OA16437A

OA16437A - Compounds and their use.

Info

Publication number: OA16437A
Application number: OA1201300221
Authority: OA
Inventors: Senthilkumar Udayampalayam Palanisamy; Maneesh Paul-Satyaseela; Shridhar Narayanan; Gopalan Balasubramanian; Aravind Appu; Senthilnathan Manickam; Hariharan Periasamy
Original assignee: Allecra Therapeutics Gmbh
Priority date: 2010-11-25
Filing date: 2011-11-25
Publication date: 2015-10-15

Abstract

Described herein are compounds and their use in the treatment of infections. The compound of formula (I), their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvates, pharmaceutically acceptable salts and pharmaceutical compositions described herein are also useful as ß -lactamase inhibitors, which restore or enhance the antibiotic spectrum of a suitable antibiotic agent. The compounds of formula (I) act as inhibitors of ß-lactamases. These compounds restore/potentiate the activities of ß-lactam antibiotics against carbapenemases. These compounds find use in diagnostic method for detecting ß-lactamases.

Description

Described herein are the use of β-lactam compounds as β-lactamase inhibitor, their analogs, dérivatives, tautomeric forms, stereoisomers, poiymorphs, solvatés, pharmaceutically acceptable salts, esters, prodrugs and métabolites thereof, for treating bacterial infections in combination with suitable antibiotic. The pharmaceutical compositions of these compounds for treating bacterial infections are described. The compounds described herein are used as diagnostic reagent for the détection of βlactamases.

Background

The β-lactam type antibiotics, namely pénicilline, cephalosporins, carbapenems, monobactams are frequently used antibiotics. It is known that βlactamases produced by microorganisms hydrolyze the p-iactam ring thereby deactivating antibiotic activity. In order to inhibit the β-lactamases, β-lactamase inhibitors are administered in combination with antibiotics. These inhibitors fonction by binding to the β-lactamase enzymes more efficiently than the β-lactam antibiotic itself. This combination helps the antibiotic to exert its antibiotic effect without being degraded by the β-lactamase enzymes. Several antibiotic/p-lactamase inhibitor combinations exist in the market for example, Ampicillin/Sulbactam, Amoxicillin/Clavulanate, Ticarcillin/Clavulanate, Piperacillin/Tazobactam, etc. These β-lactam/p-lactamase inhibitor combination antibiotics are being used for the treatment of infections caused by bacteria producing β-lactamases excepting especially carbapenemases and inhibitor-resistant β-lactamases in the community and in the hospital setting.

A growing problem by the widespread use of antimicrobiais especially βlactam antibiotics is in the development of antimicrobial résistance. A major cause for antibiotic résistance is due to β-lactamases (e.g., carbapenemases, cephaiosporinases, penicillinases, ESBLs, inhibitor-resistant β-lactamases, etc). Among many known βlactamases, Carbapenemases (e.g., KPC, Sme, NMC-A, IMI, etc.) are recently identified, which are capable of hydrolyzing ail classes of β-lactam antibiotics (Drawz, S.M. and Bonomo, R.A. Clin. Microbiol. Rev. 2010, 23(l), 160-201). These enzymes are known for their rôle in multidrug résistance (MDR). In view of the pressing need

lactamases, our research efforts in identifying potential BLIs resulted in the compound of formula (1).

To address the need for proper diagnostic method for spécifie détection of βiactamases, diagnostic method was identified using the compounds of formula (I).

Among many β-lactamase înhibitors that are known in the literature, compounds of formula (A) are disclosed in US 4,562,073,

COOR¹ wherein R¹ is hydrogen or trialkylsilyl; R² is hydrogen, trialkylsilyl or COOR2’ wherein R₂' is hydrogen, Ci._1R alkyl, C₂.₇ alkoxymethyl, C3.8 alkylcarbonyloxymethyl, C4-9 alkylcarbonyloxyethyl, (C5.7 cycioalkyl)carbonyloxymethyl, C9-14 benzylcarbonyloxyalkyl, alkoxycarbonylmethyl, C4.9 alkoxycarbonylethyl, phthalidyl, crotonolacton-4-yl, gamma-butyrolacton-4-yl, halogenated alkyl substituted with 1 to 3 halogen atoms, alkoxy- or nitro-substîtuted or unsubstituted benzyl, benzhydryl, tetrahydropyranyl, dimethylaminoethyl, dimethylchlorosilyl, trichlorosilyl, (5-substituted C]_6 alkyl or phenyl or unsubstituted-2-oxo-l,3-dioxoden4-yl)methyl, Cg-i3 benzoyloxyalkyl or group for form ing a pharmaceutically acceptable 20 sait; and R³ has the same meaning as above R₂'

Our patent, US 7,687,488 B2 (Indian équivalent IN 1217CHE2006) disclosed compounds of the formula (B). These compounds were shown to potentiate the

wherein A = C or N; Het is a three- to seven-membered heterocyclic ring; R¹ represents carboxylate anion or -COOR⁴ wherein R⁴ represents hydrogen, carboxylic acid protecting group or a pharmaceutically acceptable sait; R² and R³ may be same or different and independently represent hydrogen, halogen, amino, protected amino or optionally substituted alkyl, alkenyl, alkynyl and the like; R is represented by substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, oxo, heterocyclyl, heterocyclylalkyl groups.

There is a widespread need for β-lactamase inhibitors which are capable of inhibiting the β-lactamase enzymes, in particular, carbapenemases producing multidrug résistant bacteria. Moreover, there is a unmet medical need for combination drugs in antibiotics, specifically β-lactam antibiotics and β-lactamase inhibitors which overcome the bacterial résistance.

Objectives

One objective herein is to use the β-iactam compounds of the formula (I) as βiactamase inhibitor in combination with suitable antibiotics for treating infection caused by bacteria producing β-lactamases tike carbapenemases, cephalosporinases, penicillinases, ESBLs, inhibitor-resistant β-lactamases, ESBLs and the like.

Another objective herein is to provide a pharmaceutical composition with the compounds of formula (I) in combination with suitable antibiotics.

Yet another objective herein is to provide a method of treating or preventing bacterial infection in a host, typically an animal and most typically a human, including administering to the host a therapeutic amount of compound of formula (I) or a pharmaceutically acceptable sait and/or prodrug therein along with β-Iactam antibiotics.

Another objective herein is to provide a diagnostic reagent for the détection of β-lactamases. The said β-lactamases belong to the families of KPC (e.g., K.PC-2, KPC-3) & ESBL (e.g., SHV18) producing Enterobacteriaceae.

One more objective herein is to restore/potentiate the activity of antibiotics especially β-lactam antibiotics such as Penicillins, Cephalosporins, Carbacephem, Oxacephem, Carbapenems, Penams, Cephamycins, Penems and Monobactams towards carbapenemases and ESBLs by combining with compound of formula (I).

It is therefore an object of the présent invention to provide a compound for inhibiting β-lactamase; and/or a pharmaceutical composition comprising said compound; and/or an improved method for inhibiting β-lactamase in a cell; and/or an improved method for the treatment and/or prévention of a condition mediated by βlactamase; and/or an improved method for the treatment and/or prévention of a bacterial infection along with β-lactam antibiotic; and/or to restore/potentiate the activity of antibiotics; or at least to provide the public with a useful choice.

Su m mary

Described herein is a method or use of compound of formula (I), their dérivatives, analogs, tautomeric forms, stereoisomers, poiymorphs, solvatés, pharmaceutically acceptable compositions, métabolites, prodrugs, pharmaceutically acceptable salts and esters thereof;

In particular, provided herein are compound of formula (1), their dérivatives, analogs, tautomeric forms, stereoisomers, poiymorphs, solvatés, métabolites, prodrugs, hydrates, pharmaceutically acceptable salts and esters, for use in the inhibition of β-lactamases comprising carbapenemases, cephalosporinases, penicillinases, ESBLs, inhibitor-resistant β-lactamases, produced by bacteria; potentiating/restoring the activity of antibiotics, comprising administering a therapeutically effective amount of compound of formula (I), to a subject in need thereof; wherein A = CorN;

Het represents substituted or unsubstituted three- to seven-membered heterocyclic ring; R¹ represents carboxylate anion or -COOR⁴; wherein R⁴ represents hydrogen, , C|Côalkyl, Ce-Cioaryl, C6-CioarylC|-C6alkyl methoxybenzyl, nitrobenzyl, silyl, diphenylmethyl, proxetil, axetil, cilexetil, pivoxil, hexetil, daloxate or a pharmaceutically acceptable sait; R² and R³ may be same or different and îndependently represent hydrogen, halogen, amino, protected amino selected from the group consisting of tritylamino, acylamino such as phenylacetylamino, phenoxyacetylamino and benzoyiamino or optionally substituted Cj-Cealkyl, C2C^alkenyl and C₂-C6alkynyl;

R represents substituted or unsubstituted Ci-C^alkyl, Q-Cealkenyl, Ce-Cioaryl, C$C_I0aryiCi-Côalkyl, Cî-Cncycloalkyl, oxo, heterocyclyl and heterocyclylalkyl groups. when the groups R, R² and R³ are substituted, the substituents which may be one or more are selected from lower alkyl (Cj-Cialkyl such as methyl, ethyl, propyl and isopropyl); lower alkoxy (Ci-Qalkoxy such as methoxy, ethoxy and propoxy); lower

alkylthio (Ci-C₄alkylthio such as methylthio and ethylthio); lower alkylamino (C<C₄alkylamino such as methylamino, ethylamino and propylamino); cyclo(lower)alkyl (Cj-Côcycloalkyl such as cyclopentyl and cyclohexyl); cyclo(lower)alkenyl (C5Cecycloaikenyl such as cyclohexenyl and cyclohexadienyl); hydroxy; halogen (chloro, bromo, fluoro and iodo); amino; protected amino; cyano; nitro; carbamoyl; -CONH Cj-C₄aikyl-COO-Cj-C₄alkyl; carboxy; protected carboxy; -COO-Ci-C₄alkyl; -COheterocyclyl; sulfonyl; sulfamoyl; imino; oxo; amino(lower)alkyl such as aminomethyl, aminoethyl and aminopropyl; halo(lower)alkyl such as trifluoromethyl (CF3), fluoromethyl, fluoroethyl, bromomethyl and difluoromethyl; carboxylic acid and carboxylie acid dérivatives such as hydroxamic acid, ester and amide. Preferred substituents are Ci-C₄alkyl, C|-C₄alkoxy, Ct-C₄alkylthio, Ci-C₄alkylamino, hydroxyl, halogen and trihalomethyl. The substituents are further optionally substituted with CiC₄alkoxycarbonylCi-C4aikyl, hydroxyCi-C₄a)kyl; Ci-C₄alkyl, Ce-Cioary), heterocyclyl and esters.

In one aspect, provided herein are compound of formula (II), their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvatés, métabolites, prodrugs, hydrates, pharmaceutically acceptable salts and esters for use in inhibition of carbapenemases produced by bacteria; potentiating/restoring the activity of antibiotics, comprising administering a therapeutically effective amount of compound of formula (II), to a subject in need thereof;

•R

wherein

L = CorN;

R, R¹, R² and R³ are as defined earlier,

R^s represents hydrogen, Ci-C^lkyl, Ci-C₆alkoxy, Cj-Csalkylthio, C[-C₆alkylamino, hydroxyl, halogen and trihalomethyl; and m is 0, 1 or 2.

In another aspect, provided herein is compound for use in treating and/or preventing infections caused by carbapenemase producing bacteria, comprising administering therapeutically effective amount of compound of formula (I), to a subject in need thereof.

In yet another aspect, provided herein is compound for use in treating and/or preventing infection caused by carbapenemase producîng bacteria comprising administering therapeutically effective amount of compound of formula (I), in combination with suitable antibiotics to a subject in need thereof.

In yet other aspect, provided herein is the compound for use, for treating infections caused by β-lactamases expressed by gram négative bacteria

In yet other aspecL provided herein is the compound for use, wherein bacteria are selected from Klebsiella pneumoniae and E. coli.

