Field of the Invention

The present invention provides for an improved process for preparing

substantially pure cilastatin and pharmaceutically acceptable salts thereof.

Background of the Invention

Cilastatin, a derivatized heptenoic acid is chemically known as (2Z)-7-{[(2R)-2- amino-2-carboxyethyl]thio } -2-( { [( 1 S)-2,2-dimethylcyclopropyl]carbonyl } amino) hept-2-enoic acid. It is a renal dipeptidase I inhibitor and is co-administered as a sodium salt with imipenem in order to prevent the nephrotoxicity associated with the use of imipenem. Imipenem and cilastatin sodium (Primaxin®) combination is used as a potent broad spectrum anti-bacterial agent.

Several processes have been reported in literature for the preparation of cilastatin and pharmaceutically acceptable salts thereof.

Cilastatin is specifically claimed in U.S. Patent No. 5,147,868. It also discloses a process for preparing cilastatin, which involves reacting ethyl-7-chloro-2-ketoheptanoate with (S)-2,2-dimethylcyclopropanecarboxamide in the presence of p-toluene sulphonic acid in refluxing toluene to give ethyl(2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl] carbonyl}amino)hept-2-enoate, which upon hydrolysis gives (2Z)-7-chloro-2-({[(lS)-2,2- dimethylcyclopropyl]carbonyl}arnino)hept-2-enoic acid followed by condensation with L- cysteine in the presence of sodium hydroxide to give cilastatin. (2Z)-7-chloro-2-({[(lS)- 2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-enoic acid formed in this process has considerable amounts of (2E)-7-chloro-2-({[(l S)-2,2-dimethylcyclopropyl]

carbonyl}amino)hept-2-enoic acid (referred to as E-isomer herein after). In order to minimize the E-isomer impurity, the mixture of E and Z isomer of 7-chloro-2-({[(lS)-2,2- dimethylcyclopropyl]carbonyl}amino)hept-2-enoic acid was heated under acidic conditions. However, the process gave lower yields and inferior quality of cilastatin.

WO 2007/054771 provides a process for isolating cilastatin sodium from a solution of cilastatin acid using sodium salt of weak acid. It further provides a process for preparing cilastatin acid by condensing 7-chloro-2-[{[(lS)-2,2- dimethylcyclopropyl]carbonyl}amino]hept-2-enoic acid with L-cysteine in the presence of a base followed by isolating cilastatin acid from the aqueous solution at pH 2.0 to 4.0. However, present inventors have faced difficulty in isolating the product of desired quality of cilastatin from the reaction mixture at this particular pH range.

Taking into account the drawbacks of the aforementioned methods, the present invention provides an improved and industrially viable process for preparing

substantially pure cilastatin, and pharmaceutically acceptable salts thereof.

Summary of the Invention

In one general aspect, the present invention provides for a process for preparing cilastatin of Formula I

pharmaceutically acceptable salts thereof. The process includes the steps of:

(a) reacting ethyl 7-chloro-2-ketoheptanoate of Formula III with

dimethylcyclopropane carboxamide of Formula IV in at least one organic solvent;

Formula III Formula IV

(b) optionally adding additional first solvent;

(c) hydrolyzing the product of step a);

(d) adjusting the pH of aqueous layer to acidic;

(e) extracting the product in a second solvent;

(f) optionally adding a third solvent to step (d); (g) treating the solution with aqueous sodium hydroxide;

(h) concentrating the solution;

(i) adding a fourth solvent to obtain a slurry;

(j) isolating sodium (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl]

carbonyl } amino)hept-2-enoate;

(k) optionally converting sodium (2Z)-7-chloro-2-({[(lS)-2,2- dimethylcyclopropyl] carbonyl }amino)hept-2-enoate to the acid of Formula

V;

Formu a V

(1) optionally crystallizing (2Z)-7-chloro-2-( { [( 1 S)-2,2-dimethylcyclopropyl] carbonyl }amino)hept-2-enoic acid of Formula V; and

(m) converting (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl]

carbonyl }amino)hept-2-enoic acid of Formula V, or its sodium salt to cilastatin of Formula I, or pharmaceutically acceptable salts thereof.

Embodiments of this aspect may include one or more of the following features. For example, the first solvent is an alcohol. Suitable alcohols include methanol, ethanol, n-propanol, iso-propanol, n-butanol, or a mixture thereof. The second solvent is selected from aromatic hydrocarbons; halogenated hydrocarbons; ethers; ketone. For example, the second solvent is an aromatic hydrocarbons.

The third solvent is an alcohol and the fourth solvent is selected from aromatic hydrocarbons, aliphatic hydrocarbons, halogenated hydrocarbons, ethers, ketone, alcohols, amides, sulfoxides, and nitriles. For example, the fourth solvent is a nitrile.

In another general aspect, the present invention provides for a process for preparing cilastatin of Formula I and pharmaceutically acceptable salts thereof. The process inlcudes the steps of: (a) condensing (2Z)-7-chloro-2-({[(lS)-2,2- dimethylcyclopropyl]carbonyl}amino)hept-2-enoic acid of Formula V with L-cysteine;

(b) adjusting the pH to less than 1.5;

(c) optionally heating step (b) at 60°C to 90°C;

(d) optionally washing the aqueous layer with an organic solvent;

(e) optionally purifying aqueous layer through resin or column chromatography;

(f) extracting the aqueous layer with an organic solvent;

(g) adjusting pH of organic layer to 4.0 to 5.0;

(h) recovering cilastatin acid; and

(i) converting cilastatin acid to its pharmaceutically acceptable salt.

Embodiments of the process may include one or more of the following features. For example, the organic solvent used in step (d) is selected from aromatic hydrocarbons, halogenated hydrocarbons and ethers. The organic solvent used in step (d) may be a halogenated hydrocarbon or an ether.

