WO2009012259A1 - Pentafluorosulfanyl-substituted piperazinylpiperidine derivatives as chemokine receptor antagonists - Google Patents

Abstract

The present invention relates to pentaf luorosulfanyl substituted compounds that modulate the activity of or bind to chemokine receptors such as CCR5, rendering the compounds useful in the treatment of inflammatory diseases, immune diseases and viral infections.

Description

PENTAFLUOROSULFANYL-SUBSTITUTED PIPERAZINYLPIPERIDINE DERIVATIVES AS CHEMOKINE RECEPTOR ANTAGONISTS

FIELD OF THE INVENTION

The present invention relates to pentafluorosulfanyl substituted compounds that modulate the activity of or bind to chemokine receptors such as CCR5, rendering the compounds useful in the treatment of inflammatory diseases, immune diseases and viral infections.

BACKGROUND OF THE INVENTION

The migration and transport of leukocytes from blood vessels into diseased tissues is involved in the initiation of normal disease-fighting inflammatory responses. The process, also known as leukocyte recruitment, is also related to the onset and progression of life- threatening inflammatory, as well as debilitating autoimmune diseases. The resulting pathology of these diseases derives from the attack of the body's immune system defenses on normal tissues. Accordingly, preventing and blocking leukocyte recruitment to target tissues in inflammatory and autoimmune disease would be a highly effective approach to therapeutic intervention.

The different classes of leukocyte cells that are involved in cellular immune responses include monocytes, lymphocytes, neutrophils, eosinophils and basophils. In most cases, lymphocytes are the leukocyte class that initiates, coordinates, and maintains chronic inflammatory responses, and blockage of these cells from entering inflammatory sites is desirable. Lymphocytes attract monocytes to the tissue sites, which, collectively with lymphocytes, are responsible for most of the actual tissue damage that occurs in inflammatory disease. Infiltration of the lymphocytes and/or monocytes is known to lead to a wide range of chronic, autoimmune diseases, and also organ transplant rejection. These diseases include, but are not limited to, rheumatoid arthritis, chronic contact dermatitis, inflammatory bowel disease, lupus, systemic lupus erythematosus, multiple sclerosis, atherosclerosis, psoriasis, sarcoidosis, idiopathic pulmonary fibrosis, dermatomyσsitis, skin pemphigoid and related diseases, (e.g., Pemphigus vulgaris, P. foliacious, P. etythematosis), glomerulonephritides, vasculitides, hepatitis, diabetes, allograft rejection, and graft-versus- host disease. The process by which leukocytes leave the bloodstream, accumulate at inflammatory sites, and start disease is believed to have at least three steps which have been described as (1) rolling, (2) activation/firm adhesion and (3) transendothelial migration [Springer, T. A., Nature 346:425-433 (1990); Lawrence and Springer, Cell 65:859-873 (1991); Butcher, E. C, Cell 67: 1033-1036 (1991)]. The second step is mediated at the molecular level by chemoattractant receptors. Chemoattractant receptors on the surface of leukocytes then bind chemoattractant cytokines which are secreted by cells at the site of damage or infection. Receptor binding activates leukocytes increases the adhesiveness of the adhesion molecules that mediate transendothelial migration and promotes directed migration of the cells toward the source of the chemoattractant cytokine.

Chemotactic cytokines (leukocyte chemoattractant/activating factors) also known as chemokines, also known as intercrines and SIS cytokines are a group of inflammatory/ immunomodulatory polypeptide factors of molecular weight 6-15 kDa that are released by a wide variety of cells such as macrophages, monocytes, eosinophils, neutrophiles, fibroblasts, vascular endothelial cells, smooth muscle cells, and mast cells, at inflammatory sites (reviewed in Luster, New Eng. J Med., 338, 436-445 (1998) and Rollins, Blood, 90, 909-928 (1997)). Also, chemokines have been described in Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Schall and Bacon, Curr. Opin. Immunol, 6:865-873 (1994); Baggiolini, M., et al., and Adv. Immunol., 55:97-179 (1994). Chemokines have the ability to stimulate directed cell migration, a process known as chemotaxis. Each chemokine contains four cysteine residues (C) and two internal disulfide bonds. Chemokines can be grouped into two subfamilies, based on whether the two amino terminal cysteine residues are immediately adjacent (CC family) or separated by one amino acid (CXC family). These differences correlate with the organization of the two subfamilies into separate gene clusters. Within each gene cluster, the chemokines typically show sequence similarities between 25 to 60%. The CXC chemokines, such as interleukin-8 (IL-8), neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatory activity protein (MGSA) are chemotactic primarily for neutrophils and T lymphocytes, whereas the CC chemokines, such as RANTES, MIP- lα, MIP- lβ, the monocyte chemotactic proteins (MCP-I, MCP-2, MCP-3, MCP-4, and MCP-5) and the eotaxins (-1 and -2) are chemotactic for, among other cell types, macrophages, T lymphocytes, eosinophils, dendritic cells, and basophils. There also exist the chemokines lymphotactin-1, lymphotactin-2 (both C chemokines), and fractalkine (a CXXXC chemokine) that do not fall into either of the major chemokine subfamilies. MCP-I (also known as MCAF (abbreviation for macrophage chemotactic and activating factor) or JE) is a CC chemokine produced by monocytes/macrophages, smooth muscle cells, fibroblasts, and vascular endothelial cells and causes cell migration and cell adhesion of monocytes (see for example Valente, A. J., et al, Biochemistry, 1988, 27, 4162; Matsushima, K., et al., J. Exp. Med., 1989, 169, 1485; Yoshimura, T., et al., J. Immunol., 1989, 142, 1956; Rollins, B. J., et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 3738; Rollins, B. J., et al., Blood, 1991, 78, 11 12; Jiang, Y., et al., J. Immunol., 1992, 148, 2423; Vaddi, K., et al., J. Immunol., 1994, 153, 4721), memory T lymphocytes (see for example Carr, M. W., et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 3652), T lymphocytes (see for example Loetscher, P., et al., FASEB J., 1994, 8, 1055) and natural killer cells (see for example Loetscher, P., et al., J. Immunol., 1996, 156, 322; Allavena, P., et al., Eur. J. Immunol. , 1994, 24, 3233), as well as mediating histamine release by basophils (see for example Alam, R., et al., J. Clin. Invest., 1992, 89, 723; Bischoff, S. C, et al., J. Exp. Med., 1992, 175, 1271; Kuna, P., et al., J. Exp. Med., 1992, 175, 489). In addition, high expression of MCP-I has been reported in diseases where accumulation of monocyte/macrophage and/or T cells is thought to be important in the initiation or progression of diseases, such as atherosclerosis (see for example Hayes, I. M., et al., Arterioscler. Thromb. Vase. Biol, 1998, 18, 397; Takeya, M.. et al., Hum. Pathol., 1993, 24, 534; Yla-Herttuala, S., et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 5252; Nelken, N. A., J. Clin. Invest., 1991, 88, 1121), rheumatoid arthritis (see for example Koch, A. E., et al., J. Clin. Invest., 1992, 90, 772; Akahoshi, T., et al., Arthritis Rheum., 1993, 36, 762; Robinson, E., et al., Clin. Exp. Immunol., 101, 398), nephritis (see for example Noris, M., et al., Lab. Invest., 1995, 73, 804; Wada, T., at al., Kidney Int., 1996, 49, 761; Gesualdo, L., et al., Kidney Int., 1997, 51, 155), nephropathy (see for example Saitoh, A., et al., J. Clin. Lab. Anal., 1998, 12, 1; Yokoyama, H., et al., J. Leukoc. Biol., 1998, 63, 493), pulmonary fibrosis, pulmonary sarcoidosis (see for example Sugiyama, Y., et al., Internal Medicine, 1997, 36, 856), asthma (see for example Karma, M., et al., J. Invest. Allergol. Clin. Immunol., 1997, 7, 254; Stephene, T. H., Am. J. Respir. Crit. Care Med., 1997, 156, 1377; Sousa, A. R., et al., Am. J. Respir. Cell MoI. Biol., 1994, 10, 142), multiple sclerosis (see for example McManus, C, et al., J. Neuroimmunol., 1998, 86, 20), psoriasis (see for example Gillitzer, R., et al., J. Invest. Dermatol., 1993, 101, 127), inflammatory bowel disease (see for example Grimm, M. C, et al., J. Leukoc. Biol., 1996, 59, 804; Reinecker, H. C, et al., Gastroenterology, 1995, 106, 40), myocarditis (see for example Seino, Y., et al., Cytokine, 1995, 7, 301), endometriosis (see for example Jolicoeur, C, et al., Am. J. Pathol., 1998, 152, 125), intraperitoneal adhesion (see for example Zeyneloglu, H. B., et al., Human Reproduction, 1998, 13, 1 194), congestive heart failure (see for example Aurust, P., et al., Circulation, 1998, 97, 1 136), chronic liver disease (see for example Marra, F., et al., Am. J. Pathol., 1998, 152, 423), viral meningitis (see for example Lahrtz, F., et al., Eur. J. Immunol., 1997, 27, 2484), Kawasaki disease (see for example Wong, M.; et al., J. Rheumatol., 1997, 24,1179) and sepsis (see for example Salkowski, C. A.; et al., Infect. Immun., 1998, 66, 3569). Furthermore, anti-MCP-1 antibody has been reported to show an inhibitory effect or a therapeutic effect in animal models of rheumatoid arthritis (see for example Schimmer, R. C, et al., J. Immunol, 1998, 160, 1466; Schrier, D. J., J. Leukoc. Biol., 1998, 63, 359; Ogata, H., et al., J. Pathol., 1997, 182, 106), multiple sclerosis (see for example Karpus, W. J., et al., J. Leukoc. Biol., 1997, 62, 681), nephritis (see for example Lloyd, C. M., et al., J. Exp. Med., 1997, 185, 1371; Wada, T., et al., FASEB J., 1996, 10, 1418), asthma (see for example Gonzalo, J.-A., et al., J. Exp. Med., 1998, 188, 157; Lukacs, N. W., J. Immunol., 1997, 158, 4398), atherosclerosis (see for example Guzman, L. A., et al., Circulation, 1993, 88 (suppl.), 1-371), delayed type hypersensitivity (see for example Rand, M. L., et al., Am. J. Pathol., 1996, 148, 855), pulmonary hypertension (see for example Kimura, H., et al., Lab. Invest., 1998, 78, 571), and intraperitoneal adhesion (see for example Zeyneloglu, H. B., et al., Am. J. Obstet. Gynecol., 1998, 179, 438). A peptide antagonist of MCP-I, MCP-I (9-76), has been also reported to inhibit arthritis in the mouse model (see Gong, J.-H., J. Exp. ,4ed. , 1997, 186, 131), as well as studies in MCP-I -deficient mice have shown that MCP-I is essential for monocyte recruitment in vivo (see Lu, B., et al., J. Exp. Med., 1998, 187, 601; Gu, L., et al., Moll. Cell, 1998, 2, 275).

