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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
  • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
  • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
  • C07C69/738—Esters of keto-carboxylic acids or aldehydo-carboxylic acids
• • 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
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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
  • C07C45/56—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds from heterocyclic compounds
  • C07C45/57—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds from heterocyclic compounds with oxygen as the only heteroatom
  • C07C45/59—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds from heterocyclic compounds with oxygen as the only heteroatom in five-membered rings
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  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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  • C07C67/475—Preparation of carboxylic acid esters by splitting of carbon-to-carbon bonds and redistribution, e.g. disproportionation or migration of groups between different molecules
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
  • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
  • C12P7/26—Ketones
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
  • C07C2601/10—Systems containing only non-condensed rings with a five-membered ring the ring being unsaturated

Definitions

• the present invention relates to novel 4-vinyl-2-cyclopentene-1-one derivatives, their preparation and their use as an antibiotic agent.
• Infectious diseases are the leading cause of morbidity and mortality worldwide. Infections involving germs that are resistant to current treatments pose a major public health problem and lead to an exorbitant excess cost of treatment estimated at 62 billion euros. In Europe, 6.2% of patients who spend more than two days in intensive care contract lung infections and 3.0% of blood infections. 70% of FDA (Food and Drug Administration) -approved antibiotics and more than 80% of known antibiotics are of natural origin. These antibiotics have mainly been isolated from microorganisms (bacteria and filamentous fungi), inexhaustible resources for the discovery of antibiotics and preferentially broad-spectrum, effective against multiple community and nosocomial infections.
• the present application relates to a new family of antibiotic molecules of general formula (I)
• R 1 represents a hydrogen atom or a C 1 -C 4 alkyl radical
• R2 represents a hydroxyl or C 1 -C 4 alkoxyl radical
• R3 and R independently of one another a hydrogen atom or an alkyl radical -C 4; their enantiomers and enantiomeric mixtures, in particular in racemic form.
• the compound of formula (Ia) was isolated from a filamentous Trichoderma fungus, particularly the atrovirid species.
• the Trichoderma LMA strain used has been deposited with the CNCM. This molecule has been isolated and tested under special conditions including:
• Trichoderma in particular Trichoderma atroviride
• In-situ capture during culture of natural molecules produced by Trichoderma Natural products, secondary metabolites. This capture of the molecules is carried out thanks to a known protocol but little used in the field and called “solid phase extraction” or SPE,
• Trichoderma consists of a petri dish on which the microorganism has developed, spores and fragments of mycelium are recovered without distinction by scraping the surface of the agar plate with a sterile liquid, preferably the liquid culture medium.
• the spores and mycelium are then spread or introduced into a solid or liquid microorganism culture medium, preferably liquid and preferably PDB (Potatoes Dextrose Broth) medium consisting of potato and sugar extracts, the composition of which is for example the following: potato starch 4 g / L, dextrose 20 g / L.
• PDB Pantatoes Dextrose Broth
• the fungal strain is cultured at a temperature of between 0 ° C. and 50 ° C., preferably between 20 ° C. and 37 ° C., and preferably at 30 ° C. with rotary stirring or with a turbine driven by a motor between 25 and 25 ° C. 300 rotations per minute, preferably at a speed of 150 rotations per minute.
• the culture is constantly aerated either by spontaneous aeration by the oxygen contained in the ambient air, or by forced aeration by oxygen injection, for example that included in the compressed air.
• the culture thus described can be carried out in any solid or liquid microbiological culture device known to those skilled in the art, preferably in Erlenmeyer flasks or fermenters regardless of their size or size.
• the duration of the culture must correspond to the maximum production of the molecule, it can vary from 1 to 15 days, preferably from 3 to 7 days and preferentially another 5 days.
• In situ capture during culture, of the natural molecules produced by Trichoderma is carried out thanks to a protocol little used in the field and called "Extraction in situ and in solid phase" or in situ SPE.
• This protocol consists in introducing an inert solid element during the culture of the microorganism capable of trapping the molecules of interest released by the latter throughout the culture.
• this protocol involves resins of various natures and preferentially neutral resins such as those described in the following table and preferably apolar resins composed of polystyrene-divinylbenzene copolymers (PS-DVB) of XAD type and preferentially XAD-16 resin.
• PS-DVB polystyrene-divinylbenzene copolymers
• XAD polystyrene-divinylbenzene copolymers
• the resin is then introduced into the culture medium in a sterile manner, either before or after the sterilization thereof.
