FGFR inhibitor derivatives

Abstract

Derivatives of fibroblast growth factor receptor (FGFR) inhibitors, methods for their preparation, and methods for treating cancer are provided. The present invention provides derivatives (e.g., hydroxyl, keto, glucuronide, sulfonate, and deuterated) of FGFR inhibitors, including methods for their preparation and intermediates in their preparation, which are useful in the treatment of FGFR-mediated diseases such as cancer.

Description

[Technical Field]

[0001] This application relates to derivatives (e.g., hydroxyl, keto, glucuronide, sulfonate, and deuterated) of fibroblast growth factor receptor (FGFR) inhibitors, including methods for their preparation and intermediates in their preparation, which are useful in the treatment of FGFR-mediated diseases, such as cancer. [Background technology]

[0002] Fibroblast growth factor receptors (FGFRs) are receptor tyrosine kinases that bind to fibroblast growth factor (FGF) ligands. There are four FGFR proteins (FGFR1-4) that can bind to ligands and are involved in the control of many physiological processes, including tissue development, angiogenesis, wound healing, and metabolic regulation. Upon ligand binding, the receptor undergoes dimerization and phosphorylation, leading to stimulation of protein kinase activity and recruitment of numerous intracellular docking proteins. These interactions promote the activation of a series of intracellular signaling pathways, including Ras-MAPK, AKT-PI3K, and phospholipase C, which are important for cell growth, proliferation, and survival (reviewed in Eswarakumar et al., Cytokine & Growth Factor Reviews, 2005).

[0003] Aberrant activation of this pathway, either through overexpression of FGF ligands or FGFRs, or through activating mutations in FGFRs, can lead to tumor initiation, progression, and resistance to conventional cancer therapies. Genetic alterations, including gene amplification, chromosomal translocations, and somatic mutations leading to ligand-independent receptor activation, have been described in human cancers. Genetic alterations can include mutations, fusions, rearrangements (e.g., translocations, deletions, and inversions), and gene amplification. Large-scale DNA sequencing of thousands of tumor samples has revealed that components of the FGFR pathway are among the most frequently mutated in human cancers. Many of these activating mutations are identical to germline mutations that cause skeletal dysplasia syndromes. Mechanisms leading to aberrant ligand-dependent signaling in human diseases include overexpression of FGFs and altered FGFR splicing, leading to receptors with more promiscuous ligand-binding capacity (reviewed in Knights and Cook, Pharmacology & Therapeutics, 2010; Turner and Grose, Nature Reviews Cancer, 2010). Therefore, the development of inhibitors that target FGFRs may be useful in the clinical treatment of diseases in which FGF or FGFR activity is elevated.

[0004] Cancer types involving FGF / FGFR include, but are not limited to, carcinomas (e.g., bladder, breast, cervical, colorectal, endometrial, gastric, head and neck, kidney, liver, lung, ovarian, and prostate); hematopoietic malignancies (e.g., multiple myeloma, chronic lymphocytic lymphoma, adult T-cell leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, myeloproliferative neoplasms, and Waldenstrom's macroglubulinemia); and other neoplasms (e.g., glioblastoma, melanoma, and rhabdomyosarcoma). In addition to its role in oncogenic neoplasms, FGFR activation has also been implicated in skeletal and chondrocyte disorders, including, but not limited to, achondroplasia and craniosynostosis syndromes. Specifically, the FGFR4-FGF19 signaling axis has been implicated in the pathogenesis of many cancers, including hepatocellular carcinoma (Heinzle et al., Cur. Pharm. Des. 2014, 20:2881). Ectopic expression of FGF19 in transgenic mice has been shown to result in liver tumor formation, and neutralizing antibodies against FGF19 have been found to inhibit tumor growth in mice. Furthermore, overexpression of FGFR4 has been observed in multiple tumor types, including hepatocellular carcinoma, colorectal cancer, breast cancer, pancreatic cancer, prostate cancer, lung cancer, and thyroid cancer. Furthermore, activating mutations of FGFR4 have been reported in rhabdomyosarcoma (Taylor et al. JCI 2009, 119:3395).

[0005] Inhibitors of FGFR are currently being developed for the treatment of cancer. For example, the molecule 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one and other small molecule inhibitors of FGFR are reported in, for example, U.S. Patent Publication Nos. 2012 / 0165305; 2014-0045814; 2013-0338134; 2014 / 0171405; 2014 / 0315902; 2016 / 0115164; 2016 / 0244448; 2016 / 0244449; and 2016-0244450.

[0006] Thus, new or improved agents that inhibit FGFRs continue to be needed to develop new, more effective pharmaceuticals for treating cancer and other diseases. The compounds, including derivatives of these compounds, compositions, and methods described herein address these needs and other objectives. Summary of the Invention

[0007] The present disclosure relates, inter alia, to derivatives of Compound I: [ka] or a pharmaceutically acceptable salt thereof.

[0008] The present disclosure further relates to pharmaceutical compositions comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

[0009] The present disclosure further relates to a method of inhibiting an FGFR enzyme comprising contacting the enzyme with a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0010] The present disclosure further relates to a method for treating a disease associated with aberrant activity or expression of an FGFR enzyme, comprising administering to a patient in need thereof a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0011] The present disclosure further relates to the compounds of the present disclosure for use in treating diseases associated with abnormal activity or expression of FGFR enzymes.

[0012] The present disclosure further relates to a method for treating a disorder mediated by an FGFR enzyme, or a mutant thereof, in a patient in need thereof, comprising administering to the patient a compound of the present disclosure, or a pharmaceutically acceptable composition thereof.

[0013] The present disclosure further relates to a method for treating a disorder mediated by an FGFR enzyme, or a mutant thereof, in a patient in need thereof, comprising administering to the patient a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, in combination with another therapy or therapeutic agent described herein.

[0014] The present disclosure further relates to the use of the disclosed compounds in the preparation of a medicament for use in therapy. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a graph showing the mean cumulative percent of radioactive dose recovered in urine and feces at the indicated intervals following a single oral dose of 13 mg (250 μCi) of [C]Compound 1 to healthy male subjects. [Figure 2] 1 is a graph showing the mean radioactivity (nM equivalent) in blood or plasma and Compound 1 (nM) in plasma following a single oral dose of approximately 13 mg of [14C]Compound 1 to healthy male volunteers in the fasted state. [Figure 3] 1 shows the mass spectrum of human circulating metabolites of Compound 1. [Figure 4] 1 shows the mass spectrum of Compound 1 metabolites isolated from urine. [Figure 5] 1 shows the mass spectrum of Compound 1 metabolites isolated from feces. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure relates, inter alia, to compounds that are derivatives of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 1), as well as processes and intermediates for preparing the derivatives. The structure of Compound 1 is shown below. [ka]

[0017] Compound 1 is described in US Pat. No. 9,611,267, which is incorporated herein by reference in its entirety.

[0018] In some embodiments, the compound is a metabolite of Compound 1. In some embodiments, the compound is an active metabolite that may modulate the activity of one or more FGFR proteins and may be useful, for example, in the treatment of diseases associated with FGFR expression or activity. In some embodiments, the levels of metabolic compounds described herein are measured and profiled to assist clinicians in adjusting dosage levels of Compound 1.

[0019] Thus, the present disclosure also provides compounds of formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein: one or more OCH groups are optionally replaced with an OH group, wherein the OH group is optionally replaced with an OX group; one CH group is optionally replaced by a CX group; one CH group is optionally replaced by a C=O group; one or more CH groups of the morpholine ring are each optionally replaced with a C(OH)H group; the NH group is optionally replaced with an NX group, and where X is a group selected from: [ka] However, this compound has one or more groups selected from OH, OX, CX, C═O, and NX.

[0020] The present invention also provides a compound of formula II: [ka] or a pharmaceutically acceptable salt thereof, wherein: one CH group is optionally replaced by a CX group; one CH group is optionally replaced by a C=O group; the NH group is optionally replaced with an NX group, and The OH group is optionally replaced with an OX group; where X is a group selected from: [ka] is.

[0021] In some embodiments, the present invention provides a compound of formula II: [ka] or a pharmaceutically acceptable salt thereof, wherein one CH group is replaced by a CX group, an NH group is replaced by an NX group, or an OH group is replaced by an OX group; where X is a group selected from: [ka] is.

[0022] In some embodiments, one or more OCH groups are optionally replaced with an OH group, where the OH group is optionally replaced with an OX group. In some embodiments, the OH group is optionally replaced with an OX group. In some embodiments, one CH group is optionally replaced with a CX group. In some embodiments, one CH group is optionally replaced with a C=O group. In some embodiments, the NH group is optionally replaced with an NX group. In some embodiments, one or more CH groups on the morpholine ring are each optionally replaced with a C(OH)H group.

[0023] In some embodiments, one or more OCH groups are replaced with an OH group, where the OH group is replaced with an OX group. In some embodiments, one or more OCH groups are replaced with an OH group. In some embodiments, the OH group is replaced with an OX group. In some embodiments, one CH group is replaced with a CX group. In some embodiments, one CH group is replaced with a C=O group. In some embodiments, the NH group is replaced with an NX group. In some embodiments, one or more CH groups on the morpholine ring are each replaced with a C(OH)H group.

[0024] In some embodiments, X is [ka] In some embodiments, X is [ka] is.

[0025] The present invention provides a compound, 3-(2,6-difluoro-3-hydroxy-5-methoxyphenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 2), or a pharmaceutically acceptable salt thereof. The structure of Compound 2 is shown below: [ka] .

[0026] The present invention provides a compound, 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-4-hydroxy-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 3), or a pharmaceutically acceptable salt thereof. The structure of Compound 3 is shown below: [ka] .

[0027] The present invention provides a compound, 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 4), or a pharmaceutically acceptable salt thereof. The structure of Compound 4 is shown below. [ka] .

[0028] In some embodiments, the compound is selected from: 3-(2,6-difluoro-3-hydroxy-5-methoxyphenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one; and 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-4-hydroxy-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one, or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the compound is a compound of Formula I: [ka] Or a pharmaceutically acceptable salt thereof, wherein one CH2 group of the morpholine ring is replaced with a (C=O) group.

[0030] In some embodiments, the compound is a compound of formula (II): [ka] or a pharmaceutically acceptable salt thereof, in which one CH group is replaced by a CX group, an NH group is replaced by an NX group, or an OH group is replaced by an OX group; where X is: [ka] .

[0031] In some embodiments, the compound is a compound of formula (II): [ka] or a pharmaceutically acceptable salt thereof, in which one CH group is replaced by a CX group, an NH group is replaced by an NX group, or an OH group is replaced by an OX group; where X is: [ka] .

[0032] In some embodiments, the compound is a compound of formula I [ka] or a pharmaceutically acceptable salt thereof, wherein two CH2 groups of the morpholine ring are each replaced by a C(OH)H group.

[0033] In some embodiments, the compound is a compound of formula I: [ka] or a pharmaceutically acceptable salt thereof, wherein each OCH3 group is replaced with an OH group; and one CH2 group of the morpholine ring is replaced with a (C=O) group.

[0034] In some embodiments, the compound is a compound of formula II: [ka] Or a pharmaceutically acceptable salt thereof, wherein one CH2 group of the morpholine ring is replaced with a (C=O) group.

[0035] Also provided herein is a compound which is 3-(2,6-difluoro-3-hydroxy-5-methoxyphenyl)-1-ethyl-2-oxo-2,3,4,7-tetrahydro-1H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-8-carboxylic acid (Compound 5), or a pharmaceutically acceptable salt thereof.

[0036] The structure of compound 5 is shown below: [ka] .

[0037] Also provided herein is a compound that is 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 6), or a pharmaceutically acceptable salt thereof. The structure of Compound 6 is shown below: [ka] .

[0038] Each of the foregoing embodiments assumes that appropriate valency rules are observed.

[0039] Certain compounds described herein are metabolites of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 1). This metabolite was isolated from human blood / serum, urine, and fecal samples collected from pharmacokinetic and toxicokinetic studies of Compound 1. The metabolites of the present invention may be FGFR inhibitors and may have advantageous properties (PK, PD, toxicity, etc.) compared to the parent compound (Compound 1). For example, a metabolite of the present disclosure (e.g., Compound 2) may be a better substrate for Pgp transport compared to Compound 1. Therefore, a metabolite of the present disclosure (e.g., Compound 2) may be a more suitable candidate for intravenous administration or hepatic artery infusion for the treatment of diseases and disorders (e.g., cholangiocarcinoma) disclosed herein.

[0040] In some embodiments, the compounds of the invention are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched for the compounds of the invention. Substantial separation can include a composition containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of metabolites.

[0041] Also provided herein are compositions comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof, comprising at least about 50% by weight of the compound or salt. In some embodiments, the composition comprises at least about 60% by weight of the compound or salt. In some embodiments, the composition comprises at least about 70% by weight of the compound or salt. In some embodiments, the composition comprises at least about 80% by weight of the compound or salt. In some embodiments, the composition comprises at least about 90% by weight of the compound or salt.

[0042] Preparation process of compound 2 The present application further provides a process for preparing compound 2, which may be suitable for scale-up.

[0043] As used herein, compound 2 has the following formula: [ka] or a salt thereof, which has the following formula: [ka] or a salt thereof, wherein P 1 includes deprotecting the amino-protecting group of compound F4. 1 is a sulfonamide group. In some embodiments, P 1 is -SO2N(CH3)2.

[0044] In some embodiments, deprotecting compound F4 comprises reacting compound F4 with A1, where A1 is an acid. In some embodiments, A1 is hydrochloric acid. In some embodiments, A1 is aqueous hydrochloric acid. In some embodiments, deprotecting is carried out at 70-90°C (e.g., 80°C). In some embodiments, the deprotecting is carried out in the presence of S1, where S1 is a polar aprotic solvent. In some embodiments, S1 is 1,4-dioxane. In some embodiments, deprotecting compound F4 comprises using about 1 to about 50 molar equivalents of a deprotecting agent relative to compound F4, about 10 to about 30 molar equivalents of a deprotecting agent relative to compound F4, or about 20 molar equivalents of a deprotecting agent relative to compound F4.

[0045] Compound F4 or a salt thereof is a compound F3 having the formula: [ka] Alternatively, the salt may be produced by a process comprising reacting morpholine in the presence of RA1, where RA1 is a reducing agent. In some embodiments, RA1 is sodium triacetoxyborohydride. In some embodiments, the reaction of compound F3 with RA1 is carried out in the presence of an acid, A2. In some embodiments, A2 is an organic acid. In some embodiments, A2 is acetic acid. In some embodiments, the reaction of compound F3 with RA1 is carried out in the presence of a solvent, S2. In some embodiments, S2 is a polar aprotic solvent. In some embodiments, S2 is methylene chloride.

[0046] Compound F3 or a salt thereof may be prepared from compound F2 having the formula: [ka] or a salt thereof with DMF in the presence of a base, B1. In some embodiments, B1 is lithium diisopropylamide ("LDA"). In some embodiments, the reaction of compound F2 with DMF in the presence of B1 is carried out in the presence of S3, where S3 is a polar aprotic solvent. In some embodiments, S3 is tetrahydrofuran. The reaction of compound F2 with DMF in the presence of B1 may be carried out at a temperature between about -100°C and about -50°C (e.g., about -64°C).

[0047] Compound F2 or a salt thereof is a compound F1 having the formula: [ka] or a salt thereof, 1 It can be produced by a process comprising reacting it with an amino protecting agent comprising

[0048] In some embodiments, the amino protecting agent is P 1-X, where X is a halogen. In some embodiments, the amino protecting agent is Me2NSO2Cl. The reaction of compound F1 with the amino protecting agent may be carried out in the presence of B2, where B2 is a base. In some embodiments, B2 is a metal hydroxide base. In some embodiments, B2 is NaOH.

[0049] The reaction of Compound F1 with an amino-protecting agent may be carried out in the presence of tetrabutylammonium hydrogen sulfate. In some embodiments, the reaction of Compound F1 with an amino-protecting agent is carried out in the presence of S4, where S4 is a polar aprotic solvent. In some embodiments, S4 is tetrahydrofuran. In some embodiments, the reaction of Compound F1 with an amino-protecting agent is carried out at a temperature of about 0°C to about 50°C (e.g., about 0°C to about 30°C).

[0050] As used herein, compound 2 has the following formula: [ka] or a salt thereof, comprising: a) Compound F1 having the following formula: [ka] And, P 1 to form a compound F2 having the formula: [ka] or a salt thereof, wherein P 1 is an amino protecting group; b) reacting compound F2 with DMF in the presence of B1, wherein B1 is a base, to form compound F3 having the formula: [ka] or a salt thereof; c) reacting compound F3 with morpholine in the presence of a reducing agent RA1 to produce compound F4 having the formula: [ka] or a salt thereof; d) deprotecting compound F4 to provide compound 2 or a salt thereof.

[0051] Preparation process of compound 6 The present disclosure provides compound 6 having the following formula: [ka] or a salt thereof, comprising the step of: [ka] or a salt thereof, wherein P 2 is an amino protecting group. 2 -SO 2 -phenyl.

[0052] Deprotecting compound F5 can include treating compound F5 with B3, where B3 is a base. In some embodiments, B3 is a metal hydroxide base. In some embodiments, B3 is NaOH. In some embodiments, B3 is aqueous NaOH. In some embodiments, deprotecting compound F5 is carried out in the presence of S5, where S5 is 1,4-dioxane. In some embodiments, reacting compound F5 with B3 includes using about 1 to about 10 molar equivalents of B3 relative to compound F5, about 2 to about 8 molar equivalents of B3 relative to compound F5, or about 4 molar equivalents of B3 relative to compound F5.

