Synthesis and study of the effect of 3-substituted chromone derivatives on changes in the activity of mitochondrial complex III under experimental cerebral ischemia

Сhromone derivatives are characterized by an extensive spectrum of pharmacological activity and can be potentially effective neuroprotective agents. In this regard, this study evaluated the effect of eighteen new derivatives of 3-formylchromone on the change in the actinicity of mitochondrial complex III in ischemic and intact animals as a promising direction of neurotective therapy. The dependence of the level of pharmacological effect on changes in the quantum-chemical parameters (Mulliken Charge, Bond number, Theoretical valence, Unsaturated index, Electron density, Free valence index) of the molecules was also evaluated. The structure of the substances was confirmed by IR, UV and NMR spectroscopy, as well as by determining the melting point. The study made it possible to establish that, among the 3-substituted chromone derivatives, the most pronounced neuroprotective activity is possessed by compounds that do not contain halogen atoms in their structure. The leader compound can be considered 3 [(E) -3(3,5-di-tret-butyl-4-hydroxy-phenyl) -3-oxo-prop-1-enyl] -6-methoxy-chromen-4-one, the use of which increased the activity of mitochondrial complex III by 85.2% (p <0.05) in relation to the negative control group. To the greatest extent, the influence of the test substances on the activity of the mitochondrial complex III depended on the change of Free valence index (correlation coeficient, r=0,93701)

mitochondrial matrix and penetrate through the mitochondrial inner membrane. The process of ROS penetration through the inner membrane of mitochondria proceeds by two mechanisms: with the catalytic conversion of a superoxide radical into a peroxide-a more stable molecule, or with the participation of anionic mitochondrial channels [4].
Thus, a targeted pharmacological effect on mitochondrial complex III may be a promising direction in the treatment of oxidative stress and associated diseases, for example, ischemic stroke [5].
Chromone is known to be benzannulated 4H-pyranone-4. Chromone derivatives constitute one of the most numerous groups of natural compounds, of which the most studied derivatives are 2-arylchromoneflavones and 3-arylchromone-isoflavones. Representatives of these compounds are included in the main classes of flavonoids and have more than 5000 described structures, characterized by about 50 types of pharmacological activity [6]. It has now been established that the chromone nucleus is a promising basic structure for the targeted synthesis of new compounds with desired pharmacological properties. In particular, chromone derivatives are used in the field of neurodegenerative, inflammatory and infectious pathologies, as well as diabetes mellitus and cancer [7]. In addition, the antiallergic properties of chromone derivatives are widely known, which are associated with the stabilization of mast cells cell membranes [8]. In an earlier study, chromone derivatives had a positive effect on changes in mitochondrial function, normalizing the reactions of aerobic/anaerobic metabolism, which served as the basis for choosing representatives of a number of derivatives of 3-formylchromone as the test-objects [9] .
Considering also that the structural diversity of the types, number and position of substituents attached to the main chromone nucleus is important for a directed change in the physical, chemical and pharmacological properties of the target compounds [10], we carried out this work.

Results of assessing the effect of the studied compounds on the change in the activity of mitochondrial complex III
The effect of the test-compounds on the activity of the mitochondrial complex III was assessed in ischemic and intact animals ( Table 1). As a result, it was found that the administration of the test-substances to intact animals did not have a significant effect on changes in the activity of ubiquinolcytochrome-coxidoreductase. At the same time, in rats with cerebral ischemia, which did not receive pharmacological support, a decrease in the activity of this respiratory complex by 52% (p <0.05) was noted in relation to SO (sham-operated) animals (Table 1). On the background of the administration of the CoQ10, the catalytic properties of ubiquinolcytochrome-c-oxidoreductase in comparison with the NC (negative control) group of rats increase by 39% (p <0.05). The use of test compounds C3ACL; C3AF; C3AI; C3A6Ac and C3ACLNOH; C3AACP1; C3AACP2; C3AACP3; C3AACP4;; C3AACP5 C3AACP6; C3AACP7 also increased the activity of mitochondrial complex III by 42.3%; 64.2%; 30.9%; 25.2%; 26%; 31.7%; 43.9%; 67.5%; 53.7%; 41.5%; 46.3%; 60.2% and 85.2% respectively (all indicators p <0.05 relative to the NC group of rats). At the same time, the activity of ubiquinolcytochrome-c-oxidoreductase in animals that received compounds C3AACP2 and C3AACP7 was higher than that in rats that received CoQ10 (Table 2) by 20.4% (p <0.05) and 33.3% (p <0.05) respectively.  Table 2 shows the summary quantum-chemical parameters used in the correlation analysis. The data of the pharmacological experiment showed a significant difference in the effect of compounds that do not contain halogens compared to that of halogen-containing derivatives. In this regard, in the correlation analysis, both subgroups of test-compounds was carried out (Table 3).

