RU2287524C1 - Aspartyl derivatives of histamine, method for their preparing, pharmaceutical composition and their using as modulators of enzyme activity of antioxidant protection - Google Patents

Abstract

FIELD: bioorganic chemistry, biochemistry, pharmacy.

SUBSTANCE: invention relates to novel aspartyl derivatives of histamine of the general formula (I):

, wherein R means hydrogen atom (H),

or

, or

that are able to modulate activity of enzymes of antioxidant protection - superoxide dismutase (SOD) and catalase. Also, invention relates to using the known compounds of the general formula (I) for the same designation wherein at the same values of X the value R represents acetyl group, and to their pharmaceutically acceptable salts. Also, invention relates to a pharmaceutical composition possessing capacity to modulate activity of SOD and catalase and comprising the effective amount of compound of the general formula (I), and to a method for synthesis of compounds of the general formula (I). Method involves interaction of pentafluorophenyl ester N^α-Z-, β- or α-benzyl ester of aspartic acid with histamine followed by hydrogenolysis without isolation of intermediated protected derivatives of aspartyl histamine.

EFFECT: improved method of synthesis, valuable biochemical properties of derivatives.

12 cl, 3 tbl, 2 sch, 2 dwg, 8 ex

Description

The present invention relates to the field of bioorganic chemistry and relates to new compounds - N-aspartyl derivatives of the biogenic amine histamine, as well as a method for the synthesis of new and known compounds, their biomedical use in biology and medicine as modulators of antioxidant defense enzyme activity.

BACKGROUND OF THE INVENTION

It is known that a violation of oxidative and energy processes in the body leads to the onset and development of a number of diseases. The degree of violation of these processes correlates with the depth of the pathology, and their normalization - with the development of regenerative functions in the tissues and improvement of the condition of patients.

An important role in the regulation of the functioning of organs and tissues is played by reactive oxygen species, peroxidic compounds and antioxidant substances, in particular, antioxidant protection enzymes superoxide dismutase (SOD) and catalase. These enzymes in the body work as a balanced and coordinated system in which the functioning of one enzyme is correlated with the action of another [Halliwell B. Gutteridge J. // Free radicals in biology and medicine. / 1999. University Press. Oxford 963 p. P.171] With a change in the activity and balance of these enzymes, an increase in the number of reactive oxygen species and peroxides can occur, which, in turn, can cause destruction of proteins, nucleic acids and lipids and impaired stability of cell membranes [Halliwell B. Gutteridge J. // Free radicals in biology and medicine. / 1999. University Press. Oxford 963 p .; Boldyrev A.A. // Carnosine. Biological significance and applicability in medicine. / M.: Publishing House of Moscow State University. 1998.320 s .; Guilivi C., Cadenas E. The role of mitochondrial glutatione in DNA base oxidation. // Biochim. Biophys. Acta. 1998. V.1366. Number 3. P.265-274].

It is known that moderately increased expression of SOD in normal neuronal cells increases the lifespan of mice, and inhibition of SOD leads to apoptosis of neurons [Halliwell B. Gutteridge J. // Free radicals in biology and medicine. / 1999. University Press. Oxford 963 p. P.751]. At the same time, overexpression of Zn / Cu SOD is one of the causes of cell death, increased lipid peroxidation, and destruction of brain neurons [Y.C., Hyeok Y. Kwon, O.B. Kwon, J.H. Kang. Hydrogen peroxide-mediated Cu, Zn-superoxide dismutase fragmentation: protection by carnosine, homocarnosine and anserine. // Biochem. Biophys. Acta. 1999. V.1472. P.651-657; Halliwell B. Gutteridge J. // Free radicals in biology and medicine. / 1999. University Press. Oxford 963 p.].

The accumulation of H ₂ O ₂ in the cell can be observed with the activation of SOD and with a decrease in catalase activity.

The result of this imbalance can be a disorganization of the functioning of the cell, its apoptosis and even death, which is unacceptable for a healthy cell, but is the goal of antitumor therapy. Thus, an increased content of SOD in the cell can exert a cytotoxic effect on them, due to the fragmentation of nucleic acids under the influence of OH ^• , formed by the Fenton reaction upon decomposition of H ₂ O ₂ catalyzed by Zn, Cu-SOD [Dowja W., Kharatishvili M., Costa M. DNA and RNA strand scission by cooper, zinc and manganese superoxide dismutases. // Biometals. 1996. V.9. P.327-335].

It is known that SOD inhibitors can, by increasing the amount of O ₂ ^{- •} , cause apoptosis of tumor cells [Huang P., Feng L, Oldham EA et al. Superoxide dismutase as a target for the selective killing of cancer cells. // Nature. 2000. V.407. No. 6802. P.390-395]. In this case, the apoptosis mechanism is also associated with the formation of OH ^• from O ₂ ^{- •} followed by the destruction of nucleic acids by it.

In connection with the above, SOD and catalase enzymes are attractive targets for pharmacological effects, and modulators of their activity, both activators and inhibitors, can be used as therapeutic agents for the treatment of diseases accompanied by an increase in the level of reactive oxygen species, for example, diabetes, heart attack myocardium, cerebral ischemia, inflammatory and autoimmune diseases, etc. In addition, the regulation of the activity of antioxidant defense enzymes using biocompatible low molecular weight compounds inenia is a new strategy in the treatment of tumor diseases.

The biosynthesis of N-aspartyl derivatives of histamine (II, III, IV) was observed in the rat brain, and the biological functions of these compounds are unclear [E. Kvamme, K. L. Reichelt, P. D. Edminson. Proceedings of the tenth FEBS meeting. // 1975. P.127]. Compounds II, III, IV were isolated from brain homogenate. For aspartyl histamine derivatives, there are no published data on their chemical synthesis, physicochemical constants, and biological activity.

It is known the use of inorganic compounds, mainly salts, for example sulfides, cyanides, azides, HOCI, etc. [Halliwell B. Gutteridge J. // Free radicals in biology and medicine. / 1999. University Press. Oxford 963 p.] As SOD and catalase inhibitors.

In addition, organic compounds are used for this purpose. Thus, aminotriazole is known as a catalase inhibitor used in vitro and in vivo [Halliwell B. Gutteridge J. // Free radicals in biology and medicine. / 1999. University Press. Oxford, p. 138, 381].

