Novel Compounds

The present invention relates to novel N-benzyl-N-[(1 ,2-substituted)-ethyl]amines which are inhibitors of bacterial phenylalanyl t-RNA synthetase (FRS), processes for their preparation and their use in therapy as anti-bacterial agents. t-RNA synthetases are involved in protein biosynthesis so that inhibition thereof may be expected to lead to a cessation of cell growth. Thus, for instance, the compound mupirocin, produced by the organism Pseudomonas fluorescens, is an antibacterial agent and is used as the active ingredient in the product Bactroban, marketed by GlaxoSmithKline. Mupirocin has been shown to be an inhibitor of the isoleucyl t-RNA synthetase. Each t-RNA synthetase represents a separate target for drug discovery. t-RNA synthetase inhibitors which are selective for bacterial cells over mammalian cells are of considerable therapeutic interest as they have the potential to be used as antibacterial agents. The sequence of the t-RNA synthetase genes in organisms such as S aureus have recently been determined, see for instance US Patent no 5 756 329 (FRS alpha and beta sub-units), thereby assisting the process of identifying inhibitors. In addition, the E coli FRS has been well described (see for instance Santi et al, Biochemistry, 1971 , 10, 4804-4812). Santi et al have also described a class of inhibitors of E coli FRS which are λ/-benzyl-λ/-(1-methyl-2-phenylethyl)amines.

Other known λ/-benzyl-λ/-(1 ,2-disubstituted-ethyl)amines include λ/-benzyl-λ/-[2- cyclohexyl-1-(cyclohexylmethyl)ethyl]amine (Edgerton et al, J Am Pharm Assoc, 48, 1959, 320-3), prepared as a potential side chain for use in a penicillin G derivative and λ/-benzyl-λ/-[1-methyl-2-(4-phenylcyclohexyl)ethyl]amine (De Ivleglio et al, Farmaco, Ed Sci, 1980, 35, 191-202) which was investigated for analgesic, anorexigenic and anti- reserpine activity.

We have now found a class of compounds which are potent inhibitors of bacterial FRS, including S aureus FRS viz λ/-benzyl-λ/-(2-cyclohexyl)ethylamines.

Accordingly, the present invention provides a compound of the formula (I):

(I) in which:

R1 is hydrogen, trifluoromethyl or C(-|_6)alkyl; R2 is hydrogen or C(-|_6)alkyl; and R ³ is hydrogen or a substituent OR ⁴ in which R ⁴ is hydrogen, C(<|_g)alkyl, C(2-

6)alkenyl, C(3_7)cycloalkyl, C(3_7)cycloalkenyl or heterocyclyl, in which alkyl, alkenyl, cycloalkyl, cycloalkenyl or heterocyclyl is optionally substituted by one or two substituents selected from hydroxy, C(-|_6)alkoxy, halo, cyano, carboxy, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted aryl, and

-CONR ⁵ R ⁶ in which R ⁵ is hydrogen or C(-|_6)alkyl and R ⁶ is hydrogen or C(i_4)alkyl optionally substituted by hydroxy, -CONHC(-|_4)alkyl or heterocyclyl; and salts thereof, preferably pharmaceutically acceptable salts thereof.

Compounds of formula (I) are inhibitors of bacterial phenylalanyl tRNA synthetase, including S aureus phenylalanyl tRNA synthetase.

Compounds of formula (I) have the S configuration at the chiral centre ^* (alpha to the nitrogen) marked thus: ^* , when R ² is hydrogen, and the same absolute configuration in other compounds of formula (I) where different substituent patterns may change the order of precedence in applying the Cahn-lngold-Prelog sequence rules for determining R and S Compounds with the alternative R configuration are found to be considerably less potent as inhibitors of S aureus phenylalanyl tRNA synthetase. It will be appreciated that mixtures of the R and S compounds, for instance a racemic mixture, will have activity as inhibitors of S aureus phenylalanyl tRNA synthetase, by virtue of the presence of the compound with the S configuration and accordingly fall within the scope of the present invention.

Representative examples of R^ include hydrogen and trifluoromethyl.

Representative examples of R ² include hydrogen and methyl.

In one aspect R^ is a substituent OR** in which R^ is hydrogen, C(3_6)alkyl, C(2- 6)alkenyl, C(3_7)cycloalkyl, C(3_7)cycloalkenyl or heterocyclyl, in which alkyl, alkenyl, cycloalkyl, cycloalkenyl or heterocyclyl is optionally by one or two substituents selected from hydroxy, C(-|_6)alkoxy, halo, cyano, carboxy, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted aryl, and -CONR ⁵ R ⁶ in which R ⁵ is hydrogen or C(-|_6)alkyl and R ⁶ is hydrogen or C(i_4)alkyl optionally substituted by hydroxy, -CONHC(- _| _4)alkyl or heterocyclyl, or R^ is C(-|_2)alkyl substituted by one or two substituents selected from hydroxy, C(i_6)alkoxy, halo, cyano, carboxy, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted aryl, and -CONR ⁵ R ⁶ in which R ⁵ is hydrogen or C(-|_6)alkyl and R ⁶ is hydrogen or C(-|_4)alkyl optionally substituted by hydroxy,

Representative examples of R^ include hydrogen and OR^ in which R^ is hydrogen, methyl, allyl, methallyl, cyclopentyl, cyclohexenyl, heterocyclyl comprising one or two oxygen atoms for instance tetrahydrofuranyl, or an alkyl group, for instance methyl, ethyl, propyl or butyl optionally substituted by one or two substituents selected from nitrile, hydroxy, carboxy, optionally substituted phenyl for instance phenyl and

4-methylsulphonylphenyl, optionally substituted heteroaryl for instance pyridyl, benzimidazolyl, tetrazolyl and optionally substituted thiazolyl such as 2-chlorothiazolyl, and optionally substituted heterocyclyl for instance morpholino. If the alkyl group is disubstituted, the two substituents may be the same, for instance hydroxy, or different.

