FIELD OF THE INVENTION

The invention relates to a process for preparation of 10-propargyl-10-deazaaminopterin or its salts. The 10-propargyl-10-deazaaminopterin is also commonly known as Pralatrexate. More particularly  invention relates to a process for preparation of 10-propargyl-10-deazaaminopterin or its salts using peptide coupling agent O-(Benzotriazol-1-yl)-N N N′ N′-tetramethyluronium tetrafluoroborate  commonly known as TBTU.

BACKGROUND OF THE INVENTION

Pralatrexate is a 10-deazaaminopterin derivative which has been developed for the potential treatment of malignancies. Pralatrexate is an antifolate  structurally a folate analog inhibitor of dihydrofolate reductase (DHFR) exhibiting high affinity for reduced folate carrier-1 (RFC-1) and folylpolyglutamate synthetase (FPGS)  with antineoplastic and immunosuppressive activities  resulting in extensive internalization and accumulation in tumour cells.

10-propargyl-10-deazaaminopterin (Pralatrexate) was first reported by DeGraw et al.  "Synthesis and Antitumor Activity of 10-Propargyl-10-deazaaminopterin " J. Medical Chem. 36: 2228-2231 (1993) and was shown to act as an inhibitor of growth in the murine L1210 cell line and to a lesser extent of the enzyme dihydrofolate reductase ("DHFR").

Methods of preparation of Pralatrexate have been reported in many prior art publications including U.S. Patent No. 5 354 751 which is incorporated herein by reference. This patent also discloses use of this compound in treatment of arthritis and other proliferative diseases.

US2005354751 discloses heteroaroyl-10-deazaaminopterins and 10-alkenyl or 10-alkynyl-10-deazaaminopterins having pronounced anti-inflammatory activity  anti-leukemic and anti-tumorigenic activity  as well as a method for treatment of inflammatory diseases  leukemia and tumors. A method for preparation of 10-propargyl-10-deazaaminopterin compound is also disclosed in this document.

Journal publication Bioorganic and Medicinal Chemistry (19) 2011  page 1151  synthetic approaches to the 2009 new drugs  also discloses a method for synthesis of Pralatrexate. The method comprises alkylating dimethyl homotrephthalate with propargyl bromide in the presence of KH in THF and then with 2  4-diamino-6-(bromomethyl) pteridine hydrobromide in the presence of KH in DMF to afford crude product. Followed by hydrolysis of diester with aqueous NaOH  followed by acidification with AcOH to give crude carboxylic acid  followed by thermally induced decarboxylation in DMSO to give 10-deazapteroic acid derivative. Activation of carboxylic acid as a mixed anhydride using isobutyl chloroformate prior to coupling with diethyl L-glutamate hydrochloride in the presence of Et3N in DMF to give 10-propargyl-10-deaza-aminopterin diethyl ester. Finally  saponification of diethyl ester with aqueous NaOH in 2-methoxyethanol  followed by acidifying with AcOH giving Pralatrexate.

US6028071  US2011/190305 and US2005/267117 disclose a method wherein 4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl) benzoic acid (compound-A) is converted to the di-ester compound-B by treating the acid compound A with an optically active or racemic form of di-alkyl glutamate using a pepbenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate  commonly known as the BOP reagent or using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate commonly known as PyBOP. Both these reagents are used to prepare peptides by activating the carboxylic acid group to facilitate a nucleophilic attack on the carbonyl carbon atom by a suitable amine. Although the above mentioned BOP reagents are effective as coupling agents for making amides or peptides  they suffer from one drawback  namely  the formation of Hexamethylphosphoramide  often abbreviated as HMPA which is a known carcinogen. This has health and safety implications and should be avoided for use on a large scale. The method disclosed in these prior arts is depicted below in Scheme-2.

Scheme-2:-

Another approach known in the art for preparing compound-B is through the in-situ preparation of a mixed anhydride (Journal of Medicinal Chemistry; vol. 36; no. 15; (1993); p. 2228 - 2231). The reaction is graphically depicted below in Scheme-3.

