FORM 2
THE PATENTS ACT 1970
(Act 39 of 1970)
&
THE PATENTS RULE, 2003
COMPLETE SPECIFICATION
(SECTION 10 and Rule 13)
TITLE OF THE INVENTION
"AN IMPROVED PROCESS FOR PREPARATION OF
NEOSTIGMINE METHYLSULFATE"
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 novel process for the preparation of neostigmine
methylsulfate having desired purity. The invention especially relates to the preparation of 3-dimethylamino phenol (III), its reaction with dimethylcarbamoyl chloride in presence of 4-dimethylaminopyridine and triethylamine to give 3-[(dimethylamino)carbonyI-oxy]-N,N-dimethyIaniIine of formula (IV), which, on further reaction with dimethyl sulfate gives neostigmine methylsulfate (I) having purity conforming to regulatory specifications.
BACKGROUND OF THE INVENTION
Neostigmine methylsulfate (I), chemically known as 3-[[(dimethylamino)carbonyl]oxy]-N,N,N-trimethylbenzenaminium methylsulfate is an anticholinesterase agent, which is clinically used for symptomatic treatment of myasthenia gravis, in the treatment or prevention of post-operative non-obstructive abdominal distention and the reversal of non-depolarizing neuromuscular blocking agents (NMBAs).
Neostigmine was first developed by Hoffman-La Roche in 1933 and is available with brand names such as Bioverz (neostigmine methylsulfate), Prostigmin (neostigmine bromide) and Vagostigmin (neostigmine bromide).

US 1,905,990 discloses synthesis of neostigmine comprising reaction of 3-dimethylaminophenol with potassium hydroxide and further condensation of the phenolate salt with N,N-dimethyl carbamic acid chloride. Pure neostigmine was obtained after washing the ether solution of reaction mass with sodium hydroxide solution, followed by removal of the solvent and repeated crystallizations of the resulting residue using alcohol. Neostigmine thus obtained was converted to

neostigmine methylsulfate (I) by reaction with dimethyl sulfate in acetone and recrystallization from alcohol.
Incomplete conversion of the reactant aminophenol, coupled with repeated purification by crystallization for obtaining neostigmine free base and also for the methylsulfate salt substantially reduce the yield in the process, subsequently making it economically unviable.
GB 342237 discloses a synthetic method for neostigmine methylsulfate wherein a hazardous reagent like 3-dimethylaminophenyl chlorocarbonate, requiring anhydrous reaction conditions is employed for preparing neostigmine. The process is accompanied by low yield and purity, thus rendering it quite exorbitant for employing on a commercial scale.
US 4,948,807 discloses two synthetic processes for preparation of dialkylamino (dialkylamino)alkyl phenyl carbamates which are homologs of neostigmine. The processes comprise reaction of (dialkylamino)alkyl phenol either with isocyanate or dialkylcarbamoyl chloride in presence of a strong base like sodium hydride. From synthetic utility point of view, both of these processes pose severe limitations. The isocyanate process, despite the use of hazardous isocyanates; is limited to N-monoalkylated carbamates while the other process involves highly reactive, moisture sensitive, pyrophoric reagents like sodium hydride.
Other prior art references such as US 4,315,861 and US 2010/113819 resort to carbamoylation utilizing highly specific reagents such as N-carbamoyl saccharin derivatives or diaryl carbonates. Synthesis of these expensive reagents adds to the cost of synthesis and makes the process economically unviable.
Further, regarding 3-dimethylamino phenol, which is the starting material for present process, prior art methods as disclosed in JP 05085994, GB 1,503,392, US 3,102,913, J? 26003265, JP26003031 etc. mention that the desired compound is obtained either from resorcinol or 3-amino phenol by a high pressure reaction in an autoclave at high temperatures, which is usually followed by isolation of the resulting oil by distillation.

3-dimethylamino phenol as synthesized by above methods is likely to have impurities such as the corresponding N-mono monomethylated or O-methylated compounds, which reduce the purity of the starting material used in the present scheme.
Thus, based on the foregoing, it is evident that prior art processes utilize hazardous, moisture-sensitive, highly specific, expensive reagents and high pressure reactions. Further, majority of these processes are time-consuming and yield impure products, which are subjected to repeated crystallization to obtain neostigmine methylsulfate having desired purity.
Hence, there is a need for an industrially feasible, economical and robust process for the synthesis of neostigmine methylsulfate (I) which does not involve use of strong bases, hazardous reagents and yet specifically leads to the desired product by circumventing the formation of side products. Such a process effectively yields the final compound with high purity and in good yield.
Based on the extensive experimentation carried out by the present inventors aimed at developing an economically viable synthetic route for neostigmine methylsulfate, it was possible to design a novel synthetic strategy which could overcome the problems faced in prior art and yield a product having desired purity.
Synthesis of 3-dimethyl phenol free from the desired impurities encountered in prior art was achieved by quatemization of 3-amino phenol with dimethyl sulfate in presence of a mild base like sodium bicarbonate which circumvented the formation of the 0-methylated impurity. Further, due to the quatemization of the amino group with the methyl group, an impurity such as monomethylated amino phenol was avoided. After complete quatemization of the amino group, the quaternary ammonium compound was demethylated with ethanol amine to give dimethylamino phenol, which was then converted to neostigmine by treating with dimethyl carbamoyl chloride in presence of an organic base and catalyst to give neostigmine free base. The presence of a catalyst like 4-dimethylamino pyridine helped in achieving significant control on the formation of side products.

