1-ethanolamide pgf and pharmaceutical composition containing the same

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to 1-ethanolamide PGF of formula I useful in relaxation of mammalian intraocular pressure. Claimed substance unlike majority of ocular hypotensive prostaglandins doesn't effect through FP-receptor.

EFFECT: new effective compound for relaxation of mammalian intraocular pressure.

4 cl, 1 ex, 16 dwg, 16 tbl

 

1. The scope of the invention

The present invention relates to ocular hypotensive lipids. In particular, the present invention relates to 1-ethanolamide PGF(prostaglandin F) and related compounds, and containing these compounds, pharmaceutical compositions, and to methods of using these compounds for lowering intraocular pressure in mammals.

2. A brief description of the prior art

The prior art is well aware of numerous ocular hypotensive agents used for treatment of various hypertensive eye conditions, including primary and secondary glaucoma, posing a serious problem for human health. Drugs used for the treatment of ocular hypertension (glaucoma), include antagonists β-adrenergic receptors and various prostaglandins. There is a significant amount of scientific and patent literature concerning prostaglandins and their use for the treatment of ocular hypertension. For example, see Bito L.Z. Biological Protection with Prostaglandins, Cohen, M.M., ed., Boca Raton. Fla., CRC Press Inc., 1985, pp. 231-252; and Bito, L.Z., Applied Pharmacology in the Medical Treatment of Glaucomas Drance, S.M. and Neufeld, A.H. eds., New York, Grune & Stratton, 1984, pp. 477-505.

Patent US 5288754 contains, in addition, links to the already known prior art directed on prostaglandins and related compounds, active in the operation of the means for reducing intraocular pressure in mammals. The U.S. patent describes a "Polar s-1 Esters of Prostaglandins" ("Polar s-1-esters of prostaglandins"), including s-1-amides and-1-substituted amides of carboxylic acid compounds, known as PGF. Additional derivatives of prostaglandin related PGFthat were identified from an examination of the prior art, carried out in the research laboratories of the applicant of the present application, and which exhibit high intraocular hypotensive activity, shown below formula and identified arbitrary numbers as "well-known compound 1 " and "known connection 2". Well-known compound 1 described in patents US 5352708, 5607978 and 5688819, and the known compound 2 described in patent US 5545665.

The large majority of ocular hypotensive agents having the structure of prostaglandin or closely related structure, acting through the famous "prostaglandin" receptors. In particular, it is known that the connection PGFexerts its ocular hypotensive effect via the FP receptor. As disclosed in the work of Abramovitz et al. (J. Biol. Chem., 269:2632, 1994), incorporated herein by reference, the term "FP-receptor" refers to a receptor for prostaglandin person. The structure of PGFcontaining numbers, commonly used in nomencl the round of prostaglandins and related compounds, shown below.

PGF

The present invention is directed to a class of compounds related to 1-ethanolamide PGF2that along with the known compounds 1 and 2 were unexpectedly detected as tools, with a strong ocular hypotensive activity, but who do not show their ocular hypotensive actions or through FP-receptor, or through any other of the known prostaglandin receptor, for example, DP, EP, EP2, EP3, EP4, FP, IP and TP.

SUMMARY of the INVENTION

The present invention is directed to compounds of Formula 1, where the dotted lines indicate a lack of communication or communication provided that in the formula no two adjacent double bonds;

join wavy lines mean alpha(αdown)- or beta(βup)-configuration, where the wavy line attached to the double bond, they mean either the Z(CIS)-, or E(TRANS)configuration;

the dashed lines indicate the alpha(α)-configuration and solid triangles - beta(β)-configuration;

m is an integer from 0 to 5;

n is an integer from 1 to 6, provided that the compound represented by the formula, is not 1-ethanolamide PGF;

q and r independently from each other are the Xia integers from 0 to 6;

X represents CH2, O or S, provided that when X represents O or S, the dashed line adjacent to X, indicates the lack of communication;

R is CH3, phenyl, furyl, thienyl,3-8-cycloalkyl, or phenyl, furyl or thienyl substituted by one or two substituents selected from the group consisting of F, C1, Br, C1-6-alkyl, NO2, CN, COOH, COO-(C1-6-alkyl);

R1, R2, R3and R4independently of one another are N or C1-6-alkanoyl with normal or branched chain, benzoyl or lower With1-6-alkyl;

R5represents N or C1-6-alkyl, normal or branched chain and

R6represents N or C1-4-alkyl, normal or branched chain

or pharmaceutically acceptable salt of this compound, and these compounds reduce intraocular pressure in a mammal, but not show their ocular hypotensive activity either through FP prostaglandin receptor, or through any other well-known prostaglandin receptor.

The present invention also relates to a dedicated mainly pure 1-ethanolamide PGFwhich was unexpectedly set, is present in the major biological systems in image quality is as natural compounds. In addition, the present invention is directed to pharmaceutical compositions containing as an active ingredient 1-ethanolamide PGFand/or one or more compounds in accordance with Formula 1, and to a method of treating mammals, including humans, in need of such treatment, an effective amount of a pharmaceutical composition containing as an active ingredient 1-ethanolamide PGFand/or one or more compounds in accordance with Formula 1, to reduce intraocular pressure.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 is a graph showing the effect of increasing doses of 1-ethanolamide PGFon the smooth muscle sphincter of the iris of a cat. Dots represent averages ± SEM (standard error, standard error of the mean; n=4.

Figure 2 is a graph showing the inhibitory effect of increasing doses of 1-ethanolamide PGFon the area stimulated contraction of isolated vessel seminal duct Guinea pigs. Dots represent averages ± SEM; n=4.

Figure 3 is a graph showing the effect of increasing doses of 1-ethanolamide GFon the isolated ileum of the chicken. Dots represent averages ± SEM; n=4.

Figure 4 is a graph showing the effect of age is appropriate doses of 1-ethanolamide PGFon the isolated ileum of the Guinea pig. Dots represent averages ± SEM; n=4.

Figure 5 is a graph showing the effect of increasing doses of 1-ethanolamide PGFon isolated smooth muscle of rat aorta. Dots represent averages ± SEM; n=4.

6 is a graph showing the increase in the concentration of intracellular Ca2+[Ca2+];caused by increasing doses of PGF() and 1-ethanolamide PGF(•) in cells CRL 1497. Points are averages ± SEM; n=4.

Fig.7 is a graph showing the effect of salts 1-ethanolamide prostaglandin F() and prostaglandin F(•) on pre-reduced histamine segment of the jugular vein of a rabbit. Points are averages ± SEM; n=6 for 1-ethanolamide PGFn=7 for PGF.

