JPH054998A - Sterol compound - Google Patents

Abstract

PURPOSE:To provide the title new compound found in an extract from sponge belonging to Xestospongia, having in the molecule both cyclopropane ring and oxirane ring, having antineoplastic activity, thus useful for e.g. the therapy of leukemia. CONSTITUTION:The objective new sterol compound can be obtained by the following processes: a sponge belonging to Xestospongia collected at the coral reef in the sea near the Iriomote Island, Okinawa Pref., Japan, is immediately frozen with dry ice, the resulting frozen sample is immersed in cold methanol overnight, the methanol extract obtained is filtered, and the filtrate is concentrated under reduced pressure followed by suspension in water. The resulting suspension is extracted with ethyl acetate, the extract obtained is concentrated under a vacuum, and the ethyl acetate solubles are put to silica gel column chromatography. The resulting active fraction is then put to flush chromatography, the active fraction obtained is collected and crystallized, and the resulting crystal is filtered and then recrystallized using hexane-ethyl acetate, thus obtaining the objective new sterol compound of the formula in the form of colorless needle crystal.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sterol compound having an antitumor effect.

[0002]

2. Description of the Related Art There is no report of a compound having a structure similar to this compound and having an antitumor effect.

[0003]

The object of the present invention is to provide a novel compound having an antitumor effect.

[0004]

[Means for Solving the Problems] Xestospongi
The sponge of the genus a is collected on a coral reef near the sea near Iriomote Island, Okinawa Prefecture. The present inventor has found that the extract of sponge of the genus Xestospongia contains a novel sterol compound having an antitumor effect, and completed the present invention.

The compound of the present invention is a sterol compound represented by the following chemical formula 2.

[0006]

[Chemical 2]

The sterol compound represented by the chemical formula 2 of the present invention is obtained by extracting Xestospongia sponge as a raw material, extracting this with an organic solvent, and fractionating and purifying the extract obtained from the extract by a commonly used method. Isolated. As the organic solvent, for example, methanol, ethanol, ether, acetone, benzene, toluene, ethyl acetate or the like is used, but not limited to this, other suitable organic solvent can be used. Fractionation and purification are performed by, for example, a chromatography method such as silica gel column chromatography, preparative silica gel thin layer chromatography, high performance liquid chromatography, and the like, a recrystallization method, and the like. The elution solvent used here is, for example, ether, petroleum ether, n-hexane, ethyl acetate, benzene, toluene, acetone, methanol, ethanol, chloroform, dichloromethane or the like alone or as a mixed solvent.

[0008]

According to the present invention, Xestospongi
A novel sterol compound was isolated from a sponge of the genus a and it was found that it has an antitumor effect and is useful as a pharmaceutical.

Test Example 1 [Antitumor effect (In vitro
Screening for inhibition of proliferation of KB cells] Method 1 × 10 ³ KB cells / well in a 96-well plate with a flat bottom
0.1 ml of cell suspension (MEM containing 10% fetal bovine serum)
(Suspended in the medium) was added and cultured for 24 hours. 100 μl of a solution of the compound of the present invention dissolved in dimethyl sulfoxide and diluted with a medium (final concentration of dimethyl sulfoxide was 0.5
%) Was added and the cells were further cultured for 72 hours. After culturing 4,
5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide reagent (coloring reagent) was added, and the mixture was further cultured for 4 hours.

As a control, 100 μl of a medium (final concentration of dimethyl sulfoxide 0.5%) was added, and the cells were cultured in the same manner. After culturing, remove the medium and add 150 μl of cells.
Was dissolved in dimethylsulfoxide and the absorbance was measured, the ratio of the absorbance of the compound of the present invention to the absorbance of the control group was determined, and the 50% inhibitory concentration (IC ₅₀ ) was calculated.
As a result, IC ₅₀ was 0.042 μg / ml.

Test Example 2 [Life extension effect on in vivo P388 leukemia] Method 5 × 10 ⁵ P388 leukemia cells were intraperitoneally transplanted into DBA / 2 female mice, and tumor cells were collected from ascites on the 7th day. A cell suspension containing 5 × 10 ⁶ viable cells / ml (suspended in Hank's balanced salt solution) was prepared, and 0.2 ml (1
CDF1 female mice (7 weeks old) were intraperitoneally transplanted (× 10 ⁶ cells / mouse). The day of cell transplantation was set to day 0, and the compound of the present invention suspended in 0.5% gum arabic-physiological saline was intraperitoneally administered for 5 days from the day following cell transplantation.

Adriamycin (ADM) as a control drug
Was dissolved in physiological saline and used. The effect was obtained by calculating the median survival time (MST) of mice,

[0013]

[Equation 1] It was judged by. The results are shown in Table 1.

[0014]

[Table 1]

(Note) 1. The weight gain was represented by the difference in body weight from day 4 to day 1. 2. The control MST was 9.3 days.

Test Example 3 [Life extension effect on in vivo L1210 leukemia] Method 1 × 10 ⁵ L1210 leukemia cells were intraperitoneally transplanted into DBA / 2 female mice, and tumor cells were collected from ascites on the 7th day. Prepare a cell suspension containing 5 x 10 ⁵ viable cells / ml (suspended in Hank's balanced salt solution) and prepare 0.2 ml of the suspension.
(1 × 10 ⁵ cells / mouse) were intraperitoneally transplanted into CDF1 strain / female mice (7 weeks old). The day of cell transplantation was set to day 0, and 5 days from the day after cell transplantation, 0.5% gum arabic-
The compound of the present invention suspended in physiological saline was intraperitoneally administered.

