RU2224737C2 - Polyoxyphenylene esters, method for their preparing, biologically active substance eliciting combined antioxidant and anti-hypoxic effect - Google Patents

Abstract

FIELD: bioorganic chemistry, chemical technology, biology. SUBSTANCE: invention relates to new biologically active substances and to methods for their preparing. Invention proposes new synthesized polyoxyphenylene esters of the general formula (I)

wherein R means -OH, -S-SO₂-ONa; x = 1, 2; M means -OH, -O-C₆H₄-OH. Method for preparing polyoxyphenylene esters involves oxidation of hydroquinone with hydrogen peroxide in the presence of ferrous salts followed by addition of additional functional groups to the compound (HO)₂C₆H₃-O-C₆H₄(OH)•H₂O. New biologically active substances are nontoxic practically and they show the expressed combined antioxidant and anti-hypoxic effect. EFFECT: improved preparing method, valuable biological properties. 9 cl, 7 dwg, 9 tbl, 4 ex

Description

 The invention relates to the field of bioorganic chemistry, namely to new biologically active substances (BAS), methods for their preparation and use.

 A large group of substances is known having antioxidant (AO) and / or antihypoxic (AG) action. These include, in particular, cytochrome C, ubiquinone, methylphenazine, ionol and others (M.D. Mashkovsky. Medicines. - M .: Medicine, vol. 2, pp. 73-76; Encyclopedia of medicines. Edition. 8 , M.: RLS. 2001, 1503 pp .; VIDAL Handbook. M: Astra-PharmService, 2001, 725 pp.; Vinogradov V.M. Pharmacology and Toxicology.Sb. Kiev: Health, 1972, 7, p. 163 ) So, in particular, ubiquinone (coenzyme Q) at a dose of 200 mg / kg live weight has a normalizing effect on cells under certain hypoxia, being a natural electron carrier and occupying a central place in the electron transport enzyme chain, reacting with three enzyme systems: NAD, H-oxidase, succinate, KoQ reductase, and the cytochrome system (RF patent 1746886, 1991, CL 08P 32/06; PCT application 96/08527, 1996, CL 08 G 61/10).

 The disadvantages of most of these substances is a narrow spectrum of action, due to a significant number of contraindications, lack of effectiveness, complicated production technology.

Structural analogues are polyarylene ethers of type C ₆ H ₅ O (C ₆ H ₄ O) ₆ C ₆ H ₅ used as components of copolymers with polypropylenes or as additives that increase the thermal stability of resins and are obtained by the oxidation of phenols (US patent 3257357, 1966 , Ncl. 260-47; U.S. Patent 3,306,874, 1967, Ncl. 260-47; U.S. Patent 4,645,787, 1987, Ncl.

 The difference of these analogues from the claimed substances is the presence of other radicals, the oligomeric or polymeric nature of the compound, fundamentally different physicochemical properties.

 The closest in properties to the claimed group of inventions are derivatives of oligo-hydroxyphenylenes - drugs Olifen and others (application PCT 96/08527, CL 08 G 61/10, 1996), which have found application as antihypoxants for shock, epilepsy and chronic lung diseases, as an antioxidant in the treatment of sarcoidosis and as part of feed additives (RF patent 2082397, CL A 61 K 31/095, 1997; auth. St. USSR 1660701, CL A 61 K 31/745, 1991; RF patent 2076611, CL A 23 K 1/16, 1997; RF patent 2043765, CL A 61 K 31/095, 1995).

 Compounds of this type were obtained by polymerization of para-benzoquinone in the presence of alkaline agents in an aqueous-acetone medium with the addition of substances providing the introduction of substituent groups, for example sodium thiosulfate or guanidine, into the polymer and subsequent isolation of the target product by extraction, usually with ether.

 A disadvantage of compounds of this type is the production of a mixture of oligomers during the reaction, which leads to poor standardization of the drug, poor solubility of the compounds in water, the presence of an unpleasant aftertaste, insufficiently high efficiency for some pathologies, and the presence of complications, especially when administered intravenously.

 The problem solved by the authors was the creation of new, more effective drugs with combined antioxidant and antihypoxic properties.

