WO2015134854A2 - Small molecule vif dimerization antagonists as anti-hiv agents and for use as hiv/aids therapeutics - Google Patents

Abstract

The present invention relates to small molecule Vif dimerizatoin antagonists as anti-HIV agents. The present invention relates to the use of the disclosed small molecules and their derivatives as anti HIV agents that disrupt self association of the viral infectivity factor (Vif) found in HIV and other retroviruses. The present invention also relates to methods of using these agents, including methods of treating HIV infection or AIDS.

Description

SMALL MOLECULE VI I DIMERIZATION ANTAGONISTS

AS ANTI-HIV AGENTS AND FOR USE AS HIV/AIDS THERAPEUTICS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.

61/949,175 filed on March 6, 2014, and to U.S. Provisional Application No. 62/013,550 filed on June 18, 2014. The entire contents of the prior applications are hereby incorporated herein by references. FIELD OF THE INVENTION

[0002] The present invention relates to small molecule Vif dimerization antagonists as anti-HIV agents. The invention also relates to the use of the disclosed small molecules and their derivatives as anti-HIV agents that disrupt self-association of the viral infectivity factor (Vif) found in HIV and other retroviruses. The invention further relates to methods of using these agents, including methods of treating HIV infection or AIDS.

BACKGROUND OF THE INVENTION

[0003] HIV-1 is the causative agent of AIDS and presently infects approximately 33 million persons worldwide with approximately 1.9 million infected persons in North America alone. Recent studies have shown that HIV/AIDS has become a global epidemic that is not under control in developing nations. The rapid emergence of drug-resistant strains of HIV throughout the world has placed a priority on innovative approaches for the identification of novel drug targets that may lead to a new class of anti-retroviral therapies.

[0004] The virus contains a 10-kb single-stranded RNA genome that encodes three major classes of gene products that include: (i) structural proteins (Gag, Pol and Env); (ii) essential trans-acting proteins (Tat, Rev); and (iii) "auxiliary" proteins that are not required for efficient virus replication in permissive cells (Vpr, Vif, Vpu, Nef) (reviewed in (1)). There has been a heightened interest in Vif as an antiviral target because of the discovery that the primary function of Vif is to overcome the action of a cellular antiviral protein known as APOBEC3G or A3G (2).

[0005] A need exists for novel anti-HIV compounds and methods that can help to address the HIV/AIDS epidemic.

[0006] In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was, at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned.

SUMMARY OF THE INVENTION

[0007] Briefly, the present invention satisfies the need for novel anti-HIV compound and methods of treating HIV and AIDS. The present invention may address one or more of the problems and deficiencies of the art discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.

[0008] The present invention is based, in part, on the discovery that identifying agents that disrupt Vif self-association can lead to the identification of novel agents for use as anti-HIV therapeutics.

[0009] In one aspect, the invention provides a compound of formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Y is selected from -S0₂-NR^aR^b and -NH-S0₂-(CH₂)_n-R¹;

R^a is H; and R^b is -(CH₂)_n-R¹; or, taken together, R^a and R^b, together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring; n is 0 or 1;

R¹ is an aryl or heteroaryl ring system, wherein said aryl or heteroaryl ring system is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_₆alkyl, Ci_ ealkoxy, hydroxyl, oxo, -NHC(0)Ci_₆alkyl, and -C(0)NHCi_₆alkyl; R² is selected from halo, Ci_₆alkyl, and haloCi_₆alkyl;

R³ and R⁴ are hydrogen, or, taken together, R³ and R⁴, together with the carbon atoms to which they are attached, form a benzene ring; and

R⁵ is selected from hydrogen and methyl.

[0010] In another aspect, the invention provides a Vif dimerization antagonist comprising a compound of Formula (I).

[0011] In another aspect, the invention provides a method for treating HIV infection or AIDS in a patient, said method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I).

[0012] In another aspect, the invention provides a method for inhibiting infectivity of a lentivirus in a cell, said method comprising contacting the cell with an antiviral-effective amount of a compound of Formula (I).

[0013] In another aspect, the invention provides a method for inhibiting Vif self- association in a cell, said method comprising contacting the cell with an inhibitory-effective amount of a compound of Formula (I).

[0014] Certain embodiments of the presently-disclosed compounds, compositions, and methods have several features, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of these compounds, compositions, and methods as defined by the claims that follow, their more prominent features will now be discussed briefly. After considering this discussion, and particularly after reading the section of this specification entitled "Detailed Description of the Invention," one will understand how the features of the various embodiments disclosed herein provide a number of advantages over the current state of the art. These advantages may include, without limitation, providing novel and effective compounds and compositions, methods for treating HIV infection or AIDS, and methods of inhibiting Vif self-association in a cell. [0015] These and other features and advantages of this invention will become apparent from the following detailed description of the various aspects of the invention taken in conjunction with the appended claims and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will hereinafter be described in conjunction with the following drawing figures, wherein:

[0017] FIG. 1 provides a visual representation of the critical path associated with the

Primary in-cell quenched fluorescence resonance energy transfer (FqRET) Assay for Vif Dimerization Antagonists high throughput screening (HTS).

[0018] FIG. 2 illustrates a schematic showing the FqRET assay for use in identifying small molecules that interfere with Vif self-association.

[0019] FIGS. 3 A and 3B show fluorescence and western blot results of various combinations of N- and C-terminally tagged Vif constructs using one embodiment of the assay method of the present invention.

[0020] FIG. 4 provides images from an in-cell secondary screen for Vif-dependent

A3G degradation.

[0021] FIG. 5 depicts results from the testing six compounds according to embodiments of the invention in a Vif-dependent A3G-mCherry Degradation Screen.

[0022] FIGS. 6A and 6B are charts showing dose dependence of an anti-HIV compound according to an embodiment of the invention in both primary (FIG. 6A) and secondary (FIG. 6B) screens.

[0023] FIG. 7 depicts a chart showing testing results demonstrating Vif and A3G-

Dependent Effect on Pseudotyped HIV Infectivity Expressed as % of Control, for an embodiment of the invention.

[0024] FIG. 8 depicts a +Vif & A3G Pseudotyped Infectivity Curve for an anti-HIV agent according to an embodiment of the invention.

[0025] FIG. 9 depicts results from testing where +/-Vif virus + A3G controls and

+Vif virus + A3G produced in 293T cells in the presence of 25-0.25 μΜ of an anti-HIV agent according to an embodiment of the invention, of viral particles purified through a 20% sucrose cushion and western blotted for p24 (to show equal viral load in gel) and V5 tagged A3G. [0026] FIGS. 10A-F depict efficacy results of an anti-HIV agent according to an embodiment of the invention tested in 7-day PBMC Infections.

[0027] FIG. 11 shows toxicity results for an anti-HIV agent according to an embodiment of the invention following testing for PBMC cytotoxicity on day 7 after compound addition.

[0028] FIGS. 12A and 12B show dose dependent results for anti-HIV compounds according to embodiments of the invention in both a primary screen a secondary screen.

[0029] FIG. 13 is a chart providing data for Vif and A3G-Dependent Effect on

Pseudotyped HIV Infectivity Expressed as % of Control for several anti-HIV compounds according to embodiments of the invention.

[0030] FIG. 14 shows results from additional testing performed on anti-HIV compounds according to embodiments of the invention, of viral particles purified through a 20% sucrose cushion and western blotted for p24 (to show equal viral load in gel) and V5 tagged A3G..

[0031] FIG. 15 is a chart showing that various anti-HIV compounds according to embodiments of the invention effectively decrease fluorescence anisotropy of Vif oligomers in vitro.

[0032] FIGS. 16A and 16B are charts showing dose dependence of anti-HIV compounds according to embodiments of the invention in both a Vif-dependent A3G- mCherry Degradation Secondary Screen and a FqRET Vif Vif Interaction Primary Screen.

[0033] FIGS. 17A and 17B are charts showing dose dependence of other of anti-HIV compounds according to embodiments of the invention in both a Vif-dependent A3G- mCherry Degradation Secondary Screen and a FqRET Vif Vif Interaction Primary Screen. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention is based, in part, on the discovery that disrupting self- association of the HIV viral infectivity factor (Vif) can be a mechanism for use in identifying agents that can be used as anti-HIV agents.

[0035] Aspects of the present invention and certain features, advantages, and details thereof, are explained more fully below with reference to the non-limiting embodiments illustrated in the accompanying drawings. Descriptions of well-known materials, fabrication tools, processing techniques, etc., are omitted so as to not unnecessarily obscure the invention in detail. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the invention, are given by way of illustration only, and are not by way of limitation. Various substitutions, modifications, additions and/or arrangements within the spirit and/or scope of the underlying inventive concepts will be apparent to those skilled in the art from this disclosure, and are considered to be encompassed by the instant invention.

[0036] Vif binds to and induces the destruction of APOBEC3G (also referred to herein as "A3G"), which is a broad antiviral host-defense factor. Therefore, Vif is essential for HIV infection. Vif subunits interact to form multimers and this property has been shown to be necessary for HIV infectivity. The segment of Vif that mediates subunit interaction was previously determined to be proline-proline-leucine-proline (PPLP). However, to date, there has not been an effective high throughput screening (HTS) assay to identify agents that disrupt Vif self-association. The present invention is effective, among other things, in addressing this need.

Inhibitors of Vif Self- Association

[0037] The present invention provides various compounds that were identified the screening assay of the present invention. The compounds disclosed herein are effective as inhibitors of Vif self-association.

