RU2246941C2 - Antibacterial agents - Google Patents

Abstract

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to applying compounds of the formula (I)

for preparing an antibacterial composition and veterinary composition eliciting with the enhanced activity.

EFFECT: valuable properties of agents.

4 cl, 3 tbl, 78 ex

Description

The present invention relates to the use of N-formylhydroxylamine derivatives as antibacterial agents, to a new class of said compounds, and also to pharmaceutical and veterinary compositions containing said compounds.

The level of technology.

As a rule, pathogenic bacteria are classified as either gram-positive or gram-negative. Many antibacterial agents (including antibiotics) exhibit a specific effect against a particular gram-positive or gram-negative pathogenic microorganism. Thus, antibacterial agents that are effective against both gram-positive and gram-negative pathogenic microorganisms, as a rule, are classified as agents with a wide spectrum of action.

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

A growing problem is the resistance of bacteria to many known antibacterial agents. Accordingly, there is a continuing need for the creation of alternative antibacterial agents, especially those in which the mechanism of action is significantly different from the mechanism of action of known classes.

Among gram-positive pathogens, such as Staphylococci, Streptococci, Mycobacteria, and Enterococci, resistant strains tend to evolve / multiply, which makes them especially difficult to kill. Examples of such strains are methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative Staphylococci (MRCNS), penicillin-resistant Streptococcus pneumoniae, and many Enterococcus faecium antibiotics.

Pathogenic bacteria are often resistant to antibiotic types such as aminoglycosides, β-lactams (penicillins and cephalosporins) and chloramphenicol. Resistance includes the enzymatic inactivation of antibiotics by hydrolysis or their translation into inactive derivatives.

The family of β-lactam antibiotics (penicillins and cephalosporins) is characterized by the presence of a β-lactam ring in their structure. Resistance to this family of antibiotics in clinical cultures is most often caused by the production by resistant bacteria of the enzyme “penicillinase” (β-lactamase), which hydrolyzes the b-lactam ring, thus eliminating antibacterial activity.

A tense situation has recently been created with vancomycin-resistant enterococci strains (Woodford N. 1998 Enterococci glycopeptide-resistant: decade of experiment. Journal of Medical Microbiology. 47 (10): 849-62). Vanocycin-resistant enterococci are especially dangerous due to the fact that they often cause infections that require treatment in a hospital, and at the same time they are resistant to many antibiotics. Vancomycin works by binding to terminal D-ALa-D-ALa residues of peptidioglycan cell wall precursors. Increased resistance to vancomycin is known as VanA, and it arises from genes located on the internal element, which replaces the terminal residues with D-Ala-D-lac, thus reducing the affinity for vancomycin.

In light of the rapid emergence of multi-drug resistant bacteria, the search for antibacterial agents that are effective against an increasing number of resistant bacteria, especially enterococci resistant to vancomycin and β-lactam antibiotic resistant bacteria such as methicillin resistant Staphylococcus aureus, is crucial.

SUMMARY OF THE INVENTION

The invention is based on the discovery of the fact that certain derivatives of N-formylhydroxylamine have antibacterial activity and are a new available class of antibacterial agents. It was found that the compounds that relate to the present invention have antibacterial activity against a number of gram-positive and gram-negative organisms. Moreover, it is obvious that some compounds exhibit antibacterial activity against bacteria that are resistant to commonly used antibiotics, such as vancomycin and β-lactam antibiotics, such as methicillin-resistant Staphylococcus aureus.

It is of interest to establish the mechanism of action of the compounds that relate to the present invention, i.e. their ability to inhibit the growth of bacteria, which determines the usefulness of these compounds. However, at the moment it is believed that their antibacterial activity is provided, at least in part, due to intracellular inhibition of the polypeptide deformylase enzyme (PDF).

Bacterial polypeptide deformylases (PDF) (EC 3.5.1.31) are a conservative family of metal enzymes (Review: Meinnel T, Lazennec C, Villoing S, Blanquet S, 1997, Journal of Molecular Biology 267, 749-761), which determines bacterial viability while their function is to remove the formyl group from the N-terminal methionine residue of the eubacteria proteins synthesized on the ribosomes. Mazel et al. (EMBO J. 13 (4): 914-923, 1994) recently cloned and characterized E. coliPDF. Since PDF is necessary for bacterial growth and there is no eukaryotic analogue of PDF, each of Mazel et al. (ibid). Rajagopalan et al. (J. Am. Chem. Soc. 119: 12418-12419, 1997) and Bicker et al., (J. Biol Chem. 273 (19): 11413-11416, 1998) suggested that (PDF) is an excellent antibacterial target .

Some derivatives of N-formylhydroxylamine were previously claimed in a number of patent publications, which are listed below, however, only a small number of specific compounds have been obtained 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 use of these derivatives of N-formylhydroxylamine in these publications is determined by their ability to inhibit matrix metalloproteinases (MMPs) and, in some cases, release tumor necrosis factor (TNF) and, therefore, treat diseases or conditions created by these enzymes such as cancer or rheumatoid arthritis. However, the prior art does not disclose or imply the antibacterial activity of N-formylhydroxylamine derivatives.

In addition to the above sources of information, US-A-4,738,803 (Roques et al.) Also discloses N-formyl hydroxylamine derivatives, however, these derivatives are described as enkephalinase inhibitors and are proposed for use as antidepressants and antihypertensive agents. WO 97/38705 (Bristol-Myers Squibb) also discloses certain N-formylhydroxylamine derivatives as enkephalinase inhibitors and enzymes involved in angiotensin conversion. This information source also does not disclose or imply any antibacterial activity of the derivatives of N-formylhydroxylamine.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention, there is provided the use of a compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof for the preparation of an antibacterial composition:

where R ₁ represents hydrogen, or C ₁ -C ₆ alkyl or C ₁ -C ₆ alkyl substituted with one or more halogen atoms;

R ₂ represents a group R ₁₀ - (X) _n - (ALK) _m -, where

R ₁₀ represents hydrogen or C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, cycloalkyl, aryl, or heterocyclic groups, each of which may be unsubstituted or substituted (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy, hydroxy, mercapto, (C ₁ -C ₆ ) alkylthio, amino, halogen (including fluorine, chlorine, bromine and iodine), trifluoromethyl, cyano, nitro, -COOH, -CONH ₂ , —COOR ^A , NHCOR ^A —CONHR ^A —NHR ^A , NR ^A R ^B , or CONR ^A R ^B , where R ^A and R ^B independently represent a (C ₁ -C ₆ ) alkyl group, and ALA is linear or branched divalent C ₁ -C ₆ alkylene, C ₂ -C ₆ alkenylene, or C ₂ -C ₆ alkynylene radical, which may be broken by one or more non-adjacent -NH-, -O- or -S- bonds,

X represents —NH—, —O— or —S—,

and m and n independently represent 0 or 1;

and A represents (i) a group of formula (IA), (IB), (IC) or (ID)

where R ₃ represents hydrogen and R ₄ represents the side chain of a natural or unnatural alpha amino acid, or R ₃ and R ₄ , taken together with the nitrogen and carbon atoms to which they are respectively attached, form an optionally substituted saturated heterocyclic ring from 5 to 8 atoms which are optionally fused to a carbocyclic or second heterocyclic ring,

R ₅ and R ₆ independently represent hydrogen, or optionally substituted C ₁ -C ₈ alkyl, cycloalkyl, aryl, aryl (C ₁ -C ₆ alkyl), heterocyclyl or heterocyclyl (C ₁ -C ₆ alkyl), or R ₅ and R ₆ taken together with the nitrogen atom to which they are attached form an optionally substituted saturated heterocyclic ring of 3 to 8 atoms, a ring that is optionally fused to a carbocyclic or second heterocyclic ring, and

R ₇ represents hydrogen, C ₁ -C ₆ alkyl or an acyl group.

In accordance with another aspect, the invention provides a method for treating bacterial infections in humans and other mammals, which comprises administering to a patient suffering from such an infection an antibacterial effective amount of a compound of formula (I) as defined above.

In accordance with another aspect of the present invention, there is provided a method of treating a bacterial infection by applying an antibacterial effective amount of a compound of formula (I), as defined above, to the infection site.

The compounds of formula (I), as defined above, can be used as components of a cleaning antibacterial or disinfectant.

According to a preferred embodiment, the invention in its various aspects can be used against bacteria resistant to vancomycin, quinolone, and β-lactams, as well as the infections that they cause.

Based on the hypothesis that the compounds of formula (I) act as inhibitors of intracellular PDF, the greatest antibacterial effect can be achieved if compounds are used that efficiently penetrate the bacterial cell wall. Thus, compounds that are highly active as in vitro PDF inhibitors and which penetrate bacterial cells are preferred for use in accordance with the invention. It can be expected that the antibacterial efficacy of compounds that are potential inhibitors of PDF enzymes in vitro but do not penetrate well into the cell can be improved by using them in prodrug form, for example, in the form of their structurally modified analog, which turns into the original molecule of formula (I ), for example, under the action of an enzyme after penetration through the cell wall of a bacterium.

The invention also includes new compounds of formula (I) above, or pharmaceutically or veterinarily acceptable salts thereof,

where R ₁ represents hydrogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkyl substituted with one or more halogen atoms;

R ₂ represents a group R ₁₀ - (ALK) _m -, where

R ₁₀ represents hydrogen, or C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, cycloalkyl, aryl or heterocyclic group, each of which may be unsubstituted or substituted (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkoxy, hydroxy, mercapto, (C ₁ -C ₆ ) alkylthio, amino, halogen (including fluorine, chlorine, bromine and iodine), trifluoromethyl, nitro, -COOH, -CONH ₂ , - COOR ^A , —NHCOR ^A , —CONHR ^A , —NHR ^A , —NHR ^A R ^B , or —CONR ^A R ^B , where R ^A and R ^B independently represent a (C ₁ -C ₆ ) alkyl group,

ALA is a linear or branched divalent C ₁ -C ₆ alkylene, C ₂ -C ₆ alkenylene, C ₂ -C ₆ alkynylene radical, and it can be broken with one or more non-adjacent -NH-, -O- or -S- connections, and

m represents 0 or 1;

And represents a group of formula (IA), (IB), (IC) or (ID) above, where:

R ₃ represents hydrogen and R ₄ represents the side chain of a natural or unnatural alpha amino acid, or R ₃ and R ₄ , taken together with the nitrogen and carbon atoms to which they are respectively attached, form an optionally substituted saturated heterocyclic ring of 5 to 8 atoms which is optionally fused to a carbocyclic or second heterocyclic ring,

R ₅ and R ₆ independently represent hydrogen, or optionally substituted C ₁ -C ₆ alkyl, cycloalkyl, aryl (C ₁ -C ₆ alkyl), non-aromatic heterocyclyl, or heterocyclyl (C ₁ -C ₆ alkyl), or R ₅ and R ₆ , taken together with the nitrogen atom to which they are attached, form an optionally substituted saturated heterocyclic ring of 3 to 8 atoms, which is optionally fused to a carbocyclic or second heterocyclic ring, and

R ₈ represents hydrogen, C ₁ -C ₆ alkyl or an acyl group.

Provided that (i) when A is a group of formula (IA) or (IB) and R ₂ is C ₂ -C ₅ alkyl, then R _{4 is} not a side chain of a natural alpha amino acid or a side chain of a natural alpha amino acid wherein any functional substituents are protected, any amino groups are acylated, and any carboxyl groups are esterified;

(ii) when A is a group of formula (IA) or (IB), then R _{4 is} not a bicycloarylmethyl group; and

(iii) when A is a group of formula (IA) and R ₂ is cyclopropylmethyl, cyclobutylmethyl or cyclopentylmethyl and one of R ₅ and R ₆ is hydrogen, then R _{4 is} not tert-butyl.

As used here, the term “(C ₁ -C ₆ ) alkyl” means a linear or branched chain alkyl radical containing 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 "divalent (C ₁ -C ₆ ) alkylene radical" means a saturated hydrocarbon chain containing from 1 to 6 carbon atoms and two free valencies.

The term “(C ₂ -C ₆ ) alkenyl” means a straight or branched chain alkenyl radical containing from 2 to 6 carbon atoms and containing at least one double bond with either the E or Z configuration, where possible. The term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

The term “divalent (C ₂ -C ₆ ) alkenylene radical” means a hydrocarbon chain containing from 2 to 6 carbon atoms, at least one double bond, and two unsaturated valencies.

The term "C ₂ -C ₆ alkynyl" refers to straight or branched chains of hydrocarbon groups containing from two to six carbon atoms and having in addition one triple bond. This term will include, for example, ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentinyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.

The term "divalent (C ₂ -C ₆ ) alkynylene radical" means a hydrocarbon chain containing from 2 to 6 carbon atoms, at least one triple bond, and two free valencies.

The term "cycloalkyl" means a saturated alicyclic residue containing from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “cycloalkenyl” means an unsaturated alicyclic residue containing from 3-8 carbon atoms and includes, for example, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. In the case of cycloalkenyl rings of 5-8 carbon atoms, the ring may contain more than one double bond.

The term “aryl” refers to a mono-, bi- or tricyclic carbocyclic aromatic group and to groups consisting of two covalently linked monocyclic carbocyclic aromatic groups. Illustrative of such groups are phenyl, biphenyl and naphthyl.

The term “heteroaryl” refers to a 5- or 6-membered aromatic ring containing one or more heteroatoms and optionally fused to a benzyl or pyridyl ring; and to groups consisting of two covalently bonded 5- or 6-membered aromatic rings, each of which contains one or more heteroatoms; and to groups consisting of a monocyclic carbocyclic aromatic group covalently bonded to 5- or 6-membered aromatic rings 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, 4 - ([1,2,3] -thiadiazol-4-yl) phenyl and 5-isoxazol-3-ylthienyl.

The qualitative term “heterocyclic” or “heterocyclyl” includes “heteroaryl” as defined above and, in particular, refers to a 5-7 membered aromatic or non-aromatic heterocyclic ring containing one or more heteroatoms selected from S, N and O and optionally fused with a benzene ring, including, for example, groups such as pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, benzimide o, succinimido, phthalimido and 1,3-dioxo-1,3-dihydro-isoindol-2-yl.

The term “acyl” means a group R ₂₀ C (O) -, where R ₂₀ is (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₃ -C ₇ ) cycloalkyl, phenyl, heterocyclyl, phenyl (C ₁ -C ₆ ) alkyl, heterocyclyl (C ₁ -C ₆ ) alkyl, (C ₃ -C ₇ ) cycloalkyl (C ₁ -C ₆ ) alkyl, phenyl (C ₂ -C ₆ ) alkenyl, heterocyclyl (C ₂ -C ₆ ) alkenyl, (C ₃ -C ₇ ) cycloalkyl (C ₂ -C ₆ ) alkenyl, each of the R ₂₀ groups may be substituted.

The term “bicycloarylmethyl” means (i) a methyl group substituted by a monocyclic aryl or heteroaryl group, which in turn is substituted by a monocyclic aryl or heteroaryl group, or (ii) a methyl group substituted by a monocyclic aryl and heteroaryl group which is fused to a second monocyclic aryl or heteroaryl group; and includes both unsubstituted and substituted bicycloarylmethyl. Examples of such bicycloarylmethyl groups include naphthyl, indolyl, quinolyl and isoquinolyl.

Unless expressly stated in the context where it is used, the term “substituted” with respect to any residue in this description means substituted with up to four substituents, each of which may independently be (C ₁ -C ₆ ) alkyl, benzyl, (C ₁ -C ₆ ) alkoxy, phenoxy, hydroxy, mercapto, (C ₁ -C ₆ ) alkylthio, amino, halogen (including fluorine, chlorine, bromine and iodine), trifluoromethyl, nitro, -COOH, -CONH ₂ , -COR ^A , —COOR ^A , —NHCOR ^A , —CONHR ^A , —NHR ^A , —NR ^A R ^B , or —CONR ^A R ^B , where R ^A and R ^B independently represent a (C ₁ -C ₆ ) alkyl group. When benzyl is “substituted”, its phenyl ring itself can be substituted by any of the following substituents, with the exception of benzyl.

The terms “side chain of a natural alpha amino acid” and “side chain of a non-natural alpha amino acid” mean an R ^X group of a naturally occurring and non-natural amino acid of the formula NH ₂ —CH (R ^X ) —COOH, respectively.

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

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

Similarly, in the side chains of unnatural alpha amino acids that contain functional substituents, for example, groups such as amino, carboxyl, hydroxy, mercapto, guanidyl, imidazolyl, or indolyl, such functional substituents may optionally be protected.

The term “protected”, when used with respect to a functional substituent in the side chain of a natural or unnatural alpha amino acid, means a derivative of such a substituent that is substantially non-functional. The widely known reference book of T. W. Greene and R. G. Wuts "Protective Groups in Organic Synthesis" Second Edition. Wiley, New York, 1991, covers this subject. For example, carboxyl groups can be esterified (e.g., as C ₁ -C ₆ alkyl esters), amino groups can be converted to amides (e.g., as NHCOC ₁ -C ₆ alkylamides) or carbamates (e.g., as NHC (= O) OC ₁ -C ₆ alkyl or NHC (= O) OCH ₂ Ph carbamate), hydroxyl groups can be converted to ethers (for example, OC ₁ -C ₆ alkyl or O (C ₁ -C ₆ alkyl) phenyl ethers) or esters (e.g. SC (= O) C ₁ -C ₆ alkyl ester) and thiol groups can be converted to thioethers (e.g. tert-butyl or benzylthioether) or complex e thioethers (e.g. SC (= O) C ₁ -C ₆ alkyl thioethers).

In the compounds according to the invention there are several real or potential chiral centers, since asymmetric carbon atoms are present in them. The presence of several asymmetric carbon atoms leads to an increase in the number of diastereoisomers with R or S stereochemistry of each chiral center. The invention includes all of these diastereoisomers and mixtures thereof. Generally, the most preferred stereo configuration of a carbon atom bearing an R ₂ group is the R configuration; wherein the carbon atom bearing the R ₄ group (when there is an asymmetry) has the configuration S; and the carbon atom carrying the group R ₁ (when there is asymmetry) has the configuration R.

In the compounds of formula (I), as defined above, for use according to the invention, and in the new compounds according to the invention of formula (II), as defined above, (subject to the above conditions):

R ₁ may be, for example, hydrogen, methyl, or trifluoromethyl. Hydrogen is usually most preferred.

R ₂ may be, for example:

optionally substituted C ₁ -C ₆ alkyl, C ₃ -C ₆ alkenyl, C ₃ -C ₆ alkynyl or cycloalkyl; phenyl (C ₁ -C ₆ alkyl) -, phenyl (C ₃ -C ₆ alkenyl) - or phenyl (C ₃ -C ₆ alkynyl) - optionally substituted on the phenyl ring; cycloalkyl (C ₁ -C ₆ alkyl) -, cycloalkyl (C ₃ -C ₆ alkenyl) - or cycloalkyl (C ₃ -C ₆ alkynyl) - optionally substituted on the cycloalkyl ring; heterocyclyl (C ₁ -C ₆ alkyl) -, heterocyclyl (C ₃ -C ₆ alkenyl) - or heterocyclyl (C ₃ -C ₆ alkynyl) -, optionally substituted on the heterocyclic ring; or

CH ₃ (CH ₂ ) _p O (CH ₂ ) _q - or CH ₃ (CH ₂ ) S (CH ₂ ) _q -, where p is 0.1, 2 or 3 and q is 1, 2 or 3.

Specific examples of the R ₂ groups include methyl, ethyl, n- and isopropyl, n- and isobutyl, n-pentyl, isopentyl, 3-methyl-but-1-yl, n-hexyl, n-heptyl, n-acetyl, n- octyl, methylsulfonylethyl, ethylsulfonylmethyl, 2-methoxyethyl, 2-ethoxyethyl, 2-ethoxymethyl, 3-hydroxypropyl, allyl, 3-phenylprop-3-en-1-yl, prop-2-in-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, cyclohexyl ethyl, cyclohexylpropyl , furan-2-ylmethyl, furan-3-methyl, tetrahydrofuran-2-ylmethyl, tetra agidrofuran-2-ylmethyl, piperidinylmethyl, phenylpropyl. 4-chlorophenylpropyl, 4-methylphenylpropyl, 4-methoxyphenylpropyl, benzyl, 4-chlorobenzyl, 4-methylbenzyl, and 4-methoxybenzyl.

The most preferred R ₂ groups are n-propyl, n-butyl, n-pentyl, benzyl and cyclopentylmethyl.

For R _{3, the} most preferred example is hydrogen.

