JPH0525110A - New compound and proteolytic enzyme inhibitor containing the same compound as active ingredient - Google Patents

Abstract

PURPOSE:To obtain a new allyltetraline type lignaneamide compound useful as a treating agent for pancreatitis because of excellent proteolytic enzyme inhibiting action thereof. CONSTITUTION:A lignaneamide expressed by the formula (R is H, acetyl or 1-6C alkyl). The compound can be isolated from CANNABIS SEMEN (fruit of Cannabis sativa L . belonging to Moraceae) of a gelenical drug through (i) extraction by water, methanol, acetone, ethyl acetate and/or methylethyl ketone, (ii) partitioning extraction using a low-polar solvent, (iii) partitioning extraction using ethyl acetate, butanol and/or methylethyl ketone and (iv) chromatography and (v) recrystalization or powdering and, as necessary, further subjected to methylation, reduction or acetylation. Furthermore, the compound can be administered as an oral agent or parenteral agent without special limitation administration form. Dose of the above-mentioned compound for exhibiting its expected effects as the oral agent is normally 10mg to 1g per day for adult and preferably administered by dividing several portions.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel lignanamide and a protease inhibitor containing the lignanamide as an active ingredient.

[0002]

2. Description of the Related Art In recent years, with the advent of protease inhibitors, considerable progress has been made in therapeutic agents for pancreatitis by stopping the acute phase of pancreatitis or preventing the acute phase of pancreatitis.

However, at present, the severe form of acute pancreatitis is determined to be alive or dead within a few days, and it has been desired to develop an excellent protease inhibitor.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found a novel allyltetralin-type lignanamide compound contained in asparagus kernels, and When the pharmacological action was examined, it was found that the desired protease was contained, and based on this, the present invention was completed.

That is, the present invention provides the following formula I: (In the formula, R are the same or different and each represents a hydrogen atom, an acetyl group or an alkyl group having 1 to 6 carbon atoms.) (Hereinafter referred to as the compound of the present invention) and the compound as an active ingredient. And a protease inhibitor.

The compound of the present invention can be obtained, for example, as follows.

Asago Jin (Cannabis sativa L. dried fruits)
Water, methanol, ethanol, acetone, ethyl acetate, using a solvent alone or more selected from methyl ethyl ketone, to extract by heating from 0 ° C. to a temperature below the boiling point used, or from 0 ° C. to room temperature Ultrasonic extraction is performed to obtain an extract.

This extract was suspended in water, distributed and extracted using a low-polarity solvent such as benzene, chloroform, ether, hexane, cyclohexane, etc., and the remaining water-soluble portion from which low-polarity impurities were removed was converted to acetic acid. Partition extraction is performed using a single solvent or a mixed solvent thereof selected from ethyl, butanol, and methyl ethyl ketone to obtain an extract.

The extract obtained here is directly or dried and subjected to column chromatography using a carrier such as silica gel, ODS-silica gel, porous polymer gel, Sephadex, etc., and the eluate is fractionated to obtain a fraction. obtain.

As an elution solvent used in column chromatography, water, methanol, ethanol, acetone, ethyl acetate, tetrahydrofuran (THF), acetonitrile, benzene, ether, chloroform, petroleum ether, hexane, cyclohexane, etc., may be used alone or more. The mixed solvent of can be used.

The fraction thus obtained is treated with water or methanol, ethanol, acetone, ethyl acetate, TH.
It can be obtained by recrystallization or pulverization with a mixed solvent of F, acetonitrile, benzene, ether, chloroform, petroleum ether, hexane and the like alone or in combination.

Some of the compounds of this invention may be optionally methylated with a conventional methylating agent such as methyl iodide and potassium carbonate in acetone.
By conventional reduction methods, for example catalytic reduction with palladium-carbon saturated with hydrogen in methylene chloride, ethyl acetate and THF, or by acetylation with acetic anhydride in common acetylating agents, for example pyridine. Obtainable.

The compound of the present invention can also be obtained by the method shown below.

That is, caffeoyl tyramine and feruloyl tyramine are dissolved in an organic solvent such as acetone,
Add an aqueous ferric chloride solution and stir at room temperature for 3 to 12 hours. After completion of the reaction, an organic solvent is added to the reaction solution for partition and extraction. It can be obtained by washing the obtained organic solvent with water, drying it with a desiccant such as calcium chloride, magnesium sulfate and sodium sulfate, and then distilling the solvent off under reduced pressure.

Examples of the organic solvent used for partition extraction include ether, ethyl acetate, chloroform, butanol and the like.

If necessary, the obtained reaction product is subjected to silica gel column chromatography, medium pressure silica gel column chromatography, ODS-silica gel column chromatography, porous polymer gel, Sephadex LH.
-20, and can be obtained by recrystallization or powdering.

As an elution solvent for purification, use of a single solvent or a mixture of water, methanol, ethanol, acetone, ethyl acetate, THF, acetonitrile, benzene, ether, chloroform, petroleum ether, hexane and the like. You can

As the solvent used for recrystallization or pulverization, it is possible to use water, methanol, ethanol, acetone, ethyl acetate, benzene, ether, chloroform, petroleum ether, hexane or the like alone or as a mixed solvent thereof. it can.

Next, the fact that the compound of the present invention has excellent proteolytic enzyme inhibitory activity and is useful as a therapeutic agent for pancreatitis will be described with reference to experimental examples.

