JPH03291264A - Dimethylaniline derivative, preparation thereof and cancer cell differentiation-inducing examination reagent and cancer drug containing the same as active ingredient - Google Patents

Abstract

NEW MATERIAL:A dimethylaniline derivative of formula I. USE:Useful as a cell differentiation-inducing examination reagent or carcinogenetic agent. Since the production exhibits a strong cellular differentiation-inducing activity and has low cellular toxicity, the compound can be utilized for the clarification of cell differentiation mechanism and carcinogenesis mechanism as a biochemical reagent. Orally or parenterally administrable. PREPARATION:p-Dimethylaminobenzaldehyde of formula II is reacted with sodium cyanide and acetic acid in a solvent such as methanol at -5 deg.C to 5 deg.C to prepare a cyanhydrin ethoxyethyl ether of formula III. The compound of formula III is subsequently reacted with p-bromo-p-toluic acid methyl ester of formula IV to prepare a methyl ester of formula V. The compound of formula V is reacted with hydroxylamine in the presence of a base such as sodium hydroxide in a solvent such as methanol at the room temperature to provide the compound of formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a dimethylaniline derivative, which is a novel cell differentiation inducer, a method for producing the same, and a cell differentiation induction test reagent and an anticancer agent containing the dimethylaniline derivative as an active ingredient.

[Prior Art] Chemotherapy has traditionally been used as the main drug therapy for cancer. Chemotherapy includes methods that directly act on cancer cells to inhibit their metabolism or suppress DNA synthesis to prevent proliferation, as well as methods to differentiate undifferentiated cancer cells into functional cells and inhibit proliferation. There is a way to do it.

As cell differentiation inducers that can induce the differentiation of undifferentiated cancer cells, for example, substances that induce the differentiation of mouse friend leukemia cells have been known. For example, dimethyl sulfoxide (DMSO), hexamethylene bisacetate, etc. It is believed that HMBA and the like exert their effects by acting on the cell membrane of cancer cells, but their activity is only expressed at high concentrations, making it difficult to put them into practical use. In addition, trichostatins are attracting attention as substances that have strong cell differentiation-inducing activity at low concentrations (Japanese Patent Application Laid-Open No. 1765-1765
However, in order to obtain trichostatins, conventional methods include a) fermentation using microorganisms (Japanese Patent Application Laid-open Nos. 61-176523 and 60-149520) b) ) Synthetic methods (Japanese Unexamined Patent Publication No. 63-49595) are known, but all of them have the disadvantage of requiring a great deal of effort.

[Problem II to be Solved by the Invention] Therefore, an object of the present invention is to provide a cell differentiation-inducing substance that is easier to prepare and has a strong cell differentiation-inducing activity at a low concentration.

[Means for Solving the Problems] As a result of intensive research, the present inventors succeeded in synthesizing a compound that exhibits a strong cell differentiation-inducing activity at a low concentration, and completed the present invention.

That is, the present invention provides a dimethylaniline derivative represented by the formula [1] after converting it into cyanohydrin ethoxyethyl ether represented by the formula [1].

The present invention also provides a method for treating p-dimethylaminobenzaldehyde represented by the formula obtained by reacting with P-bromo-P-toluic acid methyl ester represented by the formula, and hydroxylamine in the presence of a base. Provided is a method for producing a dimethylaniline derivative, characterized by reacting it with a dimethylaniline derivative.

Furthermore, the present invention provides a cell differentiation induction test reagent and an anticancer agent containing the dimethylaniline derivative represented by Formula 1 [I] as an active ingredient.

[Effects of the Invention] The dimethylaniline derivatives of the present invention are potent against not only Mousefriend leukemia cells but also human chronic osteogenic leukemia cells (1,562 cells in humans), as will be clear from the Examples described below. It is a substance that moderates cell differentiation inducing activity,
It can be used as an anticancer drug.

Furthermore, the compounds of the present invention have low toxicity and are excellent in safety as drugs.

