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US005763470A United States Patent [19] [11] Patent Number: 5,763,470 Tang et a]. [45] Date of Patent: Jun. 9, 1998
[54] BENZOPYRAN COMPOUNDS AND Arteaga. C. L.. Kitten. L. J.. Coronado. E. B.. Jacobs. S.. METHODS FOR THEIR USE Kull. F. C. 1.. Allred. D. C.. and Osborne. C. K. (1989). Blockade of the type I somatomedin receptor inhibits growth [75] Inventors: Peng Cho Tang. Moraga; Gerald of human breast cancer cells in athyrnic mice. J. Clin. Invest. McMahon. Kenwood. both of Calif. 84. 1418-1423. [73] Assignee: Sugen Inc. Redwood City. Calif. Baserga. R. (1994). Oncogenes and the strategy of growth factors. Cell 79. 927-930. Baserga. R. (1995). The insulin-like growth factor I recep [21] Appl. No.1 479,876 tor: a key to tumor growth? Cancer Research 55. 249-252. [22] Filed: Jun. 7, 1995 Bolen. J. B.. Rowlcy. R. B.. Spana. C.. and Tsygankov. A. Y. (1992). The Src family of tyrosine protein kinases in [51] Int. Cl.6 ...... A61K 31/35; A61K 31/38; A61K 31/41 hemopoietic signal transduction. FASEB Journal 6. [52] US. Cl...... 514/406; 549/60; 549/404; 3403-3409. 548/364.4: 514/444; 514/456 Bolen. J. B. (1993). Nonreceptor tyrosine protein lcinases. [58] Field of Search ...... 549/404. 60; 514/456. Oncogene 8. 2025-2031. 514/406. 444; 548/364.4 Coppola. D.. et al. (1994). A functional insulin-like growth factor I receptor is required for the mitogenic and trans [56] References Cited forming activities of the epidermal growth factor receptor. Molecular and Cellular Biology 14. 4588-4595. U.S. PATENT DOCUMENTS Dati. C.. Antoniotti. S.. Taverna. D.. Perroteau. 1.. and De 4,665,202 5/1987 Rimbault et a1...... 549/402 Bortoli. M. (1990). Inhibition of c-erB-2 oncogene expres sion by estrogens in human breast cancer cells. Oncogene 5. 4,966,8495,217,9995,302,606 10/19906/19934/1994 ValleeLevitzkiSpada et etal.a1. a1...... 1001-1006. Decker. T.. and Lohmann-Mattes. M.-L. (1988). A quick 5,330,992 7/1994 Eissenstat et a1...... 514/312 and simple method for quantitation of lactate dehydrogenase FOREIGN PATENT DOCUMENTS release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. of 1mm. Methods 15. 566 226 10/1993 European Pat. 01f. . WO 91/15495 10/1991 61-69. WO 92/20642 11/1992 Dickson. R. B.. Salomon. D. S.. and Lippman. M. E. (1991). WO 92/21660 12/1992 Tyrosine kinase receptor-nuclear protooncogene interac WO 94/03427 2/1994 tions in breast cancer. Cancer Treatment and Research 61. WO 92/11202 5/1994 249-273. WO 94/14808 7/1994 Fantl. W. J.. et a1. (1992). Distinct Phosphotyrosines on a UT HER PUBLICATIONS growth factor receptor bind to speci?c molecules that medi ate di?erent signaling pathways. Cell 69. 413-423. Perrella et al.. J. Med. Chem.. Jul. 1994. 37(14). 2232-7. Fendly. B. M. (1990). Characterization of murine mono Registry No. 158521-85-8. clonal antibodies reactive to either the human epidermal Fatome et 21.. CA 85:13678 (1976). Registry Nos. growth factor receptor or HERZ/neu gene product. Cancer 57543-84-7. 57543-85-8. 57543-86-9. 57543-87-0. Research 50. 1550-1558. 57543-88-1. 57543-90-5. 59528-35-7. Fingl. E.. and Woodbury. D. M. (1975). The Pharmacologi Sakakibara et al.. CA 9038751 (1979). Registry Nos. cal Basis of Therapeutics Chapter 1. 1-46. 57543-84-7. 68792-48-3. Floege. J.. Eng. 13.. Young. B. A.. and Johnson. R. J. (1993). Factors involved in the regulation of mesangial cell prolif Deshpande et a1.. CA 106:4415 (1987). Registry Nos. eration in vitro and in vivo. Kidney International 43. 57543-84-7. 81289-21-6. 86628-45-7. S-47-S-54. Varma et al.. CA 106:49966 (1987). Registry Nos. Goldring. M. B.. and Goldring. S. R. (1991). Cytokines and 106202-43-1. 106202-44-2. 106202-45-3. 106202-46-4. cell growth control. Critical Reviews in Eukaryotic Gene 106230-54-0. 106230-55-1. Expression 1. 301-326. Greif et al.. CA 115:161210 (1991). Registry No. 136380-03-5. (List continued on next page.) Ono et al.. J. Chem. Soc.. Chem. Commun. (1993). (23). Primary Examiner-Johann Richter 1781-2 and CA 120:217189 (1994). Registry Nos. Assistant Examiner-Laura L. Stockton 57543-84-7. 57543-85-8. 57543-87-0. 153651-26-4. Attorney, Agent, or Firm-Pennie & Edmonds LLP Rene et al.. Eur. J. Med. Chem. -Chimica Therapeutica. (1975). 10(1). pp. 72-78 and CA 84:17075k(1996). [57] ABSTRACT Mathur et al.. (CA 9917052611). 1983. The present invention relates to organic molecules capable Mitchell et al.. (CA 108:166840x). 1988. of modulating tyrosine kinase signal transduction in order to Ono et 211.. (CA 1223144541‘). 1995. regulate. modulate and/or inhibit abnormal cell Akbasak. A.. and Sunar-Akbasak. B. (1992). Oncogenes: proliferation. cause or consequence in the development of glial tumors. Journal of the Neurological Sciences 111. 119-133. 8 Claims, N0 Drawings 5,763,470 Page 2
OTHER PUBLICATIONS Ozzello. L.. and Sordat. M. (1980). Behavior of tumors produced by transplantation of human mammary cell lines in Gottardis. M. M.. Robinson. S. P.. and Jordan. C. V. (1988). athymic nude mice. Europ. J. Cancer 16. 553-559. Estradiol-stimulated growth of MCF-7 tumors implanted in Plowman. G. D. et al. (1994). Receptor tyrosine kinases as athyrnic mice: A model to study the tumoristatic action of targets for drug intervention. DN&P 7. 334-339. tamoxifen. J. Steriod Biochern. 30. 311-314. Rygaard. J.. and Poulson. C. O. (1969). Heterotransplanta Honegger. A. M.. et al. (1987). Point mutation at the KI'P tion of a human malignant tumour to “nude” mice. Acta. binding site of EGF receptor abolishes protein-tyrosine path. microbiol scand. 77. 758-760. lcinase activity and alters cellular routing. Cell 51. 199-209. Sandberg-Nordqvist. A.-C.. Stahlbom. P.-A.. Reinecke. M.. Jellinek. D.. Green. L. S.. and Janjic. N. (1994). Inhibition Collins. P. V.. von Holst. H.. and Sara. V. (1993). Charac of receptor binding by high-a?inity RNA ligands to vascular terization of insulin-like growth facter l in human primary endothelial growth factor. Biochemistry 33. 10450-10456. brain tumors. Cancer Research 53. 2475-2478. Kendall. R. L.. and Thomas. K. A. (1993). Inhibition of Schlessinger. J.. and Ullrich. A. (1992). Growth factor vascular endothelial cell growth factor activity by an endog signaling by receptor tyrosine ldnases. Neuron 9. 383-391. enously encoded soluble receptor. Proc.Natl.Acad.Sci. 90. Seibert. K.. Sha?e. S. M.. Triche. T. J.. Whang-Peng. J. J.. 10705-10709. O’Brien. S. J.. Toney. J. H.. Hulf. K. K.. and Lippman. M. Kim. K. J.. Li. B.. Winer. J.. M.. Gillett. N.. E. (1983). Clonal variation of MCF-7 breast cancer cells in Phillips. H. S.. and Ferrara. N. (1993). Inhibition of vascular vitro and in athyrnic nude mice. Cancer Research 43. endothelial growth factor-induced angiogenesis suppresses 2223-2239. tumour growth in vivo. Nature 362. 841-844. Sha?e. S. M.. and Grantham. F. H. (1981). Role of hormones Kinsella. J. L.. Grant. D. 8.. Weeks. B. S.. and Kleinman. H. in the growth and regression of human breast cancer cells K. (1992). Protein lcinase C regulates endothelial cell tube (MCF-7) transplanted into athymic nude mice. JNCI 67. formation on basement membrane matrix. matrigel. Experi 51-56. mental Cell Research 199. 56-62. Skehan. P.. et a1. (1990). New Colorimetric cytotoxicity Koch. C. A.. Anderson. D.. Moran. M. R. Ellis. C.. and assay for anticancer-drug screening. Journal of the National Pawson. T. (1991). SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Sci Cancer Institute 82. 1107-1112. Slamon. D. J.. et al. (1989). Studies of the lIER-2Jneu ence 252. 668-674. Proto-oncogene in Human Breast and Ovarian Cancer. Kore. M.. et al. (1992). Overexpression of the epidermal Science 244. 707-712. growth factor receptor in human pancreatic cancer is asso ciated with concomitant increases in the levels of epidermal Songyang. Z.. et a1. (1993). SH2 domains recognize speci?c growth factor and transforming growth factor alpha. J. Clin. phosphopeptide sequences. Cell 72. 767-778. luv. 90. 1352-60. Songyang. Z.. et al. (1994). Speci?c motifs recognized by Korzseniewski. C.. and Callewaert. D. M. (1983). An the SH2 domains of Csk. 3BP2. fps/fes. GRB-Z. HCP. SHC. enzyme-release assay for natural cytotoxicity. Journal of Syk. and Vav. Molecular and Cellular Biology 14. Immunological Methods 64. 313-320. 2777-2785. Kumabe. T.. et a1. (1992). Ampli?cation of alpha-platelet Takano. S.. Gately. S.. Jiang. J. B.. and Brem. S. (1993). derived growth factor receptor gene lacking an exon coding Inhibition of angiogenesis by a novel diaminoanthraquinone for a portion of the extracellular region in a primary brain that inhibits protein kinase C. Mol. Bio. of the Cell 4. 358A. tumor of glial origin. Oncogene 7. 627-633. Torp. S. H.. Helseth. B.. Ryan. L.. Stolan. S.. Dalen. A.. and Lee. B. A.. and Donoghue. D. J. (1992). Intracellular reten Unsgaard. G. (1992). Expression of the epidermal growth tion of membrane-anchored v-sis protein abrogates auto factor receptor gene in human brain metastases. APMIS 100. crine signal transduction. The Journal of Cell Biology 118. 713-719. 1057-1070. Tuzi. N. L.. Venter. D. J.. Kumar. S.. Staddon. S. L.. Macaulay. V. M.. Everard. M. J.. Teale. J. D.. Trott. P. A.. Lemoine. N. R.. and Gullick. W. J. (1990). Expression of Van Wyk. J. J.. and Smith. I. E. (1990). Autocrine function growth factor receptors in human brain tumours. British J. of for insulin-like growth factor I in human small cell lung Cancer 63. 