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  • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms

Definitions

• This invention relates to a pyrazole-containing aryl-alkylamine compound that inhibits or modulates the activity of protein kinase B (PKB), protein kinase A (PKA), ROCK kinase or p70S6K kinase, to the use of the compound in the treatment or prophylaxis of disease states or conditions mediated by the said kinases, and to pharmaceutical compositions containing the compound.
• PKT protein kinase B
• PKA protein kinase A
• ROCK kinase or p70S6K kinase protein kinase A
• the invention relates to a single enantiomer of 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol, pharmaceutical compositions containing it and its therapeutic uses, as well as methods for its preparation and novel process intermediates.
• Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a wide variety of signal transduction processes within the cell (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Book. I and II , Academic Press, San Diego, Calif.).
• the kinases may be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine/threonine, lipids, etc.). Sequence motifs have been identified that generally correspond to each of these kinase families (e.g., Hanks, S.
• Protein kinases may be characterized by their regulation mechanisms. These mechanisms include, for example, autophosphorylation, transphosphorylation by other kinases, protein-protein interactions, protein-lipid interactions, and protein-polynucleotide interactions. An individual protein kinase may be regulated by more than one mechanism.
• kinases regulate many different cell processes including, but not limited to, proliferation, differentiation, apoptosis, motility, transcription, translation and other signalling processes, by adding phosphate groups to target proteins. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. Phosphorylation of target proteins occurs in response to a variety of extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.), cell cycle events, environmental or nutritional stresses, etc. Appropriate protein kinases function in signalling pathways to activate or inactivate (either directly or indirectly), for example, a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.
• Uncontrolled signalling due to defective control of protein phosphorylation has been implicated in a number of diseases, including, for example, inflammation, cancer, allergy/asthma, diseases and conditions of the immune system, diseases and conditions of the central nervous system, and angiogenesis.
• Apoptosis or programmed cell death is an important physiological process which removes cells no longer required by an organism. The process is important in early embryonic growth and development allowing the non-necrotic controlled breakdown, removal and recovery of cellular components. The removal of cells by apoptosis is also important in the maintenance of chromosomal and genomic integrity of growing cell populations.
• Cancerous cells consistently contain numerous mutations, errors or rearrangements in their chromosomal DNA. It is widely believed that this occurs in part because the majority of tumours have a defect in one or more of the processes responsible for initiation of the apoptotic process. Normal control mechanisms cannot kill the cancerous cells and the chromosomal or DNA coding errors continue to be propagated. As a consequence restoring these pro-apoptotic signals or suppressing unregulated survival signals is an attractive means of treating cancer.
• the enzymes of the PI3K family are activated by a range of growth and survival factors e.g. EGF, PDGF and through the generation of polyphosphatidylinositols, initiates the activation of the downstream signalling events including the activity of the kinases PDK1 and protein kinase B (PKB) also known as akt.
• PKB is a protein ser/thr kinase consisting of a kinase domain together with an N-terminal PH domain and C-terminal regulatory domain.
• the enzyme PKB alpha (akt1) itself is phosphorylated on Thr 308 by PDK1 and on Ser 473 by ‘PDK2’ now believed to be constituted from the target of rapamycin (TOR) kinase and its associated protein rictor.
• Full activation requires phosphorylation at both sites whilst association between PIP3 and the PH domain is required for anchoring of the enzyme to the cytoplasmic face of the lipid membrane providing optimal access to substrates.
• kinases have been suggested to function as a Ser 473 kinase including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Calpha (PKCalpha), PKCbeta, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product.
• MAP mitogen-activated protein
• MK2 mitogen-activated protein
• ILK integrin-linked kinase
• PKCalpha protein kinase Calpha
• mTOR mammalian target of rapamycin
• DNK-PK double-stranded DNA-dependent protein kinase
• PIK3CA somatic mutations within the PI3K catalytic subunit, PIK3CA, are common (25-40%) among colorectal, gastric, breast, ovarian cancers, and high-grade brain tumors.
• PIK3CA mutations are a common event that can occur early in bladder carcinogenesis.
• PIK3CA alterations are mainly present in lobular and ductal tumours.
• the PI3K pathway is extensively activated in endometrial carcinomas, and that combination of PIK3CA/PTEN alterations might play an important role in development of these tumors.
• Tumours activated by mutations of PI3 kinase and loss of PTEN will have sustained activation of PKB and will be as a result disproportionately sensitive to inhibition by PKA/PKB inhibitors.
• Activated PKB in turns phosphorylates a range of substrates contributing to the overall survival response. Whilst we cannot be certain that we understand all of the factors responsible for mediating the PKB dependent survival response, some important actions are believed to be phosphorylation and inactivation of the pro-apoptotic factor BAD and caspase 9, phosphorylation of Forkhead transcription factors e.g. FKHR leading to their exclusion from the nucleus, and activation of the NfkappaB pathway by phosphorylation of upstream kinases in the cascade.
• Forkhead transcription factors e.g. FKHR leading to their exclusion from the nucleus
• NfkappaB pathway by phosphorylation of upstream kinases in the cascade.
• the enzyme In addition to the anti-apoptotic and pro-survival actions of the PKB pathway, the enzyme also plays an important role in promoting cell proliferation. This action is again likely to be mediated via several actions, some of which are thought to be phosphorylation and inactivation of the cyclin dependent kinase inhibitor of p21 Cip1/WAF1 , and phosphorylation and activation of mTOR, a kinase controlling several aspects of cell size, growth and protein translation.
• the phosphatase PTEN which dephosphorylates and inactivates polyphosphatidyl-inositols is a key tumour suppressor protein which normally acts to regulate the PI3K/PKB survival pathway.
• the significance of the PI3K/PKB pathway in tumourigenesis can be judged from the observation that PTEN is one of the most common targets of mutation in human tumours, with mutations in this phosphatase having been found in ⁇ 50% or more of melanomas (Guldberg et al 1997, Cancer Research 57, 3660-3663) and advanced prostate cancers (Cairns et al 1997 Cancer Research 57, 4997).
• alpha, beta and gamma There are 3 closely related isoforms of PKB called alpha, beta and gamma (AKT1, 2 and 3), which genetic studies suggest have distinct but overlapping functions. Evidence suggests that they can all independently play a role in cancer.
• PKB beta has been found to be over-expressed or activated in 10-40% of ovarian and pancreatic cancers (Bellacosa et al 1995, Int. J. Cancer 64, 280-285; Cheng et al 1996, PNAS 93, 3636-3641; Yuan et al 2000, Oncogene 19, 2324-2330)
• PKB alpha is amplified in human gastric, prostate and breast cancer (Staal 1987, PNAS 84, 5034 ⁇ 5037; Sun et al 2001, Am. J. Pathol. 159, 431-437) and increased PKB gamma activity has been observed in steroid independent breast and prostate cell lines (Nakatani et al 1999, J. Biol. Che
• the PKB pathway also functions in the growth and survival of normal tissues and may be regulated during normal physiology to control cell and tissue function.
• disorders associated with undesirable proliferation and survival of normal cells and tissues may also benefit therapeutically from treatment with a PKB inhibitor.
• disorders of immune cells associated with prolonged expansion and survival of cell population leading to a prolonged or up regulated immune response.
• T and B lymphocyte response to cognate antigens or growth factors such as interferon gamma activates the PI3K/PKB pathway and is responsible for maintaining the survival of the antigen specific lymphocyte clones during the immune response.
• the PKB pathway contributes an important survival signal preventing the normal mechanisms by which the immune response is terminated via apoptosis of the activated cell population.
• autoimmune conditions such as multiple sclerosis and arthritis.
• Expansion of lymphocyte populations responding inappropriately to foreign antigens is a feature of another set of conditions such as allergic responses and asthma.
• inhibition of PKB could provide a beneficial treatment for immune disorders.
• PKB may play a role
• Other examples of inappropriate expansion, growth, proliferation, hyperplasia and survival of normal cells in which PKB may play a role include but are not limited to atherosclerosis, cardiac myopathy and glomerulonephritis.
• PKB pathway functions in the control of glucose metabolism by insulin.
• Available evidence from mice deficient in the alpha and beta iso forms of PKB suggests that this action is mediated by the beta iso form primarily.
• modulators of PKB activity may also find utility in diseases in which there is a dysfunction of glucose metabolism and energy storage such as diabetes, metabolic disease and obesity.
• Cyclic AMP-dependent protein kinase is a serine/threonine protein kinase that phosphorylates a wide range of substrates and is involved in the regulation of many cellular processes including cell growth, cell differentiation, ion-channel conductivity, gene transcription and synaptic release of neurotransmitters.
• the PKA holoenzyme is a tetramer comprising two regulatory subunits and two catalytic subunits.
• PKA acts as a link between G-protein mediated signal transduction events and the cellular processes that they regulate. Binding of a hormone ligand such as glucagon to a transmembrane receptor activates a receptor-coupled G-protein (GTP-binding and hydrolyzing protein). Upon activation, the alpha subunit of the G protein dissociates and binds to and activates adenylate cyclase, which in turn converts ATP to cyclic-AMP (cAMP). The cAMP thus produced then binds to the regulatory subunits of PKA leading to dissociation of the associated catalytic subunits. The catalytic subunits of PKA, which are inactive when associated with the regulatory sub-units, become active upon dissociation and take part in the phosphorylation of other regulatory proteins.
• the catalytic sub-unit of PKA phosphorylates the kinase Phosphorylase Kinase which is involved in the phosphorylation of Phosphorylase, the enzyme responsible for breaking down glycogen to release glucose.
• PKA is also involved in the regulation of glucose levels by phosphorylating and deactivating glycogen synthase.
• modulators of PKA activity may be useful in the treatment or management of diseases in which there is a dysfunction of glucose metabolism and energy storage such as diabetes, metabolic disease and obesity.
• PKA has also been established as an acute inhibitor of T cell activation
• Anndahl et al have investigated the possible role of PKA type I in HIV-induced T cell dysfunction on the basis that T cells from HIV-infected patients have increased levels of cAMP and are more sensitive to inhibition by cAMP analogues than are normal T cells. From their studies, they concluded that increased activation of PKA type I may contribute to progressive T cell dysfunction in HIV infection and that PKA type I may therefore be a potential target for immunomodulating therapy.
• -Aandahl E. M., Aukrust, P., Sk ⁇ lhegg, B. S., Müller, F., Fr ⁇ land, S. S., Hansson, V., Taskén, K. Protein kinase A type I antagonist restores immune responses of T cells from HIV - infected patients . FASEB J. 12, 855-862 (1998).
• ROCK1 and ROCK2 The ROCK kinase family comprises two known members: ROCK1 and ROCK2:
• metastasis involves a restructuring of the cytoskeleton as well as cell-cell and cell-matrix adhesions allowing cells to break away from the tumor mass, invade local tissue, and ultimately spread throughout the body. These effects on cell morphology and adhesion are regulated by members of the Rho GTPase family.
• Activated RhoA is capable of interacting with several effecter proteins including the ROCK kinases ROCK1 and ROCK2.
• ROCK1 and ROCK2 can be activated by the RhoA-GTP complex via physical association.
• Activated ROCKs phosphorylate a number of substrates and play important roles in pivotal cellular functions.
• the substrates for ROCKS include myosin binding subunit of myosin light chain phosphatase (MBS, also named MYPT1), adducin, moesin, myosin light chain (MLC), LIM kinase, and the transcription factor FHL.
• MCS myosin binding subunit of myosin light chain phosphatase
• MLC myosin light chain
• LIM kinase myosin light chain
• the phosphorylation of theses substrates modulate the biological activity of the proteins and provide a means to alter a cell's response to external stimuli.
• RhoA and RhoC are commonly observed in human cancers, including in the progression of testicular germ cell tumours, small breast carcinomas with metastatic ability, invasion and metastasis of bladder cancer, tumor progression in ovarian carcinoma.
• Rho GTPases Progression of tumors to invasive and metastatic forms requires that tumor cells undergo dramatic morphologic changes, a process regulated by Rho GTPases. Actomyosin contractility is a mechanism by which cells exert locomotory force against their environment. Signalling downstream of the small GTPase Rho increases contractility through ROCK-mediated regulation of myosin-II light chain (MLC2) phosphorylation.
