WO2011095989A2

WO2011095989A2 - An improved process for the preparation of enfuvirtide

Info

Publication number: WO2011095989A2
Application number: PCT/IN2011/000074
Authority: WO
Inventors: Ananda Kuppanna; Maria Bhaskar Reddy Komma Reddy; Debashish Datta
Original assignee: Matrix Laboratories Ltd
Priority date: 2010-02-04
Filing date: 2011-02-02
Publication date: 2011-08-11
Also published as: WO2011095989A3

Abstract

The present invention relates to a novel process for the synthesis of Enfuvirtide or pharmaceutically acceptable salts by using a hybrid approach. The fragments are synthesized by solid phase synthesis approach and the Enfuvirtide is synthesized by making use of the fragments in solution phase.

Description

AN IMPROVED PROCESS FOR THE PREPARATION OF ENFUVIRTIDE"

This application claims priority to Indian patent application 283/CHE/2010 filed on Feb 04, 2010; the contents of which are incorporated by reference in their entirety. Field of the Invention:

The present invention relates to a novel process for the synthesis of Enfuvirtide or pharmaceutically acceptable salt by using a solid and solution phase (hybrid) approach.

Background of the Invention:

Enfuvirtide (also referred to as "DP-178"; SEQ ID NO: 1 or Fuzeon or T-20), a novel anti- human immunodeficiency virus (HIV) drug, is a peptide derived from HIV-1 envelope protein gp41 C-terminal heptad repeat (CHR). This drug is a natural interfacial sequence taken from the gp41 moiety of the HIV precursor protein gp160 and represents the first case of a peptide drug derived from a protein fragment. Enfuvirtide is the first Fusion Inhibitor, as well as the first HIV entry inhibitor. Its mechanism of action is different from existing Anti HIV drugs in that the existing antiretroviral work inside T-cells to stop virus replication, but Enfuvirtide works extracellularly where it blocks Fusion between the membrane of the host immune cell T-lymphocyte and the HIV-1 virus, a late stage of the entry process. This unique function gives fusion inhibitors the potential to treat multiple-class resistant stains of HIV. Enfuvirtide consisting of 36 amino acids and is very challenging to make on a large scale synthetically and thus represents a landmark in the industrial peptide chemistry. The sequence is as follows: CH₃CO-Tyr-Thr-Ser-Leu-lle-His-Ser-Leu-lle-Glu-Glu-Ser-Gln-Asn- Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp- Phe-NH₂ (Ac-YTSLIHSLIEESQNQQEKN-EQELLELDKWASLWNWF (SEQ ID NO:1)) and the following structural formula:

Peptides are synthesized by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. The possibility of unintended reactions is obvious; therefore protecting groups are usually necessary. Chemical peptide synthesis starts at the C-terminal end of the peptide and ends at the N-terminus. This is the opposite of protein biosynthesis, which starts at the N-terminal end.

Many methods for peptide synthesis are described in the literature. In solid phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield, an amino acid or peptide group is bound to a solid support resin. Then the successive amino acids are added to the solid support until the peptide material of interest is formed. The support bound peptide is then typically cleaved from the support and subject to further processing. In some cases, the solid phase synthesis yields a final peptide of interest directly, in other cases the peptide cleaved from the support (is a peptide intermediate fragment) which is used in solution synthesis to build a longer sequences. As solid phase peptide synthesis techniques improved the rate at which a peptide could be synthesized, purification became the limiting factor in the production of high quality peptides. Further, longer peptides eventually may adopt an irregular conformation while still attached to the solid support, accordingly resulting in partial or entire loss of activity in the final product. Also, as the peptide chain becomes longer on the support resin, the efficiency of process steps such as coupling and deprotection may be compromised. This, in turn, can result in longer processing times to compensate for above problems, in addition to incremental losses in starting materials, such as co-reagents, and solvents. These problems can increase as the length of the peptide increases and therefore, it is relatively uncommon to find mature peptides of greater than 30 amino acids in length synthesized using only a solid phase procedure.

