IE51218B1 - Isophthalic acid picolylamide monohydrate,process for the preparation and pharmaceutical use thereof - Google Patents

Abstract

N,N'-bis-(3-picolyl)-4-methoxy- isophthalamide provided in crystalline form as its monohydrate, is a stable chemical structure and shows a high pharmaceutical activity for the treatment of thromboembolic disorders.

Description

This invention relates to an isophthalic acid picolylamide in the monohydrate crystal form, having pharmacological activity in inhibiting aggregation of blood platelets, combating thromboembolic disorders of the blood and delaying blood clotting. The invention also relates to a process for the production of the said new crystalline form.

In particular, the present invention provides N,Ν'-bis(3-picolyl)-4-methoxyisophthalamide monohydrate, a process for the preparation thereof and pharmaceutical compositions containing the said compound.

It is well known that N,N'-bis-(3-picolyl)-4methoxyisophthalamide, hereinafter referred to by its international approved name of picotamide, is a compound having a high fibrinolytic and anticoagulant activity (see French Patent 2100850; Chemie Therapeutique, 6, 203-7, 1971), as well as a good platelet antiaggregant activity (see U.S. Patent 3973026; Age and Ageing, 7, 246, 1978).

Picotamide as described in the above mentioned publications and patents is known in the anhydrous form and the melting point thereof, as described in French Patent 2100850 is 124°C at the Kofler bench.

According to the process described in the above mentioned patents, the compound is purified from the raw product, which contains a lot of impurities, by crystallization from anhydrous and apolar organic solvents Crystallization of the raw picotamide as obtained by the synthesis reaction, from anhydrous and apolar organic solvents, such as benzene, results in a powder-form product showing under the microscope the features of a fibrous substance, such as a voluminous down. The powder easily takes up an electrostatic charge and is relatively unstable. These characteristics are particularly detrimental when the compound is to be formulated for pharmaceutical use. The electrically charged particles repel each other and are likely to form dust when weighed and introduced into the apparatus for making up the various pharmaceutical preparations, which results in a variation of the weight of the active ingredient, making it more difficult to standardise the amount of active ingredient in the pharmaceutical composition.

It has now been found that by reacting a functional derivative of 4-methoxy-isophthalic acid with 3-picolylamine, and crystallizing the raw product from an aqueous solution, monohydrated Ν,Ν'-bis-(3picolyl)-4-methoxy-isophthalamide can be obtained, which has characteristics of chemical-physical structure and stability which make it definitely preferable to the known anhydrous picotamide described in the prior art.

It has been additionally surprisingly found that the said monohydrated picotamide thus obtained is more active and effective than anhydrous picotamide, both from the pharmacodynamic point of view and in terms of clinical pharmacology.

The present invention thus provides Ν,Ν'-bis5 (3-picolyl)-4-methoxy-isophthalamide monohydrate, C21H20N4^3^2°' m°Tecular weight 394.4.

The conventional chemical formula of the said monohydrate can be indicated as follows: The monohydrated picotamide according to the 15 present invention is a white crystalline power, which is bitter in taste, has no smell, is stable in air, can be easily crystallized from water and has a melting point of 95-97°C at the Kofler bench.

For sake of simplicity, in the following 20 description the compound as above defined will be referred to by the general name picotamide monohydrate The compound according to the invention can be differentiated from the physical-chemical point of view, from the previously well known anhydrous picotamide by an unforeseeable and definite improvement in its stability. This improvement is due to the fact that the molecule of water of hydration forms a part of the molecular structure of the compound according to the invention, being placed in the crystal lattice in a well defined position in which the oxygen atom of the water forms easily identifiable hydrogen bonds with particular atoms from different molecules of picotamide, as will be shown hereinafter, so as to build a single crystal of the compound having well defined characteristics. These characteristics, in an unforeseeable way, affect the pharmacological behaviour of the new compound and its bioavailability in mammals, with a more rapid absorption when administered to such animals, including man.

Picotamide monohydrate in the new crystalline form-according to the invention, not only avoids in a surprising way the above mentioned disadvantages of anhydrous picotamide, but in a much more surprising way, it provides a compound which is more active and effective in terms of pharmacological utility, in view of the advantageous effects observed on administration to animal and human organisms.

Although a wellgrounded theoretical explanation of said experimental results is not available at the present time, it can only be assumed that the crystalline compound according to the invention follows a different mechanism on dissolution in water to the well known anhydrous compound in the amorphous form.

The present invention also provides a process for producing the new picotamide monohydrate.

