WO2007065032A2 - Compounds and methods for inhibiting viral entry - Google Patents

Abstract

A series of geminal disulfone-containing compounds are described with utility in the treatment of HIV. An early stage inhibitor acts as a small molecule coreceptor-independent inhibitor of viral entry and can be used in combination therapy with other antiviral agents, including those described herein.

Description

COMPOUNDS AND METHODS FOR INHIBITING VIRAL ENTRY

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional application Ser. No. 60/741,996, filed December 2, 2005, the disclosure of which is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] This invention was made with Government support under Grant (or Contract) No. GM60917, awarded by the National Institute of Health. The Government has certain rights in this invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK.

[0003] NOT APPLICABLE

BACKGROUND OF THE INVENTION

[0004] Each day 14,000 people are newly infected with human immunodeficiency virus type 1 (HIV-I). With 80% of these new infections occurring by sexual transmission, there is an urgent need for a topical microbicide to prevent HIV-I from entering through the surfaces of the vagina, cervix, rectum or colon (2004 Report on the Global AIDS Epidemic. Joint United Nations Programme on HIV/ AIDS, Geneva, Switzerland). In the absence of an effective preventative vaccine, attention should be focused on alternate methods of halting infection, such as a topical microbicide that could be applied vaginally or rectally prior to intercourse. Microbicides act by preventing HIV-I infection through blocking attachment, entry, or early steps of replication (Stone, Nat Rev Drug Discov 1:977-985(2002)). Though the current arsenal of drugs used to treat HTV-I has proven to be quite effective in reducing viral loads to undetectable levels in patients, these approved drugs target viral enzymes (reverse transcriptase (RT) and protease (PR)) only after the virus has successfully invaded a host cell. The latest drug to be approved, T20 or enfuvirtide, belongs to a new class of entry inhibitors that blocks fusion of the viral membrane to the target cell membrane. Even though this drug is an entry inhibitor, it requires a specific interaction with the host before it is able to block virus entry. An ideal drug in preventing HIV-I infection would interact specifically with the virus particle and directly inactivate it, thereby rendering the particle non-infectious.

[0005] The HIV envelope glycoprotein is synthesized as a glycoprotein precursor (gplόO), which is cleaved into the surface glycoprotein (gpl20) and the transmembrane protein (gp41). GpI 2Θ and gρ41 remain noncovalently attached and are present as trimers in virions (Eckert and Kim, Annu Rev Biochem 70:777-810 (2001); Wyatt and Sodroski, Science 280:1884-1888 (1998)). This trimeric complex is located on the surface of HIV-I and is anchored to the viral envelope by the C-terminal domain of gp41. The entry process begins when gpl20 binds to the host receptor CD4. Upon interacting with CD4, gpl20 undergoes a conformational change, exposing a binding site for a chemokine coreceptor, either CCR5 (R5) or CXCR4 (X4) (Dimitrov, D. S., Cell 91:721-730 (1997)). Once the coreceptor is bound, it triggers the formation of a transient pre-hairpin intermediate structure in which the viral fusion protein gp41 inserts into the host membrane (Chan and Kim, Cell 93:681- 684(1998)). Finally, fusion of the viral membrane with the cellular membrane is driven by the restructuring of gp41 from the intermediate pre-hairpin into a trimer of hairpins which allows the viral core to enter the host cell (Chan, et al., Cell 89:263-273 (1997); Chan and Kim, Cell 93:681-684(1998); and Weissenhorn, et al., Nature 387:426-430 (1997)).

Exploiting these early stages of viral infection (viral attachment, coreceptor binding and membrane fusion) can lead to drugs that are useful prophylactically to curb the sexual transmission of HIV-I.

[0006] The present invention addresses this need.

SUMMARY OF THE INVENTION

[0007] In one aspect, the present invention provides a method for inhibiting viral entry into cells, the method comprising contacting the cells with a viral entry-inhibiting amount of a compound having the formula:

wherein each Ar is an independently selected trihydroxyphenyl group; the double bonds adjacent to the sulfone groups are each independently in either a cis or trans orientation; or a pharmaceutically acceptable salt thereof.

[0008] In a related aspect, the present invention provides a method of treating or inhibiting a HIV- viral infection, said method comprising administering to a subject in need of or at risk of such infection, a composition comprising a viral entry inhibiting amount of a compound of the formula:

in admixture with an HIV-integrase inhibiting amount of a compound of the formula

wherein the dashed line indicates an optional double bond and each Ar¹ is a substituted or unsubstituted phenyl, pyridyl or indolyl ring and is other than 3,4,5-trihydroxyphenyl.

[0009] In still another aspect, the present invention provides a pharmaceutical composition comprising an HIV viral entry inhibiting amount of a compound having the formula:

wherein each Ar is an independently selected trihydroxyphenyl group; the double bonds adjacent to the sulfone groups are each independently in either a cis or trans orientation; or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable excipient. Preferably, the composition is formulated for topical use as a gel, cream or ointment.

[0010] In a related group of embodiments, the compositions further comprise a compound of the formula:

wherein the dashed line indicates an optional double bond and each Ar¹ is a substituted or unsubstituted phenyl, pyridyl or indolyl ring and is other than 3,4,5-trihydroxyphenyl.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] Figure 1 provides the structures of L-chicoric acid and neutral disulfone-containing analogs (Compound 1 and Compound 2).

[0012] Figure 2 provides representative data from time-of-addition studies. The data shown is a representative example from 5 independent experiments. The error bars represent variation in the number of foci from the six wells per time point. [0013] Figure 3 illustrates the direct inactivation of HIV-I by Compound 2 in the absence of a cellular target. (A) The chemical structure of Compound 2. (B) The inhibitory effects of preincubating HIV-I particles with Compound 2 for 2 hours prior to the addition to HeLa H1-JC.37 cells. Infectivity was measured using the FIA. (C) A similar preincubation experiment was performed with T20 as a negative control. (D) Preincubation experiment with Compound 2 and 3TC (negative control) in HeLa Hl -JC.37, CEMxl74 and human PBMCs using p24 antigen capture ELISA to detect virus production 5 days post-infection. Data are representative of 3 independent experiments.

[0014] Figure 4 shows that Compound 2 does not bind to a cellular host factor to exert its antiviral activity. HeLa H1-JC.37 cells were incubated with drug at various concentrations for 2 hours. The drug was removed and the cells were washed two times before HIV-I NL4- 3 was added. For Compound 2 (A) and T20 (B), a normal dose-response curve was observed when the drug and virus were both added after the cells were pretreated with drug, but there was almost no inhibition when the drug was removed and only virus was added to cells. Data are representative of 3 independent experiments. [0015] Figure 5 illustrates probing the binding site of Compound 2 to ogpl40 with SPR competition studies. Overlay sensorgrams for 5OnM ogpl40 preincubated with Compound 2 injected over a Biacore CM5 chip with various HIV-I mAbs captured to the chip surface via immobilized goat anti-human IgG mAb. All responses have been normalized for the amount of mAb captured (responses <1). There was no binding interference of ogpl40 in the presence of Compound 2 with (A) gp41 mAb 5F3 (aa 526-543), (B) gp41 mAb F240 (aa 592- 606) or (C) gpl20 rnAb bl2 (CD4 binding site). Binding levels decreased with increasing concentrations of Compound 2 when ogpl40 was flowed over (D) gpl20 mAb F425 B4e8 (base of V3 loop). Binding interference was also observed for mAbs (E) 48d and (F) 17b that both target the gpl20 CD4i epitope. For these two mAbs, ogpl40 was preincubated Compound 2 along with sCD4.

[0016] Figure 6 provides a table of data for antiviral activity of Compound 2 against primary isolates and laboratory-adapted strains of HIV-I and agains SIVmac239.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0017] The term "EC₅0" refers to the concentration of compound that inhibits 50% viral activity, including infective activity, as measured in an in vitro assay, including a focal infectivity assay (described in Pincus, et al. Biotechniques 10:336 (1991)).

(0018] The term "HIV activity" refers to the ability of HIV to complete its infectious life- cycle, including binding, fusing, entering and replicating in a cell, release and/or lysis from a cell, as well as its transmission to another cell or another host. Inhibition of HIV activity can be accomplished by interfering with one or more steps of the HIV infectious life-cycle. HIV activity can be decreased or obliterated. Compounds that decrease HIV activity, measurably decrease (e.g., by 10%, 15%, 30%, 50%) detectable indicators of HIV activity (e.g., viral titer, transmission to another cell, viral nucleic acid levels) in comparison to test samples or individuals that are not contacted with the compounds.

[0019] Some compounds are "virucidal" or "a virucide." A virucidal compound prevents the completion of an HIV infectious life cycle upon contact with an HIV virion, thereby obliterating its HIV activity. A virucidal compound decreases the frequency of transmission and can prevent transmission of an HIV virus from a first cell to a second cell, and from a first infected host to a second host.

[0020] By "HIV" is intended all HTV types, subtypes, groups and clades. HIV types include, without limitation, HIV-I and HIV -2. HIV-I groups include, without limitation, Groups M (main) and O (outgroup). Distinct HIV-I subtypes, or "clades," within Group M include, without limitation, clades A, B, C, D, E, F, G, H, I, J and K. Clades of HIV are described, for example, in Coffin, et al._y Retroviruses, Cold Spring Harbor Laboratory Press (1997) and in Spira, et ah, J. Antimicrob. Chemother. 51:229 (2003).

[0021] As used herein, "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to a subject. Administration is by any route including parenteral, and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0022] The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e. C_\s means one to five carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, and the like. The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "cycloalkyl" refers to hydrocarbon rings having the indicated number of ring atoms (e.g., C_3-6 cycloalkyl) and being fully saturated or having no more than one double bond between ring vertices.

[0023] The terms "alkoxy," "acyloxy" and the like are used in their conventional sense and refer to those alkyl groups and acyl groups (respectively) that are attached to the remainder of the molecule via an oxygen atom.

[0024] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C₁-₄ haloalkyl" is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like.

[0025] The term "aryl" means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon group which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom or through a carbon atom. Non- limiting examples of aryl groups include phenyl, naphthyl and biphenyl, while non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4- thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2- pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazoIyl, benzopyrazolyl, 5- indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6- qumolyl. If not specifically stated, substituents for each of the^" above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. [0026] The above terms (e.g., "aryl" and "heteroaryl"), in some embodiments, will include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. For brevity, the terms aryl and heteroaryl will refer to substituted or unsubstituted versions as provided below.

[0027] Substituents for the aryl and heteroaryl groups are varied and are generally selected from: -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO₂, -CO₂R', -CONR'R", -C(O)R', -OC(O)NR'R", -NR"C(O)R\ -NR"C(O)₂R', ,-NR'-C(O)NR"R"% -NH-C(NH₂)=NH, -NR'C(NH₂)=NH, -NH-C(NH₂)=NR\ -S(O)R', -S(O)₂R', -S(O)₂NR³R", -NR⁵S(O)₂R", -N₃, perfluoro(Ci -C₄)alkoxy, and perfluoro(Ci-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R" and R'" are independently selected from hydrogen, C_1-8 alkyl, C_3-6 cycloalkyl, C₂.g alkenyl, C2-8 alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-Ci-₄ alkyl, and unsubstituted aryloxy-Ci-₄ alkyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkyl ene tether of from 1-4 carbon atoms.

[0028] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_r-B-, wherein A and B are independently -CH₂-, -O-, -NH-, -S-, -S(O)-, -S(O)₂-, -S(O)₂NR'- or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond.

[0029] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). [0030] The term "pharmaceutically acceptable salts" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically-acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like. Salts derived from pharmaceutically-acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally- occuring amines and the like, such as arginine, betaine, caffeine, choline, N₅N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylarninoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethyl amine, tripropylamine, tromethamine and the like. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0031] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0032] In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. [0033] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0034] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (' C). AU isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. General

[0035] The present invention derives from the surprising discovery that a small molecule inhibitor of HIV-I was able to directly inactivate HIV-I in the absence of a cellular target. Compound 2 is active against X4, R5 and dual tropic laboratory-adapted and primary strains of HIV-L This compound also binds to the HIV-I envelope glycoprotein, and competition studies map the Compound 2 binding at or near the V3 loop of gpl20. Binding to this site interferes with the sCD4 interaction. With its ability to disable the virus particle, Compound 2 represents a new class of HIV entry inhibitors that can be used as a strategy in the prevention of HW-l/AIDS. Additionally, the compound can be used as part of a combination therapy directed to both the prevention of viral cell entry and the inhibition of, for example, integrase.

