HRP970433A2 - Pharmaceutical products for curing and preventing illnesses connected with the malfunction of vascular endothelial cells - Google Patents

Description

Technical field of the invention

The invention relates to products for the treatment or prevention of diseases associated with the impaired functioning of vascular endothelial cells, which contain hydroxylamine derivatives of the general formula (I) or (II) as an effective agent.

Background of the invention

The normal functioning of vascular endothelial cells is of crucial importance for the body. These cells form the marginal surface between the circulating blood and parts of the vein wall, acting thrombogenically. The role of vascular endothelial cells in homeostasis is quite different:

- they participate in the two-way transfer of substances originating from the blood and tissues,

- they form a barrier for macromolecules,

- these cells are the site of synthesis and breakdown of mediators that regulate interactions between cellular elements of the vein wall and blood (for example, fibrinogen, collagen, proteoglycans, PGI2, EDRF (NO), EDHF, endothelin-1, angiotensin-II),

- they initiate migratory, proliferative and thrombolytic processes that contribute to tissue repair,

- they support the thromboresistance of the vein wall [Rubanyi, G., J. Cardiovasc. Pharmacol. 1993, 22 (42 Suppl.) S 1-14].

Damage to the endothelium results in atherosclerosis. Damage leading to endothelium deterioration can occur with mechanical intervention, for example catheterization, and also as a result of biochemical and immunological processes.

In the first stage of atherosclerotic plaque formation, cells filled with lipids accumulate in the intima of arteries (Steinberg D. et al., JAMA 264-304, 1990). These cells, especially blood-derived monocytes and macrophages, first adhere to the endothelium and then penetrate into the intima. Endothelial cell injury may also contribute to adhesion, although morphological changes are not evident at an early stage. Oxidation of LDL particles can result in their incorporation into monocytes located in the intima, and monocytes thus become foam cells. These foam cells form lipid layers, the earliest form of atherosclerotic changes.

In the later stages, hemorrhage, necrosis, neovascularization and sclerosis appear, and in view of this, ingrown forms of plaques are formed, which then narrow the arteries (Ip. JH, Fuster et al., J. Am. Coll. Cardiol 15:1667, 1990) .

Thrombosis can occur in different degrees of atherosclerosis. Repeated thrombosis leads to vessel damage and thromboembolic disease, such as coronary thrombosis, thrombosis of cerebral vessels, and peripheral vascular disease.

In the clinical sense, "disordered endothelial function syndrome" refers to general or localized vessel spasms, thrombosis, arteriosclerosis and restenosis. Attempts to treat these diseases have included interventional clinical techniques, bypass surgery, and medical treatment.

Only a few existing drugs may be suitable for "treating" impaired endothelial function. They fall into four main groups:

- replacement for natural "protective" endothelial substances (for example, stable analogs of PGI2, nitro-vasodilators, rt-PA/recombinant tissue plasminogen activator/)

- inhibitors or antagonists of constrictive factors produced from the endothelium (for example ACE inhibitors, angiotensin n receptor antagonists; TXA2-receptor antagonists)

- cytoprotective agents (for example free radical scavenger peroxide dismutase and probucol, and free radical production inhibitor lazaroid)

- lipid-lowering drugs.

Although none of them were originally intended for this purpose, their already proven clinically improving effects in the case of some diseases may include the protection and restoration of normal endothelial functions. The basis of new therapies in this group is the restoration of normal endothelial cells, where these cells will "do the work" themselves. Possible approaches may include stimulation of regrowth of normal endothelium, or with emerging therapeutic modalities based on recombinant DNA technology (Science 1990; 249; 1285-8). According to the available data, no recognized drug meets these criteria.

Currently, there is no known drug or drug candidate that acts directly on the endothelium, and thus none is suitable for treating impaired endothelial function.

Therefore, there is a great demand for a drug that is able to prevent, reverse or at least slow down the development of complicated symptoms, or reduce the occurrence of diseases.

Summary of the invention

During our research, we found that derivatives of hydroxylamine of the general formula (I) and (II) show a strong protective and restorative effect on vascular endothelial cells and are able to prevent their damage from different causes.

In the general formulas (I) and (II), R1 and R2 independently of each other refer to a hydrogen atom or a straight or branched alkyl group of 1 to 6 carbon atoms, or R1 and R2 together with a nitrogen atom in between form a saturated heterocyclic group with 5-7 members, which optionally contains further nitrogen and/or oxygen heteroatoms, A refers to a straight or branched alkyl group with 4 to 12 carbon atoms, a phenyl group, substituted or not, which preferably contains an alkyl, haloalkyl or nitro group as a substituent, or a heteroaromatic ring with 5 - 6 members containing nitrogen, oxygen or sulfur, in the compounds of the general formula (I), Z refers to a covalent bond, and in the compounds of the general formula (II) to a covalent bond or =NH group, in compounds of the general formula (I), X refers to a halogen atom and to the -NR3R4 group, where R3 and R4 independently of each other refer to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms, while in the compounds of the general formula (AND I) X refers to an oxygen atom, in the compounds of general formula (II), R1 refers to a hydrogen atom or a straight and branched alkyl group with 1 to 6 carbon atoms, and in general formulas (I) and (II), Y refers to hydrogen atom, a hydroxyl group or an acyloxyl group, which preferably contains an acyl part of a long fatty acid chain with 8 to 22 carbon atoms, or a cyclic aromatic carboxylic acid as its acyl component, and in compounds of the general formula (I) where X refers to the -NR3R4 group and Y refers to the hydroxyl group, the X group is condensed with the Y substituent and forms an intramolecular ring.

Salts and optically active forms of these compounds are also effective compounds.

Those compounds of the general formula (I) which have X which refers to the -NH2 group, and A which corresponds to an unsubstituted phenyl or pyridyl group, and in which Y refers to a hydroxyl group are already known from French patent application number 2362 845 A1. These compounds are, according to the above-cited patent application, selective beta-blockers, and are therefore suitable for the treatment of diabetic angiopathy.

