JPH0441429A - Seafood-derived tumor cell growth inhibitor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a tumor cell growth inhibitor collected from yellow starfish and the like.

[Conventional technology]

Currently, various substances that inhibit tumor cell proliferation are known, and numerous examples of their isolation from the natural world have been reported. However, no case of isolation from starfish has yet been reported.

On the other hand, an example of isolation of steroid saponins from starfish is Malthasterias glacialis.
Turner et al. have reported on S. ias 1acialis (Nature, 233(5316),
209-210.1971).

They determined the structure of this constituent steroid and named it Marsasterone. In addition, Kitagawa et al.
Two types of steroidal saponins were separated from er1anci, and the constituent steroids were turnastyrol A and B (
They were named Thornasterol A, B) and their structures were determined. The inventor also studied the yellow starfish (^5terias amur).
Several types of steroid saponins were isolated from the ovaries of Ovarian Asensis.
Terosaponin) (Agric, B
iol.

Chew,, 45 (3), 801-803.8
05 807.1981. same.

45 (9L 2823-2826. 1985). The pharmacological effects of some of its constituent steroids have also been reported (J, Pharmacobio-Dyn, +
EL 5-76+ 1985), including the effect of suppressing the growth of tumor cells.

[Problem to be solved by the invention]

Many types of anticancer drugs have been developed, but no one with significant efficacy has yet been developed. Therefore, by further developing substances that can suppress the proliferation of tumor cells, it is possible to provide a means for producing new anticancer agents.

[Means to solve the problem]

In order to achieve the above object, the present inventor extensively investigated the growth-inhibiting effect on AT tumor cells, mainly using starfish steroids, and as a result, succeeded in obtaining a substance with a remarkable growth-inhibiting effect.Based on this knowledge, the present inventors was able to complete.

That is, the present invention relates to a tumor cell proliferation inhibitor containing a steroid having any of the following structural formulas or a salt thereof.

Among the above steroids, the steroid saponin represented by the structural formula (1) is a starfish (Asteriasas +
urensis) and named Ovarian asterosaponin-4 (OA-4).

The physical properties of 0A-4 are melting point 207-209°C1
Specific optical rotation [α]: ”-x, s° (C=1.8, H2O
), elemental analysis S: 2.4%, ultraviolet absorption (HzO) 2
None above 30 nm. The Rf value of thin layer chromatography is 0.16 when using a silica gel 60 plate and developing with chloroform-meta/-water (10:5:1, v/v);
When developed with 50% methanol aqueous solution using a 0 plate, the value is 0.47. The method for determining the structure is Agric,
Biol, Cheta, 47(9).

2823-2826.1985, and can be determined accordingly. 0A-4 may be in the form of a salt such as a sodium salt or a calcium salt.

The steroid saponin represented by the structural formula (I[) is a desulfated product of the steroid saponin of the structural formula (1). For example, the steroid of (1) is converted into a pyridine salt and heated in dioxane to form a solvate. It can be obtained by decomposition. The physical properties of this 0A-4 desulfonated product include a melting point of 232 to 233.5°C, a specific optical rotation [,y)p
+ 7° (C=0.9, methanol), Kiesel
7 using gel 60FZ545ilanisielt
Rf value when developed with 0% methanol aqueous solution is 0.4
It is 0.

"C-NMR (C, D, N-H, O15:1) C-13
6,2 231,6 370,8 432,8 549,3 679,4 741,4 835,5 9145,8 1038,5 11116,7 1242,4 1341,5 1454,2 1523,2 1625,1 1758, 7 1813, 6 1919, 4 2074, 3 2125, 8 2323, 5 24125, 5 25131, 1 2625, 9 2717, 7 FD-Mass spectrum m/z 1187 (M”(CsbHzqzOza)+
K, 15%) 1171 (M”+ Na, base
peak) 1153 (M ” + N a Hz
0156%) 1087 (M'+Na-C,H,! (side chain + H), 12%) 1025 (M''+ Na C6
H1004 (Kinotos residue), 23%) 1007 (M”+Na-H2O-C6H1004,2
8%) 863 (M”+Na CtzHzoOq
(quinonese + glucose residue), 12%) 845 (
M'+Na HzOCtzHzoOq, 6%)
The steroid saponin represented by the structural formula (I[l) is also a desulfated steroid extracted from the starfish and named Ovarian asterosabonin-1 (OA4). For example, the separation method for 0A-1 is ^gric
, Biol, Chew, 45(3), 801
-803.1981, and the method for determining the structure is 80 of the same magazine.
5-807. A desulfated product of 0A-1 can also be obtained by similarly converting 0A-1 into a pyridine salt and heating it in dioxane to cause solvolysis.