[0053] Compound F5 may be prepared by cleaving Compound F6 having the formula: [ka] can be prepared by a process comprising reacting with CD3I in the presence of B4, where B4 is a base. In some embodiments, B4 is sodium hydride.

[0054] The reaction of compound F6 with CD3I may be carried out in the presence of a polar aprotic solvent, S6. In some embodiments, S6 is DMF. The reaction of compound F6 with CD3I may involve using about 1 to about 5 molar equivalents of CD3I relative to compound F6, about 1 to about 3 molar equivalents of CD3I relative to compound F6, or about 2 molar equivalents of CD3I relative to compound F6. The reaction of compound F6 with CD3I may involve using about 1 to about 10 molar equivalents of B4 relative to compound F6, about 2 to about 8 molar equivalents of B4 relative to compound F6, or about 4 to about 5 molar equivalents of B4 relative to compound F6.

[0055] Compound F6 can be prepared by cleaving Compound F7, which has the formula: [ka] With a Lewis acid, A3, In some embodiments, A3 is BBr3.

[0056] The reaction of compound F7 with A3 may be carried out in the presence of a polar aprotic solvent, S7. In some embodiments, S7 is methylene chloride. The reaction of compound F7 with A3 may be carried out at a temperature of about -100°C to about 30°C (e.g., about -100°C to room temperature). The reaction of compound F7 with A3 may involve using about 1 to about 20 molar equivalents of A3 relative to compound F7, about 5 to about 15 molar equivalents of A3 relative to compound F7, or about 8 to about 12 molar equivalents of A3 relative to compound F7.

[0057] As used herein, compound 6 has the following formula: [ka] or a salt thereof, comprising: a) Compound F7 having the following formula: [ka] and react with A3, a Lewis acid, where P 2 is an amino protecting group, and compound F6 has the formula: [ka] or a salt thereof; b) reacting compound F6 with CD3I in the presence of a base B4 to form compound F5 having the formula: [ka] or a salt thereof; c) deprotecting compound F5 to provide compound 6 or a salt thereof.

[0058] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13The purity of a compound can be monitored by infrared spectroscopy, spectrophotometry (e.g., UV-visible light), or mass spectrometry; or by chromatography, for example, high-performance liquid chromatography (HPLC) or thin-layer chromatography. The compound obtained by the reaction can be purified by any suitable method known in the art. For example, chromatography (medium pressure) on a suitable adsorbent (e.g., silica gel, alumina, etc.), HPLC, or preparative thin-layer chromatography; distillation; sublimation, titration, or recrystallization. The purity of a compound is generally determined by physical methods, such as measuring the melting point (if solid), obtaining an NMR spectrum, or performing HPLC separation. A compound can be said to be purified if the melting point is reduced, if undesired signals in the NMR spectrum are reduced, or if extraneous peaks in the HPLC trace are eliminated. In some embodiments, the compound is substantially purified.

[0059] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Greene's Protective Groups in Organic Synthesis, 4th Ed., John Wiley & Sons: New York, 2006, which is incorporated herein by reference in its entirety. As used herein, "amino protecting group" refers to any protecting group for protecting an amine. Examples of amino protecting groups include phenylsulfonyl, benzyloxycarbonyl (Cbz), 2,2,2-trichloroethoxycarbonyl (Troc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), 2-(4-trifluoromethylphenylsulfonyl)ethoxycarbonyl (Tsc), t-butoxycarbonyl (BOC), 1-adamantyloxycarbonyl (Adoc), 2-adamantylcarbonyl (2-Adoc), 2,4-dimethylpent-3-yloxycarbonyl (Doc), cyclohexyloxycarbonyl (Hoc), 1,1-dimethyl-2,2, Examples of alkyl groups include, but are not limited to, 2-trichloroethoxycarbonyl (TcBOC), vinyl, 2-chloroethyl, 2-phenylsulfonylethyl, allyl, benzyl, 2-nitrobenzyl, 4-nitrobenzyl, diphenyl-4-pyridylmethyl, N',N'-dimethylhydrazinyl, methoxymethyl, t-butoxymethyl (Bum), benzyloxymethyl (BOM), or 2-tetrahydropyranyl (THP), tri(Ci-4 alkyl)silyl (e.g., tri(isopropyl)silyl), 1,1-diethoxymethyl, or N-pivaloyloxymethyl (POM).

[0060] The reactions of the processes described herein can be carried out at any suitable temperature, which can be readily determined by one of ordinary skill in the art. The reaction temperature will depend, for example, on the melting and boiling points of the reagents and solvent (if present), the thermodynamics of the reaction (e.g., highly exothermic reactions may need to be carried out at lower temperatures), and the kinetics of the reaction (e.g., high activation energy barriers may require higher temperatures).

[0061] In some embodiments, concentrating a solution as described herein refers to a solution whose volume is reduced by evaporating the solvent, by heating the solution, by subjecting the solution to a reduced pressure, or any combination thereof.

[0062] The reactions of the processes described herein can be carried out in a suitable solvent, which can be easily selected by one skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, which can range, for example, from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the specific reaction step, a suitable solvent for a particular reaction step can be selected. In some embodiments, the reaction can be carried out in the absence of a solvent, such as when at least one of the reagents is a liquid or gas.

[0063] Suitable solvents may include halogenated solvents such as carbon tetrachloride, bromodichloromethane, dibromochloromethane, bromoform, chloroform, bromochloromethane, dibromomethane, butyl chloride, dichloromethane (methylene chloride), ethylene tetrachloride, trichloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethane, 2-chloropropane, α,α,α-trifluorotoluene, 1,2-dichloroethane, 1,2-dibromoethane, hexafluorobenzene, 1,2,4-trichlorobenzene, 1,2-dichlorobenzene, chlorobenzene, fluorobenzene, mixtures thereof, and the like.

[0064] Suitable ether solvents include dimethoxymethane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan, tetrahydrofuran (THF), diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether (diglyme), diethylene glycol diethyl ether, triethylene glycol dimethyl ether, anisole, tert-butyl methyl ether, mixtures thereof, and the like.

[0065] Suitable protic solvents may include, by way of example and without limitation, water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol, 2-methoxyethanol, 1-butanol, 2-butanol, iso-butyl alcohol, tert-butyl alcohol, 2-ethoxyethanol, diethylene glycol, 1-pentanol, 2-pentanol, or 3-pentanol, neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol, phenol, or glycerol.

[0066] Suitable aprotic solvents may include, by way of example and without limitation, N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), 1,3-dimethyl-2-imidazolidinone (DMI), N-methylpyrrolidinone (NMP), formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethyl sulfoxide, propionitrile, ethyl formate, methyl acetate, hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate, sulfolane, N,N-dimethylpropionamide, tetramethylurea, nitromethane, nitrobenzene, or hexamethylphosphoramide.

[0067] Suitable hydrocarbon solvents include benzene, cyclohexane, pentane, hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene, m-xylene, o-xylene, or p-xylene, octane, indane, nonane, or naphthalene.

[0068] The reactions of the processes described herein can be carried out in air or under an inert atmosphere. Typically, reactions involving reagents or products that are substantially reactive with air can be carried out using air-sensitive synthetic techniques well known to those skilled in the art.

[0069] The expressions "ambient temperature" and "room temperature," as used herein, are understood in the art and generally refer to temperatures, e.g., reaction temperatures, approaching room temperature in which the reaction is carried out, e.g., temperatures of about 20° C. to about 30° C. The term "elevated temperature," as used herein, is understood in the art and generally refers to temperatures, e.g., reaction temperatures, above room temperature, e.g., 30° C.

[0070] Provided herein is compound F2: [ka] or a salt thereof, wherein P 1 is an amino protecting group.

[0071] In some embodiments, compound F2 has the following structure: [ka] or a salt thereof.

[0072] Provided herein is compound F3: [ka] or a salt thereof, wherein P 1 is an amino protecting group.

[0073] In some embodiments, compound F3 has the following structure: [ka] or a salt thereof.

[0074] Provided herein is compound F4: [ka] or a salt thereof, wherein P 1 is an amino protecting group.

[0075] In some embodiments, compound F4 has the following structure: [ka] or a salt thereof.

[0076] Provided herein is compound F5: [ka] or a salt thereof, wherein P 2 is an amino protecting group.

[0077] In some embodiments, compound F5 has the following structure: [ka] or a salt thereof.

[0078] Provided herein is compound F6: [ka] or a salt thereof, wherein P 2 is an amino protecting group.

[0079] In some embodiments, compound F6 has the following structure: [ka] or a salt thereof.

[0080] The compounds of the present disclosure also include tautomeric forms. Tautomeric forms are obtained by the interchange of adjacent double and single bonds with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Exemplary prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position on a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

[0081] The compounds of the present disclosure also include all isotopes of atoms occurring in intermediates or final compounds. Isotopes include atoms with the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. One or more constituent atoms of the compounds of the present disclosure may be replaced or substituted with an isotope of the atom at natural or non-natural abundance. In some embodiments, the compound contains at least one deuterium atom. For example, one or more hydrogen atoms in the compounds of the present disclosure may be replaced or substituted with deuterium. In some embodiments, the compound contains two or more deuterium atoms. In some embodiments, the compound contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deuterium atoms. Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971; The Renaissance of H / D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labelling by James R. Hanson, Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in a variety of studies, such as NMR spectroscopy, metabolic experiments, and / or assays.

[0082] Substitution with heavier isotopes, such as deuterium, may confer certain therapeutic benefits, such as increased in vivo half-life or reduced dosage requirements, resulting from greater metabolic stability and, therefore, may be preferable in some cases (A. Kerekes et al. J. Med. Chem. 2011, 54, 201-210; R. Xu et al. J. Label Compd. Radiopharm. 2015, 58, 308-312).

[0083] The term "compound," as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. The term is also meant to refer to compounds of the present disclosure regardless of how they are prepared, for example, by synthetic, biological process (e.g., metabolic or enzymatic transformation), or a combination thereof.

[0084] How to use The compounds described herein can inhibit the activity of FGFR enzymes.For example, the compounds of the present disclosure can be used to inhibit the activity of FGFR enzymes in cells, individuals, or patients that require enzyme inhibition by administering an inhibitory amount of the compound to the cells, individuals, or patients.

[0085] As FGFR inhibitors, the compounds of the present disclosure are useful for treating various diseases associated with the abnormal expression or activity of FGFR enzymes or FGFR ligands. Compounds that inhibit FGFR will be useful for providing a means of preventing tumor growth or inducing apoptosis, particularly by inhibiting angiogenesis. Therefore, it is expected that the compounds of the present disclosure will prove useful for treating or preventing proliferative disorders such as cancer. In particular, tumors with activated mutants of receptor tyrosine kinases or upregulated receptor tyrosine kinases may be particularly sensitive to inhibitors.

[0086] In certain embodiments, the present disclosure provides a method for treating an FGFR-mediated disorder in a patient in need thereof, comprising administering to the patient a compound of the present disclosure or a pharmaceutically acceptable composition thereof.

[0087] For example, the compounds of the present disclosure are useful in the treatment of cancer. Examples of cancer include bladder cancer, breast cancer (e.g., hormone R positive, triple negative), cervical cancer, colorectal cancer, cancer of the small intestine, colon cancer, rectal cancer, anal cancer, endometrial cancer, gastric cancer (e.g., gastrointestinal stromal tumor), head and neck cancer (e.g., cancer of the larynx, hypopharynx, nasopharynx, oropharynx, lip, mouth, head and neck squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma, urothelial carcinoma, sarcoma, Wilms' tumor), liver cancer (e.g., hepatocellular carcinoma, cholangiocarcinoma, hepatic angiosarcoma, hepatoblastoma), lung cancer (e.g., adenocarcinoma, small cell lung carcinoma, and Non-small cell lung cancer, small cell and non-small cell carcinoma, bronchial carcinoma, bronchial adenoma, pleuropulmonary blastoma), ovarian cancer, prostate cancer, testicular cancer, uterine cancer, vulvar cancer, esophageal cancer, gallbladder cancer, pancreatic cancer (e.g., exocrine pancreatic cancer), gastric cancer, thyroid cancer, parathyroid cancer, neuroendocrine cancer (e.g., pheochromocytoma, Merkel cell carcinoma, neuroendocrine carcinoma), skin cancer (e.g., squamous cell carcinoma, Kaposi's sarcoma, Merkel cell skin cancer), and brain tumors (e.g., astrocytoma, medulloblastoma, ependymoma, neuroexocrine tumor, pineal tumor).

[0088] Further exemplary cancers include hematopoietic malignancies such as leukemia or lymphoma, multiple myeloma, chronic lymphocytic lymphoma, adult T-cell leukemia, B-cell lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia, Hodgkin's lymphoma or non-Hodgkin's lymphoma, myeloproliferative neoplasms (e.g., 8p11 myeloproliferative syndrome, polycythemia, essential thrombocythemia, and primary myelofibrosis), myelodysplastic syndromes, chronic eosinophilic leukemia, Waldenstrom's macroglobulinemia, hairy cell lymphoma, chronic myeloid lymphoma, acute lymphoblastic lymphoma, AIDS-related lymphoma, and Burkitt's lymphoma.

[0089] In certain embodiments, provided herein are methods of treating myeloid / lymphoid tumors in a patient in need thereof. In certain embodiments, the myeloid / lymphoid tumor is 8p11 myeloproliferative syndrome. As used herein, the term "8p11 myeloproliferative syndrome" (EMS) is meant to refer to myeloid / lymphoid tumors associated with eosinophilia and FGFR1 abnormalities, or myeloid / lymphoid neoplasms (MLN) with FGFR1 rearrangements. 8p11 myeloproliferative syndrome is reviewed in Jackson, Courtney C., et. al., Human Pathology, 2010, 41, 461-476. EMS is characterized by the presence of translocations involving the FGFR1 gene at the chromosome 8p11 locus, and at least 10 additional translocations and one insertion have been identified in EMS, each disrupting FGFR1 and creating novel fusion genes with various partners. See Jackson, Courtney C., et. al. Human Pathology, 2010, 41, 461-476.

[0090] In some embodiments, the myeloid / lymphoid tumor is characterized by an FGF / FGFR gene alteration. Genetic alterations can include mutations, fusions, rearrangements (e.g., translocations, deletions, inversions), and gene amplifications. In certain embodiments, the myeloid / lymphoid tumor exhibits an FGFR1 fusion. The FGFR1 fusion can be a translocation, an interstitial deletion, or a chromosomal inversion. In some embodiments, the FGFR1 fusion is an FGFR1 translocation. In certain embodiments, the myeloid / lymphoid tumor exhibits an 8p11 translocation. In certain embodiments, the 8p11 translocation is associated with FGFR1 activation. In some embodiments, the myeloid / lymphoid tumor exhibits an FGF / FGFR alteration other than an FGFR1 translocation. In certain embodiments, the patient has failed at least one previous treatment for the myeloid / lymphoid tumor (e.g., 8p11 myeloproliferative syndrome). In some embodiments, the previous treatment is surgery or radiation therapy. In some embodiments, the patient has a history of hepatitis. In some embodiments, the hepatitis is chronic hepatitis B or hepatitis C. In some embodiments, the patient has no history of hepatitis.

[0091] In certain embodiments, provided herein is a method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present disclosure. In certain embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, cancer of the small intestine, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, testicular cancer, uterine cancer, vulvar cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, thyroid cancer, skin cancer, brain cancer, leukemia, multiple myeloma, chronic lymphocytic lymphoma, adult T-cell leukemia, B-cell lymphoma, acute myeloid leukemia, Hodgkin's lymphoma or non-Hodgkin's lymphoma, Waldenstrom's macroglubulinemia, myeloproliferative neoplasms, chronic myeloid lymphoma, acute lymphoblastic lymphoma, T-lymphoblastic lymphoma, hairy cell lymphoma, Burkitt's lymphoma, glioblastoma, melanoma, rhabdomyosarcoma, lymphosarcoma, and osteosarcoma.

[0092] In certain embodiments, the cancer is bladder cancer (e.g., urothelial carcinoma, squamous cell carcinoma, adenocarcinoma).

[0093] In certain embodiments, the liver cancer is cholangiocarcinoma (e.g., intrahepatic, hilar or perihilar, distal extrahepatic). As used herein, cholangiocarcinoma is the same as cholangiocarcinoma or bile duct carcinoma. In certain embodiments, the cholangiocarcinoma is advanced or metastatic cholangiocarcinoma. In certain embodiments, the cholangiocarcinoma is surgically unresectable. In certain embodiments, the cholangiocarcinoma is intrahepatic. In certain embodiments, the cholangiocarcinoma is extrahepatic. In certain embodiments, the cholangiocarcinoma exhibits FGFR2 tyrosine kinase fusion, which defines certain molecular subtypes, as described in Arai, Yasuhito, et al. Hepatology, 2014, 59, 1427-1434. In some embodiments, the cholangiocarcinoma is characterized by FGF / FGFR gene-altered tumors. In some embodiments, the tumor exhibits FGFR2 fusion. The FGFR2 fusion can be a translocation, an interstitial deletion, or a chromosomal inversion. In some embodiments, the FGFR2 fusion is an FGFR2 translocation. The FGFR2 translocation may be selected from the group including, but not limited to, FGFR2-BICC1, FGFR2-AHCYL1, FGFR2-MACF1, and FGFR2 intron 17 rearrangement. In some embodiments, the tumor exhibits FGF / FGFR alterations other than FGFR2 translocation. In some embodiments, the cholangiocarcinoma does not exhibit FGF / FGFR gene alteration tumors.