Those compounds of the general formula (I) having X referring to a halogen atom, Y referring to a hydroxyl group, and A to a substituted or unsubstituted phenyl or pyridyl group are already known from printed PCT patent application number WO 90/04584 A1. These compounds have a selective beta-blocking effect, and are therefore suitable as effective agents in the treatment of diabetic angiopathy.

Those compounds of the general formula (II) having A referring to a phenyl (unsubstituted or substituted with a haloalkyl group), pyridyl or thienyl group, Z referring to a covalent bond, R' referring to a hydrogen atom, and Y referring to a hydroxyl group are already known from Hungarian patent application number 2385/92, printed under number T/66350. These compounds have anti-ischemic and anti-anginetic effects, and are therefore well-usable in the therapy of vein complications associated with diabetes mellitus.

Those compounds of the general formula (I) having A referring to an aromatic or heteroaromatic ring and X referring to a halogen atom, while Y referring to a hydrogen atom are already known from printed PCT patent application number WO 95/30649 A1. These compounds have an anti-ischemic effect, and can best be used in the treatment of ischemia phenomena characterized by hypertonic veins and platelet aggregation.

In none of the above cited specifications is it written that the described compounds should act on vascular endothelial cells.

As we mentioned above, our research proved that the compounds of general formulas (I) and (II) will act on the endothelial cells of the cardiovascular and cerebrovascular systems. In experiments, which will be detailed later, it was observed that these compounds are able to block or restore damage to these cells. Therefore, these compounds can be used as active substances in therapeutic products used in the treatment of diseases resulting from abnormal functioning or damage of endothelial cells, especially cardiovascular or cerebrovascular diseases, high blood pressure, hyperhomocysteinemia and peripheral vascular diseases.

Based on these observations, the invention consists in reporting hydroxylamine derivatives of the general formula (I) and (II) - where R1 and R2 independently of each other refer to a hydrogen atom or a straight or branched alkyl group of 1 to 6 carbon atoms, or R1 and R2 together with the nitrogen atom in between form a saturated heterocyclic group with 5 - 7 members, which optionally contains further nitrogen and/or oxygen heteroatoms, A refers to a straight or branched alkyl group with 4 to 12 carbon atoms, a phenyl group, substituted or no, which preferably contains an alkyl, haloalkyl or nitro group as a substituent, or a heteroaromatic ring with 5-6 members containing nitrogen, oxygen or sulfur, in the compounds of the general formula (I), Z refers to a covalent bond, and in the compounds of the general formula (H) to a covalent bond and =NH group, in compounds of the general formula (I), X refers to a halogen atom or to the -NR3R4 group, where R3 and R4 independently of each other refer to a hydrogen atom or straight or branched that alkyl group with 1 to 6 carbon atoms, while in compounds of the general formula (II) X refers to an oxygen atom, in compounds of the general formula (II), R' refers to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms , and in the general formulas (I) and (II), Y refers to a hydrogen atom, a hydroxyl group or an acyloxyl group, which preferably contains the acyl part of a long chain fatty acid with 8 to 22 carbon atoms, or a cyclic aromatic carboxylic acid as its acyl component , however, in certain types of compounds of the general formula (I) where X refers to the -NR3R4 group and Y refers to the hydroxyl group, the X group is condensed with the substituent Y and forms an intramolecular ring which is represented by the general formula (III), in in which formula A, Z, R1 and R2 have the same meaning as above, further to the salts and optically active forms of these compounds for the production of a drug used for the treatment or prevention of diseases associated with the impaired action of e endothelial cells.

The invention also relates to pharmaceutical products used for the treatment and prevention of diseases associated with the abnormal functioning of vascular cells that contain, as an active substance (apart from the usual carrier substances and auxiliary substances used in pharmaceutical preparations) a compound of the general formula (I) or (II) in 0 .5-95.5 m/m %, or in certain cases salts or optically active forms of these compounds, and in these formulas the meanings of R1, R2, A, Z, X, Y and R' are the same as above.

Those compounds of the general formula (I) and (II) are preferred for the application described in the invention, where A refers to a pyridyl, thienyl or phenyl, nitrophenyl or trifluoromethylphenyl group. These compounds of the general formula (I) are also preferred where X refers to a chlorine atom or an NH2 group. Of these last compounds, those containing an intramolecular ring formed by the condensation of groups X and Y are particularly preferred. Also preferred are those compounds of the general formula (II), where R' refers to a hydrogen atom, and those compounds of the general formula (I) or (II ), where Y refers to the hydrogen atom and the hydroxyl group. In all these mentioned groups, those compounds where NR1R2 refers to a piperidino or dialkylamino group are preferred.

The following compounds of the general formula (I) and (II) are particularly preferred for the invention:

N-[2-Benzonoxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide (Z)-2-butenedioate (1:1) (Compound No. 1)

N-[2-palmitoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide monohydrochloride (compound no. 2)

N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3-trifluoromethylbenzenecarboxyimidoyl chloride monohydrochloride

(connection no. 3)

N-[2-Hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboxyimidoyl chloride monohydrochloride (Compound No. 4)

N-[2-Hydroxy-3-(1-piperidinyl)propoxy]benzenecarboxyimidoyl chloride monohydrochloride (Compound No. 5)

N-[2-Hydroxy-3-(1-piperidinyl)propoxy]-4-pyridinecarboxyimidoyl chloride (Z)-2-butenedioate (1:1) (Compound No. 6)

N-[2-Hydroxy-3-(1-piperidinyl)propoxy]-2-nitrobenzenecarboxyimidoyl chloride monohydrochloride (Compound No. 7)

N-[3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidoyl chloride dihydrochloride (compound No. 8)

N-[3-(1-piperidinyl)propoxy]-3-nitrobenzenecarboxyimidoyl chloride monohydrochloride (Compound No. 9)