The most commonly used SOD inhibitor is diethyldithiocarbamic acid (DETCA), acting at a concentration of 10 ^-3 M [Kanie N., Kamata K. Contractile responses in spontaneously diabetic mice. I. Involvement of superoxide anion in enhanced contractile response of aorta to norepinephrine in C57BL / KSJ (db / db) mice. // General Pharmacology. 2000. V.35. No. 6. P311-318]; triethylenetetramine (TETA) with the same effective concentration [Weissmann N., Winterhalder S., Nollen M. et al. NO and reactive oxygen species are involved in biphasic hypoxic vasoconstriction of isolated rabbit lungs. // American J. Physiology. 2001. V.280. Number 4. P.638-645] .; hydroxyl and hydroxymethyl derivatives of estrogen [Huang P., Feng L, Oldham EA et al. Superoxide dismutase as a target for the selective killing of cancer cells. // Nature. 2000. V.407. No. 6802. P.390-395]; EDTA [Somani BL, Ambade V., Bulakh. PM, Sharma YV Elimination of superoxide dismutase interference in fructosamine assay. // Clinical biochemistry. 1999. V.32. Number 3. P.185-188]; diethylcarbamazine [Chen X., Catravas JD Release of a leukocyte activation inhibitor by staurosporine-treated pulmonary artery endothelial cells. // Amer. J. Physiology. 1999. V.275. P.184-192].

The main disadvantages of the known inhibitors are their toxicity, non-natural or non-endogenous origin and high active concentrations, which limits the possibility of their further study for biomedical use.

The aim of the invention is a method for the synthesis of new and known aspartyl derivatives of histamine of General formula I and their use as effective modulators of the activity of antioxidant enzymes SOD and catalase.

SUMMARY OF THE INVENTION

The present invention relates to new aspartyl derivatives of histamine of General formula I:

where R represents hydrogen, and X represents

и их фармацевтически приемлемым солям, обладающим способностью модулировать активность ферментов антиоксидантной защиты - супероксиддисмутазы (СОД) и каталазы.

and their pharmaceutically acceptable salts having the ability to modulate the activity of antioxidant defense enzymes superoxide dismutase (SOD) and catalase.

The present invention also relates to the use of compounds of the general formula I:

where R represents an acetyl group, and X represents

и их фармацевтически приемлемых солей, обладающих способностью модулировать активность ферментов супероксиддисмутазы (СОД) и каталазы.

and their pharmaceutically acceptable salts having the ability to modulate the activity of superoxide dismutase (SOD) and catalase enzymes.

Further, the present invention relates to a pharmaceutical composition having the ability to modulate the activity of SOD and catalase containing an effective amount of a compound of general formula I or a pharmaceutically acceptable salt thereof, and, if necessary, a pharmaceutically acceptable carrier.

The present invention also relates to a method for producing compounds of general formula I.

DETAILED DESCRIPTION OF THE INVENTION

New preferred compounds (II, III, IV), known preferred compounds (V, VI, VII) of general formula I are presented in table 1.

Table 1

Connection No X, name of compound R II

H α-aspartylhistamine III

H β-aspartylhistamine IV

H aminosuccinimidohistamine V

CH ₃ —CO— N-acetyl-β-aspartylhistamine VI

CH ₃ —CO— N-acetyl-β-aspartylhistamine VII

CH ₃ —CO— N-acetylaminosuccinimidohistamine

The synthesis of new (II, III, IV) and known (V, VI, VII) compounds of general formula I can be carried out using methods of peptide chemistry in solution [Gershkovich A.A., Kiberev V.K. // Chemical synthesis of peptides / Kiev. Naukova Dumka. 1992.360 p.]. It is known that in the synthesis of peptides that include the Asp-His amino acid sequence, the formation of a cyclic succinimide derivative can occur under the action of acids and bases [Ondetti MA, Deer A., Sheehan JT, Pluscec J., Kosu O. Side reaction in the synthesis of peptides containing the aspartylglycyi sequence. // J. Biochemistry. 1968. V.7. No. 11. P.4069-4075]. In order to eliminate such effects during the synthesis of new compounds of the general formula I, which are derivatives of the decarboxylated dipeptide AspHis, benzyloxycarbonyl (Z-) and benzyl (Bzl-) protective groups are used to protect the NH2- and a- or P-COOH groups of aspartic acid removed by hydrogenolysis. The amide bond is created by the method of activated pentafluorophenyl ethers as the most active of the known. The activation of the free α- or β-carboxyl group of the protected aspartic acid is carried out in ethyl acetate by the action of N, N'-dicyclohexylcarbodiimide in the presence of pentafluorophenol under cooling. Pentafluorophenyl esters of the corresponding α- or β-protected derivatives of aspartic acid are introduced into the peptide formation reaction with an equivalent amount of histamine in anhydrous dimethylformamide. Histamine is obtained from histamine dihydrochloride by the action of sodium methylate in methanol. Judging by the analysis of the reaction mixture by TLC and mass spectrometry, the reaction proceeds in high yield, but with the formation of two compounds at this stage: the target linear reaction product (Z-II (OBzl) or Z-III-OBzl) and ~ 30% of the by-product a product representing a cyclic succinimide derivative Z-IV (m / z 450.8 and 342.7, respectively).

To exclude any effects on the protected derivatives, a mixture of Z-II (OBzl) or Z-III-OBzl and Z-IV is further subjected to catalytic hydrogenolysis without separation. The separation of the desired products II or III from the impurity of the cyclic derivative IV is carried out at the final stage, using the poor solubility of the synthesized linear compounds II or III in isopropanol, in contrast to the well-soluble cyclic derivative IV. As a result of using such a scheme (Scheme 1), the yields of α-AspHA and β-AspHA, counting on the initial Z-Asp (OBzl) -OH or Z-Asp- (OH) -OBzl, are about 40%. This yield is quite high, given the unusually light cyclization of Z-II (OBzl) or Z-III-OBzl to Z-IV, which occurs even during storage and during purification by column chromatography.

