10-Propargyl-10-Deazaaminopterin polymorph and its process

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION

The present invention provides 10-Propargyl-10-Deazaaminopterin (I) crystalline polymorphic form designated as Form-SPR and its process for preparation thereof. The present application also provides 10-Propargyl-10-Deazaaminopterin (I) crystalline polymorphic Form-SPR useful as an active pharmaceutical ingredient in pharmaceutical composition comprising thereof and having anti-cancer activity.

INTRODUCTION

Anti-cancer drug Pralatrexate i.e. (2S)-2-[[4-[(lRS)-l-[(2,4-diaminopteridin-6-yl)methyl]but-3-ynyl]benzoyl]amino]pentandioic acid is represented by the following formula (I): 10-Propargyl-10-Deazaaminopterin (Pralatrexate) is an antifolate and acts as an inhibitor of dihydrofolate reductase. It is approved for treatment of patients with relapsed or refractory peripheral T-cell lymphoma. Patent US 5354751 and literature article DeGraw et al. (J. Med. Chem, 1993, 36, 2228), disclosed 10-Propargyl-l0-Deazaaminopterin. Sirotnak et al. in US 6028071B2 and EP 944389B1 disclosed 10-Propargyl-10-deazaaminopterin substantially free of 10-deazaaminopterin. Further, in US patent application US 2011/0190305A1 Gijsbertus J. Pronk discloses substantially pure diastereomers of 10-propargyl-10-deazaminopterin, or a salt thereof, wherein the diastereomer may be either (2S)-2-[[4-[(lS)-l-[(2,4-diaminopteridin-6-yl)methyl]but-3-ynyl]benzoyl]aminolpentanedioic acid or (2S)-2-[[4-[(lR)-l-[(2,4-diaminopteridin-6-yl)methyl]but-3-ynyl]benzoyl]amino]pentanedioic acid.

Occurrence of different crystal forms of same substance i.e. polymorphism, is a property of some molecules and molecular complexes. A single molecule for e.g. 10-Propargyl-10-Deazaaminopterin, may give rise to a variety of polymorphs having distinct crystal structures and physical properties like XRPD pattern, melting point, TGA, DSC and infrared absorption spectrum. Different polymorphic forms of a compound can be distinguished by using one or more of these techniques. Discovering different polymorphic forms of a pharmaceutical product can provide materials having desirable processing properties, such as ease of handling, processing and purification, storage stability. New polymorphic forms of a pharmaceutically useful compound also provide an opportunity to improve the performance characteristics of a pharmaceutical product.

It enlarges the choice of materials that a formulation scientist has available for formulation optimization, for example by providing a product with different characteristics, e.g. better processing or handling characteristics, improved dissolution profile, or improved shelf life. Therefore there is always a need for new crystalline forms of 10-Propargyl-10-Deazaaminopterin. Hence, inventors of the present application provide 10-Propargyl-10-Deazaaminopterin (I) crystalline polymorphic form designated as Form-SPR and an industrially suitable process for its preparation.

SUMMARY OF INVENTION

Particular aspects of the present specification relate to the new crystalline form of 10-Propargyl-10-Deazaaminopterin (I) designated as Form-SPR and process for its preparation. Further, the present invention of this application also relates to pharmaceutical compositions comprising of 10-Propargyl-10-Deazaaminopterin (I) Form-SPR which are useful in the treatment of various cancerous disorders. In one aspect of the present application, the present invention provides 10-Propargyl-l0-Deazaaminopterin (I) crystalline Form-SPR, characterized by X-ray powder diffraction pattern comprising at least 5 characteristic 20°peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3, 17.8, 19.1,21.5,25.2 and 25.6 ± 0.2 28° and DSC isotherm comprising atleast one endothermic peak ranging between 225 to 235 °C. In another aspect of the present application, 10-Propargyl-l0-Deazaaminopterin (I) crystalline Form-SPR is further characterized by IR absorption spectrum having characteristic peaks expressed in cm-1 at approximately 3294 cm-1, 3205 cm-1, 2114 cm-1, 1645 cm-1, 1555 cm-1 , 1538 cm-1 and 1499 cm-1.

