JPH043394B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel 7,7-dimethyl-2,3-
The present invention relates to epoxy-11-methylenespiro[5.5]undeca-3-carbaldehyde, a method for producing the same, and a liver disorder improving agent containing the compound as an active ingredient. Schizandra chinensis (Schizandrachinensis Baillon), a Japanese and Chinese medicine, is the dried fruit of Schizandrachinensis Baillon, a family of Schizandraceae, and has been added to Chinese herbal medicine since ancient times for the purposes of tonicity, fortification, and antitussiveness. The present inventors discovered and announced that the lignans contained in this gomin have an effect on improving liver damage [Pharmaceutical Journal, 102 ,
p579 (1982)]. The present inventors subsequently investigated camigrenal (7,7-dimethyl-11-methylenespiro[5.5]undec-2-ene-3-carbamate), which is the main component in gomish essential oil and has a completely different structure from lignans. We discovered that camigrenal (aldehyde) also has the same liver damage-improving effect as lignans, and by chemically converting this camigrenal, we discovered a new liver damage-improving effect that had never been reported in the literature related to camigrenal. The present invention was completed by successfully producing 7,7-dimethyl-2,3-epoxy-11-methylenespiro[5.5]undec-3-carbaldehyde, which is a compound having the following properties. The compound of the present invention has the formula (1) A novel 7,7-dimethyl-2,3-epoxy-11-methylenespiro[5.5]undec-
3-carbaldehyde [hereinafter referred to as the compound of formula (1)]. The compound of formula (1) is the compound of formula (2) It can be produced by reacting camigrenal represented by hydrogen peroxide in a solvent under alkaline conditions. In the method for producing the compound of formula (1) above, camigrenal represented by formula (2) used as a raw material is obtained by extracting, for example, gossi with an organic solvent such as petroleum ether.
Obtained by subjecting the extract to silica gel chromatography, high performance liquid chromatography, etc. [Y.Ohta and Y.Hirose, Tetrahedron
Letters No. 20, p2483 (1968)]. A specific example of the production of camigrenal is as follows:
It is as follows. Specific Example Petroleum ether 24 was added to 4.67 kg of powdered Gomici, heated under reflux at 38°C for 8 hours, and the extract was filtered. The extraction residue was subjected to the same operation twice, and the resulting extracts were combined and dried to obtain 516 g of extract.
This extract was subjected to column chromatography using 5 kg of silica gel, developed with n-hexane-ethyl acetate (94:6), and eluted in 500 ml portions to obtain fractions. Next, a part of each fraction was developed by thin layer chromatography (developing solvent: n-hexane: ethyl acetate = 17:3), and when anisaldehyde, molybdic acid, and sulfuric acid test solutions were sprayed and heated, the Rf value was approx. The fractions with spots at 0.75 were combined, the solvent was distilled off, and then high performance liquid chromatography was performed [Model: Waters
prep LC/System 500A, column: prep PAK
-500/C ₁₈ (manufactured by Waters), solvent: methanol: water = 8:3, flow rate: 0.15/min, retention time: 24 minutes], 6.91 g of a substance exhibiting the properties of a pale yellow oil (yield 0.15%). The physicochemical properties of this substance were consistent with the data for camigrenal described in the literature. The reaction between camigrenal and hydrogen peroxide represented by formula (2) is carried out by reacting hydrogen peroxide in a solvent (for example, alcohols such as methanol and ethanol) under alkaline conditions at 0 to 30°C for 30 minutes to 5 hours. This is done by letting Specific examples of the alkali added to the solvent to create alkaline conditions include sodium hydroxide, potassium hydroxide, and the like. After this reaction is completed, the reaction mixture is poured into water,
Extract with an organic solvent such as ether or ethyl acetate, and shake the organic solvent layer as is or after washing with water, shake with saturated brine, remove the saturated brine, dry, and distill off the organic solvent to obtain a crude product. A product is obtained, and this crude product is recrystallized from n-hexane to obtain a compound of formula (1). Next, the fact that the compound of formula (1) used in the present invention has a liver damage-improving effect and is useful as a pharmaceutical will be explained by showing experimental examples. In investigating the hepatopathy-improving effect of the compound of formula (1) used in the present invention, the present inventors used the method of Hikino et al. p22 (1982)]. Experimental example Effect on carbon tetrachloride liver cell damage (1) Medium Eagle minimum essential medium [manufactured by Difco, hereinafter referred to as MEM] 9.4 g, L-
Glutamine 0.292g, Sodium Bicarbonate 1.7
Dissolve 18 mg of potassium penicillin G and 50 mg of streptomycin sulfate in 1 liter of purified water, add 10 -8 M insulin and 10 ^-8 M dexamethasone.