Embodiments of the Invention

[0036] In view of the above, the present invention provides in one aspect, a method for inhibiting viral entry into cells. In this method cells are contacted with a viral entry- inhibiting amount of a compound having the formula:

wherein each Ar is an independently selected trihydroxyphenyl group; the double bonds adjacent to the sulfone groups are each independently in either a cis or trans orientation; and pharmaceutically acceptable salts thereof. In one group of preferred embodiments, the compound has the formula:

Compound 2.

[0037] In another embodiment the viral entry is HIV viral entry. In still another embodiment, the compound is provided in a topical formulation. In yet another embodiment, the compound is provided in a topical gel, cream or ointment formulation.

[0038] In some embodiments, the compound is administered to a subject with a second agent selected from an HIV-IN inhibitor, and HIV-RT inhibitor, a second viral entry inhibitor and an HlV-protease inhibitor. In some embodiments, the second agent is a non-nucleoside reverse transcriptase inhibitor. In other embodiments, the second agent is Fuseon^®, a viral entry inhibitor. In still other embodiments, the two agents are administered sequentially. In other embodiments, the two agents are administered simultaneously.

[0039] In another aspect, the present invention provides a method of treating or inhibiting a HlV-viral infection, the method comprising administering to a subject in need of or at risk of such infection, a composition comprising a viral entry inhibiting amount of a compound of the formula:

in admixture with an HIV-integrase inhibiting amount of a compound of the formula

wherein the dashed line indicates an optional double bond and each Ar¹ is a substituted or unsubstituted phenyl, pyridyl or indolyl ring and is other than 3,4,5-trihydroxyphenyl. Alternatively, the compounds can be administered separately and sequentially. In one group of embodiments, each of the Ar¹ groups is a substituted or unsubstituted phenyl, and the substituted phenyl is a phenyl ring having from 1 to 5 substituents independently selected from the group consisting of hydroxy, C-₁-₅ alkoxy, C₁.₅ acyloxy, halogen, nitro, CO₂H, SO₃H, P(O)(OH)₂, OSO3H and OP(O)(OH)₂, or wherein two substituents on adjacent carbon atoms are combined to form a fused methylenedioxy or ethylenedioxy ring, and salts and esters thereof. In still another group of embodiments, each of the Ar¹ groups is selected from:

[0040] In yet another aspect, the present invention provides a pharmaceutical composition comprising an HIV viral entry inhibiting amount of a compound having the formula:

wherein each Ar is an independently selected trihydroxyphenyl group; the double bonds adjacent to the sulfone groups are each independently in either a cis or trans orientation; or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable excipient. Preferably, the composition is formulated for topical administration.

[0041] In one particularly preferred embodiment, the compound is compound 2. The topical compositions are preferably formulated as a topical gel, cream or ointment, but can also be provided as a paste, foam or spray. In a related group of embodiments, the pharmaceutical composition further comprises a compound of the formula:

wherein the dashed line indicates an optional double bond and each Ar¹ is a substituted or unsubstituted phenyl, pyridyl or indolyl ring and is other than 3,4,5-trihydroxyphenyl. Still further preferred are those compositions wherein each of the Ar¹ groups is a substituted or unsubstituted phenyl, and the substituted phenyl is a phenyl ring having from 1 to 5 substituents independently selected from the group consisting of hydroxy, C₁-₅ alkoxy, C₁-₅ acyloxy, halogen, nitro, CO₂H, SO₃H, P(O)(OH)₂, OSO₃H and OP(O)(OH)₂, or wherein two substituents on adjacent carbon atoms are combined to form a fused methylenedioxy or ethylenedioxy ring, and salts and esters thereof. Particularly preferred are those compositions in which the Ar¹ groups are independently selected from:

General Synthetic Methods

[0042] A unique disulfone/diphosphonate reagent (3) was employed in the synthesis of the target compounds (Scheme 1, see Hadd, et al., Tetrahedron Lett., 2001, 42, 5137-5140). Combining 3 with the appropriate aromatic aldehyde in a Horner-Emmons-Wadsworth (HEW) reaction using either LiCtfBu or Hunig's base under Masamune/Roush conditions (see Blanchette, et al., Tetrahedron Lett., 1984, 25, 2183-2186) in dry THF gave the desired diolefin in good yield. Protection of phenolic groups with an appropriate protecting group such as acetate, or a methoxymethyl ether is required for coupling efficiency. After the condensation, saturated analogs are easily prepared by reducing the parent diolefin with 0 hydrogen and palladium catalyst at elevated pressure using a Parr apparatus or using super- hydride^® (see Jones, et al., Tetrahedron, 1986, 42, 6519-6534). Table 1 lists the compounds synthesized and the aldehydes reacted according to Scheme 1.

Scheme 1

5

Table 1

Geminal Disulfones Prepared by Scheme 1

Methods of treating an individual

[0043] In another aspect, the invention provides methods of treating an HIV infection, methods of decreasing the frequency of transmission of an HTV infection, and methods of inhibiting HIV activity in a host, the methods comprising administering to a subject in need thereof an effective amount of one or more of the compounds described herein that inhibit HIV activity.

[0044] In a further aspect, the invention provides methods of preventing an HIV infection, methods of preventing transmission of an HIV infection, and methods of obliterating HIV activity in a host, the methods comprising administering to a subject in need thereof an effective amount of one or more of the compounds described herein.

[0045] Selected compounds and compositions of the present invention are particularly suited for inhibiting or preventing HIV activity and for decreasing the frequency of or preventing HIV transmission of one or more HIV types and/or subtypes (clades), HIV mutants and HIV variants, and especially those that are unresponsive to currently administered anti-HIV therapies, for instance, currently used HAART therapies. Preferred compounds can inhibit or prevent the HIV activity or transmission of at least two, three, four, five, six or more HIV subtypes or clades. Preferred compounds are HIV virucides.

[0046] In some embodiments, the compounds are administered therapeutically to an HIV infected individual. In some embodiments, the compounds are administered prophylactically to an uninfected individual.

[0047] The compounds are administered to a subject through any route of administration that allows contact with an HIV virion, and particularly with an HIV envelope protein. Usually the compounds are formulated for oral administration, but can also be administered parenterally, as appropriate. For instance, the compounds can be administered by injection (intraveneously, intramuscularly, subcutaneously, intrathecally), or given transdermally, intraocularly, as an inhalant (pulmonary delivery) or intranasally. Usually, the compounds are administered orally, intravenously or topically.

[0048] In one preferred embodiment, the compounds are administered topically. Accordingly, the invention further provides for a method for prophylactically or therapeutically decreasing the frequency of or preventing the transmission of an HIV infection, the method comprising topically administering to an individual a pharmaceutical composition comprising an effective amount of one or more of the compounds of the present invention. Compounds formulated in topical pharmaceutical compositions can be prophylactically or therapeutically applied to an individual's skin or mucous membranes to decrease the frequency of or prevent the transmission of HIV infection. The topical composition is preferably introduced into the vagina, at about the time of, and preferably prior to, sexual intercourse, but may also be administered to other topically accessible skin or mucous membrane. Topical compositions can also be administered to the penis, the rectum or the mouth of an individual. The manner of administration is preferably designed to obtain direct contact of the compositions of the invention with an HIV virion. Preferably, the compounds administered for topical delivery are virucidal.

[0049] An efficacious or effective amount of one or more compounds is determined by applying methods known to those in the art, generally by first administering a low dose or small amount of compound, and then incrementally increasing the administered dose until a desired effect of inhibited HIV activity is observed in the treated subject, with minimal or no toxic side effects. Applicable methods for determining an appropriate dose and dosing schedule for administration of one or more of the compounds of the present invention are described, for example, in Goodman and Gilman 's The Pharmacological Basis of Therapeutics, 10^th Ed., Hardman, Limbird and Goodman-Gilman, Eds., McGraw-Hill (2001), and in Remington: The Science and Practice of Pharmacy, 21^st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2003). Further guidance is provided in Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^th Ed., by Ansel, Allen and Popovich, Lippencott Williams & Wilkins (2000).

[0050] A desired effect of inhibited HIV activity in a host can be measured in any of a number of ways known to those in the art. Typically, changes in HIV activity in a host are observed by measuring numbers of CD4⁺ T cells (CD4⁺ counts), HIV RNA plasma levels, usually from infected cells, such as CD4⁺ T cells, before and after treatment with a compound. Usually several HIV activity measurements are taken at designated time periods subsequent to commencing administration of the compounds, for instance, bi-weekly, weekly, bi-monthly, monthly, every 2^nd or 3^rd month, semi-annually, annually, as is appropriate. Human clinical trials of treatments against HIV activity described by Lawrence, et al. N. Engl. J. Med. 349:827 (2003); Squires, et al., Ann. Intern. Med. 139:313 (2003); Abrams, et al., Ann. Intern. Med. 139:258 (2003); Havlir, et al., J. Virol. 77: 11212 (2003) are instructive. Preferred compounds decrease HIV activity in a host, for instance, by the measured indicators of increasing CD4⁺ counts or decreasing HIV RNA levels, by at least 5- 10%, more preferably by at least 15%, 20%, 25% or 30%, and most preferably by at least 35%, 40%, 45%, 50% or more. Ϊ0051] In certain embodiments the compounds are administered to enhance the efficacy of chemotherapeutics currently administered to HIV infected individuals. For instance, the compounds can be administered in combination with one or more HIV reverse transcriptase inhibitors and/or HIV protease inhibitors.

Pharmaceutical Compositions [0052] The invention also provides for pharmaceutical compositions comprising the compounds of the present invention. Generally, the pharmaceutical compositions of the present invention comprise one or more compounds that inhibit HIV activity, as described herein, or pharmaceutically acceptable salts thereof, together with one or more pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical compositions are prepared according to methods known in the art based on the desired route of administration (e.g., oral, intravenous, intramuscular, subcutaneous, intravaginal, intrarectal, intranasal). For instance, depending on the intended route of administration, the pharmaceutical compositions can be formulated as, for example, a liquid, gel, semi-solid, solid, cream or ointment. The compositions can be aqueous, oil-based_Jvemulsified or dry (e.g., a compressed powder). In certain embodiments, the compound pharmaceutical compositions are prepared in a controlled and/or extended-release formulation (see, for example, U.S. Patent Nos. 6,235,712; 6,187,330; 6,180,608; 6,159,490 and 6,068,850, each of which is hereby incorporated herein by reference). In certain embodiments, the compounds are encapsulated for delivery. Preferred pharmaceutical compositions allow for delivery of an efficacious amount of the compounds to HIV virion repository sites in a host, and contact of the compound with an HIV virion. General principles applicable for designing pharmaceutical compositions comprising compounds are found, for example, in Goodman and Gilman 's The Pharmacological Basis of Therapeutics, supra; Remington: The Science and Practice of Pharmacy, supra. Exemplified pharmaceutical compositions of applicable for delivery of the peptide and peptide analogs of the present invention are described in U.S. Patent Nos. 6,565,879; 6,541,606; 6,506,730; 6,387,406; 6,346,242; and U.S. Patent Publication Nos. 2003/0171296 and 2003/0017203, each of which is hereby incorporated herein by reference.

[0053] In one embodiment, the compounds are prepared in pharmaceutical compositions formulated for oral administration. Formulations suitable for oral administration can consist of liquid solutions, such as an effective amount of one or more of the compounds dissolved in diluents, such as water, saline, or fruit juice; capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solid, granules or freeze-dried cells; solutions or suspensions in an aqueous liquid; and oil-in-water emulsions or water-in-oil emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Suitable formulations for oral delivery can also be incorporated into synthetic and natural polymeric microspheres, or other means to protect the agents of the present invention from degradation within the gastrointestinal tract {see, for example, Wallace et ah, Science 260, 912-915, 1993).

