Process for the synthesis of genistein

Abstract

The present disclosure provides intermediates and processes for preparing 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (genistein), or a pharmaceutically acceptable salt thereof, and for methods of purification and crystallization thereof.

Description

Attorney Docket No.: 38952-0017WO1PROCESS FOR THE SYNTHESIS OF GENTSTEIN RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 753,603, filed February 4, 2025, which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under W911 SR-23 -9-0018 awarded by the Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND) and under W81XWH-22-1-0516 awarded by the Congressionally Directed Medical Research Program (CDMRP). The government has certain rights in the invention.TECHNICAL FIELD

[0003] This disclosure relates to processes and intermediates used for making 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one, which is an isoflavone natural product.BACKGROUND

[0004] The compound 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (genistein) is an isoflavone first isolated from the brooming plant Dyer’s Genista tinctoria L. As an isoflavone found in many Fabaceae plants and a non-nutritional constituent of soybeans, genistein exerts estrogen-like functions. Several biological effects of genistein have been reported in preclinical studies, such as antioxidant, anti-inflammatory, antibacterial, anti-fibrotic, and antiviral activities, effects on angiogenesis and reduced glucose uptake in both estrogen receptor-positive MCF-7 and -negative (MDA-MB-231) breast cancer cell lines, and pharmacological activities relating to diabetes and lipid metabolism.

[0005] There are three main sources of genistein: soy processing, biotechnology, and chemical synthesis. Soybean processing provides a mixture of isoflavones, in which the composition and purity depends on the methods of extraction and isolation. In general, genistein obtained from plant sources is not a pure substance and is available without prescription as a dietary supplement. The use of plant-derived genistein for medicinal purposes presents several challenges, particularly forAttorney Docket No.: 38952-0017WO1dosage development and interpretation of clinical results as it relates to safety and efficacy. Clinical studies require high purity active pharmaceutical ingredient (API), which may be obtained more conveniently through chemical synthesis. This high purity API material requires a validated technical process adhering to, for example, Current Good Manufacturing Practice (cGMP) regulations and guidelines, or to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) specifications. The known method of synthesizing genistein, reported by Filip, K. et al. (Org. Process. Res. Dev. 2016. 20, 1354-1362) has shown multiple drawbacks, primarily safety concerns. A primary issue was found to be the use of corrosive hydrogen chloride gas in the formation of the genistein precursor and an uncontrolled exotherm in the formation of the mixed anhydride, which can be detrimental at larger scales. Additionally, solvent incompatibilities were also found such as ineffective agitation of dense suspensions and the stark difference in boiling points between methanol and water during the acidic hydrolysis preparation of the genistein precursor.SUMMARY

[0006] The compound 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one is a isoflavone natural product commonly called genistein having Formula (I):

[0007] Provided in the present disclosure are intermediates and processes for preparing the compound of Formula (I), also referred to herein as “Compound (I),” in high purity. In some embodiments, the compound of Formula (I) has a purity suitable for use in human clinical studies.

[0008] The present disclosure provides, inter alia, intermediates and processes for preparing a compound of Formula (I), or a pharmaceutically acceptable salt thereof. Accordingly, the present application provides a process including:Attorney Docket No.: 38952-0017WO1heating a compound of Formula (A3)in aqueous HQ, to afford a compound of Formula (A4)(A4) .reacting the compound of Formula (A4) with a formylating agent in the presence of a base to afford a compound of Formula (A5)wherein R is H, -C(=O)H or -C(=O)CH3;contacting the compound of Formula (A5) with concentrated HC1 to afford a crude mixture comprising the compound of Formula (I); andcontacting the crude mixture comprising the compound of Formula (I) with aqueous NaOH to form a purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0009] The present application further provides a process for preparing a compound of Formula (A3), the process including:contacting a compound of Formula (Al)Attorney Docket No.: 38952-0017WO1with a compound of Formula (A2)in the presence of 4M HC1 in 1,4-di oxane and a Lewis acid to form a reaction mixture comprisingthe compound of Formula (A3)

[0008] The present disclosure further provides a process for removing impurities from a crude mixture containing a compound of Formula (1), the process consisting of:dissolving the crude mixture comprising the compound of Formula (I) in an alcohol solvent to form a solution;contacting the solution with aqueous NaOH to form a high pH solution;filtering the high pH solution to obtain a filtered high pH solution; and contacting the filtered high pH solution with aqueous HC1 to form a slurry comprising a further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.BRIEF DESCRIPTION OF DRAWINGS

[0009] FIG. 1 shows strategies to improve the purity and appearance of Compound (I).

[0010] FIG. 2 shows a representative in-process control (IPC) HPLC chromatogram of (A3) upon completion of the reaction in step 1 of Scheme 1.

[0011] FIG. 3 shows an exemplary HPLC chromatogram of isolated material (A3) from the reaction in step 1 of Scheme 1.

[0012] FIG. 4 shows an exemplary 'H-NMR. spectrum of isolated material (A3) from the reaction in step 1 of Scheme 1.

[0013] FIG. 5 shows a representative IPC HPLC chromatogram of (A4) upon completion of reaction in step 2 of Scheme 1.Attorney Docket No.: 38952-0017WO1

[0014] FIG. 6. Shows an exemplary HPLC chromatogram of isolated material (A4) from the reaction in step 2 of Scheme 1.

[0015] FIG. 7 shows an exemplary 'H-NMR. spectrum of isolated material (A4) from the reaction in step 2 of Scheme 1.

[0016] FIG. 8 shows a representative IPC HPLC chromatogram of the crude material upon completion of the reaction in step 3 of Scheme 1.

[0017] FIG. 9 shows a representative IPC HPLC chromatogram of the crude material upon completion of the reaction in step 4A of Scheme 1.

[0018] FIG. 10 shows a representative HPLC chromatogram of crude Compound (I).

[0019] FIG. 11 shows an exemplary mass spectral analysis of crude Compound (I).DETAILED DESCRIPTION

[0020] The present disclosure provides a process for preparation of 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one, or a pharmaceutically acceptable salt thereof, which is an isoflavone natural product commonly called genistein having Formula (I):

[0021] Thus, provided in the present disclosure is a process for preparing a compound of Formula (I)or a pharmaceutically acceptable salt thereof, the process including:Attorney Docket No.: 38952-0017WO1(a) heating a compound of Formula (A3)in aqueous HC1, to afford a compound of Formula (A4)(b) reacting the compound of Formula (A4) with a formylating agent in the presence of a base to afford a compound of Formula (A5)wherein R is H, -C(=O)H or -C(=O)CH3;(c) contacting the compound of Formula (A5) with concentrated HC1 to afford a crude mixture containing the compound of Formula (I); and(d) contacting the crude mixture containing the compound of Formula (I) with aqueous NaOH to form a purified form of a compound of Formula (I).

[0022] In some embodiments, the weight ratio of the compound of Formula (A3):aqueous HC1 in step (a) is about 1:5 to about 1:10, wherein the aqueous HC1 includes concentrated HCkwater in a weight ratio of about 1 :5 to about 1:15.

[0023] In some embodiments, the weight ratio of the compound of Formula (A3):aqueous HC1 in step (a) is about 1:5 to about 1:10, e.g., about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.Attorney Docket No.: 38952-0017WO1

[0024] In some embodiments, the aqueous HC1 in step (a) includes concentrated HCkwater in a weight ratio of about 1:5 to about 1:15, e.g., about 1:5, about 1:6, about 1:7, about 1:8, about 1 :9, about 1 : 10, about 1:11, about 1 : 12, about 1:13, about 1 : 15, or about 1 : 15.

[0025] In some embodiments, the weight ratio of the compound of Formula (A3):aqueous HC1 in step (a) is about 1:6.4, wherein the aqueous HC1 includes concentrated HCkwater in a weight ratio of about 1:11.

[0026] In some embodiments, the heating in step (a) is performed at a temperature of about 80 °C to about 90 °C, e g., about 80 °C, about 82 °C, about 84 °C, about 86 °C, about 88 °C, or about 90 °C.

[0027] In some embodiments, the heating in step (a) is performed for a time of about 17 h to about 20 h, e.g., about 17 h, about 18 h, about 19h, or about 20 h.

[0028] In some embodiments, the base in step (b) includes an alkylamine. Examples of suitable alkylamines include, but are not limited to, tri ethylamine, ethyl diisopropylamine, (1,4-diazabicyclo[2.2.2]octane), diethylmethylamine, 1-methylpyrrolidine, N-methyl piperazine, or 1-methylpiperidine. In some embodiments, the base is triethylamine.

[0029] In some embodiments, the weight ratio of the compound of Formula (A4):base is about 1:2 to about 1:5, e.g., about 1:2, about 1:3, about 1:4, or about 1:5. In some embodiments, the weight ratio of the compound of Formula (A4): base is about 1:3.5.

[0030] In some embodiments, reacting the compound of Formula (A4) with a formylating agent in the presence of base in step (b) is performed with the formylating agent as the solvent.

[0031] In some embodiments, the formylating agent in step (c) is selected from the group consisting of acetic formic anhydride, triethylorthoformate, trimethylorthoformate, N,N-dimethylformamide dimethyl acetal, and N,N-dimethylformamide diethyl acetal, or any combination thereof. In some embodiments, the formylating agent in step (b) is acetic formic anhydride. In some embodiments, the formylating agent in step (b) is tri ethyl orthoformate. In some embodiments, the formylating agent in step (b) is trimethylorthoformate. In some embodiments, the formylating agent in step (b) is N,N-dimethylformamide dimethyl acetal. In some embodiments, the formylating agent in step (b) is N,N-dimethylformamide diethyl acetal.

[0032] In some embodiments, the weight ratio of the compound of Formula (A4):formylating agent is about 1:1.5 to about 1:3, e.g., about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, aboutAttorney Docket No.: 38952-0017WO11:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1 :2.3, about 1 :2.4, about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, or about 1:3.

[0033] In some embodiments, the weight ratio of the compound of Formula (A4):acetic anhydride:formic acid is about 1:1.5:2 to about 1:3:3. In some embodiments, the weight ratio of the compound of Formula (A4):acetic anhydride:formic acid is about 1:1.7:2.2.

[0034] In some embodiments, reacting the compound of Formula (A4) with a formylating agent in the presence of a base in step (b) is performed at a temperature of about 15 °C to about 20 °C, e.g., about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, or about 20 °C.

[0035] In some embodiments, the temperature during reacting the compound of Formula (A4) with a formylating agent in the presence of a base in step (b) is maintained for a time of about 15-30 h, e.g., about 15 h, about 16 h, about 17 h, about 18 h, about 19 h, about 20 h, about 21 h, about 22 h, about 23 h, about 24 h, about 25 h, about 26 h, about 27 h, about 28 h, about 29 h, or about 30 h. In some embodiments, the temperature during reacting the compound of Formula (A4) with a formylating agent in the presence of a base in step (b) is maintained for a time of about 24 h.

[0036] In some embodiments, steps (b), (c), and (d) are performed in tandem without isolation of the reaction products of steps (b) or (c).

