Patent Application
20210230111
The invention relates to clinical chemistry, in particular, to new biologically active compounds—amide derivatives of prostaglandin F2α.
Prostaglandins are the family of multifunctional biologically active substances synthesized in organism from arachidonic acid by means of cyclooxygenase (COX). These oxidized fatty acids are autocrine and paracrine lipid mediators, and they are characterized by broad-spectrum of physiological activity, for example, combination of dilatator (bronchi smooth muscles), constrictor (gastrointestinal tract smooth muscles), antiaggregatory (platelets), hypo- or hypertensive activities [Nicolau A. In: Bioactive Lipids (Eds. Nicolau A., Kokotos J.) II Bridgewater: Oily Press, 2004. —197 p]. Prostaglandin F2α is formed in organism as a result of arachidonic acid oxidative metabolism caused by cyclooxygenase at the first stage and by reductase—at the second stage of biosynthesis [Smith W. L., Marnett L. J., DeWitt D. L. Prostaglandin and thromboxane biosynthesis//Pharmacology & Therapeutics. 1991, V. 49(3), P. 153-179]. It is G-protein coupled receptor FP endogenous ligand, which activation results in intracellular calcium mobilization [Toh H, Ichikawa A, Narumiya S. Molecular evolution of receptors for eicosanoids.//FEBS Lett. 1995, V. 361, P. 17-21].
Prostaglandins are used for treatment of hypertension, thrombosis, asthma, gastric and intestinal ulcers, atherosclerosis prevention, and also in obstetrics. FP-receptor agonists are used in clinics as agents reducing intraocular pressure [Woodward D. F., Lawrence R. A., Fairbairn C. E., Shan T., Williams L. S. Intraocular pressure effects of selective prostanoid receptor agonists involve different receptor subtypes according to radioligand binding studies.//J Lipid Mediat. 1993, V. 6(1-3), P. 545-553.]
Prostamides (amides of prostaglandins and ethanolamine) are formed in organism by oxidation of arachidonic acid ethanolamide (anandamide) caused by cyclooxygenase-2 [Kozak K. R., Crews B. C., Ray J. L., Tai H. H., Morrow J. D., Marnett L. J. Metabolism of prostaglandin glycerol esters and prostaglandin ethanolamides in vitro and in vivo.//J. Biol. Chem. 2001, V.276(40), P. 36993-36998]. These derivatives appeared to be able to reduce intraocular pressure. The most known product of this class is synthetic analogue of prostamide F2-bimatoprost, wherein ethanolamine residue is replaced by ethylamine residue, and prostamide alkyl side chain is modified by introduction of phenyl radical [Woodward D. F., Krauss A. H., Chen J., Lai R. K., Spada C. S., Burk R. M., Andrews S. W., Shi L., Liang Y., Kedzie K. M., Chen R., Gil D. W., Kharlamb A., Archeampong A., Ling J., Madhu C., Ni J., Rix P., Usansky J., Usansky H., Weber A., Welty D., Yang W., Tang-Liu D. D., Garst M. E., Brar B., Wheeler L. A., Kaplan L. J. The pharmacology of bimatoprost (Lumigan)//Surv. Ophthalmol. 2001, V.45, Suppl 4, P. S337-S345].
There is known a variety of prostamide analogues, wherein as prostanoid as ethanolamine part of molecule is modified. The closest to the claimed compounds are prostamides capable of neuroprotection [V. V. Bezuglov, M. Y. Bobrov, N. M. Gretskaya, I. V. Serkov, G N Zinchenko, M. G. Akimov. Prostamides and their analogues capable of neuroprotection./Patent RU No. 2474426, priority on 26, Dec. 2011, publication 10 Feb. 2013, Bulletin No. 4]. This patent describes prostamide E analogues comprising residues of nitroxyethylamine, glycine, gamma-aminobutyric acid, hydroxyethylamine, tyramine and dopamine. However, this patent does not describe analogues of prostamide F comprising the same modified residues of ethanolamine.
There also known the analogues of prostaglandin F2α, e.g. patent [U.S. Pat. No. 7,910,767], which describes ethers, thioethers and amides of prostaglandin F2α with modified alkyl side chain, having the modified biological activity as compared to the known analogues of prostaglandin F2α, applied in the medical field (Bimatoprost, Travoprost, etc.).
