AU2871900A - G-protein coupled heptahelical receptor binding compounds and methods of use thereof - Google Patents

G-protein coupled heptahelical receptor binding compounds and methods of use thereof Download PDF

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AU2871900A
AU2871900A AU28719/00A AU2871900A AU2871900A AU 2871900 A AU2871900 A AU 2871900A AU 28719/00 A AU28719/00 A AU 28719/00A AU 2871900 A AU2871900 A AU 2871900A AU 2871900 A AU2871900 A AU 2871900A
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compound
protein coupled
moiety
disorder
coupled heptahelical
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Paul Fleming
Kazumi Shiosaki
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07C233/75—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
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    • C07C255/57—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and carboxyl groups, other than cyano groups, bound to the carbon skeleton
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Description

WO 00/46203 PCT/USOO/03042 G-PROTEIN COUPLED HEPTAHELICAL RECEPTOR BINDING COMPOUNDS AND METHODS OF USE THEREOF Background of the Invention 5 The chemokine family of peptides is defined on the basis of sequence homology and on the presence of variations on a conserved cysteine motif (Schall (1996) Cytokine 3:165-183; and Oppenheim et al. (1991) Annu. Rev. Immunol. 9:617-648). The family can be subdivided on the basis of this motif into two major subfamilies, in which members of each contain four characteristic cysteine residues. This subdivision 10 therefore defines the CC or p-chemokine family in which the first two cysteines are juxtaposed, and the CXC or a-chemokine family in which there is an intervening amino acid between the first two cysteines. Two other subfamilies have subsequently been described which have variations in the number of amino acids between the first two cysteine residues (Kelner et al. (1994) Science 266:1395-1399; Dorner et al. (1997) J. 15 Biol. Chem. 272:8817-8823; and Bazan et al. (1996) Nature 385:640-644.) Chemokines display a range of in vitro and in vivo functions ranging from proinflammatory activities on a range of cell types to proliferative regulatory activities. All of the functions of the chemokine family are believed to be signaled into a responsive cell using members of the G protein-coupled heptahelical receptor family. 20 To date several c-chemokine and p-chemokine receptors have been described. (e.g.. Neote et al. (1993) Cell 72:415-425; Ponath et al. (1996) J Exp. Med. 183:2437-2448; and Power et al. (1995) J Biol. Chem. 270:19495-19500.) Summary of the Invention 25 The present invention is based, at least in part, on the discovery of compounds which interact with G-protein coupled heptahelical receptors. The compounds of the invention can be used to treat chemokine mediated disorders, e.g. neurological, immunological, inflammatory and cancer related disorders. The present invention provides a G-protein coupled heptahelical receptor 30 (GPCR) binding compound of the formula: WO 00/46203 PCT/USOO/03042 -2 J-M (I) where J is an aromatic moiety and M is a G-protein coupled heptahelical receptor pocket interacting moiety. Preferably, the compound interacts with a p-chemokine receptor, e.g. CCR2, CCR3, CCR4, CCR5. CCR6, CCR7. CCR8, or CCR10. In a preferred 5 aspect, the compound modulates recruitment of at least one cell type. e.g. leukocytes, e.g. macrophages or eosinophils, associated with inflammation in a subject. In another aspect, the invention pertains to a method for treating a chemokine mediated disorder in a subject. The method involves administering an effective amount of a G-protein coupled heptahelical receptor binding compound such that the disorder is 10 treated, e.g. at least one symptom of the disorder is diminished or alleviated. The chemokine mediated disorder may be a neurological disorder (e.g. multiple sclerosis, Alzheimer's disease, or Parkinson's disease), an immunological disorder (e.g. AIDS, arthritis, or lupus), cancer, or an inflammatory disorder (e.g. asthma). In another aspect, the invention pertains to a compound represented by the 15 formula:
A-L
1
-B-L
2 -E (II) wherein A is selected from the group consisting of branched and straight chain alkyl, aryl, alkenyl, alkynyl, and heteroaryl moieties optionally substituted by NR'R", CN, NO2, F, 20 Cl, Br, I, CF 3 , CCl 3 , CHF2. CHCl 2 , CONR'R", S(O)NR'R", CHO, OCF 3 , OCCl 3 ,
SCF
3 , SCCl 3 , COR', CO 2 R', and OR' and wherein R' and R" are each independently hydrogen, CI-C 6 alkyl, C 2
-C
6 alkenyl, or optionally substituted aryl; LI is a linker moiety selected from the group consisting of a bond, 0, S, CHOH, CHSH, CHNH 2 , CHNHR, CHNRR', NH, NR, (CH2)n, O(CH2)n, and (CH2)nO(CH2)n, 25 an optionally substituted ring moiety of 4 to 7 atoms containing up to three heteroatoms, a chain of I to 5 atoms optionally substituted by C I -C 6 alkyl, halogens, wherein n is either 0, 1, 2, or 3, and R and R' are each independently substituted or unsubstituted C C 6 branched or straight chain alkyl, C I-C 6 branched or straight chain alkenyl, aryl, C 4 C 7 ring, optionally substituted with up to three heteroatoms; WO 00/46203 PCT/USOO/03042 B is an aromatic moiety containing from 0 to 3 heteroatoms and containing 5 to 7 members optionally substituted by NR'R", cyano, nitro, halogen, CF 3 , CHF 2 , CONR'R", S(O)NR'R", CHO, OCF 3 , SCF 3 , COR', CO 2 R', OR' where R' and R" are each independently hydrogen, halogen, Cl-C 6 alkyl, optionally substituted aryl or optionally 5 substituted aryl;
L
2 is a second linking moiety selected from the group consisting of a bond, CH 2 C=O, NHC=O, OC=0, C=0, CH 2 NHC=O, CHOH, (CH2)n, 0, NH, O(CH2)n, NH(CH2)n, CH 2 CHOH and NRC=O; and E is a G-protein coupled heptahelical receptor pocket interacting moiety. 10 In another aspect, the invention also relates to a compound represented by the formula: ZL R, (III) wherein ZI, Z 2 , Z 3 , and Z 4 are each independently N or C; 15 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R8 are each independently hydrogen,
CI-C
6 branched or straight chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl;
L
1 is 0, S, NH, NR 7 , (CH2)n, CO, CHOH, O(CH2)n, and (CH2)nO(CH2)n wherein n is either 1,2, or 3 and R 7 is C 1
-C
6 branched or straight 20 chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl; and
L
2 is a second linking moiety selected from the group consisting of a bond, CH 2 C=O, NHC=O, OC=O, C=O, CH 2 NHC=O, CHOH, (CH2)n, 0, NH, O(CH2)n, NH(CH2)n, CH 2 CHOH and NRC=O. 25 In yet another aspect, the invention relates to a pharmaceutical preparation comprised of the G-protein coupled heptahelical receptor binding compound and a pharmaceutically acceptable carrier. The invention also pertains to a packaged G protein coupled heptahelical receptor binding compound containing instructions for using the compound for treating a chemokine mediated disorder.
WO 00/46203 PCT/USOO/03042 -4 Brief Description of the Drawings Figure 1 depicts a binding curve of Compound A in a DB Assay (see Example 3 below). Figure 2 depicts a binding curve of Compound CU in a DB Assay (see Example 5 3 below). Figure 3 depicts a binding curve of Compound CV in a DB Assay (see Example 3 below). Figure 4 is a graph depicting the blockage of THP.1 cell migration by compound B in a CBIR Assay (see Example 4 below). 10 Figure 5 is a graph depicting the blockage of THP.1 cell migration by compound C in a CBIR Assay (see Example 4 below). Figure 6 shows MCP-5 induced peritoneal eosinophil recruitment in mice after administering compounds B and C in a MIR Assay (see Example 5 below). 15 Detailed Description of the Invention The present invention pertains to a G-protein coupled heptahelical receptor binding compound of the formula: J-M (1) wherein J is an aromatic moiety and M is a G-protein coupled heptahelical 20 receptor pocket interacting moiety. The language "G protein coupled heptahelical receptor" includes receptors for which belong to the G-protein coupled receptor (GPCR) superfamily of seven transmembrane domain heptahelical receptors. G-protein coupled receptors (GPCRs), along with G-proteins and effectors (intracellular enzymes and channels which are 25 modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs. These genes and gene-products are potential causative agents of disease (Spiegel et al. (1993) J. Clin. Invest. 92:1119-1125; McKusick and Amberger (1993) J. Med Genet. 30:1-26). Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have been 30 shown to cause various forms of autosomal dominant and autosomal recessive retinitis pigmentosa (see Nathans et al. (1992) Annu. Rev. Genet. 26:403-424), nephrogenic WO 00/46203 PCT/USOO/03042 -5 diabetes insipidus (Holtzman et al. (1993) Hum. Mol. Genet. 2:1201-1204 and references therein). These receptors are of critical importance to both the central nervous system and peripheral physiological processes. Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous 5 systems. The GPCR protein superfamily now contains over 250 types of paralogues, receptors that represent variants generated by gene duplications (or other processes), as opposed to orthologues, the same receptor from different species. The superfamily can be broken down into five families: Family I, receptors typified by rhodopsin and the 10 beta2-adrenergic receptor and currently represented by over 200 unique members (reviewed by Dohlman et al. (1991) Annu. Rev. Biochem. 60:653-688 and references therein); Family II, the recently characterized parathyroid hormone/calcitonin/secretin receptor family (Juppner et al. (1991) Science 254:1024-1026; Lin et al. (1991) Science 254:1022-1024); Family III, the metabotropic glutamate receptor family in mammals 15 (Nakanishi (1992) Science 258:597-603); Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum (Klein et al. (1988) Science 241:1467-1472); and Family V, the fungal mating pheromone receptors such as STE2 (reviewed by Kurjan (1992) Annu. Rev. Biochem. 61:1097-1129). Examples of GPCRs include chemokine receptors which are expressed in 20 specific tissues and leukocyte subtypes. Many chemokine receptors can bind to and be activated by more than one chemokine, and many chemokines can bind and activate more than one receptor in the nanomolar or subnanomolar range (MacKay (1996) J. Exp. Med. 184:522-549; Wells et al. (1996) Chem. Biol. 3:603-609). Chemokine receptors can be classified generally into three groups: a-chemokine receptors, P 25 chemokine receptors and c-p chemokine receptors. Preferably, the GPCR binding compounds of the present invention interact with receptors of the p-chemokine receptor family (Bonini et al. DNA Cell Biol. (1997) 16(10):1249-1256). Examples of p chemokine receptors include CCR2. CCR3, CCR4, CCR5, CCR6. CCR7, CCR8, and CCR1O.
