FIELD OF THE INVENTION

[0001] The present invention relates to a process of preparing alpha amino-acid derivatives, in particular the invention relates to manufacturing of eflornithine derivatives and their pharmaceutically acceptable salts thereof.

BACKGROUND OF THE INVENTION

[0002] Eflornithine was first prepared in 1970 and belongs to the class of organic compounds known as alpha amino acids having diverse therapeutic use. It was initially developed for cancer treatment at Merrell Dow Research Institute, but was found to be ineffective in treating malignancies but effective in reducing hair growth and in treatment of African trypanosomiasis.

[0003] Eflornithine, is a prescription drug sold under the brand name Vaniqa, Ornidyl among others, is used to treat excessive hair growth on the face in women and African trypanosomiasis (sleeping sickness).

[0004] Clinical studies shows that eflornithine acts as chemoprevention agent by lowering polyamine levels in colorectal mucosa and skin carcinogenesis. Thus eflornithine is used in combination with other medication for treating cancers.

[0005] International application WO2019243970 discloses derivatives of eflornithine derivatives among others and the methods to manufacturing the same. The present invention describes an industrially applicable process for manufacturing the eflornithine derivatives and their pharmaceutically acceptable salts and other structurally related compounds using economical reagents and chemicals for better quality, yield and stability.

SUMMARY OF THE INVENTION

[0006] In an embodiment, the present invention discloses the synthesis of eflornithine derivatives, their pharmaceutically acceptable salts thereof.

[0007] In an embodiment, the present invention discloses the synthesis of compounds having general formula I:

Formula I wherein,

RH independently represents caprylic acid, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2- oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fnmaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid , glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hydrochloric acid, hippuric acid, hydrobromic acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene- 1,5-disulfonic acid, naphthalene-2- sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, undecylenic acid, omega 3 fatty acids, omega 6 fatty acids, n-acetyl cysteine (nac), furoate, methyl furoate, ethyl furoate, aminocaproic acid, caprilic acid, alpha lipoic acid, R-lipoic acid, myristic acid, myristoleic acid, palmitoleic acid, phospholipids, phosphatidylcholine, oleic acid, elaidic acid, linoleic acid, linolenic acid, menthol, retinoic acid, vitamin A, retinol, linolelaidic acid, arachidonic acid, retinal, isotretinoin, curcumin, tretinoin, a-carotene b-carotene retinol, d2 ergosterol, ergocalciferol, 7- dehydrocholesterol, cholecalciferol, 25-hydro xycholecalciferol, calcitriol ( 1 ,25- dihydroxycholecalciferol), calcitroic acid, d4 dihydroergocalciferol, alfacalcidol, dihydrotachysterol, calcipotriol, tacalcitol, paricalcitol, tocopherol, naphthoquinone, phylloquinone (kl), menaquinones (k2), menadione (k3), menadiol (k4), thiamine, acefurtiamine, allithiamine, benfotiamine, fursultiamine, octotiamine, prosultiamine, sulbutiamine, riboflavin, niacin, nicotinamide, pantothenic acid, dexpanthenol, pantethine, pyridoxine, pyridoxal phosphate, pyridoxamine, pyritinol, biotin, folic acid, dihydrofolic acid, folinic acid, levomefolic acid, adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin, choline, dehydroascorbic acid orl-docosanol.

[0008] In an embodiment, the present invention discloses the synthesis of pharmaceutically acceptable salts of Formula I.

[0009] In an embodiment the present invention discloses a two-step process/ method of synthesizing 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride (CLX-SYN-G164A-C02).

[0010] In an embodiment, the first step comprises preparation of 2-propylpentanoyl chloride or valproic acid chloride by nucleophilic substitution reaction and the second step involves preparing final product 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride from 2-propylpentanoyl chloride.

[0011] In another embodiment the polar solvent used in the synthesis is selected from polar/non polar solvent or protic/aprotic solvent.

[0012] In another embodiment the non-limiting examples of non-polar solvent include hexane, benzene, toluene, diethyl ether, cholorform, dichloromethane (DCM), 1,4-dioxane or/and others those well known in the art.

[0013] In another embodiment the non-limiting examples of polar protic solvent include acetic acid, n-butanol, isopropanol, n-propanol, ethanol, methanol, formic acid, water and others known in the art.

[0014] In another embodiment the non-limiting examples of polar aprotic solvent include ethyl acetate, terhydrofuran (THF), dichloromethane, methyl tertiary butyl ether (MTBE), acetone, acetonitrile (MeCN), dimethylformamide (DMF) and others known in the art.

