PL190673B1 - Application of hydroxylamine derivatives - Google Patents

Abstract

1. The use of hydroxylamine derivatives of general formulas (I) and (II) wherein R1 and R2 independently represent a hydrogen atom or a straight or branched alkyl group with 1-6 carbon atoms or R1 and R2 together with the nitrogen atom between them form a saturated 5-7 membered heterocyclic group, optionally containing additional nitrogen and / or oxygen atoms as heteroatoms; A is a straight or branched alkyl group with 4-12 carbon atoms, unsubstituted or substituted a phenyl group preferably having a haloalkyl alkyl group as a substituent or a nitro or 5-6 membered heteroaromatic ring containing nitrogen, oxygen or sulfur atoms; in general of formula (I) Z is a covalent bond and in general formula (II) Z is a covalent bond or the group = NH; in general formula (I) X represents a halogen atom or a group -NR3R4, where R3 and R4 are independently a hydrogen atom or a straight or branched alkyl group of 1-6 carbon atoms, while general formula (II) X represents an oxygen atom; in general formula (II), R 'is hydrogen, either straight or branched an alkyl group of 1-6 carbon atoms; in general formulas (I) and (II) Y represents a hydrogen atom, a group hydroxy or an acyloxy group, preferably containing the acyl portion of a long chain acid a fatty acid of 8-22 carbon atoms or a cyclic aromatic carboxylic acid as part acyl; and in compounds of general formula (I), in which X is a group -NR3R4 and Y is a hydroxyl group group X is fused with the substituent Y and forms an intramolecular ring, represented by general formula (III), wherein A, Z, R1 and R2 are as defined above, and salts and the optically active forms of these compounds in the manufacture of a medicament for use in therapy or prophylaxis PL diseases resulting from damage to endothelial cells.

Description

The present invention relates to the use of hydroxylamine derivatives for the manufacture of a medicament for the treatment or prevention of diseases resulting from damage to endothelial cells.

Compounds of general formula (I), in which X is -NH ₂ , A is unsubstituted phenyl or pyridyl, and Y is hydroxy, are known from published French Patent Application No. 2 (62,845 A1) (U.S. Patent No. 4 187 220) These compounds are β-selective blockers and are therefore useful in the treatment of diabetic angiopathy.

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 known from PCT patent application publication No. WO 90 / 0.584 A1 (PL Patent No. 164 547. These compounds are β-selective blockers and therefore are useful in the treatment of diabetic angiopathy.

Compounds of general formula (II) in which A is a phenyl group (unsubstituted or substituted by haloalkyl group), pyridyl or thienyl, Z is a covalent bond, R 'is a hydrogen atom and Y is a hydroxyl group are known from Hungarian Patent Application No. 2385/92, published under number T / 66350. These compounds counteract ischemia and angina and are therefore very useful in the treatment of the venous complications associated with diabetes.

Compounds of general formula (I) in which A is an aromatic or heteroaromatic ring, X is a halogen atom and Y is a hydrogen atom are known from PCT Patent Application Publication No. WO 95/30649 A1. These compounds are anti-ischemic and can be used to treat ischemic events characterized by venous hypertension and thrombocyte aggregation.

None of the above-mentioned descriptions say that the described compounds have any effect on vascular endothelial cells.

Normal functioning of the vascular endothelial cells is essential for the body. These cells form a clearly defined surface between the circulating blood and the elements of the vein walls that act as a clotting factor. The role of vascular endothelial cells in homeostasis is varied:

- participate in the two-way transport of substances derived from blood and tissues,

- create a barrier for macromolecules,

- these molecules synthesize and break down mediators that regulate the interaction between the cellular elements of the vein walls and blood (fibrinogen, collagen, proteoglycans, PGI ₂ , EDRF (NO), EDHF, endothelin 1, angiotensin II),

- initiate migration, proliferation and dissolution of the clot, contributing to the healing of tissues,

- they maintain the resistance of the vein walls to blood clots [Rubanyi G J., Cardiovasc. Pharmacol, 1993, 22 (42 Suppl., Pp. S1-14],

Endothelial damage causes atherosclerosis. Damage leading to the deterioration of the endothelium may occur with mechanical interventions, e.g. catheterization, and may also result from biochemical and immunological processes.

In the first stage of atherosclerotic plaque formation, lipid-filled cells collect on the intima of the arteries (Steinberg D. et al., JAMA 264-304, 1990). These cells, especially blood-derived monocytes and macrophages, initially adhere to the endothelium and then penetrate the intima of the vessels. Endothelial cell damage may also contribute to adhesion, although morphological changes are not visible in the early phase. Oxidation of LDL particles can lead to their absorption by monocytes in the inner lining of the vessels, making the monocytes foam cells. Foam cells form lipid bands which are the earliest form of atherosclerotic lesions.

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In the later stages, there is bleeding, necrosis, new vascularization, and induration, during which time interstrained plaques form that gradually constrict the arteries (Ip. JH, Fuster et al., J. Am. Coli. Cardiol. 15: 1667, 1990). ).

Thrombosis can occur at different stages of atherosclerosis. Repeated thrombosis leads to vascular obstruction and thromboembolism, such as coronary thrombosis, cerebrovascular thrombosis, or peripheral vascular disease.

In a clinical sense, "endothelial dysfunction syndrome" refers to generalized or local vasoconstriction, thrombosis, atherosclerosis and restenosis. Attempts to treat these diseases include clinical intervention techniques, bypassing surgery, and drug therapy.

Only a few existing drugs may be useful in the treatment of endothelial dysfunction. They fall into four main categories:

- substitutes for natural "protective" endothelial substances (e.g. stable PG analogues !, nitro vasodilators, rt-PA / recombinant tissue plasminogen activator /)

- endothelial-derived contractile factor inhibitors or antagonists (e.g. ACE inhibitors, angitensin II receptor antagonists; TXA ₂ receptor antagonists)

- cytoprotective agents (e.g. free radical scavengers, superoxide dismutase and probucol, and free radical inhibitors, lazaroids)

- lipid-lowering drugs.

