WO2007017678A1 - Composes de pyrazolo[1,5-a] pyrimidine et leurs compositions pharmaceutiques - Google Patents

Composes de pyrazolo[1,5-a] pyrimidine et leurs compositions pharmaceutiques Download PDF

Info

Publication number
WO2007017678A1
WO2007017678A1 PCT/GB2006/002968 GB2006002968W WO2007017678A1 WO 2007017678 A1 WO2007017678 A1 WO 2007017678A1 GB 2006002968 W GB2006002968 W GB 2006002968W WO 2007017678 A1 WO2007017678 A1 WO 2007017678A1
Authority
WO
WIPO (PCT)
Prior art keywords
carbon atoms
cells
cancer
compound
apoptosis
Prior art date
Application number
PCT/GB2006/002968
Other languages
English (en)
Inventor
Finbarr Murphy
Theo Richard James
Ian Hayes
Original Assignee
Eirx Therapeutics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB0516378.7A external-priority patent/GB0516378D0/en
Application filed by Eirx Therapeutics Limited filed Critical Eirx Therapeutics Limited
Priority to EP06779092A priority Critical patent/EP1922321A1/fr
Publication of WO2007017678A1 publication Critical patent/WO2007017678A1/fr
Priority to US12/012,888 priority patent/US20080300263A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to certain pyrazolo[1 ,5-a]pyrimidine compounds, to processes for their preparation, compositions comprising them and methoda of using them. Novel screening methods are also provided.
  • Apoptosis is a genetically regulated process of cell suicide that allows for the removal of unneeded, senescent or infected cells from the body while preserving the integrity and architecture of surrounding tissue. Whereas mitosis is responsible for the generation of new cells, apoptosis in contrast is responsible for removing the cells. It is this delicate balance of mitosis versus apoptosis that maintains tissue homeostasis.
  • Apoptotic pathways can be sub-divided into two categories, either the extrinsic apoptotic signals which are initiated on the outside of the cell by ligand engagement of cell surface receptors such as Fas and TNF receptors, and/ or the intrinsic pathways activated by signals emanating from cellular damage sensors (e.g. p53) or developmental cues.
  • the pathways activated by extrinsic and intrinsic signals can overlap to some extent, receptor ligation (via an extrinsic signal) typically leads to recruitment of adapter proteins that promote caspase oligomerization and auto-processing
  • the death signal is transmitted through conformational changes in preformed receptor clusters, resulting in the recruitment of the adaptor protein FADD (Fas-associated death-domain protein) through a DD-DD interaction.
  • FADD Fas-associated death-domain protein
  • FADD binds to the prodomain of caspase-8, which results in autoactivation of caspase-8 by proteolytic processing leading to cellular destruction ⁇ reviewed in Murphy et al Curr. Opin Pharm. 2003, 3:412-419).
  • cytochrome c binding of cytochrome c to a central apoptotic regulator, Apaf-1 , promotes oligomerization of Apaf-1 and, in a reaction requiring the ATPase activity of Apaf-1 , oligomerization and activation of caspase 9).
  • Caspase 9 subsequently activates effector caspases such as 3, 6, and 7 and cellular destruction ensues (reviewed in Johnson & Jarvis 2004, Apoptosis.;9(4):423-7).
  • Apoptosis is the normal physiological response to many stimuli, including irreparable DNA damage.
  • Various diseases evolve because of hyperactivation (neurodegenerative diseases, immunodeficiency, ischaemia-reperfusion injury) or suppression of programmed cell death (cancer, autoimmune disorders).
  • Colorectal cancer is common in economically developed countries, particularly in Europe, North America and Australia and is the second leading causes of cancer-related deaths in the Western world. Every year, colorectal cancer is responsible for arr estimated 400,000 deaths worldwide. Approximately 60,000 people die from colorectal adenocarcinoma among the 150,000 new cases, which are diagnosed in Europe each year. A genetic contribution to colon cancer risk is suggested by two observations, namely a) an increased incidence of colorectal cancer among persons with a family history of colorectal cancer and b) families in which multiple family members are affected with colorectal cancer, in a pattern indicating autosomal dominant inheritence of cancer susceptibility (Burt et al 2004, Gastroenterology.
  • neoplasms present with a broad spectrum of neoplasms, ranging from benign growths to invasive cancer and are predominantly epithelial in origin (i.e adenomas or adenocarcinomas).
  • Pathologists have classified the lesion into three groups: nonneoplastic polyps, neoplastic polyps (adenomatous polyps, adenomas) and cancers.
  • carcinomas Over 95% of colorectal cancers are carcinomas, most of which are adenocarcinomas.
  • adenocarcinomas are generally considered to arise from adenomas because a) benign and malignant tissue occur within colorectal tumours (Perzin and Bridge 1981 , Cancer.48(3):799-819) and b) when patients with adenomas were followed for 20 years, the risk of cancer at the site of the adenoma was 25%, a rate much higher than the expected norm (Stryker et al 1987, Gastroenterology. 1987;93(5): 1009-13). In addition, removal of adenomatious polyps is associated with reduced colorectal cancer incidence (Muller and Sonnenberg 1995, Ann Intern Med. 15;123(12):904-10).
  • the colon is organized into compartments of cells called crypts, where stem cells that reside near the bottom give rise to transit amplyifying cells that undergo five to seven additional divisions before they become terminally differentiated into one of four cell types, namely colonocytes, goblet cells, Paneth cells and enteroendocrine cells ( Brittan
  • Paneth cells continue to migrate to the top of the crypt where they undergo apoptosis and are engulfed by stromal cells or are shed into the lumen. For tissue homeostasis to ensue, the birth rate of the colonic epithelial cells precisely equals ther rate of loss from the crypt apex. If birth/ loss ratio increases, a neoplasm occurs.
  • the adenomatous polyposis coli encodes a large multidomain protein (31OkDa) that has many different sites for interaction with other proteins expressed constitutively within the normal colonic epithelium and whose activity is modulated by the Wnt signalling pathway (Polakis 2000, Genes Dev. 2000;14(15):1837-51).
  • the APC protein has been implicated in the regulation of a number of important cellular functions, abnormalities of which have been associated with colon cancer. The best characterized roles for the APC protein in cellular function can be listed as follows:
  • APC The best-characterized function of APC is as a scaffold protein in a multi-protein complex, consisting of GSK3b, ⁇ -catenin (beta-catenin), axin and several phosphatases and kinases and whose activity is modulated by Wnt signalling.
  • Wnts are secreted glycoproteins that act as Iigands to stimulate receptor-mediated signal transduction pathways in both vertebrates and invertebrates. Activation of Wnt pathways can modulate cell proliferation, apoptosis, cell behaviours, and cell fate.
  • Wnt signaling pathways often work in a combinatorial manner with other pathways, including the fibroblast growth factor (FGF) and transforming growth factor beta (TGFb) pathways, the retinoid signaling pathways, the p53 pathway and insulin-like growth factor type 1 receptor signaling pathway.
  • FGF fibroblast growth factor
  • TGFb transforming growth factor beta
  • the Wnt/beta-catenin pathway is the best understood Wnt signaling pathway and is highly conserved during evolution.
  • GSK3b In the presence of a Wnt signal, GSK3b is inactivated, ⁇ -catenin degradation does not proceed and thus more ⁇ -catenin is available to the cell to prevent apoptosis (van Noort and Clevers 2002; Dev Biol;244(1):1-8).
  • Frat The mammalian family member of proteins termed Frat.
  • the family members are homologous to GSK-3 binding protein (GBO), a protein identified in Xenopus embryos that act as positive regulators of Wnt signaling by stabilizing ⁇ -catenin (Yost et al 1998, Cell 93, 1031-1041).
  • Wnt signaling Frat is thought to be recruited to the GSK-3/Axin/ ⁇ -catenin complex and cause a dissociation of Axin (Li et al, 1999, EMBO J. 18, 4233-4240).
  • Axin is a substrate of GSK-3
  • the dissociation of Axin from GSK-3 results in the dephosphorylation of Axin which decreases its affinity for ⁇ -catenin and allows it to be released from the GSK-3/ Axin/ ⁇ -catenin complex (Willert et al 1999, Genes Dev. 13, 1768-1773).
  • members of the Frat family of protein are normally expressed at sites of active Wnt signaling (van Amerongen et al, 2005, Genes Dev. 19(4):425-30).
  • GSK3b phosphorylates APC, ⁇ -catenin and axin which increases their interaction. This complex results in an increase of a recoginition site for ubiquitination ligases and leads to the destruction of ⁇ -catenin by the proteasome, thus decreasing intracellular levels.
  • Beta-catenin is also rapidly transported from the nucleus, thus its nuclear levels are determined by both import and export (Yokoya et al.1999; MoI Biol Cell; 10(4): 1119-31 )).
  • ⁇ -catenin forms a complex with HMG box transcription factors of the LEF and TCF classes as well as with CREB Binding Protein (CBP, refs. Takemaru and Moon, 2000; J Cell Biol 2000;149(2):249-54 Hecht et al, 2000; EMBO J; 19(8): 1839-50) leading to activation of expression of target genes.
  • CBP CREB Binding Protein
  • ⁇ -catenin The association of ⁇ -catenin with the LEF/ TCF transcription factors promote the expression of a large number of genes, some of which have been identified and have been demonstrated to be important in the development and progression of colorectal carcinoma.
  • genes include c-myc, cyclin D1 , gastrin, cyclooxygenase (COX) -2, matrix metaloproteinase (MMP)-7, urokinase-type plasminogen activator receptor ( ⁇ PAR), CD44 proteins and P-glycoproteins.
  • COX cyclooxygenase
  • MMP matrix metaloproteinase
  • ⁇ PAR urokinase-type plasminogen activator receptor
  • CD44 proteins P-glycoproteins.