Fig is a graph showing the increase in the concentration of intracellular Ca2+[Ca2+]icaused by increasing doses of PGF() and 1-ethanolamide PGF(•) in the cells of SOME 293 stably transfected recombinant FP-receptor cats. Points are average values of the ± SEM; n=3.

Fig.9 is a graph showing the total accumulation of insiststhat caused increasing doses of 17-fenprostalene F() and 1-ethanolamide PGF(•) in the cells of SOME 293 stably transfected recombinant FP-receptor cats. Given the average values ± SEM; n=3.

Figure 10 is a graph showing the total accumulation of insiststhat caused increasing doses of 17-fenprostalene F() and 1-ethanolamide PGF(•) in the cells of SOME 293 stably transfected recombinant FP-receptor of human rights. Given the average values ± SEM; n=3.

11 is a graph showing the analysis of competitive binding of labeled ligand in comparison with 5 nm3H-17-phenyl-GFusing a cell SOME of 293 stably transfected recombinant FP-receptor cats. Pictured competition performed using unlabeled 1-ethanolamide PGF(•) and unlabeled 17-phenyl-GF(). Points are averages ± SEM; n=3 experiments performed with three replicates.

Fig is a graph showing the analysis of competitive binding of labeled ligand in comparison with 5 nm3H-17-Anil-GFusing a cell SOME of 293 stably transfected recombinant FP-receptor of human rights. Pictured competition performed using unlabeled 1-ethanolamide PGF(•) and unlabeled 17-phenyl - PGF(). Points are averages ± SEM; n=3 experiments performed with three replicates.

Fig is a graph showing the effect of 0.1% 1-ethanolamide PGF(•) on intraocular pressure in dogs. Contralateral eyes to control treated filler (about). Points are averages ± SEM; n=6.

Fig is a graph showing the effect of 0.1% 1-ethanolamide PGF(•) on the diameter of the pupil of the dog. Contralateral eyes to control treated filler (about). Points are averages ± SEM; n=6.

Fig is a graph showing the effect of 1-ethanolamide PGF(•) on the surface hyperemia eye dogs. Contralateral eyes to control treated filler (about). Points are averages ± SEM; n=6.

DETAILED description of the INVENTION

In connection with the present invention it was unexpectedly found that 1 ethanolamide PGFis a natural compound and can be formed of anandamide, PR is native cannabimimetic, using recombinant MOR-2 and PGF synthase. It is known that MOR-2 (cyclooxygenase-2) is endoperoxide prostaglandin, which is inducible and is not necessarily present in the tissues (see Meade et al. (1993) J. Biol. Chem. 268: 6610-6614). Because it was not expected that MOR-2 will be present in unstimulated tissues obtained from the present invention, the data shows that a significant amount of ethanolamide PGFformed after the introduction of the white mice of anandamide, are unexpected. In addition, ethanolamide PGFalso unexpectedly been discovered in mice not receiving anandamide, which confirms the fact that 1 ethanolamide PGFis constitutive and can act as endogenous synthesized hormone.

In accordance with the invention, 1 ethanolamide GFwas synthesized in order to obtain this substance in isolated and chemically pure form and to enable the preparation of pharmaceutical compositions containing this substance. Synthesis ethanolamide PGFit also gives an opportunity to synthesize all the compounds within Formula 1, but only with such modifications in the synthesis process, which is easily accessible to the practicing organic chemist and, in particular, for a chemist working in the field of chemistry of prostaglandins and related with the joining. Thus, the present invention covers compounds of Formula 1.

Preferred compounds within the scope of legal protection of the invention is represented by Formula 1A, and particularly preferred are compounds within Formula 1A, where the dotted line between carbons 10 and 11, 8 and 12, 17 and 18 indicate the lack of communication, and between carbon atoms 5 and 6 indicate the relationship. In addition, because interest is the replacement of the hydroxyl functional groups of compounds of Formula 1A or Formula 1 (R1groups of up to R4), the currently preferred are compounds in which one or more hydroxyl groups not tarifitsirovana and not alkylated (one or more Deputy from R1to R4represents H)or, if these groups tarifitsirovana, tarifitsiruyutsya group represents alkanoyl having from 1 to 6 carbon atoms (formyl, acetyl, propanol, butanol, pentanol, hexanol or any3-6-alkanoyl branched chain). Because of interest Deputy designated as R5in Formula 1A or Formula 1, preferred are compounds in which R5is H or lower With1-3-alkyl, most preferred N. R6preferably represents H or lower With13 -alkyl, most preferred N, and n is preferably 2.

Returning again to the Formula 1A and Formula 1, the configuration of the groups OR1OR2and OR3preferable is a alpha(C-9, C-11 and C-15-α)-configuration, and the configuration of the double bond 5-6 - preferred Z(CIS)configuration.

The most preferred compounds according to the present invention are 1-ethanolamide PGFand its acylated or alkylated derivatives represented by Formula 2, where the substituents from R1to R4defined as for Formula 1, but acylated compounds are preferable alkylated.

1 ethanolamide PGF=R1=R2=R3=R4=H

The synthesis of compounds of the invention are illustrated with the synthesis of 1-ethanolamide PGFrepresented by reaction scheme 1, and as disclosed in detail below. Let us now turn to the reaction scheme. Commercially available triethanolammonium salt (salt, tromethamine, Tris(hydroxymethyl)aminomethane) carboxylic acid PGFtreated with acid to highlight the free carboxylic acid. The free carboxylic acid PGFconverted into the methyl ester by treatment meteorous agent (preferably iodide stands) in Pris is accordance acceptor acid (preferably, 1,8-diazabicyclo[5.4.0]undec-7-ene) to give the methyl ester GF. Then methyl ether PGFtreated with ethanolamine to obtain 1-ethanolamide PGF. Thus, all of the "amide" group is introduced into the molecule during the reaction between methyl ether PGFand derivative hydroxyethylamine Formula 3, where R5, R6and n are such as defined above for Formula 1 and Formula 3 represents ethanolamine, in which R5and R6mean N, and n is 2. Hydroxyethylamine Formula 3 is available through the chemical literature and successfully used in the work of the practicing organic chemist. Compounds that Formula 1 is one or more dotted lines represent communication, and/or where the configuration of the hydroxyl groups or the configuration of substituents around one or more double bonds other than 1-ethanolamide PGFcan be obtained by using reactions similar to those shown in reaction scheme 1, but using as starting compound derived From-1-carboxylic acid prostaglandin appropriate structure and stereochemistry. These initial connections are available due to the chemical literature and are used in the work of the practicing organic chemist. Compounds that Formula 1 is one or more groups of R1to R 4different from H, i.e. compounds where one or more carbon atoms of the C-9, C-11, C-15 and the hydroxy-group hydroxyalkyl amide fragment in part tarifitsirovana or alkylated, can be obtained by reaction of esterification or alkylation of the free hydroxyl" connections. Generally speaking, the modification of the a - and ω-the side chains of the compounds of Formula 1, as defined for the formula used in the work of the practicing organic chemist in accordance with U.S. patent No. 5545665, 5834498 and 5352708 or by analogy with them. Descriptions of these patents are here by reference.

