Prostaglandin F2 alpha derivatives for decreasing intraocular pressure

Abstract

The invention relates to clinical chemistry, in particular, to new biologically active compoundsamide derivatives of prostaglandin F2?. 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 F2? capable of reducing intraocular pressure.

Claims

1. A compound selected from the following compounds: ##STR00010## ##STR00011## ##STR00012##

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGURENitric oxide generation (measured as nitrite-ion by Griss's method) caused by prostaglandin F2a amide (3) in the concentration range from 25 up to 100 ?M.

(2) The object of this inventionexpansion of nomenclature of biologically active derivatives of prostaglandin F.sub.2?, capable of reducing intraocular pressure. This problem is solved by synthesis of prostaglandin F.sub.2? amide derivatives, wherein amide part is represented by amino acids, their nitro derivatives, cyclic amines and substituted ethanolamine.

(3) Thus, the subject matter of this invention is amide derivatives of prostaglandin F.sub.2? with the general formula (1):

(4) ##STR00001##
where

(5) ##STR00002##
where n=1-4 or

(6) ##STR00003##
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

(7) ##STR00004##
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

(8) ##STR00005##
where n=1, 2

(9) 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 R.sup.1+R.sup.2 could refer to heterocycle (e.g. substituted or unsubstituted piperidine, pyrrolidine, etc.); and also pharmaceutically acceptable salts for compounds with the general formula (1) with free carboxyl group; and also mixtures of two or more compounds with the general formula (1) in different ratios.

(10) These substances have low cytotoxicity and are capable of stimulating formation of endogenous nitric oxide in mammal cells.

(11) 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.

(12) Substituted means that a specific group or residue includes one or more substituents different from hydrogen atoms.

(13) Unsubstituted means that a specific group or residue does not comprise substituents different from hydrogen atoms.

(14) Alkyl means unsubstituted alkyl group with linear or branched chain comprising from 1 to 6 carbon atoms.

(15) Cycloalkyl means cyclic saturated hydrocarbon group with 3-6 ring carbon atoms (e.g. cyclopropyl, cyclobutyl and similar).

(16) Mixtureco-inclusion of two and more substances without chemical bonding, and physical properties of each component are retained unchanged.

(17) 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.

(18) Pharmaceutically acceptable cation means that this cation will not cause harm to a human organism at salt formation.

(19) 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.

(20) Biological activities of substances of this invention include absence of cytotoxicity up to concentration of 100 ?M; ability to reduce intraocular pressure, ability to increase intracellular calcium concentration, ability to increase nitrogen oxide production.

(21) The claimed substances, being analogues of natural prostaglandin F.sub.2?, are capable of interacting with specific prostanoid receptor FP, that results in stimulation of intracellular calcium mobilization.

(22) 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 F.sub.2? derivative. Moreover, compounds not comprising NO-donor groups, e.g. amide of prostaglandin F.sub.2? with serine isopropyl ester (compound 3).

(23) Therefore, the problem of this invention is solved by synthesis of new prostaglandin F.sub.2, 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 regulatornitrogen oxide into pharmacological effect mechanism.

(24) 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.

Example 1. Prostaglandin F.SUB.2? Amides with Serine and its Derivatives, Cyclopropylamine and L-Prolinol

(25) ##STR00006##

(26) Prostaglandin F.sub.2? Amide with L-Nitroserine (1).

(27) Adding 76 ?l (0.55 ?mol) of Et.sub.3N to the solution of 150 mg (0.42 ?mol) of prostaglandin F.sub.2? in 2 ml of acetonitrile under an argon atmosphere, mixing within 5 minutes and adding the solution of 71 ?l (0.55 ?mol) isobutyl chloroformate in 500 ?l 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.

(28) Adding the solution of the obtained mixed anhydride in 1 ml of DMF and 52 ?l of N-methyl morpholine to the solution of 104 mg (0.49 ?mol) 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 F2a amide with L-nitroserine, yellowish oil, yield70%.

(29) 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 [M-NO2+Na+H].sup.+, [?].sub.D.sup.25=36.0?, c=1, EtOH.

(30) Prostaglandin F.sub.2? Amide with L-Serine (2).

(31) Derived similarly from prostaglandin F.sub.2? and L-serine. Yield51%. 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). [?].sub.D.sup.25=33.5?, c=1, EtOH:H2O, 1:2.

