CD45 INHIBITORS

Abstract: Substituted naphthalenediones in accord with structural diagram (I): compositions thereof and methods for the use thereof, for the treatment of T cell mediated conditions such as autoimmune diseases and organ graft rejection. In compounds of the invention, wherein: Q<1> at each occurrence is independently selected from hydrogen, hydroxy, halogen, C(O)O(C1-C3) alkyl and C(O)phenyl, and Q<2> is selected from hydrogen, halogen, O-(C1-C3)alkyl, O-(C1-C3)alkenyl, phenyl, indolyl and naphthyl, where phenyl may be mono- or di-substituted with NO2 or halogen, and indolyl may be substituted with (C1-C3)alkyl or phenyl.

Description: CD45 INHIBITORS Background Field of the Invention Compounds, compositions and methods for the treatment of immunologically-related diseases and disorders such as autoimmune disorders and organ graft rejection. Related Art Action of the immune system is known to be involved in immunologically-related diseases and disorders such as autoimmune disorders and in organ graft rejection ("OGR"). Hematopoietic, thymus-derived cells, (so-called "T cells") have an important and pervasive role as regulators and effectors of the functions of the immune system. Hematopoietic cells, and T cells in particular have on their surfaces a major transmembrane glycoprotein designated CD45, characterized by a cluster of antigenic determinants. CD45 is also known as leukocyte common antigen ("LCA"). The cytosolic portion of CD45 has protein tyrosine phosphatase ("PTP") activity and CD45 activity is known to be essential for TCR initiated T cell activation. Studies in CD45-defιcient cell lines have shown that CD45 is a positive regulator of the T-Cell Receptor ("TCR") and that CD45 functions in TCR regulation by dephosphorylating the src kinases p56 /cλ and p59^ ' ", which allows autophosphorylation of the positive regulatory site on these enzymes; these reactions lead to downstream events and ultimately to T cell activation. Available treatments for autoimmune disorders and OGR have therapeutic disadvantages. For example, Cyclosporin A, the drug most commonly used to treat OGR, has renal and CNS toxicity. Summary of the Invention Potent inhibitors of CD45 have been discovered. Such inhibitors are useful for the treatment of various autoimmune disorders as well as for treatment of OGR. Inhibition of the phosphatase activity of CD45 by compounds of the present invention has been shown by incubating the cytosolic portion of CD45 with the compounds and /_ -nitrophenyl phosphate (pNPP), a phosphatase substrate. Spectrophotometric monitoring has shown that the liberation of -nitrophenol from the substrate by CD45 is inhibited in the presence of the compounds disclosed herein. Inhibition of the phosphatase activity of CD45 by compounds of the present invention has also been shown using a p56 / ' carboxy-terminal phosphorylated peptide as a substrate. Compounds of the present invention have also been shown to inhibit proliferation of T cells in a T-cell proliferation assay. Compounds of the present invention are naphthalenediones in accord with structural diagram I: I wherein: Q 1 at each occurrence is independently selected from hydrogen, hydroxy. halogen, C(O)O(Cι-C 3 )alkyl and C(O)phenyl, and Q is selected from hydrogen, halogen, O-(C--C 3 )alkyl, 0-(Cι-C 3 )alkenyl, phenyl, indolyl and naphthyl, where phenyl may be mono- or