Table 2. Total values of quantum chemical parameters
From the data in the table 3, it follows that the correlation coefficients for compounds that do not contain halogens are significantly higher than for halogenated derivatives. The maximum values of r are observed when analyzing such parameters as the free valence index (Fμ), electron density, and unsaturation index (IUA). Thus, when studying the structure -activity relationship, the most reliable results are achieved for chromone derivatives that do not contain halogens, and the Fμ parameters and electron density are used in the correlation equations.

DISCUSSION
Globally, ischemic stroke is the second leading cause of death and the first cause of disability in the adult population. The «gold standard» in the treatment of this pathological condition is the fastest possible recanalization of the vessel, achieved by intravenous administration of tissue plasminogen activator (t-PA). Nevertheless, this pharmacotherapeutic approach has drawbacks, which include a small «therapeutic window» and the development of post-reperfusion complications [11]. In this regard, a number of attempts to improve reperfusion treatment have been made, including through the use of adjuvants, for example, neuroprotective agents, which are currently being actively studied by the world scientific community [12]. The main direction of the action of neuroprotectors is the elimination of pathogenetic pathways of neuronal damage during ischemia, for example, oxidative stress, the activity of which directly depends on the adequate course of electron transport reactions in the mitochondrial ETC [13].
The mitochondrial respiratory chain consists of four multimeric complexes that catalyze redox reactions, with the participation of NADH and FADH2 as electron donors, which ultimately reduces molecular oxygen to water. This electron transfer is associated with the formation of a proton electrochemical gradient required for the condensation of ADP and phosphate, and, accordingly, the formation of ATP. Complex III or ubiquinolcytochrome-c-oxidoreductase is the center of the respiratory chain, transferring electrons from coenzyme Q to cytochrome C [14]. Dysfunction of complex III leads to the development of irreversible metabolic disorders that mediate a high degree of cellular damage. Thus, when the catalytic reactions of complex III are suppressed, there is overproduction of free radicals, a decrease in ATP synthesis, the formation of a mitochondrial pore of transient permeability with an increased influx of ionized calcium and cell death as a result of apoptosis or pyroptosis [15]. In this regard, the dysfunction of complex III has an extremely negative effect on the state of organs with high metabolic activity, for example, the brain. At the same time, suppression of the physiological activity of ubiquinolcytochrome c-oxidoreductase is undesirable in view of the catastrophic drop in ATP synthesis in cells with normal metabolism [16].
The study showed that the use of newly synthesized 3-substituted chromone derivatives at a dose of 40 mg / kg per os increases the activity of mitochondrial complex III in the brain of animals with reproduced cerebral ischemia, but does not change the activity of this supercomplex in rats without pathological background, which can be evidence of the regulatory action of 3-substituted chromone derivatives in relation to ubiquinolcytochrome-c-oxidoreductase. At the same time, a regularity was noted that the inclusion of halogen atoms in the target structure significantly worsens the pharmacological effect. In this connection, a correlation analysis was carried out of the dependence of the regulatory capabilities of the studied compounds in relation to complex III and quantum-chemical parameters that most fully characterize the features of the molecular structure. This analysis showed that, with a high degree of probability, halogen atoms worsen the pharmacotherapeutic response to the administration of 3-substituted chromone derivatives, with its consistently high indicators in a number of substances that do not contain halogens in their structure. At the same time, the activity of the compounds under study largely depends on the value of the free valence index (r = 0.93701). Based on the Fμ value, one can draw conclusions about the reactivity of molecules containing πbonds: the larger the Fμ value, the higher the probability for the attachment of neutral particles, which is most important for the mitochondria-oriented action of pharmacologically active substances [17]. At the same time, it is known that halogen atoms, being in the aromatic nucleus, have a strong negative inductive effect (-I effect), clearly prevailing over a weak positive mesomeric effect (+ M effect), i.e. -I >> + M, which probably distorts the π-electron cloud and leads to a decrease in therapeutic efficacy [18].