[0015] In another embodiment the final product CLX-SYN-G164A-C02 is produced in the form of amorphous or crystalline form or a combination.

[0016] In another embodiment the non- limiting examples of pharmaceutically acceptable salts of the compound of Formula I include non-toxic acid addition salts formed with inorganic acids, such as hydrochloric, hydrobromic, sulfuric, phosphoric acid and others known in art and organic acids, such as methane sulfonic, salicylic, maleic, malonic, tartaric, citric, cyclamic, ascorbic acids, short chain and long chain fatty acids which can be saturated or unsaturated and others known in art; and non-toxic salts formed with inorganic or organic bases, such as those of alkali metals, for example, sodium, potassium, and lithium, alkaline earth metals, for example, calcium and magnesium, light metals of Group III A, for example, aluminium, organic amines, such as primary, secondary, or tertiary amines, for example, cyclohexylamine, ethylamine, pyridine, (methylamino)ethanol, ethanolamine, and piperazine. The salts are prepared by conventional means

[0017] In an embodiment, present invention discloses a process for preparing 2-Amino-2- (difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride or a pharmaceutically acceptable polymorphs, stereoisomers, enantiomers thereof, comprising synthesis of 2- Propylpentanoyl Chloride and reacting the 2-Propylpentanoyl Chloride with Eflornithine HC1 in 5N Ag NaOH.

[0018] In an embodiment, the reaction process takes place in a polar or a non-polar solvents or mixtures thereof, selected from the group consisting of acetone, acetic acid, ethyl methyl ketone, methyl isobutyl ketone, isopropanol, acetonitrile, ammonia (aqueous), n-amyl acetate, amyl alcohol, aniline, benzene, 2-butanone (MEK), n-butyl acetate, n-butyl alcohol, methanol, ethanol, propanol, n-propanol, 1- butanol, 2-butanol, tert-butyl alcohol, 2-butanone, pentane, formic acid, hexane, heptane, carbon tetrachloride, carbon disulfide, chlorobenzene, chloroform, chloromethene, diethylamine, cyclohexane, o -dichlorobenzene (1,2-Dichlorobenzene), 1,2-dichloroethane, diethyelene glycol, diethyl ether, N,N-dimethyl acetamide, ethylene glycol, p-xylene, toluene, N,N- dimethylaniline, Ethyl Acetate Ethanol, ethyl acetate, 1,4-Dioxane, acetonitrile, water, heavy water or mixtures thereof in any proportion. In specific embodiment the solvent is selected from dichloromethane methanol, acetone, acetonitrile or a mixture thereof.

[0019] In an embodiment, present invention discloses the method of synthesizing 2- Propylpentanoyl Chloride by reacting valproic acid with an acid halide under suitable reaction conditions.

[0020] In another embodiment, the acid halide is selected from chlorosulfuric acid (C1S03H), sulfuryl chloride (S02C12), sulfuric acid (H2S04), thionyl chloride (SOC12), phosphorus trichloride, phosphorus oxychloride, or mixtures thereof and mixtures thereof in any proportion. In a specific embodiment acid halide is thionyl chloride.

[0021] In another embodiment, present invention discloses the reaction conditions wherein the reaction of valproic acid with the acid halide occurs at a temperature ranging from about 25-30°C over a period of about 1 to 2 hours.

[0022] In another embodiment, after reacting valproic acid with the acid halide a resulting reaction mixture is purified to obtain 2-Propylpentanoyl Chloride by removing any traces of solvent and acid halide. [0023] In another embodiment, present invention discloses the reaction of 2-Propylpentanoyl Chloride with Eflornithine HC1 in 5N Ag NaOH. wherein the reaction occurs at two different temperature ranging from about 5- 10° C for fist 12 hours and 25 to 30° C for next 36 hrs.

[0024] In another embodiment, present invention discloses the pH of the reaction mass is maintained at 1 by adding 5N Aq. HC1.

[0025] In another embodiment, present invention discloses the reaction mass is charged with methanol and filtered to remove the methanol.

[0026] In another embodiment, present invention discloses the reaction mass is charged with acetone and hexane, then filtered and washed with acetone and hexane to obtain a final reaction mass. [0027] In another embodiment, present invention discloses, the final reaction mass is dried for under vacuum at NMT 50-55°C to obtain the purified 2-Amino-2-(difluoromethyl)-5-(2- propylpentanamido) pentanoicacid acid hydrochloride.

