DESC:Field of the Invention
The present invention relates to the field of Pharmaceuticals, more particularly, the invention relates to an improved process for the synthesis of Vasopressin, chemically represented by the formula (1).

Background of the Invention
Vasopressin (1) is a peptide hormone, composed of nine amino acid residues with a molecular weight of 1084.24. It is a known antidiuretic hormone drug that promotes reabsorption of water at the collecting duct and distal renal tubular, it is also known to have an anti-diabetic effect and is used as a clinical formulation for the treatment of diabetes insipidus.

Currently Vasopressin (1) is most commonly synthesized via a Solid Phase Peptide Synthesis (SPPS), using resins to yield the crude vasopressin precursor represented by the compound of formula (2), the resultant product is further cyclized under high dilutions and purified to finally yield Vasopressin (1) as a pure product. Although there are various methods of synthesis of Vasopressin (1), the disulfide bond formation invariably involves the use of high dilution factor that is time consuming when it comes to the purification and isolation process.

For example PCT applications WO 2006041945 and WO 2006119388 discloses the linear synthesis via SPPS in the presence of rink amide resin and Fmoc protected amino acids and further cyclization by the formation of the disulfide bond wherein the process states the use of Iodine in Acetic acid, required a high dilution ratio of the linear peptide (gm/L) and purification by C18 RP-HPLC. The process involves high dilution for purification which is time consuming and not commercially viable on industrial scale. The present invention overcomes this problem as it involves the use of low dilution ratio thus reducing the amount of solvent, time and preparative column used and simultaneously obtaining a product high in purity and yield.

Further S-S Bridge cyclic and straight chain peptide analogues and methods for their preparation are described in U.S. Patent Nos. 4,310,518 and 4,235,886 discloses multiple purification processes for peptides such as Reverse phase high-performance liquid chromatography (RP-HPLC), along with the use of counterions and ion exchange columns; the final steps involve the cyclization and purification. The present invention overcomes this problem as it involves the use of low dilution ratio thus reducing the amount of solvent, time and preparative column used; also the use of an antisolvent aids in precipitation during purification which simultaneously yields a high volume product with high purity.

The article J. Am. Chem. Soc. 1991, 113(17), 6657-6662 discloses the use of aerial oxidation in DMSO method, however the process still has a high dilution and the problem of purification is not solved. The present invention overcomes this problem as it involves the use of low dilution ratio thus reducing the amount of solvent, time and preparative column used and simultaneously obtaining a product high in purity and yield.

According to Chinese patent CN 106518978 the synthesis of Vasopressin comprising the use of reverse phase high performance liquid chromatography packed with styrene - divinylbenzene copolymer wherein the use of 5% acetonitrile in water was used. The process involves a high dilution factor of ? 5 g/L and is time consuming in the HPLC purification step. The present invention overcomes this problem as it involves the use of low dilution ratio thus reducing the amount of solvent, time and preparative column used and simultaneously obtaining a product high in purity and yield.

Although the purification processes have improved over the past few years, due to the highly diluted sample concentration, the cyclization and post purification stage remains to be a bulky and highly unfavorable that still lacks an effective process for bulk drug production of peptides containing disulfide bonds. Thus there exists a need to develop an efficient method to reduce the time and wastage in the synthesis of disulfide-containing peptides such as Vasopressin (1).

The inventors of the present disclosure have surprisingly found an improved process or rather a significant time reduction process for the purification of Vasopressin (1) and also there is no need of high dilution of the reaction mixture to facilitate the disulfide bond formation. The inventors also found surprisingly a simple technique in precipitation of final product by anti-solvent addition by selecting the solvents from Acetone, methanol, isopropyl ether, Diethyl ether and methyl tertiary butyl ether.

Object of Invention
An object of the present invention is to provide an improved process for the preparation of S-S bridge formation of the compounds like Oxytocin, Desmopressin, Vasopressin, Octreotide etc. In the present scenario, inventors have developed a versatile method of S-S bridge formation particularly in Vasopressin, represented by a compound of formula (1), is an easily scalable approach.

