Discovery, synthesis, and anti-cancer activity of N-CF 3 -benzimidazoles

Discovery, synthesis, and anti-cancer activity of N-CF 3 -benzimidazoles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discovery, synthesis, and anti-cancer activity of N-CF 3 -benzimidazoles Qigang Sun, Jia-Yi Li, Qingjiang Li, Yu Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9514025/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Benzimidazole is a privileged heterocyclic pharmacophore widely used in medicinal chemistry, while the N -trifluoromethyl ( N -CF 3 ) moiety represents a valuable modification strategy to optimize the druggability of small molecules. However, systematic studies on the antitumor activity of N -CF 3 -benzimidazoles remain scarce to date. In this study, the N -CF 3 -substituted benzimidazole scaffold was identified as a core active antitumor motif through phenotypic screening of our in-house compound library. Leveraging our established synthetic methodology, a library of 39 structurally diverse N -CF 3 -benzimidazole derivatives was prepared and systematically evaluated for their in vitro antiproliferative activity against three human cancer cell lines (786-O, A498, HepG2) using the CCK-8 assay. The results showed that most compounds exhibited varying degrees of antiproliferative activity at 10 µM. Notably, the lead compound 15 displayed micromolar GI 50 values and showed the most potent activity against 786-O cells. Furthermore, in silico target prediction of representative derivatives suggested potential interactions with a panel of kinases and epigenetic modulators, providing preliminary insights into their antitumor mechanism. This work characterizes the antitumor potential of N -CF 3 -benzimidazoles, offering a critical underpinning for the development of novel antitumor drug candidates. N-CF3 moiety benzimidazole anti-cancer activity Introduction Benzimidazole, an aromatic heterocyclic framework formed by the fusion of a benzene ring with an imidazole ring, has emerged as a pivotal pharmacophore in the fields of medicinal chemistry and natural product research, owing to its distinctive structural features and physicochemical properties. 1–3 In drug discovery, the benzimidazole core exhibits remarkable druggability, serving as the central scaffold for numerous clinically approved drugs such as omeprazole (proton pump inhibitor), albendazole (antiparasitic agent), and maribavir (antiviral drug), which are widely utilized in the treatment of gastrointestinal disorders, infectious diseases, and cancers. 1,4 Furthermore, this scaffold has been demonstrated to possess broad-spectrum antimicrobial, antifungal, antiviral, and antitubercular activities, positioning it as a valuable lead structure for the development of novel agents. 1 The trifluoromethyl (CF 3 ) group represents one of the most important fluorine-containing modification units in modern medicinal chemistry, with its widespread application in drug molecule design stemming from its unique physicochemical properties. Incorporation of the CF 3 group can significantly modulate the lipophilicity, metabolic stability, and membrane permeability of small molecules, thereby enhancing their pharmacokinetic profiles and bioavailability. 5–8 Notably, 14 out of 29 (approximately 48%) of FDA-approved small-molecule drugs in 2025 contain fluorine atoms, underscoring the critical role of fluorine modification in drug development. 9 When the CF 3 group is directly attached to a nitrogen atom to form the N -trifluoromethyl ( N -CF 3 ) moiety, the resulting N -CF 3 compounds exhibit higher lipophilicity, enhanced metabolic stability, and reduced basicity compared to their traditional N-methyl ( N -CH 3 ) counterparts, rendering this moiety an important strategy for drug structural optimization. 10,11 Nevertheless, the synthesis of N -CF 3 compounds has long faced significant challenges: when CF 3 is directly linked to a nitrogen atom bearing a lone pair of electrons, the corresponding intermediates and products tend to exhibit poor stability and are prone to defluorination, rendering conventional synthetic methods largely inapplicabl. 