FIELD OF THE INVENTION

[001] The present invention relates to crystalline polymorph Form I of benzyl N ² - ((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetr ahydrofuran- 2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)-L-asparaginate and processes of preparation thereof.

BACKGROUND OF THE INVENTION

[002] Drug intermediates are the raw materials for the production of bulk drugs, or they can refer to a material produced during synthesis of a drug (API-active pharmaceutical ingredients).

[003] The purity of the drug intermediate, in the process of the synthesis of the API has high importance for the purity of the API itself.

[004] Aspacytarabine is a conjugate of cytarabine and aspartic acid wherein cytarabine is covalently attached to the carboxyl group of the side chain of aspartic acid. It is useful for treatment neoplastic diseases including hematological cancers such as leukemias thereby prolonging the survival of the patients in need of the treatment.

[005] This invention provides a crystalline form with high purity of an intermediate for the preparation of aspacytarabine and process for the preparation thereof to ensure high purity of aspacytarabine.

SUMMARY OF THE INVENTION

[006] In some aspects, provided herein a crystalline polymorph Form I of benzyl N ² - ((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetr ahydrofuran-

2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)-L-asparaginate, represented as Compound 1: (Compound 1).

[007] In some aspects, provided herein a crystalline polymorph Form I of benzyl N ² - ((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetr ahydrofuran-

2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)-L-asparaginate, represented as Compound 1 characterized by Powder X-Ray diffraction pattern comprising unique peaks at 29 ⁰ ±0.2 (d value A); °29 ±0.2 (d value A): 3.7 (23.8), 16.4 (5.4), when obtained with a Cu tube anode with Ka radiation. In other aspects the crystalline polymorph is characterized by an X-Ray Powder diffraction pattern comprising unique peaks at 29° ±0.2 (d value A); 3.7 (23.8), 5.0, (17.6), 7.3 (12.0), 14.9 (5.9), 16.4 (5.4), 18.6 (4.8), 19.5 (4.5) and 23.6 (3.8) when obtained with a Cu tube anode with Ka radiation. In other aspects the crystalline polymorph is characterized by an X-Ray Powder diffraction pattern comprising unique peaks at 29° ±0.2 (d value A); 3.7 (23.8), 5.0, (17.6), 7.3 (12.0), 8.1 (10.9), 8.5 (10.4), 13.0 (6.8), 14.9 (5.9), 16.0 (5.5), 16.4 (5.4), 18.6 (4.8), 19.5 (4.5),

21.1 (4.2), 23.6 (3.8) and 26.3 (3.4) when obtained with a Cu tube anode with Ka radiation.

[008] In some aspects, provided herein a crystalline polymorph Form I of benzyl N ² - ((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetr ahydrofuran- 2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)-L-asparaginate, represented as Compound 1 having a chemical purity greater than 95%.

[009] In some aspects, provided herein a process for the preparation of the crystalline polymorph Form I of Compound 1, wherein the process comprises removing protective groups from a compound of Formula II to obtain Compound 1, followed by crystallization of Compound 1 using an organic solvent and an anti-solvent as presented in scheme 1:

Formula II Compound 1

( Scheme 1 ) wherein Pgl and Pg2 are each independently an alcohol protecting group; Cbz is benzyloxycarbonyl; and Bn is benzyl.

[0010] In some aspects, provided herein a process for the preparation of (S)-2-amino-4-((l- ((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofura n-2-yl)-2-oxo-l,2- dihydropyrimidin-4-yl)amino)-4-oxobutanoic acid (aspacytarabine), wherein the process comprises removing Cbz group (benzyloxycarbonyl group) and Bn (benzyl) group from the crystalline polymorph Form I of Compound 1. BRIEF DESCRIPTION OF THE FIGURES

[0011] The subject matter regarded as the present invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The present invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0012] Figure 1 depicts the XRPD pattern of crystalline Compound I Form I. The X-Ray Powder diffraction pattern was performed by Bruker D8 ADVANCE ECO diffractometer using Cu Ka radiation (40 kV, 25 mA), theta to theta geometry, using LYNXEYE detector from 2 -45 20 ⁰

