DE3113150C2 - Crystalline N, N'-bis (3-picolyl) -4-methoxy-isophthalamide monohydrate, process for its preparation and pharmaceutical compositions containing it - Google Patents

Abstract

N, NΔ - bis - (3 - picolyl) - 4 - methoxy - isophthalamide monohydrate in crystalline form is a stable chemical compound and has high pharmaceutical activity for the treatment of various thromboembolic disorders.

Description

The invention relates to N, N'-Bls- (3-picolyi) -4-methoxy-isophthalamide monohydrate, elr. Process for its preparation and pharmaceutical compositions containing it.

It is known that N, N'-bis- (3-picolyl) -4-methoxy-isophthalamide (hereinafter referred to as "plcotamide" according to the internationally common name) is a compound with a high fibrinolytic and anticoagulant activity (cf. FR -PS 21 00 850. "Chimie Therapeutique", 6, 203-207, 1971), and also has good blue platelet aggregation activity (cf. US Pat. No. 3,973,026; Age and Aging, 7, 246, 1978). Plcotamide. as described in the above publications and patents. Is known in the anhydrous form, and its melting point is as specified in FK-PS 21 00 850. In the Kofier pad at 124 ° C.

This compound is made using the method given in the above-mentioned patents by crystallization of the crude product, which contains a lot of impurities, into anhydrous and Purified non-polar organic solvents.

The Crystallization of the Raw Plcotamld. like it with de. Synthesis reaction from anhydrous and non-polar organic Solvents such as benzene, results in a powdery product that is under the Microscope the characteristics of a fibrous substance, such as. B. voluminous down shows. This powder loads Slcn slightly electrostatically and is relatively unstable. These properties are particularly disadvantageous, when the compound is made for pharmaceutical use. The electrically charged particles collide w each other and fly who; when they are weighed and put into the device for the preparation of the various pharmaceuticals Preparations are introduced, which leads to a change in the particle weight, thereby reducing the Parental control is made more difficult.

It has now been found that by reacting 4-methoxy-isophthaloyldlchlorid with 3-picolylamine and by crystallization of the crude product in an aqueous solution of N.N'-bis- (3-picoIyl) -4-methoxy-isophthalamide- '5 monohydrate is obtained, which in terms of its chemical-physical properties and its stability is clearly superior to the already known anhydrous Picotamld.

In addition, it has surprisingly been found that the picotamldmohydrate obtained in this way is more active and effective Is as an anhydrous picotamld. both from the pharmacodynamic point of view and what the clinical pharmacology.

The present invention therefore relates to the crystalline N, N'-Bls- (3-plcolylM-methoxy-isophthalamide monohydrate described in claim 1).

The picotamide monohydrate according to the invention is a white, odorless, bitter one Tasting crystalline powder that is stable in the air and easily dissolvable in water.

For the sake of simplicity, this compound is referred to as "picotamide monohydrate" in the following description designated.

The compound according to the invention differs from the physico-chemical point of view from the already known connection due to the unpredictable improvement in stability. These

Improvement is due to the fact that the Molecular Hydrataitonswasser on the molecular structure the compound according to the invention takes part, since it is arranged in a precisely defined position in the crystal lattice 1st, the oxygen atom of the water of hydration having easily identifiable hydrogen bonds forms specific atoms belonging to different molecules of Plcotamld, as indicated below give, so that a single crystal of the compound with precisely defined properties is formed. These properties In unpredictable ways affect the pharmaceutical behavior of the new compound and its bioavailability in mammalian organisms with faster absorption upon administration this class of animals including humans.

The picotamide monohydrate according to the invention in the new crystalline form reports not only on obscure ones Catfish the abovementioned aftereffects of the anhydrous Plcotamlds. but also results in a connection which is more active and effective in terms of its pharmacological usefulness in view of the advantageous Effects that can be determined when administered to animals and humans.

Although a well-founded theoretical explanation for this experimental result has not yet available, it can be assumed that a solution of crystalline compound in water erflndungsgemäßen a ¹ ^ other mechanism follows as a solution of the known anhydrous compound in amorphous form in water.

