Novel 1,2,3-Triazole-Containing Quinoline–Benzimidazole Hybrids: Synthesis, Antiproliferative Activity, In Silico ADME Predictions, and Docking

The newly synthesized quinoline–benzimidazole hybrids containing two types of triazole-methyl-phenoxy linkers were characterized via NMR and elemental analysis. Additional derivatization was achieved by introducing bromine at the C-2 position of the phenoxy core. These novel hybrids were tested for their effects on the growth of the non-tumor cell line MRC-5 (human fetal lung fibroblasts), leukemia and lymphoma cell lines: Hut78, THP-1 and HL-60, and carcinoma cell lines: HeLa and CaCo-2. The results obtained, presented as the concentration that achieves 50% inhibition of cell growth (IC50 value), show that the compounds tested affect tumor cell growth differently depending on the cell line and the dose applied (IC50 ranged from 0.2 to >100 µM). The quinoline–benzimidazole hybrids tested, including 7-chloro-4-(4-{[4-(5-methoxy-1H-1,3-benzo[d]imidazol-2-yl)phenoxy]methyl}-1H-1,2,3-triazol-1-yl)quinoline 9c, 2-(3-bromo-4-{[1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl]methoxy}phenyl)-N-propyl-1H-benzo[d]imidazol-5-carboximidamide trihydrochloride 10e, 2-{4-[(1-{2-[(7-chloroquinolin-4-yl)amino]ethyl}-1H-1,2,3-triazol-4-yl)methoxy]phenyl}-N-propyl-1H-benzo[d]imidazol-5-carboximidamide trihydrochloride 14e and 2-{3-bromo-4-[(1-{2-[(7-chloroquinolin-4-yl)amino]ethyl}-1H-1,2,3-triazol-4-yl)methoxy]phenyl}-N-propyl-1H-benzo[d]imidazol-5-carboximidamide trihydrochloride 15e, arrested the cell cycle of lymphoma (HuT78) cells. The calculated ADMET properties showed that the synthesized compounds violated at most two of Lipinski’s rules, making them potential drug candidates, but mainly for parenteral use due to low gastrointestinal absorption. The quinoline–benzimidazole hybrid 14e, which was shown to be a potent and selective inhibitor of lymphoma cell line growth, obtained the highest binding energy (−140.44 kcal/mol), by docking to the TAO2 kinase domain (PDB: 2GCD).


Introduction
Nitrogen-containing heterocycles are ubiquitous in nature and represent the most common scaffold in approved drugs.An analysis of small molecule therapeutics approved by the FDA in the last five years shows that more than half of the approved drugs contain aromatic N-heterocycles [1][2][3][4][5].N-heterocycles are also a component of numerous small molecules that have been approved by the FDA for the treatment of cancer in recent years [6].
Cancer is one of the leading causes of death worldwide, with an estimated 10 million deaths in 2020 [7,8].Despite tremendous efforts, drug resistance is still one of the main reasons for cancer treatment failure [9,10].One of the latest strategies for cancer therapy is the use of molecular hybrids.The goal of molecular hybridization is the covalent binding of two or more pharmacophores in a single molecule that has the ability to eliminate drug resistance and improve selectivity [11][12][13].We aimed to apply this approach to the preparation of hybrids containing quinolines linked to benzimidazoles via a 1,4-disubstituted 1,2,3-triazole linker.To this end, we used a well-known, robust, reliable, and efficient copper-catalyzed cycloaddition of azides and terminal alkynes [14].
Click chemistry has made compounds containing 1,2,3-triazole readily available.Due to its ability to act as a linker, pharmacophore and bioisoster, the 1,2,3-triazole motif is of great importance in drug discovery and can be found in the structures of drug candidates and approved drugs [15][16][17].
Over the years, numerous triazole compounds have also been tested as potential anticancer drugs [17][18][19][20].Carboxyamidotriazole, shown in Figure 1, is a calcium channel blocker with a 1,4,5-disubstituted 1,2,3-triazole backbone.Recently, carboxyamidotriazole has been clinically investigated as an orotate prodrug salt in non-small cell lung cancer, glioblastoma, and anaplastic glioma [21,22].Considering the antiproliferative activity of compounds with the above components and continuing our work on quinoline-benzimidazole hybrids [38,39], we report here our next step in the search for anticancer agents, which involves the synthesis of triazolelinked quinoline-benzimidazole hybrids and the evaluation of their antiproliferative activity.To elucidate the possible mechanisms of the antiproliferative activities of the investigated compounds, we performed a molecular docking study on TAO2 (thousand-andone amino acids) protein kinase.In addition, in silico physicochemical and pharmacokinetic/ADMETstudies (absorption, distribution, metabolism, excretion, and toxicity) were performed to theoretically predict their behavior as drug candidates.