• the resin is introduced into the culture medium before sterilization and then sterilized at the same time as the medium.
• the amounts of resin vary from 1 g / l to 100 g / l, preferably from 10 g / l to 50 g / l and preferably to 30 g / l. Cut
• the resin is recovered alone or mixed with the biomass by any separation technique between a solid matrix and a liquid container, when the Trichoderma culture lasted the time necessary for the optimal production of the compound of formula (Ia).
• separation techniques include, without limitation, all the means and techniques of filtration, centrifugation, decantation or drying.
• the resin alone or mixed with the biomass is recovered by spontaneous or forced filtration under vacuum, preferably on a porous filter regardless of its nature and preferably on an inert fabric.
• the resin is subsequently treated either after separation from the biomass or mixed with the biomass.
• the molecules captured by the resin are recovered by bringing it into contact with a solvent whatever its nature, preferably an organic solvent, and preferably dichloromethane or methanol, and several times until the recovery of all the molecules of interest.
• the solvent having dissolved the molecules of interest is then recovered by filtration and then evaporated by various techniques known to those skilled in the art. This results in a residue that contains among others the molecule of interest of formula (Ia) mixed with other molecules produced by the microorganism, or initially present in the culture medium.
• the presence of the molecule of interest in this mixture is confirmed by chromatographic or spectrometric analyzes known to those skilled in the art, among which the various chromatography techniques, nuclear magnetic resonance or mass spectrometry.
• chromatographic or spectrometric analyzes known to those skilled in the art, among which the various chromatography techniques, nuclear magnetic resonance or mass spectrometry.
• the presence of the compound of formula (Ia) is evidenced by HPLC high performance liquid chromatography coupled with detectors of PDA, DEDL and mass spectrometry type.
• the target molecule of formula (Ia) is subsequently purified from the total residue by chromatographic techniques known to those skilled in the art, including, but not limited to, chromatography on a silica column or on a reversed phase column. atmospheric pressure or under pressure.
• the molecule of the compound of formula (Ia) is pre-purified by chromatography on a normal silica column and then purified by preparative HPLC under the following conditions: chromatography on silica in normal phase with a mixture of heptane / ethyl acetate solvent ( 9/1) for 20 min at a flow rate of 30 mL / min, followed by preparative HPLC on a C18 reverse phase column using a linear gradient of water and acetonitrile ranging from 0% to 100% acetonitrile in 10 minutes. minutes followed by a 100% acetonitrile step for 5 minutes at a flow rate of 4 mL / min. After lyophilization, 12 mg of the compound of formula (Ia) were obtained from 8L of culture.
• the NMR structural analyzes (FIGS. 1 to 5) correlated with data from the HR-ESI-MS (high resolution mass spectrometry) made it possible to determine the following empirical formula CgHi 0 O 4 of molecular mass 182.0574.
• 13 C NMR ( Figure 2) shows the presence of 9 signals (49.4, 51.7, 77.7, 120.2, 133.9, 149.2, 163.4, 166.4, 205.7 ppm), the proton spectrum ( Figure 1) shows the presence of six groups. protons (2.60, 3.74, 6.17, 6.20, 6.91, 7.33 ppm).
• the COZY spectrum (FIG. 4) shows correlations between the protons of alkenes showing the existence on the molecule of two ethylenic type unsaturations.
• the combination of NMR data and information obtained by mass spectrometry confirms that it is a cyclopentenone type molecule hydroxylated in position 4 substituted by a linear chain of methyl acrylate type.
• the infrared spectrum (FIG. 6) shows absorption bands that reinforce the presence of a ketone, an alcohol and double bonds.
• MW MicroWave (microwaves)
• Red-AI® sodium bis (2-methoxyethoxy) aluminumhybrid Reaction scheme for preparing the compound of formula (la)
• the various compounds of formula (I) may be obtained according to the above reaction scheme by choosing the starting reaction compounds substituted correspondingly to the compounds of formula (I) to be prepared.
• the groups R 3 and R 4 can be introduced in the first step by choosing the appropriate substituent on the chain of furfuryl alcohol (Microwave-gold Microreactor-Assisted Conversion of Furfuryl Alcohols into 4-Hydroxy-2-cyclopentenones, K. Ulbrich et al., Synlett 2010, No. 13, pp. 2037-2040) or later in the synthetic sequence, in particular to convert the hydroxy group to the corresponding alkoxy OR2.
