DESC:FIELD OF INVENTION
The present invention relates to an efficient process of Glucagon or a pharmaceutically acceptable salt thereof. The present invention also relates to a novel fragment as intermediate and use thereof in the preparation of Glucagon.
BACKGROUND OF THE INVENTION
Glucagon is a polypeptide hormone, secreted by the a-cells of the pancreatic islets of Langerhans. Glucagon is a single chain peptide consisting of 29 natural amino acids and is represented by the chemical formula shown below:

His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29

In the synthesis of large peptide molecules, such as Glucagon, the conformation of the growing peptide chain and its physico-chemical properties are of considerable importance. The formation of secondary structures often leads to problems of aggregation causing incomplete coupling reactions, resulting in a decrease in the synthetic yield and purity of the final compound.
Therefore, there remains a continuing need for preparation of Glucagon with better yields, more purity and low impurities; and which is commercially feasible as well.

The present invention describes a process for the preparation of Glucagon or a pharmaceutically acceptable salt thereof involving use of new fragment as an intermediate.

SUMMARY OF THE INVENTION
First aspect of present invention relates to an improved fragment based process of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising;
(a) loading of Fmoc protected Threonine with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Tyr10 in the presence of coupling agent to obtain Fmoc-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling of fragment Fmoc-Phe6-Thr7-Ser8-Asp9-OH (SEQ ID: 1) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Fmoc-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(d) deprotection of Fmoc protecting group of fragment obtained in step (c) using deprotecting agent followed by coupling of fragment Boc-His1-Ser2-Gln3-Gly4-Thr5-OH (SEQ ID: 2) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin; and
(e) deprotection and cleavage of fragment obtained in step (d) using cleaving agent to obtain Glucagon.

Second aspect of present invention relates to an improved fragment based process of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising;
(a) loading of Fmoc protected Threonine with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Asp9 in the presence of coupling agent to obtain Fmoc- Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling of fragment Fmoc-Gly4-Thr5-Phe6-Thr7-Ser8-OH (SEQ ID: 4) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Fmoc-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(d) deprotection of Fmoc protecting group of fragment obtained in step (c) using deprotecting agent followed by coupling of fragment Boc-His1-Ser2-Gln3-OH (SEQ ID: 3) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin; and
(e) deprotection and cleavage of fragment obtained in step (d) using cleaving agent to obtain Glucagon.

Third aspect of present invention relates to an improved fragment based process of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising;
(a) loading of Fmoc protected Threonine with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Asp9 in the presence of coupling agent to obtain Fmoc- Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling of fragment Fmoc-Phe6-Thr7-Ser8-OH (SEQ ID: 5) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Fmoc-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(d) deprotection of Fmoc protecting group of fragment obtained in step (c) using deprotecting agent followed by coupling of fragment Boc-His1-Ser2-Gln3-Gly4-Thr5-OH (SEQ ID: 2) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin; and
(e) deprotection and cleavage of fragment obtained in step (d) using cleaving agent to obtain Glucagon.

Fourth aspect of present invention relates to fragment compounds of SEQ ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4 and SEQ ID: 5 (as tabulated below) and process for preparing same and its use in the preparation of Glucagon or a pharmaceutically acceptable salt thereof.

Phe6-Thr7-Ser8-Asp9 SEQ ID: 1
His1-Ser2-Gln3-Gly4-Thr5 SEQ ID: 2
His1-Ser2-Gln3 SEQ ID: 3
Gly4-Thr5- Phe6-Thr7-Ser8 SEQ ID: 4
Phe6-Thr7-Ser8 SEQ ID: 5
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: 2 and its process.
In further aspect, the present invention compound of SEQ ID: 2 is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH.