  • APC contributes to mitotic spindle formation and function such that during early mitosis, APC localizes to outer kinetochores, consitent with its accumulation at microtubule ends (Kaplan et al 2001 ; Nat Cell Biol.;3(4):429-32).
  • Cells lacking APC are more prone to chromosome segregation defects and investigators have recently shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and cause a dramatic increase in mitotic abnormalities.
  • the multiple intestinal neoplasia (Min) mouse mimics FAP patients in that these mice carry a mutation in one allele of APC and lose the remaining wild-type allele in the tumours that develop (Levy et al 1994; Cancer Res.; 54(22): 5953-8).
  • the mutant APC protein is defective in its ability to promote proteolytic degradation of ⁇ -catenin and leads to an activation of ⁇ -catenin induced transcription.
  • TGF ⁇ transforming growth factor ⁇
  • SMAD4 transcription factor ⁇
  • p53 a gene that is inactivated not only in colorectal cancers but also in most other cancer types. Activation of the normal p53 gene inhibits cell growth by blocking the cell cycle and by stimulating cellular suicide (apoptosis).
  • PRL-3 which encodes a protein tyrosine phosphatase that is highly expressed in metastatic colorectal cancer cells but not in earlier stages of the disease.
  • PIK3CA which encodes a lipid kinase that is mutated in invasive forms of colorectal cancers as well as several other invasive cancer types (including cancers of the breast, stomach, brain, ovary, and lung).
  • MCP-1/CCL2 CC-chemokine Monocyte chemotactic protein-1
  • Oncogenic mutations of the APC protein or ⁇ -catenin have been found in a variety of human cancers, all of which result in a constitutively stable ⁇ -catenin protein. As well as colorectal, these cancers also include medulloblastomas, hepatoblastomas, hepatocellular carcinomas, pilomatricomas, endometrial, thyroid and Wilms' tumours (Polakis P. Genes Dev 2000 14, 1837-1851). These data imply that activation of the Wnt signalling pathway, by one means or another is one of the most common signalling abnormalities known in human cancer (Brown A, 2001 , Breast Cancer Res 3: 351-355).
  • FRAT2 (FREQUENTLY REARRANGED IN ADVANCED T-CELL LYMPHOMAS 2) Saitoh et al cloned FRAT2 cDNAs, spanning the complete coding sequence, from a human fetal lung cDNA library.
  • FRAT2 encoded 233 amino-acid protein, which showed 77.3% total amino-acid identity with Frati ; Frat2 and Frat3 were more homologous in the acidic domain (96% identity), the proline-rich domain (92% identity), and the GSK-3beta binding domain (100% identity). Elevated expression levels of Frat2 is associated with several forms of cancer, in particular gastric cancer ( Saitoh et al 2001 ; Biochem Biophys Res Commun;281(3):815-20).
  • Frati and Frat2 are intronless genes localized to the same portion of chromosome 10q24.1 and separated by only 10.7 kb (Freemantle et al, 2002; Gen;291 (1-2): 17-27). In a broad range of human tissues Frati and Frat2 are readily detected and expressed in a near identical pattern. The overlapping expression patterns suggest these two genes share a regulatory region and have similar function.
  • the proto-oncogene Frati was originally identified as a common site of proviral insertion in transplanted tumors of Moloney murine leukemia virus (M-MuLV)-infected Emu-Pirn 1 transgenic mice. Contrary to most common insertion sites implicated in mouse T cell lymphomagenesis, retroviral insertional mutagenesis of Frati constitutes a relatively late event in M-MuLV-Induced tumor development, suggesting that proviral activation of Frati contributes to progression of T cell lymphomas rather than their genesis. To substantiate this notion researchers generated transgenic mice that overexpress Frati in various organs, including lymphoid tissues.
  • Frati transgenic mice develop focal glomerulosclerosis and a nephrotic syndrome, but they do not exhibit an increased incidence of spontaneous lymphomas. Conversely, these mice are highly susceptible to M-MuLV-induced lymphomagenesis, and Frat1/Pim1 bitransgenic animals develop lymphomas with increased frequency compared to Pim1 transgenic littermates. These data support a role for Frati in tumor progression (Jonkers et al, 1999; Oncogene; 18(44):5982-90). Frati -deficient mice in which most of the coding domain was replaced by a promoterless beta-galactosidase reporter gene are normal, healthy and fertile. The pattern of LacZ expression in Frati (lacZ)/+ mice indicated Frati to be expressed in various neural and epithelial tissues.
  • Frati and Frat2 are positive regulators of the WNT- ⁇ -catenin signaling pathway.
  • 5-Fluorouracil (5-FU) and irinotecan have to date been the most widely used single agent therapies in the treatment of advanced metastatic colon cancers.
  • 5-FU Upon cell entry, 5-FU is converted to its active form 5-fluoro-20-deoxyuridine monophosphate (Allegra CJ, Grem JL (1997) Antimetabolites.
  • 5-Fluorouracil is also incorporated into RNA and interferes with RNA processing. It is an S-phase active agent with no activity in GO or G1 and causes S-phase arrest (Santi et al, 1987 , Biochemistry 26: 8606-8613).
  • Irinotecan (CPT-11) is a water-soluble camptothecin analogue, which inhibits topoisomerase I via conversion to its active metabolite SN38.
  • SN38 inhibits topoisomerase I activity by stabilising the topoisomerase I-DNA cleavable complex, which results in DNA double-strand breaks and ultimately to cell death.
  • Cells in S-phase are significantly more sensitive to camptothecins than cells in G1 or G2 (Li et al, 1972; Cancer Res 32: 2643-2650).
  • Avastin works against colon cancer by blocking a protein called vascular endothelial growth factor (VEGF). Tumors need the protein to grow and maintain their blood vessels. When VEGF is blocked, the tumor gets less blood, so it shrinks or spreads more slowly. Patients receiving Avastin lived about five months longer than patients on the standard regimen alone.
  • VEGF vascular endothelial growth factor
  • Erbitux is a genetically engineered version of a mouse antibody that contains both human and mouse components. This new monoclonal antibody is believed to work by targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, interfering with their growth.
  • EGFR epidermal growth factor receptor
  • aspirin and other NSAIDs are associated with reduced risk of colorectal carcinomas and cancer.
  • aspirin is associated with significant risk of Gastrointestinal (Gl) hemorrhage, as well as renal and hepatic side effects.
  • Gastrointestinal (Gl) hemorrhage As well as renal and hepatic side effects.
  • Recently use of aspirin among women was associated with increased risk of pancreatic cancer.
  • COX-2 specific inhibitors To overcome the nonspecific effect of aspirin, several pharmaceutics companies have developed COX-2 specific inhibitors. In 1999, the FDA granted accelerated marketing approval for celecoxib "to reduce the number of adenomatous colorectalpolyps in FAP as an adjunct to usual care" (FDA, December23, 1999). The rationale for targeting COX-2 specifically was the observation that COX isozymes were over expressed in colorectal adenomas and cancers as well as the fact that that targeted deletion of COX-2 gene prevented colorectal cancer in animals. However, despite the promise of efficacy and the widespread safe use of COX-2 specific inhibitors, long term safety of these drugs has not yet been established.
  • COX-2 inhibitors appear to share the same side effects as that of aspirin, although the Gl effects appear to be less severe than aspirin.
  • Gl effects appear to be less severe than aspirin.
  • Ml myocardial infarction
  • Ursodeoxycholic acid (ursodiol) has been shown to inhibit experimental colon carcinogenesis at least partly through decreasing toxic secondary bile acids.
  • the efficacy of ursodiol against colonic neoplasia in patients with ulcerative colitis and primary sclerosing cholangitis has been demonstrated.
  • Other activities of ursodiol that may contribute to it chemopreventative efficacy include inhibition of colon epithelium proliferation and induction of apoptosis.
  • Ursodiol is currently in a phase III study to evaluates its efficacy in adenoma prevention.
  • colorectal cancer is responsible for an estimated 400,000 deaths worldwide. Approximately 60,000 people die from colorectal adenocarcinoma among the 150,000 new cases, which are diagnosed in Europe each year.
  • Protein kinases are critical components of cellular signal transduction cascades. They are directly involved in apoptosis and survival pathways and as such are implicated in many diseases such as cancer and inflammation. Consequently they have become one of the most important target classes for drug development (Cohen P Nat Rev. Drug Discov. 1 , 309-315 (2002)).
  • imatinib (Gleevec) for chronic myeloid leukemia (CML) and gefitinib (Iressa) and erlotinib (Tarceva) for non-small cell lung cancer(NSCLC) has provided proof-of-principle that small molecule kinase inhibitors can be effective drugs.
  • CML chronic myeloid leukemia
  • Iressa gefitinib
  • Tarceva erlotinib
  • NSCLC non-small cell lung cancer
  • Pyrazolopyrimidines are known.
  • W092/18504, W002/50079, W095/35298, W002/40485, EP94304104.6, EP0628559, U. S. 6,383, 790, Chem. Pharm. Bull., (1999) 47 928, J. Med.Chem., (1977) 20,296, J. Med.Chem., (1976)19 517 and Chem. Pharm. Bull., (1962) 10 620 disclose various pyrazolopyrimidines.
  • WO2004/022561 discloses pyrazolo[1 ,5-a]pyrimidine compounds stated to be cyclin- dependent kinase inhibitors.
  • An illustrative example has the formula
  • WO2004/022560 discloses pyrazolo[1 ,5-a]pyrimidine compounds stated to be cyclin-dependent kinase inhibitors.
  • WO2004/026229 discloses pyrazolo[1 ,5-a]pyrimidine compounds stated to be cyclin-dependent kinase inhibitors.
  • WO2004/076458 discloses pyrazolo[1,5-a]pyrimidine compounds stated to be kinase inhibitors.
  • US2004/0209878 discloses pyrazolo[1 ,5-a]pyrimidine compounds stated to be cyclin-dependent kinase inhibitors.
