Regulatory Mechanisms in Biosystems

Influence of derivatives


Introduction
One of the modern approaches in creating bioregulators for clonal micropropagation of plants is the molecular design of natural and synthetic compounds that combine derivatives of nitrogen-containing heterocycles and carboxylic acid residues. Heterocyclic systems of nitrogen-containing heterocycles have highly reactive positions, which allows one to modify molecules and obtain libraries of new promising biologically active compounds (Brazhko et al., 2013).
The study of characteristic pharmacophores and molecular descriptors of the structure of derivatives of 2-((6-R-quinolin-4-yl)thio)acetic acid indicates the possibility of a fairly wide range of biological activity of these compounds. It was found that the studied derivatives (quinolin-4-ylthio) of acetic acid are promising as potential growth regulators. With the help of molecular design, potential bioactive molecules were created for experimental research (Kalinin, 1984;Kornet et al., 2021;Yakovleva-Nosar et al., 2022). It is known that Paulownia is a genus of evergreen and semievergreen deciduous trees, which is well adapted to the soil and climatic conditions of Ukraine. Popular in garden and park design, Paulownia clone in vitro 112 of Spanish selection can withstand low temperatures -25… -27 °C and has high decorative properties. This is an artificially bred and cloned tree, able to survive and develop in extreme conditions, undemanding to the soil (Bergmann, 1998).
Studies have shown that rooting in Paulownia is an auxin-dependent process (Matskevich, 2019). In microclonal propagation, Paulownia clone in vitro 112 needs to optimize and accelerate the rooting process and reduce the stress of regenerating plants when adapting to in vivo conditions. The aim of this work was to study the effect of derivatives of 2-((6-R-quinolin-4-yl)thio)acetic acid on rhizogenesis in vitro in Paulownia clone 112 explants and subsequent adaptation of microplants to in vivo conditions. Of considerable interest, in terms of creating effective stimulators of rhizogenesis for microclonal plant propagation, is the combination in one molecule of synthons such as heterocycle (6-R-Q and mercaptoalkylcarboxylic acid residue) (Brazhko et al., 2013;Kornet et al., 2021b). Today, such compounds remain poorly studied growth regulators and are a prospect for the creation of new, low-toxic and effective stimulators of rhizogenesis for microclonal plant propagation. Given the above, the preparation of derivatives of 2-((6-R-quinolin-4-yl)thio)acetic acid, and the study of the biological properties of these compounds determine the relevance of the work.
The aim of this work is to select the best among the derivatives of 2-((6-R-quinolin-4-yl)thio)acetic acid by virtual screening and experimental studies of potential stimulators of rhizogenesis for microclonal plant propagation.

Materials and methods
Derivatives of 2-((6-R-quinolin-4-yl)thio)acetic acid were synthesized to the Department of Chemistry of the Zaporizhia National University and the Department of Horticulture of the Khortytsk National Academy (Fig. 1).
The reactions and the purity of the synthesized compounds were controlled by the TLC on Sorbton-2 plates (Russia). As an eluent, mixtures of chloroform-methanol (1:1) and acetate-water (1:1) were used. Manifestations of chromatograms were performed using UV rays. The 1H NMR spectra were recorded on the "Bruker AC-300" (manufacturer Bruker, 2010, 300 MHz) device in DMSO-d6 and D2O. Chemical shifts are ex-pressed in parts per million (ppm) relative to tetramethylsilane (TMS). The coupling constants (J) are reported in Hertz (Hz). LC-MS spectra were recorded on a high-performance liquid chromatography module of the HPLC system for Agilent 1260 Infinity and a proton-ionization diodematrix probe.
Molecular descriptors of structure: gross formula, elemental composition, molecular weight, molecular refractive index, Log P, Log D, investigated compounds were determined using the computer software package ACD-I-Labs. LogP is the partition coefficient of the compound between n-octanol and water, and Log D is the lipophilicity of the compound depending on the pH of the medium Metelytsia et al., 2020). A key parameter in the study of the relationship between the structure and biological activity of organic compounds is the partition coefficient in the system of n-octyl alcohol -water. Correlations between the value of Рow and toxicity, penetration of artificial and natural membranes, biological activity of non-specific drugs, bioaccumulation, soil adsorption, etc. were found. However, the experimental determination of Рow is very time consuming. Therefore, it is generally accepted to use calculation methods for their evaluation. Adequacy of additive methods of calculation of distribution coefficients, and completeness of a set of experimental values of Рow on which this model is built were calculated.