[0037] In some embodiments, the weight ratio of the compound of Formula (A4): concentrated HC1 in step (c) is about 1 :4 to about 1:5, e.g., about 1 :4, about 1:4.1, about 1 :4.2, about 1 :4.3, about 1:4.4, about 1:4.5, about 1:4.6, about 1:4.7, about 1:4.8, about 1:4.9, or about 1:5.

[0038] In some embodiments, the weight ratio of the compound of Formula (A4): concentrated HC1 in step (c) is about 1:4.7.

[0039] In some embodiments, contacting the compound of Formula (A5) with concentrated HC1 in step (c) is performed at a temperature of about 15 °C to about 20 °C, e.g., about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, or about 20 °C.

[0040] In some embodiments, the temperature during contacting the compound of Formula (A5) with concentrated HC1 in step (c) is maintained for a time of about 15 h to about 25 h, e.g., about 15 h, about 16 h, about 17 h, about 18 h, about 19 h, about 20 h, about 21 h, about 22 h, about 23 h, about 24 h, or about 25 h. In some embodiments, the temperature during contacting the compound of Formula (A5) with concentrated HC1 in step (c) is maintained for a time of about 22 h.Attorney Docket No.: 38952-0017WO1

[0041] In some embodiments, the process further includes adding water to the crude mixture containing the compound of Formula (I) of step (c) to form an aqueous crude mixture containing the compound of Formula (I) prior to adding the aqueous NaOH in step (d).

[0042] In some embodiments, the weight ratio of the compound of Formula (A4):water added to the crude mixture containing the compound of Formula (I) is about 1:1.

[0043] In some embodiments, the temperature of the crude mixture containing the compound of Formula (I) of step (c) is maintained in a range between about 20 °C to about 25 °C during addition of the water, e.g., at about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, or about 25 °C.

[0044] In some embodiments, the aqueous crude mixture containing the compound of Formula (I) is stirred for about 15 min after addition of the water.

[0045] In some embodiments, the aqueous crude mixture containing the compound of Formula (I) is cooled to about 10 °C to about 15 °C prior to contacting with the aqueous NaOH of step (e), e.g., at about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14°C, or about 15 °C.

[0046] In some embodiments, the aqueous NaOH of step (d) includes NaOH in water in a weight ratio of NaOH:water of about 1:4 to about 1:5, e.g., about 1:4, about 1:4.1, about 1:4.2, about 1:4.3, about 1:4.4, about 1:4.5, about 1:4.6, about 1:4.7, about 1:4.8, about 1:4.9, or about 1:5. In some embodiments, the aqueous NaOH of step (d) includes NaOH in water in a weight ratio of NaOH:water of about 1:4.5.

[0047] In some embodiments, the weight ratio of the compound of Formula (A4): aqueous NaOH of step (d) is about 1:4 to about 1:5, e.g., about 1:4, about 1:4.1, about 1:4.2, about 1:4.3, about 1:4.4, about 1:4.5, about 1:4.6, about 1:4.7, about 1:4.8, about 1:4.9, or about 1:5. In some embodiments, the weight ratio of the compound of Formula (A4):aqueous NaOH of step (d) is about 1:4.3.

[0048] In some embodiments, step (d) is performed by slow addition of aqueous NaOH to the crude mixture containing the compound of Formula (I) of step (c), where the temperature of the mixture is maintained below about 30 °C.

[0049] In some embodiments, the crude mixture containing the compound of Formula (I) of step (c) is an aqueous mixture.Attorney Docket No.: 38952-0017WO1

[0050] In some embodiments, step (d) is performed by stirring the crude mixture containing the compound of Formula (I) with aqueous NaOH for about 40 min after addition of the aqueous NaOH is completed.

[0001] In some embodiments, step (d) is performed by stirring the crude mixture containing the compound of Formula (I) with aqueous NaOH for about 40 min at a temperature of at about 20 °C after addition of the aqueous NaOH is completed.

[0052] In some embodiments, the final pH of the crude mixture containing the compound of Formula (I) with aqueous NaOH is about 1 to about 3, e.g., about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3. In some embodiments, the final pH of the crude mixture containing the compound of Formula (I) with aqueous NaOH is about 2.

[0053] In some embodiments, the process further includes filtering the crude mixture containing the compound of Formula (I) with aqueous NaOH by vacuum filtration to obtain a filter cake containing a purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, the process further includes washing the filter cake containing a purified form of a compound of Formula (I) with water to form a washed filter cake containing a compound of Formula (I). In some embodiments, the process further includes washing the filter cake containing a purified form of a compound of Formula (I) two times with water to form a washed filter cake containing a compound of Formula (I), where the filter cake is vacuum filtered to remove liquids after each wash.

[0055] In some embodiments, the weight ratio of the compound of Formula (A4):water in the foregoing wash is about 1:2.

[0056] In some embodiments, the process further includes drying the foregoing washed filter cake containing a compound of Formula (I) to provide a compound of Formula (I).

[0057] In some embodiments, the foregoing drying of compound of Formula (I) is performed in a vacuum oven at about 60° C for about 15 h to about 24 h, e.g., for about 15 h, about 16 h, about 17 h, about 18 h, about 19 h, about 20 h, about 21 h, about 22 h, about 23 h, or about 24 h.Attorney Docket No.: 38952-0017WO1

[0058] In some embodiments, the purity of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, measured by HPLC % area under the curve at 205 nm is at least 97%, at least 98%, at least 99%, or greater.

[0059] In some embodiments, the process further includes preparing the compound of Formula (A3). In some embodiments, the process includes:contacting a compound of Formula (Al)with a compound of Formula (A2)in the presence of 4M HCl in 1,4-dioxane and a Lewis acid to form a reaction mixture comprisingthe compound of Formula (A3) ^3)

[0060] In some embodiments, the compound of Formula (Al) contains less than about 0.1% w / w water.

[0061] In some embodiments, the dry weight ratio of the compound of Formula (A2):compound of Formula (Al) is about 1:0.95 to about 1:1, e.g., about 1:0.95, about 1:0.96, 1 :0.97, 1 :0.98, 1 :0.99, about 1 : 1, or about 1 : 1.1. In some embodiments, the dry weight ratio of the compound of Formula (A2):compound of Formula (Al) is about 1:0.99.

[0062] In some embodiments, the weight ratio of the compound of Formula (A2):4M HCl in 1,4-dioxane is about 1:10 to about 1:15, e.g., about 1:10, about 1:11, about 1:12, about 1:13, about 1 : 14, or about 1 : 15. In some embodiments, the weight ratio of the compound of Formula (A2):4M HCl in 1,4-dioxane is about 1:11.8.Attorney Docket No.: 38952-0017WO1

[0063] In some embodiments, the Lewis acid includes a salt of a metal ion selected from the group consisting of Ga3+, Sn4+, Pb2+, Sb3+, Bi3+, Sc3+, Fe2+, Fe3+, Co2+, Co3+, Ni2+, Cu2+, Zn2+, Yb3+, Ca2, Sr2+, Al3, In3+, Sn2+, La3+, Ti4+, Zr4+, Cr3+, Ir3+, Th4+, and Pu4+. In some embodiments, the Lewis acid includes a salt of a metal ion selected from the group consisting of Ga3+, Sn4+, Pb2+, Sb3+, Bi3+, Sc3+, Fe2+, Fe3+, Co2+, Co3+, Ni2+, Cu2+, Zn2+, Yb3+, Ca2+, Sr2+, Al3+, In3+, Sn2+, La3+, Fi4+, Zr4+, Cr3+, Ir3+, Th4+, and Pu4+, or any mixture thereof.

[0064] In some embodiments, the Lewis acid includes a salt of Zn2+. In some embodiments, the Lewis acid includes ZnCh. In some embodiments, the Lewis acid is anhydrous ZnCh.

[0065] In some embodiments, the weight ratio of the compound of Formula (A2):ZnCh is about 1:0.1 to about 1:1, e.g., about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, or about 1:1. In some embodiments, the weight ratio of the compound of Formula (A2):ZnChis about 1:0.5.

[0066] In some embodiments, the reaction mixture containing the compound of Formula (Al), the compound of Formula (A2), 4M HC1 in 1,4-dioxane, and the Lewis acid is diluted with additional 1.4-dioxane to form a diluted reaction mixture. In some embodiments, the weight ratio of the compound of Formula (A4): 1,4-dioxane is about 1:1.

[0067] In some embodiments, the diluted reaction mixture is stirred at a temperature of about 20 °C to about 25 °C, e.g., at about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, or about 25 °C.

[0068] In some embodiments, the diluted reaction mixture is stirred for about 40 h to about 50 h, e.g., for about 40 h, about 41 h, about 42 h, about 42 h, about 43 h, about 44 h, about 45 h, about 46 h, about 47 h, about 48 h, about 49 h, or about 50 h. In some embodiments, the diluted reaction mixture is stirred for about 45 h.

[0069] In some embodiments, the process further includes cooling the diluted reaction mixture and adding water to form a slurry containing the compound of Formula (A3). In some embodiments, the process includes cooling the diluted reaction mixture to about 0 °C to about 10 °C (e.g., about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C or about 10 °C) and adding water in a weight ratio of the compound of Formula (A2):water of about 1:1 to about 1:2 (e.g., about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9 or about 1:2) to form a slurry containing a compound of Formula (A3).Attorney Docket No.: 38952-0017WO1

[0070] In some embodiments, the process further includes cooling the diluted reaction mixture to about 0 °C to about 10 °C (e.g., at about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, or about 10 °C), and adding water in aweight ratio ofthe compound ofFormula (A2):water of about 1:1 to about l:2 (e.g., about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9 or about 1 :2) to form a slurry containing a compound ofFormula (A3). In some embodiments, the process further includes cooling the diluted reaction mixture to about 0 °C to about 10 °C and adding water in a weight ratio of the compound ofFormula (A2):water of about 1:1.6 to form a slurry containing a compound ofFormula (A3).

[0071] In some embodiments, the water is added to the foregoing reaction mixture containing the compound ofFormula (A3) over a period of about 10 min.

[0072] In some embodiments, the foregoing slurry is held at about 0 °C to about 10 °C (e.g., at about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, or about 10 °C) for about 3 h. In some embodiments, the slurry is held at about 0 °C to about 10 °C (e.g., at about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, or about 10 °C) for about 3 h while stirring.

[0073] In some embodiments, the process further includes filtering the slurry and washing the filter cake containing the compound ofFormula (A3) with 1,4-dioxane. In some embodiments, the weight ratio of the compound of Formula (A2): 1,4-dioxane is about 1:1. In some embodiments, the filter cake is then washed with water. In some embodiments, the weight ratio of the compound ofFormula (A2):water is about 1:1 to about 1:2 (e.g., about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9 or about 1:2). In some embodiments, the process further includes fdtering the slurry and washing the filter cake containing the compound ofFormula (A3) with 1,4-dioxane in a weight ratio of the compound of Formula (A2): 1,4-dioxane of about 1:1, then washing the filter cake with water in a weight ratio of the compound ofFormula (A2):water of about 1:1.2.

