FIELD OF THE INVENTION
The present invention relates to a process for preparing pure argatroban of formula 1,
(Formula Removed)

having reduced level of impurities and to methods of identifying as well quantifying
impurities.
BACKGROUND OF THE INVENTION
Argatroban of formula 1, is an anticoagulant that is a small molecule direct thrombin inhibitor and is chemically known as (2R,4R)-4-methyl-l-[N2-(3-methyl-l,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid .
(Formula Removed)
Argatroban is used for treatment of prophylaxis or treatment of thrombosis in patients with heparin-induced thrombocytopenia (HIT) and as an anticoagulant in individuals with thrombosis and heparin induced thrombocytopenia. Argatroban has been disclosed for first time in US patents 4,201,863 and 4,258,192 (herein referred as US '863 and US '192 respectively). US '863 discloses racemic argatroban starting from 4-methyl-2-piperidine carboxylic acid, and is isolated in amorphous form but is silent about resolution of racemic argatroban. US '192 discloses synthesis of desired isomer of argatroban by reaction of (2R,4R)-4-methyl-2-piperidine carboxylic acid with thionyl chloride and ethanol to give corresponding ethyl ester which is then condensed with NG-nitro-N2-(tert-butoxycarbonyl)-L-arginine in the presence of triethylamine and isobutylchloroformate to give ethyl (2R,4R)-l-[NG-nitro-N2-(tert-butoxycarbonyl)-

L-arginyl]-4-methyl-2-piperidinecarboxylate. tert-Butoxycarbonyl group of above
intermediate is then removed using hydrochloric acid-ethyl acetate to give (2R,4R)-
l-[NG-nitro-L-arginyl]-4-methyl-2-piperidine carboxylate hydrochloride as an
amorphous solid which is further condensed with 3-methyl-8-quinolinesulfonyl
chloride in the presence of triethylamine in chloroform to give an ester intermediate
which is isolated by column chromatography followed by hydrolysis using sodium
hydroxide in ethanol to give 2R, 4R)-l-[NG-nitro-N2-(3-methyl-8-
quinolinesulfonyl)-L-arginyl]-4-methyl-2-piperidine carboxylic acid. Acid
intermediate thus obtained is further reduced and deprotected using acetic acid and palladium carbon in ethanol at 50 kg/cm2 hydrogen pressure to give argatroban, which is then recrystallized with ethanol to give pure argatroban having melting point 188-191°C. Process involves use of hazardous reagent such as thionyl chloride for the esterification step, which is an eye irritant and can generate poisonous effect on human body. Further, condensation of ethyl (2R,4R)-4-methyl-2-piperidine carboxylate with N2-nitro-N2-(tert-butoxycarbonyl)-L-arginine in the presence of strong base such as triethylamine yield impure intermediate, which is found to be contaminated with the impurities, thus require extensive purification. The process involves hydrogenation at 50 kg/cm2 which is not advisable at industrial scale due to safety reason. The patent discloses the use of strong base during hydrolysis which may lead to generation of undesired impurities, due to basicity of strong base and resulting product is having low purity and hence further purification is required as an additional step. No polymorph of argatroban is reported, only melting point is disclosed.
Several polymorphs of argatroban are reported in literature such as in US patent application 2009/0221637, PCT publication. WO 2009/124906, Chinese patent application CN 1951937 A and an article Biochem. Biophys. Res. Comm. 1981, 101, 440-446. Most of these references disclose synthesis of argatroban monohydrate, which is the marketed product.

As argatroban is product of five to six steps of reaction sequences and it is well known in the art that direct product of a chemical reaction is rarely a single compound with sufficient purity to comply with pharmaceutical standards, so it may be contaminated with impurities. The impurities that may be present in argatroban are starting materials, by-products of the reaction, products of side reactions, or degradation products. Therefore, any pharmaceutical compound such as argatroban can contain extraneous compounds or impurities, which may be the result of competing side reactions, degradation, and the like. Impurities in argatroban or any other active pharmaceutical ingredients (APIs) are undesirable and in extreme cases, might even be harmful to a patient being treated with a dosage form containing the API. In addition, in the manufacturing of APIs knowledge of the purity of the API, such as argatroban, is required before commercialization, because effectiveness of the formulated tablet is dependant upon the purity of API used. Therefore, an API like argatroban must be free from such process impurities, side products or degradation impurities or such impurities must be limited to very small amounts, before it is formulated.
As per the ICH Q7A guidelines for API manufacturers, impurities introduced during commercial manufacturing processes must be limited to very small amounts and are preferably substantially absent and in the specification impurities should be below set limits by controlling process parameters, such as temperature, pressure, time, and stoichiometric ratios, and including purification steps, such as crystallization, distillation, and liquid-liquid extraction, in the manufacturing process. The API should be free from impurities as much as possible so that it can be use for human cure. For the individual characterized impurities (identified impurities), the limits are generally 0.15% by weight. Limits for unidentified and/or uncharacterized impurities are obviously lower, generally less than 0.1 percent by weight. Therefore, regulatory authorities worldwide require that drug manufacturers should isolate, identify and characterize the impurities in their products; and pharmaceutical active

compounds must be either free from these impurities or contain impurities in acceptable Hmits.
Intermediates and by-products or any other impurities will, in most cases, be present with the API like argatroban must be analyzed for purity, by HPLC or TLC analysis, to determine the presence of any intermediates or by-products or any other impurities. The API need not be absolutely pure, as absolute purity is a theoretically idea that is typically unachievable.
The impurity present in an API can be identified by its position in the chromatogram, where the position in a chromatogram is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector. The relative position of a specific peak in the chromatogram is known as the "retention time." The retention time can vary about a mean value based upon the condition of the instrumentation, as well as many other factors. To mitigate the effects such variations have upon accurate identification of an impurity, practitioners use the "relative retention time" ("RRT") to identify impurities (Strobel p. 922). The RRT of an impurity is its retention time divided by the retention time of "a reference marker". The reference marker can be the API itself, or it may be a compound other than the API that is added to, or present already in the mixture, in an amount sufficiently large to be detectable and sufficiently low so as not to saturate the column, and to use that compound as the reference marker for determination of the RRT.
Since, argatroban is a compound of wide therapeutic use, there is an urgent need in art to provide an efficient and industrially viable method for preparation of argatroban of pharmaceutically acceptable quality. Present invention fulfills the need in the art and provides an industrial advantageous and convenient process which yield argatroban of high purity containing impurities in acceptable amount as per standard of regulatory authorities.