There also known the analogues of prostaglandin F2α comprising nitroxy groups.
U.S. Pat. No. 5,625,083 describes dinitroglycerol ethers of prostaglandins, including prostaglandin F2α, which could be used as vasodilators, antihypertensive agents, or bronchodilators.
U.S. Pat. No. 8,101,658 describes prostamides generating nitrogen oxide, which could be used for treatment of glaucoma and elevated intraocular pressure.
However, these inventions do not contain structures disclosed in this invention.
The object of this invention expansion of nomenclature of biologically active derivatives of prostaglandin F2α, capable of reducing intraocular pressure. This problem is solved by synthesis of prostaglandin F2α amide derivatives, wherein amide part is represented by amino acids, their nitro derivatives, cyclic amines and substituted ethanolamine.
Thus, the subject matter of this invention is amide derivatives of prostaglandin F2α with the general formula (1):
where
where n=1-4 or
where
where
where n=1, 2
These substances have low cytotoxicity and are capable of stimulating formation of endogenous nitric oxide in mammal cells.
Unless otherwise specified, all technical and scientific terms used in this document have the same meaning, which is common in the art, and could be understood by those skilled in the art.
“Substituted” means that a specific group or residue includes one or more substituents different from hydrogen atoms.
“Unsubstituted” means that a specific group or residue does not comprise substituents different from hydrogen atoms.
“Alkyl” means unsubstituted alkyl group with linear or branched chain comprising from 1 to 6 carbon atoms.
“Cycloalkyl” means cyclic saturated hydrocarbon group with 3-6 ring carbon atoms (e.g. cyclopropyl, cyclobutyl and similar).
“Mixture” co-inclusion of two and more substances without chemical bonding, and physical properties of each component are retained unchanged.
“Pharmaceutically acceptable salt” means that a component producing salt with a carboxyl group of the claimed substances will not cause harm to a human organism.
“Pharmaceutically acceptable cation” means that this cation will not cause harm to a human organism at salt formation.
Substances of this invention in a generic form could be synthesized by techniques known in the chemistry field. Some processes of producing the specific substances of this invention are illustrated in diagrams given in examples.
Biological activities of substances of this invention include absence of cytotoxicity up to concentration of 100 μM; ability to reduce intraocular pressure, ability to increase intracellular calcium concentration, ability to increase nitrogen oxide production.
The claimed substances, being analogues of natural prostaglandin F2α, are capable of interacting with specific prostanoid receptor FP, that results in stimulation of intracellular calcium mobilization.
Unexpected result of this invention is the ability of the claimed substances to induce endogenous synthesis of nitric oxide in mammal cells. Such properties have not been observed for any prostaglandin F2α derivative. Moreover, compounds not comprising NO-donor groups, e.g. amide of prostaglandin F2α with serine isopropyl ester (compound 3).
Therefore, the problem of this invention is solved by synthesis of new prostaglandin F2α amide derivatives with the general formula (1), capable of inducing endogenous synthesis of nitric oxide, that significantly expands opportunities of medical application of the claimed substances due to involving endogenous regulator—nitrogen oxide into pharmacological effect mechanism.
The below examples are given to illustrate this invention, and they should not be considered as limiting the scope of the invention in any way.
Prostaglandin F2α Amide with L-Nitroserine (1).
Adding 76 μl (0.55 μmol) of Et3N to the solution of 150 mg (0.42 μmol) of prostaglandin F2α in 2 ml of acetonitrile under an argon atmosphere, mixing within 5 minutes and adding the solution of 71 μl (0.55 μmol) isobutyl chloroformate in 500 μl of acetonitrile. Mixing the reaction mixture within 30 minutes at 4° C., evaporating to half of volume, diluting with 50 ml of ethyl acetate and washing with water (2×20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na2SO4. Filtering the dryer, boiling down the filtrate in rotary evaporator under water-suction pump vacuum. Drying the residue in oil pump vacuum. Obtaining 171 mg of mixed anhydride, yellow oil.
