EP1838302A2 - Peptide deformylase inhibitors, their use, and pharmaceutical compositions containing the same - Google Patents

Abstract

The present invention relates to the use of indole derivatives or of pharmaceutically acceptable salts thereof, for the manufacture of medicaments intended for the prevention or the treatment of bacterial infections or protozoan infections, or for the manufacture of herbicides.

Description

PEPTIDE DEFORMYLASE INHIBITORS, THEIR USE, AND PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME

The present invention relates to peptide deformylase inhibitors, to pharmaceutical compositions containing them, and to their use as herbicides or for treating bacterial or parasitic infections.

Peptide deformylase (PDF; EC 3.5.1.88) is an essential enzyme activity found in all Gram-positive and Gram-negative bacteria. Its cellular role is to remove the N-formyl group from all nascent proteins. This allows the action of methionine aminopeptidase, another essential bacterial enzyme responsible for N-terminal methionine excision (for a review see Giglione et al, 2004). PDF is the natural target of actinonin, a natural antibiotic substance produced by a Streptomyces sp. (Gordon et al, 1962; Chen et al, 2000). Although less potent, other natural products inhibiting PDF also produced by Streptomyces sp. were described (Chu et al, 2001).

Actinonin

Based both on sequence and structural analysis, two bacterial PDF types - PDFl and PDF2 - have been distinguished (Giglione et al, 2000a; Guilloteau et al, 2002). PDF2 are found only in Gram-positive bacteria. Because high-resolution three-dimensional (3D) structure and in depth enzymatic analyses are available, Escherichia coli PDF (PDFlB) and Bacillus stearothermophilus PDF2 (PDF2) were chosen as the representative of either PDF class (Guilloteau et al, 2002; Ragusa et al, 1998). Bacteria can have one or several functional genes encoding a PDF. For instance, Escherichia coli has one PDFl gene encoded by the def cistron, major pathogens such as Staphylo- and Streptococci spp. have only one PDF2 and Bacillus spp. have two PDF genes, the def gene encoding a PDFl and ykrB a PDF2. In B. subilis, PDF2 is the major PDF (Haas et al, 2001).

For the past decade, PDF has been selected as a target of choice for the selection of new antibiotics using state-of-the-art drug discovery methods (Giglione et al, 2000a; Meinnel, 2000; Giglione & Meinnel, 2001). During the past 4 years, a number of PDF inhibitors (PDFI) mimicking actinonin were described and several of them recently entered clinical trial (for a review see Boularot et ah, 2004). Hence, PDFI are the first success for antibacterial drug discovery arising from genomics strategies (Miesel et ah, 2003).

However, although initially thought to be a unique feature of the eubacteria (Mazel et al, 199 A), functional PDF sequences have been detected most recently in most eukaryote genomes (Giglione et al, 2000b; Bracchi-Ricard et ah, 2001). These PDF are targeted to the organelles, the mitochondrion and the plastid of plants (chloroplasts) and apicomplexan parasites (apicoplasts) such as the agent of malaria, Plasmodium falciparum. Furthermore, PDF homologs have also been evidenced in kinetoplastid protozoans (Meinnel, 2000). Based on sequence or biochemical analysis and site-directed mutagenesis data, all mitochondrial PDFs were shown to belong to a unique sub-class of type 1 PDFs (PDFlA) and their active site showed a number of singularities (Giglione et al, 2000b; Serero et al, 2001; Serero et al, 2003). Plastid PDFs are strongly related to bacterial PDFIs and are classified as PDFlB (Giglione et ah, 2004). As actinonin shows inhibitory effects against both P. falciparum and plant PDF in vitro and in vivo, this discovery first suggested that PDFI active against bacterial PDF could be used as antiparasitic agents and even as herbicides (Meinnel, 2000; Giglione & Meinnel, 2001; Wiesner et al, 2001; Serero et al, 2001; Serero et al, 2001; Dirk et al, 2001).

Nevertheless, the occurrence of a PDFlA in the human mitochondria (HsPDFl) raised a number of issues regarding the toxicity of the above-described PDFI, especially as actinonin (i) blocks efficiently in vitro HsPDFl activity and (ii) exerts cytotoxic activity in a range of concentrations (2-3 μg/ml) similar to the minimal inhibitory concentration (MIC) of the best PDFI on the most sensitive Gram-positive bacteria (Lee et ah, 2003; Serero et ah, 2003; Xu et al, 1998; Grujic et al, 2002, Lee et al 2004). Although the role of mitochondrial PDF remains unclear and the mitochondrial membrane is generally considered to be very impermeable to most compounds, the precautionary principle imposes that great caution should now be given to avoid any cross- inhibition of PDFI between PDFlA and PDFlB or PDF2. In other words, this means that new-generation PDFI should not show any inhibitory effect on mitochondrial PDF (PDFlA), including at least HsPDF.

Thus, an object of the present invention is to provide new compounds which are liable to potently inhibit peptide deformylases from bacteria, plant plastids, and protozoans such as apicocomplexan and kinetoplastid protists, but not from mitochondria, and in particular from mammalian mitochondria. Another object of the present invention relates to the use of said compounds for treating individuals with a bacterial or a parasitic infection, or as herbicides.

The present invention follows on from the unexpected finding by the Inventors that indolic derivatives could potently inhibit bacterial peptide deformylases while being essentially inactive on human mitochondrial peptide deformylase.

The present invention relates to the use of compounds having: a) the following general formula (I):

(D wherein:

- independently from each other, R₂ and R₃ represent H, an alkyl group from 1 to 5 carbon atoms, or a metal chelating group from 2 to 20 atoms comprising from 1 to 5 heteroatoms;

- independently from one another, R₄, R₅, R₆, and R₇ represent H, a halogen, or a group comprising from 1 to 10 carbon atoms;

- R₁ represents H, or a group comprising from 1 to 50 carbon atoms; provided that at least one of R₂ and R₃ represents a metal chelating group as defined above ; and

b) an IC₅₀ lower than about 1 μM with respect to the Escherichia coϊi nickel-bound peptide deformylase (SEQ ID NO: 1) and/or to the Bacillus stearothermophilm nickel- bound peptide deformylase (SEQ ID NO: 2), or of pharmaceutically acceptable salts thereof, for the manufacture of medicaments intended for the prevention or the treatment of bacterial or protozoan infections, or for the manufacture of herbicides.

A "metal chelating group" as mentioned above relates to a group which is liable to form coordination complexes with metal atoms or metallic ions (Whittaker et al, 1999 ; Boularot et al, 2004). Thus a "metal chelating group" relates, in particular, to a group bearing one or several free electronic doublets. In a preferred embodiment, the invention relates to the use of compounds of the general formula (I) as defined above, wherein, independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula:

-(CH₂)_n-(X)_nl-Y wherein:

- n is an integer from 0 to 3,

- n_\ is 0 or 1,

- X represents a group selected from-O-, -NH-, -CHOH-, -CHF-, or -CO-, and

- Y represents a group selected from the list comprising -NH₂ (amine), -NHOH (hydroxylamine), -CH=NOH (oxime), -NHCN (cyanamide), -NH-C(NOH)NH₂

(hydroxyguanidine), -SCN (thiocyanate), -SH (thiol), -B(OH)₂ (boronic acid), -COOH (carboxylic acid), -COCH₂OH (hydroxymethylketone), -COCH₂SH (thiomethylketone), -CONHOH (hydroxamic acid), -SO₂NHOH (sulfohydroxamic acid), -NO-N=O (nonoate), -NOH-COH (reverse hydroxamic acid). In another preferred embodiment, the invention also relates to the use of compounds of the general formula (I) as defined above, wherein, independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula:

-(CH₂VY wherein: - n is an integer from O to 3, and

- Y represents a group selected from the list comprising -NHOH, -CONHOH, or -NOH-COH.

In another preferred embodiment, the invention relates to the use of compounds of the general formula (I) as defined above, wherein, independently from each other, R₄, R₅ and R₆ represent H, a halogen, an alkyl, an alkoxy, a thioalkyl, a thioaryl, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, or an aryloxycarbonyl group, if adequate substituted by a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group.

The invention relates more particularly to the use of compounds of the general formula (I) as defined above, wherein, independently from each other, R₄, R₅, R₆, and R₇ represent H, a halogen, an alkyl, or an alkoxy group from 1 to 10 carbon atoms.

According to a preferred embodiment, the invention also relates to the use of compounds of the general formula (I) as defined above, wherein R₁ represents - H, or - a peptide sequence, or - an alkyl, an alkoxy, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, a thioalkyl, a thioaryl, an arylsulfonyl, an aryloxycarbonyl, an alkoxycarbonylalkyl, or an aryloxycarbonylalkyl group, if adequate substituted by a pyridine, a morpholine, a thiomorpholoine, a piperidine, a piperazine, a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group.

In another preferred embodiment, the invention relates to the use of compounds of the general formula (I) as defined above, wherein R₁ represents H, a phenylsulfonyl group of formula— SO₂-C₆H₅, or a benzyloxycarbonyl group of formula -CO-O-CH₂-C₆H₅.

According to a particularly preferred embodiment, the present invention relates to the use of compounds as defined above, of the following general formula (II) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined in claims 1 to 7, and R₁, R₂, and R₃ are as defined above. According to another particularly preferred embodiment, the present invention relates to the use of compounds as defined above, of the following general formula (III) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined above, and R₁, R₂, and Y are as defined above.

In a particularly preferred embodiment, the present invention relates to the use of compounds as defined above, of the formula (III) wherein:

- R₀ represents H, a halogen atom such as Cl, F, or Br, an alkoxy group from 1 to 10 carbon atoms such as a methoxy OCH₃ group,

- R₂ represents H or CH₃,

- Y represents -NHOH, -CONHOH, or -NOH-COH, and - R₁ represents H, -SO₂-C₆H₅, or -CO-O-CH₂-C₆H₅.

In another particularly preferred embodiment, the present invention relates to the use of compounds of the general formula (I) as defined above, characterized in that said compounds correspond to the following formulae:

6e 6Ii

Peptide deformylase (PDF) is a metallo-enzyme (i.e. its prosthetic group is a metallic ion). Thus, as intended herein, the expression "nickel-bound peptide deformylase" relates to an enzyme which contains essentially only nickel as its metallic ion. For instance, such an enzyme can be obtained by purifying it in the presence of nickel as described in Ragusa et al.

(1998).

The expression IC₅₀ relates to the concentration of compounds of the general formula

(I) which is necessary to inhibit 50% of the activity the Escherichia coli nickel-bound peptide deformylase (SEQ ID NO: 1) (EMBL/GenBank accession number P27251) and/or to the Bacillus stearothermophilus nickel-bound peptide deformylase (SEQ ID NO: 2)

(EMBL/GenBank accession number 031410).

The IC_5O for peptide deformylase can be measured by following the general method described in Serero et al. (2003).

Briefly, prior to kinetic analysis (as described in Lazennec & Meinnel, 1997), inhibitors are incubated with the enzyme studied at a set final concentration for 15 minutes at

25⁰C. Kinetic assays are started by adding a small volume of the substrate. The substrate is 1 _o o

mM Formyl-Met- AIa-S er. The kinetic analysis is performed in the presence of an enzyme concentration giving a deformylation rate of 0.5 μM/s in the absence of inhibitor. The IC₅₀ value corresponds to a concentration giving 50% inhibition.

Advantageously, the compounds of general formula (I) are liable to inhibit bacterial PDF (i.e. PDFl and PDF2), as well as chloroplastic, apicoplastic and kinetoplastic PDF, but not the mitochondrial PDF (i.e. PDFlA).

