Benzoxazepine oxazolidinone compounds and methods of use thereof

Abstract

The present application describes benzoxazepine oxazolidinone compounds having phosphoinositide-3-kinase (PI3K) regulating activity or function and stereoisomers, tautomers or pharmaceutically acceptable salts thereof, wherein the compound is selected from the following formula I structure and has substituents and structural features described in the present application. The present application also describes pharmaceutical compositions and drugs comprising compounds of formula I, as well as methods for using such PI3K regulators alone and in combination with other therapeutic agents to treat diseases or conditions mediated or dependent on PI3K disorders.

Description

[0001] This application is an application with application number 201680039251.8, application date July 1, 2016, and invention name “Benzoxipine Divisional application for the Chinese invention patent application entitled "Oxazolidinone compounds and methods of use thereof"

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This nonprovisional application, filed pursuant to 37 CFR §1.53(b), claims the benefit of U.S. Provisional Application No. 62 / 188,029, filed on July 2, 2015, pursuant to 35 USC §119(e), which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention generally relates to benzoxazepines Pharmaceutical combinations of oxazolidinone compounds having activity against hyperproliferative disorders such as cancer. The present invention also relates to methods of using said compounds for in vitro, in situ and in vivo diagnosis or treatment of mammalian cells or related pathological disorders. Background Art

[0005] Upregulation of the phosphoinositide-3-kinase (PI3K) / Akt signaling pathway is a common feature of most cancers (Yuan and Cantley (2008) Oncogene 27:5497-510). Genetic variations in the pathway have been detected in a variety of human cancers (Osaka et al. (2004) Apoptosis 9:667-76) and primarily act to stimulate cell proliferation, migration, and survival. Activation of the pathway occurs after point mutations or amplifications of the PIK3CA gene that activates the p110α (alpha) PI3K isoform (Hennessy et al. (2005) Nat. Rev. Drug Discov. 4:988-1004). Genetic deletions or loss of function mutations in the tumor suppressor PTEN (a phosphatase with opposite functions to PI3K) also increase PI3K pathway signaling (Zhang and Yu (2010) Clin. Cancer Res. 16:4325-30). These aberrations increase downstream signaling through kinases such as Akt and mTOR, and increased activity of the PI3K pathway has been proposed as a hallmark of resistance to cancer therapy (Opel et al. (2007) Cancer Res. 67:735-45; Razis et al. (2011) Breast Cancer Res. Treat. 128:447-56).

[0006] Phosphatidylinositol-3-kinase (PI3K) is the main signaling node of key survival and growth signals of lymphoma and is opposed by the activity of phosphatase PTEN. The phosphoinositide 3-dependent kinase (PI3K) signaling pathway is the most severely dysregulated pathway in hormone receptor positive breast cancer (HR+BC). The PI3K pathway is dysregulated in aggressive forms of lymphoma (Abubaker (2007) Leukemia 21:2368-2370). 8% of DLBCL (diffuse large B-cell lymphoma) cancers have PI3CA (phosphatidylinositol-3-kinase catalytic subunit α) missense mutations and 37% are PTEN negative by immunohistochemistry.

[0007] Phosphatidylinositol is one of the various phospholipids found in the cell membrane and participates in intracellular signal transduction. Cell signal transduction via 3'-phosphorylated phosphoinositide involves various cell processes such as malignant transformation, growth factor signal transduction, inflammation and immunity (Rameh et al. (1999) J.Biol Chem.274:8347-8350). The enzyme responsible for generating these phosphorylated signal transduction products, phosphatidylinositol-3-kinase (also referred to as PI3 kinase or PI3K), was initially identified as having activity associated with viral oncoproteins and growth factor receptor tyrosine kinases, which phosphorylate phosphatidylinositol (PI) and its phosphorylated derivatives at the 3'-hydroxyl of the inositol ring (Panayotou et al. (1992) Trends Cell Biol 2:358-60). Phosphoinositide-3-kinase (PI3K) is a lipid kinase (Whitman et al. (1988) Nature, 332:664) that phosphorylates lipids at the 3'-hydroxyl of the inositol ring. 3-Phospholipids (PIP3) generated by PI3 kinase act as second messengers that recruit kinases with lipid binding domains including a pleckstrin homology (PH) region such as Akt and PDK1 (phosphoinositide-dependent kinase 1) (Vivanco et al. (2002) Nature Rev. Cancer 2:489; Phillips et al. (1998) Cancer 83:41).

[0008] The PI3 kinase family includes at least 15 different enzymes subdivided by structural homology and is divided into three categories based on sequence homology and the products formed by enzyme catalysis. Class I PI3 kinases consist of 2 subunits: a 110 kd catalytic subunit and an 85 kd regulatory subunit. The regulatory subunit contains an SH2 domain and binds to tyrosine residues phosphorylated by growth factor receptors or oncogene products with tyrosine kinase activity, thereby inducing the PI3K activity of the p110 catalytic subunit, which phosphorylates its lipid substrates. Class I PI3 kinases are involved in important signal transduction events downstream of cytokines, integrins, growth factors, and immune receptors, suggesting that control of this pathway may result in important therapeutic effects such as regulation of cell proliferation and carcinogenesis. Class I PI3Ks can phosphorylate phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate (PIP2) to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-bisphosphate, and phosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ks phosphorylate PI and phosphatidylinositol-4-phosphate. Class III PI3Ks can only phosphorylate PI. As shown by recurrent oncogene mutations in p110α, the key PI3 kinase isoform in cancer is the class I PI3 kinase p110α (Samuels et al. (2004) Science 304:554; US5824492; US5846824; US6274327). Other isoforms may be important in cancer and are also implicated in cardiovascular and immunoinflammatory diseases (Workman P (2004) Biochem Soc Trans 32:393-396; Patel et al. (2004) Proc. Am. Assoc. of Cancer Res. (Abstract LB-247) 95th Annual Meeting, March 27-31, Orlando, Florida, USA; Ahmadi K and Waterfield MD (2004) "Phosphoinositide 3-Kinase: Function and Mechanisms" Encyclopedia of Biological Chemistry (Lennarz WJ, Lane MD eds) Elsevier / Academic Press). Oncogene mutations of p110α have been found very frequently in solid tumors of the colon, breast, brain, liver, ovary, stomach, lung, and head and neck. About 35-40% of hormone receptor-positive (HR + )Breast cancer tumors have PIK3CA mutations. PTEN abnormalities have been found in glioblastoma, melanoma, prostate cancer, endometrial cancer, ovarian cancer, breast cancer, lung cancer, head and neck cancer, hepatocellular carcinoma, and thyroid cancer.

[0009] PI3 kinase (PI3K) is a heterodimer composed of p85 and p110 subunits (Otsu et al. (1991) Cell 65: 91-104; Hiles et al. (1992) Cell 70: 419-29). Four different class I PI3Ks have been identified, designated as PI3Kα, β, δ and ω, each consisting of a different 110 kDa catalytic subunit and a regulatory subunit. Three of the catalytic subunits, p110α, p110β and p110δ, each interact with the same regulatory subunit p85; while p110γ interacts with a different regulatory subunit p101. The expression patterns of each of these PI3Ks in human cells and tissues are different. In each of the PI3K α, β, and δ isoforms, the p85 subunit localizes PI3 kinase to the plasma membrane by interacting through its SH2 domain with phosphorylated tyrosine residues in target proteins (present in the appropriate sequence context) (Rameh et al. (1995) Cell, 83:821-30; Volinia et al. (1992) Oncogene, 7:789-93).

[0010] The PI3 kinase / Akt / PTEN pathway is an attractive target for cancer drug development because such drugs are expected to inhibit cell proliferation, suppress signals from stromal cells that maintain cancer cell survival and chemoresistance, reverse the suppression of apoptosis, and overcome the intrinsic resistance of cancer cells to cytotoxic agents. PI3K is activated by receptor tyrosine kinase signaling as well as by mutations in the p110 catalytic subunit that activate PI3K, loss of the tumor suppressor PTEN, or by rare activating mutations in AKT.

[0011] Taselisib (GDC-0032, Roche RG7604, CAS Reg. No. 1282512-48-4, Genentech Inc.) is named 2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)-1H-pyrazol-1-yl)-2-methylpropionamide, has potent PI3K activity (Ndubaku, CO et al. (2013) J. Med. Chem. 56: 4597-4610; WO2011 / 036280; US8242104; US8343955) and is being studied in patients with locally advanced or metastatic solid tumors. Taselisib (GDC-0032) is a β-isoform-retaining inhibitor of the PI3K catalytic subunit, with a 31-fold higher selectivity for the α subunit compared to the β subunit. Taselisib exhibits higher selectivity for mutant PI3Kα isoforms compared to wild-type PI3Kα (Olivero AG et al., AACR 2013. Abstract DDT02-01). Taselisib is currently being developed for the treatment of patients with estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer (mBC) and non-small cell lung cancer (NSCLC). In a Phase Ia study using single-agent taselisib, partial responses (PR) were observed in 6 / 34 enrolled patients. All 6 responses were observed in patients with PIK3CA-mutated tumors (Juric D. et al. AACR 2013), indicating the need to determine the PIK3CA mutation status of patients treated with taselisib.

[0012] Recent clinical data of PI3K inhibitors have implicated PI3Kδ activity as a source of gastrointestinal toxicity (Akinleye et al. "Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics" Journal of Hematology & Oncology 2013, 6: 88-104; C. Saura et al. "Phase Ib Study of the PI3K Inhibitor Taselisib (GDC-0032) in Combination with Letrozole in Patients with Hormone Receptor-Positive Advanced Breast Cancer" San Antonio Breast Cancer Symposium-December 12, 2014, PD5-2; Lopez et al. "Taselisib, a selective inhibitor of PIK3CA, is highly effective on PIK3CA-mutated and HER2 / neuamplified uterine serous carcinoma in vitro and in vivo" (2014) Gynecologic Oncology).

[0013] Idelalisib (GS-1101, CAL-101, Gilead Sciences Inc., CAS Reg. No. 870281-82-6, 5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone) is a selective PI3Kδ (delta) inhibitor and is approved for the treatment of chronic lymphocytic leukemia CLL (Somoza, JRet al (2015) J. Biol. Chem. 290:8439-8446; US 6800620; US 6949535; US8138195; US 8492389; US 8637533; US 8865730; US 8980901; RE44599; RE44638). Diarrhea and colitis are among the most common adverse events reported after idelalisib treatment (Brown et al. “Idelalisib, an inhibitor of phosphatidylinositol 3-kinase p110d, for relapsed / refractory chronic lymphocytic leukemia” (2014) Blood 123(22):3390-3397; Prescribing Information 2014; REMS Fact Sheet). The significant GI toxicity observed with idelalisib treatment is consistent with the hypothesis that inhibition of PI3Kδ(delta) is the source of GI toxicity. Other serious side effects have been observed in clinical trials in combination with other therapies. Adverse events, including death, have been associated with infections such as pneumonia. In March 2016, the EMA's Pharmacovigilance Risk Assessment Committee (PRAC) issued an interim warning and recommended that patients receive combination antibiotic therapy and be regularly monitored for infections while taking Zydelig (idelalisib). In March 2016, the U.S. Food and Drug Administration issued a warning that "six ongoing trials exploring idelalisib have been suspended due to increased rates of reported adverse events, including death." Clinical trials in combination with other therapies have been suspended."

[0014] There is a need for additional PI3Kα modulators for treating cancer, particularly inhibitors of PI3Kα that are selective for tumors expressing mutant PI3Kα relative to cells expressing non-mutant PI3Kα. There is a particular need for agents that selectively inhibit PI3Kα isoforms relative to PI3Kβ, PI3Kδ, and PI3Kγ isoforms, which can be expected to result in an enhanced therapeutic window. Summary of the invention

[0015] The present invention generally relates to benzoxazepines having selective activity in modulating mutant forms of PI3K alpha isoforms and having the structure of formula I Oxazolidinone compounds:

[0016]

[0017] and stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof. Various substituents are defined in this application.

[0018] Another aspect of the present invention is a pharmaceutical composition comprising a benzoxazepine of formula I Oxazolidinone compound and pharmaceutically acceptable carrier, glidant, diluent or excipient.

[0019] Another aspect of the present invention is a method of treating cancer in a patient having cancer, comprising administering to the patient a therapeutically effective amount of a benzoxazepine of Formula I. Oxazolidinone compounds.

[0020] Another aspect of the invention is a kit for the therapeutic treatment of breast cancer comprising:

[0021] a) Benzoxazepine of formula I Oxazolidinone compounds; and

[0022] b) instructions for use in the therapeutic treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 shows the x-ray co-crystal structures of PI3Kα with A) taselisib (GDC-0032), B) compound 529 of US8242104, C) compound 101 and D) compound 103.

[0024] FIG2 shows the x-ray co-crystal structures of PI3Kα with A) taselisib (GDC-0032) and B) compound 101.

[0025] Figure 3AShown are Western blot data depicting p110α (p110a, p110 alpha) levels in HCC-1954 cells (PI3Kα mutant H1047R) after treatment with Compound 101, Compound 103, and Compound 436 of US8242104 for 24 hours.

[0026] Figure 3B Shown are Western blot data depicting p110α (p110a, p110 alpha) levels in HDQ-P1 cells (PI3Kα wild type) after treatment with Compound 101, Compound 103, and Compound 436 of US 8242104 for 24 hours. DETAILED DESCRIPTION

[0027] Certain embodiments of the present invention will now be described in detail, examples of which are illustrated in the accompanying structures and formulas. When the present invention is described in conjunction with the listed embodiments, it should be understood that the embodiments are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to include all optional forms, modified forms and equivalent forms that may be included within the scope of the present invention as defined in the claims. Those skilled in the art will recognize a variety of methods and materials that are similar or equivalent to the methods and materials described in this application and can be used to implement the present invention. The present invention is by no means limited to the methods and materials described. If one or more of the documents, patents and similar materials introduced are different or contradictory to this application (including but not limited to defined terms, term usage, described technology, etc.), the present application shall prevail. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. Although methods and materials similar or equivalent to those methods and materials described in this application can be used to implement or test the present invention, appropriate methods and materials are described below. All publications, patent applications, patents and other references mentioned in this application are fully introduced into this application by reference. Unless otherwise stated, the nomenclature used in this application is based on the IUPAC systematic nomenclature.

[0028] definition

[0029] When specifying the number of substituents, the term "one or more" refers to one substituent to the maximum possible number of substitutions, i.e., replacing one hydrogen to all hydrogens with a substituent. The term "substituent" refers to an atom or group of atoms that replaces a hydrogen atom on a parent molecule. The term "substituted" means that the specified group carries one or more substituents. When any group can carry multiple substituents and various possible substituents are provided, the substituents are independently selected and do not need to be the same. The term "unsubstituted" means that the specified group does not carry a substituent. The term "optionally substituted" means that the specified group is unsubstituted or substituted by one or more substituents independently selected from possible substituents. When specifying the number of substituents, the term "one or more" refers to one substituent to the maximum possible number of substitutions, i.e., replacing one hydrogen to all hydrogens with a substituent.

[0030] The term "alkyl" as used herein refers to a group having 1 to 12 carbon atoms (C1-C 12), wherein the alkyl group may be optionally substituted independently with one or more of the following substituents. In another embodiment, the alkyl group has 1-8 carbon atoms (C1-C8) or 1-6 carbon atoms (C1-C6). Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3) CH2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH 2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH (CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, 1-heptyl, 1-octyl, etc.

[0031] The terms "carbocycle", "carbocyclyl", "carbocyclic ring" and "cycloalkyl" refer to a cycloalkyl having 3 to 12 carbon atoms (C3-C 12) is in monocyclic form or has a non-aromatic saturated or partially unsaturated ring of a monovalence of 7-12 carbon atoms in bicyclic form. The bicyclic carbocyclic ring with 7-12 atoms can be arranged as, for example, a bicyclic [4,5] system, a bicyclic [5,5] system, a bicyclic [5,6] system or a bicyclic [6,6] system, and the bicyclic carbocyclic ring with 9 or 10 ring atoms can be arranged as a bicyclic [5,6] system or a bicyclic [6,6] system or arranged as a bridged system such as bicyclic [2.2.1] heptane, bicyclic [2.2.2] octane and bicyclic [3.2.2] nonane. Spiro carbocyclic moieties are also included in the scope of this definition. Examples of spiro carbocyclic moieties include [2.2] pentyl, [2.3] hexyl and [2.4] heptyl. Examples of monocyclic carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, etc. Carbocyclyl groups are optionally substituted independently with one or more substituents described herein.

[0032] "Aryl" means a group having 6 to 20 carbon atoms (C6-C 20 ) is a monovalent aromatic hydrocarbon radical obtained by removing a hydrogen atom from a single carbon atom in a parent aromatic ring system. Some aryl groups are represented as "Ar" in the exemplary structures. Aryl groups include bicyclic groups containing an aromatic ring fused to a saturated ring, a partially unsaturated ring, or an aromatic carbocyclic ring. Typical aryl groups include, but are not limited to, groups obtained from benzene (phenyl), substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthalene, and the like. Aryl groups may be optionally substituted independently with one or more substituents described herein.

[0033] The terms "heterocycle", "heterocyclyl" and "heterocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds in the ring) carbocyclic group having 3 to about 20 ring atoms, wherein at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, and the remaining ring atoms are C, wherein one or more ring atoms are optionally independently substituted with one or more substituents described below. The heterocycle may be a monocyclic ring having 3-7 ring members (2-6 carbon atoms and 1-4 heteroatoms selected from N, O, P and S) or a bicyclic ring having 7-10 ring members (4-9 carbon atoms and 1-6 heteroatoms selected from N, O, P and S) (e.g., a bicyclic [4,5] system, a bicyclic [5,5] system, a bicyclic [5,6] system or a bicyclic [6,6] system). Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968) (particularly Chapters 1, 3, 4, 6, 7, and 9); "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present) (particularly Volumes 13, 14, 16, 19, and 28); and J. Am. Chem. Soc. (1960) 82: 5566. "Heterocyclic" also includes groups in which a heterocyclic group is fused to a saturated ring, a partially unsaturated ring, an aromatic carbocyclic ring, or an aromatic heterocyclic ring. Examples of heterocycles include, but are not limited to, morpholin-4-yl, piperidin-1-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-1-yl, thiomorpholin-4-yl, S,S-dioxothiomorpholin-4-yl, azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thiooxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepanyl, Base, diazepine Thiazepine The term "spiro heterocyclic radical" refers to a cycloheterocyclic radical, ... Examples of heterocyclyl groups wherein 2 ring atoms are substituted with oxo (=0) moieties are pyrimidinone and 1,1-dioxo-thiomorpholinyl.

[0034] The term "heteroaryl" refers to a monovalent aromatic group in the form of a 5-, 6- or 7-membered ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur and includes fused ring systems having 5-20 atoms, wherein at least one ring is aromatic. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolyl, isoquinolyl, tetrahydroisoquinolyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.

