FIELD OF THE INVENTION

The present invention relates to a stable and pure polymorphic form of bortezomib and process for its preparation.

BACKGROUD OF THE INVENTION

Bortezomib is the adopted name for the drug compound having the chemical name [(1R)- 3-methyl-l-[[(2S)-l-oxo-3-phenyl-2-[(Pyrazinylcarbonyl)amino ]propyl]amino]butyl] boronic acid and is represented by the structural formula 1.

Bortezomib is an anti-neoplastic agent used in the treatment of multiple myeloma and mantle cell lymphoma. Bortezomib is the first therapeutic proteasome inhibitor to be tested in the humans it is introduced by Millennium Pharmaceuticals Inc., a U.S. based company. The brand name of bortezomib is VELCADE ^® which is in the form of injection, each vial contains 3.5mg of bortezomib as sterile lyophilized powder.

The lyophilized drug product contains mannitol in a ten fold excess by weight, and it is much more stable than the drug substance itself. The drug substance, drug product and the reconstituted drug product have three different molecular forms. Bortezomib drug substance exists as the trimeric boroxine in the solid state. When exposed to water, the boroxine hydrolyses to monomeric acid. The reconstituted drug product in 0.9% aqueous NaCl solution exists in equilibrium with the mannitol ester and the boronic acid. US 5,780,454 patent discloses bortezomib, its pharmaceutically acceptable salts, pharmaceutical composition and use to inhibit the proteasome function in a mammal. Further, it discloses a process for the preparation of bortezomib and its analogues. No polymorphic forms of bortezomib have been disclosed in this patent.

WO 2009/036281 discloses Form A and Form B of bortezomib. Form A is obtained by from a solvent system of methanol and water and Form B is prepared by using solvent system of either ethyl acetate or dichloromethane mixed with toluene. These two polymorphic forms Form-A and Form-B of bortezomib are identical with Form-I and Form-II of bortezomib disclosed in our earlier patent application WO 2008/075376 Al . The chemical purity (by HPLC) of these Form A and Form B is in the range 99.0 to 99.6% only even after subjecting to repeated crystallizations and impurities are in the extent of 0.2 to 0.6% in the samples. This purity is not acceptable for pharmaceutical compositions and therapeutic applications to prepare a drug product.

WO 2011/099018 discloses Form HI and also an amorphous form of bortezomib. Form HI of bortezomib is prepared by crystallization from acetonitrile. Amorphous form of bortezomib is obtained from a solvent mixture of dimethylformamide and water or by crystallization from the solvent mixture ethyl acetate and cyclohexane or heptane.

The above two forms were prepared only on 0.35g and 0.7g scale. Purity of the obtained product was not mentioned in this application. Solvents like acetonitrile, N,N-dimethyl formamide were employed for the preparation of both Form HI and amorphous form of bortezomib. These solvents have lower residual solvent limit as per ICH guide lines (410 ppm and 880 ppm respectively). Removal of these solvents requires higher temperatures at which bortezomib gets degraded resulting in a lower purity of the finished product. Incomplete removal of solvent can lead to solvate formation and is not suited for pharmaceutical applications. WO 2011/107912 application discloses Form Al and Form A2 of bortezomib: Form Al is obtained from acetonitrile and Form A2 is prepared by recrystallization from solvent system of 1,4-dioxane and water. Both Form Al and Form A2 of bortezomib are characterized by their PXRD, IR spectra and DSC thermograms. 1,4-Dioxane is not suitable for pharmaceutical applications in the final stage as it is non-volatile and leaves considerable solvent residues in the finished product and the permissible limit is only 380 ppm.

In our earlier PCT application WO 2008/075376 two polymorphic forms of bortezomib were disclosed. Polymorphic form-I is prepared by crystallization from acetone alone or dissolving bortezomib in solvents such as chloroform or acetonitrile and adding antisolvents like diisopropyl ether. Polymorphic form-II of bortezomib is obtained from recrystallization in ethyl acetate. The application also discloses the interconvertibility of the two forms. Polymorphic Form-I and Form-II are characterized by their PXRD, IR spectra and DSC thermograms.

However during scale-up work on this drug substance it has been observed that these polymorphic forms I and II are not consistently reproducible particularly on batch sizes of 50g and above. Although the chemical purity is initially >99.8% the product was found to develop impurities on storage (purity < 99.5%).

SUMMARY OF THE INVENTION

There is a need to invent stable and reproduce polymorphic form of any pharmaceutical drug substance which has great impact on bioavailability of the drug.