[0028] In another embodiment the pharmaceutically acceptable salts of the compound of Formula I is used for the treatment of cancer, trypanosomiasis and excessive hair growth.

[0029] In a specific embodiment 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride (CLX-SYN-G164A-C02) is used for the treatment of cancer, trypanosomiasis and excessive hair growth.

DETAILED DESCRIPTION OF THE INVENTION

[0030] In an embodiment, present invention provides a scalable, economical method of preparing eflornithine derivatives, pharmaceutically acceptable salts and thereof.

[0031] In an embodiment, the present invention discloses the synthesis of compounds having general formula I:

Formula I wherein

RH independently represents caprylic acid, l-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2- oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid , glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hydrochloric acid, hippuric acid, hydrobromic acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene- 1,5-disulfonic acid, naphthalene-2- sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, undecylenic acid, omega 3 fatty acids, omega 6 fatty acids, n-acetyl cysteine (nac), furoate, methyl furoate, ethyl furoate, aminocaproic acid, caprilic acid, alpha lipoic acid, R-lipoic acid, myristic acid, myristoleic acid, palmitoleic acid, phospholipids, phosphatidylcholine, oleic acid, elaidic acid, linoleic acid, linolenic acid, menthol, retinoic acid, vitamin A, retinol, linolelaidic acid, arachidonic acid, retinal, isotretinoin, curcumin, tretinoin, a-carotene b-carotene retinol, d2 ergosterol, ergocalciferol, 7- dehydrocholesterol, cholecalciferol, 25-hydroxycholecalciferol, calcitriol (1,25 dihydroxy cholecalciferol), calcitroic acid, d4 dihydroergocalciferol, alfacalcidol, dihydrotachysterol, calcipotriol, tacalcitol, paricalcitol, tocopherol, naphthoquinone, phylloquinone (kl), menaquinones (k2), menadione (k3), menadiol (k4), thiamine, acefurtiamine, allithiamine, benfotiamine, fursultiamine, octotiamine, prosultiamine, sulbutiamine, riboflavin, niacin, nicotinamide, pantothenic acid, dexpanthenol, pantethine, pyridoxine, pyridoxal phosphate, pyridoxamine, pyritinol, biotin, folic acid, dihydrofolic acid, folinic acid, levomefolic acid, adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin, choline, dehydroascorbic acid, or 1-docosanol.

[0032] In an embodiment, the present invention discloses the synthesis of pharmaceutically acceptable salts of Formula I.

[0033] In an embodiment, the present invention discloses a process for preparing 2-Amino-2- (difluoromethyl)-5-(2-propylpentanamido) pentanoic acid or pharmaceutically acceptable salts, polymorphs, stereoisomers, enantiomers thereof. [0034] In an embodiment, the present invention discloses a process for preparing 2-Amino-2- (difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride or a pharmaceutically acceptable polymorphs, stereoisomers, enantiomers thereof.

[0035] In another embodiment, the non-limiting examples of raw material used for preparing of the eflornithine derivatives and their pharmaceutically acceptable salts is selected from the non limiting examples which include acid halides, solvents, strong base, acids and other well-known raw materials in the art.

[0036] In another embodiment, the non-limiting examples of reagents used for preparing of the eflornithine derivatives broadly is selected from base namely sodium hydroxide, sodium bis(trimethylsilyl)amide, sodium hydride, sodium hydroxide, potassium hydroxide, potassium permanganate, calcium hydroxide, barium hydroxide, lithium hydroxide and other well known in the art; non-limiting examples of acids includes hydrochloric acid, sulfuric acid acid, nitric acid, acetic acid and other well known in the art; non-limiting examples of reagents includes valproic acid, valproic acid chloride, eflornithine. HC1, Thionyl chloride, dichloromethane, acetonitrile, methanol, acetone, hydrochloric acid, sodium hydroxide, hexane and 2-amino-2-(difluoromethyl)-5-(2- propylpentanamido) pentanoic acid.

[0037] In another embodiment, the non-limiting examples of acid halides include chlorosulfuric acid (CISO3H), sulfuryl chloride (SO2CI2), sulfuric acid (H2SO4) or thionyl chloride (SOCI2), the chloride of sulfurous acid; phosphorus trichloride (PCI3), the chloride of phosphorous acid; and phosphorus oxychloride (POCI3, also called phosphoryl chloride), the chloride of phosphoric acid and other well known in the art and mixtures thereof in any proportion.

