JPS6141895B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula (1) [In the formula, R ₁ and R ₂ may be the same or different, and include an amino group, an alkylamino group, (the alkyl group may be linear or branched), an aralkylamino group, an arylamino group,
represents a hydroxyalkylamino group, an alkylpiperazino group, or an aralkylpiperazino group]
The present invention relates to an antithrombotic agent containing 2,2'-dithiobis (N-substituted or unsubstituted benzamide) represented by or a salt thereof as a main component. Thrombosis is one of the main causes of stroke and myocardial infarction.
Thrombi generated for various reasons promote arteriosclerosis as wall-pressure thrombi, or cause circulatory disorders when thrombi that flow out during blood flow become emboli. These arteriosclerosis and circulatory disorders are thought to be the causes of the above-mentioned strokes and myocardial infarction attacks. Recent advances in research on platelet function have revealed that platelets play a central role in the blood clot formation process. In other words, platelets present in blood flow maintain a state of equilibrium with the normal blood vessel wall and do not form aggregates, but when an injury occurs, the platelets that are present in the blood vessel wall are in a state of equilibrium with the collagen and basement membrane scattered in the subintimal tissue. Sticky. This adhesion triggers the release of ADP and seronin from platelet dense bodies. The released ADP and serotonin cause platelets to aggregate with each other, forming platelet clumps. At the same time, it also causes the release of platelet factor 3,
Released platelet factor 3 promotes thrombin generation. As a result, it activates the blood coagulation process,
It is thought that a blood clot may form. Focusing on the important role of platelets in thrombus formation as described above, attempts have been made to use drugs that suppress platelet adhesion, release, and aggregation functions for the prevention and treatment of thrombosis. Conventionally known platelet aggregation inhibitors include nonsteroidal anti-inflammatory drugs such as aspirin and indomethatine, pyrazole derivatives such as sulfinpyrazone, pyrimidopyrimidine derivatives such as dipyridal, adenosine derivatives, and papaverine. However, in terms of pharmacological effects and safety, the development of better drugs is desired. As a result of screening and testing more than 4,000 types of compounds, the present inventors discovered that the compound represented by general formula (1) has a strong platelet aggregation inhibitory effect and low toxicity, and completed the present invention. did. The details of the present invention are shown below. The compound used in the present invention has the general formula (1)
, R ₁ and R ₂ may be the same or different, and may be an amino group, an alkylamino group having 1 to 8 carbon atoms (the alkyl group may be linear or branched), or an alkylamino group having 7 to 10 carbon atoms. Aralkylamino group, arylamino group having 6-10 carbon atoms, (W-n)-hydroxyalkylamino group having 1-6 carbon atoms (n represents zero or a positive integer), 5 carbon atoms
-8 alkylpiperazino group or carbon number 11-
2, represented by 18 aralkylpiperazino groups;
Examples include 2'-dithiobis(N-substituted benzamide or a salt thereof. Specific examples of compound names represented by the above general formula (1) include: 1 In the general formula (1), R ₁ , R ₂ are amino groups and alkylamino groups. 2,2'-dithiobis(benzamide) 2,2'-dithiobis(N-methylbenzamide) 2,2'-dithiobis(N-ethylbenzamide) 2,2' -Dithiobis(N-propylbenzamide) 2,2'-dithiobis(N-isopropylbenzamide) 2,2'-dithiobis(N-n-butylbenzamide) 2,2'-dithiobis(N-t-butylbenzamide) amide) 2,2'-dithiobis(N-hexylbenzamide) 2,2'-dithiobis(N-2-ethylhexylbenzamide) etc. 2 In general formula (1), R ₁ and R ₂ are an arylamino group, an aralkyl It is an amino group. 2,2'-dithiobis(N-benzylbenzamide) 2,2'-dithiobis(N-phenylbenzamide) 2,2'-dithiobis(N-tolylbenzamide) 2,2'- Dithiobis(N-p-methoxyphenylbenzamide) 2,2'-dithiobis(N-p-chlorophenylbenzamide) 2,2'-dithiobis(N-p-nitrophenylbenzamide) etc. 3 General formula ( In 1), R ₁ and R ₂ are hydroxyalkylamino groups. 2,2'-dithiobis(N-hydroxymethylbenzamide) 2,2'-dithiobis(N-2-hydroxyethylbenzamide) 2,2 '-dithiobis(N-3-hydroxypropylbenzamide) 2,2'-dithiobis(N-2-hydroxypropylbenzamide) etc. 4 In general formula (1), R ₁ and R ₂ are alkylpiperazino groups. 2,2'-dithiobis(N-methylpiperazinobenzamide) etc. 5 In general formula (1), R ₁ and R ₂ are aralkylpiperazino groups. 2,2'-dithiobis(N- Benzylpiperazinobenzamide) etc. The method for producing the above compound is described in, for example, Japanese Patent Publication No. 46-41399.