DETAILED DESCRIPTION OF THE 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. SPPS involves coupling an activated amino acid to a solid support. The term “solid phase synthesis” (SPPS) can also be defined as a process in which a peptide anchored by its C-terminal amino acid to a resin is assembled by the sequential addition of the optionally protected amino acids constituting its sequence.
This solid support is usually a polymeric resin bead that is functionalized (such as with an NH2 group). 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.
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.
The resin is activated by the removal of a protecting group. Resin such as Wang resin, Rink amide resin, Seiber amide 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 activated resin is then coupled with the amino acid, wherein the amino acid is protected by a terminal protecting group and optionally a side-chain protecting group. The terminal protecting group is cleaved in suitable conditions depending on its type.
The amino acids employed in the process of the present invention have the natural L- configuration; in general, such amino acids (preferably bearing a terminal protecting group) employed in the process of the present invention are commercially available. Amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code.
The term “polypeptide” and “peptide” as used herein means a compound composed of at least two constituent amino acids connected by peptide bonds (amide bond) formed by removal of water from the amino acids from which they formally are build .
The term “fragment” as used herein, refers to a portion of an amino acid sequence wherein said portion is smaller than the entire amino acid sequence of Glucagon. The fragment can be protected with N-terminal protection and side chain protection as provided herein. Fragment also refers to peptides except Glucagon as provided herein.
The term "Fmoc protected amino acid" as used herein refers to amino acids with Fmoc protection on a-amino group of the amino acids. For example “Fmoc protected Threonine” refers to Threonine with Fmoc protection on a-amino group.
The term "Boc protected amino acid" as used herein refers to amino acid with Boc protection on a-amino group of the amino acids. For example “Boc protected Histidine” refers to Histidine with Boc 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 is 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 Glucagon, or of the complete Glucagon 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). Deprotecting 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. Deprotecting 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 under basic conditions. 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. More preferably, Fmoc deprotection is carried out by using a 20% solution of piperidine in DMF. An additive such as ascorbic acid, formic acid, boric acid, citric acid can be optionally added during Fmoc deprotection to facilitate the reaction.
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.
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)tripyrrolidinophosphoniumhexafluorophosphate (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.
In a preferred aspect of present invention, the coupling steps may also be performed in the presence of an additive. The presence of an 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 (HOBt), 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 (Oxyma 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 preferred coupling agent, coupling additive and base used in present invention in any of the above process is DIC/HOBt.H2O; DIC/OxymaPure; HBTU/HOBt.H2O/DIPEA, HBTU/OxymaPure/DIPEA; HBTU/Oxy-B/DIPEA; HATU/HOAt/DIPEA or PyBOP/ HOBt.H2O/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.
Additional coupling agents may be used for recoupling of amino acids whenever Kaiser test shows little blue colour during coupling stage.
The solvent used in said coupling reaction is selected from DMF, DCM, NMP or DMSO in combination between them; preferably DMF. The coupling temperature is usually in the range of from 10 to 40 °C; preferably 25 to 30 °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 or methanol 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.
Deprotection 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, anisole, thioanisole, 1,2-ethanedithiol (EDT), phenol, water, triethylsilane (TES), DTT, triisopropylsilane (TIS/TIPS), 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS and cresol or mixture thereof and water; preferably TFA: TIS: phenol: DMS: 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.
It may be recommendable to add one or more agents selected from the group consisting of iodide salts (e.g. ammonium iodide), dimethyl sulfide, 1,4-dithiothreitol (DTT), trimethylsilylbromide, and ascorbic acid to the suspension comprising the resin. This may be done, e.g., at the onset of the incubation of peptide resin and cleavage composition, or after incubating the peptide resin with the cleavage composition for a certain time. Without wishing to be bound by any theory, it is believed that this avoids and/or reverts oxidation of the S-methyl thioether group of the methionine side chain.
In a preferred embodiment, when SPPS is used, the protected Glucagon sequence is finally deprotected and cleaved from the resin, either simultaneously or in two steps, providing crude Glucagon, which may further be purified.
The crude Glucagon 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 Glucagon or its pharmaceutically acceptable salt, provides Glucagon in better yield and high purity, which makes it suitable for large scale industrial production.
The terms "about" as used herein refer to as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value. The term "about" when used in the present application preceding a number and referring to it, is meant to designate any value which lies within the range of ±10 %, preferably within a range of ±5 %.
The terms "comprising" and "comprises" as used herein are to be construed as open ended and mean the elements recited, or their equivalents in structure or function, plus any other element or elements which are not recited.
As used herein, when an amino acid abbreviation appears with a number above the amino acid, the number refers to the corresponding amino acid position in the final Glucagon product. The numbers are provided for convenience and the appearance or absence of such numbers in a sequence does not influence the amino acid sequence or the peptide indicated in such sequence.

First aspect of the present invention relates to fragment based process of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising SEQ ID: 1 and SEQ ID: 2.
In an embodiment of first aspect, Fmoc protected Threonine as used in step (a) is Fmoc-Thr(tBu)29-OH.
In an embodiment of first aspect, Fmoc-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin in step (b) preferably is Fmoc-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.

In an embodiment of first aspect, SEQ ID: 1 (Fmoc-Phe6-Thr7-Ser8-Asp9-OH) as used in step (c) preferably is Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-OH.

In an embodiment of first aspect, (Fmoc-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin) in step (c) preferably is Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.

In an embodiment of first aspect, SEQ ID: 2 (Boc-His1-Ser2-Gln3-Gly4-Thr5-OH) as used in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH.
In an embodiment of first aspect, (Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin) in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.

Scheme 1: (Preparation of Glucagon HCl according to first aspect)

Second aspect of the present invention relates to fragment based process of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising SEQ ID: 3 and SEQ ID: 4.

