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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.
5 BACKGROUND OF THE INVENTION
Amoxicillin is a moderate-spectrum, bacteriolytic, p-Iactam 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 p-Iactam antibiotics. Amoxicillin is one of the most common antibiotics
10 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
15 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
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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. inJLuenzae, 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 multistep
process involving preparations of penicillin G and then 6-aminopenicillanic acid
which is subsequently followed by intermediate process steps viz isolation,
10 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
lS 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.
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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
5 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.
10 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
15 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-
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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
5 thereby obtaining the second reaction mixture comprising 6-APA; and
in-situ reaction of the second reaction mixture with a parahydroxyphenylglycine
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
10 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.
15 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
20 the preparation of the amoxicillin trihydrate which requires no isolation steps, no
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crystallization step, and no purification step for intermediates i.e. penicillin G and 6aminopenicillanic
acid formed during the reaction sequence.
DETAILED DESCRIPTION OF THE INVENTION
In the detailed description of the invention, numerous specific details are
5 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
10 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
15 of a beta-Iactam 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.
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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:
5 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
10 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
15 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
20 trihydrate.
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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
5 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 Le. within the system. Thus the
process of the present invention comes out to be a cost effective and an efficient
10 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
15 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, 2nd 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
20 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
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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
S 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
10 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.
lS 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
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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
5 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 HCI, H2S04, Acetic Acid and any other low pH
acid. In the present invention, HCI is added with the organic solvent to maintain the
10 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
15 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 1O-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
20 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
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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
5 the enzyme and dissolving the filtrate in aqueous HCl 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.
10 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
15 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
20 the second reaction mixture during the reaction sequence at any stage. Due to this, the
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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
5 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
10 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
15 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
20 consequence, there is a considerable improvement in the process efficacy in contrast
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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 costeffective.
The industrial applicability of the process is also enhanced significantly
5 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.
10 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
15 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
20 the running expenditure of the overall process. The in-situ process of the present
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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
5 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
10 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.
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EXAMPLES:
Example 1
Preparation of Amoxicillin trihydrate
6.2ml of 2nd generation spore for production of penicillin G was inoculated
5 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
10 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
15 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% HCI at a pH in range of 1-1.4 to form an intermediate
20 comprising 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). The
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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 lOml de-mineralized water to form the second reaction mixture. 50gm
5 equivalent of 6-APA (second reaction mixture) was charged at a temperature of 1015°
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-hydroxyphenylglycine (PHPG) ester
dissolved in lOamI 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
10 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-
15 85ml approx) to get the amoxicillin trihydrate solution. Conversion check was done
after everyone 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 HCl at a temperature of 23-25°C to remove the
20 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
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stirred for 1.5 hrs and then filtered at a temperature of a-5°C. The amoxicillin
trihydrate solution was washed with lOamI of water followed by 50ml of Isopropyl
alcohol, and then dried in TD to obtain 83-86gm of amoxicillin trihydrate salt.

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CLAIMS
We Claim:
1. A process for the preparation of Amoxicillin trihydrate wherein the process is
characterized in the steps comprising:
5 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-
20 hydroxyphenylglycine derivative m presence of the enzyme to obtain
amoxicillin trihydrate. ~
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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
5 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.
10 6. The process according to claim 1, wherein the acidic solution is selected from the
group consisting of HCl, 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
15 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
20 derivative is an ester or an amide.`