JPH0459316B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to β-carboline derivatives and salts thereof. The β-carboline derivatives and salts thereof of the present invention are novel compounds, and are represented by the following general formula (1). [In the formula, R ¹ is a hydrogen atom or a lower alkyl group,
R ² represents a vinyl group or an ethyl group. However, when R ² is an ethyl group, R ¹ must not be a hydrogen atom. ] The β-carboline derivative represented by the above general formula (1) of the present invention and its salt have antibacterial, anticancer, and antiinflammatory effects, and are useful as active ingredients of antibacterial agents, anticancer agents, and antiinflammatory agents. It can also be used as an intermediate for producing such active ingredients. In the above general formula (1), examples of the lower alkyl group represented by R ¹ include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, benzyl,
Examples include hexyl group. Representative compounds of the β-carboline derivatives of the present invention are shown below. Γ1-vinyl-3-carboxy-β-carboline Γ1-vinyl-3-methoxycarbonyl-β-carboline Γ1-vinyl-3-ethoxycarbonyl-β-carboline Γ1-vinyl-3-propoxycarbonyl-β-
Carboline Γ1-vinyl-3-tert-butoxycarbonyl-
β-Carboline Γ1-vinyl-3-hexyloxycarbonyl-
β-Carboline Γ1-ethyl-3-methoxycarbonyl-β-Carboline Γ1-ethyl-3-ethoxycarbonyl-β-Carboline Γ1-ethyl-3-isopropoxycarbonyl-
β-carboline Γ1-ethyl-3-butoxycarbonyl-β-carboline Γ1-ethyl-3-pentyloxycarbonyl-
β-Carboline Γ1-ethyl-3-hexyloxycarbonyl-
β-Carboline The β-carboline derivative of the present invention can be produced by various methods depending on the type of substituent. For example, a compound in which R ¹ is a hydrogen atom and R ² is a vinyl group in general formula (1) can be produced using a β-carboline derivative-producing bacterium belonging to the genus Nocardiopsis. Examples of the β-carboline derivative-producing bacteria include known microorganisms belonging to the genus Nocardiopsis and microorganisms newly isolated by the present inventors from Iriomote Island, Okinawa Prefecture. The latter microorganism is Nocardiopsis dassonvillei.
It has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under the name OFR1063, with application number 5909 for consignment of microorganism storage. Below is this entrusted OFR1063
Details about each stock. [Morphological characteristics] Aerial hyphae are elongated and moderately branched. The shape is linear or curved, and there are also cases where the shape is zigzag. The basal hyphae are well developed, branched irregularly, and may be quite fragmented depending on the type of medium. Spores are formed when aerial hyphae are roughly divided and then further finely divided. The size of the spores is irregular, the shape of the spores is oblong to cylindrical, and the surface is smooth. [Growth status in each medium] Table 1 shows the growth status of this strain in various representative media. All of these results were observed after culturing at 28℃ for 21 days, and the color tones are described in "Color Harmony Manual 4th Edition.
Edition, 1958, Container Corporation of
America)” labeling laws.

【table】

[Physiological properties]

1 Growth temperature range 12-46℃ (optimum growth temperature range 34-37℃) 2 Growth PH range PH4.0-9.5 (optimum growth PH range PH6.2-7.7) 3 Liquefaction of gelatin (glucose-peptone gelatin medium) Top) Positive (weak) 4 Hydrolysis of starch (on starch/inorganic salt agar medium) Positive (strong) 5 Coagulation/peptonization of skim milk Coagulation; negative Peptonization; positive (weak) 6 Production of melanin-like pigments Negative ( Peptone yeast iron agar, tyrosine agar, and tryptone yeast extract) 7 Nitrate reduction Positive (strong) 8 Cellulose decomposition Negative 9 Carbon source assimilation (on Pridham-Gotlieb agar) See Table 2 below. show. However, the evaluation of assimilability is based on the following. ;Assimilates well ±;Assimilation is questionable

[Antibacterial activity test]

The antibacterial activity of the compound of the present invention (1-vinyl-3-carboxy-β-carboline) against various bacteria (bacteria and fungi) was determined by the agar dilution plate method.
The test results are expressed as the minimum inhibitory concentration ((MIC, μg/
ml) as shown in Table 3 below. In the case of bacteria,
With Heart Infusion Medium (manufactured by Eiken Chemical Co., Ltd.)
Incubation was performed at 37°C for 18 hours, and in the case of fungi, incubation was performed at 28°C for 48 hours on potato glucose agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.).