[0035] The terms "treatment" and "treatment" refer to therapeutic treatment, wherein the purpose is to slow down (mitigate) the development or spread of undesirable physiological changes or disorders such as arthritis or cancer. For the purposes of this application, useful or desired clinical results include but are not limited to relieving symptoms, reducing the extent of the disease, stabilizing the disease state (i.e., not worsening), delaying or slowing the course of the disease, improving or alleviating the disease state and alleviating (whether partially or completely), whether detectable or undetectable. "Treatment" can also refer to extending survival compared to the survival expected without treatment. Those in need of treatment include those suffering from a disease or disorder.

[0036] The phrase "therapeutically effective amount" refers to an amount of a compound of the invention that (i) treats a specific disease, condition, or disorder; (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder; or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably stop to some extent) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. A drug can prevent cancer cell growth and / or kill existing cancer cells to some extent, and it can be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be determined, for example, by evaluating the time to disease progression (TTP) and / or determining the response rate (RR).

[0037] The term "cancer" refers to or is used to describe a physiological state in mammals that is generally characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of the above cancers include squamous cell carcinomas (e.g., epithelial squamous cell carcinomas); lung cancers, including small cell lung cancers, non-small cell lung cancers ("NSCLC"), lung adenocarcinomas, and lung squamous cell carcinomas; peritoneal cancer; hepatocellular carcinoma; stomach cancer or gastric cancer, including gastrointestinal cancer; pancreatic cancer; glioblastomas; cervical cancer; ovarian cancer; liver cancer; bladder cancer; hepatomas; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatocarcinomas; anal cancer; penile cancer; and head and neck cancer.

[0038] "Hematological malignancies" are types of cancer that affect the blood, bone marrow, and lymph nodes. Because these three are closely related through the immune system, a disease that affects one of these three will usually affect the other two as well: although lymphoma is a lymph node disease, it often spreads to the bone marrow to affect the blood. Hematological malignancies are malignancies ("cancers") and are usually treated by specialists in hematology and / or oncology. "Hematology / oncology" is a subspecialty under internal medicine in some centers, while in other centers it is considered a separate department (which also has surgical and radiation oncologists). Not all hematological diseases are malignant ("cancerous"); these other blood diseases can also be managed by hematologists. Hematological malignancies can originate from two major blood cell lineages: myeloid and lymphoid cell lineages. The myeloid cell lineage usually produces granulocytes, red blood cells, platelets, macrophages, and mast cells; the lymphoid cell lineage produces B cells, T cells, NK cells, and plasma cells. Lymphomas, lymphocytic leukemias, and myelomas arise from the lymphoid cell lineage, while acute and chronic myeloid leukemias, myelodysplastic syndromes, and myeloproliferative disorders arise from myeloid cells. Leukemias include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute monocytic leukemia (AMOL), and small lymphocytic lymphoma (SLL). Lymphomas include Hodgkin lymphoma (all four subtypes) and non-Hodgkin lymphoma (NHL, all subtypes).

[0039] "Chemotherapeutic agents" are compounds useful in treating cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, spindle-toxic plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy. Examples of chemotherapeutic agents include: ibrutinib (IMBRUVICA TM , APCI-32765, Pharmacyclics Inc. / Janssen Biotech Inc.; CAS Reg. No. 936563-96-1, US7514444), idelalisib (formerly CAL-101, GS1101, GS-1101, Gilead Sciences Inc.; CAS Reg. No. 1146702-54-6), erlotinib ( Genentech / OSIPharm.), docetaxel ( Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine ( Lilly)、PD-0325901(CASNo.391210-10-9,Pfizer)、cisplatin( (SP-4-2)-diaminedichloroplatinum(II), cis-diaminedichloroplatinum(II), CAS No.15663-27-1), carboplatin (CAS No.41575-94-4), paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, NJ), trastuzumab ( Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentaazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No.85622-93-1, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethylamine, )、doxorubicin ( CAS No.23214-92-8), Akti-1 / 2, HPPD and rapamycin.

[0040] Chemotherapeutic agents include inhibitors of B cell receptor targets, such as BTK, Bcl-2, and JAK inhibitors.

[0041] Further examples of chemotherapeutic agents include oxaliplatin ( Sanofi), bortezomib ( Millennium Pharm.), sunitinib (sutent) ( SU11248, Pfizer), letrozole ( Novartis), imatinib mesylate ( Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK 222584 (Novartis), fulvestrant ( AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, Wyeth), lapatinib ( GSK572016, Glaxo SmithKline, lonafarnib (SARASAR TM , SCH 66336, Schering Plough), sorafenib ( BAY43-9006, Bayer Labs), gefitinib ( AstraZeneca), irinotecan ( CPT-11, Pfizer), tipifarnib (ZARNESTRA TM , Johnson & Johnson), ABRAXANE TM (without Cremophor), albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus ( Wyeth), pazopanib (GlaxoSmithKline), canfosfamide ( Telik, thiotepa, and cyclosphosphamide Alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenephosphoramide sulfide, and thiamethoxam; hylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide and uracil mustard;Nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, including calicheamicin γ 1I and calicheamicin ω 1I (Angew Chem. Intl. Ed. Engl. (1994) 33: 183-186); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, rleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU);Folic acid analogs such as denopterin, methotrexate, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and floxuridine; androgens such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex ( polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine Novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine ( Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above substances.

[0042] The following are also included in the definition of "chemotherapeutic agent": (i) anti-hormonal drugs used to modulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and (toremifine citrate) and selective estrogen receptor modulators (SERDs) such as fulvestrant ( Astra Zeneca); (ii) aromatase inhibitors that inhibit aromatase (aromatase regulates estrogen production in the adrenal glands), such as 4(5)-imidazoles, aminoglutethimide, (megestrolacetate), (exemestane; Pfizer), formestanie, fadrozole, (vorozole), (letrozole; Novartis) and (anastrozole; AstraZeneca); (iii) anti-androgen drugs, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors, such as MEK inhibitors, such as cobimetinib (WO 2007 / 044515); (v) lipid kinase inhibitors, such as taselisib (GDC-0032, Genentech Inc.); (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, for example, PKC-α, Ralf and H-Ras, such as oblimersen ( Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g. ) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, e.g. and rIL-2; topoisomerase 1 inhibitors, such as and rmRH; (ix) anti-angiogenic drugs, such as bevacizumab ( Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

[0043] Therapeutic antibodies are also included in the definition of "chemotherapeutic agent", such as alemtuzumab (Campath), bevacizumab ( Genentech; cetuximab ( Imclone);panitumumab( Amgen), rituximab ( Genentech / Biogen Idec), pertuzumab (PERJETA TM ,2C4,Genentech), trastuzumab ( Genentech), trastuzumab emtansine ( Genentech Inc.) and tositumomab (BEXXAR, Corixia).

[0044] "Metabolites" are products produced by the in vivo metabolism of a particular compound or its salt. Metabolites of a compound can be identified using conventional techniques known in the art and their activity can be determined using the tests described herein. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Therefore, the present invention includes metabolites of the compounds of the present invention, including compounds produced by the following method, the method comprising contacting a compound of formula I of the present invention with a mammal for a period of time sufficient to produce its metabolites.

[0045] The term "package insert" refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or precautions concerning the use of such therapeutic products.

[0046] The term "chiral" refers to molecules that have the property of non-superimposability of their mirror image partners, whereas the term "achiral" refers to molecules that are superimposable on their mirror image partners.

[0047] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0048] "Diastereomers" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography.

[0049] "Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0050] The stereochemical definitions and common sense used in this application are generally consistent with SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. The present invention is intended to include all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers, and mixtures thereof such as racemic mixtures, forming part of the present invention. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to its chiral center. The prefixes d and l or (+) and (-) are used to specify the sign of rotation of plane polarized light by a compound, where (-) or l indicates that the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers and mixtures of the above isomers are often referred to as mixtures of enantiomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur when a chemical reaction or process is not stereoselective or stereospecific. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric substances that are not optically active. Enantiomers may be separated from a racemic mixture by chiral separation methods such as supercritical fluid chromatography (SFC). Configurational assignments at chiral centers in separated enantiomers may be tentative and are depicted in the structures of Table 1 for illustrative purposes, whereas stereochemistry is definitively established, such as from X-ray crystallographic data.

[0051] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol isomerizations and imine-enamine isomerizations. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0052] The term "pharmaceutically acceptable salt" refers to salts that are biologically or otherwise undesirable. Pharmaceutically acceptable salts include acid and base addition salts. The phrase "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients contained in the formulation and / or the mammal to be treated therewith.

[0053] The term "pharmaceutically acceptable acid addition salt" refers to those pharmaceutically acceptable salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid and organic acids selected from the aliphatic, alicyclic, aromatic, aryl-aliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid "mesylate", ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.

[0054] The term "pharmaceutically acceptable base addition salt" refers to a pharmaceutically acceptable salt formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, halamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine and polyamine resins.

[0055] "Solvate" refers to an association or complex of one or more solvent molecules with a compound of the invention. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0056] The term “EC 50 ” is the half maximal effective concentration and represents the plasma concentration of a specific compound required to obtain 50% of the maximum specific effect in vivo.

[0057] The term "Ki" is the inhibition constant and represents the absolute binding affinity of a particular inhibitor to a receptor. If there is no competing ligand (e.g., a radioligand), it is measured using a competitive binding assay and is equal to the concentration at which a particular inhibitor occupies 50% of the receptors. Ki values ​​can be logarithmically converted to pKi values ​​(-log Ki), where higher values ​​represent exponentially greater potency.

[0058] The term "IC 50 ” is the half maximal inhibitory concentration and represents the concentration of a particular compound required to obtain 50% inhibition of a biological process in vitro. IC 50 pIC values ​​were converted to logarithm 50 Value (-log IC 50 ), where higher values ​​indicate exponentially greater potency. IC 50 The values ​​are not absolute but depend on the experimental conditions and can be converted into an absolute inhibition constant (Ki), for example using the Cheng-Prusoff equation (Biochem. Pharmacol. (1973) 22:3099). Other percentage inhibition parameters, such as IC 70 、IC 90 wait.

[0059] The terms "compounds of the invention" and "compounds of the present invention" and "compounds of Formula I" include compounds of Formula I and stereoisomers, geometric isomers, tautomers, solvates, metabolites, pharmaceutically acceptable salts and prodrugs thereof.

[0060] Any formula or structure given herein (including compounds of Formula I) is also intended to represent hydrates, solvates, and polymorphs of such compounds, and mixtures thereof.

[0061] Any formula or structure given in the present application (including compounds of formula I) is also intended to represent unlabeled forms of the compounds as well as isotopically labeled forms. Isotopically labeled compounds have the structures described by the chemical formulas given in the present application, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as but not limited to 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S. 36 Cl and 125I. The present invention includes various isotopically labeled compounds of the present invention, for example, radioactive isotopes such as 3 H. 13 C and 14 C is introduced into those compounds of the present invention. The above-mentioned isotope-labeled compounds can be used for metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) including drug or substrate tissue distribution determination or radiotherapy performed on patients. Deuterium-labeled or substituted therapeutic compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetic) properties, including absorption, distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium can provide some therapeutic advantages (such as an increase in in vivo half-life or a reduction in dosage requirements) due to better metabolic stability. 18 F-labeled compounds can be used in PET or SPECT studies. Isotopically labeled compounds of the invention and their prodrugs can generally be prepared by carrying out the procedures disclosed in the following schemes or examples and preparations and substituting readily available isotopically labeled reagents for non-isotopically labeled reagents. In addition, heavier isotopes, particularly deuterium (i.e. 2 H or D) can obtain some therapeutic advantages (such as increase of half-life in vivo or reduction of dosage requirement or improvement of therapeutic index) due to better metabolic stability. It should be understood that deuterium in the present application is considered as a substituent in the compound of formula (I). The concentration of the above-mentioned heavier isotopes, especially deuterium, can be defined by the isotopic enrichment factor. In the compounds of the present invention, any atom not specifically designated as a specific isotope is intended to represent any stable isotope of the atom. Unless otherwise stated, when a position is specifically designated as "H" or "hydrogen", it should be understood that the position has a concentration of hydrogen with a natural abundance isotopic composition of hydrogen. Therefore, in the compounds of the present invention, any atom specifically designated as deuterium (D) is intended to represent deuterium.

[0062] Benzoxazepine Oxazolidinone compounds

[0063] The present invention provides a benzoxazepine of formula I Oxazolidinone compounds and pharmaceutical preparations thereof, which are potentially useful for treating cancer and have the following structure:

[0064]

[0065] and stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof, wherein:

[0066] R 1 is selected from -CH3, -CH2CH3, cyclopropyl and cyclobutyl;

[0067] R 2 Selected from -CH3, -CHF2, -CH2F and -CF3.

[0068] Exemplary embodiments of compounds of Formula I include those wherein R 1 It is cyclopropyl.

[0069] Exemplary embodiments of compounds of Formula I include those wherein R 1 It is CH3 or cyclopropyl.

[0070] Exemplary embodiments of compounds of Formula I include those wherein R 1 For CH3.

[0071] Exemplary embodiments of compounds of Formula I include those wherein R 2 is -CHF2.

[0072] Exemplary embodiments of compounds of Formula I include those wherein R 2 It is -CH2F.

[0073] Exemplary embodiments of compounds of Formula I include those wherein R 1 is cyclopropyl and R 2 is -CHF2.

[0074] Exemplary embodiments of compounds of Formula I include those wherein R 1 is cyclopropyl and R 2 It is -CH2F.

[0075] Exemplary embodiments of compounds of Formula I include those wherein R 1 CH3 and R 2 is -CHF2.

[0076] Exemplary embodiments of compounds of Formula I include the compounds in Table 1.

[0077] The compounds of formula I of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and mixtures thereof such as racemic mixtures, constitute part of the present invention. In some cases, stereochemistry has not yet been determined or has been tentatively assigned.

[0078] In addition, the present invention includes all diastereoisomers, including cis-trans (geometric) and conformational isomers. For example, if the compound of formula I contains a double bond or a fused ring, the cis- and trans-forms and mixtures thereof are included within the scope of the present invention.

[0079] In the structures shown in this application, if the stereochemistry of any particular chiral atom is not specified, the compounds of the invention are intended to include all stereoisomers. If the stereochemistry is specified by a solid wedge or dashed line representing a specific configuration, then the stereoisomer is so specified and defined.

[0080] The compounds of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc., and it is intended that the present invention embrace both solvated and unsolvated forms.

[0081] The compounds of the present invention may also exist in different tautomeric forms and all such forms are included within the scope of the present invention. The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions through the reorganization of some bonding electrons.

[0082] Biological evaluation

[0083] The relative potency of the compounds of Formula I as inhibitors of enzyme activity (or other biological activity) can be determined by determining the concentration of each compound that inhibits the activity to a predetermined degree and then comparing the results. Generally, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or "IC 50 IC can be determined using conventional techniques known in the art. 50 Typically, IC 50 It can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study. The enzyme activity values ​​obtained experimentally are then plotted against the inhibitor concentration used. The inhibitor concentration that shows 50% enzyme activity (compared to the activity in the absence of any inhibitor) is taken as the IC 50 Similarly, other inhibitory concentrations can be defined by appropriate activity determination. For example, in some cases, it may be necessary to determine the 90% inhibition concentration, i.e., IC 90 wait.

[0084] The exemplary compounds of Formula I in Table 1 were prepared, characterized and tested for binding to various isoforms and mutant forms of PI3K according to the methods of the present invention and have the following structures, corresponding names (ChemBioDraw, Version 12.0.2, CambridgeSoft Corp., Cambridge MA) and biological activities. When more than one name is associated with a compound of Formula I or an intermediate, the compound should be qualified by chemical structure.

[0085] Table 1.Compounds of formula I

[0086]

[0087]

[0088] TASELISIB

[0089] The IUPAC name of the compound known as taselisib, GDC-0032 and Roche RG7604 (CAS Reg. No. 1282512-48-4, Genentech Inc.) is: 2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide, the structure is:

[0090]

[0091] Includes stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof.

[0092] Taselesib can be prepared and characterized as described in WO 2011 / 036280, US 8242104 and US 8343955.

[0093] Pictilisib

[0094] Compounds called pictilisib, GDC-0941, Roche, RG-7321 and pictrelisib, (CAS Reg. No. 957054-30-7, Genentech Inc.,) are potent multi-target class I (pan) PI3K isoform inhibitors. GDC-0941 is currently undergoing Phase II clinical trials for the treatment of advanced solid tumors. GDC-0941 is named 4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine (US 7781433; US 7750002; Folkes et al (2008) Jour. of Med. Chem. 51(18):5522-5532) and has the following structure:

[0095]

[0096] Includes stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof.

[0097] ALPELISIB

[0098] A compound called alpelisib (BYL719, Novartis, CAS#: 1217486-61-7) is an oral selective inhibitor of the PI3Kα isoform and is undergoing clinical trials for the potential treatment of a variety of tumor types, including a Phase III study in combination with fulvestrant for second-line hormone receptor-positive, HER2-advanced metastatic breast cancer (Furet, P. et al (2013) Bioorg. Med. Chem. Lett. 23: 3741-3748; US8227462; US8476268; US8710085). Alpelisib is named (S)-N1-(4-methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide) and has the following structure:

[0099]

[0100] Biochemical inhibition of PI3K isoforms

[0101] The ability of compounds of the invention to act as inhibitors of PI3Kα with selectivity over PI3Kβ, PI3Kδ, and PI3Kγ was determined using the method of Example 901. Unless otherwise indicated, the Ki values ​​shown in Tables 2A and 2B represent the geometric mean of a minimum of three independent experiments.

[0102] Table 2A shows the biochemical inhibition of four PI3K isoforms by the compounds of formula I of Table 1. In addition, two clinically tested PI3K compounds taselisib and pictilisib are included as comparators. When compared with taselisib (GDC-0032) and pictilisib (GDC-0941), the representative compounds of the present invention show strong activity to PI3Kα, and show significantly enhanced selectivity relative to other isoforms PI3Kβ, PI3Kδ and PI3Kγ. Specifically, the selectivity ratio of the second column from the right of Table 2A shows that each compound of formula I 101-107 has a PI3Kα / δ selectivity ratio far higher than taselisib or pictilisib. In fact, both taselisib and pictilisib are more active to PI3Kδ than to PI3Kα, that is, their selectivity ratio is less than 1. The selectivity ratio of compounds of formula I 101-107 ranges from 301 times to 634 times.

[0103] Table 2B shows biochemical inhibition of two PI3K isoforms α and δ and PI3Kα to δ selectivity ratios of certain comparative compounds of US 8242104 and compounds with a dimethyloxazolidin-2-one group from US 8263633 (compound 356, column 149). The comparative compounds shown in Table 2B are examples of the broad genus described in each of US 8242104 and US 8263633. Neither US 8242104 nor US 8263633 discloses compounds within the scope of the compounds of formula I of the present invention. Although the representative comparative examples of US 8242104 as described in Table 2B show a PI3Kα (alpha) to PI3Kδ (delta) selectivity ratio>1, the maximum selectivity ratio observed is 46.9 times. Formula I compounds 101-107 therefore achieve significantly higher selectivity ratios than the examples of US 8242104. There is no teaching in US 8242104 or US 8263633 of selecting structural elements of compounds of formula I to achieve high PI3Kα selectivity over PI3Kδ. This unexpected property of greater than 300-fold PI3Kα selectivity is retained across the entire range of compounds listed in Table 1.

