A PROCESS FOR THE PREPARATION OF PRALATREXATE

FIELD OF INVENTION

The present invention relates to a process for the preparation of Pralatrexate (I) which act as an anti cancer agent.

BACKGROUND OF THE INVENTION

This invention relates to the preparation of 10-Propargyl-10-deazaaminopterin class of compounds and very particularly the preparation of Pralatrexate an anticancer agent. Pralatrexate is approved as an anti cancer agent that is used in the treatment for patients with relapsed or refractory peripheral T-cell lymphoma..

The compound (1) prepared by this invention is derived from the active ester of lO-Propargyl-4-deoxy-4-amino-10-deazapteroic acid (II) with 2-Chloro-4)6-dimethoxy-l,3,5-triazine (CDMT) (III) react with L-Glutamic acid dibenzyl ester hydrochloride (VI) followed by hydrolysis of ester to yield Pralatrexate.

Joseph I. De Graw et. al., Journal of Medicinal Chemistry, 1993, 2228-2231 and US5354751 discloses the preparation of anti-cancer agents. It involves, the reaction of 10-PropargyI-4-deoxy-4-amino-10-deazapteroic acid with isobutyl chlorofomate react with L-Glutamic acid diethyl ester followed by hydrolysis of ester to get Pralatrexate. Formation of anhydride when react with acid, the entire reaction is required to be conducted in a very dry condition which is difficult in the large scale preparations.

US patent US6028071 disclosed a method for preparing Pralatrexate, which involves the reaction of Benzotriazol-l-yloxy tris(dimethylamino)phosphonium hexafluorophosphate( BOP) is treated with I0-Propargyl-4-deoxy-4-amino-10-deazapteroic acid to obtain active ester. In this reaction, it is difficult to separate byproduct hexamethylphosphoramide (HMPA) which formed along with the product. It is mentioned that the by product (HMPA) is Carcinogenic and the separation is cumbersome at plant level.

A method for preparing Pralatrexate is disclosed in WO20I3/I77713, where 10-Propargyl-4-deoxy-4-amino-IO-deazapteroic acid treated with benzotriaol-l-yl-oxytripyrrolidinophosphonium hexafluoro phosphate (PyBOP) to obtain active ester. PyBOP is more expensive and may not be a industrially viable process.

WO2013/096800 disclosed methods for preparing Pralatrexate and polymorphs thereof in which various methods were adopted to prepare I0-Propargyl-4-deoxy-4-amino-10-deazapteroic acid and salts thereof. It is observed that there were many impurities formed along with the product and it could be difficult to separate those and making this process is not economically viable. Further HOBt and EDC.HC1 is used in the step where L-Glutamic acid ester coupling with 10-Propargyl-4-deoxy-4-amino-10-deazapteroic acid or salt to form active ester. These reagents are more sensitive towards moisture and also expensive. Hence, it is not industrially viable process.

WO2014/0I6740 (WO'740) disclosed a process for preparing Pralatrexate in which 10-Propargyl-4-deoxy-4-amino-10-deazapteroicacid treated with (benzotriazol-1 -yioxy) tris(dimethylamino) phosphonium hexafluorophosphate and then L-Glutamic acid is added to obtain active ester. Process of WO'740 is different from the process of present invention, where we have used novel reactant is used in the first step.

Therefore it is an object of present invention to provide a novel process for preparing Pralatrexate in . which a novel intermediate Compound II which further reacted with L-GIutamic acid dibenzyl ester hydrochloride followed by hydrolysis.

Further the process is economical and the reaction time is very less compare with the processes available in the art. Purity of the product obtained by the process of the present invention is not less than 99% which ensure the formation of byproduct is comparatively less, more particularly negligible. Further the novel process is industrially viable.

Advantage using L-GIutamic acid dibenzyl ester hydrochloride:
L-GIutamic acid dimethyl ester hydrochloride involves corrosive chemicals such as Thionyl chloride or acetyl chloride or Dry. Hydrochloride gas. During manufacturing eliminates lot of hydrochloride gas and it is highly acidic which require special arrangement to trap the Hcl gas. Since, L-GIutamic acid dimethyl ester does not having any chromophore which is difficult to assign the purity by normal HPLC and it requires a derivatization method.

L-Glutamic acid dibenzyl ester hydrochloride manufacturing process is simple and industrially viable process which does not involves any corrosive chemicals. We can check the purity by normal HPLC method. Hence, we have chosen L-Glutamic acid dibenzyl ester hydrochloride.