Adding the solution of the obtained mixed anhydride in 1 ml of DMF and 52 μl of N-methyl morpholine to the solution of 104 mg (0.49 μmol) of L-nitroserine nitrate in 0.500 ml of DMF at 4° C. under an argon atmosphere, and mixing the obtained mixture within 90 minutes. Boiling down the solvent, diluting the residue with 50 ml of ethyl acetate and washing with 2M water solution of NaHSO4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na2SO4. Filtering the dryer, evaporating the filtrate in rotary evaporator under water-suction pump vacuum. Obtaining 162 mg of prostaglandin F2α amide with L-nitroserine, yellowish oil, yield 70%.
PMR: 0.90 (3H), 1.29 (6H), 1.53 (3H), 1.75 (2H), 2.18 (7H), 2.41 (2H), 2.71 (1H), 3.95 (1H), 4.14 (2H), 4.33 (1H), 4.88 (2H), 5.52 (4H). Mass spectrum: 509.2832 [M+Na]+, 441.3282 [MNO2+Na+H]+, [c]D5=36.0°, c=1, EtOH.
Prostaglandin F2α Amide with L-Serine (2).
Derived similarly from prostaglandin F2α and L-serine. Yield—51%. PMR: 0.84 (3H), 1.3 (6H), 1.52 (3H), 1.95 (3H), 2.16 (6H), 2.33 (2H), 2.68 (1H), 3.89 (2H), 3.92 (1H), 4.19 (1H), 4.32 (2H), 5.34 (4H), 7.8 (1H). [α]D25=33.5°, c=1, EtOH:H2O, 1:2.
Prostaglandin F2α Amide with L-Serine Isopropyl Ether (3).
Derived similarly from prostaglandin Fa, and L-serine isopropyl ether hydrochloride. Yield—57%. PMR: 0.89 (3H), 1.22 (6H), 1.3 (9H), 1.76 (3H), 2.04 (6H), 2.39 (2H), 3.9 (2H), 4.03 (2H), 4.13 (1H), 4.63 (1H), 5.08 (1H), 5.45 (4H), 6.83 (1H). [α]D25=28.4°, c=1, EtOH.
Prostaglandin F2α Amide with D-Serine Isopropyl Ether (4).
Derived similarly from prostaglandin F2α and D-serine isopropyl ether hydrochloride. Yield—35%. PMR: 0.89 (3H), 1.21 (6H), 1.3 (9H), 1.74 (3H), 2.01 (6H), 2.4 (2H), 3.9 (2H), 4.0 (2H), 4.1 (1H), 4.6 (1H), 5.1 (1H), 5.43 (4H), 6.9 (1H). [α]D25=27.2°, c=1, EtOH.
Prostaglandin F2α Amide with L-Nitroserine Isopropyl Ether (5).
Derived similarly from prostaglandin Fa, and L-nitroserine isopropyl ether. Yield—64%. PMR: 0.84 (3H), 1.16 (6H), 1.23 (8H), 1.51 (4H), 1.96 (4H), 2.12 (4H), 3.88 (3H), 4.52 (1H), 4.9 (2H), 5.34 (5H), 7.9 (1H).
Prostaglandin F2α Amide with Cyclopropylamine (6).
Derived similarly from prostaglandin F2α and cyclopropylamine. Yield—73%. PMR: 0.50 (2H), 0.78 (2H), 0.90 (3H), 1.31 (6H), 1.61 (4H), 1.86 (4H), 2.06 (4H), 2.36 (2H), 2.70 (1H), 4.00 (1H), 4.09 (1H), 4.20 (1H), 5.41 (1H), 5.57 (2H), 5.78 (2H). [α]D25=38.0°, c=1, EtOH.
Prostaglandin F2α Amide with L-Prolinol (7).
Derived similarly from prostaglandin F2α and L-prolinol. Yield—61%. PMR: 0.91 (3H), 1.36 (6H), 1.60 (6H), 1.89 (6H), 2.08 (4H), 2.39 (1H), 2.65 (1H), 3.2 (1H), 3.45 (1H), 3.57 (1H), 3.73 (1H), 3.90 (1H), 4.06 (1H), 4.21 (2H), 5.48 (2H), 5.69 (2H). [α]D25=22.0°, c=1, EtOH.
