Description:FIELD OF THE INVENTION
The present invention relates to improved process for the preparation of Octreotide or its salt thereof.
BACKGROUND OF THE INVENTION
Octreotide is a cyclic octapeptide. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin. Octreotide is known chemically as L-Cysteinamide, D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxy-methyl) propyl], cyclic (2?7)-disulfide; [R-(R*,R*)] as depicted in Formula-I.

Formula-1
US 4,395,403 claims Octreotide. US’403 disclose preparation of Octreotide wherein of disulfide bridge between thiol groups of cysteine residues formed in liquid phase synthesis.
US 5,889,146 disclose preparation of Octreotide, wherein for the formation of disulfide bridge between thiol group of cysteine residues, Fmoc-D-Phe-Cys(Trt)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(ol)-TerephthalAcetalAmide Resin was suspended in DMF and treated with iodine.
WO 2021051766 disclose preparation of Octreotide, wherein Fmoc-Thr (tBu) -OL-2CTC resin is prepared followed by coupling of orthogonal protected cysteine- cysteine, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH and Fmoc-Phe-OH, then the orthogonal protected Alloc protecting group of cysteine, then Boc-D-Phe-OH is coupled, and then intramolecular amide reaction cyclization is carried out.
All the synthetic procedures disclosed in the literature involves use of costly resins or destructive and hazardous reagents for final deportation, Oxidation or cyclisation in the liquid phase and provides the product with impurity which are difficult to remove by purification, thus making the procedures commercially difficult to implement. The present invention provides a process which is efficient and provides consistent yield with purity more than 99%. The product when made by the process of the present invention is easy to isolate as a powder and giving prominent and consistent results thus making the process amenable for commercial scale use.
SUMMARY OF THE INVENTION
An aspect of the present invention relates to an improved process for the preparation of Octreotide or its salt thereof, comprising disulfide bridge formation by using P1-D-Phe-Cys(P2)-Phe-D-Trp(P1)-Lys(P1)-Thr(P1)-Cys(P2)-Thr(P1)-O-CTC resin, with iodine and if required quenching excess iodine with ascorbic acid followed by global cleavage of peptide from CTC resin to obtain crude peptide; wherein P1 is either same or different and independently selected from an acid labile protecting group consisting of Boc, Mmt, and or tBu; and P2 is selected from oxidative labile protecting group such as Acm, Trt.
Another aspect of the present invention relates to an improved process for the preparation of Octreotide or its salt thereof, comprising reacting Boc-D-Phe-Cys(Acm)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-O-CTC resin with iodine to obtain SEQ ID NO.1A followed by de-protecting the protecting groups/CTC resin of SEQ ID NO.1A.

SEQ ID-1A
BRIEF DESCRIPTION OF ABBREVIATIONS:
CTC Resin 2-Chlorotrityl chloride Resin
Fmoc 9-fluorenylmethyloxycarbonyl
Boc t-Butyloxycarbonyl
Trt Trityl
tBu Tert-butyl
Mmt Monomethoxytrityl
Acm acetamidomethyl
DMF Dimethylformamide
NMP N-Methyl-2-pyrrolidone
DCM dichloromethane
DIPEA Diisopropylethylamine
DIC Diisopropylcarbodiimide
HOBt 1-Hydroxybenzotriazole
TFA Trifluoro acetic acid
TIS Triisopropylsilane
DMS Dimethyl sulfide
EDT 1,2-ethanedithio
HPLC High performance liquid chromatography h/min hour/minutes
DBU l,8-Diazabicyclo[5.4.0]undec-7-ene
DMAP 4-DimethylaminopyridineTFA Trifluoroacetic acid
AC2O Acetic anhydride
DCC Dicyclohexylcarbodiimide
EDC Ethyl-dimethylaminopropyl carbodiimide
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/
OxymaPure Ethyl 2-cyano-2-hydroxyimino-acetate
DEPBT 3-(diethoxy phosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
DETAILED DESCRIPTION OF INVENTION
An aspect of the present invention relates to an improved process of preparation of Octreotide or its salt thereof comprising;
a. coupling of Fmoc-Thr(P1)-OH on CTC resin in presence of base and a suitable solvent;
b. deprotection of Fmoc-Thr(P1)-CTC resin, followed by solid-phase synthesis, amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled based on the sequence of peptide backbone of Octreotide using suitable coupling reagent and coupling additive in suitable solvent to obtain P1-D-Phe-Cys(P2)-Phe-D-Trp(P1)-Lys(P1)-Thr(P1)-Cys(P2)-Thr(P1)-O-CTC resin;
c. oxidizing P1-D-Phe-Cys(P2)-Phe-D-Trp(P1)-Lys(P1)-Thr(P1)-Cys(P2)-Thr(P1)-O-CTC resin to obtain SEQ ID NO.1; and