Another object of the invention is the method described in claim 2 for producing the new hyriral pitch

The invention also relates to the pharmaceutical agent according to claim 3.

It should be noted that in contrast to erflndungsgemäßen connection anhydrous Plcotamld a melting point of 124 ⁰ C. This difference between the melting points of the two compounds is already an indication of the different molecular structure that makes the essential difference between anhydrous picotamide and plcotamide monohydrate.

The invention is explained in more detail below with reference to the drawings, in which * indicates bell legends

1 shows a three-dimensional representation of the molecule of picotaml monohydrate, obtained from an X-ray structure analysis;

Flg. 2 is a three-dimensional representation of the single crystal of plcotamide monohydrate;

Flg. 3 the same single crystal as shown in FIG. 2 in more detail; and

Fig. 4 is a graph showing a direct comparison between the platelet aggregate concentration of Plcotamide monohydrate and anhydrous picotamld shows.

The structure of a crystal of picotamide monohydrate is apart from the physical and chemical Analysis, also characterized by the X-ray spectrum of its single crystal.

The data of the X-ray diffraction spectrum showing the spatial arrangement of the atomic centers in the new Molecule in its entirety as a dense, stable and non-hygroscopic crystal are in the List included in claim 1. In the the different atoms of the molecule by their chemical Symbols and a subsequent identification number are displayed. The special arrangement and the identification number of these atoms are from Flg. 1 can be seen, which the geometric, shows three-dimensional position of the atomic centers, as it is obtained from the table mentioned. Included the list was based on the following values:

Maximum = 92.52

Minimum = -86.27

Multiplied by 167.8829
■ *> crystal class: monoclinic

Space group: P2 / n

Number of formula units in a unit cell: 4

Number of reflections measured: 2346

Wavelength of the radiation used: Λ = 0.7107
5f »Diffrak-.ometer-type: Philips PW 1100

Computer program: SHEL X 76 (Sheldrick, G.M. 1976 "Program for Crystal Structure Determination".

U.N. Cambridge, England)
• Calculation system: Fourter difference

R value: 13.2%

Intramolecular distances between the bonds (including the symmetrically arranged atoms) In pm

1- 2 144 3- 5 140 4- 5 1.39 3- 6 150 6-11 122 4-12 151 8-12 1.23 10-12 135 14-15 149 10-16 148 14-18 1.54 20-21 137 18-24 140 21-24 139 19-25 1.33 22-26 138 20-29 136 27-29 139 26-30 1.21 20-31 108 18-33 189 9-34 131.

In particular, it can be noted that atom No. 17, indicated as 027, represents the water of hydration, while atom No. 8, indicated as 09, is an oxygen atom of the carbonyl group, the Atom number 15, indicated by NI9, is a nitrogen atom of the amide group, and atom number 21, is N224 indicated, is a nitrogen atom of the pyridine group.

In Table I, the symbols X / A, Y / B and Z / C represent the spatial coordinates of the various s Atoms.

As can be seen from Fig. 1 showing the structure of picotamld monohydrate, the oxygen atom is des!>; dralatlonswassers In the crystal lattice In a precisely defined position with respect to the picotamld moleculeQi arranged.

From the Flg. 2 It can be seen that the oxygen atom of the water of hydration inside the single crystal, represented by a rectangle, represented by hydrogen bonds (indicated by a dashed line) precisely defined atoms of different Plcotamld molecules is bound, with the molecules in an ordered arranged three-dimensional lattice and through the hydrogen bonds with the hydration oxygen atom are connected to each other.

This bond is in the wing. 3 Shown in more detail, from which it can be seen that the oxygen atom 027 IS of the water of hydration via hydrogen bonds Is bound to:

(I) The oxygen atom of the carbonyl group (O9) of a single Plcotamld molecule (bond length 281 pm);

{2} the nitrogen atom of the Amiugrüppc (Nu; a second riCuiamid-iviüicküiS, düS in the same plane as the above molecule is arranged (bond length 296 pm);

(3) the nitrogen atom of the pyramid length (N ₂₂₄ ) of a third picotamid molecule, which is arranged in the plane below or in the plane above, based on the other two bound molecules (bond length 280 pm).

