WO2019138084A1 - Dérivés de phénanthrolinone destinés à être utilisés dans le traitement d'infections bactériennes - Google Patents
Dérivés de phénanthrolinone destinés à être utilisés dans le traitement d'infections bactériennes Download PDFInfo
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- WO2019138084A1 WO2019138084A1 PCT/EP2019/050713 EP2019050713W WO2019138084A1 WO 2019138084 A1 WO2019138084 A1 WO 2019138084A1 EP 2019050713 W EP2019050713 W EP 2019050713W WO 2019138084 A1 WO2019138084 A1 WO 2019138084A1
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- 0 C*(C)(C=C1C(*)=O)c(c(*(O)c(*)cc2*)c2c(*)c2)c2C1=O Chemical compound C*(C)(C=C1C(*)=O)c(c(*(O)c(*)cc2*)c2c(*)c2)c2C1=O 0.000 description 1
- XMDLWGKZZUTGOA-UHFFFAOYSA-N CCN(C=C1C(O)=O)c(c2ncccc2c(Br)c2)c2C1=O Chemical compound CCN(C=C1C(O)=O)c(c2ncccc2c(Br)c2)c2C1=O XMDLWGKZZUTGOA-UHFFFAOYSA-N 0.000 description 1
- KPSRYRUXIKUCCU-UHFFFAOYSA-N CCN(C=C1C(OCC)=O)c(c(nccc2)c2c(Br)c2)c2C1=O Chemical compound CCN(C=C1C(OCC)=O)c(c(nccc2)c2c(Br)c2)c2C1=O KPSRYRUXIKUCCU-UHFFFAOYSA-N 0.000 description 1
- AYNHGRAPXYLSDM-UHFFFAOYSA-N CCN(C=C1C(OCC)=O)c(c2ncccc2c(N)c2)c2C1=O Chemical compound CCN(C=C1C(OCC)=O)c(c2ncccc2c(N)c2)c2C1=O AYNHGRAPXYLSDM-UHFFFAOYSA-N 0.000 description 1
- BQHQRHZJTMOKLB-UHFFFAOYSA-N CCN(C=C1C(OCC)=O)c(c2ncccc2cc2)c2C1=O Chemical compound CCN(C=C1C(OCC)=O)c(c2ncccc2cc2)c2C1=O BQHQRHZJTMOKLB-UHFFFAOYSA-N 0.000 description 1
Classifications
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- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
Definitions
- the present invention is in the field of therapeutic drugs to treat bacterial infections.
- the invention provides phenanthrolinone compounds for use in the treatment of bacterial infections, for example tuberculosis.
- Tuberculosis remains one of the most life-threatening infectious diseases worldwide with 8 million new cases and 1.5 million deaths per year. The standardized 6 months short term treatment is not always followed appropriately and results in relapses and drug resistances.
- Multi-drug resistant in particular defined as resistance to at least rifampicin (RMP) and isoniazid (INH)
- XDR-TB in particular defined as MDR-TB plus resistance to any fluoroquinolone (FQ) and at least one second line injectable agent -kanamycin (KM), amikacin (AMK) or capreomycin (CPM)
- FQ fluoroquinolone
- KM second line injectable agent -kanamycin
- AMK amikacin
- CPM capreomycin
- the mechanism of action of the gyrase resides in passing one region of duplex DNA through another, thereby introducing or removing DNA supercoils, a part of them being issued from the replication process and should be resolved for its processing.
- the reaction mechanism consists in two single stands nicks, staggered by 4 base pairs for providing a gate for double stand passage. Then nicks are resealed, thus restoring the DNA primary and secondary structure.
- the quinolone intercalates into DNA at the nicks and the quinolone C-7 ring interact with GyrB whereas the 3-carboxyl end extends into GyrA and participates in a magnesium-water bridge that stabilizes the drug-enzyme-DNA complex. It has been proven that interactions with the fluoroquinolone happen with different forms of the complex and that the quinolone C7 ring can interact alternatively with both GyrA and GyrB.
