NL8102016A - ISOPHALIC ACID PYCOLYLAMIDE MONOHYDRATE, PROCESS FOR ITS PREPARATION AND PHARMACEUTICAL USE. - Google Patents

Description

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Title: Isophthalic acid polyamide monohydrate, process for its initiation and pharmaceutical use.

The invention relates to a novel organic molecule in the monohydrate crystal form with a -Parmaceutical activity in inhibiting platelet aggregation, counteracting thromboembolic irregularities in the blood, feathet ". Slowing blood clotting. The invention also relates to a method by which said crystal 11 compound can be synthesized.

More particularly, the invention relates to N, t-bis (3-picolyl) -methylamoisophthalamide monohydrate, a process for its preparation, and to pharmaceutical preparations containing said compound.

N, U'-bis- (3-picolyl) -H-methoxyisophthalamide, hereinafter referred to by its international common name "picotamide", is known to be a compound having high fibrinolytic and anticoagulant activity (See French Pat. No. 2,100,850; Chimie Therapist Iue, 6, 203-7, 1971) as well as good platelet anti-aggregating activity. (see U.S. Patent 3,973,026; Age and Aging, 7, 2U6, 1978).

Picotamide as described in the above publications and patents is known in its anhydrous form and its melting point as described in French Patent 2,100,850 is 1.2k ° C with the Kofler bench.

This compound is purified from the crude product, which contains a large number of impurities, by crystallization from anhydrous and non-polar organic solvents in the above-described patent process.

Crystallization of the crude picotamide obtained by the synthesis reaction from anhydrous and non-polar organic solvents, such as benzene, results in a powdery product which, under a microscope, shows the properties of a "fibrous substance such as" voluminous dawn ". Said powder readily absorbs electrostatic charge and is relatively unstable. These properties are 81 0 2 0 1 6

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- 2 - without harming the compound, it is prepared for pharmaceutical use. The electrified particles repel each other and tend to volatilize when they are weighed and placed in the device to prepare the different ones; pharmaceutical formulations, resulting in a modification of the particulate matter making titer stabilization operations more difficult.

It has now been found that by reacting a functional derivative of--methoxyisophthalic acid with 3-picolylamine and allowing the crude product to crystallize from an aqueous solution of N,'T '-bis- (3-picolyl) - ^ methoxy-isophthalamide monohydrate is obtained which has such a chemical-physical structure and stability that it is clearly preferable to the known anhydrous picotamide.

Furthermore, it has also been surprisingly found that the thus obtained picotamide monohydrate is more active and efficient than vessel-free picotamide both from a pharmacodynamic point of view and in terms of clinical pharmacology.

The object of the invention is therefore N, N'-bis- (3-picolyl) -U- methoxy-isophthalamide (C.-H Hi 0_.Hn0 with molecular weight 39 ^ · ^ · 1ά go ^ j d

Its formula is stated at: the. formula sheet as formula 1.

The picotamide monohydrate of the present invention is a white, odorless bitter-tasting crystalline powder which is stable in air and easily crystallizable from water having a melting point of 95-97 ° C with the Kofler bank.

For the sake of brevity, the compound will hereinafter be referred to by the common name picotamide monohydrate.

The compound of the invention is physically chemically different from the hitherto well known compound by an unpredictable and marked improvement in stability. This improvement is due to the fact that the hydration water molecule is included in the molecular structure of the compound, in the crystal lattice in a well-defined place in which the oxygen atom of the hydration water belongs to clearly identifiable hydrogen bonds with 35 special atoms picotamidemoleouien :. establishes as will be shown in more detail, forming a single crystal 8102016 - 3 - 4 · of the bonded sheet properties. These properties do not predictably affect the pharmaceutical behavior of the new compound and its bioavailability in mammals with faster absorption when administered to said class of 5 animals, including humans.

The picotamide monohydrate in the novel crystalline form of the invention not only surprisingly avoids the above-mentioned drawbacks of vessel-free picotamide, but it provides even more surprisingly a more active and more effective compound in terms of pharmacological utility in view of the observed effects in - administration to animals and human organisms.