A representative example of a disubstituted group OR^ is 2,3-dihydroxypropoxy.

Further representative examples of OR"* when R^ is a substituted alkyl group include those in which the substituent is -CONR ⁵ R ⁶ and in which R ⁵ is hydrogen, methyl or ethyl and R ⁶ is hydrogen or C(i_2)alkyl optionally substituted by hydroxy,- CONHMe, or tetrahydrofuran-2-yl.

Salts may be formed from inorganic and organic acids. Representative examples of suitable inorganic and organic acids from which pharmaceutically

acceptable salts of compounds of formula (I) may be formed include maleic, fumaric, benzoic, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, hydrochloric, hydrobromic, sulfuric, cyclohexylsulfamic, phosphoric and nitric acids.

When used herein, the term "alkyl" and similar terms such as "alkoxy" includes all straight chain and branched isomers. Representative examples thereof include methyl, ethyl, n-propyl, /so-propyl, n-butyl, sec-butyl, /so-butyl, f-butyl, n-pentyl and n- hexyl. Preferred substituents for an alkyl group include, for example, and unless otherwise defined, halogen, cyano, azido, nitro, carboxy, (C< _| _6)alkoxycarbonyl, carbamoyl, mono- or di-(C- _| _6)alkylcarbamoyl, sulpho, sulphamoyl, mono- or di-(C-μ 6)alkylsulphamoyl, amino, mono- or di-(Ci_6)alkylamino, acylamino, ureido, (Ci_6)alkoxycarbonylamino, 2,2,2-trichloroethoxycarbonylamino, aryl, heterocyclyl, hydroxy, (C-|_6)alkoxy, acyloxy, oxo, acyl, 2-thienoyl, (Ci_6) ^alk y'thio, (C- _| _6)alkylsulphinyl, (C-μgJalkylsulphonyl, hydroxyimino, (C<|_6)alkoxyimino, hydrazino, hydrazono, benzohydroximoyl, guanidino, amidino and iminoalkylamino.

When used herein the terms "cycloalkyl" and "cycloalkenyl" include, unless otherwise defined, carbocyclic rings having from three to seven ring carbon atoms. Preferably the cycloalkyl and cycloalkenyl rings comprise five or six ring carbon atoms.

When used herein, the term "aryl" includes, unless otherwise defined, phenyl or naphthyl optionally substituted with up to five, preferably up to three substituents.

When substituted, an aryl group may have up to three substituents. Preferred substituents for an aryl group include, for example, and unless otherwise defined, halogen, cyano, (C- _| -6)alkyl, mono to perfluoro(C-|-3)alkyl, (C3-7)cycloalkyl, (C2-6)a'kenyl, (C-|-6)alkoxy, (C2-Q)a\kenoxy, arylC(-|_6)alkoxy, halo(C- _| -6)alkyl, hydroxy, amino, mono- or di-(C- _| -6)alkylamino, acylamino, nitro, carboxy, (C- _| -6)alkoxycarbonyl, (Ci-6)alkenyloxycarbonyl, (C- _| -6)alkoxycarbonyl(C- _| -6)alkyl, carboxy(C-|-6)alkyl, (C-| -6)alkylcarbonyloxy, carboxy(C-| -gjalkyloxy, (C-| -6)alkoxycarbonyl(C-| -g)alkoxy, (Ci-6)alkylthio, (C- _| -g)alkylsulphinyl, (C-|-g)alkylsulphonyl, sulphamoyl, mono- and di-(C-|-6)-alkylsulphamoyl, carbamoyl, mono- and di-(C-)-g)alkylcarbamoyl, and heterocyclyl.

When used herein, the term "heteroaryl" includes single or fused rings comprising up to four hetero-atoms in the ring selected from oxygen, nitrogen and sulphur and optionally substituted with up to three substituents. Preferably the heteroaryl ring comprises from 4 to 7, preferably 5 or 6, ring atoms. A fused heteroaryl ring system may include carbocyclic rings and need only include one heterocyclic ring.

When used herein, the term "heterocyclyl" includes non-aromatic single or fused rings comprising up to four hetero-atoms in the ring selected from oxygen, nitrogen and sulphur and optionally substituted with up to three substituents. Suitably the heterocyclic ring comprises from 4 to 7, preferably 5 to 6, ring atoms. A fused heterocyclic ring system may include carbocyclic rings and need only include one heterocyclic ring.

When substituted, a heteroaryl or a heterocyclyl group may have up to three substituents. Preferred such substituents include those previously mentioned for an aryl group as well as oxo.

When used herein, the terms 'halogen' and 'halo' include fluorine, chlorine, bromine and iodine and fluoro, chloro, bromo and iodo, respectively.