In yet other aspect, provided herein is the compound for use, wherein the carbapenemases are selected from KPC-2 and KPC-3.

In yet another aspect, provided herein is the method of treatment or prévention of infection caused by carbapenemase producîng bacteria comprising administering therapeutically effective amount of compound of formula (I).

Another aspect herein includes détection of β-lactamases expressed by Enterobacteriaceae and non- Enterobacteriaceae.

Yet another aspect herein includes use of compound of formula (I) as a diagnostic reagent for the détection of β-lactamases. The said β-lactamases belong to famîlies of KPC-2, KPC-3 and also ESBLs such as SHV18 producîng Enterobacteriaceae.

In one embodiment, provided herein is pharmaceutical composition, comprising a compound of formula (I), as an active ingrédient to treat or prevent infections caused by carbapenemase producîng bacteria.

In another embodiment, provided herein is pharmaceutical composition comprising a compound of formula (I)» as an active ingrédient to treat or prevent infections caused by carbapenemase producîng bacteria along with

a. one or more compounds of formula (I);

b. one or more antibiotics and

c. one or more pharmaceutically acceptable carrier.

In yet another embodiment, the antibiotics are selected from β-lactam antibiotics.

In yet other embodiment, provided herein are the compounds, (25,35,55)-3Methy l-3-(3-methy l-imidazol-3 -ium-1 -y I methy 1)-4,4,7-trioxo-4-th ia-1 azabicyclo[3.2.0]heptane -2-carboxylate and (25,35,55)-3 -Methy 1-3 -(4-methyl-316437 methyl-imidazol-3-ium-1 -ylmethyl)-4,4,7-trioxo-4-thia-1 -aza-bicycIo[3.2.0]heptane-2carboxylate, their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphe, solvatés, métabolites, prodrugs, pharmaceutically acceptable salts and esters.

In yet another aspect the compounds (25,35,55)-3-Methyl-3-(3-methylimidazol-3-ium-l-ylmethyl)-4,4,7-trioxo-4-thia-1 -azabicyclo[3.2.0]heptane-2carboxylate; (25,35,55)-3-Methyl-3-(4-methyl-3-methyl-imidazol-3-ium-l-ylmethyl)4,4,7-trioxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylate and their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvatés, métabolites, prodrugs, pharmaceutically acceptable salts and esters for use in the inhibition of β-lactamases, without limitation, carbapenemases, cephaîosporinases, penicillinases, ESBLs and inhibitor-resistant β-lactamases.

In yet other aspects, described herein are the compound of formula (I) for use in the treatment and/or prévention of bacterial résistance to an antibiotic.

Brief Description of Figure:

Figure 1: Double Disk Synergy Test for détection of KPC β-lactamases Detailed Description β-Lactam compounds of formula (I),

their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvatés, their pharmaceutically acceptable compositions, pharmaceutically acceptable salts and esters thereof, for use in the inhibition of carbapenemases produced by bacteria; potentiating/restoring the activity of antibiotics, wherein:

Het is a three to seven membered heterocyclic ring which may hâve suitable substituent(s) and, préférable heterocyclic group such as pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, piperidinyl, fiiranyl, thiophenyl, pyrrolidinyl, piperazinyi, oxazolidinyl, thiazolyl, pyrîdaziny), tetrazolyl (e.g. 1Htetrazolyl, 2H-tetrazo!yl, etc.), imidazolidinyl, triazolyl, 1,2,4-oxadiazolyl, 1,3,4oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl and 1,2,5-thîadiazolyl.

The defined heterocyclic groups may optionally be substituted with one or more substituents, suitable substituent(s) such as: lower alkyl (C1-C4 alkyl such as methyl, ethyl and propyl); lower alkoxy (C1-C4 alkoxy such as methoxy, ethoxy and propoxy); lower alkylthio (Ct-C4 alkyithio such as methylthio and ethylthio); lower alkylamino (C1-C4 alkyl ami no such as methylamino, ethylamino and propylamino); cyclo(lower)alkyl (Cs-C₆ cycloalkyl such as cyclopentyl and cyclohexyl); cyclo(iower)alkenyl (C₅-C₆ cycloalkenyl such as cyclohexenyl and cyclohexadienyl); hydroxyl; halogen (chloro, bromo, fluoro and iodo); amino; protected amino; cyano; nitro; carboxy; protected carboxy; sulfamoyl; imino; oxo; amino(lower)alkyl (aminomethyl, aminoethyl and aminopropyl); halogen and trihalomethyl (-CF3). Preferred substituents are C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylthio, C]-C4 alkylamino, hydroxyl, halogen and trihalomethyl. The substituents are further optionally substituted.

Typically, the moiety Het is unsubstituted or carries one or more substituents as defined above.

Preferably Het represents a five- to six-membered heterocyclic ring comprising one or two heteroatoms, including the quatemized nitrogen. More preferably, Het is selected from pyrrolyl, pyrrolinyl, imidazolyl, triazolyi, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, piperidinyl, furanyl, thiophenyl, pyrrolidinyl, piperazinyl, oxazolidinyl, thiazolyl, pyridazinyl, pyrrolidinyl and imidazolidinyl.

Preferably, Het is an aromatic ring.

More preferably, Het represents five membered heterocyclic ring;

R¹ represents carboxylate anion or -COOR⁴ wherein R⁴ represents hydrogen, Cfialkyl, Ce-Cioaryl, Ce-CioarylCi-Côalkyl, methoxybenzyl, nitrobenzyl, silyl, diphenylmethyl, proxetil, axetil, cilexetil, pivoxil, hexetil, daloxate or a pharmaceutically acceptable sait;

R² and R³ independently represent hydrogen, halogen, amino, protected amino such as tritylamino, acylamino such as phenylacetylamino, phenoxyacetylamino and benzoylamino; optionally substituted alkyl, alkenyl or alkynyl;

Preferably R is selected from -(CH2)_n-CH3, -(CHîjnCeHs, -(CH2)_n-CH=CH2, -CH2-CONH2, -CH₂-COO-(Ci-C4alkyl) comprising -CH₂COOBu', -(CH₂)_nCOheterocyclyl, -CH₂-CONH-(CH2)n-COOEt, where n is an integer ranging from 0 to 5.

More preferably, R is -fCH₂)_n-CH₃, -(CH₂)_nC₆H_s, -<CH₂)_n-CH=CH₂, -CH₂CONH₂ or -CH₂COOBu^r.

As used herein, a Ci-Csalkyl group or moiety is a linear or branched alkyl group or moiety containing from l to 6 carbon atoms. Typically a Ci-Cô alkyl group or moiety is a C1-C4 alkyl group or moiety. A C]-C4 alkyl group or moiety is a linear or branched alkyl group or moiety containing from l to 4 carbon atoms. Examples of CiCe alkyl groups and moieties include, without limitation, methyl, ethyl, n-propyl, ipropyl, n-butyl, r-butyl, t-butyl, 3-methyl-butyl, pentyl and hexyl. Examples of C_t-C4 alkyl groups and moieties include, without limitation, methyl, ethyl, n-propyl, r-propyl, n-butyl, ί-butyl and z-butyl. For the avoidance of doubt, where two alkyl moieties are présent in a group, the alkyl moieties may be the same or different, which may be optionally substituted by one or more substituents.

The term “C₂-C6alkenyl” refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched chain having about 2 to 6 carbon atoms, which may be optionally substituted by one or more substituents. Preferred alkenyl groups include, without limitation, ethenyl, l-propenyl, 2-propenyl, iso-propenyl, 2-methyl-l-propenyl, l-butenyl and 2-butenyl.

As used herein, a Cs-Cioaryl group or moiety is typically phenyl or naphthyl. Phenyl is preferred.

The term “Ce-CioarylCi-Côalkyl” refers to an aryl group directly bonded to an alkyl group, which may be optionally substituted by one or more substituents. Preferred arylalkyl groups include, without limitation, -CH₂C₆H5, -C2H4C6H5,CH(CH₃)C6H₅ and the like.

As used herein, the term “heterocyclyl” refers to a 5 to IO membered heterocyclyl group or moiety is a monocyclic non-aromatic, saturated or unsaturated C5-C10 carbocyclic ring in which one or more, for example l, 2, 3 or 4 of the carbon atoms are replaced with hetero atoms selected from N, O, S, S(O) and S(O)₂. Typically, it is a 5 to 6 membered ring. Suitable heterocyclyl groups and moieties include pyrazolidinyl, piperidinyl, piperazinyl, thiomorpholinyl, S-oxothiomorpholinyl, S,S-dioxo-thiomorpholinyl, morpholinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, 1,3-dioxolanyl, 1,4-dioxolyl and pyrazolinyl groups and moieties. Pyrazolidinyl, piperidyl, piperazinyl, pyrazolidinyl, morpholinyl and imidazolidinyl groups and moieties are preferred.

The term “heterocyclylalkyl” refers to heterocyclyl group directly bonded to an alkyl group, which may be substituted or unsubstituted.

The term “Cî-Cucycloalkyl” refers to non-aromatic mono or polycyclic ring system of about 3 to 12 carbon atoms, which may be optionally substituted by one or more substituents. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl and perhydronaphthyi.

The term “analog” includes a compound, which differs from the parent structure by one or more C, N, O or S atoms. Hence, a compound in which one of the N atoms in the parent structure is replaced by an S atom is an analog of the former.

The term “dérivative” refers to a compound obtained from a compound according to formula (I), an analog, tautomeric form, stereoisomer, polymorph, hydrate, pharmaceutically acceptable sait or pharmaceutically acceptable solvaté thereof, by a simple chemical process converting one or more functional groups, such as, by oxidation, hydrogénation, alkylation, estérification, halogénation and the like.

The term “stereoisomer” includes isomers that differ from one another in the way the atoms are arranged in space, but whose chemical formulae and structures are otherwise identicai. Stereoisomers include enantiomers and diastereoisomers.

The term “tautomers” include readily interconvertible isomeric forms of a compound in equilibrium. The keto-enol tautomerism is an example.

The term “polymorphs” include crystallographically distinct forms of compounds with chemically identicai structures.

The term “pharmaceutically acceptable solvatés” includes combinations of solvent molécules with molécules or ions of the soluté compound.

Représentative compounds (1-13) exhibiting β-lactamase inhibitory properties include but not limited to:

1. l-{[(25,35,5A)-2-Carboxy-3~methyl-4,4,7-trioxo-4-thia*l-azabicyclo[3.2.0]hept-3yl]methyl}-3-methyl-177-1,2,3-triazol-3-ium;

2. l-{[(25,3Y,57?)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2.0]hept-3yl] methyl} -3-ethyl-7/7-l ,2,3-triazol-3-ium;

3. 1 -{[(2S,3S,57î)-2-Carboxy-3-methyM,4,7-trioxo-4-thia-l -azabicyclo[3.2.0]hept-3yl]methyl}-3- n-propyl-J H-1,2,3-triazoi-3-ium;

4. 1-( [(25,35,57?)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-l -azabicycIo[3.2.0]hept-3yl]methyl}-3- allyl-/W-l ,2,3-trîazol-3-ium;

5. 1-(((25, 35,5/?)-2-Carboxy-3-methyl-4,4,7-trioxo4-thia-l-azabicyclo[3.2.0]hept-3yl]methyl}-3-(2-amino-2-oxoethyl)-77f-l,2,3-triazol-3-ium and the corresponding acid;

6. I-{[(25,35,57?)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-l -azabicyclo[3.2.0]hept-3yl]methyl}-3-(2-i-butoxy-2-oxoethyl)-7/f-l,2,3-triazol-3-ium and the corresponding acid;

7. 1-(((25,35,5/ï)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2.0]hept-3yl]methyl}-3-(2-morpholin-4-yl-2-oxoethyl)-7J7-l,2,3-triazol-3-ium and the corresponding acid;

8. 1-(((25,35,5Â)-2-Carboxy~3-methyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2.0]hept-3yl]methyl}-3-(2[(2-ethoxy-2-oxoethyl)amino]-2-oxoethyl)-777-l,2,3-triazol-3-ium and the corresponding acid;

9. l-{[(25,35,5A)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2.0]hept-3- y Ijmethy 1} -3-(2-[(3 -ethoxy-3 -oxopropy l)amino]-2-oxoethy 1 ,2,3-triazol-3 ium and the corresponding acid;

10. l-{[(25,35,57?)-2-Carboxy-3-methyl-4,4,7-trioxo4-thia-l-azabicyclo[3.2.0}hept-3yl]methyl}-3-(2-{[l-(ethoxycarbonyl)-2-hydroxypropyl]amino}-2-oxoethyl)-777l,2,3-triazol-3-ium and the corresponding acid;

11. 1 - {[(25,35,57?)-2-Carboxy-3 -methy 1-4,4,7-trioxo-4-thia-1 -azabicyclo[3.2.0]hept-3yl]methyl}-3-ben2yl-177-l,2,3-triazol-3-ium and the corresponding acid;

12. (2S,35,57î)-3-Methyl-3-(3-rnethyl-irnidazol-3-iurn- ] -ylmethyl)-4,4,7-trioxo-4-thial-azabicyclo[3.2.0]heptane-2-carboxylate and the corresponding acid; and

13. (2.S',3A',57?)-3-Methyl-3-(4-methyl-3-methyi-imidazol“3~ium~ 1 -ylrnethyl)-4,4,7trioxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylate and the corresponding acid.

