CA2282691A1 - Antibacterial heterocyclic amino acid derivatives - Google Patents

Abstract

A method of treatment of bacterial infections in humans or animals which comprises administering, in combination with a .beta.-lactam antibiotic, a therapeutically effective amount of an amino acid derivative or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof: (i) or (ii), wherein X is S, S(O)p or CH2 where p is 1 or 2; R is hydrogen, a salt forming cation or an in vivo hydrolysable ester-forming group; R1 and R2 are selected from halogen, mercapto, (C1-6)alkyl optionally substituted by 1-3 halo, phenyl, (C1-6)alkoxy optionally substituted by 1-3 halo, hydroxy(C1-6)alkyl, mercapto(C1-6)alkyl, hydroxy, CO2R7, N(R7)2 or CON(R7)2 where each R7 is independently hydrogen or (C1-6)alkyl, OCONH2, nitro, (C1-6)alkylcarbonyloxy, (C1-6)alkoxycarbonyl(C1-6)alkyl, formyl, or (C1-6)alkylcarbonyl groups; R3 is hydrogen, (C1-6)alkyl optionally substituted by up to three halogen atoms, (C3-7)cycloalkyl, fused aryl(C3-7)cycloalkyl, (C3-7)cycloalkyl(C2-6)alkyl, (C2-6)alkenyl, (C2-6)alkynyl, aryl, aryl-(CHR10)m-X1-(CHR11)n, heterocyclyl or heterocyclyl-(CHR10)m-X1-(CHR11)n, where m is 0 to 3, n is 1 to 3, each R10 and R11 is independently hydrogen or (C1-4)alkyl and X1 is O, S(O)x where x is 0-2, or a bond; R4 is hydrogen, or an in vivo hydrolysable acyl group; and R5 and R6 are independently hydrogen and (C1-6)alkyl or together represent (CH2)q where q is 2 to 5.

Description

ANTIBACTERIAL HETEROCYCLIC AMINO ACID DERIVATIVES
This invention relates to chemical compounds having metallo-~3-lactamase inhibitory and antibacterial properties. The invention also relates to methods for the preparation of such compounds, to pharmaceutical compositions containing them, and to uses thereof.
Metallo-(~lactamases confer resistance to the vast majority of ~3-lactam based therapies, including carbapenems and jeopardise the future use of all such agents. As a result of the increased use of carbapenems and other ~i-lactam antibiotics the clinical climate is becoming more favourable for the survival of clinical strains which produce metallo-(3-lactamases, and metallo-~3-lactamases have now been identified in common pathogens such as Bacillus fragilis, Klebsiella, Pseudomonas aeruginosa and Serratia marcescens. Emerging knowledge emphasises that metallo-(3-lactamases have the potential to present a crisis situation for IS antimicrobial chemotherapy.
Stanton et al., J. Med. Chem. 1983, 26, 1267, discloses tetrahydroisoquinoline carboxylic acids as ACE inhibitors. W092/14453 and EP0311362 disclose further pipecolic acid derivatives as symphatholytic agents.
W091/01724 discloses further amino acid derivatives as tyrosine hydroxyiase inhibitors.
A novel series of amino acid derivatives have now been discovered, which compounds have metallo-~i-lactamase inhibitory properties, and are useful for the treatment of infections in animals.
According to the present invention there is provided a method of treatment of bacterial infections in humans or animals which comprises administering, in combination with a (3-lactam antibiotic, a therapeutically effective amount of a compound of formula (1) or a pharmaceutically acceptable salt, solvate or in vivn hydrolysable ester thereof:
Rs Rs Rs Rs R~ X Rs X R.
R.S ~ R.S + ~ . ~ R
O N N /

COZR (i) or COzR (ii) (1) -I-SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98101278 __ wherein:
X is S, S(O)p or CH2 where p is I or 2;
R is hydrogen, a salt forming canon or an in vivo hydrolysable ester-forming group;
RI and R2 are selected from halogen, mercapto, (Cl_6)alkyl optionally substituted by 1-3 halo, phenyl, (C1_6)alkoxy optionally substituted by 1-3 halo, hydroxy(C 1 _6)alkyl, mercapto(C 1 _6)alkyl, hydroxy, C02R~, N(R~)2 or CON(R~)2 where each R~ is independently hydrogen or (Cl_6) alkyl, OCONH2, nitro, (Cl-6) alkylcarbonyloxy, (C1_~)alkoxycarbonyl(Cl_6) alkyl, formyl, or (Cl-6) alkylcarbonyl groups;
R3 is hydrogen, (C 1 _6)alkyl optionally substituted by up to three halogen atoms, (C3_~)cycloalkyl, fused aryl(C~_~)cycloalkyl, (C3_~)cycloalkyl(C2-)alkyl, (C2_6)alkenyl, (C2_6)alkynyl, aryl, aryl-(CHR 10)m-X 1-(CHR 11 )n~
heterocyclyl or heterocyclyl-(CHR 10)m-X l OCHR l l )n. where m is 0 to 3, n is 1 to 3, each R
l0 and is R11 is independently hydrogen or (Cl-4)alkyl and X1 is O, S(O)x where x is 0-2, or a bond;
R4 is hydrogen, or an in vivo hydrolysable acyl group; and RS and R6 are independently hydrogen and (C1 _6)alkyl or together represent (CH2)q where q is 2 to 5.
The compound of formula (I) may exist in a number of isomeric forms, all of which, including racemic and diastereoisomeric forms, are encompassed within the scope of the present invention.
It is preferred that the stereochemistry at the carbon atom marked * is D-.
Although racemic and other mixtures of (*) D- and L- diastereomers of known compounds of formula (I) have been described, there has been little or no attempt to isolate pure D- isomers as herein defined because the anti-hypertensive activity of the compounds has been found to reside predominantly in the L-isomer.
The preferred stereochemistry at the carbon atom marked (+) is S.
Certain compounds of formula (I) including compounds of formula (I)(i) where X is S or S(O)p or formula (I)(ii), where R~ is aryl-(CHR 10)m-X 1-(CHR
11 O~
hereafter referred to as compounds of formula (IA), compounds where RS and R6 are not hydrogen, hereafter referred to as compounds of formula (IB) and compounds of formula (I) where the stereochemistry at the carbon marked * is D-, hereafter referred to as compounds of formula (IC), are novel and as such form part of the invention.
When X1 is S(O)x, x is preferably 0.
In one aspect X 1 is O, S or a bond and R 10 and R 11 are each hydrogen.
In a preferred aspect X is S or CH2.
RI and R2 are preferably both hydrogen.

