JPH10203968A - Antitumor agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antitumor agent excellent in topoisomerase I and II inhibitory activity by including a bicyclo ring-containing compound. SOLUTION: This medicine contains a compound of the formula (R<1> to R<14> are each H, an alkyl, an alkenyl, an alkoxyl, hydroxy, a hydroxyalkyl, formyl, a halogen or amino) or its salt as an active ingredient. The compound of the formula is obtained by extracting Garcinia subelliptica, Garcinia purpurea, Garciniamangostana Linn., Garcinia dioica Bl., etc., with a solvent and isolating. A dose of the compound of the formula, in the case of human, is 0.01-1,000mg, preferably 0.1-100mg per adult daily and, in the case of animals, is 0.001-1,000mg, preferably 0.1-100mg per kg weight and the dose is administered once to four times dividedly.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a topoisomerase.
The present invention relates to compounds having I and II inhibitory activity and useful as antitumor agents, and pharmaceutically acceptable salts thereof.

[0002]

2. Description of the Related Art The life cycle of a cell, which is observed during cell division and proliferation, is called the cell cycle and is deeply involved in the regulation of cell proliferation and differentiation. Therefore, the elucidation of the control mechanism is important for cancer,
It is expected not only to elucidate the molecular mechanisms of physiological phenomena and pathological phenomena such as aging and cell death, but also to provide great clues to the development of drugs such as antitumor agents targeting the cell cycle. As a result of studying the mechanism of action of many antitumor agents that have been developed,
Many of them have been shown to have characteristic inhibitory effects on the cell cycle. For example, antimetabolites such as 5-fluorouracil, 6-mercaptopurine, and methotrexate inhibit the precursors essential for DNA synthesis, so that the cell cycle stops at the S phase. Alkaloids such as plant-derived vinplastin and vincristine, colchicine analogs colcemide, and mold-derived rhizoxin have an inhibition point in the M phase. In addition, adreamycin, etoposide, ellipticin, and the like act on topoisomerase II and inhibit the distribution (M phase) of the DNA after replication. Furthermore, camptothecin derivatives which are plant alkaloids
CPT-11 is the first type in the world to inhibit topoisomerase type I and arrests the cell cycle at the G2 phase.

[0003]

An object of the present invention is to provide a novel antitumor agent having an inhibitory activity on topoisomerase I and topoisomerase II.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that the compound represented by the general formula (1) and a salt thereof are excellent topoisomerases.
The present inventors have found that they have I and II inhibitory activities, and completed the present invention.

That is, the present invention provides the following general formula (1)

[0006]

Embedded image

Wherein R ¹ , R ⁴ , R ⁵ and R ⁸ are each a hydrogen atom and an alkenyl group, R ² , R ³ , R ⁶ and R ⁷ are each a hydrogen atom and an alkyl group, R ⁹ , R
¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ each represent a hydrogen atom or a hydroxy group) and a salt thereof as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The compound represented by the general formula (1) and a salt thereof include Garcinia subelliptica, Garcinia purpurea, Mangosteen (Garciniamangostana Linn.), And Garcinia dioica. Bl.), Calophyllum inophyllum, and Neonauclea calytcina by extraction and isolation.

In the general formula (1), R ^{1 to} R ¹⁴ each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkoxy group, a hydroxy group, a hydroxyalkyl group, a formyl group, a halogen atom or an amino group. Here, as the alkyl group, a methyl group, an ethyl group, an n-propyl group,
C 1-8 such as isopropyl group, n-butyl group, isobutyl group, t-butyl group, n-pentyl group, n-hexyl group
And a straight-chain or branched-chain alkyl group. In the present invention, among them, an alkyl group having 1 to 6 carbon atoms is preferable.

Examples of the alkenyl group include vinyl, allyl, crotyl, α-pentenyl, 2-hexenyl, 3-methyl-2-butenyl, and 1,1-dimethyl-.
2-propenyl group, 2-carboxy-1-phenylethenyl group, 5-methyl-2- (1-methylethenyl) -4
-An alkenyl group having 1 to 3 straight or branched double bonds having 2 to 14 carbon atoms, such as a hexenyl group, an isoprenyl group, a geranyl group, and a lavandril group.

Examples of the alkoxy group include a straight-chain or branched-chain alkoxy group having 1 to 6 carbon atoms such as a methoxy group, an ethoxy group and a propoxy group. In the present invention, the methoxy group is preferable.

Examples of the hydroxyalkyl group include a linear or branched alkoxy group having 1 to 6 carbon atoms such as a hydroxymethyl group, a hydroxyethyl group and a propoxy group.

[0013] Examples of the halogen atom include fluorine, chlorine, bromine and iodine.