BIOLOGICAL ACTIVITY

Compounds of the invention are unique as are potent ocular hypotensive agents, which, however, do not act via the FP receptor. This is shown using several analytical methods that are well known in the field and used to determine intraocular hypotensive activity and also to determine the receptor through which these connections. Intraocular hypotensive activity 1-ethanolamide PGFand the actual inertia in relation to FP or other known receptors of prostaglandin demonstrated several methods are especially parasital the mi and unexpected when comparing biological distribution of this connection with the "predecessor" PGF.

Description of biological research and the research results are summarized below.

RESEARCH METHODS the ISOLATED TISSUE

The tension of smooth muscles of isolated tissues were measured isometrically with sensors power offset (Grass FT-03) and recorded on a Grass Polygraphs (model 7G and E). Baths for bodies contained Krebs solution, stored at 37°and saturated With a gas mixture of 95% O2/5% CO2to obtain pH 7.4. The Krebs solution had the following composition (mm): NaCl, To 118.0; KCl, 4.7 in; KN2RHO41,2; l2, 1,9; MgSO4, 1,18; NaHCO3, 25,0; glucose, 11,7; indomethacin, 0,001.

(a) IRIS CATS

Adult domestic cats were killed by intravenous introduction of overdose of pentobarbital sodium (Anthony, Arcadia, CA). The eye was immediately nucleosomal and placed in ice. The sphincter muscle of the iris is fixed vertically with tension from 50 to 100 mg in the bath for bodies with a capacity of 10 ml with double walls. Before each experiment was provided for the stabilization period lasting 60 minutes Activity was determined by response to the reactions of reduction. Compounds were added to the bath cumulatively and at least 30 minutes after complete washing was taken to the recovery and return to the original strength. As control was determined by the response to 10-7M PGF at the beginning and end of each experiment and between the curves of dependence "dose - response". For each tissue was analyzed no more than two connections.

(b) RAT AORTA

Adult rats Sprague Dawley weighing 180-220 g were subjected to anaesthesia with inhalation of CO2after that he deceptional and was driven away. The thoracic aorta was removed and cleaned of all adjacent tissues. To remove blood lumen of the aorta was washed. The aorta was divided into three small segment length 5-8 mm Each segment was fixed with a tension of 2 g in the bath for bodies with a capacity of 10 ml with double walls, with two wire hooks are placed alongside the vessel lumen. This arrangement allows to measure the strength of contractions, develop circular layer of smooth muscle. Tissue was balanced within 1 hour before cumulative addition of compounds in the bath for bodies. As a control at the beginning and end of each experiment was determined by the response to 10-7M U-46619 (thrombocythemia). In the recovery period after a complete cleaning of the drug took 30-45 minutes. For each tissue was analyzed only one investigated the connection.

(C) the ILEUM CHICKEN

The ileum chick is a model for studying EP3-receptor. Sections of ileum chicken long the Oh of 1.5 cm were hung with tension 1, After 1 hour period balance received standard curve dose-response for PGF30 minute cleaning between individual doses. Then decumulative was added to the analyzed compound. At the end of the second control was added maximum dose of G2(10-6M). Then, for each concentration was calculated contractile activity as % of maximum response to PGE2achieved at a concentration of 10-6M

(g) the ILEUM of the Guinea PIG

The ileum of the Guinea pig as a model for studying EP1-receptor. The segment of the ileum of the Guinea pig size of about 1.5 cm was hung with a tension of 1 g in the bath for bodies with double walls. After a 1-hour period of equilibration was obtained curve of dependence "dose - response" for PGE2. Between doses was allowed a 30-minute periods of cleaning. Similarly received curve dose - response for 1-ethanolamide GF. As a control at the end of input 10-6M G2. Data were expressed as % of maximum response to the G2.

(d) the VESSEL SEMINAL DUCT Guinea PIGS

The activity against subtype of prostaglandin-sensitive EP3the receptor was determined by the ability of prostanoids inhibit convulsive reaction g is adcoy muscles, caused by the action of electric current on the isolated vessel seminal duct Guinea pigs. Parts of the vessel seminal duct Guinea pigs 1.5 cm were hung with an initial tension of 1 g and balanced for at least 30 minutes. Then every 30 seconds tissue was subjected to irritation of the electric pulse of 20 volts. After stabilization of a convulsive reaction to PGE2received cumulative standard curve of dependence "dose - response". Then use curve "cumulative dose - response was assessed by the compounds. Tissue was washed and balanced within 1 hour between adding connections. As a standard control in the end of the experiment was again estimated PGE2. Activity was calculated as percentage inhibition of the peak convulsive reactions muscles.

(e) JUGULAR VEIN of the RABBIT

New Zealand rabbits albinos of both sexes weighing 2-4 kg was administered 1000 units of heparin in marginal ophthalmic vein, and then were killed with carbon dioxide gas. The external jugular vein was purified from fat and the surrounding connective tissue was removed. The vein is dissected and each ring of 3-4 mm length was hung between two metal hooks in the bath for bodies with double walls. Tissue was balanced within 1 hour with the tension of 0.75 g, which re-regulated p the least relaxation of the tissues. To reduce tissue and determine the response used a single dose of histamine: 10-5M, then 2-3×10-6M, with washing after each dose. Then for 5 minutes was applied antagonist TR-receptor SQ29548 at a concentration of 10-6M (Ogletree et al. (1985) J. Pharmacol. Exp. Ther. 234 435-441), then added 2-3×10-6M histamine to stimulate contractile response. After 30 min after pre-treatment with histamine analyzed the relaxation response by adding the cumulative doses of the investigated compounds with 10-8-10-7M PGE2at the end of each curve of dependence "dose - response" as a control. In the recovery period after washing the fabrics took 30-50 minutes. Relaxation activity was calculated as % of the control tone caused by histamine release.