 It has been suggested that the properties of the drug should improve when replacing the —C — C— bonds between the phenyl groups of the oligomer with the —C — O — C— bond. The appearance of an oxygen atom between hybridized phenyl nuclei (FN) of an oxygen atom that has a free electron pair and ensures that phenolic nuclei come closer together due to the arrangement of individual O- (FF) bonds at an acute angle to each other should ensure the transition from individual hybridized fragments of structures to hybridization of the entire oligomer.

 Moreover, the new structure would have to have a denser electron cloud, be more stable with respect to external conditions, which made it promising for working with biosystems.

This problem was successfully solved by the creation of polyoxyphenylene ethers (PDFE) of the general formula (Fig. 1):
where R-OH, -S-SO ₂ -ONa; x = 1; 2 M = -OH, -O-C ₆ H ₄ -OH
As such compounds, in particular, were obtained:
(1) 4-hydroxyphenylene-2,4-dioxibenzene, crystalline hydrate - "DPD" (basic structure). Gross formula C ₁₂ H ₁₂ O ₄ . The molecular weight of 237 ke (analysis 240 ± 5). Mass fraction of carbon - 58.7%; mass fraction of hydrogen - 5.4%.

Formula: (HO) ₂ C ₆ H ₃ OS ₆ H ₄ ONxH ₂ O (FIG. 2).

(2) 2- (1-hydroxy-4-hydroxybenzene) -5- (sodium sulfothio) -1,4-hydroxyphenylene - “DPS”
The gross formula is C ₁₂ H ₉ O ₇ S ₂ Na. The molecular weight of 311 ke (analysis 302 ± 8).

Estimated structural formula
(HO) ₂ C ₆ H ₃ —O — C ₆ H ₃ (OH) (—S — SO ₂ ONa) (FIG. 3).

 (3) di- (1,4-hydroxyphenylene) -1,4-dioxibenzene or p-dioxi-p-diphenyl-p-hydroxyquinone - "DFT".

Gross formula C ₁₈ H ₁₄ O ₄ . The molecular weight of 294 ke (analysis 292 ± 6).

Estimated structural formula:
HO — C ₆ H ₄ —O — C ₆ H ₄ —O — C ₆ H ₄ —OH (FIG. 4).

 When dehydrated, all of these structures form needle crystals of gray (1) or dark gray (2-3) colors, highly soluble in water and practically insoluble in organic solvents: benzene, dichloromethylene, chloroform, ethyl acetate, acetone, organic alcohols (methyl, ethyl , isopropyl).

Compounds (1) and (3) are slightly soluble in acids, compound (2) is sparingly soluble in strong acids. the pH of the aqueous solution is 7.3-7.6, the density of 0.93-0.94 g / cm ³ .

The chemical structure of “PDFE” was estimated using the methods of infrared (IR) and ultraviolet spectroscopy, nuclear magnetic resonance (NMR) resonance, and a number of other methods (the IR and UV spectra of all three compounds were close to each other and had the same characteristic bands characterizing the basic structure). To assess the structure of the drug by NMR, samples of the drug were dissolved in ² H ₂ O (Isotope, 98% ² H) to a final concentration of 10 weight. % ¹ H-NMR spectra were measured at a frequency of 300 MHz. The lines in the spectra were assigned by the resonance signal of the residual protons of water (δ 4.8). NMR spectra were recorded at room temperature 20 ± 2 ^° C. The NMR spectrum is shown in FIG. 5.

 The analysis of the spectrum showed that in the resonance region of aromatic protons a multiplet with a position of δ = 6.57 ppm belonging to hydroquinone protons was found, which confirms the presence of its structure in the compound, as was expected for structures of this type. Comparison of the obtained spectrum with the spectrum of Oliphene (Fig. 6) showed the presence of a certain similarity between the spectra, due to a certain similarity of their structures (the main difference is the replacement of the -C-C- bond between benzene nuclei with the -C-O-C- bond) .

 The analysis of "PDFE" by PC spectroscopy revealed characteristic lines corresponding to the presence of hydroxyl groups, the substitution of the aromatic ring in the para position, the presence of -C-O- and -C = C- bonds (Fig. 7, Table 1), which confirms the proposed secondary structure of the claimed group of compounds.

 The study of the UV spectrum revealed the presence of a characteristic absorption band with a maximum of 289 nm, characteristic of the hydroquinoid structure.