In order to overcome the technical problem in prior arts, an object of the present invention is a method that involves the use of a solvent, a reagent, and optionally an antisolvent, to yield Vasopressin (1) in high yield with low impurities, simultaneously reducing the time taken during the production of the product.

An added object of the present invention is the cyclization stage, wherein the use of Hydrogen peroxide improves the dilution ratio and thus reducing the amount of solvent and reaction time.

Another object of the present invention is the use of antisolvents to aid in precipitation at the isolation stage thus providing a process with high yield and high purity.

Summary of the Invention
An embodiment of the present invention describes a method that involves the use of a solvent, a reagent, and optionally an antisolvent, to yield Vasopressin (1) in high yield with low impurities, simultaneously reducing the time taken during the production of the product.

The present invention also comprises of the synthesis of the Vasopressin (1), wherein the process involves the synthesis of the vasopressin precursor, represented by the formula (2)

and further the formation of the disulfide bond through the cyclization in the presence of a solvent and a reagent such as H2O2.

In an added embodiment of the present invention, the process comprises of an improved process for the synthesis of the Vasopressin (1), wherein the process involves cyclization by disulfide bond formation and further cleaving of resin to yield the crude product of Vasopressin (1).

Another embodiment of the present invention comprises of an improved process for the synthesis of the Vasopressin (1) via a one-pot method, wherein the process involves cyclization by disulfide bond formation and cleaving of resin to yield the crude product of Vasopressin (1).

In an added embodiment of the present invention, the process comprises of an improved process for the synthesis of the Vasopressin (1) via a one-pot method, wherein the process involves cleaving of resin and further cyclization by disulfide bond formation to yield the crude product of Vasopressin (1).

Another embodiment of the present invention comprises of an improved process for the synthesis of the Vasopressin (1) via a microwave synthetic method, wherein the process involves cyclization by disulfide bond formation and cleaving of resin to yield the crude product of Vasopressin (1).

In an added embodiment of the present invention, the process comprises of an improved process for the synthesis of the Vasopressin (1) via a microwave synthetic method, wherein the process involves cleaving of resin and further cyclization by disulfide bond formation to yield the crude product of Vasopressin (1).

Another embodiment of the present invention also comprises of an improved method of isolation, wherein the crude product obtained above is purified and isolated, optionally by use of an antisolvent or by preparative HPLC.

Another embodiment of the invention relates to a final purification of the compound, wherein the crude Vasopressin (1) has a lowest possible diluted sample (1g /5 ml).

Another embodiment of the invention relates to a final purification of the compound, wherein the crude Vasopressin (1) has a lowest possible diluted sample (1g /5 ml), via the use of a reagent such as H2O2 and optionally an antisolvent.

Another embodiment of the invention relates to a process of the preparation of compound Vasopressin (1) in one pot precluding the disulfide bridge formation and resin cleavage in a most efficient manner by reducing the time and increasing the yield of the product.

In final embodiment, the present invention relates to a process of the preparation of Compound Vasopressin (1) resulting in higher yields and purity, while reducing the time and amount of solvent. In addition, the use of flash chromatography reduces time, amount of solvent and the number of preparative columns as compared to the prior arts.

Detailed Description
The present invention provides an improved, economical, environment friendly and industrially scalable process for the preparation of Vasopressin (1), using a more sustainable approach. The process comprises of cyclization by the formation of disulfide bond in the presence of a solvent and a reagent, such that it reduces the g/L ratio (dilution ratio), while simultaneously improving the yield and purity with reduced time and energy.