The literature indicates that chemokines such as MCP-I and MIP- let attract monocytes and lymphocytes to disease sites and mediate their activation and thus are thought to be intimately involved in the initiation, progression and maintenance of diseases deeply involving monocytes and lymphocytes, such as atherosclerosis, restenosis, rheumatoid arthritis, psoriasis, asthma, ulcerative colitis, nephritis (nephropathy), multiple sclerosis, pulmonary fibrosis, myocarditis, hepatitis, pancreatitis, sarcoidosis, Crohn's disease, endometriosis, congestive heart failure, viral meningitis, cerebral infarction, neuropathy, Kawasaki disease, and sepsis (see for example Rovin, B. H., et al., Am. J. Kidney. Dis., 1998, 31, 1065; Lloyd, C, et al., Curr. Opin. Nephrol. Hypertens., 1998, 7, 281; Conti, P., et al., Allergy and Asthma Proc, 1998, 19, 121 ; Ransohoff, R. M., et al., Trends Neurosci., 1998, 21, 154; MacDermott, R. P., et al., Inflammatory Bowel Diseases, 1998, 4, 54). The chemokines bind to specific cell-surface receptors belonging to the family of G- protein-coupled seven-transmembrane-domain proteins (reviewed in Horuk, Trends Pharm. Sci., 15, 159-165 (1994)) which are termed "chemokine receptors." On binding their cognate ligands, chemokine receptors transduce an intracellular signal through the associated trimeric G proteins, resulting in, among other responses, a rapid increase in intracellular calcium concentration, changes in cell shape, increased expression of cellular adhesion molecules, degranulation, and promotion of cell migration.

Genes encoding receptors of specific chemokines have been cloned, and it is known that these receptors are G protein-coupled seven-transmembrane receptors present on various leukocyte populations. So far, at least five CXC chemokine receptors (CXCRl -CXCR5) and eight CC chemokine receptors (CCRl -CCR8) have been identified. For example IL-8 is a ligand for CXCRl and CXCR2, MIP-Ia is that for CCRl and CCR5, and MCP-I is that for CCR2A and CCR2B (for reference, see for example, Holmes, W. E., et al., Science 1991, 253, 1278-1280; Murphy P. M., et al., Science, 253, 1280-1283; Neote, K. et al, Cell, 1993, 72, 415-425; Charo, I. F., et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 2752-2756; Yamagami, S., et al., Biochem. Biophys. Res. Commun., 1994, 202, 1156-1162; Combadier, C, et al, The Journal of Biological Chemistry, 1995, 270, 16491-16494, Power, C. A., et al., J. Biol. Chem., 1995, 270, 19495-19500; Samson, M., et al., Biochemistry, 1996, 35, 3362- 3367; Murphy, P. M., Annual Review of Immunology, 1994, 12, 592-633). It has been reported that lung inflammation and granuroma formation are suppressed in CCRl -deficient mice (see Gao, J.-L., et al., J. Exp. Med., 1997, 185, 1959; Gerard, C, et al., J. Clin. Invest., 1997, 100, 2022), and that recruitment of macrophages and formation of atherosclerotic lesion decreased in CCR2-deficient mice (see Boring, L., et al., Nature, 1998, 394, 894; Kuziel, W. A., et al., Proc. Natl. Acad. Sci., USA, 1997, 94, 12053; Kurihara, T., et al., J. Exp. Med., 1997, 186, 1757; Boring, L., et al., J. Clin. Invest, 1997, 100, 2552).

Chemokine receptors are also known as coreceptors for viral entry leading to viral infection such as, for example, HIV infection. Reverse transcription and protein processing are the classic steps of the viral life cycle which antiretroviral therapeutic agents are designed to block. Although many new drugs that are believed to block viral entry hold promise, there is currently no agent to which HIV-I has not been able to acquire resistance. Multiple rounds of viral replication are required to generate the genetic diversity that forms the basis of resistance. Combination therapy in which replication is maximally suppressed remains a cornerstone of treatment with entry inhibitors, as with other agents. The targeting of multiple steps within the viral entry process is believed to have the potential for synergy (Starr-Spires et al., Clin. Lab. Med., 2002, 22(3), 681.)

HIV-I entry into CD4(+) cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. The TAK-779 molecule is one such antagonist of CCR5 that acts to prevent HIV-I infection. TAK-779 inhibits HIV-I replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gpl20 with CCR5. The binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7 (Dragic et al., Proc. Natl. Acad. ScL USA, 2000, 97(10), 5639).

The chemokine receptors CXCR4 and CCR5 are believed to be used as co-receptors by the T cell-tropic (X4) and macrophage-tropic (R5) HIV-I strains, respectively, for entering their host cells. Propagation of R5 strains of HIV-I on CD4 lymphocytes and macrophages requires expression of the CCR5 coreceptor on the cell surface. Individuals lacking CCR5 (CCR5 Delta 32 homozygous genotype) are phenotypically normal and resistant to infection with HIV-I . Viral entry can be inhibited by the natural ligands for CXCR4 (the CXC chemokine SDF-I) and CCR5 (the CC chemokines RANTES, MIP-I alpha and MIP-I beta). The first non-peptidic compound that interacts with CCR5, and not with CXCR4, is a quaternary ammonium derivative, called TAK-779, which also has potent but variable anti-HIV activity (De Clercq et al., Antivir. Chem. Chemother. 2001, 12 Suppl. 1, 19.

SCH-C (SCH 351125) is another small molecule inhibitor of HIV-I entry via the

CCR5 coreceptor. SCH-C, an oxime-piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-I infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. (Strizki et al, Proc. Natl. Acad. ScL USA, 2001, 98(22), 12718 or Tremblay et al., Antimicrobial Agents and Chemotherapy, 2002, 46(5), 1336).