[0074] In some embodiments, the process further includes filtering the slurry and washing the filter cake containing the compound of Formula (A3) twice with 1,4-dioxane. In some embodiments, the weight ratio of the compound ofFormula (A2): 1,4-dioxane is about 1:1.Attomev Docket No.: 38952-0017WO1

[0075] In some embodiments, the process further includes drying the fdter cake containing the compound of Formula (A3). In some embodiments, the fdter cake is dried under a nitrogen atmosphere.

[0076] In some embodiments, the process further includes oven-drying the compound of Formula (A3) at about 65 °C for about 10 to about 25 hours prior to being used as a precursor for the compound of Formula (I). In some embodiments, the process further includes oven-drying the compound of Formula (A3) at about 65 °C for about 21 hours prior to being used as a precursor for the compound of Formula (I).

[0077] In some embodiments, the purity of the compound of Formula (A3) measured by HPLC % area under the curve at 205 nm is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.

[0078] Also provided in the present disclosure is a process for removing impurities from a crude mixture containing a compound of Formula (I)or a pharmaceutically acceptable salt thereof. In some embodiments, the process includes:(a) dissolving the crude mixture containing the compound of Formula (I) in an alcohol solvent to form a solution;(b) contacting the solution with aqueous NaOH to form a high pH solution; (c) filtering the high pH solution to obtain a filtered high pH solution; and (d) contacting the filtered high pH solution with aqueous HC1 to form a slurry containing a further purified form of a compound of Formula (I).

[0079] In some embodiments, the alcohol solvent includes methanol, ethanol, or isopropanol, or any mixture thereof. In some embodiments, the alcohol solvent of step (a) includes methanol.

[0080] In some embodiments, the weight ratio of the crude mixture containing the compound of Formula (I):alcohol solvent is about 1:3 to about 1:5, e.g., about 1:3, about 1:3.5, about 1:4, about 1 :45, about 1 :5. In some embodiments, the weight ratio of the crude mixture containing the compound of Formula (I):alcohol solvent is about 1:4. In some embodiments, the weight ratio of the crude mixture containing the compound of Formula (I):alcohol solvent is about 1:4.2.Attorney Docket No.: 38952-0017WO1

[0081] In some embodiments, the process further includes cooling the solution of step (a) prior to adding the aqueous NaOH of step (b). In some embodiments, the solution of step (a) is cooled to a temperature of about 0 °C to about 10 °C (e.g., at about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, or about 10 °C) prior to adding the aqueous NaOH of step (b).

[0082] In some embodiments, the process further includes cooling the solution of step (a) for about 1 h at a temperature of about 10 °C or less, such as about 0 °C to about 10 °C (e.g., at about 0 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, or about 10 °C) prior to adding the aqueous NaOH of step (b).

[0083] In some embodiments, the aqueous NaOH includes a weight ratio of NaOH:water of about 1 : 15 to about 1 :20, e.g., about 1:15, about 1:16, about 1:17, about 1:18, about 1 : 19, or about 1:20. In some embodiments, the aqueous NaOH includes a weight ratio of NaOH:water of about 1:17. In some embodiments, the aqueous NaOH includes a weight ratio of NaOH:water of about 1:17.3.

[0084] In some embodiments, the weight ratio of the crude mixture containing the compound of Formula (I):aqueous NaOH is about 1:4 to about 1:5, e.g., about 1:1.41, about 1:1.42, about 1 : 1.43, about 1 : 1.44, about 1 : 1.45, about 1 : 1.46, about 1 : 1.47, about 1 : 1.48, about 1 : 1.48, or about 1:5.

[0085] In some embodiments, the weight ratio of the crude mixture containing the compound of Formula (I):aqueous NaOH is about 1 :4.8.

[0086] In some embodiments, step (b) is performed by slow addition of aqueous NaOH to the solution of step (a), where the temperature of the high pH solution does not exceed a temperature of about 22 °C.

[0087] In some embodiments, the high pH solution of step (b) is stirred for about 30 min to about 60 min, e.g., about 30 min, about 40 min, about 50 min, or about 60 min after all of the aqueous NaOH has been added. In some embodiments, the high pH solution of step (b) is stirred for about 50 min after all of the aqueous NaOH has been added.

[0088] In some embodiments, the high pH solution of step (b) is stirred for about 50 min at a temperature of about 10 °C to about 25 °C, e.g., about 10 °C, about 12 °C, about 14 °C, about 16 °C, about 18 °C, about 20 °C, about 22 °C, or about 25 °C after all of the aqueous NaOH hasAttorney Docket No.: 38952-0017WO1been added. In some embodiments, the high pH solution of step (b) is stirred for about 50 min at a temperature of about 20 °C after all of the aqueous NaOH has been added.

[0089] In some embodiments, the pH of the high pH solution of step (b) is about 11 to about 12, e.g., about 11.1, about 11.2, about 11.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.9, about 11.9, or about 12.

[0090] In some embodiments, the filtration of step (c) is polish filtration through a 0.1 - 1 micron filter, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 micron. In some embodiments, the polish filtration is through a 0.2 micron filter.

[0091] In some embodiments, the process further includes cooling the filtered high pH solution to a temperature of about 10 °C or less, such as about 0 °C to about 10 °C, prior to adding the aqueous HC1 of step (d).

[0092] In some embodiments, the aqueous HC1 includes a weight ratio of water: concentrated HC1 of about 1:1 to about 1:2, e.g. about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1 : 1.9 or about 1 :2. In some embodiments, the aqueous HC1 includes a weight ratio of water: concentrated HC1 of about 1:1.4.

[0093] In some embodiments, the weight ratio of the crude mixture containing a compound of Formula (I):aqueous HC1 is about 1 : 1 to about 1 :2, e.g. about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9 or about 1:2. In some embodiments, the weight ratio of the crude mixture containing a compound of Formula (I):aqueous HC1 is about 1:1.

[0094] In some embodiments, step (d) is performed by slow addition of aqueous HC1 to the cooled filtered high pH solution, where the temperature of the high pH solution does not exceed a temperature of about 25 °C.

[0095] In some embodiments, the slurry of step (d) is stirred for about 1 h to about 5 h, e.g., about 1 h, about 2 h, about 3 h, about 4 h, or about 5 h after all of the aqueous HC1 has been added. In some embodiments, the slurry of step (d) is stirred for about 3 h after all of the aqueous HC1 has been added.

[0096] In some embodiments, the slurry of step (d) is stirred for about 3 h at a temperature of about 10 to about 25 °C, e.g., about 10 °C, about 12 °C, about 14 °C, about 16 °C, about 18 °C, about 20 °C, about 22 °C, or about 25 °C after all of the aqueous HC1 has been added. In some embodiments, the slurry of step (d) is stirred for about 1 h to about 5 h, e.g., about 1 h, about 2 h,Attorney Docket No.: 38952-0017WO1about 3 h, about 4 h, or about 5 h at a temperature of about 20 °C after all of the aqueous HC1 has been added. In some embodiments, the slurry of step (d) is stirred for about 3 h at a temperature of about 20 °C after all of the aqueous HC1 has been added.

[0097] In some embodiments, the pH of the slurry of step (d) after all of the aqueous HC1 has been added is about 1 to about 2, e.g., about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.8, or about 2.

[0098] In some embodiments, the process further includes filtering the slurry of step (d) by vacuum filtration to obtain a filter cake containing the further purified form of a compound of Formula (I).

[0099] In some embodiments, the process further includes washing the filter cake containing the further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, with water to obtain a washed filter cake containing the further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0100] In some embodiments, the process further includes washing the filter cake containing the further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, with water twice with the filter cake being vacuum filtered to remove liquids after each wash to obtain a washed filter cake containing the further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0101] In some embodiments, the water used in each foregoing wash has a weight ratio of the crude mixture containing a compound of Formula (I):water of about 1:3 to about 1:5, e.g., about 1 :3, about 1 :4, or about 1 :5. In some embodiments, the water used in each wash has a weight ratio of the crude mixture containing a compound of Formula (I):water of about 1 :4.4.

[0102] In some embodiments, the process further includes drying the filter cake to provide a further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.

[0103] In some embodiments, the drying is performed in a vacuum oven at temperature of about 45 °C to about 70 °C, e.g., about 45 °C, about 50 °C, about 55 °C, about 60 °C, about 65 °C, or about 70 °C for about 26-28 h. In some embodiments, the drying is performed in a vacuum oven at temperature of about 55 °C for about 26-28 h, e.g., 26 h, 27 h, or 28 h.

[0104] In some embodiments, the crude mixture containing a compound of Formula (I) is prepared using the process described herein.Attorney Docket No.: 38952-0017WO1

[0105] In some embodiments, the purity ofthe further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, measured by HPLC % area under the curve at 205 nm is at least 99%, e.g., 99 %, 99.1% or greater.

[0106] In some embodiments, the amount of 5,7-dihydroxy-3-(4-hydroxyphenyl)-2-methyl-4H-chromen-4-one present in the further purified form of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, measured by HPLC % area under the curve at 205 nm is less than 0.2 %, such as less than 0.1%, or less than 0.05%.Definitions

[0107] The term “pharmaceutically acceptable salts” as used herein refers to salts that are prepared with relatively nontoxic acids or bases.

[0108] The term “aqueous HC1” as used herein refers to any concentration of hydrogen chloride in water.

[0109] The term “concentrated HQ” as used herein refers to about 37% w / w HC1 in water, or about 12 M HC1 in water.

[0110] The term “formylating agent” as used herein refers to a chemical reagent able to deliver a -C(O)H moiety onto a reactive position. Exemplary formylating agents include, but are not limited to, acetic formic anhydride, triethylorthoformate, trimethylorthoformate, N,N-dimethylformamide dimethyl acetal, and N,N-dimethylformamide diethyl acetal.

[0111] The term “base” as used herein refers to a substance that reacts with acids to form a salt and which releases hydroxide ions, or accepts protons.

[0112] The term “alkylamine” as used herein refers to a nitrogen with at least 1 saturated alkyl group attached. Non-limiting examples of alkylamines include triethylamine, diethylamine, ethyl diisopropylamine, diisopropylamine, ditertbuytlamine, (l,4-diazabicyclo[2.2.2]octane), diethylmethylamine, 1-methylpyrrolidine, N-methyl piperazine, and 1 -methylpiperidine.

[0113] The term “aqueous NaOH” as used herein refers to any concentration of sodium hydroxide in water.

[0114] The terms “crude” or “crude mixture” as used herein refer to an unrefined form of material. A crude material may be highly pure, or it may contain impurities.

[0115] The term “filter cake” as used herein refers to solid material remaining on a filter. For example, a slurry when filtered yields a solid (the filter cake), and a liquid (the filtrate).Attorney Docket No.: 38952-0017WO1

[0116] The term “vacuum filtration” as used herein refers to a process where a pressure differential is created by a vacuum source to remove air from the receiving chamber below the filter frit (or filter paper), allowing the liquid to pass through the porous barrier and the solid material to remain above the porous barrier.