For the synthesis of the known acetyl derivatives of aspartyl histamine, we used β- or α-benzyl esters of aspartic acid as starting compounds (Scheme 2). N ^α -acetylation was carried out by the action of acetic anhydride on a suspension of the corresponding amino-free benzyl esters of aspartic acid in water at a ratio of AC ₂ O: H ₂ O (4: 1). The activation reactions of the carboxyl group and the creation of the peptide bond are carried out under the conditions described above for Z-protected derivatives (Z-Asp (OBzl) -OH or Z-Asp- (OH) -OBzl). In the synthesis of N ^α -Ac derivatives of Asp-HA, the formation of cyclic derivative VII is also observed. Analysis of the mass and PMR spectra confirmed the absence of benzyl protection in compound VII and the presence in its IR Fourier spectrum of a strong absorption band at 1700 cm ^-1 and a weak one at 1779 cm ^-1 , corresponding to stretching vibrations of carbonyl groups in the composition of succinimide derivatives [Ondetti M .A., Deer A., Sheehan JT, Pluscec J., Kosu O. Side reaction in the synthesis of peptides containing the aspartylglycyi sequence. // J. Biochemistry. 1968. V.7. No. 11. P.4069-4075].

Due to the increased tendency to cyclize N-Ac-Asp (OBzl) onto the α- and β-structures, which occurs both during short-term storage and during hydrogenation on a palladium catalyst, mixtures of α- or β-benzyl protected peptides V (OBzl) are subjected to hydrogenation or VI-OBzl with cyclic derivative VII without isolation. The debenzylated linear peptides V or VI are separated from the cyclic derivative VII by reprecipitation from organic solvents, preferably from a mixture of methanol and acetone. The use of this synthesis scheme allows to obtain compounds V or VI with a yield of about 50%, counting on the initial α- or β-benzyl esters of aspartic acid.

Purposeful synthesis of cyclic compounds IV and VII can be carried out by processing the reaction mixtures obtained after the formation of the peptide bond in compounds Z-II (OBzl) or V (OBzl) with triethylamine (pH 8), followed by drying in vacuum, with a yield from 37 to 41% The cyclic structure of the substances thus obtained was confirmed by IR Fourier and ¹ H-NMR spectroscopy, as well as mass spectrometry.

Thus, a method for the synthesis of a family of new (II, III, IV) and known (V, VI, VII) aspartyl histamine derivatives based on Z-Asp (OBzl) OH or Z-Asp (OH) -OBzl and Ac-Asp ( OBzl) OH or Ac-Asp (OH) OBzl.

The compounds of general formula I can also be prepared as pharmaceutically acceptable salts with non-toxic acids such as fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid and the like, and bases such as sodium hydroxide, potassium hydroxide, sodium carbonate and the like.

The compounds of general formula I have the ability to modulate the activity of SOD and catalase enzymes and can be used to treat diseases associated with impaired oxidative processes and changes in the level of reactive oxygen species.

The pharmaceutical compositions contain the compound of the present invention in an amount effective to achieve the desired result, and can be administered in unit dosage forms (e.g., in solid, semi-solid or liquid forms) containing the compounds of the present invention as an active ingredient in admixture with a carrier or excipient suitable for intramuscular, intravenous, oral, sublingual, inhalation and intrarectal administration. The active ingredient may be included in the composition along with commonly used non-toxic pharmaceutically acceptable carriers suitable for the manufacture of solutions, tablets, pills, capsules, dragees, suppositories, emulsions, suspensions, ointments, gels and any other dosage forms.

Various substances can be used as fillers, such as saccharides, for example glucose, lactose or sucrose, mannitol or sorbitol, cellulose derivatives and / or calcium phosphates, for example tricalcium phosphate or calcium acid phosphate, such as starch can be used as a binder component paste, for example corn, wheat, rice, potato starch, gelatin, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidone. If necessary, disintegrating agents such as the aforementioned starches and carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, can be used.

The core of the dragee is usually coated with a layer that is resistant to the action of gastric juice. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, and suitable organic solvents or mixtures thereof.

As additives, stabilizers, thickeners, colorants and perfumes can also be used.

As an ointment base, hydrocarbon ointment bases such as white and yellow petroleum jelly (Vaselinum album, Vaselinum flavum), liquid paraffin (Oleum Vaselini), white and liquid ointment (Unguentum album, Unguentum flavum), and as additives to give more dense consistency hard paraffin and wax; absorbent ointment bases - hydrophilic petrolatum (Vaselinum hydrophylicum), lanolin (Lanolinum), cold cream (Unguentum leniens); ointment bases washed off with water - hydrophilic ointment (Unguentum hydrophylum); water-soluble ointment bases polyethylene glycol ointment (Unguentum Glycolis Polyethyleni). bentonite bases and others.

As the basis for the gels, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, polyethylene glycol or polyethylene oxide, carbopol, tylose can be used.

As a basis for a suppository, bases that are not soluble in water can be used - cocoa butter; bases soluble in water or miscible with water - gelatin-glycerol or polyethylene oxide; combined bases - soap-glycerin.

In the preparation of a unit dosage form, the amount of active ingredient used in combination with a carrier may vary depending on the recipient being treated, on the particular route of administration of the drug.

So, for example, when using the compounds of the present invention in the form of solutions for injection, the content of the active agent in them is 0.01-100 mg, preferably 1-10 mg. As diluents, a 0.9% sodium chloride solution, distilled water, novocaine solution for injection, Ringer's solution, and glucose solution can be used. When introduced into the body of the compounds of the present invention in the form of tablets and suppositories, their amount is 1.0-100 mg per unit dosage form.

Dosage forms of the present invention are obtained according to standard methods, such as, for example, mixing, granulating, dragee formation, dissolution and lyophilization.

It should be noted that the compounds of the present invention exhibit modulating activity in concentrations by orders of magnitude lower compared to known preparations. In the study of toxicity when animals were administered the studied compounds at a dose of 1000 mg / kg, the death of experimental animals was not observed orally.

A detailed description of the compounds of the present invention, their preparation and study of pharmacological activity is presented in the following examples, intended to illustrate the preferred variants of the invention, and not limiting its scope.

