NO154193B - METHOD OF PREPARING MONOHYDRATIZED PICOTAMI D - Google Patents

Description

The present invention relates to a method for the production of monohydrated N,N'-bis(3-picolyl)-4-methoxy-isophthalamine (picotamide) in crystalline form with the formula

where 4-methoxy-isophthaloyl dichloride is reacted with 3-picolylamine in tetrahydrofuran in the presence of triethylamine as proton acceptor,

and the characteristic of the method according to the invention is

a) that anhydrous tetrahydrofuran is used,

b) that the reaction product precipitates with water,

c) that the reaction product from step b is crystallized from a

solution of acetone/water (volume ratio 6:11) and

d) that the product from step c is recrystallized from water.

Picotamide in monohydrated crystal form is pharmaceutically active

in that it counteracts the aggregation of platelets, counteracts thromboembolic disorders in the blood and delays blood clotting

r ing.

It is well known that N,N<1->bis-(3-picolyl)-4-methoxy-isophthalamide

hereinafter referred to by the usual international designation

nelse "picotamide" is a compound with a high fibrinolytic and anti-coagulant activity, see French patent document 2.100.

850, further Chimie Thérapeutique 6, 203-207, 1971, as well as a good anti-aggregating effect for platelets, see US-

patent 3,973,026 and Age andAgeing, 7, 246, 1978.

Picotamide as described in the above publications is

known in the anhydrous form and the melting point is 124°C determined in

Kofler bench, as described in French patent document 2,100,850.

According to the method described in the above-mentioned patents, this compound is purified from the raw product, which contains a quantity of impurities, by crystallization from anhydrous and apolar organic solvents.

Crystallization of the crude picotamide obtained by the synthesis reaction from anhydrous and apolar organic solvents such as e.g. benzene, results in a powder product which under a microscope shows the features of a fibrous substance, with the character of voluminous down. The powder easily takes up an electrostatic charge and is relatively unstable. These properties are particularly unfortunate when the compound is prepared for pharmaceutical use. The electrified particles repel each other and tend to volatilize when they are weighed and introduced into the apparatus for the production of the various pharmaceutical preparations, and this results in a variation of the particle weight which makes the production of specific and stable doses difficult.

The recognition that underlies the present invention is that in the reaction between a functional derivative of

4-methoxy-isophthalic acid and 3-picolylamine, and by crystallization of the crude product from an aqueous solution, mono-hydrated N,N'-bis-(3-picolyl)-4-methoxy-isophthalamide is obtained with properties for chemical-physical structure and stability which makes this compound decidedly preferable to anhydrous picotamide products previously known.

It has also surprisingly been found that the thus obtained monohydrated picotamide is more active and effective than anhydrous picotamide, both from a pharmacodynamic point of view and with regard to clinical pharmacology.

The prepared monohydrated N,N'-bis-(3-picolyl)-4-methoxy-isophthalami<d><C>2i<H>20<N>4°3"<H>2° molecular weight 394.4, and The chemical structure formula can be indicated as follows:

The monohydrated picotamide produced by the method according to the invention is a white, odorless, bitter-tasting crystalline powder which is stable in air and easily crystallizes from water, with a melting point of 95°C-97°C determined in the Kofler bench.

For the sake of simplicity, the compound defined above shall be referred to in the following description with the generic term "monohydrated picotamide".

From a physico-chemical point of view, the new compound differs from the previously well-known compound with respect to an unexpected and decided improvement in stability. The improvement is due to the fact that the molecule of hydrate water participates in the molecular structure of the new compound, and is placed in the crystal lattice in a well-defined position in which the oxygen atom in the hydrate water establishes easily identifiable hydrogen bonds with special atoms belonging to different molecules of picotamide, as it should is demonstrated in the following, so that a single crystal of the compound with well-defined properties is built up. These properties surprisingly affect the pharmaceutical performance of the new compound and its bioavailability in mammalian organisms, with a faster absorption when administered to the aforementioned class of animals and humans.

Monohydrated picotamide in the new crystalline

form not only surprisingly avoids the above-mentioned disadvantages of the anhydrous picotamide, but is surprisingly more active and effective with regard to pharmacological action, on the basis of the beneficial effects observed when administered to animals and humans.

Although a reasoned theoretical explanation for the aforementioned experimental result cannot be put forward for the time being, it can

however, it is assumed that the dissolution in water of the new crystalline compound takes place by a different mechanism compared to the dissolution in water of the well-known anhydrous compound in its amorphous form.

The new compound can be included in pharmaceutical preparations in the usual pharmaceutical forms, for the clinical treatment of thromboembolic blood disorders.

As previously stated, the melting point of is monohydrated

OE OE

picotamide 95 C-97 C.