ADlOl, chemically related to SCH-C, also inhibits the entry of human immunodeficiency virus type 1 (HIV-I) via human CCR5. It has been found that ADlOl inhibits HIV-I entry via rhesus macaque CCR5 while SCH-C does not. Among the eight residues that differ between the human and macaque versions of the coreceptor, only one, methionine- 198, accounts for the insensitivity of macaque CCR5 to inhibition by SCH-C. Position 198 is in CCR5 transmembrane (TM) helix 5 and is not located within the previously defined binding site for ADlOl and SCH-C, which involves residues in TM helices 1 , 2, 3, and 7. Based on studies of amino acid substitutions in CCR5, it has been suggested that the region of CCR5 near residue 198 can influence the conformational state of this receptor. (Billick et al., 2004, J. Virol, 78(8), 4134). Accordingly, drugs which inhibit the binding of chemokines to their respective receptors can be useful as pharmaceutical agents which inhibit the action of chemokines on target cells and/or block viral entry into cells expressing these receptors. The identification of compounds that modulate the activity of chemokine receptors or block the binding of viral proteins represents an excellent drug design approach to the development of pharmacological agents for the treatment of inflammatory conditions, viral infection and other diseases associated with chemokine receptor activation. The compounds of the present invention help fulfill these and other needs

SUMMARY OF THE INVENTION The present invention provides, inter alia, CCR5-binding compounds of Formula I:

I or pharmaceutically acceptable salts thereof, wherein the variables are defined herein.

The present invention further provides compositions comprising a compound of Formula I, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

The present invention further provides methods for modulating activity of a chemokine receptor comprising contacting the chemokine receptor with a compound of Formula I, or pharmaceutically acceptable salt thereof. The present invention further provides a method of treating a disease associated with expression or activity of a chemokine receptor in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or pharmaceutically acceptable salt thereof.

The present invention further provides a method of treating a disease or condition selected from an inflammatory disease, immune disorder, and viral infection in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or pharmaceutically acceptable salt thereof.

The present invention further provides a method of treating HIV infection in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula I, or pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION

The present invention provides, inter alia, CCR5-binding compounds of Formula I:

I or pharmaceutically acceptable salts thereof, wherein:

R¹ is heteroaryl optionally substituted by one or more R⁶; R² is H, halo, cyano, nitro, Ci-C₆ alkyl, Ci-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, heteroaryl, C₃-C₇ cycloalkyl, heterocycloalkyl, SOR⁷, SO₂R⁷, COR⁸, OR⁹, SR⁹, COOR⁹, NR¹⁰R¹ ' or NR¹⁰COR⁸;

R³ is H, F, Cl, Br, I, Ci-C₄ haloalkyl, C_rC₄ haloalkoxy or heteroaryl; R⁴ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or Ci-C₆ haloalkyl; R⁵ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or C_rC₆ haloalkyl; R⁶ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ haloalkyl, Ci-C₆ alkoxy, Ci- C₆ haloalkoxy, amino, (Ci-C₆ alkyl)amino or di(Ci-C₆ alkyl)amino;

R⁷ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C)-C₆ haloalkyl, aryl, heteroaryl, C3-C7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl, (C₃-C7 cycloalkyl)alkyl, heterocycloalkylalkyl,or NR¹²R¹³;

R⁸ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Cj-C₆ haloalkyl, aryl, heteroaryl, C3-C7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl, (C₃-C₇ cycloalkyl)alkyl, heterocycloalkylalkyl, or NR¹²R¹³;

R⁹ is H, CrC₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C ,-C₆ haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl, heteroaryloxyalkyl, cycloalkyloxyalkyl, heterocycloalkyloxyalkyl, aryl, heteroaryl, C₃-C7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl; (C₃-C7 cycloalkyl)alkyl or heterocycloalkylalkyl; R^iυ and R^{1 1} are each, independently, H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci- C₆ haloalkyl, aryl, heteroaryl, C₃-C7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl; (C₃-C₇ cycloalkyl)alkyl or heterocycloalkylalkyl; or R¹⁰ and R¹¹ together with the N atom to which they are attached form a 3-, 4-, 5-, 6-, or 7-membered heterocycloalkyl group;

R¹² and R¹³ are each, independently, H, CpC₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci- C₆ haloalkyl, aryl, heteroaryl, C₃-C7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl; (C₃-C7 cycloalkyl)alkyl or heterocycloalkylalkyl; or R¹² and R¹³ together with the N atom to which they are attached form a 3-, 4-, 5-, 6-, or 7-membered heterocycloalkyl group; and r is 1, 2 or 3.

In some embodiments, R¹ is a 5-, 6-, 9- or 10-membered heteroaryl group containing at least one ring-forming N atom, wherein said 5-, 6-, 9- or 10-membered heteroaryl group is optionally substituted by 1, 2, 3 or 4 R⁶ groups. In some embodiments, R¹ is:

In some embodiments, R¹ is:

In some embodiments, R¹ is:

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is

In some embodiments, R² is H, Ci-C₆ alkyl, Ci-C₆ haloalkyl, OR , SR^y or NR 1'O^υ _nRU In some embodiments, R² is H or OR⁹. In some embodiments, R² is OR⁹. In some embodiments, R² is ethoxy.

In some embodiments, R³ is H, F, Br, CF₃, or 6- or 5-membered heteroaryl. In some embodiments, R³ is H. In some embodiments, R⁴ is Ci-C₆ alkyl. In some embodiments, R is methyl. In some embodiments, R⁵ is Ci-C₆ alkyl. In some embodiments, R is methyl. In some embodiments, R is H. In some embodiments, r is 1. In some embodiments, r is 2.

In some embodiments, the compounds of the invention have Formula II:

II.

In some embodiments, when the compound has Formula II, R¹ is:

In some embodiments, when the compound has Formula II, R is:

At various places in the present specification, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the members of such groups and ranges.

For example, the term "Ci-C ₆ alkyl" is specifically intended to individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

It is further intended that the compounds of the invention are stable. As used herein "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

As used herein, the term "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n- pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, "alkenyl" refers to an alkyl group having one or more double carbon- carbon bonds. Example alkenyl groups include ethenyl, propenyl, and the like. As used herein, "alkynyl" refers to an alkyl group having one or more triple carbon- carbon bonds. Example alkynyl groups include ethynyl, propynyl, and the like. As used herein, "haloalkyl" refers to an alkyl group having one or more halogen substituents. Example haloalkyl groups include CF₃, C₂F₅, CHF₂, CCI3, CHCl₂, C2CI5, and the like.

As used herein, "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.

As used herein, "cycloalkyl" refers to non-aromatic carbocycles including cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems, including spirocycles. In some embodiments, cycloalkyl groups can have from 3 to about 20 carbon atoms, 3 to about 14 carbon atoms, 3 to about 10 carbon atoms, or 3 to 7 carbon atoms. Cycloalkyl groups can further have 0, 1, 2, or 3 double bonds and/or 0, 1 , or 2 triple bonds. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo derivatives of pentane, pentene, hexane, and the like. A cycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. One or more ring-forming carbon atoms of a cycloalkyl group can be oxidized, for example, having an oxo or sulfido substituent. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like.

As used herein, a "heteroaryl" group refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Any ring-forming N atom in a heteroaryl group can also be oxidized to form an N-oxo moiety. Examples of heteroaryl groups include without limitation, pyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1 ,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. As used herein, "heterocycloalkyl" refers to a non-aromatic heterocycle where one or more of the ring- forming atoms is a heteroatom such as an O, N, or S atom. Heterocycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems as well as spirocycles. Example "heterocycloalkyl" groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-l,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles. A heterocycloalkyl group having one or more fused aromatic rings can be attached though either the aromatic or non-aromatic portion. Also included in the definition of heterocycloalkyl are moieties where one or more ring-forming atoms is substituted by 1 or 2 oxo or sulfϊdo groups. In some embodiments, the heterocycloalkyl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heterocycloalkyl group contains 3 to about 20, 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 triple bonds. As used herein, "heterocycloalkylene" refers to a linking heterocycloalkyl group.

As used herein, "halo" or "halogen" includes fluoro, chloro, bromo, and iodo.

As used herein, "halosulfanyl" refers to a sulfur group having one or more halogen substituents. Example halosulfanyl groups include pentahalosulfanyl groups such as SF₅.

As used herein, "alkoxy" refers to an -O-alkyl group. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.

As used herein, "alkoxyalkyl" refers to an alkyl group substituted by an alkoxy group.

As used herein, "haloalkoxy" refers to an -O-(haloalkyl) group.

