Benzimidazole-Based Derivatives as Apoptotic Antiproliferative Agents: Design, Synthesis, Docking, and Mechanistic Studies

A new class of benzimidazole-based derivatives (4a–j, 5, and 6) with potential dual inhibition of EGFR and BRAFV600E has been developed. The newly synthesized compounds were submitted for testing for antiproliferative activity against the NCI-60 cell line. All newly synthesized compounds 4a–j, 5, and 6 were selected for testing against a panel of sixty cancer cell lines at a single concentration of 10 µM. Some compounds tested demonstrated remarkable antiproliferative activity against the cell lines tested. Compounds 4c, 4e, and 4g were chosen for five-dose testing against 60 human tumor cell lines. Compound 4c demonstrated strong selectivity against the leukemia subpanel, with a selectivity ratio of 5.96 at the GI50 level. The most effective in vitro anti-cancer assay derivatives (4c, 4d, 4e, 4g, and 4h) were tested for EGFR and BRAFV600E inhibition as potential targets for antiproliferative action. The results revealed that compounds 4c and 4e have significant antiproliferative activity as dual EGFR/BRAFV600E inhibitors. Compounds 4c and 4e induced apoptosis by increasing caspase-3, caspase-8, and Bax levels while decreasing the anti-apoptotic Bcl2 protein. Moreover, molecular docking studies confirmed the potential of compounds 4c and 4e to act as dual EGFR/BRAFV600E inhibitors.