SUBSTITUTE SHEET ( rule 26 ) r i WO 98139311 PCT/EP98/01278 ._ Examples of R3 include methyl, isobutyl, phenyl-(CH2)1-5~ Phenoxyethyl, 1-indanyl, 3,4-dihydroxybenzyl, 4-hydroxycarbonyl-phenylethyl, 2-trifluoromethylquinolin-6-yl, 4-difluoromethoxy-phenylethyl and 3-methyl-2,4,5-tricarbonylimidazolidin-1-yl.
Preferably R3 is aryl-(CH2)m-Xl-(CH2)n, more preferably optionally substituted benzyl, 2-phenethyl or 3-phenylpropyl and most preferably benzyl or 2-phenethyl.
Examples of R4 include hydrogen, lower alkylcarbonyl, optionally substituted benzoyl or optionally substituted phenyl lower alkyl carbonyl, more preferably hydrogen and acetyl.
R4 is preferably hydrogen.
RS and R6 are preferably independently hydrogen or methyl.
Suitable pharmaceutically acceptable salts of the carboxylic acid group of the compound of formula (I) (or of other carboxylic acid groups which may be present as optional substituents) include those in which R is a metal ion e.g. aluminium salts, alkali metal salts (e.g. sodium, lithium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts), ammonium salts, and substituted ammonium salts, for example those with lower alkylamines (e.g.triethylamine), hydroxy-lower alkylamines (e.g. 2-hydroxyethylamine), bis-(2-hydroxyethyl)amine, tris-(2-hydroxyethyl) amine, lower-cycloalkylamines (e.g. dicyclohexyl-amine), or with procaine, dibenzylamine, N,N-dibenzyl- ethylenediamine, 1-ephenamine, ~1 methylmorpholine, ~1-ethylpiperidine, N-benzyl-~i-phenethylamine, dehydroabietylamine, ethylenediamine, N,N'-bishydroabietylethylenediamine, bases of the pyridine type (e.g. pyridine, collidine and quinoline), and other amines which have been or can be used to form quaternary ammonium salts.
Pharmaceutically acceptable salts may also be acid addition salts of any amino or substituted amino groups) that may be prcsent as optional substituents on the compound of formula (I), or of a hcterocyclic group ring nitrogcn atom.
Suitable salts include for example hydrochlorides, sulphates, hydrogen sulphates, acetates.
phosphatcs etc. and other pharmaceutically acceptable salts will be apparent to those skilled in the art. Suitable addition salts are the hydrochlorides and hydrogen sulphates.
Preferred salts are sodium salts.
Examples of suitable pharmaceutically acceptable in vivo hydrolysable ester-forming groups R include those forming esters which break down readily in the human body to leave the parent acid or its salt. Suitable groups of this type include those of part formulae (i), (ii), (iii), (iv) and (v):

SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98/01278 __ Re -CH-O.CO. Rb Rd R°-N~ Re (ii) -CH2-ORS (iii) Ra O-CO-CH -Ra (iv) -CHOCO
RkOC R
(v) R.
R
wherein Ra is hydrogen, (Cl_6) alkyl, (C3_~) cycloalkyl, methyl, or phenyl, Rb is (C 1 _6) alkyl, (C ~ _6) alkoxy, phenyl, benzyl, (C3_7) cycloalkyl, (C3_~) cycloalkyloxy, (C 1 _6) alkyl (C3_~) cycloalkyl, 1-amino (C 1 _6) alkyl, or 1-(C 1 _6 alkyl)amino (Cl_6) alkyl; or Ra and Rb together form a 1,2-phenylene group optionally substituted by one or two methoxy groups; Rc represents (Cl_6) alkylene optionally substituted with a methyl or ethyl group and Rd and Re independently represent (C 1 _6) alkyl; Rf represents (C 1 _6) alkyl; Rg represents hydrogen or phenyl optionally substituted by up to three groups selected from halogen, (Cl_6) alkyl, or (C 1 _6) alkoxy; Q is oxygcn or NH; Rh is hydrogen or (C 1 _6) alkyl; Rt is hydrogen, (C 1 _6) alkyl optionally substituted by halogen, (C2_6) alkenyl, (C 1 _6) alkoxycarbonyl, aryl or heteroaryl; or Rh and R' together form (Cl_b) alkylene; RJ
represents hydrogen, (C 1 _6) alkyl or (C 1 _6) alkoxycarbonyl; and Rk represents (C 1 _ g) alkyl, (C 1 _g) alkoxy, (C 1 _6) alkoxy(C 1 _6)alkoxy or aryl.
Examples of suitable in vivo hydrolysable ester-forming groups include, for 2o example, acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl, a-acetoxyethyl, a-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl, and (1-aminoethyl)carbonyloxymethyl; alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl, a-ethoxycarbonyloxyethyl and propoxycarbonyloxyethyl;
dialkylaminoalkyl especially di-loweralkylamino alkyl groups such as SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCTIEP98/012'78 ._ dimethylaminomethyl, dimethylaminoethyl, diethylaminomethyl or diethylaminoethyl; 2-(aikoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)pent-2-enyl and 2-(ethoxycarbonyi)but-2-enyl; and lactone groups such as phthalidyl and dimethoxyphthalidyl.
A further suitable pharmaceutically acceptable in vivo hydrolysable ester-forming group is that of the formula:
CH2 Rk O_ .O
~' I(O
wherein Rk is hydrogen, C 1 _~ alkyl or phenyl.
R is preferably hydrogen.
When used herein the term 'aryl' includes phenyl and naphthyl, each optionally substituted with up to five, preferably up to three, groups selected from halogen, mercapto, (Cl_6) alkyl optionally substituted by 1-3 halo, phenyl, (C1-6) alkoxy optionally substituted by 1-3 halo, hydroxy(Cl-6)alkyl, mercapto(Cl-6)alkyl, hydroxy, vitro, (C 1 _6) alkylcarbonyloxy, formyl, (C 1-6) alkylcarbonyl, C02R~, N(R~)2 or CON(R~)2 where each R~ is independently hydrogen or (Cl-6) alkyl, OCONH2 or (C 1 _6)alkoxycarbonyl(C 1 _6)alkyl groups.
The terms 'heterocyclyl' and 'heterocyclic' as used herein include aromatic and non-aromatic, single and fused, rings suitably containing up to four hetero-atoms in each ring selected from oxygen, nitrogen and sulphur, which rings may be unsubstituted or substituted by, for example, up to three groups selected from those mentioned above for substitution on aryl and, for non-aromatic heterocyclic rings, and oxo groups. Each heterocyclic ring suitably has from 4 to 7, preferably 5 or 6, ring atoms. The term 'heteroaryl' refers to heteroaromatic heterocyclic ring or ring system, suitably having 5 or 6 ring atoms in cach ring. A fused heterocyclic ring system may include carbocyclic rings and necd include onlv one heterocyclic ring.
Examples of heterocyclyl groups include indolyl, thienyl, isoimidazolyl, thiazolyl, furyl, quinolinyl, imidazolidinyl and benzothienyl. Compounds within the invention containing a heterocyclyl group may occur in two or more tautometric forms depending on the nature of the heterocyclyl group; all such tautomeric forms are included within the scope of the invention.
When used herein the terms 'lower alkyl', 'lower alkenyl', 'lower alkynyl' and 'alkoxy' include straight and branched chain groups containing from 1 to 6 carbon atoms, such as methyl, ethyl, propyl and butyl. A particular alkyl group is methyl.

SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98/01278 __ When used herein the term 'halogen' refers to fluorine, chlorine, bromine and iodine.
It will be appreciated that also included within the scope of the invention are pharmaceutically acceptable salts and pharmaceutically acceptable esters, including in vivo hydrolysable esters, of any carboxy groups that may be present as optional substituents in compounds of formula (I).
Some compounds of formula (I), (IA), (IB) and (IC) may be crystallised or recrystallised from solvents such as organic solvents. In such cases solvates may be formed. This invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of solvents such as water that may be produced by processes such as lyophilisation. Compounds of formula (I), (IA), (IB) and (IC) may be prepared in crystalline form by for example dissolution of the compound in water, preferably in the minimum quantity thereof, followed by admixing of this aqueous solution with a water miscible organic solvent such as a lower aliphatic ketone such as a di-(C 1 _6) alkyl ketone, or a (C 1 _6) alcohol, such as acetone or ethanol.
The compounds of formulae (I), (IA), (IB) and (IC) are metallo-~-lactamase inhibitors and are intended for use in pharmaceutical compositions. Therefore it will readily be understood that they are preferably each provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85% pure, especially at least 95% pure particularly at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions;
these less pure preparations of the compounds should contain at least 1%, more suitably at least 5% and preferably from 10 to 59% of a compound of the formula (I), (IA), (IB) or (IC) or salt, solvate or in vivo hydrolysable estcr thereof.
Compounds of fomula (I) may generally be prepared by processes analogous to those described in the prior art references listcd about.
The present invention also provides a process for the preparation of a compound of formula (IA), (IB) or (IC) as defined above, which comprises reacting a compound of formula (II) Y-C(R5'R6')-CR7(R3')-CO-W
(II) with a compound of formula (III) SUBSTITUTE SHEET ( rule 2~ ) WO 98139311 PCT/EP98/O1Z78 _._ R1, H ~ / Rz H ~ 'w,/
x C02Rx or C02R
(iii) (III) wherein W is a leaving group, Y is Y' where Y' is R4'S or a group convertible thereto and R~ is H, or Y and R~ together form a bond, Rx is R or a carboxylate protecting group, and X, R1', R2', R3', R4', RS' and R6' are R1, R2, R3, R4, RS and R6 or groups convertible thereto, wherein R, R1, R2, R3, R4, RS and R6 are as defined in formula (iA), (IB) or (IC), and thereafter, where Y and R~ form a bond, reacting the product with a nucleophilic sulphur reagent Y'H, where necessary, convening Y' into R4'S, Rx, R 1', R2', R3' R4', RS' andlor R6' into R, R 1, R2, R3, R4, RS
and/or R6 and optionally inter-converting R, Rl, R2, R3, R4, RS andJor R6.
Suitable ester-forming carboxyl-protecting groups Rx other than in vivo hydrolysable ester forming groups are those which may be removed under conventional conditions. Such groups for R" include methyl, ethyl, benzyl, p-methoxybenzyl, benzoylmethyl, p-nitrobenzyl, 4-pyridylmethyl, t5 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, s-butyl, I-amyl, allyl, diphenylmethyl, triphenylmethyl, adamantyl, 2-benzyloxyphenyl, 4-methylthiophenyl, tetrahydrofur-2-yl, tetrahydropyran-2-yl, pentachlorophenyl, acetonyl, ~-toluenesulphonylethyl, methoxymethyl, a silyl (such as trimethylsilyl), stannyl or phosphorus- containing group or an oxime radical of formula -N=CHRY where RY
is aryl or heterocyclyl, or an in_ viv hydrolysable ester radical such as defined below.
Certain compounds of formulae (II) and (III) may include an amino group which may be protected. Suitable amino protecting groups are those well known in the art which may be removed under conventional conditions if required without disruption of the remainder of the molecule.
Examples of amino protecting groups include (C1-6) alkanoyl; bcnzoyl;
benzyl optionally substituted in the phenyl ring by one or two substituents selected from (Cl-4) alkyl, (Cl-4) alkoxy, trifluoromethyl, halogen, or nitro; (C1-4) alkoxycarbonyl; benzyloxycarbonyl or trityl substituted as for benzyl above;
allyloxycarbonyl, trichloroethoxycarbonyl or chloroacetyl.
The compound of formula (III) is preferably presented as the anion prepared by treatment of the amine with an organic base such as triethylamine, pyridine or morpholine, and suitable examples of the leaving W group in the compound of formula (II) include halo such as chloro and mixed sulphonic anhydrides such as those SUBSTITUTE SHEET ( rude 26 ) WO 98/39311 PCT/EP98/01278 _._ where W is methanesulphonyloxy, toluene-p-sulphonyloxy or trifluoromethanesulphonyloxy in mixed sulphonic anhydrides.
The reaction of the compounds of formula (II) and (III) is preferably carried out at ambient temperature, for example 15-25°C, in an inert solvent such as chloroform tetrahydrofuran, dichloromethane, dioxan or dimethylformamide.
Examples of Y convertible into R4'S include halo such as bromo which may be displaced by thiobenzoic acid or thioacetic acid.
Where R~ and Y together represent a bond, the group R4'S may be introduced by addition of a nucleophilic sulphur reagent Y'H. Y' is RQ'S or a group convertible thereto. Thiolacetic acid is a suitable sulphur reagent.
Examples of groups R~', R2', R3', R4' convertible to R1, R2. R3 and R4 include those where any carboxy or amino group is protected by carboxy or amino protecting groups.
R4' in the compound of formula (II) is preferably other than hydrogen, for example acetyl.
The acid derivative of formula (II) is preferably prepared from the corresponding free acid by treatment with strong base such as sodium hydride followed by a source of the anion leaving group W, such as oxalyl chloride where W
is Cl.
The initial product of the reaction of compounds of formulae (II) and (III) is a compound of formula (IV):
Rs Rs Rs Rs R3 X R3 X R'.
Y ~ Y
N / N ~ , R2 O O _ CO2R' COZR' (IV) (v) (vi) wherein the variables are as defined in formulae (II) and (III). Novel intermediates of fomnula (IV) wherein R" is other than R when RI', R'', R3', R4', RS' and R6' are RI, R', R3, R4, RS and R6 also form pan of the invention. In one aspect Y is R4'S and R~ is H.
When Rx is other than hydrogen, the carboxy group -COORx may be deprotected, that is to say, converted to a free carboxy, carboxy salt or carboxy ester group -COOR in a conventional manner, for example as described in EP0232966A.
_g_ SUBSTITUTE SHEET ( rule 26 ) Simultaneous deprotection of -COORx and R4'S may be achieved by treatment with sodium sulphide nonahydrate in water/methanol.
When it is desired to obtain a free acid or salt of the preferred isomer of the formula (I) from an isomeric mixture, this may be effected by chromatographic separation of the diastereomers of the product. Where this is an ester and/or where R4' is other than hydrogen, the desired isomer rnay then be deprotected to give the corresponding free acid or salt. In some cases, however, it has been found V
particularly convenient first to deprotect the isomeric mixture to give an isomeric mixture of the free acid or salt of formula (I), followed by fractional recrystallisation to to give the desired acid or salt isomer. Where *D isomer of formula (I) is desired, it is preferred to use the corresponding *D isomer of the intermediate of formula (III).
When an enantiomerically pure form of (III) is used in the preparation of (I), the preferred diastereomer at position (+) of (I) can also be separated by chromatography. An enantiomerically pure form of (II) may also be used.
A carboxyl group may be regenerated from any of the above esters by usual methods appropriate to the particular Rx group, for example, acid- and base-catalysed hydrolysis, or by enzymically-catalysed hydrolysis, or by hydrogenolysis under conditions wherein the remainder of the molecule is substantially unaffected.
For example, in the case of acetonyl, by hydrolysis in acetonitrile with 0.1 M
aqueous 2o potassium hydroxide solution.
Pharmaceutically acceptable salts may be prepared from such acids by treatment with a base, after a conventional work-up if necessary. Suitable bases include sodium hydrogen carbonate to form sodium salts.
Crystalline forms of the compounds of formula (I) where R is a salt forming canon may for example be prepared by dissolving the compound (I) in the minimum quantity of water, suitably at ambient temperature, then adding a water miscible organic solvent such as a (C 1 _6) alcohol or ketone such as ethanol or acetone, upon which crystallisation occurs and which may be cncouraged for example by cooling or tnturauon.
Compounds of formulae (II) and (IIl) are known compounds or may be prcpared by procedures analogous to those described in the prior art references listed above.
RS'/R6' substituted compounds of formula (II) where Y is Y' and R~ is H may generally be prepared from an acrylic, crotonic, ~i-substituted acrylic, or (3,~3-disubstituted acrylic acid or ester of formula (V):
Rb. 3.
R5, CC2Z (V) SUBSTITUTE SHEET ( rule 26 ) WO 98139311 PCTIEP98/01278 _._ in which Z is H or a hydrolysable ester forming group and the remaining variables are as previously defined , by addition of a nucleophilic sulphur reagent Y'H. Y' is R,'S
or a group convertible thereto. Thiolacetic acid is a suitable sulphur reagent.
Subsequent conversion of the carboxylate group C02Z to a reactive acid leaving group COW, provides the compound of structure (II).
Compounds of formula (II) where Y and R7 are a bond may be obtained from compounds of formula (V) by conversion of the acid group to a leaving group COW.
Compounds of formula (V) are prepared conventionally, for example, by the reaction of a carbonyl compound RS' CORE' with a phosphorane R3'C(PPh3)C02Z.
Novel compounds of formula (III), which are cyclic a-amino acids, may be prepared by any conventional synthesis, for example:
compounds of formula (III) (iii) where X = CH2, derivatives of pipecolic acid may be synthesised from lysine derivatives by methods described in Fujii, et al, Bull. Chem.
Soc. Japan, 1975, 48, 1341;
compounds of formula (III) (iii) where X = S may be prepared as described in Heterocycles, 1985, 23, 889;
compounds of formula (III) (iv) where X = CH2 may be obtained by hydrogenation of substituted 1-isoquinoline carboxylic acids/carboxylate derivatives in the presence of Adam's catalyst as described by Adams, et al, Org. Syn., Coll. Vol.l, 1932, 463.
A compound of formula (I), (IA), (IB) or (IC) or a salt, solvate or in vivo hydrolysable ester thereof, may be administered in the form of a pharmaceutical composition together with or a pharmaceutically acceptable carrier and the invention also relates to such compositions. The compounds of formula (I) have metallo-(3-lactamase inhibitory properties, and are useful for the treatment of infections in animals, especially mammals, including humans, in particular in humans and domesticated (including farm)animals. The compounds may be used, for example, for the treatment of infections of, inter alia, the respiratory tract, the urinary tract, and soft tissues and blood, especially in humans.
The compounds may be used in combination with an antibiotic partner for the treatment of infections caused by metal lo-(3 -lactamase producing strains, in addition to those infections which are subsumed within the antibacterial spectrum of the antibiotic partner. Metallo-~3-lactamase producing strains include:- Pseudomonas aeruginosa, Klebsiella pneumoniae, Xanthomonas maltophilia, Bacteroides fragilis, Serratia marcescens, Bacteroides distasonis, Pseudomonas cepacia, Aeromonas hydrophila, Aeromonas sobria, Aeromonas salmonicida, Bacillus cereus, Legionella gormanii and Flavobacterium spp.

SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98101278 ._ It is generally advantageous to use a compound according to the invention in admixture or conjunction with a carbapenem, penicillin, cephalosporin or other ~i-lactam antibiotic and that can result in a synergistic effect, because of the metallo-~i-lactamase inhibitory properties of the compounds according to the invention. In such cases, the compound of formula (I), (IA), (IB) or (IC) and the (3-lactam antibiotic can be administered separately or in the form of a single composition containing both active ingredients as discussed in more detail below. The compositions of the invention include those in a form adapted for oral, topical or parenteral use and may be used for the treatment of bacterial infection in mammals including humans. The compounds of formula (I), (IA), (IB) or (IC) are particularly suitable for parenteral administration.
The compounds of formula (I), (IA), (IB) or (IC) may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics and other (3-lactam antibiotic/~i-lactamase inhibitor combinations.
The composition may be formulated for administration by any route, such as oral, topical or parenteral. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
The topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, eye ointments and eye or ear drops, impregnated dressings and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.
The formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1 % up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.
Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agcnts, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone;
fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine;
tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle -n-SUBSTITUTE SHEET ( ruie 26 ) WO 98/39311 PCTIEP98/01278 __ before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol;
preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents.
Suppositories will contain conventional suppository bases, e.g. cocoa-butter or other glyceride.
For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the compound can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
Advantageously, agents such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilisation cannot be accomplished by filtration. The compound can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
The compositions may contain from 0.1 ~'~ by wcight, preferably from 10-60%
by weight, of the active material, depending on the method of administration.
Where the compositions comprise dosage units, each unit will prefcrably contain from 50-S00 mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from 100 to 3000 mg per day, for instance 1500 mg per day depending on the route and frequency of administration. Such a dosage corresponds to 1.5 to SO mg/kg per day. Suitably the dosage is from 5 to 20 mg/kg per day.
No toxicological effects are indicated when a compound of formula (I), (IA), (IB) or (IC) or a pharmaceutically acceptable salt thereof is administered in the above-mentioned dosage range.

SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCTJEP98/01278 ._ A composition according to the invention may comprise a compound of formula (I), {IA), (IB) or (IC) or a salt, solvate or in vivo hydrolysable ester thereof together with one or more additional active ingredients or therapeutic agents, for example a (3-lactam antibiotic such as a carbapenem, penicillin or cephalosporin or pro-drug thereof. Carbapenems, penicillins, cephalosporins and other (3-lactam antibiotics suitable for co-administration with the compound of formula (I), (IA), (IB) or (IC) - whether by separate administration or by inclusion in the compositions according to the invention - include both those known to show instability to or to be otherwise susceptible to metailo-(3-lactamases and also those known to have a degree of resistance to metallo-~3-lactamases.
A ser-ine (3-lactamase inhibitor such as clavulanic acid, sulbactam or tazobactam may also be co-administered with the compound of the invention and the ~3-lactam antibiotic, either by separate administration, or co-formulation with one, other or both of the compounds of the invention and the (3-lactam antibiotic.
Examples of carbapenems that may be co-administered with the compounds according to the invention include imipenem, meropenem, biapenem, BMS 181139 ([4R-[4alpha,Sbeta,bbeta(R*)J]-4-[2-[(aminoiminomethyl)amino]ethyl]-3-[(2-cyanoethyl)thio]-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), B02727 ([4R-3[3S*,SS*(R*)],4alpha,Sbeta,6beta(R*)]]-6-(1-hydroxyethyl) -[[5-[1-hydroxy-3-(methylamino)propyl]-3-pyrrolidinyl]thio]-4-methyl-7-oxo-1-azabicyclo[3.2.0] kept-2-ene-2-carboxylic acid monohydrochloride), ER35786 (( 1 R, SS, 6S)-6-[1(R)-Hydroxymethyl]-2-[2(S)-[1(R)-hydroxy-1-[pyrrolidin-3(R)-yl]
methyl]pyrrolidin-4(S)-ylsulfanyl]-1-methyl-1-carba-2-penem-3-carboxylic acid hydrochloride) and S4661 ((IR,SS,6S)-2-[(3S,5S)-5-(sulfamoylaminomethyl) pyrrolidin-3-yl]thio-6-[(1R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylic acid).
Examples of penicillins suitable for co-administration with the compounds according to the invention include benzylpenicillin, phenoxymethylpenicillin, carbcnicillin, azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin.
cyclacillin, pirbenicillin, azlocillin, mezlocillin, sulbenicillin, piperacillin, and other known penicillins. The penicillins may be used in the form of pro-drugs thereof, for example as in vivn hydrolysable esters,for example the acetoxymethyl, pivaloyloxymethyl, a-ethoxycarbonyloxyethyl and phthalidyl esters of ampicillin, benzylpenicillin and amoxycillin; as aldehyde or ketone adducts of penicillins containing a 6-a-aminoacetamido side chain (far example hetacillin, metampicillin and analogous derivatives of amoxycillin); and as a-esters of carbenicillin and ticarcillin, for example the phenyl and indanyl a-esters.

SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98/01278 _ Examples of cephalosporins that may be co-administered with the compounds according to the invention include, cefatrizine, cephaloridine, cephalothin, cefazolin, cephalexin, cephacetrile, cephapirin, cephamandole nafate, cephradine, 4-hydroxycephalexin, cephaloglycin, cefoperazone, cefsulodin, ceftazidime, cefuroxime, cefmetazole, cefotaxime, ceftriaxone, and other known cephalosporins, all of which may be used in the form of pro-drugs thereof.
Examples of ~i-lactam antibiotics other than penicillins and cephalosporins that may be co-administered with the compounds according to the invention include aztreonam, latamoxef (Moxalactam - Trade Mark), and other known (3-lactam antibiotics, all of which may be used in the form of pro-drugs thereof.
Particularly suitable penicillins for co-administration with the compounds according to the invention include ampicillin, amoxycillin, carbenicillin, piperacilIin, azlocillin, mezlocillin, and ticarcillin. Such penicillins may be used in the form of their pharmaceutically acceptable salts, for example their sodium salts.
Alternatively, ampicillin or amoxycillin may be used in the form of fine particles of the zwitterionic form (generally as ampicillin trihydrate or amoxycillin trihydrate) for use in an injectable or infusable suspension, for example, in the manner hereinbefore described in relation to the compounds according to the invention. Amoxycillin, for example in the form of its sodium salt or the trihydrate, is particularly preferred for use in synergistic compositions according to the invention.
Particularly suitable cephalosporins for co-administration with the compounds according to the invention include cefotaxime and ceftazidime, which may be used in the form of their pharmaceutically acceptable salts, for example their sodium salts.
A compound of formula (I), (IA), (IB) or (IC) may be administered to the patient in conjunction with a ~3-lactam antibiotic such as a carbapenem, penicillin or cephalosporin in a synergistically effective amount.
The compounds of formula (I), (IA), (IB) or (IC) may suitably be administered to the patient at a daily dosage of from 0.7 to 50 mg/kg of body weight. For an adult human (of approximately 70 kg body weight), from 50 to 300() mg, preferably from 100 to 1000 mg, of a compound according to the invention may be administered daily, suitably in from 1 to 6, preferably from 2 to 4, separate doses. Higher or lower dosages may, however, be used in accordance with clinical practice.
When the compositions according to the invention are presented in unit dosage form, each unit dose may suitably comprise from 25 to 1000 mg, preferably from 50 to 500 mg, of a compound according to the invention. Each unit dose may, for example, be 62.5, 100, 125, 150, 200 or 250 mg of a compound according to the invention.
When the compounds of formula (I), (IA), (IB) or (IC) are co-administered with a penicillin, cephalosporin, carbapenem or other (3-lactam antibiotic, the ratio of the SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98/01278 _._ amount of the compound according to the invention to the amount of the other (3-lactam antibiotic may vary within a wide range. The said ratio may, for example, be from 100:1 to 1:100; more particularly, it may, for example, be from 2:1 to 1:30.
The amount of carbapenem, penicillin, cephalosporin or other (3-lactam antibiotic in a synergistic composition according to the invention will normally be approximately similar to the amount in which it is conventionally used ~r ~, for example from about 50 mg, advantageously from about 62.5 mg, to about 3000 mg per unit dose, more usually about 125, 250, 500 or 1000 mg per unit dose.
The present invention further provides a compound of formula (I) or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof, and in particular a compound of formula (IA), (IB) or (IC),for use in the treatment of bacterial infections.
The present invention also includes the use of a compound of formula (I), (IA), (IB) or (IC), or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof, in the manufacture of a medicament for the treatment of bacterial infections The present invention also includes the use of a compound of formula (I), (IA), (IB) or (IC), or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof as a metallo-(3-lactamase inhibitor.
All the above compositions and methods may optionally include a serine [3-lactamase inhibitor as above described.
The compounds of the present invention are active against metallo-[3-lactamase enzymes produced by a wide range of organisms including both Gram-negative organisms and Gram-positive organisms.
The following Examples illustrate compounds useful in the present invention, and intermediates in their preparation. (All temperatures are in °C).
EXAMPLES
Example 1: 3~[2'(RS)-benxyl-3'-mercaptopropionyl)-.1(RS)-carboxy~tetrahydro-1,3-thiaxine (E1) (a) 3-[S-acetyl-2'(RS)-benzyl-3'-mercaptopropionylJ-4(RS)-carboxy-tetrahydro-1,3-thiazine To a cooled (0° ), stirred solution of S-acetyl-2-benzyl-3-mercaptopropionic acid (EP0361365) (238mg, 1.0 mmol) in dry tetrahydrofuran (10 ml) containing dry dimethylformamide ( 1 drop), was added sodium hydride (44 mg of a 55%
suspension in oil, 1.0 mmol). The reaction mixture was allowed to reach room temperature and stirring was continued for a further 15 mins. The suspension was then recooled (0° ) SUBSTITUTE SHEET ( ruie 26 ) WO 98/39311 PCT/EP98/01278 __ and treated with oxalyl chloride ( l OS ul, 1.2 mmol) and stirred at room ambient temperature for 30 mins. The resulting mixture was then evaporated in vacuo and the residue suspended in dry tetrahydrofuran (S ml). The filtered solution was taken to dryness once more to afford the acid chloride as an oil A stirred, cooled (0° ) suspension of DL-4-carboxytetrahydro-1,3-thiazine (1.0 mmol) (prepared as in Heterocycles 1985, 23, 889) in dry tetrahydrofuran (10 ml) was treated with triethylamine (278 ul, 2.0 mmol) followed by a solution of the above acid chloride in dry tetrahydrofuran (S ml) added over S minutes. The mixture was stirred at ambient temperature for 3 hours and then partitioned between ethyl acetate t0 and 1M hydrochloric acid. The organic layer was washed with water and saturated brine and dried over anhydrous sodium sulphate. Evaporation of the solvent afforded crude product as an oil which was chromatographed on silica gel. Elution with 4%
methanol in chloroform, containing 0.1 % acetic acid, afforded recovered substituted propionic acid starting material followed by the title diastereomeric mixture ( 1:1 ) as a ~5 crisp foam (119 mg, 30%). 8H (CDCl3) (Isomer 1) 5.56 (1H, m), interalia.
(Isomer 2) 5.40 ( 1H, m), inter alia ppm.
(b) Title compound The S-acetyl derivative from (a) ( 119 mg, 0.32 mmol) was dissolved in water 20 (0.6 ml) and concentrated aqueous ammonia (0.4 ml, sp.gr. 0.88) and stirred at room temperature for 1 hour. The reaction mixture was diluted in ethyl acetate and washed with sufficient dilute hydrochloric acid to acidify the aqueous phase. The two-phase system was treated with saturated brine and the organic layer separated, washed with further brine, dried over sodium sulphate and evaporated to afford the crude product.
25 Chromatography on silica gel, eluting with 4% methanol in chloroform containing 0.1 % acetic acid, gave the title diastereomeric mixture as a crisp foam (SO
mg). vm~
(CHCl3) 1752, 1711, 1644, 1423 em-1. 8H (CDC13) (Isomer 1) 1.64 (1H, dd> 1 10.7 and 7.4 Hz) inter alia. (Isomer 2) 1.59 (1 H, dd,1 8.6 and 8.6 Hz) inter alia, ppm.
EIMS M+ 325.0814. Calculated for ClSH19N03S2 325.0806.
Example 2: N-[2'(RS)-benzyl-3'-mercaptopropionyl]-DL-pipecolic acid (E2) (a) N-[S-acetyl-2'(RS)-benzyl-3'-mercaptopropionyl]-DL-pipecolic acid methyl ester To a cooled (0° ), stirred solution of S-acetyl-2-benzyl-3-mercaptopropionic acid (EP0361365) (238mg, 1.0 mmol) in dry tetrahydrofuran (10 ml) containing dry dimethylformamide (1 drop), was added sodium hydride (44 mg of a SS%
suspension in oil, 1.0 mmol). The reaction mixture was allowed to reach room temperature and SUBSTITUTE SHEET ( ruie 26 ) r r WO 98/39311 PCTIEP98/01278 ._ stirring was continued for a further 15 mins. The suspension was then recooled (0° ) and treated with oxalyl chloride (105 ul, 1.2 mmol) and stirred at room ambient temperature for 30 mins. The resulting mixture was then evaporated in vacuo and the residue suspended in dry tetrahydrofuran (5 ml). The filtered solution was taken to dryness once more to afford the acid chloride as an oil A stirred, cooled (0° ) suspension of DL-pipecolic acid methyl ester (1.0 mmol) (prepared from the free acid by treatment with hydrogen chloride in methanol) in dry tetrahydrofuran (10 ml) was treated with triethylamine (278 ul, 2.0 mmol) followed by a solution of the above acid chloride in dry tetrahydrofuran (5 ml) added over 5 minutes. The mixture was stirred at ambient temperature for 3 hours and then partitioned between ethyl acetate and 1M hydrochloric acid. The organic layer was washed with water and saturated sodium bicarbonate solution and dried over anhydrous sodium sulphate. Evaporation of the solvent afforded crude product as an oil which was chromatographed on silica gel. Elution with ethyl acetate/hexane ( 1:3) afforded the title diastereomeric mixture ( 1:1 ) as an oil ( 183 mg, 50%). 8H
(CDCl3) (Isomer 1) 5.38 (1H, m), 3.71 (3H, s), 2.32 (3H, s) inter alia. (Isomer 2) 5.44 (1H, m), 3.66 (3H, s), 2.35 (3H, s) inter alia ppm.
(b) Title compound The S-acetyl derivative from (a) (183 mg, 0.50 mmol) was dissolved in water (0.6 ml) and concentrated aqueous ammonia (0.4 ml, sp.gr. 0.88) and stirred at room temperature for 0.7 hour. The reaction mixture was diluted in ethyl acetate and washed with sufficient dilute hydrochloric acid to acidify the aqueous phase.
The two-phase system was treated with saturated brine and the organic layer separated, washed with further brine, dried over sodium suiph~te and evaporated to afford the crude product. Chromatography on silica gel, cluttng with 4~k methanol in chloroform containing U.1 % acetic acid, gave the title diastereomeric mixture as a crisp foam (64 mg). vmax (CIIC131 1739. 1712. 1629, 14-i.l cm-1. 8H (CDCI3) (Isomer 1 ) 1.68 ( 1H, dd, J 10.5 and 7.0 Hz), 5.38 ( 1 H, ml inter alia.
(Isomer 2) 1.52 (1H, dd, J 7.9 and 9.6 Hz), 5.43 (1H, m) inter alia, ppm. EIMS M+ 307.
Examples 3 & 4: N-[2'(R ur S)-benzyl-3'-mercaptopropionyl]-D-pipecolic acid (E3) and N-[2'(S or R)-benzyl-3'-mercaptopropionyl]-D-pipecolic acid (E4) (a) N-[S-acetyl-2'(R or S)-benzyl-3'-mercaptopropionyl]-D-pipecolic acid methyl ester (Isomer A) and N-[S-acetyl-2'(S or R)-benzyl-3'-mercaptopropionyl]-D-pipecolic acid methyl ester (Isomer B) SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98/OI278 __ To a cooled (0° ), stirred solution of S-acetyl-2-benzyl-3-mercaptopropionic acid (EP0361365) (480mg, 2.0 mmol) in dry tetrahydrofuran (10 ml) containing dry dimethylformamide (1 drop), was added sodium hydride (88 mg of a SS%
suspension in oil, 2.0 mmol). The reaction mixture was allowed to reach room temperature and stirring was continued for a further 15 mins. The suspension was then recooled (0° ) and treated with oxalyl chloride (210 u1, 2.4 mmol) and stirred at room ambient temperature for 30 mins. The resulting mixture was then evaporated in vacuo and the residue suspended in dry tetrahydrofuran (5 ml). The filtered solution was taken to dryness once more to afford the acid chloride as an oil A stirred, cooled (0° ) suspension of D-pipecolic acid methyl ester hydrochloride (0.32g, 1.78 mmol) (prepared from the free acid by treatment with hydrogen chloride in methanol) in dry tetrahydrofuran (10 ml) was treated with triethylamine (560 ul, 4.0 mmol) followed by a solution of the above acid chloride in dry tetrahydrofuran (5 ml) added over 5 minutes. The mixture was stirred at ambient temperature for 3 hours and then partitioned between ethyl acetate and 1 M
hydrochloric acid. The organic layer was washed with water and saturated sodium bicarbonate solution and dried over anhydrous sodium sulphate. Evaporation of the solvent afforded crude product as an oil which was chromatographed on silica gel.
Elution with ethyl acetate/toluene (1:9) afforded Isomer A as an oil (160 mg, 25%).
SH (CDCl3) 5.44 (1H, d, J 4.9 Hz), 3.66 (3H, s), 2.32 (3H, s) inter alia. CIMS
MH+
364. Further elution afforded Isomer B as an oil (195mg, 30%) 8H (CDC13) 5.38 (1H, d, J 2 Hz), 3.71 (3H, s), 2.35 (3H, s) inter alia ppm. CIMS MHO' 364.
(b) N-[2'(R or S)-benzyl-3'-mercaptopropionyl)-D-pipecolic acid (E3) The S-acetyl derivative Isomer A from (a) (160 mg, 0.44 mmol) was dissolved in 0.1 M potassium hydroxide solution (8.8m1, 0.88mmol) and acetonitrile (9 ml) and stirred at room temperature for 7 hours. The reaction mixture was diluted in ethyl acetate and the organic layer discarded. The aqueous layer was acidifed with dilute hydrochloric acid and extracted with ethyl acetate The organic layer was washed with water followed by saturated brine and dried over magnesium sulphate.
Evaporation of solvent afforded E3 as a crisp foam (53 mg). vm~ (CHC13) 1710, 1629, I 444 cm' I . SH (CDCl3) 1.52 ( 1 H, dd, J 9.5 and 7.8 Hz), 5.47 ( 1 H, d, J 4.2 Hz)) inter alia, ppm. EIMS M+ 307.1241. Calculated for C16H2IN03S, 307.1242.
(c) N-[2'(S or R)-benzyl-3'-mercaptopropionyl]-D-pipecotic acid (E4) The S-acetyl derivative Isomer B from (a) (194 mg, 0.53 mmol) was dissolved in 0.1 M potassium hydroxide solution ( 10.6m1, 1.06mmo1) and acetonitrile ( 10 ml) and stirred at room temperature for 7 hours. The reaction mixture was diluted in ethyl SUBSTITUTE SHEET ( rule 26 ) r T

