FORM 2
THE PATENT ACT 1970 (39 of 1970)
& The Patents Rules, 2003 COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
"An improved process for preparation of penem compounds"
2. APPLICANT (S)
(a) NAME: Genesen Labs Ltd
(b) NATIONALITY: An Indian Company incorporated under the Indian Companies ACT 1956
(C) ADDRESS:
Genesen Labs Ltd
R-75, TTC industrial area, Thane- Belapur road ,
Rable Navi Mumbai. 400 701.
Tel No. +91-22- 66888700/1 Fax No. +91-22-66888730.
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

An improved process for preparation of penem compounds.
Field of Invention:
This invention mainly relates to an improved process for preparation of penem compounds like meropenem and it's intermediate. The pharmaceutically active agent, meropenem is mainly effective against pneumonias, gynecological insertions skin and soft structure infection, meningitis, ceptricaemia and adult fertile neutrophenila .Structure-1 represent the meropenem trihydrate.

Furthermore this invention relates to the key intermediate of meropenem structure -II

Wherein PNB represent P-nitro benzyl group and PNZ represent P- nitrobenzyloxycarbonyl group.
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Background of Invention:
(4R,5S,6S)-3-[[(3S,5S)-5-(Dimethylcarbamoyl)-3-pyrrolidinyl]thio]-6-[(lR)-l- hydroxy- ethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (referred as title compound), commonly known as meropenem (Structure- I) . Meropenem is used as antibiotic agent in the treatment of pneumonia, urinary tract infections, intra-abdominal, gynaecological, skin, and soft tissue infections, meningitis, septicemia and febrile neutropenia. In USA it is sold under the trade name of MERREM® LV. (meropenem for injection). MERREM is a sterile, pyrogen-free, synthetic, broad spectrum, carbapenem antibiotic for intravenous administration. In view of the importance of the compound of the structure-1, several synthetic procedures to prepare the compound have been reported. Meropenem firstly disclosed in US-4933,333 by Merck with it's therapeutic indications.
US patent, US 4,888,344 provides crystalline Meropenem trihydrate along with non-toxic carbonate composition, according to this patent, Meropenem was obtained by deprotecting the protecting groups of the penultimate compound by hydrogenation as per the following scheme.

Where R = PNB (p-nitrobenzyl) or allyl
R' = PNZ (p-nitrobenzyloxycarbonyl) or allyloxycarbonyl
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From above said process hydrogenation carried out under 50 Kg pressure and 10 % PDC in water and tetra hydrofuron a biphasic system, finally by column chromatography title compound was obtained.
From above said process it is very tedious due to the purification of title compound by column chromatography and furthermore process is not commercially feasible due to reaction parameter like 50 kg pressure, which is very difficult to attain and high cost of 10 % palladium on carbon. Beside the above prior art, our preferred embodiment is ,hydrogenation carried out 9-10 kg pressure in a biphasic solvent system with a simple method of isolation exerts desired yield and purity, which has been elaborately described in forgoing example.
In WO/2005/118586 patent, protected meropenem prepared by condensation of diphenyl phosphonate and mercapto pyrrolidine at temp -10 °C gives protected meropenem which furthermore crystallize with ethyl acetate yields pure crystalline meropenem with 73 % yield.
According to above said publication this intermediate is crystallized out either from concentrating the mother liquor in alkyl alkanoate such as ethyl acetate or by the addition of anti-solvent such as cyclohexane or heptane to the mother liquor in ethyl acetate. Since this patent describes the use of multiple solvent systems, the process is not commercially viable from industrial point of view owing to multiple solvent recoveries, adding further the cost of production . Furthermore process is not cost effective due low yield and unfriendly reaction parameter.
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In our preferred embodiment, protected meropenem prepared by condensing diphenyl phosphonate (compound A) and mercapto pyrrolidine (compound B) at 20 °C for 1 hour in acetonitrile exert a good yield around 99 % with purity above 97 %.
US patent 4,943,569 claims Meropenem and process for its preparation. This patent utilizes buffer like morpholinoalkyl sulfonic acid (MOPS) or its salt during deprotection stage. The description also discloses that the penultimate intermediate (diprotected Meropenem) of Meropenem can be isolated by organochemical means.