The steroid represented by structural formula (IV) is an aglycone of 0A-1, and can be obtained by treating 0A-1 with, for example, glycosidase to excise the sugar moiety. This steroid is turnastyrol A (Th
Ornasterol A) is also known as (T
etraihedron Letters No. 11
+pp967 970+1975).

As for physical property values, Rf O, 54 (Kiesel ge
l 60Fzsn, chloroform-methanol-water, 1
0:5:1, V/V), Rf O, 51 (Kiesel
gel 60Fzsn, n-butanol:acetic acid-water,
10:1:1, v/v), Rf O, 54 (Kiese
l ge160Fzs4Silanisielt, 50
% methanol), (α):”-7’ (C=I, O, methanol) and CD: [θ]!9@nll 180
0 (C=0.6, methanol).

The steroid represented by structural formula (V) is maltasterone (Nature, 233(5316), 209-2
10.1971), which is extracted from Malthasterias gracilius (Bioche+++,
J, 1970.117.543).

Ultraviolet absorption is 240n+m and infrared absorption is 169
It is located at 0 cm-'.

The steroid represented by the structural formula (VI) has the structural formula (II
), and can be obtained by treating this steroid saponin with, for example, glycosidase to remove the sugar moiety. The physical property values are as follows.

13C-NMR (CD, 0D) 37.0 32.0 71.8 33.4 51.0 69.8 43.3 37.0 146.8 39.3 11? , 7 43.6 42.4 55.1 24.0 25.7 59.6 13.7 19.7 75.6 25.9 44.8 23.5 125.8 131.8 25.9 27 17. 7 When El-mass spectrum was measured, m/z 39
8 (M”-H, 0180%), m/z 333 (M”
C6H13, 30%), m/z 315 (M”-C,
H,, -H2O, base peak) -21H),
1.61・1.68ppm (3H, S, cH, respectively)
s) 3, C-26/27H), 3.57/3.70p
pm (IH, brSC-3/6H, respectively), 5
．． 08ppn+(I Hlbroad t, H"',
C-24H), 5.30ppm (lH, broads
, )('-(, C-11H).

The steroid represented by the structural formula (■) has the structural formula (I[[
) It is an aglycone of steroid saponin represented by 1, and can be obtained by treating this steroid saponin with, for example, glycosidase to remove the sugar moiety. As for the physical property value, the specific optical rotation is [α]”+6
2.3° (C=0.9, methanol-α), CO is [θ] 287nm+7200 (C=0
．． 08, methanol) and MS is M″″332.

"C-NMR (CD, 0D) C-136,9 231,9 371,4 433,4 550,3 669,6 743,3 B 37.0 9 147.5 10 39.3 11 117.1 12 41 .6 13 43.7 14 54.9 15 23.8 16 26.4 17 64.4 1B 13.4 19 19.7 20 211.9 21 31.3 The tumor cell growth inhibitor of the present invention is a known one. It can be used in the same way as the liquid formulation, by adding excipients if necessary.
It can be used in the form of an i-drug, etc. As for the administration method, injection administration, oral administration, etc. can be used. The type and site of the tumor are not particularly limited, and it can be used for various mammals other than humans. Dosage is 0.1-100■7
The appropriate weight is about the weight of the body.