For the purpose of clarity and as an aid in the understanding of the invention, as disclosed and claimed herein, the following terms and abbreviations are as defined below:
? AcOH: Acetic acid
? tBu: Tert-butyl
? DCM: Dichloromethane
? DIC: N,N'-diisopropylcarbodiimide
? DMF: N,N'-Dimethylformamide
? DMSO: Dimethyl sulfoxide
? Fmoc: 9-fluorenylmethoxycarbonyl
? HOBt: N-hydroxybenzotriazole
? H2O2: Hydrogen peroxide
? IPA: Isopropyl alcohol
? MeOH: Methanol
? MTBE: Methyl tert-butyl ether
? Oxyma: Ethyl cyanohydroxyimino acetate
? Pbf: Pentmethyldihydrobenzofuransulfonyl
? RT: Room temperature
? SPPS: Solid phase peptide synthesis
? TFA: Trifluoroacetic acid
? TIS: Triisopropylsilane
? Trt: Trityl
? Acm: Acetamidomethyl
? DIEA: Diisopropylethylamine
? NMM: N-Methylmorpholine
? HOAT: 1-Hydroxy-7-azabenzotriazole
? RAM: Rink Amide
? DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene
? HBTU: (1H-Benzotriazol-1-yloxy)(dimethylamino)-N,N-dimethylmethaniminium hexafluorophosphate
? TBTU: (1H-Benzotriazol-1-yloxy)(dimethylamino)-N,N-dimethylmethaniminium tetrafluoroborate
? HCTU: [(6-Chloro-1H-benzotriazol-1-yl)oxy](dimethylamino)-N,N-dimethyl methaniminium hexafluorophosphate
? COMU: (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate
? HATU: (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate)

In a first embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the process comprises of the synthesis of a Vasopressin precursor represented by the compound of formula (3). The synthesis proceeds by a Solid phase peptide synthesis (SPPS) in the presence of a resin, a protecting group, a coupling reagent and capping mixture as described in Scheme 1. The synthesis of the Vasopressin precursor (3) may follow a linear or convergent synthesis.

In a second embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the process comprises of the synthesis of a Vasopressin precursor (2). The synthesis proceeds by deprotection of Vasopressin precursor (3) of the protecting groups and the resin in the presence of a deprotecting agent, a scavenger and a solvent.

In a third embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the process comprises of cyclization via disulfide bond formation between the free thiol groups of the 1st amino acid (Cys) and the 6th amino acid (Cys) of Vasopressin precursor (2), in the presence of a solvent and a reagent. The synthesis is as described in scheme 3 to obtain a crude product of Vasopressin (1) in improved yield.

In an alternate embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the process comprises of the formation of Vasopressin precursor (5) via Vasopressin precursor (4). The synthesis proceeds by the deprotection of the protecting groups on the amino acids 1st Cys and 6th Cys to yield free thiol ends of Vasopressin precursor (5). Further cyclization via disulfide bond formation between the free thiol groups of the 1st amino acid (Cys) and the 6th amino acid (Cys) of Vasopressin precursor (5) in the presence of a solvent and a reagent, yield the Vasopressin precursor (6), that is further deprotected to obtain a crude product of Vasopressin (1) in improved yield. The synthesis is as described below as Scheme 4 and may follow a linear or convergent synthesis.

In a fifth embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the resin may be selected from Rink amide resin, Rink Amide AM, Rink Amide MBHA, Seiber Amide resin and Tenta gel SRAM or combination thereof.

In the sixth embodiment, the coupling reaction may be carried out in a suitable solvent. The solvent that may be used in the coupling step include but are not limited to dichloromethane, dimethylformamide, N-methylpyrrolidone, Dimethyl acetamide or mixtures thereof. The temperature at which the coupling reaction is carried out may range from about 10°C to about 35°C.

In a seventh embodiment, the present invention describes the synthesis of Vasopressin precursor (3), wherein the protecting groups like Trityl (Trt) for Cysteine, Asparagine and Glutamine, tertiary butyl(tBu) for Tyrosine, Pentmethyldihydrobenzofuransulfonyl (Pbf) group for Arginine are selected as Fmoc-Amino Acids whereas for Boc amino acids with compatible side chain protections, but preferably Fmoc amino acids.

In an added embodiment, the present invention describes the synthesis of Vasopressin precursor (4), wherein the protecting groups like Acetamidomethyl (Acm) for Cysteine, Trityl (Trt) for Asparagine and Glutamine, tertiary butyl(tBu) for Tyrosine, Pentmethyldihydrobenzofuransulfonyl (Pbf) group for Arginine are selected as Fmoc-Amino Acids whereas for Boc amino acids with compatible side chain protections, but preferably Fmoc amino acids.

