JPH0770171A - Sterol compound - Google Patents

Abstract

(57) [Summary] [Objective] To provide a novel sterol compound having an antitumor activity. [Structure] Formula [Chemical Formula 4] [In the formula, X represents a halogen atom, R ¹ and R ² are the same or different and represent a hydrogen atom or an alkanoyl group having 2 to 5 carbon atoms, and A is —CO— or —CH (OH)
-Indicates. ] The sterol compound represented by these.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sterol compound having an antitumor activity and useful as a medicine.

[0002]

2. Description of the Related Art As a compound having a structure similar to that of the present compound, a compound described in JP-A-5-4998 (hereinafter referred to as aragusterol A) is known.

[0003]

The object of the present invention is to provide a novel sterol compound having a superior 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 that by chemically modifying it, a novel compound having an excellent antitumor effect can be obtained.
Further, they have found that a novel sterol compound having an antitumor effect can be obtained by chemically modifying known aragusterol A, and completed the present invention.

The present invention has the following formula

[0006]

[Chemical 3]

[Wherein X represents a halogen atom, R ¹ and R ² are the same or different and each represents a hydrogen atom or an alkanoyl group having 2 to 5 carbon atoms, and A is —CO— or —CH (OH) 2). -Indicates. ] It is a sterol compound represented by.

In the present invention, the halogen atom means a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a chlorine atom. The alkanoyl group is linear or branched, and examples thereof include an acetyl group, a propionyl group and a butyryl group, and preferably an acetyl group.

The compound of the present invention can be isolated and produced by the method shown below. First, among the sterol compounds of the present invention, R ¹ = R ² = H, X = Cl, A =-
The CO-compound (hereinafter referred to as aragusterol C) is isolated by extracting the sponge of the genus Xestospongia with an organic solvent, and fractionating and purifying the extract obtained from the extract by a commonly used method. 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, for example, by silica gel column chromatography, preparative silica gel thin layer chromatography, chromatographic methods such as high performance liquid chromatography, and recrystallization method. The elution solvent used here is a single solvent or a mixed solvent of ether, hexane, ethyl acetate, benzene, toluene, acetone, methanol, chloroform, dichloromethane and the like.

Further, the araguster obtained above
The sterol compound of the present invention can be obtained by chemically modifying ol C or known aragusterol A, respectively.

That is, aragusterol A is converted to a halogenating reagent (for example, Li ₂ when X is a chlorine atom).
CuCl ₄ , and when X is a bromine atom, Li ₂ CuBr _{4 and the} like) are reacted to obtain R ¹ = R ² = H, A = -C
A sterol compound of the present invention that is O- can be obtained.

The compound of the present invention in which R ¹ and / or R ² is an alkanoyl group is obtained by adding the compound obtained above to a solvent such as pyridine in the presence of a base such as 4-dimethylaminopyridine or triethylamine. Alternatively, in the absence thereof, an acid anhydride represented by the formula R ₂ O (wherein R is an alkanoyl group represented by R ¹ and R ² ) or RX ′ (in the formula, R is as defined above) And X ′ is an arbitrary halogen atom.) Or an acylating agent such as an acid halide or a dehydrating agent such as 1,3-dicyclohexylcarbodiimide or 1,1′-carbonyldiimidazole. In the presence of a compound represented by the formula ROH (wherein R has the same meaning as defined above). As an example of this reaction, for example, acetylation of aragusterol C with acetic anhydride / pyridine gives a sterol compound of the present invention in which R ¹ and / or R ² is an acetyl group.

The sterol compound of the present invention in which A is --CH (OH)-is obtained by adding the sterol compound of the present invention in which A is --CO-- to a lower alcohol such as methanol or ethanol in the presence of an acid such as acetic acid. Or in the absence,
It is obtained by reacting a reducing agent such as sodium cyanoborohydride.

When the sterol compound of the present invention is used as a medicine, it is used as a preparation for oral or parenteral administration. Examples of the dosage form include tablets, granules, powders, capsules, syrups, suspensions and injections, which can be appropriately selected depending on the patient's symptoms, age and purpose of treatment. The dose is 1 to 5 when treating an adult
The dose is 00 mg, which is administered in 2 to 3 divided doses per day. This dose can be appropriately increased or decreased depending on the age, weight and condition of the patient.

