FORMULA I COMPOUNDS, CRYSTALLINE FORM, USE OF COMPOUNDS, COMBINATION AND COMPOSITION

专利摘要:
compounds of formula i, crystalline form, use of the compounds, combination, composition and method of selection for treating cancer The present invention relates to compounds of formula (i): (i) or pharmaceutically acceptable salts thereof, wherein r1 and r2 have any of the meanings defined hereinbefore in the description; to processes for their preparation, to pharmaceutical compositions containing the same, and to their use in the treatment of cell proliferative disorders.
公开号:BR112015017331B1
申请号:R112015017331-4
申请日:2014-01-22
公开日:2022-01-11
发明作者:Bernard Christophe Barlaam；David Berry；Benedicte Delouvrie；Craig Steven Harris；Christine Marie Paul Lambert-Van Der Brempt；Gilles Ouvry；Gary Patrick Reid；Gary Peter Tomkinson
申请人:Astrazeneca Ab；
IPC主号:

专利说明:

[0001] The present invention relates to certain novel aminopyrazine derivatives, or pharmaceutically acceptable salts thereof, which possess anti-cancer activity and are therefore useful in methods of treating the body of a human or animal. The invention also relates to processes for the manufacture of said aminopyrazine derivatives, pharmaceutical compositions containing the same and their use in therapeutic methods, for example in the manufacture of medicaments for use in the prevention or treatment of cancers in a warm-blooded animal, as in man, including use in the prevention or treatment of cancer.
[0002] The present invention also relates to aminopyrazine derivatives which are selective inhibitors of the PI3 kinase family of enzymes (which is alternatively known as the 3-phosphatidylinositol kinase family or PI3K family), particularly of the PI3K- isoforms. α and PI3K-δ and are, for example, useful for antitumor therapy.
[0003] In the area of cancer, in recent years, it has been discovered that a cell can become cancerous due to the transformation of a part of its DNA into an oncogene, that is, a gene which, when activated, leads to formation of malignant tumor cells (Bradshaw, Mutagenesis, 1986, 1, 91). Several such oncogenes give rise to the production of peptides, which are kinases, a class of enzymes that are capable of phosphorylating their protein or lipid substrates. There are several classes of kinases.
[0004] First, tyrosine kinases, which can be either receptor tyrosine kinases or non-receptor tyrosine kinases. Several classes of receptor tyrosine kinases are known (Wilks, Advances in Cancer Research, 1993, 60, 43-73) based on families Petition 870190124630, 11/28/2019, p. 5/20 of growth factors, which can bind to the extracellular surface of different receptor tyrosine kinases; as an example, the classification includes Class I receptor tyrosine kinases which comprise the EGF family of receptor tyrosine kinases. Non-receptor tyrosine kinases are located intracellularly; Several classes of non-receptor tyrosine kinases are known, including the Src family, such as the Src, Lyn, Fyn, and Yes tyrosine kinases.
[0005] Second, certain kinases belong to the class of serine/threonine kinases which are also located intracellularly. Serine/threonine kinase signaling pathways include the Raf-MEK-ERK cascade and those downstream of PI3 kinases, such as PDK-1, AKT and mTOR ( Blume-Jensen and Hunter, Nature. 2001, 411, 355 ).
[0006] It is also known that some other kinases belong to the class of lipid kinases, which are located intracellularly and are, as per the kinases mentioned above, involved in the transmission of biochemical signals, such as those that influence growth and invasion. of tumor cells. Several classes of lipid kinases are known, including said family of PI3 kinases.
[0007] It is now well understood that dysregulation of oncogenes and tumor suppressor genes contributes to the formation of malignant tumors, for example, through increased cell proliferation or increased cell survival. It is also now known that signaling pathways mediated by the PI kinase family play a central role in a number of cellular processes, including proliferation and survival, and dysregulation of these pathways is a causal factor in a wide spectrum of human cancers and other diseases ( Katso et al., Annual Rev. Cell Dev. Biol. 2001, 17: 615-617 and Foster et al., J. Cell Science. 2003, 116: 3037-3040 ).
[0008] The PI3 family of lipid kinases is a group of enzymes that phosphorylate the 3-position of the inositol ring of phosphatidylinositol (PhosphatidylInositol - PI). Three major groups of PI3 kinase enzymes are known, which are classified according to their physiological substrate specificity (Vanhaesebroeck et al., Trends in Biol. Sci. 1997, 22, 267; Engleman et al., Nature Review Genetics, 2006, 7, 607). Class III PI3 kinase enzymes phosphorylate PI only. In contrast, Class II PI3 kinase enzymes phosphorylate both PI and PI4-phosphate [hereinafter abbreviated PI(4)P]. Class I PI3 kinase enzymes phosphorylate PI, PI(4)P and PI4,5-bisphosphate [hereinafter abbreviated PI(4,5)P2], although only PI(4,5)P2 is believed to be the substrate physiological cell. Phosphorylation of PI(4,5)P2 produces the lipid second messenger PI3,4,5-triphosphate [hereinafter abbreviated PI(3,4,5)P3]. More distantly related members of this superfamily are Class IV kinases such as mTOR and DNA-dependent protein kinase that phosphorylate serine/threonine residues within protein substrates. The most studied and understood of these lipid kinases are the Class I PI3 kinase enzymes.
[0009] Class I PI3 kinases are heterodimers consisting of a p110 catalytic subunit and a regulatory subunit, and the family is further divided into Class Ia and Class Ib enzymes based on regulatory partners and regulatory mechanism (Engleman et al. ., Nature Review Genetics, 2006, 7, 607). Class Ia enzymes consist of three distinct catalytic subunits (p110α, p110β and p110δ, by nomenclature define the PI3 kinase isoform as α, β or δ, respectively) that dimerize with five distinct regulatory subunits (p85α, p55α, p50α, p85β and p55Y), with all catalytic subunits being able to interact with all regulatory subunits to form a variety of heterodimers. Class Ia PI3 kinase enzymes are generally activated in response to growth factor stimulation of receptor tyrosine kinases through interaction of the SH2 domains of regulatory subunits with specific phosphotyrosine residues of activated receptor or adapter proteins, such as IRS-1. Both p110α and p110β are widely expressed in all cell types and tissues, whereas p110δ expression is more restricted to some leukocyte and epithelial cell populations. In contrast, the unique Class Ib enzyme consists of a p110Y that interacts with a p101 regulatory subunit. In addition, the Class Ib enzyme is activated in response to G-protein coupled receptor (G-Protein Coupled Receptor - GPCR) systems, as well as by the mechanisms described above.
[00010] There is now considerable evidence indicating that Class Ia PI3 kinase enzymes contribute to tumorigenesis in a wide variety of human cancers, either directly or indirectly (Vivanco and Sawyers, Nature Reviews Cancer, 2002, 2, 489-501) . In particular, the PIK3CA gene, which encodes the p110α catalytic subunit of PI3 kinases, is widely implicated in tumorigenesis. Activation point mutations, most often found in the helical or catalytic domains of p110α, increase the activity of holoenzyme PI3 kinases and can transform cells. They have been reported, particularly, as mutations that occur somatically at significant frequencies across a wide variety of tumor types (Samuels et al., Science. 2004, 304, 554; Samuels et al., Cancer Cell. 2005, 7, 561; Engleman et al., Nature Review Genetics. 2006, 7, 607; Zhao L. and Vogt PK, Oncogene 2008, 27 5486 ). Tumor-related mutations in p85α have also been identified in cancers such as those of the ovary and colon (Philp et al., Cancer Research. 2001, 61, 7426-7429). Furthermore, the p110α subunit is amplified in certain tumors, such as those of the ovary (Shayesteh et al., Nature Genetics. 1999, 21, 99-102) and cervix (Ma et al., Oncogene. 2000, 19, 2739-2744).
[00011] In addition to the direct effects, activation of Class Ia PI3 kinases is believed to contribute to tumorigenic events that occur upstream in signaling pathways, for example, through ligand-dependent or ligand-independent activation of receptor tyrosine kinases, GPCR systems or integrins ( Vara et al., Cancer Treatment Reviews. 2004, 30, 193-204 ). Examples of such upstream signaling pathways include overexpression of the Erb2 receptor tyrosine kinase in a variety of tumors, leading to activation of pathways mediated by PI3 kinases (Harari et al., Oncogene, 2000, 19, 6102-6114) and overexpression of the Ras oncogene ( Kauffmann-Zeh et al., Nature, 1997, 385, 544-548 ). Furthermore, Class Ia PI3 kinases may contribute to tumorigenesis provoked by several downstream signaling events. For example, the loss of the effect of the tumor suppressor phosphatase PTEN that catalyzes the conversion of PI(3,4,5)P3 back to PI(4,5)P2 is associated with a very wide variety of tumors through dysregulation of production. mediated by PI(3,4,5)P3 kinases PI3 ( Simpson and Parsons, Exp. Cell Res., 2001, 264, 29-41 ). Furthermore, increased effects of other signaling events mediated by PI3 kinases are believed to contribute to a variety of cancers, for example through activation of Akt (Nicholson and Anderson, Cellular Signalling, 2002, 14, 381-395) .
[00012] Consequently, the common deregulation of PI3 kinases in conjunction with that of upstream and downstream signaling pathways makes it, collectively, one of the most commonly deregulated pathways in human cancer (Hennessey et al., Nature Reviews Drug Discovery, 2005, 4, 988).
[00013] In addition to a role in mediating signaling, proliferation and survival in tumor cells, there is also good evidence that Class Ia PI3 kinase enzymes will also contribute to tumorigenesis through their function in tumor-associated stromal cells. For example, signaling to PI3 kinases is known to play an important role in mediating angiogenic events in endothelial cells in response to pro-angiogenic factors such as VEGF (Abid et al., Arterioscler. Thromb. Vase. Biol. , 2004, 24, 294-300). Since Class I PI3 kinase enzymes are also involved in motility and migration (Sawyer, Expert Opinion Investigation Drugs, 2004, 1-3, 1-19), inhibitors of PI3 kinases would provide therapeutic benefits by inhibiting the invasion of tumor cells and metastases.
[00014] In addition, Class I PI3 kinase enzymes play an important role in regulating immune cells with PI3 kinase activity contributing to pro-tumorigenic effects of inflammatory cells (Coussens and Werb, Nature, 2002, 420, 860-867 ). In fact, Class Ia PI3 kinase enzymes, PI3 δ kinases, are particularly implicated in tumorigenesis in hematologic malignancies such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (Acute Lymphoblastic Leukemia - ALL) and mantle cells (Mantle Cell Lymphoma - MCL). Elevated PI3K signaling (mainly p110δ) is reported in a wide variety of malignant lymphoid cells (Herman et al., Blood 2010, 116, 2078; Ikeda et al., Blood 2010, 116, 1460; Uddin et al., Blood, 2006, 108, 4178; Rudelius et al., Blood; 2006, 108, 1668; Garcia-Martinez. Br J Cancer 2011, 104, 1116; Renne et al., 2007 Leukemia, 2, 780). This has led to the development of PI3δ kinase targeting agents, with promising early clinical results in hematologic malignancies (Castillo et al., Expert Opinion on Investigational Drugs, 2012, 21, 15).
[00015] These results suggest that pharmacological inhibitors of Class I PI3 kinase enzymes may be of therapeutic utility for the treatment of the various forms of cancerous disease comprising solid tumors, such as carcinomas and sarcomas, and the leukemias and lymphoid malignancies.
[00016] Early studies, both preclinical and clinical, that explored the physiological and pathological roles of the PI3 kinase enzyme, largely used agents with limited kinase-inhibition selectivity, extending across the broadest kinase families, across the entire spectrum. the PI3 kinase family or across the entire Class I PI3 kinase family. Consequently, there is a need for more selective pharmaceutical inhibitors of Class I PI3 kinases to provide useful therapeutic agents with the potential to provide an optimized therapeutic margin over initial agents that came into clinical use.
[00017] In general, the compounds of the present invention possess potent inhibitory activity against a subset of the Class I PI3 kinase enzymes, in particular against the α and δ isoforms of Class Ia PI3 kinases, with relative preservation of the isoform Y and particularly β. The compounds are also selective against the broader family of PI3 kinases and the broader kinase. Such compounds possess sufficient potency against Class I PI3 kinase enzymes since they can be used in an amount sufficient to inhibit a subset of Class I PI3 kinase isoforms, particularly inhibit the α and δ isoforms of PI3 kinase enzymes of Class I, while demonstrating little activity against other kinases.
[00018] Understanding dysregulation of PI3 kinase signaling in human cancer and other diseases offers the prospect of targeting a subgroup of patients most likely to benefit from treatment with the agents described in the present patent through a known process as Personalized HealthCare (PHC) or Personalized Medicine. For these agents, patients whose disease depends on elevated or otherwise altered signaling to PI3K-α and/or PI3K-δ may particularly benefit from treatment. It is well known in the art that diagnostics can be used to provide a response predictive biomarker readout. Such diagnoses could measure one or more pathway dysregulation readings such as, but not limited to, mutation in PIK3CA, PTEN or p85 (PIK3R) genes, increase or amplification in PIK3CA gene copy number, overexpression or elevated isoform activity. α and/or δ of PI3K or use of a phosphobiomarker readout within the pathway, such as phospho-RTK or phospho-AKT. Furthermore, measurement of mutation status or activation status of additional genes, such as Kras, a potential marker of resistance in tumors with aberrant or dysregulated PIK3CA or PI3K-α (Engelman et al., Nature Medicine, 2008, 14, pages 1351-1355; Ihle et al., Cancer Research 2009, 69, pages 143-160; Janku et al., Molecular Cancer Therapeutics 2011, 10, pages 558564), could help increase the predictability of a medicine approach Custom. Alternatively, in another targeted but less specific approach, treatment could be focused on disease subsets where dysregulation of relevant PI3K isoforms is known to be more prevalent.
[00019] The compounds described can be used to target disease, either alone or in combination with another pharmaceutical agent or agents. Combining PI3 kinase inhibitors with other therapies may improve efficacy by overcoming resistance mechanisms, either innate or induced in response to the agent against PI3 kinases. There is substantial preclinical data to support such an approach (Courtney et al., J Clin Oncol 2010, 28, 1075; Engleman et al., Nature Review Genetics 2006, 7, 607). One approach is 'intra-pathway' combinations with agents that modulate other axes in the PI3 kinase signaling pathways (eg mTOR, AKT, RTK, another agent against PI3 kinases). A second approach is 'inter-pathway' combinations where inhibition of more than one signaling pathway may be beneficial over inhibition of a single pathway (e.g., combined with MEK inhibitors, Raf inhibitors, Bcl family modulators, RTK inhibitors, or DNA damage signaling modulators, such as PARP inhibitors.) Other approaches include, where the PI3 kinase inhibitor is combined with agents or regimens that are already established in clinical practice, so-called Standard of Care (SoC) approaches or combinations of agents that target mechanisms other than tumor cells, such as tumor stromal cells or through the immune system.
[00020] In addition to tumorigenesis, there is evidence that Class I PI3 kinase enzymes play a role in other diseases (Wymann et al., Trends in Pharmacological Science. 2003, 24, 366376). Both the Class Ia PI3 kinase enzymes, in particular PI3K-δ, and the single Class Ib enzyme (PI3K-Y) have important roles in cells of the immune system (Koyasu, Nature Immunology. 2003, 4, 313-319) and , thus, they are therapeutic targets for inflammatory and allergic indications. Inhibition of PI3 kinases is also, as described above, useful for treating cardiovascular disease through anti-inflammatory effects or by directly affecting cardiac myocytes (Prasad et al., Trends in Cardiovascular Medicine, 2003, 13, 206-212) . Thus, inhibitors of Class I PI3 kinase enzymes may be of great value in the prevention and treatment of a wide variety of diseases in addition to cancer.
[00021] The compounds, i.e. aminopyrazine derivatives, of the invention have been found to possess potent antitumor activity, being useful in inhibiting uncontrolled cell proliferation resulting from a malignant disease. Without intending to suggest that the compounds described in the present invention possess pharmacological activity solely by virtue of an effect on a single biological process, it is believed that the compounds confer an antitumor effect through inhibition of Class I PI3 kinase enzymes, particularly through inhibiting a subset of Class Ia PI3 kinase enzymes, more particularly through inhibition of the α and δ isoforms of PI3K.
[00022] The compounds of the present invention may also be useful in inhibiting uncontrolled cell proliferation arising from various non-malignant diseases, such as inflammatory diseases (e.g. rheumatoid arthritis and inflammatory bowel disease), fibrotic diseases (e.g. , liver cirrhosis and pulmonary fibrosis), glomerulonephritis, multiple sclerosis, psoriasis, benign prostatic hypertrophy (BPH), skin hypersensitivity reactions, blood vessel disorders (eg, atherosclerosis and restenosis), asthma allergic, insulin-dependent diabetes, diabetic retinopathy and diabetic nephropathy.
[00023] Proline amides have been described as selective agents for PI3K-α by Novartis in International Patent Applications WO2009/080705, WO2010/029082 and WO2011/000905. ATR kinase inhibitors containing aminopyrazine have been described in WO2011/143426 and WO2010/071837 (Vertex).
[00024] According to one aspect of the invention there is provided a compound of Formula (I):

[00025] wherein: R1 is methyl or ethyl; and R2 is (C2-3)alkyl substituted by hydroxyl; or a pharmaceutically acceptable salt thereof.
[00026] In another aspect of the invention, there is provided a compound of Formula (I) as defined above.
[00027] It will be understood that the term "hydroxy-substituted (C2-3)alkyl" includes both straight and branched chain groups, for example those illustrated as groups (i) to (xi) below:


[00028] It should be understood that insofar as some of the compounds of Formula (I) defined above may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the invention includes, in its definition, any one of such optically active or racemic forms which have PI3K-α and -δ inhibitory activity. The synthesis of optically active forms can be carried out by current methods of organic chemistry well known in the art, for example, through synthesis from optically active starting materials or decomposition of a racemic form. Similarly, the aforementioned activity can be evaluated using conventional laboratory techniques.
[00029] A particular enantiomer of a compound described herein may be more active than other enantiomers of the same compound.
[00030] According to a further aspect of the invention, there is provided a compound of Formula (I), or a pharmaceutically acceptable salt thereof, which is a single enantiomer being in an enantiomeric excess (% ee) of > 95, > 98% or >99%. Conveniently, the single enantiomer is present in an enantiomeric excess (%ee) of >99%.
[00031] According to a further aspect of the invention, there is provided a pharmaceutical composition which comprises a compound of Formula (I) which is a single enantiomer being in an enantiomeric excess (%ee) of >95, >98% or > 99%, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable diluent or carrier. Conveniently, the single enantiomer is present in an enantiomeric excess (%ee) of >99%.
[00032] Some compounds of Formula (I) may be crystalline and may have more than one crystalline form. It should be understood that the present invention encompasses any crystalline or amorphous form, or mixtures thereof, forms which have properties useful in inhibiting PI3K-α and -δ activity, it being well known in the art how to determine the effectiveness of a crystalline form. or amorphous for the inhibition of PI3K-α and/or -δ activity by means of the conventional assays described below.
[00033] It is generally known that crystalline materials can be analyzed using conventional techniques, such as X-Ray Powder Diffraction analysis (hereinafter XRPD - X-Ray Powder Diffraction), Differential Scanning Calorimetry (hereinafter DSC - Differential Scanning Calorimetry), Thermal Gravimetric Analysis (hereinafter TGA - ThermoGravimetric Analysis), Diffuse Reflectance Infrared Fourier Transform spectroscopy (DRIFT - Diffuse Reflectance Infrared Fourier Transform), near infrared spectroscopy (Near InfraRed - NIR ), solution and/or solid state nuclear magnetic resonance spectroscopy. The water content of such crystalline materials can be determined by means of Karl Fischer analysis.
[00034] As an example, the compound of Example 1 exhibits crystallinity and a crystalline form has been identified.
[00035] Accordingly, another aspect of the invention is 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin) Form A - 2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one.
[00036] In accordance with another aspect of the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4) -oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 5.1°.
[00037] In accordance with another aspect of the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4) -oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 18.0°.
[00038] In accordance with another aspect of the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4) -oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has an X-ray powder diffraction pattern with at least two specific peaks at about 2-theta = 5.1 and 18.0°.
[00039] According to another aspect of the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3, 4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has an X-ray powder diffraction pattern with specific peaks at about 2-theta = 5.1, 18.0, 10.2, 11.7, 19.4, 18.5, 14.8, 26, 7, 26.6, 17.8°.
[00040] In accordance with the present invention, there is provided a crystalline form, Form A, which has an X-ray powder diffraction pattern substantially equal to the X-ray powder diffraction pattern shown in Figure 1.
[00041] In accordance with another aspect of the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4) -oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta =5.1° plus or minus 0.2° 2-theta.
[00042] In accordance with another aspect of the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4) -oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 18.0° plus or minus 0.2° 2-theta.
[00043] In accordance with another aspect of the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4) -oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has an X-ray powder diffraction pattern with at least two specific peaks at about 2-theta = 5.1 and 18.0° plus or minus 0.2° 2-theta.
[00044] According to another aspect of the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4- oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one, which has a X-ray powder diffraction pattern with specific peaks at about 2-theta = 5.1, 18.0, 10.2, 11.7, 19.4, 18.5, 14.8, 26, 7, 26.6, 17.8° plus or minus 0.2° 2-theta.
[00045] Example 3 is also crystalline and three forms (A, B and C) are described here.
[00046] In accordance with the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at about 2-theta = 4.8°.
[00047] In accordance with the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at about 2-theta = 10.0°.
[00048] In accordance with the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least two specific peaks at about 2-theta = 4.8° and 10.0°.
[00049] In accordance with the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with specific peaks at about 2-theta = 4.8, 10.0, 14.6, 5.2, 19.9, 10.4, 25.4, 23.6, 24.4, 16.2°.
[00050] In accordance with the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole- 2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern X-ray powder substantially equal to the X-ray powder diffraction pattern shown in Figure 3.
[00051] In accordance with the present invention, there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole- 2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern X-ray powder with at least one specific peak at 2-theta = 4.8° plus or minus 0.2° 2-theta.
[00052] In accordance with the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at 2-theta = 10.0° plus or minus 0.2° 2-theta.
[00053] In accordance with the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least two specific peaks at 2-theta = 4.8° and 10.0°, wherein said values may be plus or minus 0.2° 2-theta.
[00054] In accordance with the present invention there is provided a crystalline form, Form A, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with specific peaks at 2-theta = 4.8, 10.0, 14.6, 5.2, 19.9, 10.4, 25.4, 23.6, 24.4, 16, 2°, wherein said values may be plus or minus 0.2° 2-theta.
[00055] In accordance with the present invention there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at about 2-theta = 5.8°.
[00056] In accordance with the present invention there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at about 2-theta = 10.9°.
[00057] In accordance with the present invention there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least two specific peaks at about 2-theta = 5.8° and 10.9°.
[00058] In accordance with the present invention there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with specific peaks at about 2-theta = 5.8, 10.9, 11.5, 25.9, 17.3, 24.0, 19.1, 12.9, 24.7, 27.2°.
[00059] In accordance with the present invention, there is provided a crystalline form, Form B of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder substantially equal to the X-ray powder diffraction pattern shown in Figure 5.
[00060] In accordance with the present invention, there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole- 2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern X-ray powder with at least one specific peak at 2-theta =5.8° plus or minus 0.2° 2-theta.
[00061] In accordance with the present invention there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at 2-theta = 10.9° plus or minus at 0.2° 2-theta.
[00062] In accordance with the present invention there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder having at least two specific peaks at 2-theta = 5.8° and 10.9°, wherein said values may be plus or minus 0.2° 2-theta.
[00063] In accordance with the present invention there is provided a crystalline form, Form B, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with specific peaks at 2-theta = 5.8, 10.9, 11.5, 25.9, 17.3, 24.0, 19.1, 12.9, 24.7, 27, 2°, wherein said values may be more or less than 0.2° 2-theta.
[00064] In accordance with the present invention there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at about 2-theta = 6.9°.
[00065] In accordance with the present invention there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at about 2-theta = 12.3°.
[00066] In accordance with the present invention there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least two specific peaks at about 2-theta = 6.9° and 12.3°.
[00067] In accordance with the present invention there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with specific peaks at about 2-theta = 6.9, 12.3, 10.5, 21.0, 24.6, 13.6, 16.4, 19.6, 20.2, 22.5°.
[00068] In accordance with the present invention, there is provided a crystalline form, Form C of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder substantially equal to the X-ray powder diffraction pattern shown in Figure 7.
[00069] In accordance with the present invention, there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole- 2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern X-ray powder with at least one specific peak at 2-theta = 6.9° plus or minus 0.2° 2-theta.
[00070] In accordance with the present invention there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with at least one specific peak at 2-theta = 12.3° plus or minus 0.2° 2-theta.
[00071] In accordance with the present invention there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder having at least two specific peaks at 2-theta = 6.9° and 12.3°, wherein said values may be plus or minus 0.2° 2-theta.
[00072] In accordance with the present invention there is provided a crystalline form, Form C, of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one which has a diffraction pattern of X-ray powder with specific peaks at 2-theta = 6.9, 12.3, 10.5, 21.0, 24.6, 13.6, 16.4, 19.6, 20.2, 22, 5°, wherein said values may be plus or minus 0.2° 2-theta.
[00073] When the present invention is stated to relate to a crystalline form of a compound of the invention, such as Example 1 or Example 3, the degree of crystallinity is conveniently greater than about 60%, more conveniently greater than about 80%, preferably greater than about 90%, and most preferably greater than about 95%. Even more preferably, the degree of crystallinity is greater than about 98%.
[00074] When the present invention is stated to relate to a crystalline form of a compound of the invention, such as in Example 1 or Example 3, the crystalline form is preferably substantially free of other crystalline forms or amorphous forms of the same compost. In this context, "substantially free" conveniently means more than about 60%, more conveniently more than about 80%, preferably more than about 90%, more preferably more than about 95%, even more preferably more than about 98% and more preferably more than about 99% of a single pure crystalline form. For example, Example 3 may be in the form of Form A and substantially free of Forms B and C; alternatively, Example 3 may be in the form of Form B and substantially free of Forms A and C; alternatively, Example 3 may be in the form of Form C and substantially free of Forms A and B. Similarly, Example 3 may be in the form of Form B and substantially free of alternative amorphous or crystalline forms.
[00075] It should be understood that the 2-theta values of X-ray powder diffraction patterns may vary slightly from one apparatus to another or from one sample to another and thus the quoted values should not be interpreted as absolute.
[00076] It is known that an X-ray powder diffraction pattern can be obtained which has one or more measurement errors, depending on the measurement conditions (such as equipment or machine used). In particular, it is generally known that intensities in an X-ray powder diffraction pattern can fluctuate depending on measurement conditions. Accordingly, it should be understood that the crystalline forms of the present invention described above, unless otherwise indicated, are not limited to crystals that provide X-ray powder diffraction patterns identical to the X-ray powder diffraction patterns shown in Figures 1, 3 and 5 and any crystals that provide X-ray powder diffraction patterns substantially similar to those shown in these Figures are within the scope of the present invention. Those skilled in the technique of X-ray powder diffraction will be able to appreciate the substantial identity of X-ray powder diffraction patterns.
[00077] Those versed in the X-ray powder diffraction technique will also realize that the relative intensity of the peaks can be affected, for example, by grains over 30 microns in size and non-unitary axes relationships, which may affect the analysis of samples. Those skilled in the art will also appreciate that the position of reflections can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer. The flatness of the sample surface can also have a small effect. Consequently, the diffraction pattern data presented are not to be understood as absolute values (see Jenkins, R. & Snyder, RL, "Introduction to X-Ray Diffractometry", John Wiley & Sons, 1996; Bunn, CW (1948) , Chemical Crystallography, Clarendon Press, London; Klug, HP & Alexander, LE (1974), X-Ray Diffraction Procedures).
[00078] In general, a measurement error of a diffraction angle in an X-ray powder diffractogram is approximately plus or minus 0.2° 2-theta and such a degree of measurement error must be taken into account when X-ray powder diffraction data is considered. Furthermore, it should be understood that intensities may fluctuate depending on experimental conditions and sample preparation (preferred orientation).
[00079] Particular compounds of the invention are each of the Examples, each of which constitutes another independent aspect of the present invention. Other specific compounds of the invention are pharmaceutically acceptable salt(s) of each of the Examples, each of which constitutes another independent aspect of the present invention.
[00080] According to a further aspect of the invention there is provided a compound of Formula (I) which is obtainable by any of the Examples as described herein.
[00081] Another characteristic is any of the scopes defined herein, provided that specific Examples, such as Examples 1, 3, 4, etc., are individually claimed.
[00082] It will be appreciated by those skilled in the art that certain compounds of Formula (I) contain asymmetrically substituted carbon atoms and, consequently, can exist and be isolated in optically active and racemic forms. Some compounds of Formula (I) may exhibit polymorphism. It should be understood that the present invention encompasses any racemic, optically active, polymorphic or stereoisomeric forms, or mixtures thereof, forms which have properties useful in inhibiting the activity of PI3K-α and -δ, being well known in the art as preparing optically active forms (e.g. by decomposing the racemic form via recrystallization techniques, via synthesis from optically active starting materials, via chiral synthesis, via enzymatic decomposition, via biotransformation or via of chromatographic separation using a chiral stationary phase) and how to determine the effectiveness of inhibiting PI3K-α and -δ activity by means of conventional assays described below.
[00083] It should be understood that certain compounds of Formula (I) defined above may exhibit the phenomenon of tautomerism. It should be understood that the present invention includes, in its definition, any tautomeric forms, or a mixture thereof, which has PI3K inhibitory activity and should not be limited to just any of the tautomeric forms used in the formula drawings or indicated in the Examples. . In general, only one such tautomeric form is indicated in the Examples which follow or is shown in any drawings of the relevant formulas which follow.
[00084] The present invention is intended to include all isotopes of atoms occurring in the present compounds. Isotopes should be understood to include atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Carbon isotopes include
[00085] A suitable pharmaceutically acceptable salt of a compound of Formula (I) is, for example, an acid addition salt of a compound of Formula (I), for example, an acid addition salt with an acid strong inorganic or organic such as hydrochloric acid, hydrobromic acid, sulfuric acid or trifluoroacetic acid. Another suitable pharmaceutically acceptable salt of a compound of Formula (I) is, for example, a salt formed within the body of a human or animal after administration of a compound of Formula (I).
[00086] It will be further understood that a suitable pharmaceutically acceptable solvate of a compound of Formula (I) also forms an aspect of the present invention. A suitable pharmaceutically acceptable solvate is, for example, a hydrate, such as a hemihydrate, a monohydrate, a dihydrate, or a trihydrate, or an alternative amount thereof.
[00087] It should further be understood that a suitable pharmaceutically acceptable prodrug of a compound of Formula (I) also forms an aspect of the present invention. Accordingly, compounds of the invention can be administered in the form of a prodrug, which is a compound that is broken down in the body of a human or animal to release a compound of the invention. A prodrug can be used to alter the physical properties and/or the pharmacokinetic properties of a compound of the invention. A prodrug can be formed when the compound of the invention contains a suitable group or substituent to which a property modifying group can be attached. Examples of prodrugs include in vivo cleavable ester derivatives that can be formed at a hydroxy group in a compound of Formula (I) in vivo cleavable amide derivatives that can be formed at an amino group in a compound of Formula (I) .
[00088] Accordingly, the present invention includes those compounds of Formula (I) as defined hereinbefore when made available through organic synthesis and when made available within the body of a human or animal by cleavage of a prodrug thereof. Accordingly, the present invention includes those compounds of Formula (I) which are produced by synthetic organic means and also those compounds which are produced in the body of a human or animal through metabolism of a precursor compound, which is a compound of Formula (I) which may be a synthetically produced compound or a metabolically produced compound.
[00089] A suitable pharmaceutically acceptable prodrug of a compound of Formula (I) is one that is based on reasonable medical judgment as being suitable for administration to the body of a human or animal without undesirable pharmacological activities and without undue toxicity.
[00090] Various forms of prodrug have been described, for example, in the following documents:
[00091] Methods in Enzymology. Vol. 42, pages 309-396, edited by K. Widder et al. (Academic Press, 1985);
[00092] Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);
[00093] A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5, "Design and Application of Pro-drugs", by H. Bundgaard pages 113-191 (1991);
[00094] H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);
[00095] H. Bundgaard et al., Journal of Pharmaceutical Sciences, 77, 285 (1988);
[00096] N. Kakeya et al., Chem. Pharm. Bull, 32, 692 (1984);
[00097] T. Higuchi and V. Stella, "Pro-Drugs as Novel Delivery Systems", A.C.S. Symposium Series, Volume 14; and
[00098] E. Roche (editor), "Bioreversible Carriers in Drug Design", Pergamon Press, 1987.
[00099] A suitable pharmaceutically acceptable prodrug of a compound of Formula (I) having a hydroxy group is, for example, an in vivo cleavable ester or ether thereof. An in vivo cleavable ester or ether of a compound of Formula (I) containing a hydroxy group is, for example, a pharmaceutically acceptable ester or ether that is cleaved in the body of a human or animal to produce the precursor hydroxy compound. Suitable pharmaceutically acceptable ester-forming groups for a hydroxy group include inorganic esters such as phosphate esters (including the cyclic phosphoramidic esters). In addition, suitable pharmaceutically acceptable ester forming groups of a hydroxy group include (1-10C) alkanoyl groups, such as acetyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups, (1-10C) alkoxycarbonyl groups, such as as ethoxycarbonyl, N,N-[di-(1-4C)alkyl]carbamoyl, 2-dialkylaminoacetyl and 2-carboxyacetyl groups. Examples of ring substituents on phenylacetyl and benzoyl groups include aminomethyl, N-aminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazin-1-ylmethyl and 4-(1-4C)alkylpiperazin-1-ylmethyl. Suitable pharmaceutically acceptable ether forming groups for a hydroxy group include α-acyloxyalkyl groups, such as acetoxymethyl and pivaloyloxymethyl groups.
[000100] A suitable pharmaceutically acceptable prodrug of a compound of Formula (I) having an amino group is, for example, an in vivo cleavable amide derivative. Suitable pharmaceutically acceptable amides of an amino group include, for example, an amide formed with (1-10C) alkanoyl groups such as acetyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups. Examples of ring substituents on phenylacetyl and benzoyl groups include aminomethyl, N-alkylaminomethyl, N,N-dialkylaminomethyl, morpholinomethyl, piperazin-1-ylmethyl and 4-(1-4C)-ylmethyl alkylpiperazin-1.
[000101] The in vivo effects of a compound of Formula (I) may be exerted, in part, by one or more metabolites that are formed within the body of a human or animal after administration of a compound of Formula (I) . As stated hereinabove, the in vivo effects of a compound of Formula (I) can also be exerted through metabolism of a parent compound (a prodrug).
[000102] The compounds of Formula (I) contain a -C(O)R2-substituted piperidine subunit, wherein R2 is hydroxy-substituted (C2-3)alkyl. A potential pathway of metabolism of these compounds is by the oxidation of the hydroxyl substituent on this group. These oxidized compounds generally retain some PI3K-α and -δ inhibitory activity.
[000103] Therefore, in accordance with a further aspect of the invention, there is provided a compound of Formula (A):

