JPS6043063B2 - Novel compound with platelet antiaggregation effect, method for producing the same, and pharmaceutical composition containing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for suppressing platelet aggregation, suppressing thrombotic disorders in the blood, and having pharmacological effects on delaying blood coagulation.
This invention relates to novel organic compounds in monohydrate crystalline form.

The invention also relates to a method allowing the synthesis of said new crystalline compounds. In particular, the present invention relates to monohydrated N-N''-bis(3-picolyl)-4-methoxyisophthalamide, processes for its preparation, and pharmaceutical compositions containing the compound.

N+N-bis(3-picolyl)-4-methoxyisophthalamide (hereinafter referred to as PicOtamide)
) is a compound with high fibrinolytic and anticoagulant activity (French patent no.
mieTlleraPeUtiqL]El6 volume, 203
7, 1971), and is well known to be a compound with good platelet antiaggregation properties (US Pat. No. 3,973,026; AgeandAgeingl.
7, p. 246, see Rev. 197).

Picotamide is known in its anhydrous form as described in the above-mentioned publications and patents, and its melting point as described in FR 21008 (branch) is similar to that of the Koffler bench (
The temperature was 124° C. according to the KOflerbench method. This compound is purified from a crude product containing a number of impurities by crystallization from an anhydrous and non-polar organic solvent according to the method described in the above-mentioned patent. Crystallization of the crude picotamide obtained by the synthetic reaction from an anhydrous, non-polar organic solvent such as benzene results in the formation of a fibrous material, e.g. bulky and fuzzy (■01L1n11n0U
It gives a powdered product with an appearance similar to Sdawr1).

The powder easily acquires an electrostatic charge and is also relatively unstable. These properties are important when the compound is prepared for pharmaceutical use.
This is a particular disadvantage when The charged particles repel each other and are dispersed when weighed and placed into equipment to make various pharmaceutical preparations, which results in changes in particle weight and makes titer stabilization operations easier. make it difficult The present inventors have prepared SO-hydrated N, by reacting a functional derivative of 4-methoxyisophthalic acid with 3-picolylamine and crystallizing the crude product from aqueous solution.
N-bis(3-picolyl)-4-methoxyisophthalamide is obtained, which possesses chemical and physical structure which make it clearly preferable compared to the previously described and known anhydrous picotamides. It was found that it has the property of stability.

Furthermore, it is surprising that the monohydrate picotamide thus obtained is more active and effective than anhydrous picotamide, both in terms of pharmaceutical efficacy and in terms of clinical pharmacology.

The subject of the invention is therefore monohydrated N,N''-bis(3-
picolyl)-4-methoxyisophthalamide C2lH
2ON4O3.H2Ol molecular weight is 394.4.

The general chemical formula can be shown as follows: Monohydrate picotamide according to the present invention is a white, odorless, bitter-tasting, crystalline powder, which is stable in air and free from water. It crystallizes easily and has a melting point of 95-9rc by Koffler bench method.

For simplicity, in the following description the compounds defined above will be represented by the generic name ゜゜picotamide monohydrate. From a physicochemical point of view, the compounds according to the invention are distinguished from the hitherto well-known compounds by an unexpected and definite improvement in stability.

This improvement is due to the fact that molecules of water of hydration take part in the molecular structure of the compounds according to the invention. It is located in a well-defined position within the crystal lattice, where the oxygen atoms of the hydrated water form clearly visible hydrogen bonds with specific atoms belonging to another molecule of picotamide, as shown below. , constitute a single crystal of a compound with well-defined properties. The properties improve the pharmacological behavior of the new compound and its bioprocessability in mammalian organs in an unexpected manner, with more rapid absorption when administered to mammals, including humans. affect. The novel crystalline form of monohydrate picotamide according to the present invention surprisingly not only avoids the disadvantages of anhydrous picotamide mentioned above, but also more surprisingly, it This results in pharmacologically more active and effective compounds in terms of the beneficial effects that they provide. Although a well-founded theoretical explanation for the experimental results is not available at present, it is possible that the aqueous solution of the crystalline compound according to the present invention follows a different mechanism from that of the aqueous solution of the known amorphous anhydrous compound. can be expected.

