The present invention relates to a process for the preparation of Treosulfan. The present invention also relates to a novel intermediate useful in the preparation of Treosulfan. BACKGROUND OF THE INVENTION

Treosulfan, chemically known as L-threitol-l,4-di-(methanesulfonate), is represented by formula I,

Formula I

Treosulfan is an active ingredient of the drug Ovastat . Treosulfan is indicated for the treatment of ovarian cancer and belongs to the class of alkylating agents, which prevents the growth and division of cancerous cells.

US3155702 discloses the preparation of Treosulfan by methanesulphonation of (2S,3S)- l,4-dibromobutane-2,3-diol with excess amount of silver methanesulphonate. The presence of free 2,3-diol in the starting material leads to side reactions and formation of undesired by-products which necessitates an additional purification step and thereby results in lower yields. Further, an additional filtration operation is also required to remove silver bromide salt generated during the process and un-reacted silver methanesulphonate, which makes the process less attractive for commercial manufacturing.

US3246012 discloses the preparation of Treosulfan by protection of hydroxyl group of dialkyl tartrates with corresponding aldehyde, ketone or a reactive derivatives to form corresponding cyclic 2,3-O-acetals and 2,3-O-ketals of butanetetrol esters followed by reduction using lithium aluminium hydride to obtain 2,3-O-acetal or ketal protected butanetetrol, which is further methanesulphonated and treated with acid. The use of highly pyrophoric and hazardous reducing agent renders the above process not ideal for industrial production. Organic Syntheses, Coll. Vol. 10, p. 297, 2004 discloses a similar reaction sequence followed by the final de-protection of methanesulphonated 2,3-O-diisopropylidene-L- threitol in methanesulfonic acid at reflux temperature, which leads to a sluggish reaction mixture and a higher number of impurities due to maintaining the reaction mixture for longer time at higher temperature.

IN 1568/MUM/2012 also discloses similar reaction sequence involving reduction of dimethyl-2,3-0-isopropylidene-L-tartrate by sodium-bis(2-methoxyethoxy) aluminium hydride followed by methanesulphonation and final deprotection with formic acid to yield Treosulfan.

KR101367641 describes reduction using lithium borohydride, which requires about 14 hours to complete the reaction and is further extended due to involvement of column chromatography purification. Tetrahedron, vol. 49, no. 30, p. 6645, 1993 describes reduction using sodium borohydride and lithium chloride, followed by flash chromatography purification. Reduction conditions as per Chem. Pharm. Bull. Vol. 42, No. 3, p. 68, 1994, are again not commercially feasible because of lithium aluminium hydride as reducing agent.

From the above, it is apparent that the processes for the preparation of Treosulfan reported in the prior arts suffer from one or more drawbacks, such as use of hazardous reducing agents, low yield, and generation of impurities, which requires additional purifications.

Thus, there still remains a need to develop an efficient and industrially useful process for the preparation of Treosulfan, which can overcome the drawback of the prior art specifically focusing on the reduction step and reaction conditions for final de-protection of 2,3-diol group. It is of particular importance to develop methods that would allow clean reaction and result in high yield and purity of the final active pharmaceutical ingredient (API) i.e. Treosulfan.

The inventors of the present invention have extensively studied various reducing agents for the reduction reaction, various 2,3-diol protecting groups based on ease of deprotection and temperature conditions for the final step so as to obtain Treosulfan in high yield and high purity. OBJECT OF THE INVENTION

It is an objective of the present invention to overcome the drawbacks of the prior art processes, as described above.

It is another objective of the present invention to provide an improved and industrially viable process for the synthesis of Treosulfan, without involving hazardous and pyrophoric reducing reagents.

It is yet another objective of the present invention to provide an improved and industrially viable process for the synthesis of Treosulfan, by avoiding harsh deprotection conditions and obtaining Treosulfan in higher purity. In yet another objective, the present invention provides a novel intermediate useful in the synthesis of Treosulfan.

SUMMARY OF THE INVENTION

The inventors of the present invention have developed a process for the preparation of Treosulfan which involves reduction reaction using non expensive and convenient reducing agent, sodium borohydride and iodine. The use of a specific combination of sodium borohydride and iodine involves surprisingly simple workup as compared to reduction using lithium aluminum hydride.

