CA1129419A - Monohydrated isophthalic acid picolylamide, process of preparation and pharmaceutical use thereof - Google Patents

Abstract

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

Description

~L12941~

This invention relates to a new organic molecule in the monohydrate crystal form, having a pharmaceutical activity in contrasting the aggregation of blood platelets, opposing thromboembolic disorders in blood and delaying the blood clotting. The invention relates also to a process by which it is possible to carry out a synthesis of said new crystalline compound.

In particular, the present invention relates to mono-hydratedN,N'-bis(3-picolyl)-4-methoxyisophthalamide, a process for the preparation thereof and pharmaceutical compositions comprising said compound.

It is well known that N,N'-bis-(3-picolyl)-4-methoxy-isophthalamide, hereinafter indicated by its international usual denomination of "picotamide", is a compound having a high fibrinolytic and anticoagulating activity (see French patent 2100850; Chimie Thérapeutique, 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 2l00850 is 124C at the Kofler bench.

This compound, according to the process describecl in the above mentioned patents, 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 product showing under a microscope the features of a fibrous substance, such as voluminous dawn. Said powder easily takes up an electrostatic charge and is relatively ~1;29419 unstable. Th~se characteristics are particularly detrimental when the compound is prepared for pharma-ceutical use. The electrized particles repulse each other and are likely to volatile when weighed and S introduced into the apparatus for making up the various pharmaceutical preparations, which results in a variation of the particle weight, making the operations of titre stabilization more difficult.

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

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

An object of the present invention is therefore mono-hydratedN,N'-bis-(3-picolyl)-4-metl-oxy-isophtllalamide C21~l20N43-l-l2J molecolar weight 394,4, The conventional chemical formulcl can l)e indicated as follows:

~` ~ C0-N ~ 2 .~ .

~1299~19 The monohydrated picot~mideaccording to the present inven-tion is a white, inodore, bitter in taste, crystalline powder, which is stable in air and easily crystallizable from water, having a melting point of 95-97C at the Kofler bench.

For sake of simplicity, in the following description the compound as above defined will be indicated by the general name "monohydratedpicotamide".
The compound according to the invention differentiates, from the physical-chemical point of view, from the previously well known compound by an unforeseeable and definite improvement in its stability. Said improvement is due to the fact that the molecule of hydration water partakes in the molecular structure of the compound according to the invention, it being placed in the crystal lattice in a well defined position, in which the oxygen atom of the hydration water est'ablishes well identifyable hydrogen bonds with particular atoms belonging to different molecules of picotamide, as it will be shown hereinafter, so as to build a single crystal of the compound having well defined characteristics. Said characteristics, in an unforeseeable way affect the pharmaceutical behavior of the new compound and its biodisposability in mammal organisms, with a more rapid absorption when administered to said class of animals, wherein man is included.

Monohydratedpicotamide in the new crystalline form according to the invention, not only avoids in a surprising way the above mentioned drawbacks of anhydrous picotamide, but in a much more surprising way, it provides a compound more active and efficaceous in terms of pharmacological utility, in view of the advantageous effects observed in the administration to animal and human organisms.

l~Z~lg Although a wellgrounded theoretical explanation of said experimental result is not available at this moment, one can only assume that a solution in water of the crystalline compound according to the invention fol-lows a different mechanism with respect to a solutionin water of the well known anhydrous compound in the amorph form.

An additional object of the present invention is a process for producing the new hydrate molecule.

It is a further object of the present invention to provide pharmaceutical compositions containing the new monohydrate molecule, in the various acceptable pharma-ceutical forms, as an active agent for the clinicaltreatment of thromboembolic disorders of blood.

As previously stated, the melting point of monohydrated picotamide is 95-97C.
It is pointed out, in this connection, that anhydrous picotamide has in contrast a melting point of 124C.
This difference o the melting points of said two compounds is already an indication of a different molecular structure, which means a substantial difference between anhydrous picotamide and monohydratedpicotamide.

The invention will be described in greater ~etail in the following specification, with reference to the accompanying drawings, wherein:

Fig. l is a tridimensional representation of the molecule of monohydratedpicotamide, as obtained from an X-ray spectrum;
Fig. 2 is a tridimensional representation of the mono-crystal of monohydratedpicotamide;

llZ94~9 Fig. 3 shows in greater detail the same monocrystal shown in Fig. 2; and Fig. 4 is a diagram which shows a direct comparison of the platelet antiaggregant activity of monohydrated picotamide and anhydrous picotamide.

The structure of a crystal of monohydrate picotamide is characterized, besides the physical and chemical analysis, also by the X-ray s~ectrum of its monocrystal.