The presence of these three bonds explains both the strength with which the water of crystallization is between different plcotamide molecules forming the crystal is retained, as well as the compactness (Strength) of the crystal itself. This compactness, caused by the molecule of crystal water, is called the reason for the improvement in bioavailability of the monohydrate form compared to the previous one known anhydrous form, which, as will be shown in more detail below, differs from the pharmacological Viewpoint from a comparison between the absorption time, the content in the blood and the j " Act of both medicines! results.

Based on the previous knowledge from the prior art about the anhydrous picotamld molecule, the improvement effect achieved by the introduction of a crystal water molecule with regard to the compactness (density) of the spatial structure of plcotamld and the improvement with regard to a noticeably better biological availability was by no means foreseeable . ^J5

The chemical elemental analysis also provided results that match the structure explained above.

Elemental analysis for C ₂₁ H ₂₀ N ₄ O ₁ · H ₂ O (molecular weight 394.4)

40

The process for making the new Plcotamldmonohydrat molecule follows what the synthesis of the compound,.

application concerned, the already known process for the production of anhydrous plcotamide. ψ

However, when the crude picotamide has been obtained, as has been surprisingly found, by ⁴ S conversion of the crude product from an aqueous mixture as opposed to an anhydrous mixture as in the previously known process, the crystalline monohydrate product as defined above is obtained .

Production example ^m

4-methoxy-isophthaioyl dichloride (molecular weight 233) 100 g (0.43 mol)

3-picolylamine (molecular weight 108) 130 g (1.2 mol)

Triethylamine 120 ml

Tetrahydrofuran (anhydrous) 120 + 200 ml ⁵⁵ _

The 3-picolylamine. the triethylamine and 120 ml of anhydrous tetrahydrofuran were in a with a | A 3 liter flask equipped with a reflux condenser, a dropping funnel and a mechanical stirrer was introduced.

The 4-methoxy-isophthalic dichloride was separately dissolved in 200 ml of anhydrous tetrahydrofuran. The |

Solution was slowly poured through the dropping funnel into the reaction mixture contained in the flask below ⁶⁰ |

Stirring introduced. The addition must take place within one and a half to two hours, whereby the following | exothermic reaction takes place:

& 5

ber. C. 63.94 H 5.62 N 14.20 O 16 .24 ge f. 63.87 5.72 14.18 16 .23%

ι π πι

After this addition of the chloride, the reaction mixture was refluxed for about 2 hours with Water slowly diluted to 2 l and stirred until a crystalline slurry separated, which '5 consisted of raw plcotamld.

This slurry was separated on a suction filter and in a wet state in 700 to 800 ml an acetone / water mixture (6 parts by volume of acetone + 11 parts by volume of water) crystallized. The thereby obtained Product was recrystallized from water, picotamldmonohydrate having a melting point of Kofler's block from 95 to 97 ° C.

Pharmacological tests

"It was found that plcotamide monohydrate had high activity as a platelet anti-aggregation agent

and flbrinolytic agent. It is therefore useful in clinical pharmacology and in Human therapy.

These activities were determined in vivo by spectrophotometric determination of the platelet aggregation activity according to Born and using the Fearnley blood clot dissolution test for determination tested for fibrinolytic activity.

example 1

Platelet aggregation activity in vivo in rabbits

New Zealand rabbits, which had fasted for 12 hours with water intake according to Belleben, were anesthetized with a 20% ethanolic solution of urethane at a dose of 0.6 ml / 100 g intraperltoneal. From Carotld-Arterle was before (control) and l '/ ₂ hours after the injection of the intraperltonealen Plcotamld monohydrate at a dose of 2S-S0-100 mg / kg 3iut eninornnien. The blood samples were made incoagulable by a 3.8% solution of sodium chloride in a volume ratio of 9: 1 and then centrifuged at 1000 rpm for 15 minutes to obtain a platelet-rich plasma (PRP). A portion of this plasma was then centrifuged at 8,000 rpm for 10 minutes to obtain a platelet poor plasma (PPP).