Instead, individual fragments may be separately synthesized on the solid phase and then coupled in solution phase to build the desired peptide. This approach requires careful selection of the peptide fragments. Thus, before starting the synthesis, the best suited fragments must be carefully selected and tested. In general protected fragments of any length can be applied, if these are of satisfying purity and solubility and fulfill a high condensation efficiency acting as C-component as well as reactive N-component to enable the subsequent condensation proceed efficiently. The resin bound C-terminal fragment is probably the most important for the success of the synthesis. While some general principles can guide fragment selection, quite often empirical testing of fragment candidates is required. Proper selection of chemical strategies is necessary for the hybrid approach as there are significant pitfalls due to poor solubility of fully protected fragments and due to the ease of epimerization in solution phase couplings. In solution phase coupling, two peptide intermediate fragments are arranged so that the N- terminal of one fragment coupled to C-terminal of other fragment, or vice versa. In addition, the side chain protecting groups which are present during solid phase peptide synthesis are retained on the fragments during the solution phase coupling to ensure the specific reactivity of the terminal ends of the fragments. Solution phase synthesis can be particularly useful in cases where the synthesis of a useful mature peptide by solid phase is either impossible or not practical.

Enfuvirtide was first disclosed in US 5464933. This patent disclosed that amidated peptides were prepared using Rink resin (Advanced Chemtech) while peptides containing free carboxy termini were synthesized on Wang (p-alkoxybenzylalcohol) resin (Bachem). First residues were double coupled to the appropriate resin and subsequent residues were single coupled. Peptides were cleaved from the resin by treatment with trifluoroacetic acid (TFA) (10 ml), H₂0 (0.5 ml), thioanisole (0.5 ml), ethanedithiol (0.25 ml) and crystalline phenol (0.75 g). Purification was carried out by reverse phase HPLC. Enfuvirtide was prepared by linear SPPS on rink amide resin. However the poor crude purities ranging 30-40% and low overall yields (6-8%) from this route were unacceptable for further development. Later an analog of Enfuvirtide, T-999, a HIV Fusion peptide was synthesized in stepwise manner using Sieber amide resin. Efficient full-length synthesis on solid-phase has not been demonstrated so far, such approach typically failing to produce more proper product than useless side products, purification of impurities posing further problems and again diminishing final yields. An improved route was needed for the synthesis of Enfuvirtide. Our initial attempts to use rink amide resin (100-200 mesh, 1.1 mmol/g) for the synthesis of Enfuvirtide were unsuccessful. Alternatively, the use of Fmoc-rink amide MBHA resin led to poor yields and purity. The super- acid labile 2-chlorotrityl chloride resin (2-CTC) was chosen as a base for the solid phase synthesis because of its ease of cleavage to protected peptide fragments under mild acidic conditions, the lack of racemization during the loading of the first amino acid , and the option of recycling the resin. In our initial plan to synthesize the EVT through fragment method following the patented procedure, we saw lower yields and less purity. The side chain of the glutamine residues at the carboxy-terminal end of fragments was not protected with trityl. It was found that the presence of trityl group on the side chain of glutamine lead to initial attachment less than 0.2 mmol/g loading. This could be due to steric hindrance of 2- CTC resin having a negative effect on the elongation, because first amino acid in this case is also very bulky. However, the absence of side chain protection facilitates the coupling with racemisation (2% reported). It has been found that the coupling of 1-16 fragment with 17-36 fragment leads to poor yield and less purity.

In the present process, the three enfuvirtide fragments are connected in five solution- phase operations while there are seven isolations in the commercial process. In the present process two enfuvirtide fragments are connected in two solution phase operations and total of 5 isolations. Generally, the approach of present process includes synthesizing two different peptide intermediate fragments using solid phase chemistry. Solution phase chemistry is then used to add additional amino acid material to the second fragment which is then coupled to the first fragment in solution.