EXAMPLES OF SYNTHESIS OF COMPOUNDS OF THE PRESENT INVENTION

The starting compounds used in the synthesis were Z-L-Asp (OBzl), Z-L-Asp-OBzl, L-Asp (OBzl) -OH, L-Asp (ОН) -OBzl manufactured by Bachem (Switzerland).

The individuality of the obtained compounds was checked by TLC on plates on Kieselgel 60 F ₂₅₄ plates (Merck, Germany) in the systems: chloroform-methanol 9: 1 (1), isopropanol-water-ammonia 6: 1: 3 (2).

Electrophoresis on paper (electrophoresis paper of Kondopoga Pulp and Paper Mill, 120 × 320 mm) is carried out in a buffer with a pH of 5.1 of pyridine-methanol-water 12: 10: 1000 in a chamber (150 × 320 × 150 mm) with a gradient of 15 V / cm for 2 hours.

Chromatograms were shown with a chlorotolidine reagent, Pauli reagent, ninhydrin, and UV light.

The optical rotation angles were measured on a Perkin Elmer 341 polarimeter (USA).

¹ H-NMR was recorded on an AMX-400 Bruker instrument (Germany).

IR Fourier spectra were recorded in KBg tablets on a Madpa 750 instrument (Nicolet USA).

The melting point was determined on a Boetius instrument (Germany).

High resolution mass spectra were obtained on a time-of-flight mass spectrometer using matrix laser desorption ionization, 2.5-dihydrooxybenzoic acid as a matrix, and REFLEX ™ III (Bruker, Germany) as a matrix.

Analytical reverse phase HPLC was performed on the following instruments:

- Breeze chromatograph, Waters detector (USA), detection at 214 nm, elution speed 1 ml / min, under conditions (1): Symmetry 300 C ₁₈ column, 3.9 × 150 mm, 5 μm, elution 0 , 1% aqueous TFA with a gradient of acetonitrile from 0 to 60% in 18 minutes

EXAMPLE 1

α-Aspartylhistamine (II)

To a solution of 2.0 g (5.6 mmol) of Z-Asp (OBzl) -OH in 20 ml of ethyl acetate, 1.10 g (5.9 mmol) of pentafluorophenol was added with vigorous stirring, the solution was cooled to 0 °, and 1.22 g (5.9 mmol) of DCC was added. Stirred for 2 hours at 0 ° and left for 12 hours at + 4 °. The DCU precipitate was filtered off.

The solvent was removed in vacuo. The oily residue was triturated with hexane. The resulting white solid precipitate was filtered off, washed with hexane and dried in vacuo over CaCl ₂ . 2.87 g (98%) of Z-Asp (OBzl) -OPfp were obtained. R _f 0.59 (1). _Mp 94-96 °. Lit. [Gross E., Mayenhofer I. // Peptides. The main methods of peptide bond formation. M .: World. 1983, p. 422.] 95.5-96 °.

- 7,47 (С 0.35; МеОН-Н₂O (1:1)). ¹H-ЯМР, D₂O δ. м.д.: 2.20-2.35 (м, 2Н, α-СН₂-НА), 2.5 (т, J 6 Гц, 2Н, β-СН₂-Asp), 3.0-3.15; 3.2-3.35 (м, 2Н, β-СН₂-НА), 3.65 (т, J 6 Гц, Н, α-CH-Asp), 6.7 (с, 1Н, 4-CH-lm), 7.6 (с, 1Н, 2-CH-lm). ИК-Фурье, δ, см^-1: 1669 (амид I); 1570 (амид II); 1081 (амид III). ВЭЖХ в условиях (1): индивидуальный пик, время удерживания 1.68 мин. Найдено, %: С 47.50; Н 6.20; N 24.32. С₉Н₁₄N₄O₃. Вычислено, %: С 47.78; Н 6.24; N 24.76. ВЭЖХ в условиях 8: индивидуальный пик, время удерживания 2.00 мин.A solution of 0.25 g (10.96 mmol) of sodium metal in 4 ml of anhydrous methanol was added to a solution of 1.0 g (5.48 mmol) of Na · 2HCl in 11.5 ml of anhydrous methanol under cooling and stirred for 20 min at 0 ° and 20 min at + 22 °. Precipitated NaCl was filtered off, washed with methanol. The solvent from the filtrate was removed in vacuo. The oily residue was dissolved in 16 ml of DMF. 2.87 g (5.48 mmol) of Z-Asp (OBzl) -OPfp was added to the resulting solution with vigorous stirring. Stirred for 2 hours at + 20 ° and left for 12 hours at + 4 °. The solvent was removed in vacuo, the residue was triturated with anhydrous ether. Maintained under ether at + 4 ° for 12 hours. The precipitate was separated by filtration and dried in vacuum over CaCl ₂ . 1.13 g (46%) of a mixture of substances with R _{f 1} 0.36 (1) Z-Asp (OBzl) -HA (Z-II (OBzl)) were obtained; R _{f 2} 0.2 (1) N ^α- benzyloxycarbonyliminosuccinimido histamine (Z-IV). Mass spectrum, m / z: [M + 1] ⁺ 451.2 Z-II (OBzl); [M + 1] ⁺ 343.1 (Z-IV). To a solution of 1.13 g of a product mixture in 12 ml of methanol, 0.80 g of 10% palladium on charcoal was added with vigorous stirring and hydrogenated in a stream of hydrogen for 1.5 h. The catalyst was filtered off. The solvent from the filtrate was removed in vacuo. The resulting white crystalline substance was triturated with isopropanol, filtered and dried in vacuo over CaCl ₂ . 0.22 g (90%) was obtained. Yield 40% based on the initial Z-Asp (OBzl) -OH. R _f 0.71 (2). E _HA ^- 0.37. _Mp 207-209 °. Mass spectrum, m / z: [M + 1] ⁺ 227.0.