SEQ ID-1
d. concomitant cleaving of CTC resin and de- protecting group of the amino acid of compound of SEQ ID-1 cleavage reagent to obtain Octreotide;
wherein P1 is either same or different and independently selected from an acid labile protecting group consisting of Boc, Mmt, and or tBu; and P2 is selected from oxidative labile protecting group such as Acm, Trt.
In an embodiment of the invention, in step (a) coupling of Fmoc-Thr(P1)-OH on CTC resin is carried out optionally in the presence of activating agent such as thionyl chloride and activated resin is treated / coupled with Fmoc-Thr(P1)-OH. The step (a) reaction is carried out in suitable solvent selected from DMF, DCM, NMP, acetonitrile, diethyl Ether, diisopropyl ether, methyl tertiary butyl ether, ethyl acetate, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran dimethyl sulphoxide, hexane, water and mixture thereof. The coupling of Fmoc-Thr(P1)-OH on CTC resin is carried out after swelling of the resin in a suitable solvent such as DCM.
In another embodiment of the invention, in step (a) the coupling reaction may be carried out in the in the presence of a base selected from base comprising diisopropylethylamine (DIPEA), triethylamine, N- methylmorpholine, N-methylpiperidine, piperdine, pyridine, etc, preferably DIPEA.
The term “solid phase synthesis” (SPPS) can 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. It comprises loading a first a-amino-protected amino acid, or peptide, onto a resin and is followed by the repetition of a sequence of steps referred to as a cycle, or as a step of elongation, consisting of the cleavage of the a-amino protecting group and the coupling of the subsequent protected amino acid. The formation of a peptide bond between two amino acids, or between an amino acid and a peptide fragment, or between two peptide fragments, may involve two steps. First, the activation of the free carboxyl group for a time ranging from 5 min. to 2 h, then the nucleophilic attack of the amino group at the activated carboxylic group.
The cycle may be repeated sequentially until the desired sequence of the peptide is accomplished.
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 de-protected. 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 de-protect Fmoc protecting groups.
In a preferred aspect of present invention, the coupling step of (a) or SPPS are carried out at a temperature which can vary in the range 10-60 °C. Preferably, the temperature varies in the range from 15 to 40 °C.
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 deprotection to facilitate the reaction.
In an embodiment of the invention, in step (b) the coupling of amino acids may be carried out in the presence of a coupling reagent and in the presence of coupling additive. The coupling reagent may be selected, among others, from the group comprising of N,N'-diisopropylcarbodiimide (DIC or DIPC), N,N'-dicyclohexylcarbodiimide (DCC), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N,N',N'-Tetramethyl-0-(benzotriazol-l-yl)uronium tetrafluoroborate (TBTU), 2-(7-Aza-lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HATU), 2-(lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HBTU), ethyl-dimethylaminopropyl carbodiimide (EDC), (3-(diethoxy phosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (DEPBT), COMU, (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate etc, preferably DIC.
In an embodiment of the invention, in step (b) coupling additive, 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), magnesium chloride, zinc chloride or copper chloride, preferably HOBt.
In an embodiment of the invention, in step (b) the coupling reaction, either involving peptides or amino acids, takes place in the presence of a solvent selected from the group comprising DMF, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran and N-methyl pyrrolidine and a mixture thereof, preferably DMF.
In an embodiment of the invention, in step (c) oxidizing P1-D-Phe-Cys(P2)-Phe-D-Trp(P1)-Lys(P1)-Thr(P1)-Cys(P2)-Thr(P1)-O-CTC comprises reacting P1-D-Phe-Cys(P2)-Phe-D-Trp(P1)-Lys(P1)-Thr(P1)-Cys(P2)-Thr(P1)-O-CTC with an oxidizing agent in the presence of solvent. The oxidizing agent is used is selected from iodine and the solvent is preferably DMF. Further, if required excess of iodine is quenched with ascorbic acid or sodium thiosulphate (Na2S2O3) in water or DMF.
In an embodiment of the invention, in step (d) concomitant cleavage in compound of SEQ ID-1 is cleavage reagent consisting on one of more of cocktail mixture of acid, scavengers and solvents to obtain crude Octreotide. The reaction temperature may range from 10 - 30 °C.
De-protection and cleavage conditions generally depend on the nature of the protecting groups and of the resin used. 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 an acid and at least one scavenger. The peptide cleavage reagent 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, EDT, triethylsilane (TES), DTT, triisopropylsilane (TIPS), 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, cresol and thiocresol or mixture thereof and water; preferably TFA: TIS: phenol: water. The relative ratio of acidic material to scavenger to water may be from about 90% to about 95% acidic material, from about 2.5% to about 5% scavenger, and from about 2.5% to about 5% water by weight. Preferably the ratio of TFA: TIPS: phenol: water is 92.5%: 2.5%: 2.5%: 2.5%.
In an embodiment of the invention, isolating pure Octreotide or its salt thereof by purification and lyophilization.
In an embodiment of the invention, the crude Octreotide or its salt thereof obtained is purified by crystallization or chromatographic techniques well known in the art. Wherein, for chromatographic techniques C-18 YMC/ Phenomenex Luna/ Kromasil/ Daisogel column can be used. Further, crude Octreotide dissolved in an aqueous solution of acetonitrile and ammonium acetate solution and purified using ammonium acetate (mobile phase A), and water (mobile phase B) to obtain acetate salt. The obtained acetate salt was purified using 0.1%-0.5% acetic acid or HCl or citric acid in water (mobile phase A), and methanol or acetonitrile (mobile phase B).

