JPH0150706B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel dibenzo-P-dioxin derivatives. The dibenzo-P-dioxin derivative of the present invention is a novel compound that has not been described in any literature and is represented by the following general formula (1). [In the formula, R represents a hydrogen atom or a lower alkanoyl group. ] The present inventors have discovered the seaweed Ecklonia kurome.
We have been conducting intensive research on the extracts of The extract contains plasmin inhibitor (plasmin
The present inventors have recognized the existence of a compound that has an inhibitory effect on the phenotypic inhibitor, and have succeeded in extracting and isolating the compound, thereby completing the present invention. In this specification, examples of lower alkanoyl groups include formyl, acetyl, propionyl,
Examples include butyryl, isobutyryl, valeryl, isovaleryl, hexanoyl, and the like. The compound of the present invention represented by the above general formula (1) is:
Alpha-2 plasmin inhibitor (α ₂ -plasmin inhibitor) is the main plasmin inhibitor in the blood.
It has the physiological activity of strongly inhibiting the activity of α 2 -macroglobulin (α ₂ -macroglobulin) and α 2 -macroglobulin. Various biological reactions such as blood coagulation and fibrinolytic phenomena (fibrinolysis) are mediated by various proteolytic enzymes, but the functions of these proteases are controlled by inhibitory protein proteins present in the body. There is. Among these inhibitor proteins, the above-mentioned plasmin inhibitor has a strong inhibitory effect on plasmin related to the fibrinolytic system, and is known to act as an inhibitor of the fibrinolytic system. Furthermore, it is known that the above-mentioned plasmin inhibitors strongly inhibit the generated plasmin in the attempt to increase fibrinolysis by activating (producing) plasmin by administering urokinase or streptokinase, which are currently used as thrombolytic agents. There is. Therefore, there is a strong desire in this field for the development of drugs that can increase fibrinolysis by inhibiting the action of these plasmin inhibitors in the blood [Nobuo Aoki et al.: Protease inhibitors in vivo; Metabolism, Vol. 14] Vol. No. 6, pp. 1099-1111 (1977), Tamotsu Matsuda: Hypercoagulable state; Small molecule dextran urokinase literature collection, pp. 1-15, edited and published by Otsuka Pharmaceutical Co., Ltd., published on January 10, 1977. reference〕. The compound of the present invention represented by the above general formula (1) is:
As is clear from the pharmacological test results described below, it has strong anti-plasmin inhibitor activity and is therefore useful as a prophylactic and therapeutic agent for thrombosis due to increased fibrinolysis, and also as an adjunct to conventional thrombosis treatment agents. is also useful. The compound of the present invention is produced, for example, according to the method shown below. Among the compounds of the present invention, the compound represented by the following formula (1a) is extracted and isolated from, for example, the seaweed Kurome in the following manner. That is, first, the seaweed Kurome is extracted using a common polar solvent such as methanol, ethanol, isopropanol, their hydrous alcohols, and ethyl acetate, and this extract is concentrated under reduced pressure to obtain a primary extract. The method for collecting the compound of general formula (1a) from the primary extract is not particularly limited, and any of various known methods utilizing physical and chemical properties can be employed. For example, the difference in solubility with impurities, common adsorbents, such as activated carbon, XAD
-2, it can be carried out by a method that utilizes the difference in adsorption affinity for silica gel, ion exchange resin, Cephadex, etc., the difference in the distribution ratio between two liquid phases, etc., or by a combination of these methods. More specifically, the above-mentioned first extract is extracted using a solvent such as ethyl acetate, chloroform, or ether by a solvent distribution method, and then this extract is concentrated under reduced pressure, and then subjected to Celite column chromatography and Cephadex LH-20.
The compound of formula (1a) can be obtained by subjecting to column chromatography or the like and eluting with a suitable solvent such as ethyl ether, acetone, methanol or the like. Further, among the compounds of the present invention, compounds other than the compound represented by the above formula (1a) [i.e., the compound of the following formula (1b)] are produced from the compound of formula (1a) according to the method shown in Reaction Scheme-1. Reaction formula-1 [In the formula, R′ represents a lower alkanoyl group. ] For the acylation of the compound of formula (1a), a wide range of reaction conditions for ordinary acylation reactions can be employed. A wide variety of conventionally known acylating agents can be used, including lower alkanoic acids such as acetic acid and propionic acid, lower alkanoic anhydrides such as acetic anhydride, and lower alkanoic acid halides such as acetyl chloride and propionyl bromide. be able to. The amount of such acylating agent to be used is not particularly limited and can be appropriately selected within a wide range, but it is usually at least an equimolar amount per hydroxyl group of the compound of formula (1a),
Preferably, it is used in an equimolar to 10-fold molar amount.