[0038] In another embodiment, the non-limiting examples of the solvents used in the reaction include polar and non-polar solvents, wherein said polar solvent can be polar protic solvent or polar aprotic solvent. Non-limiting examples of solvents include acetone, ethyl methyl ketone, methyl isobutyl ketone, acetonitrile, ammonia (aqueous), n-amyl acetate, amyl alcohol, aniline, benzene, 2- butanone (MEK), /7-butyl acetate, //-butyl alcohol, methanol, ethanol, propanol, 1- butanol, 2-butanol, tert-butyl alcohol, 2-butanone, pentane, hexane, heptane, carbon tetrachloride, carbon disulfide, chlorobenzene, chloroform, chloromethene, diethylamine, cyclohexane, o-dichlorobenzene (1,2- Dichlorobenzene), 1,2-dichloroethane, diethyelene glycol, diethyl ether, N,N-dimethyl acetamide, ethylene glycol, p-xylene, toluene, N,N-dimethylaniline, 1,4-Dioxane, acetonitrile, water, heavy water and other well solvents used in the pharmaceutical industry and mixtures thereof in any proportion. [0039] In another embodiment the non-limiting examples of non-polar solvent include hexane, benzene, toluene, diethyl ether, cholorform, dichloromethane (DCM), Ethyl Acetate Ethanol, ethyl acetate, 1,4-dioxane or other well known in the art and mixtures thereof in any proportion.

[0040] In another embodiment the non-limiting examples of polar protic solvent include acetic acid, n-butanol, isopropanol, n-propanol, ethanol, methanol, formic acid, water and other well known in the art and mixtures thereof in any proportion.

[0041] In another embodiment the non-limiting examples of polar aprotic solvent include ethyl acetate, terhydrofuran(THF), 2-methyltetrahydrofuran, dichloromethane, methyl tertiary butyl ether (MTBE), cyclopentyl methyl ether, acetone, acetonitrile(MeCN), dimethylformamide (DMF) and other well known in the art and mixtures thereof in any proportion.

[0042] In some embodiments, the reaction is conducted in the presence of a catalyst. For example, the catalyst can be an organic acid, an inorganic acid, or a combination thereof.

[0043] In certain embodiments, the process for synthesizing a compound of Formula I or a salts, polymorphs, stereoisomers, enantiomers thereof comprises the step of reacting a compound of valproic acid with an acid halide and Eflornithine HC1 or a salt thereof under reaction conditions sufficient to produce the compound of Formula I. In a certain embodiment compound of Formula I, is a salt of hydrochloric acid. Possible other salts are salts of inorganic and organic acid of Formula I is defined above.

[0044] An object of the present invention is to provide a method of preparing 2-Amino-2- (difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride (CFX-SYN-G164A-C02) having a chemical structure:

2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride (CLX-

SYN-G164A-C02) wherein the method comprises part A to prepare 2-Propylpentanoyl Chloride and Part B comprises of reacting 2-Propylpentanoyl Chloride with Eflornithine HC1 to obtain the final desired product. [0045] Another objective of the present invention is to provide a method of preparing 2- Propylpentanoyl Chloride or valproic acid chloride, which comprises reacting valproic acid in non polar solvent DCM, with an acid chloride such as SOCh at a specific temperature for a predetermined duration.

[0046] Another objective of the present invention is to provide a method comprising of adding solution of Eflornithine HC1 in NaOH along with Valproic acid chloride or 2-Propylpentanoyl Chloride in acetonitrile at specific temperature and pH for a predetermined time duration. The reaction mass was monitored for the absence of Eflornithine content by HPLC. The intermediate product 2- Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid obtained is dissolved in methanol. Methanol is completely distilled off from the crude product. The desired final product is obtain by dissolving this reaction mass/mixture with acetone and hexane.

[0047] In another embodiment the non- limiting examples of pharmaceutically acceptable salts of the compound of Formula I include non-toxic acid addition salts formed with inorganic acids, such as hydrochloric, hydrobromic, sulfuric, phosphoric acid and others known in art and organic acids, such as methane sulfonic, salicylic, maleic, malonic, tartaric, citric, cyclamic, ascorbic acids, short chain and long chain fatty acids which can be saturated or unsaturated and others known in art; and non-toxic salts formed with inorganic or organic bases, such as those of alkali metals, for example, sodium, potassium, and lithium, alkaline earth metals, for example, calcium and magnesium, light metals of Group III A, for example, aluminium, organic amines, such as primary, secondary, or tertiary amines, for example, cyclohexylamine, ethylamine, trimethylamine, ethylenediamine, pyridine, (methylamino)ethanol, ethanolamine, and piperazine. The salts are prepared by conventional means.

