JPH06287191A - Mycalamidelike substance - Google Patents

Abstract

PURPOSE:To obtain a mycalamidelike substance, capable of manifesting strong cytotoxicity, having suppressing action on tumorous cell proliferation and useful as an antitumor agent, etc., by purifying an extract from a sponge of the genus Theonella according to the column chromatography, etc. CONSTITUTION:A frozen sample of a sponge, having an yellow interior thereof, which belongs to the thenoella.sp. collected at 10-15m depth of water near HACHIJO Island is extracted with ethanol and partitoned into two layers with water and diethyl ether. The resultant diethyl ether layer is subsequently partitioned with n-hexane and 90% methanol. The hydrous methanol layer is then partitioned into two layers with carbon tetrachloride and 80% methanol to provide a carbon tetrachloride layer. The obtained carbon tetrachloride layer is subsequently fractionated by flash chromatography and the fraction eluted with 70% methanol is fractionated by the gel filtration. The prepared active fraction is purified according to the reversed phase high-performance liquid chromatography, etc. Thereby, the objective mycalamidelike substance, expressed by the formula (R is 2-hydroxy-4-methoxycarbonylbutyl, hydroxyl, etc.; Me is methyl) and having antitumor action is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel mycalamide-like substance which is an extract component from sponge and exhibits an antitumor action.

[0002]

2. Description of the Related Art Several bioactive substances have been isolated from sponges. For example, from Harichondria okadai, a fatty acid consisting of 38 carbon atoms (okadaic acid, Okadaic
acid) and norharichondrin A have been reported [J. Am. Chem. Soc.], 1981, 103, 2469-2471 and
J. Am. Chem. Soc., 1985, 107, 4796-4798], Pandaros acanthifolium reported a polyether carboxylic acid having an episulfide bond (acanthifolicin) [J. Am. Chem. Soc., 1981, 103, 2467-2468], and the Caribbean sponge Tedania ignis .
Reported a cytotoxic 18-membered ring macrolide (Tedanolide) [J. Am. Chem. Soc., 19
84, 106, 7251-7252].

[0003]

DISCLOSURE OF THE INVENTION The present inventors have conducted a search for physiologically active substances from Japanese sponges and found a spiroketal compound having an antitumor activity from sponge Discodermia calyx ( JP-A-62-178595). In addition, a macrolide substance showing strong cytotoxicity was found from sponges of the genus Mycale ( Mycale sp.), And a patent application was filed (Japanese Patent Application No. 2-1749).
54). Furthermore, when we searched for new and useful physiologically active substances from the sponges, we found that the sponge genus Teonera ( Theonera spp. )
The substance showing strong cytotoxicity was found in the lipophilic extract component of Lla sp.), and this substance was isolated to complete the present invention. That is, an object of the present invention is to provide a novel and useful physiologically active substance from sponge.

[0004]

The present invention is directed to the genus Theonella ( Theo ) that lived in the waters around Hachijojima Island at a depth of 10 to 15 m.
It was found that there are several substances showing strong cytotoxicity in the lipophilic extract of sponge of Nella sp.), and this substance was isolated to complete the present invention. The substance of the present invention is a mycalamide-like substance represented by Chemical formula 2.

[0005]

[Chemical 2] In the formula, R represents a 2-hydroxy-4-methoxycarbonylbutyl group [Chemical Formula 3], 2-hydroxytetrahydropyran-
6-yl group [Chemical Formula 4], hydroxyl group [Chemical Formula 5], 2-oxotetrahydrofuran-5-yl group [Chemical Formula 6] or 2
An -oxotetrahydropyran-6-yl group [Chemical formula 7] is shown. Further, Me represents a methyl group.

[0006]

[Chemical 3]

[Chemical 4]

[Chemical 5]

[Chemical 6]

[Chemical 7]

The mycaramide-like substance of the present invention is a sponge Th.
It can be obtained by extracting eonella sp. with ethanol, partitioning the extracted components into two layers with water and diethyl ether, and isolating from the components that migrate to the diethyl ether layer. The sponge Theonella sp., Which is the raw material, grows in the waters around Hachijojima, as well as in the waters around Japan, and can be collected and used. The sponge is frozen, crushed and then extracted. Extraction is performed at room temperature, and ethanol used for extraction is 99% or more. Extraction is performed several times using ethanol in an amount not less than twice the amount of sponge. The extract was concentrated under reduced pressure, if necessary, and then subjected to two-layer partition with water-diethyl ether, and the diethyl ether layer was mixed with n-hexane and 90%.
Two layers are distributed with methanol, and 90% methanol group is further distributed with two layers of carbon tetrachloride and 80% methanol to obtain a carbon tetrachloride layer. This carbon tetrachloride layer is subjected to flash chromatography (silica gel, solvent: hydrous methanol) using ODS to fractionate the active ingredient.