As used herein, "haloalkoxyalkyl" refers to an alkyl group substituted by a haloalkoxy group. As used herein, "arylalkyl" refers to alkyl substituted by aryl and "cycloalkylalkyl" refers to alkyl substituted by cycloalkyl. An example arylalkyl group is benzyl.

As used herein, "aryloxy" refers to -O-aryl.

As used herein, "aryloxyalkyl" refers to an alkyl group substituted by aryloxy.

As used herein, "cycloalkyloxy" refers to -O-cycloalkyl. As used herein, "cycloalkyloxyalkyl" refers to an alkyl group substituted by cycloalkyloxy.

As used herein, "heteroarylalkyl" refers to alkyl substituted by heteroaryl and "heterocycloalkylalkyl" refers to alkyl substituted by heterocycloalkyl. As used herein, "heteroaryloxy" refers to -O-heteroaryl.

As used herein, "heteroaryloxyalkyl" refers to alkyl substituted by heteroaryloxy.

As used herein, "heterocyloalkyloxy" refers to -0-heterocycloalkyl.

As used herein, "heterocycloalkyloxyalkyl" refers to alkyl substituted by heterocycloalkyloxy. As used herein, "amino" refers to NH₂.

As used herein, "alkylamino" refers to an amino group substituted by an alkyl group.

As used herein, "dialkylamino" refers to an amino group substituted by two alkyl groups.

The compounds described herein can be asymmetric (e.g., having one or more stereocenters). AU stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present invention that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms.

Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, IH- and 3H-imidazole,

IH-, 2H- and 4H- 1 ,2,4-triazole, IH- and 2H- isoindole, and IH- and 2H-pyrazole.

Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. Compounds of the invention can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. The term, "compound," as used herein is meant to include all stereoisomers, geometric iosomers, tautomers, and isotopes of the structures depicted.

In some embodiments, the compounds of the invention, and salts thereof, are substantially isolated. By "substantially isolated" is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the invention, or salt thereof. Methods for isolating compounds and their salts are routine in the art. The present invention also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17^th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Synthesis Compounds of the invention, including salts, hydrates, and solvates thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes.

The reactions for preparing compounds of the invention can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, e.g., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

Preparation of compounds of the invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T.W. Green and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

Example synthetic routes to compounds of the invention are provided in Schemes 1 and 2 below, where constituent members of the depicted formulae are defined herein.

Scheme 1 illustrates the preparation of epoxide intermediates of formula 1-8 starting with pentafluorosulfanyl-substituted anilines of formula 1-1 (Commercial Sources include Indofϊne Chemical Company, Inc., Tyger Scientific Inc., F2 Chemicals Lt., etc. ) which are reacted with N-bromosuccinimide (NBS) to yield the bromo derivatives of formula 1-2. The palladium catalyzed coupling reaction between structure 1-2 and allylborate 1-3 gives ally- pentafluorosulfanyl-substituted anilines 1-4, and the aniline functional group is then transformed to an iodo group through the routine process of a Sandmeyer reaction to form compounds of formula 1-5. Another palladium catalyzed coupling reaction between 1-5 and vinyl-tributylstannane affords the diene structures 1-6, which is cyclized using Grubbs' catalyst to provide indene derivatives 1-7. Epoxides 1-8 are subsequently obtained after treating 1-7 with /weta-chloroperbenzoic acid.

Scheme 1

NaNCWKI

Grubbs Cat.

Combining epoxides 1-8 with amines 2-1 at an elevated temperature in a solvent such as isopropyl alcohol produce a mixture of diastereomers, alcohols 2-2a and 2-2b. Alkylation of the resulting alcohol 2-2a with ethyl iodide was accomplished with base such as sodium hydride to give ethyl ether 2-3a. After removal of the Boc group in 2-3a, coupling of the resulting amine with R COOH using a coupling agent such as EDCI provides compounds of formula 2-4a.

Scheme 2

Methods In some embodiments, compounds of the invention can modulate activity of one or more chemokine receptors. The term "modulate" is meant to refer to an ability to increase or decrease activity of a receptor. Accordingly, compounds of the invention can be used in methods of modulating a chemokine receptor by contacting the receptor with any one or more of the compounds or compositions described herein. In some embodiments, compounds of the present invention can act as inhibitors of chemokine receptors. In further embodiments, the compounds of the invention can be used to modulate activity of a chemokine receptor in an individual in need of modulation of the receptor by administering a modulating amount of a compound of Formula I.

In some embodiments, compounds of the invention can bind to a chemokine receptor in such a way to block or inhibit binding of endogenous and other chemokine receptor ligands. In some embodiments, the compounds of the invention can block or inhibit binding of exogenous ligands including viral proteins involved in viral entry into cells expressing the chemokine receptor. Accordingly, compounds of the invention can block viral entry and inhibit viral infection. In some embodiments, compounds of the invention can inhibit human immuno-defϊciency virus (HIV) infection by, for example, blocking interaction of a chemokine receptor (e.g., CCR5) with HIV glycoprotein 120 (gpl20). Chemokine receptors to which the present compounds bind and/or modulate include any chemokine receptor. In some embodiments, the chemokine receptor belongs to the CC family of chemokine receptors including, for example, CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR8. In some embodiments, the chemokine receptor is CCR2. In some embodiments, the chemokine receptor is CCR5. The compounds of the invention can be selective. By "selective" is meant that the compound binds to or inhibits a chemokine receptor with greater affinity or potency, respectively, compared to at least one other chemokine receptor.

Compounds of the invention can be selective binders of CCR5, meaning that the compounds of the invention can bind to CCR5 with greater affinity than for another chemokine receptor such as at least one of CCRl, CCR2, CCR3, CCR4, CCR6, CCR7 and CCR8. In some embodiments, the compounds of the invention have binding selectivity for CCR5 over CCR2. In some embodiments, the compounds of the invention have binding selectivity for CCR5 over CCRl . In some embodiments, the compounds of the invention have binding selectivity for CCR5 over any other CCR. Selectivity can be at least about 10- fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200- fold, at least about 500-fold or at least about 1000-fold. In some embodiments, the compounds of the invention have binding affinity for CCR5 that is at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold or at least about 1000-fold greater than binding affinity for CCRl, CCR2 or any other chemokine receptor. Binding affinity can be measured according to routine methods in the art, such as according to the assays provided herein.

In some embodiments, the compounds of the invention can be selective inhibitors of CCR5, meaning that the compounds of the invention can inhibit activity of CCR5 more potently than for at least one other chemokine receptors such as, for example, CCRl, CCR2, CCR3, CCR4, CCR6, CCR7 and CCR8. In some embodiments, the compounds of the invention have inhibition selectivity for CCR5 over CCR2. In some embodiments, the compounds of the invention have inhibition selectivity for CCR5 over CCRl. In some embodiments, the compounds of the invention have inhibition selectivity for CCR5 over any other CCR. Selectivity can be at least about 10-fold, at least about 20-fold, at least about 50- fold, at least about 100-fold, at least about 200-fold, at least about 500-fold or at least about 1000-fold. In some embodiments, the compounds of the invention have inhibition affinity for CCR5 that is at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold or at least about 1000-fold greater than binding affinity for CCRl, CCR2 or any other chemokine receptor. Inhibitor potency can be measured according to routine methods in the art, such as according to the assays provided herein.

Another aspect of the present invention pertains to methods of treating a chemokine receptor-associated disease or disorder in an individual (e.g., patient) by administering to the individual in need of such treatment a therapeutically effective amount or dose of a compound of the present invention or a pharmaceutical composition thereof. A chemokine receptor-associated disease can include any disease, disorder or condition that is directly or indirectly linked to expression or activity of the chemokine receptor. A chemokine receptor- associated disease can also include any disease, disorder or condition that can be prevented, ameliorated, or cured by modulating chemokine receptor activity. A chemokine receptor- associated disease can further include any disease, disorder or condition that is characterized by binding of an infectious agent such as a virus or viral protein with a chemokine receptor. In some embodiments, the chemokine receptor-associated disease is a CCR5-associated disease such as HIV infection. Example chemokine receptor-associated diseases, disorders and conditions include inflammation and inflammatory diseases, immune disorders and viral infections. Example inflammatory diseases include diseases having an inflammatory component such as asthma, allergic rhinitis, restenosis, atherosclerosis, multiple sclerosis, Crohn's disease, ulcerative colitis, hypersensitivity lung diseases, hypersensitivity pneumonitis, eosinophilic pneumonias, delayed-type hypersensitivity, asthma, interstitial lung disease (ILD) (e.g., idiopathic pulmonary fibrosis, or ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis or dermatomyositis) and the like. Example immune disorders include rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, myastenia gravis, juvenile onset diabetes; glomerulonephritis, autoimmune throiditis, organ transplant rejection including allograft rejection and graft-versus-host disease. Example viral infections include HIV infection.