In a further aspect of the present application, it relates to 10-Propargyl-10-Deazaaminopterin (I) crystalline Form-SPR characterized by X-ray powder diffraction pattern substantially according to Fig-1, DSC isothermal pattern substantially according to Fig-2 and IR absorption spectrum substantially according to Fig-3. In yet another aspect of the present application, it relates to process for preparing 10-Propargyl-10-Deazaaminopterin (I) crystalline Form-SPR, characterized by X-ray powder diffraction pattern comprising at least 5 characteristic 29° peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3, 17.8, 19.1, 21.5, 25.2 and 25.6 ± 0.2 26° and DSC isotherm comprising atleast one endothermic peak ranging between 225 to 235 °C comprising the steps of:

1. providing a solution of 10-Propargyl-1 0-Deazaaminopterin with dimethylsulfoxide (DMSO);

2. raising the temperature of solution between 40 to 60 °C;

3. maintaining the solution mass for time duration between 10-60 min;

4. adding C3 to C8 ketone solvent;

5. cooling the solution mass up to 20-30°C;

6. isolating the crystalline Form-SPR of 10-Propargyl-10-Deazaaminopterin.

In another aspect, the present invention also relates to a composition comprising 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR, of which at least 95%, by total weight of the 10-Propargyl-10-Deazaaminopterin in the composition is 10-Propargyl-10-Deazaaminopterin crystalline form designated as Form-SPR. The composition is substantially free of any other known forms of 10-Propargyl-10-Deazaaminopterin or any other crystalline form. In a further aspect of the present invention, it relates to a pharmaceutical composition comprising 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR together with one or more pharmaceutically acceptable excipients. Further particular aspects of the invention are detailed in the description of invention, wherever appropriate.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is an example of X-ray powder diffraction ("XRPD") pattern of 10-Propargyl-l0-Deazaaminopterin crystalline Form-SPR.

Fig. 2 is an example of a differential scanning calorimetry ("DSC") curve of 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR.

Fig. 3 is an example of IR spectral pattern of 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR.

DETAILED DESCRIPTION

As set forth herein, embodiments of the present invention relate to 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR and process for preparation thereof. In one embodiment of the present application, it provides 10-Propargyl-10-Deazaaminopterin (I) crystalline polymorphic form designated as Form-SPR characterized by X-ray powder diffraction pattern comprising at least 5 characteristic 20° peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3, 17.8, 19.1, 21.5, 25.2 and 25.6 ± 0.2 26° and DSC isotherm comprising atleast one endothermic peak ranging between 225 to 235 °C. In one of the embodiments of the present application, 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR according to the present invention is, characterized by an IR absorption spectrum having characteristic peaks expressed in cm-1 at approximately 3294 cm-1, 3205 cm-1, 2114 cm-1, 1645 cm-1, 1555 cm-1,1538 cm-1 and 1499 cm-1. In a further embodiment of the present application, the 10-Propargyl-l0-Deazaaminopterin crystalline Form-SPR produced by the invention mentioned is characterized by-

i. X-ray powder diffraction pattern comprising at least 5 characteristic 20° peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3, 17.8, 19.1,21.5,25.2 and 25.6 ± 0.2 20°.

ii. DSC isotherm comprising the endothermic peak ranging between 225 to 235 °C.

iii. IR absorption characteristic peaks at approximately 3294 cm-1, 3205 cm-1, 2114 cm-1, 1645 cm-1,1555 cm-1, 1538 cm-1 and 1499 cm-1.

In another embodiment of the present application, substantially pure 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR exhibits an X-ray powder diffraction pattern as shown in FIG-1, DSC isothermal pattern as shown in Fig-2 and IR absorption spectrum as shown in Fig-3. The characteristic peaks and their d spacing values of the new crystalline Form-SPR are tabulated in the Table-1.

Table-1: Characteristic XRPD Peaks of Crystalline Form-SPR Minor variations in the observed 2 0° angles values may be expected based on the analyst person, the specific XRPD diffractometer employed and the sample preparation technique. Further possible variations may also be expected for the relative peak intensities, which may be largely affected by the non-uniformity of the particle size of the sample. Hence, identification of the exact crystalline form of a compound should be based primarily on observed 2 theta angles with lesser importance attributed to relative peak intensities. The 2 theta diffraction angles and corresponding d-spacing values account for positions of various peaks in the X-ray powder diffraction pattern. D-spacing values are calculated with observed 2 theta angles and copper K a wavelength using the Bragg equation well known to those of having skill in the art of XRPD diffractometry science.