Scheme-3:-

Both the above mentioned approaches of Scheme-2 and Scheme-3 suffer from drawbacks related to the formation of toxic byproducts or for the need of tedious separation of the required compound in a pure and enriched form.

Therefore  there is a need for an improved  simple and cost effective method for preparation of Pralatrexate which can be used for industrial scale preparation of this compound.

OBJECT OF THE INVENTION

The primary object of the invention is to provide a process for preparation of 10-propargyl-10-deazaaminopterin (Pralatrexate) or its salts by coupling a diaminopteridin derivative with a di-alkyl- glutamate using O-(Benzotriazol-1-yl)-N N N′ N′-tetramethyluronium tetrafluoroborate (TBTU) as peptide coupling agent.

Another object of the invention is to provide a commercially viable  simple and cost effective process for preparation of 10-propargyl-10-deazaaminopterin (Pralatrexate) without formation of toxic byproducts and without requiring tedious separation of the required compound in a pure and enriched form.

SUMMARY OF THE INVENTION

Accordingly  to achieve the above objectives  there is provided a process for preparation of 10-propargyl-10-deazaaminopterin (Pralatrexate) or its salts which comprises coupling diaminopteridin derivative referred herein after as compound A and a di-alkyl- glutamate  the two key components  using a peptide coupling agent O-(Benzotriazol-1-yl)-N N N′ N′-tetramethyluronium tetrafluoroborate (TBTU) in presence of an organic amine base resulting in a di-ester compound B.

Hydrolyzing the di-ester compound B and converting into 10-propargyl-10-deazaaminopterin or Pralatrexate (compound C).

The process of the invention is graphically depicted below as Scheme-1:-
Scheme-1:
The peptide coupling reagent  O-(Benzotriazol-1-yl)-N N N′ N′-tetramethyluronium tetrafluoroborate commonly referred to as TBTU is represented as below:-

TBTU
The process involves the formation of an active ester in the form of an uronium salt which reacts with an amine facilitating in the required amide bond formation.

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of the present invention are disclosed herein below. However  it is to be understood that the disclosed embodiments are merely exemplary of the invention  which can be embodied in various forms. The scope of the invention is not limited to the disclosed embodiments and terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention. The invention is defined by claims appended hereto.

The invention relates to a process for preparation of Pralatrexate or 10-propargyl-10-deazaaminopterin and its salts graphically depicted below in Scheme-01:-
Scheme-1:
Whereas  TBTU is a peptide coupling reagent  O-(Benzotriazol-1-yl)-N N N′ N′-tetramethyluronium tetrafluoroborate represented as below:-

TBTU
The process comprises treating 4-(1-(2  4-diaminopteridin-6-yl)pent-4-yn-2-yl) benzoic acid (compound A) with a di-alkyl glutamate and O-(Benzotriazol-1-yl)-N N N′ N′-tetramethyluronium tetrafluoroborate (TBTU) along with any organic amine base.

In an exemplary embodiment of the invention  the di-alkyl glutamate may be selected from any alkyl or aryl di-ester of glutamic acid. However  in a preferred embodiment  the methyl  ethyl and tert-butyl di-esters of glutamic acid may be used.

In a general embodiment of the invention  the organic amine base is a tri-alkyl amine. However  in preferred embodiment  the tri-alkyl amine is triethyl amine or di-isopropyl ethyl amine in any suitable organic solvent that allows for the dissolution of the above chemical components.

In an exemplary embodiment of the invention  the solvent is an aprotic polar solvent such as dimethyl formamide (DMF).

The above reaction mixture is allowed to stir at ambient temperature and the reaction conversion is monitored by thin layer chromatography (TLC) or HPLC. On completion  the reaction mass is subjected to conditions that allow for the efficient separation and isolation of the required product  namely a di-ester compound B.