[0104] PI3K inhibitors currently in clinical trials, such as taselisib (WO 2011 / 036280; US 8242104; US8343955) and other representative examples of US 8242104, exhibit significant activity against the PI3Kδ(delta) isoform. This lack of selectivity for PI3Kδ(delta) is consistent with the GI toxicity observed with taselisib in the clinic. There is a need for PI3Kα(alpha) inhibitors that contain the advantageous features of the representative embodiments of US8242104, while lacking activity against PI3Kδ(delta). The present invention provides compounds that satisfy this activity and selectivity profile.

[0105] The unexpected properties of PI3Kα selectivity are beneficial for eliminating the gastrointestinal toxicity observed in clinical PI3K inhibitor candidates. Recent clinical data of PI3K inhibitors have implicated PI3Kδ activity as a source of gastrointestinal toxicity (Akinleye et al., "Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics" Journal of Hematology & Oncology 2013, 6: 88-104). See Table 2 of PI3K inhibitors taselisib and pictilisib in clinical trials.

[0106] Due to the significantly higher selectivity of PI3Kα(alpha) inhibition relative to PI3Kδ(delta) inhibition, it is expected that Formula I compounds 101-107 achieve a greater margin of clinical activity driven by PI3Kα(alpha) inhibition relative to PI3Kδ(delta) driven toxicity than the clinically tested taselisib and pictilisib or any of the examples of US 8242104 or US 8263633. Therefore, the Formula I compounds of the present invention can be used as therapeutic agents with reduced toxicity characteristics relative to drugs that have a greater inhibitory effect on the normal function of PI3Kβ, PI3Kδ or PI3Kγ.

[0107] Table 2A Biochemical inhibition of PI3K isoforms by the compound of formula I and comparative compounds taselisib and pictilisib

[0108]

[0109]

[0110] Table 2B Comparison of compounds for biochemical inhibition of PI3K isoforms

[0111]

[0112]

[0113]

[0114] *Ki value represents the average of two experiments, **Ki value represents a single experiment

[0115] Interaction of compounds with PI3K

[0116] A plausible basis for the PI3K alpha selectivity of the compounds of formula I may lie in certain binding interactions.

[0117] The ability of the compounds of the invention to specifically interact with PI3Kα was determined by solving x-ray co-crystal structures of representative compounds with PI3Kα (alpha) using the method of Example 908. The optimized structural design of PI3K inhibitors with selectivity for the PI3Kα isoform relative to other isoforms may include the precise positioning and arrangement of atoms and functional groups to interact with isoform-specific residues in the binding site. Specifically, it was found that in 5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine Substitutions at positions 9 and 2 of the ring system have important effects on the specific activity of the compounds against PI3Kα.

[0118] Figure 1A -D shows the x-ray co-crystal structures of taselisib (GDC-0032), reference compound 529 (US 8242104) and two representative compounds of the present invention with PI3Kα. Figure 1A As shown, taselisib (GDC-0032) contains a primary amide functional group located in close contact with both Gln859 and Ser854, which seems to provide the possibility of hydrogen bonding interactions. Residue Gln859 is specific for the PI3Kα isoform, where different residues occupy this position in other isoforms (PI3Kβ=Asp, PI3Kδ=Asn, PI3Kγ=Lys). However, despite the close contact with the PI3Kα-specific residues, GDC-0032 has equivalent activity for both isoforms PI3Kα and PI3Kβ, and has only slightly reduced activity for isoform PI3Kγ as measured in biochemical assays (see Table 2A).

[0119] like Figure 1B As shown, reference compound 529 (US 8242104) contains a primary amide functional group in a similar position to taselisib. This functional group is within the appropriate distance and has the appropriate geometry of both Ser854 and Gln859 to make hydrogen bonding interactions. The 46.9-fold selectivity ratio of PI3Kα relative to PI3Kβ (see Table 2B) can be rationalized based on these interactions and knowing that PI3Kδ does not contain a Gln residue at position 859, these interactions should be specific for PI3Kα.

[0120] Figure 1C and 1D The primary amides of the (S)-2-aminopropionamide group of compound 101 and the (S)-2-amino-2-cyclopropylacetamide group of compound 103 each occupy a position in the binding site similar to the primary amides of GDC-0032 and reference compound 529 (US8242104). The primary amide functional groups in each representative of the present invention are within an appropriate distance and have the appropriate geometry of Ser854 and Gln859 to form ideal hydrogen bonding interactions. Despite the obvious similarities in functional group position and orientation, Figure 1C and 1DThe representative examples shown in and other compounds of the present invention with similar substituents and functional groups improve the interaction of the primary amide of taselisib and reference compound 529 (US 8242104), thereby observing that the selectivity of the compounds of the present invention for PI3Kα measured in biochemical assays relative to PI3Kδ is significantly improved. The selectivity of compound 101 is 361 times, and the selectivity of compound 103 is 634 times, which is significantly improved relative to the reference compound 529 (US 8242104) with only 46.9 times selectivity. In view of the similarity of the primary amide functional group between taselisib and other compounds of US 8242104 (as exemplified by reference compound 529), as demonstrated by the compounds of the present invention, the selectivity of PI3Kα relative to PI3Kδ is an unexpected property. There is no teaching in US 8242104 of selecting the structural elements of the compound of formula I to achieve high (>300 times) PI3Kα selectivity. The compounds of formula I of the present invention improve the interaction of the primary amide of GDC-0032, and thus it is observed that the selectivity of the compounds of the present invention for PI3Kα over PI3Kδ measured in biochemical assays is significantly improved relative to comparative compounds (see Tables 2A and 2B).

[0121] Figure 2A The x-ray structure of taselisib bound to the PI3Kα(alpha) active site is shown. The N2 atom of the triazole ring cannot directly bind to Tyr836( distance) or Ser774 (2.74 and distance, there are no complementary polar side chain interactions between the ligand and the residues. Figure 2B The x-ray structure of compound 101 bound in the PI3Kα active site is shown, and shows that the oxazolidinone ring is able to make multiple improved interactions with the protein relative to the triazole ring. The carbonyl functional group is close to the Tyr836 side chain The fluorine atom of the oxazolidinone substituent is in close contact with the hydroxyl group of Ser774. and is consistent with polar interactions or nonclassical hydrogen bonding (favorable interactions enabled by polarization of the carbon-fluorine bond) ( et al., Fluorine in Medicinal Chemistry, (2004) ChemBioChem, 5:637-643; Zhou et al., "Fluorine Bonding–How Does it Work InProtein-Ligand Interactions", (2009) J.Chem.Inf.Model., 49:2344-2355).

[0122] All compounds of the present invention contain an oxazolidinone ring and are capable of improved interactions with Tyr836 of PI3Kα (alpha). Some embodiments of the present invention also contain fluorinated substituents on the oxazolidinone ring and are capable of improved interactions with Ser774 of PI3Kα. Relative to the embodiments of US 8242104, these two binding interactions can contribute to the improved selectivity for PI3Kα observed in the embodiments of the present invention. As shown in Tables 2A and 2B, compounds containing oxazolidinone rings have higher isotype selectivity than comparable compounds containing triazole rings. Residues Ser774 and Tyr836 are not unique to PI3Kα isoforms, PI3Kδ contains the same residues at the same position, and the enhanced isotype selectivity of oxazolidinone inhibitors is not predicted by these crystal structures. Subtle changes in secondary and tertiary protein structures may lead to subtle differences in the positioning and direction of the same residue identity between different isoforms. Even in the face of x-ray crystal structures of two protein isoforms, these differences are difficult to predict and explain. Across the range of compounds exemplified in Table 1, the surprising and unexpected properties of oxazolidinone inhibitors of improved molecular interactions and enhanced isoform selectivity are retained.

[0123] Oxazolidinone differs from triazole in structure in that oxazolidinone has a carbonyl group, is more polar, and is not aromatic, whereas triazole does not have a carbonyl group, is less polar, and is aromatic.

[0124] According to the increased sp3 characteristics and the reduced number of aromatic rings, the oxazolidinone ring provides further benefits relative to the triazole ring. It is generally believed in the literature that the increase in the number of aromatic rings is associated with the increased risk of mixed binding. In contrast, the increase in the ratio of sp3 carbon (#sp3 carbon / #total carbon) is associated with improved physicochemical properties and reduced mixed binding, thereby reducing the risk of off-target toxicology. These concepts are described in reference Lovering et al., "Escape From Flatland", (2009) J.Med.Chem., 52: 6752-6756 and Ritchie and Macdonald, "Physicochemical Descriptors of Aromatic Character and Their Use in Drug Discovery", (2014) J.Med.Chem., 57: 7206-7215. The triazole aromatic ring exemplified by US 8242104 is replaced by the saturated heterocyclic oxazolidinone contained in each embodiment of the present invention, indicating a favorable reduction in off-target toxicological risk. The entirety of the exemplary compounds in US 8242104 is dominated by compounds with an aromatic ring at this position, 4 examples of carboxamide functional groups replacing the aromatic ring, and no examples of saturated cyclic or heterocyclic systems. Since the binding interactions and steric requirements of aromatic and saturated heterocyclic rings are significantly different, they are generally not interchangeable. There is no US Pat. No. 8,242,104 provides no teaching on methods for replacing aromatic rings with saturated heterocycles while retaining activity against PI3Kα.

[0125] Therefore, the compounds of the present invention are in 5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine The 9-position and 2-position of the antagonist contain optimized substituents and functional groups. These optimized compounds provide significant and hitherto unknown benefits in improving molecular interactions and increasing selective activity against PI3Kα, and have reduced activity against PI3Kδ. The compounds of the present invention can be used as therapeutic agents with an enhanced therapeutic window relative to related agents such as taselisib (GDC-0032).

[0126] Selective inhibition of mutant PI3Kα(alpha)

[0127] As described in Example 902, the ability of the compounds of the invention to act preferentially against cells containing mutant PI3Kα was determined by measuring inhibition of the PI3K pathway in the SW48 near-isogenic cell line PI3Kα wild type (parental), helical domain mutant E545K, and kinase domain mutant H1047R.

[0128] Statistical analysis: Unless otherwise stated, EC50 values ​​represent the geometric mean of at least 4 independent experiments. All statistical data were performed using KaleidaGraph software (version 4.1.3). Student t-tests were performed using unpaired data with equal variance to compare the activity against mutant cells and wild-type cells. P < 0.05 was considered significant.

[0129] Table 3A shows the inhibition of P-PRAS40 in SW48 near-isogenic cells by the compounds of Formula I of Table 1. These compounds all exhibited increased activity against mutant PI3Kα cells relative to wild-type PI3Kα cells. Relative to the activity in wild-type PI3Kα cells, the compounds of the invention showed similar activity to taselisib in SW48 mutant PI3Kα cells, with equal or higher selectivity than taselisib (see Table 3B).

[0130] Table 3B shows some comparative compounds of US 8242104, compounds with dimethyloxazolidin-2-one groups from US 8263633 (compound 356, column 149) and pictilisib inhibition of P-PRAS40 in SW48 near-isogenic cells. The comparative compounds shown in Table 3B are embodiments of the broad genus described in each of US 8242104 and US 8263633. Neither US 8242104 nor US 8263633 discloses compounds within the scope of the compounds of formula I of the present invention. The comparative compounds contain embodiments in which mutant PI3Kα cells do not significantly improve activity relative to wild-type PI3Kα cells (see comparative compounds 436 and 549, p>0.05). These compounds are structurally similar to comparative compounds (see comparative compound 529) in which mutant PI3Kα cells do not show significantly improved activity relative to wild-type PI3Kα cells. There are no common structural elements in the comparative compounds that provide teaching for selective inhibition of mutant PI3Kα (alpha) cells. More generally, there is no teaching in US8242104 or US8263633 for selecting structural elements of compounds of Formula I to achieve increased or comparable activity against mutant PI3Kα cells relative to wild-type PI3Kα cells. This unexpected property is retained across the entire range of compounds exemplified in Table 1.

[0131] Table 3A. Inhibition of P-PRAS40 in SW48 near-isogenic cells by compounds of formula I

[0132]

[0133] Table 3B. Comparison of compounds for inhibition of P-PRAS40 in SW48 near-isogenic cells

[0134]

[0135]

[0136] *EC50 refers to a single experiment

[0137] Antiproliferative activity of PI3K mutant tumor cells

[0138] The ability of the compounds of the present invention to act on PI3K mutant tumor cells was determined by measuring the cell viability EC50 in HCC1954 cells (PI3Kα mutant H1047R) and MCF7 cells (PI3Kα mutant E545K) using the method of Example 903. Table 4 shows that representative compounds of Formula I 101 and 103 of the present invention are able to inhibit the PI3K pathway and inhibit the proliferation of HCC1954 cells and MCF7 cells, and have a similar level of efficacy to the comparative compounds taselisib (compound 196, US 8242104), pictilisib and compound 436 (US 8242104).

[0139] Table 4. Antiproliferative activity of PI3K compounds in mutant PI3K-α tumor cells

[0140]

[0141] In vivo efficacy

[0142] Tables 5-8 show data from in vivo tumor growth inhibition (TGI) studies of PI3K compounds.Tumor volume changes were measured in cohorts of immunocompromised mice bearing breast cancer xenografts for 20 days or longer with daily PO (oral) administration of vehicle and PI3K compounds (Example 904).

[0143] Table 5 shows that at the maximum tolerated dose (MTD), GDC-0032 (taselisib), Compound 103, and Compound 101 are each more potent than alpelisib (BYL-719) in a PI3K mutant tumor model.

[0144] Table 6 shows that at doses below the maximum tolerated dose (ie, 25 mg / kg), Compound 101 is more potent than GDC-0032 in the PI3K mutant tumor model. Compound 101 has the potential for a higher therapeutic index (TI) because maximum efficacy is reached before maximum tolerance.

[0145] Table 7 shows that at the maximum tolerated dose, increased responses (PR and CR) were observed with Compound 101 compared to GDC-0032 in the PI3K mutant tumor model. Moreover, at the maximum tolerated dose, GDC-0032 and BYL-719 were equally effective.

[0146] Table 8 shows that at the maximum tolerated dose, GDC-0032 and Compound 101 are more potent than BYL-719 in the PI3K mutant tumor model, and Compound 101 is roughly as potent as GDC-0032.

[0147] Table 5. HCC-1954x1(ER-,PI3K H1047R Comparison of Maximum Tolerated Dose (MTD) of PI3K Compounds in Breast Cancer Xenograft Models

[0148] PI3K compounds %TGI PR CR BYL-719,40mg / kg QD,PO 80 1 1 GDC-0032, 15 mg / kg QD, PO 118 4 0 Compound 103, 100 mg / kg QD, PO 120 4 3 Compound 101, 50 mg / kg QD, PO 129 10 0

[0149] Table 6. HCC-1954x1(ER-,PI3K H1047R ) Dose ranging study of compound 101 in a breast cancer xenograft model

[0150] PI3K compounds %TGI PR CR Compound 101, 0.5 mg / kg PO, QD 19 0 0 Compound 101, 1 mg / kg PO, QD 60 2 0 Compound 101, 2.5 mg / kg PO, QD 68 1 0 Compound 101, 5 mg / kg PO, QD 84 0 0 Compound 101, 25 mg / kg PO, QD 140 7 0 Compound 101, 50 mg / kg PO, QD 149 6 0 GDC-0032, 15 mg / kg PO, QD 111 2 0

[0151] Table 7. KPL-4 (ER-, PI3K H1047R ) Dose ranging study of compound 101 in a breast cancer xenograft model

[0152]

[0153]

[0154] Table 8. HCI-003 (ER+, PI3K H1047R ) Comparison of GDC-0032, Compound 101, and BYL-719 in a breast PDX xenograft model

[0155] PI3K compounds %TGI PR CR BYL-719, 40 mg / kg PO, QD 114 2 0 GDC-0032, 15 mg / kg PO, QD 162 6 1 Compound 101, 50 mg / kg PO, QD 175 5 2

[0156] Pathway inhibition in isolated B cells

[0157] Using the method of Example 906, the ability of the compounds of the present invention to inhibit the PI3K pathway in B cells was evaluated by the effect of the compounds on the level of CD69 after agonistic a-IgM treatment. CD69 expression in B cells produced by a-IgM treatment is believed to be driven by the signaling of PI3Kδ (delta). Table 9 shows that the representative compound of Formula 1 is a more selective pathway signaling inhibitor for PI3K-mutant cell lines (SW48 (H1047R)) relative to B cells (column 3) compared to taselisib, pictilisib, alpelisib, compound-436 (US US8242104) and idelalisib.

[0158] Table 9 . Inhibition of CD69 expression in B cells by selected compounds.

[0159]

[0160]

[0161] *CD69 expression IC50 was measured in human whole blood and calculated by multiplying the human f measured from plasma protein binding experiments u Be corrected.

[0162] Pathway inhibition in PI3K mutant and wild-type tumor cells

[0163] Using the method of Example 907, the ability of the compounds of the present invention to inhibit PI3K pathway signaling in tumor cells was evaluated by measuring the p-PRAS40 levels in HCC1954 (PI3Kα mutant H1047R) and HDQ-P1 (PI3Kα wild-type) cell lines. Table 10 shows that representative Formula 1 compounds 101, 103, and 105 are able to selectively inhibit the PI3K pathway in PI3Kα mutants (HCC1954, PI3Kα mutant H1047R) relative to PI3Kα wild-type tumor cells (HDQ-P1, PI3Kα wild-type). Compared with the comparative compounds taselisib, pictilisib, alpelisib, and compound 436 (US8242104), compounds 101, 103, and 105 have greater mutant / wild-type selectivity.

[0164] Table 10 Inhibition of p-PRAS40 in HCC1954 and HDQ-P1 cell lines by selected compounds

[0165]

[0166] Degradation of p110α in PI3K mutant tumor cells

[0167] Using the method of Example 905, the ability of compounds of the invention to reduce p110a levels was determined in experiments using HCC1954 (PI3Kα mutant H1047R) and HDQ-P1 (PI3Kα wild type) cell lines. Figure 3A and 3B Representative compounds of Formula 1 101 and 103 are shown to be able to selectively promote the reduction of p110a levels in PI3K mutant (HCC1954, PI3Kα mutant H1047R) relative to PI3Kα wild-type (HDQ-P1, PI3Kα wild-type) tumor cells in a concentration-dependent manner. Figure 3A Shown are Western blot data depicting p110α (p110a, p110 alpha) levels in HCC-1954 cells (PI3Kα mutant H1047R) after treatment with Compound 101, Compound 103, and Compound 436 of US 8242104 for 24 hours. Figure 3B Shown are Western blot data depicting p110α (p110a, p110 alpha) levels in HDQ-P1 cells (PI3Kα wild type) after 24 hours of treatment with compound 101, compound 103, and compound 436 of US 8242104. Compared to compound 436 (US8242104), compounds 101 and 103 more strongly affect p110α levels.