When using an acid anhydride or a halide as an acylating agent, the acylation reaction is preferably carried out in the presence of a basic compound. Specific examples of the basic compound include alkali metals such as sodium metal and potassium metal, hydroxides, carbonates, and bicarbonates of these alkali metals, and aromatic amine compounds such as pyridine and piperidine. Further, when a lower alkanoic acid is used as the acylating agent, the acylation reaction is preferably carried out in the presence of a dehydrating agent. Specific examples of the dehydrating agent include mineral acids such as sulfuric acid and hydrochloric acid, and sulfonic acids such as para-toluenesulfonic acid, benzenesulfonic acid and ethanesulfonic acid. The acylation reaction is carried out either without a solvent or in a solvent. Examples of solvents include ketones such as acetone and methyl ethyl ketone;
Ethers such as ether and dioxane, aromatic hydrocarbons such as benzene, toluene, and xylene,
Water, pyridine, etc. can be mentioned. The reaction proceeds either under cooling, at room temperature, or under heating, but it is usually preferable to carry out the reaction at 0 to 150°C.
In particular, when an acid anhydride or a halide is used as the acylating agent, the reaction is preferably carried out at 0 to 80°C, and when a lower alkanoic acid is used as the acylating agent, the reaction is preferably carried out at 50 to 120°C. It is preferable to do so. The above reaction is generally completed in about 0.5 to 24 hours, thus producing the compound of formula (1b). Among the compounds of general formula (1) thus obtained, compounds having an acidic group include, for example, sodium hydroxide,
It can easily form salts by reacting with strong basic compounds such as potassium hydroxide and calcium hydroxide, and the present invention also includes salts of such dibenzo-P-dioxin derivatives. The target compound of the present invention thus produced can be easily isolated and purified by commonly known separation means.
Examples of the separation means include solvent distillation, solvent extraction, precipitation, recrystallization, column chromatography,
Examples include preparative thin layer chromatography. The compound of general formula (1) and its salts are usually used in the form of common pharmaceutical preparations. The formulation contains commonly used fillers, extenders, binders, wetting agents, disintegrants,
It is prepared using diluents or excipients such as surfactants and lubricants. Various forms of this pharmaceutical preparation can be selected depending on the therapeutic purpose, and representative examples include tablets, pills, powders, liquids, suspensions,
Examples include emulsions, granules, capsules, suppositories, injections (solutions, suspensions, etc.). When forming tablets, a wide variety of carriers conventionally known in this field can be used, such as lactose, sucrose,
Sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, excipients such as kegaic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, Binders such as potassium phosphate and polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, etc. Disintegrants, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, absorption enhancers such as quaternary ammonium bases, sodium lauryl sulfate, humectants such as glycerin, starch, starch, lactose, kaolin, bentonite. , adsorbents such as colloidal silicic acid, purified talc, stearate, boric acid powder, and lubricants such as polyethylene glycol. In addition, tablets may be coated with a normal coating if necessary.
For example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets can be used. When forming into a pill form, a wide variety of carriers conventionally known in this field can be used, such as glucose, lactose, starch,
Examples include excipients such as cocoa butter, hydrogenated vegetable oil, kaolin, and talc, binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol, and disintegrants such as laminalan agar. When forming into a suppository, a wide variety of conventionally known carriers can be used, such as polyethylene glycol,
Examples include cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. When prepared as injections, solutions and suspensions are preferably sterile and isotonic with blood, and when formed into solutions, emulsions, and suspensions, diluents are used. All that is customary in this field can be used, such as water, ethyl alcohol,
Examples include propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt to prepare an isotonic solution,
Glucose or glycerin may be included in the drug, and conventional solubilizing agents, buffering agents, soothing agents, etc. may also be added. Furthermore, colorants, preservatives, perfumes, flavoring agents, sweeteners, and other pharmaceuticals may be included in the drug, if necessary. The salt of the compound of general formula (1) to be contained in the drug of the present invention is not particularly limited and can be selected from a wide range, but is usually 0.1 to 70% by weight of the total composition, preferably
It is 0.5-30% by weight. There are no particular restrictions on the method of administering the drug of the present invention;
Various formulation forms, patient age, gender and other conditions,
It is administered in a manner that depends on the severity of the disease. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. In the case of injections, they are administered intravenously alone or mixed with regular fluids such as glucose and amino acids.