R ₄ can be, for example, a characteristic group of a natural a-amino acid, for example benzyl or 4-methoxyphenylmethyl, in which any functional group can be protected, any amino group can be acylated and any carboxyl group present can be amidated; or

a group of - [ALK] _n R ₉ , where ALK is a (C ₁ -C ₆ ) alkylene or (C ₂ -C ₆ ) alkenylene group, optionally broken by one or more —O— or —S — atoms or —N (R ₁₂ ) - groups [where R ₁₂ represents a hydrogen atom or a (C ₁ -C ₆ ) alkyl group], n represents 0 or 1, and R ₉ represents hydrogen or an optionally substituted phenyl, aryl, heterocyclic, cycloalkyl or cycloalkenyl group or (only when n is 1) R ₉ may further be hydroxy, mercapto, (C ₁ -C ₆ ) alkylthio, amino, halogen, trif tormetil, nitro, -COOH, -CONH ^{_2, -COOR A, -NHCOR A, -CONHR} A, -NHR A, -NR A R B, or -CONR ^A R ^B, wherein R ^A and R ^B independently represent ( C ₁ -C ₆ ) an alkyl group; or

a benzyl group substituted on the phenyl ring with a group of the formula —OCH ₂ COR ₈ , where R ₈ is hydroxyl, amino, (C ₁ -C ₆ ) alkoxy, phenyl (C ₁ -C ₆ ) alkoxy, (C ₁ -C ₆ ) alkylamino di ((C ₁ -C ₆ ) alkyl) amino, phenyl (C ₁ -C ₆ ) alkylamino; or

heterocyclyl (C ₁ -C ₆ ) alkyl group, either unsubstituted or mono- or disubstituted in the heterocyclic ring using halogen, nitro, carboxy, (C ₁ -C ₆ ) alkoxy, cyano, (C ₁ -C ₆ ) alkanoyl , trifluoromethyl (C ₁ -C ₆ ) alkyl, hydroxy, formyl, amino, (C ₁ -C ₆ ) alkylamino, di- (C ₁ -C ₆ ) alkylamino, mercapto, (C ₁ -C ₆ ) alkylthio, hydroxy ( C ₁ -C ₆ ) alkyl, mercapto (C ₁ -C ₆ ) alkyl or (C ₁ -C ₆ ) alkyl phenylmethyl; or

a group —CR _a R _b R _c in which:

each of R _a , R _b , and R _c independently represent hydrogen, (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl, phenyl (C ₁ -C ₆ ) alkyl, (C ₃ -C ₈ ) cycloalkyl; or

R _c is hydrogen and R _a and R _b independently are phenyl or heteroaryl, such as pyridyl; or

R _c is hydrogen, (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl, phenyl (C ₁ -C ₆ ) alkyl, or (C ₃ -C ₈ ) cycloalkyl, and R _a and R _b together with the carbon atom to which they are attached form a 3-8 membered cycloalkyl or 5-6 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 each independently represent (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl, phenyl (C ₁ -C ₆ ) 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 represents hydrogen, —OH, —SH, halogen, —CN, —CO ₂ H, (C ₁ –C ₄ ) perfluoroalkyl, —CH ₂ OH, —CO ₂ (C ₁ –C ₆ ) alkyl, —O (C ₁ –C ₆ ) alkyl, —O (C ₂ -C ₆ ) alkenyl, -S (C ₁ -C ₆ ) alkyl, -SO (C ₁ -C ₆ ) alkyl, -SO ₂ (C ₁ -C ₆ ) alkyl, -S (C ₂ -C ₆ ) alkenyl , -SO (C ₂ -C ₆₎ alkenyl, -SO ₂ (C ₂ -C ₆₎ alkenyl or -QW group wherein Q Representat It wishes to set up a bond or -O-, -S-, -SO- or -SO ₂ - and W represents a phenyl, phenylalkyl, (C ₃ -C ₈₎ cycloalkyl, (C ₃ -C ₈₎ cycloalkylalkyl, (C ₄ - C ₈ ) cycloalkenyl, (C ₄ -C ₈ ) cycloalkenylalkyl, heteroaryl or heteroarylalkyl group, wherein said W group may be optionally substituted with one or more substituents independently selected from hydroxyl, halogen, —CN, —CO ₂ H, —CO ₂ (C ₁ -C ₆ ) alkyl, -CONH ₂ , -CONH (C ₁ -C ₆ ) alkyl, -CONH (C ₁ -C ₆ alkyl) ₂ , -CHO, -CH ₂ OH, (C ₁ -C ₄ ) perfluoroalkyl, -O (C ₁ -C ₆ ) alkyl, -S (C ₁ -C ₆ ) alkyl, -SO (C ₁ -C ₆ ) alkyl, -SO ₂ (C ₁ -C ₆ ) alkyl, -NO ₂ , -NH ₂ , -NH (C ₁ - C ₆ ) alkyl, -N ((C ₁ -C ₆ ) alkyl) ₂ , -NHCO (C ₁ -C ₆ ) alkyl, (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl, (C ₃ -C ₈ ) cycloalkyl, (C ₄ -C ₈ ) cycloalkenyl, phenyl or benzyl.

Examples of particular values of the R ₄ group include methyl, ethyl, benzyl, 4-chlorobenzyl, 4-hydroxybenzyl, phenyl, cyclohexyl, cyclohexylmethyl, pyridin-3-ylmethyl, tert.-butoxymethyl, naphthylmethyl, isobutyl, 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. Particularly preferred values of the R ₄ group include tert.-butyl, isobutyl, benzyl and methyl.

R ₃ and R ₄ , taken together with the nitrogen and carbon atoms to which they are respectively attached, can form an optionally substituted saturated heterocyclic ring of 5 to 8 atoms. For example, R ₃ and R ₄ can form a bridge between the nitrogen and carbon atoms to which they are attached, and this bridge is a divalent radical - (CH ₂ ) _3-6 , or - (CH ₂ ) _r —O — CH ₂ ) _s , or - (CH ₂ ) _r -S- (CH ₂ ) _s -, where r and s each independently have the values 1, 2 or 3, provided that r + s = 2,3,4 or 5.

R ₅ and R ₆ may independently be, for example, hydrogen, methyl, ethyl, tert.-butyl, cyclopentyl, cyclohexyl, 1,1,3,3-tetramethylbutyl, benzyl or 2-hydroxyethyl; or R ₅ and R ₆ taken together with the nitrogen atom to which they are attached can form a saturated 5-8 membered monocyclic N-heterocyclic ring which is attached via an N atom and which optionally contains —N (R ₁₁ ) -, where R ₁₁ is hydrogen or C ₁ -C ₆ alkyl, benzyl, acyl, or an amino protecting group, O, S, SO or SO ₂ as a ring unit, and / or is optionally substituted one or more C atoms with hydroxy, C ₁ -C ₆ alkyl, hydroxy (C ₁ -C ₆ alkyl) -, C ₁ -C ₆ alkoxy, oxo, ketalized oxo, amino, mono (C ₁ -C ₆ alkyl) amino, di (C ₁ -C ₆ alkyl) amino, to arboxy, C ₁ -C ₆ alkoxycarbonyl, hydroxymethyl, C ₁ -C ₆ alkoxymethyl, carbamoyl, mono (C ₁ -C ₆ alkyl) carbamoyl, di (C ₁ -C ₆ alkyl) carbamoyl or hydroxyimino.

Examples of such rings are substituted or unsubstituted 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 or octahydroazocino.

Examples of the above substituted rings are 2- (methylcarbamoyl) -1-pyrrolidinyl, 2- (hydroxymethyl) -1-pyrrolidinyl, 4-hydroxypiperidino, 2- (methylcarbamoyl) piperidino, 4-hydroxyiminopiperidino, 4-methoxy piperidino, 4-methyl yl, 4-benzylpiperidin-1-yl, 4-acetylpiperidin-1-yl, 4-methyl-1-piperazinyl, 4-phenyl-1-piperazinyl, 1,4-dioxa-8-azaspiro [4,5] decan- 8-yl, hexahydro-3- (methylcarbamoyl) -2-pyridazinyl and hexahydro-1- (benzyloxycarbonyl) -2-pyridazinyl, decahydroisoquinolin-2-yl, and 1,2,3.4-tetrahydroisoquinolin-2-yl.

When A is a group of formula (IA), it is particularly preferred that R _{5 is} methyl or hydrogen and R _{6 is} methyl.

R ₇ may represent, for example, hydrogen, or a group R ₂₀ C (O) -, where R ₂₀ represents a (C ₁ -C ₆ ) alkyl group, such as methyl or ethyl.

Specific examples of compounds used as antibacterial agents in accordance with the invention include those included in the experimental part of the description. Preferred new compounds of the invention include

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-dimethylcarbamoyl-ethyl) -amide and

2R (or S) - [(Formyl-hydroxy-amino) -methyl] -3-cyclopentyl-propionic acid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl) -amide

and their pharmaceutically and veterinarily acceptable salts.

Compounds related to the invention can be obtained by deprotecting the O-protected N-formyl-N-hydroxyamine compound of the formula (II):

in which R ₁ , R ₂ , and A are as defined in general formula (I) and R ₂₅ is a hydroxyl protecting group removed to liberate the hydroxy group by hydrogenolysis or hydrolysis. A benzyl group is a preferred R ₂₅ group for removal by hydrogenolysis, and tert-butyl and tetrahydropyranyl groups are preferred for removal by acid hydrolysis.

Compounds of formula (II) where A is a group of formula (IA), (IB), (IC) or (ID) can be prepared by reacting an acid of formula (III) or an activated derivative thereof with an amine of formula (IVA) , (IVB), (IVC) or (IVD) respectively

where R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ have the meanings given in the general formula (I) with the exception of the —OH group in (IVB), and any substituents in R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ , which are potentially reactive in the condensation reactions, can themselves be protected from such a reaction, and R ₂₅ has the meanings defined in accordance with formula (II) above, and optionally removing the protective groups — OH groups in (IVB) and R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ .

Compounds of formula (III) can be prepared by N-formylation, for example, using acetic anhydride and formic acid, or 1-formylbenzotriazole, compounds of formula (V)

where R ₁ , R ₂ and R ₂₅ are as defined in accordance with formula (II) and X is either an auxiliary chiral substance or an OR ₂₆ group, where R ₂₆ is hydrogen or a hydroxyl protecting group. In the case where X is an OR ₂₆ group or an auxiliary chiral substance, the hydroxyl protecting group or auxiliary chiral substance is removed after the formylation step to obtain a compound of formula (V). Suitable chiral auxiliaries include substituted oxazolidinones, which can be removed by hydrolysis in the presence of a base.

In an alternative process, compounds of general formula (II) can be prepared by N-formylation, for example, using acetic anhydride and formic acid, or 1-formylbenzotriazole, compounds of formula (VI)

where R ₁ , R ₂ , R ₂₅ and A are as defined in formula (II). Compounds of formula (VI) where A is a group of formula (IA), (IB), (IC) or (ID) can be prepared by administering an acid of general formula (VII) or an activated derivative thereof

where R ₁ , R ₂ and R ₂₅ are as defined in formula (II), in contact with an amine of formula (IVA), (IVB), (IVC) or (IVD), respectively, as defined above.

Alternatively, compounds of general formula (VI) can be prepared by reducing an oxime of general formula (VIII).

Reducing agents include certain metal hydrides (e.g. sodium cyanoborohydride in acetic acid, triethylsilane or borane / pyridine) and hydrogen in the presence of a suitable catalyst.

In an alternative process, the compounds of general formula (II), where R ₁ and R ₂ are as defined in general formula (I), R ₂₅ is a hydroxyl protecting group as defined above, and A is a group of formula (IA), where R ₃ , R ₄ , R ₅ have the meanings given in general formula (IA) and R ₆ represents hydrogen, can be obtained by using the 4-component Ugi reaction of a carboxylic acid of general formula (III) as defined above, an amine of formula (IX ), an aldehyde of formula (X) and isonitrile of formula (XI).

R ₃ -NH ₂ (IX)

R ₄ -CHO (X)

R ₅ -CN (XI)

where R ₃ , R ₄ and R ₅ are as defined above.

A compound of general formula (V) can be prepared by reducing an oxime of general formula (XI)

where R ₁ , R ₂ and R ₂₅ have the meanings as defined above, and X represents either the group OR ₂₅ as defined above, or an auxiliary chiral substance. Reducing agents include certain metal hydrides (e.g. sodium cyanoborohydride in acetic acid, triethylsilane or borane / pyridine) and hydrogen in the presence of a suitable catalyst. After the reduction step, when X is an auxiliary chiral substance, it may optionally be transferred to the OR ₂₆ group.

A compound of general formula (XI) can be prepared by reacting a β-ketocarbonyl compound of general formula (XII)

where R ₁ , R ₂ , and X are as defined above with O-protected hydroxylamine.

β-ketocarbonyl compounds (XII) can be obtained as racemates by formylation or acylation of a carbonyl compound of the general formula (XIII)

where R ₂ and X have the meanings as defined above, by a compound of the general formula (XIV)

where R ₁ is as defined above and Z represents a leaving group, such as halogen or alkoxy, in the presence of a base.

Another method for preparing a compound of general formula (V) is to attach Michael's hydroxylamine derivative to α, β-unsaturated carbonyl compounds of general formula (XV)

where R ₁ , R ₂ , and X are as defined above. After the Michael addition reaction, when group X is an auxiliary chiral substance, it may optionally be transferred to OR ₂₆ group. (α, β-Unsaturated carbonyl compounds (XV) can be obtained using standard methods.

Salts of the compounds of the invention include physiologically acceptable acid addition salts, for example, hydrochlorides, bromohydrates, sulfates, methanesulfonates, p-toluenesulfonates, phosphates, acetates, citrates, succinates, lactates, tartrates, fumarates and maleates. It is also possible the formation of salts with bases, such as sodium, potassium, magnesium and calcium salts.

The compositions of the invention for any route of administration may be prepared in accordance with the pharmacokinetic properties of the active ingredient (s).

Orally administrable compositions may be in the form of tablets, capsules, powders, granules, troches, liquid or gel formulations, such as oral, topical, or parenteral sterile solutions or suspensions. Tablets and capsules for oral administration may be in the form of a single dose and contain traditional excipients, such as binding agents, for example, syrups, Arabian gum, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; other excipients, for example lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; tablet lubricant, for example magnesium stearate, talc, polyethylene glycol or silicon dioxide; disintegrants, for example potato starch, or acceptable wetting agents, such as sodium lauryl sulfate. The tablets may be coated according to a technique well known in ordinary pharmaceutical practice. Liquid orally administrable formulations may be, for example, in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or they may be in the form of a dry substance, ready to be reconstituted with water or another suitable diluent before use. Such liquid formulations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats, emulsifying agents, for example leticin, sorbitan monooleate or gum arabic; non-aqueous diluents (which may include edible fats), for example almond oil, fractionated coconut oil, oily esters such as glycerin, propylene glycol or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, ordinary perfumes and dyes.

For topical application to the skin, the active ingredient (s) can be added to creams, lotions or ointments. Compositions of creams or ointments that can be used for the manufacture of medicaments are conventional formulations well known in the art and described, for example, in standard pharmacopoeial directories such as British Pharmacopoeia.

The active ingredient (s) may also be administered parenterally in a sterile environment. Depending on the diluents used and the concentration, the drug can either be suspended in it or dissolved. Advantageously, adjuvants such as local anesthetics, preservatives or buffering agents can be dissolved predominantly in the diluent. Another route of administration of the compounds of the present invention is by intravenous infusion.

Safe and effective dosages for various types of patients and various levels of disease development will be determined during clinical trials in accordance with accepted standards in this area. It should be understood that the specific dose level for each individual patient will depend on many factors, including the activity of the particular compound used, the patient’s age, weight, general health, gender, diet, time of administration, route of administration, rate of excretion, drug combinations , the rigidity of the therapy.

The discovery that compounds with PDF inhibitory activity can inhibit or prevent bacterial growth opens up a new approach for identifying new antibacterial agents by screening test compounds for activity as PDF inhibitors in vitro, followed by confirmation of their antibacterial ability by studying bacterial growth inhibition. This discovery also makes available (i) the use of compounds with PDF inhibitory activity as antibacterial agents, and (ii) a method for treating bacterial infections or infections by applying or administering a compound that inhibits bacterial PDF activity.

Thus, according to a further aspect of the invention, there is provided a method for identifying antibacterial compounds, comprising screening the compounds for their ability to inhibit PDF in vitro, selecting those compounds that exhibit this ability and testing them for their ability to inhibit bacterial growth. The ability to inhibit bacterial growth can be demonstrated by classical studies of the inhibition of bacterial growth using cultures in liquid or solid nutrient media, such as those presented in the biological experiment described here.

Suitable in vitro PDF inhibition screening may consist of mixing together a PDF, a PDF of a substrate, preferably labeled with a detectable marker, and a test compound, and then determining, over a suitable length of time, whether or not the PDF's ability to deform the substrate is inhibited or not in the presence of the test compound.

In a preferred embodiment of the invention, the cleavable substrate is detected using a fluorogenic marker, such as fluorescamine. After removal of the formyl group from the N-terminal methionine in the PDF substrate, the free amino group reacts with fluorescamine to generate a fluorescent product.

Alternative screening involves evaluating whether a protein expressed by bacteria that express endogenous (or recombinantly expressed) PDFs, when the bacteria are grown in the presence of the test compound, is a suitable substrate for N-terminal sequencing, or it provides less substrate than the protein expressed by the same bacteria grown in the absence of the test compound. Such a method can be based on the material used in the biological description experiment.

One skilled in the art can, without departing from the scope of the invention, develop alternative methods for screening test compounds in terms of their ability to inhibit bacterial PDFs.

The natural antibiotic actinonin (see, e.g., J.C.S. Perkin I, 1975, 819) is a hydroxamic acid derivative of structure (A):

In addition to actinonin, antibacterial activity was determined for its various structural analogues (see, e.g., Broughton et al. (Devlin et al. Journal of the Chemical Society. Perkin Transactions 1 (9): 830-841, 1975; Broughton et al. Journal of the Chemical Society. Perkin Transactions 1 (9): 857-860, 1975).

However, the mechanism of action underlying the antibacterial activity of actinonin has not yet been established. The authors of the present invention found that actinonin inhibits the activity of bacterial PDF.

Matlistatin group of compounds belongs to a number of structures similar to actinonin. All of them are peptide molecules with hydroxamic acid functional groups that bind the metal. (Ogita et al., J. Antibiotics. 45 (11): 1723-1732; Tanzawa et al., J. Antibiotics. 45 (11): 1733-1737; Haruyama et ai., J. Antibiotics. 47 (12) : 1473-1480; Tamaki et al. J. Antibiotics. 47 (12): 1481-1492). Matlistatin and their close structural analogues are characterized by the presence in the molecule of a divalent piperazine-1,6-diyl group, i.e.:

Due to the fact that they are close in structure to actinonin, the observation that actinonin inhibits PDF implies that matlistatin compounds can also inhibit PDF.

According to a further aspect of the present invention, there is provided the use of a compound that inhibits the activity of a bacterial PDF for the preparation of an antibacterial composition or agent, provided

(i) that the compound is not a compound of formula (XI)

RCO-CH (W) -NH-CO-CH (Y) -CH ₂ -CO-NH-OH (XI)

where (a) R represents a cyclic amino group, W represents hydrogen, methyl, isopropyl, isobutyl or benzyl, and Y represents hydrogen, C ₁ -C ₆ alkyl, phenyl, benzyl, 4-chlorophenylmethyl, 4-nitrophenylmethyl or 4 aminophenylmethyl; or

(b) R is 2-pyridylamino or 2-thiazolylamino, W is isopropyl and Y is n-pentyl; or

(c) R is diethylamino, W is methyl or isopropyl, and Y is n-pentyl;

or (ii) that the compound is not a compound containing a divalent piperazine-1,6-diyl group, i.e., a group of formula (XII):

According to another aspect of the invention, there is provided a method of treating bacterial infections or infections by administering to a patient suffering from such infection or infection, or applying to the site of such infection or infection, an antibacterial effective amount of a compound that inhibits the activity of the bacterial PDF enzyme, provided that the compound is not to compounds included in the conditions listed in the previous paragraph.

These conditions exclude actinonin and its antibacterial active analogues disclosed in Devlin et al., Journal of the Chemical Society. Perkin Transactions 1 (9): 830-841, 1975 and Broughton et al. Journal of the Chemical Society. Perkin Transactions 1 (9): 857-860.1975, and matlistatin compounds disclosed in Ogita et al., J. Antibiotics. 45 (11): 1723-1732; Tanzawa et al., J. Antibiotics. 45 (11): 1733-1737; Haruyama et al., J. Antibiotics. 47 (12): 1473-1480 and Tamaki et al., J. Antibiotics. 47 (12): 1481-1492.

The following examples illustrate an embodiment of the invention. L-tert-leucine-N-methylamide and L-tert-leucine-N.N-dimethylamide and other amino acid derivatives are prepared according to methods known in the art. The following abbreviations were used:

DMF - N, N-Dimethylformamide, DMF

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

TLC - Thin layer chromatography

¹ H and ¹ C NMR spectra were recorded using a Bruker AC 250E spectrometer at 250.1 and 62.9 MHz, respectively; s - singlet, d - doublet, m - multiplet, t - triplet, q - quartet.

Mass spectra were obtained on a Perkin Elmer Sciex API 165 spectrometer using both positive and negative ion modes.

Infrared spectra were recorded on a Perkin Elmer PE 1600 FTIR spectrometer.