Further, the present inventors have also come up with a new 2,3-diol protecting group, which makes the process of the present invention industrially viable and avoids use of harsh deprotection reagents and column/flash chromatography. The process of present invention results in intermediates and final API throughout the process in high purity and high overall yield. The present invention also provides a novel intermediate useful in the preparation of Treosulfan.

The following aspects describe the present invention. In a first aspect, the present invention provides a process for the preparation of Treosulfan of formula I, Formula I

comprising the steps of: a) reducing a compound of formula II,

Formula II where, Ri is selected from hydrogen and alkoxy; and

R ₂ and R ₃ is independently selected from hydrogen, C _1-6 alkyl and (CO)OR' , where R' is Ci-6 alkyl; in the presence of sodium borohydride and iodine to obtain a compound of formula III,

Formula III where, Ri is selected from hydrogen and alkoxy; and b) converting the compound of formula III to Tresoulfan.

In a second aspect, the present invention provides a process for the conversion of compound of formula III to Tresoulfan, comprising the steps of: a) reacting a compound of formula III, Formula III where, Riis selected from hydrogen and alkoxy; with methanesulphonic acid or a reactive functional derivative of methanesulfonic acid, in the presence of a base to obtain a compound of formula IV;

Formula IV where, Ri is selected from hydrogen and alkoxy; and b) reacting the compound of formula IV with an acid to form Treosulfan.

In a third aspect, the present invention provides a process for the preparation of compound of formula IV, comprising the step of reacting a compound of formula III,

Formula III

where, Ri is alkoxy; with methanesulphonic acid or a reactive functional derivative of methanesulfonic acid, in the presence of a base to obtain a compound of formula IV. In a fourth aspect, the present invention provides a novel compound of formula IV,

Formula IV

where, Ri is alkoxy.

In a fifth aspect, the present invention provides a process for the preparation of Treosulfan, comprising the step of reacting a compound of formula IV,

Formula IV

where, Ri is alkoxy;

with an acid.

In a sixth aspect, the present invention provides a process for purification compound of formula II,

Formula II where, Ri is selected from hydrogen and Ci- ₄ alkoxy; and

R ₂ and R ₃ is independently selected from hydrogen and C _1-6 alkyl; a) contacting the compound of formula II with a solvent selected from the group consisting of C _1-4 alcohol, hydrocarbon solvent, a mixture thereof, a mixture of a C _1-4 alcohol with a halogenated solvent and a mixture of a hydrocarbon solvent with a halogenated solvent; and b) isolating the purified compound of formula II.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising Treosulfan, obtainable by the process of present invention, and at least one pharmaceutically acceptable excipient.

In an eighth aspect, the present invention provides a method of treating cancer, comprising administering a therapeutically effective amount of Treosulfan, obtainable by the process of present invention.

In a ninth aspect, the present invention provides a method of preparing a pharmaceutical composition, comprising a step of admixing Treosulfan obtainable by the process of present invention, with one or more pharmaceutically acceptable excipients.

DEFINITIONS

The following definitions are used in connection with the present application unless it is indicated otherwise.

The term "ambient temperature" refers to a temperature ranging from about 15°C to 35°C, preferably the term "ambient temperature" refers to a temperature from about 20°C to 30°C, more preferably to a temperature of about 25°C. The term "anti- solvent" refers to a liquid that, when combined with a solution of Treosulfan or an intermediate of Treosulfan of the present invention reduces their solubility in the solution causing crystallization or precipitation, in some instances spontaneously, and in other instances with additional steps such as seeding, cooling, scratching and/or concentrating.

The term "C _1-6 alkyl" refers to a straight chain, branched chain or cyclic hydrocarbon group, including but not limited to methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl and t-butyl.

The term refers to a straight chain, branched chain or cyclic alkyl group bonded to oxygen, including but not limited to methoxy, ethoxy, n-propyloxy, iso- propyloxy, n-butoxy, sec-butoxy and t-butoxy.

The terms "comprising" and "comprises" mean the elements recited, or their equivalents in structure or function, plus any other element or elements which are not recited.

The term "optional" or "optionally" is taken to mean that the event or circumstance described in the specification may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

As used herein, the term "contacting" includes mixing, adding, slurring, stirring or a combination thereof.

As used herein, the terms "about" are to be construed as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances. This includes, at the very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.