The data of the X-ray diffraction spectrum,which evidence the space position of the atom centers in the new molecule, taken in it.s entirety as a dense, stable and not hygroscopic crystal, are listed in the following Table I, wherein the various ato~s of the molecule are indicated by their chemical symbol fol-lowed by an identification number. The spacial place-ment and the identification number of said atoms can be observed in figure 1, which represents the geometrical tridimensional position of the atom centers, as obtained from Table I.

~1~9~19 TABLE I

SPACIAL COORDINATES OF MONOHYDRATED PICOTAMIDE (X-RAYS) MAXIMUM = 92.52 MINIMUM = -86.27 MULTIPLIED BY 167.8829 PROJECTION ATOM HEIGHT X/A Y/B Z/C S.O.F. MOLE- ELE-NUMBER CULE 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 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 C10 .2795 .6035.4219 1.0000 1 .35 Nll .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 ClS .3600 .4594-.0529 1.0000 1 .11 Nl9 .3326 .5077.1101 1.0000 1 .21 16 C22 .1082 .39961.0097 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 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 .36831.0498 1.0000 1 2.96 24 C229 .4606 .3864-.0822 1.0000 1 1.28 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 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 RELAT~D ATOMS) 1- 2 1.443- 5 1.404- 5 1.39 3- 6 1.50 6-11 1.224-12 1.518-12 1.2310-12 1.35 514-15 1.4910-16 1.4814-18 1.5420-21 1.37 18-24 1.4021-24 1.3919-25 1.3322-26 1.38 20-29 1.3627-29 1.3926-30 1.2120-31 1.08 18-33 1.899-34 1.31 It can be observed in particular that atom No. 17, indicated as 027 represents the hydratian wa1,er, while atom No. 8 indicated as 09 represents an oxygen of the methoxy group, atom No. lS indicated as Nl9 is a nitro-gen of the amide group and atom No. 21 indic~ated as N224 lS is a nitrogen atom of the pyridine group.

In Table I symbols X/A, Y/B and Z/C represent the spacial coordinates of the various atoms.

As it can be observed in Fig. l, which shows the structure of monohydrated picotamide, the oxygen atom of the hydration water is placed in the crystal lattice in a well defined position with respect to the picotamide molecule.
In Fig. 2 it can be observed that the oxygen of the hydration water inside the monocrystal represented by a rectangle is linked by hydrogen bonds (in~icated by a dashed line) to well determined atoms of different picotamide molecules, which molecules are positioned in an ordered tridimensional pattern and are linked together properly by said hydrogen bonds with the hydration oxygen.

Said linkage is shown in greater detail in Fig. 3, where it is clearly seen that oxygen 027 of the hydration water is linked by hydrogen bonds respectively to:

1~94~9 g 1) the oxygen of =C0 of the methoxy group (09) of a first picotamide molecule (bond length: 2.81 A);

2) the nitrogen of the amide group =N~I (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 (~224) of a third picotamide molecule positioned on the under-lying or overlying plane with respect to the other two bonded molecules (bond length: 2.80 A).

The presence of said three bonds gives an explanation both of the strength with which the crystallization water is inserted between different picotamide molecules forming the crystal, and the compactness of the crystal itself. Said compactness, provided by the molecule of crystallization water, is conside,red to be the reason of the improvement in biodisposability of the mono-hydrate form with respect to the previously known anhydrous form, which improvement will be evidenced hereinafter, from the pharmacological point of view, by a comparison of absorption time, blood level value and activity of both drugs.

In fact, on the ground of knowledge available from the prior art on the anhydrous picotamide molecule, an imporved effect, due to inserting a crystallization water molecule, on the compactness of the spacial structure of picotamide as well as an improvement in noticeable terms of the biodisposability was not obviously foreseeable.
The elemental centesimal chemical analysis has provided also results which are in agreement with the above illustrated structure.

llZ99~19 For C21HzoN403.l-l20 (mole 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.

S The process for the production of the new monohydrate picotamide molecule follows, as far as the synthesis of the compound is concerned, 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 mixture, in contrast to an anhydrous mixture as in the already known process, the monohydrate crystalline product as previously defined lS is obtained.

The following examples will illustrate the process for obtaining by synthesis the monohydrated picotamide, starting from 4-methoxy-isophthal,oyl dichloride, or other functional derivative of 4-methoxy-isophthalic acid, in an environment of proton acceptors.