[0048] For the purpose of the present invention, the term "pharmaceutically acceptable salt" refers to those salts of Formula I which are, within the scope of sound medical evaluation, suitable for use in humans and lower animals without any toxicity, irritation, or eliciting any allergic response. Therefore, pharmaceutically acceptable salts according to the present invention include those pharmaceutically acceptable acid addition salts formed with organic and inorganic acids and those pharmaceutically acceptable salts formed with optically active acids.

[0049] Representative acid addition salts of Formula I include, but are not limited to, acetate, aspartate, adipate, alginate, benzoate, benzenesulfonate, besylate, bicarbonate, bitartrate, bisulfate, bromide, citrate, chloride, carbonate, camsylate, camphorate, camphorsulfonate, decanoate, digluconate, edeatate, esylate, fumarate, glutamate gluceptate, gluconate, glutamate, glycolate, glycerophosphate, glycollylarsanilate, hemisulfate, hydrabamine, hydroxynaphthoate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate, iodide, isethionate, lactobionate, lactate, maleate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, methanesulfonate, nicotinate, napsylate, nitrate, 2-naphthalenesulfonate, oxalate, octanoate, oleate, pantothenate, pamoate, pectinate, persulfate, phosphate, propionate, polygalacturonate, 3-phenylpropionate, succinate, salicylate, stearate, subacetate, sulfate, tartrate, teoclate, thiocyanate, triethiiodide, p- toluenesulfonate and undecanoate.

[0050] Pharmaceutically acceptable salts of Formula I according to the present invention include: 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride, 2-Amino-2- (difluoromethyl)-5-(2-propylpentanamido) pentanoic acid phosphate, 2-Amino-2-(difluoromethyl)- 5-(2-propylpentanamido) pentanoic acid hydrobromide, 2-Amino-2-(difluoromethyl)-5-(2- propylpentanamido) pentanoic acid citrate, 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid mesylate, 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid benzenesulfonate, 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid p- toluenesulfonate, 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid bisulfate, 2- Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid maleate, 2-Amino-2- (difluoromethyl)-5-(2-propylpentanamido) pentanoic acid acetate, 2-Amino-2-(difluoromethyl)-5- (2-propylpentanamido) pentanoic acid glutamate and such others especially suitable for use as active ingredient in pharmaceutical applications.

[0051] In another embodiment the pharmaceutically acceptable salts of the compound of Formula

I is used for the treatment of cancer, trypanosomiasis and excessive hair growth.

[0052] In a specific embodiment 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid hydrochloride (CLX-SYN-G164A-C02) is used for the treatment of cancer, trypanosomiasis and excessive hair growth.

[0053] Accordingly, the methods presented in the schemes herein are illustrative rather than comprehensive.

Example 1: Route of Synthesis: [0054] Brief process for the synthesis of CLX-SYN-G164A-CQ2 t¾t Wt; 308.37

MS, Wt: 344.83

[0055] Part- A: Preparation of 2-Propylpentanoyl Chloride (Valproic acid chloride):

[0056] To a stirring solution of Valproic acid in DCM, slowly added SOC12 at 25-30°C for l-2hr, slowly raise the temperature of the reaction mass to 40-45°C and maintain for lhr. Distill off DCM atmospherically at 40-45°C and apply vacuum to remove traces of DCM & thionyl chloride to afford the desired product.

[0057] Part-B: Preparation of 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoicacid acid hydrochloride:

[0058] To a stirring solution of Fflornithine HCI in 5N Aq. NaOH added slowly Valproic acid chloride in acetonitrile at 5-10°C and maintain the reaction mass for 12hr at 5-10°C. Raise the temperature of the reaction mass to 25-30°C and maintain for 36hr. The reaction mass was monitored for the absence of Eflornithine content by HPLC. Adjust the reaction mass pH to 1 with 5N Aq. HCI and distill off water completely under vacuum at below 55°-60°C. Cool the reaction mass to RT and charge methanol into the reaction mass residue. Stir the reaction mass for lhr at RT. Filter the reaction mass. Distill of methanol completely under vacuum at below 50°C. Cool the reaction mass residue to RT. Charge acetone and hexane into the reaction mass residue and stir for l-2hr at RT. Filter the reaction mass and wash with acetone and hexane. Dry the material for 4hr under vacuum at NMT 50- 55°C to afford the desired material with purity 98.5% by HPFC. The desired material was confirmed by H-NMR, Mass and IR.

