"NOVEL METHOD FOR SYNTHESIS OF NEUROMUSCULAR BLOCKING

AGENTS" ^'

Technical Field

The present invention describes a novel method for preparing neuromuscular blocking agents.

Background and prior art

Neuromuscular-blocking agents block neuromuscular transmission at the neuromuscular junction, causing paralysis of the affected skeletal muscles. This is accomplished either by acting presynapticaUy via the inhibition of acetylcholine (ACh) synthesis or release, or by acting postsynaptically at the acetylcholine receptors of the motor nerve end-plate.

In clinical use, neuromuscular blocker is used adjunctively to anesthesia to produce paralysis, firstly to paralyse the vocal cords, and permit intubation of the trachea, and secondly to optimize the surgical field by inhibiting spontaneous ventilation, and causing relaxation of skeletal muscles. Because the appropriate dose of neuromuscular-blocking drug may paralyze muscles required for breathing (i.e. the diaphragm), mechanical ventilation should be available to maintain adequate respiration.

Neuromuscular-blocking drugs are classified into two groups viz. non-depolarizing blocking agents and depolarizing blocking agents.

Depolarizing blocking agents:

A depolarizing neuromuscular blocking agent is a form of neuromuscular blocker that depolarizes the motor end plate. An example is succinylcholine. Depolarizing blocking agents work by depolarizing the plasma membrane of the muscle fiber, similar to acetylcholine. However, these agents are more resistant to degradation by acetylcholinesterase, the enzyme responsible for degrading acetylcholine, and can thus more persistently depolarize the muscle fibers. This differs from acetylcholine, which is rapidly degraded and only transiently depolarizes the muscle.

Non-depolarizing blocking agents:

A neuromuscular non-depolarizing agent is a form of neuromuscular blocker that does not depolarize the motor end plate. These agents constitute the majority of the clinically relevant neuromuscular blockers. They act by competitively blocking the binding of ACh to its receptors, and in some cases, they also directly block the ionotropic activity of the ACh receptors. Quaternary ammonium muscle relaxants belong to the class of Nondepolarizing blocking agents.

Quaternary ammonium muscle relaxants are quaternary ammonium salts used as drugs for muscle relaxation, most commonly in anesthesia. It is necessary to prevent spontaneous movement of muscle during surgical operations. Muscle relaxants inhibit neuron transmission to muscle by blocking the nicotinic acetylcholine receptor. What they have in common, and is necessary for their effect, is the structural presence of quaternary ammonium groups, usually two. Some of them are found in nature and others are synthesized molecules.

Following are the examples of some quaternary ammonium muscle relaxants: 1. Pancuronium Bromide

Pancuronium bromide, represented by structural formula 'A' and chemically named as 1 , 1 '-((2β,3α,5α, 16β, 17β)-3 , 17-bis(acetyloxy)androstate-2, 16-diyl)bis( 1 - methylpiperidinium) dibromide, is a typical non-depolarising curare-mimetic muscle relaxant. It acts as a competitive acetylcholine antagonist on neuromuscular junctions, displacing acetylcholine (hence competitive) from its post-synaptic nicotinic acetylcholine receptors. It is, unlike suxamethonium, a non-depolarising agent, which means, that it causes no spontaneous depolarisations upon association with the nicotinic receptor in neuromuscular junction, thus producing no muscle fasciculations upon administration. Pancuronium has no hormonal activity. It exerts slight vagolytic activity (i.e. diminishing activity of the vagus nerve) and no ganglioplegic (i.e. blocking ganglions) activity.

Formula A 2. Vecuronium Bromide

Vecuronium bromide is a valuable drug having action as a muscle relaxant in the category of non-depolarizing neuromuscular blocking agents. It is an aminosteroidal competitive neuromuscular blocker. Vecuronium bromide is used to promote skeletal muscle relaxation during surgery to aid controlled respiration by increasing pulmonary compliance, and to facilitate endotracheal intubation. Vecuronium bromide has minimal histamine-release and is less likely to cause bronchospasm or cardiac adverse effects than neuromuscular blockers with significant histamine-releasing properties such as Atracurium, Mivacurium, Succinylcholine, and Tubocurarine.