Although none of these drugs were originally intended for this purpose, it has been clinically proven that their beneficial effects in certain diseases may be associated with the protection or restoration of normal endothelial function. The rationale for innovative therapies is to restore the normal lining of endothelial cells to keep endothelial cells doing their job. Potential approaches to address these problems include promoting the regrowth of normal endothelium, or new emerging therapeutic techniques based on recombinant DNA technology (Science 1990; 249: 1285-8). According to the available data, none of the recognized drugs meet these criteria.

There is currently no known drug used or proposed for use that would act directly on the endothelium, and therefore no drug suitable for the treatment of endothelial dysfunction. Thus, there is a great therapeutic need for a drug capable of preventing, inhibiting, or at least slowing down the development of complications, or reducing the incidence of the disease.

In the course of the research, it was surprisingly found that hydroxylamine derivatives of the general formulas (I) and (D) have a strong protective and regenerative effect, especially on vascular endothelial cells, and can prevent their damage caused by various reasons.

Thus, the invention relates to the use of hydroxylamine derivatives of the general formulas (I) and (II), wherein R ¹ and r independently represent a hydrogen atom or a straight or branched alkyl group with 1-6 carbon atoms, or R ¹ and R 2 together with the nitrogen atom between they form a saturated 5-7 membered heterocyclic group, optionally containing additional nitrogen and / or oxygen atoms as heteroatoms;

A is a straight or branched alkyl group with 4-12 carbon atoms, an unsubstituted or substituted phenyl group, preferably having an alkyl, haloalkyl or nitro substituent as a substituent, or a 5-6 membered heteroaromatic ring containing nitrogen, oxygen or sulfur atoms; in general formula (I), Z represents a covalent bond and in general formula (II) Z represents a covalent bond or an = NH group;

in general formula (I) X represents a halogen atom or a group -NR ³ R ⁴ , where R ³ and R ⁴ independently represent a hydrogen atom or a straight or branched alkyl group with 1-6 carbon atoms, while in general formula (II) X is oxygen atom; in general formula (II), R 'represents a hydrogen atom or a straight or branched alkyl group with 1-6 carbon atoms; in general formulas (I) and (II) Y is a hydrogen atom, a hydroxyl group or an acyloxy group, preferably having an acyl portion of a long chain fatty acid with 8-22 carbon atoms or a cyclic aromatic carboxylic acid as the acyl portion;

190 673 and in compounds of general formula (I), in which X is a group -NR ³ R ⁴ , and Y is a hydroxyl group, the group X is fused with the substituent Y and forms an intramolecular ring represented by general formula (III) in which A, Z, R ¹ and R ² are as defined above as well as the salts and optically active forms of these compounds in the manufacture of a medicament for use in the treatment or prophylaxis of diseases resulting from damage to endothelial cells.

According to the invention, the following compounds are particularly preferably used as active ingredients:

N- [2-benzoyloxy-3- (1-piperidimyl) propoxy] -3-pyridinecarboximidamide (1: 1) (Compound # 1) N- [2-palmitoyloxy-3- (1-6) monohydrochloride (Z) -2-butenedionate piperidinyl) propoxy] -3-pyridinecarboximidamide (compound No. 2) N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylbenzenecarboximidoyl chloride monohydrochloride (compound No. 2) N- [2] chloride monohydrochloride -hydroxy-3- (1-piperidinyl) propoxy] -2-thio phenocarboximidoyl (compound # 4) N- [2-hydroxy-3- (1-piperidinyl) propoxy] benzenecarboximidoyl chloride monohydrochloride (compound # 5) (Z) - N- [2-hydroxy-3 - (1-piperidinyl) propoxy] -4-pyridinecarboximidoyl chloride 2-butenedionate (1: 1) (compound No. 6) N- [2-hydroxy-3- (1-piperidinyl) chloride monohydrochloride propoxy] -2-nitrobenzenecarboximidoyl (compound No. 7) N- [3- (1-piperidinyl) propoxy] -3-pyridinecarboximidol chloride dihydrochloride (compound No. 8) N- [3- (1-piperidinyl) propoxy] chloride monohydrochloride -3-nitrobenzenecarboximido ilu (relationship # 9)

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

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

N-hexyl-N '- [3- (1-piperidinyl) propoxy] urine (compound No. 12)

5,6-dihydro-5- (1-piperidinyl) methyl-3- (3-pyridyl) -4H-1,2,4-oxadiazine (compound 13)

As mentioned above, the conducted studies proved that the compounds of general formulas (I) and (II) have an effect on endothelial cells of the cardiovascular and cerebrovascular systems. In the experiments described in detail hereinafter, it was observed that these compounds are able to block or restore the damage to these cells. As a result, these compounds can be used as active substances in therapeutic agents for the treatment of diseases resulting from the dysfunction or damage of endothelial cells, especially diseases of the cardiovascular and cerebrovascular systems, hypertension, excessive homocysteine concentration and peripheral vascular diseases.

The hydroxylamine derivatives of the general formulas (I) and (II) can be formulated as pharmaceuticals for use in the treatment and prophylaxis of diseases related to the malfunctioning of vascular cells. Such compositions contain as active ingredient (in addition to the known carriers and excipients used in pharmaceuticals) a compound of the general formula (I) or (II) in an amount of 0.5-95.5% by weight, or in some cases salts or optically active substances the forms of these compounds, in which in these formulas R1, R2, A, Z, X, Y and R 'have the meaning given above.