The organic solvent used in step (f) is selected from ethers, alcohols and esters. For example, the organic solvent is selected from ethers or alcohols.

In another general aspect, the present invention provides for a process for preparing cilastatin of Formula I and pharmaceutically acceptable salts thereof. The process includes the steps of:

(a) condensing (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl]

carbonyl}amino)hept-2-enoic acid of Formula V with L-cysteine;

(b) preparing the ammonium/amine salt of cilastatin;

(c) optionally isolating the ammonium/amine salt of cilastatin;

(d) converting the ammonium/amine salt to cilastatin acid; and

(e) isolating cilastatin sodium. In another general aspect, the present invention provides for an ammonium salt of cilastatin acid.

Embodiments of this aspect may include one or more of the following features. For example, the ammonium salt of cilastatin acid may have characteristic d-spacing (A) values selected from 17.6, 9.38, 8.85, 5.18, 4.49, 4.13, 4.10, 3.87, 3.54 or 3.18. In addition, the ammonium salt of cilastatin acid may have an XRD pattern substantially as depicted in Figure 1.

In another general aspect, the present invention provides for the methyl amine salt of cilastatin acid.

In yet another general aspect, the present invention provides for a process for preparing cilastatin of Formula I and pharmaceutically acceptable salts thereof. The process includes the steps of:

(a) preparing 7-chloro-2-(ethoxycarbonyl)heptanoic acid of Formula II

Formula II by condensing l-bromo-5-chloropentane with ethylacetoacetate in a solvent, wherein the solvent used is less than 0.5 times of l-bromo-5-chloropentane by weight; and

(b) converting 7-chloro-2-(ethoxycarbonyl)heptanoic acid of Formula II to

cilastatin or pharmaceutically acceptable salts thereof.

Embodiments of the process may include one or more of the following features. For example, the 7-chloro-2-(ethoxycarbonyl) heptanoic acid of Formula II is converted to cilastatin or pharmaceutically acceptable salts thereof.

In another general aspect, the present invention provides for substantially pure cilastatin sodium. Embodiments of this aspect may include one or more of the following features. For example, the substantially pure cilastatin sodium has a purity of more than 99.0% when determined by HPLC or a purity of more than 99.5% when determined by HPLC.

Brief Description of the Figures

Figure 1 : X-ray diffraction pattern (XRD) of cilastatin ammonium salt

Table 1 corresponds to the peak table for Figure 1.

Detailed Description of the Invention

The present invention provides intermediates and processes for preparing cilastatin and pharmaceutically acceptable salts thereof.

In one embodiment, 7-chloro-2-(ethoxycarbonyl)heptanoic acid can be prepared by reacting l-bromo-5-chloropentane with ethylacetoacetate in the presence of a base in at least one organic solvent. The base used in this process can be selected from alkali metal hydroxides (e.g., sodium hydroxide, potassium hydroxide); alkali metal carbonates (e.g., sodium carbonate, potassium carbonate); alkali metal alkoxides (e.g., sodium methoxide, sodium ethoxide, potassium t-butoxide), or a mixture thereof. The solvent used in this process can be selected from aromatic hydrocarbons (e.g., benzene, toluene, xylene); aliphatic hydrocarbons (e.g., hexane, cyclohexane, heptane); ethers (e.g., diethylethers, diisopropylether); ketones (e.g., methylisobutylketone, acetone; alcohols (e.g., methanol, ethanol, 2-propanol, n-butanol); amides (e.g., N, N-dimethylformamide, N-methyl-2-pyrrolidine); sulfoxides (e.g., dimethylsulfoxide); and nitriles (e.g., acetonitrile, propanonitrile); or a mixture thereof. It has been observed by the present inventors that the choice of solvent, the amount of solvent and the temperature of the reaction play an important role in improving the quality of the final cilastatin sodium.

In another embodiment, the process can be carried out by reacting l-bromo-5- chloropentane with ethylacetoacetate in the presence of alkali metal carbonate and at least one aliphatic hydrocarbon, such as hexane, wherein the amount of solvent can be less than 0.5 times by weight of l-bromo-5 -chloropentane.

The present invention also provides a process for preparing cilastatin of Formula I, and the pharmaceutically acceptable salts thereof. The process includes the steps of: (a) reacting ethyl 7-chloro-2-ketoheptanoate of Formula III with

dimethylcyclopropane carboxamide of Formula IV in at least one organic solvent;

Formula III Formula IV

(b) optionally adding additional first solvent;

(c) hydrolyzing the product;

(d) adjusting the pH of aqueous layer to acidic;

(e) extracting the product in a second solvent;

(f) optionally adding a third solvent;

(g) treating the solution with aqueous sodium hydroxide;

(h) concentrating the solution;

(i) adding a fourth solvent to obtain a slurry;

(j) optionally isolating sodium (2Z)-7-chloro-2-( {[(1 S)-2,2- dimethy lcyclopropyl] carbonyl } amino)hept-2-enoate ;

(k) optionally converting sodium (2Z)-7-chloro-2-({[(lS)-2,2- dimethy lcyclopropyl] carbonyl }amino)hept-2-enoate to acid of Formula V;

Formu a V

(1) optionally crystallizing (2Z)-7-chloro-2-({[(l S)-2,2-dimethy lcyclopropyl] carbonyl }amino)hept-2-enoic acid of Formula V; and (m) converting (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl] carbonyl}amino)hept-2-enoic acid of Formula V or its sodium salt to cilastatin or pharmaceutically acceptable salts thereof.

