Method for preparation quisinostat base (n-hydroxy-2-(4-((((1-methyl-1h-indol-3-yl)methyl)amino)methyl)piperidin-1-yl) pyrimidine-5-carboxamide), quisinostat hydrochloride in polymorphic form a, and intermediate compounds for their synthesis

Abstract

The present invention is directed to method of synthesis of (N-hydroxy-2-(4-((((l -methyl -1H- indol-3-yl)methyl)amino)methyl)piperidyn-l-yl)pyrimidine-5-carboxamide), Quisinostat hydrochloride in polymorphic form A, and intermediate compounds, which are useful in the treatment of diseases and disorders associated with pan-HDAC.

Description

Viriom.24.0002.WMETHOD FOR PREPARATION QUISINOSTAT BASE (N-HYDROXY-2-(4-((((l- METHYL-lH-INDOL-3-YL)METHYL)AMINO)METHYL)PIPERIDIN-l- YL)PYRIMIDINE-5-CARBOXAMIDE), QUISINOSTAT HYDROCHLORIDE IN POLYMORPHIC FORM A, AND INTERMEDIATE COMPOUNDS FOR THEIR SYNTHESISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority and benefit of U.S. Provisional Patent Application Serial No. 63 / 735,307, filed on December 17, 2024. The disclosure of this provisional application is incorporated herein by reference in its entirety for all purposes.FIELD OF INVENTION

[0002] The present invention is directed to method of synthesis of (7V-hydroxy-2-(4-((((l-methyl-lH- indol-3-yl)methyl)amino)methyl)piperidyn-l-yl)pyrimidine-5-carboxamide), Quisinostat hydrochloride in polymorphic form A, and intermediate compounds, which are useful in the treatment of diseases and disorders associated with pan-HDAC.BACKGROUND

[0003] The present invention relates to a method for synthesis a compound of formula (la), namely Quisinostat base, and Quisinostat hydrochloride (lb):

[0004] Quisinostat hydrochloride, Ar-hydroxy-2-[4-((((l-methyl-lH-indol-3- yl)methyl)amino)methyl)piperidin-l-yl]pyrimidine-5-carboxamide (compound of formula lb), is a potent, orally bioavailable second-generation pan-HDAC inhibitor (HDACi) with IC50 values ranging from 0.11 to 0.64 nM for HDACI, HDAC2, HDAC4, HDAC10, and HDACI 1.

[0005] Quisinostat exhibits broad-spectrum antitumor activity. It has demonstrated clinical efficacy both as a standalone treatment and in combination with other therapeutic agents against several types of tumors. Quisinostat suppresses cancer cell self-renewal and halts tumor progression without affecting the normal function of stem cells. It also prevents the proliferation of cells that survive1.ASBViriom.24.0002.W targeted therapies and, regardless of cancer type or resistance mechanism, inhibits the proliferation of human lung, pancreatic, and breast cancer cells. Quisinostat is not cytotoxic but can effectively prevent the growth of resistant cancer cells following cytotoxic treatment, making it a promising candidate for a novel strategy in combination cancer chemotherapy (Morales Torres, C., Wu, M.Y., Hobor, S. et al. Selective inhibition of cancer cell self-renewal through a Quisinostat-histone H1.0 axis. Nat Commun 11, 1792 (2020)).

[0006] The potent antitumor effect facilitated by Quisinostat was significantly enhanced when combined with Sorafenib. This combination therapy effectively suppressed cell proliferation and synergistically induced apoptosis. Furthermore, treatment with Quisinostat in combination with Sorafenib appears to reduce tumor volume in the HCCLM3 xenograft model. The combination of Quisinostat with Sorafenib presents a promising alternative therapy for patients with advanced hepatocellular carcinoma (He B, Dai L, Zhang X, Chen D, Wu J, Feng X, Zhang Y, Xie H, Zhou L, Wu J, Zheng S. The HDAC Inhibitor Quisinostat (JNJ-26481585) Suppresses Hepatocellular Carcinoma Alone and Synergistically in Combination with Sorafenib by G0 / G1 Phase Arrest and Apoptosis Induction. Int J Biol Sci 2018; 14(13): 1845-1858).

[0007] The methods for synthesis Quisinostat are described in the patent family “Substituted indolyl alkyl amino derivatives as novel inhibitors of histone deacetylase ”, including W02006010750, EA 010652B1, and US20150342950.

