RU2195273C2 - Pharmaceutical compounds for treatment and prophylaxis of diseases arising as result of damage of vascular endothelial cells - Google Patents

Abstract

FIELD: organic chemistry, pharmacy. SUBSTANCE: invention relates to application of definite derivatives of hydroxylamine of the general formula (I)

and (II)

Description

Field of Invention
The invention relates to products intended for the treatment and prevention of diseases resulting from damage to vascular endothelial cells that contain, as an effective agent, a hydroxylamine derivative of the general formula (I) or (II).

State of the art
The normal functioning of vascular endothelial cells is extremely important for the body. These cells form the boundary of the surface between the circulating blood and the elements of the vein wall, performing thrombogenic activity. The role of vascular endothelial cells in homeostasis is completely different:
- they take part in the two-way transfer of substances coming from blood and tissues,
- form a barrier to macromolecules,
- in these cells, the synthesis and decomposition of mediators participating in the regulation of the interaction between the cellular elements of the vein wall and blood (for example, fibrinogen, collagen, proteoglycans, PGI ₂ , EDRF (NO), EDNF, endothelin-II, angiotensin-II) is localized,
- they initiate migration, proliferative and thrombolytic processes that contribute to tissue regeneration,
- support thrombosis of venous walls [Rubanyi, G., J., Cardiovasc. Pharmacol 1933, 22 (42. Suppl., P. S1-14).

 Damage to the endothelium leads to atherosclerosis. Damage leading to endothelial wear can occur with mechanical intervention, such as catheterization, as well as biochemical and immunological processes.

 The first stage of atherosclerotic plaque formation is the filling of cells with lipids that accumulate in the inner wall of arteries (Steinberg D. Et al., JAMA 264-304, 1990). These cells - especially monocytes and macrophages coming from the blood - initially attach to the endothelium, and then penetrate into the walls of the vessel. Damage to endothelial cells can also contribute to adhesions, although no morphological changes are visible at an early stage. Oxidation of LDL (low density lipoproteins) of particles can result in their incorporation into monocytes localized in the inner walls, thus monocytes become xanthous (foamy) cells. These xantomic (foamy) cells form lipid plates, the appearance of which refers to atherosclerotic changes at an early stage.

 In the later stages, bleeding, necrosis, neovascularization, and sclerosis appear, resulting in the growth of plaques that constrict the arteries (Ir. JH, Fuster et.al., J. Am. Col. Cardiol 15: 1667, 1990).

 Thrombosis can occur at various stages of atherosclerosis. Repeated thrombosis leads to vascular blockage and thromboembolic diseases, such as coronary thrombosis, cerebral vascular thrombosis, or peripheral vascular disease.

 From a medical point of view, "endothelial dysfunctional syndrome" means a general or localized vessel spasm, thrombosis, atherosclerosis and repeated stenosis. They try to treat these diseases using methods of surgical intervention, bypass surgery and medical treatment.

Only some of the existing drugs may be suitable for the "treatment" of endothelial dysfunction. They can be divided into four main categories:
- substitutes for natural “protective” endothelial substances (for example, stable PGI ₂ analogues, nitro-vasodilators, rt-PA / recombinant tissue plasminogen activator);
- inhibitors or antagonists of contact factors produced by the endothelium (for example, ACE inhibitors, angiotensin II receptor antagonists; TXA ₂ receptor antagonists);
- protective cellular agents (for example, superoxide dismutase and probucol free radical scavengers, and free radical formation inhibitors);
- drugs that reduce the amount of lipids.

 Despite the fact that none of these drugs was originally developed for these purposes, they have already confirmed a positive clinical result of the action, which in the case of certain diseases may include the protection or restoration of normal endothelial function. Rational for innovative therapy in this category is the restoration of normal endothelial cellular behavior, in which these cells themselves will "do their job." Potential approaches may include the growth of new normal endothelium or new recombinant DNA-based therapeutic modeling technologies (Science 1990; 249: 1285-8). According to the data obtained, none of these medicines satisfies the listed requirements.

 Currently, there is no known medical tool that would be able to directly affect the endothelium, that is, there is no medical tool suitable for the treatment of endothelial dysfunction. Thus, there is a huge need for a drug that can prevent, reverse, or at least slow down the formation of complicated symptoms, or reduce the likelihood of a disease.

SUMMARY OF THE INVENTION
In the process of research, it was found that hydroxylamine derivatives of the general formulas (I) and (II) have a strong protective and regenerating effect on vascular endothelial cells and are able to prevent damage to them of any origin.

In the general formula (I) and (II), R ¹ and R ² independently from each other represent a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, or R ¹ and R ² together with a nitrogen atom, located between them, form a saturated 5-7-membered heterocyclic group, which optionally contains another heteroatom of nitrogen and / or oxygen,
A is a straight or branched alkyl group containing from 4 to 12 carbon atoms, a phenyl group substituted or unsubstituted, preferably containing 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 is a covalent bond and in the compounds of the general formula (II) a covalent bond or group = NH,
in the compounds of general formula (I), X is a halogen atom or an NR ³ R ⁴ group, where R ³ and R ^{4 are} independently a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, while in compounds of General formula (II) X is an oxygen atom,
in the compounds of general formula (II), R is a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, and
in the general formulas (I) and (II), Y is a hydrogen atom, a hydroxyl group or an acyloxy group, which preferably contains as acyl part a long chain fatty acid containing from 8 to 22 carbon atoms, or the acyl component is cyclic aromatic acid, and
in compounds of the general formula (I) in which X is an NR ³ R ⁴ group and Y is a hydroxyl group, the X group is bonded to the substituent Y and forms an intramolecular ring.

