FI74951B - FOERFARANDE FOER FRAMSTAELLNING AV EN MONOHYDRATISERAD ISOFTALSYRAPIKOLYLAMID I STABIL KRISTALLIN FORM. - Google Patents

Description

74951

This invention relates to a process for the preparation of a pharmaceutically active monohydrated stable crystalline Ν, Ν'-bis- (3-picolyl) -4-methoxyisophthalamide of the formula CH 2 -NH-NH 2 in stable crystalline form. jgj up * h2 °

c ° -nh-ch2 -fnI

0CH3 whereby a functional derivative of 4-methoxyisophthalic acid is reacted with 3-picolylamine in an anhydrous organic solvent. The compound inhibits platelet aggregation, prevents thrombotic diseases and slows blood clotting. The invention also relates to a process for synthesizing this novel crystalline compound.

In particular, the invention relates to monohydrated N, N'-bis- (3-picolyl) -4-methoxyisophthalamide, a process for its preparation and pharmaceutical preparations containing this compound.

It is well known that Ν, Ν'-bis- (3-picolyl) -4-methoxyisophthalamide, hereinafter referred to internationally as "picotamide", is a compound with high fibrinolytic and anticoagulant activity ( see FR Patent 2100850; Chimie Thärapeutique, 6, 203-7, 1971), and also a good antiplatelet effect (U.S. Patent 3,973,026; Age and Aging, 7, 246, 1978).

Picotamide, as described in the above-mentioned publications and patents, is in anhydrous form and has a melting point according to FR patent 2100850 as measured by a Kofler apparatus at 124 ° C.

2,74951 The crude product of this compound, which contains a lot of impurities, is purified according to the methods described in the above-mentioned patents by crystallization from anhydrous and apolar organic solvents.

Crystallization of the crude picotamide obtained in the synthetic reaction from anhydrous or apolar organic solvents such as benzene gives a powdery product which is fibrous when viewed under a microscope. Said powder readily takes an electro-static charge and is relatively unstable. These properties are particularly detrimental in the preparation of a compound for pharmaceutical purposes. The electrified particles repel each other and evaporate easily when weighed and exported to equipment for the manufacture of various pharmaceutical preparations, which leads to variations in particle size and which in turn makes it difficult to stabilize the titer.

It has now been found that by reacting a functional derivative of 4-methoxyisophthalic acid with 3-picolylamine, and crystallizing the crude product from an aqueous solution, a monohydrated-, Ν'-bis- (3-picolyl) -4-methoxyisophthalamide having chemical-physical structure properties and Stability, which make this compound clearly advantageous over the prior art known anhydrous picotamides.

It has further been surprisingly found that said monohydrated picotamide thus obtained is more active and effective than anhydrous picotamide, both from a pharmacodynamic and clinical pharmacological point of view.

The invention thus relates to a process for the preparation of monohydrated Ν, Ν'-bis- (3-picolyl) -4-methoxyisophthalamide, C2li20N4 <^ 3 * H2 ^ ', molecular weight 394.4.

The general chemical formula of the above compound can be represented as follows:

CO-NH-CH 2 -R 2 N

a, 2IQJ PX »·« 2 ° co-nh-ch2 0CH3

The monohydrated picotamide of the invention is a white, odorless, bitter-tasting, crystalline powder which is stable in air and easily crystallized from water and has a melting point between 95 and 97 ° C as measured by a Kofler apparatus.

In the following, for the sake of simplicity, the above compound will be referred to by the general name "monohydrated picotamide".

The compound prepared by the process of the invention differs from the previously well-known compound in physicochemical sense in that it has a surprising and clearly better stability. Said improvement in stability is due to the fact that the hydrate water molecule is part of the structure of the compound of the invention, in a well-defined position in the crystal lattice where the hydrate water oxygen atom forms clearly identifiable hydrogen bonds with certain atoms of different picotamide molecules, as shown below. crystal having properties. Said properties surprisingly affect the pharmaceutical activity of the novel compound and its bioavailability in mammals, by absorbing it more rapidly when administered to said group of animals, including man.

The monohydrated picotamide of the invention in crystalline form not only surprisingly eliminates the disadvantages associated with crystalline anhydrous picotamide, but even more surprisingly, this compound is more active and effective in pharmacological utility when considered in animal or human administration. beneficial effects.

It can be assumed that the dissolution of the crystalline compound of the invention in water follows a different mechanism than the dissolution of the well-known anhydrous compound in amorphous form in water, although no valid theoretical explanation for this experimentally obtained result is currently found.