〔Example〕

(1) Preparation of Asterosavonins 0A-1 to 0A-5 First, ovaries were removed from yellow starfish during the breeding season and freeze-dried. 100g of this dry powder was mixed with chloroform-methanol (2:
1, v/v) and homogenized with a Waring blender for extraction. The residue was roughly extracted with a mixed solvent 11 of chloroform-methanol-water (20:40:3, v/v). The extracts were combined and the chloroform-methanol-water ratio was adjusted to 10:5:3 (v
/v).

The upper layer was separated and concentrated under reduced pressure. The remaining aqueous solution was separated into layers between n-butanol and water (pH 4,0). The upper layer was separated, neutralized, and concentrated. The residue was dissolved in a small amount of water and dialyzed twice against 52°C of water. The dialysis residue was dissolved in 60 d of chloroform-methanol-water (30:60:8, ■/ν).
and applied to a DEAE-Sephadex A-25 (acetate type) column (2.2X 12.7^m). 300 d of chloroform-methanol water (3
0:60:8, v/v), then 75
Elution was carried out with 0 d of chloroform-methanol-0,8A sodium acetate M (30:60:8, v/v).

After concentrating the eluate, it was dialyzed against water to obtain a crude saponin mixture. This crude saponin mixture was placed in a silanized silica gel column (4.2 x 37 cm), and partition chromatography was performed using a 35% methanol aqueous solution.
Fraction a, fraction b, and fraction C flowed out in this order. Fraction a was added to Kieselgel (
Kiesel get P) column (1,4X 50C
I+) and chloroform-methanol-water (10
:5:1, v/v) was used as a developing solvent to obtain saponin fractions 0A-1 and 0A-2. These were each attached to a Lobar Column RP-8 (Size
B.

Merck) and chromatography was carried out using 50% methanol aqueous solution as a developing solvent to obtain 310 square centimeters of pure product 0A-1 and 140 square centimeters of pure product 0A-2. Fraction b was also separated and purified using the same Kieselgel column and Rover column under the same conditions as above to obtain 0A.
-3 pure product 30cm and 0A-4 pure product 190cm were obtained.

Fraction C was purified using the same Kieselgel column and Rover column as above under the same conditions with the order reversed.
-5 pure product 21 ■ was obtained. The above 0A-1 to 0A-5 were each confirmed by thin layer chromatography, melting point, specific rotation, etc.

(2) Preparation of each desulfated product of asterosaponin 0A-1 and 0A-4 122 mg of 0A-1 was mixed with chloroform-methanol-water (3
0:60:8, v/v) dissolved in 2d, old o-
A small column of Rad AG-50 (H”) (0.6X1.
3CI), pyridine was added thereto, and the mixture was concentrated to dryness and converted into a pyridine salt.

After thoroughly drying this pyridine salt, it was dissolved in 95% dioxane, heated under reflux for 1 hour, and desulfated to give 18 ml of desulfated product of 0A-1.

Next, 95.5 μ of 0A-4 was dissolved in chloroform-methanol-water (30:60:8, v/v), and Bio-
rad AG-50 (Ha small column (0.6X2.
0C11) was passed.

0.1 yd of pyridine was added to this decationization solution and concentrated to dryness to obtain the pyridine salt of 0A-4. This pyridine salt is 0.5 at! After dissolving in water, 9.5 m of dioxane was added and the mixture was heated under reflux for 1 hour. After distilling off the solvent 1
MN was dissolved in 90% methanol, and desalted and purified using the column chromatography described below.

Column: Rover column RP-8 (Size B) solution
Medium: 90% methanol fraction Niag Flow rate: 160 Id/m Fractions Nα8 to Nα9 were concentrated to dryness to obtain 27.7 mg of desulfated product of 0A-4. Rf O, 40 (Kie
sel ge160FZS45ilanisielt,
70% methanol) FD-Mass spectrum: Hitachi M-
Measured with 80 type 2iE focused Gc-mass spectrum.

