Green Synthesis and Anticancer Potential of 1,4-Dihydropyridines-Based Triazole Derivatives: In Silico and In Vitro Study

A library of 1,4-dihydropyridine-based 1,2,3-triazol derivatives has been designed, synthesized, and evaluated their cytotoxic potential on colorectal adenocarcinoma (Caco-2) cell lines. All compounds were characterized and identified based on their 1H and 13C NMR (Nuclear Magnetic Resonance) spectroscopic data. Furthermore, molecular docking of best anticancer hits with target proteins (protein kinase CK2α, tankyrase1, and tankyrase2) has been performed. Our results implicated that most of these compounds have significant antiproliferative activity with IC50 values between 0.63 ± 0.05 and 5.68 ± 0.14 µM. Moreover, the mechanism of action of most active compounds 13ab′ and 13ad′ suggested that they induce cell death through apoptosis in the late apoptotic phase as well as dead phase, and they could promote cell cycle arrest at the G2/M phase. Furthermore, the molecular docking study illustrated that 13ad′ possesses better binding interaction with the catalytic residues of target proteins involved in cell proliferation and antiapoptotic pathways. Based on our in vitro and in silico study, 13ad′ was found to be a highly effective anti-cancerous compound. The present data indicate that dihydropyridine-linked 1,2,3-triazole conjugates can be generated as potent anticancer agents.


Introduction
Cancer is an uncontrollable somatic cell proliferation and decline in programmed cell death, which is the subsequent foremost reason of yearly death (nearly 10 million)
Recently, in the past few years, the synthesis of hybrid bioactive compounds consisting of two or more heterocyclic scaffolds has been focused on more [27]. It has been reported that the combination of two heterocyclic scaffolds having different biological functions produce new hybrid compounds that are more medically effective than their parent molecules [28].
Keeping these observations along with our previous results in view, we aimed to prepare a library of dihydropyridines linked 1,2,3-triazoles and evaluate their therapeutic potential as antiproliferative agents against colorectal cancer Caco-2 cell lines. Moreover, the best hits of compounds were also analyzed for their ability of cell cycle arrest and induction of apoptosis to understand the mechanistic pathways of the antiproliferation activity. Furthermore, a cheminformatics study was performed to predict the inhibitory effects of best hits derivatives against protein kinase CK2α human tankyrases (tankyrase1 and tankyrase2).

Cell Culture and Cell Viability Assay
Caco-2 cell line was gifted from Zydus Research Centre, Ahmedabad, Gujarat. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) from Himedia supplemented with 20% Fetal bovine serum (FBS) from Cellclone, and 1% antibiotic (penicillinstreptomycin, Himedia) as described in our previous study [31]. Further, 1 × 10 4 cells (in 100 µL of media) were seeded in a 96-well plate for 24 h to evaluate the anti-cancerous effect of these synthesized compounds using MTT assay as reported [31,32]. After 24 h, Caco-2 cells were exposed to these synthesized drugs at different concentrations (0.05 to 100 µM) for another 24 h. Stock solutions for all drugs were prepared using dimethyl sulfoxide (DMSO). Then, media was removed, and 100 µL of media containing 10 µL of MTT was added to each well, and plates were incubated for 4 h at 37 • C. A total of 100 µL of DMSO were added to each well after removing the media containing MTT to dissolve the purple-colored formazan crystals. Using ELISA reader (Multiskan Spectrum Microplate Reader, Thermo Scientific, Waltham, MA, USA), optical density was measured at 570 nm wavelength. Along with these synthesized compounds, we have used carboplatin, gemcitabine, and daunorubicine in the range of 0.05 to 50 µM as reference drugs that are widely used in anti-cancerous therapeutic regimens. The untreated cell (without synthesized com- pounds) was used as negative control, and the growth inhibition (percentage) at various concentrations were calculated by the following equation: Percentage growth inhibition = 100 − [Mean absorbance of test group/Mean absorbance of control group] × 100 Furthermore, the IC 50 value (concentration of the inhibitor that inhibits the 50% of cell growth) was calculated through the GraphPad tool/Excel.

