Field of the Invention:
The present invention relates to an improved process for the preparation of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of formula-1, represented by the following structural formula:
The present invention also relates to new, accurate, simple HPLC methods for the
analysis of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-
1-(4-phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of
formula-1 and its intermediates.
Background of the Invention:
5-[[[(2S)-2-Amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-
(4-phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid known as
Eluxadoline and is approved in US under the brand name VIBERZI for the treatment of
irritable bowel syndrome with diarrhea. Irritable bowel syndrome is a long-term disorder of
the gut with pain or discomfort in the abdomen (belly), bloating and altered bowel habit.
European Commission granted a marketing authorization for Eluxadoline (Truberzi) on 19th
September 2016.
5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid and process for its preparation was first disclosed in US7741356 B2 herein after referred as US’356 B2.
US7994206 B2 discloses the process for the preparation of Eluxadoline by treating boc protected Eluxadoline with hydrochloric acid to provide Eluxadoline hydrochloride, which is further treated with sodium hydroxide to provide Eluxadoline with low yield and purity. The said process suffers from several draw backs such as cumbersome workup,

compound of formula-1 is obtained as gummy semi-solid which is difficult to isolate and requires tedious purifications and finally provide compound of formula-1 with low yield.
In view of the foregoing, there is an unmet need to develop cost-effective, environmental friendly and commercially viable process for the preparation of compound of formula-1 with high yield and purity.
Brief description of the Invention:
The first aspect of the present invention is to provide an improved process for the preparation of amorphous form of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethyl phenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid compound of formula-1.
The second aspect of the present invention is to provide 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl] amino]methyl]-2-methoxybenzoic acid 2-butanol solvate compound of formula-1 and process for its preparation.
The third aspect of the present invention is to provide a new, accurate and sensitive HPLC method for analyzing 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of formula-1 and its intermediates.
The fourth aspect of the present invention is to provide crystalline form of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b, herein after designated as form-S1 and process for its preparation.
The fifth aspect of the present invention is to provide 5-(((S)-2-((tert-butoxy carbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a.
Brief description of the Drawings:
Figure 1: Illustrates the PXRD pattern of crystalline form-M of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl] amino]methyl]-2-methoxybenzoic acid 2-butanol solvate compound of formula-1.

Figure 2: Illustrates the PXRD pattern of crystalline form-S1 of 5-(((S)-2-((tert-butoxy carbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b. Figure 3: Illustrates the PXRD pattern of crystalline form-S2 of 5-(((S)-2-((tert-butoxy carbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a. Figure 4: Illustrates the PXRD pattern of crystalline form-S3 of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl] amino]methyl]-2-methoxybenzoic acid 2-butanol solvate compound of formula-1.
Detailed description of the Invention:
The term “suitable base” used herein the present invention until unless specified 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; “alkali metal alkoxides” such as sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium methoxide, potassium ethoxide, potassium tert-butoxide and the like; ammonia; and organic bases such as triethyl amine, methyl amine, ethyl amine, 1,8-Diaza bicycle[5.4.0]undec-7-ene (DBU), 1,5-Diazabicyclo(4.3.0)non-5-ene (DBN), lithium diiso propylamide (LDA), n-butyl lithium, tribenzylamine, isopropyl amine, diisopropylamine, diisopropylethylamine, N-methylmorpholine, N-ethylmorpholine, piperidine, dimethylamino pyridine, morpholine, pyridine, 2,6-lutidine, 2,4,6-collidine, imidazole, 1-methyl imidazole, 1,2,4-triazole, 1,4-diazabicyclo[2.2.2]octane (DABCO) or mixtures thereof.
As used herein the term “suitable solvent” used in the present invention refers to “hydrocarbon solvents” such as n-hexane, n-heptane, cyclohexane, pet ether, toluene, pentane, cycloheptane, methylcyclohexane, m-, o-, or p-xylene, 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, t-butyl methyl ether, 1,2-dimethoxy ethane and the like; “ester solvents” such as methyl acetate, ethyl acetate,