Another aspect of the invention is a process for preparing a novel hydrate molecule.

As previously mentioned, the monohydrated picamide has a melting point of 95-97 ° C.

In this connection, it is mentioned that the anhydrous picotamide has a melting point of 124 ° C. This difference in melting points alone indicates that the molecular structures of the compounds differ, which means that anhydrous picotamide differs substantially from monohydrated picotamide.

The invention will be explained in more detail below with reference to the accompanying drawings, in which: Figure 1 shows a three-dimensional molecular model of monohydrated picotamide obtained based on an X-ray spectrum; Figure 2 shows a three-dimensional model of monohydrated picotamide monocrystal; Fig. 3 shows the crystal shown in Fig. 2 in more detail; and Figure 4 is a graph comparing the antiplatelet activity of mono- 5,74951 hydrated picotamide and crystalline unscored picotamide.

The crystal structure of monohydrated picotamide has been characterized not only by physicochemical analysis but also by its X-ray spectrum of the monocrystalline.

The following Table I shows X-ray diffraction spectral data revealing the space location of atomic centers in the new molecule, assuming that it consists entirely of dense, stable non-hygroscopic crystals, where in Table X, the various atoms of the molecule are denoted by their chemical symbol. identifioimisnumero. The space location and identification number of said atoms are shown in Figure 1, which gives the geometric three-dimensional location of the atomic centers obtained from Table I.

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Table I

Abrasive coordinates (X-ray) of monohydrated picotamide maximum = 92.52 minimum = -86.27 multiplied by 167.8829

Projection Atom Height X / AY / BZ / C SOF Mole- Ele number number requirement 1 08, 1895, 6286, 8386 1.0000 1 1.26 2 C38, 1967, 7283, 8919 1.0000 1, 27 3 Cl , 2670, 5074, 3663 1.0000 1, 82 4 C3, 2047, 4937, 6312 1.0000 1 1.60 5 C5, 2299, 4539, 4720 1.0000 1 1.44 6 C6, 2944, 4562, 2040 1.0000 1, 75 7 C7, 2164, 5904, 6832 1.0000 1 1.11 8 09, 1627, 3432, 6819 1.0000 1 2.75 9 CIO, 2795, 6035, 4219 1.0000 1, 35 10 Nil, 1385, 4642, 8824 1.0000 1 2.43 11 012, 2847, 3716, 1698 1.0000 1 1.24 12 C13, 1665, 4277, 7347 1.0000 1 2.31 13 C14, 2547, 6442, 5815 1.0000 1, 51 14 C15, 3600, 4594 -, 0529 1.0000 1, 11 15 N19, 3326, 5077, 1101 1.0000 1, 21 16 C22, 1082, 3996 1.0097 1.0000 1 3.28 17 027, 3771, 7056, 1461 1.0000 0 0.00 18 C221, 4250, 4199, 0573 1.0000 1 1.34 19 N222 -, 0616, 3513, 9680 1.0000 1 2.34 20 C223, 5422, 3476, 2266 1.0000 1 3.43 21 N224, 5202, 3510, 0056 1.0000 1 2.34 22 C227, 0384, 3880, 9075 1.0000 1 2.47 23 C228, 0004, 3683 1.0498 1.0000 1 2.96 24 C229, 4606, 3864 -, 0822 1.0000 1 1.28 25 C231 -, 0875, 3574, 7563 1.0000 1 1.23 26 C232, 0127, 3932, 6853 1.0000 1 1.33 27 C234, 4483, 4146, 2819 1.0000 1 2.47 28 C235 -, 0519 , 3732, 6042 1.0000 1, 72 29 C244, 5071, 3758, 3636 1.0000 1 3.53 30 Q 1 96., 0376, 3935, 5361 1.0000 1, 88 31 Q 2 92, 5364, 3978, 3476 1.0000 1 3.48 32 Q 3 84, 2173, 3847, 4186 1.0000 1 1.70 33 Q 4 84, 3392, 3922 -, 0552 1.0000 1, 51 34 Q 5 83 ., 3204, 6259, 3130 1.0000 1 0.00 7 74951

Bonds (including symmetrically located atoms) 1- 2 1.44 3- 5 1.40 4- 5 1.39 3- 6 1.50 6-11 1.22 4-12 1.51 8-12 1.23 10 -12 1.35 14-15 1.49 10-16 1.48 14-18 1.54 20-21 1.37 18-24 1.40 21-24 1.39 19-25 1.33 22-26 1.38 20-29 1.36 27-29 1.39 26-30 1.21 20-31 1.08 18-33 1.89 9-34 1.31

In particular, it can be seen that atom No. 17, denoted 027, represents hydrated water, while atom No. 8, denoted 09, represents oxygen of the methoxy group, atom No. 15, denoted N19, is the nitrogen of the amide group, and atom No. 21, denoted N224, is a nitrogen atom of a pyridine group.