【table】

[Cytotoxicity test for KB cells]

The human nasopharyngeal carcinoma-derived KB cells used in the test were cultured in Eagle's MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% calf serum. i.e. diameter 15mm and major axis
In a 150 mm glass test tube, 1 ml of the above medium, 0.1 ml of cell suspension, and the compound of the present invention (1-vinyl-3
0.1 ml of a dimethyl sulfoxide (DMSO) solution of -carboxy-β-carboline was added and cultured using an oblique test tube. As a control group, the same procedure as above was carried out using 0.1 ml of a physiological saline solution of mitomycin C (manufactured by Kyowa Hakko Co., Ltd.) instead of the compound of the present invention. The test group and the control group were tested in triplicate at each concentration. Cultivation was carried out at 37° C. for 72 hours using a CO ₂ -incubator (5% carbon dioxide gas flow). After the test, the protein amount was determined by the method of Oyama and Eagle [Proc.Soc.Exp.
Biol.Med., 91 , 305-307 (1956)].
reagent). In addition, the inhibition rate of protein synthesis is determined by setting the protein amount at the start of the test as Co, and the protein amount at the end of the test as T (drug administration group) and C (control group by administering solvent alone without drug).
Calculated using the formula below. In addition, the above Co is 22.1μ
g/test tube. Inhibition rate (%) = C-T/C-C ₀ ×100 The 50% inhibitory concentration (ID ₅₀ ) was determined using a logarithmic concentration response curve. The results are shown in Table 4.