N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3-trifluoromethylbenzenamide (compound No. 10)

N-hexyl-N'-[2-hydroxy-3-(1-piperidinyl)propoxy]-urea (compound no. 11)

N-hexyl-N'-[3-(1-piperidinyl)propoxy]urea (compound no. 12)

5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine (compound no. 13)

Those compounds of the general formula (I) in which X corresponds to a halogen atom can be prepared by reacting the amidoxime of the general formula 2, where A has the same meaning as above with an amino-chloro-propane derivative, where R1 and R2 and Y have the same meaning as above and - The NH2 group of the resulting intermediate compound of the general formula (1), where Z refers to a covalent bond while the other substituents have the same meaning as above, was replaced with a halogen atom by diazotization. In cases where a compound of the general formula (I) containing a hydroxyl group as a Y substituent has been produced, the required amino-chloro-propane derivative of the general formula (9) containing a hydroxyl group as a Y substituent can be produced from epichlorohydrin of the formula (3) and amine general formula (6), where R1 and R2 have the same meaning as above. An alternative procedure is the reaction of epichlorohydrin with an amidoxime of the general formula (2) first, followed by diazotization of the resulting intermediate compound of the general formula (4), where A has the meaning as above and the reacting epoxy compound of the general formula (5), where A has the same meaning as above with an amine of the general formula (6).

Those compounds of the general formula (I), where Y refers to an acyloxyl group, can be prepared by the reaction of a suitable compound of the general formula (I) containing a hydroxyl group in place of Y, and an acid chloride of the general formula (8), where R5 refers to a long alkyl chain or to an aryl group.

Compounds of the general formula (II) containing covalent bonds at position Z can be prepared in one of the following ways:

(i) a linking alkali hydroxamate of the general formula (10) where A and R' have the same meaning as above and K refers to an alkali metal cation, and a halogen compound of the general formula (9) where R1, R2 and Y have the same meaning as above, or

(ii) by the reaction of an amino compound of the general formula (II) where R1, R2, Y and R' have the same meaning as above, and an acid halide where A has the same meaning as above, while those compounds of the general formula (II) that contain a covalent bond on in place Z, and the hydrogen atom in place R1 can also be prepared, in addition to methods (i) and (ii), in the following ways:

(iii) by diazotization in a halogen-free environment of a suitable compound of the general formula (I) containing an -NH2 group at the X position and a covalent bond at the Z position, or

(iv) by hydrolysis of a suitable compound of the general formula (I) containing a halogen atom at the X position.

Those compounds of the general formula (II) where A refers to an alkyl group and Z refers to a =NH group can be prepared by reacting a compound of the general formula (II) where R1, R2, Y and R' have the same meaning as above, in an organic solvent , preferably chloroform, with an equimolar amount of alkylisocyanate where A corresponds to an alkyl group.

Compounds of general formula (II) are special cases of compounds of general formula (I) where the nitrogen-containing group X is condensed with the Y substituent to form an intramolecular ring.

Such compounds can be prepared by reacting a compound of the general formula (I) (where Y corresponds to a hydroxyl group) with an excess of thionyl chloride, followed by ring closure of the resulting intermediate compound of the general formula (I) (where Y corresponds to a chlorine atom, while the other substituents have the same meaning as above) with an excess of potassium tert-butoxide in a boiling organic solvent (preferably in t-butanol).

Hydroxylamine derivatives of the invention show surprising pharmacological properties. It is worth noting that they not only restore the endothelial cells morphologically, but also functionally, which, considering their effect, means that the endothelial cells of the cardio- and/or cerebrovascular system of the treated organism begin to function again. The good tolerability of these compounds is also considerable. These properties form the basis for the pharmaceutical use of hydroxylamine derivatives of the general formula (I) and (II). These drugs can be used to treat cardio- and cerebrovascular diseases in humans and animals, such as high blood pressure, hyperhomocysteinemia and peripheral vascular diseases.

Among cardiovascular diseases, these compounds can best be used in cases of coronary artery diseases, atherosclerosis, restenosis following catheterization and coronary bypass surgery, and among cerebrovascular diseases in cases of cerebral arterial occlusion, hydroxylamine derivatives of general formulas (I) and (II) are they can also be used in the treatment of high pressure against cases resulting from basic, renal, pulmonary and endocrine diseases, while in the therapy of peripheral vascular diseases they are best used to treat aortic stenosis in the legs.

The compounds of the invention can also be used to prevent susceptibility to diseases caused by genetic determination or occasional weakening of the protective mechanism. The usual dose depends on the treated patient and the disease being treated and may vary in the range of 0.1 - 200 mg/kg, preferably 1 - 50 mg/kg, per day. This may mean, for example, that the daily dose for human therapy is between 10 and 200 mg orally, 1 to 15 mg rectally, or 2 to 20 mg parenterally, for adult patients.

Suitable pharmaceutical compositions may be, for example, solids or liquids, in any drug preparation generally in use in human or animal therapy, such as plain or coated tablets, gel capsules, granules, solutions, syrups, suppositories, philophilized or unphilophilized injection products; they can be prepared in the usual ways. The active substance can be transferred with aids common to these types of pharmaceutical products, such as milovka, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous and non-aqueous carriers, animal or vegetable fats, paraffin derivatives, glycols, various moisturizers, dispersing or emulsifying and preserving substances.

The biological effects of the compounds of the invention represented by the general formula (I) and (II) are shown through the following achievements by experiments on the relaxation effect of the vein performed in vitro on rats. Three-month-old, genetically hypertensive (SH) Wistar Okamoto rats were treated for 1 month with the various test compounds, followed by functional and morphological examinations.