Example 1

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid- (2,2-dimethyl-1S-methyl-carbamoyl-propyl) -amide

The named compound is obtained in accordance with scheme 1 and as described in detail below:

Реагенты и условия:

Reagents and conditions:

A. piperidine, HCNO, EtOH, 80 ° C, v / n;

B. H ₂ NOBzl, 80 ° C o / n;

C. HCOOH, Ac ₂ O;

D. Pentafluorophenol, EDC, CH ₂ Cl ₂ ;

E. H-tert.-LeuNHMe, DMF, 35 ° C;

F. H ₂ , 10% Pd / C, EtOAc / EtOH.

STAGE A: 2-Butyl Acrylic Acid

Butyl malonic acid (25 g, 156 mmol) was dissolved in ethanol (250 ml) and 37% formaldehyde solution (15.45 ml, 156 mmol) was added, followed by piperidine (47 ml, 624 mmol). The mixture was stirred overnight at 80 ° C. under reflux. The solvents were removed under reduced pressure and the residue was diluted with 1 M hydrochloric acid and extracted with dichloromethane (3x30 ml). The combined organic extracts were washed with brine, dried over anhydrous magnesium sulfate, filtered and evaporated to give the desired product as a yellow oil (25 g, with residual solvent). ¹ H-NMR: δ (CDCl ₃ ), 10.04 (1H, broad s), 6.22 (1H, s), 5.57 (1H, d, J = 1.3 Hz), 2.30 (2H, t, J = 6.9 Hz), 1.38 (4H, m), and 0.91 (3H, t, J = 7.2 Hz).

STAGE B: 2RS- (Benzyloxy-amino-methyl) -hexanoic acid

A mixture of 2-butyl acrylic acid (3.43 g, 27.1 mmol) and 0-benzylhydroxylamine (5 g, 40.65 mmol) was heated at 80 ° C. overnight. The mixture was cooled to room temperature, diluted with ethyl acetate (40 ml), and washed with 1 M hydrochloric acid (3x20 ml), saturated sodium bicarbonate solution (2x20 ml) and brine (2x20 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to give the title compound as a white solid (2.62 g, 39%). ¹ H-NMR: δ (CDCl ₃ ). 8.05 (1H, broad s), 7.35 (5H, m), 5.00 (2H, m), 3.28 (2H, m), 2.98 (1H, m), 1.31 (6H, m) and 0.88 (3H, t, J = 5.0 Hz).

Stage C: 2RS - [(Benzyloxy-formyl-amino) -methyl] -hexanoic acid

2RS- (Benzyloxyamino-methyl) -hexanoic acid (2.62 g, 10.51 mmol) was dissolved in formic acid (4 ml, 105 mmol) and acetic anhydride (1.9 ml, 21.02 mmol) and stirred for nights at room temperature. The solution was diluted with ethyl acetate (40 ml), washed with water (2x20 ml), saturated sodium bicarbonate solution (20 ml) and brine (20 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to give the desired product as a yellow oil (2 9 g, 99%). ¹ H-NMR: δ (CDCl ₃ , rotamers), 8.21 (0.5H, s), 8.14 (0.5H, s), 7.37 (5H, m), 4.98 (2H, m), 3.86 (1H, m), 3.27 (0.5H, dd, J = 6.0, 14.0 Hz), 2.93 (0.5H, m), 2.77 (1H, m). 1.50 (2H, m), 1.30 (4H, m) and 0.88 (3H, m).

STAGE D: 2RS - [(Benzyloxy-formyl-amino) methyl] -hexanoic acid pentafluorophenyl ether

2RS - [(Benzyloxy-formylaminomethyl] -hexanoic acid (30.72 g, 110 mmol) and pentafluorophenol (26.31 g, 143 mmol) are dissolved in dichloromethane (150 ml) and the solution is stirred and cooled in an ice bath while adding EDC (25.3 g, 131 mmol). The reaction mixture was allowed to warm to room temperature and was stirred overnight.The solution was washed successively with 1 M hydrochloric acid (2x50 ml), 0.5 M sodium carbonate (2x50 ml) and brine (50 ml ), dried over anhydrous magnesium sulfate and filtered.The filtrate was evaporated under reduced pressure and the residue was purified by vial chromatography (silica gel, dichloromethane) to give the title compound as a colorless oil (15.0 g, 31%). ¹ H-NMR: δ (CDCl ₃ , rotamers), 8.17 (1H, broad s), 7.37 (5H, m ), 4.95-4.70 (2Н, broadened m), 4.10-3.75 (2Н, broadened m), 3.10 (1Н, broadened s), 1.88-1.55 (2Н, m), 1.39 (4Н, m) and 0.92 (3H, T, J = 7.0 Hz).

STAGE E: 2R (or S) - [(Benzyloxy-formyl-amino) -methyl] -hexanoic acid (2,2-dimethyl-1-methylcarbamoylpropyl) -amide

2RS - [(Benzyloxy-formyl-amino) methyl] hexanoic acid pentafluorophenyl ether (5 g, 11 mmol) and tert-leucine N-methylamide (1.62 g, 11 mmol) are dissolved in DMF (60 ml) and the mixture is stirred overnight at 35 ° C. The solvent was removed under reduced pressure and the residue was redissolved in dichloromethane. The solution was washed successively with 0.5 M sodium carbonate, 1.0 M hydrochloric acid and brine, dried over anhydrous magnesium sulfate and filtered. The two diastereoisomeric products are separated by flash chromatography (silica gel, elution gradient from 30% to 0%, hexane in ethyl acetate). Diastereoisomer A (higher R): ¹ H-NMR: δ (CDCl ₃ , rotamers), 8.12, 7.87 (1H, 2 broad s), 7.27 (5H, m), 6.26 (1H, d, J = 8.7 Hz ), 5.78 (1H, broadened s), 4.91-4.60 (2H, broadened m), 4.15 (1H, d, J = 2 Hz), 3.75 (2H, broadened m), 2.79 (3H, d, J = 4.8 Hz ), 2.56 (1Н, m), 1.60 -1.35 (2Н, broadened m), 1.24 (4Н, m), 0.96 (9Н, s) and 0.86 (3Н, t, J = 6.7 Hz). Diastereoisomer B (lower R _f ): ¹ H-NMR: δ (CDCl ₃ , rotamers), 8.16, 7.88 (1H, 2 broadened singlet), 7.27 (5H, m), 6.28 (1H, d, J = 8.9 Hz), 5.70-5.44 (1H, broadened s), 4.98-4.61 (2H, broadened m), 4.14 OH, d, J = 9.2 Hz), 3.78-62 (2H, broadened m), 2.85-2.60 (3Н, broadened m), 2.47 (1H, m), 1.72-1.25 (6H, broadened m), 0.98 (9H, s) and 0.88 (3H, T, J = 6.7 Hz).

STAGE F: 2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid (2,2-dimethyl-1S-methylcarbamoylpropyl) -amide.

2 - [(Benzyloxy-formylamino) methyl] -hexanoic acid- (2,2-dimethyl-1-methylcarbamoylpropyl) -amide (diastereoisomer A) (1.0 g, 2.5 mmol) was dissolved in a mixture of ethyl acetate (20 ml) and ethanol (1 ml) and the solution is placed in an argon atmosphere. 10% palladium on charcoal is added and a thin stream of hydrogen is bubbled through the suspension. After 40 minutes, TLC analysis showed that the entire starting product was consumed, with more polar positive particles of iron chloride remaining. The system was purged with argon before the catalyst was removed by filtration. The filtrate was evaporated to dryness to afford the title compound as an off-white foam (810 mg, including residual solvent). ¹ H-NMR: δ ((CD ₃ ) ₂ SO, rotamers), 9.81, 9.41 (1H, 2 broad singlets), 7.82-7.60 (3H, m), 4.04 (1H, d, J = 9.3 Hz). 3.50-3.02 (2H, m). 2.87-2.60 (1H, m), 2.41 (3H, d, J = 4.5 Hz), 1.39-0.93 6H, m), 0.75 (9H, s) and 0.67 (3H, t, J = 5.7 Hz). ¹³ C-NMR: δ ((CD ₃ ) ₂ CO), 172.5, 170.2. 157.5, 59.9, 42.8, 33.3. 29.0, 28.4, 28.2, 26.4, 24.8, 21.7 and 13.3. IR (KBr, tab.), V _max 3309, 2959, 2873, 1646 and 1540 cm ^-1 .

2 - [(Benzyloxy-formyl-amino) -methyl] -hexanoic acid- (2,2-dimethyl-1-methylcarbamoylpropyl) -amide (diastereoisomer B) (1.0 g, 2.5 mmol) was also deprotected to give diastereoisomer B of the title compound (740 mg, 97%). ¹ H-NMR: δ ((CD ₃ ) ₂ SO, rotamers), 9.75, 9.30 (1H, 2 broad singlets), 7.81-7.42 (3H, m), 4.04 (1H, d, J = 9.5 Hz), 3.53 -3.02 (2Н, m), 2.80-2.55 (1Н, m), 2.41 (3Н, d, J = 4.5 Hz), 1.33-0.82 (6Н, m), 0.72 (9Н, s) and 0.67 (3Н, t , J = 6.7 Hz). ¹³ C-NMR: δ ((CD ₃ ) ₂ SO), 172.6, 170.4, 161.7, 157.0, 59.8, 34.0, 29.4, 28.6, 26.7, 25.2, 22.1 and 14.1. IR (CVg table), v _max , 3312, 2959, 1640, 1541, 1369, and 1240 cm ^-1 .

Example 2

2R (or S) - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (2,2-dimethyl-15-tert-butylcarbamoylpropyl) -amide

The title compound is prepared analogously to the procedure of Example 1, using L-tert-leucine-N-tert-butylamide instead of L-tert-leucine-N-methylamide in Step E. Diastereoisomers not separated by flash chromatography (silica gel, ethyl acetate) in Step E, is converted into a mixture of the desired derivatives of N-formyl-hydroxylamine by hydrogenolysis. A white solid is obtained. ¹³ C-NMR: δ ((CD ₃ ) ₂ SO), 172.8, 172.5, 170.1, 169.6, 161.6, 156.9, 59.9, 59.7, 51.9, 51.7, 50.2, 49.6, 48.3, 43.2, 43.1, 42.7, 34.2, 34.0 , 29.6, 29.3, 29.2, 28.8, 28.6. 26.7, 22.2, 22.1, 20.3 and 13.9. IR (KBr), v _max , 3311, 2964, 1639, 1548, 1456, 1394, 1364, and 1226 cm ⁻¹ .

Example 3

2R (or S) - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-methyl-2-morpholin-4-yl-2-oxoethyl) -amide

A solution of 2RS - [(benzyloxy-formylamino) methyl] -hexanoic acid pentafluorophenyl ether (Example 1, Step D) (445 mg, 1 mmol) in DMF (5 ml) was added to L alanine-N-morpholinamide (158 mg, 1 mmol) in vitro and stirred at 35 ° C overnight. DMF was removed in vacuo, the residue was redissolved in dichloromethane (2 ml) and passed through a cleaning carbon briquette (cartridge) (Icolute-NH;), eluting with dichloromethane (4 ml) in order to remove pentafluorophenol. Dichloromethane was removed under reduced pressure and the residue was redissolved in formic acid (2 ml) and ethyl acetate (2 ml). Then the solution is treated with 10% palladium-carbon (200 mg) and stirred at room temperature for 2 hours. The catalyst was removed by filtration through celite, washed well with methanol and the solvent was removed in vacuo. Compounds are purified by reverse phase HPLC (elution gradient, 10-90% acetonitrile / water). Diastereoisomer A: ¹ H-NMR; δ (СО ₃ OD), 8.03 (0.5Н, s), 7.84 (0.5Н, s), 4.75 (1Н, m), 3.65 (8Н, m), 3.39 (1Н, m), 3.24 (1Н, dd, J = 4.0, 13.2 Hz), 2.84 (1H, m), 1.57 (2H, m), 1.34 (7H, m), and 0.92 (3H, m). LRMS: -ve ion 328 [M-H]. Diastereoisomer B: ¹ H-NMR; δ (CD ₃ OD), 3.66 (8Н, m), 3.41 (1Н, dd, J = 9.98, 13.1 Hz), 3.23 (1Н, m), 2.90 (0.5Н, m), 2.71 (0.5Н, m) , 1.62 (2H, m), 1.33 (7H, m), and 0.92 (3H, t, J = 6.7 Hz). LRMS: -ve ion 328 [M-H].

The compounds of Examples 4 to 12 were prepared in a similar manner to that described in Example 3, using the appropriate amine or amino component instead of L-alanine-N-morpholinamide. In the case where both diastereoisomers are obtained, diastereoisomer A elutes faster and is more active against in vitro PDF. In some cases, only the product flowing faster underwent further conversions to the final compound.

Example 4

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-dimethylcarbamoylethyl) -amide

Diastereoisomer A: ¹ H-NMR; δ (CD ₃ OD), 4.72 (1H, m), 3.53 (1H. dd, J = 8.9, 13.0 Hz), 3.23 (1H, m). 3.14 (3H, s), 2.95 (3H, s). 2.83 (0.5H, m). 2.74 (0.5H. M), 1.57 (2H, m), 1.33 (7H, m) and 0.92 (3H, m). LRMS + ve ion 288 [M + H]; -ve ion 286 [M-H].

Diastereoisomer B: ¹ H-NMR; δ (CD ₃ OD), 4.74 (1H, m), 3.41 (1H, dd, J = 9.9. 13.0 Hz), 3.25 (1H, dd, J = 4.0, 13.1 Hz), 3.15 (3H, s), 2.97 (3H, s), 2.89 (0.5H, m), 2.72 (0.5 N, m), 1.53 (2H, m), 1.33 (7H, m) and 0.93 (3H, t, J = 6.7 Hz). LRMS: + ve ion 310 [M + Na], -ve ion 286 [M-H].

Example 5

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-hydroxymethyl-3-methyl-butyl) -amide

Diastereoisomer A: ¹ H-NMR; δ (CD ₃ OD), 4.07 (1H, m), 3.55 (1H, m), 3.45 (2H, m), 3.20 (1H, m), 2.85 (0.5H, m), 2.80 (0.5H, m) , 1.60 (3H, m), 1.35 (6H, m) and 0.93 (9H, m). LRMS: + ve ion 289 [M + H], -ve ion 287 [M-H]. Diastereoisomer B: ¹ H-NMR; δ (CO ₃ OD), 4.07 (1H, m), 3.59 (1H, m), 3.45 (2H, m), 3.24 (1H, m), 2.70 (1H, m), 1.62 (3H, m), 1.35 (6H, m) and 0.93 (9H, m). LRMS: + ve ion 311 [M + H], 289 [M + H], -ve ion 287 [M-H].

Example 6

2R (or S) - [(Formylhydroxyamino) methyl] hexanoic acid (1S-hydroxymethyl-2-phenylethyl) amide

Diastereoisomer A: ¹ H-NMR; δ (СО ₃ ОD), 7.24 (5Н, m), 4.15 (1Н, m), 3.54 (2Н, d, J = 5.4 Hz), 3.38 (1Н, dd, J = 7.8, 13.1 Hz), 3.14 (1Н , dd, J = 4.7, 13.2 Hz), 2.95 (1Н, dd, J = 7.3, 13.7 Hz), 2.68 (2Н, m), 1.58 (2Н, m), 1.32 (4Н, m), and 0.91 (3Н , t, J = 6.7 Hz). LRMS: + ve ion 345 [M + Na], 323 [M + H], -ve ion 321 [M-H]. Diastereoisomer B: LRMS: + ve ion 345 [M + Na], 323 [M + H], -ve ion 321 [M-H].

Example 7

2R (or S) - [(Formyl-hydroxy-amino) methyl] rexanoic acid [2,2-dimethyl-pyridin-2-yl-carbamoyl) propyl] amide

Diastereoisomer A: a colorless oil. ¹ H-NMR; δ (СО ₃ ОD), 8.34 (1Н, m), 8.06 (1Н, m), 7.90 (1Н, m), 7.33 (1Н, m), 4.45 ОН, s), 3.55 (1Н, dd, J = 8.3 , 13.2 Hz), 3.25 (1H, m), 3.05 (1H, m), 1.61 (2H, m), 1.32 (4H, m), 1.11 (9H, s) and 0.85 (3H, m). LRMS: + ve ion 379 [M + H], -ve ion 377 [M-H].

Diastereoisomer B: a colorless oil. ¹ H-NMR; δ (СО ₃ ОD), 8.33 (1Н, m), 8.20 (0.5Н, m), 7.93 (1Н, m), 7.41 (0.5Н, m), 7.28 (1Н, m), 4.48 (1Н, s) , 3.52 (1Н, dd. J = 8.8, 13.1 Hz), 3.23 (1Н, dd, J = 3.9, 13.1 Hz), 3.05 (0.5Н, m), 2.87 (0.5Н, m), 1.62 (2Н, m ), 1.36 (4H, m), 1.11 (9H, s) and 0.93 (3H, m). LRMS: + ve ion 393 [M + Na], 379 [M + H], -ve ion 377 [M-H].

Example 8

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-dimethylcarbamoyl-2-methylpropyl) amide

Diastereoisomer A: a colorless oil. LRMS: + ve ion 338 [M + Na], -ve ion 319 [M-H].

Example 9

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-dimethylcarbamoyl-2-phenylethyl) amide

Diastereoisomer A: a colorless oil. LRMS: + ve ion 386 [M + Na], -ve ion 362 [M-H] Diastereoisomer B: colorless oil LRMS: + ve ion 386 [M + Na], -ve ion 362 [M-H]

Example 10

2R (or S) - [(Formylhydroxyamino) methyl] hexanoic acid (1S-dimethylcarbamoyl-3-methyl-butyl) amide

Diastereoisomer A: a colorless oil. LRMS: + ve ion 352 [M + Na], -ve ion 328 [M-H].

Example 11

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid [3-methyl-lS-pyrrolidine-1-carbonyl) butyl] amide

Diastereoisomer A: a colorless oil. LRMS: -ve ion 354 [M-H].

Example 12

1- {2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoyl} pyrrolidine-2S-carboxylic acid dimethylamide

Diastereoisomer A: a colorless oil. LRMS: + ve ion 336 [M + Na], -ve ion 312 [M-H]. Diastereoisomer B: a colorless oil. LRMS: + ve ion 336 [M + Na], -ve ion 312 [M-H].

Example 13

2R (or S) - [(Formyl-hydroxy-amino) methyl] hexanoic acid-1S-dimethylcarbamoyl-2,2-dimethylpropylamide

Method I

The synthesis pathway for the title compound is shown in Scheme 2 and is described in detail below.

A. HCOOEt, NaOEt;

B. HCl, NHOBzl, NaOAc, aqueous EtOH;

C. NaOH, CH ₃ OH;

D. H-TleN (CH ₃ ) ₂ ; EDC, HOAt, DMF;

E. NaCNBH ₃ , AcOH then the separation of diastereomers;

F. HCOBt, THF;

G. H ₂ , Pd / C, CH ₃ OH.

Stage A: 2RS-Formyl-heptanoic acid ethyl ester

Sodium metal (4.38 g, 0.191 mmol) is cut into small pieces and placed in a two-necked oven-dried round-bottom flask under a layer of argon. Anhydrous diethyl ether (100 ml) was added, the suspension was stirred and cooled to 0 ° C. A reflux condenser was attached to the flask before ethanol (1.03 ml, 17.3 mmol) was added dropwise. Then, a mixture of ethyl formate (15.41 g, 0.208 mmol) and ethyl caproate (25 g, 0.173 mmol) was added dropwise through a dropping funnel over about 20 minutes. The resulting orange suspension (sodium metal can still be observed) was allowed to warm to room temperature and was stirred overnight. The resulting thick orange suspension (sodium metal is not visible) is cooled to 0 ° C and diluted with ice water (100 ml). The mixture is transferred to a separatory funnel, the aqueous phase is removed, washed with diethyl ether, cooled to 0 ° C and acidified with 1 M hydrochloric acid (200 ml). The emulsion was extracted with ethyl acetate and the organic layer was separated, washed with brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give a yellow oil containing basically the title compound (11.09 g), which was used without further purification in Step B.

Stage B: 2RS- (Benzyloxyimino-methyl) -heptanoic acid ethyl ester

The Claisen solid product from Step A (11.0 g, 63.9 mmol) was dissolved in ethanol (100 ml) and water (10 ml) and cooled to 0 ° C. while sodium acetate (6.2 g, 76.6 mmol) and 0-benzylhydroxylamine hydrochloride (12.23 g, 76.6 mmol). The mixture was allowed to warm to room temperature and was stirred overnight. The resulting suspension was filtered and the filtrate was concentrated under reduced pressure. The remaining yellow paste was partitioned between ethyl acetate and water. The organic layer was washed with 1 M hydrochloric acid and brine, dried over anhydrous magnesium sulfate, filtered and evaporated to a yellow oil. The desired product is obtained by flash chromatography (silica gel, elution gradient from 10 to 25%, ethyl acetate in hexane. Yield 9.19 g (52%). ¹ H-NMR: δ (CDCl ₃ mixture of syn- and anti-isomers), 7.46 ( 0.6H, d, J = 8.0 Hz), 7.38-7.28 (5H, m), 6.79 (0.4H, d, J = 7.1 Hz), 5.11 (0.8H, s), 5.08 (1.2H, s), 4.16 (1.2H, q, J = 7.0 Hz), 4.13 (0.6H, q, J = 7.0 Hz), 3.91 (0.4H, q, J = 7.2 Hz), 3.21 (0.6H, td, J = 8.0 and 6.1 Hz), 1.90-1.48 (2H, m), 1.37-1.20 (7H, m), 0.87 (3H, t, J = 7.0 Hz).

