METHYLATION OF Mcl-1 COMPOUND

Abstract

To provide a crystalline hydrate of a compound functioning as an inhibitor of myeloid cell leukemia 1 protein (Mcl-1).SOLUTION: A crystalline hydrate of compound A, a pharmaceutical preparation comprising the crystalline hydrate of compound A, and a method for treating a subject suffering from cancer are provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 002,629, filed March 31, 2020, U.S. Provisional Patent Application No. 63 / 070,630, filed August 26, 2020, and U.S. Provisional Patent Application No. 63 / 084,367, filed September 28, 2020, each of which is hereby incorporated by reference in its entirety and for all purposes as if fully set forth herein.

[0002] The present disclosure relates to (4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-methoxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecyne-4,1'-naphthalene]-16,16,18(7H,17H)-trione (Compound A), which functions as an inhibitor of myeloid cell leukemia 1 protein (Mcl-1); or a salt or solvate thereof by methylation of ((4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-hydroxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-4,1'-naphthalene]-16,16,18(7H,17H)-trione. [Background technology]

[0003] 2. Description of Related Art The compound, (4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-methoxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-4,1'-naphthalene]-16,16,18(7H,17H)-trione (Compound A): [ka] are useful as inhibitors of myeloid cell leukemia 1 ("Mcl-1").

[0004] One common characteristic of human cancers is overexpression of Mcl-1, which prevents cancer cells from undergoing programmed cell death (apoptosis), allowing them to survive despite widespread genetic damage.

[0005] Mcl-1 is a member of the Bcl-2 family of proteins. The Bcl-2 family includes pro-apoptotic members (such as BAX and BAK) that, upon activation, form homo-oligomers in the outer mitochondrial membrane that result in pore formation and mitochondrial content disruption, steps that trigger apoptosis. Anti-apoptotic members of the Bcl-2 family (such as Bcl-2, Bcl-XL, and Mcl-1) block the activity of BAX and BAK. Other proteins (such as BID, BIM, BIK, and BAD) exhibit additional regulatory functions. Studies have shown that Mcl-1 inhibitors may be useful for the treatment of cancer; Mcl-1 is overexpressed in many cancers.

[0006] U.S. Patent No. 10,300,075, incorporated herein by reference in its entirety, discloses Compound A as an Mcl-1 inhibitor and provides methods for its preparation. However, improved synthetic methods that result in higher yields and purity of Compound A are desirable, particularly for commercial production of Compound A. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 10,300,075 Summary of the Invention [Means for solving the problem]

[0008] In one embodiment, Compound A, a salt or solvate thereof: [ka] A method for synthesizing (a) (i) a base selected from the group consisting of a non-nucleophilic base, an alkali metal hydride base, an alkali metal hydroxide base, an organolithium base, and any combination thereof; and (ii) Compound B, a salt thereof, or a solvate thereof; [ka] and an organic solvent, including an ether solvent, a non-polar solvent, or any combination thereof; and a solution comprising water, wherein the molar ratio of water to compound B is in the range of about 0.1:1 to about 3:1, to form a mixture; and (b) mixing the mixture of step (a) with MeX, wherein X is a halogen, to form a mixture comprising compound A. In some embodiments, the disclosed method further comprises quenching the mixture of step (b) with a secondary amine base. In some cases, the secondary amine base is selected from the group consisting of N,N-diethylamine, morpholine, piperidine, pyrrolidine, piperazine, and combinations thereof. In various embodiments, each mixing step occurs at a temperature ranging from about 0° C. to about 40° C. In some cases, the temperature of each mixing step is in the range of about 15° C. to about 25° C.

[0009] In some embodiments, the base comprises lithium hexamethyldisilazide ("HMDS"), sodium HMDS, potassium HMDS, lithium diisopropylamide, sodium diisopropylamide, potassium diisopropylamide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-amylate, sodium tert-amylate, potassium tert-amylate, potassium hydride, sodium hydride, potassium hydroxide, sodium hydroxide, lithium hydroxide, 2,2,6,6-tetramethylpiperidine (TMP), LiTMP, n-butyllithium (n-BuLi), n-hexyllithium, 1,1,3,3-tetramethylguanidine (TMG), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene, or any combination thereof. In some cases, the base comprises lithium hexamethyldisilazide HMDS, sodium HMDS, potassium HMDS, or any combination thereof. In embodiments, the base comprises potassium hexamethyldisilazide ("KHMDS").

[0010] In some cases, the molar ratio of base to compound B ranges from about 1:1 to about 5:1. In various cases, the molar ratio of base to compound B ranges from about 2.5:1 to about 4:1. In some embodiments, the molar ratio of base to compound B is from about 3.0:1 to about 3.5:1. In various embodiments, the molar ratio of base to compound B is about 3.2:1.

[0011] In some cases, the organic solvent is selected from the group consisting of tetrahydrofuran ("THF"), 2-methyltetrahydrofuran, cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, toluene, hexane, heptane, 1,4-dioxane, and combinations thereof. In various cases, the organic solvent comprises THF.

[0012] In some embodiments, the molar ratio of water to compound B ranges from about 0.5:1 to about 3:1. In various embodiments, the molar ratio of water to compound B ranges from about 1:1 to about 3:1. In some cases, the molar ratio of water to compound B is from about 1.4:1 to about 1.6:1.

[0013] In various instances, X is iodide. In some embodiments, the molar ratio of MeX to compound B ranges from about 1:1 to about 4:1. In various embodiments, the molar ratio of MeX to compound B is about 2.7:1.

[0014] In some embodiments, the base is mixed with the solution in step (a) for a period of about 5 seconds to about 6 hours. In various embodiments, the base is mixed with the solution in step (a) for 5 seconds or less. In some cases, the base is mixed with the solution in step (a) for 1 second or less. In some embodiments, the mixture in step (a) is stirred for about 1 second to about 12 hours. In various embodiments, the mixture in step (a) is stirred for about 1 second to about 20 minutes.

[0015] In some cases, MeX is mixed with the mixture of step (a) for a period of about 1 second to about 6 hours. In various cases, MeX is mixed with the mixture of step (a) for 5 seconds or less. In some embodiments, MeX is mixed with the mixture of step (a) for 1 second or less. In various embodiments, the mixture of step (b) is stirred for about 1 minute to 12 hours. In some cases, the mixture of step (b) is stirred for about 1 minute to about 20 minutes.

[0016] In some embodiments, Compound B is a solvate. In various embodiments, Compound B is Compound B': [ka] (wherein M is an alkali metal) In some embodiments, the alkali metal is lithium, sodium, or potassium. In various embodiments, the alkali metal is potassium.

[0017] In some embodiments, compound B' is prepared by combining compound B with an alkali hydroxide base and an organic solvent selected from the group consisting of an ethereal solvent, a nonpolar solvent, and any combination thereof to form a mixture containing compound B'. In various embodiments, the alkali hydroxide base is selected from the group consisting of potassium hydroxide, sodium hydroxide, lithium hydroxide, and combinations thereof. In some cases, the molar ratio of alkali hydroxide base to compound B ranges from about 0.5:1 to about 3:1. In various cases, the molar ratio of alkali hydroxide base to compound B is about 1.5:1. In some embodiments, the organic solvent is selected from the group consisting of tetrahydrofuran ("THF"), 2-methyltetrahydrofuran, cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, toluene, hexane, heptane, 1,4-dioxane, and combinations thereof. In various embodiments, the organic solvent includes THF. In some cases, the mixture containing compound B' is stirred for about 1 hour to about 48 hours.

[0018] 13.57, 19.13, 20.39, 24.04, 25.54, 27.75, 30.09, 31.05, 36.84, 38.27, 39.48, 43.15, 49.53, 50.30, 51.84, 54.40, 56.15, 57.28, 57.78, 60.23, 61.80, 65.65, 78.05, 85.23, 115.91, 123.10, 124.60, 128.11, 130.53, 133.18, 133.87, 134.99, 139.72, 141.47, 143.08, 151.76, and 174.30 ± 0.5 ppm in solid state 13 Also provided herein are crystalline hydrate forms of Compound A characterized by C NMR peaks.

[0019] Also provided herein is a pharmaceutical formulation comprising a crystalline hydrate form of Compound A as described herein and a pharmaceutically acceptable excipient.

[0020] Also provided herein is a method of treating a subject suffering from cancer, the method comprising administering to the subject a pharmaceutical formulation comprising a therapeutically effective amount of a crystalline hydrate form of Compound A as described herein and a pharmaceutically acceptable excipient.

[0021] Also provided herein is a crystalline hydrate form of Compound A characterized by an XRPD pattern peaks at 10.3, 16.3, and 17.1±0.2 degrees 2θ using CuKα radiation.

[0022] Also provided herein is a pharmaceutical formulation comprising a crystalline hydrate form of Compound A as described herein and a pharmaceutically acceptable excipient.

[0023] Also provided herein is a method of treating a subject suffering from cancer, the method comprising administering to the subject a pharmaceutical formulation comprising a therapeutically effective amount of a crystalline hydrate form of Compound A as described herein and a pharmaceutically acceptable excipient.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0025] Further aspects and advantages will be apparent to those skilled in the art from a review of the following detailed description taken in conjunction with the drawings. The description herein below includes specific embodiments, with the understanding that the disclosure is illustrative and is not intended to limit the invention to the specific embodiments described herein. [Brief explanation of the drawings]

[0026] [Figure 1] 1 depicts a schematic of an exemplary flow chemistry process using a plug flow reactor and a continuous stirred tank reactor (CSTR), in which compound B′ (Oct-K) and potassium hexamethyldisilazide (KHMDS) in aqueous THF as disclosed herein are pumped to a plug flow reactor (residence time: 15 seconds), followed by methylation in CSTR1 (residence time: 5 minutes), further digestion in CSTR2 (residence time: 5 minutes), and quenching in CSTR3 (residence time: 5 minutes). [Figure 2] 1 depicts a schematic of an exemplary flow chemistry process with flow skid modification, as disclosed herein, in which compound B′ (Oct-K) and potassium hexamethyldisilazide (KHMDS) in aqueous THF are mixed in CSTR0 (residence time: 5 min), followed by methylation in CSTR1 (residence time: 5 min), additional digestion in CSTR2 (residence time: 5 min), and quenching with diethylamine in CSTR3 (residence time: 5 min). [Figure 3]1 depicts the X-ray powder diffraction ("XRPD") pattern of a crystalline hydrate form of Compound A. [Figure 4] 1 depicts a DSC thermograph of the crystalline hydrate form of Compound A. [Figure 5] 1 depicts a TGA trace of a crystalline hydrate form of Compound A. [Figure 6] 1 depicts the moisture sorption profile of the crystalline hydrate form of Compound A. [Figure 7] 1 depicts the solid-state 13C NMR of the crystalline hydrate form of Compound A. [Figure 8] 1 depicts the single crystal X-ray crystal structure of the crystalline hydrate form of Compound A. DETAILED DESCRIPTION OF THE INVENTION

[0027] (4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-methoxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecyne-4,1'-naphthalene]-16,16,18(7H,17H)-trione (Compound A), a salt thereof, or a solvate thereof: [ka] ((4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-hydroxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-4,1'-naphthalene]-16,16,18(7H,17H)-trione (Compound B), a salt thereof, or a solvate thereof: [ka] Disclosed herein is a method for the synthesis of the compound by methylation of the compound.