(g) HUMAN PLATELETS

Prostanoid activity in relation to the DP, TP and IP receptors were initially identified by their ability to cause aggregation (TP-receptor activity) or inhibit ADP-induced aggregation of human platelets in vitro (DP and IP-receptor activity). Fresh whole blood was obtained from healthy donors, volunteers and was mixed with citrate-dextrose solution (ACD-a(citric) acid, (sodium) citrate, dextrose). The blood was centrifuged at a speed of 1000 Rev/min to obtain platelet-rich plasma (PRP). To 400 μl of PRP was added to 4.5 m is l prostanoid or filler, and incubated 2 min at 37° With aggregometry of Payton, watching aggregation activity. For a full aggregation of added ADP to a final concentration of 2×10-5M. Inhibition of aggregation was calculated as the percentage difference between aggregation caused 2×10-5M ADP in the absence and in the presence of a drug. Aggregation activity was calculated as the percentage of aggregation-induced prostanoid regarding aggregation caused 2×10-5M ADP. At the beginning and end of each experiment was determined by controlling the aggregation reaction only 2×10-5M ADP.

(C) CELLS CRL 1497: CA2+-The SIGNAL

Human cells CRL 1497 were seeded in culture flasks in modified, Dulbecco environment Needle (DMEM)containing 10% fetal calf serum, 2 mm 1-glutamine and 0.05 mg/ml gentamicin (all obtained from Gibco, Grand Island, NY). Cell cultures were maintained in a humid atmosphere (95% air, 5% CO2), and raised to obtain monolayer.

Cells were removed from culture flasks after approximately 1 min after treatment with a mixture of 0.05% trypsin/0.53 mm EDTA in a balanced salt Hanks solution (HBSS, Gibco, Grand Island, NY) at 37°C. Proteolytic activity inhibited by adding 5 ml of DMEM with 10% fetal calf serum. Cells are then washed with Hanks solution and the medium containing 140 mm NaCl, 50 mm is CL, 1 mm MgCl2, 1.5 mm CaCl2, 10 mm HEPES (N-2-hydroxyethylpiperazine-N-2-econsultancy acid): Tris (trihydroxystilbene), 5 mm glucose, 5 mm pyruvate, 0.1% of bovine serum albumin, pH 7.4.

Centrifugation between washes were carried out at room temperature for 15 minutes at 200 g. Cells were counted, resuspendable in the above medium and incubated with 2×10-6M mixture Fura 2 and acetoxymethyl ether to shake a water bath for 30 minutes at 37°C. the cells were washed in the above environment and resuspendable to a concentration of 2×106cells/ml and Then aliquots of 0.5 ml of cell suspension was added to the microtube with automatic covers for each experimental determination of the concentration of intracellular free CA2+([CA2+]i) accounted for 106cells.

Fluorescence was measured on a fluorescence spectrophotometer Perkin-Elmer LS-5 in the range of excitation wavelengths and emission from 340 to 492 nm, respectively, with both slits 10 nm. For each experimental determination of 106cells were washed (at 200 g for 5 min) and suspended in 2 ml cuvette with buffer containing 120 mm NaCl, 6 mm KCl, 1 mm MgSO41.5 mm l2, 20 mm HEPES, 1 mg/ml glucose and 1 mg/ml feast Vata Na. Mixing is carried out is through installed at the top blade mixer, at a temperature of 37°C. To obtain fmaxcells were literally digitonin (10 μl of 100 mg/ml DMSO). Then, to obtain fminconsistently added EGTA (etilenvinilatsetata acid) (100 mm) and 10 N NaOH to bring the pH to 8.5.

(I) RECOMBINANT FP-RECEPTOR CATS AND HUMANS; STABLE TRANSFECTANTS

Cells of SOME 293 were grown in DMEM with 10% serum fetal cow (FBS), 250 μg/ml G418 (Life Technologies) and 200 mg/ml gentamicin or penicillin/streptomycin. Selection of stable transfectants was performed using 200 μg/ml of hygromycin, and the optimal concentration was determined by examining the curves of cell death depending on the concentration of hygromycin.

Cells for transfection were grown on 10 cm plates up to 50-60% of the monolayer. Plasmid rser containing the cDNA insert for FP-receptor persons (20 μg)was added to 500 ál of 250 mm CaCl2. Then dropwise added HBSS × 2 (280 mm NaCl, 20 mm HEPES acid, 1.5 mm Na2HPO4pH 7,05-7,12) to 500 ál with constant stirring at room temperature. After 30 min, to the mixture was added 9 ml of DMEM. Then the cells previously washed with 10 ml fosfato-saline buffer (PBS), was added a mixture of DNA/PMEM/calcium phosphate.

Cells were incubated for 5 hours at 37°C in a humid atmosphere (95% air, 5% CO2). Then a solution of calcium phosphate was removed and the cells for 2 min about amityvale 10% solution of glycerol in DMEM. The glycerin solution was then changed to DMEM containing 10% serum fetal cow (FBS). Cells were incubated overnight and the medium was changed to DMEM/10% FBS containing 250 μg/ml G418 and penicillin/streptomycin. The next day was added hygromycin to a final concentration of 200 mg/ml

Ten days after transfection separated hygromycin-resistant clones and put them in a separate well on a 24-Loup night Board. After reaching monolayer each clone was transferred into one well of a 6-hole card, and then a 10 cm Cup. Cells were maintained in conditions of breeding hygromycin to use.

Stable transfectants containing FP-receptor cats were treated in a similar way, using lipofectamine. Cells were grown to 50-60% of the monolayer in 10 cm plates and with the help of lipofectamine was transfusional the plasmid rser, including insert cDNA coding FP-receptor cats. Selection using hygromycin was started 48 hours after transfection. Eight days after transfection resistant hygromycin In the clones was separated and transferred into 24-Loup nightly fee.

Research Sa2+-dependent signaling by recombinant FP-receptor cats and humans was similar to that described for cells CRL 1497.

(K) the FORMATION of INSISTSTHAT

Receptor-mediated hydrolysis of phosphoinositide (PI) was determined by the measurement of the accumulation of total insiststhat (IP) in the cells, pre-incubated with3H-myoinositol. Permanent cell lines SOME 293 expressing the FP receptor (human or cat, were seeded in 10 cm cups (106cells per plate in DMEM with 10% FBS). The next day cells were incubated with 18 µci myo-[23H]Inositol (Amersham; 10-20 µci/mmol) in 6 ml of DMEM with 10% FBS at 37°C for 24 hours. The cells are then washed twice with phosphate-saline buffer (PBS), incubated for 5 min with 1 ml trypsin-EDTA and suspended in 10 ml of DMEM containing 25 mm HEPES. Cells were besieged by centrifugation at 1000 rpm and resuspendable in DMEM/25 mm HEPES with 10 MMLiCI within 10 minutes an Aliquot of 200 µl of cell suspension were incubated with 50 µl of the drug for 30 min at 37°With (double definition), then with 750 μl of a mixture of chloroform-methanol-4 N. Hcl=100:200:2 stopped agonist stimulation, followed by adding 250 µl of chloroform and 0.5 N HCl for extraction of insiteful. 750 μl of the aqueous layer was applied to a column Packed with 0.5 ml uninominal resin Dowex AG 1-X8 (FORMATA form, 100-200 mesh, BioRad) for separating components from a radioactive label. The elution procedure consisted of three 3 ml washes with 5 mm Inositol, then two 750 μl of ELSI of 1.3 M ammonium formate with 0.1 M formic acid. The eluate was mixed with 10 ml scintillation fluid Aquasol (Packard Instrument Co.) and with the help of liquid is private scintillation counter was determined by the total concentration of 3N] insiteful.