- 7.47 (C 0.35; MeOH-H ₂ O (1: 1)). ¹ H-NMR, D ₂ O δ. ppm: 2.20-2.35 (m, 2H, α-CH ₂ -HA), 2.5 (t, J 6 Hz, 2H, β-CH ₂ -Asp), 3.0-3.15; 3.2-3.35 (m, 2H, β-CH ₂ -HA), 3.65 (t, J 6 Hz, H, α-CH-Asp), 6.7 (s, 1H, 4-CH-lm), 7.6 (s, 1H, 2-CH-lm). IR Fourier, δ, cm ^-1 : 1669 (amide I); 1570 (amide II); 1081 (amide III). HPLC under conditions (1): individual peak, retention time 1.68 min. Found,%: C 47.50; H 6.20; N, 24.32. C ₉ H ₁₄ N ₄ O ₃ . Calculated,%: C 47.78; H 6.24; N, 24.76. HPLC under conditions 8: individual peak, retention time 2.00 min.

EXAMPLE 2

β-Aspartylhistamine (III)

, - 10,52 (С 0.29; МеОН-Н₂О (1:1)). ¹H-ЯМР, δ, м.д., D₂O: 2.20-2.35 (м, 4Н, β-CH₂-Asp, α-CH₂-HA), 3.12 (т, J 5 Гц, 2Н, β-СН₂-НА), 3.61 (дд, J 4 Гц, J 6 Гц, 1Н, α-CH-Asp), 6.65 (с, Н, 4-CH-lm), 7.45 (с, Н, 2-CH-lm). ИК-Фурье, δ, см^-1: 1641 (амид I); 1528 (амид II); 1087 (амид III). ВЭЖХ в условиях (I): индивидуальный пик, время удерживания 1.90 мин. Найдено, %: С 47.78; Н 6.35; N 24.83. C₉H₁₄N₄O₃. Вычислено, %: С 47.78; Н 6.24; N 24.76.To a solution of 1.5 g (4.2 mmol) of Z-Asp (OH) -OBzl in 8.5 ml of ethyl acetate, 0.83 g (4.5 mmol) of pentafluorophenol was added with vigorous stirring, then the solution was cooled to 0 ° and 0.927 g (4.5 mmol) of DCC was added. Stirred for 3 hours at 0 ° and left for 48 hours at + 4 °. The DCU precipitate was filtered off. The solvent was removed in vacuo. The oily residue was triturated with hexane. The resulting white solid precipitate was filtered off and dried in vacuo over CaCl ₂ . 2.12 g (96.5%) of Z-Asp- (OPfp) -OBzl were obtained. R _f 0.93 (1). A solution of 0.176 g (7.64 mmol) of Na in 3 ml of anhydrous methanol was added to a solution of 0.704 g (3.82 mmol) of Na · 2HCl in anhydrous methanol under cooling and stirred for 20 min at 0 ° and 20 min at + 25 °. Precipitated NaCl was filtered off, washed with methanol. The solvent from the filtrate was removed in vacuo. The oily residue was dissolved in 20 ml of DMF. 2.0 g (3.82 mmol) of Z-Asp (OBzl) -OPfp were added to the resulting solution with vigorous stirring. Stirred for 2 hours at 20 ° and left for 20 hours at 4 °. The solvent was removed in vacuo, the residue was triturated with dry ether. It was kept at + 4 ° for 3 days. The precipitate was separated by filtration and dried in vacuum over CaCl ₂ and P ₂ O ₅ . Got a white crystalline substance. Yield 0.86 g (50%). R _{f 1} 0.54 (11) Z-Asp (HA) -OBzl (Z-III-OBzl); R _{f 2} 0.2 (11) (Z-IV). Mass spectrum, m / z: [M + 1] ⁺ 451.2 (Z-III-OBzl); [M + 1] ⁺ 343.1 (Z-IV). To a solution of 0.86 g of the product mixture in 11 ml of methanol, 0.40 g of 10% palladium on charcoal was added with vigorous stirring, hydrogenated with carbon in a stream of hydrogen for 3 h. The catalyst was filtered off. The solvent from the filtrate was removed in vacuo. The resulting white crystalline substance was triturated with isopropanol, filtered and dried in vacuo over CaCl ₂ . Yield 0.20 g (90%). The total synthesis yield is 41%. R _f 0.67 (2). E _HA ^- 0.34. _Mp 196-198 °. Mass spectrum, m / z: [M + H] ⁺ 227.1.

, - 10.52 (C 0.29; MeOH-H ₂ O (1: 1)). ¹ H-NMR, δ, ppm, D ₂ O: 2.20-2.35 (m, 4H, β-CH ₂ -Asp, α-CH ₂ -HA), 3.12 (t, J 5 Hz, 2H, β -CH ₂ -HA), 3.61 (dd, J 4 Hz, J 6 Hz, 1H, α-CH-Asp), 6.65 (s, H, 4-CH-lm), 7.45 (s, H, 2-CH -lm). IR Fourier, δ, cm ^-1 : 1641 (amide I); 1528 (amide II); 1087 (amide III). HPLC under conditions (I): individual peak, retention time 1.90 min. Found,%: C 47.78; H 6.35; N, 24.83. C ₉ H ₁₄ N ₄ O ₃ . Calculated,%: C 47.78; H 6.24; N, 24.76.

EXAMPLE 3

N-Aminosuccinimidohistamine (IV)

- 42.72 (С 0.32; МеОН-Н₂O (1:1)). ¹H-ЯМР, CD₃OD, δ, м.д.: 1.26 (дд, J=5 Гц, Н, β-CH₂-Asp), 1.85 (т, J=5 Гц, 2Н, α-СН₂-НА), 2.0 (дд, J=10 Гц, J=20 Гц, Н, β-СН₂-НА), 3.0 (т, J=5 Гц, Н, α-CH-Asp), 7.21 (с, Н, CH-4-Jm), 8.14 (с, Н, CH-2-Jm). ИК-Фурье, δ, см^-1: ИК-Фурье, δ, см^-1: 3449, 3338 (вал. NH₂); 1781, 1701 (вал. С=O в циклическом имиде); 1250 (амид III);. Найдено, %: С 51.05; Н 5.67; N 26.00. C₉H₁₂N₄O₂. Вычислено. %: С 51,92; Н 5.81; N 26,91.To a solution of 0.40 g of a mixture of Z-III-OBzl (R _f 0.54 (1)) and Z-IV (R _f 0.2 (1)) (see β-aspartyl histamine synthesis procedure) in 5 ml of chloroform-methanol 8: 2 was added triethylamine to pH 8, the solvent was removed in vacuo and dried in vacuo over anhydrous CaCl ₂ for 2 hours. Received cyclic Z-IV with Rf 0.76 (2). E _HA ^- 0.60. IR Fourier, δ, cm ^-1 : 1700, 1779 (Val. C = O in cyclic imide); 1250 (amide III). To a solution of 0.20 g (0.56 mmol) of cyclic Z-Asp- (HA) in 7 ml of anhydrous methanol, 0.14 g of 10% palladium-on-charcoal was added with vigorous stirring, it was hydrogenated in a stream of hydrogen for 1.5 h. The catalyst was filtered off. The solvent from the filtrate was removed in vacuo. The resulting oily residue was triturated with ether. A solid white precipitate was filtered off and dried in vacuo over CaCl ₂ . 0.056 g (41%) was obtained. R _f 0.76 (2). T _PL 150-152 °