 An EPR study showed the presence of a powerful signal for substance (1), associated with the presence of unpaired electrons on the phenyl group. The signal value decreases by more than half when passing to compound (2), which is associated with the addition of a thiosulfur group to the phenyl core in the para position relative to the hydroquinone structure and increased hybridization of the electron cloud of the whole compound.

The above data, along with the mechanism of formation of compounds, unambiguously show the features of the secondary structure of “PDFE”. The method of obtaining compounds "PDFE" includes:
- at the first stage, obtaining "DPD" as a result of the interaction of hydroquinone with hydrogen peroxide in the presence of ferrous salts by the reaction:
НО-С ₆ Н ₄ -ОН + Н ₂ О ₂ -> О = С ₆ Н ₄ = О + е ^- ->
О = С ₆ Н ₄ -О -> (НО) ₂ С ₆ Н ₃ -О-С ₆ Н ₄ ОН (а)
at the second stage, the introduction of thiosulfo and other groups into the structure of (1) according to standard schemes, for example
(HO) ₂ C ₆ H ₃ -O-C ₆ H ₄ OH + Na ₂ S ₂ O ₃ ->
НО-С ₆ Н ₄ -О-С ₆ Н ₂ (ОН) ₂ (-S-SO ₂ ONа) (b)
or
(HO) ₂ С ₆ Н ₃ -О-С ₆ Н ₄ ОН + НО-С ₆ Н ₄ -ОН + Н ₂ О ₂
-> НО-С ₆ Н ₄ -О-С ₆ Н ₄ -О-С ₆ Н ₄ -ОН (c)
Reaction (a) proceeds by reacting hydroquinone with an excess of hydrogen peroxide (optimum molar ratio 1: 1.5-2.0) with the introduction of 0.01-0.02 wt. % iron chloride. Excess acids formed are neutralized with alkali. At a lower ratio, the reaction rate decreases and incomplete conversion of hydroquinone is possible, which will require the introduction of additional purification of the target product from unreacted compounds.

Reaction (b) proceeds between “DPD” and sodium thiosulfate in a weight ratio of 1: 0.2-0.5 at a temperature of 40-60 ^o C and vigorous stirring for 20-60 minutes

 Reaction (c) proceeds between “DPD” and hydroquinone in a molar ratio of 1: 0.8-1.5 in the presence of 0.5-0.8 mol of hydrogen peroxide in the form of a 3% solution and 0.3-0.8 wt. .% iron chloride.

 Reactions (a) and (b) are exothermic and do not require elevated temperatures for their progress.

 An analysis of the chemical structures carried out by scanning the Beilsteins Handhuch from 1941 to the present with the aim of searching for similar compounds revealed, as analogues, derivatives of benzoquinones, for example phenyl mercantobenzoquinone and 2-phenyl-5-sulfothio-para-benzoquinone (Helvetica Chimica Acta, v. XXX, Fsaciculus 11, 1947, p. 578-584 // Beil Bd. 08/03/70 p. 2635-2637). All identified compounds had bonds between C-C or -S-S- nuclei, i.e. The claimed compounds are not described in the scientific and technical literature.

 The properties of the compounds obtained are illustrated by the following examples.

Example 1. The method of obtaining the substance "DFD". To 500 g of hydroquinone (GC), placed in a porcelain cup, add 200 ml of 30% H ₂ O ₂ (PV) and 15 mg of iron sulfate (FA) and mix thoroughly, after which 200 ml of 30% are added to the mixture another 4 times H ₂ About ₂ and 2 g of caustic soda. At the end of the reaction, a black solid is formed, which is placed for 14-16 hours in an oven at a temperature of 60-70 ^o C until the smell disappears. Yield 506 g, pH of a 5% aqueous solution of 6.8. After recrystallization from an aqueous solution according to molecular weight and spectral characteristics, the substance corresponds to the properties of "DPD".

 The effect of the ratio of ingredients on the yield of the final product is shown in table 2.

Example 2. The method of obtaining the substance "DFS". To 500 g of "DFD" placed in a porcelain cup, 150 g of sodium thiosulfate (TCH) was added and thoroughly mixed at 60 ^° C for 30 minutes. Upon completion of the reaction, a black solid forms, which is then recrystallized. Yield 780 g. After recrystallization from an aqueous solution by molecular weight and spectral characteristics, it corresponds to the properties of DFS.