In view of possibility of marginal error in the assigning 2 theta angles and d-spacing, the preferred method of comparing X-ray powder diffraction patterns in order to identify a particular crystalline form is to overlay the X-ray powder diffraction pattern of the unknown form over the X-ray powder diffraction pattern of a known form. For example, one skilled in the art can overlay an X-ray powder diffraction pattern of an unidentified crystalline form of 10-Propargyl-10-Deazaaminopterin over FIG. 1 and readily determine whether the X-ray diffraction pattern of the unidentified form is substantially the same as the X-ray powder diffraction pattern of the crystalline form SPR of this invention. If the X-ray powder diffraction pattern is substantially the same as FIG. 1, the previously unknown crystalline form of 10-Propargyl-10-Deazaaminopterin can be readily and accurately identified as the crystalline Form SPR of this invention.

In another embodiment of the present application, it provides process for preparing 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR characterized by X-ray powder diffraction pattern comprising at least 5 characteristic 26° peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3,17.8, 19.1, 21.5, 25.2 and 25.6 ± 0.2 29° and DSC isotherm comprising the endothermic peak ranging between 225 to 235 °C comprising the steps of:

1. providing a solution of 10-Propargyl-10-Deazaaminopterin with dimethylsulfoxide (DMSO);

2. raising the temperature of solution between 40 to 60 °C;

3. maintaining the solution mass for time duration between 10-60 min;

4. adding C3 to C8 ketone solvent;

5. cooling the solution mass up to 20-3 0°C;

6. isolating the crystalline Form-SPR of 10-Propargyl-10-Deazaaminopterin.

The individual steps of the process according to the present invention for preparing 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR are detailed separately herein below.

Step 1 comprises providing a solution of 10-Propargyl-10-Deazaaminopterin with dimethylsulfoxide (DMSO) 10-Propargyl-10-Deazaaminopterin is added 60-70 times by weight w.r.t. volume of DMSO used. In one particular embodiment of the present invention for 6 mL of DMSO, 400 mg of 10-Propargyl-10-Deazaaminopterin was added. 10-Propargyl-10 -Deazaaminopterin obtained from any process known in the prior art can be utilized for this reaction. The reaction is carried out at ambient temperature ranging from 20 to 35 °C. Reaction mixture is stirred for time duration ranging between 5 to 30 mins, till the clear solution is obtained.

Step 2 comprises raising the temperature of solution between 40 to 60 °C The clear solution obtained in step 1 is heated to a temperature ranging between 40 to 60 °C depending upon nature of the solution obtained in step 1.

Step 3 comprises maintaining the solution mass for time duration between 10-60 min The solution mass obtained in step 2 is maintained at the temperature of 40 to 60 °C for the time duration of 10 to 60 minutes depending upon the progress of the reaction. The reaction mass is continuously stirred for the duration mentioned at raised temperature.

Step 4 comprises adding C3 to C8 ketone solvent to the reaction mass C3 to C8 ketone solvent used in this step may be selected from acetone, methyl ethyl ketone or MIBK (Methyl Isobutyl Ketone). Ketone solvent is slowly added to the reaction mixture at the temperature range of 40 to 60 °C. Amount of Ketone solvent used in this step varies from 8 to 15 percent in volume with respect to the weight of 10-Propargyl-10-Deazaaminopterin initially used in the reaction. In one of the particular embodiment 40 mL of acetone was used for 400 mg 10-Propargyl-10-Deazaaminopterin. This reaction is maintained by stirring at the same raised temperature for time between 20 to 40 minutes depending upon the progress of the reaction and the actual reaction conditions employed.

Step 5 comprises cooling the solution mass up to 20-3 0°C The solution mass obtained from the above step is cooled to temperature ranging between 20 to 30 °C. Cooling is done slowly. A fast cooling rate of more than 10 °C/min may be avoided in view of any probable inconsistency in the further steps. It was observed that precipitation of solid starts at 35-40 °C. Reaction mass is maintained at temperature of 20-30 °C along with continuous stirring for time duration of up to not less than 10 hrs.

