DESC:Improved process for the preparation of Semalinker.

Field of The Invention
The present invention relates to an improved process for the preparation of Semalinker having the chemical Formula (I).

Formula-I

Background of the Invention

Semaglutide is a long-acting glucagon like peptide agonist developed by Novo Nordisk and approved by Food and Drug Administration for the treatment of type-2 diabetes. Semaglutide marketed under brand name “Ozempic” in the form of injection and “RYBELSUS” in the form of tablet which lowers the blood sugar level by increasing the production of insulin.

The Semaglutide peptide is chemically similar to Liraglutide, with the inclusion of two structural modifications. The first is replacement of Ala with the non-proteinogenic amino acid 2-aminoisobutyric acid (Aib) at position 2. The second is the attachment of octadecanoic diacid to the side chain of Lys-26 through a short polyethylene glycol (PEG) spacer and a ?-glutamic acid linker.

Semalinker and its process for the preparation is disclosed in CN104356224. In this process, there is a possibility of formation of several impurities which shows impact on yield as well as purity of Semalinker and additional purification techniques required to get pure Semalinker. This process is highly expensive and commercially not viable. This process have several disadvantages with lot of technical difficulties, expensive production costs and not suitable for large scale production due to complex purification methods.

In view of all these disadvantages, there is a significant need to develop a cost effective, stable, commercially viable, large scale and robust process for the preparation of highly pure Semalinker with good yield.
Summary of The Invention

The present invention provides a cost effective, novel and an efficient process for the preparation of Semalinker by making fragments followed by condensing these fragments using solution phase approach with higher yields and purity.

The present invention provides a process for the preparation of Fmoc-Lys(AEEA-NH2)-OH of Fragment-1

Fragment-1
which comprises:
a) Condensing compound of Formula-[A]

Formula-[A]

with N-(Benzyloxy carbonyloxy) hydroxy succinimide of Formula-[B]

Formula-[B]
in presence of base to obtain compound of Formula-[C];

Formula-[C]

b) coupling of compound of Formula-[C] with N-hydroxy succinimide in presence of coupling agent to obtain compound of Formula-[D];

Formula-[D]
c) coupling of compound of Formula-[D] obtained in step-b) with Fmoc-Lys-OH in presence of base to obtain compound of Formula-[E];

Formula-[E]

d) deprotection of compound of Formula-[E] using a reducing agent to obtain Fmoc-Lys(AEEA-NH2)-OH of Fragment-1.

The present invention provides a process for the preparation of Z-Glu(AEEA-OSu)-OtBu of Fragment-2

Fragment-2
which comprises:
a) coupling of compound of Formula-[F]

Formula-[F]
with N-hydroxy succinamide in presence of coupling agent to obtain compound of Formula-[G];

Formula-[G]
b) condensation of compound of Formula-[G] with compound of Formula-[A]

Formula-[A]

in presence of base and solvent to obtain compound of Formula-[H];

Formula-[H]
c) condensation of compound of Formula-[H] obtained in step-b) with N-hydroxy succinamide in presence of a reagent to obtain Z-Glu(AEEA-OSu)-OtBu of Fragment-2.

The present invention provides a process for the preparation of Fmoc-Lys[Oct-(OtBu)-Glu-(OtBu)-AEEA-AEEA]-OH of Formula-I (Semalinker)

Formula-I
which comprises;
a) condensation of compound of Fragment-3

Fragment-3

with compound of Formula-[J]

Formula-[J]
in presence of base to obtain compound of Formula-[K];

Formula-[K]

b) reacting compound of Formula-[K] with N-hydroxy succinimide to obtain compound of Formula-[L];

Formula-[L]
c) condensation of compound of formula-[L] with Fragmen-1

Fragment-1
in presence of base and solvent to obtain Semalinker of Formula-I.

The present invention provides a process for the preparation of Fmoc-Lys[Oct-(otBu)-Glu-(otBu)-AEEA-AEEA]-OH of formula-I (Semalinker)

Formula-I
Which comprises;
a) condensation of compound of Fragment-1

Fragment-1
With compound of Fragment-2

Fragment-2
in presence of base and solvent to obtain compound of Formula-[M]

Formula-[M]
b) deprotecting of compound of Formula-[M] using a reagent to obtain compound of Formula-[N];

c) condensation of compound of Formula-[N] obtained in step-b) with compound of Fragment-3;

in presence of a base to obtain Semalinker of formula-I.