OBJECTIVE OF THE INVENTION
The principal objective of the present invention is to provide pure argatroban
containing any individual identified impurity less than 0.15% and unidentified
impurity less than 0.1% by HPLC.
Still another objective of the present invention is to provide economically viable
process for the preparation of highly pure argatroban.
Another objective of the present invention is to provide a process for the preparation
of highly pure argatroban having reduced level of impurities.
Another objective of the invention is to provide a process for removal of impurities.
Still another objective of the invention is to identify, isolate and characterized
various impurities of argatroban or intermediates thereof
Yet another objective of the present invention is to provide a method for determining
identification and quantification of impurities in a sample of argatroban.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides an efficient and industrially
advantageous process for preparation of highly pure argatroban containing each
individual identified impurity less than 0.15% and unidentified impurity less than
0.1% by HPLC.
According to one embodiment, the present invention provides a process for the
preparation of pure argatroban, comprising the steps of:
a), esterifying a compound of formula II with alcoholic hydrogen chloride,
(Formula Removed)
to form an intermediate of formula III;
(Formula Removed)
wherein R is alkyl selected from methyl, ethyl and the like

b). condensing the intermediate of formula III with a reactive derivative of compound of formula IV,
(Formula Removed)
in presence of an organic base having pKa value less than 8.5 in a suitable organic solvent to form an amide intermediate of formula V;
(Formula Removed)
wherein R is as defined above
c). optionally, isolating amide intermediate of formula V;
d). deprotecting amide intermediate of formula V using a source of hydrogen chloride to form an intermediate of formula VI;
(Formula Removed)
wherein R is as defined above
e). condensing the intermediate of formula VI with a compound of formula VII,
(Formula Removed)
in the presence of a suitable base in an organic solvent to form amino-sulphonyl intermediate of formula VIII;
(Formula Removed)
wherein R is as defined above

f). optionally, isolating/recrystallizing amino-sulphonyl intermediate of formula
VIII; g). hydrolyzing amino-sulphonyl intermediate of formula VIII in presence of a weak
base to form an intermediate of formula IX; and
(Formula Removed)
h). converting the intermediate of formula IX to argatroban of formula 1. According to another embodiment, present invention provides argatroban, having less than 0.15 area % of one or more of the following impurities:
(Formula Removed)
According to another embodiment, the present invention provides a process for identification of an impurity in a sample of argatroban, selected from one or more of

impurities amongst impurity A, B, C, D or E, the process comprising performing
steps (a) and (b) in either order:
a), carrying out a chromatographic analysis on the reference sample, containing one
or more of impurities amongst A, B, C, D or E (reference marker) and argatroban, to
determine the relative retention time of the reference marker compared to
argatroban;
b). carrying out a chromatographic analysis on the sample of argatroban to
determine the relative retention time of impurities present in the sample compared to
argatroban;
and comparing the relative retention times determined in step (a) and (b); wherein if
the relative retention times determined are substantially same, the impurity of
argatroban in the sample is identified as being the same as reference marker.
According to another embodiment, the present invention provides a method of
determining the amount of an impurity, selected from one or more of impurity
among A, B, C, D and E, the method comprising performing steps (a) and (b) in
either order:
a), carrying out a chromatographic analysis on the reference sample, containing
known amount of one or more impurities amongst A, B, C, D or E (reference
standard) and argatroban, to determine the relative retention time of impurities
compared to argatroban; and measuring the area under a peak corresponding to the
reference standard;
b). carrying out a chromatographic analysis on the sample of argatroban to
determine area under HPLC peak corresponding to each and every impurity by
reference to the relative retention times thus determined compared to argatroban;
and calculating the amount of one or more of impurity among A, B, C, D and E in
argatroban, by reference to area of the HPLC peak in the sample against the area of
HPLC peak associates with the same impurity in step (a).
According to another embodiment, present invention provides novel impurities A, B
and C.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows powder X-ray diffraction pattern of argatroban after ethanol
crystallization
Figure 2 shows powder X-ray diffraction pattern of argatroban monohydrate
DETAILED DESCRIPTION OF THE INVENTION
As used herein term "pure argatroban" refers to argatroban containing any individual
identified impurity less than 0.15% and unidentified impurity less than 0.10% by
HPLC.
As used herein term "relative retention time (RRT)" refers to a relation of amount of
time a compound elutes from a column relative to argatroban.
The present invention provides an improved and industrially advantageous process
for the preparation of pure argatroban.
According to one embodiment, process involves the synthesis of pure argatroban
starting from compound of formula II i.e (2R, 4R)-4-methyl-piperidine-2-carboxylic
acid.
Generally, the process involves esterification of compound of formula II in presence
of alcoholic hydrogen chloride at temperature of about 10-50°C for few minutes to
several hours, preferably till the completion of the reaction. Alcoholic hydrogen
chloride includes methanolic-hydrogen chloride, ethanolic-hydrogen chloride and
the like; preferably, ethanolic-hydrogen chloride is employed for the reaction. The
progress of reaction can be monitored by suitable chromatographic techniques such
as high-pressure liquid chromatography (HPLC), thin layer chromatography, ultra
pressure liquid chromatography (UPLC), gas chromatography (GC) and the like.
After completion of the reaction, the intermediate of formula III can be isolated from
reaction mixture or reacted further in situ. Preferably, the intermediate of formula III
can be isolated from the reaction mixture by removal of solvent using techniques
known in prior art such as evaporation, distillation and the like. It is advantageous to
treat resulting product with a suitable base. Specifically, resulting product is
dissolved in an organic solvent and can be optionally washed with aqueous solution

