JPS6245225B2 - - 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 are a hydroxyalkylamino group (however, the hydroxy group is bonded to a carbon atom other than the terminal of the alkyl group)] The present invention relates to '-dithiobis(N-substituted benzamide) and an antithrombotic agent comprising the same. 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 cause 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 are in equilibrium with the normal blood vessel wall and do not form aggregates, but when an injury occurs, the collagen scattered in the subintimal tissue on the blood vessel wall becomes a basement membrane. Sticky. This adhesion triggers the release of ADP and serotonin 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 papaperine. However, in terms of pharmacological effects and stability, the development of better drugs is desired. As a result of screening and testing over 4000 compounds, the inventors found that the general formula (2) (In the formula, R ₁ and R ₂ may be the same or different,
2,2'- represented by an amino group, an alkylamino group, an arylamino group, an aralkylamino group, a hydroxyalkylamino group, an alkylpiperazino group, or an aralkylpiperazino group)
A patent application (Japanese Patent Application Laid-Open No. 1983-324) was filed regarding an antithrombotic agent containing dithiobis (N-substituted or unsubstituted benzamide) or a salt thereof as a main component. As a result of more detailed research, the present inventors found that
2,2'-dithiobis(N-
The present invention has been completed by discovering that substituted benzamide (substituted benzamide) has a strong platelet aggregation inhibiting effect and has low toxicity. The details of the present invention are shown below. The target compound of the present invention has R ₁ in the general formula (1),
R ₂ may be the same or different, and examples thereof include a hydroxyalkylamino group having 2 to 8 carbon atoms (however, the hydroxy group is bonded to a carbon atom other than the terminal end of the alkyl group). To specifically illustrate the compound name, in general formula (1), R ₁ and R ₂ are the following hydroxyalkylamino groups. 2,2'-dithiobis(N-2-hydroxypropylbenzamide), 2,2'-dithiobis(N-3-hydroxybutylbenzamide), 2,2'-dithiobis(N-4-hydroxyhexylbenzamide), etc. can give. Next, a method for synthesizing the compound of the present invention will be explained. The compound of the present invention is prepared by using 2,2'-dithio-bisbenzoyl halide, preferably chloride, in a solvent.
It can be obtained by reacting with hydroxyalkylamine (however, the hydroxyl group is bonded to a carbon atom other than the terminal end of the alkyl group) and recovering and purifying it by a conventional method. Examples of the inert solvent used include cyclic ethers such as dioxane and tetrahydrofuran, ethers, aromatic hydrocarbons, halogenated hydrocarbons, and hydrocarbons. The reaction is usually carried out under cooling at -20°C to 50°C, preferably 0 to 25°C.
It will be held in The reaction time is usually 1 to 5 hours. Next, experimental examples demonstrate that the compounds of the present invention have excellent platelet aggregation inhibiting effects. As control drugs, 2,2'-dithiobis(N-2-hydroxyethylbenzamide), adenosine, papaverine, aspirin, etc. were used. Experimental example 1 Platelet aggregation inhibitory effect in rabbit platelet-rich plasma (in vitro experiment) (a) Method 9 vol of blood from the common carotid artery of a domestic rabbit (2-2.5 kg) was poured into a silicone-treated flask containing 1 vol of 3.8% sodium citrate. Take blood. The obtained blood was centrifuged at 1000 rpm for 15 minutes to obtain platelet rich plasma (hereinafter abbreviated as PRP). Aggregometer for observing platelet aggregation
(manufactured by Bryston). 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 physiological saline solution containing various concentrations of drug (sample) or control inhibitor drug.
After adding 0.05 ml, let stand for 1 minute, then add various aggregation-inducing substances, observe the state of the platelet-containing solution, and measure absorbance. The agglomeration suppression rate is calculated 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. Shows changes in light transmittance. (c) Results Inhibitory effect of ADP, collagen and thrombin-induced platelet aggregation When ADP (10 ^-5 M) was added to PRP, a transient decrease in light transmittance accompanied by a change in platelet morphology was immediately followed by clear aggregation. It will be done. When collagen is added to PRP, aggregation is observed after a 1-2 minute incubation period. When thrombin is added to PRP, obvious agglutination is observed after a 30-40 second incubation period. Table 1 shows the inhibitory effects of each drug on platelet aggregation.

【table】

[Table] The results in Table 1 show that the compounds of the present invention have an inhibitory effect on platelet aggregation induced by various drugs. Furthermore, the effects of the present invention compound, 2,2'-dithiobis(N-2-hydroxypropylbenzamide), on ADP (10 ^-5 M), collagen, and thrombin (1 unit/ml) induced aggregation determined from the results in the above table. _IC50 is 7.4μg/ml, 1.6μg/ml and 2.8
It is μg/ml. Experimental Example 2 [ADF-induced platelet aggregation and dissociation promoting effect] Platelets aggregated by ADP have a property of gradually dissociating after reaching maximum aggregation.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 aggregation degree by ADP E: Aggregation degree 5 minutes after drug addition The results are shown in Table 2.