Abbreviations:
Fmoc: 9-fluorenylmethoxycarbonyl
Boc: Tert-butoxycarbonyl
DCM: dichloromethane
DMF: N, N-dimethyl formamide
DIC: N, N’-diisopropyl carbodiimide
DIEA: Diisopropylethylamine
HOBt: N-hydroxy benzotriazole
TFA: Trifluoroacetic acid
DCC: Dicyclohexyl carbodiimide

Detailed Description of the Invention

The present invention provides an improved process for the preparation of Semalinker by making appropriate fragments followed by condensing these fragments to get Semalinker with higher yields and purity.

Peptide fragments which are used in the preparation of Semalinker are as follows.
Fragment-1: Fmoc-Lys(AEEA-NH2)-OH
Fragment-2: Z-Glu(AEEA-OSu)-OtBu
Fragment-3: OtBu-C18-OSu
Fragment-4: OtBu-C18-Glu(OSu)-OtBu
Fragment-5: Fmoc-Lys(AEEA-AEEA-NH2)-OH

Coupling of amino acid is carried out in presence of a base. The base is organic or inorganic base. The inorganic base is selected from the group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide and mixture thereof; the organic base is selected from the group consisting of diisopropyl amine, N, N-diisopropyl ethylamine, triethylamine, tertiary butyl amine, dimethylamine, tri methyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine and mixture thereof.

Reagent used for deprotection in this process is preferably palladium on carbon.

Solvent used for this process is selected from the group consisting of alcoholic solvents such as methanol, ethanol, isopropanol; chlorinated solvents such as chloroform, methylene chloride; ester solvents such as ethyl acetate, butyl acetate, isopropyl acetate; ether solvents such as diethyl ether, tetrahedrofuran,2-methyl tetrahydrofuran, diisopropyl ether; aprotic solvents and protic solvents such as DMF, DMSO, DMAC, 1,4-dioxane or a mixture thereof.

The main embodiment of the present invention provides a process for the preparation of compound of Fragment-1 as shown in the Scheme-1 given below.

Scheme-1

In step-1), condensation of compound of Formula-[A] with benzyloxy carbonyl protected hydroxy succinamide in presence of a base to obtain compound of Formula-[C].

The base used in the reaction is selected from the group consisting of inorganic base such as alkali metal hydroxide, alkali metal carbonates and alkali metal bicarbonates or organic base such as triethylamine, diisopropyl ethyl amine or pyridine, preferably using sodium bicarbonate.

In step-2), coupling of compound of Formula-[C] with N-hydroxy succinamide in presence of coupling agent to obtain compound of Formula-[D].

The coupling agent used in this reaction can be selected from group consisting of Dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-hydroxy benzotriazole (HOBt), preferably using diisopropyl carbodiimide (DIC).

The solvent used in the reaction is selected from the group consisting of alcoholic solvent, chlorinated solvent, ketone solvent, ester solvent, ether solvent, preferably using methylene chloride.
In step-3), condensation of compound of Formula-[D] with Fmoc-Lys-OH in presence of a base and solvent to obtain compound of Formula-[E].
The base used in this reaction is selected from the group consisting of inorganic base such as alkali metal hydroxide, alkali metal carbonates and alkali metal bicarbonates or organic base such as triethylamine, diisopropyl ethyl amine or pyridine, preferably using sodium carbonate.
The solvent used in the reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using tetrahydrofuran.

Reaction temperature may range from 10-30°C and preferably at a temperature in the range from 25-30°C. The duration of reaction may range from 3-6 hours, preferably for a period of 4-5 hours.

In step-4), deprotection of compound of Formula-[E] carried out by using a reagent to obtain compound of Fragment-1.
The reagent used in the reaction is preferably palladium on carbon.
Solvent used for this reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using isopropanol.
The main embodiment of the present invention provides a process for the preparation of compound of Fragment-2 as shown in the Scheme-2 given below.

