FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10, rule 13)
1. Title of the invention
New process for preparation of copolymer 1
2. Applicant(s)

Name

Nationality

Address

USV Limited

Indian company incorporated under Companies Act, 1956

B. S. D. Marg Govandi, Mumbai - 400 088 Maharashtra, India.

3. Preamble to the description
The following specification describes the invention.

Field of the invention:
The present invention relates to process for preparation of polypeptides or pharmaceutically acceptable salts thereof. The present invention particularly provides one pot process for preparation of Glatiramer acetate used for treating multiple sclerosis.
Background of invention:
One of the more common neurologic diseases in human adults is multiple sclerosis. This condition is a chronic, inflammatory CNS disease characterized pathologically by demyelination. Glatiramer acetate (GA), also known as Copolymer-1, has been shown to be effective in treating multiple sclerosis (MS) (Lampert, P. W.). Daily subcutaneous injections of Glatiramer acetate (20 mg/injection) reduce relapse rates, progression of disability, appearance of new lesions by magnetic resonance imaging (MRI), (Johnson. K. P. et al.) and appearance of black holes" (Filippi, M. et al.).
Glatiramer acetate is marketed under the name Copaxone containing Glatiramer acetate as the active ingredient. Glatiramer acetate is approved for reducing the frequency of relapses in relapsing- remitting multiple sclerosis. Glatiramer acetate consists of the acetate salts of synthetic polypeptides containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine and L-lysine with an average molar fraction of 0.141, 0.427, 0.095 and 0.338 respectively. It is synthesized by chemically polymerizing the four amino acids to yield the product with the desired molecular weight range. The average molecular weight of the Glatiramer acetate is 4,700-11,000 daltons [US label for lyophilized powder] or 5,000-9,000 daltons [US label for pre-filled syringe/Summary of Product Characteristics]. Copaxone comprises a mixture of polypeptides having different molecular weights and sequences. The structural formula of Copaxone is :
(Glu, Ala, Lys, Tyr)x. *CH3COOH
(CsH9NO4.C3H7NO2C6H,4N2O2.C9H11NO3)x - XC2H4O2 The process for preparation of Glatiramer acetate is described in Euro. J. Immune. 1, 242 (1971) [Tietelbaum et al] and US3849550 [Tietelbaum et al]. The process reported consists of polymerization of N-carboxyanhydrides of L-tyrosine, L-alanine, y-benzyl L-glutamate and NE-trifluoroacetyl L-lysine in anhydrous dioxane using diethyl amine as


initiator to afford protected copolymer (Scheme I). The deblocking of y-carboxyl group of glutamic acid in the protected copolymer is carried out by treating the protected copolymer with hydrogen bromide in acetic acid. The next step is the deprotection of the NE-trifluoroacetyl group of the copolymer is achieved by stirring with IM piperidine. In the last step copoJymer-1 (Glatiramer acetate) is obtained by purification of the crude copolymer-1 through dialysis, followed by treatment with acetic acid to form its corresponding acetate salt with final purification by dialysis against water. These prior art processes involve the polymerization of four N-carboxyanhydrides, two deprotection steps, two purification steps and one acetate salt formation step.

Patents which disclose the process for preparation of copolymer-1 include US5800808, US5981589, US6054430, US6342476, US6362161 and WOOO/05250. These patents elaborate on the process for preparing copolymer-1 involving polymerization of four N-carboxyanhydrides, two deprotection steps, two purification step and one acetate salt formation step and hence involves number of steps thereby making the process economically not viable. The deprotection step is carried out by treating the protected copolymer with hydrogen bromide in acetic acid. Hydrogen bromide in acetic acid is hazardous due to release of hydrogen bromide fumes. In addition, benzyl bromide is generated as reaction by product. The free benzyl bromide is a highly reactive electrophile and reacts with nucleophiles like primary and secondary amines to generate unwanted N-