  • WO2005/063756 discloses pyrazolo[1,5-a]pyrimidine compounds stated to be corticotrophin- releasing factor antagonists.
  • J. Med. Chem 48, 2005, 7604-7614 discloses pyrazolo[1 ,5-a]pyrimidine compounds as inhibitors of human protooncogene kinase PIM-1.
  • WO2004/087707 discloses pyrazolo[1 ,5-a]pyrimidine compounds stated to be kinase inhibitors.
  • the present invention addresses / alleviates the problems of the priori: art.
  • the invention provides a compound of the formula (I):
  • Cy 1 is an optionally substituted mono or bicyclic aromatic group of from 5 to 10 ring members and 1 to 10 carbon atoms, optionally having from 1 to 5 heteroatoms independently selected from sulphur, oxygen and nitrogen;
  • R 1 , R 2 and R 3 are each independently selected from hydrogen, hydroxyl, halogen, alkyl of 1 to 6 carbon atoms, haloalkyl of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, cycloalkyl of 3 to 8 carbon atoms, alkoxy of 1 to 6 carbon atoms, cycloalkoxy of 3-8 carbon atoms, haloalkoxy of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, thioalkyl of 1 to 6 carbon atoms, sulfoxoalkyl of 1 to 6 carbon atoms, sulfonoalkyl of 1 to 6 carbon atoms, aryl of 6 to 10 carbon atoms, -COR 5 , -CO 2 R 5 , -NO 2 , -CONR 5 R 6 , -NR 5 R 6 or -N(R 5 )COR 6 , -CN
  • alkynyl of 2 to 7 carbon atoms or alkenyl of 2 to 7 carbon atoms; wherein the alkyl, heterocyclic ring, alkenyl or alkynyl moieties are optionally substituted with hydroxy!, -CN, halogen, alkyl of 1 to 6 carbon atoms, alkoxy of
  • R 4 is a group of the formula -NH-CH 2 -Cy 2 ;
  • R 5 and R 6 are each, independently hydrogen, alkyl of 1 to 6 carbon atoms or aryl of 6-10 carbon atoms;
  • Cy 2 is an optionally substituted cyclic group; or a pharmaceutically acceptable salt form thereof; for use as a medicament.
  • Cy 1 is an optionally substituted mono or bicyclic aromatic group of from 5 to 10 ring members and 1 to 10 carbon atoms, optionally having from 1 to 5 heteroatoms independently selected from sulphur, oxygen and nitrogen;
  • R 1 , R 2 and R 3 are each independently selected from hydrogen, hydroxy!, halogen, alkyl of 1 to 6 carbon atoms, haloalkyl of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, cycloalkyl of 3 to 8 carbon atoms, alkoxy of 1 to 6 carbon atoms,, cycloalkoxy of 3-8 carbon atoms, haloalkoxy of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, thioalkyl of 1 to 6 carbon atoms, sulfoxoalkyl of 1 to 6 carbon atoms, sulfonoalkyl of 1 to 6 carbon atoms, aryl of 6 to 10 carbon atoms, -COR 5 , -CO 2 R 5 , -NO 2 , -CONR 5 R 6 , -NR 5 R 6 or -N(R 5 )COR 6 , -
  • R 5 and R 6 are each, independently hydrogen, alkyl of 1 to 6 carbon atoms or aryl of 6-10 carbon atoms; Cy 2 is an optionally substituted cyclic group; or a pharmaceutically acceptable salt form thereof; for use as a medicament.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) or (II) together with at least one pharmaceutically acceptable carrier.
  • the invention provides a method of treating, inhibiting or preventing cancer in a mammal in need thereof, which comprises providing to said mammal an effective amount of a compound of formula (I) or (II).
  • the invention provides a method of inducing, inhibiting or modulating apoptosis in a mammal comprising providing to said mammal an effective amount of a compound of formula (I) or (II).
  • the invention provides the use of compound of formula (I) or (II) in a process for the preparation of a medicament for treating, inhibiting or preventing cancer.
  • the invention provides the use of compound of formula (I) or (II) in a process for the preparation of a medicament for treating, inhibiting or preventing a disease which is alleviated by the induction of apoptosis.
  • a method for detecting apoptosis- modulating activity in a candidate compound comprising the steps of: i) providing a reference cell line; ii) providing the reference cell line transformed to overexpress the FRAT2 gene (transformed cell line); iii) incubating a candidate compound with a) the reference cell line, b) the transformed cell line in the absence of GM-CSF, and c) the transformed cell line in the presence of GM-CSF; iv) quantifying the proportion of cells killed in cases a), b) and c); v) comparing the proportion of cells killed in cases a), b) and c); wherein the proportion killed in c) being greater than the proportion killed in a) and
  • Figures 1 to 20 are graphs
  • Cy 1 comprises from 1 to 5 heteroatoms (that is, at least one of the rings of Cy 1 is heterocyclic) independently ⁇ selected from the group consisting of nitrogen, oxygen, and sulphur.
  • Cy 1 comprises 1 heteroatom.
  • the heteroatom is nitrogen.
  • Cy 1 does not comprise heteroatoms (that is the ring system of Cy 1 is carbocyclic).
  • the substituents of Cy 1 may however comprise such heteroatoms.
  • At least one of the rings of Cy 1 is an aromatic ring.
  • Aromatic refers to a ring structure that has (4n+2) ⁇ electrons, where n is an integer. If Cy 1 comprises more than one ring, some, none or all of said rings may be aromatic. More preferably, all the rings Cy 1 are aromatic.
  • Cy 1 comprises at least one carbocyclic ring (i.e. a ring comprising only carbon atoms).
  • An example of a carbocyclic ring is a cycloalkyl group. Preferred cycloalkyl groups are those having from 3 to 12, more preferably from 3 to 7 carbon atoms.
  • the group Cy 1 may feature any degree of unsaturation consistent with a stable chemical structure. For example, there may be one or more than one double bond, one or more than one triple bond, or both kinds of bond.
  • Cy 1 comprises more than one ring
  • the rings may be fused in any chemically stable configuration, especially ortho-fused (two rings share two atoms), or spiro fused (two rings share one atom).
  • Bridged structures such as quinuclidine are also envisaged.
  • aromatic groups examples include thiophene, furan, isobenzofuran, chromene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalizine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, phenyl and naphthyl.
  • Cy 1 is an optionally substituted phenyl, thiophene (especially 2-thiophene), pyridine (especially 4-pyridine) or quinoline (especially 8- quinoline) group.
  • Preferred optional substituents of Cy 1 are from 1 to 5 substituents independently selected from the group consisting of hydroxyl, halogen, alkyl of 1 to 6 carbon atoms, haloalkyl of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, cycloalkyl of 3 to 8 carbon atoms, alkoxy of 1 to 6 carbon atoms, cycloalkoxy of 3- 8 carbon atoms, haloalkoxy of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, thioalkyl of 1 to 6 carbon atoms, sulfoxoalkyl of 1 to 6 carbon atoms, sulfonoalkyl of 1 to 6 carbon atoms, aryl of 6 to 10 carbon atoms, -COR 8 , -CO 2 R 8 , -NO 2 , -CONR 8 R 9 , -NR 8 R 9 or -N(R 8
  • R 8 and R 9 are each, independently hydrogen, alkyl of 1 to 6 carbon atoms or aryl of 6-10 carbon atoms.
  • Highly preferred optional substituents of Cy 1 are from 1 to 5 groups independently selected from alkoxy of 1 to 6 carbon atoms (especially methoxy), haloalkoxy of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms (especially chloromethyl), halogen (especially fluoro and chloro), -NR 8 R 9 , and a 5 or 6-membered heterocyclic ring having from 1 to 4 heteroatoms selected from O, N or S (especially morpholine, more especially 4-morpholine).
  • Cy 1 is selected from 3-methoxyphenyl, 3,4-dimethoxyphenyl, 4- methoxy phenyl, 2,4-dimethoxyphenyl, phenyl, 2-fluorophenyl, 3-(dimethylamino)phenyl, 3-quinolin-8-yl, 6-methoxy-pyridin-3-yl, 4-methyl-thiophen-2-yl, 3-chloro-4-fluorophenyl, 2-(chloromethyl)phenyl, morpholin-4-yl-phenyl and 4-pyridyl.
  • R 1 , R 2 and R 3 are hydrogen.
  • R 1 is hydrogen.
  • R 2 is hydrogen.
  • R 3 is hydrogen.
  • at least two of R 1 , R 2 and R 3 are hydrogen. More preferably, all of R 1 , R 2 and R 3 are hydrogen.
  • Preferred compounds of the invention are compounds of formula (I) wherein Cy 2 is an optionally substituted mono, bi- or tricyclic group.
  • Cy 2 comprises from 1 to 6 heteroatoms (that is, at least one of the rings of Cy 2 is heterocyclic) independently selected from the group consisting of nitrogen, oxygen, sulphur and phosphorous.
  • Cy 2 does not comprise heteroatoms (that is the ring system of Cy 2 is carbocyclic). The substituents of Cy 2 may however comprise such heteroatoms.
  • At least one of the rings of Cy 2 is an aromatic ring.
  • Aromatic refers to a ring structure that has (4n+2) ⁇ electrons, where n is an integer. If Cy 2 comprises more than one ring, some, none or all of said rings may be aromatic. More preferably, all the rings Cy 2 are aromatic.
  • Cy 2 comprises at least one carbocyclic ring (i.e. a ring comprising only carbon atoms).
  • An example of a carbocyclic ring is a cycloalkyl group.
  • Preferred cycloalkyl groups are those having from 3 to 12, more preferably from 3 to 7 carbon atoms.
  • the group Cy 2 may feature any degree of unsaturation consistent with a stable chemical structure. For example, there may be one or more than one double bond, one or more than one triple bond, or both kinds of bond.
  • Cy 2 comprises more than one ring
  • the rings may be fused in any chemically stable configuration, especially ortho-fused (two rings share two atoms), or spiro fused (two rings share one atom).
  • Bridged structures such as quinuclidine are also envisaged.
  • Cy 2 is an optionally substituted mono or bicyclic aromatic group of from 1 to 10 carbon atoms, optionally having from 1 to 5 heteroatoms selected from sulphur, oxygen and nitrogen.
  • aromatic groups examples include thiophene, furan, isobenzofuran, chromene, pyrrole,- imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalizine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, phenyl and naphthyl.
  • Cy 2 is an optionally substituted thiophene (especially 2- thiophene) or pyridine (especially 2-pyridine) group.