WO 00/46203 PCT/USOO/03042 -6 The p-chemokine receptors (CCRs) are characterized by their ability to bind to CC chemokines (also referred to as p-chemokines). The CC chemokines are characterized by a conserved cysteine motif, in which the first two cysteines are juxtaposed. The CCR family includes both specific and non-specific receptors. For 5 example, CCRI is known to bind macrophage inflammation protein-la (MIP-la), RANTES (regulation on activation normal T cell expressed and secreted), and monocyte chemoattractant protein-3 (MCP-3) (Neote et al., (1993) Cell 72:415-425). CCR2 binds MCP-1, MCP-3, and MCP-4 (Myers et al. (1995) J. Biol. Chem. 270:5786-5792; Garcia Zepeda et al. (1996) J. Immunol. 157:5613), whereas CCR3 recognizes eotaxin and 10 MCP-4 (Kitaura et al. (1996) J. Biol. Chem. 271:7725; Garcia-Zepada et al. (1996) J. Immunol. 157:5613). CCR4 is activated by macrophage inflammatory protein-la (MIP la), RANTES, and MCP-1 (Power et al. (1995) J. Biol. Chem. 270:19495). CCR5 was found to bind to and be activated by RANTES , MIP-la, and MIP-Ib (Raport et al. (1996) J. Biol. Chem. 271: 17161). CCR6 and CCR7 have recently been discovered and 15 specifically bind to liver and activation regulated chemokine (LARC) and EBI 1 -ligand chemokine (ELC) respectively (Baba et al, 1997; Yoshida et al. 1997). It is known that CCR10 binds MCP-1 and MCP-3 with high affinity. The language "aromatic moiety" includes groups with aromaticity, e.g. moieties that have at least one aromatic ring. For example, 5- and 6-membered single-ring 20 aromatic groups that may include from zero to four heteroatoms. for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole. triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to 25 as "aryl heterocycles", "heteroaryls" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato. phosphinato, cyano, amino 30 (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino. arylcarbonylamino, carbamoyl and ureido), WO 00/46203 PCT/USOO/03042 -7 amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., 5 tetralin). In a preferred embodiment, the aromatic moiety of the present invention comprises two aromatic rings, e.g. pyridyl or pyrimidyl rings, e.g. two pyrimidyl rings connected with an ether linkage. The language "G-protein coupled heptahelical receptor pocket" refers to a region of the GPCR which is capable of interacting with, e.g. binding to, the GPCR pocket 10 binding moiety. Not wishing to be bound by theory, it is believed that the GPCR pocket may be a cavity in the GPCR lined with hydrophobic amino acid residues. The language "G-protein coupled heptahelical receptor pocket interacting moiety" refers to a group which interacts with a GPCR pocket. The group may be substituted or unsubstituted aromatic, alkyl, alkenyl, cycloalkyl, etc. The interaction 15 between the GPCR pocket interacting moiety and the pocket includes any interaction which allows the compound to perform its intended function, e.g., the interaction is hydrophobic, ionic, covalent, or combinations thereof. In one aspect of the invention, the GPCR binding compound modulates the recruitment of at least one inflammatory cell type in a subject. The ability of a GPCR 20 binding compound to modulate the recruitment of at least one inflammatory cell type can be measured or observed using art-recognized techniques or assays. Examples of such assays are the Murine Inflammatory Cell Recruitment Assay (referred to herein as the MICR Assay) and the Cell Based Inflammatory Recruitment Assay (referred to herein as the CIBR Assay) as described in Examples 4 and 5, respectively. 25 The term "subject" includes any animal which expresses GPCRs, for example, mammals e.g., mice, rats, cows, sheep, pigs, horses, monkeys, dogs, cats and, preferably, humans. The language "inflammatory cell type" includes cell types associated with a chemokine mediated disorder characterized by inflammation. Examples of these cell 30 types include, but are not limited to, leukocytes, e.g., eosinophils, neutrophils, basophils, fibroblasts, monocytes, T lymphocytes, and macrophages. Leukocytes are white blood WO 00/46203 PCT/USO0/03042 -8 cells which are involved in nonspecific resistance against pathogenic microorganisms and inflammatory response. Monocytes are particularly important in the nonspecific immune response, while lymphocytes are especially important in the specific immune response. Neutrophils are the most abundant phagocytic cells in blood and are 5 continuously produced in circulating blood, affording protection against the entry of foreign materials. These leukocytes exhibit chemotaxis and are attracted to foreign substances, including invading microorganisms, which they engulf and digest along with particulate matter. Eosinophils are leukocytes which react with the acidic dye eosin. Basophils are leukocytes which stain with basic dyes. Macrophages are large ameboid 10 mononuclear phagocytic cells. The present invention also pertains to a GPCR binding compound which is an antagonist of a G-protein coupled heptahelical receptor, e.g., a p-chemokine receptor. In one embodiment, the IC 50 of the GPCR binding compound to the GPCR is about 10 pM or less, e.g., about 5 tM or less, e.g., about 1 VtM or less, e.g., about 50 nM or less. 15 The term "antagonist" includes compounds which bind to the GPCR such that the binding of a second compound to the GPCR is modulated. The ability of a compound to bind to a GPCR can be determined through using art-recognized techniques and assays. Examples of such assays include the Time Resolved Fluorescence assay (herein referred to as the TRF assay) and the Direct 20 Binding Assay (herein referred to as the DB Assay), described in Examples 2 and 3, respectively. The TRF assay determines the binding affinity of a compound to a receptor by over expressing the receptor in a culture of cells. The TRF assay determines the binding affinity of a compound to a receptor using a cell line engineered to overexpress a GPCR, e.g. CCR10. The cells are exposed simultaneously to the test 25 compound and a fluorescently labeled ligand specific for the receptor. After a predetermined amount of time, excess ligand and test compound is removed. The amount of fluorescence is measured and the percent inhibition of binding of the ligand is calculated. By repeating this experiment at multiple concentrations of test compound, it is possible to generate a dose-response curve from which an IC 5 0 can be determined.
WO 00/46203 PCT/USOO/03042 -9.
In another aspect, the invention pertains to a method for treating a chemokine mediated disorder, (e.g., a neurological disorder, an immunological disorder, a disorder characterized by inflammation, or a disorder characterized by unwanted cellular proliferation) in a subject. The method includes administering an effective amount of a 5 G-protein coupled heptahelical receptor binding compound to a subject. For example, the disorder may be treated through modulation of a p-chemokine receptor, e.g., CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, or CCRI0. Examples of preferred disorders include AIDS, multiple sclerosis, asthma. cancer, and lupus. The disorder may also be characterized by abnormal cellular signal transduction, or amounts of chemokine 10 stimulated chemotaxis. The term "administering" includes routes of administration which allow the GPCR binding compound to perform its intended function, e.g. interacting with GPCRs and/or treating a chemokine mediated disorder. Examples of routes of administration which can be used include parental injection (e.g., subcutaneous, intravenous, and 15 intramuscular), intraperitoneal injection, oral, inhalation, and transdermal. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the GPCR binding compound can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function. The GPCR binding compound can be 20 administered alone or with a pharmaceutically acceptable carrier. Further, the GPCR binding compound can be administered -as a mixture of GPCR binding compounds, which also can be coadministered with a pharmaceutically acceptable carrier. The GPCR binding compound can be administered prior to the onset of a chemokine mediated disorder, or after the onset of a chemokine mediated disorder. The GPCR 25 binding compound also can be administered as a prodrug which is converted to another form in vivo. The term "treatment" includes the diminishment or alleviation of at least one symptom associated or caused by the disorder being treated. For example, treatment can be diminishment of several symptoms of a disorder or complete eradication of a 30 disorder.
WO 00/46203 PCT/US00/03042 - 10 The language "chemokine mediated disorder" includes a disorder characterized by the participation of chemokines or association with chemokines. The language also includes disorders characterized by aberrant chemokine expression. Chemokines have a wide variety of functions. They are able to elicit chemotactic migration of distinct cell 5 types, such as monocytes, neutrophils, T lymphocytes, basophils and fibroblasts. Many chemokines have proinflammatory activity and are involved in multiple steps during an inflammatory reaction. These activities include stimulation of histamine release, lysosomal enzyme and leukotriene release, increased adherence of target immune cells to endolethial cells, enhanced binding of complement proteins, induced expression of 10 granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein -1 (MIP- 1) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 is a potent inhibitor of endolethial cell growth, interleukin-8 (IL-8) promotes proliferation of 15 keratinocytes, and GRO is an autocrine growth factor for myeloma cells. Chemokines have been proposed to participate in a number of physiological and disease conditions, including, for example, lymphocyte trafficking, wound healing, hemapoietic regulation and immunological disorders such as asthma and arthritis. The language "chemokine mediated disorder characterized by inflammation" 20 includes a disorder having inflammation as at least one of its symptoms. Examples of such disorders include anaphylaxis, systemic necrotizing vasculitis, systemic lupus erthyematosus, serum sickness syndromes, psoriasis, rheumatoid arthritis, adult respiratory distress syndrome (ARDS), allergic rhinitis, atopic dermatitis, asthma and other allergic responses, and reperfusion injury occurring after periods of ischemia such 25 as in myocardial infarction or shock. Preferably, the disorder is asthma. Other groups of possible chemokine mediated disorders include neurological related disorders, immunological related disorders and disorders characterized by unwanted cellular proliferation, e.g. cancer. The language "neurological related disorders" includes disorders of the nervous 30 system, including, but not limited to those involving the brain, the central and peripheral nervous system, and the interfaces between muscles and the nerves. Some examples of WO 00/46203 PCT/USOO/03042 - 11 neurological related disorders include Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease. "Neurological related disorders" also 5 includes neurological disorders associated with inflammation, e.g. stroke, traumatic injury to the brain, traumatic injury to the spinal cord, spinal crush, and central and peripheral nervous system trauma. The language "immunological related disorder" includes both organ-specific and systemic immunological disorders. Some examples of immunological disorders include 10 immune thyroiditis, hyperthyroidism, type I diabetes mellitus, insulin related diabetes, Addison's disease, autoimmune oophoritis, autoimmune orchitis, AIDS, autoimmune hemolytic anemia, paroxysmal cold hemoglobinuria, autoimmune thrombocytopenia, autoimmune neutropenia, pernicious anemia, autoimmune coagulopathies, myasthenia gravis, multiple sclerosis, experimental allergic encephalomyelitis, pemphigus and other 15 bullous diseases, rheumatic carditis, Goodpasture's syndrome, postcardiotomy syndrome, systemic lupus erythematosus, rheumatoid arthritis, keratitis, parotitis, polymositis, dermatomyositis, and scleroderma. Preferably, the immunological disorder is AIDS, multiple sclerosis, rheumatoid arthritis, or lupus. In another embodiment, the invention pertains to a pharmaceutical preparation 20 comprised of an effective amount of a G-protein coupled heptahelical receptor binding compound and a pharmaceutically acceptable carrier. In a preferred aspect, the effective amount is an effective amount to treat a p-chemokine mediated disorder, e.g., asthma. The language "pharmaceutically acceptable carrier" includes substances capable of being coadministered with the GPCR binding compound(s), and which allows both to 25 perform their intended function, e.g., treating a chemokine mediated disorder or preventing a chemokine mediated disorder. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the GPCR binding compound also fall within 30 the scope of the present invention.
WO 00/46203 PCT/USOO/03042 - 12 Furthermore, the language "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, 5 involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches. such 10 as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and 15 polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and 20 magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium 25 metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, c-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
WO 00/46203 PCT/USOO/03042 - 13 Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active 5 ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about I per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent. 10 Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if 15 necessary, shaping the product. Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an 20 elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste. 25 In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, 30 carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents., such as agar-agar, calcium WO 00/46203 PCT/USO0/03042 - 14 carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; 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; lubricants, such a talc, 5 calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight 10 polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium 15 carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be 20 scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized 25 by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain 30 portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
WO 00/46203 PCT/USOO/03042 - 15 The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, 5 syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluent 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, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and 10 sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert dilutents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. 15 Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar agar and tragacanth, and mixtures thereof. Formulations of the pharmaceutical compositions of the invention for rectal or 20 vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the 25 active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of a compound of this 30 invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with WO 00/46203 PCT/USOO/03042 - 16 a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils. waxes, 5 paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones. bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to a compound of this invention. excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain 10 customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can 15 also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. 20 Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may 25 contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable 30 mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating WO 00/46203 PCT/USOO/03042 - 17 materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of 5 microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as 10 aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its 15 rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. 20 Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. 25 The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration 30 is preferred.
WO 00/46203 PCT/USO0/03042 - 18 The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, 5 intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous 10 system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, 15 ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. 20 Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the 25 activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical 30 history of the patient being treated, and like factors well known in the medical arts.
