Carboxyl-containing quinazolines and related heterocycles as carriers of anti-inflammatory activity

Лікарські засоби, які б поєднували у своїй структурі ароматичний і гетероциклічний фрагменти з «фармакофорною» карбоксильною групою, широко представлені на фармацевтичному ринку. Саме ця комбінація структурних елементів міститься в молекулах нестероїдних протизапальних засобів (НПЗЗ). Детальне вивчення механізмів дії НПЗЗ дало змогу пояснити ключову роль і вплив «фармакофорної» карбоксильної групи на активність, селективність і токсичність. Упровадження в медичну практику селективних UDC 547.856.1+547.85]:615.276.015.11 DOI: 10.14739/2310-1210.2022.1.241286

The elaboration of modern conception of inflammation mechanism, evaluation of the important role of eicosanoid in the process of inflammation onset and development, estimation of structure of biological targets and known inhibitors, as well as, unprecedented development of machine learning, enhanced the role of artificial intelligence in NSAIDs design [9,10]. The abovementioned resulted in the introduction of selective COX-2-inhibitors to medicinal practice and a significant decrease of side effects and complication frequencies. However, the problem of NSAIDs toxicity has not been solved [11,12]. Thus, the search for the novel anti-inflammatory drugs using in silico methods and approaches that include structural modification of known NSAIDs by "bioisosteric" replacements of aromatic and heterocyclic fragments to other structural elements with carboxylic group as carrier of pharmacological effect is a current trend of medicinal chemistry.

Aim
The aim of the present study is to purposefully search for anti-inflammatory agents among carboxyl-containing quinazolines and related heterocycles using in silico and in vivo methods, as well as the evaluation of carboxylic group effect on the level of anti-inflammatory activity.
Molecular docking. Research was conducted by flexible 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 COX-1 enzyme in complex with DF (PDB ID -3N8Y), COX-2 in combination with DF (PDB ID -1PXX) [17]. The choice of biological targets was due to the literature about the mechanism of anti-inflammatory drugs activity [2]. Ligand preparation. Substances were drawn using Mar-vinSketch 20.19.0 and saved in mol format [18]. After that they were optimized by program Chem3D, using molecular mechanical MM2 algorithm and saved as PDB files. Molecular mechanics was used to produce more realistic geometry values for most organic molecules, owing to the fact of being и через 4 часа после инъекции флогогена. Активность соединений определяли по их способности уменьшать отек по сравнению с контрольной группой, выражали в процентах. Эксперименты проведены с соблюдением биоэтических правил и норм.
highly parameterized. Using AutoDockTools-1.5.6, PDB files were converted into PDBQT, number of active torsions was set as default [19].
In Silico Prediction. "Drug-like" characteristics were evaluated using an electronic resource [21]. Acute toxicity of the studied compounds was predicted in silico using TEST software [22,23].
Anti-inflammatory activity. Anti-inflammatory activity of the synthesized compounds was evaluated on 228 Wistar white rats (150-160 g of weight), obtained from the breeding station of "Institute of Pharmacology and Toxicology of Ukraine" (Kyiv). All experimental procedures and treatment were carried out according to the European Convention and "Regulations on the use of animals in biomedical research" [24]. Screening of the synthesized compounds with estimated anti-inflammatory activity began with the study of their effect on exudative phase of acute aseptic inflammation ("carrageenan" test) [25]. Phlogogen (1 % aqueous solution of λ-сarrageenan) was subplantarly injected in the dose of 0.1 ml in the rats' hind right paw. The left one was used as a control. The studied compounds were intragastrically administered with atraumatic probe as water solution or finely dispersed suspension stabilized by Tween-80 in a dose of 10 mg/kg, 1 hour before the injection of phlogogen. The reference drug Diclofenac sodium was administered intragastrically in a recommended dose of 8 mg/kg for pre-clinical studies. Measurement of paws volume was conducted before the experiment and 4 ("сarrageenan" test) hours after injection of phlogogen using the described methods. The activity of these substances was determined by their ability to reduce the swelling compared with control group and was expressed in percentage. It showed how the substance inhibited phlogogen swelling in relation to control swelling where the value was taken as 100 %. The activity of the studied compounds was calculated as following: where A -antiexudative activity, %; Vpe -the volume of paw edema in the experiment; Vhe -the volume of healthy paw in the experiment; Vpc -the volume of paw edema in control; Vhc -the volume of healthy paw in control.
Data were statistically processed with the licensed program Statistica for Windows 13 (StatSoft Inc., No. JPZ804I382130ARCN10-J) and "SPSS 16.0", Microsoft Office Excel 360. The results were presented as mean ± standard error of the mean. Arithmetic mean and standard error of the mean were calculated for each of the studied parameters. During verification of statistical hypothesis, null hypothesis was declined if statistical criterion was P < 0.05 [26]. 1

