EP1146868A1 - Anti-inflammatory agents - Google Patents

Abstract

Compounds of formula (I) wherein R4 is an ester or thioester group, and R, R1, R2 and R3 are as defined in the specification, inhibit intracellular leukotriene-A4 hydrolase activity and have anti-inflammatory activity.

Description

Anti-Inflammatory Agents

The present invention relates to the use of certain esters and thioesters for the treatment of diseases responsive to inhibition of intracellular leukotriene-A₄ hydrolase activity.

Background to the Invention

The leukotriene cascade of arachadonic acid is a key mechanism in many inflammatory and allergic disease states. The dihydroxy fatty acid leukotriene B₄ (LTB₄), produced by this cascade, is a key pro-inflammatory mediator. LTB₄ stimulates adhesion of circulating neutrophils to vascular endothelium, directs their migration toward sites of inflammation, and induces secretion of further inflammatory mediators. (For reviews see R.A. Lewis et al, N. Engl. J. Med. 1990, 323, 645-655 and M. -Q. Zhang, Curr. Med. Chem. 1997, 4, 67-78.) Leukotriene-A₄ hydrolase (LTA₄-hydrolase) (EC 3.3.2.6) is an enzyme that catalyses the final and rate limiting step in the synthesis of LTB₄. Inhibition of LTA₄ hydrolase selectively blocks the biosynthesis of LTB₄ which may provide an advantage over current inhibitors, such as those of 5-lipoxygenase, that block earlier in the leukotriene cascade and as a result are less selective.

Disease states associated with elevated levels of LTB₄, and which are therefore considered to be responsive to inhibition of intracellular leukotriene-A₄ hydrolase activity include asthma, inflammatory bowel disease, psoriasis and arthritis.

Peptidomimetic compounds, such as bestatin, captopril and kelatorphan exhibit LTA₄ hydrolase inhibitory activity against isolated enzyme (T.D. Penning et al, Biorg. Med. Chem. Lett., 1995, 5, p2517-2522). However, these compounds are unable to effectively penetrate cells and hence have little anti-inflammatory activity. There is therefore a need in the art for compounds which are capable of inhibiting intracellular LTA₄ hydrolase activity. Brief Description of the Invention

This invention is based on the finding that certain esters and thioesters containing an N-formyl hydroxylamine group are capable of inhibiting intracellular LTA₄ hydrolase activity, resulting in efficient attenuation of LTB₄ biosynthesis. Those esters and thioesters are therefore of use for the treatment of diseases responsive to such inhibiton, for example inflammatory and allergic conditions including asthma, rheumatoid arthritis, osteoarthritis, multiple sclerosis, ulcerative colitis, contact and atopic dermatitis, psoriasis, inflammatory bowel disease and Crohn's disease.

The following patent publications disclose compounds containing an N-formyl hydroxylamine group which are stated to be inhibitors of matrix metalloproteinases (MMPs), and thus potentially of value for the treatment of diseases involving inapproriate degradation of the extracellular matrix. However, few examples of such compounds have been specifically made and described those publications:

EP-B-0236872 (Roche)

WO 92/09563 (Glycomed)

WO 92/04735 (Syntex)

WO 95/19965 (Glycomed)

WO 95/22966 (Sanofi Winthrop)

WO 95/33709 (Roche)

WO 96/23791 (Syntex)

WO 96/16027 (Syntex/Agouron)

WO 97/03783 (British Biotech)

WO 97/18207 (DuPont Merck)

Brief Description of the Invention

This invention is based on the identification of a class of ester and thioester compounds containing an N-formyl hydroxylamine group, which are capable of inhibiting intracellular LTA₄ hydrolase activity, resulting in efficient attenuation of LTB₄ biosynthesis. Those esters and thioesters are therefore of use for the treatment of diseases responsive to such inhibiton, for example inflammatory and allergic conditions including asthma, rheumatoid arthritis, osteoarthritis, multiple sclerosis, ulcerative colitis, contact and atopic dermatitis, psoriasis, inflammatory bowel disease and Crohn's disease.

The ester and thioester compounds in question have certain structural similarities to known MMP inhibitors generically disclosed in the foregoing patent publications. Most of those prior art publications are concerned with amides rather than esters or thioesters group, but a a few (WO 92/09563, WO 95/19965 and WO 95/22966) include within their generic disclosure compounds having a carboxylate ester group in place of the amide group. The carboxylate ester compounds with which this invention is concerned thus represent a selection of a notional subclass from the compounds proposed in the art as MMP inhibitors or for other purposes, for a specific and previously unrecognised pharmaceutical utility - inhibiting intracellular LTA₄ hydrolase activity.

Detailed Description of the Invention

In its broadest aspect, the present invention provides a method for treatment of mammals suffering diseases responsive to inhibition of intracellular leukotriene-A₄ hydrolase activity, comprising administering to the mammal suffering such disease an amount of a compound of general formula (I) or a pharmaceutically acceptable salt hydrate or solvate thereof sufficient to inhibit such activity:

wherein

R is hydrogen or (C,-C₆)alkyl; R^ is hydrogen; (C C_β)all yl;

(C₂-C₆)alkenyl;

phenyl or substituted phenyl;

phenyl (C₁-C₆)alkyl or substituted phenyl(C C₆)alkyl;

phenyl (C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl

heterocyclyl or substituted heterocyclyl;

heterocyclyl(C₁-C₆)alkyl or substituted heterocyclyl(C_rC₆)alkyl;

a group BSO_nA- wherein n is 0, 1 or 2 and B is hydrogen or a (C C₆) alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C C₆)acyl, phenacyl or substituted phenacyl group, and A represents (C C₆)alkylene;

amino(C_rC₆)alkyl, (C₁-C_e)alkylamino(C₁-C₆)alkyl, di^-C^alkylaminofC,- C₆)alkyl, hydroxy(C,-C₆)alkyl, mercapto(C₁-C₆)alkyl or carboxy(C C₆) alkyl wherein the amino-, hydroxy-, mercapto- or carboxyl-group are optionally protected or the carboxyl- group amidated;

lower alkyl substituted by carbamoyl, mono(lower alkyl)carbamoyl, di(lower alkyl)carbamoyl, di(lower alkyl)amino, or carboxy-lower alkanoylamino; or

a cycloalkyl, cycloalkenyl or non-aromatic heterocyclic ring containing up to 3 heteroatoms, any of which may be (i) substituted by one or more substituents selected from C C₆ alkyl, C₂-C₆ alkenyl, halo, cyano ( -CN), -CO₂H, -CO₂R, - CONH₂, -CONHR, -CON(R)₂, -OH, -OR, oxo-, -SH, -SR, -NHCOR, and - NHCO₂R wherein R is C C₆ alkyl or benzyl and/or (ii) fused to a cycloalkyl or heterocyclic ring;

is a C C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, phenyl(C C₆ alkyl)-, heteroaryl(C C₆ alkyl)-, phenyl(C₂-C₆ alkenyl)-, heteroaryl(C₂-C₆ alkenyl)-, phenyl(C₂-C₆ alkynyl)-, heteroaryl(C₂-C₆ alkynyl)-, cycloalkyl(C_rC₆ alkyl)-, cycloalkyl(C₂-C₆ alkenyl)-, cycloalkyl(C₂-C₆ alkynyl)-, cycloalkenyl(C C₆ alkyl)-, cycloalkenyl(C₂-C₆ alkenyl)-, cycloalkenyl(C₂-C₆ alkynyl)-, phenyl(C C₆ alkyl)O(C C₆ alkyl)-, or heteroaryl(C_rC₆ alkyl)O(C₁-C₆ alkyl)- group, any one of which may be optionally substituted by

C_rC₆ alkyl,

C C₆ alkoxy, halo, cyano (-CN), phenyl or heteroaryl, or phenyl or heteroaryl substituted by C_rC₆ alkyl, C,-C₆ alkoxy, halo, or cyano (-CN); R₃ is the characterising group of a natural or non-natural α amino acid in which any functional groups may be protected; and

R₄ is an ester or thioester group,

or a pharmaceutically acceptable salt, hydrate or solvate thereof.

In another broad aspect of the invention, there is provided the use of a compound of formula (I) as defined in claim 1 in the preparation of a pharmaceutical or veterinary composition for treatment of mammals suffering diseases responsive to inhibition of intracellular leukotriene-A₄ hydrolase activity.

As used herein the term "(C,-C₆)alkyl" or "lower alkyl" means a straight or branched chain alkyl moiety having from 1 to 6 carbon atoms, including for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.

The term "(C₂-C₆)alkenyl" means a straight or branched chain alkenyl moiety having from 2 to 6 carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. This term would include, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

The term "C₂-C₆ alkynyl" refers to straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one triple bond. This term would include for example, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl- 2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.

The term "cycloalkyl" means a saturated alicyclic moiety having from 3-8 carbon atoms and includes, for example, cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl and cyclopropyl. The term "cycloalkenyl" means an unsaturated alicyclic moiety having from 4-8 carbon atoms and includes, for example, cyclohexenyl, cyclooctenyl, cycloheptenyl, cyclopentenyl, and cyclobutenyl. In the case of cycloalkenyl rings of from 5-8 carbon atoms, the ring may contain more than one double bond.

The term "aryl" means an unsaturated aromatic carbocyclic group which is moncyclic (eg phenyl) or polycyclic (eg naphthyl).

The unqualified term "heterocyclyl" or "heterocyclic" means (i) a 5-7 membered heterocyclic ring containing one or more heteroatoms selected from S, N and O, and optionally fused to a benzene ring, including for example, pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimidazolyl, maleimido, succinimido, phthalimido, 1 ,2-dimethyl-3,5-dioxo-1 ,2,4-triazolidin-4-yl, 3,4,4- trimethyl-2,5-dioxo-1-imidazolidinyl, 2-methyl-3,5-dioxo-1 ,2,4-oxadiazol-4-yl, 3- methyl-2,4,5-trioxo-1 -imidazolidinyl, 2,5-dioxo-3-phenyl-1 -imidazolidinyl, 2-oxo-1 - pyrrolidinyl, 2, 5-dioxo-1 -pyrrolidinyl or 2,6-dioxopiperidinyl, or (ii) a naphththalimido (ie 1 ,3-dihydro-1 ,3-dioxo-2H-benz[f]isoindol-2-yl), 1 ,3-dihydro-1-oxo-2H- benz[f]isoindol-2-yl, 1 ,3-dihydro-1 ,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or 2,3- dihydro-1 ,3-dioxo-1 H-benz[d,e]isoquinolin-2-yl group.