In one embodiment, step (a) involves reacting ethyl 7-chloro-2-ketoheptanoate with dimethylcyclopropane carboxamide in the presence of p-toluene sulfonic acid in at least one organic solvent. The organic solvent used in this step can be selected from aromatic hydrocarbons (e.g., benzene, toluene, xylene); aliphatic hydrocarbons (e.g., hexane, cyclohexane, heptane); halogenated hydrocarbons (e.g., dichloromethane, chlorobenzene, dichlorobenzene, chloroform, 1,2-dichloroethane); ethers (e.g., diethylether, diisopropylether, methyl tertiary butyl ether); ketones (e.g., acetone, methylisobutylketone); alcohols (e.g., methanol, ethanol, 2-propanol, n-butanol); amides (e.g., N, N-dimethylformamide, N-methyl-2-pyrrolidine); sulfoxides (e.g.,

dimethylsulfoxide); and nitriles (e.g., acetonitrile, propanonitrile) or a mixture thereof.

In another embodiment, step (b) involves optionally adding additional first solvent to the reaction mixture of step (a), wherein the first solvent can be selected from alcohols, such as methanol, ethanol, n-propanol, iso-propanol, n-butanol or a mixture thereof.

In another embodiment, step (c), involves hydrolyzing the ethyl ester obtained in the step (a) using a base, wherein the base can be selected from alkali metal hydroxides, such as sodium hydroxide, potassium hydroxide or a mixture thereof.

In another embodiment, step (d) involves maintaining the pH of the aqueous layer to 1.0 to 4.0 using an acid. The acid used in this step can be selected from an inorganic acid (e.g., sulfuric acid, hydrobromic acid, hydrochloric acid) or organic acids (e.g., acetic acid, formic acid, methanesulfonic acid), or a mixture thereof.

In another embodiment, step (e) involves extracting the product obtained in step

(d) in a second solvent. The second solvent used for the extraction can be selected from aromatic hydrocarbons (e.g., benzene, toluene, xylene); halogenated hydrocarbons (e.g., dichloromethane, chlorobenzene, dichlorobenzene, chloroform, 1,2-dichloroethane); ethers (e.g., diethylether, diisopropylether); ketone (e.g., methylisobutylketone). In another embodiment, step (f) involves optionally adding a third solvent to step (e), wherein the third solvent can be selected from alcohols such as, methanol, ethanol, 2-propanol, n-butanol, or a mixture thereof.

In another embodiment, step (g) involves treating the mixture obtained in step (e) or (f) with aqueous sodium hydroxide.

In another embodiment, step (h) involves concentrating the mixture obtained in step (g) under vacuum.

In another embodiment, step (i) involves adding a fourth solvent to the solid obtained in the step (h) to obtain slurry. The fourth solvent used in this process is selected from aromatic hydrocarbons (e.g., benzene, toluene, xylene); aliphatic hydrocarbons (e.g., hexane, cyclohexane, heptane); halogenated hydrocarbons (e.g., dichloromethane, chlorobenzene, dichlorobenzene, chloroform, 1 ,2-dichloroethane,); ethers (e.g., diethylether, diisopropylether); ketones (e.g., methylisobutylketone, acetone); alcohols (e.g., methanol, ethanol, 2-propanol); amides (e.g., N, N- dimethylformamide, N-methyl-2-pyrrolidine); sulfoxides (e.g., dimethylsulfoxide); and nitriles (e.g., acetonitrile, propanonitrile), or a mixture thereof. In a particular embodiment, the fourth solvent is selected from nitriles, such as acetonitrile or propanonitrile.

In another embodiment, step (j), involves isolating sodium (2Z)-7-chloro-2- ({[(lS)-2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-enoate from the slurry obtained in step (i). In a particular embodiment, sodium (2Z)-7-chloro-2-({[(lS)-2,2- dimethylcyclopropyl]carbonyl}amino)hept-2-enoate is filtered from the slurry in acetonitrile under inert atmosphere.

In another embodiment, step (k) involves converting sodium (2Z)-7-chloro-2- ({[(lS)-2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-enoate to (2Z)-7-chloro-2-

({[(lS)-2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-eno ic acid of Formula V using an acid, followed by extracting the product in at least one organic solvent. The acid used in this step can be selected from an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid); an organic acid (e.g., acetic acid, formic acid, p-toluenesulfonic acid) or a mixture thereof. The organic solvent used in this step can be selected from aromatic hydrocarbons (e.g., benzene, toluene, xylene); halogenated hydrocarbons (e.g., dichloromethane, chlorobenzene, dichlorobenzene, chloroform, 1,2-dichloroethane); ethers (e.g., diethylether, diisopropylether); ketones (e.g., methylisobutylketone). In a particular embodiment, sodium (2Z)-7-chloro-2-({[(lS)-2,2- dimethylcyclopropyl]carbonyl}amino)hept-2-enoate is reacted with hydrochloric acid and the product was extracted in at least one chlorinated solvent.

In another embodiment, step (1) involves crystallizing (2Z)-7-chloro-2-({[(lS)- 2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-enoic acid in at least one organic solvent. The organic solvent used in this step can be selected from aromatic

hydrocarbons (e.g., benzene, toluene, xylene); aliphatic hydrocarbons (e.g., hexane, cyclohexane, heptane); halogenated hydrocarbons (e.g., dichloromethane, chlorobenzene, dichlorobenzene, chloroform, 1,2-dichloroethane); ethers (e.g., diethylether,

diisopropylether)^ ketones (e.g., methylisobutylketone, acetone); alcohols (e.g., methanol, ethanol, 2-propanol); amides (e.g., N, N-dimethylformamide, N-methyl-2- pyrrolidine); sulfoxides (e.g., dimethylsulfoxide); and nitriles (e.g., acetonitrile, propanonitrile) or a mixture thereof. In a particular embodiment, the organic solvent is selected from dialkyl ethers; aliphatic solvent, or a mixture thereof.

In another embodiment, step (m) involves converting substantially pure (2Z)-7- chloro-2-({ [(1 S)-2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-enoic acid to cilastatin, or pharmaceutically acceptable salts thereof. In yet another embodiment, substantially pure (2Z)-7-chloro-2-({ [(1 S)-2,2-dimethylcyclopropyl]carbonyl} amino)hept-2-enoic acid is reacted with L-cysteine hydrochloride monohydrate to form cilastatin, or the pharmaceutically acceptable salts thereof.