Furthermore, it may be administered alone intramuscularly, intradermally, subcutaneously, or intraperitoneally as necessary. Suppositories are administered rectally. The dosage of the drug of the present invention is appropriately selected depending on the usage, patient's age, sex and other conditions, degree of disease, etc., but the amount of the compound of general formula (1), which is the active ingredient, is usually 1 kg of body weight per day. The amount is preferably about 0.05 to 100 mg per serving. The pharmacological test results are shown below. [Pharmacological test-1] Alpha-2 macroglobulin prepared from human plasma by the method of H. Rinderknecht et al. [Biochem.Med., 14 , 162 (1975)] was used as a plasmin inhibitor. Alpha-2 macroglobulin 17μg 0.1M
10 of the test compound at various concentrations in 0.3 ml of 0.05 M Tris-HCl buffer (PH = 7.4) containing sodium chloride.
Mix with 0.1 ml of % methanol aqueous solution and incubate at 20°C under 37°C.
Hold for minutes. Next, 0.1 ml of 10 μg/ml trypsin (manufactured by Sigma Chemical Co., Ltd., Type) was added to the above mixture, and this was maintained at 37° C. for 2 minutes. 0.5 ml of the above buffer containing 2% protamine sulfate (Sigma Chemical Co., Ltd., Grade X) was added, and the mixture was left to stand for an additional 30 minutes.
The reaction was stopped by adding 3 ml of 18% trichloroacetic acid aqueous solution, left to stand for 1 hour, centrifuged, 50 μg of supernatant was taken into a test tube, and 4 ml of 1.5N sodium hydroxide aqueous solution of 0.01% 8-hydroxyquinoline and 0.1% 8-hydroxyquinoline were added to the test tube.
% aqueous solution of bromosuccinimide was added and stirred to develop a color, and the absorbance at 500 nm was measured.
Plasmin inhibitor activity inhibition rate (%) was calculated using the following formula. Inhibition rate (%) = C-B/A-B x 100 A: Absorbance when not containing alpha-2 macroglobulin and test compound B: Containing alpha-2 macroglobulin but not containing test compound Absorbance C: Absorbance when Alpha-2 macroglobulin and the test compound are included Table 1 shows the results of determining the concentration of the test compound at which the inhibition rate determined above is 50% (50% inhibition concentration) Shown below. Test compound No. 1 Compound obtained in Example 1 below

[Pharmacology test-2]

Alpha-2 as a plasmin inhibitor
A plasmin inhibitor was used. Alpha-2 plasmin inhibitor was obtained from human plasma using the method of B.Wiman et al. [Eur.J.
Biochem., 78 , 19 (1977)], and human plasmin was prepared from human plasma using the method of DGDoutsch et al. [Science, 170 , 1095].
(1970)] was prepared by the method of M. Moroi et al. [J. Biol. Chem., 251 ,
5956 (1976)] was activated with urokinase-conjugated sepharose and used for testing. 0.06M Tris-HCl buffer (PH=7.4) containing 0.09M chloride reaction containing 3μg/ml of alpha-2 plasmin inhibitor
To 0.0 ml, 0.1 ml of test compounds at various concentrations dissolved in 10% methanol was added and held at 37°C for 20 minutes.
Then, 0.1 ml of a 0.5 casein unit/ml human plasmin solution dissolved in 0.1 M sodium phosphate buffer (PH = 7.4) containing 25% glycerin was added, and the mixture was incubated at 37°C.
After heating for 30 seconds, S- at a concentration of 3mM was added as a substrate.
2251 aqueous solution (HD-Val-L-Leu-L-Lys
-p-nitroanilide (Daiichi Kagaku) 0.1 ml was added and reacted for an additional 3 minutes. The reaction was stopped by adding 0.1 ml of 50% aqueous acetic acid. 405nm of reaction solution
The absorbance was measured and the inhibition rate (%) of plasmin inhibitor activity was calculated using the following formula. Inhibition rate (%) = C-B/A-B x 100 A: Absorbance when alpha-2/plasmin inhibitor and test compound are not included B: Alpha-2/plasmin inhibitor alone without test compound Absorbance C when containing: Absorbance when alpha-2 plasmin inhibitor and test compound are included Alpha-2 of the test compound determined by the above method
2. Inhibition rate against plasmin inhibitor is 50
% concentration (50% inhibition concentration) is shown in Table 2 below.

[Table] Examples and formulation examples are listed below. Example 1 (1) Fresh Kurome (collected in Irino, Kochi Prefecture) 600Kg
was extracted with methanol at room temperature. The extract was concentrated under reduced pressure to obtain a gummy primary extract.
This was extracted repeatedly with ethyl acetate-water (1:1 v/v) until the upper layer became colorless. The obtained upper layer was concentrated under reduced pressure to obtain 5.7 kg of a second extract. (2) 1.7 kg of the secondary extract obtained above was mixed with 3.4 kg of Celite (manufactured by Johns Manvills) and dried under reduced pressure. The obtained solid was finely ground, packed into a glass column, and eluted sequentially with benzene (18), methylene chloride (36), and ethyl ether (54), and then with methanol. 552g eluted with ethyl ether was added to Cephadex LH.