(32) Prostaglandin F.sub.2? Amide with L-Serine Isopropyl Ether (3).

(33) Derived similarly from prostaglandin F.sub.2? and L-serine isopropyl ether hydrochloride. Yield57%. 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). [?].sub.D.sup.25=28.4?, c=1, EtOH.

(34) Prostaglandin F.sub.2? Amide with D-Serine Isopropyl Ether (4).

(35) Derived similarly from prostaglandin F.sub.2? and D-serine isopropyl ether hydrochloride. Yield35%. 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). [?].sub.D.sup.25=27.2?, c=1, EtOH.

(36) Prostaglandin F.sub.2? Amide with L-Nitroserine Isopropyl Ether (5).

(37) Derived similarly from prostaglandin F.sub.2? and L-nitroserine isopropyl ether. Yield64%. 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).

(38) Prostaglandin F2? Amide with Cyclopropylamine (6).

(39) Derived similarly from prostaglandin F.sub.2? and cyclopropylamine. Yield73%. 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). [?].sub.D.sup.25=38.0?, c=1, EtOH.

(40) Prostaglandin F2a Amide with L-Prolinol (7).

(41) Derived similarly from prostaglandin F.sub.2? and L-prolinol. Yield61%. 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). [?].sub.D.sup.25=22.0?, c=1, EtOH.

Example 2. Amides of Prostaglandin F.SUB.2? with ?-Aminobutyric Acid Derivatives

(42) ##STR00007##

(43) Amide of Prostaglandin F.sub.2? with ?-Aminobutyric Acid.

(44) Adding 600 ?l of N,O-bis(trimethylsilyl) trifluoroacetamide to the solution of 103 mg (1.00 ?mol) of ?-aminobutyric acid in 1 ml of acetonitrile and mixing within 18 hours at 23? C.

(45) Adding 145 ?l (1.05 ?mol) of triethylamine and 125 ?l (0.96 ?mol) of isobutyl chloroformate to the solution of 284 mg (0.8 ?mol) of prostaglandin F.sub.2? in 3 ml of acetonitrile and mixing within 30 minutes at 23? C. Adding the obtained mixed anhydride to silylated ?-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 ?-aminobutyric acid amide, clear oil, yield60%. Mass spectrum, m/z: 462.2888 [M+Na].sup.+.

(46) Prostaglandin F.sub.2? Amide with ?-Aminobutyric Acid Nitroethanolamide (8).

(47) Adding 30 ?l (0.23 ?mol) of Et.sub.3N to the solution of 80 mg (0.18 ?mol) of prostaglandin F.sub.2? amide with ?-aminobutyric acid in 3 ml of acetonitrile under an argon atmosphere, mixing within 5 minutes and adding 30 ?l (0.22 ?mol) isobutyl chloroformate. Mixing the reaction mixture within 30 minutes at 4? C., adding the solution of 38 mg (0.37 ?mol) of nitroetanolamine nitrate and 50 ?l 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.2? with ?-aminobutyric acid nitroethanolamide (8), yield36%. 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). [?].sub.D.sup.25=35.2?, c=1, EtOH.

(48) Prostaglandin F.sub.2? Amide with ?-Aminobutyric Acid Nitroethylene Glycol Ether (9).

(49) Adding 24 mg (0.12 ?mol) of N-(3-dimethylaminopropyl)-N-ethyl carbonate hydrochloride, 3 mg (0.02 ?mol) of dimethylaminopyridine and 45 mg (0.1 ?mol) of prostaglandin F.sub.2? amide with ?-aminobutyric acid to the solution of 14 ?l (0.12 ?mol) 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 mA), 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, yield37%.

(50) 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). [?].sub.D.sup.25=24.0?, c=1, EtOH.

(51) Prostaglandin F.sub.2? Amide with ?-Aminobutyric Acid Dinitroglycerol Ester (10).