WO 00/46203 PCTIUSOO/03042 - 19 A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required 5 in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. 10 While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition. As set out above, certain embodiments of the present compounds can contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term 15 "pharmaceutically acceptable salts" is art recognized and includes relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus 20 formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1-19). 25 In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances includes relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be 30 prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such WO 00/46203 PCT/US00/03042 - 20 as the hydroxide. carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. 5 Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. The term "pharmaceutically acceptable esters" refers to the relatively non-toxic, esterified products of the compounds of the present invention. These esters can be 10 prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. Hydroxyls can be converted into esters via treatment with an esterifying agent such as alkanoyl halides. The term also includes 15 lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See, for example, Berge et al., supra.) A preferred ester group is an acetomethoxy ester group. The language "effective amount" of the compound is that amount necessary or sufficient to treat or prevent a chemokine mediated disorder, e.g. prevent the various 20 morphological and somatic symptoms of a chemokine mediated disorder. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular GPCR binding compound. For example, the choice of the GPCR binding compound can affect what constitutes an "effective amount". One of ordinary skill in the art would be able to study the aforementioned factors and make the 25 determination regarding the effective amount of the GPCR binding compound without undue experimentation. An in vivo assay as described in Example 5 below or an assay similar thereto (e.g., differing in choice of cell line or type of illness) also can be used to determine an "effective amount" of a GPCR binding compound. The ordinarily skilled artisan would select an appropriate amount of a GPCR binding compound for use in the 30 aforementioned in vivo assay.
WO 00/46203 PCT/USOO/03042 -21 The regimen of administration can affect what constitutes an effective amount. The GPCR binding compound can be administered to the subject either prior to or after the onset of a chemokine mediated disorder. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be 5 continuously infused, or can be a bolus injection. Further, the dosages of the GPCR binding compound(s) can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. In yet another aspect, the invention features a protein coupled heptahelical receptor binding compound comprising a G-protein coupled heptahelical receptor 10 binding compound packaged with instructions for using said compound for treating a chemokine mediated disorder. The invention also features a method of using a G-protein coupled heptahelical receptor binding compound to modulate the binding of a second compound to a G protein coupled heptahelical receptor. 15 In another aspect, the invention pertains to a compound represented by the formula: A-Ll-B-L 2 -E (II) where A is selected from the group consisting of branched and straight chain alkyl, aryl, alkenyl, alkynyl, and heteroaryl moieties optionally substituted by NR'R", 20 CN, NO2, F, Cl, Br, I, CF 3 , CC1 3 , CHF 2 , CHCl 2 , CONR'R", S(O)NR'R", CHO, OCF 3 , OCCl 3 , SCF 3 , SCCI 3 , COR', CO 2 R', and OR' and wherein R' and R" are each independently hydrogen, C 1
-C
6 alkyl, C 2
-C
6 alkenyl, or optionally substituted aryl;
L
1 is a linker moiety selected from the group consisting of a bond, 0, S, CHOH, CHSH, CHNH 2 , CHNHR, CHNRR', NH, NR, (CH2)n, O(CH2)n, and (CH2)nO(CH2)n, 25 an optionally substituted ring moiety of 4 to 7 atoms containing up to three heteroatoms, a chain of 1 to 5 atoms optionally substituted by C 1
-C
6 alkyl, halogens, wherein n is either 0, 1, 2, or 3, and R and R' are each independently substituted or unsubstituted C 1 C 6 branched or straight chain alkyl, C 1
-C
6 branched or straight chain alkenyl, aryl, C 4 C 7 ring, optionally substituted with up to three heteroatoms; WO 00/46203 PCT/USOO/03042 - 22 B is an aromatic moiety containing from 0 to 3 heteroatoms and containing 5 to 7 members optionally substituted by NR'R", cyano. nitro. halogen. CF 3 . CHF2. CONR'R", S(O)NR'R"., CHO, OCF 3 , SCF 3 , COR', CO2R', OR' where R' and R" are each independently hydrogen, halogen, Cl-C6 alkyl, optionally substituted aryl or optionally 5 substituted aryl;
L
2 is a second linking moiety selected from the group consisting of a bond, CH2C=O. NHC=0, OC=0, C=0, CH 2 NH C=O, CHOH. (CH2)n, 0, NH. O(CH2)n NH(CH2)n, CH2CHOH and NRC=O; and E is a G-protein coupled heptahelical receptor pocket interacting moiety. 10 In one aspect, L, is S, NH, CH 2 , or 0. In another aspect. L, is NHC=O. In one embodiment, A is represented by the following formula: R, N R, R, wherein Z, and Z-, each independently represent N or C; 15 R,, R 2 , and R 3 are independently selected from the group comprised of hydrogen, C,-C 2 branched or straight chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl. In one embodiment, both Z, and Z, are carbon, R, is alkyl (e.g.. methyl). halogen (e.g., bromine, chlorine or fluorine), or alkoxy and the L, linker is located in the meta 20 position. For example, A may be represented by the following formula: Ri R N N6 or In another embodiment, R, is carbonyl (e.g., a ketone., an aldehyde. an ester, or an amide.). Furthermore, R, may be substituted with a cyclic moiety such as piperazine, furan, or phenyl.
WO 00/46203 PCT/USOO/03042 - 23 In yet another embodiment, A is substituted or unsubstituted phenyl. Examples of substituents includes substituted or unsubstituted alkyl (e.g., methyl). alkenyl, aryl and heteroaryl moieties. Furthermore. A may be substituted with halogens (e.g., chlorine). 5 In one embodiment, L, is O. In another aspect, the invention features a compound wherein B is represented by the following formula: R R, wherein 10 Z 3 and Z 4 each independently represent N or C;
R
4 and R 5 are independently selected from the group comprised of hydrogen, C 1
-C
6 branched or straight chain alkyl, alkenyl, alkynyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl. In one embodiment, R 4 is alkyl and R 5 is hydrogen. 15 In one embodiment, B is a substituted or unsubstituted pyridyl or pyrimidyl moiety, wherein B may be represented by the following formula: N or In an aspect, the invention features a compound wherein E is represented by the formula below: 20 wherein R 6 is an electron withdrawing moiety and the aryl ring is additionally optionally substituted with zero to four halogen atoms. Preferably, E is substituted with at least one fluorine atom, e.g. two or more fluorine atoms. For example, R 6 may be alkyl, alkoxy, haloalkyl, nitro, halo, alkylamino, hydroxyalkyl, or carboxyl.
WO 00/46203 PCT/US0O/03042 - 24 In an embodiment, E is a para substituted aryl moiety represented by the formula below:
+R
6 In a further embodiment, R 6 is a halogenated alkyl moiety, e.g. a fluorinated 5 alkyl moiety, e.g. a trifluoromethyl or pentafluoroethyl. Other examples of R, include substituted or unsubstituted alkoxy moieties (e.g., methoxy. trifluoromethoxy) or thioether moieties. R 6 can be alkenyl or alkynyl (e.g., ethenyl). Furthermore, in another embodiment E is heterocyclic, e.g.. substituted or unsubstituted furanyl, imidazolyl, benzothiophenyl, benzylfuranyl, quinolinyl, 10 isoquinolinyl, benzodioxazolyl, benzoxazolyl, benzothiazolyl, benzylimidazolyl, thiazolyl, isothiaozolyl, oxazolyl, benzylthiazolyl, isooxazolyl, methylenedioxyphenyl, indolyl, thienyl, pyrimidyl, pyrazinyl, purinyl, or deazapurinyl. In yet another embodiment, E is branched or straight chain alkenyl or alkynyl. Examples include ethynyl trimethyl silane and alkenes (e.g., dienes, trienes). 15 In another aspect, the invention features a compound represented by the formula below:
R
7 R R, RZ3 R4 R, R, wherein
Z
1 , Z 2 , Z 3 , and Z 4 are each independently N or C; 20 R 1 , R2, R 3 , R 4 , R 5 , R6, R, and R, are each independently hydrogen, C,-C 6 branched or straight chain alkyl, alkenyl, alkynyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl; L, is 0, S, NH, NR,, (CHR 7 )n, CO. CR 7 0H, O(CHR 7 )n, and
(CHR
7 )nO(CHR 7 )n wherein n is either 1,2, or 3; 25 L, is a second linking moiety selected from the group consisting of a bond. CH 2 C=O, NHC=O, OC=O, C=O, CH2NHC=O. NHC=OCH 2 , CHOH, (CH 2 )n, 0, NH, O(CH 2 ), NH(CH 2
),
1 . CH 2 CHOH and NRC=O. wherein m is 0, 1, 2, or 3.
WO 00/46203 PCT/USOO/03042 - 25 Furthermore, the compound may be represented by the formula below:
R
7 R L2§ Rs R, R3 R, R5 R, (IV) wherein ZI, Z 2 , Z 3 , and Z 4 are each independently N or C; 5 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are each independently hydrogen,
C
1
-C
6 branched or straight chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl; L I is 0, S, NH, NR 7 , (CH2)n, CO, CHOH, O(CH2)n, and (CH2)nO(CH2)n wherein n is either 1,2, or 3 and R 7 is CI-C 6 branched or straight 10 chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl;
L
2 is a second linking moiety selected from the group consisting of a bond, CH 2 C=O, NHC=O, OC=O, C=0, CH 2 NHC=O, CHOH, (CH2)n, 0, NH, O(CH2)n, NH(CH2)n, CH 2 CHOH and NRC=0. 15 In one embodiment, Z, and Z 2 are both carbon. In another, R, is methyl, and R 2 and R 3 are hydrogen. In another aspect, L, is 0. In an yet another embodiment, R 4 and
R
5 are both hydrogen. In yet another embodiment, L, is NHC=0. In a preferred aspect,
R
6 is a halogenated alkyl moiety, e.g. a fluorinated alkyl moiety, e.g. a trifluoromethyl or pentafluoroethyl moiety. In another aspect, R 7 and R, are each independently fluorine or 20 hydrogen. In yet another aspect, the invention can be represented by the structure below: LRy RL L2 r R3 T if R 3 pn t r The invention also features compounds of the formula: WO 00/46203 PCT/US0O/03042 -26 R, N ~ Li '
L
2 . R6 In one embodiment, L, is 0 and L. is NHCO. In a further embodiment. L is 0, L2 is NHC=O,. and R 6 is a halogen, a halogenated alkyl group, e.g. trifluoromethyl or pentafluoroethyl), or an alkoxy group, e.g. a halogenated alkoxy group, e.g. a 5 trifluoromethoxy group. Furthermore, R 6 also can be ethenyl or a thioether moiety (e.g.
-S-CF
3 ). The invention also features binding compounds represented by the following structures: N ON C O F3 0 H N H ON N O N OCF3
OCF
3
CF
3 CF3 C0 0 T N N H o N CH 3 N (NN H OF 0 CF 3 NC N' N3' N ' O -~0 )-a O) a COCF 3 WO 00/46203 PCT/USO0/03042 - 27 0 0~~
H
3 C N N N H N 0) OCF 3 05tBu 0 C N N (NCH N t' 'Bu 0 "" CF(CFA) 0~ 0 N N5: NH N N, CCN N 0) COH(CF 3
)
2 0- L C0H(CF 3
)
2 0 1 HN
H
3 CfN N a'N H 0 COH(CF 3
)
2 CF N N' N H (To N H 01 OCF 3 0-,a C I 0 0H H Nj H N N CA 3 N' N 0' NO 2
CF
2
CF)CF
3 WO 00/46203 PCT/USOO/03042 -28 H N' N N NO O -- , a C 0 -N 0 IC I ,jH Na, N CH 3 N N CH 3 N' O O Br N FI BI rF oT 0 1NH N N C NI] ' ( N O~ne N-I -a C
CF
3
CF
3 0 0 NH N Me Ni N Me N 11-aci 0 0 K- N H NNi N Me NH~N Me NI-I CCF 3 The term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or 5 phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a straight chain or branched chain alkyl has 10 or fewer carbon atoms in its backbone (e.g., C - WO 00/46203 PCT/USOO/03042 - 29 C 10 for straight chain, C 3
-C
10 for branched chain), and more preferably 6 or fewer. Likewise, preferred cycloalkyls have from 4-7 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. Moreover, the term alkyl includes both "unsubstituted alkyls" and "substituted 5 alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, 10 cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino. sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, silyl, trialkylsilyl, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will be 15 understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "alkylaryl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term "aryl" includes aryl groups, including 5- and 6-membered single-ring 20 aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to 25 as "aryl heterocycles", "heteroaryls" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example. halogen, hydroxyl, alkoxy, alkylcarbonyloxy. arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato. phosphinato, cyano, amino 30 (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), WO 00/46203 PCT/USOO/03042 - 30 amidino. imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl. sulfonamido, nitro, trifluoromethyl, cyano, azido. heterocyclyl, alkylaryl. or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g.. 5 tetralin). The terms "alkenyl" and "alkynyl" include unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above. but that contain at least one double or triple bond, respectively. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein 10 means an alkyl group, as defined above, but having from one to three carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. The terms "alkoxyalkyl", "polyaminoalkyl" and "thioalkoxvalkyl" include alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms 15 replacing one or more carbons of the hydrocarbon backbone, e.g.. oxygen, nitrogen or sulfur atoms. The terms "polycyclyl" or "polycyclic radical" refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". 20 Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above. as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl. aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, 25 cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino. and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino. carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl. cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety. 30 The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen. sulfur and phosphorus.