Another subject of the invention is a method for producing the novel hydrated molecules.

Yet another object of the present invention is to provide pharmaceutical compositions containing the novel monohydrate molecules in various acceptable pharmaceutical forms as effective agents for the clinical treatment of thrombotic disorders of the blood. be. As mentioned above, monohydrate picotamide has a melting point of 95-97°C.

In this connection, it is pointed out that the melting point of anhydrous picotamide is 124°C.

Such a difference in the melting points of the two compounds is in itself already indicative of a different molecular structure, which means an essential difference between anhydrous picotamide and monohydrate picotamide. The invention will be explained in more detail below with reference to the accompanying drawings: FIG. 1 is a three-dimensional representation of the molecule of ropicotamide monohydrate obtained from an X-ray spectrum.

FIG. 2 is a three-dimensional representation of a single crystal of picotamide monohydrate. FIG. 3 shows the same single crystal as shown in FIG. 2 in greater detail.

FIG. 4 is a 1° rough diagram directly comparing the platelet anti-aggregation effects and blood concentrations of monohydrate picotamide and anhydrous picotamide.

The crystal structure of picotamide monohydrate has been characterized by physical and chemical analysis as well as by the X-ray spectrum of its single crystal.

X-ray diffraction spectral data confirming the spatial and r-positioning of the atomic centers in the new molecule, which is considered to be a completely dense, stable, and non-hygroscopic crystal, are shown in Table 1 below.

In the table, the various atoms of the molecule are indicated by chemical symbols with identifying numbers.5. The spatial arrangement and identification number of the atom can be seen from FIG. 1, which is taken from Table 1 and shows the three-dimensional geometrical position of the atomic center. The first atom designated as 027 represents the water of hydration, while the 8th atom designated as 09 represents the oxygen of the methoxy group, the 15th atom designated as Nl9 is the nitrogen of the amide group, and N22
It can be particularly seen that atom number 21, designated as 4, is the nitrogen atom of the pyridine group.

In Table 1, symbols X/A, . Y/B and Z/C represent the spatial coordinates of various atoms. As can be seen from Figure 1, which shows the structure of monohydrate picotamide, the oxygen atoms of the water of hydration are placed in the crystal lattice in well-defined positions relative to the picotamide molecule.

In the figure, the oxygen of the hydrated water in the single crystal, shown as a rectangle, is bound to defined atoms of each picotamide molecule by hydrogen bonds (indicated by dotted lines), and the picotamide molecules are ordered. It can be seen that they are arranged in a three-dimensional pattern and are properly bonded to each other by hydrogen bonds caused by hydrated water and oxygen.

The coupling is shown in more detail in FIG.

In Figure 3, the oxygen 027 of the hydration water is hydrogen bonded to (1) the =CO of the methoxy group of the first picotamide molecule, respectively.
with oxygen (09) (bond distance: 2.81A) (2) amide group of the second picotamide molecule in the same plane as the above molecule = nitrogen (Nl9) of NH (bond distance: 2.96A)
(3) It can be seen that it is bonded (bond distance: 2.80A) to the nitrogen (N224) of the pyridine ring of the third picotamide molecule, which is placed on the plane extending downward or upward to the two bonded molecules. .

The existence of these three bonds makes it possible to explain both the strength with which the water of crystallization fits between the different picotamide molecules forming the crystals, and the compactness of the crystals themselves.

It is believed that this compactness conferred by the water of crystallization molecules is the reason for the improvement in bioprocessability of the monohydrate relative to the previously known anhydrous form. This improvement will be explained below from a pharmacological point of view by the absorption time of the two drugs,
Confirmed by comparing blood concentration values and effects.
In fact, the improved efficacy due to the compactness of the spatial structure of picotamide and the marked improvement in biological processing due to the incorporation of water of crystallization into the molecule could not be expected based on the conventional knowledge about anhydrous picotamide molecules. It was something I couldn't do.