It has been observed by the present inventors that the isolation of sodium (2Z)-7- chloro-2-({ [(1 S)-2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-enoate, prior to its conversion to acid of Formula V and further to cilastatin helps in controlling impurities and improving purity of the final cilastatin sodium.

The present invention also provides a process for preparing cilastatin and pharmaceutically acceptable salts thereof. The process includes the steps of: (a) condensing (2Z)-7-chloro-2-({ [(1 S)-2,2-dimethylcyclopropyl]carbonyl} amino)hept-2-enoic acid with L-cysteine;

(b) adjusting the pH to less than 1.5;

(c) optionally heating step (b) at 60°C to 90°C;

(d) optionally washing aqueous layer with an organic solvent;

(e) optionally purifying aqueous layer through resin or column chromatography;

(f) optionally extracting aqueous layer with an organic solvent;

(g) adjusting pH of organic layer to 4.0 to 5.0;

(h) recovering cilastatin acid; and

(i) converting cilastatin acid to its pharmaceutically acceptable salt.

In one embodiment, step (a) involves reacting (2Z)-7-chloro-2-({[(lS)-2,2- dimethylcyclopropyl]carbonyl}amino)hept-2-enoic acid with L-cysteine in the presence of a base. The base used in this step can be selected from alkali metal hydroxide (e.g., potassium hydroxide, sodium hydroxide); alkali metal carbonates (e.g., sodium carbonate, potassium carbonate), or a mixture thereof.

In another embodiment, the pH in step (b) is adjusted using an acid. The acid used in the step (b) can be selected from inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid); organic acid (e.g., acetic acid, formic acid, p- toluenesulfonic acid), or a mixture thereof.

In another embodiment, step (d) involves washing aqueous layer of step (b) or step (c) with an organic solvent. The organic solvent can be selected from aromatic hydrocarbons (e.g., benzene, toluene, xylene); halogenated hydrocarbons (e.g., dichloromethane, dichloroethane, chlorobenzene, dichlorobenzene, chloroform, 1,2- dichloroethane); ethers (e.g., diethylether, diisopropylether) or esters (e.g., ethylacetate, ethylformate), or a mixture thereof.

In another embodiment, step (e) involves optionally purifying the aqueous layer of step (c) or (d) by resin or column chromatography. In a particular embodiment, aqueous layer may be purified by passing it through HP-20 resin. In another embodiment, step (f) involves optionally extracting the aqueous layer of step (b) or (c) by an organic solvent. The organic solvent used for extraction can be selected from ethers (e.g., diethylether, diisopropylether, tetrahydrofuran, dioxane);

alcohols (e.g., 2-propanol, n-butanol, cyclohexanol); esters (e.g., ethylacetate,

ethylformate), or a mixture thereof.

In another embodiment, step (g) involves adjusting the pH of organic layer obtained in step (f) to 4.0 to 5.0. In yet another embodiment, the pH of the organic layer is adjusted to 4.0 to 5.0 using a base. The base used in this step can be selected from organic base (e.g., triethyl amine, diethyl amine, methyl amine, ammonia), or inorganic base (e.g., alkali metal hydroxide such as, potassium hydroxide, sodium hydroxide), alkali metal carbonates such as sodium carbonate, potassium carbonate), or mixture thereof.

In another embodiment, step (h) involves recovering cilastatin acid from step (e) or (g) using a solvent. In another embodiment, the solvent used in this step can be selected from ketones (e.g., methylisobutylketone, acetone).

In another embodiment, step (i) involves converting cilastatin acid to its pharmaceutically acceptable salts thereof. In a particular embodiment, cilastatin acid is converted into its sodium salt by reacting it with sodium hydroxide.

In another embodiment, (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl] carbonyl}amino)hept-2-enoic acid of Formula V was condensed with L-cysteine in the presence of potassium carbonate in methanol, filtered to remove inorganic salts and treated with liquid ammonia or organic amine to give ammonium or amine salts of cilastatin. The ammonium or amine salt of cilastatin may be isolated and converted to cilastatin acid which upon treatment with sodium hydroxide followed by lypholization gave cilastatin sodium. The organic amine used in this reaction can be selected from methyl amine, ethyl amine, n-propyl amine, isopropyl amine, n-butyl amine or t-butyl amine, diethanolamine, tris(hydroxymethyl)methyl amine, benzyl amine, 4- methoxybenzylamine or cyclohexyl amine.

The ammonium salt of cilastatin is isolated in the solid form can be characterized by d-spacing (A) values selected from 17.6, 10.90, 9.37, 8.84, 6.80, 5.45, 5.24, 5.18, 4.81, 4.69, 4.60, 4.49, 4.43, 4.10, 4.00, 3.97, 3.87, 3.58, 3.54, 3.52, 3.39, 3.34, 3.27, 3.16, 3.05, 2.95, 2.86, 2.78, 2.69, 2.62, 2.57, 2.52, 2.50, 2.43, 2.37 and the corresponding 2-theta values selected from 5.00, 8.11, 9.44, 10.00, 13.02, 16.25, 16.92, 17.10, 18.44, 18.91, 19.30, 19.80, 21.49, 21.73, 22.24, 22.42, 22.99, 24.90, 25.12, 25.34, 26.26, 26.69, 27.23, 28.21, 29.28, 30.32, 31.24, 32.17, 33.31, 34.19, 34.86, 35.62, 35.91, 36.99, 37.92, ± 0.02, as depicted in Figure 1.

The methyl amine salt of cilastatin is isolated in a solid form, wherein the solid form can be crystalline or amorphous.

The term "lypholization", as used in the present invention, refers to freezing under vacuum.