[0054] In another embodiment, the compounds are prepared in pharmaceutical compositions formulated for intravenous delivery. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0055] In one embodiment, the compounds are prepared in pharmaceutical compositions formulated for topical administration, for instance, in a cream, a paste, a gel, a foam, an ointment, a spray, a lubricant, an emulsion or suspension. In a preferred embodiment, the pharmaceutical compositions formulated for topical administration comprise one or more compounds. In a preferred embodiment, the pharmaceutical compositions formulated for topical administration comprise one or more virucidal compounds that decrease the frequency or prevent the transmission of an HIV virus from a first infected individual to a second individual. Topical microbicidal preparations suitable for formulating pharmaceutical compositions comprising the compounds of the present invention are described in Turpin, Expert Opin. Investig. Drugs 11:1077 (2002); Garg, et al. , AIDS Patient Care Stds 17:17 (2003); Ketas, et al., AIDS Res Hum Retroviruses 19:177(2003); in U.S. Patent Nos. 6,267,985; 6,248,363 and 5,747,058, and in U.S. Patent Publication No. 2003/0049320, each of which is hereby incorporated herein by reference. Additional topical microbicidal formulations that can find use in preparing pharmaceutical compositions for the topical delivery of the compounds are described in U.S. Patent Nos. 6,635,242; 6,596,763;

6,566,095; 6,500,460; 6,428,790; 6,420,336; 6,376,504; 6,350,784; 6,165,493, each of which is hereby incorporated herein by reference.

[0056] In topical formulations, usually the compounds are included in about 0.1, 0.2, 0.5, 1.0 or 2.0 wt %, but can be included in as much as 5, 10, 15 or 20 wt % of the total formulation, or more. The compounds are formulated with one or more pharmaceutically acceptable carriers. For topical applications, the pharmaceutically acceptable carrier may additionally comprise organic solvents, emulsifϊers, gelling agents, moisturizers, stabilizers, other surfactants, wetting agents, preservatives, time release agents, and minor amounts of humectants, sequestering agents, dyes, perfumes, and other components commonly employed in pharmaceutical compositions for topical administration. Solid dosage forms for topical administration include suppositories, powders, and granules. In solid dosage forms, the compositions may be admixed with at least one inert diluent such as sucrose, lactose, or starch, and may additionally comprise lubricating agents, buffering agents and other components well known to those skilled in the art. [0057] Compound formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas. Similarly, the active ingredient can be combined with a lubricant as a coating on a condom. Indeed, preferably, the active ingredient is applied to any contraceptive device, including, but not limited to, a condom, a diaphragm, a cervical cap, a vaginal ring and a sponge. Formulations for rectal administration can be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. Other articles and delivery systems of this type will be readily apparent to those skilled in the art. EXAMPLES

[0058] General Procedures: Elemental analyses were obtained from Desert Analytics Laboratory, Tucson, AZ, USA. All materials were obtained from commercial sources and used without additional purification. The aromatic aldehydes used were bought from Aldrich Chemical Company, Inc. The THF used for reaction was 99.9% anhydrous, inhibitor free in sureseal bottles also obtained from Aldrich Chemical Company, Inc. All glassware for reactions under anhydrous conditions was flame-dried prior to use. Flash chromatography was performed on Silica Gel Geduran (40-63 um) from Merck and using EM Science ACS grade solvents. For TLC, Silica Gel 60 F₂₅₄ plates from Merck were used with detection by UV light and/or iodine chamber. ¹H NMR and ³C NMR spectra were recorded on Bruker DRX-500, Varian Mercury-300 MHz, or Varian Inova-400 MHz spectrometers at 25^°C. Chemical shifts in ppm were referenced to CDCl₃ (7.26 ppm, 77.16 ppm), DMSO-d₆ (2.50 ppm, 39.52 ppm), Acetone-dβ (2.05 ppm, 29.84 ppm) as internal standards. IR data were recorded on a Galaxy series FT-IR 3000 instrument at 25 ⁰C. Melting points were determined on a Fisher- Johns melting point apparatus.

Example 1 - Synthesis

[0059] General Procedure A: In a flame-dried flask, 1.0 eq of the disulfone reagent (3), 3.0 eq of aldehyde, and 3.3 eq of LiBr were dissolved in 4 mL of dry THF. Once in solution, 3.3 eq of Hunig's base was added. The reaction stirred overnight before being quenched by the addition of 5% HCl until pH=3-4 was attained. The solution was partitioned between ethyl acetate (80 mL) and water (50 mL) and extracted three times (50 mL). The organic phase was collected and dried over sodium sulfate. The solvent was evaporated to yield a solid material, which was purified as indicated for the individual compounds. [0060] Bis (*rα«s-β-4-carboxymethyl-styrenesulfonyl)methane (4): To a solution of 3

(300 mg, 0.6 mmol) in THF (2.5 mL) was added LiBr (156 mg, 1.8 mmol) followed by DIEA (313 μL, 1.8 mmol). Methyl 4-formylbenzoate (246 mg, 1.5 mmol) was then added and the reaction let sit for 2.5 h. Then approximately 200 μL of acetic acid was added followed by water and ethyl acetate. Hexanes is added and then whole solution is filtered and washed with water and ether to give 241 mg (86% yield) of the title compound. ¹H NMR (DMSO- d₆, 600 MHz) 3.86 (s,6H), 5.63 (s, 2H), 7.51 (d, 2H, J= 15.6 Hz), 7.62 (d, 2H, J= 15.6 Hz), 7.77 (d, 4H, J= 8.2 Hz), 7.92 (d, 4H, J= 8.2 Hz). ¹³C NMR (DMSO-^, 150 MHz) 52.33, 71.28, 128.64, 129.13, 129.57, 131.55, 136.33, 143.31, 165.48. HRFABMS (-) calc. C₂₁Hi₉O₈S₂ (M-H): 463.0521, Found: 463.0527 (M-H). Anal. Calcd. C₂IH₂₀O₈S₂: C, 54.30; H₅ 4.34. Found: C, 53.52; H, 4.79.

[0061] bis(.rαn,s-β-4-carboxy-styrenesulf<>nyl)methane (5) : To a solution of 4 in DMSO (1 mL) was added 1 mL of 1 M NaOH and heated with a heat gun for about 1 min. then concentrated HCl was added until the solution was acidic and then filtered and wash with water to give 54 mg (73%) of the title compound after drying. ¹H NMR (DMSO-d₆, 600 MHz) 5.62 (s, 2H), 7.51 (d, 2H, J= 15.6 Hz) 7.64 (d, 2H₅ J= 15.6 Hz), 7.79 (d, 2H, J= 8.3 Hz) 7.96 (d, 2H, J= 8.3 Hz). ¹³C NMR (DMSO-J₁₅, 150 MHz) 74.41, 128.40, 129.01, 129.53, 129.72, 129.77, 129.94, 132.91, 135.97, 143.39, 166.51. FABMS (+) calc. Ci₉Hi₆O₈S₂ (M+Na): 459.03, Found: 460.2 (M+Na). Anal. (Ci₉H₁₆O₈S₂) C, H.

[0062] Bis (frαrts-β-2,4,5-trimethoxy-styreiiesulfonyl)methane (6): To a solution of 3 (203 mg, 0.41 mmol) in THF (1 mL) was added 1.21 mL of 1 M potassium t-butoxide in THF followed by 2,4,5-trimethoxybenzaldehyde (239 mg, 1.2 mmol) and the reaction let sit overnight. Then approximately 2 mL of 0.1 M HCl was added followed by ethyl acetate.

The whole solution is filtered and washed with water and ether to give 110 mg (51% yield) of the title compound. ¹H NMR (DMSO-^₅ 600 MHz) 3.69 (s, 6H), 3.82 (s, 6H), 3.84 (s, 6H), 5.35 (s, 2H), 6.65 (s, 2H), 7.11 (s, 2H), 7.18 (d, 2H, J= 15.5 Hz), 7.59 (d, 2H, J= 15.5 Hz). ^!3C NMR (DMSO-4 150 MHz) 55.82, 55.95, 56.20, 97.15, 111.26, 112.06, 122.67, 139.47, 142.79, 153.20, 154.25, 219.92. HRFABMS (+) calc. C₂₃H₂₉O₁₀S₂ (M+H): 529.1124, Found: 529.1190 (M+H). Anal. (C₂₃H₂₈O₁₀S₂) C, H.

[0063] Bis (2-(3,4-dihydroxyphenyl)-ethylsuIfonyl)methane (7): A solution of 56 mg of compound 1 and 7 mg of palladium on carbon (10%) were dissolved in 5 mL of ethyl acetate and put on the parr shaker at a pressure of 57 psi. The mixture stayed on the parr shaker for 4 days, monitoring by TLC with fresh catalyst added daily. At the end of the 4 days, the mixture was filtered through a pad of celite^®. The crude oil recovered was subjected to column chromatography (30% acetone/69%toluene/l% acetic acid; Rf=0.16) to give 20 mg (39%) of the title compound. ¹H NMR (acetone-J_tf, 300 MHz) δ 3.01 (t, 4H, J= 8.4 Hz), 3.66 (t, 4H, J= 8.4 Hz), 4.89 (s, 2H), 6.64 (dod, 2H, J= 8.1 Hz, J= 1.8 Hz), 6.78 (d, 2H, J= 8.1 Hz)₅ 6.78 (s, 2H), 7.89 (brs, 4H). ¹³C NMR (acetone-^, 75 MHz) δ 27.68, 56.16, 68.77, 116.33, 116.41, 120.71, 130.03, 144.85, 146.10. FT-IR (film): v 3433 (O-H), 2916 (C-H), 1657 (arom. C=C), 1321, 1124 (SO₂). Anal Calcd for Ci₇H₂₀O₈S₂: C, 49.03; H₅ 4.84. Found: C, 50.62; H, 4.84.

[0064] Bis (tfr««s-β-3,4-methoxy-6-nitro-styrenesulfonyl)methane (8): To a solution of 3 (200 mg, 0.4 mmol) in THF (4 mL) was added 1.2 mL of 1 M lithium /-butoxide in THF followed by 3,4-dimethoxy-6-nitrobenzaldehyde (80%) (300 mg, 1.2 mmol) and the reaction let sit overnight. The solution was filtered and washed with 0.1 N HCl, THF and ether to give 93 mg of the title compound. ¹H ISIMR (OMSO-d₆, 600 MHz) 3.83 (s, 6H), 3.87 (s, 6H), 5.53 (s, 2H), 7.14 (s, 4H), 7.46 (d, 2H, J= 14.8 Hz) 7.88 (d, 2H, J= 14.8 Hz). ¹³C NMR (DMSOA 150 MHz) 56.24, 56.38, 107.77, 110.06, 128.60, 140.65, 141.59, 150.29, 152.41. HRFABMS (-) calc. C₂IH₂₁Oi₂S₂N₂ (M-H): 557.0536, Found: 557.0539 (M- H). Anal. (C₂₁H₂₂Oi₂S₂N₂) C, H, N.

[0065] Bis (*rαns-2-Bromo-6-(2-methanesulfonyl-vinyl)-pyridine) (9): The disulfone reagent (3) (182 mg, 0.36 mmol) and 6-bromo-2-pyridine-carboxaldehyde (223 mg, 1.20 mmol) were reacted according to general procedure A. After removal of the solvent, the crude product was subjected to column chromatography (hexanes/ethyl acetate 1:1, R_f=0.32) to yield 112 mg (61%) of the title compound. ¹H NMR (Acetone-^, 400 MHz) δ 5.43 (s, 2H), 7.60 (d, 2H, J=15.2 Hz), 7.63-7.78 (m, 6H), 7.87 (d, 2H, J=15.2 Hz). ¹³C NMR (Acetone-^, 100 MHz) δ 72.47, 126.21, 130.94, 131.78, 141.18, 143.03, 143.15, 152.63. FT-IR (KBr): υ 3070, 1572, 1432 (C=C str), 1331, 1119 (SO₂ str), 985 (trans C=C str). Anal (C₁₅H₁₂Br₂N₂O₄S₂) C₅ H₅ N.

