FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION
(See section 10)
"A PROCESS FOR THE PREPARATION OF
PEPTIDES"
USV Ltd, an Indian company established under the Companies Act 1956, having
office at B.S.D. Marg, Govandi, Mumbai 400 088. India
The following specification particularly describes the nature of this invention and the
manner in which it is to be performed


l


A PROCESS FOR THE PREPARATION OF PEPTIDES
Technical field
The present invention relates to an improved prócess for the preparation of
N -(aminoiminomethyl)-N2-(3-mercapto-l-oxopropyl-L-lysyIglycyl-L-a-aspartyl-L-
tryptophyl-L-prolyl-L-cysteinamide, cyclic(l→6)-disulfide of formula (1) using solid
phase Fmoc-chemistry.
Background and prior art references of the invention
US Patent No.5318899 describes N6-(aminoiminomethyl)-N2-(3-mercapto-l-
oxopropyl-L-lysylglycyl-L-a-aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic
(l→6)-disulfide of the formula (1) as a therapeutic agent for the treatment of, and
prevention of, platelet-associated ischemic disorders. It binds to the platelet receptor
glycoprotein (GP) of human platelets and inhibits platelet aggregation. Platelet
aggregation is mediated by GP complex on the surface of the platelet membrane. It
exists on the surface of unstimulated platelets in an inactive form. When platelets are
activated by adhesion and the physiological agonists, the GP also becomes activated
such that it becomes a receptor for fibrinogen, von Willebrand Factor (vWF), and
fïbronectin. However, it is the binding of fibrinogen and/or vWF that is believed to be
principally responsible for platelet aggregation and thrombus formation in vivo. This
teaches that substances which specifically inhibit the binding of fibrinogen or vWF to
GP, inhibit platelet aggregation and could be candidates for inhibiting thrombus
formation in vivo (Eric J. Topoi, Tatiana V. Byzova, Edward F. Plow and The Lancet;
Vol 353; January 16, 1999; pg 227-231). This article describes the compound having
platelet aggregation inhibition activity but does not teach the method to synthesize the
molecule.
Antagonists of platelet glycoprotein Ilb/IIIa have an approved role in reducing
the extent of thrombotic complications leading to myocardial damage during
percutaneous coronary interventions (PCI).
Compound of formula (1) is a disulphide looped cyclic heptapeptide containing
six amino acids and one mercaptopropionyl(desamino cysteinyl) residue. The disulfïde
bridge is formed between the cysteine amide and the mercaptopropionyl moieties. It is
known to be produced by solution-phase peptide synthesis and purified by preparative
2

reverse phase liquid chromatography and lyophilized
(www.fda.gov/cder/foi/label/1998/207181bl.pdf).
In terms of peptide synthesis methodology, two major synthetic techniques
dominate current practice. These are synthesis in solution (homogeneous phase) and
synthesis on solid phase (heterogeneous phase). But solution phase route is more
cumbersome as compared to the solid phase route as after each coupling the peptide
formed has to be isolated, whereas in the solid phase synthesis, the excess reagents and
by-products are washed off by simple filtration. In both, the desired peptide compound
is prepared by the step-wise addition of amino acid moieties to a building peptide
chain.
US patents 5958732 and 5318899 claim about recombinant techniques to
synthesize peptides like N6-(aminoiminomethyl)-N2-(3 -mercapto-1 -oxopropyl-L-
lysylglycyl-L-a-aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic(l→6)-disulfide
of the formula (1). The peptide obtained by this recombinant process is modified by
solution phase synthesis for conversion of lysine residue to homoarginine residue.
These patent documents also claim solid phase synthesis using Boe chemistry and the
subject matter of these patents is fundamentally different from the present invention.
As compared to Boc-chemistry, Fmoc-chemistry based synthesis utilizes a mild
procedure and because of the base lability of Fmoc group, acid-labile side-chain
protecting groups are employed providing orthogonal protection. The rationale for use
of protecting groups is that the energy of breaking a bond of a protecting group is
lower than any other group.
Patents US5686566, US5686567, US5686569, US5686570 and US5756451
deal with different PAI's in their salt or other forms of the compound of formula (1)
but do not teach the process for its preparation using Fmoc solid phase synthesis.
Likewise, patents US5759999, US5786333, US5770564, US5807825,
US5807828, US5843897, US5968902, and US5935926 describe the method of treating
platelet-associated disorders and the process for the preparation of peptide amide of
formula (1) using boe chemistry.
3

Patents US5344783 and US5851839 deal with methods for selecting and
identifying Platelet Aggregation Inhibitors (PAI) and disclose boe chemistry for the
preparation of peptide amide of formula (1).
US patent 5780595 claims antibodies specific to P AI's and also discloses boe
chemistry for the preparation of the peptide amide of formula (1).
The Fmoc route of synthesis of various other peptides is well-known in prior art
and several documents are available for their preparation. However there is a deflnite
need to develop a process for the preparation of compound of formula (1) which is
economical, involves minimal steps and also eco-friendly.
As explained earlier, Fmoc-chemistry based synthesis utilises a mild procedure
and because of the base lability of Fmoc group, acid-labile side-chain protecting
groups are employed providing orthogonal protection. The protecting groups used in
Fmoc chemistry are based on the tert-butyl moiety: tert-butyl ethers for Ser, Thr, tert-
butyl esters for Asp, Glu and Boe for Lys, His. The trt and acm groups have been
used for the protection of Cys. Theguanidine group of Arg and Har is protected by Mtr,
Pmc or Pbf. Most of the Fmoc-amino acids derivatives are commercially available.
However, a problem exists in the art for the preparation of some amino acid analogs
like peptides containing homoarginine as well as cyclic peptide compounds based on
disulfide links, because separate operations are required before purifying the end
product, which increases expense and may affect final product purity and yield. Fmoc-
homoarginine residue if purchased commercially for use in the assembly of the chain
becomes expensive. Alternatively in the peptide assembly, the Har unit is built by
guanylation of the lysine residue at the ot-NH2, which has been demonstrated to obtain
vasopressin analogues for the evaluation of its biological activity (Lindeberg et al, Int.
J. Peptide Protein Res.10, 1977, 240-244).
WO 03/093302 discloses the synthesis of the peptide of formula (1) using
Fmoc-a-nitrogen protected Ca-carboxamide cysteine. It describes the attachment of
the first amino acid, cysteine in the precipitated form to the solid support 4-
methoxytrityl polystyrene resin through its thiol side chain, followed by removing the
a-nitrogen protecting group and assembling the peptide on the said nitrogen. However,
the process uses the solid support - 4-methoxytrityl polystyrene resin which is not a
4