Vecuronium bromide which is chemically (l-[(2 ,3a,5ot, 16 , 17 )-3, 17-bis(acetyloxy)-2- (piperidin-l-yl)androstan-16-yl]-l-methylpiperidinium bromide) has a structure derived from the same aminosteroid structure as pancuronium, but missing the methyl group on the piperidine nitrogen that is attached to the 'Α' ring making it monoquaternary. It has the same configuration at all ten stereocentres as pancuronium and is a single-isomer preparation. Therefore vecuronium bromide has following structure of formula 'B'.

Formula B

3. Rocuronium Bromide

Rocuronium bromide, which is chemically l-[(2P,3a,5a,16p,17P)-17-(acetyloxy)-3- hydroxy-2-(4-morpholinyl)androstan- 16-yl] - 1 -(2-propenyl)pyrrolidinium bromide and represented by the structural formula 'C\ is an aminosteroid non-depolarizing neuromuscular blocker or muscle relaxant with a rapid to intermediate onset depending on dose and intermediate duration. It is used in modern anaesthesia, to facilitate endotracheal intubation and to provide skeletal muscle relaxation during surgery or mechanical ventilation. It acts by competing for cholinergic receptors at the motor end- plate. This action is antagonized by acetylcholinesterase inhibitors such as neostigmine or endrophonium.

Formula

4. Pipecuronium Bromide

Pipecuronium bromide, (2β,3α,5α, 16β, 17β)-3 , 17-bis(acetyloxy)-2, 16-bis(4,4- dimethylpiperazin-4-ium- 1 -yl)androstane dibromide is non-depolarizing neuromuscular blocking agent which acts as a nicotinic acetylcholine receptor antagonist. Pipecuronium bromide is represented by the structural formula 'D'

Formula D

5. Rapacuronium Bromide

Rapacuronium bromide, which is chemically l-((2p,3a,5a,16P, 17P)-3-acetoxy-17-(l- oxopropoxy)-2-( 1 -piperidinyl)androstan- 16-yl)- 1 -(2-propen- 1 -yl)piperidinium bromide and represented by structural formula Έ', is a rapidly acting, non-depolarizing neuromuscular blocker formerly used in modern anaesthesia, to aid and enable endotracheal intubation, which is often necessary to assist in the controlled ventilation of unconscious patients during surgery and sometimes in intensive care. As a nondepolarizing agent it did not cause initial stimulation of muscles before weakening them.

Formula E

It is clear from the above structures that mono- or di-acetylation of 3,17-androstanediol derivatives is one of the steps in synthesis of these neuromuscular-blocking drugs.

Specifically, acetylation refers to the process of introducing an acetyl group (resulting in an acetoxy group) into a compound, to be specific, the substitution of an acetyl group for an active hydrogen atom. A reaction involving the replacement of the hydrogen atom of a hydroxyl group with an acetyl group (CH ₃ CO) yields a specific ester, the acetate.

The literature search reveals that acetic anhydride is commonly used reagent for acetylation of hydroxyl groups.

Acetic anhydride is an irritant and flammable. Vapors of acetic anhydride are harmful. Because of its use for the synthesis of heroin by the di-acetylation of morphine, acetic anhydride is listed as a U.S. DEA List II precursor, and restricted in India and many other countries.

Acetylation of 3,17-androstanediol derivatives using acetic anhydride requires high temperature and results in coloured acetylated derivatives which are decolourised using alumina resulting in decrease in the product yield.

The inventors have surprisingly found very few reagents for mono- or di-acetylation of androstanediol or its derivatives, mentioned in the literature.