Furthermore the invention provides pharmaceutical compositions containing the new picotamide monohydrate, in various acceptable pharmaceutical forms, as an active agent for the clinical treatment of thromboembolic disorders of the blood.

As previously stated, the melting point of 10 picotamide monohydrate is 95-97°C.

It is pointed out, in this connection, that anhydrous picotamide has in contrast a melting point of 124 °C. This difference in the melting points of said two compounds is already an indication of a different molecular structure, which suggests a substantial difference between anhydrous pictoamide and picotamide monohydrate.

The invention will now be described in greater detail with reference to the accompanying drawings, in which: Figure 1 is a representation of the molecule of picotamide monohydrate as derived from an X-ray spectrum; Figure 2 is a representation of a single crystal of picotamide monohydrate; Figure 3 shows in greater detail the same single crystal shown in Figure 2; and Figure 4 is a graph which shows a direct comparison of the platelet antiaggregant activity of picotamide monohydrate and anhydrous picotamide.

The structure of a crystal of picotamide 5 monohydrate can be characterized, besides the physical and chemical analysis, by the X-ray spectrum of its monocrystal.

The data from the.Xrroy diffraction spectrum, which is evidence the spatial positions of centres of the atoms in the molecule, taken as a whole as a dense, stable and non-hygroscopic crystal, are listed in the following Table I, in which the various atoms of the molecule are indicated by their chemical symbol followed by an identification number. The spatial placement and the identification number of the atoms can be seen in Figure 1, which represents the geometrical position of the atom centres, in three dimensions as obtained from the Table I.

TABLE I Spatial Coordinates of Picotamide Monohydrate (X-Rays) Maximum = 92.52 Minimum = -86.27 Multiplied by 167.8829 Projection Atcm Number VA Y/B Z/C S.O.F. Mole- cule Ele- vation 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 BONDS (INCLUDING SYMMETRICALLY RELATED 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 It can be observed in particular that atom No. 17, indicated as 027 represents the water of hydration, while atom No. 8 indicated as 09 represents the oxygen of the methoxy group, atom No. 15 indicated as N19 is the nitrogen of the amide group and atom No. 21 indicated as N224 is the nitrogen atom of the pyridine group. 1218 In Toble I symbols X/A, Y/B and Z/C represent the spatial coordinates of the various atoms. These are defined taking into account the axis length with respect to the cell size. S.O.F. represents the spatial occupation factor of each atom. The fact that this is always equal to 1 means that the specific space is entirely occupied by the atom referred to. Molecule refers either to the compound (1) of water (0).

Elevation is the distance of the various atoms from the zero plane which is defined by the h^O oxygen atom.

As can be observed from Figure 1, which shows the structure of picotamide monohydrate, the oxygen atom of the water of hydration is placed in the crystal lattice in a well defined position with respect to the picotamide molecule.

From Figure 2 it can ba observed that the oxygen of the water of hydration inside the monocrystal represented by a rectangle is linked by hydrogen bonds (indicated by a dashed line) to well defined atoms of different picotamide molecules, which molecules are positioned in an ordered three dimensional pattern and are linked together properly by the hydrogen bonds with the oxygen of the water of hydration.

This linkage is shown in greater detail in Figure 3, from which it can be clearly seen that oxygen 027 of the water of hydration is linked by hydrogen bonds respectively to: 1) the oxygen of =CO of the methoxy group (0g) of a first picotamide molecule (bond length: 2.81 A); 2) the nitrogen of the amide group =NH (Nlg) of a second picotamide molecule placed in the same plane as the above mentioned molecule (length of the bond: 2.96 A); 3) the nitrogen of the pyridine ring (N224) a third picotamide molecule positioned on the underlying or overlying plane with respect to the other two o bonded molecules (bond length: 2.80 A).

The presence of these three bonds gives an explanation both of the strength with which the water of crystallization is inserted between different picotamide molecules forming the crystal, and the compactness of the crystal itself.

This compactness, provided by the molecule of water of crystallization, is considered to be the reason for the improvement in bioavailability of the monohydrate form as compared to the previously known anhydrous form, which improvement will be demonstrated hereinafter, from the pharmacological point of view, by a comparison of absorption time, blood levels and activities of both drugs.

In fact, on the basis of the knowledge available from the prior art relating to anhydrous picotamide, the improved effect obtained by inserting a molecule Of water of crystallization on the compactness of the spatial structure of picotamide as well as the improvement in the bioavailability was quite unpredictable.

The elemental chemical analysis has also provided results where are in agreement with the structure illustrated above.

