Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a method for improving the stability of a compound carrying at least one amine function and its ability to cross cell membranes, thus the stabilized compounds thus obtained have a better stability and a better ability to cross the membranes. cell.
• Peptides are amino acid polymers having many biological functions and finding biotechnological applications, particularly in the pharmaceutical and phytosanitary fields. Peptides have several advantages over other conventionally used molecules since they are generally water soluble, have high efficacy and selectivity for their biological targets and are associated with fewer side effects.
• pepta ⁇ bols have shown increasing pharmacological interest, because these peptides of fungal origin have antibacterial activity. This makes these peptides, for example alamethicine, good antibiotic candidates.
• peptides are generally degraded by the very acidic gastric environment and by proteases. Thus the peptides have in the body a half-life time too short that does not allow them to reach their biological target. Their mode of administration requires the use of intravenous injection, which is a constraint for patients and affects their quality of life.
• the current challenge for peptide development for both human health and crop protection is to increase their interaction with cell membranes to facilitate their uptake and to protect against degradation by peptidases.
• Another approach is to simultaneously associate with these peptides a lipophilic compound contributing to better bioavailability and PEG to increase resistance to proteolysis. Injectable forms of insulin have been developed by this approach. This method nevertheless has the disadvantage of adding two new entities on the parent molecule which can reduce its affinity for its target.
• a third approach is to replace at least one amide bond with an isosteric function.
• These isosteres are supposed to have physicochemical properties similar to the amide function. Since the atoms of the amide bond do not necessarily intervene in the recognition phenomenon, the modified peptides retain a good affinity with the biological target while being more resistant to enzymatic degradation and the extended conformation of the peptide is retained.
• 1,2,3-triazole-1,4-disubstituted heterocycle has proved particularly interesting.
• Valverde and Mindt (Chimia (Aarau) 2013: 67 (4): 262-6.) Describe that the 1,2,3-triazole heterocycle has a structure similar to the peptide bond and is stable with respect to enzymatic degradation.
• Valverde et al. (Angew Chem Int., 2013, 52, 8957 - 8960) have described nine BBN peptide analogs (7-14) each having at least one 1,2,3-triazole group in the main chain.
• the first aspect of the invention therefore aims to provide a method for improving the stability of a compound carrying at least one amino function and its ability to cross cell membranes.
• Said method consists in fixing on at least one amino function of said compound, a unit of formula (I):
• R 2 and R 3 are chosen independently of one another from a hydrogen atom and (C 1 -C 4 ) alkyl groups, especially a methyl group.
• the invention is based on the surprising effect produced by the unit of formula (I) which makes it possible to significantly increase the biological activity of a peptide.
• this motif when this motif is attached to alumethine, which is peptaibol, it increases the antimicrobial activity of this peptide by almost 10-fold.
• the unit of formula (I) improves both the resistance of a peptide to enzymatic degradation and its membrane integration.
• These advantages could be related to the fact that the unit of formula (I) does not disturb the peptide conformation, in particular the helical secondary structure of the peptide, which makes it possible to preserve the biological activities thereof.
• (C 1 -C 4) alkyl is meant a linear or branched alkyl radical of 1 to 4 carbon atoms, such as the methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl radicals.
• the substituent Ri of the unit of formula (I) is chosen from the following groups:
• the unit-attached unit has the formula ( ⁇ )
• R 2 and R 3 are chosen independently of each other from a hydrogen atom and (C 1 -C 4) alkyl groups, in particular a methyl group,
• either one is chosen from the fluorine atom and the -CF3 group, the other being a hydrogen atom
• the motif attached to a compound corresponds to formula (Ia)
• R 2 and R 3 are chosen independently of each other from a hydrogen atom and (C 1 -C 4) alkyl groups, especially a methyl group.
• the motif attached to a compound corresponds to the formula
• said compound carrying at least one amino function may be a peptide, a pseudopeptide or a compound with therapeutic activity bearing at least one amine function, said aforementioned unit of formula (I), in particular of formula ( ⁇ ), (la), (Ib), (le) or (Id), being fixed on:
• peptide means a polymer having at most 50 amino acids, advantageously at least 3 amino acids and at most 50 amino acids, more preferably at least 4 amino acids and at most 50 amino acids, linked together by peptide bonds.
• polypeptide is meant a synthetic molecule similar in functionality or overall configuration to a peptide having a biological activity and capable of interacting with the receptor (s) or target (s) of said peptide.
• a therapeutically active compound bearing at least one amine function is preferably a therapeutically active compound whose molecular weight does not exceed 5000.
• peptide bond and "amide bond” are interchangeable within the meaning of the present invention, when it relates to the -CONH- bond in a peptide.
• Said peptide may comprise proteinogenic amino acids and / or nonproteinogenic amino acids and / or non-natural amino acids.
