Design, Synthesis, Molecular Docking, ADME and Biological Evaluation Studies of Some New 1,3,4-oxadiazole Linked Benzimidazoles as Anticancer Agents and Aromatase Inhibitors

In this study, due to the potential anticancer effects of the benzimidazole ring system, a series of benzimidazole-1,3,4-oxadiazole derivatives were synthesized and characterized by 1 H NMR, 13 C NMR, and MS spectra analyses. In the in vitro anticancer assay, all the compounds tested anticancer activities using MTT-based assay against ve cancer cell lines (MCF-7, A549, HeLa, C6, and HepG2). Among them, compound 5a exhibited the most potent activity with IC 50 values of 5,165±0,211 μM and 5,995±0,264 μM against MCF-7 and HepG2 cell lines. Compound 5a was included in the BrdU test to determine the DNA synthesis inhibition effects for both cell types. Furthermore, compound 5c was also found to be more effective than doxorubicin on the HeLa cell line. The selectivity of anticancer activity was evaluated in NIH3T3 (mouse embryo broblast cell line) cell line. In vitro, enzymatic inhibition assays of aromatase enzyme were performed for compound 5a acting on the MCF-7 cell line. For compound 5a, in silico molecular docking against aromatase enzyme was performed to determine possible protein-ligand interactions and binding modes.


Introduction
Cancer is characterized as the uncontrolled growth of abnormal cells anywhere in the body, is the second leading cause of death worldwide after cardiac disease [1][2][3][4]. There are more than a hundred drugs for the cure of cancer. However, drug resistance, side effects, low selectivity, and severe toxicity are the major disadvantages of current drugs. Therefore, there is an urgent need to develop new anticancer drugs with great e ciency and high speci city [5][6][7].
Estrogen is biosynthesized from androgens by a cytochrome P450 aromatase which has been implicated in numerous diseases including breast cancers. Overexpression of aromatase has been noted in breast cancer cases. Therefore, aromatase is the target enzyme for the treatment of hormone-dependent breast cancer [8][9][10]. Third-generation aromatase inhibitors (non-steroidal aromatase inhibitors) among aromatase inhibitors, which are divided into three classes according to their clinical development, are used as rst-line therapy in the treatment of breast cancer in both early and advanced tumors (Figure 1a) [11].
In the eld of drug discovery, especially in cancer research, nitrogen-containing heterocyclic rings are extensively investigated [5].
Benzimidazole, is an important pharmacophore and a privileged structure in medicinal chemistry, especially for anti-cancer activity. It is observed that hydrogen bond donor and acceptor sites, that is, N1 and N3 in the benzimidazole core, play a critical role in binding to the biological targets [12]. In recent studies, potential anticancer activity has been noted in compounds to which benzimidazole is linked by other heterocyclic rings [13][14][15][16]. The benzimidazole ring has been applied to various marketed anticancer drugs such as bendamustine, veliparip, carbendazim, and nocodazole ( Figure 1b).
Based on the above a rmative design aspects, as shown in Figure 2, we have synthesized a new series of benzimidazole linked 1,3,4-oxadiazole derivatives which were later con rmed by 1 H NMR, 13 C NMR, and mass spectral techniques. All of the synthesized compounds (5a-5f) were tested for their anti-cancer activity against ve different cancer lines including A549, MCF-7, HeLa, HepG2, and C6. Moreover, the most active compound against the MCF-7 cell line was screened for inhibition of aromatase enzyme and DNA synthesis inhibition. Then, the in silico aromatase inhibitory activity was performed for compound 5a acting on the MCF-7 cell line.
Compounds (2a-2c) (0.018 mol) and excess of hydrazine hydrate (5 mL) were placed in the same vial and ethanol (15 mL) was added. The mixture was treated under microwave irradiation under the same reaction conditions. When the reaction was completed, the mixture was poured into iced water, the product was ltered [15].

Anticancer Activity
The anticancer activity of compounds 5a-5f was screened according to the MTT assays. The MTT assays were performed as previously described [19][20][21]. Anticancer activity of nal compounds was assessed against ve different cancer cell lines A549

Aromatase Inhibition Assay
This method was carried out according to the kit procedure (BioVision, Aromatase (CYP19A) Inhibitor Screening Kit (Fluorometric). The aromatase inhibition assay was performed as previously described [22][23].

