WO2014128538A1

WO2014128538A1 - A process for the preparation of amoxicillin trihydrate

Info

Publication number: WO2014128538A1
Application number: PCT/IB2013/056612
Authority: WO
Inventors: Rajinder Singh Gujral
Original assignee: Vardhman Chemtech Limited
Priority date: 2013-02-22
Filing date: 2013-08-13
Publication date: 2014-08-28

Abstract

The invention relates to an in-situ process for the preparation of amoxicillin trihydrate from sugarcane juice without isolation of the intermediates produced during the reaction sequence viz penicillin G and 6-aminopenicillanic acid. The invention provides an in-situ cost effective and environment friendly process in which no separate synthesis of the intermediates, no purification cost and no crystallization cost for the intermediates is required.

Description

A PROCESS FOR THE PREPARATION OF AMOXICILLIN TRIHYDRATE

FIELD OF THE INVENTION

The present invention relates to a process for in-situ preparation of Amoxicillin trihydrate from sugarcane juice. BACKGROUND OF THE INVENTION

Amoxicillin is a moderate-spectrum, bacteriolytic, β-lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms. It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics. Amoxicillin is one of the most common antibiotics prescribed for children. Amoxicillin is used to treat many different types of infections caused by bacteria, such as ear infections, bladder infections, pneumonia, gonorrhea, and E. coli or salmonella infection. Amoxicillin is also sometimes used together with another antibiotic called clarithromycin (Biaxin) to treat stomach ulcers caused by Helicobacter pylori infection. This combination is sometimes used with a stomach acid reducer called lansoprazole (Prevacid). They do not kill bacteria, but they stop bacteria from multiplying by preventing bacteria from forming the walls that surround them. The walls are necessary to protect bacteria from their environment and to keep the contents of the bacterial cell together. Bacteria cannot survive without a cell wall. Amoxicillin is effective against many different bacteria including H. influenzae, N. gonorrhoea, E. coli, Pneumococci, Streptococci, and certain strains of Staphylococci. There are certain methods in the prior art to prepare amoxicillin trihydrate. Generally, it is prepared by reaction of 6-aminopenicillanic acid (6-APA) with a hydroxyphenyl glycine salt. The method to prepare amoxicillin trihydrate is a multi-step process involving preparations of penicillin G and then 6-aminopenicillanic acid which is subsequently followed by intermediate process steps viz isolation, purification and crystallization of penicillin G and 6-APA.

These intermediate processing steps requires high usage of power consumption, machinery, water consumption, solvents, etc. The industrial scale production of these compounds entails greater investment attributable to a multi-step chemical process. This multi step process involves production of an intermediate followed by its 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 intermediate.

Hence, there is a need to provide an in-situ process for the preparation of the amoxicillin trihydrate from sugarcane juice which requires no intermediate processing steps. The present invention provides a cost effective, simplified and less laborious in-situ process for the preparation of amoxicillin trihydrate without isolation of the intermediates Penicillin G and 6- aminopenicillanic acid from the reaction sequence.

SUMMARY It is an object of the invention to provide a process for the preparation of Amoxicillin trihydrate from sugarcane juice. It is another object of the invention to provide an in-situ process for the preparation of Amoxicillin trihydrate, wherein the process is characterized in the steps comprising: in-situ 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; in-situ enzymatic reaction of the first reaction mixture to prepare a second reaction mixture wherein the preparation is characterized in the steps comprising: reacting first reaction mixture with an enzyme to form an intermediate comprising 6-APA and phenylacetic acid (PAA). Then, adding an organic solvent in presence of an acidic solution to it at a temperature of 0-40°C to form an organic layer comprising phenylacetic acid and an aqueous layer comprising 6-APA. After that, extracting the organic layer to form a third reaction mixture comprising phenylacetic acid and thereby obtaining the second reaction mixture comprising 6-APA; and in-situ reaction of the second reaction mixture with a para-hydroxyphenylglycine derivative at a temperature of 0-30°C in presence of the enzyme to obtain the amoxicillin trihydrate.