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

Those compounds of general formula (I) in which X is an NH ₂ group and A is an unsubstituted phenyl or pyridyl group and in which Y is a hydroxyl group are already known and are disclosed in French Patent Specification No. 2,362,845 A1.

 These compounds are selective beta blockers and are useful in the treatment of diabetic angiopathy as described above.

 Those compounds of general formula (I) in which X is a halogen atom, Y is a hydroxyl group, and A is an unsubstituted or substituted phenyl or pyridyl group are already known from published PCT application WO 90/04584 A1.

 These compounds have a selective beta blocking effect and are thus useful in the treatment of diabetic angiopathy.

 Those compounds of general formula (II) in which A is a phenyl (unsubstituted or substituted haloalkyl group), pyridyl or thienyl group, Z is a covalent bond, R is a hydrogen atom and Y is a hydroxyl group are already known and disclosed in the description of the Hungarian patent 2385/92 published under T / 66350. These compounds have anti-ischemic and anti-anginal effects and are thus useful in the treatment of veins in case of their disease in diabetes mellitus.

 Those compounds of general formula (I) in which A represents an aromatic or heteroaromatic ring and X represents a halogen atom, Y represents a hydrogen atom, are already known and are disclosed in PCT published application N 95/30649 A1. These compounds have an anti-ischemic effect and can be successfully used in the treatment of a disease such as ischemia, which is characterized by hypertensive veins and the appearance of platelets.

 None of the listed publications indicate that the described compounds may have an effect on vascular endothelial cells.

 As indicated above, our studies have confirmed that compounds of the general formula (I) and (II) can have an effect on endothelial cells of the cardiovascular and cerebrovascular systems. In experiments that will be described in detail, it was found that these compounds are capable of blocking or repairing damage to these cells. So, these compounds can be used as effective substances in drugs that are used in the treatment of diseases resulting from abnormal functioning or damage to endothelial cells, especially diseases of the cardiovascular or cerebrovascular system, hypertension, hyperhomocysteinemia and peripheral diseases vessels.

Based on these observations, the invention includes the use of hydroxylamine derivatives of the general formula (I) and (II), where R ¹ and R ² independently from each other represent a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, or R ¹ and R ² together with the nitrogen atom located between them, form a saturated 5-7-membered saturated heterocyclic group, which optionally contains another heteroatom of nitrogen and / or oxygen,
A is a straight or branched alkyl group containing from 4 to 12 carbon atoms, a phenyl group substituted or unsubstituted, preferably containing 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 general formula (I) Z is a covalent bond and in the compounds of general formula (II) is a covalent bond or group = NH,
in the compounds of general formula (I), X is a halogen atom or an NR ³ R ⁴ group, where R ³ and R ^{4 are} independently a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, while in compounds of General formula (II) X is an oxygen atom,
in the compounds of general formula (II), R is a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, and
in the general formulas (I) and (II), Y is a hydrogen atom, a hydroxyl group or an acyloxy group, which preferably contains as acyl part a long chain fatty acid containing from 8 to 22 carbon atoms, or the acyl component is cyclic aromatic acid, while in certain types of compounds of the general formula (I) in which X is an NR ³ R ⁴ group and Y is a hydroxyl group, the X group is linked to a substituent Y and forms an intramolecular ring represented by the general formula (III), where A, Z, R and R ² and eyut defined above.

 Moreover, salts and optically active forms of these compounds are used to obtain drugs used for the treatment and prevention of diseases associated with endothelial cell dysfunction.

The invention also relates to pharmaceutical products used for the treatment and prevention of diseases associated with the abnormal functioning of vascular cells, which contain, as an effective compound (in addition to the usual carriers and additional substances used in pharmaceutical compositions), a compound of the general formula (I) or (II) in an amount of 0.5-95.5 m / m%, or in some cases, salts or optically active forms of these compounds, with the values of R ¹ , R ² , A, X, Y and R defined above.

Most preferred for use according to the invention are those compounds of general formula (I) and (II) in which A is a pyridyl, thienyl or phenyl, nitrophenyl or trifluoromethylphenyl group. Also preferred are those compounds of general formula (I) in which X is a chlorine atom or an NH ₂ group. Of these latter compounds, compounds containing an intramolecular ring formed by linking groups X and Y are particularly preferred. Those compounds of general formula (II) in which R is a hydrogen atom and those compounds of general formula (I) and (II) are also preferred. in which Y is a hydrogen atom or a hydroxyl group. Of all the categories of compounds listed, those compounds are preferred in which the NR ¹ R ² group is a piperidino or dialkylamino group.