WO 00/46203 PCTIUS0O/03042 -31 It will be noted that the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be 5 obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. In one embodiment. the present invention includes GPCR binding compounds and/or methods of using the same which are encompassed by the formulae set forth herein and which are not described in Brombridge, et al., J. Med Chen. (1997) 40:3494, 10 U.S. 3,499,898, EP358 118, EP 344 634, and/or WO 96/23783. The contents of each of which are expressly incorporated herein by reference. In another embodiment, E or the G-protein coupled heptahelical pocket interacting moiety is not: H , and/or unsubstituted phenyl. 15 In another embodiment, L, is 0, L 2 is NHCO, and E or the G-protein coupled heptahelical pocket interacting moiety is not: NO H , and/or unsubstituted phenyl. In another embodiment, E or the G-protein coupled heptahelical pocket interacting moiety is a phenyl group having at least one substituent. In yet another 20 embodiment, E is a phenyl group having at least one substituent in the para position. In a further embodiment, E is a phenyl group having at least two substituents. The compounds of the present invention can be synthesized using standard methods of chemical synthesis and/or can be synthesized using schemes described herein. Synthesis of specific compounds is discussed in detail in the Example sections 25 below. An example of a general synthesis is outlined in the scheme below: WO 00/46203 PCT/USOO/03042 - 32 Scheme 1: General Synthesis of Test Compounds YNOY OH y 0 Y Y NO2 X I NaH, dioxane R XN' R RI R2 R3 NR N R4 N NO CI NNp N ClI N R4 R MeOH,
H
2 0 Fe, AcOH 0 x , XR0 0 Y,
R
3 R4N X RH Cl2 R N / -N C2I 3-~ R, R2H 1( N N H 2 6 R 8 Hydroxypyridine (or other hydroxyaryl precursor) is dissolved in dioxane and 5 1.5 equivalents of 95% sodium hydride is added. The mixture is stirred at room temperature for 20 minutes and 1 equivalent of 2-chloro-5-nitropyridine (or other C chloro-heterocycle) is then added. The mixture is subsequently brought to reflux for 3 hours and cooled. The reaction mixture is then quenched by addition of saturated ammonium chloride solution. Silica gel is added to the solution and the mixture is 10 rotovapped to dryness. The product is eluted from the silica gel and flash chromatographed using a mixture of ethyl acetate/hexane. The nitro group is reduced by dissolving the nitropyridine in 1:1 methanol:water. Acetic acid and iron powder is then added and the mixture is brought to reflux for 3 hours. After cooling, the iron is precipitated by addition of 20% NaOH and subsequently 15 filtered through Celite. The methanol is removed by rotary evaporation and the remaining aqueous mixture is extracted with methylene chloride. The organic layer is dried and the solvent removed by rotary evaporation to give product. The dipyridyl ether is then dissolved in methylene chloride followed the addition of polymer bound morpholine (Booth, et al., J. Am. Chem. Soc., 119, 1997, 4882-4886). 20 1.3 equivalents of acid chloride is added and the mixture shaken overnight. The excess acid chloride is scavenged using polymer bound tris-2-aminoethylamine. Filtration WO 00/46203 PCT/USOO/03042 - 33 followed by flash chromatography of the filtrate using ethyl acetate:hexane gives the product. Further examples of syntheses of compounds of the invention are included in the 5 Example section. The invention is further illustrated by the following examples which in no way should be construed as being further limiting. The contents of all references, pending patent applications and published patent applications, cited throughout this application are hereby incorporated by reference. It should be understood that the animal models 10 used throughout the examples are accepted animal models and that the demonstration of efficacy in these animal models is predictive of efficacy in humans. EXAMPLE 1: Synthesis of GPCR Binding Compounds 15 A. General Procedures Many GPCR binding compounds of the invention were made by the general reaction scheme, outlined below: 'Y NO2 X4Y OH NaH, dioxane X' O X R + R 3 NaR3 N R4N NO2 CI N N N O 2 MeOH,
H
2 0 Fe, AcOH 0 OO y0 0 Y N N 7 CH 2
C
2
R
3 N N2 R, R2 NR
NH
2 R Rg 20 I: Nucleophilic displacement of 2-chloroheterocycles WO 00/46203 PCT/USOO/03042 - 34 Method A: 50 mmol of hydroxypyridine (or other hydroxyaryl precursor) is dissolved in 500 mL of dioxane. 1.5 equivalents of 95% sodium hydride is added and the mixture stirred at room temperature for 20 minutes. 1 equivalent of 2-chloro-5-nitropyridine (or other X 5 chloro-heterocycle) is added and the mixture is brought to reflux for 3 hours. After cooling, the reaction mixture is quenched by addition of 2 mL of saturated ammonium chloride solution. 10 g of silica gel is added to the solution and the mixture is rotovapped to dryness. The product is eluted from the silica gel and flash chromatographed using a mixture of ethyl acetate/hexane. 10 Method B: 50 mmol of hydroxypyridine is dissolved in 150 mL of dry DMF. The mixture is cooled to 0 C and 1.5 equivalents of sodium hydride added. The mixture is allowed to warm to room temperature over 20 minutes, followed by addition of the a-chloro 15 heterocycle. The reaction mixture is stirred at room temperature for 16 hours, and then partitioned between ethyl acetate/water in a separatory funnel. The organic layer is dried and the solvent removed to give product which is typically > 90% pure. II: Reduction of Nitro group 20 Method A: 40 mmol of nitropyridine is dissolved in 200 mL of methanol followed by the addition of 200 mL of water. Acetic acid (8.3 mL) and iron powder (17 g) is added and the mixture is brought to reflux for 3 hours. After cooling, the iron is precipitated by 25 addition of 25 mL of 20% NaOH and the mixture filtered through Celite. Removal of the methanol by rotary evaporation is followed by extraction of the remaining aqueous mixture with methylene chloride. The organic layer is dried and the solvent removed by rotary evaporation to give product.
WO 00/46203 PCT/USOO/03042 - 35 Method B: 40 mmol of nitropyridine is dissolved in 250 mL of ethanol and treated with a mixture of tin(II) chloride (30 g) in conc. HCl (65 mL). The mixture is heated to 50 C for 1 hour. After cooling, the mixture is basified with 40% NaOH and the product 5 extracted into ethyl acetate. The solution is dried over sodium sulfate and the solvent removed to give product. III: Acylation of the amino-heterocycle 10 1 mmol of the dipyridyl ether is dissolved in 12 mL of methylene chloride followed by the addition of 0.4 g of polymer bound morpholine (loading 4 mmol/g) (Booth, et al. J. Am. Chem. Soc., 119, 1997, 4882-4886). 1.3 equivalents of acid chloride is added and the mixture shaken overnight. The excess acid chloride is scavenged using polymer bound tris-2-aminoethylamine. Filtration followed by flash chromatography of the 15 filtrate using ethyl acetate:hexane gives the product. B. Synthesis of the GPCR Binding Compounds
>NO
2 >OH >0 - + I-NaH Cl N O2 Dioxane N
NO
2 1 Fe/AcOH 3a R=CF3 MeOH/water 3b R=OCF3 3c R= Br 0 3d R=tBu N. N 3e=CF(CF3)2 0 NR NNH 2 2 20 WO 00/46203 PCT/US00/03042 - 36 (1) 2-methyl-3-hydroxy pyridine (5 g, 46 mmol) and 2-chloro-5-nitro pyridine were suspended in 500 mL of dioxane. Sodium hydride (1.7 g of 95%, 1.5 eq) was added and the mixture refluxed under argon for 3 hours. After cooling, the excess sodium hydride was quenched by addition of 2 mL of saturated ammonium chloride solution and 10 g 5 of silica gel added to the solution. The solvents were removed by rotary evaporation leaving the product adsorbed on silica. Purification was achieved by flash chromatography using 1:1 ethyl acetate/hexanes as eluent. Yield: 9.3 g, 87%. LC-MS (diode array detection at 210-300 nM) showed that the product was > 95% pure and had the expected M.W. of 232 (M+H+). 1 H NMR (CDCl 3 , shifts relative to the solvent peak 10 at 7.24 ppm): 6 8.98 (d, IH) 6 8.52 (dd, 1H) 6 8.42 (dd, 1H) 8 7.30 (dd, 1H) 6 7.2 (m, 1H) 6 7.08 (d, 1H) 6 2.38 (s, 3H). (2) Compound (1) (9.3 g) was suspended in 400 mL of 1/1 methanol/water. Acetic acid (8.3 mL) and powdered iron (17 g) were added and the mixture brought to reflux for 3 15 hours. After cooling, the iron was precipitated by addition of 25 mL of 20% NaOH. The mixture was filtered through Celite and the filter cake rinsed with methanol. The methanol was removed by rotary evaporation and the product partitioned into methylene chloride in a separatory funnel. Removal of the solvent by rotary evaporation gave 4.1 g (51 %)of product. LC-MS showed the product to be > 95% pure and to have the 20 expected M.W. of 202 (M+H+). 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 8.24 (d, 1H) 6 7.60 (d, 1H) 6 7.24 (dd, 1H) 6 7.08 (in, 2H) 6 6.88 (d, 1H) 6 3.52 (br s, 2H) 6 2.38 (s, 3H). (3a)Compound (2) (202 mg, 1 mmol) was dissolved in 10 mL of methylene chloride in a 25 20 mL scintillation vial. Polystyrene bound morpholine (0.4 g, loading 4 mmol/g) was added followed by 1.3 mmol of 4-trifluoromethylbenzoyl chloride. The mixture was shaken at room temperature for 12 hours followed by the addition if 0.5 g of polystyrene supported tris-2-aminoethylamine (loading 4 mmol/g). The mixture was shaken for an additional 12 hours and filtered through a polystyrene frit. The reaction mixture was 30 applied to a 90 g cartridge of silica gel and the product eluted with ethyl acetate.