[000104] wherein: R1A is methyl or ethyl; and R2A is (C1-2) alkyl substituted by carboxyl;
[000105] or a pharmaceutically acceptable salt thereof.
[000106] Examples of compounds of Formula (A) include Example 8, which is an identified metabolite of Example 1:
Example 8
[000107] and Example 9, which is an identified metabolite of Example 3:
Example 9
[000108] Other potential metabolites of Example 3 are two alternative oxidation products, shown below and described in Examples 10 and 11:
Example 10 Example 11
[000109] Suitable pharmaceutically acceptable salts of compounds of Formula (A) include, for example, an alkali metal or alkaline earth salt, such as a calcium or magnesium salt or an ammonium salt, or a salt with an organic base, such as methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
[000110] For the avoidance of doubt, it should be understood that where, in the present specification, a group is qualified by "hereafter defined" or "defined here before", said group encompasses the first and broadest definition that occurs , as well as each and every particular setting for this group.
[000111] Particular novel compounds of the invention include, for example, the compounds of Formula (I) or pharmaceutically acceptable salts thereof, wherein, unless otherwise stated, each of R1 and R2 has any of the defined meanings. here before or the following statements: R1 is methyl. R1 is ethyl. R2 is any one of groups (i) to (xi) as defined hereinbefore. R2 is groups (i) to (vi) as defined hereinabove. R2 is group (i).
[000112] A particular group of compounds of the invention are compounds of Formula (I) above wherein: R1 is methyl or ethyl, R2 is a group (i):

[000113] or a pharmaceutically acceptable salt thereof.
[000114] Particular compounds of the invention are, for example, the compounds of Formula (I) which are described in the examples which are given below.
[000115] For example, a particular compound of the invention is a compound of Formula (I) selected from any of the following: 1-[4-[5-[5-amino-6-(5-tert-butyl-1, 3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one (Examples 1 and 2);
[000116] 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1, 2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one (Example 3); (3R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1 ,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one (Example 4); (3S)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1 ,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one (Example 5); (2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1 ,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-2-methyl-propan-1-one (Example 6); 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4 -triazol-3-yl]-1-piperidyl]-2-hydroxy-2-methyl-propan-1-one (Example 7).
[000117] Another aspect of the present invention provides a process for preparing a compound of Formula (I) or a pharmaceutically acceptable salt thereof. A suitable process is illustrated by the following representative process variants in which, unless otherwise indicated, R1 , R2 have any of the meanings defined hereinbefore. The necessary starting materials can be obtained through current organic chemistry procedures. The preparation of such starting materials is described in conjunction with representative process variants below and within the associated Examples. Alternatively, necessary starting materials are obtained by procedures analogous to those illustrated which are within the normal competence of an organic chemist.
[000118] Suitable process variants include, for example, the following:
[000119] reaction, conveniently in the presence of a suitable activating reagent, of a compound of Formula II:

[000120] wherein R1 has any of the meanings defined hereinbefore, with the carboxylic acid R2-COOH, except that any functional group is protected if necessary, in the presence of a suitable base, whereupon any protecting group that is present is removed. Suitable coupling agents for this reaction include, for example, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, TBTU (2-(1H-benzo[d) tetrafluoroborate ][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the presence of 2-hydroxy-N-oxide pyridine.
[000121] The reaction is conveniently carried out in the presence of a suitable base. A suitable base is, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methylmorpholine, diazabicyclo[5.4.0]undec-7 -ene, diisopropylethylamine, or, for example, an alkali metal or alkaline earth carbonate or hydroxide, for example, sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide; preferably N-ethyl-N,N-diisopropylamine.
[000122] The reaction is conveniently carried out in the presence of a suitable inert solvent such as, for example, acetonitrile, N,N-dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane , benzene, toluene, xylene, methanol, ethanol, halogenated solvents such as dichloromethane, chloroform or carbon tetrachloride, and at a temperature in the range, for example, from -50°C to 100°C, preferably in the range of 0°C °C to 30 °C.
[000123] Alternatively, the carboxylic acid R2-COOH can be transformed into an activated species which can then be reacted with a compound of Formula II under conditions well known in the art.
[000124] A suitable protecting group for the hydroxyl group is the tetrahydropyran protecting group, as described in Examples 2 and 3. Suitable conditions for removing this group include mild acidic conditions in the presence of an alcohol as a solvent in a temperature between 20 to 70°C, such as methanol or ethanol. A typical mild acid used is pyridine p-toluenesulfonate.
[000125] A compound of Formula II can be obtained from the reaction of the compound of Formula III:

[000126] where P is a protecting group, such as tert-butoxycarbonyl,
[000127] with a compound of Formula R1-L, wherein L is a suitable leaving group such as, for example, a halogen group, such as a bromo group, iodo (conveniently iodine), in the presence of a suitable base, after which any protection groups that are present are removed.
[000128] A suitable base is, for example, an organic amine base, such as 1,8-diazabicyclo[5.4.0]undec-7-ene.
[000129] The reaction is conveniently carried out in the presence of a suitable inert solvent such as, for example, 2-methyltetrahydrofuran, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene, xylene, and at a temperature in the range, for example, from -50°C to 60°C, preferably in the range from -10°C to 0°C.
[000130] Suitable conditions for deprotection of tert-butoxycarbonyl include acidic conditions such as trifluoroacetic acid in an inert solvent such as dichloromethane at approximately room temperature (20-25°C).
[000131] Compound III can be obtained from a coupling reaction, in the presence of a suitable activating reagent, of the compound of Formula IV:

[000132] with a compound of Formula V:

[000133] preferably in the presence of a suitable base, followed by a cyclization reaction in the presence of a mild acid.
[000134] The coupling reaction can be carried out in the presence of a suitable coupling agent such as, for example, O-(7-azabenzotriazol-1-yl)-N,N,N',N'- hexafluorophosphate tetramethyluronium or TBTU (2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate).
[000135] The coupling reaction is conveniently carried out in the presence of a suitable base. A suitable base is, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methylmorpholine, diazabicyclo[5.4.0]undec-7-ene , diisopropylethylamine or, for example, an alkali metal or alkaline earth metal carbonate or hydroxide, for example sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide; preferably N-ethyl-N,N-diisopropylamine.
[000136] The coupling reaction is conveniently carried out in the presence of a suitable inert solvent such as, for example, N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, 1,4-dioxane , 1,2-dimethoxy ethane, benzene, toluene, xylene, methanol, ethanol, halogenated solvents such as dichloromethane, chloroform or carbon tetrachloride, and at a temperature in the range, for example, from -50°C to 100°C °C, preferably in the range of 0 °C to 30 °C.
[000137] The cyclization conditions are carried out in the presence of a mild acid, typically acetic acid. The reaction is conveniently carried out in the presence of a suitable inert solvent such as, for example, N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, tetrahydrofuran, 1,4-dioxane, 1,2- dimethoxy ethane, benzene, toluene, xylene, at a temperature in the range, for example, from 50°C to 150°C, preferably in the range from 80°C to 100°C.
[000138] Compound IV can be obtained from a reaction of the compound of Formula VI with hydrazine.

[000139] This reaction is conveniently carried out in the presence of a suitable inert solvent such as, for example, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene, xylene, or a hol, such as ethanol or isopropanol, at a temperature in the range, for example, from 20°C to 70°C, preferably about 50°C.
[000140] Compound VI can be obtained from a metal-catalyzed reaction of the compound of Formula VII with a cyanide source, such as zinc(II) dicyanide.

[000141] A suitable catalyst for the reaction includes, for example, a metal catalyst such as palladium (0), for example tetrakis(triphenylphosphine) palladium (0); or a catalyst formed in situ from a palladium (II) salt, e.g. palladium (II) acetate, palladium (II) chloride, palladium (II) bromide, bis(triphenylphosphine)palladium (II) chloride , [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) or tris(dibenzylideneacetone)dipalladium chloride, and a phosphine ligand, e.g. dicyclohexyl-(2',4',6' -triisopropylbiphenyl-2-yl)phosphine. The reaction is conveniently carried out in a suitable solvent such as N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene or xylene , and at a temperature in the range, for example, from 20°C to 150°C, preferably in the range from 60°C to 120°C. The reaction is also conveniently carried out in the presence of additional metal, such as zinc.
[000142] Suitable reactions of this type are described in "Metal-Catalyzed Cross-Coupling Reactions", Second Edition, Edited by Armin Meijere, Francois Diederich, Wiley-VCH, 2004).
[000143] Syntheses of Compound VII were described in Examples 1 and 2.
[000144] Alternatively, a compound of Formula II can be obtained via the metal-catalyzed reaction of compound VIII where R is a small alkyl, and compound IX, where P is a protecting group, such as tert-butoxycarbonyl:

[000145] A suitable catalyst for the reaction includes, for example, a metal catalyst such as palladium (0), for example tetrakis(triphenylphosphine) palladium (0); or a catalyst formed in situ from a palladium (II) salt, e.g. palladium (II) acetate, palladium (II) chloride, palladium (II) bromide, bis(triphenylphosphine)palladium (II) chloride , [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) or tris(dibenzylideneacetone)dipalladium chloride, and a phosphine ligand, e.g. dicyclohexyl-(2',4',6' -triisopropylbiphenyl-2-yl)phosphine.
[000146] The reaction is conveniently carried out in a suitable solvent such as N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene or xylene, or an alcohol, such as 4-methyl-2-pentanol, at a temperature in the range, for example, from 50°C to 180°C, preferably in the range from 120°C to 150°C.
[000147] The reaction is also conveniently carried out in the presence of additional salt, such as lithium chloride.
[000148] Suitable reactions of this type are described in "Metal-Catalyzed Cross-Coupling Reactions", Second Edition, Edited by Armin Meijere, Francois Diederich, Wiley-VCH, 2004).
[000149] A compound of Formula VIII can be obtained from the metal-catalyzed reaction of compound VII with a suitable hexaalkyl diesthane. A suitable catalyst for the reaction includes, for example, a metal catalyst, such as palladium (0), for example tetrakis(triphenylphosphine) palladium (0); or a catalyst formed in situ from a palladium (II) salt catalyst, e.g. palladium (II) acetate, palladium (II) chloride, palladium (II) bromide, bis chloride (triphenylphosphine)palladium (II), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II) chloride or tris(dibenzylideneacetone)dipalladium, and a phosphine ligand, e.g. dicyclohexyl(2') ,4',6'-tri-2-yl)phosphine. The reaction is conveniently carried out in a suitable solvent, such as N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, benzene, toluene, xylene or an alcohol. , such as 4-methyl-2-pentanol, at a temperature in the range, for example, from 50°C to 100°C, preferably in the range from 70°C to 80°C.
[000150] The compound of Formula IX can be obtained from commercially available material in a few steps as illustrated in Example 1 (with R1 = Me and P = tert-butoxycarbonyl).
[000151] It should be understood that other exchanges of the process steps in the process variants described above are also possible.
[000152] It will be understood that any compound of Formula (I) obtained by any of the processes described above may be converted to another compound of Formula (I), if necessary.
[000153] When a pharmaceutically acceptable salt of a compound of Formula (I) is required, for example an acid addition salt, it can be obtained, for example, by reacting said compound with a suitable acid. .
[000154] When a pharmaceutically acceptable prodrug of a compound of Formula (I) is required, it can be obtained using a conventional procedure. For example, an in vivo cleavable ester of a compound of Formula (I) can be obtained, for example, by reacting a compound of Formula (I) containing a hydroxy group with a pharmaceutically acceptable carboxylic acid. More information on prodrugs was provided earlier.
[000155] It will also be appreciated that, in some of the reactions mentioned hereinabove, it may be necessary or desirable to protect any sensitive groups on the compounds. Cases where protection is necessary or desirable, and methods suitable for protection, are known to those skilled in the art. Conventional protecting groups may be used in accordance with conventional practice (for illustration see T.W. Green, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991). Thus, if the reactants include groups such as amino, carboxy or hydroxy, it may be desirable to protect the group in some of the reactions mentioned herein.
[000156] A suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or/butoxycarbonyl group , an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The unprotection conditions for the above protection groups necessarily vary with the choice of protection group. Thus, for example, an acyl group, such as an alkanoyl or alkoxycarbonyl group, or an aroyl group may be removed, for example, by means of hydrolysis with a suitable base, such as an alkali metal hydroxide, for example, lithium or sodium hydroxide. Alternatively, an acyl group, such as a t-butoxycarbonyl group, may be removed, for example, by treatment with a suitable acid, such as hydrochloric acid, sulfuric acid, or phosphoric acid or trifluoroacetic acid, and a arylmethoxycarbonyl group, such as a benzyloxycarbonyl group, may be removed, for example, by hydrogenation over a catalyst, such as palladium-on-carbon, or by treatment with a Lewis acid, for example, tris(trifluoroacetate). ) of boron. A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group, which can be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
[000157] A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group, such as acetyl, an aroyl group, for example, benzoyl, or an aryl-methyl group, for example , benzyl. The unprotection conditions for the above protection groups will necessarily vary with the choice of protection group. Thus, for example, an acyl group, such as an alkanoyl group or an aroyl group, can be removed, for example, by means of hydrolysis with a suitable base, such as an alkali metal hydroxide, for example lithium hydroxide or sodium. Alternatively, an arylmethyl group, such as a benzyl group, can be removed, for example, by hydrogenation over a catalyst, such as palladium-on-carbon.
[000158] A suitable protecting group for a carboxyl group is, for example, an esterifying group, for example a methyl group or an ethyl group, which can be removed, for example, by means of hydrolysis with a base, such as sodium hydroxide or, for example, a t-butyl group, which can be removed, for example, by treatment with an acid, for example an organic acid, such as trifluoroacetic acid or, for example, a benzyl group, which can be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
[000159] Protecting groups may be removed at any convenient step in the synthesis using conventional methods well known in the chemical art.
[000160] Some of the intermediates (e.g. compounds of Formulas II, III, IV, VI, VII, VIII) defined herein are new and these are provided as an additional feature of the invention. Biological Assays
[000161] The following assays were used to measure the effects of compounds of the present invention as a) inhibitors of PI3 kinase enzymes in biochemical assays, as b) inhibitors of other kinases in biochemical assays, c) as inhibitors in vitro of phospho AKT (Thr308) in BT474 cells, d) as in vitro inhibitors of phospho AKT (Ser473) in MDA-MB-468 cells, e) as in vitro inhibitors of phospho AKT (Ser473) in Jeko cells, f) as in vitro inhibitors of phospho Chk1 (Ser345) in HT29 cells, g) as inhibitors of cell proliferation across a panel of tumor cell lines, h & i) as in vivo inhibitors of phospho AKT (Ser473) or in vivo inhibitors of growth of tumor, respectively, in SCID mice transplanted with the human breast adenocarcinoma cell line, MCF7.
[000162] Abbreviations used in assay protocols: PIP2: PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate sc: subcutaneous ATP: adenosine triphosphate DMSO: dimethyl sulfoxide TRIS: tris(hydroxymethyl)aminomethane CHAPS: 3 -[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate TDT: Dithiothreitol FBS: Fetal bovine serum DMEM: Dulbecco's Modified Eagle's Medium EDTA: ethylenediaminetetraacetic acid EGTA: ethylene glycol tetraacetic acid BSA: bovine serum albumin PBS: buffered saline with Phosphate HRP: Horseradish Peroxidase RPMI: Roswell Park Memorial Institute Medium 1640 4NQO: 4-Nitroquinoline N-oxide EMEM: Eagle's Minimum Essential Medium CO2: Carbon Dioxide PBST: Phosphate Buffered Saline/Tween Ab: Antibody MTS Reagent : [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS. In vitro enzyme inhibition assay
[000163] Inhibition of PI3K-β, PI3K-α, PI3K—Y and PI3K-δ was evaluated in a Kinase Glo-based enzyme activity assay using recombinant human enzymes. The assay platform indirectly measured ATP depletion after incubation with enzyme, PIP2 substrate, ATP and compound.
[000164] After completion of the enzymatic reaction, the remaining ATP was used in a secondary enzymatic reaction, where luciferase converted beetle luciferin to oxyluciferin under light emission. There was a direct relationship between the measured luminescence and the ATP remaining in a completed kinase reaction. Therefore, luminescence was inversely related to kinase activity. Typically, twelve different concentrations of compound were tested and raw data from inhibition of PI3K-β, PI3K-α, PI3K-Y or PI3K-δ were plotted as a function of inhibitor concentration. Method Details
[000165] Compounds in 100% DMSO were added to test plates by means of acoustic distribution. PI3K enzyme was added in a Tris buffer (50 mM Tris, pH 7.4, 0.05% CHAPS, 1 MM DTT and 10 mM magnesium chloride) and allowed to pre-incubate with the compound. for 5 minutes before adding substrate solution containing PIP2 and ATP. The enzyme reaction was stopped after 80 minutes by adding a Kinase Glo detection solution containing Luciferrin and luciferase (from the Kinase Glo(R) Plus Luminecent Kinase Assay kit (Promega #V3772). The plates were left for 30 minutes at room temperature, then read a Pherastar Instrument with a conventional luminescence filter pad.The final concentration of DMSO, ATP, and PIP2 in the assay was 1%, 8 μM, and 80 μM, respectively. Data
[000166] IC50 values were calculated using a log fit curve for a nonlinear regression fit. The IC50 value was the concentration of test compound that inhibited 50% of the enzyme activity. (b) Evaluation of kinase selectivity in addition to class 1 PDK kinase enzymes
[000167] Large kinase assay panels are offered by a variety of commercial vendors such as Millipore, Invitrogen and ProQinase. Such panels allow an assessment of the overall kinase selectivity of a given compound. The precise methods/technologies will vary depending on the supplier.
[000168] Selectivity data for some of the compounds described here were generated using enzymatic assays performed at the MRC - Division of Signal Transduction Therapy (DSTT), MRC Protein Phosphorylation Unit, Dundee, UK. Protein kinase assays were performed using a radiochemical format. Assays were performed in 384-well Multidrop plates at room temperature in a total assay volume of 25.5 μl. The compounds were pre-incubated in the presence of the enzyme and the peptide/protein substrate for 5 minutes before the start of the reaction by adding 10 μl of ATP (final concentration selected for each kinase at 5, 20 or 50 μM). Assays were performed at room temperature before quenching by adding 5 μl of phosphoric acid. The assay plate contents were then collected onto Whatman-P81-Unifilter plates by a Packard Harvester (wash buffer was 50 mM orthophosphoric acid) and air dried. The dried Unifilter plates were then sealed upon addition of MicroScint O and counted in Packard Topcount NXT scintillation counters. This protocol captures the generic format suitable for most kinases in the panel, but modifications to the protocols were necessary for a small number of kinases, as will be familiar to those skilled in the art.
[000169] Lipid kinase assays for ~18 lipid kinases were also performed in DSTT. All lipid kinase assays were performed in 384-well plates at room temperature in a total assay volume of 40 μl. The assay was performed according to the protocols provided with the ADP-GLO assay (Promega, #V9101). This protocol captures the generic format suitable for most kinases in the panel, but modifications to the protocols were necessary for a small number of kinases, as will be familiar to those skilled in the art.
[000170] Kinase selectivity was also evaluated using the KINOMEscan™ screening platform available from DiscoverX. It employs an active site-directed competitive binding assay to quantitatively measure interactions between test compounds and over 450 human kinases and disease-relevant mutant variants. KINOMEscan™ assays do not require ATP and therefore report true thermodynamic interaction affinities, as opposed to IC50 values, which may depend on ATP concentration. The methodology is based on compounds that bind to the active site of the kinase and directly (sterically) or indirectly (allosterically) prevent the kinase from binding to the immobilized ligand, thereby reducing the amount of kinase captured on a solid support. On the other hand, test molecules that do not bind kinase have no effect on the amount of kinase captured on a solid support. Screening hits are identified by measuring the amount of kinase captured in test versus control samples using a quantitative qPCR method that detects the associated DNA marker. In a similar manner, dissociation constants (Kds) for test compound-kinase interactions are calculated by measuring the amount of kinase captured on the solid support as a function of test compound concentration. (c) Protocol for assay that measures AKT (Tyr308) phosphorylated in BT474 cells
[000171] This assay was used to measure PI3K-α inhibition in cells. BT474 cells (human ductal breast carcinoma, ATCC HTB-20) were grown in 384-well black plates (Costar, #3712) at a density of 5600 cells/well in DMEM containing 10% FBS and 1% glutamine and left join overnight.
[000172] The next morning, compounds in 100% DMSO were added to the test plates by means of acoustic distribution. After a two hour incubation at 37°C and 5% CO 2 , the medium was aspirated and the cells were lysed with a buffer containing 25 mM Tris, 3 mM EDTA, 3 mM EGTA, 10% sodium fluoride. 50 mM, 2 mM sodium orthovanadate, 0.27 M sucrose, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate, 0.5% Triton X-100 and complete protease inhibitor cocktail tablets (Roche #04 693 116 001, used 1 tablet per 50 ml of lysis buffer).
[000173] After 20 minutes, cell lysates were transferred to ELISA plates (Greiner # 781077) which had been pre-coated with a total anti-AKT antibody in PBS buffer and non-specific binding was blocked with BSA at 1 % in PBS containing 0.05% Tween 20. The plates were incubated overnight at 4°C. The next day, the plates were washed with PBS buffer containing 0.05% Tween 20 and further incubated with a T308 mouse antiphospho AKT monoclonal antibody for two hours. The plates were washed again as above prior to the addition of a horse anti-mouse HRP conjugated secondary antibody. After a two hour incubation at room temperature, the plates were washed and QuantaBlu working substrate solution (Thermo Scientific #15169, prepared according to the supplier's instructions) was added to each well. The developed fluorescent product was stopped after 60 minutes by adding stop solution to the wells. Plates were read using a Tecan Safire plate reader using excitation at 325 nm and emission wavelengths at 420 nm, respectively. Unless otherwise specified, reagents contained in Cell Signaling's Path Scan Phospho AKT (Thr308) Sandwich ELISA Kit (#7144) were used in this ELISA assay. (d) Protocol for detection of phospho AKT (Ser473) in MDA-MB-468 cells as a measure for inhibition of PDK-beta kinase
[000174] This assay was used to measure PI3K-β inhibition in cells and was used, in conjunction with assay (c) above, to determine alpha versus beta selectivity in cells. MDA-MB-468 cells (human breast adenocarcinoma #ATCC HTB 132) were cultured at 1500 cells/well in 40 µl of DMEM containing 10% FBS and 1% glutamine in Greiner 384-well black flat-bottomed plates. Plates with cells were incubated for 18 hours in a 37°C incubator prior to dosing with compound s in 100% DMSO using acoustic distribution.
[000175] Compounds were dosed over a concentration range of 12 points on a randomized plaque map. Control wells were generated by dosing 100% DMSO (maximum signal) or addition of a reference compound (a PI3K-β inhibitor) that completely eliminated the pAKT signal (minimum control). The plates were incubated at 37°C for two hours, the cells were then fixed by adding 10 µl of a 3.7% formaldehyde solution. After 30 minutes, the plates were washed with PBS using a Tecan PW384 plate washer. The wells were blocked and cells permeabilized with the addition of 40 µl of PBS containing 0.5% Tween 20 and 1% Marvel™ (dry powdered milk) and incubated for 60 minutes at room temperature. The plates were washed with PBS containing 0.5% (v/v) Tween 20 and 20 µl of rabbit antiphospho AKT Ser473 antibody (Cell Signaling Technologies, #3787) in the same PBS-Tween + 1% Marvel™ added and incubated overnight at 4°C.
[000176] Plates were washed 3 times with PBS + 0.05% Tween 20 using a Tecan PW384. 20 µl of Alexa Fluor 488 anti-rabbit secondary antibody (Molecular Probes, #A11008) diluted in PBS + 0.05% Tween 20 containing 1% Marvel™ was added to each well and incubated for 1 hour at room temperature. The plates were washed three times as before, then 20 µl of PBS was added to each well and the plates sealed with a black plate sealer.
[000177] Plates were read on an Acumen plate reader as quickly as possible, measuring green fluorescence after laser excitation at 488 nm. Using this system, IC50 values were generated and the quality of the plates was determined using control wells. Reference compounds were run each time to monitor assay performance. (e) Protocol for detection of phospho AKT (Ser473) in Jeko cells
[000178] This assay was used to measure PI3K-δ inhibition in cells. Compounds at a final concentration of 10X in 10 µl of 1% DMSO (v/v) were added to the wells of a 96-well Greiner V-bottom plate (Sigma #M9686). Compounds were dosed at a concentration range of 10 points from the maximum dose of 1 μM or 10 μM, 8 compounds were dosed on a plate. There were eight maximum signal control wells per plate dosed with anti-IgM antibody (AffiniPure F(ab')2 Fragment Goat Anti-Human IgM (Stratech, #109-006-129)) and vehicle and 8 control wells signal levels dosed with anti-IgM antibody and a reference PI3K-δ inhibitor. The final vehicle concentration was 0.01% DMSO. A complete dose-response curve for a PI3K-δ-selective compound was included in each run. Jeko B cells (human mantle cell lymphoma, ATCC #CRL-3006) were cultured in 96-well V-bottom Greiner plates containing compounds. Cells were cultured at 100,000 cells/well in 70 µl of RPMI containing 1% glutamine.
[000179] Plates with cells were incubated with compound for 1 hour in a 37°C incubator. After this pre-incubation time with compound, the anti-IgM antibody described above was added to the plates at a final concentration of X5 in 20 μl of assay buffer (RPMI medium containing 1% glutamine). The final concentration of anti-IgM was 0.06 μg/ml or an equivalent EC90 dose. The plates were incubated at 37°C for 10 min, then the plates were immediately placed on ice and centrifuged at 12000 rpm for 4 min. On ice, the supernatants were carefully removed with a hand pipette and 40 µl of lysis buffer added. Plates were incubated on ice for 5 min and stored at -80°C until assayed in the Phosphor (Ser473)/Total Aki Whole Cell Lysate kit according to the manufacturer's instructions (Mesoscale Di-agnostics, #K11100D-3). (f) Protocol for detection of phospho Chk1 (Ser 345) in HT29 cells
[000180] ATR (Ataxia + Rad3 Telangiectasia Related Kinase) is a PI3 kinase related kinase which phosphorylates multiple serine or threonine substrates in response to DNA damage or replication blockage. Chk1, a protein kinase downstream of ATR, plays a key role in DNA damage checkpoint control. Chk1 activation involves the phosphorylation of Ser317 and Ser345 (the latter considered to be the preferred target for ATR phosphorylation/activation).
[000181] This is a cell-based assay to measure ATR kinase inhibition by measuring a decrease in Chk1 (Ser 345) phosphorylation in HT29 cells after treatment with compound and the UV mimetic 4NQO (Sigma #N8141) . HT29 cells (ECACC #85061109) were grown in 384-well assay plates (Costar #3712) at a density of 6000 cells/well in 40 µl EMEM medium containing 1% L-glutamine and 10% FBS and allowed to adhere. during the night. The following morning, compounds in 100% DMSO were added to the assay plates via acoustic distribution. After one hour of incubation at 37°C and 5% CO2, 40 μl of 4NQO at 3 mM in 100% DMSO were added to all wells by means of acoustic distribution, except for the minimum signal control wells. , which were left untreated with 4NQO to generate a null-response control. The plates were developed in the incubator for an additional 1 hour. Then, cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubated for 20 minutes at room temperature. Then, 20 μl of 0.1% Triton X100 in PBS was added and incubated for 10 minutes at room temperature to permeabilize the cells. Then, the plates were washed once with 50 µL/well of PBS using a Biotek EL405 plate washer.
[000182] Phospho-Chk1 Ser 345 antibody (Cell Signalling Technology #2348) was diluted 150-fold in PBS containing 0.05% polysorbate/Tween and 15 µl was added to each well and incubated overnight at room temperature. The following morning, plates were washed three times with 50 µl/well PBS using a Biotek EL405 plate washer and then 20 µl secondary Ab solution containing diluted goat anti-rabbit IgG antibody Alexa Fluor 488 500 fold (Molecular Probes #A-11008) and 0.002 mg/ml of Hoechst dye (Molecular Probes #H-3570) in PBST were added. After two hours of incubation at room temperature, the plates were washed three times with 50 µl/well of PBS using a Biotek EL405 plate washer and the plates were sealed with black plate sealers until read. Plates were read using an ArrayScan VTI instrument using an XF53 filter with a 10X objective. A set of two lasers was used to analyze nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pChk1 (488 nm). (g) Cell proliferation assays on tumor cell lines (used to demonstrate a personalized medicine hypothesis)
[000183] The sensitivity of a panel of human cancer cell lines to the effects of the compounds was determined in a standard proliferation assay. Cell lines were purchased from the AstraZeneca Cell Bank. Most cell lines are also available through Cell Bank Repositories known to those working in the art, for example ATCC, ECACC, DMSZ, RIKEN, KCLB, JCRB (HSRRB), LBNL, CLS and ICLC.
[000184] Cells were plated in 96-well plates at densities of 1000-6000 cells per well in RPMI medium containing 10% FBS. After incubation at 37°C for 16 hours, various concentrations of compound were added to the assay plates. After incubation for an additional 72 hours, viable cells were determined by adding MTS reagent (Promega #3582) to each well for two hours. MTS is a tetrazolium salt that is bioreduced by metabolically active cells in the presence of an electron-coupling reagent in formazan. The formazan product was then quantified by absorbance at 490 nm as an indicator of the relative number of live cells. In order to determine the GI50 (concentration at which cell growth was inhibited by 50%), the relative number of cells present at the time of drug addition was determined by comparing it to the MTS reading before the drug was added and this value was subtracted from the 72 hour value of untreated cells as a measure of cell growth during the assay.
[000185] Analysis of these data, described below in "Examples of Personalized Medicine/Personalized Healthcare", illustrates how these data can be analyzed to reveal that PDKα inhibitors exhibit selective growth inhibition of cell lines with mutation in the PIK3CA gene. This illustrates an opportunity for Personalized HealthCare (PHC) or Personalized Medicine where a biomarker readout for response prediction could be used to identify patients with tumors that contain mutations in the PIK3CA gene and who are most likely , would respond to the compounds described herein.
[000186] Other potential markers of response for the compounds described herein include, but are not limited to, an increase in copy number, amplification or translocation of the PIK3CA gene, and other genetic, genomic, or proteomic alterations which provide a measure of activation or dependence. the PI33 kinase pathway; for example, but not limited to, activation or mutation of one or more receptor tyrosine kinases or translocation in the PIK3R genes which encode the regulatory subunits (p85) of PI3 kinases or phosphorylation of downstream signaling markers such as pAKT, PS6 or FOXO status. Furthermore, analysis of other genes and/or the signaling of their protein products, eg Kras, can help improve the predictability of a Personalized Medicine approach. (h) Protocol for detection of phospho AKT (Ser473) from MCF-7 tumors grown in male SCID mice
[000187] This was a pharmacodynamic assay that provides a measure of PI3K-α inhibition in an animal model. Male SCID mice (AZ UK, also available from Charles River, UK) were transplanted subcutaneously (sc) with the MCF7 human breast adenocarcinoma cell line (ICRF London, also available from ATCC #HTB-22)) to determine inhibition of AKT phosphorylation with PI3 kinase inhibitors. Mice were implanted with a 0.5 mg 21-day estrogen pellet (Innovative Research of America, #E121) 24 hours prior to cell implantation. 5X 10 6 cells in 50% Matrigel (BD Bioscience) were injected into the left flank of the animals. The animals were randomly divided into groups of 8 controls and 4 treatments when the tumors reached a volume of 400 mm3 and dosing began the next day. Tumors were removed at selected time points, when blood samples were also collected for PK measurements.
[000188] Tumors excised from mice were placed in a rapid prep tube (2 ml ribbed tubes containing Lysis Matrix A, MP Biomedicals #6910-500) and immediately frozen. 1 ml lysis buffer (25 mM Tris, 3 mM EDTA, 3 mM EGTA, 50 mM sodium fluoride, 2 mM orthovanadate, 0.27 M sucrose, 10 mM beta-glycerophosphate, sodium pyrophosphate 5 mM Triton X-100 0.5%) plus phosphatase inhibitors (Sigma #P2850 and Sigma # P5726, diluted 1:100) and protease inhibitors (Sigma #P8340, diluted 1:200) were added. to each tube. Tumors were homogenized for 1 minute on a FastPrep-TM equipment (MP Biomedicals #116004500) and then left on ice for 5 minutes, followed by two additional homogenization steps, each followed by a 5 minute incubation on ice. The samples were centrifuged for 10 minutes at 13,000 rpm in a refrigerated centrifuge. The clarified lysates were then placed in fresh tubes and 10 μl used for a protein determination assay.
[000189] Detection of total and phosphorylated AKT (Ser473) was performed using an MSD Multi-spot assay kit (Meso Scale Discovery #K1510OD-3). Each well of the plate contained 4 spots; two of these were coated with mouse monoclonal antibodies supplied with the kit; one was coated with a capture antibody to total AKT and one was coated with an antibody to phosphorylated AKT (Ser473). The plates were blocked overnight in an ice chamber on a shaker with 150 μl of blocking solution per well, which was made using 20 ml of 1x wash solution plus 600 mg of Blocker A provided with the kit. Plates were washed three times with 0.3 ml per well of wash solution. An aliquot of the lysate was taken from each tumor and diluted to a concentration of 2 mg/mL with lysis buffer, 25 µl of diluted lysate was then added to each well, providing a total amount of 50 µg per well. The plates were placed on a shaker at room temperature for one hour before the plates were washed three times. A detection antibody solution was prepared using a mixture of blocking solution and washing solution plus a 1 in 50 dilution of 50x total anti-AKT antibody SULFO-TAG-TM antibody. The plates were placed on a shaker at room temperature for one hour before the plates were washed three times. 150 μl of reading buffer provided with the kit was diluted 1:4 with deionized water and added to each well and then the plate was read on the MSD plate analyzer. The reading buffer provides the correct chemical environment for electrochemiluminescence so that when the plate reader applies voltage to the plate, the electrodes on the base of the plate cause the marker attached to the detection antibody to emit light. Emitted light intensity is a quantitative measure of the amount of AKT, total or phosphorylated, that is present. To calculate the ratio of phosphorylated to total AKT, a calculation as suggested by the Meso Scale was applied: twice the phosphorylated signal divided by the total plus phosphorylated signal, then multiplied by 100 to give the % phosphoprotein. The values were converted to Log 10 and then these values were used to calculate the GEOMETRIC AVERAGE for each group plus the standard error. A Student's t-test was then applied using a doubly configured tail and unequal variance formula to verify significance. Studies have shown that a control group of 8 animals with 4 per treatment group was sufficient to carry out the study. Protocol for detection of tumor growth inhibition in MCF7 human breast adenocarcinoma cell lineage transplanted into SCID mice
[000190] This method allows evaluation of the antitumor efficacy of PI3 kinase inhibitors in vivo in a PI3-dependent model. As for the PD studies noted above, male SCID mice were transplanted s.c. with the human breast adenocarcinoma cell line, MCF7. Mice were implanted with a 0.5 mg 21-day estrogen pellet 24 hours prior to cell implantation. 5X 10 6 cells in 50% Matrigel were injected s.c. into the left flank of the animals. Animals were randomly divided into groups of 10-15 when tumors reached a volume of ~200-300 mm3 and treatment initiated. Animals were dosed for 2-4 weeks perorally, intravenously or intraperitoneally with the compound in a vehicle suitable for administration via the necessary route consistent with welfare requirements (suspension for oral dosing is in the range of pH 4-7, solution for ip/iv dosing in the pH range of 5.5-7.0) and in defined doses. Tumors were measured generally twice a week with a caliper and the tumor volume calculated using the elliptical formula (pi/6 x width x width x length).
[000191] While the pharmacological properties of compounds of Formula (I) vary with structural change as expected, in general, the activity possessed by compounds of Formula (I) can be demonstrated at the following concentrations or doses in one or more of the assays (a) and (c) above: Test (a): - IC50 against PI3K-α in the range, for example, from 1 nM - 100 nM; Test (c): - IC50 against cellular phospho AKT (Tyr308) in BT474 cells in the range, eg, 10 nM - 1 μM.
[000192] Conveniently, particular compounds of the present invention have activity at the following concentrations or doses in one or more of tests (a) and (c) above: Test (a): - IC50 against PI3K-α in the range, for example from 1 nM - 100 nM; Test (c): - IC50 against cellular phospho AKT (Tyr308) in BT474 cells in the range, eg, 10 nM - 1 μM.
[000193] Conveniently, particular compounds of the present invention have activity at the following concentrations or doses in one or more of tests (a), (c), (h) and (i) above: Test (a): - IC50 against PI3K-α in the range, for example, from 1 nM - 100 nM; Test (c): - IC50 against cellular phospho AKT (Tyr308) in BT474 cells in the range, for example, 10 nM - 1 μM; Test (h): - >50% in vivo inhibition of phospho AKT (Ser473) in the range, for example, 1-200 mg/kg/day; Test (i): - xenograft activity in the range, for example, 1-200 mg/kg/day.
[000194] The following data was generated for the Examples: Table A