[0038] In one aspect, the invention provides a compound of formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Y is selected from -S0₂-NR^aR^b and -NH-S0₂-(CH₂)_n-R¹; R^a is H; and R^b is -(CH₂)_n-R¹; or, taken together, R^a and R^b, together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring; n is 0 or 1;

R¹ is an aryl or heteroaryl ring system, wherein said aryl or heteroaryl ring system is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_₆alkyl, Ci_ 6alkoxy, hydroxyl, oxo, -NHC(0)d_₆alkyl, and -C(0)NHCi_₆alkyl;

R² is selected from halo, Ci_₆alkyl, and haloCi_₆alkyl;

R³ and R⁴ are hydrogen, or, taken together, R³ and R⁴, together with the carbon atoms to which they are attached, form a benzene ring; and R⁵ is selected from hydrogen and methyl.

[0039] Compounds of Formula (I) are useful as anti-HIV compounds.

[0040] In some embodiments, Y is -SC>₂-NR^aR^b In some of these embodiments, R^a is

H and R^b is -(CH₂)_n-R¹. In other embodiments, R^a and R^b, together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring (e.g., pyrrolidine).

[0041] In some embodiments, Y is -NH-S02-(CH2)_n-R¹.

[0042] In some embodiments, n is 0. In other embodiments, n is 1.

[0043] In some embodiments, R¹ is one of the following aryl or heteroaryl ring systems, wherein the dotted line represents the point of attachment to the parent structure, where each R¹ residue is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_₆alkyl, Ci_₆alkoxy; hydroxyl, oxo, -NHC(0)Ci_₆alkyl, and -C(0)NHCi_₆alkyl:

- 10-

[0044] In some embodiments, R¹ is a residue of the formula

wherein R ^a, R , and R ^c are independently selected from halo, Ci_6alkyl, Ci_6alkoxy; hydroxyl, oxo, -NHC(0)Ci_6alkyl, and -C(0)NHCi_6alkyl. As will be appreciated by those skilled in the art, the ^ represents the point of attachment to the parent structure. In more particular embodiments, R^6a, R^6b, and R^6c are independently selected from hydrogen, halo, Ci_ 6alkyl, and Ci_6alkoxy. In certain embodiments, R^6a, R^6b, and R^6c are independently selected from hydrogen and methyl. [0045] In some embodiments, where R¹ is substituted or unsubstituted phenyl, R¹ is not para-substituted.

[0046] In some embodiments, halo is selected from F, CI, and Br. In more particular embodiments, halo is selected from F and CI. In particular embodimetns, halo is F.

[0047] In some embodiments, R² is selected from Ci_₆alkyl (including Ci_₆Cycloalkyl, e.g., cyclopropyl), and haloCi_₆alkyl (e.g., -CF₂ or -CF₃). In some embodiments, R² is selected from methyl and ethyl. In particular embodiments, R is methyl.

[0048] In some embodiments, R³ and R⁴ are hydrogen. In other embodiments, taken together, R³ and R⁴, together with the carbon atoms to which they are attached, form a benzene ring, thereby forming a tricylic core.

[0049] In some embodiments, R⁵ is hydrogen. In some embodiments, R⁵ is methyl.

[0050] In some embodiments, the compound of Formula (I) or pharmaceutically acceptable salt thereof is a compound of the Formula (IA):

(IA) or a pharmaceutically acceptable salt thereof, where R is as defined above.

[0051] Throughout this application, unless otherwise indicated, where inventive aspects of the invention are discussed, mention of Formula (I) includes any and all of the foregoing embodiments, as well as subsets of the Markush groups describes.

[0052] In some embodiments, the compound of Formula (I) or salt thereof is selected from one of the following compounds in Table I, or is a salt thereof. As will be readily appreciated by those skilled in the art, throughout this application, certain atoms may be discussed or depicted without a necessary atom required to satisfy valency. For example, in some of the following compounds, sulfonamide linkages may show a divalent nitrogen (e.g.,

). Skilled artisans will understand that although not pictured, the nitrogen in such compounds is in fact tnvalent, and includes a hydrogen bound to the nitrogen atom. The exception would be in the case of an ion, which would be specifically noted.

TABLE I

[0053] Some compounds used in connection with certain inventive embodiments may be commercially available Through Chemical Diversity, Vitas M Labs, Enamine, Specs or 5 Chembridge.

[0054] Compounds according to embodiments of the present invention may be readily synthesized from, e.g., commercially available starting materials. For example, in accordance with some embodiments, compounds and derivatives thereof may be synthesized via boronate intermediates, as shown in Scheme 1 :

10 SCHEME 1

[0055] In Scheme 1, commercially available hydrazides 1, 2, and 3 (persons having ordinary skill in the art will recognize that other desired starting hydrazides may also be used) are reacted with commercially available intermediate 4 where the hydrazide replaces the aromatic chloride followed by intramolecular cyclization to produce boronate intermediates 5, 6, and 7. These materials may be combined in turn with, e.g., sulfonamide intermediates such as those shown in Scheme 2.

SCHEME 2

[0056] Sulfonamides 10 may include various desired amines.

[0057] Boronate intermediates 5 from Scheme 1 may be coupled to sulfonamides 10 using established carbon-carbon coupling reaction conditions (e.g., Mitsunobu) to produce a compound according to Formula (I), as shown in Scheme 3. SCHEME 3

halo Ri = Selected Am^'

Ci-saikyl (e.g., cyclopropyl)

haloCi ^'ealk ! (e.g., CF3. CF2)

[0058] In some embodiments, one compound according to Formula (I) may be further reacted to provide another compound according to Formula (I).

[0059] In describing the compounds of the present invention, various definitions known in the relevant art can be used. For example, in various embodiments, the following definitions may apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001 , which are herein incorporated by reference in their entirety.

[0060] Unless otherwise specified, alkyl is intended to include linear, branched, or cyclic hydrocarbon structures and combinations thereof. A combination would be, for example, cyclopropylmethyl. Lower alkyl refers to alkyl groups of from 1 to 6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like. Preferred alkyl groups are those of C₂o or below. Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl and the like.

[0061] Ci to C₂o hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl,

cyclohexylmethyl, camphoryl and naphthylethyl. Hydrocarbon refers to any substituent comprised of hydrogen and carbon as the only elemental constituents.

[0062] An "alkenyl" group refers to an unsaturated hydrocarbon group containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More perferably it is a lower alkenyl of from 1 to 7 carbons. The alkenyl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably hydroxyl, cyano, alkoxy, =0, =S, NO₂, N(CH₃)₂, halogen, amino, or SH.

[0063] An "alkynyl" group refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More perferably it is a lower alkynyl of from 1 to 7 carbons. The alkynyl group may be substituted or unsubstituted. When substituted, the substituted group(s) is preferably hydroxyl, cyano, alkoxy, =0, =S, NO₂, N(CH₃)₂, amino, or SH.

[0064] "Alkylene" means a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms, e.g., methylene, ethylene, 2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene, and the like.

[0065] "Alkoxyalkyl" means a moiety of the formula R^a— O— R^b— , where R^a is alkyl and R^b is alkylene as defined herein. Exemplary alkoxyalkyl groups include, by way of example, 2-methoxyethyl, 3-methoxypropyl, 1 -methyl-2-methoxyethyl, l-(2-methoxyethyl)- 3-methoxypropyl, and l -(2-methoxyethyl)-3-methoxypropyl.

[0066] The abbreviations Me, Et, Ph, Tf, Ts and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, toluenesulfonyl and methanesulfonyl, respectively. A

comprehensive list of abbreviations utilized by organic chemists (i.e., persons of ordinary skill in the art) appears in the first issue of each volume of the Journal of Organic Chemistry. The list, which is typically presented in a table entitled "Standard List of Abbreviations" is incorporated herein by reference.

[0067] Unless otherwise specified, the term "carbocycle" is intended to include ring systems in which the ring atoms are all carbon but of any oxidation state. Thus (C₃-C₁₀) carbocycle refers to both non-aromatic and aromatic systems, including such systems as cyclopropane, benzene and cyclohexene; (C₈-C₁₂) carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene. Carbocycle, if not otherwise limited, refers to monocycles, bicycles and polycycles.

[0068] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of a straight, branched or cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. A subset of alkoxy is Ci_₆alkoxy. Lower- alkoxy refers to groups containing one to four carbons. For the purpose of this application, alkoxy and lower alkoxy include methylenedioxy and ethylenedioxy.

[0069] Oxaalkyl refers to alkyl residues in which one or more carbons (and their associated hydrogens) have been replaced by oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl and the like. The term oxaalkyl is intended as it is understood in the art [see Naming and Indexing of Chemical Substances for Chemical Abstracts, published by the American Chemical Society, 196, but without the restriction of 127(a)], i.e. it refers to compounds in which the oxygen is bonded via a single bond to its adjacent atoms (forming ether bonds); it does not refer to doubly bonded oxygen, as would be found in carbonyl groups. Similarly, thiaalkyl and azaalkyl refer to alkyl residues in which one or more carbons has been replaced by sulfur or nitrogen, respectively. Examples include ethylaminoethyl and methylthiopropyl .

