PROSTAGLANDIN F2 ALPHA DERIVATIVES FOR DECREASING INTRAOCULAR PRESSURE

Abstract

The invention relates to clinical chemistry, in particular, to new biologically active compounds amide derivatives of prostaglandin F2α. These compounds have low cytotoxicity and are capable of stimulating formation of endogenous nitrogen oxide in mammal cells. Synthesis of such compounds promotes expansion of nomenclature of biologically active derivatives of prostaglandin F2α capable of reducing intraocular pressure.

Claims

1. Prostaglandin F2 alpha derivatives with the general formula ##STR00010## where R.sup.1═H, ##STR00011## where n=1-4 or R.sup.1═H, ##STR00012## where R.sup.3═CHMe.sub.2, HNCH.sub.2CH.sub.2ONO.sub.2, OCH.sub.2CH.sub.2ONO.sub.2, CH(CH.sub.2ONO.sub.2).sub.2 or R.sup.1═H, ##STR00013## where R.sup.4═H, CHMe.sub.2; R.sup.5═H, OH; R.sup.6═H, OH, ONO.sub.2, CH.sub.2OH, CH.sub.2ONO.sub.2 or R.sup.1═H, R.sup.2═CH.sub.2CH.sub.2OC(O)NHCH.sub.2CH.sub.2ONO.sub.2 or R.sup.1═H, R.sup.2═CH.sub.2CH.sub.2OC(O)OCH(CH.sub.2ONO.sub.2).sub.2 or ##STR00014## where n=1, 2 where R.sup.1 and R.sup.2 independently refer to hydrogen, alkyl, cycloalkyl, hydroxyalkyl, amino acids, ethers, and amides of amino acids, and also 12.sup.1+R.sup.2 could refer to heterocycle (e.g. substituted or unsubstituted piperidine, pyrrolidine, etc.).

2. Compounds of claim 1, wherein salt with pharmaceutically acceptable cations is produced in the presence of free carboxyl group.

3. Compounds of claim 1, capable of reducing intraocular pressure.

Description

EXAMPLE 1. PROSTAGLANDIN F.SUB.2α .AMIDES WITH SERINE AND ITS DERIVATIVES, CYCLOPROPYLAMINE AND L-PROLINOL

[0036] ##STR00006##

[0037] Prostaglandin F.sub.2α Amide with L-Nitroserine (1).

[0038] Adding 76 μl (0.55 μmol) of Et.sub.3N to the solution of 150 mg (0.42 μmol) of prostaglandin F.sub.2α 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 Na.sub.2SO.sub.4. 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.

[0039] 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 NaHSO.sub.4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na.sub.2SO.sub.4. Filtering the dryer, evaporating the filtrate in rotary evaporator under water-suction pump vacuum. Obtaining 162 mg of prostaglandin F.sub.2α amide with L-nitroserine, yellowish oil, yield 70%.

[0040] 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].sup.+, 441.3282 [MNO.sub.2+Na+H]+, [c]D.sup.5=36.0°, c=1, EtOH.

[0041] Prostaglandin F.sub.2α Amide with L-Serine (2).

[0042] Derived similarly from prostaglandin F.sub.2α 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].sub.D.sup.25=33.5°, c=1, EtOH:H2O, 1:2.

[0043] Prostaglandin F.sub.2α Amide with L-Serine Isopropyl Ether (3).

[0044] Derived similarly from prostaglandin F.sub.a, 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].sub.D.sup.25=28.4°, c=1, EtOH.

[0045] Prostaglandin F.sub.2α Amide with D-Serine Isopropyl Ether (4).

[0046] Derived similarly from prostaglandin F.sub.2α 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].sub.D.sup.25=27.2°, c=1, EtOH.

[0047] Prostaglandin F.sub.2α Amide with L-Nitroserine Isopropyl Ether (5).

[0048] Derived similarly from prostaglandin F.sub.a, 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).

[0049] Prostaglandin F.sub.2α Amide with Cyclopropylamine (6).

[0050] Derived similarly from prostaglandin F.sub.2α 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].sub.D.sup.25=38.0°, c=1, EtOH.

[0051] Prostaglandin F.sub.2α Amide with L-Prolinol (7).

[0052] Derived similarly from prostaglandin F.sub.2α 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].sub.D.sup.25=22.0°, c=1, EtOH.

EXAMPLE 2. AMIDES OF PROSTAGLANDIN F.SUB.2α .WITH γ-AMINOBUTYRIC ACID DERIVATIVES

[0053] ##STR00007##

[0054] Amide of Prostaglandin F.sub.2α with γ-Aminobutyric Acid.

[0055] 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.

[0056] 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 F.sub.2α 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 NaHSO.sub.4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na.sub.2SO.sub.4. 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 F.sub.2, and γ-aminobutyric acid amide, clear oil, yield—60%. Mass spectrum, m/z: 462.2888 [M+Na]+.

[0057] Prostaglandin F.sub.2α Amide with γ-Aminobutyric Acid Nitroethanolamide (8).

[0058] Adding 30 μl (0.23 μmop of Et.sub.3N to the solution of 80 mg (0.18 μmop of prostaglandin F.sub.2α 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 Et.sub.3N 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 NaHSO.sub.4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na.sub.2SO.sub.4. 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 F.sub.2α 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].sub.D.sup.25=35.2°, c=1, EtOH.

