HIVINHIBITORS

RELATED APPLICATIONS

[0001] The present application claims priortiy under 35 USC 119(2) to United States Provisional application serial number 60/842,219, filed September 3, 2006, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Significant progress has been made in the development of therapies and treatment regimens for HIV infection. The end of the twentieth century saw the introduction of a variety of nucleoside and nucleotide analogs (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) that, when used in combination, can have significant effectiveness against HIV. However, currently approved regimens are complex and suffer from poor patient compliance as well as a variety of undesirable side effects. Moreover, the emergence of resistance poses significant challenges, as intra-class resistance is common for all three primary classes of HIV inhibitors. There remains a need, therefore, for the identification of new HIV inhibitory agents, and particularly for new classes of agents (i.e., for agents that act by new mechanisms of action).

[0003] More recently, certain peptide agents have been identified that act as inhibitors of HIV fusion and/or entry. One such agent, enfurviritide (also known as T-20 or Fuzeon), was approved for administration in 2003. Unfortunately, enfurvitide has proven very expensive to manufacture, making it a very expensive therapeutic. Moreover, enfurvitide is given by twice-daily injection rather than by more palatable routes (e.g., oral). Injection-site reactions can be very painful, and other problematic side effects (e.g., incidence of bacterial pneumonia) have been observed.

[0004] The need for the development of new HIV inhibitors agents remains strong. Systems for identifying new agents would also be valuable.

SUMMARY OF THE INVENTION

[0005] The present invention provides a system for identifying HIV fusion inhibitors, and particularly for identifying small molecule compounds that inhibit HIV fusion. In particular, the invention provides a system for identifying small molecules that inhibit binding of a peptide that corresponds to a portion of the gp41 outer layer with its site on the gp41 inner core. Without wishing to be bound by any particular theory, we propose that such inhibitors

can interfere with the fusion of virus to host cells by blocking conformational rearrangement of gp41. Inventive inhibitors are therefore referred to herein as "HIV fusion inhibitors". [0006] The present invention provides non-peptide HIV fusion inhibitors, and furthermore provides small molecule HIV fusion inhibitors. In particular, the present invention provides HIV fusion inhibitors having the following general formula (I) as further defined herein:

(I) and pharmaceutically acceptable derivatives thereof; wherein n is an integer from 0-5;

R ¹ is hydrogen, halogen, cyano, nitro, or an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aromatic or heteroaromatic moiety; and

R is hydrogen, halogen, cyano, amino, aminoacyl, nitro, or an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aromatic or heteroaromatic moiety. [0007] The present invention also provides non-peptide HIV fusion inhibitors having the following general formula (II) as further defined below:

(II) and pharmaceutically acceptable derivatives thereof; wherein m and p are independently integers from 0-5; and

R ² are R ³ are independently hydrogen, halogen, cyano, nitro, or an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aromatic or heteroaromatic moiety. [0008] In some embodiments, inventive HIV fusion inhibitors inhibit formation of a post- fusion-like assembly with a K _app of about 5 μM or below. In some embodiments, inventive HIV fusion inhibitors inhibit envelope mediated membrane fusion in cell-cell fusion and/or in viral infectivity assays. In some embodiments, inventive HIV fusion inhibitors inhibit fusion across a broad range of clades, including both M- and T-tropic strains. In some embodiments,

inventive HIV fusion inhibitors bind in a highly conserved, hydrophobic pocket on the inner core of the gp41 trimer.

BRIEF DESCRIPTION OF THE DRAWING

[0009] Figure 1 presents a model for fusion by the HIV envelope glycoprotein. As depicted, binding of gpl20 to CD4 and co-receptor (Panel 1) triggers a conformational change that releases the grip of gpl20 (red) on gρ41 (blue). The latter extends (Panel 2) so that its fusion peptide (green) inserts into the target-cell membrane (green bar, top). As the "outer-layer" region of gp41 (dark blue) zips up along the outside of the "inner layer" (light blue: a three-chain, α-helical coiled-coil), the viral membrane (tan) and cell membrane (green) are drawn together (panels 3 and 4). It is this step that is inhibited by T- 20/enfuvirtide. The formation of a hemifusion stalk (panel 4) and a fusion pore (panel 5) complete the membrane-fusion reaction.

[0010] Figure 2 shows association of an outer-layer peptide with gp41 -5 as a screening assay. Panel A shows the sequence of a single-chain polypeptide that can fold into a model for five of the six helices in the postfusion form of the gp41 ectodomain. Panel B is a diagram illustrating that a fluoresceinated, outer-layer peptide can bind to gρ41-5. Panel C shows fluorescence-anisotropy binding curve for the association of the outer-layer peptide with gp41-5. The fraction bound, f, is plotted as a function of gp41-5 concentration (nM), at constant concentration of peptide (5 nM).

[0011] Figure 3 shows results of an inventive high-throughput screen. Panel A shows compounds identified as strong inhibitors of outer-layer peptide binding (5M038, 5M030, 5M041, and S2986), as well as compounds in the chemical library related to compound 5M038. Panel B presents a graph showing inhibition of peptide binding for five of the compounds shown in Pane; A. The fraction bound, θ, is plotted as a function of inhibitor concentration. Symbols correspond to those beneath the various compounds in Panel A. The curves show optimal sigmoidal fits to the data for 5M030 and 5M038, the only two compounds that were sufficiently soluble to yield reliable plots. Compound S2986 had an IC ₅₀ of about 5 μM in this assay (data not shown).

[0012] Figure 4 presents plots illustrating inhibition of cell-cell fusion by 5M038 (Panel A), 5M041 (Panel B), and 6M007 (Panel C). The fractional degree of fusion, determined by the lucjferase assay described in Methods, is plotted as a function of inhibitor concentration (diamonds). Squares show an experiment in which luciferase was expressed directly in the

target cells, independent of fusion, as a control for toxicity and other side effects of the inhibitors. Curves are fractional inhibition, normalized for these toxicity effects. All points are the average of 3 experiments (error bars).

[0013] Figure 5 is a graph illustrating the effect of 5M038 during the time course of HIV infection, MT-2 cells were infected with HIV in the presence and absence of either 30 μM (square) or 50 μM (circle) 5M038. Time points were taken at 3, 5 and 7 days and viral infection was measured by the amount of p24 antigen produced. Enfuvirtide, a potent peptidic viral entry inhibitor, was also tested (11 nM) and used for comparison (diamonds). All points are the average of 3 experiments (bars represent standard errors). [0014] Figure 6 presents NMR measurements of the association of 5M038 with three- chain coiled-coils containing segments of the inner core of postfusion gp41. Panel A is a chemical diagram showing the various protons detected in the spectra. Panel B shows a spectrum of free 5M038. Panels C-E show spectra of 5M038 in the presence of coiled-coils containing residues 34-50 of gρ41 (Panel C), residues 41-57 (Panel D), and residues 54-70 (Panel E). There is significant broadening and shifting of the resonance peaks only in the last of the four spectra, showing that 5M038 associates selectively with the part of the gp41 inner core formed by residues 54-70.

[0015] Figure 7 shows two possible orientations of 5M038 based on electron density maps of co-crystal structure with gp41-5 (for clarity only the inner core is shown). The pocket is formed by residues surrounding Leu-57, Trp60 and Lys63 which together form a deep cavity occupied by Trpl 17, trpl20 and Ilel24 in the post fusion structure. The density in the pocket was weak, due either to low occupancy of 5M038 or to the effects of crystal twinning (26%). The strongest density, probably corresponding to the electron-rich trifluormethyl groups, was used to position the molecule, placing one -CF ₃ group within 4.1 angstroms of Trp60 and 3.1 angstroms from Gln66, possibly forming a hydrogen bond. The second ~ CF ₃ group contacts a backbone carbonyl (3.7A) of chain A, also within 4 angstroms of Gln66. In orientation 1, the phenyl ring of 5M038 contacts Leu70 (3.8A), while in orientation 2 the phenyl ring contacts Gln64 (4.2 A). This model places considerable importance on Glnό6 in the binding of 5M038. Compound 5M038 was found to be active against 7 of 8 primary HIV isolates tested. The resistant isolate, BCF03 from group 0, has an arginine at position 66 in place of glutamine.

[0016] Figure 8 presents a flow diagram of an inventive system for designing and screening HIV fusion inhibitors. Libraries will be synthesized, and the assays will be carried

out using a fluorescence plate scanner. Cell-cell fusion assays will be done as described in the literature (Frey, Rits-Volloch et al. 2006). Viral infectivity assays will be carried out with compounds of Kd < 10 ^"6 (the question mark on round 1 indicates that compounds with suitable affinity may not yet have been found). Co-crystallization will be carried out with selected compounds having Kd < 10 ^"7 .

[0017] Figure 9 depicts compounds 5M038, 6M007, and 5M-amine.

[0018J Figure 10 depicts the general structure of library 1 of Example 8.

[0019] Figure 11 depicts the general structure of library 2 of Example 8.

[0020] Figure 12 depicts the general structure of librart 3 of Example 8.

[0021] Figure 13 depicts preliminary R groups investigated in library 2 of Example 8.

[0022] Figure 14 presents Table 1.

[0023] Figure 15 presents Table 2.

BRIEF DESCRIPTION OF THE TABLES

[0024] Table 1 shows the effects of 5M038, 5M041 and 6M007 on viral infectivity as compared with their activities in other assays. PBMCs were pre-treated with virus for 1 hour prior to addition of compound. After 5 days viral levels were determined by the amount of p24 antigen present in the culture. ³ H thymidine incorporation was used to assay possible cytotoxic effects. The TC ₅₀ S (50% toxicity concentration) for 5MO38, 5M041 and 6M007 were 43.2 μM, 58 μM, and 79.9 μM respectively. ICso". 50% inhibitory concentration. [0025] Table 2 shows the effect of 5M038 on cell-cell fusion mediated by envelope glycoproteins from various HIV and SIV isolates. The "pocket sequence" is the amino acid sequence of residues 57 to 70 (HXB2 numbering) on the inner-core helix of gp41.