Scheme-2

In step-1), compound of Formula-[F] is coupled with N-hydroxy succinamide in presence of coupling agent and solvent to obtain compound of Formula-[G].
The coupling agent used in this reaction can be selected from group consisting of Dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-hydroxy benzotriazole (HOBt), preferably using dicyclo hexyl carbodiimide (DCC).

The solvent used in the reaction is selected from the group consisting of alcoholic solvent, chlorinated solvent, ketone solvent, ester solvent, ether solvent, preferably using methylene chloride.

In step-2), compound of Formula-[G] is condensed with compound of Formula-[A] in presence of base and solvent to obtain compound of Formula-[H].

The base used in the reaction is selected from the group consisting of inorganic base such as alkali metal hydroxide, alkali metal carbonates and alkali metal bicarbonates or organic base such as triethylamine, diisopropyl ethyl amine, N-methyl morpholine or pyridine, preferably using N-methyl morpholine.

The solvent used in the reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using tetrahydrofuran.

In step-3), compound of Formula-[H] is coupled with N-hydroxy succinamide in presence of coupling agent and solvent to obtain compound of Fragment-2.

The coupling agent used in this reaction can be selected from group consisting of Dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-hydroxy benzotriazole (HOBt), preferably using dicyclo hexyl carbodiimide (DCC).

The solvent used in the reaction is selected from the group consisting of alcoholic solvent, chlorinated solvent, ketone solvent, ester solvent, ether solvent, preferably using methylene chloride

The main embodiment of the present invention provides a process for the preparation of Semalinker of Formula-1 as shown in the Scheme-3 given below.

Scheme-3
In step-1), compound of Fragment-3 is condensed with compound of Formula-[J] in presence of base and solvent to obtain compound of Formula-[K].
The base used in the reaction is selected from the group consisting of inorganic base such as alkali metal hydroxide, alkali metal carbonates and alkali metal bicarbonates or organic base such as triethylamine, diisopropyl ethyl amine, N-methyl morpholine or pyridine, preferably using Triethyl amine.

The solvent used in the reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using 2-methyl tetrahydrofuran.

In step-2, coupling of compound of Formula-[K] with N-hydroxy succinamide in presence of a coupling agent to obtain compound of Formula-[L].
The coupling agent used in this reaction can be selected from group consisting of Dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-hydroxy benzotriazole (HOBt), preferably using dicyclo hexyl carbodiimide (DCC).

In step-3, compound of Formula-[L] was condensed with Fragment-1 in presence of base and solvent to obtain Semalinker.

The base used in the reaction is selected from the group consisting of inorganic base such as alkali metal hydroxide, alkali metal carbonates and alkali metal bicarbonates or organic base such as triethylamine, diisopropyl ethyl amine, N-methyl morpholine or pyridine, preferably using Triethyl amine.

The solvent used in the reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using 2-methyl tetrahydrofuran.

In yet another embodiment of the present invention provides a process for the preparation of Semalinker of Formula-1 as shown in the Scheme-4 given below.

Scheme-4
In step-1), compound of Fragment-1 is condensed with compound of Fragment-2 in presence of base and solvent to obtain compound of Formula-[M].
The base used in this reaction is selected from the group consisting of inorganic base such as alkali metal hydroxide, alkali metal carbonates and alkali metal bicarbonates or organic base such as triethylamine, diisopropyl ethyl amine, N-methyl morpholine or pyridine, preferably using Triethyl amine.

The solvent used in the reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using 2-methyl tetrahydrofuran.
In step-2), deprotection of compound of Formula-[M] is carried in presence of a reagent and solvent to obtain compound of Formula-[N].
The reagent used for deprotection is preferably palladium on carbon.
The solvent used in the reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using methanol.
In step-3), condensation of compound of Formula-[N] is carried out with compound of fragment-2 in presence of base and solvent to obtain Semalinker of Formula-1.
The base used in this reaction is selected from the group consisting of inorganic base such as alkali metal hydroxide, alkali metal carbonates and alkali metal bicarbonates or organic base such as triethylamine, diisopropyl ethyl amine, N-methyl morpholine or pyridine, preferably using Triethyl amine.