[0028] U.S. Pat. No. 10,300,075, the entirety of which is incorporated herein by reference, discloses synthetic procedures for synthesizing Mcl-1 inhibitors, such as Compound A. The synthesis for Compound A involves multiple steps, the last of which is the methylation of Compound B to form Compound A, as shown in Scheme 1, below. Scheme 1 [ka] Traditional methylation processes for forming compound A involve low temperatures, anhydrous reaction conditions, multiple charges of base (e.g., KHMDS) and MeI to achieve complete conversion of compound B, long aging times after addition of base and methyl iodide, buffered quench steps, and elaborate work-up steps such as silica gel chromatography. Furthermore, traditional methylation processes result in excessive impurities (e.g., dimethylated compounds, such as at the bridging nitrogen atom) that are difficult to remove during purification and lower yields. Traditional methylation processes also have challenges related to reproducibility and robustness.

[0029] In contrast, the method for methylating compound B, a salt thereof, or a solvate thereof to form compound A, as described herein, results in a greatly improved yield of compound A and a significant reduction in impurities (e.g., dimethylated products) in the final product. Furthermore, the method described herein is reproducible and robust, making it suitable for producing commercial quantities, such as kilogram quantities, of compound A. In particular, it has been found that the addition of water to the methylation reaction (e.g., stoichiometric addition of water) and / or the rapid addition of base and / or methyl halide provides superior results in terms of yield, purity, reproducibility, and robustness.

[0030] As disclosed herein, a method for synthesizing Compound A includes reacting (a) (i) a base selected from the group consisting of a non-nucleophilic base, an alkali metal hydride base, an alkali metal hydroxide base, an organolithium base, and any combination thereof, with (ii) Compound B, a salt, or a solvate thereof: [ka] mixing a solution comprising an organic solvent, including an ether solvent, a non-polar solvent, or any combination thereof, and water (wherein the molar ratio of water to compound B is in the range of about 0.1:1 to about 3:1) to form a mixture; and further mixing the mixture of step (a) with MeX (wherein X is a halogen, such as F, Cl, Br, or I), to form a mixture comprising compound A, as shown in Scheme 2 below. Scheme 2 [ka]

[0031] Further provided herein are crystalline hydrate forms of Compound A, pharmaceutical formulations thereof, and methods of treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical formulation.

[0032] The compounds disclosed herein may be identified herein by either their chemical structure and / or chemical name. In the event that the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

[0033] As known to those skilled in the art, compounds with a basic and an acidic moiety can exist as zwitterions. For example, compound A can be depicted as shown below: [ka] Or it can be depicted as a zwitterion as shown below. [ka] Because it is not possible to use the same structure to represent both forms, as used herein, reference to Compound A or the first structure shown above will also refer to the zwitterionic form shown in the second structure above. For example, the crystalline forms described herein are considered to have Compound A in zwitterionic form.

[0034] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formula, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.

[0035] As used herein, dashed bonds and bold bonds [ka] Chemical structures containing one or more stereocenters, as depicted, are meant to depict the absolute stereochemistry of the stereocenters present in the chemical structure. As used herein, bonds represented by simple line symbols do not denote stereogenic preference. Unless specifically indicated to the contrary, chemical structures containing one or more stereocenters illustrated herein without depicting absolute or relative stereochemistry encompass all possible stereoisomeric forms (e.g., diastereomers, enantiomers) of the compound and mixtures thereof. Structures with a single bold or dashed line and at least one additional simple line encompass a single enantiomeric series of all possible diastereomers.

[0036] The term "about" is intended to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term "about," unless expressly stated otherwise, regardless of whether the term is explicitly used. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0037] The term "compound," as used herein, is meant to include all depicted stereoisomers, geometric isomers, tautomers, and isotopes of the structure. A compound identified herein by name or structure as one particular tautomeric form is intended to encompass other tautomeric forms unless otherwise specified.

[0038] "Treatment" or "treating" refers to any treatment of a patient's illness, such as a) preventing the illness, i.e., not allowing clinical symptoms of the illness to develop; b) arresting the illness; c) slowing or arresting the progression of clinical symptoms; and / or d) relieving the illness, i.e., causing regression of clinical symptoms. Treatment of illnesses and disorders, as used herein, is also intended to include prophylactic administration of the pharmaceutical formulations described herein to a subject (i.e., an animal, preferably a mammal, most preferably a human) believed to be in need of treatment, such as for cancer.

[0039] The term "therapeutically effective amount" means an amount effective when administered to a human or non-human patient to treat a condition; for example, a therapeutically effective amount can be an amount sufficient to treat a condition or disorder responsive to myosin activation. A therapeutically effective amount can be ascertained empirically, for example, by analyzing blood levels of a chemical or by calculating therapeutic bioavailability.

[0040] "Pharmaceutically acceptable salts" include salts with inorganic acids, such as hydrochlorides (i.e., hydrochlorides), phosphates, diphosphates, hydrobromides, sulfates, sulfinates, nitrates, and the like; and salts with organic acids, such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate, salicylate, stearate, and acetate, HOOC-(CH2) nThese salts include, but are not limited to, alkanoates such as -COOH (where n is 0-4) and similar salts. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.

[0041] The term "hydrate" refers to a chemical compound formed by the interaction of water with a compound, such as, for example, a hemihydrate, monohydrate, dihydrate, trihydrate, etc. Solvates of Compound A used in the formulations herein are within the scope of the present invention. Hydrates, as used herein, can have variable amounts of water, such as 0.6 to 2 water molecules per Compound A molecule.

[0042] "Crystalline form" and "polymorph" may be used interchangeably herein and are meant to include all crystalline and amorphous forms of a compound, such as, for example, polymorphs, pseudopolymorphs, solvates, hydrates, non-solvated polymorphs (such as anhydrates), conformational polymorphs, and amorphous forms, and mixtures thereof, unless a specific crystalline or amorphous form is referenced.

[0043] Method for methylating compound B to form compound A In one aspect, provided herein is a method for preparing Compound A by methylation of Compound B, a salt thereof, or a solvate thereof. Compound A can be prepared from Compound B, a salt thereof, or a solvate thereof in two steps according to Scheme 2 above. In the first step (Step (a)), a base is mixed with a solution containing Compound B, a salt thereof, or a solvate thereof; an organic solvent, including an ether solvent, a non-polar solvent, or any combination thereof; and water to form a mixture. In the second step (Step (b)), the mixture from Step (a) is mixed with methyl halide (MeX) to form a mixture containing Compound A. In some embodiments, Compound B is provided as a solvate, such as a hydrate. In some embodiments, Compound B is provided as a salt. Providing a salt form of Compound B prior to methylation can result in increased solubility and consistent reaction results. In some embodiments, Compound B is prepared as Compound B': [ka] wherein M is an alkali metal. In embodiments, the alkali metal can be lithium, sodium, or potassium. In some embodiments, the alkali metal is potassium. Thus, in some embodiments, Compound A is provided as a salt having the structure ((4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-hydroxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a, as shown in Scheme 3 below (where M is an alkali metal). It can be prepared by methylating 8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-4,1'-naphthalene]-16,16,18(7H,17H)-trione potassium salt (compound B'). Scheme 3 [ka]

[0044] Process (a) The first step (step (a)) of the methylation process disclosed herein comprises mixing (i) a base selected from the group consisting of a non-nucleophilic base, an alkali metal hydride base, an alkali metal hydroxide base, an organolithium base, and any combination thereof; and (ii) a solution comprising Compound B, a salt, or a solvate thereof; an organic solvent, including an ether solvent, a non-polar solvent, or any combination thereof; and water.

[0045] The base can be any base capable of deprotonating the -OH group of Compound B. In some embodiments, the base can be selected from the group consisting of a non-nucleophilic base, an alkali metal hydride base, an alkali metal hydroxide base, an organolithium base, and any combination thereof. In some cases, the base can be a non-nucleophilic base. Suitable non-nucleophilic bases can include, for example, lithium hexamethyldisilazide ("HMDS"), sodium HMDS, potassium HMDS, lithium diisopropylamide, sodium diisopropylamide, potassium diisopropylamide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-amylate, sodium tert-amylate, potassium tert-amylate, 2,2,6,6-tetramethylpiperidine (TMP), LiTMP, 1,1,3,3-tetramethylguanidine (TMG), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene, and any combination thereof. In embodiments, the non-nucleophilic base can be lithium HMDS, sodium HMDS, potassium HMDS, and any combination thereof. In some embodiments, the base can be an alkali metal hydride base. Suitable alkali metal hydride bases can include, for example, LiH, NaH, KH, RbH, CsH, BeH2, MgH2, CaH2, SrH2, BaH2, and any combination thereof. In embodiments, the alkali metal hydride base can be LiH, NaH, KH, or any combination thereof. In some cases, the base can be an alkali metal hydroxide base. Suitable alkali metal hydroxide bases can include, for example, LiOH, NaOH, KOH, RbOH, CsOH, or any combination thereof. In some embodiments, the alkali metal hydroxide base can be LiOH, NaOH, KOH, or any combination thereof. In some cases, the base can be an organolithium base.Suitable organolithium bases can include, for example, methyllithium, n-butyllithium, sec-butyllithium, isopropyllithium, tert-butyllithium, phenyllithium, or any combination thereof. In some embodiments, the organolithium base can be methyllithium, n-butyllithium, phenyllithium, or any combination thereof. In some cases, the base can include lithium hexamethyldisilazide ("HMDS"), sodium HMDS, potassium HMDS, lithium diisopropylamide, sodium diisopropylamide, potassium diisopropylamide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-amylate, sodium tert-amylate, potassium tert-amylate, potassium hydride, sodium hydride, potassium hydroxide, sodium hydroxide, lithium hydroxide, 2,2,6,6-tetramethylpiperidine (TMP), LiTMP, n-butyllithium (n-BuLi), n-hexyllithium, 1,1,3,3-tetramethylguanidine (TMG), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene, or any combination thereof. In embodiments, the base can include lithium hexamethyldisilazide HMDS, sodium HMDS, potassium HMDS (KHMDS), or any combination thereof, hi embodiments, the base is KHMDS.

