Methods for preparing tricyclic PI3K inhibitor compounds and methods for treating cancer using the same

Abstract

The present invention provides a method for preparing a tricyclic PI3K inhibitor compound with high yield and high purity in an aqueous solvent system.

Description

Technical Field

[0001] The present invention generally relates to methods for preparing compounds that inhibit PI3 kinase activity. The present invention also relates to methods for using the compounds to diagnose or treat mammalian cells or related pathological conditions in vitro, in situ and in vivo. The present invention also relates to methods for treating cancer characterized by overexpression of PI3 kinase. Background Art

[0002] Any discussion of the prior art throughout the specification should never be considered as an admission that such prior art is well known or forms part of the common general knowledge in the art. Phosphatidylinositol is one of the many phospholipids found in cell membranes, which plays an important role in intracellular signal transduction. Cell signaling through 3'-phosphorylated phosphoinositides is associated with a variety of cellular processes, such as malignant transformation, growth factor signaling, inflammation and immunity (Rameh et al. (1999) J. Biol Chem, 274: 8347-8350). The enzyme responsible for producing these phosphorylated signal products, phosphatidylinositol 3-kinase (also known as PI 3-kinase or PBK), was originally considered to be an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases, phosphorylated phosphatidylinositol (PI) and its phosphorylated derivatives at the 3'-hydroxyl of the inositol ring (Panayotou et al. (1992) Trends Cell Biol 2: 358-60).

[0003] Phosphoinositide 3-kinase (PI3K) is a lipid kinase that phosphorylates lipids at the 3-hydroxyl residue of the inositol ring (Whitmane et al. (1998) Nature, 332: 664). The 3-phosphorylated phospholipids (PIP3s) produced by PI3-kinase act as second messengers recruited by kinases with lipid binding domains (including plekstrin homology (PH) regions), such as AKT and phosphoinositide-dependent kinase-1 (PDK1). Binding of AKT to membrane PIP3s causes AKT to translocate to the plasma membrane, bringing AKT into contact with PDK1, which is responsible for activating AKT. The tumor suppressor phosphatase PTEN dephosphorylates PIP3, thus acting as a negative regulator of AKT activation. The PI3-kinases AKT and PDK1 are important in the regulation of many cellular processes, including cell cycle control, proliferation, survival, apoptosis, and motility, and are important components of the molecular mechanisms of diseases such as cancer, diabetes, and immune-inflammation (Vivanco et al. (2002) Nature Rev. Cancer 2:489; Phillips et al. (1998) Cancer 83:41).

[0004] The major PI3-kinase isoform in cancer is the class I PI3-kinase, pi 10α (alpha) (see, e.g., U.S. Pat. No. 5,824,492, U.S. Pat. No. 5,846,824, U.S. Pat. No. 6,274,327). Other isoforms have been implicated in cardiovascular disease and immune-inflammatory diseases (Workman P (2004) Biochem Soc Trans 32: 393-396; Patel et al. (2004) Proceedings of the American Association of Cancer Research (Abstract LB-247) 95th Annual Meeting, March 27-31, Orlando, Fla., USA; Ahmadi K and Waterfield MD (2004) Encyclopedia of Biological Chemistry (Lennarz WJ, Lane MD eds) Elsevier / Academic Press). The PI3 kinase / Akt / PTEN pathway is an attractive target for cancer drug development because these modulators or inhibitors are expected to inhibit proliferation, reverse inhibition of apoptosis and overcome resistance to cytotoxic drugs in cancer cells (Folkes et al. (2008) J. Med. Chem. 51:5522-5532; Yaguchi et al. (2006) Jour. of the Nat. Cancer Inst. 98(8):545-556).

[0005] Malignant glioma is the most common primary brain tumor in adults. In the most aggressive glioma subtype glioblastoma (GBM), tumor formation and growth seem to be driven by amplification or overexpression of gene products related to the signal transduction initiated by growth factors, which synergizes with gene changes to destroy cell cycle control (Holland EC (2001) Nat Rev Genet 2: 120-129). In the genome changes described in GBM, PTEN mutation and / or deletion are the most common, with an estimated frequency of 70-90% (Nutt C, Louis DN (2005) Cancer of the Nervous System (McGraw-Hill, New York), 2nd Ed, pp 837-847). These findings, together with the prognostic value of PTEN status in GBM cases (Phillips HS, et al. (2006) Cancer Cell 9:157-163), suggest the importance of the phosphoinositide 3-kinase (PI3K) / Akt pathway in promoting highly aggressive glial malignancies and treatment with PI3K inhibitors that have blood-brain barrier permeability.

[0006] Certain tricyclic PI3K inhibitor compounds of Formula III disclosed in US Pat. No. 8,883,799 (hereinafter) have been found to possess PI3 kinase modulating or inhibitory activity, anti-cancer properties, anti-inflammatory properties, and / or immunomodulatory properties.

[0007]

[0008] The compounds of formula III of U.S. Pat. No. 8,883,799 can be used to treat hyperproliferative diseases, such as cancers characterized by modulation of PI3 kinase function, such as cancers characterized by mutation or overexpression of proteins. Useful methods for preparing formula III are known. However, there is a need for improved methods for preparing compounds of formula III in high yield and high purity. Summary of the invention

[0009] In some embodiments, the present invention relates to a method for preparing a compound of formula III from a compound of formula II in a reaction mixture according to the following reaction formula,

[0010]

[0011] The method comprises: (i) forming a compound comprising formula II, an organic boron-R 4, a reaction mixture comprising a solvent system comprising at least 5 v / v% water, a base and a catalyst; (ii) reacting the reaction mixture at a temperature of less than 100° C. to form a reaction product mixture comprising a compound of formula III; (iii) isolating the compound of formula III, its stereoisomers, its geometric isomers, its tautomers or a pharmaceutically acceptable salt thereof from the reaction product mixture. The catalyst comprises palladium, and the reaction mixture comprises less than 0.05 equivalents of the catalyst per equivalent of the compound of formula II.

[0012] Furthermore, X 1 S, O, N, NR 6 , CR 1 , C(R 1 )2 or -C(R 1 )2O-. X 2 C, CR 2 or N. X 3 C, CR 3 or N. X 4 A is halogen. 2 and X 3 A fused 5-, 6- or 7-membered carbocyclyl or heterocyclyl ring, optionally substituted by one or more R 5 , R 10 and / or R 15 Group substitution. 1 , R 2 and R 3 independently selected from H, F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2, -CONH2, -NO 2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and l,l-dioxo-thiopyran-4-yl.

[0013] Furthermore, R 6 H, C1-C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12Carbocyclic), -(C1-C 12 Alkylene)(-C2-C 20 Heterocyclic), -(C1-C 12 Alkylene)-C(=O)-(C2-C 20 Heterocyclic group), (C1-C 12 Alkylene)-(C6-C 20 Aryl) and -(C1-C 12 Alkylene)-(C1-C 20 wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -(CH3)2OH, -CH2OCH3, -CN, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3) 2CONH2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and 1,1-dioxo-thiopyran-4-yl are substituted with one or more groups.

[0014] Furthermore, R 4 Selected from C6-C 20 Aryl, C2-C 20 Heterocyclic and C1-C 20 heteroaryl, each of which is optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2CH3, -CH2CN, -CN, -CF3, -CH2OH, -CO2H, -CONH2, CONH(CH3), -CON(CH3)2, -NO2, -NH2, -NHCH3, -NHCOCH3, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(O)NHCH3, -NHC(O)NHCH2CH3, -NHS(O)2CH3, -N(CH3)C(O)OC(CH3)3, -S(O)2CH3, benzyl, benzyloxy, morpholinyl, morpholinylmethyl and 4-methylpiperazin-1-yl.

[0015] Each R 5, R 10 and R 15 Independently selected from C1-C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12 Carbocyclic), -(C1-C 12 Alkylene)-(C2-C 20 Heterocyclic), -(C1-C 12 Alkylene)-C(O)-(C2-C 20 Heterocyclic group), -(C1-C 12 Alkylene)-(C6-C 20 Aryl) and -(C1-C 12 Alkylene)-(C1-C 20 heteroaryl); or two geminal R 5 , R 10 and / or R 15 The group forms a 3-, 4-, 5- or 6-membered carbocyclyl or heterocyclyl ring; wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CH2F, -CHF2, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -C The invention can be substituted with one or more of: ON(CH3)2, -C(CH3)2CONH2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NH-COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and 1,1-dioxo-thiopyran-4-yl.

[0016] Further, mor is selected from:

[0017]

[0018] wherein mor is optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, -CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH, -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C(CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2, -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -NHCH(CH3)2, -NHCH2CH2OH, -NHCH2CH2OCH3, -NHCOCH3, -NHCOCH2CH3, -NHCOCH2OH, -NHS(O)2CH3, -N(CH3)S(O)2CH3, =O, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(O)NHCH3, -NHC(O)NHCH2CH3, -S(O)CH3, -S(O)CH2CH3, -S(O)2CH3, -S(O)2NH2, -S(O)2NHCH3, -S(O)2N(CH3)2, and -CH2S(O)2CH3, one or more R 7 Group substitution.

[0019] In some other embodiments, the present invention relates to a method for preparing a compound of formula IIa from a compound of formula I according to the following reaction formula,

[0020]

[0021] The method comprises: (i) forming a reaction mixture comprising a compound of formula I, an organic halide, a solvent system, a phase transfer catalyst and a base; (ii) reacting the reaction mixture to form a reaction product mixture comprising a compound of formula IIa, a stereoisomer thereof, a geometric stereoisomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof; and (iii) separating the compound of formula IIa from the reaction product mixture.

[0022] Furthermore, the solvent system comprises at least 5 v / v% of water. X is a halide. Each R 5 , R 10 and R 15 Independently selected from H, C1-C 10or a hydrocarbon group selected from C1-C5, wherein each hydrocarbon group is optionally substituted, and the two geminal R 5 , R 10 and / or R 15 The group is oxo, or two geminal R 5 , R 10 and / or R 15 The group forms a 3, 4, 5, 6 or 7 membered carbocyclic or heterocyclic group, wherein the carbocyclic or heterocyclic group is optionally substituted.

[0023]

[0024] wherein mor is optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, -CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH, -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C(CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2, -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -NHCH(CH3)2, -NHCH2CH2OH, -NHCH2CH2OCH3, -NHCOCH3, -NHCOCH2CH3, -NHCOCH2OH, -NHS(O)2CH3, -N(CH3)S(O)2CH3, =O, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(O)NHCH3, -NHC(O)NHCH2CH3, -S(O)CH3, -S(O)CH2CH3, -S(O)2CH3, -S(O)2NH2, -S(O)2NHCH3, -S(O)2N(CH3)2, and -CH2S(O)2CH3, one or more R 7 In Formula I, R 20 -OH or -NHR 21 , R 21 For R 5 As defined above, and in Formula IIa, R 20 -O- or -NR 21 -.

[0025] In some other embodiments, the present invention relates to a method for preparing a compound of formula IIIa from a compound of formula IIa according to the following reaction formula, wherein the compound of formula IIa is prepared according to the method just described above,

[0026]

[0027] The method comprises: (i) forming a reaction mixture comprising a compound of formula IIa, an organoboron-R 4 , a solvent system comprising at least 5 v / v% water, a base and a catalyst; (ii) reacting the reaction mixture to form a reaction product mixture comprising a compound of formula IIIa; and (iii) separating the compound of formula IIIa, its stereoisomers, its geometric isomers, its tautomers, or a pharmaceutically acceptable salt thereof from the reaction product mixture by solid-liquid separation, wherein the yield of the compound of formula IIIa is at least 75%.

[0028] The catalyst comprises palladium and the reaction mixture comprises less than 0.05 equivalents of catalyst per equivalent of compound of Formula IIa.

[0029] R 4 Selected from C6-C 20 Aryl, C2-C 20 Heterocyclic and C1-C 20 R is a heteroaryl radical, each of which is optionally substituted with one or more radicals independently selected from the group consisting of F, Cl, Br, I, -CH, -CHCH, -CH(CH), -CHCH(CH), -CHCH, -CHCN, -CN, -CF, -CHOH, -COH, -CONH, CONH(CH), -CON(CH), -NO, -NH, -NHCH, -NHCOCH, -OH, -OCH, -OCHCH, -OCH(CH), -SH, -NHC(O)NHCH, -NHC(O)NHCHCH, -NHS(O)CH, -N(CH)C(O)OC(CH), -S(O)CH, benzyl, benzyloxy, morpholinyl, morpholinylmethyl, and 4-methylpiperazinyl. Each R 5 , R 10 and R 15 Independently selected from H, C1-C 10 or a hydrocarbon group selected from C1-C5, wherein each hydrocarbon group is optionally substituted, and the two geminal R 5 , R 10 and / or R 15 The group is oxo, or two geminal R 5 , R 10 and / or R 15The groups form a 3-, 4-, 5-, 6- or 7-membered carbocyclyl or heterocyclyl, wherein the carbocyclyl or heterocyclyl is optionally substituted.

[0030] In some other embodiments, the present invention relates to a method of treating cancer in a patient, wherein the cancer is characterized by overexpression of PI3 kinase, comprising administering to a person in need of such treatment a therapeutically effective amount of a PI3 kinase inhibitor compound of formula III as defined above. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown is an amide impurity profile of 5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine during crystallization from an acetic acid-water solvent system at ratios of 1:1 v / v%, 4:1 v / v%, 9:1 v / v%, and 98:2 v / v% acetic acid to water.

[0032] Figure 2 Shown is a graph of the effect of the dose of 5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine on time after administration of pAKT in normal brain tissue, expressed as the ratio of phosphorylated AKT (pAKT) to total AKT (tAKT).

[0033] Figure 3 The in vivo efficacy of 5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine against U87 MG Merchant (MG / M) human glioblastoma xenografts in subcutaneous tumor-bearing Taconic female NCR nude mice in a dose escalation study is shown, and the tumor volume versus dosing regimen (dose rate and dosing time) is described.

[0034] Figure 4 Shown is the effect of 5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine on the ratio of phosphorylated AKT (pAKT) to total AKT (tAKT) in the U87MG / M human glioblastoma xenograft model after 24 days of continuous administration at dose rates of 0.5 mg / kg, 3 mg / kg, 10 mg / kg and 18 mg / kg, wherein tumors were resected from animals at 1 hour and 4 hours after the last dose on day 24.

[0035] Figure 5 2-(2-chloro-6-morpholinyl-9H-purin-8-yl)isopropan-2-ol (Compound 5)1 H NMR (500 MHz, CDCl3) spectroscopy.

[0036] Figure 6 2-(2-chloro-6-morpholinyl-9H-purin-8-yl)isopropan-2-ol (Compound 5) 13 C NMR (125 MHz, CDCl3) spectroscopy.

[0037] Figure 7 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazine[4,3-e]purine (Compound 7) 1 H NMR (500 MHz, CDCl3) spectroscopy.

[0038] Figure 8 This is the 13C NMR (125 MHz, CDCl3) spectrum of 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purine (Compound 7).

[0039] Fig. 9 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine (boronic acid pinacol ester) 1 H NMR (500 MHz, CDCl3) spectroscopy.

[0040] Fig.10 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine (boronic acid pinacol ester) 13 C NMR (125 MHz, CDCl3) spectroscopy.

[0041] Fig.11 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) 1 H NMR (500 MHz, CDCl3) spectroscopy.

[0042] Fig.12 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) 13 C NMR (125 MHz, CDCl3) spectroscopy.

[0043] Fig.13 Graph showing mean ± daily plasma concentration vs. time profile of GDC-0084 after a single dose.

[0044] Fig.14 Figure 2 is a graph of ± single-day plasma concentration vs. time curve of GDC-0084 after multiple doses.

[0045] Fig.15 The dose (mg) for single-dose and multiple-dose regimens is max (μM) Dose ratio of GDC-0084.

[0046] Fig.16 The dose (mg) for single and multiple dose regimens vs. AUC 24 (μM*hr) of GDC-0084 dose ratio.

[0047] Fig.17 The mean single-dose plasma GDC-0084 concentration is plotted against time on a logarithmic scale.

[0048] Fig.18 The mean single-dose plasma GDC-0084 concentration versus time is plotted on a linear scale.

[0049] Fig.19 The mean single-dose plasma GDC-0084 concentration is plotted against time on a logarithmic scale.

[0050] Fig. 20 The mean single-dose plasma GDC-0084 concentration versus time is plotted on a linear scale.

[0051] Fig.21 AUC of GDC-0084 for single and multiple dose regimens 0-24 Logarithmic scale plot of (μM*hr) versus dose (mg).

[0052] Fig. 22 C for single and multiple dose regimens of GDC-0084 max Logarithmic scale plot of (μM) versus dose (mg).

[0053] Fig.23 Western blot of mouse brain probed with antibodies against pAkt, total Akt, pS6, total S6, and actin.

[0054] Fig.24 Quantification of pAkt versus total Akt and pS6 versus total S6 at 1 and 6 hours post-dose in CD-1 mice.

[0055] Fig.25ADepicted are images of GDC-0084 mouse brain distribution 1 hour after oral administration of 15 mg / kg of GDC-0084 in an orthotopic model of GS2 glioblastoma intracranial tumors. Tumor localization by cresyl violet staining and drug distribution in MALDI MS images are shown.

[0056] Fig.25B Depicted is an image of GDC-0084 mouse brain distribution 1 hour after oral administration of 15 mg / kg of GDC-0084 in an orthotopic model of U87 glioblastoma intracranial tumors. Tumor localization by cresyl violet staining and drug distribution in MALDI MS images are shown.

[0057] Fig.26A Depicted are the actual and Gaussian distributions of MALDI imaging signal intensity of GDC-0084 in intracranial tumor and non-tumor brain regions of the orthotopic mouse model U87.

[0058] Fig.26B Depicted are the actual and Gaussian distributions of MALDI imaging signal intensity of GDC-0084 in intracranial tumor and non-tumor brain regions of the orthotopic mouse model GS2.

[0059] Fig.26C Depicted are the actual and Gaussian distributions of MALDI imaging signal intensity of GDC-0084 in non-tumor areas of U87 and GS2 orthotopic mouse models.

[0060] Fig.26D Depicted are the actual and Gaussian distributions of signal intensity for GDC-0084 and the actual distribution for pictilisib in the U87 orthotopic GBM model.

[0061] Fig.27A Depicted are micro-CT images reflecting tumor size (therapeutic effect) of GDC-0084 in the U87 orthotopic mouse model following daily oral administration of 15 mg / kg GDC-0084 for two weeks compared to control treatment (GDC-0084 vehicle).

[0062] Fig.27B Figure 3 shows tumor volume (in mm) of mice treated with 15 mg / kg GDC-0084 orally administered daily in the U87 orthotopic model compared to control mice. 3 The results are expressed as the mean ± SE of ten animals.

[0063] Fig.27CDepicted are representative T-2 weighted MRI images showing the efficacy of GDC-0084 in the GS2 neurosphere tumor mouse model following four weeks of daily oral administration of 15 mg / kg GDC-0084 compared to control animals.

[0064] Fig.27D Figure 3 shows tumor volume (in mm) of mice treated with 15 mg / kg GDC-0084 orally administered daily in the U87 orthotopic model compared to control mice. 3 The results are expressed as the mean ± SE of ten animals.

[0065] Fig.28A Depicted are Western blots of PI3K pathway markers pAkt, pS6, and p4EBP1 in intracranial GS2 xenografts following four weeks of daily oral administration of 15 mg / kg GDC-0084, wherein modulation of the PI3K pathway in GS2 tumors was assessed by Western blot at the end of the 4-week dosing period and 2 and 8 hours after the final dose of 15 mg / kg GDC-0084.

[0066] Fig.28B Depicted are quantification of pAkt / total Akt, p4EBP1 / total 4EBP1, and pS6 / total S6 at 2 and 6 hours after the last 15 mg / kg dose of GDC-0084. DETAILED DESCRIPTION

[0067] Reference will now be made in detail to certain embodiments of the present invention, examples of which are shown in the accompanying structures and formulas. Although the present invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all substitutions, modifications and equivalents that may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used for the implementation of the present invention. The present invention is by no means limited to the methods and materials described. If one or more of the cited documents, patents and similar materials are different or contradictory from the present application, including but not limited to the defined terms, term usage, described technology, etc., the present application shall prevail.

[0068] The present invention provides an improved method for preparing a tricyclic PI3K inhibitor compound of formula III from a compound of formula II by Suzuki coupling according to reaction formula (1):

[0069]

[0070] Where X 1 , X 2 , X 3 ,A,mor,X4 , organic boron, R 4 , solvent systems, bases and catalysts are defined elsewhere herein. Ring A may be optionally replaced by one or more R defined elsewhere herein 5 , R 10 and / or R 15 Compared to known processes, appropriate selection of at least one process variable from the catalyst comprising Pd, the basic substance, the solvent system and / or the reaction temperature range, or appropriate selection of 2, 3 or all 4 of these process variables, provides an increased yield and / or purity of Formula III, thereby enabling the elimination of one or more process steps and / or purification steps.

[0071] The present invention further provides a method for forming a tricyclic compound of formula IIa by condensing and cyclizing a bicyclic compound precursor compound of formula I with an alkyl halide according to reaction formula (2):

[0072]

[0073] Where R 5 , R 20 , mor, X, aqueous solvent system and base are defined elsewhere herein. 1-3 may optionally be replaced by one or more R as defined elsewhere herein 10 or R 15 The ring formed may be optionally substituted with one or more R as defined elsewhere herein. 5 , R 10 and / or R 15 Compared to known processes, appropriate selection of at least one process variable from the solvent system, phase transfer catalyst, equivalent ratio of two or more reactants and / or reaction temperature range, or appropriate selection of 2, 3 or all 4 of these process variables, provides an increased yield and / or purity of the compound of formula IIa, thereby enabling the elimination of one or more process steps and purification steps.

[0074] The present invention further provides an improved method for preparing a tricyclic PI3K inhibitor compound of formula IIIa from a compound of formula IIa according to reaction formula (3):

[0075]

[0076] Where R 5 , R 10 , R 15 , R 20 , mor, X and R 4 As defined elsewhere herein. Reaction (3) is generally carried out according to reaction (1).

[0077] As described in further detail below, the present invention further provides methods of treatment using the above tricyclic PI3K inhibitor compounds.

[0078] A.SUZUKI coupling

[0079] In the Suzuki coupling reaction of reaction formula (1) and reaction formula (3), a compound of formula II or a compound of formula IIa, a solvent system comprising water, an organic boron-R 4 , a base and less than 0.05 equivalents of a catalyst containing palladium per equivalent of a compound of Formula II or a compound of Formula IIa to form a reaction product mixture containing a compound of Formula III or a compound of Formula IIIa, its stereoisomers, its geometric isomers, its tautomers, or its pharmaceutically acceptable salts.

[0080] In reaction formula (1): X 1 S, O, N, NR a , CR 1 , C(R 1 )2 or -C(R 1 )2O-, where R 1 As further defined below; X 2 C, CR 2 or N, where R 2 As further defined below; X 3 C, CR 3 or N, where R 3 As further defined below; X 4 is a halogen; the dashed line represents an optional double bond; R 4 is an optionally substituted C6-C 20 Aryl, C2-C 20 Heterocyclic or C1-C 20 Heteroaryl; A is 2 and X 3 A fused 5-, 6- or 7-membered carbocyclyl or heterocyclyl ring, optionally substituted by one or more R 5 , R 10 and / or R 15 group substituted, where each R 5 , R 10 and R 15 are independently selected from H, halogen, oxo, hydroxy, nitro, amino, hydrocarbon or substituted hydrocarbon, and the two geminal R 5 , R 10 and / or R 15 The group is oxo, or two geminal R 5 , R 10 and / or R 15The group forms a 3, 4, 5, 6 or 7 membered carbocyclic or heterocyclic group, wherein the carbocyclic or heterocyclic group is optionally substituted; mor is a morpholine ring, which is optionally replaced by one or more R defined elsewhere herein 7 Group substitution.

[0081] In some aspects of the invention, X 1 N, NR a , CR 1 , C(R 1 )2 or -C(R 1 )2O-,X 3 C or CR 3 In other respects, X 1 and X 2 N, X 3 For C.

[0082] In some aspects of the invention, A is an optionally substituted 6-membered heterocyclic ring comprising at least one heteroatom selected from N and O. In some other aspects, A is an optionally substituted morpholine.

[0083] In some aspects of the invention, R 6 H, C1-C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12 Carbocyclic), -(C1-C 12 Alkylene)(-C2-C 20 Heterocyclic), -(C1-C 12 Alkylene)-C(=O)-(C2-C 20 Heterocyclic group), (C1-C 12 Alkylene)-(C6-C 20 Aryl) and -(C1-C 12 Alkylene)-(C1-C 20wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -(CH3)2OH, -CH2OCH3, -CN, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3) 2CONH2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and 1,1-dioxo-thiopyran-4-yl. In some aspects, R 6 H or C 1-4 In other aspects, R 6 It is H or methyl.

[0084] In some respects, R 1 , R 2 and R 3 independently selected from H, F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2, -CONH2, -NO 2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and l,l-dioxo-thiopyran-4-yl.

[0085] In reaction formula (3), each R 5 , R 10 and R 15 Independently selected from H, C 1-10 Hydrocarbyl or selected from C 1-5 A hydrocarbon group, wherein each hydrocarbon group is optionally substituted, two geminal R 5 , R 10and / or R 15 The groups together form an oxo group, and the two geminal R 5 , R 10 and / or R 15 The groups together form a 3-, 4-, 5-, 6- or 7-membered carbocyclic or heterocyclic group, wherein the carbocyclic or heterocyclic group is optionally substituted. 20 -O- or -NR 21 -, R 21 For R 5 defined.

[0086] In reaction formula (1) and (3), organoboron is generally suitable for realizing any material of required yield and / or purity disclosed herein. The example of organoboron is included in A.Lennox and G Lloyd-Jones, Selection of boronreagents for Suzuki-Miyaura coupling, Chem.Soc.Rev., 2014, 412-443. Non-limiting examples of organoboron include 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl), boric acid pinacol ester, pinacol borate, boric acid and organic trifluoroborate. In some specific aspects, the organoboron is 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl).

[0087] In some aspects of the present invention, in reaction formulas (1) and (3), R 4 Independently selected from C6-C 20 Aryl, C2-C 20 Heterocyclic and C1-C 20 R is a heteroaryl radical, each of which is optionally substituted with one or more radicals independently selected from the group consisting of F, Cl, Br, I, -CH, -CHCH, -CH(CH), -CHCH(CH), -CHCH, -CHCN, -CN, -CF, -CHOH, -COH, -CONH, CONH(CH), -CON(CH), -NO, -NH, -NHCH, -NHCOCH, -OH, -OCH, -OCHCH, -OCH(CH), -SH, -NHC(O)NHCH, -NHC(O)NHCHCH, -NHS(O)CH, -N(CH)C(O)OC(CH), -S(O)CH, benzyl, benzyloxy, morpholinyl, morpholinylmethyl, and 4-methylpiperazin-1-yl. In other aspects, R 4 is an optionally substituted C6 heteroaryl group containing one or two N heteroatoms, or is an optionally substituted pyrimidine. 4is phenyl substituted with one or more groups selected from the group consisting of F, Cl, Br, I, -CH, -CHCH, -CH(CH), -CN, -CF, -CHOH, -COH, -CONH, CONH(CH), -CON(CH), -NO, -NH, -NHCH, -NHCOCH, -OH, -OCH, -OCHCH, -OCH(CH), -SH, -NHC(O)NHCH, -NHC(O)NHCHCH, -NHS(O)CH, -N(CH)C(O)OC(CH), and -S(O)CH. 4 is selected from 1H-indazole, 1H-indole, 2-indolone, 1-(indolyl)ethanone, 1H-benzo[d][1,2,3]triazole, 1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine, 1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one, 1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine, 3H-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine In another aspect, R is an optionally substituted bicyclic heteroaryl radical of 1H-pyrazol-1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine, 2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline, isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one, 3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one, quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine and pyrido[3,2-b]pyrazine. In another aspect, R is an optionally substituted bicyclic heteroaryl radical of 1H-pyrazol-1H-pyrazol-1H-pyrazol-2(4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine, 2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline, isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one, 3,4-dihydroquinolin-2(1H)-one, quinazoline-2(1H)-one, quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine and pyrido[3,2-b]pyrazine. 4 R is an optionally substituted monocyclic heteroaryl selected from the group consisting of 2-furanyl, 3-furanyl, 2-imidazolyl, 4-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-pyrazinyl, 3-pyrazinyl, 4-pyrazinyl, 5-pyrazinyl, 2-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 5-tetrazolyl, 1-tetrazolyl, 2-tetrazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-triazolyl, and 1-triazolyl. In some aspects, R 4 is an optionally substituted monocyclic heteroaryl selected from pyridyl, pyrimidinyl and pyrazolyl. 4 is an optionally substituted monocyclic pyrimidinyl. 4 is 1H-imidazol-4-yl or 2-aminopyrimidinyl. In some specific aspects, R 4is an optionally substituted moiety R described below 4 cb.

