of Phase II enzymes by the parent compound. Thus, oltipraz may have greater antiretroviral activity in vivo than in vitro, and the potential effect of metabolite III as an antagonist of tumor promoter signal transduction may account for the considerable chemopreventive activity that oltipraz is known to possess.

In summary, oltipraz, an inhibitor of human immunodeficiency virus type 1 replication in vi tro (IC₅₀ - lOμM) , undergoes extensive metabolism in vivo . Most of the orally administered drug undergoes opening of the dithiolethione ring, reduction, recyclization, and methylation to form 7-methyl-6, 8- bis (methylthio)pyrrolo [1, 2-a]pyrazine ("metabolite III") . The above experiments show that metabolite III inhibits viral replication in vi tro (ED₅₀ == 25μM) in acutely infected H9 and CEM T cell lymphoma cell lines. Although both metabolite III and oltipraz were able to inhibit phorbol-12-myristate-13- acetate-stimulated viral replication in the chronically infected Ul promonocytic leukemia cell line, only metabolite III was able to inhibit phorbol-12-myristate-13-acetate- stimulated viral replication in chronically infected ACH-2 T cell lymphoma cells. The results with ACH-2 cells suggest that oltipraz inhibits an early stage of the viral life cycle, whereas metabolite III affects human immunodeficiency virus type 1 replication at a step distal to viral integration. This is consistent with the finding that oltipraz inhibits reverse transcriptase, whereas metabolite III does not. The results wilth U293.27.2 cells demonstrates that metabolite III affects the ability of the LTR to be activated by PMA. Although the mean ED₅₀ for metabolite III in acutely infected peripheral blood mononuciear cells was 18μM, the ED₅₀ was below 5μM in three of eight independent experiments . Studies of metabolite III in combination with oltipraz in acutely infected peripheral blood mononuciear cells demonstrated significant antiviral synergy. These results raise the possibility that the in vi tro potency of oltipraz may underestimate its antiretroviral activity in vivo . Based on these results, the phar acokinetics of oltipraz and metabolite III can be compared with the pharmacodynamic effects of orally administered oltipraz in a phase I/II trial of oltipraz in patients with p24 antigenemia.

7-methyl-6 , 8 -bis (methylthio) pyrrolo- [1 , 2 -a] -pyrazine (metabolite III) , a major metabolic intermediate of oltipraz

(5-pyrazinyl-4-methyl-1, 2-dithiole-3-thione) in vivo, inhibits human immunodeficiency virus type 1 (HIV-1) replication in vi tro . Metabolite III, but not oltipraz, inhibit phorbol 12- myristate 13-acetate (PMA) -stimulated viral replication in • chronically infected ACH-2 T-cell lymphoma cells, suggesting that metabolite III disrupt viral replication at a point in the HIV-1 life-cycle that is distal to integration of the viral genome. We find that metabolite III, but not oltipraz, inhibits long-terminal repeat (LTR) -driven expression of β- galactosidase in PMA-stimulated cells (IC_{50 ^} 15 MM) . These data indicate that oltipraz may have multiple independent and beneficial actions in vivo. In the case of HIV-1, the parent drug exerts its effect by irreversibly inhibiting reverse transcriptase (RT) (9, 21), whereas the rearranged metabolite can inhibit transcription of the integrated viral genome. In the case of cancer chemoprevention, oltipraz prevents the initiation phase of carcinogenesis by diverting ultimate carcinogens from DNA (2, 22, 23), whereas the rearranged metabolite may block the growth and selection of initiated cells by mitogens and cytokines. Thus, the known antipromoting effects of oltipraz (24) may be due to the effects of rearranged metabolite, and may rationalize why oltipraz is a more effective and complete chemopreventive agent than can be appreciated by solely considering the capacity of oltipraz to act as an inducer Phase II enzymes.

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Claims

What is claimed is:

1. A pharmaceutical composition for inhibiting replication of a retrovirus in a subject infected with the retrovirus which comprises a pharmaceutically acceptable carrier and a compound having the structure:

; wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein X_j and X₂ are the same or different and are O, S, or N-R₄; and wherein R_1# R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

2. The composition of claim 1, wherein Zj and Z₂ are both N; X_j and X₂ are both S; and R_j, R₂, and R₃ are each methyl.

3. The composition of claim 1, wherein the retrovirus is ^* HIV-1, HIV-2, HTLV-1, FIV, or FLV.

4. The composition of claim 1, wherein the subject is a mammal.

5. The composition of claim 4, wherein the mammal is a human.

6. The composition of claim 4, wherein the mammal is a cat.

7. A pharmaceutical composition for inhibiting replication of a retrovirus in a subject infected with the retrovirus which comprises a pharmaceutically acceptable carrier, a first compound which inhibits replication of the retrovirus and a second compound having the structure:

wherein Z_j is either C-R_s or N; wherein Z₂ is N; wherein

Xi and X₂ are the same or different and are O, S, or N-R₄; and wherein R_l7 R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

8. The composition of claim 7, wherein Zj and Z₂ are both N; Xi and X₂ are both S; and R_l7 R₂, and R₃ are each methyl.

9. The composition of claim 7, wherein the first compound inhibits protease.

10. The composition of claim 7, wherein the first compound inhibits reverse transcriptase.

11. The composition of claim 7, wherein the retrovirus is HIV-1, HIV-2, HTLV-1, FIV, or FLV.

12. The composition of claim 7, wherein the subject is a mammal.

13. The composition of claim 12, wherein the mammal is a human.

14. The composition of claim 12, wherein the mammal is a cat.

15. The composition of claim 9, wherein the first compound is saquinavir or L-735,524.

16. The composition of claim 10, wherein the first compound is AZT, DDI, DDC, D4T, 3TC, nevirapine, or oltipraz.

17. A method of inhibiting replication of a retrovirus in a subject infected with the retrovirus which comprises administering the composition of claim 1 to the subject in an amount effective to inhibit retroviral replication.

18. A method of inhibiting replication of a retrovirus in subject infected with the retrovirus which comprises administering the composition of claim 7 to the subject in an amount effective to inhibit retroviral replication.

19. A pharmaceutical composition for inhibiting tumor- promoter initiated transcription which comprises a pharmaceutically acceptable carrier and a compound having the structure:

wherein Zj is either C-R₅ or N; wherein Z₂ is N; wherein Xj and X₂ are the same or different and are 0, S, or N-R₄; and wherein Rj, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons.

20. The composition of claim 19, wherein Zj and Z₂ are both N; X_j and X₂ are both S; and Rj, R₂, and R₃ are each methyl.

21. A method for preventing the formation of tumors in a subject which comprises administering to the subject the composition of claim 19 in an amount effective to inhibit tumor formation.

22. The method of claim 21, wherein the subject is a mammal.

23. The method of claim 22, wherein the mammal is a human.

24. The method of claim 21, wherein the subject is likely to be exposed to a tumor promoter.

25. The method of claim 21, wherein the subject is predisposed to developing tumors.

26. A compound having the structure:

wherein Z_j is either C-R₅ or N; wherein Z₂ is N; wherein Xi and X₂ are the same or different and are O, S, or N-R₄; and wherein Rj, R₂, R₃, R₄, and R₅ are the same or different and are either hydrogen or a branched or unbranched alkyl group of from 1 to 3 carbons; with the proviso that when Zj and Z₂ are both N, Xj and X₂ are both S, and R_j and R₂ are both methyl, R₃ is not methyl.