(3) Preparation of turnastyrol A sulfate 530m
g of 0^-1, conch shell (C, Lan+pas) glycosidase (Seikagaku Corporation) 800μ, 0.2M acetic acid-sodium acetate buffer solution 60d, 1M sodium chloride aqueous solution 30
d was mixed and shaken at 37°C for 64 hours. The reaction solution was extracted four times with water and disaturated n-butanol, and the n-butanol layer was washed once with water. The washings were re-extracted with water-saturated n-butanol, and all the extracted upper layers were collected and concentrated to dryness. This concentrated residue was subjected to the following reverse phase distribution column chromatography.

Carrier: Silanized silica gel 60 (Merck)
50g column: 2.3X32C11 Solvent system: 020% methanol 500d■45
% methanol 250d ■ 60% methanol 300d After concentrating the fraction ■, chloroform-methanol-water (75:25:3, v/v) 3
The following cam chromatography was carried out after dissolving in m.

Support: Kiesel gel PFzs4 (for TLC) 40g Column: Sepacol column (Seikagaku, 1.4X50cm) Flow rate: 42d/hr (pressure 6 atm) Fractionation: 15d Solution system: ■Chloroform-methanol-water (75: 25:
3, V/Ifr, 1-42 ■Chloroform-methanol-water (10:5:1, ν/v) fr, Na 43-60 As a result, turuna styrene sulfate 42■W was added to fractions 9-13. Obtained.

Rf O, 54 (Kiesel get 60FZ54
, chloroform methanol-water, 10:5:1
, v/v), Rf O,51 (Kieselget 6
0F2sn, n-butanol-acetic acid-water, 10:1
: 1, v/v), Rf 0.54 (Kiesel
gel 60F2S4.5ilanisielt :5
0% methanol) [α]: 2-t° (C = 1.0, methanol), CD: (θ) zqoall 1800
(C ~ 0.6, methanol), elemental analysis S: 5%, Ca
1c6% (as Cz, H4,0, SNa).

(4) Each steroid of OA-1 and 0A-4 and 0A-1
Preparation of 411 of 100 μ of 0A-1 with 5 d of 0.25 N sodium hydroxide
and stirred at room temperature for 20 hours. Bio-Rad
Add AG-50 (H”) to neutralize, filter to remove resin,
The filtrate was concentrated under reduced pressure. The residue was dissolved in 3d chloroform-methanol-water (30:60:8, v/v) and DE
It was applied to an AE-Sephadex A-25 (acetic acid form) column. Chloroform-methanol-water (30:60:8,
v/v) 30ad! Wash out the non-adsorbed fraction with chloroform-methanol 0.8M sodium acetate (30:6
The adsorbed fraction was eluted at 0:8, v/v). After concentrating the adsorbed fraction, it was dissolved in water and extracted four times with water-saturated n-butanol. The n-phthanol layer was washed twice with n-butanol-saturated water, and the washings were re-extracted with water-saturated n-butanol, and all the upper layers were collected and concentrated to dryness to obtain 33 ml of 0^-1 sulfate. 〇To-1 sulfate pyridine salt (30L1
g) was dissolved in 95% dioxane 10H1 and heated under reflux for 1 hour.

After concentrating with a rotary evaporator, it was partitioned between water and ethyl acetate, and the ethyl acetate layer was dehydrated and concentrated.
I got 1 steroid. Rf O, 59 (Kiese1g
e160F z S 4, chloroform-methanol-
water, 50:10:1), Rf O, 61 (Kies
el gel 60F2S45ilanisielt.

70% methanol), GLC (Silicone
0V-12%, 0.3 x 200cm, 280°C constant temperature) holding time 3.2 minutes (1MS compound), [α]:”
+62.3° (C=0.9, metal horn)
, MS: aa/z 332, 314, 299
, 281, 230 (base peak), 229
．． 211゜Next, the above DEAE-Sephadex A-
The non-adsorbed fraction (49.9cm) from the 25th column was purified by silica gel column chromatography as shown below.

Carrier: Mallin rodt CC-710g (1
, 6X12.5cm) Flow rate: 90 relatives/hr Solvent system: Chloroform-methanol-water (75:25:
3, ν/ν) Fraction: 8g Fraction Nos. 14 to 20 were concentrated to dryness, and 0A-1
4tJ! 26.0■ was obtained. Rf O, 26 (Kie
sel gel 60Ftsn, chloroform-methanol-water, 10:5:1, v/v).