alkylated products. Benzyl bromide being highly lachrymatory and handling it in large quantities on commercial scale is hazardous and unsafe. The above patents have not mentioned any method for the removal of benzyl bromide from the reaction mixtures. Thus the processes for preparation of Glatiramer acetate as disclosed above are industrially not feasible.
US6620847 describes process for preparation of copolymer-1 which comprises treating trifluoroacetyl copolymer-1 with aqueous piperidine to form a solution of copolymer-1 and purifying copolymer-1.
US 2004/0091956 describes a three step process for preparation of Glatiramer acetate comprising polymerization of a mixture of N-carboxyanhydrides of L-tyrosine, L-alanine, protected L-glutamate and protected L- lysine to obtain a protected copolymer. The deprotection of the protected copolymer is carried out by either catalytic transfer hydrogenation or palladium catalytic hydrogenation under hydrogen pressure. WO2004/043995, US 7049399 and US 2006/00172942 discloses process for preparation of Glatiramer acetate which involves polymerization of N-carboxyanhydrides of L-y-benzyl glutamate, NE-benzyl lysine, L- tyrosine and L- alanine, debenzylation is carried out either by catalytic hydrogenation under hydrogen pressure of 40-100 psi or by catalytic transfer hydrogenation (CTH). The disadvantage of this patent is that hydrogenolysis involved in the disclosed process requires pressure and high temperatures, which in turn needs additional precaution for operations on large scale, thus increasing the production cost. Additionally the product, Copaxone has to be stored at 2° to 8° C. Higher temperatures during final step may results in formation of degradants thus the quality and yields may be reduced. Further the protecting groups employed in the above patent are difficult to prepare and are not available in commercial quantities.
WO2006/050122 describes a two step process for preparing Glatiramer acetate. The process involves polymerization of a mixture of N-carboxyanhydrides of L-tyrosine, L-alanine, y-p-niethoxybenzyl/Y-benzyl L-glutamate and N-t-butoxycarbonyl L- lysine to obtain a protected copolymer or salt thereof. The deprotection of the protected copolymer or salt thereof is carried out by two different methods. First method for deprotection is by treatment with an acid and second method for deprotection is accomplished by treatment


with alkaline earth metal hydroxide. The disadvantage of this process is that use of alkaline earth metal hydroxide for deprotection of peptides result in slow reactions and formation of high levels of the diastereomer resulting from racemization/epimerization of the stereogenic centers (Tetrahedron Letters 39, 3391 (1998) [Ahmed F. Abdel-Magid et al]).
A need to develop a more efficient process was felt by the present inventors by overcoming the above said disadvantages The present invention provides a better process, by employing quaternary ammonium hydroxide compounds for the process for preparation of Glatiramer acetate which results in better yields and avoids the use of hazardous raw material and reagents such as HBr and piperidine as reported in prior art. Thus by providing new process for the preparation of Glatiramer acetate suitable for industrial scale the present inventors have addressed the problems associated with the prior art and have provided a cost effective, simple, scalable, robust process for preparation of Glatiramer acetate. The process avoids the cumbersome and tedious procedures of isolation, crystallization and purification of intermediates and hence it is economical and convenient to operate on a commercial scale. Therefore the process of the present invention is simple, cost effective, and industrially viable over the prior art procedures.
Object of the invention:
The object of the present invention is to provide a simple, commercially viable and cost effective process for the preparation of Glatiramer acetate which obviates the drawbacks of the prior art processes.
Another object of the present invention is to provide process for the preparation of Glatiramer acetate from protected copolymer by one pot deprotection of protected copolymer using quaternary ammonium hydroxide compounds having formula,

wherein Rj, R2, R3 and R, are same or different and are independently selected from alkyl,

aryl or other moiety linked via a carbon atom to the nitrogen atom; and X is a hydroxyl
moiety.
Another object of the present invention is to provide a single pot process for the
preparation of Glatiramer acetate with better yield and better purity where the
polymerization and the deprotection are carried out in a single pot.
Another object of the present invention is the synthesis of N-carboxyanhydride of the
corresponding amino acids using triphosgene, in the presence or the absence of activated
carbon (charcoal), preferably in the presence of activated carbon (charcoal).
Another object of the present invention is to avoid the use of hazardous raw material and
reagents reported in prior art.
Summary of the invention:
The present invention provides cost effective and better yielding process for the
preparation of Copolymer-1 using L-tyrosine, L-alanine, protected L-glutamic acid and
protected L-lysine or a pharmaceutically acceptable salt thereof where polymerization and
deprotection are carried out in a single pot.
According to one aspect of the present invention there is provided a process for
preparation of Copolymer-1 from the corresponding amino acids or pharmaceutically
acceptable salts thereof where deprotection of the protected copolymer are carried out in a
single pot.
According to another aspect of the present invention there is provided a process for
preparation of Copolymer-1 from the corresponding amino acids or pharmaceutically
acceptable salts thereof using quaternary ammonium hydroxide compounds.
According to another aspect of the present invention there is provided a process for the
synthesis of N-carboxyanhydride of the corresponding amino acids using triphosgene, in
the presence or the absence of activated carbon (charcoal), preferably in the presence of
activated carbon (charcoal).
Detailed description of the invention:
There is provided a process for preparation of copolymer-1, more particularly Glatiramer