- 42.72 (C 0.32; MeOH-H ₂ O (1: 1)). ¹ H-NMR, CD ₃ OD, δ, ppm: 1.26 (dd, J = 5 Hz, H, β-CH ₂ -Asp), 1.85 (t, J = 5 Hz, 2H, α-CH ₂ -HA), 2.0 (dd, J = 10 Hz, J = 20 Hz, H, β-CH ₂ -HA), 3.0 (t, J = 5 Hz, H, α-CH-Asp), 7.21 (s, H, CH-4-Jm), 8.14 (s, H, CH-2-Jm). IR Fourier, δ, cm ^-1 : IR Fourier, δ, cm ^-1 : 3449, 3338 (Val. NH ₂ ); 1781, 1701 (Val. C = O in cyclic imide); 1250 (amide III) ;. Found,%: C 51.05; H 5.67; N 26.00. C ₉ H ₁₂ N ₄ O ₂ . Calculated. %: C 51.92; H 5.81; N, 26.91.

EXAMPLE 4

N-Acetyl-α-aspartylhistamine (V)

(С 1; Н₂O). ¹H-ЯМР (CD₃OD) δ: 1.75 (с, 3Н, Ас), 2.40 (д, J 8 Гц, 2Н, β-СН₂-Asp), 2.6 (т, J 8 Гц, 2Н, α-СН₂-НА), 3.10-3.20 (м, 2Н, β-СН₂-НА), 4.31 (т, J 8 Гц, 1Н, α-CH-Asp), 6.9 (с, 1Н, 4-CH-lm), 8,05 (с, 1Н, 2-CH-lm). ИК-Фурье, ν, см^-1 (в пленке): 3319 (вал. NH); 1662 (амид I); 1570 (амид II). Найдено, %: С 49.29; Н 6.32; N 23.80. C₁₁H₁₆N₄O₄. Вычислено, %: С 49.25; Н 6.01; N 23.86. ВЭЖХ в условиях (1): время удерживания 3.35 мин.To a suspension of 2.4 g (10.75 mmol) of L-Asp (OBzl) -OH in 3 ml of water, 10 ml of acetic anhydride was added with vigorous stirring and heating to 40 °. After 30 min, 5 ml of water was added to the reaction mass, the solvent was removed in vacuo, the oily residue was dried in vacuo over NaOH, recrystallized from a mixture of 5 ml of ethanol and 0.5 ml of water, dried over CaCl ₂ . Yield 2.24 g (80%). Further synthesis was carried out starting from 1.0 g (3.77 mmol) of Ac-L-Asp (OBzl) -OH in accordance with the procedure described for compound II. Yield 0.54 g (54%) calculated on L-Asp (OBzl) -OH. R _f 0.65 (2). E _HA ^- 0.08. T _pl 112-114 °

(C 1; H ₂ O). ¹ H-NMR (CD ₃ OD) δ: 1.75 (s, 3H, Ac), 2.40 (d, J 8 Hz, 2H, β-CH ₂ -Asp), 2.6 (t, J 8 Hz, 2H, α- CH ₂ -HA), 3.10-3.20 (m, 2H, β-CH ₂ -HA), 4.31 (t, J 8 Hz, 1H, α-CH-Asp), 6.9 (s, 1H, 4-CH-lm ), 8.05 (s, 1H, 2-CH-lm). IR Fourier, ν, cm ^-1 (in film): 3319 (Val. NH); 1662 (amide I); 1570 (amide II). Found,%: C 49.29; H 6.32; N, 23.80. C ₁₁ H ₁₆ N ₄ O ₄ . Calculated,%: C 49.25; H 6.01; N, 23.86. HPLC under conditions (1): retention time 3.35 minutes

EXAMPLE 5

N-Acetyl-β-aspartylhistamine (VI)

(С 0.27; МеОН-Н₂O (1:1)). Масс-спектр, m/z: [M+H]⁺ 269.0. ¹H-ЯМР (CD₃OD) δ: 1.90 (с, 3Н, Ac), 2.45-2.70 (м, 2Н, β-CH₂-Asp), 2.85 (т, J 6 Гц, 2Н, α-CH₂-HA), 3.36-3.48 (м, 2Н, β-СН₂-НА), 4.38-4.42 (м, 1Н, α-CH-Asp), 7.20 (с, 1Н, 4-CH-lm), 8,50 (с, 1Н, 2-CH-lm). ИК-Фурье, ν/см^-1: 3271 (вал. NH), 1654 (амид I), 1551 (амид II). Найдено, %: С 46.48; Н 6.37; N 19.64. C₁₁H₁₆N₄O₄-H₂O. Вычислено, %: С 46.15; Н 6.34; N 19.57. ВЭЖХ в условиях (I): время удерживания 4.00 мин.Acetylation was carried out in accordance with the procedure described for compound VII, then the synthesis was carried out starting from 0.24 g (0.9 mmol) of Ac-L-Asp (OH) -OBzl according to the procedure for compound III. Yield 0.12 g (54%) calculated on L-Asp (OBzl) -OH. R _f 0.65 (2). E _HA ^- 0.10. Hygroscopic

(C 0.27; MeOH-H ₂ O (1: 1)). Mass spectrum, m / z: [M + H] ⁺ 269.0. ¹ H-NMR (CD ₃ OD) δ: 1.90 (s, 3H, Ac), 2.45-2.70 (m, 2H, β-CH ₂ -Asp), 2.85 (t, J 6 Hz, 2H, α-CH ₂ -HA), 3.36-3.48 (m, 2H, β-CH ₂ -HA), 4.38-4.42 (m, 1H, α-CH-Asp), 7.20 (s, 1H, 4-CH-lm), 8, 50 (s, 1H, 2-CH-lm). IR Fourier, ν / cm ^-1 : 3271 (Val. NH), 1654 (amide I), 1551 (amide II). Found,%: C 46.48; H 6.37; N, 19.64. C ₁₁ H ₁₆ N ₄ O ₄ -H ₂ O. Calculated,%: C 46.15; H 6.34; N, 19.57. HPLC under conditions (I): retention time 4.00 min.