The term "substantially pure", as used in the present invention, refers to cilastatin or pharmaceutically acceptable salts thereof having purity more than 99.0%, preferably more than 99.5% and more preferably more than 99.8% when measured by HPLC.

Having thus described the invention with the reference to the particular embodiment and illustrative examples, those in the art can appreciate the modifications to the invention as described and illustrated that do not depart from the spirit and the scope of the invention as disclosed in the specifications.

EXAMPLES

Example 1 : Preparation of Ethyl 7-Chloro-2-Ketoheptanoate

A) A mixture of l-bromo-5-chloropentane (100 g), ethylacetoacetate (84 g), pulverized potassium carbonate (178 g) in hexanes (50 mL) was stirred at 75°C to 80°C for 15 to 20 hours. After the completion of the reaction, the mixture was diluted with hexanes (200 mL), filtered through a celite bed and washed with hexanes (250 mL). The hexanes layer was cooled to -10°C to 0°C, nitrosyl sulphuric acid (40%; 256 g) was added at the same temperature and the resulting mixture was stirred for about 2 hours at about 15°C. Formaldehyde solution (-37%; 184 g) was added to the mixture and stirred for additional 2 hours. The layers were separated. The aqueous layer was diluted with water (250 mL), and extracted with hexanes (250 mL) at 30°C to 35°C. The organic layer was washed with water at 30°C to 35°C and concentrated under reduced pressure to get crude ethyl 7-chloro-2-ketoheptanoate. Yield: HO g

B) A mixture of l-bromo-5-chloropentane (100 g), ethylacetoacetate (84 g) and pulverized potassium carbonate (178 g) in hexanes (50 mL) was stirred at 75°C to 80°C for 15 to 20 hours. After the completion of the reaction, the mixture was diluted with toluene (200 mL), filtered through a celite bed and washed with toluene (250 mL). The toluene layer was cooled to -10°C to 0°C, nitrosyl sulphuric acid (40%; 256 g) was added at the same temperature and the resulting mixture was stirred for about 1 hour at about 15°C. Formaldehyde solution (-37%; 184 g) was added to the mixture and stirred for an additional 2 hours at 15°C to 20°C. The layers were separated. The aqueous layer was diluted with water (250 mL) and extracted with toluene (250 mL). Toluene layer was washed with water and concentrated under reduced pressure to get crude ethyl 7- chloro-2-ketoheptanoate.

Yield: 114.5 g

Example 2: Purification of Ethyl 7-Chloro-2-Ketoheptanoate

Crude ethyl 7-chloro-2-ketoheptanoate (Example 1 ; 110 g) was added to the solution of sodium bisulphate (230 g) in water (850 L) and stirred for about 2 hours at 25°C to 30°C. The reaction mass was washed with hexanes (140 mL). Hexanes (160 mL) were added to the aqueous layer and heated to about 60°C. Concentrated hydrochloric acid (250 g) was slowly added to the resulting mixture at 60°C to 65°C and stirred for about 2 hours at same temperature, then allowed to cool to about 35°C. The layers were separated. The aqueous layer was extracted with hexanes (80 mL) at 30°C to 35°C and the organic layer was washed with water at 30°C to 35°C. The organic layer was concentrated under reduced pressure to get pure ethyl 7-chloro-2-ketoheptanoate.

Yield: 68-70 g Example 3 : Preparation of Sodiumf 2ZV7-Chloro-2-( { IY 1 SV 2.2-Dimethylcvclopropyll carbonyl } Amino)Hept-2-Enoate

A mixture of ethyl 7-chloro-2-ketoheptanoate (Example 2; 420 g, 2.03 mole), S (+)-2,2-dimethylcyclopropane carboxamide (200 g, 1.77 mole) and p-toluenesulfonic acid (3.0 g) was refluxed for 18 hours in toluene (500 mL) and water was removed azeotropically. The reaction mixture was allowed to cool to about 25°C and successively washed with aqueous hydrochloric acid solution (10%; 1L), aqueous sodium bisulfite solution (10%; 1L) and water (1L). To the above solution was added de-natured spirit (400 mL) and water (2.6 L). The resulting mixture was cooled to about 5°C and sodium hydroxide solution (280 g in 400 mL of water) was added at 5°C to 10°C. The reaction mixture was stirred overnight at about 20°C. After the completion of the reaction, the pH was adjusted to acidic using concentrated hydrochloric acid at 5°C to 10°C. The product obtained was extracted with toluene (1.4L) and washed with water (400 mL). The organic layer was treated with active carbon and filtered. Isopropyl alcohol (400 mL) was added to the filtrate and cooled to about 5°C. The pH of this mixture was adjusted to 8.0 to 8.5 using sodium hydroxide solution (w/v; 35%) at 0°C to 5°C. The resulting solution was completely concentrated under reduced pressure at 40°C to 45 °C to obtain a residue. Acetonitrile (4 L) was added to the residue and the slurry obtained was stirred at 40°C to 45 °C for about one hour. The slurry obtained was cooled to about -15°C, filtered under nitrogen atmosphere and dried under reduced pressure to give sodium salt of (2Z)-7-chloro-2-( { [( 1 S)-2,2-dimethylcyclopropyl]

carbonyl}amino)hept-2-enoic acid.

Yield: 300 g (57.4%)

Purity: 96.56% (E-isomer: 1.37%).

Example 4: Preparation of (2Z)-7-Chloro-2-( ( Γ( 1 S -2,2-Dimethylcvclopropyll

Carbonyl} Amino)Hept-2-Enoic Acid

Concentrated hydrochloric acid was added to the solution of sodium (2Z)-7- chloro-2-( { [( 1 S)-2,2-dimethylcyclopropyl]carbonyl } amino)hept-2-enoate (Example 3 ; 100 g in 500 mL water) to adjust the pH of the solution to 2.0 to 2.5. The aqueous layer was extracted with dichloromethane (500 mL + 200 mL). The dichloromethane layer was washed with water and concentrated under vacuum to give a residue. The residue obtained was crystallized in isopropyl ether-hexanes (1:1) to give pure Z-7-chloro[(S)- 2,2-dimethylcyclo-propanecarboxamido]-2-heptenoic acid.