Adriamycin (ADM) as a control
Was dissolved in physiological saline and used. The effect was determined in the same manner as in Test Example 2. The results are shown in Table 2.

[0018]

[Table 2]

(Note) 1. The weight gain was represented by the difference in body weight from day 4 to day 1. 2. The control MST was 9.1 days.

[0020]

EXAMPLES Next, the present invention will be described more specifically by way of examples.

Example Xestosp collected from a coral reef near Iriomote Island, Okinawa Prefecture
The sponges of the genus ongia were immediately frozen on dry ice. This frozen sample (3.1 kg) was cold-soaked with methanol (6 L) overnight. The methanol extract was filtered and the filtrate was concentrated under reduced pressure to obtain a residue of 87.4 g. The same methanol extraction was performed twice more to obtain 50 g of extract. The methanol extracts were combined (137.4 g) and this was added to water 1.
It was suspended in 5 L and extracted 3 times with 1.5 L of ethyl acetate. The ethyl acetate-soluble parts were combined, and concentrated under reduced pressure to obtain ethyl acetate-soluble 26.0 g. 21 g of ethyl acetate-soluble portion was subjected to silica gel column chromatography (Fujidevison BW-8
20 MH, 500 g) to give 5 fractions (fraction 1;
Elution with 1000 ml of hexane, fraction 2; elution with hexane: ethyl acetate = 2: 1, 1000 ml, fraction 3; elution with hexane: ethyl acetate = 1: 2, 1000 ml, fraction 4; elution with 1000 ml of ethyl acetate Min 5; eluted with 1500 ml of methanol). Fraction 4 obtained here (6.56)
g) was further subjected to silica gel column chromatography (Fujidevison BW-820MH, 150 g, elution solvent; hexane: ethyl acetate = 1: 1, 1 fraction; 40 ml) to obtain 25 fractions. Fractions 13 to 20 (2.91 g) were subjected to flash chromatography (silica gel; Fujidevison BW-300, 200 g, elution solvent; hexane: ethyl acetate = 2: 1, 1 fraction; 40 ml), and 66 fractions were collected. Obtained. Fractions 41 to 47 (460 mg) crystallized, and were recrystallized from hexane-ethyl acetate to obtain 233 mg of the present compound as colorless needle crystals.

Melting point: 157 to 158 ° C. Elemental analysis value; Calculated value (%) C: 75.94, H: 10.11 Measured value (%) C: 75.64, H: 10.20 IR (KBr) cm ^-1 ; 3490, 3394, 1708, 1030

¹ H-NMR (500 MHz, CDC
l ₃ ) δ (ppm) 0.16 (1H, q, J = 4.5 Hz), 0.26 (2
H, m), 0.50 (1H, heptet, J = 5.9)
Hz), 0.73 (3H, s), 0.83 (1H,
m), 0.91 (1H, m), 0.95 (3H), 0.
96 (1H, m), 1.01 (3H, s), 1.02
(3H, d, J = 6.1 Hz), 1.07 (1H, d
d, J = 7.8 Hz, 11.0 Hz), 1.45 (1
H, m), 1.50 (1H, m), 1.62 (1H,
m), 1.71 (1H, qd, J = 3.1 Hz, 13.
1 Hz), 1.75 (1H, m), 1.79 (1H, t
d, J = 4.2 Hz, 13.0 Hz), 1.95 (1
H, m), 2.03 (1H, ddd, J = 2.2 Hz,
6.4 Hz, 14.0 HZ), 2.09 (1 H, dd
d, J = 2.1 Hz, 3.8 Hz, 15.0 Hz),
2.16 (1H, dd, J = 9.0Hz, 10.8H
z), 2.25 (1H, t, J = 14.3Hz), 2.
31 (1H, brd, J = 13.5Hz), 2.37
(1H, dt, J = 6.5Hz, 13.5Hz), 2.
92 (1H, d, J = 3.9Hz), 3.08 (1H,
d, J = 3.9 Hz), 3.39 (1H, dd, J =
4.6 Hz, 11.0 Hz), 3.46 (1 H, dd,
J = 2.4Hz, 10.6Hz)

¹³ C-NMR (125 MHZ, CDCl
₃ ) δ (ppm) 8.2 (CH ₃ ), 11.4 (CH ₃ ), 12.4 (C
H ₂ ), 12.5 (CH), 18.9 (CH ₃ ), 1
_{9.1 (CH 3), 23.7 (} CH 2), 26.8 (C
H ₂ ), 27.6 (CH), 28.8 (CH ₂ ), 2
9.4 (CH ₂ ), 31.0 (CH ₂ ), 33.7 (C
H), 34.9 (CH), 35.6 (-C-), 38.
1 (CH ₂ ), 38.4 (CH ₂ ), 40.1 (C
H ₂ ), 44.5 (CH ₂ ), 46.5 (CH), 4
8.1 (CH), 48.8 (-C-), 50.6 (CH
₂ ), 52.2 (CH), 54.3 (CH), 65.8.
(-C-), 72.2 (CH), 77.4 (CH), 2
11.8 (CO)

Claims (1)

What is claimed is: 1. A sterol compound represented by Chemical Formula 1.