No. 3, U.S. Patent Publication No. 3736280, Farmmaco
Ed.Sci, 14 , 216-239 (1959), 14 , 648-
665 (1959), etc., and can also be synthesized according to Reference Example 1 described later. In addition, 2,2'-dithiobis(N-benzylpiperazinobenzamide), 2,2'-dithiobis(N-
hydroxymethylbenzamide) 2,2'-dithiobis(N-2-hydroxypropylbenzamide), 2,2'-dithiobis(N-3-hydroxybutylbenzamide), 2,2'-dithiobis(N-2-hydroxypropylbenzamide), 2,2'-dithiobis(N-3-hydroxybutylbenzamide)
-4-hydroxybenzamide) are new compounds that have not been described in any literature. The manufacturing method of this product is shown in Reference Examples 1, 2 and 3. Examples of the salt of a disulfide compound in which R ₁ and R ₂ in the general formula (1) are an alkylpiperazino group or an aralakylpiperazino group include sulfate, hydrochloride,
Examples include mineral acid salts such as hydrobromide, and organic acid salts such as benzoic acid, fumaric acid, succinic acid, tartaric acid, and citric acid. The following drugs (sample and control drugs) are used in the following experimental examples and examples. 1 2,2'-dithiobis(N-butylbenzamide) (hereinafter referred to as compound) 2 2,2'-dithiobis(N-benzylbenzamide) (hereinafter referred to as compound) 3 2,2-dithiobis(N- 2-hydroxyethylbenzamide) (hereinafter referred to as the compound) 4 2,2'-dithiobis(N-benzylpiperazinobenzamide) hydrochloride (hereinafter referred to as the compound) 5 Adenosine 6 Papaverine 7 Aspirin 8 2,2 '-dithiobis(N-2-hydroxypropylbenzamide) (hereinafter referred to as compound) 9 2,2'-dithiobis(N-methylpiperazinobenzamide) (hereinafter referred to as compound) Next, the compound used in the present invention is The following experimental examples demonstrate that it has an inhibitory effect on platelet aggregation. Experimental example 1 Platelet aggregation inhibitory effect in rabbit platelet-rich plasma (in vitro experiment) a Method 9 vol of blood was collected from the common carotid artery of a domestic rabbit (2-2.5 kg) into a silicone-treated flask containing 1 vol of 3.8% sodium citrate. The obtained blood is
Centrifugation was performed at 1000 rpm for 15 minutes to obtain platelet rich plasma (hereinafter abbreviated as PRP). Platelet aggregation can be observed using an Aggregometer (Bryston
(manufactured by KK). The degree of aggregation after the addition of various aggregation-inducing substances was determined by the nephelometric method as an increase in light transmittance. b Procedure: Place 0.9ml of PRP into a silicone-treated cuvette for Aggregometer, and add drugs (sample) at various concentrations to this.
Alternatively, after adding 0.05 ml of a physiological saline solution containing a control inhibitory drug, leave it for 1 minute, then add various aggregation-inducing substances, observe the state of the platelet-containing solution after that, and measure the absorbance.The aggregation inhibition rate is Obtained from the following formula. Aggregation inhibition rate (%) = CD / C × 100 Here, C indicates the change in light transmittance indicating the maximum aggregation rate when physiological saline is added, and D indicates the maximum aggregation rate when the drug is added. Indicates changes in light transmittance. c Results ADP, collagen, and thrombin-induced platelet aggregation inhibitory effect When ADP (10 ^-5 M) is added to PRP, a transient decrease in light transmittance accompanied by a change in platelet morphology is immediately followed by clear aggregation. . When collagen is added to PRP, aggregation is observed after a 1-2 minute incubation period. When thrombin is added to PRP, agglutination is evident after a 30-40 second incubation period.
Table 1 shows the inhibitory effects of each drug on platelet aggregation. The results in Table 1 show that the compounds , , and have an inhibitory effect on platelet aggregation induced by various drugs.

