A novel process for the preparation of (2S)-2-{[(2S)-1-(2-aminoacetyl)-2-methylpyrrolidine-2carbonyl] amino} pentanedioic acid

Abstract

The present invention relates to a novel process for the preparation of (2S)-2-{[(2S)-1-(2- aminoacetyl)-2-methylpyrrolidine-2carbonyl] amino} pentanedioic acid (1) and its intermediates. The present invention having purity greater than 99.0% by HPLC, preferably greater than 99.5% by HPLC, more preferably greater than 99.8% by HPLC. The present invention further relates to a process for the preparation of amorphous form of (2S)-2-{[(2S)-1-(2-aminoacetyl)- 2-methylpyrrolidine-2carbonyl] amino} pentanedioic acid (1).

Description

[0001] “A NOVEL PROCESS FOR THE PREPARATION OF (2S)-2-{[(2S)-l-(2-AMINOACETYL)-2-METHYLPYRROLIDINE-2CARBONYL] AMINO} PENTANEDIOIC ACID”

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of the earlier filing date of Indian Provisional Patent Application No. 202541001130 filed on January 06, 2025, and Indian Provisional Patent Application No. 202541021073 filed on March 08, 2025.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a process for the preparation of (2S)-2-{[(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2-carbonyl] amino} pentane dioic acid (1) and its intermediates.

[0006] The present invention further relates to a process for the purification of (2S)-2-{[(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2-carbonyl] amino} pentane dioic acid (1) is having purity greater than 99.0% by HPLC, preferably greater than 99.5% by HPLC, more preferably greater than 99.8% by HPLC. The present invention further relates to a process for the preparation of amorphous form of (2S)-2-{[(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2carbonyl] amino} pentanedioic acid (1).

[0007] BACKGROUND OF THE INVENTION

[0008] Trofinetide is weak CYP3A4 inhibitor, for the treatment of Rett syndrome in adults and pediatric patients 2 years of age and older, is designated chemically as (2S)-2-{[(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2carbonyl] amino} pentane dioic acid (1) and is chemically represented by the following structural Formula (1).

[0009] o

[0010] o

[0011]

[0012] (1)

[0013] US 7,041,314 B2 discloses Trofinetide, for the first time, which discloses a process for the preparation of Trofinetide, comprising step i), reacting a compound of formula A with formula B to provide formula C ii), reacting formula C with Pd / C to provide formula D iii), reactingformula D with formula E to provide formula F, iv) reaction of formula F with Pd / C to provide formula G v), deprotecting the compound of formula G with Trifluoro acetic acid to provide Trofinetide. The above process is schematically as shown below,

[0014] OH NHBOC H2N-~ -CO2Bn

[0015]

[0016] BOC-Gly CO2Bn

[0017] Formula B Formula E Step i Step iii

[0018]

[0019]

[0020] Formula C Formula D Formula F Step iv

[0021] Step v

[0022]

[0023] Formula (1) Formula G

[0024] W02014085480 Al which discloses process for preparation of Trofinetide which as schematically as shown below, comprising step i), reacting a compound of formula a with thionyl chloride and methanol to provide formula b ii), reacting formula b with 2-(methylamino)acetic acid to provide formula d iii), reacting formula d with 1,4 dioxane to provide formula e, iv) reaction of formula e with (S)-dibenzyl 2-aminopentanedioate in presence of triethylamine to provide formula g v), compound of formula g with Pd / C to provide Trofinetide. The above process is schematically as shown below,Formula a

[0025]

[0026] US 11,827,600 B2 discloses crystalline form A and amorphous form of Trofinetide.

[0027] Hence, there is consequently a need development for new methods to sort out prior art existing methods. So, our inventors have developed a method for the preparation of(2S)-2-{[(2S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2carbonyl] amino} pentane dioic acid (1). The present invention advantageous process for preparing Trofinetide, which is relatively simple and less costly than the prior art processes and which provides an excellent yield in a high purity with low costs and industrial applicable process.

[0028] OBJECTIVE OF THE INVENTION

[0029] In main objective, the present invention provides a process for the preparation of Trofinetide of formula (1) and its intermediates.

[0030] In another objective, the present invention provides a process for the purification of Trofinetide of formula (1).

[0031] In further objective, the present invention provides a process for the preparation of amorphous form of Trofinetide of formula (1) without formation of impurity A.

[0032] In another objective, the present invention provides Trofinetide of formula (1) with purity greater than 99.5% (w / w) and comprising additional compounds selected from the group consisting of one or more formula - between 0.002 to 2%(w / w).In further objective, the present invention provides Trofinetide of formula (1) so obtained after purification is having purity greater than 99.0% by HPLC, preferably greater than 99.5% by HPLC, more preferably greater than 99.9% by HPLC.

[0033] SUMMARY OF THE INVENTION

[0034] In one aspect, the present invention provides a process for the preparation of Trofinetide of formula (1) as illustrated in scheme 1, comprising the steps of:

[0035] a) reacting (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (9) with thionyl chloride in presence of solvent to provide (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8);

[0036] b) reacting the compound of formula (8) with 2-(tert-butoxycarbonylamino) acetic acid (7) in presence of suitable coupling reagent, additive and solvent to provide (S)-m ethyl l-(2- (tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxylate (6);

[0037] c) hydrolyzing the compound of formula (6) with a base in presence of solvent to provide (S)-l-(2-(tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxylic acid (5); d) reacting the compound of formula (5) with (S)-di-tert-butyl 2-aminopentanedioate (4) in presence of suitable coupling reagent, additive and solvent to provide (S)-di-tert-butyl 2- ((S)- 1 -(2-(tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (3);

[0038] e) converting the compound of formula (3) to Trofinetide TFA (2),

[0039] f) converting the compound of formula (2) to provide crude Trofinetide(la); and optionally, purifying crude Trofinetide(la); to obtain pure Trofinetide of formula (1).

[0040] In second aspect, the present invention provides a process for the preparation of Trofinetide of formula (1) as illustrated in scheme 2, comprising the steps of:

[0041] A) reacting the compound of formula (8) with 2-(tert-butoxycarbonylamino) acetic acid (7) in presence of suitable coupling reagent, additive and solvent to provide compound of formula (6);

[0042] B) reacting the compound of formula (6) with a base in presence of solvent to provide compound of formula (5);

[0043] C) reacting the compound of formula (5) with compound of formula (10) to provide compound of formula (11);

[0044] D) converting compound of formula (11) to compound of formula (12)E) converting to the compound of formula (12) to obtain crude Trofinetide(la); optionally, purifying crude Trofinetide (la) to obtain pure Trofinetide of formula (1).