acetate.
The present invention provides an improved process for the preparation of copolymer-1 from amino acids, namely, L-tyrosine, L-alanine, L-glutamic acid and L-lysine or a pharmaceutically acceptable salt thereof using quaternary ammonium hydroxide compounds.
According to one embodiment of the present invention there is provided one pot process for preparation of copolymer-1 or pharmaceutically acceptable salts thereof comprising the steps of; combining the mixture of N-carboxyanhydride of L-tyrosine, L-alanine, protected L-glutamic acid and protected L-lysine in suitable solvent in presence of an initiator to form a protected copolymer and in situ deprotection using quaternary ammonium hydroxide salt to get copolymer-1 or a pharmaceutically acceptable salt thereof as in Scheme II.

In one of the specific embodiment of the present invention, L-amino acid NCAs [N-carboxyanhydride of L-alanine, N-carboxyanhydride of y-protected L-glutamate, N-carboxyanhydride of N-protected L-lysine, N-carboxyanhydride of L-tyrosine] dissolved in suitable solvent is added to the solution of initiator in suitable solvent with vigorous stirring over a period of about 30-40 min. The resulting mixture is stirred at room temperature for 24 hrs and quaternary ammonium hydroxide compound is added in one


lot to the reaction mixture followed by addition of water with vigorous stirring for 6 hrs at room temperature. The obtained reaction mixture is then neutralized with suitable acid to maintain pH between 5.5-6.0. The reaction mass is filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs which may be further dialyzed against suitable acid followed by water to obtain pH 5.5-6.0. The obtained solution is then lyophilized to obtain the product.
Another embodiment of the present invention provides process for preparation of Glatiramer acetate comprising the steps of;
a) subjecting to polymerization a mixture of N-carboxyanhydrides of L-tyrosine, L-alanine, protected L-glutamic acid and protected L-lysine in suitable solvent in the presence of an initiator to get protected copolymer;
b) deprotecting the protected copolymer using quaternary ammonium hydroxide compound to get Glatiramer acetate, as shown in scheme III.
After polymerization step, the deprotection step comprises single-step removal of Y-protected group from L-glutamate moiety and N-protected group from the L-lysine moiety using quaternary ammonium hydroxide compound optionally in presence of aqueous condition or in presence of suitable solvents.

PG: protecting group
Scheme III
Another embodiment of the present invention provides process for the preparation of protected copolymer comprising the following steps,


a) preparing N-carboxyanhydride of L-tyrosine, L-alanine, protected L-glutamic acid and protected L-lysine by reacting the corresponding L-amino acids with triphosgene in a suitable solvent in presence or absence of activated carbon (charcoal), preferably in the presence of activated carbon (charcoal);
b) combining the mixture of N-carboxyanhydride of L-tyrosine, L-alanine, protected L-glutamic acid and protected L-lysine, in suitable solvent in presence of an initiator to get protected copolymer.
In accordance with another embodiment, the present invention provides a process for preparation of protected L-lysine co-polymer comprising the following steps,
a) preparing N-carboxyanhydride of L-tyrosine, L-alanine, protected L-glutamic acid and protected L-lysine, by reacting the corresponding L-amino acids with triphosgene in a suitable solvent in presence or absence of activated carbon (charcoal), preferably in the presence of activated carbon (charcoal);
b) combining the mixture of N-carboxyanhydride of L-tyrosine, L-alanine, protected L-glutamic acid and protected L-lysine, in a polar aprotic solvent in the presence of an initiator to get protected copolymer.
c) deprotecting the protected L-glutamic acid of the protected copolymer by using an acid to get protected L-lysine copolymer.
Another embodiment of the present invention provides process for the preparation of copolymer-1 comprising deprotecting the protected L-lysine co-polymer by using quaternary ammonium hydroxide compound to get a copolymer-1 or a pharmaceutical ly acceptable salt thereof.
Another embodiment of the present invention provides process for preparation of Glatiramer acetate comprising the steps of;
a) preparing N-carboxyanhydride of L-tyrosine, L-alanine, protected L-glutamic acid and protected L-lysine;
b) subjecting to polymerization a mixture of N-carboxyanhydride of L-tyrosine, L-alanine,


protected L-glutamic acid and protected L-lysine in suitable solvent in the presence of an initiator to get a protected copolymer;
c) deprotecting the protected L-glutamic acid using acid to get protected L-lysine copolymer;
d) deprotecting the protected L-lysine copolymer using quaternary ammonium hydroxide compounds; and
e) isolating Glatiramer acetate, as shown in Scheme IV.