[0046] In embodiments, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is in the range of about 1:1 to about 5:1, or about 2:1 to about 5:1, or about 2:1 to about 4:1, or about 2.5:1 to about 4:1, or about 3:1 to about 3.5:1. In some embodiments, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof can be about 1:1, 1.5:1, 2:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 4:1, or 5:1. In some cases, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof can be about 3.2:1.

[0047] In embodiments, the organic solvent can include an ether solvent, a non-polar solvent, or any combination thereof. In some cases, the organic solvent can be an ether solvent. Suitable ether solvents can include, for example, tetrahydrofuran (THF), 2-methyltetrahydrofuran (MeTHF), cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, 1,4-dioxane, diethyl ether, diisopropyl ether, bis(2-methoxyethyl) ether, propylene glycol methyl ether, or any combination thereof. In embodiments, the ether solvent can be THF or 2-methyltetrahydrofuran. In some cases, the organic solvent can be a non-polar solvent. Suitable non-polar solvents can include, for example, hexane, pentane, toluene, benzene, heptane, xylene, and any combination thereof. In embodiments, the non-polar solvent can be toluene, hexane, heptane, or any combination thereof. In some cases, the organic solvent can be selected from the group consisting of THF, 2-methyltetrahydrofuran, cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, toluene, hexane, heptane, 1,4-dioxane, and any combination thereof. In some embodiments, the organic solvent comprises THF.

[0048] The methods provided herein involve adding water to a solution of Compound B, a salt thereof (e.g., Compound B'), or a solvate together with the organic solvent in step (a) before MeX is added in step (b). Adding water to step (a) of the method has been shown to significantly increase yield by 25% or more, improve the purity profile, and reduce aggregation of Compound B, a salt thereof (e.g., Compound B'), or a solvate during the methylation reaction. Without being bound by any particular theory, it is believed that the aggregation of Compound B, a salt thereof (e.g., Compound B'), or a solvate may occur due to the activation of an alkoxide (O) of one molecule of Compound B, a salt thereof (e.g., Compound B'), or a solvate. - ) moiety and a deprotonated sulfonamide (N) moiety in another molecule of Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof. - M + ) sites. Additionally, as the mixture of step (a) ages, the size of the aggregates increases. As a result, the activated alkoxide sites become sterically hindered, which can prevent MeX access to the activated hydroxyl groups and result in lower conversion rates. Aggregation in the anhydrous solution of step (a) occurs more rapidly than in solutions containing water. Without being bound by theory, it is believed that the addition of water in step (a) protects the activated alkoxide sites, in some embodiments, by forming weak bonds, thus initially slowing aggregation while simultaneously allowing MeX access for productive methylation. Thus, in some embodiments, the molar ratio of water to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof can be about 0.1:1 or greater. In some embodiments, the molar ratio of water to Compound B, a salt thereof (e.g., Compound B'), or solvate can be in the range of about 0.1:1 to about 3:1, or about 0.5:1 to about 3:1, or about 1:1 to about 3:1, or about 1.5:1 to about 3:1, or about 1:1 to about 2:1, or about 1.2:1 to about 1.8:1, or about 1.4:1 to about 1.6:1. In embodiments, the molar ratio of water to Compound B, a salt thereof (e.g., Compound B'), or solvate can be about 1.5:1.

[0049] The base can be added to the solution in step (a) over a period of time, or the base can be added to the solution in step (a) all at once. In some embodiments, the base is mixed with the solution in step (a) for a period of about 5 seconds to about 6 hours, or about 5 seconds to about 1 minute, or about 5 seconds to about 10 minutes, or about 5 seconds to about 1 hour, or about 5 minutes to about 1 hour, or about 5 minutes to about 3 hours, or about 30 minutes to about 2 hours, or about 30 minutes to about 6 hours, or 2 hours to about 4 hours, or about 2 hours to about 3 hours, about 4 hours to about 6 hours, or about 3 hours to about 5 hours. In some embodiments, the base is mixed with the solution in step (a) for a period of about 5 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, or about 6 hours. In some embodiments, the base can be mixed with the solution in step (a) all at once. In some cases, the base is mixed with the solution in step (a) within about 5 seconds, within about 4 seconds, within about 3 seconds, within about 2 seconds, or within about 1 second.

[0050] In embodiments, the mixture of step (a) can be stirred for about 1 second to about 12 hours, or about 1 second to about 6 hours, or about 1 second to about 1 hour, or about 1 second to about 20 minutes, or about 1 second to about 10 minutes, or about 1 second to 15 minutes, or about 5 minutes to about 1 hour, or about 10 minutes to 2 hours, or about 30 minutes to about 2 hours, or about 2 hours to about 6 hours, or about 2 hours to about 10 hours, or about 5 hours to about 10 hours, or about 6 hours to about 12 hours. In some embodiments, the mixture of step (a) can be stirred for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.

[0051] In embodiments, step (a) can occur at a temperature ranging from about 0° C. to about 40° C., or from about 15° C. to about 25° C. In embodiments, step (a) can occur at a temperature ranging from about 0° C. to about 40° C., or from about 15° C. to about 25° C. In some embodiments, step (a) can occur at room temperature, such as about 20° C.

[0052] Process (b) The second step (step (b)) of the methylation process disclosed herein involves combining the mixture of step (a) with MeX (where X is a halogen) to form a mixture comprising compound A.

[0053] X can be any halogen (e.g., F, Cl, Br, or I). In embodiments, X is fluoride. In some embodiments, X is chloride. In some embodiments, X is bromide. In some embodiments, X is iodide. In embodiments, the molar ratio of MeX (e.g., MeI) to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is in the range of about 1:1 to 10:1, or about 1:1 to about 5:1, or about 1:1 to about 4:1, or about 1:1 to about 3:1, or about 2:1 to about 3:1, or about 2.5:1 to about 2.9:1. In embodiments, the molar ratio of MeX (e.g., Mel) to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is about 1:1, 1.5:1, 2:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, 3.5:1, 4:1, 5:1, or 10:1. In some embodiments, the molar ratio of MeX (e.g., Mel) to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is about 2.7:1.

[0054] The MeX (e.g., MeI) can be mixed with the mixture of step (a) over a period of time, or the MeX can be mixed with the mixture of step (a) all at once. In some embodiments, the MeX is mixed with the mixture of step (a) over a period of about 1 second to about 6 hours, or about 1 second to about 1 hour, or about 1 second to about 30 minutes, or about 1 second to about 10 minutes, or about 1 minute to 1 hour, or about 30 minutes to about 2 hours, or about 1 hour to about 3 hours, or about 3 hours to about 6 hours. In embodiments, the MeX can be mixed with the mixture of step (a) over a period of about 5 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, or about 6 hours. In embodiments, the MeX can be mixed with the mixture of step (a) all at once. In some embodiments, the MeX can be mixed with the mixture of step (a) within about 5 seconds, within about 4 seconds, within about 3 seconds, within about 2 seconds, or within about 1 second.

[0055] In embodiments, the mixture in step (b) can be stirred for about 1 second to about 12 hours, or about 1 second to about 6 hours, or about 1 second to about 1 hour, or about 1 second to about 20 minutes, or about 1 second to about 10 minutes, or about 1 second to 15 minutes, or about 5 minutes to about 1 hour, or about 10 minutes to 2 hours, or about 30 minutes to about 2 hours, or about 2 hours to about 6 hours, or about 2 hours to about 10 hours, or about 5 hours to about 10 hours, or about 6 hours to about 12 hours. In some embodiments, the mixture in step (b) can be stirred for about 1 second, about 5 seconds, about 10 seconds, about 30 seconds, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.

[0056] In embodiments, step (b) can occur at a temperature ranging from about 0° C. to about 40° C., or from about 15° C. to about 25° C. In embodiments, step (b) can occur at a temperature ranging from about 0° C. to about 40° C., or from about 15° C. to about 25° C. In some embodiments, step (b) can occur at room temperature, such as about 20° C.

[0057] Isolation and purification of compound A The methods disclosed herein for methylating compound B, a salt thereof (e.g., compound B'), or a solvate thereof to form compound A can further include quenching the mixture of step (b) with a base. In embodiments, the base can be a secondary amine base. In some embodiments, the secondary amine base can be selected from the group consisting of N,N-diethylamine, morpholine, piperidine, pyrrolidine, piperazine, and any combination thereof. In some embodiments, the secondary amine base is N,N-diethylamine, morpholine, or any combination thereof. In embodiments, the molar ratio of the secondary amine base to compound B, a salt thereof (e.g., compound B'), or a solvate thereof can be in the range of about 12:1 to about 18:1, such as about 5:1 to about 25:1, or about 10:1 to about 20:1, or about 15:1.

[0058] Compound A can be isolated using specific methods suitable for those skilled in the art. In some embodiments, compound A can be isolated from the crude solution by filtration, concentrating the filtrate under vacuum, and polish-filtering the concentrated solution. Compound A can be purified by washing the polish-filtered solution with a base (e.g., 5N NaOH) and brine (e.g., 3×13 wt.% NaCl) solution. In some embodiments, compound A can be isolated by filtration and concentrating the filtrate under vacuum at about 40° C. from about 0.01 M to about 0.5 M. The concentrated solution can be further polish-filtered, and the polish-filtered solution can be washed with an alkali metal base (e.g., about 5N NaOH) and brine (e.g., 3×13 wt.% NaCl) solution.