DNA Synthesis Inhibition Assay
The BrdU cell proliferation method was performed to analyze the effects of the active compounds on the proliferation of cancer cells as previously studied [15].

Molecular Docking Analysis
The human placental aromatase cytochrome P450 protein structure was imported into UCSF Chimera 1.15 software with PDB ID: 3QEM code (https://www.rcsb.org/structure/3EQM) [24]. Heteroatoms other than HEM in the protein crystal structure have been removed. 2D ligand structures were drawn with ChemDraw Professional 17.0 software and minimized 3D structures were created using a universal force eld. Protein and ligand structures were converted to pdbqt le format with PyRx 0.8 software. Based on the ASD ligand in the protein crystal structure, active site coordinates were determined as x:85.79, y:54. 14

Chemistry
The synthesis of the target compounds (5a-5f) was shown in Figure 3. In the rst step, the compounds 1a-1c derivatives were obtained by heating 4-substituted benzaldehyde with 3,4-diamino benzoic acid in DMF and sodium bisul te. The compounds (1a-1c) were converted to a methyl ester (2a-2c) by a simple esteri cation reaction. Then, the appropriate solution of compounds 2a-2c in ethanol (95%) was treated with hydrazine hydrate to prepare compounds 3a-3c. The reaction of hydrazide derivatives (3a-3c) with carbon disul de in ethanolic potassium hydroxide gave the compounds 4a-4c. At the last reaction step, the compounds 4a-4c were reacted with acetylated piperazine derivatives in acetone to produce target compounds 5a-5f. The structures of newly synthesized compounds (5a-5f) were characterized by using various modern analytical techniques like 1 H NMR, 13 C NMR, and HRMS. 1 H NMR spectral analysis of the compounds 5a-5f demonstrated that the S-CH 2 (methylene) protons were signaled between δ 4.54-4.63 ppm, as a singlet. The protons of piperazine moiety are seen as multiplet at 2.97-3.70 ppm. In the 1 H NMR spectrum of the compounds 5c and 5d carrying 4-methoxyphenyl group in the second position of the benzimidazole ring, the protons of the methoxy substituent gave a singlet peak at 3.42-3.93 ppm. The -OC 2 H 5 group of compounds 5e and 5f on the phenyl ring, -OCH 2 protons were observed at 4.10-4.11 ppm and CH 3 protons were observed at 1.35-1.37 ppm. The benzimidazole proton was visualized in the form of doublet's doublet in 1 H NMR spectra at around 7.58-7.99, due to H-5 proton. In the 13

Anticancer Activity
In this study, all compounds were screened for their antiproliferative activity against MCF-7 (human breast adenocarcinoma cell line), A549 (lung carcinoma cell line), HepG2 (human liver carcinoma cell line) HeLa (cervical cell line), and C6 (rat glioma cell line) cell lines as well as NIH3T3 (mouse embryo broblast cell line) using MTT assay where doxorubicin was used as reference.
Results summarized in Table 1 were expressed as the mean IC 50 (half maximal inhibitory concentration) of four independent experiments.
To determine the side effects of compounds 5a and 5c, which are effective on cancer cell lines, their cytotoxic effects on the NIH3T3 cell line were investigated (Table 1).

Aromatase Inhibition Assay
The human aromatase inhibitory activity of the synthesized compounds was determined by using an in vitro uorescence-based assay [Aromatase (CYP19A) Inhibitor Screening Kit (Fluorimetric) BioVision]. The target compound was dissolved in acetonitrile, and the results were compared to letrozole, used as the reference compound. According to the result, compound 5a causes 50% aromatase enzyme inhibition effectively at 2,314±0,103 µM (Table 1).