[0094] Other cancers treatable with the compounds of the present disclosure include eye tumors, glioblastoma, melanoma, rhabdomyosarcoma, lymphosarcoma, leiomyosarcoma, urothelial carcinoma (e.g., ureter, urethra, bladder, urachus), and osteosarcoma.

[0095] Compounds of the present disclosure may also be useful in inhibiting tumor metastasis.

[0096] In some embodiments, compounds of the present disclosure may be used to treat Alzheimer's disease, HIV, or tuberculosis.

[0097] In addition to oncogenic neoplasms, the compounds of the present disclosure may be useful in the treatment of skeletal and chondrocyte disorders, including, but not limited to, achondroplasia, hypochondroplasia, dwarfism, thanatophoric dysplasia (TD) (clinical forms TD I and TD II), Apert syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Behle-Stevenson gyriform scalp syndrome, Pfeiffer syndrome, and craniosynostosis syndrome.

[0098] The compounds provided herein may further be useful in treating fibrotic diseases, such as when a disease symptom or disorder is characterized by fibrosis, including liver cirrhosis, glomerulonephritis, pulmonary fibrosis, systemic fibrosis, rheumatoid arthritis, and wound healing.

[0099] In some embodiments, the compounds provided herein can be used to treat hypophosphatemic disorders such as, for example, X-linked hypophosphatemic rickets, autosomal recessive hypophosphatemic rickets, and autosomal dominant hypophosphatemic rickets, or tumor-induced osteomalacia.

[0100] In some embodiments, provided herein is a method for increasing the survival or progression-free survival of a patient, comprising administering a compound provided herein to the patient. In some embodiments, the patient has cancer. In some embodiments, the patient has a disease or disorder described herein. In some embodiments, the patient has cholangiocarcinoma. In some embodiments, provided herein is a method for increasing the survival or progression-free survival of a patient with cholangiocarcinoma characterized by an FGFR2 fusion, comprising administering a compound provided herein to the patient. As used herein, progression-free survival refers to the length of time a patient lives with, but does not worsen, their disease during and after treatment for a solid tumor. Progression-free survival refers to the time from first administration of the compound to death or disease progression, whichever occurs first. Disease progression can be defined by RECIST v.1.1 (Response Evaluation Criteria in Solid Tumors) as assessed by an independent central radiological review committee. In some embodiments, administration of the compound results in a progression-free survival of greater than about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 9 months, about 12 months, about 16 months, or about 24 months. In some embodiments, administration of the compound results in a progression-free survival of at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 9 months, or about 12 months; and less than about 24 months, about 16 months, about 12 months, about 9 months, about 8 months, about 6 months, about 5 months, about 4 months, about 3 months, or about 2 months. In some embodiments, administration of the compound results in an increase in progression-free survival of at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 9 months, or about 12 months; and less than about 24 months, about 16 months, about 12 months, about 9 months, about 8 months, about 6 months, about 5 months, about 4 months, about 3 months, or about 2 months.

[0101] As used herein, the term "cell" refers to a cell that is in vitro, ex vivo, or in vivo. In some embodiments, an ex vivo cell may be part of a tissue sample excised from an organism, such as a mammal. In some embodiments, an in vitro cell may be a cell in cell culture. In some embodiments, an in vivo cell is a cell that resides in an organism, such as a mammal.

[0102] As used herein, the term "contacting" refers to bringing the indicated moieties together in an in vitro system or in vivo system. For example, "contacting" an FGFR enzyme with a compound described herein (e.g., Compound 1) includes administering a compound described herein to an individual or patient, such as a human, having an FGFR, as well as introducing a compound described herein (e.g., Compound 1) into a sample, including, for example, a cell preparation or purified preparation containing an FGFR enzyme.

[0103] As used herein, the terms "individual" or "patient," used interchangeably, refer to a mammal, preferably any animal, including a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, most preferably a human.

[0104] As used herein, the phrase "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent, such as any solid form or salt thereof disclosed herein, that elicits the biological or medicinal response that a researcher, veterinarian, physician, or other clinician is seeking in a tissue, system, animal, individual, or human. An appropriate "effective" amount in any individual case can be determined using techniques known to those of ordinary skill in the art.

[0105] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication, within the bounds of safe medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0106] As used herein, the phrase "pharmaceutically acceptable carrier or excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients or carriers are generally safe, non-toxic, and not biologically or otherwise undesirable, and include excipients or carriers that are acceptable for veterinary use as well as human pharmaceutical use. In one embodiment, each component is "pharmaceutically acceptable" as defined herein. For example, Remington:The Science and Practice of Pharmacy,21st ed.;Lippincott Williams & Wilkins:Philadelphia,Pa.,2005;Handbook of Pharmaceutical Excipients,6th ed.;Rowe et al.,Eds.;The Pharmaceutical Press and the American Pharmaceutical Association:2009;Handbook of Pharmaceutical Additives,3rd ed.;Ash and Ash Eds.;Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, Fla., 2009.

[0107] As used herein, the term "treat" or "treatment" refers to inhibiting a disease, e.g., inhibiting a disease, condition, or disorder (i.e., halting further development of the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder, or ameliorating a disease, e.g., ameliorating a disease, condition, or disorder (i.e., reversing the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder, e.g., reducing the severity of the disease.

[0108] It is understood that certain features of the disclosure that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment (while those embodiments are intended to be combined as if described in multiple dependent fashion). Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0109] Combination therapy One or more additional pharmaceutical agents or treatment methods, such as antiviral agents, chemotherapeutic or other anti-cancer agents, immune enhancers, immunosuppressants, radiation, antitumor and antiviral vaccines, cytokine therapy (e.g., IL2, GM-CSF, etc.), and / or tyrosine kinase inhibitors, may be used in combination with the compounds described herein for the treatment of FGFR-related diseases, disorders, or conditions, or the diseases or conditions described herein. The agents may be combined with the compounds in a single dosage form, or the agents may be administered simultaneously or sequentially as separate dosage forms.

[0110] The compounds described herein can be used in combination with one or more other kinase inhibitors for the treatment of diseases such as cancer that are affected by multiple signal transduction pathways. For example, the combination can include one or more inhibitors of the following kinases for the treatment of cancer: Akt1, Akt2, Akt3, TGF-βR, Pim, PKA, PKG, PKC, CaM-kinase, phosphorylase kinase, MEKK, ERK, MAPK, mTOR, EGFR, HER2, HER3, HER4, INS-R, IGF-1R, IR-R, PDGFαR, PDGFβR, CSFIR, KIT, FL K-II, KDR / FLK-1, FLK-4, flt-1, FGFR1, FGFR2, FGFR3, FGFR4, c-Met, Ron, Sea, TRKA, TRKB, TRKC, FLT3, VEGFR / Flt2, Flt4, EphA1, EphA2, EphA3, EphB2, EphB4, Tie2, Src, Fyn, Lck, Fgr, Btk, Fak, SYK, FRK, JAK, ABL, ALK, and B-Raf. Additionally, the solid forms of the FGFR inhibitors described herein may be combined with inhibitors of kinases associated with the PIK3 / Akt / mTOR signaling pathway, such as PI3K, Akt (including Akt1, Akt2, and Akt3), and mTOR kinase.

[0111] In some embodiments, the compounds described herein may be used in combination with one or more inhibitors of enzymes or protein receptors, such as HPK1, SBLB, TUT4, A2A / A2B, CD47, CDK2, STING, ALK2, LIN28, ADAR1, MAT2a, RIOK1, HDAC8, WDR5, SMARCA2, and DCLK1, for the treatment of diseases and disorders. Exemplary diseases and disorders include cancer, infectious diseases, inflammation, and neurodegenerative disorders.

[0112] In some embodiments, the compounds described herein can be used in combination with therapeutic agents that target epigenetic regulators.Examples of epigenetic regulators include bromodomain inhibitors, histone lysine methyltransferases, histone arginine methyltransferases, histone demethylases, histone deacetylases, histone acetylases, and DNA methyltransferases.Examples of histone deacetylase inhibitors include vorinostat.

[0113] For the treatment of cancer and other proliferative disorders, the compounds described herein may be used in combination with JAK kinase inhibitors (ruxolitinib, additional JAK1 / 2 and JAK1-selective, baricitinib or INCB39110), Pim kinase inhibitors (e.g., LGH447, INCB053914, and SGI-1776), PI3 kinase inhibitors, such as PI3K-δ selective and broad-spectrum PI3K inhibitors (e.g., INCB50465 and INCB54707), PI3K-γ inhibitors, such as PI3K-γ selective inhibitors, MEK inhibitors, CSF1R inhibitors (e.g., PLX3397 and LY3022855), TAM receptor tyrosine kinase inhibitors (Tyro-3, Axl, and Mer; e.g., INCB81776), angiogenesis inhibitors, interleukin receptor inhibitors, cyclin-dependent kinase inhibitors, BRAF inhibitors , mTOR inhibitors, proteasome inhibitors (bortezomib, carfilzomib), HDAC inhibitors (panobinostat, vorinostat), DNA methyltransferase inhibitors, dexamethasone, bromo and specific terminal family member inhibitors (e.g., bromodomain inhibitors or BET inhibitors, e.g., OTX015, CPI-0610, INCB54329, or INCB57643), LSD1 inhibitors (e.g., GSK2979552, INCB59872, and INCB60003), arginase inhibitors (e.g., INCB1158), indoleamine 2,3-dioxygenase inhibitors (e.g., epacadostat, NLG919, or BMS-986205), PARP inhibitors (e.g., olaparib, or rucaparib), and inhibitors of BTK such as ibrutinib. Additionally, to treat cancer and other proliferative diseases, the compounds described herein may be used in combination with targeted therapies such as, for example, c-MET inhibitors (e.g., capmatinib), anti-CD19 antibodies (e.g., tafasitamab), ALK2 inhibitors (e.g., INCB00928); or combinations thereof.

[0114] To treat cancer and other proliferative diseases, the compounds described herein can be used in combination with chemotherapeutic agents, nuclear receptor agonists or antagonists, or other antiproliferative agents. The compounds described herein can also be used in combination with medical therapies such as surgery or radiation therapy, e.g., gamma radiation, neutron radiation therapy, electron beam radiation therapy, proton therapy, brachytherapy, and systemic radioactive isotopes.

[0115] Examples of suitable chemotherapeutic agents include abarelix, abiraterone, afatinib, aflibercept, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amidox, amsacrine, anastrozole, aphidicolone, arsenic trioxide, asparaginase, axitinib, azacitidine, bevacizumab, bexarotene, baricitinib, bendamustine, bicalutamide, bleomycin, bortezomib, bortezomib, brivanib, buparlisib, intravenous busulfan, oral busulfan, calcitonin, and camptothecin. acetaminophen, capecitabine, carboplatin, carmustine, cediranib, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dacomitinib, dactinomycin, dalteparin sodium, dasatinib, dactinomycin, daunorubicin, decitabine, degarelix, denileukin, denileukin diftitox, deoxycoformycin, dexrazoxane, didox, docetaxel, doxorubicin, droloxafine, dromostanolone propionate, Eculizumab, enazalutamide, epidophyllotoxin, epirubicin, epothilone, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, flutamide, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, idelalisib, ifosfamide, imatinib mesylate, interferon alfa-2a, irinotecan , lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lonafarnib, lomustine, mechlorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitalamycin, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nevelbene, necitumumab, nelarabine, neratinib, nilotinib, nilutamide, niraparib, nofetumomab, oserelin, oxaliplatin, paclitaxel, pamidronate, panitumumab,Panobinostat, pazopanib, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, piralalisib, pipobroman, plicamycin, ponatinib, porfimer, prednisone, procarbazine, quinacrine, ranibizumab, rasburicase, regorafenib, reloxafin, revlimid, rituximab, rucaparib, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, tegafur, temozolomide, teniposide, testolactone, tezacitabine, thalidomide, thioguanine, thiotepa, tipifarnib, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, triapine, trimidox, triptorelin, uracil mustard, valrubicin, vandetanib, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, veliparib, talazoparib, and zoledronate.

[0116] In some embodiments, the compounds described herein may be used in combination with immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include inhibitors of immune checkpoint molecules such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3Kδ, PI3Kγ, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3 (e.g., INCAGN2385), TIM3 (e.g., INCB2390), VISTA, PD-L1, and PD-L2. In some embodiments, the immune checkpoint molecule is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, OX40 (e.g., INCAGN1949), GITR (e.g., INCAGN1876), and CD137. In some embodiments, the immune checkpoint molecule is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, and VISTA. In some embodiments, the compounds provided herein may be used in combination with one or more agents selected from a KIR inhibitor, a TIGIT inhibitor, a LAIR1 inhibitor, a CD160 inhibitor, a 2B4 inhibitor, and a TGFRbeta inhibitor.

[0117] In some embodiments, the inhibitor of an immune checkpoint molecule is a small molecule PD-L1 inhibitor. In some embodiments, the small molecule PD-L1 inhibitor has an IC50 of less than 1 μM, less than 100 nM, less than 10 nM, or less than 1 nM in the PD-L1 assay described in U.S. Patent Application Publication Nos. 20170107216, 20170145025, 20170174671, 20170174679, 20170320875, 20170342060, 20170362253, and 20180016260, each of which is incorporated by reference in its entirety for all purposes.

[0118] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1, e.g., an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is MGA012 (retifanlimab), nivolumab, pembrolizumab (also known as MK-3475), pidilizumab, SHR-1210, PDR001, ipilumimab, or AMP-224. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the anti-PD1 antibody is nivolumab. In some embodiments, the anti-PD-1 monoclonal antibody is MGA012 (retifanlimab). In some embodiments, the anti-PD1 antibody is SHR-1210. Other anti-cancer agent(s) include antibody therapeutic agents such as 4-1BB (e.g., urelumab, utomilumab).

[0119] In some embodiments, compounds of the present disclosure may be used in combination with INCB086550.

[0120] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, such as an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is BMS-935559, MEDI4736, MPDL3280A (also known as RG7446), or MSB0010718C. In some embodiments, the anti-PD-L1 monoclonal antibody is MPDL3280A or MEDI4736.

[0121] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab, AGEN1884, or CP-675,206.

[0122] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of LAG3, such as an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is BMS-986016, LAG525, or INCAGN2385.

[0123] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM3, such as an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is INCAGN2390, MBG453, or TSR-022.

[0124] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of GITR, such as an anti-GITR antibody. In some embodiments, the anti-GITR antibody is TRX518, MK-4166, INCAGN1876, MK-1248, AMG228, BMS-986156, GWN323, or MEDI1873.

[0125] In some embodiments, the inhibitor of the immune checkpoint molecule is an OX40 agonist, such as an OX40 agonist antibody or an OX40L fusion protein. In some embodiments, the anti-OX40 antibody is MEDI0562, MOXR-0916, PF-04518600, GSK3174998, or BMS-986178. In some embodiments, the OX40L fusion protein is MEDI6383.

[0126] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CD20, e.g., an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is obinutuzumab or rituximab.

[0127] The compounds of the present disclosure may also be used in combination with bispecific antibodies, in some embodiments, one domain of the bispecific antibody targets PD-1, PD-L1, CTLA-4, GITR, OX40, TIM3, LAG3, CD137, ICOS, CD3, or a TGFβ receptor.

[0128] In some embodiments, the compounds of the present disclosure can be used in combination with one or more metabolic enzyme inhibitors. In some embodiments, the metabolic enzyme inhibitor is an inhibitor of IDO1, TDO, or arginase. Examples of IDO1 inhibitors include epacadostat, NLG919, BMS-986205, PF-06840003, IOM2983, RG-70099, and LY338196.

[0129] In some embodiments, the compounds described herein may be used in combination with one or more drugs for the treatment of diseases such as cancer. In some embodiments, the drug is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of alkylating agents include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM).

[0130] Suitable antiviral agents contemplated for use in combination with the compounds of the present disclosure may include nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, and other antiviral drugs.

[0131] Examples of suitable NRTIs include zidovudine (AZT); didanosine (ddl); zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir (1592U89); adefovir dipivoxil [bis(POM)-PMEA]; lobucavir (BMS-180194); BCH-10652; emtricitabine [(-)-FTC]; beta-L-FD4 (also called beta-L-D4C, beta-L-2',3'-dicreoxy-5-fluoro-citidene); DAPD, ((-)-beta-D-2,6,-diamino-purine dioxolane); and rhodenosine (FddA). Exemplary suitable NNRTIs include nevirapine (BI-RG-587); delaviradine (BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442 (1-(ethoxymethyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione); and (+)-calanolide A (NSC-675451) and B. Exemplary suitable protease inhibitors include saquinavir (Ro 31-8959); ritonavir (ABT-538); indinavir (MK-639); nerfunavir (AG-1343); amprenavir (141W94); lasinavir (BMS-234475); DMP-450; BMS-2322623; ABT-378; and AG-1549. Other antiviral agents include hydroxyurea, ribavirin, IL-2, IL-12, pentafuside, and Yissum Project No. 11607.