-20 (3.5Kg) column chromatography and eluted with acetone (15) to obtain fractions of 2000ml each. Fraction No. 6 was concentrated under reduced pressure to obtain 150 g of a residue. Add this to Safedex LH
-20 (3.5Kg) column chromatography and eluted with methanol (20) to obtain fractions of 3000ml each. The crude crystals obtained by concentrating Fraction No. 6 under reduced pressure were recrystallized from water to obtain 1-(3,5-dihydroxyphenoxy)-6,7-[2,4 -dihydroxy-5
-(3,5-dihydroxyphenoxy)-benzo[b)furan]-dibenzo-p-dioxin-
2 g of 2,4,9-triol was obtained. λ ^M 〓 ^OH _nax : 224, 292, 244, 275, 304, 317nm IR (ν _KBr/nax ): 3350, 3300, 1605, 1470, 1360,
1290, 1260, 1190, 1140, 1130, 1080,
1055, 1035, 1005, 815cm ^-1 PMR (200MHz, DMSO−d ₆ , ppm): 5.74 (2H,
d, J=2.1), 5.79 (2H, d, J=2.1), 5.85
(2H, t, J=2.1) 6.31 (1H, s), 6.44
(1H, s), 6.73 (1H, s), 8.19 (1H, s),
9.17 (2H, s), 9.20 (2H, s), 9.43 (1H,
s), 9.84 (1H, s), 9.87 (1H, s), 10.14
(1H, s) CMR (100MHz, DMSO-d ₆ , ppm): 93.7,
93.9, 94.9, 96.5, 96.9, 98.5, 99.2, 103.4,
103.5, 120.4, 122.7, 122.8, 126.5, 134.0,
136.9, 142.0, 144.8, 146.5, 147.0, 149.6,
150.4, 150.9, 158.9, 159.0, 160.0, 160.3 Example 2 60 mg of the compound obtained in Example 1 was added to pyridine.
After reacting a mixture of 0.5 ml and 0.2 ml of acetic anhydride at room temperature for 3 hours, the reaction solution was added to ice water, and 1-(3,5-diacetyloxyphenoxy) having the following structural formula was prepared separately.
-6,7-[2,4-diacetyloxy-5-
50 mg of (3,5-diacetyloxyphenoxy)-benzo[b]furan]-2,4,9-triacetyloxy-dibenzo-p-dioxin was obtained. IR (ν ^KBr/max ): 1760, 1595, 1490, 1445, 1360,
1260, 1185, 1110, 1060, 1015, 880cm ^-1 Formulation Example 1 Sodium salt of the compound obtained in Example 1
500 mg Glucose 250 mg Distilled water for injection Appropriate amount Total volume 5 ml Dissolve the sodium salt of the compound obtained in Example 1 and glucose in distilled water for injection, and then inject into a 5 ml ampoule. After purging with nitrogen, autoclave sterilization at 121°C for 15 minutes to obtain an injection. Formulation Example 2 Compound obtained in Example 2 150.0g Citric acid 1.0g Lactose 33.5g Dicalcium phosphate 70.0g Pluronic F-65 (Pluronic F-68 30.0g Sodium lauryl sulfate 15.0g Polyvinylpyrrolidone 15.0g Polyethylene glycol (Carbowax)
1500) 4.5g polyethylene glycol (Carbowax
6000) 45.0g Cornstarch 30.0g Dry sodium lauryl sulfate 3.0g Dry magnesium stearate 3.0g Ethanol Appropriate amount of the compound obtained in Example 2, citric acid, lactose, dicalcium phosphate, Puron F-68 and sodium lauryl sulfate. Mix. The above mixture is sieved through a No. 60 screen and wet granulated with an alcoholic solution consisting of polyvinylpyrrolidone, Carbowax 1500 and 6000. Add alcohol if necessary to make the powder into a pasty mass. Add cornstarch and continue mixing until uniform particles are formed. Pass it through a No. 10 screen, put it in a tray and put it in an oven at 100℃ for 12~
Dry for 14 hours. The dry particles are sieved through a No. 16 screen, dried sodium lauryl sulfate and dried magnesium stearate are added and mixed, and compressed into the desired shape using a tablet machine. The core is treated with varnish and sprinkled with talc to prevent moisture absorption. A subbing layer is applied around the core. Apply varnish enough times for internal use. Further subbing layers and smooth coatings are applied to make the tablet perfectly round and smooth. Pigmented coatings are applied until the desired shade is obtained. After drying, the coated tablets are polished to a uniform gloss.

Claims (1)

[Claims] 1. General formula [In the formula, R represents a hydrogen atom or a lower alkanoyl group. ] A dibenzo-P-dioxin derivative and a salt thereof.