The term "heteroaryl" means a 5-7 membered substituted or unsubstituted aromatic heterocycle containing one or more heteroatoms. Illustrative of such rings are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, trizolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl.

The term "ester" or "esterified carboxyl group" means a group R₉O(C=O)- in which R₉ is the group characterising the ester, notionally derived from the alcohol R₉OH.

The term "thioester" means a group R₉S(C=O)- or R₉S(C=S)- or R₉O(C=S)-in which R₉ is the group characterising the thioester, notionally derived from the alcohol R₉OH or the thioalcohol R₉SH.

Unless otherwise specified in the context in which it occurs, the term "substituted" as applied to any moiety herein means substituted with up to four substituents, each of which independently may be (C C₆)alkyl, (C₁-C₆)alkoxy, hydroxy, mercapto, (C C₆)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), nitro, trifluoromethyl, -COOH, -CONH₂, -CN, -COOR^A , -CONHR^Aor -CONHR^AR^A wherein R^A is a (C_rC₆)alkyl group or the residue of a natural alpha-amino acid.

The term "side chain of a natural or non-natural alpha-amino acid" means the group R¹ in a natural or non-natural amino acid of formula NH₂-CH(R¹)-COOH.

Examples of side chains of natural alpha amino acids include those of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, histidine, 5- hydroxylysine, 4-hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, α-aminoadipic acid, α-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, α- methylserine, omithine, pipecolic acid, and thyroxine.

Natural alpha-amino acids which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups in their characteristic side chains include arginine, lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, and cysteine. When R₃ in the compounds of the invention is one of those side chains, the functional substituent may optionally be protected.

The term "protected" when used in relation to a functional substituent in a side chain of a natural alpha-amino acid means a derivative of such a substituent which is substantially non-functional. For example, carboxyl groups may be esterified (for example as a C C₆ alkyl ester), amino groups may be converted to amides (for example as a NHCOC,-C₆ alkyl amide) or carbamates (for example as an NHC(=O)OC_rC₆ alkyl or NHC(=O)OCH₂Ph carbamate), hydroxyl groups may be converted to ethers (for example an OC,-C₆ alkyl or a O(C C₆ alkyl)phenyl ether) or esters (for example a OC(=O)C C₆ alkyl ester) and thiol groups may be converted to thioethers (for example a tert-butyl or benzyl thioether) or thioesters (for example a SC(=0)C₁-C₆ alkyl thioester).

Examples of side chains of non-natural alpha amino acids include those referred to below in the discussion of suitable R₃ groups for use in compounds of the present invention.

Salts of the compounds of the invention include physiologically acceptable acid addition salts for example hydrochlorides, hydrobromides, sulphates, methane sulphonates, p-toluenesulphonates, phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates. Salts may also be formed with bases, for example sodium, potassium, magnesium, and calcium salts.

There are several chiral centres in the compounds according to the invention because of the presence of asymmetric carbon atoms. The presence of several asymmetric carbon atoms gives rise to a number of diastereomers with R or S stereochemistry at each chiral centre. All such diastereomers and mixtures thereof are included within the scope of the invention.

As previously stated, the compounds with which the present invention is concerned are principally distinguished from the compounds disclosed in the prior patent publications listed above by the ester or thioester group R₄. Accordingly the groups R, R.,, R_v R₂, and R₃, may include those which have been disclosed in the corresponding positions of compounds disclosed in any of those prior art patent publications listed above. Without limiting the generality of the foregoing, examples of substituents R, to R₄ are given below: The group R₁

R, may be, for example, hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl, allyl, phenylpropyl, cyclopropylmethyl, phenylprop-2-enyl, thienylsulphanylmethyl, thienylsulphinylmethyl, or thienylsulphonylmethyl; or

C C₄ alkyl, eg methyl, ethyl n-propyl or n-butyl, substituted by a phthalimido, 1 ,2-dimethyl-3,5-dioxo-1 ,2,4-triazolidin-4-yl, 3-methyl-2,5-dioxo-1- imidazolidinyl, 3,4 ,4-trimethyl-2,5-dioxo-1 -imidazolidinyl, 2-methyl-3,5-dioxo- 1 ,2,4-oxadiazol-4-yl, 3-methyl-2,4,5-trioxo-1 -imidazolidinyl, 2,5-dioxo-3- phenyl-1 -imidazolidinyl, 2-oxo-1 -pyrrolidinyl, 2, 5-dioxo-1 -pyrrolidinyl or 2,6- dioxopiperidinyl, 5,5-dimethyl-2,4-dioxo-3-oxazolidinyl, hexahydro-1 ,3- dioxopyrazolo[1 ,2,a][1 ,2,4]-triazol-2-yl, or a naphththalimido (ie 1 ,3-dihydro- 1 ,3-dioxo-2H-benz[f]isoindol-2-yl), 1 ,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl, 1 ,3-dihydro-1 ,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or 2,3-dihydro-1 ,3-dioxo- 1 H-benz[d,e]isoquinolin-2-yl group; or

cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl, cyclopropyl, tetrahydropyranyl or morpholinyl.

Presently preferred R^ groups include hydrogen, cyclopropylmethyl, n-propyl, and allyl.

The group R₂

R₂ may for example be

C C₁₂ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;

cycloalkyl(C₁-C₆ alkyl)-;

phenyl(C C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆ alkynyl)- optionally substituted in the phenyl ring; heteroaryl(C C₆ alkyl)-, heteroaryl(C₃-C₆ alkenyl)- or heteroaryl(C₃-C₆ alkynyl)- optionally substituted in the heteroaryl ring;

4-phenylphenyl(C C₆ alkyl)-, 4-phenylphenyl(C₃-C₆ alkenyl)-, 4- phenylphenyl(C₃-C₆ alkynyl)- , 4-heteroarylphenyl(C C₆ alkyl)-, 4- heteroarylphenyl(C₃-C₆ alkenyl)-, 4-heteroarylphenyl(C₃-C₆ alkynyl)-, optionally substituted in the terminal phenyl or heteroaryl ring;

phenoxy(C_rC₆ alkyl)- or heteroaryloxy(C_rC₆ alkyl)- optionally substituted in the phenyl or heteroaryl ring;

Specific examples of such groups include methyl, ethyl, n- or iso-propyl, n-, iso- or tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-nonyl, n-decyl, prop-2-yn-1-yl, cyclohexylethyl, cyclopentylmethyl, 3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop- 2-yn-1-yl, benzyl phenylpropyl, 4-chlorophenylpropyl, 4-methylphenylpropyl, 4- methoxyphenylpropyl, phenoxybutyl, 3-(4-pyridylphenyl)propyl-, 3-(4-(4- pyridyl)phenyl)prop-2-yn-1 -yl, 3-(4-phenylphenyl)propyl-, 3-(4-phenyl)phenyl)prop-2- yn-1-yl and 3-[(4-chlorophenyl)phenyl]propyk

Presently preferred R₂ groups include benzyl, n-butyl, iso-butyl, n-hexyl, and cyclopentylmethyl

The group R₃

R₃ may for example be C C₆ alkyl, phenyl, 2,- 3-, or 4-pyridyl, 2- or 3-thienyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl, 2,- 3-, or 4-pyridylmethyl, benzyl, 2,- 3-, or 4-hydroxybenzyl, 2,- 3-, or 4-benzyloxybenzyl, 2,- 3-, or 4-C_r C₆ alkoxybenzyl, or benzyloxy(C C₆alkyl)-.; or

the characterising group of a natural α-amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxyl group present may be amidated; or

a group -[Alk]_nR₆ where Alk is a (C C₆)alkyl or (C₂-C₆)alkenyl group optionally interrupted by one or more -O-, or -S- atoms or -N(R₇)- groups [where R₇ is a hydrogen atom or a (C₁-C₆)alkyl group], n is 0 or 1 , and R₆ is an optionally substituted cycloalkyl or cycloalkenyl group; or

a benzyl group substituted in the phenyl ring by a group of formula - OCH₂COR₈ where R₈ is hydroxyl, amino, (C₁-C₆)alkoxy, pheny C^C alkoxy, (C C₆)alkylamino, di((C C₆)alkyl)amino, phenyl(C₁-C₆)alkylamino, the residue of an amino acid or acid halide, ester or amide derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, α or β alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid; or

a heterocyclic(C C₆)alkyl group, either being unsubstituted or mono- or di- substituted in the heterocyclic ring with halo, nitro, carboxy, (C₁-C₆)alkoxy, cyano, (C₁-C₆)alkanoyl, trifluoromethyl (C^C^alkyl, hydroxy, formyl, amino, (C.,-C₆)alkylamino, di-(C C₆)alkylamino, mercapto, (C C₆)alkylthio, hydroxy(C C₆)alkyl, mercapto(C C₆)alkyl or (C C alkylphenylmethyl; or

a group -CR_aR_bR_c in which:

each of R_a, R_b and R_c is independently hydrogen, (C₁-C₆)alkyl, (C₂- C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl; or

R_c is hydrogen and R_a and R_b are independently phenyl or heteroaryl such as pyridyl; or

R_c is hydrogen, (C C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C C₆)alkyl, or (C₃-C₈)cycloalkyl, and R_a and R_b together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or

R_a, R_b and R_c together with the carbon atom to which they are attached form a tricyclic ring (for example adamantyl); or

R_a and R_b are each independently (C C₆)alkyl, (C₂-C₆)alkenyl, (C₂- C₆)alkynyl, phenyl(C C₆)alkyl, or a group as defined for R₀ below other than hydrogen, or R_a and R_b together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and R_c is hydrogen, -OH, -SH, halogen, -CN, -CO₂H, (C_rC₄)perfluoroalkyl, - CH₂OH, -C0₂(C_rC_β)alkyl. -0(C C_β)alkyl, -O(C₂-C₆)alkenyl, -S(C_r C₆)alkyl, -SO(C₁-C₆)alkyl, -SO₂(C_rC₆) alkyl, -S(C₂-C₆)alkenyl, -SO(C₂- C₆)alkenyl, -SO₂(C₂-C₆)alkenyl or a group -Q-W wherein Q represents a bond or -O-, -S-, -SO- or -SO₂- and W represents a phenyl, phenylalkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkylalkyl, (C₄- C₈)cycloalkenyl, (C₄-C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxyl, halogen, -CN, - CO₂H, -CO₂(C_rC₆)alkyl, -CONH₂, -CONH(C_rC₆)alkyl, -CONH(C C₆alkyl)₂, -CHO, -CH₂OH, (C_rC₄)perfluoroalkyl, -O(C_rC₆)alkyl, -S(C_r C₆)alkyl, -SO(C_rC₆)alkyl, -SO₂(C_rC₆)alkyl, -NO₂, -NH₂, -NH(C C₆)alkyl, -N((C_rC₆)alkyl)₂, -NHCO(C_rC₆)alkyl, (C C₆)alkyl, (C₂-C₆)alkenyl, (C₂- C₆)alkynyl, (C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl or benzyl.