[0132] Suitable drugs for use in combination with the compounds described herein for the treatment of cancer include chemotherapy, targeted cancer therapy, immunotherapy, or radiation therapy.The compounds described herein can be effective in combination with antihormonal agents for the treatment of breast cancer and other tumors.Suitable examples include antiestrogens, including but not limited to tamoxifen and toremifene, aromatase inhibitors, including but not limited to letrozole, anastrozole, and exemestane, adrenocorticosteroids (e.g., prednisone), progestins (e.g., megastrol acetate), and estrogen receptor antagonists (e.g., fulvestrant).Suitable antihormonal agents used in the treatment of prostate cancer and other cancers may also be combined with the compounds described herein. These include antiandrogens, including but not limited to flutamide, bicalutamide, and nilutamide, luteinizing hormone-releasing hormone (LHRH) analogs, including leuprolide, goserelin, triptorelin, and histrelin, LHRH antagonists (e.g., degarelix), androgen receptor blockers (e.g., enzalutamide), and agents that inhibit androgen production (e.g., abiraterone).

[0133] The compounds described herein may be combined or sequenced with other agents against membrane receptor kinases, particularly for patients who have developed primary or acquired resistance to targeted therapy. These therapeutic agents include inhibitors or antibodies against EGFR, Her2, VEGFR, c-Met, Ret, IGFR1, or against cancer-associated fusion protein kinases such as Flt-3, Bcr-Abl, and EML4-Alk. EGFR inhibitors include, but are not limited to, gefitinib and erlotinib, and EGFR / Her2 inhibitors include, but are not limited to, dacomitinib, afatinib, lapitinib, and neratinib. EGFR antibodies include, but are not limited to, cetuximab, panitumumab, and necitumumab. c-Met inhibitors may also be used in combination with FGFR inhibitors, including onartumuzumab, tivantinib, and INC-280. Drugs directed against Abl (or Bcr-Abl) include imatinib, dasatinib, nilotinib, and ponatinib, and drugs directed against Alk (or EML4-ALK) include crizotinib.

[0134] Angiogenesis inhibitors can be effective in some tumors in combination with FGFR inhibitors.These include antibodies against VEGF or VEGFR, or VEGFR kinase inhibitors.Antibodies or other therapeutic proteins against VEGF include bevacizumab and aflibercept.Inhibitors of VEGFR kinase and other anti-angiogenesis inhibitors include, but are not limited to, sunitinib, sorafenib, axitinib, cediranib, pazopanib, regorafenib, brivanib, and vandetanib.

[0135] Activation of intracellular signaling pathways frequently occurs in cancer, and drugs that target components of these pathways have been combined with receptor-targeting agents to increase efficacy and reduce resistance. Examples of drugs that can be combined with the compounds described herein include inhibitors of the PI3K-AKT-mTOR pathway, inhibitors of the Raf-MAPK pathway, inhibitors of the JAK-STAT pathway, and inhibitors of protein chaperones and cell cycle progression.

[0136] Drugs against PI3 kinase include but are not limited to topiralisib, idelalisib, and buparlisib.Inhibitors of mTOR, such as rapamycin, sirolimus, temsirolimus, and everolimus, may be combined with FGFR inhibitors.Other suitable examples include but are not limited to vemurafenib and dabrafenib (Raf inhibitors), and trametinib, selumetinib, and GDC-0973 (MEK inhibitors). One or more JAK inhibitors (e.g., ruxolitinib, baricitinib, tofacitinib), Hsp90 inhibitors (e.g., tanespimycin), cyclin-dependent kinase inhibitors (e.g., palbociclib), HDAC inhibitors (e.g., panobinostat), PARP inhibitors (e.g., olaparib), and proteasome inhibitors (e.g., bortezomib, carfilzomib) may be combined with the compounds described herein. In some embodiments, the JAK inhibitor is selective for JAK1 over JAK2 and JAK3.

[0137] Other suitable agents for use in combination with the compounds described herein include chemotherapy combinations such as platinum-based doublets used in lung cancer and other solid tumors (cisplatin or carboplatin plus gemcitabine; cisplatin or carboplatin plus docetaxel; cisplatin or carboplatin plus paclitaxel; cisplatin or carboplatin plus pemetrexed), or gemcitabine plus paclitaxel-bound particles (Abraxane®).

[0138] Suitable chemotherapeutic or other anti-cancer agents include, for example, alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes), such as uracil mustard, chlormethine, cyclophosphamide (Cytoxan™), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

[0139] Other suitable agents for use in combination with the compounds described herein include steroids, including 17alpha-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, and medroxyprogesterone acetate.

[0140] Other suitable agents for use in combination with the compounds described herein include dacarbazine (DTIC), optionally with other chemotherapeutic agents such as carmustine (BCNU) and cisplatin; the "Dartmouth regimen" consisting of DTIC, BCNU, cisplatin, and tamoxifen; a combination of cisplatin, vinblastine, and DTIC; or temozolomide. The compounds described herein may also be combined with immunotherapeutic agents, including cytokines such as interferon alpha, interleukin 2, and tumor necrosis factor (TNF).

[0141] Suitable chemotherapeutic or other anti-cancer agents include, for example, antimetabolites (including but not limited to antifolates, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors), such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

[0142] Suitable chemotherapeutic or other anti-cancer agents further include certain natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins), such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (TAXOL™), mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, interferons (especially IFN-α), etoposide, and teniposide.

[0143] Other cytotoxic agents include navelbene, CPT-11, anastrozole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine.

[0144] Also suitable are cytotoxic agents such as epidophyllotoxins; antitumor enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitory agents; antihormonal therapeutic agents; leucovorin; tegafur; and hematopoietic growth factors.

[0145] Other anti-cancer drug(s) include antibody therapeutics such as trastuzumab (Herceptin), antibodies against costimulatory molecules such as CTLA-4, 4-1BB, PD-L1, and PD-1 antibodies, or antibodies against cytokines (such as IL-10, TGF-β).

[0146] Other anti-cancer agents also include agents that block the migration of immune cells, such as antagonists to chemokine receptors such as CCR2 and CCR4.

[0147] Other anti-cancer agents also include agents that enhance the immune system, such as adjuvants or adoptive T-cell transfer.

[0148] Anti-cancer vaccines include dendritic cells, synthetic peptides, DNA vaccines, and recombinant viruses. In some embodiments, tumor vaccines include proteins derived from viruses involved in human cancer, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MARTI, and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

[0149] The compounds of the present disclosure may be used in combination with bone marrow transplantation for the treatment of various tumors of hematopoietic origin.

[0150] Methods for safely and effectively administering most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in standard literature. For example, the administration of many chemotherapeutic agents is described in the "Physicians' Desk Reference" (PDR, e.g., 1996 ed., Medical Economics Company, Montvale, NJ), the disclosure of which is incorporated herein by reference as if set forth in its entirety.

[0151] As provided throughout, the additional compounds, inhibitors, drugs, etc. may be combined with the present compounds in a single or sequential dosage form, or they may be administered simultaneously or sequentially as separate dosage forms.

[0152] Pharmaceutical Preparations and Dosage Forms In some embodiments, the disclosed compounds or pharmaceutical compositions thereof are suitable for oral administration. In some embodiments, the disclosed compounds or pharmaceutical compositions thereof are suitable for intravenous administration. In some embodiments, the disclosed compounds or pharmaceutical compositions thereof are suitable for arterial administration. In some embodiments, the arterial administration is hepatic artery infusion.

[0153] When used as pharmaceuticals, the compounds described herein may be administered in the form of a pharmaceutical composition, which refers to a combination of one or more compounds described herein with at least one pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure may contain 20% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 30% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 40% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 50% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 60% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 70% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 80% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 90% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 95% by weight of the compound or a salt thereof. The pharmaceutical composition of the present disclosure may contain 99% by weight of the compound or a salt thereof.

[0154] These compositions may be prepared in a manner well known in the pharmaceutical arts and administered by various routes, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including transmucosal, including ophthalmic, intranasal, intravaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including those via nebulizers; intratracheal, intranasal, epithelial, and transdermal), ocular, oral, or parenteral. Ocular delivery methods may include topical administration (eye drops), subconjunctival, periorbital, or intravitreal injection, or introduction via a balloon catheter or ocular insert surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, e.g., intrathecal, or intraventricular, administration. Parenteral administration may be in the form of a single bolus dose or, for example, via a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable.

[0155] The present disclosure also includes pharmaceutical compositions containing the compounds of the present disclosure as active ingredients in combination with one or more pharmaceutically acceptable carriers.When preparing the compositions described herein, the active ingredient is typically mixed with an excipient, diluted by an excipient, or enclosed in such a carrier, for example, in the form of a capsule, sachet, paper, or other container.When an excipient functions as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient.Thus, the composition can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or liquid media), for example, ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0156] When preparing formulation, active compound can be pulverized to obtain suitable particle size before being combined with other components.If active compound is substantially insoluble, it can be pulverized to the particle size of less than 200 mesh.If active compound is substantially water-soluble, particle size can be adjusted by pulverization to obtain substantially uniform distribution in formulation, for example, about 40 mesh.

[0157] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose.The formulation can further comprise lubricants such as talc, magnesium stearate, and mineral oil, wetting agents, emulsifying and suspending agents, preservatives such as methylbenzoate and propylhydroxybenzoate, sweeteners, and flavoring agents.The compositions described herein can be formulated to provide quick, sustained, or delayed release of active ingredients after administration to patients by using procedures known in the art.

[0158] The compositions may be formulated in unit dosage form, each dosage containing from about 5 to about 100 mg, more usually from about 10 to about 30 mg, of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce a desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0159] The active compound can be effective over a wide dosage range and is therefore generally administered in a pharmaceutically effective amount. However, it will be understood that the amount of compound actually administered will usually be determined by the physician according to the relevant circumstances, including the condition being treated, the selected route of administration, the compound actually administered, the age, weight, and response of the individual patient, the severity of the patient's condition, etc.

[0160] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical excipients to form a solid preformulation composition containing a homogeneous mixture of the disclosed compounds. When these preformulation compositions are referred to as homogeneous, the active ingredient is typically dispersed evenly throughout the composition, allowing the composition to be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above, containing, for example, about 0.1 to about 500 mg of the disclosed active ingredient.

[0161] The tablets or pills of the present disclosure can be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action.For example, the tablets or pills can comprise an inner dose and an outer dose component, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer that resists disintegration in the stomach and allows the inner component to pass intact into the duodenum or be delayed in release.A variety of materials can be used as such enteric layers or coatings, including numerous polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0162] Liquid forms into which the compounds of the present disclosure, or compositions described herein, may be incorporated for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0163] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

[0164] The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of the administration, such as prophylaxis or treatment, the condition of the patient, the method of administration, etc. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. The effective dose will depend on the disease state being treated and the judgment of the attending physician based on factors such as the severity of the disease, the age, weight, and general condition of the patient.

[0165] The compositions administered to patients may be in the form of the pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparation will typically be 3-11, more preferably 5-9, and most preferably 7-8. It will be appreciated that the use of certain of the aforementioned excipients, carriers, or stabilizers may result in the formulation of pharmaceutical salts.

[0166] Therapeutic dosages of the compounds described herein can vary according to, for example, the particular application for which the treatment is being administered, the method of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound described herein in a pharmaceutical composition can vary depending on many factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, for parenteral administration, the compounds described herein can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w / v of the compound. Some typical dosage ranges are about 1 μg / kg to about 1 g / kg of body weight per day. In some embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage can depend on variables such as the type and progression of the disease or disorder, the overall health of the particular patient, the relative bioefficacy of the selected compound, the formulation of excipients, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0167] The compounds of the present disclosure may also be formulated in combination with one or more additional active ingredients, which may include any pharmaceutical agent such as an antiviral agent, a vaccine, an antibody, an immune enhancer, an immunosuppressant, an anti-inflammatory agent, etc. [Example]

[0168] The experimental procedures for the compounds of the present invention are provided below. Preparative LC-MS purification of some of the prepared compounds was carried out on a Waters mass fractionation system. The basic instrument setup, protocols, and control software for operating these systems are described in detail in the literature. See, for example, "Two-Pump At Column Dilution Configuration for Preparative LC-MS", K. Blom, J. Combi. Chem., 4, 295 (2002); "Optimizing Preparative LC-MS Configurations and Methods for Parallel Synthesis Purification", K. Blom, R. Sparks, J. Doughty, G. Everlof, T. Haque, A. Combs, J. Combi. Chem., 5, 670 (2003); and "Preparative LC-MS Purification: Improved Compound Specific Method Optimization", K. Blom, B. Glass, R. Sparks, A. Combs, J. Combi. Chem., 6, 874-883 (2004). The separated compounds were typically subjected to analytical liquid chromatography mass spectrometry (LCMS) for purity analysis under the following conditions: Instrument: Agilent 1100 series, LC / MSD; Column: Waters Sunfire™ C 18 5 μm, 2.1 × 50 mm, buffer: mobile phase A: 0.025% TFA in water, and mobile phase B: acetonitrile; flow rate 2.0 mL / min, gradient 2% to 80% B in 3 min.

[0169] Some of the prepared compounds were also separated on a preparative scale by reversed-phase high-performance liquid chromatography (RP-HPLC) or flash chromatography (silica gel) with MS detection as indicated in the examples. Typical preparative reversed-phase high-performance liquid chromatography (RP-HPLC) column conditions are as follows: Purification at pH=2: Waters Sunfire™ C 18A 5 μm, 19 × 100 mm column was eluted with mobile phase A: 0.1% TFA (trifluoroacetic acid) in water and mobile phase B: acetonitrile at a flow rate of 30 mL / min. The separation gradient was optimized for each compound using a compound-specific method optimization protocol described in the literature [see "Preparative LCMS Purification: Improved Compound-Specific Method Optimization," K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)]. Typically, the flow rate used on a 30 × 100 mm column was 60 mL / min.

[0170] Purification at pH 10: Waters XBridge C 18 A 5 μm, 19 × 100 mm column was eluted with mobile phase A: 0.15% NH OH in water and mobile phase B: acetonitrile at a flow rate of 30 mL / min. The separation gradient was optimized for each compound using a compound-specific method optimization protocol described in the literature [see "Preparative LCMS Purification: Improved Compound-Specific Method Optimization," K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)]. Typically, the flow rate used on the 30 × 100 mm column was 60 mL / min.

[0171] Example 1 Synthesis of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 1) [ka]

[0172] Step 1: Synthesis of 4-((4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine To a 1 L flask was added 4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (50.0 g, 137 mmol) (see, e.g., Example 2) and tetrahydrofuran (THF, 266 g, 300 mL) under N2. To this mixture at -70 °C was added 2.0 M lithium diisopropylamide (77.4 g, 95 mL, 190 mmol, 1.4 equiv.) in THF / heptane / ethylbenzene. The mixture was stirred at -70 °C for 1 hour. To this mixture was added dropwise N-formylmorpholine (29.7 g, 258 mmol, 1.9 equiv.) in THF (22.2 g, 25 mL). The reaction was allowed to proceed 30 minutes after the addition. LC / MS showed clean formation of the desired product, 4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde. The reaction was quenched with acetic acid (16.4 g, 15.6 mL, 274 mmol, 2.0 equiv.), and the dry ice cooling was removed. To this mixture, morpholine (33.7 g, 33.5 mL, 387 mmol, 2.83 equiv.) was added, followed by acetic acid (74.0 g, 70 mL, 1231 mmol, 9.0 equiv.) at 0 °C (internal temperature increased from 0 °C to 18 °C) and stirred overnight. Sodium triacetoxyborohydride (52.50 g, 247.7 mmol, 1.8 equiv.) was added, and the reaction mixture temperature increased from 20 °C to 32 °C. The mixture was stirred at room temperature for 30 minutes. HPLC and LC / MS showed the reaction was complete. Water (100 g, 100 mL) was added, followed by slow addition (off-gassing!) of 2.0 M sodium carbonate (Na2CO3) in water (236 g, 200 mL, 400 mmol, 2.9 equiv). The mixture was stirred for approximately 30 minutes. The organic layer was separated, and water (250 g, 250 mL) and heptane (308 g, 450 mL) were added. The resulting slurry was stirred for 1 hour, and the solid was collected by filtration.The wet cake was washed twice with heptane (75.00 mL × 2, 51.3 g × 2) and then dried in an oven at 50 °C overnight to give the desired product, 4-((4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine as a light brown solid (52.00 grams, 81.8% yield): C. 21 H 23 ClN2O5S[M+H] + LCMS calculated for: 464.00; Found: 464.0; 1 H NMR(400MHz,DMSO-d6)δ 8.48(s,1H),8.38(m,2H),7.72(m,1H),7.64(m,2H),6.83(s,1H),6.13( s,1H),4.12(m,2H),4.00(m,2H),3.92(s,2H),3.55(m,4H),2.47(m,4H).