common commercial embodiment and also the Fmoc-a-nitrogen protected Ca-
carboxamide cysteine is not commercially available. This enables the process having
increased number of steps and also expensive with respect to the process of the present
invention. The cleavage conditions utilize ethanedithiol, which makes the process
highly toxic and non-environment fhendly requiring the use of expensive scrubbers.
The use of Fmoc-homoarginine residue in the assembly of the chain is mentioned,
which if purchased commercially, also makes the process very expensive. Overall, the
process claimed in this document is different from the process claimed in the present
invention. In addition the process of WO 03/093302 is associated with certain
limitations, which has been overcome by providing suitable modifications in the
process steps of the present invention.
Thus process of the present invention is an improved and efficiënt process over
the one described in W003/093302-A2 patent publication as herein mentioned below.
1 Does not involve the production of -SH peptide, which is susceptibïe to
aerial oxidation leading to the formation of impurities, which hamper the
purification of the fïnal product and yield.
2 Precise selection of protecting groups for amino acids to build the peptide
chain,
3 Activation of carboxylic function of the amino acid using appropriate
activating reagent to prevent the racemization of amino acids.
4 Efficiënt process for obtaining disulfide loop in the peptide amide of
formula (1) from silver-peptide salt intermediate without isolating -SH
peptide.
A considerable number of known, naturally occurring small and medium-sized
cyclic peptides as well as some of their synthetic derivatives and analogs possessing
desirable pharmacological properties have been synthesized. However, wider medical
use is often hampered due to complexity of their synthesis and purification. Therefore,
improved methods for making these compounds in simple, lesser steps and at lesser
cost are desirable and it is the need of the industry and mankind.
The purity and yield of the peptide are important aspects of any route of
synthesis. Yield, represented by the relative content of the pharmacologically active
compound in the final product, should be as high as possible. Purity is represented by
5

the degree of presence of pharmacologically active impurities, which though present in
tracé amounts only, may disturb or even render useless the beneficial action of the
peptide when used as a therapeutic agent. In a pharmacological context both aspects
have to be considered. As a rule, purification becomes increasingly difficult with larger
peptide molecules. In homogeneous (solution) phase synthesis (which is the current
method of choice for industrial production of larger amounts of peptides) repeated
purification required between individual steps provides a purer product but low yield.
Thus, improvements in yield and purification techniques at the terminal stages of
synthesis are needed. The present invention is an industrially feasible solid phase
synthesis and is a novel process to yield a high purity product with enhanced yield.
Prior art describes the use of HOBt and DIC for activation of amino acids,
which leads to the formation of the OBt ester. However, a major drawback in using this
procedure is that the preparation of the OBt ester itself takes about 20 min and also the
reaction has to be carried out at 0°C. The step-wise introduction of Na-protected
amino acids in SPPS normally involves in situ carboxyl group activation of the
incoming amino acid or the use of pre-formed activated amino acid derivatives. In
recent years, aminium and phosphonium based derivatives (HBTU, TBTU, Py Boe,
and HATU) have become the preferred tools for in situ carboxyl activation. They have
been shown to give superior results in terms of both coupling efficiency and
suppression of enantiomerization. (Fmoc Solid Phase Peptide Synthesis by Chan W.C.
and White P.D., Oxford University Press, 2000, p. 41 - 74) Use of HBTU provides
high yield and high purity. It saves time in the activation step with no cooling required.
Doublé coupling is also not required for Mpr(Acm)-OH.
Most of the Fmoc-amino acids derivatives are commercially available.
However, a problem exists in the art for the preparation of some amino acid analogs
like peptides containing homoarginine as well as cyclic peptide compounds based on
disulfide links, because separate operations are required before purifying the end
product, which increases expense and may affect final product purity and yield, Fmoc-
homoarginine residue if purchased commercially for use in the assembly of the peptide
chain becomes very expensive. Altematively the peptide assembly can be built using
lysine followed by guanylation of the lysine residue at the O-NH2 (Lindeberg et al,
Int.J. Peptide Protein Res.10, 1977, 240-244).
6

Oxidative cyclization of protected or non-protected sulthydryl groups with formation
of disulfide structures is usually carried out as the final synthetic step, the reason being
substantial thermal and chemical lability of the disulfide linkage. In few cases it is also
carried out before cleavage of the peptide molecule from the solid support. The
oxidation of open-chain peptides containing free and/or certain types of protected
sulfhydryl groups with iodine in, e.g., methanol or acetic acid is further explained in
the CRC Handbook of Neurohypophyseal Hormone Analogs, Vol. l, Part 1; Jost, K., et
al. Eds., CRC Press, Boca Raton, Fla. 1987, p. 31. Iodine, however, is not without
drawbacks as a cyclization agent. For instance, tryptophan moieties present in peptide
substrates are at risk of being iodinated, making the balance between full conversion of
starting materials and minimizing side reactions a delicate one, which, in turn, impacts
product purity. Tam (Tam J.P. et al., 1990, J. Am. Cheni. Soc., Vol. 113, p. 6657) has
demonstrated that the use of 20 - 50% solutions of PMSO in a variety of buffer
systems greatly promotes disulfide bond formation in comparison with other methods
such as aerial oxidation. DMSO is also found to greatly reduce and in some instances,
suppress completely, the aggregation and precipitation of peptides that occurred using
other oxidative procedures. Thus, the yield and purity of the disulfide looped peptide
oxidized by DMSO is much higher as compared to other known methods. In the
present invention this aspect has been rightfully tackled by not opting for Iodine route
for oxidative cyclization. Also in the present invention the silver salt of peptide amide
in place of peptide amide containing thiol group is subjected to oxidation without
isolation of SH-peptide and eliminating the formation offside products during
oxidation reaction. Thus the process steps of deprotection followed by oxidation of
guanylated peptide amide adopted in the present invention yields crude peptide amide
comprising compound of formula (1) of enhanced purity and yield. Finally purification
of the crude peptide result in enhanced yield of the final pure peptide.
Another complicating factor in known routes of synthesis is the possibility of
interaction between the desired cyclic disulfide and inofganic sulfur compounds used
for reducing excess iodine at the end of the reaction, such as sodium dithionite or
sodium thiosulfate. Such reducing sulfur-containing compounds may interact with the
disulfide linkage, which is sensitive to nucleophilic attack in general. As the process
of the present invention has avoided use of iodine, the resulting products have high
purity and related impurities are undetectable.
7