ABBREVIATIONS

XRPD X-ray powder diffraction HPLC High performance liquid chromatography

BRIEF DESCRIPTION OF FIGURES

Fig. 1; represents an X-ray powder diffraction (XRPD) pattern of Treosulfan prepared by process of present invention DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved and industrially viable process for the preparation of Treosulfan. Further, the present invention provides a novel intermediate and a process for the purification of an intermediate compound.

In a first aspect, the present invention provides a process for the preparation of Treosulfan,

Formula I

comprising the steps of: a) reducing a compound of formula II,

Formula II where, Ri is selected from hydrogen and C _1-4 alkoxy; and R ₂ and R ₃ is independently selected from hydrogen, Ci- ₆ alkyl and (CO)OR' , where R' is Ci- ₆ alkyl; in the presence of sodium borohydride and iodine to obtain a compound of formula III,

HO OH

Formula III where, Ri is selected from hydrogen and C _1-4 alkoxy; and b) converting the compound of formula III to Treosulfan. In a preferred embodiment, the process involves the compound of formula II, wherein at least one of R ₂ and R ₃ is Ci _-6 alkyl or (CO)OR' .

In another preferred embodiment, the process involves the compound of formula II, wherein both R ₂ and R ₃ are hydrogen. In yet another preferred embodiment, the process involves the compound of formula II, wherein both R ₂ and R ₃ are Ci-6 alkyl or (CO)OR' ; wherein R ₂ and R ₃ may be the same or different.

The reduction reaction may be carried out in the presence of a solvent selected from the group consisting of alcohol, ether and a mixture thereof. Preferably the reduction reaction may be carried out in the presence of a mixture of water and solvent selected from the group consisting of alcohol, ether and a mixture thereof.

The solvent may be selected from the group consisting of methanol, ethanol, isopropanol, tetrahydrofuran, diethylether, di-isopropylether, methyl tert-butyl ether and a mixture thereof. Preferably, the reaction is carried out in a mixture of water and tetrahydrofuran.

The reduction reaction may be carried out at a temperature of about 0 to 50°C. In a preferred embodiment, the reduction reaction is carried out at a temperature of about 10 to 40°C; more preferably at about 25 to 35°C. In another preferred embodiment, the reduction reaction is carried out at a temperature of about 0 to 40°C; more preferably at 0 to 35°C or at ambient temperature. The reaction may be carried out for about 15 minutes to about 10 hours; preferably for about 30 minutes to about 7 hours.

The compound of formula II, wherein at least one of R ₂ and R ₃ is (CO)OR' may be prepared from the compound of formula II, wherein at least one of R ₂ and R ₃ is hydrogen.

The process involves reaction of compound of formula II, wherein at least one of R ₂ and R ₃ is hydrogen, with alky lhalo formate in the presence of base.

Alky lhalo formate is selected from the group consisting of alkylchloroformates, preferably ethylchloro formate.

Base used in the reaction may be selected from the group consisting of diisopropylamine, diisopropylethylamine and triethylamine, preferably trie thy lamine. The reaction of compound of formula II, wherein at least one of R ₂ and R ₃ is hydrogen, with alky lhalo formate is optionally carried out in the presence of a solvent selected from the group consisting of ethers and a mixture thereof, preferably tetrahydrofuran, diethylether, di-isopropylether, methyl tert-butyl ether, more preferably tetrahydrofuran. The reaction of compound of formula II with alky lhalo formate is carried out at -10 to 40°C, preferably at -5 to 30°C.

The reaction of compound of formula II, wherein at least one of R ₂ and R ₃ is hydrogen, with alky lhalo formate is further carried forward to the reduction reaction as described above, to obtain compound of formula III. The compound of formula III thus obtained may be isolated by methods such as distillation, precipitation, cooling, filtration, centrifugation or combination thereof or may be used directly further; preferably, the compound of formula III is isolated by distillation.

The compound of formula III obtained by the process of present invention, with or without purification may be optionally dried by methods such as vacuum drying, heat drying, spray drying, freeze drying, supercritical drying or natural air drying. Any of the mentioned methods may also be used in combination to ensure removal of residual solvent. Preferably, the compound of formula III is dried by vacuum drying. Conveniently the drying is performed under vacuum and optionally under inert atmosphere.