Although no well-founded theoretical explanation of said experimental result can be given so far, it can be assumed that an aqueous solution of the crystalline compound 15 according to the invention follows a different mechanism with respect to an aqueous solution of the known anhydrous compound in the amorphous shape.

A further object of the invention is a method of preparing the new hydrated molecule.

A torch is an object of the invention to provide pharmaceutical compositions containing the new monohydrate molecule in the various acceptable pharmaceutical forms, as an active agent for the clinical treatment of blood phosphomembolic disorders.

As previously mentioned, the melting point of the picotamide monohydrate is 95-91 ° 0.

In this connection, it is noted that, on the other hand, vat-free picotamide has a melting point of 12k ° C. This difference of the melting points of both said substances is already indicative of different molecular structure, indicating an essential difference between vessel-free picotamide and picotamide monohydrate.

The invention will now be described in more detail with reference to the drawing, in which: Figure 1 is a three-dimensional image of picotaraidemono hydrate molecule obtained from an X-ray spectrum; 8102016 -1 + - fig t fig * 2 is a three-dimensional image of the mono crystal of picotajnide monohydrate; fig. 3 in larger. detail shows the same mono crystal of fig. 2 and; Fig. U is a diagram showing a direct comparison of the platelet anti-aggregation activity of picotamide monohydrate and vessel-free picotamide.

The structure of a picotamide monohydrate crystal is not only characterized by the physical and chemical analysis, but also by the X-ray spectrum of its single crystal.

The X-ray diffraction spectrum data. showing the spatial position of the atomic centers in the new molecule as a whole as a dense stable and non-hygróscopic crystal are listed in Table A below, in which the different atoms of the molecule are indicated by a chemical symbol by an identification number. The spatial arrangement and identification number of said atoms can be seen in Fig. 1, which depicts the geometric tridimensional position of the atomic centers obtained from Table A.

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Spatial coordinates of picotamide monohydrate (X-rays) »- * - 5 -

TABLE A

Maximum = 92.52 Minimum = -86.27 Multiplied by 167.8829

Projection ^ '- *. Atom Height X / A X' / B Z / C S.O.F. Molecule number 1 08 .1895 .6286 .83861 1.0000 1 1.26 2 C38 .I96T .7283 .8919 1.00001 1 .27 3 ci .2670 .5071+ .3663 1.0000 1 .82

Ij. C3 .20 ^ 7 .1 + 937 .6312 1.0000 1 1.60 5 C5 .2299 .1 + 539 .1 + 720 1.0000 1 1.½ 6 c6 .29 ^ .1 + 562 .20U0 1.0000 1 .75 <7 0y. 2161+ .590 ^. .6832 1.0000 1 1.11 8 C9 .1627 .31 + 32 .6819 1.0000 1 2.75 9 C10 .2795 .6035 .1 + 219 1.0000 1 -35 10 H11 .1385 .1 + 61 + 2 .8821 + 1.0000 1 2.1 + 3 11 012 .281 + 7 t-3716 .1698 1.0000 1 1.24 12 C13 .1665 .1 + 277 «.73U7 1.0000 1 2.31 13 C1I4. .251 + 7 .61 + 1 + 2 .5815 1.0000 1 .51 1¾ C15 .3600 .1 + 59 ^ -.0529 1.0000 1 -11 15 H19 .3326 .5077 .1101 1.0000 1 .21 16 C22 .1082 .3996 1.0097 1.0000 1 3.28 17 027 · .3771 .7056 .11 + 61 1.0000 0 0.00 18 C221 .1 + 250 .1 + 199 .0573 1.0000 1 I.3I + • jo ΪΓ222 -.0616 .3513 .9680 1.0000 1 2.34 20 C223 .51 + 22 .3I + 76 .2266 1.0000 1 3-1 + 3 21 N22U .5202 .3510 .0056 1.0000 1 2.31+ 22 C227 .0381+ .3880 .9075 1 .0000 1 2.1 + 7 23 0228 .0001+ .3683 1.01 + 98 1.0000 1 2.96 2l + C229 .I + 606 .3861+ -.0822 1.0000 1 I.28 25 C231 -.0875 .357I + .7563 1.0000 1 1.23 26 C232 .0127 .3932 .6853 1.0000 1 1.33 2T C231 + .1 + 1 + 83 .1 + 11 + 6 .2819 1-0000 1 2.1 + 7 28 0235 -.0519 -3732 .60I + 2 1.0000 1 .72 29 021 + 1 + .5071 .3758 .3636 1.0000 1 3.53 30 Q 1 96 .0376 .3935 .5361 1.0000 1 .88 31 Q 2 92. .5361+ .3978 .31 + 76 1.0000 1 3-1 + 8 32 Q 3 81+. .2173 .381 + 7 .1 + 186 1.0000 1 1-70 33 Q 1+ 81+. .3392 .3922 -.0552 1.0000 1 .51 3 ^ q 5 83. .3201+ .6259 -3130 1.0000 1 0.00 8102016 - 6 -