The active fraction eluted with 70% methanol was subjected to gel filtration using Toyopearl HW40 (trade name) with chloroform / methanol (1: 1) and hexane / chloroform / methanol (8: 7: 1). I and active fraction II were obtained. This active fraction I is Capcell pack
Reversed phase H with 60% methanol using C ₁₈ (trade name)
Purification by PLC gave compound 1. On the other hand, active fraction II
Was subjected to reverse phase HPLC using Senshupak ODS-H-4251 with 70% methanol to give compound 2. This reversed phase H
Further three active fractions A, B and C were obtained by PLC. The active fractions A and B are respectively Capcell pack C
Reverse phase HPL with 60% methanol using ₁₈ (trade name)
Purification by C gave Compound 3 and Compound 4. Active fraction C
Was fractionated on a silica gel column and purified by reverse phase HPLC with 60% methanol using Capcell pack C ₁₈ (trade name) to obtain Compound 5.

The physicochemical substances and chemical structural formulas of compounds 1 to 5 of the present invention are as follows. 1. Compound 1 (MS-1030-A-2-1-A-
1): 1) Molecular weight 573 2) Molecular formula C ₂₈ H ₄₇ NO ₁₁ 3) Light rotation [α] _D + 49.1 ° (c0.06, CHCl ₃ ) 4) Mass spectrometry (FABMS) m / z 574 (MH ⁺ ), 542 (M ⁺ -C
H ₃ O), 541 (M ⁺ -CH ₃ OH) 5) ¹ H and ¹³ C NMR (CDCl ₃ ) as shown in Table 1.

2. Compound 2 (MS-1030-A-2-1-4): 1) Molecular weight 543 2) Molecular formula C ₂₇ H ₄₅ NO ₁₁ 3) Light rotation [α] _D + 88.1 ° (c0.14, CHCl ₃ ) 4 ) Mass spectrum (FABMS) m / z 566 (M + Na ⁺ ), 494 5) ¹ H and ¹³ C NMR (CDCl ₃ ) As shown in Table 2.

3. Compound 3 (MS-1030-A-2-1-1-1) 1) Molecular weight 459 2) Molecular formula C ₂₂ H ₃₇ NO ₉ 3) Light rotation [α] _D + 136.7 ° (c0.03, CHCl ₃ ) 4 ) Mass spectrometry (FABMS) m / z 482 (M + Na ⁺ ), 428
(M ⁺ -CH ₃ O) 5) ¹ H and ¹³ C NMR (CDCl ₃ ) As shown in Table 3

[0012] 4. Compound 4 (MS-1030-A-2-1-2-1) 1) Molecular weight 527 2) Molecular formula C ₂₆ H ₄₁ NO ₁₀ 3) Light rotation [α] _D + 80.0 ° (c0.04, CHCl ₃ ). 4) Mass spectrometry (FABMS) m / z 550 (M + Na ⁺ ), 496
(M ⁺ -CH ₃ O), 478 5) ¹ H and ¹³ C NMR (CDCl ₃ ) As shown in Table 4.

5. Compound 5 (MS-1030-A-2-1-2-2) 1) Molecular weight 541 2) Molecular formula C ₂₇ H ₄₃ NO ₁₀ 3) Light rotation [α] _D + 172.0 ° (c0.03, CHCl ₃ ) 4 ) Mass spectrometry (FABMS) m / z 542 (MH ⁺ ), 510 (M ⁺
-CH ₃ O) 5) ¹ H and ¹³ C NMR (CDCl ₃ ) As shown in Table 5

[0014]

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

From the above physicochemical substances, the chemical structural formulas of compounds 1 to 5 are considered to be as follows. Compound 2 is considered to be two isomers of Chemical formula 9 and Chemical formula 10 depending on the position of the hydroxyl group at the 21st position. Compound 1 [Chemical Formula 8] Compound 2 [Chemical Formula 9] and [Chemical Formula 10] Compound 3 [Chemical Formula 11] Compound 4 [Chemical Formula 12] Compound 5 [Chemical Formula 13]

[Chemical 8]

[Chemical 9]

[Chemical 10]

[Chemical 11]

[Chemical 12]

[Chemical 13]

Next, the usefulness of the compound of the present invention will be described. The compound of the present invention has an excellent antitumor effect. In vitro inhibition of tumor cell growth and in vitro
The antitumor effect in vivo is shown together with the experimental method.