Preferred compounds of formulas (I) and (II) for the use described are those in which A is pyridyl, thienyl, or phenyl, nitrophenyl or trifluoromethylphenyl. Preferred compounds of formula (I) are also those wherein X is chlorine or NH2. Among the latter, particularly preferred compounds are those compounds which contain an intramolecular ring formed by the condensation of the X and Y groups. Preferred compounds of the general formula (II) are also compounds in which R 'represents a hydrogen atom or a hydroxyl group, and compounds of the general general formula formulas (I) and (II) wherein Y is a hydrogen atom or a hydroxyl group. In all of them

190 673 categories, preferred compounds are compounds wherein NRlR ² is piperidinoamino or dialkylamino group.

Compounds Nos. 1-13 listed above are particularly preferred.

Compounds of general formula (I) in which X is a halogen atom can be prepared by reacting an amidoxime of general formula (V) in which A is as defined above with an aminochloropropane derivative of general formula (XII) in which R ¹ , R ² and Y are as defined above and the -NH2 group of the resulting intermediate of formula (IV) wherein Z is a covalent bond and the other substituents are as defined above, is replaced by a chlorine atom by diazotization. For the preparation of compounds of general formula (I) having a hydroxyl group as Y substituent, the necessary aminochloropropane derivative of general formula (XII) having a hydroxyl group as Y substituent can be prepared from epichlorohydrin of formula (VI) and an amine of general formula (IX), wherein R ¹ and R 2 are as defined above. An alternative method consists in reacting epichlorohydrin with an amidoxime of general formula (V) followed by diazotization of the resulting intermediate of general formula (VII), wherein A is as defined above, and reacting an epoxy compound of general formula (VIII) in which A is as defined above, with an amine of general formula (IX).

Compounds of general formula (I) in which Y is an acyloxy group can be prepared by reacting a suitable compound of general formula (I) having a hydroxyl group as Y with acid chlorides of general formula (XI) in which R is long chain alkyl or an aryl group.

Compounds of general formula (II) in which Z is a covalent bond can be prepared by one of the following methods:

(i) combining a compound of general formula (XIII), wherein A and R 'are as defined above, and K is an alkali metal cation, with a halogen compound of general formula (XII), wherein R1 and Y are as defined above the meaning or (ii) reaction of the amine compound of 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, wherein compounds of general formula ( II), in which Z is a covalent bond and R 'is a hydrogen atom, can be produced, in addition to using methods (i) and (ii), also by the following methods:

(iii) diazotizing in a halogen free medium of a corresponding compound of general formula (I) in which X is -NH2 and Z is a covalent bond, or (iv) hydrolysis of a corresponding compound of general formula (I) in which X is halogen atom.

Compounds of general formula (II) in which A is an alkyl group and Z is a = NH group can be prepared by reacting a compound of general formula (II) in which R ¹ , R ² , Y, and R 'are as defined above , in an organic solvent, preferably chloroform, with an equivalent amount of an alkyl isocyanate of general formula XV wherein A is an alkyl group.

Compounds of general formula (III) are special cases of compounds of general formula (I) in which the group X containing a nitrogen atom is fused with the substituent Y to form an intramolecular ring.

Such compounds can be prepared by reacting a compound of general formula (I) (wherein Y is hydroxy) with an excess of thionyl chloride followed by ring closure of the resulting intermediate of general formula (IV) (wherein Y is chlorine and other substituents are as defined above), using an excess of refluxing potassium t-butoxide in an organic solvent (preferably t-butanol).

The hydroxylamine derivatives used according to the invention show surprising pharmacological properties. It should be noted that these compounds not only regenerate endothelial cells morphologically but also functionally. Endothelial cells are also well tolerated by their action. Such properties constitute a good basis for the pharmaceutical use of the hydroxylamine derivatives of the general formulas (I) and (II). These drugs can be used to treat diseases of the cardiovascular system 190 673

- cerebrovascular and cerebrovascular disease in humans and animals, such as hypertension, excess homocysteine and peripheral vascular disease.

Among the diseases of the cardiovascular system, these compounds can be used with good effect in the case of coronary artery disease, atherosclerosis, restenosis after plastic surgery with the use of a balloon catheter, surgery to create a connection bypassing the stenosis of the coronary artery, and in the case of cerebrovascular diseases in cases of obstruction cerebral artery.

The hydroxylamine derivatives of the general formulas (I) and (II) can also be used in the treatment of essential, renal, pulmonary and endocrine hypertension. In the treatment of peripheral vascular diseases, these compounds are particularly suitable for the treatment of aortic stenosis in the legs.

The described hydroxylamine derivatives can also be used to counteract susceptibility to genetically determined diseases or those resulting from a temporary weakening of the protective mechanism. The usual dose depends on the patient and the disease to be treated and may range from 0.1-200 mg / kg, preferably 1-50 mg / kg per day. This means, for example, that the daily dose in adult humans is 10-200 mg orally, 1-15 mg in rectal and 2-20 mg in parenteral administration.

Suitable pharmaceutical compositions can be, for example, solid or liquid substances, in any form customarily used in veterinary and medical medicine, such as tablets or coated tablets, gel capsules, granules, solutions, syrups, suppositories, and lyophilized and non-lyophilized injectables. All these agents can be prepared by known methods. The active ingredient can be found in a carrier usually used for this type of preparation, such as talc, acacia, lactose, starch, magnesium stearate, cocoa butter, aqueous and non-aqueous vehicles, animal and vegetable fats, paraffin derivatives, glycols, various wetting agents, dispersing or emulsifying agents and preservatives.

The biological activity of the compounds of formulas (I) and (II) used in the invention is illustrated by the following results of in vitro rat vascular relaxation tests and thoracic aorta morphological tests. Three-month-old Wistar Okamoto (SH) rats with essential hypertension were administered various test compounds for 1 month.