Results
The study design implied the selection of basic molecules, namely quinazolin-4(3H)-ylidene)hydrazides of mono-(di-) carboxylic acids (II) that were used as basis for construction of the virtual library of potential anti-inflammatory agents. For evaluation of promising structural modification routes, the literature data as well as our own "structure -biological activity" data were used [27][28][29][30][31][32][33][34][35][36] (Fig. 1). It should be mentioned that selected heteroaromatic basic molecules have ample opportunities for structural modification by the heterocyclization and nucleophilic degradation reactions that additionally allow to introduce various pharmacophore groups that are associated with anti-inflammatory activity (primarily carboxylic group). The general methods for the synthesis of the target quinazoline-4(3H)-ylidene)hydrazides of carboxylic and dicarboxylic acids (IIa-g), 2-R- [1,2,4] Fig. 1.
Considering the prospects of aforementioned class of the compounds and ample opportunities for their chemical modification, the in silico screening aimed at the estimation of promising objects for in vivo studies was conducted. Thus, docking studies to COX-1 and COX-2, as key enzymes of inflammation process developing, calculation of physicochemical properties, "drug-like" criteria, and toxicity parameters were performed for more than 100 candidate compounds using appropriate software and services [21][22][23]. The analysis of molecular docking results showed that calculated affinity of the most of the studied compounds to key enzymes of the inflammation were higher or comparable with reference compound. It was found that the highest affinity to enzymes were characteristic for compounds II-VI that contain the carboxylic groups. Quinazoline-4(3H)-ylidene) hydrazides of monocarboxylic acids, 2-alky-(benzyl-, phenethyl-, aryl-)-[1,2,4]triazolo[1,5-c]quinazolines were excluded from study considering their lower comparing with reference compound affinity values. Besides, studied compounds have satisfactory toxicity measures, most of them refer to non-toxic compounds (LD 50 = 585.7-2650.6 mg/kg) ( Table 1).
Results of calculation revealed that proposed compounds have the satisfiying value of "drug-like" criteria ( Table 2). Thus, for all studied compounds logP values were less than 5, molecular weight was less than 500; molecules contain no more than 10 nitrogen and oxygen atoms, less than 5 atoms -donors of hydrogen bonds, and no more than 8 rotatable bonds. The accordance to listed above parameters indicates the ability of compounds to ligand-enzyme interaction on binding site of the molecular target. Obtained data allowed to distinguish the narrower range of compounds for further synthetic and biological studies and revealed that chemical modification of carboxyl-containing heterocyclic compounds is reasonable in scope of purposeful search for agents with anti-inflammatory activity.

Number of nonhydrogens
Molecular volume, Å3    (Fig. 4В). Small and hydrophilic molecule of compound IVd forms conventional hydrogen bonds between ARG A:120 (4.83 Å), TYR A:355 (2.26 Å) and carboxylic group as well as between MET A:522 (3.29 Å) and aminophenyl fragment (Fig. 4С). Visualization of docking study of compound Vb that contains as carboxylic so ester groups (2 nd and 5 th positions) allowed to evaluate that molecule is located in hydrophilic part of the active site of enzyme and form conventional hydrogen bond between SER A:516 (1.92 Å) and GLN A:350 (2.36 Å) (Fig. 4D). Visualization of compounds IIe, IIIg, IVd, Vb docking to СОХ-2 revealed the patterns that are similar to the described above. Thus, compounds IIe, IIIg, IVd, Vb take the position which is different from Sodium Diclofenac in the active site of enzyme, as well as form alternative interactions with aminoacid moieties of protein molecule (Fig. 5).