[0015]

According to the present invention, Xestospongi
A novel sterol compound from a sponge
erol C) was isolated and further modified by chemical modification to produce a novel sterol compound. Also, known ara
chemical modification of gustanol A
terol C and new sterol compounds were prepared. These have a better antitumor effect than aragusterol A and are useful as pharmaceuticals.

[0016]

EXAMPLES Next, the present invention will be described more specifically with reference to Examples and Test Examples.

Example 1 Xestosp collected on a coral reef near the sea near Iriomote Island, Okinawa Prefecture
The sponges of the genus ongia were immediately frozen on dry ice. This frozen sample (3 kg) was cold-soaked with methanol (6 L). The methanol extract was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The same methanol extraction was performed twice more to obtain an extract, which was combined with the first extract. The remaining frozen sample was extracted in the same manner, and the methanol extract was added to a total of 26
04 g was obtained. 131 g of this methanol extract was suspended in 1.5 L of water and extracted twice with 1.5 L of ethyl acetate. The combined ethyl acetate-soluble portion was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a residue (12 g). The remainder of the methanol extract was similarly divided into 11 portions and partitioned with water and ethyl acetate to obtain a total of 267 g of ethyl acetate-soluble portion together with the previous ethyl acetate-soluble portion. The ethyl acetate-soluble part (51.3 g) was subjected to silica gel column chromatography (Fuji Devison BW-820MH 500).
g) and 3 fractions were obtained (fraction 1, hexane: ethyl acetate = 5: 1, eluted with 3000 ml; fraction 2, hexane:
Elution with 1000 ml of ethyl acetate = 1: 1; Fraction 3, 1000 ml of ethyl acetate, then 1000 ml of methanol
Elute with). The rest of the ethyl acetate-soluble portion was similarly divided into four times and separated to give a total of 34.4 g of fraction 1,
Fraction 2 of 67.8 g and fraction 3 of 138.4 g were obtained respectively. Fraction 2 (18.6 g) was subjected to activated carbon column chromatography (60 g of activated carbon) to obtain 3 fractions (fraction 2-1 and eluted with 1000 ml of methanol; fraction 2-2 and eluted with 1000 ml of ethyl acetate; Fraction 2-
3, eluted with 2000 ml of chloroform). The rest of the fraction 2 was similarly divided into four times and separated to give a total of 14.8 g of the fraction 2-1 and 25.6 g of the fraction 2-2,2.
Fractions 2-3 of 7.3 g each were obtained.

Fractions 2-3 (4.3 g) were subjected to silica gel column chromatography (Fuji Devison BW-820MH).
100 g) to give 7 fractions (fraction 2-3-1,
Elution with 200 ml of hexane: ethyl acetate = 2: 1; fraction 2-3-2, hexane: ethyl acetate = 2: 1 150
Elution with ml; fraction 2-3-3, hexane: ethyl acetate =
Elution with 2: 1 200 ml; Fraction 2-3-4, hexane: ethyl acetate = 2: eluting with 1150 ml, fraction 2-3
-5, hexane: ethyl acetate = 2: 1, eluted with 650 ml; fraction 2-3-6, hexane: ethyl acetate = 2: 1
Elution with 700 ml; fraction 2-3-7, methanol 300
Elute with ml). The rest of the fractions 2-3 was similarly divided into 5 times and separated.

Combined fraction 2-3-5 (10.52 g)
Flash chromatography (Fuji Devison BW
-300 300 g) to give 7 fractions (fraction 2-
3-5-1, hexane: acetone = 4: 1 1100m
Elution with 1; fraction 2-3-5-2, hexane: acetone =
Elution with 4: 1 500 ml; Fraction 2-3-5-3, hexane: acetone = 4: 1 Elution with 1000 ml; Fraction 2
-3-5-4, Hexane: acetone = 4: 1 400 m
Elution with 1; Fraction 2-3-5-5, hexane: acetone =
Elution with 4: 1 800 ml; Fraction 2-3-5-6, hexane: acetone = 4: 1 Elution with 400 ml; Fraction 2-
3-5-7, eluted with 500 ml of ethyl acetate).