(l) the binding of the LABELED LIGAND

Drugs plasma membrane of SOME cells 293 stably transfected with the FP-receptor, human or cat, was prepared as follows. Cells washed the TMA-buffer, scraped from the bottom of the vial and homogenized for 30 s using Brinkhman PT 10/35 of Polytron. If necessary, to bring the volume to 40 ml centrifuge tube was added TME buffer containing 100 mm Tris-base, 20 mm MgCl2, 2 mm EDTA; physiological pH value was obtained by adding 10 N. HCl.

The cell homogenate was centrifuged speed 19000 rpm for 4-6°C for 20 min on the rotor Ti-60 Beckman. The precipitate is then resuspendable in the TMA-buffer to obtain a final protein concentration of 1 mg/ml, as determined using the Biorad assay. Quantitative analysis of binding of radioactive ligands were performed in a volume of 200 µl.

Binding of [3N](N)17-phenyl-GF(specific activity 85 CI/mmol) was determined in duplicate and experiments were repeated three times. Incubation was carried out for 60 min at 25°C and stopped by adding 4 ml of ice-cold 50 mm Tris-Hcl buffer followed by rapid filtration through filters Whatman GF/B and three additional washes with 4 ml of cell manifold (Brandel). Conducted analyses of competitive binding using the final con is entrely 5 nm [ 3H](N)17-phenyl-GFand the nonspecific binding was determined using 10-5M unlabeled 17-phenyl-GF.

(m) INTRAOCULAR PRESSURE (IOP)

Studies of intraocular pressure in dogs involving pneumotonometry carried on in the minds of the breed of short-legged hound of both sexes (10-15 kg). Animals remained in the mind throughout the research and gently held his hand. Drugs were administered topically to one eye in the form of a drop with a volume of 25 μl, the other eye for the control was treated with 25 μl of filler (0.1% Polysorbate 80-10 mm Tris). For anesthesia of the cornea during tonometry used a 0.1%solution of proparacaine. Intraocular pressure was determined immediately before infusion and at 2, 4 and 6 hours after injection every day for 5 days of the study. The drug was administered immediately after registration of the first IOP values.

(h) the DIAMETER of the PUPIL

The diameter of the pupil of the dog was measured using optical rod (mm line containing a semicircular mark standard width (mm) as a control. Lightly holding the dog by hand, at normal room illumination was measured pupil diameter by selection of the semicircle to the pupil. For dogs with very dark pupils used a special spot flashlight, but t is like very quickly in order to avoid constriction of the pupil. Pupil diameter was measured at the same time that IOP and hyperemia.

(a) HYPEREMIA OCULAR SURFACE

Hyperemia ocular surface was determined visually and counted according to the system used in routine clinical practice.

Hyperemia ocular surface was evaluated simultaneously with the measurement of intraocular pressure. It should be noted that the raw eye dogs often have a pinkish-red hue. Thus, trace and even mild hyperemia not necessarily beyond the normal range.

RESULTS

Effects of 1-ethanolamide prostaglandin Fon the sphincter of the iris of a cat is depicted in figure 1 and presented in table format (table 1). 1 Ethanolamide prostaglandin F(1 ethanolamide GF) caused a dose-dependent reduction in smooth muscle sphincter of the iris of a cat. Was obtained, the magnitude of the effective concentration (EC)50equal to 58 nm. The data indicate a critical concentration in the region of 10 nm.

1 Ethanolamide prostaglandin Fwas moderately effective in the inhibition area irritation, caused the reduction of the isolated preparation of the seed vessel duct Guinea pigs. Data pokazny in figure 2 and presented in table format (table 2).

Figure 3 shows the effect of the s 1-ethanolamide GFon the isolated ileum of the chicken. Comparison of reaction with a control connection (prostaglandin E2, 10-6M) confirms that 1 ethanolamide PGFmay behave partly as a weak agonist. The data presented in Table 3. 1 Ethanolamide GFturned out to be less effective for ileum chicken (Figure 4, table 4), which, as reported, is a model for studying EP1-receptor.

Effects of increasing doses of 1-ethanolamide PGFon isolated rat aorta is shown in Figure 5 and Table 5. At high doses, 10-6and 10-5M, was observed residual activity. Because the aorta of the rat is a model for the study of TP-receptor activity against TR-receptor was further evaluated during platelet aggregation. In addition, we determined the inhibition of ADP, causing the aggregation of human platelets, in order to detect any interaction with DP or IP receptors. No effect on platelets was not observed. The data presented in Table 6; the graph is not included, as no response was.

CA2+the signal produced by increasing doses of 1-ethanolamide prostaglandin Fin fibroblasts of human skin (cells CRL 1497), is compared with the signal produced by prostaglandin F,figure 6. Data are also presented in table form (table 7). The obtained values of EC50was 16 nm for PGFand 1586 nm for 1-ethanolamide GFshowing almost a hundred-fold difference in activity relative to the FP-receptor human.

Recent studies have shown that prostaglandin Fmay cause endothelium-dependent, NO (nitric oxide)-mediated vasodilatation (Chen et al. (1995) Br. J. Pharmacol. 116: 3035-3041). Thus, the effects of 1-ethanolamide PGFcompared with the effect of PGFwhen the production of endothelium-dependent dilation of the vessels (vasorelaxation) pre-reduced under the action of histamine isolated segment of the jugular vein of a rabbit. While PGFhas produced a noticeable vasorelaxation, 1 ethanolamide PGFshowed relatively low activity (Fig.7). Data are also presented in table form (table 8). The values of EC50for the pre-reduced segment of the jugular vein of the rabbit: 1 ethanolamide PGF=2000 nm, PGF=5,3.

Since appreciable activity was observed for 1-ethanolamide GFin respect of the preparation of the sphincter of the iris of a cat, its activity was compared with the activity GFin respect of recombinant FP-receptor cats. Fig gives CPA is out activity 1-ethanolamide PGFand PGFduring stimulation of CA2+signal in the cells of SOME 293 stably transfected recombinant FP-receptor cats. Were the values obtained EC50equal to 14 nm for GFand 1458 1-ethanolamide PGFthat indicate almost a hundred-fold difference in activity. Data are also presented in table form (table 9).