In this connection, it is pointed out that anhydrous picotamide has

a melting point of 124°C. This difference in the melting points of the two compounds is already an indication of the different molecular structure, which indicates a significant difference between the structure of anhydrous picotamide and monohydrated picotamide.

The invention shall be described in more detail with reference to the attached drawings, in which: Fig. 1 is a three-dimensional sketch of the molecule of monohydrated picotamide, obtained from X-ray

spectrum

Fig. 2 is a three-dimensional sketch of the single crystal of monohydrated picotamide. Fig. 3 shows in more detail the same single crystal shown in fig. 2, and Fig. 4 is a diagram showing a direct comparison between the platelet anti-aggregation activity of monohydrated picotamide and anhydrous picotamide.

The structure of a crystal of monohydrated picotamide

is next to the physical and chemical analysis also characterized with the help of the X-ray spectrum of its single crystal.

Data from the X-ray diffraction spectrum, which shows the spatial position of the atomic centers in the new molecule,

taken in its entirety as a compact, stable and non-hygroscopic crystal, is reproduced in the subsequent Table I, in which the various atoms in the molecule are indicated by their chemical symbol followed by an identification number. The spatial location and the identification number

for the mentioned atoms can be observed in fig. 1 which represents the geometrical, three-dimensional position of the atomic centers, as obtained from Table, I.

Bonds (Including symmetrically related atoms)

It can be observed in particular that atom no. 17, indicated

as 027, represents the hydrate water, while atom no. 8 indicated as 09 represents an oxygen in the methoxy group, atom no. 15 indicated as N19 is a nitrogen atom in the amide group and atom no. 21 indicated as N2 24

is a nitrogen atom in the pyridine group.

In Table I, the symbols represent X/A, Y/B and Z/C

the spatial coordinates of the different atoms.

As can be seen from fig. 1, which shows the structure of monohydrated picotamide, the oxygen atom in the water of hydration is placed in the crystal lattice in a well-defined position in relation to the picotamide molecule.

In fig. 2 it can be seen that the oxygen in the hydrate water inside

in the single crystal represented by a rectangle, are linked by hydrogen bonds (indicated by a dotted line) to well-defined atoms of different picot-amide molecules, these molecules being in a regular three-dimensional pattern and linked systematically by means of the aforementioned hydrogen bonds with the hydrated water-oxygen.

The aforementioned binding is shown in more detail in fig. 3 from which it is clearly seen that oxygen 027 in the hydrate water is linked by hydrogen bonds to respectively:

1) The oxygen in =C0 in the methoxy group (Og) in et first picotamide molecule (bond length: 2.81 Å)2) The nitrogen in the amide group =NH (N^:g) in another picotamide molecule placed in the same plane as the above molecule (bond length: '2.96 Å ) 3) The nitrogen in the pyridine ring (N224^ of a third picotamide molecule placed on the underlying or overlying plane in relation to the two other bound molecules (bond length 2.80 A)

The presence of the aforementioned three bonds provides an explanation

both on the strength with which the crystal water is introduced between different picotamide molecules that form the crystal, and the compactness of the crystal itself. This compactness, which is provided by the molecule of crystal water, is believed to be the reason for the improvement in the bioavailability of the monohydrate form compared to the previously known anhydrous form, and this improvement will be shown below from the pharmacological point of view,

by a comparison of absorption time, blood level values and activities of both compounds.

Because of. the knowledge available from the prior art with respect to the anhydrous picotamide molecule,

could some improved effect, which is due to in-

introduction of a crystal water molecule, on the compactness of the spatial structure of the picotamide, as well as an appreciable improvement of the bioavailability are clearly not foreseen. Elemental analysis has provided results consistent with the above illustrated structure.

For C21H2()N403.H20 (molecular weight 394.4) it has been found:

C% 63.87 (theoretical 63.94) > H% 5.72 (theoretical 5.62);

N%14.18 (theoretical 14.20) and oxygen as difference.

The procedure for the production of the new monohydrate

in

picotamide molecule follows, as far as the synthesis is concerned, the already known method for the production of anhydrous picotamide.

When the crude picotamide has been obtained, it has surprisingly been found that by recrystallizing the crude product from an aqueous mixture, as opposed to an anhydrous mixture as in the previously known process, the crystalline monohydrate product as previously defined is obtained.

The following examples illustrate the procedure for the synthesis of the monohydrated picotamide, starting from 4-methoxyisophthaloyl dichloride in an environment of proton acceptors.

Example of production.

3-picolylamine, triethylamine and 120 ml of anhydrous tetrahydrofuran are introduced into a 3 liter flask equipped with a reflux condenser, dropping funnel and mechanical stirrer.