Effect of hydroxylamine derivatives on vascular relaxation in tests on the thoracic aorta of SH rats (in vitro test)

The test was performed according to the method known from the literature [Japan J. Pharmacol, 59, 339-347 (1992)]. Rats were anesthetized with nembutal (40 mg / kg, i.p. i.e. intraperitoneally), their thoracic aortas were removed and placed in an oxygenated (95% O2 + 5% CO2) Krebs Henseleit solution. Composition of the solution (mM): NaCl 118, KCl 4.7, CaCfe 2.52, MgSO4 1.64, NaHCOa 24.88, KH2PO4 1.18, glucose 5.5. Rings 3 mm long made of vessels were suspended in a 20 ml bath at 37 ° C. The tension at rest was 1 g and was kept at this level throughout the experiment. During the 1 hour equilibration period, the bath was replaced every 20 minutes. The vessels were contracted with methoxyamine (approximately 80% of the maximum contraction). After maximal contraction was achieved, acetocholine-induced vasodilation (Ach) was tested (10-104 and functional endothelial integrity was tested. Contraction strength was measured with an isomeric stress tester probe (SG-01D, Expermetria Ltd) and recorded with an OH-850 polygraph (Radelkis). The test results are given in Table 1.

Table 1

Effect of tested compounds on vascular relaxation in tests on the thoracic aorta of SH rats (in vitro test)

Substances / doses Acetocholine Doses (M) 10- 10 ' ⁵ IO- 1 2 3 4 Control group, n = 10 53.8 55.6 71.0 Compound No. 13, n = 12; 20 mg / kg 79.6 86.0 95.9 Compound No. 5, n = 11; 5 mg / kg 82.3 84.5 87.2

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c.d table 1

1 2 3 4 Compound No. 4, n = 11; 20 mg / kg 75.6 79.8 80.5 Compound No. 6, n = 10; 5 mg / kg 87.5 87.9 84.4 Compound No. 10, n = 12; 10 mg / kg 64.8 63.7 78.0 Compound No. 1, n = 12; 20 mg / kg 74.7 58.7 82.7 Compound No. 2, n = 10; 80.4 75.6 88.0 Compound No. 11, n = 12; 20 mg / kg 88.1 91.3 91.9 Compound No. 8, n = 10; 5 mg / kg 74.1 75.3 80.9 Compound No. 3, n = 8; 10 mg / kg 76.4 77.2 84.8 Compound No. 12, n = 12; 10 mg / kg 66.3 67.2 84.1 Compound No. 7, n = 11; 5 mg / kg 81.7 86.0 95.9 Captopril, n = 12; 20 mg / kg 88.7 88.2 94.2

As can be seen from Table 1, in hypertensive control animals that were not dosed with the test substances, the relaxation induced by administration of 10 µM acetocholine was reduced to 71% due to endothelial damage due to hypertension. The tested compounds significantly improved this reduced vasodilation, indicating an improvement in endothelial function.

Morphological examination of the thoracic aorta using an electron microscope

The test was performed according to a method known from the literature (Br. J. of Pharmacol, 1995; 115, 15-420). Rats' thoracic aortic wall segments with an area of 1 mm 2 were dissected and fixed at room temperature in 2.5% glutaraldehyde. An additional 1 hour fixation in 1% osmium tetroxide was then applied, after which the tissue segments were dehydrated with ethanol and embedded in Durcupan ACM. The sections were qualitatively assessed on photographs taken with a Hitachi 7100 electron microscope. The results of these tests are given in Table 2.

The results of the morphological tests were expressed on a scale of 1 to 5 depending on the extent to which the tested compounds repaired the hypertensive endothelial damage, that is, depending on the severity of the regenerative effect. On this scale, 1 represents the cases where no regeneration was observed, 2 means poor, 3 medium, 4 good and _ 5 means strong regeneration.

In comparison with the control group which was not administered the test compounds, a significant protective or regenerative effect was observed after the oral (p.o.) administration of the hydroxylamine derivatives of general formulas (I) and (II). As a result of the administration of these compounds, a thin protective layer was formed on the damaged subendothelial layer, consisting of cells containing an active nucleus and copious cytoplasm. Regeneration has proved to be quite successful in most cases.

Table 2

Evaluation of the effect of tested compounds on the thoracic aorta of SH rats (morphological test) using an electron microscope

Substance doses The degree of regeneration AND 2 Control group (saline) 1 Compound No. 13, 20 mg / kg (p.o.) 5 Compound No. 5, 5 mg / kg (p.o.) 5 Compound No. 4, 5 mg / kg (p.o.) 5 Compound No. 10, 10 mg / kg (p.o.) 4

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continued table 2

1 2 Compound No. 1, 20 mg / kg (p.o.) 3 Compound No. 11, 20 mg / kg (p.o.) 4 Compound # 8, 5 mg / kg (p.o.) 3 Compound No. 9, 5 mg / kg (p.o.) 3 Compound No. 12, 5 mg / kg (p.o.) 4 Compound No. 14, 20 mg / kg (p.o.) 3 Captopril 100 mg / kg (p.o.) 3

The experimental data support the supposition that the compounds of the general formulas (I) and (II) are capable of endothelial regeneration not only functionally, but also morphologically. With long-term treatment, these compounds produced more pronounced morphological regeneration than captopril, the reference substance.

Study of infarcted areas in rats (SH) with essential hypertension, after one month of oral administration

Experimental groups

1. Age-appropriate control group of animals

2. Verapamil (reference drug), 50 mg / kg (p.o.)

3. Compound No. 13, 2θ mg / kg (p.o.)

4. Compound No. 13, 50 mg / kg (p.o.)

5. Compound No. 5, 5 mg / kg (p.o.)

6. Compound No. 4, 5 mg / kg (p.o.)

Inducing a heart attack

Myocardial ischemia was induced by momentary occlusion of the left main coronary artery according to the method of Griswold et al (J. Pharmacol. Methods 1988, 20: 225-35). SH rats were anesthetized with pentobarbital sodium (50 mg / kg, i.p.). Following tracheotomy, animals were supplied with room air to the lungs via a small rodent ventilator (Harvard 552 model) at a stroke capacity of 1.5-2 ml of 7100 g and a rate of 55 cycles per minute to maintain normal pC> 2, pCC2 and pH parameters.