[0168] Multi-day oral administration in dogs

[0169] After multiple administrations (7-14 days) in beagle dogs, the ability of the compounds of the present invention to promote gastrointestinal and / or systemic inflammation or cause lymphoid tissue loss (lymphoid depletion) was evaluated by clinical and anatomical pathology evaluation. Formula 1 compounds 101 and 103 do not promote pro-inflammatory features determined by clinical pathology or anatomical pathology evaluations at free exposure multiples >5 times compared to TGI60 (60% inhibition of tumor growth in PI3K-mutant xenograft studies) (Table 11a, 11b). Similarly, compounds 101 and 103 only produce a small amount of lymphoid tissue loss at high exposure multiples. In contrast, experiments using the comparative compound taselisib showed significant pro-inflammatory effects and lymphoid tissue loss at free exposure multiples <0.3 times compared to TGI60 (Table 11c). Compared with compounds 101 and 103 of formula 1, the comparative compounds alpelisib (BYL-719) and compound 436 (US8242104) also caused inflammation and lymphoid tissue loss at lower exposure multiples (Tables 11d, 11e). 50The magnitude and severity of PI3Kδ(delta) inhibition found were consistent with increased exposure multiples.

[0170] Table 11 .Multi-day administration of Formula 1 and comparative compounds in dogs.

[0171] (a)

[0172]

[0173] F u Dog=0.692;F u Mouse = 0.252 (F u = unbound fraction in species plasma)

[0174] *Exposure multiples using TGI60 from a 21-day KPL4 xenograft study; TGI60@2mg / kg, 1.04μMhr total, 0.26μM hr free

[0175] **a-IgM stimulated CD69 expression (whole blood) IC 50 =67nM

[0176] + Findings related to inflammation and lymphoid organs

[0177] (b)

[0178]

[0179] F u Dog=0.507;F u Mouse = 0.03 (F u = unbound fraction in species plasma)

[0180] *Exposure multiples using TGI60 from the 21-day HCC1954 TGI study; TGI60@2mg / kg, 1.13μM hr total, 0.03μM hr free

[0181] **a-IgM stimulated CD69 expression (whole blood) IC 50 =142nM

[0182] + Findings related to inflammation and lymphoid organs

[0183] (c)

[0184]

[0185]

[0186] F uDog = 0.28; F u Mouse = 0.10 (F u = unbound fraction in species plasma)

[0187] *Exposure multiples using TGI60 from 21-day KPL4 xenograft study; TGI60 @ 4.3uM hr (total) or 0.44uM hr free

[0188] **a-IgM stimulated CD69 expression (whole blood) IC 50 =3.1nM

[0189] + Findings related to inflammation and lymphoid organs

[0190] (d)

[0191]

[0192]

[0193] F u Dog = 0.68; F u Mouse = 0.36 (F u = unbound fraction in species plasma)

[0194] *Exposure multiples using TGI60 from 21-day KPL4 xenograft study; TGI60@2.8uM hr total, 1.0uM hr free

[0195] **a-IgM stimulated CD69 expression (whole blood) IC 50 =45nM

[0196] + Findings related to inflammation and lymphoid organs

[0197] (e)

[0198]

[0199]

[0200] F u Dog=0.072;F u Mouse = 0.082 (F u = unbound fraction in species plasma)

[0201] *Exposure multiples using TGI60 from 21-day KPL4 xenograft study; TGI60 @ 72uM hr (total), 5.9uM hr (free)

[0202] **a-IgM stimulated CD69 expression (whole blood) IC 50 =613nM

[0203] + Findings related to inflammation and lymphoid organs

[0204] Administration of compounds of formula I

[0205] The compounds of the present invention can be applied by any route suitable for the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intrathecal and epidural), percutaneous, rectal, nasal, local (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal. For local immunosuppressive therapy, the compound can be applied by intralesional administration (including perfusion or contacting the graft with an inhibitor before transplantation). It should be understood that the preferred route can vary with, for example, the recipient's situation. When the compound is orally administered, it can be formulated into pills, capsules, tablets, etc. together with a pharmaceutically acceptable carrier or excipient. When the compound is administered parenterally, it can be formulated with a pharmaceutically acceptable parenteral vehicle as described below and formulated into a unit dose injection form.

[0206] The dosage for treating human patients can be from about 1 mg to about 1000 mg of the compound of formula I. A typical dosage can be from about 10 mg to about 300 mg of the compound. The dosage can be administered once a day (QID), twice a day (BID), or more frequently, depending on the pharmacokinetic and pharmacodynamic properties of the specific compound, including absorption, distribution, metabolism, and excretion. In addition, toxic factors can affect the dosage and administration regimen. When administered orally, pills, capsules, or tablets can be taken daily or at a lower frequency for a specified period of time. The regimen can be repeated for multiple treatment cycles.

[0207] Methods of treatment using compounds of formula I

[0208] The compounds of formula I of the present invention can be used to treat human or animal patients suffering from diseases or conditions such as cancer caused by abnormal cell growth, function or behavior associated with PI3K, and can therefore be treated by a method comprising administering the compounds of the present application as defined above. Human or animal patients suffering from cancer can also be treated by a method comprising administering the compounds of the present invention as defined above. The patient's condition can therefore be improved or alleviated.

[0209] The methods of the present invention also include treating a cancer selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, genitourinary tract cancer, esophageal cancer, laryngeal cancer, glioblastoma, neuroblastoma, gastric cancer, skin cancer, keratoacanthoma, lung cancer, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, lung adenocarcinoma, bone cancer, colon cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract cancer, kidney cancer, pancreatic cancer, myeloid disorders, lymphoma, hairy cell carcinoma, oral cancer, nasopharyngeal carcinoma , pharyngeal cancer, lip cancer, tongue cancer, oral cancer, small intestine cancer, colon-rectum cancer, large intestine cancer, rectal cancer, brain cancer and central nervous system cancer, Hodgkin cancer, leukemia, bronchial cancer, thyroid cancer, liver cancer and intrahepatic bile duct cancer, hepatocellular carcinoma, gastric cancer, glioma / glioblastoma, endometrial cancer, melanoma, kidney cancer and renal pelvis cancer, bladder cancer, uterine corpus cancer, cervical cancer, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, lymphocytic leukemia, chronic lymphocytic leukemia (CLL), myeloid leukemia, oral cancer and pharyngeal cancer, non-Hodgkin lymphoma, melanoma and villous colon adenoma.

[0210] Based on expression analysis, immunohistochemistry, and cell line distribution, malignancies of the colon, breast, cervix, stomach, lung, and multiple myeloma are most likely to be responsive to PI3K modulators or inhibitors.

[0211] The present invention relates to the use of a compound as described above for treating cancer in a patient.

[0212] The present invention relates to the use of a compound as described above in the preparation of a medicament for treating cancer in a patient.

[0213] The present invention relates to compounds as described above for use in treating cancer in a patient.

[0214] The present invention relates to the use of a compound as described above for treating cancer in a patient, wherein the cancer is selected from breast cancer and non-small cell lung cancer.

[0215] The present invention relates to the use of a compound as described above in the preparation of a medicament for treating cancer in a patient, wherein the cancer is selected from breast cancer and non-small cell lung cancer.

[0216] The present invention relates to compounds as described above for use in treating cancer in a patient, wherein the cancer is selected from breast cancer and non-small cell lung cancer.

[0217] The invention as described above.

[0218] Pharmaceutical preparations

[0219] In order to use the compounds of the invention for therapeutic treatment of mammals (including humans), they are generally formulated into pharmaceutical compositions according to standard pharmaceutical practice. This aspect of the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable diluent or carrier.

[0220] A typical formulation is prepared by mixing a compound of the present invention with a carrier, diluent or excipient.

[0221] Suitable carriers, diluents, additives and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water-soluble and / or swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, water, etc. The specific carrier, diluent or excipient used will depend on the means and purpose of administering the compounds of the present invention. Solvents are generally selected based on solvents that are considered safe (GRAS) by those skilled in the art for administration to mammals. Typically, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG 400, PEG 300) and mixtures thereof. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, aromatics, flavoring agents and other known additives to give the drug (i.e., the compound of the present application or its pharmaceutical composition) a high-quality appearance or to help manufacture a pharmaceutical product (i.e., a drug).

[0222] The preparation can be prepared using conventional dissolution and mixing operations. For example, bulk drug substance (i.e., a compound of the present invention or a stabilized form of the compound (e.g., a complex with a cyclodextrin derivative or other known complexing agent)) is dissolved in a suitable solvent in the presence of one or more of the above-mentioned excipients. The compound of the present invention is usually formulated into a pharmaceutical dosage form to provide an easily controllable drug dosage and enable the patient to comply with the prescribed regimen.

[0223] The pharmaceutical composition (or preparation) for administration can be packaged in a variety of ways depending on the method of administering the drug. Typically, the article for distribution includes a container in which a pharmaceutical preparation in a suitable form is stored. Suitable containers are known to those skilled in the art and include materials such as bottles (plastic and glass), pouches, ampoules, plastic bags, metal cylinders, etc. The container may also include a pry-proof device to prevent inadvertent access to the contents of the package. In addition, a label describing the contents of the container is provided on the container. The label may also include suitable warning information.

[0224] Pharmaceutical preparations of the compounds of the present application can be prepared for various routes of administration and types. For example, the compound of formula I with the desired purity can be optionally mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer into a lyophilized preparation, ground powder or aqueous solution form (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.). Preparation can be carried out as follows: at ambient temperature at a suitable pH with a suitable purity and a physiologically acceptable carrier, i.e., a carrier that is nontoxic to the recipient at the dosage and concentration used. The pH of the preparation depends primarily on the specific use and compound concentration, but can be from about 3 to about 8. Preparations in an acetate buffer at a pH of 5 are suitable embodiments.

[0225] The drug can generally be stored in the form of a solid composition, a lyophilized preparation or an aqueous solution.

[0226] The pharmaceutical compositions of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice (i.e., dosage, concentration, time schedule, course of treatment, vehicle, and route). Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of drug delivery, the method of administration, the time schedule of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the compound to be administered will depend on the above factors considered and is the minimum amount required to improve or treat a hyperproliferative disorder.

[0227] As a general suggestion, the initial pharmaceutically effective amount of the inhibitor per parenteral dose will be about 0.01-100 mg / kg, ie, about 0.1-20 mg / kg, of the patient's body weight per day, with a typical initial range of 0.3 to 15 mg / kg / day of the compound used.

[0228] Acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM Or polyethylene glycol (PEG). Active pharmaceutical ingredients can also be embedded in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. The above techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0229] Sustained release formulations of the compound of formula I can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the compound of formula I in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US 3773919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly D-(-)-3-hydroxybutyric acid.

[0230] Preparations include those suitable for the administration routes described herein. Preparations can be suitably provided in unit dosage form and can be prepared by any method known in the pharmaceutical field. Technology and preparations are generally referred to Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). The above method includes the step of combining the active ingredient with a carrier as one or more auxiliary ingredients. Generally, the preparation is prepared as follows: the active ingredient is uniformly and tightly combined with a liquid carrier or a finely dispersed solid carrier or both, and then the product is molded as required.

[0231] Formulations of compounds of Formula I suitable for oral administration can be prepared as discrete units such as pills, capsules, cachets or tablets each containing a predetermined amount of a compound of Formula I. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form such as a powder or granules and optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant in a suitable machine. Molded tablets can be prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine. The tablets can be optionally coated or scored and optionally formulated to allow slow or controlled release of the active ingredient therefrom. Tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules such as gelatin capsules, syrups or elixirs can be prepared for oral administration. Preparations of compounds of formula I intended for oral administration can be prepared according to any method known in the art of preparing pharmaceutical compositions and the above compositions may contain one or more substances including sweeteners, flavoring agents, coloring agents and preservatives to provide a palatable preparation. Tablets containing active ingredients mixed with non-toxic physiologically acceptable excipients suitable for preparing tablets are acceptable. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants such as corn starch or alginic acid; adhesives such as starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained effect over a longer period of time. For example, time-delayed substances such as glyceryl monostearate or glyceryl distearate alone or in combination with wax may be used.

[0232] For treating the eye or other external tissues such as mouth and skin, the preparation may preferably be applied in the form of a topical ointment or cream containing, for example, 0.075 to 20% w / w of the active ingredient. When formulated as an ointment, the active ingredient may be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated into a cream together with an oil-in-water cream base. If desired, the aqueous phase of the cream base may contain polyols, i.e., alcohols having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. Topical preparations may contain compounds that enhance the absorption or penetration of the active ingredient through the skin or other areas of action as required. Examples of the above-mentioned skin penetration enhancers include dimethyl sulfoxide and related analogs. The oil phase of the emulsion of the present application may be composed of known ingredients in a known manner. When the phase may contain only an emulsifier, it contains a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil as required. Preferably, a hydrophilic emulsifier is contained together with a lipophilic emulsifier as a stabilizer. It is also preferred to contain both oil and fat. At the same time, the emulsifier with or without a stabilizer constitutes a so-called emulsifying wax and the wax together with the oil and fat constitutes a so-called emulsifying cream base, which forms the oily dispersed phase of the cream. Emulsifiers and emulsion stabilizers suitable for the formulation of the present application include 60. 80, cetearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

[0233] The aqueous suspension of the compound of formula I contains a mixture of the active substance and excipients suitable for preparing aqueous suspensions. The above-mentioned excipients include suspending agents such as sodium carboxymethylcellulose, cross-linked carboxymethylcellulose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum arabic; and dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweeteners such as sucrose or saccharin.

[0234] The pharmaceutical composition of the compound of formula I may be in the form of a sterile injection such as a sterile injection aqueous or oily suspension. The suspension may be prepared using the above-mentioned suitable dispersants or wetting agents and suspending agents according to methods known in the art. Sterile injections may also be sterile injection solutions or suspensions in non-toxic parenteral acceptable diluents or solvents such as solutions in 1,3-butanediol or prepared as lyophilized powders. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils may generally be used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid may also be used to prepare injections.

[0235] The amount of active ingredient that can be combined with a carrier material to prepare a single dosage form will vary depending on the host being treated and the specific mode of administration. For example, a timed release formulation intended for oral administration to humans may contain about 1 to 1000 mg of an active substance compound and a suitable and appropriate amount of a carrier material that can account for about 5 to about 95% (weight:weight) of the total composition. A pharmaceutical composition can be prepared to provide an easily measurable amount of administration. For example, an aqueous solution intended for intravenous infusion may contain about 3 to 500 μg of active ingredient / ml solution, so that an infusion of a suitable volume can be performed at a rate of about 30 mL / hr.

[0236] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0237] Formulations suitable for topical administration to the eye also include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in the above formulations at a concentration of about 0.5 to 20% w / w, such as about 0.5 to 10% w / w, such as about 1.5% w / w.

[0238] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0239] Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

[0240] Formulations suitable for intrapulmonary or nasal administration have, for example, a particle size of 0.1 to 500 microns (including between 0.1 and 500 microns and in increments such as 0.5, 1, 30, 35 microns, etc.), which are administered as follows: rapid inhalation through the nasal passages or inhalation through the mouth to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be delivered together with other therapeutic agents, such as compounds heretofore used to treat or prevent the following conditions.

[0241] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0242] The preparation can be packaged in a unit dose or multi-dose container such as a sealed ampoule or vial and can be stored under a freeze-dried (lyophilized) state, and it only needs to add a sterile liquid carrier such as water for injection immediately before use. Instant injection solutions and suspensions are prepared by sterile powders, granules and tablets of the above-mentioned types. Preferred unit dose preparations are those preparations containing the active ingredient of the above-mentioned daily dose or unit daily subdose or its suitable fraction of the application.

[0243] The present invention also provides veterinary compositions, which thus comprise at least one active ingredient as described above and a veterinary carrier. Veterinary carriers are substances that can be used for the purpose of administering the composition and can be solid, liquid or gaseous substances that are inert or acceptable in the veterinary field and compatible with the active ingredient. These veterinary compositions can be administered parenterally, orally or by any other desired route.

[0244] Combination therapy

[0245] The compound of formula I can be used alone or in combination with other therapeutic agents for treating diseases or conditions described herein, such as inflammation or hyperproliferative conditions (e.g., cancer). In some embodiments, the compound of formula I is combined with an additional second therapeutic compound having anti-inflammatory or anti-hyperproliferative properties or can be used to treat inflammation, immune response disorders, or hyperproliferative conditions (e.g., cancer) in a pharmaceutical combination formulation or as a combination therapy administration regimen. The additional therapeutic agent may be a Bcl-2 inhibitor, a JAK inhibitor, an anti-inflammatory agent, an immunomodulator, a chemotherapeutic agent, an apoptosis enhancer, a neurotrophic factor, a cardiovascular disease therapeutic agent, a liver disease therapeutic agent, an antiviral agent, a blood disorder therapeutic agent, a diabetes therapeutic agent, and an immunodeficiency disorder therapeutic agent. The second therapeutic agent may be an NSAID anti-inflammatory agent. The second therapeutic agent may be a chemotherapeutic agent. The second compound of the pharmaceutical combination formulation or administration regimen preferably has an activity complementary to the compound of formula I so that they do not adversely affect each other. The above compounds are suitably present in an effective amount combination for the intended purpose. In one embodiment, the composition of the present application comprises a compound of Formula I or a stereoisomer, tautomer, solvate, metabolite or pharmaceutically acceptable salt or prodrug thereof in combination with a therapeutic agent such as an NSAID.

[0246] Combination therapy can be administered in a simultaneous or sequential regimen. When administered sequentially, the combination can be administered in two or more administrations. Combination administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in any order, wherein preferably there is a period of time during which both (or all) active agents simultaneously exert their biological activities.

[0247] Suitable dosages for any of the above co-administered drugs are those currently used and may be lowered due to the combined action (synergy) of the newly identified drug and other therapeutic agents or treatments.

[0248] Combination therapy can provide and be demonstrated to be "synergistic," i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects achieved when the compounds are used separately. Synergy can be achieved when the active ingredients are: (1) co-formulated in a combined unit dosage formulation and administered or delivered simultaneously; (2) delivered in separate formulations in alternation or in parallel; or (3) administered by some other regimen. When delivered in alternation therapy, synergy can be achieved when the compounds are administered or delivered sequentially, e.g., by separate injections in different syringes, separate pills or capsules, or separate infusions. Typically, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., sequentially, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[0249] In a specific embodiment of therapy, the compound of formula I or its stereoisomer, tautomer, solvate, metabolite or pharmaceutically acceptable salt or prodrug can be combined with other therapeutic agents, hormone drugs or antibody drugs such as those described in the present application and combined with surgical therapy and radiotherapy. Combination therapy of the present invention thus includes administering at least one compound of formula I or its stereoisomer, tautomer, solvate, metabolite or pharmaceutically acceptable salt or prodrug and using at least one other cancer treatment method. The amount of the compound of formula I and other pharmaceutically active therapeutic agents and the related administration schedule will be selected to achieve the desired combined therapeutic effect.