As used herein, the term "contacting" refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, "contacting" the chemokine receptor with a compound of the invention includes the administration of a compound of the present invention to an individual or patient, such as a human, having a chemokine receptor, as well as, for example, introducing a compound of the invention into a sample containing a cellular or purified preparation containing the chemokine receptor.

As used herein, the term "individual" or "patient," used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.

As used herein, the phrase "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.

As used herein, the term "treating" or "treatment" refers to one or more of (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder; and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.

One or more additional pharmaceutical agents such as, for example, anti-viral agents, antibodies, anti-inflammatory agents, and/or immunosuppressants can be used in combination with the compounds of the present invention for treatment of chemokine receptor-associated diseases, disorders or conditions. The agents can be combined with the present compounds in a single dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.

Suitable antiviral agents contemplated for use in combination with the compounds of the present invention can comprise nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors and other antiviral drugs.

Example suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir dipivoxil [Ms(POM)-PMEA]; lobucavir (BMS-180194); BCH- 10652; emitricitabine [(-)-FTC]; beta-L- FD4 (also called beta-L-D4C and named beta-L-2', S'-dicleoxy-S-fluoro-cytidene); DAPD, ((- )-beta-D-2,6,-diamino-purine dioxolane); and lodenosine (FddA).

Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine (BHAP, U- 90152); efavirenz (DMP-266); PNU- 142721; AG- 1549; MKC-442 (l-(ethoxy-methyl)-5-(l- methylethyl)-6-(phenylmethyl)-(2,4(lH,3H)-pyrimidi nedione); and (+)-calanolide A (NSC- 675451) and B.

Typical suitable protease inhibitors include saquinavir (Ro 31-8959); ritonavir (ABT- 538); indinavir (MK-639); nelrnavir (AG-1343); amprenavir (141W94); lasinavir (BMS- 234475); DMP-450; BMS-2322623; ABT-378; and AG-I 549. Other antiviral agents include hydroxyurea, ribavirin, IL-2, IL- 12, pentafuside and

Yissum Project No.11607.

In some embodiments, anti-inflammatory or analgesic agents contemplated for use in combination with the compounds of the present invention can comprise, for example, an opiate agonist, a lipoxygenase inhibitor such as an inhibitor of 5-lipoxygenase, a cyclooxygenase inhibitor such as a cyclooxygenase-2 inhibitor, an interleukin inhibitor such as an interleukin-I inhibitor, an NNMA antagonist, an inhibitor of nitric oxide or an inhibitor of the synthesis of nitric oxide, a non-steroidal antiinflammatory agent, or a cytokine- suppressing antiinflammatory agent, for example, such as acetaminophen, asprin, codiene, fentanyl, ibuprofen, indomethacin, ketorolac, morphine, naproxen, phenacetin, piroxicam, a steroidal analgesic, sufentanyl, sunlindac, tenidap, and the like. Similarly, the instant compounds can be administered with a pain reliever; a potentiator such as caffeine, an H2- antagonist, simethicone, aluminum or magnesium hydroxide; a decongestant such as phenylephrine, phenylpropanolamine, pseudophedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine; an antfϊtussive such as codeine, hydrocodone, caramiphen, carbetapentane, or dextramethorphan; a diuretic; and a sedating or non-sedating antihistamine.

In some embodiments, pharmaceutical agents contemplated for use in combination with the compounds of the present invention can comprise (a) VLA-4 antagonists such as those described in US 5,510,332, W095/15973, W096/01644, W096/06108, W096/20216, W096/229661, W096/31206, W096/4078, W097/030941, W097/022897 WO 98/426567 W098/53814, W098/53817, W098/538185, W098/54207, and W098/58902; (b) steroids such as beclornethasone, methylpi-ednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressants such as cyclosporin , tacrolimus, raparnycin and other FK506 type immunosuppressants; (d) antihistamines (HI-histamine antagonists) such as bromopheniramine, chloφheniramine, dexchlorpheniramine, triprolidine, clemastine, diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine, azatadine, cyproheptadine, antazoline, pheniramine pyrilarnine, asternizole, terfenadine, loratadine, cetirizine, fexofenadine, desearboethoxyloratadine, and the like; (e) non-steroidal anti-asthmatics such as terbutaline, metaproterenol, fenoterol, isoethaiine, albuterol, bitolterol, pirbuterol, theophylline, cromolyn sodium, atropine, ipratropium bromide, leukotriene antagonists (e.g., zafirlukast, montelukast, pranlukast, iralukast, pobilukast, SBCB- 106,203), leukotriene biosynthesis inhibitors (e.g., zileuton, BAY- 1005); (f) nonsteroidal antiinflammatory agents (NSAIDs) such as propionic acid derivatives (e.g., alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (e.g., indomethacin, acernetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenarnic acid derivatives (flufenarnic acid, meclofenamic acid, rnefenamic acid, niflumic acid and tolfenamic acid), biphenylearboxylic acid derivatives (diflunisal and flufenisal), oxicarns (isoxicarn, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone); (g) cyclooxygenase-2 (COX-2) inhibitors; (h) inhibitors of phosphodiesterase type IV (PDE-IV); (i) other antagonists of the chernokine receptors, especially CXCR-4, CCRI, CCR2, CCR3 and CCR5 ; (j) cholesterol lowering agents such as HMG-CoA reductase inhibitors (lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, and other statins), sequestrants (cholestyramine and colestipol), nicotinic acid, fenofibric acid derivatives (gemfibrozil, clofibrat, fenofibrate and benzafibrate), and probucol; (k) anti-diabetic agents such as insulin, sulfonylureas, biguanides (metformin), U.-glucosidase inhibitors (acarbose) and orlitazones (troglitazone and pioglitazone); (1) preparations of interferon beta (interferon beta- lo., interferon beta-1 P); (m) other compounds such as aminosalicylic acids, antimetabolites such as azathioprine and 6-mercaptopurine, and cytotoxic cancer chemotherapeutic agents. The weight ratio of the compound of the compound of the present invention to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of the invention can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of Formula I above in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention can be prepared by processes known in the art, for example see International Patent

Application No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient, and the like. The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral adminstration. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose- response curves derived from in vitro or animal model test systems.

The compounds of the invention can also be formulated in combination with one or more additional active ingredients which can include any pharmaceutical agent such as antiviral agents, antibodies, immune suppressants, anti-inflammatory agents and the like. In some embodiments, the compounds of the invention are formulated in combination with one or more anti-viral agents including protease inhibitors and other agents used for anti-HIV therapy. The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. EXAMPLES

Example 1

Preparation of S-K^i.SJ^-Kiiϊ^ΛVl-ethoxy-S-CpeiitafluorosulfanyO-Z^-dihydro-lH- inden-l-yl]-3-methylpiperazin-l-yl-4-methylpiperidiπ-l-yl)carbonyl]-4,6- dimethylpyrimidine

Step 1. 2-Bromo-4-(pentafluorosulfanyl)aniline.

To a solution of 4-(pentafluorosulfanyl)aniline (CAS RN# 2993-24-0, Indofme Chemical Company, Inc., cat# 26-2285) (5.00 g, 22.8 mmol) in N,N-dimethylformamide (40.0 mL, 516 mmol), N-bromosuccinimide (5.00 g, 28.1 mmol) was added in portions at room temperature. The mixture was then stirred for one hour. LC-MS indicated the reaction was complete. The reaction content was then chromatographed directly with hexane/ethyl acetate (10% to 50%) to give 5.32 g product as a reddish oil, yield 78.2%.

¹H NMR (300 MHz, CDCl₃) δ (ppm) 7.80 (d, IH), 7.47 (dd, IH), 6.69 (d, IH), 4.49 (br, IH).

MS (EI) 297.8, 299.9 (M+l).