[0013] Figure 2 depicts the XRPD pattern of amorphous Compound I. The X-Ray Powder diffraction pattern was performed by Bruker D8 ADVANCE ECO diffractometer using Cu Ka radiation (40 kV, 25 mA), theta to theta geometry, using LYNXEYE detector from 2 -45 29 ⁰ [0014] Figure 3 depicts the DSC-TGA analysis of crystalline anhydrous Compound I Form I. TG analyses were performed using a Mettler-Toledo TGA/DSC3+ analyzer. The samples were placed in aluminum pans, hermetically sealed, the lids pierced, and then inserted into the TG furnace. A weighed aluminum pan configured as the sample pan was placed on the reference platform. The furnace was heated under nitrogen with flow of 50 mL/min. The continuous line shows the weight change in a material as a function of temperature. The dotted line is a first derivative plot to help select the endpoints of the step transition or weight loss events.

[0015] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides in some embodiments a crystalline polymorph Form I of benzyl A ² -((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyr imidin-4-yl)-L-asparaginate (Compound 1) (Compound 1).

[0017] The present invention provides in some embodiments a crystalline polymorph Form I of benzyl A ² -((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5-

(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyr imidin-4-yl)-L-asparaginate (Compound 1), wherein said crystalline polymorph Form I is an anhydrous or hydrate or solvate crystalline form. In other embodiments, the crystalline polymorph Form I of Compound 1 is an anhydrous. In other embodiments, the crystalline polymorph Form I of Compound 1 is a hydrate. In other embodiments, the crystalline polymorph Form I of Compound 1 is a solvate crystalline form.

[0018] The present invention provides a crystalline polymorph Form I of benzyl N ² - ((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetr ahydrofuran- 2-yl)-2-oxo-l,2-dihydropyrimidin-4-yl)-L-asparaginate (Compound 1), wherein said crystalline polymorph Form I is characterized by Powder X-Ray diffraction pattern (see Figure 1) comprising unique peaks at 29 ⁰ ±0.2 (d value A); °29 ±0.2 (d value A): 3.7 (23.8), 16.4 (5.4), when obtained with a Cu tube anode with Ka radiation. In other embodiments, the crystalline polymorph Form I is characterized by an X-Ray Powder diffraction pattern comprising unique peaks at 29° ±0.2 (d value A); 3.7 (23.8), 5.0, (17.6), 7.3 (12.0), 14.9 (5.9), 16.4 (5.4), 18.6 (4.8), 19.5 (4.5) and 23.6 (3.8) when obtained with a Cu tube anode with Ka radiation. In other embodiments, the crystalline polymorph Form I is characterized by an X-Ray Powder diffraction pattern comprising unique peaks at 29° ±0.2 (d value A); 3.7 (23.8), 5.0, (17.6), 7.3 (12.0), 8.1 (10.9), 8.5 (10.4), 13.0 (6.8), 14.9 (5.9), 16.0 (5.5), 16.4 (5.4), 18.6 (4.8), 19.5 (4.5), 21.1 (4.2), 23.6 (3.8) and 26.3 (3.4) when obtained with a Cu tube anode with Ka radiation. In other embodiments, the crystalline polymorph Form I is characterized by an X-Ray Powder diffraction pattern as presented in Figure 1.