di-substituted with NO 2 or halogen, and indolyl may be substituted with (Cι-C 3 )alkyl or phenyl. Particular compound of the invention are 4-(4-bromo-phenyl)-[l,2]naphthoquinone; 4- (3,5-dichloro-phenyl)-[l,2]naphthoquinone; 4-(3-nitro-phenyl)-[l,2]naphthoquinone; 5,6- dioxo-5,6-dihydro-naphthalene-l-carboxylic acid methyl ester, and 5,6-dioxo-5,6-dihydro- naphthalene-2-carboxylic acid methyl ester. Compounds of the present invention are ligands of CD45 which, when bound, inhibit the activity of the protein tyrosine phosphatase (PTP) activity of the cytosolic portion of CD45. Binding of a compound of the present invention to CD45 inhibits the activity of CD45 essential for TCR initiated T cell activation. Thus, compounds of the invention inhibit the positive regulation of the TCR that leads to downstream events and T cell activation. Compounds of the present invention are useful to suppress the action of the immune system in immunologically-related diseases and disorders such as autoimmune disorders and organ graft rejection and to inhibit the action of T cells as functional regulators and effectors of the immune system. The present invention also encompasses compositions made with compounds described herein useful for the treatment of immunologically-related diseases and disorders and methods utilizing such compositions for treating such disorders. Detailed Description of the Invention As used herein, (Cι-C 4 )alkyl has its conventionally- understood meaning and particularly means linear or branched hydrocarbon chains having from one to four carbon atoms and thus includes methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and the like. As used herein, halo(Cι-C 4 )alkyl has its conventionally-understood meaning and particularly means (C--C 4 )alkyl as used herein wherein hydrogen atoms have been replaced by halogen atoms and thus includes monochloromethyl, trifluoromethyl, difluoroethyl, trifluoropropyl, perfluoro(C*-C4)alkyl, and the like. As used herein, perfluoro(Cι-C )alkyl has its conventionally-understood meaning and particularly means (Cι-C 4 )alkyl as used herein wherein each hydrogen atom has been replaced by a fluorine atom and thus includes trifluoromethyl. As used herein, (CH 2 ) n has its conventionally-understood meaning and particularly means linear hydrocarbon chains having from one to n carbon atoms and thus includes methylene, ethylene, propylene, n-butylene groups, and the like. As used herein, the terms halogen, halo, or halide have their conventionally- understood meanings and particularly mean chlorine, bromine, iodine or fluorine. As used herein, the term "from the range 1 to 6" or the like, means any integral value in the stated range, in this example 1 , 2, 3, 4, 5 or 6. Definitions of terms: DMF. N,N-dimethylformamide; THF, tetrahydrofuran; TLC, thin-layer chromatography; ΝMR, nuclear magnetic resonance; TFA. trifluoroacetic acid; HPLC, high performance liquid chromatography; DMAP, 4-dimethylaminopyridine; DMSO, dimethylsulfoxide; IC 50 , concentration giving 50% inhibition; CC 50 , concentration giving 50% cytotoxicity; ΝD, not determined. HPLC method used: Analytical HPLC using an HP 1 100 HPLC, with a C, 8 Dynamax column (5 cm x 4.6 mm, 3 μM particle size, 100 A pore size), flow rate of 0.5 mL/min, 20%- 60% CH 3 CΝ in H 2 O over 7.5 min, holding at 60% CH 3 CN for 2.5 min, while monitoring at 254 and 210 