JPH0641408B2 - Enzyme inhibitors - Google Patents

Description

TECHNICAL FIELD The present invention relates to a tyrosine kinase inhibitor. More specifically, the general formula (1) (Here, R ¹ represents hydrogen, a cyano group or a CONH ₂ group,
₂ represents a CONH ₂ group or a group represented by CONHCONH ₂ ) and relates to a tyrosine kinase inhibitor containing a 3,5-diisopropyl-4-hydroxystyrene derivative or a physiologically acceptable salt thereof as an active ingredient. is there.

The present inventors investigated the physiological activity of various 3,5-diisopropyl-4-hydroxystyrene derivatives, and found that the compound represented by the above general formula (1) exhibited excellent physiological activity, particularly tyrosine kinase inhibitory activity. The present invention has been completed based on the findings.

[Conventional technology]

Known tyrosine kinase inhibitors include, for example, quercetin, which is 3,5-diisopropyl-4.
-There is no report of such inhibitory activity for hydroxystyrene derivatives.

[Problems to be solved by the invention]

Recently, a group of cell growth factor receptors that control cell growth have tyrosine kinase activity, and a group of oncogenes that encode proteins similar to cell growth factor receptors have been reported. And their products have tyrosine kinase activity. From the above, tyrosine kinase is deeply involved in the control of cell growth, and further, canceration, which is an unlimited growth of cells, is a state in which tyrosine kinase is activated and the cell growth cannot enter the stationary phase. It is said that. Therefore, inhibiting the tyrosine kinase of cells in such a state leads to the prevention of carcinogenesis or carcinogenesis, and in turn, the 3,5-di-represented by the above-mentioned general formula (1) which is a tyrosine kinase inhibitor. The isopropyl-4-hydroxystyrene derivative can be used as a carcinostatic agent or a carcinogenic agent with few side effects.

[Means and Actions for Solving Problems]

The tyrosine kinase inhibitor, carcinostatic agent and carcinogenic agent according to the present invention are 3,5-diisopropyl-types represented by the general formula (1).
The 4-hydroxystyrene derivative or a physiologically acceptable salt thereof is used as an active ingredient. Specific examples of the compound represented by the general formula (1) include the following.

3,5-diisopropyl-4-hydroxycinnamic acid amide (hereinafter abbreviated as compound I) α-cyano-3,5-diisopropyl-4-hydroxycinnamic acid amide (hereinafter abbreviated as compound II) β, β-dicarbamoyl-3,5-diisopropyl-4-
Hydroxystyrene (hereinafter abbreviated as Compound III) β-cyano-β-carbamoyl-3,5-diisopropyl-4-hydroxystyrene (hereinafter abbreviated as Compound IV) The compound represented by the general formula (1) is a base. It is possible to form a salt, and as the base, any base capable of forming a salt with the compound represented by the general formula (1) can be selected. Specific examples of salts include, for example, (1) metal salts, particularly salts with alkali metals, alkaline earth metals, and aluminum, (2) ammonium salts, (3) amine salts, especially methylamine, ethylamine, diethylamine, triethylamine. , Pyrrolidine, piperidine, morpholine, hexamethyleneimine,
Although there are salts with pyridine, aniline, etc., as tyrosine kinase inhibitors, carcinostatic agents and carcinogenic agents, physiologically acceptable salts may be selected from these salts.

Synthesis of Compound The compound represented by the above general formula (1) can be synthesized by the following method.

(a) General formula (1) The compound represented by (R ¹ and R ² are the same as above) is 3,5
- diisopropyl-4-hydroxybenzaldehyde and R ³ -CH ₂ -R ² (wherein R ³ represents a carboxyl group, a cyano group, CO
It is synthesized by reacting a compound represented by NH ₂ group and R ² is the same as above) with a base catalyst. This synthetic method uses a reaction known as the Knoeh Energel reaction, and examples of a base that can be used as a catalyst include ammonia, primary amines, secondary amines, and salts thereof. Specific examples of bases and salts that can be used include aniline, piperidine, pyrrolidine,
Examples include ammonium acetate and piperidine acetate.

Among the compounds represented by the general formula R ³ —CH ₂ —R ² , when a compound in which R ³ is a carboxyl group is used, it is decarboxylated by selecting the conditions to obtain a compound in which R ¹ is hydrogen. You can

(b) 3,5-diisopropyl-4-hydroxycinnamic acid amide was obtained from 3,5-diisopropyl-4-hydroxybenzaldehyde and malonic acid by using the Knoehner-Engel reaction as described in (a) above. By esterifying the 3,5-diisopropyl-4-hydroxycinnamic acid obtained by a conventional method, and reacting the obtained 3,5-diisopropyl-4-hydroxycinnamic acid ester with ammonia,
Alternatively, hydrolysis of 3,5-diisopropyl-4-hydroxycinnamonitrile obtained by reacting 3,5-diisopropyl-4-hydroxybenzaldehyde with cyanoacetic acid in the presence of a base such as piperidine is carried out with an acid or an alkali. Can be synthesized by.