[0059] Example 2: Characterization of the CLX-SYN-G164A-C02

[0060] Stability data of the CLX-SYN-G164A-C02

DETERMINATION OF THE SOLUBILITY OF CLX-SYN-G164A-C02 IN PHOSPHATE BUFFERED SALINE AND 0.1N HC1 BY KINETIC METHOD [0061] The in vitro solubility of CLX-SYN-G164A-C02 was evaluated in phosphate buffer saline

(pH-2.0, 3.0, 6.0, 6.8, 7.4 and 9.0) and 0.1 N HC1 by kinetic method.

[0062] Calibration standards (1, 5, 10, 50, 100, 200 and 300 mM) were prepared in methanol: water (80:20, v/v) from a 20 mM primary stock of CLX-SYN-G164A-C02 prepared in methanol: water (80:20, v/v). The CLX-SYN-G164A-C02 (200 pM) was incubated in the respective buffers for 90 minutes at 37 °C. The solubility of the test compound was determined using HPLC-PDA and was calculated from the standard curve.

Solubility of CLX-SYN-G164A-CQ2 in 0.1 N HC1 and phosphate buffered saline at different pH [0063] CLX-SYN-G164A-C02 showed high solubility in 0.1 N HC1 and at pH 2.0 to 9.0 phosphate buffed saline under the tested conditions.

IN VITRO DETERMINATION OF THE STABILITY OFCLX-SYN-G164-C02 IN SIMULATED GASTRIC AND INTESTINAL FLUIDS

[0064] The in vitro stability of CLX-SYN-G164A-C02 was evaluated in USP simulated fluids like simulated gastric fluid (SGF; pH: 1.2), and simulated intestinal fluid (FaSSIF; pH: 6.5), (FeSSIF; pH: 5.0); and SIF with added Pancreatin (pH: 6.8. The final concentration of CLX-SYN-G164A-C02 used in the study was 5 mM. The samples were incubated at 37 ± 1°C in incubator. The reactions were terminated at 0, 30, 60 and 120 min by addition of acetonitrile containing internal standard. The supernatant from the SGF and SIF buffers was collected and analyzed for disappearance of CLX- SYN-G164A-C02 and appearance of Eflornithine and Valproic acid using LC-MS/MS method. The results are summarized in the table below.

Stability of CLX-SYN-G164A-CQ2 in simulated gastric and intestinal fluids (FaSSIF and FeSSIF):

[0065] CLX-SYN-G164A-C02 was stable in the simulated gastric fluid and simulated intestinal fluids and there in no detectable formation of Eflornithine or Valproic Acid.

[0066] Candesartan Cilexetil (reference compound) was stable in FaSSIF and FeSSIF whereas unstable in SIF (with Pancreatin); with 21%remaining at the end of 120 min incubation period.

IN VITRO INVESTIGATION OF METABOLIC STABILITY OF CLX-SYN-G164AC02 (CLX- 164A) IN LIVER MICROSOMES OF MOUSE, RAT, DOG AND HUMAN.

[0067] In vitro metabolic stability of CLX-SYN-G164A-C02 (CLX- 164 A) was determined in the mouse, rat, dog and human liver microsomes. The CLX- 164A was incubated at 1 mM with 0.5 mg/mL protein content of respective liver microsomes at 37 ± 1 °C. The reaction was initiated by the addition of NADPH (10 mM). The reaction was terminated at 0, 5, 15 and 30 min post reaction initiation, using a quenching solution containing the internal standard (warfarin). The supernatants were analyzed for CLX-164A using LC-MS/MS method. The percent disappearance of CLX-164A was calculated. The results are summarized in the table below.

[0068] Metabolic stability of CLX-164A in liver microsomes of various species:

EQUIVALENTS

[0069] The present disclosure provides among other things the large scale process of preparing , 2-Amino-2-(difluoromethyl)-5-(2-propylpentanamido) pentanoic acid derivatives and their pharmaceutically acceptable salts, polymorphs, stereoisomers, enantiomers thereof and their use as medicament for treating conditions such as cancer, trypanosomiasis and excessive hair growth. While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the process and methods herein will become apparent to those skilled in the art upon review of this specification. The full scope of the claimed systems and methods should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

[0070] All publications and patents mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.