JPH0149154B2 - - 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. R ¹ is a hydrogen atom or group

[Formula] (R is the same as above) is shown. R ² is the group

[Formula] (R is the same as above) or the same group as R. However, R ¹
When represents a hydrogen atom, R ² is a group

[Formula] and R ¹ is a group

In the case of [Formula], R ² represents the same group as R. ] The present inventors have discovered the seaweed Ecklonia Kurome (Ecklonia Kurome).
Okamura) has been intensively researched on the extract. The present inventors recognized the presence of a compound in the extract that has an inhibitory effect on plasmin inhibitors, and 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 physiological activity that strongly inhibits the activity of α 2 -macroglobulin (α ₂ -macroglobulin) and α 2 -macroglobulin (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 to develop a drug that can increase fibrinolysis by blocking 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 inhibitory activity and is therefore useful as a prophylactic and therapeutic agent for thrombosis due to increased fibrinolysis, as well as an adjunct to conventional thrombosis treatment agents. It is also useful as 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. [In the formula, R ¹ ′ is a hydrogen atom or a group

[Formula] is shown. R ² ′ is a hydrogen atom or group

[Formula] is shown. However, when R ¹ ′ represents a hydrogen atom, R ² ′ represents a group.

[Formula] and R ¹ ' is a group

In the case of [Formula], R ² ' represents a hydrogen atom. ] 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)] can be produced from the compound of formula (1a) according to the method shown in Reaction Scheme-1. . Reaction equation 1 [In the formula, R ¹ ′ and R ² ′ are the same as above. R ¹ ″ is a hydrogen atom or group

[Formula] (R' is a lower alkanoyl group). R ² ″ is a hydrogen atom or group

[Formula] (R' is the same as above) is shown. However, if R ¹ ″ represents a hydrogen atom,
R ² ″ is the base

[Formula] and R ¹ ″ is a group

[Pharmacology 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 bovine trypsin (manufactured by Sigma Chemical Co., Ltd., Type) was added to the above mixture, and this was kept at 37° C. for 2 minutes. 0.5 ml of the above buffer containing 2% protamine sulfate (Sigma Chemical Co., Ltd., Grade) 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 for 1 hour, centrifuged, 50μ of supernatant was taken into a test tube, and 0.01% 8-hydroxyquinoline in 1.5N sodium hydroxide aqueous solution 4 ml, 0.1
% 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 alpha 2 macroglobulin and the test compound are not included B: Absorbance when the test compound is not included and alpha 2 macroglobulin is included Absorbance C: Absorbance when alpha 2 macroglobulin and 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). Test compound No. 1 1-(3,5-dihydroxyphenoxy)-
2-(2,4,6-trihydroxyphenoxy)
-4,7,9-trihydroxy-dibenzo-p
-Dioxine No.2 1-{3,5-dihydroxy-4-[1-
(3,5-dihydroxyphenoxy)-2,4,
9-trihydroxy-dibenzo-p-dioxin-7-yl]oxy}phenoxy-2,4,
7,9-tetrahydroxy-dibenzo-p-dioxin