Stage C: 2RS- (Benzyloxyimino-methyl) -heptanoic acid 2RS- (Benzyloxyimino-methyl) -heptanoic acid ethyl ester (7.0 g, 25.24 mmol) was dissolved in methanol (125 ml) and the solution was cooled to 0 ° C. . 1 M sodium hydroxide (26 ml, 26 mmol) is added in portions over 2 minutes, resulting in a pale yellow emulsion. An additional amount of methanol is introduced until a clear solution forms. The solution was stirred for 90 minutes at 0 ° C, then for 5 hours at room temperature until TLC analysis showed that the entire starting product had reacted. The solvent was removed under reduced pressure and the residue was partitioned between water and ethyl acetate. The aqueous layer was cooled to 0 ° C and acidified with 1 M hydrochloric acid. The emulsion thus obtained is extracted twice with ethyl acetate. The combined organic extracts were washed with brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound as a yellow oil (5.15 g, 82%), which was used without further purification in Step D. ¹ H-NMR: δ (CDCl ₃ , mixture of syn- and anti-isomers) , 8.00 (1Н, broadened s), 7.46 (0.6Н, d, J = 7.9 Hz), 7.36-7.24 (5Н, m), 6.80 (0.4Н, d, J = 7.0 Hz), 5.13 (0.8Н, s ), 5.09 (1.2H, s), 3.94 (0.4H, q, J = 7.1 Hz), 3.27 (0.6H, td. J = 6.4 and 8.0 Hz), 1.94-1.58 (2H, m), 1.48-1.24 (4H, m) and 0.94-0.84 (3H, m).

Stage D: 2RS- (Benzyloxyimino-methyl) -heptanoic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) amide

2- (Benzyloxyiminomethyl) -heptanoic acid (5.16 g, 20.7 mmol), tert-leucine NN-dimethylamide (3.60 g, 22.77 mmol) and EDC (4.76 g, 24.84 mmol) mix it together in DMF (75 ml) and cool to 0 ° C. HOAt (250 mg, cat.) Was added and the resulting bright yellow mixture was allowed to warm to room temperature and was stirred overnight. The solvent was removed under reduced pressure and the remaining oil was partitioned between ethyl acetate and 1 M hydrochloric acid. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under reduced pressure. The title compound was obtained as a colorless oil using flash chromatography (silica gel, elution gradient from 33 to 66%, ethyl acetate in hexane). Yield 6.84 g (85%). ¹ H-NMR: δ (CDCl ₃ , mixture of syn- and anti-isomers), 7.45 (0.6H, 2d), 7.40-7.26 (5H, m), 6.72 (0.4H, 2d), 6.58 (1H, m) 5.20-4.69 (3H, m), 3.82 (0.4H, m), 3.16-3.10 (3H, m), 3.05 (0.6N. M). 2-99-2.92 (3Н, m), 1.90-1.54 (2Н, m), 1.39-1.17 (4Н, m), 0.97 (2.7Н, s), 0.96 (1.8Н. S), 0.94 (2.7Н, s), 0.92 (1.8H, s) and 0.92-0.82 (3H, m).

Stage E: 2R (or S) - (Benzyloxyamino-methyl) -heptanoic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) amide

To a solution of 2RS- (benzyloxyimino-methyl) -heptanoic acid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl) amide (5.0 g, 12.84 mmol) in acetic acid (40 ml) was added in one portion sodium cyanoborohydride (2.02 g, 32.0 mol). The reagent slowly dissolves within 1 hour with a slight evolution of gas to form a colorless solution, which is left to mix overnight. The solvent is removed by evaporation under reduced pressure and distillation in the form of an azeotrope with toluene. The remaining oil is distributed between diethyl ether and 1 M sodium carbonate (Caution - a certain amount of gas is released). The organic layer was washed with brine (70 ml), dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under reduced pressure. Two diastereoisomers of the title compound are purified and separated by flash chromatography (silica gel elution gradient from 50% to 100%, ethyl acetate in hexane). Diastereoisomer A (faster elution): colorless oil (2.27 g, 45%). ¹ H-NMR: δ (CDCl ₃ ), 7.43-7.28 (5H, m), 6.76 (1 N, broadened d, J = 9.4 Hz), 5.69 (1 N, broadened s), 4.93 (1 N, d, J = 9.4 Hz), 4.72 (2H, s), 3.15 (3H, s), 3.18-3.00 (2H, m), 2.96 (3H, s). 2.49 (1H, m), 1.66-1.49 (2H, m), 1.46-1.19 (4H, m), 0.99 (9H, s) and 0.86 (3H, t, J = 6.8 Hz).

Diastereoisomer B (slower elution): colorless oil (1.44 g, 46%). ¹ H-NMR: δ (CDCl ₃ , 7.40-7.27 (5H, m), 6.70 (1H, broad d, J = 9.0 Hz), 5.99 (1H, broad s), 4.85 (1H, d, J = 9.0 Hz ), 4.71 (2Н, d, J = 1.6 Hz), 3.16 (3Н, s), 3.06-2.97 (2Н, m), 2.95 (3Н, s), 2.57 (1Н, m), 1.74-1.21 (6Н, m), 1.00 (9H, s) and 0.88 (3H, broadened t, J = 6.7 Hz).

Stage F: 2R (or S) - [(Benzyloxy-formyl-amino) -methyl] -heptanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide

2- (Benzyloxyamino-methyl) -heptanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (diastereoisomer A) (2.02 g, 5.13 mmol) was dissolved in anhydrous THF (50 ml) and placed under argon layer. N-formyl-benzotriazole (ARKatrizky et al., Synthesis 1995, 503) (0.83 g, 5.65 mmol) was added and the mixture was stirred at room temperature for 4 hours. The solvent was evaporated under reduced pressure and the remaining oil was partitioned between dichloromethane and 1 M sodium hydroxide. The organic layer was washed once more with sodium hydroxide and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under reduced pressure. The title compound is obtained as a white crystalline solid by flash chromatography (silica gel, eluting with 33% ethyl acetate in hexane). Yield 1.60 g (74%). ¹ H-NMR: δ (CDCl ₃ ), rotamers), 8.00 (1H, broad m), 7.47-7.29 (5H, m), 6.25 (1 H, broad d, J = 9.3 Hz), 5.08-4.74 (2H , broadened m), 4.87 (1Н, d, J = 9.4 Hz), 3.89-3.52 (2Н, broadened m), 3.13 (3Н, s), 2.94 (3Н, s), 2.54 (1Н, m), 1.67- 1.11 (6H, m), 0.95 (9H, s) and 0.85 (3H, broadened t, J = 6.9 Hz).

2- (Benzyloxyamino-methyl) -heptanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (diastereoisomer B) is obtained similarly from the more slowly eluting diastereoisomer of Step E. Yield 0.38 g (41%). ¹ H-NMR: δ (CDCl ₃ , rotamers), 8.00 (1H, broad m), 7.47-7.28 (5H, broad m), 6.29 (1H, broad d, J = 9.3 Hz), 5.01-4.63 (2H, broadened m), 4.88 (1Н, d, J = 9.3 Hz), 3.82-3.51 (1.5Н, broadened m), 3.20-2.78 (6.5Н, broadened m), 2.50 (1Н, broadened m), 1.76-1.17 ( 6H, broadened m), 0.97 (9H, s) and 0.85 (3H, broadened t, J = 6.7 Hz).

Stage G: 2R (or S) - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid- (1S-dimethyl carbamoyl-2,2-dimethyl-propyl) -amide

2 - [(Benzyloxy-formyl-amino) -methyl] -heptanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide (diastereoisomer A) (1.43 g, 3.41 mol) is dissolved in methanol (50 ml) and placed under a layer of argon. A suspension of 10% palladium on carbon (100 mg, cat.) In ethyl acetate (2 ml) was added and the mixture was vigorously stirred while sparging through a solution of hydrogen. After 10 minutes, the mixture was placed in a hydrogen atmosphere and allowed to mix for 3 hours, until TLC analysis showed that all of the starting material had reacted. The system was saturated with argon and the catalyst was removed by filtration. The filtrate was concentrated under reduced pressure to give the title compound as a colorless hygroscopic foam (1.11 g, 99%). ¹ H-NMR: δ (CDCl ₃ , rotamers), 8.41 (0.35H, s), 7.83 (0.65H, broadened s), 6.80 (0.35H, broadened d.J = 8.9 Hz), 6.62 (0.65H, broadened d, J = 9.4 Hz), 4.91 (0.65 H, broadened d, J = 9.4 Hz), 4.88 (0.35 H, broadened d, J = 8.9 Hz), 4.04 (1H, dd, J = 14.7 and 7.4 Hz), 3.82 (0.65, dd, J = 14.0 and 9.7 Hz), 3.56 (0.35 H, dd, J = 14.7 and 3.3 Hz), 3.48 (0.65 H, dd, J = 14.0 and 4.0 Hz), 3.16 (1.05 N s ) 3.15 (1.95H, s), 2.98 (1.05H, s), 2.96 (1.95H, s). 2.90-2.74 (0.65H, broad m). 2.74-2.61 (0.35Н, broadened m) 1.73-1.17 (6Н, broadened m), 0.99 (3.15Н, s). 0.95 (5.85H, s) and 0.87 (3H, broadened t, J = 6.7 Hz). ¹³ C-NMR; δ (CDCl ₃ ,, 174.6, 171.2. 162.2, 157.2, 60.1, 54.5, 54.3, 52.3, 48.4, 44.8, 44.3, 35.6, 35.4, 29.6, 29.0, 26.3, 20.8, 20.2, 14.0 and 13.7. LRMS: + ve ion 352 (M + Na); -ve ion 328 (M-H).

2 - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid- (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide (diastereoisomer B) is obtained similarly from diastereoisomer B in Step E

¹ H-NMR; δ (CDCl ₃ , rotamers), 9.37 (0.5H, s), 8.40 (0.5H, s), 7.75 (0.5H, broadened s), 6.62 (0.5H, broadened s), 6.41 (0.5H, broadened d, J = 7.1 Hz), 4.87 (0.5H, broad d, J = 6.6 Hz). 4.66 (0.5H, broadened d, J = 7.6 Hz), 3.84-3.39 (2H, m), 3.21 (1.5H, broadened s), 3.14 (1.5H, broadened s), 2.98 (3H, broadened s), 2.91-2.54 (1H, m), 1.79-1.23 (6H, m) and 1.08-0.83 (12H, m). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 174.9, 173.3, 56.3, 54.8, 51.6, 50.3, 45.5, 45.1, 38.6, 38.4, 36.2, 36.0, 35.3, 34.4, 29.5, 29.4, 29.3, 29.2, 26.6, 26.5, 22.6, 22.5 and 13.9. LRMS: + ve ion 352 [M + Na], -ve ion 328 [M-H].

Method II

An alternative asymmetric synthesis route for the compound of Example 13 is shown in Scheme 3 and described below

Reagents and conditions:

A. piperdine, HCHO, EtOH, 80% C, o / n;

B. tert-BuCOCl, Et ₃ N, then 3-lithium-4-benzyl-5,5-dimethyloxazolidin-2-one;

C. H ₂ NOBzl, room temperature, o / n, then p-TsOH, EtOAc;

D. LiOH, aqueous THF, 0 ° C;

E. H-TleN (Me ₂ ) ₂ ; HOBt, EDC, DMF;

F. HCOBt, THF;

G. H ₂ , Pd / C, EtOH.

Stage A: 2-Butyl Acrylic Acid

To a solution of n-butylmalonic acid (17.2 g, 107 mmol) in ethanol (200 ml) was added piperidine (12.76 ml, 129 mmol) and a 37% aqueous formaldehyde solution (40.3 ml, 538 mmol). The solution is heated to 80 ° C and at this time a precipitate appears, which gradually dissolves within 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%). ¹ H-NMR; δ (CDCl ₃ ), 6.29 (1H, s). 5.65 (1H, s). 2.34-2.28 (2H, m), 1.54-1.26 (4H, m) and 0.94 (3H, t, J = 7.1 Hz).

Stage B: 45-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 layer of argon. Triethylamine (30 ml, 218 mmol) and pivaloyl chloride (21 ml, 168 mmol) are added at such a rate that the temperature remains below -60 ° C. The mixture was stirred at -78 ° C for 30 minutes, warmed to room temperature for 2 hours and finally cooled again to -78 ° C.

4S-benzyl-5,5-dimethyl-oxazolidin-2-one is dissolved in dry THF (500 ml) in a separate flask and cooled to -78 ° C under a layer of argon. n-Butyllithium (2.4 M solution in hexane, 83 ml, 200 mmol) was slowly added and the mixture was stirred for 30 minutes at room temperature. The resulting anion is transferred through a tube 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 bicarbonate (200 ml) and the solvents 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 showed the presence of unreacted auxiliary chiral substance in addition to the desired product. A portion of the product (30 g) was dissolved in dichloromethane and passed through a layer of silica gel to obtain the title compound in pure form, which is a yellow oil (25.3 g). ¹ H-NMR: δ (CDCl ₃ ), 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) and 0.91 (3H, t, J = 7.1 Hz). A certain amount of auxiliary chiral substance is isolated by washing the silica gel with methanol.

Stage 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 0-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 a pale yellow oil (25.3 g), which, as shown by NMR and HPLC, contained 4S-Benzyl-3- [2- (benzyloxyamino-methyl) -hexanoyl] -5,5-dimethyl -oxazolidin-2-one (ca. 82% CU) along with traces of the starting material. The product was combined with another sample (26.9 g, 76% CU) and dissolved in ethyl acetate (200 ml). Add p-toluenesulfonic acid (22.7 g, 119 mmol) and the mixture is cooled to 0 ° C. The title compound is obtained as a white crystalline solid by seeding and solidification (34%, single diastereoisomer). A second sample (14.7 g, 20%, the only diastereoisomer) was also obtained. ¹ H-NMR; δ (CDCl ₃ , 7.89 (2Н, d, J = 8.2 Hz), 7.37-7.12 (10Н, m), 7.02 (2Н, d, J = 6.9 Hz), 5.28-5.19 (2Н, m), 4.55 (1Н , m), 4.23 (1Н, m), 3.93 (1Н, m), 3.58 (1Н, m), 2.58 (1Н, m), 2.35 (3Н, s), 1.67-1.51 (2Н, m), 1.29- 1.16 (4H, m), 1.25 (3H, s), 1.11 (3H, s) and 0.80-0.75 (3H, m).

Stage D: 2R-Benzyloxyamino-methyl) -hexanoic acid 4S-Benzyl-3- [2R- (benzyloxyamino-methyl) -hexanoyl] -5,5-dimethyl-oxazolidin-2-one salt of p-toluenesulfonic acid (25.2 g , 40.2 mmol) partitioned between ethyl acetate and 1 M sodium carbonate. The organic phase is dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure. The remaining oil is 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 mass is acidified to pH 4 with 1 M citric acid and the solvents are removed. The residue was partitioned between dichloromethane and 1 M sodium carbonate. The alkaline aqueous layer was acidified to pH 4 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 afford the title compound as a colorless oil (7.4 g, 73%). ¹ H-NMR; δ (CDCl ₃ ). 8.42 (2H, broad s), 7.34-7.25 (5N.m), 4.76-4.66 (2H, m), 3.20-3.01 (2H, m), 2.73 (1 N, m), 1.70-1.44 (2H, m ), 1.34-1.22 (4Н, m) and 0.92-0.86 (3Н, m).

Stage E: 2R- (Benzyloxyamino-methyl) -hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide

2R-Benzyloxyamino-methyl) -hexanoic acid (7.98 g, 31.8 mmol) was dissolved in DMF (150 ml) and the solution was cooled to 0 ° C. EDC (6.1 g, 31.8 mmol) and HOBt (430 mg, 3.2 mmol) were added and the mixture was stirred for 15 minutes. Then tert-leucine-M, M-dimethylamide (5.53 g, 34 mmol) was added, the mixture 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 1 M hydrochloric acid, saturated sodium bicarbonate solution and brine, dried and filtered. The solvent was removed to give the title compound as a yellow oil (8.7 g, 69%), which was used without further purification in Step F. ¹ H-NMR; δ (CDCl ₃ ), 7.35-7.28 (5H, m), 6.77 (1H, broadened d, J = 9.2 Hz), 5.69 (1H, broadened s). 4.93 (1H, d, J = 9.4 Hz), 4.72 (2H, s), 3.15 (3H, s), 3.10-3.00 (2H, m), 2.95 (3H, s), 2.49 (1H, m), 1.64 -1.21 (6H, m), 0.99 (9H, s) and 0.86 (3H, t, J = 6.8 Hz)

Stage F: 2R - [(Benzyloxy-formyl-amino) methyl] hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide

2R- (Benzyloxyamino-methyl) -hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide (7.8 g, 19.9 mmol) was dissolved in dry THF (100 ml) and treated with 1-formyl -benzotriazole (3.08 g, 21.0 mmol). The reaction mass is stirred overnight at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate, washed with a 2 M sodium hydroxide solution and brine. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated to dryness under reduced pressure. The product is crystallized from ethyl ether-hexane (4.83 g, 57% two samples). ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.12 (0.6H, broad s), 7.89 (0.4H, broad s), 7.37 (5H. s), 6.25 (1 N, d, J = 9.3 Hz), 4.96 (0.6H broadened c), 4.86 (1H, d, J = 9.4 Hz). 4.80 (0.4H, broad s), 3.74 (2H, broad s), 3.13 (3H, s), 2.94 (3H. S). 2.53 (1H, m), 1.38-1.21 (6H, m), 0.95 (9H, s) and 0.85 (3H, t, J = 6.9 Hz). Note: A small amount of the sample is crystallized from ethyl ether-hexane to obtain crystals suitable for X-ray diffraction analysis. The stereochemistry given in the description is confirmed.

Step G: 2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide

2R - [(Benzyloxy-formyl-amino) -methyl] -hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-1-propyl) amide (4.72 g, 11.3 mmol) was dissolved in ethanol (80 ml) and placed under a layer of argon. A suspension of 10% palladium on carbon (940 mg) in ethyl acetate (2 ml) was added and the mixture was vigorously stirred as hydrogen was bubbled through the system. After 30 minutes, the suspension was placed under a stream of hydrogen balloon and stirred overnight at room temperature. The flask was saturated with argon before removing the catalyst by filtration. The filtrate was concentrated under reduced pressure to obtain the title compound as a colorless foam, which crystallized upon standing (3.65 g, 98%). ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.32 (0.4H, broadened s), 8.41 (0.4H, s), 7.88 (0.6H, broadened s), 7.27 (0.6H, s), 6.75 (0.4H, broadened d, J = 8.8 Hz), 6.58 (0.6H, broad d, J = 9.3 Hz), 4.89 (1H, m), 4.04 (0.4H, m), 3.82 (0.6H, m), 3.53 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.98 (1.2H, s), 2.96 (1.8H, s), 2.79 (0.6, m), 2.65 (0.4H, m), 1.78-1.58 (6H, m), 0.99 (3.6H, s), 0.95 (5.4H, s) and 0.87, 3H, t, J = 6.7 Hz). ¹³ C-NMR; δ (CDCl ₃ ; rotamers), 175.8, 173.3, 172.0, 55.4, 54.9, 52.2, 48.8, 46.3, 38.9, 38.8, 36.3, 36.1, 30.3, 30.2, 29.7, 26.9, 23.0 and 14.3. LRMS: + ve ion 352 [M + Na], -ve ion 328 [M-H]. The compounds of Examples 14 to 27 were obtained by a method similar to that described in Example 13, Method I, by replacing the ethyl ester ester with the corresponding ether in Step A. If both diastereoisomers are obtained, diastereoisomer A elutes faster and is usually more potent against PDF in vitro. In some cases, only the more rapidly leaving diastereoisomer (Step E) is transferred to the final product.

Example 14

2R (or S) - [(Formylhydroxyamino) methyl] -3-cyclopentyl-propionic acid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A. Colorless glassy substance. ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.33 (0.4H, broadened s), 8.94 (0.6H, broadened s), 8.40 (0.4H, s), 7.82 (0.6H, s), 6.82 (0.4H, broadened d, J = 8.6 Hz), 6.62 (0.6H, broad d, J = 9.3 Hz), 4.90 (1H, m), 4.06 (0.4H, broadened dd, J = 14.7, 7.3 Hz), 3.81 (0.6H, broadened dd .J = 14.0, 9.7 Hz), 3.50 (1H, m), 3.16 (1.2H, s), 3.14 (1.8H, s), 2.97 (1.2N. S), 2.95 (1.8H, s), 2.80 ( 1H, m), 1.87-1.32 (9Н, m), 1.16-0.95 (2Н, m), 0.99 (3.6Н, s) and 0.95 (5.4Н, s). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 172.9, 171.3, 55.0, 54.5, 52.0, 48.6, 45.4, 44.2, 38.5, 38.4, 37.9, 37.6, 36.4, 36.3, 35.8, 35.6, 35.5, 32.7, 32.6, 26.5, 26.4 and 25.1. LRMS: + ve ion 378 [M + Na], -ve ion 354 [M-H].