Elemental analysis also gave results consistent with the structure described above.

For C2lH9N4O3.H2O (molecular weight 394.4), C63.87% [theoretical value 63.94]; H5.
72% [theoretical value 5.62]; Nl4. The result was 18% [theoretical value 14.20] and the remainder was oxygen. The novel method for producing monohydrated picotamide follows the conventionally known method for producing anhydrous picotamide insofar as the compound is synthesized.
However, when the crude picotamide was obtained, it was surprisingly possible to obtain monohydrate crystals as defined above by recrystallizing the crude product from an aqueous mixture as opposed to an anhydrous mixture in previously known methods. It has been found that the following product can be obtained.

The example below illustrates how monohydrated picotamide can be obtained synthetically in the presence of a proton acceptor starting from 4-methoxyisophthaloyl dichloride or another functional derivative of 4-methoxyisophthalic acid. .

Preparation Example 3 - 120 ml of picolylamine, triethylamine and anhydrous tetrahydrofuran are placed in a 3' flask equipped with a reflux condenser, a dropping load and a mechanical stirrer.

4-Methoxyisophthaloyl dichloride is dissolved in portions in 200 ml of anhydrous tetrahydrofuran. This solution is slowly added to the reaction mixture in the flask from a dropwise load while stirring.

The addition reaction is 1. After the dichloride addition reaction, the following exothermic reaction proceeds:
The reaction mixture is refluxed for about 2 hours, slowly diluted with water to 2' and kept under stirring until a crystalline slurry of crude picotamide separates.

Aspirate the slurry? and acetone-water mixture (6 volumes of acetone, 11 volumes of water) 700-800
It is crystallized in a wet state from m1.

The product obtained is recrystallized from water. Monohydrate picotamide with a melting point of 95-97° C. is then obtained by the Koffler bench method. From the above, it can be seen that the method according to the invention comprises:
It is characterized by the fact that the recrystallization of the crude picotamide obtained from the synthetic reaction is carried out in an aqueous solvent.

Conversely, conventional methods using anhydrous and non-polar organic solvents such as benzene give anhydrous products. Pharmacological Tests Monohydrate picotamide was found to be highly effective as a platelet antiaggregant and fibrinolytic agent.

Therefore, it has utility in clinical pharmacology and human therapeutic applications. The effect was determined by the spectrophotometric method of Born (BOrn) for the platelet antiaggregation effect and by Huanli (1999) for the fibrinolytic effect as a whole.
Feamley's Thrombolysis Test.

Example 1 Rapid in vivo platelet aggregation effect.

A New Zealand rabbit was deprived of water for a certain period of time.
A 20% ethyl solution of urethane is administered to the peritoneal cord at a dose of 0.6 ml/100 y to cause paralysis. 25-50-10
Blood is withdrawn from the carotid artery immediately (control) and 1.5 hours after intraperitoneal injection of picotamide monohydrate at a dose of 0 mg/K9.

The blood sample was treated with sodium citrate at a volume ratio of 3.8 to 9.
% solution and then centrifuged at 1000 rpm to obtain platelet-rich plasma (PRP). A portion of the plasma is then centrifuged at 8000['Pm] to obtain platelet-poor plasma (PPP). P
PP is used to determine the zero point of the Born agglomerometer,
Then, put 1 mL of PRP into the blood of the measuring device.

Platelet aggregation is driven by disodium adenosine diphosphate (ADP) at concentrations that vary depending on platelet activity. The platelet aggregation effect is determined by the aggregation curve after treatment.
Calculated as 50% inhibition (ID5O) over that of the control.

The results will be discussed later. Example 2 Temporal effects on platelet aggregation and blood concentrations in dogs.