The following compounds of general formulas (I) and (II) are particularly preferred according to the invention:
N- [2-benzoyloxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidamide (Z) -2-butenedioate (1: 1) (compound 1)
N- [2-palmitoyloxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidamide monochlorohydrate (compound 2)
N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylphenylcarboxyimidoyl chloride monochlorohydrate (compound 3)
N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-thiophenecarboxyimidoyl chloride monochlorohydrate (compound 4)
N- [2-hydroxy-3- (1-piperidinyl) propoxy] phenylcarboxyimidoyl chloride monochlorohydrate (compound 5)
N- [2-hydroxy-3- (1-piperidinyl) propoxy] -4-pyridinecarboxyimidoyl chloride (Z) -2-butenedioate (1: 1) (compound 6)
N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-nitrophenylcarboxyimidoyl chloride monochlorohydrate (compound 7)
N- [3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidoyl chloride dichlorohydrate (compound 8)
N- [3- (1-piperidinyl) propoxy] -3-nitrophenylcarboxyimidoyl chloride monochlorohydrate (compound 9)
N- [3- (1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylbenzamide (compound 10)
N-hexyl-N '[2-hydroxy-3- (1-piperidinyl) propoxy] urea (compound 11)
N-hexyl-N '[3- (1-piperidinyl) propoxy] urea (compound 12)
5,6-dihydro-5- (1-piperidinyl) methyl-3- (3-pyridyl) -4H-1,2,4-oxadiazine (compound 13)
Those compounds of general formula (I) in which X is a halogen atom can be obtained by reacting an amidoxime of general formula (V) in which A has the above meanings with an amino-chloro-propane derivative in which R ¹ , R ² and Y have the above meanings, and the NH ₂ group of the obtained intermediate product of general formula (IV), in which Z is a covalent bond and the remaining substituents are as defined above, is replaced by a halogen atom by a diazotization reaction. In cases where a compound of general formula (I) is obtained containing a hydroxyl group as substituent Y, the amino-chloropropane derivative of general formula (XII) necessary for this reaction containing a hydroxyl group as substituent Y can be obtained from epichlorohydrin of the formula (VI) and an amine of the general formula (IX) in which R ¹ and R ² are as defined above. An alternative process involves first the reaction of epichlorohydrin with an amidoxime of general formula (V), followed by diazotization of the obtained intermediate product of general formula (VII) in which A has the above meanings, and the reaction of an epoxy compound of general formula (VIII) in which A has the above meanings with an amine of general formula (IX).

Those compounds of general formula (I) for which Y is an acyloxy group can be prepared by reacting suitable compounds of general formula (I) containing a hydroxyl group at the position of Y and acid chlorides of general formula (IX) in which R ⁵ is long an alkyl chain or an aryl group.

Compounds of general formula (II) containing, at the position of Z, a covalent bond, can be prepared by one of the following methods:
(i) linking an alkaline hydroxamate of the general formula (XIII) in which A and R are as defined above, and K is an alkali metal cation, a halide compound of the general formula (XII), in which R ¹ , R ² and Y are as defined above , or
(ii) an amino compound reaction of the general formula (XIV) in which R ¹ , R ² , Y and R are as defined above, with an acid halide in which A is as defined above,
however, these compounds of general formula (II) containing a covalent bond at the position of Z and a hydrogen atom at the position of R can also be obtained in addition to the above methods (i) and (ii) in the following ways:
(iii) by diazotization of a suitable compound of general formula (I) containing an NH ₂ group at position X and a covalent bond at position Z in a halogen-free medium, or
(iv) hydrolysis of a suitable compound of general formula (I) containing halogen at the location of X.

Those compounds of general formula (II) in which A has the meaning of an alkyl group and Z is a group = NH can be prepared by reacting a compound of general formula (II) in which R ¹ , R ² , Y and R have the above meanings, in an organic solvent medium, preferably chloroform, with an equimolar amount of alkyl isocyanate in which A represents an alkyl group.

 Compounds of general formula (III) are particular representatives of compounds of general formula (I), in which the nitrogen contained in the X group binds to the substituent Y to form an intramolecular ring.

 Such compounds can be prepared by reacting a compound of general formula (I) (in which Y is a hydroxyl group) with an excess of thionyl chloride, after which the ring closes on the resulting intermediate of general formula (IV) (in which Y is a chlorine atom, and the remaining substituents have the above values) with an excess of potassium tert-butoxide boiling in an organic solvent (preferably in tert-butanol).

 Hydroxylamine derivatives in accordance with the invention exhibit surprising pharmacological properties. It is especially necessary to emphasize that they not only restore endothelial cells morphologically, but also functionally, and this is achieved thanks to the action of these substances. It is also surprising that endothelial cells exhibit high tolerance to these compounds.

 These characteristics form the basis for the pharmaceutical use of hydroxylamine derivatives of the general formulas (I) and (II). These drugs can be used to treat cardiovascular and cerebrovascular diseases in animals and humans, such as hypertension, hyperhomocysteinemia, and peripheral vascular disease.

 Among cardiovascular diseases, these compounds can be used with great success in cases of coronary artery disease, atherosclerosis, repeated stenosis that occurs after balloon angioplasty and coronary artery bypass grafting, and among cerebrovascular diseases in case of cerebral artery occlusion, while hydroxylamine derivatives of the general formula (I) and (II) can also be used in the treatment of hypertension and are used in cases where it occurs as a result of idiopathic, renal, pulmonary and endocrine rin diseases, while in the treatment of peripheral vascular diseases they are most applicable when aortic stenosis appears on the legs.

