DESC:TECHNICAL FIELD OF THE INVENTION:
The present invention relates to a process for the convergent solid peptide synthesis of peptides having alanine in their sequence at strategic positions, via reduced racemization levels on C-termini of peptide fragments during their fragment condensation, wherein the process comprises a final coupling step in which at least two fragments are coupled at a C-terminal Ala residue via fragment condensation.

More particularly, the present invention relates to a process for the convergent solid peptide synthesis of long chain peptides such as NVG-291by fragment condensation.

BACKGROUND AND PRIOR ART OF THE INVENTION:
Peptides are on their way to becoming valuable players as possible diagnostics and therapeutics in the pharmaceutical industry as well as the dietary supplement industry. The global bioactive protein and peptides market is projected to reach around US$ 88.3 billion by the end of 2027, in terms of revenue, growing at a compound annual growth rate (CAGR) of 8.2% during the forecast period 2020-2027 (https://www.coherentmarketinsights.com/insight/request-sample/3402).It is thus obvious that peptides show a great promise as therapeutic agents.

Peptides have a relatively large molecular weight with physicochemical characteristics, requiring complexity in the process of synthesis due to the building-blocks system which may yield related by-products that are likely to be biologically active. Thus, there is a valid and growing concern over the presence of impurities in active pharmaceutical ingredients (APIs) and finished drug products (FDPs).Impurity profiling, i.e. the identity as well as the quantity of impurities in the pharmaceutical drug is now gaining critical attention from regulatory authorities in order to assure and control the quality and efficacy of the finished product.

Convergent solution and solid phase synthesis methods are the most favoured strategies for large scale production of peptides. In these methods, the peptides are constructed by the systematic joining together of fully protected fragments that have been previously assembled by solution or solid phase methods.

Fragment condensation both by the conventional and solid-phase methods is known to give a product of higher quality in comparison with the stepwise synthesis. In addition, purification of peptides that are prepared by the fragment condensation is considerably easier.

Whilst in the conventionally used step-wise synthesis, racemization or enantiomerization is generally not considered an issue, in fragment condensation the prevention of epimerization is of paramount concern. This is because, in contrast to the urethane-protected amino acids, peptides easily form chirally labile oxazolones upon C-terminal carboxyl activation that participate in amide bond formation (Scheme (A).

Scheme A: Mechanism of peptide epimerization by oxazolone formation

The normal strategy for overcoming this problem is to design the method of synthesis in such a way that, wherever possible, N-terminal fragments are selected which contain either a C-terminal Gly or Pro residue, as the former is achiral and the latter does not readily form oxazolones. However, depending on the peptide fragments this alternative is not always feasible and conducive to be implement in solid phase synthesis. Racemization of optically active amino acid residues on C-termini of peptide fragments are known to be possible during their fragment condensation.

For instance,an unexpectedly high racemization of approximately 20% of a C-terminal alanine residue in a peptide fragment, i.e. peptide sequence of ß1-Adrenoreceptor has been observed when carbodiimide method (DIC/HOBt) in DMSO was used in solid-phase fragment condensation (M. V. Sidorovaet al.,Russian Journal of Bioorganic Chemistryvol 43, 51–358(2017)). It is evident from the stated research study that a major issue with respect to high racemization levels of the C-terminal optically active amino acid residues in Convergent fragment synthesis still persists which is detrimental to the higher yield, purity and efficacy of a finished drug product.

Only the appropriate fragment method involving the C-terminal optically active amino acids can ensure the higher yield and purity of finished drug product, and reduce the generation of impurities.

NVG-291 is an investigational peptide that has shown an ability to stimulate nerve regeneration following an injury in several animal models of disease. It works by inhibiting the activity of the protein tyrosine phosphatase sigma (PTPs), a neural receptor that blocks nerve regeneration following tissue damage.

The amino acid peptide sequence of NVG-291 is as follows:
H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Asp-Met-Ala-Glu-His-Thr-Glu-Arg-Leu-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu-Ser-Gln-Glu-Tyr-Glu-Ser-Ile-NH2

NVG-291 is reported to be manufactured using well established peptide synthesis procedures however new synthetic routes for NVG-291 are still required.

NVG-291 is a polypeptide consisting of 35 amino acids. From the perspective of linear solid phase synthesis, the peptide chain is longer. It is more difficult to couple the amino acid residues stepwise with the extension of the long peptide chain due to intermolecular hydrophobic aggregation of the protected peptide chains. Also it is more prone to generate process impurities which are not favourable to the later stage purification.

US patent application US20080242836 discloses a process for preparing a peptide by solid-phase synthesis comprising combining a sequence including one or more amino acids obtainable by C-N synthesis linked to a first resin, with an amino acid sequence including one or more amino acid obtainable by N-C synthesis linked to a second resin so as to create a native peptide link between unprotected N and unprotected C terminals of said amino acid sequences.

European patent No EP0518655 discloses a solid-phase synthesis using a linear strategy starting with the coupling on a peptidyl-resin derivatized with the bi-functional spacer (Rink amide) of AzaGly with no protection for the Tyr and Ser side chains at the 4th position. This process requires that the final peptide be treated with hydrazine to hydrolyze possible side products with acylated amino-acid side chains which are incorporated in free form. This procedure provides an overall yield after purification of over 30% impurity.