[ADP-induced platelet aggregation and dissociation promoting effect]

Platelets aggregated by ADP have the property of gradually dissociating after reaching maximum aggregation, and the promoting effect of the compound of the present invention on this dissociation rate was investigated. (Method) ADP (final concentration 10 ^-5 M) is added to 0.9 ml of PRP to induce aggregation. Approximately 4 minutes after ADP addition, when maximum aggregation is reached, saline or drug is added;
The degree of dissociation after 5 minutes was calculated using the following formula. Degree of aggregation and dissociation (%)=C-E/C×100 C; Maximum degree of aggregation by ADP E; Table 2 shows the results of the degree of aggregation 5 minutes after addition of the drug.

[Mouse ADP lethal inhibitory effect (in vivo)]

It is known that when large amounts of ADP are administered intravenously to mice, the platelet aggregates generated are attracted to the pulmonary capillaries, causing respiratory distress and death of the mice. To examine the in vivo effects of drugs, oral and intravenous administration of drugs was performed.
The lethality suppressing effect of ADP was investigated. Method Roba et al.'s method [Eur.J.Pharmacol. 37 , 265]
274 (1976)]. 0.1 ml/10 g (body weight) of 0.3% CMC solution and CMC suspension of drug (sample and control drug) to mice.
One hour after oral administration, an aqueous solution of ADP (60 mg/ml) was administered at a rate of 0.1 ml/10 g (body weight)/
The animals were injected into the tail vein at a rate of 10 sec., and the mortality rate was determined. In addition, to examine the effects of intravenous administration, physiological saline and a physiological saline solution of the drug (0.5mg/
10 minutes after administering 0.2 ml/10 g (body weight) (10 mg/Kg) into the tail vein, ADP-Na was injected into the tail vein in the same manner as for oral administration, and the mortality rate was determined. . The results are shown in Table 3.

[Table] As is clear from Table 3, the mortality rate of the compound of the present invention was lower than that of the control drug both when administered orally and intravenously. On the other hand, no significant effect was observed with aspirin or papaverine used as controls. That is, it can be seen that the effect of the compound is stronger in suppressing lethality than these control drugs. Experimental Example 4 Acute Toxicity Method Male dd
0.3% of the sample for each group of 5 mice (20±1g)
The test was carried out by oral administration using a CMC suspension (specimen concentration 100 mg/ml). The results are shown in Table 4. The score of the compound is 0/5 when administered at 1 g/Kg, 1/5 when administered at 2 g/Kg,
4/5 for 38/Kg, 4/5 for 4g/Kg, 0/5 for the compound when administered at 4g/Kg (both numbers of deaths/number used), 0/5 for the compound when administered at 2g/Kg, and 0/5 for the compound at 3g/Kg.
It was 2/5 for 4g/Kg, and 4/5 for 4g/Kg.

[Mice arachidonic acid lethal inhibitory effect (in vivo)]

It is known that when a large amount of arachidonic acid is administered intravenously to mice, the platelet aggregates generated are attracted to the pulmonary capillaries, resulting in death of the mice without respiratory distress. In order to examine the in vivo effects of the drug, we investigated the lethality-inhibiting effect of arachidonic acid after oral administration of the drug. Method Uzunova AD et al.'s method (Prostaglandins 13,
P.995 (1977)]. After orally administering 0.3% CMC solution and CMC suspension of drug (sample and control drug) to mice (DD male, 22-24 g), 50 mg/Kg of sodium arachidonate was administered intravenously at 2 hours, and mortality rate was determined. I asked for The results are shown in Table 5.