nm. Examples Example 1 : [l,2]-Naphthoquinone was purchased from Acros Organics and used as received. Examples 2 to 4: The compounds of examples 2 to 4 were prepared substantially in accordance with the procedures disclosed in Takuwa, A.; Soga, O.; Iwamoto, H.; Maruyama, K. Bull. Chem. Soc. Jpn. 1986, 59, 2959-2961, which procedures are incorporated herein by reference. The physical properties of the compounds are disclosed in the reference. Example 2: 4-Ethoxy-[l,2]naphthoquinone. Example 3 : 4-Methoxy-[l ,2]naphthoquinone. Example 4: 4-Allyloxy-[l,2]naphthoquinone. Examples 5 and 6: The compounds of examples 5 and 6 were prepared substantially in accordance with the procedures disclosed in Perumal, P. T.; Bhatt, M. V. Synthesis 1980, 943-945, which procedures are incorporated herein by reference. The physical properties of the compounds are disclosed in the reference. Example 5: 4-Chloro-[l,2]naphthoquinone. Example 6: 4-Bromo-[l,2]naphthoquinone. Examples 7 and 8: The compounds of examples 7 and 8 were prepared substantially in accordance with the procedures disclosed in Henrion, J.-C; Jacquet, B.; Hocquaux, M; Barre, G.; Hedayatullah. M.; Lion, C. Bull. Soc. Chim. Belg. 1996, 105, 415-418. which procedures are incorporated herein by reference. The physical properties of the compounds are disclosed in the reference. Example 7: 4-(l -Methyl- lH-indol-3-yl)-[l ,2]naphthoquinone. Example 8: 4-(2-Phenyl-lH-indol-3-yl)-[l ,2]naphthoquinone. Example 9: 4-(2-Chloro-phenyl)-[l,2]naphthoquinone: To a solution of 4-bromo-[l ,2]naphthoquinone (350 mg, 1.48 mmol) in TΗF (20 mL) and Η?0 (5 mL) was added 2-chlorophenylboronic acid (231 mg, 1.48 mmol). followed by tri- -tolylphosphine (45 mg, 148 μmol) and K CO 3 (614 mg, 4.44 mmol). The mixture was deoxygenated with bubbling N 2 for about ten minutes, at which time the N 2 line was removed and trz ' _.(dibenzylideneacetone)dipalladium(0) (68 mg, 74 μmol) was added. The resultant mixture was heated to reflux for 2 hours under N 2 , at which point no starting bromide was detectable by TLC (hexanes: ethyl acetate, 1 : 1, v/v). The mixture was cooled to room temperature and the THF evaporated under reduced pressure. The dried material was dissolved in ethyl acetate, washed sequentially with saturated aqueous ammonium chloride, H 2 0 and brine, dried over Na 2 S0 4 , filtered, and evaporated under reduced pressure. The isolated material was chromatographed on silica gel (hexanes-ethyl acetate, 4: 1 , v/v) and dried to yield the product, 4-(2-chloro-phenyl)-[l ,2]naphthoquinone, as an orange solid. Η NMR (300 MHz, CDC1 3 ) δ 8.21 (IH, dd, J= 6.9, 2.1 Hz), 7.56-7.52 (3H. m), 7.48-7.44 (2H. m), 7.44 (IH, m), 6.91 (IH, dd, J= 2, 6 Hz), 6.39 (IH, s); HPLC: 7.45 min. Compounds of examples 10 to 14 inclusive were made by the method of Example 9, by utilizing the appropriate boronic acid. Example 10: 4-(4-Bromo-phenyl)-[l ,2]naphthoquinone: Orange solid; 'H NMR (300 MHz, CDC1 3 ) δ 8.22 (IH, dd, J= 1.8, 7.2 Hz), 7.67 (2H, d, J= 6.6 Hz), 7.63-7.60 (2H, m), 7.33 (2H, d, J= 6.6 Hz), 7.25 (IH, dd, J= 2. 8 Hz), 6.41 (IH. s); HPLC: 8.25 min. Example 1 1 : 4-Phenyl-[1.2]naphthoquinone: Orange-red solid; Η NMR (300 MHz, CDC1 3 ) δ 8.22 (IH, dd, J = 1.8. 7.2 Hz), 7.62- 7.52 (5H, m), 7.46-7.44 (2H, m), 7.30 (IH, dd, J= 1.2, 7.5 Hz), 6.43 (IH, s); HPLC: 6.90 min Example 12: [1 ,1 ']Binaphthalenyl-3 ,4-dione : Orange solid; 1H NMR (300 MHz, CDC1 3 ) δ 8.40 (IH, dd, J= 1.5, 7.5 Hz), 8.00 (IH, d, J = 8.1 Hz). 