The process involves preparation of 3-dimethylaminophenol (III) and its reaction with dimethylcarbamoyl chloride in presence of an organic base such as triethylamine using catalytic 4-dimethylaminopyridine (DMAP) to yield 3-[(dimethylamino)carbonyl-oxy]-N,N-dimethylbenzenamine (IV), which on treatment with dimethyl sulfate and subsequent purification provides neostigmine methylsulfate (I) having desired purity.
OBJECT OF THE INVENTION
An objective of the present invention is to provide an industrially applicable, cost
effective process for synthesis of neostigmine methylsulfate, which avoids use of pyrophoric, highly reactive, strong bases for introduction of carbamoyl group to 3-' dimethylaminophenol (III).
Another object of the invention is to provide a convenient method for synthesis of neostigmine methylsulfate (I) which involves facile carbamoylation of 3-dimethylaminophenol (III) using dimethylcarbamoyl chloride in presence of an organic base and a catalyst, followed by reaction with dimethyl sulfate.
A further object of the invention is to provide a method for preparation of 3-dimethyI amino phenol free from the corresponding monomethylated impurity.
SUMMARY OF THE INVENTION
An aspect of the invention relates to an improved process for the preparation of neostigmine methylsulfate (I) comprising reaction of 3-aminophenol (II) with dimethyl sulfate in presence of sodium bicarbonate, followed by treatment of the resulting quaternized salt with ethanolamine to give 3-dimethylaminophenol (III), which on reaction with dimethylcarbamoyl chloride in presence of a base and a catalyst gave 3-[(dimethylamino)carbonyl-oxy]-N,N-dimethylaniline of formula (IV), which on further reaction with dimethyl sulfate gave 3-[[(dimethylamino)carbonyl]oxy]-N,N,N-trimethylbenzenaminium methylsulfate (I) having purity conforming to regulatory specifications.
The objectives of the present invention will become apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION
While carrying out rigorous experimentation for developing an industrially viable and economical process for neostigmine methylsulfate, the present inventors observed that the reaction of the dimethylamino phenol with corresponding carbamoyl chloride was very crucial in the reaction sequence. When conventional bases such as sodium, potassium hydroxide or alkoxides of alkali metals were used, formation of phenolate ion and the subsequent reaction with the carbamoyl chloride was associated with incomplete conversion of the reactant phenol, associated side reactions during carbamoyl coupling and considerable hydrolysis of the chloride reagent. While the use
of strong bases such as sodium or potassium hydrides could ensure complete conversion of the starting material, however, it was accompanied by increased formation of undesired side products. Consequently, the yield was adversely affected and the purity of carbamoylated product was hampered. It was surprisingly observed that the aforementioned problems were considerably reduced when the carbamoylation reaction was carried out in presence of a combination of a catalyst such as dimethylamino pyridine (DMAP) and an organic base like triethylarnine. It was found that the reaction was facile and went to completion, without formation of associated impurities which were formed in prior art methods. With the use of these reagents, the need of strong, often moisture sensitive bases was eliminated and the desired reaction could be effected conveniently, with a control on formation of undesired side products. Further reaction with dimethyl sulfate and purification using organic solvents led to the final product having desired purity.
The starting material for the present scheme, 3-dimethylamino phenol was prepared by initial quaternization of the amino group in 3-amino phenol with dimethyl sulfate followed by treatment with ethanolamine to give the dimethylated product, free from impurities that are usually obtained in prior art processes.


Scheme 1: Method embodied in the present invention for the preparation of neostigmine methylsulfate (I)
In an embodiment, 3-amino phenol (II) was treated with dimethyl sulphate in presence of a mild base like sodium bicarbonate at a temperature ranging between 4 and 20°C, till complete quaternization was observed on TLC or HPLC. The reaction mixture was then warmed and ethanolamine was added to the mixture and refluxed until formation of the desired 3-dimethylamino phenol (III) was complete, as monitored by analytical methods.
Compound (III) was isolated by adding a hydrocarbon like toluene to the reaction mixture and filtering. The organic layer was separated, washed with aqueous caustic solution, and the aqueous layer was acidified to obtain compound (III).
In a further embodiment, 3-dimethylaminophenol of formula (III) was treated with dimethyl carbamoyl chloride in presence of 4-dimethylaminopyridine and triethylamine in an organic solvent to give 3-[(dimethylamino)carbonyl-oxy]-N,N-dimethylamline of formula (IV). The reaction was carried out in the temperature range of 40-70 C.

