JPH0368040B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel dammarane compounds having anticancer activity.

The compound according to the present invention is represented by the following formula (): [In the formula, glu represents a β-D-glucopyranosyl group].

As a result of various studies aimed at obtaining an excellent anticancer drug with few side effects, the present inventors discovered that there is a substance that exhibits excellent antitumor activity in the Dammaran saponins contained in Jiaogulan and filed a patent application. (Special Application No. 157925, October 1982)
As a result of further research, we found that among the prosapogenin or sapogenin obtained by hydrolyzing these saponins with acids, alkalis, or enzymes, the compound represented by the above formula () has strong antitumor activity. The present invention was completed based on the discovery that the present invention has the following properties.

The compound represented by formula () is a novel compound discovered by the present inventors. This compound () was named Mh-2 by the present inventors.

Many anticancer drugs currently in clinical use are so-called cytotoxins, which not only kill tumor cells, but also cause significant damage to normal cells, making it impossible to avoid strong side effects. The compound represented by the formula () according to the present invention is, as detailed later,
It significantly inhibits the proliferation of cervical cancer cells, melanoma tumor cells, and lung cancer cells at trace amounts of 10 μg/ml or less, and when continuously administered to mice transplanted with ascites cancer cells, it shows a clear survival effect compared to the control group. In addition, it has the remarkable feature of causing almost no damage to cells at a concentration of 10 μg/ml, which suppresses tumor cell proliferation.

As mentioned above, the compound represented by the formula () is
Prosapogenin is a compound obtained by hydrolyzing Jiaogulan saponin, and is a compound with extremely low toxicity. This is clear from the fact that Jiaogulan has been used for food and drinking since ancient times. In other words, Amachiya crane is a plant that has been described as a relief plant since the Ming dynasty in China, and has been used as food in times of famine, as a vegetable, or as a substitute for Amachiya. Its toxicity is extremely low, and the acute toxicity of the dry extract of Jiaogulan was investigated using rats, and the LD ₅₀ of oral administration was 10.
g/Kg, 1.85 g/Kg even when administered intraperitoneally, and even when administered continuously for one month at a rate of 8 g/Kg/day.
No abnormalities were observed in general symptoms, body weight, feed intake, water consumption, urine, blood, tissue weight, pathological findings, etc. In this way, the low toxicity of Jiaogulan extract indicates that the toxicity of saponin, its main component, and prosapogenin and sapogenin, which are produced when it is hydrolyzed in vivo by acids and enzymes, is extremely low. It is something.

The compound of formula () can be produced by the method described in Example 1 below.

The compound of formula () is extremely stable and can be formulated into various dosage forms for oral and parenteral use, such as tablets, pills, capsules, granules, solutions, injections, syrups, ointments, etc. be able to. To manufacture these preparations, the compound of formula () as the active ingredient is combined with lactose, starch, sucrose,
Dextrin, cellulose, agar, magnesium stearate, aluminum silicate, talc, gum arabic, gelatin, tragacanth, water,
vegetable oil, polyalkylene glycol,
It may be mixed with commonly used pharmaceutical carriers such as liquid paraffin, lanolin, petrolatum, etc. to form an appropriate dosage form. Preservatives, stabilizers, emulsifiers, solubilizing agents, etc. may be mixed as necessary. . The dosage of the compound of formula () depends on the patient's age, disease status, weight,
Although it depends on age, administration method, etc., the oral dosage for adults is usually about 50 mg to 500 mg/day. In the case of intraperitoneal administration, intramuscular administration, or direct administration to a tumor site, the above-mentioned dose may be adjusted as appropriate.

The scope of application of the compound according to the present invention includes skin cancer, uterine cancer, liver cancer, lung cancer, stomach cancer, ascites cancer, and the like.

The pharmacological experiments of the compound of formula () according to the present invention will be described below.

Test 1 Effect on cervical cancer cells Culture solution: The culture solution was a 3:1 mixture of HAM's synthetic media F-10 and L-15, with fetal bovine serum added. Serum concentration is 1/ of the maximum number of cultured cells.
We considered in advance that it would be 2%, and set it to 2%.

Operation method: Culture solution of test sample at various concentrations
Transfer 4.5 ml to a culture dish with a diameter of 60 mm, add 0.5 ml of cervical cancer cell (HeLa S3) culture suspension (containing 5 x 10 ⁴ cells), and incubate in a 5% CO ₂ incubator. The cells were cultured at 37°C for 4 days. The culture solution was removed by suction, washed with physiological saline, and 0.5 ml of 0.125% trypsin-Hank's solution was added as a stripping solution.
Cells adhering to the culture dish were detached, and 4.5 ml of physiological saline was added to suspend the cells, and the number of cells was measured using a Tor automatic hematology apparatus. The results are shown in Figure 1. As is clear from the figure, the compound () is
It can be seen that the proliferation of cervical cancer cells is significantly inhibited at a concentration of 10 μg/ml or less.