The PPP was used to adjust the zero point of a Born aggregometer, and 1 ml of PRP was introduced into the container of the measuring apparatus. Platelet aggregation caused by disodium adenosylphosphate (ADP) in concentrations which varied depending on the platelet reactivity ability. Platelet Antlaggregatlonsaktivität was calculated as 50% inhibition (inhibition) of the aggregation curve after treatment with respect to that of the control (ID _SO). The results obtained are given below.

Example 2

Cell effect on platelet aggregation and levels of the drug in blood in dogs

Picotamide monohydrate was administered orally to Beagel dogs at a dose of 100 mg / kg for 18 hours were not fed any amount of water for a long time.

Blood was drawn before (control) and 2-4-6-8-10 hours after treatment and In As a function of time, the platelet anti-aggregation activint (using the above described method) and the levels of the drug in the blood are determined.

The blood contents were determined using UV spectrophotometry according to the following procedure: 2 ml of concentrated HCl were added to 5 ml of plasma obtained by centrifugation at 100 rpm for 10 minutes and hydrolysed on a water bath at 100 ° C. for one hour . It was cooled, taken up in 2 ml of H ₂ O and filtered. The filtrate was made strongly alkaline with _{NH 4} OH and extracted _{with 30 ml CHCl 1.} After drying over anhydrous Na ₂ SO _{4, the} chloroform layer was extracted with 0.1 nH ₂ SO <, the acidic layer _{thus obtained being mixed with 0.1 HH 2} SO ₄ up to 100 ml, and the spectrophotometer was set at 228 nm read. The results are given below.

^<Λ Example 3

Fibrinolytic activity in vivo in guinea pigs

The fibrinolytic activity was determined in Italian guinea pigs after giving them picotamide

monohydrate at a dose of 100 mg / kg had been administered orally. To perform this test, the Fearnley method was applied, which was modified as follows: In maintained at 0 ⁰ C 1.7 ml tubes were Pht-sphatpuffer (pH 7.4) and 0.1 ml of a Thromblnlösung with 50 NIH / ml introduced. After adding 0.2 ml of whole guinea pig blood, the tubes were held at 0 ° C for 3 minutes to cause clumping, then placed on a 37 ° C water bath for 30 minutes to cause dissolution. Then the blood clot weight was determined.

The fibrinolyllic activity was calculated as the percentage of the decrease in blood clot weight treated animals based on the blood clot weight of the control animals.

Example 4 to

Platelet aggregation activity and flbrinolytic activity in humans (volunteers) The two in vivo activities of plcotamld monohydrate were confirmed in healthy individuals People (volunteers) of both sexes between the ages of 35 and 65 who are administered orally with an individual dose of 12 mg / kg were treated.

The inhibiting effect (the inhibitory effect) of platelet aggregation was tested using of Dnairlum ADP as an antagonist by the same procedure as in Example 1. The fibrinolytic activity was determined by determining the dissolution tent of the eugloblne. The results achieved are

Results

From the results obtained in the test of platelet anti-aggregation activity in vivo in guinea pigs (Example 1) by probit analysis, the dose capable of inhibiting platelet aggregation by 50% was calculated ) (ID ₅₀ ), and this dose was 54.10 ± 1.43 mg / kg.

A maximum effect (53.82%) in dogs (Example 2) was found in the fourth hour. The corresponding Blood volume was 22.6 A / m! Plasma, and it corresponded to a maximum value as shown in the diagram of Flg. 4 is indicated.

The inflammatory 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), the maximum platelet anti-aggregation effect occurred at a single dose of 12 mg / kg orally 4 hours after treatment and it was 81.4%; the maximum fibrinolytic activity was determined to be a 54.2% decrease in the rate of dissolution of euglobins.

35 comparative considerations

From comparing the activity and toxicity results for picotamide monohydrate with those for anhydrous plcotamide, which are given in US Pat. No. 3,973,026, it can be seen that there are significant differences In terms of activity and that these differences are favorable for the monohydrate form, what due to the fact that only one crystal water molecule characterizing the new compound was introduced was certainly not predictable.