In a ninth embodiment, the present invention describes the synthesis of Vasopressin precursor (3), wherein the coupling reagent may be selected from the group of DIC/HOBT, Oxyma/DIC, HBTU or TBTU or HCTU/HOBt/DIEA, HOAT/DIC, COMU/DIC, HATU/HOAT/DIEA, HOBT/NMM as single or a combination, preferably DIC/Oxyma.

In a tenth embodiment, the present invention describes the synthesis of Vasopressin precursor (3 and 4), wherein capping mixture consists of acetic anhydride and pyridine.

In an eleventh embodiment, the present invention describes the synthesis of Vasopressin precursor (2), wherein the deprotecting agent may be selected form 20-40% piperidine or 2% DBU or 2% DBU+2% piperidine in DMF or 20% piperidine in NMP as deprotecting agents preferably 20% piperidine in DMF.

In a twelfth embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the cleavage cocktail is selected from the group TFA/ Thioanisole/ Anisole/ Phenol in the ratio of 90:5:2:3 or TFA/Thioanisole/Phenol/EDT/Water in the ratio of 82.5:5:5:2.5:5 or TFA/Thioanisole/TIS/Phenol/Water in the ratio of 88:3:3:3:3 or TFA/EDT/Thioanisole/DCM/TIS in the ratio of 80:5:5:5:5 or TFA/TIS/Water in the ratio of 95:2.5:2.5 and isolation by Diethyl ether or methyl tertiary butyl ether, preferably MTBE.

In a thirteenth embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the solvent is selected form the group of DMSO, DMF, Water, Acetonitrile, Anisole and combination thereof, but preferably DMSO.

In a fourteenth embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the reagent is selected from the group of H2O2, Potassium dichromate, potassium permanganate or Iodine in Acetic acid as reagents, preferably H2O2.

In a fifteenth embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the isolation is carried out in the presence of an antisolvent that may be selected from the group of Acetone, Methanol, Isopropyl alcohol, Isopropyl ether, Diethyl ether or Methyl tertiary butyl ether), preferably IPA:MTBE mixture.

In a sixteenth embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the isolation and purification is carried out by Flash and preparative HPLC.

In an alternative embodiment, the present invention comprises of a one-pot synthesis via resin cleavage, followed by the disulfide bridge formation (cyclization) to obtain the crude Vasopressin (1).

In another alternative embodiment, the present invention comprises of a one-pot synthesis via formation of disulfide bridge (cyclization) and resin cleavage procedure to obtain the crude Vasopressin (1).

In another embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the process comprises of a microwave synthesis via resin cleavage, followed by the disulfide bridge formation (cyclization) to obtain the crude Vasopressin (1).

In another alternative embodiment, the present invention describes the synthesis of Vasopressin (1), wherein the process comprises of a microwave synthesis via formation of disulfide bridge (cyclization) and resin cleavage procedure to obtain the crude Vasopressin (1).
In an alternative embodiment, the present invention describes the synthesis of Vasopressin precursors (3 and 4) via the use of Liquid phase peptide synthesis (LPPS).

In another embodiment of the present invention is the synthesis of peptides comprises of the use of antisolvent to aid in precipitation during isolation and thus subsequently aiding in purification to obtain a high yield and purity of the peptide.

In a final embodiment of the present invention is the synthesis of peptides with one or more disulfide bonds by the use of key reagent like H2O2 in DMSO and subsequent purification to obtain a high yield and purity of the peptide.

Without being limited by theory, the process according to the present invention may be advantageously used to prepare the complex compounds like Vasopressin or Desmopressin. The proposed routes of the drug described in Schemes 1, 2, 3 and 4 are such that it prevents the disadvantages of the prior art. It is envisaged that by providing the alternative route improves the shelf life or stability of the product is enhanced and the impurity content of the product is decreased or rather controlled during the preparation, thereby contributing to the overall efficacy of the product.

The proposed processes according to Schemes 1, 2, 3 and 4 of the present invention are convenient to use by the end users. Furthermore, the proposed routes of the present invention for the preparation of Vasopressin reduces the medication cost due to reduction of time and energy in final step of compound of formula 1, is economic for the end user, reduces drug waste, minimizes hospital and industrial waste and eliminates risk of toxicity by producing in higher purity of compounds.