THE VEIN RELAXING EFFECT OF TESTED HYDROXYLAMINE DERIVATIVES ON THE THORAX AORTA OF SH RATS (IN VITRO TEST)

The test was carried out in a manner known from the applied literature [Japan J. Pharmacol., 59, 339-347 (1992)]. SH rats were anesthetized with Nembutal (40 mg/kg, i.p.) and their thoracic aorta was removed and placed in oxygenated (95% O2 + 5% CO2) Krebs-Henseleit solution. Solution composition (mM): NaCl 118, KCI 4.7, CaCl2 2.52, MgSO41.64, NaHCO3 24.88, KH2PO4 l, 18, glucose 5.5. Vein rings 3 mm long were suspended in a 20 mL organic bath. at 37°C. The retained tension was 1 g, which was maintained throughout the experiment. During the 1-hour equilibration period, the medium of the organic bath was changed every 20 minutes.

The veins were contracted with 10-6 M methoxamine (approximately 80% of the maximum concentration). After reaching the maximum contraction, vasodilation induced with acetylcholine (Ach) (10-6 - 10-4 M) was examined, which was informative for the state of the vein wall endothelium. Contraction force was measured with an isometric device (SG-01D, Experimetria Ltd), and recorded on an OH-850 polygraph (Radelkis). The results of the experiment are summarized in table no. 1.

Table 1

Venous relaxing effect of tested hydroxylamine derivatives on the thoracic aorta of SH rats (in vitro test)

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As can be seen from the table, in cases of untreated hypertonic control animals, the relaxation induced by the application of 10-4 M acetylcholine dropped to 71%, which is a consequence of endothelial damage caused by high pressure. The tested compounds improve this reduction in vasodilation significantly, which shows an improvement in endothelial function.

MORPHOLOGICAL EXAMINATION OF THE THORACIC AORTA WITH AN ELECTRON MICROSCOPE

This experiment was carried out according to the applied literature (Br. J. of PharmacoL, 1995; 115, 415-420). 1 mm2 parts of the wall of the aorta, the aorta of the thorax of rats were cut and then fixed at room temperature with 2.5% glutaraldehyde. This was followed by fixation with 1% osmium tetroxide, which lasted for one hour. After that, tissue sections were dehydrated with ethanol and placed in Durcupan ACM. Excision was assessed for quality based on images taken on a Hitachi 7100 electron microscope. The achievements of these experiments are shown in table no. 2.

The results of the morphological tests are expressed on a scale of 1 to 5, depending on the extent to which the treatment with the test compounds restored the endothelial damage caused by high pressure, that is, the magnitude of the restoring effect. On the scale, 1 represents cases where no restoration was observed, 2 stands for weak, 3 for medium, 4 for good, while 5 represents strong restoration.

Compared to the untreated control, a significant protective or restorative effect was observed after treatment with the hydroxylamine derivatives of the general formula (I) and (II) of the invention. Thanks to the treatment, a thin protective layer was formed over the injured subendothelium, which is composed of cells containing an active nucleus and rich cytoplasm. The restoration appeared quite effective in most cases.

Table 2.

Electron microscope evaluation of the effect of compounds from the invention on the thoracic aorta of SH rats (morphological examination)

[image]

These experimental data also support the assumption that the compounds of the general formula (I) and (II) are able to restore the endothelium not only functionally but also morphologically. After chronic treatment, these compounds resulted in a more pronounced morphological restoration than the comparative substance Captopril.

EXAMINATION OF THE INFARCTION AREA IN SPONTANEOUSLY HYPERTENSIVE (SH) RATS AFTER ORAL TREATMENT FOR ONE MONTH

Experimental groups

1. SH same old supervision

2. Verapamil (as comparator), 50 mg/kg p.o.

3. Connection no. 13, 20 mg/kg p.o.

4. Connection no. 13, 50 mg/kg p.o.

5. Connection no. 5.5 mg/kg p.o.

6. Connection no. 4, 5 mg/kg p.o.

Inducing a heart attack

Myocardial ischemia was induced with intermittent occlusion of the left main coronary artery, according to Griswold et al. (J. Pharmacol Methods 1988, 20: 225-35). SH rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). After tracheotomy, animals were ventilated with room air with a small rodent ventilator (model: Harvard 552), with a stroke volume of 1.5-2 mL/100 g and a rate of 55 strokes/min to maintain normal pO2, pCO2, and pH parameters. The right carotid artery was characterized and connected to a pressure transducer (P236B Stetham) to measure systemic arterial blood pressure (BP) with the aid of a preamplifier (Hg-O2D Experimetria®). Heart rate (HR) was measured with a cardiotachometer (HR-01, Expeimetria®). The electrocardiogram (ECG standard lead III.) was recorded on a device printer (ER-14, Micromed®) with the help of subcutaneous electrodes with a steel needle. The chest was opened with a left thoracotomy and the heart was removed with gentle pressure on the right side of the ribs.

A 4/0 silk tourniquet is quickly placed under the left main coronary artery. The heart was returned to the thorax and the animal was left to recover. Rectal temperature was monitored and maintained with a crutch at 37°C. The experimental procedure started with a stabilization period of 15 minutes, during which the observation of a constant blood pressure of less than 70 mmHg and/or the appearance of an arrhythmia led to exclusion.

Myocardial ischemia was induced with coronary artery occlusion for 1 hour and reperfusion allowed for 1 hour. Ostensibly operated animals underwent all previously described surgical procedures except coronary occlusion and reperfusion.

Quantification of myocardial infarction

At the end of the attempt, the heart was suddenly removed. The left ventricle was cut into 2 mm thick sections parallel to the atrioventricular groove. The sections were incubated in a 0.1% solution of Nitroblue tetrazole grade III, pH 7.4 for 15 minutes.

The non-infarcted area is colored blue due to the formation of a precipitate resulting from the reaction of NBT with dehydrogenase enzymes. The loss of these enzymes from the infarcted myocardium prevents the formation of precipitates; therefore, the infarcted area adjacent to the risk area remains pale yellow. LV sections were imaged (Practica) and the infarcted area was measured planimetrically. The necrotic area is expressed as a percentage of the left ventricular surface.