In another particularly preferred embodiment, the present invention relates to the use of compounds of the general formula (I) as defined above, wherein the protozoans are chosen among the apicoplast or kinetoplast bearing protozoans, and in particular are selected from the group comprising the protozoans of the following genii: Plasmodium, Toxoplasma,

Sarcocystis, Cryptosporidium, Eimera, Theileria, Babesia, Trypanosoma, Leishmania.

Advantageously, the compounds of formula (I) target and inhibit the apicoplastic or the kinetoplastic PDF and thus respectively impair the function of the apicoplast and of the kinetoplast, which inhibits the growth of protozoan parasites and/or kills them. In yet another particularly preferred embodiment, the present invention relates to the use of compounds of the general formula (I) as defined above, wherein the bacteria are selected from the group comprising the bacteria of the following genii: Staphylococcus, Streptococcus, Bacillus, Enterococcus, Pseudomonas, Acinetobacter, Enterobacter, Haemophilus. Advantageously, the compounds of general formula (I) target and inhibit both the bacterial PDFl and PDF2, which makes them broad-spectrum antibiotics which can be used both against Gram-negative and Gram-positive bacteria.

The present invention also relates to a pharmaceutical composition, characterized in that it comprises at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, as active substance, in association with a pharmaceutically acceptable vehicle.

In a preferred embodiment, the above defined pharmaceutical composition is suitable for the administration to an individual of a unit dose of about 250 mg to about 5000 mg of the compound of formula (I). In another preferred embodiment, the above defined pharmaceutical composition is suitable for the administration to an individual of a daily dose of about 250 mg to about 5000 mg of the compound of formula (I).

In another preferred embodiment, the above defined pharmaceutical composition is administered by oral, intra- venous, or intra-peritoneal route. The present invention also relates to an herbicide composition, characterized in that it comprises at least one compound of formula (I) as defined above as active substance.

In a preferred embodiment, the above defined herbicidal composition is suitable for the administration to a plant of a dose of about 1 mg/1 to about 1000 mg/1, in particular of about 10 mg/1 to about 100 mg/1, of the compound of formula (I).

Advantageously, the compounds of formula (I) target and inhibit the chloroplastic PDF, which either kills or severely impairs the growth of chloroplasts holding organisms, such as plants or algae for example.

The present invention also relates to a compound of the general formula (I), such as defined above.

The invention relates more particularly to compounds of the general formula (I) :

(I) wherein:

- independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula:

-(CH₂VOOnI-Y wherein: * n is an integer from 0 to 3,

^ n₁ is 0 or 1,

* X represents a group selected from-O-, -NH-, -CHOH-, -CHF-, or -CO-, and

* Y represents a group selected from the list comprising -NH₂ (amine), -NHOH (hydroxylamine), -CH=NOH (oxime), -NHCN (cyanamide), -NH-C(NOH)NH₂ (hydroxyguanidine), -SCN (thiocyanate), -SH (thiol), -B(OH)₂ (boronic acid), -COOH (carboxylic acid), -COCH₂OH (hydroxymethylketone), -COCH₂SH (thiomethylketone), -CONHOH (hydroxamic acid), -SO₂NHOH (sulfohydroxamic acid), -NO-N=O (nonoate), - NOH-COH (reverse hydroxamic acid),

- independently from one another, R₄, R₅, R₆, and R₇ represent H, a halogen, or a group comprising from 1 to 10 carbon atoms; - Ri represents H, or a group comprising from 1 to 50 carbon atoms; provided that :

* at least one of R₂ and R₃ represents a metal chelating group as defined above and, and * when R₃ represents a metal chelating group as defined above, then -(CH₂)_n-(X)_nl-

Y cannot represent -(CH₂)₂-NH₂ or -(CH₂)₂-SH.

The invention concerns more particularly compounds of the general formula (I) as defined above, wherein, independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula: -(CH₂VY wherein:

- n is an integer from 0 to 3, and

- Y represents a group selected from the list comprising -NHOH, -CONHOH, or -NOH-COH. Preferred compounds of the invention are those of general formula (I) as defined above, wherein, independently from each other, R₄, R₅, R₆, and R₇ represent H, a halogen, an alkyl, an alkoxy, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, a thioalkyl, a thioaryl or an aryloxycarbonyl group, if adequate substituted by a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group. In another preferred embodiment, the invention relates to compounds of the general formula (I) as defined above, wherein, independently from each other, R₄, R₅, R₆, and R₇ represent H, a halogen, an alkyl, or an alkoxy group from 1 to 10 carbon atoms. In another particularly preferred embodiment, the present invention relates to compounds of the general formula (I) as defined above, wherein Ri represents - H, or

- a peptide sequence, or

- an alkyl, an alkoxy, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, a thioalkyl, a thioaryl, an arylsulfonyl, an aryloxycarbonyl, an alkoxycarbonylalkyl, or an aryloxycarbonylalkyl group, if adequate substituted by a pyridine, a morpholine, a thiomorpholoine, a piperidine, a piperazine, a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group.

The invention aslo concerns more particularly compounds of the general formula (I) according to any of claims 16 to 20, wherein R₁ represents H, a phenylsulfonyl group of formula -SO₂-C₆H₅, or a benzyloxycarbonyl group of formula -CO-O-CH₂-C₆H₅. The invention also relates more particularly to compounds as defined above, of the following general formula (II) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined above, and R₁, R₂, and R₃ are as defined above.

In another preferred embodiment, the invention relates to compounds as defined above, of the following general formula (III) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined above, and R₁, R₂, and Y are as defined above.

In another particularly preferred embodiment, the present invention relates to compounds as defined above, of the formula (III) wherein:

- R₀ represents H, a halogen atom such as Cl, F, or Br, an alkoxy group from 1 to 10 carbon atoms such as a methoxy OCH₃ group,

- R₂ represents H or CH₃,

- Y represents -NHOH, -CONHOH, or -NOH-COH, and

- R₁ represents H, -SO₂-C₆H₅, or -CO-O-CH₂-C₆H₅.

In another particularly preferred embodiment, the present invention relates to compounds of the general formula (I) as defined above, characterized in that said compounds correspond to the following formulae:

15 16

The present invention also relates to a method for screening compounds comprising an indole structure intended for the prevention or the treatment of bacterial or protozoan infections, or for their use as herbicides, characterized in that it comprises the steps of contacting a peptide deformylase enzyme with the compounds to screen and measuring the inhibiting activity of said compounds towards said peptide deformylase enzyme.

As intended herein, "compounds comprising an indole structure" are compounds which carry the following structure:

wherein the free bonds are either linked to H or to any chemical group.

In a preferred embodiment of the above defined screening method, the inhibiting activity of the compounds to screen towards the peptide deformylase enzyme is evaluated by measuring the IC₅₀ of said compounds with respect to said peptide deformylase enzyme.

The IC₅₀ can be measured according to the above described method.

In another preferred embodiment of the above defined screening method, the compounds to screen for which the IC₅₀ is lower than about 1 μM are selected. In another preferred embodiment of the above defined screening method, the compounds to screen respond to formula (I) as defined above.

In another preferred embodiment of the above defined screening method, the peptide deformylase enzyme is Escherichia coli nickel-bound peptide deformylase (SEQ ID NO: 1) and/or Bacilus stearothermophilus nickel-bound peptide deformylase (SEQ ID NO: 2). In a particularly preferred embodiment of the above defined screening method, it is further tested that the compounds to screen have essentially no inhibiting properties towards mitochondrial peptide deformylases, in particular human mitochondrial peptide deformylases.

The synthesis of compounds having the general formula (I) can be carried out according to the following procedures or as described in the examples.

Amine, cyanamide and hydroxyguanidine indolic derivatives can be prepared as depicted in the following Scheme 1.

Scheme 1

W represents a halogen, an alkyL an aryl, an alkoxy, an aryloxy, a thioalkyl, or a thioaryl group.

Briefly, indole-3-carboxaldehyde prepared from the corresponding indole by the Vielsmeier-Haack reaction is transformed into indole-3-carbonitrile by treatment with diammonium hydrogen phosphate, 1-nitropropane and acetic acid (Jiang et ah, 2000). The nitrile is reduced into the amine with CoCl₂ / NaBH₄ (Leclerc et ah, 1998). The cyanamide is obtained by reaction of the amine with BrCN and converted into the hydroxyguanidine by reaction with hydroxylamine hydrochloride in the presence of sodium acetate (Lefevre- Groboillot et al., 2001).

Hydroxatnic acid, hydroxymethylketone and thiomethylketone indolic derivatives can be prepared as depicted in the following Scheme 2.

Scheme 2 W represents an alkyl, an aryl, an alkoxy, an aryloxy or a thioaryl group.

Briefly, an acetic acid group is introduced into the 3-indole position by reacting the zinc salt with 2-bromoacetic acid ethyl esters followed by ester hydrolysis (Dillard et ah, 1996). The hydroxymethylketone is prepared following the procedure described by Wissner (Wissner et al., 1979) by reaction of the (indol-3-yl)acetyl chloride with tris(trimethylsilyloxy) ethylene, without any Lewis acid catalyst, at room temperature (r.t), followed by hydrolysis decarboxylation of the intermediate at 8O⁰C. Thioacetyl derivatives are prepared in five steps starting from the acids which are converted into the α-diazoketones by reaction of the acylchloride with diazomethane (Salim & Capretta, 2000). Treatment with an etheral HCl(g) solution affords the chloromethylketones. Displacement of the chlorides with potassium thioacetate at r.t , in DMF yielded the corresponding thioesters which can be deprotected by hydrolysis under strictly anaerobic conditions with Na₂CO₃ in methanol followed by acidification with an etheral HCl(g) solution.

A general procedure for the preparation of hydroxamic acid indolic derivatives from the corresponding acid indolic derivatives is as follows. Some of the (indol-3-yl)acetic acid derivatives are commercially available, so a general synthetic procedure starting from the acids is described hereafter, even though the direct reaction of hydroxylamine with the ester is possible. Briefly, to a 10 ml DMF solution of the indolacetic acid derivative (0.3 mmol) are added 1-hydroxybenzotriazole (1.1 equiv., m = 44 mg), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (1.1 equiv., m = 63 mg) and N-methylmorpholine (1.1 equiv., 36 μL). This solution is stirred for one hour at r.t. After adding hydroxylamine hydrochloride (1.1 equiv., m = 23 mg) the solution is left under stirring overnight. Then DMF is evaporated under vacuo, and ethylacetate is added to the residue. This ethylacetate solution is successively washed with water, saturated aqueous NaHCO₃ solution and saturated aqueous NaCl solution. After drying over Na₂SO₄ and filtration, evaporation of the solvent to dryness affords hydroxamic acid derivatives in 40-60 % yield. A substitution at position 2 of the indolic derivatives can proceed as described in

Scheme 3.

Scheme 3

W represents a halogen, an alkyl, an aryl, an alkoxy, an aryloxy, or a thioaryl group. Briefly, reaction 2-lithio-N-benzensulfonylindole (obtained by reaction of N- benzensulfonylindole with BuLi) with dimethyloxalate (Hasan et ah, 1981) successively followed by basic hydrolysis and Wolf-kishner reduction affords the (indol-2-yl)acetic acid (Weller & Ford, 1984).

N-substituted indole can be prepared as described in Scheme 4.