These compounds (1 to 11) were prepared by foilowing the procedures provided in US 7,687,488 (Indian équivalent IN 1217CHE2006).

The compounds 12 and 13 are prepared according to reaction scheme as shown below:

wherein Het is

Compound of formula (IV) was obtained by the reaction of compound of formula (VI) with the compound of formula (V) in Step-1. In Step-2, the compound of formula (IV) was converted to the compound of formula (III). The conversion of compound of formula (III) to a compound of formula (I) may be carried out using silylatîng agent selected from hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS), trimethylsilyl iodide (TMSI), N,O-bis-(trimethylsilyl)-acetamide (BSA), methyltrimethylsilyltrifluoroacetamide (MSTFA), N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA), methyldichlorosilane, dimethyldichlorosilane, diphenyldichlorosilane, N-methylsilylacetamide (MSA), bistrimethylsiiylurea and the like in the presence of solvents like acetone, methanol, tetrahydrofuran, chloroform, dichloromethane, dichloroethane, ethyl acetate, N,Ndimethylformamide (DMF), Dimethylacetamide (DMAc) and the like or a mixture thereof. The compound of formula (1) was obtained by the reaction of compound of 20 formula (III) with a suitable R-X (X = halogen).

The β-lactam compounds described herein are preferably formed as inner salts. When the représentative substitution on R is carboxylic acid or amino group, it may be further converted to pharmaceutically acceptable salts. Bases used for making salts of carboxylic acid groups are selected from base such as sodium hydroxide, sodium 25 methoxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, calcium hydroxide, magnésium hydroxide and the like, in solvents like ether, tetrahydrofuran, methanol, /-butanol, dioxane, isopropanol, éthanol, etc. Mixture of solvents may be used. Acid addition salts could also be prepared using appropriate acid.

The stereoisomers of the compounds forming part of this invention may be prepared by using reactants in their single enantiomeric form, in the process wherever possible or by conducting the reaction in the presence of reagents or catalysts in their single enantiomeric form or by resolving the mixture of stereoisomers by conventional methods. Some of the preferred methods include use of microbial resolution, resolving the diastereomeric salts formed with chiral acids such as mandelic acid, camphorsulfonic acid, tartane acid, lactic acid and the like, wherever applicable or by using chiral bases such as brucine, cinchona alkaloids, their dérivatives and the like.

Prodrugs of the compounds of formula (I) are also contemplated by this invention. A prodrug is an active or inactive compound that is modified chemically through in-vivo physiological action, such as hydroiysis, metabolism and the like, into a compound of this invention following administration of the prodrug to a patient. The suitability and techniques involved in making, using prodrugs are well known by those skilled in the art.

Various polymorphs of compound of general formula (I) may be prepared by crystallization of compound of formula (I) under different conditions known in the prior art. For example, using different solvents commonly used or their mixtures for recrystallization; crystallizations at different températures; various modes of cooling, ranging from very fast to very slow cooling during crystallizations. Polymorphs may also be obtained by heatîng or melting the compound followed by graduai or fast cooling. The presence of polymorphs may be determined by Solid Probe NMR Spectroscopy, IR Spectroscopy, Differential Scanning Calorimetry, Powder X-ray Diffraction or such other techniques.

Pharmaceutically acceptable solvatés of the compounds of formula (I) may be prepared by conventional methods such as dissolving the compounds of formula (I) in solvents such as water, methanol, éthanol, mixture of solvents such as acetone: water, dioxane: water, N,N-dimethyiformamide:water and the like, preferably water and recrystallizing by using different crystallization techniques.

It should be noted that compounds described herein may contain groups that may exist in tautomeric forms and though one form is named, described, displayed and/or claimed herein, ail the forms are intended to be inherently included in such name, description, display and/or claim.

The β-lactam compounds disclosed herein in combination with a β-lactam antibiotic are useful for the treatment of microbial infections in humans and other warm blooded animais, under both parentéral, topical and/or orai administration. In addition to the compounds of formula (I), the pharmaceutical compositions may also contain or be co-administered with one or more known drugs selected from other clinically useful antibiotic agents such as Penicillins (Piperacillin, Ticarcillin and the like), Cephalosporins (Ceftazidime, Cefmetazole, Cefotaxime and the like), Penems (Faropenem, Meropenem, Ertapenem and the like), Carbacephem (Loracarbef and the like), Oxacephem (Moxalactam, Latamoxef, Flomoxef and the like), Cephamycins (Cefotetan and the like) Monobactams (Aztreonam, Tigemonam and the like), Aminoglycosides (Streptomycin, Gentamicin, Amikacin and the like), Bacteriocins (Colicins, Microcins and the like), Quinolones (Ciprofloxacin, Moxifloxacin and the like), Sulfonamîdes (Sulfamethoxazole and the like), Macrolïdes (Erythromycin, Roxithromycin, Azithromycîn and the like), Tetracyclines (Doxycycline, Minocycline and the like), Glycylcyclines (Tigecycline and the like), Oxazolidinones (Linezolid, Torezolid, Radezolid and the like), Lipopeptides (Daptomycin and the like), Polypeptides (Actinomycin, Bacitracin, Colistin, Polymixin B and the like), Polyene antifungals (Natamycîn, Nystatin, Amphotericin B and the like), Rifamycins (Rifampicin, Rifabutin, Rifapentine and the like), Chloramphenicol and the like or dérivatives thereof.

Antibiotics include Penicillins, Cephalosporins, Carbacephems, Oxacephems, Carbapenems, Penams, Cephamycins, Penems, Monobactams or a combination thereof.

Pencillins include, but are not limited to, Arndinocillin (Mecillinam), Amoxicillin, Ampicillin, Amylpenicillin, Apalcillin, Aspoxiciliin, Azidocillin, Azlocillin, Bacampicillin, Carbeniciliin, Carindacillin, Clometocillin, Cloxaciilin, Cyclacillin (Ciclacillin), Dicioxacillin, Epicillin, Fenbenicillin, Floxacillin (flucloxacillin), Hetacillin. Lenampicillin, Metampicillin, Methicillin, Mezlocillin, Nafcillin, Oxacillin, Penamecillin, Penethecillin, Penicillin G (Procaine Pencillin), Penicillin N, Penicillin O, Penicillin V (Phenoxymethyl Penicillin), Phenethicilün, Piperacillin, Pivampîcillin, Propicillin, Quinacillin, Sulbenicillin, Talampicillin, Temocillin, Ticarcillin, Pivmecillinam, Benzathine Penicillin, Benzyl Penicillin, Coamoxiclav, Lenampicillin or a combination thereof.

Cephalosporins include but not limited to Cephaloridin, Cephradine, Cefoxitin, Cephacetril, Cefoperazone, Cefinenoxime, Cephaloglycin, Cefonicid, Cefodizime, Cefpirome, Cefpiramide, Cefozopran, Cefoselis, Cefluprenam, Cefpimizole, Cefclidin, Cefpodoxime axetil, Cefteram pivoxil, Cefcapene pivoxil, Ceftobiprole, Ceftaroline, Cefquinome, Ceftîofur, Cefovecin, Cefadroxil, Cefalonium, Cefepime, Cefotaxime, Ceftazidime, Cefetamet pivoxil, Cefditoren pivoxil, Cephaloridine, Ceftazidime, Ceftriaxone, Cefbuperazone, Cephalothin, Cephazolin, Cephapirin, Ceftezole, Cefamandole, Cefotiam , Cefotiam hexetil, Cefiiroxime, Ceftizoxime, Cefinenoxime, Cefuzonam, Cefsulodin, Cefmetazole, Cefminox, Cephalexin, Cefradine, Cefaclor, Cefadroxil, Cefalonium, Cefprozil, Cefuroxime axetil, Cefixime, Cefpodoxime proxetil, Ceftibuten, Cefdinir, CXA-l0l(FR264205) or a combination thereof;

Penems include, without limitation, Faropenem and Carbapenems include, without limitation Meropenem, Ertapenem, Doripenem, Biapenem, Panipenem, Ritipenem, Tebipenem, Tomopenem, Sulopenem, Razupenem, Imipenem, ME1036, SM216601 or a combination thereof.

Monobactams include, without limitation, Aztreonam, Carumonam, Tigemonam, BAL19764, BAL30072 or a combination thereof.

β-lactam antibiotics in combination with compounds of the formula (I) may also be co-administered with Aminoglycosides, Bacteriocins, Quinolones, Sulfonamides, Macrolides, Tetracyclines, Glycylcyclines, Oxazolidinones, Lipopeptides, Polypeptides, Rifamycins, Chloramphenicol, Polyene antifungals and dérivatives thereof.

Compounds of the formula (I) may also contain or be co-administered with bactericidal/permeability-increasing protein product (BPI) or efflux pump inhibitors to improve activity against gram négative bacteria and bacteria résistant to antîmicrobial agents. Antiviral, antiparasitic, antifungal agents and other antibiotics can also be administered in combination with the inhibitor compounds of formula (I). The compound of formula (1) with a suitable antibiotic combination can be used for treating patients with bacteria! infections, preoperative patients, postoperative patients, patients in intensive care unit (ICU), patients with nosocomial infections and veterinaries.

The pharmaceutical composition may be in the forms normally employed, such as tablets, capsules, pills, granules, powders, syrups, lozenges, solutions, suspensions, aérosols, transdermal patches, topical creams and ointments and the like, may contain

ΙΟ flavoring agents, sweeteners, etc. in suitable solid or liquid carriers or diluents or in suitable stérile media to form injectable solutions or suspensions. The pharmaceutical composition may also contain pharmaceutically acceptable carrier that are known in the prior art.

The compounds can be lyophilized alone or in combination with antibiotic compounds/agents as described above optionally including any agents. The agents include complexing agents or anticoagulants, antioxidants, stabilizers, aminoglycosides, pharmaceutically acceptable salts and the like or mixtures thereof. The lyophilization can be performed for dilute solutions or concentrated solutions depending on the required quality of the final product. Prior to lyophilization or fireeze-drying or thawing, the lyophilizate can be degassed to optimum concentration of gas. The compounds can be filtered under stérile condition. Appropriate fllters such as ultrafiltration could also be used in order to reduce the levels of galactomannan substantially. The compounds of formula (I) could also be physically blended with a suitable antibiotic agent.

The compound of formula (I) can also be used for treating infections caused by bacteria producîng β-lactamases, in particular, K.PC-2.

In addition to the compound of formula (I), the pharmaceutical composition may also contain buffers like sodium citrate, sodium acetate, sodium tartrate, sodium carbonate, sodium bicarbonate, morpholinopropanesulfonic acid, other phosphate buffers and the like and chelating agents like ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, hydroxyethylenediaminetriacetic acid, nitrilotriacetic acid, 1,2-diaminocycIohexanetetraacetic acid, bis(2aminoethyl)ethyleneglycoltetraacetic acid, 1,6-hexamethylenediaminetetraacetic acid and the like or pharmaceutically acceptable salts thereof. Compounds of formula (I) are useful in treating or preventing a bacterial infection in a host, typically an animal and most typically a human, including administering to the host a therapeutic amount of compound of formula (I) or a pharmaceutically acceptable sait and/or prodrug therein along with β-lactam antibiotic.