4-Methoxy-isophthaloyl dichloride is dissolved separately in 200 ml of anhydrous tetrahydrofuran.

This solution is introduced slowly through the dropper

in the reaction mixture contained in the flask under stirring. The addition is carried out over one and a half to two hours, whereby an exothermic reaction takes place with the release of 2 mol of HC1.

After the aforementioned dichloride addition, the reaction mixture is refluxed for approximately two hours, slowly diluted with water to two liters and kept under stirring until separation of a crystalline thick dispersion consisting of the crude picotamide.

The aforementioned dispersion is filtered off and crystallized without drying from 700-800 ml of acetone-water mixture (6 volumes of acetone + 11 volumes of water).

The product obtained is recrystallized from water and gives monohydrated picotamide with a melting point of 95°C-97°C

on the Kofler bench. Recrystallization of the crude picotamide obtained from the synthesis reaction is thus carried out using an aqueous solvent, in contrast to the previously known technique in which anhydrous and apolar organic solvents were used, such as e.g. benzene, which gave an anhydrous product.

Pharmacological tests

Monohydrated picotamide has been found to exhibit a

high activity as a platelet aggregation and fibrinolytic agent. Consequently, it has applications in clinical pharmacology and human therapy.

D's mentioned activities were tested in vivo by spectrophotometric determination according to Born, for the platelet anti-aggregation activity and by blood clot lysis in toto tests according to Fearnley, for the fibrinolytic activity.

Example 1:

Platelet antiaggregation activity in vivo by

rabbit experiment.

New Zealand rabbits that had been kept fasting in

12 hours with water ad libitum was anesthetized with a 20% ethyl-alcohol solvent of urethane in a dose of 0.6 ml/

100 g intraperitoneally. The blood was drawn from the carotid artery before (control) and one and a half hours after the intraperitoneal injection of monohydrated picotamide, in a dose of 25-50-100 mg/kg. The blood samples were added to an anticoagulant, which was a 3.8% solution of sodium citrate in the volume ratio 9/1 and then centrifuged at 1000 revolutions per second. minute for 15 minutes to obtain a platelet-rich plasma (PRP). A portion of this plasma was then centrifuged at 8,000 rpm. minute for 10 minutes to obtain a platelet-poor plasma (PPP).

PPP was used for 0 setting of a Born aggregometer

and 1 ml of PRP was placed in the container in the measuring device.

A platelet aggregation was produced by variable concentrations of disodium adenosine diphosphate (ADP) depending on the platelet reactivity.

The platelet antiaggregation activity was calculated as

50% inhibition of the aggregation curve after treatment,

in relation to the control (ID ).

50

The results are referenced in the following.

Example 2:

The effect of time on platelet aggregation and blood levels in dogs.

Monohydrated picotamide in a dose of 100 mg/kg

was administered orally to male Beagles that were fasted for 18 hours with water ad libitum.

Blood was taken before (control) and 2/4, 6, 8, 10 respectively

hours after treatment, and as a function of time the platelet aggregation activity (using the above method) as well as blood levels of the compound were determined.

A determination of the blood levels was carried out at

using a UV spectrophotometer according to the following method: 5 ml of plasma obtained by centrifugation at 100 rpm. minute for 10 minutes, 2 ml of concentrated HCl was added and hydrolyzed on a water bath at 100°C

for 1 hour. It was cooled, added 2 ml of H 2 O and filtered. The filtrate was made strongly alkaline with NH 3 OH

and extracted with 30 ml of CHCl^. The chloroform layer, dried over anhydrous Na-jSO^, was extracted with 0.1 N ^SO^

and the acidic layer thus obtained was added CINF^SC^

to 100 ml and the spectrophotometer was read at 228 nm. The results are reproduced below.

Example 3

Fibrinolytic activity in vivo in guinea pigs.

The fibrinolytic activity was determined with Italian guinea pigs and then monohydrated picotamide was added to

administered orally at a dose of 100 mg/kg.

The Fearnley method, modified as follows, was used

for this test:

In a test tube kept at 0°C, 1.7 ml of phosphate buffer was added

(pH 7.4) and 0.1 ml of a thrombin solution with 50 NIH/ml added. After the addition of 0.2 ml guinea pig blood in toto, the tubes were kept at 0°C for 30 minutes to allow clotting and then kept in a water bath at 37°C

for 30 minutes to produce a lysis. The weight of the clot was then determined.

The fibrinolytic activity is calculated as % reduction in the clot weight of the treated animals, in relation to the clot weight of controls.

Example 4

Platelet antiaggregation and fibrinolytic activity

on voluntary subjects.