[0250] Additional therapeutic agents for use in combination with a compound of Formula I include 5-FU, docetaxel, eribulin, gemcitabine, cobimetinib, ipatasertib, paclitaxel, tamoxifen, fulvestrant, GDC-0810, dexamethasone, palbociclib, bevacizumab, pertuzumab, trastuzumab-emtansine conjugate, trastuzumab, and letrozole.

[0251] Metabolites of compounds of formula I

[0252] The in vivo metabolites of Formula I described herein also fall within the scope of the present invention. Such products may be derived, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Therefore, the present invention includes metabolites of compounds of Formula I, including compounds produced by the following method, the method comprising contacting a compound of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0253] Metabolites are typically identified by preparing a radiolabeled (e.g. 14 C or 3 H) isotope, which is administered parenterally to animals such as rats, mice, guinea pigs, monkeys or to humans at a detectable dose (e.g., greater than about 0.5 mg / kg), allowing sufficient time for metabolism to occur (usually about 30 seconds to 30 hours) and separating its conversion products from urine, blood or other biological samples. These products are easily separated because they are labeled (others are separated by using antibodies that can bind to antigenic epitopes that survive in the metabolites). The metabolite structure is determined in a conventional manner, such as by MS, LC / MS or NMR analysis. Typically, the analysis of metabolites is performed in the same manner as conventional drug metabolism studies known to those skilled in the art. Metabolites can be used for diagnostic determination of therapeutic doses of the compounds of the invention, as long as they are not otherwise present in vivo.

[0254] Products

[0255] Another embodiment of the present invention provides an article or "kit" containing materials that can be used to treat the above-mentioned diseases and conditions. In one embodiment, the kit comprises a container containing a compound of formula I or a stereoisomer, tautomer, solvate, metabolite or pharmaceutically acceptable salt or prodrug thereof. The kit may also include a label or package insert on or associated with the container. The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products, which contain information about indications, usage, dosage, administration, contraindications and / or precautions involved in using the above-mentioned therapeutic products. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container can be formed of a variety of materials such as glass or plastic. The container can hold a compound of formula I or a formulation thereof that can effectively treat the condition and can have a sterile interface (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a compound of formula I. The label or package insert indicates that the composition is used to treat a selected condition such as cancer. In addition, the label or package insert may indicate that the patient to be treated is a patient suffering from a disease such as a hyperproliferative disease, neurodegeneration, cardiac hypertrophy, pain, migraine, or a neurotraumatic disease or event. In one embodiment, the label or package insert indicates that the composition comprising the compound of Formula I can be used to treat a disease caused by abnormal cell growth. The label or package insert may also indicate that the composition can be used to treat other diseases. Alternatively or additionally, the article may also include a second container comprising a pharmaceutically acceptable buffer such as antibacterial water for injection (BWFI), phosphate buffered saline, Ringer's solution, and glucose solution. It may also include other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0256] The kit may further comprise instructions for administering the compound of Formula I and the second pharmaceutical formulation, if present. For example, if the kit comprises a first composition comprising a compound of Formula I and a second pharmaceutical formulation, the kit may further comprise instructions for administering the first and second pharmaceutical compositions simultaneously, sequentially or separately to a patient in need thereof.

[0257] In another embodiment, the kit is suitable for delivering a solid oral form of a compound of formula I, such as a tablet or capsule. The above kit preferably comprises a plurality of unit doses. The above kit may comprise a card-shaped object having a dose arranged in the order of its intended use. An example of the above kit is a "blister pack". Blister packs are known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms. A memory aid may be provided as required, for example in the form of numbers, letters or other markings or with calendar instructions indicating the days that can be administered in the treatment arrangement.

[0258] According to one embodiment, the kit may comprise (a) a first container containing a compound of formula I therein; and optionally (b) a second container containing a second pharmaceutical preparation therein, wherein the second pharmaceutical preparation comprises a second compound having anti-hyperproliferative activity. Alternatively or additionally, the kit may further comprise a third container comprising a pharmaceutical buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. It may also comprise other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0259] In some other embodiments where the kit comprises a composition of Formula I and a second therapeutic agent, the kit may comprise containers such as separate bottles or separate foil packages for accommodating the separate compositions, however the separate compositions may also be contained in a single undivided container. Typically, the kit comprises instructions for administering the separate components. The kit format is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral) or at different dosage intervals or when the attending physician requires titration of the combined components.

[0260] Preparation of compounds of formula I

[0261] The compounds of formula I can be synthesized by the following synthetic routes, which include methods similar to those known in the chemical art and especially those methods borrowed from the description of the present application and those methods for other heterocycles, such as those described in: Comprehensive Heterocyclic Chemistry II, edited by Katritzky and Rees, Elsevier, 1997, for example, Volume 3; Liebigs Annalen der Chemie, (9): 1910-16, (1985); Helvetica Chimica Acta, 41: 1052-60, (1958); Arzneimittel-Forschung, 40 (12): 1328-31, (1990), each of which is expressly incorporated by reference. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, WI) or are readily prepared using methods known to those skilled in the art (e.g., by methods outlined in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-23, Wiley, NY (1967-2006 editions) or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin (including supplements) (also available from the Beilstein online database)).

[0262] Synthetic chemistry transformations and protecting group methodology (protection and deprotection) and the necessary reagents and intermediates useful in the synthesis of compounds of Formula I are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley and Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.

[0263] The examples provide exemplary methods for preparing compounds of formula I. Those skilled in the art will recognize that other synthetic routes can be used to synthesize compounds of formula I. Although specific raw materials and reagents are described and discussed in the figures and examples, other raw materials and reagents can be easily substituted to provide a variety of derivatives and / or reaction conditions. In addition, a variety of exemplary compounds prepared by the methods described can be further modified based on the present disclosure using conventional chemical methods known to those skilled in the art.

[0264] When preparing the compound of formula I, it may be necessary to protect the remote functional group (e.g., primary amine or secondary amine) of the intermediate. The need for the above protection will vary with the properties of the remote functional group and the conditions of the preparation method. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for the above protection is easily determined by those skilled in the art. For a general description of protecting groups and their use, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0265] In the process for preparing the compound of formula I, it may be advantageous to separate the reaction products from each other and / or from the starting materials. The desired product of each step or steps is separated and / or purified to the desired uniformity by conventional techniques in the art. Typically, the above separations involve multiphase extraction, crystallization from a solvent or solvent mixture, distillation, sublimation or chromatography. Chromatography can involve a variety of methods, including, for example: reverse phase and normal phase chromatography; size exclusion chromatography; ion exchange chromatography; high, medium and low pressure liquid chromatography and apparatus; small scale analytical chromatography; simulated moving bed (SMB) and preparative thin or thick layer chromatography and small scale thin layer and flash chromatography.

[0266] Another class of separation methods involves treating the mixture with a reagent selected to bind to or otherwise render the desired product, unreacted starting material, reaction by-product, etc. separable. Such reagents include adsorbents or absorbents such as activated carbon, molecular sieves, ion exchange media, etc. Alternatively, the reagent may be an acid (in the case of alkaline substances), a base (in the case of acidic substances), a binding agent such as an antibody, a binding protein, a selective chelating agent such as a crown ether, a liquid / liquid ion extraction reagent (LIX), etc. The choice of an appropriate separation method depends on the properties of the substances involved, such as boiling point and molecular weight (in distillation and sublimation), the presence or absence of polar functional groups (in chromatography), the stability of the substance in acidic and alkaline media (in multiphase extraction), etc.

[0267] Diastereomeric mixtures can be separated into their individual diastereomers based on their physicochemical differences by methods well known to those skilled in the art, such as chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with a suitable optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acyl chloride), separating the diastereomers, and then converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. In addition, some of the compounds of the invention may be atropisomers (e.g., substituted biaryl compounds) and are considered to be part of the present invention. Enantiomers can also be separated by using a chiral HPLC column.

[0268] A single stereoisomer, such as an enantiomer, that is substantially free of its stereoisomers can be obtained by resolving the racemic mixture using, for example, an optically active resolving agent to form diastereomers (Eliel, E. and Wilen, S. "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994; Lochmuller, CH, (1975) J. Chromatogr., 113 (3): 283-302). The racemic mixture of the chiral compounds of the present invention can be separated by any suitable method, including: (1) forming ionic diastereomeric salts with chiral compounds and separating by fractional crystallization or other methods; (2) forming diastereomeric compounds with chiral derivatizing agents, separating diastereomers and converting to pure stereoisomers; and (3) directly separating substantially pure or enriched stereoisomers under chiral conditions. See "Drug Stereochemistry, Analytical Methods and Pharmacology", edited by Irving W. Wainer, Marcel Dekker, Inc., New York (1993).

[0269] In method (1), diastereomeric salts can be prepared by reacting enantiomerically pure chiral bases such as strychnine, quinine, ephedrine, strychnine, α-methyl-b-phenylethylamine (amphetamine) with asymmetric compounds having acidic functional groups, such as carboxylic acids and sulfonic acids. The diastereomeric salts can be induced to separate by fractional crystallization or ion chromatography. In order to separate the optical isomers of amino compounds, the addition of chiral carboxylic acids or sulfonic acids such as camphorsulfonic acid, tartaric acid, mandelic acid or lactic acid can lead to the formation of diastereomeric salts.

[0270] In method (1), diastereomeric salts can be formed by reacting an enantiomerically pure chiral base (such as brucine, quinine, ephedrine, strychnine, α-methyl-β-phenylethylamine (amphetamine), etc.) with an asymmetric compound having an acidic functional group (such as carboxylic acid and sulfonic acid). The diastereomeric salts can be induced to separate by fractional crystallization or ion chromatography. For the separation of optical isomers of amino compounds, the addition of chiral carboxylic acids or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid or lactic acid, can lead to the formation of diastereomeric salts.

[0271] Alternatively, by method (2), the substrate to be resolved is reacted with one enantiomer of a chiral compound to form a diastereomeric pair (E. and Wilen, S. "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed by reacting an asymmetric compound with an enantiomerically pure derivatizing agent such as a menthyl derivative, and then the diastereomers are separated and hydrolyzed to obtain pure or enriched enantiomers. The method for determining optical purity involves preparing a chiral ester of the racemic mixture, such as a menthyl ester, for example, (-) menthyl chloroformate, or Mosher ester, α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem., (1982) 47: 4165), and analyzing the presence of two atropisomers or diastereomers. 1 H NMR spectrum. Stable diastereomers of atropisomeric compounds can be separated by normal or reverse phase chromatography, followed by separation methods for atropisomeric naphthyl-isoquinolines (WO 96 / 15111). By method (3), a racemic mixture of two enantiomers can be separated by chromatography using a chiral stationary phase ("Chiral Liquid Chromatography" (1989) WJ Lough, Ed., Chapman and Hall, New York; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purified enantiomers can be distinguished by methods used to distinguish other chiral molecules with asymmetric carbon atoms, such as optical rotation and circular dichroism.

[0272] The compounds of the present invention are prepared as schematically illustrated in General Schemes 1 and 2.

[0273] Solution 1.

[0274]

[0275] a) MgCl2, triethylamine, paraformaldehyde, acetonitrile, heating; b) glyoxal, ammonium hydroxide, heating; c) cesium carbonate, 1,2-dibromoethane, DMF, heating; d) N-iodosuccinimide, DMF, heating; e) i. EtMgBr, THF, -20°C, ii. aqueous ammonium chloride solution

[0276] As shown in Scheme 1, 4-bromo-2-hydroxybenzaldehyde 2 can be obtained by formylation of commercially available 3-bromophenol. Heating 2 with glyoxal and ammonium hydroxide gave 3. The oxazepine can be formed by heating 3 with 1,2-dibromoethane. Diiodination can be induced by reaction with N-iodosuccinimide, and the 3-iodo group can be selectively removed by treatment with ethylmagnesium bromide at reduced temperature to afford 6.

[0277] Solution 2.

[0278]

[0279] f) 4-substituted oxazolidin-2-one, Cu(OAc)2, trans-N,N'-dimethylcyclohexane-1,2-diamine, potassium carbonate, dioxane, heating; g) HN(R 2 )CH(R 1 )CO2H, CuI, K3PO4, DMSO, heating; h) ammonium chloride, triethylamine, HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate)

[0280] As shown in Scheme 2, 6 can be coupled to an appropriately substituted oxazolidin-2-one using copper catalysis to afford 7. The brominated intermediate 7 can be coupled to an appropriately substituted amino acid under copper catalysis, followed by a HATU-mediated amide coupling with ammonium chloride to afford compound 8.

[0281] Example

[0282] abbreviation

[0283] DMSO Dimethyl sulfoxide

[0284] ESI Electrospray ionization

[0285] HPLC High Pressure Liquid Chromatography

[0286] LCMS Liquid chromatography mass spectrometry

[0287] min

[0288] N equivalent concentration

[0289] NMR Nuclear Magnetic Resonance

[0290] R T Retention time

[0291] LCMS Method A: Experiments were performed on a Waters Micromass ZQ2000 quadrupole mass spectrometer connected to a Waters Acquity UPLC system with a PDA UV detector. The spectrometer had an electrospray source that operated in positive and negative ion modes. The system used an Acquity BEH C18 1.7um 100x 2.1mm column maintained at 40°C or an Acquity BEH Shield RP18 1.7μm 100x 2.1mm column maintained at 40°C and a flow rate of 0.4mL / min. The initial solvent system was 95% water (solvent A) containing 0.1% formic acid and 5% acetonitrile (solvent B) containing 0.1% formic acid for the first 0.4 minutes, followed by a gradient to 5% solvent A and 95% solvent B for the next 5.6 minutes. Hold for 0.8 minutes, then return to 95% solvent A and 5% solvent B in the next 0.2 minutes. The total run time was 8 minutes.

[0292] LCMS Method B: Experiments were performed on an Agilent 1100 HPLC coupled to an Agilent MSD mass spectrometer using ESI as the ionization source. LC separations used a Phenomenex XB-C18, 1.7 mm, 50 × 2.1 mm column with a flow rate of 0.4 mL / min. Solvent A was water containing 0.1% formic acid and solvent B was acetonitrile containing 0.1% formic acid. The gradient consisted of 2-98% solvent B in 7 min, holding 97% B for 1.5 min after equilibration for 1.5 min. The LC column temperature was 40 °C. UV absorbance was collected at 220 nm and 254 nm, and a full scan of the mass spectrum was applied to all experiments.

[0293] Embodiment 101 (S)-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)propanamide 101

[0294] Step 1: 4-Bromo-2-hydroxybenzaldehyde

[0295]

[0296] A 20L 4-necked round-bottom flask was purged and maintained with an inert atmosphere of nitrogen, into which 3-bromophenol (1300g, 7.51mol), magnesium dichloride (1078g, 11.3mol), triethylamine (3034g, 30.0mol) and acetonitrile (7.8L) were placed. The mixture was stirred at 40°C for 30 minutes. At 80°C, paraformaldehyde (676g, 22.6mol) was added to the mixture. The resulting solution was stirred at 76°C for 6 hours. The reaction was repeated 5 times. The combined reaction mixture was quenched by adding 12L aqueous hydrogen chloride solution (4N). The pH value of the solution was adjusted to 5 with concentrated aqueous hydrogen chloride solution (12N). The resulting solution was extracted with 1x 20L ethyl acetate. The organic extract was evaporated in vacuo. The residue was purified by flash chromatography on silica gel (elution: 15% ethyl acetate / petroleum ether) to obtain a crude product, which was washed with 2.4L methyl tert-butyl ether: hexane (1:4). The resulting solid was collected by filtration to give 7.0 kg (78%) of the title compound as a yellow solid.

[0297] Step 2: 5-Bromo-2-(1H-imidazol-2-yl)phenol

[0298]

[0299] A solution of 4-bromo-2-hydroxybenzaldehyde (700g, 3.50mol) in methanol (7.0L) and acetaldehyde (40%) (2540g, 17.5mol) was placed in a 20L 4-neck round-bottom flask, followed by dropping ammonia (25-28%, 3500g) over 4 hours with stirring and keeping the temperature below 40°C. The resulting solution was stirred at 30-35°C for 15 hours. The reaction was repeated 9 times. The combined 9 reaction mixtures were evaporated in vacuo, keeping the temperature below 45°C. The residue was diluted with 100L ethyl acetate and stirred for 30 minutes. The solid was filtered out and the resulting solution was diluted with water. The aqueous phase was extracted with 35L ethyl acetate. The organic extract was evaporated in vacuo and the residue was purified by flash chromatography on silica gel (solvent gradient: 5-75% ethyl acetate / petroleum ether) to give 2.4kg (29%) of the title compound as a yellow solid.

[0300] Step 3: 9-Bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine

[0301]

[0302] A solution of 5-bromo-2-(1H-imidazole-2-yl)phenol (1.4kg, 5.86mol) in N,N-dimethylformamide (14L) and cesium carbonate (7.2kg, 22.1mol) were placed in a 20L 4-necked round-bottom flask. The mixture was stirred for 20 minutes. 1,2-dibromoethane (4.1kg, 21.8mol) was added to the reaction mixture. The resulting solution was stirred at 85-90°C for 4-12 hours, cooled to 15°C, and filtered. The filter cake was washed with 3.0L ethyl acetate. The filtrate was diluted with 14L ethyl acetate. The combined organic extracts were washed with brine (4x 14L), dried over anhydrous sodium sulfate, filtered and evaporated in vacuo to give 1.1kg (71%) of the title compound as a light yellow solid. LCMS(ESI): [M+H] + =265; 1 H NMR (400MHz, DMSO-d6) δ8.32 (d, J = 8.4, 1H), 7.35-7.24 (m, 3H), 7.06 (s, 1H), 4.47-4.42 (m, 4H).

[0303] Step 4: 9-Bromo-2,3-diiodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine

[0304]

[0305] In a 20L 4-necked round-bottom flask, 9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine was placed. (2.5 kg, 9.43 mol) and N, N-dimethylformamide (12.5 L), followed by addition of N-iodosuccinimide (6.0 kg, 26.7 mol) in several batches with stirring. The resulting solution was stirred at 60 ° C for 12 hours, cooled to 15 ° C with a water / ice bath, diluted with 12.5 L of water / ice and filtered. The filtered solid was recrystallized from petroleum ether to give 4.0 kg (82%) of the title compound as a yellow solid.