For €2^20^03 ·Η2θ weight 394 .4) it has been found: C% 63.87 (theory 63.94); H% 5.72 (theory 5.62); N% 14.18 (theory 14.20) and oxygen by difference.

The process for the production of the new picotamide monohydrate is as follows. As far as the synthesis of the compound picotamide is concerned, this is the already known process for the production of anhydrous picotamide.

However, when raw picotamide has been obtained, it has been found surprisingly that by recrystallizing the raw product from an aqueous solution, in contr.ast to a solution not containing water as in the already known process, the crystalline monohydrate product as previously defined is obtained.

The following examples illustrates the process for the synthesis of picotamide monohydrate, starting from 4-methoxy-isophthaloyl dichloride (or another functional derivative of 4-methoxy-isophthalic acid) in the presence 100 g (0.43 mols) 100 g (0.43 mols) 130 g (1.2 mols) 120 ml 120 + 200 ml of proton acceptors.

Preparation Example 4-methoxy-isophthaloyl dichloride (molecular weight 233) 3-picolylamine (molecular weight 108, Triethylamine Tetrahydrofuran (anhydrous) 3- picolylamine, triethylamine and 120 ml of anhydrous tetrahydrofuran are introduced in a 3 litre flask provided (O with reflux condenser, dropping funnel and mechanical stirrer. 4- methoxy-isophthaloyl dichloride is separately dissolved in 200 ml anhydrous tetrahydrofuran..

This solution is slowly introduced through the dropping IS funnel' into the reaction mixture contained in the flask, under stirring. The addition has to be effected in one and a half to two hours, carrying out the following exothermic reaction: COCl OCH, 'COCl h2n-ch2c°-“TQ “0 i-Stf A N y^CO-NH OCH II III 2.0 After said dichloride addition, the reaction mixture is refluxed for about 2 hours, slowly diluted with water to 2 litres, and maintained under stirring until separation of a crystalline slurry which consists of the raw picotamide. This slurry is recovered on a suction filter and crystallized when wet from 700-800 ml of acetone-water mixture (6 volumes acetone + 11 volumes water).

The product as obtained is recrystallized from water, thus providing picotamide monohydrate, melting point 95-97°C at the Kofler bench.

From the above process according to the invention is characterized by the fact that the recrystallization of raw picotamide, as obtained from the synthesis reaction, is carried out from an aqueous solvent, in contrast to the prior art wherein anhydrous and apolar organic solvents were used, such as benzene, which provide an anhydrous product.

Pharmacological tests It has been found that picotamide monohydrate shows a high activity as a platelet antiaggregant and as a fibrinolytic agent. It therefore has utility in application to clinical pharmacology and human therapy.

These activities have been tested in vivo through spectrophotometric determination according to Born, for platelet antiaggragant activity and through the test of whole blood clot lysis according to Fearnley, for fibrinolytic activity.

EXAMPLE 1 Platelet antiaggregant activity in vivo on rabbits.

New Zealand rabbits which had been held fasting for 12 hours with water ad libitum were anaesthetized 5 with a 20% ethanolic solution of urethane at a dose 0.6 ml/100 g intraperitoneally. The blood was withdrawn from the carotidal artery, before (control) and one and a half hour after an intraperitoneal injection of monohydrated picotamide, at a dose of 25-50-100 mg/kg. The blood samples were rendered incoagulable by a 3.8% solution of sodium citrate, in the volume ratio of 9/1 and thereupon centrifuged at 1000 rpm for 15 minutes, to obtain a platelet rich plasma (PRP). A portion of the said plasma was thereupon centrifuged at 8000 rpm for 10 minutes, to obtain a platelet lean plasma (PPP).

PPP was used for zeroing a Born aggregometer and ml of PRP was placed into the basin of the measuring apparatus. Platelet aggregation was produced with variable concentrations of disodium adenosine diphosphate (ADP), depending of the platelet reactivity.

The platelet antiaggregant activity is calculated at 50% inhibition of the aggregation curve after treatment, referred to that of control (Ιϋ^θ)· The results are given hereinafter.

EXAMPLE 2 Effect on the platelet aggregation time and blood levels in the dog.

Picotamide monohydrate at a dose of 100 mg/kg was administered orally to Beagle dogs, tf , kept unfed for 18 hours with water ad libitum.

Blood was taken before (control) and 2-4-6-8-10 I hours after treatment, and the platelet antiaggregant activity was determined as a function of time (by the method referred to above), and in addition the blood levels of the drug were determined.

