DE2506209C2 - Use of N, N'-bis (3-picolyl) -4-methoxy-isophthalamide to prevent platelet aggregation - Google Patents

Description

■ N
CO-NH-H ₂ C-

OCH ₃

Molecular weight: 376.4

w Ν, Ν'-bis (3-picolyl) -4-methoxy-isophthalamide can be prepared from 4-methoxy-isophthalic acid by two methods.

Method a

In a three-necked flask fitted with a stirrer, a reflux condenser with a silica gel tube and a Separating funnel is equipped, are given 4.7 g (0.02 mol) of 4-methoxy-isophthalic acid dichloride (mg: 233.061), dissolved in 50 ml of tetrahydrofuran, and then 4.75 g (0.044 mol) of 3-aminomethylpyrridine (3-picolylamine), dissolved in 50 ml of tetrahydrofuran, too. A slight excess is used based on the acid chloride Base. 4.4 g (0.04 <? Mol) of triethylamine are added dropwise through the separating funnel to the suspension formed in the flask, dissolved in 50 ml of tetrahydrofuran, too.

After this addition is complete, the mixture is refluxed with continuous stirring for 20 hours. The excess tetrahydrofuran is distilled off in vacuo. The residue is taken up in an excess of a saturated aqueous solution of NaHCO ₃ , filtered and the filter cake is washed with water, crystallized from ethanol and recrystallized from benzene. The melting point of the compound is 124 ° C.

Method B.

9.5 g of 3-picolylamine dissolved in 50 ml of tetrahydrofuran are placed in a 250 ml three-necked flask equipped with a stirrer, a reflux condenser, a silica gel tube and a separating funnel. 4.7 g (0.02 mol) of 4-methoxy-isophthalic acid dichloride, dissolved in 50 ml of tetrahydrofuran, are added dropwise through the separating funnel to this solution. After this addition has ended, the mixture is refluxed for 16 hours with continuous stirring. When this reaction has ended, the contents of the flask are poured into an excess of a saturated aqueous solution of NaHCO3. After a while, the mixture is separated a crystalline product forms, which are successively filtered and crystallized from benzene.The melting point of the substance is 124 ° C. t

Analysis C ₂ IH ₂₀ N ₄ O ₃ :

Found (%) C 67.19 H 6.14 N 14.68 calcd C 67.01 H 5.36 N 14.88

It has been found that Ν, Ν'-bis (3-picolyl) -4-methoxy-isophthalamide inhibits platelet aggregation. This compound is therefore useful in the pharmaceutical and therapeutic fields. These Activity was assessed in vitro and in vivo by the Bom's aggregometer, a device that uses the The degree of inhibition can be investigated.

1) In vitro aggregation inhibition activity

Rabbits fasted with water ad libitum for 12 hours were anesthetized with 20% ethyluretane, which was injected intraperitoneally at a dose of 0.6 ml / 100 g body weight. Her blood was from the Carotid artery taken and taken with 3.8% sodium citrate in a ratio of 9: 1 and is then 15 minutes at 1000 r.p.m. centrifuged. Here, a platelet-rich plasma was used obtained, hereinafter referred to as PRP.

A portion of this plasma was further 10 minutes at 8000 r.p.m. M. centrifuged, one attached to platelets poor plasma (hereinafter referred to as PPP) was obtained.

The N, N'-bis (3-picolyl) -4-methoxy-isophthalamide dissolved in a 1N HCl solution (or in a stoichiometric fumaric acid solution), which was buffered with phosphates at pH 7.4, was at various concentrations in the ratio of 0.1: 1.0, based on the plasma, was added to the PRP and incubated for 30 minutes in a 37 ° C water bath. 1.0 ml of the PRP treated in this way was placed in the container of the Born aggregometer (zero adjustment carried out with PPP). The aggregation of the platelets was caused by a concentration of 10 " ² M of the ADP disodium salt.

The activity is expressed as the inhibition of the aggregation curve obtained with the same control plasma (30 minutes in a water bath at 37 ° C after adding 0.1 ml of physiological saline solution per ml of plasma incubated), determined, the value of the control was set equal to 100.

2) In vivo aggregation inhibiting activity

Rabbits that were fasted with water ae libitum for 12 hours were anesthetized with 20% ethyluretane, which was injected intraperitoneally at a dose of 0.6 ml / 100 g body weight. The blood was before (Control) and 1 hour 30 minutes after the intraperitoneal injection of N, N'-bis (3-picolyl) -4-methoxyisophthalamide at doses of 25, 50, 100 and 200 mg / kg body weight from the carotid artery. the Blood samples were taken with 3.8% sodium citrate in a ratio of 9: 1 and then at 1000rpm Centrifuged for 15 minutes.