Statistical analysis

All values will be expressed as ±SEM. Comparison between groups will be assessed with one-way ANOVA with post hoc analysis using the Student "t" test. Statistical significance will be determined as p<0.05.

Achievements

There was no significant difference in hemodynamic parameters, left ventricle and body weight between the groups.

Table 3

Infarct size and survival rate of SH rats after coronary artery occlusion and reperfusion

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**p<0.01 vs control #p<0.01 vs verapamil

After coronary artery occlusion and reperfusion, a decrease in survival of control rats was observed. Administration of various active compounds (except compound no. 5) and the reference substance verapamil orally for one month significantly increased the resistance of rats to myocardial ischemia/reperfusion injury.

Compared to verapamil, the improvement was significantly greater after treatment with compound no. 13 (20 mg/kg) and compound no. 4. Active compounds and verapamil significantly reduce infarct size compared to control animals. Infarct size limitation was dose dependent. Higher doses significantly reduce the extent of myocardial necrosis compared to verapamil.

Our experiments indicate that the selected effective compounds significantly reduce the extension of myocardial necrosis and significantly improve the survival rate. Infarct size limitation that occurred without any noticeable changes in hemodynamic parameters, and which was significantly greater after treatment with the compounds of the invention than after administration of the reference substance verapamil.

INVESTIGATION OF DISPLACEMENT OF INJURIES

HUVEC cells were isolated and cultured according to Jaffe E.A. et al. (J. Clin. Invest, 52, 2745-2756, 1973). The test was performed as described by Vamamura S. et al. (J. Surg. Res., 63, 349-254, 1996). HUVEC cells were seeded in a 96-well plate precoated with fibronectin (2μg/well)(Sigma), and at approximately 90% confluency, the monolayer was injured along the coordinate union marked on the back of the plate. The layer was injured with a Teflon cell scraper so that it was 1 mm in width. The well was washed and filled with complete RPMI 1640 medium (containing 5% protein) for incubation (at 37°C in 5% CO2 containing air. Cells were allowed to migrate for 24 and 48 hours to the injured field and recorded or imaged through an inverted microscope (at x60 magnification) for recording.The number of cells that migrated below the reference Union was counted and evaluated with an image analyzer.

Test compound procedure

A serial tenfold dilution in medium was prepared and 5 μL/well containing 95 μL of cultured tissue was added over the injured cell monolayer. Control cultivation did not contain dilution of compound no. 13.

Achievements

After incubation for 24 hours, cells appeared spontaneously in the injured surface and even increased the number counted in the presence of the tested compound at submicromolar concentration (at 10-7 and 10-8 M). The tested compound resulted in a distinct, significant stimulation of cell migration even in a 48-hour period. The injured surface was covered about 82% compared to 47% with spontaneously transplanted human endothelial cells.

Table 4.

Achievements of testing the displacement of injuries with compound no. 13

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* Untreated control, spontaneous transfer

** Cells on the injured surface immediately after wounding, surveillance situation

The process of repairing the damaged vascular monolayer started with the displacement of endothelial cells so that the restoration of the damaged surface can then begin. All of our data indicate that the test compound can promote repair in injured human endothelial cells by directly increasing migration.

The invention is shown in the following examples without any limitation of the scope of the claims: Detailed description of preferred embodiments

Example 1

N-[2-benzoyloxy-3-(1-piperidinyl)propoxy]1-3-pyridinecarboxyimidamide (Z)-2-butenedioate (1:1) (compound no. 1)

Procedure:

20.9 g (75.0 mmol) of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide [Hung. Pat. 177,578 (1976)] was dissolved in 300 mL of benzene. 150 mL of 1 N sodium hydroxide solution was added to this solution, followed by the dropwise addition of 19.5 mL (168 mmol) of benzoyl chloride. After stirring vigorously for 2 hours, 7.1 g (67 mmol) of sodium carbonate and additional benzoyl chloride (9.75 mL; 84 mmol) were added and stirring was continued overnight. The layers were then separated, the organic portion was extracted with 1 N sodium hydroxide solution and water, dried and evaporated to dryness. The residue (41 g of oil) was dissolved in 150 mL of acetone and 8.7 g (75 mmol) of maleic acid was added. The precipitate obtained was filtered, washed with acetone and dried.

Yield: 29.0 g (78%) T.t: 194-195°C

Example 2

N-[2-palmitoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide monohydrochloride (compound no. 2)

Procedure:

14.7 g (52.8 mmol) of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide [Hung. Pat. 177,578 (1976)] was dissolved in 160 mL of chloroform. 7.7 mL (55 mmol) of triethylamine was added, followed by dropwise addition of a solution of pacnitoyl chloride (14.7 g; 56.5 mmol) in 85 mL of chloroform. The mixture was stirred overnight at room temperature. The following day, an amount of 3.8 mL of triethylamine and 7.4 g of palmitoyl chloride was added and stirring was continued for another day. The solution was then extracted successively with water, 5% acetic acid and water, dried over anhydrous sodium sulfate and evaporated to dryness. The residue (28.2 g of oil) was dissolved in ethyl acetate, and the product was precipitated by the addition of 30 mL of 1 N HCl/ethyl acetate. The thick white precipitate was filtered, washed with ethyl acetate and dried.