The processes of the prior art involve number of steps and results in side product such as benzyl bromide responsible for generation of unwanted N-alkylated products thus are not feasible on commercial scale. The advantages of the present invention are:
1) avoiding use of bromine compound,
2) increased rate of reaction,
3) better yield and lower levels of side products.
According to another embodiment of the present invention N-carboxyanhydrides of amino acids are prepared by reacting corresponding L-amino acids with triphosgene in a suitable solvent at elevated temperatures, in presence or absence of activated carbon (charcoal), preferably in the presence of activated carbon (charcoal). The present inventors surprisingly found that use of activated carbon in the preparation of N-carboxyanhydride

of amino acids leads to decrease in the reaction time and produces good yield. Preparation of N-carboxyanhydride of L-tyrosine, L-alanine, Y-Pr°tected L-glutamate and Ne-protected L-lysine is carried out at temperature 40° to 80° C for about 1 to 4 hr. The more preferable, reaction temperature is 50° to 60° C for a period of about 2-3 hr. N-carboxyanhydrides obtained by the process of the present invention has purity in the range of 95% - 99%, chloride content less than 0.5%, preferably less than 0.1%, more preferably, less than 0.05%.
The polymerization of N-carboxyanhydrides of amino acids is carried out at temperature 20° to 60° C for a period of 10 hr to 60 hr, more preferably at temperature 25° to 35° C for a period of 20 hr to 50 hr. The protecting group (PG) for the free y-carboxy group of L-glutamic acid is selected from substituted alkyl or aryl group, most preferably benzyl group or substituted benzyl group and the protecting group (PG) for the free £-amino group of L-lysine is selected from acetyl, alkyl/aryl substituted acyl and halogen substituted acetyl groups, most preferably trifluoroacetyl group.
Deprotection of the protected L-glutamic acid and/or protected L-lysine is carried out at temperature 0° to 50° C for a period of 2 hr to 24 hr, preferably at temperature 20° to 40° C for a period of 4 hr to 14 hr, most preferably at temperature 25° to 35° C for a period of5hrto8hr.
According to preferred embodiment of the present invention the undesired lower molecular weight polypeptides formed during the process are removed by filtration. Filtration techniques employed to remove the undesired molecular weight copolymer as described herein are selected from dialysis, ultrafiltration or Tangential flow filtration.
The single pot process of the present invention gives copolymer-1, Glatiramer acetate in better yield and does not require isolation of the preceding reaction mass. The reaction proceeds at an accelerated rate and completes in about 30 hours as compared to the longer duration, viz. 50-70 hours, reported in prior art.


Copolymer-1 (Glatiramer acetate) obtained according to the present invention is characterized by MALDI, IR, NMR and GPC and is found to be in accordance with the innovator sample. Further studies are under progress.
The term "one-pot" or single-pot reaction as used herein are synonymous and means
reaction in which two or more processes are conducted in a single reaction vessel without
isolating or purifying the resulting intermediates.
The term "room temperature" should be understood to mean a temperature ranging from
about 20°C to about35°C.
Quaternary ammonium hydroxide compounds used herein is represented as

wherein R1, R2, R3 and R4 may be same or different and are independently selected from alkyl, aryl or other moiety linked via a carbon atom to the nitrogen atom; and X is a hydroxyl moiety. Quaternary ammonium hydroxide compound used in the present invention is selected from group consisting of tetramethyl ammonium hydroxide, tetraethyl ammonium hydroxide, tetrapropyl ammonium hydroxide, tetrabutyl ammonium hydroxide, benzyl trimethyl ammonium hydroxide, benzyl triethyl ammonium hydroxide, or choline hydroxide or mixtures thereof, preferably tetrabutyl ammonium hydroxide or tetramethyl ammonium hydroxide or mixtures thereof. The quaternary ammonium hydroxide compounds are used in the form of solid or in aqueous solution or in organic solvents. Alternatively, ammonium hydroxide may be employed in the place of quaternary ammonium hydroxide compounds.
Polymerization initiators can be selected from bases, nucleophiles or combinations thereof. In particular, polymerization initiator can include one or more amines, alcohols, water or combinations thereof. Amines employed may be primary, secondary or tertiary amine. Suitable amines include, but are not limited to, dimethylamine, diethylamine, di-n-propylamine, di-isopropylamine, N-ethylmethylamine, di-n-butylamine, di-iso-butylamine, di-sec butylamine, di-tert-buylamine, diamylamine, di-n-octylamine, di-(2-

