Patent Application
20080269280
The present invention provides compounds, pharmaceutical compositions containing one or more of those compounds or their pharmaceutically acceptable salts, which are effective in inhibiting the binding of various chemokines, such as MIP-1α, leukotactin, MPIF-1 and RANTES, to the CCR1 receptor. As antagonists or modulators for the CCR1 receptor, the compounds and compositions have utility in treating inflammatory and immune disorder conditions and diseases.
Human health depends on the body's ability to detect and destroy foreign pathogens that might otherwise take valuable resources from the individual and/or induce illness. The immune system, which comprises leukocytes (white blood cells (WBCs): T and B lymphocytes, monocytes, macrophages granulocytes, NK cell, mast cells, dendritic cell, and immune derived cells (for example, osteoclasts)), lymphoid tissues and lymphoid vessels, is the body's defense system. To combat infection, white blood cells circulate throughout the body to detect pathogens. Once a pathogen is detected, innate immune cells and cytotoxic T cells in particular are recruited to the infection site to destroy the pathogen. Chemokines act as molecular beacons for the recruitment and activation of immune cells, such as lymphocytes, monocytes and granulocytes, identifying sites where pathogens exist.
Despite the immune system's regulation of pathogens, certain inappropriate chemokine signaling can develop and has been attributed to triggering or sustaining inflammatory disorders, such as rheumatoid arthritis, multiple sclerosis and others. For example, in rheumatoid arthritis, unregulated chemokine accumulation in bone joints attracts and activates infiltrating macrophages and T-cells. The activities of these cells induce synovial cell proliferation that leads, at least in part, to inflammation and eventual bone and cartilage loss (see, DeVries, M. E., et al., Semin Immunol 11(2):95-104 (1999)). A hallmark of some demyelinating diseases such as multiple sclerosis is the chemokine-mediated monocyte/macrophage and T cell recruitment to the central nervous system (see, Kennedy, et al., J. Clin. Immunol. 19(5):273-279 (1999)). Chemokine recruitment of destructive WBCs to transplants has been implicated in their subsequent rejection. See, DeVries, M. E., et al., ibid. Because chemokines play pivotal roles in inflammation and lymphocyte development, the ability to specifically manipulate their activity has enormous impact on ameliorating and halting diseases that currently have no satisfactory treatment. In addition, transplant rejection may be minimized without the generalized and complicating effects of costly immunosuppressive pharmaceuticals.
Chemokines, a group of greater than 40 small peptides (7-10 kD), ligate receptors expressed primarily on WBCs or immune derived cells, and signal through G-protein-coupled signaling cascades to mediate their chemoattractant and chemostimulant fanctions. Receptors may bind more than one ligand; for example, the receptor CCR1 ligates RANTES (regulated on activation normal T cell expressed), MIP-1α (macrophage inflammatory protein), MPIF-1/CKβ8, and Leukotactin chemokines (among others with lesser affinities). To date, 24 chemokine receptors are known. The sheer number of chemokines, multiple ligand binding receptors, and different receptor profiles on immune cells allow for tightly controlled and specific immune responses. See, Rossi, et al., Ann. Rev. Immunol. 18(1):217-242 (2000). Chemokine activity can be controlled through the modulation of their corresponding receptors, treating related inflammatory and immunological diseases and enabling organ and tissue transplants.
The receptor CCR1 and its chemokine ligands, including, for example MIP-1α, MPIF-1/CKβ8, leukotactin and RANTES, represent significant therapeutic targets (see Saeki, et al., Current Pharmaceutical Design 9:1201-1208 (2003)) since they have been implicated in rheumatoid arthritis, transplant rejection (see, DeVries, M. E., et al., ibid.), and multiple sclerosis (see, Fischer, et al., J Neuroimmunol. 110(1-2):195-208 (2000); Izikson, et al., J. Exp. Med. 192(7):1075-1080 (2000); and Rottman, et al., Eur. J. Immunol. 30(8):2372-2377 (2000). In fact, function-blocking antibodies, modified chemokine receptor ligands and small organic compounds have been discovered, some of which have been successfully demonstrated to prevent or treat some chemokine-mediated diseases (reviewed in Rossi, et al., ibid.). Notably, in an experimental model of rheumatoid arthritis, disease development is diminished when a signaling-blocking, modified-RANTES ligand is administered (see Plater-Zyberk, et al., Immunol Lett. 57(1-3):117-120 (1997)). While function-blocking antibody and small peptide therapies are promising, they suffer from the perils of degradation, extremely short half-lives once administered, and prohibitive expense to develop and manufacture, characteristic of most proteins. Small organic compounds are preferable since they often have longer half lives in vivo, require fewer doses to be effective, can often be administered orally, and are consequently less expensive. Some organic antagonists of CCR1 have been previously described (see, Hesselgesser, et al., J. Biol. Chem. 273(25): 15687-15692 (1998); Ng, et al., J. Med. Chem. 42(22):4680-4694 (1999); Liang, et al., J. Biol. Chem. 275(25):19000-19008 (2000); and Liang, et al., Eur. J. Pharmacol. 389(1):41-49 (2000)). In view of the effectiveness demonstrated for treatment of disease in animal models (see, Liang, et al., J. Biol. Chem. 275(25):19000-19008 (2000)), the search has continued to identify additional compounds that can be used in the treatment of diseases mediated by CCR1 signaling.
The present invention relates to compounds having formula I:
or pharmaceutically acceptable salts and N-oxides thereof; wherein each R1 is independently selected from the group consisting of C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, —X1CO2Ra, —X1SO2Ra, —X1ORa, —X1CORa, —X1CONRaRb, —X1NRaRb, —XNRaCORb, —X1CONRaRb, —X1NRaS(O)2Rb and —X1S(O)2NRaRb, wherein X1 is a bond, C1-4 alkylene, C2-4 alkenylene, or C2-4 alkynylene, and each of Ra and Rb is independently selected from the group consisting of hydrogen, C1-8 alkyl, C1-8 haloalkyl and C3-6 cycloalkyl, wherein Ra and Rb when attached to the same nitrogen atom are optionally to form a 4- to 7-membered ring having from 0-2 heteroatoms as ring members selected from N, O or S; wherein any two R1 substituents attached to the same or different carbon atoms are optionally cyclized to form a 3- to 7-membered ring; and wherein the aliphatic portions of each R1 substituent is optionally substituted with from one to three members selected from the group consisting of —OH, —ORw, —OC(O)NHRw, —OC(O)N(Rw)2, —SH, —SRw, —S(O)Rw, —S(O)2Rw, —SO2NH2, —S(O)2NHRw, —S(O)2N(Rw)2, —NHS(O)2Rw, —NRwS(O)2Rw, —C(O)NH2, —C(O)NHRw, —C(O)N(Rw)2, —C(O)Rw, —NHC(O)Rw, —NRwC(O)Rw, —NHC(O)NH2, —NRwC(O)NH2, —NRwC(O)NHRw, —NHC(O)NHRw, —NRwC(O)N(Rw)2, —NHC(O)N(Rw)2, —CO2H, —CO2Rw, —NHCO2Rw, —NRwCO2Rw, —CN, —NO2, —NH2, —NHRw, —N(Rw)2, —NRwS(O)NH2 and —NRwS(O)2NHRw, wherein each Rw is independently an unsubstituted C1-6 alkyl; the subscript m is an integer from 0-4.
The substituent Ar is an aryl or a heteroaryl group, optionally substituted with from 1-4 R2 substituents, wherein each R2 is independently selected from the group consisting of halogen, ORc, —OC(O)Rc, —NRcRd, —SRc, —Re, —CN, —NO2, —CO2Rc, —CONRcRd, —C(O)Rc, —OC(O)NRcRd, —NRdC(O)Rc, —NRdC(O)2Re, —NRc—C(O)NRcRd, —NH—C(NH2)═NH, —NReC(NH2)═NH, —NH—C(NH2)═NRe, —NH—C(NHRe)═NH, —NReC(NHRe)═NH, —NReC(NH2)═NRe, —NH—C(NHRe)═NRe, —NH—C(NReRe)═NH, —S(O)Re, —S(O)2Re, —NRcS(O)2Re, —S(O)2NRcRd, —N3, —C(NORc)Rd, —C(NRcW)═NW, —N(W)C(Rc)═NW, —NRcC(S)NRcRd, —X2(NORc)Rd, —X2C(NRcW)═NW, —X2N(W)C(Rc)═NW, —X2NRcC(S)NRcRd, —X2ORc, —O—X2ORc, —X2OC(O)Rc, —X2NRcRd, —O—X2NRcRd, —X2SRc, —X2 CN, —X2NO2, —X2CO2Rc, —O—X2CO2Rc, —X2 CONRcRd, —O—X2CONRcRd, —X2C(O)Rc, —X2OC(O)NRcRd, —X2NRdC(O)Rc, —X2 NRdC(O)2Re, —X2NRcC(O)NRcRd, —X2NH—C(NH2)═NH, —X2NReC(NH2)═NH, —X2NH—C(NH2)═NRe, —X2 NH—C(NHRe)═NH, —X2S(O)Re, —X2S(O)2Re, —X2NRcS(O)2Re, —X2S(O)2NRcRd, —X2N3, —NRd—X2ORc, —NRd—X2 NRcRd, —NRdX2CO2Rc, and —NRd—X2CONRcRd, wherein W is selected from Rc, —CN, —CO2Re and —NO2, and wherein X2 is C1-4 alkylene, C2-4 alkenylene, C2-4 alkynylene or C1-4 heteroalkylene; and each of Rc and Rd is independently selected from hydrogen, C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, or optionally Rc and Rd when attached to the same nitrogen atom can be combined to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O or S; and each Re is independently selected from the group consisting of C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl, and aryloxy-C1-4 alkyl, and each of Rc, Rd and Re is optionally further substituted with from one to three members selected from the group consisting of —OH, —ORn, —OC(O)NHRn, —OC(O)N(Rn)2, —SH, —SRn, —S(O)Rn, —S(O)2Rn, —SO2NH2, —S(O)2NHRn, —S(O)2N(Rn)2, —NHS(O)2Rn, —NRnS(O)2Rn, —C(O)NH2, —C(O)NHRn, —C(O)N(Rn)2, —C(O)Rn, —NHC(O)Rn, —NRnC(O)Rn, —NHC(O)NH2, —NRnC(O)NH2, —NRnC(O)NHRn, —NHC(O)NHRn, —NRnC(O)N(Rn)2, —NHC(O)N(Rn)2, —CO2H, —CO2Rn, —NHCO2Rn, —NRnCO2Rn, —CN, —NO2, —NH2, —NHRn, —N(Rn)2, —NRnS(O)NH2 and —NRnS(O)2NHRn, wherein each Rn is independently an unsubstituted C1-6 alkyl;
The symbol B represents an aryl or a heteroaryl ring system having 1-4 heteroatoms as ring members selected from the group consisting of N, O and S; wherein B is substituted with 0 to 5 R3 substitutents, and at each occurrence, R3 is independently selected from the group consisting of halogen, —ORf, —OC(O)Rf, —NRfRg, —SRf, —Rh, —CN, —NO2, —CO2Rf, —CONRfRg, —C(O)Rf, —OC(O)NRfRg, —NRgC(O)Rf, —NRgC(O)2Rh, —NRf—C(O)NRfRg, —NH—C(NH2)═NH, —NRhC(NH2)═NH, —NH—C(NH2)═NRh, —NH—C(NHRh)═NH, —S(O)Rh, —S(O)2Rh, —NRfS(O)2Rh, —S(O)2NRfRg, —NRfS(O)NRfRg, —N3, —C(O)NRfS(O)Rh, —C(O)NRfS(O)2Rh, —P═O(ORf)(ORg)X3Rf, —X3OC(O)Rf, —X3NRfRg, —X3SRf, —X3CN, —X3NO2, —X3CO2Rf, —X1CONRfRg, —X3C(O)Rf, —X3OC(O)NRfRg, —X3NRgC(O)Rf, —X3NRgC(O)2Rh, —XfNRf—C(O)NRfRg, —X3NH—C(NH2)═NH, —X3NRhC(NH2)═NH, —X3NH—C(NH2)═NRh, —X3NH—C(NHRh)═NH, —X3S(O)Rh, —X3S(O)2Rh, —X3NRfS(O)2Rh, —X3S(O)2NRfRg, —X3C(O)NRfS(O)2Rh, —X3C(O)NRfS(O) Rh, —X3P═O(ORf)(ORg), —Y, —X3Y and —X3N3, wherein Y is a five to ten-membered aryl, heteroaryl or heterocycloalkyl ring, optionally substituted with from one to three substitutents selected from the group consisting of halogen, —ORf, —NRfRg, —Rh, —SRf, —CN, —NO2, —CO2Rf, —CONRfRg, —C(O)Rf, —NRgC(O)Rf, —S(O)Rh, —S(O)2Rh, —NRfS(O)2Rh, —S(O)2NRfRg, —C(O)NRfS(O)2Rh, —C(O)NRfS(O)Rh, —P═O(ORf)(ORg), —X3ORf, NRfRg, —X3NRfS(O)2Rh, —X3CO2Rf, —X3CONRfRg, —X3C(O)Rf, —X3NRgC(O)Rf, —X3S(O)Rh, —X3S(O)2Rh, —X3C(O)NRfS(O)2Rh, —X3C(O)NRfS(O)2Rh, —X3P═O(ORf)(ORg) and —X3S(O)2NRfRg, any two R3 substituents on adjacent atoms may be optionally combined to form a 5- to 6-membered ring optionally having 1-2 heteroatom ring members selected from N, O and S; each X3 is independently selected from the group consisting of C1-4 alkylene, C2-4 alkenylene, C2-4 alkynylene and C1-4 heteroalkylene; each Rf and Rg is independently selected from hydrogen, C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl and aryloxy-C1-4 alkyl; and each Rh is independently selected from the group consisting of C1 g alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl and aryloxy-C1-4 alkyl, wherein the aliphatic portions of X3, Rf, Rg and Rh are optionally further substituted with from one to three members selected from the group consisting of —OH, —ORo, —OC(O)NHRo, —OC(O)N(Ro)2, —SH, —SRo, —S(O)Ro, —S(O)2Ro, —SO2NH2, —S(O)2NHRo, —S(O)2N(Ro)2, —NHS(O)2Ro, —NRoS(O)2Ro, —C(O)NH2, —C(O)NHRo, —C(O)N(Ro)2, —C(O)Ro, —NHC(O)Ro, —NRoC(O)Ro, —NHC(O)NH2, —NRoC(O)NH2, —NRoC(O)NHRo, —NHC(O)NHRo, —NRoC(O)N(Ro)2, —NHC(O)N(Ro)2, —CO2H, —CO2Ro, —NHCO2Ro, —NRoCO2Ro, —CN, —NO2, —NH2, —NHRo, —N(Ro)2, —NRoS(O)NH2 and —NRoS(O)2NHRo, wherein Ro is unsubstituted C1-6 alkyl.