sopropyl acetate, n-butyl acetate and the like; “polar-aprotic solvents such as dimethylacetamide (DMA), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP) and the like; “chloro solvents” such as dichloromethane, dichloroethane and the like; “ketone solvents” such as acetone, methyl ethyl ketone, methyl sobutylketone and the like; “nitrile solvents” such as acetonitrile, propionitrile, sobutyronitrile and the like; “alcoholic solvents” such as methanol, ethanol, n-propanol, sopropanol, n-butanol, isobutanol, t-butanol, ethylene glycol, l, 2-ethoxyethanol, diethylene glycol, 1, 2, or 3-pentanol, neo-pentyl alcohol, diethylene glycol mono ethyl ether, cyclohexanol, or glycerol and the like; “polar solvents” such as water or mixtures thereof.
The term “suitable coupling agent” is selected from Ν,Ν'- dicyclohexylcarbodiimide
DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), l-ethyl-3-(3-dimethylaminopropyl)
carbodiimide HCl (EDC.HC1), alkyl or aryl chloroformates such as ethylchloro formate,
benzylchloroformate, diphenylphosphoroazidate (DPPA), (Benzotriazol-l-yloxy)tri
pyrrolidinophosphoniumhexafluorophosphate, methanesulfonyl chloride and the like optionally in combination with l-hydroxy-7-azatriazole (HOAt), 1-hydroxybenzotriazole HOBt), 1-hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt), N-hydroxysuccinamide, N-hydroxysulfosuccinimide (Sulfo- NHS) or mixture thereof.
The first aspect of the present invention provides an improved process for the preparation of amorphous form of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethyl phenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid compound of formula-1, comprising of following steps;
a) Reacting acid addition salts of (S)-methyl-2-methoxy-5-(((1-(4-phenyl-1H-imidazol-2-yl)ethyl)amino)methyl)benzoate compound of general formula-2 with (S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)propanoic acid compound of formula-3 in presence of a suitable coupling agent and a suitable base in a suitable solvent to provide methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl) propanamido)methyl)-2-methoxy benzoate compound of formula-4,
b) treating the compound of formula-4 with lithium hydroxide in a suitable solvent and followed by treating with sodium carbonate in a suitable solvent to provide 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-

phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate sodium salt compound of formula-5a,
c) optionally purifying the compound of formula-5a with a suitable solvent,
d) treating the compound of formula-5a with a suitable hydrochloric acid source in a suitable solvent and treating with a suitable base in a suitable solvent to provide 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid compound of formula-1 or its solvates,
e) dissolving the compound of formula-1 in a suitable solvent/ mixture of solvents, followed by addition of resulting mixture to a suitable solvent at a suitable temperature to provide amorphous form of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid compound of formula-1.
Wherein, in step-a) the suitable coupling agent is selected from Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC.HC1), alkyl or aryl chloroformates such as ethyl chloroformate, benzylchloroformate, diphenylphosphoroazidate (DPPA), thionyl chloride, oxalyl chloride, phosphorous oxy chloride, phosphorouspentachloride, 4-methyl-2-oxopentanoyl chloride (i-BuCOCOCl), (Benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), methane sulfonyl chloride and the like optionally in combination with l-hydroxy-7-azatriazole (HOAt), 1-Hydroxybenzotriazole (HOBt), 1-Hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt), N-hydroxysuccinamide (HOSu), N-hydroxysulfosuccinimide (Sulfo-NHS), 4-dimethylamino pyridine and the like and suitable base is selected from organic or inorganic base; in step-d) the suitable hydrochloric acid source is selected from hydrochloric acid, aqueous hydrochloric acid, methanolic-HCl, ethanolic-HCl, IPA-HCl, hydrochloric acid gas and the suitable base is selected from organic base; in step-a) to step-e) the suitable solvent is selected from alcohol solvents, ketone solvents, ester solvents, hydrocarbon solvents, polar aprotic solvents, ether solvents, nitrile solvents, chloro solvents and polar solvents like water or mixture thereof.
The preferred embodiment of the present invention provides an improved process for the preparation of amorphous form of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethyl

phenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid compound of formula-1, comprising of following steps;
a) Reacting (S)-methyl-2-methoxy-5-(((1-(4-phenyl-1H-imidazol-2-yl)ethyl)amino) methyl)benzoate maleate salt compound of formula-2a with (S)-2-((tert-butoxy carbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)propanoic acid compound of formula-3 in presence of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl, 1-hydroxybenzotriazole and diisopropylethylamine in acetonitrile to provide methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate compound of formula-4,
b) treating the compound of formula-4 with lithium hydroxide in a mixture of isopropanol and water and followed by treating with sodium carbonate in a mixture of tetrahydrofuran, water and isopropanol to provide 5-(((S)-2-((tert-butoxycarbonyl) amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxybenzoate sodium salt compound of formula-5a,
c) treating the compound of formula-5a with hydrochloric acid in acetonitrile and then followed by treating with triethyl amine in a mixture of water and 2-butanol to provide 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid 2-butanol solvate compound of formula-1,
d) dissolving the 2-butanol solvate compound of formula-1 in a mixture of acetonitrile and water followed by addition of resulting mixture to a precooled water at 0-5°C to provide amorphous form of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoic acid compound of formula-1.
In an embodiment of the present invention, the compound of formula-4 can also treated with a suitable alkali or alkaline earth metal hydroxide or carbonates or bicarbonates or alkoxides that can form alkali or alkaline earth metal salts of compound of formula-5.
In an embodiment of the present invention the compound of formula-5a can be treated with a suitable acid in a suitable solvent to provide acid addition salts of compound of formula-1.