- Fluoroquinolones are used for many purposes, either as a monotherapy in urinary tract infections or in combination with rifampicin for severe forms of staphylococcal infections. It is a key drug for the treatment of MDR-TB in association with other second line drugs.
- a further objective of the present invention is to provide compounds able to treat bacterial infections caused by strains resistant to currently available antibiotics.
- Inventors have for the first time demonstrated that a selection of phenanthrolinone derivatives displays a strong inhibition of bacterial species, together with a low cytotoxicity.
- this series of molecules showed high efficiency against M. tuberculosis replication in vitro and in in vivo cultured macrophages, while being not toxic for mammalian cells and being active against M. tuberculosis complex strains carrying the gyrA mutations responsible for high level resistances to the fluoroquinolones.
- said compounds are active on FQ resistant M. tuberculosis strains.
- the present invention relates to a method of treating a bacterial infection which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I):
- Z represents N or N + -0 ;
- R represents:
- R represents a linear or branched Ci-C 4 alkyl group
- Ri and R 2 each independently represent an hydrogen or a linear or branched Ci-C 4 alkyl group
- R' represents:
- Ci-C 4 alkyl group a linear or branched Ci-C 4 alkyl group
- Y represents:
- R 3 and R 4 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group, in particular a -NH 2 group;
- Xi and X 2 each independently represent:
- R 5 and R 6 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group, in particular a -NH 2 group; or a piperidine, pyrrolidine, morpholine or piperazine group, optionally substituted by at least one group selected from:
- R 7 and R 8 each independently represent an hydrogen or a linear or branched C1-C4 alkyl group, in particular a -NH 2 group; providing that:
- At least one of Y and Xi does not represents an hydrogen atom
- R’ represents an hydrogen atom
- Y represents an hydrogen atom
- R’ represents an hydrogen atom
- Y represents an hydrogen atom
- R represents:
- R represents a linear or branched C 1 -C 4 alkyl group
- Ri and R 2 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group
- R' represents an hydrogen atom
- Y represents an hydrogen atom
- Xi represents a -N0 2 group
- X 2 represents an hydrogen atom.
- R’ represents a linear or branched C 1 -C 4 alkyl group; or a C3-C6 cycloalkyl group.
- Y represents:
- the present invention also relates to a method of treating a bacterial infection which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I):
- R represents:
- R represents a linear or branched C 1 -C 4 alkyl group
- Ri and R 2 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group
- R' represents:
- Y represents:
- R 3 and R 4 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group, in particular a -NH 2 group;
- Xi and X 2 each independently represent:
- R 5 and R 6 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group, in particular a -NH 2 group; or
- a piperidine, pyrrolidine, morpholine or piperazine group optionally substituted by at least one group selected from: a Ci-C 4 alkyl group;
- R 7 and R 8 each independently represent an hydrogen or a linear or branched C1-C4 alkyl group, in particular a -NH 2 group; and the stereoisomeric forms, mixtures of stereoisomeric forms and/or pharmaceutically acceptable salts forms thereof.
- the present invention also relates to a compound of formula (I) as defined above for use in the treatment of a bacterial infection.
- Xi and/or X 2 represent independently H or a -N0 2 group. In a particular embodiment, Xi and/or X 2 represent H.
- Xi and X 2 represent H.
- Xi represents a -N0 2 group and X 2 represents H.
- Y represents a -N0 2 group or a bromine atom.
- Y represents a -N0 2 group.
- R represents:
- R represents a linear or branched C 1 -C 4 alkyl group
- Ri and R 2 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group.
- R represents:
- R represents a linear or branched Ci-C 2 alkyl group
- R’ represents a linear or branched C1-C4 alkyl group.
- R’ represents a linear or branched Ci-C 2 alkyl group.
- R represents:
- R represents a linear or branched C 1 -C 4 alkyl group
- Ri and R 2 each independently represent an hydrogen or a linear or branched C 1 -C 4 alkyl group
- R' represents a linear or branched C 1 -C 4 alkyl group
- Y represents a bromine atom or a -N0 2 group
- Xi and X 2 represent an hydrogen atom or a -N0 2 group.