[0059] Compound A can be further purified by any particular method suitable to one of skill in the art. In embodiments, compound A can be purified by distillation in an organic solvent or mixture of organic solvents, followed by crystallization from the organic solvent. Compound A can be filtered and dried under vacuum. In some embodiments, compound A can be purified by distillation in denatured ethanol with 0.5% to 5% v / v toluene (e.g., 2% v / v toluene) to concentrate the solution under vacuum. In embodiments, the distillation can be at a temperature ranging from 30°C to about 100°C, e.g., 50°C, and the solution can be concentrated by distillation to about 0.01 M to about 0.5 M (e.g., about 0.13 M). In embodiments, compound A can be crystallized in an acetic acid solution by charging the concentrated solution from the distillation with acetic acid. In embodiments, the acetic acid can be about 1 N to about 5 N (e.g., 3 N). In embodiments, crystallization of compound A is accomplished by heating a concentrated solution of compound A with acetic acid to a temperature ranging from about 75° C. to about 85° C. for a period of time (e.g., about 15 minutes), optionally seeding a concentrated solution of compound A with crystals of compound A, followed by adding acetic acid and heating the seeded solution at a temperature ranging from about 75° C. to about 85° C. for a period of time (e.g., about 15 minutes), followed by cooling the solution to about room temperature (e.g., 20° C.) and aging the solution at about room temperature for a period of time (e.g., one hour or more). The aged crystallization solution is then filtered to provide purified compound A as the crystalline hydrate form described herein. In embodiments, crystallization of compound A is accomplished by combining compound A with an ethanol / water mixture to form a crystallization mixture, optionally seeding a concentrated solution of compound A with crystals of compound A, heating the crystallization mixture to a temperature ranging from about 75° C. to about 85° C. for a period of time (e.g., about 15 minutes), followed by cooling the crystallization mixture to about room temperature. The crystallization solution is then filtered to provide purified Compound A as the crystalline hydrate form described herein.In embodiments, crystallization of Compound A is accomplished by combining Compound A with about 10 volumes of 95:5 ethanol / water to form a crystallization mixture, optionally seeding a concentrated solution of Compound A with crystals of Compound A, heating the crystallization mixture to a temperature ranging from about 75° C. to about 85° C. for a period of time (e.g., about 15 minutes), and subsequently cooling the crystallization mixture to about room temperature. The crystallization solution is then filtered to provide purified Compound A as the crystalline hydrate form described herein.

[0060] crystalline hydrate form Also provided herein are crystalline hydrate forms of Compound A. The crystalline hydrate forms of Compound A include: 13.57, 19.13, 20.39, 24.04, 25.54, 27.75, 30.09, 31.05, 36.84, 38.27, 39.48, 43.15, 49.53, 50.30, 51.84, 54.40, 56.15, 57.28, 57.78, 60.23, 61.80, 65.

[0039] A solid state crystalline cellulose obtained as described in the Examples, having peaks at 65, 78.05, 85.23, 115.91, 123.10, 124.60, 128.11, 130.53, 133.18, 133.87, 134.99, 139.72, 141.47, 143.08, 151.76, and 174.30 ± 0.5 ppm. 13 In some embodiments, the crystalline hydrate form of Compound A can be characterized by C NMR. ... 13 C NMR, where "substantially" means that the reported peaks can vary by ±0.5 ppm.

[0061] The crystalline hydrate of Compound A can be further characterized by an X-ray powder diffraction pattern, obtained as described in the Examples, having peaks at 10.3, 16.3, and 17.1±0.2 degrees 2θ using CuKα radiation. The crystalline hydrate form of Compound A can optionally be further characterized by an X-ray powder diffraction pattern having additional peaks at 8.23, 24.40, 25.03, 25.49, and 32.03±0.2 degrees 2θ using CuKα radiation. The crystalline hydrate form of Compound A can optionally be further characterized by an X-ray powder diffraction pattern having additional peaks at 14.4, 14.7, 15.9, 17.7, 18.1, 19.8, 20.9, 21.7, 21.9, and 25.0±0.2 degrees 2θ using CuKα radiation. In some embodiments, the crystalline hydrate form of Compound A has an X-ray powder diffraction pattern substantially as shown in Figure 3, where "substantially" means that the reported peaks can vary by ±0.2°. It is well known in the field of XRPD that relative peak heights in a spectrum depend on many factors, such as sample preparation and instrument geometry, while peak positions are relatively insensitive to experimental details.

[0062] A differential scanning calorimetry (DSC) thermograph was obtained for the crystalline hydrate form of Compound A, as described in the Examples. The DSC curve exhibits an endothermic transition at 221° C.±3° C. Thus, in some embodiments, the crystalline hydrate form of Compound A can be characterized by a DSC thermograph having an endothermic transition with an onset between 218° C. and 224° C. For example, in some embodiments, the crystalline hydrate form of Compound A is characterized by DSC as depicted in FIG. 4.

[0063] The crystalline hydrate form of Compound A can also be characterized by thermogravimetric analysis (TGA). Thus, the crystalline hydrate form of Compound A can be characterized by a weight loss ranging from about 0% to about 3% at an onset temperature of 218°C to 224°C. For example, the crystalline hydrate form of Compound A can be characterized by a weight loss of about 2% up to about 200°C. In some embodiments, the crystalline hydrate form of Compound A has a thermogravimetric analysis substantially as depicted in Figure 5, where "substantially" means that the reported TGA characteristics can vary by ±5°C.

[0064] The crystalline hydrate form of Compound A can be characterized by a moisture sorption profile. For example, in some embodiments, the crystalline hydrate form of Compound A is characterized by a moisture sorption profile as shown in Figure 6, which shows a 3.3% weight gain at 95% RH.

[0065] The crystalline hydrate form of Compound A is further characterized by a single crystal structure substantially as shown in Figure 8 or as described in the Examples.

[0066] Further provided herein is a pharmaceutical formulation comprising a crystalline hydrate form of Compound A as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical formulation is in the form of a tablet. In some embodiments, the pharmaceutical formulation is in the form of an immediate-release tablet.

[0067] Treatment method for subjects Further provided herein is a method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation as disclosed herein, in some embodiments, the cancer is multiple myeloma, non-Hodgkin's lymphoma, or acute myeloid leukemia.

[0068] Batch Chemistry In some embodiments, the methylation of compound B, a salt thereof (eg, compound B'), or a solvate thereof to form compound A can be accomplished by batch chemistry.

[0069] In a batch process, Compound A can be prepared from Compound B, a salt thereof (e.g., Compound B′), or a solvate in two steps according to Scheme 2 above and as previously described above.

[0070] In some embodiments, the first step (step (a)) of the batch process disclosed herein comprises mixing (i) a base selected from the group consisting of a non-nucleophilic base, an alkali metal hydride base, an alkali metal hydroxide base, an organolithium base, and any combination thereof; and (ii) a solution comprising Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof; an organic solvent, including an ethereal solvent, a nonpolar solvent, or any combination thereof; and water. In some embodiments, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof ranges from about 1:1 to about 5:1. In embodiments, the molar ratio of water to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof can be about 0.1:1 or greater. In some embodiments, the base can be added to the solution in step (a) over a period of time, or can be added all at once to the solution in step (a). In some embodiments, the mixture in step (a) can be stirred for about 1 second to about 12 hours. In embodiments, step (a) can occur at a temperature ranging from about 0°C to about 40°C.

[0071] In some embodiments, the first step (step (a)) of the batch process disclosed herein comprises mixing (i) a non-nucleophilic base; (ii) Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof; an ethereal solvent; and a solution comprising water. In some embodiments, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is in the range of about 2:1 to about 4:1. In embodiments, the molar ratio of water to Compound B, a salt thereof (Compound B'), or a solvate thereof can be about 1:1 to about 3:1. In embodiments, the base can be added to the solution in step (a) over a period of about 5 seconds to about 6 hours, or the base can be added to the solution in step (a) all at once, such as within about 5 seconds. In some embodiments, the mixture in step (a) can be stirred for about 1 second to about 1 hour. In some embodiments, step (a) can occur at a temperature ranging from about 15°C to about 35°C.

[0072] In some embodiments, the first step (step (a)) of the methylation process disclosed herein comprises mixing (i) potassium HMDS; (ii) Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof; THF; and a solution comprising water. In some embodiments, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is about 3.2:1. In embodiments, the molar ratio of water to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof can be about 1.5:1. In some embodiments, the base can be added to the solution in step (a) in one go, such as within about 5 seconds. In some embodiments, the mixture in step (a) can be stirred for about 1 second to about 1 minute. In some embodiments, step (a) can occur at room temperature.

[0073] In some embodiments, the second step (step (b)) of the methylation process disclosed herein comprises combining the mixture of step (a) with MeX (wherein X is a halogen) to form a mixture containing Compound A. In embodiments, the molar ratio of MeX to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is in the range of about 1:1 to about 4:1. In some embodiments, MeX can be combined with the mixture of step (a) over a period of about 5 seconds to about 6 hours, or all at once. In embodiments, the mixture of step (b) can be stirred for about 1 second to about 12 hours. In embodiments, step (b) can occur at a temperature ranging from about 0° C. to about 40° C.

[0074] In some embodiments, the second step (step (b)) of the methylation process disclosed herein comprises combining the mixture of step (a) with MeX (where X is Cl, Br, or I) to form a mixture containing Compound A. In embodiments, the molar ratio of MeX to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is in the range of about 2:1 to about 4:1. In some embodiments, MeX can be combined with the mixture of step (a) over a period of about 5 seconds to about 1 hour or all at once; in embodiments, the mixture of step (b) can be stirred for about 1 minute to about 1 hour. In embodiments, step (b) can occur at a temperature ranging from about 15°C to about 35°C.

[0075] In some embodiments, the second step (step (b)) of the methylation disclosed herein comprises combining the mixture of step (a) with MeI to form a mixture containing compound A. In embodiments, the molar ratio of MeI to compound B is about 2.7:1. In some embodiments, MeI can be mixed with the mixture of step (a) quickly, such as within about 5 seconds. In embodiments, the mixture of step (b) can be stirred for about 5 minutes. In embodiments, step (b) can occur at a temperature of about 20° C.