DEFINITIONS

[0026] Alicyclic: The term "alicyclic", as used herein, refers to compounds which combine the properties of aliphatic and cyclic compounds and include but are not limited to cyclic, or polycyclic aliphatic hydrocarbons and bridged cycloalkyl compounds, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "alicyclic" is intended herein to include, but is not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which are optionally substituted with one or more functional groups. Illustrative alicyclic groups thus include, but are not limited to, for example, cyclopropyl, -CH ₂ -cyclopropyl, cyclobutyl, -CH ₂ -cyclobutyl, cyclopentyl, -

CH ₂ -cyclopentyl-n, cyclohexyl, -CH ₂ -cyclohexyl, cyclohexenylethyl, cyclohexanylethyl, norborbyl moieties and the like, which again, may bear one or more substituents. [0027] Aliphatic: The term "aliphatic", as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, "aliphatic" is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, the term "alkyl" includes straight and branched alkyl groups. An analogous convention applies to other generic terms such as "alkenyl", "alkynyl" and the like. Furthermore, as used herein, the terms "alkyl", "alkenyl", "alkynyl" and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, "lower alkyl" is used to indicate those alkyl groups (substituted, unsubstituted, branched or unbranched) having about 1-6 carbon atoms; in some embodiments, the term may refer to group having about 1-3 carbon atoms. In certain embodiments, alkyl, alkenyl and alkynyl groups employed in the invention contain about 1- 20 aliphatic carbon atoms. In certain other embodiments, alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-10 aliphatic carbon atoms. In yet other embodiments, alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-8 aliphatic carbon atoms. In still other embodiments, alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-6 aliphatic carbon atoms. In yet other embodiments, alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-bulyl, n-pentyl, sec- pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, moieties and the like, which again, may bear one or more substituents. Illustrative alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-proρynyl and the like.

[0028] Alkoxy: The terms "alkoxy" or "alkyloxy" as used herein refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom. In certain embodiments, the alkyl group contains about 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains about 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains about 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-4 aliphatic carbon atoms. Examples of

alkoxy groups, include but are not limited to, melhoxy, ethoxy, propoxy, isopropoxy, n- butoxy, tert-butoxy, neopentoxy and n-hexoxy.

[0029] Animal: The term "animal", as used herein, refers to humans as well as non- human animals, at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, worms and single cells. In certain exemplary embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). An animal may be a transgenic animal or a clone. [0030] Aromatic moiety: In general, the term "aromatic moiety", as used herein, refers to stable substituted or unsubstituted unsaturated mono- or polycycHc hydrocarbon moieties having preferably 3-14 carbon atoms, comprising at least one ring satisfying the Huckel rule for aromaticity. Examples of aromatic moieties include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, phenanthryl and anthracyl. It will also be appreciated that aromatic and heteroaromatic moieties, as defined herein, may be attached via an aliphatic (e.g., alkyl) or heteroaliphatic (e.g., heteroalkyl) moiety and thus also include moieties such as -(aliphatic)aromatic, -(heteroaliphatic)aromatic, -(aliphatic)heteroaromatic, - (heteroaliphatic)heteroaromatic, -(alkyl)aromatic, -(heteroalkyl)aromatic, - (alkyl)heteroaromatic, and -(heteroalkyl)heteroaromatic moieties. Thus, as used herein, the phrases "aromatic or heteroaromatic moieties" and "aromatic, heteroaromatic, - (alkyl)aromatic, -(heteroalkyl)aromatic, -(heteroalkyl)heteroaromatic, and - (heteroalkyl)heteroaromatic" are interchangeable. Substituents include, but are not limited to, any of the substituents described herein resulting in the formation of a stable compound. [0031] Aryl: In general, the term "aryl" refers to aromatic moieties, as described above, excluding those attached via an aliphatic (e.g., alkyl) or heteroaliphatic (e.g., heteroalkyl) moiety. In certain embodiments of the present invention, "aryl" refers to a mono- or bicyclic carbocyclic ring system having one or two rings satisfying the Huckel rule for aromaticity, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like. Substituenls for aryl and heteroaryl moieties include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound. [0032] Biological sample: As used herein the term "biological sample " refers to any solid or fluid sample obtained from, excreted by or secreted by any living organism, including single-celled micro-organisms (such as bacteria and yeasts) and multicellular organisms (such as plants and animals, for instance a vertebrate or a mammal, and in particular a healthy or apparently healthy human subject or a human patient affected by a

condition or disease to be diagnosed or investigated). The term therefore includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from an animal (e.g. , mammal) or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof. A biological sample can be in any form, including a solid material such as a tissue, cells, a cell pellet, a cell extract, cell homogenates, or cell fractions; or a biopsy, or a biological fluid. A biological fluid may be obtained from any site (e.g. blood, saliva (or a mouth wash containing buccal cells), tears, plasma, serum, urine, bile, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and pleural fluid, or cells therefrom, aqueous or vitreous humor, or any bodily secretion), a transudate, an exudate (e.g. fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g. a normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis). A biological sample can be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (whether primary cells or cultured cells) or medium conditioned by any cell, tissue or organ. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Biological samples may also include mixtures of biological molecules including proteins, lipids, carbohydrates and nucleic acids generated by partial or complete fractionation of cell or tissue homogenates. If desired, a biological sample may be subjected to preliminary processing, including preliminary separation techniques. In many embodiments, biological samples are taken from a human subject; however, biological samples may be from any animal, plant, bacteria, virus, yeast, etc.

[0033] Cycoalkyl: The term "cycloalkyl", as used herein, refers specifically to cyclic alkyl groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of aliphatic, heteroaliphatic or heterocyclic moieties, may optionally be substituted. An analogous convention applies to other generic terms such as "cycloalkenyl", "cycloalkynyl" and the like.

[0034] Halo or halogen: The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine, chlorine, bromine and iodine.

[0035] Haloalkyl: The term "haloalkyl" denotes an alkyl group having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

[0036] Halogenated: The term "halogenated" denotes a moiety having one, two, or three halogen atoms attached thereto.

[0037] Heteroalicyclic, heteroycloalkyl, heterocyclic: The terms "heteroalicyclic", "heterocycloalkyl" or "heterocyclic", as used herein, refer to compounds which combine the properties of heteroaliphatic and cyclic compounds and include but are not limited to saturated and unsaturated mono- or polycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl, thiofuranyl, pyrrolyl etc., which are optionally substituted with one or more functional groups, as defined herein. In certain embodiments, the term "heterocyclic" refers to a non-aromatic 5-, 6- or 7- membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, iraidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholmyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. Additionally, it will be appreciated that, in some embodiments, any of the alicyclic or heteroalicyclic moieties described above and herein may comprise an aryl or heteroaryl moiety fused thereto. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein. [0038] Heteroaliphatic: The term "heteroaliphatic", as used herein, refers to aliphatic moieties in which one or more carbon atoms in the main chain have been substituted with a heteroatom. Thus, a heteroaliphatic group refers to an aliphatic chain which contains one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, i.e., in place of carbon atoms. Thus, a 1-6 atom heteroaliphatic linker having at least one N atom in the heteroaliphatic main chain, as used herein, refers to a C ^aliphatic chain wherein at least one carbon atom is replaced with a nitrogen atom, and wherein any one or more of the remaining 5 carbon atoms may be replaced by an oxygen, sulfur, nitrogen, phosphorus or silicon atom. As used herein, a 1-atom heteroaliphatic linker having at least one N atom in the heteroaliphatic main chain refers to -NH- or -NR- where R is aliphatic, heteroaliphatic, acyl, aromatic, heteroaromatic or a nitrogen protecting group. Heteroaliphatic moieties may be branched or linear unbranched. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, any of the substituents described above.

[0039] Heteroaromatic moiety: In general, the term "heteroaromatic moiety", as used herein, refers to stable substituted or unsubstituted unsaturated mono-heterocyclic or polyheterocyclic moieties having preferably 3-14 carbon atoms, comprising at least one ring satisfying the Huckel rule for aromaticity. Examples of heteroaromatic moieties include, but are not limited to, pyridyl, quinolinyl, dihydroquinolinyl, isoquinoHnyl, quinazolinyl, dihydroquinazolyl, and tetrahydroquinazolyl. It will also be appreciated that aromatic and heteroaromatic moieties, as defined herein, may be attached via an aliphatic (e.g., alkyl) or heteroaliphatic (e.g. , heteroalkyl) moiety and thus also include moieties such as - (aliphatic)aromatic, -(heteroaliphatic)aromatic, -(aHphatic)heteroaromatic, - (heteroaliphatic)heteroaromatic, -(alkyl)aromatic, -(heteroalkyl)aromatic, - (alky l)hetero aromatic, and -(heteroalkyl)heteroaromatic moieties. Thus, as used herein, the phrases "aromatic or heteroaromatic moieties" and "aromatic, heteroaromatic, - (alkyl)aromatic, -(heteroalkyl)aromatic, -(heteroalkyl)heteroaromatic, and - (heteroalkyl)heteroaromatic" are interchangeable. Substituents include, but are not limited to, any of the previously mentioned substituents resulting in the formation of a stable compound.

[0040] Heteroaryl: The term "heteroaryl" refers to heteroaromatic moieties, as described above, excluding those attached via an aliphatic (e.g., alkyl) or heteroaliphatic (e.g., heteroalkyl) moiety, In certain embodiments of the present invention, the term "heteroaryl", as used herein, refers to a cyclic unsaturated radical having from about five to about ten ring atoms of which one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, tbiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinoHnyl, and the like. Substituents for aryl and heteroaryl moieties include, but are not limited to, any of the previously mentioned substitutents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound.

[0041] Inhibitorily active: As used herein, the term "inhibitorily active" means a compound having HIV fusion inhibition activity as defined herein. For example, an "inhibitorily active metabolite or residue" of an inventive HIV fusion inhibitor is a metabolite or residue thereof that inhibits HIV fusion. In some embodiments, the metabolite or residue

retains at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the activity of its parent compound.

[0042] Isolated: As used herein, the term "isolated", when applied to the compounds of the present invention, refers to such compounds that are (i) separated from at least some components with which they are associated in nature or when they are made and/or (ii) produced, prepared or manufactured by the hand of man.

[0043] Patient: A "patient", or "subject", as used herein, means an animal In many embodiments, the animal is a mammal, commonly a human. [0044] Pharmaceutically acceptable derivative: The phrase, "pharmaceutically acceptable derivative", as used herein, denotes any pharmaceutically acceptable salt, ester, or salt of such ester, of such compound, or any other adduct or derivative which, upon administration to a patient, provides (directly or indirectly) a compound as otherwise described herein, or a metabolite or residue thereof. Pharmaceutically acceptable derivatives thus include among others pro-drugs.

[0045] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt or salt of an ester of a compound of this invention that, upon administration to a recipient, provides, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof. Many pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et at, describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences 66:1-19, 1977 ncorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,

hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, raalate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersable products may be obtained by such quaternization. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0046] Prodrug: A "pro-drug" is a derivative of a compound, usually with significantly reduced pharmacological activity, which contains an additional moiety that is susceptible to removal in vivo yielding the parent molecule as the pharmacologically active species. An example of a pro-drug is an ester which is cleaved in vivo to yield a compound of interest. Pro-drugs of a variety of compounds, and materials and methods for derivatizing the parent compounds to create the pro-drugs, are known and may be adapted to the present invention. Certain exemplary pharmaceutical compositions and pharmaceutically acceptable derivatives will be discussed in more detail herein below.