Similarly, the fraction 2-3-6 (2.9 g) was subjected to flash chromatography (Fuji Devison BW-30).
(100 g) and eluted with hexane: acetone = 4: 1 to obtain 8 fractions (fractions 2-3-3-6-1 to 8).

Fractions 2-3-5-6, 2-3-6-3 and 2-3-6-4 were combined (2.69 g) and flash chromatographed (Fujidevison BW-300 2).
00 g, eluted with hexane: acetone = 3: 1), and the obtained crystalline fraction (2.20 g) was recrystallized from hexane: ethyl acetate to obtain colorless columnar crystals of aragusterol C (852 mg). .

Melting point: 204 to 205 ° C. Elemental analysis value; Calculated value (%) C: 70.35, H: 9.57, Cl:
7.16 Found (%) C: 70.32, H: 9.65, Cl:
7.25 IR (KBr) cm ^-1 ; 3400, 3266, 169
3 ¹ H-NMR (400 MHz, CDCl ₃ ) δ (pp
m); 0.18 (1H, q, J = 4.5Hz), 0.2
7 (2H, m), 0.53 (1H, m), 0.98 (6
H, s), 1.02 (3H, d, J = 5.4 Hz),
1.03 (3H, s), 2.03 (1H, ddd, J =
2.1 Hz, 6.4 Hz, 13.1 Hz), 2.09
(1H, ddd, J = 1.7Hz, 38Hz, 15.0
Hz), 2.38 (1H, dt, J = 6.4Hz, 1
3.5Hz), 3.44 (1H, dd, J = 4.5H
z, 11.1 Hz), 3.88 (2H, s), 3.97.
(1H, dd, J = 3.3 Hz, 11.1 Hz) ¹³ C-NMR (100 MHz, CDCl ₃ ) δ (pp
m); 9.0 (CH ₃ ), 11.5 (CH ₃ ), 12.3
(CH), 12.6 (CH ₂ ), 18.9 (CH ₃ ), 1
9.3 (CH ₃ ), 23.5 (CH ₂ ), 23.6 (CH
_{2), 28.0 (CH),} 28.9 (CH 2), 29.8
(CH ₂ ), 31.1 (CH ₂ ), 33.9 (CH), 3
5.3 (CH), 35.7 (C), 37.8 (C
H ₂ ), 38.1 (CH ₂ ), 38.6 (CH ₂ ), 4
4.6 (CH ₂ ), 46.6 (CH), 47.4 (C
H ₂ ), 49.2 (C), 52.5 (CH), 54.1
(CH), 55.0 (CH), 71.4 (CH), 7
7.1 (C), 77.8 (CH), 211.6 (CO) FABMS m / z; 495 (MH ⁺ ), 497 [(M
K + 2) ⁺ ] [α] _D ²⁶ ; ⁺ 20.1 ° (c 0.35, CHC
l ₃ ).

Example 2 Acetic anhydride (1 ml) was added to a solution of aragusterol C (101 mg) in pyridine (1 ml), and the mixture was stirred at room temperature for 7 hours.
Stir for 1 hour. The reaction solution was diluted with ether (20 ml) and washed with water (3 ml). Then, the ether solution was washed three times with a saturated aqueous solution of copper sulfate (2 ml), and then washed with water (3
ml) and washed with saturated aqueous sodium hydrogen carbonate solution (10
ml) and washed twice. Further, it was washed with water (3 ml) and then saturated saline (3 ml). The ether solution was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give a crude product (135 mg). This crude product was subjected to silica gel column chromatography (Merck, Silic
a gel 60, 15 g) and eluted with hexane: acetone = 4: 1 (13 ml / fraction),
Fractions 7 to 14 from aragusterol C dia
Cetate (113 mg) was obtained.

