DESC:FIELD OF THE INVENTION
The present invention relates to an efficient process for the preparation of Semaglutide or a pharmaceutically acceptable salt thereof. The present invention also relates to novel fragments as intermediates and use thereof in the preparation of Semaglutide.
BACKGROUND OF THE INVENTION
Semaglutide is a long-acting GLP-1 analog developed by Novo Nordisk. It is a glucagon-like peptide 1 (GLP-1) receptor agonist indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus.
H-His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[AAEAc-AAEAc-?-Glu-C18diacid] -Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-OH
Formula-I
US 8,129,343 describe Semaglutide, its analogs and process for preparation thereof. The said process involves the preparation of Semaglutide backbone using standard Fmoc solid phase peptide synthesis followed by de-protection and then coupling of the side chain fragment to the Lys20. The coupling of the side chain moiety to Lys is carried out by using 17-((S)-1- tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-l-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy) ethoxy]ethylcarbamoyl}propylcarbamoyl)heptadecanoic acid tertbutyl ester or by sequential coupling of two units of, 8-amino-3,6-dioxaoctanoic acid, ?-glutamic acid and octadecanedioic acid. US 8,637,647 describe selectively acylating lysine amino group in a peptide. The said process involves acylation of side chain amino group of Lysine using 22-carboxy-1-[(2,5-dioxo-1-pyrrolidiny)oxy]-1,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan-41-oic acid on Semaglutide backbone.
In drug substance synthesis there is always a continuing need for preparation process which can obtain drug substance in high yields, more purity and has low impurities; and which is commercially feasible as well.
SUMMARY OF THE INVENTION
An aspect of the present invention relates to an improved process of preparation of Semaglutide comprising;
a. loading of Fmoc protected glycine to a resin solid-phase support in the presence of coupling agent;
b. de-protection of Fmoc group using de-protecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Semaglutide till Phe12 in the presence of coupling agent Fmoc-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin, wherein Xn is selected from the group consisting of Alloc, Mtt, Dde, Mmt and ivDde;
c. de-protection of Fmoc protecting group of fragment obtained in step (b) using de-protecting agent followed by coupling of tetra-peptide fragment Aib8-Glu9-Gly10-Thr11-OH (SEQ ID-1) wherein, the side chain of amino acids protected with a suitable protecting group; in the presence of coupling agent, to obtain Fmoc-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin;
d. de-protection of Fmoc protecting group using de-protecting agent and coupling of N-terminal and side chain protected histidine, in the presence of coupling agent, to obtain His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin; and
e. de-protection of protecting group (Xn) from Lys26, to obtain His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin and converting the resulting peptide to Semaglutide.
In another aspect, the present invention relates to a compound of SEQ ID NO.1 and process of preparation thereof and its use in the preparation of Semaglutide,
Aib8-Glu9-Gly10-Thr11 (SEQ ID-1)
wherein the terminal amino acids are free, resin bound or protected with a suitable protecting group; and wherein, the side chain of amino acids are free or protected with a suitable protecting group.
In further aspect, the present invention relates to a compound of SEQ ID: 1.

BRIEF DESCRIPTION OF ABBREVIATIONS:
SPPS Solid phase peptide synthesis
LPPS liquid phase peptide synthesis
Fmoc 9-fluorenylmethyloxycarbonyl
Boc t-Butyloxycarbonyl
Trt Trityl
tBu Tert-butyl
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl
HPLC High performance liquid chromatography
DIPEA Diisopropylethylamine
TFA Trifluoroacetic acid
DBU l,8-Diazabicyclo[5.4.0]undec-7-ene
DMAP 4-Dimethylaminopyridine
AC2O Acetic anhydride
DMF N,N-Dimethylformamide
DCM Dichloromethane
ACN Acetonitrile
MeOH Methanol
DIPEA Diisopropylethylamine
TIS Triisopropylsilane
EDT 1,2-ethanedithiol
DMS Dimethyl sulfide
DIC/DIPC Diisopropylcarbodiimide
DCC Dicyclohexylcarbodiimide
EDC Ethyl-dimethylaminopropyl carbodiimide
HOBt 1-Hydroxybenzotriazole
HOAt l-Hydroxy-7-azabenzotriazole
TBTU N,N,N’,N'-Tetramethyl-0-(benzotriazol-l- yl)uronium tetrafluoroborate
HBTU 3-[Bis(dimethylamino)methyliumyl]-3H- benzotriazol-l-oxide hexafluorophosphate
HATU 2-(7-Aza-lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate
PyBOP (Benzotriazol-l-yloxy)-tripyrrolidinophosphoniumhexafluorophosphate
Oxyma B/
OxymaPure 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione /Ethyl 2-cyano-2-hydroxyimino-acetate
HFIP 1,1,1,3,3,3-Hexafluoroisopropanol
TFE Trifluoroethanol
TES Triethylsilane
Pd(PPh3)4 Tetrakis(triphenylphosphine)palladium(0)
h Hour/s
min Minutes
DETAILED DESCRIPTION OF INVENTION
As used herein Solid Phase Peptide Synthesis (“SPPS”) is the method and system that is most commonly used to synthesize polypeptides and amino acid sequences. This solid support is usually a polymeric resin bead that is functionalized (such as with an -OH groups). The next amino acid (which generally has its NH2 terminus protected via a Fmoc, Boc or other terminal protecting group) is reacted with the resin such that the functionalized group on the resin reacts with and binds to the activated -COOH group of the terminal amino acid. In this manner, the terminal amino acid is covalently attached to the resin through esterification.
Then, in the next step, the NH2 terminus of the terminal amino acid is deprotected, thereby exposing its NH2 group for the next reaction. Accordingly, a new amino acid (AA) is introduced. This new amino acid has its NH2 terminus protected via a protecting group (such as an Fmoc, Boc or another protecting group). As such, when this new amino acid is added, the activated ester from the new amino acid reacts with the newly deprotected NH2 group of the terminal amino acid, thereby coupling these two amino acids together. Once this new amino acid has been coupled, it likewise has a protected NH2 group that may be subsequently deprotected and reacted with the next amino acid. By doing this repetitive, iterative process over and over, the entire amino acid sequence may be constructed. Once the entire sequence has been constructed, the sequence may be uncoupled (cleaved) from the resin and deprotected, thereby producing the amino acid sequence. The amino acids side-chain functional groups are optionally protected with groups which are generally stable during coupling steps and during a-amino protecting group removal, and which are themselves removable in suitable conditions. Such suitable conditions are generally orthogonal to the conditions in which the a-amino groups are deprotected. The protecting groups of amino acids side-chain functional groups which are used in the present disclosure are generally removable in acidic conditions, as orthogonal to the basic conditions generally used to deprotect Fmoc protecting group. Those skilled in the art will appreciate how such side chains or other group may be constructed, protected, and subsequently deprotected during the synthesis process.
The solid phase synthesis (SPPS) in present invention is carried out on a resin i.e. insoluble polymer which is acid sensitive. An acid sensitive resin is selected from a group comprising Rink amide resin (RAR), Seiber amide resin, chlorotrityl resin (CTC), Sasrin, Wang Resin, 4-methytrityl chloride, TentaGel S, TentaGel TGA, NovaSyn TGT resin, HMPB-AM resin, 4-(2-(amino methyl)-5-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA include, most preferred super acid labile resin is Rink amide resin (RAR).
Resin such as Wang resin or CTC resin are swelled in suitable solvent such as but not limited to DCM, DMF or mixture thereof, by stirring at 10-40 °C, preferably at 25-30 °C for 0.5-1 h followed by washing with DCM, DMF or mixture thereof.