The symbol L1 is a linking group selected from the group consisting of C1-4 alkylene, C1-4 heteroalkylene, C2-4 alkenylene and —CH2N(Ru)X5—, optionally substituted with from 1 to 4 R4 groups, wherein R4 at each occurrence is independently selected from the group consisting of phenyl, —Rk, —X4ORi, —X4OC(O)Ri, —X4NRiRj, —X4SRi, —X4CN or —X4NO2, wherein X4 is selected from the group consisting of C1-4 alkylene, C2-4 heteroalkylene, C2-4 alkenylene and C2-4 alkynylene and each of Ri and Rj is independently selected from hydrogen, C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl, and aryloxy-C1-4 alkyl, and each Rk is independently selected from the group consisting of C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl, and aryloxy-C1-4 alkyl; each Ru is independently selected from the group consisting of hydrogen, C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl and heteroaryl, wherein Ru is optionally further substituted with from one to three members selected from the group consisting of —OH, —ORv, —OC(O)NHRv, —OC(O)N(Rv)2, —SH, —SRv, —S(O)Rv, —S(O)2Rv, —SO2NH2, —S(O)2NHRv, —S(O)2N(Rv)2, —NHS(O)2Rv, —NRvS(O)2Rv, —C(O)NH2, —C(O)NHRv, —C(O)N(Rv)2, —C(O)Rv, —NHC(O)Rv, —NRvC(O)Rv, —NHC(O)NH2, —NRvC(O)NH2, —NRvC(O)NHRv, —NHC(O)NHRv, —NRvC(O)N(Rv)2, —NHC(O)N(Rv)2, —CO2H, —CO2Rv, —NHCO2Rv, —NRvCO2Rv, —CN, —NO2, —NH2, —NHRv, —N(Rv)2, —NRvS(O)NH2 and —NRvS(O)2NHRv, wherein each Rv is independently an unsubstituted C1-6 alkyl or C1-6 haloalkyl; and X5 is selected from the group consisting of C1-2alkylene, —C(O)—, —S(O)— and —S(O)2—.
The symbol L2 is a bond or C1-3 alkylene, optionally substituted with from 1-4 R4.
In addition to the compounds provided herein, the present invention further provides pharmaceutical compositions containing one or more of these compounds, as well as methods for the use of these compounds in therapeutic methods, primarily to treat diseases associated with CCR1, CCR2 and/or CCR3 signalling activity.
The term “alkyl”, by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e. C1-8 means one to eight carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term “alkenyl” refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term “alkynyl” refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “cycloalkyl” refers to hydrocarbon rings having the indicated number of ring atoms (e.g., C3-6cycloalkyl) and being fully saturated or having no more than one double bond between ring vertices. “Cycloalkyl” is also meant to refer to bicyclic and polycyclic hydrocarbon rings such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term “heterocycloalkyl” refers to a cycloalkyl group that contain from one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized, the remaining ring atoms being C, where one or two C atoms may optionally be replaced by a carbonyl. The heterocycloalkyl may be a monocyclic, a bicyclic or a polycylic ring system. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, piperidiny, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrhydrothiophene, quinuclidine, and the like. A heterocycloalkyl group can be attached to the remainder of the molecule through a ring carbon or a heteroatom.
The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkane, having the number of carbon atoms indicated in the prefix, as exemplified by —CH2CH2CH2CH2—. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having four or fewer carbon atoms. Similarly, “alkenylene” and “alkynylene” refer to the unsaturated forms of “alkylene” having double or triple bonds, respectively.
The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, unsaturated or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, Si and S may be placed at any interior position of the heteroalkyl group. Examples include —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2, —S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —CH2—CH═N—OCH3, and —CH═CH—N(CH3)—CH3. Up to two heteroatoms may be consecutive, such as, for example, —CH2—NH—OCH3 and —CH2—O—Si(CH3)3.
The term “heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by —CH2—CH2—S—CH2CH2— and —CH2—S—CH2—CH2—NH—CH2—, —O—CH2—CH═CH—, —CH2—CH═C(H)CH2—O—CH2— and —S—CH2—C≡C—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
The terms “alkoxy,” “alkylamino”, “alkylthio” (or thioalkoxy) and alkylsilyl are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, a sulfur atom, or a silicon atom, respectively. Additionally, for dialkylamino groups, the alkyl portions can be the same or different and can also be combined to form a 3-7 membered ring with the nitrogen atom to which each is attached. Accordingly, a group represented as —NRaRb is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like. Similarly, for dialkylsilyl or trialkylsilyl groups, the alkyl portions can be the same or different and can also be combined to form a 3-7 membered ring with the silicon atom to which each is attached.
The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “C1-4 haloalkyl” is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
The term “aryl” means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon group which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalaziniyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and the like. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.
For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like).
The above terms (e.g. “aryl” and “heteroaryl”), in some embodiments, will include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. For brevity, the terms aryl and heteroaryl will refer to substituted or unsubstituted versions as provided below.
Substituents for the aryl and heteroaryl groups are varied and are generally selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO2, —CO2R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)2R′, —NR—C(O)NR″R′″, —NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH—C(NH2)═NR′, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NR′S(O)2R″, —N3, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″ and R′″ are independently selected from hydrogen, C1-8 alkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C1-4 alkyl, and unsubstituted aryloxy-C1-4 alkyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of from 1-4 carbon atoms.
Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)—(CH2)q—U—, wherein T and U are independently —NH—, —O—, —CH2— or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r—B—, wherein A and B are independently —CH2—, —O—, —NH—, —S—, —S(O)—, —S(O)2—, —S(O)2NR′— or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —(CH2), —X—(CH2)t—, where s and t are independently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)2—, or —S(O)2NR′—. The substituent R′ in —NR′— and —S(O)2NR′— is selected from hydrogen or unsubstituted C1-6 alkyl.
As used herein, the term “heteroatom” is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
As used herein, a wavy line, “”, that intersects a single, double or triple bond in any chemical structure depicted herein, represent the point attachment of the single, double, or triple bond to the remainder of the molecule.
“Protecting group” refers to a moiety, except alkyl groups, that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York, 1999, and Harrison and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8 (John Wiley and Sons, 1971-1996), which are incorporated herein by reference in their entirety. Representative hydroxy protecting groups include acyl groups, benzyl and trityl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. Representative amino protecting groups include, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (BOC), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC), and the like.
“Leaving group” has the meaning conventionally associated with it in synthetic organic chemistry, i.e., an atom or a group capable of being displaced by a nucleophile and includes halo (such as chloro, bromo, and iodo), alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g., acetoxy), arylcarbonyloxy, mesyloxy, tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g., 2,4-dinitrophenoxy), methoxy, N,O-dimethylhydroxylamino, and the like.
“Amino acid coupling reagent” refers to a reagent, such as HATU, etc., that will react with the carboxylic acid group of an amino acid to form an activated intermediate that can be used to condense with a wide variety of nucleophiles, for example, amines, alcohols and thiols, to produce other esters, thioesters or amides groups.
The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically-acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like. Salts derived from pharmaceutically-acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
The present invention derives from the discovery that compounds of formula I act as potent antagonists of the CCR1 receptor. The compounds have in vivo anti-inflammatory activity. Accordingly, the compounds provided herein are useful in pharmaceutical compositions, methods for the treatment of CCR1-mediated diseases, and as controls in assays for the identification of competitive CCR1 antagonists.
In one aspect, the present invention provides for compounds having formula I
or pharmaceutically acceptable salts and N-oxides thereof; wherein each R1 is independently selected from the group consisting of C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, —X1CO2Ra, —X1SO2Ra, —X1ORa, —X1CORa, —X1CONRaRb, —X1NRaRb, —X1NRaCORb, —X1CONRaRb, —X1NRaS(O)2Rb and —X1S(O)2NRaRb, wherein X1 is a bond, C1-4 alkylene, C2-4 alkenylene, or C2-4 alkynylene, and each of Ra and Rb is independently selected from the group consisting of hydrogen, C1-8 alkyl, C1-8 haloalkyl and C3-6 cycloalkyl, wherein Ra and Rb when attached to the same nitrogen atom are optionally to form a 4- to 7-membered ring having from 0-2 heteroatoms as ring members selected from N, O or S; wherein any two R1 substituents attached to the same or different carbon atoms are optionally cyclized to form a 3- to 7-membered ring; and wherein the aliphatic portions of each R1 substituent is optionally substituted with from one to three members selected from the group consisting of —OH, —ORw, —OC(O)NHRw, —OC(O)N(Rw)2, —SH, —SRw, —S(O)Rw, —S(O)2Rw, —SO2NH2, —S(O)2NHRw, —S(O)2N(Rw)2, —NHS(O)2Rw, —NRwS(O)2Rw, —C(O)NH2, —C(O)NHRw, —C(O)N(Rw)2, —C(O)Rw, —NHC(O)Rw, —NRwC(O)Rw, —NHC(O)NH2, —NRwC(O)NH2, —NRwC(O)NHRw, —NHC(O)NHRw, —NRwC(O)N(Rw)2, —NHC(O)N(Rw)2, —CO2H, —CO2Rw, —NHCO2Rw, —NRwCO2Rw, —CN, —NO2, —NH2, —NHRw, —N(Rw)2, —NRwS(O)NH2 and —NRwS(O)2NHRw, wherein each Rw is independently an unsubstituted C1-6 alkyl; the subscript m is an integer from 0-4; In certain embodiments, the subscripts m is an integer from 0-2.