Apoptosis/Cell Death Assay
Based on their IC 50 values, 13ab and 13ad were selected for further experiments among all synthesized compounds. Cells were exposed to 13ab and 13ad for 24 h at their IC 50 values (1.39 ± 0.04 and 0.63 ± 0.05 µM, respectively) to distinguish between apoptotic and necrotic cells using Muse™ Annexin V and Dead Cell Assay kit (Muse™ Cell Analyzer; Millipore, Billerica, MA, USA). After exposing cells to 13ab and 13ad for 24 h, annexin V and dead cell marker from a kit was used to stain treated and untreated cells as described in the manufacturer's protocol. Briefly, cells were trypsinized using trypsin-EDTA (Himedia) to get cells in suspension and washed with phosphate buffer saline (PBS) two times. Cells were resuspended in fresh media containing 1% FBS, and from that, 100 µL of cells (1 × 10 5 cells/mL) were taken. To each tube, 100 µL of Muse Annexin V & Dead Cell Reagent from the kit was added, and tubes were mixed thoroughly and kept in the dark for 20 min to stain cells. Further, results were analyzed using Muse analyzer [33].

Cell Cycle Assay
A cell cycle study was applied to assess the effectiveness of 13ab and 13ad on cell cycle arrest. The cell lines of Caco-2 (1 × 10 5 cells/mL) were treated with the respective IC 50 value concentration (1.39 ± 0.04 and 0.63 ± 0.05 µM, respectively) of 13ab and 13ad for 24 h to illustrate the impact on cell cycle using Muse ® Cell Cycle Assay Kit (Luminex, Austin, TX, USA) according to manufacturer's instructions as previously described [34]. Cells were collected in a pellet and washed twice with PBS. After that, cells were resuspended in 1 mL of ice-cold 70% ethanol to fix the cells and freeze them for at least 3 h, at −20 • C. Then, 200 µL of fixed cell suspension was taken in a test tube and centrifuged at 300× g for 5 min to get the pellet. This pellet was again washed twice with PBS and finally resuspended in 200 µL of Muse™ Cell Cycle Reagent. Tubes were incubated for 30 min in the dark prior to analyzing results on Muse™ Cell Analyzer. The outcomes were recorded in the percentage of cells available in different phases of the cell cycle.

Statistical Analysis
All data from triplicate assay were articulated as mean ± standard error of the mean (SEM), and Student's t-test was used to measure differences among control vs. experimental set. p Values < 0.05 were considered statistically significant. All experiments were performed in triplicates.

Cheminformatics Molecular Interaction Study
Autodock-Vina and PyRx tool was used for molecular docking study with the Lamarckian genetic algorithm as scoring function [35,36]. In contrast, the Discovery Studio visualizer 2021 (BIOVIA) tool was used for visualizing the interaction of molecular complex [37].
Moreover, the 3D structure of the synthesized chemical compounds was drawn using the Chemdraw tool and downloaded in a ".mol" format file. The best anticancer ligands were further energy minimized and converted into a ".pdbqt" format file. To validate the protocol, initially, the native ligands were redocked to the active sites of proteins, and after validation, all the synthesized small organic molecules were docked individually to the target proteins (3PE1, 4W6E, and 4HKI). Discovery studio visualizer was used to get the dimensions of the grid box (25 × 25 × 25 Å), and it was centered at their native ligand XYZ coordinates, that is, 22