[0306] Step 5: 9-Bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine

[0307]

[0308] A 20 L 4-necked round-bottom flask was purged and maintained with a nitrogen inert atmosphere, and 9-bromo-2,3-diiodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine was placed therein. (800g, 1.55mol) and tetrahydrofuran (2.4L), then ethylmagnesium bromide (1N solution in diethyl ether, 1.7L) was added dropwise at -20°C with stirring over 3.5 hours. The reaction mixture was stirred for 3 hours using an ice / salt bath, keeping the temperature at -15°C. The resulting mixture was quenched by adding 3.0L saturated aqueous ammonium chloride solution and extracted with ethyl acetate (2x 8.0L). The combined organic extracts were washed with brine (2x 10L), dried over anhydrous sodium sulfate, filtered and evaporated in vacuo. The crude residue was ground with 8.0L ethyl acetate: petroleum ether (1:5), filtered, and washed with petroleum ether to give 501g (83%) of the title compound as a brown solid. LCMS (ESI): [M+H] + =391; 1 HNMR (400MHz, DMSO-d6) δ8.22 (d, J = 8.7, 1H), 7.55 (s, 1H), 7.30-7.25 (m, 2H), 4.45-4.41 (m, 4H).

[0309] Step 6: (R)-2,2-Dimethyl-[1,3]dioxolane-4-carbaldehyde

[0310]

[0311] Sodium periodate (57.0 g, 270 mmol) was dissolved in hot water (115 mL) and silica gel (200 g, 220-440 mesh, particle size 35-75 μm). The mixture was stirred vigorously until a free-flowing powder was obtained. It was added to a solution of 1,2:5,6-di-O-(1-methylethylidene)-D-mannitol (50 g, 190 mmol) in dichloromethane (1.0 L) and the reaction mixture was stirred at room temperature for 1 hour. The resulting mixture was filtered through a pad of Na2SO4 and the solid was thoroughly washed with dichloromethane. The combined organic extracts were evaporated in vacuo to give 37.2 g (75%) of the title compound as a colorless oil. 1 H NMR (400MHz, CDCl3) δ9.73 (d, J=1.9Hz, 1H), 4.38 (ddd, J=7.4, 4.7, 1.9Hz, 1H), 4. 18(dd,J=8.8,7.4Hz,1H), 4.10(dd,J=8.8,4.7Hz,1H), 1.49(s,3H), 1.43(s,3H).

[0312] Step 7: (R)-4-difluoromethyl-2,2-dimethyl-[1,3]dioxolane

[0313]

[0314] To a solution of (R)-2,2-dimethyl-[1,3]dioxolane-4-carboxaldehyde (7.08 g, 54 mmol) in dichloromethane (50 mL) cooled in a water bath, diethylaminosulfur trifluoride (8.4 mL, 62.6 mmol) was added dropwise and the reaction mixture was stirred at room temperature for 3 hours. The resulting mixture was added dropwise to a rapidly stirred ice-cold saturated aqueous sodium bicarbonate solution. The mixture was further extracted with dichloromethane. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo to give 6.58 g (79%) of the crude title compound as an orange oil. 1 H NMR (400MHz, CDCl3) δ5.69 (td, J = 55.8, 4.9Hz, 1H), 4.27-4.17 (m, 1H), 4.16-4.03 (m, 2H), 1.46 (s, 3H), 1.38 (s, 3H).

[0315] Step 8: (R)-3-(tert-Butyldimethylsilyloxy)-1,1-difluoropropan-2-ol

[0316]

[0317] HCl / dioxane (4N, 10.8mL, 43.2mmol) was added to a solution of (R)-4-difluoromethyl-2,2-dimethyl[1,3]dioxolane (6.58g, 43.2mmol) in methanol (40mL) and the reaction mixture was stirred at room temperature for 30 minutes. The resulting mixture was evaporated in vacuo and azeotroped with acetonitrile. The residue was dissolved in N,N-dimethylformamide (10mL) and tert-butyldimethylchlorosilane (6.53g, 43.2mmol), triethylamine (9.0mL, 64.9mmol) and 4-(dimethylamino)pyridine) (catalytic amount) were added. The reaction mixture was stirred at room temperature for 1 hour. The resulting mixture was washed with water and then extracted with dichloromethane. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The resulting crude residue was purified by flash chromatography on silica gel (solvent gradient: 0-30% ethyl acetate in cyclohexane) to afford 3.43 g (35%) of the title compound as a yellow oil. 1 H NMR (400MHz, CDCl3) δ5.66 (td, J = 56.4, 4.6Hz, 1H), 3.76-3.60 (m, 2H), 2.46 (d, J = 6.4Hz, 1H), 0.81 (s, 9H), 0.00 (s, 6H).

[0318] Step 9: ((S)-2-Azido-3,3-difluoropropoxy)-tert-butyldimethylsilane

[0319]

[0320] Trifluoromethanesulfonic anhydride (2.9 mL, 17.4 mmol) was added dropwise to a solution of (R)-3-(tert-butyldimethylsilyloxy)-1,1-difluoropropan-2-ol (3.43 g, 15.1 mmol) and pyridine (2.0 mL, 24.2 mmol) in dichloromethane (50 mL) at -20 °C, and the reaction mixture was stirred at -20 °C for 20 minutes and then at 0 °C for 1 hour. The resulting mixture was diluted with 0.5N aqueous HCl solution and extracted with dichloromethane. The combined organic extracts were dried over magnesium sulfate and evaporated in vacuo. The crude residue was dissolved in N,N-dimethylformamide (10 mL), sodium azide (2.96 g, 45.5 mmol) was added, and the reaction mixture was stirred at room temperature for 2 hours. The resulting mixture was diluted with water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo to give 4.50 g of the crude title compound. 1 H NMR (400MHz, CDCl3) δ5.74 (td, J = 55.4, 4.4Hz, 1H), 3.81-3.71 (m, 2H), 3.58-3.47 (m, 1H), 0.81 (s, 9H), 0.00 (s, 6H).

[0321] Step 10: (S)-1-(tert-Butyldimethylsilyloxymethyl)-2,2-difluoroethylamine

[0322]

[0323] Palladium hydroxide on carbon (200 mg, 20%) was added to a solution of ((R)-2-azido-3,3-difluoropropoxy)-tert-butyldimethylsilane (4.50 g, crude, assumed to be about 15.1 mmol) in ethyl acetate (20 mL) and methanol (2.0 mL), and the reaction mixture was stirred under a hydrogen balloon for 16 hours. The reaction mixture was filtered, fresh palladium hydroxide on carbon (400 mg, 20%) was added, and the reaction mixture was stirred under a hydrogen balloon for 16 hours. The resulting mixture was filtered and the filtrate was evaporated in vacuo to give 3.08 g (90%) of the crude title product as a colorless oil. 1 H NMR (400MHz, CDCl3) δ5.66 (td, J = 57.0, 4.7Hz, 1H), 3.71-3.57 (m, 2H), 3.00-2.89 (m, 1H), 1.42 (br s, 2H), 0.82 (s, 9H), 0.00 (s, 6H).

[0324] Step 11: (S)-4-Difluoromethyloxazolidin-2-one

[0325]

[0326] HCl / dioxane (4N, 5.0 mL, 20 mmol) was added to a solution of (R)-1-(tert-butyldimethylsilyloxymethyl)-2,2-difluoroethylamine (Org. Lett., Vol. 9, No. 1, 2007, 41-44) (2.30 g, 10.3 mmol) in methanol (5.0 mL), and the reaction mixture was stirred at room temperature for 2 hours. The mixture was evaporated in vacuo, and the resulting oil was triturated with ether to give a solid, which was dried in vacuo. The solid was dissolved in a mixture of toluene (20 mL) and KOH (2.50 g, 44.6 mmol in 20 mL of water) at 0°C. Phosgene (16.3 mL, 20% in toluene) was added dropwise, the cooling bath was removed, and the reaction mixture was stirred for 1 hour. The mixture was evaporated in vacuo, the resulting residue was extracted with hot industrial methylated spirits, and the solid was collected by filtration. The filtrate was evaporated in vacuo and the resulting residue was purified by flash chromatography on silica gel (solvent gradient: 0-100% ethyl acetate / cyclohexane) to afford 830 mg (68%) of the title compound as an off-white solid. [α] D =+10.1(c=2.37,CHCl3). 1 H NMR (400MHz, CDCl3) δ5.96 (br s, 1H), 5.78 (td, J = 55.3, 4.8Hz, 1H), 4.54 (t, J = 9.2Hz, 1H), 4.42 (dd, J = 9.6, 4.4Hz, 1H), 4.17-4.06 (m, 1H).

[0327] Step 12: (S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-(difluoromethyl)oxazolidin-2-one

[0328]

[0329] 9-Bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine (250mg, 0.64mmol), (S)-4-difluoromethyloxazolidin-2-one (88mg, 0.64mmol), trans-N,N'-dimethyl-1,2-cyclohexanediamine (36mg, 0.26mmol), cuprous iodide (24mg, 0.13mmol) and potassium carbonate (177mg, 1.28mmol) in dioxane (3.0mL) were degassed with argon under ultrasound. The reaction mixture was heated at 100 ° C for 5 hours and then cooled to room temperature. The resulting mixture was diluted with 15% ammonia water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The resulting residue was triturated with methanol and then purified by preparative HPLC [C18, 60% acetonitrile (0.1% formic acid) / aqueous solution (0.1% formic acid), running for 20 minutes] to give 20mg (8%) of the title compound as a white solid. LCMS(ESI):[M+H] + =400 / 402. 1 H NMR (400MHz, CDCl3) δ8.19(d,J=9.2Hz,1H),7.29(s,1H),7.24-7.19(m,2H),6.65(ddd,J=57.8,54.5,1.0Hz,1H),4.87( ddd,J=24.0,9.2,4.0Hz,1H),4.73(dd,J=9.5,4.2Hz,1H),4.53(t,J=9.2Hz,1H),4.48-4.43(m,2H),4.38-4.33(m,2H).

[0330] Step 13: (S)-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)propanamide

[0331] (S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-(difluoromethyl)oxazolidin-2-one (600 mg, 1.50 mmol), L-alanine (267 mg, 3.00 mmol), cuprous iodide (57 mg, 0.30 mmol) and tripotassium phosphate (637 mg, 3.00 mmol) were suspended in dimethyl sulfoxide (6.0 mL). The reaction mixture was heated at 100 °C for 2 hours. After cooling to room temperature, dimethyl sulfoxide (4.0 mL), ammonium chloride (480 mg, 9.00 mmol) and triethylamine (3.1 mL, 22.5 mmol) were added. Over 5 minutes, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (5.10 g, 13.5 mmol) was added portionwise to the stirred suspension. The reaction mixture was stirred at room temperature for 1 hour and then filtered through celite washed with ethyl acetate. The organic extract was washed with saturated sodium bicarbonate aqueous solution and the aqueous phase was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and evaporated in vacuo. The crude residue was purified by silica gel flash chromatography (solvent gradient: 0-5% methanol / dichloromethane) and then purified by chiral supercritical fluid chromatography to give 294 mg (46%) 101 as an off-white solid. LCMS (ESI): R T (min)=2.89[M+H] + =408,method=A; 1 H NMR(400MHz, DMSO-d6)δ8.00(d,J=8.7Hz,1H),7.38(br s,1H),7.18(s,1H),7.00(br s,1H),6.71(t,J=55.9Hz,1H),6.41(dd,J=8.8,2.3Hz,1H),6.16(d,J=7.2Hz,1H),6.09(d,J=1.9Hz,1H),5. 02-4.89(m,1H),4.63-4.52(m,2H),4.39-4.30(m,4H),3.76(quintet,J=7.0Hz,1H),1.30(d,J=7.1Hz,3H).

[0332] Embodiment 102 (S)-2-Cyclobutyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)acetamide 102

[0333] Step 1: (R)-4-methyloxazolidin-2-one

[0334]

[0335] Phosgene (72.7 mL, 20% in toluene, 0.14 mmol) was added to a mixture of D-alaninol (8.65 g, 0.12 mmol) in toluene and aqueous KOH (124 mL, 12.5% ​​aqueous solution, 0.28 mmol) at 0°C at a rate that kept the internal temperature <5°C. The reaction mixture was stirred at 0°C for an additional 40 minutes and then evaporated to dryness. The crude residue was extracted with industrial methylated spirits, the slurry was filtered, and the filtrate was evaporated in vacuo. The resulting residue was purified by flash chromatography on silica gel (solvent gradient: 40-100% ethyl acetate / cyclohexane) to give 10.4 g (90%) of the title compound as a white solid. 1 H NMR (400MHz, CDCl3) δ6.00 (br s, 1H), 4.50 (t, J = 6.5Hz, 1H), 4.07-3.97 (m, 1H), 3.95 (dd, J = 7.8, 6.2Hz, 1H), 1.30 (d, J = 6.1Hz, 3H).

[0336] Step 2: (R)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-methyloxazolidin-2-one and (R)-3-(9-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-methyloxazolidin-2-one

[0337]

[0338] 9-Bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine A mixture of (30.0 g, 76.7 mmol), (R)-4-methyloxazolidin-2-one (7.70 g, 76.7 mmol), cuprous iodide (1.61 g, 8.40 mmol), trans-N,N'-dimethyl-1,2-cyclohexanediamine (2.7 mL, 16.9 mmol) and potassium carbonate (14.9 g, 107 mmol) was suspended in 1,4-dioxane (200 mL) and the reaction mixture was degassed with argon under ultrasound. The resulting mixture was heated at 100 °C for 16 hours. The reaction mixture was diluted with aqueous ammonia solution (about 16%) and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The resulting residue was purified by flash chromatography on silica gel (solvent gradient: 0-100% ethyl acetate / cyclohexane) to give 13.4 g (-42%) of the title compound (about 2:1 mixture of 9-Br:9-I products). 1H NMR (400MHz, CDCl3) δ8.28(d,J=7.6Hz,0.33H),8.11(d,J=6.9Hz,0.66H),7.42-7.38(m,1H),7.28-7.24(m,1.33H),7.23-7.18(m,0. 66H),4.77-4.68(m,1H),4.58(t,J=8.3Hz,1H),4.49-4.39(m,2H),4.37-4.30(m,2H),4.08(dd,J=8.4,4.5Hz,1H),1.57-1.50(m,3H).

[0339] Step 3: (R)-3-(9-bromo-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepine -2-yl)-4-methyl-oxazolidin-2-one

[0340]

[0341] 80 mg of (R)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine was separated by chiral SFC -2-yl)-4-methyloxazolidin-2-one and (R)-3-(9-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-methyloxazolidin-2-one mixture to obtain 27.6 mg of the title compound. LCMS (ESI): [M+H] + =364.0 / 366.0 / 367.2; 1 H NMR (400MHz, DMSO-d6) δ8.22(d,J=8.7Hz,1H),7.35(s,1H),7.31(dd,J=8.7,2.1Hz,1H),7.25 (d,J=2.0Hz,1H),4.65–4.54(m,2H),4.49-4.43(m,4H),4.09-4.06(m,1H),1.42(d,J=6.0Hz).

[0342] Step 4: (S)-2-cyclobutyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-amino)acetic acid methyl ester

[0343]

[0344] (4R)-3-(9-bromo-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepine A mixture of (2S)-2-amino-2-cyclobutyl-acetic acid (1.10mmol, 142mg) and tripotassium phosphate (1.37mmol, 297mg) in dimethyl sulfoxide (3mL) was heated at 120°C for 2 hours under microwave irradiation. The reaction was cooled to room temperature, iodomethane (1.4mmol, 0.086mL) was added, and the reaction mixture was extracted with dichloromethane and water. The combined organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated in vacuo. The crude product was purified by flash chromatography on silica gel (24g silica gel, solvent gradient: 5-40% 3:1 isopropyl acetate: methanol / dichloromethane) to give 100mg (85%) of the title compound.

[0345] Step 5: (S)-2-Cyclobutyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)acetamide

[0346] To (2S)-2-cyclobutyl-2-[[2-[(4R)-4-methyl-2-oxo-oxazolidin-3-yl]-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepine To a solution of methyl]-9-yl]amino]acetate (0.234mmol, 100mg) in tetrahydrofuran (5mL) was added water (0.45mL) and lithium hydroxide monohydrate (0.357mmol, 15mg). The reaction mixture was stirred at room temperature for 6 hours. The reaction mixture was evaporated in vacuo. To a solution of the resulting residue in N,N-dimethylformamide (3mL) were added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (0.353mmol, 137mg), ammonium chloride (0.71mmol, 38mg) and N,N-diisopropylethylamine (0.705mmol, 0.123mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was evaporated in vacuo, and the resulting residue was treated with water and then extracted with dichloromethane. The combined organic extracts were washed with brine, dried over magnesium sulfate and evaporated in vacuo. The crude product was purified by reverse phase HPLC followed by SFC and lyophilization to afford 15.0 mg (15%) of 102. LCMS (ESI): R T (min)=3.03,[M+H] + =412.2, method = D;1 H NMR (400MHz, DMSO-d6) δ7.96(d,J=8.8Hz,1H),7.39-7.36(brs,1H),7.13(s,1H),7.00-6.97(brs,1H),6.44(dd,J=8.9,2.3Hz,1H),6.14(d,J=2.3 Hz,1H),5.96(d,J=7.7Hz,1H),4.62–4.49(m,2H),4.38–4.28(m,4H),4.0 6-4.03(m,1H),3.70–3.61(m,1H),2.06–1.75(m,6H),1.42–1.34(m,3H).

[0347] Embodiment 103 (S)-2-Cyclopropyl-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)acetamide 103

[0348] (S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine from Example 101, Step 12 A mixture of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.28 g, 6.0 mmol) was added to the stirred suspension in portions and the reaction mixture was stirred at room temperature for 10 minutes. The resulting mixture was diluted with 15% aqueous ammonia solution and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The crude residue was purified by flash chromatography on silica gel (solvent gradient: 0-7% methanol in ethyl acetate). The residue was dissolved in a minimum amount of acetonitrile. Water was then added to precipitate the solid, which was collected by filtration and dried in vacuo to give 324 mg (75%) of 103 as an off-white solid. LCMS (ESI): R T (min)=3.21,[M+H]+ =434,method=A; 1 H NMR(400MHz,DMSO-d6)δ7.98(d,J=8.6Hz,1H),7.40(brs,1H),7.17(s,1H),7.03(br s,1H),6.71(t,J=56.0Hz,1H),6.42(dd,J=8.9,2.4Hz,1H),6.24(d,J=7.2Hz,1H),6.09(d,J=2.4Hz,1H),5.01-4.89(m,1 H), 4.63-4.51 (m, 2H), 4.38-4.29 (m, 4H), 3.15 (t, J = 7.7Hz, 1H), 1.16-1.05 (m, 1H), 0.56-0.44 (m, 3H), 0.33-0.25 (m, 1H).