In an embodiment of second aspect, Fmoc protected Threonine as used in step (a) is Fmoc-Thr(tBu)29-OH.

In an embodiment of second aspect, Fmoc-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin in step (b) preferably is Fmoc-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.

In an embodiment of second aspect, SEQ ID: 4 (Fmoc-Gly4-Thr5-Phe6-Thr7-Ser8-OH) as used in step (c) preferably is Fmoc- Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-OH.

In an embodiment of second aspect, (Fmoc-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin) in step (c) preferably is Fmoc-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.

In an embodiment of second aspect, SEQ ID: 3 (Boc-His1-Ser2-Gln3-OH) as used in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-OH.
In an embodiment of second aspect, (Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin) in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.

Scheme 2: (Preparation of Glucagon HCl according to second aspect)

Third aspect of the present invention relates to fragment based process of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising SEQ ID: 2 and SEQ ID: 5.

In an embodiment of third aspect, Fmoc protected Threonine as used in step (a) is Fmoc-Thr(tBu)29-OH.

In an embodiment of third aspect, Fmoc-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin in step (b) preferably is Fmoc-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.
In an embodiment of third aspect, SEQ ID: 5 (Fmoc-Phe6-Thr7-Ser8-OH) as used in step (c) preferably is Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-OH.
In an embodiment of third aspect, (Fmoc-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin) in step (c) preferably is Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.
In an embodiment of third aspect, SEQ ID: 2 (Boc-His1-Ser2-Gln3-Gly4-Thr5-OH) as used in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH.
In an embodiment of third aspect, t (Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin) in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(tBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-resin.

Scheme 3: (Preparation of Glucagon HCl according to third aspect)

In one embodiment, deprotection as well as cleavage of peptide from resin is preferably carried out using TFA: TIPS: phenol: water: DMS: NH4I which is having ratio of about 85 %: 5 %: 5 %: 5 %: 0.5%: 0.5% (of resin). The precipitation of crude product is preferably carried out using methyl tert-butyl ether (MTBE) followed by washing with MTBE.
In one embodiment, the resin solid-phase support as used herein any of the process can be selected from but not limited to Wang resin or 2-CTC resin.
In one embodiment, the crude Glucagon obtained is further subjected to chromatographic purification method followed by lyophilisation to obtain pure Glucagon hydrochloride salt.

Fourth aspect of present invention relates to a process of preparation of SEQ ID: 1 comprising;
(a) loading of Fmoc protected Asp9 with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc protecting group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Phe6 in the presence of coupling agent to obtain Fmoc-Phe6-Thr7-Ser8-Asp9-resin; and
(c) cleavage of fragment obtained in step (b) from the solid support using cleaving agent to obtain Fmoc-Phe6-Thr7-Ser8-Asp9-OH (SEQ ID: 1).

In an embodiment of fourth aspect, SEQ ID: 1 (Fmoc-Phe6-Thr7-Ser8-Asp9-OH) in step (c) preferably is Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-OH.
In another embodiment, the present invention relates to SEQ ID: 2 and process for preparing same and its use in the preparation of Glucagon or a pharmaceutically acceptable salt thereof.
His1-Ser2-Gln3-Gly4-Thr5 SEQ. ID: 2
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 an embodiment, the present invention SEQ ID: 2 preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH.
In an embodiment, the process of preparation of SEQ ID: No. 2 comprising;
(a) loading of Fmoc protected Thr5 with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc protecting group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Ser2 in the presence of coupling agent to obtain Fmoc-Ser2-Gln3-Gly4-Thr5-OH -resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling with Boc protected His1 with side chain protection in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-resin; and
(d) cleavage of fragment obtained in step (c) from the solid support using cleaving agent to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-OH (SEQ ID: 2).
In an embodiment, SEQ ID: 2 (Boc-His1-Ser2-Gln3-Gly4-Thr5-OH) in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH.

In another embodiment, the present invention relates to a process of preparation of SEQ ID: 3 comprising;
(a) loading of Fmoc protected Gln3 with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc protecting group using deprotecting agent and coupling of Ser2 in the presence of coupling agent to obtain Fmoc-Ser2-Gln3-OH -resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling with Boc protected His1 with side chain protection in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-resin; and
(d) cleavage of fragment obtained in step (d) from the solid support using cleaving agent to obtain Boc-His1-Ser2-Gln3-OH (SEQ ID: 3).
In an embodiment, SEQ ID: 3 (Boc-His1-Ser2-Gln3-OH) in step (d) preferably is Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-OH.
In another embodiment, the present invention relates to a process of preparation of SEQ ID: 4 comprising;
(a) loading of Fmoc protected Ser8 with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc protecting group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Gly4 in the presence of coupling agent to obtain Fmoc- Gly4-Thr5- Phe6-Thr7-Ser8-resin; and
(c) cleavage of fragment obtained in step (b) from the solid support using cleaving agent to obtain Fmoc- Gly4-Thr5- Phe6-Thr7-Ser8-OH (SEQ ID: No.4).