[0088] R 4 Non-limiting examples of moieties include the following, each of which may be optionally substituted, and wherein the wavy line indicates the site of attachment:

[0089]

[0090]

[0091]

[0092]

[0093] In some respects, each R 5 , R 10 and R 15 Independently selected from C 1-12 Alkyl, C 2-8 Alkenyl, C 2-8 Alkynyl, -(C 1-12 Alkylene)-(C 3-12 Carbocyclic), -(C 1-12 Alkylene)-(C 2-20 Heterocyclic group), -(C 1-12 Alkylene)-C(O)-(C 2-20 Heterocyclic group), -(C 1-12 Alkylene)-(C 6-20 Aryl) and -(C 1-12 Alkylene)-(C 1-20 heteroaryl); or two geminal R 5 , R 10 and / or R 15The group forms a 3-, 4-, 5- or 6-membered carbocyclyl or heterocyclyl ring; wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CH2F, -CHF2, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -C The invention can be substituted with one or more of: ON(CH3)2, -C(CH3)2CONH2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NH-COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and 1,1-dioxo-thiopyran-4-yl.

[0094] In some respects, R 5 , R 10 and R 15 Independently for C 1-12 Alkyl, which is optionally selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CH2F, -CHF2, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -N O2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and 1,1-dioxo-thiopyran-4-yl are substituted with one or more groups. In some aspects, R 10 and R 15 is hydrogen, R 5 is methyl, which is optionally substituted by one or more groups as defined herein, and in particular may be substituted by one or more substituents selected from F, OH and =0.

[0095] In some other aspects, one or more R 5, R 10 and / or R 15 The groups are independently selected from H, F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CH2F, -CHF2, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CON H2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and l,l-dioxo-thiopyran-4-yl.

[0096] Non-limiting examples of mor include the following, where the wavy line indicates the site of attachment:

[0097]

[0098] In this regard, it should be noted that one or more carbon atoms in the ring shown above may optionally be independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, -CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH, -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C(CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2, -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -NHCH(CH3)2, -NHCH2CH2OH, -NHCH2CH2OCH3, -NHCOCH3, -NHCOCH2CH3, -NHCOCH2OH, -NHS(O)2CH3, -N(CH3)S(O)2CH3, =O, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(O)NHCH3, -NHC(O)NHCH2CH3, -S(O)CH3, -S(O)CH2CH3, -S(O)2CH3, -S(O)2NH2, -S(O)2NHCH3, -S(O)2N(CH3)2, and -CH2S(O)2CH3 7 In some aspects, mor is mor-a as described above.

[0099] It will be appreciated that reference to a specific residue X as disclosed herein 1 , X 2 , X 3 , A, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 10 , R 15 Each embodiment of mor can be combined with another residue X as disclosed herein 1 , X 2 , X 3 , A, R 1 , R 2 , R 3 , R 4 , R5 , R 6 , R 7 , R 10 , R 15 and any other implementation of mor.

[0100] The catalyst comprising palladium is generally any such catalyst suitable for achieving the yields and purities disclosed herein. In some aspects, the catalyst comprising palladium is selected from chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) (e.g., "Xphos PdG1", "Xphos PdG2", and "Xphos PdG3"); 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane ("PdCl2(dppf)·CH2Cl2"); bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) ("Pd(amphos)Cl2"); bis(di-tert-butylphenylphosphine)dichloropalladium(II); bis(di-tert-butylphenylphosphine)dichloropalladium(II) ("PdCl2[( t Bu2Ph)P]2”); PdCl2(PPh3)2, the structure is Chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (Xphos pD G2); Pd(t-Bu)3; Pd(PPh3)4; Pd(OAc) / PPh3; Cl2Pd[(Pet3)]2; Pd(DIPHOS)2; Cl2Pd(Bipy); [PdCl(Ph2PCH2PPh2)]2; Cl2Pd[P(o-tol)3]2; Pd2(dba)3 / P(o-tol)3; Pd2(dba) / P(furyl)3; Cl2Pd[P(furyl)3]2; Cl2Pd(PmePh2)2; Cl2Pd[P(4-F-Ph)3]2; Cl2Pd[P(C6F6)3]2; Cl2Pd[P(2-COOH-Ph)(Ph)2]2; Cl2Pd[P(4-COOH-Ph)(Ph)2]2; and encapsulated catalyst Pd EnCat TM 30 (palladium acetate, microencapsulated in a polyurethane matrix, containing 0.4 mmol / g Pd), PdEnCat TM TPP30 (palladium acetate and triphenylphosphine, microencapsulated in a polyurethane matrix, containing 0.4 mmol / g Pd and 0.3 mmol / g phosphorus), and Pd(II)EnCat TMBINAP30 (palladium acetate and BINAP, microencapsulated in a polyurea matrix, containing 0.4 mmol / g Pd, see, e.g., U.S. Patent Application Publication No. 2004 / 0254066, which is incorporated herein by reference). In some other aspects, the catalyst comprising palladium is selected from chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) and 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane, or chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II). In some aspects, the catalyst is a complex of chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) or 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane. In other aspects, the catalyst comprising palladium is chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II).

[0101] The equivalent ratio of the catalyst comprising palladium to the compound of formula II or IIa is generally about 0.003: 1 to about 0.05: 1, about 0.003: 1 to about 0.03: 1 or about 0.004: 1 to about 0.02: 1. In some specific aspects, the catalyst is a complex of chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) or 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane, and the equivalent ratio of the catalyst comprising palladium to the compound of formula II or IIa is about 0.005: 1 to about 0.04: 1, about 0.005: 1 to about 0.03: 1, about 0.01: 1 to about 0.03: 1, or about 0.02: 1. In some other specific aspects, the catalyst is chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II), and the equivalent ratio of the catalyst comprising palladium to the compound of formula II or IIa is from about 0.004:1 to about 0.015:1, from about 0.004:1 to about 0.01:1, from about 0.004:1 to about 0.007:1, or about 0.005:1.

[0102] The base can generally be selected from any base that is soluble in the solvent system and is suitable for achieving the desired yield and purity disclosed herein. In some aspects, the base is selected from K3PO4, Cs2CO3, K2CO3, KOAc, NaOAc, Na2CO3 and KOH. In some other aspects, the base is K3PO4. The equivalent ratio of the base to the compound of formula II or IIa is generally at least 1:1, and can be in the range of about 1:1 to about 3:1 or about 1.5:1 to about 2.5:1. In a specific aspect, the ratio can be about 2:1.

[0103] In any of the various aspects of the invention, the Suzuki coupling solvent system typically comprises about 5 v / v% of water, about 10 v / v% of water, about 15 v / v% of water, about 20 v / v% of water, about 25 v / v% of water or more, and in some aspects may fall within the range of about 5 v / v% to about 25 v / v% or about 10 v / v% to about 20 v / v%.

[0104] The solvent system further comprises at least one co-solvent generally selected from non-polar solvents, polar protic solvents and polar aprotic solvents. Suitable polar aprotic solvents include, but are not limited to, N-methyl pyrrolidone, methyl isobutyl ketone, methyl ethyl ketone, tetrahydrofuran ("THF"), dichloromethane, ethyl acetate, acetone, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide. Suitable polar protic solvents include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol and acetic acid. Suitable non-polar solvents include dioxane, toluene, hexane, cyclohexane and ether.

[0105] In some aspects, the solvent system comprises water and at least one polar aprotic solvent. In other aspects, the solvent system consists essentially of water and at least one polar aprotic solvent. In other aspects, the ratio of water to the at least one polar aprotic solvent is about 1:10 v / v to about 5:1 v / v, about 1:1 v / v to about 1:10 v / v or about 1:3 v / v to about 1:7 v / v. In other aspects, the solvent system consists essentially of water and at least one polar aprotic solvent.

[0106] In some aspects, the solvent system comprises water and THF.

[0107] The temperature of the reaction is typically less than 100° C., and in some aspects can be from about 40° C. to 100° C., from about 40° C. to about 90° C., from about 40° C. to about 80° C., from about 50° C. to about 80° C., or from about 55° C. to about 75° C. The time for the reaction to be complete is typically from about 4 hours to about 48 hours, from about 4 hours to about 36 hours, or from about 4 hours to about 24 hours.

[0108] Non-limiting examples of compounds of formula III and / or IIIa include the following, wherein R 1 , R 2 , R 3 and R 4 As defined elsewhere in this document:

[0109]

[0110] In any of the above compounds of formula III, ring A may be optionally replaced by one or more R 5 , R 10 and / or R 15 The substituted amine groups (not shown) may be independently selected from those options detailed elsewhere herein.

[0111] Further non-limiting examples of compounds of formula III and IIIa include the following, wherein R 4 and R 5 As defined elsewhere in this document:

[0112]

[0113]

[0114] Further non-limiting examples of compounds of formula III and / or IIIa include:

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] In some specific aspects, the compounds of formula III and IIIa are

[0126]

[0127] In some aspects, it has been found that precipitation or crystallization of formula III and IIIa (collectively referred to as formula III) from the reaction mixture can be induced by adding water, methanol, ethanol, n-propanol or isopropanol to the reaction mixture to form formula III crystals or precipitates, which can be cleanly separated using solid-liquid separation techniques known in the art, for example. In some specific aspects, the polar protic solvent is water. In these aspects, the polar protic solvent is added to the reaction mixture to a final concentration of at least 25v / v%, at least 40v / v%, at least 50v / v%, at least 60v / v%, or at least 70v / v%. In these aspects, the volume ratio of the solvent system to the polar protic solvent added to the reaction product mixture in the reaction mixture is about 1:5v / v to about 5:1v / v, about 1:3v / v to about 3:1v / v, about 1:2v / v to about 2:1v / v, about 1:1.5v / v to about 1.5:1v / v, or about 1:1v / v. In any aspect of various aspects, polar protic solvent (such as water) can be added to the reaction mixture at the reaction temperature or at a lower temperature. After addition, the reaction mixture-polar protic solvent mixture can be optionally maintained at the reaction temperature or at a temperature of about 10°C to about 20°C lower than the reaction temperature for about 0.5 hours to about 8 hours or about 1 hour to about 4 hours. The mixture can then be further cooled to about 0°C to about 10°C and maintained for about 0.5 hours to about 8 hours or about 1 hour to about 4 hours to complete the crystallization of formula III. In some aspects, in the first step, the temperature can be reduced to about 10°C to about 30°C and maintained for about 0.5 hours to about 8 hours or about 1 hour to about 4 hours, and then cooled to 0°C to about 10°C in the second step to maintain about 0.5 hours to about 8 hours or about 1 hour to about 4 hours. In some other aspects, formula III seeds can be added to induce crystallization. Formula III solids can be separated by solid-liquid separation techniques known in the art such as filtration and centrifugation. Optionally, the collected formula III solids can be washed with other polar aprotic solvents.

[0128] Formula II, IIa, III and IIIa can be separated from the reaction mixture and purified by various methods known in the art. The reaction mixture of formula III may contain some amount of by-products (e.g., impurities 1 to 5 disclosed in the embodiments), unreacted formula II and palladium. In some aspects of the invention, the reaction mixture of formula II, IIa, III and IIIa and / or the separated compound can be purified by one or more purification methods. The desired product of each step or a series of steps is separated and / or purified (hereinafter separated) to the desired purity by the techniques commonly used in the art. Formula II, IIa and III can be separated from the reaction mixture and / or purified by various methods, and the purification method includes: (i) precipitation or crystallization, such as by evaporation, cooling and / or adding anti-solvent, optionally further combined with the addition of seed crystals; (ii) extraction, such as multiphase extraction; (iii) evaporation or distillation to form a solid residue comprising formula II, IIa or III; (iv) ultrafiltration; (v) chromatography; (vi) reverse phase HPLC; (vii) sublimation; (viii) chelation; and / or (ix) combinations thereof. Chromatography can involve many methods including, for example: reversed and normal phase, size exclusion, ion exchange, high, medium and low pressure liquid chromatography methods and apparatus, small scale analytical, simulated moving bed (SMB) and preparative thin or thick layer chromatography, as well as small scale thin layer and flash chromatography techniques.

[0129] In the chelation method, formula III is mixed with a selected reagent that binds to or separates the desired product, unreacted raw materials, reaction byproducts, etc. These reagents include adsorbents or adsorbents, such as activated carbon, molecular sieves, ion exchange media, etc. Alternatively, the reagent can be an acid in the case of an alkaline substance, a base in the case of an acidic substance, 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. In a chelation purification method, in a solvent system in which formula III is soluble, formula III is treated to remove residual palladium by mixing formula III with a metal scavenger. The temperature of the mixture is increased to dissolve the compound of formula III, the solution is maintained for a period of time, and the solution is filtered to remove the chelated palladium. Formula III can then be crystallized from the filtered solution as described elsewhere herein. In some aspects: (i) the solvent system comprises, or consists essentially of, water and acetic acid, wherein the volume ratio of acetic acid to water is from about 1:1 to about 10:1, from about 1:1 to about 5:1, or from about 1:1 to about 3:1, or about 3:1; (ii) the metal scavenger is silica-thiol; and (iii) the temperature of dissolution is from about 80°C to about 100°C, the seed crystals are combined with the filtrate at a temperature of from about 70°C to about 80°C, and the temperature of crystallization is from about 0°C to about 10°C.

[0130] In any aspect of the various aspects of the invention, the yield of the compound of formula III is at least 75%, at least 80%, at least 85% or at least 90%. The purity of the compound of formula III is at least 97%, at least 97.5% or at least 98% (area % determined by HPLC).

[0131] In one or more of the above reactions, the solvent may suitably be water and THF, the ratio of water to THF being about 1:5 w / w, the organoboron-R 4 It may be suitably 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine, the catalyst may be suitably Xphos (e.g., Xphos PdG1, Xphos PdG2, and Xphos PdG3), the base may be suitably K3PO4, and the temperature of the reaction may be suitably about 55° C. to about 75° C. About 0.8 to about 1.2 volumes of water based on the volume of the reaction mixture may be suitably mixed with the reaction product mixture, and the mixture may be suitably cooled to about 10° C. to about 30° C. to form a crystallized or precipitated reaction product.

[0132] The compounds of formula III of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. It is intended that 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.

[0133] In addition, the present invention includes all geometric and positional isomers. For example, if the compound of formula III 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. Single positional isomers and mixtures of positional isomers are also within the scope of the present invention.

[0134] In the structures shown herein, where the stereochemistry of any particular chiral atom is not specified, all stereoisomers are contemplated and included as compounds of the invention. Where stereochemistry is specified by a solid wedge or dashed line representing a specific configuration, that stereoisomer is so specified and defined.

[0135] By methods well known to those skilled in the art, for example, by chromatography and / or fractional crystallization, diastereomeric mixtures can be separated into their respective diastereomers based on their physicochemical differences. Enantiomers can be converted into diastereomeric mixtures by reacting the enantiomeric mixture with an appropriate optically active compound (e.g., a chiral auxiliary such as a chiral alcohol or Mosher's acyl chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. Moreover, some compounds of the present invention may be atropisomers (e.g., substituted biaryls) and are considered to be part of the present invention. Enantiomers may also be separated by using a chiral HPLC column.

[0136] A single stereoisomer, such as an enantiomer, substantially free of its stereoisomers can be obtained by resolving the racemic mixture using methods such as the formation of diastereomers using optically active resolving agents (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 and isolated 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 the diastereomers and converting to pure stereoisomers and (3) directly isolating the substantially pure or enriched stereoisomers under chiral conditions. See: "Drug Stereochemistry, Analytical Methods and Pharmacology," Irving W. Wainer, Ed., Marcel Dekker, Inc., New York (1993).

[0137] In the above method (1), diastereomeric salts can be formed by the reaction of an optically pure chiral base (such as strychnine, quinine, ephedrine, strychnine, α-methyl-β-phenylethylamine (amphetamine) etc.) with an asymmetric compound with 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. 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.

[0138] Alternatively, by the above 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 optically pure chiral derivatizing agent such as a methyl derivative, followed by separation of the diastereomers and hydrolysis to produce pure or enriched enantiomers. The method for determining optical purity includes preparing a chiral ester, such as a methyl ester, of the racemic mixture in the presence of a base or Mosher ester (α-methoxy-α-(trifluoromethyl)phenyl acetate), for example, using (-)methyl chloroformate (Jacob III. J. Org. Chem. (1982) 47: 4165), and analyzing 1 The presence of two atropisomeric enantiomers or diastereomers in the H NMR spectrum. Stable diastereomers of atropisomeric compounds can be separated and isolated by normal and reverse phase chromatography according to the method for separating atropisomeric naphthyl-isoquinolines (WO1996 / 015111). By the above 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.

[0139] 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.

[0140] The compounds of the present invention may also exist in different tautomeric forms, and all of these forms are included within the scope of the present invention. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also referred to as protoisomeric tautomers) include interconversions through proton migration (e.g., keto-enol and imine-enamine isomerizations). Valence tautomers include interconversions through reorganization of some bonding electrons.

[0141] The present invention also includes isotopically labeled compounds of the present invention, which are identical to those described herein, although one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. All isotopes of any particular atom or element specified are contemplated within the scope of the present compounds and their uses. Exemplary isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, and iodine, such as 2 H(D), 3 H. 11 C, 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 32 P. 33 P. 35 S. 18 F. 36 C1. 123 I and 125 I. Certain isotopically labeled compounds of the present invention (e.g., with and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 ( 14 C) isotopes are useful because they are easy to prepare and detect. In addition, heavier isotopes such as deuterium (i.e. 2 H) substitution may offer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and thus may be preferred in some circumstances. Positron emitting isotopes such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy.Isotopically labeled compounds of the invention can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0142] B. Ringing

[0143] In the annulation (ring formation) reaction of reaction formula (2), each R 5 , R 10 and R 15 Independently selected from H, C1-C 10 or a hydrocarbon group selected from C1-C5, wherein each hydrocarbon group is optionally substituted, and the two geminal R 5 , R 10 and / or R15 The groups together are oxo, or together form a 3, 4, 5, 6 or 7 membered carbocyclyl or heterocyclyl, wherein the carbocyclyl or heterocyclyl is optionally substituted. 20 -OH or -NHR 21 ; R 21 For R 5 defined; and in the ring formed in Formula IIa, R 20 -O- or -NR 21 -. Mor is an optionally substituted morpholine ring. X is a halogen (e.g., Br, I or Cl). In some aspects of the present invention, the compound of formula IIa is formed by condensing a bicyclic compound precursor compound of formula I with a nucleophilic reagent such as an organic halide derived from 1,2-ethylene glycol and an epoxide into a ring. In any of various such reactions, a reaction mixture comprising a compound of formula I, a haloalkyl, a solvent system, a phase transfer catalyst and a base is formed. The reaction mixture is reacted at high temperature to form a reaction product mixture comprising a compound of formula IIa, a stereoisomer thereof, a geometric isomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof, and then the compound of formula IIa is separated from the reaction product mixture. In some aspects of the present invention, the compound of formula IIa can be purified in one or more purification steps.

[0144] In some aspects of the invention, the solvent system comprises at least one polar protic solvent. The example of such solvent includes but is not limited to water, methanol, ethanol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, isobutyl alcohol, the tert-butyl alcohol and acetic acid (" HOAc "). In these aspects, the solvent system comprises at least 5v / v% water, at least 25v / v% water, at least 50v / v% water, at least 75v / v% water, at least 90v / v% water, or is basically composed of water.

[0145] In some aspects of the invention, the solvent system comprises at least one polar aprotic solvent. Examples of such solvents include, but are not limited to, dichloromethane, tetrahydrofuran ("THF"), ethyl acetate, acetone, N,N-dimethylformamide ("DMF"), acetonitrile ("MeCN"), dimethyl sulfoxide, N-methylpyrrolidone ("NMP"), methyl isobutyl ketone, and methyl ethyl ketone. In these aspects, the solvent comprises DMF or primarily comprises DMF.

[0146] In other aspects of the invention, the solvent system comprises at least one polar protic solvent and at least one polar aprotic solvent, and the ratio of total polar protic solvent to total polar aprotic solvent is from about 1:10 to about 10:1, from about 1:5 to about 5:1, from about 1:10 to about 1:1, from about 1:5 to about 1:1, from about 10:0 to about 1:1, or from about 5:1 to about 1:1. In some such aspects, the solvent system comprises water and THF or water and DMF.

[0147] The total solvent loading is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15 or about 20 and ranges thereof, such as about 2 to about 20, about 2 to about 15, about 2 to about 10, about 5 to about 15, about 2 to about 7, about 3 to about 7, or about 3 to about 5, by volume based on Formula I.

[0148] The nucleophile that forms annulation by condensation reaction can be a nucleophile derived from (i) 1,2-ethylene glycol or its mesylate, toluenesulfonate or its trifluoromethanesulfonate derivative or (ii) epoxide. In some aspects, the nucleophile is a halide or pseudohalide, such as an organic halide. In some aspects, the organic halide is a haloalkyl. In some specific aspects, the halide is a C1-C3 alkyl halogenated by Cl, Br or I. In some aspects, the organic halide is optionally substituted. In some aspects, the alkyl is disubstituted by halogen at the end. An example of such an organic halide is 1,2-dibromoethane. The equivalent ratio of the organic halide to the compound of Formula I is greater than 2: 1, about 2.5: 1, about 3: 1, about 3.5: 1, about 4: 1, about 4.5: 1 or about 5: 1 and ranges thereof, such as 2: 1 to about 5: 1, 2: 1 to about 4: 1, about 2.5: 1 to about 4: 1, about 2.5: 1 to about 3.5: 1 or about 3: 1. The organic halide can be combined with the reaction mixture in one or more addition steps.

[0149] In some aspects of the present invention, the reaction mixture comprises a phase transfer catalyst. In some such aspects, the phase transfer catalyst can be selected from quaternary ammonium salts and quaternary phosphonium salts. The example of this phase transfer catalyst includes but is not limited to tetra-n-butylammonium bromide (" TBAB "), benzyltrimethylammonium chloride, benzyltriethylammonium chloride, methyltrioctylammonium chloride, methyltributylammonium chloride and methyltrioctylammonium chloride. In particular aspects, the phase transfer catalyst is TBAB. The equivalent ratio of the phase transfer catalyst to the compound of formula I is about 0.1: 1, about 0.2: 1, about 0.3: 1, about 0.4: 1, about 0.5: 1 or about 0.6: 1 and its scope, such as about 0.1: 1 to about 0.6: 1, about 0.2: 1 to about 0.5: 1, about 0.2: 1 to about 0.4: 1, or about 0.3: 1.

[0150] In any of the various cyclization aspects of the present invention, the reaction mixture comprises a base. Examples of suitable bases include, but are not limited to, KOH, NaOH, K3PO4, K2CO3, NaHCO3 and Cs2CO3. In some aspects, the base is KOH, NaOH or K3PO4, and in other aspects, the base is KOH. The equivalent ratio of the base to the compound of formula I is greater than 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1 or about 5:1 and its range, for example, 2:1 to about 5:1, 2:1 to about 4:1, about 2.5:1 to about 4:1, about 2.5:1 to about 4:1, about 2.5:1 to about 3.5:1, or about 3:1. In some aspects, the base and the organic halide are present in about equimolar amounts. The base can be combined with the reaction mixture in one or more addition steps.

[0151] The temperature of the reaction is suitably about 40°C to about 90°C, about 40°C to about 70°C, about 40°C to about 60°C, or about 50°C. Based on experimental evidence to date, it has been found that the purity distribution of Formula IIa varies inversely with the reaction temperature, so that a lower reaction temperature provides an improved purity distribution measured by HPLC compared to a higher reaction temperature. The reaction time required to achieve sufficient conversion varies with the quantitative and qualitative characteristics of the reaction mixture and the reaction temperature, and is typically about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours or about 28 hours and ranges thereof, such as about 4 hours to about 28 hours, about 4 hours to about 20 hours or about 4 hours to about 12 hours.

[0152] The compound of formula IIa can be separated from the reaction mixture by various methods including precipitation, crystallization and extraction. In the example of the first extraction method, the reaction mixture can be cooled, for example, to room temperature, diluted with water (e.g., 5 to 15 volumes of water) and maintained under stirring (e.g., 5 to 15 hours). Formula IIa can then be extracted from the diluted reaction mixture into an organic solvent (e.g., 1 to 3 volumes of a suitable solvent, such as a polar aprotic solvent, such as ethyl acetate), and then the organic solvent is separated from the reaction mixture by phase separation. Multiple such extractions are within the scope of the first method. The organic phase can be appropriately washed with NaCl and concentrated (e.g., by distillation) to obtain a crude solid of formula IIa. Crude formula IIa can be optionally crystallized, for example, by dissolving in a suitable solvent at an elevated temperature (e.g., in isopropanol at 65°C), followed by crystallization by cooling. In the example of the second extraction method, water (e.g., 5 to 10 volumes) and an organic solvent (e.g., 2 to 6 volumes of ethyl acetate) can be added to the cooled reaction mixture, and the organic layer is then removed by phase separation. In some aspects, additional water (e.g., 2 to 6 volumes) and an organic solvent (e.g., 2 to 6 volumes of ethyl acetate) can be added to the aqueous phase, and the organic layer is then removed by phase separation. The combined organic layer can be concentrated (e.g., by distillation) to produce a crude solid of formula IIa. Formula IIa can optionally be mixed with a solvent (e.g., isopropanol) and subsequently subjected to a second concentration step. Crude formula IIa can optionally be crystallized, for example, by being dissolved in a suitable solvent (e.g., in 65°C isopropanol) at an elevated temperature, followed by crystallization by cooling. In aspects of the present invention in which the reaction mixture solvent system comprises water, alcohol (e.g., ethanol) can be added to the reaction product mixture and the temperature of the mixture can be reduced to induce crystallization of the compound of formula IIa. In some optional aspects, the compound seed of formula IIa can be added to the mixture. In these aspects, the volume ratio of the reaction mixture solvent system to the alcohol is from about 1:5 v / v to about 5:1 v / v, from about 1:3 v / v to about 3:1 v / v, from about 1:2 v / v to about 2:1 v / v, from about 1:1 v / v to about 1:2 v / v, or about 1:1.3 v / v. The crystallized compound of Formula IIa can be isolated by solid-liquid separation techniques known in the art.

[0153] In any of the various aspects, the yield of the compound of Formula IIa is at least 65%, at least 70% or at least 75%. The purity of the compound of Formula IIa is at least 97%, at least 97.5%, at least 98%, at least 98.5% or at least 99% (area % determined by HPLC).

[0154] Non-limiting examples of reactions within the scope of the present invention include:

[0155]

[0156]

[0157] In one or more of the above reactions, the solvent may suitably be water, the base may suitably be KOH, the phase transfer catalyst may suitably be tetra-n-butylammonium bromide, and the temperature of the reaction may suitably be about 50° C. Ethanol may suitably be mixed with the reaction product mixture, and the mixture may suitably be cooled to about 0° C. to about 10° C. in the presence of reaction product seed crystals to form a crystalline reaction product.

[0158] C. Exemplary Embodiments

[0159] In a first exemplary embodiment of the present invention, the compound of formula IIIat (GDC-0084):

[0160]

[0161] or a salt thereof prepared by a method comprising reacting a compound of formula IIa (wherein X is chlorine or bromine) in a solvent system containing at least 5 v / v% water, at a reaction temperature of less than 100° C., in the presence of a base, in the presence of less than 0.05 equivalents of a Suzuki coupling palladium catalyst per equivalent of the compound of formula IIa:

[0162] or a salt thereof is contacted with an organoboron-pyrimidin-2-amine.