WO 00/46203 PCT/US0O/03042 - 37 Removal of the solvent gave 248 mg of product. LC-MS indicated a purity > 97%and the expected M.W. of 374 (M+H+). 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 8.79 (s, lH) 6 8.19 (in, 2H) 6 7.99 (d, 1H) 6 7.84 (d, 2H) 6 7.53 (d, 2H) 6 7.21 (d, lH) 6 7.06 (in, 1H) 6 6.81 (d, I H) 6 2.20 (s, 3H). Anal. Calcd for 5 C 19
H
14 F3N 3 0 2 : C, 61.13 H, 3.78 N, 11.26 Found: C, 61.27 H, 3.76 N, 11.17. A number of similar analogs were made from intermediate (2) by acylation with other substituted benzoyl chlorides: 10 (3b) LC-MS showed the product to be > 95% pure and to have the expected M.W. of 390 (M+H+). 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm) 6 9.02 (s, 1H) 6 8.18 (d, H) 6 8.06 (d, 1H) 6 7.97 (d, 1H) 6 7.8 (in, 2H) 6 7.21 (d, 1H) 6 7.05 (in, 3H) 8 6.8 (d, IH) 6 2.20 (s, 3H). 15 (3c) LC-MS showed the product to be > 95% pure and to have the expected M.W. of 385 (M+H+). 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 9.18 (s, 1H) 6 8.24 (d, 1H) 6 8.18 (d, 1H) 6 8.04 (s, 1H) 6 7.66 (d, 2H) 6 7.45 (d, 2H) 6 7.27 (d, 1H) 6 7.08 (in, 1H) 6 6.80 (d, 1H) 6 2.21 (s, 3H). 20 (3d) LC-MS showed the product to be > 95% pure and to have the expected M.W. of 362 (M+H+). 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 9.18 (d, 1H) 6 8.22 (d, 1H) 6 8.19 (d, 1H) 6 8.17 (d, 1H) 6 7.78 (d, 2H) 6 7.36 (d, 2H) 6 7.26 (d, 1H) 6 6 7.05 M, 1H) 6 6.8 (d, 1H) 6 2.22 (s, 3H) 6 1.20 (s, 9H). 25 (3e) 4-(perfluoroisopropyl)benzoyl chloride was made as follows: 4-iodo benzoic acid (5 g, 20.2 mmol) was dissolved in DMF along with perfluoroisopropyl iodide (6 g, 20.3 mmol). Copper powder (6.35 g) and DMF (25 mL) were added and the mixture was heated at 140 C in a sealed tube for 8 hours. After cooling, the reaction mixture was partitioned between diethyl ether and 1 N HCl. The organic layer was separated and 30 dried and the resulting solid chromatographed on silica (ethyl acetate as eluent) to give a WO 00/46203 PCTUSOO/03042 - 38 mixture of compounds containing the desired product (by LC-MS.) The crude mixture was treated with oxalyl chloride (3 mL) in 25 mL of methylene chloride. After 3 hours of stirring, the solvents were removed by rotary evaporation and residual oxalyl chloride removed by azeotroping with toluene. The crude acid chloride was used to acylate 5 compound (2) according to the general procedure given above. The product was purified by preparative TLC (5:4:1 methylene chloride:ethyl acetate:methanol). LC-MS showed the product to be > 95% pure and to have the expected M.W. of 474 (M+H+) I H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 8.25 (m, 3H) 6 8.19 (s, 1H) 6 8.05 (d, 2H) 6 7.79 (d, 2H) 6 7.4 (d, IH) 6 7.2 (m, 2H) 6 6.99 (d. 1H) 6 2.2 (s, 3H). 10 C. Synthesis of "Extended Bridge" GPCR Binding Compounds r I OH N NaH, Dioxane
NO
2 Fe
NO
2 O 0 N HOAc GIN N N 4 H CF 3
NH
2 SONOO N 6 15 (4) 3-pyridinol (5g, 45.8 mmol) was dissolved in 500 mL of dioxane. Sodium hydride (1.7 g, 1.5 equivalents) was added and the mixture stirred for 10 minutes. 2-chloro-5 nitro pyridine (7.27 g, 45.8 mmol) was added and the mixture was brought to reflux for 3 hours. After cooling, the excess sodium hydride was quenched with 2 mL of saturated aqueous ammonium chloride and 10 g of silica added to the mixture. Removal of the 20 solvent left the product adsorbed on silica gel. Elution with ethyl acetate: hexane (1:1) combined with flash chromatography gave 5.4 g of product. LC-MS indicated a purity WO 00/46203 PCT/US00/03042 - 39 of> 95%. 1 H NMR (CDCl-, shifts relative to the solvent peak at 7.24 ppm): 6 9.08 (d, IH) 6 8.65-8.8 (br d, 2H) 6 8.32 (dd, lH) 6 7.79 (d, 1H) 6 7.27 (in, IH) 6 6.81 (d, 1H) 6 5.44 (s, 2H). MW=231. M+H+=232. 5 (5) The product from above (4) was dissolved in 45 mL of methanol and 45 mL of water added. Iron powder (3.6 g) and acetic acid (1.72 mL) were added and the mixture brought to reflux for 3 hours. After cooling, the iron was precipitated by addition of 20% NaOH and the mixture filtered through Celite. Removal of the methanol by rotary evaporation was followed by extraction of the product into methylene chloride. Removal 10 of the solvent gave 1.08 g of product (5). LC-MS indicated a purity of >95%. 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 8.43 (d, IH) 8 8.25 (d, 1H) 6 7.58 (d, 1H) 6 7.42 (d, 1H) 6 7.02 (dd, 1H) 6 6.8 (dd, IH) 6 6.4 (d, 1H) 6 5.1 (s, 2H) 6 3.2 (br s, 2H). MW=201. M+H+=202. 15 (6) Compound (5) from above (1 mmol, 202 mg) was dissolved in 12 mL of methylene chloride. 0.4 g of polymer bound morpholine was added, followed by 1.3 mmol of 4 trifluoromethylbenzoyl chloride. The mixture was shaken at room temperature for 12 hours, at which time 0.5 g of polymer bound tris-2-aminoethylamine was added to scavenge excess acid chloride. After an additional 12 hours of shaking, the reaction 20 mixture was filtered through a polystyrene frit and chromatographed on silica using ethyl acetate as eluent. Yield: 340 mg. LC-MS indicated the product was > 95% pure. 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 8.86 (s, 1H) 8 8.55 (d, 1H) 6 8.25, (s, 1H) 6 8.07 (s, 1H) 6 7.99 (in, 3H) 6 7.81 (d, lH) 6 7.71( d, 2H) 6 7.27 (m, 1H) 8 6.80 (d, 1H) 6 5.39 (s, 2H). MW=373 25 D. Synthesis of A-ring Pyrimidyl GPCR Binding Compounds The general procedure is analogous to those described above, the only difference being that the starting material was 5-hydroxypyrimidine rather than 3-hydroxypyridine.
WO 00/46203 PCT/USOO/03042 - 40 Br OMeOHC N N aOM e/M eOH O~ K O H N CNON O 2 K7 K--,- - + N NO N N NN0 6 7 NaH N- N N0 N O NO 2 ON N Fe/AcOH
NCF
3
NH
2 10 9 5 (6) 5-bromo pyrimidine (8 g, 50.6 mmol) was dissolved in 100 mL of methanol containing 3.14 g of sodium methoxide. The mixture was sealed in a glass pressure vessel and heated to 130 C for 3 hours. After cooling, 10 g of silica was added to the mixture and the solvent removed by rotary evaporation. The product was eluted from the 10 silica and flash chromatographed using 1:1 hexanes/ethyl acetate. Yield: 2.5 g LC-MS showed the product was > 95% pure and had the correct M.W. (M+H+= 111.0) 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 5 8.82 (s, 1H) 5 8.40 (s, 2H) 6 3.85 (s, 3H). 15 (7) 5-methoxy pyrimidine (4.0 g, 36.4 mmol) was dissolved in 30 mL of dry ethylene glycol. Powdered KOH (10 g) was added and the mixture refluxed under argon for 3 hours. Excess ethylene glycol was removed by rotary evaporation at 0.5 mm Hg/130 C. The product was extracted from the residue using several portions of boiling dioxane. Removal of the dioxane gave a viscous solution which, upon cooling, deposited white 20 needles of 5-hydroxypyrimidine. LC-MS showed the product to be > 95% pure and of the expected M.W. (M+H+=97). 1 H NMR (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm): 6 8.51 (s, IH) 6 8.20 (s, 2H).
WO 00/46203 PCT/US0O/03042 -41 (8) 5-hydroxy pyrimidine (0.35 g, 3.64 mmol) was dissolved in 15 mL of dry DMF. Sodium hydride (0.14 g, 1.6 eq) was added and the mixture stirred for /2 hour at 0 C. 2 chloro-5-nitro pyridine ((0.867 g, 1.5 eq) was added and the mixture stirred at room 5 temperature for 12 hours. The reaction mixture was partitioned between ethyl acetate and sodium bicarbonate solution and the organic layer separated and mixed with 10 g of silica gel. Removal of the solvent left the product adsorbed on silica. The product was eluted onto a flash column with ethyl acetate and fractions containing product combined and rotovapped to dryness. Yield: 0.7 g. LC-MS showed the product to be > 95% pure 10 but the expected parent ion was not observed. NMR was consistent with the expected product: 6 9.19 (s, I H) 6 9.02 (s, 1H) 6 8.89 (br s, 2H) 6 8.58 (dd, 1H) 6 7.25 (d, 1H). (9) Nitropyrimidyl-pyridine (8) was dissolved in 40 mL of 50% water/methanol containing 0.77 g of acetic acid. 1.54 g of powdered iron was added and the mixture 15 brought to reflux for 3 hours. The iron oxides were precipitated by addition of 2 mL of 20% NaOH . Filtration of the reaction mixture through Celite was followed by removal of the methanol by rotary evaporation. The product was extracted into methylene chloride and the solvent removed by rotary evaporation. Yield: 0.37 g. LC-MS showed the product to be > 90% pure and of the expected M.W. (M+H+=189). IH NMR 20 (CDCl 3 , shifts relative to the solvent peak at 7.24 ppm) 6 8.98 (s, 1H) 6 8.60 (s, 2H) 6 7.60 (d, 1H) 6 7.18 (dd, 1H) 6 6.80 (dd, 1H) 6 3.60 (br s, 2H). (10) pyrimidyl-pyridine (9) (189 mg, 1 mmol) was dissolved in 12 mL of methylene chloride along with 0.4 g of polymer bound morpholine. 4-trifluoromethylbenzoyl 25 chloride (270 mg, 1.3 mmol) was added and the mixture shaken at room temperature for 12 hours. Polymer bound tris-2-aminoethylamine (0.5 g) was added and the mixture shaken for an additional 12 hours. Filtration of the reaction mixture followed by flash chromatography using ethyl acetate as eluent gave 230 mg of the expected product. LC MS showed a purity of > 95% and the expected M.W. (M+H+=361). 1 H NMR (CDCl 3
,
WO 00/46203 PCT/USO0/03042 - 42 shifts relative to the solvent peak at 7.24 ppm): 6 9.01 (s, 1H) 6 8.68 (in, 3H) 6 8.35 (dd, 1H) 6 8.22 (d, 1H) 6 8.01 (d. 2H) 6 7.75 (d, 1H) 6 7.20 (d, IH). 5 Example 2: Identification of GPCR Binding Compounds which Interact with a GPCR Using a Time Resolved Fluorescence (TRF) Assay High-Throughput Receptor Binding Screening for Antagonists of CCR10 10 A. Tissue Culture and Production of CCR1O Cells CCR1O cells are stable recombinant K293 cells overexpressing the CCR1O receptor. The cells are routinely cultured and passaged in a growth medium composed of DMEM base medium: 10% fetal bovine serum (FBS), IX Glutamine, and 0.4 mg/ml 15 G418. 1% Pen/Strep is also included in the media when the cells are seeded into the plates on Day 1. B. Labeling of rhMCP-1 with a Europium Chelate 20 rhMCP-1 (carrier free) was supplied as a frozen stock solution in PBS at a concentration of 0.65 mg/ml. 250 pg (385 jal) was buffer exchanged on a 2.8 ml column of Sephardex G-25 (fine), equilibrated with 10mM Borax, pH 9. Fractions (250 pil) were collected and 2 pl aliquots were analyzed using a Bradford protein assay. Four fractions eluting immediately after the void volume contained the bulk of the protein and were 25 pooled (approx. 1 ml). 1 mg of Eu-labeling reagent (1.5 tmol) was dissolved in 200 ptl of water and 100 tl of this solution was added to the protein pool. The final relative concentration ratios of Eu:MCP-1 was about 26:1. The ratio of Eu:NH 2 was about 3:1. The mixture was incubated for 20 hours at 4'C and then desalted on a 15 mL column of Sephardex G-25 (fine) equilibrated with 20 mM HEPES (hemi-sodium salt), 30 pH 7.5, and 0.9% NaCl. 0.5 mL fractions were collected and I ptl aliquots were added to WO 00/46203 PCT/USOO/03042 - 43 100 pl of enhancement solution and counted on the VICTOR. The five fractions that were eluted immediately after the void volume showed a peak of Eu containing material and were pooled. 60 pl of the pool was set aside for protein assay. 36 pl of 0.1% BSA (heavy metal free; Wallac) was added as a stabilizer to the remainder. The material was 5 then stored at 4'C. The total volume was about 2.5 ml. The MCP-1 concentration was determined by a BCA protein assay with BSA as a standard to be 8.5 jaM (74 pg/ml). The Eu concentration of the material was measured on the VICTOR with a standard curve prepared from the Eu standard solution provided from the vendor, 10 Wallac. The Eu concentration was 12 p.M. The calculated stoichiometry was 1.4 Eu chelate per MCP-1 molecule. C. Preparation of Compounds for Dispensation onto Cells 15 The compounds were provided as a dried film on a polystyrene "Master Plate" containing 1 pig of compound per well. The compounds were dispensed into cell plates by preparing a 3X stock solution in binding buffer from the dried films. The cell plates were prepared according to the following method. 20 Prior to the day of dispensation of compounds onto cells, the "Master Plates" were allowed to reach room temperature. 50 pl each of 1X Binding Buffer (0.125% BSA in deionized water) and MCP-1 stock solution (40nM and 600 nM in IX Binding Buffer) was added to each well. The plates were subsequently stored at 40 C overnight. 25 D. Time-resolved fluorescence (TRF) assay Day ] Each 96-well tissue culture plate (poly-D-lysine coated) was seeded with 40,000 cells in complete media containing 0.4 mg/ml G418 (200 pl per well). The plates were 30 incubated at 370 C and 6% CO 2 and left overnight.