We performed individual stages of calculation of molecular descriptors of the structure using a number of software tools, such as: framework JSDraw, OpenBabel, PaDEL-Descriptor, McQSAR, Pandoc, АСD-I-Labs. The following molecular descriptors of the structure were calculated: gross formula, elemental composition, molecular weight, molecular refractive index, Log P, Log D, СlogP (Table 2).
Based on this, we can say that the introduction in the 6th position of the methoxy group and stoxy group in the structure of 2-((quinolin-4-yl) thio)acetic acid and its structural analogues leads to increased molar refraction. This trend is easily explained by the fact that such a change in the structure of the molecule increases the effective radii of the molecules, the molar mass, and thus increases the molar refraction. A particularly important characteristic of any biologically active substance is lipophilicity (hydrophobicity) -a model of the distribution of the substance studied between two phases that do not mix (most often used octanol -water). This characteristic is easily modulated by the use of an appropriate descriptor and is most often used to assess the ability of a substance to overcome the biological membranes of cells.
When the test substance is in the aqueous phase in the form of molecules (uncharged particles) to characterize lipophilicity we use the indicator log P (P -partition coefficient at the boundary of octane -water).
If the test substance in the aqueous solution is partially dissociated in the form of charged particles (ions), there will be a certain dynamic equilibrium between the different forms of the compound, which will vary depending on the pH of the medium.  The lipophilicity of such a system will be determined by the partition coefficient log D -the ratio of the sums of activities of all components of the organic and aqueous phases. For comparison, we obtained quantumchemical values of lipophilicity log P for neutral forms 2-((6-R-quinolin-4-yl)thio)acetic acid (compounds I-4) and the value of the partition coefficient log D at pH = 7. This characteristic is most often used to assess the ability of the substance to overcome biological membranes of cells of the root system depending on the pH of the medium. The pH of most plant cloning media is maintained in the range of 6.5 to 7.5.
It was found that the values of log D for the tested compounds are much smaller than the values of log P, this is due to the consideration in the second case of acid-base equilibrium, which is in solution of the test substances. The change in lipophilicity of the substance from the ability to dissociate into ions in aqueous solution is explained as follows. Since water is a polar solvent (μ = 1.86 D) and the dipole moment of octanol is much smaller (it can be taken as a non-polar solvent), the ions that will be formed in the aqueous medium will hardly diffuse into the organic layer and the concentration of ions in it will be caused mainly by the transition of uncharged molecules of matter, resulting in a significant reduction in the concentration of matter in the organic phase.
At introduction in 6 positions of a quinoline cycle of methoxy group insignificant increase in lipophilicity of a molecule is observed (Δ log D = 0.07-0.08). Thus, lipophilicity (log D) is an important characteristic for assessing the ability to penetrate cell biological membranes and stimulate rooting derivatives 2-((6-R-quinolin-4-yl)thio)acetic acid and its derivatives, which may exist as ions in aqueous solution.
All of the compounds tested (compounds 1-8) according to Lipinski's "rule five" can show high biological activity.
To conduct a study of the toxic effects of compounds using native material we used ejaculate of fertile men (normozoospermia). To do this, we pre-evaluated the standard spermogram according to generally accepted methods in accordance with WHO criteria (Stefanov, 2001;Tiuzikov, 2013). Measurements were performed on the sperm fertility analyzer "AFS-500-2" (NPF "Biola"). The selected ejaculate was aliquoted by 100 μL, aliquots were numbered, and the following was added: -to the first aliquot -saline solution -10 μL (intact); -to the second aliquot -ascorbic acid (AA) at a concentration of 10-6М -10 μL; -to the third aliquot -ATC at a concentration of 10 -6 М -10 μL; -to the fourth aliquot -the test substance (quinoline derivative) at a concentration of 10 -6 М -10 μL; -to the fifth -saline solution -10 μL, then hydrogen peroxide at a concentration of 200 μM -0.5 μL (reference); -to the sixth -hydrogen peroxide at a concentration of 200 μM -0.5 μL, then AA at a concentration of 10 -6 М -10 μL; -to the seventh -hydrogen peroxide at a concentration of 200 μM -0.5 μL, then ATC at a concentration of 10 -6 М; -to the eighth -hydrogen peroxide at a concentration of 200 μM -0.5 μL, then the test substance at a concentration of 10 -6 М -10 μL; -the obtained samples were incubated at 37 °C for 2 hours. Immediately after incubation, the quality criteria of sperm were studied: concentration, movement, vital activity.