of base and/or brine. Base can be selected from inorganic base sucii as alkali or alkaline metal carbonates, bicarbonates thereof such as potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate the like. The ester intermediate of formula 111 can be recovered from resulting solution by removal of organic solvent.
Ester intermediate of formula III is then condensed with a compound of formula IV or its reactive derivative thereof in the presence of an organic base having pKa value less than 8.5 in a suitable organic solvent to form amide intermediate of formula V that forms novel feature of the invention.
Generally, condensation reaction is carried out using a suitable base and activating agent at a temperature of -30 to 50°C for few minutes to several hours, preferably 5 minutes tol hour, more preferably till completion of the reaction. Reaction can be carried out by generation of reactive derivative of compound of formula IV in situ using an activating agent or can be prepared separately which is then reacted with intermediate of formula 111. It is preferable to carry out reaction at low temperature, more preferably less than 5 °C as high temperature result in the breakage of reactive derivative of compound of formula IV and converting to its corresponding acid compound. Reactive derivative of compound of formula IV employed for reaction can be selected amongst corresponding acid halide, anhydride, mixed anhydride, activated ester and the like. Activating agent used for the generation of reactive derivative of compound of formula IV includes alky or aryl chloroformate such as isobutyl chloroformate, methyl chloroformate, ethyl chloroformate, phenyl chlroformate and the like. It is advantageous to synthesis reactive derivative of formula IV in situ during the condensation reaction. Base employed in the reaction includes bases, which have pKa less than 8.5. Preferably base can be selected amongst N-methyl morpholine, pyridine, and the like, preferably N-methyl morpholine. Organic solvent can be selected from C3-6 ketones, esters, nitriles, halogenated solvents, ethers, aprotic solvents, and the like or mixture thereof, preferably acetone, methyl isobutyl ketone, methyl ethyl ketone, ethyl acetate,

acetonitrile, tetrahydrofuran, dimethylformamide, dichloromethane, chloroform and mixture thereof. Amide intermediate of formula V can be isolated from the reaction mixture using conventional methods known in the art or reaction mixture can be proceed as such for the further deprotection reaction. Specifically, intermediate of formula V can be isolated by the removal of solvent from the reaction mixture by distillation or evaporation.
During condensation reaction, it is preferable to add raw materials lot wise to get better purity. It is observed that upon addition in one lot, the starting materials remained un-reacted in the reaction mixture. Therefore, desired product of reaction is obtained in low yields and found to be contaminated with starting material as an impurity.
It is noticed that nature of base employed for the reaction is very crucial for synthesis of highly pure product in good yields. It was observed during the process development, that when reaction is carried out using a base having pka more than 8.5 such as triethyl amine (pka=10.75) as reported in prior art then it result in the formation of impure product. Amide intermediate of formula V prepared using bases such as diisopropylethylamine, triethyl amine or N-methyl piperdine (bases having pka more than 8.5) is found to contain a specific cyclized impurity of formula X,
(Formula Removed)
Dotted line (—) in cyclized impurity of formula X shows its existence in two tautomeric forms as represented by following formulas:
(Formula Removed)
Cyclized impurity may have formed due to intramolecular cyclization of starting material i.e. compound of formula IV. The possible reason for this side reaction i.e. intramolecular cyclisation may be the use of a base having higher pka which increase the nucleophilicity of amino group at C-5 carbon in compound of formula IV which further may leads to the self cyclization and results in cyclized impurity of formula X in either tautomeric form.
Above cyclised impurity, if not controlled at this stage, can result in generation of other impurities such as impurity of formula XI and/or impurity of formula XII by undergoing usual reaction sequence. Impurity of formula X in either tautomeric form may undergo deprotection reaction using source of hydrogen chloride, forming impurity of formula XI at the stage of intermediate of formula VI. The impurity of formula XI may undergo condensation with compound of formula VII which result in impurity of formula Xll. Impurity formation can be described by the following reaction scheme:
(Formula Removed)
Impurities of formula XI and Xll can also exist in two tautomeric forms similar to impurity X, and also shown by the dotted line representation in above scheme. To avoid the formation of further impurities, it is necessary to control the amount of cyclized impurity at initial stage. Therefore, it is highly advantageous to use a base having pka value less than 8.5 during condensation reaction to provide intermediate of formula V either free from above cyclized impurity of formula X or present in acceptable amounts.

Amide intermediate of formula V can optionally be purified to increase the purity of the product as well as to minimize the presence of identified impurities such as cyclized impurity of formula X along with unidentified impurities. In one more specific embodiment, the above cyclized impurity can be removed by treatment of intermediate of formula V with a suitable base.
Specifically, process involves extraction of solution of amide intermediate of formula V in water, with a water immiscible solvent such as esters, halogenated solvents, hydrocarbons and the like or mixture thereof, preferably ethyl acetate, isopropyl acetate, dichloromethane, chloroform, toluene, xylenes and the like. Thereafter, organic layer is washed with aqueous solution of base that includes alkali or alkaline metal hydroxide, carbonate, bicarbonate thereof such as potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and the like to remove impurities. Then organic layer can be optionally washed with a suitable acid and/or brine. Purified intermediate of formula V can be isolated from the resulting solution by the removal of solvent using techniques such as evaporation or distillation and the like. Amide intermediate of V is then deprotected using a source of hydrogen chloride to give intermediate of formula VI.
Specifically, process involves deprotection of intermediate of formula V using a suitable hydrogen chloride source, which includes but not limited to aqueous hydrochloric acid, hydrogen chloride gas or mixture thereof with suitable solvent selected from alcohol such as methanol, ethanol, isopropyl alcohol, tert-butanol, ester such as ethyl acetate; ether such as isopropyl ether, diethyl ether and the like or mixture thereof Preferably, the source of hydrogen chloride includes ethanolic hydrochloride. The reaction can be carried out at temperature of about -20 to 25°C for few minutes to several hours, preferably for 10 to 20 hours at temperature of about 5-20°C, more preferably till completion of the deprotection reaction. The compound of formula VI is isolated form the reaction mixture using conventional techniques known in the art such as filtration, decantation, centrifugation and the like.

