Antibacterial agents

This invention relates to novel hydroxamic acid and N-formyl hydroxylamine derivatives having antibacterial activity, to methods of treatment using such compounds, and to pharmaceutical and veterinary compositions comprising such compounds.

BACKGROUND TO THE INVENTION

Many classes of antibacterial agents are known, including the penicillins and cephalosporins, tetracyclines, sulfonamides, monobactams, fluoroquinolones and quinolones, aminoglycosides, glycopeptides, macrolides, polymyxins, lincosamides, trimethoprim and chloramphenicol. The fundamental mechanisms of action of these antibacterial classes vary.

Bacterial resistance to many known antibacterials is a growing problem. Accordingly there is a continuing need in the art for alternative antibacterial agents, especially those which have mechanisms of action fundamentally different from the known classes.

Amongst the Gram-positive pathogens, such as Staphylococci, Streptococci, Mycobacteria and Enterococci, resistant strains have evolved/arisen which makes them particularly difficult to eradicate. Examples of such strains are methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant coagulase negative Staphylococci (MRCNS), penicillin resistant Streptococcus pneumoniae and multiply resistant Enterococcus faecium.

Pathogenic bacteria are often resistant to the aminoglycoside, β-lactam (penicillins and cephalosporins), and chloramphenicol types of antibiotic. This resistance involves the enzymatic inactivation of the antibiotic by hydrolysis or by formation of inactive derivatives. The β-lactam (penicillin and cephalosporin) family of antibiotics are characterised by the presence of a β-lactam ring structure. Resistance to this family of antibiotics in clinical isolates is most commonly due to the production of a “penicillinase” (β-lactamase) enzyme by the resistant bacterium which hydrolyses the β-lactam ring thus eliminating its antibacterial activity.

Recently there has been an emergence of vancomycin-resistant strains of enterococci (Woodford N. 1998 Glycopeptide-resistant enterococci: a decade of experience. Journal of Medical Microbiology. 47(10):849-62). Vancomycin-resistant enterococci are particularly hazardous in that they are frequent causes of hospital based infections and are inherently resistant to most antibiotics. Vancomycin works by binding to the terminal D-Ala-D-Ala residues of the cell wall peptidioglycan precursor. The high-level resistance to vancomycin is known as VanA and is conferred by a genes located on a transposable element which alter the terminal residues to D-Ala-D-lac thus reducing the affinity for vancomycin.

In view of the rapid emergence of multidrug-resistant bacteria, the development of antibacterial agents with novel modes of action that are effective against the growing number of resistant bacteria, particularly the vancomycin resistant enterococci and β-lactam antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus, is of utmost importance.

BRIEF DESCRIPTION OF THE INVENTION

This invention is based on the finding that certain hydroxamic acid and N-formyl hydroxylamine derivatives have antibacterial activity, and makes available a new group of antibacterial agents. It has been found that the compounds with which this invention is concerned are antibacterial with respect to a range of bacteria, with potency against Gram-positive organisms generally being greater than against Gram-negatives. Many of the compounds of the invention show activity against bacteria responsible for respiratory infections, such as Streptococcus pneumoniae and Haemophilus influenzae.

Although it may be of interest to establish the mechanism of action of the compounds with which the invention is concerned, it is their ability to inhibit bacterial growth that makes them useful. However, it is presently believed that their antibacterial activity is due, at least in part, to intracellular inhibition of bacterial polypeptide deformylase (PDF; EC 3.5.1.31).

All ribosome-mediated synthesis of proteins starts with a methionine residue. In prokaryotes the methionyl moiety carried by the initiator tRNA is N-formylated prior to its incorporation into a polypeptide. Consequently, N-formylmethionine is always present at the N-terminus of a nascent bacterial polypeptide. However, most mature proteins do not retain the N-formyl group or the terminal methionine residue. Deformylation is required prior to methionine removal, since methionine aminopeptidase does not recognise peptides with an N-terminal formylmethionine residue (Solbiati et al., J. Mol. Biol. 290:607-614, 1999). Deformylation is, therefore, a crucial step in bacterial protein biosynthesis and the enzyme responsible, PDF, is essential for normal bacterial growth. Although the gene encoding PDF (def) is present in all pathogenic bacteria for which sequences are known (Meinnel et al., J. Mol. Biol, 266:939-49, 1997), it has no eukaryotic counterpart, making it an attractive target for antibacterial chemotherapy.

The isolation and characterisation of PDF has been facilitated by an understanding of the importance of the metal ion in the active site (Groche et al., Biophys. Biochem. Res. Commun., 245:324-6, 1998). The Fe²⁺ form is highly active in vivo but is unstable when isolated due to oxidative degradation (Rajagopalan et al., J. Biol. Chem. 273:22305-10, 1998). The Ni²⁺ form of the enzyme has specific activity comparable with the ferrous enzyme but is oxygen-insensitive (Ragusa et al., J. Mol. Biol. 1998, 280:515-23, 1998). The Zn²⁺ enzyme is also stable but is almost devoid of catalytic activity (Rajagopalan et al., J. Am. Chem. Soc. 119:12418-12419, 1997).

Several X-ray crystal structures and NMR structures of E. coli PDF, with or without bound inhibitors, have been published (Chan et al., Biochemistry 36:13904-9, 1997; Becker et al., Nature Struct. Biol. 5:1053-8, 1998; Becker et al., J. Biol. Chem. 273:11413-6, 1998; Hao et al., Biochemistry, 38:4712-9, 1999; Dardel et al., J. Mol. Biol. 280:501-13, 1998; O'Connell et al., J. Biomol. NMR, 13:311-24, 1999), indicating similarities in active site geometry to metalloproteinases such as thermolysin and the metzincins.

Recently the substrate specificity of PDF has been extensively studied (Ragusa et al., J. Mol. Biol. 289:1445-57, 1999; Hu et al., Biochemistry 38:643-50, 1999; Meinnel et al., Biochemistry, 38:4287-95, 1999). These authors conclude that an unbranched hydrophobic chain is preferred at P1′, while wide variety of P2′ substituents are acceptable and an aromatic substituent may be advantageous at the P3′ position. There have also been reports that small peptidic compounds containing an H-phosphonate (Hu et al., Bioorg. Med. Chem. Lett., 8:2479-82, 1998) or thiol (Mainnel et al., Biochemistry, 38:4287-95, 1999) metal binding group are micromolar inhibitors of PDF. Peptide aldehydes such as calpeptin (N-Cbz-Leu-norleucinal) have also been shown to inhibit PDF (Durand et al., Arch. Biochem. Biophys., 367:297-302, 1999). However, the identity of the metal binding group and its spacing from the rest of the molecule (“recognition fragment”) has not been studied extensively. Furthermore, non-peptidic PDF inhibitors, which may be desirable from the point of view of bacterial cell wall permeability or oral bioavailability in the host species, have not been identified.