 The compounds of the invention can also be used to prevent the occurrence of diseases due to a genetic predisposition or a temporary weakening of the protective mechanism. The usual dose depends on the patient who is being treated and on the development of the disease and can vary from 0.1-200 mg / kg, preferably from 1 to 50 mg / kg daily. This may mean, for example, that the daily dose for treating a person ranges from 10 to 200 mg when taken orally, from 1 to 15 mg for rectal administration and from 2 to 20 mg for parenteral administration for adult patients.

 Suitable pharmaceutical compositions can be, for example, solids or liquids, in any form of medication commonly used in human and veterinary medicine, such as coated tablets, gel capsules, granules, solutions, syrups, suppositories, lyophilized or not lyophilized products for injection; they can all be made in the usual way. An effective substance can be transferred using conventional types of excipients known for pharmaceutical products, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa bean oil, aqueous or non-aqueous carriers, animal and vegetable fats, paraffin derivatives, glycols, various moisturizers , dispersants, emulsifiers and preservatives.

 The biological efficacy of the compounds of the invention represented by the general formulas (I) and (II) is illustrated by the results of tests on the vasodilating effect obtained in experiments on rats in vitro, as well as in morphological tests on the thoracic aorta. Three-month-old genetically hypertensive (SH) Wistar Okamoto rats were treated for 1 month with various test compounds.

WASTE-EXTENDING EFFECT OF TESTED HYDROXYLAMINE DERIVATIVES ON THE RAT CHEST PORTFOLIO AORTA (EXPERIENCE IN VITRO)
The experiment is carried out in accordance with the method described in the literature [Japan J. Pharmacol., 59,339-347 (1992)]. SH rats were anesthetized with Nembutalum (40 mg / kg iv), after which their thoracic aorta was removed and placed in an oxygenated (95% O ₂ + 5% CO ₂ ) Krebs-Henseleit solution. The composition of the solution, mmol: NaCl 118, KCl 4.7, CaCl ₂ 2.52, MgSO ₄ 1.64, NaHCO ₃ 24.88, KH ₂ PO ₄ 1.18, glucose 5.5. 3 mm long thoracic aortic rings were suspended in a 20 ml organ bath at 37 ^° C. The residual stress was 1 g and remained unchanged throughout the experiment. During the equilibrium period of 1 hour, the medium in the organ bath was changed every 20 minutes. The vessels were narrowed by the action of 10 ^-6 mol of methoxyamine (approximately 80% of the maximum narrowing). After maximum narrowing was achieved, vasodilation caused by acetylcholine (Ach) (10 ^-6 -10 ^-4 mol) and the functional integrity of the endothelium were measured. The narrowing force was measured using an isometric voltage sensor (SG-01D, Experimetria Ltd) and recorded on an OH-850 polygraph (Radelkis). The results of the experiments are shown in table 1.

As follows from table 1, in the case of control animals with hypertension who did not receive the drug, the expansion caused by the use of acetylcholine at a concentration of 10 ^-4 mol is reduced to 71%, which occurs as a result of damage to the endothelium caused by hypertension. The compounds studied significantly compensate for this decrease in vasodilation, which indicates an improvement in the functioning of the endothelium.

MORPHOLOGICAL RESEARCH OF THE BREAST AORTA BY ELECTRONIC MICROSCOPY
This test was performed as described in the literature (Br. J. Of Pharmacol, 1995; 115, 415-420). The segments of the wall of the thoracic aorta of rats with an area of 1 mm ² were excised and then fixed with 2.5% glutaraldehyde at room temperature. This operation was followed by post-fixation with 1% osmium tetroxide, which lasted for 1 hour. After that, tissue segments were dehydrated with ethanol and placed in a Durcupan ACM instrument. Die cuts were quantified based on photographs taken with a Hitachi 7100 electron microscope. The results of these tests are shown in Table 2.

 The results of morphological tests were expressed on a scale of 1 to 5, depending on the degree to which treatment with the studied compounds restores endothelial damage caused by hypertension, i.e., the degree of regenerative activity. On a scale, the number 1 represents cases in which regeneration was not observed, 2 - in which it was weak, 3 - medium, 4 - good, and 5 represents strong regeneration.

 In comparison with the untreated control, a significant protective or regenerative effect was observed after treatment with hydroxylamine derivatives of the general formulas (I) and (II) according to this invention. Thanks to the treatment, a thin protective layer was formed located above the damaged subendothelium, which consisted of cells containing active nuclei and enriched cytoplasm. Regeneration seems very effective in most cases.

 These experimental data also support the assumption that the compounds of general formula (I) and (II) are able to regenerate endothelium not only functionally, but also morphologically. With prolonged use, these compounds lead to a more pronounced morphological regeneration than the captopril comparison compound.

STUDY OF THE INFARCTION AREA IN RATS WITH SPONTANEOUS HYPERTENSION (SH) AFTER ORAL ADMINISTRATION FOR A MONTH
Experimental groups
1. SH- adult matched control
2. Verapamil (preparation for comparison), 50 mg / kg orally
3. Compound 13, 20 mg / kg orally
4. Compound 5, 5 mg / kg orally
5. Compound 4, 5 mg / kg orally
Heart attack
Myocardial ischemia was caused by temporary occlusion of the main left coronary artery, as described by Griswold et al. (J. Pharmacol. Methods 1988, 20: 225-235). SH rats were anesthetized with sodium pentobarbital (50 mg / kg iv). After tracheotomy, the animals were ventilated with room air using a small rodent respirator (Harvard model 552) with a displaced piston volume of 1.5-2 ml / 100 g and a speed of 55 swings / min to maintain normal partial pressure of O ₂ , CO ₂ and pH.