In Table 1, the symbols X / A, Y / B, and Z / C denote the spatial coordinates of different atoms.

As can be seen from Figure 1, which shows the structure of the mono-hydrated picotamide, the hydrate-water oxygen atom is located at a well-defined position in the crystal lattice with respect to the picotamide molecule.

It can be seen from Figure 2 that the oxygen inside the hydrate monocrystal, indicated by a rectangle, is bound by hydrogen bonds (indicated by a dotted line) to well-defined atoms of different picotamide molecules arranged in an ordered three-dimensional pattern and bound together by hydrated oxygen.

Said binding is shown in more detail in Figure 3, which clearly shows that the oxygen 027 of the hydrate water is bound by hydrogen bonds: 1) oxygen of the methoxy group (O 2) = CO of the first picotamide molecule (bond length: 2.81 Å); 8 74951 2) nitrogen of the amide group = NH (N 2 g) of the second picotamide molecule, which second molecule is located at the same level as the above-mentioned molecule (bond length: 2.96 Å); 3) the nitrogen of the pyridine ring (N224) of the third picotamide molecule, which third molecule is located above or below the other two bound molecules (bond length: 2.80 A),

The three bonds explain both the fact that the water of crystallization is strongly bound between the different picotamide molecules forming the crystal and that the crystal itself is dense. The dense form of said crystal provided by the crystal water molecule is considered to be the reason for the improved bioavailability of the monohydrate compared to the previously known anhydrous form, the improved effect of which is shown pharmaceutically below, comparing the absorption times, blood concentration and activity of these substances.

In fact, it has not been possible to assume from the prior art data on anhydrous picotamide that the addition of a crystalline water molecule to picotamide will provide an improvement in the density of the picotamide space structure, as well as a further substantial improvement in its bioavailability.

Elemental chemical analysis has yielded results that are consistent with the structure outlined above.

For C 21 H 20 N 4 O 3 * H 2 O (m / w Weight 394.4) found: C% 63.87 (theoretical 63.94); H, 5.72 (theoretically 5.62); N, 14.18 (theoretical 14.20) and the remainder oxygen.

A process for the preparation of a novel monohydrated picotamide is carried out according to a method known from the synthesis of a compound for the preparation of anhydrous picotamide.

Once crude picamide has been obtained, it has surprisingly been found that recrystallization of the crude product from an aqueous solution gives, in contrast to the anhydrous mixture of the known method, a crystalline monohydrate product as defined above.

The following examples illustrate a process for the preparation of monohydrated picotamide, starting from 4-methoxyisophthaloyl dichloride or another functional derivative of 4-methoxyisophthalic acid, in a medium containing proton accepting agents.

Preparation Example 4-Methoxyisophthaloyl chloride 100 g (0.43 mol) (molar weight 233) 100 g (0.43 mol) 3-picolylamine (molar weight 108) 130 g (1.2 mol)

Triethylamine 120 ml

Tetrahydrofuran (anhydrous) 120 + 200 ml of 3-picolylamine, triethylamine and 120 ml of anhydrous tetrahydrofuran are added to a 3-liter flask equipped with a reflux condenser, dropping funnel and mechanical stirrer.

Separately, 4-methoxyisophthaloyl chloride is dissolved in 200 ml of anhydrous tetrahydrofuran. This solution is slowly added from the dropping funnel to the reaction mixture in the flask with stirring. The addition is carried out in 1.5-2 hours, during which the following exothermic reaction takes place: 10 74 951

COCl CO-NH

iL · *

OCH3 OCH3 SiX

I II III

After the addition of said dichloride solution, the reaction mixture is refluxed for about 2 hours, and slowly diluted with water to a volume of 2 liters, and stirring is continued until a crystalline slurry consisting of crude nicotamide separates.

Said slurry is collected by filtration and crystallized wet from 700-800 ml of an acetone-water mixture (6 volumes of acetone + 11 volumes of water).

The product obtained is recrystallized from water to give monohydrated picotamide, melting point 95-97 ° C, measured on a Kofler apparatus.

