Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
  • A61P31/06—Antibacterial agents for tuberculosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
  • A61P31/08—Antibacterial agents for leprosy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
  • A61P33/06—Antimalarials
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
  • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D211/80—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
  • C07D211/84—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen directly attached to ring carbon atoms
  • C07D211/90—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen

Definitions

• the present invention relates to bi-substrate inhibitor type molecules associating a hydrophobic substituent with an analogue of the active metabolite of isoniazid, to their process of preparation, to the pharmaceutical compositions containing them and to their use, in particular as a enoyl-ACP (Acyl Carrier Protein) reductase inhibitor, for the preparation of an anti-infective drug, in particular an antituberculous drug.
• enoyl-ACP Acyl Carrier Protein
• the invention is based on the reasoned conception of new compounds based on an improved knowledge of the mechanism of action of a reference medicine and the mechanisms of resistance.
• the fatty acid elongation system (Fatty Acid Synthase II or FAS-II System) is necessary for the biosynthesis of mycolic acids, specific constituents and essential components of the envelope of mycobacteria, especially Mycobacterium tuberculosis (or Koch's bacillus). , tuberculosis pathogen, M. leprae, leprosy agent, and other opportunistic pathogenic mycobacteria (eg M. avium, M. ulcerans, M. marinum). This potential therapeutic target is found in certain other infectious agents (ex Plasmodium falciparum) parasite responsible for malaria).
• Isoniazid is a first-line antituberculous antibiotic whose efficacy is increasingly limited by the appearance of resistant M. tuberculosis strains. It is a pro-drug that is activated by oxidation in mycobacteria by means of a catalase peroxidase (KatG) with formation of covalent adducts isonicotinoyl-NAD (INH-NAD) considered as the active metabolites of isoniazid. These adducts are excellent inhibitors of InhA, an enzyme of the FAS-II system.
• isonicotinoyl-NADP (INH-NADP)
• MabA protein which has a 3D structure related to that of InhA.
• the phenomena of resistance developed largely consist of mutations that mainly affect the enzyme KatG (lack of activation). Mutations are also found to a lesser extent at the level of the target enzyme InhA (loss of affinity of the inhibitory adduct for its target in vivo) or its promoter (overproduction of the target).
• the design of bi-substrate inhibitors that do not require a prior KatG activation step and ideally interact within the InhA active site with regions of the protein that exclude the most frequently encountered mutation sites should allow to largely bypass the current problems of resistance to NNH.
• the present inventors have therefore developed bi-substrate type InhA inhibitors combining (i) a unit derived from the active metabolites of pyridine, pyridinium or dihydropyridine type isoniazid and related structures, and (ii) a hydrophobic substituent targeting the substrate site.
• the compounds identified according to the present invention also have other targets and have inhibitory properties on unhealthy bacterial strains of InhA, making them anti-infective compounds with wide application range. .
• the present invention relates to compounds of general formula (I):
• the ring G w represents an aromatic nucleus or not aromatic, 6-membered, optionally comprising one or more nitrogen atoms, said nitrogen atom (s) possibly being substituted with an optionally substituted tetrahydrofuran group, such as 2- hydroxymethyl tetrahydrofuran-3,4-diol; or by a group -CH 2 -E where E represents an electron-withdrawing group, such as -CONRR ';-CO-SR; phenyl substituted for example with CN or NO 2 ; -CO-O-Alkyl; -CO-OAI kyle-OH; -O-Alkyl-OAc; where R and R ', which are identical or different, independently represent a hydrogen atom or an alkyl group; more preferably, the nitrogen atom is optionally substituted with a group
• W is an optionally substituted phenyl, pyridine, pyrazine or dihydropyridine ring.
• w is substituted in the ortho or meta position.