• MLC2 myosin-II light chain
• ROCK kinases are thought to participate in the induction of focal adhesions and stress fibers and to mediate calcium sensitization of smooth muscle contraction by enhancing phosphorylation of the regulatory light chain of myosin.
• ROCK inhibitors such as Y-27632 or WF-536, have been used in some studies to demonstrate these properties.
• Inhibitors of ROCKs have been suggested for use in the treatments of a variety of diseases. These include cardiovascular diseases such as hypertension, chronic and congestive heart failure, cardiac hypertrophy, restenosis, chronic renal failure and atherosclerosis. Also, because of its muscle relaxing properties, inhibitors may also be suitable for asthma, male erectile dysfunction, female sexual dysfunction and over-active bladder I syndrome.
• ROCK inhibitors have been shown to possess anti-inflammatory properties. Thus they can be used as treatment for neuroinflammatory diseases such as stroke, multiple sclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and inflammatory pain, as well as other inflammatory diseases such as rheumatoid arthritis, irritable bowel syndrome, and inflammatory bowel disease. Based on their neurite outgrowth inducing effects, ROCK inhibitors could be useful drugs for neuronal regeneration, inducing new axonal growth and axonal rewiring across lesions within the CNS.
• ROCK inhibitors are therefore likely to be useful for regenerative treatment of CNS disorders such as spinal cord injury, acute neuronal injury (stroke, traumatic brain injury), Parkinson's disease, Alzheimers disease and other neurodegenerative disorders. Since ROCK inhibitors reduce cell proliferation and cell migration, they could be useful in treating cancer and tumor metastasis. Finally, there is evidence to suggest that ROCK inhibitors suppress cytoskeletal rearrangement upon virus invasion, thus they also have potential therapeutic value in anti-viral and anti-bacterial applications. ROCK inhibitors are also useful for the treatment of insulin resistance and diabetes.
• Rho controls cell adhesion and motility through reorganization of the actin cytoskeleton and regulation of actomyosin contractility.
• Cultured rat MM1 hepatoma cells migrate in a serum-dependent, Rho-mediated manner, through a mesothelial cell monolayer in vitro.
• the ROCK kinases are thought to participate in the induction of focal adhesions and stress fibres in cultured cells, and to mediate calcium sensitization of smooth muscle contraction by enhancing phosphorylation of the regulatory light chain of myosin.
• ROCK plays an essential part in tumor cell invasion, and demonstrate its potential as a therapeutic target for the prevention of cancer invasion and metastasis.
• Rhokinase mediated this effect of RhoA, as was demonstrated by the use of Y-27632, a specific inhibitor of Rho kinase Inhibition of Rho kinase prevented the VEGF-enhanced EC migration in response to mechanical wounding but had no effect on basal EC migration.
• VEGF-induced cytoskeletal changes in ECs require RhoA and Rho kinase, and activation of RhoA/Rho kinase signalling is involved in the VEGF-induced in vitro EC migration and angiogenesis.
• Y-27632 can relax smooth muscle and increase vascular blood flow.
• Y-27632 is a small molecule that can enter cells and is not toxic in rats after oral administration of 30 mg/kg for 10 days. Effective doses for the use of this compound are approximately 30 ⁇ M. It reduces blood pressure in hypertensive rats, but does not affect blood pressure in normal rats. This has led to the identification of Rho signalling antagonists in treatment of hypertension (Somlyo, 1997 Nature 389:908; Uehata et al., 1997 Nature 389:990).
• Rho kinase inhibitor Y-27632 has been tested for the following disease applications:
• ROCKs the immediate downstream targets of the small guanosine triphosphate-binding protein Rho
• ROCKs play a central role in diverse cellular functions such as smooth muscle contraction, stress fiber formation and cell migration and proliferation.
• Overactivity of ROCKS is observed in cerebral ischemia, coronary vasospasm, hypertension, vascular inflammation, arteriosclerosis and atherosclerosis.
• ROCKs therefore, may be an important and still relatively unexplored therapeutic target in cardiovascular disease.
• Recent experimental and clinical studies using ROCK inhibitors such as Y-27632 and fasudil have revealed a critical role of ROCKs in embryonic development, inflammation and oncogenesis. This review will focus on the potential role of ROCKs in cellular functions and discuss the prospects of ROCK inhibitors as emerging therapy for cardiovascular diseases.
• Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically.
• Smooth-muscle contraction is regulated by the cytosolic Ca 2+ concentration and by the Ca 2+ sensitivity of myo filaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase.
• Rho signaling pathways in vascular smooth muscle cells are highly activated in hypertension, a condition associated with a variety of vascular diseases, including restenosis injury and atherosclerosis.
• Hypertension is a cardiovascular disorder characterized by increased peripheral vascular resistance and/or vascular structural remodeling. Recently, rapidly growing evidence from hypertensive animal models suggests that small GTPase Rho and its downstream effector, Rho-kinase, play an important role in the pathogenesis of hypertension. Activation of the Rho/Rho-kinase pathway is essential for smooth muscle contractility in hypertension. A greater RhoA expression and an enhanced RhoA activity have been observed in aortas of hypertensive rats, such as genetic spontaneously hypertensive rats and N(omega)-nitro-L-arginine methyl ester-induced hypertension.
• ROCK inhibitors therefore have potential for preventing neurodegeneration and stimulating neuroregeneration in various neurological disorders.
• Rho family of GTPases and related molecules play an important role in various aspects of neuronal development, including neurite outgrowth and differentiation, axon pathfinding, and dendritic spine formation and maintenance.
• Rho family of small GTPases Central to the regulation of the actin cytoskeleton in both neuronal and non-neuronal cells is the Rho family of small GTPases. Rho family members cycle between an inactive GDP-bound form and an active GTP-bound form. Several lines of evidence suggest that manipulating the activity state of Rho GTPases may modulate growth cone collapse and neurite outgrowth inhibition.
• Rho signaling pathway is a potential target for therapeutic interventions after spinal cord injury.
• the 70 kDa ribosomal protein kinase p70S6K (also known as SK6, p70/p85 S6 kinase, p70/p85 ribosomal S6 kinase and pp70s6k) is a member of the AGC subfamily of protein kinases.
• p70S6K is a serine-threonine kinase that is a component of the phosphatidylinositol 3 kinase (PI3K)/AKT pathway.
• PI3K phosphatidylinositol 3 kinase
• p70S6K is downstream of PI3K, and activation occurs through phosphorylation at a number of sites in response to numerous mitogens, hormones and growth factors.
• rapamycin acts to inhibit p70S6K activity and blocks protein synthesis, specifically as a result of a down-regulation of translation of these mRNA's encoding ribosomal proteins.
• p70S6K is also regulated by PI3K and its downstream target AKT. Wortmannin and rapamycin cause a decrease in p70S6K phosphorylation at sites dependent of the PI3K pathway. Mutant p70S6K is inhibited by wortmannin but not by rapamycin suggesting that the PI3K pathway can exhibit effects on p70S6K independent of the regulation of mTOR activity.
• the enzyme p70S6K modulates protein synthesis by phosphorylation of the S6 ribosomal protein.
• S6 phosphorylation correlates with increased translation of mRNAs encoding components of the translational apparatus, including ribosomal proteins and translational elongation factors whose increased expression is essential for cell growth and proliferation.
• mRNAs contain an oligopyrimidine tract at their 5′ transcriptional start (termed 5′TOP), which has been shown to be essential for their regulation at the translational level.
• p70S6K activation has also been implicated in cell cycle control, neuronal cell differentiation, regulation of cell motility and a cellular response that is important in tumor metastases, the immune response and tissue repair.
• Antibodies to p70S6K abolish the mitogenic response driven entry of rat fibroblasts into S phase, indication that p70S6K function is essential for the progression from G1 to S phase in the cell cycle.
• inhibition of cell cycle proliferation at the G1 to S phase of the cell cycle by rapamycin has been identified as a consequence of inhibition of the production of the hyperphosphorylated, activated form of p70S6K.
• the tumor suppressor LKB1 activates AMPK which phosphorylates the TSC1/2 complex in the mTOR/p70S6K pathway, therefore feeds into p70S6K through a PKB independent pathway. Mutations in LKB1 cause Peutz-Jeghers syndrome (PJS), where patients with PJS are 15 times more likely to develop cancer than the general population. In addition, 1 ⁇ 3 of lung adenocarcinomas harbor inactivating LKB1 mutations.
• PJS Peutz-Jeghers syndrome
• a role for p70S6K in tumor cell proliferation and protection of cells from apoptosis is supported based on its participation in growth factor receptor signal transduction, overexpression and activation in tumor tissues.
• Northern and Western analyses revealed that amplification of the PS6K gene was accompanied by corresponding increases in mRNA and protein expression, respectively (Cancer Res. (1999) 59: 1408-11-Localization of PS6K to Chromosomal Region 17q23 and Determination of Its Amplification in Breast Cancer).
• Chromosome 17q23 is amplified in up to 20% of primary breast tumors, in 87% of breast tumors containing BRCA2 mutations and in 50% of tumors containing BRCA1 mutations, as well as other cancer types such as pancreatic, bladder and neuroblastoma (see M Barlund, O Monni, J Kononen, R Cornelison, J Torhorst, G Sauter, O-P Kallioniemi and Kallioniemi A, Cancer Res., 2000, 60:5340-5346). It has been shown that 17q23 amplifications in breast cancer involve the PAT1, RAD51C, PS6K, and SIGMA1B genes (Cancer Res. (2000): 60, pp. 5371-5375).
• the p70S6K gene has been identified as a target of amplification and overexpression in this region, and statistically significant association between amplification and poor prognosis has been observed.
• p70S6K has been implicated in metabolic diseases and disorders. It was reported that the absence of p70S6 protects against age- and diet-induced obesity while enhancing insulin sensitivity. A role for p70S6K in metabolic diseases and disorders such as obesity, diabetes, metabolic syndrome, insulin resistance, hyperglycemia, hyperaminoacidemia, and hyperlipidmia is supported based upon the findings.
• WO 2005/061463 discloses pyrazole compounds having PKB and PKA inhibiting activity and one particular compound exemplified is 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol.
• This compound the structure of which is shown below, has a chiral centre at the carbon atom marked with an asterisk.
• Example 84 of WO 2005/061463 is a racemic mixture of the two possible enantiomers. According to Examples 106 and 107, the compound of Example 84 has IC 50 values in the in vitro PKA and PKB assays respectively of less than 1 micromolar in each case.
• WO 2005/061463 also discloses and exemplifies a number of individual enantiomers, as follows:
• Isomers A and B constitute one pair of enantiomers and Isomers C and D constitute another pair of enantiomers.
• Isomer A is 10 fold more active against PKB than its antipode Isomer B in a binding assay.
• Isomer C is about 10 fold more active than its antipode Isomer D in a binding assay.
• isomers C and D are essentially equipotent.
• the S-enantiomer of 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol is 100 fold more active (as determined by a radiometric binding assay) against PKB than the corresponding R-enantiomer.
• the isomers C and D above are essentially equipotent in the mechanistic cellular assay, and the S-enantiomer of 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol has good activity in this assay, the R-enantiomer has no measurable activity.
• the invention provides a composition comprising (S) 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol, wherein the composition is either substantially free of (R) 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol or the composition contains a mixture of the (S) and (R) enantiomers in which the (S) enantiomer predominates.
• the invention also provides a composition
• a composition comprising 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol or a salt, solvate, tautomer or N-oxide thereof, at least 75% of which is in the S-enantiomeric form.
• composition refers to a composition of matter and includes compositions which consist solely of 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol as well as compositions which contain additional components. According to the invention, at least 75% of all of the 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol present in the composition must be in the S-enantiomeric form.
• the compositions may be referred to herein for convenience as “the compositions of the invention” or “the compositions as defined herein” or “the compositions”.
• the amount of (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol present in a given composition relative to the total amount of 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol of both enantiomeric forms present in the composition may be expressed as the “enantiomeric purity”.