[0066] Bis (rr««s-β-6-bromo-3,4-dimethoxy-styrenesuIfonyl)methane (10): To a solution of 3 (300 mg, 0.6 mmol) in THF (3 mL) was added LiBr (156 mg, 1.8 mmol) followed by DIEA (420 mL, 2.4 mmol). 6-bromoveratraldehyde (441 mg, 1.8 mmol) was then added and the reaction let sit overnight. Then approximately 100 μL of acetic acid was added followed by water and ethyl acetate. The ethyl acetate layer was dried and ether was added and the solution let sit in the freezer overnight to produce a solid which is filtered to give 278 mg (75%) of the title compound. ¹H NMR (DMSO-d₆, 600 MHz) 3.75 (s, 6H)₅ 3.81 (s, 6H), 5.49 (s, 2H)₅ 7.16 (s, 2H), 7.25 (s, 2H), 7.40 (d, 2H, J= 15.3 Hz), 7.65 (d, 2H, J= 15.3 Hz). ¹³C NMR (Acetone-^, 150 MHz) 55.75, 56.08, 71.64, 110.32, 115.52, 117.56, 122.98, 126.19, 142.69, 148.45, 152.14. HRFABMS (+) calc. C₂IH₂₂O₈S₂Br₂: 625.9103, Found: 625.9131. Anal. Calcd. for C₂₁H₂₂O₈S₂Br₂ (1.1 H₂O) : C, 39.03; H, 3.78. Found: C, 38.72; H, 3.84. [0067] Bis(i>α«s-β-l-indole-5-styrenesulfonyl)methane (11): l-(t-butyloxycarbonyl)- indole-5-carboxaldehyde (202 mg, 0.82 ramol) and the disulfone reagent 3 (138 mg, 0.27 mmol) were reacted according to General Procedure A. The resulting crude solid was subjected to column chromatography (30% ethyl acetate/70% hexanes, R_f=0.22) to yield 169 mg (57%) of the desired purified product. ¹H NMR (CDCl₃, 400 MHz) 1.67 (s, 18H), 4.73 (s, 2H), 6.56 (d, 2H, 7=3.6 Hz), 7.20 (d, 2H, J=15.6 Hz), 7.48 (dd, 2H, 7=8.8 Hz, 7=2.0 Hz), 7.62 (d, 2H, 7=3.6 Hz), 7.66 (d, 2H, ./=1.6 Hz), 7.76 (d, 2H, 7=15.6 Hz), 8.14 (d, 2H, 7=8.8 Hz). ¹³C NMR (CDCl₃, 100 MHz) 28.22, 74.36, 84.56, 107.50, 115.90, 122.55, 122.95, 124.77, 126.35, 127.49, 131.07, 137.21, 147.74, 149.31.

[0068] The protected diindole (94 mg, 0.15 mmol) was then dissolved in 3 mL of dichloromethane and 0.25 mL of trifluoroacetic acid was added. This stirred for 3h at which time the solvent was evaporated and then coevaporated with toluene. 2 mL of water was then added and the solid was filtered off. The filtered solid was resuspended in 3 mL of water and this was lyophilized to give 56 mg (88%) of the desired deprotected product. ¹H NMR (DMSOrf_Λ 400 MHz) 5.45 (s, 2H), 6.50 (d, 2H, J=3.2 Hz), 7.22 (d, 2H, J=I 5.6 Hz), 7.43 (m, 6H), 7.63 (d, 2H, 7=15.6 Hz), 7.89, (s, 2H). ¹³C NMR (DMSO-d₆₎ 125 MHz) 72.37, 102.31, 112.22, 121.18, 121.97, 123.15, 123.46, 127.03, 127.86, 137.67, 146.79. FT-IR (KBr): v 2921 (C-H), 1326, 1144 (SO₂), 1199 (C-N), 974 (trans C=C). Anal. Calcd for C₂IHi₈N₂O₄S₂: C, 59.14; H, 4.25. Found: C, 58.30; H, 4.28.

[0069] Bis (*rø«s-/^3,4,5-trifluoro-styrenesulfonyI)methaiie (12): The disulfone reagent (3) (219 mg, 0.44 mmol) was combined with 3,4,5-trifluorobenzaldehyde (0.16 mL, 1.44 mmol) according to general method A. After removal of solvent, the crude material was subjected to column chromatography (8% acetone/92% toluene, R_f=O.32) to yield 149 mg (74%) of the title compound. ¹H NMR (DMSO-d₆, 500 MHz) δ 5.56 (s, 2H), 7.48 (d, 2H, 7=15.5 Hz), 7.51 (d, 2H, 7=15.5 Hz), 7.69 (m, 4H). ¹³C NMR (DMSO-d₆, 125 MHz) δ 71.98, 114.48 (d, J=16.5 Hz), 129.43, 129.71, 141.04 (d, 7=253.5 Hz), 142.83, 151.03 (dd, J=7.75 Hz, J=247.5 Hz). FT-IR (KBr): υ 3070, 1616, 1535 (C=C str), 1442 (-CH₂- str), 1336, 1149 (SO₂ str), 1247 (Ph-F str), 974 {trans C=C str). Anal (C₁₇Hi₀F₆O₄S₂): C, H.

[0070] Bis (2-(3,4,5-trifluorophenyl)-ethyIsuIfonyl)methane (13): The vinyl trifluoro derivative, 12, (106 mg, 0.23 mmol) was dissolved in dry THF and cooled to -78 ⁰C. To this was added 0.58 mL (0.58 mmol) of IM LiEt₃BH (Super-Hydride) in THF. This stirred overnight and was allowed to come to room temperature. The reaction was quenched by pouring into 50 mL of IM HCl and then extracted into EtOAc (3 x 50 mL). The combined organic fractions were then washed with water and brine. The solvent was evaporated and chromatographed in 30% EtOAc/hexanes (R_f=O.16) to give 78 mg (73%) of the title compound. ¹H NMR (DMSO-rf_& 400 MHz) δ 3.11 (4H, dt, ./=8.0 Hz, J=2.4 Hz), 3.71 (4H, dt, J=8.0 Hz, J=2.4 Hz), 5.55 (2H, s), 7.31 (4H, dd, J=8.0 Hz, J=2.4 Hz). ¹³C NMR (DMSO-^, 100 MHz) δ 26.12, 53.99, 67.67, 113.35 (dd, J_C-_F2=21.2 Hz, J_C-_F3=53 HZ), 135.42 (m), 137.60 (dt, J_C-_F=245.9 HZ, J_C._F2=16.0 HZ), 150.08 (ddd, J_c-F=245.6 Hz, J_c.

Hz). HRMS calc. Ci₇Hi₄F₆O₄S₂ (M+NH₄): 478.05814, Found:

478.058410. Anal Calcd. for Ci₇Hi₄F₆O₄S₂: C, 44.35; H, 3.06. Found: C, 45.18; H, 2.96.

[0071] Bis (frø«s-(2-MethanesuIfonyl-vinyI)-benzene) (14): The disulfone reagent (3) (103 mg, 0.21 mmol) and benzaldehyde (0.07 mL, 0.68 mmol) were combined according to general procedure A. After removal of solvent, the crude material was subjected to column chromatography (40% ethyl acetate/60% hexanes, R_f=0.43) to yield 65 mg (88%) of the title product. ¹H NMR (CDCl₃, 400 MHz) δ 4.68 (s, 2H), 7.21 (d, 2H, J=15.6 Hz), 7.38-7.54 (m, 10H), 7.67 (d, 2H, J= 15.6 Hz). ¹³C NMR (CDCl₃, 100 MHz) δ 74.11, 124.38, 129.18, 129.31, 131.77, 132.09, 147.02. FT-IR (film): υ 3060, 1612, 1448 (C=C), 1323, 1124 (SO₂ str), 976 (trans C=C str). Anal Calcd for C_nH₁₆O₄S₂: C, 58.60; H, 4.63. Found: C, 58.21; H, 4.63.

[0072] Bis (fm«5-(2-MethanesuIfonyl-ethyI)-benzene) (15): Compound 14 (64 mg, 0.18 mmol) was dissolved in 3 mL of ethyl acetate. To this was added 6 mg of palladium (10 wt. %) on carbon powder (wet) and allowed to react on the Parr shaker overnight at 60 psi. This mixture was filtered over a pad of Celite^® 503 and subjected to column chromatography (ethyl acetate/hexanes 1 :2, R_f=0.40) to yield 6 mg (10%) after spilling of some title product. ¹H NMR (CDCl₃, 400 MHz) δ 3.14 (t, 4H, J=8 Hz), 3.66 (t, 4H, J=8 Hz), 3.98 (s, 2H), 7.22- 7.35 (m, 10H). ¹³C NMR (CDCl₃, 100 MHz) δ 28.43, 55.24, 68.33, 127.62, 128.84, 129.24, 136.47. FT-IR (film): υ 3012 (C=C str), 1456 (-CH₂- str), 1311, 1128 (SO₂ str). Anal. (C₁₇H₂₀O₄S₂) C, H.

[0073] Bis (^rα«s-l-Bromo-5-(2-methanesulfonyl-vinyl)-2,3-dimethoxy-benzene) (16):

The disulfone reagent (3) (138 mg, 0.28 mmol) and 5-bromoveratraldehyde (223 mg, 0.91 mmol) were used according to general method A. Following removal of solvent, 5 mL of ethyl acetate was added from which a white solid precipitated, this was filtered off and rinsed with cold ethyl acetate to give 93 mg (53%) of the title compound (mp=238^°C). ¹H NMR (DMSO-J(_J, 400 MHz) δ 3.78 (s, 6H), 3.81 (s, 6H), 5.47 (s, 2H)₅ 7.33 (s, 2H), 7.40 (d, 2H, 7=16.0 Hz), 7.46 (d, 2H, J=16.0 Hz), 7.49 (s, 2H). ¹³C NMR (OMSO-d₆, 100 MHz) δ 56.20, 60.23, 71.36, 112.46, 116.96, 125.36, 126.47, 129.40, 143.65, 147.90, 153.33. FT-IR (KBr): υ 3064, 1616, 1491 (C=C str), 1468 (-CH₂- str), 1315, 1124 (SO₂ str), 1282, 1047 (Ph-O-C str), 1228 (C-O str), 970 (trans C=C str). Anal. (C₂IH₂₂Br₂O₈S₂) C, H.

[0074] Bis (l,2-benzyloxy-//^<α«-f-5-(2-Ethanesulfonyl-vinyl)-benzene) (17): The disulfone reagent (3) (219 mg, 0.44 mmol) and 3,4-benzyloxybenzaldehyde (419 mg, 1.44 mmol) were reacted according to general method A. After removal of solvent, 4 mL of acetone was added from which a white solid precipitated. This solid was filtered and washed with cold acetone to give 134 mg (40%) of the title compound (mp=194-195^°C). ¹H NMR (OMSO-d₆, 400 MHz) δ 5.07 (s, 4H), 5.14 (s, 4H), 5.42 (s, 2H), 7.05 (d, 2H, J=6.3 Hz), 7.18 (d, 2H, J=I 1.4 Hz), 7.22 (d, 2H, J=I 1.7 Hz), 7.32-7.43 (m, 24H). ¹³C NMR (ΩMSO-d₆, 100 MHz) δ 70.49, 70.65, 72.45, 113.86, 114.23, 124.06, 124.82, 125.65, 127.68-128.60 m, 136.83, 145.30,

148.41, 151.23. FT-IR (KBr): υ 3064, 1597, (C=C str), 1456 (-CH2- str), 1323, 1140 (SO₂ str), 1271, 1022 (Ph-O-C str), 1211 (C-O str), 979 (trans C=C str). Anal. Calcd for C₄₅H₄₀O₈S₂: C, 69.93; H, 5.22. Found: C, 69.44; H, 5.19.

[0075] Bis (l-ben2yloxy-rrαws-2-(2-Methanesulfonyl-vinyl)-benzene) (18): The disulfone reagent (3) (201 mg, 0.40 mmol) and 2-benzyloxybenzaldehyde (282 mg, 1.33 mmol) were used according to general method A. Following removal of solvent, the crude solid was purified by column chromatography (30% ethyl acetate:70% hexanes, Rf=0.36) to yield 108 mg (48%) of the title compound as a foamy solid. ¹H NMR (CDCl₃, 300 MHz) δ 4.56 (s, 2H), 5.17 (s, 4H), 6.98 (d, 2H, J=8.1 Hz), 7.32-7.49 (m, 18H), 7.93 (d, 2H, J=15.6Hz). ¹³C NMR (CDCl₃, 100 MHz) δ 70.66, 74.18, 112.98, 121.13, 121.28, 125.21, 127.41, 128.32,

128.87, 131.34, 133.28, 136.12, 142.61, 158.25. FT-IR (film): υ 3064, 1599, 1486 (C=C str), 1452 (-CH₂- str), 1317, 1124 (SO₂ str), 1250, 1051 (Ph-O-C str), 1223 (C-O str), 984 {trans C=C str). Anal. (C_3IH₂₈O₆S₂) C, H.