7.96 (IH, d. J= 8.1 Hz). 7.72 (IH, d, J = 8.4 Hz), 7.62 (IH, d, J = 7.2 Hz). 7.57 (IH, d, J- 7.8 Hz), 7.53-7.42 (4H, m), 6.83 (IH, d, J= 8.3 Hz), 6.55 (IH. s); HPLC: 8.27 min. Example 13: 4-(3.5-Dichloro-phenyl)-[l ,2]naphthoquinone: Orange solid; 1H NMR (300 MHz, CDCI3) δ 8.23 (IH, dd, J= 1.8, 7.2 Hz), 7.67-7.56 (2H. m), 7.53 (IH, m), 7.41 (IH, m), 7.33 (IH, m), 7.20 (IH. d, J = 7.5 Hz). 6.40 (IH. s): HPLC: 9.0 min. Example 14: 4-(3-Nitro-phenyl)-[1.2]naphthoquinone: Orange solid; Η NMR (300 MHz, CDC1 3 ) δ 8.41 (IH. d. J= 7.2 Hz), 8.34 (IH. s), 8.27 (IH, m). 7.78 (2H, m). 7.63 (2H, m), 7.15 (IH. d, J = 7.7 Hz), 6.46 (IH, s); HPLC: 5.78 min. Examples 15 to 17: The compounds of examples 15 to 17 were prepared substantially in accordance with the procedures disclosed in Barton, D.H.R.; Brewster, A.G.: Ley, S.V.; Read, CM.; Rosenfeld, M.N. J. Chem. Soc Perkin Trans. 1 1981, 1473-1476, which procedures are incorporated herein by reference. Example 15: 6-Benzoyl-[l,2]naphthoquinone: Orange solid; 1H NMR (300 MHz, CDC1 3 ) δ 8.23 (IH, d, J= 7.8 Hz), 7.86 (IH, dd, J = 1.5. 7.8 Hz), 7.83-7.79 (3H, m), 7.67 (IH, m), 7.57-7.51 (3H, m), 6.54 (IH, d, J= 10.2 Hz); HPLC: 5.93 min. Example 16: 5,6-Dioxo-5.6-dihydro-naphthalene-l-carboxylic acid methyl ester: This compound was prepared using 5-carbomethoxy-2-naphthol as a starting material. The starting material was prepared according to the method of Anderson, L.C.; Thomas, D.G. J. Am Chem. Soc. 1943, 65, 234-238. Orange solid; Η NMR (300 MHz, CDC1 3 ) δ 8.62 (IH, d. J= 1 1 Hz), 8.29 (IH, d, J = 7.8 Hz), 8.20 (IH. d. J= 7.8 Hz), 7.58 (IH, t, J- 7.8 Hz), 6.56 (IH. d, J= 1 1 Hz), 3.99 (3H. s); HPLC: 3.69 min. Example 17: 5,6-Dioxo-5,6-dihydro-naphthalene-2-carboxylic acid methyl ester: This compound was prepared using 6-carbomethoxy-2-naphthol as a starting material. The starting material was prepared according to the method of Anderson, L.C.; Thomas, D.G. J. Am Chem. Soc. 1943, 65, 234-238. Orange solid; Η NMR (300 MHz, CDC1 3 ) δ 8.17 (2H, m). 8.05 (IH, s), 7.52 (IH, d, J= 10.2 Hz), 6.53 (IH, d, J= 10.2 Hz), 3.99 (3H, s); HPLC: 3.61 min. Example 18: 7-Hydroxy-[1.2]naphthoquinone: This compound was prepared substantially in accordance with the procedures disclosed in Teuber, H.-.T.; Gotz, N. Chem. Ber. 1954. 1236-1251. which procedures are incorporated herein by reference. Red solid; Η NMR (300 MHz, DMSO-d 6 ) δ 10.50 (IH, broad s), 7.56 (IH, d, J= 9.8 Hz), 7.40 (IH, d, J= 8.1 Hz. 7.31 (IH, d, J= 2.7 Hz). 7.05 (IH, dd, J= 8.1, 2.7 Hz), 6.16 (IH, d, J= 9.9 Hz); Anal. Calcd. For C,oH 6 O 3 -0.1H 2 0: C, 68.26; H, 3.55. Found: C 67.96, 67.89; H, 3.64, 3.65. ASSAYS FOR BIOLOGICAL ACTIVITY Method A: Phosphatase assay using pNPP as substrate CD45 enzyme was obtained from BIOMOL (Plymouth Meeting. PA). Phosphatase activity was assayed in a buffer containing final concentrations of 25 mM imidazole at pH 7.0, 50 mM NaCl, 2.5 mM ethylenediaminetetraacetic acid ("EDTA"). 