• the (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol has an enantiomeric purity of at least 80%, more preferably at least 85%, or at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%.
• At least 99.9% of the 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol is in the S-enantiomeric form.
• substantially no (R)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol is present in the composition.
• the term “substantially no (R)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol is present in the composition” as used in this application means that no R-enantiomer can be detected using the analytical methods described herein
• the composition is a pharmaceutical composition containing the (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol or a salt, solvate, tautomer or N-oxide thereof, and a pharmaceutically acceptable carrier.
• the composition consists of (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol or a salt, solvate, tautomer or N-oxide thereof, in substantially pure form, i.e. containing less than 0.5%, more preferably less than 0.1% and most preferably less than 0.01% impurities.
• no single impurity is present in the composition in an amount corresponding to more than 0.2% by weight, and preferably no more than 0.1% by weight.
• the impurity is not present in the composition in an amount greater than 0.5%, or greater than 0.4%, or greater than 0.3%, or greater than 0.2%, or greater than 0.1%, or greater than 0.05%, or greater than 0.01%.
• the (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol is represented by formula (I) below, and may be referred to herein as the “compound of formula (I)” or “the S-enantiomer”.
• the (R)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol may be referred to for convenience herein as “the R-enantiomer”.
• R and S refer to the “R and S” nomenclature developed by Cahn, Ingold and Prelog, see Advanced Organic Chemistry by Jerry March, 4 th Edition, John Wiley & Sons, New York, 1992, pages 109-114, and see also Cahn, Ingold & Prelog, Angew. Chem. Int. Ed. Engl., 1966, 5, 385-415.
• compositions of the invention can be prepared by partially or fully resolving a mixture of (S) and (R) enantiomers of 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol, for example using chiral chromatography, as described below.
• the (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol i.e. the compound of formula (I)
• PKB protein kinase B
• PKA protein kinase A
• the invention provides a compound of the formula (I), i.e. (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol, or a salt, solvate, tautomer or N-oxide thereof, in substantially pure form, i.e. containing less than 0.5%, more preferably less than 0.1% and most preferably less than 0.01% impurities.
• a compound of the formula (I) i.e. (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol, or a salt, solvate, tautomer or N-oxide thereof, in substantially pure form, i.e. containing less than 0.5%, more preferably less than 0.1% and most preferably less than 0.01% impurities.
• the compound is other than an N-oxide and is selected from the free base or a salt, solvate or tautomer thereof.
• the compound of formula (I) or a tautomer thereof is in the form of the free base.
• the compound of formula (I) or a tautomer thereof is in the form of a salt.
• One particular salt prepared in accordance with the invention is the salt formed with hydrochloric acid.
• the invention provides:
• the invention also provides the further combinations, uses, methods, compounds and processes as set out in the claims below.
• modulation As used herein, the term “modulation”, as applied to the activity of a kinase, is intended to define a change in the level of biological activity of the protein kinase. Thus, modulation encompasses physiological changes which effect an increase or decrease in the relevant protein kinase activity. In the latter case, the modulation may be described as “inhibition”.
• the modulation may arise directly or indirectly, and may be mediated by any mechanism and at any physiological level, including for example at the level of gene expression (including for example transcription, translation and/or post-translational modification), at the level of expression of genes encoding regulatory elements which act directly or indirectly on the levels of kinase activity.
• modulation may imply elevated/suppressed expression or over- or under-expression of a kinase, including gene amplification (i.e. multiple gene copies) and/or increased or decreased expression by a transcriptional effect, as well as hyper- (or hypo-)activity and (de)activation of the protein kinase(s) (including (de)activation) by mutation(s).
• gene amplification i.e. multiple gene copies
• hyper- (or hypo-)activity i.e. multiple gene copies
• de deactivation of the protein kinase(s) (including (de)activation) by mutation(s).
• modulated modulating
• modulate are to be interpreted accordingly.
• the term “mediated”, as used e.g. in conjunction with a kinase as described herein (and applied for example to various physiological processes, diseases, states, conditions, therapies, treatments or interventions) is intended to operate limitatively so that the various processes, diseases, states, conditions, treatments and interventions to which the term is applied are those in which the kinase plays a biological role.
• the biological role played by a kinase may be direct or indirect and may be necessary and/or sufficient for the manifestation of the symptoms of the disease, state or condition (or its aetiology or progression).
• kinase activity and in particular aberrant levels of kinase activity, e.g.
• kinase over-expression need not necessarily be the proximal cause of the disease, state or condition: rather, it is contemplated that the kinase mediated diseases, states or conditions include those having multifactorial aetiologies and complex progressions in which the kinase in question is only partially involved.
• the role played by the kinase may be direct or indirect and may be necessary and/or sufficient for the operation of the treatment, prophylaxis or outcome of the intervention.
• a disease state or condition mediated by a kinase includes the development of resistance to any particular cancer drug or treatment.
• ROCK kinase(s) and “ROCK(s)” are synonomous generic terms embracing all members of the ROCK kinase family, so including both ROCK1 and ROCK2 as species within the genus. References inter alia to ROCK kinase inhibitors, ROCK kinase modulation and ROCK kinase activity are to be interpreted accordingly.
• Rho protein is a term of art used to define a large family of GTP-binding proteins that are involved in regulation of actin organization, including RhoA and RhoC.
• Rho signalling pathway defines any cellular signaling pathway in which one or more members of the Rho proteins are involved. Particularly relevant to the invention are Rho signaling pathways in which a ROCK kinase (e.g. ROCK1 and/or ROCK2) is a proximate effector (e.g. a binding partner) for one or more Rho protein(s), and such Rho signaling pathways are preferred in embodiments of the invention defined inter alia by reference to a Rho signaling pathway.
• a ROCK kinase e.g. ROCK1 and/or ROCK2
• proximate effector e.g. a binding partner
• modulation As used herein, the term “modulation”, as applied to the ROCKS as described herein, is intended to define a change in the level of biological activity of the ROCKs. Thus, modulation encompasses physiological changes which effect an increase or decrease in ROCK activity. In the latter case, the modulation may be described as “inhibition”.
• the modulation may arise directly or indirectly, and may be mediated by any mechanism and at any physiological level, including for example at the level of gene expression (including for example transcription, translation and/or post-translational modification), at the level of expression of genes encoding regulatory elements which act directly or indirectly on the levels of ROCK activity, or at the level of enzyme (e.g.
• ROCK activity for example by allosteric mechanisms, competitive inhibition, active-site inactivation, perturbation of feedback inhibitory pathways etc.
• modulation may imply elevated/suppressed expression or over- or under-expression of the ROCK, including gene amplification (i.e. multiple gene copies) and/or increased or decreased expression by a transcriptional effect, as well as hyper- (or hypo-)activity and (de)activation of the ROCK (including (de)activation) by mutation(s).
• modulated and modulate are to be interpreted accordingly.
• ROCK activity (and in particular aberrant levels of ROCK activity, e.g.
• ROCK over-expression need not necessarily be the proximal cause of the disease, state or condition: rather, it is contemplated that ROCK-mediated diseases, states or conditions include those having multifactorial aetiologies and complex progressions in which ROCK is only partially involved.
• ROCK-mediated diseases, states or conditions include those having multifactorial aetiologies and complex progressions in which ROCK is only partially involved.
• the role played by ROCK may be direct or indirect and may be necessary and/or sufficient for the operation of the treatment, prophylaxis or outcome of the intervention.
• ROCK-mediated physiological processes, diseases, states, conditions, therapies, treatments or interventions of the invention involve the Rho signaling pathway (as herein defined) and may therefore, by extension, be dubbed “Rho-mediated” physiological processes, diseases, states, conditions, therapies, treatments or interventions.
• references herein to a disease, condition, subject or patient population “in which the modulation (e.g. inhibition) of ROCK kinase is indicated” is intended to define those diseases etc. in which modulation of ROCK kinase is either clinically desirable or necessary. This might be the case, for example, where modulation of ROCK kinase would be palliative, preventative or (at least partially) curative.
• modulation As used herein, the term “modulation”, as applied to the protein kinase P70S6K described herein, is intended to define a change in the level of biological activity of P70S6K. Thus, modulation encompasses physiological changes which effect an increase or decrease in P70S6K activity. In the latter case, the modulation may be described as “inhibition”.
• the modulation may arise directly or indirectly, and may be mediated by any mechanism and at any physiological level, including for example at the level of gene expression (including for example transcription, translation and/or post-translational modification), at the level of expression of genes encoding regulatory elements which act directly or indirectly on the levels of P70S6K activity, or at the level of enzyme (e.g.
• P70S6K activity for example by allosteric mechanisms, competitive inhibition, active-site inactivation, perturbation of feedback inhibitory pathways etc.
• modulation may imply elevated/suppressed expression or over- or under-expression of P70S6K, including gene amplification (i.e. multiple gene copies) and/or increased or decreased expression by a transcriptional effect, as well as hyper- (or hypo-)activity and (de)activation of P70S6K (including (de)activation) by mutation(s).
• modulated and modulate are to be interpreted accordingly.
• the term “mediated”, as used in conjunction with P70S6K as described herein (and applied for example to various physiological processes, diseases, states, conditions, therapies, treatments or interventions) is intended to operate limitatively so that the various processes, diseases, states, conditions, treatments and interventions to which the term is applied are those in which P70S6K plays a biological role.
• the role played by P70S6K may be direct or indirect and may be necessary and/or sufficient for the manifestation of the symptoms of the disease, state or condition (or its aetiology or progression).
• P70S6K activity and in particular aberrant levels of P70S6K activity, e.g.
• P70S6K over-expression need not necessarily be the proximal cause of the disease, state or condition: rather, it is contemplated that P70S6K-mediated diseases, states or conditions include those having multifactorial aetiologies and complex progressions in which P70S6K is only partially involved.
• the role played by P70S6K may be direct or indirect and may be necessary and/or sufficient for the operation of the treatment, prophylaxis or outcome of the intervention.
• intervention is a term of art used herein to define any agency which effects a physiological change at any level.
• the intervention may comprise the induction or repression of any physiological process, event, biochemical pathway or cellular/biochemical event.
• the interventions of the invention typically effect (or contribute to) the therapy, treatment or prophylaxis of a disease or condition.
• references herein to (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol, or the compound of formula (I) or the S-enantiomer include the free base as well as ionic, salt, solvate, N-oxide, tautomeric and protected forms thereof, for example, as discussed below.
• the compound may be other than an N-oxide.
• the compound of formula (I) is other than an N-oxide and is in the form of a free base.
• the compound of formula (I) is other than an N-oxide and is in the form of a salt.
• Salt forms may be selected and prepared according to methods described in Pharmaceutical Salts: Properties, Selection, and Use , P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.
• acid addition salts may be prepared by dissolving the free base in an organic solvent in which a given salt form is insoluble or poorly soluble and then adding the required acid in an appropriate solvent so that the salt precipitates out of solution.
• Acid addition salts may be formed with a wide variety of acids, both inorganic and organic.
• acid addition salts include salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g.
• L-glutamic L-glutamic
• ⁇ -oxoglutaric glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic
• lactic e.g. (+)-L-lactic and ( ⁇ )-DL-lactic
• lactobionic maleic, malic, ( ⁇ )-L-malic, malonic, ( ⁇ )-DL-mandelic, methanesulphonic, naphthalenesulphonic (e.g.
• naphthalene-2-sulphonic naphthalene-1,5-disulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic, toluenesulphonic (e.g. p-toluenesulphonic), undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.
• toluenesulphonic e.g. p-toluenesulphonic
• undecylenic and valeric acids as well as acylated amino acids and cation exchange resins.
• One particular group of acid addition salts includes salts formed with hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulphonic, toluenesulphonic, methanesulphonic, ethanesulphonic, naphthalenesulphonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids.
• a sub-set of salts consists of salts formed with hydrochloric acid or acetic acid.
• Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-Lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulphonic, sebacic, stearic, succinic and tartaric acids.
• the compound of formula (I) may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.
• the basic pyrazole nitrogen as well as the nitrogen atom in the amino group, may take part in salt formation.