The term "Fmoc protected amino acid" as used herein refers to amino acids with Fmoc protection on a-amino group of the amino acids. “Fmoc protected serine” refers to serine with Fmoc protection on a-amino group.
As used herein the term "protecting group" refers to a labile chemical moiety which is known in the art to protect reactive groups including without limitation, hydroxyl, amino and thiol groups, against undesired reactions during synthetic procedures. Protecting groups are typically used selectively and/or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group as is or available for further reactions. Protecting groups as known in the art are described generally in Greene's Protective Groups in Organic Synthesis, 4th edition, John Wiley & Sons, New York, 2007.
The term "orthogonally protected" refers to functional groups which are protected with different classes of protecting groups, wherein each class of protecting group can be removed in any order and in the presence of all other classes (see, Barany et al., J. Am. Chem. Soc., 1977, 99, 7363-7365; Barany et al., J. Am. Chem. Soc., 1980, 102, 3084-3095). Orthogonal protection is widely used in for example automated oligonucleotide synthesis. A functional group is deblocked in the presence of one or more other protected functional groups which are not affected by the deblocking procedure. This deblocked functional group is reacted in some manner and at some point a further orthogonal protecting group is removed under a different set of reaction conditions. This allows for selective chemistry to arrive at a desired compound.
Examples of hydroxyl protecting groups include without limitation, acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, bis(2-acetoxyethoxy)methyl (ACE), 2-trimethylsilylethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, [(triisopropylsilyl)oxy]methyl (TOM), benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl, p-phenylbenzoyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMT), trimethoxytrityl, 1(2-fluorophenyl)-4-methoxypiperidin-4-yl (FPMP), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Wherein more commonly used hydroxyl protecting groups include without limitation, benzyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzoyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). Examples of amino protecting groups include without limitation, carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (Boc), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imine- and cyclic imide-protecting groups, such as phthalimido and dithiasuccinoyl. Examples of thiol protecting groups include without limitation, triphenylmethyl (Trt), benzyl (Bn), and the like.
The term “N-terminal protecting group” or “N-terminal protection” or "terminal protecting group" as used herein refers to the protecting group for the a-amino group of the amino acids or of the peptides used in the preparation of Semaglutide, or of the complete Semaglutide sequence, which is cleaved either prior to the coupling to elongate the peptide sequence or at the end of the peptide elongation. Preferably, the N-terminal protecting group is 9-fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc). De-protecting the Boc or Fmoc group from the completed-protected-coupled-product, comprises reacting the completed-protected-coupled-product with an acid in the case of Boc, or a base in the case Fmoc.
The term "deprotecting agent" as used herein refers to a reagent or reagent system (reagent(s), and solvent) useful for removing a protecting group. De-protecting agents are acids, bases or reducing agents. For example, removal of the benzyl (Bn) group is generally accomplished by reduction (hydrogenolysis), while removal of carbamates (e.g. Boc group) is generally effected by use of acids (e.g. HCl, TFA, etc.) optionally with mild heating.
In particular, the Fmoc protecting group is cleaved by treatment with a suitable secondary amine selected from the group consisting of piperidine, pyrrolidine, piperazine and DBU, preferably piperidine. More preferably, Fmoc de-protection is carried out by using a 20% solution of piperidine in DMF. An additive such as formic acid, boric acid, citric acid can be optionally added during Fmoc de-protection to facilitate the reaction. In an aspect, after de-protection of Fmoc the resulting peptide is optionally washed with HOBt.H2O, formic acid, citric acid, and ascorbic acid in presence of solvent used for coupling, preferable washing using HOBt.H2O in DMF.
The term "side chain protection" as used herein is a protecting group for an amino acid side-chain chemical function which is not removed when the terminal protecting group is removed and is stable during coupling reactions. Preferably, side-chain protecting groups are included to protect side-chains of amino acids which are particularly reactive or labile, to avoid side reactions and/or branching of the growing molecule. Illustrative examples include acid-labile protecting groups, as for instance tert-butyloxycarbonyl (Boc), alkyl groups such as tert-butyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and the like. Other protecting groups may be efficiently used as it is apparent to the person skilled in the art.
The epsilon amino group of lysine in position 26 of Semaglutide is protected with a protecting group (Xn) such as allyloxycarbonyl (Alloc), 4-methyltrityl (Mtt), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (Mmt), dichlorodiphenyl dichloroethylene (Dde), ivDde,
In one embodiment of the invention, the coupling of amino acids may be carried out in the presence of a coupling agent and optionally in the presence of coupling additive.
The term “coupling agent” as used herein, may be selected from the group comprising of N,N'- diisopropylcarbodiimide (DIC or DIPC), N,N'-dicyclohexylcarbodiimide (DCC), (Benzotriazol-1-yloxy)tripyrrolidino phosphoniumhexafluorophosphate (PyBOP), [Ethyl cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), N,N,N',N'-Tetramethyl-0-(benzotriazol-l-yl)uroniumtetrafluoroborate (TBTU), 2-(7-Aza-lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HATU), 2-(lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), ethyl-dimethylaminopropylcarbodiimide (EDC), (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (DEPBT) etc,.
Coupling additive, when used in the coupling reaction, reduces loss of configuration at the carboxylic acid residue, increases coupling rates and reduces the risk of racemization. The additive may be selected from the group comprising 1- hydroxybenzotriazole.monohydrate (HOBt.H2O), 2-hydroxypyridine N-oxide, N-hydroxysuccinimide, 1-hydroxy- 7-azabenzotriazole (HOAt), endo-N-hydroxy-5-norbornene-2, 3-dicarboxamide and ethyl 2- cyano-2-hydroxyimino-acetate (OxymaPure), 5-(Hydroxyimino)l,3-dimethylpyrimidine-2,4,6-(lH,3H,5H)-trione (Oxy-B).
The term "suitable base" as used means a base that is useful for effecting the subject reaction. One of skill in the art is aware of the many bases (organic and inorganic bases) regarded as useful in the art for the purpose of the particular reaction. Suitable bases are also exemplified by the bases disclosed herein for the specific reaction. In one embodiment of the invention, the coupling reaction may be carried out in the presence of a base selected from the group of tertiary amines comprising diisopropylethylamine (DIPEA), triethylamine, collidine, N-methylmorpholine, N-methylpiperidine etc.
In one embodiment of the invention, the coupling reaction may be carried out in the presence of an inorganic base selected from the group consisting of magnesium chloride, zinc chloride or copper chloride is optionally added during coupling stage.
The term "suitable solvent" as used herein means a solvent that is useful for effecting the subject reaction. One of skill in the art is aware of the many solvents regarded as useful in the art for the purpose of the particular reaction. Suitable solvents are also exemplified by the solvents disclosed herein for the specific reaction.
In one embodiment of the invention, the coupling reaction, either involving peptides or amino acids, takes place in the presence of a solvent selected from the group comprising dimethylformamide (DMF), dimethylacetamide (DMAc), dimethylsulfoxide (DMSO), dichloromethane (DCM), chloroform (CHCl3), tetrahydrofuran (THF), 2-methyl tetrahydrofuran (2Me-THF) and N-methyl pyrrolidine (NMP). The solvent used in said coupling reaction is selected from DMF, DCM, NMP or DMSO in combination between them; preferably DMF.