[0045] In third aspect, the present invention provides a process for the preparation of Trofinetide of formula (1) as illustrated in scheme 3, comprising the steps of:

[0046] i) the reaction of the compound of formula (9) with thionyl chloride in the presence of a solvent to provide (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8); ii) the reaction of compound of formula (8) with compound of formula (16) to provide compound of formula (15);

[0047] iii) the reaction of the compound of formula (14) with compound of formula (10) in the presence of a base, coupling reagent and additive in a solvent to provide compound of formula (13);

[0048] iv) hydrogenating the compound of formula (13) to provide Trofinetide (1).

[0049] v) the reaction of the compound of formula (13) with hydrogenation catalyst to provide crude Trofinetide (1). optionally, purifying crude Trofinetide (1) to obtain pure Trofinetide of formula (1).

[0050] In fourth aspect, the present invention provides a process for the preparation of Trofinetide of formula (1), as illustrated in scheme 4, comprising the steps of:

[0051] A) reacting (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (9) with thionyl chloride in presence of solvent to provide (S)-methyl 2-methylpyrrolidine-2- carboxylate hydrochloride (8);

[0052] B) reacting compound of formula (8) with 2-(dibenzyl amino) acetic acid (16) in presence of suitable coupling reagent, additive and solvent to provide (S)-methyl l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylate (15);

[0053] C) reacting the compound of formula (15) with a base in presence of solvent to provide compound of formula (14);

[0054] D) reacting the compound of formula (14) with (S)-di -tert-butyl 2-aminopentanedioate Hydrochloride (9) to provide (S)-di -tert-butyl 2-((S)-l-(2-(dibenzyl amino) acetyl)-2- methylpyrrolidine-2-carboxamido) pentanedioate (10A)

[0055] E) hydrolysis of compound of formula (10A) in presence of base and solvent (S)-2-((S)- l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentane dioic acid (13 A); and

[0056] F) converting to the compound of formula (1 A) to Trofinetide (1);In another aspect, the present invention provides, a novel compounds of formula 13, formula (2a), formula (2b), formula (14), formula (4a), formula (4b) formula (15), formula (5a), formula (5b), and formula (10A), formula (10a), formula (10b) and formula (13A), formula (Ila), formula (1 lb); or its pharmaceutically acceptable salts for the preparation of Trofinetide (1).

[0057] In another aspect, the present invention provides a process for the purification of Trofinetide of formula (1), comprising the steps of:

[0058] I) dissolving crude Trofinetide of formula (la) in a solvent or mixture of solvent (s), II) optionally, adding anti-solvent to step a); and

[0059] III) isolating Trofinetide of formula (1).

[0060] In further aspect, the present invention provides Trofinetide of formula (1) so obtained after purification is having purity greater than 99.0% by HPLC, preferably greater than 99.5% by HPLC, more preferably greater than 99.9% by HPLC.

[0061] BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1: Illustrates the PXRD pattern of amorphous form of Trofinetide (1).

[0063] Figure 2: Illustrates the differential scanning calorimetry (DSC) of Trofinetide (1).

[0064] Figure 3: Illustrates the Thermogravimetric Analysis (TGA) of Trofinetide (1).

[0065] Figure 4: Illustrates the FT Raman of Trofinetide (1).

[0066] Figure 5: Illustrates the FT IR Spectrum of Trofinetide (1).

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] As used herein the term “solvent” used in the present invention refers to “hydrocarbon solvents” such as n-hexane, n-heptane, cyclohexane, pet ether, toluene, pentane, cycloheptane, methyl cyclohexane, m-, o-, or p-xylene, nitromethane and the like; “ether solvents” such as dimethoxy methane, tetrahydrofuran, 1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether, anisole, methyl t-butyl ether, 1,2-dimethoxy ethane, anisole and the like; “ester solvents” such as methyl formate, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, vinyl acetate and the like; “polar-aprotic solvents such as dimethyl acetamide (DMA), dimethylformamide (DMF),dimethylsulfoxide (DMSO), N-methyl pyrrolidone (NMP) and the like; “chloro solvents” such as dichloromethane, dichloroethane, chloroform, carbon tetrachloride and the like; “ketone solvents” such as acetone, methyl ethyl ketone, acetyl acetone, methyl isobutyl ketone and the like; “nitrile solvents” such as acetonitrile, propionitrile, isobutyronitrile and the like; “alcoholic solvents” such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, 2-nitroethanol, 2-fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, polyethylene glycol, polyethylene gly col-400, 2-methoxy ethanol, 1,2-ethoxyethanol, di ethylene glycol, 1, 2, or 3 -pentanol, neo pentyl alcohol, t-pentyl alcohol, di ethylene glycol mono ethyl ether, cyclohexanol, benzyl alcohol, phenol, or glycerol and the like; “polar solvents” such as water and or mixtures thereof.

[0069] The term “base” as used in the present invention is selected from inorganic bases like “alkali metal hydroxides” such as lithium hydroxide, sodium hydroxide, potassium hydroxide and the like; “alkali metal carbonates” such as sodium carbonate, potassium carbonate, lithium carbonate and the like; “alkali metal bicarbonates” such as sodium bicarbonate, potassium bicarbonate, lithium bicarbonate and the like; “alkali metal hydrides” such as sodium hydride, potassium hydride, lithium hydride and the like; ammonia; and organic bases such as “alkali metal alkoxides” such as sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium methoxide, potassium ethoxide, potassium tert-butoxide and the like; triethyl amine, methyl amine, ethylamine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), l,5-diazabicyclo(4.3.0)non-5-ene (DBN), lithium diisopropyl amide (LDA), n-butyl lithium, tri benzylamine, isopropylamine, di isopropylamine, N, N-Diisopropylethylamine (DIPEA), N-methyl morpholine, N-ethyl morpholine, piperidine, dimethyl aminopyridine, morpholine, pyridine, 2,6-lutidine, 2,4,6-collidine, imidazole, 1 -methylimidazole, 1,2,4-triazole, 1,4-diaza bicyclo [2.2.2]octane (DABCO) or mixtures thereof.