US7741356 B2 discloses process for the preparation of 5-(((S)-2-(tert-butoxycarbonyl amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl) propanamido)methyl)-2-methoxybenzoic acid herein after referred to as “2-methoxybenzoic acid” by, reacting methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-di methylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate compound of formula-4 with aqueous lithium hydroxide in the presence of tetrahydrofuran and methanol for longer hours and followed cumbersome work-up and tedious column chromatographic purification provides 2-methoxybenzoic acid with low yield.
The said process suffers from several draw backs such as utilizing multiple solvent system, lengther reaction time and cumbersome work-up along with tedious column purification to isolate 2-methoxybenzoic acid with low yield. The said process also involves excess of solvents which leads to the generation of lot of spent solvents and solid waste which are difficult to dispose and which may lead to the pollution of the environment. Further, the said process increases the cost of the production and which is not recommended for commercial scale-up.
The present process is simple, eco-friendly and commercially viable, which involves lower mole ratio of lithium hydroxide in a single solvent and takes less time period for the completion of the reaction and further without any column purifications provides compound of formuala-5a with high yield.
5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate sodium salt compound of formula-5a is having purity greater than 98% as measured by HPLC.
5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate sodium salt compound of formula-5a obtained according to the present invention is having Boc alcohol impurity; Boc diacid impurity; RS-Isomer, Boc isopropyl ester impurity, amine impurity, Boc ester impurity and amide impurity less than 0.05% as measured by HPLC.

US7994206 B2 involves process for the preparation of compound of formula-1 by, reacting 2-methoxybenzoic acid with concentrated hydrochloric acid in acetone to provide hydrochloric acid salt of compound of formula-1, which is further neutralized by treating with aqueous sodium hydroxide to provide compound of formula-1 with low yield and purity.
On repetition of the above process, i.e. neutralization of hydrochloric acid salt of

compound of formula-1 with sodium hydroxide, the compound of formula-1 is isolated as gummy solid with low yield and purity. In order to isolate compound of formula-1 as a free solid, required multiple number of purifications and thereby making the process more uneconomical and not suitable for commercial scale-up.
The inventors of the present invention have carried out the neutralization of hydrochloride salt of compound of fornula-1 using different bases in various solvents. Surprisingly they found that the yield and purity of the final compound is very high whenever the hydrochloride salt of compound of formula-1 treated with organic base in presence of alcohol solvents.
The present inventors found that by carrying out the neutralization of hydrochloride salt of compound of formula-1 using a combination of triethyl amine and alcohol solvents preferably 2-butanol, produced the compound of formula-1 as a free solid with enhanced yield and purity. The same was illustrated in the following table:
The second aspect of the present invention provides 5-[[[(2S)-2-amino-3-[4-(amino carbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino] methyl]-2-methoxybenzoic acid 2-butanol solvate compound of formula-1.
In an embodiment of the present invention provides crystalline form of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid 2-butanol solvate.
In another embodiment of the present invention provides crystalline form of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid 2-butanol solvate compound of formula-1 herein after designated as form-M is characterized by, its powder X-Ray diffractogram having peaks at 6.2, 7.3, 8.9, 9.7, 10.3, 10.8, 12.1, 12.6, 13.6, 14.1, 14.8, 15.0, 15.6, 15.8, 17.6, 18.1, 18.7, 19.1, 19.4, 20.2, 21.1, 21.9, 22.5, 23.1, 24.0, 24.6, 25.2, 27.7, 28.0, 31.6 and 32.6 ± 0.2 degrees of two-theta as depicted in figure-1.

The crystalline form-M of compound of formula-1 obtained according to the present invention is useful in the preparation of pure 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of formula-1.
The another embodiment of the present invention provides a process for the preparation of crystalline form-M of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethyl phenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid 2-butanol solvate compound of formula-1, comprising of;
a) Dissolving 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxo propyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid hydrochloride salt compound of formula-1a in 2-butanol or aqueous 2-butanol,
b) adding a suitable organic base to the reaction mixture,
c) stirring the reaction mixture,
d) filtering the precipitated solid and drying to get the crystalline form-M of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoic acid 2-butanol solvate compound of formula-1.
The preferred embodiment of the present invention provides a process for the preparation of crystalline form-M of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethyl phenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid 2-butanol solvate compound of formula-1, comprising of;
a) Dissolving 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxo propyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid hydrochloride salt compound of formula-1a in a mixture of water and 2-butanol,
b) adding triethyl amine to the reaction mixture,
c) stirring the reaction mixture,
d) filtering the precipitated solid and drying to get the crystalline form-M of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoic acid 2-butanol solvate compound of formula-1.
The third aspect of the present invention provides a new, accurate and sensitive HPLC