It is yet another object of the invention to provide an in-situ process for the preparation of Amoxicillin trihydrate, 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 Amoxicillin trihydrate 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 less consumption of the reactant in fermentation step as well as makes the process cost effective and improves the overall efficiency of the system. It is yet another object of the invention to provide a cost effective process for the preparation of the amoxicillin trihydrate which requires no isolation steps, no crystallization step, and no purification step for intermediates i.e. penicillin G and 6-aminopenicillanic acid formed during the reaction sequence.

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 amoxicillin trihydrate detailed below.

The invention provides to the prior art, an in-situ process for the preparation of a beta- lactam antibiotic more specifically it provides a process for the preparation of amoxicillin trihydrate involving in-situ reaction. The term "in situ" is defined herein as performing two or more reaction sequences without isolating any of the intermediate that is produced during the reaction sequence.

The present invention provides an in-situ process for the preparation of Amoxicillin trihydrate from sugarcane juice in which intermediates produced during the reaction are not isolated from the reaction sequence. The process is characterized in the steps comprising: in-situ 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; in-situ enzymatic reaction of the first reaction mixture to prepare a second reaction mixture wherein the preparation is characterized in the steps comprising: reaction of the first reaction mixture with an enzyme to form an intermediate comprising 6-APA and phenylacetic acid (PAA) without isolation of penicillin G from the first reaction mixture. Then, adding an organic solvent in presence of an acidic solution to it at a temperature of 0-40°C to form an organic layer comprising phenylacetic acid and an aqueous layer comprising 6-APA. After that, extracting the organic layer to form a third reaction mixture comprising phenylacetic acid (PAA) and thereby obtaining the second reaction mixture comprising 6-APA; and in-situ reaction of the second reaction with a para-hydroxyphenylglycine derivative at a temperature of 0-30°C in presence of the enzyme to obtain amoxicillin trihydrate.

In the processes known in the prior art, penicillin G is prepared separately from sugar from which, 6-aminopenicilaanic acid (6-APA) is prepared separately. These preparations require separate isolation, purification, crystallization steps etc. Then, the 6-APA is reacted with a para-hydroxyphenylglycine derivative to form the amoxicillin trihydrate. But, in the present process, preparation of amoxicillin is carried out directly from the sugarcane juice wherein no intermediate isolation, purification, and crystallization steps are required for intermediates viz penicillin G and 6-APA. These intermediates are produced in-situ i.e. within the system. Thus the process of the present invention comes out to be a cost effective and an efficient process.

In an embodiment of the invention, the invention provides an in-situ process which avoids extra cost of purification, crystallization, equipments. During the in-situ preparation of second reaction mixture, phenylacetic acid is extracted from the system which can be recycled into the system. The recycling allows less consumption of phenylacetic acid and thereby makes the process cost effective.

In an embodiment of the invention, preparation of first reaction mixture comprising Penicillin G is done by fermentation of sugarcane juice with a salt of phenylacetic acid. In this 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 then transferred to the production fermentor. After that, 50% sugarcane juice and 17% metallic salt of phenylacetic acid is added into the production fermentor along with 20% ammonia solution to maintain the pH in range of 6.4 to 6.55 to obtain a product. The product is then, allowed to go through the purification steps for the production of the first reaction mixture comprising Penicillin G. 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, potassium, magnesium, and aluminum. 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, whereas 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 is selected from the group consisting of Penicillin G Acylase or Penicillin Amidase.

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 an intermediate 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, ammonia solution or any other alkali. The intermediate 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 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, H₂SO₄, Acetic Acid and any other low pH acid. In the present invention, HC1 is added with the organic solvent to maintain the pH in range of 1 to 3.5.