[0070] The term “coupling reagent” used in the present invention is selected from but not limited to 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, (EDC.HCl), N-Hydroxy succinimide (NHS), Dicyclohexylcarbodiimide (DCC), Benzotriazole- 1-yl-oxytri pyrrolidino-phosphonium hexafluorophosphate (BOP), N, N’ -Carbonyl diimidazole (CDI), N, N-Diisopropylcarbodiimide (DIC), 3-(Dimethylamino)propyl carbodiimide (DMP), Bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP chloride).The term “additive” used in the present invention is selected from but not limited to HOBt (1-Hydroxybenzotriazole), HOAt (l-Hydroxy-7-azabenzotriazole), TBTU (O-(Benzo triazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate), EDT (Ethyl Dimethyl aminopropylcarbodiimide), PyBOP (B enzotri azol -1-yl -oxy) Tripyrrolidinophosphonium hexafluorophosphate).

[0071] The term "alkyl" refers to straight chain or branched hydrocarbon groups, generally having specified number of carbon atoms. A " Ci-n alkyl" refers to alkyl group having 1 to 12 carbon atoms. Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, t-butyl, pent-l-yl, pent-2-yl, pent-3-yl, 3-methylbut-l-yl, 3-methylbut-2-yl,2-methylbut-2-yl, 2,2,2-trimethyleth-l-yl, n-hexyl and the like. As used herein, the term "aryl-Cl-6 alkyl" refers to an aryl group attached to the substrate through an alkyl group containing one to six carbon atoms.

[0072] The term "aryl"refers to monovalent or divalent aromatic groups respectively including 5 and 6 membered monocyclic aromatic groups that contain zero to four heteroatom independently selected from nitrogen, oxygen and sulfur. Examples of monocyclic aryl groups include, without limitation, phenyl, pyrrolyl, pyranyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyradazinyl, pyrimidinyl, and the like. The aryl groups also include bicyclic groups, tricyclic groups etc including fused 5 and 6 membered rings described above. Examples of multicyclic aryl groups include, without limitation, naphthyl, biphenyl, anthracenyl, pyrenyl, carbazolyl, benzoxazolyl, benzodi oxazolyl, benzothiazolyl, benzoimidazolyl, benzothiophenyl, quinolinyl, isoquinolinyl. indolyl, benzofuranyl, purinyl, indolizinyl and the like. The aryl groups may be attached to the substrate at any ring atom, unless such attachment would violate valence requirements.

[0073] acyl groups such as acetyl (Ac), trifluoroacetyl (TFA), benzoyl (Bz) group and the like.

[0074] In the one embodiment, the present invention provides a process for the preparation of Trofinetide (1) as illustrated in scheme 1, comprising one or more following steps:

[0075]

[0076] Scheme 1

[0077] The step a) of the forgoing process involves the reaction of (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride of formula (9) with thionyl chloride, in presence of methanol under appropriate reaction conditions to provide (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8). The step a) reaction is carried out at 60-65°C for a sufficient period till completion of the reaction.

[0078] The step b) of the forgoing process involves the reaction of compound of formula (8) with Boc-Gly-OH (7) in presence of a base, coupling reagent and additive in a solvent under appropriate reaction conditions to provide (S)-methyl l-(2-(tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxylate (6). The base used in the step b) is selected from the list as defined above, preferably DIPEA base; the coupling reagent is selected from the list mentioned as above, preferably l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, (EDC.HCl). The additive used is selected from the list mentioned as above, preferably 1-Hydroxybenzotriazole HOBt. The step b) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0079] The step c) of the forgoing process involves the hydrolyzation of compound of formula (6) with a base and a solvent under appropriate reaction conditions to provide (S)-l-(2-(tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxylic acid (5). The base used in the step c) is selected from the list as defined above, preferably sodium hydroxide base. The solvent used in step c) is selected from the list as defined above, preferably 1,4-di oxane solvent. The step c) reaction is carried out at room temperature for a sufficient period till completion of the reaction.The step d) of the forgoing process involves the reaction of compound of formula (5) with the compound of formula (4) in the presence of a base, coupling reagent and additive in a solvent under appropriate reaction conditions to provide ((S)-di -tert-butyl 2-((S)-l-(2-(tert-butoxycarbonylamino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (3). The base used in the step d) is selected from the list as defined above, preferably diisopropylethyl amine; the coupling reagent used is selected from the list mentioned as above, preferably 1-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride, (EDC.HCl), and the additive used in the step d) is selected from the list mentioned as above, preferably l-Hydroxy-7-azabenzotri azole (HOAt). The step d) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0080] The step e) of the forgoing process involves the deprotection of compound of formula (3) using cocktail solution under appropriate reaction conditions to provide the compound of formula (2). The cocktail solution is (Trifluoracetic acid water and Tri isopropyl silane). The step e) reaction is carried out at a suitable temperature of 0°C to about 5°C for a sufficient period till completion of the reaction.

[0081] The step f) of the forgoing process involves the conversion of compound of formula (2) with MP carbonate resin and water under appropriate reaction conditions to provide Trofinetide (1). The step e) reaction is carried out at a suitable temperature of 0°C to about 5°C for a sufficient period till completion of the reaction.

[0082] In the second embodiment, the present invention provides a process for the preparation of Trofinetide (1) as illustrated in scheme 2, comprising one or more following steps:

[0083]

[0084] Scheme 2

[0085] Wherein R1is a linear or branched C1-12alkyl group; or an aryl-C1-6alkyl, or (un)substituted aryl group; P is hydrogen or protecting group.

[0086] The step A) of the forgoing process involves the reaction of the compound of formula (8) with compound of formula 7 in presence of a base, coupling reagent and additive in a solvent, wherein base is selected from the list as defined above, preferably DIPEA; the coupling reagent is selected from the list mentioned as above, preferably 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl). The additive used is selected from the list mentioned as above, preferably 1-Hydroxybenzotriazole HOBt, and solvent is selected from list as defined above. The reaction is performed under appropriate reaction conditions to provide compound of formula (6), and the reaction carried out at room temperature for sufficient period till completion of the reaction.