Chemistry
The synthesis of the new quinoline-benzimidazole compounds 9a-10e and 14a-15d was carried out as shown in Scheme 1.We prepared hybrid molecules containing quinoline and benzimidazole units linked to two types of triazole methyl phenoxy linkers.The target compounds bear two non-amidine and two amidine substituents at the C-5 position of the benzimidazole molecule.We attempted additional derivatization with a bromine substituent at the C-2 position of the phenoxy core.
Triazole intermediates 6, 7, 12, and 13 were synthesized using a partially modified Quinoline is one of the preferred scaffolds in drug discovery and there are numerous reports on the antiviral, antibacterial, antifungal, antiviral, and antiparasitic activities of compounds with quinoline structure [23,24].For years, quinoline has played an important role in the discovery of anti-cancer agents [25,26].The 4-amino-7-chloroquinoline-based antimalarials chloroquine and hydroxycloroquine have been studied in more than sixty clinical trials as anticancer therapies (Figure 1) [27].Neratinib, shown in Figure 1, is a quinoline-based therapeutic that acts as a tyrosine kinase inhibitor and is used to treat the early and advanced stages of HER-2 positive breast cancer.It has also been clinically evaluated for the treatment of patients with HER-2 mutated advanced bile duct cancer [28][29][30].Benzimidazole, also known as 1H-benzimidazole and 1,3-benzodiazole, is an aromatic bicyclic N-heterocycle containing a benzene ring fused to an imidazole ring.Benzimidazolecontaining compounds have shown a wide range of biological activities and have been attracting attention in the drug discovery field for years [31,32].The benzimidazole backbone is present in several drugs approved for cancer therapy [33].A patent survey of benzimidazoles shows that more than half of the new benzimidazole compounds were developed for cancer treatment during the indicated period [34].Crenolanib (Figure 1) is in clinical trials for acute myeloid leukemia and acts as an FMS-like tyrosine kinase-3 inhibitor.Structurally, it is a compound containing a benzimidazole directly linked to a quinoline moiety [35,36].Dovitinib is also an anticancer agent containing a benzimidazole scaffold directly linked to a quinolinone.It is a tyrosine kinase inhibitor and has been used in clinical trials to treat patients with advanced pancreatic cancer [37].
Considering the antiproliferative activity of compounds with the above components and continuing our work on quinoline-benzimidazole hybrids [38,39], we report here our next step in the search for anticancer agents, which involves the synthesis of triazolelinked quinoline-benzimidazole hybrids and the evaluation of their antiproliferative activity.To elucidate the possible mechanisms of the antiproliferative activities of the investigated compounds, we performed a molecular docking study on TAO2 (thousandand-one amino acids) protein kinase.In addition, in silico physicochemical and pharmacokinetic/ADMETstudies (absorption, distribution, metabolism, excretion, and toxicity) were performed to theoretically predict their behavior as drug candidates.

Chemistry
The synthesis of the new quinoline-benzimidazole compounds 9a-10e and 14a-15d was carried out as shown in Scheme 1.We prepared hybrid molecules containing quinoline and benzimidazole units linked to two types of triazole methyl phenoxy linkers.The target compounds bear two non-amidine and two amidine substituents at the C-5 position of the benzimidazole molecule.We attempted additional derivatization with a bromine substituent at the C-2 position of the phenoxy core.The newly synthesized 1,2,3-triazole-containing quinoline-benzimidazole hybrids were tested to determine their effect on cell growth.The cell lines selected for evaluation included a range of non-tumor and tumor cells: MRC-5 (human fetal lung fibroblasts, nontumor), Hut78 (T-cell lymphoma), THP-1 (acute monocytic leukemia), HL-60 (acute pro- The required intermediates 3, 4, 5, and 11 as well as benzene-1,2-diamines 8d-e were prepared according to the previously reported procedures [40][41][42]. Triazole intermediates 6, 7, 12, and 13 were synthesized using a partially modified Cu(I)-catalyzed azide-alkyne cycloaddition previously reported by Guntai et al. [40].Target compounds 9a-10e and 14b-15d were prepared using triazole intermediates and the corresponding benzene-1,2-diamines 8a-e in the presence of sodium metabisulfite in DMSO at 165 • C [43].