Yield: 73 g (79%)

Purity: 99.2 % (E isomer: 0.05%) Example 5: Preparation of Ammonium Salt of Cilastatin

A) (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl]carbonyl}ami no)hept-2- enoic acid (Example 4; 25 g) was added to mixture of L-cysteine hydrochloride monohydrate (23 g) and potassium carbonate (76g) in methanol (375 ml) at 20°C to 22 °C. The reaction mixture was stirred at 60°C for 6 hours. The reaction mixture was allowed to cool to 35°C, filtered and washed with methanol (200 mL). The pH of the filtrate was adjusted 3.4 using methanolic HC1 (390 ml) at 0°C to 10°C. Heated the contents to 40°C to 45 °C, stirred at 40°C to 45 °C for 30 minutes, again filtered the inorganic salts and washed the salts with hot methanol (200 ml). The filtrate (190 mL) was cooled to 15°C and the pH was adjusted to 7.00 using aqueous ammonia (3 ml) at 12°C to 15°C. The mixture was stirred at 25°C to 30°C for 30 minutes. The mixture was subjected to vacuum at 40°C to 45°C. Isopropanol (25 mL) was added to the resulting residue and the mixture was concentrated under vacuum. Isopropanol (150 ml), water (2.5 ml) and aqueous ammonia (5 ml) were added to the resulting residue and heated at 50°C to 55°C for 30 minutes. The mixture was cooled to 30°C and stirred at 25°C to 30°C for 1.0 hour. The mixture was filtered, washed with isopropanol (10 ml) and dried under vacuum at 40°C to 45°C for 8.0 hours to give ammonium salt of cilastatin.

Dry Wt: 6.3 g (1.3 w/w 95%)

HPLC purity: 99.08%

B) (2Z)-7-chloro-2-( { [( 1 S)-2,2-dimethylcyclopropyl]carbonyl } amino)hept-2- enoic acid (Example 4; 25 g) was added to mixture of L-cysteine hydrochloride monohydrate (22.5 g) and potassium carbonate (75.6g) in methanol (375 ml) at 20°C to 22°C. The reaction mixture was stirred for 6 hours at 60°C to 65°C. The resultant mixture was filtered at 60°C. The filtrate (300 mL) was cooled to 10°C and aqueous hydrochloric acid (150 mL; -30%) was added at 10°C to 15°C and the reaction mixture was allowed to stir at 25°C to 30°C for 15 minutes. The resulting mixture was filtered and washed with methanol (50 mL). The filtrate was again cooled to 15°C and the pH of the solution was adjusted fo 6.6 using aqueous ammonia (-25%, 6.0 ml). The filtrate was divided into three parts 166 mL each and reacted in following manner. The filtrate (166 mL) was concentrated under vacuum. Isopropanol (25 mL) was added to the residue and again concentrated under vacuum. Isopropanol (100 mL), water (5 mL) and aqueous ammonia (-25%; 2.5 mL) were added to the resulting residue and stirred at 45°C to 50°C for one hour. The resulting mixture was allowed to cool to 25°C to 30°C and filtered to give ammonium salt of cilastatin.

Weight: 5.3 g

HPLC purity (%): 99.48

The filtrate (166 mL) was concentrated under vacuum. Isopropanol (25 mL) was added to the residue and resulting mixture was concentrated under vacuum. Isopropanol (100 mL), water (2.5 mL) and aqueous ammonia (~25%, 5 mL) were added to the residue. The resulting mixture was heated to 45°C for one hour. The reaction mixture was allowed to cool to 25°C to 30°C, filtered and dried to give ammonium salt of cilastatin.

Weight: 5.3 g

HPLC purity (%): 98.93

The filtrate ( 166 mL) was concentrated under vacuum to give a residue.

Isopropanol (25 mL) was added to the residue and again concentrated under vacuum. Isopropanol (100 mL), water (2.5 mL) and aqueous ammonia (~25%:2.5 mL) was added to the residue and heated to 45°C to 50°C for one hour. The resulting mixture was cooled to 30°C to 35°C filtered and dried to give ammonium salt of cilastatin.

Weight: 6.3 g

HPLC purity (%): 98.43

C) (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl]carbonyl}ami no)hept-2- enoic acid (Example 4; 25 g) was added to mixture of L-cysteine hydrochloride monohydrate (22.5 g) and potassium carbonate (75.1 g) in methanol (375 mL) at 20°C to 25°C. The reaction mixture was heated at 60°C to 65°C for 6 hours. The reaction mixture was allowed to cool to room temperature and filtered. The filtrate was allowed to cool to 15°C to 20°C and adjusted to pH 3.5 using methanolic hydrochloric acid at 10°C to 15°C. The mixture was filtered, washed with methanol (200 mL). The filtrate (400 mL) was cooled to 15°C and aqueous ammonia (4.0 mL) were added to adjust the pH to 6.98. The resulting mixture was concentrated under vacuum. Isopropanol (50 mL) was added to the residue. The resulting mixture was concentrated under vacuum. Isopropanol (200 mL), water (5.0 mL) and aqueous ammonia (5 mL) were added to the resulting residue and the mixture was heated to 45°C to 50°C for 90 minutes. The reaction mixture was cooled to 25°C to 30°C and stirred at the same temperature for 2 hours. The reaction mixture was filtered and dried to give ammonium salt of cilastatin.