The solvent used in the reaction is selected from the group consisting of alcoholic solvent such as methanol, ethanol, propanol, isopropanol, chlorinated solvent such as chloroform, methylene chloride, ketone solvent such as acetone, methyl isobutyl ketone, ester solvent such as ethyl acetate, isopropyl acetate, ether solvent such as tetrahydrofuran, preferably using 2-methyl tetrahydrofuran.

The key starting materials, Intermediates and reagents used in this process may be purchased or may be prepared by using prior art procedures.

EXPERIMENTAL PORTION

The details of the invention are given in the examples provided below, which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.

Example-1: Process for the preparation of Fmoc-Lys(AEEA-NH2)-OH of Fragment-1
Step-A:
Z-AEEA-OH (8 grams) was dissolved in 80 mL of Methylene dichloride at room temperature. N-hydroxy succinamide (3.71 grams) was added to the resulting reaction mixture and cooled to 5-10 °C. Slowly add 5 mL of diisopropyl carbodiimide (DIC) at 5-10 °C. Maintain for 1 hour at 5-10 °C and raise the temperature to 25-30 °C and maintain mass for 3 hours. Check the TLC/HPLC and filter the mass. Wash the filtrate with water and brine, distil off solvent below 40 °C to get Z-AEEA-OSu.

Step-B:
Fmoc- Lys-OH (3.4 grams) was dissolved in THF (17.5 mL) and water (17.5 mL). Sodium carbonate (1.5 grams) was added to the resulting reaction mixture and cooled to 5-10 °C. Z-AEEA-OSu was added to the reaction mixture and. stirr for 30 minutes at the same temperature. Raised the temperature to 25-30 °C and maintain mass for 5 hours at the same temperature. After completion of reaction, filtered the reaction mixture and washed with n-Heptane. Reaction mixture pH was adjusted to 7 with 10% aqueous HCl solution and distilled-off the solvent below 40 °C. Obtained reaction mass pH was adjusted to 3-3.5 with 10% aqueous HCl solution, extracted the mass with MDC and distil off solvent below 40 °C to get Fmoc-Lys(AEEA-Z)-OH.

Step-C:
Fmoc-Lys(AEEA-Z)-OH (5 grams) was dissolved in isopropanol. Acetic acid and palladium on carbon were added to the resulting reaction mixture under nitrogen atmosphere. Hydrogen gas was applied to the reaction mixture and maintain reaction mass up to completion of reaction at 35-40 °C. Filtered the reaction mixture and distilled-off the solvent under reduced pressure below 40 °C. Crude Fragment-1 was purified with ethyl acetate and diisopropyl ether.

Example-2: Process for the preparation of Z-Glu(AEEA-OSu)-OtBu of Fragment-2

Z-Glu(AEEA-OH)-OtBu (2 grams) was dissolved in THF (10 mL) at 25-30°C. N-hydroxy succinamide (0.52 grams) was added to the reaction mixture and cooled to 5-10 °C. Dicyclohexyl carbodiimide (0.93 grams) in THF was slowly added to the reaction mixture at 5-10 °C and stirred for 1 hour at 5-10 °C. Raised the reaction temperature to 25-30 °C and stirred for 4 hours at the same temperature.. Distilled-off the solvent under reduced pressure below 40 °C. Added methylene chloride and store in freezer for overnight. Filter the mass and distilled-off the solvent under reduced pressure at below 40 °C. crude compound was purified with ethyl acetate and Diisopropyl ether.

Example-3: Process for the preparation of OtBu-C18-OSu of Fragment-3
OtBu-C18-OH (10 grams) was dissolved in THF (80 mL) at 25-30°C. N-hydroxy succinamide (3.42 grams) was added to the resulting reaction mixture and cooled to 5-10 °C. Dicyclohexyl carbodiimide (6.18 grams) in THF was slowly added to the resulting reaction mixture at 5-10 °C and stirred for 1 hour at 5-10 °C. Raised the temperature to 25-30 °C and maintain mass for overnight. Distilled-off the solvent under reduced pressure below 40 °C. Isopropanol was added and cooled to 5-10 °C maintain for 1 hour and filtered to get the title compound

Example-4: Process for the preparation of OtBu-C18-Glu(OSu)-OtBu of fragment-4
Step-A:
OtBu-C18-OH was dissolved in methylene chloride and H-Glu(Bzl)-OtBu.HCl was added to the reaction mixture and cooled to 5-10 °C. Dicyclohexyl carbodiimide was slowly added to the resulting reaction mixture at 5-10 °C. The reaction mixture was stirred for 1 hour at 5-10 °C. Raised the temperature to 25-30 °C and maintain mass for overnight. Filtered the reaction mixture and washed the organic layer with 2.5 % aqueous sodium bicarbonate solution and 1% KHSO4 solution. Distilled-off the solvent under reduced pressure below 40 °C to get OtBu-C18-Glu(OBzl)-OtBu.