  • Optional substituents ef Cy 2 are from 1 to 5 substituents independently selected from the group consisting of hydroxyl, halogen, alkyl of 1 to 6 carbon atoms, haloalkyl of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, cycloalkyl of 3 to 8 carbon atoms, alkoxy of 1 to 6 carbon atoms, cycloalkoxy of 3-8 carbon atoms, haloalkoxy of 1 to 6 carbon atoms comprising from 1 to a maximum number of halogen atoms, thioalkyl of 1 to 6 carbon atoms, sulfoxoalkyl of 1 to 6 carbon atoms, sulfonoalkyl of 1 to 6 carbon atoms, aryl of 6 to 10 carbon atoms, -COR
  • R 8 and R 9 are each, independently hydrogen, alkyl of 1 to 6 carbon atoms or aryl of 6-10 carbon atoms.
  • Cy 2 does not have substituents.
  • Alkyl refers to an aliphatic hydrocarbon chain and includes straight and branched chains e. g. of 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, and isohexyl.
  • Alkeny alkeny!
  • Alkenyl refers to an aliphatic hydrocarbon chain having at least one double bond, and preferably one double bond, and includes straight and branched chains e. g. of 2 to 6 carbon atoms such as ethenyl, propenyl, isopropenyl,but-1-enyl, but-2-enyl, but-3-enyl, 2-methypropenyl.
  • Alkynyl refers to an aliphatic hydrocarbon chain having at least one triple bond, and preferably one triple bond, and includes straight and branched chains e. g. of 2 to 6 carbon atoms such as ethynyl, propynyl, but-1-ynyl, but-2-ynyl and but-3-ynyl.
  • Cycloalkyl refers to a cyclic, saturated hydrocarbon group having from 3 to 8 ring carbon atoms.
  • Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • Alkoxy as used herein refers to the group -O-alkyl, wherein alkyl is as defined above.
  • alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, t-butoxy, n-pentoxy, isopentoxy, neo-pentoxy, n-hexyloxy, and isohexyloxy.
  • Cycloalkoxy as used herein refers to the group -O-cycloalkyl, wherein cycloalkyl is as defined above.
  • Examples of cycloalkoxy groups are cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy, cycloheptyloxy and cyclooctyloxy.
  • Thioalkyl as used herein refers to the group -S-alkyl, wherein alkyl is as defined above.
  • Examples of thioalkyl groups are thiomethyl, thioethyl, n-thiopropyl, isothiopropyl, n- thiobutyl, isothiobutyl, sec-thiobutyl, t-thiobutyl, n-thiope ⁇ tyl, isothiopentyf, neo-thiopentyl, n-thiohexyl, and isothiohexyl.
  • Sulfoxoalkyl refers to the group -S(O)-alkyl, wherein alkyl is as defined above.
  • Sulfonoalkyl refers to a the group -S(O) 2 -aIkyl wherein alkyl is as defined above.
  • Halogen, halide or halo- refers to iodine, bromine, chlorine and fluorine.
  • Haloalkyl as used herein refers to an alkyl group as defined above wherein at least one hydrogen atom has been replaced with a halogen atom as defined above.
  • haloalkyl groups include chloromethyl, dichloromethyl, trichloromethyl, fluoromethyl, difluoromethyl and trifluoromethyl.
  • Preferred haloalkyl groups are fluoroalkyl groups ⁇ i.e. haloalkyl groups, containing fluorine as the only halogen). More highly preferred haloalkyl groups are perfluor ⁇ alkyl groups, i.e. alkyl groups wherein all the hydrogen atoms are replaced with fluorine atoms.
  • aryl refers to an unsaturated aromatic carbocyclic group of from 6 to 10 carbon atoms having a single ring (e. g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl).
  • Preferred aryl groups include phenyl, naphthyl and the like.
  • prodrugs that is compounds capable of undergoing metabolism to give compounds of formula (I) or (II) as defined above.
  • Suitable prodrugs are N-oxides and compounds having a quaternary nitrogen.
  • leaving group refers to any moiety or atom that is susceptible to nucleophilic substitution or elimination. Typically, these are atoms or moieties that when removed by nucleophilic substitution or elimination are stable in anionic form.
  • Examples of leaving groups useful in the present invention include alkyl- or arylsulphonate groups such as tosylate, brosylate, mesylate or nosylate, or halides such as fluoride, chloride, bromide, or iodide.
  • variable e.g. aryl, heterocycle, R 7 etc.
  • the compounds of the invention have therapeutic properties. They are expected to be useful in the treatment of proliferative diseases such as cancer, autoimmune diseases, viral diseases, fungal diseases, neurological/neurodegenarative diseases, arthritis, inflammation, alopecia, and cardiovascular disease.
  • proliferative diseases such as cancer, autoimmune diseases, viral diseases, fungal diseases, neurological/neurodegenarative diseases, arthritis, inflammation, alopecia, and cardiovascular disease.
  • the compounds of the invention can be useful in the treatment of a variety of cancers, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acuteiymphocytic leukemia, acutelymphoblasticleukemia, B-cell lymphoma, T- cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and
  • carcinoma including that of the bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma
  • Burkett's lymphoma Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplasia syndrome and promyelocyte leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; and other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma.
  • Compounds of the invention may induce, inhibit or modulate apoptosis.
  • the apoptotic response is aberrant in a variety of human diseases.
  • Compounds of the invention, as modulators of apoptosis will be useful in the treatment of cancer (including but not limited to those types mentioned hereinabove), viral infections (including but not limited to herpevirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus), prevention of AIDS development in HIV-infected individuals, autoimmune diseases (including but not limited to systemic lupus, erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus), neurodegenerative disorders (including but not limited to Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration
  • salts can be formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, naphthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable acids when a compound of this invention contains a basic moiety.
  • Salts may also be formed from organic and inorganic bases, preferably alkali metal salts, for example, sodium, lithium, or potassium, when a compound of this invention contains an acidic moiety.
  • the compounds of this invention may contain an asymmetric carbon atom and some of the compounds of this invention may contain one or more asymmetric centers and may thus give rise to optical isomers and diastereomers. While shown without respect to stereochemistry, the present invention includes such optical isomers and diastereomers; as well as the racemic and resolved, enantiomerically pure R and S stereoisomers; as well as other mixtures of the R and S stereoisomers and pharmaceutical acceptable salts thereof. It is recognized that one optical isomer, including diastereomer and enantiomer, or stereoisomer may have favorable properties over the other. Thus when disclosing and claiming the invention, when one racemic mixture is disclosed, it is clearly contemplated that both optical isomers, including diastereomers and enantiomers, or stereoisomers substantially free of the other are disclosed and claimed as well.
  • the present invention accordingly provides a pharmaceutical composition which comprises a compound of this invention in combination or association with a pharmaceutical acceptable carrier.
  • the present invention provides a pharmaceutical composition which comprises an effective amount of compound of this invention and a pharmaceutical acceptable carrier.
  • compositions are preferably adapted for oral administration. However, they may be adapted for other modes of administration, for example, parenteral administration for patients.
  • a composition of the invention is in the form of a unit dose.
  • Suitable unit dose forms include tablets, capsules, and powders in sachets or vials.
  • Such unit dose forms may contain from 0.1 to 100 mg of a compound of the invention.
  • the compounds of the present invention can be administered orally at a dose range of about 0.01 to 100 mg per kg.
  • Such composition may be administered from 1 to 6 times a day, more usually from 1 to 4 times a day.
  • compositions of the invention may be formulated with cOnventional excipients, such as fillers, a disintegrating agent, a binder, a lubricant, a flavouring agent, and the like. They are formulated in conventional manner.
  • the compounds of the present invention can for example be prepared according to the following reaction schemes or modification thereof using readily available starting materials, reagents and conventional synthetic procedures. It is also possible to make use of variants of these process steps, which in themselves are known to and well within the preparatory skill of the medicinal chemist.
  • reaction schemes R 1 , R 2 , R 3 , R 4 , and Cy 1 are as defined above.
  • Keto ester (XV) is suitably prepared by treatment of ester (XIII) with strong base (e.g. lithium diisopropylamide) followed by acid chloride (XIV) (scheme 2).
  • strong base e.g. lithium diisopropylamide
  • acid chloride (XIV) (scheme 2).
  • keto ester (XV) with 3-aminopyrazole (XII) gives pyridone (XVI). This is suitably converted to the corresponding chloride (XVII) by treatment with phosphoryl chloride. Reaction with amine (XVIII) gives the desired product (scheme 3), which may subsequently be converted to a pharmaceutically acceptable salt form if desired.
  • the compounds of the invention may be prepared by palladium-catalysed cross coupling of substituted 3 bromopyrazolo[1 ,5a]pyrimidines (XIX) with boronic acids (XX) to give 3-pyrazolo[1 ,5a]pyrimidines (I) or (II) (scheme 4).
  • Magnesium sulfate Alfa Aesar 33337
  • the solid residue was partitioned between 330 ml of water and 300 ml of CH2CI2.
  • the pH of the aqueous layer was readjusted to 3-4 by addition dropwise of- con. HCI.
  • the organic layer was separated and the aqueous layer extracted with further 200ml of
  • the melting point of the compound was 178-180 degree C.