• R 3 OH or -Oalkyl and R 1 and R 2 together form a single bond, or
• the compounds of formula (I) may comprise one or more asymmetric carbon atoms. They can therefore exist as enantiomers or diastereoisomers. These enantiomers, diastereoisomers, as well as their mixtures, including the racemic mixtures, form part of the invention.
• the compounds of formula (I) may exist in the form of bases or addition salts with acids. Such addition salts are part of the invention.
• salts can be prepared with pharmaceutically acceptable acids, but the salts of other acids that are useful, for example, for the purification or the isolation of the compounds of formula (I) are also part of the invention.
• the compounds of formula (I) may also exist in the form of hydrates or solvates, namely in the form of associations or combinations with one or more water molecules or with a solvent. Such hydrates and solvates are also part of the invention.
• halogen atom is understood to mean: a fluorine, a chlorine, a bromine or an iodine; alkyl group: a saturated linear or branched aliphatic group of 1 to 20 carbon atoms, unless otherwise indicated.
• alkyl group a saturated linear or branched aliphatic group of 1 to 20 carbon atoms, unless otherwise indicated.
• cycloalkyl group a cyclic alkyl group of 3 to 10 carbon atoms.
• alkenyl group a linear or branched, mono- or poly-unsaturated aliphatic group of 2 to 20 carbon atoms, comprising, for example, one or two ethylenic unsaturations
• alkynyl group a linear or branched, mono- or poly-unsaturated aliphatic group of 2 to 20 carbon atoms, comprising, for example, one or two acetylenic unsaturations
• aryl group a cyclic aromatic group comprising between 5 and 10 carbon atoms.
• aryl groups include phenyl or naphthyl; heteroaryl group: a cyclic aromatic group comprising between 5 and 10 carbon atoms and comprising between 1 and 3 heteroatoms, such as nitrogen, oxygen or sulfur.
• heteroaryl groups mention may in particular be made of pyridine.
• C A) represents a dihydropyridine group, optionally substituted with a group selected from -CH 2 COOAlkyl, -CH 2 COOAlkyleOH; is a phenyl group substituted in the ortho or meta position, preferably meta, by a group selected from C5-C20 alkyl, -Oalkyl, -Oalkenyl, -OH, phenyl optionally substituted with alkyl
• the present application also relates to the process for preparing the compounds of general formula (I).
• the compounds of general formula (I) can be prepared according to the following process.
• the compounds of general formula (I) in which R 1 and R 2 together form a single bond and R 3 represents -OH may be prepared by coupling of a compound of general formula (II):
• the coupling reaction is conducted in the presence of an organic or inorganic base such as n-BuLi or f-BuLi, in a solvent such as THF, at a temperature between -78 ° and room temperature, under an inert atmosphere.
• an organic or inorganic base such as n-BuLi or f-BuLi
• a solvent such as THF
• substitution reaction is carried out by application or adaptation of the substitution reactions known to those skilled in the art, by means of appropriate reagents. These substitution reactions are described for example in March's Advanced Organic Chemistry, 5 rd Ed., John Wiley and Sons, Inc. or Larock, Comprehensive Organic Transformations, VCH Ed. As representative, for example, there may be mentioned the substitution reaction leading to the compound of general formula (I) wherein ⁇ is substituted by an alkyl group. This reaction may in particular be carried out using alkylboronic acid, in the presence of diphenylphosphine ferrocene palladium (II) chloride, silver oxide and potassium carbonate.
• the compound of the general formula (II) is commercially available or may be prepared by the application or adaptation of known methods for substituting the nucleus as desired to obtain the compound of the formula (I) wherein the formula is substituted.
• the nucleus can be substituted by an alkyl group with the average ⁇ • of the corresponding starting material (II) in which w is substituted by a hydroxyl group, by reacting a compound of the halide type. alkyl, in the presence of a base such as potassium carbonate.