Scheme 4

W represents a halogen, an alkyl, an aryl, an alkoxy, an aryloxy or a thioaryl group. Briefly, N-carboxymethylation is achieved by reaction of the lithio derivative (prepared with two equiv. of lithium bis(trimethylsilyl)amide in THF at -78⁰C) with benzylchloroformate (Horwell et ah, 1997).

Scheme 5

The different functionalizations of the indole nitrogen (R₁) (Scheme 5) according to the invention can be prepared as described in Dillard et ah, (1996) and Eissenstat et ah, (1995) for example.

The reverse hydroxamate indolic derivatives can be prepared as depicted in Scheme 6.

Scheme 6 W represents a halogen, an alkyl, an aryl, an alkoxy, an aryloxy, a thioalkyl, or a thioaryl group.

Briefly, following routeA condensation of the aldehyde with hydroxylamine hydrochloride in pyridine affords the ketoxime which is further reduced under Borch's conditions (Borch et ah, 1971). Following route B, after reduction of the aldehyde, the hydroxymethyl derivative is either subjected to a Mitsunobu reaction (Kolpssa et ah, 1997) or brominated and subjected to a nucleophilic substitution with N,O-bis(fert- butoxycarbonyl)hydroxylarnine. Once deprotected with trifluoroacetic acid, the hydroxylamine is formylated to provide the reverse hydroxamate. Selective N-formylation is notably described in Hill et al. (2002). The adaptations which can be brought to these procedures in order to synthesize all the compounds of formula (I) according to the invention are within the ordinary skills of the man skilled in the art. Materials and Methods

All solvents and chemicals were purchased from SDS and Aldrich, respectively. DMF,

THF and CH₂Cl₂ were dried using standard procedures and stored over 4 A molecular sieves under an argon atmosphere. ¹H NMR spectra were recorded on a Bruker ARX-250 spectrometer and chemical shifts were reported in ppm downfield from TMS. IR spectra were obtained with a Perkin-Elmer Spectrum One FT-IR spectrometer equipped with a MIRacle™ single reflection horizontal ATR unit (Zirconium-Selenium crystal). FAB and CI Mass spectra were recorded at ENS in Paris. Elemental analyses were carried out by the microanalysis service at Paris VI University (France) or at Gif-sur-Yvette (CNRS, France). Experiments performed under argon were run on a vacuum line.

Scheme recapitulating chemical synthesis

1a: X = H, R = H 1b: X = Br, R = H 1c: X = H, R = CbZ 1d: X = Br₁ R = Cbz

3a: X = H, R = H 4a: X = H, R = H 5a: X = H, R = H 3b: X = Br, R = H 4b: X = Br, R = H 5b: X = Br, R = H 3c: X = H, R = Cbz 4c: X = H, R = Cbz 5c: X = H, R = Cbz

S-Carboxymeikyl-indol-l-carboxylic acid benzyl ester (Ic)

A solution of 2-(indol-3-yl)acetic acid (500 mg, M = 175.19, 2.85.mmol) and lithium bis(trimethylsilyl)amide (IM in THF, 6.27 mmol, 2.2equiv, 6,27 niL) in freshly distilled THF (10 mL) was stirred at -78°C under argon for Ih. Then benzylchloroformate (505 μL, M = 170.60, d = 1.195, 3.42.mmol, 1.2equiv) was added. The reaction mixture was stirred at -78°C for 2h, then the solvents were evaporated under vacuo. The residue was dissolved in water and extracted with diethylether. Acidification of the aqueous layer up to pH 3 with aqueous HCl (0.1N) results in the formation of 3-carboxymethyl-indol-l-carboxylic acid benzyl ester (Ic) as a white precipitate which was filtered off and washed with pentane (795 mg, yield 90%) Rf (silica gel MerckF 254, CH₂C1₂/CH₃OH 9/1 v:v mixture) - 0.4. Tf = 152°C. IR (cm⁴): 1728 and 1694 (v_c0)- ¹H NMR (250 MHz, [Do]DMSO) : £3.77 (s, 2H); 5.53 (s, 2H); 7.29-7.66 (m, 8H); 7.74 (s, IH); 8,14 (d, J= 8.1, IH). CI-MS : m/z = 327, [M+NH₄ ⁺], 100%. Anal. Calcd for Ci₈H₁₅NO₄: C, 69.89; H, 4.89; N₃ 4.53. Found: C, 69.78; H, 5.00; N, 4.55.

S-CarboxymethylS-broϊno-indol-l-carboxylic acid benzyl ester (Id) The same procedure applied to 2-(5-bromo-indol-3-yl)acetic acid (200 mg, M =254.09, 0.787 mmol) afforded (Id) (280 mg, yield: 92%). Rf (silica gel CH₂C1₂/CH₃OH 9/1 v:v mixture) = 0.5, Tf= 88°C, IR (cm^"1): 1729 and 1692 (v_co)- ¹H NMR (250 MHz, [D6] acetone): £3.80 (s, 2H); 5.51 (s,2H); 7.15 (s,lH); 7.24-7.82 (m, 7H); 8.12 (d, J= 8,1, IH); 11,16 (s, IH). CI-MS : m/z = 405, 407, [M+ NH₄ ⁺], 40%; 388, 390 [M⁺], 30%. Anal. Calcd for C₁₈Hi₄BrNO₄ -0.5 H₂O: C, 54.53; H, 3.87; N, 3.53. Found: C₅ 54.67; H, 3.72; N, 3.44.

l-(5-bromo-lH-indol-3-yl)-3-hydroxypropan-2-one (2)

Oxalyl chloride (104 μl, M = 126.93, d = 1.455, 1.18 mmol, 1.5 equiv) was added at O⁰C under argon to a solution of 2-(5-bromo-lH~indol-3-yl)acetic acid (200 mg, M = 254.09, 0.788 mmoL) in THF (10 mL) containing a few drops of DMF. The mixture was stirred at r.t. for 2 h, then evaporated under vacuo. Tris(trimethylsilyloxy)ethylene (675 μL, M = 292.59, d = 0.885, 1.94 mmol, 2.3 equiv) was added under argon to the residue dissolved in 10 mL of dioxane. After stirring at r.t. for 1O h, 10 mL of aqueous HCl (0.1N) were added and the solution was then heated at 80°C for 30 min. After adding NaCl, the solution was extracted with diethylether. The organic layer was washed with brine, dried over MgSO₄ and evaporated to dryness under vacuo After purification over column chromatography (silica gel, elution CH₂C1₂/CH₃OH 95/5 v:v mixture), l-(5-bromo-l#-indol-3-yl)-3-hydroxypropan-2-one (2) was obtained (45 mg, yield = 21%). Rf (silica gel, CH₂C1₂/CH₃OH 9/1 v:v mixture) = 0.5. Tf = 88°C. IR (cm-¹): 1702 (v_co). ¹H NMR: (250 MHz, CDCl₃): £3.00 (s, IH); 3.81 (s, 2H); 4.31 (s,2H); 7.15 (s,lH); 7.19-7.32 (m, 2H); 7.64 (s, IH); 8.21 (s, IH). CI-MS : m/z = 285, 287, [M+ NH₄ ⁺], 80%; 268, 270 [M⁺], 100%. Anal. Calcd for C₁₁Hi₀BrNO₂: C, 49.28; H, 3.76; N, 5.22. Found: C, 49.10; H, 3.75; N, 5.17.

Thioacetic acid S-[3-(indol-3-yl)-2-oxo-propyl] ester (5a)

Thionyl chloride (150 μl, M = 118.97, d = 1.63, 2.06 mmol, 1.2equiv) was added under argon to a solution (10 mL) of 2-(indol-3-yl)acetic acid (300 mg, M = 175.19, 1.72 mmol) in CH₂Cl₂. The mixture was heated at 4O⁰C for 3h, then evaporated under reduced pressure. The residue dissolved in CH₂Cl₂ (1OmL) was canulated under argon in a freshly prepared solution of diazomethane in diethylether (10.4 mL, C = 0.66 M, 6.88 mmol, 4 equiv). After stirring at O⁰C for 4h the solution was evaporated under reduced pressure to give in a 95 % yield the diazo compound (3a) as a yellow oil that was used without further purification. (3a): Rf (ethylacetate / cyclohexane 1/1 v:v mixture) = 0.65. IR (cm^"1): 2100 (V_CN) ; 1726 (v_Co)- ¹H NMR (250 MHz, CDCl₃): £3.76 (s, 2H); 5.28 (s, IH); 7.14 - 7.28 (m, 3H); 7.39 (d, J = 7.8, IH); 7.48 (d , J = 7.7, IH); 8.12 (s, IH). CI-MS: m/z = 217, [M+NH₄ ⁺], 50%; 200, ([M+H]⁺), 30%. An etheral solution of HCl(g) (630 μL, C = 6 N, 3.78 mmol, 2.2 equiv) was added to a solution of (3a) in diethylether(10 mL). 5 mL of DMF was added and diethylether was carefully evaporated. Then, a solution (5 mL) of KSAc (432 mg, M = 114.20, 3.78 mmol, 2.2equiv) in DMF was transferred via a canula under argon. After stirring overnight at r.t, evaporating the solvent under vacuo afforded (5a) that was further purified over column chromatography eluted with a 8/2 v:v mixture of ethylacetate/cyclohexane (220 mg, yield: 52%). Rf (silica gel ethylacetate/cyclohexane 1/1 v:v mixture) = 0.6. IR (cm^"1): 1788 (v_co). ¹H NMR (250 MHz, CDCl₃): £2,34 (s, 3H); 3,78 (s, 2H); 3,95 (s, 2H) ; 7.09 - 7.57 (m, 5H); 8.19 (s, IH). CI-HRMS: Calcd for C₁₃Hi₄NO₂S⁺ ([M+H]⁺), 248.0445; Found, 248.0448.

Thioacetic acid S-[3-(5-Bromo-lH-indol-3-yl)-2-oxo-propyl] ester (5b)

The same procedure applied to (5-bromo-indol-3-yl)acetic acid (300 mg, M = 254.09, 1,18 mmol) afforded 227 mg of thioacetic acid 5-[3-(5-bromo-lH-indol-3-yl)-2-oxo-propyl] ester (5b) in a 59% yield. Rf (silica gel, ethylacetate/cyclohexane 1/1 v:v mixture) = 0.5. ¹H NMR (250 MHz, CDCl₃): δ 2.36 (s, 3H); 3.78 (s, 2H); 3.92 (s, 2H) ; 7.13 - 7.29 (m, 3H); 7.72 (s, IH); 7.48 (d, J = 7.7, IH); 8.18 (s, IH). CI-HRMS: Calcd for Cj₃H₁₃BrNO₂S⁺ ([M+H]⁺), 325.9850, 327.9830; found 325.9843, 327.9837. The diazo compound (3b) was isolated in a 95 % yield: Rf (silica gel, cyclohexane / ethylacetate 3/7 v:v mixture) = 0.5. IR (cm^"1): 2101 (v_CN) ; 1715 (v_co). ¹H NMR (250 MHz, CDCl₃): £3.75 (s, 2H); 5.68 (s, IH); 7.22 (dd, J₃ = 8.5, ⁴J= 1.5, IH); 7.34 (s, IH); 7,37 (d , ³J = 8.5, IH); 7,75 (d, ⁴J = 1.5, IH). CI-MS : m/z = 278, 280, [M+], 60%; 250, 252, [M-N₂]+, 100%.