For comparison purposes, a test for the tent effect and blood levels in dogs was carried out in neutral Administration carried out under the same experimental conditions using what is already known anhydrous plcotamide to determine its bioavailability after administration.

The results of this test are shown in the graph of FIG. 4 indicated the time effect on platelet aggregation and on blood levels in dogs at a dose of 100 mg / kg p.o. shows.

The ordinal axis indicates the time in hours, and the abscissa axis indicates the blood content, measured in λ / ml plasma, as well as the platelet anti-aggregation activity, measured in%. Lines 1 and 1 'represent the anti-aggregation activity of picotamide monohydrate or anhydrous picotamide, and the solid lines Lines 2 and 2 'represent the blood hooks on picotamide monohydrate and anhydrous picotamide, respectively.

Examination of the results shown in the graph shows that anhydrous picotamide has a maximum platelet anti-aggregation effect results in the eighth hour, which corresponds to 49%, during the maximum blood content is 19.75 A / ml, which occurs in the sixth hour (is slightly shifted).

In comparison, picotamide monohydrate has the maximum platelet anti-aggregation effect in the fourth hour, and beyond that, the maximum activity peak falls rapidly with the maximum peak of the Blood content together, indicating a faster bioavailability of picotamide monohydrate, compared with anhydrous picotamide.

The results of the pharmacological tests performed with picotamide monohydrate are shown below Table I is listed in which the data of the same test with anhydrous picotamide are also given

15th

20th

Table I.

test

parameter

Picotamide- anhydrous monohydrate picotamide

Platelet aggregation in vivo (rabbit), intraperitoneally

Platelet aggregation in vivo (dog)
100 mg / kg po

tsiuipity aggregation in humans after administration of a single dose

Acute toxicity at
Rats and dogs po

ID ₅₀ (mg / kg)

maximum activity time found

54.1

Maximum inhibition of the 53.83
Aggregation (%)

4h

22.6

maximum blood content in the plasma (y / ml)

found time of 4 hours
maximum blood content

maximum inhibition 81.4

of aggregation (%)

found maximum 4 hours

Activity time

DL ₅ O (mg / kg)> 3000

108.2 48.12

8 hours

19.75 6 hours

70.11

8 hours> 3000

A comparison between the test results given in de- Table I for the two molecules shows that picotamide monohydrate has improved pharmacological effects over anhydrous picotamide having. One such unexpected improvement appears to be due to the improved bioavailability of the new one Medicinal product in the form of monohydrate crystal with a stable structure.

Therapeutic use

Picotamide monohydrate can due to its low toxicity, its high tolerability and due to the absence of adverse side effects in human therapy for the treatment of various thromboembolous disorders, in particular cerebral vascular disorders, myocardial infarctions, arteries and Flebothromboses, pulmonary embolisms, general arteriosclerotic conditions and in general cardiac surgery be used.

Various pharmaceutical preparations can be used for the stated purposes, ■ »s which contain 10 to 500 mg of the active ingredient (active agent), for which some examples are given below are:

(a) Oral: capsules. Tablets. Piilen. containing 10 to 500 mg, for a daily dose of 50 to 3000

mg / day;

S "(b) parenteral: sterilized intravenous injectable vials containing 10 to 50 mg for daily dosing from 10 to 200 mg / day.

It can also be administered rectally in the form of suppositories.

The pharmaceutical agents (preparations) according to the invention can, in addition to the active ingredient (active ingredient ^ part) still contain the usual pharmaceutically acceptable excipients and adjuvants, as on the pharmaceutical Area well known.

