JPH07278039A - Combretastatin analog compound - Google Patents

Abstract

PURPOSE:To obtain a new combretastatin analog compound useful as an anti-cancer medicine, showing strong tubulin polymerization inhibiting activity. CONSTITUTION:2-(3'-Hydroxy-4'-methoxy)phenyl-1-(3'',4'',5''-trimethoxy )phenyl-1,1- dimethoxyethane of formula 3 and 5,5-dimethyl-2-(3'-hydroxy-4'-methoxy) benzyl-2-(3'',4'',5''-trimethoxy)phenyl-1,3-dioxane of formula 4. The compound of formula 3 is obtained by reacting trimethylsilyl cyanide with 3,4,5- trimethoxybenzaldehyde to give a compound of formula I, then reacting the compound with ethyl acetate and ethanol to give a compound of formula 2, then reacting the compound with trimethyl orthoformate. The compound of formula 4 is obtained by reacting the compound of formula 3 with 2,2- dimethyl-1,3'--propanediol. The new compounds shows microtubule protein polymerization inhibiting activity and antitumor activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel compound having an antitumor activity.

[0002]

2. Description of the Related Art The anticancer agent combretastatin is considered to exhibit antitumor activity by binding to the chebulin molecule, which is the main component of intracellular microtubule proteins, and inhibiting mitosis.

Twisting of the two aromatic rings and their conformation in combretastatin are believed to have a significant effect on activity.

The synthesis of nitrogen-containing analogs of combretastatin and the antitumor activity of these compounds are described in Journal of Medicinal Ch.
embryo. Volume 34, pp. 2579-2588
Page (1991).

[0005]

The anticancer drug combretastatin is not sufficiently separated from its efficacy and toxicity, and synthesis of analogs of combretastatin has been carried out.

[0006] The present inventors have aimed to separate efficacy and toxicity in analogs of combretastatin.

[0007]

The present inventors have completed the present invention based on the findings by synthesizing combretastatin analogs and measuring the microtubule protein polymerization inhibitory activity of these compounds.

That is, the present invention relates to formula (I) and formula (II),

Formula (I)

Formula (II)

Is a combretastatin analog compound represented by:

The method for producing the combretastatin analog compound of the present invention is as shown in the following reaction routes (I) and (II).

Reaction route (I)

The effective dose of the combretastatin analog compound of the present invention is 300 mg to 5,000 per day for healthy adults.
mg, preferably 450 mg to 2,000 mg.

The composition thus obtained may be used as it is or when necessary, other known additives such as an excipient, a disintegrating agent, a binder, a lubricant, an antioxidant, a coating agent, and a coloring agent. Oral preparations such as granules, powders, capsules, tablets, dry syrups and liquids can be prepared by a conventional method by mixing agents, flavoring agents, surfactants, plasticizers and the like.

As these additives, those generally used in pharmaceuticals can be used.

[0017]

INDUSTRIAL APPLICABILITY The combretastatin analog compound of the present invention exhibits a strong tubulin polymerization inhibitory activity and is useful as an anticancer agent.

[0018]

EXAMPLES The present invention will be specifically described below with reference to Examples and Test Examples.

Example 1 Preparation of 3,4,5-trimethoxyphenyl (3'-benzyloxy-4'-methoxy) -benzyl ketone (1) Trimethylsilyl cyanide (13.8 ml, 0.11 m)
mol), 3,4,5-trimethoxybenzaldehyde (10.9 g, 0.056 mmol) and zinc iodide (several crystals) were added at room temperature, and the mixture was stirred at 50 to 70 ° C. for 2 hours and then distilled under reduced pressure (bp). 170 ° C, 0.5 mmH
g) as a pale yellow oily cyanohydrin (13.1 g, 8
0%).

Next, diisopropylamine (1.8 ml, 12.8 mmol) was added to THF (20 ml) under an argon atmosphere.
ml) and n-butyllithium (1.65N,
(7.4 ml, 12.2 mmol) was added and stirred for 30 minutes to prepare LDA.

Among these, 1-trimethylsilyloxy
3 ', 4', 5-trimethoxyphenylacetonitrile (3.0 g, 10.2 mmol) in THF (10 ml)
The solution was added at -78 ° C and stirred for 30 minutes, and then 3-benzyloxy-4-methoxybenzyl chloride (2.8
g, 10.7 mmol) in THF (10 ml) was added.

After stirring at -78 ° C for 30 minutes, 1.
Warm to room temperature over 5 hours and methanol (25 ml)
And 2N-HCl (50 ml) were added, and the mixture was stirred at room temperature overnight.