A confirmation of both activities in vivo for mono-hydrated picotamide was obtained with healthy subjects of both sexes aged 35-65 years, who were treated orally with a single dose of 12 mg/kg. The inhibitory effect on platelet aggregation was tested using disodium ADP as an antagonist, following the same method as in Example 1.

The fibrinolytic activity was assessed by determining the lysis time for euglobins.

The results obtained are listed below.

RESULTS

From the results obtained in the in vivo platelet antiaggregation activity test with rabbits (Example I) by probit analysis the dose capable of inhibiting platelet aggregation by 50% (ID^q) was judged and this dose was 54.10± 1.43 mg/kg.

A maximum effect (53.82%) in dogs (Example 2)

was found at the fourth hour. The corresponding blood level was 22.6 y/ml plasma and corresponding to a maximum value as shown in the diagram in fig. 4.

The fibrinolytic activity in guinea pigs (Example 3) tested at a dose of 100 mg/kg orally was found to

be 27.83%.

In humans (Example 4) in a single dose of 12-mg/kg administered orally, the maximum platelet aggregation effect was detected 4 hours after treatment and measured as 81.4%. The maximum fibrinolytic activity was measured as a 54.2% reduction in the lysis time of euglobins.

Comparison comments:

By comparing activity and toxicity results for monohydrated picotamide with the corresponding for

anhydrous picotamide, which is described in US patent 3,973,026, it can be concluded that significant differences exist in activity and these differences are favorable for the monohydrate form, which certainly could not be predicted on the basis of a simple introduction of a crystal water molecule which characterizes the new compound.

in

For comparison, a test was carried out with regard to time effect and blood levels in dogs, by oral administration, and under the same experimental conditions, with the previously known anhydrous picotamide to test its biological availability after administration.

The results of this test are reproduced in diagram i

fig. 4 which represents the time effect on platelet aggregation and on blood levels in dogs at a dose of

100 mg/oz.

The ordinate axis represents the time in hours and the abscissa

the axis represents the blood levels measured in µg/ml of plasma,

as well as the platelet antiaggregation activity measured in <%>.

Line 1 and 1' represent the anti-aggregation activity

of monohydrated picotamide or of anhydrous picotamide and the continuous lines 2 and 2<1>represent blood-

levels of monohydrated picotamide resp. anhydrous picotamide.

From a consideration of the results shown in the diagram, it is clear that anhydrous picotamide has a maximum anti-aggregation effect after 8 hours which is equal to 49%, while the maximum blood level is 19.75 y/ml which is shifted to the 6th hour.

In comparison, monohydrated picotamide shows a maximum platelet antiaggregation effect after 4 hours and further the maximum activity peak in time coincides with the maximum peak for blood levels and this shows a faster bioavailability in favor of monohydrated picotamide

relative to anhydrous picotamide.

The results of the pharmacological tests carried out with monohydrated picotamide are reproduced in the following table II where data for the same test with anhydrous picotamide are also listed.

A comparison of the test values shown in Table II for

the two molecules show that monohydrated picotamide has improved pharmacological effects compared to anhydrous picotamide. Such an unexpected improvement can be attributed to the improved bioavailability of the new compound in its monohydrate crystal form with stable structure.

Therapeutic applications

Monohydrated picotamide can due to its low toxicity,

high tolerability and absence of adverse side effects,

used in human therapy for the treatment of various thromboembolic disorders, especially cerebrovascular disorders, myocardial infarction, arterial and phlebo-thrombosis, pulmonary embolism, general arteriosclerotic conditions, general cardio-surgery..

For the aforementioned ailments, different pharmaceutical forms can be used containing from 10-500 mg of active agent, e.g. as follows: a) Oral: capsules, tablets, pills containing 10-500 mg, for a daily dose of 50-3000 mg/day. b) Parenteral: sterilized intravenous injectable ampoules, containing 10-50 mg, for a daily

dose of 10-200 mg/day.

The compounds can also be administered rectally in the form of suppositories.

The pharmaceutical preparations may contain common pharmaceutically tolerable carriers and additives and the delivery forms for the monohydrated picotamide and the respective dosage patterns may vary in accordance with clinical conditions and the doctor's judgement.

Claims (1)

Process for the production of monohydrated N,N'-bis-(3-picolyl)-4-methoxy-isophthalamine (picotamide) in crystalline form with formula where 4-methoxy-isophthaloyl dichloride is reacted with 3-picolylamine in tetrahydrofuran in the presence of triethylamine as proton acceptor, characterized by) that anhydrous tetrahydrofuran is used, b) that the reaction product is precipitated with water, c) that the reaction product from step b is crystallized from a solution of acetone/water (volume ratio 6:11) and d) that the product from step c is recrystallized from water.