S-β-acetylsulfanyl^-oxo-propylj-indole-l-carboxylic acid benzyl ester (5c)

The same procedure applied to 3-carboxymethyl-indol-l-carboxylic acid benzyl ester (200 mg, M = 309.32, 0.647 mmol) afforded 178 mg of (5c) in a 72% yield. Rf (ethylacetate/cyclohexane 1/1 v:v mixture) = 0.6. ¹H NMR (250 MHz, [D6] acetone): £2.34 (s, 3H); 3.98 (s, 2H); 4.07 (s, 2H); 5,50 (s, 2H); 7.15 - 7.80 (m, 9H); 8.15 (d, ⁴J= 1.5, IH). CI- HRMS: Calcd for C₂₁H₂₃NO₄S⁺ [M+NH₄ ⁺], 399.1379; found, 399.1375. The diazo compound (3c) was isolated in a 91% yield and characterized: Rf (ethylacetate/cyclohexane l/l v:v mixture) = 0.5. IR (cm^"1): 2102 (V_CN) ; 1730 and 1636 (v_Co). ¹H NMR (250 MHz, [D6] acetone): £3.78 (s, 2H); 5.50 (s, 2H); 5,82 (s, IH) ; 7.26 - 7.69 (m, 8H); 7.70 (s, IH) ; 8.17 (d, J= 8, IH). CI-MS: m/z = 351, [M+NH₄ ⁺], 100%.

2-(Indol-3-yl)-N-hydroxyacetamide (6a).

A DMF solution (10 niL) of 2-(indol-3-yl)acetic acid (500 mg, M = 175.19, 2.85 mmol), 1- hydroxybenzotriazole, 1-hydroxybenzotriazole (HOBT), (425 mg, M = 135.13, 3.14 mmol, 1.1 equiv.) and l-(3-dimethylaminopropyl)-3-ethylcarboxylimine hydrochloride, EDCI, (602 mg, M = 191.71, 3.14 mmol, 1.1 equiv) was stirred under argon for 5 min. Then N- methylmorpholine (NMM), (345 μl, M = 101.15, d = 0.92, 3.14 mmol, 1.1 equiv) was added. The reaction mixture was stirred at r.t. for 2h and hydroxylamine hydrochloride (218 mg, M = 69.5, 3.14 mmol, 1,1 equiv) was added. The solution was left under stirring overnight, then DMF was evaporated under vacuo to give a yellow oil that was dissolved in ethylacetate. This solution was successively washed with water and aqueous NaHCO₃. After the usual workup evaporating the sovent afforded (6a) as a white solid (m = 260 mg, yield = 60 %). IR (cm^"1): 1638 (vco). ¹H RMN (250 MHz, [D6] DMSO): £3.44 (s, 2H); 7.02 (t, J= 7.1, IH); 7.12 (t, J = 7.1, IH); 7.19 (s,lH); 7.39 (d, J = 7.8, IH) ; 7,62 (d, J = 7.6, IH) ; 8,74 (s, IH) ; 10.65 (s,lH) ; 11.85 (s, IH). Anal. Calcd for C₁₀H₁₀N₂O₂: C, 63.15; H, 5.30; N, 14.73. Found: C, 62.96, H₅ 5.31, N, 14.63. 2-(5-Bromo~lH-indol-3-yl)-N-hydroxyacetamide (6b)

Starting from (5-bromo-l#-mdol-3-yl)acetic acid (200 mg; M = 254.09, 0.787 mmol), 116 mg of (6b) was obtained in a 55% yield. Rf (Cl 8 silica gel, ethylacetate/methanol 1/1 v:v mixture) = 0.8. Tf = 145⁰C. IR (cm^"1): 3417 (v_0H) 3213 (v_NH), 1633 (v_co). ¹H NMR (250 MHz, [D6] DMSO): £3.49 (s, IH); 7.20-7.44 (m, 3H); 7.87 (s, IH); 8.86 (s, IH) ; 10.59 (s, IH) ; 11.14 (s, IH). CI-MS : m/z = 270, 272, [M+H]⁺, 100%. Anal. Calcd for C₁₀H₉BrN₂O₂: C, 44.63; H, 3.37; N, 10.41. Found: C, 44.52, H, 3.52; N, 10.45.

S-bromoS-hydroxycarbamoylmethyl-indole-l-carboxylic acid benzyl ester (6d) The carboxymethylation procedure described in Horwell et al. (1997) was followed from 2-(5- bromo-indol-3-yl)acetic acid (200mg, M =254,09, 0.787 mmoL) to give 3-carboxymethyl-5- bromo-indol-1-carboxylic acid benzyl ester (280 mg, yield: 92%). Starting from 5-bromo-3- carboxymethyl-indol-1-carboxylic acid benzyl ester (120 mg, M = 388.21, 0.309 mmol), 50 mg of (6d) was obtained in a 40 % yield. IR (cm^"1): 1650 and 1690 (vco). ¹H NMR (250 MHz, [D6] DMSO): <53.40 (s, 2H); 5.56 (s, 2H); 7.45-8.10 (m, 9H); 8.95 (s, IH) ; 10.75 (s, IH). Anal. Calcd for C₁₈H₁₅BrN₂O₄: C, 53.62; H, 3.75; N, 6.95. Found: C, 53.73; H, 3.54; N, 7.11.

(4-fluoro-lH4ndol-3-yl)-acetic acid ethyl ester (8e)

To a cooled solution of 4-fluoro-iH-indole 7e (M = 135.05, 300 mg, 2.22 mmol) in 3.3 mL of TΗF was added 1.39 mL (2.22 mmol) of n-BuLi 1.6 M in hexane while keeping the solution below O⁰C with an ice bath. After 15 min, 2.22 mL Of ZnCl₂ IN in Et₂O was added. The cooling bath was removed and the mixture stirred for 24 h, then evaporated under vacuo to give a wax which was further dissolved in anhydrous toluene (3.3 mL). After addition of ethyl-2-bromoacetate (246 μL, 2.22 mmol), the solution was stirred for 24 h. The mixture was then acidified with IN HCl. and poored into ethylacetate. The organic layer was wahed with brine and dried over MgSO₄. The ester was chromatographed on silica gel eluted with 10%

AcOEt / cyclohexane to give 158 mg (32 %) of (JSe). ¹H NMR (250 MHz, [D6] acetone): δ

1.24 (t, J= 7, 3H); 3.88 (s, 2H); 4.16 (q, J= 7, 2H); 6.71 (dd, ³J_HF = 11, ³J_HH = 8, IH); 7.06

(td, ³JH_H = 8, ⁴JHF = 5.4, IH); 7.23 (d, J= 8, IH); 7.28 (d, ⁵J_HF = 1.9, IH); 10.34 (s, IH).

2-(4~fluoro-lH-indol-3-yl)-N-hydroxy-acetamide (6e)

NH₂OH-HCl (497 mg, 7.15 mmol) was added as a powder to 6.4 mL of a IM solution of EtONa in EtOH. This solution was added to an ethanol solution (10 mL) of the ester (Se) (158 mg, 0.715 mmol, M = 221.03). The mixture was stirred under argon at 80 ⁰C for 24 h. Then after cooling and evaporation under vacuo, the residue was dissolved in AcOEt. This organic layer was wahed with brine, aqueous NaHCO₃, 0.1 N HCl, brine and dried over MgSO₄. After evaporation under vacuo, the hydroxamic acid (6e) was dissolved in a 1 : 1 mixture of acetone / cyclohexane. Slow evaporation of acetone under reduced pressure afforded 75 mg of (6e) as a white solid (51%). IR (cm⁴): 3354 (v_NH), 3280 (v_0H), 1631 (v_Co)- ¹H NMR (250 MHz, [D6] acetone): δ 3.72 (s, 2H); 6.70 (dd, ³J_HF = H, ³J_HH = 8, IH); 7.05 (td, ³J_HH = 8, %_F = 5.4, IH); 7.22 (d, J = 8, IH); 7.28 (s, IH); 10.00 (s, IH), 10.43 (s, IH). CI-HRMS: Calcd for Ci₀Hi₃N₃O₂F ([M+NH₄ ⁺]), 226.0992; Found 226.0991.

(5-fluoro-lH-indol-3-yl)-acetic acid ethyl ester (8β

91 mg of the ester (Sf) (yield = 44 %) was obtained from 125 mg of 5-fluoro-lH-indole (7f) (M = 135.05, 0.926 mmol). ¹H NMR (250 MHz, [D6] acetone): δ 1.24 (t, J= 7.2, 3H); 3.74 (s, 2H); 4.13 (q, J= 7.2, 2H); 6.92 (t, J= 9, IH); 7.30 (d, J= 9, IH); 7.38 (m, 2H); 10.21 (s, IH).

2-(5-fluoro-lH-indol-3-yl)-N-hydroxy-acetamide (6f)

Starting from 91mg of the ester (8f) (M = 221.23, 0.41 mmol) 21 mg (yield = 25%) of the hydroxamic acid (6J) (M = 208.19) was obtained. IR (cm^'1): 3360 (v_NH), 3175 (V_OH), 1625 (vco)- ¹H NMR (250 MHz, [D6] acetone): δ 3.57 (s, 2H); 6.91 (td, ³J_HF = ³J_HH = 9.3, ⁴J_HH = 2.3, IH); 7.33-7.41 (m, 3H); 8.00 (s, IH); 10.08 (s, IH); 10.22 (s, IH). CI-MS: m/z = 226.2, IMfNH₄ ⁴], 20%.

(5-chloro-lH-indol-S-yl)-acetic acid ethyl ester (8g)

198 mg of the ester (Sg) (yield = 43 %) was obtained from 300 mg of 5-chloro-7H-indole (7g) (M = 151.60, 1.98 mmol). ¹H NMR (250 MHz, [D6] acetone): δ 1.24 (t, J= 7, 3H); 3.77 (s, 2H); 4.14 (q, J= 7, 2H); 7.24 (d, J= 8.5, IH); 7.35-7.40 (m, 2H); 7.79 (s, IH); 10.33 (s, IH).

2-(5-chloro-lH-indol-3-yl)-N-hydroxy-acetamide (6g) Starting from 198 mg of the ester (8g) (M = 233.26, 0.85 mmol) 194 mg (yield = 95%) of the hydroxamic acid (6g) (M = 224.64) was obtained. IR (cm^"1): 3348 (v_NH), 3190 (v_0H), 1625 (vco). ¹H NMR (250 MHz, [D6] acetone): δ 3.59 (s, 2H); 7.10 (d, J= 8.5, IH); 7.35 (s, IH); 7.41 (d, J = 8.5, IH); 7.67 (s, IH); 10.00 (s, IH); 10.29 (s, IH). CI-HRMS: Calcd for C₁₀H₁₀N₂O₂Cl ([M+NH₄ ⁺]), 225.04331 (100%), 227.0461 (33%); Found 225.0432 (100%), 227.0413 (32.4%).

(5-methoxy-lH-indol-3-yl)-acetic acid ethyl ester (8h) 219 mg of the ester (8h) (yield = 46 %) was obtained from 300 mg of 5-methoxy-iH-indole (7A) (M = 147.18, 2.04 mmol). ¹H NMR (250 MHz, [D6] acetone): δ 1.23 (t, J= 7, 3H); 3.73 (s, 2H); 3.82 (s, 3H); 4.15 (q, J = 7, 2H); 6.79 (d, J = 8.5, IH); 7.11 (s, IH); 7.26-7.32 (m, 2H); 9.97 (s, IH).