[0008] The method described in these patents includes the following reaction steps:ASBViriom.24.0002.W

[0009] Hydroxamic acids of formula (I) can be obtained by reacting intermediate compounds of formula (II), where the protecting group is tetrahydropyranyloxyaminocarbonyl (referred to as intermediate compounds of formula (II-a)), with an appropriate acid, such as tri fluoroacetic acid. This reaction is carried out in a suitable solvent, such as methanol or dichloromethane.

[0010] It has been established that, alternatively, hydroxamic acids of formula (I) can be obtained by reacting intermediate compounds of formula (II), where Q represents a C1-2 alkoxycarbonyl group (referred to as intermediate compounds of formula (II-c)), with hydroxylamine in the presence of a base, such as sodium hydroxide. This reaction is carried out in a suitable solvent, such as methanol:

[0011] The scheme of the reaction is presented below:

[0012] In the commonly used synthesis method, intermediate compounds of formula (II) are utilized as core building blocks, where Q represents C1-2 alkoxy carbonyl, hydroxycarbonyl, or tetrahydropyranyloxy aminocarbonyl.

[0013] The use of these functional groups requires the introduction of protecting agents, such as (2- bromoethoxy)(l, 1 -dimethyl ethyl)dimethylsilane, 1,3-isobenzofurandione, l-[[(9H-fluoren-9- yloxy)methoxy]carbonyl]oxy-2,5-pyrrolidinedione (Fmoc-Osu), or the use of specific reagents, such as An-(ethylcarbodiimido)-A,iV-dimethyl-l,3-propanediamine monohydrochloride (EDC) and 1- hydroxy-lH-benzotriazole (HOBt).

[0014] Additionally, the synthesis of intermediate compounds of formula (II-a) involves a reductive amination reaction in the presence of a reagent such as sodium borohydride. During this reaction, free3.ASBViriom.24.0002.W hydrogen is released, posing a fire hazard. Therefore, the use of sodium borohydride is undesirable for large-scale production due to safety concerns and environmental impact considerations.

[0015] Therefore, there is a need for an improved process for obtaining Quisinostat hydrochloride. Specifically, it would be desirable to have a method that does not involve expensive specialized protecting agents or specific process-activating reagents, eliminates the step requiring sodium borohydride, and is efficient in terms of yield and chemical purity of the product, cost-effective in terms of reagents and reaction conditions, more environmentally friendly, and suitable for industrialscale application. Such a method is described in this invention.SUMMARY

[0016] The present invention relates to a method for obtaining a compound of formula (la), i.e., Quisinostat base, and Quisinostat hydrochloride (lb), which is used as an active ingredient in a pharmaceutical preparation:which involves the sequential treatment of a compound of formula (II), first by refluxing with di-terl- butyl dicarbonate (BOC2O), and then with concentrated HC1 and an aqueous solution of sodium nitrite at a temperature of 0°C.

[0017] This method of preparation Quisinostat hydrochloride has several advantages compared to the previously used method: 1) The synthetic pathway involves three stages of chemical transformation, which is one stage shorter than the previously proposed method, increasing the overall yield of the product by 10% and eliminating the use of expensive specialized protecting agents and sodium borohydride; 2) The result is the active ingredient Quisinostat hydrochloride of pharmaceutical purity in the form of an individual thermodynamically stable Polymorph A, rather than a mixture of polymorphic forms that transition into each other during storage. This has led to the production of a highly stable substance for storage.4.ASBViriom.24.0002.W

[0018] According to the new method, a compound of Formula (lb), i.e., Quisinostat hydrochloride, can be obtained from a compound of Formula (la), i.e., Quisinostat base, by treatment with an aqueous-ethanolic solution of concentrated HC1.

[0019] A compound of Formula (la), i.e., Quisinostat base, can also be obtained from an intermediate compound of Formula (III) through enzymatic synthesis using a cell-free extract of Rhodococcus erythropolis A4 with nitrilase and amidase activity.

[0020] The present invention also relates to a method for preparation the intermediate compound "- hydroxy-2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5- carboxamidine (compound of formula (II)):which includes the reaction of the intermediate compound 2-(4-((((l -methyl-lH-indol-3- yl)methyl)amino)methyl)piperi din-1 -yl)pyrimidine-5-carbonitrile (compound of formula (III)) with hydroxylamine hydrochloride under reflux in methanol.