Summary of the Invention:

The present invention relates to a novel process for the synthesis of pure Enfuvirtide or pharmaceutically acceptable salt by using a solid and solution phase (hybrid) approach. Such methods utilize solid and liquid phase synthesis procedures to synthesize and combine groups of specific peptide fragments to yield the peptide of interest. Generally, the methods of the invention comprise C-terminal amino acid of both the fragments loaded on to the 2- CTC resin in DMF in presence of excess of DIEA. Unreacted functional sites are capped with methanol in presence of DIEA. The fragment is then built using standard solid phase chemistry by first removing the N-terminal Fmoc group with piperidine in DMF and then adding a solution of the next Fmoc amino acid that has been pre-activated with DIC/HOBt in DMF. For the N-terminal fragment H-AA-(1-26)-OH, the N-terminus is acetylated with acetic anhydride and pyridine. The fragments are then cleaved from the resin using cold 1% TFA in DCM. The TFA is quenched with DIEA, and the DCM is evaporated. The peptide fragments are precipitated by the addition of DCM/IPE and isolated in high yield and purity by filtration. The solution phase assembly of the peptide begins with the coupling of H-Phe -NH₂ to the C-terminus of Fmoc-(AA-27-35)-OH in DMF using HBTU/HOBt/DIEA. The Fmoc-group is removed insitu and the amine product is isolated by precipitation with water. After washing thoroughly the H-(AA-27-36)-NH₂ is coupled with Ac-(1-26)-OH in a similar manner. The resulting Ac-(1-36)-NH2 may be isolated by precipitation the material is then subjected to a global side chain deprotection using TFA/Water/DTT and precipitated with cold MTBE. This is decarboxylated in acetonitrile as slurry at pH 4-5 with DIEA and Acetic acid. The resulting peptide is purified in the single pass by reverse phase HPLC and isolated in high yield and good purity.

Another aspect of the present invention is process for the synthesis of Ac-Tyr(tBu)-Thr(tBu)- Ser(tBu)-Leu-lle-His(Trt)-Ser(tBu)-Leu-lle-Glu(OBut)-Glu(OBut)-Ser(tBu)-Gln(Trt)-Asn(Trt)- Gln(Trt)-Gln(Trt)-Glu(OBut)-Lys(Boc)-Asn(Trt)-Glu(OBut)-Gln(Trt)-Glu(OBut)-Leu-Leu- Glu(OBut)-Leu-OH (Ac-AA (1-26)-OH, Fragment 1)

Yet another aspect of the present invention is process for the synthesis of Fmoc-Asp(OtBu)- Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)-Asn(Trt))-Trp(Boc)-OH (Fmoc-AA (27-35)-OH, Fragment 2). Yet another aspect of the present invention is process for the synthesis of Fmoc-Asp(OtBu)- Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)-Asn(Trt))-Trp(Boc)-Phe-NH2 (Fmoc-AA (27- 36)-NH₂, Fragment 3).

Yet another aspect of the present invention is an improved process for the synthesis of Ac- Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-lle-His(Trt)-Ser(tBu)-Leu-lle-Glu(OBut)-Glu(OBut)-Ser(tBu)- Gln(Trt)-Asn(Trt)-Gln(Trt)-Gln(Trt)-Glu(OBut)-Lys(Boc)-Asn(Trt)-Glu(OBut)-Gln(Trt)- Glu(OBut)-Leu-Leu-Glu(OBut)-Leu-Asp(OtBu)-Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu- Trp(Boc)-Asn(Trt))-Trp(Boc)-Phe-NH2 (protected Enfuvirtide). Yet another aspect of the present invention is process for the deprotection of the protected Enfuvirtide with TFA/DTT/Water 94/5.2/0.8 to get Ac-Tyr-Thr-Ser-Leu-lle-His-Ser-Leu-lle- Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala- Ser-Leu-Trp-Asn-Trp-Phe-NH₂ (Crude Enfuvirtide). Yet another aspect of the present invention is to provide a process to get pure Enfuvirtide from Crude Enfuvirtide.

Yet another aspect of the present invention is to provide crude Enfuvirtide having the HPLC purity more than 70%.