EXAMPLE 6

N-Acetylaminosuccinimidohistamine (VIII)

(С 0.41; МеОН-Н₂O (1:1)). Масс-спектр, m/z: [М+Н]⁺ 251.0. ¹H-ЯМР (CD₃OD+DMSO-d₆) δ: 2.20 (с, 3Н, Ас), 2.80-2.95 (м, 1Н, β-CH₂-Asp), 3.08 (т, J 9 Гц, 2Н, α-CH₂-HA), 3.25 (дд, J 10 Гц, J 22.5 Гц, 1Н, β-CH₂-Asp), 3.95 (т, J 9 Гц, 2Н, β-CH₂-HA), 4.65-4.75 (м, 1Н, α-CH₂-Asp) 7.15 (с, 1Н, 4-CH-lm), 7.90 (с, 1Н, 2-CH-lm). ИК-Фурье, ν/см^-1: 3266 (вал. NH); 1779, 1700 (вал. С=O в циклическом имиде); 1659 (С=O ацетил); 1584 (деф. NH). Найдено, %: С 52.67; Н 5.45; N 22.40. С₁₁Н₁₄O₃. Вычислено, %: С 52.79; Н 5.64; N 22.39. ВЭЖХ в условиях (I): время удерживания 4.30 мин.The synthesis was carried out starting from 1.0 g (3.77 mmol) of Ac-L-Asp (OBzl) -OH in accordance with the procedure described for compound IV. Yield 0.32 g (37%) calculated on L-Asp (OBzl) -OH. R _f 0.80 (2). E _HA ^- 0.40. _Mp 226-228 °.

(C 0.41; MeOH-H ₂ O (1: 1)). Mass spectrum, m / z: [M + H] ⁺ 251.0. ¹ H-NMR (CD ₃ OD + DMSO-d ₆ ) δ: 2.20 (s, 3H, Ac), 2.80-2.95 (m, 1H, β-CH ₂ -Asp), 3.08 (t, J 9 Hz, 2H , α-CH ₂ -HA), 3.25 (dd, J 10 Hz, J 22.5 Hz, 1H, β-CH _2- Asp), 3.95 (t, J 9 Hz, 2H, β-CH ₂ -HA), 4.65 -4.75 (m, 1H, α-CH ₂ -Asp) 7.15 (s, 1H, 4-CH-lm), 7.90 (s, 1H, 2-CH-lm). IR Fourier, ν / cm ^-1 : 3266 (Val. NH); 1779, 1700 (val. C = O in cyclic imide); 1659 (C = O acetyl); 1584 (def. NH). Found,%: C 52.67; H 5.45; N, 22.40. C ₁₁ H ₁₄ O ₃ . Calculated,%: C 52.79; H 5.64; N, 22.39. HPLC under conditions (I): retention time 4.30 min.

TEST FOR BIOLOGICAL ACTIVITY

The modulating effect of the compounds of the aspartyl histamine derivatives corresponding to the general formula I obtained by the claimed method on the activity of antioxidant protection enzymes, which was studied by known methods (FIGS. 1, 2, Tables 2, 3), was established.

EXAMPLE 7

The effect of compounds of General formula 1 on the activity of Zn / Cu SOD

In vitro, a commercial enzyme preparation (Sigma, United States), Cu / Zn-SOD (from bovine erythrocytes), was used to assess the effect on the in vitro activity of Cu / Zn-SOD compounds. SOD activity was evaluated spectrophotometrically at 550 nm by inhibiting the reduction of oxidized cytochrome c during the generation of a superoxide anion radical in the xanthine oxidase – xanthine system (50 mM KH ₂ PO ₄ buffer, pH 7.8, 10 mM EDTA, 0.01 mM Fe ³⁺ -sit. S , 0.05 mM xanthine). The amount of xanthioxidase introduced into the system should cause a cytochrome reduction rate from 0.025 op./min. The unit activity of SOD is taken as the activity of the amount of the enzyme, the content of which causes a 50% reduction in the rate of cytochrome c reduction under given conditions. The effect of the modifier was evaluated after preliminary incubation for 15 min at 25 ° [McCord JM, Fridovich I. Superoxide Dismutase. // J. Biol. Chem. 1969. V.244. P.6049-6053].

EXAMPLES OF MEDICINAL FORMS.

EXAMPLE 8

A. Tablet form

A tablet form is prepared using the following ingredients:

The compound corresponding to General formula (I) 0.5-500 mg Potato starch 20-50 mg Magnesium stearate 3 mg Aerosil 1 mg Lactose up to 300 mg

The components are mixed and compressed to form tablets weighing 300 mg each.

B. Suppositories

Suppository composition example:

The compound corresponding to General formula (I) 0.5-500 mg Cacao butter amount needed to obtain suppository.

If necessary, it is possible to produce rectal, vaginal and urethral suppositories with appropriate excipients.

V. Ointment

An example of the composition of the ointment:

The compound corresponding to General formula (I) 0.05-0.5 g Petroleum jelly 10 g

Ointments are made according to well-known technology.

G. Gels

Gel composition example:

The compound corresponding to General formula (I) 5-500 mg tylose 200 mg benzyl alcohol 0.3 mg ethanol 300 mg water up to 10 g

RESULTS

The introduction into the incubation medium containing Cu, Zn-SOD, an effector of compound V, corresponding to the general formula I, causes highly reliable inhibition of enzyme activity (50 ÷ 90%) in the range of effector concentrations of 10 ^-5 -10 ^-7 M and 10 ^-13 -10 ^-14 M. The related compound VI with another type of amide bond, on the contrary, significantly increases the activity of Cu, Zn-SOD - by 50 ÷ 70%, in the range of effector concentrations of 10 ^-10 -10 ^-14 M.