The process is accomplished in a few easy and simple steps. The use of solid
phase synthesis makes the process simpler and the use of Fmoc-chemistry eliminates
the use of harsh chemicals like HF, thereby not affecting the product stability. The
process eliminates purification of the intermediates, thereby increasing the yield and
reducing the cost. Replacement of thiols as scavengers in step (b) and (e) makes the
process more environment friendly and economical by flot having to use scrubbers for
thiols.
The choice of process often dictates the stability of the therapeutic peptide.
There has been a long awaited requirement for obtaining peptide of formula (1) which
will circumvent the limitations associated with the processes of prior art. Therefore, an
industrial process of peptide synthesis containing tryptophan, disulfide loops, s-NH2
side chain, etc demands appropriate choice of protecting groups and reaction
conditions to build up the peptide chain. This objective has been now successfully
achieved by the Applicant developing a process described in entirety in the present
application.
Glossary of terms used in the specification
AA Amino Acid
Acm Acetamidomethyl
ACT Activator
ADP Adenosine dïphosphate
AgOTf Silver trifluoromethane sulfonate
Arg Arginine
Asp Aspartic Acid
Boe / boe tert-butyloxycarbonyl
Cys Cysteine.
DCM Dichloromethane
DEP Deprotection reagent
DMF Dimethyl Formamide
DMSO Dimethyl sulphoxide
DTT Dithiothreitol
EDT Ethane dithiol
Fmoc 9-fluorenylmethyloxycarbonyl
Glu Glutamic acid
Gly Glycine
HBTU 2-( l H-Benzotriazole l -yl)-l, l ,3,3-tetramethyluronium
hexafluorophosphate
HF Hydrogen Fluoride
HIC Hydrophobic Interaction ChromatOgraphy
His Histidine
IEC Ion Exchange Chromatography
8

LC-MS Liquid Chromatography-Mass Spectroscopy
Lys Lysine
Mpr Mercaptopropionic Acid
Mtr 4-methoxy-2,3,6-trimethylbenzenesulfonyl
NMM N-methyl morpholine
Obut 0-t-butyl
Pbf 2,24,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
Pmc 2,2,5,7,8-pentamethylchroman-6-sulfonyl
PPP Platelet poor plasma
Pro Praline
PRP Platelet rich plasma
RP-HPLC Reverse Phase High Performance Liquid Chromatography.
RV Reaction Vessel
S er Serine
SOLV Solvent
SP Synthetic Peptide
TEA Triethylamine
TFA Trifluoroacetic acid
Thr Threonine
TIS Triisopropylsilane
Trp Tryptophan
Trt Trityl
Objects of the Invention
The main object of the present invention is to providean improved process to
obtain N6-(aminoiminomethyl)-N2-(3-mercapto-l-oxopropyl-L-lysyIglycyl-L-a-
aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic(l→6)-disulfide of formula (1).
Another object of the present invention is to disclose a process for obtaining
high yield and highpurity of peptide amide of formula (1)
Yet another objective of the present invention is to disclose a process of solid
phase synthesis of peptide amide of formula (1) by using Fmoc chemistry.
Still another object of the present invention is to disclose a process for the
production of peptide of formula (1), having lesser number of steps as compared to
solution phase synthesis.
Yet another object of the present invention is to design a process for the
production of peptide amide of formula (1), which is devoid of limitations associated
with prior art solid phase synthesis of compound of formula (1).
Still yet another object is to provide a process for preparing small and medium-
size peptides containing a disulfide moiety having enhanced purity
9

Still yet another object of the present invention is to select appropriate
protecting groups and reagents to minimize the formation of accompanying impurities
in process steps, thereby enhancing the yield and reducing the cost.
Summary of the Invention
The present invention relates to an improved process for the preparation of N6-
(aminoiminomethyI)-N2-(3-mercapto-l-oxopropyl-L-lysylglycyl-L-a-aspartyl-L-
tryptophyl-L-prolyl-L-cysteinamide, cyclic(l→6)-disulfide of formula (1), which
involves assembling amino acid residues and a thioalkyl carboxylic acid with an
appropriate protecting groups on a solid phase resin, cleaving the peptide thus obtained
from the resin with concomitant removal of side chain protecting groups except Acm
protecting group of thiol moiety to obtain peptide amide of formula (3), converting
lysine residue of peptide amide of formula (3) having protected thiol group to
homoarginine residue by guanylation, followed by simultaneous deprotection,
obtaining silver peptide of formula (5) and oxidation of silver peptide to obtain crude
peptide amide of formula(l) and finally subjecting to chromatographic purification.
The described process is simple, easy, environment friendly and cost effective.
Brief description of figures and Table
Fig. 1: Analytical RP-HPLC elution profile of HBTU- crude peptide from resin
(Column: PEP 300; C-18; 5μ; 150 X 3 mm; Flow rate: 0.5ml/min; Injection vol: 20μl;
Solvent System: A: 0.1% TFA, B: 100% Acetonitrile).
Fig. 2: Analytical RP-HPLC elution profile of DIC- crude peptide from resin
(Column: PEP 300; C-18; 5^; 150 X 3 mm; Flow rate: 0.5ml/min; Injection vol: 20μl;
Solvent System: A: 0.1% TFA, B: 100% Acetonitrile).
Fig. 3: Analytical RP-HPLC elution profile of crude guanylated peptide
(Column: PEP 300; C-18; 5μ; 150 X 3 mm; Flow rate: 0.5ml/min; Injection vol: 20μ1;
Solvent System: A: 0.1% TFA, B: 100% Acetonitrile).
Fig. 4: Analytical RP-HPLC elution profile of SH peptide (Column: PEP 300;
C-18; 5μ; 150 X 3 mm; Flow rate: 0.5ml/min; Injection vol: 20μl; Solvent System: A:
0.1% TFA, B: 100% Acetonitrile). Peak A- crude SH peptide.
10

Fig. 5: Analytical RP-HPLC elution profile of crude cvclic peptide (Column:
PEP 300; C-18; 5μ; 150 X 3 mm; Flow rate: 0.5ml/min; Injection vol: 20jil; Solvent
System: A: 0.1% TFA, B: 100% Acetonitrile); Peak A- crude cyclic peptide.
Fig. 6: Preparative RP-HPLC purification elution profile of crude cvclic
peptide (Column: Phenomenex Luna; C-18(2); lOμ; 250 X 50 mm; Flow rate:
50ml/min; Solvent System: A: 0.1% TFA, B: 100% Methanol).
Fig. 7: Analytical RP-HPLC elution profile of purified cvcïic peptide (Column:
PEP 300; C-18; 5μ 150 X 3 mm; Flow rate: 0.5ml/min; Solvent System: A: 0.1%
TFA, B: 100% Acetonitrile); Peak A- purified peptide.
Fig.8: MS Analysis of the pure peptide showing the mass to be 832 and
impurity to be 903
Table 1: Inhibition of ADP induced aggregation by synthesized peptide of
formula (1).
Detailed Description of the luvention
11
In accordance, the present invention provides a. process for the preparation of a
peptide N6-(aminoiminomethyl)-N2-(3-mercapto-l-oxopropyl-L-lysylglycyl-L-a-
aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic(l→6)-disulfide of formula (1)
on a solid phase, the said process comprising steps of,