Evaluation of Antiproliferative Activity of the Novel Compounds
The newly synthesized 1,2,3-triazole-containing quinoline-benzimidazole hybrids were tested to determine their effect on cell growth.The cell lines selected for evaluation included a range of non-tumor and tumor cells: MRC-5 (human fetal lung fibroblasts, non-tumor), Hut78 (T-cell lymphoma), THP-1 (acute monocytic leukemia), HL-60 (acute promyelocytic leukemia), HeLa (human cervical adenocarcinoma), and CaCo-2 (adenocarcinoma of colon).The results of the experiments, quantified according to the concentration at which 50% growth inhibition (IC 50 value) was achieved, showed different effects of the studied compounds on tumor cell growth.The results depended on factors such as the cell line tested and the dose administered.This suggests that the effects of the compounds on cell growth are influenced by a complex interplay of factors, including their chemical structure, cellular context, and dose-dependent effects.
As shown in Table 1, the IC 50 values ranged from 0.18 to over 100 µM for all compounds tested.Carcinoma cell lines were more resistant to the tested compounds compared to leukemia and lymphoma cells.In the group of non-amidine compounds without ethylamino linker (9a-9c), it was observed that compound 9c with the methoxy group on the benzimidazole moiety (R 2 ) exhibited the highest cytotoxicity and showed a non-selective effect against tumor cells compared to normal cells (Figure 2).Although 9c showed a significant effect on the growth of normal MTR-5 cells with a IC 50 of 2.71 µM, its selectivity index (SI) was 15 for THP cells (IC 50 : 0.18 µM) and 13.55 for HuT78 cells (IC 50 : 0.18 µM).Amidine compounds 9d and 9e, containing amidine and propylamidine substituents on the benzimidazole core, respectively, caused a selective and pronounced antiproliferative effect on HuT78 cells (9d: IC 50 = 2.52 µM; SI = 39.7;9e: IC 50 = 10.2 µM; SI = 9.8).No significant difference in the antiproliferative effect was observed between the normal MRC-5 and carcinoma cell lines (HeLa and CaCo2) for compounds 15a-c (Table 1).The introduction of amidine to benzimidazole (15d) resulted in a loss of antiproliferative activity, whereas the introduction of propilamidine to benzimidazole (15e) resulted in selective activity against HuT78 cells (IC50 = 7.83 µM; SI = 12.8).No significant difference in the antiproliferative effect was observed between the normal MRC-5 and carcinoma cell lines (HeLa and CaCo2) for compounds 15a-c (Table 1).The introduction of amidine to benzimidazole (15d) resulted in a loss of antiproliferative activity, whereas the introduction of propilamidine to benzimidazole (15e) resulted in selective activity against HuT78 cells (IC50 = 7.83 µM; SI = 12.8).a IC50-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.a IC50-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.a IC50-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.In the group of compounds without ethylamino linker and with added bromine at the phenoxy core (10a-e), the non-amidine compounds 10a and 10b showed increased toxicity against both normal and tumor cell lines.The replacement of hydrogen with chlorine at the benzimidazole in 10b increased antiproliferative activity in all tumor cell lines tested compared with the effect of 9b.Compound 10c, which has a methoxy group on the benzimidazole, showed lower cytotoxic activity than compound 9c, which lacked the bromine substitution at the R 1 position.Compound 10d (with an amidine group at the R 2 position) showed insignificant inhibitory potential against normal and tumor cells, while derivative 10e (with a propylamidine group at the R 2 position) showed selective activity against HuT78 cells in terms of growth of normal cells (IC 50 = 9.68 µM; SI = 9.8).
The introduction of bromine at the phenoxy core in the group of quinoline-benzimidazole hybrids containing an ethylamino linker (15a-e) significantly alters the inhibitory effects of non-amidine compounds 15a-c on the growth of leukemia and lymphoma cells compared to non-substituted non-amidine compounds 14a-c (Table 1 and Figure 2).Compared with MRC-5 cells and carcinoma cell lines, 15a-c showed significant antiproliferative activity against lymphoma cells HuT78 (IC 50 ranged from 3.98 to 7.88 µM and leukemia cells (HL-60: IC 50 ranged from 1.21 to 11.63 µM; THP1: IC 50 ranged from 1.65 and 4.66 µM).
No significant difference in the antiproliferative effect was observed between the normal MRC-5 and carcinoma cell lines (HeLa and CaCo2) for compounds 15a-c (Table 1).The introduction of amidine to benzimidazole (15d) resulted in a loss of antiproliferative activity, whereas the introduction of propilamidine to benzimidazole (15e) resulted in selective activity against HuT78 cells (IC 50 = 7.83 µM; SI = 12.8).