Examples of particular R₃ groups include benzyl, phenyl, cydohexylmethyl, pyridin-3- ylmethyl, tert-butoxymethyl, iso-propyl, iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1- methylethyl, 1-methylthio-1-methylethyl, and 1 -mercapto- 1-methylethyl.

Presently preferred R₃ groups include phenyl, benzyl, tert-butoxymethyl, iso-propyl and iso-butyl. The group R₄

Examples of particular ester and thioester groups R₄ groups include those of formula -(C=O)OR₉ , -(C=O)SR₉ , -(C=S)SR₉, and -(C=S)OR₉ wherein R₉ is (C_rC_β)alkyl, (C₂- C₆)alkenyl, cycloalkyl, cycloalky C^C^alkyl-, phenyl, heterocyclyl, phenyl(C_r C₆)alkyl-, heterocyclyl(C C₆)alkyl-, (C₁-C₆)alkoxy(C₁-C₆)alkyl-, (C_rC₆)alkoxy(C_r C₆)alkoxy(C₁-C₆)alkyl-, any of which may be substituted on a ring or non-ring carbon atom or on a ring heteroatom, if present. Examples of such R₉ groups include methyl, ethyl, n-and iso-propyl, n-, sec- and tert-butyl, 1-ethyl-prop-1-yl, 1-methyl- prop-1-yl, 1-methyl-but-1-yl, cyclopentyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- and 4-pyridylmethyl, N-methylpiperidin-4-yl, 1-methylcyclopent-1yl, adamantyl, tetrahydrofuran-3-yl and methoxyethyl.

Presently preferred are compounds of formula (IB) wherein R₄ is a carboxylate ester of formula -(C=O)OR₉, wherein R₉ is benzyl, cyclopentyl, cyclohexyl, or isopropyl.

The group R

Presently preferred R groups are hydrogen and methyl.

Specific examples of compounds of the invention include those prepared according to the Examples below, and salts, hydrates and solvates thereof.

Compounds of the invention may be prepared by deprotecting an O-protected N- formyl-N-hydroxyamino compound of formula (II):

in which R_{1 (} R₂, R₃ and R₄ are as defined in general formula (I) and R₂₅ is a hydroxy protecting group removable to leave a hydroxy group by hydrogenolysis or hydrolysis. Benzyl is a preferred R₂₅ group for removal by hydrogenolysis, and tetrahydropyranyl is a preferred group for removal by acid hydrolysis.

Compounds of formula (II) may be prepared by causing an acid of formula (III) or an activated derivative thereof to react with an amine of formula (IV)

(Hi) (IV)

wherein R R₂, R₃ and R₄ are as defined in general formula (I) except that any substituents in R_{1 f} R₂, R₃ and R₄ which are potentially reactive in the coupling reaction may themselves be protected from such reaction, and R₂₅ is as defined in relation to formula (II) above, and optionally removing protecting groups from R^ R₂, R₃ and R₄.

Compounds of formula (III) may be prepared by N-formylation, for example using acetic anhydride and formic acid, of compounds of formula (V)

wherein R^ R₂ and R₂₅ are as defined in relation to formula (II) and R₂₆ is a hydroxy protecting group, and thereafter removing the protecting group R₂₆. A compound of general formula (V) may be prepared by reduction of an oxime of general formula (VI)

wherein R_{1 τ} R₂, R₂₅ and R₂₆ are as defined above. Reducing agents include metal hydrides (eg sodium cyanoborohydride in acetic acid, triethylsilane or borane/pyridine) and hydrogenation.

A compound of general formula (VI) can be prepared by reaction of a β-keto carbonyl compound of general formula (VII)

wherein R R₂, and R₂₆ are as defined above, with an O-protected hydroxylamine.

β-keto carbonyl compounds (VI) may be prepared by acylation of the enolate derived from a carbonyl compound of formula (VII) or (VIIA)

(VII) (VIIA)

wherein R₂ and R₂₆ are as defined above, and X_c is a chiral auxiliary, with a compound of formula (VII)

wherein R is as defined above and Z is a leaving group such as chloro or alkoxy. Chiral enolates of type (VIIA) have been described by Evans (J. Am. Chem. Soc, 104, 1737, (1982)).

Another method for the preparation of a compound of general formula (IV) is by Michael addition of a hydroxylamine derivative to an α, β-unsaturated carbonyl compounds of general formula (IX)

wherein R_{1 f} R₂, and R₂₆ are as defined above. The α, β-unsaturated carbonyl compounds (IX) may be prepared by standard methods.

The compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties.

Orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, 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 before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut 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.

For topical application to the skin, the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

The active ingredient may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

The following preparative Examples describe the preparation of compounds useful in accordance with the invention The following abbreviations have been used in the examples

DCM - Dichloromethane

DMF - N,N-Dimethylformamide

HOBT - 1-Hydroxybenzotriazole

WSCDI - N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride

HCI - Hydrochloric acid

THF - Tetrahydrofuran

Example 1

2S-[2-(R,S)-Benzyl-3-(formyl-hydroxy-amino)-propionylamino]-3-phenyl-propionic acid cyclopentyl ester

Example 1 was prepared as outlined in Scheme 1 using procedures described below. Scheme 1

Reagents and conditions: A. (1) EtOH/KOH/H ₂0, reflux 5 hours, (2) piperidine, HCHO, EtOH, reflux 4 hours; B. H₂NOBzl, 80°C o/n; C. HCOOH, Ac₂0; D. WSCDI, HOBT, DMF, L-phenylalanine cyclopentyl ester r.t., 18 hours; E. H ₂(g), Pd catalyst, EtOH 90 minutes.

(a) 2-Benzyl-acrylic acid

Diethyl benzylmalonate (100g, 400mmol) was dissolved in ethanol (300mL) and treated with a solution of potassium hydroxide (134.4g, 2.4mol) in water (500mL). The mixture was heated under reflux for 5 hours and then allowed to cool. Ethanol was removed under reduced pressure and the remaining aqueous solution cooled in ice and acidified to pH1 with concentrated HCI. The product was extracted with ethyl acetate (3x200mL). The combined extracts were washed with brine, dried over magnesium sulphate, filtered and concentrated under reduced pressure to yield benzylmalonic acid as a white crystalline solid. The solid was taken-up in ethanol (250mL) and treated portionwise with piperidine (33g, 397mmol) followed by an aqueous solution of formaldehyde (37%, 150mL) which resulted in formation of a white precipitate. The reaction mixture was heated and treated with methanol (50mL) to give a homogeneous solution. Following dissolution the reaction mixture was heated under reflux for 4 hours. The reaction mixture was concentrated under reduced pressure. The aqueous residue was acidified to pH1 with 1 M HCI and the product extracted with ethyl acetate (3x150mL). The combined extracts were washed with brine, dried over magnesium sulphate, filtered and concentrated under reduced pressure to yield 2-benzyl-acrylic acid as a colourless oil which crystallized on standing (45g, 75%). ¹H-NMR; δ (CDCI₃), 7.32-7.17 (5H, m), 6.36 (1 H, s), 5.54 (1 H, d, J=1.3Hz), 3.61 (2H, s).

(b) 2-(R,S)-Benzyl-3-benzyloxyamino-propionic acid

A mixture of 2-benzyl-acrylic acid (8.0g, 55mmol) and O-benzylhydroxylamine (16.0g, 130mmol) was heated at 80°C for 18 hours. The reaction mixture was cooled, diluted with diethyl ether (100mL) and extracted with 1 M sodium carbonate (3x100mL). The combined aqueous extracts were acidified with 3M citric acid and then re-extracted with DCM (3x100mL). The combined organic extracts were washed with brine, dried over magnesium sulphate, filtered and concentrated under reduced pressure to yield 2-(R,S)-benzyl-3-benzyloxyamino-propionic acid as a white crystalline solid (7.82g, 53%). H-NMR; δ (CDCI₃), 7.61-7.15 (10H, m), 4.66 (2H, d, J=1.7Hz), 3.13-2.94 (4H, m), 2.84-2.74 (1 H, m).

(c) 2-(R,S)-Benzyl-3-(benzyloxy-formyl-amino)-propionic acid

A solution of 2-(R,S)-benzyl-3-benzyloxyamino-propionic acid (7.8g, 27.4mmol) in formic acid (40mL) was cooled in an ice-water bath and treated dropwise with acetic anhydride (15mL). The reaction was allowed to warm to room temperature and stirred for 18 hours. The reaction mixture was diluted with DCM (150mL) and partitioned with water (1 OOmL). The organic layer was separated, washed with brine, dried over magnesium sulphate, filtered and concentrated under reduced pressure to yield 2-(R,S)-benzyl-3-(benzyloxy-formyl-amino)-propionic acid as a colourless oil (7.8g, 91 %). ¹H-NMR; δ (CDCI₃), 7.87-7.16 (10H, m), 4.92-4.66 (2H, m), 4.03-3.90 (2H, m), 3.17-2.93 (2H, m), 2.78-2.70 (1 H, m).

(d) 2S-[2-(R,S)-Benzyl-3-(benzyloxy-formyl-amino)-propionylamino]-3-phenyl propionic acid cyclopentyl ester

2-(R,S)-Benzyl-3-(benzyloxy-formyl-amino)-propionic acid (2.89g, 9.3mmol) was dissolved in DMF (20mL) and treated with HOBT (1.35g, 10mmol) and WSCDI (1.91 g, 10mmol). The reaction mixture was stirred at room temperature for 1 hour before the addition of a solution of L-phenylalanine cyclopentyl ester (2.4g, 10.3mmol) in DMF (10mL). The reaction mixture was stirred at room temperature for 18 hours. DMF was removed under reduced pressure and the residue partitioned between ethyl acetate and 1 M HCI. The organic layer was separated and washed with 1 M HCI, saturated aqueous sodium bicarbonate solution and brine before drying over magnesium sulphate, filtration and concentration under reduced pressure to yield 2S-[2-(R,S)-benzyl-3-(benzyloxy-formyl-amino)-propionylamino]-3- phenylpropionic acid cyclopentyl ester (3.6g used crude in step e).