The process disclosed in the patent US4894369 employs acetyl chloride (1.13 equivalents) for acetylation of (2p,3a,5a,16p,17P)-2-(4-morpholinyl)-16-(l- pyrrolidinyl)androstan-3,17-diol. During the reaction the desired compound i.e. 17- acetate derivative is obtained in low yield which may be due to the formation of undesired 3, 17-di acetate derivative. Thus acetyl chloride is not a selective acetylating agent for androstane-diols. Purification is done by column chromatography to provide the desired compound in 48% yield. This method reduces the overall efficiency of the process and limited by the need of column chromatography which is inconvenient, time consuming and not feasible for industrial level.

The patent US5591735 discloses di-acetylation of androstanediols using acetyl chloride in presence of triethylamine. However the reaction requires 16 to 24 hours to provide diacetate derivatives, which are purified by silica gel column. The patent also discloses mono-acetylation of androstanediols using acetyl chloride in presence of triethylamine to provide 17-acetate derivatives which are purified by column chromatography. However the patent does not disclose the selectivity of mono-acetylated compounds.

The patent US5418226 discloses the preparation of rapacuronium bromide. The process involves following steps:

1. Acetylation of (2P,3a,5a,16P)-3-hydroxy-2,16-di-(l -piperidinyl)androstan-17-one by acetyl chloride to provide 3 -acetate derivative;

2. Reduction of the resulting 3-acetate derivative using sodium borohydride to provide 17-hydroxy-3 -acetate derivative;

3. Acylation of the resulting 17-hydroxy-3 -acetate derivative by propionyl chloride to provide 3-acetate- 17-propionate derivative; and

4. Mono-quaternization of the resulting 3-acetate- 17-propionate derivative by allyl bromide to provide rapacuronium bromide.

It is clear from the above process that the patent US5418226 does not disclose the procedure for regioselective acetylation or propionylation of the androstan-3,17-diol.

The patent application US20050159398 describes acetylation of (2β,3α,5α,16β,17β)-2-(4- mo holinyl)-16-(l-pyrrolidinyl)androstan-3,17-diol using molar excess of acetyl chloride or acetic anhydride to provide a reaction mixture containing mainly 3,17- diacetate derivative. The mixture is then treated with aqueous hydrochloric acid for selective deacetylation at the position 3 to yield 17-acetate derivative. Obtaining the pure 17-acetate derivative by this process is tedious involving several purification steps thus reducing the yield of desired 17-acetate derivative.

The patent US5817803 discloses acylation of androstan-3,17-diols using acylating reagents selected from CI - C5 aliphatic carboxylic acid, C1-C5 aliphatic carboxylic anhydride, and C1 -C5 claimed are acetic acid, acetic anhydride, mixture of acetic acid and acetic anhydride, and propionic acid anhydride to provide androstat-3,17-diacetate derivative. In the second aspect the patent also discloses acylation of 3-hydroxy- androstan-17-bne to provide 3-aeyl-androstan-17-one derivative which is reduced and acylated to provide androstan-3,17-diacyl derivative. In the third aspect the patent discloses acylation of 2,3-epoxy-androstan-17-ol to provide 2,3-epoxy-17-acyl- androstane derivative. Thus the patent does not provide the procedure for regioselective acylation of androstan-3, 17-diols.

The patent US7569687 discloses acetylation of (2p,3a,5a, 16p,17p)-2-(4-morpholinyl)- 16-(l-pyrrolidinyl)androstan-3,17-diol using acetic anhydride to provide 17-acetate derivative. However the time requires for acetylation is more than 22 hours and the selectivity for mono and di-acetylation is not mentioned. The crude mixture of mono- and di-acetylated products is separated using tedious method of separation to improve the selectivity which results in decrease in the yield of mono-acetylated product.