Experimental Example 100 μl of each enzyme solution [plasmin (human plasma, manufactured by Boehringer), thrombin (human plasma, manufactured by Boehringer), trypsin (bovine pancreas, manufactured by Boehringer)] in a test tube, Tris-HCl buffer ( pH 8.3) 50 μl (final concentration 33.3 mM), water (plasmin and thrombin) or 20 mM calcium chloride solution (trypsin) 25 μl, sample solution 25 μl (compound of the present invention) were taken and incubated at 37 ° C. for 5 minutes.

Next, 50 μl (15 mM) of TAME (p-toluenesulfonyl-L-arginine methyl ester) was added, and the mixture was reacted at 37 ° C. for 30 minutes, and 500 μl of an equal volume mixture of 3.5N sodium hydroxide and 3.5N hydroxylamine. The reaction was stopped by adding the solution, allowed to stand at room temperature for 25 minutes, and then the hesterin method [Reference: S.
Sterin, J. Biol. Chem. 18, 249 (1949)] was used to measure the activity.

That is, 250 μl of 6% trichloroacetic acid and 2.0 ml of 0.2 M ferric chloride solution were added for color development, the absorbance at 525 nm was measured, and the inhibition rate (%) was calculated according to the following formula II.

The control group was prepared by removing the sample solution in the same manner as described above.

Formula II

In the formula II, A is the absorbance of non-enzyme, B is the absorbance of the control group, and C is the absorbance of the sample.

50% inhibition concentration (IC ₅₀ , mg / ml) was determined from the inhibition rate at various concentrations, and the results are shown in Table 1.

Table 1

Next, the dose and formulation of the compound of the present invention will be explained.

The compound of the present invention can be administered to animals and humans as it is or together with a conventional pharmaceutical carrier. The dosage form is not particularly limited and may be appropriately selected and used as needed, and examples thereof include oral preparations such as tablets, capsules, granules, fine granules and powders, parenteral preparations such as injections and suppositories. To be

In order to exert a desired effect as an oral preparation, it varies depending on the age, body weight and degree of disease of the patient, but usually the adult compound weight of the compound of the present invention is 10 mg to 1 g several times a day. It seems appropriate to take them separately.

In the present invention, oral preparations such as tablets, capsules, granules and the like are produced by a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like.

In this type of preparation, a binder, a disintegrating agent, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a perfume, etc. may be appropriately used in addition to the above-mentioned excipients. You can
Specific examples of each are as shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose,
Ethyl cellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

[Surfactant] Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 8
0.

[Lubricant] Talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

[Flowability Accelerator] Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The compounds of the present invention can also be administered as suspensions, emulsions, syrups and elixirs, and these various dosage forms may contain flavoring agents and coloring agents. .

In order to exert the desired effect as a parenteral agent, it depends on the age, weight and degree of disease of the patient.
0.1 mg to 10 mg daily as weight of the compound of the present invention in an adult
Intravenous injection, intravenous drip infusion, subcutaneous injection, and intramuscular injection are considered appropriate.

This parenteral preparation is manufactured by a conventional method, and is generally used as a diluent in distilled water for injection, physiological saline, glucose aqueous solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol. Etc. can be used. Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. Further, from the viewpoint of stability, this parenteral preparation may be filled in a vial or the like and then frozen, the water content may be removed by a usual freeze-drying technique, and a liquid preparation may be re-prepared from the freeze-dried product immediately before use. Further, an isotonicity agent, a stabilizer, a preservative, a soothing agent and the like may be added as needed.

Other parenteral agents include external preparations, coating agents such as ointments, and suppositories for rectal administration.
It is manufactured according to a conventional method.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 10 kg of Asahi kernel was extracted with 8 liters of water-ethanol (1: 1) under reflux for 3 hours under heating for 3 times.

The obtained extracts were combined and the solvent was distilled off under reduced pressure to obtain 3.44 kg of dried extract.

This extract was suspended in 20 liters of water and chloroform 2
Partition extraction was performed 3 times with 0 l. Remaining water layer 4 with 20l butanol
The butanol extract was obtained by partitioning and extraction. The solvent of butanol extract was distilled off under reduced pressure to obtain 239.1 g of dried extract.

The dried extract was diluted with 3 l of water, subjected to a porous polymer gel (Diaion HP-20, 2 l, manufactured by Mitsubishi Kasei) column chromatography, and eluted successively with 40 l of water and 50 l of methanol.

The solvent in the methanol eluate was distilled off under reduced pressure to obtain 171.7 g of dried extract. This extract was added to silica gel (K
ieselgel 60, 70-230 mesh, Merck) column chromatography (10 cm φ × 46 cm), chloroform
-Methanol (9: 1) 10 l, chloroform-methanol (4: 1) 1
Elution was performed sequentially with a mixed solvent of 0 L and chloroform-methanol (1: 1) 10 L.

The fraction eluted with a mixed solvent of chloroform-methanol (1: 1) was subjected to preparative liquid chromatography (YMC, R-354, ODS, 5 cmφ × 50 cm, manufactured by Yamamura Chemical Co., Ltd.), and water-methanol ( By repeatedly separating and purifying with a mixed solvent of 1: 1), 8.12 g of a slightly yellow powdery substance was obtained.