European patent EP0475184 discloses a method for the synthesis of peptides including goserelin based on the synthesis in the solution of Boc-Pro-AzaGly-Bifunctional spacer which is coupled to a polymeric support. With the peptidyl-resin obtained the synthesis continues following a linear strategy with the Fmoc-t-Bu strategy. The final step consists of deprotecting the side chains with TFA:ethanethiol (90:10, v/v).

To this end, the present inventors propose to synthesize NVG-291 by fragment condensation method. In NVG-291 Sequence, “convenient” (optically inactive or slightly racemizing) amino acid residues like Gly and pro were absent, Alanine is chosen as the C-terminal amino acid for dividing into peptide blocks for convergent strategy. C-Term alanine is expected to be less prone for racemization during the fragment condensation due to less steric hindrance in the side chain of alanine residue.

The use of (14+21) convergent strategy for preparing NVG-291 according to present invention leads to a better purity product with low levels of racemization at C-Term 14 alanine residue.

OBJECTS OF THE INVENTION
It is an object of the present invention to provide a process for the solid phase synthesis of long chain peptides having alanine in their sequence at strategic positions via fragment condensation wherein the process comprises a final coupling step in which at least two fragments are coupled at a C-terminal Ala residue via fragment condensation.

It is another object of the present invention to provide an improved process for the preparation of NVG-291 (an investigational peptide) by coupling of appropriate fragments in a required sequence by convergent fragment condensation approach.

SUMMARY OF THE INVENTION
The present invention provides a process for convergent solid peptide synthesis of long chain peptides having alanine in their sequence at strategic positions(comprises at least one non terminal Ala residue, wherein the process comprises a final coupling step in which at least two fragments are coupled at a C-terminal Ala residue via fragment condensation.

The present invention particularly provides an improved process for the preparation of NVG-291 (an investigational peptide) of formula 1, by convergent fragment condensation approach.
H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Asp-Met-Ala-Glu-His-Thr-Glu-Arg-Leu-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu-Ser-Gln-Glu-Tyr-Glu-Ser-Ile-NH2
Formula 1
which comprises the following steps,
a) Synthesis of suitable fragments (protected) by solid phase peptide synthesis;
• Fmoc-1Gly-Arg(Z)-Lys(Y)-Lys(Y)-Arg(Z)-Arg(Z)-Gln-Arg(Z)-Arg(Z)-Arg(Z)-11Cys(Trt)-Asp(X)-13Met-Ala-OH
FRAGMENT A
• H-15Glu(X)-His(Trt)-Thr(X)-Glu(X)-Arg(Z)-Leu-Lys(Y)-Ala-Asn-Asp(X)-Ser(X)-Leu-Lys(Y)-Leu-Ser(?-Me,Mepro)-Gln-Glu(X)-Tyr(X)-Glu(X)-Ser(X)-Ile-Rink Amide Resin
FRAGMENT B
b) Coupling of the fragments on solid support;
c) Concurrent cleavage from the solid support and deprotection of the peptide.
d) Purification of NVG-291 (Crude) on reverse phase HPLC;
e) Isolating pure NVG-291.
wherein Y represents amino protected group of € amino group of Lys, X represents carbonyl, phenol and alcoholic protecting group, Z represents guanidine protecting group and ?-Me,Mepro represent pseudoproline protection.

DETAILED DESCRIPTION OF DRAWINGS:
The invention has other advantages and features which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:
Figure 1 depicts the synthesis of Fragment A [Protected Fragment (1-14)].
Figure 2 depicts the synthesis of NVG-291 on Rink amide MBHA resin was done according to scheme presented therein.
Figure 3depicts the HPLC chromatogram of Protected Fragment (1-14).
Figure 4 depicts the HPLC chromatogram of crude Fragment (15-35)-NH2.
Figure 5 depicts the HPLC chromatogram of Crude NVG-291.
Figure 6 depicts the HPLC chromatogram of Crude 14-D-Ala-NVG-291 impurity.
Figure 7depicts a comparison of Crude NVG-291 and 14-D-Ala-NVG-291 impurity.
Figure 8 depicts mass of NVG-291.
Figure 9 depicts a comparison of crude NVG-291 synthesis using both convergent and linear strategies.
Figure 10 depicts the HPLC chromatogram of Crude NVG-291-010620 synthesised via convergent strategy.
Figure 11 depicts the HPLC chromatogram of Crude NVG-291-010620 synthesised via linear strategy.
Figure 12 depicts a comparison of Crude NVG-291 via convergent and 14-D-Ala NVG-291 Impurity:

Abbreviations:
The specific meanings of the abbreviations used in the present invention are listed below:
ACN: acetonitrile
Ala: alanine
Arg: arginine
Asn: asparagine
Asp: aspartic acid.
Y= Boc: t-butyloxycarbonyl
CTC: Chlorotrityl chloride
DCM; dichloromethane
DIC: Diisopropylcarbodiimide
DIPEA: Diisopropylethylamine
DMF: N,N-dimethylformamide
DTT: dithiothreitol
EDT: 1,2-ethanedithiol
eq equivalents;
Fmoc: 9-fluorenylmethoxycarbonyl
Gln: glutamine
Glu: glutamic acid
Gly: glycine
His: histidine
HOBt: N-hydroxybenzotriazole
HPLC: high performance liquid chromatography
Ile: isoleucine
Leu: leucine
Lys: lysine
MBHA resin: Methylbenzylhydrylamine resin
MeOH: methanol
X= OtBu: t-butyl ester
Z= Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl
Phe: phenylalanine
RT room or ambient temperature;
Ser: serine
SPPS Solid Phase Peptide Synthesis;
tBu: t-butyl
TFA: trifluoroacetic acid
THF: Tetrahydrofuran
TIPS, TIS: triisopropylsilane
Trp: tryptophan
Trt: trityl or triphenylmethyl
Tyr: tyrosine
Val: valine

DETAILED DESCRIPTION OF THE INVENTION
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.