In a preferred embodiment, Gly, Ala, Val, Ser and Leu amino acids were coupled using DIC/OxymaPure or HBTU/HOBt.H2O/Oxymapure/DIPEA. In another preferred embodiment, Trp, Asp, Arg, Gln, Asn, Phe, Ile, Gly, Tyr, Thr, Lys and Glu were coupled using HBTU/OxymaPure/DIPEA or HBTU/HATU/DIPEA or HBTU/HOBt.H2O/Oxymapure/DIPEA. In another preferred embodiment, Aib was coupled using HATU/DIPEA or HATU/Oxyma-B/ DIPEA or HBTU/OxymaPure/DIPEA or HBTU/HATU/DIPEA. In another preferred embodiment, His was coupled using HATU/Oxyma-B/DIPEA or HATU/ Oxymapure /DIPEA. In a preferred embodiment, SEQ ID -1 was coupled to Semaglutide backbone using HATU /Oxyma- B/ DIPEA.
The amount of individual coupling agents used may range from about 1 to about 6 molar equivalents, per molar equivalent of resin with respect to resin loading capacity. Preferably, 3 molar equivalents of individual coupling agents per molar equivalent of the resin with respect to resin loading capacity may be used. The coupling temperature is usually in the range of from 10 to 50 °C; preferably 25 to 35 °C.
Additionally, the unreacted sites on the resin are optionally capped, to avoid truncated sequences and to prevent any side reactions, by a short treatment with a large excess of a highly reactive unhindered reagent, which is chosen according to the unreacted sites to be capped, and according to well-known peptide synthesis techniques. For example, capping may be performed using acetic anhydride in presence of base such as DIPEA, TEA, Pyridine and like. The aim of capping is preventing the occurrence of product like or closely eluting impurities (i.e. products lacking an amino acid building block at one position), which are probably hard to separate from the desired final product.
De-protection and cleavage conditions generally depend on the nature of the protecting groups and of the resin used. The concomitant removal of amino acid side chain protection and polymer support of the peptide from the resin involves treating the protected peptide anchored to the resin with a cleaving agent that comprises an acid and at least one scavenger. The peptide cleavage reagent (or cleaving agent) used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents. The acidic is preferably based on an acidic material such as TFA, and contains scavenger reagents including, but not limited to, water, triethylsilane (TES), DTT, triisopropylsilane (TIS/TIPS), 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, and cresol or mixture thereof and water; preferably TFA: TIS: phenol: water. The relative ratio of acidic material to scavenger to water may be from about 70 % to about 90% acidic material, from about 10 % to about 30% scavenger. Preferably the ratio of TFA: TIPS: phenol: water is 90 %: 2.5 %: 5%: 2.5 %. More, preferably the ratio of TFA: TIPS: phenol: water is 80:5:10:5.
In one embodiment of the invention, acid such as formic acid, boric acid, citric acid is optionally added along with piperidine during de-protection of Fmoc protecting group.
Orthogonal de-protection of Lys26 is carried out using hydrazine hydrate when orthogonal protecting group is Dde. When orthogonal protecting group is Mtt, de-protection is carried out using HFIP, TFE, TES in MDC. When orthogonal protecting group is Alloc, de-protection is carried out using Pd(PPh3)4. The deprotection temperature is usually in the range of from 10 to 30 °C; preferably 20 to 25 °C in suitable solvent such as but not limited to DMF, DCM. The epsilon amino group of lysine in position 26 is coupled with N-terminal and side chain protected or un-protected octadecanedioic acid -?-Glu-AEEA-AEEA-OH or by employing additional coupling/deprotection cycles as to extend the Lys26 side-chain involving Fmoc-AEEA-OH, Fmoc-Glu(OH)-OtBu and HOOC—(CH2)16-COOtBu to obtain protected Semaglutide- peptidyl resin. The coupling is performed using suitable coupling agent such as but not limited to HATU, HBTU, PyBOP, PyOxim, BOP, HCTU, COMU, and DEPBT in presence of additive such as but not limited to HOBt.H2O, OxymaPure and base such as but not limited to DIPEA, colliding base in suitable solvent such as but not limited to DMF and stirring at 10-50°C, preferably at 25-35 °C for 1 to 3 h followed by washing with solvent such as but not limited to DCM and DMF or mixture thereof.
In a preferred embodiment, Fmoc-Glu-OtBu, Fmoc-AEEA-OH, C18-diacid(OtBu)-OH were coupled using HBTU/OxymaPure/DIPEA. In another preferred embodiment, C18-diacid(OtBu)-?Glu(OtBu)-AEEA-AEEA-OH is coupled to Lys26 using HATU/Oxyma-B/DIPEA.
In a preferred embodiment, when SPPS is used, the protected Semaglutide sequence is finally deprotected and cleaved from the resin, either simultaneously or in two steps, providing crude Semaglutide, which may optionally be purified. Concomitant cleavage from the resin and side chain protecting group removal is carried out by treating the peptide with a cleaving agent comprising a mixture of TFA, TIPS, Phenol, H2O at 10-40 °C, preferably at 15-25 °C for 2 to 4 h.
The crude Semaglutide obtained may be isolated by precipitation using suitable solvents such as but not limited to MTBE, toluene, diisopropyl ether, hexane, heptane, isopropyl ether optionally purified by crystallization or chromatographic techniques well known in the art. The inventors of the present process have found that the use of the above described polypeptide fragments in preparation of Semaglutide or its pharmaceutically acceptable salt, provides Semaglutide in better yield and high purity, which makes it suitable for large scale industrial production.
In an embodiment, purification involves reverse phase chromatographic (RP-HPLC) purification. In another embodiment the RP-HPLC employs mobile phase selected from group comprising of Trifluoroacetic acid, formic acid, citric acid, Ammonium bicarbonate, Ammonium carbonate, trisaminomethane (TRIS) buffer, sodium phosphate buffer, TEAP buffer, Ammonia, acetonitrile, 2-propanol, methanol, water. In an embodiment crude Semaglutide is subjected to one or more RP-HPLC purification. In another embodiment, pure Semaglutide is isolated by subjecting the fractions collected from RP-HPLC purification to distillation, treatment with NaOH solution and followed by lyophilization.
The inventors of the present process have found that the use of the above described polypeptide fragments in preparation of Semaglutide or its pharmaceutically acceptable salt, provides Semaglutide in better yield and high purity, which makes it suitable for large scale industrial production. The term "pure Semaglutide" refers to Semaglutide which have purity over 99%, preferably over 99.5%, more preferably over 99.9%.
First aspect of the present invention relates to an improved process of preparation of Semaglutide comprising SEQ ID: 1.
In one embodiment, Fmoc protected glycine used in step (a) of first aspect is Fmoc-Gly-OH.
In one embodiment Fmoc-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin in step (b) of first aspect is preferably Fmoc-Phe12-Thr13(tBu)-Ser14(tBu)-Asp15(OtBu)-Val16-Ser17(tBu)-Ser18(tBu)-Tyr19(tBu)-Leu20-Glu21(OtBu)-Gly22-Gln23(Trt)-Ala24-Ala25-Lys26(Xn)-Glu27(OtBu)-Phe28-Ile29-Ala30-Trp31(Boc)-Leu32-Val33-Arg34(Pbf)-Gly35-Arg36(Pbf)-Gly37-Resin; wherein side chain protecting group (Xn) on lysine is selected from the group of Alloc, Mtt and Dde.
In an embodiment SEQ ID: 1 used in step (c) of first aspect specifically is Aib8-Glu9-Gly10-Thr11-OH, more specifically SEQ ID: 1 is Fmoc-Aib8-Glu9(OtBu)-Gly10-Thr11(tBu)-OH.