[0163] In one aspect of the first exemplary embodiment, the solvent system comprises water and tetrahydrofuran, wherein the ratio of water to tetrahydrofuran is from about 1:3 v / v to about 1:7 v / v, or about 1:5 v / v. In some other aspects of the first exemplary embodiment, the organoboron-pyrimidine-2-amine is 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine-2-amine. In other aspects of the first exemplary embodiment, the base is K3PO4, and the equivalent ratio of the base to the compound of formula IIa is from about 1:1 to about 3:1, or about 2:1. In other aspects of the first embodiment, the catalyst is chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II), and the equivalent ratio of the catalyst comprising palladium to the compound of formula IIa is from about 0.004:1 to about 0.007:1, or about 0.005:1. In other aspects of the first embodiment, the catalyst is a complex of 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane, and the equivalent ratio of the catalyst comprising palladium to the compound of formula IIa is from about 0.01:1 to about 0.03:1, or about 0.02:1. In other aspects of the first embodiment, the reaction temperature is from about 55°C to about 75°C. In any of the various aspects of the first embodiment, water is added to the reaction product mixture to form a mixture containing greater than 25 v / v% water to form a precipitated or crystalline compound of formula IIIa, which can be separated from the reaction product mixture. In some such aspects, the volume ratio of the solvent system to the added water is about 1:1.5 v / v to about 1.5:1 v / v, or about 1:1 v / v.

[0164] In a second exemplary embodiment of the present invention, the compound of formula IIa

[0165]

[0166] or a salt thereof by the following process, the process comprising: (a) forming a reaction mixture comprising a solvent system containing at least 5 v / v% water, 1,2-dibromoethane, a phase transfer catalyst, a base and a compound of formula I:

[0167] or a salt thereof, wherein X is bromine, chloride or iodine; and (b) reacting the reaction mixture to form a reaction product mixture comprising a compound of Formula IIa.

[0168] In one aspect of the second exemplary embodiment, the solvent system comprises at least 90v / v% of water or consists essentially of water. In some other aspects of the second exemplary embodiment, the base is KOH and the phase transfer catalyst is tetra-n-butylammonium bromide. In other aspects, the molar ratio of 1,2-dibromoethane to the compound of formula I is from about 2:1 to about 4:1, or about 3:1, and the molar ratio of 1,2-dibromoethane to the base is about 1:1. In other aspects, the reaction temperature is from about 40°C to about 60°C, for example, about 50°C. In other aspects, ethanol is mixed with the reaction product mixture, and the mixture is then cooled to form a crystalline compound of formula IIa, wherein the volume ratio of the solvent system to ethanol is from about 1:1v / v to about 1:2v / v, or about 1:1.3v / v%. In other aspects, the compound seed of formula IIa is combined with the ethanol-reaction product mixture.

[0169] D. Treatment methods

[0170] The compound of formula III of the present invention can be used by any approach suitable for the disease to be treated. Suitable approaches include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intrathecal and epidural), percutaneous, rectal, nasal, local (including oral and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal. For local immunosuppressive therapy, the compound can be used by intralesional administration, including perfusion before transplantation or otherwise contacting the transplant with an inhibitor. It is understood that preferred approaches can be changed according to, for example, the condition of the recipient. When the compound is administered orally, 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 into the injection form of a unit dose as described in detail below with a pharmaceutically acceptable parenteral carrier.

[0171] In one embodiment, the composition comprising the compound of Formula III or a salt thereof is formulated into a solid dosage form for oral administration. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In certain embodiments, a solid oral dosage form comprising a compound of Formula III or a salt thereof further comprises (i) an inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and (ii) a filler or extender, such as starch, lactose, sucrose, glucose, mannitol, or silicic acid; (iii) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose, or acacia, (iv) a humectant, such as glycerol, (v) a disintegrant, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, or sodium carbonate, (vi) a solution retarding agent, such as paraffin, (vii) an absorption promoter, such as a quaternary ammonium salt, (viii) a wetting agent, such as cetyl alcohol or glyceryl monostearate, (ix) an absorbent, such as kaolin or bentonite, and (x) a lubricant, such as one or more of talc, calcium stearate, magnesium stearate, polyethylene glycol, or sodium lauryl sulfate. In some embodiments, the solid oral dosage form is formulated into a capsule, tablet or pill. In some embodiments, the solid oral dosage form also includes a buffer. In some embodiments, this composition for solid oral dosage form can be formulated into a filler in a soft and hard filled gelatin capsule, and the capsule includes one or more excipients such as lactose or milk sugar, polyethylene glycol, etc.

[0172] In some embodiments, the tablet, dragee, capsule, pill and granule of the composition comprising the compound of formula III or its salt optionally include coating or shell, such as enteric coating.They may optionally include covering agent, and may also be such composition, which only releases active ingredient in or preferentially releases active ingredient in a certain part of intestinal tract, optionally releases active ingredient in a delayed manner.The example of embedded composition includes polymeric substance and wax, which may also be used as filler in soft and hard filled gelatin capsules using such excipient as lactose (lactose) or lactose (milk sugar) and high molecular weight polyethylene glycol etc.

[0173] In another embodiment, the composition comprises a microencapsulated compound of Formula III or a salt thereof, optionally together with one or more excipients.

[0174] In another embodiment, the composition comprises a liquid dosage form comprising a compound of formula III or a salt thereof for oral administration, optionally further comprising one or more pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In certain embodiments, the liquid dosage form optionally further comprises one or more inert diluents such as water or other solvents, solubilizers, and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol or fatty acid esters of dehydrated sorbitan, and mixtures thereof. In certain embodiments, the liquid oral composition optionally further comprises one or more adjuvants, such as wetting agents, suspending agents, sweeteners, flavoring agents and aromatics.

[0175] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among acceptable carriers and solvents, water, Ringer's solution, USP and isotonic sodium chloride solution can be used. In addition, sterile fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used to prepare injections.

[0176] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0177] In order to prolong the effect of the compound of formula III, it is usually necessary to slow down the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the compound depends on its dissolution rate, which in turn depends on the crystal size and crystalline form. Alternatively, the delayed absorption of the compound form for parenteral administration is accomplished by dissolving or suspending the compound in an oily vehicle. Injectable reservoir forms are prepared by forming a microcapsule matrix of the compound in a biodegradable polymer such as polylactide-polyglycolide. According to the ratio of the compound to the polymer and the properties of the specific polymer used, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir injectable preparations can also be prepared by embedding the compound in a liposome or microemulsion compatible with body tissues.

[0178] In certain embodiments, compositions for rectal or vaginal administration are formulated as suppositories, which can be prepared by mixing a compound of Formula III or a salt thereof with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol, or a suppository wax, for example, one that is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the compound of Formula III.

[0179] The exemplary dosage forms for topical or transdermal administration of the compound of formula III include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The compound of formula III or its salt is mixed with a pharmaceutically acceptable carrier and an optional preservative or buffer under aseptic conditions. Other examples of formulations include ophthalmic preparations, ear drops, eye drops, transdermal patches. Transdermal dosage forms can be prepared by dissolving or dispersing the compound of formula III or its salt in a medium such as ethanol or dimethyl sulfoxide. Absorption enhancers can also be used to increase the flux of the compound through the skin. Rate can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0180] Nasal aerosol or inhalation formulations of the compound of formula III or its salt can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and / or other conventional solubilizing or dispersing agents.

[0181] In certain embodiments, the pharmaceutical composition may be administered with or without food. In certain embodiments, the pharmaceutically acceptable composition is administered without food. In certain embodiments, the pharmaceutically acceptable composition of the present invention is administered with food.

[0182] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of the compound of formula III or its salt provided in the composition also depends on the specific compound in the composition.

[0183] In one embodiment, the therapeutically effective amount of the compound of the invention administered parenterally per dose is in the range of about 0.01-100 mg / kg per day, or about 0.1-20 mg / kg of patient body weight, with the usual initial range of the compound used being about 0.1 to 15 mg / kg / day, about 0.1 to 10 mg / kg / day, about 0.1 to 5 mg / kg / day, about 0.1 to 3 mg / kg / day, about 0.3 to 1.5 mg / kg / day or about 0.4 to 1 mg / kg / day. In another embodiment, oral unit dosage forms such as tablets and capsules contain about 5 to about 100 mg, about 10 to 75 mg, about 25 to 75 mg, or about 25 to 50 mg of the compound of the invention.

[0184] Exemplary tablet oral dosage forms include about 2 mg, about 5 mg, about 25 mg, about 50 mg, about 100 mg, about 250 mg or about 500 mg of a compound of formula III or a salt thereof, and also include about 5-30 mg of anhydrous lactose, about 5-40 mg of cross-linked sodium carboxymethyl cellulose, about 5-30 mg of polyvinyl pyrrolidone (PVP) K30 and about 1-10 mg of magnesium stearate. The method of preparing the tablets includes mixing the powder ingredients together and further mixing with a PVP solution. The resulting composition can be dried, granulated, mixed with magnesium stearate and compressed into tablet form using conventional equipment. Examples of aerosol formulations can be prepared by dissolving about 2-500 mg of a compound of formula III or a salt thereof in a suitable buffer solution (e.g., phosphate buffer) and adding a tonicity agent (e.g., a salt such as sodium chloride, if desired). The solution can be filtered, for example, using a 0.2 micron filter to remove impurities and contaminants.

[0185] The dosage for treating human patients can be about 10mg to about 1000mg, about 10mg to about 500mg, about 10mg to about 100mg, about 25mg to about 100mg, or about 25mg to about 75mg of the compound of formula III. Typical dosage can be about 100mg to about 300mg of the compound. According to pharmacokinetic and pharmacodynamic properties, including absorption, distribution, metabolism and excretion of a particular compound, the dosage can be once a day (QID or QD), twice a day (BID) or more frequently administered. In addition, toxic factors can affect dosage and dosage regimen. When administered orally, pills, capsules or tablets can be taken in daily or not too frequently in a specific time period. The scheme can repeat multiple treatment cycles.

[0186] In some embodiments, a total of about 0.2 mg / kg / day to about 1.5 mg / kg / day, about 0.3 mg / kg / day to about 1 mg / kg / day, or about 0.4 mg / kg / day to about 0.75 mg / kg / day of a compound of Formula III is administered once a day or twice a day. In some such embodiments, under such a dosing regimen, a single dose on the first day of the dosage cycle obtained the following pharmacokinetic results. 1 / 2 (h) is from about 10 to about 24 hours, from about 12 to about 22 hours, or from about 15 to about 20 hours. max (h) is from about 1 to about 8 hours, from about 2 to about 6 hours, from about 2 to about 4 hours, or from about 2 to about 3 hours. max (μM) is about 0.01 to about 0.5 μM, about 0.05 to about 0.4 μM or about 0.1 to about 0.3 μM. AUC inf (μM*hr) is about 0.2 to about 10 μM*hr, about 0.5 to about 10 μM*hr, about 1 to about 8 μM*hr, or about 2 to about 6 μM*hr. AUC 0-24 (μM*hr) is about 0.1 to about 10 μM*hr, about 0.5 to about 5 μM*hr, about 1 to about 5 μM*hr, or about 2 to about 4 μM*hr. In some other such embodiments, the following pharmacokinetic results were obtained after the 15th day of a single daily dose regimen of 2 mg to 30 mg or after the 8th day of a single daily dose regimen of 45 mg to 65 mg. max (h) is from about 1 to about 5 hours, from about 1 to about 3 hours, or from about 2 to about 4 hours. max (μM) is about 0.03 to about 1 μM, about 0.1 to about 1 μM, about 0.3 to about 0.8 μM, or about 0.3 to about 0.6 μM. min (μM) is about 0.005 to about 0.5 μM, about 0.01 to about 0.4 μM, about 0.05 to about 0.3 μM, or about 0.1 to about 0.3 μM. AUC 0-24 (μM*hr) is about 0.1 to about 15 μM*hr, about 0.5 to about 15 μM*hr, about 3 to about 15 μM*hr, or about 5 to about 10 μM*hr. The accumulation rate is about 1 to about 4 or about 1.5 to about 3. As used herein, T 1 / 2 T refers to the terminal half-life; max Refers to the time to reach maximum plasma concentration; C max Refers to the maximum observed plasma concentration; AUC inf Refers to the area under the concentration-time curve from time 0 to infinity; AUC 0-24 Refers to the area under the concentration-time curve from time 0 to 24 hours; C min refers to the minimum concentration; the accumulation rate refers to AUC0-24小时多次剂量 / AUC 0-24小时单剂量 In some specific embodiments, the compound of Formula III is GDC-0084 (Formula IIIat).

[0187] The compounds of formula III can be used to treat disorders of the brain and central nervous system that require crossing the blood-brain barrier. Certain compounds of formula III, such as the compound formula IIIat (GDC-0084) disclosed elsewhere herein, have favorable permeabilizing properties across the blood-brain barrier for delivery to the brain. Brain disorders that can be effectively treated with compounds of formula III include metastatic and primary brain tumors, such as gliomas (glioblastoma multiforme) and melanomas.

[0188] The compound of formula III or its salt can be used alone or in combination with other therapeutic agents. For example, the second agent of the pharmaceutical combination formulation or dosing regimen can have complementary activity to the compound of formula III so that they do not adversely affect each other. The compounds can be administered together or separately in a single pharmaceutical composition. In one embodiment, the compound or pharmaceutically acceptable salt can be co-administered with a cytotoxic agent to treat proliferative diseases and cancer.

[0189] The term "co-administration" refers to the simultaneous administration of a compound of formula III or a salt thereof and other active pharmaceutical ingredients including cytotoxic agents or any manner of separate sequential administration. If the administration is not simultaneous, the compounds are administered at a time close to each other. In addition, it does not matter whether the compounds are administered in the same dosage form, for example, one compound can be administered topically and the other compound can be administered orally.

[0190] These additional agents may be administered separately from the compounds of the present invention containing compositions as part of a multiple dosing regimen. Alternatively, those agents may be part of a single dosage form, mixed with the compounds of the present invention in a single composition. If administered as part of a multiple dosing regimen, the two active agents may be delivered simultaneously, sequentially, or within a time interval of generally 5 hours from each other.

[0191] As used herein, the terms "combination" (noun), "combination" (verb) and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the present invention. For example, a compound of the present invention can be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms or together in a single unit dosage form. Therefore, the present invention provides a single unit dosage form comprising a compound of Formula III, an additional therapeutic agent and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0192] The amount of the compounds of the invention and the additional therapeutic agent (in those compositions comprising additional therapeutic agents as described above) that can be combined with carrier materials to produce a single dosage form varies depending on the host being treated and the specific mode of administration. In certain embodiments, the compositions of the invention are formulated so that a dosage of the invention of 0.01-100 mg / kg body weight / day can be administered.

[0193] In general, any agent active against the disease or condition being treated may be co-administered. Examples of such agents can be found in Cancer Principles and Practice of Oncology by VT Devita and S. Hellman (editores), 6 th edition (February 15, 2001), Lippincott Williams & Wilkins Publishers. One of ordinary skill in the art will be able to discern which combinations of agents are useful based on the particular characteristics of the drugs and diseases involved.

[0194] In one embodiment, the method of treatment comprises co-administering a compound of Formula III or a pharmaceutically acceptable salt thereof and at least one cytotoxic agent. As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu), chemotherapeutic agents, biological inhibitors, enzymes and fragments thereof such as nucleases, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. In some embodiments, the PI3 kinase inhibitor of the present invention is administered to a patient with an additional therapeutic agent selected from chemotherapeutic agents, anti-angiogenic therapeutic agents, anti-inflammatory agents, immunomodulators, neurotropic factors, cardiovascular disease therapeutic agents, liver disease therapeutic agents, antiviral agents, blood disease therapeutic agents, diabetes therapeutic agents and immunodeficiency disease therapeutic agents. In some such embodiments, the additional therapeutic agent is bevacizumab. In some other such embodiments, the additional therapeutic agent is temozolomide.

[0195] Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, LDH-A inhibitors; fatty acid biosynthesis inhibitors; cell cycle signaling inhibitors; HDAC inhibitors, proteasome inhibitors; and cancer metabolism inhibitors.

[0196] "Chemotherapeutic agents" include chemical compounds used to treat cancer. Examples of chemotherapeutic agents include erlotinib ( Genentech / OSI Pharm.), bortezomib ( Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant ( AstraZeneca), sunitib ( Pfizer / Sugen), letrozole ( Novartis), imatinib mesylate ( Novartis、finasunate( Novartis), oxaliplatin ( Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, Wyeth), lapatinib ( GSK572016, Glaxo Smith Kline), Rhonafarmib (SCH 66336), Sorafenib ( Bayer Labs), gefitinib ( AstraZeneca), AG1478, alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, indolesulfan and azithromycin; aziridines such as chroman, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); statins; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenal corticosteroids (including prednisone and prednisolone); cyproterone; 5α-reductase inhibitors including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mosinostat; recombinant human interleukin-2 (aldesleukin), talc duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; sponge; nitrogen mustards such as chlorambucil, chlorambucil benzsulfamide, chlorphosphamide, estramustine, ifosfamide, nitrogen mustard, methoxychlor hydrochloride, mezalan hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin omega 1I (Angew Chem. Intl. Ed. Engl.1994 33:183-186); daminomycins, including daminomycin A; bisphosphonates, such as clodronate; esperamicin; and the new carcinogens and related chromoprotein enediyne antibiotic chromogens), aclacinomycins, actinomycins, authramycins, azaserine, bleomycins, cactinomycin, carabicin, carmine, carzinophilin, chromomycins, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,. (doxorubicin), morpholinodoxorubicin, cyanomo holino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, perroximate, porfibrinocin, Puromycin, quelamycin, rodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethoate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, edrenolide, cytosine ... Norcibane, floxuridine; androgens such as carrusetron, dacrotone propionate, epichlorohydrin, methenamine, testolactone; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as florin acid, acetoglucuronolide, aldehyde phosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, mecocin, sesquinone, elfomithine, elliptamide acetate, succinate, pheniramine, pheniramine, succinate ... iniumacetate, epothilones, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine; maytansine alkaloids such as maytansine and ansamycins, mitoguanidine, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losor, podophyllic acid, 2-ethylhydrazide, methylprocarbazine, polysaccharide complex (JHS Natural Products, Eugene, Oreg.), razoxane, rhizoxin, sizofuran, spirogermanium, tenuazonic acid, triimine, 2,2',2"-trichlorotriethylamine, trisporins (especially T-2 toxin, verracurin A, pyocyanin A and angiidine), urethane, vinblastine, dacarbazine, mannomustine, dibromomannitol, dibromodulanol (mitolactol), pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa; taxanes, such as TAXOL (paclitaxel, Bristol-Myers Squibb Oncology, Princeton, NJ), (without Cremophor), an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111), and (docetaxel, doxetaxel; Sanofi-Aventis), chlorambucil, (gemcitabine), 6-thioguanine, mercaptopurine, methotrexate; platinum analogs such as cisplatin and carboplatin, vinblastine, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, (Vinorelbine), cytoreduction, teniposide, edatrexate, daunorubicin, aminopterin, capecitabine Ibandronate sodium, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO); retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0197] Chemotherapeutic agents also include (i) antihormonal drugs used to modulate or inhibit tumor hormone action, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Tamoxifen citrate), raloxifene, droloxifene, iodixan, 4-hydroxytamoxifen, troloxifene, coxifen, LY117018, onapristone, and (toremifene citrate); (ii) aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, (Megestrol acetate), (exemestane, Pfizer), formestanie, fadrozole, (Vorozole), (letrozole, Novartis) and (anastrozole, AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, buserelin, tristyryl alcohol, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-α, Ralf and H-Ras; (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 rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

[0198] Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab ( Genentech), cetuximab ( Imclone), Pertuzumab ( Amgen, rituximab ( Genentech / Biogen Idee), Pertuzumab ( 2C4, Genentech), trastuzumab ( Genentech), tositumomab (Bexxar, Corixia), and antibody-drug conjugates, gemtuzumab ozogamicin ( Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents for combination with the compounds of the invention include: abozumab, asezetuzumab, alizumab, bapineuzumab, mo-bivalizumab, mo-cantuzumab, cilizumab, cilizumab, tocilizumab, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motuzumab ... motovizumab), natalizumab, nimotuzumab, nolovizumab, numavizumab, ori, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexezumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sotuzumab, tacatuzumab

[00136] The antibodies include tetraxetan, taduzumab, taduzumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, anti-CD3 monoclonal antibody (visilizumab), and anti-interleukin 12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), which is a recombinant proprietary human sequence full-length IgG1λ antibody genetically modified to recognize the interleukin-12p40 protein.

[0199] Chemotherapeutic agents also include "EGFR inhibitors", which refer to compounds that bind to or directly interact with EGFR and prevent or reduce its signaling activity, or are also referred to as "EGFR antagonists". Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimeric 225 (C225 or cetuximab; ) and remodeled human 225 (H225) (see WO 96 / 40210, Imclone Systems Inc.); IMC-11F8, a fully human EGFR-targeted antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Straghotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody against EGFR that competes with EGF and TGF-α for EGFR binding (EMD / Merck); a human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies designated E.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies can be conjugated to cytotoxic agents to generate immunoconjugates (see, e.g., EP 659,439 A2, Merck Patent GmbH). EGFR antagonists include small molecules such as the compounds described in the following U.S. Patent Nos.: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,33 2, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,391,874, 6,344,455, 5,760,041, 6,002,008 and 5,747,498 and compounds in the following PCT publications: WO98 / 14451, WO98 / 50038, WO99 / 09016 and WO99 / 24037. Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, Genentech / OSI Pharmaceuticals), PD 183805 (CI 1033, 2-acrylamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer), ZD1839, gefitinib 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca), ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca), BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-pyrimidine-2,8-diamine, Boehringer Ingelheim), PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol), (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine), CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynyl), EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth), AG1478 (Pfizer), AG1571 (SU 5271, Pfizer), dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib ( GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxyphenyl]-6[5[[[2-methylsulfonyl]ethyl]amino]methyl]-2-furyl]-4-quinazolinamine).

[0200] Chemotherapeutic agents also include "tyrosine kinase inhibitors", including the EGFR targeted drugs described in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits HER2 and EGFR overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033, Pharmacia); Raf-1 inhibitors, such as ISIS-5132, an antisense agent that inhibits Raf-1 signaling, available from ISIS Pharmaceuticals; non-HER targeted TK inhibitors, such as imatinib mesylate ( available from Glaxo SmithKline); multitargeted tyrosine kinase inhibitors, such as sunitinib ( available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloyl methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); Affinitac (ISIS 3521, Isis / Lilly); imatinib mesylate PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1Cl1 (Imclone), rapamycin (sirolimus, ); or as described in any of the following patent publications: U.S. Pat. No. 5,804,396, WO 1999 / 09016 (American Cyanamid), WO 1998 / 43960 (American Cyanamid), WO 1997 / 38983 (Warner Lambert), WO 1999 / 06378 (Warner Lambert), WO 1999 / 06396 (Warner Lambert), WO 1996 / 30347 (Pfizer, Inc), WO 1996 / 33978 (Zeneca), WO 1996 / 3397 (Zeneca), and WO 1996 / 33980 (Zeneca).

[0201] Chemotherapeutic agents also include dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, bacillus Calmette-Guérin, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon interferon alpha-2a, interferon alpha-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegaspargase, pegfilgrastim, pemetrexed disodium, pragramycin, porfil sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronic acid and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0202] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, cortisone acetate, thiocarbamide, triamcinolone acetonide, triamcinolone, mometasone, amcinonide, budesonide, desonide, fluocinolone acetonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone butyrate, hydrocortisone valerate, dipropionate propionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone butyrate, clobetasol propionate, fluocortolone hexanoate, fluocortolone pivalate and fluprednidine acetate; immunoselective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (FEG) ) (IMULAN BioTherapeutics, LLC); antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, tumor necrosis factor alpha (THFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), tocilizumab (Cimzia), golimumab (Simponi), interleukin 1 (IL-1) such as anakinra (Kineret), T cell costimulatory blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab Interleukin 13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers such as rontalizumab; 7 integrin beta-receptor blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-Mi; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTal / 2 blockers such as anti-lymphotoxin alpha (LTa); radioisotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 and radioisotopes of Lu); various experimental drugs such as thioplatin, PS-341, phenylbutyric acid, ET-18-OCH3 or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theafiavins, flavanols, proanthocyanidins, betulinic acid and its derivatives; autophagy inhibitors such as chloroquine, delta-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin and 9-aminocamptothecin); podophyllotoxin; tegafur Bexarotene Bisphosphonates such as clodronate (e.g. or ), etidronate NE-58095, zoledronic acid / zoledronate Alendronate Pamidronate Tiludronate Risedronate and epidermal growth factor receptor (EGF-R); vaccines such as Vaccines; perifosine, COX-2 inhibitors (such as celecoxib or etoricoxib), proteosome inhibitors (such as PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium Pisantron; farnesyl transferase inhibitors such as lonafa (SCH 6636, SARASAR TM ); and any pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, which is oxaliplatin (ELOXATIN TM ) in combination with 5-FU and leucovorin.

[0203] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects. Nonsteroidal anti-inflammatory drugs NSAIDs include non-selective inhibitors of cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib and valdecoxib. NSAIDs are used for symptomatic relief of rheumatoid arthritis, osteoarthritis, inflammatory joint disease, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain caused by inflammation and tissue damage, fever, intestinal obstruction, and renal colic.

[0204] In certain embodiments, chemotherapeutic agents include, but are not limited to, doxorubicin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, interferon, platinum derivatives, taxanes (e.g., paclitaxel, docetaxel), vinca alkaloids (e.g., vinblastine), anthracyclines (e.g., etoposide), cisplatin, mTOR inhibitors (e.g., rapamycin), methotrexate, actinomycin D, dolastatin 10, colchicine, trimetrexate, chlorpheniramine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents (e.g., chlorambucil), 5-fluorouracil, camptothecin, cisplatin, metronidazole, and imatinib mesylate, etc. In other embodiments, the compounds of the invention are administered in combination with biologics such as bevacizumab or panitumumab.

[0205] In certain embodiments, a compound of the invention or a pharmaceutically acceptable composition thereof is administered in combination with an antiproliferative or chemotherapeutic agent selected from any one or more of the following: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretinoin, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bacillus Calmette-Guérin, bevacuzimab, fluorouracil, bexarotene, bleomycin, bortezomib, busulfan, captestosterone, capecitabine, camptothecin, carboplatin, carmustine, cetuximab, phenylbutyric acid Nitrogen mustard, cladribine, clofarabine, cyclophosphamide, cytarabine, actinomycin D, darbepoetin alfa, daunorubicin, denileukin, dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, drostanolone propionate, epirubicin, epoetin alfa, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fulvestrant, gefitinib, gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan, Idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, megestrol acetate, melphalan, mercaptopurine, 6-MP, mesna, aminopterin, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nelarabine, nofetumomab, oprelleukin, oxaliplatin, paclitaxel, palifermin, pamidronate, pegamic acid, asparaginase, ethylenediamine Filgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, porfil sodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, sorafenib, streptozotocin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, zoledronic acid, or zoledronic acid.

[0206] E. Definition

[0207] As used herein, the term "hydrocarbyl" describes an organic compound or group consisting only of the elements carbon and hydrogen. These moieties include, but are not limited to, alkyl, alkenyl, alkynyl, and aryl moieties. These moieties also include alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenaryl, and alkynaryl. Unless otherwise indicated, these moieties preferably contain 1 to 20 carbon atoms.

[0208] As used herein, a "substituted hydrocarbyl" moiety is a hydrocarbyl moiety substituted with at least one atom other than carbon, including moieties in which carbon chain atoms are replaced with heteroatoms such as nitrogen, oxygen, silicon, phosphorus, boron, sulfur, or halogen atoms. These substituents include, but are not limited to, halogen, heterocycle, alkoxy, alkenyloxy, alkynyloxy, aryloxy, hydroxy, keto, acyl, acyloxy, nitro, tertiary amino, amido, nitro, cyano, thio, sulfinate, sulfonamide, ketal, acetal, ester, and ether.