[0076] Bis (l,2,3-benzyloxy-ϊ*rαns-(2-EthaπesulfonyI-vinyl)-benzene) (19): The disulfone reagent (3) (130 mg, 0.26 mmol) and 3,4,5-tribenzyloxybenzaldehyde were used according to general method A. After removal of the solvent, 4 mL of acetone was added to the pale yellow oil and a white solid precipitated. This solid was filtered and washed with cold acetone to give 159 mg (62%) of the title compound (mp=216^°C). ¹H NMR (Acetone-*^, 300 MHz) δ 4.94 (s, 4H), 5.09 (s, SH), 5.50 (s, 2H), 7.18-7.47 (m, 38H). ¹³C NMR (DMSO-J₁₅ ,125 MHz) δ 70.48, 70.78, 74.54, 108.22, 127.72-128.76 (m), 136.82, 136.98, 137.61, 140.01, 145.93, 152.07, 152.53. FT-IR (KBr): υ 3064, 1614, 1504 (C=C str), 1454 (-CH₂- str), 1329, 1122 (SO₂ str), 1248 (Ph-O-C str), 972 (trans C=C str). Anal. (C₅₉H₅₂O_I0S₂) C, H.

[0077] Bis (f/-α«s-l,4-Dichloro-2-(2-methanesulfonyl-vinyl)-benzene) (20): The disulfone reagent (3) (224 mg, 0.45 mmol) and 2,5-dichlorobenzaldehyde (258 mg, 1.47 mmol) were reacted according to general procedure A. After removal of solvent, 142 mg (65%) of the title compound was precipitated out of 4 mL of acetone and filtered (mp=246- 247^°C). ¹U NMR (DMSO-J_n, 400 MHz) 5.68 (s, 2H), 7.50-7.56 (m, 4H), 7.64 (d, 2H, J=15.6 Hz), 7.69 (d, 2H, J=I 5.6 Hz) δ 7.93 (d, 2H, J=2 Hz). ¹³C NMR (DMSO-J₆, 100 MHz) δ 71.05, 128.41, 130.47, 131.31, 131.71, 132.50, 132.64, 132.85, 138.74. FT-IR (KBr): υ 3068, 1608, 1462 (C=C str), 1340, 1149 (SO₂ str), 1099 (Ph-Cl str), 970 (trans C=C str). Anal. (Ci₇H₁₂Cl₄O₄S₂) C, H.

[0078] Bis (i^Irø»s-6-(2-methanesulfonyl-vinyI)-4-methoxy-benzo[l,3]dioxole) (21): The disulfone reagent (3) (224 mg, 0.44 mmol) and 7-methoxy-benzo[l,3]dioxole-5-carbaldehyde (169 mg, 0.94 mmol) were reacted according to general procedure A. After removal of solvent, 121 mg (54%) of the title compound was precipitated out of dichloromethane and filtered (mp=226-227^°C). ¹H NMR (DMSO-d₆, 400 MHz) δ 3.81 (s, 6H), 5.42 (s, 2H), 6.06 (s, 4H), 6.92 (s, 2H), 6.99 (s, 2H), 7.27 (d, 2H, J=15.6 Hz), 7.38 (d, 2H, J=15.6 Hz). ¹³C NMR (DMSO-J(S, 100 MHz) δ 56.29, 71.63, 102.03, 102.15, 110.54, 124.19, 126.66, 137.67, 143.16, 144.97, 148.77. FT-IR (KBr): υ 3057, 1595, 1448 (C=C str), 1469 (-CH₂- str), 1325, 1119 (SO₂ str), 1227, 1039 (Ph-O-C str), 970 (trans C=C). Anal. (C₂₁H₂₀O₁₀S₂) C, H. [0079] Bis (6-(2-MethanesuIfonyl-vinyl)-2,3-dihydro-benzo[l,4]dioxine) (22): The disulfone reagent (3) (221 mg, 0.44 mmol) and l,4-benzodioxan-6-carboxaldehyde (240 mg, 1.45 mmol) were reacted according to general method A. Following solvent removal, the crude mixture was subjected to column chromatography (8% acetone/92% toluene, Rf=0.26) to yield 86 mg (42%) of the title compound. ¹H NMR (CDCl₃, 500 MHz) δ 4.29 (d_appj 8H, J= 13 Hz), 4.64 (s, 2H), 6.88 (d, 2H, J=8.5 Hz), 7.00 (d, 2H₅ J=15.5 Hz), 7.05 (m, 4H). 7.53 (d, 2H, J=I 5.5 Hz). ¹³C NMR (DMSO-^₅ 75 MHz) δ 63.88, 64.40, 71.74, 117.39, 117.43, 122.81, 123.69, 125.25, 143.37, 144.40, 146.21. FT-IR (film): υ 3060, 1600, 1510 (C-C str), 1325, 1122 (SO₂ str), 1292, 1066 (Ph-O-C str), 976 {trans C=C str). Anal. (C₂IH₂₀O₈S₂) C, H.

[0080] Bis (4-Bromo-2-(2-methanesulfonyl-vinyI)-l-methoxy-benzene) (23): The disulfone reagent (3) (175 mg, 0.35 mmol) and 5-bromo-2-methoxybenzaIdehyde (248 mg, 1.15 mmol) were reacted according to general procedure A. After removal of the solvent, 188 mg (95%) of the title compound was precipitated from 5 mL of dichloromethane (mp=282^°C). ¹H NMR (DMSO-^, 300 MHz) δ 3.82 (s, 6H), 5.50 (s, 2H), 7.02 (d, 2H, J=8.7 Hz), 7.43 (d, 2H, J=15.6 Hz), 7.54 (d, 2H, J=15.6 Hz), 7.57 (dd, 4H, J=8.7 Hz, J=2.4 Hz), 7.79 (d, 2H, J=2.4 Hz). ¹³C NMR (DMS O-d₆, 100 MHz) δ 56.15, 71.23, 112.27, 114.13,

122.24, 127.71, 132.03, 135.31, 138.20; 157.44. FT-IR (KBr): υ 3091, 1614, 1487 (C=C str), 1462 (-CH₂- str), 1317, 1124 (SO₂ str), 1257 (Ph-O-C str), 1217 (C-O str), 1012 (Ph-Br str), 976 (trans C=C str). Anal. (Ci₉Hi₈Br₂O₆S₂) C, H.

Elemental Analysis:

Example 2 - Enzyme Assays

[0081] Biological Materials, Chemicals, and Enzymes. AU compounds were dissolved in DMSO and the stock solutions were stored at -20 °C. The γ[³²P]-ATP was purchased from either Amersham Biosciences or ICN. The expression systems for the wild-type IN and soluble mutant 1N^F185KC28OS were generous gifts of Dr. Robert Craigie, Laboratory of Molecular Biology, NEDDK, NIH, Bethesda, MD.

[0082] Preparation of Oligonucleotide Substrates. The oligonucleotides 2 Hop, 5'- GTGTGGAAAATCTCTAGCAGT-S¹ and 21bot, 5¹-ACTGCTAGAGATTTTCCACAC-3^l were purchased from Norris Cancer Center Microsequencing Core Facility (University of Southern California) and purified by UV shadowing on polyacrylamide gel. To analyze the extent of 3'-processing and strand transfer using 5'-end labeled substrates, 2 Hop was 5'-end labeled using T4 polynucleotide kinase (Epicentre, Madison, WI) and γ[³²P]-ATP (Amersham Biosciences or ICN). The kinase was heat-inactivated and 21bot was added in 1.5-molar excess. The mixture was heated at 95 °C, allowed to cool slowly to room temperature, and run through a spin 25 mini-column (USA Scientific) to separate annealed double-stranded oligonucleotide from unincorporated material.

[0083] Integrase Assays. To determine the extent of 3 '-processing and strand transfer, wild-type IN was preincubated at a final concentration of 200 nM with the inhibitor in reaction buffer (50 mM NaCl, 1 mM HEPES, 50 μM EDTA, 50 μM dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl₂, 0.1 mg/ml bovine serum albumin, 10 mM 2-mercaptoethanol,

10% DMSO, and 25 mM MOPS, pH 7.2) at 30 °C for 30 min. Then, 20 nM of the 5'-end ³²P- labeled linear oligonucleotide substrate was added, and incubation was continued for an additional Ih. Reactions were quenched by the addition of an equal volume (16 μl) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue). Aliquots (5 μl) were electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris-borate pH 8.3, 2 mM EDTA, 20% acrylamide, 8M urea). Gels were dried, exposed in a Phosphorlmager cassette, analyzed using a Typhoon 8610 Variable Mode Imager (Amersham Biosciences) and quantitated using ImageQuant 5.2. Percent inhibition (% I) was calculated using the following equation: % I = JOOXfJ - .(D - C)Z(N- C)]

where C, N, and D are the fractions of 21-mer substrate converted to 19-mer (3 '-processing product) or strand transfer products for DNA alone, DNA plus IN, and IN plus drug, respectively. The IC_5O values were determined by plotting the logarithm of drug concentration versus percent inhibition to obtain concentration that produced 50% inhibition. [0084] The compounds in Table 1 were first tested against the purified enzyme to determine if functional groups other than hydroxy, acetoxy, and/or methoxy could elicit activity against either 3 '-processing or strand transfer (Table 2). Compound 5 demonstrated moderate activity against both functions, 11 showed moderate activity against strand transfer only, and 7 displayed relatively potent inhibition of strand transfer and moderate activity against 3 '-processing.

Table 2: HIV-I-In Inhibition Data

Example 3 -Cell-Based Assays

[0085] Focal Infectivity Assay. Drug-susceptibility of HTV-I to inhibitors was determined with a focal infectivity assay (FIA) as previously described (see Murry, et al., J. Virol. 2003, 77, 1120-1130; and Giuffre, et al., Antimicrob. Agents Chemother. 2003, 47, 1756-1759). Immunostaining was performed using the monoclonal antibody 22-6¹³ at a 1/800 dilution. Foci were counted under a dissecting microscope at.30 to IOOX magnification. Data for drug-susceptibility assays were plotted as a percentage of control foci (no drug) versus inhibitor concentrations. The concentrations required to inhibit focus formation by 50% (EC₅0) were obtained from a best-fit line of the linear portions of those plots. EC5₀ values for each drug were determined from at least three separate experiments with six determinations per experiment.

[0086] Cytotoxicity. Toxicity was determined with the Promega CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS assay) using the manufacturer's recommended conditions. Data for cell proliferation assays were plotted as a percentage of control (no drug) versus inhibitor concentrations. The concentrations required to inhibit cell proliferation by 50% (IC₅0) were obtained from a best-fit line of the linear portions of those plots. IC5₀ values for each drug were determined from at least two separate experiments.

[0087] Time-of-Addition. The time of addition studies were performed similarly to the focal infectivity assays except that the virus was incubated with the cells for two hours at 37 °C after which unadsorbed virus was removed from the wells and replaced with fresh virus- free medium. Dextran sulfate, T20, 3TC, and test compounds were used at 10 times their respective EC₅₀ values and were added at seven different time points before or after infection: -1, 0, 2, 4, 6, 10, and 24 hours. The cells were incubated for 4 days at 37 ⁰C when they were fixed, stained and foci counted and plotted against a no drug control.

[0088] Results for antiviral, cytotoxicity and selectivity data are provided in Table 3. Table 3

Antiviral activity, cytotoxicity, and antiviral selectivity data for the IN inhibitors

Table 3 (cont'd.)

Antiviral activity, cytotoxicity, and antiviral selectivity data for the IN inhibitors

^a EC50 values are the mean ± SE from three separate determinations. ^b IC₅₀ values are the average of two experiments. ^c N/A-Cannot be calculated without cytotoxicity data.