According to the above said patents, Meropenem trihydrate was obtained by subjecting the aqueous reaction mass obtained after the deprotection of protecting group, reverse osmosis, followed by adding water-miscible organic solvent such as ethanol, iso-propanol, acetone, tetrahydrofuran (THF), dioxane, acetonitrile, etc.
In our preferred embodiment protected meropenem is prepared by simple condensation of compound A(4-nitrobenzyl (4R,5R,6S)-3-[(diphenoxyphosphoryl)oxy]-6-[(lR)-l -hydroxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate) and compound B(4-nitrobenzyl (2S',4S)-2-[(dimethylamino)carbonyl]-4- mercaptopyrrolidine-1-carboxylate) at temperature 20 °C and concomitantly deprotection is carried out under 9-10 kg pressure with simple isolation over the existing prior art.
From all above prior arts, the processes reported of meropenem trihydrate which involves lyophilization of an aqueous layer containing meropenem. The amorphous material so obtained is crystallized to get meropenem trihydrate. Alternatively the process involves column
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chromatographic purification of reaction product followed by concentration of the aqueous layer by reverse osmosis. To the concentrated aqueous layer, acetone or tetrahydrofuran or isopropanol is added for the precipitation of meropenem trihydrate. The yield of meropenem trihydrate thus obtained is generally low, and the purity is variable so, there is scope to develop an improved and efficient process for sterile meropenem without lyophilization.
Object of Invention:
The main object of the present invention is to provide the improved method for preparation of meropenam trihydrate which is commercially feasible and cost effective with desired purity and yield. Another object of said invention is to provide simple method of isolation for meropenem trihydrate over the existing prior art.
Summary of Invention:
The present invention mainly concern with industrially feasible,cost effective concomitant with green chemistry process for preparation of meropenem trihydrate. The invented process has added advantage of the improved yield and purity of meropenem trihydrate.
This method further provides substantial benefits relative to previously used or suggested production methods. For example, the starting materials, intermediates, liquid media, and catalysts used are relatively easy to handle and to dispose off, if necessary. Importantly, the present method provides high yields of the desired product or products so that substantial process efficiencies are achieved.
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The present invention carried out in two steps which has been briefly summarize below and elaborately in forgoing example.
Step-I
Preparation of protected Meropenem.
Charged 1 mole compound A (4-nitrobenzyl (4R,5R,6S)-3-[(diphenoxyphosphoryl)oxy]-6-[(lR)-l -hydroxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate) and 1.03 mole compound B (4-nitrobenzyl (2S',45)-2-[(dimethylamino)carbonyl]-4- mercaptopyrrolidine-1-carboxylate) in acetonitrile at 20 °C furthermore added 3.13 mole di-isopropylethylamine in dropwise manner at same temperature, continued the reaction up to completion monitored by HPLC . After completion of reaction added ethyl acetate and water and finally washed organic layer by sodium chloride solution and distilled out to get protected Meropenem compound which furthermore after usual treatment obtained pure protected Meropenem (4-Nitrobenzyl (4R,5S,6S)-(3-{(3S,5S)-5- [(dimethylamino)carbonyl]-l-[(4-nitrophenoxy)carbonxyl] pyrrolidin-3-yl}thio-6-[(lR)-l-hydorxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0].hept-2-ene-2-carboxylate) compound.
Step-II
Preparation of sterile Meropenem trihydrate
The purified protected meropenem(4-Nitrobenzyl (4R,5S,6S)-3-({(3S,5S)-5-
[(dimethylamino)carbonyl]-l-[(4-nitrophenoxy)carboxyl] pyrrolidin-3-yl }thio)-6- [(1R)-1-
hydorxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0].hept-2-ene-2-carboxylate) obtained from previous stage charged in pressure hydrogenetor with biphasic solvent system such as water : methyl-t-butyl ether+ N- methyl formamide or
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water : methyl-t-butyl ether+ N,N -dimethyl acetamide with 5 % palladium on activated charcoal at hydrogen pressure 9-10 Kg up to completion of reaction monitored by HPLC .