(e) 2S-[2-(R,S)-Benzyl-3-(formyl-hydroxy-amino)-propionylamino]-3-phenyl-propionic acid cyclopentyl ester

A solution of 2S-[2-(R,S)-benzyl-3-(benzyloxy-formyl-amino)-propionylamino]-3- phenyl propionic acid cyclopentyl ester (3.6g crude from step d) in ethanol (30mL) was treated with a palladium catalyst (100mg, 10%Pd on charcoal). The reaction mixture was stirred under an atmosphere of hydrogen gas for 90 minutes. Catalyst was removed by filtration and the filtrate concentrated to a colourless oil. Using reverse phase chromatography 200mg of the crude product was fractionated to provide two diastereoisomers of 2S-[2-benzyI-3-(formyl-hydroxy-amino)- propionylamino]-3-phenyl-propionic acid cyclopentyl ester. Diastereoisomer A (35mg), ¹H-NMR; δ (CDCI₃), 8.34 and 7.77 (1 H, 2xs), 7.29-6.84 (10H, m), 6.13 (1 H, d, J=7.3Hz), 5.11-5.00 (1 H, m), 4.67-4.49 (1 H, m), 3.99-3.79 (1 H, m), 3.66-3.60 and 3.51-3.47 (1 H, 2xm), 3.05-2.69 (5H, m) and 1.85-1.45 (8H, m); ¹³C-NMR; δ(CDCI₃), 174.4, 172.3, 171.4, 171.2, 138.2, 136.3, 129.8, 129.6, 129.2, 128.9, 127.5, 127.4, 127.2, 79.4, 78.9, 54.3, 54.0, 51.9, 51.1 , 48.8, 47.9, 46.6, 38.7, 38.1 , 36.8, 36.5, 33.0, 32.8 and 24.0. Diastereoisomer B (42mg), ¹H-NMR; δ (CDCI₃), 8.35 and 7.71 (1 H, 2xs), 7.33-7.15 (8H, m), 7.08-6.94 (1 H, bm), 6.83-6.73 (2H, m), 6.20 and 5.94 (2xd), 5.30-5.08 (1 H, m), 4.73-4.64 (1 H, m), 3.85-3.76 (1 H, m), 3.56-3.47 (1 H, m), 3.01-2.68 (5H, m) and 1.91-1.45 (8H, m); ¹³C-NMR; δ(CDCI₃), 174.2, 172.9, 172.4, 171.8, 138.7, 136.1 , 129.6, 129.3, 129.1 , 128.9, 128.8, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1 , 79.9, 79.2, 53.9, 53.6, 52.3, 50.3, 48.1 , 47.0, 38.3, 37.5, 36.9, 36.4, 32.9, 32.8 and 24.0.

Example 2

2S-{2R-[1-(R,S)-(Formyl-hydroxy-amino)-ethyl]-4-methyl-pentanoylamino}-3- phenylpropionic acid cyclopentyl ester

Example 2 was prepared as outlined in scheme 2 using procedures described below.

Scheme 2

Reagents and conditions: A. Sodium hexamethyldisilazide, AcCI, -60°C 3 hours; B. BzlONH₂ HCI, NaOAc, H₂0/EtOH, 50°C 18 hours; C. AcOH, NaCNBH₃, 25 hours; D. HCOOH, AcOH, 18 hours; E. LiOH, H₂0₂, THF/H₂0, 4 hours; F. L-Phenylalanine cyclopentyl ester, HOBT, WSCDI, DMF, 48 hours, G. H₂(g), Pd catalyst, EtOH, 2 hours. (a) 1 -(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-isobutyl-butane-1 ,3-dione

A solution of 4S-benzyl-3-(4-methyl-pentanoyl)-oxazolidin-2-one (31 g, 113mmol) in anhydrous THF (750mL) was cooled to -70°C under an inert atmosphere. Sodium hexamethyldisilazide (118mL of a 1 M solution, 118mmol) was added via cannula whilst maintaining the temperature below -68°C. The reaction mixture was stirred at -70°C for 30 minutes before the addition of acetyl chloride (10.2mL, 135mmoI), again maintaining the temperature below -68°C. The reaction was slowly warmed to -60°C and maintained at this temperature for 3 hours before quenching with acetic acid (6.75g, 118mmol) in diethyl ether (10mL). The solvent was removed under reduced pressure and the resulting slurry taken up in ethyl acetate and washed with brine. The organic layer was dried over sodium sulphate, filtered and concentrated under reduced pressure to leave an oil (36g) which was shown by NMR to contain the title compound contaminated with 15% of the starting material. ¹H-NMR; δ (CDCI₃), 7.35- 7.22 (5H, m), 4.69-4.65 (1 H, m), 4.63-4.57 (1 H, dd, J=3.2Hz), 4.22-4.13 (2H, m), 3.41 (1 H, dd, J=3.2Hz), 2.74 (1 H, dd, J=9.8Hz), 2.31 (3H, s), 2.10-2.04 (1 H, m), 1.68-1.60 (1 H, m), 1.49-1.39 (1 H, m) and 0.97 (6H, 2xd, J=6.5Hz).

(b) 1-(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-isobutyl-butane 1 ,3-dione 3-(O-benzyl- oxime)

1-(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-isobutyl-butane-1 ,3-dione (35.5g, 112mmol) was dissolved in water/ethanol (500mL, 10%vol/vol) and treated with benzylhydroxylamine hydrochloride (21.4g, 134mmol) and sodium acetate (18.3g, 134mmol). The reaction mixture was stirred at 50°C for 18 hours. The solution was concentrated under reduced pressure to give a white precipitate of the product which was collected by filtration (21.0g, 44%). ¹H-NMR; δ (CDCI₃), 7.38-7.08 (10H, m), 5.15-5.04 (2H, m), 4.57-4.47 (1 H, m), 4.24 (1 H, dd, J=3.6Hz), 4.07 (1 H, dd, J=8.9Hz), 3.92 (1 H, dd, J=2.6Hz), 3.16 (1 H, dd, J=2.7Hz), 2.09 (3H, s), 2.04-1.98 (1 H, m), 1.76-1.66 (1 H, dd, J=11.0Hz), 1.63-1.60 (1 H, m), 1.45-1.35 (1 H, m) and 0.94 (6H, 2xd, J=6.6Hz). (c) 4S-Benzyl-3-[2R-(1-(R,S)-benzyloxyamino-ethyl)-4-methyl-pentanoyl]-oxazolidin- 2-one

1-(4S-Benzyl-2-oxo-oxazolidin-3-yl)-2R-isobutyl-butane 1 ,3-dione 3-(O-benzyl- oxime) (21 g, 50mmol) was dissolved in acetic acid (400mL) and sodium cyanoborohydride (6.24g, lOOmmol) added portionwise. The mixture was stirred for 18 hours at room temperature then a further equivalent of sodium cyanoborohydride added. Stirring was continued for a further 7 hours then the reaction mixture concentrated under reduced pressure. The resultant oil was taken up in DCM (600mL) then carefully washed with sodium carbonate and brine. The organic layer was dried over magnesium sulphate, filtered and evaporated to a colourless oil (21 g). Column chromatography on silica gel using DCM as eluent lead to isolation of the desired product as a mixture of diastereoisomers (9.05g, 43%). ¹H-NMR; δ (CDCI₃), 7.37-7.18 (10H, m), 5.80 (1 H, bs), 4.70-4.60 (3H, m), 4.13 (1 H, m), 4.12- 4.05 (2H, m), 3.91 (1 H, m), 3.43-3.36 (1 H, m), 2.48-2.37 (1 H, m), 2.00-1.75 (1 H, m), 1.70-1.64 (1 H, m), 1.40-1.31 (1 H, m), 1.24 (3H, m) and 0.94-0.87 (6H, m).

(d) N-[2-(4S-Benzyl-2-oxo-oxazolidine-3S-carbonyl)-1-(R,S),4-dimethyl-pentyl]-N- benzyloxyformamide

4S-Benzyl-3-[2R-(1-(R,S)-benzyloxyamino-ethyl)-4-methyl-pentanoyl]-oxazolidin-2- one (12.7g, 30mmol) was taken up in formic acid (250mL) and stirred at 0°C while acetic anhydride (50mL) was added dropwise. The mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure, taken up in DCM and washed with saturated sodium bicarbonate and brine. The solution was dried over magnesium sulphate, filtered and concentrated to yield the title compound as a colourless oil (12.7g, 94%). ¹H-NMR; δ (CDCI₃, mixture of diastereoisomers), 8.35 and 8.14 (1 H, 2xbs), 7.55-7.15 (10H, m), 5.20- 4.90 (2H, bm), 4.73-4.46 (2H, m), 4.20-4.01 (3H, m), 3.31 (1H, dt, J=13.2, 3.2Hz), 2.51-2.30 (1 H, m), 1.95-1.74 (1 H, bm), 1.54-1.33 (5H, bm), 0.98-0.85 (6H, m). (e) 2R-[1 -(R,S)-(Benzyloxy-formyl-amino)-ethyl]-4-methyl-pentanoic acid

N-[2-(4S-Benzyl-2-oxo-oxazolidine-3S-carbonyl)-1-(R,S),4-dimethyl-pentyl]-N- benzyloxyformamide (7.30g, 16.1 mmol) was dissolved in THF (21 OmL) and water (60mL) and cooled to 0°C. Hydrogen peroxide (1.84mL, 30% solution, 64.5mmol) was added dropwise followed by aqueous lithium hydroxide (1.02g in10mL, 24.2mmol) and the solution stirred at 0°C for 4 hours. The reaction mixture was quenched by the addition of sodium nitrite (1.11g, 16mmol). THF was removed under reduced pressure and the chiral auxilliary removed by extraction into DCM. The aqueous solution was was neutralised (pH5) with 1 M HCI and extracted with ethyl acetate. The combined extracts were dried over magnesium sulphate, filtered and concentrated to yield the product as a yellow oil (3.78g, 80%). ¹H-NMR; δ (CDCI₃, mixture of diastereoisomers), 8.40 and 8.00 (1 H, 2xs), 7.52-7.26 (5H, m), 5.25-4.85 (2H, 2xbd), 4.45 (1 H, m), 3.85 (1 H, bm), 2.90 (1 H, bm), 1.75-1.48 (2H, bm), 1.48-1.20 (4H, bm) and 1.00-0.84 (6H, m).

(f) 2S-{2R-[1-(R,S)-(Benzyloxy-formyl-amino)-ethyl]-4-methyl-pentanoylamino}-3- phenyl-propionic acid cyclopentyl ester

A solution of 2R-[1-(R,S)-(benzyloxy-formyl-amino)-ethyl]-4-methyl-pentanoic acid (41 Omg, 1.40mmol) in DMF (1 OmL) was treated with HOBT (227mg, 1.68mmol) and WSCDI (322mg, 1.68mmol). A solution of L-phenylalanine cyclopentyl ester (394mg, 1.68mmol) in DMF (2mL) was added to the reaction mixture. The reaction was stirred at room temperature for 48 hours. DMF was removed by evaporation under reduced pressure. The residue was taken up in ethyl acetate and washed with 1 M HCI, saturated sodium bicarbonate and brine, before drying over magnesium sulphate, filtration and concentration to a colourless oil. The product was purified by column chromatography on silica gel eluting with 20-40% ethyl acetate/hexane. Product containing fractions were combined and solvent evaporated to provide the title compound as an off-white foam (571 mg, 66%). ¹H- NMR; δ (CDCI₃, mixture of diastereoisomers), 8.19 and 7.97 (1 H, 2xs), 7.50-6.90 (10H, m), 6.09 and 5.94 (1 H, 2xbd), 5.34-5.02 (2H, m), 4.95-4.74 (2H, m), 3.91-3.61 (2H, 2xbm), 3.15-2.80 (2H, m), 2.80-2.60 and 2.55-2.35 (1 H, 2xm), 1.92-1.35 (11 H, m), 1.22-1.00 (2H, m), 0.95-0.82 (6H, m).

(g) 2S-{2R-[1-(R,S)-(Formyl-hydroxy-amino)-ethyl]-4-methyl-pentanoylamino}-3- phenylpropionic acid cyclopentyl ester

A solution of 2S-{2R-[1-(R,S)-(benzyloxy-formyl-amino)-ethyl]-4-methyl-pentanoyl amino}-3-phenyl-propionic acid cyclopentyl ester (447mg, 0.88mmol) in ethanol (25mL) was treated with palladium catalyst (89mg, 10% Pd on charcoal) as a slurry in ethyl acetate (2mL). Hydrogen gas was bubbled through the resulting suspension for 2 hours. The catalyst was removed by filtration and the filtrate concentrated under reduced pressure to a white foam. The reaction product was separated by preparative reverse phase chromatography to yield two diastereoisomers. Diastereoisomer A (41 mg, 11 %), ¹H-NMR; δ (methanol-d₄), 8.60 (0.6H, d, J=8.2Hz), 8.52 (0.4H, d, J=8.2Hz), 8.24 (0.4H, s), 7.90 (0.6H, s), 7.84-7.15 (5H, m), 5.15 (1 H, m), 4.79-4.71 (1 H, m), 4.34-4.22 (0.4H, m), 3.66-3.54 (0.7H, m), 3.25-3.15 (1 H, m), 2.90 (1 H, dd, J=14.0, 10.4Hz), 2.70-2.56 (1 H, m), 1.87-1.40 (10H, bm), 1.13-0.97 (1 H, m) and 0.91-0.75 (9H, bm); ¹³C-NMR; δ(methanol-d₄), 175.9, 172.8, 138.4, 130.2, 129.5, 127.9, 59.0, 55.2, 55.1 , 54.2, 48.8, 40.4, 40.2, 38.3, 33.5, 26.6, 26.5, 24.7, 24.6, 21.7, 17.1 and16.0. Diastereoisomer B (28mg, 8%), ¹H-NMR; δ (methanol-d₄), 7.95 (0.4H, s), 7.84 (0.6H, s), 7.26-7.21 (5H, m), 5.13 (0.4H, m), 5.04 (0.6H, m), 4.73-4.66 (0.4H, m), 4.62-4.56 (0.6H, m), 4.42-4.36 (0.4H, m), 3.89-3.78 (0.6H, m), 3.18-2.61 (3H, bm), 1.77-1.44 (10H, m), 1.29-1.11 (3H, m), 1.00-0.87 (7H, m); ¹³C-NMR; δ(methanol-d₄), 172.7, 138.0, 130.4, 129.5, 127.9, 79.6, 59.0, 53.3, 49.6, 39.9, 38.8, 38.4, 33.5, 33.4, 27.1 , 26.7, 24.7, 24.6, 24.3, 24.1 , 22.1 , 21.9, 16.2 and 15.2. Example 3

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid cyclopentyl ester

The title compound was prepared as outlined in Scheme 3 and is described in detail below:

Scheme 3

Reagents and conditions: A. piperidine, HCHO, EtOH, 80°C, o/n; B. 'BuCOCI, Et₃N then 3-lithio-4-benzyl-5,5-dimethyl-oxazolidin-2-one; C. H₂NOBzl, room temp., o/n then pTsOH, EtOAc; D. LiOH, aq THF, 0°C; E. H-t-Leu-OcPentyl, HOBt, EDC, DMF; F. HCOBt, THF; G. H₂, Pd/C, EtOH.

Step A: 2-Butyl acrylic acid

To a solution of n-butylmalonic acid (17.2g, 107mmol) in ethanol (200ml) was added piperidine (12.76ml, 129mmol) and 37% aq. formaldehyde (40.3 ml, 538mmol). The solution was heated to 80°C during which time a precipitate appeared and then gradually redissolved over 1 hour. The reaction mixture was stirred at 80°C overnight then cooled to room temperature. The solvents were removed under reduced pressure and the residue was dissolved in ethyl acetate (200ml), washed successively with 1 M hydrochloric acid and brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to give the title compound as a clear oil (13.37g, 97%). ¹H-NMR; δ (CDCI₃), 6.29 (1 H, s), 5.65 (1 H, s), 2.34-2.28 (2H, m), 1.54-1.26 (4H, m) and 0.94 (3H, t, J=7.1 Hz).

Step B: 4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one

2-Butyl acrylic acid (21.5g, 168mmol) was dissolved in dry THF (500ml) and cooled to -78°C under a blanket of argon. Triethylamine (30ml, 218mmol) and pivaloyl chloride (21 ml, 168mmol) were added at such a rate that the temperature remained below -60°C. The mixture was stirred at -78°C for 30 minutes, warmed to room temperature for 2 hours and finally cooled back to -78°C.

In a separate flask, 4S-benzyl-5,5-dimethyl-oxazolidin-2-one was dissoved in dry THF (500ml) and cooled to -78°C under a blanket of argon. n-Butyllithium (2.4M solution in hexanes, 83ml, 200mmol) was added slowly and the mixture was stirred for 30 minutes at room temperature. The resulting anion was tranferred via a cannula into the original reaction vessel. The mixture was allowed to warm to room temperature and stirred overnight at room temperature. The reaction was quenched with 1 M potassium hydrogen carbonate (200ml) and the solvents were removed under reduced pressure. The residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over anhydrous magnesium sulphate, filtered and concentrated under reduced pressure to an orange oil. TLC analysis revealed the presence of unreacted chiral auxiliary in addition to the required product. A portion of the material (30g) was dissolved in dichloromethane and flushed though a silica pad to give pure title compound as a yellow oil (25.3g). ¹H-NMR;δ (CDCI₃), 7.31-7.19 (5H, m), 5.41 (2H,s), 4.51 (1 H, dd, J=9.7, 4.2Hz), 3.32 (1 H, dd, J=14.2, 4.2Hz), 2.82 (1 H, dd, J=14.2, 9.7Hz), 2.40-2.34 (2H, m), 1.48-1.32 (4H, m), 1.43 (3H, s), 1.27 (3H, s) and 0.91 (3H, t, J=7.1 Hz). Some chiral auxiliary was recovered by flushing the silica pad with methanol.

Step C: 4S-Benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin- 2-one (p-toluenesulfonic acid salt)

4S-Benzyl-3-(2-butyl-acryloyl)-5,5-dimethyl-oxazolidin-2-one (19.8g, 62.8mmol) was mixed with O-benzylhydroxylamine (15.4g, 126mmol) and stirred overnight at room temperature. The mixture was dissolved in ethyl acetate and the solution was washed with 1 M hydrochloric acid, 1 M sodium carbonate and brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to a pale yellow oil (25.3g) which was shown by NMR and HPLC analysis to contain 4S-Benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl- oxazolidin-2-one (ca. 82% d.e.) along with a trace of starting material. The product was combined with a another batch (26.9g, 76% d.e.) and dissolved in ethyl acetate (200ml). p-ToIuenesulfonic acid (22.7g, 119mmol) was added and the mixture was cooled to 0°C. The title compound was obtained as a white crytalline solid by seeding and scratching. Yield: 25.2g, (34%, single diastereoisomer). A second crop (14.7g, 20%, single diastereoisomer) was also obtained. ¹H-NMR;δ (CDCI₃), 7.89 (2H, d, J=8.2Hz), 7.37-7.12 (10H, m), 7.02 (2H, d, J=6.9Hz), 5.28-5.19 (2H,m), 4.55 (1 H, m), 4.23 (1 H, m), 3.93 (1 H, m), 3.58 (1 H, m), 2.58 (1 H, m), 2.35 (3H, s), 1.67- 1.51 (2H, m), 1.29-1.16 (4H, m), 1.25 (3H, s), 1.11 (3H, s) and 0.80-0.75 (3H, m).

Step D: 2R-Benzyloxyamino-methyl)-hexanoic acid

4S-Benzyl-3-[2R-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazolidin-2-one p-toluenesulfonic acid salt (25.2g, 40.2mmol) was partitioned between ethyl acetate and 1 M sodium carbonate. The organic phase was dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure. The residual oil was dissolved in THF (150ml) and water (50ml) and cooled to 0°C and treated with lithium hydroxide (1.86g, 44.2mmol). The solution was stirred for 30 minutes at 0°C, then overnight at room temperature. The reaction was acidified to pH4 with 1 M citric acid and the solvents were removed. The residue was partitioned between dichloromethane and 1 M sodium carbonate. The basic aqueous layer was acidified to pH4 with 1 M citric acid and extracted three times with ethyl acetate. The combined organic layers were dried over anhydrous magnesium sulfate, filtered and concentrated to provide the title compound as a colourless oil (7.4g, 73%). ¹H- NMR;δ (CDCI₃), 8.42 (2H, br s), 7.34-7.25 (5H, m), 4.76-4.66 (2H, m), 3.20-3.01 (2H, m), 2.73 (1 H, m), 1.70-1.44 (2H, m), 1.34-1.22 (4H, m) and 0.92-0.86 (3H, m).

Step E: 2S-[2R-(Benzyloxyamino-methyl)-hexanoylamino]-3,3-dimethyl butyric acid cyclopentyl ester

2R-Benzyloxyamino-methyl)-hexanoic acid (1.99g, 7.93mmol) was dissolved in DMF (50ml) and the solution was cooled to 0°. EDC (874mg, 4.56mmol) and HOBt (62mg, 0.46mmol) were added and the mixture was stirred for 15 minutes, tert- Leucine cyclopentyl ester (1.0g, 5.02mmol) was added and the reaction was allowed to warm to room temperature and stirred overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed successively with 1M hydrochloric acid, saturated sodium hydrogen carbonate and brine, dried and filtered. The solvent was removed to leave a yellow oil which was purified by flash chromatography (silica gel, 20% ethyl acetate in hexane) to afford the title compound (964mg, 28%) ¹H-NMR;δ (CDCl₃), 7.36-7.29 (5H, m), 6.62 (1 H, br d, J=9.2Hz), 5.69 (1 H, br s), 5.22-5.18 (1 H, m), 4.73 (2H, s), 4.42 (1 H, d, J=9.4Hz), 3.11-3.04 (2H,m), 2.51 (1 H, m), 1.87-1.59 (10H, m), 1.30-1.23 (4H, m), 0.97 (9H, s) and 0.87 (3H, t, J=6.7Hz). Step F: 2S-{2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid cyclopentyl ester

2S-[2R-(Benzyloxyamino-methyl)-hexanoylamino]-3,3-dimethyl butyric acid cyclopentyl ester (947mg, 2.19mmol) was dissolved in dry THF (40ml) and treated with 1 -formyl-benzotriazole (354mg, 2.41 mmol). The reaction was stirred overnight at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate and washed with 1 M sodium carbonate solution and brine. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under reduced pressure. The desired product was obtained by flash chromatography (silica gel, eluting with 25% ethyl acetate in hexane). Yield: 814mg (81%) . ¹H-NMR;δ (CDCI₃, rotamers), 8.13 (0.7H, br s), 7.88 (0.3H, br s), 7.37 (5H, br s), 6.02 (1 H, br d, J=9.1 Hz), 5.18 (1 H, m), 4.96 (1 H, br s), 4.76 (1 H, br s), 4.35 (1 H, d, J=9.2Hz), 3.74 (2H, br s), 2.53 (1 H, m), 1.87-1.59 (10H, m), 1.28-1.23 (4H, m), 0.96-0.84 (3H, m) and 0.93 (9H, s).

Step G: 2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid cyclopentyl ester

2S-{2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid cyclopentyl ester (780mg, 1.69mmol) was dissolved in ethanol (40ml) and placed under a blanket of argon. 10% palladium on charcoal (80mg) was added and the mixture was stirred vigorously as hydrogen gas was bubbled through the system. After 30 minutes the suspension was placed under a balloon of hydrogen and stirred overnight at room temperature. The flask was purged with argon before removing the catalyst by filtration. The filtrate was concentrated under reduced pressure to provide the title compound as a white foam (458mg, 73%). ¹H-NMR; δ (CD₃OD, rotamers), 0.84 (0.4H, s), 7.82 (0.6H, s), 5.19-5.15 (1 H, m), 4.27 (1 H, s), 3.82-3.61 (1.4H, m), 3.45-3.37 (0.6H, m), 3.10-2.88 (1 H, m), 1.89-1.31 (14H, m), 1.01 (3.6H, s), 0.99 (5.4H, s) and 0.92-0.87 (3H, m). ¹³C-NMR; δ (CDCI₃ rotamers), 172.8, 171.1 , 78.7, 78.4, 60.4, 60.2, 51.7, 48.0, 46.2, 44.8, 34.9, 34.7, 32.7, 32.6, 30.1 , 29.9, 29.3, 29.2, 26.6, 23.6, 22.6 and 13.8. IR (refection disc) v_max 2978, 1740, 1690, 1549, 1379, 1237, 1171 , 984, 882 cm^"1. LRMS: 393 (M+Na), 369 (M-H).

The compounds of Examples 4-6 were preparedby the method of Example 3, by using the appropriate amino acid derivative instead of te/f-leucine cyclopentyl ester in Step E:

Example 4

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenyl-propionic acid cyclo-pentyl ester

¹H-NMR; δ (CD₃OD rotamers), 8.13 (0.4H, s), 7.81 (0.6H, s), 7.36-6.92 (5H, m), 5.11-5.07 (1 H, m), 4.61 (1 H, t, J=7.6Hz), 3.65-3.46 (1 H, m), 3.42-3.30 (1 H, m), 3.19-2.92 (2H, m), 2.89-2.77 (0.6H, m), 2.74-2.51 (0.4H, m), 1.98-1.29 (14H, m) and 0.89-0.81 (3H, m). ¹³C-NMR; δ (CD₃OD rotamers), 173.0, 171.6, 136.2, 129.8, 129.6, 129.1 , 129.0, 127.7, 127.5, 79.6, 79.0, 53.9, 53.6, 51.9, 48.5, 46.4, 45.0, 38.6, 38.0, 33.0, 32.9, 30.3, 30.2, 29.6, 29.5, 24.0, 23.0 and 14.2. IR (reflection disc); v_max 3325, 2958, 1731 , 1663, 1532, 1443, 1367, 1280, 1199, 1104, 1079, 1032, 885, 749 and 699 cm-1. LRMS; +ve ion 427 (M+Na); -ve ion 403 (M-1 ). Example 5

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid methyl ester

m.p. 63.5-64.5°C. Η-NMR; δ (CD₃OD, rotamers), 8.24 (0.3H, s), 7.82 (0.7H, s), 4.33 (1 H, s), 3.82-3.58 (1.3H, m), 3.70 (3H, s), 3.45-3.37 (0.7H, m), 3.11-3.01 (0.7H, m), 2.95-2.83 (0.3H, m), 1.55-1.20 (6H, m), 1.00 (3H, s), 0.99 (6H, s) and 0.93-0.88 (3H, m). ¹³C-NMR; δ (CD₃OD), 176.6, 173.2, 62.7, 53.9, 52.5, 45.4, 35.3, 31.6, 30.6, 27.6, 24.1 and 14.7. IR (refection disc); v_max 3318, 2955, 1738, 1661 , 1642, 1549, 1530, 1465, 1443, 1352, 1216, 1165, 1104, 1040, 1008 and 879 cm ¹. LRMS; +ve ion 339 (M+Na), -ve ion 315 (M-H).

Example 6

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-methyl butyric acid cyclo-pentyl ester

Pale yellow oil. Η-NMR; δ (CD₃OD, rotamers), 8.25 (0.4H, s), 7.82 (0.6H, s), 5.19- 5.15 (1 H, m), 4.24 (1 H, d, J=6.3 Hz), 3.81-3.62 (1.4H, m), 3.49-3.38 (0.6H, m), 3.01-2.92 (0.6H, m), 2.81-2.75 (0.4H, m), 2.17-2.00 (1 H, m), 1.90-1.34 (14H, m) and 0.95-0.88 (9H, m). ¹³C-NMR; δ (CD₃OD), 176.6, 173.2, 159.8, 79.7, 60.0, 53.9, 45.8, 45.6, 34.0, 32.0, 31.5, 30.7, 25.0, 24.1 , 19.9, 19.1 and 14.7. LRMS; +ve ion 379 (M+Na), -ve ion 355 (M-H).

The compounds of Example 7-10 were prepared by the method of Example 3 by parallel synthesis, using the appropriate amino acid derivative instead of terf-leucine cyclopentyl ester in Step E. The products were purified by preparative HPLC:

Example 7

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenyl-propionic acid methyl ester

LRMS; +ve ion 373 (M+Na), -ve ion 349 (M-H). Example 8

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenyl-propionic acid ethyl ester

LRMS; +ve ion 387 (M+Na), -ve ion 363 (M-H).

Example 9

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid /so-propyi ester

LRMS; +ve ion 367 (M+Na), -ve ion 343 (M-H) Example 10

2R (or S)-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-2-phenyl acetic acid cyclpentyl ester

LRMS; +ve ion 413 (M+Na), 391 (M+H), -ve ion 389 (M-H)

Example 11 was prepared by the method of Example 3, by using the appropriate amino acid derivative instead of te/Y-leucine cyclopentyl ester in step E:

Example 11

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl-butyric acid te/f-butyl ester

m.p. 76.1-78.2°C. Η-NMR; δ CDCI₃, rotamers), 8.37 (0.4H, s), 7.80 (0.6H, s), 6.42 (0.4H, d, J=9.3Hz), 6.29 (0.6H, d, J=9.5Hz), 4.33 (1 H, d J=9.5Hz), 3.98 (0.4H, dd, J=14.6, 7.3Hz), 3.85 (0.6H, dd, J=14.1 , 9.6Hz), 3.64 (0.4H, dd, J=14.6, 3.5Hz), 3.45 (0.6H, dd, J=14.1 , 4.2Hz), 2.85-2.75 (0.6H, m), 2.73-2.66 (0.4H, m), 1.67-1.19 (6H, m), 1.47 (9H, s), 0.99 (3H, s), 0.94 (6H, s) and 0.90-0.85 (3H, m). ¹³C-NMR; δ (CDCI₃, rotamers) 176.3, 173.1 , 170.9, 82.5, 61.2, 61.0, 52.1 , 48.4, 46.7, 45.3, 35.0, 30.5, 30.4, 29.7, 29.6, 28.4, 27.0, 22.9 and 14.3. IR (reflection disc); v_ma, 3405, 2967, 1708, 1680, 1653, 1524, 1474, 1369, 1285, 1238 and 1173 cm-1. LRMS; +ve ion 381 [M+Na], -ve ion 357 [M-1].

The compounds of Examples 12 and 13 were prepared by the method of Example 3 by parallel synthesis, using the appropriate amino acid derivative instead of tert leucine cyclopentyl ester in Step E. The products were purified by preparative HPLC:

Example 12

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl-butyric acid 1-ethyl-propyl ester

LRMS; +ve ion 373 [M+1], 395 [M+Na], -ve ion 371 [M-1]. Example 13

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl-butyric acid tetrahydro-pyran-4-yl ester

LRMS; +ve ion 387 [M+1], 409 [M+Na], -ve ion 385 [M-1].