In another embodiment of the invention, the organic solvent is 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 second reaction mixture is reacted with para- hydroxyphenylglycine (PHPG) derivative in presence of the enzyme to form amoxicillin trihydrate. In this process, water is added to the second reaction mixture and the pH of the solution is maintained in range of 7.2-7.5 by the addition of 10-12% ammonia, at a temperature of 10-15°C. To this solution, a first lot of PHPG derivative is added along with the enzyme at a pH of 6.25-6.4 to form a solution of amoxicillin trihydrate. The pH is set by the addition of ammonia solution. The conversion of second reaction mixture to amoxicillin trihydrate is checked after each hour. Then a second lot of PHPG derivative is added at the same pH and conversion is checked again. After that a third lot of PHPG derivative is added and the reaction is carried out till the conversion reaches more than 95%. Then, isolation of amoxicillin trihydrate is done by filtering out the amoxicillin trihydrate layer from the enzyme and dissolving the filtrate in aqueous HC1 at a temperature of 23-25°C to remove the traces of the enzyme. Finally, isopropyl alcohol is added to the solution at a pH of 5.2-5.3. The pH is set by adding ammonia solution. Then, amoxicillin trihydrate solution is filtered, washed with Isopropyl alcohol, and dried to get amoxicillin trihydrate salt.

In another embodiment of the invention, the para-hydroxyphenylglycine derivative that reacts with the second reaction mixture is an ester or an amide.

In another embodiment of the invention, the process for the preparation of Amoxicillin and its pharmaceutical acceptable salts involves optional recycling of third reaction mixture comprising phenylacetic acid to the fermentation step. The recycling helps in less consumption of phenylacetic acid and better production of Penicillin G which in turns helps producing more 6-APA. In another embodiment of the invention, the process for the preparation of Amoxicillin and its pharmaceutical acceptable salts is an in-situ process. The process requires no isolation of Penicillin G from the first reaction mixture and 6-APA from the second reaction mixture during the reaction sequence at any stage. Due to this, the process is very useful on commercial scales. It incurs less capital costs since the cost of equipments / machinery used otherwise for purification, crystallization, etc of the intermediates is completely eliminated.

In another embodiment of the invention, the process for the preparation of the amoxicillin salt is quite efficient and cost effective. Also, it proves to generate significant feasibility for the industrial scale production of amoxicillin and pharmaceutical salt there without involving separate preparation of intermediates produced during the reaction sequence. Further, the process of invention results in saving a lot water during the process as opposed to the processes in the prior art that require lot of water in-between. In fact, lowering of water consumption starts with Cane Juice as no water is added to make Sugar solution in Pencillin G. The water in the process can be recycled, thus no special effluent treatment plant are required after every step. Also, there is no need of producing and crystallizing the Sugar during the process as Penicillin G is directly produced from sugarcane juice which helps in saving lot of power.

The process for preparation of amoxicillin as described in various embodiments of the present invention is achieved by in-situ preparations of intermediates viz Penicillin G and 6- APA, conferring high purity without the loss of the intermediates during isolation, purification and crystallization procedure. As a consequence, there is a considerable improvement in the process efficacy in contrast to the processes already available in the prior art. Also, the process of the invention eliminates the requirement of lot of chemicals required to isolate Sugar, Pencillin G, 6-APA as the process of the invention is in-situ, thus making the process cost-effective. The industrial applicability of the process is also 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. Further, the above discussed advantages of the invention make the process green; thereby help the environment in a great deal.

In another embodiment of the invention, the same process can be used to prepare ampicillin by replacing PHPG ester/amide to phenylglycine ester/amide. Ampicillin comes under same class as that of amoxicillin. In terms of spectrum and level of activity, amoxicillin is roughly treated as the successor of ampicillin. The present invention also provides an in-situ process for the preparation of ampicillin by reacting the second reacting mixture with a phenylglycine derivative in presence of the enzyme.

It is further mentioned that, if the processes of isolation, purification, crystallization of the named products (Penicillin G and 6-APA) formed in between were to be used, a lot of machinery/equipment will need to be used thereby increasing the running expenditure of the overall process. The in-situ process of the present invention avoids these un-necessary steps and hence makes the process cost effective. 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 Amoxicillin trihydrate