Diastereoisomer B. Colorless glassy substance. ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.30 (0.6H, broadened s), 8.41 (0.6H, s), 7.75 (0.4H, s), 6.52 (0.4H, broadened d, J = 8.7 Hz), 6.41 (0.6 H, broadened d, J = 7.3 Hz), 4.85 (0.4H, broadened d, J = 9.5 Hz), 4.63 ′ (0.6H, broadened d, J = 7.5 Hz), 3.85-3.40 (2H, m), 3.25 -2.95 (6H, 3 broadened singlets), 2.78 (1H, 2 broadened multiplets), 1.90-1.40 (8Н, m), 1.30 (1Н, m), 1.20-1.00 (2Н, m) and 1.05-0.95 (9Н, 2c). ¹³ C-NMR; δ (CDCl ₃ ), rotamers), 174.9, 173.3, 172.8, 56.5, 54.7, 51.5, 50.5, 44.7, 44.6, 38.6, 38.4, 38.0, 37.8, 36.2, 36.0, 35.7, 35.5, 35.3. 34.3, 33.0, 32.9, 32.4, 32.3, 30.9, 26.6, 26.5, 25.1, 25.0 and 24.9. LRMS: + ve ion 378 [M + Na], -ve ion 354 [M-H].

Example 15

2R (or S) - [(Formyl-hydroxy-amino) methyl] heptanoic acid- (1S-dimethylcarbamoyl-2.2-dimethyl-propyl) -amide

Diastereoisomer A. Dark Orange Oil. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.32 (0.33H, s), 7.76 (0.67H, broadened s), 6.78 (0.33H, broadened d, J = 9.1 Hz), 6.68 (0.67H, broadened d, J = 9.1 Hz), 4.87-4.79 (1H, m), 3.96 (0.33H, broad dd, J = 14.6, 7.6 Hz), 3.74 (0.67H, broad dd, J = 13.9, 9.7 Hz), 3.51-3.36 (1H, m), 3.09 (1H, s), 3.08 (2H, s), 2.90 (1H, s), 2.89 (2H, s), 2.86-2.55 (1H, m), 1.53-1.19 (8H, broad m), 0.92 (3H, s), 0.88 (6H, s) and 0.79 (3H, m). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 174.3, 172.0, 170.5, 170.4, 54.0, 53.5, 53.4, 50.8, 49.7, 47.4, 44.9, 43.8, 37.5. 37.4, 34.8, 34.7, 34.6, 30.6, 29.2, 29.1, 25.8, 25.5, 21.4 and 12.9. LRMS: + ve ion 344 M + H], -ve ion 342 [M-H].

Diastereoisomer B. Dark orange oil. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.36 (0.5H, s), 7.74 (0.5H, s), 6.69 (0.5H, broadened s), 6.57 {0.5H, broadened d, J = 7.6 Hz), 4.89 (0.5 H, broadened s), 4.70 (0.5Н, d, J = 7.8 Hz), 3.76-3.40 (2Н, m), 3.21 (1.5Н.с), 3.16 (1.5Н, s), 2.98 (3Н.с) , 2.81 (1H, broad s), 2.72-2.60 (1 N, m), 1.67 (2H, broad s), 1.42-1.22 (6H, m), 1.02 (4.5H, s), 0.99 (4.5H, s ), 0.90 (1.5H, s) and 0.87 (1.5H, s). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 175.2, 173.8, 173.1, 56.5, 55.1, 52.3, 51.1, 50.6, 45.8, 45.5, 39.0, 38.9, 36.6. 26.3, 35.6, 34.9, 32.1, 32.0, 30.1, 29.9, 27.4, 27.4, 27.0, 26.9, 22.9 and 14.3. LRMS: + ve ion 344 [M + H], -ve ion 342 [M-H].

Example 16

2R (or S) - [(Formyl-hydroxy-amino) methyl] pentanoic acid- (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A. White hygroscopic foam. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.40 (0.33Н, s), 7.83 (0.67Н, broadened s), 6.88 (0.33Н, broadened d, J = 8.6 Hz), 6.69 (0.67Н, broadened d. J-9.2 Hz), 4.90 (1H, t). 4.06 (0.33H, broad dd, J-14.5, 7.4 Hz), 3.82 (0.67H, broad dd, J = 13.7, 9.8 Hz), 3.57-3.44 (1H, m), 3.16 (1H, s), 3.15 ( 2H, s), 2.98 (1H, s), 2.96 (2H, s), 2.87-2.63 (1H, m), 1.64-1.26 (4H, broad m), 0.98 (3H, s), 0.94 (6H, s ) and 0.90 (3H, t, J = 7.3 Hz). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 175.8, 173.2, 172.0, 55.4, 54.9, 52.2, 48.7, 46.2, 45.0, 38.9, 38.9, 36.3, 36.1, 36.1, 32.7, 32.6, 27.0, 26.9, 20.9, 20.8 and 14.4. LRMS: + ve ion 338 [M + Na], -ve ion 314 [M-H].

Example 17

2R (or S) - [(Formyl-hydroxy-amino) -methyl] -4-methyl-pentanoic acid- (1S-dimethyl-carbamoyl-2,2-dimethylpropyl) -amide

Diastereoisomer A. White hygroscopic solid. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.41 (0.4Н, s), 7.83 (0.6Н, s), 6.65 (0.4Н, d, J = 8.6 Hz), 6.55 (0.6Н, d, J = 9.0 Hz), 4.91-4.83 (1Н, m), 4.03-3.95 (0.4Н, m), 3.84-3.74 (0.6Н, m), 3.62-3.43 (1Н.m), 3.16 (1H, s), 3.13 (2Н, s ), 2.98 (1H, s), 2.96 (2H, s), 2.89-2.79 (0.6H, m). 2.76-2.71 (0.4N.m), 1.69-1.34 (1.8N, m), 1.29-1.20 (1.2Н, m), 1.0 (3.6Н, s), 0.95 (5.4N. S) and 0.93-0.88 ( 6H, m). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 175.8, 173.3, 172.0, 171.7, 55.5, 55.0, 52.4, 49.1, 44.3, 43.2, 39.5, 39.4, 38.9, 38.8, 36.3, 36.1, 27.0. 26.9, 26.3, 26.0, 23.1, 23.0 and 22.8. LRMS: + ve ion 352 [M + Na], -ve ion 328 [M-H].

Example 18

3-Cyclohexyl-2R (or S) - [(Formylhydroxyamino) methyl] propionic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) amide

White solid. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.38 (0.25Н, s), 7.82 (0.75Н, s), 6.93 (0.25Н, d, J = 8.9 Hz), 6.74 (0.75Н, d, J = 8.9 Hz), 4.90 (1Н, d, J = 9.4 Hz), 4.02 (0.25Н, dd, J = 9.7, 14.1 Hz), 3.78 (0.75Н, dd, J = 9.7, 14.1 Hz), 3.46 (1Н, m), 3.15 (3H.s), 2.96 (3H, s), 2.92 (1H, m), 1.65 (6H, m), 1.20 (5H, m). 0.98 (9H, s) and 0.87 (2H, m). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 176.4, 174.2, 172.4, 56.0, 55.6, 53.4, 49.9, 44.0, 43.3, 39.6, 39.4, 38.7, 38.5, 36.9, 36.7, 36.6, 34.8, 34.5, 27.5, 27.4 and 27.2. LRMS: + ve ion 370 [M + H], 368 [M-H].

Example 19

2R (or S) -cyclopentyl-3- (Formyl-hydroxy-amino) -propionic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A. Foam off-white. ¹ H-NMR; δ (CD ₃ OD, rotamers), 8.22 (0.33H, s), 7.79 (0.66H, s), 4.89 OH, s), 3.87 (1H, m), 3.50 (1H, m), 3.19 (3H, s ,), 2.93 (3Н, s), 2.82 (0.66Н, m), 2.65 (0.33Н, m), 1.89 (2Н, m), 1.56 (5Н, m), 1.24 (2Н, m) and 0.98 (9Н) , from). ¹³ C-NMR; δ (CD ₃ OD, rotamers), 176.0, 56.7, 53.2, 51.1, 42.7, 39.2, 36.5, 36.4, 32.0, 27.4, 26.4 and 26.2. IR (reflective tab.) V _max 3318, 2953, 1663, 1628, 1529, 1367, 1229, 1142, 1087, 877 cm ^-1 . LRMS: + ve ion 364 [M + Na], -ve ion 340 [M-H].

Example 20

2R (or S) - [(Formyl-hydroxy-amino) methyl] octanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A. ¹ H-NMR; δ (CDCl ₃ ), rotamers), 8.40 (0.4H, s), 7.83 (0.6H. s), 6.88 (0.4H, d, J = 8.9 Hz), 6.68 (0.6H, d, J = 9.2 Hz) , 4.90 (1H, m), 4.05 (0.4H, m). 3.81 (0.6H, m), 3.50 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.97 (1.2H. S), 2.96 (1.8H, s), 2.86 (0.6 N, m), 2.69 (0.4N, m), 1.59-1.25 (10N.m), 1.14-0.95 (9N.m) and 0.89-0.77 (3N.m). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 175.2, 172.9, 171.6, 171.4, 54.9, 54.5, 54.3, 52.0, 48.4, 46.1. 45.7, 45.1, 44.7, 39.7, 38.5, 38.4, 35.8, 35.6, 35.6, 31.7, 31.5, 30.2, 30.1, 29.1, 29.1, 27.0, 26.4, 22.4 and 14.0. LRMS: + ve ion 380 [M + Na], 358 [M + H], -ve ion 356 [M-H].

Example 21

2R (or S) - [(Formyl-hydroxy-amino) methyl] nonanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A: a brown solid. ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.30 (0.4H, s), 8.41 (0.6H, s), 7.83 (0.4H, s), 6.66 (0.4H, d, J = 8.9 Hz), 6.52 (0.6H, d, J = 9.7 Hz), 4.92-4.84 (1 N, m), 4.06-3.97 (0.4Н, m), 3.87-3.77 (0.6Н, m), 3.63-3.45 (1Н.m), 3.16 (1.2 H, s), 3.14 (1.8H, s), 2.98 (1.2H, s), 2.96 (1.8H, s), 2.86-2.74 (0.6H, m), 2.66-2.63 (0.4H, m), 1.95 -1.25 (12H, m), 1.00-0.95 (9N.m), and 0.90-0.84 (3H, m). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 175.5, 172.8, 171.4, 162.2, 156.1, 55.1. 54.5, 51.3, 50.8, 48.4, 46.3, 44.9, 38.4, 38.4, 35.8, 35.7, 33.9, 31.7, 30.3, 30.2, 29.4, 29.0, 27.1, 26.5, 26.5, 24.9. 22.6 and 14.0. LRMS: + ve ion 394 [M + Na], 372 [M + H], -ve ion 370 [M-H].

Example 22

2R (or S) - [(Formyl-hydroxy-amino) methyl] -decanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A: a colorless oil. LRMS: + ve ion 408 [M + Na], 386 [M + H], -ve ion 384 [M-H].

Example 23

2R (or S) - [(Formyl-hydroxy-amino) -methyl] -5-methylhexanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A: a colorless oil. ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.31 (0.4Н, s), 8.40 (0.4Н, s), 8.17 (0.6Н, s), 6.77 (0.4Н, d, J = 7.5 Hz), 6.60 (0.6Н, d, J = 8.0 Hz). 4.89 (1H, m), 4.04 (0.4H, m), 3.83 (0.6H, m), 3.52 (1H.m), 3.16 (1.2H, s), 3.15 (1.8H. S), 2.98 (1.2H , s), 2.96 (1.8Н, s), 2.70 (1Н, m), 1.58-1.14 (5Н, m), 1.00-0.95 (9Н, m) and 0.87-0.84 (6Н, m). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 172.9, 171.5, 162.2. 156.3, 55.1, 54.6, 51.4, 48.5, 46.4, 45.0, 38.5, 38.4, 36.2, 35.9, 35.6, 29.7, 28.1, 28.0, 27.9, 26.7, 26.6, 26.5 and 22.4. LRMS: + ve ion 366 [M + Na], 344 [M + H], -ve ion 342 [M-H].

Example 24

2R (or S) - [formyl-hydroxy-amino) methyl] propanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A: ¹ H-NMR; δ (CDCl ₃ ), rotamers), 8.41 (0.55Н, s), 7.81 (0.45Н, s), 6.67 (0.45Н, d, J = 8.4 Hz), 6.51 (0.45Н, d, J = 7.2 Hz) , 4.88 (0.45Н, d, J = 9.4 Hz), 4.66 (0.55Н, d, J = 7.7 Hz), 3.76 (1Н, m), 3.55 (0.55Н, dd, J = 14.3, 9.8 Hz). 3.44 (0.45Н, dd, J = 14.2, 5.3 Hz), 3.21 (1.65Н, s), 3.14 (1.35Н, s), 2.99 (1.65Н, s), 2.97 (1.35Н, s), 2.81 (1H , m), 1.21 (1.65Н, d, J = 6.7 Hz), 1.19 (1.35Н, d.J = 6.8 Hz), 1.01 (4.95Н, s) and 0.98 (4.05Н, s). LRMS: + ve ion 310 [M + Na], -ve ion 286 [M-H].

Diastereoisomer B: ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.47 (0.4H, s), 8.41 (0.4H, s), 7.86 (0.6H, s), 6.96 (0.4H, s), 6.74 (0.6H, d, J = 7.3Hz), 4.91 (1H, m), 3.99 (0.4N. Dd, J = 14.2, 7.6 Hz), 3.83 (0.6H, dd, J = 13.8, 9.0 Hz), 3.50 (1H, m), 3.16 (1.2H, s), 3.15 (1.8H, s), 2.97 (3H, s), 2.90 (1H, m), 1.21 (1.2H, d, J = 6.8 Hz), 1.15 (1.8N. D, J = 6.5 Hz), 0.99 (3.6H, s) and 0.95 (5.4H, s). LRMS: + ve ion 310 [M + Na], -ve ion 286 [M-H].

Example 25

2R (or S) - [(Formyl-hydroxy-amino) methyl] -3-methylbutanoic acid (1S-dimethyl-carbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A: ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.33 (0.4H, s), 8.38 (0.4H, s), 7.81 (0.6H, s), 6.86 (0.4H, broadened s), 6.58 (0.6N. d, J = 8.6 Hz), 4.90 (1H, m), 4.06 (0.4H, dd, J = 14.7, 7.3 Hz), 3.91 (0.6H, dd, J = 13.8, 10.6 Hz), 3.17 (1.2H, s), 3.15 (1.8H, s), 2.98 (1.2H, s), 2.96 (1.8H, s), 2.62 (0.6H, m), 2.48 (0.4H, m), 1.90 (1H, m), 1.09-0.86 ( 15H, m). LRMS: + ve ion 338 (M + Na), -ve ion 314 (M-H).

Example 26

2R (or S) - [(Formyl-hydroxy-amino) -methyl] -3-phenyl-propionylpropionic acid- (1S-dimethylcarbamoyl-2,2-dimethylpropyl) amide

Diastereoisomer A. Colorless glassy substance. ¹ H-NMR; δ (CDCl ₃ , rotamers), 9.33 (0.3H, broadened s), 8.95 (0.7H, broadened s), 8.43 (0.3H, broadened s), 7.83 (0.7H, broadened s), 7.27-7.10 (5H, m), 6.65 (0.3H, broadened s), 6.45 (0.7H, broadened d, J = 8.2 Hz), 4.80-4.70 (1 N, m), 4.22-4.10 (0.3H, m), 3.89 (0.7 N, dd, J = 13.7, 9.6 Hz), 3.63-3.47 (1Н, m), 3.20-2.69 (3Н.m), 3.04 (3Н, broadened s), 2.86 (3Н, broadened s), and 0.87 (9Н expanded with). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 137.9, 137.7, 128.8, 128.5, 126.6, 54.9, 54.5, 51.3, 48.3, 47.3, 46.6, 38.3. 38.2. 36.2, 36.1, 35.8, 35.7, 35.6, 35.5 and 26.4. LRMS: + ve ion 386 (M + Na), -ve ion 362 (M-H).

Example 27

2R (or S) - [(Formyl-hydroxy-amino) -methyl] -3- (4-methoxyphenyl) propionic acid- (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

Diastereoisomer A: LRMS: + ve ion 416 (M + Na), 394 (M + H), -ve ion 392 (M-H)

The compounds of Examples 28 to 31 are prepared in a similar manner to that described in Example 13, Method II, replacing the corresponding amide or benzyl ether of the amino acid tert-leucine N.N-dimethylamide in Step E.

Example 28

2S- {2R- [Formyl-hydroxy-amino) methyl] hexanoylamino} -3-phenylpropionic acid

White foam. ¹ H-NMR; δ (CD ₃ OD, rotamers), 8.11 (0.35H, s), 7.80 (0.65H, s), 7.31-7.16 (5H, m), 4.68 (1H, dd, J = 8.7, 5.5Hz), 3.58 ( 1H, m), 3.39 (1H, m), 3.19 (1H, m), 2.98 (1H, m), 2.76 (1H, m), 1.55-1.26 (6H, m) and 0.90-0.85 (3H, m) . ¹³ C-NMR; (CD ₃ OD, rotamers), 176.1, 175.7, 174.7, 174.5, 138.6, 138.5, 130.3, 129.5, 129.4, 127.7, 55.0, 53.3, 49.8, 45.4, 38.4, 38.3, 31.0, 30.8, 30.1, 23.7 and 14.2. IR (reflective tab.) V _max 2932, 2359, 1727, 1660, 1551, 1454, 1381, 1221, 882, 701 cm - ¹ . LRMS: + ve ion 359 [M + Na], -ve ion 335 (M-H).

Example 29 2S- {2R- [Formyl-hydroxy-amino) methyl] hexanoylamino} -3,3-dimethylbutanoic acid

White foam. ¹ H-NMR; δ (CD ₃ OD, rotamers), 8.25 (0.3H, s), 7.82 (0.7H, s), 4.31 (1H, s), 3.83-3.29 (2H, m), 3.10-2.89 (1H, m), 1.54-1.33 (6H. M), 1.03 (3H, s), 1.01 (6H, s) and 0.92-0.87 (3H, m). ¹³ C-NMR; δ (CD ₃ OD, rotamers), 174.9, 172.9, 61.0, 52.4, 44.2, 44.0, 33.6, 30.1, 29.1, 26.2, 22.6 and 13.1. IR (reflective table) v _max 2959, 2359, 1644, 1537, 1371, 1218, 881 and 704 cm -1. LRMS: + ve ion 325 (M + Na), -ve ion 301 (M-H).

Example 30

2S- [2R- (Formyl-hydroxy-amino) -methyl] -hexanoic acid {1 - [(2S-hydroxymethyl-pyrrolidine-1-carbamoyl] -2,2-dimethyl-propyl} -amide

Colorless oil. ¹ H-NMR; δ (CD ₃ OD, rotamers), 8.26 (0.4H, s), 7.84 (0.6H, s), 4.62 (0.4H, d, J = 8.2 Hz), 4.39 (0.6H, d, J = 8.4 Hz) , 4.12 (1H, m), 3.91-3.37 (6H, broad m), 2.93 (0.6H, m), 2.78 (0.4H, m), 1.93 (5H, m), 1.45 (2H, m), 1.39 ( 3H, m), 0.97 (3H, broadened s), 0.95 (3H, broadened s), and 0.89 (3H, t, J = 6.7 Hz). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 174.8, 172.9, 65.3, 65.1, 59.6, 59.5, 55.9, 55.7, 51.9, 47.8, 44.7, 44.0, 31.5, 30.5, 29.3, 28.7, 28.1, 27.3, 23.8, 22.0, 21.2. 18.7, 18.3, 17.6, 14.7 and 13.3. LRMS: + ve ion 394 (M + Na), 372 (M + H), -ve ion 370 (M-H).

Example 31

2S- [2R- (Formyl-hydroxy-amino) -methyl] -hexanoic acid {1 - [(2-hydroxy-ethyl) methylcarbamoyl] -2,2-dimethyl-propyl} -amide

White foam. ¹ H-NMR; δ (CD ₃ OD, rotamers), 8.25 (0.25H, s), 8.03 (0.125H, s), 7.82 (0.625H, s), 4.88 (1H, m), 3.83-3.54 (4H, broadened m), 3.41 (2H, m), 3.25 (2H, s), 2.96 (2H, s and m), 1.49 (2H, m), 1.23 (4H, m), 1.00 (6H, s), 0.99 (3H, s) and 0.88 (3H. m). ₁₃ C-NMR; δ (CD ₃ OD, rotamers), 173.6, 164.4, 61.1, 61.0, 56.9, 56.5, 54.2, 53.9, 52.2, 41.8, 38.9, 36.9, 36.3, 35.3, 31.6, 30.8, 27.5, 24.1 and 14.7. LRMS: + ve ion 382 [M + Na], -ve ion 358 [M-H].