WO2009016648 discloses a novel process for preparation of rocuronium bromide using N-acetylimidazole for acetylation of (2p,3a,5a,16P,17 )-2-(4-morpholinyl)-16-(l- pyrrolidinyl)androstan-3,17-diol. However the process requires 48 hours to provide 17- acetoxy derivative with only 92% regioselectivity. The resulting 17-acetoxy derivative is crystallized using diethyl ether and petroleum ether; and further recrystallized using dichloromethane and acetonitrile to improve the selectivity. Thus the process is tedious and time consuming.

Hence there is a need in the art for an alternative reagent for the preparation of di- as well as selective mono-acylated androstanediols. Object of the invention:

The first object of the invention is to provide an alternative synthesis of neuromuscular blocking agents avoiding a need of acetic anhydride.

Another object of the invention is to provide di- as well as highly regioselective mono- acylation of androstan-3,17-diols.

Yet another object of the invention is to provide a short, easy, industrially viable, inexpensive and convenient method for synthesis of neuromuscular blocking agents.

Summary of the invention:

In accordance with the above objectives, the present invention discloses a novel method for preparing neuromuscular blocking agents comprising reacting androstan-3,17-diols with aryl esters to provide di- as well as highly regioselective mono-acylated androstan- 3,17-diols.

Detailed description of the invention:

Unless specified otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, to which this invention belongs. Although any method and material or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. To describe the invention, certain terms are defined herein specified as follows:

Unless stated to the contrary, any of the words 'having', 'including', 'includes', 'comprising' and 'comprises' mean 'including without limitations' and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but may be implemented in various combinations; The described embodiments of the invention and the disclosed examples are given for the purpose illustration rather than limitation of the invention as set forth the appended claims. Accordingly the present invention provides a novel process for preparation of neuromuscular blocking agents using aryl esters as reagents for the production of mono- as well as di-acylated of androstan-3,17-diols.

In one embodiment, the present invention provides a process for preparing compounds of formula (la) and (lb)

Formula (la) Formula (lb)

wherein R is CH ₂ or NCH3, R ¹ is hydrogen or acyl and R ² is hydrogen or acetyl.

The process comprises reacting androstane compound of (lla) or (lib) with aryl esters of formula (III)

Formula (III) Formula (lla) Formula (lib) wherein X is electron withdrawing group; Y and Z are independently hydrogen, electron withdrawing group, alkyl containing 1 to 7 carbon atoms, alkoxy containing 1 to 7 carbon atoms, halogen, NR ³ R ⁴ , mercapto, thioalkyl containing 1 to 7 carbon atoms, or Y and Z when placed ortho to each other form benzene ring; R ³ and R ⁴ are independently hydrogen, alkyl containing 1 to 7 carbons atoms, substituted or unsubstituted phenyl, or R ³ and R ⁴ together with nitrogen form 3 to 7 membered heterocyclic ring; R ⁵ is alkyl containing 1 to 7 carbon atoms; R ⁶ and R ⁷ are hydrogen or acyl; and with the proviso that R ⁶ and R ⁷ are not simultaneously acyl.

In a preferred aspect X is an electron withdrawing group selected from nitro or cyano; R ⁵ is methyl or propyl; Z and Y are hydrogen; R ² is hydrogen or acetyl; R ¹ is acetyl or propionyl. Advantageously the reaction of the compound of formula (Ila) and (lib) with the aryl ester of formula (III) is carried out presence of acid or base.

The acid used in the reaction is an organic acid containing 1 to 6 carbon atoms.

The preferred organic acid used in the reaction is trifluoroacetic acid, acetic acid, propanoic acid, butanoic acid. The most preferred organic acid used in the reaction is acetic acid.

The base which may be used in the present invention is organic base or inorganic base. The organic base is amine selected from triethylamine, diisopropyl ethyl amine or N- methylmorpholine.