RELATED PRIOR ART

Certain N-formyl hydroxylamine derivatives have previously been claimed in the patent publications listed below, although very few examples of such compounds have been specifically made and described:

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)

WO 98/38179
(GlaxoWellcome)

WO 98/47863
(Labs Jaques Logeais)

The pharmaceutical utility ascribed to the N-formyl hydroxylamine derivatives in those publications is the ability to inhibit matrix metalloproteinases (MMPs) and in some cases release of tumour necrosis factor (TNF), and hence the treatment of diseases or conditions mediated by those enzymes, such as cancer and rheumatoid arthritis.

In addition to these, U.S. Pat. No. 4,738,803 (Roques et al.) also discloses N-formyl hydroxylamine derivatives, however, these compounds are disclosed as enkephalinase inhibitors and are proposed for use as antidepressants and hypotensive agents. Also, WO 97/38705 (Bristol-Myers Squibb) discloses certain N-formyl hydroxylamine derivatives as enkephalinase and angiotensin converting enzyme inhibitors.

Our copending International Patent Application No. WO 99/39704 describes and claims, inter alia, the use of a compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof in the preparation of an antibacterial composition:
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wherein R₁represents hydrogen, C₁-C₆alkyl or C₁-C₆alkyl substituted by one or more halogen atoms; R₂represents a substituted or unsubstituted C₁-C₆alkyl, cycloalkyl(C₁-C₆alkyl)- or aryl(C₁-C₆alkyl)- group; and A represents a group of formula (IA), or (IB):

wherein R₄represents the side chain of a natural or non-natural alpha amino acid, and R₅and R₆when taken together with the nitrogen atom to which they are attached form an optionally substituted saturated heterocyclic ring of 3 to 8 atoms which ring is optionally fused to a carbocyclic or second heterocyclic ring.

Very many hydroxamic acid derivatives are known. Many have been disclosed as having matrix metalloproteinase (MMP) inhibitory activity, and thus to be potentially useful for the treatment of diseases mediated by MMPs, for example cancer, arthritides, and conditions involving tissue remodeling such as wound healing, and restenosis. In addition our International Patent Application No. WO 99/59568 describes the use of analogues of the N-formylhydroxylamine derivatives of WO 99/39704 (wherein the N-formylhydroxylamine group is replaced by a hydroxamic acid group) in the preparation of an antibacterial composition.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a group of antibacterially active hydroxamic acid and and N-formyl hydroxylamine compounds which differ in structure from those of International Patent Applications Nos. WO 99/59568 and WO 99/39704, principally in the nature of the —NR₅R₆group (see formulae (I), (IA) and (IB) above and the hydroxamic acid analogues thereof). In those applications, the term “optionally substituted” as used in relation to the saturated heterocyclic ring formed by R₅, R₆and the nitrogen to which they are attached is defined as meaning certain specific substituents. In the present compounds, the group —NR₅R₆is also an optionally substituted saturated heterocyclic ring of 3 to 8 atoms which ring is optionally fused to a carbocyclic or second heterocyclic ring, but the substituents are different from those permitted by WO 99/59568 and WO 99/39704. The group —NR₅R₆of the N-formyl hydroxylamines and hydroxamic acids of the invention is also believed to distinguish the present compounds from those known in the MMP, TNF, ACE, and enkephalinase inhibitor art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of formula (II), or a pharmaceutically or veterinarily acceptable salt, hydrate or solvate thereof
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wherein

- Q represents a radical of formula —N(OH)CH(═O) or formula —C(═O)NH(OH);
- R₁represents hydrogen, C₁-C₆alkyl or C₁-C₆alkyl substituted by one or more halogen atoms, or, except when Q is a radical of formula —N(OH)CH(═O), a hydroxy, C₁-C₆alkoxy, C₁-C₆alkenyloxy, amino, C₁-C₆alkylamino, or di-(C₁-C₆alkyl)amino group;
- R₂represents a substituted or unsubstituted C₁-C₆alkyl, cycloalkyl(C₁-C₆alkyl)- or aryl(C₁-C₆alkyl)- group;
- and A represents a group of formula (IIA), or (IIB):
  
  wherein R₄represents the side chain of a natural or non-natural alpha amino acid, and R₅and R₆when taken together with the nitrogen atom to which they are attached form a saturated heterocyclic first ring of 5 to 7 atoms which is optionally fused to a saturated or unsaturated carbocyclic or heterocyclic second ring of 5 to 7 atoms; characterised in that
(a) the said second ring is substituted by (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₈)alkoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, halo, amino, trifluoromethyl, oxo, nitro, —COOH, —CONH₂—COR^A, —COOR^A, —NHCOR^A, —CONHR^A, —NHR^A, —NR^AR^B, or —CONR^AR^Bwherein R^Aand R^Bare independently a (C₁-C₆)alkyl group; and/or
(b) the said first or second ring is substituted by a group of formula (IIC), provided that the first ring is not substituted by phenoxy, benzyl or benzyl substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, phenoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, amino, halo, trifluoromethyl, nitro, —COOH, —CONH₂—COR^A, —COOR^A, —NHCOR^A, —CONHR^A, —NHR^A, —NR^AR^B, or —CONR^AR^Bwherein R^Aand R^Bare independently a (C₁-C₆)alkyl group,

wherein
- m, p and n are independently 0 or 1;
- Z represents, a hydroxy group, or a phenyl or heterocyclic ring of 5 to 7 atoms which is optionally fused to a saturated or unsaturated carbocyclic or heterocyclic second ring of 5 to 7 atoms
- Alk¹and Alk²independently represent divalent C₁-C₃alkylene radicals;
- X represents —O—, —S—, —S(O)—, —S(O₂)—, —C(═O)—, —NH—, —NR₇— where R₇is C₁-C₃alkyl;
  
  and wherein
- Alk¹, Alk²and Z when Z is not a hydroxy group independently are optionally substituted by
  - (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl,
  - phenyl, or halophenyl,
  - trifluoromethyl,
  - monocyclic 5 or 6-membered heterocyclic,
  - benzyl, or halophenylmethyl,
  - hydroxy, phenoxy, (C₁-C₆)alkoxy, or hydroxy(C₁-C₆)alkyl,
  - mercapto, (C₁-C₆)alkylthio or mercapto(C₁-C₆)alkyl,
  - oxo,
  - nitro,
  - cyano (—CN)
  - halo (bromo, chloro, fluoro, or iodo)
  - —COOH, or —COOR^A,
  - —CONH₂, —CONHR^A, or —CONR^AR^B
  - —COR^A, —SO₂R^A,
  - —NHCOR^A,
  - —NH₂, —NHR^A, or —NR^AR^B, wherein R^Aand R^Bare independently a (C₁-C₆) alkyl group, R^Aand R^Btaken together with the nitrogen atom to which they are attached form a 5- or 6-membered heterocyclic ring which may be substituted by (C₁-C₃)alkyl, hydroxy, or
- hydroxy(C₁-C₃)alkyl.