Step-B:
OtBu-C18-Glu(OBzl)-OtBu obtained in step-A was dissolved in methanol at 25-30°C. Palladium on carbon was added to the resulting reaction mixture under nitrogen atmosphere. Hydrogen gas was applied to the reaction mixture and maintain reaction mass up to completion of reaction at 25-40 °C. Filtered the reaction mass and distilled-off the solvent under reduced pressure below 40 °C. washed the crude compound with n-Heptane to obtain OtBu-C18-Glu-OtBu.

Step-C:
OtBu-C18-Glu-OtBu obtained from step-B was dissolved in 5 volumes of methylene dichloride and N-hydroxy succinamide was added to the resulting reaction mixture and cooled to 5-10 °C. Diisopropyl carbodiimide (DIC) in methylene dichloride was slowly added to the obtained reaction mixture at 5-10 °C and stirred for 1 hour at the same temperature. Raised the reaction temperature to 25-30 °C and stirred for 3 hours at the same temperature. Filtered the reaction mixture and washed the obtained filtrate with water and brine, distil off solvent below 40 °C and recrystalize from isopropanol to obtain title compound.

Example-5: Process for the preparation of Fmoc-Lys(AEEA-AEEA-NH2)-OH of Fragment-5
Step-A:
H-AEEA-AEEA-OH was added to aqueous sodium hydroxide solution at 25-30°C and stirred for 5-10 minutes at the same temperature. Cooled the reaction mixture to 5-10°C. Boc anhydride solution in butanol was slowly added into the resulting reaction mixture at 5-10°C and stirred for 10 minutes at the same temperature. Raised the reaction mixture temperature to 25-30°C and stirred for over-night at the same temperature. Reaction mixture pH was adjusted to 7 with 20% KHSO4 and distilled-off the solvent below 40 °C. Further, Reaction mixture pH was adjusted to 3 to 3.5 with 20% KHSO4, extract the reaction mixture with methylene chloride and washed the organic layer with water and brine, distilled-off the solvent below 40 °C to yield Boc-AEEA-AEEA-OH.

Step-B:
Boc-AEEAAEEA-OH was dissolved in THF at 25-30°C.N-hydroxy succinamide was added to the reaction mixture and cooled to 5-10 °C. Dicyclohexyl carbodiimide in THF was slowly added to the reaction mixture and stirred for 1 hour at 5-10 °C. Raised the temperature to 25-30 °C and maintain the reaction mixture for 4 hours at same temperature. Filtered the reaction mixture and distilled-off the solvent under reduced pressure below 40 °C. Ethyl acetate was added to the crude compound and Filtered the reaction mixture and distilled-off the solvent under reduced pressure at below 40 °C to obtain Boc-AEEA-AEEA-OSu.

Step-C:
Fmoc-Lys-OH was dissolved in aqueous THF and 1.5 equivalent of sodium carbonate. Was added to the reaction mixture at 25-30°C. Cooled the reaction mixture to 5-10 °C and Boc-AEEA-AEEA-OSu in THF was added to the reaction mixture. The resulting reaction mixture was stirred for 15 minutes at 5-10 °C. Raised the reaction mixture temperature to 25-30 °C and stirred for overnight. Reaction mixture pH was adjusted to 7 with 20% KHSO4 and distilled-off the solvent below 40 °C. Further, reaction mixture pH was adjusted to 3-3.5 with 20% KHSO4. Reaction mixture was extracted with methylene chloride and wash the organic layer with water and brine, distilled-off solvent below 40 °C to obtain Fmoc-Lys(AEEA-AEEA-Boc)-OH.