In an embodiment the lyophilized Octreotide or its salt thereof is prepared by removal of the solvent, the removal of solvent comprises one or more of distillation, distillation under vacuum, spray drying, sublimation, agitated thin film drying ("ATFD"), and freeze drying.
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 pyridine or DIPEA and like.
Inventors, of present invention found that the peptide obtained after oxidation reaction in liquid phase is not stable for longer time. Whereas in present invention formation of disulfide bridge between thiol group of cysteine by oxidation has been carried out on the CTC resin which provide advantage in controlling the impurities in the process, reduces the number of steps and cycle time in the process which leads to cost effective process. Further, crude peptide obtained by present invention is isolated as a powder and giving prominent and consistent results while doing downstream purification, moreover, the obtained crude peptide can be stored for six months.
The terms "about" as used herein refers 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 Octreotide 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.
In one embodiment the process for preparation of Octreotide or its salt thereof of Formula-I according to the present invention is shown in scheme-1.

Scheme-1
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 Octreotide
3.5 kg of 2-CTC (0.9- 1.6 mmol/g) resin was taken in a reactor and washed with DCM for two times and followed by removal of solvents under vacuum.
The obtained resin was swelled using 5-10 volume of DCM. The first amino acid Fmoc-Thr(tBu)-OL was loaded in the presence of diisopropylethylamine (DIPEA) in DCM solvent to obtain 0.80 mmol/g loading. After first attachment, unreacted active sites on the resin were capped with methanol, DCM and DIPEA. After capping, the Fmoc protecting group of amino acid was removed with 5-20 % piperidine in DMF. After washing of residual reagents, the second amino acid, Fmoc-Cys(Acm)-OH was introduced. The Fmoc protected amino acid was activated insitu using coupling reagent such as DIC and HOBt in DMF. The obtained pre-activated mixture was added to the resin and stirred. The reaction was monitored using Kaiser test/Bromophenol blue test. After completion of reaction, resin was sequentially washed with DMF-DCM-DMF twice. However, in case of incomplete coupling, capping is carried out using acetic anhydride and DIPEA/pyridine.
Followed by sequential coupling of all the amino acids as per Octreotide backbone sequence. All amino acids used were Fmoc-Na protected except Boc-D-Phe-OH.
After completion of coupling, the disulphide bridge (S-S bridge) was formed between cysteine residues at with 2nd position and 7th position in the presence of iodine in DMF and methanol mixture. After completion of reaction, excess of iodine was quenched with ascorbic acid in water/DMF and followed by resin wash with DMF/MDC/MeOH/MTBE to obtain the SEQ ID - 1A.
The obtained SEQ ID - 1A was treated with a cocktail mixture of TFA, TIS, Phenol and H2O more preferably 92.5% TFA, 2.5% TIS, 2.5% Phenol, and 2.5% H2O solution at room temperature. After completion of the cleavage, the resin was filtered to obtain the filtrate which is concentrated and the product was precipitated using MTBE, filtered, and dried in vacuum to obtain Octreotide crude peptide (Yield: 50-60%).
Example-2: Purification of Octreotide acetate
The crude Octreotide peptide contains trifluoroacetate as a counter-ion. The crude Octreotide peptide was dissolved in an aqueous solution of acetonitrile and ammonium acetate solution. The obtained solution was loaded on a C-18 Phenomenex Luna column and using gradient elution method with Mobile phase A: (5 to 10 millimolar ammonium acetate in water); Mobile phase B: (water) for replacing the trifluoroacetate counter-ion with acetate ion and followed by water washes.
The obtained peptide was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using gradient elution method with Mobile phase A: 0.1% acetic acid in water; Mobile phase B: 100% methanol to obtain fractions containing Octreotide acetate salt. The gradient elution is performed as per the below tabular column (Table-2).
Purification conditions:
Column : C18 (Phenomenex Luna), 100 Å,10µ (DAC column- 450 mm)
Detector wavelength ?max : 214 nm
Elution time : Eluted main peak approximately at 55- 100 min.
The obtained fractions collected were passed through 0.2-micron filter and lyophilized to obtain Octreotide acetate with purity NLT 99.0 % and yield observed 40-45%.
, Claims:1. A process for preparation of Octreotide or its salt thereof comprising disulfide bridge formation by using Boc-D-Phe-Cys(Acm)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-O-CTC resin with iodine to obtain SEQ ID NO.1A followed by de-protecting the protecting groups/ cleavage of CTC resin of SEQ ID NO.1A.