The foregoing embodiments and advantages are merely exemplary and are not to be construed as limiting the scope of the present invention. The description of the exemplary embodiments of the present invention is intended to be illustrative and not to limit the scope of the invention. Various modifications and alterations, which are apparent to a person skilled in the art, are intended to fall within the scope of the invention.

The invention having been disclosed in connection with the foregoing embodiments, additional variations will now be apparent to persons skilled in the art. Various modifications and variations to the above described in the Schemes 1, 2, 3 and 4 can be made without departing from the scope of the invention.

The present invention is further described below by way of example, but not to limit the invention thus described in the embodiments. Experimental methods are without specific conditions in the examples below and may be altered in accordance with conventional methods and conditions, according to the product specification or selection.

Experimental Procedures:
Example 1: Preparation of H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 a Vasopressin precursor:

Synthesis of the peptide is carried out by a regular stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Rink amide resin (50 g). After removal of the Fmoc protecting group from the resin, the first amino acid (Fmoc-Gly-OH) is loaded on the resin in a regular coupling step (Oxyma/DIC) for 120-180 minutes to get Fmoc-Gly-resin with a loading of 0.7 mmol/g. After washing of the resin and removal of the Fmoc protecting group, the second amino acid (Fmoc-Arg(Pbf)-OH) is introduced to start the second coupling step. Fmoc protected amino acids are activated in situ using Oxyma and subsequently coupled to the resin for 90-120 minutes. Diisopropylethylamine or collidine are used during coupling as an organic base. Completion of the coupling is indicated by ninhydrin test and UV-loading test. After washing the resin with DMF & DCM, Fmoc protecting group on the a-amine is removed with 20% piperidine in DMF for 15 min each 2 times. These steps are repeated each time with another amino acid according to peptide sequence. All amino acids used are Fmoc-Na protected. Trifunctional amino acids are side chain protected as follows: Tyr(tBu), Arg(Pbf), Cys(trt), Asn (trt) and Gln(Trt). Three equivalents of the activated amino acids are employed in the coupling reactions. At the end of the synthesis the peptide-resin is washed with DMF, followed by DCM, MTBE and dried under vacuum at room temperature to obtain 120 g dry peptide-resin. The peptide, prepared as described above, is cleaved from the resin together with removal of acid-labile protecting groups using a mixture of 95% TFA, 2.5% TIS and 2.5 % of water for 3-4 hours at room temperature. Finally, the crude linear peptide was isolated from the reaction mixture by distilling the TFA to half of its volume under vacuum at below 30 0 C followed by addition of 10 V of MTBE to the mixture. Reaction mixture was filtered and dried under vacuum yielded 45 g (HPLC purity >70 %) of the linear peptide.

Example 2: Preparation of Vasopressin:

Dissolve the linear peptide H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 in 4-8 V of DMSO and added 3-6 equiv. of Hydrogen peroxide in to the reaction mixture at room temperature. The reaction mixture was stirred under continuous bubbling of air for 36 h. Progress of the reaction was followed by HPLC and after completing the reaction, the product was precipitated by adding the reaction mixture in to IPA followed MTBE addition. Crude Vasopressin was isolated by filtration and dried under vacuum yielded 40 g (HPLC purity > 70 %).

Purification and salt exchange of the crude peptide was carried out on a reverse phase preparative HPLC with a C18 column followed by lyophilization of the fractions (18 g, HPLC purity > 99 %).