[0173] Step 2: Synthesis of 4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde A 2 L reactor equipped with a thermocouple, an addition funnel, and a mechanical stirrer was charged with 4-((4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine (20.00 g, 43.1 mmol) and dichloromethane (265 g, 200 mL) at room temperature. The resulting mixture was stirred at room temperature (internal temperature was 19.5 °C) to obtain a solution. To the resulting solution was added aqueous hydrochloric acid (0.5 M, 240 g, 200.0 mL, 100 mmol, 2.32 equiv) at room temperature over 7 min. After stirring at room temperature for 23 hours or more, the two-phase reaction mixture turned into a thick, colorless suspension. When HPLC indicated the reaction was complete, the slurry was cooled to 0-5 °C and aqueous sodium hydroxide (1 N, 104 g, 100 mL, 100 mmol, and 2.32 equiv.) was added over approximately 10 min to adjust the pH of the reaction mixture to 10-11. n-Heptane (164 g, 240 mL) was added and the mixture was stirred at room temperature for 1 h. The solid was collected by filtration, and the wet cake was washed with water (2 × 40 mL) and heptane (2 × 40 mL) before being dried under vacuum in an oven at 50 °C to give the desired product, 4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde, as a light brown solid (16.9 g, 93% yield): C 19 H 19 ClN3O4S[M+H] + LCMS calculated value for: 420.00; Found value: 420.0; 1 H NMR(400MHz,DMSO-d6)δ 10.33(s,1H),8.76(s,1H),8.42(m,2H),7.74(m,1H),7.65(m,2H),6.98(s,1H),3.96(m,2H),3.564(m,4H),2.51(m,4H).

[0174] Step 3: Synthesis of N-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-2,6-difluoro-3,5-dimethoxyaniline A 2 L reactor equipped with a thermocouple, nitrogen inlet, and mechanical stirrer was charged with N,N-dimethylformamide (450 mL, 425 g), 4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (30.0 g, 71.45 mmol), and 2,6-difluoro-3,5-dimethoxyaniline (14.2 g, 75.0 mmol). To this suspension (internal temperature 20 °C) was added dropwise at room temperature (internal temperature 20-23 °C) over 10 min. Five minutes after the addition of chlorotrimethylsilane, the suspension turned into a solution. The solution was stirred at room temperature for 1.5 h and then cooled to 0-5 °C in an ice bath. Borane-THF complex in THF (1.0 M, 71.4 mL, 71.4 mmol, 64.2 g, 1.0 equiv.) was added dropwise via addition funnel over 30 min while maintaining the temperature at 0–5 °C. After the addition, the mixture was stirred for 4 h. Water (150 g, 150 mL) was added over 20 min under ice bath cooling, followed by slow addition of ammonium hydroxide solution (28% NH3, 15.3 g, 17 mL, 252 mmol, 3.53 equiv.) to pH 9–10 while maintaining the temperature below 10 °C. Additional water (250 mL, 250 g) was added via addition funnel. The slurry was stirred for 30 min, and the solid was collected by filtration. The wet cake was washed with water (90 g × 2, 90 mL × 2) and heptane (61.6 g × 2, 90 mL × 2). The product was dried under suction overnight to give the desired product N-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-2,6-difluoro-3,5-dimethoxyaniline (41.6 g, 96% yield): C 27 H 28 ClF2N4O5S[M+H] + Calculated LCMS: 593.10; Found: 593.1; 1 H NMR(400MHz,DMSO-d6)δ 8.36(m,2H),8.28(s,1H),7.72(m,1H),7.63(m,2H),6.78(s,1H),6.29(m,1H), 5.82(m,1H),4.58(m,2H),3.91(s,2H),3.76(s,6H),3.56(m,4H),2.47(m,4H).

[0175] Step 4: Synthesis of 1-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-1-(2,6-difluoro-3,5-dimethoxyphenyl)-3-ethylurea A 2 L, three-necked round-bottom flask equipped with a thermocouple, nitrogen bubbler inlet, and magnetic stir bar was charged with N-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-2,6-difluoro-3,5-dimethoxyaniline (67.0 g, 113 mmol) and acetonitrile (670 mL, 527 g). The suspension was cooled to 0-5 °C. Ethyl isocyanate (17.7 mL, 15.9 g, 224 mmol, 1.98 equiv.) was charged to the mixture over 30 seconds. After charging, the temperature remained constant at 0.7 °C. Methanesulfonic acid (16.1 mL, 23.9 g, 248 mmol, 2.2 equiv.) was added dropwise over 35 minutes, maintaining the temperature below 2 °C. The mixture was allowed to warm to room temperature and stirred overnight. 24 h after addition, the product yield was 93.7%, unreacted SM was 0.73%, and the major impurity (bisisocyanate adduct) was 1.3%. The mixture was cooled in an ice bath and quenched with sodium hydroxide (NaOH) solution (1.0 M, 235 mL, 244 g, 235 mmol, 2.08 equiv) over 20 min, followed by saturated aqueous sodium bicarbonate (NaHCO) solution (1.07 M, 85 mL, 91 g, 0.091 mol, 0.80 equiv) over 10 min. Water (550 mL, 550 g) was added, resulting in a single phase. The mixture was stirred for 2 hours and the solid was collected by filtration and washed with water (165 mL, 165 g) to give 1-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-1-(2,6-difluoro-3,5-dimethoxyphenyl)-3-ethylurea (70.3 g, 93.7% yield).

[0176] The crude 1-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-1-(2,6-difluoro-3,5-dimethoxyphenyl)-3-ethylurea (68.5 g, 103 mmol) was added to acetonitrile (616 mL, 485 g). The mixture was heated to 60-65 °C to give a pale amber suspension. The solid was filtered through Celite, and the Celite was washed with acetonitrile (68.5 mL, 53.8 g). To the pale yellow filtrate, water (685 g, 685 ml) was added to form a slurry. The slurry was stirred at room temperature overnight and filtered. The solid was added to water (685 mL, 685 g) and stirred at 60 °C for 2 hours. The solid was filtered and reslurried in heptane (685 mL, 469 g) overnight. The product was dried in an oven at 50° C. under vacuum for 48 hours to give 1-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-1-(2,6-difluoro-3,5-dimethoxyphenyl)-3-ethylurea as a colorless solid (62.2 g, 90.8% yield, 99.9% purity by HPLC area %). KF was 0.028%. Acetonitrile ( 1 H NMR) was about 1.56%, DCM ( 1 H NMR) was 2.0%: 30 H 33 ClF2N5O6S[M+H] + LCMS calculated for: EM: 664.17; Found: 664.2; 1 H NMR(400MHz,DMSO-d6)δ 8.33(m,2H),8.31(s,1H),7.72(m,1H),7.64(m,1H),6.96(m,2H),6.73(s,1H),6.43(m,1H), 4.87(s,2H),3.90(s,2H),3.77(s,6H),3.54(m,4H),3.03(m,2H),2.46(m,4H),0.95(m,3H).

[0177] Step 5: Synthesis of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one A 2000 ml flask equipped with a thermocouple, nitrogen inlet, and mechanical stirrer was charged with dry 1-((4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)methyl)-1-(2,6-difluoro-3,5-dimethoxyphenyl)-3-ethylurea (30.0 g, 45.2 mmol, KF = 0.11%) and tetrahydrofuran (1200 mL, 1063 g). To this suspension was added 1.0 M lithium hexamethyldisilazide in THF (62.3 mL, 55.5 g, 62.3 mmol, 1.38 equiv.) at room temperature. After the base was added, the mixture turned into a solution. The reaction mixture was stirred for 2 hours, and HPLC showed that no starting material was detectable. To this mixture was added 1.0 M hydrochloric acid (18.1 mL, approximately 18.1 g, 18.1 mmol, 0.4 equiv.). The solution was concentrated to 600 mL, and water (1200 mL, 1200 g) was added. After the water addition, a slurry was formed. The slurry was stirred at room temperature for 30 minutes, and the solid was collected by filtration. The wet cake was washed twice with water (60 mL x 2, 60 g x 2) and dried overnight at 50 °C to give 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one as a light brown solid (26.58 g, 93.7% crude yield): 1 H NMR showed THF 0.32%, KF 5.26%, and adjusted yield of 88.5%. 30 H 32 F2N5O6S[M+H] + LCMS calculated: EM: 628.20; Found: 628.2; 1H NMR(400MHz,DMSO-d6)δ 8.41(m,2H),8.07(s,1H),7.70(m,1H),7.63(m,2H),7.05(m,1H),6.89(s,1H),4.76( s, 2H), 4.09 (m, 2H), 3.93 (s, 2H), 3.89 (s, 6H), 3.60 (m, 4H), 2.50 (m, 4H), 1.28 (m, 3H).

[0178] Step 6: Synthesis of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one To a stirred suspension of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (10.0 g, 15.93 mmol) in 1,4-dioxane (100 mL, 103 g) in a 500 mL flask equipped with a nitrogen inlet, condenser, thermocouple, and heating mantle, was added 1 M aqueous sodium hydroxide solution (63.7 mL, 66.3 g, 63.7 mmol). The reaction mixture was heated at 75°C for 18 hours. LCMS indicated the reaction was complete. Water (100 mL, 100 g) was added to give a thick suspension. The slurry was stirred at room temperature for 1 hour and filtered. The cake was washed with water (3 × 10 mL, 3 × 10 g) and heptane (2 × 10 mL, 2 × 6.84 g). The cake was dried overnight by pulling a vacuum through the filter cake and then dried overnight in an oven under vacuum at 50 °C to give 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (6.8 g, 87.6% yield). 24 H 28 F2N5O4[M+H] + LCMS calculated for: 488.20; found: 488.2.

[0179] Example 2. Synthesis of 4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine [ka]

[0180] Step 1: Synthesis of 1H-pyrrolo[2,3-b]pyridine-7-oxide m-Chloroperoxybenzoic acid (105.5 kg, 612 mol, 1.2 equiv.) was added over 5 h to a solution of 1H-pyrrolo[2,3-b]pyridine (60 kg, 507.6 mol) in dichloromethane (600 L) with stirring at 0–10 °C. After the addition was complete, the mixture was stirred at 0–10 °C for 3 h. The resulting solid was collected by filtration, washed with heptane, and dried to give 1H-pyrrolo[2,3-b]pyridine 7-oxide. The mother liquor was concentrated, and the residue was treated with dichloromethane:heptane (2:3) and filtered to remove excess material. Crude 1H-pyrrolo-2,3-b]pyridine-7-oxide was obtained (72 kg, 96% purity), which was used in the next step without purification.

[0181] Step 2: Synthesis of 4-chloro-1H-pyrrolo[2,3-b]pyridine Crude 1H-pyrrolo[2,3-b]pyridine-7-oxide (72 kg, 253 mol) was dissolved in DMF (360 L) and heated to 50°C. A solution of methanesulfonyl chloride (85.2 kg, 746 mol, 3.0 equiv.) was added dropwise to the solution, maintaining the temperature below 70°C. After stirring at 90°C for 2 hours, the reaction solution was cooled to room temperature and added to 720 kg of ice / water. The mixture was neutralized with 6.0 M NaOH at 0°C. The resulting precipitate was collected by filtration and washed with water. The solid was mixed with 72 L of water, 48 L of ethanol, and 29 L of 30% NaOH and stirred at room temperature for 1-2 hours. Water (144 L) was added, and the mixture was treated with 37% HCl to adjust the pH to approximately 1. The product was collected by filtration and dried to give 4-chloro-1H-pyrrolo[2,3-b]pyridine (26 kg crude, 97% purity, used without purification): 1HNMR (400MHz, CDCl3) δ 11.30 (s, 1H), 8.25 (m, 1H), 7.44 (m, 1H), 7.16 (m, 1H), 6.65 (m, 1H).

[0182] Step 3: Synthesis of 4-chloro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine A solution of crude 4-chloro-1H-pyrrolo[2,3-b]pyridine (24 kg, 155.2 mol) in THF (216 L) was stirred at 0 °C, and NaH (60%, 7.56 kg, 188.6 mol, 1.3 equiv.) was added portionwise under N2. After the addition, the mixture was stirred at room temperature for 1 h. Triisopropylsilyl chloride (39.6 kg, 188.6 mol, 1.3 equiv.) was added dropwise, maintaining the temperature below 25 °C. After stirring for 20 h, the mixture was quenched with 144 L of water and extracted with 144 L of heptane. The aqueous layer was back-extracted with 72 L of methyl t-butyl ether. The combined organic layers were dried over anhydrous MgSO4 and concentrated in vacuo to give crude 4-chloro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine as a liquid, which was used without further purification, but its water content was controlled below 0.1%.

[0183] Step 4: Synthesis of 4-chloro-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde A 1000 L cryogenic reactor was charged with crude 4-chloro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine (50 kg, approximately 138 mol) and anhydrous THF (150 kg). The mixture was cooled to −75° C. and stirred under N2, while S-BuLi (1.3 M in cyclohexane, 230 L, 300 mol, 2.2 equiv.) was added dropwise over 6.0 hours, maintaining the internal temperature below −60° C. The mixture was stirred at −75° C. for an additional 2 hours. N,N-dimethylformamide (30.4 kg, 416.1 mol, 3.0 equiv.) was added dropwise over approximately 3.0 hours, controlling the internal temperature below −65° C.

[0184] After stirring at -65 to -75 °C for 2 hours, the mixture was quenched by dropwise addition of a solution of 20% HCl in isopropyl alcohol (115 kg, 635 mol, 4.5 equiv.). The mixture was then stirred overnight at room temperature (20 to 25 °C). The pH was adjusted to 7 to 8 by adding saturated NaHCO3. The formed precipitate was collected by filtration. The filter cake was washed with 76 L of water to give 4-chloro-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (14 kg, 58% yield): 1 H NMR (400MHz, DMSO-d6) δ 12.54 (s, 1H), 10.35 (s, 1H), 8.67 (s, 1H), 7.74 (m, 1H), 6.72 (m, 1H).

[0185] Step 5: Synthesis of 4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde A 500 L reactor was charged with N,N-dimethylformamide (108 L) and 4-chloro-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (10.8 kg, 59.8 mol) and cooled to 0-5 °C. To the resulting thick slurry, cesium carbonate (39 kg, 120 mol) was added at 0-5 °C. The slurry was stirred at 0 °C for approximately 20 minutes, turning the mixture into a thin, amber-colored slurry. To the thin slurry at <10 °C, benzenesulfonyl chloride (11.6 kg, 65.8 mol, 1.1 equiv.) was added dropwise through a dropping funnel. The resulting slurry was stirred at <10 °C for 1 hour, and HPLC indicated the reaction was complete. Prolonged stirring overnight at room temperature had little effect on the reaction mixture profile. Water (160 L) was added to the mixture, and the slurry was stirred for 1 hour. The solid was collected by filtration (slow). The filter cake was washed with water and dried in an oven under vacuum to give 4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde as a light brown solid (17.8 Kg, 93% yield): C 14 H 10 ClN2O3S[M+H] + LCMS calculated value: 321.00; Found value: 320.9; 1HNMR (400MHz, DMSO-d6) δ: 10.34 (s, 1H), 8.78 (s, 1H), 8.18 (m, 3H), 7.77 (m, 1H), 7.66 (m, 2H), 7.05 (m, 1H).

[0186] Step 6: Synthesis of 4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine A 1000 L reactor was charged with toluene (270 L), 4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (27 kg, 84.2 mmol), p-toluenesulfonic acid monohydrate (217 g, 1.26 mol, 0.015 equiv.), and 1,2-ethanediol (73.7 kg, 1187 mol, 14.1 equiv.). The mixture was stirred and heated to reflux for 9 hours to remove water (some ethylene glycol was also removed as the reaction progressed). (LCMS indicated the reaction was complete.) After stirring overnight at room temperature, the mixture was diluted with ethyl acetate (135 L) and washed with saturated NaHCO3 solution. The layers were separated, and the organic layer was washed with 10% aqueous NaCl and concentrated. Heptane (108 L) was added to form a slurry. The solid was collected by filtration. The solid was dissolved in dichloromethane (108 L) and filtered to remove mechanical impurities. The filtrate was concentrated and then dissolved in 67.5 L (2.5 V) of hot ethyl acetate and stirred for 2 hours. A solid formed, and the mixture was allowed to cool. The solid was collected by filtration to give 4-chloro-5-(1,3-dioxolan-2-yl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine as an off-white solid (22 kg, 70% yield): C 16 H 14 ClN2O4S[M+H] + LCMS calculated: 365.03; Found: 365.1; 1 H NMR(400MHz,DMSO-d6)δ 8.51(s,1H),8.13(m,2H),8.07(m,1H),7.73(m,1H),7.63(m,2H),6.90(m,1H),6.13(s,1H),4.12(m,2H),3.98(m,2H).

[0187] Example 3. Alternative synthesis of 4-chloro-2-(morpholin-4-ylmethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde [ka]

[0188] Step 1: Synthesis of 5-bromo-4-chloropyridin-2-amine To a slurry of 2-amino-4-chloropyridine (100 g, 777.8 mmol, 1.0 equiv.) in acetonitrile (500 mL, 5 rel. vol.) at 15–20 °C, N-bromosuccinimide (131.5 g, 738.9 mmol, 0.95 equiv.) was added portionwise over 2 h, maintaining the temperature at 15–20 °C. The reaction was stirred for 30 min, and the conversion was checked by HPLC. Depending on the conversion, 0–5 mol% additional N-bromosuccinimide was added, and the mixture was stirred for an additional 15 min. After HPLC showed complete conversion, the reaction mixture was heated, and acetonitrile (300 mL) was distilled off at atmospheric pressure. Water (250 mL) was added, and the temperature was adjusted to 50–55 °C to form a slurry. The resulting slurry was stirred for 30 min, and water (350 mL) was added over 1 h. The slurry was cooled to 20-25°C, stirred for 1 hour, and the solid was collected by filtration. The wet cake was washed with a mixture of water (75 mL) and acetonitrile (25 mL) to give the wet product, 5-bromo-4-chloropyridin-2-amine (191 g, HPLC area % purity 92.1%). The wet product was dissolved in acetic acid (500 mL, 5 rel.vol. with 2-amino-4-chloropyridine, 55-70°C), and the solution was used directly in the next step.