The compounds of Examples 32 through 59 were prepared in a similar manner to that described in Example 7, Method II, replacing the amino acids tert-leucine-NN-dimethylamide in Step E with the corresponding amine or amide / benzyl ester. In some cases, HOAt in Step E is used and carry out deprotection by hydrogenolysis (Stage G) under catalytic transfer conditions (cyclohexene, palladium on charcoal in ethanol)

Example 32

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid- (1R-dimethylcarbamoyl-2,2-dimethylpropyl) amide

Colorless oil. LRMS: + ve ion 330 [M + H], -ve ion 328 [M-H].

Example 33

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-dimethylcarbamoyl-28-methyl-butyl) -amide

White foam. LRMS: + ve ion 352 [M + Na], -ve ion 328 [M-H].

Example 34

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-

dimethylcarbamoyl-2-methoxy-2-methylpropyl) amide

From racemic p-hydroxymethylvalvin. Diastereoisomer A. Colorless oil. LRMS: + ve ion 368 [M + Na], 346 [M + H], -ve ion 344 [M-H]. Diastereoisomer B. LRMS: + ve ion 368 [M + Na], 346 [M + H], -ve ion 344 [M-H].

Example 35

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-dimethylcarbamoyl-2-hydroxy-2-methylpropyl) -amide

Colorless oil. LRMS: + ve ion 354 [M + Na], -ve ion 330 [M-H].

Example 36

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid [2- (4-chlorophenyl) -1S-dimethylcarbamoylethyl] amide

Colorless oil. LRMS: + ve ion 330 (M + H), -ve ion 328 (M-H).

Example 37

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [1S-dimethylcarbamoyl-2- (4-hydroxyphenyl) ethyl] -amide

Colorless oil. LRMS: + ve ion 402 (M + Na), 380 (M + H).

Example 38

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1S-dimethylcarbamoyl-2-naphthalen-2-yl-ethyl) -amide

Colorless oil. LRMS: + ve ion 414 (M + H), -ve ion 412 (M-H) -

Example 39

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (2-cyclohexyl-1S-dimethylcarbamoyl-ethyl) -amide

White foam. LRMS: + ve ion 392 (M + Na), 370 (M + H)

Example 40

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-dimethylcarbamoylphenylmethyl) -amide

Colorless oil. LRMS: + ve ion 350 (M + H), -ve ion 348 (M-H).

Example 41

2- {2R - [(Formyl-hydroxy-amino) -methyl] -hexanoyl} -1,2,3,4-tetrahydro-isoquinoline-3S-carboxylic acid dimethylamide

LRMS: + ve ion 398 (M + Na), 376 (M + H), -ve ion 374 (M-H).

Example 42

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (4-amino-1S-dimethylcarbamoyl-butyl) -amide

Colorless oil. LRMS: + ve ion 345 (M + H), -ve ion 343 (M-H).

Example 43

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-dimethylcarbamoyl-2-hydroxy-ethyl) -amide

Colorless oil. LRMS: + ve ion 326 (M + Na), -ve ion 302 (M-H).

Example 44

N-Hydroxy-N- [2R- (4-methylpiperazine-1-carbonyl) hexyl] formamide

LRMS: + ve ion 272 [M + H].

Example 45

N-Hydroxy-N- [2R- (morpholine-4-carbonyl) hexyl] formamide

LRMS: + ve ion 281 (M + Na), 259 (M + H), -ve ion 257 (M-H).

Example 46

N-Hydroxy-N- [2R- (2S-hydroxymethyl-pyrrolidin-1-carbonyl) -hexyl] -formamide

LRMS: -ve ion 271 (M-H).

Example 47

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-hydroxymethyl-2,2-dimethylpropyl) -amide

LRMS: + ve ion 289 (M + H), -ve ion 287 (M-H).

Example 48

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-methoxymethyl-2,2-dimethylpropyl) -amide

LRMS: + ve ion 303 (M + H), -ve ion 301 (M-H).

Example 49

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [1S- (4-benzyl-piperidin-1-carbonyl) -2,2-dimethylpropyl] amide

LRMS: -ve ion 458 (M-H).

Example 50

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [1S- (benzyl-phenylethylcarbamoyl) -2,2-dimethylpropyl] -amide

LRMS: + ve ion 496 (M + H), -ve ion 494 (M-H).

Example 51

2S - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [2,2-dimethyl-1S- (pyrrolidine-1-carbonyl) -propyl] -amide

LRMS: + ve ion 356 (M + H), -ve ion 354 (M-H).

Example 52

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [2,2-dimethyl-1S- (morpholine-4-carbonyl) -propyl] -amide

LRMS: + ve ion 372 (M + H), -ve ion 370 (M-H).

Example 53

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid [2,2-dimethyl-1S- (4-methylpiperazine-1-carbonyl) propyl] amide

LRMS: + ve ion 385 (M + H), -ve ion 383 (M-H).

Example 54

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid [2,2-dimethyl-1S- (4-methylpiperidin-1-carbonyl) propyl] amide

LRMS: + ve ion 384 (M + H), -ve ion 382 (M-H).

Example 55

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (1S-cyclohexylcarbamoyl-2,2-dimethylpropyl) -amide

LRMS: + ve ion 398 (M + H), -ve ion 396 (M-H).

Example 56

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [1S- (4-acetyl-piperidin-1-carbonyl) -2,2-dimethylpropyl] amide

LRMS: + ve ion 412 (M + H), -ve ion 410 (M-H).

Example 57

1- (2S {2R - [(Formyl-hydroxy-amino) methyl] hexanoylamino} -3,3-dimethyl-butyryl) piperidine-4-carboxylic acid methyl ester

LRMS: + ve ion 442 (M + H), -ve ion 440 (M-H).

Example 58

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid [2,2-dimethyl-1S- (octahydroquinoline-1-carbonyl) propyl] amide

LRMS: + ve ion 424 (M + H), -ve ion 422 (M-H).

Example 59

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [1S- (3,4-dihydro-2H-quinoline-1-carbonyl) -2,2-dimethylpropyl] amide

LRMS: -ve ion 416 (M-H).

Example 60

2S- {3-Ethylsulfanylmethyl-2R - [(formylhydroxyamino) methyl] propionylamino} -3,3, N, N-tetramethylbutyramide

The synthesis route for the title compound is shown in Scheme 4 and is described in detail below.

Reagents and conditions;

A. 3-lithium-4-benzyl-oxazalidin-2-one, THF, -78 ° C;

B. LiN (SiCH ₃ ) ₂ , THF, -78 ° C, then AcCl;

C. HCl, H ₂ NOBzl, NaOAc, aqueous standard;

D. NaCNBH ₃ , AcOH, room temperature;

E. HCOBt, THF;

F. LiOH, aqueous THF, 0 ° C;

G. H-Tle-N (CH ₃ ) ₂ , HOAt, EDC, DMF;

H. H ₂ Pd / C, methanol, followed by separation of diastereoisomers (HPLC).

Stage A: 2-Ethylsulfanylmethyl-acrylic acid

A mixture of malonic acid (5.2 g, 50 mmol), paraformaldehyde (3.3 g, 110 mmol), dicyclohexylamine, 9.95 ml, 50 mmol) and ethanethiol (4.06 ml, 55 mmol) in dioxane (120 ml ) heated at 70 ° C for 2 hours. The solvents were removed under reduced pressure, the residue was redissolved in ethyl acetate, and the solution was extracted with saturated aqueous sodium bicarbonate (4 x 20 ml). The combined aqueous layers were washed with ethyl acetate (20 ml), then acidified with 1 M hydrochloric acid. The resulting suspension was extracted with dichloromethane and the solution was dried over anhydrous magnesium sulfate, filtered and evaporated to give the title compound as a white solid (3.76 g, 52%). ¹ H-NMR; δ (CDCl ₃ ), 9.89 (1H, broad s), 6.35 (1H, s), 5.77 (1H, s), 3.39 (2H, s), 2.49 (2H, dd, J = 7.4, 14.5 Hz) and 1.25 (3H, t. J = 5.2 Hz).

Stage B: 4S-Benzyl-3- (2-ethylsulfanylmethyl-acryloyl) -5,5-dimethyl-oxazolidin-2-one 2-Ethylsulfanylmethyl-acrylic acid (3.76 g, 25.8 mmol) is dissolved in dry THF ( 75 ml) and cooled to -78 ° C under a layer of argon. Triethylamine (4.6 ml, 33.5 mmol) and pivaloyl chloride (3.17 ml, 25.8 mmol) are added at such a rate that the temperature remains below -60 ° C. The mixture was stirred at -78 ° C for 30 minutes, warmed to room temperature for 2 hours and finally cooled again to -78 ° C.

4S-benzyl-5,5-dimethyl-oxazolidin-2-one is dissolved in dry THF (75 ml) in a separate flask and cooled to -78 ° C under a layer of argon. n-Butyllithium (2.4 M solution in hexane, 12.9 ml, 30.9 mmol) was added slowly and the mixture was stirred for 30 minutes at room temperature. The resulting anion is transported through the tube 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 saturated sodium bicarbonate solution (20 ml) and the solvents were removed under reduced pressure. The residue was partitioned between ethyl acetate and water. The organic layer was washed sequentially with saturated sodium bicarbonate, 1 M hydrochloric acid and brine, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 20% ethyl acetate in hexane) to provide the title compound as a yellow oil (6.5 g, 76%). ¹ H-NMR; δ (CDCl ₃ ), 7.29 (5H, m), 5.58 (1H, s), 5.49 (1H. s), 4.54 (1H, dd, J = 3.9, 9.7 Hz), 3.52 (2H, dd, J = 15.8 , 3.1 Hz). 3.38 (1H, dd, J = 3.9, 14.5 Hz), 2.84 (1H, dd, J = 4.6, 14.3 Hz), 2.52 (2H, dd, J = 7.2, 14.6 Hz), 1.42 (3H, s). 1.29 (3H, s) and 1.22 (3H, t, J = 7.5 Hz). LRMS: + ve ion 356 (M + Na), 334 (M + H).

Stage C: 4S-Benzyl-3- [2 R-tert. butoxyamino-methyl) -3-ethylsulfanylmethyl-propionyl] -5,5-dimethyl-oxazolidin-2-one

4S-Benzyl-3- (2-ethylsulfanylmethyl-acryloyl) -5,5-dimethyl-oxazolidin-2-one (2.1 g, 6.3 mmol) was dissolved in ethanol (10 ml) and 0-tert .- was added. butylhydroxylamine hydrochloride (0.95 g, 7.56 mmol) and then triethylamine (1.1 ml, 7.87 mmol). The mixture was stirred at 30 ° C. overnight. The solvents were removed under reduced pressure and the residue was dissolved in ethyl acetate. The organic solution was washed sequentially with 1 M hydrochloric acid, saturated sodium bicarbonate and brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to provide the title compound as a pale yellow oil (2.42 g, 91%; HPLC single diastereoisomer). ¹ H-NMR; δ (CDCl ₃ ), 7.30 (5H, m), 5.09 (1H, broad s), 4.54 (1H, dd, J = 3.5, 9.9 Hz), 4.33 (1H, m), 3.19 (2H, m), 3.08 (1H, dd, J = 5.4, 11.8 Hz), 2.80 (3H, m), 2.56 (2H, dd, J = 7.4, 14.7 Hz), 1.41 (3H, s), 1.36 (3H, s), 1.23 ( 3H, t, J = 7.3 Hz) and 1.13 (9H. S). LRMS: + ve ion 423 (M + H).

Stage D: (2R-tert-butoxyamino-methyl) -3-ethylsulfanylmethylpropionic acid

Solution of 4S-Benzyl-3- [2R-tert.-butoxyamino-methyl) -3-ethylsulfanylmethylpropionyl] -5,5-dimethyl-oxazolidin-2-one in (2.42 g, 5.72 mmol) THF (40 ml ) is cooled to 0 ° C and a solution of lithium hydroxide (288 mg, 6.86 mmol) in water (10 ml) is added. The mixture was allowed to warm to room temperature, after which it was stirred for 5 hours. The solvent was removed under reduced pressure and the residue was partitioned between water and ethyl acetate. The aqueous layer was removed and the ethyl acetate layer was washed successively with water and saturated sodium bicarbonate solution. The combined aqueous layers were washed with ethyl acetate (20 ml) and then acidified with 1 M hydrochloric acid. The resulting emulsion was extracted with dichloromethane (3 × 20 ml) and the combined organic layers were dried over anhydrous magnesium sulfate, filtered and evaporated to provide the title compound as a colorless oil (0.68 g, 50%). ¹ H-NMR; δ (CDCl ₃ ), 8.03 (2H, broad s), 3.21 (2H, d, J = 6.1 Hz), 2.89 (2H, m), 2.75 (1H, m), 2.57 (2H, dd, J = 7.4, 14.8 Hz), 1.26 (3H, t, J = 7.4 Hz) and 1.18 (9H, s). LRMS: + ve ion 236 [M + H], -ve ion 234 M-H].

Step E: Solution 2S- [2R- (tert.-butoxyaminomethyl) -3-ethylsulfanylmethylpropionylamino} -3,3, N, N-tetramethylbutyramide 2R-tert-butoxyaminomethyl) -3-ethylsulfanylmethyl-propionic acid (340 mg, 1.44 mol) was dissolved in DMF (10 ml) and tert-leucine-N, N-dimethylamide (272 mg, 1.73 mmol), HOAt (19.6 mg, 0.14 mmol) and EDC were added. (331 mg, 1.73 mmol). The reaction mass is stirred overnight at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane. The organic solution was washed successively with 1 M hydrochloric acid, 1 M sodium carbonate solution and brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to δ (CDCl ₃ ), 6.87 (1H, d, J = 9.0 Hz), 5.11 (1H, broadened s), 4.93 (1H, d, J = 9.3 Hz), 3.15 (3H, s ), 3.11 (1H, m), 2.95 (3H. S), 2.79 (3H, m), 2.54 (3H, s), 1.22 (3H, t, J = 7.6 Hz), 1.18 (9H, s) and 1.01 (9H, s). LRMS: + ve ion 398 [M + Na], 376 [M + 1].

Step F: 2S- {2R - [(tert-butoxy-formyl-amino) methyl] -3-ethylsulfanylmethylpropionylamino} -3,3, N, N-tetramethylbutyramide.

A solution of 2S- [2R- (tert.-butoxy-amino-methyl) -3-ethylsulfanylmethyl-propionylamino} -3,3, N, N-tetramethylbutyramide (220 mg, 0.58 mmol) in dichloromethane (5 ml) is cooled to 0 ° C and treated with mixed formic acetic anhydride (0.1 ml). The reaction mass was stirred at room temperature for 4 hours, then the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (silica gel, 50% ethyl acetate in hexane as eluent) to provide the title compound as a colorless oil (120 mg, 52%). ¹ H-NMR; δ (CDCl ₃ rotamers), 8.31 (1H, broadened s), 6.56 (1H, d. J = 9.1 Hz), 4.94 (0.33H, d, J = 9.4 Hz), 4.88 (0.67H, d, J = 9.2 Hz), 4.08 (0.67Н, broadened m), 3.83 (1.34Н, broadened m), 3.13 (3Н, s), 2.95 (3Н.с), 2.80 (2Н, m). 2.61 (1H, dd, J = 6.8, 14.0 Hz), 2.49 (2H, dd, J = 7.4, 14.7 Hz), 1.29 (9H, s), 1.25 (3H, t, J = 7.2 Hz) and 0.99 (9H , from). LRMS: + ve ion 426 [M + Na], 404 [M + H].

Stage G: 2S {3-Ethylsulfanylmethyl-2R - [(formyl-hydroxy-amino) -methyl] propionyl-amino} -3,3, N, N-tetramethylbutyramide

A solution of 2S- {2R - [(tert.-butoxy-formyl-amino) methyl] -3-ethylsulfanylmethylpropionylamino} -3,3, N, N-tetramethylbutyramide (120 mg, 0.3 mmol) in deuterochloroform (1 ml) treated with trifluoroacetic acid (4 ml) and allowed to stand at 4 ° C overnight. The solvents were removed under reduced pressure and the remaining trifluoroacetic acid was removed by azeotropic distillation with toluene. The residue was purified using preparative HPLC to provide the title compound as a colorless oil (40 mg, 38%; 7: 2 mixture of diastereoisomers using HPLC). ¹ H-NMR; δ (CDCl _3. rotamers), 8.40 (0.33Н, s), 7.87 (0.67Н, s), 7.24 (0.33Н, d, J = 9.3 Hz), 6.98 (0.67Н, d, J = 9.3 Hz), 4.91 (0.67Н.d, J = 9.3 Hz), 4.90 (0.33Н, d, J = 9.3 Hz), 4.07 (0.33Н, dd, J = 7.5, 14.5 Hz), 3.86 (0.67Н, dd, J = 8.8, 14.2 Hz), 3.75 (0.67Н, m), 3.68 (0.33Н, m), 3.16 (1Н, s), 3.15 (2Н, s), 3.05 (1Н, m), 2.96 (3Н, s), 2.77 (1H, m). 2.66 (1H, m), 2.52 (2H, dd, J = 7.4, 14.8 Hz), 1.22 (3H, t, J = 7.3 Hz), 0.99 (3H, s) and 0.96 (6H, s). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 173.3, 171.6, 171.2, 55.2, 54.8, 51.1, 48.5, 45.2, 44.4, 38.5, 38.4, 35.9, 35.8, 35.7, 31.7, 31.4, 26.7, 26.6, 26.5 and 14.6. LRMS: + ve ion 370 [M + Na], 348 [M + H], -ve ion 346 [M-H]. The compound of Example 61 was prepared analogously by using piperidine instead of ethanethiol in Step A.

Example 61

2- {2 - [(Formyl-hydroxy-amino) -methyl]: - 3-piperidin-1-yl-propionylamino} -3,3, N, N-tetramethylamide of butyric acid

White solid (4: 1 mixture of diastereoisomers using HPLC). ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.29 (1H, s), 7.95 (1H, broadened s), 4.87 (1H, d, J = 9.1 Hz), 4.02 (1H, dd, J = 5.0. 14.6 Hz), 3.56 (1H, dd, J = 8.2, 14.6 Hz). 3.14 (ZN, s), 2.96 (ZN, s), 2.89 (1N.m), 2.69 (1H, m), 2.52 (5H, m). 1.65 (4H, m), 1.49 (2H, m) and 0.99 (9H, s). ¹³ C-NMR; δ (CDCl ₃ ), 172.2, 171.3, 60.4, 55.0, 54.9, 48.6, 42.4, 38.8, 36.2, 36.1, 27.0, 25.6 and 24.3. LRMS: + ve ion 371 [M + H], -ve ion 369 [M-H].

The compounds of Examples 62 to 65 were prepared in a similar manner to that described in Example 7, Method II, replacing O-tert-butylhydroxylamine 0-benzylhydroxylamine with Step B and the corresponding amines or amide / benzyl ester of the amino acid tert-leucine N, N-dimethylamide in Step E. The final deprotection is carried out by acidolysis with trifluoroacetic acid (see Example 60 above).

Example 62

2R - [(Formyl-hydroxy-amino) methyl] hexanoic acid (1R-dimethylcarbamoyl-2-methyl-2-methylsulfanylpropyl) amide

Colorless oil. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.4 (0.5H, s), 7.85 (0.5H, s), 7.11 (0.5H, d, J = 9.1 Hz), 6.93 (0.5H, d, J = 9.1 Hz), 5.15 (1H, d, J = 9.4 Hz). 3.90 (0.5N.m), 3.73 (0.5H, m), 3.64 (0.5H, d, J = 14.3 Hz), 3.48 (0.5H, dd, J = 14.0. 3.9 Hz). 3.22 (3H, s), 2.97 (3H, s), 2.83 (0.5H, m), 2.70 (0.5H, m), 2.07 (1.5H, s), 2.04 (1.5H, s), 1.58 (1 N , m), 1.36 (4Н, m), 1.32 (3Н, s), 1.28 (3Н, s) and 0.86 (3Н, t, J = 6.6 Hz). ₁₃ C-NMR; δ (CDCl ₃ ), rotamers), 175.4,173.5, 170.8, 63.6, 53.2. 53.1, 52.5,, 49.5, 47.5, 46.1, 44.9, 41.6, 37.5, 36.5, 36.4, 35.4, 30.2, 29.8, 28.0, 14.3, 12.0 and 11.9. LRMS: + ve ion 362 [M + H], -ve ion 360 [M-H].

Example 63

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid (2-benzylsulfanyl-1R-dimethylcarbamoyl-2-methyl-propyl) -amide

White foam. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.37 (0.33Н, s), 7.81 (0.66Н, s), 7.31 (5Н, m), 7.06 (0.33Н, d, J = 8.8 Hz). 6.89 (0.66Н, d, J = 9.3 Hz), 5.20 (1Н, d, J = 9.3 Hz), 3.94 (0.33Н, dd, J = 8.3, 14.6 Hz), 3.78 (2.66Н, m), 3.61 ( 0.33Н, dd, J = 3.5, 14.4 Hz), 3.42 (0.66Н, dd, J = 5.1, 14.9 Hz), 3.21 (3Н, s), 3.03 (3Н, s), 2.82 (0.66Н, m), 2.69 (0.33Н, m), 1.61 (1 Н, м), 1.42 (1 Н, м), 1.37 (3Н, s), 1.32 (3Н, s), 1.26 (4Н, m) and 0.86 (3Н, t) , J = 6.6 Hz). ¹³ C-NMR; δ (CDCl _3, rotamers), 175.3, 173.5, 171.0, 138.1, 137.4, 129.5, 129.3, 129.1, 129.0, 128.9, 127.6, 127.4, 55.9, 53.7, 52.5, 51.2, 49.6, 49.5, 46.1, 44.9, 39.0, 38.6, 36.6, 36.4, 33.9, 33.7, 30.3, 30.1, 29.7, 26.7, 26.1, 25.7, 25.5, 24.2, 22-9 and 14.3. LRMS: + ve ion 460 [M + Na], 438 [M + H], -ve ion 436 [M-H].