227-233. cancer cell lines and fresh tumor cells. Cancer Research 50. Ullrich. A.. and Schlessinger. J. (1990). Signal transduction 2511-2517. by receptors with tyrosine kinase activity. Cell 61. 203-212. Mariani. M.. et al. (1994). Inhibition of angiogenesis by Voller. A.. Bidwell. D.. and Bartlett. A. Enzyme-linked PCE26806. a potent tyrosine ln'nase inhibitor. Proceedings irnmunosorbent assay. Manual of Clinical Immunology. 2nd of the American Association for Cancer Research 35. 381. ed.. 359-371. Year Not Available. Mosmann. T. (1983). Rapid Colorimetric assay for cellular A. M.. et a1. (1991). Estrogen suppression of erbB2 growth and survival: application to proliferation and cyto expression is associated with increased growth rate of toxicity assays. Journal of Immunological Methods 65. ZR-75-l human breast cancer cells in vitro and in nude 55-63. mice. Int. J. Cancer 49. 616-623. Osborne. C. K.. Hobbs. K.. and Clark. G. M. (1985). Effect Wright. P. S.. Cross-Doersen. D.. Miller. J. A.. Jones. W. D.. of estrogens and antiestrogens on growth of human breast and Bitonti. A. J. (1992). Inhibition of angiogenesis in vitro cancer cells in athymic nude mice. Cancer Research 45. and in ovo with an inhibitor of cellular protein kinases. MDL 584-590. 27032. Journal of Cellular Physiology 152. 448-457. 5.763.470 1 2 BENZOPYRAN COMPOUNDS AND logical role of members of the P'I‘K family. their function in METHODS FOR THEIR USE differentiation processes. their involvement in tumorigenesis and in other diseases. the biochemical mechanisms under lying their signal transduction pathways activated upon 1. INTRODUCTION ligand stimulation and the development of novel drugs. The present invention relates to novel compounds capable Tyrosine kinases can be of the receptor-type (having of modulating. regulating and/or inhibiting tyrosine kinase extracellular. transmembraue and intracellular domains) or signal transduction and methods thereof. The present inven the non-receptor type (being wholly intracellular). tion is also directed to novel compounds and methods Receptor 'lyrosine Kinases. related to tyrosine kinases. whether of the receptor or 10 The KI‘Ks comprise a large family of transmembrane non-receptor class. for the prevention and/or treatment of receptors with diverse biological activities. The intrinsic disorders related to abnormal cell di?‘erentiation. prolifera function of RTKs is activated upon ligand binding. which tion and survival. results in phosphorylation of the receptor and multiple cellular substrates. and subsequently in a variety of cellular 2. BACKGROUND OF THE INVENTION responses. Ullrich & Schlessinger. 1990. Cell 6l:203-2l2. Protein tyrosine kinases (P'I‘Ks) comprise a large and At present. at least nineteen (19) distinct RI‘K subfamilies diverse class of proteins having enzymatic activity. The have been identified. One RTK subfamily. designated the PTKs play an important role in the control of cell growth and HER subfamily. is believed to be comprised of EGFR. differentiation (for review. see Schlessinger & Ullrich. 1992. HER2. HER3 and HER4. Ligands to the Her subfamily of Neuron 91383-391). receptors include epithelial growth factor (EGF). TGF-ot. For example. receptor tyrosine kinase mediated signal arnphiregulin. HB-EGF. betacellulin and heregulin. transduction is initiated by extracellular interaction with a A second family of RTKs. designated the insulin speci?c growth factor (ligand). followed by receptor subfamily. is comprised of the INS-R. the IGF- IR and the dirnerization. transient stimulation of the intrinsic protein 25 lR-R. A third family; the “PDGF” subfamily includes the tyrosine kinase activity and phosphorylation. Binding sites PDGF cc and [5 receptors. CSFIR. c-kit and FLK-Il. Another are thereby created for intracellular signal transduction subfamily of R'I‘Ks. identi?ed as the FLK family. is believed molecules and lead to the formation of complexes with a to be comprised of the Kinase insert Domain-Receptor fetal spectrum of cytoplasmic signalling molecules that facilitate liver kinase-l (KDR/FLK-l). the fetal liver kinase 4 (FLK the appropriate cellular response. (e.g.. cell division. meta 4) and the ?ns-like tyrosine kinase 1 (?t-1). Each of these bolic effects to the extrecellular microenvironment). receptors was initially believed to be receptors for hemato Schlessinger and Ullrich. 1992. Neuron 9:383-391. poietic growth factors. Two other subfamilies of Rl'Ks have With respect to receptor tyrosine kinases. it has been been designated as the FGF receptor family (FGFRl. shown also that tyrosine phosphorylation sites function as FGFR2. FGFlB and FGFR4) and the Met subfamily (c-met high-affinity binding sites for 81-12 (src homology) domains 35 and Ron). of signaling molecules. Fantl et al.. 1992. Cell 69:413-423; Because of the similarities between the PDGF and FLK Songyang et al.. 1994. Mol. Cell. Biol. 14:2777-2785); subfamilies. the two subfamilies are often considered Songyang et al.. 1993. Cell 72:767-778; and Koch et al.. together. The known KI‘K subfamilies are identi?ed in 1991. Science 252:668-678. Several intracellular substrate Plowman et aL. 1994. DN&P 7:334-339. which is incorpo proteins that associate with receptor tyrosine kinases (R'I‘Ks) 40 rated herein by reference. have been identi?ed. They may be divided into two principal The Non-Receptor Tyrosine Kinases. groups: (1) substrates which have a catalytic domain; and (2) substrates which lack such domain but serve as adapters and The non-receptor tyrosine kinases represent a collection associate with catalytically active molecules. Songyang et of cellular enzymes which lack extracellular and transmem brane sequences. At present. over twenty-four individual al.. 1993. Cell 72:767-778. The speci?city of the interac 45 tions between receptors or proteins and SR2 domains of non-receptor tyrosine kinases. comprising eleven (11) sub their substrates is determined by the amino acid residues families (Src. Frk. Btk. Csk. Abl. Zap70. Fes/Fps. Fak. lak. immediately surrounding the phosphorylated tyrosine resi Ack and LIMK) have been identi?ed. At present. the Src due. Differences in the binding a?nities between 8H2 subfamily of non-receptor tyrosine kinases is comprised of domains and the amino acid sequences surrounding the the largest number of PI‘Ks and include Src. Yes. Fyn. Lyn. phosphotyrosine residues on particular receptors are consis Lck. Blk. Hck. Fgr and Yrk. The Src subfamily of enzymes tent with the observed differences in their substrate phos has been linked to oncogenesis. A more detailed discussion phorylation pro?les. Songyang et al.. 1993. Cell of non~receptor tyrosine kinases is provided in Bolen. 1993. 72:767-778. These observations suggest that the function of Oncogene 8:2025-2031. which is incorporated herein by reference. each receptor tyrosine kinase is determined not only by its 55 pattern of expression and ligand availability but also by the Many of the tyrosine kinases. whether an KI‘K or non array of downstream signal transduction pathways that are receptor tyrosine kinase. have been found to be involved in activated by a particular receptor. Thus. phosphorylation cellular signaling pathways leading to cellular signal assays provides an important regulatory step which determines the signalling pathways leading to pathogenic conditions. selectivity of signaling pathways recruited by speci?c including cancer. psoriasis and hyper immune response. growth factor receptors. as well as di?erentiation factor Development Of Compounds To Modulate The PTKs. receptors. In view of the surmised importance of PI‘Ks to the Aberrant expression or mutations in the P'I‘Ks have been control. regulation and modulation of cell proliferation and shown to lead to either uncontrolled cell proliferation (e. g. the diseases and disorders associated with abnormal cell malignant tumor growth) or to defects in key developmental proliferation. many attempts have been made to identify processes. Consequently the biomedical community has receptor and non-receptor tyrosine ln'nase “inhibitors" using expended signi?cant resources to discover the speci?c bio a variety of approaches. including the use of mutant ligands 5 .763.470 3 4 (U.S. Pat. No. 4.966.849). soluble receptors and antibodies wherein R2 and Q are as follows: (Application No. WO 94/10202; Kendall 8: Thomas. 