Cell Cycle Perturbation
Inducing cell cycle arrest at specific checkpoints and inducing apoptosis are common strategies in cancer treatment with cytotoxic agents.Numerous studies have shown that quinoline-and benzimidazole-based compounds, as well as their hybrids, significantly affect tumor cell growth [44,45].To determine whether cell cycle arrest underscores the antiproliferative activity that some compounds exhibit at micromolar and submicromolar concentrations against lymphoma and leukemia cells, we tested the cell cycle distribution of HuT-78 treated with hybrids containing amidine or propylamidine at the C-5 position of the benzimidazole moiety (10e, 14e and 15e), which showed a selective effect on HuT78 cells growth.The effect on the cell cycle of the non-amidine compound without an ethylamino linker, compound 9c, which has a methoxy group on the benzimidazole core (R 2 ) and showed submicromolar IC 5 on leukemia and lymphoma cells (Table 1), were also investigated.As shown in Figure 3, compound 9c caused a statistically significant enrichment of the S fraction (at 31%, p < 0.05) and a significant decrease in the G1 phase (at 66.7%, p < 0.05) compared to control cells.No significant changes were observed in the other phases of cell cycle.In contrast to compound 9c, compound 10e with introduced bromine at the phenoxy core and with a propylamidine group at the R 2 position (without an ethylamino linker) caused a statistically significant increase in the aggregation of cells in the subG0/G1 phase (more than threefold, p < 0.05) with a corresponding decrease in the G1, S and G2/M phases of the cell cycle.In addition, the results of the test of the effects of quinoline-benzimidazole hybrids containing an ethylamino linker showed that the compound with propylamidine at the C-5 position of the benzimidazole moiety, 14e, and the compound with bromide at the phenoxy core, 15e, similarly affected the cell cycle by increasing the proportion of cells in subG0/G1 phase (14e: by 68%; 15e: by 36%, p < 0.05), resulting in a reduction in the number of cells in G1 and G2/M phases compared to untreated cells, while the changes in other phases of the cell cycle were not statistically significant.These results suggest that the entry of treated cells into a new cell cycle is prevented and are consistent with the results published by Zuo at al. and Zhang at al. [46,47].The subG0/G1 arrest suggests that the initiation of cell cycle arrest may be responsible for the antiproliferative potential.The subG0/G1 peak indicates DNA fragmentation and suggests that the cells die, most likely through apoptosis.The arrest of the cell cycle in the G2/M phase in cells treated with various benzimidazole derivatives has also been observed in several recently published studies [45].These compounds likely affect different phases of the cell cycle, resulting in changes in cell proliferation and growth.
Molecules 2023, 28, x FOR PEER REVIEW 8 of 24 G2/M phase in cells treated with various benzimidazole derivatives has also been observed in several recently published studies [45].These compounds likely affect different phases of the cell cycle, resulting in changes in cell proliferation and growth.

Absorption, Distribution, Metabolism, Excretion (ADME), and Toxicity Properties
Calculated ADME, pharmacokinetic and druglike properties of the analyzed compounds were presented in Table 2.According to the "Lipinski's rule of 5", the physicochemical ranges for high probability to be an oral drug are molecular weight (MW), between 150 and 500 g/mol; saturation fraction of carbons in the sp 3 hybridization, not less than 0.25; flexibility: rotatable bonds < 9; lipophilicity, XLOGP3 between −0.7 and +5.0; polarity, topological polar surface area (TPSA) between 20 and 130 Å 2 ; water solubility, log S < 6 [48].The drug similarity of all 20 quinoline-benzimidazole hybrids is schematically represented by the bioavailability radars in the Supplementary Materials (Table S1), while that for the selected molecules is presented in Figure 4