The substituent Ar is an aryl or a heteroaryl group, optionally substituted with from 1-4 R2 substituents, wherein each R2 is independently selected from the group consisting of halogen, ORc, —OC(O)Rc, —NRcRd, —SRc, —Re, —CN, —NO2, —CO2Rc, —CONRcRd, —C(O)Rc, —OC(O)NRcRd, —NRdC(O)Rc, —NRdC(O)2Re, —NRc—C(O)NRcRd, —NH—C(NH2)═NH, —NReC(NH2)═NH, —NH—C(NH2)═NRe, —NH—C(NHRe)═NH, —NReC(NHRe)═NH, —NReC(NH2)═NRe, —NH—C(NHRe)═NRe, —NH—C(NReRe)═NH, —S(O)Re, —S(O)2Re, —NRcS(O)2Re, —S(O)2NRcRd, —N3, —C(NORc)Rd, —C(NRcW)═NW, —N(W)C(Rc)═NW, —NRcC(S)NRcRd, —X2(NORc)Rd, —X2C(NRcW)═NW, —X2N(W)C(Rc)═NW, —X2NRcC(S)NRcRd, —X2ORc, —O—X2ORc, —X2OC(O)Rc, —X2NRcRd, —O—X2NRcRd, —X2SRc, —X2 CN, —X2NO2, —X2CO2Rc, —O—X2CO2Rc, —X2 CONRCRd, —O—X2CONRCRd, —X2C(O)Rc, —X2OC(O)NRcRd, —X2NRdC(O)Rc, —X2 NRdC(O)2Re, —X2NRcC(O)NRcRd, —X2NH—C(NH2)═NH, —X2NReC(NH2)═NH, —X2NH—C(NH2)═NRe, —X2 NH—C(NHRe)═NH, —X2S(O)Re, —X2S(O)2Re, —X2NRcS(O)2Re, —X2S(O)2NRcRd, —X2N3, —NRd—X2ORc, —NRd—X2 NRcRd, —NRdX2CO2Rc, and —NRd—X2CONRCRd, wherein W is selected from Rc, —CN, —CO2Re and —NO2, and wherein X2 is C1-4 alkylene, C2-4 alkenylene, C2-4 alkynylene or C1-4 heteroalkylene; and each of Rc and Rd is independently selected from hydrogen, C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, or optionally Rc and Rd when attached to the same nitrogen atom can be combined to form a five or six-membered ring having from 0 to 2 additional heteroatoms as ring members selected from N, O or S; and each Re is independently selected from the group consisting of C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl, and aryloxy-C1-4 alkyl, and each of Rc, Rd and Re is optionally further substituted with from one to three members selected from the group consisting of —OH, —ORn, —OC(O)NHRn, —OC(O)N(Rn)2, —SH, —SRn, —S(O)Rn, —S(O)2Rn, —SO2NH2, —S(O)2NHRn, —S(O)2N(Rn)2, —NHS(O)2Rn, —NRnS(O)2Rn, —C(O)NH2, —C(O)NHRn, —C(O)N(Rn)2, —C(O)Rn, —NHC(O)Rn, —NRnC(O)Rn, —NHC(O)NH2, —NRnC(O)NH2, —NRnC(O)NHRn, —NHC(O)NHRn, —NRnC(O)N(Rn)2, —NHC(O)N(Rn)2, —CO2H, —CO2Rn, —NHCO2Rn, —NRnCO2Rn, —CN, —NO2, —NH2, —NHRn, —N(Rn)2, —NRnS(O)NH2 and —NRnS(O)2NHRn, wherein each Rn is independently an unsubstituted C1-6 alkyl.
The symbol B represents an aryl or a heteroaryl ring system having 1-4 heteroatoms as ring members selected from the group consisting of N, O and S; wherein B is substituted with 0 to 5 R3 substitutents, and at each occurrence, R3 is independently selected from the group consisting of halogen, —ORf, —OC(O)Rf, —NRfRg, —SRf, —Rh, —CN, —NO2, —CO2Rf, —CONRfRg, —C(O)Rf, —OC(O)NRfRg, —NRgC(O)Rf, —NRgC(O)2Rh, —NRf—C(O)NRfRg, —NH—C(NH2)═NH, —NRhC(NH2)═NH, —NH—C(NH2)═NRh, —NH—C(NHRh)═NH, —S(O)Rh, —S(O)2Rh, —NRfS(O)2Rh, —S(O)2NRfRg, —NRfS(O)NRfRg, —N3, —C(O)NRfS(O)Rh, —C(O)NRfS(O)2Rh, —P═O(ORf)(ORg)X3Rf, —X3OC(O)Rf, —X3NRfRg, —X3SRf, —X3CN, —X3NO2, —X3CO2Rf, —X1CONRfRg, —X3C(O)Rf, —X3OC(O)NRfRg, —X3NRgC(O)Rf, —X3NRgC(O)2Rh, —XfNRf—C(O)NRfRg, —X3NH—C(NH2)═NH, —X3NRhC(NH2)═NH, —X3NH—C(NH2)═NRh, —X3NH—C(NHRh)═NH, —X3S(O)Rh, —X3S(O)2Rh, —X3NRfS(O)2Rh, —X3S(O)2NRfRg, —X3C(O)NRfS(O)2Rh, —X3C(O)NRfS(O) Rh, —X3P═O(ORf)(ORg), —Y, —X3Y and —X3N3, wherein Y is a five to ten-membered aryl, heteroaryl or heterocycloalkyl ring, optionally substituted with from one to three substitutents selected from the group consisting of halogen, —ORf, —NRfRg, —Rh, —SRf, —CN, —NO2, —CO2Rf, —CONRfRg, —C(O)Rf, —NRgC(O)Rf, —S(O)Rh, —S(O)2Rh, —NRfS(O)2Rh, —S(O)2NRfRg, —C(O)NRfS(O)2Rh, —C(O)NRfS(O)Rh, —P═O(ORf)(ORg), —X3ORf, NRfRg, —X3NRfS(O)2Rh, —X3CO2Rf, —X3CONRfRg, —X3C(O)Rf, —X3NRgC(O)Rf, —X3S(O)Rh, —X3S(O)2Rh, —X3C(O)NRfS(O)2Rh, —X3C(O)NRfS(O)2Rh, —X3P═O(ORf)(ORg) and —X3S(O)2NRfRg, any two R3 substituents on adjacent atoms may be optionally combined to form a 5- to 6-membered ring optionally having 1-2 heteroatom ring members selected from N, O and S; each X3 is independently selected from the group consisting of C1-4 alkylene, C2-4 alkenylene, C2-4 alkynylene and C1-4 heteroalkylene; each Rf and Rg is independently selected from hydrogen, C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl and aryloxy-C1-4 alkyl; and each Rh is independently selected from the group consisting of C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl and aryloxy-C1-4 alkyl, wherein the aliphatic portions of X3, Rf, Rg and Rh are optionally further substituted with from one to three members selected from the group consisting of —OH, —ORo, —OC(O)NHRo, —OC(O)N(Ro)2, —SH, —SRo, —S(O)Ro, —S(O)2Ro, —SO2NH2, —S(O)2NHRo, —S(O)2N(Ro)2, —NHS(O)2Ro, —NRoS(O)2Ro, —C(O)NH2, —C(O)NHRo, —C(O)N(Ro)2, —C(O)Ro, —NHC(O)Ro, —NRoC(O)Ro, —NHC(O)NH2, —NRoC(O)NH2, —NRoC(O)NHRo, —NHC(O)NHRo, —NRoC(O)N(Ro)2, —NHC(O)N(Ro)2, —CO2H, —CO2Ro, —NHCO2Ro, —NRoCO2Ro, —CN, —NO2, —NH2, —NHRo, —N(Ro)2, —NRoS(O)NH2 and —NRoS(O)2NHRo, wherein Ro is unsubstituted C1-6 alkyl.
The symbol L1 is a linking group selected from the group consisting of C1-4 alkylene, C1-4 heteroalkylene, C2-4 alkenylene and —CH2N(Ru)X5—, optionally substituted with from 1 to 4 R4 groups, wherein R4 at each occurrence is independently selected from the group consisting of phenyl, —Rk, —X4ORi, —X4OC(O)Ri, —X4NRiRj, —X4SRi, —X4CN or —X4N2, wherein X4 is selected from the group consisting of C1-4 alkylene, C2-4 heteroalkylene, C2-4 alkenylene and C2-4 alkynylene and each of Ri and Rj is independently selected from hydrogen, C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl, and aryloxy-C1-4 alkyl, and each Rk is independently selected from the group consisting of C1-8 alkyl, C1-8 haloalkyl, C3-6 cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, aryl, heteroaryl, aryl-C1-4 alkyl, and aryloxy-C1-4 alkyl; each Ru is independently selected from the group consisting of hydrogen, C1-8 alkylene, C1-8 haloalkylene, C3-6 cycloalkylene, C2-8 alkenylene, C2-8 alkynylene, arylene and heteroarylene, wherein Ru is optionally further substituted with from one to three members selected from the group consisting of —OH, —ORv, —OC(O)NHRv, —OC(O)N(Rv)2, —SH, —SRv, —S(O)Rv, —S(O)2Rv, —SO2NH2, —S(O)2NHRv, —S(O)2N(Rv)2, —NHS(O)2Rv, —NRvS(O)2Rv, —C(O)NH2, —C(O)NHRv, —C(O)N(Rv)2, —C(O)Rv, —NHC(O)Rv, —NRvC(O)Rv, —NHC(O)NH2, —NRvC(O)NH2, —NRvC(O)NHRv, —NHC(O)NHRv, —NRvC(O)N(Rv)2, —NHC(O)N(Rv)2, —CO2H, —CO2Rv, —NHCO2Rv, —NRvCO2Rv, —CN, —NO2, —NH2, —NHRv, —N(Rv)2, —NRvS(O)NH2 and —NRvS(O)2NHRv, wherein each Rv is independently an unsubstituted C1-6 alkyl or C1-6 haloalkyl; and X5 is selected from the group consisting of C1-2alkylene, —C(O)—, —S(O)— and —S(O)2—.
The symbol L2 is a bond or C1-3alkylene, optionally substituted with from 1-4 R4. For the compounds of the present invention, the molecular weight is typically less than or equal to 600. In some instances, preferred compounds have a molecular weight of less than 550, more preferably less than 530. In one group of particularly preferred embodiments, the compounds have a molecular weight of from about 350 to about 530. Exemplary molecular weights include, but are not limited to, about 370, 379, 406, 420, 430, 434, 435, 441, 442, 444, 446, 448, 449, 450, 455, 460, 464, 466, 468, 470, 475, 478, 479, 480, 484, 490, 491, 492, 500 and 527.
In one group of embodiments, the compounds have formula Ia:
In another group of embodiments, the compounds have formula Ia1:
In yet another group of embodiments, the compounds have formula Ia2:
wherein the subscript n is an integer from 1-4. In certain instances, Ru is selected from the group consisting of H, CH3, —CH2CH2OCH3, —CH2CH2NH2, —CH2CH2NHC(O)CH3, —CH2CH2NHC(O)NH2, —CH2CH2NHS(O)2CH3 and —CH2C(O)OCH3
In certain embodiment of compounds having Formula I the symbol B is an aryl or a heteroaryl group selected from the group consisting of:
In one embodiment of formula I, B can be attached to the remainder of the molecule through a carbon atom. Alternatively, when B is selected from the group set forth in (ii) or (iii) above, then B can be attached to the remainder of the molecule through a nitrogen atom or a carbon atom.
In a second embodiment of compounds having formula I, the symbol B is an heteroaryl group. In certain instances, B is an optionally substituted member selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl. In another instance, B is an optionally substituted member selected from the group consisting of tetrazolyl, triazolyl, imidazolyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, furanyl, oxadiazolyl, furazanyl, isothiazolyl and thienyl. In yet another instance, symbol B is an optionally substituted member selected from the group consisting of benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzotetrazolyl, benzooxazolyl, benzoisoxazolyl, benzooxadiazolyl, benzothienyl, benzoisothiazolyl, benzofuranyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pyrimidinopyridinyl, pyridazinopyridinyl, pyrazinopyridinyl, pyridinopyrimidinyl, pyrimidinopyrimidinyl, pyridazinopyrimidinyl, pyrazinopyrimidinyl, pyridinopyridazinyl, pyrimidinopyridazinyl, pyridazinopyridazinyl, pyrazinopyridazinyl, pyridinopyrazinyl, pyrimidinopyrazinyl, pyridazinopyrazinyl, pyrazinopyrazinyl, pyridinoimidazolyl, purinyl, pyridazinoimidazolyl, pyrazinoimidazolyl, pyridinooxazolyl, pyrimidinooxazolyl, pyridazinooxazolyl, pyrazinooxazolyl, pyridinoisoxazolyl, pyrimidinoisoxazolyl, pyridazinoisoxazolyl, pyrazinoisoxazolyl, pyridinooxathiadiazolyl, pyrimidinooxathiadiazolyl, pyridazinooxathiadiazolyl, pyrazinooxathiadiazolyl, pyridinooxathiazolyl, pyrimidinooxathiazolyl, pyridazinooxathiazolyl, pyrazinooxathiazolyl, pyridinothiazolyl, pyrimidinothiazolyl, pyridazinothiazolyl, pyrazinothiazolyl, pyridinopyrazolyl, pyrimidinopyrazolyl, pyridazinopyrazolyl, pyrazinopyrazolyl, pyridinopyrrolyl, pyrimidinopyrrolyl, pyridazinopyrrolyl, pyrazinopyrrolyl, isobenzofuranyl, indolizinyl, isoindolyl, indolyl, indazolyl, phthalazinyl, cinnolinyl.