The right carotid artery was catheterized and connected to it with a pressure sensor (R236V Stetham) for measurement of systemic blood pressure (BP) by means of a preamplifier (Hg-02D Experimentia ^®). Heart rate (HR) was measured using a cardiotachometer (HR-01, Experimentia ^® ). An electrocardiogram (ECG standard, lead III) was recorded on a recording instrument (ER-14, Micromed ^® ) using hypodermic steel needle electrodes. The chest was opened by left-side thoracotomy, and the heart was exposed as a result of light pressure on the right side of the chest.

 The main left coronary artery was quickly bandaged with 4/0 silk suture. The heart was returned to the place and the animal was left to recover.

The rectal temperature was determined and kept constant at 37 ^° C.

 The experimental protocol was started with a 15-min stabilization period during which all observed cases in which blood pressure was maintained below 70 mm Hg and / or arrhythmia occurred led to the exclusion of the animal.

  Myocardial ischemia was caused by coronary artery occlusion for 1 hour and subsequent reperfusion for 1 hour. Part of the operated animals was subjected to all previously described surgical procedures, with the exception of coronary occlusion and reperfusion.

Quantification of myocardial infarction
At the end of the experiment, the heart was quickly removed. The left ventricle was cut into pieces 2 mm thick parallel to the atrioventicular groove. The pieces were incubated in a 0.1% solution of nitro-blue tetrazolium dye, (NBT), grade III, pH 7.4 for 15 minutes. Non-infarcted areas turn blue as a result of the formation of a precipitate that occurs when NBT interacts with enzymes dehydrogenases. The absence of these enzymes in myocardial infarction prevents the formation of sediment; thus, the infarcted areas at risk remain pale yellow. Pieces of the left ventricle were photographed (Praktica) and myocardial regions were measured using planimetry. Areas of necrosis were expressed as a percentage of the surface of the left ventricle.

Statistical analysis
All values were expressed as means ± SEM (mean statistical deviation). Comparison between groups was evaluated using the ANOVA method on one basis, followed by a special analysis using Student's t test (Student test). Statistical significance was determined as p <0.05.

results
Among the groups there were no significant differences in hemodynamic parameters of the left ventricle and body weight (see table 3 at the end of the description).

 There is a significant decrease in the survival of rats in the control group after successive occlusion and reperfusion. The use of various active compounds (with the exception of compound 5) and a preparation for oral comparison of verapamil for a month significantly increases the resistance of rats to myocardial ischemia / reperfusion.

 Compared to verapamil, the improvements were significantly stronger after the application of compound 13 (20 mg / kg) and compound 4.

 Active compounds and verapamil significantly reduce the size of myocardial infarction in comparison with the infarction of control animals. Limiting the size of a heart attack is dose dependent. Higher doses significantly reduce the degree of myocardial necrosis compared with verapamil.

 Our experiments show that the selected active compounds significantly reduce the degree of myocardial necrosis and significantly improve survival. The limitation of the size of a heart attack occurs without any noticeable changes in hemodynamic parameters, and it was significantly greater after the use of the compounds of this invention than after the use of the comparative substance verapamil.

Wound Migration Test
HUVEC cells were isolated and cultured in accordance with Jaffe EA et al. (J. Clin. Invest., 52, 2745-2756, 1973). The analysis was performed as described by Yamamura S. et al. (J. Surg. Res., 63, 349-354, 1996). HUVEC cells were scattered in a 96-well plate pre-coated with fibronectin (2 μg / well) (Sigma) and, at approximately 90% coverage with a monolayer, they inflicted a wound along a coordinate line drawn on the underside of the plate. Damage to the layer was caused by a Teflon cell scraper, whose width was 1 mm. The cells were washed and filled with complete RPMI 1640 medium (containing 5% protein) for incubation (air containing 5% Co ₂ at 37 ^° C). The cells were allowed to migrate within 24 and 48 hours to the site of the damage to the layer and recorded changes using film or photography through an inversion microscope (at 60x magnification). The number of cells advancing beyond the control line was counted and evaluated using an image analyzer.

Test compound treatment
A series of ten-fold dilutions were prepared in culture medium and 5 μl / well was added containing 95 μl of tissue culture over the wounded cell monolayer. The control culture contained compound 13 without dilution.

results
After 24 hours of incubation, cells that spontaneously appeared on the damaged surface, as well as the increase in their number, were counted in the presence of the tested compounds in submicromolecular concentration (at 10 ^-7 and 10 ^-8 mol). The tested compounds lead to a pronounced, significant acceleration of cell migration within 48 hours. The damaged surface was covered by cells by approximately 82% compared with 47% of spontaneously migrated human endothelial cells.

 The results of the migration test for wounds with compound 13 are shown in table 4.

 The healing process of the destroyed vascular monolayer begins with the migration of endothelial cells, which, thus, can further initiate the restoration of the wounded area. All our data suggest that the tested compounds can accelerate the healing of wounded human endothelial cells with a direct increase in migration.

 The invention is illustrated by the following examples without any limitation of the claimed claims.