[0019] In some embodiments, the present invention provides a crystalline polymorph Form I of benzyl A ² -((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5

(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyr imidin-4-yl)-L-asparaginate

(Compound 1), wherein said crystalline polymorph Form I has a chemical purity greater than 90%. In other embodiments, the chemical purity is between 90% -100%. In other embodiments, the chemical purity is between 91% - 100%. In other embodiments, the chemical purity is between 92% -100%. In other embodiments, the chemical purity is between 93% - 100%. In other embodiments, the chemical purity is between 94% -100%. In other embodiments, the chemical purity is between 95% -100%. In other embodiments the chemical purity is between 96%-100%. In other embodiments the chemical purity is between 97%-100%. other embodiments the chemical purity is between 98%-100%. In other embodiments the chemical purity is between 99%-100%. In other embodiments the chemical purity is between 90% -91%. In other embodiments the chemical purity is between 90%-92%. In other embodiments the chemical purity is between 90%- 93%. In other embodiments the chemical purity is between 90%-94%. In other embodiments the chemical purity is between 90%-96%. In other embodiments the chemical purity is between 90%- 96%. In other embodiments the chemical purity is between 90%-97%. In other embodiments the chemical purity is between 90%-98%. In other embodiments the chemical purity is between 90%- 99%.

[0020] In one embodiment, the present invention provides a crystalline polymorph Form I of benzyl A^ ² -((benzyloxy)carbonyl)-A^-(l-((2R,3S,4S,5R)-3,4-dihydr oxy-5

(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyr imidin-4-yl)-L-asparaginate (Compound 1), wherein said crystalline polymorph Form I has a chemical purity greater than 95%. [0021] In some embodiments, the present invention provides a process for the preparation of the crystalline polymorph Form I of benzyl A^ ² -((benzyloxy)carbonyl)-A^-(l-((2R,3S,4S,5R)-3,4- dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1 ,2-dihydropyrimidin-4-yl)-L- asparaginate (Compound 1), wherein the process comprises removing protective groups (Pgl and Pg2) from a compound of Formula II to obtain Compound 1, followed by crystallization of

Compound 1 using an organic solvent and an anti-solvent as presented in scheme 1:

Formula II Compound 1

( Scheme 1 ) wherein Pgl and Pg2 are each independently an alcohol protecting group; Cbz is benzyloxycarbonyl; and Bn is benzyl. [0022] In some embodiments, Pgl and Pg2 are each independently an alcohol protecting group. Non limiting examples of alcohol protecting groups include acetyl ester, benzoyl ester, benzyl ether, P-methoxyethoxymethyl ether, trityl ether, silyl ether. In other embodiments, the protecting groups Pgl and Pg2 are each independently removed by an acid or by a base. In other embodiments, the protecting groups Pgl and Pg2 are both removed by an acid. In other embodiments, the protecting groups Pgl and Pg2 are both removed by a base. In other embodiments, the protecting groups Pgl and Pg2 are both removed by a fluoride anion.

[0023] In some embodiments, the present invention provides a process for the preparation of the crystalline polymorph Form I of benzyl ² -((benzyloxy)carbonyl)- ⁴ -(l-((2R,3S,4S,5R)-3,4- dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1 ,2-dihydropyrimidin-4-yl)-L- asparaginate (Compound 1), wherein the process comprises crystallization of Compound 1 using an organic solvent. In other embodiments, the present invention provides a process for the preparation of the crystalline polymorph Form I of benzyl N ² -((benzyloxy)carbonyl)-N ⁴ -(l- ((2R,3S,4S,5R)-3,4-dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2- dihydropyrimidin-4-yl)-L-asparaginate (Compound 1), wherein the process comprises crystallization of Compound 1 using an organic solvent and an anti-solvent. In other embodiments, the organic solvent is added prior to the anti-solvent.

[0024] In other embodiments, the organic solvent for crystalizing Compound 1 (with an antisolvent) comprises an alcohol, ketone, nitrile, ester, aromatic or an amide. In other embodiments, the organic solvent comprises an alcohol. In other embodiments, the organic solvent comprises 1 -butanol, 2-butanol, tert-butanol, 1 -propanol or iso-propanol. In other embodiments, the organic solvent comprises a ketone. In other embodiments, the organic solvent comprises 2-butanone (methyl ethyl ketone) or methyl isobutyl ketone (MIK). In other embodiments, the organic solvent comprises a nitrile. In other embodiments, the organic solvent comprises acetonitrile. In other embodiments, the organic solvent comprises an ester. In other embodiments, the organic solvent comprises ethyl acetate or isopropyl acetate. In other embodiments, the organic solvent is an aromatic. In other embodiments, the organic solvent comprises toluene. In other embodiments, the organic solvent comprises an amide. In other embodiments, the organic solvent comprises dimethyl-formamide (DMF) or N-methyl-2-pyrolidinone (NMP).