1994. Proc. Nat’l Acad. Sci 90:10705-09; Kim. et al.. 1993. Nature 362:841-844). RNA ligands (Jellinek. et al.. Bio chemistry 33: 10450-56); Takano. et al.. 1993. Mol. Bio. Cell 6,8-dichlcro 4-?uorophenyl 4:358A; Kinsella. et al.. 1992. Exp. Cell Res. 199:56-62; Wright. et al.. 1992. J. Cellular Phys. 152:448-57) and 6-chloro 2.4-dichlorophenyl tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; 6-chloro 4-(methylmercapto)phenyl 6-chlcro 4-(methy1sulfonyl)phenyl WO 91/15495; WO 94/14808; U.S. Pat. No. 5.330.992; 6-ch1oro 4-phenoxyphenyl Mariani. et al.. 1994. Proc. Am. Assoc. Cancer Res. H 4-isopropylphenyl 35:2268). benzo 4-chlorophenyl H thien-Z-yl More recently. attempts have been made to identify small H 2-methoxyphenyl molecules which act as tyrosine kinase inhibitors. For 6-bromo 2-metlmxyphenyl example. bis monocyclic. bicyclic or heterocyclic aryl com H berwofman-Z-yl 6-chloro Z-cyanophenyl pounds (PCF WO 92/20642). vinylene-azaindole deriva S-metboxy 2rnitrothien-4-yl tives (PCT WO 94/14808) and 1-cycloproppyl-4-pyridyl 8-methoxy 4~hydroxy-3-metlnxyphenyl quinolones (U.S. Pat. No. 5330.992) have been described 8-methcxy l-pbenylpyrawl-3-yl generally as tyrosine kinase inhibitors. Styryl compounds S-metlnxy l-methylpyrazol-El-yl S-methoxy S-bromo-Z-metlnxyphenyl (U.S. Pat. No. 5.217.999). styryl-substituted pyridyl com S-methoxy 4-cyampl1enyl pounds (U.S. Pat. No. 5.302.606). certain quinazoline B-methoxy thien-El-yl derivatives (EPApplication No. 0 566 266 Al). seleoindoles B-methoxy (tri?mmmethylphenyl and selenides (PCT WO 94/03427). tricyclic polyhydroxylic 8-tnethoxy thien-Z-yl 6,8-dichloro 2-hydroxy-3-methcxy compounds (PCT WO 92121660) and benzylphosphonic 6,8dichloro 4-chloro-l acid compounds (PCl‘ W0 91/ 15495) have been described methylpyraml-3-yl as compounds for use as tyrosine kinase inhibitors for use in 25 6,8-dichloro 3-bromothien-5-yl the treatment of cancer. 6.8-dichloro 2,5-dimethoxyphmyl 6.8-dichloro 4-t-butylphenyl The identi?cation of effective small compounds which 6.8-dichloro 4-acetamixbphenyl speci?cally inhibit tyrosine signal transduction by modulat 6,8-diehloro dioxolo[2,3-e]pheny1 ing the activity of receptor and non-receptor tyrosine kinases 30 to regulate and modulate abnormal or inappropriate cell The present invention is further directed to pharmaceuti proliferation is therefore desirable and the object of this cal compositions comprising a pharmaceutically effective invention. amount of the above-described compounds and a pharma ceutically acceptable carrier or excipient. Such a composi 3. SUMMARY OF THE INVENTION 35 tion is believed to modulate signal transduction by a tyrosine kinase. either by inhibition of catalytic activity. a?inity to The present invention relates to organic molecules ATP or ability to interact with a substrate. capable of modulating. regulating and/or inhibiting tyrosine More particularly. the compositions of the present inven kinase signal transduction. Speci?cally. the compounds of tion may be included in methods for treating diseases the present invention have the formulae: comprising proliferation or metabolic disorders. for example cancer. ?brosis. psoriasis. atherosclerosis. arthritis. and other disorders related to abnormal vasculogenesis and/or angiogenesis. such as diabetic retinopathy. 3.1. De?nitions 45 “Pharrnaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and wherein: properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid. X is O or S; 50 hydrobromic acid. sulfuric acid. nitric acid. phosphoric acid. Q is an aryl (either mono- or bicyclic) optionally substi methanesulfonic acid. ethanesulfonic acid. tuted with one or more R2; p-toluenesulfonic acid. salicylic acid and the like. n is 1. 2. 3 or 4; “Alkyl" refers to a saturated aliphatic hydrocarbon. R1 is cyano. N02. C(O)NHR2. S(O).‘.R2 or S(O)2NHR2; including straight-chain. branched-chain. and cyclic alkyl and 55 groups. Preferably. the alkyl group has 1 to 12 carbons. R2 is H. halo. trihalornethyl. alkyl. thioalkyl. hydroxy. More preferably. it is a lower alkyl of from 1 to 7 carbons. alkoxy. nitro. cyano. sulfonyl. amide. carboxyl. more preferably 1 to 4 carbons. Typical alkyl groups include sulfonarnide. or amino. methyl. ethyl. propyl. isopropyl. butyl. isobutyl. tertiary Preferably. the compounds of the present invention have butyl. pentyl. hexyl and the like. The alkyl group may be the formulae: substituted or unsubstituted. When substituted. the substi tuted group(s) is preferably. hydroxyl. cyano. alkoxy. =0. =S. N02. N(CH3)2. amino. or SH. “Alkenyl” refers to an unsaturated hydrocarbon group containing at least one carbon-carbon double bond. includ R2mm 0 Q ing straight-chain. branched-chain. and cyclic groups. Preferably. the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons. more 5.763.470 5 6 preferably 1 to 4 carbons. The alkenyl group may be It has been shown that tyrosine phosphorylation sites in substituted or unsubstituted. When substituted. the substi growth factor receptors function as high-affinity binding tutent group(s) is preferably. hydroxyl. cyano. alkoxy. =0. sites for SH2 (src homology) domains of signaling mol =S. N02. halogen. N(CH3)2 amino. or SH. An “alkynyl” ecules. Fantl et al.. 1992. Cell 69:413-423; Songyang et al.. group refers to an unsaturated hydrocarbon group containing 1994. Mol. Cell. Biol. 14:2777-2785); Songyang et al.. at least one carbon-carbon triple bond. including straight 1993. Cell 72:767-778; and Koch et al.. 1991. Science chain. branched-chain. and cyclic groups. Preferably. the 252:668-678‘. Several intracellular substrate proteins that alkynyl group has 1 to 12 carbons. More preferably it is a associate with receptor tyrosine kinases have been identi lower alkynyl of from 1 to 7 carbons. more preferably 1 to ?ed. They may be divided into two principal groups: (1) 4 carbons. The alkynyl group may be substituted or unsub substrates which have a catalytic domain; and (2) substrates stituted. When substituted. the substitutent group(s) is which lack such domain but serve as adapters and associate preferably. hydroxyl. cyano. alkoxy. =0. :8. N02. with catalytically active molecules. Songyang et al.. 1993. halogen. N(CH3)2. amino. or SH. Cell 72:767-778. The specificity of the interactions between “Alkoxy" refers to an “O-alkyl” group. where “alkyl” is receptors and SH2 domains of their substrates is determined de?ned as described above. by the amino acid residues immediately surrounding the “Aryl” refers to an aromatic group which has at least one phosphorylated tyrosine residue. Differences in the binding ring having a conjugated pi electron system and includes a?inities between SH2 domains and the amino acid carboxylic aryl. heterocyclic aryl and biaryl groups. all of sequences surrounding the phosphotyrosine residues on par which may be optionally substituted. Preferably. the aryl is ticular receptors are consistent with the observed differences a substituted or unsubstituted phenyl or pyridyl. Preferred 20 in their substrate phosphorylation pro?les. Songyang et al.. aryl substituent(s) are halogen. trihalomethyl. hydroxyl. SH 1993. Cell 72:767-778. These observations suggest that the OH. N02. amine. thioether. cyano. alkoxy. alkyl. and amino function of each receptor tyrosine lcinase is determined not only by its pattern of expression and ligand availability but groups. also by the array of downstream signal transduction path “Alkylaiyl” refers to an alkyl (as described above). 25 ways that are activated by a particular receptor. Thus. covalently joined to an aryl group (as described above). phosphorylation provides an important regulatory step Preferably. the alkyl is a lower alkyl. which determines the selectivity of signaling pathways “Carbocylic aryl” refers to a group wherein the ring atoms recruited by speci?c growth factor receptors. as well as on the aromatic ring are all carbon atoms. The carbon atoms differentiation factor receptors. are optionally substituted. 30 Tyrosine kinase signal transduction results in. among “Heterocyclic aryl” refers to groups having from 1 to 3 other responses. cell proliferation. differentiation and heteroatoms as ring atoms in the aromatic ring and the metabolism. Abnormal cell proliferation may result in a remainder of the ring atoms are carbon atoms. Suitable wide array of disorders and diseases. including the devel heteroatoms include oxygen. sulfur. and nitrogen. and opment of neoplasia such as carcinoma. sarcoma. leukemia. include furanyl. thienyl. pyridyl. pyrrolyl. N-lower alkyl 35 glioblastoma. hemangioma. psoriasis. arteriosclerosis. pyrrolo. pyn'midyl. pyrazinyl. imidazolyl and the like. all arthritis and diabetic retinopathy (or other disorders related optionally substituted. to uncontrolled angiogenesis and/or vasculogenesis). “Amide” 1 refers to an —C(O)—-NH-—R. where R is either alkyl. aryl. alkylaryl or hydrogen. This invention is therefore directed to compounds which regulate. modulate and/or inhibit disorders associated with ‘"I‘hioamide” refers to —C(S)——NH—R. where R is either abnormal cell proliferation by affecting the enzymatic activ alkyl. aryl. alkylaiyl or hydrogen. ity of the RTKs and/or the non-receptor tyrosine kinases and “Amine” refers to a —N(R")R"‘. where R" and R'". is interfering with the signal transduced such proteins. More independently either hydrogen. alkyl. aryl. or alkylaryl. particularly. the present invention is directed to compounds provided that R" and R'" are not both hydrogen. which regulate. modulate and/or inhibit the RTK and/or “Thioether" refers to ?S——-R. Where R is either alkyl. 45 non-receptor tyrosine kinase mediated signal transduction aryl. or alkylaryl. pathways as a therapeutic approach to cure many kinds of “Sulfonyl” refers to —S(O)2—R. where R is aryl. C(CN) solid tumors. including but not limited to carcinoma. =C-ary. CH-—2—CN. alkyaryl. NH-alkyl. NH-alkylaryl. or sarcoma. leukemia. erythroblastoma. glioblastoma. NH-aryl. meningioma. astrocytoma. melanoma and myoblastoma. 