Melting point: 102-103 ° C. IR (KBr) cm ^-1 ; 3490,1736,171
8,1250 ¹ H-NMR (400 MHz, CDCl ₃ ) δ (pp
m); 0.15 (1H, m), 0.23 (2H, m),
0.40 (1H, m), 0.53 (1H, m), 0.9
9 (3H, d, J = 6.7Hz), 1.00 (3H,
d, J = 6.3 Hz), 1.01 (3H, s), 1.0
7 (3H, s), 2.04 (3H, s), 2.06 (3
H, s), 2.11 (1H, ddd, J = 1.9 Hz,
4.0 Hz, 15.1 Hz), 2.25 (1 H, dd,
J = 13.9 Hz, 15.1 Hz), 2.35 (1 H,
dt, J = 6.3 Hz, 13.3 Hz), 2.80 (1
H, s), 3.76 (1H, d, J = 11.5Hz),
3.83 (1H, d, J = 11.5Hz), 4.68
(1H, dd, J = 4.8Hz, 11.1Hz), 5.
26 (1H, br d, J = 11.0 Hz) ¹³ C-NMR (100 MHz, CDCl ₃ ) δ (pp
_{m); 10.3 (CH 3)} , 11.4 (CH 3), 12.
4 (CH), 12.6 (CH ₂ ), 18.7 (CH ₃ ),
19.1 (CH ₃ ), 21.1 (CH ₃ ), 21.7 (C
H ₃ ), 23.4 (CH ₂ ), 23.9 (CH ₂ ), 2
7.5 (CH ₂ ), 27.9 (CH), 28.8 (C
H ₂ ), 30.9 (CH ₂ ), 33.9 (CH), 35.
1 (CH), 35.7 (C), 36.9 (CH ₂ ), 3
7.9 (CH ₂ ), 38.4 (CH ₂ ), 44.5 (CH
₂ ), 46.4 (CH), 47.5 (C), 47.6
(CH ₂ ), 52.1 (CH), 54.5 (CH), 5
6.6 (CH), 75.4 (CH), 76.8 (C),
80.0 (CH), 170.2 (CO), 170.8
(CO), 211.1 (CO) FABMS m / z; 579 (MH ⁺ ), 581 [(M
K + 2) ⁺ ].

Example 3 A solution of aragusterol A (66 mg) in tetrahydrofuran (1.5 ml) under an argon atmosphere was used (Tetrahedron Letters, No. 27).
Vol. 3, page 3697, 1986) prepared by the method described in Li ₂ CuCl ₄ in tetrahydrofuran (1 mol).
/ L, 0.29 ml) was added and the mixture was stirred at room temperature for 1.5 hours. Ethyl acetate was added to the reaction solution, washed successively with water and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to give a crude product. The crude product was subjected to silica gel column chromatography and eluted with dichloromethane: acetone = 6: 1.
Roll C (69 mg) was obtained. The physical properties (melting point, IR, ¹ H-NMR, ¹³ C-NMR) of this compound were the same as those of aragusterol C derived from sponge.

Example 4 Laragusterol A (30 mg) was dissolved in tetrahydrofuran (600 μl), and L was prepared according to the method described in the literature of Example 3 at room temperature under an argon atmosphere.
i ₂ CuBr ₄ in 1M tetrahydrofuran (300μ
1) was added and stirred for 30 minutes. The reaction solution was diluted with ether, washed with water and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure to give a colorless powder of a compound (18 mg) in which R ¹ and R ² were both hydrogen atoms, X was a bromine atom, and A was —CO—.

¹ H-NMR (300 MHz, CDCl ₃ )
δ (ppm); 0.16 (1H, m), 0.24 (2
H, m), 0.51 (1H, m), 0.96 (3H,
s), 1.01 (3H, s), 1.01 (1H, d, J
= 5.5 Hz), 1.02 (3H, s), 3.44 (1
H, dd, J = 4.6 Hz, 7.15 Hz), 3.74
(1H, d, J = 11.0 Hz), 3.78 (1H,
d, J = 11.0 Hz), 3.97 (1H, br d,
J = 10.6 Hz) SIMS (+ KI) m / z; 577 (MK ⁺ ), 57
9 [(MK + 2) ⁺ ].