It is preferable to carry out deprotection reaction either at ambient temperature or less than ambient temperature, as increase in temperature, will result in acidic hydrolysis of the ester group as well as deprotection of amino group present in the intermediate of formula V to give product contaminated with an impurity of formula XIII,
(Formula Removed)
The above impurity of formula Xlll, can alternatively be prepared by hydrolysis of
intermediate of formula V followed by deprotection of resulting compound, so that
presence or absence of this specific impurity in the product can be checked. In other
alternate way, it can be isolated from the reaction mixture using chromatographic
techniques. Thus isolated impurity is characterized by Mass analysis showing M+1
peak at 245.14.
Specific reaction conditions as described by the present invention result in the product
free from the impurities or present in acceptable amount.
Alternatively, intermediate of formula VI can be prepared directly from compound
of formula IV without isolation of intermediate of formula V.
Intermediate of formula VI is then made to condense with a compound of formula
VII in the presence of a suitable base in an organic solvent to form sulphonyl amino
intermediate of formula VIII.
Specifically process involves the reaction of compound of formula VI with a
compound of formula VII in the presence of a suitable base in a solvent at a
temperature of about -50 °C to room temperature for few minutes to several hours,
preferably till the completion of the reaction. Base employed for reaction can be
organic base which include substituted amine such as triethyl amine, diisopropylethyl
amine, diazabicyclo[5.4.0]undec-7-ene (DBU); or inorganic base which include alkali
or alkaline metal carbonates, bicarbonates thereof such as sodium carbonate,
potassium carbonate, sodium bicarbonate, potassium bicarbonate and the like or
combination thereof, preferably triethyl amine can be used for the reaction. Suitable

solvents include halogenated solvents, ethers, esters, ketones, hydrocarbons and the like or mixture thereof. Preferably solvent can be selected from dichloromethane, dichloroethane, chloroform, tetrahydrofuran, ethyl acetate or mixture thereof, preferably dichloromethane. After completion of reaction, it is advantageous to wash the reaction mixture with inorganic base to reduce the impurity level at intermediate stage. Inorganic base include alkali or alkaline metal carbonates, bicarbonates, hydroxides thereof such as sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, calcium carbonate, sodium hydroxide, potassium hydroxide and the like, more preferably potassium carbonate. The washing as described above is efficient to remove 3-methyl-8-quinoline sulphonic acid, which may form as by-product during the reaction. Sulphonyl amino intermediate of formula VIII can be isolated from the reaction mixture by removal of solvent or can be reacted further in situ.
Sulphonyl amino intermediate of formula VIII, thus isolated, can be optionally purified by crystallization from a suitable solvent. Suitable solvents include ethers such as diethyl ether, methyethyl ether, tetrahydrofuran, diisopropyl ether, methyl tert-butyl ether; aliphatic or aromatic hydrocarbon solvent such as cyclohexane, toluene .and the like or mixture thereof, preferably methyl tert-butyl ether. The purified intermediate of formula VIII thus crystallized can be isolated from the reaction mixture using conventional techniques known in the art such as filtration, decantation, centrifugation and the like.
Amino-sulphonyl intermediate of formula VIII is then hydrolyzed to form a compound of formula IX in presence of a weak base.
Specifically, the reaction involves reaction of the compound of formula VIII in the presence of the weak base at a temperature of about -50 to 50°C for few minutes to several hours, preferably 5 to 20°C for about 20-24 hours, preferably till the completion of the reaction. The weak base employed for hydrolysis include alkali or alkaline earth metal carbonates, hydroxides, such as sodium carbonate, potassium carbonate, calcium carbonate, lithium hydroxide, calcium hydroxide and the like

preferably lithium hydroxide. The solvent used during hydrolysis include but not limited to ether, ketones, alcohols and the like or mixture thereof or mixture with water. Preferably, solvent can be selected from tetrahydrofuran, mixture of tetrahydrofuran and water, mixture of acetone and water, mixture of alcohol and water, acetone, methanol, ethanol, isopropanol, butanol and the like. After completion of hydrolysis, intermediate of formula IX can be isolated from the reaction mixture using conventional techniques known in the art. Specifically, reaction mixture can be optionally washed with a suitable organic solvent and product can be precipitated by acidifying the aqueous layer using a suitable acid. Intermediate of formula IX thus precipitated can be isolated using techniques such as filtration, decantation, centrifugation and the like.
It is found by the present inventors that if above reaction is carried out using strong base such as sodium hydroxide or potassium hydroxide as reported in the prior art, it results in formation of impure intermediate of formula XI, i.e. contaminated with several impurities. Some of the impurities that may form during hydrolysis are identified by various spectroscopic methods like Mass analysis and found to have structures as given below;

(Structure Removed)
Mainreason for formation ol"the impurities, depend upon basicity of tiie base used for hydrolysis. If reaction is carried out using strong base, then it leads to the formation of above impurities. Therefore, to provide intermediate of formula IX free from the above impurities, present inventors have optimized the reaction condition and found that it is advantageous to use weak base in place of strong base so that product thus obtained will be free from the impurities or contain impurities in acceptable amounts. According to another embodiment, intermediate of formula IX can be prepared directly from intermediate of formula VI without isolating intermediate of formula VIII.
Intermediate of formula IX thus prepared by either of the process can be optionally purified to enhance the purity and/or to minimize the presence of impurity using a suitable purification method.
In one way, intermediate of formula IX can be purified using base acid treatment. Specifically, the intermediate of formula IX can be dissolved in solvent with subsequent addition of a suitable base. Suitable base employed can organic base such as ammonia, triethylamine, diisopropylethyl amine; or inorganic base such as alkali or alkaline metal carbonates, bicarbonates or hydroxides thereof. Thereafter, the purified intermediate of formula IX can be precipitated from the resulting solution by the addition of acid till the pH of the mixture reaches I to 2, preferably 1.5. Acid used for the acidification includes inorganic acid such as hydrochloric acid, hydrobromic acid, phosphonic acid; or organic acid such as acetic acid, citric acid and the like. The purified product thus precipitated can be isolated using techniques such as filtration, decantation, centrifugation and the like.