Yield: 10.9 g (37%)

T.t: 110-113°C

Example 3

N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxyl-3-trifluoromethylbenzenecarboxylimidoyl chloride monohydrochloride (compound No. 3)

Procedure:

Degree a)

50 g (0.245 mol) of m-trifluoromethyl-benzamidoxime and 33.7 g (0.6 mol) of potassium hydroxide were dissolved in a mixture of dimethylsulfoxide and 170 mL of water, and the mixture was cooled to 0°C. 48 mL (0.6 mol) of epichlorohydrin was added and the reaction mixture was stirred at 0°C for 5 hours and then kept in the refrigerator overnight. The next day, 250 mL of water was added and the mixture was extracted with ethyl acetate (4 x 250 mL ). The combined organics were washed with water, dried, treated with activated carbon and evaporated to dryness to give m-trifluoromethyl-N-(2,3-epoxypropoxy)-benzamidine as a colorless oil. Yield: 61 g (96%)

Degree b)

400 mL of 18% hydrochloric acid solution and 60 mL of ether were added to the obtained oil, and the mixture was cooled to -5°C with stirring. Over 40 minutes, 17.4 g (0.25 mol) of sodium nitrite dissolved in 60 mL of water was slowly added and the reaction mixture was stirred for another 20 minutes. The mixture was then extracted with ether (2 x 160 mL) and the collected organics were washed twice with water. 340 mL of 20% sodium hydroxide solution was added to the ether solution and the two-phase system was refluxed for 1 hour with stirring. The layers were then separated, the organic portion was washed with brine until neutral, dried and evaporated to dryness to give m-trifluoromethyl-N-(2,3-epoxypropoxy)-benzimidoyl chloride as a colorless oil. Yield: 30.5 g (45%)

Degree c)

A mixture of 1.19 g (4.2 mmol) of m-trifluoromethu-N-(2,3-epoxypropoxy)-benzimidoyl chloride and 0.89 mL (8.5 mmol) of tert-butylamine in 12 mL of isopropyl alcohol was refluxed through 2 an hour. The solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate and 0.98 mL of a solution of hydrochloric acid in methanol (4.3 N) was added and the mixture was concentrated to a small volume under vacuum and then diluted with ether. The resulting precipitate was isolated, washed with cold ether and dried. Yield: 0.48 g (32%)

M.p.: 150-153°C

IR (KBr): 3423, 3233,2978, 2880, 2784, 1620, 1570, 1479, 1441, 1400, 1383, 1340, 1238, 1167, 1128, 1101, 1072, 1038, 982, 930, 87897 , 714, 694 cm-1

Example 4

N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboxyimidoyl chloride monohydrochloride (compound no. 4)

Procedure:

5.0 g (15.6 mmol) of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboxyimidamide monohydrochloride was dissolved in 19 mL of water and then 6.1 mL of concentrated hydrochloric acid was added. The solution was cooled to -5°C and then a cold solution of 4.4 g (63.8 mmol) of sodium nitrite in 2.4 mL of water was added dropwise. During the reaction, the internal temperature was maintained at 0°C. After the addition was completed, the mixture was stirred for another hour. Cold benzene (60 mL) was added and the mixture was quenched by slowly adding a cold solution of 3.2 g (80 mmol) sodium hydroxide in 45 mL of water. The organic layer was separated and successively washed with portions of 20 mL of water until pH<9 (3-5 times). The organic solution was dried over anhydrous sodium sulfate, treated with activated carbon, filtered and evaporated in vacuo (t<45°C) to give 2.6 g of an oil. This residue was dissolved in 5 mL isopropyl alcohol and acidified (pH2) with isopropyl alcohol containing dry hydrochloric acid. The product was crystallized from n-hexane to give a whitish substance.

Yield: 2.0 g (38%)

M.p.: 115-123°C

Following the procedure described in the previous example, the following compounds were prepared:

Example 5

N-[2-Hydroxy-3-(1-piperidinyl)propoxy]benzenecarboxyimidoyl chloride monohydrochloride (Compound No. 5)

Starting substance: N-[2-hydroxy-3-(1-piperidinyl)propoxy]benzenecarboxyimidamide

Utilization: 23%

M.p.: 140-145°C

Example 6

N-[2-Hydroxy-3-(1-piperidinyl)propoxy]-4-pyridinecarboxyimidoyl chloride (Z)-2-butenedioate (1:1) (Compound No. 6)

Starting material: N-[2-hydroxy-3-(1-piperidinyl)propoxy]-4-pyridinecarboxynidamide In this case, the final product was isolated at the end of the work-up by dissolving the crude alkali in acetone and adding an equivalent amount of maleic acid. Utilization: 25%

M.p.: 150-154°C

Example 7

N-[2-Hydroxy-3-(1-piperidinyl)propoxy]-2-nitrobenzenecarboxyimidoyl chloride monohydrochloride (Compound No. 7)

Starting substance: N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-nitrobenzenecarboxyimidamide

Utilization: 36%

M.p.: 158-162°C

Example 8

N-[3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidoyl chloride dihydrochloride (compound No. 8)

Starting material: N-[3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide

Utilization: 33%

M.p.: 178-182°C

Example 9

N-[3-(1-piperidinyl)propoxy]-3-nitrobenzenecarboxyimidoyl chloride monohydrochloride (Compound No. 9)

Starting material: N-[3-(1-piperidino)propoxy]-3-nitrobenzenecarboximidamide

Utilization: 49%

M.p.: 173-175°C

Example 10

N-[3-[(1,1-dimethylethyl)aminol-2-hydroxypropoxy]-3-trifluoromethylbenzenamide (compound no. 10)

Procedure:

1.3 mL (15.2 mmol) of epichlorohydrin was added to a solution of 1.6 mL (15.2 mmol) of tert-butylamine in 8 mL of ethanol over 10 min with stirring, maintaining the temperature below 20°C, and allowed to stand for 3 days. Separately, 0.8 g (14.3 mmol) of potassium hydroxide was dissolved in a mixture of 20 mL of ethanol and 3 mL of water, and 3.42 g (15.2 mmol) of N-hydroxy-3-(trifluoromethyl)-benzamide were added to this solution. potassium salts and the previously prepared solution of epichlorohydrin and tert-butylamine. The reaction mixture was stirred and boiled for 10 hours and then the solvent was evaporated. The residue was triturated with 20 mL dichloromethane and 10 mL water, the organic part was separated, washed with 5 mL water and 5 mL saturated sodium chloride solution, dried over sodium sulfate, filtered and evaporated. The oily residue was crystallized from acetone-hexane to give the title compound as a white powder.