Example of preparation 4-methoxy-isophthaloyl dichloride 100 g (0.43 mols) ~mole weight 233) 100 g (0.43 mols) 3-picolylamine (mole weight 108) 130 g (1.2 mols) Triethyl-amine 120 ml Tetrahydrofuran (anhydrous) 120 + 200 ml 3-picolylamine, triethylamine and 120 ml of anhydrous tetrahydrofuran are introduced in a 3 litre flask provided with reflux cooler, dropping funnel and mechanical stirrer.
4-methoxy-isophthaloyl dichloride is separately dis-solved in 200 ml anhydrous tetrahydrofuran.

llZ9~1~

This solution is slowly introduced through the dropping 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 hexothermic reaction:

COCl CO-NH-10 ~ ~COCl ~ ~ CO ~H

I II III
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.
_ Said slurry is recovered on a suction filter and 20 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, so providing monohydrated picotamide, melting point 95-97C at the Kofler bench.

From the above the 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 by an aqueous solvent, in contrast to tlle prior art wherein anhydrous and apolar organic solvents were used, such as benzene, which provide an anhydrous product.

li2g~19 Pharmacological tests It has been found that monohydrated picotamide shows a high activity as platelet antiaggregant and fibrino-lytic agent. It has therefore a utility in applicationsto clinical pharmacology and human therapy.

Said activities have been tested in vivo through spec~ro-photometric determination according to Born, for the platelet antiaggregant activity and through the test of blood clot lysis in toto according to Fearnley, for the 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 with a 20~ ethylic solution of urethane at a dose 0.6 ml/lO0 g intraperitoneally. The blood was withdrawn from the carotidal artery, before (control) and one and a half hour after the intraperitoneal injection of monohydrated picotamide, at a dose of 2S-50-lO0 mg/kg. The blood samples were made incoagulable by a 3.8~ solution of sodium citrate, in the volume ratio of 9/1 and there-upon centrifuged at 1000 rpm for 15 minu~cs, for obtaining a platelet rich plasma (PRP). A portion of said plasma was thereupon centrifuged at 8000 rpm for lO minutes, for obtaining a platelet lean plasma (PPP) -PPP was used for zeroing a Born aggregometer and l mlof PRP was placed into the basin of the measuring apparatus. A platelet aggregation was produced by variable concentrations of disodium adenosine diphosphate (ADP), depending of the platelet reactivity.
;

llZ9419 The platelet antiaggregant activity has been calculated as 500~ inhibition of the aggregation curve after treatment, referred to that of control ~ID50).

The results are referred hereinafter.

Example 2 Time effect on the platelet aggregation and blood levels in dog.

Monohydratedpicotamide at a dose of lO0 mg/kg was administered orally to Beagle dogs, ~ , kept unfed for 18 hours with water ad libitum.
Blood was taken before (control) and 2-4-6-8-lO hours after treatment, and, in function of time, the platelet antiaggregant activity (by the above mentioned method), as well,as the blood levels of the drug were determined.

A determination of the blood levels has been effected by a UV spectrophotometer, according to the following method.

5 ml of plasma, obtained by centrifugation at lO0 rpm Eor lO minutes, were added with 2 ml of concentrated HCl and hydrolized on a water bath at 100C for l hour.
It was cooled, taken with 2 ml of 1120 and filtered. The filtrate was strongly alkalinized by N1140H and extracted by 30 ml of CHCl3. The chloroform layer, dried on anhydrous Na2S04, was extracted by 1-12S04 O.l N. the acidic layer so obtained was added with H2S04 O.l N
to lO0 ml and the spectrophotometer was read at 228 nm.
The results are displayed hereinafter.

,~

l~Z99~9 Example 3 Fibrinolytic activity in vivo on Guinea pig.

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

The Fearnley method, modified as follows, was employed ~for this test:

In tubes maintained at 0C, 1.7 ml of phosphate buffer (pH 7.4) and 0.1 ml of a thrombine solution at 50 NIH/ml were charged. After an addition of 0.2 ml of Guinea pig blood in toto, the tubes were maintained at 0C for 3~
minutes to allow clotting, then kept on a water bath at 37C 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 Plate]et antiaggregant and fibrinolytic activity on human volunteers.

A confirmation of both activities in vivo for monohydrnted picotamide was obtained on healthy human volunteers of botll sexes, aging from 35 to 65 years, which were treated orally at a single dose of 12 mg/kg.

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

~lZ9~9 The fibrinolytic activity was assessed by determining the lysis time of euglobins.

The results obtained are listed hereinafter.

Results From the results obtained in the test of the platelet antiaggregant activity in vivo on rabbit (example 1) by probit analysis the dose capable of inhibiting the platelet aggregation by 50~ (ID50) was calculated, and this dose was 54.10+1.43 mg/kg.

A maximum effect (53.82~) in dog (example 2) was found at the fourth hour. The corresponding blood level was 22.6 y/ml of plasma and it corresponds to a maximal value, as shown in the diagram of Fig. 4.