2-(5-methoxy-lH4ndol-3-yl)-N-hydroxy-acetamide (6h)

Starting from 219 mg of the ester (8g) (M = 233.26, 0.94 mmol) 90 mg (yield = 43%) of the hydroxamic acid (6h) (M = 220.22) was obtained. IR (cm⁴): 3307 (v_NH), 3160 (v_0H), 1620 (vco)- 1H NMR (250 MHz, [D6] acetone): δ 3.57 (s, 2H); 3.81 (s, 3H); 6.77 (dd, ⁵J= 8.8, ⁴J= 2.3, IH); 7.15 (d, J= 2.3, IH); 7.22 (s, IH); 7.28 (d, J= 8.8, IH); 7.93 (s, IH); 10.04 (s, IH). CI-MS: m/z = 238, [M+NH₄ ⁺], 35%.

(6-bromo-lH-indol-3-yl)-acetic acid ethyl ester (8i)

192 mg of the ester (8i) (yield = 45 %) was obtained from 300 mg of 6-bromo-iF-indole (Ji) (M = 196.05, 1.53 mmol). ¹H NMR (250 MHz, [D6] DMSO): δ 1.23 (t, J= 7.2, 3H); 3.76 (s, 2H); 4.13 (q, J= 7.2, 2H); 7.19 (dd, ³J= 8.5, ⁴J= 1.5, IH); 7.34 (s, IH); 7.55 (d, J= 8.5, IH); 7.62 (d, J= 1.5, IH); 10.28 (s, IH).

2-(6-bromo-lH-indol-3-yl)-N-hydroxy-acetamide (6i)

Starting from 192 mg of the ester (8i) (M = 282.16, 0.68 mmol) 136 mg (yield = 74%) of the hydroxamic acid (6i) (M = 269.09) was obtained. IR (cm^"1): 3335 (v_NH), 3230 (V_OH), 1615 (vco)- 1H NMR (250 MHz, [D6] acetone): δ 3.60 (s, 2H); 7.17 (dd, ⁵J = 8.5, ⁴J = 1.5, IH); 7.30 (s, IH); 7.6 (m, 2H); 8.0 (s, IH); 10.0 (s, IH); 10.27 (s, IH). CI-MS: m/z = 270, 272, [M+H]⁺, 50%.

2-(5-bromo-2-methyl-lH-indolyl)-acetic acid ethyl ester (8j)

201 mg of the ester (8J) (yield = 48%) was obtained from 300 mg of 5-bromo-2-methyl-lH- indole (7J) (M = 210.06, 1.43 mmol). ¹HNMR (250 MHz, [D6] acetone): δ 1.18 (t, J= 7, 3H); 2.33 (S, 3H); 3.66 (s, 2H); 4.06 (q, J= 7, 2H); 7.10 (d, J= 8, IH); 7.22 (d, J= 8, IH); 7.56 (s, IH); 10.11 (s, IH).

2-(5-bromo-2-methyl-lH-indolyl)-N-hydroxy-acetamide (6j) Starting from 201 mg of the ester (8J) (M = 296.16, 0.68 mmol) 160 mg (yield = 83%) of the hydroxamic acid (6J) (M = 283.12) was obtained. IR (cm^"1): 1690 (v_co). ¹H NMR (250 MHz, [D6] acetone): δ 2.44 (s, 3H); 3.69 (s, 2H); 7.14 (d, J= 8.4, IH); 7.26 (d, J= 8.4, IH); 7.67 (s, IH); 10.20 (s, IH).

15 16

5-bromo-2-methyl-lH-indole-3-carbaldehyde (9)

POCl₃ (267 μL, 2.86 mmoL, d = 1.64) was added under argon at 0°C to solution of 5-bromo- 2-methyl-liJ-indole (7J) (500 mg, 2.38 mmol) in 5 mL of DMF. After stirring at r.t. overnight, 2 mL of a 2N aqueous NaOH were added and the solution was further stirred for 2h, then poored into ethylacetate. After washing with water, drying and evaporating to dryness, 526 mg of (P) were isolated in a 95 % yield as a white solid (yield = 93 %). IR (cm^'1): 1628 (v_c=o). ¹H NMR (250 MHz, [D6] acetone): δ 2.78 (s, 3H); 7.33 (d, ³J = 8.45, IH), 7.36 (d, ³J= 8.45, IH), 8.33 (s, IH), 10.15 (s, IH), 11.04 (s, IH). CI-MS: m/z = 255, 257 [M + NH₄ ⁺], 85%; 238-240, [M⁺], 100%. Anal. Calcd for C₁₀H₈BrNO: C, 50.45; H, 3.39; N, 5.88. Found: C, 50.42, H, 3.49; N, 5.87. S-bromo^-methyl-lH-indoleS-carbaldehyde-oxime (10)

NH₂OH₅ HCl (70 mg, 1.01 mrαol) was added to a solution of (9) (200 mg, 0.84 mmoL) in 5 mL of pyridine. After stirring at r.t. for 5h, the solution was evaporated to dryness. After dissolution of the residue in ethylacetate, the solution was successively washed with IN HCl, brine and dried over MgSO₄. After evaporating the solvent 210 mg of (10) was isolated as a yellow oil in quantitative yield. IR(Cm^"1) : 1622 (V_C=_N). ¹H NMR (250 MHz, [d6] acetone): δ 2.53 (s, 3H); 7.2-7.35 (m, 2H); 7.80 (s, IH); 8.32 (s, IH); 9.69 (s, IH); 10.49 (s, IH). Anal.

Calcd for C₁₀H₉BrN₂O-0.25H₂O: C, 46.63; H, 3.72; N, 10.87. Found: C, 46.69; H, 3.66; N, 10.58.

N-[2-(5-bromo-2-methyl-lH-indole-3-yl)methyl]-hydroxylatnine(lϊ)

To a 10 mL solution of (10) (110 mg, 0.43 mmol) in methanol, were added a few crystals of methylorange then a few drops of an ethanolic solution of HCl(g) (2N). The solution turned red purple. While maintaining the red colour of the solution overtime by adding HCl, 2 equiv OfNaBH₃CN (55 mg, 0.86 mmol) in 5mL of THF were added and the solution was stirred. A few hours later, the red colour was stable and the solution was left under stirring overnight. After evaporation, the residue was dissolved in 5 mL of methanol, then 5 mL of water were added. The pH was adjusted to 9 with IN aqueous NaOH. This solution was extracted with CH₂Cl₂. After the usual workup, (11) (110 mg) was isolated as a yellow oil in a quantitative yield and characterized without further purification. ¹H NMR (250 MHz, [D6] acetone): δ 2.51 (s, 3H); 5.16 (s, 2H); 7.1-7.3 (m, 2H); 8.10 (s, IH); 10.34 (s, IH). CI-MS: m/z = 255, 257 [M+], 30%; 224, 226 ([M-NHOH]⁺), 100%. Anal. Calcd for C₁₀H₁₁BrN₂O: C, 47.08; H, 4.35; N, 10.98. Found: C, 46.97; H₅ 4.51; N, 11.13.

5-bromo-2-methyl-l-(phenylsulfonyl)-lH-indole-3-carbaldehyde (12) A solution of 5-bromo-2-methyl-iH-indole-3-carbaldehyde (9) (500 mg, 2.10 mmol) was added at 0°C under argon to a suspension OfNaH(IlO mg, 4.62 mmol, 2.2 equiv) in 10 mL of THF. After stirring for 1 h, a solution of benzenesulfonyl chloride (320 μL, 2.52 mmol, 1.2 equiv) was added. The mixture was stirred overnight at r.t.. 50 mL of water was then added and this solution was extracted with ethylacetate, the organic layer was successively washed with 0.1 N HCl₅ aqueous NaHCO₃, brine and dried over MgSO4. Filtration over celite and evaporation afforded 700 mg of (12) (95 % yield) after recrystallisation from a pentane / acetone mixture. ¹H NMR (250 MHz, [D6] acetone) : δ 3.05 (s, 3H) ; 7.57 (dd, ⁵J= 8.9, ⁴J = 2.1, IH); 7.6-8.0 (m, 5H), 8.19 (d, J= 8.9, IH), 8.41 (d, J= 2.1, IH)₃ 10.32 (s, IH). CI-MS: 111^ 378, 380 [M⁺], 100%.

2-(5-bromo-2-methyl-l-(phenylsulfonyl)-lH-indol-3-yl)ethanol (13)

To a solution of (12) (700 mg, 1.85 mmol) in 10 niL of methanol was added a solution of NaBH₄ (84 mg, 2.22 mmol) in 10 mL of methanol. After stirring for 6 h at r.t, the solution was poored into ethylacetate and washed with water and brine. The organic layer was dried over MgSO₄ and evaporated under vacuo. The alcohol was chromato graphed over silica gel eluted with cyclohexane / ethylacetate 70 / 30, yielding 700 mg of (13) in a quantitative yield as a pale yellow powder. ¹H NMR (250 MHz, [D6] acetone) : δ 2.64 (s, 3H) ; 4.02 (s, IH) ; 4.72 (s 2H) ; 7.45 (dd, ³J= 8.9, ⁴J= 2, IH) ; 7.83 (d, J= 2, IH) ; 7.6-8.0 (m, 5H) ; 8.13 (d, J= 8.9, IH). CI-MS: m/z = 380, 382 [M⁺], 100%.

Tert-butyl-2-(5-bromo-2-methyl-l-(phenylsulfonyl)-lH-indol-3yl)methyl-(tert- butoxycarbonyloxy)-carbamate (14)

To a solution of (13) (220 mg, 0.58 mmol) in 10 mL of THF, were successively added at r.t. under argon PPh₃ (1.1 equiv, 167 mg, 0.64 mmol) and after stirring for 15 min N- bromosuccinimide (1.1 equiv, 113 mg, 0.64 mmol). The mixture was stirred at r.t. overnight. The bromo derivative was not isolated and further used in this solution. A solution of N,O- bis-(tert-butoxycarbonyl)hydroxylamine (135 mg, 0.58 mmol) in 5 mL of DMF previously stirred for 15 min with NaH (1.1 equiv, 15 mg) was added to the bromo derivative. The resulting solution was stirred for 2 h. After addition of CH₂CL₂, the solution was washed with water, aqueous NH₄Cl, brine and dried over MgSO₄. After evaporation of the solvent, the residue was chromatographed over silica gel eluted with CH₂Cl₂ / C₆H₁₂ 70 / 30 and 80 /20 affording 150 mg of (14) (yield 44 %). ¹H NMR (250 MHz, [D6] acetone) : δ 1.35 (s, 9H); 1.49 (s, 9H) ; 2.67 (s, 3H) ; 4.87 (s, 2H,) ;7.47 (dd, ³J= 8.8, ⁴J= 1.8, IH) ; 7.63 (m, 3H) ; 7.78 (d, J= 1.8, IH) ; 7.9 (d, J = 7.5, 2H) ; 8.13 (d, J = 8.8, IH). CI-MS: m/z = 612, 614 [M⁺], 100%.

N-((5~bromo-2-methyl-l(phenylsulfonyl)-lH-indol-3-yl)methyl)hydroxylamine (15)

775 μL of trifluoroacetic acid (40 equiv) was added to a solution of (14) (150 mg, 0.25 mmol) in 10 mL of CH₂Cl₂. The solution was stirred for 2 h at 0°C under argon, then washed with water, aqueous NaHCO₃ and dried over MgSO4, Fitration and evaporation under reduced pressure afforded 80 mg of (15) which was tested without further purification due to its instability during the purification over silica gel. CI-HRMS : Calcd for Ci₆H₁₄O₃N₂BrS [M- H]⁺, 392.9909 (96%), 394.9889 (100%); Found 392.9911 (24.7%), 394.9895 (24.2%).

