FORM 2
THE PATENTS ACT 1970
(Act 39 of 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION
(SECTION 10 and Rule 13)
"An Improved Process for Cidofovir"
Emcure Pharmaceuticals Limited.,
An Indian company, registered under the Indian Company's Act 1957 and
having its registered office at
Emcure House, T-184, M.I.D.C, Bhosari, Pune-411026, India.
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

FIELD OF THE INVENTION
The present invention relates to a convenient, cost-effective process for the synthesis of Cidofovir. Specifically, the process relates to preparation of the key synthetic intermediate for cidofovir, namely, phosphonic acid [[(S)-2-(4-amino-2-oxo-l (2H)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy) ethyl] methyl] diethyl ester (Compound III), by reaction of (S)-N'-[(2-hydroxy-3-triphenylmethoxy)propyl]cytosine (Compound II) with diethyl-p-tosyloxymethyl phosphonate in presence of a base to obtain the intermediate compound III having desired purity.
BACKGROUND OF THE INVENTION
Cidofovir, chemically known as ({[(S)-l-(4-amino-2-oxo-l,2-dihydropyrimidin-l-yl)-3-hydroxypropan-2-yl]oxy}methyl)phosphonic acid is an antiviral compound primarily used in the treatment for cytomegalovirus (CMV) retinitis (an infection of the retina of the eye) in people having acquired immune deficiency syndrome (AIDS). The drug product, marketed under the brand name Vistide, was approved by USFDA on June 26, 1996 as an injectable formulation for the treatment of CMV retinitis in AIDS patients.
Compound (III), which is chemically known as phosphonic acid [[(S)-2-(4-amino-2-oxo-l(2h)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy)ethyl]methyl]diethyl ester, ,
Brincidofovir (CMX001), which currently is in Phase III clinical trials, is a prodrug being developed for release of cidofovir intracellularly for achieving higher intracellular and lower plasma concentrations of cidofovir effectively increasing its oral bioavailability as well as activity against DNA viruses. The prodrug being developed by Chimerix for the treatment of cytomegalovirus, adenovirus, smallpox, and ebola virus infections is an experimental antiviral drug which has received FDA permission for fast track approval.


is an important synthetic intermediate, which, after hydrolysis of phosphate ester and deprotection of the hydroxyl group, yields cidofovir.

US 5,142,051 discloses two processes for synthesis of cidofovir (Compound A; wherein B is substituted with cytosine-1-yl). The first method comprises reaction of specifically protected compound B having a free secondary hydroxyl group with the diester of p-toluenesulfonyloxy methanephosphonic acid in presence of a metal hydride base followed by subsequent cleavage of the resulting ester with trimethyl halogenosilanes to give compound A.

The second method involved treatment of unprotected compound B with chloromethane-phosphonyl dichloride (ClCH2P(O)Cl2) either in pyridine or triethyl phosphate and subsequent isomerization in mineral acid or water to give compound C, which on hydrolysis with alkali hydroxides gave the rearranged compound A.

Both the methods involve hazardous, moisture sensitive reagents like sodium hydride and chloromethane phosphonyl dichloride and require successive column chromatographic separations to obtain desired intermediates and product in pure form. These disadvantages, coupled with extended reaction times and low yields severely hamper the commercialization of these methods.

WO 9202511 discloses another method wherein cytosine is reacted with trityloxy methyloxirane in presence of sodium hydride, using anhydrous dimethylformamide as solvent, to give N-[(2-hydroxy-3-trityloxy)propyl]cytosine. Further treatment of the substituted cytosine derivative with diethyltosyloxymethane phosphonate with sodium hydride as base followed by column chromatographic purification gave N-[(2-diethyl-phosphonylmethoxy-3-triphenylmethoxy)propyl cytosine, with yield of 37.4%. Deprotection of the triphenylmethyl (trityl) group in acidic media and hydrolysis of the diethyl ester using trimethylhalogenosilanes gave cidofovir.
Although this synthetic method did not involve isomerization reactions and use of phosphonyldichloride type reagents, but its synthetic utility, especially on commercial scales is likely to be limited due to use of highly moisture sensitive, hazardous reagents like sodium hydride, which are used in excess, in more than one step of the synthetic sequence. Further, this sequence also includes column chromatographic purification, which is time consuming, requires extensive volumes of solvents and has poor reproducibility on industrial scale.
It would be evident from the foregoing that prior art methods involve hazardous, pyrophoric reagents like sodium hydride and tedious chromatographic purification procedures. Further, these routes afford cidofovir only in modest yields.
Thus, there is a need for an improved process for cidofovir which avoids the drawbacks of the known prior art processes and also eliminates need for chromatographic purification during isolation. In view of the prior art deficiencies, the present inventors have developed a process to synthesize the cidofovir key intermediate phosphonic acid [[(S)-2-(4-Amino-2-oxo-l(2h)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy) ethyl]-methyl]diethyl ester (Compound III).
The process involves reaction of (S)-NI-[(2-hydroxy-3-triphenylmethoxy)propyl] . cytosine (Compound II) with diethyl-p-tosyloxymethyl phosphonate in presence of a mild base like sodium hexamethyldisilazane (NaHMDS), which is not moisture sensitive and does not require stringent reaction conditions. It was observed that use of NaHMDS not

