JPS62263158A - Novel tetracyclic alkaloid fk separated from cocculus trilobus belonging to menispermaceae family and production thereof - Google Patents

Abstract

NEW MATERIAL:The tetracyclic alkaloid FK 1000 of formula. USE:A carcinostatic agent. PREPARATION:Small chips of the root of a plant belonging to Cocculus trilobus harvested in autumn are extracted with methanol, etc., and a water-transferable fraction of the extract is treated with a spherical resin of a porous polymer such as Amberlite XAD-2. A fraction separated by using water as mobile phase is purified by silica gel column chromatography to obtain the objective substance.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel tetracyclic alkaloid PKIO00 having an anticancer effect represented by: and a method for producing the same.

In Japan, the plant of the genus Chrysanthemum, which contains the compound FK l 000 of the present invention, is called the genus Chrysanthemum, family Chrysanthemum, and plants belonging to this family, such as Chrysanthemum albicans, Chrysanthemum japonica, Chrysanthemum japonica, Chrysanthemum japonica, and Chrysanthemum japonica, are found in Asia. , widely distributed mainly from Southeast Asia to East Asia.

As a result of extensive screening of plants, the inventors confirmed that the extracts of the genus Alocasia spp. and Alocasia spp. This invention was completed by successfully isolating the . The alkaloids conventionally isolated from plants of this family are
While most of them contain a tertiary amino group, the compound PK100O obtained in the present invention was confirmed to be a water-soluble alkaloid with a unique groove structure and a secondary amino group and an enol ether.

In the extraction of FKlooO according to the present invention, the leaves and stems of plants belonging to the genus Pygmyus can be used, but from the viewpoint of the content, it was advantageous to use pieces of the roots. Additionally, the amount of compounds contained increases or decreases depending on the time of plant harvest, but according to the findings of the inventors, those in winter usually contain less, and those in autumn usually contain more. Isolation of the target product from these pieces can be carried out by collecting the water-transferable fraction of the extract extracted with methanol using a porous polymer spherical resin such as Amberlite XAD-2 and using water as the mobile phase. Further purification was performed using silica gel column chromatography to obtain the desired product.

Another method is to directly perform a separation operation using silica gel on the methanol extract, but the target product can be isolated more efficiently by using activated carbon or ion exchange resin. did it. For extraction of the target compound, since the compound has water solubility, water may be mixed in addition to methanol alone, and the mixing ratio can be varied over a wide range as in the case of ordinary extraction of such compounds from plants. It can be adjusted to When using silica gel, a chloroform-methanol system or acetone-methanol-acetic acid can be used as an eluent; in the case of activated carbon, a basic solution is used, and then elution is performed with a water-methanol-acetic acid or methanol-acetic acid solvent. As the basic solution, an ammonia aqueous solution or the like is used.

Regarding acetic acid, it is inappropriate to use strong acids or mineral acids because they may destroy the target product, but similar organic acids can be used.

As the ion exchange resin, cation exchange resins such as DOWEX 50W-X2 (manufactured by The Dow Chemical Company), Diaion SK series (manufactured by Mitsubishi Kasei), and Amberlite rR series (manufactured by Organo) are used. It can be eluted with a basic solution such as an aqueous solution. This method can obtain the target product more efficiently by adding purification using activated carbon. According to this method, there is no need to use particularly purified water, and it is possible to use tap water etc. . In addition, since the object of the present invention has amino groups and hydroxyl groups, a protective group such as trichloroethyl carbamate is introduced into these groups in the purification process as in the conventional method, and removed after isolation. This was an effective means for preventing loss of the target product of the present invention during the isolation process.

Antitumor test In this test, P388 murine leukemia cells, Sarcoma 1
The antitumor effect in vivo was investigated using 80A cells.

Test method P388 mouse leukemia cells CDF, P388 cells 1X intraperitoneally of 1 group of 6 mice each
10.1ml of 10' cells were transplanted, and 10.1ml was transplanted 24 hours after transplantation.
Administer a prescribed amount of FK1000 intraperitoneally once a day for a total of 5 times,
The difference in survival rate between the mouse group and the mouse group to which FKlooO- was not administered was observed.

The results are shown in the table.

to 12.5±0.48 154.62
5 13.5±0.34 167.050
14.3±O0・49 177.3100
16.2±1.92 200.0 control
8.08±0.15 to.

Sarcoma! 80 Ascites-type Sarcoma 180 was transplanted into the abdominal cavity of 5-week-old male Alpino mice (ICR strain), 6 mice per group, and 7 days after transplantation,
FK100 once daily from 24 hours to 5 days after transplantation
A predetermined amount of O was administered intraperitoneally, and the difference in tumor growth rate between a group of mice and a group of mice to which PKlooO was not administered was observed.

The results are shown in the table.

10 +0.7 0.32 76.
0 -20 -0.3 0.29
83.2 -40 +1.6 0,37
56.0 +The present invention will be explained below with reference to Examples.