Furthermore, the dimethylaniline derivative of the present invention has a short synthesis process of 3 steps and can be synthesized by an efficient method. Compared to the conventional synthesis of trichostatins, the process is less than 1/4, and compared to microbial fermentation, it is practical because it can be produced in large quantities in a short time considering the time required for purification. be.

Furthermore, since this compound is easy to prepare as described above, it is easy to label with tritium or the like. In addition, as mentioned above, it has high cell differentiation inducing activity and low cytotoxicity, so it can be used as a biochemical reagent (cell differentiation inducing reagent) to elucidate cell differentiation mechanisms and carcinogenic mechanisms.

[Detailed description of the invention] The novel dimethylaniline derivative of the present invention is -H3 Hs ■ ■ Process l Process 2 ■ Process 3 Seed cow==ki Each of these steps will be explained in order below.

A crude product can be obtained by reacting p-dimethylaminobenzaldehyde (compound ①) with sodium cyanide and acetic acid. This reaction takes place at -5-5°C with a temperature of 0.3-3
The reaction is preferably carried out for a period of time, and examples of the solvent used in this reaction include methanol, ethanol, dimethylformamide, dimethyl sulfoxide, toluene, benzene, water, pentane, and ether.

Further, the mixing ratio of compound (1), sodium cyanide and acetic acid is usually 1:1:1 to 1:10:20, preferably 1
:1:1.1 to about 1:2:3. The obtained crude reaction product was immediately mixed with ethyl vinyl ether (EE) as crude reaction product 1.
3 to 5 equivalents to equivalent volume, and pyridinium para-toluene sulfone salt (PPTS) to 0.00 equivalent to crude reactant.
1 to 0.1 equivalent was added and reacted overnight at room temperature, and the resulting reaction product was subjected to column chromatography (Carrier Nijiri Gel 60 (manufactured by Merck & Co., Ltd.), elution solvent hexane: ethyl acetate = 10: O - 8: 2) can be purified to obtain cyanohydrin ethoxyethyl ether (compound ①) (step 1), and this reaction can usually be carried out in a yield of 40% or more.

Compound ■ is lithium diisopropylamide (LDA)
Alternatively, after reacting with n-butyllithium etc. to form an anion, the anion can be reacted with P-bromo-p-)ruylic acid methyl ester (compound ■) under water cooling to obtain an ester (compound ■) (Step 2 ) Preferred solvents used in the reaction include THF, dimethoxyethane, diethyl ether, hexamethylphosphoric triamide (HMPA).
, diethylene glycol dimethyl ether, toluene,
N,N'-dimethylimidazolidinone and the like can be mentioned. Among them, THF is preferably used. Also,
The reaction temperature is preferably -100°C to -20°C, more preferably -78°C to -60°C. The mixing ratio of compound (1) and compound (2) is usually 1:1 to 123, preferably 1:1.
:1 to about 1:1.1. This reaction can usually be carried out with a yield of 48% or more.

The obtained methyl ester (compound ■) can be reacted with hydroxylamine in the presence of a base at room temperature for 1 to 10 hours to obtain the dimethylaniline derivative (compound ■) of the present invention (step 3). Preferred bases to be used include potassium hydroxide, sodium hydroxide, triethylamine, sodium bicarbonate, sodium methylate, etc.1 Reaction solvents include methanol,
Ethanol, benzene, ethyl acetate, ether, THF
I can list many things. The mixing ratio of compound (1) and hydroxylua is usually 1:1 to 1:1000, preferably about 1:40.

The cancer cell differentiation-inducing activity of the above-mentioned compound of the present invention was demonstrated in Mouse Friend leukemia cells (Reference and source: C, Fr1
End et al., Canada, Cancer, conf, 8,
171, (1969) (obtained from the Institute of Applied Microbiology, University of Tokyo) (Example 3).

Friend leukemia cells are mouse cancer cells that proliferate in a state where proerythroblast differentiation has been stopped due to infection with the Friend virus. These cancer cells are undifferentiated cells and do not produce hemoglobin, but when they differentiate and become erythroblasts, they produce hemoglobin. Therefore, the degree of differentiation can be determined using hemoglobin as an indicator. That is, the differentiation induction rate can be determined from the number of benzidine-positive cells using Orkin's benzidine staining method.