The present invention provides a convergent process on solid phase for long chain peptides having Alanine in their sequence at strategic positions, wherein the process comprises a final coupling step in which at least two fragments are coupled at a C-terminal Ala residue via fragment condensation.

Accordingly, the C-Terminal ala residue is chosen to obtain less racemization levels during fragment condensation of fragments on solid phase. The said long chain peptide therefore comprises at least one non terminal Ala residue. By non-terminal Ala residue, it is meant that the long chain peptide should contain at least one Ala reside that is not at the N term or the C-term of Peptide. Nevertheless, the long chain peptide may, in addition to the non-terminal Ala residue, contain an ala residue at N- and/or at C- terminus.

The present convergent solid-phase peptide synthesis (CSPPS) process for the synthesis of long chain peptides comprising the following process steps:
(i) solid-phase synthesis of suitable protected peptides;
wherein one part of the protected peptide sequence contains the C-Terminal ala residue yielded by solid phase synthesison acid labile resin is coupled with another amino acid sequence yielded by solid phase synthesis supported on a second resin so as to provide a native peptide link between unprotected NH2 and unprotected C terminals of said amino acid sequences,
(ii) characterization of the peptides fragments;
(iii) Coupling of protected peptide fragments on solid-support;
(iv) Concurrent cleavage from the solid support and de-protection of the peptide;
(v) Purification of Crude peptide by reverse phase HPLC;
(vi) Isolation of Pure peptide.

In a preferred embodiment, the present invention provides a process for convergent solid peptide synthesis of NVG-291, following a convergent process constituting a 14 + 21 strategy.

NVG-291 peptide also contains a non-terminal Ala residue as the 14th residue from the N-terminus i.e14Ala.

H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Asp-Met-14Ala-Glu-His-Thr-Glu-Arg-Leu-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu-Ser-Gln-Glu-Tyr-Glu-Ser-Ile-NH2

In accordance with the process of the present invention, this 14Ala residue in NVG-291 enable convenient chemical ligation to form the NVG-291 peptide via fragment condensation of fragments (1-14) on (15-35) on solid phase with minimum racemization on C-terminal 14Ala. The extent of racemization at C-terminal 14Ala residue is estimated by synthesizing separately 14-D-Ala NVG-291 impurity by same 14+21 convergent strategies and its comparison with crude NVG-291 on HPLC.

In another preferred embodiment, the present invention provides a convergent solid-phase peptide synthesis (CSPPS) process for the synthesis of NVG-291 comprising the following process steps:
(i) solid-phase synthesis of protected peptides in a particular combination so that the final coupling step involves the coupling of two fragments at C-terminal 14-Ala residue for (14+21) fragment condensation,

Fmoc-1Gly-Arg(Z)-Lys(Y)-Lys(Y)-Arg(Z)-Arg(Z)-Gln-Arg(Z)-Arg(Z)-Arg(Z)-11Cys(Trt)-Asp(X)-13Met-Ala-OH
FRAGMENT A

H-15Glu(X)-His(Trt)-Thr(X)-Glu(X)-Arg(Z)-Leu-Lys(Y)-Ala-Asn-Asp(X)-Ser(X)-Leu-Lys(Y)-Leu-Ser(?-Me,Mepro)-Gln-Glu(X)-Tyr(X)-Glu(X)-Ser(X)-Ile-Rink Amide Resin
FRAGMENT B
wherein one protected Fragment (1-14) [Fragment A] of NVG-291 yielded by solid phase synthesis on an acid labile resin is combined with protected fragment (15-35) ) [Fragment B] yielded by solid phase synthesis supported on a second resin so as to provide a native peptide link between unprotected NH2 and unprotected C terminals of said amino acid sequences,
(ii) characterization of the protected peptides;
(iii) coupling of the fragments on solid support;
(iv) concurrent cleavage from the solid support and deprotection of the peptide to obtain NVG-291 Crude;
(v) purifying NVG-291 (Crude) on reverse phase HPLC;
(vi) isolating pure NVG-291.

In an embodiment, for SPPS the protecting group is selected from the group consisting of traditional Fmoc/tBu protection or Boc/benzyl protection. Other protecting groups such as Cbz, Bpoc could also be used as amino protecting group. More preferably, the protecting group for the amino acid/dipeptide pseudoproline unit is Fmoc.

In another embodiment of the present invention the carboxyl, phenolic and alcoholic groups are protected with groups selected from but not limited to a group comprising of DMT, MMT, Trt, t-butyl, t-butoxy carbonyl, and the like.