[0089] The compounds listed in Table 1 were evaluated in a focal infectivity assay (FIA) to determine anti-HIV activity. The FIA used for HIV-I has recently been described (see Murry, et al., ibid; and Giuffre, et al., ibid). It is similar to the FIA for HTV-I that was originally developed by Chesebro (Chesebro and Wehrly, J. Virol. 1988, 62, 3779-3788), except that indicator cells are HeLa Hl-JC.37 cells. These are HeLa-CD4 cells expressing human CCR5 (these cells naturally express CXCR4, see Leutenegger, et al., AIDS Research and Human Retroviruses 2001, 17, 243-251) and are permissive for infection by T-cell tropic and macrophage tropic isolates of HIV-I. [0090] As can be seen in Table 3, 5 of the 20 new compounds (7, 12, 14, 20, and 21) were found to possess antiviral activity in the low micromolar range. Compound 7 is the hydrogenated analog of 1. The data from the purified enzyme assays of 1 and 7 strongly correlate with antiviral activity: Both assays show that 7 is approximately half as active as 1. Compound 21 is a protected analog of 2, but unlike 2 it is only active in the FIA. Similarly, compounds 12, 14, and 20 do not possess IN activity but are rather potent antiviral compounds. The source of this activity apparently stems from cytoxocity (vide supra).

[0091] Using a standard cell proliferation assay (MTS assay) the cytotoxicity of the active compounds was evaluated in HeLa Hl-JC.37 cells- As can be seen in Table 3, compounds 12, 14, and 20 exhibit highly toxic effects. With 12 and 20 containing electron withdrawing substituents and 14 possessing no substituents, these compounds are possibly reacting with biological molecules via conjugate addition and that resulting toxicity was the source of their antiviral activity.

[0092] To evaluate the possibility of conjugate addition, fully saturated analogs of both 12 and 14 (13 and 15, respectively) were prepared and tested for anti-HIV activity; both of the compounds were found to be inactive. Compounds 13 and 15 were also tested in the MTS assay to see if there was a difference in cytotoxicity between the saturated and unsaturated pairs (Table 3). There was more than a 10-fold difference in toxicity between the saturated and unsaturated compounds implicating the double bond as the source of the observed toxicity. Finally, orientation effects imposed by unsaturation were examined to determine their role in cell death, similar to that observed for the cytotoxic combretastatins where cis oriented analogs possess toxicity and trans orientations are inactive (see Gaukroger, et al., J. Org. Chem., 2001, 66, 8135-8138; Lin, et al., MoI. Pharmacol, 1988, 34, 200-208; and Cushman, et al., J. Med. Chem., 1991, 34, 2579-2588). To distinguish this possibility, 12 and 14 were incubated with glutathione in PBS buffer at 37 ⁰C for four days in an analogous manner as for the FIA. After four days, the mixtures were analyzed by LC/FABMS for peaks corresponding to mono and/or double addition adducts. The mass spectra of 12 and 14 indeed showed peaks corresponding to both mono and double addition products suggesting that the compounds act as promiscuous acceptors of nucleophilic biological molecules and taken together with the previous data, but without intending to be bound by theory, the conjugate addition appears the most likely source of toxicity. [0093] The glutathione addition experiment was also conducted on the most potent disulfone compound identified thus far: the 3,4,5-trihydroxyl compound (2). In this case, neither ion peaks corresponding to the mono nor the double addition adducts were observed in the mass spectrum suggesting the resonance-donating effect of the three phenolic groups makes the double bond less susceptible to 1,4-addition. Although these studies do not discount the possibility of 1,4-addition as the mode of action for the less toxic compounds, it demonstrates that these are at least more selective in their Michael accepting capabilities, i.e. perhaps the aromatic groups serve to advantageously place the vinyl sulfone near a pertinent biological nucleophile. Example 5 -Mechanistic Investigations

[0094] Further mechanistic evaluation of the disulfone-containing analogs was conducted using 3 test compounds: the trihydroxy analog (2), the most potent compound synthesized thus far and through time-of-addition studies shown to inhibit an early stage in the viral replication process; the dihydroxy analog (1), a compound that possibly possesses a dual mode of action (see Figure 2); and the saturated analog, 7.

[0095] Time-of-addition studies were performed to determine how long after infection the addition of a drug can be delayed and still retain its activity. The earlier in the replication cycle the drug inhibits, the shorter amount of time its addition can be delayed and still retain activity. For example, entry inhibitors can be delayed for only a short time, RT inhibitors for 4 h, and protease inhibitors for 18 to 19 h (see Pluymers, et al., Antimicrob Agents

Chermothβr, 2002, 46, 3292-3297). In this study, dextran sulfate (DS), a known entry inhibitor, T20, a fusion inhibitor, 3TC, a reverse transcriptase inhibitor were used as controls. Compounds 1, 2, and 7 were compared to the controls in order to probe where in relation to the known compounds they exerted their activity. The compounds were each used at 10 times their respective EC50 values and were added at 6 different times: -1 , 0, 2, 4, 6, and 10 hours post-infection. The cells were incubated for 4 days after which they were fixed, stained and foci were counted and plotted against a no drug control (see Figure 2).

[0096] From Figure 2, it is observed that all of the compounds tested with the exception of 3TC display a sharp decrease in activity if administered following t=0. 1 was able to strongly suppress replication until the 1O h time point. This behavior is consistent with a dual mode of action: a decrease in activity followed by continued suppression but to a lesser degree followed by another decrease in activity. Compounds such as the controls, DS and T20, and test compound, 2, are all clustered towards the top of the graph indicating inhibition of an early event in viral replication; a dual mode of action for Compound 2, however, cannot be dismissed without further analysis. Compound 7 is rather ambiguous, splitting the early stage and later stage inhibitors.

[0097] A significant loss of activity beyond 2 h seems to implicate inhibition of an event such as entry/fusion as a primary mode of action. This process, while often considered one step, is a complex process including gpl20 binding to CD4, conformational changes in gpl20 exposing a coreceptor binding site, binding of gpl20 to the coreceptor, conformational changes exposing the fusogenic peptide, gp41, and fusion of the viral and cellular membranes (Miller and Hazuda, Drug Resistance Updates, 2004, 7, 89-95). To further dissect where in the entry/fusion process the compound(s) could be acting, the three test compounds were tested against three different viral strains. One of the three viral strains, NL4/3, has already been reported as this is the strain normally used in the antiviral assays. The test compounds were tested for antiviral activity against BaL, an R5 tropic strain, and 89.6, a dual-tropic strain, in the FIA. These combined data cover the breadth of coreceptor tropisms. Compounds 1, 2, and 7 inhibit all three strains of virus to a comparable degree indicating that coreceptor antagonism is not the source of their inhibition (Table 4).

Table 4

Antiviral Activity of 4 Test Compounds Agamst 3 Strains of HIV-I

COMPOUND EC₅₀ (μM) NL4/3 EC₅₀ (μM) BaL EC₅₀ (μM) 89.6 (X4) (R5) (X4R5)

1 2.4 ± 0.3 1.7 ± 0.02 1.8 + 1.0

2 0.3 ± 0.03 0.81 ± 0.1 0.4 + 0.04

7 6.2 + 1.7 3.2 ± 0.4 4.9 ± 0.5 [0098] Several peptides and peptidomimetics have been reported to inhibit the entry/fusion process (including T20, the only currently approved fusion inhbitor) and many small molecules have been reported as coreceptor antagonists (both for X4 and R5), but there have been few reports of small molecule viral entry inhibitors. Early efforts targeting HIV-I entry focused on peptide-based CD4 mimics. Soluble CD4 was shown to be effective at suppressing viral replication in several lab-adapted strains, but failed to demonstrate the same activity in primary isolates (see Daar, E.S., et al., Proc. Natl. Acad. ScL USA, 1990, 87, 6574- 6578). Later a CD4-IgG fusion protein, PRO-542, was proven to be effective against both lab-adapted strains and primary isolates, but required administration intravenously (Jacobson, et al., J. Infect. Dis., 2000, 169, 326-329). More recently, smaller peptides have been shown to be effective at blocking HIV-I entry through interference with the gpl20/CD4 interaction (Martin, L., et al., Nat. Biotechnol., 2003, 21, 71-76; and Neffe, A.T. and Meyer, B. Angew. Chem. Int. Ed., 2004, 43, 2937-2940). In contrast to the relative success of peptide and peptidomimetic CD4-based approaches to entry inhibition, small molecule inhibitors of this process have been more elusive. The only small molecule inhibitor of entry not targeted at the chemokine coreceptors reported so far has been BMS-378806, a 4-methoxy-7-azaindoIe derivative developed by Bristol-Meyers Squibb (see Figure 1, and see Lin, P.F., et al., Proc. Natl Acad. ScL USA, 2003, 100, 11013-11018). The BMS compound was originally reported to bind to gpl20 and block the initial gpl20/CD4 interaction. Subsequently, this hypothesis was refuted by Si and coworkers, who found the small molecule was still able to potently inhibit infection of cells lacking CD4 receptor (see, Si, Z., et al., Proc. Natl. Acad. ScL USA, 2004, 101, 5036-5041). In their report, Si et. al. provide evidence that BMS- 378806 prevents CD4-induced creation of the gp41 HRl coiled coil instead. While the two previous statements may seem contradictory, a model for CD4-independent viruses where the chemokine coreceptor triggers the conformational change that CD4 normally promotes in the CD4-dependent strains is possible. While the exact mechanism is still unclear, Compound 2 demonstrates potent antiviral activity inhibiting the entry/fusion process in a coreceptor- independent manner.

Example 6 - Binding of Compound 2 to gpl20 [0099] T20 and 3TC were generously provided by Dr. Raymond F. Schinazi (Emory University, Atlanta, GA).

Cells and viruses. [0100] HeLa H1-JC.37 cells were used for the focal infectivity assay (FIA) (Platt, et al., J Virol 72:2855-2864 (199S)). These cells, which naturally express CXCR4, have been engineered to stably express both CD4 and CCR5, making them permissive to all R5, X4 and dual tropic strains of HIV-I tested. In addition, they are permissive to SIV infection (Kuhmann, et al., J Virol 71 :8642-8656 (1997)). The cells were maintained in Dulbecco modified Eagle medium (DMEM; GIBCO, Invitrogen, Carlsbad, CA), supplemented with 10 % fetal bovine serum (FBS) (Omega Scientific, Tarzana, CA) that had been heat inactivated for 30 minutes at 56°C, 100 U of penicillin per ml, 100 μg of streptomycin per ml, and 2 mM L-glutamine (GIBCO). All cultures were maintained at 37⁰C with a humidified 5% CO₂ atmosphere.

[0101] CEMx 174 cells were obtained from Dr. Peter Cresswell through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NHH. They were maintained in RPMI 1640 medium (GIBCO) supplemented with 10% FBS, penicillin, streptomycin, and L- glutamine at concentrations described above. HIV-I NL4-3, 89.6, and HXBc2, and SIV stocks were grown in CEMxl74 cells. HIV-I 89.6, Ba-L, SF162 and all the primary isolates were obtained from the AIDS Research and Reference Reagent Program. HIV-I HXBc2 was

) generously provided by Dr. Mark Wainberg (McGiIl University, Montreal, QC). Infectious

NL4-3 and SIVmac239 were produced by transfecting CEMxI 74 cells using electroporation. The plasmid pNL4-3 and the two SIVmac239 half clones were kindly provided by Dr. Paul Luciw (University of California, Davis, Davis, CA) (Luciw, et al., Proc Natl Acad Sd USA 92:7490-7494 (1995)).

[0102] Human peripheral blood mononuclear cells (PBMCs) were isolated from screened donors using established protocols (see, Gallo, et al., Science 224:500-503 (1984); Jackson, et al., J CHn Microbiol 26:1416-1418 (1988); and Levy and Shimabukuro, J Infect Dis 152:734-738 (1985)). The cells were aliquoted into 24-well plates and maintained in RPMI 1640 medium supplemented with 20% FBS, 5% human IL-2, 100 U of penicillin per ml, 100 μg of streptomycin per ml, and 2 mM L-glutamine (GIBCO). The recombinant human IL-2 was obtained from Dr. Maurice Gately (Hoffman — La Roche Inc., Basel, Switzerland) through the AIDS Research and Reference Reagent Program (Lahm and Stein, J Chromatogr 326:357-361 (1985)). For virus propagation and assays using PBMCs, the cells were first stimulated with phytohemagglutinin (PHA-P; Sigma- Aldrich, St. Louis, MO) for 2-3 days. Stocks of HIV-I SF162, Ba-L, and all the HIV-I primary strains were grown in stimulated PBMCs. Virus was detected in cell culture by p24 antigen capture enzyme-linked immunosorbent assay (ELISA) or focal infectivity assay (FIA). Virus stocks were prepared by clarifying the supernatant using centrifugation at 600 x g for 10 min and then stored at - 70⁰C.