Since this compound has the physicochemical properties shown below, 6,7-dihydroxy-1- (3,4-dihydroxyphenyl) -N ¹ , N ² -bis- [2- (4- (Hydroxyphenyl)
Ethyl-2,3- (1,2-dihydronaphthalene) dicarboxamide (6,7-dihydroxy-1- (3,4-dihydroxyphenyl) -N ¹ , N ² -bis
-〔2- (4-hydroxyphenyl) ethyl-2,3- (1,2-dihydronaphth
alene) dicarboxamide}.

Mass spectrum (FAB-MS): C ₃₄ H ₃₃ N ₂ O ₈ [M + H]
⁺ Theoretical value: 597.2237 Actual value: 597.2234 Infrared absorption spectrum (IR, ν max cm ^-1 , KBr): 3364,16
56,1612,1514 UV absorption spectrum [λ max nm (log ε), MeOH]: 25
0 (4.32), 277 (4.00), 337 (4.02) Proton nuclear magnetic resonance spectrum (δ ppm in MeOH-
d ₄ ): 2.46 (2H, t, J = 7Hz), 2.68 (2H, t, J = 7Hz), 3.22 (2H, ddt,
J = 14,7,6Hz), 3.36 (2H, dt, J = 7,6Hz), 3.66 (1H, d, J = 4Hz),
4.29 (1H, d, J = 4Hz), 6.42 (1H, dd, J = 8,2Hz), 6.48 (1H, d, J = 2
Hz), 6.51 (1H, s), 6.64 (1H, d, J = 8Hz), 6.66 (2H, d, J = 8Hz),
6.69 (2H, d, J = 8Hz), 6.77 (1H, s), 6.81 (2H, d, J = 8Hz), 6.97
(2H, d, J = 8Hz), 7.15 (1H, s) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in MeOH-d ₄ ): 35.
5 (t), 35.6 (t), 42.5 (t), 42.7 (t), 47.3 (d), 51.2 (d), 116.2
(d), 116.2 (d), 116.2 (d × 2), 116.2 (d × 2), 116.8 (d), 117.
2 (d), 120.3 (d), 125.0 (s), 127.3 (s), 130.7 (d × 2), 130.8
(d × 2), 131.2 (s), 131.3 (s), 131.4 (s), 134.8 (d), 136.3
(s), 144.9 (s), 145.4 (s), 146.0 (s), 148.4 (s), 156.7 (s), 1
56.7 (s), 170.5 (s), 174.7 (s)

Example 2 The fraction eluted with the mixed solvent of silica gel column chromatography of chloroform-methanol (4: 1) obtained in Example 1 was used for preparative liquid chromatography [YMC, R-3.
54, ODS, 5 cmφ × 50 cm, manufactured by Yamamura Chemical Co., Ltd.), and repeatedly separated and purified with a mixed solvent of water-methanol-acetonitrile (7: 2: 2) to obtain 2.67 g of a slightly yellow powdery substance. .

Since this compound has the physicochemical properties shown below, 6-methoxy-7-hydroxy-1- (3,4-dihydroxyphenyl) -N ¹ , N ² -bis- [2- ( 4-hydroxyphenyl) ethyl-2,3- (1,2-dihydronaphthalene) dicarboxamide {6-methoxy-7-hydroxy-1- (3,4-dihydroxypheny
l) -N ¹ , N ² -bis- 〔2- (4-hydroxyphenyl) ethyl-2,3- (1,2-d
ihydronaphthalene) dicarboxamide}.

Mass spectrum (FAB-MS): C ₃₅ H ₃₅ N ₂ O ₈ [M + H]
⁺ Theoretical value: 611.2393 Actual value: 611.2391 Infrared absorption spectrum (IR, ν max cm ^-1 , KBr): 3324,16
44,1614,1514 UV absorption spectrum [λ max nm (log ε), MeOH]: 24
8 (4.38), 286 (4.13), 328 (4.17) Proton nuclear magnetic resonance spectrum (δ ppm in MeOH-
d ₄ ): 2.47 (2H, dd, J = 8,7Hz), 2.69 (2H, t, J = 7Hz), 3.22 (2H, d
dt, J = 14,7,6Hz), 3.37 (2H, ddt, J = 14,7,6Hz), 3.67 (1H, d, J
= 4Hz), 3.87 (3H, s), 4.31 (1H, d, J = 4Hz), 6.41 (1H, dd, J = 8,2
Hz), 6.47 (1H, d, J = 2Hz), 6.53 (1H, s), 6.64 (1H, d, J = 8Hz),
6.65 (2H, d, J = 8Hz), 6.69 (2H, d, J = 8Hz), 6.81 (2H, d, J = 8H
z), 6.85 (1H, s), 6.97 (2H, d, J = 8Hz), 7.19 (1H, s) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in MeOH-d ₄ ): 35.
5 (t), 35.6 (t), 42.4 (t), 42.7 (t), 47.3 (d), 51.0 (d), 56.6
(q), 113.3 (d), 116.1 (d), 116.1 (d), 116.2 (d × 2), 116.3 (d
× 2), 117.3 (d), 120.2 (d), 124.9 (s), 127.5 (s), 130.7 (d ×
2), 130.8 (d × 2), 131.2 (s), 131.4 (s), 132.7 (s), 134.7
(d), 136.0 (s), 145.0 (s), 146.1 (s), 148.1 (s), 149.6 (s), 1
56.7 (s), 156.8 (s), 170.4 (s), 174.5 (s)