[0189] Step 2: Synthesis of 5-bromo-4-chloro-3-iodopyridin-2-amine A solution of 5-bromo-4-chloropyridin-2-amine in acetic acid (191 g, 5-bromo-4-chloropyridin-2-amine in 500 mL of acetic acid) was distilled under reduced pressure at 40-60 °C to remove the solvent. Then, sulfuric acid (39.7 g, 96% (w / w), 388.9 mmol, 0.5 equiv.) and iodine (76.2 g, 300.3 mmol, 0.386 equiv.) were added, and the temperature was adjusted to 77-83 °C. At this temperature, a solution of periodic acid (50% (w / w), 54.89 g, 120.4 mmol, 0.155 equiv.) was added over 2-3 h. The reaction was stirred at 77-83 °C for 2-3 h, and the conversion was checked by HPLC (SM<1.0% a / a). The reaction mixture was quenched by adding solid ammonium sulfite in 4.53 g (0.05 equiv.) portions at 75-85°C until a negative KI / starch test was obtained. Typically, two portions (0.1 equiv.) of ammonium sulfite were required. The end of the quench could also be indicated by the absence of purple iodine. The reaction mixture was then diluted with water (200 mL, 2.0 rel.vol., room temperature) and the temperature was lowered to approximately 50°C. The product precipitated. At 45-60°C, the pH was adjusted to 3.0-3.5 using ammonia (25% (w / w) in water, approximately 63.6 g, 0.93 mol, 1.2 equiv. required). Neutralization was highly exothermic. The slurry was stirred at 45-50°C for 30 minutes, after which the solid was collected by filtration. The filter cake was typically washed with approximately 600 mL of water and then with 2-propanol (200 mL). The wet product was dried in a vacuum cabinet at 60° C. to give 5-bromo-4-chloro-3-iodopyridin-2-amine as a yellow to beige solid (213.5 g, 82.3% yield): CHBrClIN[M+H] + LCMS calculated for: 332.82; found: 332.8; 1 H NMR (400MHz, DMSO-d6) δ 8.09 (s, 1H), 6.60 (s, 2H).

[0190] Step 3: Synthesis of 5-bromo-4-chloro-3-(3-morpholinoprop-1-yn-1-yl)pyridin-2-amine A reactor was charged with 5-bromo-4-chloro-3-iodopyridin-2-amine (50 g, 150 mmol, 1.0 equiv.), 4-(prop-2-ynyl)morpholine (22.5 g, 180 mmol, 1.20 equiv.), diisopropylamine (18.2 g, 180 mmol, 1.2 equiv.), and 150 mL of toluene. The solution was carefully degassed using three vacuum-argon cycles. CuI (0.29 g, 1.5 mmol, 1 mol%) and Pd(PPh3)4 were then added, and the flask was purged again with argon. The mixture was stirred at 50 °C overnight (17 h). Water (50 mL, 1 vol.) was added in one portion, and the mixture was cooled to 20–25 °C. The crude product was filtered off and washed successively with 10% ammonia (50 ml, 1.0 vol), water (50 ml, 1 vol), toluene (25 ml, 0.5 vol), and 2-isopropanol (50 ml, 1.0 vol). After drying under vacuum at 50° C., 5-bromo-4-chloro-3-(3-morpholinoprop-1-yn-1-yl)pyridin-2-amine was obtained as a light brown solid (41.6 g, 87% yield): C 12 H 14 BrClIN5O[M+H] + LCMS calculated for: 329.99; Found: 330.0; 1 H NMR (400MHz, DMSO-d6) δ 8.13 (s, 1H), 6.69 (s, 2H), 3.64 (s, 2H), 3.61 (m, 4H), 2.54 (m, 4H).

[0191] Step 4: Synthesis of 4-((5-bromo-4-chloro-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine A solution of KOtBu (18.1 g, 1.4 equiv, 112.21 mmol) in tetrahydrofuran (114 mL, 3 vol) was heated to 30-35 °C, at which time 5-bromo-4-chloro-3-(3-morpholinoprop-1-yn-1-yl)pyridin-2-amine (38 g, 114.9 mmol, 1.0 equiv) was added portionwise over 1.0 h at 30-35 °C. After stirring for 2 h, the reaction was quenched with a solution of acetic acid (10.4 g, 172.4 mmol, 1.5 equiv) in water (76 mL, 2 vol), and 76 mL of THF (76 mL) was removed by distillation. The solution was then heated to reflux, MeOH (38 mL, 1 vol) was added, and the resulting suspension was cooled to 23 °C over 1 h. After stirring at 23° C. for 0.5 h, the solid was filtered off and washed with water (38 ml, 1 vol) and MeOH (38 mL, 1 vol). After drying under vacuum at 50° C., 4-((5-bromo-4-chloro-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine was obtained as a light brown powder (32.8 g, 86% yield): C 12 H 14 BrClIN5O[M+H] + LCMS calculated for: 329.99; Found: 329.8; 1 H NMR (400MHz, DMSO-d6) δ 12.22 (s, 1H), 8.34 (s, 1H), 6.40 (s, 1H), 3.65 (s, 2H), 3.58 (m, 4H), 2.42 (m, 4H).

[0192] Step 5: Synthesis of 4-((5-bromo-4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine A slurry of 4-((5-bromo-4-chloro-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine (10 g, 30.25 mmol, 1.0 equiv, assay 94% w / w) and NaH (1.69 g, 60%, 42.35 mmol, 1.4 equiv) in 38 mL of tetrahydrofuran was cooled to 0-5 °C while PhSO2Cl (7.48 g, 42.35 mmol, 1.4 equiv) was added over 1 h. After 1.5 h, HPLC indicated the reaction was not complete. Additional NaH (0.34 g, 0.3 equiv) was added and gas evolution was observed. When HPLC showed the reaction was complete, the reaction mixture was quenched with acetic acid (0.5 g) and a mixture of water (15 mL) and methanol (15 mL). The pH was adjusted to 6.5 with sodium hydroxide, and the product was isolated by filtration. The wet cake was washed with 2-isopropanol (20 mL) and water (20 mL), and the wet product (14.8 g) was dried in a vacuum cabinet to give 4-((5-bromo-4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine as a brown solid (12.57 g, 86% yield): C 18 H 18 BrClIN3O3S[M+H] + LCMS calculated for: 469.99; found: 470.0; 1 H NMR(400MHz,DMSO-d6)δ 8.56(s,1H),8.33(m,2H),7.73(m,1H),7.65(m,2H),6.83(s,1H),3.91(s,2H),3.53(m,4H),2.46(m,4H).

[0193] Step 6: Synthesis of 4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde To a suspension of 4-((5-bromo-4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)methyl)morpholine (5.0 g, 10.6 mmol, 1.0 equiv.) in 50 mL of tetrahydrofuran at −10° C. to 0° C. was added iPrMgCl (6.9 mL, 2 M in tetrahydrofuran, 13.80 mmol, 1.3 equiv.). After stirring for 2 h, N,N-dimethylformamide (1.55 g, 21.2 mmol, 2.0 equiv.) was added to the reaction solution over 0.5 h at −0.5° C. to 0° C. The mixture was stirred at −5° C. to 0° C. for 0.5 h, then warmed to 23° C. over 0.5 h and stirred at 23° C. for 1 h. The pH was adjusted to 6–7 by adding 1.5 mL of acetic acid and 10 mL of water. To the biphasic mixture, 25 mL of MeOH and 15 mL of water were added. After stirring for 1 hour, the product was filtered off and washed with 20 mL of MeOH / water (1 / 1) and 30 mL of water. After drying under vacuum at 50 °C, 4-chloro-2-(morpholinomethyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde was obtained as an off-white powder (3.39 g, 76% yield): C 19 H 19 ClN3O4S[M+H] + LCMS calculated for: 420.07; Found: 420.0; 1 H NMR(400MHz,DMSO-d6)δ 10.33(s,1H),8.76(s,1H),8.42(m,2H),7.74(m,1H),7.65(m,2H),6.98(s,1H),3.96(m,4H),3.564(m,4H),2.51(m,4H).

[0194] Example 4. Synthesis of 2,6-difluoro-3,5-dimethoxyaniline [ka] Step 1: Synthesis of methyl pentafluorobenzoate To a solution of pentafluorobenzoic acid (40 kg, 188.6 mol) in 68 L of methanol was added SOCl2 (29.2 kg, 245.2 mol, 1.3 equiv.) dropwise over 4.0 h at 20-50 °C. The mixture was then heated to reflux for 17 h. Methanol was removed by vacuum distillation, and the residue was dissolved in methyl t-butyl ether (77 L). The solution was washed with saturated NaHCO3 (37 L), dried over MgSO4, and evaporated to give methyl pentafluorobenzoate as a colorless oil (39 kg, 91% yield): 1 H NMR (400MHz, CDCl3) δ 3.90 (s, 3H).

[0195] Step 2: Synthesis of methyl-4-(benzylamino)-2,3,5,6-tetrafluorobenzoate Methyl pentafluorobenzoate (39 kg, 172.5 mol) and N,N-diisopropylethylamine (26.8 kg, 207 mol, 1.2 equiv.) were dissolved in N-methylpyrrolidinone (39 L). A solution of benzylamine (18.5 kg, 172.5 mol, 1.0 equiv.) in 19.5 L of N-methylpyrrolidinone was added dropwise over 3.5 hours, maintaining the internal temperature below 50°C. The resulting thick yellow slurry was heated to 65°C and stirred for an additional hour. The mixture was poured into 195 L of an aqueous acetic acid solution (10% acetic acid and 90% HO), and the slurry was stirred for 1 hour and filtered. The filter cake was washed with water and heptane and dried under vacuum at 35°C to give methyl-4-(benzylamino)-2,3,5,6-tetrafluorobenzoate (38 kg, 70% yield). 1 H NMR (400MHz, CDCl3) δ 7.37 (m, 5H), 4.67 (m, 2H), 4.58 (m, 1H), 3.93 (s, 3H).

[0196] Step 3: Synthesis of 4-(benzylamino)-3,5-difluoro-2,6-dimethoxybenzoic acid Methyl-4-(benzylamino)-2,3,5,6-tetrafluorobenzoate (38 kg, 121.3 mol) in methanol (72 L) was stirred at room temperature under N2, and a solution of NaOMe (25 wt%, 110.8 kg, 545.85 mol, 4.5 equiv.) in methanol was added dropwise over 3.0 h, maintaining the temperature below 50 °C. After heating to 65-70 °C for 18 h, 18 L of water was added to the reaction mixture, and the resulting solution was stirred for 1 h. The solvent was removed by vacuum distillation. Water (54 L) was added, and the resulting solution was acidified to pH 2 with 37% HCl. The mixture was extracted three times with ethyl acetate (54 kg each). The combined organic extracts were washed with water (43 L) and concentrated to dryness to form a solid. The solid was triturated with heptane (43 L) to remove impurities. The solid was collected and dried under vacuum at 40° C. to give 4-(benzylamino)-3,5-difluoro-2,6-dimethoxybenzoic acid (35 Kg, 86% yield): 1 H NMR (400MHz, CDCl3) δ 12.74 (s, 1H), 7.37 (m, 5H), 6.62 (s, 1H), 4.67 (m, 2H), 3.96 (s, 6H).

[0197] Step 4: Synthesis of n-benzyl-2,6-difluoro-3,5-dimethoxyaniline 4-(Benzylamino)-3,5-difluoro-2,6-dimethoxybenzoic acid (17 kg) was heated directly to 75-85°C under a nitrogen atmosphere for 3-4 hours. After the reaction was complete, 40 L of methyl t-butyl ether and 20 L of 1 M NaOH were added. The mixture was stirred at room temperature for 30 minutes. The organic layer was separated and washed with water (20 L) and brine (20 L). The organic phase was concentrated under reduced pressure to give the crude product. The crude material was triturated with heptane and dried under vacuum at 35°C to give N-benzyl-2,6-difluoro-3,5-dimethoxyaniline (12 kg, 82% yield): 1 H NMR (400MHz, CDCl3) δ 7.35 (m, 5H), 6.09 (m, 1H), 4.53 (m, 2H), 4.00 (s, 1H), 3.85 (s, 6H).

[0198] Step 5: Synthesis of 2,6-difluoro-3,5-dimethoxyaniline N-benzyl-2,6-difluoro-3,5-dimethoxyaniline (24 kg, 85.9 mol) was dissolved in a mixture of ethanol (120 L) and acetic acid (20 L), followed by the addition of ammonium formate (13.2 kg) and 1.68 kg of Pd / C. The mixture was heated at 50°C for 2-3 hours. The reaction mixture was then filtered through Celite®, washed with ethanol (1.2 L x 2), and concentrated. The crude product was added to 80 L of water, and the resulting slurry was filtered. The crude product was added to 60 L of methyl t-butyl ether and 2.5 kg of activated carbon, and the mixture was heated to reflux for 3 hours. After filtration and concentration, the resulting solid was added to 36 L of heptane and stirred at room temperature for 2 hours. The mixture was filtered and dried under vacuum at 35° C. to give 2,6-difluoro-3,5-dimethoxyaniline as a light brown solid (15.2 Kg, 93% yield): CH 10 F2NO2[M+H] + LCMS calculated for: 190.16; found: 190.1; 1 H NMR (400MHz, DMSO-d6) δ 6.16 (m, 1H), 5.18 (s, 2H), 3.78 (s, 6H).

[0199] Example 5. Alternative synthesis of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (compound 1) [ka]

[0200] Step 1: 4-(Ethylamino)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde [ka]

[0201] A mixture of 4-chloro-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (CAS#958230-19-8, Lakestar Tech, lot: 124-132-29: 3.0 g, 17 mmol) and ethylamine (10 M in water, 8.3 mL, 83 mmol) in 2-methoxyethanol (20 mL, 200 mmol) was heated to 130 °C and stirred overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was treated with 1 N HCl (30 mL), stirred at room temperature for 1 hour, and then neutralized with saturated aqueous NaHCO3. The precipitate was collected by filtration, washed with water, and dried to give the desired product (2.9 g, 92%). 10 H 12 NO[M+H] + LC-MS calculated for m / z: 190.1; found: 190.1.

[0202] Step 2: 5-{[(2,6-difluoro-3,5-dimethoxyphenyl)amino]methyl}-N-ethyl-1H-pyrrolo[2,3-b]pyridin-4-amine [ka]

[0203] A mixture of 4-(ethylamino)-1H-pyrrolo[2,3-b]pyridine-5-carbaldehyde (7.0 g, 37 mmol), 2,6-difluoro-3,5-dimethoxyaniline (9.1 g, 48 mmol), and [(1S)-7,7-dimethyl-2-oxobicyclo[2.2.1]hept-1-yl]methanesulfonic acid (Aldrich, catalog number 21360; 2 g, 7 mmol) in xylene (250 mL) was heated to reflux and azeotropically removed water using a Dean-Stark method for 2 days, at which point LC-MS indicated the reaction was complete. The mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in tetrahydrofuran (500 mL), and then 2.0 M lithium tetrahydroaluminate in THF (37 mL, 74 mmol) was slowly added. The resulting mixture was stirred at 50 °C for 3 hours and then cooled to room temperature. The reaction was quenched by adding water, 15% aqueous NaOH, and water. The mixture was filtered and washed with THF. The filtrate was concentrated, and the residue was washed with CH2Cl2 and then filtered to give the pure product (11 g, 82%). 18 H 21 F2N4O2[M+H] + LC-MS calculated for m / z: 363.2; found: 363.1.

[0204] Step 3: 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one [ka]

[0205] A solution of triphosgene (5.5 g, 18 mmol) in tetrahydrofuran (30 mL) was slowly added to a mixture of 5-{[(2,6-difluoro-3,5-dimethoxyphenyl)amino]methyl}-N-ethyl-1H-pyrrolo[2,3-b]pyridin-4-amine (5.6 g, 15 mmol) in tetrahydrofuran (100 mL) at 0 °C, and the mixture was then stirred at room temperature for 6 hours. The mixture was cooled to 0 °C, and then 1.0 M sodium hydroxide in water (100 mL, 100 mmol) was slowly added. The reaction mixture was stirred at room temperature overnight, and the formed precipitate was collected by filtration, washed with water, and then dried to provide the first batch of purified desired product. The organic layer in the filtrate was separated, and the aqueous layer was extracted with methylene chloride. The combined organic layers were concentrated, and the residue was triturated with methylene chloride, then filtered and dried to give another batch of product (5.5 g, 92% total). 19 H 19 F2N4O3[M+H] + LC-MS calculated for m / z: 389.1; found 389.1.

[0206] Step 4: 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one [ka]

[0207] To a solution of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (900 mg, 2.32 mmol) in N,N-dimethylformamide (20 mL) cooled to 0 °C was added sodium hydride (185 mg, 4.63 mmol, 60 wt% in mineral oil). The resulting mixture was stirred at 0 °C for 30 minutes, and then benzenesulfonyl chloride (0.444 mL, 3.48 mmol) was added. The reaction mixture was stirred at 0 °C for 1.5 hours, at which point LC-MS indicated completion of the reaction to the desired product. The reaction was quenched with saturated NH4Cl solution and diluted with water. The white precipitate was collected by filtration, then washed with water and hexanes, and dried to give the desired product (1.2 g, 98%) as a white solid, which was used in the next step without further purification. 25 H 23 F2N4O5S[M+H] + LC-MS calculated for m / z: 529.1; found: 529.1.