Yet another aspect of the present invention is to provide Enfuvirtide with purity more than 98.0%, preferably not less than 99.0%.

Detailed Description of the Invention:

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 defined below

AcOH acetic acid

Boc fe/t-butyloxycarbonyl

tBu tert-butyl

DCM Dichloromethane

DIC N,N'-diisopropylcarbodiimide

DMF Ν,Ν'-Dimethylformamide

Fmoc 9-fluorenylmethoxycarbonyl

HOBt N-hydroxybenzotriazole

MTBE Methyl tert-butyl ether

SPPS solid phase peptide synthesis

TFA trifluoroacetic acid

TIS triisopropylsilane

Trt trityl

DTT dithiothreitol The present invention relates to a novel process for the synthesis of pure Enfuvirtide Or pharmaceutically acceptable salt by using a solid and solution phase (hybrid) approach.. In one embodiment of the present invention, the process for the preparation of Enfuvirtide comprising the steps of:

a) condensing Fmoc-Asp(OtBu)-Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)- Asn(Trt))-Trp(Boc)-OH (Fragment 2) with Phe-NH₂ in /V./V-dimethylformamide containing 0-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), /V-hydroxybenzotriazole (HOBt) and /V./V-diisopropylethylamine (DIEA) to isolate Fmoc-Asp(OtBu)-Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)-Asn(Trt))- Trp(Boc)-Phe-NH₂ (Fmoc-AA (27-36)-NH₂, Fragment 3),

b) condensing Fragment 3 with Ac-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-lle-His(Trt)- Ser(tBu)-Leu-lle-Glu(OBut)-Glu(OBut)-Ser(tBu)-Gln(Trt)-Asn(Trt)-Gln(Trt)-Gln(Trt)- Glu(OBut)-Lys(Boc)-Asn(Trt)-Glu(OBut)-Gln(Trt)-Glu(OBut)rLeu-Leu-Glu(OBut)-Leu- OH (Ac-AA (1-26)-OH, Fragment 1) in DMF containing HBTU, HOBt and DIEA to isolate protected Enfuvirtide,

c) deprotecting the protected Enfuvirtide with cocktail mixture consisting of (trifluoroacetic acid/dithiothreitol/Water 94/5.2/0.8) to afford crude Enfuvirtide, and d) crystallizing the crude Enfuvirtide from a mixture of alcohol-ether followed by subsequent washings with water, acetonitrile and isopropyl ether to get enriched purity of Enfuvirtide, and

e) purifying by preparative HPLC to isolate pure Enfuvirtide.

In another embodiment, the process for the preparation of Ac-Tyr(tBu)-Thr(tBu)-Ser(tBu)- Leu-lle-His(Trt)-Ser(tBu)-Leu-lle-Glu(OBut)-Glu(OBut)-Ser(tBu)-Gln(Trt)-Asn(Trt)-Gln(Trt)- Gln(Trt)-Glu(OBut)-Lys(Boc)-Asn(Trt)-Glu(OBut)-Gln(Trt)-Glu(OBut)-Leu-Leu-Glu(OBut)- Leu-OH (Ac-AA (1-26)-OH, Fragment 1) comprising the steps of: anchoring first protected terminal amino acid to a resin,

selective deprotection of the amino group,

coupling carboxyl terminus of the next N-protected amino acid to the amine group, repeating steps c), and d) to form a peptide sequence, and

cleaving the fragment 1 from the resin using a 1% TFA in DCM. In yet another embodiment, the process for the preparation of Fmoc-Asp(OtBu)-Lys(Boc)- Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)-Asn(Trt))-Trp(Boc)-OH (Fragment- 2) comprising the steps of:

a) anchoring first protected terminal amino acid to a resin,

b) selective deprotection of the amino group,

c) coupling carboxyl terminus of the next /V-protected amino acid to the

amine group,

d) repeating steps c), and d) to form a peptide sequence, and

e) cleaving the fragment 2 from the resin using a 1% TFA in DCM.