The term “prophylaxis” or “prévention” means preventing the disease, i.e., causing the clinical symptoms of the disease not to develop.

The term “treatment*'/“treating” means any treatment of a disease in a mammal, including: (a) Inhibiting the disease, i.e., slowing or arrestîng the development of clinical symptoms; and/or (b) Relieving the disease, i.e., causing the régression of clinical symptoms.

The term “therapeutically effective amount” or “effective amount” refers to that amount of a compound or mixture of compounds of formula (I) that is sufficient to effect treatment, as defined below, when administered aione or in combination with 10 other thérapies to a mammal in need of such treatment.

The term potentiating refers to the enhancement of the effects of an agent by another agent so that the total effect is greater than the sum of the effects of either agent.

The term “compound(s) for use” as used herein embrace any one or more of the 15 following: (1) use of compound(s), (2) method of use of compound(s), (3) use in the treatment of, (4) the use for the manufacture of pharmaceutical composition / médicament for treatment/treating or (5) method of treatment / treating/ preventing / reducing t inhibiting comprising administering an effective amount of compound of formula (I) to a subject in need thereof.

The term ‘subject’ refers to patients with bacterial infections, preoperative patients, postoperative patients, patients in ICU, patients with nosocomial infections, community acquired infections and veterinaries.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and without departing from the spirit and 25 scope thereof make various changes and modifications of the invention to adapt it to various usages and conditions.

A term once described, the same meaning applies for it throughout the patent. Reference Compound-1 (Compound-1) l-{[(25,3S, 57f}-2-Carboxy-3-niethyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2.01hept-330 ylJmethyl}-3-methyl-7//-1^23-triazol-3-ium

To a suspension of (25,35,5A)-3 -methyl-7-oxo-3-(lH-1,2,3 -triazol-1 -yImethyl)4-thîa-l-azabicycio-[3.2.0]heptane-2-carboxyiic acid 4,4-dioxide (25 g) in acetone (100 mL) at 25-30 ‘C was added slowly N,O-bis(silyl)acetamide (18.6 g) with stirring.

The reaction mixture was stirred at this température (25-30 °C) for 15-20 minutes. To

the clear solution obtained, methyl iodide (100 mL) was added over a period of 15 minutes and stirred at 25-30 minutes for 24 hours. The precipitated solid was separated by filtration and washed with acetone (25 mL). Wet weight of the solid obtained was 30 g.

The above wet solid was stirred with purified water (300 mL) at 10-15 °C for 2.5 hours. To the resulted reaction mixture was added sodium thiosulfate (0.1 g) and stirred at 10-15 ’C for 10-15 minutes. To the reaction mixture, dichloromethane (300 mL) was added, stirred and the organic layer was separated, The aqueous layer was washed with a solution of Amberlite LA-2 resin (5% solution in dichloromethane twice, followed by dichloromethane twice. To the aqueous solution, actîvated carbon (1 g) was added, stirred for 15 minutes, filtered and washed with purified water (25 mL). The solution was filtered and lyophilized to get the title compound in pure form (10 g). 'H NMR (400 MHz, DMSO-d₆) δ ppm: 1.39 (s, 3H), 3.14 (dd, J = 16.0, 1.3 Hz, 1H), 3.55 (dd, J = 16.0, 4.2 Hz, 1H), 3.97 (s, 1H), 4.34 (s, 3H), 5.05 (dd, J = 4.2, 1.3 Hz, 1H), 5.29 (d, J = 14.7 Hz, 1H), 5.42 (d, J = 14.7 Hz, 1H), 8.91 (d, J = 1.3 Hz, 1H), 8.99 (d, J = 1.3 Hz, 1H). Mass m/z: M+1 peak at 315. Alternatively the solution could be subjected to spray-dry ing to yield the title compound.

Compound-12 (IS^S, 5Z?)-3-Methy l-3-(3-methyl-i midazol-3-ium-l-y 1 methyl)-4,4,7-t rioxo-4-th ial-azabicyclo|3.2.0]heptane-2-carboxyIate

Step 1: Préparation of (253^,5/î)-3-(imidazol-l-ylmethyl)-3-methyl-7-oxo-4-thia1-aza-bicyclo [3.2.0] heptane-2-carboxylic acid benzhydryl ester

To a stirred solution of imidazole (1.696 g, 24.9 mmol) in acetonitrile (75 mL) and water (25 mL) was added sodium bicarbonate (4.18 g, 49.8 mmol) and the résultant mass was stirred for 15 minutes. (2S,3S,57ï)-3-Chloromethyl-3-methyl-7oxo-4-thia-l-aza-bicyc!o[3.2.0]heptane-2-carboxylic acid benzhydryl ester (10 g, 24.8 mmol) was added to the above mixture and stirred at 25-30 °C for 24 hours. After the completion of the reaction, the reaction mass was diluted with ethyl acetate and water mixture. The organic layer was separated. The aqueous layer was again extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulphate and concentrated under vacuum to yield crude (23,3S,57Î)-3-(imidazol-l-ylmethyl)-3methyl-7-oxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester. Yield: 10 g.

Step 2: Préparation of (2333,5^)-3-(imidazol-l-ylmethyl)-3-methyM,4,7-trioxo· 4-thia -l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester

The crude (23,33,5A)-3-(imidazo!-l-ylmethyl)-3-methyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylîc acid benzhydryl ester (10 g) obtained in the previous step was dissolved in acetonitrile (50 mL). Acetic acid and water mixture was added to the above solution and was cooled to 0 - 5 °C. To the homogeneous reaction mixture potassium permanganate (14.59 g, 92.3 mmol) was added. Stirring was continued at 0 - 5 °C for another 2 hours. The reaction mass was quenched with sodium metabisulphite solution. The réaction mass was diluted with ethyl acetate and water mixture. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layer was neutralised with saturated sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. Acetone was added to the residue obtained and stirred for 30 minutes. A white solid precipitated out, which was filtered and dried. Yield: 2.60 g (22.4 %). 'H NMR (400 MHz, DMSO-dé) δ ppm: 1.09 (s, 3H), 3.35 (d, J = 16.0 Hz, 1H), 3.76 (dd, J = 16.0, 2.0 Hz, 1H), 4.42 (d, J = 15.6 Hz, 1H), 4.90 (d, J = 15.6 Hz, 1H), 5.10 (s, 1H), 5,26(m, 1H), 6.89 (s, 2 H), 6.98 (s, 1H), 7.33 - 7.50 (m, 11H). Mass m/z: 466 (M+1).

Step-3: Préparation of (2333,5R)-3-(imidazoI-l-ylmethyl)-3-methyl-4,4,7-trioxo4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid (Compound-M)

V

To a solution of (25,35,5JÎ)-3-(imidazol-l-ylmethyl)-3-methyl-4,4,7-trioxo-4thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester (900 mg, 1.9 mmol) in methanol (20 mL) was added 10% Pd/C (900 mg w/w) and stirred under hydrogen atmosphère for 2 hours. The reaction mass was filtered and washed with methanol. The filtrate was evaporated under reduced pressure. To the residue was added diethyl ether (30 mL) and stirred for 15 minutes. The white solid precipitated out was filtered and washed with diethyl ether. Yield: 530 mg (91.3 %). ^lH NMR (400 MHz, DMSO-dé) δ ppm: 1.38 (s, 3H), 3.28 (d, J = 16.4 Hz, IH), 3.68 (dd, J = 16.4,4.4 Hz, IH), 4.51 (d, 15.2Hz, IH), 4.53 (s, IH), 4.84 (d, J = 15.2Hz, IH), 5.14- 5.15 (m, IH), 7.02 (s, IH), 7.25 (s, IH), 7.85 (s, IH). Mass m/z: 300 (M+l).

Step 4: Préparation of (2S0S,5R)-3-methyl-3-(3-methyl-imidazol-3-Îum-ly!methyl)-4,4,7-trioxo-4-thia-l-azabicycIo[3.2.0Jheptane-2-carboxyIate

o.

·/ «Hj

COJ]

To a suspension of (2<S,35,57î)-3-(imidazol-l-ylmethyl)-3-methyl-4,4,7-trioxo4-thîa-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid (450 mg, 1.5 mmol) in dry acetone (1.8 mL) was added slowly N, O-bis(silylacetamide) (0.93 mL, 3.7 mmol) with stirring. The reaction mass was stirred further for 15 minutes. To the clear solution obtained, methyl iodide (1.8 mL) was added and stirred at 25 - 30 °C for 2 days. The reaction mass was concentrated and diluted with dichloromethane-water. The organic iayer was separated. The aqueous iayer was washed with a solution of Amberlïte LA-2 resin (30% solution in dichloromethane), followed by dichloromethane. The aqueous Iayer was degassed and lyophilized to obtain the title compound. Melting point: 161.37 °C. ^lH NMR (400 MHz, DiO) δ ppm: 1.53 (s, 3H), 3.47 (dd, J = 16.7, 1.36 Hz, IH), 3.70 (dd, J = 16.7, 4.2 Hz, IH), 3.94 (s, 3H), 4.41 (s, IH), 4.99 (ABquartet, J = 15.4Hz, 2H), 5.09 (m, IH), 7.53 (s, IH), 7.64 (s, IH), 8.99 (s, IH). Mass m/z: 314 (M+l). Compound 13 (25^^J?)-3-Methyl-3-(4-methyl-3-methyl-imidazol-3-iuni-l-ylmethyl)-4,4,7trioxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylate

Step 1; Préparation of (25'_r3S'^i)-3-(4-methyl-iniidazol-l-ylmethyl)-3-niethyl-7oxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester

To a stirred solution of (2S,3S,5/î)-3-chloromethyl-3-methyl-7-oxo-4-thia-laza-bicyclo[3.2.0]heptane-2-carboxylicacid benzhydryl ester (3 g, 7.4 mmol) in acetonitrile (22.5 mL)) was added sodium bicarbonate (628 mg, 7.4 mmol), water (7.5 mL) and 4-methyl-imidazole (1.22g, 7.4 mmol). The résultant mass was stirred at 25 30 °C for 42 hours. The reaction mass was diluted with ethyl acetate and water mixture. The organic layer was separated. The aqueous layer was again extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulphate and concentrated under vacuum to yield crude (2S,35',5JÎ)-3-(4-methyIimÎdazol-lylmethyl)-3-methyl-7-oxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester. Yield: 3.5 g.

Step 2: Préparation of (2S^S'^X)-3-(4-niethyl-imidazol-l-ylniethyl)-3-inethyl4,4,7-trioio-4-thia -l-aza-bicyclo[3.2.0Jheptane-2-carboxylic acid benzhydryl »

ester.

⁰A ,.O

CH,

The crude (25,3S,5/?)-3-(4-methyl-imidazol-l-yImethyl)-3-methyl-7-oxo-4thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester (3.5 g, 7.8 mmol) from the previous step was dissolved in acetonitrile (18 mL). Acetic acid (18 mL) and water (9 mL) mixture was then added to the above solution and cooled to 0 - 5 °C. To the homogeneous reaction mixture potassium permanganate (2.47 g, 15.6 mmol) was added. Stirring was continued at 0 - 5 °C for another 2 hours. The reaction mass was then quenched with sodium metabisulphite solution and diluted with ethyl acetate and water. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layer was neutralised with saturated sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. Purification of the crude compound using silicagel column chromatography (gradient elution with 40-50% ethyl acetate in hexane) yielded the pure compound as a colourless solid. Yield: 350 mg (10 %). *H

ΙΟ

NMR (400 MHz, CDCI3, δ ppm): 1.00 (s, 3H), 2.17 (s, 3H), 3.50 (dd, J = 16.2 Hz, 1.8 Hz, 1H), 3.57 (dd, J = 16.2 Hz, 4.1 Hz, 1 H), 4.24 (d, J = 15.3 Hz, 1H), 4.50 (s, 1H), 4.61-4.62 (m, 1H), 6.53 (s, 1H), 6.99 (s, 1H ),7.05 (s, 1H) 7.32-7.49 (m, 10H).