SUMMARY OF THE INVENTION:

According to the present invention, a novel process for the preparation of Pralatrexate comprising;
a) treating 10-Propargyl-4-deoxy-4-amino-IO-deazapteroic acid (II) with 2-Chloro-4,6-dimethoxy-l,3,5-triazine (CDMT) (III) in a solvent with base to prepare compound of Formula IV;
b) reacting the compound of Formula IV of step (a) with L-Glutamic acid dibenzyl ester in an organic solvent to obtain Pralatrexate ester;
c) purifying Pralatrexate obtained in step (b) using a mixture of aprotic solvents and isolate the product by adding a co solvent;
d) hydrolysis the purified compound obtained in step (c) using alkali in a protic solvent mixture;
e) Purifying the product obtained in step (d) using a mixture of aqueous protic solvents to obtain pure Pralatrexate.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 depicts the X-ay powder diffractogram of Pralatrexate.

DETAILED DESCRIPTION OF PRESENT INVENTION:

The present invention provides an industrially viable process to prepare Pralatrexate with purity is not less than 99%, which is highly economical compared with the process in the art

Methods known in the art for the preparation of Pralatrexate either the final product with impurities or much expensive of high running cost and industrially not viable process. To overcome those problems, we have employed novel reactant in the first step and also benzyl ester of L-Glutamic acid which not only reduce the time and cost, there is an improvement in the purity of the product. Since present invention is three step processes, it reduces high running cost in the large scale. The technical advancements reside in obtaining pure compound by using the novel reactant CDMT and novel ester of L-Glutamic acid.

The novel three step process for preparing Pralatrexate which involves;

a) treating 10-Propargyl-4-deoxy-4-amino-I0-deazapteroic acid (II) with 2-Chloro-4,6-dimethoxy-I,3,5-triazine (CDMT) (III) in a solvent with base to prepare compound of Formula IV (Step-I)
b) reacting the compound of Formula IV of step (a) with a solution of L-GIutamic acid dibenzyl ester in N,N-Dimethyl formamide to obtain Pralatrexate dibenzyl ester.
c) purifying the product obtained in step (b) using a mixture of aprotic solvents and isolate the product by adding a co solvent. (Step-II)
d) hydrolysis the Pure compound obtained in step (c) using alkali in a protic solvent mixture
e) purification of the product obtained in step (d) using a mixture of mixture of aqueous protic solvents.(Step-III)

The solvent used in step (a) is Dichlomethane, tetrahydrofuran, N, N-Dimethyl formamide , preferably dichloromethane.

The base used in step (a) is Triethylamine, Diisopropyl ethylamine and N-methyl morpholine, preferably triethylamine and most preferably N-methyl morpholine.

The temperature of the reaction in step (a) is between -10° and 20°C, preferably between I0°C and 15°C and most preferably between 0°C and 10°C.

Duration of the reaction in step (a) is between 1 and 2 hrs preferably between 60 and 90 min and most preferably between 45 and 60 minutes.

Reacting the compound of Formula IV of step (a) with L-Glutamic acid dibenzyl ester in N,N-Dimethyl formamide(DMF) to obtain Pralatrexate dibenzyl ester.

L-Glutamic acid ester used in the step (b) is L-Glutamic acid dibenzyl ester hydrochloride.
The temperature of the reaction in step (b) is between 0° and 30°C, preferably between 20°C and 25°C and most preferably between I0°C and I5°C.

Duration of the reaction in step (b) is between 1 and 2 hrs, preferably between 90 and 60 min and most preferably between 45 and 60 minutes.

The solvent used for purification in step(c) is selected from a mixture of ethyl acetate and dichloromethane, ethyl acetate and tetrahydrofuran, most preferably mixture of ethyl acetate and tetrahydrofuran.
The ratio of solvent mixture used in the step(c) is 1:1

The co solvent used to isolate the product in step (c) is selected from Diisopropyl ether, diethyl ether methyl tertiary butyl ether, preferably diethyl ether and most preferably methyl tertiary butyl ether.

The alkali used for hydrolysis in step (d) is sodium hydroxide, lithium hydroxide, potassium hydroxide, preferably potassium hydroxide and most preferably sodium hydroxide.

The solvent used for hydrolysis in step (d) is selected from methanol, ethanol, isopropanol, THF and mixture thereof, preferably ethanol and most preferably a mixture of methanol and tetrahydrofuran.
The ratio of solvent mixture used in the step (d) is 1:1

The temperature of the reaction in step (d) is between 0° and 30°C, preferably between 20°C and 25°C and most preferably between I0°C and 15°C.