  • the resultant mixture was stirred at RT for 72 hrs, in which a yellow suspension was formed and transferred into a 1 L one round-bottomed flask equipped with condenser and a magnetic stirrer. 500 ml of absolute ethanol was added, followed by 4-Metoxy- phenyl-2H-Pyrazol-3-ylamine (3.32g). Further 50ml of ethanol was used to rinse the flask containing the pyrazole.
  • reaction mixture was stirred, heated gradually to reflux and kept at this temperature for six hours. After concentration of the reaction mixture under vacuum Pump (water pressure), the residue was dissolved in hot water (ca 5OmI) and the mixture was filtered quickly to remove the inorganic salt. The filtrate was then acidified to pH 1with cone. HCI.
  • Nmr (DMSO-d6) demonstrated the formation of the desired material.
  • the solution was heated to 107 degree C for 3 hrs then allowed to cool overnight while stirring.
  • the reaction mixture was evaporated at reduced pressure.
  • the reaction mixture was evaporated to dryness.
  • the yellow residue was partitioned between 200ml of dichloromethane and 100ml of water.
  • the organic layer was separated and the aqueous layer extracted again with 40ml of dichloromethane.
  • the scaffold (A) was sourced commercially from Butt Park Ltd., Bath, UK.
  • the synthesis of (14) was carried out exactly as described for examples (1)-(13) using the appropriate scaffold, amine and boronic acid, giving Compound (14) in good yield, MS: m/z 324(326)7322(324) [M+H]+
  • Example 2 Generation of TF-1 that are engineered to overexpress the FRAT2 gene
  • GFP Green Fluorescent Protein
  • IRES Internal Ribosomal Entry Site
  • This example describes the cloning of the full length coding sequence (cds) of FRAT2 (GenBank Accession No. BC020165) using FRAT2 specific amplification primers and a TOPO-TA cloning kit from Invitrogen. It further describes how the FRAT2 cds is incorporated into a construct such that it is linked to a gene encoding the human recombinant Green Fluorescent Protein (hrGFP) using an internal ribosomal entry site.
  • hrGFP Green Fluorescent Protein
  • the DNA fragment is then incubated with Taq and dATPs for 5 min. at 72 0 C to add on 3'
  • A-overhangs on the amplified DNA which may have been lost during gel extraction, but which are necessary for TOPO- TA cloning.
  • the DNA fragment is "TOPO" cloned into Invitrogen's pcDNA3.1/V5-His TOPO TA expression vector according to the manufacturers instructions, and transformed into Top10 £ ⁇ coli cells. Transformed cells are plated on ampicillin containing Agar plates for selection. Colonies are screened for the FRAT2 insert using T7 forward (5'- TAATACGACTCACTATAGGG-S 1 ) and BGH reverse ( ⁇ '-TAGAAGGCACGTCGAGG-S 1 ) primers which yields a 987 bp product if FRAT2 has been successfully cloned or a 266 bp product in the absence of any insert. Colonies are screened for inserts in the sense orientation using T7 forward and FRAT2 reverse primers and in the antisense orientation using T7 forward and FRAT2 forward primers.
  • Glycerol stocks of FRAT2-containing clones are prepared and stored at -7O 0 C. Three different clones are sequenced and a clone containing no mutations is selected for further use. A large-scale preparation of the selected plasmid is purified using Qiagen's Plasmid Mid Kit (#112145). The concentration of the plasmid is adjusted to 1mg/ml for convenience during subsequent manipulations.
  • the FRAT2 gene is cloned into a modified pMSCVhyg retroviral vector (Clontech #K1062-1) using the GATEWAY System (Invitrogen), as detailed below.
  • the IRES and hrGFP genes from plREShrGFP-1a (Stratagene #240031) (position 710- 2446) is cloned into the Xho1 and Hpa1 sites of pMSCV by directional cloning, resulting in a construct called pMSIRG.
  • pMSIRG This allows the level of expression of a gene of interest to be monitored due to the expression of hrGFP on the same transcript.
  • the blunt ended attR cassette (reading frame B), from Invitrogen's GATEWAY Conversion System (#11828-19), is ligated into pMSIRG that is digested with Xho1 and blunt-ended using Klenow. Recombinant constructs are selected in which the attR cassette is in the sense orientation, namely pMSIRGattRS.
  • the FRAT2 gene In order for the FRAT2 gene to be cloned into the pMSIRGattRS expression vector, it is first cloned into the pDONR 201 vector (Invitrogen #1179-014) as follows:
  • the FRAT2 gene is amplified from pcDNA3.1/V5-His TOPO-FRAT2 plasmids using attB- linked T7 and BGH primers, i.e.T7-AttB1 forward primer (5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATACGACTCACTATAGGGS') and BGH-attB2 reverse primer (5'-
  • the resulting 1045bp product is gel extracted (using Qiagen Gel Extraction kit #28706) and cloned into pDONR201 via a GATEWAY BP cloning reaction according to Invitrogen's instructions, to generate pDONR201-FRAT2.
  • Recombinant clones are selected on kanamycin-containing agar plates.
  • Recombinant plasmids are purified using Qiagen QIAprep Spin Miniprep Kit (#27104) and the presence of the insert confirmed by restriction analysis.
  • a GATEWAY LR cloning reaction is performed using pDONR201-FRAT2 and pMSIRGattRS according to the GATEWAY Technology Instruction manual to generate pMSIRGattRS-FRAT2.
  • Recombinant clones are selected on amplicillin-containing agar plates.
  • Recombinant plasmids are purified using Qiagen QIAprep Spin Miniprep Kit (#27104) and the presence of the insert confirmed by restriction analysis.
  • a large-scale preparation of a selected clone is purified using Qiagen's Plasmid Mid Kit (#112145). The concentration of the plasmid is adjusted to 1mg/ml for convenience subsequent viral transductions into mammalian cells.
  • This example describes how the Erytholeukemic TF- 1 cell line are infected with retrovirus incorporating the FRAT2.gene, thus allowing for expression of said gene.
  • pMSIRGattRS-FRAT2 containing ecotropic retrovirus is generated by transfecting a mono-layer of 70-80% confluent Phoenix-ECO PCL (Packaging Cell Line) cells (licensed from Stanford University) with 10 ⁇ g of pMSIRGattRS-FRAT2 plasmid in a T25 flask in media containing DMEM and freshly prepared 25 ⁇ M chloroquine diphosphate (Sigma #C6628) using the Clontech CalPhos mammalian transfection kit (#PT3025-1) according to the manufacturers instructions. Cells are incubated at 37 0 C for 18 h after which the transfection medium is replaced with fresh serum-free RPMI-1640 medium. Cells are incubated at 32°C during which time virus is released into the supernatant. After 48h the supernatant is harvested by centrifugation and filtered through a 0.45 ⁇ M filter and used immediately.
  • Phoenix-ECO PCL Packaging Cell Line
  • 25 ⁇ M chloroquine diphosphate Sigma #C
  • C9M TF1 cells that have been modified to constitutively express the murine retrovirus receptor, mCAT, and thus are amenable to transduction with murine- retrovirus, are used for the infection with recombinant virus and expression of exogenous FRAT2.
  • the cells are spun down and re-suspended at a concentration of 1 x 10 5 cells/ml in normal growth medium containing 2ng/ml GM-CSF.
  • Freshly prepared polybrene [Hexadimethrine bromide] (Sigma #H9268) is added to the viral suspension to a concentration of 8 ⁇ g/ml.
  • the virus/poylbrene cocktail is added to the cells in appropriate culture vessel and these are incubated at 37 0 C for 18h after which the virus- containing supernatant is replaced with fresh RPMI-1640 growth medium, 2ng/ml GM- CSF and maintained at 37°C at a final cell density of 2x10 5 cells/ml.
  • Stably-transduced C9M-TF1 populations are selected and non-transduced populations are eliminated by adding 500-600 ⁇ g/ml hygromycin at 48 h post infection. Selected cells are monitored for cytopathic effects and re-fed with fresh selection medium every 3-4 days. Following the death of all mock-transduced cells, the selected population of 100% transduced cell are expanded for expression analysis/assay/freezing etc.
  • This example describes a procedure whereby monoclonal FRAT2 cells are isolated. Monoclonal cell lines are preferred in the current series of experiments due to their uniform response.
  • a dilute cell suspension of polyclonal FRAT2 cells is prepared at 1.5 x 10 3 cells/ml.
  • a forceps is used to partially submerge a 200 ⁇ l Gilson tip in the dilute cell suspension until the tip becomes filled with medium. The point of the tip is gently pressed against the bottom of each well of a row of a 96-well plate such that a tiny droplet forms in the centre of the well.
  • the droplets are viewed under the microscope (10X) to determine how many, if any, contain a single cell per droplet. Each droplet is independently screened by two individuals, to verify which wells contain a single cell. The process is repeated to generate several wells having a single cell per well.
  • This example describes "real time" quantitative PCR (QPCR) analysis performed on TF-1 cells that have been engineered to express the FRAT2 cds.
  • QPCR quantitative PCR
  • This example describes QPCR analysis of C9M-TF-1 cells that have been engineered to express the FRAT2 cds and where the expression of messenger RNA for FRAT2 is measured.
  • RNA is purified using Qiagen RNeasy Kits (#74104) and Qiashredders (#79654) according to manufacturers protocol.
  • cDNA is synthesized from this RNA using Superscript Il Reverse Transcriptase from Invitrogen (#18064-014) according to the manufacturers instructions.
  • Quantitative real time PCR is performed on the cDNA samples using QuantiTect SYPR Green Kit (according to the manufacturers instructions) and FRAT2 Quantitative PCR primers (QFRAT2 forward: 5'- AGCGCCGATGGACCCAAGC-3'; QFRAT2 reverse: ⁇ '-AGGGCAATGCGGTCAGGTCC- 3') on a DNA Engine Opticon system from MJ Research (PTC200DNA Engine Cycler; CFD-3200 Opticon Detector).
  • the expression level of FRAT2 is normalized- with the expression level of the housekeeping gene RPS13.
  • Figure 1 shows an example of such analysis.