N-(l-Benzenesulfonyl-5-bromo-2-methyl-lH-indol-3-ylmethyl)-N-hydroxyformamide (16). Formic acid (3.77 niL, 0.1 mol) was added dropwise at O⁰C to acetic anhydride (9.45 mL, 0.1 mol). The mixture was then heated at 50 °C for 2h and left to r.t. (IS) (80 mg, 0.2 mmol) dissolved in 10 mL of CH₂Cl₂ was added dropwise at r.t. to the previous solution. After stirring overnight, evaporation under vacuo afforded an oil that was chromatographed over silica gel eluted with CH₂Cl₂ / C₆H₁₂ 99.5 / 0.5 (vv mixture) in the presence of a few drops of acetic acid. After evaporation 50 mg of (16) (yield 60 %) was obtained upon precipitation from an ether solution into pentane. ¹H NMR (250 MHz, [D6] acetone) : δ Q272D3D ; 4.81 (s, 2H) ; 7.47 (dd, ⁵J= 8.8, ⁴J= 1.9, IH) ; 7.63 (t, J= 7.5, 2H) ; 7.73 (d, J= 7.5, IH) ; 7.8 (d, J= 1.9, IH) ; 7.94 (d, J= 7.5, 2H) ; 8.13 (d, J= 8.8, IH) ; 8.35 (s, IH) ; 9.10 (s, IH). FAB⁺- MS : 423, 425, [M+H]⁺, 10% ; 461, 463, [M+NH₄ ⁺], 10%. CI-HRMS : Calcd for C₁₇H₁₉O₄N₃BrS [M+NH₄ ⁺], 440.0280 (95.6%), 442.0260 (100%) ; Found 440.0292 (86.4%),

442.0247 (92.9%).

(l-hydroxycarbamoylmethyl-l-phenyl-ethyty-carbamic acid tert-butyl ester (18)

50 mg of (18) was prepared in 55 % yield, following the usual procedure for hydroxamic acid synthesis, starting from 3-tert-Butoxycarbonylamino-4-phenyl-butyric acid (17) (0.308 mmoL, M = 279.33, m = 86 mg). ¹H NMR (250 MHz, [D6] DMSO): δ 1.36 (s, 9H); 2.16 (m, 2H); 2.72 (m, 2H); 3.99 (m, IH), 6.71 (d, J= 8.6, IH); 7.19-7.35 (m, 5H); 8.83 (s, IH); 10. 40 (s, IH). Anal. Calcd for C₁₄H₂₀N₂O₄-0.25H₂O: C, 60.29; H, 7.59; N, 9.37. Found: C, 60.19; H, 7.38; N, 9.58. (l-hydroxycarbamoylmethyl~2-phenyl-ethyl)-carbamic acid tert-biityl ester (20)

Compound (20) was synthetised as described by Xiang et α/..[Xiang, 2002 #1007] from (1- formyl-2-phenyl-ethyl)-carbamic acid tert-butyl ester (19). IR(Cm^"1): 1682 (v_Co). ¹H NMR

(250 MHz, CDCl₃): δ 1.38 (s, IH); 2.83 (d, J = 6.7, 2H); 3.08-4.01 (m, 2H); 4.19 (m, IH); 4.61 (d, J = 3.9, IH); 7.15-7.80 (m, 5H); 8.32 (s, IH); 8.85 (s, IH). Anal. Calcd for

C₁₅H₂₂N₂O₄-O^H₂O: C, 60.47; H, 7.58; N, 9.40. Found: C, 60.67; H, 7.57; N, 9.31.

DESCRIPTION QF THE FIGURES

Figure IA, Figure IB and Figure 1C

Figure IA: HSQC (Heteronuclear Single Quantum Correlation) footprint of 2-(5-Bromo-lH- mdol-3-yl)-iV-hydroxyacetamide (inset) on ¹⁵N-labelled E. coli peptide deformylase (1 mM). The reference spectrum is shown in black contours and the spectrum in the presence of a stoichiometric amount of the compound is overlaid in gray. Amide groups that are significantly perturbed are labeled.

Figure IB: 3D structure of E. coli peptide deformylase. Aminoacid residues whose amide group NMR shift is perturbed by the binding of 2-(5-Bromo-lH-indol-3-yl)-N- hydroxyacetamide are shown in red (residues labeled in Figure IA). The catalytic metal ion is shown as a sphere. Figure 1C: NMR saturation transfer difference experiment (STD) showing transfer of magnetization from E. coli PDF (100 μM) to 2-(5-Bromo-lH-indol-3-yl)-N- hydroxyacetamide (1 mM). Top trace: reference spectrum of the mixture. Peaks labelled with stars correspond to buffer signals. The numbered peaks corresponds to signal originating from 2-(5-Bromo-lH-mdol~3-yl)-Af-hydroxyacetamide, as labeled in Figure IA. Bottom trace: STD signals. The arrow indicates the spectral region which was irradiated. Normalized STD intensities are indicated.

Figure 2

Figure 2 represents three different views of the three dimensional model of the binding of a 2- (5-Bromo-lH-indol-3-yl)-N-hydroxyacetamide compound according to the invention to the S'i pocket of Ps eudomonas aeruginosa peptide deformylase enzyme superimposed with the structures of actinonin and of a benzathiozinone-hydroxamic derivative. Figure 3 A, Figure 3B and Figure 3 C

Figures 3A and 3B respectively represent the MIC of actinonin and 2-(5-Bromo-lH-indol-3- yl)-N-hydroxyacetamide (vertical axis, μg/ml) relatively to the arabinose concentration (horizontal axis, %) for a Escherichia coli strain in which the def gene is under the control of the P_BA_D promoter.

Figure 3C represents the MIC of actinonin (black circles) and 2-(5-Bromo-lH-indol-3-yl)-N- hydroxyacetamide (black squares) (vertical axis, μg/ml) for a strain relatively to the xylose concentration (horizontal axis, %) for Bacillus subilis strains in which both the ykrB and def genes had been inactivated and in which one of the two genes was placed under the control of the PxyiA promoter.

EXAMPLES

Abbreviations used

Bacillus subtilis, Bs; Escherichia coli, Ec; 7V-formyl, Fo; Met-tRNA⁰^⁶¹ transformylase, FMT; pseudo binding constant, ICs₀; indolic derivatives, ID; knockout, KO; minimal inhibitory concentration, MIC; metalloprotease, MP; inhibition or dissociation constant, Kj, matrix metalloproteases, MMPs; peptide deformylase, PDF; peptide deformylase inhibitors, PDFI;

Protein Data Bank, PDB; structure activity relationship, SAR; saturation transfer difference,

STD; three-dimensional, 3D.

Example 1

Medium-throughput PDFI screen by NMR reveal indolic derivatives as low affinity

Iigands of PDF

Materials and methods

¹⁵N-labeled Ni-EcPDFl was obtained as described (Dardel et al, 1998) and dissolved in 10 mM HEPES-HCl pH 7.0 at a final concentration of 1 mM. NMR experiments were recorded on a Bruker Avance 600 MHz NMR spectrometer equipped with a 3 mm triple resonance flow-injection probe. The probe was connected to a Gilson liquid handler controlled by the NMR console (Bruker BEST® system). The injection protocol was as previously described (Tisne et al, 2002). For chemical shift perturbation experiments, 90 μl of 1 mM ¹⁵N-labeled EcPDFl (PDFlB) was mixed with an equal volume of the tested ligand (3 mM), dissolved in the same buffer, in a 96-well plate and refrigerated at 4°C on a Gilson 242 Peltier rack prior to injection. For saturation transfer difference (STD, Mayer et ah, 2001) experiments, unlabelled EcPDFl (PDFlB) or BsPDF2 (PDF2) were used. Similar buffer and injection volumes were used except that the final protein and ligand concentrations were 20 μM and 1 mM, respectively. STD irradiation was performed by irradiating for 1 or 2 sec with the carrier set on the protein methyl massif (0.5 ppm), using a field strength γB₁/2π = 100 Hz.

Results

The binding potency of peptide deformylase inhibitors (PDFI) with respect to PDF is essentially caused by two chemical groups: (i) a metal-binding group and

(ii) the PT group that binds the Sl' pocket of PDF (Boularot et ah, 2004).

The entropic increase due to the binding of either low affinity group creates potent PDFI with inhibition constants in the nanomolar range.

The SI' pocket of bacterial PDFs (PDFlBs and PDF2s) is known to accept with low selectivity n-butyl, n-pentyl, n-hexyl, n-phenyl (Ragusa et ah, 1999; Molteni et ah, 2004) and other cyclic side-chains (see references in Boularot et ah, 2004). hi previous analysis, mitochondrial PDF (PDFlA) were shown to display a modified Sl¹ pocket that cannot tolerate cyclic compounds such as phenyl derivatives for instance (Serero et ah, 2003; Serero et ah,

2001). In order to evidence selective PDFI (i.e. PDFI which would not target mitochondrial PDF), the approach of identifying cyclic compounds with a cyclic Pl¹ group appeared to be the most appropriate. Therefore, the Inventors endeavored to identify cyclic compounds that would bind to bacterial PDF even with a low binding constant.

Thus, a medium-scale screen of such compounds was settled to identify such chemical groups by NMR. Using a pipeting-robot connected with a flow-injection NMR probe, a library of compounds was sequentially mixed with ¹⁵N-labeled EcPDF and injected. Chemical shift perturbations were monitored by recording HSQC-spectra which were compared to the control data obtained with the unbound-enzyme, as already described (Meinnel et ah, 1996;

Meinnel et ah, 1999). Compounds, the addition of which caused resonance broadening or shifting in the (¹H-¹⁵N) HSQC protein spectrum, were identified. The affected amide groups were identified and located on the backbone of the 3D structure of EcPDF (see Figure IA, for instance).

From this screen, indolic derivatives emerged as the most interesting as they showed a chemical-change pattern that corresponded to the Sl' pocket of the enzyme (Figure IB). Reciprocally, ligand-observe saturation transfer difference (STD) experiments (Mayer et ah, 2001) were performed in order to obtain information on the mode of binding of these indolic derivatives on either EcPDFl or BsPDF (Figure 1C

For instance, similar results were obtained in both cases for compound 6b (see Example 2 below) which showed that the strongest STD effect was observed for position 4 of the indole moiety and the weakest for the methylene group attached to position 3 (Figure 1C). This confirmed that the indole group did bind to both PDFlB and PDF2 enzymes and suggested that the position 4 should be the most deeply buried, as it exhibited the strongest saturation transfer from the protein.

Indole and 5-bromoindole showed a binding constant in the millimolar range as probed by NMR titrations. This weak binding constant was confirmed by inhibition assays against bacterial (EcPDFlB and BsPDF2) and mitochondrial PDF (AtPDFlA) (see Example 4 for the procedure).

In order to improve the potency of indolic derivatives (ID), the Inventors undertook the study of their structure activity relationship. Thus, substituents have been introduced on the indolyl heterocycle at positions 1 (R₁), 2 (R₂), 3 (R₃), 4 (R₄), 5 (R₅), 6 (R₆) and 7 (R₇) as labeled below:

The 4, 5 and 6 positions which correspond to the P₁' (R₅) side chain were notably substituted by a lipophylic groups, such as a bromo group, to improve the fit into the S₁ ¹ pocket. At positions 2 and 3 were explored several metal binding groups linked to the indolic cylce optionally through a short CH₂ spacer. This included monodentate functions such as carboxylic acid, oxime and hydroxylamine and bidentate functions such as hydroxymethylketone, thiomethylketone and hydroxamic acid. Finally, position 1 was either free or protected by residues, such as a benzyloxycarbonyl residue, expected to mimic the P₂ ¹ and/or P₃ ¹ amino-acid groups (for a nomenclature of the various enzyme sub-sites see Boularot et al., 2004; Schechter et al, 1967). Two such examples of indolic derivatives, carrying a hydroxamic metal chelating group a position 3 and a bromine at position 5, are presented below, as well are their inhibitory effects on PDFs and their antibiotic properties.