The aggregation-inhibiting activity is determined in accordance with that already described above for the in vitro test Determine the method directly from the various treated plasmas after their centrifugation.

Confirmation of the in vivo inhibitory effect of the compound on platelet aggregation was made obtained by injecting the compound intramuscularly into guinea pigs at a dose of 20 mg / kg body weight and also treating volunteers orally at a dose of 12 mg / kg body weight, using the same method of determination.

Results The tests carried out show the following:

To determine the in vitro inhibiting effect of N, N'-bis (3-picolyl) -4-methoxy-isophthalamide in rabbit plasma, that concentration which causes a 50% inhibition of platelet aggregation (IC ₅₀ ) was determined using the "Probit" analysis determined. The molar concentration of the tested compound, which leads to a 50% inhibition of blood platelet aggregation, was determined to be 5.2-10 " ⁴ M (IC ₅₀ in vitro).

In the investigation of the aggregation-inhibiting effect in vivo in rabbits treated intraperitoneally, it was found that a 50% inhibition of platelet aggregation is produced at a dose of 108.2 mg / kg (IC ₅₀ in vivo).

In the guinea pigs, the activity of the compound reached a maximum of 64.50% in the fourth Hour after her injection. After 72 hours there was no longer any effect.

In humans, a maximum effect of 70.11% was found at the 8th hour after administration. After 72 hours there was no longer any effect.

To determine the acute toxicity, the compound was administered intraperitoneally to white male Swiss mice. The LD ₅₀ (FL 95%) investigations were carried out according to the method of Litchfield and Wilcoxon (J. Pharm. Exp. Thor., 96.99 / 1949). The results obtained are summarized in the table below; in this table is the compound used in the present invention with G-137

G-137

aspirin

Heparin

Flufenamic acid

Inhibition of platelet aggregation in vitro anticoagulant activity (Recalcification time) Fibrinolytic activity in vitro

Anti-inflammatory activity (edema due to carrageenin) is DL ₅₀

5.2 10 ⁴ M 4.8 10 " ³ M 8.7 10" ³ M inactive 1205 mg / kg

9.04 10 "'M.

0.398 U. IVmI

74.5 mg / kg
mg / kg

3.2 10 ³ M.

5.7 10 " ³ M 10.8 mg / kg 235 mg / kg

Due to the very low toxicity and the results presented above, it can be used according to the invention Compound used therapeutically in the form of oral or parenteral preparations in such conditions where it is advisable that there is a tendency for spontaneous aggregation of platelets, for example associated with thromboophilia and fibrinophilia. to diminish.

According to the invention, N.N'-bis (3-picolyiH-methoxy-isophthalamide and its with phosphates on one pH 7.4 buffered solution iv HCl and the non-toxic, pharmaceutically acceptable acid addition salts this connection can be used.

Claims (1)

Claim: Use of Ν, Ν'-bis (j-picolylM-methoxy-isophthalamide to inhibit platelet aggregation. The invention relates to the subject matter specified in the claim. ιυ In Chem. Ther. 71,6 (3) 203-207 (referenced in C. A. 75 (1971), 110 153 h) it is described that Ν, Ν'-bis (3-picolyI) -4-methoxy-isophthalamide Has anticoagulant and fibrinolytic activity. To put it simply, blood clotting (coagulation) takes place in three stages. The one present in the blood cells In the first stage, the enzyme thrombokinase converts prothrombin into thrombin. This transforms then in the second stage fibrinogen in fibrin. In the third stage, the fibrin threads can be pulled together blood coagulation occurs, see Ehrhardt, Ruschig: "Medicines", development, effect, presentation. Weinheim 1972. Pages 371-373. Platelet aggregation is now a different matter than that described above Blood clotting. Platelet aggregation is namely the aggregation of blood platelets into one another. Thus, anticoagulant agents inhibit the supply of thrombokinase from and thus the blood platelets : o the blood clotting. Anti-aggregating agents, on the other hand, have a completely different mechanism of action, so that anticoagulant center! are not anti-aggregating agents at the same time and vice versa. It has now surprisingly been found that a picolylamide of 4-methoxy-isophthalic acid stimulates platelet aggregation can inhibit. The invention thus relates to the use of Ν, Ν'-bis (3-picolyl) -4-methoxy-isi> phthaiamid for : 5 Inhibition of platelet aggregation. This connection corresponds to the following structural formula: