JPH06279489A - New okadaic acid derivative - Google Patents

Abstract

PURPOSE:To obtain the subject new derivative having anti-tumor activity, a strong in vitro proliferation inhibiting activity against mouse lymphatic leukemia cell and low toxicity and useful as a tumor treating agent, etc., by deriving from okadaic acid separated from a porifera. CONSTITUTION:The objective new okadaic acid derivative of formula II (R is amine residue of oligopeptide) having anti-tumor activity and low toxicity and useful as a tumor treating agent, etc., is obtained by derivatizing okadaic acid of formula I (Me is methyl) separated from Halichondria okada Kadota of a porifera to its p-nitrophenyl ester, adding glycylglycine, etc., to the ester, reacting the mixture under stirring in the presence of pyridine, triethylamine and water for 24 hours, subjecting the reaction solution to vacuum concentration after the completion of the reaction and purifying the concentrated product through silica gel chromatography.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel okadaic acid derivative. This compound has strong in vitro growth inhibitory activity against mouse lymphocytic leukemia cells (P388) which are cancer cells, and is expected to be used as an antitumor agent.

[0002]

2. Description of the Related Art In recent years, the composition of sponges has been widely searched, and compounds having various biological activities have been isolated [eg, bryostatin (GR Pettit et al., Tetrahedr
on, 41,985 (1985)), Halichondrin (Y. Hirata et
al., Pure Appl. Chem., 58, 701 (1986)]]. Among them, the following formula [II] isolated from croisokaimen

[Chemical 2] The okadaic acid represented by is known to have a very strong antitumor activity. However, its toxicity to normal cells is so strong that it cannot be used as an antitumor agent. On the other hand, in the medical field, there is always a demand for drugs that are more effective and have fewer side effects.

[0003]

The object of the present invention is to develop an antitumor active substance having low toxicity by synthesizing various derivatives of okadaic acid.

[0004]

Means for Solving the Problems As a result of extensive studies by the present inventors on various okadaic acid derivatives, a novel okadaic acid derivative represented by the following general formula [I] is found to be a cancer cell P388.
Against this, an in vitro growth inhibitory activity was found and the present invention was completed.

That is, the present invention has the general formula [I]

[0006]

[Chemical 3]

The present invention provides an okadaic acid derivative represented by the formula (wherein R represents an amine residue of an oligopeptide).

In the above general formula [I], oligopeptides in the definition of R include dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides and the like.
Peptides consisting of 6 amino acids are preferable, and specifically, glycylglycine, alanylalanine, glycylalanine, leucylglycine, glycylglycylglycine, glycylalanylalanine, alanylalanylalanine, leucyl. Glycylalanine, glycylglycylglycylglycine, glycylglycylalanylalanine,
Examples thereof include leucylglycylglycylalanylalanine, glycylglycylglycylglycylglycylglycine, and glycylglycylglycylglycylglycylglycylglycine.
The amine residue of the oligopeptide means a group formed by removing one hydrogen atom on the N-terminal amino group of the oligopeptide.

The novel okadaic acid derivative represented by the general formula [I] of the present invention can be obtained by reacting okadaic acid p-nitrophenyl ester with the above oligopeptide. Okadaic acid p-nitrophenyl ester, which is a raw material, is synthesized by a known method (JP-A-64-6215).

Specific examples of the compound contained in the above-mentioned general formula [I] of the present invention include the following compounds.

[0011]

[Chemical 4]

[0012]

EXAMPLES The present invention will be described in detail below with reference to examples and test examples.

Example 1

[0014]

[Chemical 5]

Okadaic acid p-nitrophenyl ester (2.1
glycylglycine (50.0 mg) was added to
(1 mL) and triethylamine (0.3 mL), water (0.3 mL) was added,
I stirred it for 24 hours. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography to obtain compound (1) (0.8 mg) as a white solid.