- R represents:
- R represents a linear or branched Ci-C 2 alkyl group; or a -NH 2 group;
- R' represents a linear or branched Ci-C 2 alkyl group
- Y represents a bromine atom or a -N0 2 group
- Xi and X 2 represent an hydrogen atom or a -N0 2 group.
- the compound of formula (I) is selected from:
- said bacterial infection is caused by a mycobacterium, in particular Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium bovis BCG.
- a mycobacterium in particular Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium aurum, Mycobacterium bovis BCG.
- said bacterial infection is caused by Mycobacterium tuberculosis.
- said bacterial infection is tuberculosis, leprosy, Buruli ulcer or aquarium granuloma.
- said bacterial infection is tuberculosis.
- tuberculosis is selected from the group consisting of susceptible tuberculosis, multiple drug-resistant tuberculosis and extensively drug-resistant tuberculosis.
- tuberculosis is resistant to fluoroquinolones.
- said bacterial infection is caused by a bacteria of the genus Staphylococcus, in particular Staphylococcus aureus species, more particularly Staphylococcus aureus strains susceptible or resistant to b-lactams (including methicillin- resistant strains also referred as MRSA), and/or susceptible or resistant to glycopeptides (including vancomycin-intermediate or glycopeptides- intermediate Staphylococcus aureus strains also referred as VISA or GISA), or multi-drug resistant Staphylococcus aureus strains.
- Staphylococcus aureus species more particularly Staphylococcus aureus strains susceptible or resistant to b-lactams (including methicillin- resistant strains also referred as MRSA), and/or susceptible or resistant to glycopeptides (including vancomycin-intermediate or glycopeptides- intermediate Staphylococcus aureus strains also referred as VISA or GISA), or multi-drug resistant Staphylococcus au
- said bacterial infection belongs to the group comprising:
- TSS toxic shock syndrome
- the present invention also relates to a pharmaceutical composition
- a pharmaceutical composition comprising a compound of formula (I) as defined above, or
- the compounds of formula (I) of the present invention may be administered in the form of a conventional pharmaceutical composition by any route including orally, intramuscularly, subcutaneously, topically, intranasally, intraperitoneally, intrathoracially, intravenously, epidurally, intrathecally, intracerebroventricularly and by injection into the joints.
- the dosage will depend on the route of administration, the severity of the disease, age and weight of the patient and other factors normally considered by the attending physician, when determining the individual regimen and dosage level at the most appropriate for a particular patient.
- inert, pharmaceutically acceptable carriers can be either solid or liquid.
- Solid form preparations include powders, tablets, dispersible granules, capsules, cachets, and suppositories.
- a solid carrier can be one or more substances, which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
- Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.
- Liquid form compositions include solutions, suspensions, and emulsions.
- sterile water or propylene glycol solutions of the active compounds may be liquid preparations suitable for parenteral administration.
- Liquid compositions can also be formulated in solution in aqueous polyethylene glycol solution.
- Aqueous solutions for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavoring agents, stabilizers, and thickening agents as desired.
- Aqueous solutions for oral use can be made by dispersing the finely divided active component in water together with a viscous material such as natural synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.
- the pharmaceutical composition will according to one embodiment of the present invention include 0.05% to 99% weight (percent by weight), according to an alternative embodiment from 0.10 to 50% weight, of the compound of the present invention, all percentages by weight being based on total composition.
- a therapeutically effective amount for the practice of the present invention may be determined, by the use of known criteria including the age, weight and response of the individual patient, and interpreted within the context of the disease which is being treated or which is being prevented, by one of ordinary skills in the art.
- the present invention also relates to a compound of formula (I) as defined above, and the stereoisomeric forms, mixtures of stereoisomeric forms or pharmaceutically acceptable salts forms thereof.
- a range of values in the form“x-y” or“x to y”, or“x through y”, include integers x, y, and the integers therebetween.