[0117] The term “Lewis acid” as used herein refers to chemical species that contain an empty orbital which is capable of accepting an electron pair from a Lewis base to form a Lewis acid-base complex.

[0118] The term “anhydrous” as used herein refers to a state where little to no water is present in a substance. The term is most often applied to crystalline substances after the water of crystallization is removed.

[0119] The term “neat” as used herein refers to a sample or process which is conducted in a liquid phase, without any solvent. A liquid can be described as “neat” if it is a pure substance, such as a single compound or a single element or multiple species, that happens to be in the liquid phase.

[0120] The term “one pot” as used herein refers to a process in which a reactant is subjected to successive chemical reactions in just one reactor.

[0121] The term “polish filtration” as used herein refers to a high-stream fdtering process where suspended solids are removed from a liquid stream.

[0122] The term “normal tissue” as used herein refers to tissue with no visible manifestations of disease as determined by histology.

[0123] Solid forms of Compound (I) can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure.

[0124] The process of preparing a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as disclosed herein, incorporates the steps shown in Scheme 1. While each of these steps are illustrated in Scheme 1, it is intended that the individual process steps may be claimed individually or in any combination. It is not intended that the process be limited to an overall process having each and every step in Scheme 1.Attorney Docket No.: 38952-0017WO1<> Scheme 1. Overall synthetic route for preparation of Compound (I) (genistein).

[0125] Compound (I) can be synthesized using a process shown in Scheme 1. In step 1, reagent (Al) was reacted with reagent (A2) in the presence of 4 M HC1 and a Lewis acid, e.g., ZnCh, at about ambient temperature to provide intermediate hydrochloride salt (A3). Step 1 can be carried out in 1,4 dioxane, with all reagents and the Lewis acid ideally containing minimal residual water.

[0126] In step 2, intermediate (A3) was hydrolyzed to intermediate (A4) in the presence of HC1 in water at an elevated temperature.

[0127] Compound (I) was obtained via intermediate (A5), which may be taken directly into step 4A (aqueous acid-catalyzed hydrolysis of esters) and step 4B (partial neutralization of the acid with aqueous base). Step 3 may be carried out using acetic formic anhydride as the formylating agent, which in the presence of an amine base, provides intermediate (A5) as a mixture of species, including where one or more R is hydrogen, one or more R is formyl, and one or more R is acetyl. Steps 3 and 4A / 4B may be carried out in the same reactor. Step 3 may be carried out neat, i.e., with the formylating agent and the amine base serving as the solvent.

[0128] The compound of Formula (I) of the present disclosure may act as an inhibitor of tyrosine-specific protein kinases of the epidermal growth factor (EGF) receptor and also inhibit the activity of topoisomerases. The compound of Formula (I) of the present disclosure may potentially inhibit proliferation of various cancer cells and induce cell differentiation and apoptosis, for example inducing G2 / M arrest associated with upregulated p21 expression in breast, prostate and lung cancer. The compound of Formula (I) of the present disclosure may also reduceAttorney Docket No.: 38952-0017WO1glucose uptake in both estrogen receptor-positive MCF-7 and -negative (MDA-MB-231) breast cancer cell lines. The compound of Formula (I) of the present disclosure exhibits anti angiogenic and antioxidant activities that are important for cancer prevention.

[0129] The compound of Formula (I) of the present disclosure also demonstrates significant bone sparing effects among postmenopausal women.

[0130] The compound of Formula (I) of the present disclosure is an isoflavone, which may be useful in the prevention of heart diseases, with data supporting antioxidant activity, lowering of serum cholesterol, inhibition of tyrosine kinase and / or improvement of vascular reactivity.

[0131] One of the most significant advantages of the compound of Formula (I) of the present disclosure is its low toxicity in comparison with many current chemotherapeutic drugs. The present disclosure provides methods to obtain the compound of Formula (I), or a pharmaceutically acceptable salt thereof, suitable for use in the treatment and prevention of disease.

[0132] Some embodiments provide a method for treating cancer, the method being administering to a patient in need of treatment a therapeutically effective amount of Compound (I), or a pharmaceutically acceptable salt thereof.

[0133] Some embodiments provide a method for preventing or mitigating radiation-induced toxicity to normal tissue associated with cancer radiotherapy, the method being administering to a patient in need of treatment a therapeutically effective amount of Compound (I), or a pharmaceutically acceptable salt thereof. Some embodiments provide a method for preventing or mitigating the acute and delayed effects of radiation exposure associated with radiotherapy, the method being administering to a patient in need of treatment a therapeutically effective amount of Compound (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the radiotherapy causes fibrosis. Thus, some embodiments provide a method for preventing or mitigating fibrosis associated with radiotherapy, the method being administering to a patient in need of treatment a therapeutically effective amount of Compound (I), or a pharmaceutically acceptable salt thereof.

[0134] Some embodiments provide a method for treating heart disease, the method being administering to a patient in need of treatment a therapeutically effective amount of Compound (I), or a pharmaceutically acceptable salt thereof.

[0135] Some embodiments provide a method for treating inflammatory diseases, the method being administering to a patient in need of treatment a therapeutically effective amount ofAttorney Docket No.: 38952-0017WO1Compound (T), or a pharmaceutically acceptable salt thereof. Examples of inflammatory diseases include, but are not limited to, acute respiratory distress syndrome (ARDS), asthma, idiopathic pulmonary fibrosis (IPF), inflammatory lung disease (ILD), and any other disease that induces cell adhesion factors or cytokines that are known to correlate with pathologic conditions of inflammation. In some embodiments, the inflammatory disease is inflammatory lung disease caused by viral infection.

[0136] Some embodiments provided a method for treating osteoporosis, the method being administering to a patient in need of treatment a therapeutically effective amount of Compound (I), or a pharmaceutically acceptable salt thereof.

[0137] Other embodiments, alterations, and permutations of the described embodiments are within the scope of the following claims as will be apparent to those skilled in the art. While operations are depicted in the drawings or claims in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed (some operations may be considered optional), to achieve desirable results.

[0138] Accordingly, the previously described example embodiments do not define or constrain this disclosure. Other changes, substitutions, and alterations are also possible without departing from the spirit and scope of this disclosure.EXAMPLES

[0139] The starting materials disclosed herein can be prepared in a variety of ways using commercially available materials by employing standard synthetic methods and procedures either known to those skilled in the art, or in light of the present disclosure. Bulk phloroglucinol (Al) was purchased from Thermo Fischer, received and vacuum-dried in the drying oven at 50 °C for 16 hours before sampling for water content (w / w%, Karl Fischer analysis showed <0.1% w / w H2O).

[0140] Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or several sources, classic texts such as R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis,Attorney Docket No.: 38952-0017WO1John Wiley and Sons (1994); Smith, M. B., March, J., March’ s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition, John Wiley & Sons: New York, 2001; and Greene, T.W., Wuts, P.G. M., Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons: New York, 1999, are useful and recognized reference textbooks of organic synthesis known to those in the art.AbbreviationsTerm DefinitionAUC Area under curveKF Karl -F i scher (water content)USP U.S. PharmacopeiaDME DimethoxyethaneHC1 Hydrochloric AcidSM Starting materialHPLC High pressure liquid chromatographyIPC In-process control (purity check)ELN Electronic lab notebookBR Batch recordNMR Nuclear magnetic resonanceIPA Isopropyl alcoholEtOAc Ethyl acetateMHz MegahertzDMSO Dimethyl sulfoxideOPRD Organic Process Research & Development (journal)NMT No more thanSDA Specially denatured alcoholMeOH MethanolACN acetonitrileHPLC methodsStep 1 IPC:Attorney Docket No.: 38952-0017WO1Column: Waters XBridge Shield RP18; 3.5 gm, 4.6 x 150 mmMobile Phase: A: 0.05% H3PO4 (v / v) in Water; B: ACNFlow Rate: l.O mL / minColumn temp: 40 °CUV detection: 223 nm (Bandwidth = 4 nm)Concentration^.43 mg / mLSample Diluent: MeOHInjection volume: 2.0 uLNeedle Wash: MeOHStop time: 20 minutesPost time: 0 minutesStep 1 Release:Same as step 1 LPC, except:UV detection: 210 nm (Bandwidth = 4 nm)Concentration: 0.5 mg / mLStop time: 25 minutesAttorney Docket No.: 38952-0017WO1Step 2 IPC:Same as step 1 IPC, exceptConcentration: 0.2 mg / mLStep 2 Release.Same as step 1 IPC, except:UV detection: 224 nm (Bandwidth = 4 nm)Concentration: 0.25 mg / mLStop time: 25 minutesStep 3 IPC:Same as step 1 IPC, except:UV detection: 205 nm (Bandwidth = 4 nm)Concentration: 0.38 mg / mLStop time: 25 minutesAttorney Docket No.: 38952-0017WO1Step 3 Release:Column: Agilent Zorbax Eclipse XDB-C 18; 1.8 pm; 3.0 x 150 mmMobile Phase: A: 0.05% H3PO4 (v / v) in Water; B: ACNFlow Rate: 0.5 mL / minColumn temp: 40 °CUV detection: 205 nm (Bandwidth = 4 nm)Concentration: 0.4 mg / mLSample Diluent: MeOHInjection volume: 1.0 pLNeedle Wash: MeOHStop time: 40 minutesPost time: 0 minutesMass Spectrum Analysis:Sample was freshly prepared at 62 pg / mL in 50:50 (v / v) MeOH:Water with 0.1% (v / v) Formic acid. The prepared sample solution was injected into QTRAP through direct infusion with a built-in syringe pump.Equipment:AB Sciex 3200 series QTrap with syringe pumpWater, USP purified, or equivalentAttorney Docket No.: 38952-0017WO1Methanol, LC-MS grade, or equivalentFormic Acid (FA), LC-MS grade, or equivalentMettler AT201 or MX5 balances, or equivalentQTRAP Instrument Conditions:Ion Source Probe: ESIScan Type: QI MS (QI)Polarity: PositiveScan mode: ProfileIon Source: Turbo SprayResolution: UnitScan mass range: 100 - 500 DaScans in Period: 30 (0.5167-minute duration)Threshold: 0 cpsSettling Time: 0 msecMR pause: 5.007 msecMCA: YesCenter / Width: noStep size: 0.10 DaCurtain Gas (CUR): 10 (psi, nitrogen)Collision Gas (CAD): 0.00Ion Spray Voltage (IS): 5000Source Temp (TEM): 0.00Interface heater: onDeclustering Potential: 20 VSource Probe Alignment:X-axis position: 5 mmY-axis position: 8 mmESI needle depth: 'A full counterclockwise turn up from bottom seating (black knob) Syringe Probe Program:Syringe Diameter: 4.61 mmAttorney Docket No.: 38952-0017WO1Flow Rate: 5.000 pL / min