Determination of the blood levels was effected by a UV spectrophotometer, according to the following method. ml of .plasma, obtained by centrifugation at 100 rpm for 10 minites, were added with 2 ml of concentrated HCl and hydrolysed on a water bath at 100°C for 1 hour.

The sample was cooled, 2 ml of HjO was added and the sample was filtered. The filtrate was made strongly alkaline with NH.OH and extracted with 30 ml of CHC1,. The 4 i chloroform layer, dried on anhydrous Na2SO4» was extracted by H2SO4 O.l N, the acidic layer thus obtained was made up with h2SO4 °·1 n t0 100 and a spectrophotometer reading was taken at 228 nm.

The results are given hereinafter.

EXAMPLE 3 Fibrinolytic activity in vivo in the Guinea pig.

The fibrinolytic activity was determined on Italian Guinea pigs, and picotamide monohydrate was administered orally at a dose of 100 mg/kg.

The Fearnley method, modified as follows, was employed for this test: 0.1 ml of a thrombine solution at 50 NIH/ml were charged to tubes containing 1.7 ml of phosphate buffer (pH 7.4) maintained at O’C. After.addition of 0.2 ml of Guinea pig whole blood, the tubes were maintained at O’C for 30 minutes to allow clotting, then kept on a water bath at 37°C for 30 minutes, in order to produce a lysis.

The clot weight was then determined.

The fibrinolytic activity has been calculated as percent decrease of clot weight of the treated animals, with respect to the clot weight of controls.

EXAMPLE 4 Platelet antiaggregant and fibrinolytic activity on human volunteers.

Confirmation of both activities in vivo for picotamide was obtained on healthy human volunteers of both sexes, ranging in age from 35 to 65 years, who were treated with a single dose of 12 mg/kg orally.

The inhibition effect on platelet aggregation was tested by using disodium ADP as an antagonist following the same method as in Example 1.

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

The results obtained are given hereinafter.

Results From the results obtained in the test of the platelet antiaggregant activity in vivo in the rabbit (Example 1) by probit analysis the dose capable Of inhibiting the platelet aggregation by 50% (ID^q) was calculated, and this dose was 54.10+1.43 mg/kg.

A maximum effect (53.82%) in the dog (Example 2) was found at the fourth hour. The corresponding blood level was 22.6 γ/ml of plasma which corresponds to a maximal value, as shown in the diagram of Figure 4.

The fibrinolytic activity in the Guinea pig (Example 3) tested at a dose of 100 mg/kg orally was found to be equal to 27.83%.

In man (Example 4, at a single dose of 12 mg/kg orally, the maximum platelet antiaggregant effect was shown four hours after treatment and it was measured as 81.4%; The maximim fibrinolytic activity was measured as 54.2% decrease of the lysis time of euglobins.

Comparison comments By comparing the activity and toxicity results for picotamide monohydrate with those for anhydrous picotamide, which are described in U.S. Patent 3973026, it can be seen that substantial differences in activity exist and these differences are in favour of the monohydrate, this being quite unpredictable as the result which would be obtained simply by the introduction of the molecule of water of crystallization which characterizes the new form.

For purposes of comparison a test on the blood level in the dog, as a function of time following oral administration, has been carried out under the same experimental conditions, for the already known anhydrous picotamide in order to test the bioavailability thereof after administration.

The results of this test are referred to in the graph of Figure 4, which represents the effect over a period of time on platelet aggregation and on blood levels in the dog at a dose of 100 mg/kg per os.

The ordinate axis represents the time in hours and the abscissa axis represents the blood levels measured in γ/ml of plasma, as well as the platelet antiaggregant activity measured in percent. The lines 1 and 1' represent the antiaggregant activity of picotamide monohydrate and anhydrous picotamide respectively and the continuous lines 2 and 2‘ represent the blood levels of picotamide monohydrate and anhydrous picotamide respectively.

From a consideration of the results shown on the Figure, it appears clearly that anhydrous picotamide has a maximum platelet antiaggregant effect at the 8th hour which is equal to 49%, while the maximum blood level is 19.75 γ/ml, which is offset to the 6th hour.

By comparison, picotamide monohydrate shows its maximum platelet antiaggregant effect at the 4th hour and moreover the maximum activity peak is coincident in time with the maximum peak of blood level, which is evidence of a more rapid bioavailability in favour of picotamide monohydrate as compared to anhydrous picotamide.

The results of the pharmacological tests carried out on picotamide monohydrate are listed in the following Table II, where the data of the same test for anhydrous picotamide are displayed as well.

