FR2925502A1 - New pseudopeptide compounds, useful e.g. for the therapeutic treatment or diagnosis of human or animal body, to prepare drug for pharmaceutical composition for treating microbial infections and as an antimicrobial agent - Google Patents

Abstract

Pseudopeptide compounds (I) and their derivatives having antimicrobial activity, are new. Pseudopeptide compounds of formula (I) and their derivatives having antimicrobial activity, are new. R1 : (CH 3-CH 2-CH 2-NH 2) or (CH 3-CH 2-NH-C(=NH)-NH 2); and R2, R3 : aromatic radical (optionally substituted), preferably phenyl, naphthalenyl, diphenyl, biphenyl or anthracenyl. [Image] ACTIVITY : Antimicrobial. MECHANISM OF ACTION : None given.

Description

ANTIMICROBIAL PSEUDOPEPTIDES, MEDICAMENT AND PHARMACEUTICAL COMPOSITION CONTAINING SAME

The present invention relates to the field of antimicrobials. More particularly, the present invention relates to the field of pseudopeptides having antimicrobial activity. The treatment of microbial infections, especially bacterial infections, is still a major issue today. Indeed, despite the constant advance of research and the placing on the market of new molecules, new resistance, particularly to antibiotics, appear and it becomes more and more difficult to treat certain infections, especially in hospitals with the risks. nosocomial infections. The existence and constant appearance of bacteria increasingly resistant to current antibiotics induces an important demand from the pharmaceutical industry and makes absolutely necessary the discovery of new families of antibiotics. In recent years, research has turned to a new family of antibiotic molecules still little studied, namely peptides. These molecules, usually derived from invertebrates, are molecules whose target is the bacterial membrane. Therefore, to gain resistance to this type of molecule, microorganisms should change the composition and organization of their membrane lipids. This costly solution from an evolutionary point of view explains why the resistance of microorganisms towards this type of antibiotics is only rarely referenced.

The Applicant is particularly interested in peptides from the common cuttlefish, Sepia officinalis, cephalopod mollusk belonging to Coléoïdés. Among the regulatory peptides identified in advance by the inventors, a neuropeptide having the following amino acid sequence: ALSGDAFLRFamide revealed an antibacterial activity. This neuropeptide has been described by Henry J .; Zatylny C .; Boucaud-Camou E. in Peptides, Volume 20, Number 9, September 1999, pp. 1061-1070 (10). However, since this peptide has a threshold of activity that is too high to be directly usable as a drug, the inventors have begun to synthesize a series of analogs, in order to reduce the threshold of activity of the peptide, that is to say to say to strengthen its antibacterial activity. Indeed, in recent years, many research teams have focused on synthesizing analogs of peptides or proteins that mimic the biological activities of peptides or natural proteins. By way of example, mention may be made of peptide analogues obtained by replacing one or more amino acids of the L-series with one or more corresponding amino acids of the D-series, the peptides having a modification in at least one of the peptide bonds, such as retro, inverso, retro-inverso, carba and aza bonds. The carba linkage (CH 2 -CH 2) has been described as a potential mimic of the peptide bond (Ment C. et al., European J. Pharmacol., 186, p213-222, 1990, Attwood et al., Bioorg Med. Chem Lett, 7, p429-432, 1997). Moreover, the replacement of carbon with a nitrogen atom on a peptide has made it possible to obtain interesting pseudopeptides called azapeptides and azatides respectively (Gante, J., Synthesis, p405-413, 1989, Han H. and Janda KD, J. Amer, Chem Soc., 118, p2539-2544, 1996). In general, these peptide analogues, called pseudopeptides, have the first advantage of greater metabolic stability than natural peptides or proteins due to the fact that they are not degraded by natural proteases or are slower. Moreover, the conformational changes induced by these chemical modifications can improve the biological properties of these pseudopeptides. These pseudopeptides are therefore interesting and innovative alternatives to products currently available on the market, in particular to fight against drug resistance. Thus, the Applicant, starting from the cuttlefish extract peptide, has synthesized a series of peptide analogs, or pseudopeptides. Among the many analogous peptides tested, the inventors have been able to determine that, surprisingly and unexpectedly, the substitution of aspartate D at position 5 (starting from the C-terminal) by a lysine K or an arginine R made it possible to increase strongly antibacterial activity of the peptide. In further research, the inventors further found that by substituting phenylalanines F at positions 7 and 10 (starting from the C-terminal) with aza-R31-naphthylalanines, the antibacterial activity was further increased.