Further, as preferred compounds in the present invention, R ¹ , R ³ , R ⁴ , R ⁵ and R ^{6 in the} general formula (1)
And R ⁷ are a hydrogen atom or a hydroxy group, R ²
And R ⁸ are each preferably a hydrogen atom or an alkenyl group.

Examples of the salt of compound (1) include inorganic acid salts such as hydrochloride, hydrobromide, sulfate and perchlorate, sodium salt and potassium salt. In the present invention, a sodium salt is preferable.

Compound (1) is obtained from Garcinia subel
liptica), Garcinia purpure
a), mangosteen (Garcinia mangostana Linn.), or Garcinia dioica B
l.), Calophyllum inophy
llum), Neonauclea calytci (Neonauclea calytci)
It can be obtained by extracting and isolating from one or more places (hereinafter, referred to as "the original substance") among the fruits, pericarp, bark, root bark, leaves, root materials, etc. of na). That is, the drug substance is cut into small pieces, dried and pulverized, extracted with an organic solvent such as benzene, ethyl acetate, methanol and acetone, and concentrated under reduced pressure to obtain an extract. The obtained extract is purified by recrystallization, silica gel column chromatography or the like, and the components can be isolated.

The compounds thus obtained are useful as antitumor agents because they exhibit topoisomerase I and II inhibitory activity.

When the compound which is an active ingredient of the topoisomerase inhibitor of the present invention is used as a medicament for human body, the dose is 0.01 to 1000 mg, preferably 0.1 to 100 mg per day for an adult.

The dosage for animals is in the range of 0.001 to 1000 mg, preferably 0.1 to 100 mg, per kg of body weight of the animal to be treated.

This daily dose is administered once a day or divided into 2 to 4 times.

The topoisomerase inhibitor comprising a compound which can be used as an active ingredient of the present invention can be prepared by selecting an appropriate preparation according to the administration method and adjusting various preparations which are generally used. Examples of the dosage form of the antitumor agent of the present invention include oral preparations such as tablets, powders, granules, capsules, solutions, syrups, elixirs, and oily or aqueous suspensions.

As an injection, a stabilizer, a preservative, and a solubilizing agent may be used in the preparation, and a solution containing these adjuvants may be contained in a container and then freeze-dried or the like to form a solid preparation. It may be prepared as a preparation for use. One dose may be stored in a container, or multiple doses may be stored in the same container.

[0023]

Hereinafter, the present invention will be described with reference to examples.
The present invention is not limited to these examples.

Example 1 30 kg of fresh pericarp of Garcinia subelliptica is extracted with methanol (30 L) three times for 48 hours at room temperature. About the obtained solution, the solvent was distilled off under reduced pressure, and 2.2 k
g extract was obtained. A part (1000 g) of the extract was suspended in water, and partitioned sequentially with benzene, ethyl acetate, and n-butanol to obtain benzene, ethyl acetate, n-butanol, and a water-soluble fraction, respectively. . The benzene-soluble fraction was subjected to silica gel column chromatography (n-hexane-ethyl acetate system), and the 5: 1 eluted fraction was recrystallized using n-hexane (1S, 5R, 7S). -1,7-bis (3-methyl-2-butenyl) -3- (3,
4-dihydroxybenzoyl) -8,8-dimethyl-4-hydroxy-5-{(2R) -5-methyl-2- (1-methylethenyl) -5-hexenyl} bicyclo [3.3.1] -3-none- 2,9-dione (xanthochymol) (xanthochymol) (9 g) was obtained.

[0025]

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Mp: 130-131 ° C. [α] _D ²⁴ : 138 ° (c 0.1, CHCl ₃ ). IR ν (cm ⁻¹ , KBr): 3290, 2920, 1725, 1625, 1520, 14
40, 1295, 1190. UV λ (nm, MeOH) (log ε): 230 sh, 276 (4.30), 351 sh. ¹ H NMR (400 MHz, CD ₃ OD containing 0.1% TFA) δ: 1.00
(3H, s), 1.16 (3H, s), 1.46 (2H, m), 1.48 (1H, m), 1.5
0 (3H, s), 1.53 (3H, s), 1.61 (3H, s), 1.65 (3H, s), 1.
69 (3H, s), 1.73 (3H, s), 1.85 (2H, m), 1.92 (1H, dd,
J = 13.9, 5.6Hz), 2.02 (1H, m), 2.03 (1H, m), 2.05 (1H,
m), 2.11 (1H, m), 2.25 (1H, brd, J = 14.2Hz), 2.55 (1H,
m), 2.58 (1H, m), 2.70 (1H, dd, J = 13.9, 8.8Hz), 4.5
1 (2H, brs), 4.63 (2H, brs, d, J = 6.4Hz), 4.86 (1H,
m), 5.03 (1H, m), 6.71 (1H, d, J = 8.3Hz), 6.98 (1H, d
. d, J = 8.3, 2.0Hz ), 7.19 (1H, d, J = 2.0Hz) 13 C NMR (400MHz, CD 3 OD containing 0.1% TFA) δ: 17.
8, 18.2, 18.3, 22.8, 23.2, 26.0, 26.4, 27.1, 27.4,
30.3, 32.8, 36.8, 37.7, 43.8, 44.7, 48.0, 50.2, 5
9.8, 69.6, 110.4, 113.5, 115.1, 117.4, 117.9, 12
1.3, 125.2, 125.6, 129.6, 133.6, 135.8, 147.0, 148.
9, 149.5, 152.5, 194.3, 196.0, 196.3, 210.6. EIMS m / s: 602 (5), 533 (3), 465 (42), 499 (8), 411 (5),
341 (34), 231 (34), 137 (58), 69 (100).