In still another embodiment of the present invention, the guanidine protecting groups are selected from but not limited to a group comprising of Pbf and pmc.

In yet another embodiment of the present invention, the amino protecting groups of € amino group of Lys are selected from but not limited to a group comprising of Dde, Aloc, Mtt, Cbz and boc.

In an embodiment of the present invention the solid support is a resin, selected from the group comprising 4-methylbenzhydrylamine resin (MBHA), Rink amide BHA resin or Sieber resin and 2-Chlorotrityl resin (CTC).

In another embodiment, the resin used is an acid sensitive resin, selected from the group comprising 2-Chlorotrityl resin (CTC), sasrin, Wang resin, 4-methyl trityl chloride, Tentagel S, Tentagel TGA, Rink acid resin, most probably 2-Chlorotrityl resins.

In an embodiment of the present invention the coupling agents in the process is selected from the group comprising hydroxybenzotriazole (HOBt), N, N'- diisopropylcarbodiimide (DIC), O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate (TBTU), N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU), 1,3-dicyclohexylcarbodlimide (DCC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphoniumhexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), benzotriazol-1-yloxytri(pyrrolidino)phosphoniumhexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphoniumhexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphoniumhexafluorophosphate (PyClOP), ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), 245-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) or mixtures thereof.

In an embodiment of the present invention the coupling takes place in presence of a solvent selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1 ,4- dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetone, and the like or a mixtures thereof.

Further, Fmoc is removed from the peptide in the presence of an organic base prepared in an organic solvent. The organic base is selected from the group comprising piperidine, piperazine, N-methyl morpholine, diethyl amine, triethyl amine, 1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU) and the like or a mixtures thereof.

In another embodiment of the present invention, the organic solvent is selected from the group comprising dimethyl formamide (DMF), N-Methyl-2-Pyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAC) and the like or a mixture of the listed solvents.

In an embodiment, the present process for solid phase synthesis provides deprotection of the peptide using a combination of Trifluoroacetic acid (TFA) and radical scavengers.

Accordingly, one or more radical scavengers are selected from the group comprising triisopropylsilane (TIS), dithiothreitol (DTT), 1,2-ethanedithiol (EDT), Phenol, cresol, thioanisole, ammonium iodide, DMS and water.

Simultaneous deprotection of all the protecting groups was carried out by the treatment of either of the cocktail cleavage mixtures stated below: TFA:EDT:Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) or TFA:TIS:DTT:Water (85:5:5:5 v/v) preferably with TFA:EDT:Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) cocktail mixture i.e. K type reagent.

In an embodiment of the present invention, a process for the preparation of NVG-291 comprising fragments “A” wherein, Fragment A is synthesized on solid phase.

In another embodiment of the present invention, a process for the preparation of NVG-291 comprising fragments “B” wherein, Fragment B is synthesized on solid phase.

In still another embodiment of the present invention, a process for the preparation of NVG-291 precursor peptide on solid phase by solid phase synthesis is described (Figure 2):

Fmoc-1Gly-Arg(Z)-Lys(Y)-Lys(Y)-Arg(Z)-Arg(Z)-Gln-Arg(Z)-Arg(Z)-Arg(Z)10-11Cys(Trt)-Asp(X)-13Met-Ala-15Glu(X)-His(Trt)-Thr(X)-Glu(X)-Arg(Z)-Leu-Lys(Y)-Ala-Asn-Asp(X)-Ser(X)-Leu-Lys(Y)-Leu-Ser(?Me,Mepro)-Gln-Glu(X)-Tyr(X)-Glu(X)-Ser(X)-Ile- Resin

In yet another embodiment of the present invention, cleavage and de-blocking of NVG-291 from solid support is described.

In an embodiment of the present invention, for finding a viable synthetic route, two different synthetic strategies are used.

In another embodiment of the present invention, the synthetic strategies include but not limited to linear SPPS synthesis using pseudoprolines and convergent strategy (14+21) on resin.

In still another embodiment of the present invention, the synthetic strategy is of convergent strategy (14+21) on resin.

The linear SPPS synthesis using pseudoprolines include steps of:-

H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg10-Cys-Asp-Met-Ala-Glu-His-Thr-Glu-Arg-Leu20-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu28-Ser29-Gln30-Glu-Tyr-Glu-Ser-Ile-NH2

H-Gly-Arg(pbf)-Lys(boc)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(Trt)-Asp(tbu)-Met-Ala-Glu(tbu)-His(Trt)-Thr(tbu)-Glu(tbu)-Arg(pbf)-Leu-Lys(boc)-Ala-Asn-Asp(tbu)-Ser(tbu)-Leu-Lys(boc)-Leu-Ser(?Me,Mepro)-Gln-Glu(tbu)-Tyr(tbu)-Glu(tbu)-Ser(tbu)-Ile-Rink Amide Resin

Accordingly, the linear route afforded very poor crude quality with very low area as shown in Figure 9.