Introduction
Cancer has become a global burden due to the significant increase in cancer incidence and mortality rates [1,2].Tumor resistance, drug toxicity, cancer recurrence, and the inadequate success rate of drug development reaching clinical trials are all limiting variables that exacerbate the difficulties in cancer treatment [3,4].The search for novel classes of anti-cancer medications focuses on enhancing cancer patients' treatment efficacy and survival rates.The usual "one-size-fits-all" approach of standard non-targeting medicines damages healthy cells and may not benefit all patients [5,6].Because cancer is a major focus of the precision medicine program, customized therapy approaches targeted at optimizing outcomes based on individual variability in genetic profile, lifestyle, and environmental factors are gaining popularity [7].As a result, targeted therapy is the cornerstone for precision medicine, allowing for individualized treatment targeting specific tumor oncogenic markers.
Due to their different biological properties and clinical uses, various benzimidazole compounds have recently attracted attention in anti-cancer agent research [8].Because of its unique core structure and low toxicity, benzimidazole is an ideal scaffold in the development of anti-cancer drugs [9,10].Benzimidazole is a bioactive heterocyclic molecule that is one of the top ten most commonly used five-membered nitrogen heterocycles in US Food and Drug Administration (FDA)-approved medications [11].The electron-rich nitrogen heterocycles of benzimidazole may rapidly receive or donate protons and easily facilitate the creation of different weak contacts, giving it an advantage in binding with a wide range of therapeutic targets and demonstrating broad-ranging pharmacological effects [12][13][14].A broad pharmacological profile of benzimidazole and its derivatives, substitution at the 1, 2, 5, and/or 6 positions, has been documented in numerous categories of medicinal agents with distinct features, with anti-cancer activity topping the list [8,15].
Kinases are enzymes that mediate the signaling cascade and regulate molecular functions and biological processes like growth, proliferation, differentiation, and apoptosis.The human kinome has roughly 535 protein kinases, which can be divided into tyrosine, serine/threonine, and tyrosine kinase-like enzymes [18,19].Tyrosine kinases (TKs) include both receptor tyrosine kinases (RTKs) (such as EGFR, FGFR, and PDGFR) and non-receptor tyrosine kinases (NRTKs) (such as ABL, FBK, and SRC).Serine/threonine kinases include BRAF/MEK/ERK, CDKs, and PI3K/AKT/mTOR [20].A deficiency in cellular kinase phosphorylation can eventually lead to constitutive activation of kinases in live cells, promoting the development of malignancy and tumor progression [18,21].The benzimidazole scaffold is commonly used as a template for synthesizing kinase inhibitors, as shown in Figure 1.
Molecules 2024, 28, x FOR PEER REVIEW 2 of 28 focus of the precision medicine program, customized therapy approaches targeted at optimizing outcomes based on individual variability in genetic profile, lifestyle, and environmental factors are gaining popularity [7].As a result, targeted therapy is the cornerstone for precision medicine, allowing for individualized treatment targeting specific tumor oncogenic markers.Due to their different biological properties and clinical uses, various benzimidazole compounds have recently attracted attention in anti-cancer agent research [8].Because of its unique core structure and low toxicity, benzimidazole is an ideal scaffold in the development of anti-cancer drugs [9,10].Benzimidazole is a bioactive heterocyclic molecule that is one of the top ten most commonly used five-membered nitrogen heterocycles in US Food and Drug Administration (FDA)-approved medications [11].The electron-rich nitrogen heterocycles of benzimidazole may rapidly receive or donate protons and easily facilitate the creation of different weak contacts, giving it an advantage in binding with a wide range of therapeutic targets and demonstrating broad-ranging pharmacological effects [12][13][14].A broad pharmacological profile of benzimidazole and its derivatives, substitution at the 1, 2, 5, and/or 6 positions, has been documented in numerous categories of medicinal agents with distinct features, with anti-cancer activity topping the list [8,15].
Kinases are enzymes that mediate the signaling cascade and regulate molecular functions and biological processes like growth, proliferation, differentiation, and apoptosis.The human kinome has roughly 535 protein kinases, which can be divided into tyrosine, serine/threonine, and tyrosine kinase-like enzymes [18,19].Tyrosine kinases (TKs) include both receptor tyrosine kinases (RTKs) (such as EGFR, FGFR, and PDGFR) and non-receptor tyrosine kinases (NRTKs) (such as ABL, FBK, and SRC).Serine/threonine kinases include BRAF/MEK/ERK, CDKs, and PI3K/AKT/mTOR [20].A deficiency in cellular kinase phosphorylation can eventually lead to constitutive activation of kinases in live cells, promoting the development of malignancy and tumor progression [18,21].The benzimidazole scaffold is commonly used as a template for synthesizing kinase inhibitors, as shown in Figure 1.Nazartinib (1, Figure 1) is an irreversible, third-generation EGFR TKI that targets EGFRactivating mutations [22,23].Nazartinib contains dimethylamino crotonamide, which is known as the optimal group for several covalent pan-EGFR inhibitors, as well as a racemic 3substituted azepane linker and a chloro substituent at the benzene ring of the benzimidazole nucleus, which contributes to its improved solubility, oral bioavailability, selectivity, and high affinity for EFGR [24,25].In xenograft models, nazartinib inhibits EGFR signaling and the MAPK pathway, inducing cell cycle arrest, apoptosis, and tumor regression [26].Nazartinib is now being tested in a phase I/II clinical trial in patients with EGFR-mutant non-small cell lung cancer [22].
Ramurthy and colleagues reported the anti-BRAF V600E activity of two benzimidazolebased compounds, 2 and 3 (Figure 1), with IC 50 values of 0.002 and 0.014 mmol/L, respectively [27].The rapidly accelerated fibrosarcoma (RAF) oncogene is associated with cancer cellular processes such as proliferation, invasion, metastasis, and cell survival.Both 2 and 3 can decrease the proliferation of SK-MEL-28 melanoma cells containing B-RAF V600E proteins [27].Ramurthy and colleagues demonstrated that including a methyl group at the NH of benzimidazole increases the binding affinity to RAF kinase compared to the unmethylated homolog.
Recently [10], we reported on the design and synthesis of a novel series of 1,3,4oxadiazole/benzimidazole chalcones as antiproliferative agents inducing apoptosis with dual inhibitory effect against EGFR and BRAF V600E .Compound 4 (Figure 1) was shown to be the most potent antiproliferative agent, with a GI 50 value of 1.20 µM against four cancer cell lines, when compared to the reference doxorubicin, which had a GI 50 value of 1.10 µM.Compound 4 was tested for inhibitory efficacy against EGFR and BRAF V600E as prospective antiproliferative targets.Compound 4 inhibited both targets significantly, with IC 50 values of 0.55 ± 0.10 µM and 1.70 ± 0.20 µM, respectively, when compared to the reference erlotinib IC 50 values of 0.08 ± 0.01 and 0.06 ± 0.01 µM, respectively.
Motivated by the promising anti-cancer activity of some benzimidazole-based derivatives and as part of our ongoing effort to develop a dual or multi-targeted anti-cancer agent [28][29][30][31][32][33], we present here the design, synthesis, and antiproliferative activity of a novel series of benzimidazole-based derivatives (4a-j, 5, and 6, Figure 2).NCI chose all newly synthesized compounds for an in vitro one-dose inhibitory experiment against a panel of 60 cancer cell lines.The most active compounds were chosen for five-dose experiments and mechanistic studies against two potential targets, EGFR and BRAFV 600E .Furthermore, the most effective derivatives were tested for apoptosis-inducing activity against caspase-3, caspase-8, Bax, and anti-apoptotic Bcl2.Finally, docking analyses were undertaken for the most potent derivatives to evaluate their binding mechanisms and interactions with the selected receptors.
Nazartinib (1, Figure 1) is an irreversible, third-generation EGFR TKI that targets EGFR-activating mutations [22,23].Nazartinib contains dimethylamino crotonamide, which is known as the optimal group for several covalent pan-EGFR inhibitors, as well as a racemic 3-substituted azepane linker and a chloro substituent at the benzene ring of the benzimidazole nucleus, which contributes to its improved solubility, oral bioavailability, selectivity, and high affinity for EFGR [24,25].In xenograft models, nazartinib inhibits EGFR signaling and the MAPK pathway, inducing cell cycle arrest, apoptosis, and tumor regression [26].Nazartinib is now being tested in a phase I/II clinical trial in patients with EGFR-mutant non-small cell lung cancer [22].
Ramurthy and colleagues reported the anti-BRAF V600E activity of two benzimidazolebased compounds, 2 and 3 (Figure 1), with IC50 values of 0.002 and 0.014 mmol/L, respectively [27].The rapidly accelerated fibrosarcoma (RAF) oncogene is associated with cancer cellular processes such as proliferation, invasion, metastasis, and cell survival.Both 2 and 3 can decrease the proliferation of SK-MEL-28 melanoma cells containing B-RAF V600E proteins [27].Ramurthy and colleagues demonstrated that including a methyl group at the NH of benzimidazole increases the binding affinity to RAF kinase compared to the unmethylated homolog.
Recently [10], we reported on the design and synthesis of a novel series of 1,3,4-oxadiazole/benzimidazole chalcones as antiproliferative agents inducing apoptosis with dual inhibitory effect against EGFR and BRAF V600E .Compound 4 (Figure 1) was shown to be the most potent antiproliferative agent, with a GI50 value of 1.20 µM against four cancer cell lines, when compared to the reference doxorubicin, which had a GI50 value of 1.10 µM.Compound 4 was tested for inhibitory efficacy against EGFR and BRAF V600E as prospective antiproliferative targets.Compound 4 inhibited both targets significantly, with IC50 values of 0.55 ± 0.10 µM and 1.70 ± 0.20 µM, respectively, when compared to the reference erlotinib IC50 values of 0.08 ± 0.01 and 0.06 ± 0.01 µM, respectively .
Motivated by the promising anti-cancer activity of some benzimidazole-based derivatives and as part of our ongoing effort to develop a dual or multi-targeted anti-cancer agent [28][29][30][31][32][33], we present here the design, synthesis, and antiproliferative activity of a novel series of benzimidazole-based derivatives (4a-j, 5, and 6, Figure 2).NCI chose all newly synthesized compounds for an in vitro one-dose inhibitory experiment against a panel of 60 cancer cell lines.The most active compounds were chosen for five-dose experiments and mechanistic studies against two potential targets, EGFR and BRAFV 600E .Furthermore, the most effective derivatives were tested for apoptosis-inducing activity against caspase-3, caspase-8, Bax, and anti-apoptotic Bcl2.Finally, docking analyses were undertaken for the most potent derivatives to evaluate their binding mechanisms and interactions with the selected receptors.