It is apparent from the above that the process according to the invention is characterized by recrystallization of the crude picotamide obtained by the synthetic reaction from an aqueous solvent, in contrast to the prior art using anhydrous and non-polar organic solvents such as benzene to give an anhydrous product.

Pharmacological experiments

Picotamide monohydrate has been found to have high activity as an antiplatelet agent and as a fibrinolytic agent.

These activities were studied in vivo using a Born spectrophotometric assay for antiplatelet activity and 11,74951

Total dissolution test for Fearnley blood clot for fibrinolytic activity.

Example 1

In vivo antiplatelet activity of rabbit platelets.

New Zealand rabbits fasted for 12 hours but given free water were anesthetized by intraperitoneal administration of urethane in 20% ethyl solution at a dose of 0.6 ml / 100 g. Blood was collected from the carotid arteries of the animals before (control) and one and a half hours after administration of monohydrated picotamide by intraperitoneal injection at a dose of 25-50-100 mg / kg. Blood samples were non-coagulated with 3.8% sodium citrate by the addition of 9/1 by volume and then centrifuged at 1000 rpm for 15 minutes to obtain platelet-rich plasma (PRP). A batch of this plasma was then centrifuged at 8000 rpm for 10 minutes to obtain platelet-poor plasma (PPP).

PPP was used to reset the Horn aggregometer and 1 ml of PRP was added to the measuring chamber of the device. Addition of disodium adenosine diphosphate at various concentrations of disodium (ADP) resulted in platelet aggregation, which aggregation was dependent on platelet reactivity. The antiplatelet activity was calculated as the 50% inhibition of the aggregation curve after treatment compared to the control group (Id5q).

The results are shown below.

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Example 2

Time dependence of platelet aggregation in dogs.

Male Beagle dogs fasted for 18 hours with free water were orally administered 100 mg / kg picotamide monohydrate.

Blood samples were taken before (control) and 2-4-6-8-10 hours after treatment, and the antiplatelet activity as a function of time (as described above) and the concentration of the administered substance in the blood were determined.

The blood concentration was determined with a UV spectrophotometer according to the following method.

To 5 ml of plasma obtained by centrifugation at 100 rpm for 10 minutes was added 2 ml of concentrated HCl and hydrolyzed in a water bath at 10 ° C for 1 hour. The mixture was cooled and 2 ml of water were added and filtered. The filtrate was made strongly basic with NH 4 OH and extracted with 30 mL of CHCl 3. The chloroform layer was dried over Na 2 SO 4 and extracted with 1 SO 4 to a volume of 100 ml and a spectrometer reading was taken at 228 nm. The results are shown below.

Example 3

Fibrinolytic activity is in vivo in guinea pigs.

Fibrinolytic activity was determined in Italian guinea pigs by oral administration of 10 mg / kg monohydrated picotamide.

The Fearnley method modified as follows was used in this experiment;

To test tubes maintained at 0 ° C were added 1.7 ml of phosphate buffer (pH 7.4) and 0.1 ml of thrombin solution 50 NIH / ml. The tubes were kept at 0 ° C for 30 minutes after the addition of 0.2 ml of guinea pig blood to form a clot, and then the tubes were kept at 37 ° C for 30 minutes to effect dissolution. The weight of the clot was then determined.

Fibrinolytic activity was calculated as the percentage reduction in clot weight of treated animals.

Example 4

Antiplatelet and fibrinolytic activity of human platelets.

The above in vivo activities of picotamide monohydrate were observed in healthy subjects of both sexes aged 35-65 years, with a single oral dose of 12 mg / kg.

Inhibition of platelet aggregation was determined using disodium ADP as an antagonist according to the method described in Example 1.

Fibrinolytic activity was determined by measuring the disintegration time of euglobins (a protein in serum).

The results obtained are shown below.

Score

In experiments to determine the inhibition of platelet aggregation in rabbits in vivo (Example 1), the dose that inhibited platelet aggregation by 50% (ID, -q) was calculated by probit analysis to be 54.10 μl, 43 mg / kg.

Dogs had a maximal effect (Example 2} after 4 hours. The corresponding concentration in the blood was 22.6 γ / ml plasma and this corresponds to the maximum value as shown in Figure 4.

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The fibrinolytic activity determined in guinea pigs (Example 3) at a dose of 100 mg / kg orally was 27.83%.

In humans (Example 4), a single dose of 12 mg / kg orally four hours after dosing resulted in maximal inhibition of platelet aggregation and was determined to be 81.4%. A maximal tibrinolytic activity resulted in a 54.2% decrease in euglobin degradation.