5 mM dithiothreitol ("DTT") and 10 μg/mL bovine serum albumin ("BSA") using /?NPP as a substrate. Compounds were tested in a range from 30 to 0.01 μM, with a final concentration of 1 or 5% dimethylsulfoxide ("DMSO"), depending on the compound solubility. Activity was measured by following the increase in absorbance at 405 nm using a SpectraMax Plus spectrophotometric plate reader (Molecular Devices, Sunnyvale, CA). Method B: Cytotoxicity Assay: Calcein-AM (Molecular Probes, Eugene, OR) uptake, as a quantitative measure of cell viability, was used to evaluate the toxic effect of compounds on T cells. Briefly, PBMC were treated for 3-7 days with 3-10 μg/ml PHA, a potent T-cell mitogen. to preferentially expand the T-cell population. (Bradley, Linda M. Cell Poliferation in Selected Methods in Cellular Immunology. Eds. Mishell. B.B. and Shiigi. S.M., W.H. Freeman and Co.. San Francisco. 1980.) The T-cell lymphoblasts were purified by separation over Lymphoprep, plated at 2 x 10 ' Jwell in a round bottom 96-well plate containing RPMI with compound and incubated overnight at 37 °C in an incubator containing 5% CO 2 . The dilution scheme and culture media were the same as those used in the T-cell proliferation assay. After the incubation period, cells were washed with Dulbecco's phosphate- buffered saline (D-PBS) and incubated with lμM Calcein-AM for 30-45 min in D-PBS as described in the technical sheet provided with The LIVE/DEAD Viability/Cytotoxicity Kit from Molecular Probes. Percent viability was assessed on a fluorescent plate reader (excitation filter 485/20 nm: emission filter 530/25 nm) where the 100% control value is the fluorescence intensity observed in the absence of test compound. Method C: Phosphatase assay using lck 10-mer as substrate. Phosphatase activity was assayed in 96 well plates in a buffer containing final concentrations of 25 mM HEPES at pH 7.2. 5 mM DTT and 10 μg/mL BSA, using the lck carboxy-terminal peptide TEGQpYQPQP as the substrate (Cho, H., Krishnaraj. R.. Itoh. M.. Kitas, E., Bannwarth, W.. Saito, H.. Walsh, C.T. 1993. Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTPb. LAR. and CD45) toward the phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors. Protein Science 2:977-984). Compounds were tested in a range from 30 to 0.01 μM in a final concentration of 5% DMSO. Enzyme was incubated with substrate, with or without compound, at room temperature for 1.5 h. At the end of the incubation period, BIOMOL "Green Reagent" (BIOMOL, Plymouth Meeting, PA) was added to each well, the plates incubated at room temperature for 30 min and absorbance read at 620 nm. Method D: Cell isolation and T cell proliferation assay: Whole blood was obtained from healthy human blood donors. Peripheral blood mononuclear cells ("PBMC") were isolated using Lymphoprep density-gradient centrifugation (Nycomed Amersham, Oslo, Norway), washed, counted and resuspended at 2 X 10 6 cells/ mL in RPMI 1640 medium containing glutamine, 0.1 mg/mL gentamycin and 10% heat inactivated human serum. PBMC were transferred to 96- well plates (2 X 10 5 cells/well) containing compound or vehicle control, with the final concentration of DMSO not to exceed 0.3%), and incubated for 1 hour before addition of the activating anti-CD3 antibody, OKT3 (30 ng/mL). After 24 hours in culture, the cells were pulsed with [ 3 H]thymidine (1 μCi/well) overnight and harvested the next day onto 96-well Packard GF/C filter plates using a Packard Cell Harvester (Packard Instruments, Meriden, CT). The filter plate was dried, the bottom of the plate sealed, 25 μL of Microscint 20 scintillation fluid added to each well, the top of the plate sealed with TopSeal-A, and the plate counted on a Packard TopCount. The data from the TopCount is transferred into Excel 5 (Microsoft, Redmond, WA) and formatted for EC 50 determination using Prism software (GraphPad Software, San Diego, CA). Table 1 shows the inhibition of CD45 activity in the /. NPP asssay and the lck assay certain compounds of the present invention. Inhibition in the T cell proliferation assay, as well as results from T cell cytotoxicity assay are shown. Table 1 :