method for analyzing 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxo
propyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid
compound of formula-1 and it intermediates.
The technique of choice for the analysis of APIs or pharmaceutical compositions (e.g. a tablet or capsule) is usually High Performance liquid Chromatography (HPLC) coupled with a UV-Visible detector. The API and the impurities present, if any, are separated on the HPLC stationary phase and they can be detected and quantified using their response obtained from the UV-Visible detector.
The likely impurities in APIs and pharmaceutical compositions include residual quantities of synthetic precursors (intermediates), by-products which arise during the synthesis of the API, residual solvents, isomers of the API (e.g. geometrical isomers, diastereomers or enantiomers), contaminants which are present in materials used in the synthesis of the API or in the preparation of the pharmaceutical composition, and unidentified adventitious substances. Other impurities which may appear on storage include degradants of the API, for instance formed by hydrolysis or oxidation.
However, the current HPLC methods are not suitable for the detection and estimation of all total synthetic impurities and other related substances that are present, especially with respect to known and unknown impurities that are present in Eluxadoline. Studies by the present inventors have culminated in the development and validation of a new, accurate, efficient, reproducible and simple HPLC method for the analysis of Eluxadoline, particularly with respect to the related substances formed during the synthetic process, whilst avoiding the typical problems associated with the prior art methods.
In an embodiment of the present invention provides a new, accurate and sensitive HPLC method for the analyzing of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethyl phenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid compound of formula-1.
In another embodiment of the present invention the purity of the 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl) ethyl]amino]methyl]-2-methoxy benzoic acid compound of formula-1 can be determined using chiral HPLC method using Lux i-Cellulose-5 (Make: Phenomenex), Chiralpak® IC-3 column.

The fourth aspect of the present invention provides crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b and the P-XRD pattern was depicted in figure-2.
In another embodiment of the present invention provides a process for the preparation of crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-di methylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b, comprising of:
a) Adding a suitable solvent to methyl 5-(((S)-2-(tert-butoxycarbonylamino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl) propanamido)methyl)-2-methoxybenzoate compound of formula-4,
b) cooling the reaction mixture,
c) adding aqueous lithium hydroxide to the reaction mixture,
d) heating and stirring the reaction mixture at a suitable temperature,
e) cooling the reaction mixture and optionally purifying the obtained compound using a suitable solvent to provide crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl) amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b.
Wherein, in step-b) the suitable temperature is ranging from 0°C to 10°C; in step-d) the suitable temperature is ranging from 25°C to 65°C; in step-a) and e) the suitable solvent is selected from alcohol solvents, ketone solvents, ester solvents, hydrocarbon solvents, polar aprotic solvents, ether solvents, nitrile solvents, chloro solvents and polar solvents like water or mixture thereof.
The preferred embodiment of the present invention provides a process for the preparation of crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b, comprising of:
a) Adding isopropanol to methyl 5-(((S)-2-(tert-butoxycarbonylamino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido) methyl)-2-methoxybenzoate compound of formula-4,

b) cooling the reaction mixture to 0-5°C,
c) adding aqueous lithium hydroxide to the reaction mixture,
d) raising the temperature of the reaction mixture to 25-30°C and stirring the reaction mixture,
e) heating the reaction mixture to 40-45°C and stirring the reaction mixture,
f) cooling the reaction mixture and purifying the obtained compound using isopropanol to provide crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propan amido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b. The fifth aspect of the present invention provides 5-(((S)-2-((tert-butoxy carbonyl)
amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl) propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a.
The another embodiment of the present invention provides crystalline form of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a, herein after designated as form-S2 and the P-XRD pattern was depicted in figure-3.
The another embodiment of the present invention provides process for the preparation of crystalline form-S2 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-di methylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a, comprising of:
a) Adding a suitable solvent to 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido) methyl)-2-methoxy benzoate lithium salt,
b) adding a suitable sodium source to the reaction mixture,
c) heating and stirring the reaction mixture,
d) cooling the reaction mixture and optionally, purifying the obtained compound using a suitable solvent to provide crystalline form-S2 of 5-(((S)-2-((tert-butoxycarbonyl) amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a.

Wherein, in step-b) the suitable sodium source is selected from sodium carbonate, sodium bicarbonate, sodium hydroxide, sodium methoxide; in step-c) the suitable temperature is ranging from ambient temperature to reflux temperature of the solvent used in the reaction; in step-d) the suitable temperature is ranging from 20-35°C; in step-a) and d) the suitable solvent is same as defined in step-a) of the fourth aspect of the present invention.
The preferred embodiment of the present invention provides process for the preparation of crystalline form-S2 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a, comprising of:
a) Adding a mixture of tetrahydrofuran and water to 5-(((S)-2-((tert-butoxycarbonyl) amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido) methyl)-2-methoxy benzoate lithium salt,
b) adding sodium carbonate to the reaction mixture,
c) heating the reaction mixture to 40-45°C and stirring the reaction mixture,
d) cooling the reaction mixture to 25-30°C,
e) purifying the obtained compound by, adding a mixture of tetrahydrofuran and water and then adding isopropanol to the obtained reaction mixture to provide crystalline form-S2 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethyl phenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a.
The 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-
((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate sodium
salt compound of formula-5a can be prepared by, hydrolyzing methyl 5-(((S)-2-(tert-butoxy
carbonylamino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)
ethyl)propanamido)methyl)-2-methoxybenzoate compound of formula-4 using a suitable
sodium source in a suitable solvent.
The crystalline form-S1 and form-S2 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido) methyl)-2-methoxy benzoate are useful in the preparation of pure compound of formula-1.
5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of formula-