• the compounds of general formula (I) in which R 1 and R 2 together form a single bond and R 3 represents -OH may be prepared by cyclization of compounds of general formula (V):
• B is a hydrogen atom when n'est is unsubstituted or is the substituent corresponding to the general formula (I) desired (in both cases, the compound (IV) corresponds to the compound (I)), or a halogen atom, the cyclization then being followed in this case by the substitution reaction of the halogen atom of the compound (IV) with the appropriate substituent of v ⁇ corresponding to the general formula (I) desired.
• this reaction is carried out, without isolation of the intermediate acyl chloride, at a temperature between room temperature and the boiling temperature of the reaction mixture.
• the compound of formula (V) can be obtained from a corresponding compound of formula (VI):
• this reaction can be carried out using a reducing agent such as sodium tetraborohydride, in a solvent such as an alcohol, for example ethanol.
• a reducing agent such as sodium tetraborohydride
• a solvent such as an alcohol, for example ethanol.
• the compound of formula (VII) may be prepared by coupling of the compounds of formula (VIII) and (IX) corresponding:
• ⁇ -, ⁇ ⁇ and X are as defined in formula (VII) and Alk, Alk ', which are the same or different, independently represent an alkyl group.
• this reaction is carried out in the presence of a base such as a lithium-based compound, for example lithium diisopropylamide, in a suitable organic solvent, such as diethyl ether.
• a base such as a lithium-based compound, for example lithium diisopropylamide
• a suitable organic solvent such as diethyl ether.
• this reaction is carried out in an anhydrous medium, under an inert atmosphere, by adding the base to the compound of formula (IX) and then adding the compound of general formula (VIII).
• this reaction is conducted at temperature between -75 ⁇ C and 0 ⁇ C,
• the compound of formula (VIII) can be obtained by coupling of the compounds of formula (X) and (Xl) corresponding: B) in which ⁇ , X, Alk and Alk 'are defined as in general formula (VIII).
• this reaction is carried out using EDCI type coupling reagents such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (hydrochloride salt) and HOBT (1-hydroxy-7H-benzotriazole hydrate) and in the presence of a base such as triethylamine.
• the preparation process further comprises the step of
• R-HaI (XII) in which HaI represents a halogen atom, such as bromine and R represents a group of the -CH 2 E type, E being an electron-withdrawing group defined as in general formula (I).
• this reaction is conducted in a solvent such as anhydrous tetrahydrofuran, at a temperature between room temperature and the reflux temperature of the reaction mixture.
• the compound of general formula (I) can be obtained by reduction from the compound of general formula ( I) wherein ( ⁇ A- ⁇ ) represents a pyridinium ring.
• a reducing agent there may be mentioned sodium triacetoxyborohydride in an acetic medium. Generally, this reaction is conducted at a temperature between -10 ° and room temperature.
• the method according to the invention comprises the subsequent step of isolating the desired product obtained.
• the starting compounds (II), (III), (IX), (X), (X1) and the appropriate reagents are commercially available or can be prepared by application or adaptation of methods known to those skilled in the art.
• the compounds according to the invention exhibit remarkable anti-infectious properties.
• the subject of the invention is medicaments which comprise a compound of formula (I)
• the ring G w represents an aromatic or nonaromatic 6-membered ring optionally comprising one or more nitrogen atoms, the said nitrogen atom (s) being able to optionally substituted with an optionally substituted tetrahydrofuran group, such as 2-hydroxymethyl-tetrahydrofuran-3,4-diol; or by a group -CH 2 -E where E represents an electron-withdrawing group, such as -CONRR ';-CO-SR; -CO-OAI kyle; -CO-OAI kyle-OH; -O-Alkyl-OAc; phenyl substituted for example with CN or NO 2 ; where R and R ', which are identical or different, independently represent a hydrogen atom or an alkyl group; and / or the said nitrogen atom (s) which may be present in the form of pyridinium salts, the counterion then being the anion of a halogen atom, such
• R3 represents -OH or -Oalkyl and R2 forms with R1 a single bond linking the nitrogen atom to the carbon atom substituted by R3,
• the compounds according to the invention can be used, in humans or in animals, as anti-infectives, in particular in the treatment or prevention of:
• mycobacteriosis more particularly tuberculosis, leprosy or opportunistic diseases, such as those related to Mycobacterium avium, M. bovis, M. marinum and / or M. ulcerans;
• any pathogen-related infection having an Enoyl-Acyl Carrier Protein Reductase enzyme, or a structurally related enzyme, belonging to the Short Chain Dehydrogenase Reductases (SDR) superfamily.