In yet another embodiment Fmoc-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin used in step (c) of first aspect is Aib8-Glu9-Gly10-Thr11-OH to obtain Fmoc-Aib8-Glu9(OtBu)-Gly10-Thr11(tBu)-Phe12-Thr13(tBu)-Ser14(tBu)-Asp15(OtBu)-Val16-Ser17(tBu)-Ser18(tBu)-Tyr19(tBu)-Leu20-Glu21(OtBu)-Gly22-Gln23(Trt)-Ala24-Ala25-Lys26(Xn)-Glu27(OtBu)-Phe28-Ile29-Ala30-Trp31(Boc)-Leu32-Val33-Arg34(Pbf)-Gly35-Arg36(Pbf)-Gly37-Resin.
In one embodiment, N-terminal and side chain protected histidine used in step (d) of first aspect is Boc-His7(Trt)-OH.
In still another embodiment His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin of step (d) of first aspect is preferably Boc-His7(Trt)-Aib8-Glu9(OtBu)-Gly10-Thr11(tBu)-Phe12-Thr13(tBu)-Ser14(tBu)-Asp15(OtBu)-Val16-Ser17(tBu)-Ser18(tBu)-Tyr19(tBu)-Leu20-Glu21(OtBu)-Gly22-Gln23(Trt)-Ala24-Ala25-Lys26(Xn)-Glu27(OtBu)-Phe28-Ile29-Ala30-Trp31(Boc)-Leu32-Val33-Arg34(Pbf)-Gly35-Arg36(Pbf)-Gly37-Resin.
In one embodiment of first aspect, in step (e) side chain de-protection on lysine from fragment obtained in step (d) is carried out using1-5% hydrazine hydrate in DMF, wherein Xn is Dde. Wherein Xn is Mtt, de-protection is carried out using HFIP, TFE, TES in MDC. Where in Xn is Alloc, de-protection is carried out using Pd(PPh3)4.
In another embodiment of first aspect, the conversion of His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin to Semaglutide comprises reacting His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin with side chain protected or un-protected fragment octadecanedioic acid -?-Glu-AEEA-AEEA-OH OR sequentially reacting the resin with AEEA, AEEA, ?-glutamic acid(OtBu), octadecanedioic acid(OtBu) OR sequentially reacting the resin with AEEA-AEEA, ?-glutamic acid(OtBu), octadecanedioic acid(OtBu) followed by concomitant cleavage of solid support and protecting group of ensuing peptide using a cleaving agent.
In one embodiment, octadecanedioic acid(OtBu) is also referred as C18-diacid(OtBu), C18-monoester or 18-(tert-butoxy)-18-oxoicosanoic acid and octadecanedioic acid is also referred as C18-diacid or C18-acid.
In another embodiment His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[(AEEA-AEEA-Glu(OtBu)-C18-diacid(OtBu)]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin is subjected to concomitant cleavage.
In yet another embodiment Boc-His7(Trt)-Aib8-Glu9(OtBu)-Gly10-Thr11(tBu)-Phe12-Thr13(tBu)-Ser14(tBu)-Asp15(OtBu)-Val16-Ser17(tBu)-Ser18(tBu)-Tyr19(tBu)-Leu20-Glu21(OtBu)-Gly22-Gln23(Trt)-Ala24-Ala25-Lys26[AEEA-AEEA-Glu(OtBu)-C18-diacid(OtBu)]-Glu27(OtBu)-Phe28-Ile29-Ala30-Trp31(Boc)-Leu32-Val33-Arg34(Pbf)-Gly35-Arg36(Pbf)-Gly37-Resin is subjected to concomitant cleavage using a cleaving agent.
In one embodiment of first aspect, Semaglutide obtained according to this invention optionally subjected to chromatographic purification methods and lyophilisation.
In further aspect, the present invention relates to a compound of SEQ ID NO.1 and process of preparation thereof.
Aib8-Glu9-Gly10-Thr11-OH (SEQ ID-1)
In further aspect, process for the preparation of SEQ ID-1 comprising;
a. Loading of Fmoc protected threonine to resin solid-phase support, wherein side chain may be protected with suitable protecting group, in the presence of coupling agent;
b. de-protection of Fmoc protecting group using de-protecting agent and coupling of Fmoc protected glycine in the presence of coupling agent;
c. de-protection of Fmoc protecting group using de-protecting agent and coupling of Fmoc protected glutamic acid 2-aminoisobutyric acid, wherein side chain may be protected to obtain backbone of SEQ ID-1; and
d. Cleavage of SEQ ID-1 from the solid support and removal of amino acid protecting group using cleaving reagent to obtain Aib8-Glu9-Gly10-Thr11-OH (SEQ ID-1).
In one embodiment of the invention, SEQ ID: 1 specifically is be Fmoc-Aib8-Glu9(OtBu)-Gly10-Thr11(tBu)-OH.
In one embodiment of the invention, de-protecting agents for Fmoc de-protection is base in suitable organic solvent. The base used may be an inorganic or organic base. Preferably the base is an organic base selected from the group comprising piperidine, pyrrolidine, piperazine, tert-butylamine, DBU and diethylamine, preferably piperidine in DMF. In one embodiment of the invention, additive for Fmoc de-protection is selected from the group comprising formic acid, citric acid and boric acid or mixture thereof. In one embodiment of the invention, Fmoc de-protection is carried out with de-protecting agents with additive wherein Semaglutide is synthesized by solid phase peptide synthesis (SPPS) or liquid phase peptide synthesis (LPPS). Further, approach to solid phase synthesis can be liner synthesis or fragment synthesis.
The invention is further exemplified by the following non-limiting examples, which are illustrative representing the preferred modes of carrying out the invention. The invention's scope is not limited to these specific embodiments only but should be read in conjunction with what is disclosed anywhere else in the specification together with those information and knowledge which are within the general understanding of the person skilled in the art.
In one embodiment the process for preparation of Semaglutide of Formula-I according to the present invention is shown in scheme-1.

Scheme 1
Examples:
Example 1: Synthesis of Semaglutide using two fragment condensation strategy using Wang resin
Wang resin (100 g, 0.50 mmol/g substitution) was washed with DCM in a reaction flask, solvent was removed using vacuum and allowed to swell in DCM. Fmoc-Gly-OH (2.0 eq.), DIC (3.0 eq.) and DMAP (0.05 eq.) were dissolved in DMF/ DCM, and added to the resin and stirred for 2-3 h. The resin was washed with DMF. Substitution obtained after first amino acid loading was 0.40 mmol/g. Further, capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, de-protection was performed using 5 % piperidine in DMF with 0.5 % boric acid mixture for 10 min. and reaction completion was monitored using Kaiser test. The obtained resin was washed twice with DMF containing 0.5 % formic acid followed by fresh DMF and washings were monitored by Chloranil test.
The rest of the amino acids and fragments were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent and additive were dissolved in DMF and the reaction mixture was added to the resin and stirred for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. Resin was washed thrice with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Gly, Ala, Val, Ser and Leu amino acids were coupled using DIC/OxymaPure
ii) Trp, Asp, Arg, Gln, Asn, Phe, Ile, Gly, Tyr, Thr, Lys, C18-diacid(OtBu)-OH, Fmoc-Glu-OtBu, Fmoc- AEEA-OH were coupled using HBTU/OxymaPure/DIPEA
iii) Fragment Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH and Boc-His(Trt)-OH were coupled using HATU /Oxyma- B/ DIPEA
iv) Mtt was removed using HFIP/TFE/TES/MDC
All Fmoc de-protection reactions were performed by using 5- 20 % piperidine in DMF with 0.5 % boric acid. Solvents were removed using vacuum.
As per the sequence, coupling and de-protection reactions were performed to obtain
Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((2xAEEA-?Glu-C18-diacid(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.