Effects of 1-ethanolamide GFand 17-phenyl-GFthe total accumulation of insiststhat in the cells of SOME 293 stably transfected recombinant FP-receptor cats shown in Fig.9. Again there is a great difference in the activity. Values of EC50was 9 nm for 17-phenyl-PGFand 524 nm for 1-ethanolamide PGF. Data are also presented in tabular form (table 10).

In addition, they investigated the effects of 1-ethanolamide PGFand 17-phenyl-PGFthe total accumulation of insiststhat in the cells of SOME 293 stably transfected recombinant FP-receptor of human rights. Were the values obtained EC50equal to 8 nm for 17-phenyl PGFand 1003 nm. The data presented in Figure 10 and in Table 11. The results were similar to the results obtained for recombinant FP-receptor cats.

According to functional studies using stable transfetsirovannyh FP-receptor drugs, 1-PGFhad a relatively moderate affinity (chemical affinity) to the FP receptor. Thus, 1-ethanolamide PGF(known compound 1) had only a weak affinity for the receptor cats, while 17-phenyl - PGFshowed high affinity (11; table 12). the 50%inhibitory concentration (IC50for the obtained 1-ethanolamide PGFamounted to 882 nm, compared with the value IR50equal 43,5 to 17-phenyl-GF. Similar results were obtained for recombinant FP-receptor person: in the case of 1-ethanolamide GFIR50=2015 nm, 17-phenyl - GF-IR50=2,3 nm. These data on the binding of labeled ligand is depicted in Fig and presented in the form of a table (table 13).

Eye step 1-ethanolamide GFstudied on dogs. 1 Ethanolamide PGF0.1%dose caused a marked and highly significant reduction of intraocular pressure (Fig; table 14). The effect was maintained throughout the 24-hour experimental period. 1 Ethanolamide PGFalso caused a marked constriction of the pupil (miosis) (Fig; table 15). In contrast to ocular hypotensive effect, reducing the diameter of the pupil eliminated by 24 h time point. 1 Ethanolamide PGFcaused slight redness of the eye surface, comparable with trace tlanepantla, throughout the 24-hour experimental period (Fig; table 16).

In the following Tables, unless otherwise indicated, the concentration of the investigational or control joints are shown as logarithmic exponent. In line with this and as an example, "-8" in the tables means a concentration of 10-8M investigational or control connection.

Table 1
The concentration of 1-ethanolamide prostaglandin F, log [M]
-8-7-6-5
% The average value of the maximum contractile response χ±SEM2,7±1,777±5,5119,2±6,0126,0±3,6

TABLE 1. Effect of increasing doses of 1-ethanolamide PGFon isolated smooth muscle sphincter of the iris of a cat. Control reduction was created using the 10-7M PGF. Given the average values ± SEM; n=4.

Table 2
The concentration of 1-ethanolamide prostaglandin F, log [M]
-8 -7-6-5
% The mean value of the maximum inhibition of abbreviations χ±SEM2,2±1,312,5±6,237,7±8,498,5±1,0

TABLE 2. The inhibitory effect of increasing doses of 1-ethanolamide PGFon the area stimulated contraction of isolated vessel seminal duct Guinea pigs.

Table 3
The concentration of 1-ethanolamide prostaglandin F, log [M]
-8-7-6-5
% The average value of the maximum contractile response χ±SEM0±015,0±8,450,7±4,066,5±9,4

TABLE 3. Effect of increasing doses of 1-ethanolamide PGFon the isolated ileum of the chicken. Control reduction was created using the 10-6M PGE2. Given the average values ± SEM; n=4.

Table 4
The concentration of 1-ethanolamide prostaglandin F, log [M]
-8-7-6-5
% Average value of the maximum response χ±SEM0±00±01,7±1,015,8±1,6

TABLE 4. Effect of increasing doses of 1-ethanolamide PGFon the isolated ileum of the Guinea pig. Control reduction was created using the 10-6M PGE2. Given the average values ± SEM; n=4.

Table 5
The concentration of 1-ethanolamide prostaglandin F, log [M]
-8-7-6-5
% The average value of the maximum contractile response χ±S0±00±04,0±2,69,7±4,2

TABLE 5. Effect of increasing doses of 1-ethanolamide PGFon isolated rat aorta. Control reduction was created using the 10-7M U-46619. Given the average values ± SEM; n=4.

Table 6
The concentration of 1-ethanolamide prostaglandin F, log [M]
-7-6-5
% Platelet aggregation0±00±00±0
% Inhibition of platelet aggregation0±00±00±0

TABLE 6. Effect of increasing doses of 1-ethanolamide prostaglandin Fon platelet aggregation and inhibition of aggregation induced by ADP. The control response is called with 2×10-5M ADP. Given the average values ± SEM; n=4.

Table 7
The concentration of 1-ethanolamide prostaglandin F, log [M]
-9-8-7-6-5
The average value of the Δ[CA2+]iχ±SEM-3,4±1,513,5±3,945,8±11,597,9±10,8
The concentration of prostaglandin F, log [M]
-9-8-7-6-5
The average value of the Δ[Ca2+]iχ±SEM5,± 2,447,7±9,4of 124.1±6,5127,4±7,6136,7±11,2

TABLE 7. The increase in intracellular concentration of free Ca2+[Ca2+];caused by increasing doses of 1-ethanolamide prostaglandin Fand prostaglandin Fin CRL 1497 skin fibroblasts.

Table 8
The concentration of 1-ethanolamide prostaglandin F, log [M]
-10-9-8-7-6-5
% average relaxation χ±SEM100±099,7±0,393,2±2,280,3±3,254,3±10,238,4± and 11.3
The concentration of prostaglandin F, log [M]
-11-10-9-8-7-6
% average relaxation χ±S98±1,192,5±2,371,9±7,225,8±7,88,3±3,06,7±2,7

TABLE 8. Vasorelaxation p is evritania reduced segment of the jugular vein of a rabbit under the action of 1-ethanolamide prostaglandin Fand prostaglandin F. Control expansion vessels provide with 10-7M PGE2. Given the average values ± SEM; n=6 for 1-ethanolamide PGFn=7 for GF.