[0208] Step 5: 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-2-oxo-7-(phenylsulfonyl)-2,3,4,7-tetrahydro-1H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-8-carbaldehyde [ka]

[0209] To a solution of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (1.75 g, 3.31 mmol) in tetrahydrofuran (80 mL) at −78° C. was added freshly prepared lithium diisopropylamide (1 M in tetrahydrofuran (THF), 3.48 mL, 3.48 mmol). The resulting mixture was stirred at −78° C. for 30 minutes, and then N,N-dimethylformamide (1.4 mL, 18 mmol) was slowly added. The reaction mixture was stirred at −78° C. for 30 minutes, then quenched with water and extracted with EtOAc. The combined organic extracts were then washed with water and brine. The organic layer was dried over NaSO and concentrated. The residue was purified by flash chromatography eluting with 0-20% EtOAc in DCM to give the desired product as a white solid (1.68 g, 91%). 26 H 23 F2N4O6S(M+H) + LC-MS calculated for m / z: 557.1; found: 556.9.

[0210] Step 6: 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one [ka]

[0211] To a solution of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-2-oxo-7-(phenylsulfonyl)-2,3,4,7-tetrahydro-1H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-8-carbaldehyde (1.73 g, 3.11 mmol) in dichloromethane (50 mL) was added morpholine (0.95 mL, 11 mmol), followed by acetic acid (2 mL, 30 mmol). The resulting yellow solution was stirred at room temperature overnight, and then sodium triacetoxyborohydride (2.3 g, 11 mmol) was added. The mixture was stirred at room temperature for 3 hours, at which point LC-MS indicated the reaction was complete to the desired product. The reaction was quenched with saturated NaHCO3 and then extracted with ethyl acetate (EtOAc). The organic extracts were combined and then washed with water and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography eluting with 0-40% EtOAc in DCM to give the desired product as a white solid (1.85 g, 95%). 30 H 32 F2N5O6S(M+H) + LC-MS calculated for m / z: 628.2; found: 628.0.

[0212] Step 7: 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one To a solution of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholin-4-ylmethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (1.5 g, 2.4 mmol) in tetrahydrofuran (40 mL) was added tetra-n-butylammonium fluoride (1 M in THF, 7.2 mL, 7.2 mmol). The resulting solution was stirred at 50°C for 1.5 hours, then cooled to room temperature and quenched with water. The mixture was extracted with dichloromethane (DCM), and the organic extracts were combined and then washed with water and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography eluting with 0-10% MeOH in DCM to give the desired product as a white solid, which was further purified by preparative HPLC (pH=2, acetonitrile / HO). 24 H 28 F2N5O4(M+H) + LC-MS calculated for m / z: 488.2; found: 488.0. 1 H NMR(500MHz,DMSO)δ 12.09(s,1H),8.06(s,1H),7.05(t,J=8.1Hz,1H),6.87(s,1H),4.78(s,2H),4.50(s,2H),4.17(q,J=6. 8Hz,2H),3.97(br,2H),3.89(s,6H),3.65(br,2H),3.37(br,2H),3.15(br,2H),1.37(t,J=6.8Hz,3H).

[0213] Example 6 Synthesis of 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-2-morpholin-4-ylmethyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide [ka]

[0214] Step 1. Synthesis of 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide [ka]

[0215] A 200 L glass reactor was assembled with overhead stirring, a condenser, a thermocouple, a dropping funnel, and a nitrogen inlet, and each apparatus was purged with nitrogen. Potable water (3.1 L) and sodium hydroxide (3093 g) were charged to the reactor, and the mixture was stirred at approximately 71 °C until a solution was obtained. The reaction mixture was cooled to approximately 30 °C, and THF (15.0 L) was added. The reaction mixture was cooled to 10 °C, and 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (3000 g) and n-Bu4N were added. + HSO4 - (262 g) was added continuously while maintaining the temperature at about 10° C. The material was rinsed into the reactor with THF (15.0 L) while maintaining the temperature at about 7° C. N,N-dimethylsulfamoyl chloride (1.244 L) was added while maintaining the temperature at about 7° C. The reaction mixture was heated to about 17° C. and stirred at about 22° C. for 7 hours. Potable water (120.0 L) was charged while maintaining the temperature at about 20° C., and the reaction mixture was stirred at about 18° C. for 1 hour. The reaction mixture was filtered, and the filter cake was washed four times with potable water (30.0 L each wash). The product was air-dried on the filter for 14.5 hours to give crude 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-N,N-dimethyl-2-oxo-1,2,3,4-tetrahydro-7H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-7-sulfonamide (4694 g).

[0216] Purification: A 100 L glass reactor was assembled with overhead stirring, a condenser, a thermocouple, a dropping funnel, and a nitrogen inlet, and each apparatus was purged with nitrogen. A chromatography column was loaded with CHCl (37.5 L) and silica gel (15,000 g), thoroughly mixed, and eluted onto the surface of the silica gel. Sea sand (4000 g) and magnesium sulfate (6000 g) were sequentially loaded onto the column. Crude 3-(2,6-difluoro-3,5-dimethoxy-phenyl)-1-ethyl-N,N-dimethyl-2-oxo-1,2,3,4-tetrahydro-7H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-7-sulfonamide (6118 g) and CHCl (22.5 L) were thoroughly mixed until a solution was obtained, and the solution was then loaded onto the column. The elution rate was found to be too slow, so the magnesium sulfate and solution were removed from the column and filtered. The filter cake was washed with CHCl (20 L), and the filtrate was loaded onto the column. The vessel was rinsed with CHCl (2.5 L), and the rinse was loaded onto the column. The column was eluted with EtOAc / CHCl (prepared separately with 9.4 L of EtOAc and 178.1 L of CHCl). The desired fractions were partially concentrated under vacuum (using two rotavapors for convenience) at approximately 45 °C to a target total volume remaining of 24 L (approximately 4 L per kg of loaded 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-N,N-dimethyl-2-oxo-1,2,3,4-tetrahydro-7H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-7-sulfonamide). The distillation residue (24 L) was charged to a 100 L reactor and the temperature was adjusted to about 28° C. Heptane (54 L) was charged and the reaction mixture was stirred at about 24° C. for 1 hour. The reaction mixture was filtered and the filter cake was washed with heptane (24 L). The product was air-dried on the filter for about 3 hours to give the product (5382 g). 21 H 23 F2N5O5S[M+H] + LCMS calculated for: 495.5; found: 495.1. 1H NMR (400MHz, DMSO-d6) δ8.12(s, 1H), 7.72(s, 1H), 7.07(t, 1H), 6.89(s, 1H), 4.83(s, 2H), 4.14(t, 2H), 3.91(s, 6H), 2.96(s, 6H), 1.35(t, 3H).

[0217] Step 2. Synthesis of 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-2-formyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide [ka]

[0218] A 22 L glass reactor and a 200 L glass reactor were assembled with overhead stirring, a condenser, a thermocouple, an addition funnel, and a nitrogen inlet, and each apparatus was purged with nitrogen. THF (2.38 L) and N,N-diisopropylamine (0.82 L) were charged to the 22 L glass reactor, and the mixture was cooled to -72°C. A 2.5 M solution of n-BuLi in hexane (2.13 L) was charged while maintaining the temperature at approximately -70°C. The reaction mixture was stirred at -71°C for approximately 7 minutes and warmed to -2°C over 3.5 hours to form a 1 M LDA solution.

[0219] 3-(2,6-Difluoro-3,5-dimethoxyphenyl)-1-ethyl-N,N-dimethyl-2-oxo-1,2,3,4-tetrahydro-7H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-7-sulfonamide (gross 1075 g, 93.07 wt %, net 1001 g) and THF (10.0 L) were charged to a first rotavapor and rotated at approximately 63°C for approximately 28 minutes without collecting solvent until a solution was obtained. 3-(2,6-Difluoro-3,5-dimethoxyphenyl)-1-ethyl-N,N-dimethyl-2-oxo-1,2,3,4-tetrahydro-7H-pyrrolo[3',2':5,6]pyrido-[4,3-d]pyrimidine-7-sulfonamide (gross 1075 g, 93.07 wt %, net 1001 g) and THF (10.0 L) were charged to a second rotavapor and rotated at about 61 °C for about 35 minutes without collecting solvent until a solution was obtained. Both mixtures were concentrated under reduced pressure at about 50 °C. THF (10.0 L per rotavapor) was charged to each rotavapor, and both mixtures were concentrated under reduced pressure at about 50 °C. THF (10.0 L) was charged to the first rotavapor, and the mixtures were rotated at about 64 °C for 14 minutes without collecting solvent until a solution was obtained. THF (10.0 L) was charged to a second rotavapor, and the mixture was spun at approximately 64°C for 14 minutes without collecting the solvent until a solution was obtained. A PCT by GC for residual CHCl passed. Both solutions were transferred to a 200 L reactor using THF (26 L) to aid in the transfer. The reaction mixture was cooled to -65°C. While maintaining the temperature at approximately -64°C, 1 M LDA solution (4.84 L) was charged over approximately 1 hour, and the reaction mixture was stirred at -65°C for 2.25 hours. While maintaining the temperature at approximately -65°C, DMF (1.56 L) was charged over 30 minutes. The reaction mixture was stirred at -64°C for 31 minutes and warmed to -12°C over approximately 2 hours. Separately, an aqueous ammonium chloride solution was prepared by thoroughly mixing ammonium chloride (160 g) with drinking water (1.6 L). Aqueous ammonium chloride was charged over 17 minutes while maintaining the temperature at about −5° C., and the reaction mixture was allowed to warm to 19° C. over about 7.5 hours. The reaction mixture was partially concentrated under vacuum at about 45° C. (using two rotavapors for convenience).A total of 36 L was collected by distillation (approximately 18 L collected per kg of 3-(2,6-difluoro-3,5-dimethoxy-phenyl)-1-ethyl-N,N-dimethyl-2-oxo-1,2,3,4-tetrahydro-7H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-7-sulfonamide charge). The distillation residue was charged to a 200 L reactor, the temperature was adjusted to approximately 23°C, and the reaction mixture was stirred at 23°C for approximately 2 hours. Potable water (20.0 L) was added over approximately 1 hour, and the reaction mixture was stirred at approximately 23°C. The reaction mixture was filtered, and the filter cake was washed twice with potable water (10.0 L each wash). The product was air-dried on the filter for approximately 3.5 hours to yield crude product (2028 g). The crude product was slurried in MTBE at 46-53°C for 1 hour, then cooled to room temperature, filtered, and washed with more MTBE to give 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-2-formyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide: C. 22 H 23 F2N5O6S[M+H] + LCMS calculated for: 524.13; Found: 524.80. 1 H NMR(400MHz,DMSO-d6)δ 10.24(s,1H),8.31(s,1H),7.50(s,1H),7.05(m,1H),4.86(s,2H),4.17(m,2H),3.86(s,6H),3.04(s,6H),1.29(m,3H).

[0220] Step 3. Synthesis of 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-2-morpholin-4-ylmethyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide A 200 L glass reactor was assembled with overhead stirring, a condenser, a thermocouple, a dropping funnel, and a nitrogen inlet, and each apparatus was purged with nitrogen. Methylene chloride (20.0 L) and 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-2-formyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide (2000 g) were added to the reactor, and the mixture was stirred at 18°C ​​until a solution was obtained. Morpholine (1.7 L) was added to the reaction mixture. Acetic acid (2.0 L) was added over 34 minutes while maintaining the temperature at approximately 32°C. The reaction mixture was stirred at approximately 27°C for 4 hours. Sodium triacetoxyborohydride (1620 g) was added over 40 minutes while maintaining the temperature at approximately 28°C. The reaction mixture was stirred at approximately 24°C for 2.5 hours. Separately, a solution was prepared by thoroughly mixing sodium bicarbonate (2800 g) and drinking water (40.0 L) until a solution was obtained. The solution was added over 36 minutes, maintaining the temperature at approximately 19°C, until the pH reached 8-9. The reaction mixture was stirred at approximately 18°C ​​for 30 minutes. The phases were separated. The organic phase was extracted three times with methylene chloride (10.0 L per extraction). The combined organic phase was washed with drinking water (20.0 L). Separately, a solution was prepared by thoroughly mixing sodium chloride (2001 g) and drinking water (20.0 L) until a solution was obtained. The organic phase was washed with sodium chloride solution and dried over MgSO4 (600 g). The mixture was filtered, and the filter cake was washed with methylene chloride (6.0 L). The combined filtrate and washings were concentrated under reduced pressure at 38° C. to give crude 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-2-morpholin-4-ylmethyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide (2200 g).Chromatography with 0-40% EtOAc in DCM gave 7-(2,6-difluoro-3,5-dimethoxy-phenyl)-9-ethyl-2-morpholin-4-ylmethyl-8-oxo-6,7,8,9-tetrahydro-3,4,7,9-tetraaza-cyclopenta[a]naphthalene-3-sulfonic acid dimethylamide: C. 26 H 32 F2N6O6S[M+H] + LCMS calculated for: 595.21; found: 474.2. 1 H NMR(400MHz,DMSO-d6)δ 8.10(s,1H),7.05(t,1H),6.81(s,1H),4.80(s,2H),4.12(m,2H),3.91( s, 6H), 3.79 (s, 2H), 3.57 (m, 4H), 3.10 (s, 6H), 2.50 (m, 4H), 1.35 (m, 3H).

[0221] Example 7. Synthesis of 3-(2,6-difluoro-3-hydroxy-5-methoxyphenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 2) [ka]

[0222] A mixture of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-N,N-dimethyl-8-(morpholin-4-ylmethyl)-2-oxo-1,2,3,4-tetrahydro-7H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-7-sulfonamide (44.88 g, 75.48 mmol) in 1,4-dioxane (400 mL, 4000 mmol) was stirred under N as a mixture of 12.0 M hydrogen chloride in water (1260 mL, 1510 mmol), and water (1260 mL, 6990 mmol) was added via addition funnel (internal temperature reached 35 °C). The resulting solution was heated at 80 °C for 18 h. LC-MS showed no starting material remained. The reaction mixture was cooled to room temperature, the organic solvent was concentrated, and the resulting aqueous HCl solution was diluted with 200 mL of 2N HCl solution. The resulting aqueous solution was extracted with DCM (3×80 mL), and the combined DCM phases were re-extracted with 6N HCl (80 mL). The combined aqueous HCl solution was stirred and cooled to 0-5°C with ice water. The acidic solution was neutralized by the dropwise addition of 25% NaOH (~200 mL) to pH >12. The resulting solid (deprotected unreacted starting material or compound 1) was filtered and washed with water (3×250 mL). The basic aqueous solution was acidified to pH ∼6 with aqueous HCl and extracted with DCM (3×30 mL). The combined DCM solution was dried over Na2SO4 and concentrated. The crude product was then slurried with MTBE, filtered, washed with more MTBE, and dried in a vacuum oven at 50° C. overnight to give 3-(2,6-difluoro-3-hydroxy-5-methoxyphenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3′,2′:5,6]pyrido[4,3-d]pyrimidin-2-one (277 mg): C 23 H 26 F2N5O4[M+H] + LCMS calculated for: 474.47; found: 474.2. 1H NMR(400MHz,DMSO-d6)δ 11.40(s,1H),9.95(s,1H),7.99(s,1H),6.78(m,1H),6.42(s,1H),4.76( s, 2H), 4.09 (m, 2H), 3.80 (s, 3H), 3.60 (m, 6H), 2.41 (m, 4H), 1.28 (m, 3H).

[0223] Example 8. Synthesis of 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (Compound 6) [ka]

[0224] Step 1. Synthesis of 3-(2,6-difluoro-3,5-dihydroxyphenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one A solution of 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (2.0 g, 3.2 mmol) in 25.0 mL of DCM was stirred at −78 °C while BBr3 (3.10 mL, 10.0 equiv.) (neat) was added dropwise. After 30 min, the dry ice bath was removed and the reaction was allowed to warm slowly to room temperature. After 2 h, HPLC indicated the disappearance of starting material. The reaction was then cooled to 0 °C, carefully treated with 20 mL of ice water, and stirred for 30 min. The resulting solid was filtered, washed with additional water, and allowed to dry in the funnel overnight. The crude product was treated with 20% MeOH in DCM (10 mL), stirred for 20 min, then filtered and dried to give 3-(2,6-difluoro-3,5-dihydroxyphenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one as a white solid (1.208 g, 61% yield): C 28 H 28 F2N5O6S[M+H] + LCMS calculated for: 600.17; found: 600.4. This material was used in the next step without further purification.

[0225] Step 2: Synthesis of 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one

[0226] 3-(2,6-Difluoro-3,5-dihydroxyphenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (1.20 g, 2.00 mmol) and NaH (60% dispersion in mineral oil, 216 mg, 9.01 mmol) in DMF (12.4 mL, 160 mmol) were stirred at room temperature under N for 15 min (total solution), followed by MeI-D 3 (0.263 mL, 4.20 mmol) was added dropwise. After stirring at room temperature for 1 hour, the reaction was cooled to 0 °C, 30 mL of ice water was added, and the resulting solid was stirred for 30 minutes. The resulting solid was filtered, washed with water, and dried. The crude product was purified by Biotage chromatography using 0-6% MeOH in DCM to give 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one as a white solid (950 mg, 75% yield): C 30 H 26 D6F2N5O6S[M+H] + LCMS calculated for: 634.23; Found: 634.5. 1 H NMR (400 MHz, DMSO-d6) δ chromatography), 8.07 (s, 1H), 7.70 (m, 1H), 7.63 (m, 2H), 7.05 (m, 1H), 6.89 (s, 1H), 4.76 (s, 2H), 4.09 (m, 2H), 3.93 (s, 2H), 3.60 (m, 4H), 2.50 (m, 4H), 1.28 (m, 3H).