[0025] In other embodiments, the anti-solvent for crystalizing Compound 1 comprises an ether, a hydrocarbon or a chlorinated solvent. In other embodiments, the anti-solvent comprises ether. In other embodiments, the anti-solvent comprises diethyl ether, cyclopentyl methyl ether or methyl tert-butyl ether (MTBE). In other embodiments, the antisolvent comprises a hydrocarbon. In other embodiments, the anti-solvent comprises hexane or heptane. In other embodiments, the antisolvent comprises chlorinated solvents. In other embodiments, the anti-solvent comprises chloroform or dichloromethane.

[0026] In other embodiments, the volume ratio between the organic solvent and the anti-solvent is between 99:1 and 1:99. In other embodiments, the volume ratio between the organic solvent and the anti-solvent is between 99:1 and 80:20. In other embodiments, the volume ratio between the organic solvent and the anti-solvent is between 99:1 and 70:30. In other embodiments, the volume ratio between the organic solvent and the anti-solvent is between 99:1 and 60:40. In other embodiments, the volume ratio between the organic solvent and the anti-solvent is between 99:1 and 50:50. In other embodiments, the volume ratio between the organic solvent and the antisolvent is between 99:1 and 40:60; 99:1 and 30:40; and 99:1 and 20:80; and 99:1 and 10:90.

[0027] In some embodiments, the present invention provides a process for the preparation of the crystalline polymorph Form I of benzyl A ² -((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4- dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1 ,2-dihydropyrimidin-4-yl)-L- asparaginate (Compound 1), wherein the process comprises crystallization of Compound 1 using butanol and the anti-solvent is MTBE.

Process for the preparation of aspacytarabine

[0028] In some embodiments, provided herein a process for the preparation of (S)-2-amino-4- ((l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro furan-2-yl)-2-oxo-l,2- dihydropyrimidin-4-yl)amino)-4-oxobutanoic acid (aspacytarabine) or salt thereof, wherein the process comprises removing Cbz group (benzyloxycarbonyl group) and Bn group from the crystalline polymorph Form I of benzyl A ² -((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4- dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1 ,2-dihydropyrimidin-4-yl)-L- asparaginate (Compound 1).

[0029] In another embodiment the aspacytarabine-salt comprises a strong acid salt. In another embodiment the salt is selected from the group consisting of, hydrochloride salt, hydrobromide salt, TFA salt, methanesulfonate salt, phosphate salt, toluenesulfonate salt, benzenesulfonate salt, bisulfate salt and sulfate salt. In one embodiment, the salt is a hydrochloride salt. In one embodiment, the salt is a hydrobromide salt. In one embodiment, the salt is a TFA salt. Each possibility represents a separate embodiment of the invention. In another embodiment, the aspacytarabine-salt is soluble in water. [0030] Compound 1 is used as an intermediate for the preparation of aspacytarabine. Compound 1, if precipitated as amorphous (Figure 2), the amorphous form is less pure, with lower yield than the crystalline Compound 1. Therefore, the use of crystalline Compound 1 for the preparation of aspacytarabine results in a higher yield and high purity of aspacytarbine.

[0031] In some embodiments, provided herein a process for the preparation of (S)-2-amino-4- ((l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro furan-2-yl)-2-oxo-l,2- dihydropyrimidin-4-yl)amino)-4-oxobutanoic acid (aspacytarabine), wherein the process comprises removing Cbz group (benzyloxycarbonyl group) and Bn group simultaneously from the crystalline polymorph Form I of benzyl A ² -((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4- dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo- 1 ,2-dihydropyrimidin-4-yl)-L- asparaginate (Compound 1).