EXAMPLE 2 Dried Peel of Garciniapurea 850
g is extracted 5 times with methanol (3 L) for 48 hours at room temperature. The obtained solution was concentrated under reduced pressure to obtain an extract (305 g) from which the solvent was removed. A part (300 g) of the extract was suspended in water, and partitioned sequentially using benzene, ethyl acetate, and n-butanol. The benzene-soluble fraction is subjected to vacuum liquid chromatography (n-hexane-ethyl acetate system) (10: 1)
The eluted fraction was recrystallized from n-hexane using (1R, 5S, 7R) -1,7-bis (3-methyl-2-butenyl) -3.
-(3,4-Dihydroxybenzoyl) -8,8-dimethyl-4-hydroxy-5-{(2S) -5-methyl-2- (1-methylethenyl) -4-hexenyl} bicyclo [3.3.1]- 3-none-2,9-dione (garcinol) (garcinol) (5 g) was obtained.

[0028]

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Mp: 123-124 ° C. [α] _D ²⁴ : -138 ゜ (c 0.1, CHCl ₃ ). IR ν (cm ⁻¹ , KBr): 3560, 2960, 2920, 1730, 1635, 16
05, 1530, 1450, 1295, 1195. UV λ (nm, MeOH) (log ε): 233sh, 278 (4.43), 348sh. ¹ H NMR (400 MHz, CD ₃ OD containing 0.1% TFA) δ: 1.00
(3H, s), 1.15 (3H, s), 1.45 (1H, m), 1.50 (3H, s), 1.
58 (3H, s), 1.59 (3H, s), 1.65 (3H, s), 1.65 (3H, s),
1.70 (3H, s), 1.73 (3H, s), 1.92 (1H, dd, J = 15.5, 5.0
Hz), 1.98 (1H, m), 2.02 (1H, m), 2.02 (1H, m), 2.05 (1
H, m), 2.08 (1H, m), 2.25 (1H, brd, J = 13.7Hz), 2.62
(1H, m), 2.63 (1H, brd, J = 13.2Hz), 2.71 (1H, dd, J = 1
3.2, 9.7Hz), 4.46 (2H, brs), 4.88 (1H, m), 5.03 (1H,
m), 5.05 (1H, m), 6.69 (1H, d, J = 8.3Hz), 6.96 (1H, d
. d, J = 8.3, 2.0Hz ), 7.19 (1H, d, J = 2.0Hz) 13 C NMR (400MHz, CD 3 OD containing 0.1% TFA) δ: 8.2,
18.1, 18.2, 18.3, 23.2, 25.90, 25.94, 26.4, 27.1,
27.4, 30.3, 33.5, 37.3, 43.8, 45.2, 48.0, 50.2, 5
9.8, 69.7, 113.0, 115.1, 117.3, 117.8, 121.3, 124.
1, 125.2, 125.5, 129.5, 132.7, 133.6, 135.8, 146.2,
149.5, 152.4, 194.1, 195.5, 196.1, 210.6. EIMS m / s: 602 (42), 533 (19), 465 (100), 499 (14), 411
(15), 341 (64), 231 (63), 137 (47), 69 (65).

Test Example 1 Measurement of Topoisomerase Type I Inhibitory Activity Topoisomerase I (6 units / ml) [Takara Shuzo Co., Ltd.] was diluted 1 / 5-fold with a buffer (1 × Topoisomerase I buffer / 0.01% BSA). Used for measurement.

1 μl of each concentration of the compound (1) obtained in Examples 1 and 2 was placed in an Eppendorf tube, and the required amount of Mi was added.
lli Q, 10 × Topoisomerase I buffer (350 mM Tris-HCl
<pH8.0> / 720 mM KCl / 50 mM DTT / 50 mM spermidine / 50
12 μl of a mixture of mM MgCl ₂ ), 0.1% BSA and supercoil DNA (pUC 19) was added to each tube, followed by vortexing and light centrifugation. Next, 2 μl of the diluted topoisomerase I was added to each tube, followed by vortexing and gentle centrifugation (however, Milli Q 2 μl was used for the reaction system containing no enzyme and compound (1) prepared as a control reaction system).
And 1 μl of 50% DMSO, and 1 μl of 50% DMSO instead of the reaction system containing no compound (1)).