After completion of reaction, aqueous layer extracted with methyl-t-butyl ether+ N- methyl formamide or methyl-t-butyl ether+ N,N -dimethyl acetamide and added acetone dropwise in aqueous layer at controlled temperature 5-8 °C, obtained the title compound (4R,5S,6S)-3-[[(3S,5S)-5-(dimethylcarbamoyl)-3-pyrrolidinyl]thio]-6-[(lR)-l- hydroxy- ethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid).
Details description of invention:
In our preferred embodiment, the above said method is commercially viable with green chemistry. The method comprising the condensation of compound A(4-nitrobenzyl (4R,5R,6S)-3-[(diphenoxyphosphoryl)oxy]-6-[(lR)-l -hydroxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate) with compound B (4-nitrobenzyl (2S',4S)-2-[(dimethylamino)carbonyl]-4-mercaptopyrrolidine-1-carboxylate) at temperature 20 °C to produce protected meropenam with desired purity and yield, which has been schematically represented as follow.
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In our scope of invention , the condensation reaction carried out at 20 °C added advantage of desired yield above 99% with purity above 98 % as such type of condensation not covered in any prior art
In deprotection of protected meropenem hydrogenation carried out under pressure 9-10 kg for 3 hours with palladium on activated charcoal at pH 7.0 to obtain meropenem trihydrate . Isolation of meropenem trihydrate from hydrogenated reaction mass comprises:
1) washed the aqueous layer twice by methyl-t-butyl ether+ N- methyl formamide so as to remove impurity p-touludine and colour impurity.
2) Aqueous layer subjected to under distillation to get clear solution for filtration purpose through micron filters.
3) Cooled the reduced aqueous layer up to 5-8 °C and added cold acetone and sodium bi¬carbonate solution without exceeding the temperature and pH 7.0 to precipitate out meropenem trihydrate and finally washed by cold acetone.
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The compound A and compound B dissolved in acetonitrile and stirred for 10 minutes at temperature 20 °C further to this reaction mass, added di-isopropylamine dropwise with controlled temperature and continued the reaction at the same temperature for about 50 minutes up to completion. The reaction mixture poured in mixture of ethyl acetate, water and 2N hydrochloric acid .After separating the layer, organic layer washed by phosphate buffer and sodium chloride solution, distilled off the organic layer to get solid of protected meropenem. Furthermore purification with water at 5 °C and subsequently purified by methanol and obtained purified protected meropenem.
In our scope of invention, hydrogenation carried out in a biphasic system(water: methyl-t-butyl ether+ N- methyl formamide) offers several advantages. Firstly, side products such as p-toluidine and coloring impurities remain in the organic layer whereas deprotected meropenem is present in aqueous layer. Secondly, isolation of meropenem from aqueous layer is easy, as removal of impurities present in organic layer is facilitated by layer separation. Use of sodium bicarbonate
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solution as a buffer offers advantage in terms of cost and availability. The buffer system also serves as a tool for carrying out the reaction at a pH of about 7.
After completion of reaction, the aqueous layer containing the product was washed with an organic solvent and combined aqueous layers was added to miscible organic solvent so as to precipitate meropenem trihydrate from the solution thereof. The precipitation carried out at lower temperature of about 5-8°C. The separated meropenem trihydrate is then isolated by means of filtration or centrifuge and dried suitably to get pure meropenem trihydrate having desired purity. Furthermore for sterile meropenem, after hydrogenation aqueous layer containing meropenem concentrate to get clear solution which can be easily filtered through series of .45 \x and .2 u filter paper under aseptic condition and addition of sterile acetone under the same condition obtained sterile meropenem trihydrate.
Following examples are offered to provide the person skill in the art with sufficiently clear and complete explanation of this invention, but should not consider as a limitation to the essential aspect of the object thereof, as they have been explained in below.
Example-I
Preparation of protected meropenem.