Table 9
The concentration of 1-ethanolamide prostaglandin F; log [M]
-10-9-8-7-6-5
The average value of the Δ[CA2+]iχ±SEM--0±028,5±3,145,2±6,477,2±14,7
The concentration of prostaglandin F, log [M]
-10-9-8-7-6-5
The average value of the Δ[Ca2+]iχ±SEM6,5±5,022,2±6,445,7±5,378,8±7,387,9±9,085,0±9,6

TABLE 9. The increase in intracellular concentration of free CA2+[CA2+]icaused by increasing doses of 1-ethanolamide prostaglandin Fsub> 2αand prostaglandin Fin SOME cells-293 stably transfected recombinant FP-receptor cats. Given the average values ± SEM; n=3.

Table 10
The concentration of 1-ethanolamide prostaglandin F, log [M]
-9-8-7-6-5
% The mean value of the maximum education of insiststhat χ±SEM-3,4±3,214,9±4,763,0±16,489,2±4,8
The concentration of 17-fenprostalene F, log [M]
-9-8-7-6-5
% The mean value of the maximum education of insiststhat χ±SEM17,8±6,45B,8±10,889,7±2,499,9±1,1-

TABLE 10. The total accumulation of insiststhat caused increasing doses 1-ethanolamide prostaglandin Fand 17-fenprostalene Fin SOME cells-293 stably transfected recombinant FP-receptor Ko is Ki. Control irritation create using the 10-6M 17-phenyl-GF. Given the average values ± SEM. n=3 for 1-ethanolamide GFand 4 for 17-phenyl-GFin experiments performed with two replicates.

Table 11
The concentration of 1-ethanolamide prostaglandin F, log [M]
-9-8-7-6-5
% The mean value of the maximum accumulation insiststhat χ±SEM-0,3±0,610,0±1,648,4±3,385,0±1,9
The concentration of 17-fenprostalene F, log [M]
-9-8-7-6-5
% The mean value of the maximum accumulation insiststhat χ±SEM14,7±3,357,8±2,092,3±0,9100,0±1,3-

TABLE 11. The total accumulation of insiststhat caused increasing doses 1-ethanolamide prostaglandin F17-fenprostalene Fin the cells of SOME 293, hundred the ilen transfected with the recombinant FP-receptor of human rights. Control irritation create using the 10-6M 17-phenyl-GF. Given the average values ± SEM. n=3 for 1-ethanolamide PGFand 4 for 17-phenyl-PGFin the experiments, repeated twice.

Table 12
The concentration of 1-ethanolamide prostaglandin F, log [M]
-9-8-7-6-5
% The average value of specific binding χ±SEM89,6±3,293,7±3,889,7± and 4.948,0±2,98,3±4,8
The concentration of 17-fenprostalene F, log [M]
-10-9-8-7-6-5
% The average value of specific binding χ±SEMof 92.7±6,980,7±7,143,7±1,517,0±6,510,3±8,90±0

TABLE 12. Study competitive binding of labeled ligand (1-ethanolamide GF17-Anil-GFagainst 5 nm3H-17-phenyl-GFwith recombinant FP-receptor cats. Given the average values ± SEM; n=3 experiments, repeated twice.

Table 13
The concentration of 1-ethanolamide prostaglandin F, log [M]
-9-8-7-6-5
% The average value of specific binding χ±SEM94,7±1,4103,3±0,395,9±1,563,7±2,719,7±1,2
The concentration of 17-fenprostalene F, log [M]
-10-9-8-7-6-5
% The average value of specific binding χ±SEM98±1,165,0±1,024,0±1,25,3±1,51,7±1,70 0

TABLE 13. Study competitive binding of labeled ligand (1-ethanolamide PGFand 17-phenyl - PGFagainst 5 nm3H-17-phenyl-PGFwith recombinant FP-receptor of human rights. Given the average values ± SEM; n=3 experiments performed twice.

Table 14
Time measurement of intraocular pressure (hour)
024624
The average intraocular pressure (mm RT. Art.) χ±SEMThe tested eye19,1±0,916,2±

1,1**
14,6±

0,9**
14,0±

0,9**
15,2±

0,7**
Control eyes20,3±0,720,4±1,020,0±1,020,8±0,620,0±0,9

TABLE 14. The effect of 0.1%solution of 1-ethanolamide prostaglandin Fon the intraocular pressure of the dog. Given the average values ± SEM; n=6. **p<0,01 significant deviation compared to the baseline intraocular pressure according to t-student distribution.

Table 15


The time measurement of the pupil diameter (h)


024b24
Pupil diameter (mm)The tested eye6,7±0,21,0±0,4**1,0± 0,3**1,5± 0,4**7,0±0,3
Control eyes6,3±0,26,3±0,26,3±0,26,3±0,26,3±0,2

TABLE 15. The effect of 0.1%solution of 1-ethanolamide prostaglandin Fthe diameter of the pupil of the dog. Given the average values ± SEM; n=6. **p<0,1 significant deviation compared with the base diameter of the pupil according to the t-student distribution.

Table 16
The measurement time hyperemia ocular surface (hour)
024624
Figure hyperemia ocular surfaceThe test is th eyes 0,3±0,11,3±0,21,2±0,21,2±0,21,1±0,2
Control eyes0,2±0,10,5±00,5±00,5±00,4±0

TABLE 16. The effect of 0.1%solution of 1-ethanolamide prostaglandin Fthe hyperemia of the palpebral surface of the dog. Given the average values ± SEM; n=6.

PHARMACEUTICAL COMPOSITIONS, METHODS of USE

The pharmaceutical compositions can be prepared by combining a therapeutically effective amount of at least one compound according to the present invention or its pharmaceutically acceptable salt as an active ingredient with a standard acceptable in ophthalmology, pharmaceutical excipients, and by preparation of standard dosage forms suitable for local ophthalmic use. Therapeutically effective amount is usually from about 0.0001 to 5% (wt./vol.), preferably, from about 0.001 to 1.0 percent (weight/about.) for liquid dosage forms.

For ophthalmic applications it is preferable to prepare the solutions, using as a basic filler physiological saline solution. It is preferable to maintain the pH of such OFTAL the lithological solutions in the range between 6.5 and 7.2 using a suitable buffer system, most preferred is a neutral pH value. Medicines can also contain standard, pharmaceutically acceptable preservatives, stabilizers and surfactants.

Preferred preservatives that can be used in the pharmaceutical compositions of the present invention include, but are not limited to list, benzalkonium chloride, chlorobutanol, thimerosal (sodium salt utilitarianismlol acid), retaliatation and retinirovannyh. Preferred surface-active substance is, for example, Tween 80 (monooleate polyoxyethylene(20)sorbitan). It is preferred to use in ophthalmic preparations of the present invention, various fillers. These fillers include, but are not limited to list, polyvinyl alcohol, povidone (polyvinylpyrrolidone), hypromellose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purified water.

If necessary or for convenience can be added sliders for tone. They include, but are not limited to list, salts, particularly sodium chloride, potassium chloride, mannitol and glycerin, or any other suitable ophthalmologist acceptable tone regulator.