Example 64

2R - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [2-benzylsulfanyl-2-methyl-1R- (morpholine-4-carbonyl) propyl] amide

White foam. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.44 (0.5H, s), 8.37 (0.5H, s). 7.30 (5N.m), 6.88 (0.5H, d, J = 8.3 Hz), 6.78 (0.5H, d, J = 9.2 Hz), 5.12 (1H, d, J = 9.5 Hz), 3.91 (1H, dd , J = 8.2, 14.6 Hz), 3.78 (10Н, m), 3.45 (1Н, dd, J = 4.5, 14.2 Hz), 2.80 (0.5Н, m), 2.64 (0.5Н, m), 1.61 (1Н, m), 1.41 (1H, m), 1.36 (3H, s), 1.33 (3H, s), 1.29 (4H, m) and 0.87 (3H, t, J = 6.8 Hz). ¹³ C-NMR; δ (CDCl ₃ , rotamers), 175.5, 173.4, 169.4, 137.8, 129.5, 129.3, 129.1, 129.0, 127.8, 127.5, 67.1, 67.0, 53.3, 53.2, 51.99, 49.6, 49.5, 49.2, 47.9, 46.5, 45.0, 43.2. 43.0, 34.0, 30.3, 30.2, 29.7, 26.8, 26.5, 25.9, 25.8, 22.9 and 14.3. LRMS: + ve ion 502 [M + Na], 480 [M + H], -ve ion 478 [M-H].

Example 65

2 - [(Formyl-hydroxy-amino) -methyl] -hexanoic acid [2-benzylsulfanyl-2-methyl-1R (or S) - (4-methylpiperidin-1-carbonyl) propyl] amide

Diastereoisomer A. White solid. LRMS: + ve ion 514 [M + Na], 492 [M + H], -ve ion 490 [M-H].

Diastereoisomer B. Colorless resin. LRMS: + ve ion 514 [M + Na], 492 [M + H], -ve ion 490 [M-H].

The compounds of Examples 66 to 68 were prepared in a manner analogous to that described in Example 7, Method II, replacing the corresponding malonic acid with butyl malonic acid in Step A, O-tert-butyl hydroxylamine with O-Benzylhydroxylamine in Step C. The stereo selectivity in the Michael addition reaction was varied. The final deprotection is carried out by acidolysis with trifluoroacetic acid (see example 60, above).

Example 66

2R - [(Formyl-hydroxy-amino) -methyl] -pent-4-enoic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) -amide

The only diastereoisomer. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.40 (0.25Н, s), 7.84 (0.75Н, s), 7.05 (0.35Н. d, J = 9.0 Hz), 6.74 (0.65Н, d, J = 9.3Iu), 5.70 (1H, m), 5.03-5.24 (2H, m), 4.88 (1H, dd, J = 9.4, 6.7 Hz), 3.98 (0.5H, m). 3.81 (0.5H, m), 3.55 (1H, m), 3.14 (3H, s). 2.97 (1.3H, s), 2.96 (1.7H, s), 2.75-2.92 (1H, m), 2.16-2.50 (2H, m), 0.98 (4.5H, s) and 0.94 (4.5H, s). LRMS: + ve ion 336 [M + Na], -ve ion 312 [M-H].

Example 67

2R - [(Formyl-hydroxy-amino) -methyl] -hex-5-enoic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) -amide

Diastereoisomer A: a colorless oil. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.42 (0.45Н, s), 7.84 (0.55Н, s), 6.78 (0.45Н, d, J = 8.4 Hz), 6.60 (0.55Н, d, J = 9.3 Hz), 5.74 (1H, m), 5.03 (2H, m), 4.88 (1H, m), 4.14 (0.4H, m), 3.81 (0.6H, m), 3.55 (1H, m), 3.16 (1H, s) , 3.15 (2H, s), 2.98 (1H, s), 2.97 (2H, s), 2.85 (0.7H, m), 2.68 (0.3H, m), 2.07 (2H. M), 1.73 (1.6H, m), 1.50 (0.4H, m), 0.99 (4H, s) and 0.95 (5H, s). LRMS: + ve ion 350 [M + Na], -ve ion 326 [M-H]. Diastereoisomer B: a colorless oil. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.41 (0.5Н, s), 7.75 (0.5Н, s), 6.58 (0.5Н, d, J = 9.1 Hz), 6.36 (0.5Н, d, J = 9.1 Hz), 5.75 (1H, m), 5.01 (2H, m), 4.86 (0.5H, d, J = 9.5Iu), 4.64 (0.5H, d, J = 7.5In), 3.42-3.82 (2H, m), 3.22 (1.5Н, s), 3.07 (1.5Н, s), 2.99 (3Н, s), 2.87 (0.5Н, m), 2.66 (0.5Н, m), 2.13 (2Н, m), 1.81 (1Н, m ), 1.49 (1H, m), 1.02 (4.5H, s) and 1.00 (4.5H, s). LRMS: + ve ion 350 [M + Na], -ve ion 326 [M-H].

Example 68

2R - [(Formyl-hydroxy-amino) -methyl] -hex-4-inic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) -amide

Diastereoisomer A: a colorless oil. ¹ H-NMR; δ (CDCl ₃ , rotamers), 8.39 (0.4H. s), 7.87 (0.6H, s), 7.20 (0.4H, d, J = 8.4 Hz), 6.94 (0.6H, d, J = 9.3 Hz), 4.90 (1H, m), 3.66-4.14 (2H, m), 3.16 (2H, s), 3.14 (2H, s), 2.96 (3H, s), 2.88 (1H, m). 2.41 (2H, m). 1.77 (3H, m), 1.00 (3.5H, s) and 0.96 (5.5H, s). LRMS: + ve ion 348 [M + Na], -ve ion 324 [M-H].

Diastereoisomer B: a colorless oil. ¹ H-HMP; δ (CDCl ₃ , rotamers), 8.37 (0.5H, s), 7.81 (0.5H, s), 6.87 (1H, m), 4.91 (0.5H, d, J = 9.4 Hz), 4.79 (0.5H, d , J = 8.2 Hz). 3.76 (1.5H, m), 3.63 (0.5H, m), 3.19 (1.5H, s), 3.14 (1.5H, s), 2.98 (3H, s), 2.85 (1H, s), 2.41 (2H, m), 1.77 (3H, m), 1.03 (4.5H, s) and 1.01 (4.5H, s). LRMS: + ve ion 348 [M + Na], -ve ion 324 [M-H].

Example 69

2R- [1R (or S) - (Formyl-hydroxy-amino) ethyl] hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethyl-propyl) -amide

The named compound is obtained according to the synthesis pathway presented in Scheme 5 and described in detail below:

Scheme 5

Reagents and conditions:

A. (CHO) n, EtSH, dicyclohexylamine, dioxane, 70 ° C, 2 hours;

B. Tert-BuCOCl, triethylamine, then 3-lithium-4-benzyl-5,5-dimethyloxazolidin-2-one;

C. HCl, H ₂ NO-tert-Bu, triethylamine, o / n;

D. LiOH, aqueous THF, 0 ° C;

E. H-tert.-LeuN (Me ₂ ) ₂ , HOAt, EDC, DMF;

F. HCOAc, CH ₂ Cl ₂ ;

G. trifluoroacetic acid, chloroform.

Stage A: 4-Benzyl-3-hexanoyl-oxazolidin-2-one 4S-Benzyl-oxazolidin-2-one (14.5 g, 81.7 mmol) was dissolved in dry THF (75 ml) under argon. The solution was cooled in an ice bath before the slow addition of n-butyllithium (1.6 M in hexane, 56 ml, 89.2 mmol). A lithium salt crystallizes from the solution in the form of a solid mass, and it is allowed to warm to room temperature overnight. The resulting orange suspension was again cooled in an ice bath while adding a cooled solution of hexanoyl chloride (10.4 ml, 74.3 mmol) in dry THF (50 ml). The mixture was allowed to warm to room temperature and then stirred for 3 hours. The reaction was quenched with a 1 M sodium carbonate solution (5 ml) and the solvent was removed under reduced pressure. The residue was partitioned between 1 M sodium carbonate solution (100 ml) and ethyl acetate (150 ml). The organic layer was removed and the aqueous layer was extracted with additional ethyl acetate. The combined organic layers were washed successively with water, 1 M sodium carbonate solution and brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to give an orange oil. Purification by flash chromatography afforded the title compound as a yellow oil (10.21 g, 50%). ¹ H-NMR; δ (CDCl ₃ ), 7.38-7.24 (3H, m). 7.24-7.16 (2H, m), 4.68 (1H, m), 4.24-4.12 (2H, m), 3.30 (1H, dd, J = 13.4, 3.2 Hz), 3.02-2.86 (2N.m), 2.77 ( 1H, dd, J = 13.4, 9.6 Hz), 1.77-1.63 (2H, m), 1.44-1.30 (4H, m) and 0.92 (3H, broadened t, J = 6.9 Hz).

Stage B: 1- (4S-Benzyl-2-oxo-oxazolidin-3-yl) -2R-butyl-butane-1,3-dione 4-Benzyl-3-hexanoyl-oxazolidin-2-one (10.2 g , 37.1 mmol) is dissolved in THF (150 ml) in an argon atmosphere and cooled to -78 ° C. Lithium hexamethyldisilazide (1 M in THF, 41 ml, 41 mmol) was introduced through the tube for several minutes and the resulting green solution was stirred at -78 ° C for 2 hours. Acetyl chloride (3.3 ml, 46.3 mmol) was added slowly and the reaction mixture was stirred for 3.5 hours. A solution of citric acid (3.0 g, 14 mmol) in water (15 ml) is quickly added and the reaction is quenched. The solvent was removed under reduced pressure, and the residue was partitioned between ethyl acetate and water, washed with brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated to provide the title compound as a yellow oil (12.11 g, residual solvent contained), which was used without further purification in Step C. ¹ H-NMR; δ (CDCl ₃ ,), 7.37-7.21 (5Н, m), 4.68 (1Н, m), 4.53 (1Н, dd, J = 9.6, 3.7 Hz), 4.23-4.13 (2Н, m), 3.43 (1Н, dd, J = 13.5, 3.3 Hz), 2.75 (1-N, dd, J = 13.5, 9.9 Hz), 2.33 (3H, s), 2.03 (1H, m), 1.77 (1 N, m), 1.46- 1.26 (4H, m) and 0.98-0.86 (3H, m).

Stage C: 1- (4S-Benzyl-2-oxo-oxazolidin-3-yl) -2K-butylbutan-1,3-dione 3- (0-benzyloxy)

To a solution of 1- (4S-benzyl-2-oxo-oxazolidin-3-yl) -2R-butyl-butan-1,3-one (12, 11 g, 38.15 mmol) in water (10 ml) and ethanol (90 ml) sodium acetate (3.75 g, 45.78 mmol) and 0-benzylhydroxylamine hydrochloride (7.31 g, 45.78 mmol) are added. The resulting suspension was allowed to stir at room temperature overnight. The product (7.3 g, 45%, the only oxime isomer) is crystallized directly from the reaction mixture and filtered, washed with aqueous ethanol (1: 1) and dried under vacuum. An additional product (5.31 g, 33%, a mixture of oxime isomers) was obtained as a yellow mother liquor by acid-base extraction and then column chromatography. ¹ H-NMR; δ (CDCl ₃ , basic oxime isomer), 7.34-7.20 (8Н, m), 7.12-7.07 (2Н.m), 5.14-5.02 (2Н, m), 4.53 (1Н, m), 4.13 (1Н, dd, J = 9.4, 4.0 Hz), 4.04 (1H, broad t, J = 8.4 Hz), 3.91 (1H, dd, J = 9.0, 2.7 Hz), 3.16 (1H, dd, J = 13.4, 2.9 Hz), 2.09 (3H, s), 1.97 (1H, m), 1.75 (1H, dd J = 13.4, 10.8 Hz), 1.67 (1H, m), 1.45-1.22 (4H, m) and 0.91 (3H, broadened t, J = 6.9 Hz).

Stage D: 4S-Benzyl-3- [2R- (1R (or S) -benzyloxyamino-ethyl) -hexanoyl] -oxazolidin-2-one

The mixture of the oximes obtained in Stage C (5.31 g, 12.5 mmol) was dissolved in acetic acid (30 ml) and cooled in an ice water bath, and then sodium cyanoborohydride (0.8 g, 12.5 mmol) was added. in one serving. After a few minutes, the rapid evolution of gas ceases, after which an additional portion of cyanoborohydride (0.8 g) is introduced. The reaction mass is allowed to warm to room temperature and it is stirred overnight. Acetic acid was removed under reduced pressure and the residue was distilled as an azeotrope with toluene. The resulting oil was dissolved in ethyl acetate, washed with water, 1 M sodium carbonate and brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated to give a pale yellow oil, which was purified by flash chromatography (silica gel, 10 to 25%, ethyl acetate in hexane as eluent). Yield 3.43 g, (64%). ¹ H-NMR; δ (CDCl ₃ , mixture of diastereoisomers), 7.36-7.17 (10Н, m), 5.80 (0.45Н, broadened s), 5.55 (0.55Н, broadened d, J = 8.9 Hz), 4.72-4.59 (ЗН, m), 4.20-4.05 (2H. M), 3.97 (0.45H, m), 3.82 (0.55H, m), 3.47-3.22 (2H, m), 2.45 (1H, m), 1.90-1.48 (2H, m). 1.40-1.14 (7H, m) and 0.95-0.84 (3H, m).

Stage E: M- [2R- (4S-Benzyl-2-oxo-oxazolidine-3-carbonyl) -1R (or S) -methyl-hexyl] -N-benzyloxyformamide

4S-Benzyl-3- [2R- (1 R (or S) -benzyloxyamino-ethyl) -hexanoyl] -oxazolidin-2-one (3.08 g, 7.3 mmol) was dissolved in dry THF and treated with N-formylbenzotriazole (1.60 g, 10.9 mmol). The reaction mass is stirred for 4 hours at room temperature. The solvent was removed under reduced pressure and the remaining oil was partitioned between dichloromethane (40 ml) and 1 M sodium hydroxide solution (30 ml). The organic layer was removed, washed with an additional amount of sodium hydroxide solution, then with brine, dried over anhydrous magnesium sulfate, filtered and evaporated. Purification by flash chromatography (silica gel, 20 to 50%, ethyl acetate in hexane gives the title compound as a pale yellow solid (2.50 g, 76%. ¹ H-NMR; δ (CDCl ₃ , mixture of diastereoisomers and rotamers ), 8.22 (1H, broad m), 7.54-7.13 (10H, m), 5.22-3.92 (7H, broad, m), 3.30 (1H, m), 2.48 (1H, broad, m), 1.85-1.13 ( 9H, broad m) and 0.93-0.83 (3H, m).

Stage F: 2R- [1R (or S) - (Benzyloxy-formyl-amino) -ethyl] -hexanoic acid N- [2R- (4S-Benzyl-2-oxo-oxazolidine-3-carbonyl) -1R (or S ) -methyl-hexyl] -N-benzyloxy-formamide (1.50 g, 3.31 mmol) was dissolved in THF (25 ml) and water (5 ml) and the solution was cooled in an ice water bath. A solution of hydrogen peroxide (27% v / v), 13.26 mmol) was added, and then lithium hydroxide (167 mg, 3.98 mmol) was immediately added. The reaction mixture was allowed to warm to room temperature and was stirred for an additional 3 hours. The solution was cooled again before adding sodium nitrite (0.92 g, 13.3 mmol). After 10 minutes, most of the solvent was removed under reduced pressure to give a white paste, which was partitioned between ethyl acetate (25 ml) and 1 M sodium carbonate (30 ml). The organic layer was washed with an additional sodium carbonate solution and the combined aqueous extracts were washed with ethyl acetate. The aqueous layer was cooled and acidified with 1 M hydrochloric acid and extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate, filtered and evaporated to provide the title compound as a green oil (839 mg, 86%). ¹ H-NMR; δ (CDCl ₃ , mixture of diastereoisomers and rotamers), 8.40-7.64 (2Н, broad m), 7.48-7.27 (5Н, m), 5.23-4.80 (2Н, m), 4.16 (1Н, broadened m). 2.79 (1H, m), 1.67-1.47 (2H, m), 1.47-1.18 (7H, m) and 0.95-0.82 (3H, m).

Stage G: 2R- [1R (or S) - (Benzyloxy-formyl-amino) ethyl] -hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) -amide 2R- [1R (or S) - (Benzyloxy- formyl amino) ethyl] hexanoic acid (839 mg, 2.86 mmol), tert-leucine NN-dimethylamide (498 mg, 3.15 mmol) and EDC (658 mg, 3.43 mmol) are dissolved together in DMF (15 ml) and a catalytic amount of HOAt (60 mg) was added. The solution was allowed to mix for several days at room temperature. The solvent was removed under reduced pressure and the remaining oil was partitioned between ethyl acetate and 1 M hydrochloric acid (75 ml). The organic layers are washed successively with 1 M hydrochloric acid, 1 M sodium carbonate solution and brine, dried over anhydrous magnesium sulfate, filtered and evaporated to give a yellow foam (1.08 g, 82%). ¹ H-NMR; δ (CDCl ₃ , mixture of diastereoisomers and rotamers), 8.13 (1H, broadened m), 7.52-7.31 (5H, m), 6.28 (1H, broadened m), 5.36-4.67 (3H, broadened m), 4.09 (1H, broad m), 3.14 (3H, s), 2.95 (1.2H, s), 2.93 (1.8H, s), 2.48 (1H, broad m), 1.61-1.04 (9H, m), 0.99 (3.6H, s ), 0.95 (5.4Н, s) and 0.89-0.75 (3Н, m).

Stage H: 2R- [1R (or S) - (Formyl-hydroxy-amino) -ethyl] -hexanoic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) -amide

2R- [1R (or S) - (Benzyloxy-formyl-amino) ethyl] hexanoic acid (tS-dimethylcarbamoyl-2,2-dimethylpropyl) -amide (200 mg, 0.46 mmol) is dissolved in methanol (15 ml ) and placed under a layer of argon. A suspension of 10% palladium on carbon (20 mg) in ethyl acetate was added and the mixture was stirred in a hydrogen atmosphere for 3 hours. The catalyst was removed by filtration and the filtrate was evaporated to give a colorless oil (163 mg, quantitative);

Two diastereoisomeric products are separated by preparative HPLC. Diastereoisomer A (27 mg): ¹ H-NMR; δ (CDCl ₃ , mainly one rotamer), 8.67 (0.9H, broadened s), 7.92 (1H, s), 6.74 (0.1 N, broadened m), 6.54 (0.9H, d, J = 9.4 Hz), 4.93 (0.9Н, d, J = 9.4 Hz), 4.64 (0.1 Н, broadened m), 3.89 (1Н, dd, J = 6.6, 2.6 Hz), 3.16 (3Н, s), 2.96 (3Н, s), 2.62 -2.48 (1Н, m), 1.52-1.06 (6Н.m), 1.35 (3Н, d, J = 6.6 Hz), 1.00 (9Н, s) and 0.82 (3Н, t, J = 6.9 Hz). ¹³ C-NMR; δ (CDCl ₃ ), 173.0, 171.3, 57.2, 54.4, 50.4, 38.4, 35.6, 29.9, 29.1, 26.6, 22.5, 17.2 and 13.9. LRMS: + ve ion 366 [M + Na], -ve ion 342 [M-H]. Diastereoisomer B (42 mg): ¹ H-NMR; δ (CDCl ₃ , mixture of rotamers), 9.15 (0.6H, s), 8.60 (0.4H, broadened s), 8.42 (0.6H, s), 7.84 (0.4H, s), 6.83 (0.6H, d, J = 9.2 Hz), 6.55 (0.4H, d, J = 9.4 Hz), 4.91 (0.6H, d, J = 9.2 Hz), 4.89 (0.4H, d, J = 9.4 Hz), 4.69 (0.6H, qdd , J = 7.0, 4.3 Hz), 3.92 (0.4 N. dq, J = 9.1, 6.8 Hz), 3.15 (3H, s), 2.97 (1.8H, s), 2.95 (1.2H, s), 2.59 (0.4 N, td, J = 9.8, 4.3 Hz), 2.39 (0.6Н, td, J = 7.4, 4.3 Hz), 1.92-1.07 (6Н, m), 1.37 (1.2Н, d, J = 6.8 Hz), 1.31 (1.8H, d, J = 7.0 Hz), 1.01 (5.4H, s), 0.96 (3.6N. S), 0.85 (1.8H, t, J = 7.2 Hz) and 0.83 (1.2H, t, J = 7.2 Hz). ¹³ C-NMR; δ (CDCl ₃ , mixture of rotamers), 175.7, 173.2, 171.3, 170.7, 56.7, 55.0, 54.4, 53.2, 50.8,49.9, 38.3, 35.7, 35.6, 35.5, 35.4, 30.3, 29.5, 29.3, 26.5, 26.4, 22.5 , 22.4, 16.0, 15.4 and 13.8. LRMS: + ve ion 366 [M + Na], -ve ion 342 [M-H].