[0021] The present invention also relates to a method for preparation the intermediate compound 2- (4-((((l -methyl- lH-indol-3-yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carbonitrile (compound of formula (III)):which includes the reaction of (l-methyl-lH-indol-3-yl)methanamine (compound of formula (V)):5.ASBViriom.24.0002.Wwherein X is Cl, Br, I, methyl sulfonate, or trifluoromethylsulfonate, in a suitable solvent in the presence of a base.

[0022] The present invention also relates to a method for purifying a compound of formula (lb), i.e., Quisinostat hydrochloride, to obtain a solid form of Quisinostat hydrochloride (Form A), characterized by an XRPD pattern disclosed herein.

[0023] In some aspects, the present disclosure provides a method of preparing the compound of the present disclosure, comprising one or more steps described herein.

[0024] In some specific non-limiting aspects, the present disclosure provides methods of preparing the compound of the present disclosure, described in the examples.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. In the case of conflict between the chemical structures and names of the compounds disclosed herein, the chemical structures will control.6.ASBViriom.24.0002.W

[0026] Other features and advantages of the disclosure will be apparent from the following detailed description, claims and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows XRPD profde of solid Form A of the compound of Formula (lb), Quisinostat hydrochloride.

[0028] Figure 2 showsJH NMR spectrum of the compound of formula (lb), Quisinostat hydrochloride.

[0029] Figure 3 shows13C NMR spectrum of the compound of formula (lb), Quisinostat hydrochloride.

[0030] Figure 4 shows IR spectrum of solid Form A of the compound of formula (lb), Quisinostat hydrochloride.

[0031] Figure 5 shows mass spectrum of the compound of formula (lb), Quisinostat hydrochloride.

[0032] Figure 6 shows UV absorption spectrum of the compound of formula (lb), Quisinostat hydrochloride.DETAILED DESCRIPTION

[0033] The present invention relates to a method for preparation a compound of Formula (la), i.e., Quisinostat base, and Quisinostat hydrochloride (lb) in polymorphic Form A, which involves three stages of chemical transformation.

[0034] The details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, illustrative methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated herein by reference in their entireties.Definitions7.ASBViriom.24.0002.W

[0035] The articles "a" and "an" are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0036] The term "and / or" is used in this disclosure to mean either "and" or "or" unless indicated otherwise.

[0037] The term “optional” or “optionally” means that the subsequently described event or circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

[0038] As used herein, the term “substituted” means that the specified group or moiety bears one or more suitable substituents wherein the substituents may connect to the specified group or moiety at one or more positions. For example, an aryl substituted with a cycloalkyl may indicate that the cycloalkyl connects to one atom of the aryl with a bond or by fusing with the aryl and sharing two or more common atoms.

[0039] The term "solvate" refers to a complex of variable stoichiometry formed by a solute and solvent. Such solvents for the purpose of the disclosure may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, MeOH, EtOH, and AcOH. Solvates wherein water is the solvent molecule are typically referred to as hydrates. Hydrates include compositions containing stoichiometric amounts of water, as well as compositions containing variable amounts of water.

[0040] The present disclosure also contemplates isotopically-labelled compounds of Formula (I) (e.g., those labeled with2H and14C). Deuterated i.e.,2H or D) and carbon-14 (i.e.,14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labelled compounds of Formula {I) can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or in the Examples herein below, by substituting an appropriate isotopically labelled reagent for a non-isotopically labelled reagent.

[0041] The term “salt” refers to pharmaceutically acceptable salts.

[0042] The term “pharmaceutically acceptable salt” also refers to a salt of the compositions of the present disclosure having an acidic functional group, such as a carboxylic acid functional group, and a base.8.ASBViriom.24.0002.W

[0043] The isolation and purification procedures described herein can be carried out, if desired, using any suitable separation or purification method, such as filtration, extraction, crystallization, column chromatography, thin layer chromatography, or preparative chromatography, or a combination of these techniques. Specific examples of suitable separation and isolation methods are provided below. However, other equivalent techniques may also be used.Methods of the Present Disclosure

[0044] The preparation of Quisinostat hydrochloride (lb) involves the sequential treatment of a compound of formula (II), first by refluxing with di-Ze / v-butyl dicarbonate (BOC2O), and then with concentrated HC1 and an aqueous solution of sodium nitrite at a temperature of 0°C.

[0045] Alternatively, a compound of formula (lb), i.e., Quisinostat hydrochloride, can be obtained from a compound of Formula (la), i.e., Quisinostat, by treatment with an aqueous-ethanolic solution of concentrated HC1.