Measured indicators: total sperm concentration; total number of sperm in the ejaculate; rapid progressive motility (A); slow progressive motility (B); progressive motility (A + B); relative number of sperm with normal morphology; concentration of functional sperm; concentration of sperm with progressive motility; concentration of immotile sperm; the total number of sperm with progressive motility; total number of functional sperm; total number of immotile and non-progressive sperm; average speed (A + B) of motile sperm; index of normal motile sperm.
To address the issues of differentiation of living and dead sperm, Bloom's supravital staining is performed. The researchers evaluated the presence or absence of cell membrane permeability for eosin dye (EO; 1% aqueous solution) according to WHO guidelines, followed by counting living and dead cells. Live sperm were not stained (transparent), dead were stained in pink. To prepare a smear, 1 drop of ejaculate and 1 drop of eosin dye are applied to a medical glass, the drops are mixed with each other with another glass just like the blood sample, and a smear is complete. After the smear were dried in air, the number of live and dead sperm was counted by microscopy under an immersion lens (х 100) with х 10 binoculars. 100 stained and unstained sperm were counted and the percentage of living and dead sperm was determined.
The study of rhizogenesis was performed in vitro, with the addition of synthesized compounds at a concentration of 1 mg/L in the nutrient medium. Murashige-Skuga nutrient medium was prepared for rhizogenesis (Murashige, 1962), containing half the concentration of macrosalts and trace elements and 2% sucrose. The compounds were added before sterilization of the nutrient medium. The control was nutrient media without growth regulators (МС 0). The nutrient medium was sterilized by autoclaving under a pressure of 0.11 MPa for 30 minutes. The explants were cultivated at an air temperature of 22-24 °С with a photoperiod of 16 hours, a relative humidity of 65-70% and an illumination of 2.5 thousand lux. The results were recorded for 28 days and took into account the number, length of roots, frequency of rhizogenesis.
The tables and figures show the arithmetic mean values and their standard error (x ± SE). The certainty of differences between the samples was assessed using ANOVA (P < 0.05).

Results
Toxicity study of 4-thioquinolines using the GUSAR program (Germany) and TEST (USA) showed that they are low-toxic (Table 3). Among the derivatives of 2-((6-R-quinolin-4-yl)thio)acetic acid the most toxic compounds were those that did not have in the 6-th position of the quinoline cycle alkoxy substituents (QAC-1 and QAC-5). Sodium salts are more toxic than the corresponding acids. This is due to the increased bioavailability of ionized compounds.
The study of rhizogenesis was performed in vitro, with the addition of synthesized compounds QAC-1-8 at a concentration of 1 mg/L in the nutrient medium. For rhizogenesis we prepared nutrient medium Murashige-Skuga, which contained half the concentration of macrosalts and trace elements and 2% sucrose. The compounds were added before sterilization of the nutrient medium. The control was nutrient media without growth regulators (MS0). The nutrient medium was sterilized by autocla-ving under a pressure of 0.11 MPa for 30 minutes. The explants were cultivated at an air temperature of 22-24 °С with a photoperiod of 16 hours, a relative humidity of 65-70% and an illumination of 2.5 thousand lux. The results were recorded for 28 days and took into account the number, length of roots, frequency of rhizogenesis.
In vitro rhizogenesis was studied with the obtained compounds, with the addition of synthesized compounds QAC-1-8 at a concentration of 1 mg/L in the nutrient medium. Murashige T. Scoog medium was prepared for rhizogenesis, which contained half the concentration of macrosalts and trace elements and 2% sucrose. The compounds were added before sterilization of the nutrient medium. The control was nutrient media without growth regulators (MS0). Comparison drug -2-(naphthalene-5yl)acetic acid (NAA). The nutrient medium was sterilized by autoclaving under a pressure of 0.11 MPa for 30 minutes The explants were cultivated at an air temperature of 22-24 °С with a photoperiod of 16 hours, a relative humidity of 65-70% and an illumination of 2.5 thousand lux. The results were recorded for 28 days and took into account the number, length of roots, frequency of rhizogenesis. The data obtained show that the compounds of all tested compounds when added to the nutrient medium for rhizogenesis containing ½ MS and 1 mg/L of the compound caused the maximum increase in rhizogenesis of Pavlovna clone 112. Note: differences compared to controls (MS0 medium without growth stimulants): * -P < 0.05; ** -P < 0.01; *** -P < 0.001.