Examples of inorganic base include alkali metal carbonate, alkaline earth metal carbonate, alkali metal bicarbonate, alkaline earth metal bicarbonate and mixture thereof. Examples of alkali metal carbonate include sodium carbonate and potassium carbonate. Examples of alkali metal bicarbonate include sodium bicarbonate and potassium bicarbonate. Examples of alkaline earth metal carbonate include calcium carbonate and magnesium carbonate. Examples of alkaline earth metal bicarbonate include calcium bicarbonate and magnesium bicarbonate.

The process of the present invention may be carried out at suitable temperature. To minimize the decomposition of products and impurity formation the reaction is carried out at 55 to 150°C, more preferably at 60 to 130°C. The most preferred temperature for acetylation is 80 to 110°C.

The reaction normally completes in 3 to 13 hours, more preferably 4 to 10 hours and the most preferably 6 to 8 hours.

The aryl acetate of formula (III) used for the synthesis is 2 to 10 equivalent of the androstane compound of formula (Ila) or (lib), more preferably 4 to 8 equivalents. The most preferred quantity of the aryl ester to carry out the reaction rapidly is 6 to 7 equivalents. Surprisingly it is observed that only 12 - 15% of the aryl ester is utilized for the reaction and the remaining can be easily recycled and reused; thus making the process economical. In a preferred aspect the reaction of the androstanediol of formula (lib) and (Ila) wherein R ⁶ and R ⁷ are hydrogen; with the aryl ester of formula (III) wherein R ⁵ is methyl, is carried out in basic conditions using triethylamine for 7 to 8 hours to provide the compounds of formula (lb) and (la) wherein R ¹ is methyl and R ² is hydrogen (i.e. C-17 acetylated diol) with more than 98% regioselectivity. The di-acetylated product is formed in less than 2% and can easily be removed by simple crystallization.

In another aspect the reaction of androstanediol of formula (lib) and (Ila) wherein R ⁶ and R ⁷ are hydrogen; with the aryl ester of formula (III) wherein R ⁵ is methyl, is carried out in acidic conditions using acetic acid for 6 hours to provide the compounds of formula (lb) and (la) wherein R ¹ and R ² are acetyl (i.e. 3,17-diacetate compound), in more than 95% yield.

Thus aryl acetate is found to be an efficient acetylating agent for androstane-diols under acidic as well as basic conditions. It provides di-acylated product under acidic conditions and regioselective C-17 mono-acetylated product under basic conditions.

The process of the present invention also avoids the tedious procedure of purification mentioned in the prior art. Hence the process is feasible, economical and industrially applicable.

Another embodiment of the present invention is to provide a process for preparing neuromuscular blocking agents via quaternization of (la) and (lb).

The pure mono- and di-acetylated androstanediols of formula (la) and (lb) obtained by the process of present invention can be quaternized with alkyl bromide (e.g. methyl bromide) or allyl bromide using known procedure to afford neuromuscular blocking agents in 60 - 80% yield with more than 99% purity.

The neuromuscular blocking agents obtained by the process of the invention show desirable pharmacological activity, broad safety margins without toxicity or unfavourable side effects, and may be formulated into a dosage form by combining with one or more pharmaceutically acceptable excipients using known techniques.

Further details of the process of the present invention will be apparent from the examples presented below highlighting the selectivity and yield of the products. Examples presented are purely illustrative and should not be construed as limiting the scope of the invention in any manner.

Examples

Example 1

A clean and dry four neck round bottom flask was charged with 2,16-di(piperidin-l- yl)androstane-3,17-diol (100 gm), acetic acid (300 ml) and p-nitrophenyl acetate (237 gm). The flask was heated to 100 - 1 10°C and maintained for 6 hours under HPLC monitoring. The HPLC analysis of the reaction mixture showed diacetate 98 - 99%, mono acetate 0.1 - 0.3 % and 3,17-diol - nil. The reaction mixture was quenched into water; followed by work-up to obtain 2,16-di(piperidin-l-yl)androstane-3,17-diol 3,17-diacetate.

