FIELD OF THE INVENTION

The present invention relates to a process for the preparation of Isoxazolyl penicillins from sugarcane juice. More specifically it relates to an in-situ process for the preparation of Isoxazolyl penicillins involving no separate synthesis of intermediates viz Penicillin G and 6- APA.

BACKGROUND OF THE INVENTION

Penicillin compounds are widely used as antibacterial drugs. These compounds consist of a β-lactam ring fused to a thiazolidine ring; hence they are referred as β-lactam antibiotics. Typical examples of penicillins which are commonly used in clinical practice are benzylpenicillin (penicillin G), phenoxylmethylpenicillin (penicillin V), ampicillin and carbenicillin.

Despite of their wide usage as chemo therapeutic usage they suffer a major drawback that some of the microorganisms on which they are acted upon produce β-lactamase which cleave the β-lactam ring of penicillin compounds to produce compounds which are devoid of antibacterial activity. There are certain compounds which are well capable to deal with such problem by inhibiting β-lactamases. These compounds are known as semi synthetic antibiotics or isoxazolyl penicillins in specific which are in the same class as penicillin. Some of the examples of the semi synthetic antibiotics are cloxacillin, dicloxacillin, flucloxaciUin, oxacillin and their respective pharmaceutical salts thereof. There are certain methods in the prior art which produces isoxazolyl penicillin derivatives such as oxacillin, cloxacillin, dicloxacillin or flucloxacillin. Generally, the method to prepare these isoxazolyl penicillins is a multi-step process which involves preparation of intermediates viz Penicillin G and then 6-aminopenicillanic acid which is subsequently followed by isolation of the intermediates and thereby purification and crystallization of the intermediates. After that, 6-aminopenicillanic acid is reacted with a carboxylic acid chloride to form the isoxazolyl penicillin.

Main problem faced during this process is the high usage of power consumption, stability and purity of the intermediates. The industrial scale production of these compounds entails greater investment attributable to a multi-step process. This multi step process involves production of intermediates followed by their addition of an organic solvent to commence the purification and crystallization procedure. Filtration, purification and crystallization procedure are extensive, costly and time consuming processes. This multi step process demands an increased cost of production primarily due to isolation and complex purification methods employed for the intermediates.

Hence, there is a need to provide an in-situ process for the preparation of the isoxazolyl penicillins to avoid these issues of concern. The present invention provides a cost effective, simplified and less laborious in-situ process for the preparation of isoxazolyl penicillins viz cloxacillin, dicloxacillin, flucloxacillin, oxacillin and their respective pharmaceutical salts thereof without isolation of the intermediates Penicillin G and 6-aminopenicillanic acid.

SUMMARY It is an object of the invention to provide a process for the preparation of Isoxazolyl Penicillins from sugarcane juice.

It is another object of the invention to provide an in-situ process for the preparation of Isoxazolyl Penicillins wherein the process is characterized in the steps comprising: preparation of a first reaction mixture comprising Penicillin G, by fermentation of sugarcane juice with a salt of phenylacetic acid to form a product comprising Penicillin G and thereby purification of the product to form the first reaction mixture which is used for the preparation of a second reaction mixture; preparation of second reaction mixture by enzymatic reaction of the first reaction mixture wherein the preparation is characterized in the steps comprising: reacting first reaction mixture with an enzyme to form a solution comprising 6- APA and phenylacetic acid (PAA). Then, adding a first organic solvent in presence of an acidic solution to it at a temperature of 0-40°C to form a first organic layer comprising phenylacetic acid and a first aqueous layer comprising 6- APA. After that, extracting the first organic layer to form a third reaction mixture comprising phenylacetic acid and thereby obtaining the second reaction mixture comprising 6-APA; in-situ addition of a second organic solvent and a carboxylic acid chloride to the second reaction mixture at a temperature of 0-30°C to obtain a second organic layer and a second aqueous layer; and in-situ separation of the second organic layer and addition of a sodium salt complex to form the Isoxazolyl Penicillin. It is yet another object of the invention to provide an in-situ process for the preparation of Isoxazolyl Penicillins wherein the process comprises preparation of a first reaction mixture comprising Penicillin G, then preparation of a second reaction mixture by enzymatic reaction of the first reaction mixture without isolation of Penicillin G and finally, using the second reaction mixture for the preparation of the Isoxazolyl penicillins without isolation of 6-APA from the second reaction mixture.