Step 3: Préparation of (25,3>S\5/?)-3-inethyl-3-(4-methyl-3-methyl-imidazol-3-iuml-ylmethyl)-4,4,7-trioxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester

Ι5

έο^Η

Το a suspension of (2S,35,5/i)-3-(4-methyl-imidazol-l-ylmethyl)-3-methyl4,4,7-trioxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester (350 mg, 0.6 mmol) in dry acetone (4 mL) was added methyl iodide (4 mL) and stirred at 25 - 30 °C for 15 hours. The reaction mass was concentrated and purified using silica gel column chromatography (gradient elution with 0-10 % MeOH in dichloromethane) to yield the product as a pale yellow solid. Yield: 320 mg (96%). ’H NMR (400 MHz, CDC1₃, δ ppm): 1.35 (s, 3H), 2.30 (s, 3H), 3.47 (dd, J = 16.4 Hz, 1.7 Hz 1H), 3.58 (dd, J = 16.4 Hz, 4.4 Hz 1H), 3.89 (s, 3H), 4.6 (s, 1H), 4.69 (m, 1H), 4.89 (ABquartet, J = 15.9 Hz, 2H), 7.01 (s, 1H), 7.26 (s, 1H ), 7.32-7.49 (m, 10H), 9.83(s, 1H).

Step 4: Préparation of (25^S'^jR)-3-methyl-3-(4-methyl-3-methyl-imidazoL3-iuml-ylmethyl)~4,4,7-trioxo-4-thîa-l-aza-bicyclo[3.2.0|heptane-2-carboxylate

i

To a suspension of (25,3S,5R)-3-methyl-3-(4-methyl-3-methyl-imidazol-3-iuml-ylmethyl)-4,4,7-trioxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl ester (310 mg, 0.62 mmol) was added m-cresol (3 mL) and stirred at room température ovemight. Hexane (3 x 25mL) was added to the reaction mixture and stirred for 5 minutes then decanted. Diethyl ether (15 mL) was added to it. The solid obtained was dtluted with water and treated with Amberlite LA-2 resin (30% solution in dichloromethane), followed by dichloromethane. The aqueous layer was lyophilised to yield the product as a pale yellow solid. Yield: 130 mg (75%). *H NMR (400 MHz,

DaO) 8 ppm: 1.52 (s, 3H), 2.31 (s, 3H), 3.47 (dd, J = 16.7Hz, 1.3Hz 1H), 3.71 (dd, J = 16.7Hz, 4.1 Hz 1H), 3.80 (s, 3H), 4.39 (s, 1H), 4.92 (ABquartet, J = 15.4 Hz, 2H), 5.08 (m, 1H), 7.38 (s, 1H ), 8.86 (s, 1H). Mass m/z: 328 (M+l).

The examples below are provided by way of illustration only and should not be considered to limit the scope of the invention. Variation and changes, that are obvious to one skilled in the art, are intended to be within the scope and nature of the invention. Biology:

Détection of KPC/ESBL producing Enterobacteriaceae

In this experiment, Compound-1 is used as a diagnostic reagent for the détection of β-lactamases belonging to the families KPC & ESBL (e.g., SHV18) producing Enterobacteriaceae. A simple set of absorbent paper disks impregnated with antibiotic on agar medium is used for the détection. When the bacterial strain expresses β-lactamases, the zone of inhibition in combination with Compound-1 will be significantly larger than the antibiotic alone.

Methodology 1:

• 0.5 McFarland of the test organism was inoculated at 1:10 dilution on Muller Hinton Agar plates o Test organisme: Klebsiella pneumoniae (K.p) ATCC BAA-1705, Klebsiellapneumoniae ATCC 700603, Escherichia coli (E.c) Ecoli233 • Carbapenem (e.g., Imipenem [IPM] 10 pg) and cephalosporin (e.g., Ceftazidime [CAZ] 30 pg) paper disks (7 mm) were placed on the inoculated agar plates • Compound-1 (60 pg) disk was placed at a distance of 7 & 10 mm from the carbapenem and cephalosporin disks.

• The presence of expressed carbapenemases or ESBLs was measured as the expansion of Imipenem or Ceftazidime’s inhibition zones due to synergy in the presence of Compound-1.

Results:

Synergy was observed as an increase of the Imipenem or Ceftazidime zone adjacent to Compound-1 containing disk (Figure 1).

Methodology 2:

The methodology remains the same as Methodology 1 except the following change · Compound-1 (60 gg) was added on the same disk in combination with a carbapenem (e.g., Imipenem 10 gg) or cephalosporin (e.g., Ceftazidime 30 gg) and placed on the înoculated agar plate.

• The presence of the expressed carbapenemases or ESBLs was measured as an increase in Imipenem or Ceftazidime’s zone diameter in combination with

Compound-1 comparée! to antibiotic alone.

Results

The inhibitoiy activity of Compound-1 on carbapenemases or ESBLs was demonstrated by an increase in the zone diameter (Table 1) of Imipenem or Ceftazidime in combination with Compound-1 compared to antibiotic alone (Figure 1).

Methodology 1 in figures (A), (B) & (C) show an increase in the zone of inhibition of Imipenem or Ceftazidime adjacent to compound-1 containing disk when kept at a distance of 10 mm and 7 mm, Methodology 2 in figures (A), (B) & (C) show an increase in the zone of inhibition of Imipenem or Ceftazidime in combination with compound-1 (IT & CT) rather than the antîbîotic alone (I & C). Both the methods in 20 (D) do not show increase in the zone of inhibition and that compound-1 does not show any zone of inhibition, due to the absence of β-iactamase in the strain.

Results for isolâtes with KPC enzymes are shown in Table 1.

Table 1: Zone of inhibition (ZOI) for clinical isolâtes with class A carbapenemases and ESBL

Phenotype	Organism	Strain ID	ZOI(	mm)
CAZ	1PM
Alone	+ Compound-1	Alone	+ Compound-1
KPC2	K. pneumoniae	ATCC BAA-1705	12	18.5	14.5	20
KPC3	E. coli	Ecoli233	11	22.5	14	20
SHV18	K. pneumoniae	ATCC 700603	12	23	25	25
β-lac-ve	E. coli	ATCC 25922	25	25	26.5	26.5

• Compound-1 increased the zone of inhibition of Ceftazidime from 12 to 18.5 mm and 11 to 22.5 mm against the tested KPC2 and KPC3 producîng strains respectively.

• Compound-1 increased the zone of inhibition of Imipenem from 14.5 to 20 mm and 14 to 20 mm against the tested KPC2 and KPC3 producîng strains respectively.

• Compound-1 increased the zone of inhibition of Ceftazidime from 12 to 23 mm against the tested SHV18 producing strain whîle there was no change in the diameter of Imipenem with or without Compound-1 indicating the inhérent activity of Imipenem against this strain.

• Against the β-lactamase négative strain, there is no impact of Compound-1 either on Ceftazidime or Imipenem since both these antibiotics hâve inhérent activity against this strain.

Conclusion:

Compound-1 can be used as a diagnostic tool for the détection of β-lactamases including KPC.

In vitro Testing

The β-lactam compounds of formula (I) described herein were assessed in combination with β-lactam antibiotics for its potential as β-lactamase inhibitor against carbapenemase enzymes. The compounds described herein were assessed in vitro for antibacterial activity against for example KPC producing & KPC expressîng bacterial gram négative strains, β-lactamase inhibitory assay with these enzymes. The β-lactam compounds having a substitution on the heterocyclyl nitrogen atom(s) show significant β-lactamase inhibiting property. For comparative studies, Tazobactam, Clavulanic acid and Sulbactam were used along wîth the β-lactam antibiotics. Carbapenems, Cephalosporins, Monobactams and Penems (including those of veterinaiy use) were chosen as the antibacterial agents.

In vitro Antimicrobiai Testing by determining the Minimum Inhibitory concentration (MIC): Broth micro dilution method

The β-lactam compound was tested for in vitro antibacterial activitîes by the broth micro-dilution or agar dilution method as specified in documents published by Clinical and Laboratory Standards Institute (CLSI), USA (formerly NCCLS). Approved standard M7-A7, Jan 2006, CLSI, Wayne, Pennsylvania, USA and Ml 00S18, Januaiy 2008, CLSI, Wayne, Pennsylvania, USA.

Synergistic broth micro-dilution MIC was done in checkerboard format with a range of concentrations of the antibacterial agents along with several concentrations of the BLI compounds and other comparator BLI agents in 96 well microtitre plates. Briefly, stock solutions (e. g. 2560 & 1280 pg/mL) of the β-lactam antibiotics is made in water, 0.1 M Phosphate buffer, pH 6.0 or pH 7.0 or appropriate solvents accordingly. Similarly stock solutions of the BLI agents including the compound-1 were made. β-lactam antibiotics were screened in a concentration range of 0.06-128 pg/mL. BLI agents including the BLI compounds were tested in a concentration range of 1-64 pg/mL. Working solutions of ail were made by appropriate dilutions in cation adjusted Mueller Hinton broth (caMHB). Two fo!d dilutions of the antibacterial agents were done from the working solutions in caMHB serially in the wells of the 96 well microtitre plates. BLI agents including the BLI compounds were also serially diluted and then each concentration to be tested was added to each of the different antibacterial concentration. The BLI compounds, other comparator BLIs and ail the antibacterial agents were also tested individually. The bacterial inoculum was prepared by picking 3 to 5 well isolated bacterial colonies with the same morphological appearance from an 18-24 h old culture and adjusting the turbidity of the saline suspension to 0.5 McFarland turbidity standard équivalent to a bacterial population of -lxlO⁸ colony forming units (CFU) per mL of suspension. The suspension was diluted 1:100 in caMHB to get a bacterial population of ~1χ10^ό CFU/mL as inoculum. This bacterial inoculum was added into the wells of the microtitre plate containing caMHB with antibacterîals or antibacterials + BLI agents in equal volume to the volume of the caMHB with antibacterials or antibacterials + BLI agents. Hence, the final inoculum becomes half (-5x10⁵ CFU/mL) and the concentrations of the tested antibacterials and combinations also becomes half. The inoculated plates were incubated at 35 °C in an ambient atmosphère for 18-20 h. The plates after incubation were observed with naked eye with the aid of optical mirror and MIC was recorded as the concentration, which showed no growth or Visual turbidity of the inoculated culture.

In agar dilution method briefly, stock solutions of the cephalosporins for veterinary use (e.g. 2 mg/mL) was made in water, 0.1 M phosphate buffers or appropriate solvents and the solution was serially two fold diluted. Compound-1 was dissolved in water and Tazobactam (comparator BLI) in 0.1 M phosphate buffer, pH 6.0 to get a solution of l mg/mL. Cephalosporins were screened in a concentration range of 0.5-32 pg/mL. For combination, Tazobactam or the Compound-1 described herein were tested at a fîxed concentration of 4 pg/mL along with the cephalosporins concentration ranging from 0.5 to 32 pg/mL. Cephalosporins alone and in combination with the compound-1 or Tazobactam from each concentration was added to 20 mL of molten Mueller Hinton agar that has been cooled to 40-50 °C and poured in pétri dishes. The compound of formula (I) and Tazobactam were also tested individually. The bacterial inoculum was prepared by picking 3 to 5 well isolated bacterial colonies with the same morphologicai appearance from an 18-24 h old culture and adjusting the turbidity of the saline suspension to 0.5 McFarland turbidity standard équivalent to a 10 bacterial population of ~lxl0⁸ CFU per mL of suspension. The suspension was diluted 1: 10 in saline to get a bacterial population of ~lxl0⁷ CFU/mL as inoculum. This bacterial inoculum was inoculated onto the prepared pétri dishes by a multipoint inoculator with each inoculum spot containing ~lxl0⁴ CFU of the bacterial strain. The inoculated pétri dishes were incubated at 35 °C in an ambient atmosphère for 18-20 h.