4-nitrobenzyl(4R,5R,6S)-3-[(diphenoxyphosphoryl)oxy]-6-[(lR)-l -hydroxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10 gm) and 4-nitrobenzyl (2S,4S)-2-[(dimethylamino)carbonyl]-4- mercaptopyrrolidine-1-carboxylate (6.122 gm) was dissolved in
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acetonitrile at 20 °C. The solution was stirred under same condition for 10 minutes to get clear
solution .Di-isopropylamine(9.15ml)was added dropwise under stirring condition to above solution
at controlled temperature and continued the reaction for 50 minutes at same reaction parameter.
After completion of reaction ,added 150 ml ethyl acetate , 75 ml water and 10 ml 2N hydrochloric
acid to above reaction mass , separated the layer and washed organic layer by 50 ml phoshate
buffer and 50 ml of 25 % sodium chloride solution ,dried over sodium sulphate and distilled off up
to free solid .Added 200 ml water at ambient temperature and stirred for 15 minutes ,cool to 5 °C.
and added 50 ml methanol at same reaction parameter for 1 hour, filtered the solid and washed with
10 ml chilled water to obtain pure 4-Nitrobenzyl (4R,5S,6S)-3-({(3S,5S)-5-
[(dimethylamino)carbonyl]-l-[(4-nitrophenoxy)carbonxyl]pyrrolidin-3-yl}thio)-6-[(lR)-l-hydorxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0].hept-2-ene-2-carboxylate (referred as protected meropenem) Yield = 12.00 gm.
Example-II
Preparation of Meropenem trihydrate (Non sterile).
Charged4-Nitrobenzyl(4R,5S,6S)-3-({(3S,5S)-5-[(dimethylamino)carbonyl]-l-[(4-nitrophenoxy) carbonxyl]pyrrolidin-3-yl}thio)-6-[(lR)-l-hydorxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0].hept-2-ene-2-carboxylate (referred as protected meropenem ) obtained from previous stage in 120 ml of water methyl-t-butyl ether+ N- methyl formamide with 12 gms 5 % palladium on activated charcoal added with 120 ml of aqueous solution of sodium bi carbonate , pH about 7 . The above said biphasic reaction mixture hydrogenated for 3 hours under 9-10 kg hydrogen pressure at
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ambient temperature. After completion of reaction , the mixture was filtered and separated aqueous layer.
Wash the above said aqueous layer by mixture of (methyl-t-butyl ether+ N- methyl formamide) (10+10) ml twice and finally by 10 ml ethyl acetate. Concentrated under vacuum up to l/4th of total volume , 200 ml acetone was added to aqueous layer slowly at temperature about 5-8 °C and resultant mass was stirred for 3 hours at same temperature .The separated solid ware filtered, washed with chilled acetone and dried at ambient temperature to get meropenem trihydrate Yield= 6.00 gm.
Example-Ill
Preparation of sterile Meropenem trihydrate.
Charged 4-Nitrobenzyl(4R,5S,6S)-3-({(3S,5S)-5-[(dimethylamino)carbonyl]-l-[(4-nitrophenoxy) carbonxyl]pyrrolidin-3-yl}thio)-6-[(lR)-l-hydorxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0].hept-2-ene-2-carboxylate (referred as protected meropenem ) obtained from previous stage in 120 ml of water methyl-t-butyl ether+ N- methyl formamide with 12 gms 5 % palladium on activated charcoal and added 120 ml of aqueous solution of sodium bi carbonate , pH about 7 . The above said biphasic reaction mixture hydrogenated for 3 hours under 9-10 kg hydrogen pressure at ambient temperature. After completion of reaction, the mixture was filtered and separate aqueous layer. Washed the above said aqueous layer by mixture of (methyl-t-butyl ether+ N- methyl formamide) (10+10) ml twice and finally by 10 ml ethyl acetate. Concentrated under vacuum up to l/4th of total volume make pH of concentrate layer 7 by using sterile sodium bicarbonate solution and filtered through series of 0.45 u and 0.2 u filter paper under aseptic condition.
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200 ml acetone was added to aqueous layer slowly at temperature about 5-8 °C and resultant mass was stirred for 3 hours at same temperature under aseptic condition .The separated solid were filtered, washed with chilled acetone and dried at ambient temperature to get meropenem trihydrate Yield= 6.00 gm.