ethylhexyl)-amine, di-isononyiamine, diallylamine, N-methylaniline, diphenylamine, aziridine, pyrrole, pyrrolidine, imidazole, indole, piperidine, purine, and combinations thereof, most preferable being diethylamine. Other polymerization initiators which can be used include K-tOBu, NaH, KH, triethylamine, tetramethyl piperdine, dicyclohexylamine, dicyclohexylundecane, lithiumdiisopropyl amine, t- BuLi or combinations thereof. The suitable solvent used is selected from the group consisting of water, alcohols, polar aprotic solvent and chlorinated solvents. The polar aprotic solvent are selected from methyl acetate, ethyl acetate, dimethyl furan, dimethylformamide, 1,4-dioxane, tetrahydrofuran or mixtures thereof. Chlorinated solvent can be selected from methylene dichloride, chloroform or ethylene dichloride. Alcohols can be selected from straight or branched chain alcohols. The preferred solvent used for deprotection is 1,4-dioxane, water or mixture thereof. In reactions where 1,4-dioxane and water are used as a solvent mixture, any ratio can be employed, preferable being 1:1.
The acid used may be inorganic acid or organic acid and is selected from the group consisting of hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphorous acid, phosphoric acid, fluorosulfonic acid, chlorosulfonic acid, acetic acid, formic acid, propionic acid, benzene sulfonic acid, methane sulfonic acid, p-toluenesulfonic acid and p-(n-dodecyl)benzene sulfonic acid.
Advantages of the present invention :
1) It provides an improved process for the preparation of Glatiramer acetate, where polymerization and deprotection are carried out in a single pot.
2) It provides a process for the preparation of Glatiramer acetate from the protected copolymer where the deprotection is carried out in a one pot process.
3) It provides the use of quaternary ammonium hydroxide compounds for the
simultaneous removal of the both the protecting groups of the protected copolymer.
4) Use of quaternary ammonium hydroxide compounds for deprotection of protected
copolymer helps to reduce the reaction time.


5) Preparation of N-carboxy anhydride of amino acids using triphosgene in presence of activated charcoal decreases the reaction time and provides the product with better yield and purity.
6) The present invention is devoid of hydrogenation for deprotection of protected copolymer as mentioned in the prior art, which involves pressure and high temperature, thereby reducing the production cost and minimizing safety operations.
7) The present invention employs protecting groups which are easy to prepare and are available in commercial quantities.
8) The present invention provides a cost effective, simple, scalable, robust process, for obtaining glatiramer acetate, with better yield and purity.
While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are included within the scope of the present invention. The examples are provided to illustrate particular aspects of the disclosure and do not limit the scope of the present invention.
Examples: Example 1:
25 g (0.28 moles) of L-alanine and activated charcoal (1.25 g) were suspended in 550 ml of tetrahydrofuran (THF). The reaction mixture was heated to 50°-55°C for 1 hr. 41.5 g (0.14 moles) of triphosgene, dissolved in 200 ml of THF, was added to the obtained suspension over a period of about 10 min at 50-55° C. The reaction mixture was stirred for 2 hr at 50-55° C. After the completion of reaction, the reaction mixture was cooled to room temperature, filtered through hyflo bed. The obtained filtrate was then concentrated under vacuum to get an oil. 200 ml ethyl acetate was added to the obtained oil, stirred for 10 min and filtered. 400 ml of hexane was added to the obtained filtrate to get solid. The obtained solid was filtered, washed with hexane and dried. Yield : 16.25 g (65% w/w w.r.t. amino acid).