Bonds (including symmetrically related atoms) t1_ 2 .1.IA 3- 5 .1.U0 V 5 1.39 3- 6 1.50 6-11 1.22 lf-12 1.51 8-12 1.23 10-12 l'.35 1U-15 1 .U9 10-16 1Λ8 1U-18 1.5U 20-21 1.31 5 18-2U 1.U0 21-2U 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 9-3 ^ 1.31

It can be observed in particular that atomic number 17 denoted as 027 represents the water of hydration, while atomic number 8 TQ denoted as 09 represents an oxygen of the methoxy group, atomic number 15 denoted as H19 is a nitrogen of an amide group and atomic number 21 denoted as N22 ^ is a nitrogen atom of the pyridine group.

In table A, the symbols X / A, Y / B and Z / C are the spatial coordinates of the different atoms.

As can be seen from Figure 1, which shows the structure of pxcotaoide monohydrate, the oxygen atom of the hydration water in the crystal lattice is placed in a well-defined place. relative to the picotamide molecule.

In Figure 2 it can be seen that the oxygen of the hydration water within the single crystal imaged by a rectangle through hydrogen bonds (indicated by a dotted line) is connected to well-defined atoms of different picotamide molecules, which are molecules in a regular tridimensional pattern are placed and well interconnected by said hydrogen bonds with the hydration oxygen.

Said bond is depicted in more detail in Fig. 3, in which it is clearly seen that the oxygen O27 of the hydration water with hydrogen bonds is respectively connected to: 1) the oxygen of .00. of the methoxy group (0 ^) of a first picotamide molecule (bond length: 2.81 A °); 2) the nitrogen of the amide group = NH of a second picotamide molecule placed in the same plane as the above-mentioned molecule (bond length 2.96 A °); 3) the nitrogen of the pyridine ring (N ^) of a third picotamide molecule placed on the underlying or overlying surface with respect to the other two bonded molecules (bond length 2.80 A °).

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The presence of said three bonds explains both the strength with which the water of crystallization is present between different picotamide molecules to form the crystal and the compactness of the crystal itself. This consistency, provided by the molecular crystallization water, is considered to be the reasons for the improvement in bioavailability of the monophydrate form with respect to the previously known vessel-free form, which improvement will be apparent from a pharmacological point of view by comparing absorption time, blood level value and activity of both 10 connections.

Indeed, based on the available knowledge of the prior art vessel-free picotamide molecule, an improved effect due to the insertion of a crystallized water molecule on the compactness of the spatial structure of the picotamide 15 as well as on the bioavailability was unforeseeable .

The basic chemical analysis has also provided results consistent with the above-illustrated structure.

Cg ^ HgQÏT ^ O ^. HgO (molecular weight 39 ^ Λ) has been found: 20 C% 63.37 (theory 63.9 * 0; H # 5.72 (theory 5.62); U # 1 ^ 18 (theory 1 ^, 20) and oxygen by difference).

The process for the preparation of the new picotamide monohydrate molecule follows, insofar as the synthesis of the compound is concerned, the already known process for the preparation of anhydrous picotamide.

However, after obtaining the crude picotamide by the method, it was found that by recrystallizing the crude product from an aqueous mixture as opposed to an anhydrous mixture, in the known method, the crystalline monohydrate product is obtained as previously described.

