Synthesis of novel thiourea - /urea - benzimidazole derivatives as anticancer agents

: A new series of urea and thiourea derivatives containing benzimidazole group as potential anticancer agents have been designed and synthesized. The struc tures of the synthesized compounds were characterized and con ﬁ rmed by spectroscopic techniques such as 1 H NMR, 13 C NMR, and mass spectrometry. In vitro anti cancer assay against two breast cancer ( BC ) cell lines, MDA - MB - 231 ER ( − ) /PR ( − ) and MCF - 7 ER (+) /PR (+) , revealed that the cytotoxicity of 1 -( 2 -( 1 H - benzo [ d ] imidazol - 2 - yla mino ) ethyl )- 3 - p - tolylthiourea ( 7b ) and 4 -( 1 H - benzo [ d ] imidazol - 2 - yl )- N -( 3 - chlorophenyl ) piperazine - 1 - carbox amide ( 5d ) were higher in MCF - 7 with IC 50 values of 25.8 and 48.3 µM, respectively, as compared with MDA MB - 231 cells. Furthermore, 7b and 5d were assessed for their apoptotic potential using 4 ′ ,6 - diamidino - 2 - phenylin -dole, acridine orange/ethidium bromide staining, and Caspase - 3/7. After incubation with MCF - 7, the com pounds 7b and 5d induced apoptosis through caspase - 3/7 activation. In conclusion, the compounds 7b and 5d are potential candidates for inducing apoptosis in di ﬀ erent geno typic BC cell

According to the World Health Organization (WHO), the number of people diagnosed with cancer increased from 10 million in 2000 to 19.3 million in 2020. Breast cancer (BC) is the world's most diagnosed cancer as reported by International Agency for Research on Cancer (IARC) in 2020 (WHO, 2021) with metastasis and drug resistance being the main challenge for successful treatments [31]. Moreover, the drug resistance of cancer cells exerts more pressure to look for new effective chemotherapeutic agents. BC is subtyped or classified based on their genotypic differences and metastasis characteristics. The toxicity of the drug against different types of BC cells differs significantly due to variation in the metabolic outcome of their genotype [32,33].
Apoptosis is an important biological event that takes place in multicellular organisms to remove unwanted or damaged cells [34]. Apoptosis has been extensively used for therapeutic applications and biological studies and huge effort has been dedicated to the discovery of apoptosis-inducing molecules that may have antitumor potential [35].

Light microscopy
To investigate the effects of active compounds on the morphology of MCF-7, the cells were seeded in DMEM. The cells were treated with 2 × IC 50 value for 24 h, and imaged using a light microscope (Leica, Germany) at 200× magnification.
2.5 4′,6-Diamidino-2-phenylindole (DAPI) staining assay DAPI staining was carried according to ref. [39] with modifications. Cells were cultured in a 24-well culture  plate (Nist, China) for 24 h. After incubation with 2 × IC 50 of 7b and 5d for 24 h, cells were washed with PBS, fixed with ice cold methanol for 5 min and then were again washed with PBS. After washing, the cells were stained with DAPI (1 μg mL −1 ) and incubated in dark for 30 min.
Methanol was used as a control. The cells were photographed using a fluorescence microscope (Evos, USA).

Caspases-3/7
The assessment of caspase-3/7 was carried out based on the manufacturer's manual. Briefly, the cells were cultured in a 24-well culture plate (Nist, China) for 24 h. The cells were treated as described in the Section 2.5. After treatment, 1 μg mL −1 of caspase-3/7 reagent (Invitrogen, USA) was added and shaken for 30 s and incubated in dark for 30 min. Methanol was used as a control. The cells were photographed using a fluorescence microscope.

Results and discussion
The synthesis of thiourea and urea derivatives of benzimidazole is outlined in Scheme 1. The synthesis of target thiourea 4a-j and urea 5b-e was achieved by reacting 2-(piperazin-1-yl)-1H-benzo[d]imidazole hydrochloride 1 [30,31] with substituted isothiocyanates 2a-j and isocyanates 3b-e, respectively, in the presence of an excess of triethylamine to obtain good yields. Similarly, two derivatives thiourea 7b and urea 8b were prepared by reacting N1-(1H-benzo[d]imidazol-2yl)ethane-1,2-diamine hydrochloride 6 [38] with isothiocyanates 2b and isocyanates 3b, respectively. The purity of the prepared compound is confirmed by recrystallization, its melting point, chromatographic technique, and spectroscopy data. The newly synthesized compounds (4a-j, 7b) and (5b-e, 8b) were characterized by MS, 1 H and 13 C NMR, and IR spectral data. These data, detailed in the experimental part, are consistent with the suggested structures. The 13 C NMR data revealed the presence of a signal between 181.1 and 182.7 ppm for compounds (4a-j, 7b) and between 155.3 and 157.5 ppm for compounds (5b-e, 8b). This signal corresponds to quaternary carbons C]S and C]O, respectively. In the IR spectra, absorption bands associated with C]S and C]O stretching vibrations are appearing between 1,523 and 1,596 cm −1 for compounds (4a-j, 7b) and between 1,634 and 1,642 cm −1 for compounds (5b-e, 8b), respectively.
The HRMS of the synthesized compounds are in good agreement with the calculated values.
The diversity of the proposed compounds in Scheme 1 is evident from the different functional groups (urea and thiourea), substitution on the phenyl ring including activating and deactivating groups, and also from using the piperazine and ethylenediamine as spacers. These structure variations can help investigating the structureactivity relationship.
The newly synthesized thiourea 4a-j and urea 5b-e were investigated for cytotoxicity using MTT cell viability assay in two human BC cell lines, MCF-7 and MDA-MB-  Figures 2 and 3, only the compounds 7b and 5d were cytotoxic to the tested cancer cell lines. For the remaining compounds, the IC 50 values were not calculated because they were higher than 1,000 µg mL −1 . However, the cytotoxicity of compounds 7b and 5d was higher in MCF-7 with IC 50 values of 25.8 and 48.3 µM, respectively, as compared with their activity on MDA-MB-231 cells. The IC 50 values for 7b and 5d compounds were 54.3 and 89.5 µM, respectively, on MDA-MB-231 cells ( Table 1).