Yield - 115 gm (97%)

HPLC Purity - more than 99%

Example 2

A clean and dry four neck round bottom flask was charged with 2, 16-di(piperidin- 1 - yl)androstane-3,17-diol (4.58 gm), acetic acid (20 ml) and o-nitrophenyl acetate (10.86 gm). The flask was heated to 100 - 110°C and maintained for 6 hours under HPLC monitoring. The HPLC analysis of the reaction mixture showed diacetate 98 - 99%, mono acetate 0.1 - 0.3% and 3,17-diol - nil. The reaction mixture was quenched into water; followed by work-up provide 2,16-di(piperidin-l-yl)androstane-3,17-diol 3,17-diacetate.

Yield - 5.2 gm (96.6%)

HPLC Purity - more than 99%

Example 3

A clean and dry four neck round bottom flask was charged with 2-(4-morpholinyl)-16- (pyrrolidin-l-yl)androstane-3,17-diol (50.0 gm), triethyl amine (47.58 ml), p-nitrophenyl acetate (91.04 gm) and dichloroethane (500 ml). The flask was heated to 80 - 85°C and maintained for 7 to 8 hours under HPLC monitoring. The HPLC analysis of the reaction mixture showed 17-acetate (mono) 94 - 95%, diacetate 5 - 6% and 3,17-diol 0.2 - 0.3%. The reaction mixture was quenched in water; followed by work-up to obtain 2-(4- mo holinyl)-16-(pyrrolidin-l-yl)androstane-3,17-diol 17-acetate.

Yield - 51.3 gm (94.1%)

HPLC Purity -

1) 17-acetate - more than 98%

2) diacetate - upto 2.0%

The isolated 17-acetate was further crystallized from acetonitrile to achieve more than 99.5% purity.

Example 4

A clean and dry four neck round bottom flask was charged with 2,16-di(piperidin-l- yl)androstane-3,17-diol (5 gm), triethyl amine (6.12 ml), p-nitrophenyl acetate (1 1.36 gm) and dichloroethane (50 ml). The flask was heated to 80 - 85°C and maintained for 8 to 10 hours under HPLC. The HPLC analysis of the reaction mixture showed 17-acetate (mono) 85 - 90%, diacetate 10 - 15% and 3,17-diol 0.2 - 0.3%. The reaction mixture was quenched in water; followed by work-up to obtain 2,16-di(piperidin-l-yl)androstane- 3, 17-diol 17-aceate derivative.

Yield - 5.3 gm (97%)

HPLC

1) 17-acetate - more than 98%

2) diacetate - upto 2.0%

Example 5

A 250 ml round bottom flask was charged with (2β,3α,5α, 16β, 17β)-2, 16-bispiperidino- 3,17-diacetoxy-5-androstane (30 gm) and THF (360 ml) at room temperature. Methyl bromide gas_was purged, reaction mixture was stirred for 48 hours, and followed by conventional work-up to obtain vecuronium bromide.

Yield - 27.8 gm (79%)

HPLC - 99.5% Example 6

A 250 ml round bottom flask was charged with (2β,3α,5α, 16β, 17β)-2, 16-bispiperidino-

3,17-diacetoxy-5-androstane (15 gm) and acetonitrile (180 ml) at 40 °C. Methyl bromide gas was purged, the reaction mixture was stirred for 96 hours, and worked-up to obtain pancuronium bromide.

Yield - 12.55 gm (62%)

Assay - 99.5% (potentiometry)

Example 7

A 250 ml round bottom flask was charged with (2 ,3a,5a,16 ,17P)-2-(4-morpholinyl)- 16-(l-pyrrolidinyl)androstan-3-ol-17-acetate (10 gm), allyl bromide (8.16 ml), distilled water (5.0 ml) and dichloromethane (100 ml) at room temperature. The mixture was stirred for 24 hours; followed by conventional work-up to provide rocuronium bromide. Yield - 9.58 gm (77%)

HPLC - 99.65%