Without wishing to be bound by any theory, the inventors are of the opinion that the substitution of aspartate D by a positively charged Lysine K or arginine R would promote the interaction between the peptide and the bacterial membrane. Similarly, the aza-R3-amino acids would provide better stability to the molecule and thus a better activity.

Thus, the present invention relates to a pseudopeptide of formula (I) H 2 N (I) wherein: R 1 represents or HN / NH 2 R 3 and R 3 are the same or different and each independently represents a substituted or unsubstituted aromatic radical, the radical aromatic being preferably selected from phenyl, naphthalenyl, diphenyl, biphenyl and anthracenyl. and its derivatives having antimicrobial activity. In the present invention, the term "substituted aromatic radical" means an aromatic radical substituted with at least one group chosen in particular from a linear or branched C2-C20 methyl, ethyl, alkyl or alkenyl, or any other substituent n not altering the antimicrobial activity of the pseudopeptide. By phenyl is meant a radical of the formula By naphthalenyl is meant a radical of the formula By biphenyl is meant a radical of the formula By diphenyl is meant a radical of the formula or by anthracenyl is meant a radical of the formula or of the particular pseudopeptides of the invention comprising radicals of phenyl, naphthalenyl, diphenyl, biphenyl and anthracenyl type are represented in the following formulas (Ia), (Ib), (Ic), (Id), (Ie) and (If). H2N (Ia) (Ib) (Ic) (Id) in which R1 is as previously defined. Moreover, any other combination of aromatic radicals is possible as long as the antimicrobial activity of the pseudopeptide is not impaired. The invention particularly relates to a pseudopeptide as described above, characterized in that it is chosen from the following pseudopeptides of formula (II), (III), (IV) and (V) H2N (Ie) (If) H2N (V) and their derivatives having antimicrobial activity. The subject of the invention is also a pseudopeptide as described above, characterized in that it is chosen from the following pseudopeptides of formula (VI), (VII), (VIII) and (IX) H2N HN (VI) (VII) HN H2N (VIII) H2N H2N H2N (Ix) and their derivatives having antimicrobial activity. "Derivatives" within the meaning of the invention are understood to mean, in particular, pseudopeptides which have undergone at least one addition, and / or at least one substitution, and / or at least one deletion of an amino acid, and which exhibit antimicrobial activity. The derivatives according to the invention may consist of pseudopeptides in which one or more amino acids are substituted by their enantiomers, natural amino acids of D or L conformation, rare amino acids, in particular hydroxyproline, hydroxylysine or allohydrolysine. , 6-N-methylsilyl, N-ethylglycine, N-methylglycine, N-ethylasparagine, alloisoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, amino-butyric acid and synthetic amino acids including ornithine, norleucine, norvaline, cyclohexyl-alanine, and omega-amino acids. The derivatives according to the invention also cover pseudopeptides whose side chain of one or more of the amino acids is substituted by groups which do not deteriorate the antimicrobial activity of the pseudopeptide of the invention. Pseudopeptides which exhibit a posttranslational modification and / or a chemical modification, in particular glycosylation, amidation, acylation, acetylation, methylation as well as the pseudopeptides which carry a protecting group, may also be mentioned as derivatives. Protective group according to the present invention is understood to mean any group which makes it possible to avoid the degradation of the peptide of the invention, such as in particular tert-butyloxycarbonyl and fluorenemethoxycarbonyl. The pseudopeptides of the invention and their derivatives can be synthesized chemically according to techniques known to those skilled in the art. These techniques are in particular those of resin-type polymeric support synthesis, by BOC (t-butoxycarbonyl) strategy and / or FMOC (9-fluorenylmethyloxycarbonyl) strategy. The invention also aims to apply the antimicrobial properties of the pseudopeptide of the invention or of one of its derivatives for preventing and / or treating microbial infections in both humans and animals. In fact, the antimicrobial activity, in particular the antibacterial activity, of the pseudopeptide of the invention was demonstrated by means of the in vitro tests described hereinafter in the examples. The tests carried out revealed a broad-spectrum antibacterial activity, both on Gram + bacteria and on Gram - bacteria.

In the context of the present invention, antimicrobial properties are understood to mean antibacterial properties as well as antiviral, antiparasitic and antifungal properties.

The subject of the invention is thus a pseudopeptide as described above or one of its derivatives for the implementation of a method of therapeutic treatment or diagnosis of the human or animal body. The invention also relates to a medicinal product characterized in that it comprises as active agent a pseudopeptide as described above or one of its derivatives. Conventionally, the term "medicament" is intended to mean any substance or composition presented as possessing curative or preventive properties with regard to human or animal diseases, as well as any product that can be administered to humans or animals in order to establish a medical diagnosis or to restore, correct or modify their organic functions.

The invention also relates to a pharmaceutical composition characterized in that it comprises as active agent a pseudopeptide as described above or one of its derivatives and a pharmaceutically acceptable vehicle. The pharmaceutical composition of the invention may further comprise another active ingredient such as another antimicrobial agent, especially an antibiotic. The pharmaceutical composition according to the invention may also comprise several pseudopeptides and derivatives as described above.

By "pharmaceutically acceptable carrier" is meant according to the present invention, any substance which is added to the pseudopeptide or to one of its derivatives to promote their transport, and / or to avoid their substantial degradation in said composition and / or to preserve their properties. antimicrobial. The vehicle is chosen according to the type of application of the composition. In particular, when the composition is applied to a pharmaceutical use in human and animal health, a person skilled in the art will choose the pharmaceutically acceptable vehicle adapted to the route of administration of the pharmaceutical composition of the invention. Thus, the pharmaceutical compositions according to the invention consist of at least one pharmaceutically acceptable vehicle and the pseudopeptide or a derivative thereof in free form or in the form of an addition salt with a pharmaceutically acceptable acid, pure state or associated with any other pharmaceutically compatible product. The pharmaceutical compositions according to the invention can be used orally, parenterally, rectally or topically. As solid compositions for oral administration, mention may be made of tablets, hard capsules, powders, etc. In these compositions, the pseudopeptide or one of its derivatives are mixed with one or more inert diluents conventionally used, and optionally to other substances, such as, for example, a lubricant, a colorant, a coating, etc.

As liquid compositions for oral or ocular administration, mention may be made of suspensions, solutions, emulsions, pharmaceutically acceptable syrups containing inert diluents conventionally used, and possibly other substances such as wetting agents, sweeteners, thickeners, etc. Sterile compositions for parenteral administration may be aqueous or non-aqueous solutions, suspensions or emulsions. As the solvent or vehicle, water, propylene glycol, vegetable oils or other suitable organic solvents may be employed. These compositions may also contain adjuvants, such as wetting agents, isotonic agents, emulsifiers, etc.

The compositions for topical administration may be, for example, creams, lotions, mouthwashes, nasal or eye drops or aerosols. In the pharmaceutical compositions object of the present invention, the amount of pseudopeptide or one of its derivatives object of the invention advantageously used is between 0.01 and 90% by weight. However, it is obvious that one skilled in the art will be able to adapt this quantity according to the type of pharmaceutical compositions and according to the mode of administration of said compositions. Another subject of the invention is the use of a pseudopeptide as described above or of one of its derivatives for the manufacture of a medicament or a pharmaceutical composition intended for the treatment of microbial infections. Finally, the subject of the invention is a pseudopeptide as described above or one of its derivatives for use as an antimicrobial. For the purposes of the invention, the term antimicrobial antimicrobial as well as anti-viral, antiparasitic and antifungal. The present invention also relates to a method for preventing and / or treating microbial infection. The present method comprises administering to a subject an effective amount of a pseudopeptide or a derivative thereof according to the present invention. By subject is meant in the present invention any animal or any human for which a microbial infection and, more particularly, a bacterial infection, has been diagnosed, but also any animal or any human likely to suffer from this infection. An effective amount within the meaning of the invention means an amount of active agent sufficient to prevent, combat or cure a microbial infection. The invention provides in particular the daily administration to a subject of an amount of between 50 mg and 2000 mg of the pseudopeptide of the invention or of one of its derivatives, for a period of between 6 and 10 days.

Preferably, the invention provides for the administration three times a day for eight days of approximately 500 mg of the peptide, or one of one of its derivatives or fragments, or the polypeptide of the invention.

The following examples illustrate the present invention and can not be interpreted as limiting its scope.

EXAMPLE 1 Synthesis of the Pseudopeptides of the Invention The synthesis of the pseudopeptides of the invention was carried out using a continuous flow Pionner automatic synthesizer (Applied Biosystems), according to the protocol of use supplied with the apparatus. A Fmoc synthesis strategy was used: the N-terminal end of each amino acid is protected with 9-fluorenylmethyloxycarbonyl (Fmoc). The amine group of the Fmoc-lysine side chain was further protected by a t-butoxycarbonyl (Boc) group. The Fmoc-amino acid deprotection was performed using a mixture of DMF (dimethylformamide, stock solution containing no trace of amines) and 20% piperidine (99% pure stock solution).

In addition, the mixture N-Hydroxybenzotriazole / O-benzotriazole-N, N, N ', N'-tetramethyl-uronium-hexafluorophosphate / N-Ethyldiisopropyl amine (HOBt / TBTU / DIEA) was used in proportions (1/1 / 2) dissolved in DMF (stock solution containing no traces of amines) as activator and coupling agent. The resin (Rink amide resin, 200 mg) preloaded (at -0.6 mmol / g) was placed in the synthesizer reactor. The couplings were carried out with eight times the stoichiometry as amino acid or aza-β-amino acids and a coupling time of 30 minutes for the amino acids and 60 minutes for the α-α-amino acids. coupling and at the end of synthesis, the N-terminal end of the last residue grafted automatically with a 20% solution of piperidine in DMF was deprotected Resin cleavage and deprotection of the functional groups of the side chains were carried out at the same time by the action of the mixture (95% TFA (trifluoroacetic acid), 2.5% water, 2.5% TIS or TrilsopropylSilane) The resin was then extracted from the reactor and rinsed with dichloromethane and then dried in a desiccator. was then placed in a flask and then the freshly prepared cleavage cocktail was added and the reaction medium was allowed to stir for 3 h at room temperature and the solution containing the peptide was then recovered. After filtration of the resin on sintering After evaporation of the solvent under reduced pressure to a volume of about 2 mL, the crude peptide was isolated by precipitation in ice-cold ether and sinter filtration. It was then purified by HPLC on a XTerra C18 reverse phase column (300 × 19 mm) according to an elution gradient (solvent A: water + 0.1% TFA and solvent B acetonitrile + TFA 0.08%) 0% B to 70% % B in 20min then 70% B at 0% B in 5 min, with a flow rate of 8 mL / min. A UV detection was then carried out at 210 nm, and then the purity of the hybrid peptides synthesized by HPLC was checked on a XTerra C18 reverse phase column (250 × 4.6 mm) and mass spectrometry by the ESI technique in an acetonitrile / acetonitrile mixture. water (50/50). The synthesis protocol used is summarized in the following reaction scheme: activation, HOBt, TBTU aa O orplageJ Fmoc, NH H2N deprotectioi FmocHN 20% piperidine DMF 20% piperidine DMF H2N Fmoc, NHR 'O 20% piperidine HN DMF NHO NH2 Resin Aa = Amino acid Example 2: Antibacterial activity of a pseudopeptide of the invention The minimal bacterial growth inhibitory concentration (MIC) of the following pseudopeptide of formula (II) was measured for various bacterial strains: NH, N H2N The Minimal Inhibitory Concentration of Bacterial Growth (MIC) was measured as follows: The bacteria tested (Table 1) were suspended in a suitable Poor Broth nutrient medium, corresponding to a Bactotryptone solution. 10 g / l added with NaCl 5 g / l prepared in ultrapure water. The antimicrobial activities were then detected by a growth inhibition test in a liquid medium in microtiter plates (Hétru, C., & Bulet, P. (1997), Strategies for the isolation and characterization of antimicrobial peptides of invertebrates , Methods in Molecular Biology, Vol 78. 35-49). 10 μl of each concentration of pseudopeptide (II) of the invention were plated in microtiter plates in the presence of 100 μl of culture medium containing the bacteria (at a final concentration equivalent to 1 milliDO at 595 nm).

The incubation was then carried out at 30 ° C. for 18 h while stirring, and the growth of the microorganisms was measured by following the absorbance at 595 nm using a microtiter plate reader spectrophotometer.

By Minimal Inhibitory Concentration of Bacterial Growth (MIC) is meant the lowest concentration of peptide that inhibits any visible culture of a bacterial strain after 18 hours of culture at 30 ° C. Table 1 gives the spectrum of activity of the pseudopeptide of formula (II). This table shows that the pseudopeptide has a broad-spectrum antibacterial activity, on both Gram-positive and Gram-negative bacteria, and has extremely low activity thresholds, sometimes even lower than ampicillin (Pseudomonas aeruginosa).

Table 1 Spectrum of activity of pseudopeptide (II) Bacteria MIC (pg / mL) Pseudopeptide Ampicillin Gram-positive bacteria Bacillus megaterium 10-20 10-20 Listeria monocytogenesis 40-80 1-2,5 Staphylococcus aureus 10-20 1- 2.5 Gram-negative bacteria Escherichia coli 20-40 1-2.5 Salmonella typhimurium 40-80 2.5-5 Pseudomonas aeruginosa 80-160 160-320 Klebsiella pneumoniae NA 1-5 Vibrio harveyi 320-640> 90 Vibrio Example 3: Cytotoxic activity of the pseudopeptide of formula (II) Cell types: Chinese hamster ovary cells (CHO-K1) and murine mesothelioma cells (302-sp). Both cell types were placed in the presence of the pseudopeptide at different concentrations for 24 hours. At the end of this incubation, a cell survival test was carried out using the CELL GROWTH DETERMINATION kit KIT, MTT BASED (SIGMA-Aldrich). The principle of this kit is based on an absorbance measurement corresponding to the mitochondrial activity of living cells.