  • Quantitative Real-time PCR shows that increased levels of FRAT2 mRNA are detected in FRAT2 expressing cells compared to wild type (WT) C9M-TF1 cells whether they are grown in the presence or absence of GM-CSF.
  • WT wild type
  • FRAT2 results in an almost 5 fold increase in mRNA levels in cells grown in the presence of GM-CSF, and 3 fold increase in cells grown in the absence of GM-CSF.
  • Figure 1 shows levels of ⁇ -catenin in FRAT2 C9M-TF1 cells compared with wild type C9M-TF1 following GMCSF withdrawal.
  • This example describes the effect of withdrawing GM-CSF from C9M-TF-1 cells on levels of ⁇ -catenin, and how expression of FRAT2, as exemplified in FRAT2 C9M-TF1 ceils, is able to maintain levels of the ⁇ -catenin protein.
  • RNA Either wild type C9M- TF1 cells or FRAT2-C9M-TF1 are cultured in the presence or absence of GMCSF for 24h after which time RNA is isolated.
  • the RNA is purified using Qiagen RNeasy Kits (#74104) and Qiashredders (#79654) according to manufacturers protocol.
  • cDNA is synthesized from this RNA using Superscript Il Reverse Transcriptase from Invitrogen (#18064-014) according to the manufacturers instructions.
  • Quantitative real time PCR is performed on the cDNA samples using QuantiTect SYPR Green Kit (according to the manufacturers instructions) and the quantitative RCR primers as indicated below are used with a DNA Engine Opticon system from MJ Research (PTC200DNA Engine Cycler; CFD-3200 Opticon Detector).
  • the expression level of all genes is normalized with the expression level of the housekeeping gene RPS13.
  • Figure 2 shows that withdrawal of GM-CSF for 24h causes a dramatic decrease in the level of ⁇ -catenin expression; however overexpression of FRAT2 is able to rescue this decrease.
  • FRAT2 in known as a positive regulator of the wnt signaling pathway by displacing GSK3 ⁇ from the axin/beta-catenin complex, thereby facilitating the translocation of beta-catenin to the nucleus and the transcriptional activation of several downstream genes including c-myc, cyclin D1 and cox2.
  • RNA Either wild type C9M- TF1 cells or FRAT2-C9M-TF1 are cultured in the presence or absence of GMCSF for 24h after which time RNA is isolated.
  • the RNA is purified using Qiagen RNeasy Kits (#74104) and Qiashredders (#79654) according to manufacturers protocol.
  • cDNA is synthesized from this RNA using Superscript Il Reverse Transcriptase from Invitrogen (#18064-014) according to the manufacturers instructions.
  • Quantitative real time PCR is performed on the cDNA samples using QuantiTect SYPR Green Kit (according to the manufacturers instructions) and the quantitative PCR primers as indicated below are used with a DNA Engine Opticon system from MJ Research (PTC200DNA Engine Cycler; CFD-3200 Opticon Detector).
  • the expression level of all genes is normalized with the expression level of the housekeeping gene RPS13.
  • Figures 3, 4 and 5 withdrawal of GM-CSF can affect expression of the ⁇ -catenin regulated genes c-myc, Cyclin D1 and Cox2.
  • overexpression of FRAT2 reduces the decrease in expression, thus demonstrating that FRAT2 can positively affect ⁇ -catenin regulated genes.
  • Example 4 Demonstration that FRAT2 C9M-TF1 are more resistant to apoptosis than wild type C9M-TF-1
  • This example describes the assays used to determine whether FRAT2 C9M-TF1 engineered cells are more resistant to apoptosis than their wild type C9M-TF1 cells.
  • FSC/SSC analysis utilised in this example represents an apoptosis assay that can distinguish accurately between cells that have undergone apoptosis or necrosis from viable cells. This is based on the characteristic changes in cell size and granularity associated with viable, apoptotic and necrotic morphology.
  • FRAT2 C9M-TF1 and C9M- TF- 1 cells are analysed utilising the flow cytometer. The position of the cell population on the FSC and SSC scale are noted by isolating the population with a gate. There is no difference in FSC/SSC parameters for either FRAT2 C9M-TF1 or C9M-TF1 in healthy cultures.
  • the effect of removing GMCSF on cell viability is ascertained by observing the movement of the cell population from the viable gate previously recorded. Necrotic cells increase in size before disintegrating and therefore are noted to shift up and to the right of the viable cell population initially while next shifting completely into the debris section of the FSC/SSC plot (bottom left) when disintegrated. Apoptotic cells however simply move to the left of the viable population gradually reducing in size, and then shift gradually down the SSC scale as the granularity of the cells increases in late stage apoptosis.
  • the changes in FSC/SSC parameters can be used to identify cells undergoing apoptosis post GMCSF withdrawal.
  • Cells are cultured for 72h with or without GMCSF (2ng/ml) after which they are harvested, and acquired immediately by a FacsCalibre (Bectorf Dickenson). Forward and Side Scatter parameters are assessed using Cell Quest software.
  • the FSC/SSC parameters of the cell population cultured in the presence of GMCSF is compared to the FSC/SSC parameters of the cells cultured in the absence of GMCSF.
  • the percentage of cells remaining within the viable cell gate post 72h GMCSF starvation is ascertained for both FRAT2 C9M-TF1 or C9M-TF1 populations.
  • Fig 6 shows that when GMCSF is withdrawn from either FRAT2 C9M-TF1 or C9M-TF1, the cells undergo apoptosis.
  • the extent of cells undergoing apoptosis is significantly less in the FRAT2 C9M-TF1 relative to the C9M-TF1 cells (28% c.f. 61%, respectively), thus the presence of FRAT2 makes cells refractive to apoptosis.
  • This example demonstrates that the protective effective of FRAT2 expression can also be examined using the addition of Alamar Blue to cultures of cells with and without GMCSF.
  • the addition of Alamar Blue to a culture of cells enables the practitioner to measure the viability of the cells.
  • the internal environment of the proliferating cell is more reduced than that of non-proliferating cells.
  • the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN, and NADH/NAD increase during proliferation.
  • Compounds such as resazurin, the active component of Alamar Blue can be reduced by these metabolic intermediates and are useful in monitoring cell proliferation because their reduction is accompanied by a measurable shift in colour.
  • resazurin accepts electrons from these compounds, it changes from the oxidized indigo blue, non- fluorescing state to the reduced fluorescent pink state resorufin. The reduction of resazurin appears to require uptake into the cell.
  • resazurin is not reduced by compounds in the cell culture medium and implicates metabolism within the cells under study as the site of resazurin reduction. Whether the cellular location of reduction of resazurin is cytoplasmic or mitochondrial has yet to be definitively determined. However, resazurin continues to have great utility in measurements of cell viability and proliferation.
  • Cultures of both FRAT2 C9M-TF1 or C9M-TF1 cells are washed twice in PBS to remove any residual culture medium, prior to being counted and resuspended in culture medium with GMCSF (2ng/ml) being present or absent at a concentration of 2x10 5 cells/ ml.
  • GMCSF GMCSF (2ng/ml) being present or absent at a concentration of 2x10 5 cells/ ml.
  • One hundred microlitres of cells are pipetted into wells of a 96 well plate such that there are 2 x10 4 cells per well. Cells are cultured at 37 0 C in an environment of 5% CO 2 .
  • Percentage survival is calculated by comparing the fluorescence of cells cultures in the absence of GMCSF to their cognate cultures cultured in the presence of GMCSF.
  • Figure 7 shows that removai of GMCSF from culture medium in C9M-TF1 cells induces apoptosis, such that the percentage of cells surviving is only approximately 60% of their cognate cells in the presence of GMCSF.
  • FRAT2 C9M-TF1 appear to be resistant to apoptosis induced by GMCSF withdrawal as after 72h, there is not a reduction in the percentage of viable cells, as determined by Alamar Blue conversion.
  • This example describes an assay to examine the effect of the PI3K inhibitor LY294002 on cell survival for both FRAT2-C9M-TF1 or C9M-TF1 cells, as determined by Alamar Blue.
  • the growth factor GMCSF mediates its survival effect through the P13/ AKT pathway. Consequently, inhibitors of this pathway will increase apoptosis in pathway- dependent cell lines.
  • Both FRAT2-C9M-TF1 or C9M-TF1 are split 24 hours prior to drug treatment in order to bring the cultures into the log phase of growth.
  • the cells are plated directly into a 96 well microtitre plates containing 1OuL volumes of a serial dilutions of LY294002, such that there was a final cell number of 5000 cells per well in 9OuL volumes thus reducing the concentration of the compounds to their final test concentration.
  • the plates are incubated for 72h at 37 0 C and 5% CO 2 after which time they are assayed by Alamar Blue.
  • Figure 8 shows the results obtained from one such Control Plate where the PI3-Kinase inhibitor LY294002 was titrated against C9M-TF1 cells and FRAT2-C9M- TF1 cells illustrating that FRAT2 expressing cells are very much more resistant to the effects of LY294002 than are their C9M counterparts.
  • the EC50 for FRAT2 cells is approximately 5OuM whereas the value for C9M cells is approximately 6uM.
  • FRAT2 leads to the stabilization ⁇ -catenin through disassociation of GSK3 ⁇ from Axin and through the inhibition of ⁇ -catenin phosphorylation which results in an increased level of ⁇ -catenin in the nucleus of the cell where it forms a complex with the HMB box transcription factors LEF and TCF as well as with CREB Binding Protein.
  • This association of ⁇ -catenin with the LEF/ TCF transcription factors promotes the expression of a large number of genes, many of which have been identified and shown to be important in the development and progression of colorectal carcinoma.
  • AKT/PKB As LY294002 inhibits the phosphorylation of AKT/PKB by PI3-Kinase, AKT/PKB is no longer able to inhibit GSK3 ⁇ and thus prevent ⁇ -catenin phosphorylation, leading to the degradation of ⁇ -catenin and the subsequent induction of apoptosis.