• the acid has a pKa of less than about 3 (e.g. an acid such as hydrochloric acid, sulphuric acid or trifluoroacetic acid)
• the compound of formula (I) will typically form salts with 2 molar equivalents of the acid.
• the salt forms of the compound of formula (I) are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci ., Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compound of formula (I), also form part of the invention.
• the compound of formula (I) may also form N-oxides and such N-oxides are within the scope of the definition of the compound of formula (I).
• the compound of formula (I) is not an N-oxide.
• N-Oxides can be formed by treatment of the parent amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry , by Jerry March, 4 th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady ( Syn. Comm. 1977, 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.
• MCPBA m-chloroperoxybenzoic acid
• the compound of formula (I) can be prepared from a racemic mixture of the S-enantiomer and the R-enantiomer by using a suitable separation technique such as chiral chromatography (chromatography on a chiral support) and such techniques are well known to the person skilled in the art.
• the enantiomers can be separated by forming diastereoisomeric salts with chiral acids such as (+)-tartaric acid, ( ⁇ )-pyroglutamic acid, ( ⁇ )-di-toluloyl-L-tartaric acid, (+)-mandelic acid, ( ⁇ )-malic acid, and ( ⁇ )-camphorsulphonic, separating the diastereoisomers by preferential crystallisation, and then dissociating the salts to give the individual enantiomer of the free base.
• chiral acids such as (+)-tartaric acid, ( ⁇ )-pyroglutamic acid, ( ⁇ )-di-toluloyl-L-tartaric acid, (+)-mandelic acid, ( ⁇ )-malic acid, and ( ⁇ )-camphorsulphonic
• the compound of formula (I) includes variants with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element.
• a reference to hydrogen includes within its scope 1 H, 2 H (D), and 3 H (T).
• references to carbon and oxygen include within their scope respectively 12 C, 13 C and 14 C and 16 O and 18 O.
• the isotopes may be radioactive or non-radioactive.
• the compounds contain no radioactive isotopes. Such compounds are preferred for therapeutic use.
• the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.
• any polymorphic forms of the compounds solvates (e.g. hydrates), complexes (e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals) of the compounds, and pro-drugs of the compounds.
• solvates e.g. hydrates
• complexes e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals
• pro-drugs is meant for example any compound that is converted in vivo into a biologically active composition as defined herein.
• some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (—C( ⁇ O)OR) is cleaved to yield the active drug.
• esters may be formed by esterification, for example, of any of the hydroxyl groups (—C( ⁇ O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required.
• metabolically labile esters include those of the formula —C( ⁇ O)OR wherein
• prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in Antibody-directed Enzyme Prodrug Therapy (ADEPT), Gene-directed Enzyme Prodrug Therapy (GDEPT), Polymer-directed Enzyme Prodrug Therapy (PDEPT), Ligand-directed Enzyme Prodrug Therapy (LIDEPT), etc.).
• the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
• the substituted benzophenone (10) is converted to the epoxide (11) by reaction with trimethylsulphonium iodide in dimethylsulphoxide in the presence of a base (e.g. a hydride base such as sodium hydride).
• a base e.g. a hydride base such as sodium hydride.
• the epoxide (11) is then reacted with ammonia in an alcoholic solvent such as methanol, typically with heating, to give the amine (12) as a racemic mixture of R- and S-enantiomers.
• the amine (12) can be reacted directly with a pyrazole boronate (such as 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyrazole) in the presence of a palladium catalyst (such as tetrakistriphenylphosphine palladium (0)) under Suzuki coupling conditions to give the racemic compound (15).
• a pyrazole boronate such as 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyrazole
• a palladium catalyst such as tetrakistriphenylphosphine palladium (0)
• racemic mixture can be resolved by methods well known to those skilled in the art, for example using the chiral chromatography methods and other methods described herein.
• the invention provides a process for the preparation of a compound of the formula (15), which process comprises the removal of a protecting group PG from a compound of the compound (14) and thereafter optionally separating the optical isomers of compound (15) and isolating the S-enantiomer thereof.
• the invention also provides a compound preparable by the foregoing process, as well as a compound of the formula (15) whenever prepared by the said process.
• the invention provides a process for the preparation of a compound of the formula (15), which process comprises (i) reacting a compound of the formula (13) with a pyrazole derivative of the formula (16):
• X is a group B(OH) 2 or a boronate ester group (such as a 4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl group) in the presence of a palladium catalyst (such as tetrakistriphenylphosphine palladium (0)) under Suzuki coupling conditions to give a compound of the formula (14); (ii) removing the protecting group PG from the compound of the compound (14) and thereafter (iii) optionally separating the optical isomers of compound (15) and isolating the S-enantiomer thereof.
• a palladium catalyst such as tetrakistriphenylphosphine palladium (0)
• a preferred intermediate (14) is the compound wherein PG is a Boc group.
• the compound of formula (I) can be prepared by following the method described in Example 84 of WO 2005/061463 (Astex) and then isolating the S-enantiomer using the separation methods described above and elsewhere herein.
• the substituted benzophenone (10) is converted to the epoxide (11) by reaction with trimethylsulphonium iodide in dimethylsulphoxide in the presence of a base as described in Scheme 1 above, except that the sodium hydride base is replaced with potassium tert-butoxide.
• the potassium tert-butoxide is added to a rapidly stirred mixture of the benzophenone (10) and trimethylsulphonium iodide, typically at room temperature.
• the use of potassium tert-butoxide as the base rather than sodium hydride confers significant advantages.
• the tert-butoxide can be added to a preformed mixture of the benzophenone (10), trimethylsulphonium iodide and dimethylsulphoxide. This means that the dimsyl anion is consumed very quickly after it is formed and therefore only small amounts of the dimsyl anion are present in the reaction mixture at any given time.
• the use of potassium tert-butoxide avoids the formation of large concentrations of the relatively viscous and somewhat hazardous dimsyl sodium.
• the absence of large concentrations of the viscous dimsyl sodium means that the reaction mixture is much easier to stir allowing more efficient mixing of the reactants and the avoidance of localised pockets of unreacted or incompletely reacted materials, an advantage which is enhanced by the fact that the tent-butanol formed during the reaction is a good solvent for the reactants and product.
• reaction sequence shown in Scheme 1 the epoxide (11) is reacted with ammonia in an alcoholic solvent such as methanol with heating, to give the amine (12).
• Reactions of this type may be carried out in a microwave reactor, typically under pressure and give good yields and purity on relatively small scale reactions.
• reaction of the epoxide (11) with the sodium azide is typically carried out in a polar solvent, e.g. an aqueous solvent comprising water and a water-miscible solvent such as acetone.
• a polar solvent e.g. an aqueous solvent comprising water and a water-miscible solvent such as acetone.
• the reaction is usually carried out with heating, for example to the reflux temperature of the solvent system.
• Conversion of the azido alcohol (17) to the amino alcohol (12) may be achieved by reaction with triphenyl phosphine followed by treatment with an aqueous acid and particularly an aqueous solution of a substituted sulphonic acid, preferably an alkyl- or arylsulphonic acid such as methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid or camphorsulphonic acid.
• a substituted sulphonic acid preferably an alkyl- or arylsulphonic acid such as methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid or camphorsulphonic acid.
• 4-toluenesulphonic acid is particularly preferred.
• the reaction is believed to proceed by initial cyclisation to form an aziridine followed by ring opening in the presence of the acid to give the amino alcohol.
• the amino-alcohol can be isolated as a stable, easy to handle salt and readily purified.
• an optically active form of camphorsulphonic acid e.g. d-camphorsulphonic acid
• fractional crystallisation of the salt can be carried out to separate the individual salts of the two enantiomers of the amino alcohol (12).
• Treatment of the salts with base then gives the individual enantiomers of the amino alcohol (12).
• the azide compound (17), the amino-alcohol (12) and its individual enantiomers and acid addition salts of the amino-alcohol (12) and its enantiomers are believed to be novel and, as such, form further aspects of the invention.
• the invention provides 2-amino-1-(4-chloro-phenyl)-1-[4-iodo-phenyl]-ethanol and acid addition salts thereof as defined herein.
• the invention provides (R) 2-amino-1-(4-chloro-phenyl)-1-[4-iodo-phenyl]-ethanol, and acid addition salts thereof as defined herein.
• the invention provides (5) 2-amino-1-(4-chloro-phenyl)-1-[4-iodo-phenyl]-ethanol, and acid addition salts thereof as defined herein.
• preferred acid addition salts are salts formed with methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid or camphorsulphonic acid (e.g. d-camphorsulphonic acid).
• a particularly preferred salt is the salt formed with 4-toluenesulphonic acid.
• the compound of the formula (12) and its acid addition salts have activity against the kinase PKB and, as such, should be useful in therapy, and in particular for the uses (e.g. anti-cancer uses) described herein for the compound of formula (I).
• Pharmaceutical compositions containing the compound of formula (12) or an acid addition salt thereof as defined herein and a pharmaceutically acceptable carrier, and the therapeutic uses of the compound of formula (12) or its acid addition salts constitute further aspects of the invention.
• the invention provides a method of preparing an optically active form of a compound of the formula (12), which method comprises the fractional crystallisation of an acid addition salt of the compound of the formula (12), wherein the salt is derived from an optically active acid (e.g. d-camphorsulphonic acid).
• an optically active acid e.g. d-camphorsulphonic acid
• the invention provides a process for the preparation of a compound of the formula (12) as defined herein, which process comprises the reaction of a compound of the formula (17) with a tertiary phosphine such as triphenylphosphine in a polar aprotic solvent such as tetrahydrofuran at a temperature above room temperature (for example at the reflux temperature of the solvent) followed by treatment with aqueous acid, for example a substituted sulphonic acid such as 4-toluenesulphonic acid.
• a tertiary phosphine such as triphenylphosphine
• a polar aprotic solvent such as tetrahydrofuran
• triphenylphosphine As an alternative to triphenylphosphine, other tertiary phosphines may be used and these include other triarylphosphines such as tritolylphosphine, trialkylphosphines such as tributylphosphine, tri-cycloalkyl phosphines such as tricyclohexylphosphine, and tertiary phosphines containing mixtures of aryl and/or alkyl and/or cycloalkyl groups. However, triphenylphosphine is preferred.
• substituted sulphonic acids may be used; for example alkyl- and arylsulphonic acid such as methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, and camphorsulphonic acid, as described above.
• the invention provides a process for the preparation of a compound of the formula (17); which process comprises the reaction of an epoxide compound of the formula (11) with an alkali metal azide (e.g. sodium azide) or trimethylsilylazide (TMS-azide) in a polar solvent (e.g. an aqueous organic solvent such as aqueous acetone), preferably with heating (for example to the reflux temperature of the solvent).
• an alkali metal azide e.g. sodium azide
• TMS-azide trimethylsilylazide
• the invention provides a process for the preparation of a compound of the formula (12), which process comprises the steps of:
• alkali metal azides e.g. lithium azide, potassium azide and sodium azide
• sodium azide is most preferred.
• the invention provides a process for the preparation of a compound of the formula (15), 2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol; which method comprises:
• composition of the invention is a pharmaceutical composition (e.g. formulation) comprising a compound of formula (I) together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.
• the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing the compound of formula (I) together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials, as described herein.
• pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• a subject e.g. human
• Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
• compositions containing the composition as defined herein can be formulated in accordance with known techniques, see for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA.
• the invention provides the composition as defined herein in the form of a pharmaceutical composition.
• compositions can be in any form suitable for oral, parenteral, topical, intranasal, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration.
• compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery.
• the delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilisation of a suitable infusion pump.
• compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilisation protectants and combinations of agents for, inter alia, stabilising the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient.
• aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels,
• compositions for parenteral administration may also take the form of aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents (R. G. Strickly, Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, Vol 21(2) 2004, p 201-230).
• Liposomes are closed spherical vesicles composed of outer lipid bilayer membranes and an inner aqueous core and with an overall diameter of ⁇ 100 ⁇ m.
• moderately hydrophobic drugs can be solubilized by liposomes if the drug becomes encapsulated or intercalated within the liposome.