• Amino acids are the amino acids incorporated into proteins when translating messenger RNA through ribosomes. There are a total of 22 protein amino acids, namely Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Pyl, Salt, Ser, Thr.
• Non-proteinogenic amino acids are those amino acids that can not be incorporated into proteins when translating messenger RNA through ribosomes. These amino acids can be acids natural amines.
• non-proteinogenic amino acids there may be mentioned ⁇ -aminoisobutyric acid (Aib), isovaline (Iva), homoserine (Hse), dihydro-2-aminobutyric acid (Dhb) , 2,4-diaminobutyric acid (Dab) or sarcosine (Sar).
• Non-natural amino acids are amino acids that can not be naturally produced by a living organism.
• the peptide may be a peptide extracted from a biological organism or an artificially synthesized peptide.
• the invention relates to a method for improving the stability of a peptide and its capacity to cross the cell membrane, in which the unit of formula I as described above, in particular of formula ( ⁇ ) , (Ia), (Ib), (Ie) or (Id), is attached to the amino function of the amino acid placed at the N-terminus or, when said peptide comprises at least one lysine, to the function amine of the side chain of said at least one lysine.
• said unit replaces the amino acid in the N-terminal position of said peptide.
• Another aspect of the invention relates to compounds that can be obtained by the method of the invention.
• These compounds are analogs of compounds carrying at least one amino function, especially analogs of peptides, pseudopeptides or compounds with therapeutic activity. These analogs have better stability and ability to cross the cell membrane compared to the molecules from which they were prepared.
• such an analogue comprises a unit of formula I:
• - Ri is chosen from a phenyl group substituted with one to three identical or different substituents chosen independently of one another from the fluorine atom and the -CF3 or -OCF3 groups and, the biphenyl, pyrenyl, naphthyl and phenanthrenyl groups, optionally substituted with a substituent selected from -
• R2 and R3 are chosen independently of each other from a hydrogen atom and (C1-C4) alkyl groups, in particular a methyl group,
• said unit being attached to at least one amino function of said compound, in particular, said pattern being fixed on:
• the invention relates to the analogs of a peptide represented by formula II
• Said peptide contained in said analog of formula II is linked to the -CO group of the unit of formula I by the amino function of its amino acid in the N-terminal position or, when it comprises at least one lysine, by an amino function of the side chain of said at least one lysine
• said peptide is peptaibol.
• peptaibol refers to a class of linear peptides acetylated at the N-terminus and having antibacterial and antifungal activity, the peptaibols being characterized, on the one hand, by a high number of non-proteionogenic amino acids essentially the acid a- aminoisobutyric acid (Aib) or isovaline (Iva) and, on the other hand, the presence of the C-terminal side of an aminoalcohol, phenylalaninol and leucinol being the most common.
• Aub acid a- aminoisobutyric acid
• Iva isovaline
• Peptibols consist essentially of 7 to 21 encoded and uncoded amino acids. With the exception of the Aib and Gly residues which do not have asymmetric carbon, these amino acids are of (L) configuration except isovaline which can be found in the form (D).
• pepta ⁇ bols mention may be made, for example, of alamethicine or bergofungine D.
• the invention relates to analogs of an alamethicin.
• Alamethicine is a family of several tens of 20 amino acid peptides classified into two main groups: alamethicin F30 and alamethicin F50, the two groups being distinguished by the amino acid in position 18, a glutamic acid (Glu) for F30 alamethicins and glutamine (Gin) for F50 alamethicins.
• Glu glutamic acid
• Gin glutamine
• Alamethicin F30 / 5 is represented by the amino acid sequence hereinafter: ## STR1 ##
• Alamethicin F50 / 5 is represented by the amino acid sequence: AcUPUAUAQUVUGLUPVUUQQFol (SEQ ID NO: 2).
• the invention relates to the analogs of alamethicine, corresponding to the formula (IIa)
• R 2 and R 3 are independently selected from hydrogen and methyl
• Pepl is the peptide of sequence SEQ ID NO: 3 (PUAUAQUVUGLUPVUUEQFol) or the peptide of sequence SEQ ID NO: 4 (PUAUAQUVUGLUPVUUQQFol).
• the Pepl peptide is linked to the -CO group of the unit of formula (Ia) or (Ib) by the amino function of the amino acid in the N-terminal position.
• the invention relates to an alamethicin analog F50 / 5 of formula (Ile)
• Another object of the invention is to provide a method for the preparation of the above analogs, in particular analogs of a compound bearing at least one amino function, in particular analogs of a peptide, a pseudopeptide or a compound with therapeutic activity carrying at least one amino function.