50 4. DETAILED DESCRIPTION OF THE Indications may include. but are not limited to brain cancers. INVENTION bladder cancers. ovarian cancers. gastric cancers. pancreas cancers. colon cancers. blood cancers. lung cancers and bone The present invention relates to compounds capable of regulating and/or modulating tyrosine kinase signal trans cancers. duction and more particularly receptor and non-receptor 55 4.1. The Compounds tyrosine kinase signal transduction The invention is generally directed to compounds and/or Receptor tyrosine kinase mediated signal transduction is compositions comprising compounds having the formulae: initiated by extracellular interaction with a speci?c growth factor (ligand). followed by receptor dimerization. transient stimulation of the intrinsic protein tyrosine kinase activity and phosphorylation. Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signalling molecules that facilitate the appropriate cellular response (e.g.. cell division. metabolic elfects to the extra 65 cellular microenvironment). See. Schlessinger and Ullrich. wherein: 1992. Neuron 9:383-391. X is O or S; 5 4.763.470 7 8 Q is an aryl (either mono- or bicyclic) optionally substi transduction may be determined using available techniques. tuted with one or more R2; Preferentially. a compound is subjected to a series of screens nis 1.2.3 or4; to determine the compound’s ability to modulate. regulate R‘ is cyano. N02. C(0)NHR2. s(o)2R2 or S(O)2NHR2; and/or inhibit cell proliferation. These screens. in the order and in which they are conducted. include biochemical assays. R2 is H. halo. trihalomethyl. alkyl. thioalkyl. hydroxy. cell growth assays and in vivo experiments. alkoxy. nitro. cyano._sulfonyl. amide. carboxyl. sul fonamide or amino. 4.2. Indications Preferably. the compounds of the present invention have the formulae: Cell proliferative disorders which can be treated or further studied by the present invention include cancers. blood N vessel proliferative disorders. ?brotic disorders. and mesan | \ 02 gial cell proliferative disorders. ' Blood vessel proliferation disorders refer to angiogenic R; O Q and vasculogenic disorders generally resulting in abnormal 15 proliferation of blood vessels. The formation and spreading wherein R2 and Q are as follows: of blood vessels. or vasculogenesis and angiogenesis. respectively. play important roles in a variety of physiologi cal processes such as embryonic development. corpus R; Q luteurn formation. wound healing and organ regeneration. They also play a pivotal role in canca development. Other 6,8-dichloro 4-?uorophenyl B-methoxy 4-?tiorophenyl examples of blood vessel proliferation disorders include 6-chloro 2,4-dichloroplenyl arthritis. where new capillary blood vessels invade the joint échloro 4-(methylmercapto)phenyl and destroy cartilage. and ocular diseases. like diabetic 6-chloro 4-(methylsulfonyl)phenyl retinopathy. where new capillaries in the retina invade the 6-chloro 4-phenoxyphenyl 25 vitreous. bleed and cause blindness. Conversely. disorders H 4-isopiopylphenyl benzo 4-chloroplierlyl related to the shrinkage. contraction or closing of blood H thien-l-yl vessels. such as restenosis. are also implicated. H 2-methoxyphenyl Fibrotic disorders refer to the abnormal formation of ?-bromo Z-methoxyplmryl H benmfurau-Z-yl extracellular matrix. Examples of ?brotic disorders include 6-chloro 2*:yamphenyl hepatic cirrhosis and mesangial cell proliferative disorders. B-methoxy 2-nitrothien-4-yl Hepatic cirrhosis is characterized by the increase in extra 8~n1etl1oxy 4-hydroxy-3 cellular matrix constituents resulting in the formation of a methoxyphenyl 8-methoxy l-phenylpyrazol-B-yl hepatic scar. Hepatic cirrhosis can cause diseases such as 8-methoxy l -methylpyrazol-3-yl 35 cirrhosis of the liver. An increased extracellular matrix 8-methoxy S-bromo-Z-methoxyphenyl resulting in a hepatic scar can also be caused by viral S-methoxy 4-cyamphenyl infection such as hepatitis. Lipocytes appear to play a major S-methoxy thien-B-yl S-methoxy 3-(tri?wmmethyl)pbenyl role in hepatic cirrhosis. Other ?brotic disorders implicated B-rnetboxy thicn-Z-yl include atherosclerosis (see. below). 6,8-dichloro 2-hydroxy-3-methoxy Mesangial cell proliferative disorders refer to disorders 6,8-dicl1loro 4-chloro-1 methylpyrazol-B-yl brought about by abnormal proliferation of mesangial cells. 6,3-dichloro 3-bromothien-5-yl Mesangial proliferative disorders include various human 6,8-dichloro 2,5-dimethoxyphenyl renal diseases. such as glomerulonephritis. diabetic 6.8-dichloro 4-t-bmylphenyl nephropathy. malignant nephrosclerosis. thrombotic 6.8-dich1oro 4-acetamidophenyl 6.8-dichloro dioxolo[2,3-c]phenyl 45 microangiopathy syndromes. transplant rejection. and glom erulopathies. The PDGF-R has been implicated in the main tenance of mesangial cell proliferation. Floege et al.. 1993. The chemical formulae referred herein may exhibit the Kidney International 43:847-854. phenomenon of tautomerism. As the formulae drawing The cell proliferative disorders which are indications of within this speci?cation can only represent one of the possible tautomeric forms. it should be understood that the the present invention are not necessarily independent For invention encompasses any tautomeric form which pos example. ?brotic disorders may be related to. or overlap. sesses the ability to regulate and/or modulate cell prolifera with blood vessel proliferative disorders. For example. ath tion and is not limited to any one tautomeric form utilized erosclerosis results. in part. in the abnormal formation of within the formulae drawing. ?brous tissue within blood vessels. In addition to the above compounds and their pharma 55 P'I'Ks have been associated with such cell proliferative ceutically acceptable salts. the invention is further directed. disorders. For example. some members of the RTK family where applicable. to solvated as well as unsolvated forms of have been associated with the development of cancer. Some the compounds (e.g. hydrated forms) having the ability to of these receptors. like the EGFR ('Iuzi et al.. 1991. Br: J. regulate and/or modulate cell proliferation. Cancer 632227-233; Torp et al.. 1992. APMIS 100:713-719) The compounds described above may be prepared by any HERZ/neu (Slamon et al.. 1989. Science 244:707-712) and process known to be applicable to the preparation of the PDGF-R (Kumabe et al.. 1992. Oncogene 7:627-633) chemically-related compounds. Suitable processes are illus are overexpressed in many tumors and/or persistently acti trated by the following representative examples. Necessary vated by autocrine loops. In fact. in the most common and starting materials may be obtained by standard procedures of severe cancers these receptor over-expressions Akbasak and organic chemistry. 65 Sunar-Akbasak. 1992. J. Neural. Sci. 111:119-133; Dickson An individual compound’s relevant activity and e?iciency et al.. 1992. Cancer Treatment Res. 6l:249-273; Korc et al.. as an agent to affect receptor tyrosine kinase mediated signal 1992. J. Clin. Invest. 90:1352-1360) and autocriue loops 5 1.763.470 9 10 (Lee and Donoghue. 1992. J. Cell. Biol. 11821057-1070; and a possible involvement in related disorders. Similarly. Korc et al.. supra; Akbasak and Sunar-Akbasak. supra) have Zap 70 is implicated in T-cell signalling. been demonstrated. For example. the EGFR receptor has The identi?cation of CTK modulating compounds to been associated with squamous cell carcinoma. astrocytoma. augment or even synergize with R'I'K aimed blockers is an glioblastoma. head and neck cancer. lung cancer and bladder aspect of the present invention. cancer. HERZ has been associated with breast. ovarian. gastric. lung. pancreas and bladder cancer. The PDGF-R has Finally. both R'I‘Ks and non-receptor type kinases have been associated with glioblastoma. lung. cancm ovarian. been connected to hyperimmune disorders. cancer melanoma and prostate cancer. The KI‘K c-met has 4.3. Pharmaceutical Formulations and Routes of been generally associated with hepatocarcinogenesis and 10 Administration thus hepatocellular carcinoma. Additionally. c-met has been linked to malignant tumor formation. More speci?cally. the The identified compounds can be administered to a human RFK c-met has been associated with. among other cancers. patient. by itself. or in pharmaceutical compositions where colorectal. thyroid. pancreatic and gastric carcinoma. leuke it is mixed with suitable carriers or excipient(s) at doses to mia and lymphoma. Additionally. over-expression of the treat or ameliorate a variety of disorders. A therapeutically c-met gene has been detected in patients with Hodgkins effective dose further refers to that amount of the compound disease. Burkitts disease. and the lymphoma cell line. sufficient to result in amelioration of symptoms. Techniques The IGF-IR. in addition to being implicated in nutritional for formulation and administration of the compounds of the support and in type-II diabetes. has also been associated with instant application may be found in “Remington’s Pharma several types of cancers. For example. IGF-I has been ceutical Sciences.” Mack Publishing Co.. Easton. Pa.. latest implicated as an autocrine growth stimulator for several edition. tumor types. e.g. human breast cancer carcinoma cells (Arteaga et al.. 1989. J. Clin. Invest. 