The compounds according to the invention are suitably provided in substantially pure form, for example at least 50% pure, suitably at least 60% pure, advantageously at least 75% pure, preferably at least 85% pure, more preferably at least 95% pure, especially at least 98% pure, all percentages being calculated as weight/weight. An impure or less pure form of a compound according to the invention may, for example, be used in the preparation of a more pure form of the same compound or of a related compound (for example a corresponding derivative) suitable for pharmaceutical use.

Preferred compounds of formula (I) include the compounds of Examples 6, 21 , 24, 28, 30 and 31. A compound of formula (I) may be prepared by a process which comprises reacting an aldehyde compound of formula (II):

(II) in which R^ is as hereinbefore defined; with an amine compound of formula (III):

in which RI and R^ are as hereinbefore defined; or a salt thereof under reductive alkylation conditions.

Reductive alkylation conditions are well known in the art and include, for instance, adding the aldehyde of formula (II) to a stirred solution of the amine of formula

(III) in a solvent such as methanol, and thereafter adding a borohydride reducing agent, for instance, sodium cyanoborohydride or sodium triacetoxyborohydride, or, more preferably, a resin supported reagent such as (polystyrylmethyl)-trimethylammonium cyanoborohydride.

Aldehydes of formula (II) and amines of formula (III) are available from commercial sources or may be readily prepared therefrom by adapting well known synthetic transformations. It will be appreciated that the above reductive alkylation may be performed in the reverse orientation by coupling a cyclohexylpropanone with a benzylamine derivative.

The compounds of this invention are active against both Gram positive and Gram negative organisms, including Haemophilus, for instance H. influenzae Q1 ; Moraxella, for instance M. catarrhalis 1502; Streptococci, for instance S. pyogenes CN 10 and S. pneumoniae R1629 and N 1387; Staphylococci, for instance S. aureus Oxford; Escherichia, for instance E. CoIi DCO, and Enterococci, for instance Ent. faecalis I. In addition, compounds of this invention are active against Staphylococci organisms such as S. aureus and coagulase-negative strains of Staphylocci such as S. epidermidis which are resistant (including multiply-resistant) to other anti-bacterial agents, for instance, D-lactam antibiotics such as, for example, methicillin; macrolides; aminoglycosides, and lincosamides. Compounds of the present invention are therefore useful in the treatment of MRSA, MRCNS and MRSE. Compounds of the present invention are also active against strains of E. faecalis including vancomycin resistant strains and therefore of use in treating infections associated with VRE organisms. Furthermore, compounds of the present invention are useful in the treatment of Staphylococci organisms which are resistant to mupirocin.

Bacterial infections which may be treated include respiratory tract infections, otitis media, meningitis, endocarditis, skin and soft tissue infections and urinary tract infections in man, mastitis in cattle, and respiratory infections in animals such as pigs and cattle. Accordingly, in a further aspect, the present invention provides a method of treating bacterial infection in human or non-human animals, which method comprises administering a therapeutically effective amount of a compound of formula (I) as hereinbefore defined, to a human or non-human animal in need of such therapy.

The present invention provides a pharmaceutical composition comprising a compound of formula (I) together with a pharmaceutically acceptable carrier or excipient. The present invention also provides a method of treating bacterial infections in animals, especially in humans and in domesticated mammals, which comprises administering a compound of formula (I), or a composition according to the invention, to a patient in need thereof.

The invention further provides the use of a compound of formula (I) in the preparation of a medicament composition for use in the treatment of bacterial infections. The compounds and compositions according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics.

The compounds and compositions according to the invention may be formulated for administration by any route, for example oral, topical or parenteral. The compositions may, for example, be made up in the form of tablets, capsules, powders, granules, lozenges, creams, syrups, or liquid preparations, for example solutions or suspensions, which may be formulated for oral use or in sterile form for parenteral administration by injection or infusion. Tablets and capsules for oral administration may be in unit dosage form, and may contain conventional excipients including, for example, binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrollidone; 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; and pharmaceutically acceptable wetting agents, for example 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 another suitable vehicle before use. Such liquid preparations may contain conventional additives, including, for example, 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 (for example glycerine), propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavouring and colour agents. Compositions according to the invention intended for topical administration may, for example, be in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, impregnated dressings, and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1 % to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.

Compositions according to the invention may be formulated as suppositories, which may contain conventional suppository bases, for example cocoa-butter or other glycerides.

Compositions according to the invention intended for parenteral administration may conveniently be in fluid unit dosage forms, which may be prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, may be either suspended or dissolved in the vehicle. In preparing solutions, the compound may be dissolved in water for injection and filter-sterilised before being filled into a suitable vial or ampoule, which is then sealed. Advantageously, conventional additives including, for example, local anaesthetics, preservatives, and buffering agents can be dissolved in the vehicle. In order to enhance the stability of the solution, the composition may be frozen after being filled into the vial, and the water removed under vacuum; the resulting dry lyophilized powder may then be sealed in the vial and a accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions may be 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 may instead be sterilised by exposure to ethylene oxide before being suspended in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in such suspensions in order to facilitate uniform distribution of the compound.

A compound or composition according to the invention may suitably be administered to the patient in an antibacterially effective amount.