After diluting with water, the mixture was extracted with ether, washed with a saturated aqueous solution of sodium hydrogencarbonate, 1N-NaOH, water and a saturated saline solution in this order, dried over anhydrous magnesium sulfate, and then distilled under reduced pressure.

Recrystallization from ethanol-hexane gave colorless needle crystals (3.4 g, 79%, mp 84-85 ° C).

Elemental analysis; calculated value (as C25H26O6)
C: 71.07, H: 6.20. Measured value C: 70.8
3, H: 6.13.

FABMS; m / z: 423 (M ++
H). NHR (500MHz, CDCl3) δ: 3.84 (3
H, s), 3.85 (6H, s), 3.90 (3H,
s), 4.13 (2H, s), 5.10 (2H, s),
6.81 (1H, dd, J = 1.9, 8.3Hz),
6.83 (1H, d, J = 1.9 Hz), 6.84 (1
H, d, J = 8.3 Hz), 7.27 (1H, t, J =
7.0 Hz), 7.33 (2H, t, J = 7.0H
z), 7.40 (2H, d, J = 7Hz).

IR (CHCl3) cm-1; 3000w,
2950w, 2920w, 2820w, 1670s, 1
580s, 1455s, 1405s, 1330s, 12
50m, 1120m, 1020m, 995m.

Example 2 Preparation of 3,4,5-trimethoxyphenyl (3'-hydroxy-4'-methoxy) benzyl ketone (2) 3,4,5-Trimethoxyphenyl (3'-benzyloxy-4) '-Methoxy) -benzyl ketone (1) 84
Ethyl acetate (20 ml) and ethanol (10 ml) were added to 5 mg and 2.0 mmol to dissolve, and 10% P was added.
d / C (0.1 g) was added, and the mixture was vigorously stirred under a hydrogen stream at normal pressure. The catalyst was filtered off and the filtrate was concentrated under reduced pressure. The obtained white solid was recrystallized from ethanol to give colorless prism crystals (518 mg, 78%, mp. 156-158 ° C).

Elemental analysis; calculated value (as C18H20O6)
C: 65.07, H: 6.07. Measured value C: 65.0
1, H: 6.09.

FABMS; m / z: 333 (M ++
H).

NMR (500 MHz, CDCl3) δ:
3.85 (3H, s), 3.88 (6H, s), 3.9
0 (3H, s), 4.14 (2H, s), 5.67 (1
H, s), 6.74 (1H, dd, J = 1.7, 8.1)
Hz), 6.80 (1H, d, J = 8.1 Hz), 6.
84 (1H, d, J = 1.7 Hz). IR (CHCl3) cm-1; 3550w, 3000w,
2950w, 2930w, 2900w, 2830w, 1
670s, 1585s, 1505s, 1460s, 14
10s, 1330s, 1265m, 1150m, 112
5m, 1025m, 995m.

Example 3 2- (3'-hydroxy-4'-methoxy) phenyl-
1- (3 ", 4", 5 "-trimethoxy) phenyl-
Production of 1,1-dimethoxyethane (3) 3,4,5-trimethoxyphenyl (3'-hydroxy-4'-methoxy) benzyl ketone (2) 20 mg,
0.06 mmol was dissolved in methanol (2 ml), trimethyl orthoformate (1 ml, excess amount) and p-toluenesulfonic acid (1 crystal) were added, and the mixture was heated under reflux for 1 hr.

After adding a saturated aqueous sodium hydrogen carbonate solution, the mixture was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure to give a colorless oil (22 mg, 97%).

FABMS; m / z 347 (M ++ OCH3).

HRFABMS; calculated value (C19H23O
6): 347.1495, found: 347.1516.

NMR (500 MHz, CDCl3) δ:
3.04 (2H, s), 3.29 (6H, s), 3.7
2 (6H, s), 3.79 (3H, s), 3.83 (3
H, s), 5.40 (1H, s), 6.17 (1H, d)
d, J = 2.1, 8.2HZ), 6.44 (2H,
s), 6.49 (1H, d, J = 2.1 Hz), 6.5
5 (1H, d, J = 8.2 Hz).

IR (CHCl3) cm-1; 3550w,
3000w, 2950w, 2930w, 2850w, 1
590s, 1500m, 1460s, 1405m, 13
35m, 1265s, 1120s, 1040m, 102
5 m, 1000 m.