[0070] Unless otherwise specified, acyl refers to formyl and to groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. One or more carbons in the acyl residue may be replaced by nitrogen, oxygen or sulfur as long as the point of attachment to the parent remains at the carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, benzyloxycarbonyl and the like. Lower-acyl refers to groups containing one to four carbons. The double bonded oxygen, when referred to as a substituent itself is called "oxo".

[0071] Aryl and heteroaryl mean (i) a phenyl group (or benzene) or a monocyclic 5- or 6-membered heteroaromatic ring containing 1-4 heteroatoms selected from O, N, or S; (ii) a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-4 heteroatoms selected from O, N, or S; or (iii) a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-5 heteroatoms selected from O, N, or S. The aromatic 6- to 14-membered carbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin, and fiuorene and the 5- to 10-membered aromatic heterocyclic rings include, e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole. As used herein aryl and heteroaryl refer to residues in which one or more rings are aromatic, but not all need be. [0072] Arylalkyl refers to a substituent in which an aryl residue is attached to the parent structure through alkyl. Examples are benzyl, phenethyl and the like. Hetero arylalkyl refers to a substituent in which a heteroaryl residue is attached to the parent structure through alkyl. In one embodiment, the alkyl group of an arylalkyl or a heteroarylalkyl is an alkyl group of from 1 to 6 carbons. Examples include, e.g., pyridinylmethyl, pyrimidinylethyl and the like.

[0073] Heterocycle means a cycloalkyl or aryl carbocycle residue in which from one to three carbons is replaced by a heteroatom selected from the group consisting of N, O and S. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Unless otherwise specified, a heterocycle may be non- aromatic or aromatic. Examples of heterocycles (which may also referred to as "heterocyclic ring systems" or "heterocyclic rings") that fall within the scope of the invention include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like. It is to be noted that heteroaryl is a subset of heterocycle in which the heterocycle is aromatic. Examples of heterocyclyl residues additionally include piperazinyl, 2- oxopiperazinyl, 2-oxopiperidinyl, 2-oxo-pyrrolidinyl, 2-oxoazepinyl, azepinyl, 4-piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl,

isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinylsulfone, oxadiazolyl, triazolyl and tetrahydroquinolinyl.

[0074] As used herein, the term "optionally substituted" may be used interchangeably with "unsubstituted or substituted". The term "substituted" refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to alkyl, aryl, cycloalkyl, or heterocyclyl wherein one or more H atoms in each residue are replaced with halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxyloweralkyl, carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, loweralkoxy, haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl [- C(=0)0-alkyl], alkoxycarbonylamino [ HNC(=0)0-alkyl], carboxamido [-C(=0)NH₂], alkylaminocarbonyl [-C(=0)NH-alkyl], cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, (alkyl)(aryl)aminoalkyl, alkylaminoalkyl (including cycloalkylaminoalkyl), dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino, alkylsulfinyl, alkylsulfonyl, acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl, heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy, hydroxyimino, alkoxyimino, oxaalkyl, aminosulfonyl, trityl, amidino, guanidino, ureido, benzyloxyphenyl, and benzyloxy. "Oxo" is also included among the substituents referred to in "optionally substituted"; it will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g. on phenyl). In one embodiment, 1, 2 or 3 hydrogen atoms are replaced with a specified radical. In the case of alkyl and cycloalkyl, more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine.

[0075] The terms "haloalkyl" and "haloalkoxy" mean alkyl or alkoxy, respectively, substituted with one or more halogen atoms. The terms "alkylcarbonyl" and

"alkoxycarbonyl" mean -C(=0)alkyl or -C(0)alkoxy, respectively.

[0076] The term "halogen" (or "halo") means fluorine, chlorine, bromine or iodine.

In one embodiment, halogen may be fluorine or chlorine.

[0077] It is understood that any alkyl, alkenyl, alkynyl, cycloalkyl and cycloalkenyl moiety described herein can also be an aliphatic group, an alicyclic group or a heterocyclic group. An "aliphatic group" is non-aromatic moiety that may contain any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and optionally contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched or cyclic and preferably contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted. It is understood that aliphatic groups may be used in place of the alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylene groups described herein.

[0078] Substituents ⁿ are generally defined when introduced and retain that definition throughout the specification and in all independent claims.

[0079] As used herein, and as would be understood by the person of skill in the art, the recitation of "a compound" - unless expressly further limited - is intended to include salts, solvates and inclusion complexes of that compound. Unless otherwise stated or depicted, structures depicted herein are also meant to include all stereoisomeric (e.g., enantiomeric, diastereomeric, and cis-trans isomeric) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and cis-trans isomeric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays. The term "solvate" refers to a compound of Formula I in the solid state, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. Inclusion complexes are described in Remington: The Science and Practice of Pharmacy 19^th Ed. (1995) volume 1 , page 176-177, which is incorporated herein by reference. The most commonly employed inclusion complexes are those with cyclodextrins, and all cyclodextrin complexes, natural and synthetic, are specifically encompassed within the claims.

[0080] The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. When the compounds of the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric,

ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p-toluenesulfonic, and the like. When the compounds contain an acidic side chain, suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine,

chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.

[0081] While it may be possible for the compounds of the invention to be

administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutical carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0082] In another aspect, the invention provides a Vif dimerization antagonist comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0083] In another aspect, the invention provides a method for treating HIV infection or AIDS in a patient, said method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Persons having ordinary skill in the art will recognize that, accordingly, the invention necessarily also provides a compound of Formula (I), or a salt thereof, for use in treating HIV or AIDS. The same holds true for other methods discussed herein.

[0084] In some embodiments, the inventive method further comprises administering a therapeutically effective amount of at least one other agent for treating HIV selected from the group consisting of HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, ΗΓ protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CC 5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, and HIV integrase inhibitors. [0085] In another aspect, the invention provides a method for inhibiting infectivity of a lentivirus in a cell, said method comprising contacting the cell with an antiviral-effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments, the compound of Formula (I) or salt thereof is administered with a pharmaceutically acceptable carrier.

[0087] In some embodiments, the compound of Formula (I) or salt thereof is effective to disrupt or inhibit multimerization of Vif in the cell, thereby inhibiting infectivity of the lentivirus.

[0088] In some embodiments, the lentivirus is selected from the group consisting of HIV-1 and HIV-2.

[0089] In another aspect, the invention provides a method for inhibiting Vif self- association in a cell, said method comprising contacting the cell with an inhibitory-effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0090] In some embodiments, the compound of Formula (I) or pharmaceutically acceptable salt thereof is administered with a pharmaceutically acceptable carrier.

[0091] In some embodiments, the compound of Formula (I) or salt thereof is effective to disrupt or inhibit multimerization of Vif in the cell, thereby inhibiting Vif self-association in the cell.

[0092] These and other aspects are discussed in greater detail below.

[0093] As used herein, the term "physiologically functional derivative" refers to any pharmaceutically acceptable derivative of a compound of the present invention that, upon administration to a mammal, is capable of providing (directly or indirectly) a compound of the present invention or an active metabolite thereof. Such derivatives, for example, esters and amides, will be clear to those skilled in the art, without undue experimentation.

Reference may be made to the teaching of Burger 's Medicinal Chemistry And Drug

Discovery, 5 ¹¹¹ Edition, Vol 1 : Principles and Practice, which is incorporated herein by reference to the extent that it teaches physiologically functional derivatives.

[0094] As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. The term

"therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For use in therapy, therapeutically effective amounts of a compound of the present invention, as well as salts, solvates, and physiological functional derivatives thereof, may be administered as the raw chemical. Additionally, the active ingredient may be presented as a pharmaceutical composition.

[0095] Pharmaceutical compositions of the present invention comprise an effective amount of one or more compound of the present invention, or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one compound of the present invention, or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0096] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0097] The term "lentivirus" as used herein may be any of a variety of members of this genus of viruses. The lentivirus may be, e.g., one that infects a mammal, such as a sheep, goat, horse, cow or primate, including human. Typical such viruses include, e.g., Vizna virus (which infects sheep); simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), chimeric simian human immunodeficiency virus (SHIV), feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV). "HIV," as used herein, refers to both HIV-1 and HIV-2. Much of the discussion herein is directed to HIV or HIV-1; however, it is to be understood that other suitable lentiviruses are also included.

[0098] The term "mammal" as used herein refers to any non-human mammal. Such mammals are, for example, rodents, non-human primates, sheep, dogs, cows, and pigs. The preferred non-human mammals are selected from the rodent family including rat and mouse, more preferably mouse. The preferred mammal is a human.

[0099] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptide, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[00100] "Pharmaceutically acceptable" means physiologically tolerable, for either human or veterinary applications. In addition, "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be

administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. Essentially, the pharmaceutically acceptable material is nontoxic to the recipient. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. For a discussion of pharmaceutically acceptable carriers and other components of pharmaceutical compositions, see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, 1990.

[00101] As used herein, "pharmaceutical compositions" include formulations for human and veterinary use. [00102] "Test agents" or otherwise "test compounds" as used herein refers to an agent or compound that is to be screened in one or more of the assays described herein. Test agents include compounds of a variety of general types including, but not limited to, small organic molecules, known pharmaceuticals, polypeptides; carbohydrates such as oligosaccharides and polysaccharides; polynucleotides; lipids or phospholipids; fatty acids; steroids; or amino acid analogs. Test agents can be obtained from libraries, such as natural product libraries and combinatorial libraries. In addition, methods of automating assays are known that permit screening of several thousands of compounds in a short period.