Step 6 comprises isolating the crystalline Form-SPR of 10-Propargyl-10-Deazaaminopterin The crystalline material obtained in step 5 is filtered and then washed with a C3 to C8 ketone solvent. C3 to C8 ketone solvent system employed for the washing of the final reaction mass in this step may be selected from non-limiting example of acetone, methyl ethyl ketone or the like with suitable polarity according to the reaction requirements. This reaction sequence of washing with the C3 to C8 ketone solvent may be repeated according to the requirements to achieve the desired purity level. The crystalline material obtained is dried under reduced pressure conditions at a temperature between 50-70°C, to afford pure crystalline form-SPR. Reduced pressure conditions for recovering the solvents may be suitably utilized by person skilled in the art in order to achieve the dried material. Drying of the crystalline material at the temperature of 50-70 °C is done for time duration of up to not less than 10 hrs.

Process of isolating 10-Propargyl-10-Deazaaminopterin Form-SPR comprise processes but not limited to conventional processes including scrapping, if required filtering from slurry and optional drying, which may be carried out at room temperature for the suitable durations to retain the 10-Propargyl-10-Deazaaminopterin crystalline Form-SPR characteristics. The merit of the process according to the present invention resides in that - product obtained after recovery is obtained in 10-Propargyl-10-Deazaaminopterin Form-SPR. Said material is found to be a very stable crystal lattice which is adequately stable to handle and store for longer time without any significant or measurable change in its morphology and physicochemical characteristics. 10-Propargyl-10-Deazaaminopterin Form-SPR retains its purity upto more than 99.2% during accelerated storage conditions. This stable form thus, offers various advantages in terms of storage, shelf life and favorable impurity profile.

The process related impurities, including unreacted intermediates, side products, degradation products and other medium dependent impurities, that appear in the impurity profile of the 10-Propargyl-10-Deazaaminopterin may be substantially removed by the process of the present invention resulting in the formation of substantially pure - crystalline 10-Propargyl-10-Deazaaminopterin Form-SPR. In view of maintaining the equilibrium to the impurity profile compliance, the process may require in-process quality checks to avoid unnecessary repetitions of the same process steps. Pure 10-Propargyl-10-Deazaaminopterin in the crystalline Form-SPR obtained according to the process of the present invention results in the final API purity by HPLC of more than 99% w/w , which is substantially free from 10-deazaaminopterin ( also known as Impurity -A). In the context of substantially free from 10-deazaaminopterin, it may be construed that minor baseline ripple in the expected region (or which may not be quantifiable by known methods to person skilled in the art) shall not be construed the limiting scope of the invention.

The 10-Propargyl-10-Deazaaminopterin Form-SPR described herein may be characterized by X-ray powder diffraction pattern (XRPD) and Thermal techniques such as differential scanning calorimetry (DSC) analysis. The samples of 10-Propargyl-10-Deazaaminopterin Form-SPR were analyzed by XRPD on a Bruker AXS D8 Advance Diffractometer using X-ray source - Cu Kα radiation using the wavelength 1.5418 A and lynx Eye detector. DSC was done on a Perkin Elmer Pyris 7.0 instrument. Illustrative examples of analytical data for the 10-Propargyl-10-Deazaaminopterin Form-SPR obtained in the Examples are set forth in the Figs. 1-3. In a further embodiment according to the specification, the invention also relates to a composition containing 10-Propargyl-10-Deazaaminopterin Form-SPR of which at least 95%, by total weight of the 10-Propargyl-10-Deazaaminopterin in the composition, is the crystalline Form- SPR. In yet another embodiment of the invention, the composition may be substantially free of any other known forms of 10-Propargyl-10-Deazaaminopterin or any other crystalline form.

The 10-Propargyl-10-Deazaaminopterin Form-SPR obtained by the process of the present application may be formulated as solid compositions for oral administration in the form of capsules, tablets, pills, powders or granules. In these compositions, the active product is mixed with one or more pharmaceutically acceptable excipients. The drug substance can be formulated as liquid compositions for oral administration including solutions, suspensions, syrups, elixirs and emulsions, containing solvents or vehicles such as water, sorbitol, glycerin, propylene glycol or liquid paraffin. In one embodiment of the present invention, it also includes premix comprising one or more pharmaceutically acceptable excipients in the range of 1 to 50% w/w with 10-Propargyl-10-Deazaaminopterin Form-SPR, while retaining the nature of the premix. The compositions for parenteral administration derived from 10-Propargyl-10-Deazaaminopterin Form-SPR can be suspensions, emulsions or aqueous or non-aqueous sterile solutions or lyophilized powder. As a solvent or vehicle, propylene glycol, polyethylene glycol, vegetable oils, especially olive oil, and injectable organic esters, e.g. ethyl oleate, may be employed. These compositions can contain adjuvants, especially wetting, emulsifying and dispersing agents. The sterilization may be carried out in several ays, e.g. using a bacteriological filter, by incorporating sterilizing agents in the composition, by irradiation or by heating. They may be prepared in the form of sterile compositions, which can be dissolved at the time of use in sterile water or any other sterile injectable medium.