[0076] Flow Chemistry In some embodiments, the methylation of compound B, a salt thereof (e.g., compound B'), or a solvate to form compound A can be accomplished by flow chemistry. Flow chemistry allows for rapid mixing and residence times, as well as a small footprint and efficient throughput throughout production. Flow chemistry can be advantageous for processes synthesizing compound A because there is a narrow kinetic window available to produce compound A within drug substance specifications. Flow chemistry can also advantageously provide precise reagent stoichiometry in processes for synthesizing compound A. Rapid mixing times allow the compound B, a salt thereof (e.g., compound B'), or a solvate, and base deprotonation reaction (step (a)) to occur rapidly, on the order of seconds, so that compound B, a salt thereof (e.g., compound B'), or a solvate, is not aged with the base for an extended period of time. The compound B, a salt thereof (e.g., compound B'), or a solvate, and base stoichiometry can be adjusted by the flow rates of each reagent, which is another advantageous aspect of flow chemistry. This rapid residence time provides the necessary contact time and stoichiometry for deprotonation while preventing the lower conversions that result from long digestion times with strong bases, such as KHMDS.

[0077] In some embodiments, the methylation of compound B, a salt thereof (e.g., compound B'), or a solvate thereof to form compound A by flow chemistry can comprise a plug flow reactor, a continuous stirred tank reactor, or any combination thereof. In embodiments, the methylation of compound B, a salt thereof (e.g., compound B'), or a solvate thereof to form compound A by flow chemistry can comprise multiple continuous stirred tank reactors.

[0078] For example, Example 3 describes a flow chemistry process for the methylation of Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof to form Compound A. Figure 1 is a schematic of the flow chemistry process. Figure 1 shows a schematic in which a solution of Compound B' and water in an organic solvent along with a solution of KHMDS is added to a plug flow reactor, which then charges the activated solution to a continuous stirred tank reactor, which is followed by the addition of MeI to the continuous stirred tank reactor to form a solution containing Compound A, which flows to a collection and diethylamine quench tank.

[0079] As another example, Example 4 describes a flow chemistry process for the methylation of Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof to form Compound A, and FIG. 2 is a schematic representation of the flow chemistry process. FIG. 2 shows a schematic in which a solution of Compound B' and water in an organic solvent, along with a solution of KHMDS, is added to a continuous stirred tank reactor (CSTR0). This solution flows to another continuous stirred tank reactor (CSTR1) simultaneously with the addition of a solution of MeI. The solution in CSTR1 then flows to an aging continuous stirred tank reactor (CSTR2), and the solution in CSTR2 flows to a quenching continuous stirred tank reactor (CSTR3), where it is quenched with diethylamine. The quenched solution from CSTR3 flows to a collection tank as a settled solution containing Compound A.

[0080] In a flow chemistry process, compound A can be prepared from compound B, a salt thereof (e.g., compound B), or a solvate thereof in multiple steps. In the first step (step (a)), a base is mixed with a solution comprising compound B, a salt thereof (e.g., compound B'), or a solvate thereof; an organic solvent, including an ether solvent, a nonpolar solvent, or any combination thereof; and water, optionally in a plug flow reactor, to form a mixture. In the second step (step (b)), the mixture from step (a) and a methyl halide (MeX) are mixed, optionally in a continuous stirred tank reactor, to form a mixture comprising compound A.

[0081] In some embodiments, the first step (step (a)) of the flow chemistry process disclosed herein comprises combining (i) a base selected from the group consisting of a non-nucleophilic base, an alkali metal hydride base, an alkali metal hydroxide base, an organolithium base, and any combination thereof; and (ii) a solution comprising Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof; an organic solvent, including an ethereal solvent, a nonpolar solvent, or any combination thereof; and water, in a reactor, such as a plug flow reactor or a continuous stirred tank reactor. In some embodiments, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof ranges from about 1:1 to about 5:1. In embodiments, the molar ratio of water to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof can be about 0.1:1 or greater. In some embodiments, the base can be added to the solution in step (a) over a period of time, or can be added all at once to the solution in step (a). In some embodiments, the mixture of step (a) can be stirred for about 1 second to about 12 hours. In embodiments, step (a) can occur at a temperature ranging from about 0° C. to about 40° C.

[0082] In some embodiments, the first step (step (a)) of the flow chemistry process disclosed herein comprises mixing (i) a non-nucleophilic base; (ii) a solution comprising Compound B, a salt thereof, or a solvate thereof; an ethereal solvent; and water in a plug flow reactor or a continuous stirred tank reactor. In some embodiments, the molar ratio of base to Compound B, a salt thereof (e.g., Compound B'), or a solvate thereof is in the range of about 2:1 to about 4:1. In embodiments, the molar ratio of water to Compound B can be from about 1:1 to about 3:1. In some embodiments, the base can be added to the solution in step (a) in one go, such as within about 5 seconds. In some embodiments, the mixture in step (a) can be stirred for from about 1 second to about 1 hour. In some embodiments, step (a) can occur at a temperature ranging from about 15°C to about 35°C.

[0083] In some embodiments, the first step (step (a)) of the flow chemistry process disclosed herein comprises pumping (i) potassium HMDS; (ii) a solution comprising Compound B'; THF; and water into a plug flow reactor with a residence time of about 5 to about 25 seconds. In some embodiments, the molar ratio of base to Compound B, its salt (e.g., Compound B'), or solvate is about 3.2:1. In embodiments, the molar ratio of water to Compound B can be about 1.5:1. In some embodiments, the base can be added to the solution in step (a) in one go, such as within about 5 seconds. In some embodiments, the mixture of step (a) can optionally reside in the plug flow reactor for about 1 second to about 1 minute. In some embodiments, step (a) can occur at room temperature, such as 20°C.

[0084] In some embodiments, the first step (step (a)) of the flow chemistry process disclosed herein comprises combining (i) potassium HMDS; (ii) a solution comprising Compound B'; THF; and water into a continuous stirred tank reactor. In some embodiments, the molar ratio of base to Compound B, its salt (e.g., Compound B'), or solvate is about 3.2:1. In embodiments, the molar ratio of water to Compound B can be about 1.5:1. In embodiments, the base can be added to the solution in step (a) in one go, such as within about 5 seconds. In some embodiments, the mixture of step (a) can be stirred for about 5 minutes and then transferred to a second continuous stirred tank reactor. In embodiments, step (a) can occur at room temperature.

[0085] In some embodiments, the second step (step (b)) of the flow chemistry process disclosed herein comprises combining the mixture of step (a) with MeX (wherein X is a halogen) in a reactor, such as a continuously stirred tank reactor, to form a mixture comprising compound A. In some embodiments, the mixture comprising compound A can be transferred to one or more reactor tanks, such as a continuously stirred reactor tank. In embodiments, the molar ratio of MeX to compound B ranges from about 1:1 to about 10:1. In some embodiments, MeX can be combined with the mixture of step (a) over a period of about 1 second to about 6 hours or burst. In embodiments, the mixture of step (b) can be stirred for about 1 second to about 12 hours. In embodiments, step (b) can occur at a temperature ranging from about 0° C. to about 40° C.

[0086] In some embodiments, the second step (step (b)) of the flow chemistry process disclosed herein comprises combining the mixture of step (a) with MeX (where X is Cl, Br, or I) in a reactor, such as a continuously stirred reactor tank, to form a mixture containing compound A. In some embodiments, the mixture containing compound A is transferred to one or more reactor tanks, such as a continuously stirred reactor tank. In embodiments, the molar ratio of MeX to compound B ranges from about 2:1 to about 4:1. In some embodiments, MeX can be combined with the mixture of step (a) over a period of about 1 second to about 1 hour or burst. In embodiments, the mixture of step (b) can be stirred for about 1 minute to about 1 hour. In embodiments, step (b) can occur at a temperature ranging from about 15° C. to about 35° C.

[0087] In some embodiments, the second step (step (b)) of the flow chemistry process disclosed herein includes mixing the mixture of step (a) with MeX (where X is iodide) in a continuously stirred reactor tank to form a mixture containing compound A, and the mixture containing compound A is transferred to a second continuously stirred reactor tank. In embodiments, the molar ratio of MeX to compound B is about 2.7:1. In some embodiments, the MeX can be mixed with the mixture of step (a) in one go, such as within about 5 seconds. In embodiments, the mixture of step (b) can be stirred for about 5 minutes, then transferred to the second continuously stirred reactor tank and stirred again for about 5 minutes. In embodiments, step (b) can occur at a temperature of about 20° C.

[0088] In embodiments, the flow chemistry process for methylating compound B can have a third step. In the third step, the mixture from step (b) can be added to a reactor, such as a continuous stirred tank reactor, and quenched with a base (e.g., a secondary amine base). In embodiments, the mixture from the third step can be stirred for about 1 minute to about 48 hours. In embodiments, step (b) can occur at a temperature ranging from about 0°C to about 40°C. In some embodiments, the third step comprises transferring the mixture from step (b) to a new reactor, such as a continuous stirred tank reactor, and quenching with a secondary amine base. In embodiments, the mixture from the third step can be stirred for 1 minute to about 1 hour. In embodiments, step (b) can occur at a temperature ranging from about 15°C to about 35°C. In some embodiments, the third step comprises transferring the mixture from step (b) to a continuous stirred tank reactor and quenching with diethylamine. In embodiments, the mixture from the third step can be stirred for about 5 minutes. In embodiments, step (b) can occur at a temperature of about 20° C., or at room temperature.

[0089] Preparation of Compound B' Compound A can be prepared by methylating a salt form of compound B, such as compound B'. In certain embodiments, it can be advantageous to synthesize compound A from a salt form of compound B, such as compound B'. In some embodiments, compound B, which is a free acid, can have low solubility in certain solvents, which can present challenges for reaction reproducibility. Compound B' can provide improved reproducibility and consistency because it is more easily solubilized in certain solvents. Compound B' can be prepared by mixing compound B with a base and an organic solvent to form a mixture containing compound B'. In embodiments, the base can be an alkali hydroxide base. In embodiments, the organic solvent can be selected from the group consisting of an ether solvent, a non-polar solvent, and any combination thereof.

[0090] In embodiments, the alkali hydroxide base can be selected from the group consisting of potassium hydroxide, sodium hydroxide, lithium hydroxide, and any combination thereof. In some embodiments, the alkali hydroxide base is potassium hydroxide. In embodiments, the molar ratio of alkali hydroxide base to compound B ranges from about 0.5:1 to about 10:1, or from about 0.5:1 to about 5:1, or from about 0.5:1 to about 3:1, or from about 0.5:1 to about 2:1, or from about 1:1 to about 3:1, or from about 1:1 to about 2:1. In embodiments, the molar ratio of alkali hydroxide base to compound B is about 0.5:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In embodiments, the molar ratio of alkali hydroxide base to compound B is about 1.5:1.