  • the increased survival of the FRAT2 overexpressing cell line indicates that the cells are using a particular pathway that is very commonly associated with colon cancer. Consequently, identifying hit molecules that induce apoptosis in the FRAT2 cells suggests that the molecules may be targeting that pathway.
  • the current example describes how the candidate compounds were screened against a number of cell lines, including both FRAT2-C9M-TF1 and C9M-TF1 cells.
  • Adherent cells are plated at a pre-determined seeding density (Table 1) in flat-bottom 96 well plates 24 hours prior to drug treatment.
  • the cell lines are plated in 100 ⁇ l volumes in each well.
  • Cells are plated in all wells apart from those in Column 12 which contain 100 ⁇ L normal culture medium alone. These plates will be known as the Test Plates.
  • the Test Plates are labelled with a Daughter Plate ID.
  • Daughter Plates containing 10 ⁇ l of a 100 ⁇ M solution of test compound selected from among the candidate compounds stored at -80 0 C are thawed at room temperature.
  • a 10 ⁇ L volume of 0.1%DMSO in PBS is added to all wells of Column 12 and the wells in Row A-F of Column 1.
  • a 10 ⁇ l volume of a pre-determined concentration of the PI3- Kinase inhibitor LY294002 is added to wells in Row G and H of Column 1.
  • a 90 ⁇ l volume of the appropriate cell culture medium is then added to each well and mixed by repeated aspiration/dispensing. The compounds are at a final test concentration of 10 ⁇ M.
  • the media is removed from all wells of the cell culture plate using an 8-Channel Vacuum Aspirator.
  • the contents of the Daughter Plates are then transferred to the appropriate wells of their relevant Test Plates and the Test Plates are incubated for 72h at 37°C and 5% CO 2 .
  • Test Plates are assayed by Alamar Blue.
  • a 10 ⁇ l volume of Alamar Blue is added to all wells of the Test Plate.
  • the Test Plates are incubated for 3-4 h at 37 0 C and 5% CO 2 and finally read using the BioTek SYNERGY plate reader (Excitation 530/25nm - Emission 590/35nm). Percentage inhibition is determined using Microsoft Excel spreadsheet calculations.
  • Suspension cells are split 24 hours prior to drug treatment in order to bring the cultures into the log phase of growth.
  • the cells are plated directly into the Daughter Plates at the appropriate pre-determined seeding density (Table 1) in 90 ⁇ l volumes, the concentration of the test compounds thus being reduced to 10 ⁇ M.
  • Cells are plated in all wells apart from those in Column 12 which contain 90 ⁇ l normal culture medium alone. The plates are incubated for 72h at 37 0 C and 5% CO 2 after which time they are assayed by Alamar Blue as per the preceding example.
  • C9M Derived from TF1 (Human erythroleukaemia)
  • Example 6 Determination of the EC 50 for the compounds across cancer and normal cells
  • This Example describes how the EC 50 was calculated for the claimed compounds across a breadth of cell lines including the C9M-TF-1 and FRAT2-C9M-TF1 , and the colon cancer cell line HT29 and the breast cancer cell line MCF7. Also included in this example is the EC 50 of the compounds across the non-transformed breast cell line MCF10A, which have a significantly higher EC 50 than their transformed counterparts. This demonstrates that the claimed compounds are more active in cancer cells than their non cancerous counterparts.
  • a 90 ⁇ l volume of the appropriate cell culture medium is then added to each well of the Titration Plates and mixed by repeated aspiration/dispensing, thus taking the compounds to their final test concentration.
  • the media is removed from all wells of the cell culture plate and the contents of the Titration Plates are then transferred to the appropriate wells of their relevant Test Plates and incubated for 72h at 37 0 C and 5% CO 2 after which time they are assayed by Alamar Blue.
  • EC50s are determined .
  • Suspension cells are split 24 hours prior to drug treatment in order to bring the cultures into the log phase of growth. On the day of the assay the cells are plated directly into the
  • Titration Plates at the appropriate pre-determined seeding density (Table 1) in 90 ⁇ I volumes thus reducing the concentration of the compounds to their final test concentration.
  • the plates are incubated for 72hours at 37 0 C and 5% CO 2 after which time they are assayed by AIamar Blue.
  • the EC 50 values are calculated using the values obtained from the AIamar Blue readout which are converted into a "Percentage of the Untreated Control" and using Graph Pad Prism these values are plotted against the logarithm of the test concentration. Using a non-linear regression analysis a sigmoidal dose-response curve is generated from which the EC 50 is determined.
  • Table 3 indicates the EC 50 values for example compounds when titrated against a range of cell lines.
  • the EC 50 of C9M-TF1 cells for the indicated compounds was significantly greater (range 2- 9 fold greater) than the EC 50 values for the engineered cells expressing FRAT2, screened either in the presence (FRAT2+) or absence (FRAT-) of growth factor, confirming our earlier observation from the primary screen that FRAT2-C9M-TF1 are more sensitive to the indicated compounds.
  • the EC 50 of both cells lines to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. is also given.
  • the FRAT2-C9M-TF1 cells are more resistant to LY294002, as demonstrated by the significantly different EC 50 1 S concentrations (approx. 8.5 fold difference).
  • EC 50 concentrations for the indicated compounds for a transformed colon cell line HT29 and a transformed breast cell line MCF7 are also described in the table.
  • the EC 50 for the breadth of compounds range from 0.7 ⁇ M to 4 ⁇ M in the cell based assay.
  • the table lists the EC 50 for the compounds on the non transformed breast cell line MCF10A. As can be seen, the EC 50 for the compounds are approximately 6 -10 fold greater in MCF10A cells than for their transformed counterparts MCF7, thus these compounds are more active in transformed cancer cells than normal cells.
  • Table 3 EC 50 values for example compounds when titrated against a selection of cell lines.
  • Figures 9, 10 and 11 show dose-response curves generated for compounds of examples 4 and 5.
  • Figures 19 and 20 show dose-response curves generated for compounds of examples 2 and 3.
  • Example 7 Claimed compounds induce cell death through apoptosis
  • This Example demonstrates that the indicated molecules are able to induce cell death through apoptosis.
  • sub G1 analysis is used, the principal of which is based on the fact that as cells undergo apoptosis, their DNfA is fragmented and lost from the cell. Thus, measurement of the amount of DNA in a cell will identify cells that have less DNA and are undergoing apoptosis.
  • the second way we demonstrate that the mechanism of cell death is via apoptosis is by examining the percentage of cells that have a compromised mitochondrial polarization potential.
  • Propidium Iodide is a fluorescent stain and therefore the intensity of the fluorescence detected by the flow cytometer correlates with the amount of DNA within that cell.
  • the percentage of the cells with a Sub G1 profile and hence the percentage of cells undergoing apoptosis can be ascertained.
  • the indicated cell lines are plated in a 6 well plate such there is 100,000 cells per well in full culture medium. Cells are treated with the indicated small molecule at a final concentration of 25 ⁇ M for 72h. After this time the cells are stained by resuspending the cells in staining buffer (0.1% Sodium Citrate, 0.1% TritonX-100, 200 ⁇ l of Propidium Iodide at 5mg/ml made up to 2OmIs in PBS) for 24 hours at 4 0 C in the dark. The Pl stained cells aFe then acquired by the flow cytoraeter. Analysis of FL2 fluorescence is performed on Cell Quest software to allow quantification of the Sub-G1 phase of the cell.
  • staining buffer 0.1% Sodium Citrate, 0.1% TritonX-100, 200 ⁇ l of Propidium Iodide at 5mg/ml made up to 2OmIs in PBS
  • Mitochondrial Membrane Potential is quantified utilising the JC-1 Dye acquired from Molecular Probes and used according to manufacturers instructions.
  • the indicated cell lines are plated in a 6 well plate such there is 100,000 cells per well in full culture medium. Cells are exposed for the indicated small molecule at a final concentration of 25 ⁇ M for 72h.
  • JC1 Cells are harvested and incubated with the JC1 dye as per manufacturers protocol. When this dye is incubated with viable cells its remains in an aggregate form that fluoresces red on activation with an argon laser. However when apoptosis is occurring the JC-1 dye shifts to a predominantly monomer form that fluoresces green on activation. Therefore a shift from a red to a red/green or green fluorescent population indicates an apoptotic population. Analysis of fluorescence is performed on Cell Quest software to allow quantification of the apoptosis occurring in the population of cells examined. The percentage of cells undergoing apoptosis post treatment with molecules is compared directly to apoptosis induced in control wells.
  • Figures 12, 13 and 14 show apoptosis modulation of Cancer Cells by example compounds of the invention as determined by sub G1 analysis.
  • Figures 15 to 18 show apoptosis modulation of cancer cells by example compounds as determined by JC-1 analysis
  • the Sub G1 Analysis measures DNA Fragmentation, a characteristic of cells undergoing apoptosis. The percentage Sub G1 correlates positively with cells undergoing apoptosis. As can be seen in Fig 12-14, treatment of cells with the compounds increases the percentage of cells undergoing apoptosis as demonstrated by. the increase in cells having a Sub G1 profile. Of note however is the fact that MCF10A cells have reduced number of cells with a sub G1 profile, relevant to its transformed counterpart MCF7 (Fig13 vs.