A subset of compounds of the invention consists of those of formula (II) as defined above wherein:

(a) the said second ring is substituted by (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₁-C₈)alkoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, amino, trifluoromethyl, oxo, nitro, —COOH, —CONH₂—COR^A, —COOR^A, —NHCOR^A, —CONHR^A, —NHR^A, —NR^AR^B, or —CONR^AR^Bwherein R^Aand R^Bare independently a (C₁-C₆)alkyl group; and/or
(b) the said first or second ring is substituted by a group of formula (IIC), provided that the first ring is not substituted by phenoxy, benzyl or benzyl substituted by (C₁-C₆)alkyl, (C₁-C₆)alkoxy, phenoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, amino, halo, trifluoromethyl, nitro, —COOH, —CONH₂—COR^A, —COOR^A, —NHCOR^A, —CONHR^A, —NHR^A, —NR^AR^B, or —CONR^AR^Bwherein R^Aand R^Bare independently a (C₁-C₆)alkyl group,

wherein
- m, p and n are independently 0 or 1;
- Z represents, a hydroxy group, or a phenyl or heterocyclic ring of 5 to 7 atoms which is optionally fused to a saturated or unsaturated carbocyclic or heterocyclic second ring of 5 to 7 atoms
- Alk¹and Alk²independently represent divalent C₁-C₃alkylene radicals;
- X represents —O—, —S—, —S(O)—, —S(O₂)—, —C(═O)—, —NH—, —NR₇— where R₇is C₁-C₃alkyl;
  
  and wherein
- Alk¹, Alk²and Z when Z is not a hydroxy group independently are optionally substituted by
  - (C₁-C₆)alkyl, (C₂-C₆)alkenyl, or (C₂-C₆)alkynyl,
  - phenyl, or halophenyl,
  - trifluoromethyl,
  - monocyclic 5 or 6-membered heterocyclic,
  - benzyl,
  - hydroxy, phenoxy, or (C₁-C₆)alkoxy,
  - mercapto, or (C₁-C₆)alkylthio,
  - oxo,
  - nitro,
  - —COOH, or —COOR^A,
  - —CONH₂, —CONHR^A, or —CONR^AR^B
  - —COR^A,
  - —NHCOR^A,
  - —NH₂, —NHR^A, or —NR^AR^B, wherein R^Aand R^Bare independently a (C₁-C₆) alkyl group.

In another aspect, the invention provides a method for the treatment of bacterial infections in humans and non-human mammals, which comprises administering to a subject suffering such infection an antibacterially effective dose of a compound of formula (II) as defined above.

In a further aspect of the invention there is provided a method for the treatment of bacterial contamination by applying an antibacterially effective amount of a compound of formula (II) as defined above to the site of contamination.

The compounds of formula (II) as defined above may be used as component(s) of antibacterial cleaning or disinfecting materials.

On the hypothesis that the compounds (II) act by inhibition of intracellular PDF, the most potent antibacterial effect may be achieved by using compounds which efficiently pass through the bacterial cell wall. Thus, compounds which are highly active as inhibitors of PDF in vitro and which penetrate bacterial cells are preferred for use in accordance with the invention. It is to be expected that the antibacterial potency of compounds which are potent inhibitors of the PDF enzyme in vitro, but are poorly cell penetrant, may be improved by their use in the form of a prodrug, ie a structurally modified analogue which is converted to the parent molecule of formula (II), for example by enzymic action, after it has passed through the bacterial cell wall.

As used herein the term “(C₁-C₆)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.

As used herein the term “divalent (C₁-C₃)alkylene radical” means a saturated hydrocarbon chain having from 1 to 3 carbon atoms and two unsatisfied valencies.

As used herein 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. The term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

As used herein the term “C₂-C₆alkynyl” 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.

As used herein the term “cycloalkyl” means a saturated alicyclic moiety having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term “heteroaryl” refers to a 5- or 6-membered aromatic ring containing one or more heteroatoms;. Illustrative of such groups are thienyl, furyl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl.

As used herein the unqualified term “heterocyclyl” or “heterocyclic” includes “heteroaryl” as defined above, and in particular means a 5-7 membered aromatic or non-aromatic heterocyclic ring containing one or more heteroatoms selected from S, N and O, including for example, pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzofuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, maleimido and succinimido groups.

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 (C₁-C₆)alkyl, (C₁-C₆)alkoxy, hydroxy, mercapto, (C₁-C₆)alkylthio, amino, halo (including fluoro, chloro, bromo and iodo), trifluoromethyl, nitro, —COOH, —CONH₂—COR^A, —COOR^A, —NHCOR^A, —CONHR^A, —NHR^A, —NR^AR^B, or —CONR^AR^Bwherein R^Aand R^Bare independently a (C₁-C₆)alkyl group.

As used herein the terms “side chain of a natural alpha-amino acid” and “side chain of a non-natural alpha-amino acid” mean the group R^xin respectively a natural and non-natural amino acid of formula NH₂—CH(R^x)—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.

In natural alpha-amino acid side chains which contain functional substituents, for example amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups as in arginine, lysine, glutamic acid, aspartic acid, tryptophan, histidine, serine, threonine, tyrosine, and cysteine, such functional substituents may optionally be protected.

Likewise, in the side chains of non-natural alpha amino acids which contain functional substituents, for example, amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl groups, such functional substituents may optionally be protected.

The term “protected” when used in relation to a functional substituent in a side chain of a natural or non-natural alpha-amino acid means a derivative of such a substituent which is substantially non-functional. The widely used handbook by T. W. Greene and P. G. Wuts “Protective Groups in Organic Synthesis” Second Edition, Wiley, New York, 1991 reviews the subject. For example, carboxyl groups may be esterified (for example as a C₁-C₆alkyl ester), amino groups may be converted to amides (for example as a NHCOC₁-C₆alkyl amide) or carbamates (for example as an NHC(═O)OC₁-C₆alkyl or NHC(═O)OCH₂Ph carbamate), hydroxyl groups may be converted to ethers (for example an OC₁-C₆alkyl or a O(C₁-C₆alkyl)phenyl ether) or esters (for example a OC(═O)C₁-C₆alkyl ester) and thiol groups may be converted to thioethers (for example a tert-butyl or benzyl thioether) or thioesters (for example a SC(═O)C₁-C₆alkyl thioester).