a) assembling a peptide chain comprising of six amino acids and a thioalkyl
carboxylic acid in a required sequence on a solid support resin by coupling
to directly join one another by peptide bonds to obtain peptide of formula
(2);
(Acm)Mpr-Lys(Boc)-Gly-Asp(Obut)-Tip-Pro-Cys(Acm)-Resin
Formula (2)
b) capping the ftee amino groups of step(a) after each coupling with acetic
anhydride;
c) cleaving and deprotecting, all groups except acm group in the peptide of
step (b) from the solid support resin to obtain peptide-amide of formula (3);
(Acm)Mpr-Lys-Gly-Asp-Trp-Pro-Cys(Acm)-CONH2
Formula (3);
d) guanylating the peptide of step (c) at ε-lysine-NH2 in an organic solvent
followed by precipitating with an another solvent to obtain peptide-amide
of formula (4);
(Acm)Mpr-Homoarg-Gly-Asp-Trp-Pro-Cys(Acm)-CONH2
Formula (4)
e) treating the peptide amide of Formula (4) of step(d) with a heavy metal salt
in an appropriate solvent, followed by precipitating using an organic solvent
to obtain the heavy metal-peptide salt of formula (5);

Formula (5)
f) treating the heavy metal-peptide salt of step (e) with an appropriate
nucleophilic reagent to obtain the crude peptide amide of formula (1); and
g) purifying the crude peptide amide of step (f) by chromatographic
techniques.
An embodiment of the present invention involves reaction of amino and
carboxylic equivalent of compounds to form the said peptide bond.
12

Another embodiment of the present invention provides C-terminal of the
protected fïrst amino acid bound to a solid phase resin through a linker to obtain a solid
phase bound amino acid.
Yet another embodiment of the present invention uses solid support has any
amide resin, preferably Rink Amide Resin.
Still another embodiment of the present invention uses first protected amino
acid as thiol protected Fmoc cysteine.
Yet another embodiment of the present invention uses HBTU as the coupling
agent.
Still yet another embodiment of the present invention provides a cleavage of
the resin with the linker leading to release of assembled peptide amide.
Yet another embodiment of the present invention provides peptide amide
compound of formula (1) obtained by linking each of terminal functionaliry, which is
an amino or carboxylic acid group or derivatives thereof.
Stil! another embodiment of the present invention uses amino acids selected
from the group consisting of Cys, Pro, Trp, Asp, Lys, Gly, Arg, Har, Leu, Glu.
An embodiment of the present invention uses thioalkyl carboxylic acid 2-
thiopropionic acid.
Another embodiment of the present invention provides the use of protecting
groups for amino function of an amino acid as Fmoc or Boe.
Yet another embodiment of the present invention provides the use of carboxyl
function as unprotected or protected O-tBu ester.
Still another embodiment of the present invention uses the protecting group for
thiol-function as Acm group.
Still yet another embodiment of the present invention provides cleavage of the
peptide from solid support resin using the reagents TFA, TIS, EDT, DCM, Phenol and
water in a defmed ratio, preferably TFA(85-98%) : TIS(0-5%) : H2O(0-5%) : EDT(0-
5%): Phenol(0-5%), more preferably TFA(94.5-95.5%) : TIS(0-2.5%) : H2O(0-3%) :
EDT(0-2.5%).
.13

Another embodiment of the present invention utilizes an organic solvent for
guanylation selected from a group consisting of DMF, ethanol and methanol.
Yet another embodiment of the present invention the guanylation of peptide is
performed preferably by using the solvent DMF.
Still another embodiment of the present invention the precipitation of the
peptide amide of formula (4) is performed using a solvent selected from the group
consisting of acetone, acetonitrile, methanol, ethers, pentane, hexane and mixture
thereof.
Still yet another embodiment of the present invention the precipitation is
preferably performed using acetonitrile.
Another embodiment of the present invention) the purification of the peptide of
formula (4) can be achieved by RP-HPLC.
Yet another embodiment of the present invention the peptide amide of formula
(1) obtained has purity more than 99%.
Still yet another embodiment of the present invention the preparation of the
peptide of formula (1) by solid phase synthesis is carried out using Fmoc chemistry.
Further embodiment of the present invention uses heavy metal salt for removal
of acm selected from thallium trifluoromethane sulphonate, mercuric acetate or silver
trifluoromethane sulphonate, preferably silver trifluoromethane sulphonate.
Another embodiment of the present invention the heavy metal peptide salt is
obtained by preferably treating peptide of formula (4) with silver trifluoromethane
sulphonate in TFA.
Yet another embodiment of the present invention the precipitation of the heavy
metal-peptide salt of Formula (5) is preferably carried out using an etheral solvent and
more preferably disopropyl ether.
Still another embodiment of the present invention the heavy metal-peptide salt
may be treated with HC1 and DMSO to simultaneously remove the heavy metal and to
oxidize the resulting peptide to yield crude peptide amide of formula (1).
Still yet another embodiment of the present invention the crude peptide amide
of formula (1) may be purified by RP-HPLC.
14

Another embodiment of the present invention the purification of crude peptide
amide of formula (1) is preferentially performed by RP-HPLC using C-4, C-8 or C-l8
silica or polymer reverse phase columns using methanol and/or acetonitrile in
combination with aqueous TFA(0-0.5%) as mobile phase
Still another embodiment of the present invention uses methanol (AR grade) for
purification of crude peptide enabling the process inexpensive.
Yet another embodiment of the present invention provides process for
preparation of an intermediate peptide of formula (2) as given under:
(Acm)Mpr-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-Resin
Formula (2)
Still another embodiment of the present invention provides process for
preparation of an intermediate peptide of amide formula (3) as given under:
(Acm)Mpr-Lys-Gly-Asp-Trp-Pro-Cys(Acm)-CONH2
Formula (3)
Still yet another embodiment of the present invention provides process for
preparation of a peptide amide of formula (4) as given below:
(Acm)Mpr-Homoarg-Gly-Asp-Trp-Pro-Cys(Acm)-CONH2
Formula (4)
Yet another embodiment of the present invention provides process for preparation
of an intermediate peptide amide silver salt of formula (5) as given under:

Formula (5)
The following examples are illustrative of the present invention and not to be construed
to limit the scope of the invention.
15