Chemistry
Scheme 1 depicts the synthesis method for compounds 4a-j, 5, and 6.Condensation of 3,4-aminobenzoic acid with p-chlorobenzaldehyde in the presence of sodium metabisul-phite and DMF resulted in the synthesis of benzimidazole-5-carboxylic acid (1) in a good yield [34,35]. Compound 1's infrared spectra showed that C=O was present at 1662 cm −1 .Furthermore, a prolonged peak at 2683-3358 cm −1 was identified as the COOH group's OH.Furthermore, the 1 HNMR spectra of sample 1 showed the existence of seven aromatic protons at δ 7.63-8.20 ppm and a singlet signal at δ 13.32 ppm that is attributable to the COOH proton, which is exchangeable with D 2 O.The Fischer esterification of carboxylic acid 1 was accomplished using methanol and a few drops of conc.H 2 SO 4, and the mixture was heated at reflux for 17 h to obtain compound 2 in 60% yield [36,37]. Compound 2's IR spectra showed that the OH peak vanished and the C=O group shifted to 1716 cm −1 as a result of ester production.Compound 2's 1 HNMR spectra revealed a singlet peak associated with CH 3 at 3.87 ppm.The hydrazide 3 was prepared in 58% yield by heating the ester 2 at reflux with 99% hydrazine monohydrate in ethanol [38].Compound's 3 IR spectra revealed forked peaks in the infrared spectrum corresponding to the NH 2 and NH protons, respectively, at 3316, 3292, and 3423cm −1 .In addition" 1 H NMR spectra revealed that the methyl protons signal vanished and singlet signals appeared at δ: 4.46 and 9.81 ppm, corresponding to NH2 and NH, respectively.The target compounds 4a-j were prepared in high yield by heating the hydrazide 3 at reflux with substituted benzaldehyde in absolute ethanol, catalyzed by a few drops of glacial acetic acid.The structure of hydrazone derivatives 4a-j was confirmed using different spectroscopic techniques.The IR spectra of 4a-j exhibited the disappearance of forked NH 2 and the presence of absorption bands at υ max ; 3423-3225 and at υ max ; 1441-1566 cm -1 , corresponding to NH and C=N, respectively.The 1 H NMR spectra of compounds 4a-j revealed the disappearance of the NH 2 proton at δ: 4.46-5.66ppm and the presence of two signals at δ: 8.32-8.86 and δ: 11.60-13.41ppm attributed to azomethine proton CH=N and hydrazone NH, respectively.Moreover, the 13  The thiosemicarbazide 5 was prepared via heating at reflux of the hydrazide 3 ethyl isothiocyanate in absolute ethanol [39].The 1,2,4-triazole-3-thione 6 was synthes Scheme 1. Synthetic steps for compounds 4a-j, 5, and 6.The thiosemicarbazide 5 was prepared via heating at reflux of the hydrazide 3 with ethyl isothiocyanate in absolute ethanol [39].The 1,2,4-triazole-3-thione 6 was synthesized by cyclization of the 1,4-disubstituted thiosemicarbazide 5 in the presence of 2N sodium hydroxide solution followed by acidification with hydrochloric acid until pH = 3.The proposed mechanism for forming 1,2,4-triazole derivative 6 is through the nucleophilic addition of terminal nitrogen atoms to the carbonyl group; then, the species eliminate water to afford the triazole product 6.