[00103] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[00104] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like are encompassed within the term "treating," and refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[00105] "Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[00106] "Viral infectivity" as that term is used herein means any of the infection of a cell, the replication of a virus therein, and the production of progeny virions therefrom.

[00107] A "virion" is a complete viral particle; nucleic acid and capsid, further including and a lipid envelope in the case of some viruses. [00108] In another aspect, the invention provides a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[00109] In some aspects, the compounds of Formula (I) and pharmaceutical compositions disclosed herein are for use in providing a Vif dimerization antagonist, inhibiting Vif self-association in a cell, inhibiting infectivity of a lentivirus in a cell, and/or treating FHV infection or AIDS in a patient.

Methods of Using the Inhibitors of Vif Self- Association

[00110] The inhibitors of Vif self-association described herein can be used for various uses.

[00111] In one embodiment, the inhibitors of Vif self-association described herein can be used in a method for treating or preventing HIV infection or AIDS in a patient. This method involves administering to a patient in need of such treatment or prevention a therapeutically effective amount of a compound of described herein, or a pharmaceutically acceptable salt thereof. The method can further include administering a therapeutically effective amount of at least one other agent for treating HIV selected from the group consisting of HIV reverse transcriptase inhibitors, non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, and HIV integrase inhibitors.

[00112] In one embodiment, the inhibitors of Vif self-association described herein can be used in a method for inhibiting infectivity of a lentivirus in a cell. This method involves contacting a cell with an antiviral-effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

[00113] In one embodiment, the inhibitors of Vif self-association described herein can be used in a method for inhibiting Vif self-association in a cell. This method involves contacting a cell with an inhibitory-effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

[00114] The present invention further provides various methods of using the Vif self- association inhibitors, where the first step involves conducting the screening assay of the present invention to identify the agents as being inhibitors of Vif self-association. Such methods are described below. [00115] In one embodiment, the present invention provides a method for inhibiting infectivity of a lentivirus. This method involves identifying an agent that disrupts Vif self-association by performing the screening method of the present invention, and contacting a cell with an antiviral-effective amount of said agent under conditions effective to disrupt or inhibit multimerization of Vif in the cell, thereby inhibiting infectivity of the lentivirus. In one embodiment, the agent is effective to inhibit dimerization by direct or indirect inhibition of binding of Vif dimers at the Vif dimerization domain.

[00116] In one embodiment, the present invention provides a method for inhibiting Vif self-association in a cell. This method involves identifying an agent that disrupts Vif self-association by performing the screening method of the present invention, and then contacting a cell with an inhibitory-effective amount of said agent under conditions effective to disrupt or inhibit multimerization of Vif in the cell, thereby inhibiting Vif self-association in the cell.

[00117] In one embodiment, the present invention provides a method for treating or preventing HIV infection or AIDS in a patient. This method involves identifying an agent that disrupts Vif self-association by performing the screening method of the present invention, and then administering to a patient in need of such treatment or prevention a therapeutically effective amount of the agent.

Methods of Treatment

[00118] In one embodiment, the present invention provides methods of treating a disease, disorder, or condition associated with a viral infection. Preferably, the viral infection is HTV. The method comprises administering to a subject, such as a mammal, preferably a human, a therapeutically effective amount of a pharmaceutical composition that inhibits Vif self-association.

[00119] The invention includes compounds identified using the screening methods discussed elsewhere herein. Such a compound can be used as a therapeutic to treat an HIV infection or otherwise a disorder associated with the inability to dissociate Vif: Vif complexes.

[00120] The ability for a compound to inhibit Vif self-association can provide a therapeutic to protect or otherwise prevent viral infection, for example HIV infection.

[00121] Thus, the invention includes pharmaceutical compositions. Pharmaceutically acceptable carriers that are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey), the disclosure of which is incorporated by reference as if set forth in its entirety herein.

[00122] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic peritoneally-acceptable diluent or solvent, such as water or 1 ,3 -butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di- glycerides.

[00123] Pharmaceutical compositions that are useful in the methods of the invention may be administered, prepared, packaged, and/or sold in formulations suitable for oral, rectal, vaginal, peritoneal, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.

[00124] The compositions of the invention may be administered via numerous routes, including, but not limited to, oral, rectal, vaginal, peritoneal, topical, pulmonary, intranasal, buccal, or ophthalmic administration routes. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[00125] As used herein, "peritoneal administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Peritoneal administration thus includes, but is not limited to, administration of a

pharmaceutical composition by injection of the composition, by application of the

composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like. In particular, peritoneal administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. [00126] A pharmaceutical composition can consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[00127] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[00128] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical

administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of

pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the

pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[00129] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[00130] Formulations of a pharmaceutical composition suitable for peritoneal administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for peritoneal administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for peritoneal administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g. , sterile pyrogen- free water) prior to peritoneal administration of the reconstituted composition.

[00131] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic peritoneally-acceptable diluent or solvent, such as water or 1 ,3 -butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di- glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[00132] Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in- water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically- administrable formulations may, for example, comprise from about 1 % to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

[00133] Typically, dosages of the compound of the invention which may be

administered to an animal, preferably a human, will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.

[00134] The compound can be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like. Preferably, the compound is, but need not be, administered as a bolus injection that provides lasting effects for at least one day following injection. The bolus injection can be provided intraperitoneally.

Method of Screening

[00135] The current invention relates to a method of screening for an agent (e.g., a small molecule compound) that disrupts Vif self-association (also referred to herein as Vif dimerization and Vif multimerization).

[00136] In one aspect, the present invention provides a method of identifying an agent that disrupts Vif self-association. This method involves (i) providing a Vif: Vif complex comprising a first Vif protein or fragment associated with a second Vif protein or fragment; (ii) contacting the Vif:Vif complex with a test agent under conditions effective to generate a detectable signal when the Vif:Vif complex is disrupted; and (iii) detecting the detectable signal to determine whether or not the test agent disrupts the Vif: Vif complex, wherein disruption of the Vif: Vif complex by the test agent identifies an agent that disrupts Vif self- association.

[00137] A suitable test agent can include a small molecule, a peptide, a polypeptide, an oligosaccharide, a polysaccharide, a polynucleotide, a lipid, a phospholipid, a fatty acid, a steroid, an amino acid analog, and the like. In one embodiment, the test agent is from a library of small molecule compounds.

[00138] In one embodiment, the contacting step comprises incubating the Vif:Vif complex with one type of test agent or more than one type of test agent.

[00139] In another embodiment, the contacting step comprises associating the test agent with the Vif: Vif complex either directly or indirectly.

[00140] The detactable signal may be detected using a detection technique selected from the group consisting of fluorimetry, microscopy, spectrophotometry, computer-aided visualization, and the like, or combinations thereof.

[00141] The detectable signal may be selected from the group consisting of a fluorescent signal, a phosphorescent signal, a luminescent signal, an absorbent signal, and a chromogenic signal.

[00142] In one embodiment, the fluorescent signal is detectable by its fluorescence properties selected from the group consisting of fluorescence resonance energy transfer (FRET), fluorescence emission intensity, fluorescence anisotropy, and fluorescence lifetime (FL).

[00143] In one embodiment, the Vif: Vif complex is provided with a first detection moiety attached to the first Vif protein or fragment and a second detection moiety attached to the second Vif protein or fragment.

[00144] In one embodiment, the first detection moiety and the second detection moiety generate a detectable signal in a distance-dependent manner, so that disruption of the VifVif complex is sufficient to separate the first detection moiety and the second detection moiety a distance effective to generate the detectable signal.

[00145] In one embodiment, the first detection moiety and the second detection moiety comprise a fluorescence resonance energy transfer (FRET) pair, wherein the first detection moiety is a FRET donor and the second detection moiety is a FRET acceptor. The FRET donor and the FRET acceptor can comprise a fluorophore pair selected from the group consisting of EGFP-REACh2, GFP-YFP, EGFP-YFP, GFP-REACh2, CFP-YFP, CFP- dsRED, BFP-GFP, GFP or YFP-dsRED, Cy3-Cy5, Alexa488-Alexa555, Alexa488-Cy3, FITC- Rhodamine (TRITC), YFP-TRITC or Cy3, and the like.

[00146] In one embodiment, the Vif: Vif complex is provided in a host cell co-transfected with a first plasmid encoding the first Vif protein or fragment and a second plasmid encoding the second Vif protein or fragment.

[00147] In one embodiment, the ratio of the first plasmid to the second plasmid is effective to optimize the generation of the detectable signal when the VifVif complex is disrupted. The optimized ratio of the first plasmid to the second plasmid may be about 1 :4, wherein the first plasmid further comprises a signal donor moiety and the second plasmid further comprises a signal quencher moiety.

[00148] In one embodiment, the host cell is stably or transiently co-transfected with the first and second plasmids.

[00149] In one embodiment, the host cell is selected from the group consisting of a mammalian cell, an insect cell, a bacterial cell, and a fungal cell. A suitable mammalian cell can include a human cell.

[00150] In one embodiment, the host cell is a cell culture comprising a cell line that is stably co-transfected with the first and second plasmids. [00151] The method of identifying an agent that disrupts Vif self-association of the present invention can be configured as a high throughput screening assay. The high throughput screening assay can have a Z'-factor of between about 0.5 and about 1.0.