The examples below illustrate the process for preparing the picotamide monohydrate from if-methoxyisophthaloyl dichloride or other functional derivative of U-methoxyisophthalic acid in an environment of proton acceptors.

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Preparation example U-methoxy isophthaloyl dichloride 100 g (0.1 + 3 mol) (mol wt. 233) 100 g (0.1 + 3 mol) 3-picolylamine (mol wt. 108) 130 g (1.2 mol ) 3 5 triethylamine 120 cm

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tetrahydrofuran (anhydrous) 120 + 200 cm-3

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3-picolylamine, triethylamine and 120 cm anhydrous tetrahydrofuran are placed in a 3 liter flask equipped with reflux cooler drop funnel and mechanical stirrer.

10 L + methoxyisophthaloyl dichloride is separately dissolved in 200 cm anhydrous tetrahydrofuran.

This solution is slowly added to the mixture in the flask by means of the dropping funnel while stirring. The addition should be made in an hour and a half to two hours with the isothermal reaction proceeding as indicated by the reaction scheme of the formula sheet.

After the dichloride is added, the reaction mixture is refluxed for about two hours, diluted slowly with water to two liters, and maintained with stirring to separate a crystalline knit consisting of the raw picotamide.

This knit is recovered on a suction filter e & in a wet state

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crystallized from 700-800 cm0 acetone water mixture (6 volume acetone + 11 volumes what is).

The product obtained is recrystallized from water to obtain picotamide monohydrate, m.p. 95-97 ° C, with the Kofler bank.

From the above, it follows that the process is characterized by the fact that the recrystallization of the crude picotamide, as obtained by the synthesis reaction, is carried out by an aqueous / solvent as opposed to the prior art, in which anhydrous non-polar organ Organic solvents were used as benzene, whereby an anhydrous product was obtained.

Pharmacological tests

Picotamide monohydrate has been found to have high activity as a platelet anti-aggregating agent. and fibrinolytic agent.

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It therefore finds application in clinical pharmacology and human therapy.

Said activities have been "tested in vivo by spectrophotometric" assay by Bom for the "platelet anti-aggregation activities" and by the test of the "blood coagulation lysis in Toto by Fearaley, for the fibrinolytic activity.

Example I

Platelet anti-aggregation activity in vivo on rabbits.

New Zealand rabbits that had fasted for 12 hours with water 10 ad libitum were anesthetized with a 20% ethyl solution of

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urethane intraperitoneally with a dose of 0.6 cm / 100 g. Blood was drawn from the carotid artery before (control) and an hour and a half after the intraperitoneal injection of picotamide monohydrate at a dose of 25-50-100 mg / kg. The blood samples were rendered uncoagulable by a 3.8% solution of sodium citrate in the volume ratio of 9/1 and then centrifuged at 1000 rpm for 15 minutes to obtain a platelet rich plasma (PEP). Part of said plasma was then centrifuged at 8000 rpm. for 10 minutes to obtain a platelet arm. plasma (PPP).

20 PP? was used to zero. setting of. a .Bom aggregator and 1 cm 3 prp were placed in the vessel of the measuring device. Platelet aggregation was produced by varying concentrations of disodium adenosine diphosphate (ADP) depending on platelet reactivity.

Platelet anti-aggregation activity was calculated as a 50% retardation of the aggregation curve after treatment, based on the control (ID ^).

The results will be discussed later.

Example II

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

Picotamide monohydrate at a dose of 100 mg / kg was administered orally to Beagle dogs, Cf, who had been fasted with water ad libitum for 18 hours.

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Blood was drawn before (control) and 2-4-6-8-10 hours after treatment and as a function of time platelet aggregation activity (by the above method) and blood levels of the drug were determined.

Blood levels were determined with a UV spectrophotometer according to the method below.

3 5 cm plasma obtained by centrifugation at 100 rpm.

for 10 minutes were added with 2 ml of concentrated HCl and hydrolyzed on a water bath at 100 ° C for 1 hour. It was cooled, taken up with 2 cm of water and filtered. The filtrate was made highly alkaline with 11% OH and extracted with 30 cm CHCl 4.