In another embodiment in compounds having Formula I, the symbol B has the formula selected from the group set forth in
In yet another embodiment, the symbol B has the formula selected from the group set forth in
Within compounds having formula I, in certain embodiments, Ar is a phenyl group having from 1-3 R2 substituents. In certain instances, R2 is F, Cl, Br, I, Me, Et, propyl, butyl, OMe, CN or CF3. In another embodiment, Ar is a phenyl group having the formula
wherein R1a, R2b, R2c, R2d and R2e at each occurrence are independently selected from the group consisting of hydrogen, halogen, —ORc, —OC(O)Rc, —NRcRd, —SRc, Re, —CN, —NO2, —CO2Rc, —CONRcRd, —C(O)Rc, —OC(O)NRcRd, —NRdC(O)Rc, —NRc(O)2Rc, —NRcC(O)NRcRd, —NH—C(NH2)═NH, —NReC(NH2)═NH, —NH—C(NH2)═NRe, —NH—CHRe)═NH, —NReC(NHRe)═NH, —NReC(NH2)═NRe, —NH—C(NHRe)═NRe, —NH—C(NReRe)═NH, —S(O)Re, —S(O)2Re, —NRcS(O)2Re, —S(O)2NRcRd, —N3, —C(NORc)Rd, —C(NRcW)═NW, —N(W)C(Rc)═NW, —NRcC(S)NRcRd, —X2C(NORc)Rd, —X2C(NRcW)═NW, —X2N(W)C(Rc)═NW, —X2 NRcC(S)NRcRd, —X2 ORc, —O—X2ORc, —X1OC(O)Rc, —X2NRcRd, —O—X2NRcRd, —X2 SRc, —X2CN, —X2NO2, —X2CO2Rc, —O—X2CO2Rc, —X2CONRCRd, —O—X2CONRcRd, —X2C(O)Rc, —X2OC(O)NRcRd, —X2NRdC(O)Rc, —X2NRdC(O)2Re, —X2NRcC(O)NRcRd, —X2 NH—C(NH2)═NH, —X2NReC(NH2)═NH, —X2 NH—C(NH2)═NRe, —X1NH—C(NHRe)═NH, —X2S(O)Re, —X2S(O)2Re, —X2NRcS(O)2Re, —X2S(O)2NRcRd, —X2N3, —NR2X2 ORc, —NRd—X2NRcRd, —NRd—X2CO2Rc, and —NRd—X2CONRcRd.
In a second embodiment, R2a and R2b are each independently selected from group consisting of hydrogen, halogen, cyano, heteroaryl, —NO2, —CO2Rc, —CONRcRd, —C(O)Rc, —S(O)Re, —S(O)2Re, —Re, —C(NORc)Rd, —C(NRcW)═NW, N(W)C(RcC)═NW, —X2C(NORc)Rd, —X2C(NRcW)═NW, —X2N(W)C(RcC)═NW, —X2NRcRd, —X2SRc, —X2CN, —X2NO2, —X2CO2Rc, —X2CONRcRd, —X2C(O)Rc, —X2OC(O)NRcRd, —X2NRdC(O)Rc, —X2NRdC(O)2Re, —X2NRcC(O)NRcRd, —X2 NH—C(NH2)═NH, X2NReC(NH2)═NH, —X2 NH—C(NH2)═NRe, —X2 NH—C(NHRe)═NH, —X2S(O)Re, —X2S(O)2Re, —X2NRcS(O)2Re, —X2S(O)2NRcRd and —X2N3; R2d and R2e are each hydrogen; and R2c is selected from the group consisting of hydrogen halogen, —ORc, —SRc, —Re, —CN, —N2, CO2Rc, C(O)Rc, —NRdC(O)Rc, —NRdC(O)2Rc, —S(O)2Re, —S(O)2NRcRd, —X2ORc, —O—X2ORc, —X2NRcRd, —O—X2NRcRd and —NRd—X2 CO2Rc. In certain instances, R2c, R2d and R2e are each hydrogen; and R2a and R2b are each independently selected from the group consisting of hydrogen, halogen, —ORc, —SRc, —Re, —CN, and —NO2. In certain other instances, R2a and R2b are each independently F, Cl, Br, I, Me, Et, propyl, butyl, OMe, CN or CF3.
In a third embodiment, R2a, R2b, R2d and R2e are each hydrogen; and R2c is selected from the group consisting of hydrogen halogen, —ORc, —SRc, —Re, —CN, —NO2, —CO2Rc, —C(O)Rc, —NRdC(O)Rc, —NRdC(O)2Re, —S(O)2Re, —S(O)2NRcRd, —X2CRc, —O—X2CRc, —X2 NRcRd, —O—X2 NRcRd and —NRd—X2CO2Rc. In certain instances, R2c is F, Cl, Br, I, Me, Et, propyl, butyl, OMe, CN or CF3.
In a fourth embodiment, R2a and R2c are each independently selected from group consisting of hydrogen, halogen, cyano, heteroaryl, —NO2, —CO2Rc, —CONRcRd, —C(O)Rc, —S(O)Re, —S(O)2Re, —Re, —C(NORc)Rd, —C(NRcW)═NW, N(W)C(RcC)═NW, —X2C(NORc)Rd, —X2C(NRcW)═NW, —X2N(W)C(RcC)═NW, —X2NRcRd, —X2SRc, —X2CN, —X2NO2, —X2CO2Rc, —X2CONRcRd, —X2C(O)Rc, —X2OC(O)NRcRd, —X2NRdC(O)Rc, —X2NRdC(O)2Re, —X2NRcC(O)NRcRd, —X2 NH—C(NH2)═NH, X2NReC(NH2)═NH, —X2 NH—C(NH2)═NRe, —X2 NH—C(NHRe)═NH, —X2S(O)Re, —X2S(O)2Re, —X2NRcS(O)2Re, —X2S(O)2NRcRd and —X2N3; R2b and R2d are each hydrogen; R2e is selected from the group consisting of hydrogen halogen, —ORc, —SRc, —Re, —CN, —NO2, —CO2Rc, —C(O)Rc, —NRdC(O)Rc, —NRdC(O)2Re, —S(O)2Re, —S(O)2NRcRd, —X2ORc, —O—X2ORc, —X2NRcRd, —O—X2NRcRd and —NRd—X2CO2Rc.
In a fifth embodiment, R2b and R2d are each independently selected from group consisting of hydrogen, halogen, cyano, heteroaryl, —NO2, —CO2Rc, —CONRcRd, —C(O)Rc, —S(O)Re, —S(O)2Re, —Re, —C(NORc)Rd, —C(NRcW)═NW, N(W)C(RcC)═NW, —X2C(NORc)Rd, —X2C(NRcW)═NW, —X2N(W)C(RcC)═NW, —X2NRcRd, —X2SRc, —X2CN, —X2NO2, —X2CO2Rc, —X2CONRcRd, —X2C(O)Rc, —X2OC(O)NRcRd, —X2NRdC(O)Rc, —X2NRdC(O)2Re, —X2NRcC(O)NRcRd, —X2 NH—C(NH2)═NH, X2NReC(NH2)═NH, —X2 NH—C(NH2)═NRe, —X2 NH—C(NHRe)═NH, —X2S(O)Re, —X2S(O)2Re, —X2NRcS(O)2Re, —X2S(O)2NRcRd and —X2N3; R2a and R2c are each hydrogen; R2e is selected from the group consisting of hydrogen halogen, —ORc, —SRc, —Re, —CN, —NO2, —CO2RcC(O)Rc, —NRdC(O)Rc, —NRdC(O)2Re, S(O)2Re, —S(O)2NRcRd, —X2ORc, —O—X2ORc, —X2NRcRd, —O—X2NRcRd and NRd—X2CO2Rc.
In a sixth embodiment, R2b and R2c are each independently selected from group consisting of hydrogen, halogen, cyano, heteroaryl, —NO2, —CO2Rc, —CONRcRd, —C(O)Rc, —S(O)Re, —S(O)2Re, —Re, —C(NORc)Rd, —C(NRcW)═NW, —N(W)C(Rc)═NW, —X2C(NORc)Rd, —X2C(NRcW)═NW, —X2N(W)C(Rc)═NW, —X2 NRcRd, —X2SRc, —X2CN, —X2NO2, —X2CO2Rc, —X2CONRcRd, —X2C(O)Rc, —X2OC(O)NRcRd, —X2NRdC(O)Rc, —X2NRdC(O)2Re, —X2NRcC(O)NRcRd, —X2NH—C(NH2)═NH, —X2NReC(NH2)═NH, X2NH—C(NH2)═NRe, —X2NH—C(NHRe)═NH, —X2S(O)Re, —X2S(O)2Re, —X2NRcS(O)2Re, —X2S(O)2NRcRd and —X2N3; R2a, R2d and R2e are each hydrogen.
In a seventh embodiment, R2a, R2b, R2c and R2e are each hydrogen; and R2d is selected from the group consisting of hydrogen halogen, —ORc, —SRc, —Re, —CN, —NO2, —CO2Rc, —C(O)Rc, —NRdC(O)Rc, —NRdC(O)2Re, S(O)2Re, —S(O)2NRcRd, —X2ORc, —O—X2ORc, —X2NRcRd, —O—X2NRcRd and —NRd—X2CO2Rc. In certain instances, R2d is F, Cl, Br, I, Me, Et, propyl, butyl, OMe, CN or CF3.
Within compounds having formula I, in certain embodiments, L1 is selected from the group consisting of optionally substituted C2 alkenylene, —CH2—, —CH2O—, —CH2NRk—, —CH2NH—, —CH2N(Ru)C(O)—, —CH2N(Ru)S(O)—, —CH2N(Ru)S(O)2— and —CH2N(Ru)CH2—. Ru is independently selected from the group consisting of hydrogen, C1-8 alkylene, C1-8 haloalkylene, C3-6 cycloalkylene, C2-8 alkenylene, C2-8 alkynylene, arylene and heteroarylene, wherein Ru is optionally further substituted with from one to three members selected from the group consisting of —OH, —ORv, —OC(O)NHRv, —OC(O)N(Rv)2, —SH, —SRv, —S(O)Rv, —S(O)2Rv, —SO2NH2, —S(O)2NHRv, —S(O)2N(Rv)2, —NHS(O)2Rv, —NRvS(O)2Rv, —C(O)NH2, —C(O)NHRv, —C(O)N(Rv)2, —C(O)Rv, —NHC(O)Rv, —NRvC(O)Rv, —NHC(O)NH2, —NRvC(O)NH2, —NRvC(O)NHRv, —NHC(O)NHRv, —NRvC(O)N(Rv)2, —NHC(O)N(Rv)2, —CO2H, —CO2Rv, —NHCO2Rv, —NRvCO2Rv, —CN, —NO2, —NH2, —NHRv, —N(Rv)2, —NRvS(O)NH2 and —NRvS(O)2NHRv, wherein each Rv is independently an unsubstituted C1-6 alkyl or C1-6 haloalkyl; ad X5 is selected from the group consisting of C1-4alkylene, —C(O)—, —S(O)— and —S(O)2—. In certain instances, Ru is selected from the group consisting of H, C1-4alkyl, —CH2CH2OC1-4alkyl, —CH2CH2NH2, —CH2CH2NHC(O)(C1-4alkyl), —CH2CH2NHC(O)NH2, —CH2CH2NHS(O)2(C1-4alkyl) and —CH2C(O)OC1-4alkyl. In certain other instances, Ru is selected from the group consisting of H, CH3, —CH2CH2OCH3, —CH2CH2NH2, —CH2CH2NHC(O)CH3, —CH2CH2NHC(O)NH2, —CH2CH2NHS(O)2CH3 and —CH2C(O)OCH3.
Within compounds having formula I, in some embodiments, L2 is a bond. In certain other embodiments, L2 is C1-3alkylene selected from the group consisting of —CH2—, —CH2CH2—, CH2CH2CH2—, —CH2CH(CH3)— and —CH(CH2CH3)—. In yet certain other embodiments, L2 is C1-3 alkylene substituted with a member selected from the group consisting of phenyl, C1-4 alkyl, —X4ORi, —X4OC(O)Ri, —X4NRiRj, —X4SRi, —X4CN or —X4NO2. In certain instances, X4 is a bond. In certain other instances, X4 is C1-2 alkylene.
As shown in the examples below, there are variety of synthetic routes by which a skilled artisan can prepare compounds and intermediates of the present invention. Schemes 1, 1A-1D, 2 and 2A illustrate several methods for the preparation of certain oxo-imidazolidine derivatives of the invention. In each of these schemes, P is a protecting group, X is a leaving group, such as a halogen atom; and non-interfering substituents are provided as —R, —R′, —R″, and —R′″.
The schemes below provide certain synthetic routes that can be followed to access certain oxo-imidazolidine derivatives of the present invention. Other routes or modification of the routes presented below would be readily apparent to a skilled artisan and within the scope of the present invention.