In another way, intermediate of formula IX can be purified using a suitable solvent which includes alcohol such as methanol, isopropanol; ketone such as acetone, methyl isobutyl ketone; ester such as ethyl acetate, isopropyl acetate and the like or mixture thereof. Specifically, the intermediate of formula IX in a suitable solvent can be heated to a temperature of 40 to70 °C for few minutes to few hours. Purified intermediate of formula IX can be obtained from the reaction mixture by suitable techniques such as filtration or centrifugation.
Intermediate of formula IX thus prepared is then converted to argatroban of formula 1 by subsequent removal of nitro group and reduction of quinoline ring Specifically, the process involves the reaction of the compound of formula IX with a suitable reducing agent in the presence of a suitable acid in an organic solvent at a temperature of about 0 to 100°C. Suitable reducing agent includes catalyst such as palladium on carbon. Solvent employed for the reaction includes alcohol such as ethanol, methanol and the like. Acid used for the reaction can be organic acid such as acetic acid, citric acid; or inorganic acid such as hydrochloric acid and the like. Reduction is usually carried out using a catalyst under hydrogen pressure of about 0-20 kg/cm2, preferably 2-7 kg/cm2. The reduction reaction is preferably carried out using palladium on carbon in the presence of acetic acid in the reaction solvent such as ethanol. After the completion of the reaction, solvent can be removed from reaction and product can be isolated by neutralizing the reaction mixture in a water immiscible solvent by the addition of suitable base. Base includes inorganic base such as alkali or alkaline metal carbonate such as potassium carbonate, sodium carbonates and the like. Water immiscible solvent includes halogenated solvent such as dichloromethane, chloroform; ester such as ethyl acetate, propylacetate; hydrocarbon solvent such as toluene and the like or mixture thereof It is desirable to add a water miscible solvent along with water to ensure the complete separation of the layers. Argatroban can be isolated from the resulting organic layer by the removal of solvent using suitable techniques such as evaporation or distillation. Argatroban thus prepared has been characterized by XRD which shows it's amorphous nature.

Argatroban thus prepared can be crystallized using a solvent such as alcohol or alcohol in mixture with water. Crystalline nature of argatroban is characterized by X-ray diffractogram, which shows the unique characteristic peaks. It has been found that argatroban displays different polymorph on crystallization with ethanol (as shown in Figure 1) as compared to argatroban monohydrate, which is obtained after crystallization from mixture of ethanol and water (as shown in Figure 2). X-ray diffraction patterns of argatroban is measured on a PANalytical X'Pert Pro diffractometer with Cu radiation and expressed in terms of two-theta, d-spacings and relative intensities. One ordinarily skilled in the art understands that experimental differences may arise due to differences in instrumentation, sample preparation or other, which can alter the 20 values, d-spacings and relative intensities slightly. Crystallization process can be repeated or can be used along with other purification processes till the product of desired purity is obtained. Argatroban thus prepared have displays purity of more than 99.0% by HPLC, preferably 99.5% by HPLC, more preferably 99.9%. Argatroban prepared by the present invention contain identified and unidentified impurities less than 0.15%, preferably less than 0.10 %, respectively more preferably free from impurities.
Various regulatory authorities put emphasis on the identification, characterization of the impurities present in pharmaceutical compounds. As is known by those skilled in the art, the management of process impurities is greatly enhanced by understanding their chemical structures, and by identifying the parameters that influence the amount of impurities in the final product. Moreover, it is important that the presence or absence of an impurity in a product be identified in any quality control process in order to ensure that the process complies with the required standards set down in the regulatory approval of that product prior to it being released for commercial sale. To comply with the pharmacopeias requirement, present inventors have identified impurities that may form during reduction at the final stage; some impurities may form due to partial or incomplete reduction of intermediate of formula IX or by the reduction of impurities that may present in intermediate of formula IX.

Impurity of the present invention can be characterized by various spectroscopic techniques like 1H and 13C Nuclear magnetic resonance (NMR), Ultraviolet spectroscopy (UV), Mass spectrometry (MS), Infrared spectroscopy (IR). The percentage of impurity present in argatroban can be identified by chromatographic techniques like thin layer chromatography (TLC) or high-pressure liquid chromatography (HPLC) preferably, by high pressure liquid chromatography. The impurities that may form during reduction are identified and found to have structures as given below;
(Structure Removed)

In addition to above impurities, some other impurities may also form during reduction reaction or some may carried forward from intermediates but those impurities are removed during work up process and are not detected in final product.
Reason for the formation of impurity A is partial reduction of intermediate of formula IX wherein removal of nitro group takes with incomplete reduction of the quinoline ring. The impurity B may form due to partial reduction of intermediate of formula IX wherein nitro group alone partially reduced to hydroxylamine group. Impurity C may form due to carry over of impurity of formula XV which gets reduced and result in the formation of impurity C. Impurity D may form during the reduction of intermediate of formula IX wherein only removal of nitro group takes place without any reduction of quinoline ring. In contrast to this, impurity E may form only by the complete reduction of quinoline ring without removal of nitro group of intermediate of formula IX.
According to another embodiment, present invention provides novel impurities A, B and C of argatroban.
All the impurities as described in the present invention can be either isolated from reaction mixture or can be synthesized so that they can be used as reference standard as well as reference marker. The isolation as well as synthesis of each of impurities can be carried out by the reaction sequence as described in the present invention. In addition to isolation and characterization, the presence or absence of an impurity in a product should be identified in any quality control process in order to ensure that process complies with the required standards set down in the regulatory approval of that product prior to it being released for commercial sale. Therefore,