• SDR Short Chain Dehydrogenase Reductases
• the compounds according to the invention can be used in the treatment of tuberculosis.
• the present invention relates to pharmaceutical compositions comprising, as active principle, a compound according to the invention.
• These pharmaceutical compositions contain an effective dose of at least one compound according to the invention, or a pharmaceutically acceptable salt, a hydrate or solvate of said compound, as well as at least one pharmaceutically acceptable excipient.
• Said excipients are chosen according to the pharmaceutical form and the desired mode of administration, from the usual excipients which are known to those skilled in the art.
• compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal or rectal administration the active ingredient of formula (I) above, or its salt, solvate or hydrate, may be administered in unit dosage form, in admixture with conventional pharmaceutical excipients, to animals and humans for the prophylaxis or treatment of the above disorders or diseases.
• Suitable unit dosage forms include oral forms such as tablets, soft or hard capsules, powders, granules and oral solutions or suspensions, sublingual, oral, intratracheal, intraocular, intranasal forms of administration. by inhalation, topical, transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms and implants.
• the compounds according to the invention can be used in creams, gels, ointments or lotions.
• compositions according to the invention may comprise, as active ingredient, from 10 to 800 mg of a compound of general formula (I) according to the invention.
• a unitary form of administration of a compound according to the invention in tablet form may comprise the following components: Compound according to the invention 50.0 mg
• the dose of active ingredient administered per day may reach 0.01 to 100 mg / kg, in one or more doses, preferably 0.02 to 50 mg / kg.
• the dosage appropriate to each patient is determined by the physician according to the mode of administration, the weight and the response of said patient.
• the present invention also relates to combinations of a compound of general formula (I) according to the invention and an active principle with anti-infectious activity.
• an anti-infective compound there may be mentioned isoniazid, rifampicin, pyrazinamide, ethambutol, chloroquine, or any other antibiotic molecule currently used clinically.
• the present invention also relates to the process for preparing the molecules of general formula (I) above.
• the present invention also relates to the use of the compounds of general formula (I) in therapy.
• Compound 1a has the following formula:
• Compound 1b has the following formula:
• the reaction medium is allowed to return to ambient temperature with stirring and the reaction is monitored by TLC (eluent: dichloromethane / methanol 95/5). Then 50 mL of distilled water is added to the solution and the reaction mixture is extracted with ethyl acetate (4 x 20 mL). The organic phases are combined, washed with a saturated solution of sodium chloride, dried over anhydrous sodium sulphate and concentrated on a rotary evaporator. The oily residue obtained is purified by chromatography on a column of silica gel (eluent: dichloromethane / methanol gradient 100/0 to 98/2), to give 330 mg (28%) of the ketoamide 1b in the form of a yellow solid.
• Step 3 carbonyl reduction of amidocétone 1b obtained in the 2nd step.
• keto amide 1b (627 mg, 1.6 mmol) in absolute ethanol (80 ml) is added sodium tetraborohydride (421 mg, 8.8 mmol). After stirring for 3 hours at room temperature, 50 ml of distilled water are added. The reaction mixture is extracted with dichloromethane (3 ⁇ 20 ml). The organic phases are combined, dried over anhydrous sodium sulphate and concentrated on a rotary evaporator to give 631 mg (85%) of the two rotamers of the hydroxyamide 1c as a yellow paste.