As shown in
Morphological alternations in the cells were observed using phase contrast microscope after incubation with 7b and 5d for 24 h. Cells in the control group retained the morphological form, most of which were adherent to the surface, tightly packed, and distinctively monolayer. Exposure of the BC cells to 7b and 5d compounds led to rounding, shrinkage of the cells, loss of contact, and debris formation (Figures 2b and c and 3b and c).
The potential of inducing apoptosis by the 7b and 5d was assessed by DAPI staining. The results indicated that the number of apoptotic cells was higher in 7band 5dtreated cells than DMSO control. The changes that took place in MCF-7 cells as a result of 7b and 5d treatments are represented in Figures 4a and b and 5a and b. The treatment resulted in chromatin condensation and nuclear fragmentation which are apoptotic indicators. The apoptotic morphology was detected by AO/EB fluorescent staining of MCF-7 cells incubated with 7b and 5d compounds. As evident in Figures 4c and d and  5c and d, nuclei stained with green color demonstrate live cells, while greenish yellow and orange-red nuclei show apoptotic cells. The red color shows dead cells. The negative control (untreated cells) cells appeared bright green.
In apoptotic cells, caspase-3/7 activity cleaves the substrate, releasing a DNA dye, which moves to the nucleus and binds with DNA that gives shiny green fluorescence. The treated cells (Figures 4e and f and 5e and f) showed an increase in the caspase-3/7 activity after 24 h of exposure to 7b and 5d, indicating that the apoptosis induced is caspase-3/7 dependent.
Apoptosis is a highly organized physiological mechanism to destroy abnormal cells [40] and it is an attractive endpoint in anticancer agents' research. It is induced by many effective anticancer agents [41]. A variety of compounds have been known to induce apoptosis in various human cancer cells. So it is important to screen for apoptotic inducers from synthetic anticancer compounds. There are many ways of detecting apoptosis. Detection of apoptosis using fluorescence microscope has many advantages over other methods because it does not include an enzymatic reaction that is affected by many variables such as pH, temperature, and composition buffers [42]. Single and double staining methods for detecting apoptosis (AO/EB), produce reliable and reproducible results. The results of the apoptotic cell identified by the AO/EB method are highly reproducible [43].
Induction of apoptosis is often linked to the activation of caspases. Incubation of MCF-7 cells with 7band 5d-induced caspase-3/7 activity is evident by a bright green fluorescence in treated cells after 24 h. Thus, apoptosis induced by 7b and 5d was caspase dependent. Both pathways, intrinsic and extrinsic pathways, activate downstream effector caspases mainly caspase-3 and caspase-7 [44]. Apoptosis is the main target in treatment strategies of cancer. Anticancer compounds that induce apoptosis are promising candidate for cancer therapy. Further studies are required to assess other pathways for apoptosis. These data provide a scientific foundation for the discovery and development of novel anticancer compounds from benzimidazole thiourea and benzimidazole urea derivatives.

Conclusion
In summary, we have designed and synthesized a series of 16 novel compounds (4a-j), (5b-e), 7b, and 8b that contain benzimidazole, piperazine, ethylenediamine, and urea or thiourea moieties. These compounds were characterized by using suitable spectroscopic techniques including 1 H and 13 C NMR, IR, and high-resolution mass spectrometry. In vitro anticancer assay against breast cancer cell lines, MCF-7 and MDA-MB-231, revealed that the cytotoxicity of compounds 7b and 5d were higher in MCF7 with IC 50 values of 25.8 and 48.3 µM, respectively, as compared with their activity in MDA-MB-231 cells. These results could be a lead to prepare new benzimidazole derivatives with potent anticancer activity. Author contributions: Lamia Ali Siddig carried out the synthesis experiments and characterization. Abdelouahid Samadi carried out the spectral analysis and characterization. Mohammad A Khasawneh and Haythem A. Saadeh conceived and planned the synthesis. Nael Abutaha and Mohammad A. Wadaan conceived, planned, and carried out the experiments in bioassay. Haythem A. Saadeh was in charge of overall direction and planning, and wrote the manuscript with input from all authors. All authors have read and agreed to publish the manuscript.

Conflict of interest:
The authors declare no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: All data are included in this published article.