[000195] Test protocol a: these are the average values calculated from a given number of test repetitions. ** Test protocol c: these are the average values calculated from a given number of test repetitions. # Test protocol f: one test replicate performed only. Combination Studies Materials and Methods
[000196] MCF7 is an estrogen receptor positive breast tumor cell line that carries a mutation in the PIKC3CA gene (E545K). Male SCID mice (AZ UK) were transplanted subcutaneously (s.c.) with an MCF7 human breast adenocarcinoma cell line (ICRF London) to determine the antitumor activity of PI3 kinase inhibitors. Mice were implanted with 0.5 mg of a 21-day-old estrogen pellet (Innovative Research of America) 24 hours before cell implantation. 5X 10 6 cells in 50% Matrigel (BD Bioscience) were injected s.c. into the left flank of the animals.
[000197] BT474 is an estrogen receptor positive breast tumor cell line with high expression of Her2 and carries a mutation in the PIK3CA gene (K111NM). Female athymic nude Swiss mice (swiss nu/nu - AZUK) were transplanted subcutaneously with the human epithelial breast ductal carcinone cell line BT474c (AZ-derived from BT474 - ATCC HTB-20). Mice were implanted with 0.36 mg of 60-day estrogen pellets (Innovative Research of America) 24 hours prior to cell implantation. 5X 10 6 cells in 50% Matrigel (BD Bioscience) were injected s.c. into the left flank of the animals.
[000198] HCC70 is a breast tumor cell line which is deficient in the expression of the PTEN gene. Female athymic nude Swiss mice (nu/nu Swiss - AZUK) were transplanted subcutaneously with the HCC70 epithelial ductal breast tumor cell line (ATCC - CRL2315). 1X 10 6 cells in 50% Matrigel (BD Bioscience) were injected s.c. into the left flank of the animals.
[000199] The animals were randomly divided into groups of 10-15 when the tumors reached a volume of ~200-300 mm3 and treatment started. Animals were dosed for 3-4 weeks perorally with the compound in a suitable vehicle at defined doses and times. Tumors were measured two - three times a week with a caliper and the tumor volume calculated using the elliptical formula (pi/6 x width x width x length).
[000200] When administered alone, AZD5363 was formulated in 10% DMSO, 25% Kleptose solution. (Kleptose is supplied by Roquette-Pharma (Trademark) - hydroxypropyl beta-cyclodextrin - suitable for in vivo use and formulations).
[000201] When co-administered with Example 3, AZD5363 was formulated in HPMC/Tween (0.5% Methocel (hydroxypropyl methyl cellulose)/0.1% Polysorbate 80). The suspension was milled overnight.
[000202] Example 3 was formulated in HPMC/Tween (0.5% Methocel (hydroxypropyl methyl cellulose)/0.1% Polysorbate 80).
[000203] AZD8186 was formulated in HPMC/Tween (0.5% Methocel (hydroxy propyl methyl cellulose)/0.1% Polysorbate 80).
[000204] When co-administered with Example 3, AZD8186 was formulated in HPMC/Tween (0.5% Methocel (hydroxy propyl methyl cellulose)/0.1% Polysorbate 80). The suspension was milled overnight.
[000205] Olaparib was formulated in 10% DMSO/30% Kleptose solution. Inhibition of Tumor Growth by Example 3 in Combination with AKT Inhibitor (AZD5363) - Sequential Administration
[000206] Studies were performed on the BT474 xenograft model. Example 3 and AZD5363 were dosed twice daily (BID) 6-8 hours apart in a sequential 2-day treatment/5-day interruption weekly cycle, so that AZD5363 was dosed on days 1 and 2 of weekly cycle and Example 3 was dosed on days 3 and 4 of the weekly cycle. Example 3 was dosed at doses of 50 mg/kg BID and AZD5363 was dosed at doses of 170 mg/kg BID in HPMC/Tween and DMSO/Kleptose, respectively.
[000207] The tumor growth curve (as shown in Figure 9) indicates that intermittent dosing of Example 3 or AZD5363 partially inhibited tumor growth relative to vehicle-only control (HPMC/Tween). The combination of Example 3 plus AZD5363 induced tumor regression. Inhibition of Tumor Growth by Example 3 in Combination with AKT Inhibitor (AZD5363) - Co-administration
[000208] Studies were performed on the BT474 xenograft model. Example 3 and AZD5363 were dosed twice daily (BID) 6-8 hours apart and concomitantly on a weekly cycle of 2 days treatment/5 days off. Example 3 was dosed at doses of 25 mg/kg BID and AZD5363 was dosed at doses of 100 mg/kg BID, both in HPMC/Tween.
[000209] The tumor growth curve (shown in Figure 10) indicates that intermittent dosing of Example 3 or AZD5363 partially inhibited tumor growth relative to vehicle-only control (HPMC/Tween). The combination of Example 3 plus AZD5363 induced tumor regression during the dosing period, although followed by tumor regrowth during the dosing period. Inhibition of Tumor Growth by Example 3 in Combination with PARP Inhibitor (Olaparib)
[000210] Studies were performed on the BT474 xenograft model. Example 3 and Olaparib were dosed every day throughout the study, Example 3 twice daily (BID) 6-8 hours at 25 mg/kg each dose and Olaparib once daily (QD) at 100 mg/kg 1 hour after the first daily dose of Example 3. Both agents were administered in HPMC/Tween.
[000211] The tumor growth curve (Figure 11) indicates that olaparib alone had no significant effect on tumor growth, Example 3 only partially inhibited the growth, but the combination of Example 3 plus olaparib induced tumor regression. mor Inhibition of Tumor Growth by Example 3 in Combination With PARP Inhibitor (Olaparib)
[000212] Studies were performed on the MCF7 xenograft model. Example 3 and Olaparib were dosed every day throughout the study, twice daily, Example 3 6-8 hours at 25 mg/kg each dose and Olaparib once daily (QD) at 100 mg/kg 1 hour post-dose. first daily dose of Example 3. Both agents were administered in HPMC/Tween.
[000213] The tumor growth curve (Figure 12) indicates that olaparib only had a minimal effect on tumor growth, Example 3 only caused some tumor regression, but the combination of Example 3 plus olaparib induced a regression stronger than the tumour. Inhibition of Tumor Growth by Example 3 in Combination With PI3Kbeta/delta Inhibitor (AZD8186)
[000214] Studies were performed on the HCC70 xenograft model. Example 3 and AZD8186 were dosed every day, twice daily (bid), throughout the study, Example 3 at 25 mg/kg each dose and AZD8186 at 50 mg/kg each dose. Both agents were administered in HPMC/Tween. The tumor growth curve (Figure 13) indicates that AZD8186 partially inhibited tumor growth, Example 3 only inhibited tumor growth more strongly, but the combination of Example 3 plus AZD8186 induced tumor growth. tumor progression.
[000215] According to a further aspect of the invention, there is provided a pharmaceutical composition which comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in association with a pharmaceutically acceptable diluent or carrier. acceptable.
[000216] Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents, granulating and disintegrating agents, binding agents, lubricating agents, preservatives and antioxidants. Tablet formulations may be uncoated or coated, either to modify their disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract, or to improve their stability and/or appearance, in any case using conventional coating agents and procedures well known in the art.
[000217] The compositions for oral use may alternatively be in the form of hard gelatine capsules, in which the active ingredient is mixed with an inert solid diluent, or as soft gelatine capsules, in which the active ingredient is mixed with water. or an oil.
[000218] Aqueous suspensions generally contain the active ingredient in a finely powdered form together with one or more suspending agents, dispersing agents or wetting agents. Aqueous suspensions may also contain one or more preservatives, antioxidants, coloring agents, flavoring agents and/or sweetening agents.
[000219] Oil suspensions can be formulated by suspending the active ingredient in a vegetable oil or a mineral oil. Oil suspensions may also contain a thickening agent. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant.
[000220] Dispersible powders and granules suitable for preparing an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Additional excipients, such as sweetening, flavoring and coloring agents, may also be present.
[000221] The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or a mixture of either. Emulsions may also contain sweetening, flavoring and preservative agents.
[000222] Syrups and elixirs may be formulated with sweetening agents and may also contain a demulcent, preservative, flavoring and/or coloring agent.
[000223] The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the suitable dispersants or wetting agents and suspending agents that have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
[000224] Compositions for administration by inhalation may be in the form of a conventional pressurized aerosol prepared to dispense the active ingredient as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants, such as volatile fluorinated hydrocarbons, can be used and the aerosol device is conveniently arranged to deliver a metered amount of active ingredient.
[000225] For more information on formulation the reader should refer to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of the Editorial Board), Pergamon Press 1990.
[000226] The amount of active ingredient that is combined with one or more excipients to produce an individual dosage form will necessarily vary depending on the host treated and the particular route of administration. For example, oral administration to humans will generally require, for example, 1 mg to 2 g of active agent (more suitably 100 mg to 2 g, e.g. 250 mg to 1.8 g, such as 500 mg to 1.8 g, particularly from 500 mg to 1.5 g, conveniently from 500 mg to 1 g), to be administered in admixture with an appropriate and convenient amount of excipients which may vary from about 3 to about 98 percent by weight of the total composition. It will be understood that if a large dosage is required, several dosage forms may be required, for example two or more tablets or capsules, with the dose of active ingredient conveniently divided between them. Conveniently, a single solid dosage form may contain between 1 and 300 mg of active ingredient.
[000227] The dose size for therapeutic or prophylactic purposes of a compound of Formula (I) will naturally vary according to the nature and severity of the pathological state, the age and sex of the animal or patient and the route of administration, in accordance with with well-known principles of medicine.
[000228] When using a compound of Formula (I) for therapeutic or prophylactic purposes, it will generally be administered so that a daily dose in the range, for example, from 1 mg/kg to 5 100 mg/kg of body weight- ral, is received, if necessary administered in divided doses. In general, smaller doses will be administered when a parenteral route is used. Thus, for example, for intravenous administration, a dose in the range, for example, from 1 mg/kg to 25 mg/kg of body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, from 1 mg/kg to 25 mg/kg of body weight will be used. Oral administration, however, is preferred, particularly in tablet form. Typically, unit dosage forms will contain about 10mg to 0.5g of a compound of the present invention.
[000229] The compounds of the invention can be administered daily or more than once a day. Compounds of the invention may also be administered at a suitable dosing schedule, for example, compounds of the invention may be administered one or more times per day (e.g., once, twice, or three times per day) for a period of time. certain number of days, followed by a period of days when no dose is given. This dosing cycle (consisting of dosing days and days off dosing) can then be repeated. Conveniently, a dosing cycle is a period of 5-14 days, such as 5, 7, 10 or 14 days, more conveniently 7 days. In one aspect, compounds of formula (I) are dosed for one day or two or three consecutive days, followed by 3, 4, 5 or 6 days without any doses in a dosing cycle.
[000230] In one aspect, the compound of formula (I) is administered for 1 day, followed by no dose for 2, 3 or 4 days.
[000231] In another aspect, the compound of formula (I) is administered for 2 days, followed by no dose for 4, 5 or 6 days.
[000232] In another aspect, the compound of formula (I) is administered for 3 days, followed by no dose for 3, 4 or 5 days.
[000233] In another aspect, the compound of formula (I) is administered for 4 days, followed by no dose for 2, 3 or 4 days.
[000234] In another aspect, the compound of formula (I) is administered for 5 days, followed by no dose for 1, 2 or 3 days.
[000235] In another aspect, the compound of formula (I) is administered every other day.
[000236] The above dosing schedules are conveniently applied when the compounds of the invention are used as monotherapy. Other examples of possible dosing schedules for administering the compounds of the invention, such as combination therapy, are described below.
[000237] As stated above, PI3K-α and —δ enzymes are known to contribute to tumorigenesis by one or more of the effects of mediating cancer and other cell proliferation, mediating angiogenic events, and mediating motility, migration, and invasion of cancer cells. The compounds of the present invention have been found to possess potent antitumor activity, which is believed to be achieved through inhibition of PI3K-α and -δ enzymes that are involved in the signal transduction steps leading to tumor cell proliferation and survival. and the invasiveness and migratory capacity of metastatic tumor cells.
[000238] Accordingly, the compounds of the present invention are of value as antitumor agents, in particular as selective inhibitors of proliferation, survival, motility, diffusion and invasiveness of mammalian cancer cells that lead to inhibition of tumor growth and survival and inhibition of metastatic tumor growth. Particularly, the compounds of the present invention are of value as anti-proliferative and anti-invasive agents in the containment and/or treatment of solid tumor diseases. Particularly, the compounds of the present invention are expected to be useful in the prevention or treatment of those tumors which are sensitive to the inhibition of PI3K-α and/or -δ enzymes and which are involved in the signal transduction steps leading to proliferation and survival of tumor cells and the migratory capacity and invasiveness of metastatic tumor cells. Furthermore, the compounds of the present invention are expected to be useful in the prevention or treatment of those tumors which are mediated solely or in part by the inhibition of PI3K-α and/or -δ enzymes, i.e. the compounds can be used to produce a PI3K-α and/or -δ enzyme inhibitory effect in a warm-blooded animal in need of such treatment.
[000239] According to a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use as a medicament in a warm-blooded animal, such as The man.
[000240] In accordance with another aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in producing an antiproliferative effect in an animal of hot-blooded, just like man.
[000241] In accordance with a further feature of this aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in a warm-blooded animal, such as the man, as an anti-invasive agent in the containment and/or treatment of solid tumor diseases.
[000242] According to another aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for producing an antiproliferative effect in a warm-blooded animal, just like man.
[000243] According to a further feature of this aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the production of a antiproliferative effect on a warm-blooded animal such as man.
[000244] According to another feature of this aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in a blood vessel. animal, such as man, as an anti-invasive agent in the containment and/or treatment of solid tumor diseases.
[000245] According to another feature of this aspect of the invention, there is provided a method for producing an antiproliferative effect in a warm-blooded animal, such as man, in need of such treatment comprising administering to said animal, an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000246] According to another feature of this aspect of the invention, there is provided a method for producing an anti-invasive effect for containing and/or treating solid tumor disease in a warm-blooded animal such as man. , which necessitates such treatment comprising administering to said animal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000247] In accordance with another aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the prevention or treatment of cancer in an animal of hot-blooded, just like man.
[000248] According to a further aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the preparation of a medicament for use. in the prevention or treatment of cancer in a warm-blooded animal such as man.
[000249] According to another feature of this aspect of the invention, there is provided a method for preventing or treating cancer in a warm-blooded animal, such as man, in need of such treatment comprising administering to said animal, of an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000250] According to another aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the prevention or treatment of solid tumor diseases in a warm-blooded animal, just like man.
[000251] According to a further aspect of the invention there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in prevention or treatment of solid tumor disease in a warm-blooded animal such as man.
[000252] According to another feature of this aspect of the invention, there is provided a method for preventing or treating solid tumor diseases in a warm-blooded animal, such as man, in need of such treatment comprising administering to said animal , an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000253] In accordance with a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the prevention or treatment of those tumors which are sensitive to inhibition of PI3K-α and/or -δ enzymes that are involved in signal transduction steps that lead to proliferation, survival, invasiveness and migratory capacity of tumor cells.
[000254] According to a further feature of this aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the prevention or treatment those tumors that are sensitive to the inhibition of PI3K-α and/or -δ enzymes that are involved in signal transduction steps that lead to proliferation, survival, invasiveness and migratory capacity of tumor cells.
[000255] In accordance with another feature of this aspect of the invention, there is provided a method for preventing or treating tumors that are sensitive to inhibition of PI3K-α and/or -δ enzymes that are involved in signal transduction steps that lead to to the proliferation, survival, invasiveness and migratory ability of tumor cells which comprises administering to said animal an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000256] According to a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in providing an inhibitory effect on PI3K- α and -δ.
[000257] In accordance with a further feature of this aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the preparation of a medicament for use in providing a inhibitory effect of PI3K-α and -δ enzymes.
[000258] According to a further aspect of the invention, there is also provided a method for providing an inhibitory effect on PI3K-α and/or -δ enzymes comprising administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000259] As stated hereinabove, certain compounds of the present invention have substantially better potency against PI3K-α and -δ enzymes than against other PI3 kinase enzymes or other kinases. Such compounds possess sufficient potency against the PI3K-α and -δ enzymes that they can be used in sufficient amounts to inhibit the PI3K-α and -δ enzymes, while demonstrating little activity against the PI3K-β enzyme and against most other kinase enzymes. Such compounds are likely to be useful for selective inhibition of PI3K-α and -δ enzymes and are likely to be useful for the effective treatment, for example, of tumors activated by PI3K-α and/or -δ enzymes.
[000260] In accordance with this aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in providing a selective inhibitory effect on PI3K-α and -δ enzymes. .
[000261] In accordance with a further feature of this aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in providing a selective inhibitory effect of PI3K-α and -δ enzymes.
[000262] According to a further aspect of the invention, there is also provided a method for providing a selective inhibitory effect on PI3K-α and -δ enzymes which comprises administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000263] By "a selective inhibitory effect on PI3K-α and -δ enzymes" is meant that the compound of Formula (I) is more potent against PI3K-α and -δ enzymes than against other class 1 PI3 kinases and, in general, exhibit good selectivity towards other members of the PI3 kinase family and broader classes of kinase enzymes comprising tyrosine and ser/thr kinases.
[000264] In accordance with a further feature of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the treatment of breast cancer, stomach (gastric ) and esophageal cancer, non-small cell lung cancer (NSCLC), including squamous cell carcinoma (SCC) and adenocarcinoma, head and neck squamous cell carcinoma (H&N), gynecological cancers (including endometrial, ovarian and cervical cancers) and hematological cancers such as multiple myeloma, lymphomas and leukemias (including chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (Acute Lymphoblastic Leukaemia) - ALL) and mantle cell lymphoma (Mantle Cell Lymphoma - MCL).
[000265] In accordance with a further feature of this aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the treatment of bladder, brain/CNS cancer. , colorectal, lung (all other forms), gallbladder and bile ducts, and skin.
[000266] In accordance with a further feature of this aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, for use in the treatment of prostate, bone, kidney cancer. , liver, mela noma, gastrointestinal, pancreas, testes, thyroid, penis, vulva, and other types of tumors with a dependence on PI3 kinases through mutation, amplification, or other aberrations.
[000267] In accordance with a further feature of this aspect of the invention, there is provided a method for treating breast, stomach (gastric) and esophageal cancer, NSCLC, including SCC and adenocarcinoma, H&N SCC, gynecological cancers (including endometrium, ovary and cervix) and hematological cancers such as multiple myeloma, lymphomas and leukemias (including CLL, ALL and MCL) in a warm-blooded animal such as man who needs such treatment comprising administration of an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000268] In accordance with another feature of this aspect of the invention, there is provided a method for treating bladder, brain/CNS, colorectal, lung (all other forms), gallbladder and bile ducts and skin cancer in a warm-blooded animal, such as man, in need of such treatment comprising administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000269] According to another feature of this aspect of the invention, there is provided a method for treating prostate, bone, kidney, liver, melanoma, gastrointestinal, pancreas, testes, thyroid, penis, vulva and other types of cancer. tumors with a dependence on PI3 kinases through mutation, amplification or other aberrations, in a warm-blooded animal, such as man, in need of such treatment comprising administering an effective amount of a compound of Formula ( I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore.
[000270] According to a further feature of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the treatment of cancer. breast, stomach (gastric) and esophagus, NSCLC including SCC and adenocarcinoma, H&N SCC, gynecologic cancers (including endometrial, ovarian and cervical cancers) and hematologic cancers such as multiple myeloma, lymphomas and leukemias ( including CLL, ALL and MCL).
[000271] According to a further feature of this aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the treatment of cancer bladder, brain/CNS, colorectal, lung (all other forms), gallbladder and bile ducts, and skin.
[000272] According to a further feature of this aspect of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined hereinbefore, in the manufacture of a medicament for use in the treatment of cancer prostate, bone, kidney, liver, melanoma, gastrointestinal, pancreas, testes, thyroid, penis, vulva and other types of tumors with a dependence on PI3 kinases through mutation, amplification or other aberrations.
[000273] In a feature of the invention, the cancer to be treated is breast cancer. In another aspect of this feature, breast cancer is estrogen receptor positive. In one embodiment of the present aspect, the compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered in combination with an anti-hormonal agent, as defined herein. In another embodiment of this aspect, Example 3 is administered in combination with an anti-hormonal agent, as defined herein. In another embodiment of this aspect, Example 3 is administered in combination with olaparib or a pharmaceutically acceptable salt thereof, and optionally further in combination with an anti-hormonal agent, as defined herein. In another embodiment of this aspect, Example 3 is administered in combination with AZD5363 or a pharmaceutically acceptable salt thereof, and optionally further in combination with an anti-hormonal agent, as defined herein.
[000274] In an aspect where the treatment of cancer is indicated, it should be understood that this may refer to the prevention of metastases and the treatment of metastases, ie the spread of cancer. Consequently, the compounds of the present invention can be used to treat a patient who has no metastases to prevent them from occurring, or to prolong the period of time before they occur, and in a patient who already has metastases to treat them. . In addition, cancer treatment may refer to the treatment of an established primary tumor or tumors and the development of a primary tumor or tumors. Therefore, in one aspect, cancer treatment is about preventing metastases. In another aspect of the invention, the treatment of cancer relates to the treatment of metastases. In another aspect of the invention, the treatment of cancer refers to the treatment of an established primary tumor or tumors or the development of a primary tumor or tumors.
[000275] As stated hereinabove, the in vivo effects of a compound of Formula (I) may be exerted, in part, by one or more metabolites (such as compounds of formula A, as defined hereinbefore) which are formed within the body of a human or animal following administration of a compound of Formula (I).
[000276] Particular compounds of the invention have better potency against PI3-α and -δ kinases than against other class I PI3 kinase isoforms, such as -β and -y. In one aspect, the compounds of the invention are selective for PI3K-α and -δ compared to PI3K-β or -y.
[000277] The present invention, therefore, also contemplates a method for inhibiting PI3-α kinases in a patient comprising administering to a patient an effective amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof. in the inhibition of PI3-α kinase in the patient.
[000278] The present invention therefore also contemplates a method for inhibiting PI3-α and -δ kinase in a patient comprising administering to a patient an amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof , effective in inhibiting PI3-α and -δ kinases in the patient.
[000279] The compound of Formula (I), or a pharmaceutically acceptable salt thereof, being an inhibitor of PI3 kinases, also has potential therapeutic uses in a variety of other pathological conditions. For example, PI3 kinases play an important role in promoting smooth muscle proliferation in the vascular tree, i.e. vascular smooth muscle cells (Thyberg, European Journal of Cell Biology, 1998, 76(1), 33-42) , and in the lungs (airway smooth muscle cells), Krymskaya, VP, BioDrugs. 2007, 21(2), 85-95. Excessive proliferation of vascular smooth muscle cells plays an important role in the formation of atherosclerotic plaques and the development of neointimal hyperplasia after invasive vascular procedures (Scwartz et al., Progress in Cardiovascular Disease, 1984, 26, 355-372; Clowes et al., Laboratory Investigations, 1978,39, 141-150). Furthermore, excessive proliferation of airway smooth muscle cells leads to the development of COPD in the context of asthma and chronic bronchitis. Inhibitors of PI3 kinase activity, therefore, can be used to prevent vascular restenosis, atherosclerosis and COPD.
[000280] PI3 kinases also play an important role in leukocyte function (Fuller et al., The Journal of Immunology, 1999, 162(11), 6337-6340; Eder et al., The Journal of Biological Chemistry, 1998, 273 (43), 28025-31) and lymphocyte function (Vicente-Manzanares et al., The Journal of Immunology, 1999, 163(7), 40014012). For example, leukocyte adhesion to inflamed endothelium involves the activation of endogenous leukocyte integrins through a PI3 kinase-dependent signaling process. In addition, oxidative burst (Nishioka et al., FEBS Letters, 1998, 441(1). 6366 and Condliffe, AM et al., Blood, 2005, 106(4). 1432-40) and cytoskeleton reorganization (Kirsch et al, Proceedings National Academy of Sciences USA, 1999, 96(11), 6211-6216) in neutrophils appears to involve signaling PI3 kinases. Migration and directional movement of neutrophils are also dependent on the activity of PI3 kinases (Camps, M., et al, Nat Med. 2005, 11(9).936-43 and Sadhu, C. et al, J Immunol. 2003, 170 (5), 2647-54). Thus, inhibitors of PI3 kinases may be useful in reducing the adhesion and activation of leukocytes at sites of inflammation and, consequently, may be used to treat acute and/or chronic inflammatory diseases. PI3 kinases also play an important role in lymphocyte proliferation and activation (Fruman et al., Science, 1999, 283 (5400), 393-397). In particular, PI3K-δ is essential for B cell development and function, including IgM-specific antibody-induced B cell proliferation ( Okkenhaug K. et al., Science, 2002, 297(5583). 1031-1034 ), receptor-induced DNA synthesis of B cells, and IL-4-induced proliferation and survival (Bilancio A. et al., Blood, 2006, 107, 642-650). These observations indicate that PI3K-δ has a crucial and non-redundant role in B cell function, which is not compensated by other class I PI3Ks. Given the important role of lymphocytes in autoimmune diseases, an inhibitor of PI3 kinase activity can be used in the treatment of such devices ( Rommel C, Camps M and Ji H, Nat Rev Immunol. 2007, 1038, 191-201 ).
[000281] The anticancer treatment defined hereinabove may be applied as a single therapy or may involve, in addition to the compound of the invention, conventional surgery or conventional radiotherapy and chemotherapy. Such chemotherapy may include one or more of the following categories of antitumor agents: (i) antiproliferative/antineoplastic drugs and combinations thereof as used in medical oncology, such as alkylating agents (e.g. cisplatin, oxaliplatin, carboplatin , cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, temozolamide and nitrosoureas); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea); antitumor antibiotics (for example anthracyclines such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example, vinca alkaloids, such as vincristine, vinblastine, vindesine, and vinorelbine, and taxoids, such as taxol and taxotere, and polokinase inhibitors); and topoisomerase inhibitors (for example, epipodophyllotoxins such as etoposide and teniposide, ansacrine, topotecan and camptothecin); (ii) antihormonal agents such as antiestrogens (eg tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxifene), antiandrogens (eg bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or agonists LHRH compounds (eg, goserelin, leuprorelin, and buserelin), progestogens (eg, megestrol acetate), aromatase inhibitors (eg, anastrozole, letrozole, vorazol, and exemestane) and 5α-reductase inhibitors , such as finasteride; (iii) inhibitors of growth factor function and its downstream signaling pathways: Included are Ab modulators of either growth factor or growth factor receptor targets (re-reviewed by Stern et al. Critical Reviews in Oncology/Haematology , 2005, 54, pages 11-29); small molecule inhibitors of such targets are also included, for example kinase inhibitors - examples include the anti-ErbB2™ antibody trastuzumab [Herceptin], the anti-EGFR antibody panitumumab, the anti-EGFR antibody cetuximab [Erbitux, C225] and tyrosine kinase inhibitors, including inhibitors of the erbB receptor family, such as epidermal growth factor family (EGFR/erbB1) receptor tyrosine kinase inhibitors, such as gefitinib or erlotinib, erbB2 tyrosine kinase inhibitors, such as lapatinib, and mixed erb1/2 inhibitors such as afatanib; similar strategies are available for other classes of growth factors and their receptors, eg inhibitors of the hepatocyte growth factor family or their receptors, including ron and c-met; Insulin inhibitors and inhibitors of the insulin growth factor family and its receptors (IGFR, IR), inhibitors of the platelet-derived growth factor family or its receptors (PDGFR) and inhibitors of signaling mediated by other receptor tyrosine kinases, such as c-kit, AnLK and CSF-1R; also included are modulators that target signaling proteins in the broader PI3 kinase signaling pathway, e.g. inhibitors of other isoforms of PI3 kinases, such as PI3K-β, and ser/thr kinases, such as AKT, mTOR, PDK, SGK, PI4K or PIP5K; also included are serine/threonine kinase inhibitors not listed above, for example, raf inhibitors such as vemurafenib, MEK inhibitors such as selumetinib (AZD6244), Abl inhibitors such as imatinib or nilotinib, inhibitors Btk inhibitors such as ibrutinib, Syk inhibitors such as fostamatinib, aurora kinase inhibitors (e.g. AZD1152), inhibitors of other Ser/Ter kinases such as JAKs, STATs and IRAK4, and cyclin-dependent kinase inhibitors; (iv) modulators of DNA damage signaling pathways, eg PARP inhibitors (eg olaparib), ATR inhibitors or ATM inhibitors; (v) modulators of apoptotic pathways and cell death, such as modulators of the Bcl family (e.g. ABT-263/Navitoclax, ABT-199); (vi) anti-angiogenic agents, such as those that inhibit the effects of vascular endothelial growth factor [e.g., the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin™) and, for example, a receptor tyrosine kinase inhibitor VEGF, such as sorafenib, axitinib, pazopanib, sunitinib, and vandetanib (and compounds that work through other mechanisms (eg, linomide, inhibitors of αvβ3 integrin function, and angiostatin)]; (vii) vascular damaging agents, such as Combretastatin A4; (viii) anti-invasion agents, e.g. inhibitors of the c-Src kinase family (dasatinib, Med. Chem., 2004, 47, 6658-6661) and bosutinib (SKI-606), and inhibitors of metalloproteinase such as marimastat, inhibitors of urokinase plasminogen activator receptor function, or heparanase antibodies]; (ix) immunotherapy approaches including, for example, in vivo and ex vivo approaches, to enhance immunogenicity of patient's tumor cells, such as transfection with cytokines, such as interleukin 2, interleukin 4, or granulocyte-macrophage colony stimulating factor, approaches to decreasing T cell anergy, approaches using transfected immune cells, such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines and approaches using anti-idiotypic antibodies. Specific examples include monoclonal antibodies that target PD-1 (eg, BMS-936558) or CTLA4 (eg, ipilimumab and tremelimumab); (x) antisense or RNAi-based therapies, for example, those that target the listed targets; (xi) gene therapy approaches including, for example, approaches to replace aberrant genes, such as aberrant p53 or aberrant BRCA1 or BRCA2, gene-directed enzyme prodrug therapy approaches (Gene-Directed Enzyme Pro-Drug The-rapy - GDEPT), such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy, such as multidrug resistance gene therapy.
[000282] In accordance with this aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and another antitumor agent, in particular any of the antitumor agents listed under (i) - (xi) above. In particular, the antitumor agent listed in (i) - (xi) above is the standard treatment for the specific cancer being treated; Those skilled in the art will understand the meaning of "standard care".
[000283] Therefore, in another aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with another antitumor agent, in particular an antitumor agent selected from one listed in (i) - (xi) up here.
[000284] In a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with another antitumor agent, in particular an antitumor agent selected from one listed in (i) above.
[000285] In another aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and any of the antitumor agents listed under ( i) above.
[000286] In a further aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and a taxoid such as, for example , taxol or taxotere, conveniently taxotere.
[000287] In a further aspect of the invention, there is provided a compound of Formula (I) or is a pharmaceutically acceptable salt thereof in combination with another antitumor agent, in particular an antitumor agent selected from one listed in (ii) herein above.
[000288] In another aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and any of the listed anti-hormonal agents. under (ii) above, for example any of the antiestrogens listed under (ii) above.
[000289] In a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an mTOR inhibitor, such as those described in WO2008/023161, for example:

[000290] In a further aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and an inhibitor of mTOR, such as those described in WO2008/023161, for example:

[000291] In particular, the mTOR inhibitor is AZD2014, which has the following structure:

[000292] In one aspect, the above combination of the compound of formula (I) and AZD2014 is suitable for use in the treatment of ER-positive breast cancer, optionally in combination with the standard treatment of hormone therapy.
[000293] In a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with a PI3K-β inhibitor.
[000294] The combination of a compound of formula (I) with a PI3K-β inhibitor may be particularly useful in the treatment of tumors, e.g. prostate, breast (e.g. triple negative breast), squamous cell NSCLC and kidney cancer, based on PTEN loss.
[000295] In another aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and a PI3K-β inhibitor.
[000296] In one aspect, the PI3K-β inhibitors described herein also have some PI3K-δ inhibitory activity.
[000297] In a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with a PI3K-β inhibitor, such as any of the examples in International Patent Application WO2011/051704 .
[000298] In another aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and a PI3K-β inhibitor, such as such as any of the examples in International Patent Application WO2011/051704.
[000299] In another aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an inhibitor of PI3K-β and PI3K-δ, such as 8-((1R)-1- (3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide (Example 3.06b in International Patent Application WO2011/051704, also known as AZD8186) or a pharmaceutically acceptable of the same:

[000300] In another aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and an inhibitor of PI3K-β and PI3K -δ, such as 8-((1R)-1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide (Example 3.06b in International Patent Application WO2011/051704, also known as AZD8186) or a pharmaceutically acceptable salt thereof:

[000301] In a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an AKT kinase inhibitor, such as (S)-4-amino-N-(1- (4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide (AZD5363) or a pharmaceutically acceptable salt thereof (see for example , WO2009/047563 ).
[000302] The combination of a compound of Formula (I) and an AKT inhibitor may be particularly useful in the treatment of tumors with a higher prevalence of PIK3CA gene mutations, such as ER-positive breast cancer, endometrial cancer, ovarian cancer, squamous cell NSCLC, gastric cancer, bladder cancer and biliary tract cancer.
[000303] In another aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and an AKT kinase inhibitor such as (S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamide (AZD5363 ) or a pharmaceutically acceptable salt thereof (see, for example, WO2009/047563).
[000304] In a further aspect of the invention, there is provided a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with olaparib (4-[3-(4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4- fluoro-benzyl]-2H-phthalazin-1-one) or a pharmaceutically acceptable salt thereof.
[000305] The combination of a compound of Formula (I) and olaparib may be particularly useful in both wild-type or BRCA-deficient triple negative breast cancer and estrogen receptor positive (ER+) breast cancer ve), particularly those with mutations in the PIK3CA gene.
[000306] In another aspect of the invention, there is provided a combination suitable for use in the treatment of cancer comprising a compound of Formula (I) as defined hereinbefore or a pharmaceutically acceptable salt thereof and olaparib (4-[3-( 4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one) or a pharmaceutically acceptable salt thereof.
[000307] Particular combinations of the invention comprise any of the compounds of the Examples herein (or a pharmaceutically acceptable salt thereof) and an mTOR inhibitor, PI3K-β inhibitor, AKT kinase inhibitor or olaparib, as described herein above. Other particular combinations of the invention comprise Example 3 (or a pharmaceutically acceptable salt thereof) and an mTOR inhibitor, PI3K-β inhibitor, AKT kinase inhibitor, or olaparib, as described herein above. Other particular combinations of the invention comprise Example 3 (or a pharmaceutically acceptable salt thereof) and a PI3K-β inhibitor, AKT kinase inhibitor, or olaparib (or a pharmaceutically acceptable salt of any of the same), as described herein above. More particularly, examples of combinations of the invention comprise Example 3 (or a pharmaceutically acceptable salt thereof) and any of AZD8186, AZD5363 and olaparib (or a pharmaceutically acceptable salt of any of them). Another example of a combination of the invention comprises Example 3 and AZD2014.
[000308] In all of the above combinations, it should be understood that the combination may also be dosed with the standard treatment, as understood by those skilled in the art, such as other treatments (i) to (xi) hereinabove. For example, when any of the above combinations are intended to be used for the treatment of ER+ve breast cancer, standard treatment with hormone therapy (e.g., the agents listed in (ii) above) may be used in conjunction with along with the combination of the invention. In other respects, suitably, the standard treatment may be selected from (i) above.
[000309] Therefore, in another aspect of the invention, there is provided a triple combination suitable for use in the treatment of cancer of: a compound of formula (I) (such as Example 3) or a pharmaceutically acceptable salt thereof; an mTOR inhibitor, PI3K-β inhibitor, AKT kinase inhibitor or olaparib or a pharmaceutically acceptable salt thereof; and standard treatment of therapy for the cancer being treated.
[000310] Suitably, standard treatment of therapy may be dosed in accordance with the usual dosing regimen, as understood by those skilled in the art.
[000311] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from a list of in (i) - (xi) hereinabove, in association with a pharmaceutically acceptable diluent or carrier.
[000312] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising Example 3 or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from one listed under (i) - (xi) herein above in association with a pharmaceutically acceptable diluent or carrier.
[000313] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising Example 3 or a pharmaceutically acceptable salt thereof in combination with AZD5363, AZD8186 or olaparib (or a pharmaceutically acceptable salt of any one thereof) in association with a pharmaceutically acceptable diluent or carrier.
[000314] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from one listed in (i) - (xi) hereinabove in association with a pharmaceutically acceptable diluent or carrier for use in the treatment of cancer.
[000315] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising Example 3 or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from one listed under (i) - (xi) herein above in association with a pharmaceutically acceptable diluent or carrier for use in the treatment of cancer.
[000316] According to a further aspect of the invention, there is provided a pharmaceutical composition comprising Example 3 or a pharmaceutically acceptable salt thereof in combination with AZD5363, AZD8186 or olaparib (or a pharmaceutically acceptable salt of any of the same) in association with a pharmaceutically acceptable diluent or vehicle for use in the treatment of cancer.
[000317] According to another feature of the invention, there is provided the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from those listed in (i) - ( xi) above, in the manufacture of a medicament for use in the treatment of cancer in a warm-blooded animal such as man.
[000318] According to another feature of the invention, there is provided the use of Example 3 or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from one listed in (i) - (xi) hereinabove in the manufacture of a drug for use in the treatment of cancer in a warm-blooded animal such as man.
[000319] According to another feature of the invention, there is provided the use of Example 3 or a pharmaceutically acceptable salt thereof in combination with AZD5363, AZD8186 or olaparib (or a pharmaceutically acceptable salt of any of the same). mos) in the manufacture of a drug for use in the treatment of cancer in a warm-blooded animal such as man.
[000320] Thus, in a further feature of the invention, there is provided a method of treating cancer in a warm-blooded animal, such as man, in need of such treatment, which comprises administering to said animal an amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from one listed in (i) - (xi) hereinabove.
[000321] Thus, in a further feature of the invention, there is provided a method of treating cancer in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of Example 3 or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from one listed in (i) - (xi) hereinabove.
[000322] Thus, in a further feature of the invention, there is provided a method of treating cancer in a warm-blooded animal, such as man, in need of such treatment which comprises administering to said animal an effective amount of Example 3 or a pharmaceutically acceptable salt thereof in combination with AZD5363, AZD8186 or olaparib (or a pharmaceutically acceptable salt of any of the same).
[000323] According to another aspect of the present invention, there is provided a kit comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof in combination with an antitumor agent selected from those listed in (i) - (xi) here above.
[000324] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an antitumor agent selected from one listed in (i) - (xi) above in a second unit dosage form; and container means for containing said first and second dosage forms.
[000325] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an antitumor agent selected from one listed in (i) - (xi) hereinabove, in a second unit dosage form; container means for containing said first and second dosage forms; and, optionally, instructions for use.
[000326] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an mTOR inhibitor, such as those described in WO2008/023161, for example:

[000327] in a second unit dosage form; and
[000328] c) container means for containing said first and second dosage forms.
[000329] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; a PI3K-β inhibitor such as any one of the examples in International Patent Application WO2011/051704 or a pharmaceutically acceptable salt thereof in a second unit dosage form; and container means for containing said first and second dosage forms.
[000330] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; a PI3K-β inhibitor such as any one of the examples in International Patent Application WO2011/051704 or a pharmaceutically acceptable salt thereof in a second unit dosage form; container means for containing said first and second dosage forms; and, optionally, instructions for use.
[000331] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an inhibitor of PI3K-β and PI3K-δ, which is 8-((1R)-1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene -6-carboxamide (Example 3.06b in International Patent Application WO2011/051704, also known as AZD8186) or a pharmaceutically acceptable salt thereof in a second unit dosage form; and container means for containing said first and second dosage forms.
[000332] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an inhibitor of PI3K-β and PI3K-δ, which is 8-((1R)-1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene -6-carboxamide (Example 3.06b in International Patent Application WO2011/051704, also known as AZD8186) or a pharmaceutically acceptable salt thereof in a second unit dosage form; container means for containing said first and second dosage forms; and, optionally, instructions for use.
[000333] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an AKT kinase inhibitor, such as (S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl )piperidine-4-carboxamide or a pharmaceutically acceptable salt thereof (AZD5363, see, for example, WO2009/047563), in a second unit dosage form; container means for containing said first and second dosage forms; and, optionally, instructions for use.
[000334] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an AKT kinase inhibitor, such as (S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl )piperidine-4-carboxamide or a pharmaceutically acceptable salt thereof (AZD5363, see, for example, WO2009/047563), in a second unit dosage form; and container means for containing said first and second dosage forms.
[000335] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; an AKT kinase inhibitor, such as (S)-4-amino-N-(1-(4-chlorophenyl)-3-hydroxypropyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl )piperidine-4-carboxamide or a pharmaceutically acceptable salt thereof (AZD5363, see, for example, WO2009/047563), in a second unit dosage form; container means for containing said first and second dosage forms; and, optionally, instructions for use.
[000336] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; olaparib (4-[3-(4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one) or a pharmaceutically acceptable salt thereof, in a second dosage form unitary; container means for containing said first and second dosage forms.
[000337] According to another aspect of the present invention, there is provided a kit comprising: a compound of Formula (I) or a pharmaceutically acceptable salt thereof in a first unit dosage form; olaparib (4-[3-(4-cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one) or a pharmaceutically acceptable salt thereof, in a second unit form unit dosage; container means for containing said first and second dosage forms; and, optionally, instructions for use.
[000338] In all of the above combinations, uses, treatment methods and kits, AZD5363, AZD8186 and olaparib may be in the form of the free base or in the form of a pharmaceutically-acceptable salt. Accordingly, in one embodiment, AZD5363 is in the form of a free base; in another embodiment, AZD5363 is in the form of a pharmaceutically acceptable salt. In another embodiment, AZD8186 is in the form of a free base; in another embodiment, AZD8186 is in the form of a pharmaceutically acceptable salt. In another fashion, olaparib is in the form of a free base; in another embodiment, olaparib is in the form of a pharmaceutically acceptable salt.
[000339] While the compounds of Formula (I) are primarily valuable as therapeutic agents for use in warm-blooded animals (including man), they are also useful whenever it is necessary to inhibit the effects of PI3-α and - δ. Thus, they are useful as pharmacological standards for use in the development of new biological tests and in the research of new pharmacological agents.
[000340] Here, where the term "combination" is used, it should be understood that it refers to simultaneous, separate or sequential administration. In one aspect of the invention, "combination" refers to simultaneous administration. In another aspect of the invention, "combination" refers to separate administration. In a further aspect of the invention, "combination" refers to sequential administration. When administration is sequential or separate, the delay in administration of the second component should not be such as to lose the beneficial effect.
[000341] In one embodiment, sequential treatment involves administration of each component of the combination within an 11-day period. In another modality, this period is 10 days. In another modality, this period is 9 days. In another modality, this period is 8 days. In another modality, this period is 7 days. In another mode, this is a period of 6 days. In another modality, this period is within 5 days. In another embodiment, this period is within 4 days. In another embodiment, this period is within 3 days. In another embodiment, this period is within 2 days. In another embodiment, this period is within 24 hours. In another mode, this period is within 12 hours.
[000342] Sequential administration and co-administration are both exemplified here in the combination experiments with Example 3 and AZD5363 in model BT474. In this example, sequential administration is illustrated by dosing AZD5363 for 2 days, followed by Example 3 for 2 days, then 3 days with no dose of either agent before the pattern is repeated ("dose cycle" ). Co-administration is illustrated with a dosing regimen in which both AZD5363 and Example 3 are dosed for 2 days, followed by 5 days with no dose of either agent. In these two examples, sequential administration appears to be more effective in causing tumor regression, which illustrates the potential importance of regimen optimization.
[000343] Other potential co-dosing regimens include: 1) a dosing cycle in which both AZD5363 and Example 3 are dosed for 2 days, followed by 3 days with no dose of either agent; 2) a dosing cycle in which both AZD5363 and Example 3 are dosed for 3 days, followed by 4 days with no dose of either agent; 3) a dosing cycle in which both AZD5363 and Example 3 are dosed for 4 days, followed by 3 days with no dose of either agent; 4) a dosing cycle in which both AZD5363 and Example 3 are dosed for 5 days, followed by 2 days with no dose of either agent; 5) a dosing cycle in which AZD5363 and Example 3 are dosed every other day; 6) a dosing cycle wherein AZD5363 and Example 3 are dosed every three days; 7) a dosing cycle wherein AZD5363 and Example 3 are dosed on a weekly schedule with 3 and 4 day intervals between dosing (eg, Monday/Thursday); 8) a dosing cycle in which AZD5363 and Example 3 are dosed on a weekly schedule with 2 and 3 day gaps between dosing (eg, Monday/Wednesday/Friday).
[000344] Combinations of compounds of formula (I), particularly in Example 3, with an mTOR inhibitor, such as AZD2014, or a PI3K-β inhibitor (such as the AZD8186 inhibitor) may suitably be administered in a similar scheme to those described above for the combination of Example 3 and AZD5363.
[000345] A combination of a compound of formula (I) and olaparib may be dosed according to a regimen where olaparib is dosed daily and the compound of formula (I) is dosed according to an intermittent dosing schedule (such as, for example, 2 days of dosing, followed by 3 to 5 days of no dosing).
[000346] Each of these illustrative dosage regimens comprises a further aspect of the invention. Each of these illustrative dosage regimens can also be applied to combinations with other antitumor agents listed in (i) to (xi) above.
[000347] It may be advantageous, within a given dosing cycle, to administer one specific component of the combination before the other - i.e., sequential dosing.
[000348] Therefore, in one embodiment, the sequential administration comprises sequential administration of a compound of formula (I) (particularly Example 3) prior to administration of the other antitumor agent listed in (i) to (xi) above, particularly an agent anti-tumor selected from AZD5363, AZD8186 and olaparib, within one dosing cycle.
[000349] In another embodiment, the sequential administration comprises the sequential administration of the antitumor agent listed in (i) to (xi) above, in particular an antitumor agent selected from AZD5363, AZD8186 and olaparib, prior to administration of the compound of formula (I) (particularly Example 3) within a dosing cycle.
[000350] In one embodiment, the antitumor agent listed in (i) to (xi) above and the compound of formula (I) are dosed up to 2 days apart. In another embodiment, the antitumor agent listed in (i) to (xi) above and the compound of formula (I) are dosed at 1 day intervals. In another embodiment, the antitumor agent listed in (i) to (xi) above and the compound of formula (I) are administered up to 18 hours apart. In another embodiment, the antitumor agent listed in (i) to (xi) above and the compound of formula (I) are administered up to 12 hours apart. In another embodiment, the antitumor agent listed in (i) to (xi) above and the compound of formula (I) are dosed up to 6 hours apart. In another embodiment, the antitumor agent listed in (i) to (xi) above and the compound of formula (I) are dosed up to 3 hours apart.
[000351] In other embodiments, the dosing cycle may be 5 to 10 days in duration.
[000352] In accordance with other embodiments, the dosing cycle may be 6 to 10 days in duration.
[000353] In other embodiments, the dosing cycle may be 7 to 9 days in duration.
[000354] In other embodiments, the dosing cycle may be 6 to 8 days in duration.
[000355] In other embodiments, the dosing cycle may be 10 days in duration.
[000356] In other embodiments, the dosing cycle may be 9 days in duration.
[000357] According to other embodiments, the dosing cycle may be 8 days long.
[000358] In other embodiments, the dosing cycle may be 7 days in duration.
[000359] In other embodiments, the dosing cycle may be 6 days in duration.
[000360] In other embodiments, the dosing cycle may be 5 days in duration.
[000361] In other embodiments, the dosing cycle may involve the compound of formula (I) (particularly in Example 3) being dosed for 2-4 consecutive days, not being dosed during the other days of a 6 to 4 dosing cycle. 9 days in duration.
[000362] In other embodiments, the dosing cycle may involve the compound of formula (I) (particularly in Example 3) being dosed for 3-4 consecutive days, not being dosed during the other days within a cycle dose of 6 to 9 days in duration (eg 7 days in duration).
[000363] In other embodiments, the dosing cycle may involve the compound of formula (I) (particularly in Example 3) being administered for 3-5 consecutive days and not being dosed during the other days within a dosing cycle from 7 to 10 days of duration.
[000364] In other embodiments, the dosing cycle may involve the compound of formula (I) (particularly in Example 3) being dosed for 5 consecutive days, not being dosed during the other days during a 6 to 9 day dosing cycle of duration.
[000365] In accordance with other embodiments, the dosing cycle may involve the compound of formula (I) (particularly in Example 3) being dosed for 4 consecutive days and not being dosed during the other days during a dosing cycle of 6 to 9 days duration (eg 7 days duration).
[000366] In other embodiments, the dosing cycle may involve the compound of formula (I) (particularly in Example 3) being dosed for 3 consecutive days, not being dosed for the other 15 days within a 6-day dosing cycle. 9 days in duration.
[000367] Dosing cycles can be separated by a number of days where none of the active components of the combination are administered.
[000368] Combination therapy as described above may be added to standard therapy treatment typically performed according to the usual prescription dosing schedule. Personalized Medical Assistance
[000369] Another aspect of the present invention is based on the identification of a link between the status of the gene encoding phosphoinositide 3-kinase, alpha catalytic polypeptide (PIK3CA), and susceptibility to treatment with a compound of Formula ( I). This therefore offers opportunities, methods and tools to screen patients for treatment with a compound of Formula (I), particularly cancer patients, and/or avoid treating patients who are less likely to respond to treatment. therapeutic, thereby avoiding unnecessary treatment and any side effects that may be associated with such ineffective treatment.
[000370] The present invention relates to patient selection tools and methods (including personalized medicine). Selection is based on whether the tumor cells to be treated have the wild-type or mutant PIK3CA gene. PIK3CA gene status, therefore, can be used as a biomarker of susceptibility to treatment with an inhibitor of PI3K-α and -δ.
[000371] There is a clear need for biomarkers that will enrich or select patients whose tumors respond to treatment with an inhibitor of PI3K-α and -δ, such as a compound of Formula (I). Patient selection biomarkers that identify patients most likely to respond to an agent are ideal for treating cancer as they reduce unnecessary treatment of patients with unresponsive tumors in relation to the potential side effects of such agents. .
[000372] A biomarker can be described as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention". A biomarker is any identifiable and measurable indicator associated with a particular condition or disease where there is a correlation between the presence or level of the biomarker and some aspect of the condition or disease (including the presence of, the level or level of change, the type of, the stage of, the susceptibility to the condition or disease or the responsiveness to a drug used to treat the condition or disease). Correlation can be qualitative, quantitative or qualitative and quantitative. Typically, a biomarker is a compound, compound fragment or group of compounds. Such compounds can be found in any compounds in or produced by an organism, including proteins and peptide(s), nucleic acids and other compounds.
[000373] Biomarkers can have predictive power and as such can be used to predict or detect the presence, level, type or stage of particular conditions or diseases (including the presence or level of particular microorganisms or toxins), the susceptibility (including genetic susceptibility) to particular conditions or diseases or the response to particular treatments (including drug treatments). It is believed that biomarkers will play an increasingly important role in the future of drug discovery and development, improving the efficiency of research and development programs. Biomarkers can be used as diagnostic agents, disease progression monitors, treatment monitors, and clinical outcome predictors. For example, several biomarker research projects are trying to identify specific cancer markers and specific cardiovascular and immune disease markers. It is believed that the development of new validated biomarkers will lead to significant reductions in both healthcare and drug development costs and significant improvements in the treatment of a wide variety of diseases and conditions.
[000374] In order to optimally design clinical trials and obtain maximum information from these trials, a biomarker may be necessary. The marker may be measurable in tumor and surrogate tissues. Ideally, these markers also correlate with efficacy and, therefore, could ultimately be used for patient selection.
[000375] Thus, the technical problem underlying this aspect of the present invention is the identification of means for stratifying patients for treatment with a compound of Formula (I). The technical problem is solved by the arrangement of the modalities characterized in the claims and/or description.
[000376] As detailed in the examples herein, cells which have a mutation in PIK3CA have been found to be, in general, more susceptible to growth inhibition by the compound of Formula (I).
[000377] The invention provides a method for determining the sensitivity of cells to a compound of Formula (I). The method comprises determining the status of the PIK3CA gene in said cells. Cells are identified as likely to be sensitive to a compound of Formula I if the cells carry a mutated PIK3CA gene. Consequently, patients with a mutated PIK3CA gene are predicted to be particularly susceptible to treatment with a compound of Formula (I). A cell is defined as sensitive to a compound of Formula (I) if it inhibits the increase in cell number in a cell growth assay (either through inhibition of cell proliferation and/or through increased cell death). The methods of the invention are useful for predicting which cells are most likely to respond to a compound of Formula (I) by inhibiting growth.
[000378] The present invention is further based, in part, on methods that can be used to determine a patient's response to a compound of Formula (I), including determining whether to administer a compound of Formula (I). Specifically, the methods of the present invention include determining the status of the PIK3CA gene. The presence of a mutated PIK3CA gene indicates that tumor cells are more likely to respond to growth inhibition when contacted with a compound of Formula (I). PIK3CA gene status can therefore be used to select patients for treatment with a compound of Formula (I).
[000379] Furthermore, an in vitro method for identifying a patient likely to be sensitive to a compound of Formula (I) is described. Also described are uses of oligoor polynucleotide primers or probes capable of detecting the mutation status of the PIK3CA gene. Also described is the use of PIK3CA mutation detection kits including, but not limited to, PIK3CA mutation detection kits marketed by diagnostic companies including Qiagen and Roche Molecular Systems. In another embodiment, the invention relates to an in vitro method for determining whether a patient suffering from cancer is likely to be a responder to treatment with a compound of Formula (I), said method. all comprising the steps of: (i) obtaining a representative sample of the tumor that has been previously collected from said patient; and (ii) determining whether the PIK3CA genes contain a mutation in said sample. A mutation in the PIK3CA 5 gene is indicative of an increased likelihood of response to treatment with a compound of Formula (I). As a single gene biomarker test, the identification of tumors that contain a mutation in PIK3CA will enrich the response to a compound of Formula (I). Individual tumors that contain a mutation in PIK3CA are more likely to respond to a compound of Formula (I).
[000380] A "tumor representative" sample can be the actual isolated tumor sample or can be a sample that has been further processed, for example, a PCR amplified nucleic acid sample from the tumor sample. Definitions:
[000381] In this Personalized Health Care section:
[000382] "Allele" refers to a particular form of a genetic locus that is distinguished from other forms by its particular nucleotide or amino acid sequence.
[000383] "Amplification reactions" are nucleic acid reactions which result in specific amplification of target nucleic acids relative to non-target nucleic acids. Polymerase Chain Reaction (PCR) is a well-known amplification reaction.
[000384] "Cancer" is used herein to refer to neoplastic cell growth resulting from transformation into a neoplastic phenotype. This cellular transformation often involves genetic mutation.
[000385] "Gene" is a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including a promoter, exons, introns and other sequence elements which may be located in regions of 5' or 3' flanks (not within the transcribed portions of the gene) that control expression.
[000386] "Gene status" refers to whether or not the gene is wild-type (ie, mutant).
[000387] "Label" refers to a composition capable of producing a detectable signal indicative of the presence of the target polynucleotide in a test sample. Suitable labels include radioisotopes, chromophores, nucleotides, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent units and so on. As such, a marker is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
[000388] "Non-synonymous variations" refers to a variation (deviation) in or overlap of the coding sequence of a gene that results in the production of a distinct (altered) polypeptide sequence. These variations may or may not affect the function of the protein and include missense variants (resulting in substitution of one amino acid for another), nonsense variants (resulting in a truncated polypeptide due to the generation of a premature stop codon -ro) and insert/delete variants.
[000389] "Synonymous variation" refers to a variation (deviation) in the coding sequence of a gene that does not affect the encoded polypeptide sequence. These variations can indirectly affect protein function (eg, by altering gene expression), but, in the absence of evidence to the contrary, are generally accepted as harmless.
[000390] "Nucleic acid" refers to single or double stranded DNA and RNA molecules, including naturally occurring nucleic acids found in nature and/or modified nucleic acids having modified backbones or bases, as known in the art.
[000391] "Primer" refers to a sequence of single-stranded DNA oligonucleotides capable of acting as an initiation point for the synthesis of a primer extension product that is complementary to the nucleic acid strand to be copied. The length and sequence of the primer should be such that it is capable of initiating the synthesis of extension products. A typical primer contains at least about 7 nucleotides in length of a sequence substantially complementary to the target sequence, but slightly longer primers are preferred. Typically, primers contain about 1526 nucleotides, but longer or shorter primers can also be used.
[000392] "Polymorphic site" is a position within a locus at which at least two alternative sequences are found in a population.
[000393] "Polymorphism" refers to the variation in sequence observed in an individual at a polymorphic site. Polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites and may, but not necessarily, result in detectable differences in gene expression or protein function. In the absence of evidence of an effect on protein expression or function, common polymorphisms, including non-synonymous variants, are generally considered to be included in the wild-type gene sequence definition. A catalog of human polymorphisms and associated annotation, including validation, observed frequencies, and disease association, is maintained by the NCBI (dbSNP: http://www.ncbi.nlm.nih.gov/projetos/SNP/). Please note that the term "polymorphism", when used in the context of gene sequences, should not be confused with the term "polymorphism" when used in the context of the solid state form of a compound, which is the crystalline or amorphous nature of a compound. compound. Those skilled in the art will understand the meaning intended by its context.
[000394] "Probe" refers to single-stranded sequence-specific oligonucleotides that have a sequence that is exactly complementary to the target sequence of the allele to be detected.
[000395] "Response" is defined by measurements made in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST) which involves classifying patients into two main groups: those who show a partial response or stable disease and those who show signs of progressive disease.
[000396] "Stringent hybridization conditions" refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate and 20 pg/ml cut denatured salmon sperm DNA, followed by washing the filters in 0.1X SSC at about 65°C.
[000397] "Survival" encompasses the overall survival of patients as well as progression-free survival.
[000398] "Overall Survival - OS" is defined as the time from initiation of drug administration to death from any cause. "Progression-Free Survival" (PFS) is defined as the time from initiation of drug administration to the first onset of disease progression or death from any cause.
[000399] According to one aspect of the invention, there is provided a method for selecting a patient for treatment with a compound of Formula (I), the method comprising providing a sample containing tumor cells from a patient; determining whether the PIK3CA gene in the sample containing the patient's tumor cells is wild-type or mutant; and selecting a patient for treatment with a compound of Formula (I) based thereon.
[000400] The method can include or exclude the actual patient sample isolation step. Thus, in accordance with one aspect of the invention, there is provided a method for selecting a patient for treatment with a compound of Formula (I), the method comprising determining whether the PIK3CA gene in a sample containing tumor cells previously isolated from the patient is wild type or mutant; and selecting a patient for treatment with a compound of Formula (I) based thereon.
[000401] In one embodiment, the patient is selected for treatment with a compound of Formula (I) if the tumor cell DNA has a mutant PIK3CA gene. In other embodiments, a patient whose tumor cell DNA has a wild-type PIK3CA gene is not selected for treatment with a compound of Formula (I).
[000402] According to another aspect of the invention, there is provided a method for predicting a patient's response to a compound of Formula (I), the method comprising determining whether the PIK3CA gene in the patient's tumor cells is wild-type or muted. -tant and, based thereon, predict the responsiveness of a patient to treatment with a compound of Formula (I).
[000403] In accordance with another aspect of the invention, there is provided a method for determining the likelihood of efficacy of treatment with a compound of Formula (I) in a human patient afflicted with cancer comprising: determining whether the gene(s) of PIK3CA in the patient's tumor cells is wild-type or mutant and, based on this, predict a patient's responsiveness to treatment with a compound of Formula (I).
[000404] For purposes of the present invention, a wild-type gene status is intended to indicate normal or adequate gene expression and normal function of the encoded protein. In contrast, mutant status is intended to indicate abnormal or inappropriate gene expression or expression of a protein with altered function, consistent with the known roles of mutant PIK3CA in cancer (as described here). Any number of genetic or epigenetic alterations, including, but not limited to, mutation, amplification, deletion, genomic rearrangement, or alterations in the methylation profile, can result in a mutated state. However, if such changes result in proper expression of the normal protein or a functionally equivalent variant, then the gene status is considered to be wild-type. Examples of variants that would not normally result in a functional mutant gene state include synonymous coding variants and common polymorphisms (synonymous or non-synonymous). As discussed below, gene status can be assessed by a functional assay or can be inferred from the nature of the detected deviations from a reference sequence.
[000405] In certain embodiments, the wild-type or mutant status of the PIK3CA gene is determined by the presence or absence of non-synonymous nucleic acid variations in the genes. Observed non-synonymous variations that correspond to known common polymorphisms with no functional effects noted do not contribute to a mutated gene state.
[000406] Other variations in the PIK3CA gene that signify a mutant state include variations in splicing sites that decrease recognition of an intron/exon junction during processing of pre-mRNA into mRNA. This can result in an exon skipping or the inclusion of the normally intronic sequence in mRNA splicing (intron retention or use of cryptic splice junctions). This can, in turn, result in the production of aberrant protein with insertions and/or deletions relative to the normal protein. Thus, in other embodiments, the gene has a mutant state if there is a variant that alters the splicing site recognition sequence at an intron/exon junction.
[000407] Furthermore, measurement of the mutation status or activation status of additional genes, such as Kras, a potential marker of resistance in tumors with aberrant or dysregulated PIK3CA or PI3K-α, could help to increase the predictability of a personalized medicine approach.
[000408] In a survey that was conducted at AstraZeneca in breast cancers (based on the COSMIC database (Welcome Trust Sanger Institute, September 2011), > 55 different mutations in the PIK3CA gene were identified by crossing over with a set of data that covered > 5K of human tumors. Most mutations occurred at a frequency of <1%, 3 occurred at a frequency of 1-3%, but four mutations accounted for ~88% of the total mutations in PIK3CA. These were kinase domain missense mutations in the C-terminal kinase domain, H1047R (55%) and H1047L (5%), and in the helical domain residues, E545K (18%) and E542K (11%). of other mutations prevalent in breast cancer, although not exhaustive, includes R38H, R38C, R88Q, N345K, C420R, E453Q, P539R, E542K, E545K, E545A, Q546K, Q546P, M1043I, M1043V, H1047R, H1047L, H1047Y. Consequently, diagnostic assays can be constructed that focus on detecting the most common, thus allowing the identification of most mutations in PIK3CA. For example, the Cobas(TM) PIK3CA Mutation Test from Roche Molecular Systems is designed to detect 17 mutations in exons 1, 4, 7, 9 and 20 of the PIK3CA gene (E542K, E545A, E545G, E545K, E545D, Q546K, Q546R , Q546E, Q546L, N345K, C420R, R88Q, H1047L, H1047R, H1047Y, G1049R and M10431) on DNA isolated from formalin-fixed paraffin-embedded tumor samples. This kit is capable of capturing up to ~95% of mutations in ER+ve breast cancer. The distribution of mutations differs among other tumor types and the diagnostic strategy can be adapted accordingly. For example, in endometrial cancer, there is a more uniform distribution of mutations spread throughout the coding sequence of the PIK3CA gene and a greater number of mutations in the N-terminal region of the protein (communicated by Douglas A. Levine, MD, TCGA 2nd Annual Symposium, November 28, 2012), compared to breast cancers.
[000409] For PIK3CA, reference sequences are available for the gene (GenBank accession number: NG_012113), mRNA (GenBank accession number: NM_006218) and protein (GenBank accession number: NP006209 or Swiss-Prot accession: P42336 ). The reference gene sequences (genomic region) include 5000 bases of upstream sequence and 2000 bases of downstream sequence. Mutations within PIK3CA are well known (COS-MIC database - Welcome Trust Sanger Institute) and those skilled in the art will be able to determine the state of the PIK3CA gene, that is, whether a particular PIK3CA gene is wild-type or mutant, based on DNA or protein sequence comparison with wild-type.
[000410] It will be apparent that the gene and mRNA sequences described for PIK3CA and the catalytic subunit P110a of the PI3 alpha kinase protein sequence are each a representative sequence. In normal individuals, there are two copies of each gene, a maternal and a paternal copy, which are likely to have some sequence differences. Furthermore, within a population there will be numerous allelic variants of the gene sequence. Other sequences considered wild-type include those which have one or more synonymous changes in the nucleic acid sequence (changes which do not change the encoded protein sequence), common non-synonymous polymorphisms (eg, germline polymorphisms) that alter protein sequence but do not affect protein function, and splice site and intronic sequence changes.
[000411] According to another aspect of the invention, there is provided a method for determining the likelihood of effectiveness of treatment with a compound of Formula (I) in a human patient afflicted with cancer comprising: detecting the presence or absence of at least one non-synonymous nucleic acid variation in said patient's PIK3CA gene relative to the wild-type gene, wherein the presence of at least one non-synonymous nucleic acid somatic variation in the PIK3CA gene indicates that the trait is likely -treatment with the compound of Formula (I) is effective.
[000412] In accordance with another aspect of the invention, there is provided a method for assessing a subject's susceptibility to treatment with a compound of Formula (I), which method comprises: determining the non-synonymous mutation status of the gene of PIK3CA in the individual's tumor cell DNA; and (ii) determining the susceptibility of the susceptible subject to treatment with a compound of Formula (I) as a function of the non-synonymous mutation status of the PIK3CA gene in the tumor cells.
[000413] There are numerous methods available to those skilled in the art to determine the status of the PIK3CA gene. Gene status can be determined by determining the nucleic acid sequence. This could be done through direct sequencing of the full-length gene or analysis of specific sites within the gene, for example commonly mutated sites.
[000414] An alternative means of determining whether the PIK3CA gene is wild-type or mutant or not is to assess the function of the transcribed gene. Functional mutation of this PIK3CA gene produces a protein that has increased lipid kinase activity, resulting in increased downstream signaling pathway in cells including, but not limited to, activation of Akt and S6 kinases. Assays to assess the functional status of PIK3CA variants when expressed in cells include, but are not limited to: (i) increased production of the kinase activity product of the PIK3CA gene, phosphatidylinositol triphosphate (PI(3,4,5 )P3); (ii) increased levels of phosphorylated Akt or S6 kinase; (iii) increased focus and colony formation of NIH-3T3 cells transfected with the PIK3CA variant; (Ikenoue T. et al., Cancer Res. 65, 2005, 4562-4567 ). samples
[000415] The patient sample to be tested for gene status may be any tumor tissue or tumor cells containing the sample obtained or obtained from the individual. The test sample is conveniently a sample of blood, buccal swab, biopsy or other body fluid or tissue obtained from an individual. Particular examples include: circulating tumor cells, circulating DNA in plasma or serum, cells isolated from the ascitic fluid of patients with ovarian cancer, pulmonary sputum from patients with lung tumors, a fine needle aspirate from a patient with breast cancer, urine, peripheral blood, a cell scraping, a hair follicle, a patch of skin, or a mouth sample.
[000416] It will be appreciated that the test sample may also be a nucleic acid sequence which corresponds to the sequence in the test sample, i.e. all or a part of the nucleic acid region in the sample may first be amplified using any convenient technique eg Polymerase Chain Reaction (PCR) before analysis. The nucleic acid can be genomic DNA or fractionated or whole cellular RNA. In particular embodiments, the RNA is whole cellular RNA and is used directly as a template for labeling a first strand of cDNA using random primers or poly A primers. The nucleic acid or protein in the test sample can be extracted from the sample. according to conventional methodologies (see Green & Sambrook, Eds., Molecular Cloning: A Laboratory Manual, (2012, 4th edition, Vol. 1-3, ISBN 9781936113422), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The diagnostic methods of the invention can be performed using a sample previously taken from the patient or individual. Such samples may be preserved by freezing or fixed and embedded in formalin-paraffin or other means. Alternatively, a sample containing fresh tumor cell can be obtained and used.
[000417] The methods of the invention can be applied using the cells of any tumor. Tumors suitable for treatment with a compound of Formula (I) have been described hereinabove.
[000418] Mutations in PIK3CA are widely found in clinical tumors, but the prevalence of mutations in each of the genes varies significantly by tumor tissue type. For example, mutations in PIK3CA are relatively common in breast cancer but relatively rare in kidney tumors. Table 1



[000419] Table 1: Prevalence of PIK3CA mutations in clinical samples. Source of information about PIK3CA is the COSMIC database (release v62). The patient selection methods of the invention may be particularly useful in disease (tissue) segments where there is a high incidence of PIK3CA mutations (e.g. breast, urinary tract, endometrial, large intestine, cervical, etc.).
[000420] As will be evident to those skilled in the art, these frequency data are continually being refined and updated as new and more comprehensive data emerge from the Human Cancer Genome Profiling consortium, such as TCGA (The Cancer Genome Atlas) and ICGC (International Cancer Genome Consortium). Consequently, additional PIK3CA-dependent tumor types may be identified and eligible for treatment with the compounds described herein. Methods for Detection of Nucleic Acids
[000421] Detection of mutant PIK3CA nucleic acids can be employed, within the scope of the present invention, to predict response to drug treatment. Since mutations in these genes occur at the DNA level, the methods of the invention can be based on detecting mutations or variations in genomic DNA, as well as transcripts and proteins themselves. It may be desirable to confirm mutations in genomic DNA by analyzing transcripts and/or polypeptides, in order to ensure that the detected mutation is, in fact, expressed in the individual.
[000422] It will be apparent to those skilled in the art that there are a large number of analysis procedures that can be used to detect the presence or absence of variant nucleotides at one or more positions in a gene. In general, detection of an allelic variation requires a mutation discrimination technique, optionally an amplification reaction (such as one based on polymerase chain reaction) and, optionally, a signal generation system. There are a multitude of mutation detection methods available in the art and these can be used in combination with a signal generation system, of which there are numerous available in the art. Many methods for detecting allelic variation are reviewed by Nollau et al., Clin. Chem., 1997, 43, 1114-1120; Anderson S.M. Expert Rev Mol Diagn. 2011, 11, 635-642; Meyerson M. et al., Nat Rev Genet., 2010, 685-696; and in standard textbooks, for example, "Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford University Press, 1996 and "PCR", 2nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.
[000423] As noted above, determining the presence or absence of a special variation or plurality of variances in the PIK3CA gene in a cancer patient can be accomplished in a variety of ways. Such tests are commonly performed using DNA or RNA from biological samples collected, for example, tissue biopsies, urine, feces, sputum, blood, cells, tissue scrapings, breast aspirates or other cellular materials, and may be performed using a variety of methods including, but not limited to, PCR, hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatches, mass spectrometry, or DNA sequencing, including minisequencing.
[000424] Suitable mutation detection techniques include the Amplification Refractory Mutation System (ARMS™), Amplification Refractory Mutation System Linear Extension (ALEX™), Competitive Oli-gonucleotide Priming System (COPS), Taqman, Molecular Beacons, restriction fragment length polymorphism (RFLP) and restriction site-based PCR and Fluorescence Resonance Energy Transfer (FRET) techniques.
[000425] In particular embodiments, the method used to determine the nucleotide(s) within a biomarker gene is selected from: allele-specific amplification (allele-specific PCR) - such as the Amplification Refractory Mutation System (ARMS) ), sequencing, allelic discrimination assay, hybridization, restriction fragment length polymorphism (Restriction Fragment Length Polymorphism - RFLP) or an oligonucleotide binding assay (Oligonucleotide Ligation Assay - OLA).
[000426] In particular embodiments, hybridization with allele-specific probes can be performed by: (1) allele-specific oligonucleotides bound to a solid phase (eg, glass, silicon, nylon membranes) with the labeled sample in solution , for example, as in many DNA chip applications; or (2) ligated sample (often cloned DNA or PCR amplified DNA) and solution-labeled oligonucleotides (allele-specific or short, to allow hybridization sequencing). Diagnostic tests may involve a panel of variances, often on a solid support, which allows the simultaneous determination of more than one variance. Such hybridization probes are well known in the art (see, for example, Green & Sambrook, Eds., Molecular Cloning: A Laboratory Manual, (2012, 4th edition, Vol. 1-3, ISBN 9781936113422), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and may span two or more sites of variation.
[000427] Thus, in one embodiment, detection of the presence or absence of at least one mutation requires contacting the PIK3CA nucleic acid containing a putative mutation site with at least one nucleic acid probe. The probe preferentially hybridizes to a nucleic acid sequence that includes a site of variance and contains complementary nucleotide bases at the site of variance under selective hybridization conditions. Hybridization can be detected with a detectable marker using markers known to those skilled in the art. Such labels include, but are not limited to, radioactive, fluorescent, dye, and enzymatic labels.
[000428] In another embodiment, detection of the presence or absence of at least one mutation requires contacting the PIK3CA nucleic acid containing a putative mutation site with at least one nucleic acid primer. The primer preferentially hybridizes to a nucleic acid sequence that includes a site of variance and contains nucleotide bases complementary to the site of variance under selective hybridization conditions.
[000429] Oligonucleotides used as primers for specific amplification can bring the nucleotide base complementary to the mutation of interest at the center of the molecule (so that amplification depends on differential hybridization; see, for example, Gibbs et al. , 1989, Nucl. Acids Res., 17, 2437-248) or at the 3'-terminal end of a primer where, under appropriate conditions, incompatibility can prevent or reduce polymerase extension (see, for example, Prossner, 1993, Tibtech. 11,238).
[000430] In yet another embodiment, detecting the presence or absence of at least one mutation comprises sequencing at least one nucleic acid sequence and comparing the obtained sequence with the known wild-type nucleic acid sequence.
[000431] Alternatively, the presence or absence of at least one mutation comprises determination, by mass spectrometry, of at least one nucleic acid sequence.
[000432] In one embodiment, detecting the presence or absence of at least one nucleic acid variance comprises performing a Polymerase Chain Reaction (PCR). The target nucleic acid sequence that contains the hypothetical variation is amplified and the nucleotide sequence of the amplified nucleic acid is determined. Determining the nucleotide sequence of the amplified nucleic acid comprises sequencing at least one segment of nucleic acid. Alternatively, the amplification products can be analyzed using any method capable of separating the amplification products according to their size, including automated and manual gel electrophoresis and so on.
[000433] Mutations in the genomic nucleic acid are advantageously detected by means of techniques based on mobility shift in amplified nucleic acid fragments. For example, Chen et al., Anal Biochem 1996, 239, 61-9, describe the detection of single base mutations by a competitive mobility shift test. In addition, essays based on the technique of Marcelino et. al., BioTechniques 1999, 26, 1134-1148, are commercially available.
[000434] In one particular example, capillary heteroduplex analysis can be used to detect the presence of mutations based on shift in duplex nucleic acid mobility in capillary systems as a result of the presence of mismatches.
[000435] Generating nucleic acids for sample analysis generally requires nucleic acid amplification. Many amplification methods rely on an enzyme chain reaction (such as a polymerase chain reaction, ligase chain reaction, or a self-sustaining sequence replication) or replication of all or part of the vector into which it has been cloned. Preferably, the amplification according to the invention is exponential amplification as demonstrated, for example, by the polymerase chain reaction.
[000436] Many methods of signal and target amplification have been described in the literature, for example, overviews of these methods in Landegren U. et. al., Science, 1988 242, 229-237 and Lewis R., Genetic Engineering News 1990, 10, 54-55. These amplification methods can be used in the methods of the present invention and include polymerase chain reaction (PCR), in situ PCR, ligase amplification reaction (LAR), ligase hybridization, bacteriophage QP replicase, Transcription-based Amplification System (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (Nucleic Acid Sequence-Based Amplification - NASBA) and in situ hybridization. Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
[000437] Polymerase Chain Reaction (PCR) is a nucleic acid amplification method described, inter alia, in United States Patent Nos. 4,683,195 and 4,683,202. PCR consists of repeated cycles of primer extension reactions generated with DNA polymerase. The target DNA is thermally denatured and two oligonucleotides, which span the target sequence on opposite strands of the DNA to be amplified, are hybridized. These oligonucleotides become primers for use with DNA polymerase. The DNA is copied by extension of the primer to make a second copy of both strands. By repeating the cycle of thermal denaturation, primer annealing, and extension, the target DNA can be amplified a million-fold or more, in about two to four hours. PCR is a molecular biology tool that must be used in conjunction with a detection technique to determine amplification results. An advantage of PCR is that it increases sensitivity by amplifying the amount of target DNA by 1 million to 1 billion times in approximately 4 hours. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., Gynaecologic Oncology, 1994, 52: 247-252).
[000438] An allele-specific amplification technique, such as the amplification Refractory Mutation System (ARMS™) (Newton et al., Nucleic Acids Res.. 1989, 17, 2503-2516, can also be used to detect mutations of a Under the appropriate PCR amplification conditions, a single base mismatch located at the 3' end of the primer is sufficient for preferential amplification of the perfectly matched allele (Newton et al., 1989, supra), allowing species discrimination closely. The basis of an amplification system using the primers described above is that oligonucleotides with a mismatched 3' residue will not function as primers in PCR under the appropriate conditions. This amplification system allows genotyping solely by inspection of agarose gel reaction mixtures after electrophoresis.
[000439] Analysis of the amplification products can be performed using any method capable of separating the amplification products according to their size, including automated and manual gel electrophoresis, mass spectrometry, and so on.
[000440] Methods of isolation, amplification, and nucleic acid analysis are routine for those skilled in the art, and examples of protocols can be found, for example, in Green & Sambrook, Eds., Molecular Cloning: A Laboratory Manual, ( 2012, 4th edition, Vol. 1-3, ISBN 9781936113422), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). A particularly useful protocol source for methods used in PCR amplification is PCR (Basics: From Background to Bench) by M.J. McPherson, S.G. Mailer, R. Beynon, C. Howe, Springer Verlag; 1st edition (October 15, 2000), ISBN: 0387916008.
[000441] The present invention also provides diagnostic kits comprising predictive and degenerate primers for amplifying a target nucleic acid in the PIK3CA gene and instructions, amplification protocol, and analysis of the results. The kit may alternatively also comprise buffers, enzymes and containers for carrying out amplification and analyzing the amplification products. The kit may also be a component of a screening or diagnostic kit comprising other tools, such as DNA microarrays or other carriers. Preferably, the kit also provides one or more control templates, such as nucleic acids isolated from normal tissue samples, and/or a series of samples representing different variances in the reference genes.
[000442] In one embodiment, the kit provides two or more pairs of primers, each pair capable of amplifying a different region of the reference gene (PIK3CA) (each region a site of potential variance) thereby providing a kit for analyzing the expression of several gene variances in a biological sample in one reaction or several parallel reactions.
[000443] The primers in the kits can be labeled, eg fluorescently labeled, to facilitate detection of amplification products and consequent analysis of nucleic acid variances. The kit may also allow more than one deviation to be detected in an analysis. A combined kit, therefore, will comprise primers capable of amplifying different segments of the reference gene. Primers can be differentially labeled, for example using different fluorescent labels, in order to differentiate between variances.
[000444] Kits for use in detecting PIK3CA mutations are also described, including, but not limited to, the PIK3CA mutation detection kits marketed by diagnostic companies, including Qiagen and Roche Molecular Systems.
[000445] In another aspect, the invention provides a method of treating a patient who is suffering from cancer comprising: determining the wild-type or mutant status of the PIK3CA gene in the patient's tumor cells and, whether the PIK3CA gene is mutant, administering to the patient an effective amount of a compound of Formula (I).
[000446] As used herein, the terms "effective" and "efficacy" include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of the treatment to achieve a desired biological effect on the patient. Physiological safety refers to the level of toxicity or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side effects) resulting from the administration of the treatment. "Less effective" means that the treatment results in a lower therapeutically significant level of pharmacological efficacy and/or a therapeutically higher level of adverse physiological effects.
[000447] According to another aspect of the invention, there is provided the use of a compound of Formula (I) for treating a cancer patient whose tumor cells have been identified as having a mutant PIK3CA gene.
[000448] According to another aspect of the invention, there is provided a compound of Formula (I) for treating cancers with tumor cells identified as carrying a mutant PIK3CA gene.
[000449] In still other embodiments, the invention relates to a pharmaceutical composition comprising a compound of Formula (I) for use in the prevention and treatment of cancer with tumor cells identified as carrying a mutant PIK3CA gene.
[000450] For all of the above, mutant forms of PIK3CA are determined/identified at all positions throughout the gene.
[000451] For all of the above, using tumors such as breast cancer as an example, certain mutant forms of PIK3CA determined/identified are those at positions R38, R88, N345 5, C420, E453, P539, E542K, E545K, Q546, M1043, M1043 and H1047R,
[000452] For all of the above, using tumors such as breast cancer as an example, certain mutant forms of PIK3CA determined/identified are those at positions E542, E545 and H1047. Examples of Personalized Health Care/Personalized Medicine Cell Proliferation Assay In Tumor Cell Lines
[000453] The sensitivity of a panel of human cancer cell lines to the effects of the compounds was determined in a standard proliferation assay. Details of the assay protocol are given under biological assay(s) (g), above. Mutation Correlation Analysis Methods
[000454] Measurement of pharmacological data of cell growth inhibition in response to treatment with Example 3 was obtained for a set of 209 cancer cell lines from a variety of tissues and from various sources. Each cell line was classified as sensitive (GI50 <= 1.0 μM) or resistant (GI50 > 1.0 μM).
[000455] The mutation status for the genes in each cell line was obtained by integrating results from internal (Astra-Zeneca) and public sources. Public data included all cell line data from the Genomics of Drug Sensitivity in Cancer Project databases, release 3 (Garnett MJ et al. Nature, 2012, Mar 483, 570-5), Cancer Cell Line Encyclopedia Project (Barretina J. et al., Nature 2012, 483, 603-7) and Catalog of Somatic Mutations In Cancer (COSMIC) (release v61; http://www.sanger.ac.uk/genetics/CGP/cosmic/; Forbes SA et al. Nu-cleic Acids Res. 2011,39 (database edition): D945-50; Forbes SA et al., Curr Protoc Hum Genet. 2008; Chapter 10: Unit 10.11) and selected journal articles . Silent mutations in coding regions (synonymous variants) and non-synonymous polymorphisms were excluded and, for the purposes of this analysis, the zygosity of mutations was ignored. For each cell lineage and gene combination, status was summarized as mutant (MUT), wild-type (WT), or inconsistent (INCON). Some initially inconsistent cases (independent WT and MUT observations for the same gene in the same cell lineage) were resolved by weighting internal observations and those for the COSMIC Cancer Cell Line Pro-ject (CCLP) subset or selecting a state after manual review. In cases where inconsistent observations could not be resolved, the INCON identifier was retained and the gene status was considered unknown during analysis.
[000456] Associations between mutation status and response were identified by building contingency tables for each gene and determining odds ratios and p-values by doubly configured Fisher's exact test. Cell lines classified as marginal for the response were excluded from the initial analysis to identify candidate biomarkers. For mutation status, MUT or WT found were counted and genes with less than 4 WT cell lines or 4 MUT cell lines were also excluded. Results and discussion
[000457] Associations between mutation status and response were identified as described in Methods. The cell line response to Example 3 and the corresponding genetic status of the PIK3CA gene are shown in Table 3. Table 3. Pharmacology, response classification and mutation status of the PIK3CA gene for the PIK3CA gene lines. cells used in this study.