Test 2 Effect on black tumor cells Test 1 except that 0.5 ml of black tumor cell (B16) culture suspension (containing 1 x 10 ⁵ cells) was used.
conducted a similar experiment. The results are shown in Figure 2.

Test 3 Effect on lung cancer cells The same experiment as Test 1 was conducted except that 0.5 ml of lung cancer cell (3LL) culture suspension (containing 1×10 ⁴ cells) was used. The results are shown in Figure 3.

Test 4 Effect on ascites cancer 0.5 ml of ascites suspension (containing 1 x 10 ⁶ cells) of ascites cancer cells induced in white mice (BALB/c SrCL) using 2-methylcholanthrene was administered to white mice ( BALB/c, SrCL (6 weeks old, male) was injected intraperitoneally, and 0.5 ml of a physiological saline solution (concentration: 40 mg/50 ml) of the test sample was injected every other day from the next day until the end of the experiment.
was injected intraperitoneally (dose: 20 mg/Kg/2 days), and the number of days the mice survived was examined. The control group was injected with physiological saline. The results are shown in Figure 4. As is clear from the figure, a clear survival effect was observed in the group administered with the compound of formula () compared to the control group.

Test 5 Cytotoxicity Effect of ascites cancer cells obtained in the same manner as in Test 4.
A Hank's solution suspension (containing 1×10 ⁶ cells) was prepared, and 10 µ of an ethanol solution (10 µg/ml) of the test sample was added to 5 ml of this suspension, followed by incubation at 37°C. 1 from 30 minutes after starting incubation
100μ of the cell suspension was collected every hour, stained with 100μ of 0.25% trypan blue solution, and the total cell number and the number of unstained viable cells were counted using hemocytes to determine the cell viability. The results are shown in Figure 5. As is clear from the figure, when the test sample concentration was 10 μg/ml, although cell proliferation was suppressed, almost no cell damage effect was observed.

The following is a compound () (Mh-2) according to the present invention.
Examples of the production of this compound and examples of anticancer compositions containing this compound are given below.

Example 1 Production of Compound (2) (Mh-2) 2g of Gypsophila total saponin containing 2-α-hydroxyprotopanaxadiol saponin as a main component was added to a 0.005M NaH ₂ PO ₄ aqueous solution (pH 4.0)
Dissolve in 100ml, add 1g of cellulase, 37-38
After stirring for 24 hours at 10% aqueous methanol 200 °C
ml and 2 g of cellulase and stir for 6 days. This reaction solution was placed in a 20 g column of μ Bonda Pack C ₁₈ (manufactured by Nippon Waters), washed with 50% aqueous methanol 2, and then eluted with 100% methanol 2. The eluate is concentrated under reduced pressure to obtain 1.2 g of residue. This residue was subjected to column chromatography using 100 g of silica gel (developing solvent: dichloromethane/methanol/ethyl acetate/water (2:2:4:1)), and 43 mg of Mh-2 compound () and gynosaponin TN-1 were obtained. 780mg is obtained.

Mh-2 is a colorless crystalline powder, and its physicochemical properties are as follows.

Melting point: 184-6°C Light exposure: [α] _D = +20.6° (C = 1.7, methanol) Solubility: Soluble in water, methanol and ethanol, sparingly soluble in ethyl acetate, insoluble in benzene and hexane.

1R (ν ^KBr _nax cm ^-1 ): 3400, 1645, 1155, 1075,
1030, 985 (see Figure 6) Elemental analysis (as C ₃₆ H ₆₂ O ₉ 2H ₂ O): C H calculated value (%): 64.07 9.86 Actual value (%): 64.35 9.81 Example 2 Tablet Mh-2 5.0g Lactose 12.4g Microcrystalline cellulose 12.4g Cornstarch 0.1g Magnesium stearate 0.1g Mix the above ingredients well and make 100 plain tablets by direct compression method.
Make locks. Contains Mh-250mg per tablet (300mg).

Example 3 Capsules Mh-2 5.0g Lactose 45.0g The above ingredients were mixed well and capsules were prepared using a conventional method.
Manufacture 250 pieces. One capsule contains 20mg Mh-2
Contains.

Example 4 Granules Mh-2 20.0g Lactose 376.0g Magnesium stearate 4.0g The above ingredients are thoroughly mixed and 400g of granules are produced using a dry granulator. 1 g of granules contains Mh-250 mg.

[Brief explanation of drawings]

Figures 1, 2, and 3 are graphs showing the inhibitory effect of compound () on the proliferation of cervical cancer cells, melanoma tumor cells, and lung cancer cells, respectively. Figure 4 is the survival extension of compound () on mice with ascites cancer. A graph showing the effect, Figure 5 is a graph showing the degree of cytotoxic effect of compound (), and Figure 6 is an infrared absorption spectrum of compound () measured by the KBr method.

Claims (1)

[Claims] 1 Formula: [In the formula, glu represents a β-D-glucopyranosyl group] A compound represented by the following.