In an embodiment of the present invention, the convergent (14+21) on resin strategy for NVG-291 include a process of:-

H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg10-Cys-Asp-Met-Ala-Glu-His-Thr-Glu-Arg-Leu20-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu28-Ser29-Gln30-Glu-Tyr-Glu-Ser-Ile-NH2

H-Gly-Arg(pbf)-Lys(boc)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(Trt)-Asp(tbu)-Met-Ala-Glu(tbu)-His(Trt)-Thr(tbu)-Glu(tbu)-Arg(pbf)-Leu-Lys(boc)-Ala-Asn-Asp(tbu)-Ser(tbu)-Leu-Lys(boc)-Leu-Ser(?Me,Mepro)-Gln-Glu(tbu)-Tyr(tbu)-Glu(tbu)-Ser(tbu)-Ile-Rink Amide Resin

Fmoc-1Gly-Arg(pbf)-Lys(boc)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)10-11Cys(Trt)-Asp(tbu)-13Met-14Ala-OH[Fragment (1-14) SPPS on CTC]
+
H-15Glu(tbu)-His(Trt)-Thr(tbu)-Glu(tbu)-Arg(pbf)-Leu-Lys(boc)-Ala-Asn-Asp(tbu)-Ser(tbu)-Leu-Lys(boc)-Leu-Ser(?Me,Mepro)-Gln-Glu(tbu)-Tyr(tbu)-Glu(tbu)-Ser(tbu)-Ile-Rink Amide Resin [Fragment (15-35) SPPS on Rink Amide]
[Coupling of two fragments on Resin]

In another embodiment of the present invention, the convergent (14+21) on resin strategy for NVG-291 is done via fragment coupling of protected fragments (1-14) on (15-35)-Rink amide MBHA Resin.

In still another embodiment of the present invention, coupling of fragments (1-14) + (15-35)-Rink amide was accomplished with low racemisation at 14-Ala position, also the formed 14-D-Ala NVG impurity is well separable from NVG-291 crude.

In yet another embodiment of the present invention, the process of synthesis of crude NVG-291 via convergent strategy is much better as compare to crude NVG-291 via linear strategy. (shown in Figure 9, 10 and 11).

In another embodiment of the present invention, the area and purity of Crude NVG-291 synthesised via convergent strategy is much better as compare to synthesis of crude NVG-291 via linear strategy.

In another embodiment of the present invention, convergent strategy synthesis crude NVG-291 with a purity and area of 59% and 3376909 respectively (shown in Figure 9).

In another embodiment of the present invention, linear strategy synthesis crude NVG-291 with a purity and area of 43% and 1093574 respectively (shown in Figure 9).

In another embodiment of the present invention, the level of racemization in crude NVG-291 via convergent stratergy is low compared to14-D-Ala NVG-291 impurity (shown in Figure 12).

Examples: Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1 Synthesis of NVG-291.Synthesis of NVG-291 on Rink amide MBHA resin was done according to Scheme 1:

Synthesis of Fmoc-1Gly-Arg(pbf)-Lys(boc)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(Trt)-Asp(tbu)-Met-14Ala-OH on CTC resin (Fragment A)

Stage-1: Synthesis of Fmoc–Ala14–CTC Resin:
Fmoc-Ala-OH (35.2 mm, 1.76 eq.) is dissolved in DCM (200 ml) in a beaker, added DIEA (30mm, 1.5eq.) and stirred for 5 min.
Meanwhile 25 gm CTC resin (40 mm, f = 1.6) was taken in a cartel bottle, added the Fmoc-Ala-OH solution in it and stirred for 2 min followed by addition of DIEA (60mm, 3 eq.) in dry Dichloromethane (10 ml) and stirred for 40 min at room temperature, after 40 min the resin was capped with ml of Methanol (20 ml) for 20 min and drained. Thereafter, transfer the whole contents of resin in a SPPS vessel and washed the resin with of DCM:DIEA:MeOH (85:5:5) (6 times) followed by DMF (10 times).

Stage-2: Synthesis of Fmoc-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid (Fmoc–Ala14–CTC Resin) was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Met-OH (60mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 min at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-3: Synthesis of Fmoc-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid i.e. (Fmoc–Met13- Ala14–CTC Resin) was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Asp(tbu)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-4: Synthesis of Fmoc-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid i.e. Fmoc-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Cys(trt)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-5: Synthesis of Fmoc-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid i.e. Fmoc-Cys(Trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-6: Synthesis of Fmoc- Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid Fmoc-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH dipeptide (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-7: Synthesis of Fmoc- Arg(pbf)8-Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid Fmoc-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-8: Synthesis of Fmoc-Gln7-Arg(pbf)8-Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid Fmoc- Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gln-OH (60 mm, 3eq.) was coupled using HOBt (120 mm, 6 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-9: Synthesis of Fmoc-Arg(pbf)6-Gln7-Arg(pbf)8-Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid Fmoc-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-10: Synthesis of Fmoc-Arg(pbf)5-Arg(pbf)6-Gln7-Arg(pbf)8-Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid Fmoc-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-11: Synthesis of Fmoc-Lys(Boc)4-Arg(pbf)5-Arg(pbf)5-Gln7- Arg(pbf)8Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin: Fmoc-deprotection of the loaded amino acid Fmoc-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Lys(Boc)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-12: Synthesis of Fmoc-Lys(Boc)3-Lys(Boc)4-Arg(pbf)5-Arg(pbf)5-Gln7- Arg(pbf)8Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met 13- Ala14–CTC Resin: Fmoc-deprotection of the loaded amino acid Fmoc-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Lys(Boc)-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-13: Synthesis of Fmoc-Gly1-Arg(pbf)2-Lys(Boc)3-Lys(Boc)4-Arg(pbf)5-Arg(pbf)5-Gln7-Arg(pbf)8Arg(pbf)9-Arg(pbf)10-Cys(trt)11-Asp(tbu)12-Met13- Ala14–CTC Resin:
Fmoc-deprotection of the loaded amino acid Fmoc-Arg(pbf)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC Resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gly-OH (60 mm, 3eq.) was coupled using HOBt (60 mm, 3 eq) and DIC (60 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
Yield = 112.5 gm