WO 00/46203 PCT/USOO/03042 -44 Day 2 The cells were approximately 90% confluent in the wells by Day 2. The cells were washed twice with Binding Buffer (no BSA) using 200 pl per wash. 40 pl of 5 Binding Buffer was left behind following the second wash. To this final 40 p1l. 100 Pl of Binding Buffer was then added. Excess Binding Buffer from each well was removed leaving about 15 pl in each well. 15 p1 of compound solution was dispensed into each well followed by 15 pl of 30 nM Eu-MCPI working solution. The plates were then incubated at room temperature for 3 hours. 10 The plates were then washed twice with 150 pl of Wash Buffer (250 mM Hepes, pH 7.4; 1mM CaCl 2 ; 1. 15 M NaCl in deionized water). 15 Vt1 remained in each well following the washes. Then, the plates were washed ten times with Wash Buffer using 200 pl per wash. After the final wash, about 40 pl of Wash Buffer was left behind. To this residual Wash Buffer, 100 pl of Wash Buffer was added. The excess Wash Buffer 15 was then removed, leaving about 15 pl behind. 100 pl of Enhancement Solution was added to each well. Time-resolved fluorescence was read by the Wallac/Victor Plate Reader 60 minutes later. Time-resolved fluoremetry measurements were taken from 50 psec to 400 psec. 20 E. Method of Identification of GPCR Interacting Compounds Controls were included on each 96-well assay plate. The controls were (1) wells with no compounds and no unlabeled MCP 1, (2) wells with no compounds and unlabeled MCP1 (13.3 nM) corresponding to the IC 50 , and (3) wells with no compounds and unlabeled MCP1 (200 nM) corresponding to complete inhibition. Each control was 25 set up in duplicates. Hits were defined by compounds that reduce binding of Eu-MCP I below the level of the 50% inhibition control containing unlabeled MCPI at the IC 50 concentration. Percent inhibition was expressed by the following formula: % inhibition = [1 - (test well cps - background cps) (no inhibition control cps 30 background cps)] x 100%; WO 00/46203 PCT/USOO/03042 - 45 where background is defined as cps from control set #3 (wells with complete inhibition) and the no inhibition control is defined as cps from control set #1 (wells with no compounds or unlabeled MCP 1). Hits showing greater than 50% inhibition were retested using other assay 5 methods. The results of this assay are summarized in the Tables below. In Table 1, *** represents IC50's less than 5 pM, ** represents IC 50 's of between 5 and 15 pM, * represents IC 50 's greater than 15 pM. In Table 2, *** represents very high binding affinity. ** represents high binding 10 affinity, and * represents some binding affinity for the CCR10 receptor.
WO 00/46203 PCT/USOO/03042 - 46 Table 1: Time-resolved fluorescence (TRF) assay Results for Very High Binding Affinity Compounds Compound IC 50 Rating A E F CU Cw CV DG DO EO EQ B ** C ** G ** T ** U ** w ** DR ** DS ** DP ** H * I* Y * AB * DH * WO 00/46203 PCT/US00/03042 - 47 Table 2: Time Resolved Fluorescence (TRF) Assay Results 0 I -al 0 N RN N )-a R Compound # R, R, Affinity A Me
CF
3 B H
CF
3 C H Cl E Me
OCF
3 F H
OCF
3 G Me t-Bu H H t-Bu I Me
CF(CF
3
)
2 J H COH(CF 3
)
2 ** K Me COH(CF 3
)
2 ** L Me NO 2 ** M H NO 2 ** N H (CF 2 )2CF 3 ** 0 H Me * P H N(Me) 3 C * Q Me N(Me) 3 C1 * R H CN * CV Me I CW Me Br CY CONH 2
CF
3 * DG Cl
CF
3 DH OMe
CF
3 DO Me Cl WO 00/46203 PCT/USOO/03042 -48 DP Me CHCH, DQ Me H ** DR Me
CH
2
CH
3 DS Me
CH(CH
3
)
2 * DT Me CC1 3 ** DX Me Me ** EA Br CF 3 ** EO Me
S-CF
3 EQ Me
CF
2
CF
3 zI L - NN
R
2 N R R6 Comp.# Z [ R 1
R
2
L
1
R
6 Affinity S N H H 0 CF 3 ** T CH H Me 0 CF 3 U CH H Me 0 OCF 3 V CH H Me 0 COH(CF 3 )2 ** W CH H H CH 2 0 CF 3 X CH H H CH 2 0 OCF 3 ** CX N H H 0 OCF 3 ** CZ N H H CH 2 N(CO CF3 * (p-CF 3 C 6
H-
4 )) DA N H H CH 2 NH CF 3 * EB CH I Me 0 CF 3 * ED CH H H NH CF, ** EJ CH H CH,-Ph 0 CF 3
*
WO 00/46203 PCT/USOO/03042 - 49 R3 OR L2
R
8 Comp. Z 1
R
1
R
3 J Z 3
L
2
R
6 ' R 7
R
8 Afni Y CH H H N NHCO Cl H H Z N H H N NHCO Cl H Cl * CU N Me H N NHCO CF 3 F H AA N Me H N NHCO Cl H Cl * AB N H H N NHCO Cl Cl H AC N H H N NHCH, CF3 H H * AD N H H N NHCIH2 Cl H H * AE CH H H N NHCO CF3 H H ** AF CH H H CH NHCO Cl H H * AG CH H H CH NHCO CF 3 H H * AH N H H N CONH CF 3 H H * Al N H H N NHSO 2 Me H H * AJ N H Cl N NHCH H H H * (COOMe) NHCO AK N H H N NHCONH H H H * AL N H H N NHCONH Cl H H * AM N H H N NHSO 2 F Cl H * AN N H Cl N NHSO 2 H Cl H * AO N H H N (NHCO) 2 H H H * AP N H H N NHSO 2 Cl H H * AQ N H H N NHSO 2 Cl Cl H * AR N H H N NHCO O(CH-,) H H * Me AS N H H N NHCO H H H * CH,O AT N H H N NHCO H CN H * WO 00/46203 PCT/USO0/03042 -50 AU CH H H CH NHCO CF 3 H H * AV CH H H CH NHCO Ph H H * AW CH H H CH NHCO O(CH 2
)
5 H H * Me AX CH H H CH NHCO H H H * CH0 AY CH H H CH NHCO H CN H * DC CH H H CH NHCO t-Bu H H * DD CH H H CH NHCO CN H H * DE N H H N N(Me)CO CF, H H * DI N H COOMe N NHCO CF 3 H H * DJ N H COOEt N NHCO CF 3 H H * DV N Me H N NHCO H H H * CH) DY N H CO-(N- N NHCO CF 3 H H * piprazinyl) EF N H H N CH2NII CF 3 H H * Co WO 00/46203 PCT/USO0/03042 - 51 R3 OR4 N N R L 2 -E Comp R, R3 R 4 L2 E Affinity BA H Cl H NHCOMe * BB H H Me NHCO p-CF 3
-C
6
H
4 ** BC H H Me NHCO p-OCF 3
-C
6
H
4 ** BD H H H NHSO 2 4-CF 3 - * cyclohex[ 1 ]enyl BE H Cl H NHCO(CH 2
)
2 COOH - * BF H H H NO, * BG H H H NHCH 2 CH(CN)COOEt - * BH H Cl H NCHN(Me) 2 BI H Cl H NHCH 2 CH(CN)COOEt - * BJ H Cl H NHSO 2 Me - * BK H Cl H NHCHNOH - * BL H Cl H 1-pyrolyl - * BM H Cl H NHCOEt - * BN H Cl H NHCO- * transCH=CHCOOH BO H Cl H NHCONHCO m-Cl-C 6
H
4 * BP H Cl H NHCONHCO 2,6 * dimethoxyphenyl BQ H H H NHCO 3-pyridyl * BR H H H NHCO 3-phenyl phenyl * BS H H H NHCO 3-napthyl * BT H H H NHCO 3,5-(CF 3 )2-C 6
H
3 * BU H H H NHCO cyclohexyl * BV H Cl H NHCO m-Cl-C 6
H
4
*
WO 00/46203 PCTIUSOO/03042 -52 DN H H H NHCO - t-Bu \ / DU Me H H NHCOCH, / ** DW Me H H NHCO * \/ DZ H H H NHCO CF, EL Me H H NHCO/ EM Me H H NHCO * EN Me H H NHCO * ER Me 0 H NHCO * -~ s6 WO 00/46203 PCT/USOO/03042 -53 0 R3 L 2 -E Comp R 3
L
2 E Affinity BW H NHCO 2-pyridyl * BX H NHCO 3-napthyl * BY H NHCO 3,5-(CF 3
)
2
-C
6
H
3 BZ H NHCO cyclohexyl * CA CF 3 OCO p-tBu-C 6
H
4 * CB CF 3 OCO 3-pyridyl * CC CF 3 OCO (p-C 6
H
5
)-C
6
H
4 * CD CF 3 OCO 3-napthyl * CE CF 3 OCO p-(O(CH 2
)
5
CH
3
)-C
6
H
4 * CF CF 3
OCOCH
2 0 phenyl * CG CF 3 OCO m-cyanophenyl * CH CF 3 OCO p-cyanophenyl * CI CF 3 OCO 3,5-(CF 3
)
2
-C
6
H
3 CJ CF 3 OCO cyclohexyl * WO 00/46203 PCT/USOO/03042 -54 A N N 0 N N H Compound A E Affinity CK Me p-tBu-phenyl * CL Me 3-pyridyl * CM Me (p-C 6
H
5
)-C
6
H
4 * CN Me 3-napthyl * CO Me p-(O(CH 2
)
5 *
CH
3
)-C
6
H
4 CP Me
-CH
2 -0-C 6
H
5 * CQ Me m-CN-C 6
H
4 * CR Me p-CN-C 6
H
4 * CS Me 3,5-(CF 3
)
2
-C
6
H
3 * CT Me cyclohexyl * DF ' p-CF 3 -phenyl * S N DM 0 p-CF 3 -phenyl * EG p-CF 3 -phenyl ** WO 00/46203 PCT/USOO/03042 -55 NXO N a 0 a N N, H
R
6 Compound R, R6, Affinity DB H CF3 * DL Me
O-CF
3 * 5
L
1 Z 0 L Z 1 R, N H
CF
3 Compound Z, Z 2 R, L, Z 3
Z
4 Affinity DK CH N H S N CH * EC CH CCJ H CH 2 N N * EH N CH Me 0 N CMe ** El N CH H 0 N CMe * EK CH COMe H CF N N * N EP CH cOme H NH N N * WO 00/46203 PCT/USOO/03042 - 56 Example 3: Direct Binding Assay (DB Assay) for Identification of GPCR Binding Compounds CCR10 Manual Binding Assay Method 5 CCRI0 receptors were expressed in stably transfected K293 cells, and the cells were maintained in DMEM base medium, 10% FBS, IX glutamine, and 0.4 mg/ml G418, and in the incubator set at 37 0 C, 6.0 % CO2 and 90% relative humidity. The day before the experiment, the cells were trypsinized and 200 pl of the cell suspension 10 (150,000 cells/ml) was deposited into 96-well Biocoat plates (poly-D-lysine-coated). The binding assay was performed 24 hours later. 8-point dose response curves were generated as follows: compounds to be tested were dissolved in DMSO at 10 mg/ml concentration and diluted to 100 pg/ml into n butanol. 0.75, 1.5, 3, 6, 12, 18, 24, and 30 pl of the compounds were dispensed into 8 15 wells of a 96-well Costar plate. In the case of more potent compounds, further dilution was applied to generate 10 pg/ml compound solutions in n-butanol, and the solutions were used to make the compound plates by dispensing 1, 2, 4, 8, 10, 20 pl of the diluted solutions, and 4 and 8 pl of the 100 pg/ml solutions, into 8 wells of the plate. The compound plates were placed in the hood overnight to evaporate the butanol, leaving 20 dried films of the compounds. On the day of the binding assay, 50 p1 of filtered binding buffer (25 mM HEPES, pH 7.4, 75 pM EDTA, 11.5 mM KCl, 115 mM NaCl, 6 mM MgSO4, and 1.8 mM CaCl 2 ) was added into each well, and the plates were stored at 4 oC for about two hours. Before performing the binding assay, the compounds were thoroughly resuspended in the binding buffer. 