EXAMPLES
EXAMPLE (1) Chemical synthesis of linear peptide
(Acm)Mpr-Lys(Boc)-Gly-Asp(OBut)-Trp-Pro-Cys(Acm)-Resin
Formula (2)
General Procedure:
The assembly of the peptide chain is carried out in the following manner. The
resin is transferred to the RV of the peptide synthesizer [CS936, CS BIO, Calif.
Peptide Synthesizer] and the linear peptide is assembled on it using l .5 - 4.0 times
mole excess amino acid derivatives, on the peptide synthesizer. The first amino acid,
Fmoc-Cys (C), is coupled to the resin by deprotecting the Fmoc-group on the resin,
followed by activating the Fmoc-Cys(C) by HBTU in the presence of NMM. For
coupling of the next amino acid, Proline, the a-nitrogen of the first amino acid i.e.
Fmoc-Cys(C), is deprotected followed by activating the Fmoc-Pro by HBTU in the
presence of NMM. This process is repeated with all the amino acids till the entire
linear peptide chain is assembled on the solid support. The Mpr is assembled at the
end. Each coupling is carried out for a time range of 45-90 min. The coupling steps are
followed by capping with acetic anhydride for 30-60 min. After the coupling are
complete, the resin is washed with organic solvent/s which may be selected from the
range of DMF, N-methyl pyrrolidone or DCM, preferably DMF followed by DCM,
and then dried under vacuüm. The linear peptide of formula (2) is obtained.
The peptide was synthesized as peptide amide by solid phase peptide synthesis
technology on rink amide resin using Fmoc chemistry.

Instrument CS936, CS BIO, Calif. Peptide synthesizer
Resin Rink amide resin (0.65mm/g)
Activator HBTU/0.4MNMM
Solvent Dimethyl Formamide
Deprotection 20%Piperidine
The resin (15,38g-rink amide, 10 mmole) was transferred to the RV of the
CS936 and swollen in DMF.
16

(i) Synthesis of Fmoc Cys(Acm)-resin by coupling of Fmoc-Cys(Acm)/HBTU to
the resin. The pre-swollen resin (l0mmole) was washed twice with DMF
followed by removal of Fmoc by treatment with 20% piperidine twice. The
resin was washed 6 times with DMF. Fmoc Cys(Acm)(20mmoles) and HBTU
(equimole to amino acid) were dissolved in 0.4M NMM and added to the resin.
Coupling was carried out for 60min under optimized stirring. The resin was
washed once again with DMF thrice. After the coupling, the free amino groups
were capped by acetic anhydride (2.5M) for 45 min followed by washing with
DMF three times. This HBTU process is a one-step process wherein ester is
not isolated.
The synthesis cycle was programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV l0min X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Fmoc-Cys (Acm)/HBTU
5 AA 45min XI Fmoc-Cys (Acm) COUPLING
6 SOLV 30sec X3 WASHES RESIN
(ii) Synthesis of Fmoc-Pro-Cys(Acm)-resin by coupling Fmoc-Pro/HBTU to Fmoc-
Cys(Acm)-resin. The reaction was carried out as in step l. The synthesis cycle was
programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Fmoc-Pro/HBTU
5 AA 45min XI COUPLING Fmoc-Pro
6 SOLV 30sec X3 WASHES RESIN
(iii)Synthesis of Fmoc-Trp-Pro-Cys(Acm)-resin by coupling Fmoc-Trp/HBTU to
Fmoc-Pro- Cys(Acm)-resin. The reaction was carried out as in step 1. The
synthesis cycle was programmed as follows:
Step Reagent Time Repeat Activity
l SOLV 30sec X3 WASHES RESIN
17

2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Fmoc-Trp/HBTU
5 AA 45min XI COUPLING Fmoc-Trp
6 SOLV 30sec X3 WASHES RESIN
(iv)Synthesis of Fmoc-Asp(Obut)-Trp-Pro-Cys(Acm)-resin by coupling Fmoc-Asp
(Obut)/HBTU to Fmoc-Trp-Pro- Cys(Acm)-resin. The reaction was carried out as
in step 1. The synthesis cycle was programmee as follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Fmoc-Asp(Obut)/HBTU
5 AA 45min XI COUPLING Fmoc-Asp(Obut)
6 SOLV 30sec X3 WASHES RESIN
(v) Synthesis of Fmoc-Gly-Asp (Obut)-Trp-Pro-Cys(Acm)-resin by coupling
Fmoc-Gly/HBTU to Fmoc-Asp(Obut)-Trp-Pro-Cys(Acm)-resin . The reaction was
carried out as in step l. The synthesis cycle was programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Fmoc-Gly/HBTU
5 AA 45min XI COUPLING Fmoc-Gly
6 SOLV 30sec X3 WASHES RESIN
(vi)Synthesis of Fmoc-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-resin by
coupling Fmoc-Lys(Boc)/HBTU to Fmoc -Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-
resin . The reaction was carried out as in step l.The synthesis cycle was
programmed as follows:
Step Reagent Time Repeat Activity
l SOLV 30sec X3 WASHES RESIN
18

2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Fmoc-Lys(Boc)/HBTU
5 AA 45min XI COUPLING Fmoc-Lys(Boc)
6 SOLV 30sec X3 WASHES RESIN
(vii) Synthesis of Mpr(Acm)-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-resin
by coupling Mpr(Acm)/HBTU to Fmoc-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-
Cys(Acm)-resin. The reaction was carried out as in step 1. The synthesis cycle was
programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 ACT 30sec XI DISSOLVES Mpr(Acm)/HBTU
5 AA 45min XI COUPLING Mpr(Acm)
6 SOLV 30sec X3 WASHES RESIN
EXAMPLE (2) Chemical synthesis of linear peptide
(Acm)Mpr-Lys(Boc)-Gly-Asp(OBut)-Trp-Pro-Cys(Acm)-Resin
Formula (2)
General Procedure:
The assembly of the peptide chain is carried out in the following marmer. The
resin is transferred to the RV of the peptide synthesizer [PS3, Protein Technologies,
Peptide Synthesizer] and the linear peptide is assembled on it using 1.5 - 4.0 times
mole excess amino acid derivatives, on the peptide synthesizer. The first amino acid,
Fmoc-Cys (C), is coupled to the resin by deprotecting the Fmoc-group on the resin,
followed by activation of Fmoc-Cys(C). Fmoc-Cys(C) (1.3 mmole) and HOBt (2.6
mmole) were dissolved in DMF (5.0ml) and cooled to less than 10°C in an ice bath.
DIC (1.74 mmole) was added to the reaction mixture as a single aliquot. The mixture
was then agitated for 6 minutes before being charged to the damp resin in the reaction
vessel. The coupling reaction takes place for 60mins.
19

For coupling of the next amino acid, Proline, the a-nitrogen of the first amino
acid i.e. Fmoc-Cys(C), is deprotected. This is followed by activation of Fmoc-Pro by
DIC/HOBt in cold conditions as described above and then transfer of this mixture to
the reaction vessel. This process is repeated with all the amino acids till the entire
linear peptide chain is assembled on the solid support. The Mpr is assembled at the
end. Each coupling is carried out for a time range of 45-90 min. Coupling of Mpr is
repeated. The coupling steps are followed by capping with acetic anhydride for 30-60
min. After the coupling are complete, the resin is washed with organic solvent/s which
may be selected frorn the range of DMF, N-methyl pyrrolidone or DCM, preferably
DMF followed by DCM, and then dried under vacuüm. The linear peptide of formula
(2) is obtained.
The peptide was synthesized as peptide amide by solid phase peptide synthesis
technology on rink amide resin using Fmoc chemistry.