Duration of the reaction in step (d) is between I and 4 hrs, preferably between 2 and 3hrs and most preferably between 2 hrs.

Solvent mixture used for purification of the product obtained in step(d) is mixture of THF, methanol and water, THF, ethanol and water, THF, acetonitrile and water, preferably THF, Methanol and water and most preferably THF, acetonitrile and water.

Ratio of Acetonitrile, water and THF is 50:25:25.The purity of the product is greater than 99.0%

Scheme:

Example-I:
To a suspension of 10-Propargyl-4-deoxy-4-amino-10-deazapteroic acid (lOOgm) in MDC (1 lit), CDMT (80.6 gms) and N-Methyl Morpholine was added at 0-10°C and stir for 1 hr. To that the solution of L-Glutamic acid dibenzyl ester. HC1 (I12gms) in DMF (600 ml) was added at 0-5°C under nitrogen atmosphere. The reaction mixture was stirred for lhrat 10 -I5°C. The completion of reaction was monitored by HPLC. After completion of reaction, the reaction mixture was washed with water, and the organic layer was concentrated to obtain the crude. The crude obtained was dissolved in a mixture of Tetrahydrofuran and ethyl acetate. To that methyl tert-butyl ether was added with stirring. The solid formed was filtered and dried under vacuum to yield 10-Propargyl-10-deazaaminopterin dibenzyl ester, having purity greater than 97%. Yield: 90%w/w.

IR(KBr,cnr'): 2949(C-H), 1735.15, (CO for ester), 1627.92(C=0 for amide), 1589.31, 1562.34, 1533.41 (C=C- Aromatic) 3442.94, 3138.18 (-NH2), 1359.82 (C-O)
'H NMR: (DMSO-d6; 300 MHz): 8 7.42 (d,2H): 7.75 (d,2H): 2.11-2.22 (m,1H): 2.61 (d,2H): 2.80 (s,lH): 3.15-3.34 (m,2H): 8.41 (d,lH): 6.57(s,2H): 7.60 (s,2H): 8.73 (d,lH): 4.474.54 (m,lH): 1.982.08 (m,2H); 2.51 (t,2H): 5.08 (s,2H): 5.14 (s,2H): 7.33-7.34 (m,10H)
l3C NMR: (DMSO-d6; 300 MHz): 8 121.47, 127.38, 127.68, 131.96, 42.99, 24.87, 82.53, 72.82, . 42.99, 146.83, 147.01, 154.52, 162.64, 162.77, 150.55,52.06, 166.78,30.11,25.61, 171.63,65.51, 135.95, 127.86, 127.97, 128.05, 136.09, 128.36, 128.39

Example-Il:
10-Propargyl-10-deazaaminopterin dibenzyl ester (100 gm) was dissolved in mixture of Tetrahydrofuran and Methanol (31it), reaction mixture was cooled to 0 -5°C.To that NaOH solution (40 gms in 1. lit water) was added at 0 -5°C. The reaction mixture was stirred for 3hrs at 10 -15°C. The completion of reaction was monitored by HPLC. After completion of reaction, the pH of the reaction mixture was adjusted with diluted Acetic acid and the solid formed was filtered and dried under vacuum to yield 10-Propargyl-10-deazaaminopterin (Pralatrexate Crude) having purity greater than 98%.Yield: 70%w/w

Example-IH
The 10-Propargyl-10-deazaaminopterin (Pralatrexate Crude) was suspended in mixture of Tetrahydrofuran (350ml) and Acetonitrile (700ml) at 25-30°C. Then reaction mixture temperature was raised to 60 -65°C and water (350ml) was added at 60-65°C. The clear solution obtained was cooled to 25-30°C. The solid formed was filtered and dried under vacuum to yield Pralatrexate having purity greater than 99%. Yield: 60 %w/w.
IR(KBr,cm"'):2949(C-H), 1735.15, (C=0 for ester), 1627.92(C=0 for amide), 1589.31, 1562.34, 1533.41. (C=C- Aromatic) 3442.94, 3138.18 (-NH2) 1359.82 (C-O) ■ 'H NMR (DMSO-d6; 300MHz): 8 7.39 (d,4H): 7.74 (d,2H): 2.11-2.22 (m,lH): 2.61 (d,2H): 2.80 (s,lH): 3.15-3.34 (m,2H):8.4 1(d,lH): 6.57 (s,2H): 7.60 (s,2H): 8.73 (dJH): 4.47-4.54 (m,lH): 1.982.08 (m32H): 2.51 (t,2H): 5.08 (s,2H): 5.14 (s,2H): 7.33-7.34 (m,10H) I3C NMR (DMSO-d6; 300MHz): 5 121.51, 127.34, 127.68, 132.27,43.07,24.87,82.56,72.84, 39.63, 146.66, 147.30, 154.08, 162.37, 162.78, 150.57,52.01, 166.42,30.52,26.04, 173.57, 173.97, 65.51, 135.95, 127.86, 127.97, 128.05, 136.09, 128.36, 128.39 XRD (°2Theta; Cu): Powder x-ray diffraction of Pralatrexate having peaks at 7.42, 14.9, 15.8, 17.4, 22.5, 22.5, 23.5, 23.96, 28.86, 32.94 ± 0.2° theta ■