Number of rotatable bonds
Carboxylic group of compound IIe does not form any interactions with amino acid moieties of enzyme, but there is conventional hydrogen bond between hydrazide group and SER A:516 (3.08 Å) (Fig. 5А). Compound IIIg, despite the location in hydrophilic part, forms conventional hydrogen bond of carboxylic group with ARG C:106 (2.85 Å) (Fig. 5В).
Visualization of compound IVd interaction with active site of COX-2 (Fig. 5С) allowed to evaluate the position,

5A
5C 5B 5D similar to previous compounds, in active site of enzyme and the presence of conventional hydrogen bond between carboxylic group and TYR C:341 (2.34 Å). It should be mentioned that compound Vb form more conventional hydrogen bonds comparing to the listed above compounds. It may be explained by the presence of both carboxylic and ester groups in same molecule, aforementioned fragments form interactions with SER С:516 (3.10 Å), Tyr С:371 (3.09 Å) and ARG C:106 (3.06 Å).
Reconstruction of [1,2,4]triazolo [1,5-c]quinazoline cycle with additional introduction of carboxyalkyl group to position 5 (V) caused the significant loss of anti-inflammatory activity (AA = 0.47-22.93 %) independently determined by the substituent in the 2 nd position (Fig. 3). The exception was compound Vb, that inhibited the development of the edema by 37.55 %. Above mentioned compound contains ethoxycarbonyl and carboxyethyl fragments in the 2 nd and 5 th positions correspondingly.
The formation of more complex heterocyclic system also did not lead to the increasing of anti-inflammatory activity. Thus, dihydropyrrolo[1,2-a] [1,2,4]triazolo [1,5-c] quinazolines that contain carboxylic group (VIa, VIb) or propanoic acid moiety (VIc, VId) in angular position 4a were not effective and reduce paw edema on 0.94-17.16 % (Fig. 3). Therefore, anti-inflammatory activity of the studied compounds significantly depends on molecule conformation, the nature of pharmacophore, its position in heterocyclic fragment, and length of linker alkyl fragment that effects on the lipophilicity of molecule.
The conducted visualization of molecular docking results proved our assumption about dependence of anti-inflammatory activity level on spatial location of molecule in active center (i. e. conformation) and lipophilicity (the length of carboxyalkyl fragment). Thereby, studied compounds take the position that differs from that of the classic COX-inhibitor (Sodium Diclofenac) in active site, and therefore form alternative enzyme-ligand interactions between carboxylic group and amino-acid moieties of protein. It should be mentioned that in some cases studied compounds do not form the abovementioned type of interaction (Figs. 4,5). Although studied compounds are promising anti-inflammatory agents, they cannot be referred to classic COX inhibitors and require the further investigations of mechanism of action (PLA-inhibiting activity, LOX-inhibiting activity, etc.) and feasibilities of structural optimization.

Conclusions
1. The predicted affinity values, calculated "drug-like" criteria and toxicity parameters, visualization of the docking in active site of biological targets as well as experimental studies results showed that investigated compounds are promising in scope of purposeful search for anti-inflammatory drugs.
2. The conducted in vivo screening of anti-inflammatory activity among carboxyl-containing quinazolines and related heterocyclic compounds allowed to detect series of substances that by the level of anti-inflammatory activity compete with reference-compound "Sodium diclofenac" on the carrageenan-induced paw edema model. 3. Presented data may be considered as theoretical basis for further structural modification of studied compounds aimed at the elaboration of novel anti-inflammatory agents and the evaluation of their activity mechanism (lipoxygenase inhibitors, phospholipase inhibitors, etc.).