Instrument PS3, Protein Technologies, Peptide synthesizer
Resin Rink amide resin (0.65mm/g)
Acti vator DIC/HOBT
Solvent Dimethyl Formamide
Deprotection 20%Piperidine
The resin (Ig-rink amide, 0.65 mmole) was transferred to the RV of the PS3
and swollen in DMF.
Synthesis of Fmoc Cys(Acm)-resin by coupling of activated Fmoc-Cys(Acm)
to the resin. The pre-swollen resin (0.65mmole) was washed twice with DMF followed
by removal of Fmoc by treatment with 20% piperidine twice. The resin was washed 6
titnes with DMF. Fmoc Cys(Acm)(1.3mmoles) and HOBt (2.6 mmole) were dissolved
in DMF (S.Oml) and cooled to less than 10°C in an ice bath. DIC (1.74 mmole) was
added to the reaction mixture as a single aliquot. The mixture was then agitated for 6
minutes before being charged to the damp resin. Coupling was carried out for 60min
under optimized stirring. The resin was washed once again with DMF thrice. After the
coupling, the free amino groups were capped by acetic anhydride (2.5M) for 45 min
followed by washing with DMF three times. This DIC/HOBt process is a manual and
multistep process.
20

The synthesis cycle was programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV l0min X3 WASHES RESIN

2 DEP 5min X2 DEP N-TERMINUS

3 SOLV 30sec X6 WASHES RESÏN,
4 Manual addition of activated Fmoc amino acid.
5 AA 45min XI Fmoc-Cys (Acm) COUPLING
6 SOLV 30sec X3 WASHES RESIN
(ii) Synthesis of Fmoc-Pro-Cys(Acm)-resin by coupling activated Fmoc-Pro to
Fmoc - Cys(Acm)-resin. The reaction was carried out as in step 1. The synthesis
cycle was programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 Manual addition of activated Fmoc amino acid.
5 AA 45min XI COUPLING Fmoc-Pro
6 SOLV 30sec X3 WASHES RESIN
(iii)Synthesis of Fmoc-Trp-Pro-Cys(Acm)-resin by coupling activated Fmoc-Trp to
Fmoc-Pro- Cys(Acm)-resin. The reaction was carried out as in step 1. The
synthesis cycle was programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 Manual addition of activated Fmoc amino acid.
5 AA 45min XI COUPLING Fmoc-Trp
6 SOLV 30sec X3 WASHES RESIN
(iv)Synthesis of Fmoc-Asp(Obut)-Trp-Pro-Cys(Acm)-resin by coupling Fmoc-Asp
to Fmoc-Trp-Pro- Cys(Acm)-resin. The reaction was carried out as in step 1. The
synthesis cycle was programmed as follows:
21

Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 Manual addition of activated Fmoc amino acid.
5 AA 45min XI COUPLING Fmoc-Asp(Obut)
6 SOLV 30sec X3 WASHES RESIN
(v) Synthesis of Fmoc-Gly-Asp (Obut)-Trp-Pro-Cys(Acm)-resin by coupling
Fmoc-Gly to Fmoc-Asp(Obut)-Trp-Pro-Cys(Acm)-resin . The reaction was carried
out as in step l. The synthesis cycle was programmed as follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 Manual addition of activated Fmoc amino acid.
5 AA 45min XI COUPLING Fmoc-Gly
6 SOLV 30sec X3 WASHES RESIN
(vi)Synthesis of Fmoc-Lys(Boc)-GIy-Asp(Obut)-Trp-Pro-Cys(Acm)-resin by
coupling Fmoc-Lys(Boc) to Fmoc -Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-resin. The
reaction was carried out as in step l.The synthesis cycle was programmed as
follows:
Step Reagent Time Repeat Activity
1 SOLV 30sec X3 WASHES RESIN
2 DEP 5min X2 DEP N-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 Manual addition of activated Fmoc amino acid.
5 AA 45min XI COUPLING Fmoc-Lys(Boc)
6 SOLV 30sec X3 WASHES RESIN
(vii) Synthesis of Mpr(Acm)-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-resin
by coupling Mpr(Acm) to Fmoc-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-
22

resin. The reaction was carried out as in step 1. The synthesis cycle was
programmed as follows:

Step
1 Reagent Time Repeat
SOLV 30sec X3 Activity
WASHES RESIN
2 DEP 5min X2 DEPN-TERMINUS
3 SOLV 30sec X6 WASHES RESIN
4 Manual addition of activated Fmoc amino acid.
5 AA 45min XI COUPLING Mpr(Acm)
6 SOLV 30sec X3 WASHES RESIN
In the synthesis coupling of Mpr(Acm) had to be carried out rwice to complete the
coupling reaction.
EXAMPLE (3) CLEAVAGE OF THE PEPTIDE FROM THE RESIN TO YIELD
PEPTIDE AMIDE (Acm)Mpr-Lys-Gly-Asp-Trp-Pro-Cys(Acm)-CONH2
(Formula (3))
The assembled peptide resin (from Example l or 2) is treated with 500 ml of
cleavage cocktail consisting of TFA (95%): TIS(2.5%) : H2O(2.5%) : EDT(0%) :
Phenol (0%) for 2 hrs at R.T in CS936. The reaction mixture is filtered through RV,
and TFA was evaporated on Rotavap. Precipitation of the peptide was carried out at -
20°C by addition of 300 ml of cold di isopropyl ether with constant stirring. The crude
peptide precipitate in the solvent is let to stand at -20°C for 10 hrs. The peptide was
isolated by filtering through Whatman paper no. 5, followed by cold solvent wash
(lOOml x 3) to remove the scavengers used in the cleavage cocktail. The crude peptide
precipitate is dried under vacuüm over P2Os, and characterized by RP-HPLC (Fig.l
and 2).
Example l Example 2
Yield: 58.73 Yield: 48.73
% purity of peptide: 90% %purity of peptide: 79.68%
EXAMPLE (4) GUANYLATION OF CRUDE PEPTIDE TO YIELD
(Acm)Mpr-Homoarg-Gly-Asp-Trp-Pro-Cys(Acm)-CONH2 (formula (4))
The peptide (lg, 1.157mmole) isdissolved in 15 ml of DMF, thepH adjustedto
9.0 with TEA. The reagent 3,5-dimethylpyrazole-l-carboxamidine nitrate (931.5mg) in
23