The pétri dishes after incubation were placed on a dark non-reflecting surface and the MIC was recorded as the concentration, which showed no growth of the inoculated culture.

Table 2: Minimum inhibitory concentration (MIC) of Imipenem in combination with β-lactamase inhibitor (BLI) Compound-12 against Klebsiella pneumoniae carbapenemase (KPC) producing strains

KPC type	Strains	MIC (pg/mL) of imipenem in combination with BLI at 4 or 16 pg/mL
No BLI	Compound-13	Compound-M	Compound-12	Compound-1	Tazobactam
4	16	4	16	4	16	4	16	4	16
KPC2	K. pneumoniae ATCC BAA1705	32- 64	2	2	8	8	4	4	4	<0.5-1	16	8
KPC2	K. pneumoniae UMM3	8	NA	NA	8	NA	2	NA	1	NA	4	NA
KPC2*	E. cloacae 01 MGH 049	8	NA	NA	8	NA	8	NA	4	NA	8	NA
KPC3	E. colt Ecoli233	16	8	1	16	16	4	1	4	1	8	8
KPC3	K. pneumoniae NCTC 13438	128	NA	NA	64	NA	64	NA	32	NA	64	NA

NA: Not available * Presence of AmpC was observed phenotypically

Compound-12 shows improved activity against the KPC (2 & 3) producing strains within the range similar to Compound-1, while Compound-M is only 25 moderately active not within the expected range of activity.

Table-3a: MIC of Penems or Monobactam/Compound-1 against KPC-2 producing K. pneumoniae (ATCC B AA-1705)

BLI	BLI conc. (pg/mL)	IMP	MER	ERT	FAR AZT	BLI
MIC (pg/mL)
32-64	32-64	>64	>64	>64	>64
+ Compound-1	2	4-8	16	NA	NA	NA
4	4	4-8	32	>64	>64
8	2	2-4	16	>64	NA
16	1	1	8	64	64
64	<0.5-1	<0.5	<0.5	4	<0.5
+ Tazobactam	2	16	32	NA	NA	NA	>64
4	16	32	>64	>64	>64
8	8-16	16-32	>64	>64	NA
16	8	8-16	64	>64	>64
64	4-8	4	32	>64	>64
+ Clavulanic acid	2	8-16	16	32	>64	NA	>64
4	8-16	4-8	64	>64	>64
8	4-8	4	NA	NA	NA
16	2	4	32	>64	>64
64	<0.5	2	8	>64	64
+ Sulbactam	2	16-32	32	NA	NA	NA	>64
4	16-32	32	>64	>64	>64
8	16	32	>64	>64	NA
16	8	32	>64	>64	>64
64	8	16	64	>64	>64

IMP: Imipenem, MER: Meropenem, ERT: Ertapenem, FAR: Faropenem & ΑΖΎ: Aztreonam.

Compound-1 synergized with Imipenem and Meropenem better than 10 Tazobactam or Clavulanic acid or Sulbactam against KPC-2 producing strain ATCC BAA1705 at >4 pg/mL concentration. It also showed better synergy with Ertapenem, Faropenem and Aztreonam than the above comparators at 64 pg/mL concentration (Table-3a). Similarly, the following compounds in the sériés showed the restoratîon of antibacterial activity of Imipenem & Meropenem (Table- 3b).

Table 3b: Penems BLI compounds against KPC-2 producing JC. pneumoniae (ATCC BAA-1705)

BLI	BLI conc. (pg/mL)	MIC (pg/mL)
Imipenem	Meropenem	BLI
32-64	32-64
Compound-4	4	4	8	>16
16	1	4
Compound-2	4	4	8	>16

	16	1	2
Compound-6	4	8	16	>16
16	8	4
Compound-5	4	8	8	>16
16	1	2
Compound-8	4	8	8	>16
16	4	4
Compound-1	4	4	8	>16
16	1	1
Tazobactam	4	16	32	>16
16	8	8-16

Table-4: Human cephalosporins/compound-1 against KPC-2 producing K. pneumoniae (ATCC BAA-1705)

BL1	BLI conc. (pg/mL)	CEF CTX	CTZ CTB BLI
MK	C (pg/mL)
>128	>128	>128	>64	>64
+ Compound-1	2	64	32	>64	>64
4	32	16	>64	>64
8	16	16	64	>64
16	4	4	4	64
64	<0.5	<0.5	1	<0.5
+ Tazobactam	2	64	>64	>64	>64	>64
4	64	32-64	>64	>64
8	32	32	>64	>64
16	32	32	>64	>64
64	32	32	64	>64
+ Clavulanic acid	2	64	32-64	>64	>64	>64
4	64	32-64	>64	>64
8	32	16-32	>64	>64
16	32	16	64	>64
64	8	8	16	64
+ Sulbactam	2	>64	>64	>64	>64	>64
4	>64	>64	>64	>64
8	64	64	>64	>64
16	64	32	>64	>64
64	64	32	64	>64

CEF: Cefepime, CTX: Cefotaxime, CTZ: Ceftazidime & CTB: Ceftobiproie

Compound-1 synergized with Cefepime better than Tazobactam or Clavulanic 10 acid or Sulbactam against KPC-2 producing strain ATCC BAA1705 at >16 pg/mL concentration. It also showed better synergy with Cefotaxime & Ceftazidime than the above comparators at 64 pg/mL concentration. Ceftobiproie did not show any synergy at the tested conc. against ail the compared compounds (Table-4).

Table-5: Veterinary cephalosporins/compound-l against KPC-2 producing K. pneumortiae (ATCC BAA-1705)

BLI	BLI conc. (pg/mL)	CFQ CFF CFD CFL BLI
MIC (pg/mL)
>64	>64	>64	>64	>64
+ Compound-1	1	>64	>64	NA	NA
2	>64	>64	NA	NA
4	64	>64	>64	>64
8	64	64	NA	NA
16	8-16	16-32	>64	32
32	2-4	4	NA	NA
64	1	2-4	64	8
+ Tazobactam	1	>64	>64	NA	NA	>64
2	>64	>64	NA	NA
4	>64	>64	>64	>64
8	64	>64	NA	NA
16	64	>64	>64	>64
32	64	64	NA	NA
64	64	64	>64	>64

CFQ: Cefquinome, CFF: Ceftiofur, CFD: Cefadroxil & CFL: Cefalonium

Compound-1 synergized with cephalosporins for veterinary use Cefquinome and Ceftiofur better than Tazobactam against KPC-2 producing strain ATCC 10 BAA1705 at >32 pg/mL concentration. Cefadroxil & Cefalonium did not show the desired synergy at the tested concentrations (Table-5).

Table-6a: Carbapenems & human cephalosporins/compound-l against KPC-3 expressing E. coli (J53 R6206)

BLI	BLI conc. (pg/mL)	1MP MER CEF CTX CTZ CTB BLI
MIC (pg/mL)
2-4	2-4	>16	>32	>32	>32	>8
+ Compound- 1	2	1	<0.06	1	1	2	2
4	0.5	<0.06	0.25	<0.25	1	0.5
8	0.5	<0.06	<0.125	<0.25	1	<0.25
+ Tazobactam	2	2	1	16	8	32	32	>8
4	2	0.5	8	8	32	16
8	1	0.5	4	4	32	16
+ Clavulanîc acid	2	1	1	8	4	32	8	>8
4	1	1	8	1	32	8
8	0.5	0.5	1	<0.25	8	2
+ Sulbactam	2	4	2	16	>32	32	32	>8
4	2	1	16	16	32	32
8	2	1	8	8	32	16

IMP: Imipenem, MER: Meropenem, CEF: Cefepime, CTX: Cefotaxime, CTZ: Ceftazidime & CTB: Ceftobiprole

Compound-1 synergized with Imipenem, Meropenem, Cefepime, Cefotaxime, Ceftazidime and Ceftobiprole better than Tazobactam or Clavulanic acid or Sulbactam against KPC-3 expressing E. coli strain J53 R6206 at >2 μg/mL concentration (Table6a). Sîmilarly, the following compounds in the sériés showed the restoration of antibacterial activity of Imipenem & Meropenem (Table- 6b)

Table-6b: Carbapenems BLI compounds against KPC-3 expressing E. coli (J53 R6206)

BLI	BLI conc. (pg/mL)	M	IC (pg/mL)
Imipenem	Meropenem	BLI
2-4	2-4
Compound-4	4	0.5	<0.125	>16
16	0.25	<0.125
Compound-2	4	0.5	<0.125	>16
16	0.25	<0.125
Compound-6	4	1	0.5	>16
16	0.5	<0.125
Compound-5	4	0.5	<0.125	>16
16	0.25	<0.125
Compound-8	4	1	0.25	>16
16	0.25	<0.125
Compound-1	4	0.5	<0.125	>16
16	0.25	<0.125
Tazobactam	4	2	0.5	>16
16	1	<0.125

Table-7: Veterinary cephalosporins/Compound-1 against KPC-3 expressing E coli (J53 R6206)

	BLI conc.	CFQ	CFF	CFD	CFL	BLI
BLI	MIC (pg/mL)
	(pg/mL)	16	16	>32	32	>16
+ Compound-1	4	<0.5	<0.5	>32	4
+ Tazobactam	4	8	2	>32	32	>16

CFQ: Cefquinome, CFF: Ceftiofur, CFD: Cefadroxil & CFL: Cefalonium

Compound-1 synergized with Cephalosporins for veterinary use Cefquinome Ceftiofur & Cefalonium better than Tazobactam against KPC-3 expressing E. coli strain J53 R6206 at 4 pg/mL concentration. Cefadroxil did not show the desired synergy at the tested concentrations (Table-7).

β-Lactamase inhibitory assay with carbapenemases

The Compound-1 was subjected to β-lactamase inhibitory assay to déterminé IC50 and to compare that with the comparator BLI agents as described elsewhere (Bebrone et. al., Antimicrob. Agents. Chemother, 2001, 45(6): 1868-1871; Jamieson et. al, Antimicrob. Agents. Chemother, 2003, 47(5): 1652-1657). Briefly, enzyme extracts 10 from KPC-2 producing and K.PC-3 expressing bacterial gram négative strains were used to study the β-lactamase inhibitory activity and détermination of ICjo using CENTA as the substrate for β-lactamase.

Table-8: β-lactamase enzyme inhibitory assay of Compound-1 with carbapenemases

BLI	1C_5O (pM)
KPC2	KPC2
Compound-l	190	10.7
Tazobactam	980	71
Clavulanic acid	330	92
Sulbactam	1400	235

The IC50 of compound-1 is lower than the compared BLIs against the crude

KPC-2 & 3 enzyme extracts indicating its superior binding hence potency of the BLI of compound-1 of formula (I) (Table-8).

TabIe-9: Comparison of MIC (pg/mL) of Piperacillin in combination with standard Tazo and Novel Inhibitor compounds against spécifie Extended spectrum β-lactamase (ESBLs) producing Gram-negative isolâtes from ATCC

ATCC strains		MIC of Piperacillin with different concentrations of Tazo or Compound-I2
Inhibitor Conc.:				2 pg/mL	4	pg/mL
ESBL Phenotype!	Tazo	Compound-M	Compound-12	Tazo	Compound-M	Compound-12	Tazo	Compound-M	Compound-12
E.coli BAA-201	TEM-3	4	4	2	4	4	4	2	4	4
E.coli BAA-197	TEM-12	64	4	4	8	8	4	4	4	4
E.coli BAA-198	TEM-26	4	4	4	4	4	4	4	4	4
P.mirabilis BAA-663	TEM-89	8	64	8	8	>128	16	4	>128	>128
E.coli BAA-199	SHV-3	>128	>128	>128	>128	>128	>128	>128	>128	2
E.coli BAA-200	SHV-4	>128	>128	32	>128	>128	8	4	>128	4
Kpneumoniae 700603	SHV-18	16	16	8	16	32	16	16	16	8

Itt vivo eflicacy of Compound-1 against KPC carbapenemase producîng strains

The Compound-1 is a potent inhibitor of ESBLs and its inhibitory activity against KPC enzymes was demonstrated in vitro. Compound-1 was evaluated against KPC2 producîng K. pneumoniae ATCC BAA 1705 in the pharmacodynamies models of mice systemic infection and thigh infection for in vivo translation of its inhibitory activity against KPC2. In these models, eflicacy of β-lactams as single agents deteriorated due to KPC2 mediated hydroiysis. By combining compound-1 with βlactams, its potential to restore or enhance the eflicacy of β-Iactams was assessed.