• Said method comprises the following steps:
• R2 and R3 are chosen independently of each other from a hydrogen atom and (C1-C4) alkyl groups, especially a methyl group,
• Ri is chosen from:
• a phenyl group substituted with one to three identical or different substituents chosen independently of one another from the fluorine atom and the -CF3 or -OCF3 groups and, the biphenyl, pyrenyl, naphthyl and phenanthrenyl groups, optionally substituted with a substituent selected from - OCHs, fluorine atom, -CF 3 or -OCFs
• Ri is chosen from:
• a phenyl group substituted with one to three identical or different substituents chosen independently of one another from the fluorine atom and the groups -CF3 or -OCF3 and, biphenyl, pyrenyl, naphthyl and phenanthrenyl groups,
• R2 and R3 are chosen independently of each other from a hydrogen atom and (C1-C4) alkyl groups, in particular a methyl group, - Re is the hydroxyl group or a chlorine,
• step (ii) reacting, optionally in the presence of at least one coupling agent, between a compound of formula III obtained in step (i) and at least one amino group of a compound to be stabilized to obtain an analogue of said compound .
• This reaction is carried out between a precursor carrying the alkyne function and a precursor bearing the azide function in the presence of a catalyst. It makes it possible to produce 1,2,3-triazoles-1-4-disubstituted at room temperature while increasing the kinetics of reaction.
• the hydroxyl group of a compound of formula (III) can form an activated ester by reactions with different coupling agents known in the art or be converted into acyl chloride. All these techniques are known to those skilled in the art.
• FIGS. 1A, 1B, 1C and 1D respectively illustrate the analytical HPLC results for alumethicin F50 / 5 (FIG. 1A) and the alamethicin F50 / 5 analogs, namely compound 5 (FIG. 1B), the compound 6 (figure
• FIGS. 2A, 2B and 2C respectively illustrate the spectra of
• Figure 3 shows the chemical shifts of proton amide resonances for each amino acid between compound 6 or 7 and alamethicin.
• Figure 4 shows the circular dichroism spectra of alamethicin and compounds 6 and 7.
• HPLC High-performance liquid chromatography (high performance liquid chromatography)
• the analysis is carried out on a PhenomenexKinetex C-18 column (100 ⁇ 300 mm) using a gradient consisting of buffer A (aqueous solution A comprising 0.1% AF) and buffer B (ACN comprising 0 , 1% AF).
• the elution is carried out at a flow rate of 0.5 ml / min with 10% of buffer B up to 100% of the buffer B in 30 min followed by a return to 10% of the buffer B in 7 min.
• Analytical HPLC is performed by the Waters® 2695 HPLC system with a Vydac® 218MS 5 ⁇ M C-18 column (250 x 4.6 mm) from Grace and a light scattering detector.
• the eluents used are buffer A (0.1% FA in water) and buffer B (0.1% FA in MeOH).
• Imidazole-1-sulfonyl azide hydrochloride (36mmol) is added to ⁇ -aminoisobutyric acid (Aib) (30mmol), K2CO3 (90mmol) and CUSO4.5H2O (0.3mmol) in MeOH (300ml) .
• the mixture is stirred at ambient temperature for 12 hours.
• the mixture is concentrated and then diluted with diethyl ether (100 mL) and extracted with saturated NaHCO3 (3x50 mL).
• the remaining aqueous phase is acidified to pH 1 with 1M HCl solution and extracted three times with ethyl acetate (3 x 50 mL).
• the organic extract is washed with saturated NaCl solution (2 x 30 mL) and dried with MgSO 4 , filtered and concentrated under reduced pressure to obtain an oily yellowish product. This product is used in the next step without further purification.
• R 4 and R 5 are both hydrogen (compound 2); either R 4 and R 5 are both fluorine (compound 3); either R 4 is the group -CF 3 and R 5 is the hydrogen atom.
• step 1.4 The compound 1 obtained in step 1.4. (1 mmol) and the respective alkynes (1 mmol) are dissolved in degassed acetonitrile (10 mL). 2,6-lutidine (2 mmol) and diisopropylethylamine (2 mmol) are added to this solution under argon. The copper iodide (0.1 mmol) is then added to this solution. The reaction is carried out under stirring under argon for 6 h. The mixture is diluted with EtOAc (150 mL). The solution is washed with a mixture of saturated NH 4 CI and NH 4 OH with a proportion of 9: 1.
• 2,4-difluorophenyl Aib-OH (compound 3): A white solid of 2.5 g (80%) is obtained by reacting 1.5 g (11.6 mmol) ) of compound 1 and 1.6 g (11.6 mmol) of 1-ethynyl-2,4-difluorobenzene. Rf 0.5 (cHex / EtOAc / AcOH 4.9: 4.9: 0.2 (v / v)).