Scheme 1B shows the synthesis of certain oxo-imidazoline derivatives of the present invention from intermediate 2.
Scheme 1C shows the synthesis of certain oxo-imidazoline derivatives of the present invention from intermediate 3.
Scheme 1D shows the synthesis of certain oxo-imidazoline derivatives of the present invention from intermediate 4.
Scheme 2 shows the synthesis of an oxo-imidazolidine intermediate used to prepare the compounds of the present invention.
Scheme 2A shows the synthesis of certain oxo-imidazoline derivatives of the present invention from intermediate 5.
In addition to having a compound(s) of formula I provided above, the compositions for modulating CCR1, CCR2 and CCR3 activity in humans and animals will typically contain a pharmaceutical carrier or diluent.
The term “composition” as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The pharmaceutical compositions for the administration of the compounds of this invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy and drug delivery. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions and self emulsifications as described in U.S. Pat. No. 6,451,339, hard or soft capsules, syrups, elixirs, solutions, buccal patch, oral gel, chewing gum, chewable tablets, effervescent powder and effervescent tablets. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, antioxidants and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example PVP, cellulose, PEG, starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated, enterically or otherwise, by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Additionally, emulsions can be prepared with a non-water miscible ingredient such as oils and stabilized with surfactants such as mono-diglycerides, PEG esters and the like.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxy-ethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrin, PEG and surfactants.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. Additionally, the compounds can be administered via ocular delivery by means of solutions or ointments. Still further, transdermal delivery of the subject compounds can be accomplished by means of iontophoretic patches and the like. For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. As used herein, topical application is also meant to include the use of mouth washes and gargles.
The compounds of the invention may be formulated for depositing into a medical device, which may include any of variety of conventional grafts, stents, including stent grafts, catheters, balloons, baskets or other device that can be deployed or permanently implanted within a body lumen. As a particular example, it would be desirable to have devices and methods which can deliver compounds of the invention to the region of a body which has been treated by interventional technique.
In exemplary embodiment, the inhibitory agent of this invention may be deposited within a medical device, such as a stent, and delivered to the treatment site for treatment of a portion of the body.
Stents have been used as delivery vehicles for therapeutic agents (i.e., drugs). Intravascular stents are generally permanently implanted in coronary or peripheral vessels. Stent designs include those of U.S. Pat. No. 4,733,655 (Palmaz), 4,800,882 (Gianturco), or 4,886,062 (Wiktor). Such designs include both metal and polymeric stents, as well as self-expanding and balloon-expandable stents. Stents may also used to deliver a drug at the site of contact with the vasculature, as disclosed in U.S. Pat. No. 5,102,417 (Palmaz) and in International Patent Application Nos. WO 91/12779 (Medtronic, Inc.) and WO 90/13332 (Cedars-Sanai Medical Center), U.S. Pat. No. 5,419,760 (Narciso, Jr.) and U.S. Pat. No. 5,429,634 (Narciso, Jr.), for example. Stents have also been used to deliver viruses to the wall of a lumen for gene delivery, as disclosed in U.S. patent application Ser. No. 08/746,404, filed Nov. 8, 1996 (Donovan et al.).
The term “deposited” means that the inhibitory agent is coated, adsorbed, placed, or otherwise incorporated into the device by methods known in the art. For example, the inhibitory agent may be embedded and released from within (“matrix type”) or surrounded by and released through (“reservoir type”) polymer materials that coat or span the medical device. In the later example, the inhibitory agent may be entrapped within the polymer materials or coupled to the polymer materials using one or more the techniques for generating such materials known in the art. In other formulations, the inhibitory agent may be linked to the surface of the medical device without the need for a coating by means of detachable bonds and release with time, can be removed by active mechanical or chemical processes, or are in a permanently immobilized form that presents the inhibitory agent at the implantation site.
In one embodiment, the inhibitory agent may be incorporated with polymer compositions during the formation of biocompatible coatings for medical devices, such as stents. The coatings produced from these components are typically homogeneous and are useful for coating a number of devices designed for implantation.
The polymer may be either a biostable or a bioabsorbable polymer depending on the desired rate of release or the desired degree of polymer stability, but a bioabsorbable polymer is preferred for this embodiment since, unlike a biostable polymer, it will not be present long after implantation to cause any adverse, chronic local response. Bioabsorbable polymers that could be used include, but are not limited to, poly(L-lactic acid), polycaprolactone, polyglycolide (PGA), poly(lactide-co-glycolide) (PLLA/PGA), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid), poly(D-lactic acid), poly(L-lactic acid), poly(D,L-lactic acid), poly(D,L-lactide) (PLA), poly (L-lactide) (PLLA), poly(glycolic acid-co-trimethylene carbonate) (PGA/PTMC), polyethylene oxide (PEO), polydioxanone (PDS), polyphosphoester, polyphosphoester urethane, poly(amino acids), cyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonate), copoly(ether-esters) (e.g., PEO/PLA), polyalkylene oxalates, polyphosphazenes and biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen and hyaluronic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, cross linked or amphipathic block copolymers of hydrogels, and other suitable bioabsorbable popolymers known in the art. Also, biostable polymers with a relatively low chronic tissue response such as polyurethanes, silicones, and polyesters could be used and other polymers could also be used if they can be dissolved and cured or polymerized on the medical device such as polyolefins, polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymers and copolymers, vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinylpyrrolidone; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile, polyvinyl ketones; polyvinyl aromatics, such as polystyrene, polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; pyran copolymer; polyhydroxy-propyl-methacrylamide-phenol; polyhydroxyethyl-aspartamide-phenol; polyethyleneoxide-polylysine substituted with palmitoyl residues; polyamides, such as Nylon 66 and polycaprolactam; alkyd resins, polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins, polyurethanes; rayon; rayon-triacetate; cellulose, cellulose acetate, cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; and carboxymethyl cellulose.
Polymers and semipermeable polymer matrices may be formed into shaped articles, such as valves, stents, tubing, prostheses and the like.
In one embodiment of the invention, the inhibitory agent of the invention is coupled to a polymer or semipermeable polymer matrix that is formed as a stent or stent-graft device.
Typically, polymers are applied to the surface of an implantable device by spin coating, dipping or spraying. Additional methods known in the art can also be utilized for this purpose. Methods of spraying include traditional methods as well as microdeposition techniques with an inkjet type of dispenser. Additionally, a polymer can be deposited on an implantable device using photo-patterning to place the polymer on only specific portions of the device. This coating of the device provides a uniform layer around the device which allows for improved diffusion of various analytes through the device coating. In preferred embodiments of the invention, the inhibitory agent is formulated for release from the polymer coating into the environment in which the medical device is placed. Preferably, the inhibitory agent is released in a controlled manner over an extended time frame (e.g., months) using at least one of several well-known techniques involving polymer carriers or layers to control elution. Some of these techniques were previously described in U.S. Patent Application 20040243225A1, the entire disclosure of which is incorporated in its entirety.
Moreover, as described for example in U.S. Pat. No. 6,770,729, which is incorporated herein in its entirety, the reagents and reaction conditions of the polymer compositions can be manipulated so that the release of the inhibitory agent from the polymer coating can be controlled. For example, the diffusion coefficient of the one or more polymer coatings can be modulated to control the release of the inhibitory agent from the polymer coating. In a variation on this theme, the diffusion coefficient of the one or more polymer coatings can be controlled to modulate the ability of an analyte that is present in the environment in which the medical device is placed (e.g. an analyte that facilitates the breakdown or hydrolysis of some portion of the polymer) to access one or more components within the polymer composition (and for example, thereby modulate the release of the inhibitory agent from the polymer coating). Yet another embodiment of the invention includes a device having a plurality of polymer coatings, each having a plurality of diffusion coefficients. In such embodiments of the invention, the release of the inhibitory agent from the polymer coating can be modulated by the plurality of polymer coatings.
In yet another embodiment of the invention, the release of the inhibitory agent from the polymer coating is controlled by modulating one or more of the properties of the polymer composition, such as the presence of one or more endogenous or exogenous compounds, or alternatively, the pH of the polymer composition. For example, certain polymer compositions can be designed to release a inhibitory agent in response to a decrease in the pH of the polymer composition. Alternatively, certain polymer compositions can be designed to release the inhibitory agent in response to the presence of hydrogen peroxide.
In yet another aspect, the present invention provides methods of treating CCR1-, CCR2- and/or CCR3-mediated conditions or diseases by administering to a subject having such a disease or condition, a therapeutically effective amount of a compound of formula I above. The “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.
CCR1 provides a target for interfering with or promoting specific aspects of immune cell functions, or more generally, with functions associated with CCR1 expression on a wide range of cell types in a mammal, such as a human. Compounds that inhibit CCR1, are particularly useful for modulating monocyte, macrophage, lymphocyte, granulocyte, NK cell, mast cells, dendritic cell, and certain immune derived cell (for example, osteoclasts) function for therapeutic purposes. Accordingly, the present invention is directed to compounds which are useful in the prevention and/or treatment of a wide variety of inflammatory and immunoregulatory disorders and diseases (see Saeki, et al., Current Pharmaceutical Design 9:1201-1208 (2003)).
For example, an instant compound that inhibits one or more functions of CCR1 may be administered to inhibit (i.e., reduce or prevent) inflammation or cellular infiltration associated with an immune disorder. As a result, one or more inflammatory processes, such as leukocyte emigration or infiltration, chemotaxis, exocytosis (e.g., of enzymes, histamine) or inflammatory mediator release, can be inhibited. For example, monocyte infiltration to an inflammatory site (e.g., an affected joint in arthritis, or into the CNS in MS) can be inhibited according to the present method.
Similarly, an instant compound that promotes one or more functions of CCR1 is administered to stimulate (induce or enhance) an inflammatory response, such as leukocyte emigration, chemotaxis, exocytosis (e.g., of enzymes, histamine) or inflammatory mediator release, resulting in the beneficial stimulation of inflammatory processes. For example, monocytes can be recruited to combat bacterial infections.
Diseases and conditions associated with inflammation, immune disorders and infection can be treated using the method of the present invention. In a preferred embodiment, the disease or condition is one in which the actions of immune cells such monocyte, macrophage, lymphocyte, granulocyte, NK cell, mast cell, dendritic cell, or certain immune derived cell (for example, osteoclasts) are to be inhibited or promoted, in order to modulate the inflammatory or autoimmune response.
In one group of embodiments, diseases or conditions, including chronic diseases, of humans or other species can treated with modulators of CCR1, CCR2 or CCR3 function. These diseases or conditions include: (1) allergic diseases such as systemic anaphylaxis or hypersensitivity responses, drug allergies, insect sting allergies and food allergies, (2) inflammatory bowel diseases, such as Crohn's disease, ulcerative colitis, ileitis and enteritis, (3) vaginitis, (4) psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria and pruritus, (5) vasculitis, (6) spondyloarthropathies, (7) scleroderma, (8) asthma and respiratory allergic diseases such as asthma, allergic asthma, allergic rhinitis, hypersensitivity lung diseases and the like, (9) autoimmune diseases, such as fibromyalagia, scleroderma, ankylosing spondylitis, juvenile RA, Still's disease, polyarticular juvenile RA, pauciarticular juvenile RA, polymyalgia rheumatica, Takuyasu arthritis, rheumatoid arthritis, psoriatic arthritis, osteoarthritis, polyarticular arthritis, multiple sclerosis, systemic lupus erythematosus, type I diabetes, type II diabetes, type I diabetes (recent onset), optic neuritis, glomerulonephritis, and the like, (10) graft rejection including allograft rejection and acute and chronic graft-vs-host disease, (11) fibrosis (e.g. pulmonary fibrosis (i.e. idiopathic pulmonary fibrosis, interstitial pulmonary fibrosis), fibrosis associated with end-stage renal disease, fibrosis caused by radiation, tubulointerstitial fibrosis, subepithelieal fibrosis, scleroderma (progressive systemic sclerosis), hepatic fibrosis (including that caused by alcoholic or viral hepatitis), primary and secondary cirrhosis), (12) acute and chronic lung inflammation (chronic obstructive pulmonary disease, chronic bronchitis, adult respiratory distress syndrome, respiratory distress syndrome of infancy, immune complex alveolitis) and (13) other diseases in which undesired inflammatory responses or immune disorders are to be inhibited, such as cardiovascular disease including atherosclerosis, vascular inflammation resulting from tissue transplant or during restenosis (including, but not limited to restenosis following angioplasty and/or stent insertion), other acute and chronic inflammatory conditions such as myositis, neurodegenerative diseases (e.g., Alzheimer's disease), encephalitis, meningitis, hepatitis, nephritis, sepsis, sarcoidosis, allergic conjunctivitis, otitis, sinusitis, synovial inflammation caused by arthroscopy, hyperuremia, trauma, ischaemia reperfusion injury, nasal polyosis, preeclampsia, oral lichen planus, Guillina-Barre syndrome, granulomatous diseases, conditions associated with leptin production, Behcet's syndrome and gout and in wound healing applications (14) immune mediated food allergies such as Celiac disease.