only afforded intermediate compound III with desired purity but also resulted in significant improvement in the yield for the intermediate and thereafter cidofovir as compared to prior art. Further, by avoiding use of hydride reagent, the synthetic route resulted in a robust process applicable on commercial scale with significantly enhanced ease of operation.
OBJECT OF THE INVENTION
An objective of the present invention is to provide the intermediate phosphonic acid [[(S)-2-(4-amino-2-oxo-l(2h)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy) ethyl]-methyl] diethyl ester (Compound III) by a safe, cost-effective process which does not involve use of environmentally hazardous, moisture sensitive reagents such as sodium hydride.
Another object of the present invention is to provide an efficient, convenient process for preparation of cidofovir using the aforementioned intermediate, which is easily applicable on commercial scale.
SUMMARY OF THE INVENTION
The present invention relates to a novel process for the preparation of cidofovir intermediate, phosphonic acid [[(S)-2-(4-amino-2-oxo-l(2h)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy)ethyl]-methyl]diethyl ester (Compound III) by overcoming the problems faced in the prior art.
Yet another aspect of the invention relates to an improved process for the preparation of cidofovir comprising reaction of (S)-N1-[(2-hydroxy-3-triphenylmethoxy)propyl] cytosine with diethyl-p-tosyloxymethyl phosphonate (DETMP) in presence of sodium hexamethyldisilazane as base to yield compound III, followed by reaction with trimethylsilyl bromide and subsequent treatment with hydrochloric acid.
These objectives of the present invention will become more apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION
While working on the development and optimization of the synthetic process for cidofovir, present inventors have surprisingly found that the reaction of trityl protected cytosine derivative (compound II) with diethyl-p-tosyloxy methyl phosphonate could be effectively and conveniently carried out using sodium hexamethyldisilazane as base.
During experimentation in case of the aforementioned reaction, wherein prior art methods disclose use of hazardous, pyrophoric and moisture sensitive bases like sodium hydride, the present inventors observed that.
a) The hydride base was required in excess, and after completion of the reaction, unreacted hydride had to be carefully hydrolyzed using alcohol, which, due to the liberated hydrogen, was a potentially hazardous reaction on commercial scale.
b) Poor solubility of sodium hydride in most of the organic solvents necessitated use of phase transfer catalysts like tetrabutyl ammonium bromide in the reaction, which added to cost and unit operations for their separation from the reaction mass.
c) Owing to the pyrophoric nature and high moisture sensitivity of the hydride reagent, wherein hydrogen gas is liberated due to reaction of hydride with moisture, there were serious concerns over the industrial scale operations involving the said reaction.
The safety concerns were supported by the literature reports regarding thermal runaway reactions of pilot plant reactors containing sodium hydride and dimethylformamide as disclosed in Buckey, J. et al., Chem. Eng. News, 1982, 60(28), p.5 and De Wall, G. Chem. Eng. News, 1982, 60(37), p.5).
The inventors, during their extensive experimentation envisaged the development of a safe and efficient synthetic method for preparation of intermediate compound III. It was surprisingly observed that when sodium hexamethyldisilazane was used as a base during the reaction of introduction of methanephosphonate group to the secondary hydroxyl group in compound II, the reaction was facile and the intermediate compound III was obtained in good yield without resorting to any additional purification step like column chromatographic separation.

Also, since sodium hexamethyldisilazane is soluble in organic solvents ranging from toluene to tetrahydrofuran and other ethers, various solvents could be tried and optimized for the reaction. It also eliminated the need of a phase transfer catalysts like tetrabutyl ammonium bromide. Further, it was also observed that NaHMDS was required in lesser quantities as compared to sodium hydride.
Other bases such as sodium methoxide, potassium methoxide, sodium tertiary butoxide were also tried by the inventors and NaHMDS was surprisingly found superior to all the other bases.
The scheme for synthesis of intermediate compound III using sodium hexamethyldisilazane and its further conversion to cidofovir dihydrate is given below.

Scheme 1: Method embodied in the present invention for the preparation of cidofovir (I)
In an embodiment, reaction of (S)-N1-[(2-hydroxy-3-triphenylmethoxy) propyl] cytosine (Compound II), with diethyl- p-tosyloxy methyl phosphonate in presence of sodium hexamethyldisilazane and an organic solvent yielded the desired intermediate, phosphonic acid [[(S)-2-(4-amino-2-oxo-l(2H)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy)ethyl] methyl]diethyl ester (Compound III).
Sodium hexamethyldisilazane was gradually added to the reaction mass at low temperature, either directly or as solution in tetrahydrofuran.