84:1418-1423) and 4.3.1. Routes of Administration small lung tumor cells (Macauley et al.. 1990. Cancer Res. Suitable routes of administration may. for example. 50:2511-2517). In addition. IGF-L integrally involved in the 25 include oral. rectal. transmucosal. or intestinal administra normal growth and ditferentiation of the nervous system. tion; parenteral delivery. including intramuscular. appears to be an autocrine stimulator of human gliomas. subcutaneous. intramedullary injections. as well as Sandberg-Nordqvist et al.. 1993. Cancer Res. intrathecal. direct intraventricular. intravenous. 53:2475-2478. The importance of the IGFJR and its ligands intrapen'toneal. intranasal. or intraocular injections. in cell proliferation is further supported by the fact that many 30 Alternately. one may administer the compound in a local cell types in culture (fibroblasts. epithelial cells. smooth rather than systemic manner. for example. via injection of muscle cells. T-lymphocytes. myeloid cells. chondrocytes. the compound directly into a solid tumor. often in a depot or osteoblasts. the stem cells of the bone marrow) are stimu sustained release formulation. lated to grow by IGF-I. Goldring and Goldring. 1991. Furthermore. one may administer the drug in a targeted Eukaryotic Gene Expression 1:301-326. In a series of recent 35 publications. Baserga even suggests that IGF-I-R plays a drug delivery system. for example. in a liposome coated central role in the mechanisms of transformation and. as with tumor-speci?c antibody. The liposomes will be targeted such. could be a preferred target for therapeutic interven to and taken up selectively by the tumor. tions for a broad spectrum of human malignancies. Baserga. 4.3.2. Composition/Formulation 1995. Cancer Res. 55:249-252; Baserga. 1994. Cell 791927-930‘, Coppola et al.. 1994. Mol. Cell. Biol. The pharmaceutical compositions of the present invention 14:4588-4595. may be manufactured in a manner that is itself known. e.g.. The association between abnormalities in R'I‘Ks and dis by means of conventional mixing. dissolving. granulating. ease are not restricted to cancer. however. For example. dragee-making. levigating. emulsifying. encapsulating. entrapping or lyophilizing processes. RI‘Ks have been associated with metabolic diseases like 45 psoriasis. diabetes mellitus. wound healing. in?ammation. Pharmaceutical compositions for use in accordance with and neurodegenerative diseases. For example. the EGF-R is the present invention thus may be formulated in conven indicated in corneal and dermal wound healing Defects in tional manner using one or more physiologically acceptable the Insulin-R and the IGF- 1R are indicated in type-II dia carriers comprising excipients and auxiliaries which facili betes mellitus. Amore complete correlation between speci?c 50 tate processing of the active compounds into preparations RI‘Ks and their therapeutic indications is set forth in Plow which can be used pharmaceutically. Proper formulation is man et al.. 1994. DN&P 7:334—339. dependent upon the route of administration chosen. Not only receptor type tyrosine kinases. but also many For injection. the agents of the invention may be formu cellular tyrosine kinases (Cl‘Ks) including src. abl. fps. yes, lated in aqueous solutions. preferably in physiologically fyn. lyn. lck. blk. hck. fgr. yrk (reviewed by Bolen et al.. 55 compatible bu?ters such as Hanks’s solution. Ringer’s 1992. FASES J. 6:3403-3409) are involved in the prolifera solution. or physiological saline bu?’er. For transmucosal tive and metabolic signal transduction and thus in indica administration. penetrants appropriate to the barrier to be tions of the present invention. For example. mutated src permeated are used in the formulation. Such penetrants are (v-src) has been demonstrated as an oncoprotein (pp6?v‘m) generally known in the art. in chicken. Moreover. its cellular homolog. the proto For oral administration. the compounds can be formulated oncogene ppGON" transmits oncogenic signals of many readily by combining the active compounds with pharma receptors. For example. overexpression of EGF-R or HER?! ceutically acceptable carriers well known in the art. Such neu in tumors leads to the constitutive activation of pp60c’ carriers enable the compounds of the invention to be for m. which is characteristic for the malignant cell but absent mulated as tablets. pills. dragees. capsules. liquids. gels. from the normal cell. On the other hand. mice de?cient for 65 syrups. slurries. suspensions and the like. for oral ingestion the expression of c-src exhibit an osteopetrotic phenotype. by a patient to be treated. Pharmaceutical preparations for indicating a key participation of c-src in osteoclast function oral use can be obtained by combining the compounds with 5 .763.470 11 12 a solid excipient. optionally grinding a resulting mixture. the suspension may also contain suitable stabilizers or and processing the mixture of granules. after adding suitable agents which increase the solubility of the compounds to auxiliaries. if desired. to obtain tablets or dragee cores. allow for the preparation of highly concentrated solutions. Suitable excipients are. in particular. ?llers such as sugars. Alternatively. the active ingredient may be in powder including lactose. sucrose. mannitol. or sorbitol; cellulose form for constitution with a suitable vehicle. e.g.. sterile preparations such as. for example. maize starch. wheat pyrogen-free water. before use. starch. rice starch. potato starch. gelatin. gum tragacanth. The compounds may also be formulated in rectal com methyl cellulose. hydroxypropylrnethyl-cellulose. sodium positions such as suppositories or retention enemas. e.g.. carboxymethylcellulose. and/or polyvinylpyrrolidone containing conventional suppository bases such as cocoa (PVP). If desired. disintegrating agents may be added. such butter or other glycerides. as the cross-linked polyvinyl pyrrolidone. agar. or alginic In addition to the formulations described previously. the acid or a salt thereof such as sodium alginate. compounds may also be formulated as a depot preparation. Dragee cores are provided with suitable coatings. For this Such long acting formulations may be administered by purpose. concentrated sugar solutions may be used. which implantation (for example subcutaneously or may optionally contain gum arabic. talc. polyvinyl 15 intramuscularly) or by intramuscular injection. Thus. for pyrrolidone. carbopol gel. polyethylene glycol. and/or tita example. the compounds may be formulated with suitable nium dioxide. lacquer solutions. and suitable organic sol polymeric or hydrophobic materials (for example as an vents or solvent mixtures. Dyestu?s or pigments may be emulsion in an acceptable oil) or ion exchange resins. or as added to the tablets or dragee coatings for identi?cation or sparingly soluble derivatives. for example. as a sparingly to characterize different combinations of active compound 20 soluble salt. doses. A pharmaceutical carrier for the hydrophobic compounds Pharmaceutical preparations which can be used orally of the invention is a cosolvent system comprising benzyl include push-?t capsules made of gelatin. as well as soft. alcohol. a nonpolar surfactant. a water-miscible organic sealed capsules made of gelatin and a plasticizer. such as 25 polymer. and an aqueous phase. The cosolvent system may glycerol or sorbitol. The push-?t capsules can contain the be the VPD co-solvent system. VPD is a solution of 3% w/v active ingredients in admixture with ?ller such as lactose. benzyl alcohol. 8% w/v of the nonpolar surfactant polysor binders such as starches. and/or lubricants such as talc or bate 80. and 65% w/v polyethylene glycol 300. made up to magnesium stearate and. optionally. stabilizers. In soft volume in absolute ethanol. The VPD co-solvent system capsules. the active compounds may be dissolved or sus 30 (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose pended in suitable liquids. such as fatty oils. liquid paraf?n. in water solution. This co-solvent system dissolves hydro or liquid polyethylene glycols. In addition. stabilizers may phobic compounds well. and itself produces low toxicity be added. All formulations for oral administration should be upon systemic administration. Naturally. the proportions of in dosages suitable for such administration. a co-solvent system may be varied considerably without For buccal administration.the compositions may take the 35 destroying its solubility and toxicity characteristics. form of tablets or lozenges formulated in conventional Furthermore. the identity of the co-solvent components may manner. be varied: for example. other low-toxicity nonpolar surfac For administration by inhalation. the compounds for use tants may be used instead of polysorbate 80; the fraction size according to the present invention are conveniently deliv of polyethylene glycol may be varied; other biocompatible ered in the form of an aerosol spray presentation from polymers may replace polyethylene glycol. e.g. polyvinyl pressurized packs or a nebuliser. with the use of a suitable pyrrolidone; and other sugars or polysaccharides may sub propellant. e.g.. dichlorodi?uoromethane. stitute for dextrose. trichloro?uoromethane. dichlorotetra?uoroethane. carbon