Example 70

N-Cyclohexyl-2- {2 - [(formyl-hydroxy-amino) -methyl] -3-phenyl-propionylamino} -3,3-dimethylamide butyric acid

Stock solutions of 1 M ammonia in methanol (1 ml, 1 mmol) and 1 M trimethylacetaldehyde in methanol (1 ml, 1 mmol) were mixed in a test tube and allowed to stand for 1 hour. A 1 M solution of cyclohexyl isocyanide in methanol (1 ml, 1 mmol) was added, followed by 0.5 M 2RS - [(benzyloxyformylamino) methyl] hexanoic acid in methanol (2 ml, 1 mmol). The reaction mixture was stirred at room temperature for 2 days. The solvent was removed using a Savant centrifuge and the reaction mixture was crystallized from ethyl acetate-hexane to give 2- {2 - [(benzyloxy-formyl-amino) methyl] -3-phenyl-propionylamino} -N-cyclohexyl-3,3-dimethylamide acid in the form of a white solid (93 mg, 18%), which was deprotected by catalytic exchange hydrogenolysis (hydrogen, 10% palladium-carbon, methanol-ethyl acetate), which made it possible to obtain the named compound (75 mg, 99%) . White solid. LRMS: + ve ion 440 [M + Na], 418 [M + H], -ve ion 416 [M-H].

The compounds of Examples 71 to 77 were prepared in parallel using the Ugi 4-component condensation reaction, as described above. All products are obtained with a purity> 85%, as shown by HPLC.

Example 71

2- {2 - [(Formyl-hydroxy-amino) -methyl] -3-phenyl-propionylamino} -3,3-dimethylhexanoic acid cyclohexylamide

White solid (90 mg). ¹ H-NMR; δ (CD ₃ OD), 7.82 (1H, s), 7.29-7.08 (5H, m), 4.20 (1H, d, J = 5.0 Hz), 3.89 (1H, m), 3.19 (1H, m), 2.95 -2.67 (2H, m). 1.88-1.58 (5H. Broad m), 1.44-1.05 (9H, broad m) and 0.89 (9H, s). LRMS: + ueion 468 [M + Na], 446 [M + H], -ve ion 444 [M-H].

Example 72

2- {2 - [(Formyl-hydroxy-amino) -methyl] -3-phenyl-propionylamino} -3,3-dimethylhexanoic acid phenylmethylamide

White solid (77 mg). ¹ H-NMR; δ (CD ₃ OD), 7.82 (1H, s), 7.35-7.11 (10H, m), 4.38-4.19 (3H, m), 3.85 (1H, m), 3.52 (1H, m), 2.97-2.63 ( 3H, m), 1.37-1.11 (4H, m) and 0.93-0.78 (9H, m). LRMS: + ve ion 476 [M + Na], 454 [M + H].

Example 73

2- {2 - [(Formyl-hydroxy-amino) -methyl} -3-phenylpropionylamino} -3,3-dimethylbutanoic acid tert-butylamide

White solid (47 mg). ¹ H-NMR; δ (CD ₃ OD), 7.82 (1Н, s), 7.45 (1Н.m), 7.30-7.09 (5Н, m), 4.12 (1Н, d, J = 7.2 Hz), 3.89 (1Н, m), 3.41 (1N.m), 3.15 (1N.m), 2.97-2.68 (2H, m), 1.28 (9H, s) and 0.92 (9H, s). LRMS: + ve ion 414 [M + Na], 392 [M + H], -ve ion 390 [M-H].

Example 74

2 {2 - [(Formyl-hydroxy-amino) -methyl] -3-phenyl-propionylamino} -3,3-dimethylhexanoic acid (1,1,3,3-tetramethyl) butyramide.

White solid (65 mg). ¹ H-NMR; δ (CD ₃ OD), 7.79 (1H, s), 7.42-7.21 (1H, m), 7.20-7.10 (5H, m), 4.23 (1H, d, J = 9.1 Hz), 3.86 (1H, m) , 3.51 (1Н, m), 3.23 (1Н, m), 3.00-2.56 (2Н, m), 1.50-1.15 (12Н, m) and 1.02-0.83 (18Н, m). LRMS: + ve ion 498 [M + Na], 476 [M + H], -ve ion 474 [M-H],

Example 75

N- (Cyclohexyl-cyclohexylcarbamoyl-methyl) -2 - [(formyl-hydroxy-amino) -methyl] -3-phenylpropionamide

White solid (98 mg). ¹ H-NMR; δ (CD ₃ OD), 7.38-7.08 (5Н, m), 4.01 (1Н.m), 3.81 (1Н, m), 3.68-3.35 (2Н, m), 3.15 (1Н, m), 2.98-2.65 ( 2H, m), 1.88-1.49 (10H, broad m) and 1.45-0.83 (11 N, broad m). LRMS: + ve ion 466 [M + Na], 444 [M + H], -ve ion 442 [M-H].

Example 76

N- (Cyclohexyl-phenylmethylcarbamoyl-methyl) -2 - [(formyl-hydroxy-amino) -methyl] -3-phenylpropionamide

White solid (34 mg). ¹ H-NMR; δ (CD ₃ OD), 7.35-7.10 (10H, m), 4.44-4.23 (2H, m). 4.05 (1H, m), 3.87-3.35 (2H, m), 3.09 (1H, m), 2.85-2.72 (2N. M), 1.65-1.46 (4H, m), 1.38-0.93 (5H, broadened m) and 0.75-0.51 (2H, broad m). LRMS: + ve ion 474 [M + Na], -ve ion 450 [M-H].

Example 78

N- [Cyclohexyl- (1,1,3,3-tetramethyl-butylcarbamoyl) methyl] -2 - [(formyl-hydroxy-amino) methyl] -3-phenylpropionamide

White solid (51 mg). ¹ H-NMR; δ (CD ₃ OD), 7.80 (1H. s), 7.36-7.10 (5H, m), 4.05 (1H, m), 3.85 (1H, m), 3.49 (1H, m), 3.15 (1H, m) , 2.91 (1H, m), 2.68 (1H, m), 1.90 (1H, m), 1.80-1.48 (7H, m), 1.40-1.12 (UN, m) and 1.08-0.83 (10H, m). LRMS: + ve ion 496 [M + Na], 474 [M + H], -ve ion 472 [M-H].

Biological Example A.

Demonstration of the antibacterial effects of compound 1 (Example 1) and compound 2 (Example 13).

a).

Minimum inhibitory concentrations (MIC) of inhibitors against E. coli strain DH5a (Genotype F-f80d / acZDM15D (lacZYA-argF) U169 deoR recA1 endA1 hsdR17 (r $\genfrac{}{}{0ex}{}{\text{-}}{\text{k}}$ , m $\genfrac{}{}{0ex}{}{\text{+}}{\text{k}}$ ) phoA supE441 ^- thi-1 gyrA96 relA1) obtained from GibcoBRL Life Technologies, Enterobacter cloacae (U.S. culture type collection number 13047), Klebsiella pneumoniae (U.S. culture type collection number 13883) or Staphylococcus capitzs (U.S. culture type collection number 35661) determined in the following way. Stock solutions of the test compound (Compounds 1 and 2 of Examples 1 and 13, respectively (both isomers A)) and three standard laboratory antibiotics carbenicillin (Sigma, catalog number C3416), kanamycin (Sigma, catalog number K4000) and chloramphenicol (Sigma, catalog number C1919) were obtained by dissolving each compound in dimethyl sulfoxide at a concentration of 10 mM. To determine the minimum inhibitory concentration, two serial dilutions were prepared in 2xYT nutrient medium (typtone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L, obtained from BIO 101Inc, 1070 Joshua Way, Vista, CA92083 , USA) to obtain 0.05 ml of the compound containing medium per well. Inoculums were obtained from cultures grown overnight in 2xYT culture medium at 37 ° C. The cell density was adjusted to absorb at 660 nm (A ₆₆₀ ) = 0, l, the compositions standardized in optical density were diluted 1: 1000 with 2xYT growth medium, and 0.05 ml of diluted bacteria were inoculated into each well. Microtiter plates were incubated at 37 ° C for 18 hours in a wet incubator. MIC (mM) was defined as the minimum drug concentration at which visible growth was inhibited.

b)

Minimum Inhibitory Concentrations (MIC) of inhibitors against Mycobacterium ranae (American culture type collection number 110), Pseudomonas aeruginosa (American culture type collection number 9027), Klebsiella pneumoniae (American culture type collection number 10031), Helicobacter pylori (American culture type collection number 43504), clinical cultures resistant to Streptococcus pneumoniae aminoglycosides and erythromycins and methicillin resistant (MR) Staphylococus aureus (US culture type collection number 33591) were determined as following manner, stock solutions of test compounds 1 and 2 (both isomers A) three standard laboratory antibiotics gentamycin (G), ampicillin (A) and erythromycin (E), were prepared by dissolving each compound in dimethylsulfoxide at a concentration of 10 mM. The same methods were used as for a), except that Brain Heart Infasion (GIBCO) was used as the medium for Mycobacterium ranae and incubated at 37 ° C for 48 hours for Staphylococcus aureus (MR), Klebsiella pneumoniae and Pseudomonas aeruginosa used Nutrient nutrient medium (DIFCO), incubated them at 37 ° C for 20 hours, Columbia agar-based medium (OXOID) containing 7% sheep blood was used for Helicobacter pylori and incubated at 37 ° C in within 72 hours, Streptococcus pneumoniae used tryptone soybean nutrient medium (DIFCO) containing 7% calf serum, and incubated them at 37 ° C for 48 hours. MIC (mM) was defined as the minimum drug concentration at which visible growth was inhibited.

With a positive control of organisms (1% DMSO, without test compound), the growth of all microorganisms was ensured.

In the negative empty control (absence of microorganisms + test compound), microorganisms did not grow.

In another experiment, the minimum inhibitory concentrations of compound 1 and the product from Example 13 (compound 3) for gram-positive and gram-negative bacteria were determined using the Microdilution Broth Method according to the approved standard methodology of the National Committee for Clinical Laboratory Standards (Methids for dilution antimicrobial susceptibility tests for bacteria that grow aerobically- Fourth Edition ISBN 1-56238-309-4).

Activity against gram-positive bacteria.

Activity against gram-negative bacteria.

The activity of compound 3 and the product from Example 14 (compound 4) against Enterococcus faecalis clinical cultures resistant to many antibiotics, which were evaluated using the method used for the above results, are summarized in the following table, and comparison with the results obtained in the same way for known antibacterial agents:

Biological Example B

i) Cloning of the PDF gene of Escherichia coli .

The E. coli PDF gene was cloned into pET24a (+) (designated pET24-PDF) and used to transform BL21 DE3 cells from Novagen Inc. (Madison, Wisconsin). Clones were selected at 37 ° C on YT agar plates (tipton 8 g / l, yeast extract 5 g / l, NaCl 5 g / l, agar 15 g / l) with the addition of kanamycin.

i) PDF Expression

20 ml of overnight culture of BL21 DE3 cells containing pET24-PDF was used to infect 500 ml of 2xYT culture medium (tipton 16 g / l, yeast extract 10 g / l, NaCl 5 g / l) containing 30 μg / ml kanamycin, A 2-liter flask with a septum, and grown at 37 ° C with stirring to OD _{600 of} 0.6. The culture was then induced by adding to the medium to a concentration of 1.0 mM isopropyl-bD-thiogalactopyranoside (IPTG). Induction continued for the next 3 hours at 37 ° C, the cells were collected by centrifugation, the cell pellet was washed with 250 ml of saline phosphate buffer (PBS) and stored at -70 ° C.

iii) Preparation of a soluble protein fraction.

Cells from 1 L of the expression solution were resuspended in 2x25 ml of ice-cold saline phosphate buffer. The cell suspension in ice was sonicated using an MSE Soniprep 150 calibrated to the medium at an amplitude of 20-25 microns at 6 x 20 pulses per second. The resulting suspension was then purified by centrifugation at 20,000 xg for 15 minutes. Then the supernatant was used to further purify the enzyme.

iv) PDF Cleanup.

E. coli lysate from 1 L of culture in saline phosphate buffer (PBS) was added to 2 M ammonium sulfate. A 15 ml phenylsepharose column was equilibrated with PBS / 2M ammonium sulfate at 4 ° C. The lysate was loaded onto the column and washed with equilibration buffer. The column was washed with a decrease in the concentration of ammonium sulfate from 2 M to 0 M with a buffer volume equal to 10 column volumes. 5 ml fractions were collected and analyzed using SDS-PAGE. Fractions containing most of the 20 kDa PDF were combined. The pooled fractions were concentrated using a 3 kDa membrane to a volume of 5 ml. The fraction was then loaded onto a Superdex 75 column (molecular size exclusion chromatography) equilibrated with PBS. Concentrated pooled PDF fractions were eluted at 1 ml / min at 4 ° C, 5 ml fractions were collected and analyzed using SDS-PAGE. The purest fractions were combined and stored at -70 ° C.

(v) In vitro PDF study.

The study was carried out in a single 96-well plate in a final volume of 100 μl containing:

- 20 μl PDF (4 μg / ml)

- 20 μl 100 mM Hepes pH 7.0 + 1 M KCl + 0.05% Brij

- 10 μl of serial dilution of the test compound in 20% DMSO

- 50 μl formyl-Met-Ala-Ser (8 mm)

The plate was incubated at 37 ° C for 30 minutes. The free amino groups of the deformed product (Met-Ala-Ser) were determined using fluorescamine, adding the following components:

- 50 μl 0.2 M borate pH 9.5

- 50 μl of fluorescamine (150 μg / ml in dry dioxane)

Quantification of fluorescence was performed on an SLT Fluostar plate reader at an exciting wavelength of 390 nm and an emitted wavelength of 485 nm. The standard control reactions were those without inhibitor, which gave a zero inhibition schedule, and the enzyme-free and without inhibitor, which gave a 100% inhibition schedule. Data were analyzed by converting fluorescence units to% inhibition and a plot of% inhibition versus inhibitor concentration was plotted. The data were optimized as a sigmoid function: y = A + ((BA-A) / 1 + (C / x) ^D ))), where A represents zero inhibition, B represents 100% inhibition and C represents IC ₅₀ , D represents the angle of inclination. IC ₅₀ is the concentration of inhibitor (nM), which is required to reduce enzyme activity by 50%. It was found that the compounds of the invention inhibit bacterial PDF in vitro. Additionally, it was found that actinonin (Sigma, cat. No. A-6671) inhibits bacterial PDF in vitro.

Biological Example C.

The demonstration that compound 2 inhibits PDF in vivo.

1. Stopping the mPHKi-Met reformation reaction leads to stability with respect to compound 2 (diastereomer / isomer A).

Trimethoprim specifically inhibits dihydrofolate reductase, thereby reducing the reserves of tetrahydrofolate derivatives (THF), including formyl tetrahydrofolate (fTHF), a methionyl tRNA formyl transferase substrate (EC 2.1.2.9). If all essential metabolites whose biosynthesis includes THF derivatives, for example pantothenate, methionine, glycine, purine nucleotides and thymidine, are supplied externally in the form of precursor compounds in an enriched medium supplemented with thymidine, then bacteria grown in an enriched medium with thymidine (0.03 mM) and trimethoprim (100 μg / ml) can synthesize all chemical components of normal cells, with the exception of fMet-TPHKi (Baumstark et al., J. Bacteriol. 129 ^ 457-471, 1977). Instead, unformed Met-tRHKi is used, which leads to the formation of polypeptides that do not contain a formyl group at the N-terminus, regardless of the effect of deformylases. As predicted by the inventors, it was found that DH5a cells grown in LB medium (tipton 10 g / l, yeast extract 5 g / l, NaCl 10 g / l pH 7.5) with the addition of trimethoprim and thymidine are resistant to to compound 2 (diastereomer A). The demonstration that the growth of cells in which the expressed proteins undergo a normal deformation process is inhibited by Compound 2A, while the unformilated proteins that are produced by cells grown under these conditions are not inhibited by Compound 2A, shows that Compound 2A probably acts by inhibiting the deformylation reaction carried out by the PDF.

Table 3 Growing conditions The minimum inhibitory concentration, μm Lb fifteen LB trimethoprim (SOmkg / ml), thymidine (0.3 mM) > 200

2. Treatment of bacteria with compound 2A leads to the accumulation of proteins with a blocked N-terminus.

If the compounds of the invention actually inhibit PDF in vivo, the consequence of treating the bacteria with compound 2 (Example 2, diastereomer A) will be the accumulation of newly synthesized proteins with N-formylmethionine at the N-terminus. Such proteins will be blocked at the N-terminus and cannot be used as a substrate for sequencing by N-terminal chemical degradation according to Edman.

To verify this assumption, the desired protein is expressed in the presence or absence of the test compound. The protein is isolated, purified and then sequenced, subjecting it to degradation according to Edman, using techniques known to the person skilled in the art.

Bacterial cells transformed with an expression vector capable of expressing the small regulatory subunit of human calpain were grown to an OD _{600 of} 0.6 and then exposed to IPTG to induce the expression of a heterologous protein in the presence of 200 μM compound 2A, in the presence of 240 μM carbenicillin or in the presence of carrier control for 2.5 hours. Protein extracts were separated by SDS-PAGE, the calpain subunit was eluted and the protein sequence was determined by Edman chemical degradation using an ABI automatic protein sequencer. Sequenced equal amounts of protein. The inventors found that the protein yield during treatment with compound 2A was significantly reduced by 85% compared with the controls of the carrier and carbenicillin.

The calpainic small regulatory subunit was cloned from messenger RNA obtained from a biopsy of a stomach tumor using the InVitrogen Micro Fast Track ™ mRNA isolation kit, series 2.2 (catalog number K1520-02). A DNA copy of this RNA was synthesized using the Promega Riboclone ™ M-MLV-RT (H-), Noti cDNA synthesis system (Promega, catalog number C 1660), in accordance with the manufacturer's instructions. Two oligonucleotide primers for use in the polymerase chain reaction (PCR) were synthesized by Applied Biosystems, Inc., Custom Services based on the published sequence of the small calpain subunit (EMBL access number X04104).

The HindIII / Xhol calpain fragment was then cloned into the HindIII and Xhol cut expression vector pET24d (+) from Novagen Inc, (Madison, WI, USA) using standard techniques. A ligation mixture was used to transform competent DH5a cells (Life Technologies, Inc, Grand Island, NY, USACat # 18265-017). Colonies were selected by growing overnight at 37 ° C on YT plates with 30 μg / ml kanamycin. A plasmid DNA was obtained using the Promega Plus SV miniprep kit, and clones with a calpain insert were identified using standard techniques. The DNA sequence was confirmed using the Applied Biosystems PE sequencing cycle as described above.

The E. coli gene cloned into pET24d (+) (designated as pET24-CANS) was used to transform BL21 DE3 cells from Novagen Inc, (Madison, Wisconsin). Clones were selected at 37 ° C on agar plates (tipton 8 g / l, yeast extract 5 g / l, NaCl 5 g / l, agar 15 g / l) with the addition of 30 μg / ml kanamycin.

Claims (4)

1. The use of a compound of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof for the preparation of an antibacterial composition:

where R _{1 is} hydrogen or C ₁ -C ₆ alkyl; R ₂ is a group - (ALA) mR, where R is C ₁ -C ₆ alkyl or cycloalkyl; ALA - linear or branched divalent C ₁ -C ₆ alkylene; m is 0 or 1; A

R ₄ represents the side chain of a natural or unnatural alpha amino acid, R ₅ and R ₆ independently represent hydrogen or optionally substituted C ₁ -C ₈ alkyl, cycloalkyl, aryl, aryl (C ₁ -C ₆ alkyl), heterocyclyl or heterocyclyl (C ₁ -C ₆ alkyl). 2. The use of claim 1, wherein the compound of formula I is 2R (or S) - [formyl-hydroxyamino) methyl] hexanoic acid (1S-dimethylcarbamoylethyl) amide or a pharmaceutically or veterinarily acceptable salt thereof. 3. The use according to claim 1, wherein the compound of formula I is 2R (or S) - [(formyl-hydroxyamino) methyl] -3 cyclopentylpropionic acid (1S-dimethylcarbamoyl-2,2-dimethylpropyl) -amide or its pharmaceutically or a veterinarily acceptable salt. 4. An antibacterial pharmaceutical or veterinary composition containing a compound of formula I with the substituent values given in claim 1, together with a pharmaceutically or veterinarily acceptable excipient or carrier. According to claim 1 of the claims, in terms of features R ₅ and R ₆ - hydrogen or C ₁ -C ₆ -alkyl, priority is set on 02/07/1998 according to the first application GB 98025497. For the rest of the features of claim 1 of the formula and in paragraphs. 2-4 established priority from 02/05/1999 according to the international application PCT / GB 99/00386.