[0091] In embodiments, the organic solvent can be selected from the group consisting of an ether solvent, a non-polar solvent, and any combination thereof. In some cases, the organic solvent can be an ether solvent. Suitable ether solvents can include, for example, tetrahydrofuran (THF), 2-methyltetrahydrofuran (MeTHF), cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, 1,4-dioxane, diethyl ether, diisopropyl ether, bis(2-methoxyethyl) ether, propylene glycol methyl ether, and any combination thereof. In embodiments, the ether solvent can be THF or 2-methyltetrahydrofuran. In some cases, the organic solvent can be a non-polar solvent. Suitable non-polar solvents can include, for example, hexane, pentane, toluene, benzene, heptane, xylene, and any combination thereof. In embodiments, the non-polar solvent can be toluene, hexane, heptane, or any combination thereof. In embodiments, the organic solvent can be selected from the group consisting of THF, 2-methyltetrahydrofuran, cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, toluene, hexane, heptane, 1,4-dioxane, and any combination thereof, hi some embodiments, the organic solvent is THF.

[0092] In embodiments, the mixture containing compound B' can be stirred for about 1 hour to about 48 hours, or about 2 hours to about 6 hours, or about 2 hours to about 10 hours, or about 5 hours to about 10 hours, or about 6 hours to about 12 hours, or about 12 hours to about 24 hours, or about 15 hours to about 24 hours, or about 10 hours to about 20 hours, or about 24 hours to about 48 hours, or about 30 hours to about 40 hours, or about 30 hours to about 48 hours, or about 40 hours to about 48 hours. In some embodiments, the mixture containing compound B' can be stirred for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours, about 15 hours, about 20 hours, about 24 hours, about 30 hours, about 35 hours, about 40 hours, or about 48 hours.

[0093] Other embodiments While the present disclosure is to be read in conjunction with the detailed description thereof, it should be understood that the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims, for example, as set forth in Examples 1-6. [Example]

[0094] The following examples are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0095] Materials and Methods Unless otherwise stated, commercially available reagents are used as is without further purification. A 1.0 M solution of MeI in THF is prepared by weight. Batch and flow chemistry equipment (reactors, tubing, pumps, connections and accessories) are from commercial suppliers.

[0096] The synthesis of the starting material (Compound B) for the following synthetic method is disclosed in U.S. Patent No. 10,300,075. The starting materials, intermediates, and final products of the reactions can be isolated and purified, if necessary, using conventional techniques, including, but not limited to, filtration, distillation, crystallization, chromatography, etc. Such materials can be characterized using conventional means, including physical constants and spectral data.

[0097] Unless specified to the contrary, the reactions described herein are conducted at atmospheric pressure and at temperatures ranging from about -78°C to about 150°C, or from about 0°C to about 50°C, or from about 15°C to about 25°C.

[0098] Example 1: Preparation of Compound B' from Compound B [ka] ((4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-hydroxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-4,1'-naphthalene]-16,16,18(7H,17H)-trione potassium salt (chemical Compound B': To a round-bottom flask containing solid potassium hydroxide (28 g, 0.43 mol, 1.5 eq.) was added compound B (214 g, 0.285 mol, 1.0 eq.), followed by anhydrous tetrahydrofuran (2.8 L, 0.10 M). The solution was stirred at 20 °C for at least 12 hours. The solution was then filtered, rinsed with tetrahydrofuran (211 mL), and the filtrates were combined. The filtrate was azeotropically dried by distillation to yield a 0.090-0.10 M solution in THF containing 1.4-1.6 eq. of water. The compound B' solution was then used directly in the next step. 1H NMR(600MHz,THF-d8):δ 7.726(d,J=8.4Hz,1H),7.314(br s,1H),7.181(br d,J=7.5Hz,1H),7.074(dd,J=8.4,2.0Hz,1H),7.010(d,J=2.0Hz,1H),6.569(br d,J=7.5Hz,1H),6.080(m,1H),5.705(br d,J=15.4Hz,1H),4.032(br d,J=13.6Hz,1H),3.893(m,2H),3.662(m,1H)3.639(m,1H),3.317(br d,J=14.1Hz,1H),2.976(m,1H),2.957(m,1H),2.741(m,1H),2.709(m,1H),2.679(m,1H),2.641(br d,J=10.5Hz,1H),2.455(br d,J=9.9Hz,1H),2.433(m,1H),2.419(m,1H),2.307(m,1H),2.242(m,1H),2.193(m,1H),2.178(m,2H),2.121(m,1H),2.055(m,1H),1.956(m,1H),1.929(m,1H),1.877(m,1H),1.868(m,2H),1.859(m,1H),1.811(m,1H),1.652(m,1H),1.639(m,1H),1.630(m,1H),1.520(m,1H),1.482(m,2H),1.340(m,1H),1.299(m,1H),1.239(br d,J=7.0Hz,3H),1.197(m,1H),1.076(m,1H),0.948(br d,J=6.6Hz,3H); 13 C NMR(151MHz,THF-d8):δ 175.4,151.4,141.7,140.6,140.0,135.8,134.2,132.6,131.0,129.9,129.2,127.4,120.5,120.3,116.3,81.1,76.3,67.1,63.2,62.3,61.3,60.9,56.9,56.5,56.3,49.5,43.6,38.9,35.8,34.6,31.2,30.9,29.3,27.5,27.0,25.3,22.4,20.3,17.1,8.1.

[0099] Example 2: Batch preparation of Compound A from Compound B' [ka] A solution of compound B' in THF containing 1.5 equivalents of water is prepared according to Example 1.

[0100] To a solution of compound B' (100 mg, 0.1 mmol, 1.0 equiv., containing 1.5±0.1 equiv. of water) in THF (1.3 mL, 13 vol.) was added anhydrous THF (1.2 mL, 12 vol.). 1.0 M KHMDS in THF (0.32 mL, 0.32 mmol, 3.2 equiv.) was then rapidly charged in one go to the stirring solution of compound B' within 15 seconds. Immediately after the KHMDS solution was charged, a 1.0 M solution of MeI in THF (0.27 mL, 0.27 mmol, 2.7 equiv.) was then rapidly charged in one go to the stirring reaction mixture within 15 seconds. After stirring for 5 minutes, the reaction was quenched with diethylamine (0.16 mL, 1.5 mmol, 15.0 equiv.).

[0101] Example 3: Flow Chemistry Procedure 1 - Plug Flow Reactor and Continuously Stirred Tank Reactor Methylation Reaction Flow Process (4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-methoxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1,19-etheno-16l6-cyclobuta[i][1,4] Oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecyne-4,1'-naphthalene]-16,16,18(7H,17H)-trione (Compound A): A solution of Compound B' in THF (1.0 equivalent, 80 g / min), a 1.0 M solution of KHMDS in THF (3.2 equivalents), and anhydrous THF (1.4 L) were pumped into a plug flow reactor (PFR) at 20 °C with a residence time of 15 seconds. A solution of Compound B' in THF containing 1.5 equivalents of water was prepared from Compound B according to Example 1. The PFR effluent and a 1.0 M solution of iodomethane in THF (2.7 equivalents) were simultaneously added to Continuous Stirred Tank Reactor 1 (CSTR1). The reaction slurry was aged in CSTR1 for 5 minutes at 20 °C and then transferred to a second reactor (CSTR2). The slurry was aged in CSTR2 at 20° C. for 5 minutes. The resulting slurry was transferred from CSTR2 to CSTR3. N,N-diethylamine (15 equivalents) was added simultaneously in CSTR3. The slurry was aged in CSTR3 at 20° C. for 5 minutes. The crude stream was collected in fractions from CSTR3 for analysis. The collected fractions containing the crude stream at greater than 95% conversion were carried through the isolation process. FIG. 1 shows an illustrative flow process diagram of this process.

[0102] Example 4: Flow Chemistry Procedure 2-4× Continuous Stirred Tank Reactor Methylation Reaction Flow Process (4S,7aR,9aR,10R,11E,14S,15R)-6'-chloro-10-methoxy-14,15-dimethyl-10-{[(9aR)-octahydro-2H-pyrido[1,2-a]pyrazin-2-yl]methyl}-3',4',7a,8,9,9a,10,13,14,15-decahydro-2'H,3H,5H-spiro[1 ,19-Etheno-1616-cyclobuta[i][1,4]oxazepino[3,4-f][1,2,7]thiadiazacyclohexadecine-4,1'-naphthalene]-16,16,18(7H,17H)-trione (Compound A): A solution of Compound B' in THF (116 g, 0.15 mol, 1.0 eq) was diluted with anhydrous THF (1.4 L). A solution of Compound B' in THF containing 1.5 eq of water was prepared from Compound B according to Example 1. Compound B' in THF (1.0 eq, 16.7 mL / min) and a 1.0 M solution of KHMDS (3.2 eq) in THF were added simultaneously to CSTR0. The resulting solution was aged in CSTR0 at 20 °C for 5 min and then transferred to CSTR1. A 1.0 M solution of iodomethane in THF (2.7 eq) was added simultaneously to CSTR1. The reaction slurry was aged in CSTR1 at 20° C. for 5 minutes and then transferred from CSTR1 to CSTR2. The slurry was aged in CSTR2 at 20° C. for 5 minutes and then transferred from CSTR2 to CSTR3. N,N-diethylamine (15 equivalents) was added simultaneously in CSTR3. The slurry was aged in CSTR3 at 20° C. for 5 minutes. The crude stream was collected in fractions from CSTR3 for analysis. The collected fractions containing the crude stream at greater than 95% conversion were carried through the isolation process. Figure 2 shows an illustrative process flow diagram of this process.