60

For this purpose 3 sheets of drawings

Claims (3)

j 31 13 150 (2) (3) (4) X / A (41Y / B H ₂ O the following atomic parameters from the 6) Π) 1 Patent claims: 08 _ 0.1895 0.6286 up to 97 ° C and I. U6 I. C38 - 0.1967 0.7283 (5) ( 1 0.72 Cl - 0.2670 0.5074 (41Z / C 1.0000 1 0.82 I. C3 - 0.2047 0.4937 0.8386 1.0000 1 1.60 ύ
I Iu C5 - 0.2299 0.4539 0.8919 1.0000 1.44 1. Crystalline N, N'-bis (3-picoIyI) -4-methoxy-isophthalamide monohydrate of the formula C6 - 0.2944 0.4562 0.3663 1.0000 0.75 I. C7 - 0.2164 0.5904 0.6312 1.0000 1.11 • 09 - 0.1627 0.3432 0.4720 1.0000 2.75 !
ϊ ClO . - 0.2795 0.6035 0.2040 1.0000 0.35 CO-NH-CH ₂ - Nile - 0.1385 0.4642 0.6832 1.0000 - 2.43 012 - 0.2847 0.3716 0.6819 1.0000 1.24 C13 - 0.1665 0.4277 0.4219 1.0000 I. 2.31 I. I CO-NH-CH ₂ - /
OCH ₃ ijj C14 - 0.2547 0.6442 0.8824 1.0000 0.5. j C15 - 0.3600 0.4594 0.1698 1.0000 0.11 N19 - 0.3326 0.5077 0.7347 1.0000 0.21 I. C22 - 0.1082 0.3996 0.5815 1.0000 3.28 with a melting point on the Kofler block of 95
X-ray structure analysis of its single crystal: 027 - 0.3771 0.7056 -0.0529 1.0000 ) 0.00 j 35 C221 - 0.4250 0.4199 0.1101 1.0000 U4 (D N222 - -0.0616 0.3513 1.0097 1.0000 ( 2.34 1 C223 - 0.5422 0.3476 0.1461 1.0000 3.43 2 N224 - 0.5202 0.3510 0.0573 1.0000 2.34 i 40 3 C227 - 0.0384 0.3880 0.9680 1.0000 2.47 4th C228 - 0.0004 0.3683 0.2266 1.0000 2.96 5 C229 - 0.4606 0.3864 0.0056 1.0000 1.28 6th C231 - -0.0875 0.3574 0.9075 1.0000 1.23 : 4> 7th C232 - 0.0127 0.3932 1.0498 1.0000 1.33 8th C234 - 0.4483 0.4146 -0.0822 1.0000 2.47 9 C235 - -0.0519 0.3732 0.7563 1.0000 0.72 10 C244 = 0.5071 0.3758 0.6853 1.0000 3.53 11th Ql 96.0 0.0376 0.3935 0.2819 1.0000 0.88 12th Q2 92.0 0.5364 0.3978 0.6042 1.0000 3.48 13th Q3 84.0 0.2173 0.3847 0.3636 1.0000 1.70 j 55 14th 0.5361 1.0000 15th 0.3476 1.0000 16 0.4186 17th i ^W) 18th i 19th I. 20th I. 21 22nd 23 24 25th 26th 27 28 29 30th 31 32, (1) (2) C) (4) X / A (4} Y / B (4) Z / C (5) (6) (7) 33 Q4 84.0 0.3392 0.3922 -0.0552 1.0000 1 0.51 34 Q5 83.0 0.3204 0.6259 0.3130 1.0000 1 0.00 (1) Serial number in the computer printout (2) Identification numbers of the atoms according to FIG. I. (3) height (4) Space coordinates of the atoms (5> Space occupation factor of the atoms (6) Affiliation of the atom to the organic (1) or water molecule (0) (7) Distance to the plane 0.00 through the oxygen atom 2. A process for the preparation of the compound according to claim 1 by reacting 4-methuxy-isophthaloyl dichloride with 3-picololyamine in tetrahydrofuran and in the presence of triethylamine as proton acceptor, characterized in that one a) works in anhydrous tetrahydrofuran. b) the reaction product precipitates with water, c) the crystalline slurry obtained in this way in the moist state in an acetone-water mixture (Volume ratio 6:11) crystallized and d) the product thus obtained is recrystallized from water. 3. Pharmaceutical agent for the treatment of thromboembolic disorders in mammals as Consequence of an increase in platelet aggregation and an increase in thrombin activity in the blood, containing the compound according to claim 1 and customary carriers or auxiliaries.