[0046] The method for obtaining Quisinostat salts, described in WO 2006 / 010750, published on February 2, 2006, discloses the amorphous forms of Quisinostat salts - the C2HF3O2 salt and the dihydrochloride salt, as well as methods for their preparation, which are presented in Scheme below:ASBViriom.24.0002.W

[0047] According to this scheme, at stage 1, the intermediate product of formula (V) was obtained by reacting the intermediate product of formula (I) with the carboxaldehyde of formula (II) in the presence of sodium borohydride in methanol (a reductive amination reaction).

[0048] At stage 2, the intermediate product of formula (IV) was obtained by reacting the intermediate product of formula (V) with sodium hydroxide in ethanol.

[0049] At stage 3, the intermediate product of formula (VII) was obtained by reacting the intermediate product of formula (IV) with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (VI) in the presence of appropriate reagents such as V-(ethylcarbonyldiimido)-V,V-dimethyl-l,3-propanediamine monohydrochloride (EDC) and 1-hydroxy-lH-benzotriazole (HOBT). The reaction was carried out in a mixture of dichloromethane and tetrahydrofuran.

[0050] At stage 4, the C2HF3O2 salt of hydroxamic acid of formula (VI) was obtained by reacting the intermediate product of formula (VII) with trifluoroacetic acid. The reaction was carried out in methanol.

[0051] The method for obtaining the compound of formula (lb), i.e., Quisinostat hydrochloride, as described in WO 2008 / 138918, is depicted in Scheme below. The intermediate compound of formula (Ila) can be converted into the HC1 salt of formula (lb) by adding hydrochloric acid in a suitable solvent, such as ethanol or methanol, at room temperature with vigorous stirring of the reaction mixture:

[0052] Unlike the methods known in the prior art, the method of the present invention is depicted in the following scheme:10.ASBViriom.24.0002.W

[0053] To a solution of JV-hydroxycarboximidamide (II) in dioxane, a double excess of Boc anhydride (BOC2O) is added in small portions under cooling. The reaction mixture is then stirred at 50°C for 3 hours. Subsequently, the reaction mixture is cooled to 0°C, concentrated HC1 is added, and while maintaining the reaction temperature at 0°C, an aqueous solution of sodium nitrite is added dropwise with vigorous stirring. The mixture is stirred at 0°C for 45 minutes. After the reaction is complete, the reaction mixture is diluted with water, and the precipitate is filtered, washed on the filter with acetonitrile, and dried. A powder of white or nearly white color with a light brown tint is obtained.

[0054] The compound of formula (la), i.e., Quisinostat, can be obtained from the intermediate compound of formula (III) through enzymatic synthesis using a cell-free extract of Rhodococcus erythropolis A4 with nitrile hydratase and amidase activity.

[0055] The cell-free extract from Rhodococcus erythropolis A4 was used as a catalyst in the first stage of the reaction (nitrile hydration), which was carried out at 20°C and pH 8 in a Tris / HCl buffer (50 mM, pH 8). The buffer contained 15 mM substrate, methanol (3% v / v) as a cosolvent, and an appropriate amount of nitrile hydratase. Ammonium sulfate was added to a final concentration of 240 mM when using the cell-free extract from the A4 strain. After 48 hours, hydroxylamine (from a 2 M stock solution) was added to a final concentration of 500 mM, the temperature was increased to 28°C, and the reaction was continued by adding another portion of the cell-free extract. The formation of hydroxamic acid was monitored spectrophotometrically. Upon completion of the reaction, the reaction mixture was centrifuged and extracted with ethyl acetate. The ethyl acetate fractions were combined, concentrated under reduced pressure, and the solid residue was recrystallized from ethanol.11.ASBViriom.24.0002.W

[0056] Alternatively, the compound of formula (lb), i.e., Quisinostat hydrochloride, can be obtained from the compound of formula (la), i.e., Quisinostat, by adding an aqueous solution of concentrated HC1 to an alcoholic solution of Quisinostat (la) at room temperature. The resulting precipitate is filtered, washed on the filter with acetonitrile, and dried. A powder of white or nearly white color with a light brown tint is obtained.

[0057] The synthesis of the intermediate compound of formula (II) can be carried out by reacting the intermediate compound of formula (III) with hydroxylamine hydrochloride:

[0058] To a solution of the nitrile (111) in isopropanol, a 1.1 -fold excess of hydroxylamine hydrochloride is added. Then, with vigorous stirring for 1 hour at room temperature, a 1.2-fold excess of NaHCCh is added in small portions. The reaction mixture is then heated to 50°C and stirred for an additional hour. After the reaction is complete, the mixture is cooled to room temperature, filtered to remove inorganic salts, and the filtrate is evaporated to dryness under reduced pressure. The solid residue is recrystallized from ethanol.

[0059] The synthesis of the intermediate compound of formula (III) can be carried out by reacting the intermediate compounds of formulas (IV) and (V) in the presence of a base:

[0060] To a solution of the intermediate compounds of formulas (V) and (IV) in dry dioxane, potassium carbonate is added while stirring, and the mixture is heated to 60-80°C. Heating is continued for 4-6 hours. The reaction mixture is then cooled and diluted with water. The reaction12.ASBViriom.24.0002.W product — the intermediate compound of formula (III) — precipitates as a solid. It is fdtered, washed with cold water, and recrystallized from 80% isopropanol.

[0061] The intermediate compounds of formulas (V) and (IV) are commercially available.Preparation and characterization of the solid Form A of Quisinostat hydrochloride (lb)

[0062] A method for synthesizing Quisinostat hydrochloride (lb) has been proposed, in which only one solid form of Quisinostat hydrochloride (Form A) is obtained, namely recrystallization from methanol. Thus, the production process for Quisinostat hydrochloride (lb) excludes the formation of any other polymorphic forms, except for Form A.

[0063] The solid form, Form A, of the compound of formula (lb) can be characterized by an XRPD pattern containing peaks at approximately 7.5, 12, 12.5, 15, 17, 23.5, 24.5, 25, 26, and 28. The solid form can be further characterized by an XRPD pattern (Fig. 1), containing the peaks described in Table 1

[0064] Table 1 Characteristic peaks of the XRPD pattern of Quisinostat hydrochloride, Form A, of the compound of formula (lb).13.ASBViriom.24.0002.W

[0065] The solid form, Form A, of the compound of formula (Tb) exhibits improved stability properties during storage. The reduced hygroscopicity and slower accumulation of impurities during storage have allowed the shelf life of the substance to be extended to 5 years (see Table 2).

[0066] Table 2. The stability study results of Quisinostat hydrochloride, Form A, in primary and secondary packaging (high-density polyethylene bag, combined material bag based on aluminum foil) under the conditions: temperature 25±2°C, humidity 60±5% for 5 years.

[0067] In some embodiments, the compound of Formula (I) is an isotopic derivative of any one of the compounds disclosed herein.

[0068] It is understood that the isotopic derivative can be prepared using any of a variety of art- recognized techniques. For example, the isotopic derivative can generally be prepared by carrying out the procedures disclosed in the Schemes and / or in the Examples described herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0069] In some embodiments, the isotopic derivative is a deuterium labeled compound.14.ASBViriom.24.0002.W

[0070] In some embodiments, the isotopic derivative is a deuterium labeled compound of any one of the compounds of the Formulae disclosed herein.

[0071] The term “isotopic derivative”, as used herein, refers to a derivative of a compound in which one or more atoms are isotopically enriched or labelled. For example, an isotopic derivative of a compound of Formula (I) is isotopically enriched with regard to, or labelled with, one or more isotopes as compared to the corresponding compound of Formula (I). In some embodiments, the isotopic derivative is enriched with regard to, or labelled with, one or more atoms selected from2H,13C,14C,15N,18O,31P, and34S. In some embodiments, the isotopic derivative is a deuterium labeled compound (z.e., being enriched with2H with regard to one or more atoms thereof).

[0072] It is understood that the deuterium labeled compound comprises a deuterium atom having an abundance of deuterium that is substantially greater than the natural abundance of deuterium, which is 0.015%.

[0073] In some embodiments, the deuterium labeled compound has a deuterium enrichment factor for each deuterium atom of at least 3500 (52.5% deuterium incorporation at each deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation). As used herein, the term “deuterium enrichment factor” means the ratio between the deuterium abundance and the natural abundance of a deuterium.

[0074] It is understood that the deuterium labeled compound can be prepared using any of a variety of art-recognized techniques. For example, the deuterium labeled compound can generally be prepared by carrying out the procedures disclosed in the Schemes and / or in the Examples described herein, by substituting a deuterium labeled reagent for a non-deuterium labeled reagent.

[0075] A compound of the disclosure or a pharmaceutically acceptable salt or solvate thereof that contains the aforementioned deuterium atom(s) is within the scope of the disclosure. Further, substitution with deuterium (z.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.

[0076] It is to be understood that the compounds of any Formula described herein include the compounds themselves, as well as their salts, and their solvates, if applicable. A salt, for example, can be formed between an anion and a positively charged group (e.g., amino) on a substituted compound disclosed herein. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate,15.ASBViriom.24.0002.W sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., tri fluoroacetate).

[0077] It is to be understood that the compounds of the present disclosure, for example, the salts of the compounds, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Nonlimiting examples of hydrates include monohydrates, dihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0078] As used herein, the term “solvate” means solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H2O.

[0079] As used herein, the term “analog” refers to a chemical compound that is structurally similar to another but differs slightly in composition (as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog is a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.

[0080] It is also to be understood that certain compounds of any one of the Formulae disclosed herein may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. A suitable pharmaceutically acceptable solvate is, for example, a hydrate such as hemi-hydrate, a mono-hydrate, a di-hydrate or a tri-hydrate. It is to be understood that the disclosure encompasses all such solvated forms that possess inflammasome inhibitory activity.

[0081] It is also to be understood that certain compounds of any one of the Formulae disclosed herein may exhibit polymorphism, and that the disclosure encompasses all such forms, or mixtures thereof, which possess inflammasome inhibitory activity. It is generally known that crystalline materials may be analysed using conventional techniques such as X-Ray Powder Diffraction analysis, Differential Scanning Calorimetry, Thermal Gravimetric Analysis, Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy, Near Infrared (NIR) spectroscopy, solution and / or solid state nuclear magnetic resonance spectroscopy. The water content of such crystalline materials may be determined by Karl Fischer analysis.16.ASBViriom.24.0002.W

[0082] Compounds of any one of the Formulae disclosed herein may exist in a number of different tautomeric forms and references to compound of the disclosure include all such forms. For the avoidance of doubt, where a compound can exist in one of several tautomeric forms, and only one is specifically described or shown, all others are nevertheless embraced by Formulae disclosed herein. Examples of tautomeric forms include keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto / enol (illustrated below), imine / enamine, amide / imino alcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, and nitro / aci-nitro.keto enol enolatePreparation of Compounds

[0083] The compounds of the present invention can be prepared in a number of ways well known to those skilled in the art of organic synthesis. By way of example, compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art. Suitable methods include but are not limited to those methods described below. Compounds of the present invention can be synthesized by following the steps outlined in the part “Examples” which comprise different sequences of assembling intermediates or compounds. Starting materials are either commercially available or made by known procedures in the reported literature or as illustrated below.EXAMPLESGeneral synthetical procedures and examples of the compound’s preparation.

[0084] All reagents were commercial and were used without further purification. Yields refer to purified and spectroscopically pure compounds. Thin layer chromatography (TLC) was performed using Merck TLC Aluminum sheets silica gel 60 F254 plates and visualized by fluorescence quenching under UV light. Flash chromatography was performed using silica gel (Chromatorex, MB 70-40 / 75, 40-75 pm) purchased by Fuji Silysia Chemicals.

[0085] NMR spectra were recorded on a Varian-400MR operating at 400 MHz for 'H. Chemical shifts are reported in ppm with the solvent resonance as the internal standard. Data is reported as follows: s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets; coupling constants in Hz; integration.

[0086] Abbreviations used in the following examples and elsewhere herein are:17.ASBViriom.24.0002.WACN acetonitrile aq. aqueousDMSO dimethyl sulfoxide eq equivalent g gram, gas h hour(s)HPLC high pressure (or performance) liquid chromatographyL literLCMS liquid chromatography mass spectrometryM molarMHz megahertz min minutes mL milliliterNMR nuclear magnetic resonance rt room temperature t temperature, tripletTLC thin layer chromatographySynthesis of V-Hydroxy-2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin-l- yl)pyrimidine-5-carboxamide hydrochloride (lb)

[0087] Experiment 1. Synthesis of ;V-Hydroxy-2-(4-(((( l -methyl- l H-indol-3- yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboxamide hydrochloride (lb).

[0088] To a solution of JV-hydroxycarboximidamide (II) (1 eq) in dioxane, 2 eq of Boc anhydride (BOC2O) were added in small portions under cooling. The reaction mixture is then stirred at 50°C for 3 h. Subsequently, the reaction medium is cooled to 0°C, concentrated HC1 was added, and an aq. solution of sodium nitrite was added dropwise under vigorous stirring while maintaining the temperature of the reaction medium at 0°C. The mixture was stirred at 0°C for 45 min. After the reaction was complete, the reaction mixture was diluted with water, the resulting precipitate was18.ASBViriom.24.0002.W filtered, washed on the filter with acetonitrile, and dried. A crystalline powder of white or nearly white color with a light brown hue was obtained.Synthesis of \-Hydroxy-2-(4-(((( 1 -methyl- 111-indol-3-yl)methyl)amino)methyl)piperidin-l- yl)pyrimidine-5-carboxamide hydrochloride (lb)

[0089] Experiment 2. Synthesis of A-Hydroxy-2-(4-((((l-methyl-lH-indol-3- yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboxamide hydrochloride (lb).

[0090] A-Hydroxy-2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin-l- yl)pyrimidine-5-carboxamide (compound of formula (la)) was dissolved in acetonitrile. The calculated amount of concentrated HC1 was added dropwise with vigorous stirring over the course of an hour. The reaction mixture was then stirred for an additional hour. A gradual formation of a precipitate, white or nearly white in color, was observed, and the reaction mixture thickens. The reaction medium was then diluted with water, the precipitate was filtered, washed on the filter with acetonitrile, and dried. A crystalline powder was obtained, ranging in color from white to light brown with pinkish or grayish hues.

[0091] 'I I-NMR. of lb:JH NMR (599 MHz, DMSO-tfc), 5: 11.21 (s, 1H), 9.25 (s, 1H), 8.71 (s, 2H),7.83 (d, J= 7.9 Hz, 1H), 7.64 (s, 1H), 7.49 (d, .7 = 8.2 Hz, 1H), 7.23 (t, J= 7.6 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 4.70 (d, J = 13.4 Hz, 2H), 4.30 (t, J ------ 5.2 Hz, 2H), 3.83 (s, 3H), 2.99 - 2.91 (m, 2H),2.84 (q, <7= 6.4 Hz, 2H), 2.16 - 2.09 (m, 1H), 1.90 - 1.84 (m, 2H), 1.14 (qd, .7= 12.5, 4.1 Hz, 2H).

[0092] 13C-NMR ofIb:13C NMR (151 MHz, DMSO- s), 6: 162.40, 161.73, 157.66, 136.87, 132.18,127.76, 122.20, 119.88, 1 19.47, 114.77, 110.49, 104.40, 51.08, 43.52, 41.90, 40.55, 40.43, 40.29,40.16, 40.02, 39.88, 39.74, 39.60, 33.41, 33.17, 29.65.

[0093] Correlation of absorption bands for Quisinostat hydrochloride, compound of formula (lb)19.ASBViriom.24.0002.WSynthesis of 7V-hydroxy-2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyI)piperidin- yl)pyrimidine-5-carboximidamide (II)yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboximidamide (compound of formula (II)).

[0095] A mixture of compound (III) (1 eq), hydroxylamine hydrochloride (3 eq), and sodium carbonate (3 eq) in ethanol was refluxed overnight with stirring. The mixture was then cooled to rt, and the precipitate was filtered. The filtrate was concentrated under vacuum using a rotary evaporator. Diethyl ether was added to the residue, the precipitate was filtered, and the filtrate was again concentrated under vacuum using a rotary evaporator. Compound (II) was obtained as a yellow oil with a yield of 90-95%. 'H NMR (400 MHz, DMSO-< / 6), 5: 1.00 - 1.17 (m, 2H, piperidine), 1.73 - 1.90 (m, 3H, piperidine), 2.46 (m, 2H, CH2), 2.97 (td, J= 12.9, 2.7 Hz, 2H, piperidine), 3.69 (s, 3H, N-Me), 3.80 (s, 2H, CH2N), 4.63 (d, J = 13.3 Hz, 2H, piperidine), 6.88 - 7.03 (m, 1H, Ar-H), 7.03 - 7.12 (m, 1H, Ar-H), 7.13 (s, 1H, indole-H), 7.32 (d, J= 8.2 Hz, 1H, Ar-H), 7.57 (d, J= 7.9 Hz, 1H, Ar-H), 8.64 (s, 2H, pyrimidine-H), 10.05 (s, 1H, N-OH).Synthesis of 2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine- 5-carbonitrile (III)20.ASBViriom.24.0002.W

[0096] Experiment 4. Synthesis of 2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin- l-yl)pyrimidine-5-carbonitrile (compound of formula (III)).

[0097] A solution of (l-methyl-lH-indol-3-yl)methanamine (V) (1 eq) and 2-(4-(chloromethyl)piperidin-l-yl)pyrimidine-5-carbonitrile (IV) (1 eq) in ACN was heated in the presence of K2CO3 (5 eq) for 20 h. The completion of the reaction was monitored by LCMS analysis. The reaction mixture was then concentrated using a rotary evaporator, and methanol was added. The resulting crystals are filtered and dried. 'H NMR (400 MHz, DMSO-t / (>), 8: 0.88 - 1.15 (m, 2H, piperidine), 1.66 - 1.91 (m, 3H, piperidine), 2.43 (m, 2H, CH2), 2.96 (td, J = 12.7, 2.7 Hz, 2H, piperidine), 3.69 (s, 3H, N-Me), 3.79 (s, 2H, CH2N), 4.56 - 4.74 (m, 2H, piperidine), 6.88 - 7.02 (m, 1H, Ar-H), 7.02 - 7.11 (m, 1H, Ar-H), 7.13 (s, 1H, indole-H), 7.31 (d, J= 8.2 Hz, 1H, Ar-H), 7.56 (d, J= 7.9 Hz, 1H, Ar-H), 8.64 (s, 2H, pyrimidine-H).

[0098] Experiment 5. Synthesis of Quisinostat hydrochloride (lb) in solid Form A.

[0099] The substance Quisinostat hydrochloride (lb) in solid form A is obtained from A-hydroxy-2- (4-((((l -methyl- lH-indol-3-yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboxamide (compound of formula (la)) by recrystallization from methanol.Equivalents

[0100] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.21.ASB

Claims

Viriom.24.0002.WCLAIMSWhat is claimed is:

1. A method for synthesis of A-hydroxy-2-(4-((((l-methyl-lH-indol-3- yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboxamide hydrochloride (lb), which comprises (1) the reaction of 2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin-l- yl)pyrimidine-5-carbonitrile (compound of formula (III)) with hydroxylamine hydrochloride:the reaction of A -hydroxy-2-(4-((((l -methyl- lH-indol-3-yl)methyl)amino)methyl )piperi din- 1- yl)pyrimidine-5-carboximidamide with di- / c / 7-butyl dicarbonate and sodium nitrite:

2. The method according to claim 1, wherein A-hy droxy-2-(4-((((l -methyl- lH-indol-3- yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboxamide hydrochloride (lb) is in in polymorphic form A, characterized by major peaks at 7.5; 12; 12.5; 15; 17; 23.5; 24.5; 25; 26; 28.

3. The method according to claim 1, wherein the reaction of 2-(4-((((l-methyl-lH-indol-3- yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carbonitrile (compound of formula (III)) with hydroxylamine hydrochloride (1) is carried out in ethanol in the presence of a base.22.ASBViriom.24.0002.W4. The method according to claim 3, wherein the base is sodium carbonate.

5. The method according to claim 1, wherein the reaction of A^-hydroxy-2-(4-((((l-methyl-lH- indol-3-yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboximidamide with di-Z / 'Z-butyl dicarbonate and sodium nitrite Boc anhydride is added in small portions under cooling to dioxane solution of V-hydroxy-2-(4-((((l -methyl- lH-indol-3-yl)methyl)amino)methyl)piperidin-l - yl)pyrimidine-5-carboximidamide.

6. The method according to claim 5, wherein 2 equivalents of Boc anhydride are added.

7. The method according to claim 5, wherein concentrated HC1 is added, and an aqueous solution of sodium nitrite is added dropwise under vigorous stirring while maintaining the temperature of the reaction medium at 0°C after Boc anhydride was added.

8. A method for synthesis of jV-hydroxy-2-(4-((((l-methyl-lH-indol-3- yl)methyl)amino)methyl)piperidin-l-yl)pyrimidine-5-carboximidamide (II):which comprises the reaction of 2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin-l- yl)pyrimidine-5-carbonitrile (III) with hydroxylamine hydrochloride.

9. A method for synthesis of 2-(4-((((l-methyl-lH-indol-3-yl)methyl)amino)methyl)piperidin-l- yl)pyrimidine-5-carbonitrile (III):23.ASBViriom.24.0002.Wwhich comprises the reaction of (l-methyl-lH-indol-3-yl)methanamine (V) and compound (IV) in in the presence of base, wherein X is selected from Cl, Br, I CH3SO2O- and CF3SO2O-.

10. A solid Form A of Quisinostat hydrochloride, defined by its XRPD pattern shown in Figure 1.

11. The solid form according to claim 10, defined by its XRPD pattern comprising the peaks described in Table below:24.ASBViriom.24.0002.W25.ASB

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