Example 5 Aragusterol C (25 mg) was dissolved in methanol (1 ml), and acetic acid (40 μl) was added thereto. After cooling this solution to 0 ° C., sodium cyanoborohydride (20 mg) was added with stirring, and the mixture was stirred at 0 ° C. for 3 hours. The reaction solution was diluted with ether, washed with water and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained crude product was recrystallized from ethyl acetate-hexane, whereby R ¹ and R ² of the colorless powder were both a hydrogen atom, X was a chlorine atom and A.
A compound (19 mg) in which is —CH (β-OH) — was obtained.

Melting point: 115 to 118 ° C. IR (KBr) cm ⁻¹ ; 3381 ¹ H-NMR (300 MHz, CDCl ₃ ) δ (pp
m); 0.16 (1H, m), 0.24 (2H, m),
0.47 (1H, m), 0.69 (3H, s), 0.7
9 (3H, s), 0.94 (3H, s), 1.01 (1
H, d, J = 6.0 Hz), 1.92 (1 H, m),
2.13 (1H, t, J = 9.2Hz), 2.91 (1
H, d, J = 4.1 Hz), 3.07 (1H, d, J =
4.1Hz), 3.36 (1H, dd, J = 4.6H)
z, 11.0 Hz), 3.46 (1H, dd, J = 2.
7 Hz, 11.2 Hz), 3.58 (1 H, m) SIMS (+ KI) m / z; 535 (MK ⁺ ), 53
7 [(MK + 2) ⁺ ] [α] _D ²⁸ ; ⁺ 3.75 ° (c 0.16, CHC
l ₃ ).

Example 6 The compound obtained in Example 4 in which R ¹ and R ² are both hydrogen atoms, X is a bromine atom and A is —CO— is treated in the same manner as in Example 5, Both R ¹ and R ² of the colorless powder are hydrogen atoms, X is a bromine atom, and A is —CH (β-O.
H) -is obtained.

¹ H-NMR (300 MHz, CDCl ₃ )
δ (ppm); 0.16 (1H, m), 0.24 (2
H, m), 0.51 (1H, m), 0.82 (3H,
s), 0.94 (3H, s), 0.97 (3H, s),
1.01 (1H, d, J = 5.9Hz), 3.43 (1
H, dd, J = 4.6 Hz, 11.3 Hz), 3.59
(1H, m), 3.73 (1H, d, J = 10.8H
z), 3.79 (1H, d, J = 10.8Hz), 3.
96 (1H, br d, J = 10.6 Hz) SIMS (+ KI) m / z; 579 (MK ⁺ ), 58
1 [(MK + 2) ⁺ ].

Test Example [In Vivo L1210 Leukemia Life-Prolonging Effect] 1 × 10 ⁵ L1210 leukemia cells were intraperitoneally transplanted into DBA / 2 female mice, and tumor cells were collected from ascites on day 6 or 7. A cell suspension containing 5 × 10 ⁵ viable cells / ml (suspended in Hank's balanced salt solution) was prepared.
2 ml (1 × 10 ⁵ cells / mouse) was intraperitoneally transplanted into CDF ₁ female mice (8 weeks old). The day of cell transplantation was set to day 0, and aragusterol C and aragusterol A suspended in 0.5% acacia-physiological saline were intraperitoneally administered for 5 days from the day following cell transplantation.

The effect is the median survival time of mice (Medi
an Survival Time (MST),
It was determined by T / C = (MST of drug administration group) / (MST of control group) × 100 (%).

The results are shown in Table 1.

[0035]

[Table 1]

(Note) Weight gain = (weight of day4)-(weight of day1)

Claims (2)

[Claims] 1. The formula: [In the formula, X represents a halogen atom, R ¹ and R ² are the same or different and represent a hydrogen atom or an alkanoyl group having 2 to 5 carbon atoms, and A is —CO— or —CH (OH)
-Indicates. ] The sterol compound represented by these. 2. The formula: [In the formula, X represents a halogen atom, R ¹ and R ² are the same or different and each represents a hydrogen atom or an acetyl group, and A represents —CO— or —CH (OH) —. ] The sterol compound represented by these.