Alternatively. other delivery systems for hydrophobic dioxide or other suitable gas. In the case of a pressurized pharmaceutical compounds may be employed. Liposomes aerosol the dosage unit may be determined by providing a 45 and emulsions are well known examples of delivery vehicles valve to deliver a metered amount. Capsules and cartridges or carriers for hydrophobic drugs. Certain organic solvents of e.g. gelatin for use in an inhaler or insu?lator may be such as dimethylsulfoxide also may be employed. although formulated containing a powder mix of the compound and a usually at the cost of greater toxicity. Additionally. the suitable powder base such as lactose or starch. compounds may be delivered using a sustained-release The compounds may be formulated for parenteral admin 50 system. such as semipermeable matrices of solid hydropho istration by injection. e.g.. by bolus injection or continuous bic polymers containing the therapeutic agent. Various infusion. Formulations for injection may be presented in unit sustained-release materials have been established and are dosage form. e.g.. in ampoules or in multi-dose containers. well known by those skilled in the art. Sustained-release with an added preservative. The compositions may take such capsules may. depending on their chemical nature. release forms as suspensions. solutions or emulsions in oily or SS the compounds for a few weeks up to over 100 days. aqueous vehicles. and may contain formulatory agents such Depending on the chemical nature and the biological sta as suspending. stabilizing and/or dispersing agents. bility of the therapeutic reagent. additional strategies for Pharmaceutical formulations for parenteral administra protein stabilization may be employed tion include aqueous solutions of the active compounds in The pharmaceutical compositions also may comprise suit water-soluble form. Additionally. suspensions of the active able solid or gel phase carriers or excipients. Examples of compounds may be prepared as appropriate oily injection such carriers or excipients include but are not limited to suspensions. Suitable lipophilic solvents or vehicles include calcium carbonate. calcium phosphate. various sugars. fatty oils such as sesame oil. or synthetic fatty acid esters. starches. cellulose derivatives. gelatin. and polymers such as such as ethyl oleate or triglycerides. or liposomes. Aqueous polyethylene glycols. injection suspensions may contain substances which 65 Many of the P'TK modulating compounds of the invention increase the viscosity of the suspension. such as sodium may be provided as salts with pharmaceutically compatible carboxymethyl cellulose. sorbitol. or dextran. Optionally. counterions. Pharmaceutically compatible salts may be 5 ,763.470 13 14 formed with many acids. including but not limited to weight. the severity of the affliction. the manner of admin hydrochloric. sulfuric. acetic. lactic. tartaric. malic. succinic. istration and the judgment of the prescribing physician. etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. 4.3.4. Packaging 4.3.3. Effective Dosage The compositions may. if desired. be presented in a pack Pharmaceutical compositions suitable for use in the or dispenser device which may contain one or more unit present invention include compositions wherein the active dosage form containing the active ingredient. The pack may ingredients are contained in an effective amount to achieve for example comprise metal or plastic foil. such as a blister its intended purpose. More speci?cally. a therapeutically pack. The pack or dispenser device may be accompanied by 10 effective amount means an amount effective to prevent instructions for administration. Compositions comprising a development of or to alleviate the existing symptoms of the compound of the invention formulated in a compatible subject being treated. Determination of the effective pharmaceutical carrier may also be prepared. placed in an amounts is well within the capability of those sldlled in the appropriate container. and labelled for treatment of an indi art. especially in light of the detailed disclosure provided cated condition. Suitable conditions indicated on the label 15 herein. may include treatment of a tumor. inhibition of For any compound used in the method of the invention. angiogenesis. treatment of ?brosis. diabetes. and the like. the therapeutically effective dose can be estimated initially from cell culture assays. For example. a dose can be for 5. EXAMPLE mulated in animal models to achieve a circulating concen 20 COMPOUND SYNTHESIS tration range that includes the ICs0 as determined in cell culture (i.e.. the concentration of the test compound which The compounds of the present invention may be synthe achieves a half-maximal inhibition of the PTK activity). sized according to known techniques. The following repre Such information can be used to more accurately determine sent preferred methods for synthesizing the compounds of useful doses in humans. 25 the claimed invention: A therapeutically e?ective dose refers to that amount of the compound that results in amelioration of symptoms or a 5.1. Synthesis Of 6.8-Dichloro-2-(4-?uorophenyl) prolongation of survival in a patient. Toxicity and therapeu 3-nitro-l.2-benzopyran tic e?icacy of such compounds can be determined by stan The preferred method for synthesizing 6.8-Dichloro-2-(4 dard pharmaceutical procedures in cell cultures or experi 30 ?uorophenyl)-3-nitro- 1 .Z-benzopyran is as follows: mental animals. e.g.. for determining the LD50 (the dose 4-Fluoro-[3-nitrostyrene was prepared by condensation of lethal to 50% of the population) and the EDso (the dose 4-?uorobenzaldehyde and nitromethane in the presence of therapeutically effective in 50% of the population). The dose base (Henry reaction). 3 .5 -Dichloro-5 ratio between toxic and therapeutic effects is the therapeutic hydroxybenzaldehyde (0.382 g) and 4-?uoro-[5-nitrostyrene index and it can be expressed as the ratio between [D50 and 35 (367 mg. 1.1 eq) were stirred in 3 ml of pyridine for 48 hours ED50. Compounds which exhibit high therapeutic indices at room temperature. The reaction was partitioned in a are preferred. The data obtained from these cell culture mixture of ethyl acetate and 2N hydrochloric acid. The ethyl assays and animal studies can be used in formulating a range acetate layer was washed once with 2N hydrochloric acid. of dosage for use in human. The dosage of such compounds once with saturated sodium bicarbonate. once with brine. lies preferably within a range of circulating concentrations dried over anhydrous magnesium sulfate. ?ltered and evapo that include the ED50 with little or no toxicity. The dosage rated to dryness. Chromatography of the residue on silica gel may vary within this range depending upon the dosage form in methylene chloride and methanol gave 0.2 g of 6.8 employed and the route of administration utilized. The exact dichloro-2-(4-?uorophenyl)-3-nitro-1.2-benzopyran. a yel formulation. route of administration and dosage can be low solid. chosen by the individual physician in view of the patient’s 45 condition. (See e.g. Fingl et al.. 1975. in "The Pharmaco 5.2. Synthesis Of 2-(4-Fluorophenyl)-8-methoxy-3 logical Basis of Therapeutics”. Ch. 1 p1). nitro- 1 .2 -benzopyran Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are The preferred method for synthesizing 2-(4 sui?cient to maintain the lu'nase modulating effects. or 50 Fluorophenyl)-8-methoxy-3-nitro- 1.2-benzopyran minimal elfective concentration (MEC). The MEC will vary (Compound A) is as follows: A mixture of 2-hydroxy-3 for each compound but can be estimated from in vitro data; methoxybenzaldehyde (304 mg) and 4-?uoro-[5-nitrostyrene e. g. the concentration necessary to achieve 50—90% inhibi (367 mg. 1.1 eq) was stirred in 3 ml of pyridine for 48 hours tion of the kinase using the assays described herein. Dosages at room temperature as disclosed in section 5.1. The reaction was partitioned in a mixture of ethyl acetate and 2N hydro necessary to achieve the MEC will depend on individual 55 characteristics and route of administration. However. HPLC chloric acid. The ethyl acetate layer was washed once with assays or bioassays can be used to determine plasma con 2N hydrochloric acid. once with saturated sodium centrations. bicarbonate. once with brine. dried over anhydrous magne Dosage intervals can also be determined using MEC sium sulfate. ?ltered and evaporated to dryness. Chroma value. Compounds should be administered using a regimen tography of the residue on silica gel in methylene chloride which maintains plasma levels above the MEC for 10-90% and methanol gave 0.2 g of 2-(4-?uorophenyl)-8-methoxy of the time. preferably between 30-90% and most preferably S-nitro- 1.2-benzopyran. a yellow solid between 50—90%. In cases of local administration or selec 5.3. Synthesis Of 6-Chloro-2-(2.4-dichlorophenyl) tive uptake. the effective local concentration of the drug may 3-nitro- 1.2-ben zopyran not be related to plasma concentration. 