Detailed description of a preferred embodiment of the invention
Example 1
N- [2-benzoyloxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidamide (Z) -2-butenedioate (1: 1) (compound 1)
Methodology:
20.9 g (75.0 mmol) of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -3-pyridine-carboxyimidamide [Hung. Pat. 177578 (1976)] dissolved in 300 ml of benzene. To this solution was added 150 ml of a 1 N sodium hydroxide solution, and then 19.5 ml (168 mmol) of benzoyl chloride were added dropwise. After vigorously stirring the mixture for 2 hours, 7.1 g (67 mmol) of sodium carbonate and an additional portion (9.75 ml; 84 mmol) of benzoyl chloride are added, followed by stirring overnight. The phases are separated, the organic layer is extracted with a solution of 1 N sodium hydroxide solution and water, dried and evaporated to dryness. The residue (41 g, oil) was dissolved in 150 ml of acetone and 8.7 g (75 mmol) of maleic acid was added. The resulting precipitate was filtered off, washed with acetone and dried. Yield: 29.0 g (78%). T. pl.: 194-195 ^o C.

Example 2
N- [2-palmitoyloxy-3- (1-piperidinyl) propoxy] -3-pyridine-carboxyimidamide monochlorohydrate (compound 2)
Methodology:
14.7 g (52.8 mmol) of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -3-pyridine-carboxyimidamide [Hung. Pat. 177578 (1976)] dissolved in 160 ml of chloroform. To this solution was added 7.7 ml (55 mmol) of triethylamine, and then a solution (14.7 g, 56.5 mmol) of palmitoyl chloride in 85 ml of chloroform was added dropwise. The mixture was stirred at room temperature overnight. The next day, an additional 3.8 ml of triethylamine and 7.4 g of palmitoyl chloride are added and stirring is continued for another day. The solution was then extracted with water, 5% acetic acid and water sequentially, dried with anhydrous sodium sulfate and evaporated to dryness.

 The residue (28.2 g, oil) was dissolved in ethyl acetate and the product was precipitated by the addition of 30 ml of 1N HCl in ethyl acetate. The voluminous white precipitate was filtered off, washed with ethyl acetate and dried.

Yield: 10.9 g (37%). T. pl.: 110-113 ^o C.

Example 3
N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylphenylcarboxyimidoyl chloride monochlorohydrate (compound 3)
Methodology:
Stage a)
50 g (0.245 mol) of m-trifluoromethylbenzamidoxime and 33.7 g (0.6 mol) of potassium hydroxide are dissolved in a mixture of dimethyl sulfoxide and 170 ml of water, the mixture is cooled to 0 ^{° C.} and 48 ml (0.6 mol) of epichlorohydrin are added . The reaction mixture was stirred at 0 ^{° C.} for 5 hours and left 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 organic layer was washed with water, dried, treated with activated carbon and evaporated to dryness, resulting in m-trifluoromethyl-N- (2,3-epoxypropyl) benzamidine as a colorless oil.

 Yield: 61 g (96%).

Stage b)
400 ml of a solution of 18% hydrochloric acid and 60 ml of ether are added to the resulting oil, the mixture is cooled to -5 ^° C with stirring. A solution of 17.4 g (0.25 mmol) of sodium nitrite in 60 ml of water is added slowly over 40 minutes and the reaction mixture is stirred for another 20 minutes. The mixture was extracted with ether (2 x 160 ml) and the combined organic phase was washed twice with water. 340 ml of a 20% sodium hydroxide solution are added to the ether solution, and the biphasic system is boiled for 1 hour with stirring. Then the phases are separated, the organic layer is washed with brine to a neutral pH, dried and evaporated to dryness, obtaining m-trifluoromethyl-N- (2,3-epoxypropoxy) benzimidoyl chloride as a colorless oil.

 Yield: 30.5 g (45%).

Stage c)
A mixture of 1.19 g (4.2 mmol) of N - [(2,3-epoxy-) propoxy] -3-trifluoromethyl-phenylcarboxyimidoyl chloride and 0.89 ml (8.5 mmol) of tert-butylamine in 12 ml of isopropyl alcohol boil for 2 hours. The solvent is removed in vacuo. The residue was dissolved in ethyl acetate, a solution of 0.98 ml of a methanol (4.3 N) solution of hydrogen chloride was added, and the mixture was concentrated in vacuo to a small volume, after which it was diluted with ether. The precipitate formed is separated, washed with cold ether and dried.

Yield: 0.48 g (32%). Mp: 150-153 ^o C.

IR spectrum, (KBr): 3423, 3233, 2978, 2880, 2784, 1620, 1570, 1479, 1400, 1383, 1340, 1238, 1167, 1128, 1101, 1072, 1038, 982, 930, 897, 804, 787, 714, 694 cm ^-1 .

Example 4
N- [2-Hydroxy-3- (l-piperidinyl) propoxyl] -2-thiophenecarboxyimidoyl chloride monochlorohydrate (compound 4)
Methodology:
5.0 g (15.6 mmol) of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-thiophenecarboxyimidamide monochlorohydrate was dissolved in 19 ml of water, then 6.1 ml of concentrated hydrochloric acid was added. The solution was cooled to -5 ^{° C.} and a cooled solution of 4.4 g (63.8 mmol) of sodium nitrite in 2.4 ml of water was added dropwise to it. During the reaction, the temperature was maintained at exactly 0 ^° C. After the addition was complete, the mixture was stirred for another 1 hour. Cold benzene (60 ml) was added to the mixture and slowly alkalized with a cold solution of 3.2 g (80 mmol) of sodium hydroxide in 45 ml of water. The organic layer is separated and washed successively with 20 ml portions of water until the pH is less than 9 (3-5 times). The organic layer was dried with anhydrous sodium sulfate, treated with activated carbon, filtered, evaporated in vacuo (t <45 ^° C) and 2.6 g of oil were obtained. It is dissolved in 5 ml of isopropyl alcohol and acidified (pH 2) with isopropyl alcohol containing dry hydrogen chloride. The product is crystallized from n-hexane to give a white substance.