Comparing the concentration required to obtain a 50% differentiation induction rate with trichostatin A, the concentration of the compound of the present invention was about 100 times higher.

In addition, 1562 cells (manufactured by Dainippon Pharmaceutical Co., Ltd., P
,T,Rowley,EXP,Herg+at,9,3
2. (1981)) was investigated. In humans, 1,562 cells are undifferentiated cancer cells that produce hemoglobin when they differentiate and become functional cells, so their differentiation-inducing activity can be examined in the same manner as Mouse Friend leukemia cells. As a result, it was found that the compound of the present invention had the same differentiation-inducing activity as trichostatin A at 1/10 times the concentration (Example 4).

Furthermore, the toxicity of the present invention was investigated using human cervical cancer HeLa cells (obtained from Professor Masaatsu Yamada, Faculty of Pharmaceutical Sciences, University of Tokyo). This H
When eLa cells survive and repeat cell division, they form colonies. Therefore, the degree of toxicity can be determined from the colony formation inhibiting concentration. As a result, it was found that the toxicity of the compound of the present invention was about 1/10 times lower than that of trichostatin A (Example 5).

Thus, it can be seen that the compounds of the present invention have strong cancer cell differentiation-inducing activity and low toxicity.

The anticancer agent of the present invention containing the substance represented by formula [I] as an active ingredient can be administered either orally or parenterally. For oral administration, soft and hard capsules, tablets,! It can be administered as W granules, fine granules, powders, etc. In the case of parenteral administration, it can be administered in the form of an injection, an infusion, or a solid or suspended viscous solution, such as a liniment that allows for sustained mucosal absorption. The manufacturing method of these dosage forms and the selection of excipients, disintegrants, suspending agents, etc. used in the manufacturing thereof can be easily made by those in the field. Furthermore, it is also possible to incorporate compounds having an anticancer effect other than the compound represented by the above formula into the anticancer agent of the present invention.

The proportion of the active ingredient in the anticancer agent of the present invention varies depending on the dosage form, and is usually 0.1 to 50% by weight, regardless of the dosage form.

The dosage depends on the age, gender, symptoms, and desired ft1 of the fever patient.
The decision is made by the doctor taking into account the therapeutic effect, administration period, etc., but it is usually desirable to make the decision based on an adult daily dose of 0.01 to 100 μg (appropriate dose).

[Examples] Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

Preparation of Indigo Negative Yu Formulation 2.5 mg of the compound of the present invention was dissolved in a mixture of 1 g of purified sesame oil and 100 g of aluminum stearate gel. Each 0.5 ml of the produced solution was dispensed into capsules to prepare oral capsules.

Confectionery Wataru Synthesis of the compound of the present invention 6.0 g (40 g) of commercially available p-dimethylaminoaldehyde
(1) was dissolved in 200 parts of 99% ethanol, and 3.2 g (64-1 ol) of sodium cyanide was suspended therein. 4 ml of acetic acid was added to this mixture while cooling with water and keeping the temperature below 10°C. After 1 hour, 300 sl of cold water was added and extraction was repeated with chloroform. The chloroform layer was washed with water and brine, dried over magnesium sulfate, and immediately ethyl vinyl ether (10) and pyridinium p-toluenesulfone salt (500B) were added and reacted overnight at room temperature.

The resulting reaction solution was washed with water and brine, dried over magnesium sulfate, and then concentrated under reduced pressure. The obtained phase acid was subjected to column chromatography (column: silica gel 60 (manufactured by Merck), developing solvent: hexane: ethyl acetate = 10
:0 to 8:2 to obtain 4 g (yield: 40%) of cyanohydrin ethoxyethyl ether (compound 2) as a yellow oil.

The physical properties of the obtained compound are as follows.