[000458] The gene for which mutations were most strongly correlated with sensitivity to Example 3 was PIK3CA. Only 12 of 177 PIK3CA WT cell lines (7.7%) were sensitive to Example 3, while 15 of 32 cell lines (46.9%) that are PIK3CA mutants were sensitive, which corresponds to a proportion of odds of 12.1 and a p-value of 1.2 x 10-7 (see Table 4). Table 4. Contingency table for PIK3CA mutation status and response to Example 3.
Odds Ratio: 12.1 p-value: 1.2 x 10-7
[000459] As already mentioned, it has been reported that measurement of the mutation status or activation status of additional genes, such as KRAS, a potential marker of resistance in tumors with aberrant or dysregulated PIK3CA or PI3K-α, could help to increase the predictability of a personalized medicine approach.
[000460] This was exemplified for the above dataset comparing enrichment of KRAS mutations in PIK3CA mutant cells with the response to cell line inhibition. The analysis was limited to cell lines containing "hotspot" mutations of the two genes (at codons E542, E545 and H1047 for PIK3CA and at codons K12, 13 and Q61 for KRAS). This demonstrated that, in PIK3CA mutant cell lines, mutations in KRAS conferred resistance to inhibition by Example 3.
[000461] - Twenty-eight cell lines contained activating mutations in PIK3CA.
[000462] - 6 of 19 cell lines (31.6%) contained an activating mutation in PIK3CA and a wild-type KRAS gene was resistant to Example 3.
[000463] - 7 of 9 PIK3CA mutant cell lines (77.8%) contained coexisting KRAS mutations and were resistant to Example 3.
[000464] This translates to an odds ratio of 7.5 and a p-value of 0.042 (see Table 5).
Odds Ratio: 7.5 p-value: 0.042 Table 5. Contingency table for mutation status in PIK3CA and KRAS and response to Example 3. EXAMPLES
[000465] The invention will now be illustrated in the following Examples in which, in general: (i) the operations were carried out at room temperature, that is, in the range of 17 to 25°C, and under an atmosphere of a gas inert, such as nitrogen, unless otherwise indicated; (ii) evaporations were carried out by rotary evaporation or using Genevac in vacuo equipment and processing procedures were carried out after removal of residual solids through filtration; (iii) purifications by means of flash chromatography were performed on an automated Armen Glider Flash: Spot II Ultimate system (Armen Instrument, Saint-Ave, France) using Merck Normal Phase Si60 Silica cartridges (particle size: 15-40 or 40-63 μmm) obtained from Merck, Darmstad, Germany; (iv) preparative chromatography was performed on a Waters instrument (600/2700 or 2525) equipped with ZMD or ZQ ESCi mass spectrometers and a Waters X-Terra or Waters X-Bridge or Waters SunFire (C- 18.5 micron, silica, 19 mm diameter, 100 mm long, flow rate 40 mL/minute), using decreasingly polar mixtures of water (containing 0.2% ammonium carbonate) and acetonitrile as eluent ; (v) yields, when present, are not necessarily the maximum attainable; (vi) in general, the structures of the final products of formula I were confirmed by means of nuclear magnetic resonance (Nu-clear Magnetic Resonance - NMR); NMR chemical shift values were measured on the delta scale [roton magnetic resonance spectra were determined using a Bruker Avance 500 instrument (500 MHz)]; measurements were performed at room temperature, unless otherwise indicated; the following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublet; dt, doublet of triplets; bs, broad signal; (vii) in general, general by-products of Formula I were also characterized by means of mass spectroscopy after liquid chromatography (Liquid Chromatography Mass Spec-troscopy - LCMS); LCMS was performed using a Waters Alliance HT (2790 & 2795) equipped with a Waters ZQ ESCi or ZMD ESCi mass spectrometer and a 5 μm (2.1 x 50 mm) C-18 X Bridge column with a flow rate of 2.4 ml/min, using a solvent system of 95% A + 5% C to 95% B + 5% C for 4 minutes, where A = water, B = methanol, C = methanol:water at 1:1 (containing 0.2% ammonium carbonate); (viii) the intermediates, in general, were not fully characterized and the purity was evaluated by means of thin layer chromatography, mass spectrometry, HPLC and/or NMR analysis;
[000466] (ix) X-ray powder diffraction spectra were determined (using a Bruker D4 Analytical Instrument) by mounting a sample of the crystalline material on a single Bruker silicon crystal (SSC) and spreading the sample in a thin layer with the aid of a microscope slide. The sample was centrifuged at 30 revolutions per minute (to improve counting statistics) and irradiated with X-rays generated by a long (copper ino) focusing tube operated at 40 kV and 40 mA, with a wavelength of 1, 5418 angstroms The collimated X-ray source was passed through an automatic variable divergence slit fixed at V20 and the reflected radiation directed through a 5.89 mm anti-scatter slit and a 9.55 mm slit detector. The sample was exposed for 0.03 seconds in 0.00570° increments 2-theta (continuous scan mode) over the range 2 degrees to 40 degrees 2-theta in theta-theta mode. The operating time was 3 minutes and 36 seconds. The instrument was equipped with a position-sensitive detector (Lynxeye). Control and data capture were performed using a Dell Optiplex 686 NT 4.0 Workstation operating with Diffrac+ software. in the X-ray powder diffraction technique will notice that the relative intensity of the pi cos can be affected, for example, by grains over 30 microns in size and non-unit proportions that can affect sample analysis. Those skilled in the art will also appreciate that the reflection position can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer. The flatness of the surface of the sample may also have a small effect. Thus, the diffraction pattern data presented should not be understood as absolute values; (x) Differential Scanning Calorimetry was performed using a TA Instruments DSC Q1000 instrument. Typically, less than 5 mg of material contained in a standard aluminum container equipped with a lid was heated over the temperature range of 25°C to 300°C at a constant heating rate of 10°C per minute. A purge gas using nitrogen was used at a flow rate of 50 mL per minute; and
[000467] (xi) the following abbreviations have been used: aq. aqueous CDCl3 deutero-chloroform CHCl3 chloroform DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCM dichloromethane DEA diethylamine DIPEA N-ethyl-N-isopropylpropan-2-amine DMA N,N-dimethylacetamide DMF N-dimethylformamide DMSO dimethyl sulfoxide DSC Differential Scanning Calorimetry DtAD (E)-di-tert-butyl diazene-1,2-dicarboxylate EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Diethyl ether EtOH ethanol EtOAc ethyl % Ee % enantiomeric excess HOPO 2-hydroxy-pyridine N-oxide HPLC High performance liquid chromatography IPA isopropyl alcohol MCN acetonitrile MeOH methanol MIBK methyl isobutyl ketone MTBE methyl tert-butyl ether NMP 1-methyl-2-pyrrolidone at temp sat environment saturated sun. THF solution tetrahydrofuran TEA triethyl amine TBTU 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium v/v volume/volume TFA trifluoroacetic acid Example 1: 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-Methyl- 1 H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

[000468] 3-Hydroxy-propanoic acid (30% v/v soln in water) (200 µl, 47.0 mg, 0.52 mmol) was evaporated to dryness, then azeotroped with toluene. The acid was dissolved in NMP (1 mL) and molecular sieves (100 mg, 0.26 mmol), N-ethyl-N-isopropylpropan-2-amine (0.136 mL, 0.78 mmol) were added, followed by the addition of 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (209 mg, 0.65 mmol). After 30 minutes of stirring, 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2 ,4-triazol-5-yl)pyrazin-2-amine (100mg, 0.26mmol) was added and the mixture was stirred for 2 hours. The reaction mixture was purified by preparative HPLC using a reversed-phase Waters X-Bridge column (C-18, 5 micron, silica, 19 mm diameter, 100 mm long, flow rate 40 ml/minute) and decreasingly polar mixtures of water (containing 0.2% ammonium carbonate) and acetonitrile as eluent.
[000469] Fractions containing the desired compound were evaporated to dryness to afford 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin) -2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (45.0 mg, 37.9%) as a pale yellow solid: 1 H NMR spectrum (CDCl 3 ) 1.52 (9H, s), 1.79-1.94 (2H, m), 2.07-2.15 (2H, m), 2.58 (2H, 10 t), 2.84-2.94 (1H, m), 3.00 - 3.10 (1H, m), 3.17-3.26 (1H, m), 3.53 (1H, t), 3.86-3.94 (3H, m), 4.30 (3H, s), 4.56 -4.62 (1H, m), 9.02 (1H, s); Mass Spectrum [M+H]+ = 456.
[000470] 3-(5-tert-Butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4- triazol-5-yl)pyrazin-2-amine (Example 1.1) was prepared as follows.
[000471] At 20°C, tert-butyl 4-carbamoylpiperidine-1-carboxylate (47 g, 205.88 mmol) in dichloromethane (500 mL) was added dropwise to a stirred solution of triethylaxon hexafluorophosphate (V) (56.2 g, 226.47 mmol) in dichloromethane (500 mL) over a period of 45 minutes, under nitrogen. The resulting solution was stirred at 20°C overnight. A saturated aqueous solution of Na2CO3 was then added until a pH of 8 was obtained. The phases were decanted and the aqueous phase was extracted again with 200 ml of CH2Cl2, then the organic phases were dried over MgSO4, filtered and concentrated to obtain tert-butyl-4-(ethoxy(imino)methyl)1-carboxylate piperidine (51.0 g, 97%) as a colorless liquid: 1 H NMR spectrum (CDCl3 ) 1.28 (3H, t), 1.46 (9H, s), 1.47 (2H, m), 1 .79-1.93 (2H, m), 2.28 (1H, m), 2.73 (2H, m), 4.10 (2H, q), 4.13-4.18 (2H, m ); Mass Spectrum [M+H]+ = no mass ion.
[000472] To a stirred solution of tert-butyl 4-(ethoxy(imino)methyl)piperidine-1-carboxylate (51 g, 198.95 mmol) in dioxane (500 mL) was added formohydrazine (17.92 g). , 298.43 mmol). This solution was allowed to stir at 40°C overnight under N2 resulting in the precipitation of a white solid (intermediate hydrazide). The reaction mixture was then heated at 80°C for 6 hours, cooled to room temperature and concentrated. The residue was dissolved in 500 ml of CH2Cl2 and 300 ml of water were added. The phases were decanted and the organic phase was washed with brine, dried over MgSO4, filtered and concentrated to obtain tert-butyl-4-(1H-1,2,4-triazol-3-yl)1-carboxylate. piperidine (46.0 g, 92%) as a white solid: 1 H NMR Spectrum (CDCl 3 ) 1.47 (9H, s), 1.76 (2H, m), 1.98-2.11 ( 2H, m), 2.91 (2H, s), 2.97-3.08 (m, 1H), 4.06-4.23 (2H, m), 8.05 (1H, s); Mass Spectrum [M+H]+ = no mass ion.
[000473] To a stirred solution of tert-butyl 4-(1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (22 g, 87.19 mmol) in dichloromethane (250 mL) was 2N sodium hydroxide (131 mL, 261.58 mmol) is added. The reaction mixture was vigorously stirred with a mechanical stirrer and a solution of benzyltrimethylammonium tribromide (37.4 g, 95.91 mmol) in dichloromethane (250 mL) was then added dropwise, maintaining the temperature at about 15°C. The reaction mixture was allowed to stir at room temperature for one hour and 2N HCl was added to provide a pH of 5 (keeping the temperature at about 15°C). The phases were decanted and the organic phase was washed with H2O (2x1 L), dried over MgSO4, filtered and concentrated to obtain 4-(5-bromo-1H-1,2,4-triazol-3- tert-butyl yl)piperidine-1-carboxylate (25.00 g, 87%) as an off-white solid: 1 H NMR spectrum (CDCl 3 ) 1.46 (9H, s), 1.67 -1.84 ( 2H, m), 1.90-2.13 (2H, m), 2.77-2.96 (2H, m), 2.98-3.10 (1H, m), 3.94-4, 35 (2H, m); Mass Spectrum [M+H]+ = no mass ion.
[000474] To a stirred suspension of tert-butyl 4-(5-bromo-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (26 g, 78.50 mmol) in toluene (200 mL) and methanol (50 mL) was added dropwise (diazomethyl)trimethylsilane (2M solution) in hexane (43.2 mL, 86.35 mmol) under N2, maintaining the temperature at about 20°C. °C: Gas release and a small exotherm were observed. The yellow solution obtained was stirred at room temperature for one hour. The solvent was evaporated and the resulting oil was purified on silica eluting with 40% EtOAc in petroleum ether to give 4-(5-bromo-1-methyl-1H-1,2,4-triazol-3-yl) tert-butyl piperidine-1-carboxylate (15.00 g, 55.3%) as an oil: 1 H NMR spectrum (CDCl 3 ) 1.46 (9H, s), 1.65-1.78 (2H, m), 1.90-2.01 (2H, m), 2.68-3.02 (3H, m), 3.83 (3H, s), 3.94-4.31 (2H, m) ; Mass Spectrum [M+H]+ = no mass ion.
[000475] Hydrazine monohydrate (34 ml, 1094.95 mmol) was added gradually to a stirred suspension of ethyl 3-aminopyrazine-2-carboxylate (21.3 g, 139.09 mmol) in ethanol (65 mL) at room temperature The resulting slurry was stirred at 60°C for two hours, cooled to room temperature and filtered. The solid was washed with ice-cold ethanol (2 x 25 ml) and dried to constant weight to provide 3-aminopyrazine-2-carbohydrazide (20.75 g, 97%) as a beige solid: 1 H NMR spectrum (DMSO-d6 ) 4.49 (2H, d), 7.46 (2H, br s), 7.78 (1H, d), 8.17 (1H, d), 9.79 (1H, t) ; Mass Spectrum [M+H]+ = 154.
[000476] 2-(1H-Benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (47.7 g; 148.69 mmol) was added gradually over 15 minutes to a stirred suspension of N-ethyl-N-isopropylpropan-2-amine (70.6 mL, 405.51 mmol), pivalic acid (17.08 mL, 148.69 mmol) and 3 -aminopyrazine-2-carbohydrazide (20.7 g, 135.17 mmol) in acetonitrile (350 mL) and the reaction mixture was stirred at 80°C for 20 minutes (a solution was obtained). The reaction mixture was cooled to 0°C and N-ethyl-N-isopropylpropan-2-amine (70.6 mL, 405.51 mmol), followed by 4-methylbenzene-1-sulfonyl chloride (77 g, 405 .51 mmol) were added over a period of 15 minutes. The reaction mixture (yellow suspension) was brought to reflux (solubilization) and then allowed to stir at room temperature for 14 hours, yielding a dark orange solution. The solution was concentrated. The residue was diluted with dichloromethane, washed with water, brine, dried over magnesium sulfate and concentrated. The crude product was purified by flash chromatography on silica gel eluting with 0 to 40% ethyl acetate in dichloromethane. The solvent was evaporated to dryness. The resulting mixture was triturated with ether (100 mL), filtered, washed with minimal ether and dried to obtain 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2- amine (20.8 g, 70.2%) as a pale yellow solid: 1 H NMR spectrum (CDCl 3 ) 1.53 (9H, s), 1.58-1.68 (2H, m), 6.67 (2H, s), 8.13 (2H, dt); Mass Spectrum [M+H]+ = 220.
[000477] 1-Bromopyrrolidine-2,5-dione (18.57 g, 104.36 mmol) was added gradually to a solution of 3-(5-tert-butyl-1,3,4-oxadiazole-2- yl)pyrazin-2-amine (20.8 g, 94.87 mmol) in THF (320 mL) and the solution was stirred at room temperature for 16 hours. The reaction mixture was concentrated and the residue was dissolved in dichloromethane (300 mL), washed with water (2 x 150 mL), brine, dried over magnesium sulfate and concentrated. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel, eluting with 0 to 10% ethyl acetate in dichloromethane. The solvent was evaporated to dryness to obtain 5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (25.5 g, 90%) as a beige solid: 1 H NMR spectrum (CDCl 3 ) 1.52 (9H, s), 8.23 (1H, s); Mass Spectrum [M+H]+ = 300.
[000478] To a suspension of 5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (45 g, 150.94 mmol) in toluene (450 mL) 1,1,1,2,2,2-hexamethyldiesthanane (37.6 mL, 181.12 mmol) and bis(triphenylphosphine)palladium (II) chloride (5.30 g, 7.55 mmol) were added. . The reaction mixture was degassed with argon and heated at 80°C for two hours (solubilization after heating, orange solution then reprecipitated and turned black, indicating the reaction was complete). The reaction mixture was cooled, concentrated and the residue was dissolved in CH2Cl2 and filtered over Decalite to remove insoluble impurities. The filtrate was concentrated and purified on silica eluting with 0-10% EtOAc in CH2Cl2. The solvent was concentrated to obtain 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(trimethylstannyl)pyrazin-2-amine (22.63 g, 39.2%) as an orange solid: 1 H NMR spectrum (CDCl 3 ) 0.38 (9H, s), 1.53 (9H, s), 6.49 (2H, broad s), 8.13 (1H, s); Mass Spectrum [M+H]+ = 384.
[000479] To a stirred suspension of tert-butyl 4-(5-bromo-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate 20 (2.700 mg, 7.82 mmols) and 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(trimethylstannyl)pyrazin-2-amine (2.988 mg, 7.82 mmols) in 4-methyl-2 -pentanol (28 ml) were added lithium chloride (995 mg, 23.46 mmol) and bis(triphenylphosphine)palladium (II) chloride (220 mg, 0.31 mmol). The mixture was degassed with argon and heated at 140°C for two hours. The reaction was cooled and the resulting precipitate was collected by filtration, washed with isopropanol (25 mL), water (25 mL) and dried under suction. The organic fraction was concentrated with isopropanol and the precipitate formed was collected and combined with the main precipitate, providing 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazole- tert-Butyl 2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (3.0 g, 79%): 1H NMR (DMSO-d6) 1.41 (9H, s), 1.45 (9H, s), 1.50 30-1.68 (2H, m), 1.95 (3H, dd), 2, 78-3.05 (1H, m), 3.96 (3H, d), 4.21 (3H, s), 8.86 (1H, s); Mass Spectrum [M+H]+ = 484.
[000480] A solution of 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H- tert-Butyl 1,2,4-triazol-3-yl)piperidine-1-carboxylate (3g, 6.20mmols) in TFA (15ml) and CH 2 Cl 2 (15ml) was stirred at 25°C for one hour. hour. The mixture was subjected to azeotropic distillation with toluene, a 7N solution of ammonia in methanol and dichloromethane were added and the mixture was adsorbed onto silica gel. The crude product was purified by flash chromatography on silica gel eluting with methanol in 0-8% dichloromethane, followed by methanolic ammonia in 0-10% dichloromethane. The solvent was evaporated to dryness to obtain 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1 ,2,4-triazol-5-yl)pyrazin-2-amine (2.040 g, 86%) as a yellow crystalline solid: 1 H NMR spectrum (DMSO-d6) 1.45 (9H, s), 1.55 -1.66 (2H, m), 1.86 (2H, dd), 2.52-2.61 (2H, m), 2.69-2.78 (1H, m), 2.95-3 .02 (2H, m), 4.20 (3H, s), 8.86 (1H, s); Mass Spectrum [M+H]+ = 384. Isolation of single crystalline form from Example 1
[000481] X-ray powder diffraction spectra of the isolated material showed that the material was crystalline but a mixture of polymorphic forms. This material had a melting point of 226.4°C (onset).
[000482] Form A material was produced by mixing the parent material in acetonitrile at 25°C. Approximately 20 mg of the original material was placed in a flask with a magnetic stir bar and approximately 2 mL of acetonitrile added. The vial was then hermetically sealed with a lid and allowed to stir on a magnetic stir plate. After about 5 days, the sample was removed from the plate, the lid removed and the suspension allowed to dry under ambient conditions before being analyzed by XRPD and DSC. This form (Form A) was determined to be crystalline by XRPD. This material had a melting point of 227.2°C (onset).
[000483] The same crystalline form can be obtained by stirring the raw material in acetonitrile overnight at room temperature, then filtering the resulting solid, washing with cold acetonitrile and drying.
[000484] In one aspect of the invention, there is provided a process for forming a crystalline form of Example 1 (Form A) by mixing a sample of the compound in acetonitrile. Ten X-ray powder diffraction peaks are shown in the Table below: Ten X-ray powder diffraction peaks for Form A of Example 1

[000485] An X-ray powder diffraction spectrum of Example 1, Form A, is shown in Figure 1. DSC analysis of Example 1, Form A, shows a melting endotherm with an onset at 227.2°C and a peak at 228.6°C (Figure 2).
[000486] Thus, DSC analysis shows that Example 1, Form A, is a solid with an onset of melting at about 227.2°C and a melting peak at 228.6°C.
[000487] A DSC of Example 1, Form A, is shown in Figure 2. X-Ray Powder Diffraction
[000488] Analytical instrument: Bruker D4.
[000489] The X-ray diffractogram was determined by placing a sample of the crystalline material on a Bruker single crystal silicon (SSC) mounting bracket and spreading the sample in a thin layer with the aid of a microscope slide. The sample was centrifuged at 30 revolutions per minute (to improve counting statistics) and irradiated with X-rays generated by a long-thin copper focusing tube operated at 40 kV and 40 mA with a wavelength of 1.5418 angstroms. . The collimated X-ray source was passed through an automatic variable divergence slit fixed at V20 and the reflected radiation directed through a 5.89 mm anti-scatter slit and a 9.55 mm slit detector. The sample was exposed for 0.03 seconds per 0.00570° 2-theta increment (continuous scan mode) over the range 2 degrees to 40 degrees 2-theta in theta-theta mode. The operating time was 3 minutes and 36 seconds. The instrument was equipped with a position-sensitive detector (Lynxeye). Control and data capture were via a Dell Optiplex 686 NT 4.0 Workstation running Diffrac+ software. Those versed in the technique of X-ray powder diffraction will realize that the relative intensity of peaks can be affected, for example grains over 30 microns in size and non-unit proportions can affect sample analysis. Those skilled in the art will also appreciate that the reflection position can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer. The flatness of the surface of the sample may also have a small effect. Consequently, the diffraction pattern data presented should not be taken as absolute values. Differential Scanning Calorimetry
[000490] Analytical instrument: TA Instruments DSC Q1000.
[000491] Typically, less than 5 mg of material contained in a standard aluminum container equipped with a lid was heated over the temperature range of 25°C to 300°C at a constant heating rate of 10° C per minute. A purge gas using nitrogen was used at a flow rate of 50 ml per minute.
[000492] An alternative synthesis of the compound of Example 1 is given below as Example 2. Example 2: 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4) -oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

[000493] Pyridine 4-methylbenzenesulfonate (3.58 g, 14.25 mmol) was added to a suspension of 1-(4-(5-(5-amino-6-(5-tert-butyl-1) ,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro- 2H-pyran-2-yloxy)propan-1-one (37 g, 68.57 mmol) in methanol (275 mL) under nitrogen. The mixture was stirred at 60°C for 1.5 hours. The mixture was soluble after 5 minutes. The mixture was kept at 50°C overnight, during which time a precipitate formed. The reaction mixture was dissolved in dichloromethane (400 ml), washed with water (300 ml) and brine (100 ml). The aqueous extracts were washed with DCM (100 mL) and the combined organic layers were dried over MgSO4 and concentrated. The crude product was purified by flash chromatography on silica gel eluting with 100% ethyl acetate in 10:50:40 methanol/ethyl acetate/DCM. The product-containing fractions were evaporated to dryness to obtain a beige solid (24.5 g). The solid was slurried overnight in acetonitrile (500 mL), filtered and dried under high vacuum to obtain 1-(4-(5-(5-amino-6-(5-tert-butyl-1, 3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one ( Example 2) (24 g, 78%) as a cream solid: 1H NMR spectrum (DMSO-d6) 1.51 (9H, s), 1.55-1.68 (1H, m), 1.68- 1.84 (1H, m), 1.96-2.13 (2H, m), 2.78-2.93 (1H, m), 2.98-3.1 (1H, m), 3. 19-3.3 (1H, m), 3.71 (2H, q), 3.93-4.04 (1H, m), 4.27 (3H, s), 4.35 -4.48 ( 1H, m), 4.54 (1H, t), 7.96 (2H, s), 8.92 (1H, s); Mass Spectrum [M+H]+ = 456.
[000494] (4-(5-(5-Amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1, 2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one (Example 2.1) was prepared as follows.
[000495] Hydrazine hydrate (23.59 mL, 480.75 mmol) was added dropwise to a stirred mixture of methyl 3-amino-6-bromopyrazine-2-carboxylate (100 g, 418.04 mmol) in EtOH (2 L). The mixture was heated to 50°C under nitrogen. The resulting slurry was stirred at 50°C for 16 hours. In addition, hydrazine (2.5 ml) was added in one portion and the suspension was stirred at 50°C for a further 24 hours. Ethanol (500 mL) was charged to the thick reaction mixture and the mixture was allowed to cool to room temperature. The resulting suspension was filtered and the solid was washed with ethanol (1 L) and dried in vacuo to provide 3-amino-6-bromopyrazine-2-carbohydrazide (98 g, quantitative) as a cream solid: 1 H NMR spectrum (DMSO-d6) 4.52 (2H, s), 7.59 (2H, s), 8.30 (1H, s), 9.74 (1H, s); Mass Spectrum [M+H]+ = 232.
[000496] Pivalic anhydride (165 mL, 815.38 mmol) was added to a stirred mixture of 3-amino-6-bromopyrazine-2-carbohydrazide (172 g, 741.26 mmol) in acetonitrile (1.8 L ) and the mixture was heated at 80°C for one hour. The reaction was allowed to stir for 16 hours. The required yellow solid material was isolated by filtration. The filtrate was partitioned between EtOAc (2 L) and aqueous sodium bicarbonate (2 L). The organic layer was washed with saturated brine and dried over MgSO4 . The solution was filtered and concentrated to obtain a sticky orange solid, which was triturated with MTBE (250 mL). The insoluble yellow solid was isolated by filtration and this material was shown to be identical to the first solid. The combined solids were dried in a vacuum oven at 50°C for 3 days to provide 3-amino-6-bromo-N-pivaloylpyrazine-2-carbohydrazide (224 g, 96%) as a solid. yellow: 1H NMR spectrum (DMSO-d6) 1.17 (9H, s), 7.62 (2H, s), 8.37 (1H, s), 9.42-9.56 (1H, m) , 10.09-10.23 (1H, m); Mass Spectrum [M+H]+ = 318.
[000497] Toluenesulfonyl chloride (164 g, 861.60 mmol) was added gradually to a suspension of 3-amino-6-bromo-N-pivaloylpyrazine-2-carbohydrazide (227 g, 718.00 mmol ) and N,N-diisopropylethylamine (297 m1,1795.01 mmol) in acetonitrile (2200 ml). The mixture was stirred for two hours at 70°C. The reaction was allowed to cool to room temperature overnight. The reaction mixture was partitioned between ethyl acetate (2 L) and sodium bicarbonate solution (2 L). The organic layer was washed with saturated brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting brown/beige solid was triturated with hot MTBE (1000 mL) and isolated by filtration and dried to provide 5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl) pyrazin-2-amine as a yellow solid (187 g, 87%). The mother liquors were evaporated to dryness. The crude solid was triturated with MTBE (500 mL), filtered and washed with 100 mL of MTBE. The resulting solid was air-dried overnight to obtain a second crop of 5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (36 g , 17%) : 1 H NMR spectrum (DMSO-d6) 1.43 (9H, s), 7.70 (2H, s), 8.39 (1H, s); Mass Spectrum [M+H]+ = 298.
[000498] In an alternative preparation, to 3-amino-6-bromo-N-pivaloylpyrazine-2-carbohydrazide (2.301 g, 7.28 mol) in MeCN (10.8 L) was added DIPEA (3.044 L, 17.48 mol) and p-toluenesulfonyl chloride (1.665 g, 8.73 mol) gradually (~280 gx 6) at 50°C over a period of 30 minutes. The reaction temperature was maintained between 65-70°C by controlling the rate of addition. After the addition was complete, the reaction mixture was stirred at 70°C for one hour. The mixture was cooled to room temperature and quenched with 5% NaHCO3 (aqueous, 24.2 L). The resulting suspension was stirred for 30 minutes and then filtered. The product was washed with water (14.8 L), dried at 50°C for 16 hours. The product was dissolved in DCM (12 L) and the phases were separated. The organic phase was loaded onto a silica block (6 kg) and the product was eluted with 20% EtO-Ac/DCM (8 x 10L). Concentration of the product containing fractions provided 1987 g (92% yield) with a purity of 99.8% by HPLC.
[000499] A stream of nitrogen gas was passed through a solution of 5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (89.35 g , 239.75 mmol) in DMA (1.2 L) for 20 minutes. Dicyclo(2',4',6'-tri-2-yl)phosphine (11.43 g, 23.98 mmol), tris(dibenzylideneacetone)dipalladium (0) (5.49 g, 5.99 mmol ), zinc (1.568 g, 23.98 mmol) and dicyanozinc (16.89 g, 143.85 mmol) were added sequentially to the stirred mixture. The mixture was heated to 100°C and stirred for one hour. The mixture was cooled and partitioned between DCM (3L) and water (1L). The black mixture was filtered through celite and the organic layer was separated. The solution was washed with water and then with brine. The solution was dried over magnesium sulfate and concentrated under reduced pressure. The residue was triturated with MTBE and isolated by filtration, washing with MTBE. The filter cake was dried in vacuo to provide 5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile (55.7 g, 95%) as a light orange solid: 1H NMR spectrum (DMSO-d6) 1.46 (9H, s), 6.02 (1H, s), 8.38 (2H, s); Mass Spectrum [M-H]- = 242.
[000500] The product can be slurried in heptanes, then filtered and dried as an alternative to MTBE grinding.
[000501] Hydrazine hydrate (82 mL, 1.69 mol) was added to 5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile (55 g, 225.18 mmol) in IPA (200 mL) and the mixture was heated at 50°C under nitrogen for 16 hours. The mixture was cooled in an ice bath. The resulting precipitate was collected by filtration, washed with IPA and diethyl ether and dried to constant weight to obtain (Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazole- 2-yl)pyrazine-2-carbohydrazonamide (49.2 g, 79%) as a yellow solid: 1H NMR spectrum (DMSO-d6) 1.45 (9H, s), 5.26 (2H, s ), 5.58 (2H, s), 7.56 (2H, s), 8.75 (1H, s); Mass Spectrum [M+H]+ = 277.
[000502] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (74.3 g, 195.44 mmol) was added to a solution of N-Boc-acid -isonipecotic (41.1 g, 179.15 mmol) and 4-methylmorpholine (35.9 mL, 325.74 mmol) in DMA (800 mL). The mixture was stirred for 10 minutes, then (Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide (45 g, 162.87 mmol) was added to the solution in one portion (exotherm observed from 22°C to 27°C). After a few minutes, the product crystallized from the reaction mixture. The reaction mixture was removed from the vessel and filtered through sintering. Additional DMA was added to wash the product from the sides of the vessel (150 mL) and this was poured onto the filter cake. Isopropanol (600 ml) was added to the vessel and the remaining part of the product in the vessel was suspended in this solvent under vigorous stirring. The isopropanol suspension was used to wash the filter cake once the DMA was removed by suction. The filter cake was suction dried, then washed with MTBE and suction dried once more. The filter cake was dried in vacuo to give 4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl) (Z)-tert-butyl methylene)hydrazinecarbonyl)piperidine-1-carboxylate (76 g, 95%) as a yellow solid: 1 H NMR spectrum (DMSO-d6) 1.40 (9H, s), 1.46 (9H, s), 1.63-1.9 (2H, m), 2.33-2.6 (2H, m, obscured by DMSO signal), 2.63 - 3.03 (2H, m), 3.18-3.48 (4H, m, dimmed by water signal), 3.88-4.11 (2H, m), 6.43 (2H, s), 7.76 ( 2H, br), 8.84 (0.5H, s), 8.87 (0.5H, s), 9.85 (1H, s): Mass Spectrum [M+H]+ = 488.
[000503] In an alternative preparation, N-Boc-isonipecotic acid can be made in situ as follows. Isonipecotic acid (858g, 3.74 mol) was dissolved in DMA (25.3 L) and 4-methylmorpholine (393 ml, 3.74 mol) added. Stir for 5 minutes and add isobutyl chloroformate (489 mL, 3.74 mol). The reaction mixture was stirred at 25°C for 2h and cooled to 15°C before (Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl) pyrazine-2-carbohydrazonamide (940 g, 3.4 mol) was added gradually over 10 minutes. The reaction mixture was stirred for 1-2h at 15°C. Water (20.5 L) was added gradually over 1h and stirred for a further 1h before being filtered. The filter cake was then washed with water (4 x 4 L) and dried on the filter before being dried in a vacuum oven at 50°C until dry to provide the desired product.
[000504] Acetic acid (200 mL) was added to dioxane (500 mL) in a fixed 3L double lined vessel and the solution was heated to 70°C under nitrogen. 4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)methylene)hydrazinecarbonyl)-piperidine-1-carboxylate (Z)-tert-butyl (74.5 g, 152.80 mmol) was added gradually to the hot mixture. After 10 minutes, the temperature was increased to 100°C (slight reflux). The reaction mixture was stirred at 100°C for 1.5 hours (suspension), then kept at 80°C overnight (solution formed after holding overnight). The resulting solution was concentrated under reduced pressure, then diluted with toluene, evaporated to dryness, taken up in toluene and concentrated again. The residual oil was mixed with a little ethyl acetate and concentrated to dryness. A solid crystallized from the solution, which was triturated with MTBE (200 mL) and isolated by filtration. The filter cake was washed with water and MTBE to obtain 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl) tert-Butyl -1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (50 g, 70%) as a gray solid.
[000505] The filtrate was concentrated under reduced pressure to provide a yellow solid. This material was triturated with MTBE and filtered. The filter cake was washed with ethyl acetate and then MTBE to obtain a second crop as a pale yellow solid (4.93 g, 7%). This material was identical to that of the first crop: 1H NMR spectrum (DMSO-d6) 1.17 (9H, s), 1.22 (9H, s), 1.29 -5 1.47 (2H, m), 1.671.78 (2H, m), 2.57-2.87 (3H, m), 3.57-3.92 (2H, m), 7.56 (2H, br), 8.56 (1H, m). s), 13.47 (2H, broad s); Mass Spectrum [M+H]+ = 470.
[000506] 1,8-diazabicyclo[5.4.0]undec-7-ene (19.87 mL, 132.90 mmol) was added to a suspension of 4-(5-(5-amino-6-(5- tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate tert-butyl (48 g, 102.23 mmol) in 2-methylTHF (300 mL). A dark solution was obtained after 5 minutes, treated with charcoal and filtered through a pad of celite, washing the charcoal and additional charcoal with 2-methylTHF (100 mL). The filtrate was stirred with an air stirrer at a temperature of -5°C in a fixed 3 L double-lined vessel under an atmosphere of nitrogen. 2-MethylTHF (100 mL) was added to help stir the yellow suspension. Iodomethane (7.96 mL, 127.78 mmol) was added dropwise over 15 minutes. The mixture was stirred for two hours and the reaction mixture was warmed to room temperature. After 16 hours more iodomethane was added (6 ml) and DBU (20 ml) was added and stirring was continued for 16 hours. The mixture was poured into water and stirred for 5 minutes. The insoluble material was isolated as a beige solid and dried in vacuo to provide 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin- tert-Butyl 2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (24.77 g, 50.1%). The source liquors were concentrated under reduced pressure and the residue was purified by flash silica column chromatography using MTBE as eluent. A second crop of the desired product (13.04 g, 26%) was thus obtained as a yellow solid: 1 H NMR spectrum (DMSO-d6) 1.47 25 (9H, s), 1.51 (9H, s), 1.57-1.76 (2H, m), 1.94-2.1 (2H, m), 2.87-3.09 (3H, m), 3.9-4.08 ( 2H, m), 4.26 (3H, s), 7.97 (2H, br, s), 8.92 (1H, s); Mass Spectrum [M+H]+ = 484.
[000507] 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2 tert-Butyl ,4-triazol-3-yl)piperidine-1-carboxylate (36.81 g, 76.12 mmol) was added to a solution of 2,2,2-trifluoroacetic acid (100 ml, 1305.87 mmols) in DCM (100 mL). The mixture was stirred for 3 hours at room temperature. The mixture was concentrated under reduced pressure. The residue was dissolved in DCM (1.5 L) and vigorously stirred concentrated ammonia (150 mL) in water (400 mL) added. The aqueous phase was washed with DCM (400 mL) and the combined organic solutions were dried over magnesium sulfate, filtered and concentrated to dryness to obtain 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl )-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine (30.0 g, 103%) as a solid yellow:
[000508] 1H NMR spectrum (DMSO-d6) 1.44 (9H, s), 1.54-1.69 (2H, m), 1.8-1.92 (2H, m), 2.53 -2.63 (2H, m), 2.68-2.83 (1H, m), 2.933.05 (2H, m), 4.19 (3H, s), 7.89 (2H, br), 8.85 (1H, s); Mass Spectrum [M+H]+ = 384.
[000509] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30.4 g, 80.04 mmol), was added to a stirred solution of 3-acid (tetrahydro-2H-pyran-2-yloxy)propanoic acid (12.67 g, 72.76 mmol) and N-ethyl-N-isopropylpropan-2-amine (25.3 mL, 145.52 mmol) dissolved in acetonitrile (200 mL) at 25°C. The resulting solution was stirred at 25°C for 20 minutes and then 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-( piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine (30 g, 72.76 mmol) was added gradually, washing the last portion into the mixture as a slurry in acetonitrile (100 ml). After stirring for 1 hour, the precipitate was collected by filtration, washed with acetonitrile and dried in vacuo to obtain 1-(4-(5-(5-amino-6-(5-tert-butyl-1) ,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro- 2H-pyran-2-yloxy)-propan-1-one (35.0 g, 89%) as a beige solid. The filtrate was diluted with DCM (600 mL), washed with water, dried over magnesium sulfate and concentrated. The residue was purified by flash chromatography on silica gel eluting with a gradient of 7N ammonia in methanol with 2 to 2.5% dichloromethane. A second crop of product (3.31 g, 6.13 mmol, 8.43%) was also obtained as a cream solid. Both samples were combined to provide a beige solid: 1 H NMR spectrum (DMSO-d6) 1.44 (9H, s), 1.52-1.79 (4H, m), 1.88-2.04 ( 2H, m), 2.53-2.73 (2H, m), 2.73-2.87 (1H, m), 2.91-3.05 (1H, m), 3.133.24 (1H, m). m), 3.37-3.47 (1H, m), 3.53-3.65 (1H, m), 3.7-3.8 (1H, m), 3.81-3.89 ( 1H, m), 3.89-3.99 (1H, m), 4.20 (3H, s), 4.29-4.4 (1H, m), 4.54-4.61 (1H, m). m), 7.60-8.20 (2H, br), 30 8.85 (1H, s); Mass Spectrum [M+H]+ = 540. Example 3: 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin) -2-yl-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