It is yet another object of the invention to provide a process in which phenylacetic acid (PAA) is recovered and thereby recycled to the fermentation step. This recycling helps in better yield of the Isoxazolyl Penicillins and less consumption of the reactant in fermentation step, hence makes the process cost effective.

It is yet another object of the invention to provide a process for the preparation of Isoxazolyl Penicillins such as cloxacillin, dicloxacillin, flucloxacillin, oxacillin and their respective pharmaceutical acceptable salts thereof.

It is yet another object of the invention to provide a cost effective process for the preparation of isoxazolyl penicillin which requires no isolation step, no crystallization step, and no purification step for intermediates i.e. penicillin G and 6-aminopenicillanic acid formed during the reaction.

DETAILED DESCRIPTION OF THE INVENTION

In the detailed description of the invention, numerous specific details are described to provide a thorough understanding of the various embodiments of the invention. However, one skilled in the relevant art will recognize that an embodiment of the invention can be practiced without one or more of the specific details, or with other apparatus, systems, assemblies, methods, components, materials, parts, and/or the like. In other instances, well-known structures, materials, or operations are not specifically shown or described in detail to avoid obscuring aspects of embodiments of the invention.

The various aspects of the present invention leading to a process for the preparation of sodium salt of various isoxazolyl penicillins are detailed below.

The invention provides to the prior art, an in-situ process for the preparation of beta- lactamase inhibitor semi synthetic antibiotics more specifically it provides a process for the preparation of isoxazolyl penicillins. Isoxazolyl penicillins come in same class as penicillin and are used as penicillinase-resistant penicillin compounds. Penicillinase or beta-lactamase enzyme is an enzyme which is produced by certain strains of bacteria that can deactivate certain types of penicillin antibiotics. Thus, penicillinase-resistant antibiotics such as isoxazolyl penicillins and their pharmaceutical acceptable salts are useful in fighting certain bacterial infections such as Staphylococcus when other penicillins wouldn't work.

Isoxazolyl penicillins such as cloxacillin, dicloxacillin, flucloxacillin, oxacillin and their respective pharmaceutical acceptable salts thereof are used against bacteria that produce beta lactamase or penicillinase, which affects the antimicrobial activity of the antibiotics.

The invention provides a process for the preparation of sodium salt isoxazolyl penicillins such as cloxacillin sodium, dicloxacillin sodium, flucloxacillin sodium, and oxacillin sodium involving in-situ reaction. The term "in situ" is defined herein as performing two or more reaction sequences without isolating any of the intermediates that is produced during the reaction sequence. The invention provides an in-situ process for the preparation of isoxazolyl penicillins, which is quite efficient and cost effective and which proves to generate significant feasibility for an industrial scale production of sodium salt of isoxazolyl penicillins without involving separate preparation of the intermediates like Penicillin G and 6-aminopenicillanic acid. The present invention provides an in-situ process for the preparation of various

Isoxazolyl Penicillins from sugarcane juice in which intermediates like penicillin G and 6- aminopenicillanic acid produced during the reaction are not isolated from the reaction sequence. The process is characterized in the steps comprising: preparation of a first reaction mixture comprising Penicillin G by fermentation of sugarcane juice with a salt of phenylacetic acid to form a product comprising Penicillin G and thereby purification of the product to form the first reaction mixture which is used for the preparation of second reaction mixture wherein the preparation is in-situ; preparation of a second reaction mixture by enzymatic reaction of the first reaction mixture wherein the preparation is in-situ and is characterized in the steps comprising: reaction of the first reaction mixture with an enzyme to form a solution comprising 6-APA and phenylacetic acid (PAA) without isolation of penicillin G from the first reaction mixture. Then, adding a first organic solvent in presence of an acidic solution to it at a temperature of 0-40°C to form a first organic layer comprising phenylacetic acid and a first aqueous layer comprising 6- APA. After that, extracting the second organic layer to form a third reaction mixture comprising phenylacetic acid (PAA) and thereby obtaining the second reaction mixture comprising 6-APA; in-situ addition of a second organic solvent and a carboxylic acid chloride to the second reaction mixture (without isolation of 6-APA) at a temperature of 0-30°C to obtain a second organic layer and a second aqueous layer; and in-situ separating the second organic layer and then adding a sodium salt complex to it to form the Isoxazolyl Penicillin.