I Ry 1lax 298G (s), 2900 (g
), 1615 (s), 1530 (S), 815 (
s) cm-'IHN pJ R81) P Garden 1.25.1.28 (3H, t, J=7.0 Hz
), 1.39.1.42 (3H, d, J=6.2H
z), 3.00 (6H, s), 3.5-3.8 (2H
), 4.88.5.08 (IH, q, J-6, 2H
z), 5.32.5.45 (IH, s), 6.71
(2H, d, J=8.0 Hz), 7.35, 7.3
7 (2)1. d, J-8,0Hz) Next, 1.5 g of cyanohydrin ethoxyethyl ether (compound ■)
(6,1s■ol) is dry tetrahydrofuran 151
1, and an equal amount of n-butyllithium was added at -78°C to react. After 15 minutes, 1.52 g (6-7 g) of p-bromo-p-toluic acid methyl ester was dissolved in 15 g of dry tetrahydrofuran. -70℃2
After stirring for an hour, an aqueous ammonium chloride solution was added, and the reaction mixture was heated to room temperature. The reaction mixture was extracted with ether, washed with water and brine, dried over magnesium sulfate, and concentrated under reduced pressure. 2.8 g of the obtained crude ester was immediately mixed with 50 ml of acetic acid,
50 ml of water and 50 ml of THF were added and stirred at 50°C for 1 hour. The resulting reaction solution was neutralized with sodium bicarbonate and then extracted with 50 parts of ethyl acetate. To the ethyl acetate layer, 50+ wl of 5% sodium hydroxide solution was added and stirred for 1 hour at 0°C.The reaction mixture was separated into layers, the aqueous phase was extracted with ethyl acetate, the ethyl acetate layer was washed with water and brine, and magnesium sulfate was added. The residue was dried under reduced pressure and concentrated under reduced pressure to obtain 0.855 g (yield: 48%) of methyl ester (compound ①) as a yellow amorphous solid. Further recrystallization was performed with ethyl acetate to obtain fine needle-like crystals. The physical properties of the obtained compound are as follows.

11.1),: 142-144℃ I Rv KBr 1720 (s), 1670 (s
), 1600 (S), +275 (s), 815 (
s) c+++-''HN M R5ppm CDCh
/CDJD (1:1)3.06 (6H,s), :1
．． 89 (3H, s), 4.24 (2H, s), 5.
55 (2H, d, J-9,0Hz), 7.35 (
2)1. d, J-11,2)1z), 7.91 (
2H, d, Jl=9.0 Hz), 7.97 (2)
1. d.

Jsen81 Hz) Methyl ester (compound) 1001g (0.34m
5 ol) was added to 5 ml of a methanol solution of hydroxylamine prepared from 3.2 g of hydroxylamine hydrochloride, 2.58 g of potassium hydroxide, and 23 μl of methanol. Once heated to dissolve the crystals, 0.03 g of potassium hydroxide was added.
g was added thereto, and the mixture was stirred at room temperature for 4 hours. 1 Add hydrochloric acid and p
The resulting crystals were collected, thoroughly washed with water, and concentrated to obtain 66 mg (yield: 65 tons) of a powdery solid. Recrystallization with chloroform:methanol (1:1) gave the compound of the present invention (compound ①) in the form of almost colorless fine needle-like crystals. The physical properties of the obtained compound of the present invention are as follows.

s, p,: 155-160℃ (decomposition) IRy■a
X 3200 (br), 161 (1 (s), 1530 (s)
, 1370(S), 945(r) C Rho 1 1HNMRδl @ CDCl3/CD, OD (1:1
)2.95 (6H,s), 4.21 (2H,s),
6.66 (28, d.

J=9.0 Hz), 7.33 (2H, d, J-8
, 3Hz), 7.42 (2H.