[0032] In some embodiments, provided herein a process for the preparation of (S)-2-amino-4- ((l-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydro furan-2-yl)-2-oxo-l,2- dihydropyrimidin-4-yl)amino)-4-oxobutanoic acid (aspacytarabine), wherein the process comprises removing Cbz group (benzyloxycarbonyl group) and Bn group from the crystalline polymorph Form I of benzyl A ² -((benzyloxy)carbonyl)-A ⁴ -(l-((2R,3S,4S,5R)-3,4-dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-l,2-dihydropyrimi din-4-yl)-L-asparaginate

(Compound 1), wherein the removal of the Cbz group is prior to the removal of the Bn group. In other embodiments, the removal of the Bn group is prior to the removal of the Cbz group.

[0033] In some embodiments, the Cbz (benzyloxycarbonyl) group and the Bn (benzyl) group of Crystalline polymorph Form I of Compound 1 are removed by hydrogenolysis to obtain aspacytarabine.

[0034] In some embodiments, provided herein aspacytarabine prepared from crystalline polymorph Form I of Compound 1. In some embodiments, provided herein aspacytarabine prepared from crystalline polymorph Form I of Compound 1 characterized by Powder X-Ray diffraction pattern comprising unique peaks at 29 ⁰ ±0.2 (d value A); °29 ±0.2 (d value A): 3.7 (23.8), 16.4 (5.4), when obtained with a Cu tube anode with Ka radiation.

[0035] In some embodiments, provided herein aspacytarabine prepared from crystalline polymorph Form I of Compound 1, wherein the aspacytarabine has a chemical purity of between 95% -100%. In other embodiments, the aspacytarabine has a chemical purity of between 96% - 100%. In other embodiments, the aspacytarabine has a chemical purity of between 97% -100%. In other embodiments, the aspacytarabine has a chemical purity of between 98% -100%. In other embodiments, the aspacytarabine has a chemical purity of between 99% -100%. In other embodiments, the aspacytarabine has a chemical purity of between 99% -100%

[0036] The following examples are to be considered merely as illustrative and non-limiting in nature. It will be apparent to one skilled in the art to which the present invention pertains that many modifications, permutations, and variations may be made without departing from the scope of the invention.

EXAMPLES

EXAMPLE 1

A process for preparing crystalline polymorph Form I of Compound 1

[0037] To a 500ml reactor equipped with mechanical stirrer were added 33g of Formula II [Pgl=Pg2= (Me3C)Me2Si-] and 120ml of EtOAc. The temperature was set to 20°C and 47ml of triethylamine trihydrofluoride were added. The reaction mixture was stirred at 20°C till reaction completion. The batch temperature was set to 5°C and 200ml of 10% Na2COa solution were charged. The batch temperature was adjusted to 20°C and the reaction mixture was stirred for 30 minutes. The reaction mixture was filtered. The aqueous layer was removed, the organic phase was washed with 170ml of 5% NaHCCh followed by 2x60ml water washes. The organic phase was concentrated to 120ml by vacuum distillation with jacket temperature of NMT 60°C. To the concentrated reaction mixture were added 100ml of 1 -butanol and the reaction mixture was concentrated again to 120ml by vacuum distillation with jacket temperature of NMT 60°C. The reaction mixture was stirred for 12 hours at25°C during which slurry appeared. 100ml of 1-butanol were added and the reaction mixture was concentrated to 185ml by vacuum distillation with jacket temperature of NMT 60°C. The reaction mixture was cooled down to 25°C 330ml of MTBE were added and the reaction mixture was stirred for 5 hours at 25 °C. The solid was filtered and dried under vacuum oven to give 21g Compound 1.

[0038] It is appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove. Rather the scope of the present invention includes both combinations and sub-combinations of various features described hereinabove as well as variations and modifications. Therefore, the invention is not to be constructed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by references to the claims, which follow.