1 obtained according to the present invention having the following impurities less than 0.05% as measured by HPLC.
a). 5-(((R)-2-Amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid {diastereomers impurity}, b). 4-((S)-2-Amino-3-((3-carboxy-4-methoxybenzyl)((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl )amino)-3-oxopropyl)-3,5-dimethyl benzoic acid {diacid impurity},
c). Isopropyl 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido) methyl)-2-methoxy benzoate {isopropyl impurity}, d). Sec-butyl 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido) methyl)-2-methoxy benzoate {butyl ester impurity}, and e). 5-(((R)-2-Amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((R)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoic acid {Enantiomer impurity}.
5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of formula-1 obtained according to the present invention having purity greater than 99.85%, preferably greater than 99.95%, most preferably greater than 99.98% as measured by HPLC.
5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-

phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of formula-1 produced by the present invention can be further micronized or milled in a conventional techniques to get the desired particle size to achieve desired solubility profile based on different forms of pharmaceutical composition requirements. Techniques that may be used for particle size reduction include, but not limited to ball, roller and hammer mills, and jet mills. Milling or micronization may be performed before drying, or after the completion of drying of the product. The process of the present invention is schematically represented as below:
P-XRD Method of Analysis:
PXRD analysis of compounds produced by the present invention were carried out using BRUKER D8 ADVANCE/AXS X-Ray diffractometer using Cu Kα radiation of wavelength 1.5406 A° and continuous scan speed of 0.03°/min.
The invention also encompasses pharmaceutical compositions comprising compound

of formula-1 or salts thereof of the present invention. As used herein, the term "pharmaceutical compositions" or "pharmaceutical formulations" include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, or injection preparations.
The process described in the present invention was demonstrated in examples illustrated below. These examples are provided as illustration only and therefore should not be construed as limitation of the scope of the invention.
Examples:
Example-1: Preparation of methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carba
moyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)
methyl)-2-methoxybenzoate: (Formula-4)
Hydroxybenzotriazole (20.0 gms) and N-(3-dimethylaminopropyl)-N′-ethyl
carbodiimide hydrochloride (56.8 gms) were added to a mixture of acetonitrile (500 ml) and (S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)propanoic acid (100 gms) compound of formula-3 at 10-15°C and stirred for 30 minutes at the same temperature. (S)-methyl-2-methoxy-5-(((1-(4-phenyl-1H-imidazol-2-yl)ethyl)amino)methyl)benzoate maleate (142.8 gms) compound of formula-2a was added to the reaction mixture at 10-15°C. Diisopropylethylamine (76.8 gms) was slowly added to the reaction mixture at 10-15°C. Raised the temperature of the reaction mixture to 25-30°C and stirred for 90 minutes at the same temperature. N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (28.4 gms) was added to the reaction mixture at 25-30°C and stirred for 1 hour at the same temperature. Water (1.0 lts) and ethyl acetate (600 ml) were added to the reaction mixture at 25-30°C and stirred for 20 minutes at the same temperature. Both the organic and aqueous layers were separated and aqueous layer was extracted with ethyl acetate. Combined the organic layers and washed twice with aqueous sodium carbonate solution followed with hydrochloric acid solution at 25-30°C. Further, organic layer was washed with aqueous sodium chloride solution. Distilled off the solvent completely from the organic layer under reduced pressure. Isopropanol (200 ml) was added to the obtained solid at 40-45°C. Distilled off the solvent completely from the reaction mixture under reduced pressure. Yield: 172.8 gms.