Step-D:
Fmoc-Lys(AEEA-AEEA-Boc)-OH obtained in step-C was dissolved in methylene chloride. Cooled the reaction mixture to 5 to15 °C. 1,4-dioxane in HCl was added to the resulting reaction mixture and maintain reaction mass up to completion of reaction at 25-30 °C. Distilled-off the solvent under reduced pressure below 40 °C and washed the obtained crude with diisopropyl ether to get fragment-5.

Example-6: Process for the preparation of Semalinker
Step-A:
H-Glu(AEEA-OH)-OtBu.HCl (0.7 grams) dissolved in THF (3.5 mL) and water (3.5 mL). Aqueous sodium carbonate solution was added to the reaction mixture and cooled the reaction mixture to 5-10 °C. OtBu-C18-OSu (0.68 grams) in THF was slowly added to the reaction mixture at 5-10°C and stirred for 30 minutes at the same temperature. Raised the reaction temperature to 25-30 °C and stirred the reaction mixture overnight at the same temperature. Filtered the reaction mixture and washed with diisopropyl ether. Reaction mixture pH was adjusted to 7 with 10% aqueous HCl solution and distilled-off solvent below 40 °C. Further, its pH was adjusted to 3-3.5 with 10% aqueous HCl solution. Extracted the reaction mixture with methylene chloride and washed the methylene chloride layer with water, distilled-off the solvent under reduced pressure below 40 °C and obtained crude was purified using Heptane to obtain OtBu-C18-Glu(AEEA-OH)-OtBu.

Step-B:
OtBu-C18-Glu(AEEA-OH)-OtBu (0.5 grams) was dissolved in THF and N-hydroxy succinamide was added to it at 25-30°C. Cooled the reaction mixture to 5-10 °C. Dicyclohexyl carbodiimide (0.147 grams) was slowly added to the resulting reaction mixture at 5-10 °C and stirred for 30 minutes at same temperature. Raised the reaction temperature to 25-30 °C and stirred for 3 hours at the same temperature. Filtered the reaction mixture. Distilled-off the solvent under reduced pressure below 40 °C. methylene dichloride was added to the crude and kept in freezer for overnight. Filtered the reaction mass and distilled-off solvent under reduced pressure below 40 °C. Recrystalize the crude in ethyl acetate and diisopropyl ether to get OtBu-C18-Glu(AEEA-OSu)-OtBu.

Step-C:
Fmoc-Lys (AEEA-NH2)-OH (0.256 grams) was dissolved in THF and water. Sodium carbonate was added to the resulting reaction mixture at 25-30°C. Cooled the reaction mixture to 5-10 °C. OtBu-C18-Glu (AEEA-OSu)-OtBu (0.15 grams) in THF (0.52 mL) was added to the reaction mixture at 5-10°C and stirred for 15 minutes at the same temperature. Raised the temperature to 25-30 °C and stirred for 4 hours at the same temperature. filtered the reaction mixture and washed with n-heptane. The reaction mixture pH was adjusted to 7 with 20% KHSO4 and distilled-off solvent below 40 °C. Further, reaction mixture pH was adjusted to 3 to 3.5 with 20% KHSO4. Extracted the reaction mixture with methylene chloride and wash with water. Distilled-off the solvent below 40 °C to get Semalinker.