[0059] Prostaglandin F.sub.2α Amide with γ-Aminobutyric Acid Nitroethylene Glycol Ether (9).

[0060] 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 F.sub.2α 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 NaHSO.sub.4 (20 MJI), with water (2×20 ml) and with saturated water solution of NaCl (20 ml) and drying above anhydrous Na.sub.2SO.sub.4. 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 (9), white oil, yield 37%.

[0061] 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].sub.D.sup.25=24.0°, c=1, EtOH.

[0062] Prostaglandin F.sub.2α Amide with γ-Aminobutyric Acid Dinitroglycerol Ester (10).

[0063] 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 F.sub.2α 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 NaHSO.sub.4 (20 ml), with water (2×20 ml) and with saturated water solution of NaCl (20 ml) and drying above anhydrous Na.sub.2SO.sub.4. 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 F.sub.2α 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].sup.+. [α].sub.D.sup.25=30.2°, c=1, EtOH

EXAMPLE 3. PROSTAGLANDIN F.SUB.2α .AMIDE WITH 2-AMINOETHYL-[2-(NITROXY)ETHYL]CARBAMATE (11)

[0064] ##STR00008##

[0065] Adding 93 mg (0.37 μmol) of disuccinimidyl carbonate and 52 μl (0.37 μmol) of Et.sub.3N to the solution of 150 mg (0.37 μmol) of prostaglandin F.sub.2α, 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 Et.sub.3N 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 NaHSO.sub.4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na.sub.2SO.sub.4. 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].sub.D.sup.25=34.6°, c=1, EtOH

[0066] Prostaglandin F2α amide with 2-aminoethyl-2-(nitroxy)-1-[(nitroxy)methyl]ethyl]carbonate (12)

##STR00009##

[0067] Adding 32 mg (0.12 μmop of disuccinimidyl carbonate and 20 μl (0.14 μmop of Et.sub.3NK to the solution of 55 mg (0.14 μmop of prostaglandin F.sub.2α 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 NaHSO.sub.4 (20 ml) and saturated water solution of NaCl (20 ml) and drying above anhydrous Na.sub.2SO.sub.4. 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].sup.+.

EXAMPLE 4. CYTOTOXIC ACTIVITY OF PROSTAGLANDIN F.SUB.2α .DERIVATIVES

[0068] 3T3-L1 cells were incubated at 95% humidity, in the 5% CO.sub.2 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%).

[0069] Assessment of Substance Effect on Cell Culture

[0070] 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.

[0071] Assessment of Cell Survival in the Culture Using the MTT Assay

[0072] 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 CO.sub.2-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).

[0073] All test substances did not have noticeable cytotoxicity up to the concentration of 100 μM (Table 1).

TABLE-US-00001 TABLE 1 Cytotoxicity of prostaglandin F.sub.2a derivatives relative to cultivated mice 3T3-L1 cells Compound number Cytotoxicity, EC50 μM 1 226.4 ± 2 2 >200 3 143.6 ± 3 5 195.2 ± 2 6 124.4 ± 2 7 104.4 ± 2 8 193.9 ± 4 11 184.5 ± 3 20 .sup. 137.5 ± 1.5

EXAMPLE 5. REDUCING INTRAOCULAR PRESSURE CAUSED BY PROSTAGLANDIN F.SUB.2α DERIVATIVES

[0074] 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). [0075] 2 groups of rabbets were used for testing each sample: [0076] 5 animals instilled with a single dose of sample into both eyes. [0077] 5 animals instilled with a single dose of normal saline into both eyes.

[0078] 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.

TABLE-US-00002 TABLE 2 Reducing intraocular pressure in normotensive rabbits after instillation of prostaglandin derivative solutions (maximum value of 5 animals' average data) Compound number IOP reduction, mm Hg 3 3.20 5 1.20 6 3.30 8 2.00 11 1.50 19 1.90

EXAMPLE 6. INCREASING OF THE INTRACELLULAR CALCIUM CONCENTRATION CAUSED BY PROSTAGLANDIN F.SUB.2α .DERIVATIVES

[0079] 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% CO.sub.2, 95% humidity at 37° C.

[0080] 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.

[0081] Results

[0082] The addition of test substances to 3T3-L1 cells causes increase of intracellular calcium concentration. Thus, when adding reference substances: 67 μM of prostaglandin F.sub.2α and prostamide F2 (prostaglandin F.sub.2α 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.

EXAMPLE 7. NITROGEN OXIDE GENERATION CAUSED BY PROSTAGLANDIN F.SUB.2α .DERIVATIVES

[0083] 3T3-L1 cells were incubated at 95% humidity, in the 5% CO.sub.2 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%).

[0084] Determination of NO Generation Level

[0085] 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.

[0086] 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.

TABLE-US-00003 TABLE 3 Nitrogen oxide (NO) generation by mice 3T3-L1 cells caused by prostaglandin F.sub.2a derivatives at concentration of 100 μM. Compound number NO generation, % to control 1 7 2 25 3 15 6 5 7 15 8 22 11 28 20 57

[0087] However, for prostaglandin F.sub.2α 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 FIG. 1.