[0141] The following examples demonstrate the methods described herein. As shown in Scheme 1, the compound of Formula (I), also referred to as “Compound (I)” was prepared in 4 steps. While the products of the first two steps were isolated, the product of step 3 (A5) was telescoped into steps 4A and 4B to obtain Compound (I) in acceptable yield and purity. In the event of lower purity of the final product, Example 4 discloses a procedure for increasing the purity of Compound (I) by introducing a base / acid treatment protocol. Example 5 discloses crystallizations with and without darco decolorizing charcoal followed by wet milling.Example 1. Synthesis of 2-(4-hydroxyphenyl)-l-(2,4,6-trihydroxyphenyl)ethan-l-iminium chloride (A3)A3

[0142] Multiple experiments were carried out in order to optimize production of (A3), which are summarized in Table 1 and discussed below. It was previously reported (Filip, K. et al. Org. Process. Res. Dev. 2016. 20, 1354-1362) that the reaction of step 1 could be run using DME as the solvent with addition of HC1 gas into the reaction mixture. The use of HC1 gas on large scale is problematic in that it allows for more variability into the process and introduces safety concerns (HC1 is a very high pressure gas and is corrosive and toxic). Accordingly, an alternative source of HC1 was sought. Various acidic solvents that were commercially available were tested such as 1 M HC1 in EtOAc, 4 M HC1 in dioxane, and 6 M HC1 in IPA. The idea to use FIC1 in IPA was abandoned when analysis of the reaction stream revealed multiple new impurities. In the case of 1 M HC1 in EtOAc, the reaction profile was cleaner than HC1 in IPA, but at the expense of a slower rate with the reaction eventually stalling after 98 hours. When 4 M HC1 in dioxane was utilized, the reaction profile was better than the experiments reproducing the conditions of Filip, K. et al., so this was the solvent utilized for further development. The reaction was repeated with mechanical stirring to ensure translation into further scale-up and was found to be able to scale-up.Attorney Docket No.: 38952-0017WO1Table 1. Summary of step 1 optimization experiments

[0143] In both dioxane and DME, residual amounts of (Al) (despite being the limiting reagent) and other small impurities were observed in variable amounts. These impurities were found to be the hydrolysis products of the benzyl nitrile starting material (A2), i.e., the corresponding amide and carboxylic acid; suggesting the consumption of the nitrile (A2) towards the reaction and towards side reaction, allowing for excess (Al) to remain. To mitigate formation of these byproducts, (Al) was dried to prevent moisture from entering the reaction. In a comparison between dried (Al), i.e., (< 0.1% KF) and the undried (5.2% KF), there was a stark difference between the two reaction profiles and was thus implemented as a point of control to ensure low moisture content in the starting materials. Dilution of the reaction with dioxane was incorporated in an effort to further slow byproduct formation but was found to have no effect. The benzyl nitrileAttorney Docket No.: 38952-0017WO1(A2) was thus made the limiting reagent as this was the control point that would affect the overall yield and quality of the step. The addition of a slight excess of (Al) was found to lower the byproduct formation in the reaction and was thus implemented into the lab confirmation batch, where it was found to have similar yield and quality as previous smaller pilots.

[0144] During synthesis of a demonstration batch, it was found that (A3) had deliquesced with the solvent and washes. The overall quality of the material was found to be good, but this phase change required further investigation. A solubility study was conducted, and the results are shown in Table 2.Table 2. Solubility study of compound of Formula (A3)<

[0145] Despite low solubility in each solvent individually, a mixture of the two solvents surprisingly increased the solubility of the product dramatically. It was thus speculated that a slower fdtration of the material would allow for the two washes (first dioxane, then water) to mix and thus make the material dissolve and potentially pass through the filter, which would result in product losses to the filtrate. A smaller pilot study was conducted, where the material was filtered at room temperature and was washed with additional dioxane instead of water. This pilot was found to contain similar quality material as the demo batch, but also allowed for a faster filtration. Accordingly, the water wash was removed and substituted with an additional dioxane wash while also filtering at room temperature rather than a cooled slurry.Exemplary procedure:

[0146] To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added 4 M HC1 in dioxane (41.94 kg). Stirring was initiated and phloroglucinol (3.35 kg), 4-hydroxyphenylacetonitrile (A2, 3.40 kg) and anhydrous ZnCh (1.70 kg) were sequentially added into the reaction vessel. A dioxane rinse of 2.95 kg was applied to the charge bags to ensure all solids were added to the reactor. A slight exotherm up to 26 °C was observed after charging all materials. The bulk suspension was stirred for 45 hours, 18 minutes at 20-25 °C when IPC analysisAttorney Docket No.: 38952-0017WO1indicated reaction completion criteria were met (4-hydroxyacetonitrile <1% relative to (A3), see FIG. 2).

[0147] The reactor contents were adjusted to between 0-10 °C and USP H2O (1.41 kg) was added over the course of 10 minutes. The mixture was held at this temperature while stirring for 3 hours before vacuum fdtration on an 18” benchtop filter funnel equipped w / polypropylene filter cloth. Mother liquors were looped / recirculated into the reactor to collect residual product before final deposit onto the filter cake.

[0148] The solids were sequentially washed with dioxane (3.55 kg) and USP H2O (4.06 kg) and dried on the funnel under a nitrogen atmosphere for 1 hr 20 minutes.

[0149] The solids were transferred from the filter funnel onto polyethylene-lined drying trays, whereupon partial deliquescence on the drying trays was noted. The bulk material was sampled for HPLC (FIG. 3) and 'H NMR analysis (FIG. 4) before vacuum-drying all the collected product at 25 °C overnight (17 hours).

[0150] The observed solids showed no evidence of deliquescence upon standing in air. Nonetheless, the bulk material was oven-dried under vacuum at 65 °C for a further 21 hours before sampling / packaging.

[0151] 6.49 kg of 2-(4-hydroxyphenyl)-l-(2,4,6-trihydroxyphenyl)ethan-l-iminium chloride (A3) was isolated as a dark yellow solid. 85.8% isolated yield, with purity of 94.7% AUC (FIG.3)- Table 7. Materials used in Example 1 exemplary procedureAttomev Docket No.: 38952-0017WO1Example 2. Synthesis of 2-(4-hydroxyphenyl)-l-(2,4,6-trihydroxyphenyl)ethan-l-one (A4)A4

[0152] Multiple experiments were carried out in order to optimize production of (A4), which are summarized in Table 3 and discussed below. Development of Step 2 began with familiarization using the conditions previously reported (Filip, K. et al. Org. Process. Res. Dev. 2016. 20, 1354-1362). Overall, the yield and quality of the product was acceptable, however some factors such as temperature and solvent ratio were examined. The reaction solvent was initially about 5% methanol in water (conditions reported by Filip, K. et al.) and the reaction mixture was heated to about 95 °C, which is above the boiling point of methanol. Removal of the methanol allowed for a similar reaction profile. Increasing the methanol content provided a similar reaction profile; however, lowering the temperature caused reaction stalling, suggesting temperature is a key factor in the reaction. Heating the reaction to 100 °C allowed for a slight increase in conversion to product. Optimal reaction conditions were achieved using no methanol as described in the exemplary procedure.Table 3. Summary of step 2 optimization experimentsAttorney Docket No.: 38952-0017WO1Exemplary Procedure:

[0153] To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added USP water (36.0 kg). Stirring was initiated and 6.18 kg of 2-(4-hydroxyphenyl)-l-(2,4,6-trihydroxyphenyl)ethan-1-iminium chloride (A3) was added as a solid in one portion. Cone, hydrochloric acid (3.28 kg, 1.6 eq.) was added, and the reaction mixture stirred briefly. The internal temperature was adjusted to 80-90 °C and the mixture stirred for 17 hours before IPC analysis indicated a completed reaction ((A3) <3% remaining, see FIG. 5). After a total of 20 hours at the temperature specified above, the vessel contents were cooled over the course of 2 hours to an internal temperature of 10 °C. The mixture was aged for a further 2 hours before the bulk slurry was filtered on an 18” benchtop filter funnel. The solids on the filter funnel were washed twice with USP water (24.9 kg per wash). The filter cake was further vacuum filtered for 22 minutes to remove all filtrate before transferring the material to polyethylene-lined trays. The solids were dried in a vacuum oven at 65°C for 13 hours. Solids were weighed and redried for a further 3 hours at the same temperature to ensure a constant weight (<1% difference between consecutive weighing.)

[0154] 4.65 kg of 2-(4-hydroxyphenyl)-l-(2,4,6-trihydroxyphenyl)ethan-l-one (A4) was isolated as a yellow solid. 85.3% isolated yield, with a purity of 98.7% AUC (FIG. 6).

[0155] rH NMR analysis was conducted using a Bruker 400 MHz in d6-DMSO. Analysis shows that the observed structure conformed to the expected product (FIG. 7).Table 8. Materials used in Example 2Attorney Docket No.: 38952-0017WO1Example 3. Synthesis of 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one (I)

[0156] Three batch experiments were carried out in order to optimize the combination of steps 3 with 4A and 4B, which are summarized in Table 4 and discussed below. A safety analysis of the previously reported conditions (Filip, K. et al. Org. Process. Res. Dev. 2016. 20, 1354-1362) indicated that the charging of formic acid into acetic anhydride could potentially lead to a deleterious exotherm. Precautions were made by actively cooling during the charging. A pilot scale experiment showed minimal effect on the quality of Compound (I) crude.

[0157] As shown in Scheme 1, the compound of Formula (A5) is drawn within square brackets indicating that it was an intermediate that was not isolated. It was also a mixture of chemical species, some of which had one or more R as a hydrogen, others with one or more R as a formyl, and others with one or more R as acetyl. The species where all R were hydrogen is Compound (I). The species where one or more R was a formyl- or acetyl ester were removed from (A5) to form Compound (I) by the steps of 4 A and 4B. Steps 4 A and 4B ensured efficient isolation of Compound (I). While step 4A cleaved any formyl- or acetyl esters via acid-catalyzed de-esterification, step 4B neutralized the acid and ensured Compound (I) was not in a deprotonated form (i.e., as a sodium phenylate) because the final pH was about 2. Compound (I) formed a precipitate while the triethyl amine (or other base or bases) formed HC1 salts that remained in solution at low pH.

[0158] As shown in Scheme 2, formation of an acetyl ester on the phenolic OH adjacent to the benzyl ketone, upon cyclization gave a 2-methyl bi-product, which upon treatment with step 4A and 4B gave 5,7-dihydroxy-3-(4-hydroxyphenyl)-2-methyl-4H-chromen-4-one, “methyl impurity (II).” This was particularly problematic because methyl impurity (II) was difficult to separate from Compound (I).Attorney Docket No.: 38952-0017WO1Scheme 2. Formation of methyl impurity (IT). Conditions: 4A) Cone. HC1, 20 °C; 4B) Aqueous NaOH, 20 °C.

[0159] An alternative strategy for the formation of Compound (I) from (A4) is to utilize other formylating agents such as triethylorthoformate, or other reagents such as trimethylorthoformate, N,N-dimethylformamide dimethyl acetal, andN,N-dimethylformamide diethyl acetal. This would eliminate the formation of methyl impurity (II). The use of triethylorthoformate, for example, would allow only for ethanol to be formed as a byproduct rather than a mixture of anhydrides leading to methyl impurity (II).

