Exploring the Synergistic Anticancer Potential of Benzofuran–Oxadiazoles and Triazoles: Improved Ultrasound- and Microwave-Assisted Synthesis, Molecular Docking, Hemolytic, Thrombolytic and Anticancer Evaluation of Furan-Based Molecules

Ultrasound- and microwave-assisted green synthetic strategies were applied to furnish benzofuran–oxadiazole 5a–g and benzofuran–triazole 7a–h derivatives in good to excellent yields (60–96%), in comparison with conventional methods (36–80% yield). These synthesized derivatives were screened for hemolysis, thrombolysis and anticancer therapeutic potential against an A549 lung cancer cell line using an MTT assay. Derivatives 7b (0.1%) and 5e (0.5%) showed the least toxicity against RBCs. Hybrid 7f showed excellent thrombolysis activity (61.4%) when compared against reference ABTS. The highest anticancer activity was displayed by the 5d structural hybridwith cell viability 27.49 ± 1.90 and IC50 6.3 ± 0.7 μM values, which were considerably lower than the reference drug crizotinib (IC50 8.54 ± 0.84 μM). Conformational analysis revealed the spatial arrangement of compound 5d, which demonstrated its significant potency in comparison with crizotinib; therefore, scaffold 5d would be a promising anticancer agent on the basis of cytotoxicity studies, as well as in silico modeling studies.


Introduction
In recent years, natural-and synthetic furan-based ( Figure 1) based chemotherapeutic agents have attracted the attention of medicinal chemists to develop novel pharmacological agents with diverse pharmacophores andhave expanded the scope and treatment of various diseases [1][2][3]. Heterocyclic benzofurans are the basic structural units of biologically active families of natural products such as egonol, homoegonol and moracin [4]. Natural and synthetic furan derivatives exhibit profound physiological and chemotherapeutic potential against a wide variety of pathogens by displaying antimicrobial, antioxidant [5], antitubulin [6], anti-inflammatory [7], antiviral [8], antihyperglycemic [9], analgesic [10,11], anticancer [11,12], antifungal [13] and antipyretic activities [14]. The therapeutic profile of furan-based molecules has attracted medicinal researchers to design and develop anticancer agents which could play a pivotal role in cancer therapy [1,15,16]. In addition to this, oxadiazole and triazole ring systems, which are part of many natural products, have become the focus of pharmacologists and medicinal chemists on account of their medicinal and pharmacology activities, especially in the field of oncology [17,18].
Cancer is a notably complex, prominent and lethal disease which poses a serious human health problem; ithasresulted in 7.6 million deaths globally, and this number is expected to reach 13 million by 2030 [19][20][21]. Naturally occurring benzofuran derivatives such as denthyrsin and HIBE (1-(6-hydroxy-2-isopropenyl-1-benzofuran-5-yl)-1-ethanone) displayed cytotoxic potential against ovarian cancer and human breast cancer cell lines [22,23]. Sulfonamide scaffolds of benzofuran-imidazopyridines showed anticancer activity against ovarian cancer, MCF-7, lung cancer and colon cancer [24]. Benzofuran-oxadiazole hybrids exhibited significant cytotoxicity against pancreatic cancer and colon cancer cell lines in vitro [25]. The 2-aryl benzo furan-appended 4-aminoquinazoline structural motifs significantly inhibited the epidermal growth factor receptortyrosine kinase phosphorylation in vitro against human lung cancer, cervical cancer, colorectal adenocarcinoma and hepatocellular carcinoma cell lines [26]. The benzofuran-carboxylic acid hybrids proved to be the best antiproliferative agents against breast cancer cell lines and significant inhibitors of carbonic anhydrases [27]. Thiazolodin-4-one benzofuran derivatives exhibited remarkable antitumor activity against human HEPG2 cell lines [28]. Different furan-based anticancer agents are listed in Figure 2   Continuing our previous research work towards the synthesis of benzofuran-based oxadiazole and triazole hybrids using a conventional approach [29], here, our group applied green synthetic strategies such as ultrasonic-and microwave-assisted synthetic approaches to achieve environmentally friendly synthesis with maximum production of benzofuranoxadiazole and triazolestructural hybrids over shorter reaction times. The synthesized molecules were further evaluated for their hemolytic, thrombolytic and anticancer potential.