Color development was performed using sulfuric acid. It gave a yellow spot when heated.

190■ 0A-4 and Naringinase (Taisho Pharmaceutical) 320■ were mixed in acetic acid-sodium acetate buffer (pH 4,
0) Dissolved in 40ml and shaken at 38-40°C for 86 hours. After the reaction was completed, the reaction solution was extracted four times with 40 d of water-saturated n-butanol.

This n-butanol extract was dissolved in 3d water, CD:(θ:+2117 na++7200(C
~0.08, column chromatography shown below was performed.

Column: 2.3X15C11 Support: 5ilanized 5ilica gel (
Merck) 30g Solvent system: 025% methanol
350d■45% methanol 350d■
300 d of 100% methanol was concentrated to dryness to obtain 91.3 d of enzymatically decomposed product.

This enzymatically decomposed product was dissolved in 5d chloroform-methanol-water (40:10:1), and the column chromatography shown below was obtained.

Column: 1.7X33cm Support: Wakogel C-20030g Solvent system: Chloroform-methanol-water (40:10:
1) Fraction: 20 g Flow rate: 109 Ill/m Fractions 12 to 21 were concentrated to dryness to obtain 27.2 μ of 0A-4 sulfate. Dissolve 14μ of this 0A-4 sulfate in 1μ of methanol, and
0.5 d of pyridine passed through a small column (0.5 x 1 cm) of AG-50 (H,'') was added and concentrated to dryness on a rotary evaporator to convert it into a pyridine salt. was dissolved in 95% dioxane and heated under reflux for 1 hour. After removing the solvent, it was dissolved in ethyl acetate, washed with water three times, dehydrated with sodium sulfate, and concentrated to dryness to obtain 10.5d of 0A-4 steroid. .Rf O,51
(Kiesel gel F254, chloroform-
methanol-water, 50:10:1), Rf 0.31 (
Kiesel gel 60FZS4silanisi
elt, 70% methanol), as: rm/z
398.383.380.365.355.333.
316.315.297.287.285゜(5) Preparation of 0^-4 Prosavogenin 1-3 210■
0A-4 and conch glycosidase (Seikagaku Corporation) 40
■, Naringinase 220■ was dissolved in 0.1 M acetic acid-sodium acetate buffer (pH 4,0) 100-
The reaction was carried out for 84 hours on a rotary shaker at °C.

The reaction solution was extracted with n-butanol, and the extract was made into a 20% methanol solution and subjected to column chromatography under the following conditions.

Column: 1.7X17cm Support: Silanized silica gel (Merck) Solvent system: 0
20% methanol 100d■45% methanol 200d■60% methanol
200 dl was concentrated to dryness to obtain 113.7 dl of enzymatically decomposed product.

The entire amount of this enzymatic decomposition product was dissolved in chloroform-methanol-water (10:10:1, v/v), and Bio-
It was passed through a small column of Rad AG50()I').

Add pyridine to this decationized solution to make a pyridine salt,
Desulfurization was carried out by heating under reflux for 1 hour in 95% dioxane.

After the reaction solution was concentrated to dryness, it was dissolved in 3d chloroform-methanol-water (50:10:1, v/v), and column chromatography was performed under the following conditions.

Column: Sepacol column (Seikagaku, 1.4X50cta) Support: Kiesel gel PFzs<40g Solvent system: Chloroform-methanol-water (50:10:1,
v/v) Flow rate: 42 d, /hrc pressure 6 atm) Fraction: 5 g As a result, Prosavogenins 1-3 (P-1-3) were isolated as shown below.

” TLC (1): Kiesel gel 60
F2S4, chloroform methanol-water, 50:10:
1” TLC (2): Kiesel gel 60
F2545ilanisielt, 70% methanol Prosapogenin-L (P-1) is the steroid of 0A-4 bound to quinobose, and Prosabogenin-2 (P-2) is the steroid of 0A-4 bound to quinovose xylose. Prosavogenin-3
(P-3) is 0A-4 steroid plus quinovose xylose-quinovose 3IJ! are combined.