The obtained resin was washed with DMF, DCM, MeOH, and MTBE and allowed to dry. Cleavage cocktail mixture of TFA/TIPS/Phenol/H2O (90.0:2.5:5.0:2.5) was added to the resin and stirred for 3-4 h. The obtained reaction mass was filtered and the filtrate was evaporated and crude peptide was isolated using chilled MTBE. The obtained peptide was washed with MTBE and dried to obtain crude Semaglutide (125 g and product content 30.5 g). The crude peptide obtained was purified in a manner analogous to that as described in example-1 to get pure Semaglutide.
Example 2: Synthesis of Semaglutide using two fragment condensation strategy using Wang resin (Linear + side chain)
Wang resin (100 g, 0.50 mmol/g substitution) was washed with DCM in a reaction flask, solvent was removed using vacuum and allowed to swell in DCM. Fmoc-Gly-OH (2.0 eq.), DIC (3.0 eq.) and DMAP (0.05 eq.) were dissolved in DMF/ DCM, and added to the resin and stirred for 2-3 h. The resin was washed with DMF. Substitution obtained after first amino acid loading was 0.40 mmol/g. Further, capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, de-protection was performed using 5 % piperidine in DMF with 0.5 % boric acid mixture for 10 min and reaction completion was monitored using Kaiser test. The obtained resin was washed twice with DMF containing 0.5 % formic acid followed by fresh DMF and washings were monitored by Chloranil test.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent and additive were dissolved in DMF and the reaction mixture was added to the resin and stirred for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. Resin was washed thrice with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Gly, Ala, Val, Ser and Leu amino acids were coupled using DIC/OxymaPure
ii) Trp, Asp, Arg, Gln, Asn, Phe, Ile, Gly, Tyr, Thr, Lys were coupled using HBTU/OxymaPure/DIPEA
iii) Aib was coupled using HATU/DIPEA
iv) His was coupled using HATU/Oxyma-B/DIPEA
v) Mtt was removed using HFIP/TFE/TES/MDC
vi) C18-diacid(OtBu)-?Glu(OtBu)-AEEA-AEEA-OH is coupled using HATU/Oxyma-B/DIPEA
All Fmoc deprotection reactions were performed by using 5- 20 % piperidine in DMF with 0.5 % boric acid. Solvents were removed using vacuum.
As per the sequence, sequential coupling and de-protection reaction performed to obtain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu) Asp(OtBu) -Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys ((2xAEEA-?Glu-C18-diacid(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.
The obtained resin was washed with DMF, DCM, MeOH, and MTBE and allowed to dry. Cleavage cocktail mixture of TFA/TIPS/Phenol/H2O (90.0:2.5:5.0:2.5) was added to the resin and stirred for 3-4 h. The obtained reaction mass was filtered and the filtrate was evaporated and crude peptide was isolated using chilled MTBE. The obtained peptide was washed with MTBE and dried to obtain crude Semaglutide (119 g and product content 29.75 g). The crude peptide obtained was purified in a manner analogous to that as described in example-1 to get pure Semaglutide.
Example 3: Synthesis of Semaglutide using three fragment condensation strategy using Wang resin
Wang resin (100 g, 0.50 mmol/g substitution) was washed with DCM in a reaction flask, solvent was removed using vacuum and allowed to swell in DCM. Fmoc-Gly-OH (2.0 eq.), DIC (3.0 eq.) and DMAP (0.05 eq.) were dissolved in DMF/ DCM, and added to the resin and stirred for 2-3 h. The resin was washed with DMF. Substitution obtained after first amino acid loading was 0.40 mmol/g. Further, capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, de-protection was performed using 5 % piperidine in DMF with 0.5 % boric acid mixture for 10 min and reaction completion was monitored using Kaiser test. The obtained resin was washed twice with DMF containing 0.5 % formic acid followed by fresh DMF and washings were monitored by Chloranil test.
The rest of the amino acids and fragments were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent and additive were dissolved in DMF and the reaction mixture was added to the resin and stirred for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. Resin was washed thrice with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Gly, Ala, Val, Ser and Leu amino acids were coupled using DIC/OxymaPure
ii) Trp, Asp, Arg, Gln, Asn, Phe, Ile, Gly, Tyr, Thr, Lys were coupled using HBTU/OxymaPure/DIPEA
iii) Fragment Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH and Boc-His(Trt)-OH were coupled using HATU/ Oxyma- B/ DIPEA
iv) C18-diacid(OtBu)-?Glu(OtBu)-AEEA-AEEA-OH is coupled using HATU/ Oxyma- B/ DIPEA
v) Mtt was removed using HFIP/TFE/TES/MDC
All Fmoc de-protection reactions were performed by using 5- 20 % piperidine in DMF with 0.5 % boric acid. Solvents were removed using vacuum.
As per the sequence, coupling and de-protection reactions were performed to obtain
Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((2xAEEA-?Glu-C18-diacid(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.
The obtained resin was washed with DMF, DCM, MeOH, and MTBE and allowed to dry. Cleavage cocktail mixture of TFA/TIPS/Phenol/H2O (90.0:2.5:5.0:2.5) was added to the resin and stirred for 3-4 h. The obtained reaction mass was filtered and the filtrate was evaporated and crude peptide was isolated using chilled MTBE. The obtained peptide was washed with MTBE and dried to obtain crude Semaglutide (129 g and product content 30.75 g). The crude peptide obtained was purified in a manner analogous to that as described in example-1 to get pure Semaglutide.
Example 4: Synthesis of Semaglutide using two fragment condensation strategy using 2-CTC resin
CTC resin (100 g, 0.50 mmol/g substitution) was washed with DCM in a reaction flask, solvent was removed using vacuum and allowed to swell in DCM. Fmoc-Gly-OH (2.0 eq.), DIPEA (3.0 eq.) were dissolved in DMF/ MDC was added to the resin and stirred for 2-3 h. The resin was washed with DMF. Substitution obtained after first amino acid loading was 0.40 mmol/g. Further, capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, de-protection was performed using 5 % piperidine in DMF with 0.5 % boric acid mixture for 10 min and reaction completion was monitored using Kaiser test. The obtained was washed twice with DMF containing 0.5 % formic acid followed by fresh DMF and washings were monitored by Chloranil test.
The rest of the amino acids and fragments were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent and additive were dissolved in DMF and the reaction mixture was added to the resin and stirred for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. Resin was washed thrice with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Gly, Ala, Val, Ser and Leu amino acids were coupled using DIC/OxymaPure
ii) Trp, Asp, Arg, Gln, Asn, Phe, Ile, Gly, Tyr, Thr, Lys, Fmoc- AEEA-OH, Fmoc-Glu-OtBu, C18-diacid(OtBu) were coupled using HBTU/OxymaPure/DIPEA
iii) Fragment Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH and Boc-His(Trt)-OH were coupled using HATU/Oxyma- B/ DIPEA
iv) Dde was removed using NH2-NH2/ DMF
All Fmoc deprotection reactions were performed by using 5- 20 % piperidine in DMF with 0.5 % boric acid. Solvents were removed using vacuum.
As per the sequence, coupling and deprotection reactions were performed to obtain
Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys((2xAEEA-?Glu-C18-diacid(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC Resin.
The obtained resin was washed with DMF, DCM, MeOH, and MTBE and allowed to dry. Cleavage cocktail mixture of TFA/ TIPS/ Phenol/ H2O (85.0: 5.0:5.0: 5.0) was added to the resin and stirred for 3-4 h. The obtained reaction mass was filtered and the filtrate was evaporated and crude peptide was isolated using chilled MTBE. The obtained peptide was washed with MTBE and dried to obtain crude Semaglutide (135 g and product content 31.5 g). The crude peptide obtained was purified in a manner analogous to that as described in example-1 to get pure Semaglutide.