Pharmaceutically acceptable excipients used in the compositions comprising 10-Propargyl-10-Deazaaminopterin Form-SPR of the present application include, but are but not limited to diluents such as starch, pregelatinized starch, lactose, powdered cellulose, microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, mannitol, sorbitol, sugar and the like; binders such as acacia, guar gum, tragacanth, gelatin, pre-gelatinized starch and the like; disintegrants such as starch, sodium starch glycolate, pregelatinized starch, Croscarmellose sodium, colloidal silicon dioxide and the like; lubricants such as stearic acid, magnesium stearate, zinc stearate and the like; glidants such as colloidal silicon dioxide and the like; solubility or wetting enhancers such as anionic or cationic or neutral surfactants, waxes and the like. Other pharmaceutically acceptable excipients that are of use include but not limited to film formers, plasticizers, colorants, flavoring agents, sweeteners, viscosity enhancers, preservatives, antioxidants and the like. Pharmaceutically acceptable excipients used in the compositions of 10-Propargyl-10-Deazaaminopterin (I) Form-SPR of the present application may also comprise to include the pharmaceutically acceptable carrier used for the preparation of solid dispersion, wherever utilized in the desired dosage form preparation. Certain specific aspects and embodiments of the present application will be explained in more detail with reference to the following examples, which are provided by way of illustration only and should not be construed as limiting the scope of the invention in any manner.

EXAMPLE

Reference example 1: Preparation of 10-Propargyl-10-Deazaaminopterin 10-Propargyl-10-Deazaaminopterin can be prepared by process substantially according to the process disclosed in literature article J.. Med. Chem, 1993, 36, 2228 and US patent nos. 5354751 and 6028071, by following the below mentioned reaction scheme:

Example-01: Preparation of 10-Propargyl-10-Deazaaminopterin (I) crystalline polymorphic Form- SPR

STEP A: Preparation of crude 10-Propargyl-l0-Deazaaminopterin (I) crystalline polymorphic Form- SPR 6.0 mL DMSO was charged in to a 50 ml RBF at 25-30°C. Further at same temperature, 400 mg 10-Propargyl-l0-Deazaaminopterin was added to the reaction mixture. Reaction mixture is stirred for 10-15 minutes. After obtaining the clear solution, the reaction mass was heated to 50-55 °C. Maintaining the temperature at 50-55 °C stirring was performed for 30 min. This was followed by slow addition of 40.0 mL acetone. Reaction mass was further stirred for 30 min. Then slowly cooled the reaction temperature to 25-30°C, where stirring was performed for 16 h. After completion of the reaction, the reaction mass was filtered through Buckner funnel. The wet cake obtained was further washed with 2.0 mL acetone. The material obtained was suck dried, unloaded and dried for 30 mins at 25-30°C by air drying to afford 360 mg crude 10-Propargyl-10-Deazaaminopterin (I) crystalline polymorphic Form- SPR. Yield: 90%