(I) Action in vitro Cell growth inhibition test against leukemia cells P388 RPMI containing tumor cells P388 in 10% fetal calf serum
Using the solution added to 1640 culture medium, the number of cells in the culture medium is adjusted to 1 × 10 ⁵ cells / ml. Dispense 1 ml into a plastic well. The mycalamide-like substance is dissolved in dimethylsulfoxide (hereinafter abbreviated as DMSO) and added to the cell suspension culture solution so that the final concentration of DMSO is 0.4% by volume and the mycaramide-like substance is at a predetermined concentration. The cells were cultured in air containing gas for 3 days. As a control, a cell suspension culture solution containing 0.4% by volume of DMSO was similarly cultured. After culturing, the cells were stained with Trivan blue staining solution, the number of viable cells was counted, and the IC ₅₀ value (50% cell growth inhibitory concentration) was determined from the inhibition rate relative to the control. Mitomycin C was used as a control drug. The results are shown in Table 6.

[0018]

[Table 6] ──────────────────────────────────── Compound number MS number IC ₅₀ (ng / ml ) ──────────────────────────────────── 1 MS-1030-A-2-1-A- 1 0.11 2 -4 0.053 3 -1-1 9.1 4 -2-1 1.1 5 -2-2 0.71 ───────────────── ──────────────────── Mitomycin C 10 or 9.2 ──────────────────────── ─────────────

(Ii) In vivo action Antitumor test against leukemia cells P388 P38 on day 7 after intraperitoneal transplantation in DBA / CRJ mice
4 × 10 ⁵ cells of 8 cells were intraperitoneally transplanted into 6 BDF ₁ mice (5 weeks old, male) per group. The active ingredient was intraperitoneally administered 24 hours after transplantation according to the schedule shown in Table 7. The intermediate survival days of each active ingredient administration group were calculated for the tumor growth inhibitory effect, and the survival rate (T / C) of each administration group to the control group was calculated.
%) Was calculated from the following formula. The results are shown in Table 7. T / C% = T / C × 100 T: intermediate survival days of administration group C: intermediate survival days of control group

[0020]

[Table 7]

In order to use the mycalamide-like substance of the present invention as a medicine, it is administered in a form convenient for exhibiting an antitumor effect. The micamalamide-like substance may be a medicinal product as it is, but may also be provided in a composition according to pharmaceutical practice as a mixture with a pharmaceutically acceptable diluent and / or other pharmacologically active substance. Therefore, the mycalamide-like substance of the present invention can appropriately take a form for oral or parenteral administration. Eg powders, granules,
Tablets, dragees, capsules, pills, suppositories, suspensions, solutions,
Emulsions, injections and aerosols. The dosage of the mycaramide-like substance of the present invention varies depending on various causes such as sensitivity difference, age, sex, body weight, administration method, administration timing, interval, medical condition, physical condition, property of pharmaceutical preparation, kind of preparation.

The compounds of the present invention and the process for producing them will be further described with reference to Examples.

[Examples] A frozen sample of the sponge Theonella sp. With a yellow inside was collected at a water depth of 10 to 15 m around Hachijojima, and 15 kg of the sample was extracted with ethanol and partitioned into two layers with water and diethyl ether. After the diethyl ether layer was partitioned with n-hexane and 90% methanol, the hydrous methanol layer was further partitioned into two layers with carbon tetrachloride and 80% methanol to give a carbon tetrachloride layer of 2.1 g.
Got The carbon tetrachloride layer was fractionated by flash chromatography using ODS and hydrous methanol. The active fraction eluted with 70% methanol was added to TOYOPEARL
) HW40 with chloroform / methanol (1: 1) and hexane / chloroform / methanol (8: 7:
The fractions were sequentially fractionated by gel filtration using 1) to obtain active fractions I and II. Active Fraction I is a capsule pack (Capcell p
ak) Purified by reverse phase HPLC using C ₁₈ and 60% methanol to give 2.1 mg of compound 1. On the other hand, the active fraction II was treated with Senshupak ODS-H.
Reversed phase HPLC with -4251 and 70% methanol
Then, 4.8 mg of Compound 2 was obtained and three active fractions A, B and C were obtained. Active fractions A and B were purified by reverse phase HPLC using Capcell pak C ₁₈ and 60% methanol, respectively, to give 0.6 mg of compound 3 and 0.8 mg of compound 4. The active fraction C was fractionated with a silica gel column and then Capcell pak C
Purification by reverse phase HPLC using ₁₈ and 60% methanol gave 0.5 mg of compound 5. The physicochemical properties and chemical structural formulas of Compounds 1 to 5 were as described above.

Claims (1)

[Claims] 1. A mycalamide-like substance represented by Chemical Formula 1 In the formula, R represents 2-hydroxy-4-methoxycarbonylbutyl group, 2-hydroxytetrahydropyran-6-yl group, hydroxyl group, 2-oxotetrahydrofuran-
5-yl group or 2-oxotetrahydropyran-6-
Represents an yl group. Further, Me represents a methyl group.