Example-7: Alternative process for the preparation of Semalinker
Step-A:
Fmoc-Lys(AEEA-NH2)-OH was dissolved in THF and water at 25-30°C. Sodium carbonate (0.18 grams) was added to the reaction mixture and cooled to 5-10 °C. Z-Glu (AEEA-OSu)-OtBu (0.67 grams) in THF was slowly added to the resulting reaction mixture at 5-10°C and stirred for 15 minutes at the same temperature. Raised the reaction mixture temperature to 25-30 °C and stirred for overnight at same temperature. Filtered the reaction mixture and washed with n-heptane. Reaction mixture pH was adjusted to 7 with 20% KHSO4 and distilled-off the solvent under reduced pressure below 40 °C. Further, the reaction mixture pH was adjusted to 3 to 3.5 with 20% KHSO4. Extracted the reaction mixture with methylene chloride and organic layer was washed with water and brine solution, distilled-off the solvent under reduced pressure below 40 °C to get Fmoc-Lys(Z-Glu(AEEA-AEEA)-OtBu)-OH.
Step-B:
Fmoc-Lys[Z-Glu(AEEA-AEEA)-OtBu]-OH obtained in step-A was dissolved in isopropanol. Acetic acid and palladium on carbon was added to the reaction mixture under nitrogen atmosphere. hydrogen gas was purged in autoclave and maintain the reaction mixture up to completion of reaction at 35-40 °C. Filtered the reaction mixture and concentrated HCl was added to the reaction mixture. Distilled-off the solvent under reduced pressure below 40 °C and obtained crude was washed with diisopropyl ether.
Step-C:
Fmoc-Lys [NH2-Glu(AEEA-AEEA)-OtBu]-OH.HCl obtained in step-B was dissolved in THF and water. Sodium carbonate solution was added the reaction mixture at 25-30°C.. Cooled the reaction mixture to 5-10 °C. OtBu-C18-OSu (Fragment-3) was added to the resulting reaction mixture at 5-10°C and stirred for 15 minutes at the same temperature. Raised the temperature to 25-30 °C and maintain mass for overnight. Filtered the mass and washed the reaction mixture with n-Heptane followed by mixture of diisopropyl ether and heptane (30:70). The reaction mixture pH was adjusted to 7 with 20% KHSO4 and distilled-off the solvent below 40 °C. Further, the reaction mixture pH was adjusted to 3-3.5 with 20% KHSO4. Extracted the reaction mass with methylene chloride and washed the organic layer with water. Distilled-off the solvent below 40 °C to get Semalinker.
,CLAIMS:We claim:

1. A process for the preparation of Fmoc-Lys[Oct-(otBu)-Glu-(otBu)-AEEA-AEEA]-OH (Semalinker) having the structural Formula I

which comprises;
a) condensation of compound of Fragment-3

with compound of Formula-[J]

in presence of base to obtain compound of Formula-[K];

b) reacting compound of Formula-[K] with N-hydroxy succinimide to obtain compound of Formula-[L];

c) condensation of compound of formula-[L] with Fragment-1

in presence of base and solvent to obtain Semalinker of Formula-I.

2. The process for the preparation of Fmoc-Lys(AEEA-NH2)-OH of Fragment-1 as claimed in claim 1, which comprises:

a) Condensing compound of Formula-[A]

with N-(Benzyloxy carbonyloxy) hydroxy succinimide of Formula-[B]

in presence of base to obtain compound of Formula-[C];

b) coupling of compound of formula-C with N-hydroxy succinimide in presence of coupling agent to obtain compound of Formula-[D];

c) coupling of compound of Formula-[D] obtained in step-b) with Fmoc-Lys-OH in presence of base to obtain compound of Formula-[E];

d) deprotection of compound of formula-[E] using a reducing agent to obtain Fmoc-Lys(AEEA-NH2)-OH of Fragment-1.

3. The process as claimed in claim 1, wherein said base is selected from the group consisting of potassium carbonate, lithium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, ammonium hydroxide and mixture thereof; the organic base is selected from the group consisting of diisopropyl amine, N, N-diisopropyl ethylamine, triethylamine, tertiary butyl amine, dimethylamine, tri methyl amine, isopropyl ethylamine, pyridine, N-methyl morpholine or a mixture.

4. The process as claimed in claim 1, wherein said solvent is selected from the group consisting of alcoholic solvents such as methanol, ethanol, isopropanol; chlorinated solvents such as chloroform, methylene chloride; ester solvents such as ethyl acetate, butyl acetate, isopropyl acetate; ether solvents such as diethyl ether, tetrahedrofuran, 2-methyl tetrahydrofuran, di isopropyl ether; aprotic solvents and protic solvents such as DMF, DMSO, DMAC, 1,4-dioxane or a mixture .

5. The process as claimed in claim 1, wherein said coupling agent is selected from group consisting of Dicyclohexyl carbodiimide (DCC), di isopropyl carbodiimide (DIC), 1-hydroxy benzotriazole (HOBt).

6. The process as claimed in claim 1, wherein said reducing agent is selected from group consisting palladium on carbon.