In the processes known in the prior art, 6-aminopenicillanic acid (6-APA) is prepared separately and is reacted with carboxylic acid chlorides to form Isoxazolyl penicillins but in the present process, 6-APA is produced in-situ (within the system) by enzymatic reaction of the first reaction mixture comprising penicillin G generated after the fermentation of sugarcane with a salt of phenylacetic acid. This 6-APA is then reacted with the carboxylic acid chlorides to the Isoxazolyl Penicillins. The in-situ preparation avoids the extra cost of purification, crystallization, equipments, storage of separately produced 6-APA, thus this process comes out to be cost effective. During the in-situ preparation of second reaction mixture, phenylacetic acid is recovered from the system which is recycled to the fermentation step. This recycling makes the process cost effective and allows less consumption of the reactant.

In an embodiment of the invention, the preparation of isoxazolyl penicillins is carried out directly from the sugarcane juice.

In an embodiment of the invention, first reaction mixture comprising Penicillin G is prepared by fermentation of sugarcane juice with a salt of phenylacetic acid. In the fermentation process, 2 ^nd generation spore is inoculated into a seed fermentor having sterilized media at a pH of 6 to 6.1 to form a broth. 10% of the broth is transferred to the production fermentor. Into the production fermentor, 50% sugarcane juice and 17% metallic salt of phenylacetic acid is added along with 20% ammonia solution to maintain the pH in range of 6.4 to 6.55 to obtain a product which is allowed to go through the purification steps for the production of the first reaction mixture comprising Penicillin G. In the processes known in prior art, penicillin G is produced from sugar solution. In another embodiment of the invention, the salt of the phenylacetic acid used is the metallic salt and the metal of the metallic salt is selected from the group comprising of sodium, magnesium, aluminum and potassium.

In another embodiment of the invention, the purification of the product produced after fermentation is done by ultra-filtration and nano filtration. The ultra filtration is done to remove the unwanted mycelium and microorganisms from the product. After that, nano-filtration is done to concentrate the purified product to form the first reaction mixture comprising penicillin G.

In an exemplary embodiment of the invention, the ultra filtration is done twice followed by nano filtration to purify and concentrate the product produced after the fermentation step.

In another embodiment of the invention, the enzyme used to react with the first reaction mixture is selected from the group consisting of Penicillin G Acylase or Penicillin Amidase. In present invention, the enzyme used is Penicillin G Acylase and the reaction between the enzyme and the first reaction mixture takes place at a temperature of 28-29°C.

In another embodiment of the invention, the enzymatic reaction of the first reaction mixture takes place at a pH of 6.8 to 8.5 to form a solution comprising 6-APA and PAA. An alkali solution is added to the first reaction mixture along with the enzyme to maintain the pH in range of 6.8 to 8.5. The alkali is selected from the group comprising of sodium bicarbonate, ammonium carbonate, caustic solution and ammonia solution. The solution comprising 6-APA and PAA is allowed to settle and is filtered. The filtrate is then washed with de-mineralized water and is cooled at a temperature of 0-30°C. In present invention the alkali added is 10% ammonia solution. In another embodiment of the invention, the acidic solution is added with the first organic solvent till the pH of the solution reaches in range of 1 to 3.5 and is selected from the group consisting of HC1, H ₂ SO ₄ , Acetic Acid and any other low pH acid. In the present invention, HC1 is added with the first organic solvent to maintain the pH in range of 1 to 3.5.

In another embodiment of the invention, the first organic solvent and the second organic solvent are independently selected from the group consisting of toluene, butyl acetate, methylene chloride, methyl isobutyl ketone, ethyl acetate and any other water immiscible solvent.

In another embodiment of the invention, the carboxylic acid chloride that reacts with the aqueous layer formed after addition of first organic solvent is selected from the group consisting of 3-(2-chlorophenyl)-5-methylisoxazole-4-carbonyl chloride (CMIC chloride), 3-(2,6- dichlorophenyl)-5-methylisoxazole-4-carbonyl chloride (DICMIC chloride), 3-(2-cholor-6- florophenyl)-5methylisoxazole-4 carbonyl chloride (FCMIC chloride), phenyl-5- methylisoxazole-4-carbonyl chloride (PMIC chloride) and any other side chain isoxazoles depending upon the isoxazolyl penicillin to be prepared.

In an exemplary embodiment of the invention, the carboxylic acid chloride used for the preparation of cloxacillin sodium is CMIC chloride. In an exemplary embodiment of the invention, the carboxylic acid chloride used for the preparation of dicloxacillin sodium is DICMIC chloride.