• the molecule of the compound of formula (Ia) is redissolved in appropriate solvents and at an appropriate concentration, preferably in dimethyl sulfoxide DMSO at a concentration of 10 mg / ml. This initial solution is then diluted according to the different biological tests to be performed. Two series of tests were carried out.
• FIG. 7 shows that the antibiotic activity of the compound of formula (Ia) is dependent dose.
• Figure 7 illustrates the antibiotic activity of the compound of formula (Ia) on various target pathogenic microorganisms in amounts of 1 to 100 g in comparison with chloramphenicol and gentamycin, respectively at 30 g and 10 g. It shows, for example in the case of Escherichia coli ATCC 25922, that the compound (Ia) has a much higher activity than that of chloramphenicol. In fact, the 1 g inhibition of the compound of formula (la) is equivalent to that obtained with 30 g of chloramphenicol.
• FIG. 7 shows that, in addition to the broad spectrum of sensitive pathogenic bacteria, the molecule of the compound of formula (Ia) is effective at very low doses.
• TLC analysis The reaction crude obtained, the fractions and the purified products were analyzed by thin layer chromatography (TLC) on gel plates. Silica 60 F 2 54 with a thickness of 0.2 mm on an aluminum support (Merck). The plates are observed under a UV lamp (at 254 and 312 nm) before being revealed by spraying a solution of ammonium molybdate ( ⁇ ) 2 ⁇ 2 ⁇ 7 (100 g / l in 10% sulfuric acid) and heating.
• the frontal ratio is defined as the ratio between the distance traveled by the compound on the plate and the solvent front.
• the high resolution mass spectra were performed on a mass spectrometer equipped with an atmospheric pressure ionization source (ESI) and a TOF time-of-flight type mass analyzer (LCT®, Waters).
• ESI atmospheric pressure ionization source
• LCDT® TOF time-of-flight type mass analyzer
• the nuclear magnetic resonance experiments were carried out on Bruker Avance 300 and 500 units using deuterated chloroform CDCl3.
• the chemical shifts are expressed in ppm (parts per million) and calibrated relative to the reference solvent.
• the coupling constants are expressed in Hertz (Hz).
• the multiplicity of signals is expressed by the following abbreviations: s (singlet), bs (wide singlet), d (doublet), dd (doublet split), t (triplet), m (multiplet), q (quadruplet).
• the rotatory powers of the compounds were measured using a Jasco TM P1010 polarimeter equipped with Spectro Manager software.
• Source monochromatic luminous is the D-line of sodium.
• the experiments were carried out with a 100 mm quartz tank of 350 ⁇ , and the products were solubilized in methanol.
• the infrared (IR) absorption spectra of the described compounds were measured on the Perkin-Elmer Spectrum 100 FT-IR spectrometer. The device is equipped with Spectrum software (version 6.3.5) from Perkin-Elmer. The compounds were prepared in solution in methanol and then dried with compressed air. The absorption bands are given in cm -1 .
• N-Butyl lithium (1.6 M in hexane, 2.14 ml, 1.1 eq) is added to a solution of diisopropylamine (529 ⁇ l, 1.2 eq) in 10 ml of THF and maintained at -78 ° C. After 30 minutes, the methyl propiolate (277 ⁇ l, 1.0 eq) is added to the previously obtained LDA solution. The reaction is stirred for 1 h at -78 ° C. Compound 3 (660 mg, 3.1 mmol) is solubilized in 5 ml of THF at -78 ° C. and the solution is slowly added to the previously generated lithium acetylide solution. The reaction is stirred at -78 ° C for 1 hour.
• Compound 7 was analyzed by chiral HPLC equipped with a UV detector in order to highlight the two enantiomers and to be able to separate them. (Alliance Waters 2695 HPLC chain coupled to a Waters 996 PDA detector, chiral IC Daicel Chiral 4.6 x 250 mm (5 ⁇ ) column, eluent n-Heptane / Isopropanol 80:20).
• the present invention relates to the compounds of formula (I) or (la) for their use as an antibiotic agent in particular as a broad-spectrum antibiotic agent and in particular against gram-positive and gram-negative pathogenic bacteria that are multidrug-resistant.
• the present invention relates to a pharmaceutical composition containing as active principle at least one compound of formula (I) or (la) associated with a pharmaceutically acceptable excipient, which can be determined easily according to the general knowledge of the skilled person according to the chosen route of administration.

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• 2014
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