Step 2. 2-Allyl'4-(pentafluorosulfanyl)aniline

2-Bromo-4-(pentafluorosulfanyl)aniline (4.0 g, 0.013 mol), cesium fluoride (4.1 g,

0.027 mol) and tetrakis(triphenylphosphine)palladium(0) (0.9 g, 0.0008 mol) were mixed together in tetrahydrofuran (50.0 mL, 0.616 mol) and stirred for 30 min. 2-Allyl-4,4,5,5- tetramethyl-l,3,2-dioxaborolane (4.0 g, 0.024 mol) was then added and the resulting reaction mixture was heated to reflux over night. The reaction mixture was diluted with Et₂O (150 niL) followed by water (100 niL). The layers were separated and the aqueous layer was extracted with Et₂O (2x100 mL). The combined organic layers were then washed with water, brine, dried over Mg₂SO₄, and concentrated. The residue was purified by Combiflash (5% ether in hexanes) to give 2.43 g desired product, yield 70%.

¹H NMR (300 MHz, CDCl₃) δ (ppm) 7.45 (d, IH), 7.43 (s, IH), 6.62 (d, IH), 5.92 (m, IH), 5.10-5.24 (m, 2H), 4.02 (br, 2H), 3.30 (d, IH).

MS (EI) 260.0 (M+l).

Step 3. 2-Allyl-l-iodo-4-(pentafluorosulfanyl)benzene.

A cold (0 ⁰C) solution of sodium nitrite (0.60 g, 0.0087 mol) in water (5.0 mL, 0.28 mol) was added slowly to a cold (0 ⁰C ) solution of 2-allyl-4-(pentafluorosulfanyl)aniline (2.0 g, 0.0077 mol) in 12.5 M of hydrogen chloride in water (3.5 mL) and ice 10 g. The mixture was stirred at 0 ⁰C for another 5 min before being added to a cold solution of potassium iodide (12.7 g, 0.0767 mol) in water (50 mL, 3 mol). The mixture was then kept below 10 ⁰C for 30 min before being warmed up to rt. To the reaction mixture, hexanes were added to extract the product. After drying over MgSO₄ and being filtered and concentrated, the residue was chromatographed (eluted with 100% heaxane) to give 1.8 g product as a reddish oil, yield 63%; 1H NMR (300 MHz, CDCl₃) δ (ppm) 7.93 (d, IH), 7.58 (d, IH), 7.29 (dd, IH), 5.93

(m, IH), 5.10-5.30 (m, 2H), 3.55 (d, IH).

Step 4. 2-Allyl-4-(pentqfluorosulfanyl)-l-vinylbenzene

A solution of 2-allyl-l-iodo-4-(pentafluorosulfanyl)benzene (1.50 g, 0.00405 mol), tributylethenylstannane (3.00 mL, 0.0103 mol), 2,6-di-tert-butyl-4-methylphenol (10.0 mg, 0.0000454 mol) and tetrakis(triphenylphosphine)palladium(0) (200.0 mg, 0.0001731 mol) in toluene (20 mL, 0.2 mol) was heated to reflux for 1.5 h. After the mixture was cooled to rt, it was diluted with Et₂O (100 mL) and then 2 M aqueous KF solution (100 mL) was added. The resulting mixture was stirred overnight. The organic layer was separated from the sludge and the aqueous layer, after drying over Mg₂SO₄. The concentrated crude material was purified by Combiflash (100% hexanes) twice to give the desired product (0.76 g) as a colorless oil, yield 69 %.

¹H NMR (400 MHz, CDCl₃) δ (ppm) 7.53-7.62 (m, 3H), 6.92 (dd, IH), 5.95(m, IH), 5.72 (d, IH), 5.44 (d, IH), 5.14 (d, IH), 5.02 (d, IH), 3.49 (d, 2H).

Step 5. 6-(pentafluorosulfanyl)-lH-indene

To a solution of 2-allyl-4-(pentafluorosulfanyl)-l-vinylbenzene (0.660 g, 0.00244 mol) in methylene chloride (10.0 niL, 0.156 mol), bis(tricyclohexylphosphine)benzylidine ruthenium(IV) dichloride (50.0 nig, 0.0000608 mol) (Grubbs catalyst, 1st generation) was added. The mixture was heated to reflux for one hour. The reaction content was then injected to the top of a Combiflash column, and eluted with 100% hexane. After concentration, 0.464 g of product was obtained as a colorless oil.

¹H NMR (300 MHz, CDCl₃) δ (ppm) 7.86 (s, IH), 7.70 (d, IH), 7.43 (d, IH), 6.91 (m, IH), 6.78 (m, IH), 3.48 (brs, IH).

Step 6. 4-(pentafluorosulfanyl)-6, 6a-dihydro-laH-indeno[l,2-b]oxirene

To 6-(pentafluorosulfanyl)-lH-indene (0.400 g, 0.00165 mol) in dichloromethane (5.0 mL, 0.078 mol) was added m-chloroperbenzoic acid (0.700 g, 0.00312 mol) (max. 77%) and the resulting mixture was stirred at rt overnight. The reaction mixture was diluted with 50 mL of methylene chloride and then washed with sat. NaHCO₃ solution, brine, and then dried over MgSO₄. After being filtered, the solution was used directly for next step.

Step 7. t-Butyl 4-(3S)-4-[(lR,2R)-2-hydroxy-5-(pentafluorosulfanyl)-2,3-dihydro-lH-inden-l- yl]-3-methylpiperazin-l-yl-4-methylpiperidine-l-carboxylate

4-(Pentafluorosulfanyl)-6,6a-dihydro-laH-indeno[l,2-b]oxirene (0.426 g, 1.65 mmol) and tert-butyl-4-methyl-4-[(35)-3-methylpiperazin-l-yl]piperidine-l-carboxylate (1.5 g, 5.0 mmol) (see, US. Pat. App. Pub. No. 2005/0261310) were dissolved in IPA. The mixture was heated to 90 ⁰C overnight. LCMS (M+H) = 556.2. The reaction mixture was concentrated and the residue was chromatographed (silica gel) with EtOAc/hexane (20-100%) to give two diastereomers A: 132 mg [Rf = 0.5, ethyl acetate], B, 1 10 mg [Rf = 0.7, ethyl acetate]. The yield of A, the title compound was 14.4% over two steps.

Step 8. t-Butyl 4-(3S)-4-[(lR,2R)-2-ethoxy-5-(pentafluorosulfanyl)-2,3-dihydro-lH~inden-l- yl]-3-methylpiperazin-l-yl-4-methylpiperidine-l-carboxylate

To a solution of tert-butyl 4-(3S)-4-[(7R,2#>2-hydroxy-5-(pentafluorosulfanyl)-2,3- dihydro- 1 H-inden- 1 -yl]-3 -methylpiperazin- 1 -yl-4-methylpiperidine- 1 -carboxylate (Isomer A from Step 7, 132.0 mg, 0.0002376 mol) in N,N-dimethylformamide (5.0 mL, 0.064 mol) at 0

⁰C, was added sodium hydride (50.0 mg, 0.00125 mol) followed by iodoethane (0.1000 mL,

0.001250 mol). The mixture was stirred overnight. LC-MS indicated the reaction was complete. The reaction mixture was then injected directly to the top of a silica column and eluted with ethyl acetate/hexanes (10% to 70%) to give 88.0 mg product (yield 63%). MS (EI) 584.2 (M+l).

Step 9. 5-[(4-(3S)-4-[(lR,2R)-2-ethoxy-5-(pentafluorosulfanyl)-2,3-dihydro-lH-inden-l-yl]- 3-methylpiperazin-l-yl-4-methylpiperidin-l-y^[)carbonyl]-4,6-dimethylpyrimidine tert-Butyl 4-(35)-4-[(7R,2Λ>2-ethoxy-5-(pentafluorosulfanyl)-2,3-dihydro-lH-inden- l-yl]-3-methylpiperazin-l -yl-4-methylpiperidine- 1 -carboxylate from Step 8 (88.0 mg,

0.000151 mol) was treated with 4.0 M of hydrogen chloride in 1,4-dioxane (5.0 mL) at rt for

1 h. The mixture was concentrated to dryness in vacuo resulting in (S)-l-((]R,2R)-5- pentafluorosulfanyl-2-ethoxy-2,3-dihydro-lH-inden-l-yl)-2-methyl-4-(4-methylpiperidin-4- yl)piperazine hydrochloride. LC/MS: 484.2 (M+H).