Example 3: Preparation of H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 a Vasopressin precursor:

Synthesis of the peptide is carried out by a regular stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from Rink amide resin (50 g). After removal of the Fmoc protecting group from the resin the first amino acid (Fmoc-Gly) is loaded on the resin in a regular coupling step to provide loading of about 0.7 mmol/g. The free amino group of the resultant product by capping in the presence of acetic anhydride: Pyridine in the ratio 1:3, this step is repeated after every coupling stage to increase the purity of the final peptide. After washing of the resin and removal of the Fmoc protecting group the second amino acid (Fmoc-Arg (Pbf)) is introduced to start the second coupling step. Fmoc protected amino acids are activated in situ using Oxyma and subsequently coupled to the resin for 50 minutes. Diisopropylethylamine are used during coupling as an organic base. Completion of the coupling is indicated by Kaiser test. After washing of the resin, the Fmoc protecting group on the a-amine is removed with 20% piperidine in DMF for 20 min. These steps are repeated each time with another amino acid according to peptide sequence. All amino acids used are Fmoc-Na protected. Trifunctional amino acids are side chain protected as follows: Tyr (tBu), Arg (Pbf), and Cys (Trt). Asn (Trt) and Gln (trt) are used. Three equivalents of the activated amino acids are employed in the coupling reactions. At the end of the synthesis the peptide-resin is washed with DMF, followed by DCM, and dried under vacuum to obtain Crude dry peptide-resin.

Example 4: Isolation of Linear crude peptide as TFA salt:

The peptide, prepared as described above Example 1 is cleaved from the resin together with removal of acid-labile protecting groups using a 95% TFA, 2.5% TIS, 2.5% water solution for 2 hours at room temperature. The product is precipitated by distilling out TFA and by the addition of 10 volumes of MTBE to the distilled crude, filtered and dried in vacuum to obtain 36 g product. Residual TFA <0.25%. HPLC purity: 70-75% Assay By HPLC: 25-35

Example 5: Cyclisation of Linear peptide to form vasopressin crude:

The linear peptide, prepared as described above Example 2 is
Dissolved in 10 vol of DMSO and added hydrogen peroxide 3.0 eq. reaction mass was for stirred for 36 hrs at Room temperature ,after completion of the reaction material was isolated by using IPA and MTBE mixture. The completion of the reaction is test by the Elmann’s test for free thiol groups. Crude purity: 70-75% Assay: 35-40.

Example 6: Purification of crude peptide by using Flash chromatography:

Crude peptide is purified by using Flash chromatography technique in which sample is dissolved in water and filtered through 0.2 micron filter and sample is loaded on to the C18 cartridge and eluted with Acidic buffer in Mobile phase A and solvent as Mobile phase B and collected the peak and lyophilized to get the purified peptide. HPLC purity: 80-85% Assay By HPLC: 70-80.

Example 7: Preparation of pure Vasopressin:

H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 crude peptide (prepared as described in Example 3) is purified on a preparative C18 RP-HPLC column. Fractions containing >95% pure product are combined and diluted to a concentration of about 1 g/L. The resulting solution is loaded on a C18 RP-HPLC column and purified to obtain fractions containing Vasopressin trifluoroacetate at a purity of >98.0%. After exchange of the counterion with acetate (on RP-HPLC), the fractions are collected and lyophilized to obtain final dry peptide, HPLC purity: >98.0%

Example 8: Preparation of Vasopressin (1) by one pot method:

Synthesis of the peptide is carried out by a regular stepwise Fmoc SPPS (solid phase peptide synthesis) using Rink amide AM resin (50 g). After removal of the Fmoc protecting group from the resin (20% piperidine in DMF for 15 min, 2 times), the first amino acid (Fmoc-Gly) is loaded on the resin using Oxyma/DIC mixed anhydride method for 90-120min coupling time, to get Fmoc-Gly-Resin, with a loading 0.7 mmol/g. After washing of the resin and removal of the Fmoc protecting group, the subsequent amino acids from 2nd to 9th, (i.e., Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Cys(trt)-OH, Fmoc-Asn(trt)-OH, Fmco-Gln(trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Cys(trt)-OH)) are coupled to the resin using Oxyma and Diisopropylethylamine or collidine are used during coupling as an organic base for 90-120minutes. Completion of the coupling is indicated by ninhydrin test and UV-loading test. Wash the resin with DMF & DCM. Fmoc protecting group on the a-amine is removed by 20% piperidine in DMF for 15 min, 2 times. Three equivalents of the activated amino acids are employed in the coupling reactions. At the end of the synthesis the peptide-resin is washed with DMF, followed by DCM, and MTBE, and then dried under vacuum to obtain dry peptide-resin. One pot global deprotection and S-S bond formation was carried out by stirring the reaction mass in a mixture of 90% TFA, 5% Triisopropyl silane and 5% water for 6 hrs at room temperature. Finally, the crude Vasopressin was isolated from the reaction mixture by distilling the TFA to half of its volume under vacuum at below 30 0 C followed by addition of 10 V of MTBE to the mixture. Reaction mixture was filtered and dried under vacuum yielded 40 g (HPLC purity >60 %) of crude vasopressin.