DMF (15ml) is added to the peptide. The reaction mixture is stirred at 30°C for 4 days

with multiple additions of one time excess of reagent 3,5 -dirnethyjpyrazole-1 -
carboxamidine nitrate.
The peptide is precipitated from the reaction mixture by the addition of 280ml
of acetonitrile (pH adjusted to 8.0 with TEA). The mix is further kept at -20°C for 10
hrs. It is fïltered through Whatman no. 5 filter paper and washed with acetonitrile (pH
8.0) 3 times, followed by plain acetonitrile to neutralize the pH. The precipitate is dried
under high vacuüm overnight. Yield: 85%. The peptide was characterized by RP-
HPLC (Fig.2).
EXAMPLE (5) DE-ACM OF THE GUANYLATED PEPTIDE FOLLOWED BY
OXIDATION TO YIELD

TFA (134.9ml) and anisole (2.7ml) are mixed, cooled in ice, added to 658 mg
of pre-cooled peptide from example 3 and saturated with nitrogen. This is followed by
addition of AgOTf (3.47g) and stirred for 2hrs in an ice bath. TFA is removed under
high vacuüm and silver salt of the peptide was precipitated by addition of diisopropyl
ether (~400ml). The reaction mixture is filtered through G-4 sintered funnel and
precipitate (silver-peptide) is re-suspended in diisopropyl ether (60ml x3), washed as
above and dried over P2Ü5 under vacuüm.
The oxidation silver peptideis carried out by dissolving 10 mg of the silver-
peptide salt in 15.6ml of 50% DMSO / IM HCI in ice-cold condition. The reaction
mixture is stirred for 3 hrs at 25 °C. The precipitate is filtered through a G-4 sintered
tunnel or Hyflo bed to remove silver chloride. The filtrate is checked for completion of
oxidation (Fig.4).On completion of the reaction crude peptide of formuïa (1) is
obtained. Percentage purity: 85%
EXAMPLE (6) PURIFICATION OF S-S PEPTIDE
The crude disulfide looped peptide of formuïa (I) is loaded on to prep C-18
column (50 x 250mm, 100A). The peptide is purifïed by using aqueous TFA (0.1%)
and methanol in a gradiënt program (Fig.5). This is followed by an isocratic run using
the above said solvent systems on a Shimadzu preparative HPLC System consisting of
24

a controller, 2 LC8A pumps, UV-Vis detector. The purified peptide amide of formula
(1) is analysed by analytical RP-HPLC (Fig.6). The mass is determined, by Mass
Spectrophotometer (Fig. 7).

EXAMPLE (7): PURIFICATION OF S-S PEPTIDE'
The purifïcation was carried out in the same manner as Example 5, except that
Acetonitrile was used instead of methanol to obtain peptide amide of formula (1).
EXAMPLE (8): DE-ACM OF THE GUANYLATED PEPTIDE USING
MERCURIC (II) ACETATE
Same as in Example (4), except that the Acm group protection of cysteineis
removed from the guanylated peptide by treatment with mercury (II) acetate.

The peptide (13.4mg) es'timated by Lowry's method, of Cys-Acm) is dissolved
in 400(il of acetic acid (10%). Ten times excess of mercury (II) acetate (82.96mg) is
added to it, the reaction mass vortexed and kept at R.T. for 5 hrs. 100 times excess of
β-mercaptoethanol(I81.37(μl)isadded, the solution vortexed and let to stand overnight
at room temperature. The reaction mixture is centrifuged for 4min, and supernatant
collected. The precipitate is extracted with 400μl x 3 of 10% acetic acid by
centrifugation. The filtrates are pooled and percentage purity determined by RP-HPLC
is 55% (Fig 3).
EXAMPLE (9): DE-ACM OF THE GUANYLATED PEPTIDE USING IODINE
Same as in Example (4), except that the Acm group protection of cysteine is
removed from the guanylated peptide by treatment with iodine.
The peptide (9.18mg, estimated by Lowry's method, of Cys-Acm)is dissolved
in 17.8ml of acetic acid (80%) and purged with NI for 15mins. ImM solution of I2 (in
80%acetic acid, -4ml)is added to the peptide solution, over a period of 1 hr, till there is
a persistent yellow color. The mixture is stirred for an additional 30-mins followed by
neutralisation with IN Na2 S2O3, till the yellöw color disappeared, and lyophilized.
Estimation of 'SH' is done by Ellman Test, which is negative indicating that removal of
ACM has not been achieved.
EXAMPLE (lOj: PURIFICATION OF THE DE-ACM PEPTIDES
The mercury (II) acetate treated and k treated peptide samples were desalted by
RP-HPLC, using the hyperprep (250 x lOmm, 12μ ,C-18 column).
25

EXAMPLE (11): PLATELET AGGREGATION INHIBITION ASSAY TO CHECK
THE BIOACTIVITY OF FORMULA (1)
The bioactivity of peptide of formula (1) is checked using platelet aggregation
inhibition assay using 4X Laser Aggregometer (EMA). Freshly venous blood from
consented human donors are drawn and collected in citrated buffer. The platelet rich
plasma (PRP) and platelet poor plasma are separated by centrifugation. Platelet count
in PRP is adjusted to 2-3 xlO8 platelets / ml. After adjusting the baseline aggregation
with PPP, the PRP was treated with 10-20 mM ADP and checked the percent total
aggregation. The PRP is then first incubated with varying concentrations of reference
standard and synthesized peptide of formula (1). ADP is then added to check the
inhibition of aggregation. The reproducibility of bioactivity of synthesized peptide of
formula lis checked several times and compared with reference standards. Table l
represents one of many experiments (from 12 experiments). The ICso dose for
synthesized peptide (SP) was less than 140nM as compared to commercial reference
standard. There is more than 50% inhibition of ADP induced platelet aggregation with
SP seen in most of the samples and results are comparable with commercial reference
standard.
26

Table 1: Inhibition ofADP induced aggregation by synthesized peptide
(SP) of formula l