Weight: 12.5 g

HPLC purity (%): 99.03

D) (2Z)-7-chloro-2-( { [( 1 S)-2,2-dimethylcyclopropyl] carbonyl } amino)hept-2- enoic acid (Example 4; 25 g) was added to mixture of L-cysteine hydrochloride monohydrate (22.5 g) and potassium carbonate (76 g) in methanol (375 ml) at 10°C to 15°C. The reaction mixture was heated at 60°C to 65°C for 4 hours. The reaction mixture was filtered and washed with methanol (200 ml). The pH of filtrate was adjusted to 3.2 using aqueous hydrochloric acid (-30%; 55 mL) at 10°C to 15°C. The mixture was again filtered. The pH of the resulting filtrate was adjusted to 7.1 using aqueous ammonia

(-25%; 10 mL) at 12°C to 15°C. The mixture was concentrated and isopropanol (125 mL) was added to the residue and again concentrated under vacuum. Isopropanol (600 mL), water (25 mL) and aqueous ammonia (12.5 mL) were added to the resulting residue and heated to 45°C to 50°C for one hour. The mixture was allowed to cool to 30°C and stirred at the same temperature for one hour. The mixture was filtered and dried to give ammonium salt of cilastatin.

Weight: 26 g

HPLC purity (%): 98.97

Example 6: Preparation of Cilastatin Acid

A) (2Z)-7-chloro-2-({[(lS)-2,2-dimethylcyclopropyl]carbonyl}ami no)hept-2- enoic acid (Example 4; 200 g; 0.73 mol) was added to a pre-cooled solution of sodium hydroxide (152 g, 3.8 mol in 400 mL water) and L-cysteine hydrochloride monohydrate (179.6 g; 1.023 mol; in 1.2 L water)) at about 5°C. The reaction mixture was stirred for about 24 hours at about 25°C. After the completion of the reaction, pH of the reaction mixture was adjusted to 1 using hydrochloric acid (concentrated; 240 mL) at 10°C to 15°C and then stirred at 70°C to 75°C for about one hour. The reaction mixture was cooled, washed with dichloromethane and passed through HP -20 resin column using methanol and water as eluent. The fractions containing the product were concentrated under vacuum to give a solid residue. Acetone (2 L) was added to the solid residue and stirred at room temperature to filter cilastatin acid.

Yield: 170 g

Purity: 99.8%

B) (2Z)-7-chloro-2-({ [(1 S)-2,2-dimethylcyclopropyl]carbonyl}amino)hept-2- enoic acid (Example 4; 10 g; 36.56 mmol) was added to a pre-cooled solution of sodium hydroxide (10.23 g; 400 mL water) and L-cysteine hydrochloride monohydrate (12.88 g in 40 mL water) at about 5°C. The reaction mixture was stirred for about 12 hours at about 25°C. After the completion of the reaction, pH of the reaction mixture was adjusted to about 0.48 using hydrochloric acid (concentrated; 15 mL) and

tetrahydrofuran (60 mL) was added. The reaction mixture was stirred for about 15 minutes. The layers were separated and the pH of the organic layer was readjusted to about 4.3 using ammonia solution (10 mL). The organic layer was separated and concentrated under vacuum to give solid residue. Acetone (100 mL) was added to the solid residue and stirred at room temperature to filter cilastatin acid.

Yield: 13.14 g

Purity: 97.24%

C) (2Z)-7-chloro-2-( { [( 1 S)-2,2-dimethylcyclopropyl]carbonyl } amino)hept-2- enoic acid (Example 4; 10 g; 36.56 mmol) was added to a pre-cooled solution of sodium hydroxide (10.23 g in 400 mL water) and L-cysteine hydrochloride monohydrate (12.6 g; 73.04 mmol in water 120 mL) at -4°C to to 0°C. The reaction mixture was stirred for about 12 hours at about 25°C. After the completion of the reaction, pH of the reaction mixture was adjusted to about 0.55 using hydrochloric acid (concentrated; 15 mL) and n- butanol (50 mL) was added. The reaction mixture was stirred for about 15 minutes. The layers were separated and the pH of the organic layer was readjusted to about 4.16 using ammonia solution (10 mL). The organic layer was separated and concentrated under vacuum until about 30 mL of residue is left. The concentrated organic layer was stirred at room temperature to filter cilastatin acid.

Yield: 8.4 g

Purity: 98.22%

D) (2Z)-7-chloro-2-({ [( 1 S)-2,2-dimethylcyclopropyl]carbonyl} amino)hept-2- enoic acid (Example 4; 20 g) was added to a pre-cooled solution of sodium hydroxide (20.47 g; 0.52 mol in water 80 mL) and L-cysteine hydrochloride monohydrate (25.66 g; 0.07 mol) in water (240 mL) at 0°C to 5°C. The reaction mixture was stirred for about 24 hours at about 25°C. After the completion of the reaction, pH of the reaction mixture was adjusted to about 1 using hydrochloric acid (concentrated; 90 mL) at 10°C to 15°C and the reaction mixture was stirred at 70°C to 75°C for about one hour. The reaction mixture was washed with dichloromethane (200 mL) and the aqueous layer was extracted with tetrahydrofuran (300 mL). The pH of the organic layer was readjusted to about 4.13 using ammonia (20 mL). The organic layer was separated and concentrated under vacuum to give solid residue. Acetone (500 mL) was added to the solid residue and stirred at room temperature to filter cilastatin acid.

Yield: 22 g

Purity: 97.5%

E) (2Z)-7-chloro-2-( { [( 1 S)-2,2-dimethylcyclopropyl]carbonyl } amino)hept-2- enoic acid (Example 4; 50 g; 0.15mol) was added to a pre-cooled solution of sodium hydroxide (51.2 g; 1.28 mol in 200 mL water) and L-cysteine hydrochloride

monohydrate (94.15 g; 0.36 mol in 600 mL water) at 5°C. The reaction mixture was stirred for about 24 hours at about 25°C. After the completion of the reaction, pH of the reaction mixture was adjusted to about 1 using hydrochloric acid (concentrated; 100 mL) at 10°C to 15°C and reaction mixture was stirred at 70°C to 75°C for about one hour.