The compounds of Examples 14 and 15 were prepared from 2R-(.erf-butoxyamino- methyl)-hexanoic acid and the appropriate amino acid derivative by anology with methods described for Example 3:

Example 14

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenyl-propionic acid benzyl ester

Η-NMR; δ CDCI₃, rotamers) 8.33 (0.4H, s), 7.79 (0.6H, s), 7.37-7.21 (8H, m), 7.01- 6.98 (2H, m), 6.24-6.15 (1 H, m), 5.21-5.07 (2H, m), 4.94-4.84 (1 H, m), 3.91 (0.4H, dd, J=14.7, 7.4Hz), 3.77 (0.6H, dd, J=14.3, 9.7Hz), 3.55 (0.4H, dd, J=14.5, 3.4Hz), 3.43 (0.6H, dd, J=14.2, 4.4Hz), 3.25-2.98 (2H, m), 2.71-2.64 (0.6H, m), 2.52-2.51 (0.4H, m), 1.56-1.25 (6H, m) and 0.84 (3H,br.s). ¹³C-NMR; δ (CDCI₃, rotamers) 157.7, 172.5, 171.2, 135.5, 135.3, 135.0, 134.8, 129.3, 129.2, 128.7, 128.6, 127.4, 127.2, 67.7, 67.4, 53.4, 50.8, 48.0, 46.1 , 44.6, 38.0, 37.6, 29.9, 29.8, 29.1 , 29.0, 22.5 and 13.7. LRMS; +ve ion 427 [M+1], 449 [M+Na].

Example 15

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl-butyric acid benzyl ester

Η-NMR; δ (CDCI₃, rotamers) 8.35 (0.2H, s), 7.74 (0.8H, s), 7.35 (5H, s), 6.50 (1 H, d, J=9.3Hz), 5.21-5.15 (2H, m), 4.45 (1 H, d, J=9.3Hz), 3.98 (0.2H, dd, J=14.6, 7.4Hz), 3.82 (0.8H, dd, J=14.1 , 9.8Hz), 3.62 (0.2H, dd, J=14.7, 3.5Hz), 3.42 (0.8H, dd, J=14.1 , 3.9Hz), 2.88-2.74 (0.8H, m), 2.71-2.63 (0.2H, m), 1.66-1.16 (6H, m), and 0.98-0.83 (12H, m). ¹³C-NMR; δ (CDCI₃, rotamers)176.4, 173.4, 171.6, 135.6, 129.0, 128.9, 67.7, 67.4, 60.9, 60.7, 52.6, 48.3, 46.5, 45.0, 35.3, 35.1 , 30.5, 30.3, 29.6, 29.5, 27.0, 26.9, 22.9 and 14.3. LRMS; +ve ion 393 [M+1], 415 [M+Na], -ve ion 391 [M-1]. Example 16 was prepared from 3-benzyloxyamino-2R-cyclopentylmethyl-propionic acid and phenylalanine cyclopentyl ester by analogy with methods described in patent number WO 99/39704:

Example 16

2S-[2R-Cyclopentylmethyl-3-(formyl-hydroxy-amino)-propionylamino]-3-phenyl- propionic acid cyclopentyl ester

m.p. 76.8-78.5°C. Η-NMR; δ CDCI₃, rotamers) 8.34 (0.3H, s), 7.78 (0.7H, s), 7.34- 7.09 (5H, m), 6.38-6.33 (1 H, m), 5.24-5.19 (0.4H, m), 5.15-5.11 (0.6H, m), 4.83-4.71 (1 H, m), 3.91 (0.4H, dd, J=14.6, 7.4Hz), 3.76 (0.6H, dd, J=14.1 , 9.5Hz), 3.55 (0.4H, dd, J=14.6, 3.4Hz), 3.42 (0.6H, dd, J=14.1 , 4.3Hz), 3.23-2.92 (2H, m), 2.82-2.73 (0.7H, m), 2.58-2.56 (0.3H, m), 1.87-1.27 (17H, m) and 1.05-1.03 (2H, m). ¹³C-NMR; δ (CDCI₃, rotamers) 176.0, 173.1 , 171.7, 171.5, 136.2, 136.1 , 129.8, 129.6, 129.1 , 128.9, 127.7, 127.5, 79.6, 79.0, 53.9, 53.6, 52.4, 48.8, 45.7, 44.4, 38.6, 38.0, 37.9, 37.7, 36.9, 36.8, 33.4, 33.3, 33.0, 32.9, 32.8, 32.7, 25.6, 25.5 and 24.0. IR (reflection disc); V_max 3329, 2946, 2864, 1732, 1682, 1644, 1525, 1445, 1345, 1252, 1196 and 881 cm-1. LRMS; +ve ion 431 [M+1], -ve ion 429 [M-1]. Examples 17-28

Further compounds useful in accordance with the invention are those specifically named and characterised in International patent application WO 99/41232, as follows:

Example 17

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-2-phenyl acetic acid ethyl ester (Example 10 of WO 99/41232.)

Example 18

S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-2-phenyl acetic acid iso- propyl ester (Example 11 of WO 99/41232.)

Example 19

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid ethyl ester (Example 12 of WO 99/41232.)

Example 20

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid cyclobutyl ester (Example 14 of WO 99/41232.)

Example 21

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid cyclohexyl ester (Example 15 of WO 99/41232.)

Example 22

S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid -methoxy-ethyl ester (Example 16 of WO 99/41232.)

Example 23

S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid -methyl-piperidin-4-yl ester (Example 17 of WO 99/41232.)

Example 24

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3,3-dimethyl butyric acid cyclopentylmethyl ester (Example 18 of WO 99/41232.)

Example 25

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenyl-propionic acid /^'so-propyl ester (Example 19 of WO 99/41232.)

Example 26

S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-2-phenyl acetic acid methyl ester (Example 20 of WO 99/41232.)

Example 27

S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-2-phenyl acetic acid tert- butyl ester (Example 21 of WO 99/41232.)

Example 28

2S-{2R-[(Formyl-hydroxy-amino)-methyl]-hexanoylamino}-3-phenyl-propionic acid tert- butyl ester (Example 22 of WO 99/41232.)

Biological Example

Inhibition of Leukotriene Synthesis by Rat Basophilic Leukaemia Cells

The rat basophilic leukaemia cell line RBL-1 was obtained from ATCC or ECACC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, non-essential amino acids, sodium pyruvate, penicillin, streptomycin and 2mM glutamine, at 37°C in an atmosphere of 5% CO₂ in air, as recommended by the culture collection.

RBL-1 cells were harvested during log growth, washed and resuspended in Hanks Balanced Salt Solution, without calcium chloride and magnesium chloride, supplemented with 10% foetal bovine serum and penicillin, streptomycin, and 2mM glutamine and adjusted to a final cell concentration of 1.25X10⁵ cells/ml. Cells incubated in the presence of the appropriate concentration of inhibitor or vehicle for 4.75 hours at 37°C. Cells then transferred to ice for 15 minutes. Cells washed by centrifugation and resuspension in pre-chilled Phosphate Buffered Saline, with calcium chloride and magnesium chloride, supplemented with the appropriate concentration of test sample. After the PBS wash, cells resuspended at a final cell concentration of equalivent to 2.35X10⁶ cell/ml in PBS/sample buffer. Cells transferred to a microfuge tube on ice prior to ionophore stimulation.

Treated cells then exposed to 10μM A23187 for 15 minutes at 37°C . Cells transferred to ice, thereby stopping the reaction. Cell free supernatant harvested by centrifugation. The levels of leukotriene B₄ then determined in the cell free supernatant using the Leukotriene B₄ [³H] assay system from Amersham Pharmacia Biotech.

Using this methodology the IC₅₀ value for the compound of example 4 was estimated as 0.7nM and for Kelatorphan as 350nM.

Claims

Claims

1. A method of treatment of mammals suffering diseases responsive to inhibition of intracellular leukotriene-A₄ hydrolase activity, comprising administering to the mammal suffering such disease an amount of a compound of general formula (I) or a pharmaceutically acceptable salt hydrate or solvate thereof sufficient to inhibit such activity:

wherein

R is hydrogen or (C C₆)alkyl;

R., is hydrogen;

(C C_β)alky1;

(C₂-C₆)alkenyl;

phenyl or substituted phenyl;

phenyl (C C₆)alkyl or substituted phenyl(C C₆)alkyl;

phenyl (C₂-C₆)alkenyl or substituted phenyl(C₂-C₆)alkenyl

heterocyclyl or substituted heterocyclyl; heterocyclyl(C C₆)alkyl or substituted heterocyclyl(C,-C₆)alkyl;

a group BSO_nA- wherein n is 0, 1 or 2 and B is hydrogen or a (C^C_β) alkyl, phenyl, substituted phenyl, heterocyclyl substituted heterocyclyl, (C C₆)acyl, phenacyl or substituted phenacyl group, and A represents (C_rC₆)alkylene;

amino(C_rC₆)alkyl, (C₁-C₆)alkylamino(C₁-C₆)alkyl, di(C C₆)alkylamino(C_r C₆)alkyl, hydroxy(C C₆)alkyl, mercapto(C C₆)alkyl or carboxy(C C₆) alkyl wherein the amino-, hydroxy-, mercapto- or carboxyl-group are optionally protected or the carboxyl- group amidated;

lower alkyl substituted by carbamoyl, mono(lower alkyl)carbamoyl, di(lower alkyl)carbamoyl, di(lower alkyl)amino, or carboxy-lower alkanoylamino; or

a cycloalkyl, cycloalkenyl or non-aromatic heterocyclic ring containing up to 3 heteroatoms, any of which may be (i) substituted by one or more substituents selected from C_rC₆ alkyl, C₂-C₆ alkenyl, halo, cyano ( -CN), -CO₂H, -CO₂R, - CONH₂, -CONHR, -CON(R)₂, -OH, -OR, oxo-, -SH, -SR, -NHCOR, and - NHCO₂R wherein R is C.,-C₆ alkyl or benzyl and/or (ii) fused to a cycloalkyl or heterocyclic ring;

is a C.,-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, phenyl(C_rC₆ alkyl)-, heteroaryl(C,-C₆ alkyl)-, phenyl(C₂-C₆ alkenyl)-, heteroaryI(C₂-C₆ alkenyl)-, phenyl(C₂-C_e alkynyl)-, heteroaryl(C₂-C₆ alkynyl)-, alkyl)-, cycloalkyl (C₂-C₆ alkenyl)-, cycloalkyl(C₂-C₆ alkynyl)-, cycloalkenyl(C C₆ alkyl)-, cycloalkenyl(C₂-C₆ alkenyl)-, cycloalkenyl(C₂-C₆ alkynyl)-, phenyl(C_rC_β alkyl)O(C C₆ alkyl)-, or alkyl)O(C C₆ alkyl)- group, any one of which may be optionally substituted by C C_β alkyl, C,-C₆ alkoxy, halo, cyano (-CN), phenyl or heteroaryl, or phenyl or heteroaryl substituted by C_rC₆ alkyl, C C₆ alkoxy, halo, or cyano (-CN);

R₃ is the characterising group of a natural or non-natural α amino acid in which any functional groups may be protected; and

R₄ is an ester or thioester group.

2. The use of a compound of formula (I) as defined in claim 1 in the preparation of a composition for treatment of mammals suffering diseases responsive to inhibition of intracellular leukotriene-A₄ hydrolase activity.