Reagent and reaction conditions
The chemical structure of compound 6 was elucidated by spectroscopic techniques and elemental analyses.The IR spectrum of 6 exhibited a band at υ max : 3201-3361 cm −1 and υ max : 1501-1537 cm −1 , corresponding to NH of triazole and C=S, respectively.The 1 H NMR spectrum of the target compound 6 revealed an exchangeable signal at δ: 13.96-14.36ppm, attributed to NH triazole, and a triplet-quartet pattern was observed at δ: 1.16-1.20 and δ: 4.08-4.15ppm, attributed to the ethyl protons.The 13 C NMR spectrum of compound 6 revealed the presence of a signal at δ: 166.69 ppm related to C=S carbon and at δ: 13.91, 18.93 related to CH 3 and CH 2 moieties.

Biology 2.2.1. In Vitro Anti-Cancer Activity
The final compounds were submitted to the National Cancer Institute, Bethesda, USA, for screening in vitro for anti-cancer [40,41].Eleven compounds, 4a-j, 5, and 6, were selected according to the Development Therapeutic Program (DTP) protocol.The selected compounds were evaluated for their in vitro cytotoxic activity against a panel of sixty cancer cell lines (NCI-60 cell line panel) derived from six cell lines panel of leukemia, nine cell lines of lung cancer, six cell lines of CNS cancer, seven cell lines of colon cancer, eight cell lines of melanoma, six cell lines of ovarian cancer, eight cell lines of renal cancer, two cell lines of prostate cancer, and eight cell lines of breast cancer, representing the full nine human systems as leukemia, melanoma, and cancers of the lung, colon, brain, breast, ovary, kidney, and prostate.