[00152] The method of identifying an agent that disrupts Vif self-association of the present invention can further involve (i) quantitating the detectable signal; (ii) amplifying the detectable signal; and (iii) attaching a first epitope tag to the first Vif protein or fragment and attaching a second epitope tag to the second Vif protein or fragment, wherein said first and second epitope tags are different from one another.

[00153] In one embodiment, the first and second epitope tags are selected from the group consisting of AU1 epitope tags, AU5 epitope tags, Beta-galactosidase epitope tags, c- Myc epitope tags, ECS epitope tags, GST epitope tags, Histidine epitope tags, V5 epitope tags, GFP epitope tags, HA epitope tags, and the like.

[00154] The method of identifying an agent that disrupts Vif self-association of the present invention can further involve subjecting the test agent identified as disrupting the Vif: Vif complex to a validation assay effective to confirm disruption of Vif self-association by the test agents.

[00155] The method of identifying an agent that disrupts Vif self-association of the present invention can further involve subjecting the test agent identified as disrupting the Vif: Vif complex to toxicity, permeability, and/or solubility assays.

[00156] Other methods, as well as variation of the methods disclosed herein will be apparent from the description of this invention. For example, the test compound may be either fixed or increased, a plurality of compounds or proteins may be tested at a single time.

[00157] Based on the disclosure presented herein, the screening method of the invention is applicable to a robust Forster quenched resonance energy transfer (FqRET) assay for high-throughput compound library screening in microtiter plates. The assay is based on selective placement of chromoproteins or chromophores that allow reporting on Vif: Vif complex disruption. For example, an appropriately positioned FRET donor and FRET quencher will results in a "dark" signal when the quaternary complex is formed between Vif dimers, and a "light" signal when the Vif: Vif complex is disrupted.

[00158] The skilled artisan would also appreciate, in view of the disclosure provided herein, that standard binding assays known in the art, or those to be developed in the future, can be used to assess the disruption of Vif self-assocation in the presence or absence of the test compound to identify a useful compound. Thus, the invention includes any compound identified using this method.

[00159] The screening method includes contacting a mixture comprising recombinant

Vif dimers with a test compound and detecting the presence of the Vif:Vif complex, where a decrease in the level of Vif: Vif complex compared to the amount in the absence of the test compound or a control indicates that the test compound is able to inhibit Vif self-association. In certain embodiments, the control is the same assay performed with the test compound at a different concentration (e.g. a lower concentration), or in the absence of the test agent, etc.

[00160] Determining the ability of the test compound to interfere with the formation of the Vif: Vif complex, can be accomplished, for example, by coupling the Vif dimers with a tag, radioisotope, or enzymatic label such that the Vif: Vif complex can be measured by detecting the labeled component in the complex. For example, a component of the complex

32 125 35 14 3

(e.g., a single Vif protein) can be labeled with P, I, S, C, or H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, a component of the complex can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label is then detected by determination of conversion of an appropriate substrate to product.

[00161] Determining the ability of the test compound to interfere with the Vif self- association can also be accomplished using technology such as real-time Biomolecular

Interaction Analysis (BIA) as described in Sjolander et al., 1991 , Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705. BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore, BIAcore International AB, Uppsala, Sweden). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[00162] In more than one embodiment of the methods of the present invention, it may be desirable to immobilize particular Vif dimers to facilitate separation of complexed from uncomplexed forms of one or both of the molecules, as well as to accommodate automation of the assay. The effect of a test compound on the Vif:Vif complex, can be accomplished using any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized micrometer plates, which are then combined with the other corresponding component of the Vif:Vif complex in the presence of the test compound. The mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound material, the matrix is immobilized in the case of beads, and the formation of the complex is determined either directly or indirectly, for example, as described above.

[00163] The test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam et al., 1997, Anticancer Drug Des. 12:45).

[00164] Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al, 1993, Proc. Natl. Acad. USA 90:6909; Erb et al, 1994, Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al, 1993, Science 261 : 1303; Carrell et al, 1994, Angew. Chem. Int. Ed. Engl. 33 :2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061 ; and Gallop et al, 1994, J. Med. Chem. 37: 1233.

[00165] Libraries of compounds may be presented in solution (e.g., Houghten, 1992,

Biotechniques 13 :412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991 , J. Mol. Biol. 222:301 -310; and Ladner supra).

[00166] In situations where "high-throughput" modalities are preferred, it is typical that new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. The current trend is to shorten the time scale for all aspects of drug discovery.

[00167] In one embodiment, high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[00168] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the described invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[00169] Publications discussed herein are provided solely for their disclosure prior to the filing date of the described application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

EXAMPLES & TESTING

[00170] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. EXAMPLE 1

Assay Development for High Throughput Molecular Screening

I. Specific Aims

[00171] The proposed research seeks to develop a novel high throughput screen based on quenched FRET to identify small molecules that bind to the HIV protein known as Viral Infectivity Factor (Vif) and disrupt its self-association. The primary function of Vif is to bind to the host-defense factor known as APOBEC3G (A3G) and induce A3G degradation through a polyubiquitination-dependent proteosomal pathway. Although Vif was discovered more than a decade ago, its requirement was only known as 'being essential for infection of non- permissive cells'. The function of Vif was revealed in the discovery of A3 G as a host-defense factor. A3G binds to single-stranded replicating HIV DNA and introduces multiple dC to dU mutations in the negative strand that templates dG to dA mutations in the protein-coding strand of HIV in the absence of Vif. During the late phase of HIV infection, A3G can become packaged with virions such that it is in position to interact with nascent DNA during viral replication upon infection. Vif prevents A3G viral packaging while also reducing the cellular abundance of A3G thereby promoting viral infectivity.

[00172] Research by our lab and others revealed that multimerization of Vif through a small C-terminal motif, ¹⁶¹PPLP¹⁶⁴, was required for the interaction of Vif with A3G. The critical importance of Vif self-association through this motif was demonstrated with Vif multimerization antagonist peptides that also contained the HIV TAT membrane transduction motif in order to penetrate cells. This peptide prevented co-immunoprecipitation of Vif, markedly reduced Vif-dependent A3G destruction and restored A3G antiviral activity in the presence of Vif. Ultimately small molecules with Vif multimerization antagonistic activity are of greater long-term value in the drug industry. Given the antiviral capacity of the peptide in living cells we believe Vif multimerization is an accessible target in vivo with significance equal to the A3G-Vif interaction. In fact, the C-terminal self-association motif is relatively small and does not overlap with any of the other Vif or A3G interaction domains making it perhaps a more attractive target than the relatively large A3G-Vif interaction domain (residues 40-44 and 52-72) in the N-terminus of Vif.

[00173] We seek to develop a primary and secondary screen and apply 'hit' validation assays for small molecules that disrupt Vif s ability to multimerize (directly or allosterically) in order to protect A3G antiviral activity from Vif mediated inhibition. Given the increasing preponderance of HIV strains that are resistant to the current antiviral drugs on the market, a therapeutic against a novel target such as Vif multimerization would have a significant impact on the worldwide epidemic of HIV/AIDS.

II. Background and Significance

[00174] The virus contains a 10-kb single-stranded RNA genome that encodes three major classes of gene products that include: (z) structural proteins (Gag, Pol and Env); (z^'z) essential trans-acting proteins (Tat, Rev); and (z^'z^'z) "auxiliary" proteins that are not required for efficient virus replication in permissive cells (Vpr, Vif, Vpu, Nef) (reviewed in (1)). There has been a heightened interest in Vif as an antiviral target because of the discovery that the primary function of Vif is to overcome the action of a cellular antiviral protein known as APOBEC3G or A3G (2). In permissive cells (e.g. 293T, SUPT1 and CEM-SS T cell lines) vz -deleted HIV- 1 clones replicate with an efficiency that is essentially identical to that of wild-type virus. However in non-permissive cells (e.g. primary T cells, macrophages, or CEM, H9 and HUT78 T cell lines), vzj-deleted HIV-1 clones replicate with 100- to 1000-fold reduced efficiency (3-8). The failure of Vz/^deficient HIV-1 mutants to accumulate reverse transcripts and generate integrating provirus in the non-permissive cells is due to the ability of A3G to interact with viral replication complexes and impair their progression as well as A3G mutagenic activity on nascent proviral single-stranded DNA (2,9-11).

[00175] Discovery of A3G. The function of A3 G (formerly named CEM 15) as an antiviral host factor was discovered in 2002 in experiments designed to identify host cell factors in non-permissive cells that would necessitate the expression of Vif (2).

Heterokaryons consisting of non-permissive and permissive cells retained the non-permissive phenotype for Vif-deficient virus, demonstrating expression of a dominant neutralizing factor in non-permissive cells (3,4). Subtractive transcriptome analysis identified a cDNA encoding A3G (2) as a member of the APOBEC family of cytidine deaminases active on single- stranded nucleic acids (12,13). Transfection of permissive cells with A3G cDNA was necessary and sufficient for conversion to the non-permissive phenotype for Vif-deficient HIV-1 infectivity (2).