The process described above may be varied, for example in terms of the quantity of the starting compound of formula II that is treated, the volume of the solvent or a mixture of solvents, the temperature of the treatment, cooling phases and/or drying conditions.

The inventors of the present invention have found that the use of a specific combination of sodium borohydride with iodine provides advantages of using non-expensive and convenient reagents as compared to hazardous and highly pyrophoric lithium aluminium hydride. It has been realized that the reduction of the compound of formula II using sodium borohydride with iodine results in a corresponding reduced compound in very good yield and favors a clean reaction with minimal amounts of side products. The conventional reducing agents suffer from drastic reaction conditions, low to moderate yield and occurrence of several side reactions. The combination of iodine and sodium borohydride helps to carry out the reaction under neutral and mild conditions and in high yield. In addition, the work-up procedure is surprisingly simple and the reaction can be performed at 0 ^° C to ambient temperature. The inventors of the present invention have found that the addition of iodine with sodium borohydride, advantageously reduces the reaction time and improves the quality of reaction which ultimately results in an industrially viable process. Also, the process of the present invention avoids tedious purification processes such as column/flash chromatography and thus drastically reduces the time for commercial manufacturing while still being able to obtain the compound of formula III with high purity.

The process of the present invention provides the compound of formula III with more than 95% purity, preferably more than 97% purity, more preferably more than 99% purity.

In a second aspect, the present invention provides a process for the conversion of the compound of formula III to Treosulfan, comprising the steps of: a) reacting a compound of formula III,

Formula III

where, Ri is selected from hydrogen

with methanesulphonic acid or a reactive functional derivative of methanesulfonic acid, in the presence of a base to obtain a compound of formula IV,

Formula IV where, Ri is selected from hydrogen alkoxy; and b) reacting the compound of formula IV with an acidto form Treosulfan.

The reaction of step a) may be carried out using methanesulphonic acid or a reactive functional derivative of methanesulfonic acid. The reactive functional derivative of methanesulphonic acid may be selected from methanesulphonic acid anhydride or methanesulphonyl halide. Preferably, the reaction is carried out using methanesulphonyl chloride.

The base used in step a) may be an organic base selected from the group consisting of pyridine, diisopropylamine, diisopropylethylamine and trie thy lamine; an inorganic base selected from the group consisting of alkali and alkaline metal hydroxides, alkoxides and carbonates thereof; or a mixture of an organic base or an inorganic base as specified above.

The base used in step a) is preferably selected from diisopropylamine, pyridine and alkali metal carbonates; preferably diisopropylamine.

The reaction maybe optionally carried out in the presence of a solvent selected from the group consisting of halogenated solvent, ketone anda mixture thereof. Preferably, the solvent is selected from the group consisting of dichloromethane, dichloroethane, chloroform, acetone, methylisobutylketone, methylethyl ketone and a mixture thereof, preferably the solvent is dichloromethane.

The compound of formula IV may be optionally purified by crystallization from a solvent. The solvent may beselected from the group consisting of halogenated solvent, alcohol, ketoneanda mixture thereof, preferably the solvent is selected from the group consisting of methanol, ethanol, isopopanol, acetone, methylisobutylketone, dichloromethane and a mixture thereof. The precipitation can be effected by cooling the mixture of the compound of formula IV in a solvent or by addition of an anti-solvent. The purification of compound of formula IV may be repeated to obtain the compound of formula IV with desired purity.

The compound of formula IV, with or without purification, may be isolated by methods such as distillation, precipitation, cooling, filtration, centrifugation or acombination thereof or may be used directly for further treatment with acid. The compound of formula IV obtained by the process of the present invention, with or without purification may be optionally dried by methods such as vacuum drying, heat drying, spray drying, freeze drying, supercritical drying or natural air drying. Any of the mentioned methods may also be used in combination to ensure removal of residual solvent. Preferably, the compound of formula IV is dried by vacuum drying. Conveniently, the drying is performed under vacuum and optionally under an inert gas atmosphere.

The process described above may be varied, for example in terms of the quantity of the starting compound of formula III, methanesulfonic acid or its reactive functional derivative, that is treated, the volume of the solvent or a mixture of solvents, the temperature of the treatment, cooling phases and/or drying conditions.