In Vitro One-Dose Assay
The selected compounds 4a-j, 5, and 6 were tested at a single concentration of 10 µM.The screening results for the selected compounds are illustrated in Table 1.
The screening results of the target compounds revealed that benzimidazole-bearing hydrazone derivatives 4a-j have more potent anti-cancer activity than triazole derivatives 6.Among hydrazone derivatives, compounds 4c, 4d, 4e, 4f, 4g, 4h, and 4j showed promising anti-cancer activity in a one-dose assay at 10 µM.
Compound 4c revealed significant to complete cell death against the leukemia cell line with cell inhibition percentages of 90.In Vitro Five-Dose Assay Compounds 4c, 4e, and 4g were selected for advanced five-dose testing against the full panel of 60 human tumor cell lines [42,43], which represents nine tumor subpanels screened at five concentrations (0.01, 0.1, 1, 10, and 100 µM).The results are used to construct a dose-response curve log of concentration versus percentage growth inhibition (Figures 3-5) and calculate three response parameters for each cell line (GI 50 , TGI, and LC 50 ).The GI 50 value (concentration of maximum 50% cell proliferation inhibition) relates to compound concentration causing inhibition by 50% in total cell growth, the TGI value (cytostatic activity) refers to the concentration of the compound resulting in a reduction of total growth, and the LC 50 value (cytotoxic activity) indicates the concentration of the compound causing a net 50% inhibition of initial cells at the end of the incubation period of 48 h.The standard for a compound's selectivity depends on the ratio resulting from dividing the entire MID panel (the average sensitivity of all cell lines to the test agent) (µM) by its individual MID (µM) subpanel.Ratios between 3 and 6 indicate moderate selectivity; ratios > 6 refer to high selectivity towards the respective cell line, whereas compounds that do not meet either of these criteria are rated nonselective [44].In vitro five-dose assay screening results are illustrated in Tables 2-4.The standard for a compound's selectivity depends on the ratio resulting from dividing the entire MID panel (the average sensitivity of all cell lines to the test agent) (µM) by its individual MID (µM) subpanel.Ratios between 3 and 6 indicate moderate selectivity; ratios >6 refer to high selectivity towards the respective cell line, whereas compounds that do not meet either of these criteria are rated nonselective [44].In vitro five-dose assay screening results are illustrated in Tables 2-4.
Compounds 4c, 4e, and 4g achieved complete cell death for different cancer cell lines.These compounds were thus selected for five-dose testing to screen their activity at five different concentrations.In addition, these compounds have a broad and potent spectrum of anti-cancer activity without selectivity toward any of the tested cancer cell lines.
Compound 4c revealed a GI 50 value ranging from 0.420 to 8.99 µM toward the majority of the examined cell lines, a selectivity ratio from 0.57 to 5.96, a TGI from 2.41 to >100 µM, and a LC 50 from 9.61 to >100 µM.Compound 4c showed moderate selectivity toward leukemia cell lines as the ratio between selectivity towards cancer subpanels = 5.96 (Table 2 and Figure 3).
In the same context, compound 4e was found to have broad-spectrum cell growth inhibition activity against most of the tested tumor subpanels, with GI 50 values ranging from 0.97 to 4.93 µM, selectivity ratio ranging from 0.79 to 1.35, TGI from 2.13 to 9.03 µM, and LC 50 from 5.73 to 77.90 µM (Table 3 and Figure 4).
Finally, compound 4g exhibited potent antitumor activity toward all the examined cell lines, with GI 50 values ranging between 0.997 and 7.81 µM, selectivity ratio ranging from 0.87 to 1.39, TGI from 1.64 to >100 µM, and LC 50 between 6.33 and >100 µM (Table 4 and Figure 5).

EGFR Inhibitory Assay
The most effective derivatives from in vitro anti-cancer assay (4c, 4d, 4e, 4g, and 4h) were tested for EGFR inhibition as a potential target for antiproliferative action [45,46].Table 5 shows the results as IC 50 values versus erlotinib as the control drug.The results showed that the investigated compounds had potential EGFR inhibitory action, with IC 50 values ranging from 0.09 to 0.68 µM compared to erlotinib, which has an IC 50 value of 0.08 µM.Furthermore, the findings of this investigation are consistent with those of an in vitro five-dose inhibition assay in which compound 4e was the most potent EGFR inhibitor with an IC 50 value of 0.09 µM, equivalent to the reference erlotinib.Compound 4c was 1.2-fold less potent than 4e, having an IC 50 value of 0.11 µM.Compounds 4d, 4g, and 4h inhibited EGFR in a weak to moderate approach, with IC 50 values of 0.68 µM, 0.27 µM, and 0.56 µM, respectively, being seven-fold, three-fold, and six-fold less effective than 4e.These findings suggested that compounds 4c and 4e could be promising antiproliferative agents that target the EGFR inhibitory pathway.
Compounds 4c and 4e, which were the most potent derivatives in the antiproliferative and EGFR suppressive assays, were also the most effective derivatives as anti-BRAF V600E , with IC 50 values of 0.31 ± 0.07 µM and 0.20 ± 0.02 µM, respectively.These findings suggest that compounds 4c and 4e have significant antiproliferative activity as dual EGFR/BRAFV 600E inhibitors, implying that additional structural modifications may be required to develop a more potent lead molecule for future development.