Stage-14: Selective cleavage of 2-Chlorotrityl resin:
From the Fmoc-Gly-Arg(pbf)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–CTC was performed with the mixture of 20% TFE in DCM. The above peptidyl was taken in a RBF and treated with 20% TFE-DCM (15 ml/gm of total resin) for 3 hrs at RT. After 3 Hrs the resin is filtered and filtrate was concentrated under reduced pressure followed by precipitating in diethyl ether.
Yield = 84 gm (103%)
Purity = 94%
HPLC chromatogram of Protected Fragment (1-14) is depicted in FIG 3.

Example 2: Preparation method on resin of NVG-291 fragment (15-35) on resin:
H-15Glu(tbu)-His(Trt)-Thr(tbu)-Glu(tbu)-Arg(pbf)-Leu-Lys(boc)-Ala-Asn-Asp(tbu)-Ser(tbu)-Leu-Lys(boc)-Leu-Ser(?Me,Mepro)-Gln-Glu(tbu)-Tyr(tbu)-Glu(tbu)-Ser(tbu)-Ile-Rink Amide Resin

Stage-1: Synthesis of Fmoc–IIe35–Rink amide MBHA Resin:
Fmoc-Rink amide MBHA resin with a Loading of ~0.33 mmol/gram (about 30.3 gm resin, 10 mmol) was swelled in DMF for 30 mins by agitation under nitrogen, decanting the solvent, washing the resin twice by using DMF. Fmoc-deprotection of the Rink amide resin was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The coupling of the first amino acid Fmoc-Ala-OH (10 mm, 2.5 eq), was carried out by addition of HOBt (10 mm, 2.5 eq) and DIC (10mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-2: Synthesis of Fmoc-Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ser(tbu)-OH (10mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-3: Synthesis of Fmoc-Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Glu (tbu)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-4: Synthesis of Fmoc-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Tyr(tbu)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-5: Synthesis of Fmoc- Glu(tbu)31- Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Glu(tbu)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-6: Synthesis of Fmoc- Gln30-Glu(tbu)31- Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gln-OH dipeptide (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-7: Synthesis of Fmoc-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu)34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-Ser(psiMe,Mepro)-OH (10 mm, 2eq.) was coupled using HOBt (10 mm, 2 eq) and DIC (10 mm, 2 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-8: Synthesis of Fmoc-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Lys(boc)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-9: Synthesis of Fmoc-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-10: Synthesis of Fmoc-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ser(tbu)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-11: Synthesis of Fmoc-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Asp(tbu)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-12: Synthesis of Fmoc-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Asn-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.
Stage-13: Synthesis of Fmoc-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Ala-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-14: Synthesis of Fmoc-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32- Glu(tbu)33- Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Lys(Boc)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-15: Synthesis of Fmoc-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Leu-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-16: Synthesis of Fmoc-Arg(pbf)19-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Arg(pbf)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-17: Synthesis of Fmoc-Glu(tbu)18-Arg(pbf)19-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu) 34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Glu(tbu)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-18: Synthesis of Fmoc-Thr(tbu)17-Glu(tbu)18-Arg(pbf)19-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu) 34- IIe35–Rink amide MBHA Resin: Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Thr(tbu)-OH (10 mm, 2.5eq.) was coupled using HOBt (10 mm,2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-19: Synthesis of Fmoc-His(trt)16-Thr(tbu)17-Glu(tbu)18-Arg(pbf)19-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu)34-IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-His(trt)-OH (10 mm, 3eq.) was coupled using HOBt (10 mm, 3 eq) and DIC (10 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-20: Synthesis of Fmoc-Glu(tbu)15-His(trt)16-Thr(tbu)17-Glu(tbu)18-Arg(pbf)19-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu)34- IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. Fmoc-Glu(tbu)-OH (10 mm, 3 eq.) was coupled using HOBt (10 mm, 3 eq) and DIC (10 mm, 3 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Stage-21: Synthesis of Fmoc-Glu(tbu)15-His(trt)16-Thr(tbu)17-Glu(tbu)18-Arg(pbf)19-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu)34-IIe35–Rink amide MBHA Resin:
Fmoc-deprotection of the loaded amino acid was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF. The Fmoc-Gly-OH (10 mm, 2.5 eq.) was coupled using HOBt (10 mm, 2.5 eq) and DIC (10 mm, 2.5 eq) in DMF solvent. The mixture was stirred via Nitrogen for 40-50 mins at RT. Upon completion of coupling of the amino acid confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF.

Fmoc-deprotection of the loaded amino acid (0.5 mm, Example 2) was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.

HPLC chromatogram of crude Fragment (15-35)-NH2 is depicted in FIG 4.