Polymorphic form in the case of heat and moisture sensitive products like bortezomib has an impact on the chemical stability as well. A stable polymorph on a time scale of six months at specified temperature is essential to maintain the chemical purity of the drug substance (>99.5%). There is thus a need to investigate the polymorphism of bortezomib with the following objectives:

A stable polymorph that remains stable over a period of time and reproducible on larger scale runs (50g and above).

A polymorph which is also chemically stable and retains the polymorphic integrity over a period of time. A polymorph which is chemically stable and retains the polymorphic integrity on scale- up.

Surprisingly, during the larger scale production of bortezomib (about 1 OOg scale) a new polymorphic form is obtained which is chemically pure, stable and reproducible.

1) In one aspect, the present invention provides a novel crystal form of bortezomib designated as Form N. The novel polymorphic form N of bortezomib of the present invention is characterized by a) Peaks in the Powder X-ray diffraction spectrum (PXRD) having 2e values at about 3.6, 4.7, 5.5, 8.8, 9.3, 9.5, 1 1.1, 14.6, 15.5, 16.4, 16.7, 17.8, 18.5, 18.8, 19.5, 20.16, 20.46, 21.6 and 22.3 ±0.2 degrees. b) Infrared absorption bands in the IR spectrum (KBr):

446, 511, 606, 701, 749, 870, 927, 1021, 1048, 1082, 1113, 1153, 1201, 1273, 1323, 1402, 1466, 1518, 1581, 1661, 2868, 2928, 2954, 3029, 3063, 3281 and 3391 crn ¹ c) DSC thermogram exhibiting two endotherms between about 70°C and 95°C and about 125°C and 150°C. 2) In another aspect, the prevention invention provides a process for the preparation of polymorphic Form N of bortezomib having high chemical purity by the following synthetic scheme

Scheme

3) According to another aspect of the present invention, the process provides novel and chemically pure polymorphic form N of bortezomib and comprises, a) coupling of (lS,2S,3R,5S)-Pinanediol-P-(l-phenyl)-L-alanine-L-Leucine boronate HCI of formula 2 with with 2-Pyrazine carboxylic acid of formula 3 in the presence of TBTU and Ν,Ν-diisopropylethyl amine to get (IS, 2S,3R,5S)-Pinanediol-N-(2- Pyrazine carbonyl)-L-Phenylalanine-L-leucine boronate of formula 2 b) deprotecting of pinanediol moiety of compound of formula 3 using isobutyl boronic acid in the presence of aq. hydrochloric acid to yield bortezomib (formula 1) c) crystallizing crude bortezomib from acetone at room temperature followed by recrystallization from ethyl acetate d) leaching the resulting product of step-c with n-heptane and drying at room temperature for 25-30h under high vacuum (750 mmHg) to afford of high chemical purity bortezomib (> 99.8%) containing total chemical and chiral impurities of less than 0.2%.

Brief description of drawings:

FIG.1 : Powder X-ray diffraction pattern of bortezomib polymorphic Form-N

FIG.2: DSC thermogram of bortezomib polymorphic Form-N

FIG.3: Infrared absorption spectrum of bortezomib polymorphic Form-N

X-ray powder diffraction spectra were measured on a Siemens d5000 x-ray powder diffracto-meter having a copper-ka radiation (1.5406a).

DSC data was collected from mettler toledo; model: DSC 823 ^e

IR spectra were recorded on perkin elmer; model: ftir paragon 1000

The details of the process of the invention are provided in the examples given below which are provided by way of illustration only and therefore should not be construed to limit the scope of the invention. List of Abbreviations

AGR: All glass reactor

TBTU: 0-(Benzotriazol- 1 -yl)-N,N,N' ,Ν' -tetramethyluronium tetrafluoroborate

HPLC: High performance liquid chromatography

PXRD: Powder X-ray diffraction

I.R: Infrared spectroscopy

DSC: Differential scanning calorimetry EXAMPLE

Preparation of (lS,2S,3R,5S)-Pinanediol-N-(2-Pyrazine carbonyl)-L-Phenylalanine- L-leucine boronate (4)

(lS,2S,3R,5S)-Pinanediol-p-(l-phenyl)-L-alanine-L-Leucine boronate HC1 (2, 320g, 0.71 moles), pyrazine carboxylic acid (3, 95.36g, 0.76 moles), TBTU (252.4g, 0.78 moles) and methylene chloride (3.80L) were charged in to 10L AGR. The reaction mass was cooled to -2 to 2°C, and Ν,Ν-Disiopropyl ethyl amine (320g, 2.48 moles) was added drop wise. The reaction mass was then warmed to 15°C and stirred for 80 min at 15-25°C.