A composition according to the invention may suitably contain from 0.1% by weight, preferably from 10 to 60% by weight, of a compound according to the invention (based on the total weight of the composition), depending on the method of administration.

The compounds according to the invention may suitably be administered to the patient at a daily dosage of from 1.0 to 50 mg/kg of body weight. For an adult human (of approximately 70 kg body weight), from 50 to 3000 mg, for example about 1500 mg, of a compound according to the invention may be administered daily. Suitably, the dosage for adult humans is from 5 to 20 mg/kg per day. Higher or lower dosages may, however, be used in accordance with normal 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, preferable from 50 to 500 mg, of a compound according to the invention.

The following Examples illustrate the present invention.

Intermediate 1. 1-(3-Trifluoromethylcyclohexyl)prop-2-ylamine acetate salt.

To 1-(3-trifluoromethylphenyl)prop-2-ylamine (1.11 g, 5.46 mmol) in AcOH (8 ml) was added platinum (IV) oxide (670 mg, 2.95 mmol) and the mixture hydrogenated at 50 p.s.i. for 24 hours. The mixture was filtered through celite, then concentrated in vacuo to give the title compound (as a mixture of diastereomers) as a cream solid (1.14 g, 5.46 mmol, 100%); m/z (ES ⁺ ) 210 (100%, M+H ⁺ ).

Intermediate 2. i-Cyclohexylbut-2-ylamine acetate salt.

To 1-phenylbut-2-ylamine (1.73 g, 11.6 mmol) in AcOH (35 ml) was added platinum (IV) oxide (527 mg, 2.32 mmol) and the mixture hydrogenated at 50 p.s.i. for 24 hours. The mixture was filtered through celite, then concentrated in vacuo to give the title compound as a white solid (2.50 g, 11.6 mmol, 100%); m/z (ES ⁺ ) 156 (100%, M+H ⁺ ).

Intermediate 3. 2-(4-Methanesulfonylbenzyloxy)benzaldehyde. To a stirred mixture of 2-hydroxybenzaldehyde (3.4 ml, 32 mmol), and 4- methanesulfonylbenzyl bromide (1.99 g, 8.00 mmol) in DMF (3 ml) was added K ₂ CO ₃ (4.4 g, 32 mmol). After 24 hours the mixture was concentrated in vacuo, the residue taken up in EtOAc and NaOH (aq). The organic layer was dried (MgSO ₄ ), filtered, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel eluting with 0-60% EtOAc in 40-60 petroleum ether to give the title compound as a white solid (1.11 g, 3.81 mmol, 48%); m/z (ES ⁺ ) 313 (100%, M+Na ⁺ ).

Intermediate 4. (2-Formylphenoxy)acetonitrile. To a stirred mixture of 2-hydroxybenzaldehyde (3.4 ml, 32 mmol), and iodoacetonitrile (1.34 g, 8.00 mmol) in DMF (3 ml) was added K ₂ CO ₃ (4.4 g, 32 mmol). After 24 hours the mixture was concentrated in vacuo, the residue taken up in EtOAc and NaOH (aq). The organic layer was dried (MgSO ₄ ), filtered, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel eluting with 0-60% EtOAc in 40-60 petroleum ether to give the title compound as a yellow solid (319 mg, 1.91 mmol, 24%); D (CDCI ₃ ) 4.97 (s, 2H), 7.11 (d, 1 H), 7.20 (t, 1 H), 7.63 (t, 1 H), 7.90 (d, 1 H), 10.44 (s, 1 H).

Intermediate 5. 2-(Pyridin-4-ylmethoxy)benzaldehyde. To a stirred mixture of 2-hydroxybenzaldehyde (3.4 ml, 32 mmol), and 4- chloromethylpyridine hydrochloride (1.31 g, 8.00 mmol) in DMF (3 ml) was added K ₂ CO ₃ (5.5 g, 40 mmol). After 24 hours the mixture was concentrated in vacuo, the residue taken up in EtOAc and NaOH (aq). The organic organic layer was dried (MgSO ₄ ), filtered, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel eluting with 0-60% EtOAc in 40-60 petroleum ether to give the title compound as an orange/brown solid (936 mg, 4.39 mmol, 55%); m/z (ES ⁺ ) 214 (100%, M+H ⁺ ).

Intermediate 6. 2-(2-Formylphenoxy)-λ/-(2-hydroxyethyl)-λ/-methylacetamide .

To a stirred mixture of (2-formyl-phenoxy)-acetic acid (685 mg, 3.8 mmol), and 1- hydroxy-7-azabenzotriazole (569 mg, 4.2 mmol) in DMF (10 ml) was added 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (728 mg, 3.8 mmol) and, after 20 minutes 2-methylamino-ethanol (305 μl, 3.8 mmol). The solvent was removed in vacuo after 85 hours. The resulting residue was taken up in NaHCO ₃ (aq) and DCM, added to hydromatrix gel, then eluted with DCM. The organic eluant was concentrated in vacuo, and the residue purified by flash column chromatography on silica gel eluting with 0-4% MeOH in DCM to give the title compound as a cream solid (481 mg, 2.03 mmol, 53%); m/z (ES+) 260 (100%, M+Na ⁺ ).