[0209] The term "alkyl" as used herein refers to a group consisting of 1 to 12 carbon atoms (C 1-12 ) is a saturated straight or branched monovalent hydrocarbon group, wherein the alkyl group may be optionally substituted independently by one or more of the following substituents. In another embodiment, the alkyl group is 1 to 8 carbon atoms (C 1-8 ), or 1 to 6 carbon atoms (C 1-6 ). Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like.

[0210] The term "alkylene" as used herein refers to a group consisting of 1 to 12 carbon atoms (C 1-12 ) is a saturated straight or branched divalent hydrocarbon group, wherein the alkylene group may be optionally substituted independently by one or more of the following substituents. In another embodiment, the alkylene group is 1 to 8 carbon atoms (C 1-8 ), or 1 to 6 carbon atoms (C 1-6 ). Examples of the alkylene group include, but are not limited to, methylene, ethylene, propylene, and the like.

[0211] The term "alkenyl" refers to a group having at least one site of unsaturation (ie, carbon-carbon, sp 2 Double bond) 2-8 carbon atoms (C 2-8 ), wherein the alkenyl group may be optionally substituted independently with one or more substituents as described herein, and includes groups with "cis" and "trans" orientations, or "E" and "Z" orientations. Examples include, but are not limited to, ethylenyl or vinyl, allyl, and the like.

[0212] The term "alkenylene" refers to a group having at least one site of unsaturation (i.e., carbon-carbon sp 2 double bond) of 2 to 12 carbon atoms (C2-12 ) is a straight or branched divalent hydrocarbon radical of the type alkylene group, wherein the alkylene group may be optionally substituted, and includes groups having "cis" and "trans" orientations, or "E" and "Z" orientations. Examples include, but are not limited to, ethylenylene or vinylene, allyl, and the like.

[0213] The term "alkynyl" refers to a 2 to 8 carbon atom (C 2-8 ) is a linear or branched monovalent hydrocarbon group, wherein the alkynyl group may be optionally substituted independently by one or more substituents described herein. Examples include, but are not limited to, ethynyl, propynyl, etc.

[0214] The terms "carbocycle", "carbocyclic group", "carbocyclic ring" and "cycloalkyl" refer to a cycloalkyl group having 3 to 12 carbon atoms (C 3-12 ) as a monocyclic ring or 7 to 12 carbon atoms as a bicycle. Bicyclic carbocyclic rings with 7 to 12 atoms can, for example, be arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, and bicyclic carbocyclic rings with 9 or 10 ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or bridged systems such as bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane and bicyclo [3.2.2] nonane. 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, and the like.

[0215] "Aryl" refers to a 6-20 carbon atom (C 2 - 4) radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. 6-20 ) is a monovalent aromatic hydrocarbon radical. Some aryl groups are represented as "Ar" in the exemplary structures. Aryl groups include bicyclic groups containing an aromatic ring fused to a saturated, partially unsaturated ring or an aromatic carbocyclic ring. Typical aryl groups include, but are not limited to, groups derived from benzene (phenyl), substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, and the like. Aryl groups are optionally substituted independently with one or more substituents described herein.

[0216] The terms "herocycle", "heterocyclyl" and "herocyclic ring" are used interchangeably herein and refer to a saturated or partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic group of 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 of the following substituents. The heterocycle can be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P and S), such as a bicyclo [4,5], [5,5], [5,6] or [6,6] system. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to date), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. SOC. (1960) 82: 5566. "Heterocyclyl" also includes groups in which a heterocyclic group is fused to a saturated ring, a partially unsaturated ring, or an aromatic carbocyclic or 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-dioxothiomorpholin-4-yl, oxocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, pyrrolyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thiooxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxacan-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl, [1,4]diazepan-1-yl, The term "butylene" refers to butylene, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepanyl, diazepanyl, thiazepanyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindole, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothiophenyl, dihydrofuranyl, pyrazolidinyl imidazolinyl, imidazolidinyl, 3-azabicyclo [3.1.0] hexanyl, 3-azabicyclo [4.1.0] heptane, azabicyclo [2.2.2] hexanyl, 3H-indolylquinolizinyl and N-pyridylurea. Spirocyclic moieties are also included within the scope of this definition. Examples of heterocyclic groups where 2 ring atoms are substituted with oxo (=0) moieties are pyrimidinone and 1,1-dioxo-thiomorpholinyl. The heterocyclic groups herein are optionally substituted independently with one or more substituents described herein.

[0217] The term "heteroaryl" refers to a monovalent aromatic group of 5, 6 or 7 membered rings and includes fused ring systems of 5-20 atoms (at least one of which is aromatic) containing one or more atoms independently selected from nitrogen, oxygen and sulfur heteroatoms. 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. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[0218] Where possible, the heterocycle or heteroaryl may be carbon (carbon-linked) or nitrogen (nitrogen-linked) bonded. By way of example and not limitation, the carbon-bonded heterocycle or heteroaryl is bonded at position 2, 3, 4, 5, or 6 of pyridine, at position 3, 4, 5, or 6 of pyridazine, at position 2, 4, 5, or 6 of pyrimidine, at position 2, 3, 5, or 6 of pyrazine, at position 2, 3, 4, or 5 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, at position 2, 4, or 5 of oxazole, imidazole, or thiazole, at position 3, 4, or 5 of isoxazole, pyrazole, or isothiazole, at position 2 or 3 of aziridine, at position 2, 3, or 4 of azetidine, at position 2, 3, 4, 5, 6, 7, or 8 of quinoline, at position 1, 3, 4, 5, 6, 7, or 8 of isoquinoline.

[0219] By way of example and not limitation, the nitrogen-bonded heterocycle or heteroaryl is bonded at the 1 position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, at the 2 position of isoindole or isoindoline, at the 4 position of morpholine, and at the 9 position of carbazole or β-carboline.

[0220] The term "organoboron" refers to an organic derivative of boron. Examples of organoboron compounds include esters of the following structures:

[0221]

[0222] Wherein R 'is suitably a hydrocarbon group, a substituted hydrocarbon group, an alkyl group, an alkylene group, a carbocycle, a heterocycle, an aryl group, a heteroaryl group and an aryl-alkyl group. Examples of R 'include ethylene (-CH2CH2-), neopentyl (-CH2C(CH3)(CH3)CH2-) and pinacol (-C(CH3)(CH3)C(CH3)(CH3)C-). R "is suitably an optionally substituted hydrocarbon group, a substituted hydrocarbon group, an alkyl group, an alkylene group, a carbocycle, a heterocycle, an aryl group, a heteroaryl group and an aryl-alkyl group. Examples of R "include substituted heteroaryl compounds, including 2-hydroxypyridine and 2-aminopyrimidine. In some specific aspects, R 'is pinacol and R "is 2-aminopyrimidine. Other examples of organoboron compounds include triorganoboranes and hydrides, boric acids and boric acids and esters, carboranes and borane compounds.

[0223] The term "volume" refers to the amount of a first liquid compound relative to the volume of a second compound or mixture of second compounds with which it is combined. For example, four volumes of a first liquid added to one volume of a second compound is associated with a volume percentage of the first liquid of 80% calculated by the following formula: (4 volumes of first liquid) / (4 volumes of first liquid+1 volume of second compound)*100=80 v / v%.

[0224] The term "chiral" refers to molecules that have the property of non-superimposability on their mirror image partner, whereas the term "achiral" refers to molecules that are superimposable on their mirror image partner.

[0225] The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0226] "Diastereomers" refers to stereoisomers with 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 under high resolution analytical procedures such as electrophoresis and chromatography.

[0227] "Enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0228] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as protomeric tautomers) include interconversions via proton migration (e.g., keto-enol and imine-enamine isomerizations). Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0229] The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugarates, formates, benzoates, glutamates, methanesulfonates, "mesylate", ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-(2-hydroxy-3-naphthoate)) salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt can have more than one charged atom in its structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0230] The phrase "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0231] "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.

[0232] The terms "inventive compounds" and "compounds of the present invention" and "compounds of formula II, IIa, III and IIIa" include compounds of formula II, IIa, III and IIIa and stereoisomers, geometric isomers, tautomers, solvates, metabolites and pharmaceutically acceptable salts and prodrugs thereof.

[0233] The terms "treat" (verb) and "treatment" (name) refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as the development or spread of cancer. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" also means prolonging survival compared to expected survival if not receiving treatment. Patients in need of treatment include those already suffering from the disease or disorder as well as those susceptible to the disease or disorder or those in need of prevention of the disease or disorder.

[0234] The phrase "therapeutically effective amount" refers to an amount of a compound of the invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells, reduce the size of a tumor, inhibit (i.e., slow down to some extent and preferably stop) the infiltration of cancer cells into peripheral organs, inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent that a drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer treatment, efficacy may be determined, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (R).

[0235] The term "cancer" refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer (gastroc cancer) or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, 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, liver cancer, anal cancer, penile cancer, and head and neck cancer.

[0236] Example

[0237] method

[0238] The chemical reactions described in the examples can be readily adapted to prepare many other PI3K inhibitors of the invention, and alternative methods for preparing the compounds of the invention are considered to be within the scope of the invention. For example, the synthesis of non-exemplary compounds according to the invention can be successfully carried out by modifications obvious to those skilled in the art, such as by utilizing other suitable reagents known in the art in addition to those described, and / or by conventional changes in reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be considered to have applicability to preparing other compounds of the invention.

[0239] In the examples described below, all temperatures are expressed in degrees Celsius unless otherwise stated. Unless otherwise stated, reagents were purchased from commercial suppliers such as Sigma Aldrich Chemical Company, JT Baker, Boron Molecular, Mallinckrodt, and used without further purification. The reactions listed below are generally carried out in anhydrous solvents under a positive pressure of nitrogen or argon or with a drying tube (unless otherwise stated), and the reaction flasks are usually equipped with rubber septa to introduce substrates and reagents by syringe. Glassware is dried and / or heated to dryness. Column chromatography is performed on a Biotage system (manufacturer: Dyax Corporation) with a silica gel column or a silica gel SEP column. (Waters). Chloroform was used as the reference standard (7.25 ppm) and the wavelengths were obtained at 400 MHz in deuterated CDCl3, d6-DMSO, CH3OD or d6-acetone solutions (reported in ppm). 1 H NMR spectra. When peak multiplicities are reported, the following abbreviations are used: s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublet), dt (doublet of triplet). Coupling constants, when given, are reported in Hertz (Hz).

[0240] HPLC can be performed by the following exemplary method:

[0241]

[0242]

[0243]

[0244]

[0245] HPLC analysis of 2-(2-chloro-6-morpholinyl-9H-pyran-purin-8-yl)propan-2-ol (Compound 5), 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine (Compound 7) and 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) can be performed as follows. Diluent: acetonitrile. Mobile phase A: 0.05% TFA / water. Mobile phase B: 0.05% TFA / acetonitrile. Column: Ace 3C18HL column, 3×50 mm 3.0 μm. Column temperature: 35°C. Detector wavelength: 220 nm. Injection volume: 2 μL. Flow rate: 1 mL / min. Sample concentration: 0.5-1.0 mg / mL in 50% acetonitrile / water. Program: 0.0 min 5.0% B, 0.3 min 5.0% B, 2.0 min 60.0% B, 4.0 min 90% B, 5.0 min 90% B, 5.1 min 5.0% B, 6.5 min 5.0%. Typical retention times: 5 (RT 2.61 min), 7 (RT 2.20 min), GDC-0084 (RT 2.67 min).

[0246] The structure of compound 5 was confirmed by NMR (see Figure 5 and 6 The structure of compound 7 was confirmed by NMR (see Figure 7 and 8The structure of GDC-0084 was confirmed by NMR (see Fig.11 and 12 ).

[0247] Example 1: The preparation of 2,6-dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (Compound 2) is as follows:

[0248]

[0249] 2,6-dichloro-9H-purine (Compound 1) (28.50 kg, 150.79 mol, 100 mol%), EtOAc (285.00 L, 10 vol) and p-toluenesulfonic acid pyridinium salt (PPTS) (568.5 g, 2.26 mol, 1.5 mol%) were added to the reactor, followed by the slow addition of 3,4-dihydro-2H-pyran (34.25 kg, 407.16 mol, 270 mol%) at 20 to 25 ° C. The mixture was slowly heated to 50 to 55 ° C and maintained until HPLC analysis showed that Compound 1 did not exceed 1.0A% (3 h). The reaction mixture was then cooled to 20 ° C, washed with saturated NaHCO3 aqueous solution (81 L, 2.8 vol) and brine (81.00 L, 2.8 vol), dried with Na2SO4 (14.30 kg), filtered and vacuum distilled to remove EtOAc (230 L). The crude product was filtered and charged into a reactor. Hexane (142 L) was added and the mixture was stirred for 15 min. The mixture was filtered and then vacuum dried at 55 °C for 8 hours to obtain 39.90 kg of compound 2 as a green solid (yield 97%, HPLC 99A%): melting point 116 °C; 1 H NMR (500MHz, DMSO-d6) δ8.92(s,1H), 5.72(dd,1H), 4.01(m,1H), 3.72(m,1H), 2.25(m,1H), 1.98(m,2H), 1.74(m,1H), 1.58(m,2H); 13 C NMR (125MHz, DMSO-d6) δ153.2, 151.7, 150.4, 146.9, 131.1, 82.2, 68.2, 30.2, 24.8, 22.5. HRMS[M+H] + Calculated as C 10 H 10 Cl2N4O 273.0304; found 273.0300.

[0250] Example 2: The preparation of 4-(2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)morpholine (Compound 3) is as follows:

[0251]

[0252] Compound 2 (39.90 kg, 146.09 mol, 100 mol%) and MeOH (399 L) were added to the reactor and then cooled to 0 ° C. Morpholine (38.20 kg, 438.48 mol, 300 mol%) was added at 0-5 ° C. The reaction mixture was warmed to 20 to 25 ° C until HPLC analysis showed that compound 2 did not exceed 1.0A% (24 h). The reaction mixture was then cooled to 0 to 5 ° C, maintained for 1 h, and then filtered. The filter cake was washed with hexane (200 L, 5 vol) and vacuum dried at 55 ° C for 14 h to obtain 44.40 kg of compound 3 as a green solid (yield 94%, HPLC 99A%): melting point 139 ° C; 1 H NMR (500MHz, DMSO-d6) δ8.39(s,1H), 5.60(dd,1H), 4.20(bs,4H), 4.01(m,1H), 3.71(m,4H), 2.17(m,1H), 1.94(m,2H), 1.74(m,1H), 1.58(m,2H); 13 C NMR (125MHz, DMSO-d6) δ153.9, 153.2, 151.6, 139.0, 118.4, 83.3, 68.2, 66.5, 45.8, 30.6, 24.9, 22.7. HRMS[M+H] + Calculated as C 14 H 18 ClN5O2 324.1222; found 324.1222.

[0253] Example 3: Preparation of 2-(2-chloro-6-morpholinyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-8-yl)propan-2-ol

[0254] As shown below, 2-(2-chloro-6-morpholinyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-8-yl)propan-2-ol (Compound 4) was prepared from 4-(2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)morpholine (Compound 3).

[0255]

[0256] Compound 3 (310 g, 100 mol%) and tetrahydrofuran (4.0 L, 13 vol.) were added to a 12 L round bottom flask and a reaction solution was formed. The solution was cooled to -10 ° C, i-PrMgCl (287 mL, 2.0 M in THF) was added, while keeping the temperature below or equal to -5 ° C. A reaction mixture was formed by merging n-BuLi (421 mL, 2.5 M in hexane) with the reaction solution while keeping the temperature below or equal to -5 ° C. The reaction mixture was maintained at less than or equal to -5 ° C until HPLC analysis showed that compound 3 did not exceed 5 area percentages ("A%") by HPLC determination. Acid was added to the reaction mixture, and then quenched by adding H2O (8.0 L, 0 ° C). Layering, the aqueous phase was extracted with MTBE (1.5 L). The combined organic extracts were washed with saturated NaCl aqueous solution (1.5 L), and then concentrated to near dryness. i-PrOH (1.5L) is combined with the organic extract and the mixture is concentrated to near dryness. The concentrate is transferred to a 2L round-bottom flask, combined with 2-propanol (1L), and heated at 50°C for 3h. The mixture is cooled to 25°C, slurries are formed by crystallization and kept at this temperature for 12h. The slurries are then cooled to 0°C, kept at this temperature for 3h, filtered to separate the filter cake, and washed with cold 2-propanol (200mL). The filter cake is vacuum dried at 50°C for 12h. The method obtains 329g of compound 4 (yield 90%) as an off-white solid. It is noted that approximately 3% of compound 4 is lost during the crystallization process.

[0257] In the scale-up evaluation, i-PrMgCl (1.0M in THF solution, 82L, 82.00mol, 65mol%) was added to a solution of compound 3 (40.80kg, 126.01mol, 100mol%) in anhydrous THF (408L, 10vol) at -15°C in N2, followed by n-BuLi (2.5M in hexane, 55L, 137.50mol, 109mol%) between -10°C and -5°C. After 30min at this temperature, acetone (16.20kg, 278.92mol, 221mol%) was slowly added to the solution. The reaction mixture was stirred at -10±5°C for 2h until HPLC analysis showed that compound 3 did not exceed 1.0A% (2h). The mixture was then transferred to a reactor containing water (40.80kg, 1vol) at 0 to 5°C, the mixture was stirred for 5min, and the layers were separated. The aqueous phase was extracted with MTBE (136 L, 3.3 vol). The combined organic extracts were washed with brine (119 L, 2.9 vol), dried over Na2SO4 (34 kg), filtered and evaporated to provide a crude product. The yellow residue was suspended in 2-propanol (204 L, 5 vol), and the resulting slurry was heated to 50°C for 60 minutes. The slurry was cooled to 25°C over 2 h, and then cooled to 0 to 5°C over 1 h. The product was collected by filtration, washed with cold 2-propanol (40.8 L, 1 vol), and dried under vacuum at 55°C for 12 h to give 42.50 kg of compound 4 as an off-white powder (yield 91%): melting point 181°C; 1 H NMR (500MHz, DMSO-d6) δ6.27(dd,1H), 5.81(s,1H), 4.15(vbs,4H), 4.05(m,1H), 3.69( m,4H), 3.55(m,1H), 1.97(m,1H), 1.70(m,1H), 1.60(m,3H), 1.56(m,3H), 1.53(s,3H); 13 C NMR (125MHz, DMSO-d6) δ155.2, 154.0, 153.7, 152.3, 117.1, 84.3, 70.0, 68.5, 66.5, 45.8, 30.5, 29.7, 28.0, 25.0, 23.5. HRMS[M+H] + Calculated as C 17 H 24 ClN5O3 382.1640; found 382.1644.

[0258] Example 4: Preparation of 2-(2-chloro-6-morpholinyl-9H-purin-8-yl)propan-2-ol

[0259] 2-(2-Chloro-6-morpholinyl-9H-purin-8-yl)propan-2-ol (Compound 5) was prepared from 2-(2-chloro-6-morpholinyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-8-yl)propan-2-ol (Compound 4) as shown below.

[0260]

[0261] Compound 4 (327 g, 100 mol%), p-toluenesulfonic acid monohydrate (4.88 g, 3 mol%) and methanol (2.6 L, 8 vol) were combined in a 5 L round bottom flask to form a solution. The solution was heated to 50 ° C and then kept at this temperature for 3 h, so that the content of compound 4 determined by HPLC did not exceed 1A%. The solution was cooled to ambient temperature, concentrated to dryness and combined with water (1.0 L). The solid product was separated by filtration and washed with water (2 × 500 mL) and then with heptane (2 × 500 mL), and then vacuum dried at 50 ° C for 12 h. This method obtained 246 g of compound 5 as a light yellow fluffy solid.

[0262] Example 5: Preparation of 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine

[0263] As shown below, 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine (Compound 7) was prepared from 2-(2-chloro-6-morpholinyl-9H-purin-8-yl)propan-2-ol (Compound 5).

[0264]

[0265] Compound 5 (240 g, 100 mol%), DMF (806 mL, 3.6 vol), cesium carbonate (303 g, 115 mol%) and 1,2-dibromoethane (139 mL, 200 mol%) were combined in a 5 L round bottom flask. The resulting heterogeneous mixture was heated to 90 ° C for 3 hours, whereupon HPLC analysis showed that compound 5 was 30 A% and compound 7 was 49 A%. Additional 1,2-dibromoethane (69.4 mL, 100 mol%) was added and reacted for 3 hours, whereupon HPLC analysis showed that compound 5 was 28 A% and compound 7 was 54 A%. Additional cesium carbonate (355 g, 135 mol%) and 1,2-dibromoethane (34.7 mL, 50 mol%) were then added and reacted for 3 hours, whereupon HPLC analysis showed that compound 5 was 2 A% and compound 7 was 87 A%. The mixture was cooled to ambient temperature, water (2.2 L) was added, and the mixture was stirred for 12 h. Attempts to crystallize the product after adding water to quench the reaction were unsuccessful because a gummy solid was produced. EtOAc (1.3 L) was added, the layers were separated, and the organic phase was washed with a saturated aqueous NaCl solution (3×1.2 L). The organic phase was concentrated to a minimum stirring volume to produce a viscous solid. Isopropanol (300 mL) was added to the solid, the mixture was heated to 65° C. and maintained at this temperature for 2 hours, and then the mixture was cooled to ambient temperature. The resulting solid was filtered and dried under vacuum at 50° C. for 12 h. This method obtained 147 g of compound 7 as a yellow crystalline solid.

[0266] Example 6: Preparation of 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine (GDC-0084)

[0267] 5-(6,6-Dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) was prepared from 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine (Compound 7) as shown below.

[0268]

[0269] In the Suzuki cross-coupling reaction, compound 7 (138 g, 100 mol%), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine ("pinacol boronate") (113 g, 120 mol)), dioxane (1.7 L), and potassium phosphate (3.0 M solution, 284 mL, 200 mol%) were combined in a 3 L round bottom flask. The structure of the rosin boronate was confirmed by NMR (see Fig. 9 and 10 ). The contents were bubbled with N2 for 30 min, then PdCl2dppf·CH2Cl2 (6.96 g, 2 mol%) was added and the mixture was bubbled with N2 for 10 min. The mixture was heated to 80°C and then maintained at this temperature for 2 h, after which analysis by HPLC showed complete consumption of compound 7. The mixture was transferred to a 12 L round-bottom flask, water (6.8 L) was added, the mixture was cooled to 5°C, filtered, and the filter cake was washed with water (2×500 mL). The filter cake was dried under vacuum at 50°C for 12 h. The resulting crude GDC-0084 was transferred to a 5 L round-bottom flask and combined with THF (3.2 L), HOAc (1.6 L) and water (480 mL). The mixture was heated to 50°C, and Si-(CH2)3SH ("Si-Thiol") (80 g) was added to the resulting solution. The mixture was maintained at 50°C for 3 h, cooled to ambient temperature and maintained for 12 h. The mixture was filtered through a diatomaceous earth / silica gel pad and the filtrate was concentrated to dryness. The resulting light brown solid was transferred to a 5L round-bottom flask and combined with THF (3.2L), HOAc (1.6L) and water (480mL). The mixture was heated to 50°C and THF (100mL) was added to obtain a clear solution. In order to improve the color of the solution, Si-thiourea (80g) was added, the mixture was kept at 50°C for 2h, and then filtered through a 2mm Teflon filter while hot. The resulting light yellow solution was distilled to dryness, slurried with n-butanol (3L), filtered, and washed with heptane (2×1L). The washed filtrate was dried under vacuum at 70°C for 24h. The method obtained 149g of GDC-0084 (two-step yield was 91%) as a yellow crystalline solid. The purity by HPLC was 98.9A%, the main impurities were determined as impurities-1 and impurities-2 as shown below, and the residual Pd level was 12ppm.

[0270]

[0271] The total yield of GDC-0084 from the series of reaction steps of Examples 1 to 4 was 44%, impurity-1 was present at 0.52A% (HPLC) and impurity-2 was present at 0.18A% (HPLC).

[0272] Example 7: Cyclization of amino alcohol compound 5 to generate fused morpholine compound 7

[0273] The effects of solvent, base and reaction temperature on the alkylation of compound 5 with 1,2-dibromoethane to form compound 7 were evaluated. Table 1 below lists the method parameters and reagents of experiments 1 to 10 and the combination of the relevant reaction transformations, wherein experiments 1 to 8 were carried out on a 0.3 g scale, experiment 9 was carried out on a 60 g scale, and experiment 10 was carried out on a 100 g scale. Experiments 1 to 8 were carried out by combining the reagents (1.0 mmol of compound 5) with 1.5 mL of solvent in a vial, mixing with a stirring bar at ambient temperature, and then raising to the reaction temperature. The reactions of experiments 9 and 10 were completed by mixing the reagents at ambient temperature and mechanically mixing, and then raising to the reaction temperature. For experiment 9, 1,2-dibromoethane was added dropwise to the heated reaction mixture over 1 hour and an exotherm was observed. For experiment 10, compound 5 was added to the heated reaction mixture in two portions, with an interval of 1 hour between additions. The total reaction time for each experiment was 12 hours. The results are recorded in Table 5 below, where "Exp" refers to the experimental number, "DBU" refers to diazabicycloundecene, "Temp" refers to the reaction temperature, "A%sub" refers to the area percentage (HPLC) of the substrate 2-(2-chloro-6-morpholinyl-9H-purin-8-yl)propan-2-ol, "A%product" refers to the area percentage (HPLC) of 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine, "A%Imp.3" refers to the area percentage (HPLC) of impurity 3 (described below - area percentage determined by HPLC after 12 hours reaction time) and "A%Imp.4" refers to the area percentage (HPLC) of impurity 4 (described below - area percentage determined by HPLC).

[0274]

[0275] Table 1

[0276] Exp Alkali Solvent Temp A%Sub A%prob A%Imp.3 A%Imp.4 1 <![CDATA[Cs2CO3]]> DMF 90℃ 1 86 13 0 2 <![CDATA[K2CO3]]> DMF 90℃ 1 87 12 0 3 <![CDATA[Cs2CO3]]> MIBK 90℃ 4 82 13 1 4 <![CDATA[K2CO3]]> MIBK 90℃ 33 56 7 4 5 <![CDATA[Cs2CO3]]> THF 90℃ 5 79 15 1 6 <![CDATA[K2CO3]]> THF 90℃ 46 36 7 11 7 <![CDATA[Cs2CO3]]> <![CDATA[CH3CN]]> 90℃ 3 84 14 0 8 <![CDATA[K2CO3]]> <![CDATA[CH3CN]]> 90℃ 4 81 11 4 9 <![CDATA[K2CO3]]> DMF 100℃ 0 85 11 0 10 <![CDATA[K2CO3]]> DMF 110℃ 0 85 11 0

[0277] It was found that cesium carbonate and potassium carbonate had similar effects on conversion, with the latter providing a slightly lower amount of impurity 3. The ratio of product to impurity 3 was relatively insensitive to base, solvent and temperature. The reaction was found to be exothermic on a larger scale, and dose-controlled additions were investigated to mitigate potential safety risks. Slow addition of 1,2-dibromoethane to a mixture of compound 5 and K2CO3 (experiment 9) did not adversely affect conversion, but did result in the formation of impurity 5 (see below). Adding compound 5 in batches to a mixture of 1,2-dibromoethane and K2CO3 (experiment 10) suppressed the formation of impurity 5 to less than 1 area % (by HPLC) without negatively affecting conversion.

[0278]

[0279] The exothermic ring-forming reaction was further examined by reaction calorimetry. In the first experiment, the Advanced Reaction System Screening Tool (ARSST TM ) found that the reaction was exothermic at 45 °C and the exothermic decomposition temperature (TD) was 185 °C 24 -Temperature at which the time to maximum rate is 24 hours). ARSTT methodology is known in the art and is available from Fauske & Associates, Inc. See, for example, James P. Burlebach, "Advanced Reactive System Screening Tool (ARSST)", North American Thermal Analysis Society, 28 th Annual Conference, Orlando, Oct. 4-6, 2000. In the second calorimetric experiment, integration of the reaction heat flow curve in a 90°C isothermal reaction calorimeter showed an adiabatic temperature rise of 76.5°C, which was a dose-controlled exotherm by the batch addition of compound 5. In summary, the calorimetric data showed that the exotherm can be effectively controlled by using dose-controlled addition.