SEQ ID-1A
2. A process for preparation of Octreotide or its salt thereof comprising the steps of;
a. coupling of Fmoc-Thr(P1)-OH on CTC resin in presence of base and a suitable solvent;
b. Fmoc de-protection from Fmoc-Thr(P1)-CTC resin, followed by solid-phase synthesis, amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled based on the sequence of peptide backbone of Octreotide using suitable coupling reagent and coupling additive in suitable solvent to obtain P1-D-Phe-Cys(P2)-Phe-D-Trp(P1)-Lys(P1)-Thr(P1)-Cys(P2)-Thr(P1)-O-CTC resin;
c. oxidizing P1-D-Phe-Cys(P2)-Phe-D-Trp(P1)-Lys(P1)-Thr(P1)-Cys(P2)-Thr(P1)-O-CTC resin to obtain SEQ ID NO.1; and

SEQ ID-1
d. concomitant cleaving of CTC resin and de- protecting group of the amino acid of compound of SEQ ID-1 in the presence of cleavage reagent to obtain Octreotide.
wherein P1 is either same or different and independently selected from an acid labile protecting group consisting of Boc, Mmt and or t-Bu; and P2 is selected from oxidative labile protecting group such as Acm, Trt.
3. The process as claimed in step (a) of claim 2, wherein base is selected from diisopropylethylamine (DIPEA), triethylamine, N- methylmorpholine, N-methylpiperidine, piperdine, pyridine.
4. The process as claimed in step (a) of claim 2, wherein suitable solvent selected from DMF, DCM, NMP, acetonitrile, diethyl ether, diisopropyl ether, methyl tertiary butyl ether, ethyl acetate, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran dimethyl sulphoxide, hexane, water and mixture thereof
5. The process as claimed in step (b) of claim 2, wherein Fmoc de-protection is carried out in presence of suitable organic base selected from piperidine, pyrrolidine, piperazine, tert-butylamine, DBU and diethylamine in suitable solvent selected from the group comprising DMF and NMP.
6. The process as claimed in step (b) of claim 2, wherein coupling reagent selected from N,N'- diisopropylcarbodiimide (DIC or DIPC), N,N'-dicyclohexylcarbodiimide (DCC), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N,N',N'-Tetramethyl-0-(benzotriazol-l-yl)uronium tetrafluoroborate (TBTU), 2-(7-Aza-lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HATU), 2-(lH-benzotriazole-l-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HBTU), ethyl-dimethylaminopropyl carbodiimide (EDC), (3-(diethoxy phosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (DEPBT).
7. The process as claimed in step (b) of claim 2, coupling additive selected from 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), magnesium chloride, zinc chloride or copper chloride.
8. The process as claimed in step (b) of claim 2, suitable solvent selected from DMF, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran and N-methyl pyrrolidine and a mixture thereof.
9. The process as claimed in step (c) of claim 2, wherein oxidation is carried out in presence of iodine in DMF followed by quenching with ascorbic acid or sodium thiosulphate.
10. The process as claimed in step (d) of claim 2, wherein cleavage reagent is selected from TFA, EDT, triethylsilane (TES), DTT, triisopropylsilane (TIPS), 2, 2’-(ethylenedioxy)diethane, acetyl cystein, DMS, phenol, cresol, thiocresol and water mixture thereof.