Amide of Prostaglandin F2α with γ-Aminobutyric Acid.
Adding 600 μl of N,O-bis(trimethylsilyl) trifluoroacetamide to the solution of 103 mg (1.00 μmop of γ-aminobutyric acid in 1 ml of acetonitrile and mixing within 18 hours at 23° C.
Adding 145 μl (1.05 μmop of triethylamine and 125 μl (0.96 μmop of isobutyl chloroformate to the solution of 284 mg (0.8 μmop of prostaglandin F2α in 3 ml of acetonitrile and mixing within 30 minutes at 23° C. Adding the obtained mixed anhydride to silylated γ-aminobutyric acid solution and mixing within 4 hours at 23° C. Adding methanol (5 ml) to the reaction mixture, mixing within 20 minutes and evaporated in rotary evaporator under water-suction pump vacuum. Dissolving the residue in ethyl acetate (20 ml) and washing with 2M water solution of NaHSO4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na2SO4. Filtering the dryer, evaporating the filtrate in rotary evaporator under water-suction pump vacuum. Purifying the residue by silica gel column chromatography. Obtaining 132 mg of prostaglandin F2, and γ-aminobutyric acid amide, clear oil, yield—60%. Mass spectrum, m/z: 462.2888 [M+Na]+.
Prostaglandin F2α Amide with γ-Aminobutyric Acid Nitroethanolamide (8).
Adding 30 μl (0.23 μmop of Et3N to the solution of 80 mg (0.18 μmop of prostaglandin F2α amide with γ-aminobutyric acid in 3 ml of acetonitrile under an argon atmosphere, mixing within 5 minutes and adding 30 μl (0.22 μmop isobutyl chloroformate. Mixing the reaction mixture within 30 minutes at 4° C., adding the solution of 38 mg (0.37 μmop of nitroetanolamine nitrate and 50 μl of Et3N in 2 ml of dichloromethane and mixing within 12 hours at 23° C. Boiling down the solvent, diluting the residue with 20 ml of ethyl acetate and washing with 2M water solution of NaHSO4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na2SO4. Filtering the dryer, evaporating the filtrate in rotary evaporator under water-suction pump vacuum. Purifying the residue by silica gel column chromatography. Obtaining 35 mg of amide of prostaglandin F2α with γ-aminobutyric acid nitroethanolamide (8), yield—36%. PMR: 0.88 (3H), 1.21 (6H), 1.65 (3H), 1.77 (4H), 2.11 (7H), 2.38 (1H), 2.62 (1H), 3.33 (2H), 3.55 (2H), 3.97 (1H), 4.18 (2H), 4.53 (2H), 4.68 (2H), 5.47 (4H). [α]D25=35.2°, c=1, EtOH.
Prostaglandin F2α Amide with γ-Aminobutyric Acid Nitroethylene Glycol Ether (9).
Adding 24 mg (0.12 μmol) of N-(3-dimethylaminopropyl)-N′-ethyl carbonate hydrochloride, 3 mg (0.02 μmop of dimethylaminopyridine and 45 mg (0.1 μmol) of prostaglandin F2α amide with γ-aminobutyric acid to the solution of 14 μl (0.12 μmol) of nitroethylene glycol in 1 ml of dichloromethane and mixing within 90 minutes at 23° C. Diluting the reaction mixture with chloroform (20 ml) and washing with 2M water solution of NaHSO4 (20
PMR: 0.89 (3H), 1.38 (11H), 1.88 (9H), 2.27 (2H), 2.87 (2H), 3.22 (2H), 3.81 (1H), 3.99 (1H), 4.07 (1H), 4.37 (2H), 4.67 (2H), 5.36 (4H), 7.57 (1H). [α]D25=24.0°, c=1, EtOH.
Prostaglandin F2α Amide with γ-Aminobutyric Acid Dinitroglycerol Ester (10).