[0087] The step B) of the forgoing process involves the hydrolyzation of compound of formula (6) with a base and a solvent. The base used in the step b) is selected from the list as defined above, preferably sodium hydroxide base. The solvent used in step b) is selected from the list as defined above, preferably 1,4-di oxane solvent. The reaction is performed under appropriate reaction conditions to provide compound of formula (5) and the reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0088] The step C) of the forgoing process involves the reaction of the compound of formula (5) with compound of formula 10 to provide compound of formula (11) The step c) reaction is carriedout at room temperature for a sufficient period till completion of the reaction. In step C) protecting group ‘P’ is selected from but not limited to tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyl (Bn), carbamate group, p-m ethoxyphenyl (PMP), p-m ethoxybenzyl (PMB), 3,4-dimethoxy benzyl (DMPM), trityl (Tr), alkyl / aryl sulfonyl such as methanesulfonyl, ethanesulfonyl, benzenesulfonyl, p-toluenesulfonyl (tosyl) and the like;

[0089] The step D) of the forgoing process involves the reaction of the compound of formula (11) using cocktail solution under appropriate reaction conditions to provide the compound of formula (12). The cocktail solution is (Trifluoracetic acid water and Tri isopropyl silane (TIPS). The step d) reaction is carried out at a suitable temperature for a sufficient period till completion of the reaction.

[0090] The step E) of forgoing process involves the reaction of the compound of formula (12) with hydrogenation catalyst to provide Trofinetide. The step e) hydrogenation catalyst is preferably Pd / C and H2. The step e) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0091] The forgoing process involves the purification of crude Trofinetide(la) with MP carbonate resin and water under appropriate reaction conditions to provide pure Trofinetide (1). The step f) reaction is carried out at a suitable temperature of for a sufficient period till completion of the reaction.

[0092] In the third embodiment, the present invention provides a process for the preparation of Trofinetide (1), as illustrated in scheme 3, comprising one or more following steps:Step 1) ■HC1 OH Step 3) (9)

[0093] Step 5)

[0094]

[0095] (1)

[0096] (13) Scheme 3

[0097] Wherein R1is a linear or branched C1-12alkyl group; or an aryl-C1-6alkyl, or (un)substituted aryl group; P is hydrogen or protecting group.

[0098] The step a) of the forgoing process involves the reaction of (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride of formula (9) with thionyl chloride in the presence of a solvent under appropriate reaction conditions to provide (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8). The solvent selected from the list defined above preferably alcoholic solvent and more preferably methanol. The step a) reaction is conducted at 60-65°C for a sufficient period till completion of the reaction.

[0099] The step b) of the forgoing process involves the reaction of compound of formula (8) with 2-(dibenzyl amino) acetic acid (16) in the presence of a base, coupling reagent and additive in a solvent under appropriate reaction conditions to provide (S)-methyl l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylate (15). The base used in step b) is selected from the list as defined above, preferably organic base and more preferably diisopropylethyl amine (DIPEA); the coupling reagent is selected from the list mentioned as above, preferably 1-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl). The additive used is selected from the list mentioned as above, preferably 1 -Hydroxybenzotriazole (HOBt). The solvent selected from the list defined above preferably alcoholic solvent and more preferably methanol. The step b) reaction is carried out at room temperature for a sufficient period till completion of the reaction.The step c) of the forgoing process involves the hydrolyzation of compound of formula (15) with a base and a solvent under appropriate reaction conditions to provide (S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (14). The base used in the step c) is selected from the list as defined above preferably alkali metal hydroxide and more preferably lithium hydroxide. The solvent used in step c) is selected from the list defined above preferably alcoholic solvent and more preferably methanol. The step c) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0100] The step d) of the forgoing process involves the reaction of compound of formula (14) with the L-glutamic acid dibenzyl ester 4-toluenesulfonate (10) in the presence of a base, coupling reagent and additive in a solvent under appropriate reaction conditions to provide (S)-dibenzyl 2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (13). The solvent used in step d) is selected from the list as defined above, preferably dichloromethane. The base used in the step d) is selected from the list as defined above, preferably organic base and more preferably diisopropylethyl amine; the coupling reagent used is selected from the list mentioned as above, preferably 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) or Bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP chloride), and the additive used in the step d) is selected from the list mentioned as above, preferably 1 -Hydroxybenzotriazole (HOBt). The step d) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0101] The step e) of the forgoing process involves the deprotection of compound of formula (13) to provide Trofinetide (1). The hydrogenation catalyst used in step e) is selected from the list as defined above, preferably on palladium carbon (Pd / C). The step e) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0102] In another embodiment, the compound of formula (8), and compound of formula (15) can be prepared in-situ and proceeds to next steps without further purification.

[0103] In the fourth embodiment, the present invention provides a process for the preparation of Trofinetide (1), comprising one or more following steps:Step 6a) Trofinetide (1)

[0104]

[0105] Scheme 4

[0106] The step A) of the forgoing process involves the reaction of (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride of formula (9) with thionyl chloride in the presence of a solvent under appropriate reaction conditions to provide (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8). The solvent selected from the list defined above preferably alcoholic solvent and more preferably methanol. The step A) reaction is conducted at 60-65°C for a sufficient period till completion of the reaction.

[0107] The step B) of the forgoing process involves the reaction of compound of formula (8) with 2-(dibenzyl amino) acetic acid (16) in the presence of a base, coupling reagent and additive in a solvent under appropriate reaction conditions to provide (S)-methyl l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylate (15). The base used in step b) is selected from the list as defined above, preferably organic base and more preferably diisopropylethyl amine (DIPEA); the coupling reagent is selected from the list mentioned as above, preferably 1-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride, (EDC.HCl). The additive used is selected from the list mentioned as above, preferably 1 -Hydroxybenzotriazole (HOBt). The solvent selected from the list defined above preferably alcoholic solvent and more preferably methanol. The step B) reaction is carried out at room temperature for a sufficient period till completion of the reaction.The step C) of the forgoing process involves the hydrolyzation of compound of formula (15) with a base and a solvent under appropriate reaction conditions to provide (S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (14). The base used in the step c) is selected from the list as defined above preferably alkali metal hydroxide and more preferably lithium hydroxide. The solvent used in step c) is selected from the list defined above preferably alcoholic solvent and more preferably methanol. The step C) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0108] The step D) of the forgoing process involves the reaction of compound of formula (14) with the L-glutamic acid di-tertiary butyl hydrochloric acid (9 A) in the presence of a base, coupling reagent and additive in a solvent under appropriate reaction conditions to provide (S)-di-tert-butyl 2-((S)-l-(2-(dibenzylamino)acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (10A). The solvent used in step D) is selected from the list as defined above, preferably dichloromethane. The base used in step D) is selected from the list as defined above, preferably using organic base and more preferably using diisopropylethyl amine; the coupling reagent used in step D) is selected from the list mentioned as above, preferably l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, (EDC.HCl) or Bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP chloride), and the additive used in the step d) is selected from the list mentioned as above, preferably l-Hydroxy-7-azabenzotriazole (HOAt). The step D) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0109] The step E) of the forgoing process involves the deprotection of compound of formula (10 A) using cocktail solution under appropriate reaction conditions to provide the compound of formula (13A). The cocktail solution is a mixture of Trifluoracetic acid, Tri isopropyl silane (TIPS) and water at the ratio of (95%:2.5%:2.5%). The step E) reaction is carried out at a suitable temperature of 0°C to about 5°C for a sufficient period till completion of the reaction. Formula (10A) can be used next step without isolation.