(0047] Small Molecule: In general, a small molecule is understood in the art to be an organic molecule that is less than about kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, the small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, small molecules are non-polymeric. In some embodiments, small molecules are not proteins, peptides, or amino acids. In some embodiments, small molecules are not nucleic acids or nucleotides. In some embodiments, small molecules are not saccharides or polysaccharides.

[0048] Stable: The term "stable", as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.

[0049] Substituted: It is understood that compounds as described herein, may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic, carbon and heteroatom substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible subslituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment and prevention, for example of disorders, as described generally above. Examples of substituents include, but are not limited to aliphatic; heteroaliphatic; alicycHc; heteroalicycHc; aromatic, heteroaromatic; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; -NO ₂ ; -CN; -CF ₃ ; -CH ₂ CF ₃ ; -CHCl ₂ ; -CH ₂ OH; - CH ₂ CH ₂ OH; -CH ₂ NH ₂ ; -CH ₂ SO ₂ CH ₃ ; - or -GR ^GI wherein G is -O-, -S-, -NR ⁰² -, -C(O)-, - S(O)-, -SO ₂ -, -C(O)O-, -C(O)NR ⁰² -, -OC(O)-, -NR ⁰² C(O)-, -OC(O)O-, - OC(O)NR ⁰² -, -NR ⁰² C(O)O-, -NR ⁰² C(O)NR ⁰² -, -C(=S>, -C(=S)S-, -SC(=S)-, - SC(=S)S-, -C(=NR ^G2 X -C(-NR ^G2 )O-, -C(=NR ^G2 )NR ⁰³ -, -OC(=NR ^O2 >, -NR ⁰² C(^NR ⁰³ )-, - NR ⁰² SO ₂ -, -NR ⁰² SO ₂ NR ⁰³ -, or -SO ₂ NR ⁰² -, wherein each occurrence of R ^GI , R° ² and R ⁰³ independently includes, but is not limited to, hydrogen, halogen, or an optionally substituted aliphatic, heteroaliphatic, alicyclic, heteroalicycHc, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein. [0050] Suffering from: An individual who is "suffering from" HIV infection has been diagnosed with HIV infection (or, if not a human, a corresponding retroviral infection such as SIV infection, FIV infection, etc.)

[0051] Susceptible to: An individual who is "susceptible to" HIV infection is one who has been exposed to HIV, or is likely to become exposed to HIV (e.g., through close or

initiraate contact with another individual infected with HIV). If the individual is not a human, "susceptible to HIV infection" means susceptible to infection with a corresponding immunoideficiency retrovirus (e.g., SIV, FIV, etc.).

[0052] Unit Dosage Form: The expression "unit dosage form" as used herein refers to a physically discrete unit of pharmaceutical composition (e.g., a single table or capsule, etc.), generally containing a single "dose" of active ingredient. Those of ordinary skill in the art, however, will appreciate that a doctor or veterinarian may well adjust dosing of a particular individual, for example, prescribing a whole or fractional number of unit dosage forms to be taken or administered as a single dose.

[0053J Therapeutically Effective Amount: The term "therapeutically effective amount" means an amount of inventive HIV fusion inhibitor that is sufficient, when administered to a subject suffering from or susceptible to HIV infection and/or one or more associated diseases, disorders or conditions to treat the HIV infection and/or associated disease(s), disorder(s) or condition(s).

[0054] Therapeutically Active Agent: This term refers to an agent that, when administered to a subject, has a therapeutic effect. The term encompasses "active pharmaceutical ingredients" as understood in the pharmaceutical industry,

[0055] Thioalkyl: The "thioalkyl" as used herein refers to an alkyl group, as previously defined, attached to the parent molecular moiety through a sulfur atom. In certain embodiments, the alkyl group contains about 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains about 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains about 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-4 aliphatic carbon atoms. Examples of thioalkyl groups include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0056] Treating: The terms "treat" or "treating," as used herein, refer to partially or completely alleviating, inhibiting, preventing, delaying the onset of, reducing the incidence of, ameliorating and/or relieving one or more symptoms or features of a particular disease, disorder or condition (e.g., HIV infection).

[0057] It will be appreciated that, as used herein, the terms "aliphatic", "heteroaliphatic",

"alkyl", "alkenyl", "alkynyl", "heteroalkyl", "heteroalkenyl", "heteroalkynyl", and the like encompass substituted and unsubstituted, saturated and unsaturated, and linear and branched groups. Similarly, the terms "alicyclic", "heterocyclic", "heterocycloalkyl", "heterocycle"

and the like encompass substituted and unsubstituted, and saturated and unsaturated groups. Additionally, the terms "cycloalkyl", "cycloalkenyl", "cycloalkynyl", "heterocycloalkyl", "heterocycloalkenyl", "heterocycloalkynyl", "aromatic", "heteroaromatic", "aryl", "heteroaryl" and the like, used alone or as part of a larger moiety, encompass both substituted and unsubstituted groups.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

HIV Fusion

[0058] Enveloped viruses such as HIV and influenza virus enter cells by membrane fusion (1). The gplόO envelope glycoprotein of HIV-I, its fusion protein, is synthesized as a single polypeptide chain with a C-terminal membrane anchor, and it requires a processing cleavage (to gpl20 and gρ41) for activation (2, 3). The "fusion peptide", a glycine-rich, hydrophobic sequence required for interaction with the target-cell membrane, lies near the N- terminus of gp41 that is created by this cleavage (4), Like other "class 1" fusion proteins, such as influenza virus haemagglutinin, gpl60 is a homotrimer, and its fusion-promoting fragment, gρ41, is also trimeric (1).

[0059] The cleaved, mature trimer of gpl20/gp41 can be triggered by receptor binding to undergo an irreversible conformational change (5-9). In effect, cleavage of the gpl60 precursor produces a metastable conformation, but the barrier to its rearrangement is high. The image of a "spring-loaded" device has been used to describe this situation, and the triggering event then corresponds to releasing the catch on the spring (10) . [0θ60] The structure of the gp41 ectodomain in the postfusion conformation is a trimer of α-helical hairpins (11, 12). Its "inner core" is an α-helical coiled-coil formed by amino- terminal segments of the three polypeptide chains; the helices of its "outer layer" are the carboxy-terminal segments of those chains. The 30 residue loop that connects inner core and outer-layer helices (also called "heptad repeats 1 and 2 " or "HRl" and "HR2", respectively) is absent from the crystal structures; sequence conservation suggests that it resembles the loop seen in the ectodomain of the TM subunit from MoMuLV and in the Gp2 ectodomain of Ebola virus (13, 14). One feature of the contact between each outer-layer helix and the inner core is a hydrophobic pocket on the latter, containing residues Leu57, TrpθO and Lys63, which receives the side chains of Trpl 17, Trpl20 and Ilel24.

[0061] The sequence of molecular events during the fusion-promoting conformational rearrangement (refolding) of gpl20/gp41 is thought to be as follows (Fig. 1); (1) The gpl20

fragment dissociates, allowing the gp41 fragment to rearrange. (2) An initial reorganization of the gρ41 polypeptide chain pulls the fusion peptide from a buried position and projects it against the target-cell membrane. The result of this process is probably a conformation with significant half-life, the so-called "prefusion intermediate". (3) In a subsequent step, each fusion-promoting fragment folds back upon itself, probably by zippering up the outer layer along the surface of the inner core. This step brings together the transmembrane segment at the C-terminus of the fusion-promoting fragment, anchored in the viral bilayer, and the fusion peptide at the N-terminus, bound to the target membrane. The two membranes are thus induced to approach each other and ultimately to merge.

Identification and/or Characterization of HIV Fusion Inhibitors

[0062] The present invention provides a new sytem for identifying HIV fusion inhibitors.

In particular, the present invention provides a system for identifying inhibitors that interfere with binding of the gp41 outer helix with the gp41 inner core. Such inhibitors may block the gp41 conformational change associated with fusion.

[0063J The inventive system includes a first component corresponding to a gp41 outer helix and a second component corresponding to a gp41 inner core. Binding of the first and second components to one another is detected in the presence or absence of test agent. Test agents that reduce binding of first and second components to one another are considered to be

HIV fusion inhibitors according to the present invention.

(0064] In some embodiments, inventive HIV fusion inhibitors reduce binding of first and second components by at least about 10%, 20%, 30%, 30%, 40%, 60%, 70%, 80%, 90% or more as compared with the level of binding observed under otherwise identical conditions lacking test compound.

[0065] In some embodiments, inventive HIV fusion inhibitors reduce binding of the first and second components with an IC$o below about 10 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM or less. In some embodiments, inventive HIV fusion inhibitors reduce binding of the first and second comopnents with an ICso below about 0.5 μM, 0.1 μM, 50 nM, 20 nM, 10 nM or less.

The first and second components utilized in the inventive system for identifying HIV fusion inhibitors may represent any entities with sufficient structural similarity to the gp41 outer helix (first component) or the gp41 inner core (second component) to act as an appropriate model for gp41 outer helix - inner core interaction. In particular, such first and second

components include features corresponding to the interacting elements of the gρ41 outer helix and inner core.

[0066] For instance, as discussed above, gp41 residues Leu57, TrpόO and Lys63 all participate in a hydrophobic pocket located on the gp41 inner helix. This outer pocket receives the side chains of Trpl 17, Trpl20, and Ilel24. Thus, in certain embodiments of the invention, a useful first component according to the present invention includes features corresponding to Trpl 17, Trpl20, and Ilel24, and a useful second component according to the present invention includes features corresponding to Leu57, TrpόO and Lys63, Those of ordinary skill in the art will appreciate that the features corresponding to interacting residues of the gp4l outer helix and inner core should desirably share those characteristics (e.g., location and/or orientation of hydrogen-bonding partners, hydrophobicity, three-dimensional contours, etc.) relevant to interaction.

[0067] In some embodiments, at least one such component is or includes a polypeptide including appropriate interacting sequences from the gp41 outer helix (first component) or the gp41 inner core (second component). For example, in some embodiments, the first component comprises a polypeptide chain including a tryptophan in a position and orientation corresponding to gp41 Trpl 17, a tryptophan in a position and orientation corresponding to gp41 Trpl20, and an isoleucine in a position and orientation corresponding to gp41 Ilel24. Comparably, in some embodiments, the second component comprises a polypeptide chain including a leucine in a position and orientation corresponding to gp41 Leu57, a tryptophan in a position and orientation corresponding to gp41 TrpόO, and a lysine corresponding to gp41 Lys63.

[0068] In some embodiments one or both of the first and second components comprises a polypeptide including at least one contiguous stretch of amino acids (e.g., of about 2, 5, 10, 15, 20, 25, 30, 35, 40, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or more amino acids that are identical to those found in a contiguous stretch of gp41. In some embodiments, one or both of the first and second components comprises a polypeptide including at least one contiguous stretch of amino acids that is at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or less identical to a contiguous stretch of amino acids found in gp41.