The mixture was mixed to a concentration of 10 ⁻⁶ M and oversterilized. This medium was prepared by mixing inactivated calf serum (left at 56°C for 30 minutes) to a concentration of 10% [this medium is referred to as 1% CS-MEM solution]. (2) Preparation of hepatocytes After anesthetizing the rat with ether, the rat's abdomen was opened, a cannula was inserted into the portal vein, and a mixed gas of 5% carbon dioxide and 95% oxygen gas was insufflated.
1% bovine serum albumin fraction V (manufactured by Sigma) and 0.5mM ethylene glycol-bis(β-aminoethyl ether) kept at ℃.
Calcium-free Hank's solution containing N,N,N',N'-tetraacetate acid [Reference: JH
Hanks and REWallace, Proc.Soc.Exp.
Biol.Med., 71 , p196 (1949)] [30 ml/min], and the inferior vena cava below the liver was cut to remove sufficient blood. The severed inferior vena cava is ligated, and the perfusion fluid is drained from the cannula previously inserted into the inferior vena cava above the diaphragm from the right atrium.
Calcium-free Hank's solution containing 50mg of collagenase and 4mM calcium chloride warmed to 37℃
ml/min] and circulated for 1 to 15 minutes. Thereafter, the liver was transferred to a shear dish containing calcium-free Hank's solution, and the hepatocytes were dispersed using two forceps. The cell dispersion was passed through three layers of gauze and centrifuged at 4°C [50G, 1 minute]. Thereafter, the supernatant was removed, calcium-free Hank's solution was added, and centrifugation was repeated 3 to 4 times until the supernatant became clear, and the cells were suspended in 10% CS-MEM. The average survival rate of cells according to the trypan blue method [using the trypan blue metabolic ability of the cells as an index, and calculating the survival rate by counting the number of viable cells that are not stained by trypan blue (dead cells are stained) using a hemocytometer] is 85%, cell number was 2-4 x ¹⁰⁸ cells/200g body weight. (3) Culture the cell suspension 5x with 10% CS-MEM solution.
Dilute to 10 ⁵ cells/ml, sprinkle 1 ml each on a 35 mm shear plate, and incubate with 5% carbon dioxide gas.
Cultured at ℃. (4) Bioassay Carbon tetrachloride [10mM, final concentration]
Dissolved in ethanol [1%, final concentration],
After mixing with the culture medium to make a carbon tetrachloride medium, the test drug was dimethylsulfoxide (hereinafter referred to as DMSO).
[1%, final concentration] was dissolved in the above carbon tetrachloride medium [concentration of test drug is 1 mg in 1 ml of medium]. Next, remove the medium from the primary culture medium described in (3) that was cultured for 24 hours, and replace it with carbon tetrachloride medium 1 of the test drug.
ml in a 5% carbon gas incubator.
After culturing at 36.5°C for 1 hour to induce hepatocyte damage by carbon tetrachloride, 0.5ml of the medium was taken and centrifuged at 4°C [1500G, 10 minutes], and 0.2ml of the supernatant was collected. [Glutamic acid-
Pyruvic acid-transaminase] activity was measured. GPT is one of the amino acid transferases contained in hepatocytes, and low GPT activity in the medium means less destruction of hepatocytes.
In other words, it means improvement of hepatocyte damage. Measurement was performed using the Karmen method [Reference: A.
Karmen, F. Wroblewski and J. Ladue, J. Clin.
Invest., 34 , p126 (1955)] using an automatic analyzer [RaBA-SUPER]. All results are expressed as the mean ± standard error of three experiments and were tested using the one-dimensional variance method. The results are shown in Table 1 below.

[Table] The control shows the GPT value in the medium of primary cultured rat hepatocytes cultured in the presence of carbon tetrachloride as 100. The numerical value indicates how much the drug reduces the GPT value in the medium compared to the control when 1 mg of the drug is administered to 1 ml of the medium. As shown in Table 1, the compound of formula (1), which is a compound of the present invention, is
It was confirmed that it has an effect on improving liver damage by suppressing the rise in GPT levels. In addition, as an acute toxicity test of the compound of formula (1), it was administered orally to mice at 2000 mg/Kg, and intravenously administered to mice.