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Claims:
1) An improved process for preparation of meropenem trihydrate comprising steps:
a) Condensation of compound A(4-nitrobenzyl (4R,5R,6S)-3-[(diphenoxyphosphoryl)oxy]-6-[(lR)-l-hydroxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate)and compound B(4-nitrobenzyl (2S,4S)-2-[(dimethylamino)carbonyl]-4- mercaptopyrrolidine-1-carboxylate) in selected solvent such as acetonitrile at temperature 20 °C to get protected meropenem.
b) Isolation of protected meropenem comprising the extraction of mixture of ethyl acetate , water and 2N hydrochloric acid .
c) Furthermore , organic layer washed with phosphate buffer and 25% sodium chloride solution and distilled off to get solid of protected meropenem.
d) Finally solid obtained from above said process treated with mixture of water and methanol to obtain pure protected meropenem.
e) Hydrogenation of protected meropenem carried out in biphasic solvent system such as water: methyl -t- butyl ether + N- methyl formamide under pressure 9-10 kg by using reusable palladium catalyst to exerts a desired yield and purity.
2) The process as claimed in claim 1 wherein, condensation of compound A(4-nitrobenzyl (4R,5R,6S)-3- [(diphenoxyphosphoryl)oxy] -6- [(lR)-l-hydroxyethyl] -4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylate) and compound B (4-nitrobenzyl (2S,4S)-2-[(dimethylamino)carbonyl]-4- mercaptopyrrolidine-l-carboxylate)carried out in polar non protic
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solvent such as acetonitrile as reaction media at temperature 18-20 °C for 50-60 minutes to exerts a desired yield and purity.
3) The process as claimed in claim 1 wherein, solvent used for purification in step (d), selected from
aliphatic alcohol and polar protic solvent or mixture thereof.
4) The process as claimed in claim 1 wherein, solvent used for purification in step (d) aliphatic
alcohol is methanol and polar protic solvent is water.
5) The process as claimed in claim 1 wherein, biphasic solvent system for hydrogenation in step (e) selected from water: methyl-t-butyl ether + N- methyl formamide , water : toluene + N- methyl formamide, water : methyl-t-butyl ether + N,N dimethyl accetamide or mixture thereof.
6) The process as claimed in claim 1 wherein, preferably biphasic solvent system in step (e) is water: methyl-t-butyl ether + N- methyl formamide.
7) The process as claimed in claim 1 wherein, hydrogenation in step (e) carried out with reusable palladium on activated charcoal with biphasic solvent system under 9-10 kg pressure for 3 hours.
8) The process as claimed in claim 1 wherein, isolation of meropenem trihydrate from reaction mass after hydrogenation carried out by subsequent extraction of biphasic solvent system and
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ethyl acetate and concomitantly dropwise addition of acetone in aqueous layer with controlled temperature 5-8 ° C exerts precipitation of meropenem trihydrate in yield 75-80 % with purity 99% above.
9) A process for preparation of sterile meropenem trihydrate comprises:
a) Filtrate of reaction mass after hydrogenation filter through series of 0.45 p. and 0.2 \i filter under aseptic condition .
b) Slowly Addition of water miscible solvent i.e. sterile acetone to filtrate with controlled temperature under aseptic condition to isolate pure sterile meropenem trihydrate .
10 ) The improved process as claimed in claim 1 to 9 wherein said process is used to manufacture sterile meropenem trihydrate as substantially described herein with reference to the examples in our scope of invention to obtain purity above 99 % with yield 75- 80%.

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Abstract:
The present invention relates to improved process for preparation of meropenem trihydrate Structure-I (4R,5S,6S)-3-[[(3S,5S)-5-(Dimethylcarbamoyl)-3-pyrrolidinyl]thio]-6-[(lR)-l- hydroxy-ethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid) and its penultimate stage intermediate(4-Nitrobenzyl(4R,5S,6S)-3-({(3S,5S)-5-[(dimethylamino)carbonyl]-l-[(4-nitrophenoxy)carbonxyl]pyrrolidin-3-yl}thio)-6-[(lR)-l-hydorxyethyl]-4-methyl-7-oxo-l-azabicyclo[3.2.0].hept-2-ene-2-carboxylate).

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