The solvent was distilled off at 40-45 °C to obtain a brown coloured oil and the resulting product was dissolved in ethyl acetate (4.0L), washed with 5% aq. phosphoric acid solution (2.2L), 2% aq. potassium carbonate solution (2.2L) followed by 10% brine solution (2.5L). The organic layer was dried over sodium sulphate, and solvent was evaporated under reduced pressure to get a pale yellow coloured foam of the title compound.

Wt. of the compound: 365g; purity by HPLC: about 85%.

Above compound was taken to next step without further purification.

Preparation of bortezomib polymorphic forni-N

(l S,2S,3R,5S)-Pinanediol-N-(2-Pyrazine carbonyl)-L-Phenylalanine-L-leucine boronate (4, 360g, 0.69 moles), methanol (2.9L), isobutyl boronic acid (129.4g, 1.27 moles) and n- heptane (2.9L) were charged into 10L AGR and stirred for 10 min at room temperature. Aq. hydrochloric acid solution (218 ml cone, hydrochloric acid and 1.96L water) was added to the above reaction mass by maintaining the temperature of the reaction mass between 25-30°C. The reaction mass was stirred for 4h at room temperature. The layers were separated, lower aq. layer was washed with n-heptane (2xlL) and layers were separated. The aq. layer was concentrated under reduced pressure at 40-45°C to obtain a cream colured suspension. The resulting suspension was dissolved in 2N aq. sodium hydroxide solution (prepared from 176g sodium hydroxide and 2L water) and washed with dichloromethane (3x1.5L). The aq. layer was separated and pH adjusted to 5.8 to 6.2 using aq. hydrochloric acid to separate the gummy material from the solution. The precipitated material was extracted into dichloromethane (2x1.8L) and layers were separated. The organic layer was dried over sodium sulphate and solvent was distilled off under reduced pressure to obtain pale yellow coloured oil. Ethyl acetate (2.16L) was charged to the oil and the solvent distilled off as above to yield bortezomib as off white coloured foamy solid (270g).

To the above solid, acetone (1.08L) was added and the reaction mass was allowed to stir for about lh at room temperature to form white coloured suspension. Acetone (0.54L) was charged further to the above suspension and stirred for another 2h. T he product was filtered off under suction with the help of chilled acetone (0.54L, 0-5°C), and dried under suction for lh, wt. of the solid: 1 18g.

Ethyl acetate (1.65L) was charged into a 5L AGR and warmed to 70-72°C, above solid (118g) was charged. The resulting solution was filtered through sintered funnel. The filtrate was allowed to stir at room temperature for 2.0-2.5h and the obtained slurry was filtered. The product was dried under suction for 2-3h under N ₂ atmosphere, wt. of the solid: 90g.

The above solid was leached with heptane (2x1.8L) at room temperature for 1.5h and then filtered. The resulting solid was dried under high vacuum (750mmHg) for 30h to afford bortezomib polymorphic form-N of high chemical purity. Wt. of the product: 80g Purity of bortezomib polymorphic form-N by HPLC > 99.8%. Related and chiral impurities <0.2%. STABILITY OF BORTEZOMIB

Bortezomib prepared according to the present invention is subjected for stability studies in both long term (-20±5°C) and accelerated (5±3°C) storage conditions. Compound is found to be stable for the testing period of six months. Bortezomib (Form-N) of 99.96 chemical purity and 99.99% chiral purity was used in these studies and the results are tabulated (Table- 1).

Table-1: Stability studies of Form-N of bortezomib

Stability data of Form-II of bortezomib disclosed in WO 2008/075376 is also summarized for comparison (Table-2). Here also Bortezomib (Form-II) of 99.96 chemical purity and 99.99%) chiral purity was employed for studies in both long term (- 20±5°C) and accelerated (5±3°C) storage conditions. Table-2: Stability of our Form-II of bortezomib (WO 2008/075376)

ADVANTAGES OF PRESENT INVENTION:

1. Present invention discloses novel crystal form of bortezomib designated as Form-N which is stable, reproducible on larger scale runs, and useful for the treatment of multiple myeloma. 2. Processes for the preparation of the novel crystalline form, namely Form-N of bortezomib is simple and easy to adopt on a commercial scale.

3. Polymorphic Form-N of bortezomib obtained by the present invention is of high purity in terms of related substances as well as enantiomeric purity.

4. Polymorphic Form-N of bortezomib retains the polymorphic integrity and chemical/chiral purity over a long period of time (6 months).