Apoptosis Induction Assays
One method of treating cancer is to regulate or stop the uncontrolled proliferation of cancer cells.Using the natural dying process of the cell is a highly successful strategy.Targeting apoptosis is effective for many types of cancer because apoptosis evasion is a hallmark of cancer that is not particular to the etiology or type of cancer.Many anticancer drugs target distinct stages of the intrinsic and extrinsic apoptotic pathways [48][49][50].Compounds 4c and 4e, the most effective derivatives in all in vitro investigations, were investigated for their capacity to initiate apoptosis.

Assay for Caspase-3 Activation
Caspase-3 is a key caspase that cleaves numerous cell proteins, resulting in apoptosis [51].Compounds 4c and 4e were tested as activators of caspase-3 against the prostatic cancer (PC-3) cell line, compared to staurosporine as the control drug [52], with the results presented in Table 6.Compounds 4c and 4e showed high caspase-3 protein overexpression levels of 365 ± 12 and 470 ± 15 Pg/mL, respectively.They elevated the protein caspase-3 in the PC-3 cancer cell line by around seven to nine times compared to untreated control cells.Compared to reference staurosporine, which had a caspase-3 level overexpression of 260 ± 5 pg/mL, compounds 4c and 4e were more active as caspase-3 activators.These findings suggested that the compounds under investigation have apoptotic potential, which could provide additional support for their antiproliferative impact.Table 6.Caspase-3, caspase-8, Bax, and Bcl-2 levels for compounds 4c, 4e, and staurosporine on prostatic cancer (PC-3) cell line.

Caspase-3
Caspase- Assays for Caspase-8, Bax, and Bcl2 Levels Using staurosporine as a control, compounds 4c and 4e were investigated further for their impact on caspase-8, Bax, and anti-apoptotic Bacl-2 levels against the prostate cancer (PC-3) cell line.Table 3 lists the results.Compound 4e (2.20 ng/mL) had the highest level of caspase-8 overexpression, followed by compound 4c (1.90 ng/mL) and the reference drug staurosporine (1.65 ng/mL).Compounds 4c and 4e, increased caspase-8 levels by 16 and 18 times, respectively, compared to the untreated control cell.
Compared to untreated PC-3 cancer cells, compounds 4c and 4e increased Bax levels by 26-and 31-fold (185 pg/mL and 220 pg/mL, respectively).Both are more active than staurosporine, which induces Bax levels up to 170 pg/mL, and a 24-fold increase over untreated cells.Finally, compounds 4c and 4e induced equipotent down-regulation of anti-apoptotic Bcl-2 protein levels in the PC-3 cancer cell line when compared to stau-rosporine.These findings suggest that compounds 4c and 4e act as caspase-3, caspase-8, and Bax activators and down-regulators of the anti-apoptotic Bcl-2, classifying them as apoptotic triggers.

Docking Study
The most active compounds, 4c and 4e, have multiple structural options regarding their probable binding mechanism and interactions with EGFR and BRAF active sites.These possibilities were investigated by molecular modeling studies using the Molecular Operating Environment (MOE) software (version 09.2022) [53].Furthermore, the docking methodology was validated, and the co-crystallized ligand erlotinib, GW572016, was redocked into the active site using the EGFR domain (PDB ID: 1 M17).On the other hand, the structure of BRAF kinase in a complex with the inhibitor vemurafenib was used to validate the docking protocol for BRAF V600 (PDB ID: 3OG7) [54].With a docking score (S) of −13.4442, the re-docked ligand displayed an RMSD of 0.8429 between the docked pose and the co-crystallized ligand for EGFR.When the co-crystallized ligand was docked, the RMSD between the two was 0.2808 (docking score (S) = −10.2469)for the BRAF V600 .
The most potent molecules, 4c and 4e, were docked into the EGFR's active site along with erlotinib (as a positive control).Tables 7 and 8 present the results of the docking experiments together with the kind of interactions, the distances (in Å) between the interacting residues, and the binding affinity (in kcal mol −1 ).Compounds 4c and 4e demonstrated substantial interaction with Met793 and other important interacting amino acids required for inhibition of EGFR when compared to codocked ligand erlotinib as a positive control.Compared to erlotinib, which has a binding affinity of −1.9 kcal mol −1 , compounds 4c and 4e had strong interaction scores of −0.5 and −1.5 kcal mol −1 , respectively.The many binding interactions with the critical amino acid within the EGFR active site, particularly the gatekeeper Met793 and LEU 694 residues, can account for these significant binding affinities (Figures 6-8).