To a solution of above hydrochloride salt together with 4,6-dimethyl-pyrimidine-5- carboxylic acid (50.0 mg, 0.000329 mol) in methylene chloride (5.0 niL, 0.078 mol) was added 1-hydroxybenzotriazole (50.0 mg, 0.000370 mol), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (6.0El mg, 0.00031 mol) and triethylamine (0.50 mL,

0.0036 mol) . The resulting mixture was stirred at rt overnight. The reaction mixture was diluted with methylene chloride, then washed with IN NaOH, brine, dried (MgSO₄), filtered and concentrated. The crude material was purified by Prep-HPLC and then freeze-dried to give the title compound 33 mg (yield 35.4% over two steps).

¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.93 (d, IH), 7.58 (m, IH), 7.52 (m, IH), 7.35 (m, IH), 4.7 (t, IH), 4.41 (m, IH), 4.11 (m, IH), 3.57-3.68 (m, 1.5H), 3.44-3.57 (m, 1.5H), 3.27-3.44 (m, IH), 3.16-3.27 (m, IH), 2.91-3.06 (m, IH), 2.70-2.91 (m, 2.5H), 2.56-2.70 (m, IH), 2.49-2.56 (m, IH), 2.46 (m, 6H), 2.15-2.39 (m, 2H), 1.66-2.15 (m, 3H), 1.37-1.54 (m, IH), 1.07-1.37 (m, IH), 0.96 (s, 3H). MS (EI) 618.2 (M+l).

Example 2 CCR5 Expression A leukophoresis (Biological Specialty, Colmar, PA) was obtained from normal, drug free donors and peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation. Monocytes were further isolated via centrifugal elutriation. After being washed, the monocytes were re-suspended at 10⁶ cells/ ml with RPMI (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT) and 10-20 ng/mL of recombinant human IL-10 (R&D systems, Minneapolis, MN) and incubated in the same medium at 37 ⁰C with 5% CO₂ for 24-48 hr. CCR5 expression on the IL-10 - treated monocytes was then verified by staining the cells with a PE-conjugated anti-human CCR5 antibody ((PharMingen, San Diego, CA), followed by FACS analysis using FACSCalibur (BD Biosciences, Bedford, MA).

Example 3

CCR5 Binding Assay In a 96 well MultiScreen^1M filter plate (Millipore Systems, Billerica, MA), 3x10⁵ IL- 10-treated monocytes (Example 2) in 150 μL RPMI (Invitrogen, Carlsbad, CA) with 20 mM HEPES (Invitrogen, Carlsbad, CA) and 0.3% BSA (Sigma, St Louis, MO) were incubated at room temperature for 1 hr. with 0.2 nM ' ²⁵I-MIP- lβ (Perkin Elmer, Boston, MA) and a series concentrations of the compound of Example 1 (5-[(4-(5iS)-4-[(7i?,2Λ)-2-ethoxy-5- (pentafluorosulfanyl)-2,3-dihydro- 1 H-inden- 1 -yl]-3-methylpiperazin- 1 -yl-4-methylpiperidin- l-yl)carbonyl]-4,6-dimethylpyrimidine). Non-specific binding was determined by incubating the cells with 0.3 μM MIP- lβ (R&D Systems, Minneapolis, MN). The binding reaction was terminated by harvesting the cells onto the filter in the plate on a vacuum manifold (Millipore Systems, Billerica, MA). The filter was then washed 5 times with RPMI (Invitrogen, Carlsbad, CA) supplemented with 20 mM HEPES (Invitrogen, Carlsbad, CA), 0.3% BSA (Sigma, St Louis, MO) and 0.4 M NaCl on the vacuum manifold, air dried, and peeled from the plate. The filter dishes corresponding to the sample wells in a filter plate were punched out using the Millipore Punch System (Millipore Systems, Billerica, MA). The amount of bound radioactivity on each filter dish was determined by counting on a gamma counter. Specific binding was defined as the total binding minus the non-specific binding. The binding data were evaluated with Prism (GraphPad Software, San Diego, CA).

The compound of Example 1 was found to be a potent binder of CCR5 according this assay, with an IC₅₀ value of less than 500 nM.

Example 4

HIV-I Entry Assay

Replication defective HJV-I reporter virions are generated by cotransfection of a plasmid encoding the NL4-3 strain of HIV-I (which has been modified by mutation of the envelope gene and introduction of a luciferase reporter plasmid) along with a plasmid encoding one of several HIV-I envelope genes as described by, for example, Connor et al, Virology, 206 (1995), 935-944. Following transfection of the two plasmids by calcium phosphate precipitation, the viral supernatants are harvested on day 3 and a functional viral titer determined. These stocks are then used to infect U87 cells stably expressing CD4 and the chemokine receptor CCR5 which have been preincubated with or without test compound. Infections are carried out for 2 hours at 37 ⁰C, the cells washed and media replaced with fresh media containing compound. The cells are incubated for 3 days, lysed and luciferase activity determined. Results are reported as the concentration of compound required to inhibit 50% of the luciferase activity in the control cultures.

Example 5 HIV-I Replication Assay in MT-4 Cells

Inhibition of HIV-I NL4.3 (or III_B) replication assays can be carried out as previously described (Bridger, et al., J. Med. Chem. 42:3971-3981 (1999); De Clercq, et al., Proc. Natl. Acad. Sci. 89:5286-5290 (1992); De Clercq, et al., Antimicrob. Agents Chemother. 38:668- 674 (1994); Bridger, et al. J. Med. Chem. 38:366-378 (1995)). To summarize, anti-HIV activity and cytotoxicity measurements are carried out in parallel and are based on the viability of MT-4 cells that are infected with HIV in the presence of various concentrations of the test compounds. After the MT-4 cells are allowed to proliferate for 5 days, the number of viable cells are quantified by a tetrazolium-based calorimetric 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) procedure in 96-well microtrays. Results can be quanitited to yield EC₅₀ values which represent the concentration required to protect 50% of the virus-infected cells against viral cytopathicity.

Example 6

Chemokine Receptor Inhibition/Binding Assays The capacity of the compounds of the invention to antagonize chemokine receptor

(e.g., CCR2) function can be determined using a suitable screen (e.g., high through-put assay). For example, an agent can be tested in an extracellular acidification assay, calcium flux assay, ligand binding assay or chemotaxis assay (see, for example, Hesselgesser et al., J Biol. Chem. 273(25): 15687- 15692 (1998); WO 00/05265 and WO 98/02151, each of which is incorporated herein by reference in its entirety).

In an example assay, a chemokine receptor which can be isolated or recombinantly derived is used which has at least one property, activity or functional charateristic of a mammalian chemokine receptor. The specific property can be a binding property (to, for example, a ligand or inhibitor), a signalling activity (e.g., activation of a mammalian G protein, induction of rapid and transient increase in the concentration of cytosolic free calcium [Ca ]i, cellular response function (e.g., stimulation of chemotaxis or inflammatory mediator release by leukocytes), and the like. In one embodiment, a composition containing a chemokine receptor or variant thereof is maintained under conditions suitable for binding. The receptor is contacted with a compound to be tested, and binding is detected or measured.

In further embodiments, the assay is a cell-based assay in which cells are used that are stably or transiently transfected with a vector or expression cassette having a nucleic acid sequence which encodes the receptor. The cells are maintained under conditions appropriate for expression of the receptor and are contacted with an agent under conditions appropriate for binding to occur. Binding can be detected using standard techniques. For example, the extent of binding can be determined relative to a suitable control. Also, a cellular fraction, such as a membrane fraction, containing the receptor can be used in lieu of whole cells.

Detection of binding or complex formation between compounds of the invention and chemokine receptors can be detected directly or indirectly. For example, the compound can be labeled with a suitable label (e.g., fluorescent label, label, isotope label, enzyme label, and the like) and binding can be determined by detection of the label. Specific and/or competitive binding can be assessed by competition or displacement studies, using unlabeled agent or a ligand as a competitor.

The antagonist activity of test agents can be reported as the inhibitor concentration required for 50% inhibition (IC₅₀ values) of specific binding in receptor binding assays using, for example, I-labeled MCP-I, as ligand, and Peripheral Blood Mononuclear Cells (PBMCs) prepared from normal human whole blood via density gradient centrifugation. Specific binding is preferably defined as the total binding (e.g., total cpm on filters) minus the non-specific binding. Non-specific binding is defined as the amount of cpm still detected in the presence of excess unlabeled competitor (e.g., MCP-I).