Example 3 Fractions eluted with a mixed solvent of chloroform-methanol (9: 1) for silica gel column chromatography in Example 1 were subjected to medium pressure silica gel column chromatography (CIG column, 22 mmφ × 30 cm, Kusano Science Co., Ltd.). The product was recrystallized from methanol to give 598 mg of colorless needle crystals. Since this compound has the physicochemical properties shown below, 6-methoxy-7-hydroxy-1- (3
-Methoxy-4-hydroxyphenyl) -N ¹ , N ² -bis- 〔2- (4-
Hydroxyphenyl) ethyl-2,3- (1,2-dihydronaphthalene) dicarboxamide {6-methoxy-7-hydroxy-1- (3-met
hoxy-4-hydroxyphenyl) -N ¹ , N ² -bis- 〔2- (4-hydroxyphen
yl) ethyl-2,3- (1,2-dihydronaphthalene) dicarboxamid
e} was decided.

[0054] mp: 165 to 168 ° C Mass spectrum _{(FAB-MS): C 36} H 37 N 2 O 8 [M + H] + theory: 625.2550 Found: 625.2554 Infrared absorption spectrum (IR, ν max cm ^{- 1} , KBr): 3340,16
60,1606,1514 UV absorption spectrum [λ max nm (log ε), MeOH]: 24
6 (4.36), 285 (4.04), 336 (4.09) Proton nuclear magnetic resonance spectrum (δ ppm in MeOH-
d ₄ ): 2.48 (2H, dt, J = 8,2Hz), 2.70 (2H, t, J = 7Hz), 3.23 (2H, d
dt, J = 14,7,6Hz), 3.36 (1H, t, J = 7Hz), 3.40 (1H, t, J = 7Hz),
3.68 (1H, d, J = 4Hz), 3.75 (3H, s), 3.89 (3H, s), 4.35 (1H, d, J
= 4Hz), 6.41 (1H, dd, J = 8,2Hz), 6.53 (1H, s), 6.64 (1H, d, J = 8
Hz), 6.65 (2H, d, J = 8Hz), 6.68 (2H, d, J = 8Hz), 6.69 (1H, d, J =
2Hz), 6.82 (2H, d, J = 8Hz), 6.87 (1H, s), 6.97 (2H, d, J = 8Hz),
7.20 (1H, s) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in MeOH-d ₄ ): 35.
4 (t), 35.6 (t), 42.4 (t), 42.7 (t), 47.6 (d), 51.0 (d), 56.3
(q), 56.6 (q), 112.5 (d), 113.2 (d), 116.0 (d), 116.2 (d ×
2), 116.2 (d × 2), 117.2 (d), 121.4 (d), 124.9 (s), 127.6
(s), 130.7 (d × 2), 130.8 (d × 2), 131.1 (s), 131.4 (s), 132.
6 (s), 134.6 (d), 135.9 (s), 146.2 (s), 148.1 (s), 148.8 (s),
149.6 (s), 156.7 (s), 156.8 (s), 170.4 (s), 174.5 (s)

Example 4 360 mg of the compound obtained in Example 1 was dissolved in 10 ml of dry acetone, 1 mg of potassium carbonate was added, and 2 ml of methyl iodide was gradually added with a dropping funnel while heating at 60 ° C. and stirred overnight. .

After the completion of the reaction, the reaction solution was cooled to room temperature,
The mixture was left standing in 0 ml, and partitioned and extracted with 100 ml of chloroform × 2. The chloroform layer was washed with water and dried over calcium chloride, and then the solvent was distilled off under reduced pressure. CIG column (22mmφ × 30c
m), and the fraction separated with benzene-acetone (7: 3) was recrystallized from methanol to obtain 130 mg of colorless needle crystals.

Since this compound has the physicochemical properties shown below, 6,7 dimethoxy-1- (3,4-dimethoxyphenyl) -N ¹ , N ² -bis- [2- (4-methoxy Phenyl) ethyl-
2,3- (1,2-dihydronaphthalene) dicarboxamide (6,7-d
imethoxy-1- (3,4-dimethoxyphenyl) -N ¹ ,, N ² -bis- 〔2- (4-
methoxyphenyl) ethyl-2,3- (1,2-dihydronaphthalene) di
carboxamide} was decided.