Example 2:
25 g (0.105 moles) of Y-benzyl L-glutamate and activated charcoal (1.25 g) were suspended in 300 ml of tetrahydrofuran (THF). The reaction mixture was heated to 50°-55°C for 1 hr. 15.43 g (0.052 moles) of triphosgene dissolved in 200 ml of THF was added to the obtained suspension over a period of about 10 min at 50-55° C. The reaction mixture was stirred for 1 hr at 50-55° C. After the completion of reaction, the reaction mixture was cooled to room temperature, filtered through hyflo bed. The obtained filtrate was then concentrated under vacuum to get an oil. 200 ml hexane was added to the obtained oil to get solid. The obtained solid was filtered, washed with hexane and dried. Yield : 18.5 g (74% w/w w.r.t. amino acid).
Example 3:
25 g (0.102 moles) of NE-trifluoroacetyl L-lysine and activated charcoal (1.25 g) were suspended in 300 ml of tetrahydrofuran (THF). The reaction mixture was heated to 50°-55°C for 1 hr. 15.13 g (0,051 moles) of triphosgene dissolved in 200 ml of THF was added to the obtained suspension over a period of about 10 min at 50-55° C. The reaction mixture was stirred for 1 hr at 50-55° C. After the completion of reaction, the reaction mixture was cooled to room temperature, filtered through hyflo bed. The obtained filtrate was then concentrated under vacuum to get an oil. 200 ml hexane was added to the obtained oil to get solid. The obtained solid was filtered, washed with hexane and dried. Yield : 22.5 g (90% w/w w.r.t. amino acid).
Example 4:
25 g (0.137 moles) of L-tyrosine and activated charcoal (1.25 g) were suspended in 300 ml of tetrahydrofuran (THF). The reaction mixture was heated to 50°-55°C for 1 hr. 20.33 g (0.068 moles) of triphosgene dissolved in 200 ml of THF was added to the obtained suspension over a period of about 10 min at 50-55° C. The reaction mixture was stirred for 1 hr at 50-55° C. After the completion of reaction, the reaction mixture was cooled to room temperature, filtered through hyflo bed. The obtained filtrate was then concentrated under vacuum to get an oil. 200 ml hexane was added to the obtained oil to


get solid. The obtained solid was filtered, washed with hexane and dried. Yield : 17.5 g (70% w/w w.r.t. amino acid).
Example 5:
A solution of diethylamine (0.7 ml) in 1.5 L of anhydrous dioxane was stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (50 g), N-carboxyanhydride of Y-benzyl L- glutamate (35 g), N-carboxyanhydride of N£-trifluoroacetyl L-lysine (83 g), N-carboxyanhydride of L-tyrosine (18 g)] dissolved in 2 L of anhydrous dioxane was added to it over a period of about 30 min. The resulting mixture was stirred at room temperature for 24 hrs. Deionized water was added to the reaction mixture to get white solid. The obtained solid was filtered under vacuum and dried under nitrogen atmosphere. Yield 130 g (69.9% w/w).
Example 6:
A solution of diethylamine (0.19 ml) in 150 ml of anhydrous dioxane was stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (5 g). N-carboxyanhydride of y-benzyl L- glutamate (3.5 g), N-carboxyanhdride of N£-trifluoroacetyl L-lysine (8.3 g), N-carboxyanhdride of L-tyrosine (1.8 g)] dissolved in 200 ml of anhydrous dioxane was added to it over a period of about 30 min. The resulting mixture was stirred at room temperature for 24 hrs. Deionized water was added to the reaction mixture to get white solid. The obtained solid was filtered under vacuum and dried under nitrogen atmosphere. Yield 12.0 g (64.5% w/w).
Example 7:
A solution of diethylamine (0.26 ml) in 150 ml of anhydrous dioxane was stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (5 g), N-carboxyanhydride of y-henzyl L- glutamate (3.5 g), N-carboxyanhydride of NE-trifluoroacetyl L-lysine (8.3 g), N-carboxyanhydride of L-tyrosine (1.8 g)] dissolved in 200 ml of anhydrous dioxane was added to it over a period of about 30 min. The resulting mixture was stirred at room temperature for 24 hrs. Deionized water was added to the


reaction mixture to get white solid. The obtained solid was filtered under vacuum and dried under nitrogen atmosphere. Yield 11.5 g (61.8% w/w).
Example 8:
A solution of diethylamine (0.54 ml) in 150 ml of anhydrous dioxane was stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (5 g), N-carboxyanhydride of Y-benzyl L- glutamate (3.5 g), N-carboxyanhydride of NE-trifluoroacetyl L-lysine (8.3 g), N-carboxyanhydride of L-tyrosine (1.8 g)] dissolved in 200 ml of anhydrous dioxane was added to it over a period of about 30 min. The resulting mixture was stirred at room temperature for 24 firs. Deionized water was added to the reaction mixture to get white solid. The obtained solid was filtered under vacuum and dried under nitrogen atmosphere. Yield 10.9 g (58.6% w/w).
Example 9:
Protected copolymer (1.0 g) of Example 5 was treated with 33% HBr in acetic acid (8.0 ml) and stirred at room temperature for 17 hr. Ice-cold water (100 ml) was added to the reaction mixture to get a white solid. The solid was filtered and washed with water. Yield 0.6 g ( 60% w/w).
Example 10:
Protected copolymer (14 g) of Example 5 was treated with 33% HBr in acetic acid (112 ml) and stirred at room temperature for 17 hr. Ice-cold water (700 ml) was added to the reaction mixture to get a white solid. The solid was filtered and washed with water. Yield 9.4 g( 67.14% w/w).
Example 11:
Trifluoroacetyi copolymer (0.2 g) of Example 9 was suspended in water (10 ml). Tetrabutyl ammonium hydroxide (0.4 g) was added to the suspension in one lot and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a
2 3 APR 2009