Chemistry
Ultrasonic irradiation and microwave-assisted experiments were performed, respectively, in an ultrasonic cleaner bath (Model 1510, 115 v, 1.9 L) with a mechanical timer and heater switch and at a frequency of 47 kHz, and with microwave apparatus (Model EA-180M) witha frequency of 2450 Hz and power consumption of 1150 watt. Analytical-grade solvents and reagents were used; these were purchased from Merck, Alfa Aesar and Sigma-Aldrich through local suppliers. The reaction progress was monitored using pre-coated silica gel plates. The synthesized scaffolds were purified using the column chromatographic technique and by the process of recrystallization in ethanol and methanol solvents.

Biological Evaluation
Oxadiazole-and triazole-based benzofuran derivatives were evaluated for hemolysis, thrombolysis and anticancer activity.

Hemolysis Assay
A fresh blood sample (5 mL) from a healthy donor was collected in an EDTA tube. The blood was transferred to microcentrifuge tubes and centrifugedfor 5 min at 1000 rpm to obtain the red blood cells (RBCs). The supernatant was then discarded, and the RBCpellet was washed three times with phosphate-buffered saline (PBS). The RBC pellet was collected after washing and 20µL of sample solution in DMSO was added. The tubes were incubated at 37 • C for 60 min. Having been removed from the incubator, the tubes were recentrifuged at 13,000 rpm for 5 min. The supernatant was collected and diluted with chilled PBS solution. Absorbance was recordedat 517 nm. ABTS was used as a positive control, while DMSO was employed as a negative control in this protocol. The experiment wasconducted in triplicate, and the RBC lysis percentage was calculated using this formula [40]: % Age hemolysis = Absorbance of sample − Absorbance of negative control × 100 Absorbance of positive control

Thrombolysis Assay
The thrombolytic assay was performedaccording tomethodologyin the literature [41]. A blood sample (3 mL) was collected from a healthy human donor, and 500 µL was transferred to pre-weighed, clean Eppendorf tubes. The tubes containing the blood wereweighed again andincubatedfor 1 h at 37 • C to induce clot formation. The serum was then discarded, and the tube containing theclot was weighed. An amount of 40 µL of sample solution in DMSO was added to the clot.The tubes were again incubated for 3 h at 37 • C, and the lysis resultswere observed. ABTS was used as a positivecontrol, while DMSO was used as a negative control in this assay. The experiment was performed in triplicate, and the lysis percentage was calculated using the formula below: % Age clot lysis = Initial clot weight − Final clot weight × 100 Initial clot weight

MTT Assay Preparation of Cell Culture
The human lung cancer cell line A549 was cultured in Dulbecco's modified Eagle medium (DMEM), composed of 10% FBS (fetal bovine serum), 100 units/mL of penicillin (1%) and 100 µg/mL of streptomycin (1%), with 5% CO 2 (carbon dioxide) at 37 • C in a moistened atmosphere. The A549 cell line was treated with synthesized compounds in a final concentration of DMSO (dimethyl sulfoxide) of less than 1%.

Determination of Cell Viability
The cytotoxic therapeutic potential of synthesized structural hybrids was evaluated usinga standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The cells of theA549 cell line were grown, overnight, in a microculture of 96-well plates. The A549 cells were supplemented with different concentrations of compounds for 48 h and further incubated with 20 µL of MTT mixture (5 mg/mL) for 4 h at 37 • C. Following this, 150 µL of control DMSO was added to formazan crystals.The percentage of cell viability was determined viaquantified absorbance in a microplate readerat 490 nm wavelength [42].

Molecular Docking Studies
The compound 5d with a higher pharmacological therapeutic potential was subjected to in silico studies to delineate the mechanism of anticancer activity against lung cancer A549cell lines. The molecular docking studies of these synthesized compounds at a targeted protein were carried out using auto dock Vina 1.1.2 software intermitted with PyRx. The runs of docking were achieved with grid box coordinates of −7.65, −28.27 and 43.34 for x, y and z, respectively. Considering the important role of signaling parameters in cancer, receptors of ALK (anaplastic lymphoma kinase) present an established target to evaluate the anticancer therapeutic potential of synthesized compounds [43,44]. To accomplish computational studies against this target, the crystallographic structure of anaplastic lymphoma kinase in conjunctionwith crizotinib having PDB ID: 2XP2 was obtained from the RCSB protein data bank; it was saved in Pdb formatafter the removal of water molecules and the co-crystallization ofthe ligand with the addition of polar hydrogen. The structure of the co-crystallized ligand crizotinib has been nominated as a potential therapeutic moiety against ALK targets;the structure of the synthesized compound 5d was selected because of its marked experimental results. The structures were drawn on Chemdraw 20 professional software (chemoffice) and saved as a mol file [45,46]. Following this, energy minimization and polar hydrogen addition were performed, and structures were saved in Pdb format through Bio via Discovery Studio Visualizer (DSV), as shown in Figure 3. The results in the form of pictures were processed through the latest versions of Pymol and Ligplot plus software.