In an embodiment, SEQ ID: 4 (Fmoc-Gly4-Thr5- Phe6-Thr7-Ser8-OH) in step (d) preferably is Fmoc- Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-OH.
In another embodiment, the present invention relates to a process of preparation of SEQ ID: 5 comprising;
(a) loading of Fmoc protected Ser8 with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc protecting group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Phe6 in the presence of coupling agent to obtain Fmoc- Phe6-Thr7-Ser8-resin; and
(c) cleavage of fragment obtained in step (b) from the solid support using cleaving agent to obtain Fmoc-Phe6-Thr7-Ser8-OH (SEQ ID: 5).

In an embodiment, SEQ ID: 5 (Fmoc-Phe6-Thr7-Ser8-OH) d in step (d) preferably is Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-OH.

Fifth aspect of present invention involves linear process of preparing Glucagon or its pharmaceutically acceptable salts comprising:
(a) loading of Fmoc protected Threonine with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Ser2 in the presence of coupling agent to obtain Fmoc-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling with Boc protected His1 with side chain protection in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(d) deprotection and cleavage of fragment obtained in step (c) using cleaving agent to obtain Glucagon; and
(e) purification of resulting peptide obtained in step (d) to obtain Glucagon or a pharmaceutically acceptable salt thereof.

The present invention provides essentially pure Glucagon or a pharmaceutically acceptable salt thereof containing not more than 0.5% of any individual impurity or peptidic impurity, preferably not more than 0.2% of any individual impurity or peptidic impurity.

Particularly preferred types of impurities are formed during synthesis and storage of glucagon and may exemplarily be selected from the group consisting of amino acids, peptides and derivatives thereof. In particular encompassed are impurities selected from the group consisting of amino acids, peptides, and derivatives thereof, which may result from processes such as premature chain termination during peptide synthesis, omission or unintended addition of at least one amino acid during peptide synthesis, incomplete removal of protecting groups, side reactions occurring during amino acid coupling or Fmoc deprotection steps, inter- or intramolecular condensation reactions, side reactions during peptide cleavage from a solid support, racemization, any other type of isomer formation, deamidation, (partial) hydrolysis, and aggregate formation. Peptidic contaminations resulting from such processes as outlined above are sometimes referred to as “related substances”.

In an aspect of present invention, the impurity or peptide impurity comprises one or more of the following compounds:
Impurity Sequence
Met(O27)- glucagon
L-histidyl-L-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-S-(O)-L-asparagyl-L-threonine
Met(O27)- glucagon
L-histidyl-L-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-R-(O)-L-asparagyl-L-threonine
(D-Ser8)-glucagon
L-histidyl-L-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-D-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
(Des-Gly4)- glucagon
L-histidyl-L-seryl-L-glutaminyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
(Des-Thr5)-glucagon
L-histidyl-L-seryl-L-glutaminyl-glycyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
Des-Ser2)-glucagon
L-histidyl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
(Glu3)-glucagon
L-histidyl-L-seryl-L-glutamyl -glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
(D-Ser2)-glucagon
L-histidyl-D-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
(Asp28)-glucagon
L-histidyl-L-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-alpha-aspartyl-L-threonine
(D-His1)-glucagon
D-histidyl-L-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
(Glu20)-glucagon
L-histidyl-L-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutamyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine
(Glu24) glucagon
L-histidyl-L-seryl-L-glutaminyl-glycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-alpha-aspartyl-L-tyrosyl-L-seryl-L-lysyl-L-tyrosyl-L-leucyl-L-alpha-aspartyl-L-seryl-L-arginyl-L-arginyl-L-alanyl-L-glutaminyl-L-alpha-aspartyl-L-phenylalanyl-L-valyl-L-glutamyl-L-tryptophyl-L-leucyl-L-methionyl-L-asparagyl-L-threonine

The relative content of said impurities in the solid-phase conjugated glucagon is preferably determined by measuring the relative peak area (peak area of the impurity divided by total peak area of all peaks observed) in analytical method of the corresponding crude peptide.

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.