Intermediate 7. 3-(2-Formylphenoxy)-λ/-(methylcarbamoylmethyl)propionamide. To a stirred mixture of 3-(2-formylphenoxy)propionic acid (458 mg, 2.36 mmol), and 1- hydroxy-7-azabenzotriazole (385 mg, 2.83 mmol) in DMF (10 ml) was added 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (452 mg, 2.36 mmol) and, after 20 minutes 2-methylaminoacetamide hydrochloride (324 mg, 2.60 mmol) followed by (diethylaminomethyl)-polystyrene (1.1 g, 3.52 mmol The solvent was removed in vacuo after 85 hours. The resulting residue was taken up in NaHCO ₃ (aq) and DCM, added to hydromatrix gel, then eluted with DCM. The organic eluant was concentrated in vacuo, and the residue purified by flash column chromatography on silica gel eluting with 0-4% MeOH in DCM to give the title compound as a white solid (260 mg, 984 μmol, 42%); m/z (ES+) 287 (100%, M+Na ⁺ ); d (CDCI ₃ ) 2.79-2.82 (m, 5H), 3.96 (d, 2H), 4.39 (t, 2H), 6.04 (br s, 1 H), 7.02 (d, 1 H), 7.05 (br s, 1 H), 7.10 (t, 1 H), 7.56 (t, 1 H), 7.79 (d, 1 H).

Intermediate s. 4-(2-Formylphenoxy)-λ/-(tetrahydrofuran-2-ylmethyl)butyrami de. To a stirred mixture of 4-(2-formylphenoxy)butyric acid (791 mg, 3.8 mmol), and 1- hydroxy-7-azabenzotriazole (52 mg, 384 μmol) in DMF (10 ml) was added 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (882 mg, 4.6 mmol) and, after 20 minutes tetrahydro-furan-2-ylmethylamine (465 mg, 4.6 mmol). The solvent was removed in vacuo after 85 hours. The resulting residue was taken up in NaHCO ₃ (aq) and DCM, added to hydromatrix gel, then eluted with DCM. The organic eluant was concentrated in vacuo, and the residue purified by flash column chromatography on silica gel eluting with 0-4% MeOH in DCM to give the title compound as an orange/brown gum (667 mg, 2.29 mmol, 60%); m/z (ES+) 292 (100%, M+H ⁺ ).

Intermediate 9. 2-(2-Morpholin-4-yl-ethoxy)benzaldehyde

To a stirred mixture of 2-hydroxybenzaldehyde (3.4 ml, 32 mmol), and 4-(2- chloroethyl)morpholine hydrochloride (1.49 g, 8.00 mmol) in DMF (3 ml) was added K ₂ CO ₃ (5.5 g, 40 mmol). After 24 hours the mixture was concentrated in vacuo, the residue taken up in EtOAc and NaOH (aq). The organic layer was dried (MgSO ₄ ), filtered, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel eluting with 0-60% EtOAc in 40-60 petroleum ether to give the title compound as a golden brown oil (1.43 g, 8.1 mmol, 76%); d _H (400 MHz, CDCI ₃ ) 2.58-2.60 (m, 4H), 2.87 (t, 2H), 3.71-3.73 (m, 4H), 4.24 (t, 2H), 6.99 (d, 1 H), 7.04 (t, 1 H), 7.54 (t, 1 H), 7.84 (d, 1 H), 10.49 (s, 1 H).

Intermediate 10. 2-(2-Benzimidazolylmethoxy)benzaldehyde

To a stirred mixture of 2-hydroxybenzaldehyde (3.4 ml, 32 mmol), and 2- chloromethylbenzimidazole (1.33 g, 8.00 mmol) in DMF (3 ml) was added K ₂ CO ₃ (4.4 g, 32 mmol). After 24 hours the mixture was concentrated in vacuo, the residue taken up in EtOAc and NaOH (aq). The organic layer was dried (MgSO ₄ ), filtered, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel eluting with 0-60% EtOAc in 40-60 petroleum ether to give the title compound as a pale yellow solid (1.25 g, 4.9 mmol, 62%); d _H (400 MHz, CDCI ₃ ) 5.51 (s, 2H), 7.12 (d, 1 H), 7.18 (t, 1 H), 7.25-7.30 (m, 2H), 7.53-7.58 (m, 1 H)

7.61 (t, 1 H), 7.74-7.79 (m, 1 H), 7.82 (d, 1 H), 10.31 (s, 1 H), 10.57 (br s, 1 H).

General Procedure for the preparation of orf/io-alkoxybenzaldehydes

The alcohol (10 mmol) in dry DMSO (2 ml.) was added dropwise to a suspension of a 60% dispersion of sodium hydride (10 mmol) in mineral oil in dry DMSO (10 ml_) at room temperature and under an argon atmosphere. After stirring for 1 h f-butyl-(2-fluoro- benzylidene)amine (10 mmol) was added and the resultant mixture stirred for 16 h. Water (30 ml_) was added and the organic material extracted into diethyl ether (3 x 30 ml_). The combined organic phases were washed with water, dried and evaporated. The residue was dissolved in a mixture of 2% aqueous acetic acid (100 ml_) and THF (30 ml.) and stirred at room temperature. After 24 h the THF was evaporated and aqueous layer extracted with diethyl ether (3 x 30 ml_), the combined ether extracts were dried and evaporated to yield the corresponding aldehyde.