In another group of embodiments, diseases or conditions can be treated with modulators of CCR1 function. Examples of diseases to be treated with modulators of CCR1 function include cancers (both primary and metastatic) (e.g., multiple myeloma; Hata, H., Leukemia & Lymphoma, 2005, 46(7); 967-972), cardiovascular diseases, diseases in which angiogenesis or neovascularization play a role (neoplastic diseases, retinopathy and macular degeneration), infectious diseases (viral infections, e.g., HIV infection, and bacterial infections) and immunosuppressive diseases such as organ transplant conditions and skin transplant conditions. The term “organ transplant conditions” is meant to include bone marrow transplant conditions and solid organ (e.g., kidney, liver, lung, heart, pancreas or combination thereof) transplant conditions.
Pharmaceutical compositions of this invention can also inhibit the production of metalloproteinases and cytokines at inflammatory sites, either directly or indirectly (as a consequence of decreasing cell infiltration) thus providing benefit for diseases or conditions linked to these cytokines.
The compounds of the present invention are accordingly useful in the prevention and treatment of a wide variety of inflammatory and immunoregulatory disorders and diseases. Depending on the disease to be treated and the subject's condition, the compounds of the present invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
In the treatment or prevention of conditions which require chemokine receptor modulation an appropriate dosage level will generally be about 0.001 to 100 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.01 to about 25 mg/kg per day; more preferably about 0.05 to about 10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. Within this range the dosage may be 0.005 to 0.05, 0.05 to 0.5 or 0.5 to 5.0 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.
It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, hereditary characteristics, general health, sex and diet of the subject, as well as the mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition for the subject undergoing therapy.
Diseases and conditions associated with inflammation, immune disorder, infection and cancer can be treated or prevented with the present compounds, compositions, and methods.
The compounds and compositions of the present invention can be combined with other compounds and compositions having related utilities to prevent and treat the condition or disease of interest, such as inflammatory or autoimmune disorders, conditions and diseases, including inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, polyarticular arthritis, multiple sclerosis, allergic diseases, psoriasis, atopic dermatitis and asthma, and those pathologies noted above.
For example, in the treatment or prevention of inflammation or autimmunity or for example arthritis associated bone loss, the present compounds and compositions may be used in conjunction with an anti-inflammatory or analgesic agent such as an opiate agonist, a lipoxygenase inhibitor, such as an inhibitor of 5-lipoxygenase, a cyclooxygenase inhibitor, such as a cyclooxygenase-2 inhibitor, an interleukin inhibitor, such as an interleukin-1 inhibitor, an NMDA antagonist, an inhibitor of nitric oxide or an inhibitor of the synthesis of nitric oxide, a non steroidal anti-inflammatory agent, or a cytokine-suppressing anti-inflammatory agent, for example with a compound such as acetaminophen, aspirin, codeine, fentanyl, ibuprofen, indomethacin, ketorolac, morphine, naproxen, phenacetin, piroxicam, a steroidal analgesic, sufentanyl, sunlindac, tenidap, and the like. Similarly, the instant compounds and compositions may be administered with an analgesic listed above; a potentiator such as caffeine, an H2 antagonist (e.g., ranitidine), simethicone, aluminum or magnesium hydroxide; a decongestant such as phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, ephinephrine, naphazoline, xylometazoline, propylhexedrine, or levo desoxy ephedrine; an antitussive such as codeine, hydrocodone, caramiphen, carbetapentane, or dextromethorphan; a diuretic; and a sedating or non sedating antihistamine.
Likewise, compounds and compositions of the present invention may be used in combination with other drugs that are used in the treatment, prevention, suppression or amelioration of the diseases or conditions for which compounds and compositions of the present invention are useful. Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound or composition of the present invention. When a compound or composition of the present invention is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound or composition of the present invention is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients or therapeutic agents, in addition to a compound or composition of the present invention. Examples of other therapeutic agents that may be combined with a compound or composition of the present invention, either administered separately or in the same pharmaceutical compositions, include, but are not limited to: (a) VLA-4 antagonists, (b) corticosteroids, such as beclomethasone, methylprednisolone, betamethasone, prednisone, prenisolone, dexamethasone, fluticasone, hydrocortisone, budesonide, triamcinolone, salmeterol, salmeterol, salbutamol, formeterol; (c) immunosuppressants such as cyclosporine (cyclosporine A, Sandimmune®, Neoral®), tacrolimus (FK-506, Prograf®), rapamycin (sirolimus, Rapamune®) and other FK-506 type immunosuppressants, and mycophenolate, e.g., mycophenolate mofetil (CellCept®); (d) antihistamines (H1-histamine antagonists) such as bromopheniramine, chlorpheniramine, dexchloipheniramine, triprolidine, clemastine, diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine, azatadine, cyproheptadine, antazoline, pheniramine pyrilamine, astemizole, terfenadine, loratadine, cetirizine, fexofenadine, descarboethoxyloratadine, and the like; (e) non steroidal anti asthmatics (e.g., terbutaline, metaproterenol, fenoterol, isoetharine, albuterol, bitolterol and pirbuterol), theophylline, cromolyn sodium, atropine, ipratropium bromide, leukotriene antagonists (e.g., zafmlukast, montelukast, pranlukast, iralukast, pobilukast and SKB-106,203), leukotriene biosynthesis inhibitors (zileuton, BAY-1005); (f) non steroidal anti-inflammatory agents (NSAIDs) such as propionic acid derivatives (e.g., alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, rniroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and tioxaprofen), acetic acid derivatives (e.g., indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin and zomepirac), fenamic acid derivatives (e.g., flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (e.g., diflunisal and flufenisal), oxicams (e.g., isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (e.g., acetyl salicylic acid and sulfasalazine) and the pyrazolones (e.g., apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone and phenylbutazone); (g) cyclooxygenase-2 (COX-2) inhibitors such as celecoxib (Celebrex®) and rofecoxib (Vioxx®); (h) inhibitors of phosphodiesterase type IV (PDE IV); (i) gold compounds such as auranofin and aurothioglucose, (j) etanercept (Enbrel®), (k) antibody therapies such as orthoclone (OKT3), daclizumab (Zenapax®), basiliximab (Simulect®) and infliximab (Remicade®), (l) other antagonists of the chemokine receptors, especially CCR5, CXCR2, CXCR3, CCR2, CCR3, CCR4, CCR7, CX3CR1 and CXCR6; (m) lubricants or emollients such as petrolatum and lanolin, (n) keratolytic agents (e.g., tazarotene), (o) vitamin D3 derivatives, e.g., calcipotriene or calcipotriol (Dovonex®g), (p) PUVA, (q) anthralin (Drithrocreme®), (r) etretinate (Tegison®) and isotretinoin and (s) multiple sclerosis therapeutic agents such as interferon β-1β (Betaseron®), interferon (β-1α (Avonex®), azathioprine (Imurek®, Imuran®), glatiramer acetate (Capoxone®), a glucocorticoid (e.g., prednisolone) and cyclophosphamide (t) DMARDS such as methotrexate (u) other compounds such as 5-aminosalicylic acid and prodrugs thereof; hydroxychloroquine; D-penicillamine; antimetabolites such as azathioprine, 6-mercaptopurine and methotrexate; DNA synthesis inhibitors such as hydroxyurea and microtubule disrupters such as colchicine. The weight ratio of the compound of the present invention to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the present invention is combined with an NSAID the weight ratio of the compound of the present invention to the NSAID will generally range from about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. Combinations of a compound of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
The following examples are offered to illustrate, but not to limit the claimed invention.
Reagents and solvents used below can be obtained from commercial sources such as Aldrich Chemical Co. (Milwaukee, Wis., USA). 1H-NMR were recorded on a Varian Mercury 400 MHz NMR spectrometer. Significant peaks are provided relative to TMS and are tabulated in the order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet) and number of protons. Mass spectrometry results are reported as the ratio of mass over charge, followed by the relative abundance of each ion (in parenthesis). In tables, a single m/e value is reported for the M+H (or, as noted, M−H) ion containing the most common atomic isotopes. Isotope patterns correspond to the expected formula in all cases. Electrospray ionization (ESI) mass spectrometry analysis was conducted on a Hewlett-Packard MSD electrospray mass spectrometer using the HP1100 HPLC equipped with an Agilent Zorbax SB-C18, 2.1×50 mm, 5μ column for sample delivery. Normally the analyte was dissolved in methanol at 0.1 mg/mL and 1 microlitre was infused with the delivery solvent into the mass spectrometer, which scanned from 100 to 1500 daltons. All compounds could be analyzed in the positive ESI mode, using acetonitrile/water with 1% formic acid as the delivery solvent. The compounds provided below could also be analyzed in the negative ESI mode, using 2 mM NH4OAc in acetonitrile/water as delivery system.
The following abbreviations are used in the Examples and throughout the description of the invention:
NMP: N-methyl-2-pyrrolidinone
DMF: Dimethyl formamide
EtOAc: Ethyl acetate
BOC2O: di-tertbutyl dicarbonate or BOC anhydride
BOP: O-Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate
PyBOP: (Benzotriazol-1-yloxy)tripyrrolidino-phosphonium Hexafluorophosphate
HATU: O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
rt: room temperature
LDA: Lithium diisopropyl amide
DIPEA: Diisopropyl ethylamine
Compounds within the scope of this invention can be synthesized as described below, using a variety of reactions known to the skilled artisan. One skilled in the art will also recognize that alternative methods may be employed to synthesize the target compounds of this invention, and that the approaches described within the body of this document are not exhaustive, but do provide broadly applicable and practical routes to compounds of interest.
Certain molecules claimed in this patent can exist in different enantiomeric and diastereomeric forms and all such variants of these compounds are claimed. The detailed description of the experimental procedures used to synthesize key compounds in this text lead to molecules that are described by the physical data identifying them as well as by the structural depictions associated with them.
Two regioisomers can sometimes exist for certain compounds of the invention. For these cases, both regioisomeric types have demonstrated biological properties and are meant to be within the scope of all the appended claims, whether explicitly drawn or not.
Those skilled in the art will also recognize that during standard work up procedures in organic chemistry, acids and bases are frequently used. Salts of the parent compounds are sometimes produced, if they possess the necessary intrinsic acidity or basicity, during the experimental procedures described within this patent.
Step 1: To a mixture of 2-methyl-3-chloroaniline (14.16 g, 100 mmol, 1 equiv), Boc-glycine (17.52 g, 1 equiv), BOP reagent (48.6 g, 1.1 equiv) in 50 mL of DMF was added TEA (42 mL, 3 equiv). After overnight, standard aqueous workup followed by flash chromatography gave 20 g of [(3-chloro-2-methyl-phenylcarbamoyl)-methyl]-carbamic acid tert-butyl ester, which was treated with ˜300 mL of 4N HCl in dioxane overnight to afford 2-amino-N-(3-chloro-2-methyl-phenyl)-acetamide.
Step 2: A mixture of 2-amino-N-(3-chloro-2-methyl-phenyl)-acetamide (74) (1.18 g, 1 equiv), paraformaldehyde (HCHO)n (0.77 g, 1.5 equiv) and 3 mL of acetic acid was heated at 90° C. for 3 h and evaporated to dryness to afford 3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one as acetic acid salt.
Step 3: To a mixture of 3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one acetic acid salt (422 mg, 1 equiv) and K2CO3 (552 mg) in 5 mL of DCM/water (1:1 v/v) was added chloroacetyl chloride (240 μL, 1.5 equiv). One hour later, the organic layer was separated and dried over sodium sulfate to give 1-(2-chloro-acetyl)-3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one.
Step 4: A mixture of 1-(2-chloro-acetyl)-3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one (572 mg, 2 mmol, 1 equiv), K2CO3 (276 mg, 1 equiv) and 2-methoxyethylamine (520 μL, 1 equiv) in 2 mL of DMF was stirred at rt overnight. Dilution with EtOAc, washing with water followed by drying the organic layer over sodium sulfate, filtration and evaporation of the volatile gave crude 3-(3-chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one.
Step 5: A mixture of 3-(3-Chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (650 mg, 2 mmol, 1 equiv), 3-chloro benzoyl chloride (350 mg, 1 equiv) and pyridine (330 μL, 1 equiv) in 5 mL of DCM Standard aqueous workup followed by purification on silica gel gave 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide. LCMS observed for (M+H)+: 464.1
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 464.1
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+:
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 446.2.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 450.1.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 460.2.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 466.1.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 490.2.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 464.1.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 475.1.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 480.2.