STEP B: Purification of crude 10-Propargyl-10-Deazaaminopterin (I) crystalline polymorphic Form- SPR 200 mg of above obtained material (STEP A) is charged in to a 25 ml RBF at 25-30°C. Further 6mL acetone was added to the RBF. Reaction mass was stirred for 10-15 minutes, followed by heating to 50-55 °C. The reaction mass was maintained at this temperature for 30 mins along with continuous stirring. Then reaction mixture was slowly cooled to the temperature of 25-30 °C, where stirring was performed for 1h. On completion of stirring the reaction mass was filtered through Buckner funnel and the wet cake obtained was washed with 1.0 mL acetone. The wet material is unloaded and suck dried for 10-15 min. This reaction sequence was repeated twice to achieve the desired purity level. Final material obtained is dried under vacuum at temperature of 60-65°C for 16h to afford 110 mg of pure 10-Propargyl-lO-Deazaaminopterin (I) crystalline polymorphic Form- SPR which is characterized by M. Pt. of 221-224 °C and has XRPD pattern as per Fig-1; DSC isotherm as per Fig-2 and IR spectrum as per Fig-3. Yield: 55.0% Purity: 99.3% While the foregoing provides a detailed description of the preferred embodiments of the invention, it is to be understood that the descriptions are illustrative only of the principles of the invention and not limiting. Furthermore, as many changes can be made to the invention without departing from the scope of the invention, it is intended that all material contained herein be interpreted as illustrative of the invention and not in a limiting sense.

We Claim:

1) Crystalline 10-Propargyl-10-Deazaaminopterin (I) Form-SPR characterized by X-ray powder diffraction pattern comprising at least 5 characteristic 20° peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3, 17.8, 19.1, 21.5, 25.2 and 25.6 ± 0.2 28° and DSC isotherm comprising atleast one endothermic peak ranging between 225 to 235 °C.

2) 10-Propargyl-l 10-Deazaaminopterin (I) crystalline Form-SPR according to claim-1, having IR absorption spectrum characterized by peaks expressed in cm-1 at approximately 3294 cm-1, 3205 cm-1, 2114 cm-1, 1645 cm-1, 1555 cm=1, 1538 cm"1 and 1499 cm-1.

3) 10-Propargyl-l 10-Deazaaminopterin (I) crystalline Form-SPR characterized by X-ray powder diffraction pattern comprising at least 5 characteristic 20° peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3, 17.8, 19.1, 21.5, 25.2 and 25.6 ± 0.2 20°, DSC isotherm comprising the endothermic peak ranging between 225 to 235 °C and IR absorption characteristic peaks at approximately 3294 cm-1, 3205 cm-1, 2114 cm-1,1645 cm-1,1555 cm-1,1538 cm-1 and 1499 cm-1.

4) 10-Propargyl-l 10-Deazaaminopterin (I) crystalline form-SPR according to any of claim 1 to 3, characterized by X-ray powder diffraction pattern substantially according to Fig-1, DSC isothermal pattern substantially according to Fig-2 and IR absorption spectrum substantially according to Fig-3 .

5) A process for preparing 10-Propargyl-l 10-Deazaaminopterin (I) crystalline form-SPR, characterized by X-ray powder diffraction pattern comprising at least 5 characteristic 20° peaks selected from the XRPD peak set of 8.3, 12.0, 12.3, 14.1, 15.8, 16.7, 17.3, 17.8, 19.1, 21.5, 25.2 and 25.6 ± 0.2 20° and DSC isotherm comprising atleast one endothermic peak ranging between 225 to 235 °C, comprising the steps of:

a) providing a solution of 10-Propargyl-10-Deazaaminopterin with dimethylsulfoxide (DMSO);

b) raising the temperature of solution between 40 to 60 °C;

c) maintaining the solution mass for time duration between 10-60 min;

d) adding C3 to C8 ketone solvent;

e) cooling the solution mass up to 20-30°C;

f) isolating the crystalline Form-SPR of 10-Propargyl-10-Deazaaminopterin.

6) A process for preparing 10-Propargyl-l0-Deazaaminopterin (I) crystalline Form-SPR according to claim 5, wherein ketone solvent used in Step d) is selected from acetone, methyl ethyl ketone or MIBK.

7) A process for preparing 10-Propargyl-l 10-Deazaaminopterin (I) crystalline Form-SPR according to claim 5, wherein Step f) of isolating the crystalline form-SPR further comprises the steps of-

i. filtering the crystalline material;

ii. Washing with C3 to C8 ketone solvent;

iii. drying the crystalline material under vacuum at temperature between 50-70°C; iv. collecting the material as pure crystalline form-SPR.

8) A process for preparing 10-Propargyl-l 10-Deazaaminopterin (I) crystalline form-SPR according to claim 7, wherein Step iii. comprises drying up to more than 10 hours.

9) A pharmaceutical composition comprising 10-Propargyl-l 10-Deazaaminopterin (I) crystalline form-SPR according to any of the preceding claims, together with one or more pharmaceutically acceptable excipients.