 The effect of the ratio of ingredients on the yield of the final product is shown in table 3.

 Example 3. The method of obtaining the substance "DFT".

To 500 g of hydroquinone (GC) and 1000 g of "DPD", placed in a porcelain cup, add 100 ml of 30% hydrogen peroxide (PV) and 7.5 g of iron chloride (LC), mix thoroughly, and then into the mixture another 14 times make 100 ml of 30% PV and 4 g of caustic soda. At the end of the reaction, a black solid forms, which is placed for 10 hours in an oven at a temperature of 60-70 ^o C until the smell disappears. Yield: 506 g.

 After recrystallization from an aqueous solution, the substance corresponds to the properties of "DFT" in molecular weight and spectral characteristics.

 The effect of the ratio of ingredients on the yield of the final product is shown in table 4.

 Example 4. The study of the biological properties of the drugs. The study of the biological properties of preparations of the type "PDFE" was carried out in accordance with the guidelines (Methodological guidelines for the formulation of studies to substantiate the sanitary standards of harmful substances in the air of the working area. 2163. M., 1980, 19 pp .; Determination of the safety and effectiveness of biologically active additives to food MUK 2.3.2.721-98. Ministry of Health of the Russian Federation., M. 1999, 87 pp.) White mice, rats, guinea pigs and rabbits of standard weight were used in the experiments. For intragastric administration of powders of the substances "PDFE" and Olifen ex tempore, aqueous suspensions were prepared.

 The dynamics of the weight of rats and the relative mass of internal organs to body weight was determined on the scales of VLR-500.

 Daily urine was collected using exchange cells for rats of the Italian company Texnoplast. Blood for biochemical studies was obtained by puncture of the tail vein of rats or from the orbital sinus of guinea pigs.

 The activity of Alt, AcT, alkaline phosphatase, thymol test, the content of total protein and serum lipid phosphorus, as well as urine protein were determined using Bio-Lat-Test kits of the Czech company Lahema.

 SPP was determined by S.V. Speransky (Speransky S.V. Pharmacology and Toxicology, 1965, 1, p. 123), and the remaining indicators are generally accepted methods.

 In conditions of free behavior, the behavior of animals was studied using the "open field" method (Y. Buresh, O. Bureshova, J.P. Houston Methods and basic experiments on the study of brain and behavior. - M.: Higher School, 1991, 450 p.). Animals slaughtered by decapitation underwent autopsy. For histological examination, lungs, heart, liver, kidneys, stomach and skin were taken. The material was fixed in 15% formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin and Sudan IV for fat.

 The study of the gonadotoxic effect of Epofen was carried out according to indicators required in the initial assessment of chemicals. The study of mutagenic activity was carried out according to the frequency of micronuclei formation in polychromatophilic bone marrow erythrocytes (from the femur) of white mice in accordance with the methodological recommendations. (Methods of an experimental study to establish the threshold of action of industrial poisons on the generative function. Ministry of Health of the USSR, Moscow, 1969, 25 pp .; Methods of experimental research to establish the thresholds of action of industrial poisons on generative fiction for the purpose of hygienic regulation. Methodological recommendations. Ministry of Health of the USSR, 1744 , M., 1978, 35 pp.; Assessment of the mutagenic activity of chemicals by the micronucleus method. Guidelines. MH USSR 28/10. M., 1984. 21 pp .; Evaluation criteria and methods for predicting the long-term effects on the organ low level of harmful substances in the air due to the action on the body of harmful substances in the air of the working zone. Information letter. Research Institute of gigantic labor and prof. zabol., M., 1985, 15 pp.)

 Statistical processing of the experimental results was performed according to Student-Fisher.

 Determination of indicators of acute toxicity included experiments on rodents (mice, rats). Animals were randomly assigned to groups using randomization. The criteria for the acceptability of randomization were considered to be the absence of external signs of diseases and the homogeneity of groups by body weight (± 20%).

 The substances were ground into powder, from which aqueous solutions (suspensions) were prepared for intragastric (v / f) administration. The introduction was carried out orally through a metal atraumatic probe in increasing doses but to Litchfield-Unloxon.