• the two rotamers are obtained in proportion 65/35, but for the sake of convenience the 1 H and 13 C spectra are described as if the protons or carbons of the minority rotamer had the same intensity as the protons or carbons corresponding to the majority rotamer.
• the reaction mixture is concentrated on a rotary evaporator.
• the residue obtained is purified by chromatography on a column of silica gel (eluent: gradient dichloromethane / methanol: 100/0 to 80/20), to give 312 mg (85%) of the keto acid 1d in the form of a solid yellow.
• keto acid 1d (331 mg, 1.1 mmol) in thionyl chloride (20.0 mL, 300.0 mmol) is stirred at 600 ° C for 2 hours.
• the reaction medium is then concentrated under reduced pressure, the residue is taken up in dichloromethane and is again evaporated. This operation is repeated twice.
• the residue is finally taken up in acetone (17 ml) and then a 32% aqueous solution of ammonium hydroxide (9.2 ml, 3.0 mmol) is added dropwise.
• the resulting solution is stirred at room temperature for 1 h.
• the reaction medium is concentrated on a rotary evaporator.
• the residue obtained is purified by column chromatography on silica gel (eluent: dichloromethane / methanol gradient: 100/0 to 95/5) to give 268 mg (80%) of hemiamidal 1 in the form of a beige powder. .
• Step 2 Synthesis of compound 3 by condensation of 3,4-pyridinedicarboximide and Compound 3a obtained in Step 1.
• reaction is treated with a saturated solution of ammonium chloride (10 ml) and the reaction medium is extracted with ethyl acetate (3 ⁇ 8 ml). .
• organic phases are recovered, dried over sodium sulphate and concentrated under vacuum.
• the white powder obtained is purified by chromatography on a column of silica gel (eluent: gradient dichloromethane / methanol: 100/0 to 93/7) to give a mixture of para / meta compounds.
• Para A product is recovered cleanly after 3 days of recrystallization in acetone.
• the reaction is treated with a saturated solution of ammonium chloride (30 ml) and the reaction medium is extracted with ethyl acetate (3 ⁇ 20 ml). mL). The organic phases are recovered, dried over sodium sulphate and concentrated under vacuum.
• the white powder obtained (720 mg) is purified by chromatography on a column of silica gel (eluent: gradient dichloromethane / methanol: 100/0 to 93/7) to obtain 128 mg (48%) of the mixture of para / meta compounds .
• the para product is recovered (32.3 mg, 28%) after 3 days of recrystallization from acetone. This recrystallization product is still contaminated with 23% of the meta compound.
• EXAMPLE 18 Demonstration of the inhibitory effect of the compounds claimed on the activity of InhA.
• InhA protein was expressed in E. coli after cloning the inhA gene into a pET plasmid vector.
• the growth of the resulting strain and the induction of inhA gene expression by 1 mM IPTG (isopropyl ⁇ -D-thiogalactoside) leads to the production of the 28.5 KDa (28,368 Da) water-soluble InhA protein (monomer) which is purified by standard protein purification techniques.
• the enzyme exists as a tetramer in solution and is stored in 50 mM Hepes buffer, 50% glycerol at -20O.
• the enoyl reductase activity of InhA is followed by UV spectrometry following the disappearance of the signal of the reduced cofactor NADH at 340 nm as a function of time.
• the enzymatic reaction is carried out in a final volume of 100 ⁇ l (in quartz vat, optical path 1 cm).
• the absorption of each reaction mixture is determined with a spectrophotometer UVIKON 293 (Bio-Tek Kontron Instruments) connected to a thermostatic bath for regulating the temperature of the tank to 25O.
• a baseline is performed during preincubation just before the start of the measurements. The measurements are carried out for 3 minutes after preincubation for 5 minutes or 2 hours.