1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until pH 5,5-6.0 is achieved. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield 0.10 g (50% vv/w).
Example 12:
Trifluoroacetyl copolymer (9.4 g) of Example 10 was suspended in water (470 ml). Piperidine (54.52 ml) was added to the suspension in one lot and stirred vigorously. The reaction mixture was stirred for 24 hrs at room temperature. The reaction mixture was filtered, passed through tangential flow filtration having molecular weight cut off (MWCO) cassette of 1 KD and concentrated to half the volume. Added 0.3% aqueous acetic acid to the concentrated solution till pH 5-6, then lyophilized to dryness to obtain a white fluffy solid. Yield 6 g (63.8% w/w).
Example 13:
Protected copolymer (0.2 g) of Example 5, was taken in a single neck flask provided with a magnetic stirrer. Dioxane (7 ml) was added to the flask and stirred for about 10 min followed by addition of water (7 ml). 0.8 g of tetrabutyl ammonium hydroxide was added in one lot to the reaction mass and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until a pH 5.5-6.0 was obtained. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield 0.045 g (22.5% w/w).
Example 14:
Protected copolymer (2 g) of Example 5, was taken in a single neck flask provided with a magnetic stirrer. Dioxane (70 ml) was added to the flask and stirred for about 10 min followed by addition of water (70 ml). 8 g of tetrabutyl ammonium hydroxide was added


in one lot to the reaction mass and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, and the filtrate was subjected to ultrafiltration using a 1 KD membrane and concentrated to half the volume. The obtained concentrated solution was then lyophilized to dryness to obtain a white fluffy solid. Yield 0.54 g (27% w/w).
Example 15:
Protected copolymer (10 g) of Example 5 is taken in a single neck flask provided with a magnetic stirrer. Dioxane (350 ml) is added to the flask and stirred for about 10 min followed by addition of water (350 ml). 40 g of tetrabutyl ammonium hydroxide is added in one lot to the reaction mass and stirred vigorously. The reaction mixture is stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture is diluted with 300 ml of deionized water and passed through tangential flow filtration having MWCO cassette of 1 KD and concentrated to half the volume. The obtained concentrated solution is then lyophilized to dryness to obtain a white fluffy solid.
Example 16:
Protected copolymer (0.5 g) of Example 6, was taken in a single neck flask provided with a magnetic stirrer. Dioxane (17.5 ml) was added to the flask and stirred for about 10 min followed by addition of water (17.5 ml). 2 g of tetrabutyl ammonium hydroxide was added in one lot to the reaction mass and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until a pH 5.5-6.0 was achieved. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield: 0.12g (24%w/w)
Example 17:
Protected copolymer (0.2 g) of Example 7, was taken in a single neck flask provided with a magnetic stirrer. Dioxane (7 ml) was added to the flask and stirred for about 10 min


followed by addition of water (7 ml). 0.8 g of tetrabutyl ammonium hydroxide was added in one lot to the reaction mass and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until a pH 5.5-6.0 was achieved. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield: 0.05g (25%w/w)
Example 18:
Protected copolymer (0.2 g) of Example 8, was taken in a single neck flask provided with a magnetic stirrer. Dioxane (7 ml) was added to the flask and stirred for about 10 min followed by addition of 7 ml of water. Tetrabutyl ammonium hydroxide (0.8 g) was added to the above mixture in one lot and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until pH 5.5-6.0 was achieved. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield: 0.055 g (27.5% w/w).
Example 19:
Protected copolymer (0.2 g) of Example 5, was taken in a single neck flask provided with a magnetic stirrer. Dioxane (5 ml) was added to the flask and stirred for about 10 min followed by addition of water (6 ml). 1.6 ml of 25 % aqueous tetramethyl ammonium hydroxide was added to the reaction mass and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until a pH 5.5-6.0 was achieved. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield: 0,064 g (32% w/w).