The organic solvent was selected from a group comprising aromatic hydrocarbons such as toluene, xylene mixture, cyclohexane, nitrobenzene etc. or mixtures thereof.
After completion of the reaction, as monitored by HPLC, the organic layer was washed with aqueous sodium hydroxide solution and concentrated to give the compound 3-[(dimethylamino)carbonyl-oxy]-N,N-dimethylaniline of formula (IV) which was then stirred with or passed through aluminium oxide and filtered.
Compound (IV) was reacted with dimethyl sulfate using acetone as a solvent at 25 to 45 C. After completion of the reaction, as monitored by TLC, the product neostigmine methylsulfate was filtered off.
Further treatment of neostigmine methylsulfate with a mixture of organic solvents, optionally at higher temperature, followed by cooling gave neostigmine methylsulfate having desired purity.
The organic solvents were selected from the group comprising alcohols such as methanol, n-butanol, isobutanol, tertiary butanol, propanol and isopropanol and esters like butyl acetate, ethyl acetate, isopropyl acetate, etc. The purification was preferably carried out using methanol and ethyl acetate.
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.
EXAMPLES
Example 1: Synthesis of 3-[(dimethylamino)carbonyl-oxy]-N^V-dimethylaniline (IV)
Dimethyl carbamoyl chloride (97.9 g) was gradually added to a stirred mixture of 3-dimethylaminophenol (III) (100.7g), 4-dimethylamino pyridine (8.9 g) and triethylamine (110.5 g) in 550 ml toluene at 25-35°C and the reaction mass was stirred at 45-65°C. After completion of the reaction, as monitored by HPLC, water was added to the reaction mass, and the organic layer was separated. The toluene layer was washed with aqueous sodium hydroxide and aqueous dithionite solutions, and the organic layer

was concentrated to obtain 3-[(dimethylamino)carbonyl-oxy]-N,N-dimethylaniline as residue, which was stirred or passed through aluminium oxide along with a mixture of ethyl acetate and cyclohexane. The mixture was filtered and concentrated to give neostigmine free base. Yield: 130.4 g (85.9%) Purity: 99.5%
Example 2: Synthesis of neostigmine methylsulfate (I)
Dimethyl sulfate (96.9 g) was slowly added to a stirred mixture of 3-[(dimethylamino)carbonyl-oxy]-N,N-dimethylaniline (100.2g) and acetone (450ml) at 25 to 30°C and stirring was continued at 35 to 45°C till completion of the reaction. The reaction mass was filtered to give compound (I).
Compound (I; 150.5 g) was dissolved in a 1:1 mixture of methanol and ethyl acetate (520 ml), heated to around 55°C. and diluted further with ethyl acetate to provide neostigmine methylsulfate (I) having desired purity. Yield: 125.6 g (77.9%) Purity: NLT 99.8 %.
Example 3: Synthesis of 3-dimethylamino phenol (III)
Dimethyl sulfate (1735.2 g) was gradually added to a stirred mixture of 3-aminophenol (500.3g), water (1500 ml) and sodium bicarbonate (1077.6 g) and stirred at 5 tol5°C till completion of the reaction, as monitored by HPLC. The reaction mass was then refluxed with ethanolamine (2000 g). Toluene was then added to the reaction mass after cooling, and the mass was stirred and filtered. The filtrate was washed with aqueous sodium hydroxide solution and the separated aqueous layer was neutralized with concentrated hydrochloric acid to give 3-dimethylamino phenol of formula (III), which was then filtered, washed with water and dried. Yield: 498.5 g (79%) Purity: 99.9%.

Claims
1. An improved process for the preparation of neostigmine methylsulfate (I)
comprising reaction of 3-aminophenol (II) with dimethyl sulfate in presence of
sodium bicarbonate followed by treatment of the resulting quaternary salt with
ethanolamine to give 3-dimethylaminophenol (III), which on reaction with
dimethylcarbamoyl chloride in presence of a base and a catalyst gave 3-
[(dimethylamino)carbony]-oxy]-N,N-dimethylaniline of formula (IV), which on
further reaction with dimethyl sulfate and isolation gave 3-
[[(dimethylamino)carbonyl]oxy]-N,N,N-trimethylbenzenaminium methylsulfate
(I).
2. A process as claimed in claim 1, wherein the catalyst is 4-dimethyl amino pyridine and base is triethylamine.
3. A process as claimed in claim 1, wherein the isolation comprises treatment of neostigmine methylsulfate with a mixture of organic solvents selected from acetates and alcohols.
4. A process as claimed in claim 3, wherein the acetate solvent is selected from a group comprising of n-butyl acetate, ethyl acetate, isobutyl acetate while the alcohol is selected from a group comprising of methanol, ethanol, propanol, isopropanol, butanol, isobutanol.
5. A process as claimed in claim 1, wherein the purity of neostigmine methylsulfate so obtained is at least 99.7 %.
6. A process for preparation of 3-dimethylaminophenol of formula (III) comprising reaction of 3-aminophenol with dimethylsulfate in presence of sodium bicarbonate and ethanolamine.