 To study each dose of the drug, groups of 6 animals of the same sex were used. In addition, there were similar in number groups of control animals of each sex, to which equivalent volumes of solvent — distilled water — were introduced along the same route. The observation period was 14 days. The results are shown in tables 5-7.

 As can be seen from the tables, preparations of the "PDFE" type are significantly less toxic than "Oliphene". The administration of substances in toxic doses after 20-30 minutes was accompanied by the development of inhibition, physical inactivity, ataxia, tremor, ruffled hair, and hypersalivation. Within an hour, attacks of clonic-tonic seizures with a violation of the act of breathing developed. The death of animals was observed within 3-5 hours with symptoms of shortness of breath and paralysis. The surviving animals were inhibited, hypodynamic, had an untidy appearance.

 At autopsy a day after acute administration, the following was revealed. The pleura and organs of the chest are not changed. Light pale pink, airy, without condensation to the touch. The size of the heart is within normal limits. In the cavities of the heart contains a small amount of liquid blood. The heart muscle is dense, brown in color. The stomach is filled with a small amount of solid food. The mucous membrane is shiny, folded, slightly pinkish in color. The mucosa of the small intestine is shiny, smooth, pinkish in color.

 The size and shape of the liver do not differ from the control. The surface of the liver is smooth. The capsule is thin, transparent. The liver pattern in the section is not changed. Liver tissue is moderately full-blooded. The kidneys are of normal size and shape, brownish in color, dense, with a distinct cortical and medullary substance in the section. The thyroid gland, adrenal glands and pancreas do not differ in appearance from the control. When examining the histological preparations of the stomach and parenchymal organs, differences in the structure between the experimental and control groups were not found.

 The vessels of the lungs were moderately full-blooded. The epithelium of the alveoli and intrapulmonary bronchi did not represent any changes. The alveoli were filled with air. Edema or pneumonia was not observed. The myobifrils of the left ventricle of the heart and interventricular septum had a distinct transverse striation, the nuclei of cardiomyocytes were light. Myocardial vessels are moderately full-blooded. The lobular structure of the liver persisted. The boundaries of hepatocytes were clear, the hepatocyte cytoplasm was weakly basophilic, granular, with a sufficient chromatin content and a thin nuclear membrane.

 The epithelium of the convoluted tubules of the kidneys did not represent any changes. Its cytoplasm was granular, oxyphilic, and its nuclei were bright. Glomerular vessels are moderately full-blooded.

 The structure of the mucous membrane of the fundus of the stomach did not differ from the control. The nuclei of the epithelium of the mucosa and glands were light. In the basal parts of the glands, granularity in the cytoplasm was preserved. The epithelium of the villi and crypts of the small intestine mucosa was preserved; mitosis was present in the crypts.

 When staining frozen sections of the liver with Sudan IV, signs of fatty degeneration were not observed. Subdural and perivascular hemorrhages were observed in the brain.

 The analysis of the values of the mass coefficients of the internal organs of rats did not reveal any significant differences between the groups of animals that received the substances being compared, and in relation to the control group.

 According to the results of studies of the substance of preparations, “PDFE” belong to the IV class of low toxic drugs (N. Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning. Ed. IV, Baltimore, 1975, 427 p.).

 In chronic daily (for 30 days) iv administration of substances to rats at a dose of 100 mg / kg (a dose that is 2 times higher than the maximum therapeutic for humans) (Encyclopedia of Medicines, ed. 7. Edited by Yu.F. Krylov, M., 2000, p. 678) no lethal effects were observed. The data in the tables indicate that no statistically significant differences with the control (water) were revealed for any of the indicators used (p> 0.05 at a 95% probability level) or pathological deviations beyond the limits of physiological norm variation.

 When opening rats slaughtered the day after the last injection of substances, the size, shape and color of the internal organs did not have macroscopic changes compared to the control. The mucous membranes of the stomach and small intestine were shiny, pale pink, with no signs of irritation or inflammation.

 Histological examination of preparations of the lungs, myocardium, liver, kidneys and gastric mucosa of experimental animals showed no dystrophic, inflammatory, or necrobiotic changes in the above organs.