There are several actual or potential 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 diastereoisomers with R or S stereochemistry at each chiral centre. The invention includes all such diastereoisomers and mixtures thereof. Currently, the preferred stereoconfiguration of the carbon atom carrying the R₂group is R; that of the carbon atom carrying the R₄group (when asymmetric) is S; and that of the carbon atom carrying the R₁group (when asymmetric) is R.

In the compounds of the invention:

- R₁may be, for example, hydrogen, methyl, or trifluoromethyl. Hydrogen is currently preferred.
- R₂may be, for example:
  - optionally substituted C₁-C₆alkyl, C₃-C₆alkenyl, C₃-C₆alkynyl or cycloalkyl;
  - phenyl(C₁-C₆alkyl)-, phenyl(C₃-C₆alkenyl)- or phenyl(C₃-C₆alkynyl)- optionally substituted in the phenyl ring;
  - cycloalkyl(C₁-C₆alkyl)-, cycloalkyl(C₃-C₆alkenyl)- or cycloalkyl(C₃-C₆alkynyl)- optionally substituted in the cycloalkyl ring;
  - heterocyclyl(C₁-C₆alkyl)-, heterocyclyl(C₃-C₆alkenyl)- or heterocyclyl(C₃-C₆alkynyl)- optionally substituted in the heterocyclyl ring; or
  - CH₃(CH₂)_pO(CH₂)_q— or CH₃(CH₂)_pS(CH₂)_q—, wherein p is 0, 1, 2 or 3 and q is 1, 2 or 3.

Specific examples of R₂groups include

- methyl, ethyl, n- and iso-propyl, n- and iso-butyl, n-pentyl, iso-pentyl 3-methyl-but-1-yl, n-hexyl, n-heptyl, n-acetyl, n-octyl, methylsulfanylethyl, ethylsulfanylmethyl, 2-methoxyethyl, 2-ethoxyethyl, 2-ethoxymethyl, 3-hydroxypropyl, allyl, 3-phenylprop-3-en-1-yl, prop-2-yn-1-yl, 3-phenylprop-2-yn-1-yl, 3-(2-chlorophenyl)prop-2-yn-1-yl, but-2-yn-1-yl, cyclopentyl, cyclohexyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, furan-2-ylmethyl, furan-3-methyl, tetrahydrofuran-2-ylmethyl, tetrahydrofuran-2-ylmethyl, piperidinylmethyl, phenylpropyl, 4-chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl, benzyl, 4-chlorobenzyl, 4-methylbenzyl, and 4-methoxybenzyl.
- Presently preferred groups at R₂are (C₁-C₆)alkyl-, cycloalkylmethyl-, (C₁-C₃)alkyl-S—(C₁-C₃)alkyl-, or (C₁-C₃)alkyl-O—(C₁-C₃)alkyl-, especially n-propyl, n-butyl, n-pentyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl or cyclohexylethyl.

R₄may be, for example

- the characterising group of a natural α amino acid, for example benzyl, or 4-methoxyphenylmethyl, 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]_nR₉where Alk is a (C₁-C₆)alkylene or (C₂-C₆)alkenylene group optionally interrupted by one or more —O—, or —S— atoms or —N(R₁₂)— groups [where R₁₂is a hydrogen atom or a (C₁-C₆)alkyl group], n is 0 or 1, and R₉is hydrogen or an optionally substituted phenyl, aryl, heterocyclyl, cycloalkyl or cycloalkenyl group or (only when n is 1) R₉may additionally be hydroxy, mercapto, (C₁-C₆)alkylthio, amino, halo, trifluoromethyl, nitro, —COOH, —CONH₂—COOR^A, —NHCOR^A, —CONHR^A, —NHR^A, —NR^AR^B, or —CONR^AR^Bwherein R^Aand R^Bare independently a (C₁-C₆)alkyl group; or
- a benzyl group substituted in the phenyl ring by a group of formula —OCH₂COR₆where R₆is hydroxyl, amino, (C₁-C₆)alkoxy, phenyl(C₁-C₆)alkoxy, (C₁-C₆)alkylamino, di((C₁-C₆)alkyl)amino, phenyl(C₁-C₆)alkylamino; or
- a heterocyclic(C₁-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 (C₁-C₆)alkyl, hydroxy, formyl, amino, (C₁-C₆)alkylamino, di-(C₁-C₆)alkylamino, mercapto, (C₁-C₆)alkylthio, hydroxy(C₁-C₆)alkyl, mercapto(C₁-C₆)alkyl or (C₁-C₆)alkylphenylmethyl; or
- a group —CR_aR_bR_cin which:
  - each of R_a, R_band R_cis independently hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, (C₃-C₆)cycloalkyl; or
  - R_cis hydrogen and R_aand R_bare independently phenyl or heteroaryl such as pyridyl; or
  - R_cis hydrogen, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or (C₃-C₆)cycloalkyl, and R_aand R_btogether 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_band R_ctogether with the carbon atom to which they are attached form a tricyclic ring (for example adamantyl); or
  - R_aand R_bare each independently (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, phenyl(C₁-C₆)alkyl, or a group as defined for R_cbelow other than hydrogen, or R_aand R_btogether with the carbon atom to which they are attached form a cycloalkyl or heterocyclic ring, and R_cis hydrogen, —OH, —SH, halogen, —CN, —CO₂H, (C₁-C₆)perfluoroalkyl, —CH₂OH, —CO₂(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —O(C₂-C₆)alkenyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)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)alkyl, —CONH₂, —CONH(C₁-C₆)alkyl, —CONH(C₁-C₆alkyl)₂, —CHO, —CH₂OH, (C₁-C₄)perfluoroalkyl, —O(C₁-C₆)alkyl, —S(C₁-C₆)alkyl, —SO(C₁-C₆)alkyl, —SO₂(C₁-C₆)alkyl, —NO₂, —NH₂, —NH(C₁-C₆)alkyl, —N((C₁-C₆)alkyl)₂, —NHCO(C₁-C₆)alkyl, (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, (C₄-C₈)cycloalkenyl, phenyl or benzyl.