[0110] The step F) of forgoing process involves the reaction of the compound of formula (13 A) with hydrogenation catalyst to provide Trofinetide (1). The hydrogenation catalyst used in step f) is selected from the list as defined above, preferably on palladium carbon (Pd / C). The step f) reaction is carried out at room temperature for a sufficient period till completion of the reaction.In any of the foregoing embodiments, the compound of formula (13 A) can be prepared in-situ and proceeds to next steps without further purification.

[0111] In the fifth embodiment, the present invention provides a process for the preparation of compound of formula (16), as illustrated in scheme 5, comprising the steps of:

[0112] Aqu KOH

[0113] Ethanol

[0114] Water Aqu HC1

[0115] step a)

[0116] (17) (18)

[0117]

[0118] Scheme 5

[0119] The step a) of the forgoing process involves the reaction of 2-aminoacetic acid of formula (17) with (chloromethyl)benzene of formula (18) in the presence of a base and solvent under appropriate reaction conditions to provide 2-(dibenzylamino)acetic acid (16). The base is selected from inorganic bases like “alkali metal hydroxides” such as lithium hydroxide, sodium hydroxide, potassium hydroxide and the like; The solvent is selected from the list defined above preferably alcoholic solvent and more preferably isopropanol, “polar-aprotic solvents such as dimethyl acetamide (DMA), dimethylformamide (DMF), dimethylsulfoxide (DMSO), N-methyl pyrrolidone (NMP) and the like; “chloro solvents” such as dichloromethane, di chloroethane, chloroform, carbon tetrachloride and the like; “polar solvents” such as water and or mixtures thereof. The step a) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0120] In the sixth embodiment, the present invention provides a process for the preparation of compound of formula (10) as illustrated in scheme 6, comprising the steps of:

[0121] ^^2 step a)

[0122] (19)

[0123]

[0124] Scheme 6

[0125] The step a) of the forgoing process involves the reaction of the compound of formula (19) with compound of formula (20) and p-Toluene sulfonic acid monohydrate in a solvent under appropriate reaction conditions to provide compound of formula (10). The solvent is selectedfrom the group “hydrocarbon solvents” such as n-hexane, n-heptane, cyclohexane, pet ether, toluene, pentane, cycloheptane, methyl cyclohexane, m-, o-, or p-xylene, nitromethane and the like; “ester solvents” such as methyl formate, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, vinyl acetate and the like; “polar-aprotic solvents” such as dimethyl acetamide (DMA), dimethylformamide (DMF), dimethylsulfoxide (DMSO), N-methyl pyrrolidone (NMP) and the like; “polar solvents” such as water and or mixtures thereof. The step a) reaction is carried out at room temperature for a sufficient period till completion of the reaction.

[0126] In another aspect, the present invention provides, a novel compounds of formula 13, formula (2a), formula (2b), formula (14), formula (4a), formula (4b) formula (15), formula (5a), formula (5b), and formula (10A), formula (10a), formula (10b) and formula (13A), formula (Ila), formula (1 lb); or its pharmaceutically acceptable salts

[0127]

[0128] V£l o o

[0129] II H I]

[0130] I »i o 1x° / H°'A^z(s)r 'oHHN^G Ox ' \

[0131] / s c /

[0132] o" O

[0133] qoi *01 IIK^O AM HN^U.°r^. OK' \ o-^ oy J

[0134] (Z'ZoI) o

[0135] o o

[0136] K a. (9) o^x^o 'o

[0137] 0=4?? H

[0138] OxrtST\

[0139] •^N

[0140] VAL^^Z MMNY*U VOI

[0141] qs HN^O o r \=vV >^ 7, L

[0142] ^0 ° b o (si). X 1^11 / o - o JU7 XS)N^^ >

[0143]

[0144] 6

[0145]

[0146] In another embodiment, the present invention provides a process for the purification of Trofinetide of formula (1), comprising the steps of:

[0147] I) dissolving crude Trofinetide of formula (la) in a solvent or mixture of solvent (s), II) optionally, adding anti-solvent to step a); and

[0148] III) isolating Trofinetide of formula (1).

[0149] In another embodiment, Trofinetide (1) obtained in the present invention could be crystalline or amorphous form.

[0150] In another embodiment, Trofinetide (1) obtained in the present invention stable amorphous form.

[0151] The present invention provides amorphous form of Trofinetide (1) and its PXRD pattern.

[0152] In another embodiment, Trofinetide (1) obtained in the present invention could be anhydrous or hydrate forms viz., monohydrate, dihydrate, trihydrate and the like; preferably anhydrous form.

[0153] In further objective, the present invention provides Trofinetide of formula (1) so obtained after purification is having purity greater than 99.0% by HPLC, preferably greater than 99.5% by HPLC, more preferably greater than 99.9% by HPLC.In another embodiment, the present invention provides Trofinetide of formula (1) and its intermediates having purity greater than 99.0% by HPLC, preferably greater than 99.5% by HPLC, more preferably greater than 99.9% by HPLC.

[0154] In another embodiment, the present invention provides Trofinetide of formula (1) so obtained is having free of formula A represented by the following structure.

[0155]

[0156] Formula A

[0157] Yet, in another embodiment, Trofinetide (1) produced after purification is having purity more than 99.0% by HPLC and total impurities with below structures less than 0.01 % (w / w), preferably less than 0.05% (w / w).

[0158] Formula B Formula C

[0159]

[0160] 13A

[0161] Formula DIn another embodiment, the present invention provides Trofinetide of formula (1) so obtained is having impurity of formula D, E, F, and formula G represented by the following structure between 0.001 % (w / w) to 0.15%(wt)

[0162] In another embodiment, the present invention provides Trofinetide of formula (1) so obtained is having impurity of formula D, E, F, G, H, I, J, K, L, M and formula N represented by the following structure between 0.001 % (w / w) to 0.15%(wt).