presence of the above impurities present at intermediate stage or argatroban may be identified by chromatographic techniques like thin layer chromatography (TLC) or high-pressure liquid chromatography (HPLC) and preferably using HPLC. According to another embodiment, the present invention provides a process for identification of an impurity in a sample of argatroban, selected from one or more of impurities amongst A, B, C, D, or E.
The process for identification of impurities involves the chromatographic analysis of reference sample containing one or more of impurities amongst A, B, C, D, or E (reference marker) and argatroban. The same analysis is also carried out on the sample of argatroban. Thereafter, relative retention times of the impurities present in reference sample and sample of argatroban is carried out. Then by comparing the relative retention times calculated in the two samples, presence of impurity can be determined. The reference sample can be prepared by adding any one or more of the identified impurities of present invention to argatroban sample. If the relative retention times thus determined in the two samples are substantially same, the impurity of argatroban in the sample is identified as being the same as reference marker. The chromatographic analysis can be carried out by HPLC or TLC. According to another embodiment, the present invention provides a method of determining the amount of an impurity, selected from one or more of impurity among A, B, C, D, or E.
The process for quantification of impurities involves the chromatographic analysis of the reference sample containing known amount of one or more of impurities amongst A, B, C, D, or E (reference marker) and argatroban. Same analysis is also carried out on sample of argatroban. Thereafter, relative retention times of the impurities present in reference sample and sample of argatroban is calculated and also area under the peak with the specific impurity or impurities of present invention is calculated. Then by comparing the area under the peaks (referring to RRT of the specific impurity) amount of one or more of impurity among A, B, C, D, and E in argatroban can be calculated. The reference sample can be prepared by adding

known concentration of one or more of the identified impurities of present invention to argatroban sample. The chromatographic analysis can be carried out by HPLC. Major advantages realized in the present invention are that process can be easily and conveniently scaled-up for industrial large-scale production and that the process is simple, economical, high throughput and provides highly pure argatroban. The other advantage of the present invention is that it avoids the formation impurities at intermediate level by employing use of base suitable for the specific reaction that yields the corresponding product with fewer amounts of impurities or free from the impurities. The present invention also provides novel impurities that may be present in argatroban and their isolation, characterization. The present invention also provides identification and quantification of impurities present in final product. Although, the following examples illustrate the present invention in more detail, but should not be construed as limiting the scope of the invention. Example 1: Preparation of ethyl (2R, 4R)-4-methyl-piperidine-2-carboxvlate To a solution of ethanolic hydrochloride (500ml), (2R, 4R)-4-methyl - piperidine-2-carboxylic acid (50 g, 0.35 mol) was added at ambient temperature and stirred for 1 hour. Thereafter, reaction mixture was refluxed for 3 hours. After completion of reaction, the solvent was evaporated to give residue which was dissolved in ethyl acetate (1500ml), washed with 20% aqueous potassium carbonate solution (1500ml) and brine solution (200 ml). The resulting solution was dried over anhydrous sodium sulphate and evaporated under vacuum to give 50 g (84%) of title compound. Example 2: Preparation of ethvl-(2R, 4R)-l-[NG-nitro-L-arginvll-4-methvl-2-piperidine carboxylate hydrochloride
Method A: Step I: preparation of_ethyl-(2R, 4R)-l-[NG-nitro-N2 -(tert-butoxy carbonyl) -L- arginyl]-4-methyl-2-piperidine carboxylate
To a stirred solution of NG-nitro-N2 (tert-butoxycarbonyl)-L-arginine (30 g, 0.09 mol) in dry tetrahydrofuran (450 ml), A'-methylmorpholine (22.6 g, 0.21 mol) was added at room temperature and stirred to get a clear solution. The reaction mixture was cooled to -15 to -20°C followed by addition of isobutyl chloroformate (22.98 g,

0.17 mol) at -15 to -20°C under nitrogen atmosphere. Thereafter, a solution of ethyl (2R, 4R)-4-methyl-piperidine-2-carboxylate (22.6 g, 0.13 mol) in dry tetrahydrofuran (226 ml) was added to the reaction mixture at -15 to -20 °C and stirred for 10 minutes at the same temperature. Then, temperature of the reaction mixture was slowly raised to room temperature and the solvent was evaporated from reaction mixture to give title compound having purity 98.0% and cyclised impurity: 0.3 % by HPLC. Water (300ml) was added to resulting product and then mixture was extracted with ethyl acetate (300ml). The organic layer was washed successively with aqueous potassium hydroxide solution (100 ml), aqueous citric acid solution (100 ml) and finally with brine solution. Resulting organic layer was dried over anhydrous sodium sulphate and evaporated to give 40 g (90%) of the title compound having purity 98.5 %; and cyclised impurity not detected by HPLC. Step II: Preparation of ethyl-(2R, 4R)-l-[NG-nitro-L-arginyl]-4-methyl-2-piperidine carboxylate hydrochloride
A solution of ethyl-(2R,4R)-l-[NG-nitro-N2-(tert-butoxy carbonyl)-L-arginyl]-4 -methyl-2-piperidine carboxylate (10g, 0.02 mol) in ethanolic hydrochloride (20 ml 20%) was stirred for 10-15 hour at 10-15°C. The precipitated solid thus was filtered and washed with ethyl acetate to give 6.1 g (70%) of the title compound as white solid having purity 99.0 % by HPLC.
Method B: To a stirred solution of NG-nitro-N2 (tert-butoxycarbonyl)-L-arginine (30 g, 0.09 mol) in dry tetrahydrofuran (450 ml), N-methylmorpholine (22.6g, 0.22 mol) was added at ambient temperature and stirred to get a clear solution. Thereafter, reaction mixture was cooled to -15 to -20°C followed by slow addition of isobutyl chloroformate (22.98 g, 0.17 mol) at -15 to -20°C under nitrogen atmosphere. Thereafter, a solution of ethyl (2R, 4R)-4-methyl-piperidine-2-carboxylate (22.6 g, 0.13 mol) in dry tetrahydrofuran (220 ml) was added to the reaction mixture at -15 to -20°C and the stirred for 10 minutes at the same temperature. Then, temperature of the reaction mixture was slowly raised to ambient temperature. Solvent was evaporated from the resulting reaction mixture to give residue. Water (300 ml) was