[Table] Production examples of the compounds of the present invention are shown below as Examples. Example 1 100 ml of a medium having the following composition was placed in a 500 ml Erlenmeyer flask, and Nocardiopsis dasonvillei strain OFR1063 was cultured in this medium with rotational shaking at 30° C. and pH 7.0 for 3 days. <Medium composition> Starch 30g/Glucose 5 Polypeptone S 10 MgSO ₄・7H ₂ O 0.5 KH ₂ PO ₄ 0.02 Na ₂ HPO ₄ 0.05 CoCl ₂・6H ₂ O 1mg/ FeSO ₄・7H ₂ O 1 MnCl ₂・4H ₂ O 1 〃 ZnSO ₄・7H ₂ O 1 〃 25 ml of the seed culture obtained above was rotated for 2 days at a culture temperature of 30°C in a 2-volume Sakaguchi flask containing 500 ml of the medium with the above composition. Perform shaking culture. This preculture 8 was added with 600 medium of the above composition.
Culture temperature is 30℃ in 1t capacity tank, air flow rate is 600/min,
Cultivate for 64 hours at a stirring speed of 120 rpm. The obtained culture solution is centrifuged at 8000 rpm to remove bacterial cells, and this supernatant is
Charge a column packed with 50% HP20 (manufactured by Mitsubishi Kasei Corporation), wash with water, elute with 100% ethyl acetate, and concentrate the eluate to obtain 210 g of a brown residue.
80 g of this residue was subjected to column chromatography using 800 g of silica gel and developed with chloroform:methanol=10:1 to obtain 200 ml fractions each of No. 1 to No. 33. Next, the mixture was developed with chloroform:methanol=5:1 to obtain 200 ml fractions each of No. 34 to No. 51. Testing the antibacterial activity of each fraction No.17 to No.37
activity is observed in Fractions No. 17 to No. 37 are combined, concentrated, and crystallized. Take this 3.23
Obtain crude crystals of g. 500mg of crude crystals in methanol
Recrystallization from acetonitrile yields 250 mg of the antibacterial active substance 1-vinyl-3-carboxy-β-carboline in the form of yellow columnar crystals. The production of this compound is confirmed by the following physical and chemical properties. Rf value (silica gel thin layer chromatography) 0.56 (n-butanol: acetic acid: water = 4:1:1) 0.60 (n-propanol: 2N-ammonia =
7:3) Melting point 254-256℃ (decomposition) Dragendorff reagent positive (red-orange) Bromcresol green reagent positive (yellow) Solubility Soluble in dimethyl sulfoxide, dimethylformamide, alkaline water, methanol, ethanol, tetrahydrofuran It is poorly soluble in water,
Insoluble in chloroform, ether and dioxane. Elemental analysis value (as C ₁₄ H ₁₀ N ₂ O ₂ ) C H N Calculated value (%) 70.58 4.23 11.76 Analysis value (%) 70.36 4.46 11.86 UV spectrum λ ^EtOH _nax = 225 nm (sh, ε = 19700) 281 nm (ε =41300), 358nm (ε=6970),
365nm (sh, ε=6500) IR spectrum ν ^KBr _nax =3200, 3050, 3000, 2900, 2850, 1710,
1620, 1580, 1500, 1455, 1360, 1310,
1250, 1140, 1105, 1070, 995, 935, 895,
815, 795, 740, 655, 595, 545, 500 cm ^-1 NMR spectrum δ ^DMSO-d _6ppn = 12.23 (1H, br, s), 8.86 (H, s), 8.39 (1H, d, J = 7.6Hz) ,
7.69 (1H, d, J = 7.6Hz), 7.61 (1H, t, J = 7.6Hz), 7.48 (1H, dd, J = 16.9, 10.6Hz), 7.31 (1H, t, J = 7.6Hz), 6.71 (1H, dd,
J=16.9, 1.2Hz), 5.73(1H, dd, J=10.6,
1.2Hz) Example 2 1-vinyl-3-carboxy-β-carboline
Suspend 400 mg in 15 ml of methanol, and while cooling in ice water (0°C), add an ethereal solution of diazomethane with stirring until no bubbles are generated from the reaction solution. The solvent is evaporated to give a tan residue.
This residue was chromatographed on 10 g of silica gel and developed with benzene:ethyl acetate=2:1 to obtain fractions of 3 ml each. Fractions No. 4 to No. 10 are combined and the solvent is distilled off to obtain 340 mg of crude crystals. Recrystallization from benzene-ethyl acetate gave 65 mg of 1-vinyl-3-methoxycarbonyl-β-carboline in the form of pale yellow needles. Melting point: 213-215°C (decomposition) Example 3 (1) Production of 1-ethyl-3-carboxy-β-carboline Dissolve 30 mg of 1-vinyl-3-carboxy-β-carboline in 20 ml of methanol, and add 10% palladium on carbon. Add 15 mg and perform catalytic reduction at normal pressure for 1 hour. The catalyst was filtered off from the reaction solution, and the solvent was distilled off to obtain 28 mg of a residue. This was recrystallized from methanol-acetonitrile to obtain 18 mg of the desired compound in the form of pale yellow-green columnar crystals. Melting point 270-272℃ (decomposed) (2) 1-ethyl-3-methoxycarbonyl-β-
Production of carboline 1-ethyl-3-carboxy- obtained in (1) above
The target compound is obtained in the same manner as in Example 2 using β-carboline. Pale yellow columnar crystals (benzene-ethyl acetate) Melting point 223-225℃ Example 4 325 mg of 1-vinyl-3-methoxycarbonyl-β-carboline was suspended in 14 ml of ethanol, and while cooling in ice water, 90 mg of sodium ethylate was added to ethanol. Add 2 ml of the solution dropwise while stirring. Return to room temperature and stir for 1 hour. The solvent was distilled off from the reaction solution, and the resulting residue was chromatographed on 8 g of silica gel and developed with benzene:ethyl acetate=3:1. Take 5 ml of fractions, combine fractions No. 4 and 5, and distill off the solvent to obtain crude crystals.
Get 210mg. Recrystallization from benzene-ethyl acetate gave 54 mg of 1-vinyl-3-ethoxycarbonyl-β-carboline in the form of pale yellow needles. Melting point: 198-220°C (decomposition) Example 5 1-ethyl-3-methoxycarbonyl-β-carboline was converted to 1-ethyl-3-methoxycarbonyl-β-carboline in the same manner as Example 4.
3-Ethoxycarbonyl-β-carboline is obtained. Pale yellow needle crystals (ethanol) Melting point: 208-210°C Below, examples of preparing preparations containing the compound of the present invention as an active ingredient will be given. Formulation Example 1 Sodium salt of 1-vinyl-3-carboxy-β-carboline 200 mg Glucose 250 mg Distilled water for injection Appropriate total amount 5 ml Dissolve the compound of the present invention and glucose in distilled water for injection, then inject into a 5 ml ampoule. . After purging with nitrogen, sterilize under pressure at 121°C for 15 minutes to obtain an injection. Production example 2 1-ethyl-3-methoxycarbonyl-β-carboline 100g Avisiel (trade name, manufactured by Asahi Kasei Corporation) 40g Cornstarch 30g Magnesium stearate 2g TC-5 (trade name, hydroxypropyl methyl cellulose) 10g Polyethylene glycol-6000 3g castor oil 40g methanol 40g The compound of the present invention, Abysiel, cornstarch and magnesium stearate were mixed, polished and sugar-coated.
Compress the tablets with an R10mm kine. The obtained tablets were TC-
5. Coat with a film coating agent consisting of polyethylene glycol-6000, castor oil and methanol to produce film coated tablets. Formulation example 3 1-vinyl-3-methoxycarbonyl-β-carboline 100g Abysiel 40g Cornstarch 30g Magnesium stearate 2g Methyl acrylate-methacrylic acid copolymer
5.7g Triacetin 0.6g Ethanol 50.4g The compound of the present invention, Abysiel, cornstarch and magnesium stearate were mixed, polished and sugar-coated.
Compress the tablets with an R10mm kine. The resulting tablets are coated with a film coating agent consisting of methyl acrylate-methacrylic acid copolymer, triacetin, and ethanol to produce enteric-coated tablets.

Claims (1)

[Claims] 1. General formula [In the formula, R ¹ is a hydrogen atom or a lower alkyl group,
R ² represents a vinyl group or an ethyl group. However, when R ² is an ethyl group, R ¹ must not be a hydrogen atom. ] A β-carboline derivative represented by these and its salt.