- the phrases“1-6”, or“1 to 6” or“1 through 6” are intended to include the integers 1, 2, 3, 4, 5, and 6.
- Preferred embodiments include each individual integer in the range, as well as any subcombination of integers.
- preferred integers for“1-6” can include 1, 2, 3, 4, 5, 6, 1-2, 1-3, 1-4, 1- 5, 2-3, 2-4, 2-5, 2-6, etc.
- alkyl refers to a straight-chain, or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert-butyl, pentyl, isoamyl, neopentyl, l-ethylpropyl, 3-methylpentyl, 2,2- dimethylbutyl, 2,3-dimethylbutyl, hexyl, etc.
- alkyl moiety of alkyl-containing groups such as alkoxy, alkoxycarbonyl, and alkylaminocarbonyl groups, has the same meaning as alkyl defined above.
- Lower alkyl groups which are preferred, are alkyl groups as defined above which contain 1 to 4 carbons.
- a designation such as“Ci-C 4 alkyl” refers to an alkyl radical containing from 1 to 4 carbon atoms.
- cycloalkyl refers to a saturated or partially saturated mono- or bicyclic alkyl ring system containing 3 to 10 carbon atoms.
- a designation such as“C 4 -C 7 cycloalkyl” refers to a cycloalkyl radical containing from 4 to 7 ring carbon atoms.
- Preferred cycloalkyl groups include those containing 5 or 6 ring carbon atoms. Examples of cycloalkyl groups include such groups as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, pinenyl, and adamantanyl.
- the term“patient” or“subject” refers to a warm blooded animal such as a mammal, preferably a human, or a human child, which is afflicted with, or has the potential to be afflicted with one or more diseases and conditions described herein.
- bacterial infection is in particular meant a bacterial infectious disease, i.e. a proliferation of a harmful strain of bacteria on or inside the body.
- susceptible strains strains whose growth is inhibited by antibiotic concentrations usually achievable in human serum and at the site of infection when the recommended dosage is used.
- resistant strains strains whose growth is not inhibited by antibiotic concentrations achievable in human serum and at the site of infection when the recommended dosage is used.
- susceptibility and resistance of a bacterial strain to a given antibiotic are defined by determining the MIC of this antibiotic for that strain and analyzing results according to lower and higher breakpoints and interpretative standards of the Clinical and Laboratory Standards Institute in the USA (Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. M100-S19, CLSI, PA, USA) or equivalent standards in other countries.
- An MIC inferior to the lower breakpoint indicates susceptibility.
- An MIC superior to the higher breakpoint indicates resistance.
- An MIC between the two breakpoints indicates intermediate susceptibility.
- multi-drug resistant TB is defined as resistance to at least rifampicin (RMP) and isoniazid (GNH) and extensively drug resistant TB (XDR-TB) is defined as MDR-TB plus resistance to any fluoroquinolone (FQ).
- RMP rifampicin
- GH isoniazid
- XDR-TB extensively drug resistant TB
- FQ fluoroquinolone
- a “therapeutically effective amount” refers to an amount of a compound of the present invention effective to prevent or treat the symptoms of particular disorder.
- the term“pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
- the present invention is directed to pharmaceutically acceptable salts of the compounds described above.
- pharmaceutically acceptable salts includes salts of compounds of the present invention derived from the combination of such compounds with non-toxic acid or base addition salts.
- Acid addition salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric and phosphoric acid, as well as organic acids such as acetic, citric, propionic, tartaric, glutamic, salicylic, oxalic, methanesulfonic, para-toluenesulfonic, succinic, and benzoic acid, and related inorganic and organic acids.
- inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric and phosphoric acid
- organic acids such as acetic, citric, propionic, tartaric, glutamic, salicylic, oxalic, methanesulfonic, para-toluenesulfonic, succinic, and benzoic acid, and related inorganic and organic acids.
- Base addition salts include those derived from inorganic bases such as ammonium and alkali and alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, as well as salts derived from basic organic amines such as aliphatic and aromatic amines, aliphatic diamines, hydroxy alkamines, and the like.