The di-ester compound B is then subjected to conditions known in the art that allow for the hydrolysis of the ester without epimerization of the amino acid to obtain the desired Pralatrexate (compound C). In one general embodiment  the alkyl group is a tert-butyl group and trifluoro acetic acid is used in a suitable organic solvent such as dichloromethane (DCM).

In another embodiment  the alkyl group is an ethyl or methyl group and both these groups are removed using methods known in the art for base induced ester hydrolysis using metal hydroxides in a suitable mixed aqueous and organic solvent systems  preferably alcoholic solvents at temperatures that range between ambient to elevated temperatures.

Examples

Various embodiments of the invention are further exemplified with the help of given examples. However  these examples are only for the purpose of explanation of the process to a person of ordinary skill and various parameters of the examples are illustrative only and should not be taken as limitation to the claims.

Example-1:
Preparation of di-tert-butyl 2-(4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl) benzamido) pentanedioate (B):

A B
In a clean and dry 2 liter 3 neck round bottom flask (3N RBF)  equipped with magnetic stirrer  nitrogen inlet  addition funnel  25g of AL/B056/ S09 (4-(1-(2  4-diaminopteridin-6-yl) pent-4-yn-2-yl) benzoic acid) was charged in 625 ml of anhydrous dimethyl formamide (DMF) at ambient temperature. Stirring was started and 25.3g of TBTU and 20.46g of di tert. butyl glutamate ester were charged simultaneously in one portion under nitrogen atmosphere. Then reaction mixture was cooled to 0-5oC and finally 29.05g of triethyl amine was added drop wise to the reaction mixture at the same temperature. Then the overall reaction was brought to ambient temperature and was stirred for 14 hr at that temperature under nitrogen atmosphere. Progress of the reaction was monitored by TLC (10% Methanol in chloroform). After completion of the reaction  the reaction mixture was poured in to 2.5 liter of water and stirred for 45minutes. The precipitated solid was filtered and dried. The product S14 was obtained with a yield of above 80% and purity above 99%.

Example-2:
Preparation of 2-(4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl)benzamido)pentane dioic acid (C):
B C
In a clean and dry 50 ml 3N RBF equipped with magnetic stirrer  addition funnel and guard tube  2g of the compound S14 (di-tert-butyl 2-(4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl) benzamido) pentanedioate) and 20 ml of dichloromethane (DCM) were charged. Temperature was cooled to 0oC and 8 ml of trifluoro acetic acid was added drop wise at 0oC. The reaction mixture was brought to ambient temperature and stirred for 4 hours at the same temperature. 20ml of water was added to it and the layers were separated. The aqueous layer was extracted with IPA in DCM (10%) (10 X 10ml). The aqueous layer pH was adjusted to 3 using saturated ammonium acetate solution and the reaction mixture was stirred for 15 minutes at that pH and was filtered over celite bed. The filtrate was taken and its pH was adjusted to 4 with saturated ammonium acetate solution and was stirred overnight at ambient temperature. The pale yellow colored solid was thrown out which was filtered and dried to give the compound S11 with a yield of around 70% and purity above 99%.

Example-3:
Preparation of diethyl 2-(4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl)benzamido)pentanedioate:

A B
In a clean and dry 2 liter 3 neck round bottom flask (3N RBF)  equipped with magnetic stirrer  nitrogen inlet  addition funnel  10g of AL/B056/ S09 (4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl)benzoic acid) was charged in 250ml of anhydrous dimethyl formamide (DMF) at ambient temperature. Stirring was started and 10.05g of TBTU and 6.9g of di ethyl glutamate ester were charged simultaneously in one portion under nitrogen atmosphere. Then reaction mixture was cooled to 0-5oC and finally 16.13g of triethyl amine was added drop wise to the reaction mixture at the same temperature. Then the overall reaction was brought to ambient temperature and was stirred for 14 hours at that temperature under nitrogen atmosphere. Progress of the reaction was monitored by TLC (10% Methanol in chloroform). After completion of the reaction  the total reaction contents were poured in to 400ml of water and stirred for 45minutes and then extracted with dichloromethane DCM (3 X 50ml)  organic layer was separated and dried over sodium sulphate and concentrated under reduced pressure to give 12g of the crude product which was purified by column to give 8.1g of the desired product as yellow colored solid.