Since no deaths were observed even after administration of 240 mg/Kg, it was confirmed that the compound of formula (1) used in the present invention is extremely safe. The effective dosage of the compound of formula (1) varies depending on the age, weight, and severity of the disease of the patient, based on experimental data on its liver damage-improving effect and the results of acute toxicity tests. The daily dose for adults is approximately 50 mg to 200 mg, which can be administered once or in divided doses. For parenteral administration, 10 mg ~
50mg is made into an injection according to the conventional manufacturing method, and can be injected subcutaneously, intravenously, or intramuscularly. As an oral dosage form, the compound of formula (1) can be used as is as a liver disorder improving agent, but it can be used by adding excipients, adjuvants, etc. that are used in ordinary formulations and using the conventional method for manufacturing formulations. Therefore, it can be used in the form of preparations such as powders, granules, tablets, and capsules. EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 300 mg of camigrenal obtained in the above specific example,
Add 0.11 ml of 6N sodium hydroxide to a mixture of 0.4 ml of 30% hydrogen peroxide and 2 ml of methanol,
This was stirred at 20-25°C for 2 hours. After the reaction is complete,
The reaction mixture was poured into water and extracted with diethyl ether. After washing the diethyl ether layer with water, it was shaken with saturated brine, the saturated brine was removed, dried over anhydrous sodium sulfate, and the solvent was distilled off. A crude product was obtained. This crude product was recrystallized using n-hexane to obtain 110 mg of the compound of formula (1). The physicochemical properties of the compound of formula (1) are as follows. Melting point: 92-93℃ Optical rotation: +17.5 (c.0.8, CHCl ₃ ) Infrared absorption spectrum (IR): ν ^CHCl3 _nax cm ^-1 : 1715, 1623, 895 Proton nuclear magnetic resonance spectrum ( ¹ H- NMR): δppm (CDCl ₃ ): 0.80 (3H, s), 0.85 (3H,
s), 1.0-2.4 (12H, m), 3.43 (1H, bd, J=
5), 4.35 (1H, bs), 4.87 (1H, bs), 8.83 (1H,
s) Mass spectrum (Ms): m/z (%): 234 (m ⁺ 82), 216 (43), 205 (88)
，
173 (73), 109 (98), 69 (100) Example 2 10 g of the compound of formula (1) obtained in Example 1 was added to lactose 89
g and 1 g of magnesium stearate,
This mixture was compressed into tablets with a diameter of 20 mm using a single-shot tablet machine.
A slug tablet having a diameter of 1.5 mm and a weight of approximately 23 g was prepared, crushed with an oscillator, and sorted to obtain granules of 20 to 50 meshes. Take 0.5-2g of this granule per day. Example 3 50 g of the compound of formula (1) obtained in Example 1 was mixed with 290 g of potato starch, water was added and kneaded,
The mixture was granulated into granules using a screen with a mesh size of 1 mm x 1 mm, dried, and sized using a No. 16 mesh sieve. Add this to 10g of magnesium stearate.
were mixed and compressed using a tablet machine to produce tablets each weighing 350 mg. One tablet of this tablet contains 50 mg of the compound of formula (1) obtained in Example 1, and one to four tablets are taken per day depending on the symptoms. Example 4 Lactose was added to 50 g of the compound of formula (1) obtained in Example 1.
190 g and 10 g of magnesium stearate were mixed and 250 mg each was filled into hard capsules. Each capsule contains 50 mg of the compound of formula (1) obtained in Example 1, and the amount of the compound of formula (1) obtained in Example 1 is 1 to 4 times per day depending on the symptoms.
Take capsules. Example 5 50g of the compound of formula (1) obtained in Example 1 was added to 170g of crystalline cellulose and 5g of magnesium stearate.
This mixture was compressed using a single-shot tablet machine to produce tablets with a diameter of 7 mm and a size of 225 mg. One tablet contains 50% of the compound of formula (1) obtained in Example 1.
It contains 1 to 4 tablets per day, depending on the symptoms. Example 6 5 g of the compound of formula (1) obtained in Example 1 was dissolved in 1 part of distilled water for injection heated to 60°C according to the usual method for manufacturing injections, and the solution was made isotonic with sodium chloride. After that, it was sealed in an ampoule. 1 ml of this injection contains 5 mg of the compound of formula (1) obtained in Example 1. This injection is injected subcutaneously, intravenously, or intramuscularly at 2 to 10 ml per day depending on the symptoms.

Claims (1)

[Claims] 1 Formula (1) 7,7-dimethyl-2,3-epoxy-11-methylenespiro[5.5]undeca-3-carbaldehyde. 2 Formula (2) Formula (1) characterized in that hydrogen peroxide is applied to camigrenal expressed in a solvent under alkaline conditions. A method for producing 7,7-dimethyl-2,3-epoxy-11-methylenespiro[5.5]undeca-3-carbaldehyde. 3 Formula (1) A liver disorder improving agent containing 7,7-dimethyl-2,3-epoxy-11-methylenespiro[5.5]undec-3-carbaldehyde as an active ingredient.