Claims: CLAIMS 1. A method for treating immunologically-related diseases, autoimmune disorders and organ graft rejection comprising administering to a subject suffering therefrom an effective amount of a compound in accord with structural diagram I: I or tautomers thereof or pharmaceutically-acceptable salts thereof: wherein: Q 1 at each occurrence is independently selected from hydrogen, hydroxy, halogen, C(O)O(C ! -C 3 )alkyl and C(O)phenyl. Q 2 is selected from hydrogen, halogen, O-(Cι-C 3 )alkyl, O-(Cι-C 3 )alkenyl, phenyl, indolyl and naphthyl, where phenyl may be mono- or di-substituted with NO 2 or halogen, and indolyl may be substituted with (CrC 3 )alkyl or phenyl. 2. A method according to Claim 1, wherein in a compound in accord with structural diagram I: Q 2 is selected from O-methyl, O-ethyl, O-CH 2 CH=CH 2 , 3-(N-methyl-indole), 3-(2- phenyl-indole), 1-naphthyl, phenyl, 2-chlorophenyl, 4-bromophenyl, 3,5-dichlorophenyl and 3-nitrophenyl, and Q 1 is hydrogen. 3. A pharmaceutical composition for use in the method of Claim 1, comprising a pharmaceutically-acceptable excipient or diluent together with a compound in accord with structural diagram I: I or tautomers thereof or pharmaceutically-acceptable salts thereof: wherein: Q 1 at each occurrence is independently selected from hydrogen, hydroxy, halogen, C(O)O(Cι-C 3 )alkyl and C(O)phenyl. Q 2 is selected from hydrogen, halogen, O-(Cι-C 3 )alkyl, O-(Cι-C 3 )alkenyl, phenyl, indolyl and naphthyl, where phenyl may be mono- or di-substituted with NO 2 or halogen, and indolyl may be substituted with (Cι-C 3 )alkyl or phenyl. 4. A compound selected from: 4-(4-bromo-phenyl)-[l,2]naphthoquinone; 4-(3,5-dichloro-phenyl)-[l,2]naphthoquinone; 4-(3-nitro-phenyl)-[l,2]naphthoquinone; 5,6-dioxo-5,6-dihydro-naphthalene-l-carboxylic acid methyl ester, and 5,6-dioxo-5,6-dihydro-naphthalene-2-carboxylic acid methyl ester. 5. A pharmaceutical composition comprising an effective amount of a compound according to Claim 4, and a pharmaceutically-acceptable excipient or diluent. 6. A method for treating immunologically-related diseases, autoimmune disorders and organ graft rejection comprising administering to a subject suffering therefrom an effective amount of a compound according to Claim 4.

Inventors: CHAPDELAINE MARC JEROME, KNAPPENBERGER KATHERINE, STEELMAN GARY, SUCHARD SUZANNE, SYGOWSKI LINDA, URBANEK REBECCA, VEALE CHRIS ALLAN, CHAPDELAINE, MARC, JEROME, KNAPPENBERGER, KATHERINE, STEELMAN, GARY, SUCHARD, SUZANNE, SYGOWSKI, LINDA, URBANEK, REBECCA, VEALE, CHRIS, ALLAN
Applicants: ASTRAZENECA AB, ASTRAZENECA UK LTD, CHAPDELAINE MARC JEROME, KNAPPENBERGER KATHERINE, STEELMAN GARY, SUCHARD SUZANNE, SYGOWSKI LINDA, URBANEK REBECCA, VEALE CHRIS ALLAN, ASTRAZENECA UK LIMITED, CHAPDELAINE, MARC, JEROME, KNAPPENBERGER, KATHERINE, STEELMAN, GARY, SUCHARD, SUZANNE, SYGOWSKI, LINDA, URBANEK, REBECCA, VEALE, CHRIS, ALLAN
Application year: 2000
Main classification: A61K31
IPC codes: A61K31, A61P37, A61P43, C07C46, C07C50, C07C69, C07C205, C07D209