The right carotid artery was catheterized and connected to a pressure transducer (Stetham P236B) to measure systemic arterial blood pressure (BP) using a preamplifier (Hg-02D Experimetria2). Heart rate (HR) was measured with a cardiotachometer (HR-01, ExperimetriaR). The electrocardiogram (ECG standard lead ID) was recorded on a recorder (ER-14, Micromedr) using electrodes in the form of subcutaneous steel needles. The thorax was opened by thoracotomy. on the left side and the heart was extracted by gentle pressure on the right side of the costal system. A 4/0 silk garter was quickly placed under the left main coronary artery. The heart was returned to the ribcage and allowed to regain consciousness. 37 ° C.

The experiments were started with a 15 minute stabilization period during which the observation of a sustained blood pressure of less than 9333 Pa (70 mm Hg) and / or the appearance of arrhythmias led to the animal being excluded from the study.

Myocardial ischemia was induced by occluding the coronary artery for 1 hour and allowing reperfusion within 1 hour. The simulated operated animals were subjected to all the surgical procedures described above, except for coronary occlusion and reperfusion.

Quantitative assessment of the extent of myocardial infarction

At the end of the experiment, the heart was quickly removed. The left ventricle was cut into 2 mm thick slices parallel to the coronary sulcus of the heart. The slices were incubated for 15 minutes in 0.1% nitro blue tetrazolium blue (NBT) class III, pH 7.4. Non-infarcted areas turned blue as a result of the sediment that was formed

190 673 by reaction of NBT with the enzymes dehydrogenases. The absence of these enzymes in the infarction areas of the myocardium prevents plaque formation, so that the infarct areas in the risk region remain pale yellow. Left ventricular sections were photographed (Practica) and infarct areas were measured with a planimeter. The area of necrosis was expressed as a percentage of the area of the left ventricle.

Statistical analysis

All values are expressed as mean ± SEM. Data for the comparison of individual groups were analyzed by means of one-way ANOVA, followed by Post-hoc Student's t-test. The significance level was defined as p <0.05.

Results

There were no significant differences in haemodynamic parameters, left ventricle, and body weight for the different groups of animals.

Table 3

The extent of infarction and the survival rate of SH rats after coronary occlusion and reperfusion

Group The extent of infarction (%) Survival rate (%) Control group n = 9 42.7 + 1.37 28.13 Verapamil, 50 mg / kg, n = 8 24.3 ± 2.87 '** 53.3 ** Compound No. 13, 20 mg / kg, n = 7 22.3 ± 3.6 **, # 77.8 **, # Compound No. 13, 50 mg / kg, n = 5 15.2 ± 3.7 ** 60.0 ** Compound No. 5, 5 mg / kg, n = 3 29.3 ± 2.9 ** 30.0 Compound No. 4, 5 mg / kg, n = 5 25.6 ± 4.0 ** 71.4 **, #

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

After coronary occlusion and reperfusion, a marked decrease in the survival rate was observed in the control group of rats. Oral administration of the various active compounds (except compound # 5) and the reference substance verapamil for one month significantly increased the resistance of rats to myocardial ischemia and reperfusion injury.

The improvement was significantly greater after the administration of Compound # 13 (20 mg / kg) and Compound # 4 as compared to verapamil.

The active compounds and verapamil significantly reduced the infarct size compared to the control animals group. The limitation of the infarct severity was dose-related. The higher doses significantly reduced the extent of myocardial necrosis as compared to verapamil treated animals.

These experiments indicate that the selected active compounds significantly reduce the extent of myocardial necrosis and significantly improve the survival rate. The reduction in infarct severity was without any change in the hemodynamic parameters and was significantly higher after administration of the compounds used in the invention than after administration of the reference substance verapamil.

Migration test through damaged cell monolayer

HUVEC cells were isolated and cultured according to the method described by Jaffe E.A. et al. (J. Clin. Invest., 52, 2745-2756, 1973). The assay was performed as described by Yamamura S. et al. (J. Surg. Res., 63, 349-354, 1996). HUVEC cells were seeded in a 96-well plate pre-coated with fibronectin (2 µg / well) (Sigma) and at about 90% pourable, the monolayer was damaged along the line drawn on the underside of the plate. The layer was damaged with a 1 mm wide Teflon cell scraper. The wells were washed and filled with complete RPMI 1640 medium (containing 5% protein) for incubation (at 37 ° C, in air containing 5% CO2). Cells were allowed to migrate for 24 and 48 hours to the damaged field and pictures were taken through an operating microscope

190,673 in inverted position (at x60 magnification). The number of cells transported off the baseline was counted and evaluated using an image analyzer.

Action with the test compound

A series of ten-fold dilutions in the medium was prepared and added at 5 µl / well containing 95 µl of tissue culture per damaged cell monolayer. The control culture did not contain diluted Compound # 13.

Results

After 24 hours incubation, cells appeared spontaneously in the lesion area and their increased numbers were counted even in the presence of less than a micromolar concentration of the test compound (at ^10.7 and ^10.8 M), which led to a clear, significant promotion of cell migration during 48 hours. The injured area was covered for approximately 82%, compared to 47% for the spontaneously migrating human endothelial cells.

Table 4

Results of the cell migration test through the damaged monolayer using Compound # 13

10-x (M) doses Area covered Cells / mm2 % / mm2 24 h 48 h 24 h 48 h 7 689 1009 56 82 8 750 948 61 80 Without adding compound * 480 578 39 47 Adherent cells * 180 6

* Control sample, spontaneous migration ** Cells on the damaged surface immediately after the damage occurred

The repair process of the damaged vascular monolayer begins with the migration of endothelial cells and this way the damaged area can be further reconstructed. All the obtained data suggest that the tested compounds can stimulate the repair of damaged human endothelial cells by directly increasing migration.

The invention is illustrated by the following examples in which the abbreviation m.p. means melting point.