Statistical Data
The statistical data analysis was performed by using prism. All the measurements were carried out in triplicate, and the results are depicted as mean ±SD. Ultrasound-and microwave-assisted green reactions are environmentally friendly, time saving, economical and good yield methodologies, and were adopted as depicted in Scheme 1. The conventional approach of synthesizing benzofuran-oxadiazole was already reported [29] as affording compounds in moderate to good (36-80%) yields but required a longer period of time to complete the reaction. This study was initiated to increase the yield and reduce the time of synthesized derivatives by applying ultrasoundand microwave-assisted approaches to achieve benzofuran-oxadiazole derivatives. In the ultrasound-and microwave-assisted approaches, the scaffold 5-(benzofuran-2-yl)-1,3,4oxadiazole-2-thiol 3 was coupled with substituted bromoacetanilide derivatives 4a-h to synthesize corresponding S-alkylated products of benzofuran-oxadiazole 5a-g in good to excellent yields, as depicted in Table 1. The ultrasound-assisted strategy yielded products of 60-86% in 30 min at 40 • C, compared withthe conventional method, which afforded products of 36-80% in 24 h. The microwave approach was much more efficient in that the maximum yield of products (69-94%) was obtained in the very short duration of 60 s. The benzofuran-oxadiazole derivative 5d was achieved withthe maximum yield, while the derivative 5f was obtained withthe least yield via ultrasonication and microwave irradiation approaches, as mentioned in Table 1. Benzofuran-triazole derivatives were synthesized via green synthetic methodologies, namelyultrasound-and microwave-assisted protocols. Benzofuran-triazole derivatives (7a-h) were obtained in low to moderate (38-79%) yields in 36 h by utilizing conventional methodology [28], while the yield was increased (60-90%) in the ultrasound-assisted synthetic protocol, in which the scaffold 5-(benzofuran-2-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol 6 was coupled with substituted bromoacetanilide derivatives 4a-h to synthesize corresponding S-alkylated benzofuran-triazole derivatives 7a-h in good to excellent yield at 40 • Cwithin 30 min. Similarly, the microwave approach was even more efficient in that the S-alkylated benzofuran-triazole structural motifs were obtained in maximum yield (68-96%) within the very short duration of 70 s.The benzofuran-triazole derivative 7h was achieved in the maximum yield (90% and 96%) in ultrasound-and microwave-assisted synthetic approaches, while the derivative 7c was obtained in the least yield(60% and 68%) in ultrasound-assisted andmicrowaveirradiation approaches, as shown in Table 2.

Structure-Activity Relationship (SAR)
The structure-activity relationship ofhemolysis, thrombolysis and the anticancer potential of benzofuran-oxadiazole and triazole hybrids can be interpreted on the basis of substitution patterns present on the phenyl ring of N-(substituted-phenyl)-acetamide. The electron-donating ethoxy group atthe para position of the phenyl ring in benzofuranoxadiazole compound 5e showed 0.5% toxicity against RBCs, whereas benzofuran-triazole derivative 7b exhibited 0.1% toxicity, which was significantly lower than the other derivatives. On the other hand, compounds 5b and 7g (22.12% and 23.4%) proved to be the most toxic compounds among the benzofuran-oxadiazole and benzofuran-triazole series, respectively (Figure 4a). In the case of thrombolytic activity, the benzofuran-oxadiazole scaffold 5a with unsubstituted phenyl displayed better thrombolytic potential (56.8%) compared withdiethyl amine moiety containing the 5g benzofuran-oxadiazole hybrid. The phenyl group increased the thrombolytic potential of 5a, while the diethylamine resulted in a decrease in thrombolytic activity (48.3%) of the 5g hybrid. The benzofuran-triazole hybrid 7f was the most active thrombolytic agent among all the screened derivatives of benzofuranoxadiazole and benzofuran-triazole, displaying the highest thrombolysis potential (61.4%) due to the presence of two methyl groups adjacently attached on the phenyl ring at ortho para positions. The benzofuran derivative 7h showed the least thrombolytic potential (48.1%) compared with all thetested compounds. The thrombolysis activity of 7h was significantly decreased due to the presence of two chloro EWD groups adjacently attached on the phenyl ring at the ortho and para positions, as depicted in Figure 4b.
The benzofuran-oxadiazole derivative 5d exhibited excellent anticancer activity (cell viability 27.49 ± 1.90% and IC 50 6.3 ± 0.7 µM) compared withall the screened derivatives, due to the presence of a methoxy group at the meta position on the phenyl ring;because of the presence of the ethoxy group at the para position on the phenyl ring, however, the benzofuran-oxadiazole derivative 5e displayedmoderate anticancer activity (cell viability 34.47 ± 2.19% and IC 50 17.9 ± 0.46 µM) in comparison with the reference drugs crizotinib and cisplatin (28.22 ± 3.88 and 15.34 ± 2.98 µM), respectively. The structural hybrid 5d is more potent than crizitonib and showed less cytotoxic potential than cisplatin. Due to the presence of two chloro EWD groups adjacently attached on the phenyl ring at the ortho and para positions, benzofuran-triazole scaffold 7h exhibited slightly less potency (cell viability 29.29 ± 3.98% and IC 50 10.9 ± 0.94 µM) against lung cancer cell line A549 compared with 5d scaffold, which was the most active compound among all the screened derivatives. The benzofuran-triazole hybrid 7f was an inactive compound (cell viability 99.1%) against the A549 cell line due to the presence of two methyl groups adjacently attached on the phenyl ring at the ortho and para positions (Figure 4c).