CONCLUSION
The study showed that among the studied 3-substituted chromone derivatives, the highest regulatory activity in relation to ubiquinolcytochrome-c-oxidoreductase is possessed by 3 -[(E) -3-(3,5-di-tert-butyl-4hydroxy-phenyl) -3-oxo-prop-1-enyl] -6-methoxy-chromen-4-one, the use of which contributed to the restoration of mitochondrial complex III (the activity was 85.2% higher (p <0.05) in relation to the group of negative control). The calculated correlation coefficients of the activity level from changes in the quantumchemical parameters of molecules showed that the inclusion of halogens in the target structure leads to a significant decrease in the pharmacological effect. The above facts allow us to assert that, in order to achieve mitochondrial effects in predicting new compounds that are 3-substituted chromone derivatives, it is not advisable to introduce halogens into the aromatic nucleus, be it annelated or as an acetophenone fragment.
The structural formulas of the test-compounds are given in Table 4.

Cerebral ischemia model
Cerebral ischemia was modeled by the method of irreversible occlusion of the middle cerebral artery. The course of the operation: in anesthetized animals (chloral hydrate 350 mg / kg, intraperitoneally), on the depilated area below and to the right of the eye, the skin was dissected and the muscles were moved apart. Then the process of the zygomatic bone was removed and the skull was exposed. Next, a burr was used to make a trepanation hole above the intersection of the middle cerebral artery and the olfactory tract, the dura mater was removed, and the artery was electrocoagulated, followed by cutting to avoid vessel recanalization, and the wound was sutured in layers. The seam was treated with an antiseptic solution. Before awakening, the animals were left under a warming lamp [21].
CoQ10 (Hunan Warrant Pharm, China) at a dose of 200 mg / kg (per os) was used as a reference drug [22]. The compounds under study were administered at a dose of 40 mg / kg orally for 3 days after modeling brain ischemia (the reference was administered in the same way) [9]. The compounds under study were administered to intact animals for 10 days [9].

Biomaterial preparation
When studying the activity of mitochondrial complex III, the brain of animals was used as a biomaterial, which was homogenized in the isolation medium (1 mmol EGTA + 215 mmol mannitol + 75 mmol sucrose + 0.1% BSA solution + 20 mmol HEPES with pH 7.2). The resulting homogenate was centrifuged at 1100 g for 2 minutes. Secondary supernatant in the amount of 700 μl was transferred into Eppendorf tubes, mixed with 75 μl of 10% Percol and centrifuged at 18000g for 10 minutes. The pellet was resuspended in 1 ml of isolation medium and centrifuged for 5 minutes at 10,000 g [23].

Methods for assessing the activity of the mitochondrial complex III
The activity of complex III was assessed by a spectrophotometric method based on an increase in the optical density (550 nm) of a solution containing succinate, cytochrome C, rotenone, and an analyzed sample. The activity of the complex was expressed in terms of the protein concentration in the analyzed sample. The protein content was estimated according to the Bradford method [24].

Statistical Methods
Statistical processing of the research results was carried out using the applied software packages MS Excel 2013 and STATISTICA 6.0 (StatSoft, USA) for Windows. Data were expressed as M (mean) ± SEM (standard error of the mean). Statistically significant differences between groups were assessed by one-way analysis of variance (ANOVA) with post-Newman-Keuls post-processing at a significance level of p <0.05. Correlation analysis was performed using Spearman's test.