Example 1 N- [2-benzoyloxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboximidamide (1: 1) (Compound No. 1)

20.9 g (75.5 mmoles) of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -3-pyramidal-carboimidamide [Hungarian patent no. 177 578 (1976)] were dissolved in 300 ml of benzene. . To this solution was added 150 ml of a 1N sodium hydroxide solution, followed by dropwise addition of 19.5 ml (168 mmol) of benzoyl chloride. After vigorous stirring for 2 hours, 7.1 g (67 mmol) of sodium carbonate and a further portion of benzoyl chloride (9.75 ml; 84 mmol) were added and stirring was continued overnight. The phases were then separated, the organic phase was extracted with 1N sodium hydroxide solution and water, then 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 to the solution.

The resulting precipitate was filtered off, washed with acetone and dried. Yield: 29.0 g (78%), mp: 194-195 ° C.

Example 2

N-P-palmitoyloxy-S-O-piperidinyljpropoxy-S-pyridinecarboximidamide monohydrochloride (Compound # 2)

14.7 g (52.8 mmol) of N- [2-hydroxy-3- (1-piperidinium) ^ (^] ^ (^ <^ S ^) ^) 3 ^ ^ ^ <^> t ^ Z / n (^ l <^^^ ol ^^ S ^^ and ^^^ <^ oamidLl [Hungarian Patent No. 177,578 (1976)].

190 673

To this solution was added 7.7 ml (55 mmol) of triethylamine, followed by dropwise addition of a solution of palmitoyl chloride (14.7 g; 56.5 mmol) in 85 ml of chloroform. The mixture was stirred overnight at room temperature. The next day, an additional 3.8 ml of triethylamine and 7.4 g of palmitoyl chloride were added and stirring was continued for the next day. The solution was extracted sequentially 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 adding 30 ml of 1N HCl in ethyl acetate. A thick white precipitate was filtered off, washed with ethyl acetate and dried. Yield: 10.9 g (37%), mp: 110-113 ° C.

Example 3

N- [3 - [(1,1-DimethylethylaminoJ-J-hydroxypropoxy] -j-trifluoromethylbenzenecarboximidoyl chloride monohydride (compound # 3)

Stage a)

50 g (0.245 mol) of m-trifluoromethylbenzamidoxime and 33.7 g (0.6 mol) of potassium hydroxide were dissolved in a mixture of dimethylsulfoxide and 170 ml of water, and the resulting mixture was cooled to 0 ° C. To this mixture 48 ml (0.6 mol) of epichlorohydrin was added and the mixture was stirred for 5 hours at 0 ° C and then stored overnight in a refrigerator. The next day, 250 ml of water was added and the mixture was extracted with ethyl acetate (4x250 ml). The combined organic phases were washed with water, dried, treated with charcoal and evaporated to dryness to give m-trifluoromethyl-N- (2,3-epoxypropoxy) benzamidine as a colorless oil. Yield: 61 g (96%)

Stage b)

400 ml of 18% hydrochloric acid solution and 60 ml of ether were added to the resulting oil, and the resulting mixture was cooled to -5 ° C with stirring. A solution of 17.4 g (0.25 mol) of sodium nitrite in 60 ml of water was then slowly added over 40 minutes and the reaction mixture was stirred for an additional 20 minutes. The mixture was extracted with ether (2 x 160 ml) and the combined organic phases were washed twice with water. 340 ml of 20% sodium hydroxide solution was added to the ethereal solution, and the stirring biphasic system was heated under reflux for 1 hour. The phases were then separated, the organic layer 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%)

Stage c)

A mixture of 1.19 g (4.2 mmol) of N - [(2,3-epoxy) propoxy] -3-trifluoromethylbenzenecarboximidoyl chloride and 0.89 ml (8.5 mmol) of t-butylamine in 12 ml of isopropyl alcohol was heated to 2 hours at reflux. The solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate and 0.98 ml (4.3N) of methanolic hydrogen chloride was added, then the mixture was concentrated to a small volume in vacuo and diluted with ether. The resulting precipitate was separated, washed with cold ether and dried. Yield: 0.48 g (32%); mp: 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, 897, 804, 787.714 , 694 cm ⁴ .

Example 4

N- [2-hydroxy-3- (1-pipeyidinyl) propoxy] -2-thiophenecarboximidoyl chloride monohydrochloride (compound No. 4)

5.0 g (15.6 mmol) of N-p-hydroxyr3- (1-pipfrsidinyl) propoxy ·] -2-thio-enecarboximidamide monohydrochloride were dissolved in 19 ml of water and 6.1 ml of concentrated hydrochloric acid were added. The solution was cooled to -5 ° C and a cold solution of 4.4 g (63.8 mmol) of sodium nitrite in 2.4 ml of water was added dropwise. The internal temperature was kept at 0 ° C throughout the reaction. After the addition of the reagent was complete, the mixture was stirred for an additional hour. Then 60 ml of cold benzene was added and the mixture was made basic by adding a cold solution of 3.2 g (80 mmol) of sodium hydroxide in 45 ml of water. The organic phase was separated and washed sequentially with 20 ml portions of water until the pH was <9 (3-5 times). The organic solution was dried over anhydrous sodium sulfate, treated with charcoal, filtered and evaporated under reduced pressure (t <45 ° C) to give 2.6 g of an oil. The residue was dissolved in 5 ml of isopropyl alcohol and acidified (pH 2) with isopropyl alcohol containing anhydrous hydrogen chloride. The product was crystallized from n-hexane to give an off-white substance. Yield: 2.0 g (38%); mp: 115-123 ° C.

The following compounds were prepared according to the method described in the previous example. Example 5

N- [2-Hzdroxsh - (- (1-pipezcdzl-yl) propoxy] berzrenokiirboximidoyl chloride monohydrochloride (compound No. 5)

Starting material: N- [2-Hygro.sy-3- (1-pipei · ydynyio) propoxy] benzenecarboxyimidamide. Yield: 23%; mp: 140-145 ° C.