Can be used in various buffers and means for adjusting the pH when the conditions is, the obtained product is ophthalmologist acceptable. Accordingly, the buffer composition include acetate buffers, citrate buffers, phosphate and borate buffers. To regulate the pH of such medicinal products, if necessary, can be used in acid or base.

Similarly, ophthalmologist acceptable antioxidant for use in the present invention includes, but is not limited to the list, sodium metabisulfite, sodium thiosulfate, acetylcysteine, bottled hydroxyanisol and bottled hydroxytrol.

Other fillers that may be included in ophthalmic preparations are chelating agents. The preferred chelating agent is the disodium salt of ethylenediaminetetraacetic acid, although other chelating agents may also be used instead or in conjunction with it.

Components are typically used in the following quantities:

Component amount (% weight/vol.)

the active ingredient about 0.001 to 5

preservative 0-0,10

filler 0-40

the tone regulator 0-10

the buffer of 0.01-10

the pH regulator, a number of which bring the pH to

4,5-7,5

the antioxidant, if necessary

surfactant, if necessary

purified water, if necessary, to bring the volume to 100%

Factual is the first dose of the active compounds of the present invention depends on the specific compound and the conditions of administration; the choice of an appropriate dose is successfully carried out within the knowledge of a qualified professional.

Ophthalmic drug of the present invention is conveniently packaged in a form suitable for metered application, for example, in containers fitted with a dropper for easy introduction into the eye. Containers suitable for drip application, usually made of a suitable inert non-toxic plastic material and generally contain from 0.5 to about 15 ml.

Solutions, especially not contain preservatives, often prepared in containers for disposable use, containing up to about ten, preferably, approximately five standard doses, where the usual standard dose contains from one to about 8 drops, preferably from one to about 3 drops. The volume of one drop is usually about 20 to 35 mm.

DETAILED description of the SYNTHESIS METHODS

Example 1.

GF(free carboxylic acid)

To a mixed solution of 20 g tromethamine salt PGF(tromethamine-Tris(hydroxymethyl)aminomethan) (51,9 mmol) in 150 ml of N2Oh was added concentrated Hcl so that the pH of the solution was brought to 2. The solution was extracted with 3×200 ml of chloroform and concentrated in vacuum. The free carboxylic acid PGFpaaul is stayed, mainly in the form of sediment, nasloivshihsya between aqueous and organic phases. The precipitate from this layer was collected and combined with the precipitate obtained by filtration of the concentrated chloroform solution, after which the combined precipitates were washed with a 50% mixture of chloroform/methanol.

Methyl ether PGF; methyl-(5Z,8α,9α,11α,13E,15S)-9,11,15-trihydroxypregn-5,13-Dien-1-oat

To a stirred solution of the free carboxylic acid PGF(2,528 g, 7,13 mmol) in 50 ml of acetone was added 3.2 ml of 1,8-diazabicyclo[5.4.0]undec-7-ene (21,39 mmol) and of 1.33 ml of methyl iodide (21,39 mmol). The solution was stirred over night, diluted with 150 ml ethyl acetate, washed 2×50 ml of 0.5 m LiOH, 1×50 ml of brine and concentrated in vacuo to obtain the specified connection, pure methyl ester (purity checked by NMR1N).

1H NMR (DCl3): 5.56mm-of 5.34 (m, 4H), to 4.01 (m, 1H), a 3.87 (m, 1H), 3,63 (s, 1H), 3,18 (t, 2H, J=5.8 Hz), 2,31-2,02 (m, 8H), 1,69-1,25 (m, N), 0,849 (t, 3H, J=6,7 Hz).

1 Ethanolamide PGF; (5Z,8α,9α,11α,13E,15S)-9,11,15-trihydroxy-N-(2-hydroxy-ethyl)simple-5,13-Dien-1-amide

To a stirred solution of methyl ether PGF(235,4 mg, 0,639 mmol) in 4.5 ml of anhydrous methanol was added 771 μl of ethanolamine (12,78 mmol). The tube containing the mixture was sealed and during the night was heated at 50°C. Otbi the Ali of the sample, concentrated in vacuum by heating to remove excess ethanolamine and analyzed by NMR1H. it Was shown that the reaction was completed by 80%. Solution to remove residual ethanolamine was concentrated in high vacuum at 65°C for 1.5 hours, then at 60°during the night using distillation Kugehlrohr'a.1H NMR and TLC in 5% methanol/ethyl acetate showed the destruction of ethanolamine (methyl ester: Rf=0,41, 1 ethanolamide: Rf=0,04). Technical product was subjected to flash chromatography with a mixture of 5% methanol/ethyl acetate to extract the remaining unreacted educt, methyl ester, in fractions 4-8, and a 50% mixture of methanol/ethyl acetate to obtain the desired specified connection in fractions 11-18. Additional flash chromatography fractions 11-18 mixture of 15% methanol/ethyl acetate gave the specified connection that does not contain the original substance, but still contaminated by epimer 15 in some fractions (fractions 7-10), and the desired product in pure form (fractions 11-20). Spectra1H NMR and13With NMR specified compound and its 15-epimer identical. Theoretical yield = 254,0 mg actually received output 205,6 mg, 81%.

1H NMR (CD3OD): 5,4-5,2 (m, 4H), 3,98 (m, 1H), with 3.89 (m, 1H), and 3.72 (m, 1H), 3,47 (t, 2H, J=5.8 Hz), 3,18 (t, 2H, J=5.8 Hz), 2,24-1,90 (m, 8H), 1,50-to 1.21 (m, N), coefficient was 0.796 (t, 3H, J=6.5 Hz).

1. 1-Ethanolamin the GFformula:

or its pharmaceutically acceptable salt, a reducing intraocular pressure in mammals exhibiting ocular hypotensive activity not through FP prostaglandin receptor.

2. 1 Ethanolamide PGFaccording to claim 1, characterized in that it is a dedicated mostly clean chemical substance.

3. Pharmaceutical composition effective to reduce intraocular pressure in a mammal in need of such treatment a composition comprising as an active ingredient an effective amount of 1-ethanolamide PGFformula:

or its pharmaceutically acceptable salt, a reducing intraocular pressure in mammals, but also showing ocular hypotensive activity not through FP prostaglandin receptor, and pharmaceutically acceptable filler.

4. The pharmaceutical composition according to claim 3, suitable for local injection into the eye of a mammal.



 

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< / BR>
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< / BR>
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< / BR>
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< / BR>
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< / BR>
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< / BR>
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< / BR>
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2 ex, 3 tbl

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1 ex, 1 tbl

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