• Mycobacterium smegmatis mc 2 155 (Ro) strain is cultured in Middelbrook 7H9 (Difco) + glycerol 0.2% + 0.05% tween 80 to 37O culture medium with shaking (200 rpm) in order to avoid the formation of bacterial veil. After 3 days, the culture R 0 is left standing for 10 min, the time that the larger aggregates sediment. The supernatant is taken to launch a new Ri culture, in the same conditions, seeded at 1/100. After stirring for 3 days at 37 °, and 10 min of rest, the supernatant is removed. The optical density of the bacterial suspension is measured at 650 nm and reduced to a value between 0.002 and 0.003 by dilution in the culture medium (without tween 80), it is the R 2 .
• the antimycobacterial activity is evaluated by colorimetric MTT reduction tests (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide) on the M. smegmatis mc 2 strain. 155.
• MTT yellow tetrazolium salt
• the reduction can no longer take place and the solution remains yellow.
• a reading of the optical density at 570 nm makes it possible to observe the formation of the reduced purple formazan MTT. Percent inhibition is calculated by subtracting from 100 the ratio of ODs without and with inhibitor multiplied by 100: 100 - (OD ⁇ nh - / DOtem * 100)
• the tests are carried out on NUNC microplates (Merk-eurolab) 96 wells. Each product is tested over a concentration range starting from 5 mM by carrying out successive two-fold dilutions. Each well contains 100 ⁇ L of compound in solution in the culture medium (7H9 + 0.2% Gro with 1% (v / v) of final DMSO). 100 ⁇ L of bacterial suspension R 2 is then added. The plate is closed with parafilm and incubated at 37 ° C. After 24 hours of incubation, 50 ⁇ L of a 1 mg / mL solution of MTT are added to each well. After incubation for 3 hours at 37 ° C., 100 ⁇ l of lysis buffer are added. in each well and the plate is stirred at room temperature until the solution is well homogeneous.
• optical density is measured at 570 nm with a microplate reader spectrophotometer ⁇ Quant Bio-tek instruments, INC.
• INH has no activity on C. glutamicum at 5mM.
• the compounds according to the invention are capable of inhibiting the growth of the bacterium C. glutamicum at a Cl 50 less than or equal to at 10 ⁇ M.
• the bacteria belonging to this strain have the particularity of not having a FAS-II elongation cycle in the biosynthesis of fatty acids. There is no equivalent of InhA in C. glutamicum.
• the compounds according to the invention which are active on this strain of bacteria thus also have another target than the FAS-II system.
• the Corynebacterium glutamicum strain is grown in BHI (Brai Heart Infusion) medium at 300 with shaking at 200 rpm. The OD of the culture is directly measured and adjusted to a value of 0.002-0.003 by dilution in LB culture medium. C. glutamicum also grows in the medium LB, it is used in place of the BHI because the latter is very colored and disturbs the reading of the OD at 570 nm.
• BHI Brain Heart Infusion
• the stock solutions are prepared in DMSO at a concentration 100 times higher than the highest concentration to be tested. Evaluation of the growth of bacterial suspension of C. / lutamicum
• test runs in the same way as for the test on M. smegmatis.
• the tests are carried out on 96-well NUNC (Merck-eurolab) microplates. All concentrations were tested at least twice.
• Each well contains 100 ⁇ L of compound in solution in the culture medium (LB with 1% final DMSO). 100 ⁇ l of bacterial suspension of 0.002 OD at 650 nm are then added.
• the plate is closed with parafilm and incubated at 30 ° C. for C. glutamicum. After two hours of incubation, 50 ⁇ L of a 1 mg / mL solution of tetrazolium salt (MTT) are added to each well. After one hour incubation at 30O for C. glutamicum, 100 .mu.l of lysis buffer are added to each well and the plate is stirred at room temperature until the solution is well homogeneous.
• MTT tetrazolium salt
• optical density is measured at 570 nm with a spectrophotometer ⁇ Quant Bio-tek Instruments, INC, 96-well plate reader.

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