JPH03291263A - 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 and a carcinogenic agent. Since the compound exhibits a strong cell differentiation-inducing activity in a low concentration and has a low cellular toxicity, the compound can be utilized for the clarification of cellular differentiation mechanism and carcinogenesis mechanism. The compound can be readily manufactured in large amounts. Orally or parenterally administrable. PREPARATION:A grignard reagent of formula II is reacted with glutaric anhydride in a ratio of 1:1-1:10, preferably 1:1-1:2, and the prepared carboxylic acid is esterified into methyl ester thereof. The prepared ester of formula III is reacted with hydroxylamine in the presence of a based to prepare the compound of formula I.

Description

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

[Prior Art] Conventionally, chemotherapy has been mainly used as 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.

Examples of cell differentiation inducers that can induce the differentiation of cancer cells, which are undifferentiated cells, include substances that induce the differentiation of mouse friend leukemia cells, such as dimethyl sulfoxide (DMSO), hexamethylene bisacetate Although HMBA and the like are thought to exert their effects by acting on the cell membranes of cancer cells, 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 a strong cell differentiation-inducing activity at low concentrations (Japanese Patent Application Laid-Open No. 1755-1983).
However, in order to obtain trichostatins, conventional methods a) Fermentation using microorganisms (Japanese Patent Application Laid-open No. 61-176523, JP 60-149520) b ) Synthesis method (Japanese Patent Application Laid-Open No. 63-49595) is known, but both methods have the disadvantage of requiring a great deal of effort.

[Problems 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 strong cell differentiation-inducing activity at low concentrations.

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

That is, 1 the present invention is 1 formula [I] [I] Provided is a dimethylaniline derivative represented by

Furthermore, the present invention involves methyl esterifying a carboxylic acid obtained by reacting a Grignard reagent represented by the formula with glutaric anhydride to obtain a methyl ester represented by the formula, and then reacting it with hydroxylamine in the presence of a base. A method for producing a dimethylaniline derivative is provided.

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

[Effects of the Invention] The dimethylaniline derivative of the present invention can be used not only in mouse friend leukemia cells but also in human chronic myeloid leukemia cells (human chronic myeloid leukemia cells), as will become clear in the Examples described later.
It is also a substance that has a strong cell differentiation-inducing activity against cancer, and can be used as an anticancer agent.

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 can be synthesized using a short and efficient method of only three steps. Compared to the conventional synthesis of trichostatins, it takes 174 steps or less, and compared to microbial fermentation, considering the labor involved in making silk, it can be produced in a short time and in large quantities, making it practical. .

Furthermore, since the present compound is easy to prepare as described above, it is also easy to label with tritium or the like. Furthermore, 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] It can be manufactured by the method shown in .

Process 2 ■ Hx CH.

■ ■ ■ ■ ■ Process 1 ■ Process 3 Base E=1 Hereinafter, each of these steps will be explained in order.

Mende to P-bromomethylaniline (compound)
method (A, Mendel, J, Organo
Grignard reagent (compound ■
) can be obtained. Examples of the nonaqueous solvent used in this reaction include tetrahydrofuran (THF) and ether, and THF is preferably used in the present invention.

Compound (1) can be obtained by reacting glutaric anhydride (compound (2)) with Grignard reagent (2) in a non-aqueous solvent under water cooling (Step 1). Examples of the nonaqueous solvent used in this reaction include THF, 1.4 dioxane, dimethoxyethane, diethyl ether, and toluene. Among them, THF is preferably used. Further, the reaction temperature is preferably 18°C to room temperature, more preferably 17°C to room temperature.
The temperature is 8°C to -30°C. The mixing ratio of compound (1) and compound (2) is usually 1:1 to 1:10, preferably 1:1 to 1:1.
: About 2. This reaction can usually be carried out with a yield of 20% or more.

Compound (1) is prepared by incubating the obtained compound (2) with dimethyl sulfate and a base in a solvent at 0°C to 60°C, preferably 40 to 60°C.
It can be obtained by methyl esterification by reacting at 0°C for 10 minutes to 1 hour (Step 2) Examples of solvents used in the reaction include methanol, ethanol, acetone, 1,4 dioxane, etc. . Furthermore, examples of preferable bases used in the reaction include triethylamine, sodium hydroxide, potassium carbonate, dicyclohexylethylamine, and the like. Also, the mixing ratio of compound ■ and dimethyl sulfate is usually 1:1 to 1:10.
．． Preferably 1:1.05 to 1:1. :] It is about .