Picotamide monohydrate at a dose of 100 mg/Kg, 18
, administered orally to a vehicle dog (male) that is deprived of water ad libitum. Before treatment (control) and 2-4-6-
After 8-1C, blood is drawn and platelet aggregation and blood concentration of drug are determined as a function of time (by the methods described above).

Measurement of blood concentration is performed using a W spectrophotometer according to the following method.

Plasma obtained by centrifugation at 100 r'Pm for 10 minutes 5
Add 2 ml of concentrated hydrochloric acid to WLI and hydrolyze it in a 100°C water bath for 1 hour.

This is cooled, dissolved in 2 mL of water, and filtered.
The deep water was made strongly alkaline with NH4OH, and 30
Extract with m1(7)CHCI3. Anhydrous Na2sO
The chloroform layer dried in step 4 was soaked in 0.1N (7) H2S.
Extracted with O4 and added to the acid layer so obtained
．． Add 1N (7) H2SO4 to make 100ml and make 22
Measure with a spectrophotometer at 8nn1. The results will be discussed later. Example 3 Fibrinolytic action in the living body of guinea pigs.

Fibrinolytic activity was measured in Italian guinea pigs,
Monohydrate picotamide was administered orally continuously at 100 m9/K.
Administered at a dosage of 9. The Fernley method, modified as follows, was used for this test: In a test tube kept at 0°C, add 1.7 ml of phosphate buffer (pH 7).
．． 4) and 0.1 m of 50 NIH/Mt thrombin solution
Enter 1.

After adding a total of 0.2 ml of guinea pig blood, the test tube was kept at O ℃ for 30 minutes to coagulate, and then kept in a water bath at 3 TC for 3 minutes to cause the dissolution phenomenon. Next, measure the weight of the coagulum. The fibrinolytic effect is calculated as the percentage decrease in clot weight of treated animals compared to the clot weight of controls.

Example 4 Platelet aggregation and fibrinolytic effects in humans.

Confirmation of the dual effects of picotamide monohydrate in vivo was obtained by orally treating 35-657 healthy men and women with a single dose of 12m9/K9. Platelet aggregation was inhibited using AD as an antagonist according to the same method as in Example 1.
Tested using P disodium. The fibrinolytic effect was evaluated by measuring the dissolution time of euglopin.

The results obtained are shown in the table below.

Results By statistical analysis, the dose (ID5O) that can suppress platelet aggregation by 50% was calculated from the results obtained from the in vivo platelet aggregation test (Example 1) for Rapid, which was 54.10. ±1.43m9/K9.

The maximum effect (53.82%) in dogs (Example 2) was seen at the 4th hour.

The corresponding blood concentration was 22.6 γ/Tn9 per ml of plasma, which corresponds to the maximum value as shown in the graph of FIG. The fibrinolytic effect in guinea pigs (Example 3) tested orally at a dose of 100 mg/K9 was 27
．． It was found to be equal to 83%.

Person with a single dose of 12m9/Kg orally (Example 4)
In , the maximum platelet aggregation effect was seen 4 hours after treatment, and this was determined to be 81.4%; the maximum fibrinolytic effect was due to a decrease in the dissolution time of euglopin.
．． It was measured to be 2%.

Comparative Findings on the Effects and Toxicity Results of Monohydrate Picotamide and U.S. Patent No. 3
A comparison of these anhydrous picotamides described in No. 973,026 shows that there is a substantial difference in action, and that this difference favors the monohydrate form.

This could not have been expected based on the simple introduction of crystalline water molecules, which is a feature of the new compound.
For comparison, studies on time effects and blood concentrations in dogs by oral route were carried out under the same experimental conditions as for the already known anhydrous picotamide in order to test the biological processing after its administration.