(6) Preparation of Marsasterone Prepared by the method described in Biochem, J, 1970.117.543.

(7) II Measurement of inhibitory effect on tumor cell proliferation Tumor cells were cultured in the medium supplemented with steroid saponins obtained in (1) to (6) above, and the inhibitory effect on tumor cell proliferation (cytolytic effect) was examined. Ta. Steroid saponins, their solvolysis products, and prosapogenin were water-soluble and were added to the medium as they were, but the three steroids, namely 0A-1 steroid, 0A-4 steroid, and malsasterone, were dissolved in ethanol and the final ethanol concentration was 1. It was added so as not to exceed %. All solutions were subjected to autoclave treatment.

ES medium (Tamizu Pharmaceutical Co., Ltd.) containing 2% tallow serum (Armour) was added to 0.4 dBuf 24-well plate) (Fal
FM from mouse breast cancer.
3A cells were suspended at 1:2.0 x 10' cells in each well. While supplying air containing 5% carbon dioxide gas,
It was incubated for 4 days at 7°C. As a result, the number of cells in each well had grown to 3×10 6 cells. Next, the steroid saponin etc. obtained in (1) to (6) were added to each well at 1100 p/at! (7) Add at a concentration of 37
Incubated for an additional 1 hour at °C. Table 1 shows the results of counting the number of cells in each well using the trivan blue staining method.

For comparison, a culture medium to which 1% ethanol was added instead of steroid saponin etc. was incubated in the same manner and the cell number was counted. The results are also shown in the same table.

Table 1 Additives Number of cells (X 105 cells) OA-
1 OA-2 OA-3 OA-4 OA-5 OA-1 Desulfated product 0A-4 Desulfated product Tournastyrol A sulfate 0A-1 Steroid 0A-1 41! -4 Steroid Malsasterone 7.5 6.8 8.3 0.09 6.5 2.9 7.4 7.2 6.6 6.3 7.7 0.5 Ethanol 7.4 Example 2 Turnastyrol A sulfate, 0A-1 steroid, 0A-1 4m and malsasterone 1μg/1td
l, 3.15! g/relative, 10ag/af and 31, 5
Each medium was added at four levels of ttg/Id as in Example 1.
It was incubated for 4 days in the same manner as above. Table 2 shows the results of similarly counting the number of cells remaining in each well.

For comparison, a medium to which 0.01 to 1% ethanol was added was incubated in the same manner and the cell number was counted. The results are also shown in the same table.

Table 2 Example 3 OA-4 STAY F O, OO1 pg/af, 0.0
1! g/af, 0.1aged, I ttg/d,
Incubation was carried out for 4 days in the same manner as in Example 2 at IO μg/d(7) 5 level. Table 3 shows the results of similarly counting the number of cells remaining in each well. For comparison, a culture medium to which 0.0001 to 1% ethanol was added was incubated in the same manner and the number of cells was counted. The results are also shown in the same table.

Table 3 OA-4 Steroid 5.3 3.9 4.2 1.6 0.3 Turnastyrol A Sulfate 0A-1 Steroid 0A-1 Tetrasaccharide Malsasterone 11.0 11.0 7.5 2.30. 6 0.
9 0 0 5.6 14.0 12.0 7.04.2 5.
1 1.1 0 [Effects of the Invention] Various antitumor agents have been developed, but no example of isolation of tumor cell growth inhibitors from starfish has been reported (the present invention further extends the scope of developing anticancer agents). Special ↓This steroid saponin anticancer drug has not yet been commercialized, and it is hoped that a new anticancer drug will be commercialized.

Claims (1)

[Scope of Claims] A tumor cell growth inhibitor containing a steroid or its salt having any of the following structural formulas ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [I] ▲ Numerical formulas, chemical formulas, tables, etc. Yes ▼ [II] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [III] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [IV] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [V] ▲ Mathematical formulas, chemical formulas, tables, etc. There are ▼ [VI] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [VII]