25 The cells were washed three times with the binding buffer by adding and then decanting the medium or binding buffer and drying the plates on paper towels. Then the buffer was added slowly to the side wall of the wells to avoid disturbing the cells. After the third wash, the plates were dried over paper towels to leave about 5 p1 of the binding buffer in each well. An additional 10 pl of binding buffer was added to each well. 15 p1 30 of the compound solutions, and then 15 pl of the Eu 3 +-labeled MCP-1 ligand solution WO 00/46203 PCT/USOO/03042 - 57 (15 nM) in binding buffer with 0.1% BSA was added. The binding reaction mixtures were maintained at RT for three hours. After three-hour of incubation, 150 pl of the wash buffer (25 mM HEPES, pH 7.4, 0.1 mM CaCl 2 and 115 mM NaCl) was added into each well and the solution was 5 decanted and the plates were dried over paper towels. Subsequently, three sets of three washes were performed. Fresh wash buffer was used at each step. and pipette tips were also changed to avoid cross-contamination of the Eu 3 +-labeled ligand. The buffer solution of the final wash was decanted and the plate thoroughly dried on a paper towel. For each set, 100 pl of the enhancement buffer (Wallac) was added, and incubated with 10 the cells at RT for one hour. Time-resolved fluorescence (excitation wavelength 320 nM, emission 615 nM) was measured on the Wallac Victor fluorescence reader. Inhibition of MCP-l binding was determined according to the following formula: % inhibition = [I - (test well cps - background cps) ( (no inhibition control cps background cps)] (100%) 15 Figures 1, 2, and 3 depict the binding curves for Compounds A, CU and CV, respectively. Example 4: Cell Based Inflammatory Recruitment Assay (CBIR Assay) 20 THP- 1 Cell Motility Assays A THP-1 cell line was used that expresses both CCR-10 and CCR-2. The cells were placed in the top half of a chamber (Transwell plate, 24-well, 5 [M pore size, purchased from Costar) separated in the middle by a membrane. A gradient of chemokine (MCP-1 25 or MCP-3, purchased from R&D) was established which, in the absence of inhibitors, leads to migration of cells across the membrane. Quantitation of cell migration was done using a FACScan machine. The % migration was calculated as the number of migrated cells divided by the number of input cells. As shown in Figures 4 and 5, compounds B and C blocked chemokine induced migration of cells. 30 WO 00/46203 PCTUSOO/03042 - 58 Example 5: Modulation of Recruitment of Inflammatory Cell Types using GPCR Binding Compounds B and C in a Murine Inflammatory Recruitment Assay (MIR Assay) 5 Mouse Peritoneal Infiltration Studies CCR-10 and its ligands MCP-1, MCP-3 (MCP-5 in mice) have been demonstrated to mediate the recruitment of eosinophils and a variety of other leukocytes to tissue where it is expressed. Antibodies against CCR- 10 have been shown to block the effects of these ligands. This example demonstrates that compounds B and C are capable of 10 blocking MCP-5 induced peritoneal eosinophil recruitment. Mice and in vivo procedures: 8-1 0-wk-old C57BL/6J mice were purchased from the Jackson laboratory (Bar Harbor, ME) and kept in Millennium Pharmaceuticals Inc. Specific Pathogen Free 15 mouse facility. Peritoneal recruitment assays in vivo with MCP-5 or mMCP-1 (mJE) protein were performed after injection of 1mg/mouse i.p. of either MCP-5 or mMCP-1. Two hours after chemokine injection, peritoneal lavage was performed and leukocytes from this organ were collected and enumerated. In one series of blocking experiments, mice 20 were injected i.v. either with 50 nmol/Kg (7 mM/mouse) or 100 nmol/Kg (15 mM/mouse) of compound B or C 30 min before MCP-5 or mMCP-l administration. Immunohistochemical phenotyping and quantitation of leukocytes. Total peritoneal cell counts were performed and aliquots (5x105 cells/slide) were 25 pelleted onto glass slides by cytocentrifugation. To determine the number of eosinophils and mononuclear cells, slides were stained with Wright-Giemsa (Fisher Diagnostics, Pittsburgh, PA). T-lymphocytes, B-lymphocytes and mononuclear phagocytes were identified by Thy 1.2 (53-2.1) (PharMingen, San Diego, CA), IgM (11/41) (PharMingen, San Diego, CA) and Moma-2 (Biosource Int. Camarillo, CA) staining. Percentage of 30 eosinophils, lymphocytes, neutrophils and macrophages was determined by counting their number in eight high power fields (40x magnification; total area 0.5 mm2) per area WO 00/46203 PCTIUSOO/03042 - 59 randomly selected and dividing this number by the total number of cells per high power field. To obtain the absolute number of each leukocyte subtype in the lavage, these percentages were multiplied by the total number of cells recovered from the peritoneal fluid. 5 As shown in Figure 6, mice pretreated with compounds B and C prior to MCP-5 challenge showed significantly reduced levels of eosinophil recruitment than untreated mice. Control experiments with eotaxin demonstrate that the compounds are acting through CCR- 10 rather than by inhibition of cytoskeletal function. 10 EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. 15

Claims (97)

1. A G-protein coupled heptahelical receptor binding compound wherein said compound is of the formula: 5 J-M wherein J is an aromatic moiety; and M is a G-protein coupled heptahelical receptor pocket interacting moiety. 10
2. The G-protein coupled heptahelical receptor binding compound of claim 1, wherein said compound interacts with a p-chemokine receptor.
3. The G-protein coupled heptahelical receptor binding compound of claim 2, wherein said compound interacts with a chemokine receptor selected from the group 15 consisting of CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, and CCR1O.
4. The G-protein coupled heptahelical receptor binding compound of claim 1, wherein said compound modulates recruitment of at least one inflammatory cell type upon administration to a subject. 20
5. The G-protein coupled heptahelical receptor binding compound of claim 4, wherein said compound modulates leukocyte recruitment.
6. The G-protein coupled heptahelical receptor binding compound of claim 5, 25 wherein said compound modulates eosinophil recruitment.
7. The G-protein coupled heptahelical receptor binding compound of claim 1, wherein said compound binds to a G-protein coupled heptahelical receptor with an IC 5 0 of about 10 pM or less. 30 WO 00/46203 PCT/USOO/03042 - 61
8. The G-protein coupled heptahelical receptor binding compound of claim 7, wherein said compound binds to a G-protein coupled heptahelical receptor with an IC 5 0 of about I VtM or less. 5
9. The G-protein coupled heptahelical receptor binding compound of claim 8, wherein said compound binds to a G-protein coupled heptahelical receptor with an IC 5 0 of about 50 nM or less.
10. The G-protein coupled heptahelical receptor binding compound of claim 7, 10 wherein said G-protein coupled heptahelical receptor is a p-chemokine receptor.
11. The G-protein coupled heptahelical receptor binding compound of claim 1, wherein said compound is an antagonist of a G-protein coupled heptahelical receptor. 15
12. The G-protein coupled heptahelical receptor binding compound of claim 11, wherein said antagonist is an antagonist of a p-chemokine receptor.
13. The G-protein coupled heptahelical receptor binding compound of claim 1, wherein said compound comprises at least one pyridyl moiety. 20
14. The G-protein coupled heptahelical receptor binding compound of claim 13, wherein said compound comprises two pyridyl moieties.
15. The G-protein coupled heptahelical receptor binding compound of claim 1, 25 wherein said compound comprises at least one pyrimidyl moiety.
16. The G-protein coupled heptahelical receptor binding compound of claim 15, wherein said compound comprises two pyrimidyl moieties. WO 00/46203 PCT/USO0/03042 - 62
17. The G-protein coupled heptahelical receptor binding compound of claim 1, wherein said compound further comprises an amide bridging moiety.
18. The G-protein coupled heptahelical receptor binding compound of claim 1, 5 wherein said compound further comprises an ether bridging moiety.
19. The G-protein coupled heptahelical receptor binding compound of claim 1, wherein said G-protein coupled heptahelical receptor pocket interacting moiety is a para substituted aryl moiety. 10 WO 00/46203 PCT/USOO/03042 - 63
20. A compound represented by the formula: A-L 1 -B-L 2 -E wherein A is selected from the group consisting of branched and straight chain 5 alkyl, aryl., alkenyl, alkynyl, and heteroaryl moieties optionally substituted by NR'R", CN, NO 2 , F, Cl, Br, I, CF 3 , CCl 3 , CHF 2 , CHCl 2 , CONR'R", S(O)NR'R", CHO, OCF 3 , OCCl 3 , SCF 3 , SCCl 3 , COR', CO2R', and OR' and wherein R' and R" are each independently hydrogen, C I-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or optionally substituted aryl; 10 L 1 is a linker moiety selected from the group consisting of a bond, 0, S, CHOH, CHSH, CHNH 2 , CHNHR, CHNRR', NH, NR, (CH2)n, O(CH2)n, and (CH2)nO(CH2)n, an optionally substituted ring moiety of 4 to 7 atoms containing up to three heteroatoms, a chain of 1 to 5 atoms optionally substituted by CI-C 6 alkyl, halogens, wherein n is either 0, 1, 2, or 3, and R and R' are each independently substituted or unsubstituted C 15 C 6 branched or straight chain alkyl, C 1 -C 6 branched or straight chain alkenyl, aryl, C 4 C 7 ring, optionally substituted with up to three heteroatoms; B is an aromatic moiety containing from 0 to 3 heteroatoms and containing 5 to 7 members optionally substituted by NR'R", cyano, nitro, halogen, CF 3 , CHF 2 . CONR'R", S(O)NR'R", CHO, OCF 3 , SCF 3 , COR', CO 2 R', OR' where R' and R" are each 20 independently hydrogen, halogen, CI-C6 alkyl, optionally substituted aryl or optionally substituted aryl; L 2 is a second linking moiety selected from the group consisting of a bond, CH 2 C=O, NHC=O, OC=0, C=O, CH 2 NHC=O, CHOH, (CH2)n, 0, NH, O(CH2)n, NH(CH2)n, CH 2 CHOH and NRC=O; and 25 E is a G-protein coupled heptahelical receptor pocket interacting moiety. WO 00/46203 PCT/USO0/03042 - 64
21. The compound of claim 20, wherein A is represented by the following formula: R, R 3 wherein Z, and Z, each independently represent N or C; 5 RI, R2, and R 3 are independently selected from the group comprised of hydrogen, C 1 -C 6 branched or straight chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl.