[0349] Embodiment 104 (S)-2-Cyclopropyl-2-((2-((R)-4-methyl-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)acetamide 104

[0350] (R)-3-(9-bromo-5,6-dihydroimidazo[1,2-d][1,4]benzoxazepine A mixture of (2S)-2-amino-2-cyclopropyl-acetic acid (3.295 mmol, 379.3 mg) and tripotassium phosphate (4.393 mmol, 951.5 mg) in dimethyl sulfoxide (35 mmol, 2.5 mL) was heated at 110 °C for 2 hours under microwave irradiation. The reaction mixture was cooled to room temperature. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (12.08 mmol, 4260 mg), ammonium chloride (12.08 mmol, 646 mg) and triethylamine (1.53 mL, 11.0 mmol) were added to the reaction mixture. After 20 minutes at room temperature, the reaction mixture was treated with water and then extracted with dichloromethane. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The crude product was purified by reverse phase HPLC and lyophilized to give 110 mg (25% over 2 steps) of 104. LCMS (ESI): R T (min)=2.588,[M+H] + =398.2, method = B; 1H NMR(400MHz,DMSO-d6)δ7.96(d,J=8.8Hz,1H),7.39(d,J=2.2Hz,1H),7.13(s,1H), 7.02(d,J=2.3Hz,1H),6.42(dd,J=8.9,2.4Hz,1H),6.20(d,J=7.1Hz,1H),6.09(d,J =2.4Hz,1H),4.61–4.49(m,2H),4.40–4.27(m,4H),4.10–3.99(m,1H),3.22–3.09( m,1H),1.42–1.36(m,3H),1.16–1.04(m,1H),0.56–0.42(m,3H),0.32-0.27(m,1H).

[0351] Embodiment 105 (S)-2-Cyclopropyl-2-((2-((S)-4-(fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)acetamide 105

[0352] Step 1: (R)-1-(tert-Butyldimethylsilyloxy)-3-fluoropropan-2-ol

[0353]

[0354] Tert-butyldimethylsilyl chloride (1.60 g, 10.63 mmol) was added to a solution of (R)-3-fluoropropane-1,2-diol (1.00 g, 10.6 mmol), triethylamine (1.93 mL, 13.8 mmol) and a catalytic amount of 4-(dimethylamino)pyridine in dichloromethane, and the reaction mixture was warmed to room temperature and stirred at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with dichloromethane. The combined organic fractions were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The resulting crude residue was purified by flash chromatography on silica gel (solvent gradient: 0-40% ethyl acetate / cyclohexane) to give 1.80 g (81%) of the title compound as a colorless oil. 1 H NMR (400MHz, CDCl3) δ 4.45-4.36 (m, 1H), 4.34-4.25 (m, 1H), 3.87-3.73 (m, 1H), 3.66-3.56 (m, 2H), 2.30 (d, J = 6.0Hz, 1H), 0.82 (s, 9H), 0.00 (s, 6H).

[0355] Step 2: (S)-2-Azido-3-fluoropropoxy)-tert-butyldimethylsilane

[0356]

[0357] Trifluoromethanesulfonic anhydride (1.67 mL, 9.93 mmol) was added dropwise to a solution of (R)-1-(tert-butyldimethylsilyloxy)-3-fluoropropan-2-ol (1.80 g, 8.60 mmol) and pyridine (1.2 mL, 13.8 mmol) in dichloromethane, and the reaction mixture was stirred at -20 °C for 20 minutes and then at 0 °C for 30 minutes. The reaction mixture was diluted with 0.5N aqueous HCl and extracted with dichloromethane. The combined organic extracts were dried over magnesium sulfate, filtered and evaporated in vacuo. The residue was dissolved in N,N-dimethylformamide (5.0 mL) and sodium azide (1.68 g, 25.9 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The resulting mixture was diluted with water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo to give the crude title compound, which was used further without purification. 1 H NMR (400MHz, CDCl3) δ4.58-4.26(m,2H),3.75-3.63(m,2H),3.62-3.46(m,1H),0.80(s,9H),0.00(s,6H).

[0358] Step 3: (S)-1-(tert-Butyldimethylsilyloxymethyl)-2-fluoroethylamine

[0359]

[0360] Palladium hydroxide (400 mg, 20% on carbon) was added to a solution of ((S)-2-azido-3-fluoropropoxy)-tert-butyldimethylsilane (crude, assumed to be 8.60 mmol) in ethyl acetate (15 mL) and methanol (5.0 mL), and the reaction mixture was stirred under a hydrogen balloon for 16 hours. The resulting mixture was filtered, fresh palladium hydroxide (400 mg, 20% on carbon) was added, and the reaction was stirred under a hydrogen balloon for an additional 16 hours. The resulting mixture was filtered and the filtrate was evaporated in vacuo to give the title compound as a ca. 2:1 mixture of product: starting material, which was used further without purification.

[0361] Step 4: (S)-4-Fluoromethyloxazolidin-2-one

[0362]

[0363] HCl / dioxane (4N, 2.0 mL, 8.00 mmol) was added to a solution of (S)-1-(tert-butyldimethylsilyloxymethyl)-2-fluoroethylamine (crude, assumed to be 8.60 mmol) in methanol (3.0 mL) and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated in vacuo. The resulting residue was dissolved in a mixture of toluene (20 mL) and KOH (2.89 g, 51.6 mmol, 12.5% ​​aqueous solution) at 0°C. Phosgene (13.6 mL, 20% in toluene) was added dropwise to the mixture, the cooling bath was removed and the resulting mixture was stirred for 1 hour. The reaction mixture was evaporated in vacuo and the resulting residue was extracted with hot industrial methylated spirits. The filtrate was evaporated in vacuo and the resulting residue was purified by flash chromatography on silica gel (solvent gradient: 50-100% ethyl acetate / cyclohexane) to give 450 mg (44%, 3 steps) of the title compound as an off-white solid. 1 H NMR (400MHz, CDCl3) δ5.69(br s,1H),4.59-4.42(m,2H),4.42-4.32(m,1H),4.25-4.08(m,2H).

[0364] Step 5: (S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-(fluoromethyl)oxazolidin-2-one and (S)-3-(9-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-(fluoromethyl)oxazolidin-2-one

[0365]

[0366] 9-Bromo-2-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine A mixture of 4-(4-(4-(4-(4-nitro-2-ol)-1-nitropropene)-2-nitropropene (722 mg, 1.85 mmol), (S)-4-fluoromethyloxazolidin-2-one (220 mg, 1.85 mmol), 3,4,7,8-tetramethyl-1,10-phenanthroline (131 mg, 0.55 mmol), Cu(OAc)2.H2O (74 mg, 0.37 mmol), potassium carbonate (510 mg, 3.70 mmol) and dioxane (6.0 ml) was sealed in a tube and the mixture was degassed with argon under ultrasonic conditions. The reaction mixture was heated at 100°C for 72 hours. The resulting reaction mixture was diluted with 15% aqueous ammonia and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The crude residue was purified by flash chromatography on silica gel (solvent gradient: 0-100% ethyl acetate / cyclohexane) to give 390 mg (53%) of the title compound (approximately 2:1 mixture of 9-Br and 9-I products). LCMS(ESI):[M+H] + =382 / 384 / 430; 1 H NMR(400MHz, CDCl3)δ8.22(d,J=9.3Hz,0.7H),8.05(d,J=8.8Hz,0.3H),7.43-7.37(m,0.6H),7.29(s,1.2H),7.23-7.18 (m,1.2H),5.03-4.66(m,3H),4.60(t,J=8.5Hz,1H),4.54(dd,J=8.6,4.3Hz,1H),4.47-4.43(m,2H),4.37-4.33(m,2H).

[0367] Step 6: (S)-2-Cyclopropyl-2-((2-((S)-4-(fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)acetamide

[0368] (S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-(fluoromethyl)oxazolidin-2-one and (S)-3-(9-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine A mixture of 1-(4-(fluoromethyl)oxazolidin-2-one (195 mg, about 2:1 mixture Br:I, about 0.49 mmol), L-cyclopropylglycine (104 mg, 0.90 mmol), cuprous iodide (17 mg, 0.09 mmol) and tripotassium phosphate (190 mg, 0.90 mmol) in dimethyl sulfoxide (1.5 mL) was degassed with argon under ultrasonic conditions. The reaction mixture was heated at 100 ° C for 16 hours and then cooled to room temperature. The resulting mixture was diluted with dimethyl sulfoxide (1.0 mL), and ammonium chloride (144 mg, 2.70 mmol) and triethylamine (950 μL, 6.75 mmol) were added. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (1.54 g, 4.05 mmol) was then added to the mixture in portions, and the reaction mixture was stirred at room temperature for 1 hour. The resulting mixture was diluted with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The resulting crude residue was purified by silica gel flash chromatography (solvent gradient: 0-5% methanol / dichloromethane) and then further purified by silica gel flash chromatography (solvent gradient: 0-100% methyl acetate / cyclohexane) to obtain 90 mg (48%) 105 as an off-white solid. LCMS (ESI): R T (min)=2.76[M+H] + =416,method=A; 1 H NMR (400MHz, DMSO-d6) δ7.94 (d, J=8.8Hz, 1H), 7.40 (br s, 1H), 7.17 (s, 1H), 7.03 (br s,1H),6.41(dd,J=8.8,2.3Hz,1H),6.22(d,J=7.1Hz,1H),6.09(d,J=2.2Hz,1H),4.99(ddd,J=48.3,9.8,2.5Hz,1H),4.81-4.56(m, 3H), 4.40 (dd, J=8.6, 3.9Hz, 1H), 4.37-4.29 (m, 4H), 3.15 (t, J=7.6Hz, 1H), 1.16-1.05 (m, 1H), 0.54-0.43 (m, 3H), 0.33-0.25 (m, 1H).

[0369] Embodiment 106 (S)-2-((2-((S)-4-(Fluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)propanamide 106

[0370] (S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -2-yl)-4-(fluoromethyl)oxazolidin-2-one and (S)-3-(9-iodo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine A mixture of 9-Br:9-I (195 mg, about 2:1 mixture of 9-Br:9-I, about 0.49 mmol), L-alanine (87 mg, 0.98 mmol), cuprous iodide (17 mg, 0.09 mmol) and tripotassium phosphate (208 mg, 0.98 mmol) in dimethyl sulfoxide (3.0 mL) was degassed with argon under ultrasonic conditions. The reaction mixture was heated at 100 ° C. for 4 hours and then cooled to room temperature. The resulting mixture was diluted with dimethyl sulfoxide (3.0 mL), and ammonium chloride (157 mg, 2.94 mmol) and triethylamine (683 μL, 4.8 mmol) were added. 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (1.10 g, 2.94 mmol) was then added to the mixture in portions, and the reaction mixture was stirred at room temperature for 30 minutes. The resulting mixture was diluted with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered and evaporated in vacuo. The resulting residue was purified by silica gel flash chromatography (solvent gradient: 0-5% methanol / dichloromethane) and then further purified by chiral supercritical fluid chromatography to give 36 mg (19%) 106 as an off-white solid. LCMS (ESI): R T (min)=2.43[M+H] + =390,method=A; 1 H NMR (400MHz, DMSO-d6) δ7.96 (d, J=8.8Hz, 1H), 7.37 (br s, 1H), 7.17 (s, 1H), 7.00 (br s,1H),6.39(dd,J=8.6,1.6Hz,1H),6.15(d,J=7.0Hz,1H),6.09(d,J=1.6Hz,1H),5.08 -4.55(m,5H),4.42-4.28(m,4H),3.76(quintet,J=7.2Hz,1H),1.30(d,J=7.2Hz,3H).

[0371] Embodiment 107(S)-2-((2-((S)-4-(difluoromethyl)-2-oxooxazolidin-3-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine -9-yl)amino)butanamide 107

[0372] (S)-3-(9-bromo-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepine A mixture of (S)-2-aminobutyric acid (124 mg, 1.19 mmol), cuprous iodide (22.8 mg, 0.119 mmol), tripotassium phosphate (255 mg, 1.19 mmol) and dimethyl sulfoxide (6.0 mL) was stirred at 100 °C under argon for 6 hours. The resulting mixture was allowed to cool to room temperature before the addition of ammonium chloride (188 mg, 3.52 mmol) and triethylamine (1.2 mL, 8.80 mmol). To the stirred suspension was added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (2.01 g, 5.28 mmol) in portions and the reaction mixture was stirred at room temperature for 1 hour. The resulting mixture was diluted with ethyl acetate, washed with saturated ammonium chloride, dried over magnesium sulfate, filtered and evaporated in vacuo. The crude product was purified by silica gel flash chromatography (solvent gradient: 0-10% methanol / ethyl acetate), further purified by reverse phase HPLC, and then purified by chiral supercritical fluid chromatography to afford 73.6 mg (30%) of 107 as a white solid. LCMS (ESI): R T (min)=3.13,[M+H] + =422,Method=A; 1 H NMR (400MHz, DMSO-d6) δ7.99 (d, J = 8.8Hz, 1H), 7.40 (s, 1H), 7.17 (s, 1H), 7. 03(s,1H),6.71(t,J=56.0Hz,1H),6.44(dd,J=8.8,2.2Hz,1H),6.13(d,J=2 .2Hz,1H),6.09(d,J=7.6Hz,1H),5.02-4.89(m,1H),4.62-4.53(m,2H),4.4 1-4.27(m,4H),3.65-3.60(m,1H),1.72-1.59(m,2H),0.94(t,J=7.3Hz,3H).

[0373] Embodiment 901 p110α(alpha) PI3K binding assay

[0374] The PI3K binding assay is intended to determine the biochemical potency of small molecule PI3K inhibitors. The PI3K lipid kinase reaction is performed in the presence of PIP2:3PS lipid substrate (Promega #V1792) and ATP. After the kinase reaction is terminated, the kinase activity is determined using Promega ADP-Glo TM (Promega #V1792) assay detects whether the kinase reaction converts ATP to ADP through phosphorylation of a phospholipid substrate. As shown in Table 12, the following conditions were used for each PI3K isoform.

[0375] Table 12.

[0376] Kinase source Final kinase concentration ATP(uM) PIP2:3PS(uM) Reaction time (min.) PI3Kα Millipore#14-602-K 0.2nM 40 50 120 PI3Kβ Promega#V1751 0.6nM 40 50 120 PI3Kδ Millipore#14-604-K 0.25nM 40 50 120 PI3Kγ Millipore#14-558-K 0.4nM 25 50 120

[0377] After 120 minutes of reaction, the kinase reaction is terminated. Any ATP remaining after the reaction is consumed, leaving only ADP. The kinase detection reagent is then added to convert ADP to ATP, which is used in the coupled luciferin / luciferase reaction. The luminescent output is measured and correlated with kinase activity.

[0378] All reactions were performed at room temperature. For each PI3K isotype, 3 μl mixture (1:1) of enzyme / lipid substrate solution was added to a 384-well white assay plate (Perkin Elmer #6007299) containing only 50 nl test compound or DMSO, for untreated controls only. The reaction was started by adding 2 μl ATP / MgCl2. The kinase reaction buffer contained 50 mM HEPES, 50 mM NaCl, 3 mM MgCl2, 0.01% BSA, 1% DMSO, and the enzyme and substrate concentrations shown in the table above. 10 μL ADP-Glo ​​reagent was added to terminate the reaction. The plate was read in the Perkin Elmer Envision system using the luminescence mode. A 10-point dose response curve was generated for each test compound. The Ki value of each compound was determined using the Morrison equation.

[0379] Binding assays: Initial polarization experiments were performed on an Analyst HT 96-384 (Molecular Devices Corp, Sunnyvale, CA.). Samples for fluorescence polarization affinity measurements were prepared as follows: 1:3 serial dilutions of p110αPI3K (Upstate Cell Signaling Solutions, Charlottesville, VA) starting at a final concentration of 20 μg / mL in polarization buffer (10 mM Tris pH 7.5, 50 mM NaCl, 4 mM MgCl2, 0.05% Chaps, and 1 mM DTT) were added to a final concentration of 10 mM PIP2 (Echelon-Inc., Salt Lake City, UT.). After incubation at room temperature for 30 minutes, the reaction was stopped by the addition of GRP-1 and PIP3-TAMRA probes (Echelon-Inc., Salt Lake City, UT.) at final concentrations of 100 nM and 5 nM, respectively. The rhodamine fluorophore was read using a standard cutoff filter (λ 激发 =530nm;λ 发射 =590 nm). The fluorescence polarization values ​​were plotted as a function of protein concentration. 50 The value is obtained as follows: Use The data were fitted to a four-parameter equation using Synergy software (Reading, PA). This experiment also determined the protein concentration appropriate for subsequent inhibitor competition experiments.

[0380] Inhibitor IC 50 The value was determined as follows: 0.04 mg / mL p110αPI3K (final concentration) was added to the wells containing a 1:3 serial dilution of the antagonist in ATP (Cell Signaling Technology, Inc., Danvers, MA) / polarization buffer at a final concentration of 25 mM. After incubation at room temperature for 30 minutes, the reaction was stopped by adding GRP-1 and PIP3-TAMRA probes (Echelon-Inc., Salt Lake City, UT) at final concentrations of 100 nM and 5 nM, respectively. The reaction was performed in a 384-well black low-volume The rhodamine fluorophore was read using a standard cutoff filter (λ 激发 =530nm;λ 发射=590 nm). Fluorescence polarization values ​​were plotted as a function of antagonist concentration and IC 50 Values ​​were obtained by fitting the data to a four-parameter equation in Assay Explorer software (MDL, San Ramon, CA.).

[0381] Alternatively, inhibition of PI3K was determined in a radioassay using purified recombinant enzyme and ATP at a concentration of 1 μM (micromolar concentration). Compounds were serially diluted in 100% DMSO. The kinase reaction mixture was incubated at room temperature for 1 h and the reaction was terminated by adding PBS. A sigmoidal dose-response curve fit (variable slope) was then used to determine the IC 50 value.

[0382] Embodiment 902 Selective inhibition of mutant PI3Kα(alpha)

[0383] The ability of the compounds of the invention to preferentially act against cells containing mutant PI3Kα (alpha) was determined by measuring inhibition of the PI3K pathway in the SW48 near-isogenic cell line: PI3Kα wild type (parent), helical domain mutant E545K, and kinase domain mutant H1047R. The following assay is intended to determine the cellular potency and mutant selectivity of small molecule PI3Kα inhibitors. The assay utilizes near-isogenic cell lines expressing PI3Kα WT, PI3Kα mutant E545K / + (Horizon Discovery 103-001), or PI3Kα mutant H1047R / + (Horizon Discovery 103-005). The potency of pPRAS40 inhibition of PI3Kα was measured in each cell line after 24 hours of compound treatment. The mutation selectivity of the PI3Kα inhibitor is determined by EC in the WT relative to the E545K cell line and the WT relative to the H1047R cell line. 50 Effectiveness ratio determined.

[0384] Cell culture: The cell lines were maintained in a cell culture incubator at 37°C and 5% in a cell culture medium containing RPMI1640 (prepared at Genentech), 10% FBS (Gibco 16140-071), 2mM L-glutamine (prepared at Genentech) and 10mM HEPES pH 7.2 (prepared at Genentech). The cells were split at a ratio of 1:8 every 72 hours using 0.25% trypsin-EDTA (Gibco 25200).