[00176] A3G antiviral mechanism. Multiple labs have characterized a deaminase- dependent antiviral function of A3G and its packaging into HIV virions (9-11). Sequencing of proviral genomes revealed that cells infected with virions containing A3G had dG to dA hypermutations throughout the protein encoding positive strand (9-11), consistent with A3G dC to dU mutation of the negative strand during reverse transcription (1 1). Furthermore, A3G acts processively 3' to 5' along the minus-strand HIV DNA template (14,15) with mutations occurring in regions where the HIV DNA is single-stranded for the longest period of time during HIV reverse transcription (16,17). The hypermutations introduce multiple premature stop codons and codon sense changes that negatively affect the virus (9-11). The dU mutations in minus-strand viral DNA can trigger the uracil base excision pathway mediated by uracil DNA glycosylase (UDG) that is recruited into virions (18,19), leading to cleavage of viral DNA before integration into host DNA (10). Reduction in proviral DNA can also occur through what has been proposed to be a physical block to reverse transcription by A3G (5,6,20-22).

[00177] Vif-dependent inhibition of A3G antiviral activity. Vif-expressing viruses overcome A3G by suppressing viral packaging of A3G and targeting it for proteosomal degradation (23-26). Vif promotes A3G degradation through its ability to bind to the ubiquitination machinery. A consensus SOCS (suppressor of cytokine signaling)-box in the C-terminus of Vif binds to the Elongin C subunit of the E3 ubiquitin ligase complex that also contains Cullin 5 and Elongin B (26). Vif also contains a zinc binding HCCH motif that confers an interaction with Cullin 5 (27). Vif serves as a bridge for A3G to Elongin C and Cullin 5 in the E3 ubiquitin ligase complex, leading to polyubiquitination of both Vif and A3G (25-27). Recent studies have shown that only polyubiquitination of Vif on one or more of its 16 lysine residues is required for proteosomal degradation of A3G and Vif (28). Site- directed mutagenesis demonstrated that alteration of a single amino acid within A3G could affect Vif interaction (29-31). An aspartic acid at position 128 in A3G is required for HIV-1 Vif to degrade human A3G whereas a lysine at position 128 is required for simian immunodeficiency virus from African green monkey (SIVagm) Vif to degrade agmA3G (29- 31). Alanine scanning mutation analysis of A3G revealed that residues adjacent to D128 are also crucial for Vif interaction with A3G, specifically proline 129 and aspartic acid 130 (32). On the other hand, relatively large regions within the N-terminus of Vif are involved in its interaction with A3G. Deletion and point mutation analyses of Vif identified residues 40- YRHHY-44 and 52-72 as being critical regions within Vif responsible for A3G interaction and degradation (33-36).

[00178] Vif self-association. An analysis of Vif deletion mutants in the Zhang lab at

Thomas Jefferson University in 2001 revealed that residues 151-164 were critical for Vif multimerization, an interaction that was required for infectivity of non-permissive cells (37). Subsequent phage display revealed that peptides with a P P motif bound to PPLP within Vif, and in doing so blocked Vif multimerization in vitro (38). Upon linkage of a cell transducing peptide to PPLP containing peptides of Vif, both the Zhang lab (using antennapedia homeodomain, RQIKIWFQNRRM WKK (SEQ ID NO:l)) and our lab (using HIV TAT transduction domain, YGRKKRRQRRRG (SEQ ID NO:2)) revealed that these peptide chimeras transduced cells and blocked live HIV infectivity (38,39). A3G incorporation into viral particles was enhanced in the presence of the peptide resulting in marked suppression of HIV infectivity (39). Donahue et al. demonstrated that mutating the PPLP motif to AAAP enabled A3G antiviral activity. More importantly, they showed through co- immunoprecipitation analysis that the Vif multimerization mutant had significantly reduced interaction with A3G. On the other hand, the Vif mutant retained interactions with Elongin C and Cullin 5 in a manner equivalent to wild-type Vif (40). The data reveal that Vif self- association is essential for both viral infectivity and Vif interaction with A3G. Moreover, the Vif multimerization domain can be disrupted in vivo, demonstrating its potential as a drug target.

[00179] Advantage of targeting Vif self-association. To date, four characteristics of

Vif-A3G interaction have been studied in enough detail to make them of potential interest as drug targets. These are: (i) Vif self-association, ( ) the Vif surface and (Hi) the A3G surface that contribute to the interface of Vif-A3G complexes, and (zv) Vif polyubiquitination.

[00180] Vif polyubiquitination may be the most difficult functionality of Vif to selectively target, because there are 16 lysines on Vif that are capable of being

polyubiquitinated (28). Small molecules that affect ubiquitination of Vif are likely to be toxic given that ubiquitin-mediated degradation is an essential part of the cell and 'hits' on this target are likely to have off-target effects leading to toxicity. Moreover, Vif bound to A3G that is not degraded would likely still prevent A3G viral packaging.

[00181] There has been some promising work involving the Vif-A3G interface. The

Gabuzda lab evaluated 1 -mer peptides of Vif regions for their ability to antagonize the Vif- A3G interaction. A peptide containing amino acids 57-71 of Vif was identified that blocked Vif-A3G interaction in vitro (41). However the efficacy of this peptide as an antiviral in vivo is yet to be determined. The Rana lab has identified a small molecule that is capable of blocking Vif-dependent degradation of A3G in HEK 293T cells through HTS based on Vif- dependent degradation of a fluorescently tagged A3G. The molecular target of the small molecule and its mechanism of action are unclear (42). [00182] Considering A3G as a drug target, the major caveat to targeting the N-terminal region of A3G involved in Vif binding is the fact that the same region of A3G is also involved in crucial interactions for its cellular and antiviral activity. Deletion analysis revealed that residues 104-156 of A3G were crucial for HIV Gag binding and viral packaging (43,44). Also, scanning alanine mutagenesis demonstrated that amino acids 124-YYFW-127 were especially important for viral packaging (32). The Smith lab recently showed that there is a cytoplasmic retention signal in residues 113-128 of A3 G that interacts with an as-of-yet unidentified cytoplasmic partner that prevents A3G from entering the nucleus (45). The related proteins, APOBEC1 and AID, must traffic to the nucleus but their nuclear import and access to genomic DNA are strictly regulated (46) to prevent their potential genotoxicity due to unregulated DNA deaminase activity (47-53). Therefore, small molecules that prevent A3G binding to Vif at residues 128-130 of A3G (32) have the potential negative outcome of affecting A3G viral packaging or enabling A3G access to the genome.

[00183] We propose that the Vif multimerization domain is an attractive target for drug development. Blocking the Vif self-association has proven to be an accessible target in vivo and disrupting Vif self-association prevents Vif-A3G interaction in a manner that will prevent the degradation of A3G and preserve its antiviral activity (38,39). Preliminary data will demonstrate the practicality of using Vif for the development of HTS that are biased for Vif multimerization.

[00184] Based on these considerations, the goal of this proposal is to develop a human cell-based homogenous assay as a primary HTS and an orthogonal secondary screen for small molecules that antagonize Vif dependent A3G degradation. Viral infectivity assays and A3G viral encapsidation will serve as functional endpoints to validate hits obtained from a preliminary library screening.

EXAMPLE 2

Vif Self-Association as a Target and Related Assays

[00185] Studies with a peptide containing the Vif multimerization motif and the HIV

TAT transduction motif demonstrate that Vif self-association is accessible in vivo. The peptide prevents live HIV viral infection of H9 and MT-2 T cell lines that endogenously express A3G. After twenty days of infection the peptide blocks viral infectivity, reducing reverse transcriptase (RT) activity in cell supernatants to levels that were on par with those from no virus cell control or cells treated with the potent antiviral AZT. The reduction in infectivity is dependent on the presence of Vif and A3G (39) and the peptide specifically allows 2.6-fold more A3G to enter viral particles as evident when the A3G western blot signals of (+) and (-) peptide are normalized for p24 gag recovery. This demonstrates that targeting Vif self-association alleviates the Vif-dependent inhibition of A3G viral packaging.

[00186] Development of the quenched FRET primary screen. FIG. 1 provides a visual representation of the critical path associated with the Primary in-cell quenched FRET Assay for Vif Dimerization Antagonists (HTS). EGFP is a FRET donor and REACh2 (Resonance Energy Accepting Chromoprotein 2) is a non-fluorescent FRET acceptor (54). The non- fluorescent REACh2 is able to quench EGFP signal in a distance-dependent manner when they are linked to interacting domains. However, if there is no interaction, EGFP and

REACh2 are not proximal and quenching will not occur. This is an ideal system for HTS in which the default condition is quenched signal due to interacting Vif molecules linked to the FRET pair. As illustrated in the schematic provided in FIG. 2, a small molecule 'hit' will produce a positive fluorescent signal by interfering with Vif self-association and alleviating the quench.