The chloroform layer dried over anhydrous H 2 SO 2 was extracted with 0.1 N HgSO 2 and the acidic layer thus obtained was made up to 100 cm 2 with 0.1 N HgSO 2 and the spectrophotometer read at 228 n.m.

The results are reported below.

Example III

In vivo fibrinolytic activity in the guinea pig.

Fibrinolytic activity was determined on Italian guinea pigs and picotamide monohydrate was administered orally successively at a dose of 100 mg / kg.

The Fearaley method modified as follows was used for this test:

Tubes maintained at 0 ° C were charged 1.7 cur phosphate buffer 3 3 (pH 7.4) and 0.1 cmJ of a thrombin solution with 50 HIH / em.

3. .

After adding 0.2 cm of guinea blood in total, the tubes were maintained at 0 ° C for 30 minutes to cause clotting, then maintained on a water bath at 3 ° C for 30 minutes to cause lysis. The solidification weight was then determined.

Fibrinolytic activity was calculated as a 30 percent decrease. coagulation weight of the treated animals relative to the coagulation weight of control animals.

Example IV

Platelet anti-aggregation and fibrinolytic activity in humans.

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Confirmation of both activities in vivo for picotamide monohydrate was obtained in healthy people of both sexes aged 35 to 65 years treated orally with a single dose of 12 mg / kg.

The retarding effect of the plaque aggregation was tested using disodium ADP as an antagonist by the same method as in Example I.

Fibrinolytic activity was estimated by determining the lug time of euglobins.

The results obtained are listed below.

Results

From the results obtained in the rabbit platelet antiaggregation activity in vivo test (Example 1) d.Gor "probit" analysis, the dose was allowed to slow platelet aggregation by 50% (ID ^ q) calculated and this dose was 5 ^ 10-1.3 mg / kg.

A may clearing effect ($ 53.82) in the dog (Example II) was found at the fourth hour. The corresponding blood level was

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22.6 µg / cm plasma and it corresponds to a maximum value as shown in the diagram of Fig. B as 20.

The fibrinolytic activity in the guinea pig (Example III) tested at a dose of 100 mg / kg orally was found to be $ 27.83.

In humans (Example IV) at a single dose of 12 mg / kg orally, the maximum platelet anti-aggregation effect was found four hours after treatment and was determined to be 81.9%. Maximum fibrinolytic activity was determined to decrease the lug time of euglobins.

Commentary on the comparisons

When comparing the activity and toxicity results of picotamide monohydrate with those of anhydrous picotamide described in U.S. Patent 3,973,026, it can be said that significant differences in activity exist and these differences are favorable for the monohydrate form which was certainly unpredictable from 81 0 2 0 1 6

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- 12 - the fact that only a crystallization water molecule was introduced into the new compound.

For comparison, a time effect and blood levels test in the dog was conducted orally under the same test conditions for the previously known anhydrous picotamide to test its bioavailability after administration.

The results of this test are explained by the diagram of Figure h-, which represents the time effect on platelet aggregation and on blood levels in the dog at a dose of 100 mg / kg per os.

The ordinate, the time in hours and the abscissa, the blood levels determined in γ / cm 2 plasma as well as the platelet disaggregation activity determined in percent. Curves 1 and 1 * represent the anti-aggregation activity of picotamide monohydrate pico-15-anhydride, respectively, and solid lines 2 and 2 'represent the blood levels of picotamide monohydrate, picotamide anhydrous, respectively.

A review of the results clearly shows that anhydrous picotamide has a maximal platelet anti-aggregation effect. .

has at the eighth hour, which is equal to ^ 9%, while the maximum blood level is 19.75 µ / cm, which is evident at the sixth hour.

In contrast, picotamide monohydrate exhibits the maximal platelet aggregation effect in the fourth hour, and in addition, the peak activity peak over time coincides with the peak peak blood level indicating faster bioavailability in favor of picotamide monohydrate compared to anhydrous picotamide.

The results of the pharmacological tests conducted with picotamide monohydrate are summarized in Table B below, which also lists the results of the same test for anhydrous picotamide.