Example-4:
Preparation of 2-(4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl)benzamido)pentane dioic acid (C):

B C
In a clean and dry 50 ml 3 neck round bottom flask (3N RBF) equipped with magnetic stirrer  addition funnel and guard tube  3g of S14 (diethyl 2-(4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl) benzamido) pentanedioate) and 100 ml of ethanol were charged. The reaction mixture was cooled to 0oC and NaOH solution (0.506g of NaOH dissolved in 10Ml of water) was added drop wise at 10-15oC. The reaction mixture was brought to ambient temperature and stirred for 36 hours at the same temperature. Reaction was monitored by HPLC. After completion of the reaction  ethanol was stripped off below 40o C and the obtained residue was poured in to 25ml of water and was extracted with dichloromethane DCM (5X 20ml). The aqueous layer pH was adjusted to 3 using saturated ammonium acetate solution and was stirred for 15 minutes at that pH and then it was filtered over celite bed. The filtrate was taken and its pH was adjusted to 4 with saturated ammonium acetate solution and was stirred overnight at ambient temperature. The pale yellow colored solid was thrown out which was filtered and dried to give the compound S 11 with a yield of 74%.

The obtained 10-propargyl-10-deazaaminopterin or Pralatrexate may be converted into pharmaceutically acceptable salts for preparation of pharmaceutical compositions suitable for therapeutic applications. The pharmaceutical compositions may oral formulations or intravenous injectables for the treatment of prostate cancer  T-cell lymphoma  breast cancer  lung cancer  hematologic malignancies  head and neck cancer  cancer of the gastrointestinal tract  ovarian cancer  osteosarcoma and rheumatoid arthritis.

We claim:
1. A process for preparation of 10-propargyl-10-deazaaminopterin comprising:

i). treating a diaminopteridin derivative (compound A) with a di-alkyl-glutamate and
a coupling agent in presence of an organic amine base to obtain a di-ester compound (B);

ii). hydrolyzing the di-ester compound and converting into 10-propargyl-10- deazaaminopterin (compound C) .

2. A process as claimed in claim 1  wherein said coupling agent is a peptide coupling agent.

3. A process as claimed in claim 2  wherein said peptide coupling agent is O-(Benzotriazol-1-yl)-N N N′ N′-tetramethyluronium tetrafluoroborate (TBTU).

4. A process as claimed in claim 1  wherein said diaminopteridin derivative is 4-(1-(2 4-diaminopteridin-6-yl)pent-4-yn-2-yl) benzoic acid (compound A).

5. A process as claimed in claim 1  wherein said di-alkyl glutamate is any alkyl or aryl di-ester of glutamic acid selected from methyl  ethyl and tert-butyl di-esters of glutamic acid.

6. A process as claimed in claim 1  wherein said organic amine base is a tri-alkyl amine selected from triethyl amine and di-isopropyl ethyl amine.

7. A process as claimed in claim 6  wherein said tri-alkyl amine is present in an aprotic polar solvent.

8. A process as claimed in claim 7  wherein said aprotic polar solvent is dimethyl formamide (DMF).

9. A process as claimed in claim 1  wherein said hydrolysis and conversion of the di-ester compound comprises hydrolysis of the ester without epimerization of the amino acid to obtain 10-propargyl-10-deazaaminopterin (compound C).

10. A process for preparation of 10-propargyl-10-deazaaminopterin such as herein described in the description with reference to the examples.

Dated this 26th day of November 2012.

Afzal Hasan
Of HASAN AND SINGH
IN/PA-1328