Oxygen Inhibitory Action The tyrosine kinase inhibitor according to the present invention has the general formula
The compound represented by (1) or a salt thereof is used as an active ingredient. The enzyme inhibitory action and toxicity of these compounds are as shown in the following experimental examples. Specifically, the tyrosine kinase inhibitory effect was measured by the method described below.

The tyrosine kinase inhibitory action of the compound of the present invention is
S. Cohen et al.'S method for measuring tyrosine kinase activity [The. Journal of Biological Chemistry (J. Bi
ol.Chem.), 257, 1523 (1982)] for reference.
That is, human cancer cell-derived established strain A-431 was added to fetal bovine serum 10
%, Streptomycin (50 μg / m), penicillin G (50 international units / m) and kanamycin (50 μm
g / m) in a Dulbecco's modified Eagle medium [Nippon Pharmaceutical Co., Ltd.] at 37 ° C. under 5% CO ₂ condition. The obtained cells were treated according to the method of S. Cohen et al. Above to obtain a membrane preparation containing the epidermal growth factor receptor-tyrosine kinase complex (hereinafter abbreviated as a membrane preparation). The term "membrane preparation" as used herein refers to a cell membrane fraction of A-431 cells, and unlike normal cells, the A-431 cells have a very large amount of epidermal growth factor-receptor (hereinafter referred to as E-cell).
Abbreviated as GF-receptor. ) Is expressed and the membrane preparation of the cell is used, the tyrosine kinase activity can be sufficiently measured without purifying the receptor. This membrane preparation was used for the following measurements without being solubilized.

N-2-hydroxyethylpiperazino-N'-2-ethanesulfonic acid buffer solution (20 mM, pH 7.4) MnCl ₂ (1 mM),
Bovine serum albumin (7.5 μg), membrane preparation (10 as protein)
μg) to a sample dissolved in dimethyl sulfoxide,
After incubation at 0 ° C for 5 minutes, epidermal growth factor (hereinafter abbreviated as EGF) (100 ng) was added, and the mixture was added at 0 ° C.
Incubated for 15 minutes. EGF here
Can be added to activate the tyrosine kinase of the EGF-receptor present in the membrane preparation. EG
The F-receptor is one of the typical receptor tyrosine kinases and shows almost no activity unless EGF binds to the EGF binding site. However, when EGF binds to the EGF binding site, it changes to an activated form that exerts strong tyrosine kinase activity. The activated EGF-receptor specifically phosphorylates the receptor itself and the tyrosine residue of the protein substrate. The phosphorylation increased by adding EGF to the reaction system is tyrosine-specific, and the tyrosine kinase activity can be measured by quantifying the phosphorylation amount.

To quantify the amount of phosphorylation referred to here, [γ- ³² P] ATP, which is one of the substrates for measuring tyrosine kinase activity, is added. Since tyrosine kinase catalyzes the reaction of transferring the phosphate group at the γ-position of ATP to the hydroxyl group of the tyrosine residue in the protein, [γ- ³² P] ATP in which the phosphate at the γ-position is radiolabeled as ATP When used, the activity of tyrosine kinase can be detected as an increase in radioactivity of the substrate protein (EGF-receptor itself in the method for measuring inhibitory activity of the present invention). Therefore, [γ- ³² P] ATP (3000 Ci / m mol, 0.1 μC
i) was added to the final volume of 70 μm, and the mixture was further incubated at 0 ° C. for 15 minutes, and the reaction liquid of 50 μm was added to Whatman 3.
Immediately after soaking in MM paper, the reaction was stopped with 10% trichloroacetic acid-10 mM sodium pyrophosphate aqueous solution. The paper was thoroughly washed with the same solution, then washed with ethanol, dried, and the radioactivity remaining on the paper was measured using a liquid scintillation counter, and this value was designated as A. At the same time, as a control, a reaction without addition of EGF, a reaction without addition of a sample, and a reaction without addition of EGF and a sample were carried out, and the same measurement was carried out to obtain B, C and D, respectively.

The tyrosine kinase inhibition rate was calculated by the following formula.

Here, the term CD represents the tyrosine kinase activity of the control without the test compound, and the term AB represents the tyrosine kinase activity in the presence of the test compound.

Therefore Shows the tyrosine kinase activity or% inhibition that was inhibited by the test compound relative to the control tyrosine kinase activity.

Table 1 shows the tyrosine kinase inhibitory action of the compounds according to the present invention. From this result, it can be seen that the compound according to the present invention strongly inhibits tyrosine kinase.

Acute toxicity ICR female mice (body weight 23 to 26 g) were used, and each group consisted of 6 mice. Compounds (I) to (IV) suspended in a 2.5% aqueous solution of gum arabic containing 0.2% Tween 80 were added to 0.1 m /
Oral administration was performed at a rate of 10 g body weight. Observe general symptoms for 2 weeks after administration to determine the number of dead / tested cases, and
The% lethal dose LD ₅₀ (mg / Kg) was estimated. As a result, no deaths were observed with the compounds (I) to (IV) of the present invention even after administration of 1000 mg / Kg, and the LD ₅₀ of the compounds (I) to (IV) was 1000 mg / K.
It was estimated to be over g and was found to have low toxicity.