Examples of particular R₄groups include methyl, ethyl, benzyl, 4-chlorobenzyl, 4-hydroxybenzyl, phenyl, cyclohexyl, cyclohexylmethyl, pyridin-3-ylmethyl, tert-butoxymethyl, naphthylmethyl, iso-butyl, sec-butyl, tert-butyl, 1-benzylthio-1-methylethyl, 1-methylthio-1-methylethyl, 1-mercapto-1-methylethyl, 1-methoxy-1-methylethyl, 1-hydroxy-1-methylethyl, 1-fluoro-1-methylethyl, hydroxymethyl, 2-hydroxyethyl, 2-carboxyethyl, 2-methylcarbamoylethyl, 2-carbamoylethyl, and 4-aminobutyl. Presently preferred R₄groups include tert-butyl, iso-butyl, benzyl, isopropyl and methyl.

R₅and R₆taken together with the nitrogen atom to which they are attached form a saturated 5- to 7-membered monocyclic N-heterocyclic first ring which is attached via the N atom, and which is optionally fused to a saturated or unsaturated carbocyclic or heterocyclic second ring of 5 to 7 atoms. One or more additional ring hetero atoms such as nitrogen may be present in the first ring. Examples of such first rings are 1-pyrrolidinyl, piperidin-1-yl, 1-piperazinyl, hexahydro-1-pyridazinyl, morpholin-4-yl, tetrahydro-1,4-thiazin-4-yl, tetrahydro-1,4-thiazin-4-yl 1-oxide, tetrahydro-1,4-thiazin-4-yl 1,1-dioxide, hexahydroazipino, thiomorpholino, diazepino, thiazolidinyl or octahydroazocino. Presently preferred are piperidin-1-yl and 1-piperazinyl. The substituent (IIC) may be present on a ring carbon atom or a ring nitrogen atom of the first or second rings.

In the substituent (IIC) (from whose definition benzyl, certain substituted benzyls, and phenoxy are excluded) Alk¹and Alk²may independently represent, for example —(CH₂)— or —(CH₂CH₂)—. In the case where m is 0 and p is 1, X may be, for example —C(═O)— or —S(O₂)—. In such cases n may be 0 or 1, and when the —NR₅R₆first ring contains a second ring nitrogen, the —C(═O)— or —S(O₂)— of (IIC) may be linked to that ring nitrogen in an amide or sulphonamide bond.

In the substituent (IIC) m, n and p may all be 0, so that the group Z is directly linked to the —NR₅R₆first ring.

In a preferred subset of the compounds of the invention, the substituent (IIC) has the formula —CH₂Z, —OZ, or —(C═O)Z wherein (subject to the exclusion of benzyl, certain substituted benzyls, and phenoxy) Z is a phenyl, 3,4-methylenedioxyphenyl, morpholinyl, pyrimidinyl, 1,2,3-thiadiazolyl, 1,4-thiazolyl, benzofuranyl, furanyl, thienyl, pyranyl, pyrrolyl, pyrazolyl, isoxazolyl, or pyridyl ring which may optionally be substituted as specified. In particular, Z may be a phenyl, 3,4-methylenedioxyphenyl, morpholinyl, pyrimidin-2-yl, 1,2,3-thiadiazol-5-yl, 1,4-thiazol-5-yl, benzofuran-2-yl, 2- or 3-furanyl, 2- or 3-thienyl, 2- or 3-pyranyl, 2-, 3- or 4-pyrrolyl, 3-, 4- or 5-pyazolyl, 3-, 4- or 5-isoxazolyl, or 2-, 3- or 4-pyridyl ring any of which may optionally be substituted as specified in the broad description of the compounds of the invention.

In the compounds of formula (II) as defined above wherein Q is a radical of formula —C(═O)NH(OH) the radicals R₁, R₂, and A may be any of those disclosed above in relation to compounds (II) wherein Q is a radical of formula —N(OH)CH(═O). However, in addition, R₁may be, for example, a hydroxy, methoxy, ethoxy, n-propyloxy, allyloxy, amino, methylamino, dimethylamino, ethylamino, or diethylamino group.

Specific examples of substituents (IIC) include those present in the compounds specifically named, and/or exemplified herein.

Examples of specific compounds of the invention are those of the Examples herein. In those Examples, where a compound of formula (II) above wherein Q is an N-formylhydroxylamine radical —N(OH)CH(═O) is disclosed, it is to be understood that the equivalent compound wherein Q is a hydroxamate radical —C(═O)NH(OH) is also a specific compound of the invention, and vice versa.

Preferred compounds of the invention include those selected from the group consisting of compounds of formulae (IID)-(IIG) and (IIW)-(IIZ):
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wherein

- R₂is n-propyl, n-butyl, n-pentyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl or cyclohexylethyl;
- R₄is tert-butyl, iso-butyl, benzyl or methyl;
- Y is —CH₂—, —O— or —(C═O)—; and
- Z is a phenyl, 3,4-methylenedioxyphenyl, morpholinyl, pyrimidinyl, 1,2,3-thiadiazolyl, 1,4-thiazolyl, benzofuranyl, furanyl, thienyl, pyranyl, pyrrolyl, pyrazolyl, isoxazolyl, or pyridyl ring; in particular, a phenyl, 3,4-methylenedioxyphenyl, morpholinyl, pyrimidin-2-yl, 1,2,3-thiadiazol-5-yl, 1,4-thiazol-5-yl, benzofuran-2-yl, 2-or 3-furanyl, 2- or 3-thienyl, 2- or 3-pyranyl, 2-, 3- or 4-pyrrolyl, 3-, 4- or 5-pyazolyl, 3-, 4- or 5-isoxazolyl, or 2-, 3- or 4-pyridyl ring, which may optionally be substituted as specified in the general description of compounds of the invention.

Particular compounds of the invention, preferred for their potency against organisms which infect the respiratory system, include N-[1S-(4-benzo[1,3]dioxol-5-ylmethyl-piperazine-1-carbonyl)-2,2-dimethyl-propyl]-2R-cyclopentylmethyl-3-(formyl-hydroxyamino)-propionamide and N-[1S-(4-Benzo[1,3]dioxol-5-ylmethyl-piperazine-1-carbonyl)-2,2-dimethyl-propyl]-2R-cyclopentylmethyl-N-hydroxy-succinamide Compounds of the invention in which Q is an N-formylhydroxyamino group may be prepared by deprotecting an O-protected N-formyl-N-hydroxyamino compound of formula (III):
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in which R₁, R₂, and A are as defined in general formula (I) and R₂₅is a hydroxy protecting group removable to leave a hydroxy group by hydrogenolysis or hydrolysis. Benzyl is a preferred R₂₅group for removal by hydrogenolysis, and tert-butyl and tetrahydropyranyl are preferred groups for removal by acid hydrolysis.

Compounds of the invention in which Q is a hydroxamic acid group may be prepared by reacting the parent compound wherein Q is a carboxylic acid group (IIIA)
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with hydroxylamine or an N- and/or O-protected hydroxylamine, and thereafter removing any O- or N-protecting groups.