Example 3: Synthesis of Protected NVG-291 on resin
Synthesis of Fmoc-Gly-Arg(pbf)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala–Glu(tbu)15-His(trt)16-Thr(tbu)17-Glu(tbu)18-Arg(pbf)19-Leu20-Lys(Boc)21-Ala22-Asn23-Asp(tbu)24-Ser(tbu)25-Leu26-Lys(Boc)27-Leu28-Ser29(psiMe,Mepro)-Gln30-Glu(tbu)31-Tyr(tbu)32-Glu(tbu)33-Ser(tbu)34-IIe35–Rink amide MBHA Resin:
Fmoc-Gly-Arg(pbf)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-Ala-OH (4 gm, 2 eq.) and HOBT (0.3 gm, 4 eq) were dissolved in DMF (15 ml) and cooled to 0±2oC. while cooling, addition of DIC (0.3 ml, 4eq.) is done and stirred for 30 secs and added in to the above peptidyl resin (0.5 mm, Example 2). The mixture was stirred via Nitrogen for overnight at RT. Upon completion of coupling of the fragment is confirmed by Kaiser Test, the excess reagents were drained and washed the peptidyl resin four times by DMF. Further Fmoc-deprotection was carried out by washing the resin using 20 % piperidine in DMF two times for 2 and 10 min, followed by washing the resin four times with DMF.
Weight of the peptidyl resin: 4 gm

Example 4: Preparation of Crude NVG-291
H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-Asp-Met-Ala-Glu-His-Thr-Glu-Arg-Leu-Lys-Ala-Asn-Asp-Ser-Leu-Lys-Leu-Ser-Gln-Glu-Tyr-Glu-Ser-Ile-NH2:
Global deprotection of all the protecting groups and Resin was carried out by the treatment of TFA:TIS:DTT:Water (90:5:2.5:2.5 V/v;w/v). The cleavage was carried out at <10°C for initial 15 minutes followed by the stirring of the peptidyl resin for 3 hours at ambient temperature. The crude cleavage mixture was then filtered, the resin washed thoroughly with TFA. The filtrate was dropped on to 12 mL of cold dry Diethyl ether per mL of cocktail and further 6 additional washing with diethyl ether were done to the product. Product was dried under vacuum for 16 hrs.

The isolated yield of the crude NVG-291 peptide: 1.4 gm
The Purity of the crude NVG-291 peptide: 50%
HPLC chromatogram of Crude NVG-291 is depicted in FIG 5.

Example 5: Determiningthe extent of racemization in crude NVG-291 by comparison of Crude NVG-291 withCrude 14-D-Ala-NVG-291 impurity
To check the presence of racemization in fragment condensation i.e. 14-D-Ala-NVG-291 formed in the convergent coupling of coupling of Protected fragment (1-14) on H-15Glu----35Ile-Rink amide MBHA resin, the present inventors synthesized 14-D-Ala-NVG-291 impurity following the same Convergent (14+21) strategy i.e. by coupling of Fmoc-Gly-Arg(pbf)-Lys(boc)-Arg(pbf)-Arg(pbf)-Gln-Arg(pbf)-Arg(pbf)-Arg(pbf)-Cys(trt)-Asp(tbu)-Met-D-Ala-OH fragment on Glu(tbu)15-His(trt)-Thr(tbu)-Glu(tbu)-Arg(pbf)-Leu-Lys(Boc)-Ala-Asn-Asp(tbu)-Ser(tbu)-Leu-Lys(Boc)-Leu-Ser(psiMe,Mepro)-Gln-Glu(tbu)-Tyr(tbu)-Glu(tbu)-Ser(tbu)-IIe–Rink amide MBHA Resin.

HPLC chromatogram of Crude 14-D-Ala-NVG-291 impurity is depicted in FIG 6.
A comparison of Crude NVG-291 and 14-D-Ala-NVG-291 impurity is depicted in FIG 7.
The mass of the NVG-291 is depicted in FIG.8.

Advantages of the present invention:
• The process of the present invention enable convenient chemical ligation of peptides having alanine in their sequence at strategic position via reduced racemization levels at C-Term ala during fragment condensation.
• High purity of the peptide obtained by the present invention reduces the burden on downstream processing steps.
• The process of the present invention is an added advantage as compared to the conventionally used step wise linear peptide synthesis.

,CLAIMS:
1. A process for the synthesis of NVG-291 by convergent solid-phase peptide synthesis (CSPPS) comprising the steps of:
(i) solid-phase synthesis of suitable protected peptides;
wherein one part of the protected peptide sequence (1-14) contains a C-Terminal Ala residue on acid labile resin, is coupled with another amino acid sequence (15-35) supported on a second resin so as to provide a native peptide link between a unprotected NH2 and the unprotected C terminals of the amino acid sequences,
(ii) coupling of protected peptide fragments on a solid support;
(iii) concurrent cleavage from the solid support and deprotection of the NVG-291 crude;
(iv) purification of the NVG-291 crude by reverse phase HPLC;
(v) isolation of the Pure NVG-291.

2. The process as claimed in Claim 1, wherein the process is a convergent process.

3. The process as claimed in Claim 1, wherein the convergent process constituting a 14 + 21 strategy.

4. The process as claimed in Claim 1, wherein the peptides fragments are fragment A and fragment B respectively.

5. The process as claimed in Claim 1, wherein the peptides fragments are coupled at the C-terminal 14-Ala residue.

6. The process as claimed in Claim 1, wherein the process include condensation of fragments with minimum racemization on C-terminal Ala.

