Substituted pyrrolo[1,2-a][1,2,4]triazolo-(triazino-)[c]quinazolines – a promising class of lipoxygenase inhibitors

The modern strategy of potential biologically active molecules search (“drug-design”) is based on several innovation approaches. The method of high thrоughрut biological screening and method of molecular modeling deserves the most attention among such approaches. Lipoxygenase (LOX) is one of the most perspective biological target for the substituted pyrrolo[1,2-a][1,2,4]triazolo-(triazino-)[c]quinazolines. So, molecular docking towards LOX and enzyme activating activity was investigated.

Цель работы -направленный поиск потенциальных ингибиторов ЛОГ среди неизвестных пирроло [1,2- The modern strategy of potential biologically active mole cules search ("drug-design") underwent significant changes and became the most important part of modern medical chemistry [1][2][3]. Now it is based on several innovation approaches, such as virtual screening, combinatory che mistry, high thrоughрut screening, molecular modeling, frag mentoriented design, optimization of the leading structure, etc. Among the abovementioned approaches, the method of high thrоughрut biological screening deserves the most attention. This method allows estimating activity of many compounds against a known biological target in short terms. Structures of biological targets are known. With the help of them, the molecular mechanism of interaction of the ligand with protein could be explained. So, they are used for mo lecular docking [4][5][6]. Protein threedimensional structure (at the current stage of technology development, as a rule conformationally rigid) and structure of ligand (the known inhibitor and synthesized compound) are used as a starting information for docking. The optimal ligand conformation with a specific binding energy value for the biological target is the docking result. Using this results perspective objects for further high thrоughрut screening could be revealed. In view of many approximations, the binding energy does not always correlate with the relevant experimental data. Howe ver, it gives an understanding of the mechanism and ligand activity efficiency.

Aim
So, the purpose of work is the directed search of lipoxy ge nases potential inhibitors among the unknown pyrrolo [1,2a]

Materials and methods
The research of lipoxygenase activity has been conducted for a number of original pyrrolo [1,2a] Fig. 1), which were synthesized at the Department of Organic and Bioorganic Chemistry of the Zaporizhzhia state medical university (the Head of the Department, Dr.hab., Professor, S. I. Kovalenko). The features of the structures of the synthesized compounds were evaluated by IR-, NMR spectroscopy, and chromatog raphymass spectrometry and were discussed in detail [18]. Molecular docking. The research was conducted by flex ible molecular docking, as an approach of finding molecules with affinity to a specific biological target. Macromolecules from Protein Data Bank (PDB) were used as biological targets, namely LOX (soybean) enzyme in complex with protocatechuic acid (PDB ID -1N8Q) [19]. The choice of biological targets was due to the literature on the mechanism of anti-inflammatory drug action [17].
Ligand preparation. Substances were drawn using Marvin Sketch 19.24 and saved in mol format [20]. After that, they were optimized by program Chem3D, using the molecular mechanical MM2 algorithm and saved as pdb-files. Molecular mechanics was used to producing more realistic geometry values for the majority of organic molecules, owing to the fact of being highly parameterized. Using AutoDockTools1.5.6 pdb-files were converted into PDBQT, the number of active torsions was set as default [21].
Lipinski's rule of five. Druglike characteristics (Log P, molecular polar surface area, number of nonhydrogens, num ber of hydrogen bond acceptors (groups N and O), number of hydrogen bond donors (groups NH and OH) and number of rotatable bonds) were evaluated and optimized using an electronic resource [23].
Soybean LOX inhibition study in vitro. In vitro study was evaluated as it was reported previously [24,25]. To 3.880 ml of borate buffer, 40 µl 2 × 10 5 w/v solution of LOX in the buffer and 40 µl of 100 uM studied compound (or nordihydroguaiaretic acid (NDGA)) solution were added. The formed mixture was shaken and incubated at ambient temperature for 5 min. After incubation, the 40 µl of 0.01 M solution of sodium linoleate was added. After 20 min. incubated at ambient temperature absorption at 234 nm was recovered. The results are calculated by the formula: LOX inhibiting activity, % = (А control -А test compound ) / А control × 100 %

Results
The results of molecular docking have shown, that substituted pyrrolo [1,2a] However, high affinity to specified enzymes is not always the main factor for activity revealing. It may be due to the influence of additional factors (lipophilicity, metabolism, etc.), which are described by the «druglike» criteria (Table 2). Analysis of "druglike" results indicates, that the test compounds have no deviations from Lipinski's rules (LogP ≤5; molecular weight ≤500; ability to be a proton acceptor ≤10; ability to be a proton donor ≤5; bond rotation ≤8), as

Disscussion
Among lipooxygenases (LOX), six isoforms are most known (LOX-5, 15-LOX, 15-LOX-2, 12-LOX, 12R-LOX and eLOX-3), which play an important role in the development of various pathological processes [19]. 5-LOX is a precursor for the synthesis of B 4 leukotrienes (LTB 4 ), peptidyl leukotrienes (LTC 4 , LTD 4 or LTE 4 ) and lipoxins that cause inflammatory processes. Compounds were analyzed with the use of molecular docking considering the structural similarity of LOX-5 to soybean lipoxygenase LOX (sLOX) type 1b and its role in processes of inflammation. Especially, as 1b sLOX is the molecular biological target, and the high affinity of ligands (the synthesized compounds) to lipoxygenases is one of the desirable characteristics of anti-inflammatory agents.
The visualization of complexes was conducted for evaluation of the effects of structural features of ligands on the level of binding with molecular target. The analysis of the types of main interactions with aminoacid moieties of protein was performed as well (Table 1, Fig. 2). So, visualization of the structure of NDGA with the active site to soybean LOX (Fig. 2) allows to establish, that it has hydrogen and hydrophobic interactions with the amino-acid residues:

The main interactions types between compounds, pharmacological standards and amino acid residues of enzymes
Proto-catechuic acid --  18 has the highest affinity to the soybean LOX target, among the investigated ones. Visua lization of this structure with the soybean LOX active site (Fig. 2) showed, that it is characterized by four hydrogen bonds with the amino acid residues: SER281 (3.16Å), GLY569 (2.69Å), GLY570 (3.40Å), GLY570 (3.45Å), hydrophobic interaction with LEU563 (5.46Å) and quite strong π-Sulfur interaction with HIS219 (4.82Å). So, an important aspect of compounds' high affinity to soybean LOX is the presence of several hydrogen bonds, hydrophobic interactions, donor-acceptor interactions due to sulfur and fluorine lone electron pairs ( Table 1).
The comparative analysis of "drug-like" results and soybean LOX inhibition has shown, that the lipoxygenase activity depends on molecule lipophilicity and availability of acceptors and donors of hydrogen bond. The last statement agreed with the data of molecular docking (Table 1, Fig. 2). So, substituted pyrrolo[1,2-a] [1,2,4]triazino [2,3-c]quinazolines with a fluorine atom (2.13) and a 2-thienyl fragment (2.18) in the molecule inhibit lipoxygenase by 36.33 % and 39.83 % respectively. The increase of lipophilicity promotes higher ability to inhibit soybean LOX (