Example 5: Synthesis of Semaglutide using sequential strategy by 2- CTC resin
CTC resin (100 g, 0.50 mmol/g substitution) was washed with DCM in a reaction flask, solvent was removed using vacuum and allowed to swell in DCM. Fmoc-Gly-OH (2.0 eq.), DIPEA (3.0 eq.) were dissolved in DMF/ DCM, and added to the resin and stirred for 2-3 h. The resin was washed with DMF. Substitution obtained after first amino acid loading was 0.40 mmol/g. Further, capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, de-protection was performed using 5 % piperidine in DMF with 0.5 % boric acid mixture for 10 min and reaction completion was monitored using Kaiser test. The obtained resin was washed twice with DMF containing 0.5 % formic acid followed by fresh DMF and washings were monitored by Chloranil test.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent and additive were dissolved in DMF and the reaction mixture was added to the resin and stirred for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. Resin was washed thrice with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Gly, Ala, Val, Ser and Leu amino acids were coupled using DIC/OxymaPure
ii) Trp, Asp, Arg, Gln, Asn, Phe, Ile, Gly, Tyr, Thr, Lys, Fmoc- AEEA-OH, Fmoc-Glu-OtBu, C18-diacid(OtBu) were coupled using HBTU/ OxymaPure/ DIPEA
iii) His and Aib were coupled using HATU/Oxyma-B/ DIPEA
iv) Dde was removed using NH2-NH2/DMF
All Fmoc de-protection reactions were performed by using 5- 20 % piperidine in DMF with 0.5 % boric acid. Solvents were removed using vacuum.
As per the sequence, sequential coupling and de-protection reaction performed to obtain Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)Asp(OtBu) -Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys ((2xAEEA-?Glu-C18-diacid(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-CTC Resin.
The obtained resin was washed with DMF, DCM, MeOH, and MTBE and allowed to dry. Cleavage cocktail mixture of TFA/ TIPS/ Phenol/ H2O (85.0:5.0:5.0:5.0) was added to the resin and stirred for 3-4 h. The obtained reaction mass was filtered and the filtrate was evaporated and crude peptide was isolated using chilled MTBE. The obtained peptide was washed with MTBE and dried to obtain crude Semaglutide (115 g and product content 29.75 g). The crude peptide obtained was purified in a manner analogous to that as described in example-1 to get pure Semaglutide.
Example 6: Synthesis of Fmoc-Aib-Glu(OtBu)-Gly-Thr(tBu)-OH
Fmoc-Thr(tBu)-OH was loaded to 2-CTC resin (100 g, substitution of 1.0 mmol/g) in presence of DIPEA/ DCM.
The rest of the amino acids were coupled sequentially as per the sequence using following coupling conditions:
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, and additive were dissolved in DMF and the reaction mixture was added to the resin and stirred for 1.5 – 2.0 h.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Gly was coupled using DIPC/ OxymaPure/ DMF
ii) Glu and Aib were coupled using HBTU/ OxymaPure/ DIPEA
iii) All Fmoc-deprotection were performed by using 10 % piperidine in DMF
The obtained peptide was cleaved using mixture of 1 % TFA/ DCM and precipitated with MTBE to get crude protected peptide (80.3g) and crystallized using EtOAc/ ACN/ n-hexane (72.59 g).
Example 7: Synthesis of C18-diacid(OtBu)-?Glu(OtBu)-2xAEEA-OH
Fmoc-AEEA-OH was loaded to 2-CTC resin (100 g) to achieve substitution of 1.0 mmol/g in DIPEA/DCM.
The rest of the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
Fmoc-AA-OH, coupling reagent, and additive were dissolved in DMF and the reaction mixture was added to the resin and kept for stirring for 1.5 – 2.0 h.
i) AEEA, ?Glu(OtBu) and C18-diacid(OtBu) were coupled using HBTU/ Oxyma B/ DIPEA
ii) Fmoc-deprotection were performed by using 10 % piperidine in DMF
The obtained peptide was cleaved using mixture of 1% TFA/ DCM and precipitated with MTBE to get crude peptide (110.5 g) and crystallized using EtOAc/ ACN/ n-hexane (85.92g).
Example 8: Synthesis of Semaglutide using sequential strategy
Wang resin (100 g, 0.50 mmol/g substitution) was washed with DCM in a reaction flask, solvent was removed using vacuum and allowed to swell in DCM. Fmoc-Gly-OH (1.0-3.0 eq.), DIC (2.0-6.0 eq.) and DMAP (0.02-0.10 eq.) were dissolved in DMF/ DCM, was added to the resin and kept for stirring for 2-4 h. The resin was washed with DMF. Substitution obtained after first amino acid loading was 0.30 to 0.50 mmol/g. further, capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After 1st amino acid attachment, de-protection was performed using 5-20 % piperidine in DMF stir for 10 min and reaction completion was monitored using Kaiser test. After the reaction complies, resin was washed thrice 0.1 M HOBt.H2O in DMF followed by thrice fresh DMF and washings were monitored by Chloronil test.
The rest of the amino acids were coupled as per the sequence using following coupling conditions:
Fmoc-AA-OH/Boc-AA-OH, coupling reagent and additive were dissolved in DMF and the reaction mixture was added to the resin and kept for stirring for 1.5 – 3.0 h at 20 to 40 °C temperature. Coupling was monitored using Kaiser test. Resin was washed thrice with DMF.
Fmoc-AA-OH/Boc-AA-OH, coupling reagent, additive details given below:
? Ala, Val, Ser and Leu , Trp, Asp, Arg, Gln, Asn, Phe, Ile, Gly, Tyr, Thr, Lys, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, C18-diacid(OtBu)-OH are coupled using HBTU/HOBt.H2O/Oxymapure/DIPEA
? Gly, Aib is coupled using HBTU/HATU/DIPEA at 35 °C
? His is coupled using HATU/ Oxymapure-B /DIPEA at 25 °C
? Mtt removal using HFIP/TFE/TES/MDC at 27 °C
All Fmoc de-protection reactions were performed by using 5- 20 % piperidine in DMF at 18 to 25 °C temperature. Solvents were removed using vacuum.
As per the sequence, sequential coupling and de-protection reaction performed to achieve Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp (OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys ((2xAEEA-?Glu-C18-diacid(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg (Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.
Resin was washed with DMF followed by DCM, MeOH, and MTBE and allowed to dry. Cleavage cocktail mixture of TFA/TIPS/Phenol/H2O (80:5:10:5) was added to the resin and kept for stirring for 3-4 h. Filtered the mass and filtrate was evaporated and chilled MTBE was used to isolate the crude peptide. MTBE washes were repeated and crude peptide was dried to obtain crude Semaglutide (120 to 130 g and product content 30-40 g.)
Example 9: Purification of Crude Semaglutide
The crude peptide was purified by RP-HPLC as follows. In the first cycle, 10.0 g of crude Semaglutide was weighed and dissolved in 2.0 L of 0.3% liq.NH3 solution in water.
Purification cycle-1 (RP-1)
The crude Semaglutide was purified by RP-HPLC system with 100 X 300 mm reverse phase C8 DAC column using given purification methods and detection wavelength of 220 nm. Wash the DAC (stationary phase C8-10 µ-100 Å) with 90:10 (acetonitrile: water containing 0.1% TFA) for two column volumes followed by equilibrating with 100 % mobile phase A [80% of 0.1 % aqueous TFA and 20% (v/v) buffer B] for two column volumes. Filtered the above crude solution and injected into the column.
Buffer A: 80% of 0.1 % aqueous TFA and 20% (v/v) buffer B
Buffer B: Mixture of methanol, acetonitrile, water and isopropyl alcohol in ratio of 65:20:10:5, respectively
Run the gradient method containing mobile phase B from 30 % to 100 % over a period 130 min. In which, product elutes at 90 % to 100 % near about 110 – 120 min. Above 90 % pure fractions pooled for RP-2 (second cycle). The column is regenerated by given wash with 90:10 [acetonitrile: water containing 0.1% TFA] for two column volumes and re equilibrated with 100% mobile phase A [80% of 0.1 % aqueous TFA and 20% (v/v) buffer B] for two column volumes prior to next injection sequence. Followed the above procedure until the completion of the crude. Fractions having purity more than 50.0 % pooled and reinjected to obtain above 90 % pure fractions by following the same procedure for elution mentioned in the above. Pooled all main fractions having purity more than 90 % and subjected (injected) to stationary phase C8-10 µ-100 Å for second cycle (purification-2).
Purification cycle-2 (RP-2, Salt conversion)
Buffer A: 30% (v/v) buffer B and 70% sodium phosphate buffer (pH: 7.5 ± 0.2)
(Mixture of Na2HPO4 and NaH2PO4)
Buffer B: Mixture of methanol, acetonitrile, water and isopropyl alcohol in ratio of 65:20:10:5, respectively
Run the gradient method containing mobile phase B from 20% to 100 % over a period 140 min. In which, product elutes at 25 % to 95 % near about 90 – 100 min. Above 99 % pure fractions pooled. The column is regenerated by given wash with 90:10 (acetonitrile: water containing70% sodium phosphate buffer) for two column volumes and re-equilibrated with 100% mobile phase A [30% (v/v) buffer B and 70% sodium phosphate buffer (pH: 7.5 ± 0.2)] for two column volumes prior to next injection sequence. Fractions having purity more than 60.0 % was pooled and reinjected to obtain above 99.0 % pure fractions by following the same procedure for elution mentioned in the above. All pooled passing fractions again loaded on to pre-equilibrated column.
The target fraction was collected and concentrated and lyophilized. Purified Semaglutide was obtained with HPLC purity of >99.0%, yield: 10.0%.
Example 10: Purification of Crude Semaglutide
The crude peptide was purified by RP-HPLC as follows. In the first cycle, 10.0 g of crude Semaglutide was weighed and dissolved in 2.0 L of 0.3% liq.NH3 solution in water. The crude Semaglutide was purified by RP-HPLC system with 50 X 250 mm reverse phase C8 DAC column using given purification methods and detection wavelength of 220 nm.
Purification cycle-1 (RP-1)
Mobile phase description:
Buffer A: TEAP buffer (pH: 7.5 ± 0.2) 50 mL of triethylamine and 14 mL of orthophosphoric acid in 20 L of water.
Buffer B: Mixture of methanol, acetonitrile, water and isopropyl alcohol in ratio of 65:20:10:5, respectively.
Run the gradient method containing mobile phase B from 50 % to 100 % over a period 130 min. In which, product elutes at 50 % to 80 % near about 50 – 70 min. Above 90 % pure fractions pooled for RP-2 (second cycle). The column is regenerated by given wash with 90:10 [acetonitrile: buffer A] for two column volumes and re equilibrated with 100 % mobile phase A for two column volumes prior to next injection sequence. Followed the above procedure until the completion of the crude. Fractions having purity more than 50.0 % pooled and reinjected to obtain above 90 % pure fractions by following the same procedure for elution mentioned in the above. Pooled all main fractions having purity more than 90 % and subjected (injected) to stationary phase C8-10 µ-100 Å for second cycle (purification-2). Combined 3 run main fractions were collected from RP-1 and dilute with water and carried forward to purification cycle-2.
Purification cycle-2 (RP-2)
The target fractions were collected and reload in RP-HPLC system equipped with 50 X 250 mm reverse phase C8 DAC column using given purification stations and detection wavelength of 220 nm.
Buffer A: 80 % of 0.1 % aqueous TFA and 20 % (v/v) buffer B
Buffer B: Mixture of methanol, acetonitrile, water and isopropyl alcohol in ratio of 65:20:10:5, respectively.
Run the gradient method containing mobile phase B from 30 % to 100 % over a period 150 min. In which, product elutes at 55 % to 65 % near about 80 – 95 min. Above 98 % pure fractions pooled. The column is regenerated by given wash with 90:10 (acetonitrile: buffer A) for two column volumes and re-equilibrated with 100% mobile phase A [80 % of 0.1 % aqueous TFA and 20 % (v/v) buffer B] for two column volumes prior to next injection sequence. Fractions having purity more than 60.0 % was pooled and reinjected to obtain above 98.0 % pure fractions by following the same procedure for elution mentioned in the above.
All pooled passing fractions again loaded on to pre-equilibrated column.
Purification cycle-3 (RP-3, Salt conversion)
The target fraction was collected from RP-2 and dilute with water, reload in RP-HPLC system equipped with 50 X 250 mm reverse phase C8 DAC column using given purification stations and detection wavelength of 220 nm.
Buffer A: 30 % (v/v) buffer B and 70 % sodium phosphate buffer (pH: 7.5 ± 0.2), (Mixture of Na2HPO4 and NaH2PO4)
Buffer B: Mixture of methanol, acetonitrile, water and isopropyl alcohol in ratio of 65:20:10:5, respectively
Run the gradient method containing mobile phase B from 24 % to 100 % over a period 100 min. In which, product elutes at 50 % to 60 % near about 70– 80 min. Above 99 % pure fractions pooled. The column is regenerated by given wash with 90:10 (acetonitrile: buffer A) for two column volumes and re-equilibrated with 100% mobile phase A for two column volumes prior to next injection sequence. Fractions having purity more than 60.0 % was pooled and reinjected to obtain above 99.0 % pure fractions by following the same procedure for elution mentioned in the above.
The targeted fraction was collected to give the purified peptide with a purity greater than 99.0 %. The fractions were collected and concentrated and lyophilized. Purified Semaglutide was obtained with HPLC purity of >99.0 %, yield: 10.0 %.
Example 11: Purification of Crude Semaglutide
The crude peptide was purified by RP-HPLC as follows. In the first cycle, 10.0 g of crude Semaglutide was weighed and dissolved in 2.0 L of 0.3% liq.NH3 solution in water.
Purification cycle-1 (RP-1)
The crude Semaglutide was purified by RP-HPLC system with 100 X 300 mm reverse phase C8 DAC column using given purification methods and detection wavelength of 220 nm. Wash the DAC (stationary phase C8-10 µ-100 Å) with 90:10 (acetonitrile: water containing 0.1% TFA) for two column volumes followed by equilibrating with 100 % mobile phase A [ 0.1 % aqueous TFA & 25% (v/v) buffer B] for two column volumes. Filtered the above crude solution and injected into the column.
Buffer A: 0.1 % aqueous TFA
Buffer B: Acetonitrile
Run the gradient method containing mobile phase B from 30 % to 50 % over a period 120 min. In which, product elutes at 40 to 45 % near about 80 – 100 min. Above 90 % pure fractions pooled for RP-2 (second cycle). The column is regenerated by given wash with 90:10 [acetonitrile: Buffer A] for two column volumes and re equilibrated with mobile phase A [75% of 0.1 % aqueous TFA] and buffer B [25% (v/v)] for two column volumes prior to next injection sequence. Followed the above procedure until the completion of the crude. Fractions having purity more than 50.0 % pooled and reinjected to obtain above 90 % pure fractions by following the same procedure for elution mentioned in the above. Pooled all main fractions having purity more than 90 % and subjected (injected) to stationary phase C8-10 µ-100 Å for second cycle (purification-2).