The following three aldehydes were prepared by the General Procedure for the preparation of ort/70-alkoxybenzaldehydes

Intermediate 11 2-Cyclopentyloxybenzaldehyde δ _H (CDCI ₃ ) 10.50 (1 H, s), 7.82 (1 H, dd), 7.50 (1 H, td), 6.99-6.96 (2H, m), 4.91-4.87 (1 H, m), 1.96-1.91 (4H, m), 1.84-1.79 (2H, m), 1.68-1.66 (2H, m).

Intermediate 12 2-(Tetrahydrofuran-3-yloxy)benzaldehyde δ _H (CDCI ₃ ) 10.48 (1 H, s), 7.85 (1H, dd), 7.53 (1 H, t), 7.04 (1 H, t), 6.91 (1 H, d), 5.08- 5.04 (1 H, m), 4.09-3.92 (4H, m), 2.34-2.20 (2H, m).

Intermediate 13 2-(Cyclohex-2-enyloxy)benzaldehyde δ _H (CDCI ₃ ) 10.52 (1 H, s), 7.83 (1 H ₁ dd), 7.53 (1 H, d), 7.04-6.98 (2H, m), 6.03-5.99 (1 H, m), 5.89-5.86 (1 H, m), 4.91 (1 H, t), 2.26-1.68 (6H, m).

Intermediate 14 2-(2-Formylphenoxy)propionitrile

2-Hydroxybenzaldehyde (9 g, 73.7 mmol) was dissolved in dry dimethylformamide (70 ml). Potassium carbonate (10.19 g, 73.7 mmol) was added followed by 2- chloropropionitrile (1.65 g, 18.4 mmol). The yellow suspension was stirred at room temperature and the course of the reaction monitored every 14 hours. Four aliquots of

2-chloropropionitrile (1.65 g, 18.4 mmol) were added to complete the reaction. The reaction mixture was concentrated and the residue partitioned between ethyl acetate and 1 N aqueous sodium hydroxide. The organic phase was dried and concentrated and the residue was chromatographed on Kieselgel 60 eluting with dichloromethane to yield the title compound (8.7 g); d _H (CDCI ₃ ) 1.89 (3H, d, J=6.7 Hz, CH ₃ ), 5.03 (1 H, q, J=6.7 Hz, OCH), 7.14 (1 H ₁ d, J=8.4 Hz, CH), 7.23 (1 H, t, J=7.6 Hz, CH), 7.59 (1 H, bt, J=7.4 Hz, CH), 7.90 (1 H, bd, J=7.7 Hz, CH), 10.45 (1 H, bs, CHO).

General Procedure for Reductive Alkylation. To a stirred solution of the amine (200 μmol) in dry MeOH (2 ml) was added the aldehyde (200 μmol), followed by AcOH (2 drops). (Polystyrylmethyl)trimethyl- ammonium cyanoborohydride resin (190 mg, 700 μmol) was added after 20 minutes. Once the reaction had gone to completion the resin was removed by filtration and the solvent removed in vacuo. The residue was purified by flash column chromatography on silica gel eluting with 0-20% EtOH/NH ₃ (aq) (9:1 ) in DCM to give the desired compound.

The tabulated examples were prepared by the General Procedure for Reductive Alkylation using amines and aldehydes from commercial sources, or prepared by literature procedures, or where indicated, by the procedures outlined above.

Example 28 ((S)-I -Cyclohexylprop-2-yl){2-[1 -(1 H-tetrazol-5-yl)ethoxy]benzyl}amine

The compound of Example 27 (0.15 g, 0.5 mmol) was dissolved in 1 ,2-dichlorobenzene (5 ml) and tributyltin azide (0.82 g, 2.5 mmol) was added. After stirring at 125°C for 1 h, the reaction mixture was chromatographed on Kieselgel 60 eluting with 0-5% methanol ammonia (9:1 ) in dichloromethane to yield the title compound (0.124 g); m/z (ES ⁺ ) 344 (MH ⁺ , 100%).

Example 29 3-{2-[((S)-1-Cyclohexylprop-2-ylamino)methyl]phenoxy}butane- 1,2-ol

a) 2-(1-Methylallyloxy)benzaldehyde To a solution of 2-hydroxybenzaldehyde (5.00 g, 40.9 mmol) in dry DMF (50 ml.) at 5 ⁰ C was added, portionwise, a 60% dispersion of sodium hydride in mineral oil (1.64 g, 40.9 mmol). After stirring at room temperature for 0.25 h a solution of 3-chloro-1-butene (5.8 ml_, 58.4 mmol) was added and the resultant mixture left to stir for 48 h. The reaction was quenched by pouring onto 1 M hydrochloric acid (400 ml_) and the organic material extracted with the diethyl ether (2 x 75 ml_). The combined organic phases were washed with 2M sodium hydroxide solution, dried and evaporated to yield the title compound; δ _H (CDCI ₃ ) 10.60 (1 H, s), 7.83 (1 H, dd), 7.51 (1 H, d), 7.03-6.97 (2H ₁ m), 5.94-5.87 (1 H, m), 5.35-5.20 (2H, m), 4.94-4.90 (1 H ₁ m), 1.51 (3H, d). b) ((S)-I -Cyclohexylprop-2-yl)-[2-(1-methylallyloxy)benzyl]amine The title compound was prepared from the compound of 29a as described in the General Procedure for Reductive Alkylation; m/z (ES+) MH ⁺ 302 (100%). c) ((S)-I -Cyclohexylprop-2-yl)-[2-(1 -methylallyloxy)benzyl]carbamic acid dimethylethyl ester A solution of the compound of 29b (1.5 g, 5 mmol) and sodium bicarbonate (0.46 g, 6.0 mmol) in dioxane (40 mL) and water (20 ml.) was treated with di-f-butyl carbonate (1.3 g, 6.0 mmol). After stirring at room temperature for 16 h the mixture was partitioned between diethyl ether and water, the organic phase was separated, dried and evaporated to yield the crude product. Chromatography over silica