DNA Synthesis Inhibition Assay
According to the MTT assay, compound 5a for MCF7 and HepG2 cell lines were selected for the DNA synthesis inhibition assay.
MCF-7 and HepG2 cells were incubated with three different concentrations (2xIC 50 , IC 50 , and IC 50/2 ) of the compounds for 24 and 48 h periods. The tested compounds showed time-and dose-dependent inhibitory activity on DNA synthesis of the tumor cells.
Doxorubicin was used as the positive control. Figure 4 shows the DNA % synthesis inhibitory activity of the compound 5a and standard drug doxorubicin on MCF7 cells. Compound 5a was found to have 92.44, 82.12, and 75.48 % DNA synthesis inhibition after 24 h of incubation whereas the same compound was found to have 44.63, 55.71, and 50.54 % DNA synthesis inhibition after 48 h. Figure 5 shows the DNA % synthesis inhibitory activity of compounds 5a and standard drug on HepG2 cells. DNA % inhibition was increased with the increasing incubation period (24h and 48 h).

Molecular Docking Analysis
Molecular docking analyzes are frequently used in drug design and development stages. It is useful in predicting how the protein and ligand interact at the atomic level [17,18]. In this study, the interaction of compound 5a compound, which is the most active derivative, with the aromatase enzyme was investigated by the molecular docking method. The suitability of the protein crystal structure (PDB ID: 3EQM, resolution: 2.90 Å) bond angles between the atoms was determined by creating a Ramachandran Plot through the PROCHECK server (https://servicesn.mbi.ucla.edu/PROCHECK/), and it was observed that 98.2% of the amino acid angles were in the allowed region. Validation of the molecular docking process is important for the success of the study.
Accordingly, the ASD compound, which is the natural ligand of the 3EQM crystal structure, was removed from the area where it was located and re-docked to the active site. The RMSD was measured as 0.46 Å between the crystalline ASD and the docked ASD, and it was concluded that the docking process was successful and made accurate predictions. Then, the synthesized compounds were molecular docking to the active site. As shown in Figure 6, compound 5a is positioned right next to the HEM structure. As given in Table 2, the compound 5a human placental aromatase enzyme formed hydrogen bonding with residues Arg115, Leu372, Leu477, and Val369 and hydrophobic interactions with some other amino acids at its active site.

ADME Prediction
Many drug molecule candidates remain in phase studies without a drug molecule due to poor ADME properties. Making some theoretical ADME calculations of the designed and newly synthesized compounds can provide convenience in advanced in vitro and in vivo studies of the compound. Therefore, some properties of the designed compounds such as physiochemical, lipophilicity, water-solubility, pharmacokinetics, drug-likeness, and medicinal chemistry properties were calculated using SwissADME online tools, and some properties most active compound 5a was given in Table 3. The molecular weight of compound 5a is slightly greater than 500 (518.63 g/mol). The logP value is less than 5 in all lipophilicity calculations. Its solubility in water is between moderate and poor. Its gastrointestinal absorption was calculated as low and also not able to pass through the blood-brain barrier. It has the potential to inhibit Cyp enzymes, that is, to interact with other drug molecules. While druglikeness is low according to Ghose and Egan, it is appropriate according to the restrictive rules of Lipinski, Veber, and Muegge. The leadlikeness property shows two deviations from the shortening rules. Other medicinal chemistry parameters are suitable.

Conclusion
In conclusion, we have prepared some new novel benzimidazole-1,3,4-oxadiazole derivatives and their cytotoxicity pro le were evaluated on A549, MCF-7, C6, HepG2, and HeLa cell lines. Among these derivatives, compound 5a showed potent anticancer activity against MCF7 (5,165±0,211 µM) and HepG2 (5,995±0,264 µM) cell lines. Compound 5c (7,316±0,276 µM) showed the most anticancer activity against the HeLa cell line. Furthermore, compound 5c (17,193±0,784 µM) showed anticancer activity against HepG2 cell line similar to doxorubicin. Compound 5a tested against human aromatase in an in vitro uorescence enzymatic assay. Further detailed biological studies including DNA synthesis inhibition assay and aromatase inhibition studies delivered promising results. Molecular docking experiments returned for compound 5a novel compound a binding mode comparable to letrozole. On the other hand, drug target prediction studies con rmed the role of compound 5a for aromatase activity inhibition and open new perspectives for further studies.

Declarations
Con ict of interest statement: The authors declared no con ict of interest.  Synthesis pathway of the designed compounds 5a-5f.