• Hydrophobic drugs can also be solubilized by liposomes if the drug molecule becomes an integral part of the lipid bilayer membrane, and in this case, the hydrophobic drug is dissolved in the lipid portion of the lipid bilayer.
• the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• sterile liquid carrier for example water for injections
• the pharmaceutical formulation can be prepared by lyophilising a compound of formula (I). Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
• compositions of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
• suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
• Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion.
• the solution can be dosed as is, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose), before administration.
• the pharmaceutical composition is in a form suitable for sub-cutaneous (s.c.) administration.
• Pharmaceutical dosage forms suitable for oral administration include tablets, capsules, caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches and buccal patches.
• tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, eg; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g.
• swellable crosslinked polymers such as crosslinked carboxymethylcellulose
• lubricating agents e.g. stearates
• preservatives e.g. parabens
• antioxidants e.g. BHT
• buffering agents for example phosphate or citrate buffers
• effervescent agents such as citrate/bicarbonate mixtures.
• Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form.
• Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.
• the solid dosage forms can be coated or un-coated, but typically have a coating, for example a protective film coating (e.g. a wax or varnish) or a release controlling coating.
• a protective film coating e.g. a wax or varnish
• the coating e.g. a EudragitTM type polymer
• the coating can be designed to release the active component at a desired location within the gastro-intestinal tract.
• the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the compound in the stomach or in the ileum or duodenum.
• the drug can be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract.
• a release controlling agent for example a release delaying agent which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract.
• the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract.
• the active compound can be formulated in a delivery system that provides osmotic control of the release of the compound. Osmotic release and other delayed release or sustained release formulations may be prepared in accordance with methods well known to those skilled in the art.
• compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient.
• Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, dragées, tablets or capsules.
• compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.
• compositions of the invention can also be formulated as solid dispersions.
• Solid dispersions are homogeneous extremely fine disperse phases of two or more solids.
• Solid solutions molecularly disperse systems
• one type of solid dispersion are well known for use in pharmaceutical technology (see (Chiou and Riegelman, J. Pharm. Sci., 60, 1281-1300 (1971)) and are useful in increasing dissolution rates and increasing the bioavailability of poorly water-soluble drugs.
• Solid dosage forms include tablets, capsules and chewable tablets.
• Known excipients can be blended with the solid solution to provide the desired dosage form.
• a capsule can contain the solid solution blended with (a) a disintegrant and a lubricant, or (b) a disintegrant, a lubricant and a surfactant.
• a tablet can contain the solid solution blended with at least one disintegrant, a lubricant, a surfactant, and a glidant.
• the chewable tablet can contain the solid solution blended with a bulking agent, a lubricant, and if desired an additional sweetening agent (such as an artificial sweetener), and suitable flavours.
• the pharmaceutical formulations may be presented to a patient in “patient packs” containing an entire course of treatment in a single package, usually a blister pack.
• Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions.
• the inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.
• compositions for topical use include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.
• formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active compound.
• compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known.
• the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose.
• compositions will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity.
• a formulation may contain from 1 nanogram to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient.
• particular sub-ranges of compound are 0.1 milligrams to 2 grams of active ingredient (more usually from 10 milligrams to 1 gram, e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).
• a unit dosage form may contain from 1 milligram to 2 grams, more typically 10 milligrams to 1 gram, for example 50 milligrams to 1 gram, e.g. 100 milligrams to 1 gram, of active compound.
• the active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.
• the activity of the compound of formula (I) as inhibitors of protein kinase A and protein kinase B can be measured using the assays set forth in the examples below and the level of activity exhibited by a given compound can be defined in terms of the IC 50 value.
• the compound of the formula (I) is an inhibitor of protein kinase A and protein kinase B.
• the compositions of the invention will therefore prove useful in treating or preventing proliferative disorders such as cancers.
• tumours with deletions or inactivating mutations in PTEN or loss of PTEN expression or rearrangements in the (T-cell lymphocyte) TCL-1 gene may be particularly sensitive to PKB inhibitors.
• Tumours which have other abnormalities leading to an upregulated PKB pathway signal may also be particularly sensitive to inhibitors of PKB.
• abnormalities include but are not limited to overexpression of one or more PI3K subunits, over-expression of one or more PKB iso forms, or mutations in PI3K, PDK1, or PKB which lead to an increase in the basal activity of the enzyme in question, or upregulation or overexpression or mutational activation of a growth factor receptor such as a growth factor selected from the epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), platelet derived growth factor receptor (PDGFR), insulin-like growth factor 1 receptor (IGF-1R) and vascular endothelial growth factor receptor (VEGFR) families.
• EGFR epidermal growth factor receptor
• FGFR fibroblast growth factor receptor
• PDGFR platelet derived growth factor receptor
• IGF-1R insulin-like growth factor 1 receptor
• VEGFR vascular endothelial growth factor receptor
• compositions of the invention will also be useful in treating other conditions which result from disorders in proliferation or survival such as viral infections, and neurodegenerative diseases for example.
• PKB plays an important role in maintaining the survival of immune cells during an immune response and therefore PKB inhibitors could be particularly beneficial in immune disorders including autoimmune conditions.
• PKB inhibitors could be useful in the treatment of diseases in which there is a disorder of proliferation, apoptosis or differentiation.
• PKB inhibitors may also be useful in diseases resulting from insulin resistance and insensitivity, and the disruption of glucose, energy and fat storage such as metabolic disease and obesity.
• cancers which may be inhibited include, but are not limited to, a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
• a carcinoma for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
• the disease or condition comprising abnormal cell growth in one embodiment is a cancer.
• cancers include breast cancer, ovarian cancer, colon cancer, prostate cancer, oesophageal cancer, squamous cancer and non-small cell lung carcinomas.
• a further subset of cancers includes breast cancer, ovarian cancer, prostate cancer, endometrial cancer and glioma.
• compositions of the invention can also be used in combination with other anticancer agents. Examples of such combinations are set out below.
• Immune disorders for which the compositions of the invention may be beneficial include but are not limited to autoimmune conditions and chronic inflammatory diseases, for example systemic lupus erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus, Eczema hypersensitivity reactions, asthma, COPD, rhinitis, and upper respiratory tract disease.
• PKB plays a role in apoptosis, proliferation, differentiation and therefore the compound of formula (I) could also be useful in the treatment of the following diseases other than cancer and those associated with immune dysfunction; viral infections, for example herpes virus, pox virus, Epstein-Barr virus, Sindbis virus, adenovirus, HIV, HPV, HCV and HCMV; prevention of AIDS development in HIV-infected individuals; cardiovascular diseases for example cardiac hypertrophy, restenosis, atherosclerosis; neurodegenerative disorders, for example Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotropic lateral sclerosis, retinitis pigmentosa, spinal muscular atropy and cerebellar degeneration; glomerulonephritis; myelodysplastic syndromes, ischemic injury associated myocardial infarctions, stroke and reperfusion injury, degenerative diseases of the musculoskeletal system, for example, osteoporosis and arthritis, aspirin-sensitive rhinosinusitis, cystic fibro
• the compounds of formula (I) modulate (e.g. inhibit) the activity of ROCK kinase.
• the compounds therefore find application in: (a) the treatment or prophylaxis of a disease or condition in which the modulation (e.g. inhibition) of ROCK kinase is indicated; and/or (b) the treatment of a subject or patient population in which the modulation (e.g. inhibition) of ROCK kinase is indicated; and/or (c) the treatment or prophylaxis of a disease or condition in which the modulation (e.g. inhibition) of the Rho signalling pathway is indicated; and/or (d) the treatment of a subject or patient population in which the modulation (e.g. inhibition) of the Rho signalling pathway is indicated.
• tumour metastasis e.g. tumour metastasis
• tumour invasion e.g. tumour invasion
• tumour adhesion e.g. tumour cell adhesion
• actinomycin contractility-dependent tumour metastasis invasion or progression
• cell transformation e.g. ROCK-mediated tumour metastasis, invasion, progression or adhesion
• ROCK-mediated actinomycin contractility-dependent tumour metastasis invasion or progression
• ROCK-mediated cell transformation e.g.
• the invention also finds application in relation to cancer (e.g. ROCK-mediated cancer), especially where the cancer (for example being a ROCK-mediated cancer) is selected from: (a) testicular germ cell tumours; (b) small breast carcinomas with metastatic ability; (c) bladder cancer; (d) ovarian cancer; (e) prostate cancer; and (f) hepatocellular carcinoma.
• cancer e.g. ROCK-mediated cancer
• cardiovascular diseases or conditions particularly those selected from: (a) hypertension; (b) heart dysfunction (e.g. chronic and congestive heart failure); (c) cardiac hypertrophy; (d) restenosis; (e) renal dysfunction (e.g. chronic renal failure); (f) atherosclerosis (arteriosclerosis); (g) cardioprotection; (h) allograft survival; (i) cerebral ischemia; (j) coronary vasospasm; and (k) vascular inflammation.
• cardiovascular diseases or conditions particularly those selected from: (a) hypertension; (b) heart dysfunction (e.g. chronic and congestive heart failure); (c) cardiac hypertrophy; (d) restenosis; (e) renal dysfunction (e.g. chronic renal failure); (f) atherosclerosis (arteriosclerosis); (g) cardioprotection; (h) allograft survival; (i) cerebral ischemia; (j) coronary vasospasm; and (k) vascular inflammation.
• muscle e.g. smooth muscle
• diseases and conditions include muscle (e.g. smooth muscle) dysfunction, for example selected from: (a) asthma; (b) penile erectile dysfunction; (c) female sexual dysfunction; (d) over-active bladder I syndrome; and (e) abnormal smooth muscle (e.g. associated with hypertension).
• inflammation comprises or is manifest by: (a) rheumatoid arthritis; (b) irritable bowel syndrome; (c) inflammatory bowel disease; (d) vascular inflammation, and (e) a neuroinflammatory disease or condition.
• neuroinflammatory diseases or conditions these may be selected from: (a) stroke; (b) multiple sclerosis; (c) Alzheimer's disease; (d) Parkinson's disease; (e) amyotrophic lateral sclerosis; and (f) inflammatory pain.
• CNS diseases or conditions including those selected from: (a) spinal cord injury or trauma; (b) brain injury or trauma; (c) acute neuronal injury (e.g. stroke or traumatic brain injury); (d) Parkinson's disease; (e) Alzheimer's disease; (f) neurodegenerative conditions or diseases; (g) stroke (e.g. associated with hypertension); (h) cerebral vasospasm; (i) inhibition of neurite growth and sprouting; (j) inhibited neurite regeneration; (k) compromised post-trauma functional recovery; (l) demyelinating diseases or disorders; (m) inflammatory CNS diseases or disorders; (n) neuropathic pain; and (o) neurodegeneration.
• CNS diseases or conditions including those selected from: (a) spinal cord injury or trauma; (b) brain injury or trauma; (c) acute neuronal injury (e.g. stroke or traumatic brain injury); (d) Parkinson's disease; (e) Alzheimer's disease; (f) neurodegenerative conditions or diseases; (g) stroke (e.
• CNS diseases or conditions include those selected from: Downs syndrome and ⁇ -amyloid angiopathy, such as but not limited to cerebral amyloid angiopathy, hereditary cerebral hemorrhage, disorders associated with cognitive impairment, such as but not limited to MCI (“mild cognitive impairment”), Alzheimer Disease, memory loss, attention deficit symptoms associated with Alzheimer disease, neurodegeneration associated with diseases such as Alzheimer Disease or dementia including dementia of mixed vascular and degenerative origin, pre-senile dementia, senile dementia and dementia associated with Parkinson's Disease, progressive supranuclear palsy or cortical basal degeneration, Parkinson's Disease, Frontotemporal dementia Parkinson's Type, Parkinson dementia complex of Guam, HIV dementia, diseases with associated neurofibrillar tangle pathologies, dementia pugilistica, amyotrophic lateral sclerosis, corticobasal degeneration, Down syndrome, Huntington's Disease, postencephelatic parkinsonism, progressive supranuclear palsy, Pick's Disease, Niemann-
• graft protection e.g. cardiovascular or inflammatory graft protection
• diabetes e.g. diabetes fibroses
• asthma e.g. pulmonary vasoconstriction
• glaucoma e.g. glaucoma
• fibroses e.g. liver fibrosis and kidney fibrosis
• infectious diseases and conditions include infectious diseases or conditions, including metazoan, protozoan, fungal, prion, viral or bacterial infestations, diseases or infections.