Thus, on the nutrient medium QAC-1 and QAC-5 was there a maximum stimulation of rhizogenesis, which exceeded the effect of the comparison drug -2-(naphthalene-5-yl)acetic acid. Paulownia clones formed 4.92 ± 0.54 and 4.32 ± 0.43 roots (P < 0.05), respectively. This environment contributed the most to the formation of 7-8 roots and the frequency of rhizogenesis was more than 80%. Significantly the longest roots were observed on the medium QAS-1 (P < 0.05), QAC-5 (P < 0.001) in comparison with the control and the comparison drug. The engraftment of plants on the substrate peat universal:sand:vermiculite in a ratio of 2:1:1 was 73%. Paulownia clone 112 on hormone-free nutrient medium initiates the minimum number and length of roots from all studied variants of media. In contrast, with media containing compounds, all tested compounds caused a significantly higher number of roots (P < 0.05), and media with compounds QAC-1, QAC-5 had the maximum number (P < 0.001) of roots compared to control. The length of the roots was dominated by nutrient media containing compounds QAC-1 and QAC-5 (P < 0.001) in comparison with the comparison drug (Table 1, Fig. 3).
Thus, the addition of sodium salts to the nutrient medium for rhizogenesis of QAC-1-5 compounds significantly increased the number and length of roots (P < 0.001) with the maximum percentage of rhizogenesis frequency (Fig. 3) compared to the corresponding starting acids.

Discussion
The selection of substances for the substrate during microclonal propagation of plants is an urgent problem. Stimulation of rhizogenesis is the most problematic task for the efficiency of plant reproduction (Amer et al., 2019;Anjos et al., 2021). It is known that each plant has its own individual characteristics that affect the composition of the nutrient medium for explants. The nutrient medium was sterilized by autoclaving under a pressure of 0.11 MPa. Therefore, the compounds that are added to the nutrient medium must have the necessary chemical resistance to decomposition (Arteta et al., 2018;Awada et al., 2020).
The most important moment in the clonal micropropagation of any culture is the planting of plants in the substrate, it is at this stage that there is a danger of the death of plants -regenerants, therefore it is important to obtain an optimal root system that will provide nutrition and growth of regenerants (Ivashchuk et al., 2018;Grishchenko et al., 2020). It is known that when explants of plants without roots or with poorly developed root systems were planted in the substrate, 34% of plants took root, which made production unprofitable. Stimulation of root formation is the result of the interaction of a substance with plant cells. It depends on the characteristics of the substance (molecular structure, physical and chemical properties), biological object and mode of action. It is known that the toxicological problem is associated with the use of synthetic biologically active substances. It is associated with the presence of many of them as side effects, that is, undesirable effects (Vostrikova, 2020;Koprulu, 2021). A significant number of works by domestic and foreign authors are devoted to the study of virtual and experimental methods for obtaining low-toxic substances and methods of reducing the toxicity of quinoline derivatives Yepes et al., 2021;Lenin et al., 2022).
Various quinoline derivatives are used both as synthons in organic synthesis and molecular design, and as known effective biologically active compounds. During the synthesis of a new compound, there is a need to calculate the molecular descriptors of the structure, physical and chemical properties, the main constants that affect the manifestation of toxic effects. Such problems are solved using modern chemometric research methods.
The studied derivatives of 2-((6-R-quinolin-4-yl)thio)acetic acid are synthetic analogues of known growth stimulants, such as 2-(naphthalene-5-yl)acetic acid. From the change in rhizogenesis indicators, it is clear that the studied compounds exhibit auxin properties. Therefore, the conducted research has a high practical potential for obtaining new effective, lowtoxic, less expensive substances for plant propagation, in the conditions of microlonal production.

Conclusion
The results of the study spread the idea of the influence of 2-((6-Rquinolin-4-yl)thio)acetic acid on rhizogenesis. It was determined that the molecular descriptors correspond to Lipinski's rule, the compounds are low-toxic and have optimal bioavailability, so they can be used as a basis for further research as growth stimulators of rhizogenesis in the micro-clonal propagation of plants of other species, especially ornamental plants, the propagation of which is very costly under standard conditions.