Molecular Docking Study
Molecular docking of 20 quinoline-benzimidazole hybrids was performed on the binding site of the TAO2 kinase domain defined according to the bonded ligand staurosporine (STU) (PDB: 2GCD).STU is a well-known inducer of apoptosis in a wide range of the tumor cell lines.The crystal structure of this complex was chosen because it provides the details of the interactions between TAO2and staurosporine, which explains the relatively low potency of staurosporine against TAO2.Although STU is too toxic to be used directly as a therapeutic agent, it could serve as a template for the design of inhibitors specific to TAO2 [51].Compounds were ranked by the total energy of the predicted pose in the binding site (Table 3), and the docking scores were compared with the docking results of the staurosporine.The highest binding energy on the TAO2 kinase domain, even before standard ligand staurosporine, was observed for compound 14e (−140.44 kcal/mol), which has also exhibited a strong selective antiproliferative effect on HuT78 cells (IC50 = 10.86 µM).The docking result of STU is followed by strong selective inhibitors of HuT78 cells: 9e, 14d, 9d, and 10e.The energies of the main interactions between compound 14e and residues in the binding site of the TAO2 kinase are shown in Table 4.The position of this compound into the binding site of the TAO2 kinase domain represented as a hydrophobic surface is shown in Figure 5a, while its interactions with amino acid residues are presented using a 2D diagram (Figure 5b).Six physicochemical properties are taken into account: lipophilicity, size, polarity, solubility, flexibility and saturation [49].Compound 9c displayed antiproliferative affinities against all tested tumor cell lines, especially on Hut78 cells (IC 50 = 0.2 µM) (Table 1).It also exhibited a toxic effect on normal MTR-5 cells but with great selectivity toward lymphoma cell line Hut78.That compound does not violate either one of the Lipinski rules, making it an ideal drug candidate.According to its bioavailability radar, compound 9c has optimal range of all properties, except for the fact that it is low in saturation index.It means that the ratio of sp 3 hybridized carbons over the total carbon count of the molecule (fraction Csp 3 ) is lower than 0.25.Compound 9c is poorly soluble in water, but it has high gastrointestinal absorption (GIA), which is important for effective oral drugs.It also has low blood-brain barrier permeation (BBBP), which means that is safe for the unwanted effects of peripheral drugs on the brain (Table 2) [50].The ability to be transported by P-glycoprotein (P-gp) out of the cell is an important ADME property of compound 9c.It means that it could be eliminated from cells, thereby preventing intracellular accumulation and decreasing toxicity.Compound 14e showed strong selective activity against HuT78 cells (IC 50 = 10.86 µM) (Table 1).It breaks only one Lipinski rule since it is MW > 500 g/mol (Table 2).The bioavailability radar of compound 14e reveals its inappropriate drug properties: large size (MW > 500 g/mol); low in saturation index (fraction Csp 3 = 0.19); flexibility (number of rotatable bonds > 9).Since that compound is poorly soluble in water, has a low GIA index, and cannot act as a P-glycoprotein substrate, it could only be used for parenteral drug administration.