Drug Susceptibility Assays. [0103] The procedures for the FIA (Chesebro and Wehrly, J Virol 62:3779-3788 (1988); Giuffre, et al., Antimicrob Agents Chemother 47:1756-1759 (2003); and Meadows, et al., J Med Chem 48:4526-4534 (2005)) and the HIV-I p24 antigen capture ELISA (Higgins, et al., J CHn Microbiol 24:424-430 (1986)) were previously described. Both assays were used to monitor virus replication in cell cultures and to quantify the susceptibility of HIV-I and SIV to antiviral drugs. Briefly for the FIA, 4.5 x 10³ HeLa Hl-JC.37 cells per well were seeded into a 96- well microtiter plate and incubated overnight at 37°C. The medium was then removed and replaced with 100 μl per well of DMEM supplemented with 0.1% FBS with or without drug, and incubated for 1 hour at 37°C. The medium was removed and the cells were then incubated for 1-2 hours with 100 μl per well of DMEM plus 0.1% FBS₅ 20-60 focus- forming units (ffu) of HIV-I and the appropriate drug concentration. After virus adsorption, an additional 100 μl of growth medium, FBS, and drug was added to bring the FBS concentration to 10% while maintaining the starting drug concentration. The cells were incubated for 4 days at 37°C and a humidified 5% CO₂ atmosphere. Cells were fixed and immunostained according to previously described methods (Meadows, et al., ibid; and Murry, et al., J Virol 77: 1120-1130 (2003)). Data were plotted as a percentage of control foci (no drug) versus inhibitor concentration. Within each experiment, each value represents the mean of at least five replicate wells. Concentrations required to inhibit focus formation by 50% (EC50S) were obtained directly from the linear portion of these plots by using a computer-generated regression line. Results from three or more independent experiments were used to derive the EC50 values plus or minus standard error.

Drug-virus preincubation studies using the FIA.

[0104] Aliquots (100 μl) of concentrated HIV-I NL4-3 virus stock (10⁵ ffu/ml) were added to a 48-weIl microtiter plate. Concentrated drug stock was added to each aliquot at the appropriate volume to achieve the desired drug concentration. For Compound 2, the concentrations tested were 0, 0.1, 0.3, 1, 3, 10 and 30 μM, while the concentrations for T20 were 0, 2.5, 5, 10, 20, 40, 80, and 240 nM. , The drug-virus solutions were incubated for 2 hours at 37⁰C. The solutions were then diluted 400-fold with DMEM plus 0.1% FBS, resulting in levels of drug in solution well below inhibitory levels. The virus solution (100 μl per well) was added to a 96-well microtiter plate seeded with HeLa H1-JC.37 as described for the FIA. For each drug concentration, there were 5 replicate wells. The virus was allowed to adsorb for 2 hours at 37⁰C₃ followed by the addition of 100 μl of growth medium and FBS to bring the FBS concentration to 10%. The standard procedure for the FIA was followed for immuno staining the cells, quantification of foci, and data analysis (see above).

Drug-virus preincubation studies using p24 ELISA.

[0105] Similar preincubation experiments were performed with other cells using a p24 ELISA assay to determine antiviral activity. The drug- virus preincubation step was also performed with a p24 ELISA assay similar to those described in the previous section using the FIA. 3TC was used in these experiments as the negative control and was tested at the same concentrations as Compound 2. After the virus was incubated with drug, the solutions were diluted 400- fold in the appropriate medium containing 0.1% FBS, and then added to CEMxl74 cells (5 x 10⁴ cells/well), PBMCs (1 x 10⁴ cells/well), or HeLa cells (4.5 x 10⁴ cells/well). After incubation for 2 hours, the virus solution was removed and the cells were washed two times with media using centrifugation to remove unadsorbed virus. Finally, the cells were resuspended in media containing 10% FBS and aliquoted into a 96-well microtiter plate at 225 μl per well. Following 4 days of incubation at 37°C, 200 μl per well of supernatant was removed and added to p24 ELISA plates. The ρ24 ELISA was used to measure virus replication (Higgins, et al., J CHn Microbiol 24:424-430(1986)), and data were analyzed as described for the FIA (see above).

Drug-cell preincubation studies using the FIA.

[0106] HeLa H1-JC.37 cells (4.5 x 10³ cells per well) were seeded into 96-well microtiter plates and incubated overnight at 37°C. The medium was removed and 100 μl per well of DMEM containing 0.1 % FBS and the desired drug concentration was added and incubated for 2 hours. Compound 2 and T20 concentrations in this experiment were the same as in the drug-virus preincubation experiment (see above). The medium with drug was then removed and the cells were washed twice with DMEM plus 0.1% FBS. 100 μl per well of medium containing 0.1% FBS and 20-60 ffu/well of HIV-I NL4-3 was added and incubated for 2 hours. A positive control was also included in which drug was added along with virus to the HeLa cells as is performed in the normal drug susceptibility FIA. The volume was then brought up to 200 μl and 10% FBS and the plates were incubated for 4 days at 37°C in a humidified 5% CO₂ atmosphere: The FIA protocol was then followed for immunostaining, quantification of foci, and data analysis.

Surface PIasmon Resonance (SPR) Competition Binding Studies.

[0107] All binding experiments were performed on a Biacore 3000 optical sensor and research-grade CM5 sensor chips (Biacore AB, Uppsala, Sweden) at 25°C. The running buffer for all experiments, 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% v/v Surfactant P20 (HBS-P, Biacore AB), was vacuum filtered and degassed immediately prior to use. The instrument was primed 5 times with running buffer immediately before experiments were performed. Goat anti-human Ig capture antibody (Southern Biotech, Birmingham, AL) was covalently immobilized using a standard coupling procedure with an amine coupling kit

(Biacore AB) (Johnsson, et al., Anal Biochem 198:268-277 (1991)). The four flow cells (Fes) on a CM5 chip were activated simultaneously by injecting a 1 : 1 mixture of 100 mM N- hydroxysuccinimide (NHS) and 400 mM l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 7 min at 20 μl/min. A solution of 2.5 μg/ml purified goat anti-human Ig in 10 mM sodium acetate, pH 4.5 was injected for 6 min over all 4 Fes, resulting in -1400 response units (RU) of antibody immobilized on each. Residual reactive groups were deactivated with a 7 min injection of 1 M ethanolamine hydrochloride, pH 8.5 (20 μl/min). The surface was then conditioned by three 15 μl injections of 146 mM phosphoric acid (H3PO₄) at 100 μl/min to remove any noncovalently bound antibody. [0108] The immobilized goat anti-human Ig was used to capture all the monoclonal antibodies (mAbs). Several of these mAbs were provided as crude supernatant or contained other additives that would have made direct immobilization of the mAbs difficult (Canziani, et al., Anal Biochem 325:301-307 (2004)). All mAbs and sCD4 were obtained from the AIDS Research and Reference Reagent Program. The antibodies used in the competition studies were 5F3 (gp41 - aa 526-543), F240 (gp41 - aa 592-606), bl2 (gpl20 - CD4 binding site), F425 B4e8 (gpl20 - base of V3 loop), 48d (gpl20 - CD4i epitope), and 17b (gpl20 - CD4i epitope). The mAbs were diluted to 10 μg/ml in running buffer and injected for 1 min at 20 μl/min over Fc2 or Fc4. Only 2 mAbs were captured per binding cycle as FcI was used as a reference surface and Fc3 was used as a control surface. The amount of antibody captured was different for each mAb. The antigen, 50 nM ogpl40 (Srivastava, et al., J Virol 77:11244-11259 (2003)), was preincubated with 0, 1, 5, or 10 μM Compound 2 for 30 min then injected over all Fes for 1 min at 20 μl/min. For 48d and 17b, which bind to the CD4- induced epitope, the I min analyte injection consisted of 50 nM ogpl40 preincubated with Compound 2 and 150 nM sCD4. For all antigen injections, dissociation was monitored for 2 min and the surfaces were regenerated simultaneously with a 2 min injection of 146 mM H₃PO₄ at 20 μl/min. A blank injection was also included (1 min injection with 2 min dissociation) prior to the analyte injection and was used to double reference the binding data (Myszka, DG, JMoI Recognit 12:279-284 (1999)). All data were analyzed using BIAevaluation 4.1 software (Biacore) and Scrubber software 2.0 (Center for Biomolecular Interaction Analysis, University of Utah).

[0109] Antigen binding data were analyzed by aligning all the sensorgrams, including the buffer response, on the x axis and zeroing on the y axis. Systematic noise and other artifacts that occurred in all 4 flow cells were removed by subtracting the antigen responses with the response from the reference flow cell, FcI . Antigen data were then double referenced by subtracting the buffer injection (Myszka, ibid). To be able to compare the responses from different injection cycles, the antigen responses were normalized for the differences in the amount of antibody captured on the surface (Canziani, et al., Anal Biochem 325:301-307 (2004)). Normalization was performed by dividing the antigen response data by the mAb capture level within the injection cycle. The antibody capture level was determined by the baseline level immediately prior to the antigen injection (Canziani, et al., ibid).

Results Binding to ogpl40 and Direct Inactivation of HIV-I by Compound 2.

[0110] Having established that Compound 2 exerts its antiviral activity early in the viral replication process (Meadows, et al., J Med Chem 48:4526-4534 (2005)), surface plasmon resonance (SPR) was used to determine whether the compound would directly bind to the HIV-I envelope glycoprotein. For these experiments a well characterized soluble trimeric oligomerized envelope glycoprotein ogpl40 (Srivastava, et al., J Virol 77:11244-11259

(2003)) was used. Ogpl40 contains gpl20 (with a partial deletion in the V2 loop) and the ectodomain of gp41, was employed. Ogpl40 was used in these studies, rather than the monomelic gpl20, because it represents a more biologically relevant target. The protein has been shown to resemble the native glycoprotein on the virus surface through antibody- binding and structure characterization (Srivastava, et al., J Virol 77:11244-11259 (2003)). In addition, ogpl40 also contains the ectodomain of gp41 which has proven to be important as a target for entry inhibition (Bianchi, et al., Proc Natl Acad Sci USA 102:12903-12908 (2005); and Chan and Kim, Cell 93:681-684 (1998)). Using SPR, ogpl40 was immobilized directly on a CM5 chip (Biacore) surface and varying concentrations of Compound 2 were flowed over it. Initial studies showed such tight binding to ogpl40 that the interaction appeared irreversible (data not shown) as the bound Compound 2 could not be removed from ogpl40. To evaluate whether Compound 2 was binding to the virus particle directly and inactivating it, a preincubation study was performed in which concentrated cell-free HIV-I NL4-3 was first incubated with varying concentrations of Compound 2. The Compound 2- virus solution was then diluted to levels where the concentration of Compound 2 in solution was well below inhibitory levels, following which viral infectivity was determined by the FIA. A similar experiment was performed using T20 as a negative control. Compound 2 was able to prevent infectivity of HTV-I following drug-pretreatment (see Figure 3B). T20 was not able to inactivate virus in the absence of cells (see Figure 3C), consistent with its mode of action as a fusion inhibitor that requires virus binding to target cells. Accordingly, Compound 2 appears to bind to a site on the virus that blocks the first stage of viral entry - attachment.

[0111] To eliminate the possibility that a cellular factor was the target for the antiviral activity of Compound 2, a similar experiment was performed in which the cells were first incubated with Compound 2, washed and then infected with HTV-I NL4-3. There was no inhibition observed at any drug concentration tested (see Figure 4), supporting the conclusion that Compound 2 targets the virus directly.

[0112] . To determine whether Compound 2 could directly inactivate virus infectivity in more biologically relevant cells, a p24 antigen capture ELISA was used to detect virus replication, and a Compound 2-virus preincubation experiment was run using PBMCs, CEMx 174 cells, and HeLaHl-JC.37 cells as a control. 3TC was tested with CEMxI 74 cells as a negative control for the experiment. As seen in Figure 3D, Compound 2 inactivated HTV-I infectivity in all 3 cell lines.

Compound 2 Binds at or Near the Base of the V3 Loop of gpl20 and Blocks CD4 Attachment.