In an exemplary embodiment of the invention, the carboxylic acid chloride used for the preparation of flucloxacillin sodium is FCMIC chloride. In an exemplary embodiment of the invention, the carboxylic acid chloride used for the preparation of oxacillin sodium is PMIC chloride.

In another embodiment of the invention, separation of second organic layer after the addition of second organic solvent is aided with the addition of a salt solution and a solid salt. In the present invention, the salt solution is selected from the group consisting of sodium bicarbonate or any other suitable alkali, whereas the solid salt used is sodium chloride. After the separation of the second organic layer de-moisturization of the second organic layer is done by the addition of sodium chloride. The addition of sodium chloride in excess of saturation point in the mixture of second organic layer and the second aqueous layer helps in removal of excess water in the second organic layer and thereby helps in improving the yield as second organic layer dissolved in second aqueous layer comes out.

In another embodiment of the invention, the sodium salt complex added to the de- moisturized second organic layer to form Isoxazolyl Penicillin is selected from any sodium salt, preferably sodium 2-ethylhexanoate. Sodium salt complex is added till the pH of the solution reaches to 7.5-8.5. The solution obtained after the addition of the sodium salt complex is filtered, washed with ethyl acetate (250*3ml) and finally dried at a temperature of 50-55°C to form the Isoxazolyl Penicillin. In another embodiment of the invention, the process for the preparation of Isoxazolyl Penicillins is an in-situ process in which, neither Penicillin G produced after fermentation of sugarcane with the salt phenylacetic acid in the first reaction mixture is isolated for further reactions, nor 6-aminopenicillanic acid produced after the enzymatic reaction of the first reaction mixture is isolated for further reactions.

In another embodiment of the invention, the yield of the isoxazolyl penicillins is increased by reducing the water content from the second organic layer formed by addition of the second organic solvent. The reduction of water content from the second organic layer is done by recovering the second organic solvent from the second organic layer and adding a lot of salt (sodium chloride) to it.

In general organic layers are soluble in water to some extent. So, by reducing the water content decreases the solubility of the organic layer and thus increases the yield of the product (Isoxazolyl penicillins).

In another embodiment of the invention, the process for the preparation of Isoxazolyl Penicillins involves recycling of third reaction mixture comprising phenylacetic acid to the fermentation step. This recycling of phenylacetic acid helps in better production of Penicillin G which in turns helps producing more 6-APA. This more 6-APA then reacts with the carboxylic acid chloride to produce more Isoxazolyl Penicillin. Also, due to recycling, recovery of the reactant i.e. phenylacetic acid can be done which in turns less consumption of the reactant; hence the whole process becomes a cost effective process.

In an exemplary embodiment of the present invention, cloxacillin sodium is prepared from sugarcane juice. The process comprises: in-situ preparation of a first reaction mixture comprising Penicillin G wherein the process comprises fermentation of sugarcane juice with a salt of phenylacetic acid to form a product and thereby purification of the product by ultrafiltration and nano-filtration to form the first reaction mixture; in-situ preparation of the second reaction mixture by enzymatic reaction of the first reaction mixture to prepare a second reaction mixture comprising 6-APA wherein the preparation comprising: reacting the first reaction mixture with Penicillin acylase enzyme to form a solution comprising 6-APA and phenylacetic acid; adding an alkali to maintain the pH in range of 6.8-8.5 preferably 8; filtering and washing the filtrate; adding a first organic solvent in presence of HC1 to the filtrate to obtain a first aqueous and a first organic layer; extracting the first organic layer comprising phenylacetic acid and recycling it to the fermentation step and thereby obtaining the second reaction mixture comprising 6-APA; adding a second organic solvent and CMIC chloride to the second reaction mixture without isolating 6-APA from it to obtain a second aqueous layer and a second organic layer; adding sodium bicarbonate and sodium chloride to the solution formed for better separation of the second organic layer; de-moisturizing the second organic layer and reacting it with sodium 2-ethylhexanoate to form the cloxacillin sodium.