Molecular Docking Study
Molecular docking of 20 quinoline-benzimidazole hybrids was performed on the binding site of the TAO2 kinase domain defined according to the bonded ligand staurosporine (STU) (PDB: 2GCD).STU is a well-known inducer of apoptosis in a wide range of the tumor cell lines.The crystal structure of this complex was chosen because it provides the details of the interactions between TAO2and staurosporine, which explains the relatively low potency of staurosporine against TAO2.Although STU is too toxic to be used directly as a therapeutic agent, it could serve as a template for the design of inhibitors specific to TAO2 [51].Compounds were ranked by the total energy of the predicted pose in the binding site (Table 3), and the docking scores were compared with the docking results of the staurosporine.The highest binding energy on the TAO2 kinase domain, even before standard ligand staurosporine, was observed for compound 14e (−140.44 kcal/mol), which has also exhibited a strong selective antiproliferative effect on HuT78 cells (IC 50 = 10.86 µM).The docking result of STU is followed by strong selective inhibitors of HuT78 cells: 9e, 14d, 9d, and 10e.The energies of the main interactions between compound 14e and residues in the binding site of the TAO2 kinase are shown in Table 4.The position of this compound into the binding site of the TAO2 kinase domain represented as a hydrophobic surface is shown in Figure 5a, while its interactions with amino acid residues are presented using a 2D diagram (Figure 5b).Compound 14e is located in the hydrophobic surface of the ATP-binding cleft of the TAO2 due to the presence of plenty of hydrogen bonds and van der Waal interactions.Compound 14e creates major hydrogen bonds through the N atom of benzimidazole scaffold with His36 (3.09 Å), and the N atom of propilamidine moiety with Asp114 (2.79 Å) and Glu117 (3.02 Å).Van der Waals interactions are very similar to those observed in staurosporine complex with TAO2 kinase (PDB: 2GCD): Gly35; Ile34; Phe39; Glu76; Gln90; Ile89; Gly168; Phe170; Met312.The other interactions are as follows: π-σ interactions (Leu80; Val42); π-anion (Met105; Asp169); π-alkyl interactions (Lys57).We note the very tight π-σ interaction of the quinolone moiety with Leu80 (3.23 Å), leading to a strong interaction energy (11.43 kcal/mol).The interactions between TAO2 and staurosporine involve three hydrogen bonds with Glu106, Cys108, and Gly155 [51].
Mitogen-activated protein kinases (MAPKs) are involved in signal transduction pathways in eukaryotic cells, controlling multiple cellular programs.MAPKs are activated by MAPK kinases.TAO2 is a MAP3K level kinase that activates p38 MAPKs, which regulate the production of cytokines.TAO2 is activated in response to apoptosis-inducing agents and acts as regulators of apoptosis; it represents a potential drug target for the treatment of cancer [52].TAO2 is thought to be a physiological regulator of p38 because it is activated Compound 14e is located in the hydrophobic surface of the ATP-binding cleft of the TAO2 due to the presence of plenty of hydrogen bonds and van der Waal interactions.Compound 14e creates major hydrogen bonds through the N atom of benzimidazole scaffold with His36 (3.09 Å), and the N atom of propilamidine moiety with Asp114 (2.79 Å) and Glu117 (3.02 Å).Van der Waals interactions are very similar to those observed in staurosporine complex with TAO2 kinase (PDB: 2GCD): Gly35; Ile34; Phe39; Glu76; Gln90; Ile89; Gly168; Phe170; Met312.The other interactions are as follows: π-σ interactions (Leu80; Val42); π-anion (Met105; Asp169); π-alkyl interactions (Lys57).We note the very tight π-σ interaction of the quinolone moiety with Leu80 (3.23 Å), leading to a strong interaction energy (11.43 kcal/mol).The interactions between TAO2 and staurosporine involve three hydrogen bonds with Glu106, Cys108, and Gly155 [51].
Mitogen-activated protein kinases (MAPKs) are involved in signal transduction pathways in eukaryotic cells, controlling multiple cellular programs.MAPKs are activated by MAPK kinases.TAO2 is a MAP3K level kinase that activates p38 MAPKs, which regulate the production of cytokines.TAO2 is activated in response to apoptosis-inducing agents and acts as regulators of apoptosis; it represents a potential drug target for the treatment of cancer [52].TAO2 is thought to be a physiological regulator of p38 because it is activated during the differentiation of C2C12 myoblasts in parallel with activation of p38 [53].Therefore, TAO2 is a potential target for the treatment of p38 MAPK-associated diseases such as leukemia, breast cancer, prostate cancer, bladder cancer, liver cancer, lung cancer, thyroid cancer, and many others.Selective inhibitors of TAO2, which inhibit the regulation of MAPK signaling pathway, are used for the development of potential cancer therapy [54].

Chemistry
All solvents and reagents were used without purification from commercial sources.To monitor the progress of a reaction and for comparison purposes, thin layer chromatography (TLC) was performed on precoated Merck silica gel 60F-254 plates using an appropriate solvent system, and the spots were detected under UV light (254 nm).Melting points (uncorrected) were determined using the Buchi 510 melting point instrument. 1 H and 13 C NMR spectra were recorded using a Bruker Avance DPX-300 or Bruker AV-600 spectrometer.All data were recorded in DMSO-d6 at 298 K.Chemical shifts were related to the residual solvent signal of DMSO at d 2.50 ppm for 1 H and d 39.50 ppm for 13 C. Elemental analyses for carbon, hydrogen and nitrogen were performed using the Perkin-Elmer 2400 elemental analyzer.The analyses are given as symbols of the elements.The analytical results obtained are within 0.4% of the theoretical value.