[0163] Formula E

[0164] formula F

[0165]

[0166] formula H

[0167] formula I

[0168]

[0169] formula J formula Kformula M

[0170]

[0171] formula N

[0172] In another embodiment, the present invention provides Trofinetide of formula (1) so obtained is having purity greater than 99.0% by HPLC, preferably greater than 99.5% by HPLC, more preferably greater than 99.9% by HPLC.

[0173] In another embodiment, the anhydrous form of Trofinetide of formula (1) obtained in the present invention is having water content less than 1.0% (w / w), preferably less than 0.5% (w / w).

[0174] In another embodiment, the Trofinetide of formula (1) obtained in the present invention is having water content less than 10.0% (w / w), preferably less than 5.0% (w / w) and more preferably less than 1.0% (w / w).

[0175] In another embodiment, Trofinetide of formula (1) obtained according to the present invention has a particle size distribution of D90 less than 200μm, preferably less than 150 μm, more preferably less than 100 μm and still more preferably less than 50 μm.

[0176] DEFINITIONS:

[0177] Abbreviation Description

[0178] DIPEA N, N-Diisopropylethylamine

[0179] EDC.HCl 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

[0180] BOP chloride Bis(2-oxo-3-oxazolidinyl)phosphonic chloride

[0181]

[0182] HOBt 1-Hydroxybenzotriazole

[0183] HOAt 1-Hydroxy-7-azabenzotriazole

[0184] TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate)

[0185] EDT (Ethyl Dimethyl aminopropylcarbodiimide)

[0186] Boc tert-butyloxycarbonyl

[0187] Bn benzyl

[0188] Fmoc 9-fluorenylmethyloxycarbonyl

[0189] DMF dimethylformamide

[0190] DMA dimethyl acetamide

[0191] DMSO dimethylsulfoxide

[0192] TFA trifluoroacetyl

[0193] Tri isopropyl silane TIPS

[0194] MeOH methanol

[0195] DCM Dichloromethane

[0196] 1H NMR Proton Nuclear Magnetic Resonance

[0197] 13C NMR Carbon- 13 Nuclear Magnetic Resonance

[0198] IR Infrared Spectroscopy

[0199] HPLC High-Performance Liquid Chromatography

[0200]

[0201] In certain specific embodiments of the present application will be explained in greater detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the application in any manner.

[0202] Examples:

[0203] Experimental procedures according to Scheme 1

[0204] Example 1: Preparation of (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8)

[0205] (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (9) (100g) was added to methanol at 30°C. Cooled the mixture to 0 to 5°C. Thionyl chloride (86.1g) was added for 30 minutes and stirred. The reaction temperature was raised to 60-65°C and stirred till completion. After completion of reaction, the reaction mass was cooled to 40-45°C, then distilled off to get the title compound. Yield: 80%Example 2: Preparation of (S)-methyl l-(2-(tert-butoxycarbonylamino) acetyl)-2-methylpyrrolidine-2-carboxylate (6)

[0206] Boc-Gly-OH (7) (9.75g) was added to Dichloromethane (100mL) at 30-35°C. Cooled the mixture to 0-5 °C. 1 -Hydroxybenzotriazole (2.25g) and (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8) (10g) were added at 0-5°C and then Diisopropylethylamine was added dropwise to the reaction mass.l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (17g) was added to the reaction mixture and stirred. The reaction temperature was raised to 25-30°C and stirred till completion. After completion of reaction, water was added and stirred, then layers were separated. Water was added to the organic layer and stirred, again layers were separated and aqueous HC1 solution was added to the organic layer followed by sodium bicarbonate was added and stirred. Distilled the organic layer to get the crude material. Hexane was added to the crude at room temperature and stirred. Filtered the solid, washed with hexane (50 ml) and dried to get the title compound. Yield: 100%.

[0207] Example 3: Preparation of (S)-l-(2-(tert-butoxycarbonylamino) acetyl)-2-methylpyrrolidine-2-carboxylic acid (5)

[0208] (S)-methyl l-(2-(tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxylate (100 g) (6) was added to 1,4 Dioxane (100 mL), then and water (500mL) at 25-30°C. Cooled the mixture to 0-5°C. Sodium Hydroxide solution was added dropwise and stirred. The reaction temperature was raised to 25-30°C and stirred till completion. After completion of reaction, ethyl acetate was added and stirred, layers were separated. Dichloromethane was added to the aqueous layer and stirred. Layers were separated. Ethyl acetate was added to the aqeous layer at 0-5°C, then pH was adjusted to 2-3 with Cone. HC1 and stirred. Combined the total organic layer distilled to get the title compound. Yield: 79.9%

[0209] Example 4: Preparation of ((S)-di-tert-butyl 2-((S)-l-(2-(tert-butoxycarbonylamino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (3).

[0210] NH2-Glu-(OtBu)2-OH (4) (100g) was added to dichloromethane (lOOOmL) at 30-35°C and cooled to 0-5°C. l-Hydroxy-7-azabenzotriazole (14.1g), and Diisopropylethylamine (135g) was added dropwise at 0-5°C. To this 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (106g) was added and stirred. The reaction temperature was raised to 25-30°C stirred till completion. After completion of reaction, water was added and stirred. Layers were separated, water was added to the organic layer and stirred. Again, layers were separated and aqueous HC1 solution was added to the organic layer followed by sodium bicarbonate was added and stirred. Distilled the organic layer to get the crude material. Hexane was added tothe crude at room temperature and stirred. Filtered the solid, washed with hexane (50 ml) and dried to get the title compound. Yield: 95%

[0211] Example 5: Preparation of (S)-2-((S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioic acid Trifluoroacetic acid (2)

[0212] (S)-di -tert-butyl 2-((S)- 1 -(2-(tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (3) (100g) was added to cocktail solution (Trifluoracetic acid (950mL), water (25 mL) and Triisopropyl silane (25 mL)) at 0-5°C and stirred. The obtained solution was distilled to get the crude. Methyl tert-butyl ether(500mL) was added to the crude at 10-15°C and stirred. Decant the MTBE layer and dried to get the title compound. Yield:89%.

[0213] Example 6: Preparation of Trofinetide (1).

[0214] (S)-2-((S)-l-(2-aminoacetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioic acid Trifluoroacetic acid (2) (100 g) was added to water (1000 mL) and MP carbonate resin at room temperature and stirred till the completion. After completion of the reaction, the reaction mass was filtered and washed the resin, then distilled to get the crude material. To this crude material Methyl tert-butyl ether was added and stirred. Decant the Methyl tert-butyl ether layer and dried to get the foam solid. Acetonitrile was added to the obtained solid and stirred, the acetonitrile layer was removed and dried to get the title compound. Yield:85.0%; Purity: 99.9%

[0215] Experimental procedures according to Scheme 3:

[0216] Example 7: Preparation of (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8).