This reaction can usually be carried out with a yield of 90% or more.

The obtained methyl ester (compound ■) can be reacted with hydroxylamine in the presence of a base at room temperature for 1 to 24 hours to obtain the dimethylaniline derivative (compound ■) of the present invention (step 3), which can be used in the present invention. Preferred bases include potassium hydroxide, triethylamine, sodium hydroxide, 1% sodium hydroxide, sodium methylate, etc. Examples of the reaction solvent include methanol, ethanol, benzene, ethyl acetate, ether, etc. I'll come. The mixing ratio of compound (1) and hydroxylamine is usually 1:1 to 1:1000, preferably about 1:40. This reaction can usually be carried out with a yield of 54% or more.

The cancer cell differentiation-inducing activity of the above-mentioned compound of the present invention was demonstrated in Mouse Friend leukemia cells (References and source C, Fr1en).
d et al., Canada, Cancer, Conf, 8.17
1. (1969), University of Tokyo, Institute of Applied Microbiology, obtained from Professor Michio Oishi) (Example 3).

Friend leukemia cells are mouse cancer cells in which proerythroblasts have stopped differentiating and proliferate due to infection with the Friend virus. These cancer cells are undifferentiated cells and do not produce hemoglobin, or they differentiate into erythroblasts and 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 ventisin-positive cells using Orkin's ventisin staining method.

When the concentration required to obtain a 50% differentiation induction rate was compared with that of trichostatin A, the concentration of the compound of the present invention was about 10 times higher.

In addition, 1562 cells (manufactured by Dainippon Pharmaceutical Co., Ltd., 11
Ding, Rowley, EXP, Herwat, 9, 32.
(1981)) was investigated. In humans, 1,562 undifferentiated cancer cells produce hemoglobin when they differentiate and become functional cells, so it is possible to examine their differentiation-inducing activity using the same method as mouse-friend leukemia cells. As a result, it was found that the compound of the present invention has the same differentiation-inducing activity as trichostatin A (Example 4)
.

Furthermore, the toxicity of the present invention was investigated using human uterine carcinoma 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 concentration inhibiting colony formation. As a result, it was found that the toxicity of the compound of the present invention was 1/100 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 [1] as an effective acid can be administered either orally or parenterally. When administered orally, it can be administered in the form of soft or hard capsules, tablets, granules, fine granules, powders, etc. In the case of parenteral administration, it can be administered in the form of an injection, a drip, or a solid or suspended viscous liquid, such as a suppository that allows for sustained mucosal absorption. A person skilled in the art can easily select the method for producing these dosage forms and the excipients, disintegrants, suspending agents, etc. used in the production. 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 effective acid content of 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 should be determined by the doctor, taking into account the patient's age, sex, symptoms, desired therapeutic effect, administration period, etc.The usual daily dose is 0.01 to 100 mg (the amount of active ingredient )
It is desirable to make decisions based on

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

Example 1 Preparation of the formulation The compound of the invention @2.5B was dissolved in a mixture of 1 g of purified sesame oil and 100 g of aluminum stearate gel.

0.511 portions of the resulting solution were dispensed into capsules to prepare oral capsules.

Synthesis of the compound of the present invention Commercially available p-bromodimethylaniline 1.0 g (5 mm
The Grignard reagent obtained by reacting magnesium (ol) with magnesium in THFaml was mixed with glutaric anhydride 1 at -78°C.
, was added dropwise over 10 minutes to THF solution 511 in which 2 g of the solution was dissolved. After stirring at -78°C for 1.5 hours, the temperature was raised to 0°C and
Stir for hours. A saturated ammonium chloride solution was added to the resulting reaction solution and extracted with ether to obtain 242 mg of crude reaction product (
A yield of 21z) was obtained. The obtained crude reaction product was methanol 1
0.28 ml of triethylamine was added, and 0.127 ml of dimethyl sulfate was added, and the mixture was heated under reflux for 1 hour. After the 0 reaction, it was extracted with ethyl acetate, washed with water and brine, dried, and concentrated. 280 mg of ester was obtained. 280 g of the obtained methyl ester was added to 5 ml of a methanol solution of hydroxyamine prepared from 1.6 g of hydroxyamine hydrochloride, 1.29 g of potassium hydroxide, and 12 ml of methanol, and once heated to dissolve the crystals, water was added. 0.02 g of potassium oxide was added, and the mixture was stirred at room temperature for 4 hours. Concentrated hydrochloric acid was added to adjust the pH to 4.5, methanol was removed under reduced pressure, extracted with ethyl acetate, washed with water and brine, dried, and concentrated to give 190 mg of powdery solid (yield 6).
8z) was obtained. Recrystallization from chloroform:methanol (1.1) gave the compound of the present invention as an almost colorless amorphous solid.