General Procedure for the Synthesis of Compounds 6, 7, 12 and 13
Aldehyde 3 or 4 (1 mmol) and the appropriate azide 5 or 11 (1 mmol) were dissolved in 5 mL DMF and, while stirring at 65 • C, 0.4 mL of 1M sodium ascorbate water solution and 0.2 mL of 1M CuSO 4 water solution were added.The reaction mixture was then stirred at 65 • C for 24 h.The reaction mixture was filtered through a short Al2O3 column, with the addition of water, and the product was precipitated.

General Procedure for the Synthesis of Compounds 9a-10e and 14a-15e
A solution of an aldehyde 6, 7, 12 and 13 (1 mmol) appropriate benzene-1,2-diamine 9a-d (1 mmol) and Na 2 S 2 O 5 (0.5 mmol) in DMSO (15 mL) was heated at 165 • C for 15 min.The mixture was cooled down to room temperature.The addition of water (5 mL) resulted in precipitation.The resulting residues for compounds 10a-c, 11a-c, 12a-c, 13a-c, 14a-c, 15a-c were collected with filtration and products were obtained via recrystallization using methanol.The resulting residues for compounds 9d-e, 10d-e, 14d-e and 15d-e were collected with filtration and products were obtained via recrystallization using methanol and converted to hydrochloride salts using anhydrous methanol saturated with HCl(g).

Proliferation Assay
Growth-inhibitory activity was assessed using a slightly modified procedure based on the National Cancer Institute's protocol [55].Briefly, the cells were seeded in 96-well microtiter plates and incubated for 24 h.They were then treated with 10 −7 to 10 −4 M concentrations of the tested compounds for an additional 72 h.After the treatment period, the effects of the tested compounds on the growth rate of the cells were examined using the MTT assay [56].Absorbance was measured at 595 nm using a microplate reader.The IC 50 value, which represents a 50% inhibition of cell growth, and QC calculation were performed using the GraphPadPrism and Excel software.The selectivity index was calculated according to the following formula: SI = IC 50 value of normal cell line IC 50 value of cancer cell line The effect of each concentration was analyzed by plotting the logarithm of the concentration of the evaluated compound against the corresponding percentage inhibition value using least squares.

Cell Cycle Analysis
HuT78 cells (1 × 10 5 cells/mL) were seeded in 24-well plates (Falcon, Durham, SAD) and treated with previously determined IC 50 values for compounds 10e, 14e, and 15e (5 × 10 −6 mol/dm 3 ) and 9c (0.5 × 10 −6 mol/dm 3 ).The cells were exposed to the compounds for 24 h, collected, washed with PBS, fixed with 70% ethanol and stored at −20 • C until analysis.On the day of analysis, the cell pellets were washed twice with PBS, resuspended in 1 mg/mL PI and 0.2 mg/mL RNase A and left in a dark/cold atmosphere for 30 min.The stained cells were analyzed using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer.Tests were performed in duplicate and repeated twice.FlowJo software, version 10.2., was adapted for the analysis of DNA histograms.STATISTICA 13.5 (TIBCO Software Inc., Tulsa, USA) was used for the statistical analysis of the results.Student's t test was used for data analysis.Differences were considered statistically significant at p < 0.05.

Calculation of ADME Properties
SwissADME web tool (http://www.swissadme.ch,accessed on 25 August 2023) gives free access to a pool of fast yet robust predictive models for physicochemical properties, pharmacokinetics, drug-likeness and medicinal chemistry friendliness parameters through the input of molecular structure [49].