[0160] It bears noting that step 3 disclosed in Example 3 used the formylating agent as the solvent, which minimized cost and negated the need for solvent removal. Addition of an aprotic solvent in step 3 is envisioned to be within the disclosed process.

[0161] In addition, as noted above, the compound of Formula (A5) may be regarded as an intermediate and was not isolated in the exemplary procedure. Isolation of the compound of Formula (A5) is envisioned to be within the disclosed process. The practice of combining two or more steps in a reactor (“one-pot”) otherwise termed “tandem reactions” or sometimes referred to as “telescoping” reduces operational steps and saves time. The combination of steps 3 and 4A and 4B may be carried out in the same reactor or different reactors.Table 4. Summary of step 3 optimization experiments*ND= not determinedExemplary procedure:

[0162] To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added acetic anhydride (7.13 kg). The mixture was cooled to < 15 °C. Maintaining the lowered temperature, formic acid (9.31 kg) was added to the reaction vessel. 4.295 kg of 2-(4-Hydroxyphenyl)-l -(2,4,6-trihydroxyphenyl) ethan-l-one (A4) was added and the mixture cooled to < 15 °C. Triethylamine (14.81 kg) was slowly added to the reaction mixture, maintaining an internal temperature of < 20 °C. The mixture was then allowed to stir at 20 °C for 24 hours before IPC analysis. IPC analysisAttorney Docket No.: 38952-0017WO1indicated completion of the reaction ((A4) was < 5%, see FIG. 8). The mixture was then cooled to > 15 °C and HC1 (19.79 kg) was added, maintaining an internal temperature below 22 °C. The mixture was then allowed to stir at 20 °C for 22 hours before IPC analysis. IPC analysis indicated completion of the reaction (< 5% of (A4) remained, see FIG. 9). Water (4.21 kg, USP) was added, maintaining an internal temperature below 25 °C, and the slurry was stirred for at least 15 minutes before the pH was checked (value = -0.7). In an adj acent reactor, water (15.10 kg, USP) and sodium hydroxide (3.38 kg) were added and the contents stirred until dissolution. The slurry mixture was cooled to < 15 °C. The sodium hydroxide solution was then slowly added maintaining an internal temperature below 30 °C. The mixture was then stirred at 20 °C for 39 minutes before the pH was checked (value = 2.2). The slurry was then fdtered on an 18” benchtop filter funnel. The solids were washed with water (8.80 kg) and pulled dry for 40 minutes. The solids were washed again with water (8.22 kg) and pulled dry for 48 minutes before transferring the bulk material to polyethylene-lined trays. The solids were dried in a vacuum oven at 60 °C for 15 hours. Solids were weighed and redried for a further 6 hours at the same temperature to ensure a constant weight (< 1% difference between consecutive weighing).

[0163] 4.00 kg of crude Compound (I) was isolated as an off-white solid in 89.7% yield, with a purity of 96.8% AUC. A representative HPLC analysis is shown in FIG. 10.

[0164] Mass spectral analysis showed the (M+H)+and (M+Na)+peaks for Compound (I) (FIG.11).1H NMR analysis was conducted using a Bruker 400 MHz Spectrometer, with the compound dissolved in d6-DMSO. Analysis showed that the observed structure conformed to Compound (I). Table 9. Materials used in Example 3Attorney Docket No.: 38952-0017WO1Example 4. Base / Acid purification of 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one, Compound (I)

[0165] In a demonstration batch of Compound (I) prepared according to Scheme 1 and described herein, it was found that the amount of methyl impurity (II) was 0.18% AUC, which was very close to the specification limit for API (0.20% AUC). This prompted development of a remediation protocol should the scenario arise where the methyl impurity (II) was not below acceptable levels. Thus, half of the demonstration batch of Compound (I) was taken downstream, and the other half was reprocessed in an additional base / acid purification protocol to examine the extend of further purging of methyl impurity (II) (see FIG. 1 for flow chart). The base / acid purification protocol reduced the level of methyl impurity (II) from 0.18%AUC to 0.12%AUC; thus, a successful remediation was in place should methyl impurity (II) be above specifications.

[0166] An additional batch of Compound (I) was subjected the foregoing base / acid purification protocol to reduce the level of the methyl impurity (II) from 0.15% down to 0.10% AUC; however this was on a small scale and scalability was not established. Incorporation of an IPC of no more than 0.20% AUC of the methyl impurity (II) would determine whether the foregoing remediation process would be implemented in additional batches.Table 5. Summary of Base / Acid purification experimentsExemplary Procedures:Batch A:

[0167] To a vessel was added water (17.00 kg, USP) and sodium hydroxide (0.98 kg). The contents were stirred until dissolved. To another vessel was added water (1.75 kg, USP) and cone. HC1 (2.44 kg). The contents were stirred until dissolved. To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added crude Compound (I) (3.70 kg), i.e., a material obtained from the procedure described in Example 3 but of lesser purity. Methanol (15.21 kg) was added, and the mixture was stirred for 1 hour while cooling the mixture to < 10 °C. The above sodium hydroxide solution was slowly added, maintaining an internal temperature below 22 °C. The mixture wasAttomev Docket No.: 38952-0017WO1then stirred for 50 minutes at 20 °C before the pH was verified (value = 11.3). The mixture was then polish filtered using a 0.2-micron cartridge filter into an adjacent reactor. The reactor and cartridge filter were rinsed with water (3.62 kg, USP). The filtrate was then cooled to < 10 °C. The above HC1 solution was then slowly added, maintaining an internal temperature below 25 °C. The mixture was then stirred at 20 °C for 3 hours before verifying the pH (value = 1.4). The resultant slurry was filtered on an 18” benchtop filter funnel. Water (16.11 kg, USP) was added to the reactor and used to wash the product cake. The solids were then washed again with water (16.80 kg, USP). The solids were pulled dry on the funnel for 15 hours. The product was then transferred to polyethylene-lined trays and dried in a vacuum oven at 55 °C for 26 hours. Solids were weighed and redried for a further 2 hours at the same temperature to ensure a constant weight (< 1% difference between consecutive weighing).

[0168] 3.64 kg of base / acid purified Compound (I) was isolated as an off-white solid (99.42% AUC). The amount of 5,7-dihydroxy-3-(4-hydroxyphenyl)-2-methyl-4H-chromen-4-one, i.e., methyl impurity (II), was found to be 0.18% AUC. (IPC: NMT 0.20% AUC methyl impurity (II) present).Table 10. Materials used in Example 4, Batch ABatch B:

[0169] To a vessel was added water (8.60 kg, USP) and sodium hydroxide (0.49 kg). The contents were stirred until dissolved. To another vessel was added water (0.90 kg, USP) and cone. HC1 (1.29 kg). The contents were stirred until homogeneous. To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added crude Compound (I) (1.80 kg), i.e., a material obtained from the procedure described in Example 3 but of lesser purity. Methanol (7.51 kg) was added, and the mixture was stirred for 1 hour while cooling the mixture to < 10 °C. The above sodium hydroxide solution was slowly added while maintaining an internal temperature below 22 °C. The mixtureAttorney Docket No.: 38952-0017WO1was then stirred for 50 minutes at 20 °C before the pH was verified (value = 11.3). The mixture was then polish filtered using a 0.2-micron cartridge filter into an adjacent reactor. The reactor and cartridge filter were rinsed with water (1.85 kg, USP). The filtrate was then cooled to < 10 °C. The above HC1 solution was then slowly added while maintaining an internal temperature below 25 °C. The mixture was then stirred at 20 °C for 3 hours before verifying the pH (value = 1.4). The slurry was filtered on an 18” benchtop filter funnel. Water (7.97 kg, USP) was added to the reactor and used to wash the product cake. The solids were then washed again with water (8.12 kg, USP). The solids were pulled dry on the funnel for 15 hours. The product was then transferred to polyethylene-lined trays and dried in a vacuum oven at 55 °C for 23 hours. Solids were weighed and redried for a further 2 hours at the same temperature to ensure a constant weight (< 1% difference between consecutive weighing).

[0170] 1.57 kg of Compound (I) base / acid purified product was isolated as an off-white solid (99.67% AUC). The methyl impurity (II) was found to be 0.12% AUC.Table 11. Materials used in Example 4, Batch BExample 5. Recrystallization of Compound (I)

[0171] Multiple experiments were carried out in order to optimize the recrystallization and final API isolation protocol, which are summarized in Table 6 and discussed below. The material in a demonstration batch of Compound (I) was found to be outside of the specifications (i.e., it was not white to off-white). One portion of the batch proceeded to milling in an effort to determine the effect of milling on color, while the other half was recrystallized with introduction of decolorizing charcoal, i.e., darco (see FIG. 1). The milled material was found to have the same color, while the darco-treated material provided the intended white color within specification. This allowed for introduction of the darco treatment into the cGMP process. However, there was concern that darco could leach through during filtration. Despite darco levels being well underAttorney Docket No.: 38952-0017WO1genotoxic levels, a remediation through a secondary recrystallized lacking the darco was installed to ensure filtration of carbon. Additionally, it was noticed that the methyl impurity also further purged during each of these recrystallizations.Table 6. Summary of recrystallization experiments

[0172] These studies focused primarily on color assessment because a previous batch of Compound (I) contained residual amounts of black particulates that were determined to be carbonaceous, i.e., darco. Despite the amount of black particulates being well under ICH specifications (2.8 ppm), the aim to remove them was sought. The primary focus was to assess whether darco was necessary for the process. In a side-by-side analysis, recrystallized products with and without darco experiments were conducted and it was found that the non-treated material was an off-white color while the darco-treated Compound (I) was white. This was later implemented on scale to assess the effectiveness of scalability.