In another embodiment of the invention, the preparation of the isoxazolyl penicillins is done without isolation of the intermediates i.e. penicillin G and 6-APA at any stage i.e. reaction is carried out in an in-situ manner, which is very useful on commercial scales. It incurs less capital costs since the cost of equipments / machinery used otherwise for purification of the intermediates is completely eliminated. The process for preparation of isoxazolyl penicillins as described in various embodiments of the present invention is achieved by in-situ preparations of intermediates i.e. Penicillin G and 6-APA conferring high purity without the loss of the intermediate during its isolation, purification and crystallization procedure. As a consequence, there is a considerable improvement in the process efficacy in contrast to the preparation process already available in the prior art. Also, the industrial applicability of the process is enhanced significantly since it can be easily set up at an industrial scale without the use of complex purification methods or equipments, which thereby increase the efficiency and substantially reduce the cost of industrial production. It is further mentioned that, if the processes vide the prior art were to be used to isolate, purify, crystallize the Penicillin G and 6-APA formed during the reaction sequence, all or most of the machinery/equipment will need to be used thereby increasing running expenditure on the bulk production of the named products. It is classified here, that the improvements are not limited to just the factors as mentioned herein. Several variations in the processes and the methods herein disclosed will suggest themselves to those skilled in the art. However, it is to be understood that the present disclosure relates to the preferred embodiment of the invention which is for purposes of illustration only and not to be construed as limiting the scope of the invention.

A pharmaceutical composition containing the product obtained according to the process of the invention has no need to be formulated with additional auxiliaries.

The present invention will now be illustrated in greater detail with reference to Examples, but the present invention should not be interpreted as being restricted thereto. EXAMPLES: Example 1

Preparation of Cloxacillin Sodium

6.2ml of 2nd generation spore was inoculated into a seed fermentor and 16L media is prepared for it at a pH of 5.9-6.1. Batch was matured till log 55 hr with pH 5.25 and 10% of it was transferred to the production fermentor. In the production fermentor 100L media was prepared at a pH of 6.9. At temperature 25C matured seeds were transferred to it. From log 3 hr onwards, 41 L of 50% sugarcane juice was fed along with 1.4 L of 17% sodium phenylacetate into the production fermentor. From log 4 hr onwards 20 % ammonia solution was added to maintain the pH in the range of 6.4 to 6.55 to obtain a product comprising penicillin G. After ultra- filtration and nano-filtration the concentration of the product was made to 31 mg/ml to form the first reaction mixture. 50gm equivalent of first reaction mixture comprising Penicillin G was dissolved in 450ml of de-mineralized water. At 28-29°C Penicillin acylase enzyme was added. The pH was maintained at around 8 by the addition of 10% ammonia solution. Reaction was continued till completion. The solution was allowed to settle for 5 min and then filtered. The filtrate was then washed with 90ml de-mineralized water. The temperature of the resulting solution was then cooled to 4-5°C and 170ml of toluene was added in presence of HC1 at a pH in range of 1-1.4 to obtain a solution comprising 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). The solution obtained was stirred for 5 min to form a first organic layer and a first aqueous layer. The first organic layer comprising PAA was separated to form a third reaction mixture and recycled into the system whereas the first aqueous layer was filtered and wash with 10ml de-mineralized water to form the second reaction mixture. The temperature of the solution was maintained at 0-5°C and 400ml ethyl acetate and 31.5gm of CMIC chloride were added to the filtered/washed second reaction mixture. The pH of the solution was raised to 1.85 with 9% NaHC0 ₃ solution in 2 hrs and then further raised to 2.4 in next 2 hrs. After the completion of 4hrs, NaCl was dissolved to obtain a second aqueous layer and a second organic layer. The second organic layer was then separated. After the separation of the second organic layer, ethyl acetate is recovered and NaCl is added to the second organic layer to remove the excess water content. This removal of water decreases the solubility of the product and thereby increases the yield. The temperature was raised to 15-18°C and 50-60 ml of 50% Sodium 2- ethylhexanoate was added to the de-moisturized second organic layer till the pH of the final solution reaches to 8-8.2 to get the solution of cloxacillin sodium. The resultant solution was then filtered, washed with ethyl acetate (250ml*3) and dried at a temperature of 50-55°C. The yield of the Cloxacillin sodium obtained was more than 50gm. The product obtained complies with the pharmacopeia requirement.

RAW MATERIALS AND THEIR INPUTS REQUIRED FOR EXAMPLE 1

Raw material Input

2 ⁿ Generation spore 6.2ml

Media 16L

50 % Sugarcane juice 20L

17% Phenylacetic acid 4L

20% Ammonia solution (Till pH is adjusted) De-mineralized water 550 ml

Toluene 170ml

30% HC1 44 ml

Enzyme lOOgm (wet)

Ethyl acetate 1 150ml

CMIC chloride 31.5gm

Sodium chloride 70 gm

10% NH ₃ (Ammonia) 27-30ml

9% NaHC0 240-250ml

50% Sodium 2-ethylhexanoate 50-60ml

Yield 50gm-minimum