[0069] In some embodiments of the invention, the first component comprises a single polypeptide chain. In some embodiments, this polypeptide chain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acids long. In some embodiments, the

polypeptide chain includes an α helix corresponding to a gp41 outer helix. In some embodiments, the polypeptide comprises a stretch about 25-60 amino acids long that includes an a helix corresponding to a gp41 outer helix. In some embodiments, the polypeptide chain comprises amino acids corresponding to gp41 sequences between about residues 115 and 160. For instance, in some embodiments, the first component is a polypeptide comprising gp41 amino acids 117-154.

|0070] In some embodiments of the invention, the second component comprises at least one polypeptide chain. In some embodiments, this polypeptide chain is at least about 5, 10, 15, 20, 25, 30, 35 _j 40, 45, 50, 55, 60 or more amino acids long. In some embodiments, the second component comprises at least one α helix corresponding to an inner core a helix. In some embodiments, the polypeptide chain comprises amino acids corresponding to gp41 sequences between about residues 30 and 80. For instance, in some embodiments, the second component is a polypeptide comprising gp41 amino acids 35-70.

[0071] In some embodiments of the invention, the first or second component comprises at least 2, 3, 4, 5 or more α helices. In some embodiments, the a component comprising multiple α helices comprises an α helical coiled-coil structure comprised of interacting α helices.

[0072J In some embodiments of the invention, either the first or the second component comprises a single gp41 α helix (i.e., the first component is a polypeptide chain comprising a single gp41 outer layer α helix or the second component is a polypeptide chain comprising a single gp41 inner core α helix) and the other component comprises a plurality of gp41 α helices; in some such embodiments, the other component comprises all of the remaining gp41 α helices. For instance, in some embodiments of the invention, the first component comprises a single outer layer α helix and the second component comprises three inner core α helices and two outer layer a helices. Conversely, in some embodiments, the second component comprises a single inner core a helix and the first component comprises two inner core a helices and three outer layer a helices. According to the present invention, binding of the first and second components can be detected by any available means. In some embodiments one or both of the first and second components is labeled with a detectable marker (e.g., a radioisotopic, chemiluminescent, or fluorescent moiety). Those of ordinary skill in the art are well familiar with a variety of different direct or indirect labeling strategies and will appreciate those that can readily be employed in the practice of the present invention.

HIV Fusion Inhibitors

[0073] The present invention provides small molecule inhibitors of HIV fusion. Prior to the present invention, there was considerable debate over whether it would be possible to identify or develop small molecule fusion inhibitors that block the gp41 conformational change. In particular, the coiled-coil is a relatively special kind of protein interface. Some studies had identified peptides that could interfere with the gp41 coiled-coil interactions (see, for example, 15-19; see also 22-25). Indeed, one such peptide (enfurvitide; T-20) has now been approved as a therapeutic for use in the treatment of HIV infection. However, identification of polypeptides that can disrupt protein-protein interactions does not suggest the possibility of identifying small molecules that can disrupt the same interactions, Particularly for a complex interacting protein structure like a coiled-coil, it might be expected that a peptide corresponding to one of the interacting regions could disrupt the overall interaction (e.g., behaving as a dominant negative interaction partner), but no such expectation could apply to a small molecule. Indeed, it might be expected that the multiple interactions within a coiled-coil structure could readily overcome any interference from a small molecule. The present invention demonstrates that suitably designed, sensitive, structure-based assays can detect interference by small molecules with gp41 coiled-coil interactions, and indeed identifies small molecule compounds that achieve such interference with ICsos in at least the low micromolar range.

[0074] It will be understood that the present invention provides small molecule HIV fusion inhibitor having chemical structures as depicted herein, and further encompasses all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of such structures; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. The present invention therefore provides single stereochemical isomers (e.g., pure preparations of individual stereoisomers) as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of depicted structures or formulae are within the scope of the invention. Furthermore, unless otherwise stated, all tautomeric forms of the structures or formulae of the invention are within the scope of the invention.

[0075] It should further be understood that the present invention encompasses compounds that differ from depicted structures only in the presence of one or more isotopically enriched atoms. For example, compounds having the depicted sturcutres or formulae except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³ C- or

¹⁴ C-enriched carbon are within the scope of this invention. Such compounds may be useful, for example, as analytical tools or probes in biological assays.

[0076] It should also be understood that the present invention encompasses pharmaceutically acceptable derivatives, and in particular prodrugs, metabolites, and pharmaceutically acceptable salts of the depicted compounds.

[0077] As described herein, the present invention specifically provides HIV fusion inhibitors of the general formula (I) as further defined below:

(D and pharmaceutically acceptable derivatives thereof; wherein n is an integer from 0-5;

R ¹ is hydrogen, halogen, cyano, m ^' tro, or an aliphatic (including, e.g., heteroaliphatϊc, alicyclic, heteroalicyclic, etc.), aromatic (including e.g., heteroaromatic) moiety; and

R is hydrogen, halogen, cyano, amino, aminoacyl, nitro, or an aliphatic (including, e.g., haloaliphatic, heteroaliphatic, alicyclic, heteroalicyclic), aromatic (including, e.g., heteroaromatic) moiety; in certain embodiments R is an aromatic or heteroaromatic moiety; in certain embodiments, R is an aryl or heteroaryl moiety, optionally subsituted with halogen or haloaliphatic (e.g., haloalkyl).

[0078] In certain embodiments, inventive HIV fusion inhibitors have the general formula (II) as further defined below:

(II) and pharmaceutically acceptable derivatives thereof; wherein m and p are independently integers from 0-5; and

R ² are R ³ are independently hydrogen, halogen, cyano, nitro, or an aliphatic (including, e.g., heteroaliphatic, alicyclic, heteroalicyclic) or aromatic (including, e.g., heteroaromatic) moiety,

[0079] In certain embodiments, inventive HIV fusion inhibitors have one of the following structures:

[0080] In other embodiments, compounds having one of the following structures are excluded:

[0081] In certain embodiments, the present invention defines particular classes of compounds which are of special interest. For example, one class of compounds of special interest includes compounds of formulae (I ^A ):

(i ^A ) wherein R ^1A are R ^1B are independently hydrogen, hydroxyl, halogen, cyano, nitro, or an aliphatic, heteroaliphatic, alicyclic, heteroalicycHc, aromatic or heteroaromatic moiety; and R is as previously defined above and in classes and subclasses as described herein. [0082] Another class of compounds of special interest includes compounds of formula (I ^B ):

(i ^B ) wherein R ^1A is hydrogen, hydroxyl, halogen, cyano, nitro, or an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aromatic or heteroaromatic moiety; and R is as previously defined above and in classes and subclasses as described herein. [0083] Another class of compounds of special interest includes compounds of formula (I ^c ):

wherein q is an integer from 0-5; and R ⁴ is hydrogen, hydroxyl, halogen, cyano, nitro, or an aliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aromatic or heteroaromatic moiety. [0084] Another class of compounds of special interest includes compounds of formula

(1°):

d ^D ) wherein R is as defined above and in classes and subclasses as described herein. [0085] Another class of compounds of special interest includes compounds of formula

(I ^E ):

on ^■ Ex wherein R is as defined above and in classes and subclasses as described herein. [0086] Another class of compounds of special interest includes compounds of formula

(f):

(I ^F ) wherein R and R ¹ are as defined above and in classes and subclasses as described herein.

[0087] Another class of compounds of special interest includes compounds of formula (I ^G ):

d ^g ) wherein R is as defined above and in classes and subclasses as described herein.

[0088] Another class of compounds of special interest includes compounds of formula

(I ^H ):

d ^H ) wherein R and R ¹ are as defined above and in classes and subclasses as described herein. [0089] Another class of compounds of special interest includes compounds of formula

(I ¹ ):

(I ¹ ) wherein R is as defined above and in classes and subclasses as described herein. [0090] Another class of compounds of special interest includes compounds of formula (I ^J ):

(I ^J ) wherein R and R ^IA are as defined above and in classes and subclasses as described herein.

[0091] Another class of compounds of special interest includes compounds of formula <π ^A ):

(H ^A )

[0092] A number of important subclasses of each of the foregoing classes deserve separate mention; these subclasses include subclasses of the foregoing classes in which: [0093] i) R ¹ is hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl, -(heteroalkyl)heteroaryl moiety; or haloryl,

[0094] ii) R ¹ is hydrogen, C _!-6 alkyl or C _1-6 haloalkyl; [0095] iii) R ¹ is hydrogen or C^haloalkyl; [0096] iv) R ¹ is hydrogen or -CF ₃ ; [0097] v) n is i; [0098] vi) n is 2; [0099] vii) n is 3;

[00100] viii) n is 1 and R ¹ is C^haloalkyl; [00101] ix) n is 1 and R ¹ is -CF ₃ ;

[00102] x) n is 2 and each occurrence of R ¹ is independently C^ ₆ haloalkyl; [θ0103] xi) n is 2 and each occurrence of R ¹ is -CF ₃ ;

[00104J xii) R ² is hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, heteroaryl, haloaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl or - (heteroalkyl)heteroaryl moiety;

[00105] xiii) R ² is hydrogen, Cj.ealkyl, Ci-ealkoxy or Ci^haloalkyl; [00106J xiv) R ² is hydrogen; [0θ107] xv) m is l; [00108] xvi) m is 2;

[00109] xvii) m is 3;

[00110] xviii) R ³ is hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, heteroaryl, haloaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl or - (heteroalkyl)heteroaryl moiety;

[00111] xix) R ³ is hydrogen, hydroxyl, nitro, Ci-ealkyl, Ci. ₆ alkoxy or Cj _-6 haloalkyl;

[001121 xx) R ³ is hydrogen, hydroxyl, nitro or d. ₆ alkoxy;

[00113] xxi) R ³ is hydrogen, hydroxyl, nitro or methoxy;

[00114] xxii) p is l;

[00115] xxiii) p is 2;

[00116] xxiv) p is 3;

[00117] xxv) p is 1 and R ³ is hydrogen, hydroxyl, nitro or

[00118] xxvi) p is 1 and R ³ is hydrogen, hydroxyl, nitro or methoxy;

[00119] xxvii) p is 2 and each occurrence of R ³ is independently hydrogen, hydroxyl, nitro or Cμealkoxy;

[00120] xxviii) p is 2 and each occurrence of R ³ is hydrogen, hydroxyl, nitro or methoxy;

[00121] xxix) P is 3 and each occurrence of R ³ is independently hydrogen, hydroxyl, nitro or C|- ₆ alkoxy;

[00122J xxx) p is 3 and each occurrence of R ³ is hydrogen, hydroxyl, nitro or methoxy;

[00123] xxxi) R ⁴ is hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, haloaryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl or -