Example-2: Preparation of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt: (Formual-5b)
Aqueous lithium hydroxide solution was slowly added to a mixture of methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate (172.8 gms) and isopropanol (864 ml) at 0-5°C. Raised the reaction mixture temperature to 25-30°C and stirred for 12 hours at the same temperature. Heated the reaction mixture to 40-45°C and stirred for 3 hours at the same temperature. Cooled the reaction mixture to 25-30°C. Isopropanol (4150 ml) was added to the reaction mixture at 25-30°C and stirred for 45 minutes at the same temperature. Filtered the precipitated solid, washed with isopropanol and dried to get the title compound. Yield: 145.0 gms; Purity by HPLC: 99.45%; Boc alcohol impurity: 0.13%; Boc diacid impurity: 0.13%; Boc isopropyl ester impurity: 0.18%; Boc ester impurity: 0.01%; Acid impurity: 0.01%; SMUI: 0.13%. The P-XRD pattern of the obtained compound was depicted in figure-3.
Example-3: Preparation of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate sodium salt: (Formula-5a)
Tetrahydrofuran (870 ml) and water (435 ml) was added to 5-(((S)-2-((tert-butoxy carbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxybenzoate lithium salt (145 gms) at 25-30°C. Sodium carbonate (27.3 gms) was added to the reaction mixture at 25-30°C. Heated the reaction mixture to 40-45°C and stirred for 45 minutes at the same temperature. Cooled the reaction mixture to 25-30°C. Isopropanol (2175 ml) was added to the reaction mixture at 25-30°C and stirred for 3 hours at the same temperature. Filtered the precipitated solid, washed with tetrahydrofuran. To the obtained solid, tetrahydrofuran (840 ml) and water (420 ml) was added at 25-30°C. Heated the reaction mixture to 40-45°C and stirred for 45 minutes at the same temperature. Cooled the reaction mixture to 25-30°C. Isopropanol was added to the reaction mixture at 25-30°C and stirred for 3 hours at the same temperature. Filtered the precipitated solid, washed with tetrahydrofuran and dried to get the title compound.

Yield: 130 gms; Purity by HPLC: 99.84%; Boc alcohol impurity: 0.02%; Boc diacid impurity: 0.03%; Boc isopropyl ester impurity: 0.01%; Boc ester impurity: Not detected; Acid impurity: Not detected%; SMUI: 0.1%. The P-XRD pattern of the obtained compound was depicted in figure-2.
Example-4: Preparation of crystalline form-M of Eluxadoline 2-butanol solvate:
Acetonitrile (200 ml) was added to 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido) methyl)-2-methoxy benzoate sodium salt (100 gms) at 25-30°C. Cooled the reaction mixture to 0-5°C. Acetonitrile (100 ml) and hydrochloric acid solution was slowly added to the reaction mixture at 0-5°C. Raised the temperature of the reaction mixture to 20-25°C and stirred for 4 hours at the same temperature. Water was added to the reaction mixture at 20-25°C. Cooled the reaction mixture to 0-5°C. Neutralizing the reaction mixture using triethyl amine at 0-5°C. 2-Butanol (3000 ml) was added to the reaction mixture at 0-5°C. Raised the temperature of the reaction mixture to 25-30°C and stirred for 6 hours at the same temperature. Cooled the reaction mixture to 0-5°C and stirred for 2 hours at the same temperature. Filtered the precipitated solid, washed with 2-butanol and dried to get the title compound. Yield: 76.5 gms; M.R: 185-190°C; 2-Butanol content: 13.35%.
Purity by HPLC: 99.92%; Diacid impurity: 0.06%; Amide alcohol impurity: Not detected; Isopropyl ester impurity: Not detected; Boc ester impurity: 0.02%; HIUI: 0.02%; Diastereomer impurity: Not detected. Chiral purity by HPLC: 99.97%; Enantiomer impurity: 0.03%; The P-XRD pattern of the obtained compound was depicted in figure-1.
Example-5: Preparation of Amorphous form of Eluxadoline:
A mixture of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid butanol solvate (100 gms), acetonitrile (150 ml) and water (150 ml) was stirred for 10 minutes at 25-30°C. Heated the reaction mixture to 40-45°C and stirred for 15 minutes at the same temperature. Filtered the reaction mixture through hy-flow bed and washed with a mixture of acetonitrile and water. The filtrate was added to pre-cooled water at 0-5°C and stirred for 2 hours at the same temperature. Filtered the precipitated solid, washed with chilled water and dried to get the title compound. Yield: 70.0 gms; M.R: 180-186°C; Water content: 5.17% w/w