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Test Parameter Picotamide Anhydrous monohydrate picotamide

Platelet aggre-low in vivo 5 (rabbit) intraperitoneal_IDmg / kg_5 ^ -1_108.2_

Platelet aggre- maximum slight in vivo delay% (dog) 100 mg / kg of aggregation 53.83 1 * 8.12 ----- TO per os - max 1 act ivit éiis- - time found every hour 8th hour maximum blood- level γ / cmr 15 in plasma 22.6 19.75 maximum blood level time found at the 6th hour

Platelet aggregation maximum decreases human traging% after single aggregation '81, b 70.11 administration - maximum activity time 25 found Ue hour 8th hour

Acute toxicity he rat and dog per os DL .mg / kg> 3,000> 3,000

A comparison of the test values shown in tahel B for the heather molecules shows that picotamide monohydrate has an improved pharmacological effect compared to anhydrous picotamide. Such an unexpected improvement appears to be due to the increased bioavailability of the new drug in its stable monohydrated crystal structure.

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Therapist is applications-

Due to its low toxicity, high tolerance and absence of adverse side effects, picotami demonohydrate is useful in human therapy for the "treatment of various thromboembolic disorders, especially" cerebrovascular disorders, myocarditis infarctio, arterial and vein thrombosis, pulmonary embolism, general arte ^ io riosclerotic conditions and general cardiac surgery.

For that application, various pharmaceutical forms can be used, containing 10 - 500 mg of the active ingredient, of which T0 follow the examples below: a) Oral: capsules, tablets, pills with 10-500 mg, for a daily dose of 50-3000 mg / day ; b) Parenterally: sterilized endovenous vials of 10-50 mg for a daily dose of 10-200 / day.

The drug may also be administered rectally in the form of a suppository.

It will be understood that the pharmaceutical preparations may contain, in addition to the active ingredient, the usual pharmaceutically acceptable carriers and additives as is customary in pharmaceuticals. It will also be understood that the dosage forms of the picotamide monohydrate and the respective dosing patterns may be changed depending on the clinical conditions and experience of .......

the doctors.

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Claims (8)

1. N, Nr-bis- (3-picolyl) - - - methoxy-i-phthalamide monohydrate in crystalline form of the formula 1 of the formula sheet, characterized in that it has a melting point of 95-97 ° C on the Kofler bank and an X-ray diffraction pattern of its single crystal as listed in Table A. 2. Process for the preparation of N, N * -bisl (3-picolyl) - - - methoxy - by reacting a functional derivative of k-methoxyisophthalic acid with 3-picolylamine in an anhydrous organic solvent characterized in that the reaction product is precipitated with water and the crude product is recrystallized from an aqueous solution and then from water. 3. Process according to claim 2, characterized in that after precipitation of the reaction product with water, the product is crystallized from a solution of acetone and water and then recrystallized from water. k. Pharmaceutical composition for the treatment of mammalian thromboembolic disorders, due to an increase in platelet aggregation and an increase in thrombin activity in the blood, characterized in that the active ingredient is a therapeutically effective amount. Ν * -bis- (3-picolyl) isophthalaraidaonohydrate *. in crystalline form, as well as pharmaceutically acceptable carriers. 5 · Preparation according to claim h. in dosage units with 10-500 mg of the active ingredient. Pharmaceutical composition according to claim 5, for oral administration containing 10-500 mg of the active ingredient. 7. Pharmaceutical composition according to claim 5 for parenteral administration containing 10-50 mg of the active ingredient. 8. Active agent for the treatment of mammalian thromboembolic disorders resulting from increased platelet aggregation and blood thrombin activity, consisting of N, N1-bis- (3-picolyl) - - - metho: xy- isoft aalamide monohydrate in crystalline form. Active agent according to claim 8, characterized in that it is administered orally in a dose of 50-3000 mg / day. Active agent according to claim 8, characterized in that the parenteral is administered in a dose of 10-200 mg / day. 81 0 2 0 1 6 Af VCO-NH-CH2 Λ U, η20 CO-Ν Η-CHj-r ^^ h OCH k J1 J COCl ~ h2n-ch2- ^ || _ \ β kxAvcod * 2 kN> 2HCt kyAco-NH-ti ^ OCH, W J OCH3 3 3 N A 2 8102016 ® Societa Italo-Britannica L. Manetti-II. Roberts & C.