[00187] The system employs the use of HEK 293T cells due to their high transfection efficiency (up to 90% with Turbofect (Thermo Fisher) transfection reagent) and Vif s established functionality in these cells demonstrated by many investigators (24,29,32,42). Transient transfection is necessary for EGFP-Vif since over expression of Vif inhibits the cell-cycle and stable cell lines are difficult to establish.. In addition, transiently transfected cells have the ability to maintain an expression level of Vif-HA-REACh2 that is higher than EGFP-V5-Vif to ensure maximum amount of quenched protein in the cell. HTS analysis of Vif-Vif multimerization through quenched FRET utilizes Vif-HA-REACh2 (quencher) and EGFP-V5-Vif (fiuorophore) transfected at an optimized ratio. The interaction of Vif molecules enables quenching of EGFP signal by REACh2. Control experiments with either mutations within the PPLP domain crucial for Vif-Vif interaction (4A mutant) or peptides that mimic this domain prevented Vif-Vif interaction and consequently resulted in a stronger fluorescence signal (a hit produces a positive signal) (see FIG. 3A). The positive control peptide confirmed that Vif-Vif multimerization was responsible for the quenched FRET along with western blotting showing equivalent expression of mutants or peptide treated cells when compared to control (see FIG. 3B). These confirmations established that the assay was optimized for HTS. The screen has been optimized to yield CVs less than 3% and a Z' factor greater than 0.6 in 96-well and 384-well formats. [00188] FIG. 4 provides images from an in-cell secondary screen for Vif-dependent

A3G degradation. A3G-mCherry is stably expressed in 293T cells under puromycin selection. 50 ng of Vif was transiently transfected into the cells in 384-well format with Turbofect. 4 hours after transfection the chemistries were added to cells. 24 hours after chemistries were added the mCherry signal was read on a Biotek Synergy 4 plate reader. The signal from plated cells not transfected with Vif was averaged and set at 100% (FIG. 4, left image), and cells transfected with Vif and treated with DMSO only were averaged and set at 0% (FIG. 4, right image). A chemistry that inhibits Vif s ability to chaperone A3G to the proteasomal degradation pathway would result in an increased mCherry signal compared to the DMSO only control, and any signal that is much higher than the no-Vif positive control is likely to be due to autofluorescence from the chemistry itself.

Exemplary Compounds According to and for Use in the Inventive Methods

[00189] Compounds according to embodiments of the present invention were tested in a Vif-dependent A3G-mCherry Degradation Screen. FIG. 5 depicts results from the testing, respective to six compounds (CPD #'s 42, 88, 43, 89, 44, and 78 from Table I). As shown in FIGS. 6A and 6B, CPD 78 shows dose dependence in both the primary screen (FIG. 6A, showing a dose dependent disruption of Vif: Vif FRET) and the secondary screen (FIG. 6B, showing dose dependent inhibition of A3G-mCherry degradation through Vif). The primary screen is represented as a change in RFU over the quenched control (ARFU). The secondary screen is compared to the +Vif (0%) and -Vif (100%) controls.

[00190] FIG. 7 depicts a chart showing testing results demonstrating Vif and A3G-

Dependent Effect on Pseudotyped HIV Infectivity Expressed as % of Control, for CPD 78. Pseudotyped infectivity experiments were performed in 6 well format in order to obtain enough virus to do viral particle purifications for western blot detection of A3G in the viral particle. The antiviral activity of the hits in a single-round infection with pseudotyped HIV were conducted using HEK293T producer cells +/- A3G and viruses that are +/- Vif. The wild type HIV proviral vector codes for all HIV genes except nef (replaced with EGFP) and env. The AVif proviral vector is identical to wild type except that it contains a stop codon early within the Vif gene. AVif +A3G is a strong positive control for this assay because without Vif present, A3G is able to be encapsidated within viral particles and have strong antiviral activity(see FIG. 7). Alternatively, in the absence of A3G, both wild type and AVif viruses should have high infectivity (see FIG. 7). Single-round infectivity assays utilized transient transfection of the viral vectors with VSV-G coat protein vector and V5-A3G in the +A3G conditions with Fugene HD (Promega). Proviral DNA:VSV-G:A3G were added to cells with a ratio of 1 :0.5 :0.08 which establishes levels of A3G that are comparable to endogenous A3G. These virus producer cells were dosed with compounds four hours after transfection and viral particles were harvested from the media 24 hours after transfecting by filtering through a 0.45 -micron syringe filter. Viral load was then normalized with a p24 ELISA (Perkin Elmer).

[00191] The infections utilized TZM-bl reporter cells that contain stably integrated luciferase that is driven by the HIV-LTR promoter, therefore luciferase is expressed upon successful HIV infection. Triplicate infections in 96-well plates at 10,000 cells/well with 500 pg p24/well proceeded for 48 hours before the addition of SteadyGlo™ Reagent (Promega) to each well for 30 minutes. Luminescence was measured as a quantitative metric for changes in infectivity with each compound as compared to controls, in which relative luminescence units (RLU) with no compounds are set to 100%.

[00192] Although all compounds to this point in screening have shown little to no toxicity in the Primary and Secondary Screens, when cells are challenged with viral production they are more sensitive to toxicity from compounds since cells have to combat viral proteins overtaking of cellular pathways and any adverse effects from the compounds. This manifests through cells unable to make virus along with a lifting off of the adherent cells from the plate; with not enough virus to move into infectivity assays and cytotoxicity displayed by cells in the presence of compounds during viral production the compound is flagged as toxic and off target. If the producer cells are healthy and make enough virus for infectivity testing there are three possible outcomes and two are undesirable for any compound to go forward. First is a lack of antiviral activity and second is antiviral activity that is independent of Vif and A3G expression. Finally, The gold standard is antiviral activity that is Vif and A3G dependent. This is best represented as a differential between infectivity in the presence of Vif and A3G vs the absence of Vif and A3G.

[00193] FIG. 8 depicts a CPD 78 +Vif & A3G Pseudotyped Infectivity Curve.

[00194] FIG. 9 depicts results from testing where +/-Vif virus + A3G controls and +Vif virus + A3G produced in 293T cells in the presence of 25-0.25 mM of CPD 78 were purified through a 20% sucrose cushion and western blotted for p24 (to show equal viral load in gel) and V5 tagged A3G. As demonstrated, CPD 78 increases A3G in Viral Particles. [00195] FIGS. 10A-F depict efficacy results of CPD 78 tested in 7-day PBMC

Infections. Methods involve stimulating human purified PBMCs with PHA & IL-2. The stimulated PBMCs were infected with a panel of HIV- 1 isolates encompassing various clades, as well as various isolates within a specific clade. The infected PBMCs werer treated with various concentrations of test compounds. Virus replication was measured by cell free reverse-transcriptase activity in the culture supernatant. The potency of the compound is assessed by the IC50 and level of maximum inhibition.

[00196] Table II depicts additional efficacy results of CPDS 40, 86, 41, and 87 (along with results for AZT), in 7-day PBMC infections. As shown, CPDS 40 and 86 stand out as the best among all clades with CPD 41 being slightly less effective and CPD 87 showing low to no antiviral activity across all clades (except Subtype A). Toxicity is not an issue in PBMC up to 100 μΜ for all tested compounds.

[00197] FIG. 11 shows toxicity results for CPD 78 following testing for PBMC cytotoxicity on day 7 after compound addition as measured by a tetrazoiium compound that is bioreduced by cells to a colored formazan product by dehydrogenase enzymes in

metaboiically active ceils.

[00198] Table III provides additional toxicity data relating to CPD 78. In the table, NR

= No Response (up to 50 μιη).

TABLE III: CPD 78 Toxicity

Tests AC50 Results (Day)

[00199] Table IV shows comparative plasma protein binding (PPB) results for CPD 78 as compared to propanolol and warfarin. TABLE IV m n pm M& plasma

Test frmstittm f act on

C mt ID C me.

o ranolol mouso 18.7% 83.3% 102% arfarin mouse 8.0% 84 J%

CPD 78 mo se 3.0%? 96.5% 88,2%

[00200] FIGS. 12A and 12B show dose dependent results for anti-HIV compounds according to embodiments of the invention in both a primary screen (FIG. 12A, showing a dose dependent disruption of Vif: Vif FRET) and a secondary screen (FIG. 12B, showing dose dependent inhibition of A3G-mCherry degradation through Vif). The primary screen is represented as a change in RFU over the quenched control (ARFU). The secondary screen is compared to the +Vif (0%) and -Vif (100%) controls.

[00201] FIG. 13 is a chart providing data for Vif and A3G-Dependent Effect on Pseudotyped HIV Infectivity Expressed as % of Control for several anti-HIV compounds according to embodiments of the invention. As shown, compounds 40, 86, 41, 87, and 78 all demonstrated high differentials between +/- A3G and Vif.

[00202] FIG. 14 shows results from additional testing performed on compounds 40, 86,

41, 87, and 78. Specifically, +/-Vif virus + A3G controls and +Vif virus + A3G produced in 293T cells in the presence of 25, 12.5 & 6.25 μΜ of CPDS 40, 86, 41, 87, and 78 were purified through a 20% sucrose cushion and western blotted for p24 (to show equal viral load in gel) and V5 tagged A3G. As can be seen, while all compounds were active, CPDS 40, 86, 41, and 87 were particularly efficacious in increasing A3G in viral particles.

Tables V and VI show results from testing anti-HIV compounds according to embodiments of the invention in Primary, Secondary and Toxicity Screens. The Primary Screen and Secondary Screen A were performed as described above (see FIGS. 3&4) at concentrations of 20, 10, 2 and 1 μΜ. Secondary Screen B is a version of the Vif-dependent A3G-mCherry Degradation screen (FIG. 4) in which Vif is expressed from a stably integrated lentivirus with Vif expression from the TET-ON inducible promoter that is turned on in the presence of doxycycline (as opposed to being transiently tranfected) screened at concentrations of 20, 10, 2 and 1 μΜ. Secondary Screen C is a FqRET based screen as in the Primary Screen (FIG. 3), but instead of detecting an interactions between EGFP-VIF and VIF-REACh2 it detects an interaction between EGFP-A3G and a mutant of VIF called AC Vif that has the SOC Box deleted so that it can still interact with A3G but is unable to shuttle A3G to the proteasome since it cannot bind to EloC through the SOCS Box. This screen was also performed at concentrations of 20, 10, 2 and 1 μΜ for each compound. 293T Tox Screen tests 48 hour toxicity at 40, 20, 4, and 2 μΜ, measured by a tetrazolium compound that is bioreduced by ceils to a colored formazan product by dehydrogenase enzymes in metabolically active cells, percentages are based on the DMSO control set to 100%.