Chemistry Optimization and Synthesis of Compounds
The optimization of copper source and reductant was explored, and the findings are reported in Table 1. CuSO 4 ·5H 2 O was found to be successful in producing Cu(I) in the presence of sodium ascorbate as a reductant and yielded the required product of 13aa (84% yield) among the Cu sources evaluated ( Table 1, entry 2). However, Cu(OAc) 2 was found to be the more effective and yielded the desired product 13aa in 96% yield (Table 1, entry 6). The major difference was observed in the rate of the reaction. In the presence of copper sulfate and sodium ascorbate/hydrazine hydrate, more time was consumed to complete the reaction as compared to copper acetate and hydrazine hydrate. 1.95 Å resolution, and human tankyrase 2 (TNKS2) (PDB Id: 4HKI) at 2.15 Å resolutio in complex to their native inhibitors were obtained from PDB (Protein Data Bank) dat base [38][39][40]. The heteroatoms were removed from proteins, and adding of polar hydro gens was performed to prepare it for molecular docking work, thereafter converting th ".pdb" format file to Autodock suitable ".pdbqt" format.
Moreover, the 3D structure of the synthesized chemical compounds was drawn u ing the Chemdraw tool and downloaded in a ".mol" format file. The best anticancer lig ands were further energy minimized and converted into a ".pdbqt" format file. To val date the protocol, initially, the native ligands were redocked to the active sites of pr teins, and after validation, all the synthesized small organic molecules were docked in dividually to the target proteins (3PE1, 4W6E, and 4HKI). Discovery studio visualiz was used to get the dimensions of the grid box (

Optimization and Synthesis of Compounds
The optimization of copper source and reductant was explored, and the finding are reported in Table 1. CuSO4.5H2O was found to be successful in producing Cu (I) the presence of sodium ascorbate as a reductant and yielded the required product 13aaˈ (84% yield) among the Cu sources evaluated ( Table 1, entry 2). Howeve Cu(OAc)2 was found to be the more effective and yielded the desired product 13aa' in 96 yield (Table 1, entry 6). The major difference was observed in the rate of the reaction. I the presence of copper sulfate and sodium ascorbate/hydrazine hydrate, more time wa consumed to complete the reaction as compared to copper acetate and hydrazine hy drate. The synthesis of a 1,4-dihydropyridines-based substituted 1,2,3-triazoles 13aa -13ag and 14ba -14bg was achieved by following the synthetic route as shown in Scheme 1. sources, and 1 mmol reductants were stirred in 1 mL water in a sealed tube for the allocated time mentioned in the above table. b Isolated yields.

In Vitro Cytotoxicity
The cytotoxic efficacy of these synthesized compounds and reference drug carboplatin, gemcitabine, and daunorubicin in terms of IC 50 is shown in Figure 2. From IC 50 values, it is very much clear that most compounds have significant cytotoxicity in a dose-dependent manner (Figures S15 and S16; Table S1).

Cell Cycle Inhibition
Results showed a significant increase in G2/M phases and decreased in the G0/G1 phase of the cell cycle when treated with these compounds for 24 h. All the results were statistically significant (p < 0.05) as compared to untreated cells (Figure 3), suggesting the regulating effect of these compounds on the cell cycle by arresting cell proliferation and progression in the G2/M phase resulting in the increased apoptosis in Caco-2 cells.

Cell Cycle Inhibition
Results showed a significant increase in G2/M phases and decreased in the G0/G1 phase of the cell cycle when treated with these compounds for 24 h. All the results were statistically significant (p < 0.05) as compared to untreated cells (Figure 3), suggesting the regulating effect of these compounds on the cell cycle by arresting cell proliferation and progression in the G2/M phase resulting in the increased apoptosis in Caco-2 cells.

Induction of Apoptosis
The results (Figure 4) revealed that control cells have a very low percentage of annexin V staining showing a higher percentage of viable cells. Both compounds exhibited a significant effect in decreasing the number of viable cells, while late apoptotic and necrotic cells were observed to be increased, which is suggestive of the fact that these compounds have the potential to induce apoptosis in Caco-2 cells. Nevertheless, in early apoptotic cells, no significant difference was observed.

Induction of Apoptosis
The results ( Figure 4) revealed that control cells have a very low percentage of annexin V staining showing a higher percentage of viable cells. Both compounds exhibited a significant effect in decreasing the number of viable cells, while late apoptotic and necrotic cells were observed to be increased, which is suggestive of the fact that these compounds have the potential to induce apoptosis in Caco-2 cells. Nevertheless, in early apoptotic cells, no significant difference was observed.

Discussion
The synthetic strategy was previously developed in our laboratory, in which various DHP's linked with heterocyclic moieties were reported [24][25][26][42][43][44][45]. Keeping this observation in view, we aimed to improvise and prepare a library of dihydropyridines linked 1,2,3-triazoles via a simple, cost-effective, and expedient route in high yield, which was further evaluated for anticancer activity, and the mechanism of action was studied.