Example 20:
Protected copolymer (10 g) of Example 5, is taken in a single neck flask provided with a magnetic stirrer. Dioxane (250 ml) is added to the flask and stirred for about 10 min followed by addition of water (300 ml). 80 ml of 25 % aqueous tetramethyl ammonium hydroxide is added to the reaction mass and stirred vigorously. The reaction mixture is stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture is diluted with 450 ml of deionized water and passed through tangential flow filtration having MWCO cassette of 1 KD and concentrated to half the volume. The obtained concentrated solution is then lyophilized to dryness to obtain a white fluffy solid. Yield: 3.7 g (37% w/w).
Example 21:
A solution of diethylamine (0.007 ml) in 15 ml of anhydrous dioxane was stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (0.5 g), N-carboxyanhydride of y-benzyl L- glutamate (0.35 g), N-carboxyanhydride of NE-trifluoroacetyl L-lysine (0.83 g), N-carboxyanhydride of L-tyrosine (0.18 g)] dissolved in 20 ml of anhydrous dioxane was added to it over a period of about 30 min. The resulting mixture was stirred at room temperature for 24 hrs. 25 ml of water was added to the resulting mixture followed by addition of tetrabutyl ammonium hydroxide (5.2 g) in one lot and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until pH 5.5-6.0 was achieved. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield: 0.68g (36% w/w)
Example 22:
A solution of diethylamine (0.07 ml) in 150 ml of anhydrous dioxane is stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (5 g), N-carboxyanhydride of y-benzyl L- glutamate (3.5 g), N-carboxyanhydride of NE-


trifluoroacetyl L-lysine (8.3 g), N-carboxyanhydride of L-tyrosine (1.8 g)] dissolved in 200 ml of anhydrous dioxane is added to it over a period of about 30 min. The resulting mixture is stirred at room temperature for 24 hrs. 250 ml of water is added to the resulting mixture followed by addition of tetrabutyl ammonium hydroxide (52 g) in one lot and stirred vigorously. The reaction mixture is stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture is filtered, passed through tangential flow filtration having MWCO cassette of 1 KD and concentrated to half the volume. The obtained concentrated solution is then lyophilized to dryness to obtain a white fluffy solid.
Example 23:
A solution of diethylamine (0.026 ml) in 15 ml of anhydrous dioxane was stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (0.5 g), N-carboxyanhydride of y-benzyl L- glutamate (0.35 g), N-carboxyanhydride of NE-trifiuoroacetyl L-lysine (0.83 g), N-carboxyanhydride of L-tyrosine (0.18 g)] dissolved in 20 ml of anhydrous dioxane was added to it over a period of about 30 min. The resulting mixture was stirred at room temperature for 24 hrs. 25 ml of water was added to the resulting mixture followed by addition of tetrabutyl ammonium hydroxide (5.2 g) in one lot and stirred vigorously. The reaction mixture was stirred for 6 hrs at room temperature, neutralized with acetic acid to pH 5.5-6.0. The reaction mixture was filtered, loaded into a 1 KD dialysis bag, dialyzed against water for about 3 hrs. It was then dialyzed against about 0.3% acetic acid and again with water until a pH 5.5-6.0 was achieved. The obtained solution was then lyophilized to dryness to obtain a white fluffy solid. Yield: 0.70 g (37.6% w/w).
Example 24:
A solution of diethylamine (0.26 ml) in 150 ml of anhydrous dioxane is stirred vigorously and a mixture of L-amino acid NCAs [N-carboxyanhydride of L-alanine (5 g), N-carboxyanhydride of y-benzyl L- glutamate (3.5 g), N-carboxyanhydride of N£-trifluoroacetyl L-lysine (8.3 g), N-carboxyanhydride of L-tyrosine (1.8 g)] dissolved in


200 ml of anhydrous dioxane is added to it over a period of about 30 min. The resulting mixture is stirred at room temperature for 24 hrs. 250 ml of water is added to the resulting mixture followed by addition of tetrabutyl ammonium hydroxide (52 g) in one lot and stirred vigorously. The reaction mixture is stirred for 6 hrs at room temperature, neutralized with acetic acid to pH=5.5-6.0. The reaction mixture is filtered, passed through tangential flow filtration having MWCO cassette of 1 KD and concentrated to half the volume. The obtained concentrated solution is then lyophilized to dryness to obtain a white fluffy solid.
Dated this 20th day of April 2009.

Dr. K. G.Rajendran Head-Knowledge Cell USV LIMITED