Comparing the activity and toxicity results obtained for the monohydrated picotamide with the anhydrous picotamide described in U.S. Patent 3,973,026, it can be seen that there is a significant difference in activity between these, with the monohydrated picotamide being more active. This is surprising given that the improvement has been achieved by introducing a crystal water molecule into the compound.

To compare the above compounds under the same experimental conditions, the time dependence and concentration of anhydrous picotamide in the blood of dogs when administered orally was determined to determine the bioavailability of this compound after dosing.

The results of this experiment are shown in the curve of Figure 4, which shows the time dependence of platelet aggregation and the blood concentration of the administered compound when administered to dogs at 100 mg / kg orally.

The ordinate describes the blood concentration of the test compound in hours and the abscissa, expressed in γ / ml plasma, and also the antiplatelet activity in percent. Curves 1 and 1 'depict the antiplatelet activity of monohydrated picotamide platelets and anhydrous picotamide, respectively, and continuous curves 2 and 2' depict blood levels of monohydrated picotamide and anhydrous picotamide, respectively.

It is clear from the graph that anhydrous picotamide has a maximal platelet inhibitory effect after 8 hours at 49% and a maximum blood concentration of 19.75 γ / ml, which then begins to decrease after 6 hours,

The maximum antiplatelet effect of monohydrated picotamide, on the other hand, is reached after four hours and, in addition, the maximum activity occurs at the same time as the maximum blood concentration, which proves the faster bioavailability of this compound compared to anhydrous picotamide.

The results of the pharmacological tests performed on monohydrated picotamide are shown in Table II below, which also shows the results of the tests performed on anhydrous picotamide.

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Table XI

Experiment Parameter Monohydrated Anhydrous picotamide picotamide

Platelet aggregation in vivo (rabbit) intraperitoneally ID5Q «mgAg 54.1 108.2

Platelet Aggregation Aggregation Maximum Inhibition% 53.83 48.12 in vivo (dog) - 100 mg / kg oral Determined maximum duration of activity 4 hours 8 hours

Maximum plasma concentration γ / ml 22.6 19.75 Specified maximum concentration time 4 hours 6 hours

Platelet Aggregation Aggregation Maximum Inhibition% 81.4 70.11 In Human Single - Dose Determined Maximum After Time 4 Hours 8 Hours

Acute oral toxicity to rat and dog -mg / kg> 3,000> 3,000

Comparing the results shown in Table II for said two compounds, it is seen that the pharmaceutical effect of monohydrated picotamide is better than that of anhydrous picotamide. This unexpected improvement appears to be due to the improved bioavailability of this new compound in the form of a stable monohydrate crystal.

Due to its low toxicity, good tolerability and lack of undesirable side effects, picohydrate monohydrate can be used in humans for the treatment of various thrombotic diseases, in particular cerebrovascular diseases, myocardial infarction, vascular thrombosis, pulmonary thrombosis in general, congestive heart failure.

Various pharmaceutical preparations containing 10 to 5 mg of active substance can be used for said uses, examples of which are given as follows: a) orally: capsules, tablets, pills containing 1 to 500 mg, where the daily dose is 50 to 3000 mg / day; b} parenterally: sterilized for intravenous injection in vials containing 1Q-5Q mg, with a daily dose of 1Q-2Q mg / day.

The pharmaceutical preparation may also be administered through the intestinal suppositories.

The pharmaceutical preparation may also contain, in addition to the active ingredient, conventional pharmaceutically acceptable carriers and excipients well known in the pharmaceutical art. It will also be appreciated that the routes of administration and dosages of the monohydrated picamide may be varied according to clinical practice and the judgment of the physician.

Claims (2)

74951 A process for preparing a pharmaceutically active monohydrated, stable crystalline form of N, N1-bis- (3-picolyl) -4-methoxyisophthalamide / having the formula CO-NH-CH2-H2O CO-NH-CH2 OCH3 reacting the functional derivative of 4-methoxyisophthalic acid with 3-picolylamine in an anhydrous organic solvent, characterized in that the reaction product is precipitated with water and the crude product thus obtained is recrystallized from an aqueous solution and then from water to 95-97 ° C. measured by a Kofler apparatus and the X-ray diffraction spectrum of the monocrystal is as shown in Table 1 of the description. Process according to claim 1, characterized in that said product, after being precipitated with water, is crystallized from acetone / water and recrystallized from water.