Adding 44 mg (0.43 μmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride in 0.5 ml of dichloromethane, 5 mg (0.04 μmop of N-dimethylaminopyridine and 42 mg (0.22 μmol) of glycerol dinitrate to the solution of 84 mg (0.2 μmol) of prostaglandin F2α amide with γ-aminobutyric acid in 1 ml of dichloromethane under an argon atmosphere, and mixing within 2 hours at 23° C. Evaporating the solvent, diluting the residue with ethyl acetate (20 ml) and washing with 2M water solution of NaHSO4 (20 ml), with water (2×20 ml) and with saturated water solution of NaCl (20 ml) and drying above anhydrous Na2SO4. Filtering the dryer, evaporating the filtrate in rotary evaporator under water-suction pump vacuum. Purifying the residue by silica gel column chromatography. Obtaining 38 mg of prostaglandin F2α amide with γ-aminobutyric acid dinitroglycerol ester (10), yield 33%. PMR: 0.84 (3H), 1.43 (14 H), 1.98 (8H), 2.34 (2H), 3.04 (2H), 3.88 (2H), 4.31 (1H), 4.46 (2H), 4.77 (2H), 5.36 (5H), 7.75 (1H). Mass spectrum, m/z: 626.2968 [M+Na]+, 586.3943 [M+H−H2O]+. [α]D25=30.2°, c=1, EtOH
Adding 93 mg (0.37 μmol) of disuccinimidyl carbonate and 52 μl (0.37 μmol) of Et3N to the solution of 150 mg (0.37 μmol) of prostaglandin F2α, amide with ethanolamine in 3 ml of acetonitrile and mixing within 1.5 h at 23° C. Then, adding 62 mg (0.37 μmol) of nitroetanolamine nitrate and 37 mg (0.37 μmol) of Et3N and mixing within 18 hours at 4° C. Evaporating the solvent, diluting the residue with 20 ml of ethyl acetate and washing with 2M water solution of NaHSO4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na2SO4. Filtering the dryer, evaporating the filtrate in rotary evaporator under water-suction pump vacuum. Purifying the residue by silica gel column chromatography. Obtaining 38 mg of amide (11), white oil, yield 19%. PMR: 0.90 (3H), 1.31 (6H), 1.55 (4H), 1.76 (3H), 2.15 (5H), 2.53 (2H), 3.34 (2H), 3.53 (2H), 4.01 (1H), 4.18 (4H), 4.56 (2H), 5.40 (2H), 5.57 (2H), 5.71 (1H), 6.25 (1H). Mass spectrum, m/z: 552.2983 [M+Na]+. [α]D25=34.6°, c=1, EtOH
Prostaglandin F2α amide with 2-aminoethyl-2-(nitroxy)-1-[(nitroxy)methyl]ethyl]carbonate (12)
Adding 32 mg (0.12 μmop of disuccinimidyl carbonate and 20 μl (0.14 μmop of Et3NK to the solution of 55 mg (0.14 μmop of prostaglandin F2α amide with ethanolamine in 1.5 ml of dichloromethane and mixing within 40 minutes at 23° C. Then, adding 25 mg (0.14 μmop of glycerol dinitrate and 16 mg (0.14 μmop of N-dimethylaminopyridine and mixing within 18 hours at 4° C. Evaporating the solvent, diluting the residue with 20 ml of ethyl acetate and washing with 2M water solution of NaHSO4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na2SO4. Filtering the dryer, evaporating the filtrate in rotary evaporator under water-suction pump vacuum. Purifying the residue by silica gel column chromatography. Obtaining 20 mg of amide (12), white oil, yield 19%. PMR: 0.84 (3H), 1.24-1.53 (12H), 2.01 (8H), 2.39 (1H), 2.51 (1H), 2.64 (2H), 3.64 (2H), 4.00 (2H), 4.18 (1H), 4.46 (2H), 4.81 (2H), 5.31 (5H), 7.95 (1H). Mass spectrum, m/z: 628.2770 [M+Na]+, 588.2843 [M+H−H2O]+.
3T3-L1 cells were incubated at 95% humidity, in the 5% CO2 atmosphere, at 37° C. Cultivation was performed in DMEM medium comprising 7% FCS, 2 mM of glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin and 0.25 μg/ml of amphotericin B. Cells were subcultured every 48-72 hours. For the purpose of suspending the cells were incubated in Versene's solution within 1 minute and in Trypsin-EDTA solution within 1 minute (0.25%).