Example 6

N- [2-hydroxy - (- (1-pipeyydinylpropoxy] -4-pirycy nocarboxshimidoyl chloride (1: 1) (compound No. 6)

Starting material: N- [2-hydroxy-3- (1-pipiizidinyl) propoxy] -4-predominantb-oximidamide.

In this case, the end product was isolated at the end of the work-up by dissolving the crude base in acetone and adding an equivalent amount of maleic acid. Yield: 25%; mp: 150-154 ° C.

Example 7

N- [2-Hzdroxy - (- (1pperidinyl) propoxy] -2-nitrobenaenecarboxshimidoyl chloride monohydrochloride (compound No. 7)

Starting material: N- [2-hydroxy - (- (1-pipisidinyl) propoxy] -2-nilrobenzenecarboxshimidamide Yield: 36% mp: 158-162 ° C.

Example 8

N- [3- (1-piperidinyl ^) J ^. ^ I ^^^^. S; zj chloride dihydrochloride (compound No. 8)

Starting material: N- [3- (1-pipezidinyl) propoxy] -3-pyridin-) carboximidoamide Cd. Yield: 33%; mp: 178-182 ° C.

Example 9

N - [(- (1-pipezidinyl) proxy] - (- nitrc> benzenecarboxyimidoyl chloride monohydrochloride (compound No. 9)

Starting material: N - [(- (1-plpeiydnzlo) propoxide] - (- n -trobenzene carboxshimidamide Yield: 49%, mp: 173-175 ° C.

Example 10

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

While stirring and keeping the temperature below 20 ° C, to 1.6 ml (15.2 mmol) of tert-butylamine in 8 ml of ethanol was added 1.3 ml (15.2 mmol) of epichlorohydrin in 10 minutes. 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 to this solution was added 3.42 g (15.2 mmol) of potassium salt of N-hzdroxsh (- (trifluiomethyl) ] mzamide and a previously prepared solution of epichlorohydrin and t-butylamine. The reaction mixture was stirred and refluxed for 10 hours, then the solvent was evaporated. The residue was triturated with 20 ml of dichloromethane and 10 ml of water, the organic phase was separated, washed with 5 ml of water and 5 ml. ml of saturated sodium chloride solution, dried over sodium sulfate, filtered and evaporated The oily residue was crystallized from a mixture of acetone and hexane to give the title compound as a white powder Yield: 0.85 g (17.3%); : 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-plperidinzlo) propoxide] urea (compound No. 11)

4.9 mL (45.9 mmol) of hexyl isocyanate was added to a solution of 8.0 g (45.9 mmol) of 1-ammox-2-hzdroxsh - (- (1-plperidinyl) propane in 60 ml of chloroform, and the reaction mixture was stirred for 20 hours at room temperature After addition of a further 1.6 ml (15 mmol) of hexyl isocyanate, stirring was continued for a further 2 hours and the dissolution was evaporated.

190 673 under reduced pressure. A white crystalline product was obtained by trituration with petroleum ether. Yield: 9.9 g (72%); mp: 50-52 ° C.

IR (KBr): 3310, 2932, 2858, 2804, 1666, 1551, 1454, 1377, 1306, 1092, 1040, 995, 791.725, 604 cm '!

The following compounds were prepared according to the method described in the previous example.

Example 12

N-Hexyl-N '- [3- (1-piperidinyl) propoxy] urea (compound No. 12)

Starting material: 1-aminoxy-3- (1-piperidinyl) propane Yield: 85% (oil)

IR (KBr): 3354, 2932, 2856, 2810, 2777, 1666, 1543, 1486, 1377, 1308, 1155, 1134, 1076 cm ^'r.

Example 13

5,6-Dihydro-5- (1-piperidinyl) methyl-3- (3-pyridyl} 4H-1,2,4-oxadiazine (compound 13).

Stage a)

17.5 g (0.05 mol) of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -3-pyridine carboximidamide dihydrochloride was dissolved in 50 ml of thionyl chloride, the resulting mixture was refluxed for 1 hour and evaporated. to dry. The residue was dissolved in 300 ml of methanol, treated with charcoal, and after filtration, the solvent was evaporated under reduced pressure. The residue was dissolved in the minimum amount of ethanol and the solution was cooled in a refrigerator to yield crystalline N- [2-chloro-3- (1-pipendhnyl] propoxy-d-pyrimidinecarboximidamide dihydrochloride) dihydrochloride as intermediate. Yield: 13.2 g (71%) ); mp: 127-145 ° C.

Stage b)

To a solution of 16.5 g (143.5 mmol) of potassium t-butoxide dissolved in 150 ml of t-butanol was added 13.2 g (35.7 mmol) of N- [2-chloro-3- (1-piperidinyl) propoxy] -3-pyridine carboximidamide. The mixture was refluxed for 6 hours and evaporated under reduced pressure, then 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 afford the title compound as white crystals.

Yield: 3.5 g (38%); mp 157.5-158 ° C.

Example 14. Tablets

For the production of tablets weighing 200 mg, containing 50 mg of active substance, the following were used:

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

129 mg microcrystalline cellulose (eg "Avicel ph 102") mg polyvinylpyrrolidone (eg "Polyplasdone XL") mg of magnesium stearate.

Example 15. Capsules

The following was used to produce a 300 mg capsule:

mg of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-nitrobenzenecarboximido chloride monohydrochloride number of mg yellow beeswax mg of soybean oil

130 mg of vegetable oil

100 mg of preformed capsule.

Example 16. Solution

To prepare 100 ml of the solution, the following was used:

500 mg of N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-thiophenecarboximidoyl chloride monohydrochloride g of sorbitol

0.05 g of sodium saccharin in double-distilled water to make up to 100 ml.