EXAMPLES
Example 1: Synthesis of Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4–Thr(tBu)5-OH (SEQ ID: 2)
Fmoc-Thr(tBu)-OH was loaded to 2-CTC resin (100 g) to obtain substitution of 1.0 mmol/g in DIPEA/DCM. All the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
The rest of the amino acids were coupled as per the sequence using following coupling
conditions:
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) Gly, Gln and Ser were coupled using DIC/ OxymaPure
ii) His was coupled using HBTU/Oxyma B/DIPEA
iii) Fmoc-deprotection were performed by using 10 % piperidine in DMF with 0.5 % ascorbic acid

Peptide was cleaved using mixture of 1% TFA/DCM and precipitated with MTBE to get crude peptide and crystallized using EtOAc/ACN/n-hexane to obtained product.
Example 2: Synthesis of Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-OH (SEQ ID: 5)
Fmoc-Ser(tBu)-OH was loaded to 2-CTC resin (100 g) to obtain substitution of 1.0 mmol/g in DIPEA/DCM. All the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
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 – 2.0 h.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Thr and Phe were coupled using HBTU/ OxymaPure/ DIPEA
ii) All Fmoc-deprotection were performed by using 10 % piperidine in DMF with 0.5 % ascorbic acid
Peptide was cleaved using mixture of 1% TFA/DCM and precipitated with MTBE to get crude protected peptide and crystallized using EtOAc/ACN/n-hexane to obtained product.
Example 3: Synthesis of Glucagon HCl using SEQ ID: 2 and SEQ ID: 5 on Wang resin
Wang resin (100 g; 0.50 mmol/g) was washed with DCM/DMF in a reaction flask, solvent removed using vacuum and allowed to swell in DCM/DMF. Fmoc-Thr(tBu)-OH (1.5 eq.), DIC (2.0 eq.) and DMAP (0.05 eq.) dissolved in DMF were added to the resin and kept for stirring for 2-3 h. Coupling was monitored using Kaiser test. After reaction complies, resin was washed with DMF. Substitution obtained after first amino acid loading was 0.36 mmol/g. Capping was performed using acetic anhydride and DIPEA in DMF. Resin was washed with DMF.
After first amino acid attachment, Fmoc deprotection was performed using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid and reaction completion was monitored using Kaiser test. After reaction complies, resin was washed with DMF containing 0.5 % formic acid followed by DMF 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 kept for stirring for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. After reaction complies, resin was washed thrice with DMF to obtain Fmoc-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(OtBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-wang resin.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Met, Ala, Val, and Leu amino acids were coupled using DIC/ OxymaPure
ii) Trp, Asp, Arg, Gln, Asn, Phe, Tyr and Ser were coupled using HBTU/ Oxyma B/ DIPEA
iii) Lys, Asp, SEQ ID: 5 (Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-OH) and SEQ ID: 2 (Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH) were coupled using HATU/ Oxyma B/ DIPEA.
All Fmoc-deprotection steps were performed by using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid. Solvents were removed using vacuum.
As per the sequence all fragments were coupled to obtain Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(OtBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-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/ammonium iodide/DMS (84: 5: 5: 5: 0.5: 0.5 %) was added to the resin and kept for stirring for 3-4 h. After reaction complies, the reaction mixture was filtered and filtrate was evaporated and crude peptide was isolated using chilled MTBE. Crude peptide was washed with MTBE and dried to obtain H-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-OH.
Example 4: Synthesis of Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3- OH (SEQ ID: 3)
Fmoc-Gln(Trt)-OH was loaded to 2-CTC resin (100 g) to obtain substitution of 1.0 mmol/g in DIPEA/DCM. All the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
The rest of the amino acids were coupled as per the sequence using following coupling
conditions:
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) Ser was coupled using DIC/OxymaPure
ii) His was coupled using HBTU/DIPEA/OxymaPure
iii) Fmoc-deprotection were performed by using 5- 15 % piperidine in DMF with 0.5 % boric acid

Peptide was cleaved using mixture of 1% TFA/DCM and precipitated with MTBE to get crude peptide and crystallized using EtOAc/ACN/n-hexane to obtained product.

Example 5: Synthesis of Fmoc- Gly4–Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-OH (SEQ ID: 4)
Fmoc-Ser(tBu)-OH was loaded to 2-CTC resin (100 g) to obtain substitution of 1.0 mmol/g in DIPEA/DCM. All the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
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 – 2.0 h.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Gly, Thr and Phe were coupled using HBTU/ OxymaPure/ DIPEA
ii) All Fmoc-deprotection were performed by using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid

Peptide was cleaved using mixture of 1% TFA/DCM and precipitated with MTBE to get crude protected peptide and crystallized using EtOAc/ACN/n-hexane to obtained product.