[000510] Pyridine 4-methylbenzenesulfonate (11.62 g, 46.24 mmol) was added to a suspension of 1-(4-(5-(5-amino-6-(5-tert-butyl-1) ,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro- 2H-pyran-2-yloxy)propan-1-one (128 g, 231.19 mmol) in methanol (1 L) under nitrogen atmosphere. The mixture was stirred at 60°C for 1.5 hours. The mixture was soluble after 5 minutes. The mixture was kept at 50°C overnight, during which time a precipitate formed. The solid material was isolated by filtration and washed with water and acetonitrile. This material still contained minor impurities from the previous stage and further purification was required. The material was dissolved in dichloromethane and purified by flash chromatography on silica gel (0% to 10% methanol/DCM in methanol/DCM). The desired product, 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H 1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (Example 3) (92 g, 85%) was thus isolated as a cream solid (Form A) : 1 H NMR spectrum (DMSO-d6) 1.41.51 (12H, m), 1.51-1.78 (2H, m), 1.89-2.05 (2H, m), 2.72 -2.86 (1H, m), 2.91-3.05 (1H, m), 3.12-3.24 (1H, m), 3.64 (2H, q), 3.83-4 .01 (1H, m), 4.29-4.41 (1H, m), 4.47 (1H, t), 4.58 (2H, q), 8.26 (2H, s), 8. 85 (1H, s); 1H NMR spectrum [M+H]+ = 470.
[000511] 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H- 1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one (Example 3.1) was prepared as follows.
[000512] 1,8-diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) was added to a suspension of 4-(5-(5-amino-6-(5-tert- tert-butyl butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (150 g, 319, 46 mmol) in 2-methylTHF (1.2 L). Iodoethane (46 mL, 575.03 mmol) was added and the mixture was stirred for 16 hours at 35°C. More iodoethane (46 ml, 575.03 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (76 ml, 511.14 mmol) were added and stirring was continued for 24 hours at 35°C. °C The mixture was poured into water and the insoluble material was isolated by filtration, washed with water and MTBE and dried in vacuo to give 4-(5-(5-amino-6-(5-tert-butyl-1,3) tert-Butyl ,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (116 g, 73.0 %) as a yellow solid. The filtrate was extracted with DCM and the organic solution was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica using a gradient elution (30% MTBE/heptane in 100% MTBE). A second crop of the desired product (12 g, 24.12 mmol, 7.55%) was thus isolated as a yellow solid, which was then combined with the first crop: 1H NMR spectrum (DMSO -d6) 1.41 (9H, s), 1.44 (9H, s), 1.48 (3H, t), 1.52-1.69 (2H, m), 1.87-2.04 (2H, m), 2.79-3.03 (3H, m), 3.86-4.03 (2H, m), 4.59 (2H, q), 7.89 (2H, s), 8.85 (1H, s); Mass Spectrum [M+H]+ = 498. TFIF may also be a suitable solvent for the above reaction.
[000513] TFA (400 mL) was added gradually to a solution of 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2 tert-butyl -yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (126 g, 253.22 mmol) in DCM (400 mL). The mixture was stirred for 16 hours at room temperature. The mixture was concentrated under reduced pressure, dissolved in DCM (1 L) and slowly added to a vigorously stirred solution of concentrated aqueous ammonia (500 ml) in water at 0°C. The organic solution was separated from the aqueous phase and concentrated under reduced pressure to obtain 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-( piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine (101 g, 100%) as a yellow solid: 1 H NMR spectrum (DMSO-d6) 1.4 -1.52 (12H, m), 1.57-1.73 (2H, m), 1.83-1.93 (2H, m), 2.57 - 2.7 (2H, m), 2 .71-2.84 (1H, m), 2.96-3.09 (2H, m), 4.58 (2H, q), 8.06 (2H, s), 8.84 (1H, s ); Mass Spectrum [M+H]+ = 398.
[000514] *In another experiment on a similar scale (about 170 g of starting material), the following isolation procedure was used: the layers were separated and the top layer (emulsion with a solid) was filtered. The solid was washed with DCM (0.5 L) and the filtrate transferred to a separatory funnel. The layers were separated and the aqueous layer was extracted with DCM (0.5 L). The organic layers were dried over MgSO4 , filtered and concentrated. The product was dried at 50°C overnight (81.75 g). The solid from the extraction was suspended in water (200 mL) for 30 min at room temperature and filtered. The product was dried at 50°C in vacuo (61.8 g).
[000515] Another variation is as follows: a suspension of 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl) tert-Butyl -1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (3009.5 g, 6.05 mol) in DCM (9L) was cooled to 5 -10°C under N2. TFA (9L) was slowly added to the suspension, keeping the temperature <30°C. The reaction mixture was stirred at room temperature for one hour. The mixture was concentrated, the resulting residue was dissolved in water (30 L) and slowly added to a 35% aqueous ammonia solution (12 L) at 0-5°C. The suspension was stirred for 30 min, then the product was filtered off and washed with water (2 x 6 L). The product was dried at 50°C under vacuum for 2 days (2496 g).
[000516] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluophosphate (HATU, 106 g, 279.51 mmol), was added gradually to a stirred solution. of 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (44.3 g, 254.10 mmol) and N,N-ethyl-N-isopropylpropan-2-amine (89 mL, 508.21 mmols) dissolved in acetonitrile (600 mL) at 25°C. The resulting solution was stirred at 25°C for 20 min, then 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin- 4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine (101 g, 254.10 mmol) was added gradually, washing the last portion into the mixture as a slurry in acetaminophen. tonitrile (300 ml). After stirring for 1 hour, the precipitate was collected by filtration, washing with acetonitrile and dried in vacuo to give 1-(4-(5-(5-amino-6-(5-tert-butyl-1) ,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro- 2H-pyran-2-yloxy)propan-1-one (128 g, 91%) as a beige solid. The filtrate was diluted with DCM (600 ml), washed with water, dried over magnesium sulfate and concentrated. The residue was purified by flash chromatography on silica gel eluting with a gradient of 7N ammonia in methanol with 2 to 2.5% dichloromethane. A second crop of the desired product (40 g, 72.2 mmol, 28.4%) was obtained as a cream solid, which was combined with the first crop: 1 H NMR Spectrum (DMSO-d6) 1.29-1 .48 (16H, m), 1.48 - 1.75 (4H, m), 1.83-1.99 (2H, m), 2.48-2.68 (2H, m), 2.68 -2.79 (1H, m), 2.87-2.99 (1H, m), 3.07-3.19 (1H, m), 3.32-3.42 (1H, m), 3 .47-3.6 (1H, m), 3.64-3.75 (1H, m), 3.75-3.84 (1H, m), 3.84-3.95 (1H, m) , 4.24-4.39 (1H, m), 4.47-4.6 (3H, m), 7.84 (2H, s), 8.79 (1H, s): Mass Spectrum [M + Na] + = 577.
[000517] Alternative Preparation:
[000518] To a solution of 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (48.80 g 0.2774 mol) and N-ethyl-N-isopropylpropan-2-amine (86.96 mL, 0.4993 mol) in THF (552 mL), add O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (115.73 g, 0. 3051 mol) gradually at room temperature under nitrogen. The resulting mixture was stirred for 10 minutes, then 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)- 1H-1,2,4-triazol-5-yl)pyrazin-2-amine (122.5 g (110.25 g active), 0.2774 mol) was added gradually over one hour. After 3.5 h, the mixture was concentrated and the residue was suspended in MeCN (275 mL) for 15 minutes at room temperature. The product was filtered off, washed with MeCN (3 x 110 mL) and dried overnight at 50°C under vacuum. This provided 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H 1 , 2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one (131.9 g, 96%). In another alternative preparation, HBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) in THF can be used as a coupling agent instead of HATU. Alternative Preparation of Example 3
[000519] To a suspension of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1- ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one (131.9 g, 0 .2382 mol) in methanol (1045 ml) was added pyridinium p-toluenesulfonate (11.97 g, 47.7 mmol) under N 2 . The reaction mixture was stirred at 60°C for 5.5 h, then at 50°C overnight. The reaction mixture was cooled to 0°C and the solid was filtered off. The product was suspended in water (250 mL) for 20 min at room temperature, filtered, washed with water (3 x 40 mL) and dried at 50°C in vacuo. This provided 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1 ,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (21.4 g) as Form A (see below).
[000520] The methanol liquors were concentrated and the resulting solid was mixed in water (0.6 L) for 20 min at room temperature. The solid was isolated by filtration and washed with water (3x100 mL). The filter cake was suspended a second time in water (0.5 L) for a further 20 minutes. The product was isolated by filtration, washed with water (100 x 5 ml) and dried at 50°C in vacuo. This provided 81.9 g of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1- ethyl-1H 1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (81.9 g) as Form A.
[000521] Both crops were combined (103.3 g), grown with Form B (16.68 g) and suspended in MeCN (826 mL) at room temperature overnight. This provided 117.4 g of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1- ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one as a pale yellow solid (117.4 g), Form B (see below). This material was further purified by suspending it in heptane (7.5 vols rel) for 1 hour. The mixture was filtered, dried on the filter and dried at 50°C in a vacuum oven overnight to provide 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3, 4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (102.5g ) as Form B.
[000522] Form B can also be made by slurrying Form A in MeCN without culture.
[000523] Form A or B can also be converted to Form C as follows.
[000524] A suspension of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl -1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (e.g. Form B made by the processes described above) in IPA (12 vol ) was heated at reflux until the solid dissolved. The solution was filtered hot, then cooled to room temperature. This provided 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1 ,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one as a light yellow solid (99.3 g, 97%) as Form C.
[000525] Form C can also be converted to Form B as follows.
[000526] In a 10 L flanged flask, Form C (377.8 g portion 1) in MIBK (7900 mL) was heated to 110-115°C to provide a solution. The solution was allowed to cool to 97-103°C and immediately filtered into a 50L vessel containing a seed of Form B (0.8g) in acetonitrile (8220ml) with stirring at -15°C. During the addition, the temperature in the 50 L vessel was maintained between -15 and 25°C by jacket cooling. Three more portions of the compound dissolved in MIBK were added using a similar method. To the resulting suspension was added a seed of Form B (0.8 g) and the mixture was then stirred at 10-20°C overnight. In the process, analysis confirmed the desired form (Form B) with no visible Form C or amorphous. The mixture was filtered and washed with acetonitrile (3340 mL). The solid was dried in an oven for 2 days (solid was broken up during drying into a powder and a mixture of small lumps ~1 mm to 3-4 mm in size) to a constant weight. Yield = 1532.8 g (93.5%).
[000527] 3-(Tetrahydro-2H-pyran-2-yloxy)propanoic acid was prepared as follows.
[000528] To a stirred solution of methanol (2.4 L) and concentrated sulfuric acid (44.4 mL, 832.61 mmol) at 0°C under nitrogen was added, dropwise, beta-propiolactone ( 175 mL, 2.78 mol). This solution was allowed to stir at room temperature for 2 days. The reaction mixture was cooled to 10°C before gradually adding sodium bicarbonate (145 g, 1.72 mol). The resulting suspension was allowed to stir at room temperature for 75 minutes. This solution was filtered, the filter cake was washed with methanol (800 mL). The filtrate was evaporated to an oil, which was redissolved in dichloromethane (1.2 L) and stirred for 60 minutes before further filtration. This solution was filtered before evaporation to obtain methyl 3-hydroxypropanoate hydrochloride (219 g, 76%) as an oil. 1 H NMR spectrum (CDCl 3 ) 2.50 (2H, t), 3.63 (3H, s), 3.78 (2H, t).
[000529] Pyridinium p-toluenesulfonate (7.65 g, 30.45 mmol) was added to a clear solution of methyl 3-hydroxypropanoate (63.4 g, 609.00 mmol) and 3,4-dihydro -2H-pyran (78 mL, 852.60 mmol) in dichloromethane (650 mL) at room temperature under nitrogen to provide a cloudy solution. This was allowed to stir at room temperature overnight. The reaction mixture was washed with water (250 mL) and brine (250 mL) before drying (MgSO4 ) and evaporating to an oil. This crude product was purified by flash silica chromatography, gradient elution of 15 to 30% EtOAc in heptane. The pure fractions were evaporated to dryness to afford methyl 3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.7 g, 59.0%) as a colorless oil: 1 H NMR spectrum (CDCl 3 ) 1.47 (4H, dddd), 1.55 - 1.84 (2H, m), 2.55 30 (2H, t), 3.33-3.53 (1H, m), 3.53-3 .7 (4H, m), 3.78 (1H, ddd), 3.93 (1H, dt), 4.42 - 4.72 (1H, m); 1 H NMR spectrum [MH] + = 189.
[000530] Sodium hydroxide (2M, 349 mL, 697.58 mmol) was added to a solution of methyl 3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.68 g, 359.58 mmol) in THF (680 mL) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. The THF was removed in vacuo, the aqueous layer was then washed with ethyl acetate (260 mL) before cooling to 0°C and carefully acidifying to a pH of 5 by adding hydrochloric acid ( 2 M). The product was extracted with ethyl acetate (3 x 250 mL) before drying (MgSO 4 ) and evaporation to afford 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (57.0 g, 91%) like a clear oil. This material was dissolved in ethyl acetate (750 mL) and then washed with water (3 x 250 mL) and brine (250 mL) to remove remaining acetic acid. The organic solution was dried (MgSO4) and evaporated to afford 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (45.67g, 72.9%) as a colorless oil: 1H NMR spectrum (CDCl3) 1.43-1.67 (4H, m), 1.65-1.95 (2H, m), 2.68 (2H, t), 3.48-3.58 (1H, m), 3. 73 (1H, dt), 3.88 (1H, ddd), 4.02 (1H, dt), 4.59-4.7 (1H, m); 1 H NMR spectrum [M-H]- = 173. Example 3, as hereinabove, a crystalline solid was isolated in three different crystalline forms, described as Forms A, B and C.
[000531] The crystal structure of Form A of Example 3 can be characterized by XRPD and DSC.
[000532] The methods for performing these techniques are as described for Example 1. Ten X-ray powder diffraction peaks for Example 3, Form A

[000533] The XRPD for Example 3, Form A, is shown in Figure 3.
[000534] DSC analysis of Example 3, Form A, shows an endotherm with an onset at 27.0°C and a peak at 63.0°C. Other endothermic shifts are seen with onsets and peaks at the following temperatures: 166.5°C and 168.7°C, 172.2°C and 173.2°C and a final melting at 174.8°C and a peak at 175.7°C (Figure 4).
[000535] Thus, DSC analysis shows that Example 3, Form A, is a solvated material with an onset of desolvation at about 27.0°C and a peak at about 63.0°C.
[000536] X-ray diffraction spectra for Example 3 (Form A) showed the material to be crystalline. This material had a desolvation point of 28.0°C (onset).
[000537] Example 3 may also exist in an alternative polymorphic form, referred to herein as Form B. Preparation of Form B was described above.
[000538] This material had a melting point of 172.5°C (onset).
[000539] In another aspect of the invention, there is provided a process for making Form B of Example 3 by mixing a sample of Example 3 in acetonitrile. In a further aspect of the invention, there is provided a process for making Form B of Example 3 from a solution of Form C of Example 3 in MIBK. Ten X-ray powder diffraction peaks for Example 3, Form B

[000540] The XRPD for Example 3, Form B, is illustrated in Figure 5.
[000541] DSC analysis of Example 3, Form B, shows a melting endotherm starting at 172.5°C and peaking at 174.2°C (Figure 6).
[000542] Thus, DSC analysis shows that Example 3B is a solid with an onset of melting at about 172.5°C and a peak melting point at about 174.2°C.
[000543] Example 3 may also exist in a third crystalline form, referred to herein as Form C. A process for producing Form C from, for example, Form B material, has been described above via crystallization from alcohol. isopropyl (IPA).
[000544] Therefore, in another aspect of the invention, there is provided a process for making Form C of Example 3 by crystallizing Example 3 from IPA.
[000545] Example 3, Form C, is characterized by providing at least one of the following 2θ values measured using CuKa radiation: 6.9 and 12.3. Example 3, Form C, is characterized by providing an X-ray diffraction pattern substantially as shown in Figure A. Ten X-ray powder diffraction peaks are shown below. Ten X-ray powder diffraction peaks for Example 3, Form C

[000546] DSC analysis of Example 3, Form C, shows a melting endotherm starting at 183.0°C and peaking at 185.6°C (Figure B).
[000547] Thus, DSC analysis shows that Example 3, Form C, is a high melting solid with melting onset at about 183.0°C and a peak at about 185.6°C. Details of the techniques used for the analysis of Form C X-Ray Powder Diffraction
[000548] Analytical instrument: PANalytical Cubix.
[000549] The X-ray powder diffractogram was determined by placing a sample of the crystalline material on a single-crystal silicon (SSC) PA-Nalytical mounting bracket and spreading the sample in a thin layer with the aid of a foil blade. microscope. The sample was centrifuged at 30 revolutions per minute (to improve counting statistics) and irradiated with X-rays generated by a long-thin copper focusing tube operated at 40 kV and 40 mA with a wavelength of 1.5418 angstroms. The collimated X-ray source was passed through a 0.04rad Soller slit, then an automatic variable divergence set at V20, finally a 20 mm beam mask. The reflected radiation is directed through a 20 mm anti-scatter slit and a 0.04rad Soller slit. The sample was exposed for 1.905 seconds per 0.0025067° 2-theta increment (continuous scan mode) over the range 2 degrees to 40 degrees 2-theta in theta-theta mode. The instrument was equipped with an X-Celerator detector. Control and data capture were performed using a Dell Pen-tium 4HT Workstation operating with X'Pert Industry software. Those versed in the technique of X-ray powder diffraction will realize that the relative intensity of peaks can be affected, for example grains over 30 microns in size and non-unit proportions can affect sample analysis. Those skilled in the art will also appreciate that the reflection position can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer. The flatness of the surface of the sample may also have a small effect. Consequently, the diffraction pattern data presented should not be taken as absolute values. Differential Scanning Calorimetry
[000550] Analytical instrument: TA Instruments DSC Q1000.
[000551] Typically, less than 5 mg of material contained in a standard aluminum container fitted with a lid was heated over the temperature range of 25°C to 300°C at a constant heating rate of 10° C per minute. A purge gas using nitrogen was used at a flow rate of 50 ml per minute. Example 4:
[000552] (3R)-1-[4-[5-[5-amino-6-(5-tert-butyl,1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1- methyl-1,2,4-triazol-3-yl-1-piperidyl]-3-hydroxy-butan-1-one

[000553] 2-(1H-Benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (201 mg, 0.63 mmol) was added to a stirred suspension of 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4- triazol-5-yl)pyrazin-2-amine (200 mg, 0.52 mmol, described in Example 1), N-ethyl-N-isopropylpropan-2-amine (0.273 mL, 1.56 mmol) and acid (R )-3-hydroxybutanoic acid (65.2 mg, 0.63 mmol) in N,N-dimethylformamide (3 mL). The resulting suspension was stirred at room temperature for two hours. The resulting mixture was purified by preparative HPLC using a reversed-phase Waters X-Bridge column (C-18, 5 microns, silica, 30 mm diameter, 150 mm long, flow rate 60 mL/min ) using an isocratic mixture of 31% acetonitrile in water (containing ammonium carbonate (2 g/l). Fractions containing the desired compound were evaporated to dryness to give a pale yellow solid. This solid was stirred in acetonitrile (3 mL). ) at room temperature. The resulting solid was filtered, washed with ice water, acetonitrile and dried to obtain the title compound (125 mg, 51.0%) as a pale yellow solid. 1 H NMR spectrum (CDCl 3 ) 1, 24 (3H, d), 1.52 (9H, s), 1.85 (2H, m), 2.10 (2H, m), 2.35 (1H, dd), 2.55 (1H, d ), 2.90 (1H, m), 3.05 (1H, m), 3.20 (1H, m), 3.90 (1H, m), 4.25 (1H, m), 4.31 (3H, s), 4.6 (1H, m), 9.03 (1H, s); 1H NMR spectrum [M+H]+ = 470. Example 5: (3S)-1 -[4-[ 5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1 -methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one

[000554] Using a procedure similar to that described in Example 4, 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl) )-1H-1,2,4-triazol-5-yl)pyrazin-2-amine was reacted with (S)-3-hydroxybutanoic acid to obtain the title compound (167 mg, 68.2%) as a pale yellow solid. 1 H NMR spectrum (CDCl 3 ) 1.24 (3H, d), 1.52 (9H, s), 1.85 (2H, m), 2.10 (2H, m), 2.35 20 (1H, m) dd), 2.55 (1H, d), 2.90 (1H, m), 3.05 (1H, m), 3.20 (1H, m), 3.90 (1H, m), 4, 25 (1H, m), 4.31 (3H, s), 4.6 (1H, m), 9.03 (1H, s); Mass Spectrum [M+H]+ = 470. Example 6 (2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2- yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-2-methyl-propan-1-one

[000555] Using a procedure similar to that described in Example 4, 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl) )-1H-1,2,4-triazol-5-yl)pyrazin-2-amine was reacted with (R)-3-hydroxy-2-methylpropanoic acid to obtain the title compound (87 mg, 47.4 %) as a pale yellow solid. 1 H NMR spectrum (CDCl 3 ) 1.55 (9H, s), 1.61 (3H, broad s), 1.8-2.0 (2H, m), 2.10-2.25 (2H, 10 m), 2.90 (2H, m), 3.10 (1H, m), 3.3 (2H, m), 3.77 (2H, m), 4.33 (3H, s), 4.6 ( 1H, m), 9.05 (1H, s); Mass Spectrum [M+H]+ = 470. Example 7: 1 -[4-[5-[(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]- 6-(5-amino-1-methyl-1,2,4-[5-triazol-3-yl]-1-piperidyl]-2-hydroxy-2-methyl-propan-1-one

[000556] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (100 mg, 0.52 mmol) was added in one portion to 2-hydroxy-2-methylpropanoic acid (38.0 mg, 0.37 mmol) , 3-(5-tert-butyl1-,3,4-oxadiazol-2-yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5 -yl)pyrazin-2-amine (100 mg, 0.26 mmol) and 2-hydroxy-pyridine-N-oxide (57.9 mg, 0.52 mmol), dissolved in NMP (1.2 mL) under atmosphere of argon. The resulting solution was stirred at 25°C for 3 hours. Pyridine (100 µL, 1.24 mmol) was added and the mixture was stirred for 18 hours. More 2-hydroxypyridine 1-oxide (57.9 mg, 0.52 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (100 mg, 0.52 mmol) were added. The mixture was then heated to 70°C for 48 hours, plus 2-hydroxy-2-methylpropanoic acid (15 mg, 0.14 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (50. 0 mg, 0.26 mmol) and 2-hydroxypyridine 1-oxide (25.0 mg, 0.23 mmol) were added and the mixture was then held at 70°C for 8 hours. The solution was purified by preparative HPLC using a Waters X-Bridge reversed phase column (C-18, 5 micron, silica, 19 mm diameter, 100 mm long, flow rate 40 ml/minute) and decreasingly polar mixtures of water containing ammonium carbonate (0.2%) and acetonitrile as eluent to obtain the title compound (71 mg, 58%) as a pale yellow solid. 1 H NMR spectrum (CDCl 3 ) 1.55 (15H, s/g), 1.90 (2H, m), 2.15 (2H, m), 3.05-3.3 (4H, m), 4, 32 (3H, s), 4.6 (1H, m), 9.03 (1H, s); Mass Spectrum [M+H]+ = 470. Example 8: 3-[4-[5-[5-Amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl) acid pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-oxo-propanoic