d, J-9,0)! ri, 7.63 (2H, d, J
-8,3J(z) Eika Negative Ufriend Differentiation-inducing effect on leukemia cells
MNo. 1 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) 9.4 g, L-
A culture medium prepared by dissolving 0.3 g of glutamine and 0.7 g of sodium bicarbonate in 11 distilled water, and then adding beef tallow serum (manufactured by HyC1one) to a final concentration of 12% by volume was placed in a 6 cm diameter Petri dish (Nunc). The solution was dispensed into 5 ml portions (manufactured by Sekisui Chemical Co., Ltd.). Furthermore, 5×10' Friend leukemia cells/ml and a predetermined amount of the compound of the present invention were added and cultured for 6 days at 37° C. in an acid gas incubator. Trichostatin A was used as a comparison agent. After counting the number of cells on a hemocytometer,
Cells were sorted by centrifugation and stained with benchcin. 0.5 benzidine hydrochloride to be 21g/I
A solution prepared by dissolving z in acetic acid solution and adding 0.5 z of 30% hydrogen peroxide solution was placed in a 96-well microtiter plate, and the same amount of cell suspension was added thereto. After 5 minutes, 200 ml of each cell was added under a microscope.
The cells were counted, and the percentage of cells stained blue with benzidine was determined. In addition, each value was converted by taking the activity of 1,8z dimethyl sulfoxide (DIISO) as 100z.

The activities of the compounds of the invention and of trichostatin A are shown in Table 1.

As is clear from Table 1, the compound of the present invention exhibited approximately 1/100 the activity compared to trichostatin A.

Differentiation-inducing effect on Bun1D44-562 cells RPM1164ONo, 1 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.)
Dissolve 10.4g and 0.8g of baking soda in distilled water of step 11,
Next, an amount of beef tallow serum (Hy
A medium prepared by adding CIone (manufactured by CIone) was placed into a 6 cm diameter
1 ml was dispensed into a petri dish (manufactured by Nunc). Furthermore, 1X10' of -562 cells/quantity 1 and a predetermined amount of the compound of the present invention were added and cultured for 6 days in an acid gas incubator at 37°C. Trichostatin A was used as a comparison agent. After counting the number of cells on a hemocytometer in the same manner as in Example 3, benchcine staining was performed to determine the differentiation induction rate. Note that each value was calculated using the activity of 10-'M mitomycin C as 100%.

The activities of the compounds of the invention and trichostatin A are shown in Table 2.

As is clear from Table 2, the cell differentiation inducing activity against human 1562 cells was found to be about 10 times that of trichostarin A, which is 1 more potent.

Eagle MEM No. 1 medium (manufactured by Hinaga Seiyaku 1) 9 that inhibits colony formation of HeLa cells
．． 4g, L-glutamine 0.3g and fit t 0
．． A culture medium prepared by dissolving 7 g in distilled water and adding fetal bovine serum (manufactured by LycIone) in an amount to give a final concentration of 10% by volume was placed in a 6 cm diameter Petri dish (Nunc).
Company! V) 5a1 aliquot was dispensed at °. Furthermore, HeLa disease cells
OO pieces and a predetermined amount of the compound of the present invention were added for 10 days.
The cells were cultured in a 5% carbon dioxide incubator at 7°C.

Trichostatin A was used as a comparison agent. After culturing, discard the culture solution and add 5 parts of methanol and 1 part of methanol and fix for 15 minutes.
% Giemsa solution for 30 minutes. Colonies with 10 or more cells were counted, and the colony formation rate of the petri dish at each drug concentration was determined, with the number of colonies in the petri dish to which no drug was added as 100.

In addition, the colony formation inhibition rate was determined using the following formula.

Colony formation inhibition rate (2) = 100 - Colony formation rate (%) The results are shown in Table 3.

As is clear from the table, the toxicity of the present invention was found to be lower than that of trichostatin A.

Claims (4)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ Dimethylaniline derivative represented by (2) After converting p-dimethylaminobenzaldehyde shown by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ to cyanhythrin ethoxyethyl ether shown by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, There are chemical formulas, tables, etc. ▼ The formula obtained by reacting with p-bromo-p-toluic acid methyl ester shown in ▲ There are mathematical formulas, chemical formulas, tables, etc. 2. The method for producing a dimethylaniline derivative according to claim 1, which comprises reacting with a dimethylaniline derivative. (3) A cell differentiation induction test reagent containing the dimethylaniline derivative according to claim 1 as an active ingredient. (4) An anticancer agent containing the dimethylaniline derivative according to claim 1 as an active ingredient.