In yet another embodiment, the resin used for synthesis of peptide undergoes swelling in presence of solvent selected from methylene chloride, tetrahydrofuran, N,N- dimethylformamide, /V-methylpyrrrolidone or mixture thereof. The resin is then treated with N-terminus protected amino acid in presence of coupling agent for a desired period of time to affect the coupling.

In yet another embodiment, the resin is selected from 2-chloro tritylchloride resin, TentaGel S RAM, 4-methoxy tritylchloride resin, SASARIN Resin, Siber amide resin or HMPB Resin.

In yet another embodiment, deprotection of the protected amino acid anchored to the resin is done selectively in the presence of nucleophilic base such as 20% piperidine in dimethylformamide, methylene chloride or N-methyl pyrrolidine.

In yet another embodiment, before proceeding to next step, the unreacted linkers on the resin (polymer) are protected to avoid the undesired peptide chain formation. Preferably the free groups on resin are protected with acetylating reagents such as a solution of 5% DIEA and 10% methanol in DCM. This process of capping is performed after anchoring the first protected amino acid to resin.

In yet another embodiment, the coupling agents used for the coupling are selected from DIC and HOBt or HOBt, HBTU and DIEA. The amount of protected amino acid used in the present invention is selected from 1 to 3M with respect to resin loading capacity. In yet another embodiment, the coupling reaction is carried out in the presence of solvents selected from dichloromethane, tetrahydrofuran, dimethylformamide, N-methylpyrolidone or mixtures thereof. In yet another embodiment, the resin after completion of the reaction is optionally washed with solvents such as DMF and DCM to remove residual reagents and byproducts.

In yet another embodiment, adding a solution of next Fmoc amino acid that has been pre- activated with DIC/HOBt. The process is repeated till the completion of all the amino acids in stepwise manner and the reactions were monitored using Kaiser Test.

The functional group present on the amino acids used in the process of the present invention may be appropriately protected to avoid any undesired by products. Suitable protecting groups are described in the literature (See for example, P Wuts, and T.W. Greene, Protective Groups in Organic Synthesis John Wiley & Sons, 4th Edition 2007). The protecting group may vary depending upon the particular amino acid which may include but are not limited to tBu or Trt or Boc.

In yet another embodiment, the N-terminus is acetylated with acetylating agent such as acetic anhydride in presence of a base such as pyridine before cleavage of peptide from the resin.

In yet another embodiment, cleavage of the peptide from the resin and deprotecting the functional protecting groups on amino acid groups is carried out using TFA and a cocktail mixture of carbocation scavengers to provide crude Enfuvirtide with an HPLC purity of approximately 70%. Cleaving the peptide from the resin involves treating the protected peptide anchored to the resin with an acid having at least one scavenger.

According to the present invention, the peptide cleavage and global deprotection reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents. Scavengers are selected from EDT, DDM, TIPS, TES, Phenol, thioanisole or mixture thereof. The cocktail mixture used for the reaction is selected from group comprising of TFA/DTT/Water-94/5.2/0.8 respectively. In yet another embodiment, after the completion of the reaction, the reaction mixture may optionally be filtered and washed with acid or an organic solvent. The crude Enfuvirtide is isolated by combining the reaction mass with an organic solvent. The organic solvent is selected from diethyl ether, isopropyl ether, tert-butyl methyl ether, tert-butyl ethyl ether, isopropyl acetate, ethyl acetate or mixture thereof.

In yet another embodiment, the peptide cleavage and global deprotection reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents. Scavengers are selected from EDT, DDM, TIPS, TES, Phenol, thioanisole or mixture thereof. The cocktail mixture used for the reaction is selected from group comprising of TFA/DTT/Water-94/5.2/0.8 respectively.

In yet another embodiment, resin used in the present invention are of standard mesh size which is about 100-200 mesh, more preferably 200 mesh.

In yet another embodiment, purification of the crude Enfuvirtide is carried out by preparative RP-HPLC to produce pure Enfuvirtide or its pharmaceutically acceptable salts.