Compounds of formula (III) or (IIIA) may be prepared by causing an acid of formula (IV) or (IVC) or an activated derivative thereof to react with an amine of formula (IVA) or (IVB)
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wherein R₁R₂, R₄, R₅, and R₆are as defined in general formula (II) except that any substituents in R₁R₂, R₄, R₅, and R₆which are potentially reactive in the coupling reaction may themselves be protected from such reaction, and R₂₅is as defined in relation to formula (III) above, and optionally removing protecting groups R₁R₂, R₄, R₅, and R₆.

Compounds of formula (IVA), (IVB) and (IVC) are prepared by standard literature methods, and many are commercially available.

Compounds of formula (IV) may be prepared by N-formylation, for example using acetic anhydride and formic acid, or 1-formylbenzotriazole, of compounds of formula (V)
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wherein R₁, R₂and R₂₅are as defined in relation to formula (III) and Y is either a chiral auxiliary or an OR₂₆group wherein R₂₆is hydrogen or a hydroxy protecting group. In the case where Y is an OR₂₆group or a chiral auxiliary the hydroxy protecting group or auxiliary is removed after the formylation step to provide the compound of formula (IV). Suitable chiral auxiliaries include substituted oxazolidinones which may be removed by hydrolysis in the presence of base.

A compound of general formula (V) may be prepared by reduction of an oxime of general formula (VII)
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wherein R₁, R₂, and R₂₅are as defined above, and Y is either an OR₂₆group as defined above or a chiral auxiliary. Reducing agents include certain metal hydrides (eg sodium cyanoborohydride in acetic acid, triethylsilane or borane/pyridine) and hydrogen in the presence of a suitable catalyst. Following the reduction when the group Y is a chiral auxiliary it may be optionally converted to a OR₂₆group.

A compound of general formula (VII) can be prepared by reaction of a β-keto carbonyl compound of general formula (VIII)
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wherein R₁, R₂, and Y are as defined above, with an O-protected hydroxylamine.

β-keto carbonyl compounds (VIII) may be prepared in racemic form by formylation or acylation of a carbonyl compound of general formula (IX)
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wherein R₂and Y are as defined above, with a compound of general formula (X)

wherein R₁is as defined above and Q is a leaving group such as halogen or alkoxy, in the presence of a base.

The Examples herein provide further details of routes and methods for the preparation of compounds of the 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.

Compositions with which the invention is concerned may be prepared for administration by any route consistent with the pharmacokinetic properties of the active ingredient(s).

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.

Safe and effective dosages for different classes of patient and for different disease states will be determined by clinical trial as is required in the art. 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 examples illustrate embodiments of the invention. Note that the “Preparative Example A” does not describe the preparation of a compound of the invention, but is included to provide details of synthetic routes and methods for the preparation of compounds of the invention.

¹H and ¹³C NMR spectra were recorded using a Bruker DPX 250 spectrometer at 250.1 and 62.9 MHz, respectively. Mass spectra were obtained using a Perkin Elmer Sciex API 165 spectrometer using both positive and negative ionisation modes. Infra-red spectra were recorded on a Perkin Elmer PE 1600 FTIR spectrometer. Analytical HPLC was performed on a Beckman System Gold, using Waters Nova Pak C18 column (150 mm, 3.9 mm) with 20 to 90% solvent B gradient (1 ml/min) as the mobile phase. [Solvent A: 0.05% TFA in 10% water 90% methanol; Solvent B: 0.05% TFA in 10% methanol 90%], detection wavelength at 230 nm. Preparative HPLC was performed on a Gilson autoprep instrument using a C18 Waters delta prep-pak cartridge (15 μm, 300 A, 25 mm, 10 mm) with 20 to 90% solvent B gradient (6 ml/min) as the mobile phase. [Solvent A water; Solvent B: methanol], UV detection was at 230 nm.

The following abbreviations have been used throughout:

DCM
Dichloromethane

DEAD
Diethyl-azo-dichlorocarboxylate

EDC
N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride

HOAt
1-Hydroxy-7-aza-benzotriazole

HOBt
1-Hydroxybenzotriazole

HPLC
High performance liquid chromatography

LRMS
Low resolution mass spectrometry

NMR
Nuclear magnetic resonance

RT
Retention Time

TLC
Thin layer chromatography

TFA
Trifluoroacetic acid

THF
Tetrahydrofuran

EXAMPLE 1

2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid {1S-[4-(4-methoxy-benzoyl)-piperidine-1-carbonyl]-2,2-dimethyl-propyl}-amide
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The title compound was prepared as detailed below (see also Scheme 1)
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Reagents and conditions: A. piperidine, HCHO, EtOH,80° C., O/N; B. ^tBuCOCl, 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. formic acetic anhydride, Et₂N, THF; F. PfpOH, EDC, HOBt, THF; G. amine, CH₂Cl₂; H. cyclohexene, Pd/C, EtOH.

Step A: 2-Butyl acrylic acid

To a solution of n-butylmalonic acid (17.2 g, 107 mmol) in ethanol (200 ml) was added piperidin (12.76 ml, 129 mmol) and 37% aq. formaldehyde (40.3 ml, 538 mmol). The solution was heated to 80° C. during which time a precipitate appeared and 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 (200 ml), 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 (13.37 g, 97%). ¹H-NMR; δ (CDCl₂), 6.29 (1H, s), 5.65 (1H, s), 2.34-2.28 (2H, m), 1.54-1.26 (4H, m), 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 (21.5 g, 168 mmol) was dissolved in dry THF (500 ml) and cooled to −78° C. under a blanket of argon. Triethylamine (30 ml, 218 mmol) and pivaloyl chloride (21 ml, 168 mmol) 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 dissolved in dry THF (500 ml) and cooled to −78° C. under a blanket of argon. n-Butyllithium (2.4 M solution in hexanes, 83 ml, 200 mmol) was added slowly and the mixture was stirred for 30 minutes at room temperature. The resulting anion was transferred via a cannula into the original reaction vessel. The mixture was allowed to warm to room temperature and was stirred overnight at room temperature. The reaction was quenched with 1 M potassium hydrogen carbonate (200 ml) 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 sulfate, filtered and concentrated under reduced pressure to give an orange oil. TLC analysis revealed the presence of unreacted chiral auxiliary in addition to the required product. A portion of the material (30 g) was dissolved in dichloromethane and flushed through a silica pad to give pure title compound as a yellow oil (25.3 g). ¹H-NMR; δ (CDCl₂), 7.31-7.19 (5H, m), 5.41 (2H,s), 4.51 (1H, dd, J=9.7 & 4.2 Hz), 3.32 (1H, dd, J=14.2 & 4.2 Hz), 2.82 (1H, dd, J=14.2 & 9.7 Hz), 2.40-2.34 (2H, m), 1.48-1.32 (4H, m), 1.43 (3H, s), 1.27 (3H, s), 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 (19.8 g, 62.8 mmol) was mixed with O-benzylhydroxylamine (15.4 g, 126 mmol) 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 afford a pale yellow oil (25.3 g) which was shown by NMR and HPLC analysis to contain 4S-benzyl-3-[2-(benzyloxyamino-methyl)-hexanoyl]-5,5-dimethyl-oxazoldin-2-one (ca. 82% d.e.) along with a trace of starting material. The product was combined with another batch (26.9 g, 76% d.e.) and dissolved in ethyl acetate (200 ml). p-Toluenesulfonic acid (22.7 g, 119 mmol) was added and the mixture was cooled to 0° C. The title compound was obtained as a white crystalline solid by seeding and scratching. Yield: 25.2 g, (34%, single diastereoisomer). A second crop (14.7 g, 20%, single diastereoisomer) was also obtained. ¹H-NMR;δ (CDCl₃), 7.89 (2H, d, J=8.2 Hz), 7.37-7.12 (10H, m), 7.02 (2H, d, J=6.9 Hz), 5.28-5.19 (2H, m), 4.55 (1H, m), 4.23 (1H, m), 3.93 (1H, m), 3.58 (1H, m), 2.58 (1H, 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), 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 (25.2 g, 40.2 mmol) 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 (150 ml) and water (50 ml), cooled to 0° C. and treated with lithium hydroxide (1.86 g, 44.2 mmol). 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 1M 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 compounds as a colourless oil (7.4 g, 73%). ¹H-NMR;δ (CDCl₃), 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 (1H, m), 1.70-1.44 (2H, m), 1.34-1.22 (4H, m) and 0.92-0.88 (3H, m).

Step E: 2R-[(Benzyloxy-formylamino)methyl)]-hexanoic acid

To a solution of 2R-(Benzyloxyamino-methyl)-hexanoic acid (30.6 g, 0.12 mol) in dry THF (300 ml) was added formic acetic anhydride (26.8 ml, 0.31 mol) at 0° C. Triethylamine (18.5 ml, 0.13 mol) was added and the reaction was stirred for 1 h at 0° C. and 60 h at room temperature. The solvent was removed in vacuo to yield the title compound as a yellow oil (33.6 g, 99%) which was used in Step F without further purification. ¹H-NMR; (CDCl₃, rotamers), 8.20-8.08 (0.7H, br s), 8.07-7.92 (0.3H, br s), 7.50-7.25 (5H, br m), 5.07-4.70 (2H, br m), 3.95-3.52 (2H, br m), 2.90-2.66 (1H, br s), 1.72-1.20 (6H, br m), 1.00-0.78 (3H, br s). LRMS: +ve ion 280 [M+1].

Step F: 2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid pentafluorophenyl ester

To a solution of 2R-[(Benzyloxy-formylamino)-methyl)]-hexanoic acid (7.8 g, 19.9 mmol) in dry THF (500 ml) was added pentafluorophenol (44.3 g, 0.24 mol), EDC (27.7 g, 0.14 mol) and HOBt (16.2 g, 0.12 mol). The reaction was stirred overnight at room temperature. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate, washed successively with 1 M sodium carbonate (3×500 ml) and water (1×500 ml), dried over anhydrous magnesium sulfate and filtered. The solvent was removed in vacuo to yield a yellow oil (60 g) that was purified by flash chromatography (5:1, hexane:ethyl acetate→1:2 hexane:ethyl acetate) to yield a clear oil (42.0 g, 79%). ¹H-NMR; δ (CDCl₃, rotamers), 8.20-8.09 (0.7H, br s), 8.09-7.92 (0.3H, br s), 7.60-7.21 (5H, br m), 5.00-4.70 (2H, br m), 4.04-3.72 (2H, br m), 3.18-3.00 (1H, br s), 1.85-1.57 (2H, br m), 1.50-1.26 (4H, br m), 1.00-0.82 (3H, br m); LRMS: 466 [M+H].

Step G: 2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid {1S-[4-(4-methoxy-benzoyl)-piperidine-1-carbonyl]-2,2-dimethyl-propyl}-amide

2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid pentafluorophenyl ester (231 mg, 0.52 mmol) and 2S-amino-1-[4-(4-methoxy-benzoyl)-piperidin-1-yl]-3,3-dimethyl-butan-1-one (prepared from N-benzoyloxycarbonyl-L-tert-leucine) (259 mg, 0.78 mmol) were dissolved in dichloromethane (6 ml) and the mixture was stirred overnight at 27° C. An excess of Amberlyst A-21 ion exchange resin was added and the mixture stirred for 2.5 hrs before filtration. The resulting solution was then treated with methyl isocyanate polystyrene resin for 5 hrs. The mixture was filtered and solvent was removed under reduced pressure. Mass spectrometric analysis showed presence of pentafluorophenol, so the residue was dissolved in methanol (5 ml) and an excess of A-26 carbonate resin was added. The mixture was stirred overnight before filtration and removal of solvent under reduced pressure to afford the title compound as a brown oil (358 mg, 0.60 mmol). LRMS: +ve ion 594 [M+H].

Step H: 2R-[(Formyl-hydroxy-amino)-methyl]-hexanoic acid {1S-[4-(4-methoxy-benzoyl)-piperidine-1-carbonyl]-2,2-dimethyl-propyl}-amide

2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid {1S-[4-(4-methoxy-benzoyl)-piperidine-1-carbonyl]-2,2-dimethyl-propyl}-amide (358 mg, 0.60 mmol) was dissolved in ethanol (6 ml). Cyclohexene (0.60 ml) was added and the mixture placed under a blanket of argon. A suspension of 10% palladium on charcoal (40 mg) in ethyl acetate (1 ml) was added and the mixture was stirred at 70° C. for 5 hrs. The reaction mixture was cooled and the catalyst removed by filtration. The filtrate was concentrated to provide the title compound as a brown oil (294 mg, 0.58 mmol). Characterising data are provided in Table 1.

The compounds of Examples 2-13 were prepared by the synthetic route outlined in Scheme 1 and as described in detail for Example 1. Steps G and H were carried out in parallel for all examples. L-tert-leucine derivatives were prepared according to established literature methods. Purification of the final compounds, where necessary, was carried out by preparative HPLC.

TABLE 1

Mass Spec.