In a third aspect, the present invention provides a process for the preparation of compound of formula IV comprising the step of reacting a compound of formula III with methanesulphonic acid or a reactive functional derivative of methanesulfonic acid, in the presence of a base to obtain a compound of formula IV. In a fourth aspect, the present invention provides a novel intermediate of formula IV, where Ri is

In a preferred embodiment, the present invention provides a compound of formula IV, wherein Ri is selected from methoxy, ethoxy, propoxy and butoxy; preferably Ri is selected from methoxy and ethoxy; more preferably Ri is methoxy. The compound of formula IV is preferably the compound of formula IVa,

Formula IVa

In a fifth aspect, the present invention provides a process for the preparation of Treosulfan, comprising the step of reactinga compound of formula IV, where Ri is Q. ₄ alkoxy; with an acid. The process of reacting the compound of formula IV with an acid results in the deprotection of 2,3-diol protecting group.

The acid used in process, may be selected from the group consisting of hydrochloric acid, methanesulphonic acid, formic acid, phosphomolybdic acid, trifluoro acetic acid, acetic acid, p-toluenesulfonic acid anda mixture thereof; preferably, the acid is hydrochloric acid.

The step of reacting the compound of formula IV with an acid maybe optionally carried out in the presence of a solvent selected from the group consisting of alcohol, ether ester, carboxylic acid anda mixture thereof; preferably, the reaction is carried out in the presence of a solvent selected from methanol, tetrahydrofuran, ethylacetate, formic acid, acetic acid or a mixture thereof; more preferably methanol.

The mixture of the compound of formula IV and a solvent is maintained at 15 to 90°C, preferably at 20 to 70°C till the completion of the reaction. Preferably, the reaction mixture is stirred for 1 to 16 hours, more preferably for 4to 12 hours, even more preferably for 5 to 12 hours. Treosulfan thus obtained may be isolated by methods known to the person skilled in the art followed by optional washing with the initially used pre-cooled solvent and drying under vacuum.

Treosulfan thus obtained may be optionally purified by contacting Treosulfan with a solvent selected from the group consisting of alcohol, ketone andmixtures thereof. Preferably, the solvent is selected from the group consisting of methanol, ethanol, isopropanol, acetoneandmixtures thereof. The purification can be repeated with the same or different solvents to achieve the final API with high purity and minimal impurities, preferably substantially free from impurities.

The mixture of Treosulfan and solvent may be maintained at 10°C to ambient temperature to obtain a clear reaction mixture devoid of any insoluble particles. Additional operations such as normal filtration or microne filtration or both may be further carried out to ensure the complete removal of any insoluble particles.

The precipitation of the compound may be carried out by cooling the reaction mixture or by addition of an anti-solvent selected from the group consisting of ether solvents; preferably di-isopropyl ether. Treosulfan thus obtained may be isolated by methods such as distillation, precipitation, cooling, filtration, centrifugation or combination thereof; preferably Treosulfan is isolated by filtration.

Treosulfan isolated by the process of present invention may be optionally dried by the methods such as vacuum drying, heat drying, spray drying, freeze drying, supercritical drying or natural air drying. Any of the mentioned methods may also be used in combination to ensure complete removal of residual solvent. Preferably Treosulfan is dried by vacuum drying. As will be recognized, the drying time will be dependent upon, amongst other things, the amount of material to be dried, and the particular drying method used. Generally, a drying time of 1 hour to 30 hours, preferably 2 to 25 hours, most preferably 5 to 22 hours, is used to dry Treosulfan. Conveniently, the drying is performed under vacuum and optionally under an inert gas atmosphere, for example by passing a stream of warm inert gas such as nitrogen over or through the material.

In a sixth aspect, the present invention provides a process for the purification of the compound of formula II,

Formula II where, Ri is selected from hydrogen and C _1-4 alkoxy; and R ₂ and R ₃ is independently selected from hydrogen, Ci- ₆ alkyl comprising the steps of: a) contacting the compound of formula II with a solvent selected from the group

consisting of C _1-4 alcohol, hydrocarbon solvent, a mixture thereof, a mixture of a C _1-4 alcohol with a halogenated solvent, and a mixture of a hydrocarbon solvent with a halogenated solvent; and b) isolating purified compound of formula II. The process involves contacting the compound of formula II with a solvent selected from the group consisting of C _1-4 alcohol, hydrocarbon solvent, a mixture thereof, a mixture of a Ci-4 alcohol with a halogenated solvent, and a mixture of a hydrocarbon solvent with a halogenated solvent at -5 to 90°C preferably at 0 to 70 °C. The reaction mixture may be stirred for 30 minutes to 8 hours, preferably 1 to 5 hours.