Method:

Mice systemic infection model

Female Swiss Albino mice, weighing 18 - 22 g were used for ali studies. For each dose group, 5 or 6 mice were included. Study protocole were reviewed and approved by Institutional Animal Ethical Committee, Orchid Research Laboratories Limited. Mice were housed in individually ventilated cages provided with food and water ad libitum, throughout the study period. From ovemight culture in Brain-Heart Infusion agar medium, challenge inoculum with required bacterial density was prepared in normal saline containing Hog gastric mucin. In studies involved with doripenem, washed bacterial cells was used. Each mouse was infected with of challenge inoculum by intra-peritoneal injection.

Piperacillin with β-lactamase inhibitor (BLI) combinations; Increasing concentrations of piperacillin and BLIs (Compound-1 or tazobactam) as single agents or piperacillin in combination with BLIs at 1:1 ratio were prepared in aqueous agar (Bacto agar). Infected mice were dosed sub-cutaneously, with drug préparations at three different time points post-infection.

Imipenem with BLI combinations: Increasing concentrations of Imipenem as single agent or in combination with fixed concentration of BLIs were used to dose infected mice, sub-cutaneously. In this experiment Imipenem was always administered along with cilastatin.

Doripenem with BLI combinations: Increasing concentrations of doripenem as single agent or in combination with fixed concentration of BLIs were used to dose infected mice, sub-cutaneously.

Survival of the treated mice were monitored twice a day, up to 7 days postinfection. Efficacy dose 50 (ED50) was calculated by Reed and Muench method (Reed, L.J.; Muench, H.. A simple method of estimating fifty percent endpoints. The American Journal ofHygiene, 1938,27:493-497.).

Neutropénie mice thlgh infection model (Human adapted model)

Female Swiss Albino mice weighing 24 - 30 g were used for ail studies. Study protocols were reviewed and approved by Institutional Animal Ethical Committee, Orchid Research Laboratories Limited. Mice were rendered neutropénie by intraperitoneal cyclophosphamide injections. Log phase culture in brain heart infusion broth was injected in to mice thighs. Imipenem or Doripenen alone or in combination were administered sub-cutaneously in decreasing fractionated doses every 15 minutes over the period of 5.5 h (Flückiger, U. et al.. “Intégration of pharmacokinetics and pharmacodynamies of Imipenem in a human-adapted mouse model*. Antimicrobial Agents and Chemother, 1991,35(9): 1905-1910). The Compound-1 or tazobactam was administered sub-cutaneously as bolus dose at the start of dosing. Efficacy end point was 6 h in Imipenem studies and 8 h in Doripenen studies.

Efficacy of piperacillin restored by Compound-1 against KPC2 K. pneumoniae ATCC BAA 1705 in mice System ic infection model

Piperacillin alone was not efficacious up to 800 mg/kg. The Compound-1 restored the efficacy of piperacillin as piperacillin demonstrated an ED50 of 50 mg/kg in combination with Compound-1 at 1:1 ratio. As Compound-1 atone was not efficacious, piperacillin efficacy in combination was attributed to KPC2 enzyme inhibitory activity of Compound-1. The clinically used tazobactam however could not restore the piperacillin efficacy as its combination with piperacillin at 1:1 ratio up to >200 : >200 mg/kg did not show efficacy (Table 10).

Table 10: Comparison of efficacy of piperacillin in combination with Compound1 versus tazobactam

Dose group	EDso (mg/kg)
Piperacillin	>800
Compound-1	>64
Tazobactam	>200
Piperacillin: Compound-1 at 1:1 ratio	50:50
Piperacillin:Tazobactam at 1:1 ratio	>200 : >200

Efficacy of Imipenem enhanced by Compound-1 against KPC2 A' pneumoniae ATCC BAA 1705 in mice systemic infection model

Imipenem alone showed an EDso of 8.9 mg/kg. Combining compound-1 at fixed 64 mg/kg resulted in enhancement of efficacy with ED50 of 2.2 mg/kg. Addition of tazobactam at same dose with Imipenem resulted in ED50 of 4 mg/kg. Significant increase in efficacy of Imipenem by compound-1 was due to its inhibitory activity on KPC2 enzyme (Table 11).

Table 11: Comparison of efficacy of Imipenem in combination with Compound-1 versus tazobactam

Dose group	ED.» (mg/kg)
Imipenem	8.9
Imipenem + Compound-1 (64 mg/kg)	2.2
Imipenem + Tazobactam (64 mg/kg)	4

Efficacy of Doripenen enhanced by Compound-1 against KPC2 K. pneumoniae ATCC BAA 1705 in mice systemic infection model

Doripenem alone and Doripenen in combination with compound-1 or tazobactam were evaluated. The compound-1 or tazobactam were tested at 20 mg/kg and 64 mg/kg. Doripenem alone showed an ED50 of 14.14 mg/kg. Its efficacy was significantly enhanced by compound-1 at 20 mg/kg with an ED50 of 1.4 mg/kg and at 64 mg/kg with an ED50 of 1.62 mg/kg. Tazobactam improved the efficacy of Doripenen marginally with an ED50 of 11.89 mg/kg and 6.48 mg/kg at tazobactam doses of 20 and 64 mg/kg respectively (Table 12)

These results suggest the potent inhibitory activity of compound-1 on KPC2 resulting in protection of Doripenen form KPC2 mediated hydrolysis thus restoring the efficacy of Doripenen.

Table 12: Comparison of efficacy of Doripenen in combination with Compound-1 versus tazobactam

Dose group	ED_m (mg/kg)
Doripenem	14.14
Doripenem + Compound-l (20 mg/kg)	1.4
Doripenem + Compound-1 (64 mg/kg)	1.62
Doripenem + Tazobactam (20 mg/kg)	11.89
Doripenem + Tazobactam (64 mg/kg)	6.48

Eflîcacy of Imipenem enhanced by Compound-1 against KPC2 K. pneumoniae ATCC BAA 1705 in neutropénie mice thigh infection model

The mean initial bacterial load at the start of therapy was 1.8E+06 CFU/thigh. Imipenem 140 mg/kg administered as fractionated doses over the period of 5.5 h was not efficacious; bacteria grew to 6.8E+06 CFU/thigh after 6 h of therapy. Combining Imipenem with compound-1 at 140 mg/kg as bolus dose restored the efïïcacy as bacterial load reduced to 2.1E+05 CFU/thigh (Table 13). This experimental resuit showed that compound-1 demonstrated inhibitory potential in the tough mice thigh infection model.

Table 13: In vivo pharmacodynamies (Thigh infection model) of Imipenem in combination with Compound-1

Dose group	Bacterial load (CFU/thigh)
Initial bacterial load	1.81E+06
Infection control	4.6E+07
Imipenem 140 mg/kg treated	6.8E+06
Imipenem (140 mg/kg ) + Compound-1 (140 mg/kg)	2.1E+05

Efïicacy of Doripenem enhanced by compound-1 against KPC2 K. pneumoniae ATCC BAA 1705 in neutropénie mice thigb infection model

Three experiments were carried out where Doripenen alone or in combination with compound-1 or tazobactam (two experiments) were evaluated. Doripenem 70 mg/kg was administered in fractionated doses over the period of 5.5 h (Flückiger, U. et al. “Intégration of pharmacokinetics and pharmacodynamies of Imipenem in a humanadapted mouse model*. Antimicrobial Agents and Chemother,. 1991, 35(9): 19051910). Compound-1 or tazobactam was administered as bolus dose at initiation of therapy. Efïicacy end point was 8 h after initiation of therapy.

The initial bacterial load ranged from 1.4E+07 - 3.1E+O7 CFU/thigh. Doripenem 70 mg/kg alone exerted a static effect on the bacterial load. Mice treated with Doripenen alone showed bacterial load of 5.4E+06 - 2.6E+07 CFU/thigh. Combining compound-1 at 35 mg/kg with Doripenen brought the bacterial load down ίο 7.9E+05 - 1.3E-H16 CFU/thigh. Doripenem was also combined with tazobactam at 35 mg/kg in two experiments. Tazobactam dîd not hâve impact on efïicacy of Doripenen as mice showed bacterial load of 1.2 E+07-1.6E+07 CFU/thigh (Table 14).

Table 14: In vivo pharmacodynamies (Thigh infection mode!) of Doripenem in combination with Compound-1

Dose group	Bacterial load (range) (CFU/thigh)
Initial bacterial load	1.4E+07-3.1 E+07
Infection control	7.3 E+07- 1.6 E+08
Doripenem 70 mg/kg treated	5.4 E+06-2.6 E+07
Doripenem (70 mg/kg) + Compound-1 (35 mg/kg)	7.9 E+05 - 1.4 E+06
Doripenem (70 mg/kg) + Tazobactam (35 mg/kg)	1.19 x 10'-1.6 x 10'

Conclusion

In ali these experiments, efficacy of β-lactams as single agents deteriorated as they were not stable to KPC2. Being an inhibitor of KPC2, compound-1 restored or io significantly enhanced the efficacy of β-lactams.

Claims

1. A compound of formula (I), their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphe, solvatés, métabolites, prodrugs, hydrates, pharmaceutically acceptable salts and esters, for use in the inhibition of βlactamases produced by bacteria; restoring/potentiating the activity of antibiotics, 10 comprising administering a therapeutically effective amount of compound of formula (I), to a subject in need thereof;

wherein

A = CorN;

Het represents optionally substituted three- to seven-membered heterocyclic ring;

15 R¹ represents carboxylate anion or -COOR⁴ wherein R⁴ represents hydrogen, CiCealkyl, Co-Cioaryl, Cô-CtoarylCi-Cfialkyl, methoxybenzyl, nitrobenzyl, silyl, diphenylmethyl, proxetil, axetil, cilexetil, pivoxil, hexetil, daloxate or a pharmaceutically acceptable sait;

R² and R³ independently represent hydrogen, halogen, amino, protected amino or 20 optionally substituted Ci-Cealkyl, C2-Cealkenyl and C2-C₆alkynyl;

R represents substituted or unsubstituted Ci-Côaikyl, C2-Céalkenyl, C₆-C|oaryl, C₆CioarylCi-Cealkyl, C₃-Ci2cycloalkyl, oxo, heterocyclyl and heterocyclylalkyl groups;

when the groups R, R² and R³ are substituted, the substituents which may be one or 25 more are selected from lower alkyl (Ci-C₄alkyl selected from the group consisting of methyl, ethyl, propyl and isopropyl); lower alkoxy (Ci-C4alkoxy selected from the group consisting of methoxy, ethoxy and propoxy); lower alkylthio (C|C₄alkylthio selected from the group consisting of methylthio and ethylthio); lower alkylamino (C|-C₄alkylamino selected from the group consisting of methylamino, 30 ethylamino and propylamino); cyclo(lower)alkyl (Cs-Cftcycloalkyl selected from the group consisting of cyclopentyl and cyclohexyl); cyclo(lower)alkenyl (CsCecycloalkenyl selected from the group consisting of cyclohexenyl and cyclohexadienyl); hydroxy; halogen (chloro, bromo, fluoro and iodo); amino;

5 protected amino; cyano; nitro; carbamoyl; -CONHCi-C4alkyl-COO-Ci-C4alkyl; carboxy; protected carboxy; -COO-Ci-C4alkyl; -CO-heterocyclyl; sulfonyl; sulfamoyl; imino; oxo; amino(lower)alkyl selected from the group consisting of aminomethyl, aminoethyl and aminopropyl; halo(lower)alkyl selected from the group consisting of trifluoromethyl (-CF3), fluoromethyl, fluoroethyl, bromomethyl 10 and difluoromethyl; carboxylic acid and carboxylic acid dérivatives selected from the group consisting of hydroxamic acid, ester and amide.