The cyclic succinimide derivative VII weakly affects the activity of Cu, Zn-SOD, but significantly activates catalase (60%) in a wide range of concentrations of 10 ^-12 -10 ^-4 M effector.

Deacetylated compounds corresponding to the general formula 1 (R = H), to a much lesser extent affect the activity of Cu, Zn SOD and catalase - the effects do not exceed 30%. For these compounds, a multidirectional effect with respect to catalase is also observed: compound II weakly inhibits catalase in a wide range of concentrations (10 ^-4 -10 ^-12 M), and compound III activates it, as well as SOD at an effector concentration of 10 ^-8 -10 ⁴ M.

CONCLUSION

New and known compounds corresponding to general formula I have a modulating effect on antioxidant enzymes — Cu, Zn SOD and catalase, and a high percentage of their inhibition (or activation) is achieved, including at very low concentrations and / or in a wide range of concentrations effector.

These properties of the proposed series of new and known compounds corresponding to general formula I can be widely used, including in oncology (antitumor effect), immunology (modulation of immune responses, radioprotective effect), cardiology (anti-ischemic and antihypoxic effect), and ophthalmology (treatment of cataracts), to correct the consequences of diabetes, disorders of the central nervous system, as well as antihypoxants, including when using hyperbaric oxygenation, wound healing agents; in cosmetology - as additives that prevent the destruction and aging of skin cells.

Thus, effective modulators of antioxidant protection enzyme activity — Cu, Zn SOD and catalase — have been proposed that correspond to general formula I, including new and known derivatives of aspartyl histamine, including in the form of pharmaceutically acceptable salts, as well as a simple method for their preparation.

table 2
The effect of compound VI on the activity of SOD in vitro (basic activity of SOD 7.8 ± 0.14 U / mg) The concentration of the drug (M) SOD activity, U / mg protein one 2 10 ^-4 6.8 ± 0.10 10 ^-5 5.8 ± 0.16 10 ^-6 7.8 ± 0.12 10 ^-7 7.8 ± 0.14 10 ^-8 8.8 ± 0.10 10 ^-9 7.8 ± 0.11 ^10-10 13.6 ± 0.12 ^10-11 11.7 ± 0.12 10 ^-12 11.7 ± 0.10 10 ^-13 11.7 ± 0.10 ^10-14 11.7 ± 0.10 10 ^-15 8.8 ± 0.12 10 ^-16 10.7 ± 0.10 10 ^-17 12.8 ± 0.12

Table 3
The effect of compound VII on the activity of catalase in vitro (basic activity of catalase 262 ± 11 U / mg) The concentration of the drug (M) Catalase Activity, U / mg Protein 10 ^-3 304 ± 11 10 ^-4 399 ± 11 10 ^-5 420 ± 14 10 ^-6 451 ± 13 10 ^-7 441 ± 12 10 ^-8 430 ± 10 10 ^-9 430 ± 11 ^10-10 315 ± 10 ^10-11 462 ± 11 10 ^-12 283 ± 12 10 ^-13 304 ± 11 ^10-14 252 ± 14 10 ^-15 220 ± 10 10 ^-16 367 ± 11 10 ^-17 304 ± 10

Scheme 1

Synthesis of Compounds II, III, IV

Scheme 2

Synthesis of Compounds V, VI, VII

Claims (12)

1. Aspartyl derivatives of histamine of General formula I

where at R = H,

or

or

and their pharmaceutically acceptable salts. 2. The compounds according to claim 1, with the ability to modulate the activity of antioxidant enzymes - superoxide dismutase and catalase. 3. The compound according to claim 2, where at R = H,

with the ability to inhibit catalase, including in the concentration range from 10 ^-12 M to 10 ^-4 M. 4. The compound according to claim 1, where at R = H,

possessing the ability to activate superoxide dismutase, including in the concentration range from 10 ^-14 M to 10 ^-10 M, and catalase, including in the concentration range from 10 ^-8 M to 10 ^-4 M. 5. A pharmaceutical composition having the ability to modulate the activity of superoxide dismutase and catalase, comprising as an active agent an effective amount of a compound of general formula I

where R represents hydrogen or an acetyl group, X represents

or

or

and their pharmaceutically acceptable salts, and a pharmaceutically acceptable carrier. 6. The use of compounds of General formula I

where R represents an acetyl group, X represents

or

or

and their pharmaceutically acceptable salts for modulating the activity of superoxide dismutase and catalase enzymes. 7. The use of compounds according to claim 6, where

to inhibit the activity of superoxide dismutase, including in the concentration range from 10 ^-6 M to 10 ^-7 M and from 10 ^-13 M to 10 ^-14 M. 8. The use of compounds according to claim 6, where

for activation of superoxide dismutase, including in the concentration range from 10 ^-10 M to 10 ^-14 M. 9. The use of compounds according to claim 6, where

to activate catalase, including in the concentration range from 10 ^-12 M to 10 ^-4 M. 10. The method of obtaining N-aspartyl derivatives of histamine of the general formula (I), which consists in the fact that the activated pentafluorophenyl ether of the N ^α -protected derivative of the α- or β-benzyl ester of aspartic acid is reacted with histamine in an organic solvent, and the products formed are N-protected derivatives of aspartylhistamine α- or β-esters without separation of the N-protected cyclic succinimide derivative of aspartylhistamine are hydrogenolized in the presence of a palladium catalyst on activated carbon for the removal of protections followed by separation of the target products H-Asp (HA) -OH or H-Asp (OH) -HA from

crystallization from an organic solvent. 11. The method of claim 10, where benzyloxycarbonyl or acetyl groups are used as the N ^α -protection. 12. The method of claim 10, wherein the amide bond formation reaction is carried out in dimethylformamide, and the crystallization of the linear products of H-Asp (HA) -OH or H-Asp (OH) -HA is carried out from isopropanol.

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