[0173] Despite efforts to remove darco from the process, it appeared that after the recrystallization, the material turned a tan color rather than staying as off-white. Milling of the tancolored Compound (I) did not improve the color. Only when darco was introduced into the process, did the color improve. A small sample (10 g) was then dissolved in DMSO / Acetone (1 : 1 v / v) and no black particulates were found. Upon scale-up to a 50 g, a very small amount of black particulates were present. To ensure that no black particulates would pass through the filtration, it was implemented that a secondary recrystallization without darco would be incorporated in order to have a second polish filtration after darco.Exemplary procedures:Batch A:

[0174] To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added water (7.01 kg) and ethanol (5.56 kg). The contents were stirred for 5 minutes, then 1.64 kg of crude Compound (I) was then added. Ethanol (40.64 kg) was added, and the solution was heated to 75 °C and keptAttorney Docket No.: 38952-0017WO1at temperature until dissolution of Compound (I). The contents were cooled to 55 °C and stirred for 15-30 minutes. The solution was polish fdtered into an adjacent reactor. The filtrate was then concentrated to 10 volumes under reduced pressure, keeping the jacket temperature below 60 °C. The contents were then heated to 75 °C and water (28.81 kg) was added, maintaining the internal temperature. The slurry was then allowed to cool ambiently overnight. The slurry was then filtered on a benchtop filter. The product was then washed with an ethanol / water mixture (3.105 kg, 1:1 v / v), with a repeat wash with a ethanol / water mixture (2.86 kg, 1:1 v / v). The solids were then dried under reduced pressure at 55 °C until constant weight to give 1.34 kg (81.7% yield) of recrystallized Compound (I) as a light tan solid with a purity of 99.71% AUC. The amount of methyl impurity (II) was 0.15% AUC.Batch B:

[0175] To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added water (7.01 kg) and ethanol (5.56 kg). The contents were stirred for 5 minutes, then 1.58 kg of crude Compound (I) was added. Ethanol (39.50 kg) was added and the solution was heated to 75 °C and kept at temperature until dissolution of Compound (I). The contents were cooled to 55 °C and stirred for 15-30 minutes. The solution was polish filtered into an adjacent reactor. The filtrate was then concentrated to 10 volumes under reduced pressure keeping the jacket temperature below 60 °C. The contents were then heated to 75 °C and water (27.37 kg) was added maintaining the internal temperature. The slurry was then allowed to cool ambiently overnight. The slurry was then filtered on a benchtop filter. The product was then washed with an ethanol / water mixture (2.92 kg, 1:1 v / v) with a repeat wash with a ethanol / water mixture (3.00 kg, 1:1 v / v). The solids were then dried under reduced pressure at 55 °C until constant weight to provide 1.42 kg (89.9% yield) of recrystallized Compound (I) as a light tan solid with a purity of 99.76% AUC. The amount of methyl impurity (II) was 0.08% AUC.Batch C:

[0176] To a 25-gallon Pfaudler glass-lined reactor under nitrogen was added water (5.72 kg) and ethanol (4.64 kg). The contents were stirred for 5 minutes. 1.32 kg crude Compound (I) was added. Ethanol (33.00 kg) was added and heated to 75 °C and kept at temperature until dissolution of Compound (I). A slurry of darco decolorizing charcoal (0.14 kg) in ethanol (2.03 kg) was prepared and added to the reactor. The vessel was rinsed with ethanol (0.69 kg). The contents were cooled to 55 °C and stirred for 15-30 minutes. The solution was polish filtered into an adjacentAttorney Docket No.: 38952-0017WO1reactor. The filtrate was then concentrated to 10 L / kg under reduced pressure, keeping the jacket temperature below 60°C. The contents were then heated to 75 °C and water (21.16 kg) was added, maintaining the internal temperature. The slurry was then allowed to cool ambiently over 16 hours. The slurry was then filtered on a benchtop filter. The product was then washed with an ethanol / water mixture (2.41 kg, 1:1 v / v), with a repeat wash with an ethanol / water mixture (2.43 kg, 1:1 v / v). The solids were then transferred to a vacuum-oven on polyethylene-lined trays and dried under reduced pressure at 55 °C until constant weight to provide 1.11 kg (84.1% yield) of recrystallized Compound (I) as a white solid with a purity of 99.80% AUC. The amount of methyl impurity (II) was 0.11% AUC.Batch D:

[0177] To a 50-gallon Pfaudler glass-lined reactor under nitrogen was added water (2.87 kg) and ethanol (1.50 kg). The contents were stirred. In a separate reactor was added ethanol (4.25 kg) and water (8.40 kg). Recrystallized Compound (I), 1.360 kg, Batch B, was added. Water (0.22 kg) was added to rinse the walls, and the slurry was held at 20 °C. The slurry was then milled for 45 turnovers over 41 minutes. The milled material was rinsed with an ethanol / water mixture (0.42 kg, 4:6 v / v). The slurry was then fdtered and washed with an ethanol / water mixture (2.49 kg, 4:6 v / v). The solids were then collected and dried under vacuum at 55 °C to provide 1.30 kg (95.5% yield) of Compound (I) as a light tan solid with a purity of 99.74% AUC. The amount of methyl impurity (II) was 0.08% AUC.Batch E:

[0178] To a 50-gallon Pfaudler glass-lined reactor under nitrogen was added water (2.10 kg) and ethanol (1.03 kg). The contents were stirred. In a separate reactor was added ethanol (4.89 kg) and water (8.95 kg). Recrystallized Compound (I), 1.07 kg, Batch C, was added. Water (0.44 kg) was added to rinse the walls, and the slurry was held at 20 °C. The slurry was then milled for 45 turnovers over 45 minutes. The milled material was rinsed with an ethanol / water mixture (0.34 kg, 4:6 v / v). The slurry was then fdtered and washed with an ethanol / water mixture (1.92 kg, 4:6 v / v). The solids were then collected and dried under vacuum at 55 °C to provide 0.98 kg (91.5% yield) of Compound (I) as a white solid with a purity of 99.76% AUC. The amount of methyl impurity (II) was 0.12% AUC.

Claims

Attorney Docket No.: 38952-0017WO1WHAT IS CLAIMED IS:A process for preparing a compound of Formula (I)or a pharmaceutically acceptable salt thereof, the process comprising:(a) heating a compound of Formula (A3)in aqueous HC1, to afford a compound of Formula (A4)(b) reacting the compound of Formula (A4) with a formylating agent in the presence of a base to afford a compound of Formula (A5)wherein R is H, -C(=O)H or -C(=0)CH3;(c) contacting the compound of Formula (A5) with concentrated HC1 to afford a crude mixture comprising the compound of Formula (I); and(d) contacting the crude mixture comprising the compound of Formula (I) with aqueous NaOH to form a purified form of a compound of Formula (I).Attorney Docket No.: 38952-0017WO12. The process of claim 1, wherein the weight ratio of the compound of Formula (A3):aqueous HC1 in step (b) is about 1 :5 to about 1:10, wherein the aqueous HC1 comprises concentrated HC1: water in a weight ratio of about 1 :5 to about 1:15.

3. The process of claim 1 or 2, wherein the weight ratio of the compound of Formula (A3):aqueous HC1 in step (b) is about 1:6.4, wherein the aqueous HC1 comprises concentrated HC1: water in a weight ratio of about 1:11.

4. The process of any one of claims 1-3, wherein the heating in step (b) is performed at a temperature of about 80 °C to about 90 °C.

5. The process of any one of claims 1-4, wherein the heating in step (b) is performed for a time of about 17 h to about 20 h.

6. The process of any one of claims 1-5, wherein the base in step (c) comprises an alkylamine.

7. The process of claim 6, wherein the base is triethylamine.

8. The process of any one of claims 6-7, wherein the weight ratio of the compound of Formula (A4): base is about 1 :2 to about 1:5.

9. The process of any one of claims 6-8, wherein the weight ratio of the compound of Formula (A4): base is about 1:3.5.

10. The process of any one of claims 1-4, wherein the reacting of the compound of Formula (A4) with a formylating agent in the presence of base in step (c) is performed with the formylating agent as the solvent.Attorney Docket No.: 38952-0017WO111. The process of any one of claims 1-10, wherein the formylating agent in step (c) is selected from the group consisting of acetic formic anhydride, triethylorthoformate, trimethylorthoformate, N,N-dimethylformamide dimethyl acetal, and N,N-dimethylformamide diethyl acetal.

12. The process of any one of claims 1-11, wherein the formylating agent in step (c) comprises acetic formic anhydride.

13. The process of claim 12, wherein the weight ratio of the compound of Formula (A4):acetic anhydride :formic acid is about 1:1.5:2 to about 1:3:3.

14. The process of claim 12 or 13, wherein the weight ratio of the compound of Formula (A4):acetic anhydride: formic acid is about 1:1.7:2.2.

15. The process of any one of claims 1-14, wherein the reacting of the compound of Formula (A4) with a formylating agent in the presence of a base in step (c) is performed at a temperature of about 15 °C to about 20 °C.

16. The process of claim 15, wherein the temperature is maintained for a time of about 24 h.

17. The process of any one of claims 1-16, wherein steps (c), (d), and (e) are performed in tandem without isolation of the reaction products of steps (c) or (d).

18. The process of any one of claims 1-17, wherein the weight ratio of the compound of Formula (A4): concentrated HC1 in step (d) is about 1:4 to about 1:5.

19. The process of any one of claims 1-18, wherein the weight ratio of the compound of Formula (A4): concentrated HC1 in step (d) is about 1:4.7.Attorney Docket No.: 38952-0017WO120. The process of any one of claims 1-19, wherein the contacting the compound of Formula (A5) with concentrated HC1 in step (d) is performed at a temperature of about 15 °C to about 20 °C.

21. The process of claim 20, wherein the temperature is maintained for a time of about 22 h.

22. The process of any one of claims 1-21, further comprising adding water to the crude mixture comprising the compound of Formula (I) of step (d) to form an aqueous crude mixture comprising the compound of Formula (I) prior to adding the aqueous NaOH in step (e).

23. The process of claim 22, wherein the weight ratio of the compound of Formula (A4): water added to the crude mixture comprising the compound of Formula (I) is about 1:1.

24. The process of claim 22 or 23, wherein the temperature of the crude mixture comprising the compound of Formula (I) of step (d) is maintained in a range between about 20 °C to about 25 °C during addition of the water.

25. The process of any one of claims 22-24, wherein the aqueous crude mixture comprising the compound of Formula (I) is stirred for about 15 min after addition of the water.

26. The process of any one of claims 22-25, wherein aqueous crude mixture comprising the compound of Formula (I) is cooled to about 10 °C to about 15 °C prior to contacting with the aqueous NaOH of step (e).

27. The process of any one of claims 1-26, wherein the aqueous NaOH of step (e) comprises NaOH in water in a weight ratio of NaOH:water of about 1 :4 to about 1:5.

28. The process of any one of claims 1-27, wherein the aqueous NaOH of step (e) comprises NaOH in water in a weight ratio of NaOH:water of about 1 :4.5.Attorney Docket No.: 38952-0017WO129. The process of any one of claims 1-28, wherein the weight ratio of the compound of Formula (A4):aqueous NaOH of step (e) is about 1 :4 to about 1 :

530. The process of any one of claims 1-29, wherein the weight ratio of the compound of Formula (A4):aqueous NaOH of step (e) is about 1:4.3.

31. The process of any one of claims 1-30, wherein step (e) is performed by slow addition of aqueous NaOH to the crude mixture comprising the compound of Formula (I) of step (d), wherein the temperature of the mixture is maintained below about 30 °C.

32. The process of any one of claims 1-31, wherein step (e) is performed by stirring the crude mixture comprising the compound of Formula (I) with aqueous NaOH for about 40 min after addition of the aqueous NaOH is completed.

33. The process of claim 32, wherein the stirring is performed at about 20 °C.

34. The process of claim 32 or 33, wherein the final pH of the crude mixture comprising the compound of Formula (I) with aqueous NaOH is about 1 to about 3.

35. The process of any one of claim 32 - 34, wherein the final pH of the crude mixture comprising the compound of Formula (I) with aqueous NaOH is about 2.

36. The process of any one of claims 1-35, further comprising filtering the crude mixture comprising the compound of Formula (I) with aqueous NaOH by vacuum filtration to obtain a filter cake comprising a purified form of a compound of Formula (I).