Yield: 2.0 g (38%). Mp: 115-123 ^o C.

 Following the procedure described in the previous example, the following compounds were obtained.

Example 5
N- [2-hydroxy-3- (1-piperidinyl) propoxy] phenylcarboxyimidoyl chloride monochlorohydrate (compound 5)
Starting material: N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-phenylcarboxyimidamide
Yield: 23%. Mp: 140-145 ^o C.

Example 6
N- [2-hydroxy-3- (1-piperidinyl) propoxy] -4-pyridinecarboxyimidoyl chloride (Z) -2-butenedioate (1: 1) (compound 6)
Starting material: M- [2-hydroxy-3- (1-piperidinyl) propoxy] -4-pyridinecarboxyimidamide. In this case, the final product was isolated at the end of the experiment by dissolving the crude product in acetone and adding an equivalent amount of maleic acid. Yield: 25%. Mp: 150-154 ^o C.

Example 7
N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-nitrophenylcarboxyimidoyl chloride monochlorohydrate (compound 7)
Starting material: N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-nitrophenylcarboxyimidamide
Yield: 36%. Mp: 158-162 ^o C.

Example 8
N- (3- (1-piperidinyl) propoxy) -3-pyridinecarboxyimidoyl chloride dichlorohydrate (compound 8)
Starting material N- [3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidamide
Yield: 33%. Mp: 178-182 ^o C.

Example 9
N- [3- (1-piperidinyl) propoxy] -3-nitrophenylcarboxyimidoyl chloride monochlorohydrate (compound 9)
Starting material: N- [3- (1-piperidinyl) propoxy] -3-nitrophenylcarboxyimidamide
Yield: 49%. Mp: 173-175 ^o C.

Example 10
N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylbenzamide (compound 10)
Methodology:
1.3 ml (15.2 mmol) of epichlorohydrin is added to a solution of 1.6 ml (15.2 mmol) of tert-butylamine in 8 ml of ethanol for 10 minutes with stirring, while maintaining the temperature below 20 ^o C, and left for 3 days . Separately, 0.8 g (14.3 mmol) of potassium hydroxide is dissolved in a mixture of 20 ml of ethanol and 3 ml of water, and 3.42 g (15.2 mmol) of the potassium salt of N-hydroxy-3- (trifluoromethyl) - are added to this solution. benzamide and, previously prepared, a solution of epichlorohydrin and tert-butylamine. The reaction mixture is boiled with stirring for 10 hours, after which the solvent is distilled off. The residue was triturated with 20 ml of methylene chloride and 10 ml of water, the organic phase was separated, washed with 5 ml of water and 5 ml of a saturated solution of sodium chloride, dried over sodium sulfate, filtered and evaporated. The oily residue was crystallized from acetone-hexane to give a white powder of the title compound.

Yield: 0.85 g (17.3%). T. pl.: 156-158 ^o C.

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

Example 11
N-hexyl-N '- [2-hydroxy-3- (l-piperidinyl) propoxy] urea (compound 11)
Methodology:
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, 4.9 ml (45.9 mmol) of hexyl isocyanate and reaction the mixture is stirred for 20 hours at room temperature. After adding an additional 1.6 ml (15 mmol) of hexyl isocyanate, stirring was continued for another 2 hours, after which the solvent was distilled off in vacuo. A white crystalline product is obtained by trituration with petroleum ether.

Yield: 9.9 g (72%). T. pl.: 50-52 ^o C.

IR Spectrum (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 compounds were obtained.

Example 12
N-hexyl-N '- [3- (1-piperidinyl) propoxy] urea (compound 12)
Starting compound: 1-aminooxy-3- (1-piperidinyl) propane
Yield: 85% (oil)
IR Spectrum (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-pyridinyl) -4H-1,2,4-oxadiazine
Methodology:
Stage a)
17.5 g (0.05 mol) of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidamide dichlorohydrate was dissolved in 50 ml of thionyl chloride, boiled for 1 hour, 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 distilled off under reduced pressure. The residue was dissolved in a minimal amount of ethanol and, after cooling, a yellow crystalline N- [2-chloro-3- (1-piperidinyl) propoxy] pyridinecarboxyimidamide dihydrochloride was obtained as an intermediate.

Yield: 13.2 g (71%). T. pl.: 127-145 ^o C.

Stage b)
13.2 g (35.7 mmol) of N- [2-chloro-3- (1-piperidinyl) propoxy] pyridinecarboxyimidamide dichlorohydrate are added to a solution of 16.5 g (143.5 mmol) of potassium tert-butylate dissolved in 150 ml of tert-butanol. The mixture is boiled for 6 hours, after which it is evaporated in vacuo. 100 ml of a 5% sodium hydroxide solution are added and the mixture is extracted three times with 300 ml 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 white crystals of the title compound.