The human PBMCs described above can be used in a suitable binding assay. For example, 200,000 to 500,000 cells can be incubated with 0.1 to 0.2 nM ¹²⁵I-labeled MCP-I, with or without unlabeled competitor (1OnM MCP-I) or various concentrations of compounds to be tested. ¹²⁵I-labeled MCP-I, can be prepared by suitable methods or purchased from commercial vendors (Perkin Elmer, Boston MA), The binding reactions can be performed in 50 to 250 μl of a binding buffer consisting of IM HEPES pH 7.2, and 0.1% BSA (bovine serum albumin), for 30 min at room temperature. The binding reactions can be terminated by harvesting the membranes by rapid filtration through glass fiber filters (Perkin Elmer) which can be presoaked in 0.3% polyethyleneimine or Phosphate Buffered Saline (PBS). The filters can be rinsed with approximately 600 μL of binding buffer containing 0.5 M NaCl or PBS, then dried, and the amount of bound radioactivity can be determined by counting on a Gamma Counter (Perkin Elmer).

The capacity of compounds to antagonize chemokine receptor function can also be determined in a leukocyte chemotaxis assay using suitable cells. Suitable cells include, for example, cell lines, recombinant cells or isolated cells which express a chemokine receptor (e.g., CCR2) and undergo chemokine receptor ligand-induced (e.g., MCP-I) chemotaxis. The assay utilizes human peripheral blood mononuclear cells, in a modified Boyden Chamber (Neuro Probe). 500,000 cells in serum free DMEM media (In Vitrogen) are incubated with or without the inhibitors and warmed to 37 ⁰C. The chemotaxis chamber (Neuro Probe) is also prewarmed. 400 μL of warmed 10 nM MCP-I is added to the bottom chamber in all wells expect the negative control which has DMEM added. An 8 micron membrane filter (Neuro Probe) is place on top and the chamber Hd is closed. Cells are then added to the holes in the chamber lid which are associated with the chamber wells below the filter membrane. The whole chamber is incubated at 37 ⁰C, 5% CO₂ for 30 minutes. The cells are then aspirated off, the chamber lid opened, and the filter gently removed. The top of the filter is washed 3 times with PBS and the bottom is left untouched. The filter is air dried and stained with Wright Geimsa stain (Sigma). Filters are counted by microscopy. The negative control wells serve as background and are subtracted from all values. Antagonist potency can be determined by comparing the number of cells that migrate to the bottom chamber in wells which contain antagonist, to the number of cells which migrate to the bottom chamber in MCP-I control wells.

Compounds of the present invention can be considered active if they have IC₅₀ values in the range of about 0.01 to about 500 nM for the above binding assay. In chemotaxis assays, active compounds have IC50 values in the range of about 1 to about 3000 nM.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application, including all patents, publications and books, is incorporated herein by reference in its entirety.

Claims

What is claimed is:

1. A compound of Formula I:

I or pharmaceutically acceptable salt thereof, wherein:

R¹ is heteroaryl optionally substituted by one or more R ;

R² is H, halo, cyano, nitro, Ci-C₆ alkyl, Ci-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, heteroaryl, C₃-C₇ cycloalkyl, heterocycloalkyl, SOR⁷, SO₂R⁷, COR⁸, OR⁹, SR⁹, COOR⁹, NR¹⁰R¹¹ Or NR¹⁰COR⁸;

R³ is H, F, Cl, Br, I, Ci-C₄ haloalkyl, Ci-C₄ haloalkoxy or heteroaryl;

R⁴ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or C_rC₆ haloalkyl;

R⁵ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl or Ci-C₆ haloalkyl;

R⁶ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ haloalkyl, C_rC₆ alkoxy, Ci- C₆ haloalkoxy, amino, (Ci-C₆ alkyl)amino or di(Ci-C₆ alkyl)amino;

R⁷ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ haloalkyl, aryl, heteroaryl, C₃-C₇ cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl, (C₃-C₇ cycloalkyl)alkyl, heterocycloalkylalkyl,or NR¹²R¹³;

R⁸ is H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ haloalkyl, aryl, heteroaryl, C₃-C₇ cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl, (C₃-C₇ cycloalkyl)alkyl, heterocycloalkylalkyl, Or NR¹²R¹³;

R⁹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci-C₆ haloalkyl, alkoxyalkyl, haloalkoxyalkyl, aryloxyalkyl, heteroaryloxyalkyl, cycloalkyloxyalkyl, heterocycloalkyloxyalkyl, aryl, heteroaryl, C₃-C7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl; (C₃-C₇ cycloalkyl)alkyl or heterocycloalkylalkyl;

R¹⁰ and R¹¹ are each, independently, H, Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Ci- C₆ haloalkyl, aryl, heteroaryl, C₃-C7 cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl; (C₃-C₇ cycloalkyl)alkyl or heterocycloalkylalkyl; or R¹⁰ and R¹ ' together with the N atom to which they are attached form a 3-, A-, 5-, 6-, or 7-membered heterocycloalkyl group; R¹² and R¹³ are each, independently, H, C_I-C_O alkyl, C₂-C6 alkenyl, C₂-C_O alkynyl, Ci- C₆ haloalkyl, aryl, heteroaryl, C₃-C₇ cycloalkyl, heterocycloalkyl, arylalkyl, heteroarylalkyl; (C₃-C₇ cycloalkyl)alkyl or heterocycloalkylalkyl; or R¹² and R¹³ together with the N atom to which they are attached form a 3-, 4-, 5-, 6-, or 7-membered heterocycloalkyl group; and r is 1, 2 or 3.

2. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R¹ is a 5-, 6-, 9- or 10-membered heteroaryl group containing at least one ring- forming N atom, wherein said 5-, 6-, 9- or 10-membered heteroaryl group is optionally substituted by 1, 2, 3 or 4 R⁶ groups.

3. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R is:

4. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R¹ is:

5. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R is:

6. The compound of claim 1 , or pharmaceutically acceptable salt thereof, wherein R is H, C₁-C₆ alkyl, Ci-C₆ haloalkyl, OR⁹, SR⁹ or NR¹⁰R¹¹.

7. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R is H or OR⁹.

8. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R³ is H, F, Br, CF₃, or 6- or 5-membered heteroaryl.

9. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R³ is H.

10. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R⁴ is Ci-C₆ alkyl.

11. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R⁴ is methyl.

12. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R⁵ is Ci-C₆ alkyl.

13. The compound of claim 1, or pharmaceutically acceptable salt thereof, wherein R⁵ is methyl.

14. The compound of claim 1 having Formula II:

II or pharmaceutically acceptable salt form thereof.

15. The compound of claim 13, or pharmaceutically acceptable salt thereof, wherein R

16. The compound of claim 13, or pharmaceutically acceptable salt thereof, wherein R is:

17. The compound of claim 1 which is 5-[(4-(35>4-[(7/?,2Λ)-2-ethoxy-5- (pentafluorosulfanyl)-2,3-dihydro- 1 H-inden- 1 -yl]-3-methylpiperazin- 1 -yl-4-methylpiperidin- l-yl)carbonyl]-4,6-dimethylpyrimidine, or pharmaceutically acceptable salt thereof.

18. A composition comprising a compound of claim 1 , or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

19. A method of modulating activity of a chemokine receptor comprising contacting said chemokine receptor with a compound of claim 1, or pharmaceutically acceptable salt thereof.

20. The method of claim 19 wherein said chemokine receptor is CCR5.

21. The method of claim 19 wherein said modulating corresponds to inhibiting.

22. The method of claim 19 wherein said compound is a selective inhibitor of CCR5.

23. The method of claim 19 wherein said compound is a selective binder of CCR5.

24. A method of treating a disease associated with expression or activity of a chemokine receptor in a patient comprising administering to said patient a therapeutically effective amount of a compound of claim 1, or pharmaceutically acceptable salt thereof.

25. The method of claim 24 wherein said chemokine receptor is CCR5.

26. The method of claim 25 wherein said compound is a selective inhibitor or binder of CCR5.

27. A method of treating a disease or condition selected from an inflammatory disease, immune disorder, and viral infection in a patient comprising administering to said patient a therapeutically effective amount of a compound of claim 1, or pharmaceutically acceptable salt thereof.

28. The method of claim 27 wherein said disease or condition is an inflammatory disease.

29. The method of claim 28 wherein said disease or condition is an immune disorder.

30. The method of claim 28 wherein said disease or condition is a viral infection.

31. The method of claim 30 wherein said viral infection is HIV infection.

32. A method of treating HIV infection in a patient comprising administering to said patient a therapeutically effective amount of a compound of claim 1, or pharmaceutically acceptable salt thereof.

33. The method of claim 32 further comprising administering to said patient at least one anti-viral agent.