¹ H-NMR (MeOH-d ₄ ): 5.56 (2H), 5.36 (1H),
5.29 (1H), 5.06 (1H), 4.55 (1H), 4.19 (1H), 4.17 (1H),
4.09 (1H), 4.05 (2H), 3.97 (1H), 3.82 (1H), 3.80-3.24
(4H), 3.60 (1H), 3.51 (2H), 3.42 (1H), 3.35 (1H) 3.27
(1H), 2.31 (1H), 2.19 (1H), 2.00-1.20 (27H), 1.78 (3
H), 1.33 (3H), 1.06 (3H), 1.01 (3H), 0.93 (3H)

Example 2

[0018]

[Chemical 6]

Okadaic acid p-nitrophenyl ester (2.1
After adding glycylglycylglycine (74.3 mg) to (mg),
Pyridine (1 mL) and triethylamine (0.3 mL), water (0.3 mL)
Was added and the mixture was stirred for 2 hours. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography to obtain compound (2) (1.2 mg) as a white solid.

¹ H-NMR (MeOH-d ₄ ): 5.54 (2H), 5.35 (1H),
5.27 (1H), 5.07 (1H), 4.57 (1H), 4.23 (1H), 4.19 (1H),
4.07 (1H), 4.03 (2H), 4.00-3.75 (6H), 3.95 (1H), 3.82
(1H), 3.60 (1H), 3.51 (1H), 3.41 (1H), 3.33 (1H) 3.26 (1
H), 2.32 (1H), 2.17 (1H), 2.00-1.20 (25H), 1.76 (3H),
1.56 (3H), 1.18 (3H), 1.08 (3H), 1.01 (3H), 0.95 (3H)

Example 3

[0022]

[Chemical 7]

Okadaic acid p-nitrophenyl ester (2.3
alanylalanine (60.4 mg) to (mg) and then pyridine
(1 mL) and triethylamine (0.3 mL), water (0.3 mL) was added,
Stirred for 2 hours. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography to obtain compound (3) (1.0 mg) as a white solid.

¹ H-NMR (MeOH-d ₄ ): 5.57 (2H), 5.35 (1H),
5.28 (1H), 5.07 (1H), 4.51 (1H), 4.24 (1H), 4.05 (1H),
4.03 (2H), 3.91 (1H), 3.58 (1H), 3.50-3.00 (2H), 3.48
(1H), 3.41 (1H), 3.33 (1H), 3.26 (1H), 3.13 (1H), 2.32
(1H), 2.00-1.20 (29H), 1.88 (1H), 1.76 (3H), 1.31 (3
H), 1.08 (3H), 0.97 (3H), 0.95 (3H), 0.94 (3H), 0.93 (3
H)

Example 4

[0026]

[Chemical 8]

Okadaic acid p-nitrophenyl ester (2.1
alanylalanylalanine (84.7 mg) to (mg),
Pyridine (1 mL) and triethylamine (0.3 mL), water (0.3 mL)
Was added and the mixture was stirred for 2 hours. The reaction solution was concentrated under reduced pressure and then purified by silica gel column chromatography to obtain compound (4) (1.2 mg) as a white solid.

¹ H-NMR (MeOH-d ₄ ): 5.49 (1H), 5.42 (1H),
5.35 (1H), 5.29 (1H), 5.07 (1H), 4.52 (1H), 4.22 (1H),
4.06 (1H), 3.96 (1H), 3.59 (1H), 3.51 (1H), 3.50-3.00
(3H), 3.41 (1H), 3.35 (1H), 3.26 (1H), 2.26 (1H), 2.19
(1H), 2.00-1.30 (32H), 1.76 (3H), 1.54 (3H), 1.18 (3
H), 1.08 (3H), 0.96 (9H), 0.94 (3H)

Example 5

[0030]

[Chemical 9]

Glycylglycylglycylglycine (380.2 mg) was added to okadaic acid p-nitrophenyl ester (7.6 mg), and then pyridine (4.0 m).
L), triethylamine (0.9 mL), water (1.9 m
L) was added and the mixture was stirred for 7 hours. The reaction solution was concentrated under reduced pressure and then purified by liquid chromatography to obtain compound (5) (8.7 mg) as a white solid.

¹ H-NMR (MeOH-d ₄ ): 5.62 (2H), 5.33 (1H), 5.2
9 (1H), 5.05 (1H), 4.53 (1H), 4.05 (2H), 4.00-3.75 (8H), 3.4
9 (1H), 2.31 (1H), 2.16 (1H), 2.05-1.31 (33H), 1.74 (3H), 1.
30 (3H), 1.05 (3H), 0.99 (3H), 0.92 (3H).