[0217] (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (9) (100g) was dissolved in methanol (500 mL) at 25-30°C and stirred. The reaction mass was cooled to 0 to 5°C, then thionyl chloride (53 mL) was added and stirred. The reaction temperature was raised to 60-65°C and stirred till completion of the reaction. After completion of reaction, the reaction mass was cooled to 40-45°C, then distilled off the compound using methanol and toluene to get the title compound. Yield: 109.49%; Purity:99.5%

[0218] Example 8: Preparation of (S)-methyl l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylate (15)

[0219] 2-(Dibenzylamino)acetic acid (16) (100 g) was dissolved in dichloromethane (5000 mL). To the reaction mixture, 1 -hydroxybenzotriazole (HoBt) (75 g) was added at a temperature of 25-30°C. The reaction mixture was then cooled to 0-5°C. Diisopropylethylamine (DIPEA) (360g) was added, followed by (S)-methyl 2-methylpyrrolidine-2-carboxylate hydrochloride (8) (142 g) at 0-5°C. To the reaction mass, 1 -ethyl -3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC HC1) (170 g) was added. The temperature was then raised to 25-30°C, and the reaction mixture was stirred. After completion of the reaction, water was added. The layers were separated. Water was added to the organic layer, stirred, and the layers were separated again. An aqueous hydrochloric acid (HC1) solution was added to the organic layer, followed by sodium bicarbonate (NaHCO₃), and the mixture was stirred. The organic layer was then distilled and washed with water, followed by drying to obtain the title compound. Yield:

[0220] 70.49%. Purity: 99%

[0221] Example 9: Preparation of (S)-l-(2-(dibenzyl amino) acetyl)-2-m ethyl pyrrolidine-2-carboxylic acid hydrochloride (14)

[0222] (S)-methyl l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylate (100 g) (15) was added to methanol (7 volumes) at 25-30°C. The mixture was cooled to 0-5°C. A solution of lithium hydroxide (133.6 g) was added dropwise, and the reaction was stirred. The temperature was then raised to 65-70°C and the reaction was stirred until completion. After the reaction was complete, the mixture was distilled and cooled to 25-30°C. The pH was adjusted using hydrochloric acid (HC1). The mixture was stirred and then filtered to remove the wet material. The wet material was washed with dichloromethane. The solid was filtered and dried to obtain the title compound. Yield: 149.20% Purity:99.9%

[0223] Example 10: Process for the preparation of (S)-dibenzyl 2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (13)

[0224] L-Glutamic acid dibenzyl ester 4-toluenesulfonate (10) (34.18 g) was added to dichloromethane (1500 mL) at 25-30°C. To the reaction mixture, (S)-l-(2-(dibenzylamino)acetyl)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (14) (100g) was added, and the mixture was cooled to 0-5°C. 1 -Hydroxybenzotriazole anhydrous (HoBt) was added at the same temperature, and the mixture was stirred. To the reaction mixture, diisopropylethylamine (DIPEA) (57.31 g) and l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) (18.49 g) were added dropwise at 0-5°C and stirred. The reaction temperature was then raised to 25-30°C, and the mixture was stirred until the reaction was completed. After completion, Citric acid, sodium bicarbonate, sodium chloride, hydrochloric acid, sodium bicarbonate, sodium chloride is dissolved in water, separate the organic layer and aqueous layer at 25-30°C,take the organic layer, washed and stir the reaction mass, filter the reaction through 7.5 g of hyflo bed at 25-30°C, washed with methyl tert butyl ether take the organic layer and dried with sodium sulphate, distilled under vacuum at 35-45°C,added (lOOmL) ethyl acetate, (50 mL) water at 25-30°C, stir the reaction mass and separate the organic layer, organic layer dried with sodium sulphate, washed with ethyl acetate, distilled, and dried to obtain the title compound. Yield: 85 % Purity:99%

[0225] Example 11: Alternative Preparation of (S)-dibenzyl 2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (13).

[0226] L-Glutamic acid dibenzyl ester 4-toluenesulfonate (10) (74.06 g) was added to dichloromethane (1300 mL) at 25-30°C. To the reaction mixture, (S)-l-(2-(dibenzylamino)acetyl)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (14) (65 g) was added, and the mixture was cooled to 0-5°C. Triethylamine (TEA) (112.11 mL) was then added, followed by Bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP chloride) (53.38 g), which was added dropwise at 0-5°C and stirred. The reaction temperature was raised to 25-30°C, and the mixture was stirred until the reaction was completed. After completion, the mixture was washed with water, then distilled and dried to obtain the title compound. Yield:

[0227] 91.72% Purity:98%

[0228] Example 12: Preparation of Trofinetide (1).

[0229] (S)-Dibenzyl 2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (13) (100 g) was added to a mixture of ethyl acetate (55 mL) and water (275 mL) at room temperature in the presence of palladium on carbon (Pd / C). The reaction mixture was stirred until completion of reaction. After completion of the reaction, the solid was washed with water, separate the layer with ethyl acetate, and dichloromethane was added to the reaction mass, and the mixture was stirred for 10-15°C, The layers were separated, take the aqueous layer into RB flask, degas the reaction mass for Ihr at temperature to remove dichloromethane, and then filtered the reaction mass through 0.2 pm under nitrogen atmosphere. After completion, the obtained solid was transferred into a clean and dry round bottom flask, acetonitrile (2.0 Vol), ethanol 0.5 Vol, tert-butyl alcohol were added at room temperature. Cooled the reaction mass temperature to 5-10°C and stirred, solid was filtered under vacuum at temperature, washed with mixture of ethanol and tertiary butyl acetate, and dried to obtain the title compound. Yield:85.0%. purity: 99.95%. The PXRD pattern of the obtained compound is illustrated in figure- 1, 2, 3, 4 and 5.