Donor
Number Percent
Aggregation by
ADP Concentration
(nM) % Inhibition by

SP of
formula 1 Reference
Standard
1. 89.4% 70 ND ND

140 44.6 31.20

280 61.40 52.50

2. 92.13 70 46.66 30.31

140 51.16 45.18

280 67.74 60.92

3. 54.14 70 57.42 27.41

140 60.28 49.20

280 ND ND

4. 68.11 70 ND ND

140 20.52 23.06

280 42.73 40.53

5. 66.50 70 52.63 45.68

140 87.21 57.44

280 ND ND

6. 66.17 70 ND ND

140 82.92 70.56

280 ND ND
27

We Claim:
1. A process for the preparation of a peptide N6-(aminoiminomethyI)-N2-(3-
mercapto-1-oxopropyl-L-lysylglycyl-L-α-aspartyl-L-tryptophyl-L-prolyl-L-
cysteinamide, cyclic(1→6)-disulfide of formula (1) on a solid phase,

the said process comprising:
a. assembling a peptide chain comprising of six amino acids and a
mercaptoalkyl thioalkyl carboxylic acid in a required sequence on a
solid support resin, by coupling to directly join one another by peptide
bonds to obtain a peptide bound resin of formula (2) as given below:
(Acm)Mpr-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-Cys(Acm)-Resin
Formula (2)
b. capping the free amino groups after each coupling with acetic
anhydride;
c. cleaving and deprotecting, all groups except acm group, the peptide of
step (a) from the resin to obtain peptide amide of formula (3) as given
below:

28

d. guanylating the peptide at ε-lysine-NH2 in an organic solvents followed
by precipitating with another solvent to obtain peptide of formula (4) as
given below, by precipitation with another solvent;

e. treating the peptide of Formula (4) with a heavy metal salt in an
appropriate solvent, followed by precipitation of the heavy metal-
peptide salt using an organic solvent to obtain the heavy metal-peptide
salt of formula (5);

f. treating the heavy metal-peptide salt of step (e) with a thiol followed by
desalting and oxidation to obtain a peptide of formula (1); and
g. purifying the crude peptide of step (f) or (g) by RPHPLC in isolation or
in combination with HIC and IEC chromatographic techniques.
2. A process as claimed in claim l, wherein the reaction of amino and carboxylic
equivalent of compounds forms the said peptide bond.
3. A process of claim l, wherein the C-terminal of the protected first amino acid is
bound to a solid phase through a linker to obtain a solid phase bound amino acid.
4. A process of claim l, wherein the solid support used is any amide resin,
preferably Rink Amide Resin.
5. A process of claim l, wherein the first protected amino acid is a thiol protected
Fmoc Cysteine.
6. A process of claim l, wherein in the cleavage of the resin with the linker leads to
the release of assembled peptide amide.
7. A process of claim l, wherein the peptide amide compound is a compound joined
to each of the terminal functionalities by a peptide bond and wherein each
terminal functionalities is an amino or carboxylic acid group or derivatives
thereof.
29

8. A process of claim l, wherein the amino acids used are selected from the group
consisting of Cys, Pro, Trp, Asp, Lys, Gly, Arg, Har, Leu and Glu.
9. A process of claim l, wherein the mercaptoalkyl thioalkyl carboxylic acid used is
2-mercaptopropionic thiopropionic acid.
10. A process of claim l wherein, the protecting group for -NH2 functional group of
an amino acid is Fmoc or Boc.
11. A process of claim l, wherein the protecting group for -COOH functional group
is unprotected or protected O-tBu esteror free.
12. A process of claim l, wherein the protecting group for SH-function is Acm
group.
13. A process of claim l, wherein in step (c), the peptide is cleaved from solid
support resin using the reagents TFA, TIS, EDT, DCM, Phenol and water in a
defined ratio, preferably TFA(85-98%) : TIS(0-5%) : H2O(0-5%) : EDT(0-5%):
Phenol(0-5%), more preferably TFA(94.5-95.5%) : TIS(0-2.5%) : H2O(0-3%) :
EDT(0-2.5%).
14. A process of claim l, wherein in step (d), the organic solvent used for
guanylation is selected from the group consisting of DMF, ethanol and methanol.
15. A process of claim l, wherein in the step (d), the precipitation of the peptide of
formula 4 is carried out by performed using a solvent selected from the group
consisting of acetone, acetonitrile, methanol, ethers, pentane, hexane and mixture
thereof.
16. A process as claimed in claim 15, wherein the precipitation is carried out in
preferably performed using acetonitrile.
17. A process of claim l, wherein in the step (g), the peptide of formula (1) obtained
has purity more than 99%.
18. A process as claimed in claim l, wherein the preparation of the peptide of
formula (1) by solid phase synthesis is carried out using Fmoc chemistry.
19. A process as claimed in claim l, wherein the assembly of the amino acids gives a
peptide bound resin of formula 2,
30


20. A process as claimed in claim l, wherein in the step (d), the guanylation of
peptide of formula 3 is carried out performed preferably by using the solvent in
DMF.
21. A process as claimed in claim l, wherein the purification of the peptide of
Formula (4) may be carried out achieved by RP-HPLC.
22. A process as claimed in claim l, wherein the heavy metal salt used for the
treatment of peptide of Formula (4) is silver trifluoromethane sulphonate in TFA.
23. A process as claimed in claim l, wherein the precipitation of the heavy metal-
peptide salt of Formula (5) is preferably carried out using an ethereal solvent and
more preferably diisopropyl ether.

24. A process as claimed in claim l, wherein in step (f), the heavy metal-peptide salt
may be treated with HC1 and DMSO to simultaneously remove the heavy metal
and to oxidize the resulting peptide to yield a crude peptide amide of Formula
(1).
25. A process as claimed in claim l, wherein the crude peptide amide of Formula (1)
is may be purified by RP-HPLC.
26. A process as claimed in claim l, wherein the purification of crude peptide amide
of formula (1) is preferentially performed by RP-HPLC using C-4, C-8 or C-18
silica or polymer reverse phase columns using methanol and/or acetonitrile in
isolation or combination with aqueous TFA(0-0.5%) as the mobile phase.
27. An intermediate peptide of formula (2)
(Acm)Mpr-Lys(Boc)-Gly-Asp(Obut)-Trp-Pro-Cys("Acm)-Resin
Formula 2
31

28. An intermediate peptide of formula (3)
(Acm)Mpr-Lys-GIy-Asp-Trp-Pro-Cys(Acm)-CONH2
Formula (3)
29. An intermediate peptide of formula (4)
(Acm)Mpr-Homoarg-Gly-Asp-Trp-Pro-Cys(Acm)-CONH2
Formula (4)
30. An intermediate peptide salt of the formula (5)

Dated this 8Ih day of October 2004.
To
The Controller of Patents,
The Patent Office,
At Mumbai
32