Example 1 6 kg of dried roots of Aotsudurafuji were mixed with 45 L of methanol.
669 g of extracted extract was obtained from the solution extracted twice, and this extract was suspended in 1.3 L of water, washed three times with two portions of n-hexane, and then distributed three times with two portions of ethyl acetate using a separating funnel. , the aqueous layer was concentrated to obtain 553 g of residue. This residue was applied to an column packed with ion exchange resin Amberlite and chloroform-diethylamine (7:
3), the fractions in which spots were observed around Rr value of 0.32 were collected and concentrated again to obtain 52 g of the target product as a pale brown, indestructible powder.

Specific optical rotation [α] 6° knee 77.0° (c=0.1.Me
OH) I n : ν! ! ! Scm-' 3400
(br)U■:λuni! ”nm (ε) 215(
16000), 234 (8000), 283 (2700
) MSIII/Z (%): 333 (M”l 2),
318 (19), 258 (+00), 244 (16),
200 (3) 115 (5), 83 (7) CD (c = 9.67 X 10'S, MeOI-1) (
θ) ”(nm): +62100 (238)(po
sitive maximum)' II -N M
R (CD y OD ) δ: 6.75 (IH, d, J
=8.3l-1z) [i, 53 (th I, d, J=8.
3Hz), 4.35 (II-I, d, J=6.1H
z), 4.28 (IH, d, J=3.5Hz), 3.8
4 (IH, ddd, J=13.3.3.7, 3.5Hz
), 3.82(311,!3), 3.68(31(,
s), 3.1 5 (I H, dd, j = 17.7
, 6.111z), 2.90 (IH, dd, J=13.
3゜3, 711z>, 2.89 (I H, d, J =
17.7) fz), 2,73 (I H, dd, J
=13.1.4.7H2), 2.63(IH.

ddd, J=13.1, 12.5, 3.4Hz), 2.
17 (IH.

t, J=13.31-! z), 2.01 (IH, dd,
J=12.7°3.4Hz), 1.88(IH, d
dd, J=12.7, 12.5°4, 7 Hz) 13C-NMrt (CD30D, δ, ppm):
I 47.67(s), 147°B l(s)-14
5-72(s), I 30-/l 0(s), 1299
5(s), 121.47(s), 119.51(d),
110°96(d), 68.57(d), 66.47(
d), 57.26 (Q) 56.95 (q), 47.15
(d), 41.00 (L), 39°57 (8), 37.
16(t), 36.78(t), 36.09(t) Example 2 800g of the whole plant of Houzanthuzurahuji was added to 2L of 10% methanol water.

120 ml of 28% ammonia water was added to 12 L of the solution extracted with 2 UX 5 water to make it alkaline, and 100 g of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was added to this liquid and stirred for about 30 minutes.

The extract containing activated carbon was filtered, washed with water until the filtrate became neutral, eluted with methanol-acetic acid (100:1), and concentrated at about 40°C. The concentrated solution was poured into a DOWEX 50W-X2 column, which is a strongly acidic cation exchange resin, and the acidic and neutral components in the non-adsorption area were washed away with purified water. It was washed with normal ammonia water, and the two fractions eluted at 1.5 normal were collected. 20 g of activated carbon was added to 2 L of this solution, stirred, and then filtered. After washing with water until the filtrate becomes neutral, it is eluted with a methanol-acetic acid (100:1) solution, and the
2.8 g of FKlooo is concentrated at °C to give a light brown amorphous form.
I got it.

IR: Shi! :cm-' 3400 (br), 1
670.1600, Uv:λ Engineering! ! ”nm (g)
260 (3000), 287 (3580)'H-NMR
(DtO-DSS) δ: 6.89 (IH, d, J=8
．． 3Hz), 6.65 (IH, d, J=8.3Hz)
, 4.41 (IH.

d, J=6.1H2), 4.38(IH, d, J=3.
9H2), 3.99 (IH, ddd, J=13.2,3
．． 9,3.7Hz), 3°82 (3H,s), 3.6
8 (3H, s), 3.19 (IH, dd.

J = 18.3, 6.1 Hz), 2.92 (IH,
d, J=18°3Hz), 2.93-2.43(3H,
complex), 2.06 (IH, dd, J=13.
4,13.4H2), 1.95-1.82 (2H, c
complex) ”C-NMR (DtO-DSS, δ, 1) I) Il1)
: 149.4 (s), 147, 3 (s), 1
47, 1 (s), 132.5 (s), 132.1 (
s), 127.1(s), 121.0(d), 113.
3(d), 69.6(d), 67.5(d), 60.2
(Q), 59, 1 (Q), 47.8(d), 41.
8(t), 40.9(S), 39.0(t), 37.7
(t), 37.7 (t) Example 3 0.8Kg of dried rhizomes of P. elegans were immersed in 3L of methanol at room temperature, and the resulting extract was treated in the same manner as in Example 2 to obtain 2.0g of Obtained FK 1000.

Example 4 In the same manner as in Example 3, 0 and 8 kg of PKlooO were treated to obtain 2.4 g of PKlooO as an acetate salt.

Example 5 1.9 g of FK 1 000 was obtained from the extract extracted using 10% methanol water (2 L) and then water (2 L x 5) instead of methanol in Example 3.

Claims (1)

[Claims] 1) FK1000 compound represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ 2) A formula characterized by separating and purifying an extract obtained by extracting from a plant of the genus Trifoliaceae ▲There are mathematical formulas, chemical formulas, tables, etc.▼ Manufacturing method of FK1000 compound represented by