TABLE V

TABLE VI

[00203] Fluorescence anistropy. Fluorescence anisotropy of 150 nM AlexaFluor-488 labeled Vif was measured after incubation with anti-HIV compounds according to embodiments of the invention. FIG. 15 depicts results for CPDS 78, 86, 87, and 38 (37 °C, 1 hr) with a Synergy4 plate reader (BioTek). A decrease in anisotropy is indicative of smaller- sized particles, suggesting disruption of Vif oligomers. CPD 87 has a reduced effect on anisotropy compared to CPD 78 and CPD 86. CPD 38 had an even further reduced effect consistent with its reduced activity in primary and secondary screens (see FIG. 16 A & B).

[00204] As discussed above, the pseudotyped infectivity screen showed that antiviral activity is linked to A3G and Vif expression and increased A3G in viral particles. Since some of the anti-HIV compounds according to embodiments of the invention share some similar chemical features compared to the N RTi Rescriptor (see below), in order to rule out anti-RT activity an in vitro RT assay was performed. As summarized in Table VII, all tested compounds demonstrated weak or no anti-RT activity compared to NNRTi controls.

Rescriptor TABLE VII

[00205] FIGS. 16A and 16B are charts showing dose dependence of CPDS 78, 35, 81 , 36, 82, 37, 83, and 38 in both a Vif-dependent A3G-mCherry Degradation Screen (Fig. 16A) and a FqRET VifVif Interaction Screen (FIG. 16 B).

[00206] FIGS. 17A and 17B are charts showing dose dependence of CPDS 80, 91 , 79, 90, 82, and 45 in both a Vif-dependent A3G-mCherry Degradation Screen (Fig. 17A) and a FqRET VifVif Interaction Screen (FIG. 17 B) and reveal that a methyl at position R⁵ has more activity then a hydrogen.

[00207] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprise" (and any form of comprise, such as "comprises" and "comprising"), "have" (and any form of have, such as "has" and "having"), "include" (and any form of include, such as "includes" and "including"), "contain" (and any form contain, such as "contains" and "containing"), and any other grammatical variant thereof, are open-ended linking verbs. As a result, a method or device that "comprises", "has", "includes" or "contains" one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more steps or elements. Likewise, a step of a method or an element of a device that "comprises", "has", "includes" or "contains" one or more features possesses those one or more features, but is not limited to possessing only those one or more features. Furthermore, a device or structure that is configured in a certain way is configured in at least that way, but may also be configured in ways that are not listed.

[00208] As used herein, the terms "comprising," "has," "including," "containing," and other grammatical variants thereof encompass the terms "consisting of and "consisting essentially of."

[00209] The phrase "consisting essentially of or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

[00210] All publications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

[00211] Subject matter incorporated by reference is not considered to be an alternative to any claim limitations, unless otherwise explicitly indicated.

[00212] Where one or more ranges are referred to throughout this specification, each range is intended to be a shorthand format for presenting information, where the range is understood to encompass each discrete point within the range as if the same were fully set forth herein.

[00213] While several aspects and embodiments of the present invention have been described and depicted herein, alternative aspects and embodiments may be affected by those skilled in the art to accomplish the same objectives. Accordingly, this disclosure and the appended claims are intended to cover all such further and alternative aspects and embodiments as fall within the true spirit and scope of the invention.

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[00214] Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

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Claims

WHAT IS CLAIMED IS:

1. A method for treating HIV infection or AIDS in a patient, said method comprising administering to the patient a therapeutically effective amount of a compound of Formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Y is selected from -S0₂-NR^aR^b and -NH-S0₂-(CH₂)_n-R¹; R^a is H; and R^b is -(CH₂)_n-R¹; or, taken together, R^a and R^b, together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring; n is 0 or 1;

R¹ is an aryl or heteroaryl ring system, wherein said aryl or heteroaryl ring system is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_₆alkyl, Ci_ ₆alkoxy, hydroxyl, oxo, -NHC(0)Ci_₆alkyl, and -C(0)NHCi_₆alkyl;

R² is selected from halo, Ci_₆alkyl, and haloCi_₆alkyl;

R³ and R⁴ are hydrogen, or, taken together, R³ and R⁴, together with the carbon atoms to which they are attached, form a benzene ring; and

R⁵ is selected from hydrogen and methyl.

The method according to claim 1 , wherein R is methyl The method according to claim 1, wherein R is a residue formula:

wherein R ^a, R , and R ^c are independently selected from hydrogen, halo, Ci_₆alkyl, and Ci_ ₆alkoxy.

4. The method according to claim 3, wherein R ^a, R , and R ^c are independently selected from hydrogen and methyl.

5. The method according to any one of the preceding claims, wherein the compound of Formula (I) or pharmaceutically acceptable salt thereof is a compound of the Formula (IA):

(IA) harmaceutically acceptable salt thereof.

6. The method according to claim 1, wherein said compound or pharmaceutically acceptable salt thereof is administered with a pharmaceutically acceptable earner.

7. The method according to claim 1 further comprising: administering a therapeutically effective amount of at least one other agent for treating HIV selected from the group consisting of HIV reverse transcriptase inhibitors, non- nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV attachment inhibitors, CCR5 inhibitors, CXCR4 inhibitors, HIV budding or maturation inhibitors, and HIV integrase inhibitors.

8. A method for inhibiting infectivity of a lentivirus in a cell, said method comprising:

contacting a cell with an antiviral-effective amount of a compound of

Formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein:

Y is selected from -S0₂-NR^aR^b and -NH-S0₂-(CH₂)_n-R¹;

R^a is H; and R^b is -(CH₂)n-R¹; or, taken together, R^a and R^b, together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring; n is 0 or 1 ; R¹ is an aryl or heteroaryl ring system, wherein said aryl or heteroaryl ring system is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_₆alkyl, Ci_ ₆alkoxy, hydroxyl, oxo, -NHC(0)d_₆alkyl, and -C(0)NHCi_₆alkyl; selected from halo, Ci_₆alkyl, and haloCi_₆alkyl;

R³ and R⁴ are hydrogen, or, taken together, R³ and R⁴, together with the carbon atoms to which they are attached, form a benzene ring; and selected from hydrogen and methyl.

9. The method according to claim 8, wherein said compound or pharmaceutically acceptable salt thereof is administered with a pharmaceutically acceptable carrier.

10. The method according to claim 8, wherein said compound or pharmaceutically acceptable salt thereof is effective to disrupt or inhibit multimerization of Vif in the cell, thereby inhibiting infectivity of the lentivirus.

11. The method according to claim 8, wherein the lentivirus is selected from the group consisting of HIV- 1 and HIV-2.

A method for inhibiting Vif self-association in a cell, said method comprising:

contacting a cell with an inhibitory-effective amount of a compound of

Formula (I):

(I) or a pharmaceutically acceptable salt thereof, wherein: Y is selected from -S0₂-NR^aR^b and -NH-S0₂-(CH₂)_n-R¹;

R^a is H; and R^b is -(CH₂)_n-R¹; or, taken together, R^a and R^b, together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring; n is 0 or 1;

R¹ is an aryl or heteroaryl ring system, wherein said aryl or heteroaryl ring system is substituted with 0, 1, 2, or 3 substituents individually selected from halo, C^alkyl, Ci_ ealkoxy, hydroxyl, oxo, -NHC(0)Ci_₆alkyl, and -C(0)NHCi_₆alkyl;

R² is selected from halo, Ci_₆alkyl, and haloCi_₆alkyl;

R³ and R⁴ are hydrogen, or, taken together, R³ and R⁴, together with the carbon atoms to which they are attached, form a benzene ring; and

R⁵ is selected from hydrogen and methyl.

13. The method according to claim 12, wherein said compound or pharmaceutically acceptable salt thereof is administered with a pharmaceutically acceptable earner.

14. The method according to claim 12, wherein said compound of Formula (I) or pharmaceutically acceptable salt thereof is effective to disrupt or inhibit

multimerization of Vif in the cell, thereby inhibiting Vif self-association in the cell.

A Vif dimerization antagonist comprising a compound of Formula (I)

(I) or a pharmaceutically acceptable salt thereof, wherein:

Y is selected from -S0₂-NR^aR^b and -NH-S0₂-(CH₂)_n-R¹;

R^a is H; and R^b is -(CH₂)n-R¹; or, taken together, R^a and R^b, together with the nitrogen atom to which they are attached, form a 5- or 6-membered heterocyclic ring; n is 0 or 1;

R¹ is an aryl or heteroaryl ring system, wherein said aryl or heteroaryl ring system is substituted with 0, 1, 2, or 3 substituents individually selected from halo, Ci_₆alkyl, Ci_ ₆alkoxy, hydroxyl, oxo, -NHC(0)d_₆alkyl, and -C(0)NHCi_₆alkyl; R is selected from halo, Ci_₆alkyl, and haloCi_₆alkyl;

R³ and R⁴ are hydrogen, or, taken together, R³ and R⁴, together with the carbon atoms to which they are attached, form a benzene ring; and

R⁵ is selected from hydrogen and methyl.