added to resulting residue and mixture was extracted with ethyl acetate (300 ml). The organic layer was washed successively with aqueous potassium hydroxide solution (100 ml), aqueous citric acid solution (100 ml) and finally with brine solution. The resulting organic layer was dried over anhydrous sodium sulphate and treated with ethanolic hydrochloride (80 ml 20 %w/v) for 10-15 hour at 10-15°C. The solid thus precipitated was filtered and washed with ethyl acetate to give 24 g of title compound as white solid having purity 99.0 % by HPLC. Example 3: Preparation of (2R.4R)-l-[NG-nitro-N2-(3-methvl-8-quinolinesulfonvl)-L-arginvll-4-methvl-2-piperidinecarboxvlic acid Method A: Step I: Preparation of ethyl (2R,4R)-l-[NG-nitro-N2-(3-methyI-8-quinolinesulfonyl) -L-arginyl]-4-methyl-2-piperidine carboxylate To a stirred suspension of ethyl-(2R, 4R)-l-[NG-nitro-L-arginyl]-4-methyl-2-piperidine carboxylate hydrochloride(25 g, 0.06 mol) in dichloromethane (250 ml), triethylamine (5.45 g, 0.53 mol) was added at 5-10 °C and stirred for 30 minutes to get a clear solution. 3-Methyl-8-quinoline sulphonyl chloride (16.3 g, 0.07 mol) was added to the reaction mixture at 0-5 ° C and then stirred for 3 hour at 10-15 °C. After completion of reaction, demineralized water (160 ml) was added to the reaction mixture and layers were separated. Organic layer was washed successively with 10 % aqueous potassium carbonate solution (125 ml) and brine solution (100 ml). Resulting organic layer was dried over anhydrous sodium sulphate and solvents were evaporated to give a residue which was crystallized with methyl tert-butyl ether to give 31.7 g (90 %) of title compound as off-white solid having purity 98.0 % by HPLC.
Step II: Preparation of (2R,4R)-l-[NG-nitro-N2-(3-methyl-8-quinolinesuIfonyl)-L-arginyl]-4-methyl-2-piperidine carboxylic acid
To a stirred solution of ethyl (2R,4R)-l-[NG-nitro-N2-(3-methyl-8-quinolinesulfonyl) -L-arginyl]-4-methyl-2-piperidine carboxylate (25 g, 0.04 mol) in a mixture of tetrahydrofuran (50 ml) and water (150 ml), a solution of lithium hydroxide (3.64 g, 0. 09 mol) in water (50 ml) was added at 10-15 °C and reaction mass was stirred at

ambient temperature for 20-24 hours. Thereafter, reaction mass was washed with ethyl acetate (125 ml) and pH of aqueous layer was adjusted to 1.2 by the addition of 1N hydrochloric acid (125 ml). The solid thus precipitated was filtered and dissolved in water (250 ml) by the addition of aqueous ammonia (50 ml, 25%). The pH of the reaction mixture was adjusted to 1.5 with 1N hydrochloric acid (125ml) to precipitate the product. The precipitated solid was filtered and purified with methanol (250 ml) to give 19 g (80%) of the title compound having purity 98.5 % by HPLC.
Method B: To a stirred suspension of ethyl-(2R,4R)-l-[NG-nitro-L-arginyl]-4-methyl-2-piperidine carboxylate hydrochloride(25 g, 0.06 mol) in dichloromethane (250 ml), triethylamine (5.4 g, 0.05 mol) was added at 5-10°C and stirred for 30 minutes to get a clear solution. 3-Methyl-8-quinoline sulphonyl chloride (16.3 g, 0.07 mol) was added at 0-5 °C to the reaction mixture and then stirred for 3 hours at 10-15°C. After completion of reaction, demineralized water (160 ml) was added to the reaction mixture and layers were separated. Organic layer was washed successively with 10 % aqueous potassium carbonate solution (125 ml) and brine solution (100 ml). Resulting organic layer was dried over anhydrous sodium sulphate and evaporated. A mixture of tetrahydrofuran (50ml) and water (150ml) was added to resulting reaction mass followed by addition of lithium hydroxide solution (3.64 g, 0.09 mol) in water (50 ml) at 10-15°C and reaction mixture was stirred the at room temperature for 20-24 hours. Thereafter, resulting reaction mixture was washed with ethyl acetate (125 ml) and pH of aqueous layer was adjusted to 1.3 with IN hydrochloric acid (125 ml). The solid thus precipitated was filtered and purified with methanol (250 ml) to give (20 g (84%) of title compound as white solid having purity 98.5 % by HPLC. Example 4; Preparation of argatroban monohydrate
To a solution of [NG-nitro-N2-(3-methyl-8-quinoline sulfonyl )-L-arginyl-4-methyl-2-piperidine carboxylic acid (15 g, 0.03 mol) in a mixture of ethanol (375 ml) and acetic acid (60 ml), 10% palladium on carbon ( 3.75 g, ~50% wet) was added. The
reaction mass was shaken under hydrogen pressure (4-5 kg/cm ) at 30-40 °C for 30 hours. After the completion of the reaction, reaction mixture was filtered through hyflow bed to remove the catalyst and evaporated to give residue. Resulting residue was stirred in a mixture of dichloromethane (225 ml) and pH of the mixture was adjusted to 6-7 by the addition of 20% aqueous potassium carbonate solution (75 ml). Layers were separated and organic layer was treated with demineralized water (75 ml) and methanol. The organic layer was evaporated to give amorphous argatroban.
Resulting argatroban was re-crystallized from ethanol to give crystalline title compound (showing XRD as shown in figure 1) which was further dissolved in a mixture of water (225 ml) and ethanol (100 ml) at 90-100°C and maintained at same temperature for 3 hours. The reaction mass was very slowly cooled to 15-20 °C. The precipitated solid was filtered, washed with demineralized water (30 ml) and dried at 50-55 °C under vacuum for 12 hours to give 9 g (63%) of title compound as white crystalline powder having purity 99.9% by HPLC (isomer ratio 63.4 : 36.6 : R: S) and displays XRD pattern as shown in figure 2. Example 5: Preparation of impurity of formula XIII.
To a stirred solution of ethyl-(2R,4R)-l-[NG-nitro-N2-(tert-butoxy carbonyl)-L-arginyl]-4 -methyl-2-piperidine carboxylate (5 g) in methanol (40 ml), potassium hydroxide (0.85 g) was added at room temperature. Thereafter, reaction mixture was heated to 50 °C and stirred for 10 hours. Solvents were distilled off from the reaction mixture tom give a residue. Water (25 ml) was added to the resulting residue and pH of the mixture was adjusted to 3 by the addition of 1 N hydrochloric acid (5 ml), the resulting product was extracted with ethyl acetate (50 ml). Ethyl acetate layer was washed with brine solution and dried over anhydrous sodium sulfate. Organic layer was evaporated to give 2 g (55 %) of the title compound.