65 The amount of composition administered will. of course. The preferred method for synthe sizing be dependent on the subject being treated. on the subject’s 4-(3-chlorophenyl)-3-cyano-2-(pyrido-2-sulfonyl)thiophene 5,763,470 15 16 is as follows: Reaction of 2-hydroxy-5-chlorobenzaldehyde Reaction of 4-bromo-2-hydroxybenzaldehyde and (0.313 g) and 2.4-djchloro-B-nitrostyrene (0.480 g) in pyri Z-methoxy-B-nitrostyrene as described in section 5.1 gave dine as described in section 5.1 gave 6-chloro-2-(2.4 6-bromo-2-(2-methoxyphenyl)-3 -nitro- 1 .Z-benzopyran. dichlorophenyl)-3-nitr0-l.Z-benzopyran. 5.12. Synthesis Of 2-(Benzofuran-2-yl)-3-nitro-1.2 5.4. Synthesis Of 6-Chloro-2-[4-(methylmercapto) pheny1]-3-nitro-1.2-benzopyran benzopyran The preferred method for synthesizing 6-Chloro-2-[4 The preferred method for synthesizing 2-(Benzofuran-2 (methyhnercapto)phenyl]-3-nitro-1.2-benzopyran is as fol yl)-3-nitro-l.Z-benzopyran is as follows: Reaction of lows: Reaction of 2-hydroxy-5-chlorobenzaldehyde and 2-hydroxybenzaldehyde and l-(benzofuran-2-yl)-2 4—methylmercapto-[5-nitrostyrene as described in section 5.1 nitroethene as described in section 5.1 gave 2~(benzofnran gave 6-ch1oro-2-[4-(rnethylmercapto)phenyl]-3-nitro-1.2 2-yl)-3-nilro- l .Z-benzopyran. benzopyran. 5.13. Synthesis Of 6-Chloro-2-(2-cyanophenyl)-3 5.5. Synthesis Of 6-Chloro-2-[4(methylsulfonyl) nilro-LZ-benzopyran 15 phenyl]-3-nitro-l.2-benzopyran The preferred method for synthesizing 6-Chloro-2-(2 The preferred method for synthesizing 6-Chloro-2-[4 cyanophenyl)-3-nitro-1.2-benzopyran is as follows: Reac (methylsulfonyl)phenyl]-3-niu'o-l.Z-benzopyran is as fol tion of 2-hydroxy-S-chlorobenzaldehyde and 4-cyano-B lows: Reaction of 2-hydroxy-S-chlorobenzaldehyde and nitrostyrene as in described 5.1 gave 6-chloro-2-(2 4—methoxysulfonyl-B-nitrostyrene as described in section cyanophenyl)-3-nitro-1.2-benzopyran. 5.1 gave 6-chloro-2-[4-(methylsulfonyl)phenyl]-3-nitro-1.2 benzopyran. 5.14. Synthesis Of 8-Methoxy-3-nitro-2-(2 nitrothien-4-yl)-1 .Z-benzopyran 5.6. Synthesis Of 6-Chloro-3-nitro-2-[4-(phenoxy) phenyl] - 1 .2-benzopyran The preferred method for synthesizing 8-Methoxy-3 25 The preferred method for synthesizing 6-Chloro-3-nitro nitro-2-(2-nitIothien-4-yD-1.2-benzopyran is as follows: 2-[4-(phenoxy)phenyl]-1.2-benzopyran is as follows: Reac Reaction of 2-hydroxy-3-methoxybenzaldehyde and tion of Z-hydroxy-S-chlorobenzaldehyde and 4-phenoxy-B l-nitro-2-(2-nitrothien-4-yl)ethene as described in section 5.1 gave 8-methoxy-3-nitro-2-(2-nitrothien-4-yl)-1.2 nitrostyrene as described in section 5.1 gave 6-chloro-3 benzopyran. niu-o-Z- [4- (phe noxy)ph enyl] —1 .Z-benzopyran. 30 5.7. Synthesis Of 2-(4-isopropylphenyl)—3-nitro-1.2 5.15. Synthesis Of 2-(4-Hydroxy-3-methoxyphenyl) benzopyran 8-methoxy-3-nin'o-l2-benzopyran The preferred method for synthesizing 2-(4 The preferred method for synthesizing 2-(4-Hydroxy-3 isopropylphenyl)-3-nitro-1.2-benzopyran is as follows: 35 methoxyphenyD-8-methoxy-3-nin'o-1.2-benzopyran is as Reaction of Z-hydroxybenzaldehyde and 4-isopropyl-[5 follows: Reaction of 2-hydroxy-3-methoxybenzaldehyde nitrostyrene as described in section 5.1 gave 2-(4 and 4-hydroxy-3-meth0xy-B-niu'ostyrene as described in isopropylphenyl)-3-nilro-l.2-benzopyran section 5.1 gave 2-(4-hydroxy-3-methoxyphenyl)-8 methoxy-3-nitro-12-benzopyran. 5.8. Synthesis Of 2-(4-chlorophenyl)-3-nitr0-[6.7] benzo-lZ-benzopyran. 5.16. Synthesis Of 8-Meth0xy-3-nitro-2-( 1 The preferred method for synthesizing 2-(4-chlorophenyl) phenylpyrazol-3 -yl)-l .2-benzopyran -3-nitro-[6.7]benzo- 1 .2-benzopyran: Reaction of The preferred method for synthesizing 8-Methoxy-3 Z-hydroxy- l-naphthaldehyde and 4-chloro-B-nitrostyrene as nitro-2-(1-phenylpyrazo1-3-yl)-1,2-benzopyran is as fol described in section 5.1 gave 2-(4-chlorophenyD-3-nitro-[6. 45 lows: Reaction of 2-hydroxy-3-methoxybenzaldehyde and 7]benzo- 1.2-benzopyran. l-nitro-2-(1-phenylpyrazole-3-yl)ethene as described in sec 5.9. Synthesis Of 3-Nitro-2-(thien-2-yl)-1.2 tion 5.1 gave 8-methoxy-3-nitro-2-(l-phenylpyrazol-3-yl) benzopyran 1.2-benzopyran. The preferred method for synthesizing 3-Nitro-2-(thien 50 5.17. Synthesis Of 8-Methoxy-3-nitro-2-(1 2-yl)-1.2-benzopyran is as follows: Reaction of methylpyraz ol-3-y1)- LZ-benzopyran 2-hydroxybenzaldehyde and l-nitro-2-(thien-2-yl)ethene as described in section 5.1 gave 3-nitro-2-(thien-2-yl)-l.2 The preferred method for synthesizing 8-Methoxy-3 benzopyran. nitro-2-(l-methylpyrazol-3-yl)-1.2-benzopyran is as fol 55 lows: Reaction of 2-hydroxy-3-methoxybenzaldehyde and 5.10. Synthesis Of 2-(2-MethoxyphenyD-3-nin'o-l. 1-nitro-2-(1-methylpyrazole-3-yl)ethene as described in 2-benzopyran section 5.1 gave 8-methoxy-3-nitro-2-(1-methylpyrazol-3 The preferred method for synthesizing 2-(2 yl)-l.2-benzopyran. Methoxyphenyl)-3-nitro-1.2-benzopyran is as follows: Reaction of Z-hydroxybenzaldehyde and Z-methoxy-B 5.18. Synthesis Of 2-(5-Bromo-2-rnethoxyphenyh nitrostyrene as described section 5.1 gave 2-(2 8-methoxy-3-nitro-1.2-benzopyran meth0xyphenyl)-3-nitro-l.2-benzopyran. The preferred method for synthesizing 2-(5-Bromo-2 5.11. Synthesis Of 5-Bromo-2-(2-methoxyphenyl) methoxyphenyD-8-rnethoxy-3-nitro-1.2-benzopyran is as 3- nitro- 1.2-benzopyran follows: Reaction of 2-hydroxy-3-methoxybenzaldehyde 65 and 5-bromo-2-methoxy-[$-nitrostyrene as described in sec The preferred method for synthesizing 5-Bromo-2-(2 tion 5.1 gave 2-(5-bromo-2-methoxyphenyl)-8-methoxy-3 methoxyphenyl)-3-nitro-1.2-benzopyran is as follows: nitro- 1.2-benzopyran. 5.763.470 17 18 5.19. Synthesis Of 2-(4-Cyanophenyl)-8-rnethoxy lows: Reaction of 3.5-dichloro-2-hydroxybenzaldehyde and 3-nitro- 1.2-benzopyran 2.5-dimethoxy-B-nitrostyrene as described in section 5.1 gave 6.8-dichloro-3-nitro-2- [2 .5-dimethoxyphenyl]-1.2 The preferred method for synthesizing 2-(4 benzopyran. Cyanophenyl)-8-methoxy-3-nitro-1.2-benzopyran is as fol lows: Reaction of 2-hydroxy-3-methoxybenzaldehyde and 5.27. Synthesis Of 6.8-Dichloro-3-nitro-2-(4-t 4-cyano-B-nitrostyrene as in described in section 5.1 gave butylphenyl)- 1 .2-benzopyran 2-(4-cyanophenyl)-8-methoxy-3-nitro-1.2-benzopyran. The preferred method for synthesizing 6.8-Dichloro-3 5.20. Synthesis Of 8-Methoxy-3-nitro-2-(thien-3 nitro-2-(4-t-butylphenyl)-1.2-benzopyran is as follows: yl)- 1.2-benzopyran Reaction of 3.5-dichloro-2-hydroxybenzaldehyde and 4-t The preferred method for synthesizing 8-Methoxy-3 butyl-[S-nitrostyrene as described in section 5.1 gave 6.8 nitro-2-(thien-3-yl)-1.2-benzopyran is as follows: Reaction dichloro-3-nitro-2-(4-t-butylphenyl)-1 .2-be nzopyran. of 2-hydroxy-3-methoxybenzaldehyde and 1-nitro-2-(thien 5.28. Synthesis Of 6.8-Dichloro-3-nitro-2-(4 3-y1)ethene as described in section 5.1 gave 8-rnethoxy-3 acetamidophenyl)- 1 .2-benzopyran nitro-2-(thien-3-yl)- 1 .2-benzopyran. The preferred method for synthesizing 6.8-Dichloro-3 5.21. Synthesis Of 8-Methoxy-3-nitro-2-[3 nitro-2-(4-acetamidophenyl)-1.2-benzopyran is as follows: (tri?uoromethyhpheny? - 1.2-benzopyran Reaction of 3.5-dichloro-2-hydroxybenzaldehyde and The preferred method for synthesizing 8-Methoxy-3 4-acetamido-[3-nitrostyrene as in Example 1 gave 6.8 nit1'o-2-[3-(tri?uoromethyl)phenyl]-1.2-benzopyran is as dichloro-3-nitro-2-(4-acetamidophenyb-1.2-benzopyran. follows: Reaction of 2-hydroxy-3-methoxybenza1dehyde 5.29. Synthesis Of 6.8-Dichloro-3-nitro-2-(dioxolo and 3-tri?uoromethyl-B-nitrostyrene as described in section {4.5-c]phenyl)-1.2-benzopyran 5.1 gave 8-methoxy-3-nitro-2-[3-(tri?uoromethyl)phenyl] 1.2-benzopyran. The prefm'red method for synthesizing 6.8-Dichloro-3 nitro-2-(dioxolo[4.5-c]phenyl)-1.2-benzopyran is as fol 5.22. Synthesis Of 8-Methoxy-3-nitro-2-(thien-2 lows: Reaction of 3.5-dichloro-2-hydroxybenzaldehyde and yl)- 1.2-ben zopyran 1-(dioxolo[4.5—c]phenyl)-2-nitroethene as described in sec The preferred method for synthesizing 8-Methoxy-3 tion 5.1 gave 6.8dichloro-3-nitro-2-(dioxolo[4.5-c]phenyl) nitro-2-(thien-2-yl)-1.2-benzopyran is as follows: Reaction 1.2benzopyran. of 2-hydroxy-3-methoxybenzaldehyde and l-nitro-2-(thien 6. EXAMPLES 2-yl)ethene as described in section 5.1 gave 8-methoxy-3 nitro-2-(thien-2-yl)- 1.2-benzopyran. RI‘K Assays 5.23. Synthesis Of 6.8-Dichloro-3-nitro-2-[2 35 The following assays and animal models may be used to hydroxy-3-methoxyphenyl1- 1.2-benzopyran determine the level of activity and effect of the di?erent The preferred method for synthesizing 6.8-Dichloro-3 compounds of the present invention on one or more of the nitro-2-[2-hyd.roxy-3-methoxyphenyl]-1.2-benzopyran is as Rl‘Ks. Similar assays can be designed along the same lines follows: Reaction of 3.5-d.ichloro-2-hydroxybenzaldehyde for any tyrosine kinase using techniques well known in the and 2-hydroxy-3-methoxy-[3-nitrostyrene as described in art. section 5.1 gave 6.8-dichloro-3-nitro-2-[2-hydroxy-3 6.1. Enzyme Linked Immunosorbent Assay methoxyphenyl]-l.2-benzopyran. (ELISA) 5.24. Synthesis Of 6.8-Dichloro-3-nitro-2-(4-chloro Enzyme linked immunosorbent assays (ELISA) may be l-methylpyrazol-3 -yl)- 1 .2-benzopyran used to detect and measure the presence of tyrosine kinase The preferred method for synthesizing 6.8-Dichloro-3 activity. The ELISA may be conducted according to known nitro-2-(4-chloro-1-methylpyrazol-3-yl)- 1.2-benzopyran is protocols which are described in. for example. Voller. et al.. as follow s: Reaction of 3 .5 -di chloro-2 1980. “Enzyme-Linked Immunosorbent Assay.” In: Manual of Clinical Inmunology, 2d ed.. edited by Rose and hydroxybenzaldehyde and 1~(4-chloro- l-methylpyrazol-fi 50 yl)-2-nitroethene as described in section 5.1 gave 6.8 Friedman. pp 359-371 Am. Soc. Of Microbiology. dichloro-3-nitro-2-(4-chloro-1-methylpyrazol-3-yl)- 1.2 Washington. DC. benzopyran. The disclosed protocol may be adapted for determining activity with respect to a specific RTK. As example. the 5.25. Synthesis Of 6.8-Dichloro-3-nitro-2-(3 55 preferred protocols for conducting the ELISA experiments brornothien-S-yD- l .2-benzopyran for speci?c RTKs is provided below. Adaptation of these The preferred method for synthesizing 6.8-Dichloro-3 protocols for determining a compound’s activity for other nitro-2-(3-bromothien-5-yl)-1.2-benzopyran is as follows: members of the RTK family. as well as non-receptor tyrosine Reaction of 3.5-d.ichloro-2-hydroxybenzaldehyde and 1-6 kinases. are within the scope of those in the art. bromothien-5-yl)-2-nitroethene as described in section 5.1 gave 6.8-dichloro-3-nitro-2-(3-bromothien-5-y1)-1.2 6.1.1. FLK-l ELISA benzopyran. An ELISA assay was conducted to measure the kinase activity of the FLK-l receptor and more speci?cally. the 5.26. Synthesis Of 6.8-Dichloro-3-nitro-2-[2.5 inhibition or activation of protein tyrosine kinase activity on dimethoxypheny?