In yet another embodiment, the present invention is to provide a process to get pure Enfuvirtide from crude Enfuvirtide.

In yet another embodiment, the present invention is to provide crude Enfuvirtide having the HPLC purity more than 70%. In yet another embodiment, the present invention is to provide Enfuvirtide with purity more than 98.0%, preferably not less than 99.0%.

The process for the preparation of Enfuvirtide is summarized in synthetic scheme-l depicted below:

Examples: Preparation of Enfuvirtide (N-Acetyl-L-tyrosyl-L-threonyl-L-seryl-L-leucyl-L- isoleucyl-L-histadyl-L-seryl-L-leucyl-L-isoleucyl-L-glutaminyl-L-glutamyl-L-seryl-L-glutaminyl- L-asparaginyl-L-glutaminyl-L-glutaminal-L-glutamyl-L-lysyl-L-asparaginyl-L-glutamyl-L- glutaminyl-L-glutamyl-L-leucyl-L-leucyl-L-glutamyl-L-leucyl-L-aspartyl-L-lysyl-L-tryptophyl-L- alanyl-L-seryl-L-leucyl-L-tryptophyl-L-asparginyl-L-tryptophyl-L-phenylalaninamide). Preparation of Fragment 1

The 2-chloro tritylchloride resin (10g, loading 0.7mmol/g) was allowed to swell for 30 min. in DCM (50 mL). The mixture was filtered and fresh DMF (50 mL), Fmoc-Leu (6.8g, 19.3 mmol) and diisopropylethylamine (DIEA, 14mL, 78.6mmol) were added. The mixture was stirred at room temperature for 2 h., filtered and washed with DMF and DCM. Subsequently, the mixture was stirred with a solution of 5% DIEA and 10% methanol in DCM for 30 min. The resin was washed with DMF and DCM and dried in vacuum to yield the loaded resin. The loading was determined using Beer-Lambert law and found to be 0.5mmol/g. After resin loading and prior to first deprotection, the resin is allowed to swell in DMF for 1 h. The Fmoc group was deprotected with 20% piperidine in DMF and subsequent amino acid couplings were done using DIC/HOBt as coupling agent. After every coupling and deprotection through washings ware done prior to proceed for next amino acid. All the couplings and deprotections were monitored using ninhydrin test.

Preparation of Fragment 2

The 2-chloro tritylchloride resin (10g, loading 0.7mmol/g) was allowed to swell for 30 min. in DCM (50 mL). The mixture was filtered and fresh DMF (50 mL), Fmoc-Trp (Boc) (8.4g, 16.02mmol) and diisopropylethylamine (DIEA, 13ml_, 73.02 mmol) were added. The mixture was stirred for 2 h. at room temperature, filtered and washed with DMF and DCM. Subsequently the mixture was stirred with a solution of 5%-DIEA and 10% methanol in DCM for 30 min. The resin was washed with DMF and DCM and dried in vacuum to yield the loaded resin. The loading was determined using Beer-Lambert law and found to be 0.5mmol/g .After resin loading and prior to first deprotection; the resin is allowed to swell in DMF for 1 h. The Fmoc group was deprotected with 20% piperidine in DMF and subsequent amino acid couplings were done using DIC/HOBt as coupling agent. After every coupling and deprotection through washings was done prior to proceed for next amino acid .All the couplings and deprotections were monitored using ninhydrin test.

Preparation of Fragment 3

To a stirred solution of Fragment 2 (1.0 eq) and HOBt (1.2 eq) in DMF, was added DIEA (3.0eq) and lowed to cool to 10^°C. HBTU (1.0 eq) was added to the reaction mixture, stirred for 15 min at 10^°C, then Phe-NH2 was added (1.0 eq) and stirred for over night at room temperature. The reaction mixture was cooled and then water was added, stir for 30 min's. The precipitated solid was filtered, washed with water and dried under vacuum at 45 C for 2 h. The material was washed with 50% hexane /IPE followed by IPE and dried. The Fmoc was then deprotected with DMF/ piperidine and carried for next coupling.