Purity by HPLC: 99.95%; Diacid impurity: 0.07%; Alcohol impurity: 0.01%; Isopropyl ester impurity: 0.02%; Boc ester impurity: 0.01%; HIUI: 0.07%. Chiral purity by HPLC: 99.98%; Enantiomer impurity: 0.02%. The P-XRD of the obtained compound is matching with the P-XRD pattern of amorphous form disclosed in our co-pending Indian patent application no. 201641007801.
Example-6: Preparation of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate lithium salt: (Formula-5b)
Aqueous lithium hydroxide solution was slowly added to a mixture of methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate (172.8 gms) and isopropanol (864 ml) at 0-5°C. Raised the reaction mixture temperature to 25-30°C and stirred for 12 hours at the same temperature. Heated the reaction mixture to 40-45°C and stirred for 3 hours at the same temperature. Cooled the reaction mixture to 25-30°C. Isopropanol (4150 ml) was added to the reaction mixture at 25-30°C and stirred for 45 minutes at the same temperature. Filtered the precipitated solid, washed with isopropanol and dried to get the title compound. Yield: 145.0 gms; Purity by HPLC: 99.45%; Boc alcohol impurity: 0.13%; Boc diacid impurity: 0.13%; Boc isopropyl ester impurity: 0.18%; Boc ester impurity: 0.01%; Acid impurity: 0.01%; SMUI: 0.13%.
Example-7: Preparation of crystalline form-S3 of Eluxadoline 2-butanol solvate:
Acetonitrile (600 ml) and hydrochloric acid solution (600 ml) were added to 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt (200 gms) at 10-15°C and stirred for 20 minutes at the same temperature. Raised the temperature of the reaction mixture to 25-30°C and stirred for 4 hours at the same temperature. Water (600 ml) and 2-butanol (300 ml) were added to the reaction mixture at 25-30°C and stirred for 15 minutes at the same temperature. Reaction mixture was neutralized by using triethylamine (750 ml) at 25-30°C and stirred for 2 hours at the same temperature. Filtered the precipitated solid, washed with water and dried for 24 hours at 60-65°C to get the title compound. Yield: 122 gms; Purity by HPLC: 99.88%. The P-XRD pattern of the obtained compound was depicted in figure-4.

We Claim:
1. An improved process for the preparation of amorphous form of 5-[[[(2S)-2-amino-3-[4-
(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)
ethyl]amino]methyl]-2-methoxy benzoic acid compound of formula-1, comprising of
following steps;
a) Reacting (S)-methyl-2-methoxy-5-(((1-(4-phenyl-1H-imidazol-2-yl)ethyl)amino) methyl)benzoate compound of general formula-2 or its acid addition salts with (S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)propanoic acid compound of formula-3 in presence of a suitable coupling agent and a suitable base in a suitable solvent to provide methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl) propanamido)methyl)-2-methoxy benzoate compound of formula-4,
b) treating the compound of formula-4 with lithium hydroxide in a suitable solvent and followed by treating with sodium carbonate in a suitable solvent to provide 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate sodium salt compound of formula-5a,
c) optionally purifying the compound of formula-5a with a suitable solvent,
d) treating the compound of formula-5a with a suitable hydrochloric acid source in a suitable solvent and treating with a suitable organic base in a suitable solvent to provide 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid compound of formula-1 or its solvates,
e) dissolving the compound of formula-1 in a suitable solvent / mixture of solvents, followed by addition of resulting mixture to a suitable solvent at a suitable temperature to provide amorphous form of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid compound of formula-1.
2. The process as claimed in claim 1, wherein, in step-a) the suitable coupling agent is
selected from Ν,Ν'- dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide

(DIC), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC.HC1), alkyl or aryl
chloroformates such as ethyl chloroformate, benzylchloroformate, diphenylphosphoro
azidate (DPPA), thionyl chloride, oxalyl chloride, phosphorous oxy chloride, phosphorous
pentachloride, 4-methyl-2-oxopentanoyl chloride (i-BuCOCOCl), (Benzotriazol-l-yloxy)
tripyrrolidinophosphoniumhexafluorophosphate (PyBOP), methane sulfonyl chloride and
the like optionally in combination with l-hydroxy-7-azatriazole (HOAt), 1-hydroxybenzo
triazole (HOBt), 1-hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt), N-hydroxy
succinamide (HOSu), N-hydroxysulfosuccinimide (Sulfo-NHS), 4-dimethylamino pyridine
and the like and suitable base is selected from organic or inorganic base;
in step-b) the compound of formula-4 can also treated with a suitable alkali or alkaline
earth metal hydroxide or carbonates or bicarbonates or alkoxides that can
form alkali or alkaline earth metal salts of compound of formula-5, which is further treated
with a suitable acid in a suitable solvent to provide acid addition salts of compound of
formula-1.
in step-d) the suitable hydrochloric acid source is selected from hydrochloric acid, aqueous
hydrochloric acid, methanolic-HCl, ethanolic-HCl, IPA-HCl, hydrochloric acid gas;
in step-a) to step-e) the suitable solvent is selected from alcohol solvents, ketone solvents,
ester solvents, hydrocarbon solvents, polar aprotic solvents, ether solvents, nitrile solvents,
chloro solvents and polar solvents like water or mixture thereof.
3. The process as claimed in claim 1, wherein an improved process for the preparation of
amorphous form of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxo
propyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxy benzoic acid
compound of formula-1, comprising of following steps;
a) Reacting (S)-methyl-2-methoxy-5-(((1-(4-phenyl-1H-imidazol-2-yl)ethyl)amino)
methyl)benzoate maleate salt compound of formula-2a with (S)-2-((tert-butoxy carbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)propanoic acid compound of formula-3 in presence of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl, 1-hydroxybenzotriazole and diisopropylethylamine in acetonitrile to provide methyl-5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-