2. The compound for use as claimed in claim 1, wherein the compounds are selected from formula (II),

R¹

15 their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvatés, métabolites, prodrugs, hydrates, pharmaceutically acceptable salts and esters, for use in the inhibition of carbapenemases produced by bacteria; restoring/potentiating the activity of antibiotics, comprising administering a therapeutically effective amount of compound of formula (I), to a subject in need 20 thereof;

wherein:

L = CorN;

R¹ represents carboxylate anion or -COOR⁴ wherein R⁴ represents hydrogen, CjCealkyl, Ce-Cioaryl, Ce-CioarylCi-Côalkyl, methoxybenzyl, nitrobenzyl, silyl,

25 diphenylmethyl, proxetil, axetil, cilexetil, pivoxil, hexetil, daloxate or a pharmaceutically acceptable sait;

R² and R³ may be same or different and independently represent hydrogen, halogen, amino, protected amino, optionally substituted Ct-Cealkyi, Cî-Côalkenyl and Cî-Cealkynyl;

30 R represents substituted or unsubstituted Ci-Cftalkyl, C2-C₆alkenyl, Cô-Cioaryl, C#· Cioaryl Cj-Céalkyl, C3-Ct2cycloalkyl, oxo, heterocyclyl and heterocyclylalkyl;

R⁵ is hydrogen, C|-C«alkyl, Ci-C^aikoxy, C|-C6alkylthio, Ci-Côalkylamîno, hydroxyl, halogen and trihalomethyl; and m is 0, 1 or 2.

3. The compound for use as claimed in claim 1 or 2, wherein the compound is selected from:

1 - {[(25,35,55)-2-Carboxy-3 -methy 1-4,4,7-trioxo-4-th ia-1 azabicyclo[3.2.0]hept-3-yl]methyl}-3-methyl-177-l,2,3-triazol-3-ium;

1 - {[(25,35,5A)-2-Carboxy-3-methy l-4,4,7-trioxo-4-th ia-1 azabicyclo[3.2.0]hept-3-yl]methyl} -3-ethyl-7/7-1,2,3-triazol-3-ium;

1 - {[(25,35,55)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-1 azabicyclo[3,2.0]hept-3-y l]methyl }-3 -n-propy l-l H-1,2,3 -triazol-3 -ium ;

1 - {[(25,35,55)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thîa-1 azabicyclo [3.2.0]hept-3-y 1] methyl }-3 -al ly l-l H-1,2,3-triazol-3 -ium ;

1 - {[(25,35,55)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-1 azabicyclo[3.2.0]hept-3-yl]methyl}-3-(2-amino-2-oxoethyl)-777-l,2,3triazol-3-ium and the corresponding acid;

1 - {[(25,35,55)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-1 azabicyclo[3.2,0]hept-3-yl] methyl} -3-(2-Abutoxy-2-oxoethy Ï)-1H-1,2,3triazol-3-ium and the corresponding acid;

1 - {[(25,35,55)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-1 azabicyclo[3.2.0]hept-3-yl]methyl}-3-(2-morpholin-4-yl-2-oxoethyl)-7Hl,2,3-triazol-3-ium and the corresponding acid;

1 -{[(25,35,57?)-2-Carboxy-3-methyM,4,7-trioxo-4-thia-1 azabicyclo[3.2.0]hept-3-yl]methyl}-3-{2[(2-ethoxy-2-oxoethyl)amino]-2oxoethyl}-7#-l,2,3-triazol-3-ium and the corresponding acid;

1 - {[(25,35,57î)-2-Carboxy-3 -methyl -4,4,7-trioxo-4-thia-1 azabicyclo[3.2.0]hept-3-yl]methyl}-3-{2-[(3-ethoxy-3-oxopropyl)amino]-2oxoethyl}-7iAl,2,3-triazol-3-ium and the corresponding acid;

1 - {[(25,35,5Æ)-2-Carboxy-3 -methy 1-4,4,7-trioxo-4-thia-1 azabicyclo[3.2.0] hept-3 -yl] methyl} -3-(2- {[ 1 -(ethoxycarbonyi)-2hydroxypropy(]amino}-2-oxoethyl)-7/f-l,2,3-triazol-3-ium and the corresponding acid;

1 - {[(25,35,55)-2-Carboxy-3 -methy 1-4,4,7-trioxo-4-th ia-1 azabicyclo[3.2.0]hept-3-yl]methyl}-3-benzyl-17f-l ,2,3-triazol-3-ium and the corresponding acid;

(25,35,57?)-3-Methyl-3-(3-methyl-imidazol-3-ium-l-ylmethyl)-4,4,7-trioxo4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate and the correspondîng acid; and (25,35,5/?)-3-Methyl-3-(4-methyl-3-methyl-imidazol-3-ium-1 -ylmethyl)4,4,7-trioxo-4-thia-l-aza-bicyclo[3.2.0]heptane-2-carboxylate and the correspondîng acid, their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvatés, métabolites, prodrugs, hydrates, pharmaceutically acceptable salts and esters.

4. The compound for use as claimed in claim l or 2, wherein the compound is,

1 - {[(2S, 3S, 5Æ)-2-Carboxy-3-methyl-4,4,7-trioxo-4-thia-1 -azabicyclo[3.2.0]hept-3yl]methyl}-3-methyl-lH-l,2,3-triazol-3-ium and its dérivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvatés, métabolites, prodrugs, hydrates, pharmaceutically acceptable salts and esters.

5. The compound of formula (I) for treating and/or preventing infections caused by bacteria producing β-lactamases comprising carbapenemases, cephalosporinases, penicilünases, ESBLs, inhibitor-resistant β-lactamases, in combination with suitable antibiotic, comprising administering therapeutically effective amount of compound of formula (I), to a subject in need thereof.

6. The compound for use as claimed in any one of the claims 1-4, for treating and/or preventing infection caused by carbapenemase producing bacteria comprising administering therapeutically effective amount of compound of formula (I), in combination with suitable antibiotics to a subject in need thereof.

7. The compound for use as claimed in any one of the claims 1-4, wherein bacteria are selected from Gram-negative bacteria.

8. The compound for use as claimed in any one of the claims 1-4, wherein the carbapenemases are selected from KPC.

9. A pharmaceutical composition comprising a compound of formula (I) as claimed in any one of the claims 1-4, as an active ingrédient for inhibiting β-lactamases.

10. A pharmaceutical composition comprising a compound of formula (I) as claimed in any one of the claims 1-4, as an active ingrédient along with

a. one or more compounds of claim 1 ;

b. one or more antibiotics and

c. one or more pharmaceutically acceptable carrier.

11. The compound for use as claimed in any one of the claims 1-4, wherein the antibiotics are β-lactam antibiotics.

12. The compound for use as claimed in claim 11, wherein the antibiotics are selected from Penicillins, Cephalosporins, Carbacephem, Oxacephem, Carbapenems, Cephamycins, Penems, Monobactams or a combination thereof.

13. The compound for use as claimed in claim 12, wherein the penicillins are selected from Amdinocillin (Mecillinam), Amoxicillin, Ampicillin, Amylpenicillin, Apalcillin, Aspoxicillin, Azidocîllin, Azlocillin, Bacampicillin, Carbenicillin, Carindacillin, Clometocillin, Cloxacillin, Cyclacillin (Ciclacillin), Dicloxacillin, Epicillin. Fenbenicillin, Floxacillin (flucloxacillin), Hetacillin, Lenampiciilin, Metampicillin, Methicillin, Mezlocillin, Nafcillin, Oxacillin, Penamecillin, Penethecillin. Penicillin G (Procaine Pencillin), Penicillin N, Penicillin O, Penicillin V (Phenoxymethyl Penicillin), Phenethicillin, Piperacillin, Pivampicillin, Propicillin, Quinacillin, Sulbenicillin, Talampicillin, Temocillin, Ticarcillin, Pivmecillinam, Benzathine Penicillin, Benzyl Penicillin, Co-amoxiclav and Lenampiciilin.

14. The compound for use as claimed in claim 12, wherein the cephalosporins are selected from Cephaloridin, Cephradine, Cefoxitin, Cephacetril, Cefinenoxime, Cephaloglycin, Cefonicid, Cefodizime, Cefpirome, Cefpiramide, Cefozopran, Cefoselis, Cefluprenam, Cefpimizole, Cefclidin, Cefpodoxime axetil, Cefteram pivoxil, Cefcapene pivoxil, Ceftobiprole, Ceftaroline, Cefoperazone, Cefquinome,

Ceftiofur, Cefovecin, Cefadroxil, Cefalonium, Cefepime, Cefotaxime, Ceftazidime, Cefetamet pivoxil, Cefditoren pivoxil, Cephaloridine, Ceftazidime, Ceftriaxone, Cefbuperazone, Cephaiothin, Cephazolin, Cephapirin, Ceftezole, Cefamandole, Cefotiam, Cefotiam hexetil, Cefuroxime, Ceftizoxime, Cefmenoxime, Cefuzonam, Cefsulodin, Cefmetazole, Cefmînox, Cephalexin, Cefradine, Cefaclor, Cefadroxil, Cefalonium, Cefprozil, Cefiiroxime axetil, Cefixime, Cefpodoxime proxetil, Ceftibuten, CXA-10l(FR264205), and Cefdinir.

15. The compound for use as claimed in claim 12, wherein the carbapenems are selected from Meropenem, Ertapenem, Doripenem, Biapenem, Panipenem, Ritipenem, Tebipenem, Tomopenem, Sulopenem, Razupenem, Imipenem, ME1036 and SM216601.

16. The compound for use as claimed in daim 12, wherein the Monobactams are selected from Aztreonam, Carumonam, Tigemonam, BAL19764 and BAL30072.

17. The compound for use as claimed in anyone of claims 1 to 4, wherein the antibiotics are selected from Imipenem, Faropenem, Doripenem, Meropenem, Ertapenem, Aztreonam, Cefepime, Cefotaxime, Ceftazidime, Ceftobiprole, Cefquinome, Ceftiofur, Cefadroxil and Cefalonium.

18. The compound of formula (I) for use as a diagnostic reagent for the détection of βlactamases including carbapenemases.

19. The compound for use as claimed in claim 18 as a diagnostic reagent for the détection of β-lactamases belonging to the familles of KPC and ESBL producing Enterobacteriaceae.

20. (2S'₎3S’,57î)-3-Methyl-3“(3-methyl-imidazol-3-ium“l-ylmethyI)-4,4,7-trioxo-4-Üiial-azabicyclo[3.2.0]heptane-2-carboxylate; and (2S,3S',57î)-3-Methyl-3-(4-methyl-

3-methyl-imidazol-3-ium-l -ylmethyl)-4,4,7-trioxo-4-thia-1 -azabicyclo[3.2.0]heptane-2-carboxylate, their dérivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvatés, métabolites, prodrugs, hydrates, pharmaceutically acceptable salts and esters.

21. The compound for use as claimed in claim 1 or 2, wherein the ‘subject’ is selected from patients with bacterial infections, preoperative patients, postoperative patients, patients in ICU, patients with nosocomial infections, community acquired infections and veterinaries.

22. The compound for use as claimed in anyone of daims 1,5-13 and 15 substantially as hereinbefore described with reference to the examples and/or the figures of the accompanying drawings.

23. The Compound for use as claimed in any one of daims 2-4, substantially as herein described or exemplifîed.

24. The pharmaceutical composition as claimed in daim 10, wherein the antibiotics are selected from the group consisting of Penicillins, Cephalosporins, Penems, Carbacephem, Carbapenems, Oxacephem, Monobactams, Aminoglycosides, Bacteriocins, Quinolones, Sulfonamides, Macrolides, Tetracyclines, Glycylcyclines, Oxazolidinones, Lipopeptides, Polypeptides, Rifamycins, Chloramphenicol, Polyene antifungals and dérivatives thereof.