Assessment of Substance Effect on Cell Culture
Cells were inoculated by 30 thousand into wells of 96-well plate (monolayer) in 100 μl of medium for MTT-test. After 24 hours of cultivation 100 μl of test substances in the medium used for cell culture were added to the cells with replacement of culture medium with a new one. Cells were incubated with test substances for 24 hours. Cells, to which 100 μl of medium used for cell culture were added to, were used as positive control.
Assessment of Cell Survival in the Culture Using the MTT Assay
After the incubation, the medium was removed from each well, and then 100 μl of MTT solution were added to each well (0.5 mg/ml of MTT, 3.5 mg/ml of D-glucose in Hanks' solution). The plate was placed into CO2-incubator (Ependorf, Germany) for 1.5 h. After that, MTT was removed and 100 μl of DMSO were added to each well and mixed for 2 minutes in a shaker at 550 rpm. Then, the solution optical density was determined at the wavelength of 576 nm and 620 nm using a plate reader (EFOS 9305, OAO MZ Sapphir).
All test substances did not have noticeable cytotoxicity up to the concentration of 100 μM (Table 1).
137.5 ± 1.5
The study was performed on chinchilla rabbits with a mass of 2.5-3 kg. IOP was measured by automatic manual veterinary tonometer Tonovet (Icare, Finland).
All test samples were instilled by 2 drops by a disposable pipette. IOP was measured before instillation and further, every hour within 6 hours. The results are given in Table 2. All tested substances were capable of reducing intraocular pressure in normotensive rabbits. This effect is more prominent for the compounds 3 and 6.
3T3-L1 cells were cultivated in DMEM medium with addition of 10% fetal calf serum (FCS), 4 mM of L-glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin, 2.5 μg/ml of amphotericin B. Cells were cultured in the atmosphere of 5% CO2, 95% humidity at 37° C.
Cell density at the time of experiment was 7000 per well of a 96-well plate, Calcium Green dye (1.5 mM, loading in 0.4% Pluronic F-62 in incubation medium without BSA for 1 hour at 37° C., triple washing of 200 μl of incubation medium after loading), incubation medium: Hanks' solution, 1 g/l of D-glucose, 1 mg/ml of fat-free BSA, introduction of substances in ethanol (<0.5% final concentration), incubation at 25° C., detecting at the excitation wavelength 485 nm, emission wavelength 535 nm.
Results
The addition of test substances to 3T3-L1 cells causes increase of intracellular calcium concentration. Thus, when adding reference substances: 67 μM of prostaglandin F2α and prostamide F2 (prostaglandin F2α ethanolamide), the calcium sensor fluorescence increases by 195 and 185 AU relative to control respectively for 10 minutes; while in the presence of the substance 3, such increase was 405 AU.
3T3-L1 cells were incubated at 95% humidity, in the 5% CO2 atmosphere, at 37° C. Cultivation was performed in DMEM medium comprising 7% FCS, 2 mM of glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin and 0.25 μg/ml of amphotericin B. Cells were subcultured every 48-72 hours. For the purpose of suspending the cells were incubated in Versene's solution within 2-3 minutes and in Trypsin-EDTA solution (0.25%).
Determination of NO Generation Level
Cells were inoculated by 30 thousand into wells (monolayer) of 96-well plate in 100 μl of medium. Incubation with substances lasted 20 hours. After that, medium aliquot was sampled from each well, and NO concentration was determined by modified Griess method. 12.5 μl of 0.04% sulfanilamide water solution were added to 75 μl of test medium in wells of 96-well plate for EIA, held within 10 minutes at room temperature protected from light, then, 12.5 μl of 2% naphthylethylenediamine solution in 3M of HCl were added, held again for 10 minutes at room temperature protected from light, then the optical absorption was determined at the wavelength of 540 nm.
Maximum activity of the most tested compounds by induction of nitrogen oxide generation was observed at the tested substance concentration of 100 μM. The results are given in Table 3.
However, for prostaglandin F2α amide (3) the most effective concentration was 25 μM. At this concentration, nitric oxide level in 3T3-L1 cells increased by 60% relative to control indicated in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018118157 | May 2018 | RU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/RU2019/050062 | 5/15/2019 | WO | 00 |