(heteroalkyl)heteroaryl moiety;

[00124] xxxii) R ⁴ is hydrogen, halogen, Q^alkyl, Ci-ealkoxy or Cj-ghaloalkyl;

[00125] xxxiii) R ⁴ is hydrogen;

[00126] xxxiv) R ⁴ is Ci. ₆ alkyl;

[00127] xxxv) R ⁴ is halogen;

[00128] xxxvi) R ⁴ is F or Cl;

[00129] xxxvϋ) R ⁴ is hydrogen, halogen or methyl;

[00130] xxxiv) q is l;

[00131] xxxv) q is 2;

[00132] xxxvi) q is 3;

[00133] xxxvii)q is 1 and R ⁴ is hydrogen, halogen or Cμgalkyl;

[00134] xxxviii) q is 1 and R ⁴ is hydrogen, F, Cl or methyl;

[00135] xxxix)q is 2 and each occurrence of R ⁴ is independently hydrogen, halogen or C]- ealkyl;

[00136] xl) q is 2 and each occurrence of R ⁴ is hydrogen, F, Cl or methyl;

[00137] xli)q is 3 and each occurrence of R ⁴ is independently hydrogen, halogen or Cj- ealkyl;

[00138] xlii) q is 3 and each occurrence of R > 4 i ^■ s hydrogen, F, Cl or methyl; [0100] xliii) R is aryl, haloaryl, or heteroaryl; [0101] xliv) R is one of:

N-N N-N

_? 4A- ⁷ >!- ^, 4A^ ^X O^ ^f - wherein q is an integer from 0 to 3; each occurrence of R ^4A is independently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, -(alkyl)aryl or -(alkyl)heteroaryl, -OR ^4C , -SR ^4C , -NR ⁴ Y ^C , -SO ₂ NR ^4B R ^4C , -C(=O)NR ^4B R ^4C , halogen, -CN, -NO ₂ , -C(=O)OR ^4C , - N(R ^4B )C(=O)R ^4C , wherein each occcurrence of R ^4B and R ^4C is independently hydrogen, lower alkyl, lower heteroalkyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl or acyl, or R ^4B and R ^4C taken together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclic ring;

[0104J xlvii) R is < ^R V

[0105] It will be appreciated that for each of the classes and subclasses described above and herein, any one or more occurrences of aliphatic or heteroaliphatic may independently be

substituted or unsubstituted, cyclic or acyclic, linear or branched, saturated or unsaturated and any one or more occurrences of aryl, heteroaryl, cycloaliphatic, cycloheteroaliphatic may be substituted or unsubstituted.

[0106] The reader will also appreciate that any and all possible combinations of the variables described in i)- through xlvii) above (e.g., R ¹ , R ² , R ³ , R ⁴ , among others) are considered part of the invention. Thus, the invention encompasses any and all compounds of formula I or II generated by taking any possible permutation of variables R ¹ , R ² , R ³ , R ⁴ , n, m, p, q, and other variables/substituents (e.g., R ^1A , R ^!B , R ^3A , R ^3B , R ^3C , R ^4A , etc.) as further defined for R ¹ , R ² , R ³ , R ⁴ , described in i)- through xlvii) above.

[0107] For example, an exemplary combination of variables described in i)- through xlvii) above includes those compounds of Formula I wherein:

R ¹ is hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl or -(heteroalkyl)heteroaryl moiety; n is 1 ;

q is 1 ; and

R ⁴ is hydrogen, halogen, Ci-galkyl, Ci-ealkoxy or Ci-ehaloalkyl.

[0108] Other exemplary combinations of substituents are illustrated by compounds of the following subgroups I through XVI:

[0109] I. Compounds having the structure (and pharmaceutically acceptable derivatives thereof):

wherein R ^1A is R ^!B are independently hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl or - (heteroalkyl)heteroaryl moiety; and R is aryl or heteroaryl. In certain embodiments, R ^1A is

R ^1B are independently hydrogen, or C[. ₆ haloalkyl. In certain embodiments, R ^1A is R ^1B are independently hydrogen or In certain embodiments, R ^IA is R ^1B are independently hydrogen or -CF ₃ . In certain embodiments, R is optionally substituted phenyl, 2-thienyl, 3-thienyl, N-pyrrolyl. In certain embodiments, R has one of the following structure:

wherein R ⁴ is hydrogen or and R ^4A and R ^4B are indepently halogen. In certain embodiments, R ⁴ is hydrogen or Ci _-6 alkyl; and R ^4A and R ^4B are indepently F or Cl. [0110] H. Compounds having the structure (and pharmaceutically acceptable derivatives thereof):

wherein R ^1A is hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl or -(heteroalkyl)heteroaryl moiety; and R is aryl or heteroaryl. In certain embodiments, R ^iA is hydrogen, d-ealkyl or C _1- ghaloalkyl. In certain embodiments, R ^1λ is hydrogen or Ci-shaloalkyl. In certain embodiments, R ^1A is -CF ₃ . In certain embodiments, R is optionally substituted phenyl, 2- thienyl, 3-thienyl, N-pyrrolyl. In certain embodiments, R has one of the following structure:

wherein R ⁴ is hydrogen or Ci-ealkyl; and R ^4A and R ^4B are indepently halogen. In certain embodiments, R ⁴ is hydrogen or C^alkyl; and R ^4A and R ^4B are indepently F or Cl. [0111] HI. Compounds having the structure (and pharmaceutically acceptable derivatives thereof):

wherein R ^3A , R ^3B and R ^3C are independently hydrogen, halogen, hydroxyl, cyano, nitro, or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, aryl, heteroaryl, -(alkyl)aryl, -(alkyl)heteroaryl, -(heteroalkyl)aryl or - (heteroalkyl)heteroaryl moiety, In certain embodiments, R , R and R are independently hydroxyl, nitro, Q-ealkyl, Cj-ealkoxy or Q-ehaloalkyl. In certain embodiments, R ^3A , R ^3B and R ^3C are independently hydroxyl, nitro, C^alkoxy or Ci-βhaloalkyl, In certain embodiments, R ^3A , R ^3B and R ^3C are independently hydroxyl, nitro or Q^alkoxy. In certain embodiments, R ^3A , R ^3B and R ^3C are independently hydroxyl, nitro or methoxy.

[0112] It will also be appreciated that for each of the subgroups I-III described above, a variety of other subclasses are of special interest, including, but not limited to those classes described above i)- xlvii) and classes, subclasses and species of compounds described above and in the examples herein.

[0113] Some of the foregoing compounds can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., stereoisomers and/or diastereomers. Thus, inventive compounds and pharmaceutical compositions thereof may be in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the invention are enantiopure compounds. In certain other embodiments, mixtures of stereoisomers or diastereomers are provided.

(0114] Furthermore, certain compounds, as described herein may have one or more double bonds that can exist as either the Z or E isomer, unless otherwise indicated. The invention additionally encompasses the compounds as individual isomers substantially free of other isomers and alternatively, as mixtures of various isomers, e.g., racemic mixtures of stereoisomers. In addition to the above-mentioned compounds per se, this invention also encompasses pharmaceutically acceptable derivatives of these compounds and compositions comprising one or more compounds of the invention and one or more pharmaceutically acceptable excipients or additives.

[0115] Compounds of the invention may be prepared by crystallization of compound of formula (I) or (II) under different conditions and may exist as one or a combination of

polymorphs of compound of general formula (I) or (II) forming part of this invention. For example, different polymorphs may be identified and/or prepared using different solvents, or different mixtures of solvents for recrystallization; by performing crystallizations at different temperatures; or by using various modes of cooling, ranging from very fast to very slow cooling during crystallizations. Polymorphs may also be obtained by heating or melting the compound followed by gradual or fast cooling. The presence of polymorphs may be determined by solid probe NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffractogram and/or other techniques. Thus, the present invention encompasses inventive compounds, their derivatives, their tautomeric forms, their stereoisomers, their polymorphs, their pharmaceutically acceptable salts their pharmaceutically acceptable solvates and pharmaceutically acceptable compositions containing them.

Pharmaceutical Compositions

[0116] As discussed above, the present invention provides HIV fusion inhibitors, and in particular provides small molecule HIV fusion inhibitors. These compounds are useful for the treatment of HIV infection and/or associated diseases, disorders and conditions. The present invention provides pharmaceutical compositions comprising a therapeutically effective amount of inventive HIV fusion inhibϊtor(s) appropriately formulated for administration to a subject suffering from or susceptible to HIV infection and/or an associated disease, disorder or condition. Such pharmaceutical compositions include one or more pharmaceutically acceptable excipients and may optionally include one or more additional therapeutically active agents.

[0117] It will be appreciated that HIV fusion inhibitors according to the present invention may be administered in free form or, where appropriate, as a pharmaceutically acceptable derivative thereof. In some embodiments, an HIV fusion inhibitor is administered in a salt form; in some embodiments an HIV fusion inhibitor is administered in a prodrug form. [0118] Appropriate excipients for use in pharmaceutical compositions of the present invention may include, for example, one or more carriers, binders, fillers, vehicles, disintegrants, surfactants, dispersion or suspension aids, thickening or emulsifying agents, isotonic agents, preservatives, lubricants, and the like, or combinations thereof, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in

formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof.

[0119] Inventive pharmaceutical compositions may be formulated for any desirable route of delivery including, for example, parenteral, oral, rectal, intracisternal, intravaginal, intraperitoneal, topical, bucal, nasal, intraocular, etc.

[0120] To give but a few examples, parenteral formulations are typically sterile injectable aqueous or oleaginous suspensions. Such formulations may be prepared according to the known art using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water (including water for injection), Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition,, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0121] Inventive injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0122] In order to prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form can be accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0123) Oral formulations are typically either liquid or solid. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to any active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydroforfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0124] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Such solid dosage forms typically include one or more carriers, binders, fillers, disintegrating agents, surfactants, humectants (e.g., glycerol), solution retarding agents (e.g., paraffin), absorption accelerators, wetting agents, absorbents, buffering agents, gildants, plasticizers and/or lubricants, etc.

[0125] For example, suitable carriers may include, for example, sodium citrate or dicalcium phosphate.

[0126] Suitable binders include but are not limited to starch, PVP (polyvinyl pyrrolidone), low molecular weight HPC (hydroxypropyl cellulose), microcrystalline cellulose, low molecular weight HPMC (hydroxypropyl methylcellulose), low molecular weight carboxymethyl cellulose, ethylcellulose, alginates, gelatin, polyethylene oxide, acacia, dextrin, sucrose, magnesium aluminum silicate, and polymethacrylates. [0127] Fillers include agents selected from the group consisting of microcrystalline cellulose, starch, lactitol, lactose, a suitable inorganic calcium salt, sucrose, glucose, mannitol, silicic acid, or a combination thereof. In some embodiments, the core comprises a binder or filler.