Analysis of Amino acid: The Amino acid content was evaluated and the results are shown below:
S. No. Name RT uM Mole Ratio
1. Asp 1.12 0.76 1.01
2. Glu 1.17 0.83 1.09
3. Gly 1.08 0.85 1.12
4. Cys 1.26 1.55 2.04
5. Tyr 2.07 0.77 1.01
6. Phe 3.66 1.09 1.04
7. Pro 1.35 0.79 1.04
8. Arg 1.35 0.70 0.92

MALDI TOF Results: The Mass Spectroscopy was evaluated via MALDI TOF and the results are shown below:
Mass Spectroscopy Spectrum:

M/Z Charge Actual Mass Weight Intensity
1085.141 1 1.00011 1084.134 0.816 18798856

MSMS Spectrum:

From the foregoing, it may be understood that the embodiments of the present invention described above are well suited to provide the advantages set forth; and since many possible embodiments may be made of the various features of this invention, all without departing from the scope of the invention, it is to be understood that all matter hereinbefore set forth or shown in the description and synthetic schemes is to be interpreted as illustrative and that in certain instances some of the features may be used without a corresponding use of other features, all without departing from the scope of the invention.
,CLAIMS:We Claim:
1. An improved process for the preparation of Vasopressin (1),

wherein the process comprises of:
(i) Synthesis of a linear peptide Vasopressin precursor (3) by a Solid phase peptide synthesis (SPPS) in the presence of a resin,

(ii) Cleaving of the linear peptide Vasopressin precursor (3) from the resin,
(iii) Isolation of the linear crude peptide,
(iv) Cyclization by disulfide bond formation in the presence of H2O2 and DMSO as solvent at low dilution ratio,
(v) Isolation and purification of the final product of Vasopressin (1).
2. An improved process for the preparation of Vasopressin (1),

wherein the process comprises of:
(i) Synthesis of a linear peptide Vasopressin precursor (3) by a Solid phase peptide synthesis (SPPS) in the presence of a resin,

(ii) Cleaving of the linear peptide Vasopressin precursor (3) from the resin,
(iii) Isolation of the linear crude peptide in the presence of antisolvent,
(iv) Cyclization by disulfide bond formation in the presence of H2O2 and DMSO as solvent at low dilution ratio,
(v) Isolation and purification of the final product of Vasopressin (1) in the presence of antisolvent.
3. An improved process for the preparation of Vasopressin (1) according to claims 1 or 2, wherein the resin is rink amide resin.
4. An improved process for the preparation of Vasopressin (1) according to claim 1 or 2, wherein the linear crude peptide is isolated in the presence of 90-97% TFA, 1.5-3% TIS,1.5-3% water as a TFA salt.
5. An improved process for the preparation of Vasopressin (1) according to claim 1 or 2 involves the reaction conditions, wherein the linear peptide (Vasopressin Precursor) is dissolved in 4-8 volumes of DMSO and 3-6 equivalents of H2O2 and DMSO at room temperature.
6. An improved process for the preparation of Vasopressin (1) according to the preceding claims, wherein the H2O2 reduces the dilution ratio to 2.5 to 7.5ml/gm.
7. An improved process for the preparation of Vasopressin (1) according to claim 2, wherein the antisolvent used is IPA and MTBE, preferably in the ratio of 1:2.
8. An improved process for the preparation of Vasopressin (1), wherein the process comprises of the use of antisolvent during the isolation and purification process.
9. An improved process for the preparation of Vasopressin (1) according to the preceding claims, wherein the purity of Vasopressin (1) is >98%.
10. An improved process for the preparation of Vasopressin (1) according to claim 1 or 2 can proceed by one pot synthesis.