We claim:
1. A novel process for the preparation of Pralatrexate using the compound (V) comprising
a) treating 10-Propargyl-4-deoxy-4-amino-10-deazapteroic acid (II) with 2-Chloro-4,6-dimethoxy-l,3,5-triazine (CDMT) (III) in a solvent with base to prepare compound of Formula IV;
b) reacting the compound of Formula IV of step (a) with L-Glutamic acid dibenzyl ester hydrochloride in an organic solvent to obtain Pralatrexate ester;
c) purifying Pralatrexate dibenzyl ester obtained in step (b) using a mixture of aprotic
■ solvents and isolate the product by adding a co solvent;
d) hydrolysis the purified compound obtained in step (c) using alkali in a protic solvent mixture
e) purifying the product obtained in step (d) using a mixture of aqueous protic solvents to obtain pure Pralatrexate.

2. The process as claimed in claim 1, wherein solvent used in step (a) is Dichlomethane, tetrahydrofuran, N, N-Dimethyl formamide, preferably dichloromethane.
3. The process as claimed in claim 1, wherein base used in step (a) is Triethylamine, Diisopropyl ethylamine and N-methyl morpholine, preferably triethylamine and most preferably N-methyl morpholine.
4. The process as claimed in claim 1, wherein temperature of the reaction in step (a) is between -10° and 20°C, preferably between 10°C and 15°C and most preferably between 0°C and 10°C; duration of the reaction in step (a) is between 1 and 2 hrs preferably between 60 and 90 min and most preferably between 45 and 60 minutes.
5. The process as claimed in claim I, wherein the solvent used in step (b) is N,N-Dimethyl formamide(DMF).
6. The process as claimed in claim 1, wherein L-Glutamic acid diester used in the step (b) is L-
. Glutamic acid dibenzyl ester hydrochloride.

7. The process as claimed in claim 1, wherein the temperature of the reaction in step (b) is between 0° and 30°C, preferably between 20°C and 25°C and most preferably between 10°C and 15°C; the reaction time is between 1 and 2 hrs, preferably between 90 and 60 min and most preferably between 45 and 60 minutes.
8. The solvent used for purification in step(c) "is a mixture of ethyl acetate and dichloromethane, ethyl acetate and tetrahydrofuran, most preferably mixture of ethyl acetate and tetrahydrofuran and the ratio of solvent mixture used in the step(c) is 1:1.
9. The co solvent used to isolate the product in' step (c) is selected from Diisopropyl ether, diethyl ether methyl tertiary butyl ether, preferably methyl tertiary butyl ether.
10. The process as claimed in claim 1, wherein the alkali used for hydrolysis in step (d) is sodium hydroxide, lithium hydroxide, potassium hydroxide, preferably potassium hydroxide and most preferably sodium hydroxide.
11. The solvent used for hydrolysis in step (d) is selected from methanol, ethanol, isopropanol, THF and mixture thereof, preferably ethanol and most preferably a mixture of methanol and tetrahydrofuran and the ratio of solvent mixture used in the step (d) is 1:1.
.12. The temperature of the reaction in step (d) is between 0° and 30°C, preferably between 20°C and 25°C and most preferably between 10°C and 15°C; the reaction time in step (d) is between I and 4 hrs, preferably between 2 and 3hrs and most preferably between 2 hrs;
13. The solvent mixture used for purification of the product obtained in step(d) is mixture of THF, methanol and water, THF, ethanol and water, THF, acetonitrile and water, preferably THF, methanol and water and most preferably THF, acetonitrile and water and ratio of Acetonitrile, water and THF is 50:25:25.The purity of the product is greater than 99.0%

14. Pralatrexate having X-ray powder diffraction peaks at 7.42, 14.9, 15.8, 17.4, 22.5, 22.5, 23.5, 23.96, 28.86, 32.94 ± 0.2° theta.