The fibrinolytic activity in Gui~ea pig (example 3) tested at a dose of 100 mg/kg orally was found 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 maximum fibrinolytic activity was measured as 54.2 decrease of the lysis time of euglobins.

Comparison comments By comparing the activity and toxicity results for mono-hydrated picotamide with those for anhydrous picotamide, which are described in U.S. patent 3973026, it can be considered that substantial differences in activity exist and said differences are favorable to the mono-hydrate form, which was certainly unforeseeable on the ground of the simple introduction of a crystallization ;~ .

l~Z941g - 16 ~

water molecule characterizing the new compound.

For sake of comparison a test on the time effect and the blood levels in dog, orally, has been effected, under the same experimental conditions, for the already known anhydrous picotamide in order to test the bio-disposability thereof after administration.

The results of this test are referred to in the diagram of Fig. 4, which represents the time effect on platelet aggregation and on blood levels in 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 y/ml of plasma, as well as the platelet antiaggregant activity measured in percent. The lines 1 and 1' represent the antiaggregant activity of monohydrated picotamide andanhydrous picotamid,e respectively and the continuous lines 2 and 2' represent the blood levels of monohydrated picotamide and anhydrous picotamide respectively.

From a consideration of the results shown on the diagram, 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 y/ml, which is offset at the 6th hour.

In comparison, monohydrated picotamide shows the maximum platelet antiaggregant effect at the 4th hour and more-over the maximum activity peak is coincident in time with the maximum peak of blood leve~ which evidences a more rapid biodisposability in favor of monohydrated picotamide with respect to anhydrous picotamide.

llZ9~19 The results of the pharmacological tests carried out on monohydrated picotamide are listed in the following Table II, where the data of the same test for anhydrous picotamide are displayed as well.
TABLE II

Monohydrated Anhydrous Test Parameter Picotamide picotamide Platelet ag-gregation in vivo (rabbit) intraperiton-eally ID50.mg/kg 54.1 108.2 Platelet ag- maximum gregation in inhibition ~
vivo (dog) of aggregation 53.83 48.12 100 mg/kg per os maximum activity time, found 4th hour 8th hour maximum blood level ~/ml in plasma 22.6 19.75 maximum blood level time, found 4th hour 6th hour Platelet ag- maximum gregation in inhibition ~
man after of aggregation_ 81.4 70.11 single ad-ministration maximum activity time, found 4th hour 8th hour Acute toxicity in rat and dog, per os D~50.mg/kg >3,000 >3,000 A comparison of the test values shown in Table II
for the two molecules, shows that monohydrated picotamide has improved pharmacological effects with respect to anhydrous picotamide. Such an unexpected improvement ~Z~419 appears to be due to the improved biodisposability of the new drug in its form of monohydrate crystal of stable structure.
Therapeutical utilizations Monohydrated picotamide, in view of its low toxicity, high tollerability and absence of unfavorable side effects, can be useful in human therapy for treatment of various thromboembolic disorders, particularly cerebrovascular disorders, myocardial infarction, artery and flebo thrombosis, pulmonary embolism, general arteriosclerotic conditions, general cardio-surgery.

For said use, various pharmaceutical forms can be employedJ containing from 10 to 500 mg of active agent, examples of which can be mentioned as follows:

a) orally: capsules, tablets, pills containing 10-500 mg, for a daily dosage of 50-3000 mg/day;

b) parenterally: sterilized endovenous injectable vials, containing 10-50 mg, for a daily dosage of 10-200/day.
It can be also administered rectally, in the form of suppository.

The pharmaceutical compositions may obviously contain, besides tlle active agent, usual pharmaceutically acceptable vehicles and adiuvants, as is well known in the pharmaceutical field. It is also obvious that the administration forms of monohydrated picotamide and the respective dosage patterns can be varied according to the clinical circumstances and experience of physicians.

Claims (3)

Claims

1. A process for the production of monohydrated N,N'-bis-(3-picolyl)-4-methoxy-isophthalamide of formula having a melting point of 95-97°C at the Kofler bench and showing an X-ray diffraction spectrum of its mono-crystal, as referred to in Table I of the specification, wherein a functional derivative of 4-methoxy-isophthalic acid is reacted with 3-picolylamine, in a proton acceptor environment, in an anhydrous organic solvent, characterized in that the reaction product is precipitated by water and the raw product is recrystallized from an aqueous solution and successively from water.

2. A process according to claim 2, wherein, after the precipitation of the reaction product by water, said product is crystallized from a solution of acetone/water and recrystallized from water.

3. A compound of formula (I) as defined in claim 1 whenever obtained in a process as claimed in claim 1 or claim 2 or by an obvious chemical equivalent thereof.