-1.2-benzopyran 65 the FLK-l receptor. Speci?cally. the following assay was The preferred method for synthesizing 6.8-Dichloro-3 conducted to measure kinase activity of the FLK-l receptor nitro-2-[2.5-dimethoxyphenyl1-1.2-benzopyran is as fol in FLK—1/NIH3T3 cells. 5.763.470 19 20 Materials And Methods 7. Dilute Compounds/Extracts 1:20 in polypropylene 96 Materials. The following reagents and supplies were used: well plates using starvation media. Dilute dimethylsul a. Corning 96-well ELISA plates (Corning Catalog No. foxide 1:20 for use in control wells. 25805-96); 8. Remove starvation media from 96 well cell culture b. Cappel goat anti-rabbit IgG (catalog no. 55641); plates and add 162 pl of fresh starvation media to each well. c. PBS (Gibco Catalog No. 450-1300EB); 9. Add 18 pl of 1:20 diluted Compound/Extract dilution d. TBSW Butler (50 Mm Tris (Ph 7.2). 150 Mm NaCl and (from step 7) to each well plus the 1:20 dimethylsul 0.1% Tween-20); foxide dilution to the control wells (iVEGF). for a ?nal e. Ethanolarnine stock (10% ethanolamine (Ph 7.0). stored 10 dilution of 1:200 after cell stimulation. Final dimeth at 4° C.); ylsulfoxide is 0.5%. Incubate the plate at 37° C.. 5% f. HNTG bu?er (20mM HEPES bu?’er (Ph 7.5). 150 rnM CO2 for two hours. NaCl. 0.2% Triton X-l00. and 10% glycerol); 10. Remove unbound antibody from ELISA plates by g. EDTA (0.5M (Ph 7.0) as a 100x stock); 15 inverting plate to remove liquid. Wash 3 times with h. Sodium ortho vanadate (0.5M as a 100x stock); TESW+0.5% ethanolamine. pH 7.0. Pat the plate on a i. Sodium pyro phosphate (0.2M as a 100>
TABLE 1
ELISA Assay Results
IGR- HER-2 HER-2 FLK-l # IR IR FBFR PDGFR BT474 3T3 Cell. FLK-l
A >100 >100 >50 3.9 11 B 23.8 <10O >100 8 3 .7
2. Resuspend the cells in fresh DMEM+10% FBS-H-2mM 6.2. Cell Growth Assays L-Glutamlne. and transfer to 96-well tissue culture plate (Corning. 25806-96) at 20.000 cells/well (100 [Ll/Well). The 20 The following assays may be conducted to measure the cells are then incubated for 1 day. Following such effect of the claimed compounds upon cell growth as a result incubation. 0.01% serum medium (90/ul) replaces the old of the compound’s interaction with one or more RTKs. media and the cells incubate in 5% CO2 and 37° C. over Unless otherwise speci?ed. the following assays may be night. generally applied to measure the activity of a compound B. ELISA Plate Coating and Blocking 25 against any particular RI‘K. To the extent that an assay. set l. Coat the ELISA plate (Corning 25805-96) with Anti-IR forth below. refers to a speci?c RTK. one skilled in the art Antibody at 0.5 ugwell in 100 |.Ll PBS at least 2 hours. would be able to adapt the disclosed protocol for use to 2. Remove the coating solution. and replace with 100 u; measure the activity of a second RTK. blocking Buffer. and shake for 30 minutes. Remove the blocking bu?’er and wash the plate just before adding lysate. 30 6.2.1. Soft Agar Assay C. Assay Procedures 1. The drugs are tested in semm-free condition. The soft agar assay may be used to measure the effects oil 2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM substances on cell growth. Unless otherwise stated the soft in 96-well poly-propylene plate. and transfer 10 pl/well of agar assays were carried out as follows: this solution to the cells to achieve ?nal drug dilution 1:100. 35 Material And Reagents. The following materials and and ?nal DMSO concentration of 1.0%. Incubate the cells in reagents were used: 5% CO2 at 37° C. for 2 hours. a. A Water bath set at 39° C. and another water bath at 37° 3. Prepare fresh cells lysis buffer (HNTG*) C. HNTG (5x) 2 ml b. 2X assay medium is comprised of 2>< Dulbeoco’s EDTA 0.1 ml Modi?ed Eagle’s Medium (DMEM) (Gibco Cat. # Na3VO4 0.1 ml CA400-4AN03) supplemented by the following: Na4P2O7 0.1 ml 20% Fetal Bovine Serum (PBS) 2 mM sodium pyruvate H2O 7.3 ml 4 mM glutamine amine; and HNTG* 10 ml 20 mM HEPES Non-essential Amino Acids (1:50 froc. 4. After drug incubation for two hours. transfer 10 til/well 45 100x stock). of 1 pM insulin in PBS to the cells (Final cont=l00 nM) . 0. 1x assay medium made of 1x DMEM supplemented and incubate at 5% CO2 at 37° C. for 10 minutes. 5. Remove media and add 100 til/well HNTG* and shake with 10% FES. 1 mM sodium pyruvate. 2 mM for 10 minutes. Look at cells under microscope to see if they glutamine. 10 mM HEPES. non-essential amino acid are adequately lysed. (1:100 from 100>< stock). 6. Using a 12-channel pipette. scrape the cells from the 50 (1. 1.6% SeaPlaque Agarose in autoclave bottle. plate. and homogenize the lysate by repeat aspiration and e. Sterile 35 mm Corning plates (FMC Bioproducts Cat. dispense. Transfer all the lysate to the antibody coated #50102). ELISA plate. and shake for 1 hour. f. Sterile 5 ml glass pipets (individually wrapped). 7. Remove the lysate. wash the plate. transfer anti-pTyr (1:3.000 with TEST) 100 pl/well. and shake for 30 minutes. 55 g. Sterile 15 m1 and 50 ml conical centrifuge tubes. 8. Remove anti-pTyr. wash the plate. transfer Tago (1:3. h. Pipets and sterile lips. . 000 with TEST) 100 pl/well. and shake for 30 minutes. i. Sterile microcentrifuge tubes. 9. Remove detection antibody. wash the plate. and transfer j. Cells in T75 ?asks: SKOV-3 (AT CC HTB77). fresh ABTS/H2O2 (1.2 ul H202 to 10 ml AE'I‘S) 100 Ill/Well k. 0.25% Trypsin solution (Gibco #25200-015). to the plate to start color development. Procedure. The following procedure was used to conduct 10. Measure OD in Dynatec MRSOOO. which is connected the soft agar assay: to Ingres. All following steps should follow Ingres instruc A. Procedure for making the base layer tion. 1. Have all the media warmed up in the 37°C. water bath. 6.1.7. Experimental Results From ELISA Assays 65 2. To make 1>< of assay medium+0.8% agar: make a 1:2 The experimental results for Compounds 4158 and 4350 (volzvol) dilution of melted agar (cooled to 39° C.). using the above-described protocols are set forth at Table l: with 2x assay medium. 5.763.470 31 32 3. Keep all media with agar warm in the 39° C. water bath described by Skehan et al.. 1990. J. Natl. Cancer Inst. when not in use. 82:1107-1112. The assay measures protein content of acid 4. Dispense 1 ml of 1x assay medium+0.8% agar into ?xed cells using the counterion binding dye sulforhodamine dishes and gently swirl plate to form a uniform base B (SRB. Sigma). The compounds were solubilized in layer. Bubbles should be avoided. DMSO (Sigma. cell culture grade) and diluted into appro 5. Refrigerate base layers to solidify (about 20 minutes). priate growth medium at two-fold the desired ?nal assay Base layers can be stored overnight in the refrigerator. concentration. In assays using C6 cells. compounds (100 pl) B. Procedure for collecting cells were added to 96-well plates containing attached cellular 1. Take out one ?ask per cell line from the incubator; monolayers (2000 cells/well in 100 1). C6 cells were main aspirate off medium; wash once with PBS and aspirate 10 tained in Ham’s F10 supplemented with 5% fetal bovine off; add 3 ml of trypsin solution. serum (FBS) and 2 mM glutamine (GLN). After 4 days (37° C.. 5% CO2) the monolayers were washed 3 times with PBS 2. After all cells dissociate from the flask. add 3 ml of 1x and ?xed with 200 pl ice-cold 10% TCA (Fisher Scienti?c). assay media to inhibit trypsin activity. Pipet the cells up and kept at 4° C. for 60 min. The TCA was removed and the and down. then transfer the suspension into a 15ml ?xed monolayers were washed 5 times with tap water and tube. allowed to dry completely at room temperature on absorbent 3. Determine the concentration of cells using a Coulter paper. The cellular protein was stained for 10 min. with 100 counter. and the viability by trypan blue exclusion. u] 0.4% SRB dissolved in 1% acetic acid. After 5 washes 4. Take out the appropriate volume needed to seed 3300 with tap water. the dye was solubilized in 10 mM Tris base viable cells per plate and dilute it to 1.5 ml with (100 pl per well) and absorbance read at 570 nm on a 1>