[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. [Prostaglandins 13 ,
P.995 (1977)]. After oral administration of 0.3% CMC solution and CMC suspension of drug (replacement and control drug) to mice (DD strain, male, 22-24 g), 50 mg/Kg of sodium arachidonate was administered intravenously at 2 hours, resulting in death. The rate was calculated. The results are shown in Table 3.

[Table] The results in Table 3 show that the compound of the present invention exhibits an excellent preventive effect against mortality mainly due to respiratory depression. Experimental example 4 Acute toxicity method Based on the Lebens-Kerbet 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 score of 2,2'-dithiobis(N-2-hydroxypropylbenzamide) was 0/5 at 2g/Kg, 2/5 at 3g/Kg, and 4/5 at 4g/Kg.
(Number of deaths/Number used), LD ₅₀ is 3g/
Kg or more. Therefore, the compound of the present invention is a safe drug with a high LD ₅₀ value. 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 the general formula (1) can be used in the form of tablets, powders, capsules, injections, etc. by conventional formulation. At this time, conventional excipients, disintegrants, binders, spreading agents, lubricants, pigments, coating components, diluents, etc. are used. Preferably used excipients include glucose,
Lactose, disintegrants include starch, sodium alginate, lubricants include magnesium stearate, paraffin sulfate, talc, binders include simple syrup, ethanol, gelatin, and coatings include dispersants and plasticizers. Examples of dispersants include methyl cellulose and ethyl cellulose, and examples of plasticizers include glycerin and starch. 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 a single dose. 10-500mg/
Be an adult. Example 1 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]] 10.3 g (0.03 mol) of 2,2'-dithio-1,1'-bis(benzoyl chloride) was suspended in 50 ml of dioxane, and cooled to 10-12°C in an ice-water bath. do. While 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 Actual 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): 3rd
As shown in the figure. Example 2 Preparation of 2,2'-dithiobis(N-2-hydroxybutylbenzamide) 1-Amino-
A solution of 3.57 g of 2-butanol in 10 ml of dioxane
Add dropwise at below 15℃. After the dropwise addition, continue to stir at room temperature for 2 hours. After the reaction is complete, pour into 200 ml of ice water. Separate the precipitated crystals by filtration, dry and recrystallize from ethanol to obtain 3.96 g of the desired product as white crystals (88.4
%). Melting point 169-171℃ Elemental analysis value (as C ₂₂ H ₂₈ N ₂ O ₄ S ₂ ) C H N Actual value (%) 58.88 6.30 6.20 Calculated value (%) 58.90 6.29 6.24 Infrared absorption spectrum (potassium bromide tablet method) :cm
⁻ , max 3290(NH), 2965( _CH3 ), 2940( _CH2 ), 1635
(c=0) ¹ H-NMR (DMSO-d ₆ ) δ: 8.43 (2H, t,
NH), 7.17−7.69 (8H, m, ph), 4.66 (2H,
d, OH), 3.0−3.82 (6H, m, CH ₂ CH),
1.17−1.83 (4H, m, CH ₂ CH ₃ ), 0.96 (6H,
t, CH ₃ ) Example 3 Tablet manufacturing method 1 Materials used 2.2'-dithiobis(N-2-hydroxypropylbenzamide) (hereinafter referred to as compound 1)
2000g Lactose 100g Starch powder 150g Carboxymethylcellulose calcium (CMC-Ca) 150g Polyvinyl alcohol (PVA) 100g Magnesium stearate 25g 2525g 2 Method Weigh the above amounts of Compound 1, lactose, starch, and CMC-Ca, and place them in a mixing machine. Mix well in a bowl to make a mixed powder. A kneading solution containing PVA is added to this powder and granulated according to a conventional wet granulation method, followed by drying and sizing. Magnesium stearate is added and mixed to produce granules for tablets. Using a rotary tablet press and a 9 mm 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 4 Capsule manufacturing method 1 Materials used (shell) Hydroxypropyl methyl cellulose 48 g Polyethylene glycol-4000 10 g Titanium oxide (TiO ₂ ) 30 g 2 Method The above shell components were mixed with an acetone-dichloromethane mixture (volume ratio 1:1). ) to produce a coating solution. The tablets obtained in Example 3 are coated with the coating liquid in a conventional manner to obtain coated tablets.

[Brief explanation of the drawing]

Figure 1 shows 2,2'-dithiobis(N-2-hydroxypropylbenzamide) obtained in Example 1.
The infrared absorption spectrum obtained by the KBr tablet method is shown.

Claims (1)

[Claims] 1. General formula 2,2'-dithiobis (wherein, R is a hydroxyalkylamino group having 2 to 8 carbon atoms (however, the hydroxy group is bonded to a carbon atom other than the terminal of the alkyl group)] N-substituted benzamide).