(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate compound of formula-4,
b) treating the compound of formula-4 with lithium hydroxide in a mixture of isopropanol and water and followed by treating with sodium carbonate in a mixture of tetrahydrofuran, water and isopropanol to provide 5-(((S)-2-((tert-butoxycarbonyl) amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxybenzoate sodium salt compound of formula-5a,
c) treating the compound of formula-5a with hydrochloric acid in acetonitrile and then followed by treating with triethyl amine in a mixture of water and 2-butanol to provide 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid 2-butanol solvate compound of formula-1,
d) dissolving the 2-butanol solvate compound of formula-1 in a mixture of acetonitrile and water followed by addition of resulting mixture to a precooled water at 0-5°C to provide amorphous form of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoic acid compound of formula-1.
4. Crystalline forms of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid, which includes:
a) Crystalline form-M of 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid 2-butanol solvate which is characterized by its powder X-Ray diffractogram having peaks at 6.2, 7.3, 8.9, 9.7, 10.3, 10.8, 12.1, 12.6, 13.6, 14.1, 14.8, 15.0, 15.6, 15.8, 17.6, 18.1, 18.7, 19.1, 19.4, 20.2, 21.1, 21.9, 22.5, 23.1, 24.0, 24.6, 25.2, 27.7, 28.0, 31.6 and 32.6 ± 0.2 degrees of two-theta as depicted in figure-1.
b) Crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b and which is characterized by its P-XRD pattern as depicted in figure-2.

c) Crystalline form-S2 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a and which is characterized by its P-XRD pattern as depicted in figure-3.
5. 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl]amino]methyl]-2-methoxybenzoic acid 2-butanol solvate compound of formula-1.
6. 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a.
7. A process for the preparation of crystalline form-M of 5-[[[(2S)-2-amino-3-[4-(amino carbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2yl)ethyl] amino]methyl]-2-methoxy benzoic acid 2-butanol solvate compound of formula-1, comprising of: treating 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-

dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a with suitable hydrochloric acid source in a suitable solvent and followed by in-situ treating with organic base in 2-butanol or aqueous 2-butanol to provide crystalline form-M of 5-(((S)-2-amino-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoic acid 2-butanol solvate compound of formula-1,
8. A process for the preparation of crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl)
amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)
propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b,
comprising of:
a) Adding methyl 5-(((S)-2-(tert-butoxycarbonylamino)-3-(4-carbamoyl-2,6-dimethyl phenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxybenzoate compound of formula-4 to a suitable solvent selected from alcohol solvents, ketone solvents, ester solvents, hydrocarbon solvents, polar aprotic solvents, ether solvents, nitrile solvents, chloro solvents and polar solvents like water or mixture thereof,
b) cooling the reaction mixture to 0°C to 10°C,
c) adding aqueous lithium hydroxide to the reaction mixture,
d) heating the reaction mixture to temperature ranging from 30°C to 65°C and stirring the reaction mixture,
e) cooling the reaction mixture and optionally purifying the obtained compound using alcohol solvents to provide crystalline form-S1 of 5-(((S)-2-((tert-butoxycarbonyl) amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl) ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt compound of formula-5b.
9. A process for the preparation of crystalline form-S2 of 5-(((S)-2-((tert-butoxycarbonyl)
amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)
propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a,
comprising of:

a) Adding 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate lithium salt to a suitable solvent selected from alcohol solvents, ketone solvents, ester solvents, hydrocarbon solvents, polar aprotic solvents, ether solvents, nitrile solvents, chloro solvents and polar solvents like water or mixture thereof,
b) adding a suitable sodium source selected from sodium carbonate, sodium bicarbonate, sodium hydroxide, sodium methoxide to the reaction mixture,
c) heating the reaction mixture to a temperature ranging from 30°C to reflux temperature of the solvent used and stirring the reaction mixture,
d) cooling the reaction mixture to 25-30°C and filtering the obtained compound,
e) optionally, purifying the obtained compound by, adding a mixture of tetrahydrofuran and water and then adding isopropanol to the obtained reaction mixture to provide crystalline form-S2 of 5-(((S)-2-((tert-butoxycarbonyl)amino)-3-(4-carbamoyl-2,6-dimethylphenyl)-N-((S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl)propanamido)methyl)-2-methoxy benzoate sodium salt compound of formula-5a.
10. 5-[[[(2S)-2-amino-3-[4-(aminocarbonyl)-2,6-dimethylphenyl]-1-oxopropyl][(1S)-1-(4-phenyl-1H-imidazol-2-yl)ethyl]amino]methyl]-2-methoxybenzoic acid compound of formula-1 obtained according to the preceding claims having Boc alcohol impurity; Boc diacid impurity; RS-Isomer, Boc isopropyl ester impurity, amine impurity, Boc ester impurity and amide impurity less than 0.05% as measured by HPLC.