Chemistry
Because of dual action, the nature of Cu(I) salt is very essential for CuAAC reaction. In CuAAC, Cu(I) functions both as π and σ-electrophilic Lewis acid [46]. Several published reports show different copper sources were used individually or in combination with catalyst or ligand or via a green chemistry approach for the synthesis of triazole ring-based derivatives [30,[47][48][49]. The CuI was found to be a better copper source in the presence of the ligand and DMF as solvent through the conventional method [49], while in the green chemistry approach, the CuSO 4 ·5H 2 O and Cu(OAc) 2 in the presence of reductants sodium ascorbate and hydrazine hydrate, respectively was earlier reported for better yield of click adduct in a short period of time [30,50,51]. From the previous literature, we hypothesized that hydrazine hydrate could be used as a reductant for CuSO 4 ·5H 2 O or base for CuI and CuBr. Moreover, we also hypothesized that sodium ascorbate could be used as a reductant for Cu(OAc) 2 because these combinations of copper sources and reductants might get a better yield in a minimum period of time via the green chemistry approach, and they were not reported earlier. We optimized the copper source (Cu(OAc) 2 ) and reductant (hydrazine hydrate) for the synthesis of title compounds on the basis of higher yield within a short period of time. This reaction combination was earlier reported by Jiang et al. [30], but optimization of the various copper sources with different reductants was not reported. Therefore, in this study, several copper sources were investigated considering these facts, and the results are reported in Table 1. CuSO 4 ·5H 2 O was found to be successful in producing Cu(I) in the presence of sodium ascorbate as a reductant and yielded the required product of 13aa (84% yield) among the Cu sources evaluated (Table 1, entry 2). However, Cu(OAc) 2 was found to be the more effective and yielded desired product 13aa in 96% yield (Table 1, entry 6). The major difference was observed in the rate of the reaction. In the presence of copper sulfate and sodium ascorbate/hydrazine hydrate, more time was consumed to complete the reaction as compared to copper acetate and hydrazine hydrate. Hence, it was confirmed that hydrazine hydrate is working as a better reductant as compared to sodium ascorbate. In the absence of a reductant, the reaction did not progress, and traces were discovered at the end (Table 1, Entry 1, 4, 7, and 9). On the other hand, CuI and CuBr as different sources provide inferior yields in the existence of hydrazine hydrate.
The key 1,4-dihydropyridine component (11) was obtained by the condensation 2aminocrotononitrile (9) with substituted propargylated benzaldehyde (10) in acetic acid by following the Hantzsch pyridine synthesis methodology [29]. The desired title compounds 13aa -13ag and 14ba -14bg were synthesized by the 1,3-diploar cycloaddition reaction between propargylated 1,4-dihydropyridines and substituted azides derivatives under click chemistry conditions in quantitative yields [30]. The existence of the propargyl group in 1,4-dihydropyridines (11) was confirmed by the presence of a signal at δ 5.20-5.26 for two protons of the -CH 2 group. Similarly, the formation of the 1,2,3-triazole ring in the title compound was confirmed by the resonance of the proton in the triazolyl ring at a δ 8.70-9.00 (s, 1H, ArH) as a singlet.
Several reports suggested that the orthogonal analytical method ( 1 H NMR) can be considered as a primary analytical tool for the detection of purity of the compounds in addition to structural identification in which the overlapping of the peaks or extra peaks suggests the presence of impurities in the compound [52,53]. In this study, the NMR spectra did not exhibit any extra peaks or any overlapping of the peaks in several compounds, but we observed a few compounds having extra peaks in very low intensity, and thus, we interpreted that the compounds are pure enough for the biological screening. We obtained the excellent yield (84-96%) of final compounds in most of the cases without any requirement of further purification through chromatography (Table 2).