[0230] Experimental procedure according to Scheme 4

[0231] Example 13: Preparation of (S)-di-tert-butyl 2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (10A).(S)-di-tert-butyl 2-aminopentanedioate hydrochloride (9) (40.29 g) was added to dichloromethane (950 mL) at 25-30°C. Dimethylformamide (DMF) (50 mL) and hydroxybenzotriazole (HoBt) (9.20 g) were then added at the same temperature, and the reaction mixture was cooled to 0-5°C. To the reaction mass, diisopropylethylamine (DIPEA) (116 mL) was added, followed by (S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (14) (50 g). l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HC1) (41.67 g) was added, and the mixture was stirred. The reaction temperature was then raised to 25-30°C, and the mixture was stirred until completion of reaction. After completion, the obtained solid was washed with water, filtered, and dried to obtain the title compound. Yield: 100.77%, Purity: 99% (HPLC)

[0232] Example 14: Preparation of (S)-2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentane dioic acid (13A).

[0233] (S)-Di-tert-butyl 2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (10A) (69 g) was added to a cocktail solution comprising TFA: TIPS: water in the ratio of 95:2.5:2.5 at 25-30°C. The reaction mixture was stirred until the reaction was completed. The resulting solid was isolated and purified using MTBE (10 volumes) to obtain the title compound. Yield: 49.77% Purity: 99.5% (HPLC)

[0234] Example 15: Preparation of Trofinetide (1).

[0235] ((S)-di -tert-butyl 2-((S)-l-(2-(tert-butoxy carbonylamino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentanedioate (10A) (100g) was added to The cocktail solution is a mixture of Trifluoracetic acid, Tri isopropyl silane (TIPS) and water at the ratio of (95%:2.5%:2.5%) to provide (S)-2-((S)-l-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxamido) pentane dioic acid (13A) was added to a mixture of ethyl acetate (275 mL) and water (275 mL) at room temperature in the presence of palladium on carbon (Pd / C). The reaction mixture was stirred until the reaction was completed. After completion of the reaction, the obtained solid was washed with ethyl acetate and water, then filtered and dried to obtain the title compound Yield: 93%; Purity: 99.9%(by HPLC)

Claims

We claim:

1. A process for the preparation of Trofinetide of formula (1), having purity greater than 99.0% by High-Performance Liquid Chromatography (HPLC), comprising the steps of:a) reacting (S)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (9)with thionyl chloride in presence of solvent to provide compound of formula (8) in- situWherein R1 is a linear or branched Cl -12 alkyl group; or an aryl-Cl-6 alkyl, or (un)substituted aryl group;b) reacting the compound of formula (8) with 2-(dibenzyl amino) acetic acid (16)in presence of suitable coupling reagent, additive and solvent to provide compound of formula (15) in-situc) hydrolyzing the compound of formula (15) with a base in presence of solvent to provide (S)-1-(2-(dibenzyl amino) acetyl)-2-methylpyrrolidine-2-carboxylic acid hydrochloride (14);d) reacting the compound of formula (14) with compound of formula (10)Wherein P is hydrogen or protecting group,in presence of suitable coupling reagent, additive and solvent to provide compound of formula (13);(13)e) hydrogenating the compound of formula (13) to provide crude Trofinetide (1). f) purifying crude to obtain pure Trofinetide (1) having purity greater than 99.0%. wherein the Trofinetide (1) obtained in the present according to the process is having purity greater than 99.5% by HPLC and comprising formula D, E, F and formula G present between 0.001% to 2 %(wt)2. The process as claimed in claim 1, wherein the base is selected from methyl amine, ethyl amine, dimethylamine, diethylamine, diisopropyl amine, diisopropylethyl amine (DIPEA), diisobutylamine, triethyl amine, tert.butyl amine, pyridine, 4- dimethylaminopyridine (DMAP), N-methyl morpholine (NMM), 2,6-lutidine, n-methyl pyridine (NMP), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo[2.2.2]octane (DABCO), imidazole; “alkali metal hydroxides” such as lithium hydroxide, sodium hydroxide, potassium hydroxide and the like;3. The process as claimed in claim 1, wherein the solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, 2-nitroethanol, 2- fluoroethanol, 2,2,2-trifluoroethanol, ethylene glycol, polyethylene glycol, polyethylene glycol -400, 2-m ethoxy ethanol, 1,2-ethoxyethanol, di ethylene glycol, 1, 2, or 3 -pentanol, neo pentyl alcohol, t-pentyl alcohol, diethylene glycol mono ethyl ether, cyclohexanol, benzyl alcohol, phenol, or glycerol and the like; “chloro solvents” such as dichloromethane, di chloroethane, chloroform, carbon tetrachloride and the like;4. The process as claimed in claim 1, wherein the “coupling reagent” used is selected from but not limited to 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), N-Hydroxy succinimide (NHS), Dicyclohexylcarbodiimide (DCC), Benzotri azole- 1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (BOP), N, N’- Carbonyldiimidazole (CDI), N, N-Diisopropylcarbodiimide (DIC), 3- (Dimethylamino)propyl carbodiimide (DMP). Bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP chloride)5. The process as claimed in claim 1, wherein the “additive” is selected from but not limited to HOBt (1-Hydroxybenzotriazole), HOAt (1-Hydroxy-7-azabenzotriazole), TBTU (O- (Benzo triazol- l-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate), EDT (Ethyl Dimethyl aminopropylcarbodiimide), PyBOP (Benzotriazol-l-yl-oxy) Tripyrrolidinophosphonium hexafluorophosphate).

6. The process as claimed in claim 1, wherein the protecting group ‘P’ is selected from tertbutyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyl (Bn), carbamate group, p -methoxy phenyl (PMP), p-methoxybenzyl (PMB), 3,4-dimethoxy benzyl (DMPM), trityl (Tr), alkyl / aryl sulfonyl such as methanesulfonyl, ethanesulfonyl, benzenesulfonyl, p-toluenesulfonyl (tosyl) and the like;7. A novel compounds of formula (13), formula (2a), formula (2b), formula (14), formula (4a), formula (4b) formula (15), formula (5a), formula (5b), formula (6), and formula (10A), formula (10a), formula (10b) and formula (13A), formula (11a), formula (11b), formula (12); or its pharmaceutically acceptable salts2a14(12)10b13Allawherein R1 and P are defined above8. A composition comprising a compound of formula (1):o(1)Or a stereoisomer, or pharmaceutically acceptable salt thereof, and additionally comprising one or more compounds selected from the group consisting of formula D, E, F and formula G present between 0.00 lwt% and about 0.15%.

9. Trofinetide of formula (1):Or a stereoisomer, or pharmaceutically acceptable salt thereof, and additionally comprising one or more compounds selected from the group consisting of formula D, E, F, formula G, H, I, J, K, L, M, and formulaN present between 0.001wt% and about 0.15 wt%.Formula Eformula Fformula Hformula Iformula Jformula N

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