gel eluting with petroleum ether containing increasing concentrations of diethyl ether gave the title compound; m/z (ES+) MH ⁺ 402 (100%). d) ((S)-I -Cyclohexylprop-2-yl)-[2-(1 -oxiranylethoxy)-benzyl]carbamic acid dimethylethyl ester The compound of 29c (100 mg, 0.33 mmol) was added to a solution of 3-chloroperoxybenzoic acid (63 mg, 0.37 mmol) in dichloromethane (20 mL) at room temperature. After stirring for 16 h the residual peracid was reduced by addition of aqueous sodium bisulfite solution, and the dichloromethane layer separated and washed with sodium bicarbonate solution, dried and evaporated to yield the title product; m/z (ES+) [M-Boc] 317 (100%). e) 3-{2-[((S)-1-Cyclohexylprop-2-ylamino)methyl]phenoxy}butane- 1,2-ol The compound of 29d (81 mg, 1.93 mmol) was suspended in 10% aqueous sulfuric acid. After stirring for 12 h at 100 ⁰ C the homogeneous mixture was cooled and poured into aqueous sodium bicarbonate solution. The organic material was extracted with diethyl ether (2 x 25 mL) and the combined extracts dried and evaporated to yield the crude product. Chromatography over silica gel eluting with dichloromethane containing increasing concentrations of methanol/ammonium hydroxide gave the title compound; m/z (ES+) MH ⁺ 336 (100%).

Example 30 (S)-3-{2-[((S)-1 -Cyclohexylprop-2-ylamino)methyl]phenoxy}propane- 1,2-diol

a) (R)-2-(2,2-dimethyl-1,3-dioxolan-4-ylmethoxy)benzaldehyde To a stirred mixture of sodium hydride (0.084 g, 60% in oil, 2.1 mmol) in DMF (5 ml) at O ⁰ C under argon was added dropwise 2-hydroxybenzaldehyde (0.234 g, 1.92 mmol), in DMF (10 ml). Stirring was continued for 30 min then (S)-2,2-dimethyl-1 ,3-dioxolan-4-ylmethyl p- toluenesulfonate (0.5 g, 1.75 mmol) in DMF (4 ml) was added dropwise. The mixture was heated to 100 ⁰ C for 24 h, then cooled and poured into ice water. Extraction with ethyl acetate, drying and evaporation in vacuo gave a crude product which was purified by flash column chromatography on silica gel eluting with 20% EtOAc in 40-60 petroleum ether to give the title compound as a yellow oil (0.257 g, 62%); D ₀ (400 MHz, CDCI ₃ ) 1.41 , (s, 3H), 1.46 (s, 3H), 3.97 (dd, 1 H), 4.09 (dd, 1 H), 4.18 (m, 2H), 4.53 (quintet, 1 H), 7.00 (d, 1 H), 7.06 (t, 1 H), 7.55 (dt, 1 H), 7.84 (dd, 1 H), 10.52 (s, 1 H). b) (R)-4-{2-[((S)-1-Cyclohexylprop-2-ylamino)methyl]phenoxymeth yl}-2,2-dimethyl- 1,3-dioxolane The compound of 30a (0.094 g, 0.40 mmol) was reacted with (S)-1- cyclohexylprop-2-ylamine (0.056 g, 0.40 mmol) using the general procedure for reductive amination to give the title product as a colourless gum (0.137 g); m/z (ES ⁺ ) 362 (MH ⁺ , 100%). c) (S)-3-{2-[((S)-1 -Cyclohexylprop-2-ylamino)methyl]phenoxy}propane-1 ,2-diol The compound of 30b was dissolved in 4 M hydrogen chloride in dioxan (1 ml) and water (1 ml) added with stirring. After 10 min the solvents were removed in vacuo and the residual material loaded onto an SCX cartridge. After washing with methanol the

cartridge was eluted with 2 M ammonia in methanol to give the title product (0.114 g, 89%); m/z (ES ⁺ ) 322 (MH ⁺ , 100%).