【table】

[Table] From the results in Table 5, it can be seen that the compounds of the present invention exhibit an excellent preventive effect against mortality due to respiratory depression. As explained above, the drug provided by the present invention inhibits and suppresses platelet aggregation, and is expected to be used as an antithrombotic agent. Incidentally, the compound represented by general formula (1) can be used in the form of tablets, powders, capsules, injections, etc. by conventional processing methods. At this time, conventional excipients, disintegrants, binders, spreading agents, lubricants, pigments, coating components, diluents, etc. are used. Preferably used excipients include glucose,
Disintegrants such as lactose, starch and sodium alginate, lubricants such as magnesium stearate, paraffin sulfate and talc, binders such as simple syrup, ethanol, and gelatin, and coatings such as dispersants and plasticizers. Dispersants include methyl cellulose, ethyl cellulose, etc., and plasticizers include glycerin,
Starch etc. are used. Crystalline cellulose is also used as having disintegrating, lubricating, binding and excipient properties. The administration method is oral or injection, and since the compound represented by general formula (1) is a safe compound with an extremely high LD ₅₀ value, it is possible to administer a fairly high dose, but the lowest effective dose is 100 mg/dose. ~500mg/
Be an adult. Example 1 Tablet manufacturing method 1 Materials used Compound 2000g Lactose 100g Starch 150g Carboxymethylcellulose calcium 150g (CMC-Ca) Polyvinyl alcohol (PVA) 100g Magnesium stearate 25g 2525g 2 Method Compound, lactose, starch and CMC- Weigh the above amount of Ca and mix well in a mixer to make a mixed powder. Add a mixture containing PVA to this powder,
Granulate according to the usual wet granulation method, then dry,
Sort the particles. Magnesium stearate is added and mixed to produce granules for tablets. Using a rotary tablet press and a 9 mm diameter sugar-coated punch, tablets with a diameter of 9 mm, a thickness of 4 mm, and a weight of 252.5 mg are produced. Example 2 Capsule manufacturing method 1 Materials used (shell) Hydroxypropyl 48g Methylcellulose polyethylene glycol-4000 10g Titanium oxide (TiO ₂ ) 30g 2 Method The above shell components were mixed with an acetone-dichloromethane mixture (volume ratio 1: 1) to produce a coating liquid. Example 1
The tablets obtained in step 1 are coated to obtain coated tablets. Example 3 Method for producing injection solution 500 mg of the compound and 50 g of glucose are weighed out, and distilled water is added to dissolve them to bring the total amount to 1. This solution was filtered using a membrane filter with a pore size of 0.22μ (Millipore, FGLP14200) under pressure (0.5Kg/cm ² ) with N ₂ gas.
Dispense into 20ml white ampoules and seal. Example 4 In Examples 1 and 2, the compound (), that is, 2,
2,2′-dithiobis(N-2
-Hydroxypropylbenzamide) gives similar tablets and capsules. Reference Example 1 Production of 2,2'-dithiobis(N'-benzylpiperazinobenzamide) hydrochloride [In general formula (1), R ₁ = R ₂ = N'-benzylpiperazino group