The results of this test are shown in the graph of Figure 4, where the glass
00mg/K9, shows the time effect of platelet aggregation and blood concentration in dogs at the oral dose. The horizontal axis shows time in hours, and the vertical axis shows blood concentration in γ units per plasma Tnt and platelet aggregation effect in percent units. line 1
and 1'' represent the anti-aggregation effect of picotamide monohydrate and picotamide anhydride, respectively, and continuous lines 2 and 2'' represent blood concentrations of picotamide monohydrate and picotamide anhydride, respectively. Considering the results shown in the figure, anhydrous picotamide has a maximum platelet aggregation effect of 49% at the 8th hour, while the maximum blood concentration is 19.75γ/Mt, which can occur at the 6th hour. It's obvious.

In comparison, monohydrate picotamide exhibits a maximum platelet aggregation effect at the fourth hour, and furthermore, the maximum peak of effect coincides with the same time as the maximum peak of blood concentration.

This evidence provides evidence of more rapid biological processing favoring monohydrate picotamide compared to anhydrous picotamide. The results of pharmacological studies conducted on monohydrate picotamide are summarized in the following table ■.

Data from the same test for anhydrous picotamide are also presented here. A comparison of the test values for the two molecules, shown in Table ■, shows that monohydrate picotamide was improved compared to anhydrous picotamide. Indicates that it has pharmacological effects.

This unexpected improvement is believed to be due to the improved bioprocessability of this new drug in the form of a stable structural monohydrate crystal. Uses in the treatment Monohydrate picotamide, in view of its low toxicity, high tolerability and absence of untoward side effects, is widely used in various thrombotic disorders, especially cerebrovascular disorders, myocardial infarction, arterial and venous thrombosis. It may be useful in the treatment of humans for pulmonary embolism, general atherosclerotic conditions, and general cardiac surgical procedures.

For this use, various drug forms containing from 10 to 500 m9 of active agent may be employed.

An example of this could be something like the following: (
a) Orally: capsules, tablets, pills containing 10-500 m9 of active agent, for doses of 50-3000 m9/day; (b) Parenterally; for doses of 10-200 mg/day. A small sterile intravenous pin containing 10-50 m9 of active agent.

It can also be administered rectally in the form of suppositories.

The pharmaceutical compositions obviously may contain, in addition to the active agent, such vehicles and excipients as are normally pharmaceutically acceptable and well known in the pharmaceutical art.

It is also clear that the dosage form of picotamide monohydrate and the respective dosing pattern can be varied according to the clinical conditions and the experience of the physician.

[Brief explanation of the drawing]

FIG. 1 is a three-dimensional representation of the molecule of monohydrate picotamide.

Claims (1)

[Claims] 1 Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ A monohydrated N,N'-bis-(3 -picolyl)-4-methoxy-isophthalamide. 2 Monohydrated N reacting a functional derivative of 4-methoxy-isophthalic acid with 3-picolylamine in an anhydrous organic solvent
, N'-bis-(3-picolyl)-4-methoxy-isophthalamide, characterized in that the reaction product is precipitated with water and the crude product is recrystallized from an aqueous solution and subsequently from water. manufacturing method. 3. Process according to claim 2, characterized in that after precipitation of the reaction product with water, the product is crystallized from an acetone/water solution and recrystallized from water. 4. A therapeutically effective amount of monohydrated N,N'- crystalline form as an active agent in a pharmaceutical composition for the treatment of thrombosis in mammals by increasing platelet aggregation and increasing thrombin action in the blood. A pharmaceutical composition for treating thrombosis in mammals, comprising bis(3-picolyl)4-methoxy-isophthalamide and a pharmaceutically acceptable vehicle. 5. A pharmaceutical composition for the treatment of thrombosis in a mammal according to claim 4 in the form of a dosage unit containing from 10 to 500 mg of active agent. 6. The pharmaceutical composition for treating thrombosis in mammals according to claim 5, which contains 10 to 500 mg of the active agent and is administered orally. 7. The pharmaceutical composition for treating thrombosis in mammals according to claim 5, which contains 10 to 500 mg of active agent and is administered parenterally.