Melting point: 196-197 ° C. Mass spectrum (FD-MS) m / z: 681 [M + H] ⁺ Elemental analysis: C ₄₀ H ₄₄ N ₂ O ₈ Theoretical value; C: 70.57, H: 6.51, N: 4.12 Measured value; C: 70.34, H: 6.49, N: 4.10 Infrared absorption spectrum (IR, ν max cm ^-1 , KBr): 3260,16
40,1608,1512 UV absorption spectrum [λ max nm (log ε), MeOH]: 24
7 (4.46), 284 (4.08), 331 (4.12) proton nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ):
2.58 (1H, dt, J = 14,7Hz), 2.65 (1H, dt, J = 14,7Hz), 2.71 (2H,
t, J = 7Hz), 3.34 (2H, ddt, J = 14,7,6Hz), 3.45 (2H, ddd, J = 14,
7,6Hz), 3.57 (1H, d, J = 2Hz), 3.74 (3H, s), 3.74 (3H, s), 3.76
(3H, s), 3.78 (3H, s), 3.80 (3H, s), 3.87 (3H, s), 4.66 (1H, d,
J = 2Hz), 6.17 (1H, t, J = 6Hz), 6.34 (1H, dd, J = 8,2Hz), 6.57 (1
H, d, J = 2Hz), 6.63 (1H, d, J = 8Hz), 6.65 (1H, s), 6.70 (1H, s),
6.74 (2H, d, J = 8Hz), 6.78 (2H, d, J = 8Hz), 6.93 (2H, d, J = 8H
z), 6.99 (2H, d, J = 8Hz), 7.02 (1H, s), 7.19 (1H, t, J = 6Hz) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ): 34.6
(t), 34.6 (t), 41.0 (t), 41.3 (t), 44.6 (d), 49.6 (d), 55.1
(q), 55.2 (q), 55.6 (q), 55.6 (q), 55.6 (q), 56.0 (q), 111.0
(d), 111.1 (d), 111.3 (d), 112.1 (d), 113.8 (d × 2), 114.0 (d
× 2), 119.6 (d), 123.6 (s), 126.6 (s), 129.5 (d × 2), 129.6
(d × 2), 130.6 (s), 130.7 (s), 130.9 (s), 131.9 (d), 135.4
(s), 147.7 (s), 148.0 (s), 148.6 (s), 150.8 (s), 158.0 (s), 1
58.3 (s), 168.5 (s), 171.4 (s)

Example 5 282 mg of the compound obtained in Example 2 was dissolved in 10 ml of dry acetone, 1 mg of potassium carbonate was added, and the mixture was heated at 60 ° C. and stirred overnight while gradually adding 1 ml of methyl iodide with a dropping funnel. did.

After the completion of the reaction, the reaction solution was cooled to room temperature,
The mixture was allowed to stand in 0 ml and partitioned and extracted with chloroform (100 ml × 2).
After washing the chloroform layer with water and drying over magnesium sulfate,
The solvent was distilled off under reduced pressure. CIG column (22 mmφ ×
(30 cm), and the fraction separated with benzene-acetone (7: 3) was recrystallized from methanol to obtain 130 mg of colorless needle crystals.

This compound had the same physicochemical properties as the compound obtained in Example 4.

Example 5 122 mg of the compound obtained in Example 2 was dissolved in 10 ml of dry acetone, 1 mg of potassium carbonate was added, and 1 ml of methyl iodide was gradually added with a dropping funnel while heating at 60 ° C. overnight. It was stirred.

After the completion of the reaction, the reaction solution was cooled to room temperature,
The mixture was allowed to stand in 0 ml and partitioned and extracted with chloroform (100 ml × 2).
After washing the chloroform layer with water and drying over magnesium sulfate,
The solvent was distilled off under reduced pressure. CIG column (22 mmφ ×
(30 cm) and the fraction separated with benzene-acetone (7: 3) was recrystallized from methanol to obtain 48 mg of colorless needle crystals.

The physicochemical properties of this compound were the same as those of the compound obtained in Example 4.

Example 6 200 mg of the compound obtained in Example 3 was dissolved in 20 ml of THF. 5 in advance
The above sample solution was added to 5 ml of a THF solution containing 37 mg of% palladium-carbon on a hydrogen saturated reaction vessel, and the mixture was vigorously stirred overnight at room temperature.

After completion of the reaction, palladium-carbon was removed by filtration with a filter paper, and the solvent of the obtained filtrate was distilled off under reduced pressure.
A reaction product was obtained. The reaction product was applied to a CIG column (22 mmφ × 30 cm), and the fraction separated with benzene-ethyl acetate (7: 3) was recrystallized from methanol to obtain 160 mg of colorless needle crystals.

Since this compound has the physicochemical properties shown below, 6,7 dimethoxy-1- (3,4-dimethoxyphenyl) -N ¹ , N ² -bis- [2- (4-methoxy Phenyl) ethyl-
2,3- (1,2,3,4-tetrahydronaphthalene) dicarboxamide (6,7-dimethoxy-1- (3,4-dimethoxyphenyl) -N ¹ , N ² -bis
-(2- (4-methoxyphenyl) ethyl-2,3- (1,2,3,4-tetrahydr
onaphthalene) dicarboxamide}.