Purification cycle-2 (RP-2)
Buffer A: 20mm Ammonium bicarbonate (pH: 7.5 ± 8.0)
Buffer B: Acetonitrile,
Run the gradient method containing mobile phase B from 30% to 40 % over a period 90 min. In which, product elutes at 35 % to 40 % near about 45 – 75 min. Above 99 % pure fractions pooled. The column is regenerated by given wash with 90:10 (acetonitrile: buffer A) for two column volumes and re equilibrated with mobile phase A [90% of 20mm Ammonium bicarbonate] and buffer B[ 10% (v/v)] for two column volumes prior to next injection sequence. Fractions having purity more than 80.0 % was pooled and reinjected to obtain above 99.0 % pure fractions by following the same procedure for elution mentioned in the above. All pooled passing fractions again loaded on to pre-equilibrated column.
Na-Salt conversion: The target fraction was collected and concentrated, add 0.04 M NaOH solution to the product solution obtained after distillation under continuous agitation over a period of 15 to 30 min under controlled temperature and lyophilized. Purified Semaglutide was obtained with HPLC purity of >99.0%, yield: 10.0%.
Example 12: Purification of Crude Semaglutide
The crude peptide was purified by RP-HPLC as follows. In the first cycle, 10.0 g of crude Semaglutide was weighed and dissolved in 2.0 L of 0.3% liq.NH3 solution in water.
Purification cycle-1 (RP-1)
The crude Semaglutide was purified by RP-HPLC system with 100 X 300 mm reverse phase C8 DAC column using given purification methods and detection wavelength of 220 nm. Wash the DAC (stationary phase C8-10 µ-100 Å) with 90:10 (acetonitrile: water containing 0.1% TFA) for two column volumes followed by equilibrating with 100 % mobile phase A [0.1 % aqueous TFA & 25% (v/v) buffer B] for two column volumes. Filtered the above crude solution and injected into the column.
Buffer A: 0.1 % aqueous TFA
Buffer B: 0.1 % TFA in Acetonitrile
Run the gradient method containing mobile phase B from 40 % to 50 % over a period 120 min. In which, product elutes at 45 to 50 % near about 60 – 90 min. Above 90 % pure fractions pooled for RP-2 (second cycle). The column is regenerated by given wash with 90:10 [acetonitrile: Buffer A] for two column volumes and re equilibrated with mobile phase A [75% of 0.1 % aqueous TFA] and buffer B [25% (v/v)] for two column volumes prior to next injection sequence. Followed the above procedure until the completion of the crude. Fractions having purity more than 50.0 % pooled and reinjected to obtain above 90 % pure fractions by following the same procedure for elution mentioned in the above. Pooled all main fractions having purity more than 90 % and subjected (injected) to stationary phase C8-10 µ-100 Å for second cycle (purification-2).
Purification cycle-2 (RP-2, Salt conversion)
Buffer A: Mixture of Na2HPO4 and NaH2PO4 (pH: 8.0 ± 0.5)
Buffer B: Acetonitrile,
Run the gradient method containing mobile phase B from 30% to 40 % over a period 120 min. In which, product elutes at 35 % to 40 % near about 65 – 95 min. Above 99 % pure fractions pooled. The column is regenerated by given wash with 90:10 (acetonitrile: buffer A) for two column volumes and re-equilibrated with mobile phase A [90% (v/v) ] and buffer B [10% (v/v) ] for two column volumes prior to next injection sequence. Fractions having purity more than 80.0 % was pooled and re-injected to obtain above 99.0 % pure fractions by following the same procedure for elution mentioned in the above. All pooled passing fractions again loaded on to pre-equilibrated column.
The targeted fraction was collected to give the purified peptide with a purity greater than 99.0 %. The fractions were collected and concentrated and lyophilized. Purified Semaglutide was obtained with HPLC purity of >99.0 %, yield: 11.0 %.
,CLAIMS:We claim:
1. A peptide fragment having amino acids sequence SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof.
2. A process for the preparation of Semaglutide or a pharmaceutically acceptable salt thereof, comprising use of SEQ ID: 1 or a pharmaceutically acceptable salt thereof.
3. A process for the preparation of Semaglutide or a pharmaceutically acceptable salt thereof, comprising;
a) loading of Fmoc protected glycine to a resin solid-phase support in the presence of coupling agent and a catalyst;
b) de-protection of Fmoc group using de-protecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Semaglutide till Phe12 in the presence of coupling agent to obtain Fmoc-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin, wherein Xn is selected from the group consisting of Alloc, Mtt, Dde, Mmt and ivDde;
c) de-protection of Fmoc protecting group of fragment obtained in step (b) using de-protecting agent followed by coupling of tetra-peptide fragment Aib8-Glu9-Gly10-Thr11-OH (SEQ ID-1) wherein, the side chain of amino acids protected with a suitable protecting group; in the presence of coupling agent, to obtain Fmoc-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin;
d) de-protection of Fmoc protecting group using de-protecting agent and coupling of N-terminal and side chain protected histidine, in the presence of coupling agent, to obtain His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26[Xn]-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin; and
e) de-protection of protecting group (Xn) from Lys26, to obtain His7-Aib8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Arg34-Gly35-Arg36-Gly37-Resin and converting the resulting peptide to Semaglutide.
4. A process for the preparation of preparation of peptide fragment having amino acids sequence SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, comprising;
a) Loading of Fmoc protected threonine to resin solid-phase support, wherein side chain may be protected with suitable protecting group, in the presence of coupling agent;
b) de-protection of Fmoc protecting group using de-protecting agent and coupling of Fmoc protected glycine in the presence of coupling agent;
c) de-protection of Fmoc protecting group using de-protecting agent and coupling of Fmoc protected glutamic acid and 2-aminoisobutyric acid, wherein side chain may be protected to obtain backbone of SEQ ID-1; and
d) cleavage of SEQ ID-1 obtained in step (d) from resin using cleaving agent to obtain SEQ ID-1 or a pharmaceutically acceptable salt thereof.
5. The process as claimed in claims 3-4, wherein resin solid-phase support is selected from Wang resin and 2-Chlorotrityl resin (CTC).
6. The process as claimed in claims 3-4, wherein coupling agent is selected from group comprising of DIC/OxymaPure, HBTU/OxymaPure/DIPEA, HATU/DIPEA, HATU/Oxyma-B/DIPEA, HBTU/HOBt.H2O/Oxymapure/DIPEA, HBTU/HATU/ DIPEA and HATU/ Oxymapure /DIPEA.
7. The process as claimed in claims 3-4, wherein Fmoc de-protection is carried out using base selected from piperidine, pyrrolidine, piperazine, tert-butylamine, DBU and diethylamine optionally in presence of additive selected from ascorbic acid, formic acid, boric acid and citric acid.
8. The process as claimed in claim 3, wherein de-protection of protecting group (Xn) from Lys26 comprises using hydrazine hydrate when Xn group is Dde, using HFIP, TFE, TES when Xn group is Mtt, using Pd(PPh3)4 when Xn group is Alloc.
9. The process as claimed in claim 3, wherein conversion to Semaglutide comprises concomitant cleavage from the resin and side chain protecting groups removal using mixture of TFA, TIPS, Phenol, H2O.
10. The process as claimed in claim 4, wherein cleaving agent comprises TFA and DCM.