7. The process as claimed in Claim 4, wherein the fragment A is
Fmoc-1Gly-Arg(Z)-Lys(Y)-Lys(Y)-Arg(Z)-Arg(Z)-Gln-Arg(Z)-Arg(Z)-Arg(Z)-11Cys(Trt)-Asp(X)-13Met-Ala-OH

8. The process as claimed in Claim 4, wherein the fragment B is
H-15Glu(X)-His(Trt)-Thr(X)-Glu(X)-Arg(Z)-Leu-Lys(Y)-Ala-Asn-Asp(X)-Ser(X)-Leu-Lys(Y)-Leu-Ser(?-Me,Mepro)-Gln-Glu(X)-Tyr(X)-Glu(X)-Ser(X)-Ile-Rink Amide Resin

9. The process as claimed in Claim 7 and 8, wherein the Y include but not limited to amino protected group of € amino groups of Lys.

10. The process as claimed in Claim 7 and 8, wherein the X include but not limited to carbonyl, phenol and alcoholic protecting group.

11. The process as claimed in Claim 7 and 8, wherein the Z include but not limited to guanidine protecting groups.

12. The process as claimed in Claim 8, wherein the ?-Me,Mepro include but not limited to pseudoproline protection groups.

13. The process as claimed in Claim 9, wherein the amino acid protecting group is selected from Fmoc/tBu protection, Boc/benzyl protection, Cbz protection or Bpoc protection.

14. The process as claimed in Claim 10, wherein the carbonyl, phenolic and alcoholic groups are protected with groups selected from the group comprising DMT, MMT, Trt, t-butyl, or t-butoxy carbonyl.

15. The process as claimed in Claim 11, wherein the guanidine protecting groups are selected from a group comprising Pbf and pmc.

16. The process as claimed in Claim 13, wherein the protecting group for the amino acid/dipeptide pseudoproline unit is Fmoc.

17. The process as claimed in Claim 4, wherein Fragment A and Fragment B are synthesized on a solid phase.

18. The process as claimed in Claim 17, wherein the solid support is selected from the group comprising 4-methylbenzhydrylamine resin (MBHA), Rink amide BHA resin or Sieber resin and 2-Chlorotrityl resin (CTC).

19. The process as claimed in Claim 1, wherein the coupling of the peptide fragments on solid-support are done using coupling agents.

20. The process as claimed in Claim 19, wherein the coupling agent is selected from the group comprising hydroxybenzotriazole (HOBt), N, N'- diisopropylcarbodiimide (DIC), O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate (TBTU), N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU), 1,3-dicyclohexyl carbodlimide (DCC), 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl), benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphoniumhexafluorophosphate (BOP), N,N-bis-(2-oxo-3-oxazolidinyl)phosphonic dichloride (BOP-C1), benzotriazol-1-yloxytri(pyrrolidino)phosphoniumhexafluorophosphate (PyBOP), bromotri(pyrrolidino)phosphoniumhexafluorophosphate (PyBrOP), chlorotri(pynolidino)phosphoniumhexafluorophosphate (PyClOP), ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma Pure), O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TCTU), 245-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), 2-succinimido-1,1,3,3-tetramethyluronium tetrafluoro borate (TSTU), 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT)or mixtures thereof.

21. The process as claimed in Claim 1, wherein the coupling takes place in presence of a solvent selected from the group comprising of DMF, DCM, THF, NMP, DMAC methanol, ethanol, isopropanol, dichloroethane, 1 ,4- dioxane, 2-methyl tetrahydrofuran ethyl acetate, acetonitrile, acetoneand the like or a mixtures thereof.

22. The process as claimed in Claim 7, wherein Fmoc is removed from the peptide in the presence of an organic base prepared in an organic solvent.

23. The process as claimed in Claim 22, wherein the organic base is selected from the group comprising piperidine, piperazine, N-methyl morpholine, diethyl amine, triethyl amine, 1,8-Diazabicyclo [5.4.0]undec-7-ene (DBU) and the like or a mixtures thereof.

24. The process as claimed in Claim 22, wherein the organic solvent is selected from the group comprising dimethyl formamide (DMF), N-Methyl-2-Pyrrolidone (NMP), dichloromethane (DCM), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAC) and the like or a mixture of the listed solvents.

25. The process as claimed in Claim 1, wherein the deprotection of the peptide is carried out in the presence of a combination of Trifluoroacetic acid (TFA) and radical scavengers.

26. The process as claimed in Claim 25, wherein the radical scavengers are selected from the group comprising triisopropylsilane (TIS), dithiothreitol (DTT), 1,2-ethanedithiol (EDT), Phenol, cresol, thioanisole, ammonium iodide, DMS and water.

27. The process as claimed in Claim 1, wherein the simultaneous deprotection of all the protecting groups is carried out by the treatment of either of the cocktail cleavage mixtures stated below: TFA:EDT:Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) or TFA:TIS:DTT:Water (85:5:5:5 v/v) preferably with TFA:EDT:Phenol:Thioanisole:water (82.5:2.5:5:5:5 v/v) cocktail mixture i.e. K type reagent.