The title compound was prepared according to the procedure described for the synthesis of 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide by using appropriate reagents. LCMS observed for (M+H)+: 444.2.
Step 1: In a scintillation vial was taken 809 mg 4-chloroaniline (6.35 mmol, 1.00 eq), 1.20 g BOC-Gly-OH (6.98 mmol, 1.10 eq), 3.40 g BOP (7.61 mmol, 1.20 eq), 3.50 mL TEA (25.38 mmol, 4.00 eq) and 16 mL NMP. The mixture was stirred overnight at room temperature, after which the solvent was removed in vacuo and the crude product used “as is” for the next step.
Step 2: The crude material from ES033-187 was slurried in 30 mL of 4.0M HCL/p-dioxane at room temperature for two hours. The slurry was diluted with 10 mL ethyl ether and the solid material was collected by vacuum filtration and dried in vacuo to get 2-amino-N-(4-chloro-phenyl)-acetamide hydrochloride.
Step 3: Took 1.86 g 2-amino-N-(4-chloro-phenyl)-acetamide hydrochloride (7.41 mmol, 1.00 eq), 444 mg paraformaldehyde (˜2 eq), and 5 mL acetic acid in a scintillation vial. The mixture was allowed to stir at 90 C overnight, after which the solvent was removed in vacuo. The crude material was used “as is” in the next step.
Step 4: Took 37 mg [(3-chloro-benzoyl)-(2-methoxy-ethyl)-amino]-acetic acid (0.136 mmol, 1.00 eq), 53 mg 3-(4-chloro-phenyl)-imidazolidin-4-one (0.272 mmol, 2.00 eq), 90 mg BOP (0.204, 1.5 eq), 76 uL TEA (0.545 mmol, 4.00 eq), and 1 mL NMP in a 4 mL vial. The mixture was stirred overnight at room temperature and the crude product was purified by reversed phase HPLC (acetonitrile-H2O with 0.1% TFA as the eluent) to yield 3-chloro-N-{2-[3-(4-chloro-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-benzamide. MS (ES) M+H expected=450.0, found=450.1.
The following compounds were prepared according to the procedure for the synthesis of EXAMPLE 13 using the appropriate amines:
MS (ES) found for (M+H)+=434.1.
MS (ES) found for (M+H)+=430.1.
MS (ES) found for (M+H)+=446.1.
MS (ES) found for (M+H)+=480.1.
MS (ES) found for (M+H)+=484.0.
MS (ES) found for (M+H)+=434.1.
MS (ES) found for (M+H)+=480.1.
MS (ES) found for (M+H)+=441.1.
MS (ES) found for (M+H)+=460.1.
MS (ES) found for (M+H)+=464.1.
MS (ES) found for (M+H)+=448.1.
MS (ES) found for (M+H)+=494.1.
MS (ES) found for (M+H)+=464.1.
In a 4 mL vial was taken 100 mg of 3-(3-chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (0.31 mmol, 1.00 eq), 52 mg of 3-chloro-4-methylbenzoic acid (0.31 mmol, 1.00 eq), 163 mg BOP (0.37 mmol, 1.20 eq), 171 uL (1.23 mmol, 4.00 eq), and 1.5 mL NMP. The mixture was stirred overnight at room temperature and the crude product was purified by reversed phase HPLC (acetonitrile —H2O with 0.1% TFA as the eluent) to yield 3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-N-(2-methoxy-ethyl)-4-methyl-benzamide. MS (ES) M+H expected=478.0, found=478.1.
In a 4 mL vial was taken 100 mg of 3-(3-chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (0.31 mmol, 1.00 eq), 52 mg of 3-hydroxy-4-methylbenzoic acid (0.31 mmol, 1.00 eq), 163 mg BOP (0.37 mmol, 1.20 eq), 171 uL (1.23 mmol, 4.00 eq), and 1.5 mL NMP. The mixture was stirred overnight at room temperature and the crude product was purified by reversed phase HPLC (acetonitrile —H2O with 0.1% TFA as the eluent) to yield N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-3-hydroxy-N-(2-methoxy-ethyl)-4-methyl-benzamide. MS (ES) M+H expected=480.0, found=480.1.
In a 4 mL vial was taken 100 mg of 3-(3-chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (0.31 mmol, 1.00 eq), 61 mg of 3-cyanobenzoyl chloride (0.37 mmol, 1.20 eq), and 500 uL NMP. The mixture was stirred overnight at room temperature and the crude product was purified by reversed phase HPLC (acetonitrile —H2O with 0.1% TFA as the eluent) to yield N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-3-cyano-N-(2-methoxy-ethyl)-benzamide. MS (ES) M+H expected=455.0, found=455.1.
In a 4 mL vial was taken 100 mg of 3-(3-chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (0.31 mmol, 1.00 eq), 61 mg of 3-cyanobenzoyl chloride (0.37 mmol, 1.20 eq), and 500 uL NMP. The mixture was stirred overnight at room temperature and the crude product was purified by reversed phase HPLC (acetonitrile —H2O with 0.1% TFA as the eluent) to yield N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-3-trifluoromethyl-N-(2-methoxy-ethyl)-benzamide. MS (ES) M+H expected=498.0, found=498.1.
In a 4 mL vial was taken 100 mg of 3-(3-chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (0.31 mmol, 1.00 eq), 61 mg of 3-cyanobenzoyl chloride (0.37 mmol, 1.20 eq), and 500 uL NMP. The mixture was stirred overnight at room temperature and the crude product was purified by reversed phase HPLC (acetonitrile —H2O with 0.1% TFA as the eluent) to yield N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-3-methyl-N-(2-methoxy-ethyl)-benzamide. MS (ES) M+H expected=444.0, found=444.2.
A mixture of 3-(3-Chloro-2-methyl-phenyl)-imidazolidin-4-one (360 mg, 1.7 mmol, 1 eq), (3-Chloro-benzoylamino)-acetic acid (365 mg, 1.7 mmol, 1 eq), BOP (909 mg, 2.05 mmol, 1.2 eq), and TEA (0.88 ml) in DMF (5 ml) was stirred at room temperature overnight. It was purified with HPLC to give the desired product. LCMS observed for (M+H)+: 406.1
The title compound was prepared by procedure analogous to that described in Example 12 using (4-Chloro-5-methyl-3-oxazol-2-yl-pyrazol-1-yl)-acetic acid. LCMS observed for (M+H)+: 434.2
The title compound was prepared by procedure analogous to that described in Example 12 using (6-Chloro-2-oxo-benzooxazol-3-yl)-acetic acid. LCMS observed for (M+Na)+: 442
The title compound was prepared by procedure analogous to that described in Example 12 using (4-Chloro-phenoxy)-acetic acid. LCMS observed for (M+H)+: 379.2
A mixture of 1-(2-Chloro-acetyl)-3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one (230 mg, 0.8 mmol, 1 eq), 1H-Pyrazolo[3,4-b]pyridine (96 mg, 0.8 mmol, 1 eq), Cesium carbonate (653 mg, 2.0 mmol, 2.5 eq) in DMF (6 ml) was stirred at room temperature overnight. It was diluted with ethyl acetate, washed with water and purified with HPLC to give the desired product. LCMS observed for (M+H)+: 370.1
A mixture of 1-(2-Chloro-acetyl)-3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one (230 mg, 0.8 mmol, 1 eq), 1H-Pyrazolo[3,4-b]pyridine (96 mg, 0.8 mmol, 1 eq), Cesium carbonate (653 mg, 2.0 mmol, 2.5 eq) in DMF (6 ml) was stirred at room temperature overnight. It was diluted with ethyl acetate, washed with water and purified with HPLC to give the desired product. LCMS observed for (M+H)+: 370.1
Step 1: A mixture of 1-(2-Chloro-acetyl)-3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one (2.0 g, 7 mmol, 1 eq), 2-Methoxy-ethylamine (3.15 g, 42 mmol, 6 eq), potassium carbonate (1.9 g, 14 mmol, 2.0 eq) in DMF (20 ml) was stirred at room temperature overnight. It was diluted with ethyl acetate, washed with water and purified with flash chromatography to give the desired product: 3-(3-Chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one. LCMS observed for (M+H)+:
Step 2: A mixture of 3-(3-Chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (100 mg, 0.3 mmol, 1 eq), 3-Chloro-benzenesulfonyl chloride (84 mg, 0.4 mmol, 1.3 eq), TEA (0.12 ml) in THF (2 ml) was stirred at room temperature overnight. It was diluted with ethyl acetate, washed with water and purified with flash chromatography to give the desired product. LCMS observed for (M+H)+: 500.1
A mixture of 3-(3-Chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (100 mg, 0.3 mmol, 1 eq), 1-Chloro-3-isocyanato-benzene (56 mg, 0.36 mmol, 1.2 eq), in THF (2 ml) was stirred at room temperature overnight. It was concentrated, dissolved in acetonitrile and purified with HPLC to give the desired product. LCMS observed for (M+H)+: 479.3
A mixture of 3-(3-Chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (100 mg, 0.3 mmol, 1 eq), 1-Chloro-4-isocyanato-benzene (56 mg, 0.36 mmol, 1.2 eq), in THF (2 ml) was stirred at room temperature overnight. It was concentrated, dissolved in acetonitrile and purified with HPLC to give the desired product. LCMS observed for (M+H)+: 479.1
Step 1: The step 1 compound: (2-{2-[3-(3-Chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethylamino}-ethyl)-carbamic acid tert-butyl ester was prepared by procedure analogous to that described in Example 13, step 1, using (2-Amino-ethyl)-carbamic acid tert-butyl ester.
Step 2: The step 2 compound: [2-((3-Chloro-benzoyl)-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-amino)-ethyl]-carbamic acid tert-butyl ester was prepared by procedure analogous to that described in Example 13, step 2, using 3-Chloro-benzoyl chloride.
Step 3: The step 2 compound: [2-((3-Chloro-benzoyl)-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-amino)-ethyl]-carbamic acid tert-butyl ester was deprotected in HCl-Dioxane to give the desired product. LCMS observed for (M+H)+: 449.4.
A mixture of 3-(3-Chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one (160 mg, 0.49 mmol, 1 eq), 1-Chloro-3-chloromethyl-benzene (95 mg, 0.59 mmol, 1.2 eq), Cesium carbonate (325 mg, 1 mmol, 2 eq) in DMF (2 ml) was stirred at room temperature overnight. It was concentrated, dissolved in acetonitrile and purified with HPLC to give the desired product. LCMS observed for (M+H)+: 450.2
The title compound was prepared by procedure analogous to that described in Example 12 using (4-Chloro-5-methyl-3-trifluoromethyl-pyrazol-1-yl)-acetic acid. LCMS observed for (M+H)+: 435.2
The title compound was prepared by procedure analogous to that described in Example 12 using 3-(3-Chloro-2-methyl-phenyl)-1-[2-(2-methoxy-ethylamino)-acetyl]-imidazolidin-4-one and 5-Chloro-thiophene-2-carboxylic acid. LCMS observed for (M+H)+: 470.2
Step 1: To 1-(2-Chloro-acetyl)-3-(3-chloro-2-methyl-phenyl)-imidazolidin-4-one (250 mg, 087 mmol, 1 eq) in THF (2 ml) was added methylamine (4.3 ml, 2M in THF), stirred for 1 hour, then evaporated to dryness to give the step 1 compound: 3-(3-Chloro-2-methyl-phenyl)-1-(2-methylamino-acetyl)-imidazolidin-4-one.
Step 2: the product of step 1 was dissolved in DCM 2 ml and treated with 3-Chloro-benzoyl chloride (230 mg, 1.3 mmol, 1.5 eq), TEA (0.0.58 ml), stirred for 2 hours, concentrated, dissolved in acetonitrile and purified with HPLC to give the desired product. LCMS observed for (M+H)+: 420.1
The title compound was prepared by procedure analogous to that described in Example 25 using butylamine. LCMS observed for (M+H)+: 462.4
A mixture of N-(2-Amino-ethyl)-3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-benzamide (80 mg, 0.16 mmol, 1 eq), Acetyl chloride (0.014 ml, 0.19 mmol, 1.2 eq), TEA (0.07 ml) in THF (1 ml) was stirred at room temperature overnight. It was purified with HPLC to give the desired product. LCMS observed for (M+H)+: 491.2
A mixture of N-(2-Amino-ethyl)-3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-benzamide (80 mg, 0.16 mmol, 1 eq), Trimethylsilyl isocyanate (0.027 ml, 0.19 mmol, 1.2 eq), in THF (1 ml) was stirred at room temperature overnight. It was purified with HPLC to give the desired product. LCMS observed for (M+H)+: 492.1
A mixture of N-(2-Amino-ethyl)-3-chloro-N-{2-[3-(3-chloro-2-methyl-phenyl)-4-oxo-imidazolidin-1-yl]-2-oxo-ethyl}-benzamide (80 mg, 0.16 mmol, 1 eq), methanesulfonyl chloride (0.015 ml, 0.19 mmol, 1.2 eq), TEA (0.07 ml) in THF (1 ml) was stirred at room temperature overnight. It was purified with HPLC to give the desired product. LCMS observed for (M+H)+: 527.2
The title compound was prepared by procedure analogous to that described in Example 25 using Amino-acetic acid methyl ester. LCMS observed for (M+H)+: 478.1.