[0280] Example 8: Study of Suzuki coupling method parameters

[0281] Various method parameters for the production of GDC-0084 by the Suzuki cross-coupling reaction of compound 7 with pinacol boronate were studied. The reaction of each experiment was completed by mixing the reagents shown in Table 2 at ambient temperature and then heating under mixing. The reaction was carried out by mixing the reagent (0.9mmol of compound 7) in a solvent (3.9mL) at a solvent ratio of 10:1 in a vial at ambient temperature, evacuating and filling with nitrogen, sealing the vial, and then heating the sealed vial. Each of experiments 1, 2 and 4 to 7 was reacted at 80°C for 2 hours. Experiment 3 was reacted for 10 hours. The results are recorded in Table 2 below, where "Exp" refers to the experimental number, "Cat%" refers to the molar percentage of the catalyst, and "Ratio" refers to the molar ratio of the product to the substrate.

[0282] Table 2

[0283]

[0284]

[0285] 1 After 10 hours, the product to substrate ratio of Experiment 9 was 99:1.

[0286] With regard to catalyst loading, the conversion was found to be slow at 1 mol%. High conversion was found in most solvents.

[0287] Example 9: Solubility Test

[0288] The solubility of GDC-0084 in various single and ternary solvent systems was measured at 50° C., wherein each ternary solvent mixture had a solvent ratio of 67:24:9 on a volume basis. The solubility results are shown in Table 3 below in wt % (mg / g).

[0289] Table 3

[0290] Solvent Solubility Solvent Solubility DMF 0.9wt.% <![CDATA[DMF / HOAc / H2O]]> 0.3wt.% THF 0.7wt.% <![CDATA[DMF / HOAc / H2O]]> 3.7wt.% MeTHF 0.3wt.% <![CDATA[MeTHF / HOAc / H2O]]> 2.7wt.% MeOH 0.2wt.% <![CDATA[MeOH / HOAc / H2O]]> 0.4wt.% EtOH 0.1wt.% <![CDATA[EtOH / HOAc / H2O]]> 1wt.% n-PrOH 0.1wt.% <![CDATA[n-PrOH / HOAc / H2O]]> 1.8wt.% i-BuOH 0.1wt.% <![CDATA[i-BuOH / HOAc / H2O]]> 2.1wt.% n-BuOH 0.1wt.% <![CDATA[n-BuOH / HOAc / H2O]]> 2.4wt.% PhMe 0.1wt.% <![CDATA[Toluene / HOAc / H2O]]> 3.8wt.% HOAc 2.8wt.% <![CDATA[HOAc / HOAc / H2O]]> 2.1wt.%

[0291] The results showed that toluene / HOAc / water (67:24:9) was the best solvent system for GDC-0084 solubility. A ratio of 69:30:1 was selected for scale-up evaluation.

[0292] Example 10: Study on Pd Removal

[0293] Crude GDC-0084 prepared according to Example 8 was found to contain elevated levels of residual palladium. In this example, the removal of palladium from crude GDC-0084 was investigated. In a series of experiments, 5 g of a solution of GDC-0084 containing 2400 ppm Pd in ​​THF / HOAc (2:1 ratio) was exposed to various metal scavengers (at a loading of 20 wt%) at 55°C for 14 hours, followed by filtration and concentration of the filtrate, and the resulting purified GDC-0084 was analyzed for palladium content. The scavengers included: 2 Porous carbon beads of 0.3-0.8 mm with a surface area of ​​1 / g ("Quadrapure C"), 100 mesh activated carbon (Darco G-60), activated carbon larger than 45 μm ("Darco KB-G"), ("SiTAACoNa"), Si-(CH2)3NHC(=S)NHCH3 ("Si-Thiourea"), Si-thiol (Si-Thiol), and powdered synthetic magnesium-silicon ("Si-Thiol / Florisil"). Si-thiol and Si-thiourea are proprietary immobilized resins obtained from Silicycle. The results are listed in Table 4 below, where Si-thiourea and Si-thiol are the most effective scavengers.

[0294] Table 4

[0295] Cleaners ppm Pd Cleaners ppm Pd None (control) 2400 SiTAAcONa 900 Quadrapure C 2200 Si-Thiourea 16 Darco G-60 1600 Si-Thiol 6 Darco KB-G 900 Si-Thiol / Florisil 7

[0296] Example 11: Preparation of pure GDC-0084

[0297] Pure GDC-0084 was prepared from compound 5 in three steps as shown below:

[0298]

[0299] In the first step, compound 7 was prepared from compound 5. DMF (20.0 L, 3.43 vol), 1,2-dibromoethane (7.36 kg, 39.2 mol, 200 mol%), potassium carbonate (6.76 kg, 48.9 mol, 250 mol%) and the first portion of compound 5 (3.01 kg, 10.1 mol, 52 mol%) were added to a 100 L reactor. 1 H NMR (500MHz, DMSO-d6): δ13.02(s,1H), 5.55(s,1H), 4.44-3.91(m,4H), 3.83-3.57(m,4H), 1.52(s,6H). 13C NMR (125 MHz, DMSO-d6): δ 157.7, 153.4, 153.0, 151.7, 117.2, 68.4, 66.0, 45.3, 29.3). The mixture was heated to 103°C, maintained at this temperature for 2 h, and then cooled to 50°C. HPLC IPC showed that compound 5 was 0.05A%, and impurity 4 was 3.9A%. A second portion of compound 5 (2.82 kg, 9.47 mol, 48 mol%) was charged to the reactor. The mixture was heated to 85°C, maintained at this temperature for 16 h, and then cooled to 53°C. HPLC IPC showed that compound 5 was 0.05A% and impurity 4 was 0.05A%. The reaction was cooled to 21°C, and ethyl acetate (19.5 L) and purified water (40.0 L) were added. The water layer was removed and additional ethyl acetate (19.5 L) and purified water (20.0 L) were added to the reactor. Remove the second aqueous phase. GC IPC shows that DMF is 0.7wt% in the organic solution. The solution is transferred from the 100L reactor to the 50L reactor together with ethyl acetate rinse (1.0L). Distill to the minimum stirring volume (9L) and add 2-propanol (12L). Distill again to the minimum stirring volume (9L) and add 2-propanol (30L). GC IPC shows that ethyl acetate is 0.06wt%. The 2-propanol suspension (39L) is then heated to 65°C, kept at this temperature for 2h, cooled to 5°C over 1h, and the mixture is kept at a reduced temperature for 2h. HPLC IPC shows that the product concentration in the supernatant is 14mg / g. Filter the mixture with an Aurora filter and wash the filter cake with isopropanol (16L). After the filtrate can no longer be collected from the filter, the filter cake is dried on a filter at 50±5°C (cooling water temperature in the water jacket) under a nitrogen-purged indoor vacuum. HPLC IPC shows that impurity 3 is <1.0A%. Drying was continued until GC IPC showed 0.91 wt% of 2-propanol. This process yielded 4.27 kg of compound 7 product as a light yellow solid (yield 67%; 97.8 A% by HPLC). 1 H NMR (500MHz, DMSO-d6): δ4.75-4.00(m,8H), 3.73-3.71(m,4H), 1.56(s,6H). 13 C NMR (125MHz, DMSO-d6): δ152.9, 151.7, 151.6, 151.5, 117.3, 73.6, 66.0, 57.6, 45.3, 41.6, 27.2.

[0300] In the second step, crude GDC-0084 was prepared from compound 7. A glass acid bottle was charged with K3PO4·H2O (5.84 kg, 24.8 mol, 200 mol%) in purified water (8.45 L). The contents were stirred until homogeneous and then sparged with nitrogen for ≥ 1 h. A 100 L reactor was charged with K3PO4 aqueous solution, compound 7 from the first step (4.10 kg, 12.7 mol, 100 mol%), pinacol borate (3.36 kg, 15.2 mol, 120 mol%) ( 1 H NMR (500MHz, DMSO-d6): δ8.38(s,2H), 7.02(s,2H), 1.27(s,12H). 13C NMR (125 MHz, DMSO-d6): δ 164.6, 163.9, 107.8, 83.4, 24.6) and THF (55.8 L). The contents were bubbled with N2 for 45 min, then PdCl2dppf·CH2Cl2 catalyst (0.207 kg, 0.25 mol, 2 mol%) was added. The mixture was bubbled with N2 for 10 min, heated to ≥61°C, and maintained at this temperature for 4 h. HPLC IPC showed compound 7 at 0.027 mg / mL. Purified water (45.8 L) was added and the reaction mixture was cooled to 7°C and maintained at this temperature for 1 h. The reaction mixture was filtered through an Aurora filter and the filter cake was washed with purified water (4×30.0 L). After no more filtrate could be collected from the filter, the filter cake was dried on the filter under N2-purged house vacuum at 70°C (cooling water temperature in the water jacket). Drying was continued for 8 h. HPLC IPC impurity 2 is 0.4A%. The contents of the Aurora filter (crude GDC-0084) are transferred to a 100L reactor and mixed with Si-Thiol (2.22kg), Si-Thiourea (2.22kg), acetic acid (17.5L), toluene (7.5L) and pure water (0.25L) to form a mixture. The mixture is heated to 90°C and maintained at this temperature for 3h. The mixture is transferred to a Nutsche filter together with acetic acid (7.5L) / toluene (3.5L) rinses (2x), and the hot filtrate (about 70°C) is directly passed through an online filter (polishing filtration) into a 50L reactor. Metal analysis IPC shows residual Pd≤3ppm. Distillation is performed to a minimum stirring volume (10L), and the contents are then heated to 70°C. 2-Propanol (40.0 L) was added to the reactor through an in-line filter (polishing filtration) and the resulting suspension was heated to 70°C, maintained at this temperature for 1 h, cooled to 18°C ​​over 4 h, and maintained at the reduced temperature for 9 h. HPLC IPC showed a concentration of 1.5 mg / g of GDC-0084 in the supernatant. The mixture was filtered through a filter dryer and the filter cake was washed with isopropanol (48 L). After no more filtrate could be collected from the filter, the filter cake was dried in a filter dryer at 70°C (cooling water temperature in the water jacket) under room vacuum purged with N2. GC IPC showed 0.52 wt% isopropanol and 0.44 wt% acetic acid. The process gave 3.87 kg of GDC-0084 as a light yellow solid (80% yield in step 2; HPLC 99.4A%). 1 H NMR (500MHz, DMSO-d6): δ9.09(s,2H), 7.03(s,2H), 4.32-4.17(m,4H), 4.17-4.04(m,4H), 3.84-3.65(m,4H), 1.58(s,6H). 13C NMR (125MHz, DMSO-d6): δ163.8, 157.6, 154.2, 152.5, 151.3, 151.0, 120.3, 117.3, 73.7, 66.2, 57.8, 45.2, 41.5, 27.3.

[0301] The combined total synthesis of the first and second steps yielded 3.87 kg (99.6% purity) of GDC-0084, with a yield of 55%. The measured impurities included impurity 2 (0.08A%) and impurity 6 (0.24A%) (as shown below); total undetermined impurities <0.05A%. The final solvent content was 1 wt%, including 0.57 wt% (i-PrOH) and 0.43 wt% HOAc, the final water content was 0.09 wt%, and the final residual Pd level was <3 ppm.

[0302]

[0303] Example 12: Preparation of 2-chloro-6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine

[0304] The method of converting compound 5 to compound 7 using a phase transfer catalyst was evaluated. More specifically, compound 5 was reacted with 1,2-dibromoethane in a toluene / alkaline water solvent system in the presence of a phase transfer catalyst Aliquat 336 (containing C8 and a lesser amount of C 10 Compound 7 was prepared by condensation in the presence of a quaternary ammonium salt of a mixture of chains; 1-octanium, N-methyl-N,N-dioctyl chloride), and about 50% conversion to compound 7 was achieved.

[0305] Solvent screening was then completed by condensation of compound 5 with 1,2-dibromoethane in each of chlorobenzene, THF, Me-THF, DCM, and DCE to prepare compound 7. No conversion was achieved in DCE, partial conversion was achieved in chlorobenzene, and for the remaining solvents, the conversion was lower than that in toluene / alkaline water.

[0306] The preparation of compound 7 by condensation of compound 5 with 1,2-dibromoethane in a basic aqueous solvent in the absence of an organic solvent and in the presence of a phase transfer catalyst, tetrabutylammonium bromide (TBAB) was evaluated. The condensation reaction was found to be complete in an aqueous solvent without an organic co-solvent. Base screening experiments were performed using the bases KOH, NaOH, K2CO3, and NaHCO3, and each base was found to show similar reactivity and provide complete conversion with a similar purity spectrum. KOH was selected for further evaluation due to its highest water solubility.

[0307] The reagent stoichiometry evaluation was performed as summarized in Table 5 below, and 1 equivalent of compound 5 was converted to compound 7 by condensation with 1,2-dibromoethane in a basic aqueous solvent in the presence of TBAB phase transfer catalyst. In the reaction, 0.34 mmol of compound 5, KOH, TBAB, and 1,2-dibromoethane were combined with 10 mL of solvent in a vial at ambient temperature, and the reaction mixture was heated to 90° C. with vigorous stirring for 17 hours. The conversion was determined by HPLC. In Table 5, "Exp" refers to experimental, "1,2-DBE" refers to 1,2-dibromoethane, "equiv" refers to equivalent, and "Conv.%" refers to the conversion of 2-(2-chloro-6-morpholinyl-9H-purin-8-yl)propan-2-ol.

[0308] Table 5

[0309] Exp 1,2-DBE mol% KOH mol% TBAB mol% Conv.% 1 200 200 15 52 2 200 400 30 45 3 400 200 30 68 4 400 400 15 87 5 400 400 30 100 6 300 300 30 100

[0310] The data show that complete conversion is achieved with a combination of equimolar amounts of 1,2-dibromoethane and KOH with a catalytic amount of TBAB (0.3 equiv.). Optimization of the reagent stoichiometry helps drive the reaction to completion. One set of optimized conditions was determined to be equal amounts of 1,2-dibromoethane and KOH (300 mol % each) and a catalytic amount of TBAB (30 mol %) in water at 90°C for 17 hours. It is believed that the use of excess base and 1,2-dibromoethane minimizes the competitive formation of vinyl bromide.

[0311] Example 13: Reaction Temperature Study

[0312] Reaction temperature evaluation was performed as summarized in Table 6 below, and compound 5 was converted to compound 7 by condensation with 1,2-dibromoethane (400 mol%) in a basic water solvent (400 mol% KOH) in the presence of TBAB phase transfer catalyst (30 mol%) for 22 hours. The results are also recorded in Table 6 below, where "Exp" refers to experimental and "A%" refers to the area % of 2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine analyzed by HPLC.

[0313] Table 6

[0314] Exp. temperature A％ Exp. temperature A％ 1 90.0 80.9 4 60.0 92.9 2 80.0 89.9 5 50.0 96.0 3 70.0 91.2 --- --- ---

[0315] The results show that a decrease in reaction temperature from 90°C to 50°C leads to an improved impurity profile.

[0316] Example 14: Crystallization Solvent Studies

[0317] The phase transfer catalyst reactions of Examples 12 and 13 resulted in the separation of the compound 7 product as an oil containing a significant 1,2-dibromoethane concentration from the aqueous layer. In those reactions, 2-propanol was added to promote product precipitation (crystallization). Seeding with 1% of the product seed crystals slightly improved the crystallization, but oil was observed in some of the products. It was found that replacing isopropanol with ethanol allowed a substantially clean separation of the product in solid form from the product mixture, with a water to ethanol ratio of 1.3:1 providing the cleanest separation.

[0318] Example 15: Study of reaction conditions

[0319] The reaction conditions for preparing compound 7 found in Examples 12 to 14 that provide relatively high conversions were evaluated by the reaction of compound 5 (480 g), 1,2-dibromoethane (300 mol%), KOH (300 mol%), TBAB (30 mol%), and H2O (5 vol), wherein the amount of equivalents of the reactants is based on the equivalents of the starting materials. The reaction was carried out at 50° C. for 12 hours, after which EtOH (5.5 vol) was added to crystallize the product. Compound 7 was formed with a yield of 64% and a purity of 99.4A% as determined by HPLC.

[0320] Example 16: Pd Catalyst Studies

[0321] Various palladium-containing catalysts for preparing GDC-0084 from compound 7 were evaluated. For each experimental reaction, 3 mmol of compound 7 was mixed with THF (8 mL), water (1.2 mL), K3PO4 (6 mmol-200 mol%) and pinacol borate (4.0 mmol-130 mol%). The reaction was carried out at 65°C for 5 hours. For product crystallization, water (7.1 mL) was added to the reaction product mixture at 50°C, solidified at this temperature for 1 hour, and cooled to 20°C. The reaction product was separated by filtration and washed 3 times with 1 volume of water. Each time with water. The results are recorded in Table 7, where "Exp" refers to the experimental number; "Cat.Eq." refers to the equivalent of the catalyst; "Add.Lig." refers to the mol% of the added ligand; "Lig.Eq." refers to the equivalent of the ligand; "BaseEq." refers to the equivalent of the base; and "Cov.%" refers to the conversion percentage of compound 7 determined by HPLC.

[0322] Table 7

[0323]

[0324]

[0325] PdXPhos was the most active catalyst, providing full conversion, and the catalytic activity was maintained at a concentration of 0.5 mol% (0.5 mol%) even in the absence of added ligand (Experiment 6). Reduction of the PdXPhos catalyst loading to 0.3 mol% (0.3 mol%) decreased the conversion. Pd(amphos)C 12 (Experiment 2) and PdCl2(t-Bu2PhP)2 catalysts were inactive at the concentrations evaluated.

[0326] Example 17: Catalyst Optimization Study

[0327] The reaction conditions found in Example 16 that provided the highest conversion for the preparation of GDC-0084 from a reaction mixture comprising compound 7 (78 g) starting material, pinacol borate (120 mol%), THF (8 vol %), H2O (1.2 vol), PdXphos (0.5 mol%), and K3PO4 (200 mol%) were evaluated. The reaction was carried out at 65°C for 4 hours. The reaction mixture was purified by the Si-thiol purification method of Example 6, but only 10 g of Si-Thiol was used. The Si-Thiourea purification step of Example 6 was not performed in this example. GDC-0084 was formed in a yield of 94% and a purity of 99.3A% as determined by HPLC, with residual Pd of 815 ppm.

[0328] Compared to Example 6, this example replaces the PdCl2dppf·CH2Cl2 catalyst with a more reactive PdXPhos catalyst, reduces the palladium loading from 2 mol% (2 mol%) to 0.5 mol% (0.5 mol%), eliminates the Si-Thiourea scavenger, reduces the total scavenger loading by approximately 90%, and reduces the total solvent by 71%, while providing comparable yield and purity.

[0329] Example 18: Purification by crystallization

[0330] Purification of crude GDC-0084 by crystallization can be suitably accomplished by crystallization from an acetic acid-water solvent. However, GDC-0084 may react with acetic acid to form the acetamide impurity 6. In a first evaluation, the formation of impurity 6 in a solution with GDC-0084 (2.6 mmol), acetic acid (3.6 mL), toluene (1.6 mL) and trace water (0.01 mL) was evaluated over time at 90°C. The results are recorded in Table 8 below, where "% Acet." refers to the acetamide percentage.

[0331] Table 8

[0332]

[0333]

[0334] In the second evaluation, toluene was removed and the formation of impurity 6 of GDC-0084 over time was evaluated in a solution of 11 volumes of acetic acid and water at 70°C. Four ratios of acetic acid to water were evaluated, including 98:2, 9:1, 4:1, and 1:1. The results are shown in Figure 1 As shown, it is shown that with the increase of water amount, the amount of acetamide (impurity 6) formed after 6 hours is reduced by 6 times, from 0.6 area % (by HPLC) to 0.1 area %. The results further show that when the ratio of acetic acid to water is less than 4:1 at 70°C, the acetamide impurity is less than 0.15A%.

[0335] Further development showed that GDC-0084 was completely soluble in 10 volumes of acetic acid: water (3:1) at 90° C. and crystallized out at 60° C. The 30° C. temperature width of the metastable zone was considered sufficient for polishing filtration at 90° C. Based on the reduction in Pd loading from 2 to 0.5 mol %, it was found that treatment with only 10 wt % of Si-thiol was sufficient to reduce residual Pd to below 10 ppm.

[0336] Under the conditions obtained in Examples 12-18 for the preparation of GDC-0084 from compound 5, including (i) cyclization of compound 5 with 1,2-dibromoethane in water using a phase transfer catalyst to generate compound 7, (ii) Suzuki cross-coupling with pinacol boronate using 0.5 mol% of XPhos Pd G2 catalyst to provide crude GDC-0084; and (iii) final cleanup / recrystallization from acetic acid / water to obtain GDC-0084 in 52% yield and 99.7 area% purity in polymorphic form. By adjusting the crystallization solvent composition, the acetamide impurity 6 was reduced from 0.25 area% to less than 0.05 area% (HPLC).

[0337] Compared to the preparation of GDC-0084 from compound 5 according to Examples 5 and 6, under the conditions of preparing GDC-0084 from compound 5 in Examples 12 to 18: (i) in stage 1 (preparation of compound 7 from compound 5), DMF and EtOAc were removed from the process, the total solvent volume was reduced by 54%, and all extractions and solvent exchanges were eliminated; (ii) in stage 2 (compensation of crude GDC-0084 from compound 7), the total solvent volume was reduced from 58 to 17 volumes (reduced by 71%); and (iii) in stage 3 (crude GDC-0084 purification process), toluene was removed and the total solvent volume of recrystallization was reduced from 33 to 21 volumes (reduced by 37%). In general, the total number of unit runs was reduced from 21 to 7, and the total solvent volume of the three stages was reduced by 64%, eliminating toluene and DMF. As a result, the process quality intensity (PMI-see Concepcion. J., et al., "Using the Right Green Yardstick: Why Process Mass Intensity is Used in the Pharmaceutical Industry to Drive More Sustainable Processes", Org. Process Res. Dev., 2011, 912-917 (see Henderson, RK et al., "Lessons Learned through Measuring Green Chemistry Performance: The Pharmaceutical Experience", American, Dev., 2011, 912-917) was reduced from 140 to 70, which is within the practical range of commercial processes (see Henderson RK, et al., "Lessons Learned through Measuring Green Chemistry Performance: The Pharmaceutical Experience", American Chemical Society, Green Chemistry Institute, Pharmaceutical Roundtable: www.acs.org / green-chemistry ,2008).

[0338] Example 19: Preparation of 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine

[0339] Purified GDC-0084 was prepared from compound 5 in a three-step process as shown below:

[0340]

[0341] In the first step, water (45.1 kg, 4.8 volumes), potassium hydroxide (5.4 kg, 96.2 mol, 304 mol%), compound 5 (9.40 kg, 31.6 mol, 100 mol%), tetrabutylammonium bromide (2.92 kg, 9.06 mol, 28.7 mol%) and 1,2-dibromoethane (17.7 kg, 94.2 mol, 298 mol%) were added to a 100 L reactor. The mixture was heated to 47 ° C and kept at this temperature for 20 h. HPLC IPC showed that compound 5 was 3.1A%. Ethanol (46.2 kg, 6.2 vol) and compound 7 seeds (96.6 g, 1 wt%) in ethanol (0.60 kg, 0.06 volume) were added. The reactor contents were kept for 2 h, then cooled to 5 ° C and kept for 1 h over 2 h. The mixture was filtered through a filter dryer and washed with water (27.4 kg, 2.9 volumes). After no more filtrate could be collected from the filter, the filter cake was dried on the filter at 60°C (cooling water temperature in the water jacket) under nitrogen purged house vacuum. This process afforded 6.85 kg of compound 7 as a light yellow solid (6.85 kg, 67% yield; 98.5 A% by HPLC). The following impurities were detected at low levels: compound 5 (0.43 A%); impurity-3 (0.22 A%); impurity-4 (0.1 A%); and impurity-3 (0.22 A%). Melting point 147°C; 1 H NMR (500MHz, DMSO-d6) δ4.75-4.00(m,8H), 3.73-3.71(m,4H), 1.56(s,6H); 13 C NMR (125MHz, DMSO-d6) δ152.9, 151.7, 151.6, 151.5, 117.3, 73.6, 66.0, 57.6, 45.3, 41.6, 27.2. HRMS[M+H] + Calculated as C 14 H 18 ClN5O2 324.1222; found 324.1225.

[0342] In the second step, water (8.00 kg, 1.2 volumes), THF (39.2 kg, 6.5 volumes), potassium phosphate monohydrate (9.51 kg, 40.4 mol, 194 mol%), compound 7 (6.75 kg, 20.85 mol, 100 mol%) and pinacol borate (5.50 kg, 24.88 mol, 119 mol%) were added to a 100 L reactor to form a mixture. The mixture was cycled from vacuum to nitrogen three times, then Xphos Pd G2 (82.0 g, 0.104 mol, 0.5 mol%) was added. The mixture was cycled from vacuum to nitrogen three times, heated to ≥ 67 ° C, and kept at this temperature for 5 h. HPLC IPC analysis showed complete conversion. Pure water (48.1 kg, 7.1 volumes) was added and kept at 50 ° C for 1 h. The reaction was cooled to 20 ° C over 2 h, kept at 20 ° C for more than 2 h, cooled to 5 ° C, and kept at this temperature for 2 h. The mixture was filtered on a filter dryer and the filter cake was washed with water (37.1 kg, 5.5 volumes). After no more filtrate could be collected from the filter, the filter cake was dried on the filter under nitrogen purged house vacuum at 60° C. (cooling water temperature in the water jacket). Crude GDC-0084 (7.49 kg, 94% yield, 99.4A% by HPLC) was obtained as an off-white solid.

[0343] In the third step, water (7.75 kg, 1 volume), acetic acid (60.8 kg, 7.5 volumes), crude GDC-0084 (7.70 kg, 20.13 mol, 100 mol%) and Si-Thiol (770 g, 10 wt%) were added to a 100 L reactor to form a mixture. The mixture was heated to 90° C. and then maintained at that temperature for 3 h. The contents were filtered through an Aurora filter and then through a 1 μm polishing filter, and the filter was rinsed with hot acetic acid (7.10 kg, 0.9 volumes). The resulting solution was then cooled to 77° C., and GDC-0084 seeds (82 g, 1.1 wt%) were added as a slurry in acetic acid (69 g) and water (87 g). The contents were maintained at 68° C. for 1 h. Purified water (12.0 kg, 1.6 volumes) was added to the slurry, and the slurry was cooled to 45°C, maintained at 45°C for 1 hour, cooled to 20°C over 2 hours, maintained at 20°C for 6 hours, cooled to 5°C over 2 hours, and maintained at 5°C for 2 hours. The slurry was filtered on a filter dryer and the filter cake was washed with water (69.9 kg, 9.1 volumes). After no more filtrate could be collected from the filter, the filter cake was dried on a filter at 60°C (cooling water temperature in the water jacket) under a nitrogen-purged house vacuum. GDE-0084 (6.41 kg, 83% yield, 99.7 A%) was obtained as an off-white solid. Melting point 211°C: 1H NMR (500MHz, DMSO-d6) δ9.09(s,2H), 7.03(s,2H), 4.32-4.17(m,4H), 4.17-4.04(m,4H), 3.84-3.65(m,4H), 1.56(s,6H); 13 C NMR (125MHz, DMSO-d6) δ163.8, 157.6, 154.2, 152.5, 151.3, 151.0, 120.3, 117.3, 73.7, 66.2, 57.8, 45.2, 41.5, 27.3. HRMS[M+H] + Calculated as C 18 H 22 N8O2 383.1938; found 383.1945. Residual Pd level was less than 10 ppm.

[0344] Compared to the scavenging method of Example 6, THF was removed from the solvent system, eliminating the Si-thiourea scavenging step and reducing the loading of the Si-thiol scavenger by 90%.

[0345] Example 20: Determination of blood-brain barrier penetration of 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine

[0346] The ability of GDC-0084 (Compound IIIat) to penetrate the mouse blood-brain barrier (BBB) ​​was determined by evaluating the ratio of unbound brain to unbound plasma concentration (Bu / Pu) in female CD-1 mice. The ability of Compound IIIat to penetrate the rat blood-brain barrier (BBB) ​​was determined by evaluating the concentration of Compound IIIat in the cerebrospinal fluid of male Sprague-Dawley rats. The results are listed in Table 9 below.