The solvent used for the purification may be selected from the group consisting of methanol, ethanol, isopropanol, butanol, toluene, hexane, heptanes and mixture thereof. Halogenated solvent may be selected from the group consisting of dichloromethane, dichloroethane and chloroform. Preferably the compound of formula II is purified using solvent selected from methanol, ethanol, isopropanol, a mixture of toluene and isopropanol, a mixture of dichloromethane and isopropanol, a mixture of dichloromethane and hexane and a mixture of dichloromethane and heptane.

The compound of formula II thus obtained may be isolated by methods such as distillation, precipitation, cooling, filtration, centrifugation or combination thereof or may be used directly further; preferably compound of formula II is isolated by filtration.

The compound of formula II thus isolated may be further washed with the pre-cooled solvent used initially in the step a).

The compound of formula II isolated by the process of present invention may be optionally dried by methods such as vacuum drying, heat drying, spray drying, freeze drying, supercritical drying or natural air drying. Any of the mentioned methods may also be used in combination to ensure removal of residual solvent. Preferably, the compound of formula II is dried by vacuum drying. As will be recognized, the drying time will be dependent upon, amongst other things, the amount of material to be dried, and the particular drying method used. Generally a drying time of 1 hour to 30 hours, preferably 2 to 25 hours, most preferably compound of formula II is dried for 5 to 22 hours. Conveniently the drying is performed under vacuum and optionally under an inert gas atmosphere, for example by passing a stream of warm inert gas such as nitrogen over or through the material.

The process of the present invention provides a simple purification process for the compound of formula II, as compared to prior art processes which involves use of solvent- antisolvent methodology {Tetrahedron, vol. 46, No. 12, p. 4165, 1990) and use of dichloromethane and petroleum ether for isolation and crystallization respectively {Synthesis, No. 15, p. 2488-90, 2008).

The present inventors have found that by following the purification of the present invention, the compound of formula II is obtained in higher purity as compared to the processes of the prior art. Comparative data is presented below.

Thus, the process of the present invention results in an intermediate (compound of formula II) of high purity, which thereby results in high purity of the final API.

The compound of formula II, wherein R ₂ and R ₃ is independently selected from same or different C _1-6 alkyl, as used in the process of the present invention may be obtained by the processes known in the art, wherein the process comprises protecting dialkyl-L-taratrate with p-anisaldehyde-dimethylacetal in the presence of a catalytic amount of p-toluene sulfonic acid and an azeotropic solvent such as toluene. The compound of formula II thus obtained may be isolated by methods known by a person skilled in art or may be used directly for the purification step.

The compound of formula II, with or without purification, is further used for the preparation of Tresoulfan.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising Treosulfan, obtainable by the process of the present invention and at least one pharmaceutically acceptable excipient.

In an eighth aspect, the present invention provides a method of treating cancer, comprising administering a therapeutically effective amount of Treosulfan, obtainable by the process of present invention. Preferably the present invention provides a method of treating epithelial ovarian cancer.

In a ninth aspect, the present invention provides a method of preparing a pharmaceutical composition, comprising step of admixing Treosulfan obtainable by the process, with one or more pharmaceutically acceptable excipients.

INSTRUMENT

XRPD: X-ray diffraction data is obtained using a Bruker AXS D8 advance powder X-ray Diffractometer, CuKa radiation, wavelength 1.54A.

EXAMPLES Detailed experimental parameters suitable for the preparation of Treosulfan or intermediates according to the present invention are provided by the following examples, which are intended to be illustrative and not limiting.