Example 6

[0034]

[Chemical 10]

Okadaic acid p-nitrophenyl ester (1
Glycylglycylglycylglycylglycylglycine (150.0 mg) was added to 0.1 mg), and then pyridine (5.0
mL) and triethylamine (3.0 mL), water (4.0
(mL) was added and the mixture was stirred for 24 hours. The reaction solution was concentrated under reduced pressure and then purified by liquid chromatography to obtain compound (6) (1.7 mg) as a white solid.

¹ H-NMR (MeOH-d ₄ ): 5.47 (2H), 5.28 (1H), 5.2
3 (1H), 4.99 (1H), 4.48 (1H), 4.02 (1H), 3.95-3.65 (10H), 3.
44 (1H), 3.34 (1H), 2.25 (1H), 2.09 (1H), 2.00-1.20 (32H),
1.69 (3H), 1.22 (3H), 0.99 (3H), 0.94 (3H), 0.86 (3H).

Example 7

[0038]

[Chemical 11]

Okadaic acid p-nitrophenyl ester (1
Glycylglycylglycylglycylglycylglycylglycine (150.0 mg) was added to 0.2 mg), and then pyridine (5.0 mL), triethylamine (3.0 mL) and water (5.0 mL) were added, and 24.5 I stirred it for a while. The reaction solution was concentrated under reduced pressure and then purified by liquid chromatography to obtain compound (7) (1.2 mg) as a white solid.

¹ H-NMR (MeOH-d ₄ ): 5.54 (2H), 5.34 (1H), 5.2
9 (1H), 5.06 (1H), 4.54 (1H), 4.00-3.70 (12H), 3.40 (1H), 2.
32 (1H), 2.16 (1H), 2.06-1.23 (33H), 1.76 (3H), 1.31 (3H),
1.06 (3H), 1.01 (3H), 0.93 (3H).

Test Example 1 Mouse lymphocytic leukemia cells (P388) were treated with 2-hydroxy
Ciethyl disulfide 5 μM, kanamycin sulfate 100
RPMI containing 10% fetal bovine serum supplemented with μg / ml
In addition to -1640 medium, the cultured cells were added at 1x10.^Four Pieces / ml
And prepare the compounds (1) to (4) at a predetermined concentration.
To add CO₂ Incubator (CO₂5%, humidity 10
The cells were cultured at 0%, 37 ° C) for 4 days. By MTT colorimetric method
Measure the number of viable cells and calculate the growth inhibition rate compared to the control group.
50% cell growth inhibitory concentration (IC ₅₀) Was asked. Table of results
Shown in 1.

[0042]

[Table 1]

Test Example 2 Adriamycin-resistant mouse lymphocytic leukemia cells (P3
88 (ADM)) was added to RPMI-1640 medium containing 10% fetal calf serum containing 2-hydroxyethyl disulfide (5 μM) and kanamycin sulfate (100 μg / ml) to prepare cultured cells at 5 × 10 ⁴ cells / ml. 2) or (4) was added to a predetermined concentration, and a CO ₂ incubator (CO ₂ 5%, humidity 100%, 37
C.) for 4 days. The number of surviving cells was measured by the MTT colorimetric method, and the 50% cell growth inhibitory concentration (IC ₅₀ ) was determined from the growth inhibition rate relative to the control group. The results are shown in Table 2.

[0044]

[Table 2]

Test Example 3 Mouse lymphocytic leukemia cells (P388) were treated with 2-hydroxyethyl disulfide 5 μM and kanamycin sulfate 100.
RPMI containing 10% fetal bovine serum supplemented with μg / ml
-1640 medium, add 1 × 10 ⁴ cells / ml
And adding the compounds (5) to (7) to a predetermined concentration, and adding a CO ₂ incubator (CO ₂ 5%, humidity 10).
The cells were cultured at 0%, 37 ° C) for 4 days. The number of viable cells was measured by the MTT colorimetric method, and the 50% cell growth inhibitory concentration (IC ₅₀ ) was determined from the growth inhibition rate relative to the control group. The results are shown in Table 3.

[0046]

[Table 3]

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Claims (1)

[Claims] 1. A general formula: (In the formula, R represents an amine residue of an oligopeptide), an okadaic acid derivative.