The above-mentioned Eppendorf tube was placed at 37 ° C. for 30 minutes.
After leaving it on the ice, SDS / PK / BJ (0.66% SDS / 0.33
mg / ml Proteinase K / 0.88 × BJ <20% ficoll / 0.25% BPB>)
Was added to each tube, and the mixture was left at 50 ° C. for 5 minutes, vortexed and centrifuged, left again at 50 ° C. for 30 minutes, and vortexed and centrifuged.

Test Example 2 Measurement of Topoisomerase Type II Inhibitory Activity A, Extraction of Crude Topoisomerase II from Rat Infant Brain Nuclei (IBN) , 1 minute) to remove glycerol. Transfer the pellet to a nuclear extraction buffer (20 mM T
ris-HCl <pH7.5>, 0.3 M NaCl, 140 mM b-Me, 50 ml / ml B
(SA, 20 mM PMSF) and suspended in ice for 30 minutes. Next, the supernatant was separated by centrifugation (10,000 × g, 10 minutes) to obtain crude topoisomerase II. This was diluted according to the activity.

B, Purified topoisomerase II (Top GEN, In
c) Dilution Topoisomerase II (Top GEN, Inc) was diluted with Nuclear extension buffer to 0.75 unit / ml.

1 μl of each concentration of compound (1) was placed in an Eppendorf tube, and required amount of Milli Q, 4 × Topoisomerase II
buffer (250 mM Tris-HCl <pH 7.9>, 600 mM KCl, 50 mM
MgCl ₂ , 0.5 mM EDTA, 2.5 mM ATP, 150 mg / ml BSA), k
18 μl of a buffer mixed with DNA (312 ng / ml) was added, and vortexing and light centrifugation were performed. Next, diluted crude topoisomerase II or diluted purified topoisomerase II
1 μl was added, and vortexing and light centrifugation were again performed (however, 1 μl of Milli Q and 50% DMSO 1 were added to the reaction system containing no enzyme and compound (1) prepared as a control reaction system).
μl and 50% DMSO 1 in the reaction system without compound (1).
μl was added instead).

The above Ependorf tube was allowed to stand at 30 ° C. for crude topoisomerase II and at 37 ° C. for purified topoisomerase II for 30 minutes, and 4 μl of SDS / PK / BJ was added on ice.
I put each. Then, in both cases, leave at 50 ° C for 5 minutes, vortex and perform light centrifugation.
After standing for 30 minutes, vortexing and light centrifugation were performed.

C, 1 × TBE buffer for agarose gel electrophoresis and densitometry electrophoresis (89 mM Tris Base / 89 mM boric acid / 2
agarose gel electrophoresis (-EtBr
After electrophoresis, the gel was stained with 1 × TBE (0.1 ml / ml EtBr), and after decolorization of the gel, UV irradiation was performed, and the band was observed. In the measurement of topoisomerase type I inhibitory activity, the concentration of the agarose gel was 1.2%, and in the type II activity measurement, it was 0.8%.

The reaction amount of the enzyme was determined by quantifying the band density of the agarose gel using a computer <Gel Plotting Macros> attached to NIH Image. In the type I activity measurement, the band of supercoil DNA was
In the mold activity measurement, the band of the minicircle DNA was loaded into a computer, quantified and calculated to obtain a residual activity ratio. A graph is created from the values, and the concentration of the compound (1) that inhibits the activity of topoisomerase I and II by 50% (IC ₅₀ )
Expressed as Table 1 shows the results.

[0039]

[Table 1]

From Table 1, it is apparent that the compound (1) of the present invention has an activity of inhibiting topoisomerase I and II.

[0041]

The compound (1) of the present invention exhibits excellent topoisomerase I and II inhibitory activity and is useful as an antitumor agent.

Claims (2)

[Claims] 1. A compound of the general formula (1) (Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R
⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ each represent a hydrogen atom, an alkyl group, an alkenyl group, an alkoxy group, a hydroxy group, a hydroxyalkyl group, a formyl group, a halogen atom or an amino group) An antitumor agent comprising the represented compound and a pharmaceutically acceptable salt thereof as an active ingredient. 2. A compound of the general formula (1) (Wherein R ¹ , R ⁴ , R ⁵ and R ⁸ are each a hydrogen atom and an alkenyl group; R ² , R ³ , R ⁶ and R ⁷ are each a hydrogen atom and an alkyl group, R ⁹ , R ¹⁰ , R ¹¹ , R
¹² , R ¹³ and R ¹⁴ each represent a hydrogen atom or a hydroxy group) and a pharmaceutically acceptable salt thereof as an active ingredient.