3. A method as claimed in claim 1 or the use as claimed in claim 2 wherein the stereochemical configuration of the carbon atom carrying the groups R₃ and R₄ is S.

4. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R., is:

hydrogen, methyl, ethyl, n-propyl, n-butyl, isobutyl, cyclopropylmethyl, allyl, phenylpropyl, phenylprop-2-enyl, thienylsulphanylmethyl, thienylsulphinylmethyl, or thienylsulphonylmethyl; or

C C₄ alkyl.eg methyl, ethyl n-propyl or n-butyl, substituted by a phthalimido, 1 ,2-dimethyl-3,5-dioxo-1 ,2,4-triazolidin-4-yl, 3-methyl-2,5-dioxo-1- imidazolidinyl, 3,4,4-trimethyl-2,5-dioxo-1 -imidazolidinyl, 2-methyl-3,5-dioxo- 1 ,2,4-oxadiazol-4-yl, 3-methyl-2,4,5-trioxo-1 -imidazolidinyl, 2,5-dioxo-3- phenyl-1 -imidazolidinyl, 2-oxo-1 -pyrrolidinyl, 2, 5-dioxo-1 -pyrrolidinyl or 2,6- dioxopiperidinyl, 5,5-dimethyl-2,4-dioxo-3-oxazolidinyl, hexahydro-1 ,3- dioxopyrazolo[1 ,2,a][1 ,2,4]-triazol-2-yl, or a naphththalimido (ie 1 ,3-dihydro- 1 ,3-dioxo-2H-benz[f]isoindol-2-yl), 1 ,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl, 1 ,3-dihydro-1 ,3-dioxo-2H-pyrrolo[3,4-b]quinolin-2-yl, or 2,3-dihydro-1 ,3-dioxo- 1 H-benz[d,e]isoquinolin-2-yl group; or

cyclohexyl, cyclooctyl, cycloheptyl, cyclopentyl, cyclobutyl, cyclopropyl, tetrahydropyranyl or morpholinyl.

5. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R^ is hydrogen, cyclopropylmethyl, n-propyl, or allyl.

6. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₂ is:

C C₁₂ alkyl, C₃-C₆ alkenyl or C₃-C₆ alkynyl;

phenyl(C|-C₆ alkyl)-, phenyl(C₃-C₆ alkenyl)- or phenyl(C₃-C₆ alkynyl)- optionally substituted in the phenyl ring; heteroaryl(C₁-C₆ alkyl)-, heteroaryl(C₃-C₆ alkenyl)- or heteroaryl(C₃-C₆ alkynyl)- optionally substituted in the heteroaryl ring;

4-phenylphenyl(C C₆ alkyl)-, 4-phenylphenyl(C₃-C₆ alkenyl)-, 4- phenylphenyl(C₃-C₆ alkynyl)- , 4-heteroarylphenyl(C₁-C₆ alkyl)-, 4- heteroarylphenyl(C₃-C₆ alkenyl)-, 4-heteroarylphenyl(C₃-C₆ alkynyl)-, optionally substituted in the terminal phenyl or heteroaryl ring; or

phenoxy(C C₆ alkyl)- or heteroaryloxy(C C₆ alkyl)- optionally substituted in the phenyl or heteroaryl ring.

7. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₂ is: methyl, ethyl, n- or iso-propyl, n- , iso- or tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-nonyl, n-decyl, prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yI, benzyl phenylpropyl, 4-chlorophenylpropyl, 4- methylphenylpropyl, 4-methoxyphenylpropyl, phenoxybutyl, 3-(4- pyridylphenyl)propyl-, 3-(4-(4-pyridyl)phenyl)prop-2-yn-1-yl, 3-(4- phenylphenyl)propyl-, 3-(4-phenyl)phenyl)prop-2-yn-1-yl or 3-[(4- chlorophenyl)phenyl]propyl-.

8. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₂ is benzyl, n-butyl, iso-butyl, n-hexyl, or cyclopentylmethyl.

9. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₃ is C,-C₆ alkyl, phenyl, 2,- 3-, or 4-pyridyI, 2- or 3- thienyl, 2,- 3-, or 4-hydroxyphenyl, 2,- 3-, or 4-methoxyphenyl, 2,- 3-, or 4- pyridyl methyl, benzyl, 2,- 3-, or 4-hydroxybenzyl, 2,- 3-, or 4-benzyloxybenzyl, 2,- 3-, or 4-C C₆ alkoxybenzyl, or benzyloxy(C C₆alkyl)-.

10. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₃ is the characterising group of a natural α amino acid, in which any functional group may be protected, any amino group may be acylated and any carboxyl group present may be amidated.

11. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₃ is a group -[Alk]_nR₆ where Alk is a (C C₆)alkyl or (C₂- C₆)alkenyl group optionally interrupted by one or more -O-, or -S- atoms or -N(R₇)- groups [where R₇ is a hydrogen atom or a (C₁-C₆)alkyl group], n is 0 or 1 , and R₆ is an optionally substituted cycloalkyl or cycloalkenyl group.

12. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₃ is a benzyl group substituted in the phenyl ring by a group of formula -OCH₂COR₈ where R₈ is hydroxyl, amino, (C C₆)alkoxy, phenyl(C C₆)alkoxy, (C C₆)alkylamino, di((C_rC₆)alkyl)amino, phenyl(C_rC₆)alkylamino, the residue of an amino acid or acid halide, ester or amide derivative thereof, said residue being linked via an amide bond, said amino acid being selected from glycine, α or β alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid.

13. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₃ is a heterocyclic(C₁-C₆)alkyl group, either being unsubstituted or mono- or di-substituted in the heterocyclic ring with halo, nitro, carboxy, (C_rC₆)alkoxy, cyano, (C C₆)alkanoyl, trifluoromethyl (C C₆)alkyl, hydroxy, formyl, amino, (C^C^alkylamino, di-(C C₆)alkylamino, mercapto, (C C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or (C^C alkylphenylmethyl.

14. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₃ is a group -CR_aR_bR_c in which: each of R_a, R_b and R_c is independently hydrogen, (C^C^alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, (C₃-C₈)cycloalkyl; or R_c is hydrogen and R_a and R_b are independently phenyl or heteroaryl such as pyridyl; or

R_c is hydrogen, (C_rC_β)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C_rC₆)alkyl, or (C₃-C₈)cycloalkyl, and R_a and R_b together with the carbon atom to which they are attached form a 3 to 8 membered cycloalkyl or a 5- to 6-membered heterocyclic ring; or

R_a, R_b and R_c together with the carbon atom to which they are attached form a tricyclic ring (for example adamantyl); or

R_a and R_b are each independently (C C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or a group as defined for R_c below other than hydrogen, or R_a and R_b together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and R_c is hydrogen, -OH, -SH, halogen, -CN, - CO₂H, (C₁-C₄)perfluoroalkyl, -CH₂OH, -CO₂(C C₆)alkyl, -O(C_rC₆)alkyl, -O(C₂- C₆)alkenyl, -S(C_rC₆)alkyl, -SO(C_rC₆)alkyl, alkyl, -S(C₂- C₆)alkenyl, -SO(C₂-C₆)alkenyl, -SO₂(C₂-C₆)alkenyl or a group -Q-W wherein Q represents a bond or -O-, -S-, -SO- or -SO₂- and W represents a phenyl, phenylalkyl, (C₃-C₈)cycloalkyl, (C₃-C₈)cycloalkylalkyl, (C₄-C₈)cycloalkenyl, (C₄- C₈)cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, which group W may optionally be substituted by one or more substituents independently selected from, hydroxyl, halogen, -CN, -CO₂H, -CO₂(C₁-C₆)alkyI, -CONH₂, -CONH(C_r C₆)alkyl, -CONH(C₁-C₆alkyl)₂, -CHO, -CH₂OH, (C_rC₄)perfluoroalkyl, -O(C_r C₆)alkyl, -S(C_rC₆)alkyl, -SO(C_rC₆)alkyl, -SO₂(C_rC₆)alkyl, -NO₂, -NH₂, -NH(C_r C₆)alkyl, -N((C₁-C₆)alkyl)₂, -NHCO(C C₆)alkyl, (C_rC₆)alkyl, (C₂-C₆)alkenyl, (C₂- C₆)alkynyl, (C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl or benzyl.

15. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₃ is phenyl, benzyl, tert-butoxymethyl, iso-propyl or iso- butyl.

16. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₄ is a group of formula -(C=O)OR₉ , -(C=O)SR₉ , - (C=S)SR₉, and -(C=S)OR₉ wherein R₉ is (C,-C_β)alkyl, (C₂-C₆)alkenyl, cycloalkyl, cycloalkyl(C C₆)alkyl-, phenyl, heterocyclyl, phenyl(C C₆)alkyl-, heterocycly C^ C₆)alkyl-, (C_rC₆)alkoxy(C_rC₆)alkyl-, or (C₁-C₆)alkoxy(C₁-C₆)alkoxy(C₁-C₆)alkyl-, any of which may be substituted on a ring or non-ring carbon atom or on a ring heteroatom, if present.

17. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₄ is a group of formula -(C=O)OR₉ wherein R₉ is methyl, ethyl, n-or iso-propyl, n-, sec- or tert-butyl, 1 -ethyl-prop- 1 -yl, 1-methyl-prop-1-yl, 1- methyl-but-1-yl, cyclopentyl, cyclohexyl, allyl, phenyl, benzyl, 2-, 3- and 4- pyridylmethyl, N-methylpiperidin-4-yl, 1-methylcyclopent-1yl, adamantyl, tetrahydrofuran-3-yl or methoxyethyl.

18. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R₄ is a group of formula -(C=O)OR₉ wherein R₉ is benzyl, cyclopentyl, cyclohexyl or isopropyl.

19. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R is hydrogen or methyl.

20. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in claim 3, wherein R is hydrogen; R., is hydrogen, cyclopropylmethyl, n- propyl or allyl; R₂ is benzyl, n-butyl, iso-butyl, n-hexyl or cyclopentylmethyl; R₃ is phenyl, benzyl, tert-butoxymethyl, isopropyl or iso-butyl; and R₄ is a group of formula -(C=O)OR₉ wherein R₉ is benzyl, cyclopentyl, cyclohexyl or isopropyl.

21. A method as claimed in claim 1 , the use or claim 2, or a method or use as claimed in any of claims 3-20, wherein the disease is asthma, rheumatoid arthritis, osteoarthritis, multiple sclerosis, ulcerative colitis, contact or atopic dermatitis, psoriasis, inflammatory bowel disease or Crohn's disease.