- bases useful in preparing the salts of this invention thus include ammonium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, methylamine, diethylamine, ethylenediamine, cyclohexylamine, ethanolamine and the like.
- salts are included in the invention. They may serve as intermediates in the purification of the compounds, in the preparation of other salts, or in the identification and characterization of the compounds or intermediates.
- the pharmaceutically acceptable salts of compounds of the present invention can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared.
- the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. Such solvates are within the scope of the present invention.
- compounds of the present invention may exist in various stereoisomeric forms.
- the compounds of the present invention include both diastereomers and enantiomers.
- the compounds are normally prepared as racemates and can conveniently be used as such, but individual enantiomers can be isolated or synthesized by conventional techniques if so desired. Such racemates and individual enantiomers and mixtures thereof form part of the present invention.
- Stereoisomers can be prepared by stereospecific synthesis using enantiomerically pure or enantiomerically enriched starting materials.
- the specific stereoisomers of either starting materials or products can be resolved and recovered by techniques known in the art, such as resolution of racemic forms, normal, reverse-phase, and chiral chromatography, recrystallization, enzymatic resolution, or fractional recrystallization of addition salts formed by reagents used for that purpose.
- the compounds of the present invention may be prepared in a number of methods well known to those skilled in the art, including, but not limited to those described below, or through modifications of these methods by applying standard techniques known to those skilled in the art of organic synthesis.
- the appropriate modifications and substitutions will be readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art. In particular, such methods can be found in R.C. Larock, Comprehensive Organic Transformations, Wiley-VCH Publishers, 1999.
- the compounds of the present invention may contain one or more asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms.
- optically active or racemic forms all chiral, diastereomeric, racemic forms, isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
- mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.
- reagents and starting materials are commercially available, or readily synthesized by well-known techniques by one of ordinary skill in the arts.
- Figure 1 concerns the toxicity of compounds 13, 23, 24 and rifampicin against mammalian cells (VERO cells).
- Positive control (C+) cells without compound.
- Negative control (C-) lysed cells after 24 hours incubation.
- Figure 2 is related to the cytotoxicity of compounds 12, 13, 23 and 24 in human macrophages after 6 days of incubation. Results are expressed in Relative luminescence units. Rifampicin and Moxifloxacin (Moxi) are used. Positive control (C+) : cells without compound. Negative control (C-) : lysed cells after 24 hours incubation.
- FIG. 3 E. coli and M. smegmatis display altered morphology after treatment by compound 23 (MR34504).
- the morphology of E. coli (A) and M. smegmatis (B) was visualized by confocal fluorescence microscopy after a combined staining with FM4-64 (membrane in red) and DAPI (DNA in blue).
- Bacterial cells was treated with 2.5 pg/mL of 23 or 2 pg/mL of ofloxacine for 1.5 hours for E. coli and for 6 hours for M. smegmatis. Elongation of bacterial cells in presence of 23 or ofloxacin is observed.
- Procedure A general method for the synthesis of enamines
- Procedure B general method for cyclization of the enamine in a tricycle
- the precipitate formed is filtered and recrystallized in a DMF-ethyl acetate mixture (50/50).
- Procedure F General method for halogenation of 1,10-phenanthrolinone
- Procedure G general hydrolysis method of ester to acid
- Procedure H general method for the synthesis of amides and hydrazides
- Method B To a suspension of the appropriate acid derivative in 50 ml of anhydrous THF are added oxalyl chloride (1,5 Eq) and DMF (2 drops). The reaction medium is stirred at room temperature for 3 hours, then evaporated to dryness to lead to the corresponding acyl chloride. The obtained acyl chloride is solubilized in 25 ml of anhydrous acetonitrile and then added dropwise to a solution of the amine or hydrazine (1,2 Eq) in 25 ml of anhydrous acetonitrile. This mixture is kept under agitation at room temperature for 2 hours. The formed precipitate was purified by petroleum ether washing, recrystallization or column chromatography.