6.2ml of 2nd generation spore for production of penicillin G 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 the ultra-filtrations 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 taken for reaction. At 28-29°C Penicillin acylase enzyme was added. The pH was maintained at around 8 by the addition of 10% ammonia solution. The solution was allowed to settle for 5 min and then filtered. The filtrate (Enzyme) 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 30% HC1 at a pH in range of 1-1.4 to form an intermediate comprising 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). The intermediate obtained was stirred for 5 min to form an organic layer and an aqueous layer. The organic layer comprising PAA was separated to form a third reaction mixture and recycled into the system whereas the aqueous layer was filtered and wash with 10ml de- mineralized water to form the second reaction mixture. 50gm equivalent of 6-APA (second reaction mixture) was charged at a temperature of 10-15°C and a pH of 7.2-7.5. The pH was maintained by adding 40-45ml of 10-12% ammonia solution. Then, 37.5 gm of para- hydro xyphenylglycine (PHPG) ester dissolved in 100ml of water was added to it. After that, 85- 88gm of penicillin G acylase enzyme was added to it to start the reaction at a pH of 6.25-6.4. The pH was maintained by the addition of 20-25ml of 10-12% ammonia solution. Then, second lot of PHPG ester (18.75gm) in 50ml of water was added to it at the pH of 6.25-6.4 and conversion of 6-APA to amoxicillin trihydrate was checked. Finally, a third lot of PHPG ester (18.75gm) in 50ml of water was added to the solution and same pH of 6.25-6.4 was maintained throughout by the addition of the ammonia solution (80-85ml approx) to get the amoxicillin trihydrate solution. Conversion check was done after every one hour of the reaction and the reaction was carried out till the conversion reaches to 95%. Then, isolation of amoxicillin trihydrate was done by filtering out the amoxicillin trihydrate layer from the enzyme and dissolving the filtrate in 125-130ml of 15% aqueous HC1 at a temperature of 23-25°C to remove the traces of the enzyme. Finally, 140- 150ml isopropyl alcohol was added to the solution at a pH of 5.2-5.3. The pH was set by adding the ammonia solution. Solution was stirred for 1.5 hrs and then filtered at a temperature of 0- 5°C. The amoxicillin trihydrate solution was washed with 100ml of water followed by 50ml of Isopropyl alcohol, and then dried in TD to obtain 83-86gm of amoxicillin trihydrate salt.

Claims

CLAIMS We Claim:

1. A process for the preparation of Amoxicillin trihydrate wherein the process is characterized in the steps comprising:

a) in-situ preparation of a first reaction mixture comprising penicillin G from sugarcane juice by fermentation of the sugarcane juice with a salt of phenylacetic acid to obtain a product comprising Penicillin G and thereby purification of the product to form the first reaction mixture;

b) in-situ enzymatic reaction of the first reaction mixture to prepare a second reaction mixture, wherein the characterized in the steps comprising:

i) reacting the first reaction mixture with an enzyme to form an intermediate comprising 6-aminopenicillanic acid and phenylacetic acid;

ii) adding an organic solvent in presence of an acidic solution to the intermediate to form an organic layer and an aqueous layer; and

iii) extracting the organic layer to form a third reaction mixture comprising phenylacetic acid and thereby obtaining the second reaction mixture comprising 6-aminopenicillanic acid; and

c) in-situ reaction of the second reaction mixture with para-hydroxyphenylglycine derivative in presence of the enzyme to obtain amoxicillin trihydrate.

2. The process according to claim 1, wherein the salt of phenylacetic acid in step (a) is a metallic salt and wherein the metal of the metallic salt is selected from the group comprising of sodium, potassium, magnesium, and aluminum.

3. The process according to claim 1, wherein the purification of the product obtained after fermentation is done by ultra-filtration followed by nano-filtration.

4. The process according to claim 1 , wherein the enzymatic reaction in step (i) takes place at a pH of 6.8-8.5.

5. The process according to claim 1 , wherein the enzyme used is selected from Penicillin G Acylase and Penicillin Amidase.

6. The process according to claim 1, wherein the acidic solution is selected from the group consisting of HC1, Acetic Acid, Sulphuric acid and any low pH acid.

7. The process according to claim 1 , wherein the acidic solution is added till the pH reaches in the range of 0.3-4.5.

8. The process according to claim 1, wherein the organic solvent is selected from the group consisting of toluene, Butyl acetate, Methylene chloride, Methyl isobutyl ketone, Ethyl acetate and any other water immiscible solvent.

9. The process according to claim 1, wherein the third reaction mixture is recycled into the step (a).

10. The process according to claim 1, wherein the para-hydroxyphenylglycine derivative is an ester or an amide.