The reaction mixture was washed with dichloromethane (200 mL) and the aqueous layer was extracted with n-butanol (500 mL). The pH of the organic layer was readjusted to about 4.16 using ammonia (50 mL). The organic layer was separated and concentrated under vacuum to give solid residue. Acetone (500 mL) was added to the solid residue and stirred at room temperature to filter cilastatin acid. Yield: 60 g

Purity: 93.5%

F) The pH of the solution of cilastatin ammonium salt (5 g) in methanol (lOOmL) was adjusted to 3.4 using cationic exchange resin (Indion-225) at 15°C. The reaction mixture was filtered through a celite bed to remove resin and the filtrate was concentrated under vacuum. Acetone (50 mL) was added to the resulting residue and the mixture was heated 40°C for 30 minutes. The reaction mixture was allowed to cool to 25°C to 30°C and mixture was stirred at the same temperature for 30 minutes. The mixture was filtered and dried under vacuum at 40°C to 45°C.

Weight: 3.2 g

HPLC purity (%): 99.27

G) The pH of the solution of cilastatin ammonium salt (5 g) in methanol (100 mL) was adjusted to 3.0 using cationic exchange resin (Indion-225) at 15°C. The reaction mixture was filtered to remove the resin and filtrate was concentrated under vacuum. Methanol (75 mL) and activated carbon (0.5 g) were added to the resulting residue and heated to 40°C to 45°C for 30 minutes, filtered through a celite bed and washed with methanol (amount). The resulting filtrate was concentrated under vacuum. Acetone (75 mL) and water (5 mL) were added to the resulting residue and heated to 40°C to 45°C for 30 minutes. The mixture was allowed to cool to 25°C to 30°C and stirred at the same temperature for one hour. The resulting mixture was filtered and dried under vacuum at 40°C to 45°C to give cilastatin acid.

Weight: 3.2 g

HPLC purity (%): 99.27

H) The pH of the solution of cilastatin ammonium salt (5.1 g) in methanol (100 mL) was adjusted to 3.5 using cationic exchange resin (Indion-225) at 20°C to 25°C.

The reaction mixture was stirred at 25°C to 30°C for 20 minutes, filtered to remove the resin and washed with methanol (50 mL) at temperature 25°C to 30°C. The filtrate was concentrated under vacuum. Acetone (75 mL) and water (2.5 mL) were added to the residue and heated at 45°C for 30 minutes. The mixture was allowed to cool to 25°C to 30°C filtered and dried to give cilastatin acid. Weight: 3.92 g

HPLC purity (%): 99.15

I) The pH of the solution of ammonium salt of cilastatin (5.1 g) in methanol (100 mL) and water (5.0 mL) were adjusted to 2.78 using cationic exchange resin (Indion-225) at 20°C to 25°C. The reaction mixture was filtered to remove the resin and washed with methanol (20 mL). The filtrate was concentrated under vacuum. Water (20 mL) was added to the resulting residue. The resulting mixture was washed with dichloromethane (20 mL). The aqueous layer was extracted with n-butanol (20 mL) at 40°C. The pH of the n-butanol layer was adjusted to 3.6 using an aqueous ammonia solution (-25%, 0.5 ml). The n-butanol layer was washed with water (20 mL) and concentrated under vacuum. Acetone (75 mL) and water (2.5 mL) were added to the resulting residue and heated to 40°C for 30 minutes. The resulting mixture was allowed to cool 25°C to 30°C, filtered and dried to give cilastatin acid.

Weight: 3.2 g

HPLC purity (%): 99.21

Example 7: Preparation of Cilastatin Methyl Amine Salt

The pH of the solution of cilastatin acid (10 g) in methanol (20 mL) was adjusted to 6.9 using aqueous methyl amine solution (-40%; 4.0 ml) at 25°C to 30°C. The resulting mixture was concentrated under vacuum. Isopropanol (200 mL), water (10 ml) and aqueous ammonia (-25%; 5.0 ml) were added to the resulting residue and heated at 40°C to 45°C for one hour. The mixture was allowed to cool to 25°C to 30°C and stirred at the same temperature for one hour. The mixture was filtered and dried to give methyl amine salt of cilastatin.

Weight: 8.8 g

HPLC purity (%): 99.26

Example 8: Preparation of Cilastatin Sodium

A) Pulverized sodium hydroxide (0.544 g, 13.62 mmol) was added to a solution of cilastatin acid (5 g) in methanol (15 mL). Activated carbon (0.25 g) was added, stirred for about 30 minutes, filtered through a hyflo bed and washed with methanol (5 mL). The filtered solution was dispersed slowly into n-butanol (200 mL) at 20°C to 25°C and stirred for about 2 hours. The product was filtered, and washed with n-butanol (10 mL) followed by acetone (10 mL) and dried at 45°C to 50°C under vacuum to get cilastatin sodium.

Yield: 4.2 g

B) Triethylamine (0.65 g) was added to cilastatin acid (2 g) in n-butanol (40 mL). Activated carbon (0.1 g) was added, stirred for about 30 minutes and filtered the reaction mass through a hyflo bed. Pulverised sodium hydroxide (0.22 g) was added to filtrate and stirred for about 5 hours. The product was filtered, washed with n-butanol (5 mL) followed by acetone (5 mL) and dried at 45°C to 50°C under vacuum to get cilastatin sodium.

Yield: 1.6 g

C) Aqueous sodium hydroxide (3.9 g in 105 mL of water) was added to a solution of cilastatin acid in water (175 mL) and stirred at room temperature for 30 minutes. The resulting mixture was filtered through a micron filter. The filtrate was lyophilized for 36 to 40 hours to give cilastatin sodium.

Yield: 36 g

HPLC purity (%): 99.70