Example 2

In vitro assay of indolic-hydroxamic derivatives

The in vitro inhibition profile of compounds 6b and 6d of the invention vis-a-vis PDF of various origins was assessed and compared to that of actinonin and of RAS270 and RAS238, two synthetic inhibitors of PDF. RAS358 was synthesized as described previously (WO 02/070653). Compound RAS358 is a reverse hydroxamic compound. RAS270 corresponds to the hydroxamate derivative of RAS358.

50 mg of RAS270 was prepared in a 55 % yield, following the usual procedure for hydroxamic acid synthesis, starting from 3-tert-Butoxycarbonylamino-4-phenyl-butyric acid (commercially available) (0.308 mmoL, M = 279.33, m = 86 mg).

RAS270 RAS358

Materials and methods All chemicals and enzymes for protein analysis were purchased from Sigma. The peptides were from Bachem AG. Enzymes for DNA manipulation were purchased from New England Biolabs. Oligonucleotides were synthesized by MWG-AG Biotech

Escherichia coli PDF (EcPDFlB) and Bacillus stearothermophilus PDF2 (BsPDF2) were used as the representative of either bacterial PDF class and purified as described in the presence of nickel ions (Ragusa et ah, 1998). Arabidospis thaliana mitochondrial PDF (AtPDFlA) and human mitochondrial PDF (HsPDFl) were purified as described (Serero et al., 2001; Serero et al., 2003). An assay coupling PDF activity and formate dehydrogenase activity to assess PDF activity was used. The absorbance at 340 nm of NADH (ε_M=6,300 M .cm^"1) was monitored at 37⁰C essentially as previously described (Lazennec et al, 1997). The reaction was started by adding 5-15 μl of purified enzyme. In each case, the kinetic parameters were derived from iterative non-linear least square fits of the Michaelis-Menten equation, using the experimental data (Dardel et al, 1994) as previously described (Serero et al, 2001). All inhibitors were diluted in dimethyl sulfoxide and the final assay buffer contained 10 % of this solvent.

Results

The results are presented in the following Table 1:

Compound Actinonin 6b 6ύ RAS270 RAS358 in vitro assay IC₅₀ (nM)"

EcPDFlB 4 (TB) 35 26 44 50

BsPDF2 <24 (TB) <21 (TB) <27 (TB) 300 400

AtPDFlA <27 (TB) 111000 14000 83000 150000

HsPDFIm* <100 (TB) 360000 ND ND ND

HsPDFl <100 (TB) 120000 ND ND ND ^a TB is tight binding i.e. the associated IC₅₀ measured corresponded to half of the enzyme concentration put in the assay. This concentration was identical for each enzyme type and corresponded to 40, 20 25, 600 and 13.000 nM for EcPDFl, BsPDF2, AtPDFl, HsPDFIm and HsPDFl, respectively. ^b HsPDFIm which corresponds to variant E91L/C43G of HsPDFl was described in Serero et al (2003). Similar results were obtained with the wild-type enzyme but the sensitivity was reduced as this enzyme is poorly active. This is in contrast with the double mutant which is two orders of magnitude more active.

ND not determined

The IC50 values of 6b and 6d with respect to PDFlB are approximately one order of magnitude greater than that of Actinonin and of the same order of magnitude than RAS270 and RAS358. For PDF2, the IC50 values of 6b and 6d are of the same order than that of actinonin and are more than one order of magnitude lower than that of RAS270 and RAS358. As for the mitochondrial PDFs, whereas actinonin demonstrates a potent inhibitory effect, the inhibitory effects of 6b and 6d are particularly low, so that the compounds can be said to be devoid of any inhibitory activity.

Further, it should be noted that the fact that 6b shows slow-binding mode to both PDF types, is indicative of an inhibitor induced-fit conformational change of the enzyme. Example 3

In vivo assay of indolic-hydroxamic derivatives

The in vivo inhibition profiles of compounds 6b and 6d of the invention were assessed and compared to that of actinonin.

Materials and methods

AU bacterial strains were grown in Luria-Bertani (LB) medium (1% bacto-tryptone, 0.5 % yeast extract, 0.5% NaCl).

Strains. Two wild-type or pseudo wild-type B. subtilis strains were used, MO3482, a prototrophic skin prophage-curated derivative of JH642 (a kind gift of P. Stragier, IBPC, France) and 168 (trpC2) (Anagnostopoulos et al, 1961). Strains MHYlOl (Ayb'B::nm, trpCI) MHDlOl (Δrfe/l:nm, trpCI) were derived from 168 and previously described (Haas et al, 2001). Two further B. subtilis strains in which both deformylase wild-type loci (def and ykrB) are deleted and one defomylase version is conditionally expressed under the control of xylose were also used (Haas et αl., 2001): MHY103 (Δde/::nm; ΔykrB ::erm; AthrC::xylR-P_xy]_A-yki'B- spc, trpC2) and MHD103 (Δrfe/::erm; ΔykrB::nm; AthrC::xylR-P_Xy]_A-def-spc, trpCl). 138 and its derivatives were a kind gift of C. Freiberg (Bayer AG, Germany). E. coli strains JMlOlTr (gαlK, rpsL, recA56, srl-300::Tnl 0) and CAG1284 (λ\ tolC210::ΥnlO, rph-) have been described elsewhere (Hirel et αl., 1988; Singer et αl., 1989). E. coli CAG 1284 susceptibility tests. A susceptibility test was designed to determine the susceptibility to drugs of the tolC strain CAG1284. The EcPDFl open reading frame was cloned into the pBAD vector (Invitrogen) with its C-terminus in frame with the 6-His tag. This cloning renders the synthesis of EcPDFl dependent on arabinose concentration (Guzman et αl., 1995). The CAG1284 strain, which is highly susceptible to several antibiotics including chloramphenicol (Sulavik et αl., 2001), was first transformed with the pBAD construct and selected on LB medium supplemented with 50 μg/ml ampicillin, 3.4 μg/ml chloramphenicol and 0.5% glucose. Bacteria were cultured overnight in this medium at 37°C and the culture was then diluted 1:100 and used to inoculate 3 ml of medium. When the OD₆₀₀ reached 0.9, the suspension was diluted appropriately and 2 x 10⁴ of bacteria in 100 μl were layered on 30 ml of solid LB supplemented with 50 μg/ml ampicillin, actinonin (0.1-3 μM) and either glucose (0.5%) or arabinose (0.0002-0.2%) in Petri dishes. The minimum inhibitory concentration was defined as the lowest concentration of actinonin causing no growth after 18 h of incubation at 37 °C. Results

1. Inhibition of Gram-negative bacteria

Two E. coli derivatives (JMlOlTr and CAG1284) were first used to get an insight on the minimal inhibitory concentration (MIC) values of compound 6b and to compare them with that of the natural potent PDFI actinonin (Chen et al , 2000) and with that of RAS270.

With a WT strain (JMlOlTr), MIC of both actinonin and 6b were similar and in the range of 30-40 μg/ml, whereas the MIC of RAS270 was above 300 μg/ml (Table 2).

As actinonin is strongly detoxified by the AcrAB-tolC efflux pump, a tolC mutant

(strain CAG1284) was used to check whether this could be the same with 6b. It was observed that the MIC of 6b was slightly reduced by a factor of only 5.5 , in contrast to that of actinonin which was reduced 300-fold (Table 2). This indicates that the PDF inhibitors according to the invention are poorly detoxified by the bacterial efflux system.

Table 2: E. coli MIC

Compound Actinonin 6b RAS270 in vivo assay MIC (μg/ml)

E. co/z-JMIOlTr 27 40 >300 E. cσ//-CAG1284 0.1 7 32

MIC was also measured in a tolC context in which the expression of the def gene was under the control of the P_BAD promoter. Thus, the impact of increased PDF concentration on MIC was checked thanks to this promoter being induced by increasing arabinose concentrations (Guzman et al, 1995; Chen et al, 2000). In the case of actinonin, there is an almost linear increase of MIC with actinonin concentration (Figure 3A) which is indicative that (i) PDF is the primary target of actinonin, and (ii) PDF concentration is the main factor limiting the MIC in the absence of an efflux pump. When 6b was challenged in the same genetic background, a two-order MIC curve was observed (Figure 3B). The first part of the curve was linear which indicated that PDF was the target of 6b. A saturation curve was observed above 65 μg/ml. This value is similar to that of the MIC of the WT which indicates that membrane permeability is the limiting step of the action of 6b.

2. Inhibition of Gram-positive bacteria

In a second series of experiments, a Gram-positive model organism which expresses both a PDFlB and PDF2, B. subtilis, was tested. Again the MIC of actinonin was compared to that of 6b and 6d. In a WT context (strains 168 and MO3482) the MIC of 6b and 6d (20 μg/ml) was one order of magnitude higher than that of actinonin, whereas, as could be expected from their low binding potency against PDF2, both phenyl derivatives RAS270 and RAS358 had no antibiotic properties against B. subilis (Table 3).

Then, the impact of 6b and 6d on two B. subtilis strain derivatives (MHDlOl and MHYlOl) that expressed either PDF was also studied (Table 3).

It was found that the MIC of actinonin, 6b and 6d followed the same trend, with a reduced value in the yJcrB gene-inactivated context. This confirmed that the def gene product was less expressed than ykrB (Haas et αl., 2001) and suggested that 6b and 6d targeted indeed both PDFlB and PDF2.

Table 3: B. subtilis MIC

Compound Actinonin 6b (Hl RAS270 RAS358 in vivo assay MIC (μg/ml)

B. subtilis-168 1.2 20 20 >300 >300 B. subtilis-MO34S2 1 5.4 16 >300 >300 B. subtilis-MHDlOV 1.2 2.7 12 >300 >300 B. subtilis-MHY101^c 0.2 1.6 6 8 9 ^c MHD is deficient in PDFl (def gene) and MHY is deficient in PDF2 (ykrB gene) as described in Haas et αl., (2001).

To prove that 6b and 6d targeted specifically PDFs in B. subilis, strains in which both the ykrB and def genes had been inactivated and in which one of the two gene was placed under the control of the XyIR promoter, P_xyi_A, were used. Under these conditions, PDFlB or PDF2 expression is dependent of L-xylose (Haas et αl., 2001). It was observed that the MIC of both actinonin and 6b depended upon L-xylose concentration in either MHD 103 or MYD 103 strain (Figure 3C).

Finally, the appearance of resistance to 6b and actinonin in B. subilis were determined and compared. A similar value (5.10^"7 vs 6.10^"7) was obtained, in keeping with previous measurements of others and the by-pass of the formylation-defoππylation pathway induced by tranformylase gene inactivation in Bacillus spp (reviewed in Giglione et al, 2001). This data was another strong argument indicating that the target of 6b was PDF. Example 4

Three-dimensional modeling of the binding of a hydroxamic indolic derivative to a PDF

Materials and methods Three-dimensional (3D) modeling was achieved using InsightII software (Accelrys).