Example 17. Vial for injection

The following was used to prepare a 2 ml injection vial containing 2 mg of the active ingredient:

190,673 mg of 5,6-gihydro 15- (1-pipelidinyl °) melol-3- (3-pyridyl) -4H-1,2,4-oxadiazinv sterile pyrogen-free physiological saline to make up to 2.0 ml.

Example 18 Infusion solution To prepare 500 ml of infusion solution, the following was used:

mg of N-hexyl-N '- [3- (1-yinry4ynelo) yropoxide] urea in sterile pyrogen-free physiological saline to make up to 500 ml.

R ^{1 A} \

From 'CH.

^ CH

CH, ^ R ² (I)

XYR ¹

(II)

AR ³ \

/

CH \

CH, —N

R '\\

CH.

(III)

N - O

190 673

ΝΗ,

Υ ί

CH

ΝΗ,

Α Ν - ΟΗ (V)

CH,

CH, (IV)

CH ₂

Cl CH \

(VI)

- ch ₂ /

CH,

ΝΗ,

CH -CH, \ / 'Ο (VII)

190 673

Hal (VIII)

CH - CHj \ /

ABOUT

HN (IX) ^c, x ₂

CH, CH, R (XI) (xii)

(X)

190 673

Ο

R ’(XIII) ο

A - Ν zz c rz ο r

A Hal ^(XV) (XVI)

NH

CH,

Y

CH

CH

- ^N r ² (XIV)

Publishing Department of the UP RP. Mintage 50 copies. Price PLN 4.00.

Claims (14)

Patent claims 1. Use of a hydroxylamine derivatives, the general formulas (I) and (II) in which R ¹ and R ² independently represent a hydrogen atom or a straight or branched alkyl group having 1-6 carbon atoms, or R1 and R2 together with the nitrogen atom between they form a saturated 5-7 membered heterocyclic group, optionally containing additional nitrogen and / or oxygen atoms as heteroatoms; A is a straight or branched alkyl group with 4-12 carbon atoms, an unsubstituted or substituted phenyl group, preferably having an alkyl, haloalkyl or nitro substituent as a substituent, or a 5-6 membered heteroaromatic ring containing nitrogen, oxygen or sulfur atoms; in general formula (I), Z represents a covalent bond and in general formula (II) Z represents a covalent bond or an = NH group; in general formula (I) X represents a halogen atom or a group -NR ³ R ⁴ , where R ³ and R ⁴ independently represent a hydrogen atom or a straight or branched alkyl group with 1-6 carbon atoms, while in general formula (II) X is oxygen atom; in general formula (II), R 'represents a hydrogen atom or a straight or branched alkyl group with 1-6 carbon atoms; in general formulas (I) and (II) Y is a hydrogen atom, a hydroxyl group or an acyloxy group, preferably having an acyl portion of a long chain fatty acid with 8-22 carbon atoms or a cyclic aromatic carboxylic acid as the acyl portion; and in compounds of general formula (I) in which X is a group -Nr3r4 and Y is a hydroxyl group, the group X is fused with the substituent Y to form an intramolecular ring represented by general formula (III), wherein A, Z, R ^r and R2 are as defined above, as well as the salts and optically active forms of these compounds in the manufacture of a medicament for use in the treatment or prophylaxis of diseases resulting from damage to endothelial cells. 2. Use according to claim 1 The method of claim 1, wherein the active ingredient is N- [2-benzoyloxy-3- (1-piperidinyl) propoxy] -3-pyridinecarboximidamide (Z) -2-butenedionate (1: 1). 3. Use according to claim 1 The compound of claim 1, wherein the active ingredient is N- [2-palmitoyloxy-3- (1-piperidinyl) propoxy] -3-pyridine carboximidamide monohydrochloride. 4. Use according to claim 1 The method of claim 1, wherein the active ingredient is N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylbenzenecarboximidoyl chloride monohydrochloride. 5. Use according to claim 1 The method of claim 1, wherein the active ingredient is N- [2-hydroxy-3- (1-piperidinyl) propoxy] -2-thiophenecarbosyimidoyl chloride monohydrochloride. 6. Use according to claim 1 A method according to claim 1, characterized in that the active ingredient is N- [2-hydroxy-3- (1-pipey / dyly) propoxy] benene carboximidoyl chloride monohydrochloride. 7. Use according to claim 1 2. A method according to claim 1, characterized in that the active ingredient is N- [2-hydroxy-3- (1-pi-pericyl) propyl-4-pridinecarboximidoyl chloride (Z) -2-butenedionate. 8. Use according to claim 1 The method of claim 1, wherein the active ingredient is N- [2-hydroxy-3- (1-pipeayidinyl) propoxy] -2-nitrorenzenokaboxyimidoyl chloride monohydrochloride. Use according to claim 1 2. A method as claimed in claim 1, characterized in that the active ingredient is N- [3- (1-piperidinio) propoxy] -3-pyridinecaboximidoyl chloride dihydrochloride. Use according to claim 1 A process as claimed in claim 1, characterized in that the active ingredient is N- [3- (1-piperidinic)) propoxy] -3-nitrobenzene-oxy-ar- oximidoyl chloride monohydrochloride. 11. The use according to claim 1 The method of claim 1, wherein the active ingredient is N- [3 - [(1,1-dimethylethyl) amino] -2-hydroxypropoxy] -3-trifluoromethylbenzamide. 12. The use according to claim 1 2. The process of claim 1, wherein the active ingredient is N-hexyl-N '- [2-hydroxy-3- (1-piperidinyl) propoxy] urea. 13. Use according to claim 1 2. The process of claim 1, wherein the active ingredient is N-hexyl-N'- [3- (1-piperidinyl) propoxy] urea. 190 673 14. Use according to claim 1 6. The method of claim 1, wherein the active ingredient is 5,6-dihydro-5- (1-pipeyrthi} methyl) non-yl-3- (3-pyriyl) -4H-1,2,4-oxadiazine.