 The epithelium of the alveoli and intrapulmonary bronchi did not represent any changes, the alveoli were air. Atelectasis or pulmonary edema was not observed. The transverse striation of the myocardial myofibrils was distinct. The structure of the liver was not a violation. Borders of hepatocytes were distinct, granular cytoplasm, weakly basophilic, nuclei bright with a thin membrane and distinct nucleoli. Necroepithelium with oxyphilic granular cytoplasm and clear, clear nuclei. The epithelium of the gastric mucosa is preserved, the main and parietal cells of the glands of the stomach are not changed.

 The chronic intake of PDFE substances into the body of mice and rats through the stomach does not cause dystrophic or destructive measurements of parenchymal organs and is not accompanied by irritation of the mucous membranes of the gastrointestinal tract.

 Example 5. Evaluation of antihypoxic and antioxidant properties of drugs.

 Evaluation of the effectiveness of new biologically active substances was evaluated on the model of acute hypoxia. (Guidance on physiology. Ecological physiology of man. Adaptation of a person to extreme environmental conditions, under the editorship of OG Gazenko. M .: Nauka, 1979, pp. 333-336; Guide to practical exercises in pathological physiologists. Edited by O M. Pavlenko, Moscow: Medicine, 1974.P. 174-175).

The animals were placed in a 250 ml jar tightly closed with a glass lid lubricated with sealant. The maximum lifespan and symptoms of thanatogenesis were recorded using a stopwatch. Cans with animals during the study were in the air conditioner, ensuring the constancy of the experimental conditions (temperature 20 ^o C, humidity 65-70%, atmospheric pressure). The controls were intact animals.

After death, the brain was obtained ex tempore in each animal, its homogenesis was performed (400 rpm, 10 strokes of the pestle), and the catalase activity, the level of diene conjugates and lipid hydroperoxides were determined in the homogenate (Tyson CA, Luman KO, Stephens RJ, Arch. Env . Health, 1982, Vol. 37., 3, p. 167-176; Modern methods in biochemistry. Edited by VN Orekhovich. M.: Medicine, 1977. S. 62-64). These indicators allow you to evaluate the antioxidant properties of the drug (table 8)
It turned out that the drugs significantly increase the life time of mice, while the lipid peroxidation rates stabilized (the levels of malondialdehyde and brain lipid hydroperoxides decreased) and antioxidant (catalase) activity was restored, which indicates an increase in the reserve antioxidant activity of the brain.

 The drug "DFS" was compared by the effectiveness of antihypoxic effects with ubiquinone, cytochrome and Oliphene in various types of hypoxia in rats using experimental and published data. The test results are shown in table 9.

 The data obtained show that compounds of the "PDFE" type are practically non-toxic and have pronounced antihypoxant and antioxidant properties, which makes them promising for further use in the medical, food and cosmetic industries, as well as in veterinary medicine and agriculture.

Claims (9)

1. Polyoxyphenylene ethers of the General formula

where R = —OH, —S — SO ₂ —ONa; x = 1, 2; M = —OH, —O — C ₆ H ₄ —OH. 2. Polyoxyphenylene ethers according to claim 1, having a combined antioxidant and antihypoxic effect. 3. The compound according to claim 1 of the General formula

4. The compound according to claim 1 of the General formula

5. The compound according to claim 1 of the General formula

6. A method of producing polyoxyphenylene ethers, which consists in the oxidation of hydroquinone with hydrogen peroxide at a molar ratio of these reagents 1: (1.5-2), respectively, in the presence of ferrous salts, and then neutralization of the formed acid with alkali to obtain the compound according to claim 3, and, if necessary, additional functional groups are introduced into the obtained ether. 7. The method according to claim 6, which consists in the fact that iron chloride is used as a ferrous salt in a concentration of 0.01-0.02 wt.%. 8. The method according to claim 6 or 7, which consists in the fact that upon receipt of the compound according to claim 4, the compound according to claim 3 is mixed with sodium thiosulfate in a weight ratio of 1: (0.2-0.5), respectively, at a temperature 40-60 ° C with stirring for 20-60 minutes 9. The method according to claim 6 or 7, which consists in the fact that upon receipt of the compound according to claim 5, the compound according to claim 3 is mixed with hydroquinone in a molar ratio of 1: (0.8-1.5), respectively, in the presence of 0.5-0.8 moles of hydrogen peroxide and 0.3-0.8 wt.% Salts of ferrous iron.

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