Example 6: Synthesis of Glucagon HCl using SEQ ID: 3 and SEQ ID: 4 on Wang resin
Wang resin (100 g; 0.50 mmol/g) was washed with DCM/DMF in a reaction flask, solvent
removed using vacuum and allowed to swell in DCM/DMF. Fmoc-Thr(tBu)-OH (1.5 eq.), DIC (2.0 eq.) and DMAP (0.05 eq.) dissolved in DMF were added to the resin and kept for stirring for 2-3 h. Coupling was monitored using Kaiser test. After the reaction
complies, resin was washed with DMF. Substitution after first amino acid loading was 0.36 mmol/g. Capping was performed using acetic anhydride and DIPEA in DMF and resin was washed with DMF.
After first amino acid attachment, deprotection was performed using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid and reaction completion was monitored using Kaiser test. After the
reaction complies, resin was washed with DMF containing 0.5 % formic acid followed by DMF 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 kept for stirring for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. After the reaction complies, resin was washed thrice with DMF to obtain Fmoc-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(OtBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-wang resin.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Met, Ala, Val, and Leu amino acids were coupled using DIC/ OxymaPure
ii) Trp, Arg, Gln, Asn, Phe, Tyr and Ser were coupled using HBTU/ Oxyma B/ DIPEA
iii) Lys, Asp, SEQ ID: 4 (Fmoc- Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-OH) and SEQ ID: 3 (Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-OH) were coupled using HATU/ Oxyma B/ DIPEA.
All Fmoc-deprotection steps were performed by using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid. Solvents were removed using vacuum.
As per the sequence all fragments were coupled to obtain Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(OtBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-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/ammonium iodide/DMS (84: 5: 5: 5: 0.5: 0.5 %) was added to the resin and kept for stirring for 3-4 h. After reaction complies, the reaction mixture was filtered, filtrate was evaporated and crude peptide was isolated using chilled MTBE. Crude peptide was washed with MTBE and dried to obtain H-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-OH.
The crude peptide was purified by two cycles RP-HPLC. In the first cycle, crude Glucagon was dissolved in 8 volumes of 10 % acetonitrile in water containing 0.01% HCl in water. The resulting solution was loaded on a C8 RP-HPLC column, performed first step of purification (RP1) with 20 mM ammonium chloride in water and 30% mobile phase A in 70% acetonitrile. Collected the main fractions and carried out second cycle of purification (RP2) with 90 % (20 mM Ammonium bicarbonate in water and Acetonitrile, purified to obtain fractions containing Glucagon at a purity of NLT 98.00%., the fractions were collected, converted to HCl salt through RP-HPLC and lyophilized to obtain final dry Glucagon, NLT 98.00% and any individual unspecified impurities NMT 0.20 %.

Example 7: Synthesis of Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9OH (SEQ ID: 1)
Fmoc-Asp(OtBu)-OH was loaded to 2-CTC resin (100 g) to obtain substitution of 1.0 mmol/g in DIPEA/DCM. All the amino acids were coupled sequentially to the resin with Fmoc-/tBu strategy.
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 – 2.0 h.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
i) Ser, Thr and Phe were coupled using HBTU/ OxymaPure DIPEA
ii) All Fmoc-deprotection were performed by using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid
Peptide was cleaved using mixture of 1% TFA/DCM and precipitated with MTBE to get crude protected peptide and crystallized using EtOAc/ACN/n-hexane to obtained product.

Example 8: Synthesis of Glucagon HCl using SEQ ID: 1 and SEQ ID: 2 on Wang resin
Wang resin (100 g; 0.50 mmol/g) was washed with DMF/DCM in a reaction flask, solvent removed using vacuum and allowed to swell in DMF/DCM. Fmoc-Thr(tBu)-OH (1.5 eq.), DIC (3.0 eq.) and DMAP (0.05 eq.) dissolved in DMF were added to the resin and kept for stirring for 2-3 h. Coupling was monitored using Kaiser test. After reaction complies, resin was washed with DMF. Substitution obtained after first amino acid loading was 0.36 mmol/g. Capping was performed using acetic anhydride and DIPEA in DMF. Resin was washed with DMF.
After first amino acid attachment, Fmoc deprotection was performed using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid and reaction completion was monitored using Kaiser test. After reaction complies, resin was washed with DMF containing 0.5 % formic acid followed by 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 kept for stirring for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. Resin was washed with DMF to obtain Fmoc-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(OtBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-CTC resin.
Fmoc-Axx-OH/ Boc-Axx-OH, coupling reagent, additive details given below:
i) Met, Gly, Ala, Val, and Leu amino acids were coupled using DIC/ OxymaPure
ii) Trp, Asp, Arg, Gln, Asn, Phe, Tyr and Ser were coupled using HBTU/ Oxyma B/ DIPEA
iii) Lys, Asp, SEQ ID: 1 (Fmoc-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9OH) and
SEQ ID: 2 (Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH) were coupled using HATU/ Oxyma B/ DIPEA

All Fmoc-deprotection steps were performed by using 5- 15 % piperidine in DMF with 0.5 % ascorbic acid. Solvents were removed using vacuum.
As per the sequence all fragments were coupled to obtain Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(OtBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-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/ammonium iodide/DMS (84: 5: 5: 5: 0.5: 0.5 %) was added to the resin and kept for stirring for 3-4 h. After reaction complies, the reaction mixture was filtered and filtrate was evaporated and crude peptide was isolated using chilled MTBE. Crude peptide was washed with MTBE and dried to obtain H-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-OH.