Molecular Docking
Molecular docking of quinoline-benzimidazole hybrids, which exhibited antiproliferative activities, was performed on the MAP3K TAO2 kinase.The crystal structure of the MAP3K TAO2 kinase domain was taken with bounded inhibitor staurosporine from the PDB base (PDB ID: 2GCD).Protein structure was prepared sing BIOVIA Discovery Studio Visualizer 4.5 (Dassault Systèmes, Paris, France).The 3D structures of ligands were optimized using Spartan '08 (Wavefunction, Inc.; Irvine, CA, USA, 2009), using the molecular mechanics force field (MM+) [57], and subsequently via the semiempirical AM1 method [58].The molecular-docking-optimized molecular structures of 12 compounds were performed with iGEMDOCK (BioXGEM, Hsinchu, Taiwan) using generic evolutionary method (GA).The GA parameters were set as follows: population size 200; generations 70; number of poses 3; and binding site radius 8 Å).After each compound was docked into the binding site, iGEMDOCK generated protein-compound interaction profiles of electrostatic (E), hydrogen-bonding (H), and van der Waals (V) interactions.iGEMDOCK infers pharmacological interactions and clusters for post-screening analysis based on these profiles and compound structures.Finally, the docked compounds were ranked by total energy of a predicted pose in the binding site that is defined as: (E/kcal mol −1 ) is: E = vdW + Hbond + Elec, where the vdW is van der Waal energy, Hbond is the hydrogen bonding energy, and Elect terms are electro statistic energy [59].
The results of in vitro studies of antiproliferative activity against one non-tumor and seven cancer cell lines indicate that the introduction of a linker into bromine-substituted non-amidine compounds increases the selectivity and decreases the activity against the carcinoma cell lines tested (HeLa and CaCo2).Chlorine derivatives with bromine and without the linker have selective activity against all cell lines tested.All three non-amidine derivatives with bromine and with a linker showed selective activity against leukemia and lymphoma cells.Non-amidine compounds with a linker showed higher activity against leukemia and lymphoma cells after the introduction of bromine.These compounds affect different phases of the cell cycle, leading to changes in cell proliferation and growth.Cell cycle inhibition may be a key factor in the potential anti-cancer properties of these compounds.
A molecular docking study revealed that the quinoline-benzimidazole hybrid with propylamine at the C-5 position of the benzimidazole moiety, compound 14e, had the highest affinity to inhibit TAO2 protein kinases, whose activity has been linked to DNA damage and cancer proliferation.This compound showed high and selective antiproliferative activity against lymphoma cell line.Despite its excellent drug-like properties, compound 14e is not suitable for oral administration due to some deviations in its bioavailability.
Further research and investigation into the mechanisms of action and potential therapeutic applications of these hybrids would be necessary to fully understand their effects on the cell cycle and their ability to induce apoptosis as a mode of treated cell death.

Figure 2 .
Figure 2. Structure activity relationship for antiproliferative activity of the 1,2,3-triazole-containing quinoline-benzimidazole hybrids.2.2.2.Cell Cycle PerturbationInducing cell cycle arrest at specific checkpoints and inducing apoptosis are common strategies in cancer treatment with cytotoxic agents.Numerous studies have shown that quinoline-and benzimidazole-based compounds, as well as their hybrids, significantly

a
IC50-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC 50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.

Figure 3 .
Figure 3. Flow cytometry analysis of the cell cycle distribution of HuT-78 cells exposed to compounds 9c (0.5 µM) and 10e, 14e and 15e (5 µM) for 24 h.(a) DNA histograms present changes in cell cycle.(b) Data are expressed as mean ± standard deviation.A statistically significant p value is defined as p < 0.05 (*).

Figure 3 .
Figure 3. Flow cytometry analysis of the cell cycle distribution of HuT-78 cells exposed to compounds 9c (0.5 µM) and 10e, 14e and 15e (5 µM) for 24 h.(a) DNA histograms present changes in cell cycle.(b) Data are expressed as mean ± standard deviation.A statistically significant p value is defined as p < 0.05 (*).

Table 1 .
Sensitivity of human tumor and normal cells to investigated compounds expressed as IC 50 a value.

Table 1 .
Sensitivity of human tumor and normal cells to investigated compounds expressed as IC50 a value.

Table 1 .
Sensitivity of human tumor and normal cells to investigated compounds expressed as IC50 a value.
a IC50-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.a IC50-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.a IC50-concentration of the compound that inhibits cell growth by 50%.Data represent mean IC50 (µM) values ± standard deviation (SD) from three independent experiments.Exponentially growing cells were treated with compounds during 72 h.Cytotoxicity was analyzed using MTT survival assay.5-FU: 5-fluorouracil.

Table 2 .
Calculated ADME, pharmacokinetic and druglike properties of the analyzed compounds.

Table 4 .
The energies (kcal/mol) of the main interactions between TAO2 kinase domain and compound 14e.