[0347] For mouse studies, the [brain] / [plasma] ratio was determined after oral administration of 25 mg / kg Compound III to female CD-1 mice. MCT refers to the indicated drug dose in 0.5% methylcellulose and 0.2% Tween 80. [Brain] u and [plasma] uRefers to the unbound concentration measured in brain and plasma, respectively. [Brain] / [Plasma] ratio is the average value of 3 animals at each time point measured at 1 hour and 6 hours after administration. Data show that the Bu / Pu ratio is 0.41 at 1 hour and 6 hours, thus proving that compound IIIat can penetrate the brain in large quantities. For male Sprague-Dawley rat studies, the concentration of compound IIIat in cerebrospinal fluid (CSF) was determined, and the [brain] / [plasma] ratio was evaluated after administering an oral dose of 15 mg / kg of compound IIIat as an MCT suspension. [Brain] / [plasma] and 1 animal was measured in each of 0.25 hours and 2 hours and 3 were measured at 8 hours, and the recorded data were the range across three time points (the average value of 3 animals at 8 hours).

[0348] By using HTDialysis 96-well blocks ( The extent of protein binding was determined in vitro in mouse plasma (Bioreclamation, Inc., Hicksville, NY) by equilibrium dialysis with ELISA kit (Bioreclamation, Inc., Hicksville, NY). Compounds were added to pooled plasma from multiple animals (n≥3) at a total concentration of 10 μM. Plasma samples were equilibrated with phosphate-buffered saline (pH 7.4) at 37°C, 90% humidity and 5% CO2 for 4 hours. After dialysis, the concentrations of compounds in plasma and buffer were measured by LC-MS / MS. The percentage of unbound in plasma was determined by dividing the concentration measured in the post-dialysis buffer by the concentration measured in the post-dialysis plasma and multiplying by 100. Incubations were performed in triplicate with a coefficient of variation of no greater than 30%.

[0349] The free fraction in mouse brain was determined as described by Kalvass. Briefly, brain tissue was homogenized in 3 volumes of phosphate buffered saline and compounds were added at a final concentration of 10 μM. 300 μl aliquots were dialyzed in 500 μl buffer at 37°C for 4 h in an incubator at 90% humidity and 5% CO2 in a RED apparatus (Thermo Scientific, Rockford, IL). After dialysis, tissue and buffer samples were analyzed as described for plasma protein binding studies.

[0350] Twelve female CD-1 mice (Charles River Laboratories, Hollister, CA) were given oral (PO) doses of the indicated compounds in 0.5% methylcellulose / 0.2% Tween 80 (MCT). Two blood samples of approximately 0.15 mL were collected from each mouse (n=3 mice at each time point) by retro-orbital bleeding or terminal cardiac puncture while the animals were anesthetized with isoflurane. Blood samples were collected in tubes containing K2EDTA as an anticoagulant, pre-dosing and 0.083, 0.25, 0.5, 1, 3, 6, 9 and 24 h after dosing. Samples were centrifuged within 1 h of collection, and plasma was collected and stored at -80 ° C until analysis. After plasma protein precipitation using acetonitrile, the total concentration of the compound was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the supernatant was injected onto a column, a Varian MetaSil AQ C18 column (50×2 mm, 5 μm particle size). A CTC HTS PAL autosampler (LEAP Technologies, Chapel Hill, NC) connected to a Shimadzu SCL-10A controller (Shimadzu, Columbia MD) with an LC-10AD pump was used for LC-MS / MS analysis with an AB Sciex API 4000 triple quadrupole mass spectrometer (AB Sciex, Foster City, CA). The aqueous mobile phase was water containing 0.1% formic acid, and the organic mobile phase was acetonitrile containing 0.1% formic acid. The lower and upper limits of quantification determined were 0.005 μM and 10 μM, respectively. The total run time was 1.5 min, and ionization was performed in positive ion mode. In the case of determining brain concentration, brains were collected at 1 and 6 hours after doses in 3 different animals at each time point, rinsed with ice-cold saline, weighed and stored at -80°C until analysis. For compound quantification, mouse brains were homogenized in 3 volumes of water. The homogenate was extracted by protein precipitation with acetonitrile. LC-MS / MS analysis was performed as described for plasma. Brain homogenate concentrations were converted to brain concentrations to calculate brain to plasma ratios.

[0351] The results are shown in Table 9. The total brain to plasma ratio was 1.4 for mice and 1.9-3.3 for rats. u / [plasma] u is 0.41, [CSF] / [plasma] in rats u0.73-1.0. Although rat brain protein binding has not been measured, CSF concentration has been determined as a surrogate for unbound brain concentration. See Liu, X., et al., Unbound Drug Concentration in Brain Homogenate and Cerebral Spinal Fluid at Steady State as a Surrogate for Unbound Concentration in Brain Interstitial Fluid, Drug Metab. Dispos. 2009, 37, 787-793. [CSF] / [Plasma] u The concentration ratio was 0.73-1.0, indicating that compound IIIat effectively crossed the BBB of rats.

[0352] Table 9

[0353] type [Brain] / [Plasma] <![CDATA[[Brain] u / [Plasma] u > <![CDATA[[CSF] u / [plasma] u ]]> Mouse 1.4 0.41 ---- Rat 1.9-3.3 ---- 0.73-1.0

[0354] The effect of compound IIIat on pAKT in normal brain tissue was evaluated, expressed as the ratio of phosphorylated AKT (pAKT) to total AKT (tAKT). AKT is critical for proliferation and anti-apoptotic signaling pathways, and increased AKT activation by phosphorylation has been found to be involved in a variety of tumor formations. In the evaluation, female CD-1 mice were given a single PO dose of the specified compound. Brain and plasma were collected from 3 animals at each time point at the specified time after administration. The brain was divided into half for PD analysis and compound concentration measurement. The samples were stored at -70 ° C and analyzed for total concentration. For PD analysis, a cell extraction buffer (Invitrogen, Camarillo, CA) containing 10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS and 0.5% deoxycholate was supplemented with phosphatase, protease inhibitors (Sigma, St. Louis, MO) and 1 mM PMSF and added to frozen brain biopsies. The brain was homogenized with a small pestle (Konte Glass Company, Vineland, NJ), briefly sonicated on ice, and centrifuged at 20,000 g for 20 min at 4°C. Protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). Proteins were separated by electrophoresis and transferred to NuPage nitrocellulose membranes (Invitrogen, Camarillo, CA). The Licor Odyssey infrared detection system (Licor, Lincoln, NE) was used to assess and quantify protein expression. ser473 PI3K pathway markers were assessed by immunoblotting with antibodies against AKT and total Akt (Invitrogen, Camarillo, CA and Cell Signaling, Danvers, MA). Inhibition (%) of pAkt was calculated by comparing the pAkt signal to that measured in untreated mice.

[0355] Figure 2 Results of oral administration of compound IIIat at a dose of 3 mg / kg or 10 mg / kg are shown, where pAKT in normal mouse brain tissue was measured and determined to be inhibited 1 hour after administration. At 4 hours after administration, the 3 mg / kg dose no longer resulted in inhibition of pAKT compared to the 10 mg / kg dose. The results indicate that compound IIIat engages its target behind a completely intact BBB and therefore freely penetrates the mouse brain.

[0356] Example 21: Evaluation of 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) on glioblastoma

[0357] The in vivo efficacy of compound GDC-0084 (Compound IIIat) against U87 MG Merchant (MG / M) human glioblastoma xenografts was evaluated in a dose escalation study in Taconic female NCR nude mice bearing subcutaneous tumors.

[0358] All in vivo studies were conducted in compliance with the requirements of Genentech's Institutional Animal Care and Use Committee. PTEN-null U-87MG / M human glioblastoma cancer cells (an in-house derivative of U-87MG cells from the American Type Culture Collection (Manassas, VA)) were cultured in RPMI 1640 medium containing 1% L-glutamine and 10% fetal bovine serum (Hy Clone; Waltham, MA). Cells growing in the logarithmic phase were harvested and resuspended in HBSS:Matrigel (BD Biosciences; Franklin Lakes, NJ) (1:1, v:v) for injection into 20-week-old female NCr nude mice (Taconic Farms, Cambridge City, IN). Animals received 5 million cells subcutaneously in the right thorax in 0.1 mL. The established tumors ranged from 200-500 mm 3 Mice with tumors of equal size were divided into groups (n=6-7 / group) to receive increasing doses of 16. The inhibitor was prepared weekly in 0.5% methylcellulose and 0.2% Tween-80 at the concentration required for the target dose in a volume of 0.2 mL. All preparations were stored in a refrigerator and allowed to reach room temperature and mixed thoroughly by vortexing, then administered by gavage once a day for 23 days before oral administration. The formula used was: tumor volume (mm 3 )=0.5×(length×width 2 ) Tumor volume was calculated from vertical length and width thickness measurements. Changes in body weight were recorded as percentage change from starting body weight.

[0359] Adopt mixed modeling method to analyze the tumor volume of repeated measurement of same animal over time, because this method solves the appropriate dropout before repeated measurement and research end (Pinheiro etc., 2008).For dosage and fixed time effect, Log2 (tumor volume) growth trace is applicable to each dosage group with restricted cubic spline.In R 2.13.0 version (R Development Core Team 2008; R Foundation for Statistical Computing; Vienna, Austria), use R package nlme (version 3.1-97) to fit by linear mixed effect model.The tumor volume of assembly is plotted with natural scale in Prism (version 5.0b of Mac version) (GraphPad Software; La Jolla, CA).Also use linear mixed effect analysis, it uses R to analyze the repeated measurement of weight change from same animal over time.

[0360] Mice bearing tumor xenografts were administered with a dose rate of 0.45 mg / kg, 2.2 mg / kg, 4.5 mg / kg, 8.9 mg / kg, 13.4 mg / kg or 17.9 mg / kg of compound IIIat on days 0, 2, 4, 7, 9, 11, 13, 16, 19 and 22, wherein compound IIIat was a suspension in a vehicle (0.5% methylcellulose / 0.2% Tween-80). A control group of mice was administered a vehicle in the absence of drug under the same dosing schedule. Figure 3 The dose changes in tumor volume over time for each compound are depicted as cubic spline fits generated by linear mixed effects analysis of log-transformed volumes.

[0361] Compound IIIat achieved significant and dose-dependent tumor growth inhibition. Tumor growth inhibition was first observed at the 2.2 mg / kg dose level. Higher doses resulted in greater tumor growth inhibition, including tumor regression at the 17.9 mg / kg dose level. Each dose was well tolerated during the study. Compound IIIat was found to have an antiproliferative EC of 740 nM in U87 cells. 50 .

[0362] Example 22: Evaluation of the therapeutic effect of 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine on pAKT

[0363] The effect of compound IIIat on the pharmacodynamic (PD) marker pAKT in the U87 MG / M human glioblastoma xenograft model was evaluated after continuous administration for 24 days at dose rates of 0.5 mg / kg, 3 mg / kg, 10 mg / kg and 18 mg / kg. Tumors were removed from animals 1 hour and 4 hours after the last dose on day 24 and processed for analysis of pAKT and total AKT. Results Figure 4 pAKT was measured by electrochemiluminescence using the Meso Scale Discovery according to the manufacturer's instructions (Gaithersburg, MD). Ser473 and total AKT levels.

[0364] Compound IIIat was found to have a significant PD effect in U87 tumors. Dose- and concentration-dependent inhibition of pAKT was observed at 1 and 4 hours after administration, indicating that tumor growth inhibition was a result of targeted inhibition.

[0365] Example 23: Assessment of Kinase Inhibition by GDC-0084

[0366] GDC-0084 (ie, compound IIIat) was evaluated for its inhibition of 229 kinases of class I PI3K Kiapp. The percentage of inhibition of 229 kinases by 1 μM GDC-0084 is recorded in Table 10 below:

[0367] Table 10

[0368]

[0369]

[0370]

[0371] The selectivity of GDC-0084 for class I PI3K kinases was evaluated and the results are recorded in Table 11 below:

[0372] Table 11

[0373] Class I PI3K kinases <![CDATA[Selective (Ki app )]]> PI3Kα 2nM PI3Kβ 46nM PI3Kδ 3nM PI3Kγ 10nM mTOR 70nM

[0374] Example 24: Stability Evaluation of 5-(6,6-dimethyl-4-morpholinyl-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine

[0375] The hepatocyte stability of compound IIIat was evaluated in preclinical species. Certain in vivo pharmacokinetic parameters were also evaluated. In this example, the in vivo t 1 / 2 Methods Prediction of liver clearance from hepatocyte incubation. 1 mg / kg of Compound IIIat prepared in 60% PEG400 / 10% ethanol was administered intravenously to male Sprague-Dawley rats, female CD-1 mice, male cynomolgus monkeys, and beagle dogs. Compound IIIat was administered orally (PO) at the specified doses in 0.5% methylcellulose and 0.2% Tween 80 (MCT). The results are recorded in Table 12 below, where "Cyno" refers to cynomolgus monkeys; "Cl hep " refers to the hepatocyte clearance rate, in mL / min / kg; "in vivo Cl" refers to the in vivo clearance rate after IV administration, in mL / min / kg; the IV dose is 1 mg / kg; "Vss" refers to the apparent distribution volume at steady state, in L / kg; oral doses are recorded in mg / kg; "Cmax" refers to the peak serum concentration expressed in μm; "AUC" refers to the area under the concentration of compound IIIat in plasma versus time curve and is expressed in μm·h; "F%" refers to the percentage of drug bioavailability; "PPB%" refers to the percentage of drug bound to plasma proteins.

[0376] Table 12

[0377]

[0378] With the exception of rats, there was good correlation between the predicted clearance based on hepatocyte stability and the in vivo clearance.

[0379] Example 25: Human Phase I Trial

[0380] An open-label, multicenter, phase I dose-escalation study was conducted using a standard "3+3" design to evaluate the safety, tolerability, and pharmacokinetics of GDC-0084 (compound formula IIIat). GDC-0084 is a potent, oral, selective, small molecule inhibitor of class I PI3K and mTOR kinases, with average apparent inhibition constants (Ki) of 2.2, 41, 2.7, and 9.7 nM for ρ110α / ρ85α, ρ110β / ρ85α, ρ110δ / ρ85α, and ρ110γ, respectively.

[0381] GDC-0084 was orally administered once daily in a 28-day continuous dosing cycle to a group of 47 patients with progressive or recurrent high-grade gliomas (WHO grade III-IV) who were treated during or after prior treatment with at least one glioma regimen containing radiation therapy and / or were not suitable for regimens known to provide clinical benefit. GDC-0084 is provided in three doses in a capsule formulation: 1 mg, 5 mg, and 25 mg. GDC-0084 capsules are stored at room temperature (59°F-86°F [15°C and 30°C]). Plasma samples for pharmacokinetic ("PK") analysis were collected on days 1 and 8 or 15 of cycle 1. Fluorodeoxyglucose positron emission tomography ("FDG-PET") was performed at baseline and during treatment.

[0382] The median time to first diagnosis was 40.5 months (range: 11-190 months). At enrollment in the study, 33 patients (70.2%) were classified as WHO grade IV gliomas and 14 patients (29.8%) were classified as WHO grade III gliomas. Of these patients, 55.3% had progressive disease, 40.4% had stable disease, 1 patient was not evaluable, and data for one patient were missing. Overall, all patients had received cancer surgery, radiotherapy, and systemic treatment. The median number of prior cancer surgeries was 2.0 (range: 1 to 6), the median number of prior radiotherapy was 1.0 (range: 1 to 2), and the median number of prior systemic treatments was 3.0 (range: 1 to 5).

[0383] Patients received GDC-0084 daily in a 28-day cycle (daily dosing for 4 weeks). On Day 1 of Cycle 1, GDC-0084 was administered in a clinical setting, which was adjusted to accommodate frequent blood draws up to 24 hours after the morning dose. When the study drug was taken at home, patients took GDC-0084 on the same day (±2 hours). The dosing time may have been adjusted to accommodate a time offset from the home dosing schedule (e.g., a clinical visit for PK sampling or movement), but the time should not exceed 4 hours at a time.

[0384] Unless otherwise indicated by the patient, GDC-0084 was taken on an empty stomach (i.e., approximately 1 hour before or 2 hours after a meal), except for those days when dosing was performed under fasting conditions (e.g., extensive PK sampling was performed during Cycle 1 as described elsewhere herein). For dosing under fasting conditions, patients fasted overnight for at least 8 hours before dosing and 2 hours after dosing. GDC-0084 capsules were swallowed whole (without chewing) with 240 mL (8 ounces) of water.

[0385] Dose escalation continued according to the dose escalation rule until the maximum tolerated dose (“MTD”) was exceeded, excessive medication burden (defined as the inability of 2 or more patients in the cohort to take ≥90% of the doses consisting of at least eight capsules) was declared, or analysis of available PK data indicated that exposure was unlikely to increase with further escalation of the GDC-0084 dose.

[0386] In the PK assessment, patients were treated with 8 dose groups of GDC-0084 at the following dose levels in a 28-day (once a day) cycle: Group 1 (2 mg); Group 2 (4 mg); Group 3 (8 mg); Group 4 (15 mg); Group 5 (20 mg); Group 6 (30 mg); Group 7 (45 mg); and Group 8 (65 mg). PK assessments were performed after administration of GDC-0084 in the fasting state on study days 1 and 8 (for groups 7-8) or day 15 (for groups 1-6). A single dose of GDC-0084 was administered orally on day 1 of cycle 1, followed by frequent blood sampling up to 72 hours for groups 1-6 and up to 24 hours for groups 7-8 to determine the PK characteristics of a single dose of GDC-0084. For groups 1-6 (2-30 mg), a single dose was given on day 1 of cycle 1, followed by a 7-day washout period, after which dosing continued once a day for 28 consecutive days starting on day 8. Blood samples for groups 1-6 for PK analysis were collected on day 15. For groups 7 and 8 (45-65 mg), subjects were dosed once a day starting on day 1 of cycle 1 for 28 days, and blood samples were collected on day 8 for multiple-dose PK analysis. The concentration of GDC-0084 in plasma samples was quantified using a validated LC-MS / MS assay with a lower level of quantification (LLOQ) of 0.00052 μM.

[0387] Plasma concentration-time data for GDC-0084 were tabulated and descriptive statistics were calculated and compared between groups. Mean plasma GDC-0084 concentration data were plotted by group versus nominal time. All plasma concentration-time data collected in cycle 1 were analyzed using the PCR amplification software (version 6.4, Pharsight Corp, Mountain View, CA) to estimate PK parameters including, but not limited to, AUC 0-最后 (where AUC is the area under the concentration-time curve) and / or AUC inf , C max , C min ,t max, half-life, CL / F, and cumulative ratio. Estimates of each PK parameter and summary statistics (mean, standard deviation, coefficient of variation, median, minimum, and maximum) are presented by dose level and schedule. Nominal time data were used in the analysis, and AUC was calculated using the linear up / down trapezoidal method.

[0388] The pharmacokinetic parameters of GDC-0084 after single and multiple doses are listed in Tables 13 and 14, respectively, where SD refers to standard deviation; %CV refers to coefficient of variation; ND refers to not determined; T 1 / 2 T refers to the terminal half-life; max Refers to the time to reach maximum plasma concentration; C max Refers to the maximum observed plasma concentration; AUC inf refers to the area under the concentration-time curve from time 0 to infinity; CL / F refers to apparent oral clearance; AUC refers to 0-24 Refers to the area under the concentration-time curve from time 0 to 24 hours; C min refers to the minimum concentration; and the accumulation ratio refers to AUC 0-24小时多剂量 / AUC 0-24小时单剂量 .

[0389] Table 13: PK parameter results after a single dose (Cycle I, Day 1)

[0390]

[0391]

[0392] Table 14: PK parameter results after multiple doses (Cycle 1 Day 15 for Groups 1-6, Cycle 1 Day 8 for Groups 7 & 8)

[0393]

[0394] a For Group 6 (30 mg), n=5.

[0395] The concentration-time curves of GDC-0084 after single and multiple doses are shown in Fig.13 and Fig.14 . Fig.13 Plotted are mean ± SD plasma concentrations of GDC-0084 versus time following a single dose (Cycle 1, Day 1). Fig.14 Figure 2 is a graph of ±SD plasma concentrations of GDC-0084 versus time after multiple doses (Cycle 1 Day 15 for Groups 1 to 6 and Cycle 1 Day 8 for Groups 7 and 8). After a single oral dose, GDC-0084 was rapidly absorbed with a median T max About 2 hours (range 1 to 8 hours). After reaching peak plasma concentration, the concentration decreases and the apparent terminal phase t 1 / 2Approximately 18.73 hours (range 3.41 to 47.3 hours; calculated across Groups 1-6 (2 to 30 mg) after a single dose).

[0396] Fig.15 The dose (mg) of single-dose ("SD") and multiple-dose ("MD") regimens is max (μM) Dose ratio of GDC-0084. Fig.16 Dose (mg) versus AUC for single-dose ("SD") and multiple-dose ("MD") regimens 24 (μM*11) GDC-0084 dose ratio diagram. The data show that the cumulative ratio (AUC 0-24小时多剂量 / AUC 0-24小时单剂量 ) ranged from 0.577 to 4.84, with a mean of 2.12 ± 0.896. For both single and multiple doses, in all groups, C on day 1 of cycle 1 max and AUC 0-24 Both increased in a dose-proportional and dose-linear manner. Fig.17 Mean single-dose (SD) plasma GDC-0084 concentration versus time on a logarithmic scale (Cycle 1, Day 1). Fig.18 Figure 2 is a plot of mean single-dose (SD) plasma GDC-0084 concentration versus time on a linear scale (Cycle 1, Day 1). Fig.19 Mean single-dose (SD) plasma GDC-0084 concentration versus time on a logarithmic scale (Cycle 1, Day 8 / 15). Fig. 20 Figure 2 is a plot of mean single-dose (SD) plasma GDC-0084 concentration versus time on a linear scale (Cycle 1, Day 8 / 15). Fig.21 AUC of GDC-0084 single-dose (SD) and multiple-dose (MD) regimens 0-24 Logarithmic scale plot of (μM*hr) versus dose (mg). Fig. 22 Figure 2 is a logarithmic scale plot of Cmax (μM) versus dose (mg) for GDC-0084 for single dose (SD) and multiple dose (MD) regimens.

[0397] In summary, concentrations from brain tumor tissues indicate that GDC-0084 crosses the blood-brain barrier and is evenly distributed throughout the brain. The experimental results show that GDC-0084 inhibits human mTOR with an average apparent Ki of 70 nM. GDC-0084 is rapidly absorbed and exhibits a linear and dose-proportional increase in exposure with a half-life (t 1 / 2approximately 19 hours) supports once-daily dosing. The MTD was determined to be 45 mg when GDC-0084 was taken orally once daily in a 28-day cycle. At a dose of 45 mg, steady-state concentrations were consistent with the antitumor activity observed in the xenograft model. In patients who underwent FDG-PET imaging, 7 of 27 patients had a metabolic partial response based on predefined criteria. In the 34 patients with exploratory MRI results, none of the MRI-derived indices (Ktrans, cerebral blood volume, apparent diffusion coefficient) showed any significant trends in drug plasma exposure. GDC-0084 had a median T of approximately 2 hours after a single dose. max The mean accumulation ratio was 2.1 ± 0.90, and the degree of accumulation was consistent with theoretical accumulation based on half-life estimates and daily dosing intervals. After single and multiple doses (2 mg to 65 mg once daily) in all groups, GDC-0084 showed C max and AUC 0-24 The increase was approximately linear and dose proportional.

[0398] Tumor response was determined by evaluation of FDG-PET or by the Response Assessment in Neuro-Oncology (RANO).

[0399] Inhibition of glucose uptake was assessed using FDG-PET assessment and will be used as a surrogate test to address whether GDC-0084 is able to exert a biological effect in tumor tissue. The outcome measure for this objective was based on the maximum standardized uptake value (SUV max ). Tumor regions of interest (ROIs) were determined for each patient in pre-treatment PET imaging and corresponded to a subset of target lesions from the pre-treatment tumor assessment scan used to analyze the patient. PET response determination was performed according to the modified European Organization for Research on Cancer Therapy (EORTC) definition (Young H, Baum R, Cremerius H, et al., "Measurement of clinical and subclinical tumor response using [18F]-fluorodeoxyglucose and positron emission tomograph: review and 1999 EORTC recommendations", European Organization for Research and Treatment of Cancer (EORTC) PET Study Group. Eur J Cancer 1999; 35: 1773-82). Specifically, the SUV of each ROI scanned on treatment was calculated. maxSUV with corresponding pre-processed scans max Compare and determine the percent change. If there is more than one ROI, SUV max The overall percentage change for each ROI (mSUV max ) max The arithmetic mean of the percent change. PET responses were defined as follows. Progressive disease (PET-PD): mSUV max Percent increase > 20% or SUV max Development of new lesions above background and not explained by another cause (e.g. infection). Stable disease (PET-SD): mSUV max Increase percentage ≤ 20% or mSUV max Percent reduction ≤ 20%. Partial response (PET-PR): mSUV max Percent reduction >20%. Complete response (PET-CR): SUV in all ROIs max Indistinguishable from background (ie, complete disappearance of all PET lesions).

[0400] Tumor responses according to the RANO guidelines are generally based on the Wen approach (Wen PY, Macdonald DR, Reardon DA, et al. "Updated response assessment criteria for high-grade gliomas: Response Assessment in Neuro-Oncology Working Group", J Clin Oncol 2010; 28: 1963-72), where disease is categorized as "complete response", "partial response", "stable disease" and "progression". A complete response requires, among other factors, all of the following: complete disappearance of all enhanced measurable and non-measurable disease for at least 4 weeks; no new lesions; stable or improved non-enhancing (T2 / FLAIR) lesions. A required partial response is a ≥50% reduction from baseline in the sum of the products of the perpendicular diameters of all measurable enhancing lesions (e.g., as measured by MRI) for at least 4 weeks; no progression of measurable disease; no new lesions. If a patient does not qualify for a complete response, partial response, or progression, stable disease will occur. Progression was defined by any of the following: an increase of ≥25% in the sum of the products of the perpendicular diameters of all enhancing lesions (compared with the smallest tumor measurement at baseline [before treatment] or after the start of treatment [i.e., if not a decrease, compared with baseline]) on stable or increasing doses of corticosteroids; a significant increase in T2 / FLAIR nonenhancing lesions on stable or increasing doses of corticosteroids compared with the baseline scan or best response after the start of treatment not due to comorbid events; the appearance of any new lesions; progression of unequivocal nonmeasurable lesions; or unequivocal clinical worsening not attributable to causes other than neoplasia or to a reduction in the dose of corticosteroids.

[0401] Some patients underwent FDG-PET and additional exploratory MPJ assessments to investigate potential pharmacodynamic effects of GDC-0084. 18A decrease in F-FDG uptake suggests decreased glucose metabolic activity and PI3K pathway inhibition may be a PD response. A total of 27 patients underwent FDG-PET imaging at baseline, on Cycle 2 Day 1, and for those participating in the 45 mg and 65 mg dose levels, also on Cycle 1 Day 8. Based on FDG-PET, 5 of the 27 patients (18.5%) had a metabolic partial response according to predefined criteria. A trend toward decreased median survival in normal brain tissue was observed at doses of at least 45 mg GDC-0084 per day, suggesting CNS penetration of GDC-0084. GDC-0084 was detected at similar levels in brain tumors and brain tissue, with brain tissue / tumor to plasma ratios greater than 1 and greater than 0.5 for total drug and free drug, respectively. Among the evaluable patients, 26 (55.3%) patients had the best overall response with progressive disease, and 19 (40.4%) patients had stable disease. FDG-PET and concentration data from brain tumor tissues indicated that GDC-0084 crossed the blood-brain barrier and was evenly distributed throughout the brain.