- WO 98/39311 PCT/EP98/012'78 _._ acetate and the organic layer discarded. The aqueous layer was acidifed with dilute hydrochloric acid and extracted with ethyl acetate The organic layer was washed with water followed by saturated brine and dried over magnesium sulphate.
Evaporation of solvent afforded E4 as a crisp foam (60 mg). vmax (CHCl3) 1743, 1710, 1628, cm-1. SH (CDC13) 1.70 (1H, dd, J 10.8 and 6.8 Hz), 5.39 (1H, d, m)) inter alia, ppm.
EIMS M'~ 307.
Example 5:
N-(2-Benzyl-3-mercaptopropionyl)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic to acid (ES) (a) 1,2,3,4-Tetrahydroisoquinoline-1-carboxylic acid A solution of 1-isoquinolinecarboxylic acid (2.0g; Aldrich) in acetic acid (30m1) was hydrogenated in the presence of Adam's catalyst ( Pt02; cf. R.
Adams, et al, Org. Syn. Coll. Vol. 1932, 1, 463.] (320mg) at 60 psi, at room temperature for 7h.
The catalyst was filtered off and washed with further portions of acetic acid.
The combined filtrate and washings were evaporated and the residue recrystallised from water to afford the title acid (l.U2g); vmax (CHC13) 3104, 2360br and 1616 cm-1, SH [(CD3)2S0] 2.88 (2H,m), 3.20 (2H, m), 4.43 (1H, s), 7.U7-7.20 (3H,m) and 7.71 (lH,m).
(b) Methyl 1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid hydrochloride Hydrochloric acid gas was passed into a stirred suspension of the acid from (a) (730mg) in methanol (25m1) with water bath cooling until the solution was saturated.
The mixture was left at room temperature for 40h and the solvent was evaporated to give a residue, which was then partitioned between water and ether. the aqueous phase was neutralised with potassium carbonate and the ether phase separated.
The aqueous phase was extracted with two further portions of ether and the combined cther layers wcrc washed with brine, dricd ovcr anhydrous magnesium sulphate and then evaporated to 2Uml. The solution was saturated with HCI gas and the product precipitated as an oil. The ether was decanted and the residue triturated with methanol, followed by ether and was left for 48h to crystallise. The solid was filtered off, washed with a small portion of ether and dried in vacuo to give the title hydrochloride (334mg): vmax(CHC13) 3410, 2400-28UObr and 1750 cm-l.
(c) Methyl N-(S-acetyl-2'-benzyl-3'-mercaptopropionyl)-1,2,3,4-tetrahydroisoquinoline-1-carboxylate SUBSTITUTE SHEET ( rule 26 ) WO 98/39311 PCT/EP98/01278 _._ Oxalyl chloride was added to a stirred soloution of S-acetyl-2-benzyl-3-mercaptopropionic acid (EP0361365) (238mg) in dichloromethane (lOml).
Dimethylformamide ( 1 drop) was added and the mixture was stirred for 1.5h.
The solvents were evaporated and the residue was triturated with further portions of dichloromethane and evaporated (x 2) to give the acid chloride. The hydrochloride from (c) (228mg) was partitioned between water and ether, with stirring, and an excess of potassium carbonate added portionwise.The ethereal layer was separated and the aqueous layer extracted with ether twice. The combined organic layers~were washed with brine, dried (MgS04) and evaporated. To the residue, in dichloromethane (lOml) was added a solution of the previously-prepared acid chloride in dichloromethane (2m1), followed by triethylamine (0.28m1). The mixture was washed successively with citric acid solution, water, sodium hydrogencarbonate solution and brine. It was dried (MgS04) and evaporated and the residue chromatographed on Kieselgel. Elution with ethyl acetate-hexane (1:3) gave the title ester, a mixture of racemic diastereomers (2:1 ), as a gum (369mg, 89%), vmax (CHC13) 1741, 1683 and 1636cm-l; HRMS Found: m/z 411.1508. C23H25N04S
requires M+ 411.1504.
(d) Title compound Water (5m1) was added to a stirred solution of the ester from (c) (369mg) in methanol (5m1). Sodium sulphide 9.H20 (645mg) was added and the mixture was stirred for lh 35min. Dilute hydrochloric acid (2m1) was added and the mixture partitioned between ethyl acetate and water. The organic phase was washed with water, followed by brine and dried (MgS04). Evaporation, followed by column chromatography of the residue (Kieselgel, eluting 5% methanol in chloroform), gave the title mercaptocarboxylic acid (E5) (156mg) as a 2:1 mixture of racemic diastereomers, vmax (CHCl3) 1753,1717 and 1636 cm-1; 8H [(CD3)2S0] 2.25-3.0 (7H,m), 3.30-4.15 (4H,m), 5.53 and 5.19 (2:1 ratio, together, 2H) and 7.05-7.55 (9H, m, ArH); HRMS Found: m/z 355.1243. C20H24N03S requires M+ 355.1242.
BIOLOGICAL ACTIVITY
ISO screen:-The inhibitory activity of the compounds of the invention was measured in 25mM PIPES pH 7 buffer at 10 concentrations (1000, 333, 111, 37, 12.3, 4.1, .1.4, 0.46, 0.15 and O.OSItM) at 37oC using nitrocefin (91 ~tM, final concentration) as the reporter substrate. The assays were performed with a 5 minute preincubation of enzyme and inhibitor and were conducted in the presence of added zinc sulphate (Zn2+ 1001tM, final concentration). The methodology is described in detail in the SUBSTITUTE SHEET ( rule 26 ) r ?