The crude peptide was purified by two cycles RP-HPLC. In the first cycle, crude Glucagon was dissolved in 0.025 – 0.10 % HCl aqueous solution, added 0.25 -1.0 % acetonitrile to get clear solution. The resulting solution was loaded on a C8 RP-HPLC column, performed first step of purification (RP1) with 20 -50 mM ammonium bicarbonate in water and Acetonitrile. Collected the main fractions and carried out second cycle of purification (RP2) with 0.05 – 010 % Acetic acid in water and Acetonitrile, purified to obtain fractions containing Glucagon at a purity of NLT 98.00%., the fractions were collected, converted to 0.008 % HCl salt through RP-HPLC and lyophilized to obtain final dry Glucagon, NLT 98.00% and any individual unspecified impurities NMT 0.20 %.

Example 9: Synthesis of Glucagon HCl using sequential strategy
Wang resin (100 g; 0.50 mmol/g) was washed with DCM in a reaction flask, solvent removed using vacuum and allowed to swell in DCM. Fmoc-Thr(tBu)29-OH (2.0 eq.), DIC (3.0 eq.) and DMAP (0.075 eq.) dissolved in DMF were added to the resin and kept for stirring for 2-3 h. Coupling was monitored using Kaiser test. After reaction complies, resin was washed with DMF. Substitution obtained after first amino acid loading was 0.41 mmol/g. Capping was performed using 5% v/v acetic anhydride and 10% DIPEA in DMF. Further resin was washed with DMF.
After first amino acid attachment, Fmoc deprotection was performed using 10% piperidine in DMF with 1% formic acid and reaction completion was monitored using Kaiser test. After reaction complies, resin was washed with 0.1M HOBt in DMF. 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 kept for stirring for 1.5 – 2.0 h. Coupling was monitored using Kaiser test. Resin was washed with DMF.
Fmoc-AA-OH/ Boc-AA-OH, coupling reagent, additive details given below:
All amino acids used (3.0 eq.) for all positions except Thr(29).
i) Met(27), Leu(26), Val(23), Phe(22), Asp(21), Ala(19), Asp(15) and Leu(14) amino acids are coupled using DIC(7.5 eq) /HOBt.H2O (3.0 eq.), temperature of reaction is 25±2°C.
ii) Asn(28), Trp(25), Gln(24), Gln(20), Arg(18), Arg(17), Ser(16), Tyr(13), Lys(12), Ser(11), Tyr(10), Asp(9), Ser(8), Thr(7), and Phe(6) are coupled using HBTU(3.0 eq.)/ HOBt.H2O (3.0 eq.)/DIPEA(5.0 eq.), temperature of reaction at 25±2°C
iii) Thr(5), Gln(3), Ser(2) and His(1),coupled using HATU(3.0 eq.)/ HOBt.H2O (3.0 eq.)/ DIPEA(5.0 eq.), temperature of reaction at 25±2°C
iv) Gly(4) coupled using HATU(3.0 eq.) / HOBt.H2O (3.0 eq.)/ DIPEA(5.0 eq.) and temperature of reaction at 35±2°C
v) For recoupling of amino acids, all amino acids used 1.5 (equiv.) except Thr (29) and His(1). In addition, all coupling reagents and additives used as per their sequence. However, their equivalent was half of coupling step, like DIC (3.75 equiv.), HOBt.H2O (1.5 equiv.), HBTU (1.5 equiv.), HATU (1.5 equiv.), DIPEA (2.5 equiv.)
For amino acid Thr(29) to Gln(20) deprotection reaction step is performed by using 10% piperidine in DMF with 1% formic acid at 25±2 °C.
For amino acids Ala(19) to Thr(7) deprotection reaction step is performed by using solution of 15% piperidine in DMF with 1% formic at 25±2 °C.
For amino acids from Phe(6) to His(1) de-protection reaction step is performed by using 15 % piperidine in DMF with 0.5 % DBU in DMF at 25±2 °C.
Solvent was removed using vacuum.
As per the sequence, sequential coupling and deprotection reaction was performed to obtain
Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-Phe6-Thr(tBu)7-Ser(tBu)8-Asp(OtBu)9-Tyr(tBu)10-Ser(tBu)11-Lys(Boc)12-Tyr(tBu)13-Leu14-Asp(OtBu)15-Ser(tBu)16-Arg(Pbf)17-Arg(Pbf)18-Ala19-Gln(Trt)20-Asp(OtBu)21-Phe22-Val23-Gln(Trt)24-Trp(Boc)25-Leu26-Met27-Asn(Trt)28-Thr(tBu)29-Wang resin.
Resin washed with DMF followed by DCM, and MTBE and allowed to dry. Cleavage cocktail mixture of TFA/TIPS/phenol/H2O/ammonium iodide/DMS (80: 7.5: 5: 7.5: 0.5: 0.5 %) was added to the resin and kept for stirring for 3-4 h. Filtered the mass and filtrate was evaporated by using rotary evaporation. Concentrated mass precipitated by using chilled MTBE followed by washing with MTBE. Wet peptide obtained is dried by using vacuum to obtain crude peptide H-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-OH.

Dated this: 23rd day of August 2024 Dr. S. Ganesan
Alembic Pharmaceutical Ltd.

,CLAIMS:WE CLAIM:

1. A process for the preparation of Glucagon or a pharmaceutically acceptable salt thereof, comprising use of at least one compound selected from group consisting of SEQ ID: 1, SEQ ID: 2, SEQ ID: 3, SEQ ID: 4, SEQ ID: 5 or a pharmaceutically acceptable salt thereof.

2. An improved process for of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising;
(a) loading of Fmoc protected Threonine with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Tyr10 in the presence of coupling agent to obtain Fmoc-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling of fragment Fmoc-Phe6-Thr7-Ser8-Asp9-OH (SEQ ID: 1) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Fmoc-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(d) deprotection of Fmoc protecting group of fragment obtained in step (c) using deprotecting agent followed by coupling of fragment Boc-His1-Ser2-Gln3-Gly4-Thr5-OH (SEQ ID: 2) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin; and
(e) deprotection and cleavage of fragment obtained in step (d) using cleaving agent to obtain Glucagon or a pharmaceutically acceptable salt thereof.

3. An improved process for of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising;
(a) loading of Fmoc protected Threonine with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Asp9 in the presence of coupling agent to obtain Fmoc- Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling of fragment Fmoc-Gly4-Thr5-Phe6-Thr7-Ser8-OH (SEQ ID: 4) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Fmoc-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(d) deprotection of Fmoc protecting group of fragment obtained in step (c) using deprotecting agent followed by coupling of fragment Boc-His1-Ser2-Gln3-OH (SEQ ID: 3) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin; and
(e) deprotection and cleavage of fragment obtained in step (d) using cleaving agent to obtain Glucagon or a pharmaceutically acceptable salt thereof.

4. An improved process for of preparation of Glucagon or a pharmaceutically acceptable salt thereof comprising;
(a) loading of Fmoc protected Threonine with side chain protection to a resin solid-phase support in the presence of coupling agent;
(b) deprotection of Fmoc group using deprotecting agent and sequential coupling of amino acids with N-terminal protection and side chain protection based on the sequence of peptide backbone of Glucagon till Asp9 in the presence of coupling agent to obtain Fmoc- Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(c) deprotection of Fmoc protecting group of fragment obtained in step (b) using deprotecting agent followed by coupling of fragment Fmoc-Phe6-Thr7-Ser8-OH (SEQ ID: 5) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Fmoc-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin;
(d) deprotection of Fmoc protecting group of fragment obtained in step (c) using deprotecting agent followed by coupling of fragment Boc-His1-Ser2-Gln3-Gly4-Thr5-OH (SEQ ID: 2) wherein, the side chain of amino acids are protected with a suitable protecting group; in the presence of coupling agent; to obtain Boc-His1-Ser2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Lys12-Tyr13-Leu14-Asp15-Ser16-Arg17-Arg18-Ala19-Gln20-Asp21-Phe22-Val23-Gln24-Trp25-Leu26-Met27-Asn28-Thr29-resin; and
(e) deprotection and cleavage of fragment obtained in step (d) using cleaving agent to obtain Glucagon or a pharmaceutically acceptable salt thereof.

5. The process as claimed in any of claims 2 to 4, wherein resin solid-phase support is selected from Wang resin and chlorotrityl resin (CTC).

6. The process as claimed in any of claims 2 to 4, wherein coupling agents selected from group comprising of DIC/ OxymaPure; HBTU/OxymaB/DIPEA and HBTU/OxymaPure/ DIPEA.

7. The process as claimed in any of claims 2 to 4, wherein Fmoc deprotection 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 any of claims 2 to 4, wherein deprotection and cleavage using cleaving agent comprising of mixture of TFA, TIPS, phenol, H2O, ammonium iodide, DMS.

9. Compound of SEQ ID: 2:

Boc-His(Trt)1-Ser(tBu)2-Gln(Trt)3-Gly4-Thr(tBu)5-OH or pharmaceutically acceptable salt thereof.

Dated this: 23rd day of August 2024 Dr. S. Ganesan
Alembic Pharmaceutical Ltd.