• the infectious disease or condition may comprise pathogen-mediated cytoskeletal rearrangement.
• Proliferative Disorders including cancers: The invention also finds application as a means of preventing the growth of or inducing apoptosis of neoplasias. It is therefore anticipated that the invention will prove useful in treating or preventing proliferative disorders such as cancers.
• abnormalities include but are not limited to overexpression of one or more of the Rho signalling pathway members, or mutations in said members which lead to an increase in the basal activity of ROCK kinase(s) or the Rho signalling pathway (which may for example be associated with upregulation or overexpression or mutational activation of a growth factor receptor such as a growth factor selected from the epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), platelet derived growth factor receptor (PDGFR), insulin-like growth factor 1 receptor (IGF-1R) and vascular endothelial growth factor receptor (VEGFR) families).
• EGFR epidermal growth factor receptor
• FGFR fibroblast growth factor receptor
• PDGFR platelet derived growth factor receptor
• IGF-1R insulin-like growth factor 1 receptor
• VEGFR vascular endothelial growth factor receptor
• the invention will be useful in treating other conditions which result from disorders in proliferation or survival such as viral infections, and neurodegenerative diseases for example.
• the invention therefore finds broad application in the treatment of diseases in which there is a disorder of proliferation, apoptosis or differentiation.
• cancers which may be inhibited include, but are not limited to, a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
• a carcinoma for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
• a further example of a hematopoietic tumour of lymphoid lineage is multiple myeloma.
• cancers include breast cancer, ovarian cancer, colon cancer, prostate cancer, oesophageal cancer, squamous cancer and non-small cell lung carcinomas.
• a further subset of cancers includes breast cancer, ovarian cancer, prostate cancer, endometrial cancer and glioma.
• Another example of a disorder of proliferation is myeloproliferative syndrome.
• Immune disorders for which the invention may be beneficial include but are not limited to autoimmune conditions and chronic inflammatory diseases, for example systemic lupus erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus, Eczema hypersensitivity reactions, asthma, COPD, rhinitis, and upper respiratory tract disease.
• ROCK-mediated physiological processes play a role in apoptosis, proliferation, differentiation and therefore the invention could also be useful in the treatment of the following diseases other than cancer and those associated with immune dysfunction; viral infections, for example herpes virus, pox virus, Epstein-Barr virus, Sindbis virus, adenovirus, HIV, HPV, HCV and HCMV; prevention of AIDS development in HIV-infected individuals; cardiovascular diseases for example cardiac hypertrophy, restenosis, atherosclerosis; neurodegenerative disorders, for example Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotropic lateral sclerosis, retinitis pigmentosa, spinal muscular atropy and cerebellar degeneration; glomerulonephritis; myelodysplastic syndromes, ischemic injury associated myocardial infarctions, stroke and reperfusion injury, degenerative diseases of the musculoskeletal system, for example, osteoporosis and arthritis, aspirin-sensitive rhinosinusitis, cyst
• the invention may also be useful in diseases resulting from insulin resistance and insensitivity, and the disruption of glucose, energy and fat storage such as metabolic disease and obesity.
• the invention contemplates ROCK-mediated intervention, treatment or prophylaxis of any kind.
• the invention finds application in relation to treatment or prophylaxis comprising: (a) the modulation (e.g. inhibition) of ROCK kinase; or (b) intervention at the level of the activity of ROCK kinase; or (c) intervention at the level of the Rho signalling pathway (e.g. at the level of RhoA and.or RhoC).
• Interventions which effect: (a) muscle (e.g. smooth muscle) relaxation; (b) vascular muscle relaxation (e.g. to increase vascular blood flow); (c) nerve cell modulation; (d) reduction of cell proliferation; (e) reduction of cell migration; (f) suppression of cytoskeletal rearrangement upon pathogen invasion or infection; (g) acceleration of tissue regeneration; and (h) enhancement of post-traumatic functional recovery.
• the nerve cell modulation may comprise: (a) neuronal regeneration; (b) new axonal growth induction; (c) axonal rewiring across lesions within the CNS; (d) neurite outgrowth; (e) neurite differentiation; (f) axon pathfinding; (g) dendritic spine formation; (h) dendritic spine maintenance; (i) modulation of neurite growth cone collapse; and (j) modulation of neurite outgrowth inhibition.
• transplantation therapy e.g. comprising graft protection
• Yet other applicable methods comprise a method of diagnosis and treatment of a disease state or condition, which method comprises: (i) screening a patient to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against ROCK kinase; and (ii) where it is indicated that the disease or condition from which the patient is thus susceptible, thereafter administering to the patient a compound according to the invention.
• the subject or patient population may be selected from: (a) those in which ROCK kinase is dysfunctional (for example, hyperactive); and (b) those which have been subject to diagnostic tests for ROCK dysfunction (e.g. for ROCK hyperactivity); (c) those in which the Rho signalling pathway is dysfunctional; and (d) those which have been subject to diagnostic tests for Rho signalling pathway dysfunction.
• the compounds of formula (I) modulate (e.g. inhibit) the activity of protein kinase p70S6K.
• the compounds therefore find application in: (a) the treatment or prophylaxis of a disease or condition in which the modulation (e.g. inhibition) of protein kinase p70S6K is indicated; and/or (b) the treatment of a subject or patient population in which the modulation (e.g. inhibition) of protein kinase p70S6K is indicated.
• the invention therefore finds application in relation to conditions selected from: (a) cancer (e.g. p70S6K-mediated cancer); (b) tumour metastases; (c) immune dysfunction; (d) tissue damage (e.g. arising from inflammation); (e) chromosome 17q23 amplification (or conditions arising therefrom or associated therewith); (f) Peutz-Jeghers syndrome (or conditions arising therefrom or associated therewith); (g) LKB1 mutation(s) (or conditions arising therefrom or associated therewith); (h) BRCA1 mutation(s) (or conditions arising therefrom or associated therewith); (i) BRCA2 mutation(s) (or conditions arising therefrom or associated therewith); (j) dysfunctional apoptotic programmes; (k) growth factor receptor signal transduction, overexpression and activation in tumour tissue; (l) a metabolic disease or disorder; (m) those associated with abnormal cell proliferation and/or metabolism; and (n) neuronal disorders.
• cancer e.g. p70S6K-mediated
• the disease or condition arising from or associated with chromosome 17q23 amplification may be selected from: (a) primary breast tumours; (b) tumours (e.g. breast tumours) containing BRCA2 mutations; (c) tumours (e.g. breast tumours) containing BRCA1 mutations; (d) pancreatic tumours; (e) bladder tumours; and (f) neuroblastomas.
• the disease or condition arising from or associated with LKB1 mutation(s) may be lung adenocarcinoma containing LKB1 mutation(s) (e.g. inactivating LKB1 mutation(s)).
• the disease or condition arising from or associated with BRCA1/2 mutation(s) may be breast cancer.
• the metabolic disease or disorder may be selected from: (a) obesity (for example age-induced obesity or diet-induced obesity); (b) diabetes; (c) metabolic syndrome; (d) insulin resistance; (e) hyperglycemia; (f) hyperaminoacidemia; and (g) hyperlipidmia.
• Proliferative Disorders including cancers: The invention also finds application as a means of preventing the growth of or inducing apoptosis of neoplasias. It is therefore anticipated that the invention will prove useful in treating or preventing proliferative disorders such as cancers. Examples of such abnormalities include but are not limited to overexpression of p70S6K (or the other syndromes described herein).
• the invention will be useful in treating other conditions which result from disorders in proliferation or survival such as viral infections, and neurodegenerative diseases for example.
• the invention therefore finds broad application in the treatment of diseases in which there is a disorder of proliferation, apoptosis or differentiation.
• cancers which may be inhibited include, but are not limited to, a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
• a carcinoma for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g.
• a further example of a hematopoietic tumour of lymphoid lineage is multiple myeloma.
• cancers include breast cancer, ovarian cancer, colon cancer, prostate cancer, oesophageal cancer, squamous cancer and non-small cell lung carcinomas.
• a further subset of cancers includes breast cancer, ovarian cancer, prostate cancer, endometrial cancer and glioma.
• Another example of a disorder of proliferation is myeloproliferative syndrome.
• Immune disorders for which the invention may be beneficial include but are not limited to autoimmune conditions and chronic inflammatory diseases, for example systemic lupus erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus, Eczema hypersensitivity reactions, asthma, COPD, rhinitis, and upper respiratory tract disease.
• p70S6K-mediated physiological processes play a role in apoptosis, proliferation, differentiation and therefore the invention could also be useful in the treatment of the following diseases other than cancer and those associated with immune dysfunction; viral infections, for example herpes virus, pox virus, Epstein-Barr virus, Sindbis virus, adenovirus, HIV, HPV, HCV and HCMV; prevention of AIDS development in HIV-infected individuals; cardiovascular diseases for example cardiac hypertrophy, restenosis, atherosclerosis; neurodegenerative disorders, for example Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotropic lateral sclerosis, retinitis pigmentosa, spinal muscular atropy and cerebellar degeneration; glomerulonephritis; myelodysplastic syndromes, ischemic injury associated myocardial infarctions, stroke and reperfusion injury, degenerative diseases of the musculoskeletal system, for example, osteoporosis and arthritis, aspirin-sensitive rhinosinus
• the invention may also be useful in diseases resulting from insulin resistance and insensitivity, and the disruption of glucose, energy and fat storage such as metabolic disease and obesity.
• the invention contemplates protein kinase p70S6K-mediated intervention, treatment or prophylaxis of any kind.
• the invention finds application in relation to treatment or prophylaxis comprising: (a) the modulation (e.g. inhibition) of protein kinase p70S6K; (b) intervention at the level of the activity of protein kinase p70S6K; (b) inhibition of progression from G1 to S phase in the cell cycle in vivo; (c) inhibition of cell cycle proliferation at the G1 to S phase of the cell cycle; (d) use of a compound of formula (I) as a rapamycin surrogate; (e) use of a compound of formula (I) as a wortmannin surrogate; (f) the re-establishment of appropriate apoptotic programmes; (g) the inhibition of growth factor receptor signal transduction, overexpression and activation in tumour tissue; (h) modulation of neuronal cell differentiation; (i) modulation of cell motility
• the treatment or prophylaxis may also comprise a method of diagnosis and treatment of a disease state or condition, which method comprises: (i) screening a patient to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against protein kinase p70S6K; and (ii) where it is indicated that the disease or condition from which the patient is thus susceptible, thereafter administering to the patient a compound of formula (I) as herein defined.
• the subject or patient population may be selected from: (a) those in which protein kinase p70S6K is dysfunctional (for example, hyperactive); (b) those which have been subject to diagnostic tests for p70S6K is dysfunction (e.g. for p70S6K hyperactivity); (c) those in which chromosome 17q23 is amplified; and (d) those which have been subject to diagnostic tests for amplification of chromosome 17q23; (e) those in which BRCA1 mutation(s) are present; (f) those which have been subject to diagnostic tests for BRCA1 mutation(s); (g) those in which BRCA2 mutation(s) are present; (h) those which have been subject to diagnostic tests for BRCA2 mutation(s); (i) those in which LKB1 mutation(s) are present; (j) those which have been subject to diagnostic tests for LKB1 mutation(s); and (k) those which have been screened as defined herein.
• compositions of the invention have physiochemical properties suitable for oral exposure.
• composition as defined herein should exhibit improved oral bioavailability relative to prior art compounds.
• Oral bioavailability can be defined as the ratio (F) of the plasma exposure of a compound when dosed by the oral route to the plasma exposure of the compound when dosed by the intravenous (i.v.) route, expressed as a percentage.
• compositions having an oral bioavailability (F value) of greater than 30%, more preferably greater than 40%, are particularly advantageous in that they may be administered orally rather than, or as well as, by parenteral administration.