This example illustrates the evaluation of the biological activity associated with compounds of interest of the invention.
A. Cells
1. CCR1 Expressing Cells
a) THP-1 Cells
THP-1 cells were obtained from ATCC (TIB-202) and cultured as a suspension in RPMI-1640 medium supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, 0.05% 2-mercaptoethanol and 10% FBS. Cells were grown under 5% CO2/95% air, 100% humidity at 37° C. and subcultured twice weekly at 1:5 (cells were cultured at a density range of 2×105 to 2×106 cells/mL) and harvested at 1×106 cells/mL. THP-1 cells express CCR1 and can be used in CCR1 binding and functional assays.
Monocytes were isolated from human buffy coats using the Miltenyi bead isolation system (Miltenyi, Auburn, Calif.). Briefly, following a Ficoll gradient separation to isolate peripheral blood mononuclear cells, cells were washed with PBS and the red blood cells lysed using standard procedures. Remaining cells were labeled with anti-CD14 antibodies coupled to magnetic beads (Miltenyi Biotech, Auburn, Calif.). Labeled cells were passed through AutoMACS (Miltenyi, Auburn, Calif.) and positive fraction collected. Monocytes express CCR1 and can be used in CCR1 binding and functional assays.
B. Assays
1. Inhibition of CCR1 Ligand Binding
CCR1 expressing cells were centrifuged and resuspended in assay buffer (20 mM HEPES pH 7.1, 140 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, and with 0.2% bovine serum albumin) to a concentration of 5×106 cells/mL for THP-1 cells and 5×105 for monocytes. Binding assays were set up as follows. 0.1 mL of cells (5×105 THP-1 cells/well or 5×104 monocytes) was added to the assay plates containing the compounds, giving a final concentration of ˜2-10 μM each compound for screening (or part of a dose response for compound IC50 determinations). Then 0.1 mL of 125I labeled MIP-1α (obtained from Perkin Elmer Life Sciences, Boston, Mass.) or 0.1 mL of 125I labeled CCL15/leukotactin (obtained as a custom radiolabeling by Perkin Elmer Life Sciences, Boston, Mass.) diluted in assay buffer to a final concentration of ˜50 pM, yielding ˜30,000 cpm per well, was added (using 125I labeled MIP-1α with THP-1 cells and 125I labeled CCL15/leukotactin with monocytes), the plates sealed and incubated for approximately 3 hours at 4° C. on a shaker platform. Reactions were aspirated onto GF/B glass filters pre-soaked in 0.3% polyethyleneimine (PEI) solution, on a vacuum cell harvester (Packard Instruments; Meriden, Conn.). Scintillation fluid (40 μl; Microscint 20, Packard Instruments) was added to each well, the plates were sealed and radioactivity measured in a Topcount scintillation counter (Packard Instruments). Control wells containing either diluent only (for total counts) or excess MIP-1α or MIP-1β (1 μg/mL, for non-specific binding) were used to calculate the percent of total inhibition for compound. The computer program Prism from GraphPad, Inc. (San Diego, Calif.) was used to calculate IC50 values. IC50 values are those concentrations required to reduce the binding of labeled MIP-1α to the receptor by 50%. (For further descriptions of ligand binding and other functional assays, see Dairaghi, et al., J. Biol. Chem. 274:21569-21574 (1999), Penfold, et al., Proc. Natl. Acad. Sci. USA. 96:9839-9844 (1999), and Dairaghi, et al., J. Biol. Chem. 272:28206-28209 (1997)).
2. Calcium Mobilization
To detect the release of intracellular stores of calcium, cells (THP-1 or monocytes) were incubated with 3 μM of INDO-1AM dye (Molecular Probes; Eugene, Oreg.) in cell media for 45 minutes at room temperature and washed with phosphate buffered saline (PBS). After INDO-1AM loading, the cells were resuspended in flux buffer (Hank's balanced salt solution (HBSS) and 1% FBS). Calcium mobilization was measured using a Photon Technology International spectrophotometer (Photon Technology International; New Jersey) with excitation at 350 nm and dual simultaneous recording of fluorescence emission at 400 nm and 490 nm. Relative intracellular calcium levels were expressed as the 400 nm/490 nm emission ratio. Experiments were performed at 37° C. with constant mixing in cuvettes each containing 106 cells in 2 mL of flux buffer. The chemokine ligands may be used over a range from 1 to 100 nM. The emission ratio was plotted over time (typically 2-3 minutes). Ligand blocking compounds of interest (up to 10 μM) were added at 10 seconds, followed by chemokines at 60 seconds (i.e., MIP-1α; R&D Systems; Minneapolis, Minn.) and control chemokine (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) at 150 seconds.
3. Chemotaxis Assays
Chemotaxis assays were performed using 5 m pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). CCR1 chemokine ligands (i.e., MIP-1α, CCL15/Leukotactin; R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of CCR1 mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber was loaded with 29 μt of chemokine (i.e., 0.1 nM CCL15/Leukotactin) and varying amounts of compound; the top chamber contained 100,000 THP-1 or monocyte cells in 20 μl. The chambers were incubated 1-2 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
C. Identification of Inhibitors of CCR1
1. Assay
To evaluate small organic molecules that prevent the receptor CCR1 from binding ligand, an assay was employed that detected radioactive ligand (i.e, MIP-1α or CCL15/Leukotactin) binding to cells expressing CCR1 on the cell surface (for example, THP-1 cells or isolated human monocytes). For compounds that inhibited binding, whether competitive or not, fewer radioactive counts are observed when compared to uninhibited controls.
THP-1 cells and monocytes lack other chemokine receptors that bind the same set of chemokine ligands as CCR1 (i.e., MIP-1α, MPIF-1, Leukotactin, etc.). Equal numbers of cells were added to each well in the plate. The cells were then incubated with radiolabeled MIP-1α. Unbound ligand was removed by washing the cells, and bound ligand was determined by quantifying radioactive counts. Cells that were incubated without any organic compound gave total counts; non-specific binding was determined by incubating the cells with unlabeled ligand and labeled ligand. Percent inhibition was determined by the equation:
% inhibition=(1−[(sample cpm)−(nonspecific cpm)]/[(total cpm)−(nonspecific cpm)])×100.
2. Dose Response Curves
To ascertain a compound of interest's affinity for CCR1 as well as confirm its ability to inhibit ligand binding, inhibitory activity was titered over a 1×10−10 to 1×10−4 M range of compound concentrations. In the assay, the amount of compound was varied; while cell number and ligand concentration were held constant.
3. CCR1 Functional Assays
CCR1 is a seven transmembrane, G-protein linked receptor. A hallmark of signaling cascades induced by the ligation of some such receptors is the pulse-like release of calcium ions from intracellular stores. Calcium mobilization assays were performed to determine if the CCR1 inhibitory compounds of interest were able to also block aspects of CCR1 signaling. Compounds of interest able to inhibit ligand binding and signaling with an enhanced specificity over other chemokine and non-chemokine receptors were desired.
Calcium ion release in response to CCR1 chemokine ligands (i.e., MIP-1α, MPIF-1, Leukotactin, etc.) was measured using the calcium indicator INDO-1. THP-1 cells or monocytes were loaded with INDO-1/AM and assayed for calcium release in response to CCR1 chemokine ligand (i.e., MIP-1α) addition. To control for specificity, non-CCR1 ligands, specifically bradykinin, was added, which also signals via a seven transmembrane receptor. Without compound, a pulse of fluorescent signal will be seen upon MIP-1α addition. If a compound specifically inhibits CCR1-MIP-1α signaling, then little or no signal pulse will be seen upon MIP-1α addition, but a pulse will be observed upon bradykinin addition. However, if a compound non-specifically inhibits signaling, then no pulse will be seen upon both MIP-1α and bradykinin addition.
One of the primary functions of chemokines is their ability to mediate the migration of chemokine receptor-expressing cells, such as white blood cells. To confirm that a compound of interest inhibited not only CCR1 specific binding and signaling (at least as determined by calcium mobilization assays), but also CCR1 mediated migration, a chemotaxis assay was employed. THP-1 myelomonocytic leukemia cells, which resemble monocytes, as wells as freshly isolated monocytes, were used as targets for chemoattraction by CCR1 chemokine ligands (i.e., MIP-1α, CCL15/leukotactin). Cells were place in the top compartment of a microwell migration chamber, while MIP-1α (or other potent CCR1 chemokine ligand) and increasing concentrations of a compound of interest was loaded in the lower chamber. In the absence of inhibitor, cells will migrate to the lower chamber in response to the chemokine agonist; if a compound inhibited CCR1 function, then the majority of cells will remain in the upper chamber. To ascertain a compound of interest's affinity for CCR1 as well as to confirm its ability to inhibit CCR1 mediated cell migration, inhibitory activity was titered over a 1×10−10 to 1×10−4 M range of compound concentrations in this chemotaxis assay. In this assay, the amount of compound was varied; while cell number and chemokine agonist concentrations were held constant. After the chemotaxis chambers were incubated 1-2 hours at 37° C., the responding cells in the lower chamber were quantified by labeling with the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content, and by measuring with a Spectrafluor Plus (Tecan). The computer program Prism from GraphPad, Inc. (San Diego, Calif.) was used to calculate IC50 values. IC50 values are those compound concentrations required to inhibit the number of cells responding to a CCR1 agonist by 50%.
4. In Vivo Efficacy
a) Rabbit Model of Destructive Joint Inflammation
To study the effects of compounds of interest on inhibiting the inflammatory response of rabbits to an intra-articular injection of the bacterial membrane component lipopolysaccharide (LPS), a rabbit model of destructive joint inflammation is used. This study design mimics the destructive joint inflammation seen in arthritis. Intra-articular injection of LPS causes an acute inflammatory response characterized by the release of cytokines and chemokines, many of which have been identified in rheumatoid arthritic joints. Marked increases in leukocytes occur in synovial fluid and in synovium in response to elevation of these chemotactic mediators. Selective antagonists of chemokine receptors have shown efficacy in this model (see Podolin, et al., J. Immunol. 169(11):6435-6444 (2002)).
A rabbit LPS study is conducted essentially as described in Podolin, et al. ibid., female New Zealand rabbits (approximately 2 kilograms) are treated intra-articularly in one knee with LPS (10 ng) together with either vehicle only (phosphate buffered saline with 1% DMSO) or with addition of a compound of interest (dose 1=50 μM or dose 2=100 μM) in a total volume of 1.0 mL. Sixteen hours after the LPS injection, knees are lavaged and cells counts are performed. Beneficial effects of treatment were determined by histopathologic evaluation of synovial inflammation. Inflammation scores are used for the histopathologic evaluation: 1-minimal, 2-mild, 3-moderate, 4-moderate-marked.
b) Evaluation of a Compound of Interest in a Rat Model of Collagen Induced Arthritis
A 17 day developing type II collagen arthritis study is conducted to evaluate the effects of a compound of interest on arthritis induced clinical ankle swelling. Rat collagen arthritis is an experimental model of polyarthritis that has been widely used for preclinical testing of numerous anti-arthritic agents (see Trentham, et al., J. Exp. Med. 146(3):857-868 (1977), Bendele, et al., Toxicologic Pathol. 27:134-142 (1999), Bendele, et al., Arthritis Rheum. 42:498-506 (1999)). The hallmarks of this model are reliable onset and progression of robust, easily measurable polyarticular inflammation, marked cartilage destruction in association with pannus formation and mild to moderate bone resorption and periosteal bone proliferation.
Female Lewis rats (approximately 0.2 kilograms) are anesthetized with isoflurane and injected with Freund's Incomplete Adjuvant containing 2 mg/mL bovine type II collagen at the base of the tail and two sites on the back on days 0 and 6 of this 17 day study. A compound of interest is dosed daily in a sub-cutaneous manner from day 0 till day 17 at a efficacious dose. Caliper measurements of the ankle joint diameter were taken, and reducing joint swelling is taken as a measure of efficacy.
Claims the benefit of Provisional Application Ser. No. 60/895,211, filed Mar. 16, 2007 which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60895211 | Mar 2007 | US |