Yield: 0.85 g (17.3%)

M.p.: 156-158°C

IR(KBr): 2976, 2858, 1612, 1556, 1379, 1352, 1313, 1273, 1165, 1130, 1072, 694 cm-1

Example 11

N-hexyl-N'-[2-hydroxy-3-(1-piperidinyl)propoxy]-urea (compound no. 11)

Procedure:

4.9 mL (45.9 mmol) of hexyl isocyanate was added to a solution of 8.0 g (45.9 mmol) of 1-aminooxy-2-hydroxy-3-(1-piperidinyl)-propane dissolved in 60 mL of chloroform, and the reaction mixture was stirred for 20 hours at room temperature. After addition of an additional 1.6 mL (15 mmol) of hexyl isocyanate, stirring was continued for another two hours, when the solvent was evaporated in vacuo. The white crystalline product was obtained by trituration with petroleum ether.

Yield: 9.9 g (72%)

T.t: 50-52°C

IR(KBr): 3310, 2932, 2858, 2804, 1666, 1551, 1454, 1377, 1306, 1092, 1040, 995, 791, 725, 604 cm-1

Following the procedure described in the previous example, the following compound was prepared:

Example 12

N-hexyl-N'-[3-(1-piperidinyl)propoxy]-urea (compound no. 12)

Starting substance: 1-aminooxy-3-(1-piperidinyl)-propane

Utilization 85% (oil)

IR (KBr): 3354, 2932, 2856, 2810, 2777, 1666, 1543, 1486, 1377, 1308, 1155, 1134, 1076 cm-1

Example 13

5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridiyl)-4H-1,2,4-oxadiazine (compound no. 13)

Procedure:

Degree a)

17.5 g (0.05 mol) of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxymidamide dihydrochloride was dissolved in 50 mL of thionyl chloride, boiled for one hour and then the mixture was evaporated to dryness . The residue was dissolved in 300 mL of methanol, treated with activated carbon and, after filtration, the solvent was evaporated under reduced pressure. The residue was dissolved in a minimal amount of ethanol and refrigerated to give crystalline N-[2-chloro-3-(1-piperidinyl)propoxy]-3-pyridinecarboxymidamide dihydrochloride as an intermediate.

Yield: 13.2 g (71%)

M.p.: 127-145°C

Degree b)

13.2 g (35.7 mmol) of N-[2-chloro-3-(1-piperidinyl)propoxy]-3-pyridinecarboxymidamide dihydrochloride was added to a solution of 16.5 g (143.5 mmol) of potassium tert-butoxide dissolved in 150 mL tert-butanol. The mixture was boiled for 6 hours, then evaporated in a vacuum. 100 mL of 5% sodium hydroxide solution was added and the mixture was extracted three times with 300 mL portions of ethyl acetate. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. The residue was triturated with diethyl ether to give the title compound as white crystals.

Yield: 3.5 g (38%)

M.p.: 157.5-158°C

Example 14

Pills

For the production of 200 mg tablets containing 50 mg of the active ingredient, use:

50 mg of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-benzenecarboxyimidoyl chloride monohydrochloride

129 mg of microcrystalline cellulose (for example "Avicel ph 102")

20 mg polyvinyl-pyrrolidone (for example "polyplasdone XL")

1 mg of magnesium stearate

Example 15

Capsules

For 300 mg capsules, use:

50 mg N-[2-hydroxy-3-(1-piperidyl)propoxy]-2-nitro-benzenecarboxyimidoyl chloride monohydrochloride

10 mg of yellow beeswax

10 mg soybean oil

130 mg of vegetable oil

100 mg of capsular cells

Example 16

Solution

For 100 mL of solution, use:

500 mg N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboxyimidoyl chloride monohydrochloride

10 g of sorbitol

0.05 g sodium saccharin

add 100 mL of double distilled water

Example 17

Injection ampoule

For each 2 mL injection ampoule containing 2 mg of the active substance, use:

2 mg 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine

add 2.0 mL of physiological saline solution, pyrogenic, sterile.

Example 18

Infusion solution

For 500 mL of infusion solution, use:

20 mg of N-hexyl-N'-[3-(1-piperidinyl)propoxy]-urea

add 500 mL of physiological saline solution, pyrogenic, sterile.

Claims (16)