[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. Alpha 2 plasmin inhibitor 3μg
Contains 0.06M Tris containing 0.09M Sodium Chloride
Add 0.1 ml of test compounds at various concentrations dissolved in 10% methanol to 0.7 ml of hydrochloric acid buffer (PH = 7.4),
It was kept at 37°C for 20 minutes. Next, 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 after heating at 37°C for 30 seconds, S was added as a substrate at a concentration of 3 mM. -2251 aqueous solution (H-D-
0.1 ml of Val-L-Leu-L-Lys-p-nitroanilide (Daiichi Kagaku) was added thereto, and the mixture was further reacted at 37°C for 3 minutes.
The reaction was stopped by adding 0.1 ml of 50% aqueous acetic acid. The absorbance of the reaction solution at 405 nm 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: When alpha-2/plasmin inhibitor alone is included without the test compound Absorbance C: Absorbance when alpha-2/plasmin inhibitor and test compound are included The inhibition rate of the test compound against alpha-2/plasmin inhibitor determined by the above method 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, and 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 and packed into a glass column, and benzene (18), methylene chloride (36), and ethyl ether (54)
After sequential elution with methanol. Sephadex 552g eluted with ethyl ether
It was subjected to LH-20 (3.5Kg) column chromatography and eluted with acetone (15) to obtain fractions of 2000ml each. Fraction No. 5 was concentrated under reduced pressure, and the resulting crude crystals were recrystallized from hot water to give 1-
(3,5-dihydroxyphenoxy)-2-(2,
4,6-trihydroxyphenoxy)-4,7,
5 g of 9-trihydroxy-dibenzo-p-dioxin was obtained. Melting point 206-207℃, colorless plate-like crystal λ ^MeOH _nax : 232 (ε, 40000), 292 (ε, 3400) nm IR (ν ^KBr _nax ): 3250, 1615, 1480, 1375, 1260,
1200, 1148, 1120, 1085, 1060, 1005,
815cm ^-1 EIMS (m/z): 496 (M ⁺ ) 478, 372, 354, 338,
264, 232, 126, 69 High-resolution mass spectrum Actual value 496.0646 Theoretical value 496.0642 (as C ₂₄ H ₁₆ O ₁₂ ) PMR (200MHs, DMSO−d ₆ , ppm): 5.80 (1H,
d, J=2.6), 5.83 (1H, s) 5.85 (1H,
t, J=2.1) 5.85 (2H, s) 5.88 (2H,
d, J=2.1) 5.98 (1H, d, J=2.6) 8.97
(1H, s) 9.07 (2H, s) 9.13 (2H, s)
9.18 (1H, s) 9.40 (1H, s) 9.51 (1H,
s) CMR (100MHz, DMSO-d ₆ , ppm): 93.9,
94.2, 94.9, 96.2, 96.4, 98.7, 122.6,
122.7, 122.9, 124.9, 137.3, 141.5,
142.0, 146.0, 147.6, 151.0, 153.0,
154.7, 158.6, 160.4 Example 2 Fraction No. 6 obtained in Example 1(2) was concentrated under reduced pressure to obtain 150 g of a residue. This was subjected to Sephadex LH-20 (3.5 kg) column chromatography and eluted with methanol (20) to obtain fractions of 3000 ml each. The crude crystals obtained by concentrating fraction No. 5 under reduced pressure were recrystallized from water to obtain 1-
{3,5-dihydroxy-4-[1-(3,5-dihydroxyphenoxy)-2,4,9-trihydroxy-dibenzo-p-dioxin-7-yl]
10 g of phenoxy-2,4,7,9-tetrahydroxy-dibenzo-p-dioxin was obtained. Melting point 300℃ or higher, colorless needle crystals λ ^MeOH _nax : 235 (ε, 34000), 292 (ε, 3500) nm IR (ν ^KBr _nax ): 3250, 1605, 1480, 1360, 1260,
1190, 1140, 1110, 1080, 1040, 1010,
800cm ^-1 MS spectrum FDMS (m/z): 742 (M ⁺ , C ₃₆ H ₂₂ O ₁₈ ), 726 EIMS (m/z): 264, 248, 232, 142, 126, 69,
44, 28 PMR (200MHs, DMSO-d ₆ , ppm): 5.73 (2H,
d, J=2.1), 5.80 (1H, d, J=2.9),
5.82 (1H, t, J = 2.1), 5.83 (1H, d,
J=2.6), 5.96 (2H, s), 5.99 (1H, d,
J=2.9), 6.03 (1H, d, J=2.6), 6.14
(1H, s), 6.17 (1H, s), 9.13 (2H,
s), 9.18 (1H, s), 9.20 (1H, s), 9.25
(1H, s), 9.33 (2H, s), 9.42 (1H,
s), 9.47 (1H, s), 9.57 (1H, s), 9.67
(1H, s) CMR (100MHz, DMSO-d ₆ , ppm): 93.7,
93.9, 94.0, 94.8, 96.4, 98.5, 98.6, 98.7,
122.6, 122.7, 122.8, 123.4, 123.5,
124.3, 124.6, 131.7, 137.3, 141.8,
141.9, 142.5, 142.7, 146.0, 151.0,
153.0, 154.3, 155.9, 158.7, 160.3 Example 3 1-(3,5-dihydroxyphenoxy)-2-(2,4,6-trihydroxyphenoxy)-4 obtained in Example 1, 7,9-trihydroxy-
Dibenzo-p-dioxin 200mg, pyridine 2ml
After reacting a mixture of 1 ml of acetic anhydride and 1 ml of acetic anhydride at room temperature for 20 hours,
The reaction solution was added to ice water, and the resulting precipitate was separated and recrystallized from ethanol to give 1-(3,5-diacetyloxyphenoxy)-2-(2,4,6-triacetyloxyphenoxy). c)-4,7,9-triacetyloxy-dibenzo-p-dioxin 285
I got mg. Melting point 186-188℃, colorless plate crystal IR (ν ^KBr _nax ): 3075, 2925, 1760, 1600, 1485,
1455, 1365, 1170, 1115, 1105, 1074,
1040, 1010, 885, 830, 690, 650, 575,
545, 510cm ^-1 EIMS (m/z): 832 (M ⁺ -42), 790, 748, 706,
664, 622, 580, 538, 496, 370, 43 Example 4 1-{3,5-dihydroxy- obtained in Example 2
4-[1-(3,5-dihydroxyphenoxy)-
2,4,9-trihydroxy-dibenzo-p-dioxin-7-yl]oxy}phenoxy-2,
4,7,9-tetrahydroxy-dibenzo-p-
From dioxin, 1-{3,
5-Diacetyloxy-4-[1-(3,5-diacetyloxyphenoxy)-2,4,9-triacetyloxy-dibenzo-p-dioxin-7-
yl]oxy}phenoxy-2,4,7,9-tetraacetyloxy-dibenzo-p-dioxin was obtained. Colorless amorphous IR (ν ^KBr _nax ): 3075, 1760, 1630, 1600, 1490,
1465, 1440, 1365, 1280, 1185, 1115,
1095, 1075, 1040, 1020, 965, 880, 840
cm ^-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 amount 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-68 30.0g (Pluronic F-68) Sodium lauryl sulfate 15.0g Polyvinylpyrrolidone 15.0g Polyethylene glycol 4.5 g (Carbowax 1500) Polyethylene glycol 45.0g (Carbowax 6000) Cornstarch 30.0g Dry sodium lauryl sulfate 3.0g Dry magnesium stearate 3.0g Ethanol Appropriate amount Compound obtained in Example 2, citric acid, lactose, dicalcium phosphate , Purone F-68 and sodium lauryl sulfate. 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, mixed with dry sodium lauryl sulfate and dry magnesium stearate, 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. R ¹ represents a hydrogen atom or a group [Formula] (R is the same as above). R ² represents the group [Formula] (R is the same as above) or the same group as R. However, when R ¹ represents a hydrogen atom, R ² represents a group [formula], and when R ¹ represents a group [formula], R ² represents the same group as R. ] A dibenzo-p-dioxin derivative represented by these and its salt.