[0103] Example 5: General isolation process (e.g., for Examples 2, 3, and 4) Compound A was isolated from the crude solutions of Examples 2, 3, and 4 as follows: The crude solution was filtered at 20°C. The solution was then concentrated under vacuum at 40°C to approximately 0.13 M. The concentrated solution was polish filtered. The filtered solution was charged with 51 mL of 5N NaOH, followed by 206 mL of 13 wt% NaCl solution. The solution was stirred at 20°C for at least 5 minutes. Agitation was stopped, and the phases were allowed to separate at 20°C for at least 5 minutes. The aqueous layer was removed. 206 mL of 13 wt% NaCl solution was charged to the organic layer, and the batch was stirred at 20°C for at least 5 minutes. Agitation was stopped, and the phases were allowed to separate at 20°C for at least 5 minutes. The aqueous layer was removed. 946 mL of 2% v / v toluene in denatured ethanol was charged to the organic layer. The batch was distilled under vacuum at 50°C to approximately 0.13 M. 2% v / v toluene in denatured ethanol (997 mL) was charged to the distilled solution, and the batch was distilled under vacuum at 50°C to approximately 0.13 M. Another charge of 2% v / v toluene in denatured ethanol (997 mL) was charged to the distilled solution, and the distillation was repeated once more. The batch was polish filtered and then heated to 80±5°C. After reaching temperature, 3N AcOH (22 mL, 0.14 mol, 0.5 equiv) was charged to the heated solution. The solution was aged for at least 15 minutes at 80±5°C. In a separate container, a slurry of Compound A seeds (0.51 g) in 2% v / v toluene in denatured ethanol (2.6 mL) was prepared. The seed slurry was then charged to the heated solution. The solution was aged for at least 15 minutes at 80±5°C. 3N AcOH (108 mL, 0.70 mol, 2.5 equiv) was then charged over a period of 1 h at 80° C. The batch was then slowly cooled to 20° C. over a period of 1 h. The slurry was aged for at least an additional 1 h at 20° C., followed by filtration of the solid. The cake was washed with 2% v / v toluene in denatured ethanol (2×308 mL). The filtered solid was dried under vacuum at 65° C. for at least 12 h to give Compound A (75.6 g, 96.1 wt %, 76% yield): IR (thin film, cm -1):2936.63,1507.87,1341.57,1308.78,1257.25,1212.75,1184.65,1095.64,1008.98,812.23,777.10,568.64; 1 H NMR(600.13MHz,DMSO-d6):δ 7.671(d,J=8.5Hz,1H),7.241(dd,J=8.5,2.4Hz,1H),7.190(br d,J=1.9Hz,1H),7.153(d,J=2.4Hz,1H),7.018(dd,J=8.1,1.9Hz,1H),6.825(d,J=8.1Hz,1H),5.633(dt,J=16.2,5.5Hz,1H),5.430(br d,J=16.2Hz,1H),4.031(d,J=12.3Hz,1H),3.981(d,J=12.3Hz,1H),3.874(br d,J=15.0Hz,1H),3.810(q,J=6.5Hz,1H),3.579(d,J=14.2Hz,1H),3.254(s,3H),3.212(d,J=14.2Hz,1H),3.019(br d,J=10.7Hz,1H),2.953(br dd,J=15.0,10.4Hz,1H),2.907(m,1H),2.796(m,1H),2.793(m,1H),2.717(m,1H),2.610(m,1H),2.580(m,1H),2.489(m,1H),2.447(br d,J=14.2Hz,1H),2.431(m,1H),2.303(m,1H),2.300(m,1H),2.268(br d,J=14.2Hz,1H),2.253(m,1H),2.219(m,1H),2.202(m,1H),2.059(m,1H),2.031(m,1H),2.001(m,1H),1.858(m,2H),1.781(m,1H),1.688(br d,J=15.3Hz,1H),1.654(m,1H),1.634(m,1H),1.612(m,1H),1.524(m,1H),1.519(m,1H),1.445(m,1H),1.403(m,1H),1.303(m,1H),1.227(d,J=7.1Hz,3H),1.191(m,1H),0.945(d,J=6.7Hz,3H); 13C NMR (150.90 MHz, DMSO-d): δ 169.0,150.5,139.8,139.1,130.6,130.2,129.3,128.7,127.9,126.0 ,119.0,117.2,115.4,80.9,79.5,60.8,59.9,59.6,58.9,57.9,55.7, HRMS(ESI):C42H57ClN4O5S+H Calculated value for 765.38, Measured value: 765.38.

[0104] Example 6 - Stoichiometric addition of water The method of Example 2 was followed, except that the amount of water and the amount of base used were varied in each of the three reactions. Various equivalents of water (0.6 to 3.0 equivalents) were tested to determine the amount of water tolerated in the reaction and the amount required for the reaction to proceed at greater than 98% conversion (Table 1). As long as the equivalents of base offset the increase in the equivalents of water, the reaction proceeded at greater than 97% conversion. Using more than 1.0 equivalent of water provided greater than 99% conversion and less than 0.05% dimethyl impurity. Without water, inconsistencies were observed in the conversion to Compound A across replicate trials.

[0105] [Table 1]

[0106] Example 7 - Rapid addition of KHMDS to Compound B' followed by immediate addition of MeI The rate of addition of both KHMDS and MeI was tested to determine whether the rate of addition of either component played a role in reaction efficiency (Table 2). A more efficient reaction was found to occur when the addition of KHMDS was rapid (within seconds). A decrease in conversion was observed when KHMDS was added to compound B' over 20 minutes. The rate of addition of MeI did not appear to affect reaction conversion.

[0107] [Table 2]

[0108] Example 8: Preparation and characterization of crystalline hydrate forms of Compound A A crystalline hydrate form of Compound A was formed by combining Compound A with approximately 10 volumes of 95:5 ethanol / water, thermocycled to 70° C. for 15 minutes in a sealed vial, and then cooled.

[0109] X-ray powder diffraction: X-ray powder diffraction data were obtained on a PANalytical X'Pert PRO X-ray diffraction system equipped with a RTMS detector. Samples were scanned in continuous mode from 5 to 45° (2θ) using CuKα radiation (1.54 Å) at 45 kV and 40 mA with a step size of 0.0334°. The incident beam path included a 0.02 Radsolar slit, a 15 mm mask, a 4° fixed anti-scatter slit, and a programmable divergence slit. The diffraction beam included a 0.02 Radsolar slit, a programmable anti-scatter slit, and a 0.02 mm nickel filter. Samples were prepared on a low-background sample holder and placed on a rotation stage with a 2-second rotation time. For variable temperature studies, samples were prepared on a flat sample holder and placed on a TTK-450 temperature-controlled stage. For variable humidity studies, a modular humidity generator (ProUmid) was used to control the atmosphere in the THC humidity sample chamber. The XRPD pattern of the crystalline hydrate form material of Compound A is shown in FIG. 7 and the XRPD peaks are listed in Table 3.

[0110] Thermal Analysis: Differential scanning calorimetry (DSC) was performed on a TA Instruments Q1000 / 2000 calorimeter in aluminum Tzero pans under dry nitrogen flowing at 50 ml / min. The DSC of the crystalline hydrate form of Compound A is shown in Figure 4. Thermogravimetric analysis (TGA) was performed on a TA Instruments Q500 analyzer in platinum pans under dry nitrogen flowing at 60 ml / min. The DSC and TGA of the crystalline hydrate form of Compound A are shown in Figures 4 and 5.

[0111] Moisture Sorption: Moisture sorption data were collected using a Surface Measurement Systems DVS Advantage instrument. Equilibrium criteria were set at ±0.001% weight change over 10 minutes with a maximum equilibration time of 360 minutes. The moisture sorption profile of the crystalline hydrate form of Compound A is shown in Figure 6.

[0112] [Table 3]

[0113] [Table 4]

[0114] Single Crystal Data: A dry powder sample of Compound A crystalline hydrate form was used for single crystal structure determination. The specimen selected for data collection had approximately dimensions of 0.002 x 0.008 x 0.025 mm 3 The crystals were needle-shaped crystals of 1000 nm. The crystals were mounted in a MiTeGen™ fixture with mineral oil (STP Oil Treatment). The first diffraction pattern showed that the crystals were of marginal quality, producing smeared, elongated, and split reflections and only weakly diffracting.

[0115] Diffraction data (φ- and ω-scans) were collected at 100 K on a Bruker-AXS X8 Kappa diffractometer coupled to a Bruker APEX2 CCD detector using CuKα radiation (λ = 1.54178 Å) from a 1 μS microsource. Data reduction was performed using the program SAINT [1], and equivalent-based semi-empirical absorption corrections were performed using the program SADABS [2]. A summary of the crystal properties and data / fine-tuning statistics is shown in Table 5.

[0116] The structure of Compound A crystalline hydrate was determined at 100 K in the monoclinic chiral space group P21 with one molecule of Compound A and 80% water molecules in the asymmetric unit.

[0117] [Table 5]

[0118] The foregoing description is set forth for clarity of understanding only, and no unnecessary limitations should be understood therefrom, since modifications within the scope of the invention may be apparent to those skilled in the art.

[0119] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0120] Throughout this specification, when a composition is described as comprising ingredients or raw materials, it is understood that the composition can also consist essentially of, or consist of, any combination of the listed ingredients or raw materials, unless otherwise stated. Similarly, when a process is described as comprising particular steps, it is understood that the process can also consist essentially of, or consist of, any combination of the listed steps, unless otherwise stated. The inventions illustratively disclosed herein may suitably be practiced in the absence of any element or step not specifically disclosed herein.

[0121] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope and spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0122] The methods disclosed herein, and their individual steps, can be performed manually and / or with the aid of automation provided by electronic equipment. While the methods have been described with reference to specific embodiments, those skilled in the art will readily understand that other ways of performing the acts associated with the methods may be used. For example, the order of various steps may be changed without departing from the scope or spirit of the methods, unless otherwise noted. In addition, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

[0123] In the context of this disclosure (especially in the context of the claims), the use of the terms "a," "an," "the," and similar referents should be construed to include both the singular and the plural unless otherwise stated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein by reference as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended to better explain the disclosure herein and is not a limitation on the scope of the disclosure herein unless otherwise stated. No language herein should be construed as indicating any non-claimed element as essential to the practice of the disclosure herein.

[0124] All patents, publications, and references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict between the present disclosure and the incorporated patents, publications, and references, the present disclosure shall control.

Claims

1. Compound A, a salt or solvate thereof: 【Chemical 1】 A method for synthesizing (a) (i) a base selected from the group consisting of a non-nucleophilic base, an alkali metal hydride base, an alkali metal hydroxide base, an organolithium base, and any combination thereof; (ii) Compound B, a salt or solvate thereof: 【Chemistry 2】 and mixing a solution comprising an organic solvent, including an ether solvent, a non-polar solvent, or any combination thereof; and water, wherein the molar ratio of water to Compound B is in the range of about 0.1:1 to about 3:1, to form a mixture; (b) combining the mixture of step (a) with MeX, where X is a halogen, to form a mixture comprising Compound A; A method comprising:

2. 2. The method of claim 1, wherein the base comprises lithium hexamethyldisilazide ("HMDS"), sodium HMDS, potassium HMDS, lithium diisopropylamide, sodium diisopropylamide, potassium diisopropylamide, lithium tert-butoxide, sodium tert-butoxide, potassium tert-butoxide, lithium tert-amylate, sodium tert-amylate, potassium tert-amylate, potassium hydride, sodium hydride, potassium hydroxide, sodium hydroxide, lithium hydroxide, 2,2,6,6-tetramethylpiperidine (TMP), LiTMP, n-butyllithium (n-BuLi), n-hexyllithium, 1,1,3,3-tetramethylguanidine (TMG), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene, or any combination thereof.