[0402] Thirty-four patients were evaluated by exploratory magnetic resonance imaging ("MRI"). Dynamic contrast-enhanced (DCE) MRI data showed that in the four patients receiving the highest drug exposure (AUC 0-24小时 >8uMhr), a decrease in tumor Ktrans, a measure of tumor permeability reflecting tumor angiogenesis, was observed in patients with MRI-derived MRI (Ktrans, cerebral blood volume, apparent diffusion coefficient) exposure. The Ktrans changes were within the possible noise range of the measurement (based on the changes in the low exposure group). Overall, none of the MRI-derived indices (Ktrans, cerebral blood volume, apparent diffusion coefficient) showed any significant trend in drug plasma exposure.

[0403] Example 26: Transport assay in cell monolayers

[0404] Martin-Darby canine kidney (MDCK) cells expressing human P-gp, human BCRP or mouse Bcrp1, and LLC-PK1 cells transfected with mouse P-gp (mdr1a) were used to determine whether GDC-0084 is a substrate for these transporters. MDC1-MDCKI cells were obtained with permission from NCI (National Cancer Institute, Bethesda, MD), and Bcrp1-MDCKII, BCRP-MDCKII, and Mdr1a-LLC-PK1 cells were obtained from the Netherlands Cancer Institute (Amsterdam, the Netherlands). For transport studies, 2.5 × 10 5 The seeding density was 1.3 × 10 cells / mL (except for MDR1-MDCKI, which was 1.3 × 10 5Cells were plated on 24-well Millicell plates (Millipore, Billerca, MA) 4 days before use (polyethylene terephthalate membrane, 1 μm pore size). GDC-0084 was tested at 5 μM in the apical to basolateral (AB) and basolateral to apical (BA) directions. The compounds were dissolved in a transport buffer consisting of Hank's balanced salt solution (HBSS) and 10 mM HEPES (Invitrogen, Grand Island, New York). Lucifer yellow (Sigma-Aldrich, St. Louis, MO) was used as a paracellular and monolayer integrity marker. GDC-0084 concentrations in the donor and receiving compartments were determined by LC-MS / MS analysis. The apparent permeability (P) in the apical direction of the AB and BA directions was calculated after 2 hours of culture. app )for:

[0405] P app =(dQ / dt)·(1 / AC0)

[0406] Where: dQ / dt = apparent rate of compound in the receiving chamber; A = surface area of ​​the insert; C0 = initial substrate concentration at T0. The efflux ratio (ER) is calculated as (P app,B-A / P app,A-B ).

[0407] The results are shown in Table 15 below.

[0408] Table 15: Apparent permeability of GDC-0084 in transfected cells (P app )

[0409]

[0410] Apparent permeability (P app ) is high and is comparable to metoprolol (the high P app In cells transfected with MDCK or LLC-PK1, the efflux ratio (P app,B-A / P app,A-B ) was not significantly different from 1, indicating that GDC-0084 is a poor substrate for the efflux transporters P-gp and BCRP.

[0411] Example 27: Determination of plasma protein binding to brain

[0412] GDC-0084 protein binding was determined in vitro in mouse plasma (Bioreclamation, Inc., Hicksville, NY) using 300 μL plasma and 500 μL phosphate buffered saline in two chambers of the device by equilibrium dialysis. GDC-0084 was added to the pooled plasma (n>3) at a total concentration of 5 μM. Plasma samples were balanced for 4 hours with phosphate buffered saline (pH 7.4) at 37°C, 90% humidity and 5% CO2. After dialysis, the concentration of GDC-0084 in plasma and buffer was measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The unbound GDC-0084 percentage in plasma was determined by dividing the concentration measured in the post-dialysis buffer by the concentration measured in the post-dialysis plasma and multiplying by 100. Incubation was performed in triplicate. Parameters are expressed as mean ± standard deviation.

[0413] The free fraction of GDC-0084 in the mouse brain was determined as described by Kalvass et al. (Kalvass JC, Maurer TS, Pollack GM, "Use of plasma and brain unbound fractions to assess the extent of brain distribution of 34 drugs: comparison of unbound concentration ratios to in vivo p-glycoprotein efflux ratios", Drug Metab. Dispos. 2007; 35(4): 660-666). Briefly, brain tissue was homogenized in 3 volumes of phosphate-buffered saline and GDC-0084 was added at a final concentration of 5 μM. A 300 μl aliquot was dialyzed against a 500 μl volume of buffer in a RED apparatus (Thermo Scientific, Rockford, IL) at 37°C for 4 hours in an incubator with 90% humidity and 5% CO2. After dialysis, tissue and buffer samples were analyzed as described for plasma protein binding studies.

[0414] The results showed that when tested at 5 μM, GDC-0084 had low binding to plasma proteins, with a free fraction (%) of 29.5±2.7 (n=3) in mouse plasma. Binding to brain tissue was higher, with a free fraction of 6.7% (±1; n=3).

[0415] Example 28: Regulation of pAkt and pS6 in the brain

[0416] Inhibition of the PI3K pathway was assessed in the brain of healthy mice by measuring two markers, pAkt and pS6. Female CD-1 mice were dosed PO with 25 mg / kg of GDC-0084. Brain and plasma were collected from 3 animals at 1 hour and 6 hours after dosing at each time point. For PD analysis and GDC-0084 concentration measurement, individual brains were divided into halves. Samples were stored at -80°C and analyzed for total GDC-0084 concentration. For PD analysis, a cell extraction buffer (Invitrogen, Camarillo, CA) containing 10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS and 0.5% deoxycholate was supplemented with phosphatase and protease inhibitors (Sigma, St. Louis, MO) and added to frozen brain biopsies. The brain was homogenized with a small pestle (Konte Glass Company, Vineland, NJ), briefly sonicated on ice, and centrifuged at 20,000 g for 20 minutes at 4°C. Protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). Proteins were separated by electrophoresis and transferred to NuPage nitrocellulose membranes (Invitrogen, Camarillo, CA). The Licor Odyssey infrared detection system (Licor, Lincoln, NE) was used to assess and quantify protein expression. Ser473 , total Akt, pS6 Ser235 / 236 PI3K pathway markers were assessed by immunoblotting with antibodies to 547, 571, 592, 504, 512, 524, 536, 547, 551, 576, 581, 592 ...

[0417] After a single oral dose of GDC-0084 (25 mg / kg), pAkt and pS6 levels were significantly lower than those detected in control animals ( Fig.23 ). Inhibition of pAkt and pS6 reached 90% 1 hour after administration and remained greater than 70% 6 hours after administration ( Fig.24 ).

[0418] Example 29: First Method for Measuring Efficacy of U87 and GS2 in a Brain Tumor Model

[0419] Six female nude mice (Charles River Laboratories) were implanted with U87 MGM human glioblastoma cancer cells (described elsewhere herein) or GS2 tumor cells (Gunther HS, Schmidt NO, Phillips HS, et al., "Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria", Oncogene 2008; 27(20): 2897-2909)) and injected into the right striatum (subcortical portion of the forebrain) by stereotaxic surgery in a volume of 3 to 5 μL (250K U87 cells and 100K GS2 cells). A single oral dose of 15 mg / kg GDC-0084 (also containing 0.5% methylcellulose / 0.2% Tween 80 (MCT)) was administered 19 to 21 days after implantation. Mice were euthanized by exsanguination by perfusion under anesthesia 1 hour and 6 hours after administration. Brains were removed, snap frozen in liquid N2 and stored in a -80°C freezer until analysis. Fresh frozen tissue sections were obtained on a 12 μm thick cryostat (Leica CM3050S, Buffalo Grove, IL) and thaw-mounted onto indium tin oxide-coated slides (Bruker Daltonics, Billerica, MA). Tissue sections were analyzed by MALDI MS imaging, providing signal intensity (rather than absolute quantification), followed by histological examination with cresyl violet staining.

[0420] For MALDI MAS analysis, 40 mg / mL of 2,5-dihydroxybenzoic acid (Sigma-Aldrich, St.Louis, MO) solution was prepared in methanol: water (70:30 v / v). The internal standard [D6] GDC-0084 of stable labeling was incorporated into the MALDI matrix solution at 2 μM and then deposited on the tissue sections. The matrix solution was evenly sprayed onto the tissue using an HTX TM-sprayer (HTX technologies, Chapel Hill, NC). The matrix-coated tissue sections were transferred to a MALDI mass spectrometer (SolariX7T FT-ICR, Bruker Daltonics, Bremen, Germany) for imaging analysis. Imaging data were collected in positive ionization mode with a 100 μm pixel resolution under continuous accumulation of a selected ion (CASI) window optimized for a 50 Da window centered at m / z 383 (m / z 358-408). Laser intensity and number of shots were optimized for sensitivity to the parent drug (1200 shots) while collecting ions in the mass range of m / z 150-3000. Drug images were generated based on the exact mass of the parent drug (GDC-0084 m / z 383.1938) using FlexImaging v4.0 64-bit (Bruker Daltonics, Billerica, MA) with a mass tolerance of ±2 mDa and normalized to the internal standard response.

[0421] After completing the imaging experiment, the matrix coating was removed by rinsing the slide in 100% methanol for 30 seconds or until the entire matrix was clearly removed. Use freshly prepared 0.5% cresyl violet staining solution (Chaurand P, Schwartz SA, Billheimer D, Xu BJ, Crecelius A, Caprioli RM, "Integrating histology and imagingmass spectrometry", Anal. Chem. 2004; 76 (4): 1145-1155) by immersing the slide for 30 seconds to stain the tissue section, and then rinse it again for 30 seconds in two cycles of 100% ethanol. Microscope images were obtained at 10x magnification on Olympus BX51 (Tokyo, Japan) and stitched using MicroSuite Analytical v3.0 software (Olympus, Tokyo, Japan). Subsequently, the stained images were aligned with the optical images in Flexlmaging for visualization and annotation of tumor areas and non-tumor areas for drug images.

[0422] To evaluate drug distribution, MALDI MS data from U87 and GS2 tumor models were co-aligned to cresyl violet-stained microscopy images in Flexlmaging regions of interest (ROIs), which were selected based on anatomical features defined in histological images, including tumor and non-tumor areas. Drug intensity for each pixel within the defined ROI was extracted and output. Drug intensity was binned in 0.1 increments from 0.0 to 2.0. Histograms were created in GraphPad Prism 5 to visualize the distribution of pixel intensity frequencies.

[0423] Example 30: Second Method for Measuring Efficacy of U87 and GS2 in Brain Tumor Models

[0424] U87 glioblastoma cancer cells (described elsewhere herein) and GS2 glioblastoma cells were selected to test the efficacy of GDC-0084 in mouse brain models. These U87 and GS2 models are PTEN-deficient, and the GS2 cell line presents copy number deletion at the PTEN locus (Gunther HS et al.), and no PTEN protein was detected by Western blotting (Carlson BL, Pokorny JL, Schroeder MA, Sarkaria JN., "Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery", Curr Protoc Pharmacol. 2011; Chapter 14: Unit 14 16). Two cell lines were confirmed by STR analysis (DNA Diagnostic Center) of cells within 5 generations of those cells used for in vivo research. U87 (250K) and GS2 (100K) tumor cells were injected into the right striatum (the lower part of the forebrain cortex) in a volume of 3-5 μl by stereotaxic surgery. For each experiment, mice were randomly divided into 10 groups to obtain equivalent average tumor volumes between the treatment and control groups of each model. Treatment began 7 days (U87) or 14 days (GS2) after tumor cell inoculation, and GDC-0084 (15 mg / kg) was given daily or vehicle (MCT) was given PO daily for 2 or 4 weeks, respectively. Mouse weights were recorded twice a week during the study, and animals were euthanized if weight loss was greater than 20% of their initial body weight. Tumor volume was monitored by in vitro micro-computed tomography (micro-CT) imaging and T2 MRI of GBM models U87 and GS2, respectively. Differences between treatment groups were assessed using the Student's t test in Prism (Prism 5, GraphPad). MRI was performed on a Varian 9.4T MRI system with a 30mm orthogonal volume coil. During imaging, animals were anesthetized with 2% isoflurane in air. Body temperature was continuously monitored using a rectal probe and maintained at 37°C by a heated air flow system regulated by an internal LabVIEW controller software. Lesions were detected by MRI using a T2-weighted fast spin echo with a multi-slice (FSEMS) sequence. 12–20 axial 0.5–0.8 mm thick slices were acquired with a 20 × 20 mm field of view (FOV) and a 128 × 128 matrix, zero-filled to 256 × 256 images.TR = 3500-4000 ms, TE = 9-10 ms, ETL = 8, k-0 = 4, NEX = 8. Tumor volumes were calculated from T2-weighted FSEMS images using the intensity threshold-based region growing tool in MRVision software. Brain sample preparation, micro-CT scanning, and image analysis for ex vivo micro-CT imaging were performed as previously described (de Crespigny A, Bou-Reslan H, Nishimura MC, Phillips H, Carano RA, D'Arceuil HE, "3D micro-CT imaging of the postmortem brain", J. Neurosci. Methods. 2008; 171(2): 207-213).

[0425] In studies with GS2 tumor-bearing mice, plasma and brain were also collected at the end of treatment to measure GDC-0084 and assess PI3K pathway modulation in tumors. Each brain was dissected to separate the tumor from healthy tissue. Plasma and normal brain were processed and analyzed by LC-MS / MS. GS2 tumors isolated from brain were processed as described above and PI3K pathway markers pAkt, pS6, and p4EBP1 were measured.

[0426] Example 31: MALDI imaging results

[0427] The distribution of GDC-0084 in the brain and intracranial U87 and GS2 tumors after a single PO dose (15 mg / kg) was studied by MALDI imaging. Brains were collected 1 hour after the dose, and Fig.25A and 25B The images presented in Figure 2 show that GDC-0084 is readily and fairly evenly distributed throughout the brain, including GS2 ( Fig.25A ) and U87( Fig.25B ) tumors. In addition, the uniformity and distribution pattern of GDC-0084 in brain tumor and non-tumor areas were further analyzed. The signal intensity frequency (pixel intensity frequency) appeared to follow a normal distribution in the healthy brain, which was consistent with the signal intensity frequency observed in U87 tumors ( Fig.26A ); mean pixel intensity 0.54)) overlap (mean pixel intensity 0.54). A Gaussian distribution of the signal was also observed in GS2 tumors ( Fig.26B ), however the mean pixel intensity was slightly lower (0.34 vs. 0.55), indicating that the concentration of GDC-0084 in GS2 tumors was generally lower than that in normal brain. Comparison of the homogeneity of GDC-0084 signal in non-tumor areas between U87 and GS2 tumor-bearing mice showed the same distribution ( Fig.26C), confirming the reproducible and consistent brain penetration properties of GDC-0084. Similar results were obtained in the brain collected 6 hours after administration. In addition, in order to compare the distribution of GDC-0084 with non-brain-penetrating compounds, MALDI images previously obtained with pictilisib in the U87 tumor model (Salphati L, Shahidi-Latham S, Quiason C, et al., "Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib (GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastoma models-assessment by matrix-assisted laser desorption ionization", Drug Metab Dispos. 2014; 42 (7): 1110-1116) were reanalyzed using the method used here. In a comparative analysis, reanalysis of data previously obtained with the non-brain-penetrating compound PictilisB (Salphati et al.) revealed a non-uniform (non-Gaussian) intratumoral distribution of pixel intensities ( Fig.26D ). While the signal intensity of GDC-0084 in U87 tumors can be fitted to a Gaussian curve, the signal from pictilisib is concentrated in the low-intensity bins and its distribution appears more uneven ( Fig.26D ). GDC-0084 provided improved uniform and undifferentiated compound distribution throughout healthy brain tissue and tumor tissue compared to pictilisib. Based on the brain tumor model results, it is believed that GDC-0084 provides improved treatment not only to the core of the tumor but also to invasive glioma cells that are protected by an intact BBB or blood-tumor barrier.

[0428] Example 32: Brain Tumor Model Results

[0429] The efficacy of GDC-0084 was tested in the U87 and GS2 intracranial models. GDC-0084 was administered PO to U87 and GS2 tumor-bearing mice at 15 mg / kg per day for 2 and 4 weeks, respectively. The effect of treatment on U87 and GS2 tumor volume was assessed at the end of the dosing period. Fig.27A Images of U87 tumors obtained by micro-CT are shown. Fig.27B ) showed a reduction of approximately 70% in U87 tumor volume compared to GS2 tumors as measured by MRI. Fig.27C) in treated mice was significantly (p < 0.01) less than (≈40%) in the control group ( Fig.27D At the end of the study, plasma and healthy brain concentrations of GDC-0084 were measured in mice bearing GS2 tumors and are presented below together with the brain-to-plasma ratios in Table 16. Brain concentrations and brain-to-plasma ratios in the normal portion of the brain were comparable to those obtained previously (Table 9). Modulation of the PI3K pathway in GS2 tumors was assessed by Western blot at the end of the dosing period, 2 and 8 hours after the final administration of GDC-0084 ( Fig.28A ). pAkt levels were significantly reduced by 90% and 70% at 2 and 8 hours, respectively. Inhibition of pS6 and p4EBP1 was less pronounced at 2 hours, reaching 35% and 43%, respectively. Both markers returned to baseline levels 8 hours after administration ( Fig.28B ).

[0430] Table 16 Plasma concentrations, brain concentrations, and brain-plasma ratios (non-tumor half of the brain) measured 2 hours and 8 hours after PO administration of GDC-0084 (15 mg / kg) to GS2 tumor-bearing mice

[0431] Time after dose (h) Brain (μM) Plasma (μM) Brain-to-plasma ratio 2 5.51±1.58 3.64±2.05 1.67±0.51 8 2.48±1.25 2.01±1.19 1.29±0.16

[0432] When introducing elements of the present invention or the preferred embodiments thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements besides the listed elements.

[0433] This written description uses embodiments to disclose the invention, including the best mode, and also enables those skilled in the art to practice the invention, including making and using any device or system and performing any combined method. The patentable scope of the invention is defined by the claims, and may include other embodiments that occur to those skilled in the art. If these other embodiments have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that are not substantially different from the literal language of the claims, then these other embodiments are intended to be within the scope of the claims.

[0434] Implementation

[0435] A. A method for preparing a compound of formula III from a compound of formula II in a reaction mixture according to the following reaction formula,

[0436]

[0437] The method comprises:

[0438] (i) forming a reaction mixture comprising a compound of formula II, an organoboron-R 4 , a solvent system comprising at least 5 v / v% water, a base, and a catalyst;

[0439] (ii) reacting the reaction mixture at a temperature of less than 100° C. to form a reaction product mixture comprising a compound of Formula III;

[0440] (iii) separating the compound of formula III, its stereoisomers, its geometric isomers, its tautomers or its pharmaceutically acceptable salt from the reaction product mixture;

[0441] in,

[0442] The catalyst comprises palladium, and the reaction mixture comprises less than 0.05 equivalents of the catalyst per equivalent of the compound of Formula II;

[0443] X 1 S, O, N, NR 6 , CR 1 , C(R 1 )2 or -C(R 1 )2O-;

[0444] X 2 C, CR 2 or N;

[0445] X 3 C, CR 3 or N;

[0446] X 4 is a halogen;

[0447] A is the same as X 2 and X 3 A fused 5-, 6- or 7-membered carbocyclyl or heterocyclyl ring, optionally substituted by one or more R 5 , R 10 or R 15 Group substitution;

[0448] R 6 H, C1-C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12 Carbocyclic), -(C1-C 12 Alkylene)(-C2-C 20 Heterocyclic), -(C1-C 12 Alkylene)-C(=O)-(C2-C 20 Heterocyclic group), (C1-C 12 Alkylene)-(C6-C 20Aryl) and -(C1-C 12 Alkylene)-(C1-C 20 wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -(CH3)2OH, -CH2OCH3, -CN, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3) 2CONH2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and 1,1-dioxo-thiopyran-4-yl;

[0449] R 1 , R 2 and R 3 independently selected from H, F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2, -CONH2, -NO 2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and l,l-dioxo-thiopyran-4-yl;

[0450] R 4 Selected from C6-C 20 Aryl, C2-C 20 Heterocyclic and C1-C 20heteroaryl, each optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CH, -CHCH, -CH(CH), -CHCH(CH), -CHCH, -CHCN, -CN, -CF, -CHOH, -COH, -CONH, CONH(CH), -CON(CH), -NO, -NH, -NHCH, -NHCOCH, -OH, -OCH, -OCHCH, -OCH(CH), -SH, -NHC(O)NHCH, -NHC(O)NHCHCH, -NHS(O)CH, -N(CH)C(O)OC(CH), -S(O)CH, benzyl, benzyloxy, morpholinyl, morpholinylmethyl, and 4-methylpiperazin-1-yl;

[0451] Each R 5 , R 10 and R 15 Independently selected from C1-C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12 Carbocyclic), -(C1-C 12 Alkylene)-(C2-C 20 Heterocyclic), -(C1-C 12 Alkylene)-C(O)-(C2-C 20 Heterocyclic), -(C1-C 12 Alkylene)-(C6-C 20 Aryl) and -(C1-C 12 Alkylene)-(C1-C 20 heteroaryl); or two geminal R 5 , R 10 and / or R 15The group forms a 3-, 4-, 5- or 6-membered carbocyclyl or heterocyclyl ring; wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CH2F, -CHF2, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -C ON(CH3)2, -C(CH3)2CONH2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NH-COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholinyl and l,l-dioxo-thiopyran-4-yl;

[0452] mor is selected from:

[0453]

[0454] wherein mor is optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, -CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH, -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C(CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2, -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -NHCH(CH3)2, -NHCH2CH2OH, -NHCH2CH2OCH3, -NHCOCH3, -NHCOCH2CH3, -NHCOCH2OH, -NHS(O)2CH3, -N(CH3)S(O)2CH3, =O, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(O)NHCH3, -NHC(O)NHCH2CH3, -S(O)CH3, -S(O)CH2CH3, -S(O)2CH3, -S(O)2NH2, -S(O)2NHCH3, -S(O)2N(CH3)2, and -CH2S(O)2CH3, one or more R 7 Group substitution.

[0455] A1. A method according to embodiment A, wherein the solvent system further comprises at least one polar aprotic solvent selected from N-methylpyrrolidone, methyl isobutyl ketone, methyl ethyl ketone, tetrahydrofuran, dichloromethane, ethyl acetate, acetone, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide.

[0456] A2. A method according to embodiment A1, wherein the ratio of water to the at least one polar aprotic solvent is about 1:10 v / v to about 5:1 v / v, about 1:1 v / v to about 1:10 v / v, or about 1:3 v / v to about 1:7 v / v.

[0457] A3. A method according to embodiment A1 or A2, wherein the solvent system comprises water and tetrahydrofuran.

[0458] A4. A method according to any one of embodiments A1-A3, wherein the solvent system essentially consists of water and the at least one polar aprotic solvent.

[0459] A5. The method according to any one of embodiments A-A4, wherein the organoboron-R 4 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-R 4 .

[0460] A6. A method according to any one of embodiments A-A5, wherein the base is selected from K3PO4, Cs2CO3 and KOH.

[0461] A7. A method according to any one of embodiments A-A6, wherein the base is K3PO4.

[0462] A8. A method according to any one of embodiments A-A7, wherein the equivalent ratio of base to compound of formula II is at least 1:1, about 1:1 to about 3:1, or about 2:1.

[0463] A9. A method according to any one of embodiments A to A8, wherein the catalyst containing palladium is selected from chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) ("PdXphos"); 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane ("PdCl2dppfCH2Cl2"); bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) ("Pd(amphos)Cl2"); bis(di-tert-butylphenylphosphine)dichloropalladium(II) ("Pd 122"); PdCl2(PPh3)2; Pd(t-Bu)3; Pd(PPh3)4; Pd(Oac) / PPh3; Cl2Pd[(Pet3)]2; Pd(DIPHOS)2 ;Cl2Pd(Bipy);[PdCl(Ph2PCH2PPh2)]2;Cl2Pd[P(o-tol)3]2;Pd2(dba)3 / P(o-tol)3;Pd2(db a) / P(furyl)3; Cl2Pd[P(furyl)3]2; Cl2Pd(PmePh2)2; Cl2Pd[P(4-F-Ph)3]2; Cl2Pd[P(C6F6)3]2; Cl2Pd[P(2-COOH-Ph)(Ph)2]2; Cl2Pd[P(4-COOH-Ph)(Ph)2]2, palladium acetate containing 0.4mmol / g Pd microencapsulated in a polyurea matrix; palladium acetate and triphenylphosphine containing 0.4mmol / g Pd and 0.3mmol / g phosphorus microencapsulated in a polyurea matrix; and palladium acetate and BINAP containing 0.4mmol / g Pd microencapsulated in a polyurea matrix.

[0464] A10. A method according to embodiment A9, wherein the palladium-containing catalyst is selected from a complex of chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) and 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane, or chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II).

[0465] A11. A method according to any one of embodiments A-A10, wherein the equivalent ratio of the catalyst comprising palladium to the compound of Formula II is about 0.003:1 to 0.05:1, about 0.003:1 to about 0.03:1, or about 0.004:1 to about 0.02:1.

[0466] A12. A method according to any one of embodiments A-A11, wherein the catalyst is chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II), and the equivalent ratio of the catalyst containing palladium to the compound of formula II is about 0.004:1 to about 0.015:1, about 0.004:1 to about 0.01:1, about 0.004:1 to about 0.007:1, or about 0.005:1.

[0467] A13. A method according to any one of embodiments A to A11, wherein the catalyst is a complex of chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) or 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane, and the equivalent ratio of the catalyst containing palladium to the compound of formula II is about 0.005:1 to about 0.04:1, about 0.005:1 to about 0.03:1, about 0.01:1 to about 0.03:1, or about 0.02:1.

[0468] A14. A method according to any one of embodiments A-A13, wherein the temperature of the reaction is about 40°C to 100°C, about 40°C to about 90°C, about 40°C to about 80°C, about 50°C to about 80°C, or about 55°C to about 75°C.

[0469] A15. A method according to any one of embodiments A-A14, wherein the method further comprises adding a polar protic solvent to the reaction product mixture to form a mixture containing greater than 25 v / v% water and separating the compound of formula III from the reaction product mixture by solid-liquid separation.

[0470] A16. A method according to embodiment A15, wherein the polar protic solvent is selected from water, methanol, ethanol, isopropanol, n-propanol and acetic acid.

[0471] A17. A method according to embodiment A16, wherein the polar protic solvent is water.

[0472] A18. A method according to embodiment A17, wherein the volume ratio of the solvent system to the water added to the reaction product mixture is about 1:5 v / v to about 5:1 v / v, about 1:3 v / v to about 3:1 v / v, about 1:2 v / v to about 2:1 v / v, about 1:1.5 v / v to about 1.5:1 v / v, or about 1:1 v / v.

[0473] A19. The method according to embodiment A17 or A18, wherein the method further comprises adding seed crystals of the compound of formula III to the mixture of the reaction product mixture and water.

[0474] A20. The method of any one of embodiments A to A19, further comprising a purification step comprising:

[0475] (i) mixing a compound of formula III with a metal scavenger in a solvent system comprising at least one polar protic solvent;

[0476] (ii) heating the mixture to dissolve the compound of formula III;

[0477] (iii) filtering the heated mixture;

[0478] (iv) reducing the temperature of the filtrate and mixing seed crystals of the compound of formula III with the cooled filtrate;

[0479] (v) reducing the temperature of the filtrate and seed mixture to induce crystallization of the purified compound of Formula III; and

[0480] (vi) collecting purified crystals of the compound of formula III.

[0481] A21. A method according to embodiment A20, wherein

[0482] (i) the solvent system comprises or consists essentially of water and acetic acid, wherein the volume ratio of acetic acid to water is from about 1:1 to about 10:1, from about 1:1 to about 5:1, or from about 1:1 to about 3:1, or about 3:1;

[0483] (ii) the metal scavenger is a silicon-thiol; and

[0484] (iii) the dissolving temperature is about 80°C to about 100°C, the seed crystals are combined with the filtrate at a temperature of about 70°C to about 80°C, and the crystallization temperature is about 0°C to about 10°C.

[0485] A22. The method of any one of embodiments A-A21, wherein the yield of the compound of formula III is at least 75%, at least 80%, at least 85% or at least 90%.

[0486] A23. A method according to any one of embodiments A-A22, wherein the purity of the compound of Formula III is at least 97%, at least 97.5%, or at least 98% (area % determined by HPLC).