The 3D structures of Ps eudomonas aeruginosa PDF (PaPDFl) bound to a benzathiozinone- hydroxamic derivative (BTH) (PDB entry IS 17, Molteni et al, 2004) and PaPDFl bound to actinonin (PDB entry ILRY, Guilloteau et al, 2002) were aligned. Compound 6b was constructed with the Sketcher module, aligned on the structure of both actinonin and compound BTH using the hydroxamate group as a fixed anchor and used to replace either compound in the 3D structure. The 6b structure docked to PaPDFl was further minimized with the CharmM forcefield and the lowest energy structure selected.

Results The 3D modeling of compound 6b in the active site of PDFl revealed that it fitted perfectly the Si¹ pocket with the bromide group mimicking the C₄ methyl group of n-butyl or methionine side-chain, i.e. the optimal side-chain accepted in the Sl¹ pocket (Figure 2). Furthermore, this model placed position 4 of the indole moiety deep inside the S 1 ' pocket, buried under the side chain of His¹³², in keeping with the STD data discussed in Example 1. Together, these data indicated that indolic derivatives should display antibiotic activity but not inhibit animal mitochondrial PDFs.

Example 5

Growth inhibition of Arabidopsis thaliana Arabidopsis thaliana (ecotype WS4) growth inhibition is observed at concentrations higher than 50 μM for 6b or 6d. Besides, AtPDFlB ICs₀ value is similar to that observed with EcPDFlB in the case of compounds 6b and 6d. Details of the experimental procedure are given in Serero et al. (2001) and Giglione et al. (2003).

Thus, the Inventors have demonstrated, through two examples (6b and 6d) that indolic compounds according to the invention:

(i) target specifically PDFl and PDF2, notably in Gram-negative and Gram-positive bacteria,

(ii) show essentially no inhibitory activity towards mitochondrial PDFs. Results obtained with compounds 6a, 6g, 6f, 6i, 6e, 6h, 6j, 11, 15, and 16, are shown on the following Table 4

Table 4

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Claims

1. The use of compounds having: a) the following general formula (I):

(D wherein:

- independently from each other, R₂ and R₃ represent H, an alkyl group from 1 to 5 carbon atoms, or a metal chelating group from 2 to 20 atoms comprising from 1 to 5 hetero atoms;

- independently from one another, R₄, R₅, R₆, and R₇ represent H, a halogen, or a group comprising from 1 to 10 carbon atoms;

- R₁ represents H, or a group comprising from 1 to 50 carbon atoms; provided that at least one of R₂ and R₃ represents a metal chelating group as defined above ; and

b) an IC₅₀ lower than about 1 μM with respect to the Escherichia coli nickel-bound peptide deformylase (SEQ ID NO: 1) and/or to the Bacillus stearothermophilus nickel- bound peptide deformylase (SEQ ID NO: 2), or of pharmaceutically acceptable salts thereof, for the manufacture of medicaments intended for the prevention or the treatment of bacterial or protozoan infections, or for the manufacture of herbicides.

2. The use of compounds of the general formula (I) according to claim 1, wherein, independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula: wherein:

- n is an integer from 0 to 3, - ni is 0 or 1, - X represents a group selected from-0-, -NH-, -CHOH-, -CHF-, or -CO-, and

- Y represents a group selected from the list comprising -NH₂ (amine), -NHOH (hydroxylamine), -CH=NOH (oxime), -NHCN (cyanamide), -NH-C(NOH)NH₂ (hydroxyguanidine), -SCN (thiocyanate), -SH (thiol), -B(OH)₂ (boronic acid), -COOH (carboxylic acid), -COCH₂OH (hydroxymethylketone), -COCH₂SH (thiomethylketone), -CONHOH (hydroxamic acid), -SO₂NHOH (sulfohydroxamic acid), -NO-N=O (nonoate), -NOH-COH (reverse hydroxamic acid).

3. The use of compounds of the general formula (I) according to claim 1 or 2, wherein, independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula:

-(CH₂)_n-Y wherein:

- n is an integer from O to 3, and - Y represents a group selected from the list comprising -NHOH, -CONHOH, or

-NOH-COH.

4. The use of compounds of the general formula (I) according to any of claims 1 to 3, wherein, independently from each other, R₄, R₅, R₆, and R₇ represent H, a halogen, an alkyl, an alkoxy, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, a thioalkyl, a thioaryl or an aryloxycarbonyl group, if adequate substituted by a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group.

5. The use of compounds of the general formula (I) according to any of claims 1 to 4, wherein, independently from each other, R₄, R₅, R₆, and R₇ represent H, a halogen, an alkyl, or an alkoxy group from 1 to 10 carbon atoms.

6. The use of compounds of the general formula (I) according to any of claims 1 to 5, wherein Ri represents - H, or

- a peptide sequence, or

- an alkyl, an alkoxy, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, a thioalkyl, a thioaryl, an arylsulfonyl, an aryloxycarbonyl, an alkoxycarbonylalkyl, or an aryloxycarbonylalkyl group, if adequate substituted by a pyridine, a morpholine, a thiomorpholoine, a piperidine, a piperazine, a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group.

7. The use of compounds of the general formula (I) according to any of claims 1 to 6, wherein R₁ represents H, a phenylsulfonyl group of formula -SO₂-C₆H₅, or a benzyloxycarbonyl group of formula -CO-O-CH₂-C₆H₅.

8. The use of compounds according to any of claims 1 to 7, of the following general formula (II) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined in claims 1 to 7, and R₁, R₂, and R₃ are as defined in claims 1 to 7.

9. The use of compounds according to any of claims 1 to 8, of the following general formula (IH) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined in claims 1 to 7, and R₁, R₂, and Y are as defined in claims 1 to 7.

10. The use of compounds according to claim 9, of the formula (III) wherein:

- R₀ represents H, a halogen atom such as Cl, F, or Br, an alkoxy group from 1 to 10 carbon atoms such as a methoxy OCH₃ group, - R₂ represents H or CH₃, - Y represents -NHOH, -CONHOH, or -NOH-COH, and

- R₁ represents H, -SO₂-C₆H₅, Or-CO-O-CH₂-C₆H₅.

11. The use of compounds of the general formula (I) according to any of claims 1 to 10, characterized in that said compounds correspond to the following formulae:

6e 6h

12. The use of compounds of the general formula (I) according to any of claims 1 to 11, wherein the protozoans are chosen among the apicoplast or kinetoplast bearing protozoans, and in particular are selected from the group comprising the protozoans of the following genii: Plasmodium, Toxoplasma, Sarcocystis, Cryptosporidium, Eimera, Theileria, Babesia, Trypanosoma, Leishmania.

13. The use of compounds of the general formula (I) according to any of claims 1 to 11, wherein the bacteria are selected from the group comprising the bacteria of the following genii: Staphylococcus, Streptococcus, Bacillus, Enter ococcus, Pseudomonas, Acinetobacter, Haemophilus, Enterobacter.

14. A pharmaceutical composition, characterized in that it comprises at least one compound according to any of claims 1 to 11, or a pharmaceutically acceptable salt thereof, as active substance, in association with a pharmaceutically acceptable vehicle.

15. An herbicide composition, characterized in that it comprises at least one compound according to any of claims 1 to 11 as active substance.

16. A compound of the general formula (I) :

(I) wherein: - independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula: wherein:

* n is an integer from 0 to 3, * n_\ is 0 or 1,

* X represents a group selected from-O-₅ -NH-, -CHOH-, -CHF-, or -CO-, and

* Y represents a group selected from the list comprising -NH₂ (amine), -NHOH (hydroxylamine), -CH=NOH (oxime), -NHCN (cyananiide), -NH-C(NOH)NH₂ (hydroxyguanidine), -SCN (thiocyanate), -SH (thiol), -B(OH)₂ (boronic acid), -COOH (carboxylic acid), -COCH₂OH (hydroxymethylketone), -COCH₂SH (thiomethylketone),

-CONHOH (hydroxamic acid), -SO₂NHOH (sulfohydroxamic acid), -NO-N=O (nonoate), -

NOH-COH (reverse hydroxamic acid),

- independently from one another, R₄, R₅, R₆, and R₇ represent H, a halogen, or a group comprising from 1 to 10 carbon atoms; - R₁ represents H, or a group comprising from 1 to 50 carbon atoms; provided that :

* at least one of R₂ and R₃ represents a metal chelating group as defined above and, and

* when R₃ represents a metal chelating group as defined above, then -(CH₂)_n-(X)_ni~ Y cannot represent -(CH₂)₂-NH₂ or -(CH₂)₂-SH.

17. Compounds of the general formula (I) according to claim 16, wherein, independently from each other, R₂ and R₃ represent H, methyl, ethyl, propyl, or a metal chelating group of the following formula: -(CHz)_n-Y wherein:

- n is an integer from 0 to 3, and

- Y represents a group selected from the list comprising -NHOH, -CONHOH, or -NOH-COH.

18. Compounds of the general formula (I) according to claim 16 or 17, wherein, independently from each other, R₄, R₅, R₆, and R₇ represent H, a halogen, an alkyl, an alkoxy, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, a thioalkyl, a thioaryl or an aryloxycarbonyl group, if adequate substituted by a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group.

19. Compounds of the general formula (I) according to any of claims 16 tol8, wherein, independently from each other, R₄, R₅, R₆, and R₇ represent H, a halogen, an alkyl, or an alkoxy group from 1 to 10 carbon atoms.

20. Compounds of the general formula (I) according to any of claims 16 to 19, wherein Ri represents

- H, or

- a peptide sequence, or - an alkyl, an alkoxy, an acetyl, an alkoxycarbonyl, an aryl, an aryloxy, a thioalkyl, a thioaryl, an arylsulfonyl, an aryloxycarbonyl, an alkoxycarbonylalkyl, or an aryloxycarbonylalkyl group, if adequate substituted by a pyridine, a morpholine, a thiomorpholoine, a piperidine, a piperazine, a hydroxyl, a thiol, a thioalkyl, a thioaryl, or an amino group.

21. Compounds of the general formula (I) according to any of claims 16 to 20, wherein Rj represents H, a phenylsulfonyl group of formula -SO₂-C₆H₅, or a benzyloxycarbonyl group of formula -CO-O-CH₂-C₆H₅.

22. Compounds according to any of claims 16 to 21, of the following general formula (II) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined in claims 16 to 21, and R₁, R₂, and R₃ are as defined in claims 16 to 21.

23. Compounds according to any of claims 16 to 22, of the following general formula (III) :

wherein R₀ corresponds to R₄, R₅, R₆, or R₇ as defined in claims 16 to 21, and R₁, R₂, and Y are as defined in claims 16 to 21.

24. Compounds according to claim 23, of the formula (III) wherein:

- R₀ represents H, a halogen atom such as Cl, F, or Br, an alkoxy group from 1 to 10 carbon atoms such as a methoxy OCH₃ group, - R₂ represents H or CH₃,

- Y represents -NHOH, -CONHOH, or -NOH-COH, and

- R₁ represents H, -SO₂-C₆H₅, or -CO-O-CH₂-C₆H₅.

25. Compounds of the general formula (I) according to any of claims 16 to 24, characterized in that said compounds correspond to the following formulae:

e 6h

26. A method for screening compounds comprising an indole structure intended for the prevention or the treatment of bacterial or protozoan infections, or for their use as herbicides, characterized in that it comprises the steps of contacting a peptide deformylase enzyme with the compounds to screen and measuring the inhibiting activity of said compounds towards said peptide deformylase enzyme.