- Benzaldehyde (1,1 Eq) is added to a solution of the appropriate hydrazine (1 Eq) in MeOH.
- the reaction mixture is heated to reflux for 6 hours. After cooling, the obtained product is purified by recrystallization in the appropriate solvent.
- This compound was obtained by following protocol B with 1.8 g of ethyl [(Quinoleine 8- ylamino) methylene] malonate (leq, 6.71 mmol) in 40 ml of diphenyl ether. This compound was obtained as light brown crystals with a 71% yield.
- This compound was obtained by following protocol C from 1 g of derivative 4 (lEq, 3.73 mmol). This compound was obtained as a milky white solid with a cotton-like aspect and in a yield of 60%.
- This derivative was obtained by following protocol E with 1 g of derivative 8 (lEq, 3.05 mmol). This compound was obtained as a yellow powder in a yield of 60%.
- This derivative was obtained by following protocol E with 1 g of derivative 9 (lEq, 2.92 mmol). This compound was obtained as a yellow powder with a yield of 70%.
- This derivative was obtained by following protocol F using 1 g of derivative 5 (lEq, 3.54 mmol). This compound was obtained as a white solid after recrystallization in acetonitrile with a 49% yield.
- This derivative was obtained by following protocol F using 1 g of derivative 6 (lEq, 3.37 mmol). This compound was obtained as a white solid after recrystallization in acetonitrile with a yield of 48%.
- This derivative was obtained by following protocol G using 1 g of derivative 8 (lEq, 3.055 mmol). This compound was obtained as a yellow solid with a yield of 94%.
- This derivative was obtained by following protocol G using 1 g of derivative 9 (lEq, 2.929 mmol). This compound was obtained as a yellow solid in a yield of 91%.
- This derivative was obtained by following protocol G using 1 g of derivative 12 (lEq, 2.665 mmol). This compound was obtained as a white solid with a 95% yield.
- This derivative was obtained by method B of protocol H using 1 g of derivative 19_(lst, 3.341 mmol). This compound was obtained as yellow solid with a 38% yield after washing in petroleum ether.
- This derivative was obtained by method B of protocol H using 1 g of derivative 20 (leq, 3,194 mmol). This compound was obtained as a sufficiently pure yellow solid with a 45% yield after washing in petroleum ether.
- This derivative was obtained by method B of protocol H using 1 g of derivative 19 (lst, 3.341 mmol). This compound was obtained as a yellow solid with a 54% yield after washing in petroleum ether.
- This derivative was obtained by method B of protocol H using 1 g of derivative 20 (leq, 3,194 mmol). This compound was obtained as a yellow solid with a 52 % yield after washing in petroleum ether.
- yV-oxide derivative 27 was synthesized in 67% yield starting from 4 and using meta chloroperbenzoic acid in dichloromethane. Nitration of the latter took place this time selectively in 7 position and 28 was obtained in 34% yield.
- M.smegmatis, M. aurum, M.marinum, and BCG were grown in Middlebrook 7H9 broth (Difco) supplemented with ADC.
- M. tuberculosis strains H37Rv were grown in Middlebrook 7H9 broth (Difco) supplemented with ADC and 0,05% Tween 80, except when referred, or in 7H11 Middlebrook broth (Difco) supplemented with OADC and 0,5% glycerol for CFU analysis.
- Staphylococcus aureus was grown in Mueller Hinton Broth. These isolates consisted of clinical isolates from the Bichat-Claude Bernard Hospital in Paris.
- the assays were carried out as described by Banfi et al. (J. of Antimicrobial Chemotherapy. 2003. 52, 796-800) with some modifications.
- the microdilution tests were performed in 96- well plates. Two-fold dilutions of each drug were prepared in the test wells in complete 7H9 and Sauton broth, the final drug concentrations were as follows: Rifampicin, Ofloxacin and Amikacin were prepared in a stock solution with a final concentration ranging from 10 to 0.0048 pg/ml). Five microliters of each bacterial suspension was added to 100 pL of drug- containing culture medium. Control wells were prepared with culture medium only and bacterial suspension only.