Computational Modeling Studies of the Most Active Compound 5d
The synthesized compound 5d (B) and co-crystallized ligand crizotinib were docked against the anaplastic lymphoma kinase (ALK) receptors to understand better the mechanisms of attachment and inhibition. The compound-docked conformations were interpreted by binding interactions, energies, polar bonding and ligand-receptor binding.
The molecular comprehension of docking experimentssuggest that compound 5d (B) is more prominent as it produces excellent results with binding energies of −9.925 Kcal/mol in the best binding mode. This exceeds the average energy threshold of −8.985 Kcal/mol given by crizotinib, thereby commending the improved affinity of synthesized compound 5d (B) against the ALK receptor (Table 4). Crizotinib was found to have noticeable contacts with these active site residues, but compound 5d (B) displayed more effective binding at these active amino acid AA residues, representing more effective working of this synthesized chemical moiety. The binding of compound 5d (B) and crizotinib with receptors are elaborated in Figure 5. The phenyl and heterocyclic rings of compound 5d (B) showed π-sigma interaction with LEU A: 1256 and VAL A: 1130, while the NH group gave conventional hydrogen bonds with GLY A: 1201. Another prominent interaction appeared with GLU A: 1210 through π-anion linkage.  Evaluation through docking studies provides good evidence that synthesized compound 5d presents excellent interaction inside the binding pocket of a receptor, showing its better anticancer potential as compared withthe co-crystallized ligand crizotinib. These computational studies are reliable and appropriate for performing binding interaction prediction. The conformational analysis revealed thespatial arrangement of compound 5d, justifying it to be more potent, in comparison with crizotinib. Interestingly, compound 5d interactions were more realistic, with better results. Therefore, scaffold 5d would be a promising anticancer agent on the basis of both cytotoxicity and in silico studies.

Conclusions
The target S-alkylated structural motifs of benzofuran-oxadiazole and -triazole were synthesized in good to excellent yields by utilizing ultrasound-and microwave-assisted green synthetic protocols. The compounds were evaluated for anticancer, hemolytic and thrombolytic activities, and it wasrevealed that most of tested derivatives displayed significant hemolytic, thrombolytic and anticancer activities. The hemolysis assay indicated that benzofuran-triazole derivative 7b and benzofuran-oxadiazole 5e proved to be the least toxic compounds among all the synthesized derivatives. The benzofuran-oxadiazole hybrid 5d with cell viability 27.49 ± 1.90 and IC 50 (6.3 ± 0.7 µM) values was recognized as the most potent anticancer candidate against lung cancer cell line A549 compared with the reference drugs crizotinib (IC 50 8.54 ± 0.84 µM) and cisplatin with (IC 50 3.88 ± 0.76 µM). The docking studies explored the mode of action and inhibition mechanism of compound 5d againstcancer cells; the results of the comprehensive docking analysis of compound 5d are consistent and in agreement with biological diagnostic findings. Overall, the current studies suggest that benzofuran-linked oxadiazole and triazole hybrids are capable of being established as lead compounds in cancer therapy; in particular, scaffold 5d could be a promising anticancer agent on the basis of cytotoxicity andin silico studies. The SAR studies suggested that more modifications to oxadiazole and triazole derivatives of benzofuran may lead to advanced anticancer candidates in cancer therapy.