[0227] Step 3: Synthesis of 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one A 25 mL flask was charged with 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-7-(phenylsulfonyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (1000 mg, 1.578 mmol) in 1,4-dioxane (10.0 mL) and 1 N sodium hydroxide in water (6312 μL, 6.31 mmol). The solution was heated to 74 °C (internal temperature) for 15 h. LCMS indicated the reaction was complete. The clear, pale yellow solution was cooled to room temperature (a solid began to precipitate from solution as the mixture cooled) to give an off-white suspension. Water (10.0 mL) was added at 20–25 °C, and the resulting solid was stirred for 30 min. The solid was filtered and washed six times with water, checking the pH of the final wash (pH = ca. 7). Crude HNMR of the product (0.70 g) showed the presence of some dioxane.

[0228] Crude 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one (0.70 g, 1.418 mmol) in DCM (3.88 ml, 60.3 mmol) was heated to 22-36 °C to obtain a clear solution. After approximately 5 min, MTBE (0.693 ml, 5.82 mmol) was added. After stirring for approximately 1 h, a precipitate formed, and the mixture was stirred for an additional 30 min. The mixture was filtered, washed with MTBE, washed with heptane, and dried in a vacuum oven under N at 50 °C to obtain the product (0.63 g, 81% yield, 96% HPLC purity). The crude solid was purified using Biotage chromatography using 0-10% MeOH in DCM, and the fractions were combined, concentrated, and dried in a vacuum oven at 50 °C to give 3-(2,6-difluoro-3,5-bis(methoxy-d3)phenyl)-1-ethyl-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidin-2-one in 99.5% HPLC purity: C 24 H 22D6F2N5O4[M+H] + LCMS calculated for: 494.24; found: 494.2. 1 H NMR (400 MHz, DMSO-d6) δ 11.75 (chromatographically determined 9 (s, 1H), 7.05 (m, 1H), 6.89 (s, 1H), 4.76 (s, 2H), 4.09 (m, 2H), 3.60 (m, 6H), 2.50 (m, 4H), 1.28 (m, 3H).

[0229] Compound 6 may also be prepared according to Example 1, using 2,6-difluoro-3,5-bis(methoxy-d3)aniline instead of 2,6-difluoro-3,5-dimethoxyaniline. 2,6-Difluoro-3,5-bis(methoxy-d3)aniline can be produced according to Example 4, Step 3, using sodium methoxide-d3 instead of sodium methoxide.

[0230] Example A FGFR enzyme assay Inhibitor potency of example compounds was determined in an enzymatic discontinuous assay measuring peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and 0.2 μL volumes were transferred to wells of a 384-well plate. For FGFR isoforms (FGFR-1, FGFR-2, FGFR-3 wild-type, and the mutant isoform, FGFR-4), including phosphorylated and non-phosphorylated proteins, 5 μL / well of enzyme diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and preincubated with the inhibitor for 5–15 min at ambient temperature. Appropriate controls (enzyme blank and enzyme without inhibitor) were included in the plate. Reactions were initiated by the addition of 5 μL / well of a volume containing both biotinylated EQEDEPEGDYFEWLE (SEQ ID NO: 1) peptide substrate and ATP in assay buffer. The 10 μL / well reaction concentration of peptide substrate was 500 nM, while the ATP concentration was maintained near or below the Km of ATP for each FGFR isoform. ATP Km values ​​were previously determined for each FGFR isoform in a series of separate experiments. Reaction plates were incubated at 25°C for 1 hour, and the reactions were terminated by the addition of 5 μL / well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg / mL BSA, pH 7.8; 45 mM EDTA, 600 nM staurosporine, 3.75 nM Eu antibody PY20, and 180 nM APC-streptavidin, Perkin Elmer Lance reagent). Plates were allowed to equilibrate at ambient temperature for approximately 10 minutes before scanning on a PheraStar plate reader (BMG Labtech) instrument.

[0231] Data were analyzed using either GraphPad prism or XLfit. IC values ​​were calculated by fitting the data to a four-parameter logistic equation to generate sigmoidal dose-response curves with variable Hill coefficients. 50The value was derived from the Prism equation: Y = Bottom + (Top - Bottom) / (1 + 10^((Log IC 50 -X)*Hillslope)); XLfit equation: Y=(A+((BA) / (1+((X / C)^D)))) where X is the logarithm of the inhibitor concentration and Y is the response.

[0232] FGFR inhibition data for various compounds of the present disclosure are shown in Table 1 below. Symbol: "+" indicates IC 50 "++" indicates IC ≥ 10 nM but < 100 nM 50 "+++" indicates an IC greater than or equal to 100 nM but less than 500 nM 50 and "++++" indicates an IC of 500 nM or greater but less than 1000 nM. 50 Shows. [Table 1]

[0233] Example B: KATO III Whole Blood pFGFR2α ELISA Assay To measure tyrosine-phosphorylated fibroblast growth factor receptor 2 alpha (FGFR2α) in the KATO III spiked whole blood assay, KATO III cells were purchased from ATCC and maintained in Iscove's medium containing 20% ​​FBS (Gibco / Life Technologies). To measure the inhibition of FGFR2α activity by test compounds, cells were cultured at 5 × 10 in Iscove's 0.2% FBS. 6Cells were resuspended at 1000 cells / ml. 50 μL of cells were then spiked into a 96-deep-well, 2-ml polypropylene assay block (Costar) in the presence or absence of a range of test compound concentrations and 300 μL of human heparinized whole blood (Biological Specialty Corp, Colmar, PA). After 4 hours of incubation at 37°C, red blood cells were lysed using Qiagen EL buffer, and the cell lysate was resuspended in lysis buffer (Cell Signaling) containing a standard protease inhibitor cocktail (Calbiochem / EMD) and PMSF (Sigma) for 30 minutes. The lysate was transferred to a standard V-bottom polypropylene tissue culture plate and frozen overnight at -80°C. Samples were tested using the R&D Systems DuoSet IC Human Phospho-FGF R2α ELISA, and plates were read using a SpectraMax M5 microplate reader set at 450 nm with wavelength compensation at 540. Calculate the IC by fitting the curve of percent inhibition of the inhibitor versus the logarithm of the inhibitor concentration using GraphPad Prism 5.0 software. 50 The decision was implemented.

[0234] KATOIII whole blood pFGFR2α ELISAIC 50 The data are shown in Table 2 below. Symbol: "+" indicates IC 50 "++" indicates IC ≥ 50 nM but < 250 nM 50 "+++" indicates IC greater than 250 nM but less than 500 nM 50 "++++" indicates an IC of 500 nM or greater but less than 1000 nM 50 "+++++" indicates an IC of 1000 nM or greater but less than 3000 nM. 50 Shows. [Table 2]

[0235] Example C: Determination of permeability and P-gp-mediated transport in Caco-2 cells Caco-2 cells were grown in 96-well transwell plates at a seeding density of 14,000 cells / well in DMEM medium. To measure permeability in the absorptive (AB) direction, test compounds in HBSS were added to the donor compartment (apical side), and 4% BSA in HBSS was added to the receiver compartment (basolateral side). To determine whether a compound was a P-gp substrate, permeability values ​​were measured in both the AB and BA directions in the absence of 4% BSA (bidirectional transport assay). Digoxin and cyclosporine A were included as positive controls for P-gp substrate and inhibitor, respectively, to ensure P-gp function in the bidirectional transport assay. Varying concentrations of test compounds were added to the donor compartment (apical side for AB transport and basolateral side for BA transport), while HBSS solution was added to the receiver compartment (basolateral side for AB transport and apical side for BA transport). For the AB permeability studies, the donor volume was 0.075 mL and the receiver volume was 0.25 mL. For the AB permeability studies, the donor volume was 0.25 mL and the receiver volume was 0.075 mL. Incubation was performed at 37°C for 120 min. Transepithelial electrical resistance (TEER) was measured before and after the 120 min incubation to confirm the integrity of the cell monolayer. At the end of the incubation period, samples were removed from both the donor and receiver sides and mixed with acetonitrile for protein precipitation. The supernatant was collected after centrifugation for analysis using an LC-MS / MS method. Permeability coefficient (Papp) values ​​from the Caco-2 studies were determined using the following equation: Papp(cm / s)=(F*VD) / (SA*MD) where the flux rate (F, mass / time) is calculated from the slope of the cumulative amount of the compound of interest on the receiver side, SA is the surface area of ​​the cell membrane, VD is the volume of the donor, and MD is the initial volume of solution in the donor chamber.

[0236] The efflux ratio from the Caco-2 study was calculated as the ratio of Papp measured in the BA direction divided by Papp in the AB direction.

[0237] Permeability data in Caco-2 cells for various compounds of the present disclosure are shown in Table 3 below. [Table 3]

[0238] P-gp-mediated transport data in Caco-2 cells for various compounds of the present disclosure are shown in Tables 4 and 5 below. [Table 4] [Table 5]

[0239] The efflux ratio for compound 1 at 30 μM is 0.64. In contrast, the efflux ratio for compound 2 at 30 μM is 19. This indicates that compound 2 is a more effective substrate for Pgp transport than compound 1 and that compound 2 is not well absorbed into the bloodstream. Compound 2 may be a candidate for IV administration or hepatic artery infusion for the treatment of diseases and disorders (e.g., cholangiocarcinoma).

[0240] Furthermore, the permeability of Compound 1 in Caco-2 cells is 11 P m, whereas the permeability of Compound 2 in Caco-2 cells is 0.5 P m. Considering the slight differences in compound structure, the large differences in permeability and efflux rates between Compound 1 and Compound 2 are unexpected.

[0241] Example D: Human Absorption, Metabolism, and Excretion Studies of Compound 1 An open-label study was conducted to evaluate the mass balance, pharmacokinetics, and metabolite profile of a single oral dose of [14C]Compound 1. [ka]

[0242] Seven male subjects each received a single oral dose of an 11 mg Compound 1 tablet along with an [14C]Compound 1 solution (approximately 250 μCi) after an overnight fast. Blood / plasma, urine, and feces were collected from participants for 4 to 10 days after dosing.

[0243] Human release criteria include that at least 90% of the administered radioactive dose is recovered, and that not more than 1% of the administered radioactive dose is recovered in the excreta (urine and feces combined) in two consecutive 24-hour urine and fecal collection samples.

[0244] 12.6% of the administered dose was recovered in the urine, and 82.4% of the administered dose was recovered in the feces. Overall recovery of radioactive decay in urine and feces was 95.1% over the 240-hour study. Rapid absorption was observed, with peak total radioactivity and plasma Compound 1 concentrations at approximately 2.0 hours post-dose.

[0245] FIG. 1 shows the mean cumulative percent of radioactive dose recovered in urine and feces at the indicated intervals after a single oral dose of 13 mg (250 μCi) of [C]Compound 1 to healthy male subjects.

[0246] Figure 2 shows the mean radioactivity (nM equivalent) in blood or plasma and the concentration of Compound 1 (nM) in plasma after a single oral dose of approximately 13 mg of [C]Compound 1 to healthy male volunteers in a fasted state. The total radioactive decay in plasma and the half-life of Compound 1 were approximately 10 hours. The ratio of plasma Compound 1 concentration to total plasma radioactivity was approximately 0.7, indicating that Compound 1 was the major circulating component in plasma. The blood-to-plasma radioactivity ratio (approximately 0.8) indicated that the radioactivity was poorly associated with blood cells.

[0247] Four minor circulating metabolites (<10% of compound-related substances) were observed, as shown below. [ka]

[0248] Compound 2-1 is the glucuronide derivative of Compound 2. Compound 2-2 is the sulfonic acid derivative of Compound 2. Compound 1-1 is the keto derivative of Compound 1. Circles around certain parts of the chemical structures indicate options for attachment of the glucuronide, sulfonic acid, and keto groups.

[0249] The percentage of each compound dose isolated from the blood / plasma samples is shown in the table below. [Table 6]

[0250] FIG. 3 shows the mass spectrum of the human circulating metabolites of Compound 1.

[0251] 12.6% of the administered dose was recovered in the urine. Two metabolites of Compound 1 were isolated from urine, as shown below. [ka]

[0252] Circles around certain parts of the chemical structure indicate attachment options for glucuronide and sulfonate groups. The percentage of each compound dose isolated from urine samples is shown in the table below. [Table 7]

[0253] FIG. 4 shows the mass spectrum of metabolites of Compound 1 isolated from urine.

[0254] 82.4% of the administered dose was recovered in the feces. The following metabolites of Compound 1 were isolated from the feces: [ka]

[0255] Compound 4-1 is a keto derivative of compound 4. Compound 2-3 is a keto derivative of compound 2. Compound 1-2 is a dihydroxylated derivative of compound 1. Circles around certain parts of the chemical structure indicate attachment options for the hydroxyl and keto groups.

[0256] [Table 8]

[0257] FIG. 5 shows the mass spectrum of metabolites of Compound 1 isolated from feces.

[0258] Various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Each reference, including all patents, patent applications, and publications cited in this application, is hereby incorporated by reference in its entirety.

Claims

1. A compound that is 3-(2,6-difluoro-3,5-dimethoxyphenyl)-1-ethyl-4-hydroxy-8-(morpholinomethyl)-1,3,4,7-tetrahydro-2H-pyrrolo[3',2':5,6]pyrido[4,3-d]pyrimidine-2-one, or a pharmaceutically acceptable salt thereof.

2. Formula II: 【Chemistry 1】 [In the formula, one C-H group is replaced by a C-X group, an N-H group is replaced by an N-X group, or an O-H group is replaced by an O-X group; where X is 【Chemistry 2】 [A base selected from] A compound of or a pharmaceutically acceptable salt thereof.

3. X 【Transformation 3】 The compound according to claim 2 or a pharmaceutically acceptable salt thereof.

4. X 【Chemistry 4】 The compound according to claim 2 or a pharmaceutically acceptable salt thereof.

5. A compound according to any one of claims 2 to 4, wherein one C-H group is substituted with a C-X group, or a pharmaceutically acceptable salt thereof.

6. A compound according to any one of claims 2 to 4, wherein an N-H group is substituted with an N-X group, or a pharmaceutically acceptable salt thereof.

7. A compound according to any one of claims 2 to 4, wherein the O-H group is substituted with an O-X group, or a pharmaceutically acceptable salt thereof.

8. A substantially isolated compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. A composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

10. The composition according to claim 9, which is suitable for oral administration.

11. The composition according to claim 9, which is suitable for intravenous administration.

12. The composition according to claim 9, which is suitable for arterial administration.

13. The composition according to claim 12, wherein the arterial administration is hepatic artery injection.

14. A pharmaceutical agent for inhibiting FGFR enzymes, comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 9 to 13.

15. A pharmaceutical for treating cancer in a patient requiring treatment for cancer, comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 9 to 13.

16. The pharmaceutical product according to claim 15, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, thyroid cancer, skin cancer, leukemia, multiple myeloma, chronic lymphocytic lymphoma, adult T-cell leukemia, B-cell lymphoma, acute myeloid leukemia, Hodgkin lymphoma or non-Hodgkin lymphoma, Waldenström macroglobulinemia, hairy cell lymphoma, Burkitt lymphoma, glioblastoma, melanoma, and rhabdomyosarcoma.

17. A pharmaceutical for treating myeloproliferative disorders in patients requiring treatment for myeloproliferative disorders, comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 9 to 13.

18. The pharmaceutical product according to claim 17, wherein the myeloproliferative disorder is selected from polycythemia vera, essential thrombocythemia, and primary myelofibrosis.

19. A pharmaceutical for treating bone disorder or chondrocyte disorder in a patient requiring treatment for such disorder, comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 9 to 13.

20. The pharmaceutical product according to claim 19, wherein the bone disorder or chondrocyte disorder is selected from achondroplasia, hypochondrodysplasia, dwarfism, thanatophoric dysplasia (TD), Apert syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Behle-Stevenson gyroscal syndrome, Pfeiffer syndrome, and craniosynostosis syndrome.

21. A pharmaceutical for treating hypophosphatemic disorders in patients requiring treatment for hypophosphatemic disorders, comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 9 to 13.

22. The pharmacopoeia according to claim 21, wherein the hypophosphatemic disorder is X-linked hypophosphatemic rickets, autosomal recessive hypophosphatemic rickets, autosomal dominant hypophosphatemic rickets, or tumor-induced osteomalacia.

23. A pharmaceutical for treating bile duct cancer in a patient requiring treatment for bile duct cancer, comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 9 to 13.

24. The pharmaceutical product according to claim 23, wherein the cholangiocarcinoma is progressive or metastatic cholangiocarcinoma.

25. A pharmaceutical for treating myeloid / lymphoid tumors in patients requiring treatment for myeloid / lymphoid tumors, comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 9 to 13.

26. The pharmaceutical product according to claim 25, wherein the myeloid / lymphoid tumor is 8p11 myeloproliferative syndrome.

27. The pharmacopoeia according to claim 25, wherein the myeloid / lymphoid tumor is related to eosinophilia.

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