Yield: 3.5 g (38%). Mp: 157.5-158 ^o C.

Example 14: Tablets
For the manufacture of tablets with a mass of 200 mg containing 50 mg of the active substance, use:
50 mg N- [2-hydroxy-3- (1-piperidinyl) propoxy] phenylcarboxyimidoyl chloride monochlorohydrate
129 mg microcrystalline cellulose (for example, the brand "Avicel pH 102")
20 mg polyvinylpyrrolidone (for example, the brand "Polyplasdone XL"
1 mg magnesium stearate
Example 15: Capsules
For the manufacture of capsules with a mass of 300 mg use:
50 mg N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-nitrophenylcarboxyimidoyl chloride monochlorohydrate
10 mg yellow beeswax
10 mg soybean oil
130 mg of vegetable oil
100 mg capsule shell
Example 16: Solution
For 100 ml of solution use:
500 mg N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-thiophenecarboxyimidoyl chloride monochlorohydrate
10 g sorbitol
0.05 g sodium saccharin
100 ml twice distilled water.

Example 17: Solution for injection in an ampoule
For every 2 ml of injection, containing 2 mg of active substance, use:
2 mg 5,6-dihydro-5- (1-piperidinyl) methyl-3- (3-pyridyl) -4H-1,2,4-oxadiazine
in 2.0 ml of physiological saline sterile pyrogen-free solution.

Example 18: Solution for intravenous administration
For 500 ml of solution for intravenous administration use:
20 mg N-hexyl-N- [3- (1-piperidinyl) propoxy] urea
in 500 ml of physiological saline sterile pyrogen-free solution.

Claims (14)

1. The use of hydroxylamine derivatives of General formulas I and II

where R ¹ and R ² independently from each other represent a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, or R ¹ and R ² together with the nitrogen atom located between them form a saturated 5-7-membered a heterocyclic group, which optionally contains another nitrogen and / or oxygen heteroatom;
A is a straight or branched alkyl group containing from 4 to 12 carbon atoms, a phenyl group substituted or unsubstituted, preferably containing 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 IZ is a covalent bond, and in the compounds of the general formula II, the covalent bond or group = NH, in the compounds of the general formula I, X is a halogen atom or a group NR ³ R ⁴ , where R ³ and R ^{4 are} independent each other is a hydrogen atom or a straight or branched alkyl group containing from 1 to 6 carbon atoms, while in the compounds of the general formula II X is an oxygen atom, in the compounds of the general formula II R 'is a hydrogen atom or a straight or branched an alkyl group containing from 1 to 6 carbon atoms, and in compounds of the general formulas I and II, Y is a hydrogen atom, a hydroxyl group or an acyloxy group, which preferably contains as acyl part a long chain fatty acid containing th from 8 to 22 carbon atoms or an acyl component is an aromatic cyclic acid, and compounds of general formula I, in which X is NR ³ R ⁴ group and Y is a hydroxyl group, the X group is bonded to Y substituent and forms an intramolecular ring, represented by general formula III

in which A, Z, R ¹ and R ² have the above meanings,
as well as salts and optically active forms of these compounds for the preparation of drugs for the treatment and prevention of diseases resulting from damage to endothelial cells.  2. The use according to claim 1, wherein the hydroxylamine derivative is N- [2-benzoyloxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidamide (Z) -2-butenedioate (1: 1).  3. The use according to claim 1, wherein the hydroxylamine derivative is N- [2-palmitoyloxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidamide monochlorohydrate.  4. The use according to claim 1, wherein the hydroxylamine derivative is N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylphenylcarboxyimidoyl chloride monochlorohydrate.  5. The use according to claim 1, wherein the hydroxylamine derivative is N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-thiophenecarboxyimidoyl chloride monochlorohydrate.  6. The use according to claim 1, wherein the hydroxylamine derivative is N- [2-hydroxy-3- (1-piperidinyl) propoxy] phenylcarboxyimidoyl chloride monochloride.  7. The use according to claim 1, wherein the hydroxylamine derivative is N- [2-hydroxy-3- (1-piperidinyl) propoxy] -4-pyridinecarboxyimidoyl chloride (Z) -2-butenedioate.  8. The use according to claim 1, wherein the hydroxylamine derivative is N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-nitrophenylcarboxyimidoyl chloride monochlorohydrate.  9. The use according to claim 1, wherein the hydroxylamine derivative is N- [3- (1-piperidinyl) propoxy] -3-pyridinecarboxyimidoyl chloride dichlorohydrate.  10. The use according to claim 1, wherein the hydroxylamine derivative is N- [3- (1-piperidinyl) propoxy] -3-nitrophenylcarboxyimidoyl chloride monochlorohydrate.  11. The use according to claim 1, wherein the hydroxylamine derivative is N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylbenzamide.  12. The use according to claim 1, wherein the hydroxylamine derivative is N-hexyl-N '[2-hydroxy-3- (1-piperidinyl) propoxy] urea.  13. The use according to claim 1, wherein the hydroxylamine derivative is N-hexyl-N '[3- (1-piperidinyl) propoxy] urea.  14. The use according to claim 1, wherein the hydroxylamine derivative is 5,6-dihydro-5- (1-piperidinyl) methyl-3- (3-pyridyl) -4H-1,2,4-oxadiazine.

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