(52) Adding 44 mg (0.43 ?mol) of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride in 0.5 ml of dichloromethane, 5 mg (0.04 ?mol) of N-dimethylaminopyridine and 42 mg (0.22 ?mol) of glycerol dinitrate to the solution of 84 mg (0.2 ?mol) of prostaglandin F.sub.2? amide with ?-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.2? amide with ?-aminobutyric acid dinitroglycerol ester (10), yield33%. 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].sup.+, 586.3943 [M+H-H2O].sup.+. [?].sub.D.sup.25=30.2?, c=1, EtOH

Example 3. Prostaglandin F.SUB.2? Amide with 2-Aminoethyl-[2-(Nitroxy)Ethyl]Carbamate (11)

(53) ##STR00008##

(54) Adding 93 mg (0.37 ?mol) of disuccinimidyl carbonate and 52 ?l (0.37 ?mol) of Et.sub.3N to the solution of 150 mg (0.37 ?mol) of prostaglandin F.sub.2? amide with ethanolamine in 3 ml of acetonitrile and mixing within 1.5 h at 23? C. Then, adding 62 mg (0.37 ?mol) of nitroetanolamine nitrate and 37 mg (0.37 ?mol) 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, yield19%. 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].sup.+. [?].sub.D.sup.25=34.6?, c=1, EtOH

(55) Prostaglandin F2? Amide with 2-Aminoethyl-2-(Nitroxy)-1-[(Nitroxy)Methyl]Ethyl]Carbonate (12).

(56) ##STR00009##

(57) Adding 32 mg (0.12 ?mol) of disuccinimidyl carbonate and 20 ?l (0.14 ?mol) of Et.sub.3NK to the solution of 55 mg (0.14 ?mol) of prostaglandin F.sub.2? amide with ethanolamine in 1.5 ml of dichloromethane and mixing within 40 minutes at 23? C. Then, adding 25 mg (0.14 ?mol) of glycerol dinitrate and 16 mg (0.14 ?mol) 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, yield19%. 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].sup.+, 588.2843 [M+H-H2O].sup.+.

Example 4. Cytotoxic Activity of Prostaglandin F.SUB.2? .Derivatives

(58) 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 ?g/ml of streptomycin and 0.25 ?g/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%).

(59) Assessment of Substance Effect on Cell Culture

(60) Cells were inoculated by 30 thousand into wells of 96-well plate (monolayer) in 100 ?l of medium for MTT-test. After 24 hours of cultivation 100 ?l 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 ?l of medium used for cell culture were added to, were used as positive control.

(61) Assessment of cell survival in the culture using the MTT assay After the incubation, the medium was removed from each well, and then 100 ?l 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 ?l 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).

(62) All test substances did not have noticeable cytotoxicity up to the concentration of 100 ?M (Table 1).

(63) TABLE-US-00001 TABLE 1 Cytotoxicity of prostaglandin F.sub.2? derivatives relative to cultivated mice 3T3-L1 cells Compound number Cytotoxicity, EC50 ?M 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 20 137.5 ? 1.5

Example 5. Reducing Intraocular Pressure Caused by Prostaglandin F.SUB.2? Derivatives

(64) 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).

(65) 2 groups of rabbets were used for testing each sample:

(66) 5 animals instilled with a single dose of sample into both eyes.

(67) 5 animals instilled with a single dose of normal saline into both eyes.

(68) 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.

(69) 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 F2a Derivatives

(70) 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 ?g/ml of streptomycin, 2.5 ?g/ml of amphotericin B. Cells were cultured in the atmosphere of 5% CO.sub.2, 95% humidity at 37? C.

(71) 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 ?l 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.

(72) Results

(73) The addition of test substances to 3T3-L1 cells causes increase of intracellular calcium concentration. Thus, when adding reference substances: 67 ?M of prostaglandin F.sub.2? and prostamide F2 (prostaglandin F.sub.2? 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. Nitric Oxide Generation Caused by Prostaglandin F2? Derivatives

(74) 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 ?g/ml of streptomycin and 0.25 ?g/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%).

(75) Determination of NO Generation Level

(76) Cells were inoculated by 30 thousand into wells (monolayer) of 96-well plate in 100 ?l 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 ?l of 0.04% sulfanilamide water solution were added to 75 ?l of test medium in wells of 96-well plate for EIA, held within 10 minutes at room temperature protected from light, then, 12.5 ?l 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.

(77) Maximum activity of the most tested compounds by induction of nitrogen oxide generation was observed at the tested substance concentration of 100 ?M. The results are given in Table 3.

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

(79) However, for prostaglandin F.sub.2? amide (3) the most effective concentration was 25 ?M. At this concentration, nitric oxide level in 3T3-L1 cells increased by 60% relative to control indicated in the FIGURE.