• Solid phase peptide synthesis is performed by an automated peptide synthesizer (CEM liberty one, Orsay, France) using Fmoc / O-tbutyl chemistry. All the amino acids protected by Fmoc, DIC, Oxyma, the 2-chlorotrityl chloride resin preloaded with phenylalaninol (loaded 0.67 mmol / g or 0.36 mmol / g) come from the company Iris Biotech (Germany). DCM, DMF, CHex, DIEA, AC2O, TFA, TIS, FA, CAN and piperidine for peptide synthesis are obtained from Aldrich (USA). The synthesis of peptides in the solid phase is carried out according to the known methods (Ben Haj Salah et al., Org Lett 2014, 16, 1783-1785).
• Alamethicin F50 / 5 and its compound analogs 5, 6 and 7 are synthesized by this method.
• the crude almethicin F50 / 5 is purified by semi-preparative HPLC using a linear gradient of 50% to 100% of buffer B in buffer A for 35 min at a flow rate of 3 mL / min to obtain alamethicin F50 / 5.
• purified 89 mg, 45% yield,> 98% purity).
• Compound 5 is an analogue of alamethicin F 50/5 having the following sequence: ⁇ ⁇ [ ⁇ ⁇ ] ⁇ ⁇ ⁇ ⁇ ⁇ 3 ⁇ ⁇ 36 ⁇ ⁇ ⁇ ⁇ ⁇ 3 ⁇ ⁇ £ ⁇ ⁇ 3 ⁇ ⁇ ⁇ ⁇ ⁇ 6 ⁇ ⁇ ⁇ .
• the crude compound 6 is purified by semi-preparative HPLC using a linear gradient of 20% to 70% of buffer B in buffer A for 40 min at a flow rate of 3 mL / min to obtain compound 6 (85 mg, 42%). yield,> 98% purity).
• Compound 7 corresponds to formula (IId) and is represented by the following sequence:
• Alamethicin and its analogs 5, 6 and 7 are all analyzed respectively by analytical HPLC according to the protocol described in section 1.2. using a gradient of 10% to 100% of buffer B with a flow rate of 0.9 mL / min for 50 min.
• Circular dichroism spectra are measured according to the protocol described in Biopolymers. 2015 Mar 18. doi: 10.1002 / beep.22641. [Epub ahead of print]).
• KB cells (ATCC CCL 17 human oral epidermoid carcinoma,
• the trypsinized cells were suspended at 2 ⁇ 10 5 cells / ml suspended in supplemented BME and then 50 ⁇ l of this suspension was deposited in each well of a 96-well microplate, (Nunclon delta). ground, Thermo Scientific Nunc). After a 48 h incubation, 50 ⁇ L of each sample (alamethicin F50 / 5, compounds 5, 6 and 7) was added to the 5 ⁇ of initial cell suspension. These samples were tested at a final 5% (v / v) methanol content in supplemented BME at concentrations ranging from 3 to 400 ⁇ g / mL.
• a methanoic solution at 5% (v / v) in the BME was used as a control.
• the cell viability was evaluated by the colorimetric MTT colorimetric (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium, Sigma-Aldrich) test. All technical and biological in vitro assays were performed three times.
• a sequential dilution culture assay was performed to compare the activity of the alamethicin analogs, compounds 5, 6 and 7 to alumethine F50 / 5 using concentrations of: 100, 50, 25, 12.5, 6.25, 3.1, 1.5 ⁇ g / mL.
• the bacterial strains of Bacillus subtilis (CIP 103406) and Staphylococcus aureus were cultured and maintained in LB culture medium and tryptic soy agar at 28 ° C. Growth phase exponential culture in LB medium (after incubation for 6 h in fresh medium) was diluted to 1x10 4 CFU / ml in LB medium.
• Alamethicin F50 / 5 and analogs 5, 6 and 7 are respectively analyzed by analytical HPLC according to the protocol described in section 1.2. above. The results show that these synthesized compounds are purified to a satisfactory level by semi-preparative HPLC.
• Tables 2, 3, 4, 5, 6 and 7 below show respectively the chemical shifts of * H, 13 C for alamethicin F50 / 5, compound 6 and compound 7.
• Table 8 shows the coupling constant of 3 _7 (HN, Ha) of alamethicin, compound 6 and compound 7 in Hz.
• Table 1 Results of the tests of compounds 5, 6 and 7 on the cell growth of KB cells and two gram-positive bacteria.
• the compounds of the invention listed in Table 9 below are synthesized according to the method described in the application and analyzed by LC-MS according to the protocol described in Part 1.1. above. The results of LC-MS confirm that these analogs are pure and present the mass expected molecular.
• the compounds listed in Table 9 below are tested on Bacillus subtilis. The respective MIC (minimum inhibitory concentration) value of these compounds with respect to this bacterium is also found in Table 9.

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• 2015
  • 2015-09-28 FR FR1559130A patent/FR3041638A1/fr not_active Withdrawn
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