Melting point: 236 to 238 ° C Mass spectrum (FD-MS): 683 [M + H] ⁺ infrared absorption spectrum (IR, ν max cm ^-1 , KBr): 1646,16
14,1514,1250 UV absorption spectrum [λ max nm (log ε), EeOH]: 27
8 (4.07), 284 (4.07) proton nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ):
2.26 (1H, dt, J = 14,7Hz), 2.50 (1H, dt, J = 14,7Hz), 2.62 (1H,
t, J = 11Hz), 2.67 (1H, dt, J = 14,7Hz), 2.74 (1H, dt, J = 14,7H
z), 2.83 (2H, m), 3.01 (1H, dt, J = 7,6Hz), 3.25 (1H, dd, J = 14,
11Hz), 3.43 (2H, dd, J = 7,6Hz), 3.46 (1H, m), 3.58 (3H, s), 3.
76 (3H, s), 3.78 (3H, s), 3.79 (3H, s), 3.84 (3H, s), 3.87 (3H,
s), 4.27 (1H, d, J = 11Hz), 5.29 (1H, t, J = 6Hz), 5.93 (1H, t, J =
6Hz), 6.20 (1H, s), 6.55 (1H, s), 6.56 (1H, d, J = 2Hz), 6.74 (4
H, s), 6.75 (1H, dd, J = 8,2Hz), 6.80 (1H, d, J = 8Hz), 6.85 (2H,
d, J = 8Hz), 7.12 (2H, d, J = 8Hz) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ): 32.2
(t), 34.8 (t), 34.9 (t), 40.7 (t), 41.0 (t), 45.3 (d), 48.2
(d), 54.7 (d), 55.2 (q), 55.3 (q), 55.8 (q), 55.9 (q), 55.9
(q), 55.9 (q), 110.6 (d), 110.9 (d), 111.4 (d), 112.2 (d), 11
3.9 (d × 2), 114.1 (d × 2), 121.9 (d), 126.6 (s), 129.5 (d ×
2), 129.7 (d × 2), 129.9 (s), 130.5 (s), 130.7 (s), 136.7
(s), 147.4 (s), 147.5 (s), 148.0 (s), 149.2 (s), 158.2 (s), 1
58.3 (s), 172.9 (s), 173.7 (s)

Example 8 To a solution of 105 mg of the compound obtained in Example 1 in 1 ml of dry pyridine was added 500 μl of acetic anhydride, and the mixture was stirred at room temperature overnight. After the reaction,
The reaction solution was allowed to stand in 50 ml of water and partitioned and extracted with ethyl acetate (50 ml × 2). The ethyl acetate layer was washed with water and dried over magnesium sulfate, and the reaction product obtained by distilling off the solvent under reduced pressure was used for CIG column (22 mm
φ × 30 cm) and separated and purified with ethyl acetate to obtain 140 mg of amorphous powder.

Since this compound has the physicochemical properties shown below, 6,7 diacetoxy-1- (3,4-diacetoxyphenyl) -N ¹ , N ² -bis- [2- (4- (Acetoxyphenyl)
Ethyl-2,3- (1,2-dihydronaphthalene) dicarboxamide (6,7-diacetoxy-1- (3,4-diacetoxyphenyl) -N ¹ , N ² -bis
-〔2- (4-acetoxyphenyl) ethyl-2,3- (1,2-dihydronaphth
alene) dicarboxamide}.

Mass spectrum (FD-MS) m / z: 849 [M + H] ⁺ elemental analysis [C ₄₆ H ₄₄ N ₂ O ₁₆ ]: theoretical value; C: 65.09, H: 5.22, N: 3.30 measured value ; C: 64.86, H: 5.22, N: 3.30 Infrared absorption spectrum (IR, ν max cm ^-1 , KBr): 1766,16
48,1618,1536,1198 UV absorption spectrum [λ max nm (log ε), MeOH]: 26
7 (4.19), 296 (4.24) proton nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ):
2.22 (3H, s), 2.24 (3H, s), 2.26 (3H, s), 2.27 (3H, s), 2.27 (3
H, s), 2.30 (3H, s), 2.66 (1H, dt, J = 14,7Hz), 2.74 (1H, m), 2.
74 (2H, t, J = 7Hz), 3.31 (1H, dt, J = 14,7Hz), 3.42 (1H, m), 3.4
2 (2H, m), 3.43 (1H, d, J = 2Hz), 4.90 (1H, d, J = 2Hz), 6.53 (1H,
t, J = 6Hz), 6.72 (1H, d, J = 2Hz), 6.77 (1H, s), 6.93 (2H, d, J = 8
Hz), 6.97 (2H, d, J = 8Hz), 7.00 (1H, dd, J = 8,2Hz), 7.04 (1H,
s), 7.05 (1H, d, J = 8Hz), 7.07 (2H, d, J = 8Hz), 7.10 (2H, d, J = 8
Hz), 7.10 (1H, s), 7.79 (1H, t, J = 6Hz) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ): 20.5
(q), 20.5 (q), 20.7 (q), 20.9 (q), 21.1 (q), 21.1 (q), 34.7
(t), 34.7 (t), 40.6 (t), 41.0 (t), 43.6 (d), 49.4 (d), 121.3
(d × 2), 121.5 (d × 2), 122.8 (d), 123.1 (d), 123.4 (d), 124.
2 (s), 125.8 (d), 129.4 (d), 129.4 (s), 129.7 (d × 2), 129.7
(d × 2), 130.7 (s), 135.3 (s), 136.4 (s), 136.6 (s), 139.9
(s), 141.3 (s), 141.6 (s), 141.6 (s), 142.9 (s), 149.0 (s), 1
49.2 (s), 167.7 (s), 168.0 (s), 168.1 (s), 168.5 (s), 169.5
(s), 169.6 (s), 169.6 (s), 169.8 (s)

Example 9 To a solution of 115 mg of the compound obtained in Example 2 in 5 ml of dry pyridine was added 2 ml of acetic anhydride, and the mixture was stirred overnight at room temperature. After completion of the reaction, the reaction solution was left in 100 ml of water and partitioned and extracted with ethyl acetate (100 ml × 2). The ethyl acetate layer was washed with water and dried over magnesium sulfate, and the reaction product obtained by distilling off the solvent under reduced pressure was used for CIG column (22 mm
(φ × 30 cm) and separated and purified with hexane-ethyl acetate (1: 2) to obtain 113 mg of amorphous powder.