[0128] Incorporation of suitable disintegrant(s) into a solid dosage form may facilitate breakdown. Addition of disintegrant may facilitate release of active compound and achievement of concentration equilibration in the GI tract. Suitable disintegrants are also known in the art and include but are not limited to, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, crospovidone (cross-linked PVP), sodium carboxymethyl starch (sodium starch glycolate), cross-linked sodium carboxymethyl

cellulose (croscarmellose), pregelatinlzed starch (starch 1500), microcry stall ine starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum) or a combination thereof.

[0129] Suitable surfactants are also known in the art and include, e.g., poloxamer, polyoxyethylene ethers, polyoxyethylene sorbitan fatty acid esters polyoxyethylene fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ether, polysorbates, cetyl alcohol, glycerol fatty acid esters (e.g., triacetin, glycerol monostearate, and the like), polyoxymethylene stearate, sodium lauryl sulfate, sorbitan fatty acid esters, sucrose fatty acid esters, benzalkonium chloride, polyethoxylated castor oil, and docusate sodium, and the like, and combinations thereof. In some embodiments the core may further comprise a surfactant. [0130] Solution retarding agents such as paraffin, absorption accelerators such as quaternary ammonium compounds, wetting agents such as, for example, cetyl alcohol and glycerol monostearate, absorbents such as kaolin and bentonite clay, and/or lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0131] Solid compositions may also be employed as fillers in soft and hard-filled gelatin capsules, for example using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0132] Solid compositions may also be prepared in a micro-encapsulated form with one or more excipϊents as noted above. For example, tablets, dragees, capsules, pills, and/or granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets

and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0133] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0134] Dosage forms for topical or transdermal administration of a compound of this invention can include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. Generally, the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be prepared, for example, by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0135] Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention.

Methods of Use and Treatment

[0136] Inventive HIV fusion inhibitors are useful in the treatment of HIV infection and/or associated diseases, disorders and conditions. Thus, pharmaceutical compositions containing one or more inventive HIV fusion inhibitors may be administered to one or more individuals suffering from or susceptible to HIV infection. The present invention therefore encompasses methods of inhibiting gp41 conformational change and/or HIV fusion in a biological sample, as well as methods of treating HIV infection in subjects.

[0137] Inventive HIV fusion inhibitors or compositions containing them can be administered according to any appropriate dosing regimen. Typically, about 1 mg/kg to about 25 mg/kg, of subject body weight is administered, one or more times a day, to obtain

the desired therapeutic effect. As is known in the art, the exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. Compounds of the invention are preferably formulated in unit dosage form for ease of administration and uniformity of dosage.

[0138] It will be understood, however, that the total daily usage of the compounds and pharmaceutical compositions of the present invention will be decided by the attending physician or veterinarian within the scope of sound medical judgment. The specific effective dose level for any particular patient or subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient or subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts.

[0139] It will also be appreciated that the compounds and pharmaceutically acceptable compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutically acceptable compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another agent used to treat the same disorder), or they may achieve different effects (e.g., control of any adverse effects). (0140] In will further be appreciated that therapeutically active agents utilized in combination may be admininstered together in a single composition, or alternatively may be administered separately in different compositions.

[0141] For example, inventive HIV fusion inhibitors may be administered in combination with one or more other HIV inhibitors including, for example, with one or more nucleotide analogs (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and/or fusion inhibitors (e.g., peptidic fusion inhibitors), other fusion inhibitors (e.g., enfurviritide/T-20/Fuzeon), etc.

[0142] Know nucleotide analogs include, for example, Abacavir (Ziagen ^® ); AZT -

Zidovudine (Retrovir ^® ); ddC - Zalcitabine (Hivid ^φ ); ddl - Didanosine (Videx ^® ); d4T -

Stavudine (Zerit ^® ); FTC- Emtricitabine (Emtriva ^® ); 3TC - Lamivudine (Epivir ^® ) Tenofovir

(Viread ^® ), and combinations thereof. Particularly useful such combinations include, for example, AZT+3TC; TDF+3TC; TDF+FTC; ABC+3TC; Abacavir+3TC; etc.

[0143] Known non-nucleoside reverse transcriptase inhibitors include, for example,

Nevirapine (Viramune ); Efavirenz (Sustiva ); Delavirdine (Rescriptor ); and combinations thereof.

[0144] Known protease inhibitors include, for example, Amprenavir (Agenerase );

Atazanavir (Reyataz ^® ); Fosamprenavir (Telzir®, Lexiva®); Indinavir (Crixivan );

Lopinavir/r (Kaletra ^® ); Nelfinavir (Viracept ^® ); Ritonavir (Norvir ^® ); Saquinavir (Invirase ^® ,

Fortovase ^® ) and combinations thereof. As is known in the art, it is often desirable to "boost" protease inhibitors by combination with one or more inhibitors of cytochrome P450. For example, ritonavir is a very potent inhibitor of the isoenzyme 3A4, a subunit of cytochrome

P450. Small doses of ritonavir allow boosting of pharmacokinetic parameters sue as maximum concentration (Cmax), trough levels (Ctrough), and/or half-life, and can simplify daily dosing regimens. Appropriate boosting combinations include, for example, saquinavir/r; indinavir/r; lopinavir/r; etc.

[0145] Alternatively or additionally, inventve HIV fusion inhibitors may be administered in combination with one or more anti-infectives (e.g., antibiotics, etc.), pain relievers, or other agents intended to address symptoms of one or more diseases, disorders, or conditions that afflict an immunocompromised individual but are not directly caused by HIV.

[0146] In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

TREATMENT KITS

[0147] In other embodiments, the present invention relates to a kit for conveniently and effectively carrying out the methods in accordance with the present invention. In general, an inventive pharmaceutical pack or kit comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such kits are especially suited for the delivery of solid oral forms such as tablets or capsules. In some embodiments, such a kit includes a number of unit dosages, and may also include a card

having the dosages oriented in the order of their intended use. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered. Alternatively, placebo dosages, or calcium dietary supplements, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, can be included to provide a kit in which a dosage is taken every day. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

EXEMPLIFICATION

[0148] The representative Examples that follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art.

[0149] The following Examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and the equivalents thereof. It will be appreciated, however, that these examples do not limit the invention. Variations of the invention, now known or further developed, are considered to fall within the scope of the present invention as described herein and as hereinafter claimed.

Example 1; Identification of HlV Fusion Inhibitors Using Inventive Screen [0150] The present Example describes the development and utilization of a system for identifying HIV fusion inhibitors. In particular, this Example describes an inventive screen in which potential HIV fusion inhibitors are screened for their ability to compete with a peptide corresponding to a gρ41 outer helix for binding to a gp41 inner core construct.

Materials and Methods

[0151] Gp41-5 cloning, expression and purification: We have expressed a single- chain model for five of the six gp41 helices (Fig. 2a). The construct contains residues 35 through 70 of HRl and residues 117 thru 150 of HR2 from HXB2. The amino acid sequence for HRl is SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARJL, and for HR2, WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL. The C-terminal end of each of the first two inner-core segments is connected to the N-terminal end of the succeeding outer- layer helix by the 6 residue linker, SGGRGG. The C-terminal end of each outer-layer helix is connected to the N-terminal end of the succeeding inner-core segment by the 6 residue linker GGKGGS. The DNA fragment encoding gp41-5 was subcloned into the expression vector pRSET (Invitrogen) and transformed into Escherichia coli cells BL21 DE3/pUBS. For purification, cell pellets were dissolved in cold (4°C) glacial acetic acid and incubated on ice for 30 minutes. Cell debris was removed by centrirugation (RCF=I 8000, 30 min). The supernatant was diluted to 10% acetic acid with deionized water and loaded onto a r/+eversed phase Cl 8 column (Vydac) . The column was eluted with an acetonitrile gradient (30% to 90%). The protein eluted at 50% acetonitrile; it was >90% pure as judged by SDS PAGE. [0152] Gp41»5 refolding: Lyophilized protein was dissolved in 6M guanidine HCl at a concentration of lmg/ml and dialyzed successively against 10OmM glycine pH3.5 and PBS pH 7.4. The precipitate was removed by centrifugation, and the protein (>98% pure as judged by SDS PAGE) was used without further purification.

[0153] Peptide synthesis: All peptides were synthesized using fmoc chemistry on PAL supports using an Applied Biosystems model 431 peptide synthesizer. Peptides were cleaved using Reagant R (TFA/thioanisole/EDT/anisole 90:5:3:2), precipitated into cold diethyl ether and purified by reversed phase C18 HPLC (0.1%TFA:acetonitrile gradient). All peptides were characterized by electrospray mass spectrometry at the Mass Spectrometry Facility in the Department of Chemistry and Chemical Biology at Harvard University, Labeling of peptides at the N-terminus was achieved as follows. Synthetic peptide, still attached to the resin and with side chains protected but N-terminus deprotected, was suspended in a small volume of NMP. 5-FAM (Molecular Probes) was dissolved in NMP and added to the peptide suspension followed by the addition of lOul 4-methyl morpholine. The reaction was allowed to proceed under slow stirring for approximately two days. Cleavage and deprotection of labeled peptides was performed as described above.

[0154] High-throughput screening: All screens were performed at the Harvard Medical School Institute for Chemistry and Chemical Biology (ICCB). Gp41-5 at a concentration of

7.4 nM in PBST was loaded into 384 well microtitre plates (30 μl volume per well), followed by transfer of 0,1 μl of compound (5 mg/ml in DMSO). After incubation for 1 hour at room temperature, fluorescein-labeled C38 was added to each well (final concentration 5-10 nM). After a 90 minute incubation period at room temperature, fluorescence polarization measurments were recorded on an Analyst HTS. A 12 hour time point revealed no major differences from the 90 minute time point. Compounds that appeared to be active were retested independently. Commercial libraries screened included those from CEREP ₅ Maybridge, Bionet, and Chembridge. The hits described in this paper were from Bionet (the 5M series) and CEREP (S2986).

Results

[0155] We designed and expressed a soluble, single-chain protein construct we call gp41 -

5, in order to provide a suitable target for a high-throughput assay for HIV fusion inhibitors

(Fig. 2). This strategy takes advantage of the unusually simple structure of gp41 in its postfusion state - a trimer of hairpins that can be linked into a single, covalent polypeptide that folds into a six-helix bundle. A related construct (called "five-helix") was used as a fusion inhibitor, in studies mentioned above (25). Our gp41-5 contains three inner-core segments (residues 35-70), alternating with two outer-layer segments (residues 117-150).

Short linkers connect the segments. After folding, the molecule contains five of the helices present in the six-helix bundle. It is relatively soluble, and it binds with high affinity peptides that contain part or all of sequence in the sixth helix. Moreover, molecules of any sort that can bind the groove exposed by the absence of the sixth HR region should also be able to bind the pre-hairpin intermediate.