Example 4 Of 5,5-dimethyl 2- (3'-hydroxy-4'-methoxy) benzyl-2- (3 ", 4", 5 "-trimethoxy) phenyl 1,3-dioxane (4) Production 3,4,5-trimethoxyphenyl (3′-hydroxy-4′-methoxy) benzyl ketone (2) (100 m
g, 0.30 mmol) was dissolved in benzene (50 ml), and 2,2-dimethyl-1,3-propanediol (4
7 mg, 0.45 mmol) and p-toluenesulfonic acid (10 mg) were added, and the mixture was heated under reflux for 6 hours while azeotropically dehydrating.

After adding a saturated aqueous sodium hydrogen carbonate solution, the mixture was extracted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous magnesium sulfate, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1) and colorless. An oil (56 mg, 44%) was obtained.

FABMS; m / z 419 (M +).

HRFABMS; calculated value (C23H31O
7): 419.2070, found: 419.2070.

NMR (CDCl3) δ: 0.58 (3
H, s), 1.13 (3H, s), 2.90 (2H,
s), 3.36 (2H, d, J = 10.8Hz), 3.
46 (2H, d, J = 10.8Hz), 3.74 (6
H, s), 3.83 (3H, s), 3.86 (3H,
s), 5.42 (1H, s), 6.38 (1H, J =
2.1 Hz), 6.42 (2H, s), 6.63 (1
H, d, J = 8.0 HZ), 6.71 (1 H, d, J =
2.1 Hz).

IR (CHCl3) cm -1; 3550w,
2800w, 2750w, 2650w, 2620w, 1
590s, 1500m, 1460s, 1405m, 13
25s, 1265s, 1160m, 1125s, 108
0m, 1020m, 1000m.

Test Example 1 [Microtubule protein polymerization inhibitory activity] [Sample] Compound (2); 3 mg, Compound (3); 7 m
g, compound (4); 10 mg.

[Microtubule protein (Microtubul
Preparation of c protein] Microtubule protein was prepared from pig brain by two cycles of polymerization-depolymerization. Seven fresh pig brains were prepared and the cerebellum was removed. MESbu
FFer (100 mM MES, 1 mlM EGTA,
0.5 mM MgCl2, 1 mM, 2-mercaptoethanol, 1 mM GTP, pH = 6.5) was added, and the mixture was homogenized with a Warning Blender under ice cooling.

Then, the mixture was centrifuged at 4 ° C. and 50,000 g for 30 minutes, and the resulting supernatant was mixed with the same volume of glycerol.
buffer (100 mM MES, 1 mlM EGT
A, 0.5 mM MgCl2, 1 mM, 2-mercaptoethanol, 1 mM GTP, 8M glycero
1, pH = 6.5) was added.

After incubating at 37 ° C. for 40 minutes, it was centrifuged at 25 ° C. at 100,000 g for 45 minutes, and the polymerized microtubule protein was collected as a pellet. MESbuffer was added to this pellet, and the mixture was allowed to stand for 30 minutes under ice cooling for polymerization.

It was centrifuged at 4 ° C. at 100,000 g for an hour, and the resulting supernatant and the body volume of glycerol b
A microtubule protein was obtained by adding a buffer and repeating the above-mentioned polymerization centrifugation operation and decentrifugation operation.

[Measurement of Microtubule Protein Polymerization Inhibitory Activity] Polymerization inhibitory activity was measured according to the turbidity method.

The microtubule protein prepared by the above method was diluted to 2 mg / ml with MESbuffer under ice cooling. Transfer the protein solution to a quartz cell for absorbance measurement, add each drug dissolved in DMSO, and at the same time, add 37
℃.

The absorbance at 400 nm was measured, and the degree of polymerization of microtubule proteins (IC50 value) was measured.

The results are shown in Table 1.

[0053]

[Table 1]

Test Example 2 Compounds (2), (3) and (4) were dissolved in methanol and diluted with sterilized physiological saline so as to have a desired concentration.

2 × 10 4 HL-60 (human leukemia) cancer cells were prepared using a culture medium containing RPMI-1640 medium.
It was adjusted to -1 x 104 / ml, and 2 ml was dispensed into a 6-well petri dish having a diameter of 35 mm. Then, the test cells to which 50 μl of the sample prediluted to the target concentration were added at the same time as the start of the culture were cultured at 37 ° C. in a 5% carbon dioxide gas incubator for 3 to 4 days, and then the viable cells were measured. And inhibition rate from IC
50 values were determined.

The results are shown in Table 2.

[0057]

[Table 2]

[0058]

Front Page Continuation (72) Inventor Shigeo Morimoto 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd.

Claims (2)

[Claims] 1. Combretastatin analogs represented by 2. Combretastatin analogs represented by