TABLE II Test Parameter Picotamide monohydrate . Anhydrous piootanide Platelet aggregation in vivo (rabbit) intraperitoneally ID50.mg/kg 54.1 108.2 Platelet aggregation in vivo (dog) 100 mg/kg per os maxinum inhibition % of aggregation 53.83 48.12 maxinum activity time, found 4th hour 8th hour maxinum blood level γ/ml in plasma 22.6 19.75 maxinum blood level tine, found 4th hour 6th hour Platelet aggregation in man after single administration maximum inhibition I of aggregation 81.4 70.11 maxinun activity time, found 4th hour 8th hour Acute toxicity in rat and dog, per os DL,.0.mg/kg 3,000 3,000 A comparison of the test values shown in Table II for the two molecules, shows that picotamide monohydrate has improved pharmacological effects with respect to anhydrous picotamide. Such an unexpected improvement appears to be due to the improved bioavailability of the drug in the new form of its crystalline monohydrate of stable structure.

Therapeutic use Picotamide monohydrate, in view of its low toxicity, high tolerance level and absence of unfavourable side effects, can be useful in human therapy for the treatment of various thromoembolic disorders, particularly cerebrovascular disorders, myocardial infarction, artery and flebo thrombosis, pulmonary embolism, general arteriosclerotic conditions, general cardio-surgery.

For these indications, various pharmaceutical forms can be employed, containing from 10 to 500 mg of active ingredient, examples of which can be mentioned as follows: a) oral administration: capsules, tablets, pills containing 10-500 mg, for a total daily dosage of 50-3000 mg/day; b) parenteral administration : sterilized endovenous injectable vials, containing 10-50 mg, for a total 5 daily dosage of 10-200 mg/day.

The active ingredient can be also administered rectally, in the form of a suppository.

The pharmaceutical compositions may also contain, in addition to the active ingredient, conventional 10 pharmaceutically acceptable vehicles and adjuvants, as is well known in the pharmaceutical field. It is also obvious that the form in which picotamide monohydrate is administered and the respective dosage patterns can be varied according to the clinical circumstances and experience of the physician.

Claims (14)

CLAIMS:

1. Ν,Ν'-bis-(3-picolyl)-4-methoxy-isophthalamide monohydrate in crystalline form of formula: 2. 6. A pharmaceutical composition as claimed in Claim 2 or 3.

2. A process for the production of N,N'-bis-(

3. -picolyl)4-methoxy-isophthalamide monohydrate which comprises

4. N,N’-bis-(3-picolyl)-4-methoxy-isophthalamide 2q monohydrate when produced by a process as claimed in Claim 5. Accompanying Preparation Example. 5, in unit dosage form each unit dose containing 10 to 500 mg of the active ingredient.

5. A pharmaceutical composition containing, as active ingredient, N-N'-bis-(3-picolyl)-isophthalamide monohydrate in crystalline form, together with at least one pharmaceutically acceptable carrier or vehicle. 5 having a melting point of 95-97’C at the Kofler bench and showing an X-ray diffraction spectrum of its monocrystal, as set out in Table I of the specification.

6. , adapted for oral administration, each unit dose Ιθ containing 10 to 500 mg of the active ingredient.

7. A pharmaceutical composition as claimed in Claim

8. A pharmaceutical composition as claimed in Claim 6, adapted for parenteral administration, each unit dose containing 10 to 50 mg of the active agent.

9. N,N 1 -bis-(3-picolyl)-4-methoxy-isophthalamide 15 monohydrate in crystalline form.

10. N,N'-bis-(picolyl)-4-methoxy-isophthalamide monohydrate in crystalline form for use in the treatment of thromboembolic disorders in mammals. 10 reacting a functional derivative of 4-methoxy-isophthalic acid with 3-picolylamine in an anhydrous organic solvent, precipitating the reaction product with water and recrystallizing the raw product from an aqueous solution and then from water. 15 3. A process as claimed in Claim 2, wherein after precipitation of the reaction product with water, the product is crystallized from a solution of acetone/water and recrystallized from water.

11. A pharmaceutical composition according to any one 2o of claims 5-8 for use in the treatment of thromboembolic disorders in mammals.

12. A process according to Claim 2 or Claim 3 for the production of N,Ν'-bis-(3-picolyl)-4-methoxyisophthalamide monohydrate, substantially as hereinbefore described with particular reference to the

13. N ,N'-bis-(3-picolyl)-4-methoxy-isophthalamide monohydrate whenever produced by a process claimed in a preceding claim.

14. A pharmaceutical composition according to any 10 one of claims 5-8 and 11; substantially as hereinbefore described.