• the compound of formula (I) is both more potent and more selective in its activities against different kinases, and demonstrates enhanced selectivity for and potency against PKB in particular.
• the compound of formula (I) is significantly more potent than its R-enantiomer at inhibiting PKB in vitro and in cells.
• the IC 50 for the compound of formula (I) against the isolated PKB enzyme in an in vitro radiometric assay is 0.01 ⁇ M compared to 0.96 ⁇ M for the R-enantiomer. This approximate 100-fold difference in potency is also observed in a cell-based mechanistic assay which measures phosphorylation of GSK3 ⁇ , a direct downstream substrate of PKB.
• the compound of formula (I) displays an IC 50 of 1.1 ⁇ M, compared to a value for the R-enantiomer of >50 ⁇ M.
• the compound of formula (I) is advantageous over prior art compounds in that it has different susceptibilities to P450 enzymes and in that it exhibits improvements with regard to drug metabolism and pharmacokinetic properties.
• the compound of formula (I) has IC 50 values of greater than 10 ⁇ M against each of the cytochrome P450 enzymes 1A2, 2C9, 2C19, 3A4 and 2D6.
• the compound of formula (I) should exhibit reduced dosage requirements.
• the compound of formula (I) is potentially less toxic than prior art compounds.
• the hERG channel is one of a family of potassium ion channels the first member of which was identified in the late 1980s in a mutant Drosophila melanogaster fruitfly (see Jan, L. Y. and Jan, Y. N. (1990). A Superfamily of Ion Channels. Nature, 345(6277):672).
• the biophysical properties of the hERG potassium ion channel are described in Sanguinetti, M.
• HERG encodes the Ikr potassium channel. Cell, 81:299-307, and Trudeau, M. C., Warmke, J. W., Ganetzky, B., and Robertson, G. A. (1995). HERG, a Human Inward Rectifier in the Voltage-Gated Potassium Channel Family. Science, 269:92-95.
• the compounds of formula (I) has negligible hERG ion channel blocking activity.
• composition as defined herein will be useful in the prophylaxis or treatment of a range of disease states or conditions mediated by protein kinase A and/or protein kinase B and/or a ROCK kinase and/or p70S6K kinase. Examples of such disease states and conditions are set out above.
• compositions are generally administered to a subject in need of such administration, for example a human or animal patient, preferably a human.
• composition will typically be administered in amounts that are therapeutically or prophylactically useful and which generally are non-toxic.
• benefits of administering a composition as defined herein may outweigh the disadvantages of any toxic effects or side effects, in which case it may be considered desirable to administer compounds in amounts that are associated with a degree of toxicity.
• compositions may be administered over a prolonged term to maintain beneficial therapeutic effects or may be administered for a short period only. Alternatively they may be administered in a pulsatile or continuous manner.
• a typical daily dose of the compound of formula (I) can be in the range from 100 picograms to 100 milligrams per kilogram of body weight, more typically 5 nanograms to 25 milligrams per kilogram of bodyweight, and more usually 10 nanograms to 15 milligrams per kilogram (e.g. 10 nanograms to 10 milligrams, and more typically 1 microgram per kilogram to 20 milligrams per kilogram, for example 1 microgram to 10 milligrams per kilogram) per kilogram of bodyweight although higher or lower doses may be administered where required.
• the composition as defined herein can be administered on a daily basis or on a repeat basis every 2, or 3, or 4, or 5, or 6, or 7, or 10 or 14, or 21, or 28 days for example.
• the compound of formula (I) may be administered orally in a range of doses, for example 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, e.g. 2 to 200 mg or 10 to 1000 mg, particular examples of doses including 10, 20, 50 and 80 mg.
• the compound may be administered once or more than once each day.
• the compound can be administered continuously (i.e. taken every day without a break for the duration of the treatment regimen).
• the compound can be administered intermittently, i.e. taken continuously for a given period such as a week, then discontinued for a period such as a week and then taken continuously for another period such as a week and so on throughout the duration of the treatment regimen.
• treatment regimens involving intermittent administration include regimens wherein administration is in cycles of one week on, one week off; or two weeks on, one week off; or three weeks on, one week off; or two weeks on, two weeks off; or four weeks on two weeks off; or one week on three weeks off—for one or more cycles, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more cycles.
• a patient will be given an infusion of a composition as defined herein for periods of one hour daily for up to ten days in particular up to five days for one week, and the treatment repeated at a desired interval such as two to four weeks, in particular every three weeks.
• a patient may be given an infusion of a composition as defined herein for periods of one hour daily for 5 days and the treatment repeated every three weeks.
• a patient is given an infusion over 30 minutes to 1 hour followed by maintenance infusions of variable duration, for example 1 to 5 hours, e.g. 3 hours.
• a patient is given a continuous infusion for a period of 12 hours to 5 days, an in particular a continuous infusion of 24 hours to 72 hours.
• the quantity of compound administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.
• the compound of formula (I) can be administered as the sole therapeutic agent or they can be administered in combination therapy with one of more other compounds for treatment of a particular disease state, for example a neoplastic disease such as a cancer as hereinbefore defined.
• a neoplastic disease such as a cancer as hereinbefore defined.
• other therapeutic agents or treatments that may be administered together (whether concurrently or at different time intervals) with the compounds of the formula (I) include but are not limited to:
• Each of the compounds present in the combinations of the invention may be given in individually varying dose schedules and via different routes.
• the compounds of the formula (I) can be administered simultaneously or sequentially.
• they can be administered at closely spaced intervals (for example over a period of 5-10 minutes) or at longer intervals (for example 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).
• the compound of formula (I) may also be administered in conjunction with non-chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery and controlled diets.
• non-chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery and controlled diets.
• composition as defined herein and one, two, three, four or more other therapeutic agents can be, for example, formulated together in a dosage form containing two, three, four or more therapeutic agents.
• the individual therapeutic agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.
• a patient Prior to administration of a composition as defined herein, a patient may be screened to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against a particular target kinase (e.g. protein kinase A and/or protein kinase B and/or ROCK kinase and/or P70S6K kinase).
• a target kinase e.g. protein kinase A and/or protein kinase B and/or ROCK kinase and/or P70S6K kinase.
• a biological sample taken from a patient may be analysed to determine whether a condition or disease, such as cancer, that the patient is or may be suffering from is one which is characterised by a genetic abnormality or abnormal protein expression which leads to up-regulation of PKA and/or PKB or to sensitisation of a pathway to normal PKA and/or PKB activity, or to upregulation of a signal transduction component upstream of PKA and/or PKB such as, in the case of PKB, P13K, GF receptor and PDK 1 & 2.
• a biological sample taken from a patient may be analysed for loss of a negative regulator or suppressor of the PKB pathway such as PTEN.
• loss embraces the deletion of a gene encoding the regulator or suppressor, the truncation of the gene (for example by mutation), the truncation of the transcribed product of the gene, or the inactivation of the transcribed product (e.g. by point mutation) or sequestration by another gene product.
• the patient may be screened for dysfunction in ROCK activity (e.g. elevated or up-regulated ROCK expression, mutations in ROCK genes or ROCK gene regulatory elements) or Rho signalling dysfunction (as described herein).
• dysfunction in ROCK activity e.g. elevated or up-regulated ROCK expression, mutations in ROCK genes or ROCK gene regulatory elements
• Rho signalling dysfunction as described herein.
• up-regulation includes elevated expression or over-expression, including gene amplification (i.e. multiple gene copies) and increased expression by a transcriptional effect, and hyperactivity and activation, including activation by mutations.
• the patient may be subjected to a diagnostic test to detect a marker characteristic of up-regulation of a kinase (e.g. PKA and/or PKB and/or ROCK kinase and/or P70S6K kinase).
• diagnosis includes screening.
• marker we include genetic markers including, for example, the measurement of DNA composition to identify mutations of the kinase (e.g. PKA and/or PKB and/or ROCK kinase and/or P70S6K kinase).
• marker also includes markers which are characteristic of up regulation of the kinase (e.g. PKA and/or PKB and/or ROCK kinase and/or P70S6K kinase) and/or other factors which lead to an upregulation of the relevant pathways, including enzyme activity, enzyme levels, enzyme state (e.g. phosphorylated or not) and mRNA levels of the aforementioned proteins.
• markers which are characteristic of up regulation of the kinase e.g. PKA and/or PKB and/or ROCK kinase and/or P70S6K kinase
• other factors which lead to an upregulation of the relevant pathways, including enzyme activity, enzyme levels, enzyme state (e.g. phosphorylated or not) and mRNA levels of the aforementioned proteins.
• tumour biopsy samples selected from tumour biopsy samples, blood samples (isolation and enrichment of shed tumour cells), stool biopsies, sputum, chromosome analysis, pleural fluid, peritoneal fluid, bone marrow or urine.
• Identification of an individual carrying a mutation in PKA and/or PKB or a rearrangement of TCL-1 or loss of PTEN expression may mean that the patient would be particularly suitable for treatment with a PKA and/or PKB inhibitor.
• Tumours may preferentially be screened for presence of a PKA and/or PKB variant prior to treatment. The screening process will typically involve direct sequencing, oligonucleotide microarray analysis, or a mutant specific antibody.
• Screening methods could include, but are not limited to, standard methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) or in-situ hybridisation.
• RT-PCR reverse-transcriptase polymerase chain reaction
• telomere amplification is assessed by creating a cDNA copy of the mRNA followed by amplification of the cDNA by PCR.
• Methods of PCR amplification, the selection of primers, and conditions for amplification, are known to a person skilled in the art.
• Nucleic acid manipulations and PCR are carried out by standard methods, as described for example in Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc., or Innis, M. A. et-al., eds. PCR Protocols: a guide to methods and applications, 1990, Academic Press, San Diego.
• FISH fluorescence in-situ hybridisation
• in situ hybridization comprises the following major steps: (1) fixation of tissue to be analyzed; (2) prehybridization treatment of the sample to increase accessibility of target nucleic acid, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization, and (5) detection of the hybridized nucleic acid fragments.
• the probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters.
• Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions.
• Standard methods for carrying out FISH are described in Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc and Fluorescence In Situ Hybridization: Technical Overview by John M. S. Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd ed.; ISBN: 1-59259-760-2; March 2004, pps. 077-088; Series: Methods in Molecular Medicine.
• the protein products expressed from the mRNAs may be assayed by immunohistochemistry of tumour samples, solid phase immunoassay with microtitre plates, Western blotting, 2-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and other methods known in the art for detection of specific proteins. Detection methods would include the use of site specific antibodies. The skilled person will recognize that all such well-known techniques for detection of upregulation of the kinase (e.g. PKB and/or PKA and/or ROCK kinase and/or P70S6K kinase), or detection of kinase variants could be applicable in the present case.
• kinase e.g. PKB and/or PKA and/or ROCK kinase and/or P70S6K kinase
• PKB beta has been found to be upregulated in 10-40% of ovarian and pancreatic cancers (Bellacosa et al 1995, Int. J. Cancer 64, 280-285; Cheng et al 1996, PNAS 93, 3636-3641; Yuan et al 2000, Oncogene 19, 2324-2330). Therefore it is envisaged that PKB inhibitors, and in particular inhibitors of PKB beta, may be used to treat ovarian and pancreatic cancers.
• PKB alpha is amplified in human gastric, prostate and breast cancer (Staal 1987, PNAS 84, 5034-5037; Sun et al 2001, Am. J. Pathol. 159, 431-437). Therefore it is envisaged that PKB inhibitors, and in particular inhibitors of PKB alpha, may be used to treat human gastric, prostate and breast cancer.
• PKB inhibitors and in particular inhibitors of PKB gamma, may be used to treat steroid independent breast and prostate cancers.
• Detection of ROCK may be carried out at either the mRNA or protein level. Specific examples of methods where levels of Rho and ROCK have been determined in clinical samples include:
• Detection of p70S6K may be carried out at either the mRNA or protein level. Exemplary methods are described for example in J Naltl Cancer Inst (2000): 92, pp. 1252-9 (which describes detecting the activation of Ribosomal Protein S6 Kinase by complementary DNA and tissue microarray analysis uses comparative genomic hybridization (CGH) and cDNA and tissue microarray analyses to identify amplified and overexpressed genes).
• CGH comparative genomic hybridization
• the compounds prepared were characterised by liquid chromatography and mass spectroscopy using the systems and operating conditions set out below. Where chlorine is present, the mass quoted for the compound is for 35 Cl. The operating conditions used are described below.
• step 1B The amino alcohol product of step 1B (9.55 g, 25.56 mmol) was suspended in 1,4-dioxane (220 ml) and 2M NaOH added (16.6 ml, 33.24 mmol). The mixture was stirred vigorously until homogenous. Di-tert-butyl dicarbonate (6.14 g, 28.11 mmol) was added and the reaction mixture was stirred at 45 C for 20 hours. Upon cooling, the reaction mixture was concentrated and partitioned between EtOAc (150 ml) and water (150 ml). The organic layer was separated and washed with Brine (150 ml), dried (MgSO 4 ) and concentrated to yield a yellow oil (14.08 g).
• the solution was degassed with nitrogen and tetrakistriphenylphosphine palladium (0) (0.612 g, 0.53 mmol) was added.
• the mixture was degassed with nitrogen and then heated at 85° C. under nitrogen for 2 hours.
• the reaction mixture was then allowed to cool to room temperature. Additional batches of reagents were then added: potassium phosphate (7.88 g, 37.10 mmol) and pyrazole boronate (4.1 g, 21.11 mmol).
• the reaction mixture was degassed with nitrogen and a further batch of tetrakistriphenylphosphine palladium (0) (0.101 g, 0.087 mmol) was added.
• the reaction mixture was degassed and then heated at 85° C.
• optical activities of both the S enantiomer and the R enantiomer were determined using an AA-10 automatic polarimeter (Optical Activity Limited).
• the S-enantiomer had a retention time of 15.283 minutes.
• This example describes the preparation of 2-amino-1-(4-chloro-phenyl)-1-[4-iodo-phenyl]-ethanol, intermediate compound 1B in Example 1.
• Potassium tert-butoxide (18.48 g, 165.0 mmol) was added to a rapidly stirred suspension of 4-chloro-4′-iodobenzophenone (51.38 g, 150.0 mmol) and trimethylsulphonium iodide (33.66 g, 165.0 mmol) in dimethylsulphoxide (200 ml) and the resulting mixture was stirred at room temperature for 3 hours. The mixture was diluted with ethyl acetate (500 ml), washed with water (3 ⁇ 500 ml) and then with brine (500 ml).
• Triphenylphosphine 31.44 g, 120.0 mmol was added to a solution of (RS)-2-azido-1-(4-chlorophenyl)-1-(4-iodophenyl)ethanol (47.94 g, 120.0 mmol) in tetrahydrofuran (400 ml) and the mixture was stirred and held at reflux for 5 hours whereupon toluene-4-sulphonic acid monohydrate (22.8 g, 120.0 mmol) and water (40 ml) were added and the mixture was stirred and held at reflux for a further 16 hours. Upon cooling to room temperature the mixture was evaporated to dryness in vacuo.
• Example 2C can be converted into the N-Boc derivative by the method of Example 1C (but using an additional equivalent of sodium hydroxide to take account of the toluene sulphonic acid salt) and then subjected to a Suzuki coupling reaction followed by removal of the Boc protecting group as described in Example 1D and Example 1E and the resulting mixture of enantiomers resolved by the method of Example 1F to give (S)-2-amino-1-(4-chloro-phenyl)-1-[4-(1H-pyrazol-4-yl)-phenyl]-ethanol.
• the biological activity of the compound of formula (I) is described in the following examples.
• the biological properties of the compound of formula (I) are described in the poster by John F. Lyons et al., page 3512s, Poster Session B, Abstract B251, AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics, Oct. 22-26, 2007, San Francisco, Calif. (copy available on the Astex Therapeutics website: www.astex-therapeutics.com or www.astex-therapeutics.com/investorsandmedia/publications)
• the compound of formula (I) can be tested for PK inhibitory activity using the PKA catalytic domain from Upstate Biotechnology (#14-440) and the 9 residue PKA specific peptide (GRTGRRNSI), also from Upstate Biotechnology (#12-257), as the substrate.
• a final concentration of 1 nM enzyme is used in a buffer that includes 20 mM MOPS pH 7.2, 40 ⁇ M ATP/ ⁇ 33 P-ATP and 50 mM substrate.
• Compounds are added in dimethylsulphoxide (DMSO) solution to a final DMSO concentration of 2.5%. The reaction is allowed to proceed for 20 minutes before addition of excess orthophosphoric acid to quench activity. Unincorporated ⁇ 33 P-ATP is then separated from phosphorylated proteins on a Millipore MAPH filter plate. The plates are washed, scintillant is added and the plates are then subjected to counting on a Packard Topcount.
• DMSO dimethylsulphoxide
• the % inhibition of the PKA activity is calculated and plotted in order to determine the concentration of test compound required to inhibit 50% of the PKA activity (IC 50 ).
• PKT protein kinase B
• a final concentration of 0.6 nM enzyme is used in a buffer that includes 20 mM MOPS pH 7.2, 30 ⁇ M ATP/ ⁇ 33 P-ATP and 25 ⁇ M substrate.
• Compounds are added in DMSO solution to a final DMSO concentration of 2.5%.
• the reaction is allowed to proceed for 20 minutes before addition of excess orthophosphoric acid to quench activity.
• the reaction mixture is transferred to a phosphocellulose filter plate where the peptide binds and the unused ATP is washed away. After washing, scintillant is added and the incorporated activity measured by scintillation counting.
• the % inhibition of the PKB activity is calculated and plotted in order to determine the concentration of test compound required to inhibit 50% of the PKB activity (IC 50 ).
• the activity of compound of formula (I) against the hERG K + ion channel can be determined using the assay described in the article by M. H. Bridgland-Taylor et al., Journal of Pharmcaological and Toxicological Methods, 54 (2006), 189-199.
• the potency of the compound of Example 1 against cytochrome P450 (CYP450) enzymes 1A2, 2C9, 2C19, 3A4 and 2D6 was determined using the Pan Vera Vivid Cyp450 screening kits available from Invitrogen (Paisley, UK).
• the CYP450s were supplied in the form of baculosomes containing the CYP450 and NADPH reductase and the substrates used were the fluorescent Vivid substrates.
• the final reaction mixtures were as follows:
• the excitation and emission wavelengths were 390 nm and 460 nm for 1A2, 2C19 and 3A4, 390 nm and 485 nm for 2D6 and 485 nm and 530 nm for 2C9. Initial rates were determined from progress curves.
• test compound was made up in acetonitrile and tested against the CYP450s at a concentration of 10 ⁇ M.
• the compound of Example 1 had an IC 50 greater than 10 ⁇ M against 1A2, 2C9, 2C19, 3A4 and 2D6.
• the effect on inhibiting PKB in U87MG cells is determined by the ability of compounds to inhibit phosphorylation of the direct downstream substrate GSK3 ⁇ on serine 9.
• Cells are plated in 96 well plates and allowed to recover overnight prior to addition of the inhibitor compound for 1 hour. After 1 hour, cell are fixed and blocked with 3% paraformaldehyde, 0.25% gluteraldehyde, 0.25% Triton X100 and 5% Marvel in TBS-T. Following this cells are incubated with primary antibody directed to the phosphorylated form of GSK3 ⁇ (Cell Signaling) overnight at 4° C.
• ROCK-II (h) (5-10 mU) is incubated with 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 ⁇ M KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, 10 mM MgAcetate and [ ⁇ - 33 P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required).
• the reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 ⁇ l of a 3% phosphoric acid solution. 10 ⁇ l of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
• P70S6 enzyme is bought from Upstate and used at 2 nM in the assay.
• the substrate S6 cocktail (AKRRRLSSLRA) is used at 2504 (Km has not been determined).
• AKRRRLSSLRA The substrate S6 cocktail
• the 33 P- ⁇ phosphate from ATP is transferred to the serine residue.
• the reaction mixture is transferred to a phosphocellulose filter plate where the peptide binds and the unused ATP is washed away. After washing, scintillant is added and the incorporated activity measured by scintillation counting.
• Assay Buffer 10 mM MOPS pH 7.0 0.1 mg/ml BSA 0.001% Brij-35 0.5% glycerol 0.2 mM EDTA 10 mM MgCl 2 0.01% ⁇ -mercaptoethanol Made as a 10X stock, stored at 20° C. in 2 ml aliquots 15 ⁇ M ATP
• ATP 10 mM stock
• ATP ATP (10 mM stock) added fresh from concentrated stocks. ATP will break down over time, keep on ice as far as possible and use small aliquots to ensure the stock is fresh.
• the amount of 33 P-ATP added assumes it is on its reference date. The exact amount needs to be adjusted with time.
• ATP concentration is approximately 1504. KM for ATP calculated to 47 uM radiometrically. Controls are “no compound” (DMSO only) and “no enzyme” (use 10 ⁇ l of the enzyme mix prior to adding enzyme). Cover with a plate seal (TopSeal A—Packard) or plastic lid from filter plate (moderate radiation barrier). Mix by gentle shaking Incubate at room temperature for 50 minutes. Stop the reaction by adding 20 ⁇ l of 2% orthophosphoric acid.
• a tablet composition containing a composition as defined herein is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg magnesium stearate as a lubricant and compressing to form a tablet in known manner.
• BP lactose
• a capsule formulation is prepared by mixing 100 mg of a composition as defined herein with 100 mg lactose and filling the resulting mixture into standard opaque hard gelatin capsules.
• a parenteral composition for administration by injection can be prepared by dissolving a composition as defined herein (e.g. in a salt form) in water containing 10% propylene glycol to give a concentration of active compound of 1.5% by weight. The solution is then sterilised by filtration, filled into an ampoule and sealed.
• a parenteral composition for injection is prepared by dissolving in water a composition as defined herein (e.g. in salt form) (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.
• a composition as defined herein e.g. in salt form
• mannitol 50 mg/ml
• a formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (I) (e.g. in a salt form) in water at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
• a formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (I) (e.g. in a salt form) in water containing a buffer (e.g. 0.2 M acetate pH 4.6) at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
• a buffer e.g. 0.2 M acetate pH 4.6
• a composition for sub-cutaneous administration is prepared by mixing a composition as defined herein with pharmaceutical grade corn oil to give a concentration of 5 mg/ml.
• the composition is sterilised and filled into a suitable container.
• Aliquots of formulated compound of formula (I) are put into 50 ml vials and lyophilized.
• the compositions are frozen using a one-step freezing protocol at ( ⁇ 45° C.).
• the temperature is raised to ⁇ 10° C. for annealing, then lowered to freezing at ⁇ 45° C., followed by primary drying at +25° C. for approximately 3400 minutes, followed by a secondary drying with increased steps if temperature to 50° C.
• the pressure during primary and secondary drying is set at 80 millitor.

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  • 2008-03-14 AU AU2008224657A patent/AU2008224657B2/en not_active Ceased
  • 2008-03-14 PT PT87190252T patent/PT2134339E/pt unknown
  • 2008-03-14 ES ES08719025.2T patent/ES2511817T3/es active Active
  • 2008-03-14 KR KR1020097020516A patent/KR20100014720A/ko not_active Application Discontinuation
  • 2008-03-14 RU RU2009137779/04A patent/RU2527151C2/ru not_active IP Right Cessation
• 2009
  • 2009-09-08 IL IL200822A patent/IL200822A/en not_active IP Right Cessation
  • 2009-09-10 ZA ZA2009/06303A patent/ZA200906303B/en unknown
• 2010
  • 2010-01-08 HK HK10100202.2A patent/HK1133828A1/zh not_active IP Right Cessation
• 2011
  • 2011-07-11 US US13/180,127 patent/US8497294B2/en not_active Expired - Fee Related
• 2014
  • 2014-08-25 JP JP2014170885A patent/JP2015028033A/ja active Pending
  • 2014-10-27 HR HRP20141034AT patent/HRP20141034T1/hr unknown
  • 2014-11-12 CY CY20141100938T patent/CY1115877T1/el unknown

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