4c
−0. Compounds 4c and 4e demonstrated substantial interaction with Met793 and other important interacting amino acids required for inhibition of EGFR when compared to codocked ligand erlotinib as a positive control.Compared to erlotinib, which has a binding affinity of −1.9 kcal mol −1 , compounds 4c and 4e had strong interaction scores of −0.5 and −1.5 kcal mol −1 , respectively.The many binding interactions with the critical amino acid within the EGFR active site, particularly the gatekeeper Met793 and LEU 694 residues, can account for these significant binding affinities (Figures 6-8).Additionally, vemurafenib was docked into the BRAF V600E active site among the three most active molecules, 4c and 4e.The docking study data are compiled in Table 8, includ- Additionally, vemurafenib was docked into the BRAF V600E active site among the three most active molecules, 4c and 4e.The docking study data are compiled in Table 8, including information on the kind of interactions, binding affinity (in kcal mol −1 ), and distances (in Å) from the interacting residues.A co-docked ligand, vemurafenib, was a positive control in comparing molecules 4c and 4e.Strong interactions between these drugs and CYS 532 and other essential interacting amino acids required for BRAF V600 were shown.With binding affinities of −5.4 and −1.9 kcal mol −1 , respectively, compounds 4c and 4e showed considerable interaction scores compared to Vemurafenib, which had a binding affinity of −3.5 kcal mol −1 .The high binding scores of these compounds were established by a significant interaction between the synthesized compounds and the essential amino acid in the BRAF active site, with CYS 532 (Figures 9-11).Additionally, vemurafenib was docked into the BRAF V600E active site among the three most active molecules, 4c and 4e.The docking study data are compiled in Table 8, including information on the kind of interactions, binding affinity (in kcal mol −1 ), and distances (in Å) from the interacting residues.A co-docked ligand, vemurafenib, was a positive control in comparing molecules 4c and 4e.Strong interactions between these drugs and CYS 532 and other essential interacting amino acids required for BRAF V600 were shown.With binding affinities of −5.4 and −1.9 kcal mol −1 , respectively, compounds 4c and 4e showed considerable interaction scores compared to Vemurafenib, which had a binding affinity of −3.5 kcal mol −1 .The high binding scores of these compounds were established by a significant interaction between the synthesized compounds and the essential amino acid in the BRAF active site, with CYS 532 (Figures 9-11).

Conclusions
Finally, a new class of novel compounds with benzimidazole scaffolds (4a-j, 5, and 6) was synthesized and tested for anticancer activity.NCI chose compounds 4c, 4e, and 4g for five dose tests, with selectivity ratios ranging from 0.60 to 5.80 at the GI50 levels.Compounds 4c and 4e showed significant EGFR and BRAF V600E inhibition.Apoptotic marker assay results indicate that compounds 4c and 4e act as caspase-3, caspase-8, and Bax activators as well as down-regulators of the anti-apoptotic Bcl-2, classifying them as apoptotic triggers.Molecular docking simulations of 4c and 4e also revealed good binding modes within the EGFR and BRAF V600 active sites.To obtain a more effective lead compound for future development, compounds 4c and 4e require further structural modifications as well as the synthesis of more derivatives.

Chemistry
The melting point was obtained utilizing a Thomas-Hoover capillary device and is uncorrected.The infrared (IR) spectra were recorded as films on KBr plates using the FT-IR spectrometer.Thin-layer chromatography was used to evaluate the reaction mixture, homogeneity, and purity of the synthesized compounds (Merck, Darmstadt, Germany).
General Details: See Supplementary Materials.
The key intermediates 1-3 were prepared according to previously reported procedures [34,36,38].5.1.1.General Procedure for Synthesis of N'-(substituted benzylidene)-2-(4-chlorophenyl)-1H-benzimidazole-5-carbohydrazide (4a-j) Equimolar quantities of the appropriate compound 3 (2 mmol, 0.50 g) and the appropriate substituted benzaldehyde in absolute ethanol (25 mL) were refluxed for 6 h in the presence of a catalytic amount of glacial acetic acid.The resulting crude solid was filtered off and recrystallized from absolute ethanol to afford compounds 4a-j in a good yield.