[0113] To map the binding site of Compound 2 to the envelope glycoprotein, a SPR was carried out to perform competition studies in which the binding of ogpl40 to well- characterized human mAbs targeting gpl20 or gp41 was measured in the presence of varying concentrations of Compound 2. The mAbs targeting gp41, 5F3 (Buchacher, et al., AIDS Res Hum Retroviruses 10:359-369 (1994)) and F240 (Cavacini, et al., AIDS Res Hum Retroviruses 14:1271-1280 (1998)), were captured to the chip in different flow cells. Ogpl40 preincubated with 0, 1, 5 and 10 μM of Compound 2 was then injected over the surface and binding to each mAb was measured simultaneously. At all concentrations of Compound 2, the binding levels observed for each mAb were equal to ogpl40 alone (Figure 5, A and B), indicating that gp41 is not the target of Compound 2.

[0114] Next, antibodies were captured that targeted gpl20 — bl2, which is directed at the CD4 binding site (Burton, et al., Proc Natl Acad Sci USA 88:10134-10137 (1991)), and F425 B4e8, which recognizes the base of the V3 loop (Cavacini, et al., Aids 17:685-689 (2003)). As in the case with the gp41 antibodies, there was no change in binding levels of ogpl40 preincubated with Compound 2 to bl2 (Figure 5C), indicating that the CD4 binding site is not the target. However, a decrease in binding was observed with F425 B4e8 (Figure 5D) with increasing concentrations of Compound 2 preincubated with ogpl40. As a control experiment, sCD4 was immobilized to a CM5 chip and injected Compound 2 over the surface. As expected, there was no binding observed between sCD4 and Compound 2 (data not shown). These results demonstrate that Compound 2 binds to gpl20 and not to the CD4 receptor, confirming that a host cell factor is not the target of Compound 2.

[0115] To further define the binding site, two human monoclonal antibodies, 48d and 17b, were captured using the same experimental setup as in the previous experiments. 48d and 17b both target the CD4-induced epitope located on the V3 loop of gpl20 and neutralize virus by interfering with the chemokine coreceptor binding (Thali, et al., J. Virol. 67:3978- 3988 (1993)). In these competition studies, ogpl40 was incubated with Compound 2 along with sCD4 (Garlick, et al., AIDS Res Hum Retroviruses 6:465-479 (1990)). For each antibody, there was a decrease in binding with increasing concentrations of Compound 2 (Figure 5, E and F). The level of ogpl40 binding to each antibody in the presence of 10 μM Compound 2 was equivalent to the binding level of ogpl40 with no sCD4 present. These data are further evidence that Compound 2 acts by binding to the envelope glycoprotein and prevents the binding events or conformational changes needed for HIV-I entry.

Aπti-HIV Spectrum. [0116] Due to the diversity of the envelope glycoprotein, it was important to determine whether Compound 2 could inhibit a broad range of HIV-I isolates. Using the FIA, Compound 2 showed antiviral activity against a panel of HIV-I laboratory-adapted strains and a number of clinical isolates from diverse subtypes. The results are summarized in Table 5 (see Figure 6). Against the laboratory-adapted strains, Compound 2 inhibited HIV-I with EC₅₀ values ranging from 330 nM to 850 nM. It showed a wider range of potency against primary strains of HIV-I, with EC₅₀ values from 190 to 2300 nM, with no correlation of potency with virus subtype or coreceptor usage. Compound 2 was most potent against both a B clade dual tropic virus (92TH014) and a C clade R5 tropic virus (98TZO13) with an EC50 value of 190 nM. The fact that Compound 2 is effective against R5, X4 and dual tropic viruses, indicates that the inhibitory activity does not involve a coreceptor. hi addition to these HIV strains, Compound 2 exhibited antiviral activity against SIVmac239, though with less potency (EC50 = 1200 nM). The activity against SIVmac239 indicates that Compound 2 may be interfering with a conserved region involved in the interaction between these viruses and their target cells.

Discussion

[0117] Entry inhibitors are attractive candidates for prophylactic microbicide development to prevent the sexual transmission of HIV-I . Inhibitors that block the early stages of the replication cycle (attachment, coreceptor and fusion inhibitors) can have an advantage over existing therapeutic approaches that target viral enzymes RT and PR because they can prevent HIV-I entry into target cells, thereby preventing establishment of a long-term infection. Anti-HIV agents that have moved into Phase III clinical trials for the development of HIV-I microbicides are detergents (Savvy) or other substances (PRO 2000) that do not specifically target the virus . A number of other microbicide candidates target virus replication (tenofovir), bind to gp41 (2F5), or target host-cell structures, such as CD4 (TNX- 355) or CCR5 (PSC-RANTES) (Lederman, et al., Nat Rev Immunol 6:371-382 (2006)). Although these strategies are promising, many involve large molecules (mAbs or long peptides) that are expensive to produce in large scale quantities necessary for microbicide use. This is a particularly important consideration since it has been suggested that inhibitor concentrations need to be orders of magnitude higher than the observed EC₅₀ values obtained in vitro to completely protect against HIV-I infection (Veazey, et al., Nature 438:99-102 (2005)). [0118] The results above demonstrate that Compound 2 is a novel small molecule inhibitor that directly inactivates HIV-I in the absence of a cellular target. It is able to inactivate virus in HeLa cells, CEMxI 74 cells and PBMCs. Its specificity to gpl20 is demonstrated by the direct binding of Compound 2 to ogpl40 and its ability to inhibit R5, X4 and dual tropic HIV-I strains, indicating that its mechanism of action is independent of coreceptor usage. Furthermore, there was no direct binding observed between Compound 2 and sCD4 in SPR studies and pretreatment of drug with cells prior to infection of HIV-I showed no virus inhibition. The ability of Compound 2 to bind to gpl20 and inactivate virus further demonstrates that gpl20 is a viable target for small molecule inhibitors to block HIV-I entry.

[0119] The evidence above indicates that Compound 2 exerts antiviral activity through an interaction with gpl20 that either prevents binding of CD4 or prevents gpl20 from undergoing conformation changes after CD4 binding that are needed for membrane fusion and entry. Competition studies using SPR revealed that Compound 2 binds at the base of the V3 loop and prevents the CD4i epitope from being exposed. Since Compound 2 binds to the V3 region of gpl20, it is notable that it is able to inhibit a diverse range of both laboratory- adapted and primary strains of HIV-I from all the major clades tested. This suggests that Compound 2 binds to a conserved region within the V3 loop. Although the V3 loop has been characterized as a hypervariable region, much of the V3 loop, including the tip and the crown, are highly conserved (Stanfield, et al., J. Virol. 80:6093-6105 (2006)). Studies to structurally define the interaction between Compound 2 and gpl20 are underway, including selections of resistant variants.

[0120] There remains an urgent need to develop an efficacious topical microbicide to help curb the sexual transmission of HIV-I from infected individuals to uninfected individuals. The ideal microbicide should meet the following requirements: (i) be highly potent against HIV-I, (ii) act directly on the virus and inactivate it without the need for metabolic activation, (iii) be effective against a range of HIV-I strains, (iv) have minimal cytotoxic effects, and (v) be relatively inexpensive to manufacture. Cost-effectiveness favors the development of small molecule inhibitors, such as Compound 2. Only a few small molecule entry inhibitors have been reported (Zhao, et al., Virology 339:213-225 (2005)); one of particular note is BMS-378806 which has been shown to inhibit viral entry by blocking the gρl20-CD4 interaction (Lin, et al., Proc Natl Acad Sd USA 100:11013-11018 (2003); and Luciw, et al., Proc Natl Acad Sd USA 92:7490-7494 (1995)). However, it is not clear whether or not BMS-378806 can inactivate the virus without the presence of the host-cell receptor, though its use as a microbicide in combination with two other compounds, CMPD 167 and C52L, was protective when applied vaginally in the rhesus macaque/SHTV model (Veazey, et al., Nature 438:99-102 (2005)). This experiment showed that small molecule entry inhibitors have the potential to be effective at inhibiting HIV when applied topically to the vaginal surface, especially when used in conjunction with other compounds.

[0121] Compound 2 is a prototype for a new class of small molecule entry inhibitors that can disarm HIV-I by direct inactivation through a specific interaction with gpl20 without the presence of cellular target. These characteristics make this compound particularly useful as a topical microbicide.

[0122] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting viral entry into cells, said method comprising contacting said cells with a viral entry-inhibiting amount of a compound having the formula:

wherein each Ar is an independently selected trihydroxyphenyl group; the double bonds adjacent to the sulfone groups are each independently in either a cis or trans orientation; and pharmaceutically acceptable salts thereof.

2. A method in accordance with claim 1, wherein the compound has the formula:

3. A method in accordance with claim 1, wherein the viral entry is HIV viral entry.

4. A method in accordance with claim 1, wherein the compound is provided in a topical formulation.

5. A method in accordance with claim 1, wherein the compound is provided in a topical gel, cream or ointment formulation.

6. A method in accordance with claim 1, wherein said compound is administered to a subject with a second agent selected from the group consisting of an HIV- IN inhibitor, and HIV-RT inhibitor, a second viral entry inhibitor and an HIV-protease inhibitor.

7. A method in accordance with claim 6, wherein said compound and said second agent are administered sequentially.

8. A method in accordance with claim 6, wherein said compound and said second agent are administered simultaneously.

9. A method in accordance with claim 6, wherein said HIV-RT inhibitor is a non-nucleoside reverse transcriptase inhibitor.

10. A method in accordance with claim 6, wherein said second viral entry inhibitor is Fuseon^®.

11. A method of treating or inhibiting a HIV- viral infection, said method comprising administering to a subject in need of or at risk of such infection, a composition comprising a viral entry inhibiting amount of a compound of the formula:

in admixture with an HIV-integrase inhibiting amount of a compound of the formula

wherein the dashed line indicates an optional double bond and each Ar¹ is a substituted or unsubstituted phenyl, pyridyl or indolyl ring and is other than 3,4,5-trihydroxyphenyl.

12. A method in accordance with claim 11, wherein each of said Ar¹ groups is a substituted or unsubstituted phenyl, and said substituted phenyl is a phenyl ring having from 1 to 5 substituents independently selected from the group consisting of hydroxy, Ci-5 alkoxy, Ci_-S acyloxy, halogen, nitro, CO₂H, SO₃H, P(O)(OH)₂, OSO₃H and OP(O)(OH)₂, or wherein two substituents on adjacent carbon atoms are combined to form a fused methylenedioxy or ethylenedioxy ring, and salts and esters thereof.

13. A method in accordance with claim 11, wherein each of said Ar¹ groups is selected from the group consisting of:

14. A pharmaceutical composition comprising an HIV viral entry inhibiting amount of a compound having the formula:

wherein each Ar is an independently selected trihydroxyphenyl group; the double bonds adjacent to the sulfone groups are each independently in either a cis or trans orientation; or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable excipient.

15. A pharmaceutical composition of claim 14, formulated for topical administration.

16. A pharmaceutical composition of claim 14, wherein said compound is

17. A pharmaceutical composition of claim 16, formulated as a topical gel, cream or ointment.

18. A pharmaceutical composition of claim 14, further comprising a compound of the formula:

wherein the dashed line indicates an optional double bond and each Ar¹ is a substituted or unsubstituted phenyl, pyridyl or indolyl ring and is other than 3,4,5-trihydroxyphenyl.

19. A pharmaceutical composition of claim 18, wherein each of said Ar¹ groups is a substituted or unsubstituted phenyl, and said substituted phenyl is a phenyl ring having from 1 to 5 substituents independently selected from the group consisting of hydroxy, Ci-5 alkoxy, C_1-5 acyloxy, halogen, nitro, CO₂H, SO₃H, P(O)(OH)₂, OSO₃H and OP(O)(OH)₂, or wherein two substituents on adjacent carbon atoms are combined to form a fused methylenedioxy or ethylenedioxy ring, and salts and esters thereof.

20. A pharmaceutical composition of claim 19, wherein each of said Ar¹ groups is selected from the group consisting of:

21. A pharmaceutical composition of claim 14, further comprising an HW-ESf inhibitor.

22. A pharmaceutical composition of claim 14, further comprising an HIV-RT inhibitor.

23. A pharmaceutical composition of claim 22, wherein the HIV-RT inhibitor is a non-nucleoside reverse transcriptase inhibitor.

24. A pharmaceutical composition of claim 14, further comprising an HIV-protease inhibitor.

25. A pharmaceutical composition of claim 14, further comprising a second viral entry inhibitor.

26. A pharmaceutical composition of claim 25, wherein said second viral entry inhibitor is Fuseon^®.