Preparation and Dosage As the preparation of the tyrosine kinase inhibitor, carcinostatic agent, or carcinogen-preventing agent according to the present invention, any of oral, enteral and parenteral administration preparations can be selected. Specific formulations include tablets, capsules, fine granules, syrups, suppositories,
Examples include ointments and injections. As a carrier for the preparation of the tyrosine kinase inhibitor, carcinostatic agent, or carcinogen-preventing agent according to the present invention, an organic or inorganic solid or liquid, which is suitable for oral, enteral or other parenteral administration, is usually inert. A pharmaceutical carrier material is used. Specific examples include crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats and oils, gums, and polyalkylene glycols. The tyrosine kinase inhibitor of the present invention, a carcinostatic agent, for the carrier in the formulation,
The proportion of carcinogenic agent can vary between 0.2 and 100%. Also, a tyrosine kinase inhibitor according to the present invention,
The carcinostatic agent and carcinogenic agent can include other tyrosine kinase inhibitors, carcinostatic agents, carcinogenic agents, and other drugs that are compatible with this. In this case, it goes without saying that the tyrosine kinase inhibitor, the anticancer agent and the carcinogenic agent of the present invention may not be the main components in the preparation.

The tyrosine kinase inhibitor, anti-cancer agent, and anti-tumor agent according to the present invention are generally administered in a dosage that achieves a desired action without causing side effects. Although this specific value should be determined by the doctor's judgment, it is generally 10 per adult per day.
It will normally be administered in the range of 10 mg to 10 g, preferably 20 mg to 5 g. The tyrosine kinase inhibitor, carcinostatic agent and carcinogenic agent of the present invention are used as active ingredients in an amount of 1 mg to 5 mg.
It may be administered as a pharmaceutical preparation in the unit of g, preferably 3 mg to 1 g.

〔Example〕

Next, the present invention will be specifically described with reference to Production Examples and Examples of the compound of the present invention, but these Examples do not limit the present invention.

Production Example 1 Synthesis of compound II 3.18 g of 3,5-diisopropyl-4-hydroxybenzaldehyde and 2.78 g of α-cyanoacetamide were dissolved in 40 m of ethanol, 1 m of piperidine was added, and the mixture was heated under reflux for 5.5 hours. After cooling, the solvent was distilled off, the residue was dissolved in ethanol, and a small amount of water was added to form a residue. The crystals obtained were recrystallized from benzene to give 4.2 g of compound II.
Obtained.

Production Example 2 Synthesis of compound III 3.09 g of 3,5-diisopropyl-4-hydroxybenzaldehyde and 1.53 g of malonamide were dissolved in 30 m of ethanol, 0.2 m of piperidine and 0.6 m of acetic acid were added, and the mixture was heated under reflux for 6 hours. Chloroform was added to the reaction solution, followed by washing with water, separating the chloroform layer, distilling off the solvent, and subjecting the residue to column chromatography with silica gel as a carrier, with chloroform-ethanol (4: 1 v / v). Elution gave 450 mg of compound III.

Example 1 A mixture of 100 g of compound II, 55 g of lactose and 41 g of dried potato starch was kneaded with 20 m of water, then extruded through a 16 mesh screen and dried at 40 ° C. to granulate. Then, it is uniformly mixed with 4 g of magnesium stearate and compressed by a conventional method to give 100 tablets per 200 mg.
A tablet containing mg of compound II was obtained.

Example 2 Instead of the compound II of Example 1, the compound IV is used.
In the same manner as in the above, 100 mg of compound IV in 200 mg per tablet is used.
A tablet containing was obtained.

Example 3 196 g of granules obtained in exactly the same manner as in Example 1 was mixed with 4 g of magnesium stearate, and then 200 mg each was filled into a No. 2 hard capsule, and a hard capsule containing 100 mg of Compound II in one capsule. did.

Example 4 Example 3 using compound IV instead of compound II of example 3
By the same method as described above, a hard capsule containing 100 mg of Compound IV in one capsule was prepared.

Example 5 Compound II 10.0 g Lactose 85.0 g Crystalline cellulose 4.5 g Magnesium stearate 0.5 g The above ingredients were mixed well to obtain a powder containing 100 mg of Compound II in 1 g.

Example 6 Example 5 using compound IV instead of compound II of example 5
A powder containing 100 mg of compound IV in 1 g was obtained in the same manner as in.

Continuation of the front page (56) Reference JP-A-53-86043 (JP, A) JP-A-53-82721 (JP, A) JP-A-60-54315 (JP, A) JP-A-58-79920 (JP , A) Actual development Sho 58-79919 (JP, U)

Claims (1)

[Claims] 1. A tyrosine kinase inhibitor containing a 3,5-diisopropyl-4-hydroxystyrene derivative represented by the following general formula (1) or a physiologically acceptable salt thereof as an active ingredient. (Here, R ¹ represents hydrogen, a cyano group, or a CONH ₂ group, and R ₂ represents a CONH ₂ group or a group represented by CONHCONH _2. )