Conclusions
Finally, a new class of novel compounds with benzimidazole scaffolds (4a-j, 5, and 6) was synthesized and tested for anticancer activity.NCI chose compounds 4c, 4e, and 4g for five dose tests, with selectivity ratios ranging from 0.60 to 5.80 at the GI 50 levels.Compounds 4c and 4e showed significant EGFR and BRAF V600E inhibition.Apoptotic marker assay results indicate that compounds 4c and 4e act as caspase-3, caspase-8, and Bax activators as well as down-regulators of the anti-apoptotic Bcl-2, classifying them as apoptotic triggers.Molecular docking simulations of 4c and 4e also revealed good binding modes within the EGFR and BRAF V600 active sites.To obtain a more effective lead compound for future development, compounds 4c and 4e require further structural modifications as well as the synthesis of more derivatives.

Chemistry
The melting point was obtained utilizing a Thomas-Hoover capillary device and is uncorrected.The infrared (IR) spectra were recorded as films on KBr plates using the FT-IR spectrometer.Thin-layer chromatography was used to evaluate the reaction mixture, homogeneity, and purity of the synthesized compounds (Merck, Darmstadt, Germany).
General Details: See Supplementary Materials.
The key intermediates 1-3 were prepared according to previously reported procedures [34,36,38].Equimolar quantities of the appropriate compound 3 (2 mmol, 0.50 g) and the appropriate substituted benzaldehyde in absolute ethanol (25 mL) were refluxed for 6 h in the presence of a catalytic amount of glacial acetic acid.The resulting crude solid was filtered off and recrystallized from absolute ethanol to afford compounds 4a-j in a good yield.5.1.2.General procedure for the synthesis of 2-aryl-1H-benzimidazole-5-carbonyl hydrazine-1-carbothioamide (5a) Equimolar quantities (2 mmol, 0.50 g) of compound 3 and appropriate isothiocyanate in absolute ethanol (25 mL) were heated under reflux for 6 h.The resulting crude solid was filtered off, dried, and used for the following step without further purification.Appropriate thiosemicarbazide 5a (10 mmol) was dissolved in 2N sodium hydroxide solution (20 mL), and the resulting solution was stirred under reflux for 4 h.The solution was acidified with dilute HCl until (pH 3); the formed precipitate was filtered off, washed with distilled water, dried, and then recrystallized from ethanol to afford corresponding 1,2,4-triazole 6a in a good yield.

Assay for EGFR Inhibitory Effect
Compounds 4c, 4d, 4e, 4g, and 4h were investigated for EGFR inhibitory action using erlotinib as the control drug [45].The detailed methods are described in the Supplementary Materials to this article.

Assay for BRAF V600E Inhibitory Action
Compounds 4c, 4d, 4e, 4g, and 4h were tested as possible BRAF V600E inhibitors, compared to erlotinib and vemurafenib as control drugs [47].Refer to the Supplementary Materials for more details.

Apoptotic Markers Assays
Compounds 4c and 4e, the most effective derivatives in all in vitro investigations, were investigated for their capacity to initiate apoptosis through their effect on caspase-3, caspase-8, Bax, and anti-apoptotic Bcl2 [52].For more details, see Supplementary Materials.

Figure 3 .
Figure 3. Dose-response curves for all cell lines for compound 4c.

Figure 4 .
Figure 4. Dose-response curves for all cell lines for compound 4e.

Figure 4 .
Figure 4. Dose-response curves for all cell lines for compound 4e.

Figure 5 .
Figure 5. Dose-response curves for all cell lines for compound 4g.

Figure 5 .
Figure 5. Dose-response curves for all cell lines for compound 4g.

Table 2 .
Screening results of in vitro five doses in µM for compound 4c.

Table 2 .
Screening results of in vitro five doses in µM for compound 4c.
a : Average sensitivity of all cell lines in µM.MID b : Average sensitivity of all subpanel cell lines in µM.

Table 3 .
Screening results of in vitro five doses in µM for compound 4e.
a : Average sensitivity of all cell lines in µM.MID b : Average sensitivity of all subpanel cell lines in µM.

Table 4 .
Screening results of five doses in µm for compound
a : Average sensitivity of all cell lines in µM.MID b : Average sensitivity of all subpanel cell lines in µM.

Table 7 .
The target compound's binding energy score throughout the EGFR docking study.

Table 8 .
The target compounds' binding energy scores throughout the BRAF V600 docking study.

Table 8 .
The target compounds' binding energy scores throughout the BRAF V600 docking study.

Table 8 .
The target compounds' binding energy scores throughout the BRAF V600 docking study.