Abstract

L'invention concerne certains composés de pyrazolo[1,5-a]pyrimidine et leurs procédés préparation, des compositions comprenant lesdits composés et des procédés d'utilisation. Ces composés sont utilisés pour traiter le cancer. L'invention concerne également de nouveaux procédés de criblage.
PCT/GB2006/002968 2005-08-05 2006-08-08 Composes de pyrazolo[1,5-a] pyrimidine et leurs compositions pharmaceutiques WO2007017678A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP06779092A EP1922321A1 (fr) 2005-08-09 2006-08-08 Composes de pyrazoloý1,5-a¨pyrimidine et leurs compositions pharmaceutiques
US12/012,888 US20080300263A1 (en) 2005-08-05 2008-02-06 Pyrazolo [1,5-a] pyrimidine compounds and pharmaceutical compositions containing them

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US70659305P 2005-08-09 2005-08-09
GBGB0516378.7A GB0516378D0 (en) 2005-08-09 2005-08-09 Compound
US60/706,593 2005-08-09
GB0516378.7 2005-08-09

Publications (1)

Publication Number Publication Date
WO2007017678A1 true WO2007017678A1 (fr) 2007-02-15

Family

ID=37036525

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2006/002968 WO2007017678A1 (fr) 2005-08-05 2006-08-08 Composes de pyrazolo[1,5-a] pyrimidine et leurs compositions pharmaceutiques

Country Status (2)

Country Link
EP (1) EP1922321A1 (fr)
WO (1) WO2007017678A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2049539A1 (fr) * 2006-06-21 2009-04-22 Bayer Schering Pharma Aktiengesellschaft Pyrazolopyrimidines et leurs sels, compositions pharmaceutiques les comprenant, procédés de préparation de celles-ci et leurs utilisations.
US8937064B2 (en) 2007-12-19 2015-01-20 Vertex Pharmaceuticals Incorporated Pyrazolo[1,5-a]pyrimidines useful as JAK2 inhibitors
WO2016207217A1 (fr) * 2015-06-23 2016-12-29 Les Laboratoires Servier Nouveaux dérivés bicycliques, leur procédé de préparation, et compositions pharmaceutiques les contenant

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997029109A1 (fr) * 1996-02-07 1997-08-14 Janssen Pharmaceutica N.V. Pyrazolopyrimidines en tant qu'antagonistes du recepteur de crf
WO2001023387A2 (fr) * 1999-09-30 2001-04-05 Neurogen Corporation PYRAZOLO[1,5,-a]-1,5-PYRIMIDINES A SUBSTITUTION ALKYLENE-DIAMINE ET PYRAZOLO[1,5-a]-1,3,5-TRIAZINES
WO2002040485A1 (fr) * 2000-11-17 2002-05-23 Ishihara Sangyo Kaisha, Ltd. Medicaments preventifs ou therapeutiques contre le diabete, contenant des composes a heterocycle fusionne ou leurs sels
WO2004022561A1 (fr) * 2002-09-04 2004-03-18 Schering Corporation Pyrazolopyrimidines tenant lieu d'inhibiteurs de kinases dependantes de la cycline
WO2004022560A1 (fr) * 2002-09-04 2004-03-18 Schering Corporation Pyrazolopyrimidines utilisees en tant qu'inhibiteurs des kinases cycline-dependantes
WO2004076458A1 (fr) * 2003-02-28 2004-09-10 Teijin Pharma Limited Derives de pyrazolo[1,5-a]pyrimidine
US20040209878A1 (en) * 2002-09-04 2004-10-21 Schering Corporation And Pharmacopeia, Inc. Novel pyrazolopyrimidines as cyclin dependent kinase inhibitors
WO2005063756A1 (fr) * 2003-12-22 2005-07-14 Sb Pharmco Puerto Rico Inc Antagonistes du recepteur de crf et procedes correspondants

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997029109A1 (fr) * 1996-02-07 1997-08-14 Janssen Pharmaceutica N.V. Pyrazolopyrimidines en tant qu'antagonistes du recepteur de crf
WO2001023387A2 (fr) * 1999-09-30 2001-04-05 Neurogen Corporation PYRAZOLO[1,5,-a]-1,5-PYRIMIDINES A SUBSTITUTION ALKYLENE-DIAMINE ET PYRAZOLO[1,5-a]-1,3,5-TRIAZINES
WO2002040485A1 (fr) * 2000-11-17 2002-05-23 Ishihara Sangyo Kaisha, Ltd. Medicaments preventifs ou therapeutiques contre le diabete, contenant des composes a heterocycle fusionne ou leurs sels
EP1334973A1 (fr) * 2000-11-17 2003-08-13 Ishihara Sangyo Kaisha, Ltd. Medicaments preventifs ou therapeutiques contre le diabete, contenant des composes a heterocycle fusionne ou leurs sels
WO2004022561A1 (fr) * 2002-09-04 2004-03-18 Schering Corporation Pyrazolopyrimidines tenant lieu d'inhibiteurs de kinases dependantes de la cycline
WO2004022560A1 (fr) * 2002-09-04 2004-03-18 Schering Corporation Pyrazolopyrimidines utilisees en tant qu'inhibiteurs des kinases cycline-dependantes
US20040209878A1 (en) * 2002-09-04 2004-10-21 Schering Corporation And Pharmacopeia, Inc. Novel pyrazolopyrimidines as cyclin dependent kinase inhibitors
WO2004076458A1 (fr) * 2003-02-28 2004-09-10 Teijin Pharma Limited Derives de pyrazolo[1,5-a]pyrimidine
WO2005063756A1 (fr) * 2003-12-22 2005-07-14 Sb Pharmco Puerto Rico Inc Antagonistes du recepteur de crf et procedes correspondants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHIOTA T ET AL: "SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIP OF A NEW SERIES OF POTENT ANGIOTENSIN II RECEPTOR ANTAGONISTS: PYRAZOLO[1,5-A]PYRIMIDINE DERIVATIVES", CHEMICAL AND PHARMACEUTICAL BULLETIN, PHARMACEUTICAL SOCIETY OF JAPAN, TOKYO, JP, vol. 47, no. 7, 1999, pages 928 - 938, XP001156827, ISSN: 0009-2363 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2049539A1 (fr) * 2006-06-21 2009-04-22 Bayer Schering Pharma Aktiengesellschaft Pyrazolopyrimidines et leurs sels, compositions pharmaceutiques les comprenant, procédés de préparation de celles-ci et leurs utilisations.
US8937064B2 (en) 2007-12-19 2015-01-20 Vertex Pharmaceuticals Incorporated Pyrazolo[1,5-a]pyrimidines useful as JAK2 inhibitors
WO2016207217A1 (fr) * 2015-06-23 2016-12-29 Les Laboratoires Servier Nouveaux dérivés bicycliques, leur procédé de préparation, et compositions pharmaceutiques les contenant
FR3037959A1 (fr) * 2015-06-23 2016-12-30 Servier Lab
CN108137497A (zh) * 2015-06-23 2018-06-08 法国施维雅药厂 新的二环衍生物、其制备方法以及含有它们的药物组合物
JP2018527297A (ja) * 2015-06-23 2018-09-20 レ ラボラトワール セルヴィエ 新規な二環式誘導体、それらを調製するためのプロセス及びそれらを含有する医薬組成物
US10323041B2 (en) 2015-06-23 2019-06-18 Les Laboratoires Servier Bicyclic derivatives, a process for their preparation and pharmaceutical compositions containing them
EA033566B1 (ru) * 2015-06-23 2019-11-05 Servier Lab Новые бициклические производные, способ их получения и фармацевтические композиции, содержащие их
US10618909B2 (en) 2015-06-23 2020-04-14 Les Laboratoires Servier Bicyclic derivatives, a process for their preparation and pharmaceutical compositions containing them
CN108137497B (zh) * 2015-06-23 2021-06-15 法国施维雅药厂 新的二环衍生物、其制备方法以及含有它们的药物组合物

Also Published As

Publication number Publication date
EP1922321A1 (fr) 2008-05-21

Similar Documents

Publication Publication Date Title
EP3204377B1 (fr) Composés d'aminopyridyloxypyrazole
US7491829B2 (en) RAF inhibitor compounds and methods
KR101903305B1 (ko) 피라졸로피리미딘 ｊａｋ 억제제 화합물 및 방법
KR20080085154A (ko) 티로신 키나제 모듈레이터로서의 트리아졸로피리다진
KR20180033194A (ko) 키랄 디아릴 매크로사이클 및 이것의 용도
WO2010139966A1 (fr) Dérivés d'azole en tant qu'inhibiteurs de la voie wnt
KR20210065097A (ko) 치환된 인돌 및 이의 사용 방법
Lu et al. An activated Notch1 signaling pathway inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma cell line EC9706
JPWO2006073202A1 (ja) ＧＳＫ３β阻害効果に基づくがんの抑制および抗がん剤の評価方法
KR20210013071A (ko) Erbb 수용체 억제제
AU2016247476B2 (en) Prognostic biomarkers for TTK inhibitor chemotherapy
Bata et al. Inhibitors of the Hippo pathway kinases STK3/MST2 and STK4/MST1 have utility for the treatment of acute myeloid leukemia
Wang et al. Structure-activity relationship investigation for benzonaphthyridinone derivatives as novel potent Bruton's tyrosine kinase (BTK) irreversible inhibitors
Mah et al. Identification of 4-phenoxyquinoline based inhibitors for L1196M mutant of anaplastic lymphoma kinase by structure-based design
Sviripa et al. Par-4 secretion: stoichiometry of 3-arylquinoline binding to vimentin
Zhu et al. Addressing the Enzyme-independent tumor-promoting function of NAMPT via PROTAC-mediated degradation
WO2007017678A1 (fr) Composes de pyrazolo[1,5-a] pyrimidine et leurs compositions pharmaceutiques
Lai et al. Expression of xeroderma pigmentosum complementation group C protein predicts cisplatin resistance in lung adenocarcinoma patients
US20080300263A1 (en) Pyrazolo [1,5-a] pyrimidine compounds and pharmaceutical compositions containing them
El-Wakil et al. Transforming Type II to Type I c-Met kinase inhibitors via combined scaffold hopping and structure-guided synthesis of new series of 1, 3, 4-thiadiazolo [2, 3-c]-1, 2, 4-triazin-4-one derivatives
Wang et al. SOMCL-863, a novel, selective and orally bioavailable small-molecule c-Met inhibitor, exhibits antitumor activity both in vitro and in vivo
Fang et al. Palbociclib and Michael-acceptor hybrid compounds as CDK4/6 covalent inhibitors: Improved potency, broad anticancer spectrum and overcoming drug resistance
Guantay et al. Deoxycytidine kinase (dCK) inhibition is synthetic lethal with BRCA2 deficiency
KR100554221B1 (ko) Ｒｅｔ／ｐｔｃ와 ｓｔａｔ3의 효소―기질 특이성을이용한 항암물질 선별방법
Myers Discovery and optimisation of small-molecule ERK5 inhibitors as cancer therapeutics

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2006779092

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2006779092

Country of ref document: EP