[0487] A24. The method according to any one of embodiments A to A23, wherein X 1 N, NR 6 , CR 1 , C(R 1 ) 2 or C(R 1 )2O;X 3 C or CR 3 .

[0488] A25. The method according to embodiment A24, wherein X 1 and X 2 N; X 3 For C.

[0489] A26. A method according to any one of embodiments A-A25, wherein A is an optionally substituted 6-membered heterocycle comprising at least one heteroatom selected from N and O.

[0490] A27. The method according to embodiment A26, wherein X 2 is N, and A is optionally substituted morpholine.

[0491] A28. The method of any one of embodiments A-A27, wherein mor is optionally substituted morpholine.

[0492] A29. The method according to any one of embodiments A to A28, wherein R 4 is selected from optionally substituted C6 aryl, optionally substituted C6 heterocycle and optionally substituted C6 heteroaryl.

[0493] A30. The method according to embodiment A29, wherein R 4 is an optionally substituted C6 heteroaryl group containing one or two N heteroatoms.

[0494] A31. A method according to embodiment A30, wherein R4 is an optionally substituted pyrimidine.

[0495] A32. The method according to any one of embodiments A-A31, wherein the compound of formula III is

[0496]

[0497] B. A method for preparing a compound of formula IIa from a compound of formula I in a reaction mixture according to the following reaction formula,

[0498]

[0499] The method comprises:

[0500] (i) forming a reaction mixture comprising a compound of formula I, an organic halide, a solvent system, a phase transfer catalyst and a base; (ii) reacting the reaction mixture to form a reaction product mixture comprising a compound of formula IIa, a stereoisomer thereof, a geometric stereoisomer thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof; and (iii) isolating the compound of formula IIa from the reaction product mixture;

[0501] in,

[0502] The solvent system comprises at least 5 v / v% water;

[0503] X is a halide;

[0504] Each R 5 , R 10 and R 15 Independently selected from H, C1-C 10 A hydrocarbon group or a hydrocarbon group selected from C1-C5 hydrocarbon groups, wherein each hydrocarbon group is optionally substituted, two geminal R 5 , R 10 and / or R 15 The group is oxo, or two geminal R 5 , R 10 and / or R 15 The group forms a 3-, 4-, 5-, 6- or 7-membered carbocyclic or heterocyclic group, wherein the carbocyclic or heterocyclic group is optionally substituted;

[0505] mor is selected from:

[0506]

[0507] wherein mor is optionally independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, -CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH, -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C(CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2, -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -NHCH(CH3)2, -NHCH2CH2OH, -NHCH2CH2OCH3, -NHCOCH3, -NHCOCH2CH3, -NHCOCH2OH, -NHS(O)2CH3, -N(CH3)S(O)2CH3, =O, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(O)NHCH3, -NHC(O)NHCH2CH3, -S(O)CH3, -S(O)CH2CH3, -S(O)2CH3, -S(O)2NH2, -S(O)2NHCH3, -S(O)2N(CH3)2, and -CH2S(O)2CH3, one or more R 7 group substitution;

[0508] Wherein in Formula I, R 20 -OH or -NHR 21 , where R 21 For R 5 defined above, and wherein in Formula IIa, R 20 -O- or -NR 21 -.

[0509] B1. A method according to embodiment B, wherein the solvent system comprises at least 50 v / v% water, at least 75 v / v% water, at least 90 v / v% water, or essentially consists of water.

[0510] B2. A method according to embodiment B or B1, wherein the base is selected from K3PO4, Cs2CO3, K2CO3, KOAc, NaOAc, Na2CO3 and KOH.

[0511] B3. A method according to embodiment B2, wherein the base is KOH.

[0512] B4. A method according to any one of embodiments B-B3, wherein the phase transfer catalyst is selected from quaternary ammonium salts and quaternary phosphonium salts.

[0513] B5. A method according to embodiment B4, wherein the phase transfer catalyst is selected from tetra-n-butylammonium bromide, benzyltrimethylammonium chloride, benzyltriethylammonium chloride, methyltrioctylammonium chloride, methyltributylammonium chloride and methyltrioctylammonium chloride.

[0514] B6. A method according to embodiment B5, wherein the phase transfer catalyst is tetra-n-butylammonium bromide.

[0515] B7. A method according to any one of embodiments B-B6, wherein the molar ratio of the organic halide dibromoethane to the compound of Formula I is greater than 2:1 to about 4:1, 2:1 to about 4:1, or about 3:1.

[0516] B8. A method according to any one of embodiments B-B7, wherein the organic halide and the base are present in approximately equimolar amounts.

[0517] B9. A method according to any one of embodiments B-B8, wherein the temperature of the reaction is about 40°C to about 90°C, about 40°C to about 70°C, about 40°C to about 60°C, or about 50°C.

[0518] B10. A method according to any one of embodiments B-B9, wherein the method further comprises mixing a polar protic solvent with the reaction product mixture, and then lowering the temperature of the mixture to induce crystallization of the compound of formula IIa in the reaction product mixture, wherein the crystallized compound of formula IIa is separated from the reaction product mixture.

[0519] B11. A method according to embodiment B10, wherein the polar protic solvent is selected from water, methanol, ethanol, isopropanol, n-propanol and acetic acid.

[0520] B12. A method according to embodiment B11, wherein the polar protic solvent is ethanol.

[0521] B13. A method according to embodiment B12, wherein the volume ratio of the solvent system to ethanol is about 1:5 v / v to about 5:1 v / v, about 1:3 v / v to about 3:1 v / v, about 1:2 v / v to about 2:1 v / v, about 1:1 v / v to about 1:2 v / v, or about 1:1.3 v / v.

[0522] B14. The method according to embodiment B12 or B13, wherein the method further comprises adding seed crystals of the compound of formula IIa to the mixture of the reaction product mixture and ethanol.

[0523] B15. The method of any one of embodiments B-B14, wherein the yield of the compound of Formula II is at least 60%, at least 65%, at least 70%, or at least 75%.

[0524] B16. A method according to any one of embodiments B-B15, wherein the purity of the compound of Formula II is at least 97%, at least 97.5%, at least 98%, at least 98.5% or at least 99% (area % determined by HPLC).

[0525] B17. The method according to any one of embodiments B-B16, wherein each R 5 , R 10 and R 15 are independently selected from H and optionally substituted C 1-5 Alkyl, or two geminal R 5 , R 10 and / or R 15 The groups together are oxo or form a 3- to 6-membered cycloalkyl or heterocycloalkyl having one or two heteroatoms selected from N and O.

[0526] B18. The method according to embodiment B17, wherein each R 5 , R 10 and R 15 Independently selected from H, C 1-5 alkyl and C substituted by at least one of deuterium, halogen and hydroxyl 1-5 alkyl.

[0527] B19. The method according to any one of embodiments B-B18, wherein in Formula I, R 20 -OH, -NH2 or -NH-C 1-5 alkyl.

[0528] B20. The method according to embodiment B19, wherein in Formula I, R 20 It is -OH.

[0529] B21. A method according to any one of embodiments B-B20, wherein the organic halide is 1,2-dibromoethane.

[0530] B22. The method according to any one of embodiments B-B21, wherein the compound of formula IIa is

[0531]

[0532] C. The method according to any one of embodiments B-B22, wherein the method further comprises a method for preparing a compound of formula IIIa from a compound of formula IIa in a reaction mixture according to the following reaction formula,

[0533]

[0534] The method comprises:

[0535] (i) forming a reaction mixture comprising a compound of formula IIa, an organoboron-R 4 , a solvent system comprising at least 5 v / v% water, a base, and a catalyst;

[0536] (ii) reacting the reaction mixture to form a reaction product mixture comprising a compound of Formula IIIa; and

[0537] (iii) separating the compound of formula IIIa, its stereoisomers, its geometric isomers, its tautomers, or its pharmaceutically acceptable salt from the reaction product mixture by solid-liquid separation, wherein the yield of the compound of formula IIIa is at least 75%;

[0538] in,

[0539] The catalyst comprises palladium and the reaction mixture comprises less than 0.05 equivalents of the catalyst per equivalent of the compound of Formula IIa;

[0540] R 4 Selected from C6-C 20 Aryl, C2-C 20 Heterocyclic and C1-C 20 heteroaryl, each of which is optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CH, -CHCH, -CH(CH), -CHCH(CH), -CHCH, -CHCN, -CN, -CF, -CHOH, -COH, -CONH, CONH(CH), -CON(CH), -NO, -NH, -NHCH, -NHCOCH, -OH, -OCH, -OCHCH, -OCH(CH), -SH, -NHC(O)NHCH, -NHC(O)NHCHCH, -NHS(O)CH, -N(CH)C(O)OC(CH), -S(O)CH, benzyl, benzyloxy, morpholinyl, morpholinylmethyl, and 4-methylpiperazinyl.

[0541] C1. A method according to embodiment C, wherein the solvent system further comprises at least one polar aprotic solvent selected from tetrahydrofuran, dichloromethane, ethyl acetate, acetone, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide.

[0542] C2. A method according to embodiment C1, wherein the ratio of water to the at least one polar aprotic solvent is about 1:10 v / v to about 5:1 v / v, about 1:1 v / v to about 1:10 v / v, or about 1:3 v / v to about 1:7 v / v.

[0543] C3. A method according to embodiment C1 or C2, wherein the solvent system comprises water and tetrahydrofuran.

[0544] C4. A method according to any one of embodiments C1-C3, wherein the solvent system consists essentially of water and at least one polar aprotic solvent.

[0545] C5. A method according to any one of embodiments C-C4, wherein the organoboron-R 4 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-R 4 .

[0546] C6. A method according to any one of embodiments C-C5, wherein the base is selected from K3PO4, Cs2CO3 and KOH.

[0547] C7. A method according to any one of embodiments C-C6, wherein the base is K3PO4.

[0548] C8. A method according to any one of embodiments C-C7, wherein the equivalent ratio of base to the compound of Formula IIa is at least 1:1, about 1:1 to about 3:1, or about 2:1.

[0549] C9. A method according to any one of embodiments C to C8, wherein the catalyst containing palladium is selected from chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) ("PdXphos"); 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane ("PdCl2dppfCH2Cl2"); bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) ("Pd(amphos)Cl2"); bis(di-tert-butylphenylphosphine)dichloropalladium(II) ("Pd 122"); PdCl2(PPh3)2; Pd(t-Bu)3; Pd(PPh3)4; Pd(Oac) / PPh3; Cl2Pd[(Pet3)]2; Pd(DIPHOS)2 ;Cl2Pd(Bipy);[PdCl(Ph2PCH2PPh2)]2;Cl2Pd[P(o-tol)3]2;Pd2(dba)3 / P(o-tol)3;Pd2(db a) / P(furyl)3; Cl2Pd[P(furyl)3]2; Cl2Pd(PmePh2)2; Cl2Pd[P(4-F-Ph)3]2; Cl2Pd[P(C6F6)3]2; Cl2Pd[P(2-COOH-Ph)(Ph)2]2; Cl2Pd[P(4-COOH-Ph)(Ph)2]2, palladium acetate containing 0.4mmol / g Pd microencapsulated in a polyurea matrix; palladium acetate and triphenylphosphine containing 0.4mmol / g Pd and 0.3mmol / g phosphorus microencapsulated in a polyurea matrix; and palladium acetate and BINAP containing 0.4mmol / g Pd microencapsulated in a polyurea matrix.

[0550] C10. A method according to embodiment C9, wherein the catalyst containing palladium is selected from a complex of chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) and 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane.

[0551] C11. A method according to any one of embodiments C-C10, wherein the equivalent ratio of the catalyst comprising palladium to the compound of Formula IIa is about 0.003:1 to 0.05:1, about 0.003:1 to about 0.03:1, or about 0.004:1 to about 0.02:1.

[0552] C12. A method according to any one of embodiments C-C11, wherein the catalyst is chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II), and the equivalent ratio of the catalyst containing palladium to the compound of Formula IIa is about 0.004:1 to about 0.015:1, about 0.004:1 to about 0.01:1, about 0.004:1 to about 0.007:1, or about 0.005:1.

[0553] C13. A method according to any one of embodiments C to C11, wherein the catalyst is a complex of chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) and 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane, and the equivalent ratio of the catalyst containing palladium to the compound of formula IIa is about 0.005:1 to about 0.04:1, about 0.005:1 to about 0.03:1, about 0.01:1 to about 0.03:1, or about 0.02:1.

[0554] C14. A method according to any one of embodiments C-C13, wherein the method further comprises adding a polar protic solvent to the reaction product mixture to form a mixture comprising at least 25 v / v% water, and separating the compound of formula IIIa from the reaction product mixture.

[0555] C15. A method according to embodiment C14, wherein the polar protic solvent is selected from water, methanol, ethanol, isopropanol, n-propanol and acetic acid.

[0556] C16. A method according to embodiment C15, wherein the polar protic solvent is water.

[0557] C17. A method according to embodiment C16, wherein the volume ratio of the solvent system to the water added to the reaction product mixture is about 1:5 v / v to about 5:1 v / v, about 1:3 v / v to about 3:1 v / v, about 1:2 v / v to about 2:1 v / v, about 1:1.5 v / v to about 1.5:1 v / v, or about 1:1 v / v.

[0558] C18. A method according to embodiment C16 or C17, wherein the method further comprises adding seed crystals of a compound of formula IIIa to a mixture of the reaction product mixture and water.

[0559] C19. The method of any one of embodiments C-C18, wherein the method further comprises a purification step comprising:

[0560] (i) mixing a compound of formula IIIa with a metal scavenger in a solvent system comprising at least one polar protic solvent;

[0561] (ii) heating the mixture to dissolve the compound of formula IIIa;

[0562] (iii) filtering the heated mixture;

[0563] (iv) reducing the temperature of the filtrate and mixing seed crystals of the compound of Formula IIIa with the cooled filtrate;

[0564] (v) reducing the temperature of the mixture of the filtrate and the seed crystals to induce crystallization of the purified compound of Formula IIIa; and

[0565] (vi) collecting purified crystals of the compound of formula IIIa.

[0566] C20. A method according to embodiment C19, wherein

[0567] (i) the solvent system comprises or consists essentially of water and acetic acid, wherein the volume ratio of acetic acid to water is from about 1:1 to about 10:1, from about 1:1 to about 5:1, or from about 1:1 to about 3:1, or about 3:1;

[0568] (ii) the metal scavenger is a silicon-thiol; and

[0569] (iii) the dissolving temperature is about 80°C to about 100°C, the seed crystals are combined with the filtrate at a temperature of about 70°C to about 80°C, and the crystallization temperature is about 0°C to about 10°C.

[0570] C21. A method according to any one of embodiments C-C20, wherein the yield of the compound of formula IIIa based on the compound of formula IIa is at least 75%, at least 80%, at least 85% or at least 90%.

[0571] C22. A method according to any one of embodiments C-C21, wherein the purity of the compound of Formula IIIa is at least 97%, at least 97.5%, or at least 98% (area % determined by HPLC).

[0572] C23. The method according to any one of embodiments C-C22, wherein the compound of formula IIIa is

[0573]

[0574] D. A method of treating cancer in a patient, wherein the cancer is characterized by overexpression of PI3 kinase, comprising administering to a human in need of such treatment a therapeutically effective amount of a PI3 kinase inhibitor compound of Formula III according to Embodiment A.

[0575] D1. The method according to embodiment D, characterized in that the PI3 kinase inhibitor compound is a compound IIIat of the following formula:

[0576]

[0577] D2. A method according to embodiment D or D1, wherein the dose of the PI3 kinase inhibitor compound is about 0.2 mg / kg / day to about 1.5 mg / kg / day, about 0.3 mg / kg / day to about 1 mg / kg / day, or about 0.4 mg / kg / day to about 0.75 mg / kg / day.

[0578] D3. A method according to any one of embodiments D-D2, wherein the terminal half-life of the PI3 kinase inhibitor compound in multiple cancer cells after administration of a single dose on the first day of the dosage cycle is about 10 hours to about 24 hours, about 12 hours to about 22 hours, or about 15 hours to about 20 hours.

[0579] D4. A method according to any one of embodiments D-D3, wherein the time to reach the maximum plasma concentration of the PI3 kinase inhibitor after administration of a single dose on the first day of a dosage cycle is about 1 hour to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, or about 2 hours to about 3 hours.

[0580] D5. A method according to any one of embodiments D-D4, wherein the maximum plasma concentration of the PI3 kinase inhibitor after administration of a single dose on the first day of a dosage cycle is about 0.01 μM to about 0.5 μM, about 0.05 μM to about 0.4 μM, or about 0.1 μM to about 0.3 μM.

[0581] D6. A method according to any one of embodiments D-D5, wherein, after administration of a single dose on the first day of a dosage cycle, the area under the concentration-time curve of the PI3 kinase inhibitor from time 0 to infinity in multiple cancer cells is about 0.2 μM*hr to about 10 μM*hr, about 0.5 μM*hr to about 10 μM*hr, about 1 μM*hr to about 8 μM*hr, or about 2 μM*hr to about 6 μM*hr.

[0582] D7. A method according to any one of embodiments D-D6, wherein, after administration of a single dose on the first day of a dosage cycle, the area under the concentration-time curve of the PI3 kinase inhibitor from time 0 to 24 hours in multiple cancer cells is about 0.1 μM*hr to about 10 μM*hr, about 0.5 μM*hr to about 5 μM*hr, about 1 μM*hr to about 5 μM*hr, or about 2 μM*h to about 4 μM*hr.

[0583] D8. A method according to any one of embodiments D-D7, wherein the PI3 kinase inhibitor is administered orally.

[0584] D9. A method according to any one of embodiments D-D8, wherein the PI3 kinase inhibitor is administered orally without food or under fasting conditions.

[0585] D10. The method of any one of embodiments D-D9, wherein the cancer is brain cancer.

[0586] D11. The method of any one of embodiments D-D10, wherein the cancer is glioma.

[0587] D12. The method of any one of embodiments D-D10, wherein the cancer is glioblastoma.

[0588] D13. A method according to any one of embodiments D-D12, wherein the method further comprises administering an additional therapeutic agent to the patient; the additional therapeutic agent is selected from chemotherapeutic agents, anti-tumor angiogenesis therapeutic agents, anti-inflammatory agents, immunomodulators, neurotropic factors, cardiovascular disease therapeutic agents, liver disease therapeutic agents, antiviral agents, blood disease therapeutic agents, diabetes therapeutic agents and immunodeficiency disease therapeutic agents.

[0589] D14. The method according to embodiment D13, wherein the additional therapeutic agent is bevacizumab.

[0590] D15. The method of embodiment D13, wherein the additional therapeutic agent is temozolomide.

Claims

1. A method for preparing a compound of formula IIa from a compound of formula I in a reaction mixture according to the following reaction formula, The method comprises: (i) forming a reaction mixture comprising a compound of Formula I, an organic halide, a solvent system, a phase transfer catalyst and a base; (ii) reacting the reaction mixture to form a reaction product mixture comprising a compound of Formula IIa, a stereoisomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof; and (iii) separating the compound of formula IIa from the reaction product mixture; in, The solvent system consists essentially of water; X is a halogen; Each R 5 , R 10 and R 15 Independently selected from H, C1-C5 alkyl; mor is selected from: Wherein in Formula I, R 20 is -OH, and wherein in Formula IIa, R 20 It is -O-.

2. The method according to claim 1, characterized in that: The base is selected from K3PO4, Cs2CO3, K2CO3, KOAc, NaOAc, Na2CO3 and KOH.

3. The method according to claim 1, characterized in that The phase transfer catalyst is selected from quaternary ammonium salts and quaternary phosphonium salts.

4. The method according to claim 3, characterized in that The phase transfer catalyst is selected from tetra-n-butylammonium bromide, benzyltrimethylammonium chloride, benzyltriethylammonium chloride, methyltrioctylammonium chloride, methyltributylammonium chloride and methyltrioctylammonium chloride.

5. The method according to claim 1, characterized in that The organic halide and the base are present in equimolar amounts.

6. The method according to claim 1, characterized in that The reaction temperature is 40°C to 90°C.

7. The method according to claim 6, characterized in that The temperature of the reaction was 50°C.

8. The method according to claim 1, characterized in that The method further comprises mixing a polar protic solvent with the reaction product mixture and then reducing the temperature of the mixture to induce crystallization of the compound of Formula IIa in the reaction product mixture, wherein the crystallized compound of Formula IIa is separated from the reaction product mixture.

9. The method according to claim 8, characterized in that The polar protic solvent is selected from water, methanol, ethanol, isopropanol, n-propanol and acetic acid.

10. The method according to claim 9, characterized in that The polar protic solvent is ethanol.

11. The method according to claim 10, characterized in that The volume ratio of the solvent system to ethanol is 1:5 v / v to 5:1 v / v.

12. The method according to claim 11, characterized in that The method further comprises adding seed crystals of a compound of Formula IIa to the mixture of the reaction product mixture and ethanol.

13. The method according to claim 1, characterized in that The yield of the compound of formula IIa is at least 60%.

14. The method according to claim 1, characterized in that The purity of the compound of Formula Ha was at least 97% by area % as determined by HPLC.

15. The method according to claim 1, characterized in that The organic halide is 1,2-dibromoethane.

16. The method according to claim 15, characterized in that The molar ratio of 1,2-dibromoethane to the compound of formula I is greater than 2:1 to 4:

1.

17. The method according to any one of claims 1 to 16, characterized in that The compound of formula IIa is 18. The method according to claim 1, characterized in that The method further comprises preparing a compound of formula IIIa from a compound of formula IIa in a reaction mixture according to the following reaction formula, The method comprises: (i) forming a compound comprising formula IIa, an organoboron-R 4 , a reaction mixture of a solvent system, a base and a catalyst, wherein the solvent system comprises at least 5 v / v% water; (ii) reacting the reaction mixture to form a reaction product mixture comprising a compound of Formula IIIa; and (iii) isolating the compound of formula IIIa, its stereoisomer, its tautomer, or a pharmaceutically acceptable salt thereof from the reaction product mixture by solid-liquid separation, wherein the yield of the compound of formula IIIa is at least 75%; in, The catalyst comprises palladium and the reaction mixture comprises less than 0.05 equivalents of the catalyst per equivalent of the compound of Formula IIa; R 4 Selected from C6-C 20 Aryl, C2-C 20 Heterocyclic and C1-C 20 heteroaryl, each of which is optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CH, -CHCH, -CH(CH), -CHCH(CH), -CHCH, -CHCN, -CN, -CF, -CHOH, -COH, -CONH, CONH(CH), -CON(CH), -NO, -NH, -NHCH, -NHCOCH, -OH, -OCH, -OCHCH, -OCH(CH), -SH, -NHC(O)NHCH, -NHC(O)NHCHCH, -NHS(O)CH, -N(CH)C(O)OC(CH), -S(O)CH, benzyl, benzyloxy, morpholinyl, morpholinylmethyl, and 4-methylpiperazinyl.

19. The method according to claim 18, characterized in that The solvent system further comprises at least one polar aprotic solvent selected from tetrahydrofuran, dichloromethane, ethyl acetate, acetone, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide.

20. The method according to claim 19, characterized in that The ratio of water to the at least one polar aprotic solvent is from 1:10 v / v to 5:1 v / v.

21. The method according to claim 18, characterized in that The solvent system consists essentially of water and at least one polar aprotic solvent.

22. The method according to claim 21, characterized in that The solvent system comprises water and tetrahydrofuran.

23. The method according to claim 18, characterized in that The organic boron-R 4 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-R 4 .

24. The method according to claim 18, characterized in that The base is selected from K3PO4, Cs2CO3 and KOH.

25. The method according to claim 24, characterized in that The base is K3PO4.

26. The method according to claim 18, characterized in that The equivalent ratio of base to compound of formula IIa is at least 1:

1.

27. The method according to claim 26, characterized in that The equivalent ratio of the base to the compound of formula IIa is 1:1 to 3:

1.

28. The method according to claim 26, characterized in that The equivalent ratio of the base to the compound of formula IIa is 2:

1.

29. The method according to claim 18, characterized in that The equivalent ratio of the catalyst comprising palladium to the compound of Formula IIa is from 0.003:1 to 0.05:

1.

30. The method according to claim 18, characterized in that The method further comprises adding a polar protic solvent to the reaction product mixture to form a mixture comprising at least 25 v / v% water, and isolating the compound of Formula IIIa from the reaction product mixture.

31. The method according to claim 30, characterized in that The polar protic solvent is selected from water, methanol, ethanol, isopropanol, n-propanol and acetic acid.

32. The method according to claim 31, characterized in that The polar protic solvent is water.

33. The method according to claim 32, characterized in that The volume ratio of the solvent system to the water added to the reaction product mixture is 1:5 v / v to 5:1 v / v.

34. The method according to claim 33, characterized in that The volume ratio of the solvent system to the water added to the reaction product mixture is 1:3 v / v to 3:1 v / v.

35. The method according to claim 33, characterized in that The volume ratio of the solvent system to the water added to the reaction product mixture was 3:1 v / v.

36. The method according to claim 32, characterized in that The method further comprises adding seed crystals of a compound of Formula IIIa to the mixture of the reaction product mixture and water.

37. The method according to any one of claims 18 to 36, characterized in that The method further comprises a purification step, the purification step comprising: (i) mixing a compound of formula IIIa with a metal scavenger in a solvent system comprising at least one polar protic solvent; (ii) heating the mixture to dissolve the compound of formula IIIa; (iii) filtering the heated mixture; (iv) reducing the temperature of the filtrate and mixing seed crystals of the compound of Formula IIIa with the cooled filtrate; (v) reducing the temperature of the mixture of the filtrate and the seed crystals to induce crystallization of the purified compound of Formula IIIa; and (vi) collecting purified crystals of the compound of formula IIIa.

38. The method according to claim 37, characterized in that (a) the solvent system comprises water and acetic acid, wherein the volume ratio of acetic acid to water is 1:1 to 10:1; (b) the metal scavenger is silicon-thiol; and (c) the dissolving temperature is 80°C to 100°C, the seed crystals are combined with the filtrate at a temperature of 70°C to 80°C, and the crystallization temperature is 0°C to 10°C.

39. The method according to claim 18, characterized in that The yield of the compound of the formula IIIa is at least 80%, based on the compound of the formula IIa.

40. The method according to claim 18, characterized in that The purity of the compound of formula IIIa was at least 97% by area % as determined by HPLC.

41. The method according to claim 18, characterized in that The compound of formula IIIa is 42. The method according to claim 18, characterized in that The palladium-containing catalyst is selected from chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II); [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane ("PdCl2 dppf CH2Cl2"); bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) ("Pd(amphos)Cl2"); bis(di-tert-butylphenylphosphine)dichloropalladium(II) ("Pd 122"); chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (Xphos Pd G2); PdCl2(PPh3)2; Pd(t-Bu)3; Pd(PPh3)4; Pd(Oac) / PPh3; Cl2Pd[(Pet3)]2; Pd(DIPHOS)2; Cl2Pd(Bipy); [PdCl(Ph2PCH2PPh2)]2; Cl2Pd[P(o-tol)3]2; Pd2(dba)3 / P(o-tol)3; Pd2(dba ) / P(furyl)3; Cl2Pd[P(furyl)3]2; Cl2Pd(PmePh2)2; Cl2Pd[P(4-F-Ph)3]2; Cl2Pd[P(C6F6)3]2; Cl2Pd[P(2-COOH-Ph)(Ph)2]2; Cl2Pd[P(4-COOH-Ph)(Ph)2]2; palladium acetate containing 0.4mmol / g Pd microencapsulated in a polyurea matrix; palladium acetate and triphenylphosphine containing 0.4mmol / g Pd and 0.3mmol / g phosphorus microencapsulated in a polyurea matrix; and palladium acetate and BINAP containing 0.4mmol / g Pd microencapsulated in a polyurea matrix.

43. The method according to claim 42, characterized in that The catalyst is chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II), and the equivalent ratio of the catalyst comprising palladium to the compound of formula IIa is 0.004:1 to 0.015:

1.

44. The method according to claim 42, characterized in that The catalyst is a complex of chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) or [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane, and the equivalent ratio of the catalyst containing palladium to the compound of formula IIa is 0.005:1 to 0.04:1.

Images