Since this compound has the physicochemical properties shown below, 6-methoxy-7-acetoxy-1- (3,4-diacetoxyphenyl) -N ¹ , N ² -bis- [2- (4-acetoxyphenyl) ethyl-2,3- (1,2-dihydronaphthalene) dicarboxamide (6-methoxy-7-acetoxy-1- (3,4-diacetoxyphe
nyl) -N ¹ , N ² -bis- 〔2- (4-acetoxyphenyl) ethyl-2,3- (1,2
-dihydronaphthalene) dicarboxamide}.

Mass spectrum (FD-MS) m / z: 821 [M + H] ⁺ infrared absorption spectrum (IR, ν max cm ^-1 , KBr): 1764,16
60,1620,1532,1198 UV absorption spectrum [λ max nm (log ε), MeOH]: 29
5 (4.19), 319 (4.00) proton nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ):
2.22 (3H, s), 2.25 (3H, s), 2.26 (3H, s), 2.27 (3H, s), 2.29 (3
H, s), 2.68 (1H, dt, J = 14,7Hz), 2.74 (1H, m), 2.78 (2H, t, J = 7
Hz), 3.34 (1H, dt, J = 14,7Hz), 3.43 (1H, m), 3.46 (2H, dt, J = 1
4,7Hz), 3.39 (1H, d, J = 2Hz), 3.85 (3H, s), 4.83 (1H, d, J = 2H
z), 6.51 (1H, t, J = 6Hz), 6.69 (1H, d, J = 2Hz), 6.80 (1H, brs),
6.83 (1H, brs), 6.89 (1H, s), 6.93 (2H, d, J = 8Hz), 6.98 (2H,
d, J = 8Hz), 7.00 (1H, dd, J = 8,2Hz), 7.04 (1H, d, J = 8Hz), 7.08
(2H, d, J = 8Hz), 7.11 (2H, d, J = 8Hz), 7.76 (1H, t, J = 6Hz) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ): 20.5
(q), 20.6 (q), 20.9 (q), 21.1 (q), 21.1 (q), 34.7 (t), 34.8
(t), 40.7 (t), 40.9 (t), 43.3 (d), 49.7 (d), 56.0 (q), 112.2
(d), 121.3 (d × 2), 121.5 (d × 2), 123.0 (d), 123.2 (d), 123.
8 (d), 125.8 (d), 129.3 (s), 129.6 (s), 129.7 (d × 2), 129.7
(d × 2), 129.9 (s), 130.5 (d), 136.6 (s), 136.6 (s), 140.4
(s), 140.5 (s), 140.9 (s), 141.5 (s), 149.0 (s), 149.2 (s), 1
50.5 (s), 168.1 (s), 168.4 (s), 168.5 (s), 169.5 (s), 169.5
(s), 169.8 (s), 170.2 (s)

Example 10 To a solution of 150 mg of the compound obtained in Example 3 in 1 ml of dry pyridine was added 0.5 ml of acetic anhydride, and the mixture was stirred at room temperature overnight. After the reaction,
The reaction solution was allowed to stand in 100 ml of water and partitioned and extracted with ethyl acetate (100 ml × 2). The ethyl acetate layer was washed with water and dried over magnesium sulfate, and then the reaction product obtained by distilling the solvent under reduced pressure was used for CIG column (22
mmφ × 30 cm) and separated and purified with hexane-ethyl acetate (4: 6) to obtain 160 mg of amorphous powder.

Since this compound has the physicochemical properties shown below, 6-methoxy-7-acetoxy-1- (3-methoxy-4-acetoxyphenyl) -N ¹ , N ² -bis- [2 -(4-acetoxyphenyl) ethyl-2,3- (1,2-dihydronaphthalene)
Dicarboxamide {6-methoxy-7-acetoxy-1- (3-methoxy-
4-acetoxyphenyl) -N ¹ , N ² -bis- 〔2- (4-acetoxyphenyl) et
hyl-2,3- (1,2-dihydronaphthalene) dicarboxamide}.

Mass spectrum (FD-MS) m / z: 793 [M + H] ⁺ infrared absorption spectrum (IR, ν max cm ^-1 , KBr): 1768,16
68,1621,1508,1196 UV absorption spectrum [λ max nm (log ε), MeOH]: 21
9 (4.57), 283 (4.08), 324 (3.91) proton nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ):
2.26 (3H, s), 2.26 (3H, s), 2.28 (3H, s), 2.28 (3H, s), 2.66 (1
H, dt, J = 14,7Hz), 2.72 (1H, dt, J = 14,7Hz), 2.80 (2H, dt, J = 7
Hz), 3.32 (1H, dt, J = 13,7Hz), 3.44 (1H, m), 3.49 (2H, dt, J = 1
4,6Hz), 3.69 (1H, d, J = 2Hz), 3.70 (3H, s), 3.84 (3H, s), 4.71
(1H, d, J = 2Hz), 6.33 (1H, t, J = 6Hz), 6.44 (1H, dd, J = 8,2Hz),
6.65 (1H, d, J = 2Hz), 6.81 (1H, brs), 6.82 (1H, d, J = 8Hz), 6.8
6 (1H, s), 6.93 (2H, d, J = 8Hz), 6.96 (1H, brs), 6.99 (2H, d, J =
8Hz), 7.05 (2H, d, J = 8Hz), 7.10 (1H, t, J = 6Hz), 7.12 (2H, d, J
= 8Hz) ¹³ C-nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ): 20.6
(s), 20.6 (s), 21.1 (s), 21.1 (s), 34.7 (t), 34.8 (t), 40.6
(t), 41.1 (t), 44.2 (d), 49.1 (d), 55.8 (q), 56.0 (q), 112.0
(d), 112.4 (d), 119.7 (d), 121.4 (d × 2), 121.7 (d × 2), 122.
5 (d), 124.0 (d), 129.3 (s), 129.4 (s), 129.6 (s), 129.7 (d ×
2), 129.7 (d × 2), 131.3 (d), 136.4 (s), 136.5 (s), 138.4
(s), 141.0 (s), 141.5 (s), 149.0 (s), 149.0 (s), 149.3 (s), 1
50.5 (s), 168.3 (s), 168.5 (s), 169.0 (s), 169.6 (s), 169.7
(s), 171.0 (s)

Example 11 Caffeoyl tyramine 1.579 g in an acetone solution of 30 ml, 2.
10 ml of a 5% ferric chloride aqueous solution was added, and the mixture was stirred overnight at room temperature. Ethyl acetate (50 ml × 2) was added to the reaction mixture for partition extraction. The obtained ethyl acetate layer was washed with water, dried over sodium sulfate, the solvent was distilled off under reduced pressure, the obtained reaction product was subjected to a CIG column (22 mmφ × 30 cm), and chloroform-methanol (7:
After separation in 1), gel filtration with Sephadex LH-20 (ethanol) gave 135 mg of amorphous powder.

This compound was in agreement with the physicochemical properties of the compound obtained in Example 1.

Example 12 To 4 ml of an acetone solution containing 86 mg of feruloyltyramine, 2.5%
4 ml of an aqueous ferric chloride solution was added, and the mixture was stirred at room temperature overnight. Ethyl acetate (50 ml × 2) was added to the reaction mixture for partition extraction. The obtained ethyl acetate layer was washed with water and dried over sodium sulfate,
The solvent was distilled off under reduced pressure, and the resulting reaction product was subjected to CIG column (22
(mmφ × 30 cm), and the fraction separated with chloroform-methanol (95: 5) was recrystallized with methanol to obtain 25 mg of amorphous needle crystals.

This compound was in agreement with the physicochemical properties of the compound obtained in Example 3.

Example 13

According to the above formulation, the ingredients (1) to (4) were uniformly mixed and compression-molded with a tableting machine to give tablets (200 mg each).
20 mg of the compound obtained in Example 1 is contained in one tablet, and 3 to 10 tablets for adults are to be taken in several divided doses per day.

Example 14

According to the above formulation, a part of and was uniformly mixed, compression-molded, pulverized, and the remaining amount of and was added and mixed, and the mixture was compression-molded with a tableting machine to give one tablet.
0 mg tablets were obtained.

One tablet of this compound was prepared from compound 2 obtained in Example 4.
It contains 0mg, and adults take 3-10 tablets daily in several divided doses.

Example 15

According to the above formulation, and were uniformly mixed, the mixture was kneaded by a conventional method, granulated by an extrusion granulator, dried and crushed, and then mixed, and compressed by a tableting machine. It was molded to obtain a tablet of 200 mg each.

One tablet of this compound was prepared from compound 2 obtained in Example 4.
It contains 0mg, and adults take 3-10 tablets daily in several divided doses.

Example 16

According to the above formulation, the components (1) to (4) are uniformly mixed, compression-molded by a compression molding machine, and then crushed by a crusher,
Sieve to obtain granules.

The compound 1 obtained in Example 7 was added to 1 g of this granule.
It contains 00mg, and 0.6 to 2g for adults is divided into several doses.

Example 17

According to the above recipe, the ingredients (1) to (4) were mixed uniformly and kneaded. After granulating with an extrusion granulator, it was dried and sieved to obtain granules.

Compound 1 obtained in Example 8 was added to 1 g of this granule.
It contains 00mg, and 0.6 to 2g for adults is divided into several doses.

Example 18

According to the above-mentioned formulation, 1 to 3 were mixed uniformly, and 200 mg was filled in a No. 2 capsule.

One capsule of this capsule contains 20 mg of the compound obtained in Example 9, and 3 to 10 capsules for an adult are to be taken in several divided doses per day.

Example 19 After dissolving and in distilled water for injection, the mixture was poured into a 5 ml ampoule and autoclaved at 121 ° C. for 15 minutes to obtain an injection.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masao Chen             3586 Yoshiwara, Ami-cho, Inashiki-gun, Ibaraki             Within Mura

Claims (2)

[Claims] 1. The following formula I (In the formula, Rs are the same or different and each represents a hydrogen atom, an acetyl group or an alkyl group having 1 to 6 carbon atoms.). 2. The following formula I (In the formula, Rs are the same or different and each represents a hydrogen atom, an acetyl group, or an alkyl group having 1 to 6 carbon atoms.) A proteolytic enzyme inhibitor containing lignanamide as an active ingredient.