[0156] A peptide with the sequence of the missing outer helix (C38, residues 117-154) was labeled at its N-terminus with fluoresceine. Binding of the labeled peptide (C 38*) to gp41-5 was monitored by fluorescence polarization (Fig. 2C). The assay is sensitive, enabling us to perform high-throughput screens.

[0157] We screened several libraries of small molecules (MW<500), searching for compounds that interfered with binding of C38*. As a positive control, C38 (6 μM) was added to one well on each plate.

[0158] Of the 34,800 compounds screened, four (5MO3O, 5M038, 5M041, and S2986,

Fig. 3A) completely blocked C38* binding to gp41-5 under our screening conditions (library compound concentration = 40 μM; C38* = 10 nM). Three contained a 2,4-

bis(trifluoromethyl)[l,2,4] triazolo[4,3-a][l,8] naphthyridine ring, with different substituents in the 9-position. Follow-up analysis of structure activity relationships revealed two additional compounds structurally similar to 5M038 (Fig. 3A). Only compounds 5M038 and 5M030 had sufficient solubility and inhibitory activity to determine accurate IC5 ₀ S in our fluorescence polarization assay (~5 μM and 9 μM, respectively: Fig. 3B). Compounds 5M041 and 6K061 also had significant inhibitory activity (30% and 40 % inhibition, respectively, at 7.5 μM). Compound 6M007, which contains a similar ring system with the exception of a trifluoromethyl substituent in the 6-ρosition rather than the 2 and 4 positions, showed no activity in the fluorescence polarization assay.

Example 2: HIV Fusion Inhibitors Block Cell-Cell Fusion

[0159] The present Example describes further characterization of compounds identified herein, and illustrates the ability of certain compounds to block gp41 -mediated membrane fusion.

Materials and Methods

[0160] Further to the Materials and Methods included in other Examples, the following

Materials and Methods were employed:

[0161] Cell-cell fusion assay: Assays were performed as described previously (23, 27) with the following modifications. Target and effector cells were mixed briefly at equal concentrations prior to transfer to 96 well plates. All inhibitors were dissolved in DMSO and diluted 100 fold in the final assay. After the addition of inhibitor cells were incubated at 30 C for 6 hours, a time point previously determined to be in the linear range of the assay. Assays were stopped by aspiration of media followed by addition of reporter lysis buffer (Promega) and transfer to -20 ⁰ C.

Results

[0162] To determine the effects of compounds identified in herein on gp41 -mediated membrane fusion, we used a cell-cell fusion assay (23, 27). Compound toxicities were initially determined using Trypan blue staining. In addition, to control for non-specific effects of added compound, we prepared doubly transfected cells with plasmids encoding T7 polymerase and luciferase. Compounds 5M038 and 5M041 strongly inhibited gp41-mediated membrane fusion. At their solubility limits (90 μM for 5M038; 50 μM for 5M041), both

compounds reduced the level of cell-cell fusion by more than 80% (Fig. 4). Although compound 6M007 had little activity in the fluorescence polarization assay, it was as active as 5M038 against envelope-mediated membrane fusion. We discuss below this difference in assay response. Compounds 6K061 and 5M030 were too toxic to be tested properly.

Example 3: Inventive HIV Fusion Inhibitors Block HIV Infection

[0163] The present Example describes further characterization of compounds identified herein, and illustrates the ability of certain compounds to block HIV infection.

Materials and Methods

(0164] Further to the Materials and Methods presented in other Examples, the following Materials and Methods were employed:

[0165] Viral-infectivity assay; Peripheral blood mononuclear cells (PBMCs) were exposed to HIV-2076 (1000 TCID50/10 ⁶ cells) for 1 hour. Cells were washed once with medium, and 0.1 ml of HIV-infected PBMCs (2x10 ⁵ cells) were added to each well of a 96 flat- well cell plate followed by the addition of compound. The final concentration of DMSO in each well was 0.5%. Supernatants were harvested after 5 days and assayed for HIV-I p24 antigen production. For time course assays, 10 ⁶ MT-2 cells were cultured in 1 ml of RPMI- 1640 with 10% fetal calf serum in individual wells of a 24 well plate. In selected wells, 5M038 or enfuvirtide was added to each well to achieve final concentrations of 30 or 50 μM for 5M038 or 50 ng/ml (11 nM) enfuvirtide. Each control or experimental condition was conducted in triplicate. After one hour, 500 TCID _5O HIV-IIIB was added and cells were incubated for 7 days at 37° C in 5% CO ₂ . 100 μL of supernatant fluid was removed from each well on days 3, 5 and 7 for HIV-I p24 Ag quantification. The medium was replaced with culture medium with the appropriate concentrations of inhibitor. HIV-I p24 Ag was quantified using kits according to the manufacturer's instructions (Perkin Elmer).

Results

[00139 J We determined the effects on HIV infectivity of compounds described herein, by exposing peripheral blood mononuclear cells to HIV-I (2076) in the presence and absence of each compound. When cells were pretreated with a compound prior to HIV infection, compounds 5M038 and 5M041 blocked infection with IC ₅₀ S of 19 and 18 μM respectively (Table 1). 6M007 had a weaker effect, showing 50% inhibition at a concentration greater

than 30 μM. To demonstrate that, like enfuvirtide, 5M038 can inhibit cell-to-cell spread of virus, we compared the two directly over a 7 day period as shown in Fig 5. At a concentration as low as 30 μM, 5M038 nearly completely suppressed appearance of p24 antigen over the course of the experiment.

Example 4: Binding Site of Inventive BIV Fusion Inhibitors

[0166) The present Example describes studies to pinpoint the binding site of certain inventive HIV Fusion Inhibitors within gp41.

Materials and Methods

[0167] Further to the Materials and Methods presented in other Examples, the following Materials and Methods were employed:

[0168] Gp41 chimeras: All chimeras were synthesized using fmoc chemistry and purified as described above. The peptide sequence for construct 1 is Ac- RMKQIEDKIEEIESKQKKIEN EIARIKKLLSQIVQQQNNLLRAIEA-NH ₂ . The peptide sequence for construct 2 is Ac-RMKQIE

DKIEEIESKQKKIENEIARJKKLQNNLLRAIEAQQHLLQL-NH ₂ . The peptide sequence for constuct 3 (IQNl 7) is Ac-RMKQIE

DKIEEIESKQKKIENEIARIKKLLQLTVWGIKQLQARIL-NH ₂ . All constructs were analyzed by CD spectroscopy and analytical ultracentrifugation. Constructs 1 and 3 were fully helical; construct 2 was >60% helical. Constructs 1 and 3 gave apparent molecular weights consistent with a trimer, while construct 2 gave a slightly lower apparent molecular weight (11,000 as compared to expected 16,782). We interpreted this last result to indicate a mixture of monomer and trimer. Size exclusion chromatography supported this interpretation. [0169] Proton NMR experiments: All assays were performed on a Varian 400MHz spectrophotometer with triple resonance probe. Samples were prepared in PBS/D2O. Compound stocks were prepared in DMSO-de at 10mg/ml (-26 mM) and diluted in PBS/D20 to a final concentration of 200μM (final DMSO < 1%). Spectra were in the presence or absence of protein, also at 200 μM (based on MW of trimer).

Results

[0170] Proton NMR experiments show that 5M038 gives significantly broadened proton resonances in the presence of gρ41 -5 (data not shown). To use this property to pinpoint the location of binding, we synthesized a set of three peptides containing 17-residue segments of

the gρ41 inner core attached to a 29-residue trimerization domain derived from the coiled- coil of GCN4 (Fig. 6). The latter helps to solubilize and trimerize the short segment of gp41 core and permits its use at concentrations suitable for NMR.

[0171] Proton NMR spectra were recorded from compound 5M038 (200 μM) in the presence of each of the three peptides (also at 200 μM). Resonances remain sharp in the presence of constructs 1 and 2, which contain residues 34-50 and 41-57, respectively. In the presence of peptide 3, which contains residues 54-70, the proton lines from 5M038 broaden significantly and shift upfield. This segment of the inner core contains a deep cavity formed by residues surrounding Leu57, Trp60 and Lys 63 and occupied in the post-fusion structure by Trpl 17, Trpl20 and lie 124 from the outer-layer helix. This pocket has been exploited previously (22, 23), and it appears likely that 5M038 binds it as well. [0172] To probe further the binding site for these molecules, we performed competition assays using peptides missing key residues that insert into the inner-core pocket. While 5M038 is able to compete against a "full-length" peptide containing residues 117-154, it shows no activity against a shorter peptide containing residues 119-154 (data not shown). The latter lacks the key binding residue Trpl 17; the result provides further evidence that 5M038 occupies the Trpl 17 pocket. T-20/enfuvirtide does not include Trpl 17, and 5M038 and enfuvirtide are indeed weakly synergistic in viral infectivity experiments (data not shown).

Example 5: Broad Spectrum Activity of Inventive HIV Fusion Inhibitors

[0173 J The present Example demonstrates that inventive HIV fusion inhibitors can have broad activity across different HIV isolates.

Materials and Methods

[0174] As will be clear from context, various Materials and Methods described herein were utilized in this Example.

Results

[0175] The hydrophobic pocket formed by Leu57, Trp60 and Lys 63 is a very highly conserved region among the different isolates of HIV-I group M (see Table 2). We expected inventive HIV fusion inhibitors that target this region to show activity across a broad range of

isolates. Conversely, broad activity would be consistent with our identification of the site and mode of action.

[0176] We selected a number of Env sequences derived from primary isolates and assayed them for cell-cell fusion (see Materials and Methods herein) in the presence of compound 5M038. The sequences include members from clades A,B ₅ D,E and F and include both T- and M-tropic strains. We also included two divergent strains BCF03 from group O and SIV mac32H. The compound was active against the all of the group M isolates tested while exhibiting much weaker activity towards the group O strain (only 40% inhibition at 100 μM) and SIV (35% inhibition at 100 μM).

Example 6: Use of Inventive Screen to Characterize Peptidic HIV Fusion Inhibitors [0177J The present Example descrbes characterization of known peptide HIV fusion inhibitors in an inventive binding competition assay.

Materials and Methods

[0178] As will be clear from context, various Materials and Methods described herein were utilized in this Example.