22. The compound of claim 21, wherein Z, and Z 2 are both carbon. 10
23. The compound of claim 22, wherein A is represented by the following formula: R, N
24. The compound of claim 23, wherein R, is methyl. 15
25. The compound of claim 23, wherein A is represented by the following formula: R, N
26. The compound of claim 25, wherein R 1 is alkyl. 20
27. The compound of claim 26, wherein R 1 is methyl.
28. The compound of claim 25, wherein R, is a halogen. 25
29. The compound of claim 28, wherein R, is chlorine. WO 00/46203 PCT/USOO/03042 - 65
30. The compound of claim 25, wherein R, is alkoxy.
31. The compound of claim 20, wherein L, is selected from the group consisting of 5 S, NH, and CH,.
32. The compound of claim 20, wherein L, is 0.
33. The compound of claim 20, wherein B is represented by the following formula: Z- ~Z4 10 R4 R5 wherein Z 3 and Z 4 each independently represent N or C; R 4 and R 5 are independently selected from the group consisting of hydrogen, C 1 -C 6 branched or straight chain alkyl, alkoxy, thioalkyl, hydroxyalkyl, halo, 15 haloalkyl, amino, alkylamino, or carboxyl.
34. The compound of claim 33, wherein B is a substituted or unsubstituted pyridyl moiety. 20
35. The compound of claim 33, wherein B is a substituted or unsubstituted pyrimidyl moiety.
36. The compound of claim 33, wherein B is represented by the following formula: N 25 WO 00/46203 PCT/USOO/03042 - 66
37. The compound of claim 34, wherein B is represented by the following formula:
38. The compound of claim 20, wherein L, is NHC=O. 5
39. The compound of claim 20, wherein E is represented by the formula below: RQ wherein R 6 is an electron withdrawing moiety and the aryl ring is additionally optionally substituted with zero to four halogen atoms. 10
40. The compound of claim 39, wherein E is substituted with at least one fluorine atom.
41. The compound of claim 40, wherein E is substituted with two fluorine atoms. 15
42. The compound of claim 39, wherein R is alkyl, alkoxy. haloalkyl, nitro, halo, alkylamino, hydroxyalkyl, thioether or carboxyl.
43. The compound of claim 39, wherein E is represented by the formula below: 20
44. The compound of claim 43, wherein R 6 is unsubstituted alkyl.
45. The compound of claim 43, wherein R 6 is a substituted alkyl moiety. 25 WO 00/46203 PCT/US00/03042 - 67
46. The compound of claim 43, wherein R 6 is a fluorinated alkyl moiety.
47. The compound of claim 46, wherein R6 is perfluorinated. 5
48. The compound of claim 47, wherein R 6 is trifluoromethyl.
49. The compound of claim 20, wherein E is heterocyclic.
50. The compound of claim 49, wherein E is substituted or unsubstituted furanyl. 10
51. The compound of claim 20, wherein E is branched or straight chain, substituted or unsubstituted alkenyl.
52. The compound of claim 20, wherein E is branched or straight chain, substituted 15 or unsubstituted alkynyl.
53. A compound represented by the formula below: RL, Z' LL2 -E R8 R, R 3 R4 R, R6 Z,, Z 2 , Z 3 , and Z 4 are each independently N or C; 20 RI, R 2 ,R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are each independently hydrogen, C,-C 6 branched or straight chain alkyl, alkenyl, alkynyl, alkoxy, thioalkyl, hydroxyalkyl, halo, haloalkyl, amino, alkylamino, or carboxyl; L, is 0, S, NH, NR 7 , (CHR 7 )n, CO, CR 7 OH, O(CHR 7 )n, and (CHR 7 )nO(CHR 7 )n wherein n is either 1,2, or 3; 25 L, is a second linking moiety selected from the group consisting of a bond, CH 2 C=0, NHC=0, OC=O, C=0, CH 2 NHC=O, NHC=OCH 2 , CHOH, (CH 2 )n, 0, NH, O(CH 2 )m, NH(CH 2 )m, CH 2 CHOH and NRC=0, wherein m is 0, 1, 2, or 3.
54. The compound of claim 53, wherein Z, and Z, are both carbon. WO 00/46203 PCT/USOO/03042 - 68
55. The compound of claim 54. wherein R, is a halogen. alkyl or alkoxy, and R 2 and R 3 are both hydrogen. 5
56. The compound of claim 53, wherein L, is 0.
57. The compound of claim 53, wherein R 4 and R. are both hydrogen.
58. The compound of claim 53, wherein L, is NHC=O. 10
59. The compound of claim 53, wherein R 6 is a substituted or unsubstituted alkyl moiety.
60. The compound of claim 59, wherein R 6 is perhalogenated. 15
61. The compound of claim 60, wherein R 6 is trifluoromethyl or pentafluoroethyl.
62. The compound of claim 53, wherein R 7 and R, are each independently fluorine or hydrogen. 20
63. A compound of claim 53, represented by the structure below: R 7 R, L L 2 R, R 1 N NR NR R 2 R 3 R 4 6R 5
64. A compound of claim 63, represented by the structure below: R, N L N N~ Li L 2 . 25 WO 00/46203 PCT/USOO/03042 - 69
65. A compound of claim 64, wherein L, is 0.
66. A compound of claim 64, wherein L, is NHC=O. 5
67. A compound of claim 64, wherein R, is a halogenated alkvl. alkoxy, thioether. or a halogen.
68. A compound of claim 67, wherein R. is trifluoromethyl or pentafluoroethyl. 10
69. A compound of claim 64, wherein R 6 is a substituted alkoxy group.
70. A compound of claim 69, wherein R, is a trifluoromethoxy group. 15
71. A compound of claim 69, wherein R 6 is a trifluoromethyl thioether group.
72. A compound of claim 53, selected from the group consisting of: N O N CF OCF30 CN C N N H H HO CF HCF 3 0 N CH 3 N NN0 ( N) N 0 CF- WO 00/46203 PCT/UJSOO/03042 - 70 00 Na'H N N H3 N N' SOCF 3 0 C 0 -r0 N- a,' H N a,' H N H 3 C'N N' o -5 Ia 0' ~ CF 3 'a CF 3 0 0 H 3 C "( T N a N NC N f 0')0CF 3 tu ~ 0 -"H N-1 ( NaN' NCH N t'-a u " - CF(CF 3 ) 2 0 0 H 1 N N N C'NH 3 N' 0) COH(CF 3 ) 2 O' C0H(CF 3 ) 2 0 - N H H 3 C N N' N~ H " NI ' COHI(CF 3 ) 2CF WO 00/46203 PCTUSOO/03042 -71 N H NH O NH N N o 0, O N OCF 3 N cl a0 0 I, INal-r N CH 3 NN N H NO 2 CF 2 CF 2 CF 3 0~ ~ 0 H ON H CH N N N O O B0 SN C NOH 00 N CH 3 N02~ N N H 'N H N B 0 Ny~ 0 F N C N H X Me NII Cl N~ aoNF 00 0 N Me NH (N Me NH X 0 0 0 N Me NH -N Me NH Ka WO 00/46203 PCT/US0O/03042 - 72 0 0 NH~a CF N I CF 3
73. A method for treating a chemokine mediated disorder in a subject afflicted with said disorder, comprising administering an effective amount of a G-protein coupled heptahelical receptor 5 binding compound such that said disorder is treated, in that at least one symptom of the disorder is diminished or alleviated, wherein said compound is of the formula: J-M wherein J is an aromatic moiety; and 10 M is a G-protein coupled heptahelical receptor pocket interacting moiety.
74. The method of claim 73, wherein said disorder is treated through modulation of a P-chemokine receptor. 15
75. The method of claim 74, wherein said p-chemokine receptor is selected from the group consisting of CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, and CCRIO.
76. The method of claim 73, wherein said disorder is a neurological disorder. 20
77. The method of claim 76, wherein said neurological disorder is selected from the group consisting of Alzheimer's disease, dementias related to Alzheimer's disease, Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease. 25
78. The method of claim 76, wherein said neurological disorder is associated with inflammation. WO 00/46203 PCT/USOO/03042 - 73
79. The method of claim 76, wherein said neurological disorder is selected from the group consisting of stroke, traumatic injury to the brain, traumatic injury to the spinal cord, spinal crush, and central and peripheral nervous system trauma. 5
80. The method of claim 73, wherein said disorder is a immunological disorder.
81. The method of claim 80, wherein said immunological disorder is selected from the group consisting of immune thyroiditis, hyperthyroidism, type I diabetes mellitus, 10 insulin related diabetes, Addison's disease, autoimmune oophoritis, autoimmune orchiitis, autoimmune hemolytic anemia, paroxysmal cold hemoglobinuria, autoimmune thrombocytopenia, autoimmune neutropenia, pernicious anemia. autoimmune coagulopathies, myasthenia gravis, allergic encephalomyelitis, pemphigus and other bullous diseases, rheumatic carditis, Goodpasture's syndrome, postcardiotomy 15 syndrome, rheumatoid arthritis, keratitis, parotitis, polymositis, dermatomyositis, and scleroderma.
82. The method of claim 80, wherein said immunological disorder is AIDS. 20
83. The method of claim 80, wherein said immunological disorder is lupus.
84. The method of claim 80, wherein said immunological disorder is multiple sclerosis. 25
85. The method of claim 73, wherein said disorder is characterized by inflammation.
86. The method of claim 85, wherein said disorder is asthma.
87. The method of claim 73, wherein said disorder is characterized by unwanted 30 cellular proliferation. WO 00/46203 PCT/USOO/03042 - 74
88. The method of claim 87, wherein said disorder is cancer.
89. The method of claim 73, wherein said disorder is characterized by unwanted cellular migration. 5
90. The method of claim 73, wherein said disorder is characterized by abnormal cellular signal transduction.
91. The method of claim 73, wherein said disorder is characterized by abnormal 10 amounts of chemokine stimulated chemotaxis.
92. A pharmaceutical preparation comprised of an effective amount of a G-protein coupled heptahelical receptor binding compound and a pharmaceutically acceptable carrier, wherein said compound is of the formula: 15 J-M wherein J is an aromatic moiety; and M is a G-protein coupled heptahelical receptor pocket interacting moiety. 20
93. The pharmaceutical preparation of claim 92, wherein said effective amount is effective to treat a p-chemokine mediated disorder.
94. The pharmaceutical preparation of claim 93, wherein said effective amount is an effective amount to treat asthma. 25 WO 00/46203 PCT/US00/03042 - 75
95. A packaged G-protein coupled heptahelical receptor binding compound comprising a G-protein coupled heptahelical receptor binding compound packaged with instructions for using said compound for treating a p-chemokine mediated disorder, wherein said compound is of the formula: 5 J-M wherein J is an aromatic moiety; and M is a G-protein coupled heptahelical receptor pocket interacting moiety. 10
96. The packaged G-protein coupled heptahelical receptor binding compound of claim 95, wherein said p-chemokine mediated disorder is asthma.
97. A method of using a G-protein coupled heptahelical receptor binding compound, comprising using said G-protein coupled heptahelical receptor binding compound to 15 modulate the binding of a second compound to a G-protein coupled heptahelical receptor, wherein said G-protein coupled heptahelical receptor binding compound is of the formula: J-M wherein 20 J is an aromatic moiety; and M is a G-protein coupled heptahelical receptor pocket interacting moiety.
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