Anticancer Activity
All compounds were screened against Caco-2 (colorectal adenocarcinoma) cell line to evaluate in vitro cytotoxicity. We noticed that all the title compounds except five compounds (13aa , 13ag , 13af , 14ba , and 14bd ) provided better cytotoxic activity than all the three reference drugs. Out of those compounds, 13ab (IC 50 = 1.39 ± 0.04 µM) have second highest cytotoxic effect, which is around 5.37-fold, 3.22-fold, and 10.11-fold more active than carboplatin (IC 50 = 7.49 ± 0.29 µM), gemcitabine (IC 50 = 4.51 ± 0.19 µM) and daunorubicin (IC 50 = 14.12 ± 0.37 µM). While 13ad provided the highest cytotoxic effect (IC 50 = 0.63 ± 0.05 µM) is 11.75-fold, 7.06-fold, and 22.13-fold more active against the Caco-2 cell line than carboplatin, gemcitabine, and daunorubicin, respectively. Overall, all compounds have a significant (* p < 0.05) cytotoxic effect against the Caco-2 cell line and thus could be used as a promising target for developing anticancer drugs. Moreover, we noticed that the potency of 13ad is much better than the 13ab . From these compounds, we have used 13ab and 13ad further cellular assays. Conventional chemotherapeutic drugs are already known to modulate cancer cells growth by targeting cells in different phases of the cell cycle [54,55]. Thus, to investigate the underlying relationship of cytotoxicity of these compounds and cell cycle arrest, DNA accumulation at different phases of the cell cycle was analyzed using Muse TM analyzer-based flow cytometric analysis. The significant increase in G2/M phases and decreased in G0/G1 phase of the cell cycle when treated with 13ab and 13ad compounds for 24 h, suggesting the regulating effect of these compounds on the cell cycle by arresting cell proliferation and progression in the G2/M phase resulting in the increased apoptosis in Caco-2 cells.
Furthermore, in apoptosis assay, we observed a significant decrease in the number of viable cells, while late apoptotic and necrotic cells were observed to be increased (Figure 4), which is suggestive of the fact that these compounds have the potential to induce apoptosis in Caco-2 cells. Several studies have reported the same cell cycle phase arrest in different cell lines [56,57]. Dhanasekhar et al. [58] described that lanatoside C has the potential to induce G2/M cell cycle arrest, which is leading toward suppressed cancer cell growth in three different cell lines, i.e., breast (MCF7), lung (A549), and liver (HepG2). They further described lanatoside C has the potential to arrest cells in the G2/M phase of the cell cycle by hindering the MAPK/Wnt/PAM signaling pathway, which is involved in its regulation. They also showed its capacity to induce apoptosis through targeting signaling pathways regulating DNA damage such as PI3K/AKT/mTOR. Another study revealed that dioscin inhibits the growth cancerous growth of osteosarcoma through G2/M cell cycle phase arrest and apoptosis by targeting the JNK/p38 pathway [59]. Thus, these compounds could be further used to check their efficiency to target different signaling pathways involved in cell cycle regulation as well as apoptosis for better insight into their mechanism of action.