Example 31 (R)-3-{2-[((S)-1 -Cyclohexylprop-2-ylamino)methyl]phenoxy}propane- 1,2-diol

a) (S)-2-(2,2-dimethyl-1,3-dioxolan-4-ylmethoxy)benzaldehyde To a stirred mixture of sodium hydride (0.084 g, 60% in oil, 2.1 mmol) in DMF (5 ml) at O ⁰ C under argon was added dropwise 2-hydroxybenzaldehyde (0.234 g, 1.92 mmol), in DMF (10 ml). Stirring was continued for 30 min then (R)-2,2-dimethyl-1 ,3-dioxolan-4-ylmethyl p- toluenesulfonate (0.5 g, 1.75 mmol) in DMF (4 ml) was added dropwise. The mixture was heated to 100 ⁰ C for 24 h, then cooled and poured into ice water. Extraction with ethyl acetate, drying and evaporation in vacuo gave a crude product which was purified by flash column chromatography on silica gel eluting with 20% EtOAc in 40-60 petroleum ether to give the title compound as a yellow oil (0.169 g, 40%); d _H (400 MHz, CDCI ₃ ) 1.41 , (s, 3H), 1.46 (s, 3H), 3.96 (dd, 1 H), 4.09 (dd, 1 H), 4.19 (m, 2H), 4.52 (quintet, 1 H), 7.00 (d, 1 H), 7.07 (t, 1 H), 7.54 (dt, 1 H), 7.84 (dd, 1 H), 10.52 (s, 1 H). b) (S)-4-{2-[((S)-1-Cyclohexylprop-2-ylamino)methyl]phenoxymeth yl}-2,2-dimethyl- 1,3-dioxolane The compound of 31a (0.094 g, 0.40 mmol) was reacted with (S)-1- cyclohexylprop-2-ylamine (0.056 g, 0.40 mmol) using the general procedure for reductive amination to give the title product as a colourless gum (0.086 g); m/z (ES ⁺ ) 362 (MH ⁺ , 100%). c) (R)-3-{2-[((S)-1 -Cyclohexylprop-2-ylamino)methyl]phenoxy}propane-1 ,2-diol The compound of 31b was dissolved in 4 M hydrogen chloride in dioxan (1 ml) and water (1 ml) added with stirring. After 10 min the solvents were removed in vacuo and the residual material loaded onto an SCX cartridge. After washing with methanol the cartridge was eluted with 2 M ammonia in methanol to give the title product (0.077 g, 62%); m/z (ES ⁺ ) 322 (MH ⁺ , 100%).

Biological Data

1. Enzyme Inhibition (S. aureus FRS) - aminoacylation assay

Compounds of the present invention may be assayed for their ability to inhibit the enzyme phenylalanyl tRNA synthetase (FRS), using recombinant S. aureus FRS, as follows:

Steady-state kinetics. Aminoacylation reactions were performed using modifications to existing methods (1 ) as previously described (2, 3). All assays were carried out at 22 ⁰ C in 30 mM Tris-HCI pH 7.9, 10 mM MgCI ₂ , 75 mM KCI, 2 mM DTT and 2 mg/ml (final concentration) E. coli MRE 600 tRNA (Boehringer Mannheim; equivalent to ~1 μM tRNA ^Pne ). The assay was performed using ca 1-2 nM PheRS, [2,3,4,5,6- ³ H] L- Phenylalanine (Amersham; Specific activity = 4.37 TBq/mol) and 2.5 mM ATP in a total volume of 50 μl. After 10 min the reactions were quenched with an appropriate amount of Yttrium silicate beads (PDE Assay Kit, RPNQ 0150, Amersham) suspended in 167 mM sodium citrate, pH 2. The assays were performed in 96-well or 384-well opaque plates. The beads are allowed to settle for at least 45 minutes or spun for 5 min at 2500rpm, using a MISTRAL 3000E centrifuge before counting on the TopCount (Packard) or Microbeta Trilux (Wallac). The least squares fitting was performed using GRAFIT (4).

References

1. Calender, R. and Berg, P. 1966 Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis Biochemistry, 5, 1681 - 1690

2. Pope, A.J., Moore, K.J., McVey, M., Mensah, L., Benson, N., Osbourne, N., Broom, N., Brown, J. B. and O'Hanlon, P. 1998. Characterization of Isoleucyl- tRNA synthetase from Staphylococcus aureus. I. Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques. J.

Biol. Chem. 273 (4B): 31680 - 31690.

3. Pope, A.J., Moore, K.J., McVey, M., Mensah, L., Benson, N., Osbourne, N., Broom, N., Brown, J. B. and O'Hanlon, P. 1998 Characterization of Isoleucyl-tRNA synthetase from Staphylococcus aureus. II. Mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady- state kinetics. J. Biol. Chem. 273 (4B): 31691 - 31701.

Pure recombinant S. aureus FRS (US Patent no 5 756 329) was obtained using standard purification procedures. The enzyme is diluted in Dilution Buffer which consists of 10 mM Tris / Cl, 2 mM DTT pH 7.9.

Results

Enzyme inhibition

All the examples were tested in the S. aureus FRS assay; compounds 4-12, 16, 17, 20 and 21-31 had IC50 values <200 nM.

Examples 1 , 5-21 , 26, 30 and 31 were tested in an E. coli lysate FRS assay; compounds 1 , 6-8, 10-13, 15-17, 20, 21 and 30 had IC50 values <20 nM.

2. Antibacterial Activity Compounds of the present invention were assayed for antibacterial activity against a range of pathogenic organisms (strains of S aureus, S pneumoniae, E faecalis, H influenzae and M catarrhalis) in a standard MIC assay modified by the inclusion of cyclodextrin, to assist with solubility.

Examples 1 , 6-13, 17, 18 and 21-31 had MIC values <16 μg/ml against some strains of the organisms S. pneumoniae, H. influenzae and/or M. catarrhalis.