WE CLAIM
1) A process for the preparation of highly pure argatroban, comprising the steps of: a).esterifying a compound of formula II with alcoholic hydrogen chloride,
(Formula Removed)
to form an intermediate of formula III;
(Formula Removed)
wherein R is alkyl selected from methyl, ethyl and the like b).condensing the intermediate of formula III with a reactive derivative of compound of formula IV,
(Formula Removed)
in presence of an organic base having pKa value less than 8.5 in a suitable organic solvent to form an amide intermediate of formula V;
(Formula Removed)
wherein R is as defined above c). optionally, isolating amide intermediate of formula V; d).deprotecting amide intermediate of formula V using a source of hydrogen
chloride to form an intermediate of formula VI;
(Formula Removed)
wherein R is as defined above e). condensing the intermediate of formula VI with a compound of formula VII,

(Formula Removed)
in the presence of a suitable base in an organic solvent to form amino-sulphonyl intermediate of formula VIII;

(Formula Removed)

wherein R is as defined above f). optionally, isolating/recrystallizing amino-sulphonyl intermediate of formula
VIII; g).hydrolyzing amino-sulphonyl intermediate of formula VIII in presence of a
weak base to form an intermediate of formula IX; and

(Formula Removed)

h).converting the intermediate of formula IX to argatroban of formula I.
2) The process according to claim 1, wherein in step a) alcoholic hydrogen chloride is selected from methanolic-hydrogen chloride, ethanolic-hydrogen chloride and the like; preferably, ethanolic-hydrogen chloride and the like.
3) The process according to claim 1, wherein in step b) reactive derivative of compound of formula IV is selected from anhydride, mixed anhydride, activated ester and the like; organic base includes -methyl morpholine, pyridine, and the like; and organic solvent includes C3-6 ketones such as acetone, methyl isobutyl ketone, methyl ethyl ketone; esters such as ethyl acetate; nitriles such as acetonitrile; halogenated solvents such as dichloromethane, chloroform; ethers

such as tetrahydrofuran; aprotic solvents such as dimethylformamide and the like or mixture thereof.
4) The process according to claim 3, wherein in reactive derivative of compound of formula IV is prepared by reacting compound of formula IV with a suitable activating agent selected from alky or aryl chloroformate such as isobutyl chloroformate, methyl chloroformate, ethyl chloroformate, phenyl chlroformate and the like.
5) The process according to claim 1, wherein step d) aqueous hydrochloric acid, hydrogen chloride gas or mixture thereof with suitable solvent selected from alcohol such as methanol, ethanol, isopropyl alcohol, tert-butanol, ester such as ethyl acetate; ether such as isopropyl ether, diethyl ether and the like or mixture thereof; in step e) suitable base is selected from organic base which includes substituted amine such as triethyl amine, diisopropylethyl amine, diazabicyclo[5.4.0]undec-7-ene (DBU); or inorganic base which includes alkali or alkaline metal carbonates, bicarbonates thereof such as sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and the like or combination thereof; and organic solvent includes halogenated solvents such as dichloromethane, dichloroethane, chloroform; ethers such as tetrahydrofuran; esters such as ethyl acetate; ketone, hydrocarbons and the like or mixture thereof
6) The process according to claim 1, wherein step g) weak base is selected from alkali or alkaline earth metal carbonates, hydroxides, such as sodium carbonate, potassium carbonate, calcium carbonate, lithium hydroxide, calcium hydroxide and the like.
7) Argatroban having less than 0.15 % of one or more of the following impurities:
(Formula Removed)

(Formula Removed)

8) A process for identification of an impurity in a sample of argatroban, selected
from one or more of impurities amongst impurity A, B, C, D or E, the process
comprising performing steps (a) and (b) in either order:
a), carrying out a chromatographic analysis on the reference sample, containing one or more of impurities amongst A, B, C, D or E (reference marker) and argatroban, to determine the relative retention time of the reference marker compared to argatroban; b). carrying out a chromatographic analysis on the sample of argatroban to determine the relative retention time of impurities present in the sample compared to argatroban;
and comparing the relative retention times determined in step (a) and (b);
wherein if the relative retention times determined are substantially same, the
impurity of argatroban in the sample is identified as being the same as
reference marker.
9) A method of determining the amount of an impurity, selected from one or more
of impurity among A, B, C, D and E, the method comprising performing steps
(a) and (b) in either order:

a), carrying out a chromatographic analysis on the reference sample, containing known amount of one or more impurities amongst A, B, C, D or E (reference standard) and argatroban, to determine the relative retention time of impurities compared to argatroban; and measuring the area under a peak corresponding to the reference standard; b). carrying out a chromatographic analysis on the sample of argatroban to determine area under HPLC peak corresponding to each and every impurity by reference to the relative retention times thus determined compared to argatroban; and calculating the amount of one or more of impurity among A, B, C, D and E in argatroban, by reference to area of the HPLC peak in the sample against the area of HPLC peak associates with the same impurity in step (a). 10) Impurity of formula,
(Formula Removed)