Cheminformatics Molecular Interaction Analysis
For the computational molecular docking of ligand (13ab and 13ad ) with target proteins, the selection of PDB files (3PE1, 4W6E, and 4HKI) for the current study was due to their high resolution and their availability with their respective native ligand, which are the inhibitors and bound to the catalytic active site [6,[60][61][62][63].
The molecular docking with protein kinase CK2α subunit (3PE1) predicted the binding score of interacted molecules that revealed the binding energy and binding affinity of 13ad (−11 kcal/mol and 1.16 × 10 8 M −1 ) is better than 13ab (−9.3 kcal/mol and 6.56 × 10 6 M −1 ). Our results are in correspondence with previous studies where it was re-ported that the residues of protein kinase CK2α subunit such as LYS68 and VAL116 formed hydrogen bonds, whereas VAL66, HIS160, and MET163 via hydrophobic bond interacted with CX-4945 [64,65]. Moreover, Oramas-Royo et al. docked some synthesized compounds with protein kinase CK2α and reported that the compounds fully occupied the active site in the adenine region (VAL53, VAL66, VAL116, and MET163), the hydrophobic region I (PHE113, ILE95, and ILE174), and hydrophobic region II (LEU45, and HIS115) [66]. Furthermore, the interaction of docked ligands and tankyrase1 (4W6E), as illustrated in Table 3 and Figure 6, revealed the binding energy and binding affinity of 13ad (−10 kcal/mol and 2.14 × 10 7 M −1 ) is better than 13ab (−8.6 kcal/mol and 2.01 × 10 6 M −1 ) are in correspondence with a previous study where it was reported that the 1,2,4-triazole derivative stabilized in adenosine and nicotinamide binding pocket of tankyrase1, which interacted with SER1186, PHE1188, ALA1191, ILE1192, LYS1195, HIS1201, MET1207, and TYR1213 [67]. Another study revealed that mainly GLU1291, SER1221, GLY1185, GLY1196, and ASP1198 residues were involved in stabilizing the tankyrase1 with natural compounds [63]. Our results demonstrate that the complex of flavone (native ligand) and human tankyrase2 (4HKI) redocked in the active site was stabilized by the three hydrogen-bonded residues (GLY1032, SER1068, and HIS1031) and seven hydrophobic interactions with residues HIS1031, ALA1062, LYS1067, TYR1060, and TYR1071 of adenosine and nicotinamide subsites, which are in correspondence to the previous reports that validate the protocol [4]. The molecular docking also predicted the binding score of interacted molecules with 4HKI protein, which revealed the binding energy and binding affinity of 13ad (−10.4 kcal/mol and 4.20 × 10 7 M −1 ) is better than 13ab (−9 kcal/mol and 3.90 × 10 6 M −1 ) (Table 3 and Figure 7).
Numerous proteins are involved in the progression of cancer where protein kinase CK2α and tankyrases (tankyrase1 and tankyrase2) were reported earlier as potential molecular targets to treat various human pathophysiological disorders and found to be overexpressed in several cancer cells [3,4]. The CK2α involved in tumorigenic, antiapoptotic, and failure of cell cycle arrest process by reducing the caspase activation potentiates the cancer drug-resistant controls unfolded protein response, reduces the tumor suppressor functions, and regulates the chaperone activity [3,5,68]. Whereas tankyrases (tankyrase1 and tankyrase2) are involved in various cellular pathways to promote cell proliferation, telomerase maintenance, helps in mitosis, and are involved in Wnt signaling. Recently, inhibition of tankyrases and/or protein kinase CK2α has emerged as an alluring approach for the finding of novel anticancer drugs [4,6,60,62,63,66,67]. Several inhibitors of CK2α were reported earlier, where CX-4945 exhibited the ATP-competitive mode of inhibition and showed anti-proliferative activity by promoting cell cycle arrest, inducing the caspase activity and apoptosis; hence, it was approved as an orphan drug by FDA for cancer treatment [60,62,66]. Therefore, the cheminformatics approach was applied in this study to understand the mechanism of action of antiproliferative and apoptotic properties of the best active compounds by inhibiting the target proteins involved in cell proliferation and antiapoptotic pathways. It is now evident that 13ad have better antiproliferation activity than the other compounds due to their better inhibition of target proteins involved in cancer proliferation.

Conclusions
In conclusion, using alkyne dihydropyridines and substituted azides, we have effectively established a novel, operationally easy, inexpensive, and environmentally friendly copper-catalyzed azide alkyne cyclization (CuAAC) reaction for the synthesis of functionalized triazoles. Among all the synthesized derivatives, 13ad was found to be the most favorable compound as it was showing 7.06-fold, 11.75-fold, and 22.13-fold better activity than gemcitabine, carboplatine, and daunorubicin against Caco-2 cell lines, respectively. Moreover, compound 13ad showed a significant effect on apoptosis in terms of cellular death and had the potential to arrest cells in G2/M phases of the cell cycle, resulting in increased apoptosis. The molecular docking study concluded that 13ad possess better binding affinity and interacted with the catalytic residues of target proteins involved in cell proliferation and antiapoptotic pathways. Nevertheless, more experiments may be needed to have a better idea about their mechanism of action against cancerous cells.