Table 2 gives the cytotoxic activity of the pseudopeptide of formula (II) of the invention expressed as a percentage of cell viability. The pseudopeptide of the invention exhibits a cytotoxicity only for the dose of 640 μg / ml, ie a dose 4 to 64 times higher than the active concentrations on the bacteria (Table 1).

TABLE 2 Cytotoxic activity of the pseudopeptide of formula (II) expressed as a percentage of cell viability Doses pg / mL CHO cells 302-sp cells 100% 116.10% 94.40% 114.15% 160 53.77% 94, 63% 640 3.16% 6.34% Example 4: Hemolytic activity of the pseudopeptide of formula (II)

Haemolytic tests were performed on erythrocytes obtained from rabbit blood. The erythrocytes were rinsed three times in phosphate buffered saline (137mM NaCl, 2. 7mM KCl, 10mM Na2HPO4, 2mM KH2PO4, pH 7.4) and then taken up in the same buffer to obtain a 1-erythrocyte solution. %. The erythrocytes thus prepared were placed in the presence of different concentrations of the pseudopeptide of formula (II) for 1 hour at 37 ° C. The mixture was then centrifuged and then the absorbance at 415 nm of the supernatant was measured for percent hemolysis by reference to the 100% hemolysis obtained with 1% triton.

The tests carried out revealed no hemolytic effect of the pseudopeptide for the doses tested.

Table 3 Hemolytic activity of the pseudopeptide of formula (II) on rabbit erythrocytes expressed as a percentage of haemolysis Doses pg / mL Hemolysis% 0.00% 40 0.79% 160 0.31%

Claims (9)

1. Pseudopeptide of formula (I) HZN (I) wherein: R 1 represents NH 2 or HN NH 2 R 2 and R 3 are identical or different and each independently represents a substituted or unsubstituted aromatic radical, the aromatic radical preferably being chosen from phenyl, naphthalenyl, diphenyl, biphenyl and anthracenyl and its derivatives having antimicrobial activity. 2. Pseudopeptide according to claim 1, characterized in that it is chosen from the following molecules of formula (II), (III), (IV) and (V) H2N H2N HZN O (IV) H2N (V) and their derivatives having antimicrobial activity. 3. Pseudopeptide according to claim 1, characterized in that it is chosen from the following molecules of formula (VI), (VII), (VIII) and (IX) HZN (VI) (VII) H2N (VIII) H2N (IX ) and their derivatives having antimicrobial activity. HN 4. Pseudopeptide according to any one of Claims 1 to 3, characterized in that the said derivatives are chosen from: the pseudopeptides which have undergone at least one addition, and / or at least one substitution, and / or at least one deletion of a amino acid, and having antimicrobial activity, - pseudopeptides in which one or more amino acids are substituted by their enantiomers, natural amino acids of D or L conformation, rare amino acids including hydroxyproline, hydroxylysine, the allohydrolysine, 6-N-methylsilyl, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, amino-butyric acid and synthetic amino acids, in particular ornithine, norleucine, norvaline, cyclohexyl-alanine, and omega-amino acids, and having antimicrobial activity, pseudopeptides whose side chain of one or more Amino acids are substituted by groups which do not deteriorate the antimicrobial activity of the pseudopeptide of the invention, pseudopeptides which exhibit a post-translational modification and / or a chemical modification, in particular glycosylation, amidation, acylation. , an acetylation, a methylation as well as the pseudopeptides which carry a protecting group chosen in particular from tert-butyloxycarbonyl and fluorenemethoxycarbonyl, and having an antimicrobial activity. 5. Pseudopeptide or a derivative thereof according to any one of claims 1 to 4 for the implementation of a method of therapeutic treatment or diagnosis of the human or animal body. 6. Medicinal product characterized in that it comprises as active agent a pseudopeptide or a derivative thereof according to any one of claims 1 to 4. 7. Pharmaceutical composition characterized in that it comprises as active agent a pseudopeptide or a derivative thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable vehicle. 25 30 8. Use of a pseudopeptide or a derivative thereof according to any one of claims 1 to 4 for the manufacture of a medicament or a pharmaceutical composition for (e) treatment of microbial infections. The pseudopeptide or a derivative thereof according to any one of claims 1 to 4 for use as an antimicrobial.