1. Use of hydroxylamine derivatives of the general formula (I) and (II) - where R1 and R2 independently of each other refer to a hydrogen atom or a straight or branched alkyl group of 1 to 6 carbon atoms, or R1 and R2 together with a nitrogen atom between form a saturated heterocyclic group with 5 - 7 members, which optionally contains more nitrogen and/or oxygen heteroatoms, A refers to a straight or branched alkyl group with 4 to 12 carbon atoms, a phenyl group, substituted or not, which preferably contains an alkyl, haloalkyl or nitro group as a substituent, or a 5-6 membered heteroaromatic ring containing nitrogen, oxygen or sulfur, in the compounds of the general formula (I), Z refers to a covalent bond, and in the compounds of the general formula (II) to a covalent bond or =NH group, in the general formula (I), X refers to a halogen atom or to the -NR3R4 group , where R3 and R4 independently refer to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms, while in the general formula (II) X refers to an oxygen atom, in the general formula (II), R' refers to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms, and in the general formulas (I) and (II), Y refers to a hydrogen atom, a hydroxyl group or an acyloxy group, which preferably contains the acyl part of a long fatty acid chain with 8 to 22 carbon atoms, or a cyclic aromatic carboxylic acid as its acyl component, and in compounds of the general formula (I) where X refers to the -NR3R4 group and Y refers to . to the hydroxyl group, the X group is condensed with the Y substituent and forms an intramolecular ring represented by the general formula (II), in which the formulas A, Z, R1 and R2 have the same meaning as above, and salts and optically active forms of these compounds, characterized in that it is used for the production of a drug useful for treating or preventing diseases associated with impaired endothelial cell function. 2. The use of N-[2-benzoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide (Z)-2-butenedioate (1:1), characterized by the fact that it is an effective ingredient in the process from claim 1. 3. The use of N-[2-palmitoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidamide monohydrochloride, indicated by the fact that it represents an effective ingredient in the process from claim 1. 4. The use of N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3-trifluoromethylbenzenecarboxyimidoyl chloride mono hydrochloride, indicated by the fact that it represents an effective ingredient in the process from claim 1. 5. The use of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboxyimidoyl chloride monohydrochloride, characterized by the fact that it is an effective ingredient in the process of claim 1. 6. Use of N-[2-hydroxy-3-(1-piperidinyl)propoxy]benzenecarboxynidoyl chloride monohydrochloride, indicated by the fact that it is an effective ingredient in the process of claim 1. 7. Use of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-4-pyridinecarboxyimidoyl chloride (Z)-2-butenedioate, characterized by the fact that it is an effective ingredient in the process of claim 1. 8. The use of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-nitrobenzenecarboxyimidoyl chloride monohydrochloride, indicated by the fact that it is an effective ingredient in the process of claim 1. 9. The use of N-[3-(1-piperidinyl)propoxy]-3-pyridinecarboxyimidoyl chloride dihydrochloride, indicated by the fact that it is an effective ingredient in the process of claim 1. 10. The use of N-[3-(1-piperidinyl)propoxy]-3-nitrobenzenecarboxyimidoyl chloride monohydrochloride, indicated by the fact that it is an effective ingredient in the process of claim 1. 11. The use of N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3-trifluoromethylbenzamide, indicated by the fact that it is an effective ingredient in the process of claim 1. 12. Use of N-hexyl-N'-[2-hydroxy-3-(1-piperidinyl)propoxy]-urea, characterized by the fact that it is an effective ingredient in the process of claim 1. 13. Use of N-hexyl-N'-[3-(1-piperidinyl)propoxy]-urea, characterized by the fact that it is an effective ingredient in the process of claim 1. 14. The use of 5,6-dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine, indicated by the fact that it is an effective ingredient in the process of claim 1. 15. A method of treating or preventing diseases associated with impaired functioning of endothelial cells, indicated by the fact that the patient is given hydroxylamine derivatives of the general formulas (I) and (II) - where R1 and R2 independently of each other refer to a hydrogen atom or straight or branched an alkyl group of 1 to 6 carbon atoms, or R1 and R2 together with a nitrogen atom in between form a saturated heterocyclic group with 5 - 7 members, which optionally contains further nitrogen and/or oxygen heteroatoms, A refers to a straight or branched alkyl group with 4 to 12 carbon atoms, a phenyl group, substituted or not, which preferably contains an alkyl, haloalkyl or nitro group as a substituent, or a heteroaromatic ring with 5-6 members containing nitrogen, oxygen or sulfur, in compounds of the general formula (I), Z refers to a covalent bond, and in the compounds of the general formula (II) to a covalent bond or =NH group, in the general formula (I), X refers to a halogen atom or to the -NR3R4 group, where R3 and R4 independently of each other refer to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms, while in the general formula (II) X refers to an oxygen atom, in the general formula (II), R' refers to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms, and in the general formulas (I) and (II), Y refers to a hydrogen atom, a hydroxyl group or an acyloxy group, which preferably contains the acyl part of a long fatty acid chain with 8 to 22 carbon atoms, or a cyclic aromatic carboxylic acid as its acyl component, and in the compounds of the general formula (I) where X refers to the -NR3R4 group and Y refers to the hydroxyl group, the X group is condensed with the substituent Y and forms an intramolecular ring that is represented by the general formula (III), in which formula A, Z , R1 and R2 have the same meaning as above or their salts and optically active forms. 16. Pharmaceutical products for the treatment and prevention of diseases associated with the impaired functioning of vascular endothelial cells, indicated by the fact that they include as an effective ingredient (apart from the usual carriers and auxiliary substances used in pharmaceutical preparations) 0.5-95.5 m/m% of hydroxylamine derivatives general formulas (I) or (II), or their salts and optically active forms, where R1 and R2 independently of each other refer to a hydrogen atom or a straight or branched alkyl group of 1 to 6 carbon atoms, or R1 and R2 together with with a nitrogen atom in between they form a saturated heter o cyclic group with 5 - 7 members, which optionally contains more nitrogen and/or oxygen heteroatoms, A refers to a straight or branched alkyl group with 4 to 12 carbon atoms, a phenyl group, substituted or not, which preferably contains an alkyl, haloalkyl or nitro group as a substituent, or a 5-6 membered heteroaromatic ring containing nitrogen, oxygen or sulfur , in compounds of general formula (I), Z refers to a covalent bond, and in compounds of general formula (II) to a covalent bond or =NH group, in the general formula (I), X refers to a halogen atom or to the -NR3R4 group, where R3 and R4 independently of each other refer to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms, while in the general formula ( II) X refers to an oxygen atom, in the general formula (II), R' refers to a hydrogen atom or a straight or branched alkyl group with 1 to 6 carbon atoms, and in the general formulas (I) and (II), Y refers to a hydrogen atom, a hydroxyl group or an acyloxy group, which preferably contains the acyl part of a long fatty acid chain with 8 to 22 carbon atoms, or a cyclic aromatic carboxylic acid as its acyl component, and in the compounds of the general formula (I) where X refers to the -NR3R4 group and Y refers to the hydroxyl group, the X group is condensed with the substituent Y and forms an intramolecular ring that is represented by the general formula (III), in which formula A, Z , R1 and R2 have the same meaning as above.