Reference Example 1 (repetition of Tetrahedron, vol. 46, No. 12, p. 4165, 1990):

A reaction mixture of dimethyl-L-tartrate (10. Og), p-toluene sulfonic acid (0.013g) and p- anisaldehydedimethylacetal (l l.Og) in toluene (150ml) was refluxed and the azeotropical mixture of toluene-methanol was continuously removed from the reaction mixture for 3-5 hours. The reaction mixture was cooled to ambient temperature, diluted with dichloromethane (50ml) and neutralised by addition of potassium carbonate (5.0g) followed by stirring for an hour . The reaction mixture was filtered and filtrate was evaporated to give yellow crude compound, which was further dissolved in dichloromethane (25ml) followed by addition of petroleum ether (100ml) and stirred for an hour at ambient temperature. The solid was filtered, washed with petroleum ether (20ml) and dried under vacuum at 35-40°C for 15-20 hours to obtain 16.63g (72.15%) of dimethyl (4R,5R)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5-dicarboxylate having purity 98.4% by HPLC.

Reference Example 2 (repetition of Synthesis, No. 15, p. 2488-90, 2008):

A reaction mixture of dimethyl-L-tartrate (5.0g), p-toluene sulfonic acid (0.0064g) and p- anisaldehyde dimethylacetal (5.35g) in toluene (25ml) was refluxed and the azeotropical mixture of toluene-methanol was continuously removed from the reaction mixture for 3-5 hours. The reaction mixture was cooled to ambient temperature, diluted with dichloromethane (25ml) and neutralised by addition of potassium carbonate (5.0g) followed by stirring for an hour. The reaction mixture was filtered and filtrate was evaporated to give yellow crude residues. The crude was further re-crystallized in petroleum ether (25ml), filtered the solid and washed with petroleum ether (15ml) followed by drying under vacuum at 35-40°C for 15-20 hours to obtain 7.4g (89.15%) of dimethyl (4R,5R)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5-dicarboxylate having purity 98.8% by HPLC. Example-1: Preparation of dimethyl (4R,5R)-2-(4-methoxyphenyl)-l,3-dioxolane- 4,5-dicarboxylate

A reaction mixture of dimethyl-L-tartrate (500g), p-toluene sulfonic acid (5.38g) and p- anisaldehyde dimethylacetal (665g) in toluene (2250ml) was refluxed to 110-115°C. The azeotropical mixture of toluene-methanol was continuously removed from the reaction mixture till the completion of the reaction. The reaction mixture was cooled to ambient temperature and quenched with aq. saturated sodium bicarbonate solution (2500ml), layers were separated. Resulting organic layer was washed with water (2500ml x 2) followed by evaporation of organic layer. Isopropyl alcohol (3500ml) was charged to the residue and heated to 60-70°C followed by cooling at ambient temperature. Reaction mixture was stirred at 0-5°C for 1-2 hours and filtered. The solid thus obtained was washed with pre- cooled isopropyl alcohol and dried under vacuum at 35-40°C for 15-20 hours to obtain 767.0g (92.93%) of dimethyl (4R,5R)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5- dicarboxylate having purity 99.97% by HPLC.

Example-2: Preparation of (4S,5S)-2-(4-methoxyphenyl)-l ₅ 3-dioxo!ane-4,5- diyifdimethanol

Method-l :To a mixture of dimethyl (4R,5R)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5- dicarboxylate (765g), Iodine (13. lg) in tetrahydrofuran (3750ml) and water (76ml), sodium borohydride (146.52g) was added at 0-15°C and stirred for 1 -2 hours at ambient temperature. The reaction was quenched with 30% aq. ammonium chloride (6100ml) solution and dichloromethane (7650ml). The layers were separated and the aqueous layer was extracted by dichloromethane (3800ml x 3) followed by washing of combined organic layers with water (3800ml), The resulting organic layer was evaporated at 35-65°C to obtain 525.0g (83.9%) of (4S,5S)-2-(4-methoxyphenyl)-l,3- dioxolane-4,5-diyl]dimethanol having purity 99.72% by HPLC. Method-2: To a mixture of dimethyl (4R,5R)-2-(4-methoxyphenyl)-l,3-dioxolane- 4,5-dicarboxylate (765g), Iodine (13.10g) in tetrahydrofuran (3750ml) and water (76.5ml), sodium borohydride (146.52g) was added at 0-10°C and stirred for Ihours at 0-5°C and stirred for 3-4 hours at ambient temperature. The reaction was quenched with 30% aq. ammonium chloride (6120ml) solution and dichloromethane (7650ml) at ambient temperature. The layers were separated and the aqueous layer was extracted by dichloromethane (3825m! x 3) followed by washing of combined organic layers with water (3825ml). The resulting organic layer was evaporated at 50-60°C to obtain 525 g (84.7%) of (4S,5S)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5-diyl]dirnethai iol having purity 99.72% by HPLC. Example-3: Preparation of (4S,5S)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5- diyl]bis(methylene) dimethanesulfonate