3. 3. The method of claim 2, wherein the base comprises lithium hexamethyldisilazide HMDS, sodium HMDS, potassium HMDS, or any combination thereof.

4. 4. The method of claim 3, wherein the base comprises potassium hexamethyldisilazide ("KHMDS").

5. 5. The method of claim 1, wherein the molar ratio of base to compound B ranges from about 1:1 to about 5:

1.

6. 6. The method of claim 5, wherein the molar ratio of the base to compound B ranges from about 2.5:1 to about 4:

1.

7. 6. The method of claim 5, wherein the molar ratio of the base to compound B is from about 3.0:1 to about 3.5:

1.

8. 6. The method of claim 5, wherein the molar ratio of the base to compound B is about 3.2:

1.

9. 9. The method of any one of claims 1 to 8, wherein the organic solvent is selected from the group consisting of tetrahydrofuran ("THF"), 2-methyltetrahydrofuran, cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, toluene, hexane, heptane, 1,4-dioxane, and combinations thereof.

10. 10. The method of claim 9, wherein the organic solvent comprises THF.

11. 11. The method of any one of claims 1 to 10, wherein the molar ratio of water to compound B ranges from about 0.5:1 to about 3:

1.

12. 12. The method of claim 11, wherein the molar ratio of water to compound B ranges from about 1:1 to about 3:

1.

13. 12. The method of claim 11, wherein the molar ratio of water to compound B is from about 1.4:1 to about 1.6:

1.

14. 14. The method of any one of claims 1 to 13, wherein X is iodide.

15. 15. The method of any one of claims 1 to 14, wherein the molar ratio of MeX to compound B ranges from about 1:1 to about 4:

1.

16. 16. The method of claim 15, wherein the molar ratio of MeX to compound B is about 2.7:

1.

17. 17. The method of any one of claims 1 to 16, wherein the base is mixed with the solution in step (a) for a period of from about 5 seconds to about 6 hours.

18. 17. The method of any one of claims 1 to 16, wherein the base is mixed with the solution in step (a) for no more than 5 seconds.

19. 20. The method of claim 18, wherein the base is mixed with the solution in step (a) for 1 second or less.

20. 20. The method of any one of claims 1 to 19, wherein the mixture of step (a) is stirred for about 1 second to about 12 hours.

21. 21. The method of claim 20, wherein the mixture of step (a) is stirred for about 1 second to about 20 minutes.

22. 22. The method of any one of claims 1 to 21, wherein the MeX is mixed with the mixture of step (a) for a period of from about 1 second to about 6 hours.

23. 22. The method of any one of claims 1 to 21, wherein the MeX is mixed with the mixture of step (a) for no more than 5 seconds.

24. 24. The method of claim 23, wherein the MeX is mixed with the mixture of step (a) for 1 second or less.

25. 25. The method of any one of claims 1 to 24, wherein the mixture of step (b) is stirred for about 1 minute to 12 hours.

26. 26. The method of claim 25, wherein the mixture of step (b) is stirred for about 1 minute to about 20 minutes.

27. The method of any one of claims 1 to 26, wherein compound B is a solvate.

28. Compound B is compound B': 【Chemistry 3】 wherein M is an alkali metal. The method according to any one of claims 1 to 27, wherein the compound is a salt having the structure:

29. 29. The method of claim 28, wherein the alkali metal is lithium, sodium, or potassium.

30. 30. The method of claim 29, wherein the alkali metal is potassium.

31. 31. The method of any one of claims 28 to 30, wherein compound B' is prepared by mixing compound B with an alkali hydroxide base and an organic solvent selected from the group consisting of an ether solvent, a non-polar solvent, and any combination thereof to form a mixture comprising compound B'.

32. 32. The method of claim 31 , wherein the alkali hydroxide base is selected from the group consisting of potassium hydroxide, sodium hydroxide, lithium hydroxide, and combinations thereof.

33. 33. The method of claim 31 or 32, wherein the molar ratio of alkali hydroxide base to compound B ranges from about 0.5:1 to about 3:

1.

34. 34. The method of claim 33, wherein the molar ratio of the alkali hydroxide base to compound B is about 1.5:

1.

35. 35. The method of any one of claims 29 to 34, wherein the organic solvent is selected from the group consisting of tetrahydrofuran ("THF"), 2-methyltetrahydrofuran, cyclopentyl methyl ether, tert-butyl methyl ether, 1,2-dimethoxyethane, toluene, hexane, heptane, 1,4-dioxane, and combinations thereof.

36. 36. The method of claim 35, wherein the organic solvent comprises THF.

37. 37. The method of any one of claims 31 to 36, wherein the mixture containing compound B' is stirred for about 1 hour to about 48 hours.

38. 38. The method of any one of claims 1 to 37, further comprising quenching the mixture of step (b) with a secondary amine base.

39. 39. The method of claim 38, wherein the secondary amine base is selected from the group consisting of N,N-diethylamine, morpholine, piperidine, pyrrolidine, piperazine, and combinations thereof.

40. 40. The method of any one of claims 1 to 39, wherein each mixing step occurs at a temperature in the range of from about 0°C to about 40°C.

41. 41. The method of claim 40, wherein the temperature of each mixing step ranges from about 15°C to about 25°C.

42. 13.57, 19.13, 20.39, 24.04, 25.54, 27.75, 30.09, 31.05, 36.84, 38.27, 39.48, 43.15, 49.53, 50.30, 51.84, 54.40, 56.15, 57.28, 57.78, 60.23, 61.80, 65.65, 78.05, 85.23, 115.91, 123.10, 124.60, 128.11, 130.53, 133.18, 133.87, 134.99, 139.72, 141.47, 143.08, 151.76, and 174.30 ± 0.5 ppm solid state １３ A crystalline hydrate form of Compound A, characterized by a C NMR peak.

43. 43. The crystalline hydrate form of Compound A of claim 42, further characterized by XRPD pattern peaks at 10.3, 16.3, and 17.1±0.2 degrees 2θ using CuKα radiation.

44. 44. The crystalline hydrate form of Compound A of claim 43, further characterized by XRPD pattern peaks at 8.1, 10.7, 12.5, 13.3, 15.1, and 20.3±0.2 degrees 2θ using CuKα radiation.

45. 45. The crystalline hydrate form of Compound A of claim 44, further characterized by XRPD pattern peaks at 14.4, 14.7, 15.9, 17.7, 18.1, 19.8, 20.9, 21.7, 21.9, and 25.0±0.2 degrees 2θ using CuKα radiation.

46. 46. ​​The crystalline hydrate form of Compound A according to any one of claims 42 to 45, having an XRPD pattern substantially as shown in Figure 3.

47. 47. The crystalline hydrate form of Compound A of any one of claims 42 to 46, having an endothermic transition at 218°C to 224°C as measured by differential scanning calorimetry.

48. 48. The crystalline hydrate form of Compound A of claim 47, wherein the endothermic transition is at 224°C ± 3°C.

49. 49. The crystalline hydrate form of Compound A of claim 48, having a thermogravimetric analysis ("TGA") substantially as shown in Figure 5.

50. 50. The crystalline hydrate form of Compound A of any one of claims 42 to 49, having a single crystal structure substantially as shown in Figure 8.

51. 51. A pharmaceutical formulation comprising the crystalline hydrate form of Compound A according to any one of claims 42 to 50 and a pharmaceutically acceptable excipient.

52. 52. A method of treating a subject suffering from cancer, comprising administering to said subject a therapeutically effective amount of the pharmaceutical formulation of claim 51.

53. 53. The method of claim 52, wherein the cancer is multiple myeloma, non-Hodgkin's lymphoma, or acute myeloid leukemia.

54. A crystalline hydrate form of Compound A characterized by XRPD pattern peaks at 10.3, 16.3, and 17.1±0.2 degrees 2θ using CuKα radiation.

55. 55. The crystalline hydrate form of Compound A of claim 54, further characterized by XRPD pattern peaks at 8.1, 10.7, 12.5, 13.3, 15.1, and 20.3±0.2 degrees 2θ using CuKα radiation.

56. 56. The crystalline hydrate form of Compound A of claim 55, further characterized by XRPD pattern peaks at 14.4, 14.7, 15.9, 17.7, 18.1, 19.8, 20.9, 21.7, 21.9, and 25.0±0.2 degrees 2θ using CuKα radiation.

57. 55. The crystalline hydrate form of Compound A of claim 54, having an XRPD pattern substantially as shown in Figure 3.

58. 58. The crystalline hydrate form of Compound A of any one of claims 54 to 57, having an endothermic transition at 218°C to 224°C as measured by differential scanning calorimetry.

59. 59. The crystalline hydrate form of Compound A of claim 58, wherein the endothermic transition is at 224°C ± 3°C.

60. 60. The crystalline hydrate form of Compound A of claim 59, having a thermogravimetric analysis ("TGA") substantially as shown in Figure 5.

61. 61. The crystalline hydrate form of Compound A of any one of claims 54 to 60, having a single crystal structure substantially as shown in Figure 8.

62. 62. The crystalline hydrate form of Compound A of any one of claims 42-61, wherein said crystalline hydrate form of Compound A is formed in the presence of acetic acid.

63. 62. The crystalline hydrate form of Compound A of any one of claims 42-61, wherein said crystalline hydrate form of Compound A is formed in the presence of water and ethanol.

64. 64. A pharmaceutical formulation comprising the crystalline hydrate form of Compound A according to any one of claims 54 to 63 and a pharmaceutically acceptable excipient.

65. 65. A method of treating a subject suffering from cancer, comprising administering to said subject a therapeutically effective amount of the pharmaceutical formulation of claim 64.

66. 66. The method of claim 65, wherein the cancer is multiple myeloma, non-Hodgkin's lymphoma, or acute myeloid leukemia.

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