Results

[0179] We used the fluorescence polarization assay to examine other inhibitors that target gp41. A peptide corresponding to the part of T-20 contained in gρ41-5 (i.e., residues 127- 154) and a peptidic entry inhibitor (23) both failed to compete with C38 at concentrations up to 100 μM and 150 μM respectively under our assay conditions ([*C38]=5 nM), even though the peptidic entry inhibitor blocks cell-cell fusion. These results are similar to our observations with 6M007. The in vitro fluorescence polarization assay is an equilibrium measurement; whereas the inhibition of fusion (cell-cell or virus-cell) is probably a kinetic one. Moreover, there are three potential sites per envelope trimer in the cellular assays, but only one in our gp41-5 measurement. For both these reasons, competitive inhibition of binding of outer helix to gp41-5 should be a more stringent criterion of interaction than inhibition of fusion. The present invention therefore provides a new system for identifying HIV fusion inhibitors. The spectrum of potential fusion inhibitors, and particularly small molecule fusion inhibitors is likely broader than the particular range of compounds that we have already detected and described herein. Also, limited solubility of some of the

compounds, such as 6M007, and its variation with buffer, ionic strength, or other factors, may also account for differences between cell-based and protein-based assays.

Example 7: Crystallization of an Inventive HIV Fusion Inhibitor with a gp41 Model Polypeptide

[0180] The present Example describes crystallization of a gρ41 model with model 5M038. To improve the solubility of gp41-5, we added various lengths of HR2 to the C- terminus of the expressed protein. Crystals were obtained with at least two different such constructs. In one form, the Trpl l7 pocket was occluded by a crystal contact. In another, which appeared to contain the compound, substantial twinning precluded confident placement of 5M038. Two strong peaks, likely to mark the locations of the electron-dense - CF ₃ groups, restrict possible models, as shown in Fig. 7.

Example 8: Design of Additional HIV Fusion Inhibitors

[0181] The present Example describes a system for designing and screening potential small molecule compounds that inhibit HIV fusion as described herein.

a. Starting point

[0182] i. The starting point for the initial libraries will be compound 5M038, as this compound shows favorable solubility and low toxicity in cells. From preliminary SAR based on compounds screened, we have determined that compound 5M038, the active compound, contains trifluoromethyl groups in the 2 and 4 positions and a phenyl group in the 9 position (Fig. 9). The other active compounds of this set also have the 2 trifluoromethyl groups but with different aromatic groups in the 9 position (Fig. 9). Compound 6M007 is less active, missing the 2 trifluoromethyl groups but with an additional trifluormethyl in the 6 position. The compound SM-amine, contains both trifluoromethyl groups, but has the aromatic group in the 9 position replaced with an amine. It also has lower activity. Thus, very similar compounds show different affinities. The importance of the trifluormethyl groups in the 2 and 4 position will be explored in initial library 1 (section d. i). The nature of the group in position 9 will be explored in library 2(d ii). The addition of substituents at position 6 will be explored in library 3 (d iii). See figure 9.

[0183] ii. Binding Pocket, Prior to round I, we will screen 5M038 and 5M041 (both active) and 6M007 (less active), as well as most or all commercially available analogs, by computational docking into the known binding pocket (fig. 7). These computations will guide our design of the initial libraries. The docking approach and the software we will use (Glide) are described in d.iv, below. We will use the results of this first computational screen to refine our hypotheses about critical SAR and to modify the design of libraries to test the hypotheses more critically.

b. Initial libraries

[0184] In the first stage of chemical synthesis, we will generate three small libraries aimed at identifying the minimum pharmacophore necessary for activity and at establishing SAR trends. The variable R groups in each of the libraries will be chosen both to explore SAR trends and to test hypotheses based on docking studies (see section d.iv). The libraries will be screened using our FP assay (see section d.i), and the results will be used to rank each chemical modification. If any of the initial compounds has Kd<500 nM, it will also be tested for activity against viral infectivity (see section d.ii). Active compounds from the initial libraries will be studied by molecular modeling/docking, to aid in the generation of subsequent rounds of chemical syntheses. Compounds with Kd<100 nM will be candidates for co-crystallization with gp41-5α (see section d.v).

[0185] i. Exploration of the importance of the trifluoromethyl group (initial library 1 Fig 10). In our published screens, the active set contains trifluoromethyl groups at positions 2 and 4, and one less active compound (6M007) lacks both, while containing instead a trifluoromethyl group at position 6. Our first library will therefore focus on the systematic removal and/or replacement of the two trifluoromethyl groups at positions 2 and 4 of compound 5M038 (Fig. 9). The selected replacement groups will represent a diverse set of isoelectronic and isosteric equivalents to the trifluoromethyl group (15-20 analogs, 2 rounds). [0186] ii. Exploratin o/ the R group at position 9 (initial library 2). This library (15-20 analogs, 2 rounds) will focus on the systematic variation of the R group in the 9 position in an attempt to establish SAR trends (fig. 11). All of the active compounds that have been screened contain a small aromatic group in this position. R will therefore be selected to represent a diverse set of electron withdrawing, electron donating and steric substituents. Aldehydes and water solubilizing groups will also be considered in order to improve solubility. Initial results indicate that compounds of structure A (see fig. 13) showed good activity and compounds of structure B showed less.

[0187] iii. Exploration of Rl group at position 6 (initial library 3 fig. 12). This library (15-20 analogs, 2 rounds) will look at derivatives of 5M038 at the 6 position to establish SAR trends. At this time all active compounds contain a hydrogen at position 6. Rl will be selected to represent a diverse set of electron withdrawing, electron donating and steric substituents. R will remain a phenyl group throughout this library, as in 5M038, in order to obtain SAR trends specific to Rl .

c. Subsequent steps

[0188] i. Generation of round 2 libraries. Results from the initial libraries will be used to generate a second round (2 libraries anticipated). These libraries will combine successful aspects of the initial 3 libraries. For example, the highest affinity R from library 2 and Rl from library 3 will be combined. They will also be designed to refine our hypotheses about SAR trends discovered in round 1. As with the initial libraries, these compounds will be tested in our FP assay (see section d.l) and the results will be used in combination with round 1 to rank each chemical modification and construct SAR trends. Compounds with Kd<500 nM will be tested for activity against viral infectivity (see section d ii). Active compounds will also be studied by molecular modeling/docking to aid in the creation of round 3 library.

[0189] ii. Generation of round 3 library. As in c.i, results from rounds 1 and 2 will be used to generate a final library,

[0190] iii. Co-crystallization of high affinity compounds with gp415 alpha. Compounds with affinities <100nM will be co-crystallized with the gp41 pre-fusion mimetic gp41-5alρha (see section d. v).

d. Methods

[0191] i. FP. All of the compounds synthesized for this study are tested in the FP assay as previously described (Frey, Rits-Volloch et al. 2006), with slight modifications. Stock solutions of each compound are made by dissolving it in DMSO at a concentration of 5mg/ml. Assays are performed in 384 well microtiter plates (Nunc), and fluorescence polarization measurement are made on an Analyst HTS (Molecular Devices). Gp41-5 is added to each well at a final concentration of 7.4nM in PBST (5OmM phosphate buffer pH7.4, 15OmM NaCl 0.05% Tween), followed by transfer of 0.5 μl of compound (~150uM). After incubation for 1 hour at room temperature, fluorescein-labeled C38 is added to each well (final concentration 5-10 nM). After a 90 minute incubation period at room temperature,

fluorescence polarization measurements are recorded. A 12 hour time point is also recorded to ensure that equilibrium has been reached. Active compounds are re-tested by titration to determine IC50s.

[0192] ii. Viral infectivity. We use an assay designed to report inhibition of early steps in the viral life cycle (Li, Gao et al. 2005). The panel of reporter viruses we employ has been selected to represent a range of isolates and clades. It is the panel used for standardized antibody neutralization assays: like our compounds, neutralizing antibodies prevent viral entry. Activity is measured as the decrease in luciferase (Luc) reporter-gene expression after a single round of virus infection in JC53-BL cells, a genetically engineered HeLa cell line expressing CD4, CCR5, CXCR4, and a Tat responsive reporter gene for firefly luciferase. Briefly, 200 TC1D50 of virus is incubated with various dilutions of test samples for 1 hour at 37 C. Freshly trypsinized cells are then added to each well. After a 48 hour incubation, cells are treated with Bright GIo reagant After a 2 minute incubation at room temperature to allow for cell lysis, samples of lysate are transferred to black 96 well plates for luminescence measurements.

[0193] iii. Cell-cell fusion. As a complement to the viral infectivity assays, compounds with significant increases in affinity (Ko<500nM) are tested for activity against envelope mediated membrane fusion in a cell cell fusion assay. Assays are performed as described previously (Nussbaum, Broder et al. 1994); Ferrer, Kapoor et al. 1999). Target and effector cells are mixed briefly at equal concentrations prior to transfer to 96 well plates. All inhibitors are dissolved in DMSO and diluted 100 fold in the final assay. After the addition of inhibitor, cells are incubated at 30° C for 6 hours, a time point previously determined to be in the linear range of the assay. Assays are stopped by aspiration of media followed by addition of reporter lysis buffer (Promega) and transfer to -20 ⁰ C.

[0194] iv. Computational docking. We will use the Glide software package (Schrodinger Inc) (Friesner, Banks et al. 2004) to predict docking interactions. The Glide package is one of the most accurate commercially available molecular docking packages (Halgren, Murphy et al. 2004). It is widely used in the pharmaceutical industry (Perola, Walters et al. 2004), and it has been used successfully to identify lead compounds for the treatment of ALS (Ray, Nowak et al. 2005). Computational docking will provide hypotheses on how the compounds interact with the pocket and guide our compound selection at each stage of chemical library synthesis. The four steps in performing a docking study using Glide are (1) ligand preparation, (2) protein preparation, (3) docking analysis, (4) optimization/energy minimization. In the course of our previous work, we obtained a crystal structure of 5MO38

at 0.9A resolution (unpublished). These coordinates give us a very accurate structure for this ligand, including the orientation of rotatable bonds. These coordinates are used in the ligand preparation step. For the target-protein structure, we use coordinates derived from crystals of gp41-5α (unpublished), which contains a single exposed binding pocket. Some of the neighboring side chains rearrange, with respect to their positions in the complete, post-fusion gp41 structure (with all pockets filled with outer-layer Trpl 17). For comparison, we also carry out some calculations with the coordinates derived from complete gp41, with the outer- layer "peeled away" from the pocket but without rearrangement of neighboring side chains. [0195] v. Crystallization and x-ray structure determination. We have designed a mimetic of the pre-fusion intermediate, which we call gρ41-5α. Crystallization of gp41-5, the mimetic on which we base our FP assay, is difficult, because of its limited sollubility. The design of gp41-5α overcomes this problem by adding a portion of the remaining outer- layer helix, to cover a substantial part of the hydrophobic core, while leaving the binding pocket exposed. We crystallize gp41-5α (unpublished), and phases are obtained by molecular replacement. The structural studies are carried out in parallel with medicinal chemistry, in an effort to accelerate the process with a fully structure-based approach.

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EQUIVALENTS

[0196] While we have described a number of embodiments of this invention, it is apparent that the examples and embodiments specifically detailed herein may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been particularly represented herein.