all, : 1266C (decomposition) l Ry wax/KBr 3:] 60 (m), 3215 (s), 1650 (S),
1615(s), 820(s)C Peng 1'HNMR8pp Seal CDCh/CDJD (1:1
)2.96 (6)1. S), 4.21 (2)
1. s), 6.66 (2H, d.

J=9.0)1z),7.33(2H,d,
J-8, 3Hz), 7.42 (2H.

d, J=9.0) 1z), 7.63 (2H
, d, J wealth 8.3 Hz) Example 3 ・t Differentiation-inducing effect on Friend leukemia cells Eagle ME
MNo. 1 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) 9.4 g, L-
A culture medium prepared by dissolving 0.3 g and 3110.7 g of glutamine in 11 distilled water and adding fetal bovine serum (manufactured by HyCIone) to a final concentration of 12% by volume was placed in a Petri dish with a diameter of 6 cm (Nunc). The solution was dispensed into 5 ml portions (manufactured by Sekisui Chemical Co., Ltd.). Further, 5×10 layers/ml of Friend's leukemia cells 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, the cells were separated by centrifugation and stained with benzidine. Add benzidine hydrochloride to 2mg/g+1
, sx#l: Dissolve in acid solution and add 30% hydrogen peroxide solution to 0.
5 was added to a 96-well microtiter plate, and the same amount of cell suspension was added.After 5 minutes, 200 cells were counted at each point under a microscope, and the percentage of cells that were stained blue by Bencin was counted. I asked for In addition, each value is 1.8
z Dimethyl sulfoxide (DMSO) activity to 100
It was converted as z.

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

As is clear from the table, the compound of the present invention exhibited an activity of about 171O compared to trichostatin A.

(differentiation inducing effect on -562 cells at birth 4) RPM1164ONo, 1 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.)
10.4 g and 0.8 g of sodium bicarbonate were dissolved in distilled water in Step 11, and then fetal bovine serum (H
A medium prepared by adding yCIone) to a diameter of 6 cm
1 ml of the solution was dispensed into a petri dish (manufactured by Nunc). 1xlO' cells/ml of Zarani-562 cells 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, benzidine 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 of trichostatin A are shown in Table 2.

As is clear from the table, the cell differentiation inducing activity against human 1562 cells was equivalent to that of trichostatin A, indicating that it was strong.

Colony formation inhibitory effect on HeLa cells with 194-fold Ecle MEM No. 1 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) 9.
4g, L-curcumin 0.3g and baking soda 0.7g in 1
A culture medium prepared by dissolving 1 in distilled water and then adding fetal bovine serum (manufactured by HyClone) in an amount to give a final concentration of 10% by volume was placed in a Petri dish with a diameter of 6 cm (manufactured by Nunc).
ml was dispensed. 100 HeLa cells and a predetermined amount of the compound of the present invention were added to a colander and cultured in a 5% carbon dioxide gas incubator at 37°C for 10 days. Trichostatin A was used as a specific drug. After culturing, discard the culture solution, add 5 ml of methanol, fix for 15 minutes, and add 30 ml of 2% Giemsa's solution.
Stained for minutes. Count cells or colonies of 10 or more,
The colony formation rate of the petri dish at each drug concentration was determined, setting 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 (Umugi) = 100-Colony formation rate (%) The results are shown in Table 3.

As is clear from Table 3, the compound of the present invention was found to be less toxic than trichostatin A.

Claims (4)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ Dimethylaniline derivative represented by (2) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc.▼ The carboxylic acid obtained by reacting the Grignard reagent shown by the formula with glutaric anhydride is methyl esterified, and the formula ▲ There are mathematical formulas, chemical formulas, tables, etc.▼ Claim 1, characterized in that the methyl ester shown above is prepared and then reacted with hydroxylamine in the presence of a base.
A method for producing the dimethylaniline derivative described above. (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.