Example
Structure
Data
HPLC

1

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[M + H] = 504
RT = 21.7 mins 88% pure

2

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[M + H] = 487
RT = 20.1 mins 85% pure

3

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[M + H] = 505 [M − H] = 503
RT = 17.3 mins 83% pure

4

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[M + H] = 447 [M − H] = 445
RT = 21.5 mins 90% pure

5

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[M + H] = 478 [M + Na] = 500 [M + H] = 476
RT = 20.8 mins 95% pure

6

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[M + H] = 465 [M − H] = 463
RT = 20.6 mins 93% pure

7

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[M + H] = 502 [M + Na] = 524 [M − H] = 500
RT = 19.3 mins 94% pure

8

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[M + Na] = 496 [M − H] = 472
RT = 21.2 mins 91% pure

9

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[M + H] = 537 [M − H] = 535
RT = 19.8 mins 95% pure

10

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[M + H] = 475 [M − H] = 473
RT = 24.3 mins 87% pure

11

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[M + H] = 477 [M − H] = 475
RT = 20.3 mins 84% pure

12

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[M + Na] = 487 [M − H] = 463
RT = 18.7 mins 94% pure

13

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[M + H] = 502
RT = 21.0 mins 88% pure

14

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[M + H] = 512 [M − H] = 510
RT = 21.7 mins 84% pure

15

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[M + H] = 491 [M − H] = 489
RT = 18.7 mins 98% pure

16

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[M + Na] = 650 [M − H] = 626
RT = 21.5 mins 86% pure

17^#

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[M + H] = 546 [M − H] = 544
RT = 22.8 mins 88% pure

^#
¹H-NMR; δ (CD₃OD, rotamers), 8.26(0.4H, s), 7.84(0.6H, s), 7.69(2H, m), 7.39(2H, m), 6.49(0.4H, s), 6.42(0.6H, s), 5.01(0.6H, s), 4.96(0.4H, s), 4.64(0.6H, d, J=13.1 Hz), 4.51(0.4H, d, J=13.2 Hz), 4.36(0.6H, d, J=13.2 Hz), 4.29(0.4H, d, J=13.6 Hz), 3.10(1H, m), 3.43(0.4H, m), 3.32 (0.6H, m), 3.00(2H, m), 2.86(2H, m), 2.09(2H, m), 1.59(4H, m), 1.27(4H, m), 1.02(9H, m), 0.90(1.4H, s) and 0.79(1.6H, s).

The compounds of Examples 18 to 40 were prepared from 2R-[(Benzyloxy-formyl-amino)-methyl]-hexanoic acid pentafluorophenyl ester in a similar way to Example 1 but with the following modifications.

Step G: Generic experimental procedure for the synthesis of an array of amides

The coupling of amines to the pentafluorophenyl ester were carried out on a Zymate XPII laboratory robot. To a solution of the pentafluorophenyl ester (55.8 mg, 0.12 mmol) in dichoromethane (2 ml) were added the individual amines (0.25 mmol) and the reaction mixtures were stirred at room temperature for 60 h. Purification was effected by removing excess amine and pentafluorophenol using scavenger resins. The pentafluorophenol was removed using a three fold excess (0.36 mmol) of A-26 carbonate resin (3.5 mmol loading). The resin was added to the reaction mixtures and agitated for 24 h, after which time it was filtered off. The excess amines were removed using a three fold excess (0.36 mmol) of methylisocyanate polystyrene resin (1.2 mmol loading). The resin was added to the reaction mixtures and agitated for 4 h, after which time it was filtered off. The solvent was removed in vacuo using a Savant Speed Vac Plus to yield the coupled products. Yields were not calculated and the purity and integrity of each compound was verified using HPLC and LRMS.

Step H: Generic Transfer Hydrogenation Procedure

Coupled products from Step G were taken up in an ethanol-cyclohexene solution (3 ml, 10% in cyclohexene) and Pd/C (20% w/w) was added and the reactions stirred at 80° C. for 24 h. The Pd/C was filtered off and the solvent was removed in vacuo using a Savant Speed Vac Plus to yield the title compounds (Examples 18 to 40, Table 2). Yields were not calculated and the purity and integrity of each compound was verified using HPLC and LRMS.

TABLE 2

Example
Structure
Mass Spectral Data
HPLC
Purification

18

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336 (M + 1, 70)
RT 7.5 min 100%
ion exchange resin, Prep HPLC

19

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368 (M + 1, 100)
RT 21.8 min 80%
Resins, Prep HPLC

20

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392 (M + 1, 100)
RT 8.2 min 100%
Resins Prep HPLC

21

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378 (M + 1, 40) 362 ([M + 1]-Me, 100)
RT 12.0 min and 12.2 min (diastereomers) >98%
Resins Prep HPLC

22

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378 (M + 1, 100)
RT 18.5 min 100%
Resins

23

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424 (M + 1, 30), 258 ([M + 1]- [C₆H₅]₂CH₂100)
RT 17.5 min 95%
Resins

24

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333 (M + 1, 30)
RT 21.6 min 100%
Resins

25

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421 (M + 1-H₂O, 50) 437 (M − 1, 60)
RT 22.3 min 100%
Resins prep HPLC

26

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334 (M + 1, 100)
RT 17.7 min 100%
Resins

27

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458 (M + 1, 20) 258 ([M + 1]- [C₆H₅]C₆H₄ClCH, 100)
RT 26.4 min 100%
Resins prep HPLC

28

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368 (M + 1, 100)
RT 22.1 min 100%
Resins prep HPLC

29

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346 (M + 1, 100)
2 peaks, RT 3.2 min and 3.6 min 100%
Resins

30

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316 (M + 1, 100)
2 peaks, RT 3.1 min and 3.5 min 100%
Resins

31

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283 ([M + 1]-H₂O), 90)
1 peak with shoulder, RT 16.8 min
Resins

32

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402 (M + 1, 100)
RT 15.8 min >95%
Resins

33

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364 (M + 1, 100)
RT 11.7 min >95%
Resins

34

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403 (M + 1, 100)
RT 14.7 min 95%
Resins

35

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373 (M + 1, 100)
RT 14.5 min 95%
Resins

36

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460 (M + 1, 100)
RT 13.3 min >95%
Resins, Prep HPLC

37

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379 (M + 1, 100),
RT 13.6 min >95%
Resins

38

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352 (M + 1, 100)
RT 5.9 min >95%
Resins

39

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352 (M + 1, 100)
RT 11.3 min >95%
Resins

40

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362 (M + 1, 100)
15.7 min >95%
Resins

The compounds of Examples 1-40 are named as follows:

EXAMPLE 1