[Production of compound represented by formula] 6.9 g (0.02 mol) of 2,2'-dithio-1,1'-bis(benzoyl chloride) was added to 50 ml of dioxane.
Disperse in water and cool to 10°C with ice water. 7.0 g (0.04 mol) of benzylpiperazine is dissolved in 50 ml of dioxane and the solution is added dropwise over about 30 minutes. After further stirring at 10°C for 1 hour, the reaction was continued at 70°C for 2 hours with stirring.
After the reaction is completed, the reaction solution is filtered, the filtered product is washed with acetone, and then recrystallized with ethanol to obtain 11.1 g of white crystals (yield: 80%) of the desired product. The physical properties of this product are as follows 1 Elemental analysis value C H N Cl S Experimental value (%) 61.30 5.85 8.15 9.87 8.97 Calculated value (%) 62.14 5.82 8.05 10.19 9.22 (as 2HCl salt) 2 Melting point 230℃ or higher (decomposition) 3 Infrared absorption spectrum (potassium bromide tablet method shown in Figure 1) 4 Solubility Easily soluble in water, slightly soluble in methanol and ethanol. Insoluble in ether, benzene and chloroform. Reference example 2 2,2'-dithiobis Production of (N-hydroxymethylbenzamide) [Production of a compound in which R ₁ = R ₂ is represented by a hydroxymethyl group (-CH ₂ -OH) in general formula (1)] 3 g of 2,2'-dithiobis(benzamide)
(0.01 mol) is dissolved in 30 ml of dimethyl sulfoxide, and 1.6 g (0.02 mol) of formalin (37% formaldehyde aqueous solution) is added little by little while stirring at room temperature. After the addition is complete, add 2 drops of 1N aqueous sodium hydroxide solution and continue stirring at room temperature for 1 hour. Next, raise the temperature on the water bath and heat and stir at 65-70°C for 2 hours. After cooling to room temperature, pour the reaction into 300 ml of cold water. After leaving it for one night, the white crystals formed are vacuumed and separated.
Repeat washing with water three times, take out the crystals, and air dry. Yield 3.0g (yield 82.5%) Melting point 181℃ (decomposition) (experimental value) (calculated value) Elemental analysis values: C 52.04% 52.72% H 4.66% 4.42% N 7.74% 7.68% S 17.20% 17.59% Infrared absorption spectrum : Shown in Figure 2. Solubility: Soluble in DMSO, DMF, poorly soluble in ethanol water, insoluble in ether, benzene, chloroform, etc. Reference Example 3 Production of 2,2'-dithiobis(N-2-hydroxypropylbenzamide) [In general formula (1), R ₁ = R ₂ = N-2-hydroxypropylamino group

Production of compound represented by [Formula]] 2,2'-dithiobis(benzoyl chloride)
10.3 g (0.03 mol) is suspended in 50 ml of dioxane and cooled to 10-12°C with ice water. Under stirring,
A solution of 9.0 g (0.12 mol) of 2-hydroxypropylamine in 50 ml of dioxane is added dropwise over about 60 minutes. After the addition was completed, the mixture was allowed to react at room temperature for 2 hours. After the reaction is complete, pour the reaction solution into 300 ml of ice water with stirring to form a white precipitate. Filter and dry the precipitate. The obtained precipitate was dioxane-water (80:20,
Volume ratio) When recrystallized from 30ml of the mixed liquid, the melting point is 175~
10.1 g of white crystals at 7° C. are obtained. Elemental analysis value C H N S Measured value (%) 57.03 5.51 6.73 15.11 Calculated value (%) 57.11 5.76 6.66 15.25 Infrared absorption spectrum (potassium bromide tablet method): Shown in FIG.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of 2,2'-dithiobis(N-benzylpiperazinobenzamide) hydrochloride obtained in Reference Example 1 by the KBr tablet method. Figure 2 shows the 2,2'-dithiobis(N-hydroxymethylbenzamide) obtained in Reference Example 2.
The infrared absorption spectrum obtained by the KBr tablet method is shown.
Figure 3 shows the 2,2'-dithiobis(N-2-hydroxypropylbenzamide) obtained in Reference Example 3.
Infrared absorption spectrum obtained by KBr tablet method is shown.

Claims (1)

[Claims] 1 General formula (1) (In the formula, R ₁ and R ₂ may be the same or different, and include an alkylamino group, an aralkylamino group,
(represents a hydroxyalkylamino group, an alkylpiperazino group, or an aralkylpiperazino group)
An antithrombotic agent containing 2,2'-dithiobis(N-substituted benzamide) or a salt thereof as an active ingredient.