Method-l:To a solution of (4S,5S)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5- diyl]dimethanol (145g) in dichloromethane (2175ml), pyridine (191g) and methanesulphonyl chloride (190. l g) was added at 0-5 ^° C. The reaction mixture was stirred for 2-3 hours at ambient temperature followed by quenching with water (1450ml). The organic layer was washed with water (1450ml x 4) and evaporated. The resulting residue was added to isopropanol (725ml) and stirred for 1-2 hours at ambient temperature and further for 1-2 hours at 0-5 C. The solid was filtered and washed with pre-cooled isopropanol (145ml). The resulting product was dissolved in acetone (1300ml) followed by addition of isopropanol (2610ml). Resulting reaction mixture was stirred for 1-2 hours at ambient temperature and then cooled at 0-5 ^° C. The solid thus obtained was filtered and washed with pre-cooled isopropanol (145ml x 2) and dried under vacuum at 30-35°C for 15-20 hours to give 190.8g (79.4%)of (4S,5S)-2-(4- methoxyphenyl)-l,3-dioxolane-4,5-diyl]bis(methylene) dimethanesulfonate. Method-2: To a solution of (4S,5S)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5- diyl]dimethanol (525, Og) in dichloromethane (7350ml), di-isopropylamine (663. Og) was added at ambient temperature followed by addition of methanesulphonyl chloride solution (624. Og in 525ml dichloromethane) at 0-10°C. The reaction mixture was stirred for 1-2 hours at 0-10 ^° C followed by stirring for 3-4 hours at ambient temperature. The organic layer was washed with water (2 x 5250ml) and evaporated. The residues were dissolved in acetone (4725ml) followed by addition of isopropanol (9450ml), stirred for about 1-2 hour at ambient temperature and then at 0-5 ^° C for 1-2 hours. The resulting solid was filtered, washed with pre-cooled isopropanol (525 x 2 ml)and dried under vacuum at 35-45°C for 15-20 hours to give 705.0g (81.45%) of (4S,5S)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5-diyl]bis(methy lene)

dimethanesulfonate having purity 99.92% by HPLC.

Example-4: Preparation of Treosulfan

Method-1: To a solution of (4S,5S)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5- diyl]bis(methylene) dimethanesulfonate (745. Og) in methanol (7450ml), concentrated hydrochloric acid (260ml) was added at 15-25°C followed by stirring for 10-15 hours at ambient temperature. The reaction mixture was cooled to 0-5°C and further stirred for 1-2 hours at 0-5°C followed by filtration and washing the solid with pre-cooled methanol (745ml). The solid thus obtained was dissolved in acetone (3725ml) followed by microne filtration. Di-isopropyl ether (7450ml) was added to the filtrate and stirred for 1-2 hours at ambient temperature and then cooled at 0-5°C. The solid thus obtained was filtered and washed with di-isopropyl ether (745ml x 2) followed by drying at 30-35°C for 15-20 hours to obtain 96.5g of Treosulfan having purity 99.9% by HPLC.

XRPD of Treosulfan obtained by above process is shown in Fig. 1. Method-2:To a solution of (4S,5S)-2-(4-methoxyphenyl)-l,3-dioxolane-4,5- diyl]bis(methylene)dimethanesulfonate (650. Og) in methanol (6500ml), 9N hydrochloric acid (227.5ml) was added at 0-10°C followed by stirring for 6-8 hours at ambient temperature. The reaction mixture was cooled to 0-5°C and further stirred for 1-2 hours followed by filtration and washing the solid with pre-cooled methanol (2 x 650ml). The solid thus obtained was dissolved in acetone (3250ml). Di-isopropyl ether (6500ml) was added to the resulting solution, stirred for 1-2 hours at ambient temperature and then cooled at 0-5°C. The solid thus obtained was filtered and washed with di- isopropyl ether (650ml x 2) followed by drying at 30-35°C for 15-20 hours to obtain 312g (68.4) of Treosulfan having purity 99.81% by HPLC.