37. The process of any one of claims 1-36, further comprising washing the filter cake comprising a purified form of a compound of Formula (I) with water to form a washed filter cake comprising a compound of Formula (I).Attomev Docket No.: 38952-0017WO138. The process of claim 37, wherein the washing of the filter cake is performed twice, and wherein the filter cake is vacuum filtered to remove liquids after each wash.

39. The process of any one of claims 36 - 38, wherein the weight ratio of the compound of Formula (A4):water in each wash is about 1 :2.

40. The process of any one of claims 1-39, further comprising drying the washed filter cake comprising a compound of Formula (I) to provide a compound of Formula (I).

41. The process of claim 40, wherein the drying is performed in a vacuum oven at about 60° C for about 15 h to about 24 h.

42. The process of any one of claims 1-41, wherein the purity of the compound of Formula (I) measured by HPLC % area under the curve at 205 nm is at least 97%.

43. The process of any one of claims 1-42, further comprising preparing the compound of Formula (A3), the process comprising:contacting a compound of Formula (Al)with a compound of Formula (A2)Attorney Docket No.: 38952-0017WO1in the presence of 4M HC1 in 1,4-dioxane and a Lewis acid to form a reaction mixture HO^^OHTOC|- X OH comprising the compound of Formula (A3)44. The process of claim 43, wherein the compound of Formula (Al) contains less than about 0.1% w / w water.

45. The process of claim 43 or 44, wherein the dry weight ratio of the compound of Formula (A2):compound of Formula (Al) is about 1:0.95 to about 1:1.

46. The process of any one of claims 43-45, wherein the dry weight ratio of the compound of Formula (A2):compound of Formula (Al) is about 1:0.99.

47. The process of any one of claims 43-46, wherein the weight ratio of the compound of Formula (A2):4M HC1 in 1,4-dioxane is about 1:10 to about 1:15.

48. The process of any one of claims 43-47, wherein the weight ratio of the compound of Formula (A2):4M HC1 in 1,4-dioxane is about 1:11.8.

49. The process of any one of claims 43-48, wherein the Lewis acid comprises a salt of a metal ion selected from the group consisting of Ga3+, Sn4+, Pb2+, Sb3+, Bi3+, Sc3+, Fe2+, Fe3+, Co2+, Co3+, Ni2+, Cu2+, Zn2+, Yb3+, Ca2+, Sr2*, Al3+, In3, Sn2+, La3+, Ti4+, Zr4+, Cr3+, Ir3+, Th4+, and Pu4+, or any mixture thereof.

50. The process of any one of claims 43-49, wherein the Lewis acid comprises anhydrous ZnCl2.Attorney Docket No.: 38952-0017WO151. The process of claim 50, wherein the weight ratio of the compound of Formula (A2):ZnCh is about 1:0.1 to about 1:1.

52. The process of claim 50 or 51, wherein the weight ratio of the compound of Formula (A2):ZnCh is about 1:0.5.

53. The process of any one of claims 43-52, wherein the reaction mixture comprising the compound of Formula (Al), the compound of Formula (A2), 4M HC1 in 1,4-di oxane, and the Lewis acid is diluted with additional 1.4-dioxane in a weight ratio of the compound of Formula (A4): 1,4-di oxane of about 1 : 1 to form a diluted reaction mixture.

54. The process of claim 53, wherein the diluted reaction mixture is stirred at a temperature of about 20 °C to about 25 °C.

55. The process of any one of claims 53 or 54, wherein the diluted reaction mixture is stirred for about 40 h to about 50 h.

56. The process of any one of claims 53-55, wherein the diluted reaction mixture is stirred for about 45 h.

57. The process of any one of claims 53-56, further comprising cooling the diluted reaction mixture to about 0 °C to about 10 °C and adding water in a weight ratio of the compound of Formula (A2):water of about 1 : 1 to about 1 :2 to form a slurry comprising a compound of Formula (A3).

58. The process of claim 57, wherein the weight ratio of the compound of Formula (A2):water is about 1:1.6.

59. The process of claim 57 or 58, wherein the water is added to the reaction mixture comprising the compound of Formula (A3) over a period of about 10 min.Attorney Docket No.: 38952-0017WO160. The process of any one of claims 57-59, wherein the slurry is held at about 0 °C to about 10 °C for about 3 h.

61. The process of any one of claims 43-60, further comprising filtering the slurry and washing the filter cake comprising the compound of Formula (A3) with 1,4-dioxane in a weight ratio of the compound of Formula (A2): 1,4-dioxane of about 1:1, then washing the filter cake with water in a weight ratio of the compound of Formula (A2):water of about 1 : 1 to about 1 :2.

62. The process of claim 61, wherein the weight ratio of the compound of Formula (A2):water is about 1:1.2.

63. The process of any one of claims 43-60, further comprising filtering the slurry and washing the filter cake comprising the compound of Formula (A3) twice with 1,4-dioxane in a weight ratio of the compound of Formula (A2): 1 ,4-dioxane of about 1:1.

64. The process of any one of claims 61-63, further comprising drying the filter cake under a nitrogen atmosphere.

65. The process of any one of claims 43-64, wherein the compound of Formula (A3) is oven dried at 65 °C for about 10 to about 25 hours prior to being used as a precursor for the compound of Formula (I).

66. The process of claim 65, wherein the compound of Formula (A3) is oven dried for about 21 hours.

67. The process of any one of claims 43-66, wherein the purity of the compound of Formula (A3) measured by HPLC % area under the curve at 205 nm is at least 95%.

68. A process for removing impurities from a crude mixture comprising a compound of Formula (1)Attorney Docket No.: 38952-0017WO1the process comprising:(a) dissolving the crude mixture comprising the compound of Formula (I) in an alcohol solvent to form a solution;(b) contacting the solution with aqueous NaOH to form a high pH solution;(c) filtering the high pH solution to obtain a filtered high pH solution; and(d) contacting the filtered high pH solution with aqueous HC1 to form a slurry comprising a further purified form of a compound of Formula (I).

69. The process of claim 68, wherein the alcohol solvent comprises methanol, ethanol, or isopropanol, or any mixture thereof.

70. The process of claim 68 or 69, wherein the alcohol solvent of step (a) comprises methanol.

71. The process of any one of claims 68-70, wherein the weight ratio of the crude mixture comprising the compound of Formula (I):alcohol solvent is about 1:3 to about 1:5.

72. The process of claim 71, wherein the weight ratio of the crude mixture comprising the compound of Formula (I):alcohol solvent is about 1:4.

73. The process of any one of claims 68-72, further comprising cooling the solution of step (a) to a temperature of about 10 °C or less prior to adding the aqueous NaOH of step (b).

74. The process of claim 73, wherein the cooling is for a period of about 1 h.

75. The process of any one of claims 68-74, wherein the aqueous NaOH comprises a weight ratio ofNaOH:water of about 1:15 to about 1:20.Attorney Docket No.: 38952-0017WO176. The process of claim 75, wherein the aqueous NaOH comprises a weight ratio of NaOH:water of about 1:17.

77. The process of any one of claims 68-76, wherein the weight ratio of the crude mixture comprising the compound of Formula (I):aqueous NaOH is about 1 :4 to about 1 :5.

78. The process of claim 77, wherein the weight ratio of the crude mixture comprising the compound of Formula (I):aqueous NaOH is about 1:4.8.

79. The process of any one of claims 68-78, wherein step (b) is performed by slow addition of aqueous NaOH to the solution of step (a), wherein the temperature of the high pH solution does not exceed a temperature of about 22 °C.

80. The process of any one of claims 68-79, wherein the high pH solution of step (b) is stirred for about 50 min after all of the aqueous NaOH has been added.

81. The process of claim 80, wherein the stirring is performed at a temperature of about 20 °C.

82. The process of any one of claims 68-81, wherein the pH of the high pH solution of step (b) is about 11 to about 12.

83. The process of any one of claims 68-82, wherein the filtration of step (c) is polish filtration through a 0.1 - 1 micron filter.

84. The process of claim 83, wherein the polish filtration is through a 0.2 micron filter.

85. The process of any one of claims 68-84, further comprising cooling the filtered high pH solution to a temperature of about 10 °C or less prior to adding the aqueous HC1 of step (d).Attorney Docket No.: 38952-0017WO186. The process of any one of claims 68-85, wherein the aqueous HC1 comprises a weight ratio of water: concentrated HC1 of about 1:1 to about 1:2.

87. The process of claim 86, wherein the aqueous HC1 comprises a weight ratio of waterconcentrated HC1 of about 1:1.4.

88. The process of any one of claims 68-87, wherein the weight ratio of the crude mixture comprising a compound of Formula (I):aqueous HC1 is about 1 : 1 to about 1 :2.

89. The process of claim 88, wherein the weight ratio of the crude mixture comprising a compound of Formula (I):aqueous HC1 is about 1:1.

90. The process of any one of claims 68-89, wherein step (d) is performed by slow addition of aqueous HC1 to the cooled filtered high pH solution, wherein the temperature of the high pH solution does not exceed a temperature of about 25 °C.

91. The process of any one of claims 68-90, wherein the slurry of step (d) is stirred for about 3 h after all of the aqueous HC1 has been added.

92. The process of claim 91, wherein the stirring is performed at a temperature of about 20 °C.

93. The process of any one of claims 68-92, wherein the pH of the slurry of step (d) after all of the aqueous HC1 has been added is about 1 to about 2.

94. The process of any one of claims 68-93, further comprising filtering the slurry of step (d) by vacuum filtration to obtain a filter cake comprising the further purified form of a compound of Formula (I).Attomev Docket No.: 38952-0017WO195. The process of any one of claims 68-94, further comprising washing the filter cake comprising the further purified form of a compound of Formula (I) with water to obtain a washed filter cake comprising the further purified form of a compound of Formula (I).

96. The process of claim 95, wherein the washing of the filter cake is performed twice, and wherein the filter cake is vacuum filtered to remove liquids after each wash.

97. The process of claim 96, wherein the water used in each wash has a weight ratio of the crude mixture comprising a compound of Formula (I):water of about 1 :3 to about 1:

598. The process of claim 96 or 97, wherein the water used in each wash has a weight ratio of the crude mixture comprising a compound of Formula (I):water of about 1 :4.4.

99. The process of any one of claims 68-98, further comprising drying the filter cake comprising the further purified form of a compound of Formula (I) to provide a further purified form of a compound of Formula (I).

100. The process of claim 99, wherein the drying is performed in a vacuum oven at temperature of about 55 °C for about 26-28 h.

101. The process of any one of claims 68-100, wherein the crude mixture comprising a compound of Formula (I) is prepared using the process of any one of claims 1-67.

102. The process of any one of claims 68-101, wherein the purity of the further purified form of a compound of Formula (I) measured by HPLC % area under the curve at 205 nm is at least 99%.

103. The process of any one of 68-102, wherein the amount of 5,7-dihydroxy-3-(4-hydroxyphenyl)-2-methyl-4H-chromen-4-one present in the further purified form of a compound of Formula (I) measured by HPLC % area under the curve at 205 nm is less than 0.2 %.

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