- the plates were sealed and incubated for 7 days at 37°C. After the incubation time, 30 pL of 0,01 % resazurin solution were added per well, coloring them blue. Plates were incubated at 37°C for additional 24, h. After incubation, plates were red for color change from blue to pink in live bacteria-containing wells. MIC was defined as the lowest drug concentration that prevented resazurin color change from blue to pink. Each MIC was determined three times in duplicate experiments. Viable bacteria from control wells and from tests wells were plated onto 7H11 agar medium in order to determine Minimal bactericidal concentration (MBC).
- MBC Minimal bactericidal concentration
- Vero monkey cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mm glutamine and Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture. When the cell monolayer became confluent, Vero cells were removed from the flask and seeded at 25000 cells per well in a tissue-culture treated 96- well plate. The mixture was incubated for 24 hours after the compound was added while keeping the final DMSO concentration at 1%. The plates were incubated 24 and 48 hours before ATP content determination. The CellTiter-Glo kit from Promega was used to measure ATP content of the cell represented by a luminescent signal as an indicator of viability. IC50 was defined as the concentration of compound that caused a 50% decrease in the ATP signal compared to the DMSO control.
- DMEM Modified Eagle Medium
- FBS fetal bovine serum
- PSN Penicillin-Streptomycin-Neomycin
- monocytes were isolated from healthy donors by CD 14 positive selection using CD 14 microbeads (Miltenyi Biotec).
- cells were differentiated in RPMI-1640 medium containing 20 ng/mL GM-CSF (R&D Systems) at 10% 0 2 , 5% C0 2 . After 1 week, cells were plated on 24 well plates and infected with wild- type M. tuberculosis H37Rv at multiplicity of infection (MOI) of 0.5. After 24 h of infection, test compound was added.
- MOI multiplicity of infection
- Infected human macrophages containing drug or not were then incubated for 6 days. Macrophages were visually inspected for viability and CellTiter-Glo assay was used as control. If drug-treated macrophages looked healthy, they were lysed with 0.1% Triton-X for enumeration of intracellular bacteria by plating on Middlebrook 7H11 agar plates containing OADC supplement.
- Tables 1 and la show activities of a series of molecules of 4-oxo-2, 3-dihydro- 1,10- phenanthroline-3-carboxylic acids carrying or not a nitro group at position 6.
- the inhibitory effects of the molecules on bacterial growth were measured using the dilution procedure based on cell viability using the resazurin microtiter assay (REMA) plate method.
- Resazurin is a blue dye, which reduces to the pink colored and fluorescent resorufin proportionally to aerobic respiration.
- Molecules inhibiting the growth have been identified by a dual wavelength measurement (570 and 604 nm) at a time determined during preliminary experiments with known antibiotics like ofloxacin, rifampicin, and kanamycin.
- Table 1 Antibacterial activities of phenanthrolmone analogues against M. tuberculosis.
- Table la Antibacterial activities of phenanthrolmone analogues against M. tuberculosis. The activity of the molecules against other mycobacterial species and bacteria from other genera are indicated in Table 2. Compounds showed some activity against S. aureus. No activity was observed with Pseudomona aeruginosa, Enterobacter cloacae, Klebsiella or Acinetobacter Baumanii.
- the toxicity of the molecules was investigated on Vero cells after 48 hours incubation, using Rifampicin as a control. All the compounds are non-toxics in Vero cells showing an IC50 > 100 pg/ml (approximately 64 times MIC) ( Figure 1). Toxicity was also assayed on macrophages after 6 days incubation, the same cells were used for testing the activity of the molecules on M. tuberculosis replicating in infected cells. In this case moxifloxacin and rifampicin were used as control. No major toxicity was observed, compound 12 showing for instance an IC50 > 50 pg/ml ( Figure 2).
- Molecules of the invention were investigated for their inhibitory effect on the growth of M. tuberculosis replicating in in vitro cultured macrophages. Molecules showing in vitro activity against M. tuberculosis are also efficient in in vivo conditions of replication of the bacilli inside macrophages (Tables 3 and 4).