[000557] Ethyl 3-chloro-3-oxopropanoate (0.037 mL, 0.29 mmol) was added dropwise to a stirred solution of 3-(5-tert-butyl-1,3,4-oxadiazole-2- yl)-5-(1-methyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine (100 mg, 0.26 mmol) and triethylamine (0.047 mL, 0.34 mmol) dissolved in CH2Cl2 (1.5 mL) over a period of 2 minutes at 0°C under nitrogen. The mixture was stirred at 0°C for 10 minutes and then allowed to warm to room temperature and stirred for 1 hour. The mixture was evaporated, dissolved in DMF. The white solid was filtered and the filtrate was purified by preparative HPLC using a Waters X-Terra reversed phase column eluting with a mixture of water (containing 0.2% ammonium carbonate) and acetonitrile to obtain 3-( 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-methyl-1H-1,2,4- ethyl triazol-3-yl)piperidin-1-yl)-3-oxopropanoate (80 mg, 61.7%) as a yellow solid. This material was suspended in THF (2 mL). 2N sodium hydroxide (0.235 mL, 0.47 mmol) and water (0.5 mL) were added. The mixture was stirred at room temperature overnight. 2N hydrochloric acid (230 µL) was added to the mixture. Solvents were evaporated. The residue was diluted with CH2Cl2 (30 mL) and water (5 mL). The organic phase was washed with brine and dried over MgSO4 . Solvents were evaporated. The resulting foam was triturated in ether. The resulting yellow solid was filtered, dried, triturated in acetonitrile (3 mL). The yellow solid was collected by filtration, dried at 40°C to provide the title compound (50 mg, 68%) as a yellow solid. 1H NMR spectrum (DMSO-d6) 1.46 (9H, s), 1.58 (1H, m), 1.74 (1H, m), 1.98 (2H, m), 2.84 (1H , m), 3.0 (1H, m), 3.21 (1H, m), 3.46 (2H, m), 3.83 (1H, m), 4.22 (3H, s), 4.34 (1H, m), 7.8-8.2 (1H, m), 8.87 (1H, s); Mass Spectrum [M+H]+ = 470. Example 9: 3-[4-[5-[5-Amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl) acid pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-oxo-propanoic

[000558] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (474 mg; 1.25 mmol) was added over 30 seconds gradually at a stirred solution of 3-ethoxy-3-oxopropanoic acid (150 mg, 1.13 mmol), N-ethyl-N-isopropylpropan-2-amine (0.394 mL, 2.26 mmol) and 3-(5-(tert- butyl)-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2 -amine (450 mg, 1.13 mmol) dissolved in DMF (20 mL) at 50°C. The resulting solution was collected after 1 min (reaction complete) and immediately allowed to cool to room temperature. The reaction mixture was concentrated and diluted with EtOAc (100 mL) and washed sequentially with water (20 mL) and saturated brine (20 mL). The organic layer was dried over MgSO4 , filtered and evaporated to give 3-(4-(5-(5-amino-6-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl) Crude ethyl )pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-oxopropanoate (850 mg).
[000559] Some of the material (780 mg) was dissolved in THF (20 mL). To this solution were added 2N aqueous sodium hydroxide (2.3 ml, 4.57 mmol) and water (5 ml), followed by methanol (5 ml) to provide a clear solution. The mixture was stirred at room temperature for 3 hours. The THF was evaporated. The aqueous layer was acidified to pH 3 with 2N aqueous hydrochloric acid (2.5 ml). Dichloromethane (50 ml) was added and the organic phase extracted. The organic phase was washed with brine (10 ml) and dried over MgSO4 . Solvents were evaporated. The resulting gum was purified by preparative HPLC (X-Bridge Waters Prep C18 OBD column, 5 μ, silica, 50 mm in diameter, 100 mm in length), using decreasingly polar mixtures of water (containing 1% ammonia). ) and acetonitrile as eluents. Fractions containing the desired compound were evaporated to dryness to obtain the pure ammonium salt. This was solubilized in water and acidified to pH 3 with 2N hydrochloric acid (~0.3 mL). Dichloromethane (50 ml) was added and the organic phase separated, washed with brine (5 ml) and dried over MgSO4 . After filtration, the resulting solution was evaporated to dryness and the residue was triturated with diethyl ether (5 ml) and filtered to obtain 3-(4-(5-(5-amino-6-(5-(tert) -butyl)-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3- oxopropanoic acid (195 mg, 26.5%) as a cream solid. 1H NMR spectrum (DMSO-d6) 1.45 (9H, s), 1.48 (3H, m), 1.55-1.62 (1H, m), 1.70 -25 1.80 (1H , m), 1.95-2.05 (2H, m), 2.80-2.90 (1H, m), 2.95-3.05 (1H, m), 3.15-3.25 (1H, m), 3.45 (2H, s), 3.78-3.85 (1H, m), 4.30-4.40 (1H, m), 4.55-4.65 (2H , m), 7.80 -8.00 (2H, broad s), 8.88 (1H, s), 12.60 (1H, s); Mass Spectrum [M+H]+ = 484. Example 10: (2S)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-2,3-dihydroxy-propan-1-one
[000560] To a mixture of 3-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H- 15, 2,4-triazol-5-yl)pyrazin-2-amine (257 mg, 0.50 mmol, TFA salt), (S)-2,2-dimethyl-1,3-dioxolane-4- potassium carboxylate (101 mg, 0.55 mmol) and EDCI (105 mg, 0.55 mmol) in DCM (5 mL) were added 1-hydroxy-1H-benzotriazole hydrate (85 mg, 0.56 mmol) and DIPEA (194 mg, 1.50 mmol). The mixture was stirred for 16 hours at room temperature. Water was added to the mixture and the mixture was extracted with DCM. The organic layers were washed with brine and dried over Na2SO4, filtered and concentrated to provide (S)-(4-(5-(6-amino-(5-(tert-butyl)-1,3,4-oxadiazole-2 -yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)(2,2-dimethyl-1,3-dioxolan-4-yl) ) methanone (320 mg). Mass Spectrum [M+H]+ = 526. To a mixture of (S)-(4-(5-(5-amino-6-(5-(tert-butyl)-1,3,4-oxadiazole- 2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)(2,2-dimethyl-1,3-dioxolan-4- yl) methanone (320 mg) in DCM (10 mL) at room temperature, TFA (1.6 mL, 20.77 mmol) was added dropwise. The mixture was stirred for 16 h at room temperature, concentrated and purified by preparative HPLC (Waters XBridge Prep C18 OBD column, 5 μ, silica, 19 mm diameter, 100 mm long) using decreasingly polar mixtures of water (containing 0.1% NH 3 ) and MeCN as the eluent. Fractions containing the desired compound were evaporated to dryness to obtain the title compound (142 mg, 48%) as a white solid. 1 H NMR spectrum (400 MHz, DMSO-d6, 30°C): 1.45 (12H, m), 1.56 (1H, m), 1.70 (1H, m), 1.98 (2H, m). m), 2.85 (1H, m), 3.00 (1H, m), 3.20 (1H, m), 3.45 (1H, s), 3.55 (1H, s), 4, 05 (1H, m), 4.35 (2H, m), 4.60 (2H, m), 4.70 (1H, m), 4.85 (1H, m), 7.90 (2H, m ), 8.85 (1H, s); Mass Spectrum [M+H]+ = 486. Example 11: (2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazole-2) - yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-2,3-dihydroxy-propan-1-one
[000561] 3-(5-(tert-butyl)-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2, 4-triazol-5-yl)pyrazin-2-amine was reacted with potassium (R)-2,2-dimethyl-1,3-dioxolane-4-carboxylate using a procedure similar to that described in Example 10 to provide the compound of the title (0.145 g, 40%) as a solid. 1 H NMR spectrum (400 MHz, DMSO-d6, 30°C): 1.45 (12H, m), 1.60 (2H, m), 1.98 (2H, m), 2.85 (1H, m). m), 3.00 (1H, m), 3.17 (1H, m), 3.45 (1H, s), 3.55 (1H, s), 4.05 (1H, m), 4, 35 (2H, m), 4.60 (2H, m), 4.70 (1H, m), 4.85 (1H, m), 7.90 (2H, m), 8.85 (1H, s ); Mass Spectrum [M+H]+ = 486. Brief Description of Figures
[000562] Figure 1 shows an X-ray powder diffraction pattern for Example 1, Form A.
[000563] Figure 2 shows a DSC thermogram for Example 1, Form A.
[000564] Figure 3 shows an X-ray powder diffraction pattern for Example 3, Form A.
[000565] Figure 4 shows a DSC thermogram for Example 3, Form A.
[000566] Figure 5 shows an X-ray powder diffraction pattern for Example 3, Form B.
[000567] Figure 6 shows a DSC thermogram for Example 3, Form B.
[000568] Figure 7 shows an X-ray powder diffraction pattern for Example 3, Form C.
[000569] Figure 8 shows a DSC thermogram for Example 3, Form C.
[000570] Figure 9 shows tumor growth inhibition by Example 3 in combination with AKT inhibitor (AZD5363) - sequential administration
[000571] Figure 10 shows the inhibition of tumor growth by Example 3 in combination with AKT inhibitor (AZD5363), co-administration.
[000572] Figure 11 shows the inhibition of tumor growth by Example 3 in combination with PARP inhibitor (Olaparib) in the BT474 xenograft model.
[000573] Figure 12 shows the inhibition of tumor growth by Example 3 in combination with PARP inhibitor (Olaparib) in the MCF7 xenograft model.
[000574] Figure 13 shows by inhibition of tumor growth by Example 3 in combination with (AZD8186).

权利要求:
Claims (21)
[0001]
1. Compound of Formula (I):
[0002]
2. A compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that R2 is selected from groups (i) to (xi):
[0003]
A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, characterized in that R2 is selected from groups (i) to (vi), as defined in claim 2.
[0004]
A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, characterized in that R2 represents a group (i), as defined in claim 2.
[0005]
A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, characterized in that R1 is methyl.
[0006]
A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, characterized in that R1 is ethyl.
[0007]
7. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that it is selected from: 1-[4-[5-[5-amino-6-(5-tert-butyl- 1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one ; 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4 -triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one; (3R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1 ,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one; (3S)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1 ,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-butan-1-one; (2R)-1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1 ,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-2-methyl-propan-1-one; 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-methyl-1,2,4 -triazol-3-yl]-1-piperidyl]-2-hydroxy-2-methyl-propan-1-one.
[0008]
Compound according to claim 1, characterized in that it is 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin -2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one, or a pharmaceutically acceptable salt thereof.
[0009]
Compound according to claim 1, characterized in that it is 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin -2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one;
[0010]
10. A compound according to claims 1 to 9, or a pharmaceutically acceptable salt thereof, characterized in that it is in a crystalline form.
[0011]
11. Crystalline form of 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl compound -1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one (form A) characterized by the fact that it has an X-ray diffraction with specific peaks of approximately 2 - theta= 4.8°, 5.2°, 10.0°, 10.4°, 14.6°, 16.2°, 19.9°, 23.6°, 24.4° and 25, 4° wherein said values may be plus or minus 0.2° 2-theta, as measured using CuKα radiation, and additionally having an initial Differential Scanning Calorimetry endotherm with an onset at 27.0°C and a peak at 63 .0°C.
[0012]
12. Crystalline form of 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl compound -1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one (form B), characterized by the fact that it has an X-ray diffraction with specific peaks of approx. -mally 2-theta= 5.8°, 10.9°, 11.5°, 12.9°, 17.3°, 19.1°, 24.0°, 24.7°, 25.9° and 27.2° wherein said values may be plus or minus 0.2° 2-theta, as measured using CuKα radiation, and additionally having a Differential Scanning Calorimetry endotherm with an onset at 172.5°C and a peak at 174.2°C.
[0013]
13. Crystalline form of 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl compound -1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one (C form), characterized by the fact that it has an X-ray diffraction with at least two peaks approximately 2-theta = 6.9°, 10.5°, 12.3°, 13.6°, 16.4°, 19.6°, 20.2°, 21.0°, 22.5° ° and 24.6° wherein said values may be plus or minus 0.2° 2-theta, as measured using CuKα radiation, and additionally having a Differential Scanning Calorimetry endotherm with an onset at 183.0°C and a peak at 185.6°C.
[0014]
14. A compound according to claims 1 to 13, or a pharmaceutically acceptable salt thereof, characterized in that it is for use as a medicine.
[0015]
15. Use of the compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that the use is in the manufacture of a medicament for preventing or treating cancer in an animal warm-blooded, just like the man.
[0016]
16. Use of the compound of formula (I) according to claim 15, characterized in that the compound is 1-[4-[5-[5-amino-6-(5-tert-butyl-1,3) ,4-oxadiazol-2-yl)pyrazin-2-yl]-1-ethyl-1,2,4-triazol-3-yl]-1-piperidyl]-3-hydroxy-propan-1-one or a salt pharmaceutically acceptable thereof.
[0017]
17. Use of the compound of formula (I) according to claim 15, characterized in that the cancer is breast cancer.
[0018]
18. A combination suitable for use in the treatment of cancer, characterized in that it comprises a compound of Formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof and another antitumor agent selected from AZD2014, AZD8186, AZD5363 or olaparib.
[0019]
19. Pharmaceutical composition characterized in that it comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, as defined in claim 1, and a pharmaceutically acceptable diluent or carrier.
[0020]
20. Use of a compound of Formula (I), characterized in that it is in the preparation of a medicament to treat a patient suffering from cancer, wherein the patient is selected for treatment with a compound of formula (I) if a patient sample containing tumor-derived DNA or tumor cells was provided and the PIK3CA gene in the tumor-derived DNA or tumor cells of the patient was determined to be wild-type or mutant.
[0021]
21. Use of a compound of Formula (I) as defined in claim 1, characterized in that it is in the preparation of a medicament for the treatment of cancers with tumor cells identified as carrying a mutant PIK3CA gene.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112015017331B1|2022-01-11|FORMULA I COMPOUNDS, CRYSTALLINE FORM, USE OF COMPOUNDS, COMBINATION AND COMPOSITION

---

JP6286031B2|2018-02-28|Compound

---

Cohen et al.2021|Kinase drug discovery 20 years after imatinib: Progress and future directions

---

AU2016296878B2|2020-12-17|Chiral diaryl macrocycles and uses thereof

---

ES2780650T3|2020-08-26|Dihydroimidazopyrazinone derivatives used in the treatment of cancer

---

BR112019015389A2|2020-03-10|ESTROGEN RECEPTOR MODULATORS

---

WO2017080980A1|2017-05-18|Dihydropyrrolopyrazinone derivatives useful in the treatment of cancer

---

EA038233B1|2021-07-28|DEUTERATED IMIDAZO[4,5-c]QUINOLIN-2-ONE COMPOUNDS AND THEIR USE IN TREATING CANCER

---

WO2021178296A1|2021-09-10|Therapeutic uses of macrocyclic compounds

---

EA037533B1|2021-04-09|6,7,8,9-TETRAHYDRO-3H-PYRAZOLO[4,3-f]ISOQUINOLINE DERIVATIVES USEFUL IN THE TREATMENT OF CANCER

同族专利:

---

公开号 | 公开日

---

EP2948447B1|2016-09-21|

---

RU2015132370A|2017-03-02|

---

KR102202516B1|2021-01-12|

---

MA38287B1|2018-08-31|

---

HK1214592A1|2016-07-29|

---

DOP2015000170A|2015-08-16|

---

CL2015002027A1|2015-11-13|

---

UY35275A|2014-07-31|

---

AU2014208964B2|2016-09-01|

---

KR20150109385A|2015-10-01|

---

JP2016508484A|2016-03-22|

---

CA2897279A1|2014-07-31|

---

NI201500096A|2016-02-16|

---

CN104936953A|2015-09-23|

---

ES2608395T3|2017-04-10|

---

BR112015017331A2|2017-07-11|

---

RU2644769C2|2018-02-14|

---

US9156831B2|2015-10-13|

---

MA38287A1|2018-01-31|

---

AP2015008604A0|2015-07-31|

---

BR112015017331A8|2019-11-12|

---

CR20150367A|2015-10-20|

---

CN104936953B|2017-03-08|

---

CA2897279C|2020-12-29|

---

US20140206700A1|2014-07-24|

---

SG11201505631PA|2015-08-28|

---

JP6307096B2|2018-04-04|

---

IL239890D0|2015-08-31|

---

AR094553A1|2015-08-12|

---

TN2015000313A1|2017-01-03|

---

PE20151979A1|2016-01-15|

---

MX2015009465A|2015-09-28|

---

TWI606047B|2017-11-21|

---

WO2014114928A1|2014-07-31|

---

MX361136B|2018-11-28|

---

PH12015501584A1|2015-10-05|

---

EP2948447A1|2015-12-02|

---

AU2014208964A1|2015-08-06|

---

TW201522329A|2015-06-16|

---

US9657008B2|2017-05-23|

---

US20160137634A1|2016-05-19|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

NL7112373A|1970-09-25|1972-03-28|

---

US4256887A|1978-04-06|1981-03-17|Merck & Co., Inc.|1,2,4-Triazoles and a method for their preparation|

---

US4683202B1|1985-03-28|1990-11-27|Cetus Corp|

---

US4683195B1|1986-01-30|1990-11-27|Cetus Corp|

---

JP3028168B2|1993-03-29|2000-04-04|住化ファインケム株式会社|Method for producing benzenesulfonamide derivative|

---

US5656416A|1994-12-22|1997-08-12|Eastman Kodak Company|Photographic processing composition and method using organic catalyst for peroxide bleaching agent|

---

AT197300T|1995-02-02|2000-11-15|Smithkline Beecham Plc|INDOLDER DERIVATIVES AS A 5-HT RECEPTORANTAGONISTE|

---

GB9517559D0|1995-08-26|1995-10-25|Smithkline Beecham Plc|Novel compounds|

---

ZA9610738B|1995-12-22|1997-06-24|Warner Lambert Co|Subtype selective nmda receptor ligands and the use thereof|

---

EP0915877A1|1996-07-25|1999-05-19|MERCK SHARP &amp; DOHME LTD.|SUBSTITUTED TRIAZOLO PYRIDAZINE DERIVATIVES AS INVERSE AGONISTS OF THE GABA A?$g5 RECEPTOR SUBTYPE|

---

GB9711753D0|1997-06-06|1997-08-06|Merck Sharp & Dohme|Therapeutic agents|

---

CN1443184A|2000-07-19|2003-09-17|霍夫曼-拉罗奇有限公司|Pyrimidine derivatives|

---

AT447561T|2001-11-21|2009-11-15|Pharmacia & Upjohn Co Llc|SUBSTITUTED ARYL 1,4-PYRAZINE DERIVATIVES|

---

US7012077B2|2001-12-20|2006-03-14|Hoffmann-La Roche Inc.|Substituted cyclohexane derivatives|

---

WO2003062224A1|2002-01-17|2003-07-31|Eli Lilly And Company|Aza-cyclic compounds as modulators of acetylcholine receptors|

---

US20030195192A1|2002-04-05|2003-10-16|Fortuna Haviv|Nicotinamides having antiangiogenic activity|

---

JP4901102B2|2002-05-03|2012-03-21|エクセリクシス，インク．|Protein kinase modulator and method of use thereof|

---

GB0306097D0|2003-03-15|2003-04-23|Elam T Ltd|Electroluminescent complexes|

---

WO2004084813A2|2003-03-21|2004-10-07|Smithkline Beecham Corporation|Chemical compounds|

---

DE10348044A1|2003-10-15|2005-05-19|Imtm Gmbh|Dual alanyl aminopeptidase and dipeptidyl peptidase IV inhibitors for the functional influence of different cells and for the treatment of immunological, inflammatory, neuronal and other diseases|

---

CN100465173C|2004-01-12|2009-03-04|西托匹亚研究有限公司|Selective kinase inhibitors|

---

AU2005252160A1|2004-04-13|2005-12-22|Astellas Pharma Inc.|Polycyclic pyrazines as potassium ion channel modulators|

---

JP2006028056A|2004-07-14|2006-02-02|Agro Kanesho Co Ltd|Pyrimidine derivative and pest controlling agent containing the same|

---

AU2005275183B2|2004-07-26|2011-03-17|Eli Lilly And Company|Oxazole derivatives as histamine H3 receptor agents, preparation and therapeutic uses|

---

BRPI0515488A|2004-09-20|2008-07-29|Xenon Pharmaceuticals Inc|heterocyclic derivatives and their use as therapeutic agents|

---

WO2006034315A2|2004-09-20|2006-03-30|Xenon Pharmaceuticals Inc.|Heterocyclic derivatives for the treatment of diseases mediated by stearoyl-coa desaturase enzymes|

---

US7767677B2|2004-09-20|2010-08-03|Xenon Pharmaceuticals Inc.|Heterocyclic derivatives and their use as stearoyl-CoA desaturase inhibitors|

---

AU2005316826A1|2004-12-15|2006-06-22|Merck Sharp & Dohme Corp.|Inhibitors of Akt activity|

---

US7834181B2|2005-02-01|2010-11-16|Slaon-Kettering Institute For Cancer Research|Small-molecule Hsp90 inhibitors|

---

KR100959771B1|2005-06-10|2010-05-28|머크 샤프 앤드 돔 코포레이션|Inhibitors of Akt activity|

---

WO2007096764A2|2006-02-27|2007-08-30|Glenmark Pharmaceuticals S.A.|Bicyclic heteroaryl derivatives as cannabinoid receptor modulators|

---

EP2004625B1|2006-03-22|2009-12-30|Vertex Pharmaceuticals Incorporated|C-met protein kinase inhibitors for the treatment of proliferative disorders|

---

TW200808800A|2006-05-05|2008-02-16|Astrazeneca Ab|MGluR5 modulators V|

---

EP2044051B1|2006-06-22|2010-01-27|BIOVITRUM AB |Pyridine and pyrazine derivatives as mnk kinase inhibitors|

---

DK2034839T3|2006-06-30|2017-12-04|Sloan-Kettering Institute For Cancer Res|TREATMENT OF NEURODEGENERATIVE DISEASES BY INHIBITION OF HSP90|

---

PE20080405A1|2006-07-21|2008-06-18|Irm Llc|COMPOUNDS AND COMPOSITIONS AS INHIBITORS OF ITPKb|

---

PL2057156T3|2006-08-23|2017-08-31|Kudos Pharmaceuticals Limited|2-methylmorpholine pyrido-,pyrazo- and pyrimido-pyrimidine derivatives as mtor inhibitors|

---

US8119671B2|2007-03-07|2012-02-21|Nissan Chemical Industries, Ltd.|Isoxazoline-substituted benzamide compound and pest control agent|

---

KR20100053556A|2007-08-14|2010-05-20|바이엘 쉐링 파마 악티엔게젤샤프트|Fused bicyclic pyrimidines|

---

WO2009024825A1|2007-08-21|2009-02-26|Astrazeneca Ab|2-pyrazinylbenzimidazole derivatives as receptor tyrosine kinase inhibitors|

---

AU2008309383B2|2007-10-11|2012-04-19|Astrazeneca Ab|Pyrrolo [2, 3 -D] pyrimidin derivatives as protein kinase B inhibitors|

---

AU2008315746A1|2007-10-25|2009-04-30|Astrazeneca Ab|Pyridine and pyrazine derivatives useful in the treatment of cell proliferative disorders|

---

US8268754B2|2007-12-07|2012-09-18|Nissan Chemical Industries, Ltd.|Substituted dihydroazole compound and pest control agent|

---

EP2234618A4|2007-12-21|2011-04-27|Scripps Research Inst|Benzopyrans and analogs as rho kinase inhibitors|

---

JP2011506563A|2007-12-20|2011-03-03|ノバルティスアーゲー|Bisthiazole derivatives, process for their preparation and their use as pharmaceuticals|

---

EP2310391A1|2008-06-27|2011-04-20|SBIO Pte Ltd|Pyrazine substituted purines|

---

BRPI0918496A2|2008-09-02|2019-09-24|Novartis Ag|bicyclic kinase inhibitor compound, its use, pharmaceutical composition and method for inhibiting pim kinase activity in a cell|

---

JP2010083883A|2008-09-08|2010-04-15|Nissan Chem Ind Ltd|Isoxazoline-substituted benzamide compound and harmful organism-controlling agent|

---

NZ590754A|2008-09-10|2012-08-31|Novartis Ag|Pyridin-4-yl substituted 2-carboxamide pyrrolidine-amide urea derivatives and their use in the treatment of phosphatidylinositol 3-kinase mediated diseases|

---

JP2010063863A|2008-09-11|2010-03-25|Mugen:Kk|Golf practice club indicating hitting point position which has a plurality of pipes or flat plates with different acoustic vibration on back of face portion of head, and method of manufacturing the same|

---

CN101676271B|2008-09-17|2011-12-14|山东轩竹医药科技有限公司|Tetracycline derivatives containing unsaturated heterocyclic amine|

---

AU2009313198B2|2008-11-10|2016-03-24|Vertex Pharmaceuticals Incorporated|Compounds useful as inhibitors of ATR kinase|

---

NZ592792A|2008-11-14|2013-07-26|Bayer Schering Pharma Ag|Heterocyclically substituted aryl compounds as hif inhibitors|

---

EP2376485B1|2008-12-19|2017-12-06|Vertex Pharmaceuticals Incorporated|Pyrazine derivatives useful as inhibitors of atr kinase|

---

JP2012513409A|2008-12-23|2012-06-14|アボット・ラボラトリーズ|Antiviral compounds|

---

WO2010086613A1|2009-01-30|2010-08-05|Betagenon Ab|Compounds useful as inhibitors as ampk|

---

US20130310362A1|2009-02-13|2013-11-21|Bayer Pharma Aktiegesellschaft|Fused pyrimidines|

---

EA201101186A1|2009-02-13|2012-04-30|Байер Фарма Акциенгезельшафт|CONDENSED PYRIMIDINES|

---

EP2408780A2|2009-03-20|2012-01-25|Pfizer Inc.|3-oxa-7-azabicycloý3.3.1¨nonanes|

---

GB0909441D0|2009-06-02|2009-07-15|Univ Sheffield|Novel indole derivatives|

---

US8293753B2|2009-07-02|2012-10-23|Novartis Ag|Substituted 2-carboxamide cycloamino ureas|

---

EP2470021B1|2009-08-27|2014-10-22|Merck Sharp & Dohme Corp.|Novel pyrrolidine derived beta 3 adrenergic receptor agonists|

---

EA022120B1|2009-10-20|2015-11-30|Целльзом Лимитид|Heterocyclyl pyrazolopyrimidine analogues as jak inhibitors|

---

US8399460B2|2009-10-27|2013-03-19|Astrazeneca Ab|Chromenone derivatives|

---

EP2338888A1|2009-12-24|2011-06-29|Almirall, S.A.|Imidazopyridine derivatives as JAK inhibitors|

---

EP2526102B1|2010-01-22|2017-03-08|Fundación Centro Nacional de Investigaciones Oncológicas Carlos III|Inhibitors of PI3 kinase|

---

WO2011095196A1|2010-02-05|2011-08-11|Merck Patent Gmbh|Hetaryl-[1,8]naphthyridine derivatives|

---

JP2011178673A|2010-02-26|2011-09-15|Nissan Chem Ind Ltd|Control method for non-agricultural/horticultural insect pest|

---

NZ603156A|2010-03-30|2014-10-31|Verseon Corp|Multisubstituted aromatic compounds as inhibitors of thrombin|

---

WO2011133920A1|2010-04-23|2011-10-27|Cytokinetics, Inc.|Certain amino-pyridines and amino-triazines, compositions thereof, and methods for their use|

---

WO2011143426A1|2010-05-12|2011-11-17|Vertex Pharmaceuticals Incorporated|Compounds useful as inhibitors of atr kinase|

---

CN102311396B|2010-07-05|2015-01-07|暨南大学|Pyrazine derivative and preparation method as well as application thereof to pharmacy|

---

EP2616469B1|2010-09-14|2016-10-26|Exelixis, Inc.|9h-purine compounds as pi3k-delta inhibitors and methods for their manufacture|

---

WO2012034526A1|2010-09-16|2012-03-22|Hutchison Medipharma Limited|Fused heteroaryls and their uses|

---

WO2012068589A2|2010-11-19|2012-05-24|Constellation Pharmaceuticals|Modulators of methyl modifying enzymes, compositions and uses thereof|

---

CN102718745A|2011-03-30|2012-10-10|中国科学院上海药物研究所|Novel amino pyridine compound, its preparation method, pharmaceutical composition containing compound and application thereof|

---

WO2012162635A1|2011-05-26|2012-11-29|Sunovion Pharmaceuticals Inc.|Metabotropic glutamate receptors 5 modulators and methods of use thereof|

---

WO2013049591A2|2011-09-29|2013-04-04|Verseon Corporation|Dual inhibitor compounds and methods of use thereof|

---

DK2833973T3|2012-04-05|2018-01-02|Vertex Pharma|Compounds useful as ATR kinase inhibitors and combination therapies thereof|WO2016146591A1|2015-03-16|2016-09-22|Astrazeneca Ab|Combination treatment|

---

TW201726140A|2015-09-17|2017-08-01|瑞典商阿斯特捷利康公司|Novel biomarkers and methods of treating cancer|

---

TW201813963A|2016-09-23|2018-04-16|美商基利科學股份有限公司|Phosphatidylinositol 3-kinase inhibitors|

---

TW201825465A|2016-09-23|2018-07-16|美商基利科學股份有限公司|Phosphatidylinositol 3-kinase inhibitors|

---

TW201815787A|2016-09-23|2018-05-01|美商基利科學股份有限公司|Phosphatidylinositol 3-kinase inhibitors|

---

CN107137405A|2017-04-14|2017-09-08|黄德莲|A kind of pharmaceutical composition for treating ephritis|

---

CN110317190A|2018-03-28|2019-10-11|首都医科大学|A kind of application of triazole-ramification of carboxylic esters in field of medicaments|

---

WO2020094363A1|2018-11-05|2020-05-14|Syngenta Participations Ag|Pesticidally active azole-amide compounds|

---

JP2022506508A|2018-11-05|2022-01-17|アイオバンス バイオセラピューティクス，インコーポレイテッド|Expanded culture of TIL using AKT pathway inhibitors|

---

EP3935053A1|2019-03-08|2022-01-12|Syngenta Crop Protection AG|Pesticidally active azole-amide compounds|

---

EP3941912A1|2019-03-20|2022-01-26|Syngenta Crop Protection AG|Pesticidally active azole amide compounds|

---

CN113597426A|2019-03-22|2021-11-02|先正达农作物保护股份公司|N- [1-ethyl ] -2-cyclopropyl-6-pyridine-4-carboxamide derivatives and related compounds as insecticides|

---

US20210330665A1|2020-04-24|2021-10-28|Astrazeneca Ab|Pharmaceutical formulations|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2019-08-20| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

---

2021-07-13| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

---

2021-11-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2022-01-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/01/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

EP13305078.1|2013-01-23|

---

EP13305078|2013-01-23|

---

PCT/GB2014/050163|WO2014114928A1|2013-01-23|2014-01-22|Chemical compounds|

---