WO 98/39311 PCT/EP98/01278 _ following references: Payne et al (1991), J. Antimicrob. Chemother., 28:255:
Payne et al (1994), Antimicrob. Agents and Chemother., 38:767.
Results:-Compounds of the Examples exhibit I50 values against Bacteroides fragilis CfiA metallo-(3-lactamase of between 0.5 and 21 ~tM and I50 values against Stenotrophomonas maltophilia L1 metallo-~3-lactamase of between 0.07 and 2 p.M.
Antibacterial activity of the compounds of the invention in combination with l0 meropenem against a metallo-(3-lactamase producing strain of Bacteroides fragilis:-The antibacterial activity of meropenem was potentiated as follows:-A concentration of 8pg/ml of compounds E3 and ES each reduced the MIC of meropenem against B.fragilis 262 from >128~tg/ml to 32~tg/ml.
[MIC = minimum inhibitory concentration (itg/ml)]

SUBSTITUTE SHEET ( rule 26 )

Claims (19)

Claims

1. A method of treatment of bacterial infections in humans or animals which comprises administering, in combination with a .beta.-lactam antibiotic, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof:

wherein:
X is S, S(O)p or CH2 where p is 1 or 2;
R is hydrogen, a salt forming cation or an in vivo hydrolysable ester-forming group;
R1 and R2 are selected from halogen, mercapto, (C1-6)alkyl optionally substituted by 1-3 halo, phenyl, (C1-6)alkoxy optionally substituted by 1-3 halo, hydroxy(C1-6)alkyl, mercapto(C1-6)alkyl, hydroxy, CO2R7, N(R7)2 or CON(R7)2 where each R7 is independently hydrogen or (C1-6) alkyl, OCONH2, nitro, (C1-6) alkylcarbonyloxy, (C1-6)alkoxycarbonyl(C1-6) alkyl, formyl, or (C1-6) alkylcarbonyl groups;
R3 is hydrogen, (C1-6)alkyl optionally substituted by up to three halogen atoms, (C3-7)cycloalkyl, fused aryl(C3-7)cycloalkyl, (C3-7)cycloalkyl(C2-6)alkyl, (C2-6)alkenyl, (C2-6)alkynyl, aryl, aryl-(CHR10)m-X1-CHR11)n, heterocyclyl or heterocyclyl-(CHR10)m-X1-(CHR11)n, where m is 0 to 3, n is 1 to 3, each R10 and R11 is independently hydrogen or (C1-4)alkyl and X1 is O, S(O)x where x is 0-2, or a bond;
R4 is hydrogen, or an in vivo hydrolysable acyl group; and R5 and R6 are independently hydrogen and (C1-6)alkyl or together represent (CH2)q where q is 2 to 5.

2. A compound of formula (IA) which is a compound of formula (I)(i) where X
is S or S(O)p or formula (I)(ii) as defined in claim 1, where R3 is aryl-(CHR10)m-X1-(CHR11)n.

3. A compound of formula (IB) which is a compound of formula (I) as defined in claim 1 where where R5 and R6 are not hydrogen.

4. A compound of formula (IC) which is a compound of formula (I) as defined in claim 1 where the stereochemistry at the carbon marked * is D-.

5. A method according to claim 1 wherein X1 is O, S or a bond and R10 and R11 are each hydrogen.

6. A method according to claim 1 or 5 wherein X is S or CH2.

7. A method according to any of claims 1, 5 or 6 wherein R1 and R2 are both hydrogen.

8. A method according to any of claims 1 or 5 to 7 wherein R3 is benzyl or 2-phenethyl.

9. A method according to any of claims 1 or 5 to 8 wherein R4 is hydrogen.

10. A method according to any of claims 1 or 5 to 9 wherein R5 and R6 are independently hydrogen or methyl.

11. A method according to any of claims 1 or 5 to 10 wherein the stereochemistry at the carbon atom marked (+) is S.

12. A compound selected from 3-[2'(RS)-benzyl-3'-mercaptopropionyl]-4(RS)-carboxy-tetrahydro-1,3-thiazine N-[2'(RS)-benzyl-3'-mercaptopropionyl]-DL-pipecolic acid N-[2'(R or S)-benzyl-3'-mercaptopropionyl]-D-pipecolic acid N-[2'(S or R)-benzyl-3'-mercaptopropionyl]-D-pipecolic acid and N-(2-Benzyl-3-mercaptopropionyl)-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof.

13. A process for the preparation of a compound according to claim 2, 3 or 4, which comprises reacting a compound of formula (II) Y-C(R5'R6')-CR7(R3')-CO-W (II) with a compound of formula (III) wherein W is a leaving group, Y is Y' where Y' is R4'S or a group convertible thereto and R7 is H, or Y and R7 together form a bond, R x is R or a carboxylate protecting group, and X, R1', R2', R3', R4', R5' and R6' are R1, R2, R3, R4, R5 and R6 or groups convertible thereto, wherein R, R1, R2, R3, R4, R5 and R6 are as defined in formula (IA), (IB) or (IC), and thereafter, where Y and R7 form a bond, reacting the product with a nucleophilic sulphur reagent Y'H, where necessary, converting Y' into R4'S, R x, R1', R2', R3' R4', R5' and/or R6' into R, R1, R2, R3, R4, R5 and/or R6 and optionally inter-convening R, R1, R2, R3, R4, R5 and/or R6.

14. A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof, as defined in claim 1, together with a .beta.-lactam antibiotic in a synergistically effective amount and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising a compound according to claim 2, 3, 4 or 12 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition according to claim 15 which additionally comprises a .beta.-lactam antibiotic in a synergistically effective amount.

17. A composition according to claim 14 or 16 wherein the .beta.-lactam antibiotic is a carbapenem selected from imipenem, meropenem, biapenem, BMS 181139 ([4R-[4alpha,5beta,6beta(R*)]]-4-[2-[(aminoiminomethyl)amino]ethyl]-3-[(2-cyanoethyl)thio]-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0] kept-2-ene-2-carboxylic acid), BO2727 ([4R-3[3S*,5S*(R*)],4alpha,5beta,6beta(R*)]]-6-(1-hydroxyethyl)-[[5-[1-hydroxy-3-(methylamino)propyl]-3-pyrrolidinyl]thio]-4-methyl-7-oxo-1-azabicyclo[3.2.0] kept-2-ene-2-carboxylic acid monohydrochloride), ER35786((1R, 5S,6S)-6-[1(R)-Hydroxymethyl]-2-[2(S)-[1(R)-hydroxy-1-[pyrrolidin-3(R)-yl]
methyl]pyrrolidin-4(S)-ylsulfanyl]-1-methyl-1-carba-2-penem-3-carboxylic acid hydrochloride) and S4661 ((1R,5S,6S)-2-((3S,5S)-5-(sulfamoylaminomethyl) pyrrolidin-3-yl]thio-6-[(1R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylic acid).

18. A compound of formula (I) or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof, as defined in claim 1 for use in the treatment of bacterial infections.

19. The use of a compound of formula (I) or a pharmaceutically acceptable salt, solvate or in vivo hydrolysable ester thereof, as defined in claim 1 in the manufacture of a medicament for the treatment of bacterial infections.