The organic solvent was selected from the group comprising tetrahydrofuran, diethyl ether, di-tertiary butyl ether, toluene etc., preferably tetrahydrofuran or mixtures thereof.
The reaction was carried out at a temperature of-15 to 30°C but preferably in the range of-10to+10°C.
After completion of the reaction, as monitored by HPLC, ammonium chloride solution was added to the reaction mixture, followed by extraction of the mass with ethyl acetate. Separation and concentration of the organic layer gave compound III, which was subjected to further reaction to yield cidofovir.
In another embodiment, phosphonic acid [[(S)-2-(4-amino-2-oxo-l(2H)-pyrimidinyl) -1 -(hydroxymethyl)-2-(triphenylmethoxy)ethyl]methyl]diethyl ester (Compound III) was treated with trimethyl silyl bromide using dichloromethane as solvent in the temperature rangeof20to50°C.
After completion of the reaction, as monitored by HPLC; the reaction mass was concentrated, followed by treatment with aqueous hydrochloric acid. After completion of the trityl deprotection reaction, the resulting mass was extracted with dichloromethane. After separation of the organic layer, aqueous sodium hydroxide solution was added to the aqueous layer till pH of 3.0 to 3.5 was attained, followed by addition of isopropyl alcohol. Filtration of the obtained solid gave cidofovir dihydrate.
Compound II was prepared following methods disclosed in prior art. (R)-(+)-Glycidol was treated with trityl chloride using triethylamine and solvent dichloromethane and the resulting trityl protected alcohol was reacted with cytosine at 110-140°C in presence of sodium hydroxide and solvent dimethylformamide to give compound II.
The following examples are meant to be illustrative of the present invention. These examples exemplify the invention and are not to be construed as limiting the scope of the invention.
Example 1: Synthesis of [[(S)-2-(4-amino-2-oxo-l(2H)-pyrimidinyl)-l-hydroxymethyl)-2-(triphenylmethoxy)ethyl] methyl] diethyl ester (Compound III)

Sodium hexamethyldisilazane (467 ml of 1.0 M solution in tetrahydrofuran) was gradually added to the mixture of (S)-N1-[(2-hydroxy-3-triphenylmethoxy) propyl] cytosine (Compound II; 100.6 gms) and tetrahydrofuran (200 ml) at -15 to 0°C and the resultant mass was stirred for some time. Diethyl-p-tosyloxymethylphosphonate, (DETMP) (143.2 gms) was carefully added to the reaction mixture at -15 to 0°C and stirring was continued.
After completion of the reaction, as monitored by HPLC, aqueous solution of ammonium chloride (60 gms in 300 ml water) was added to the stirred reaction mixture, followed by extraction of the mass with ethyl acetate. Separation and concentration of the organic layer gave the desired intermediate, phosphonic acid [[(S)-2-(4-amino-2-oxo-l(2H)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy) ethyl] methyl] diethyl ester, (compound III). Yield: 94.1 gms (70%)
Example 2: Synthesis of cidofovir dihydrate (la).
Trimethylsilyl bromide (159.2 gms) was gradually added to the stirred mixture of dichloromethane (500ml) and [[(S)-2-(4-amino-2-oxo-l(2H)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy)ethyl] methyl] diethyl ester, (compound III; 100.3
o
gms). The reaction mixture was heated to 40-42 C with continued stirring till completion of the reaction as monitored by HPLC.
After completion, the reaction mass was concentrated till a thick mass was obtained. Aqueous hydrochloric acid solution (350ml) was added to it and the resultant mass was stirred at ambient temperature till completion of the deprotection reaction as monitored by HPLC. After completion of reaction, the reaction mass was extracted with dichloromethane and the organic layer was separated. Aqueous sodium hydroxide solution (35 gms sodium hydroxide in 120 ml water) was added to the aqueous layer till pH of 3.0 to 3.5 was attained. Isopropyl alcohol (400 ml) was then gradually added, along with stirring, followed by filtration of the obtained solid to give cidofovir dihydrate. Yield: 45.7 gms (83.5%)

We claim:
1. A process for the preparation of cidofovir dihydrate (la), comprising reaction of
(S)-Nl-[(2-hydroxy-3-triphenylmethoxy) propyl] cytosine, (compound II) with diethyl-p-tosyloxymethyl phosphonate (DETMP) in presence of sodium hexamethyldisilazane, in an organic solvent to give phosphonic acid [[(S)-2-(4-amino-2-oxo-l(2H)-pyrimidinyl)-l-(hydroxymethyl)-2-(triphenylmethoxy)ethyl] methyl] diethyl ester (compound III), which is then hydrolyzed with trimethylsilyl bromide and subsequently treated with aqueous hydrochloric acid to provide cidofovir dihydrate.
2. A process as claimed in claim 1, wherein the solvent is selected from the group
comprising of tetrahydrofuran, diethyl ether, di-tertiarybutyl ether, toluene and mixtures thereof.
3. A process as claimed in claim 1, wherein the reaction is carried out at a temperature of -15 to30°C.
4. A process as claimed in claim 1, wherein further to aqueous hydrochloric acid treatment, isopropyl alcohol was added to the aqueous mixture of cidofovir after the pH was adjusted between 3.0 to 3.5.