Purification of fragment 3

The peptide was dissolved in a minimum amount of ethyl acetate and hexane was added slowly till the precipitate forms. The reaction mixture was stirred for 30 min. The separated solid was filtered and dried under vacuum at 45^°C for 1 h to afford fragment 3 in 80% yield and 83% HPLC purity. Preparation of protected Enfuvirtide

To a stirred solution of Fragment 3 (1.0 eq) and HOBt (1.2 eq) in DMF, was added DIEA (3.0 eq) and lowed to cool to 10^°C. HBTU (1.0 eq) was added to the reaction mixture and stirred for 15 min at 10 C and then added Fragment 1 (1.0 eq). The resulting solution was stirred for over night at room temperature. The reaction mixture was cooled and water was added under stirring and maintained for 30 min. The precipitated solid was filtered, washed with water and dried under vacuum at 45 C for 2 h. The material was washed with 50% hexane /IPE followed by IPE and dried. Purification of protected Enfuvirtide

The peptide was dissolve in a minimum amount of ethyl acetate and hexane was added slowly till the precipitate forms. The resultant reaction mixture was stirred for 30 min., the separated solid was filtered and dried under vacuum at 45°C for 1 h to afford fragment 4 in 80% yield and 83% HPLC purity.

Preparation of crude Enfuvirtide

Deprotection of the side chain functional groups of enfuvirtide are cleaved using cocktail mixture consisting of (TFA/DTT/Water 94/5.2/0.8). The cocktail mixture was cooled and a solution of protected peptide in DCM was added at 10°C. The resultant reaction mass was stirred for 5 h at room temperature, cooled, the chilled IPE was added and stirred for 30 min. The solid was filtered and washed with IPE.

Purification of Enfuvirtide

The crude Enfuvirtide was dissolved in minimum amount of methanol, cooled to 10 C, IPE was added and stirred for 30 min. The separated solid was filtered, washed with IPE and dried. It is further washed with water, ACN and IPE and dried to get Enfuvirtide in 85% yield and 70% HPLC purity.

Claims

We claim: 1 A process for the preparation of Enfuvirtide comprising the steps of:

a) condensing Fmoc-Asp(OtBu)-Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)- Asn(Trt))-Trp(Boc)-OH (Fragment 2) with Phe-NH₂ in Λ/,/V-dimethylformamide containing 0-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), A/-hydroxybenzotriazole (HOBt) and A/,/V-diisopropylethylamine (DIEA) to isolate Fmoc-Asp(OtBu)-Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)-Asn(Trt))- Trp(Boc)-Phe-NH₂ (Fmoc-AA (27-36)-NH₂, Fragment 3),

b) condensing Fragment 3 with Ac-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-lle-His(Trt)- Ser(tBu)-Leu-lle-Glu(OBut)-Glu(OBut)-Ser(tBu)-Gln(Trt)-Asn(Trt)-Gln(Trt)-Gln(Trt)- Glu(OBut)-Lys(Boc)-Asn(Trt)-Glu(OBut)-Gln(Trt)-Glu(OBut)-Leu-Leu-Glu(OBut)-Leu- OH (Ac-AA (1-26)-OH, Fragment 1) in DMF containing HBTU, HOBt and DIEA to isolate protected Enfuvirtide,

e) purifying by preparative HPLC to isolate pure Enfuvirtide. 2 A process for the preparation of Ac-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-lle-His(Trt)-Ser(tBu)- Leu-lle-Glu(OBut)-Glu(OBut)-Ser(tBu)-Gln(Trt)-Asn(Trt)-Gln(Trt)-Gln(Trt)-Glu(OBut)- Lys(Boc)-Asn(Trt)-Glu(OBut)-Gln(Trt)-Glu(OBut)-Leu-Leu-Glu(OBut)-Leu-OH (Ac-AA ( 1 - 26)-OH, Fragment 1) comprising the steps of:

a) anchoring first protected terminal amino acid to a resin,

b) selective deprotection of the amino group, ^{" ~}

c) coupling carboxyl terminus of the next /V-protected amino acid to the amine group, d) repeating steps c), and d) to form a peptide sequence, and

e) cleaving the fragment 1 from the resin using a 1% TFA in DCM. 3 A process for the preparation of Fmoc-Asp(OtBu)-Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu- Trp(Boc)-Asn(Trt))-Trp(Boc)-OH (Fragment-2) comprising the steps of:

a) anchoring first protected terminal amino acid to a resin, b) selective deprotection of the amino group,

c) coupling carboxyl terminus of the next W-protected amino acid to the

amine group,

d) repeating steps c), and d) to form a peptide sequence, and

e) cleaving the fragment 2 from the resin using a 1% TFA in DCM.

4 The process according to claim 2 and 3, wherein said the said resin is selected from 2- chloro tritylchloride resin, TentaGel S RAM, 4-methoxy tritylchloride resin, SASARIN Resin, Siber amide resin or HMPB Resin.

5 The process according to claim 2 and 3, wherein said swelled resin is then treated with /V-terminus protected amino acid in presence of coupling agent for a desired period of time to affect the coupling. 6 The process according to claim 5, wherein said coupling agent used for the coupling is selected from DIC and HOBt or HOBt, HBTU and DIEA. The amount of protected amino acid used in the present invention is selected from 1M to 3M with respect to resin loading capacity. 7 The process according to claim 2 and 3, wherein said resin capping is performed after anchoring the first protected amino acid to resin.

8 The process according to claim 2 and 3, wherein said coupling reaction in step c) is carried out in the presence of solvents selected from dichloromethane, tetrahydrofuran, dimethylformamide, /V-methylpyrolidone or mixtures thereof. 9 The process according to claim 2 and 3, wherein said adding a solution^" of next Fmoc amino acid in step d) has been pre-activated with DIC/HOBt and process is repeated till the completion of all the amino acids in stepwise manner and the reactions were monitored using Kaiser Test.

10 The process according to claim 2 and 3, wherein said cleavage of the peptide from the resin and deprotecting the functional protecting groups on amino acid groups is carried out using TFA and a cocktail mixture of carbocation scavengers to provide crude Enfuvirtide with an HPLC purity of more than 70%. The process according to claim 2 and 3, wherein said peptide cleavage and deprotection reagent used in the process of the present invention is a cocktail mixture of acid, scavengers and solvents. The process according to claim 11 , wherein said cocktail mixture is comprising of TFA/DTT/Water-94/5.2/0.8 respectively and scavenger is selected from EDT, DDM, TIPS, TES, Phenol, thioanisole or mixture thereof. The process according to claim 2 and 3, wherein said crude Enfuvirtide is isolated by combining the reaction mass with an organic solvent selected form diethyl ether, isopropyl ether, tert-butyl methyl ether, tert-butyl ethyl ether, isopropyl ether, ethyl acetate or mixture thereof, The process according to claim 2 and 3, wherein said purification of the crude Enfuvirtide is carried out by preparative RP-HPLC to produce pure Enfuvirtide or its pharmaceutically acceptable salts. Ac-Tyr(tBu)-Thr(tBu)-Ser(tBu)-Leu-lle-His(Trt)-Ser(tBu)-Leu-lle-Glu(OBut)-Glu(OBut)- Ser(tBu)-Gln(Trt)-Asn(Trt)-Gln(Trt)-Gln(Trt)-Glu(OBut)-Lys(Boc)-Asn(Trt)-Glu(OBut)- Gln(Trt)-Glu(OBut)-Leu-Leu-Glu(OBut)-Leu-OH (Ac-AA (1-26)-OH, Fragment 1) is having the HPLC purity more than 80%. Fmoc-Asp(OtBu)-Lys(Boc)-Trp(Boc)-Ala-Ser(tBu)-Leu-Trp(Boc)-Asn(Trt))-Trp(Boc)-OH (Fragment-2) is having -the HPLC purity more than 90%. ^" Enfuvirtide with purity more than 98.0%.