- Table 3 Activity of compounds 23 and 13 inside human macrophages at 0 and 6 days of incubation. Results are expressed in logUFC/ml. Cells infected without treatment were considered as the positive control. The antibacterial activity is of the range as observed for rifampicin.
- Table 4 Activity of compounds 12 and 24 inside human macrophages at 0 and 6 days of incubation. Results are expressed in logCFU/ml. Cells infected without treatment were considered as the positive control.
- Table 4a Activity of compounds 12, 13, 23 and 24 and moxifloxacin inside M. tuberculosis- infected human macrophages at 0 and 6 days of incubation. Results are expressed in logCFU/ml. Infected cells without treatment were considered as the positive control. The antibacterial activity is of the range as observed for moxifloxacin.
- FQ resistant bacteria The activity of the molecules were analyzed with M. tuberculosis (H37Rv) mutants resistant to fluoroquinolones (FQ). These mutants carrying either the mutations at position 88, 90, 91, at position 94 of the gene gyrA . Also position 500 at position 540 of the gene gyrB (table 6).
- Example 3 Phenanthrolinic analogs inhibit multiplication of mycobacteria and E. coli Imaging methodology was chosen for investigating the mode of action of the compounds of the invention.
- confocal fluorescent microscopy and combined staining with DAPI (fluorescent stain that binds DNA) and FM 4-64 (lipophilic dye that stains membrane) have been used.
- Escherichia coli ATCC 25922 were cultivated in LB broth and Mycobacterium smegmatis in 7H9 broth. 2 mL of exponential-phase cell cultures was treated with antibiotic ofloxacine (2 pg/mL final) or compound 23 (2.5 pg/mL final) and grown at 37°C during 1.5 hour in the case of E. coli, 6 hours in the case of M. smegmatis. Then, treated culture were harvested, pelleted by centrifugation, washed with PBS, and fixed with 4% PFA in PBS during 10 min.
- the pellet was resuspended in PBS containing 2 pg/mL DAPI and 1 mg/mL FM 4-64 (invitrogen) and incubated at room temperature for 20 minutes in order to stain DNA and cell membrane. After a final washing in PBS, 5 m ⁇ stained culture was placed between glass slide and coverslip for microscopy. Fluorescence signals were detected by using Olympus FV1200 confocal microscope equipped with an oil immersion 60x objective. Images were analyzed using Olympus Fluoview software.
- E. coli cells treated by 23 appear elongated and filamentous in comparison with untreated cells and have altered DNA morphology (Figure 3).
- E. coli treated by fluoroquinolone ofloxacin display also elongated morphology than untreated cells but however shorter than the cells in presence of 23. These morphologies seem indicate that bacteria in presence of these compounds failed to divide and are consistent with the fact that 23 target DNA gyrase, thus leading to replication fork stalling.
- M. smegmatis has been used in imaging experiments. M. smegmatis cells treated by 23 or ofloxacin were slightly longer than untreated cells and appear more segmented as visualized by membrane staining with FM 4-64. These observations can also indicate an inhibition of cell division.
- Mutants resistant to the compounds were selected by using different mycobacterial species. No mutant could be isolated either from M. tuberculosis or M. smegmatis. However, several mutants could be isolated from M. marinum. Their genomes were entirely sequenced and compared to the wild type strain used for the selection of the mutants. No mutations were observed in the gyrase genes or any enzymatic component of the replication process. Only mutations likely affecting permeation or efflux were found. This is at difference to the easy selection of mutants resistant to other quinolones and fluoroquinolones. These finding show the usefulness of molecules blocking bacterial multiplication without provoking the selection of resistant mutants that will impair treatment using them during chemotherapy.
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Abstract
La présente invention s'inscrit dans le domaine des médicaments thérapeutiques pour traiter des infections bactériennes. En particulier, l'invention concerne des composés de phénanthrolinone destinés à être utilisés dans le traitement d'infections bactériennes, par exemple de la tuberculose.
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