[0385] Assay operation: harvest cells and plate them in 384-well tissue culture treated assay plates (Greiner cat#781091) and incubate overnight at 37°C, 5% CO2. Three cell lines (WT, E545K and H1047R) were plated in parallel and assayed. The next day, the test compound was serially diluted in dimethyl sulfoxide (DMSO) and added to the cells (the final DMSO concentration was 0.5%). The cells were then incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, the cells were lysed and pPRAS40 levels were measured using a Meso-Scale custom pPRAS40384w assay kit (Meso Scale Discovery, cat#L21CA-1). Cell lysates were added to an assay plate pre-coated with an antibody for phosphorylated PRAS40. Phosphorylated PRAS40 in the sample was bound to the capture antibody overnight at 4°C. The detection antibody (anti-total PRAS40 labeled with electrochemiluminescent SULFO-TAG) was added to the bound lysate and incubated at room temperature for 1 hour. MSD reading buffer was added so that when a voltage was applied to the plate electrode, the label bound to the electrode surface emitted light. The MSD sector instrument measures the intensity of the light and quantitatively measures the amount of phospho-PRAS40 in the sample. Relative to the untreated control, the percentage inhibition of PRAS40 phosphorylation by the test compound at different concentrations was calculated. EC was calculated using a 4-parameter logistic nonlinear regression dose-response model. 50 value.

[0386] Statistical analysis: EC 50 Values ​​represent the geometric mean of at least 4 independent experiments. All statistics were performed using KaleidaGraph software (version 4.1.3). Student t-tests were performed using unpaired data with equal variance to compare the activity of mutant cells and wild-type cells. P < 0.05 was considered significant.

[0387] Embodiment 903 In vitro cell viability assay

[0388] Cells (1500 per well) were seeded in 384-well plates for 16 hours. On the second day, 9 serial 1:3 compound dilutions were prepared in DMSO in 96-well plates. The compounds were then further diluted into growth medium using a Rapidplate robot (Zymark Corp.). The diluted compounds were then added to quadruplicate wells of a 384-well cell plate and incubated at 37°C and 5% CO2. After 4 days, the relative number of viable cells was measured by luminescence using Cell Titer-Glo (Promega) according to the manufacturer's instructions and read on a Wallac Multilabel Reader (Perkin-Elmer). EC was calculated using Prism 6.0 software (GraphPad) 50 value.

[0389] Embodiment 904 In vivo mouse tumor xenograft efficacy

[0390] Mouse : Female severe combined immunodeficient mice (CB-17SCID.bg Charles River Labs, SanDiego), NOD.SCID (Charles River Labs, Hollister) or NCR. nude mice (Taconic) were 8 to 9 weeks old and had a body weight range of 18-26 grams on day 0 of the study. Animals received water and laboratory autoclaved rodent diet 5010 (LabDiet St.Louis, MO) ad libitum. Mice were housed in static microisolators (12-hour light cycle). Genentech specifically complies with the requirements of the laboratory animal care and use guidelines in terms of restrictions, management, surgical procedures, feeding and fluid supervision and veterinary care. Genentech's animal care and use procedures are approved by the International Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC), which ensures compliance with accepted laboratory animal care and use standards. Mice were placed in standard rodent microisolator cages at Genentech and adapted to the research conditions for at least 3 days before the study tumor cell implantation. Only animals that appeared healthy and had no obvious abnormalities were used in the research.

[0391] Tumor implantationXenografts were initiated with cancer cells (HCC1954x1 or KPL4) or passaged tumors (HCI-003). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units / mL penicillin, 100 μg / mL (micrograms per milliliter) streptomycin sulfate, and 25 μg / mL gentamicin, harvested in logarithmic phase growth and resuspended at a concentration of 3×10 6 or 5×10 6 The tumors were then implanted in 50% phenol red-free Matrigel (Becton Dickinson Bioscience; San Jose, CA) and Hank's balanced salt solution at 10 cells / mL. For the HCI-003 patient-derived model, 30 mg of beeswax pellets containing approximately 1 mg of 17β (beta)-estradiol were implanted subcutaneously 3 days prior to implantation of tumor fragments. Tumor cells or fragments were implanted in the 2 / 3 mammary fat pad and when the average size approached the target range of 100-250 mm 3 Once the majority of tumors reached the target size, mice were divided into groups of 7-10 mice based on tumor volume.

[0392] Therapeutic agents : PI3K compounds are provided as free bases in dry powder form and stored at room temperature in the dark. The vehicle for taselisib (GDC-0032) and BYL719 is 0.5% methylcellulose: 0.2% Tween 80 (MCT) in deionized water. The vehicle control for compound 101 is 0.5% methylcellulose / 0.2% Tween 80 (MCT) nanosuspension. MCT nanosuspension is prepared by initially preparing MCT suspension. Once prepared, the MCT suspension is ground for about 24 hours using 1mm glass beads and a rare earth magnetic stirring bar to make a fine nanosuspension. The final particle size is checked using a particle size analyzer. Drug doses are prepared weekly and stored at 4°C.

[0393] treat Mice are dosed PO daily by gavage in a volume of 100 μL, microliters (5 mL / kg) with either vehicle or PI3K compounds expressed as free base equivalents for 21-28 days.

[0394] end Tumor volume was measured in two dimensions (length and width) using Ultra Cal IV calipers (Model 54 10 111; Fred V. Fowler Company) as follows: Tumor volume (mm 3 )=(length×width 2)×0.5 and analyzed using Excel version 11.2 (Microsoft Corporation). The linear mixed effects (LME) modeling method is used to analyze repeated measurements of tumor volume over time from the same animal (Pinheiro, J. et al. nlme: linear and nonlinear mixed effects models, 2009; R package version 3.2.5). This method takes into account repeated measurements and appropriate withdrawals due to any non-treatment-related animal deaths before the end of the study. Cubic regression splines are used to fit nonlinear distributions to the time course of log2 tumor volume at each dose level. These nonlinear distributions are then associated with the dose in the mixed model. Tumor growth inhibition (%TGI) in the form of vehicle control percentage is calculated using the following formula as the area under the curve (AUC) fitted for each dose group per day relative to the vehicle: %TGI=100×(1-AUC 药物 / AUC 媒介物 ). Using this formula, a TGI value of 100% indicates tumor stasis, a TGI value greater than (>) 1% but less than (<) 100% indicates tumor growth delay, and a TGI value greater than (>) 100% indicates tumor regression. A partial response (PR) for an animal is defined as a tumor regression greater than (>) 50% but less than (<) 100% of the initial tumor volume. A complete response (CR) is defined as 100% tumor regression (i.e., no measurable tumor) on any day during the study.

[0395] toxicity Animals were weighed daily for the first 5 days of the study and then twice weekly. Animal weights were measured using Adventurer AV812 scale (Ohaus Corporation) was used to measure the body weight. The body weight change percentage was calculated as follows: Body weight change (%) = [(weight 新一天 -weight 第0天 ) / weight 第0天 ]×100. Mice were frequently observed for obvious signs of any adverse treatment-related side effects and clinical signs of toxicity were recorded when observed. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 1 treatment-related (TR) death in 10 treated animals. Any dosing regimen that resulted in greater toxicity was considered to be above the maximum tolerated dose (MTD). If clinical signs and / or autopsy indicated that the death was attributed to treatment side effects, the death was classified as TR or if the death was caused by unknown reasons during the dosing period or within 10 days after the last dose, the death could also be classified as TR. If there was no evidence that the death was related to treatment side effects, the death was classified as NTR.

[0396] Example 905. Cell culture I and in vitro inhibitor experiments

[0397] Cell lines were grown under standard tissue culture conditions in RPMI medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. HCC-1954 and HDQ-P1 are breast cancer cell lines (American Type Culture Collection; Manassas, VA). HCC-1954 and HDQ-P1 cells were plated at 80,000 cells / well in each well of a 6-well tissue culture plate and incubated overnight at 37°C. The cells were incubated with the indicated concentrations of each compound for 24 hours. After incubation, the cells were washed once with cold phosphate-buffered saline (PBS) and incubated in Biosource supplemented with protease inhibitors (F. Hoffman-LaRoche; Mannheim, Germany), 1 mM phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich; St. Louis, MO). TM Cells were lysed in cell extraction buffer (Invitrogen; Carlsbad, CA). Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific; Rockford, IL).

[0398] Protein determination

[0399] Protein concentrations were determined using the Pierce BCA protein assay kit (Rockford, IL). For immunoblotting, equal protein amounts were separated by electrophoresis through NuPage Bis-Tris 4-12% gradient gels (Invitrogen; Carlsbad, CA); proteins were transferred to nitrocellulose membranes using the IBlot system and protocol from InVitrogen. Antibodies against p110α and phospho-Akt (Ser473) were obtained from Cell Signaling (Danvers, MA). Antibodies against β-actin and GAPDH were from Sigma.

[0400] Embodiment 906 CD19 + CD27 - B cell CD69 expression in B cells, human whole blood measurement of CD69 expression

[0401] Cell culture: Human whole blood was dispensed into 96 deep well plates at 100 μl per well. Compounds were diluted in DMSO to produce the desired stock concentration, then further diluted in PBS to the desired working concentration and added at a volume of 5.5 μL per well. Samples were then incubated at 37°C under 5% CO2 for 1 hour and at 37°C under 5% CO2 for 18 hours before adding 5 μg (10 μl per well) of goat anti-IgM F(ab')2 (Southern Biotech, AL). All treatments were tested in duplicate.

[0402] Cell separation and staining procedures : After incubation, CD19 was determined by staining whole blood samples with a mixture of CD27; 10 μl / well (clone L128; BD Biosciences, NJ) CD19; 7.5 μl / well (clone SJ25C1; BD Biosciences, NJ) and CD69; 10 μl (clone FN50; BD Biosciences, NJ) + The level of CD69 expression on CD27- cells. In addition, human whole blood from each donor was stained with isotype-matched fluorescent control antibodies. After adding the appropriate antibody mixture, the whole blood samples were stained in the dark for 30 minutes and then cracked with BD Pharm Lysis (BD Bioscience, NJ). The resulting samples were then washed with FACS buffer (phosphate buffered saline (Ca / Mg++-free), 1mM EDTA, 25mM HEPES pH 7.0, 1% fetal bovine serum (heat inactivated)) and fixed in FACS buffer supplemented with 0.1% formaldehyde (Polysciences Inc, PA) and 0.1% Pluronic F-68 (Sigma, MO). Data were obtained using BD LSR-II (BD Biosciences) with BD FACSDiva software.

[0403] CD19 + CD27 - CD69 expression on B cells The cells were evaluated for CD19, CD27, and CD69 levels by flow cytometry using BD FACSDiva software, and CD19 + CD27 - The CD69 MFI-mean values ​​of the lymphocyte populations were determined using Genedata software (Genedata Screener, MA). The compound concentration that resulted in 50% inhibition of the mean CD69 MFI (IC 50 ).

[0404] Embodiment 907 HCC1954 and HDQP1 pPRAS40 EC 50

[0405] The cells were plated in 384-well tissue culture treated assay plates and incubated overnight. The next day, the cells were treated with compounds and incubated for 24 hours. After 24 hours, the cells were lysed and pPRAS40 levels were measured using the Meso-Scale assay platform. These cell lines are very useful for characterizing the selectivity of PI3Ka inhibitors for mutant PI3Ka. The HCC1954 cell line expresses mutant PI3K PI3Ka (E545K) vs WT in HDQP1.

[0406] Measurement principle :The MSD platform provides a method for measuring the phosphorylation level of pPRAS40 in a single sample. Cell lysate is added to an assay plate pre-coated with an antibody against full PRAS40. After cell lysis, PRAS40 in the sample is allowed to bind to the capture antibody. A detection antibody (anti-phospho-PRAS40) labeled with the electrochemiluminescent compound MSD SULFO-TAG is added to the bound lysate. MSD reading buffer is added so that when a voltage is applied to the plate electrode, the label bound to the electrode surface emits light. The MSD sector meter measures the intensity of light and quantitatively measures the amount of phospho-EGFR in the sample (Meso Scale assay principle).

[0407] Material:

[0408]

[0409]

[0410] operate :

[0411] - Prepare compounds at 2 mM concentration in DMSO. Prepare DMSO compound titration plates, 1:3 in neat DMSO.

[0412] The DMSO master plate contained 72 μl of 13 compounds.

[0413] • Mutant Selective Control Compound: Add 72ul of 2mM control compound to well B2 on each assay plate. This control compound exhibits approximately 20-fold greater potency in the HCC1954 cell line relative to the HDQP1 cell line.

[0414] Using a multichannel pipette, transfer 36 ul from each compound well to the well just below (eg B2 to C2) to set up duplicate dose-response curves.

[0415] • Use the Biomek Fx method titled "SLS_serial dilution / 1plate_384_3_13_3x" to perform serial dilutions of compounds in a master plate.

[0416] When not in use, seal and store the DMSO mother and daughter plates with a heat sealer.

[0417] Day 1: Cell plating

[0418] 1. For each cell line, seed 12,500 cells in 45 ul of culture medium. Allow cells to settle / attach to the plate for 15-20 minutes at room temperature.

[0419] 2. Incubate the cells overnight in a 37°C humidity and CO2 controlled incubator.

[0420] Day 2: Compound plate preparation and compound treatment

[0421] 1. For 10X intermediate dilution plate: add 95ul serum-free medium to a standard format Greiner 384-well polypropylene plate.

[0422] 2. Add intermediate compound dilutions in media and add to cells using the biomek Fx protocol: "SLSIntermed Dil Add 5ul to Cells July 13 2012." The Biomek protocol transfers 5ul from the DMSO daughter plate to the intermediate dilution plate containing 95ul media and mixes media + compound. This method then transfers 5ul from the intermediate dilution plate to the appropriate cell plate.

[0423] 3. Incubate the treated cells at 37 degrees in a 5% CO2 humidified incubator. 24 hours .

[0424] Day 3: Cell lysis and addition to MSD plates

[0425] Block the MSD assay plate with 50ul 3% Blocker A / 1X MSD Wash Buffer at room temperature for 1-2 hours. This solution can be stored at 4°C for up to one month. Blocker Buffer A contains 1X MSD Wash Buffer, 20mL 1X Tris Wash Buffer, and 600mg Blocker A.

[0426] Prepare lysis buffer:

[0427]

[0428] Aspirate the medium and lyse the cells

[0429] 1. Lyse cells in 50 μl of lysis buffer. Lyse for 10-20 minutes at room temperature on a plate shaker.

[0430] 2. While cells are lysing, wash the blocked plate with 1x MSD Wash Buffer.

[0431] 3. Transfer 42ul lysate (21+21μL) to blocked MSD pPRAS40 assay plate.

[0432] 4. Seal the MSD plate and shake at 4°C overnight.

[0433] Day 4: MSD determination / detection

[0434] 8. Prepare a 1% Blocker A solution in 1X MSD Wash Buffer. (20 mL 1X Tris Wash Buffer and 200 mg Blocker A (1% w / v). This solution can be stored at 4°C for one month.

[0435] 9. Wash the MSD plate with 1x MSD Wash Buffer.

[0436] 10. Add 10 μl of diluted SULFO-TAG detection antibody to the plate and incubate at room temperature with shaking for 1 hour.

[0437]

[0438] 11. Wash the plate 4 times with 1X MSD Wash Buffer.

[0439] 12. Add 35 μl 1X Reading Buffer using reverse phase pipetting to avoid foaming.

[0440] 13. Read the plate immediately on the MSD SECTOR instrument.

[0441] Embodiment 908 Cocrystal structure with p110α(alpha)

[0442] N-terminally truncated p110α(alpha) was generated according to Chen et al. and Nacht et al. (Chen, P., YLDeng, S. Bergqvist, MD Falk, W. Liu, S. Timofeevski and A. Brooun "Engineering of anisolated p110alpha subunit of PI3Kalpha permits crystallization and provides a platform for structure-based drug design," (2014) Protein Sci 23(10):1332-1340; Nacht, M. et al (2013) "Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kalpha," J. Med. Chem. 56 (3): 712-721).

[0443] In the presence of the project compound, standard protocols were used to produce crystals. Harvested crystals were preserved by immersion in liquid nitrogen for diffraction data collection and placed on a synchrotron beamline producing monochromatic X-rays. Diffraction data were collected, reduced, and merged using standard protocols. The crystal unit cell and space group were isomorphous to those reported previously (Nacht, 2013; Chen, 2014). The project compound was placed into the electron density map and crystallographically refined to the resolution limit of #imgpt180# using standard protocols.

[0444] In the present application, the units ul, uMol, etc. mean μl, μMol, etc.

[0445] Although the above invention has been described in some detail by way of illustration and examples for the purpose of clear understanding, the description and examples should not be construed as limiting the scope of the invention. The entire disclosures of all patents and scientific literature cited in this application are expressly incorporated into this application by reference.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from Formula I: in: R 1 is selected from -CH3, cyclopropyl and cyclobutyl; R 2 Selected from -CH3, -CHF2, -CH2F and -CF3.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 It is CH3 or cyclobutyl.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 2 It is -CHF2 or -CF3.

4. A pharmaceutical composition, consisting of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable excipient is selected from a pharmaceutically acceptable carrier, a glidant or a diluent.

6. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable excipient is selected from the group consisting of silicon dioxide, powdered cellulose, microcrystalline cellulose, metal stearates, sodium aluminosilicate, sodium benzoate, calcium carbonate, calcium silicate, magnesium carbonate, asbestos-free talc, stearowet C, starch, magnesium lauryl sulfate, magnesium oxide, and combinations thereof.

7. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable excipient is corn starch.

8. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable excipient is starch 1500.

9. A method for preparing a pharmaceutical composition, comprising combining a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient.

10. The method of claim 9, wherein the pharmaceutically acceptable excipient is selected from a pharmaceutically acceptable carrier, a glidant or a diluent.

11. Use of a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer in a patient having cancer, wherein the cancer is selected from breast cancer and non-small cell lung cancer.

12. The use of claim 11, further comprising administering to the patient an additional therapeutic agent selected from the group consisting of 5-FU, docetaxel, eribulin, gemcitabine, cobimetinib, ipatasertib, paclitaxel, tamoxifen, fulvestrant, GDC-0810, dexamethasone, palbociclib, bevacizumab, pertuzumab, trastuzumab-emtansine conjugate, trastuzumab, and letrozole.

13. The use according to claim 11, wherein the cancer is breast cancer. The use according to claim 13 , wherein the breast cancer is estrogen receptor positive breast cancer.

15. The use of claim 13, wherein the breast cancer subtype is basal or tubular epithelial.

16. The use of claim 11, wherein the cancer expresses a PIK3CA mutant selected from the group consisting of: E542K, E545K, Q546R, H1047L, and H1047R.

17. The use of claim 11, wherein the cancer expresses a PTEN mutant.

18. The use of claim 13, wherein the cancer is HER2 positive.

19. The use of claim 13, wherein the patient is HER2 negative, estrogen receptor negative, and progesterone receptor negative.

20. A kit for therapeutic treatment of breast cancer, comprising: a) A pharmaceutical composition according to any one of claims 4 to 8; and b) Instructions for use for the therapeutic treatment of breast cancer.

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