12 Against this backdrop, the incorporation of the N-trifluoromethyl moiety into the benzimidazole framework to construct N -CF 3 -benzimidazole derivatives holds promise for further enhancing the druggability of the parent core and expanding its application potential in medicinal chemistry. In recent years, the significance of this unique scaffold has garnered increasing attention; however, existing research has primarily focused on synthetic methodology, while systematic investigations into its pharmacological activities remain scarce, with its potential antitumor, antimicrobial, and other medicinal values yet to be fully explored. 13,14 Leveraging an efficient synthetic strategy previously established by our research group, 15 we have successfully constructed a series of N -CF 3 -substituted benzimidazole derivatives and conducted evaluations of their antitumor activities. Building upon these findings, we aim to further advance the synthesis and biological activity studies of N -CF 3 -benzimidazole compounds, exploring their application prospects in antitumor drug development and laying a foundation for the in-depth investigation and potential utilization of N -CF 3 -benzimidazole derivatives. Results and Discussion 2.1 Hit Discovery for Potential Anti-Cancer Agents . To identify novel anticancer therapeutic candidates, we conducted phenotypic screening of our in-house library comprising natural products and synthetic derivatives via the Cell Counting Kit-8 (CCK-8) cell viability assay. The screening outputs demonstrated that a panel of structurally diverse chemotypes exerted measurable antiproliferative activity against tumor cells. Notably, all active hit scaffolds were found to harbor a conserved benzimidazole core motif. Building on our well-established synthetic methodology for N -CF 3 -benzimidazoles from prior work, we therefore set out to systematically characterize the antitumor potency of N -CF 3 -benzimidazoles, as well as to comprehensively dissect their pharmacological profiles and translational application potential in cancer therapy. 2.2. Chemistry. Recently, we developed a mild and efficient one-pot method for the synthesis of 2-iodo- N -CF 3 benzimidazoles via fluorinative cyclization of readily accessible ortho -diisocyanoarenes. 15 In the present study, the tested compounds 1 – 27 were selected from our established library of 2-iodo- N -CF 3 benzimidazole, with their structures illustrated in Scheme 1A. Four new 2-iodo- N -CF 3 benzimidazole derivatives ( 36 – 39 ) were additionally synthesized using the same protocol (Scheme 1C). A broad range of substituents on the benzene ring of the benzimidazole core were investigated, including methyl ( 2 , 19 – 22 ), chloro ( 3 ), bromo ( 4 , 21 – 22 , 25 – 26 ), phenyl ( 6, 12 ), substituted phenyl groups ( 7 – 9 , 13 – 15 , 25 – 27 , 36 – 39 ), and methoxy ( 23 – 24 ). Furthermore, two benzimidazoles incorporating naphthalene and thiophene moieties ( 5 , 10 ) were evaluated. An N -CF 3 dihydroimidazole derivative ( 11 ) and three diazepine analogs ( 16 – 18 ) were also included in the screening panel. Finally, a series of C2-substituted N -CF 3 benzimidazole derivatives ( 28 – 35 ) were examined. These compounds were prepared via further functionalization of the C-I bond in compound 15 through nucleophilic aromatic substitution (S N Ar) and palladium-catalyzed cross-coupling reactions (Scheme 1B). 2.3 Cytotoxicity Evaluation. The in vitro cytotoxicity of all synthesized N -CF 3 -benzimidazole derivatives was determined via the CCK-8 assay against three human cancer cell lines: renal carcinoma 786-O, A498, and hepatocellular carcinoma HepG2, with staurosporine (STS) serving as the positive control. We first assessed the tumor cell growth inhibition rate of each derivative at 10 µM (Table 1), and the results showed that the tested N -CF 3 -benzimidazoles exhibited varying levels of anti-proliferative activity against the tested cancer cell lines. Table 1 The cell viability of the N -CF 3 -benzimidazoles Cpd. Cell Viability (100%) a Cpd. Cell Viability (100%) a 786-O A498 HepG2 786-O A498 HepG2 STS 53.8 0.1 60.6 20 114.6 116.0 94.5 1 96.3 88.6 94.3 21 111.0 115.5 97.7 2 100.1 91.4 98.3 22 113.6 102.9 111.0 3 82.2 98.0 83.8 23 114.3 105.6 106.8 4 99.7 101.6 83.6 24 129.1 106.3 113.8 5 97.3 103.7 99.0 25 104.0 85.5 83.9 6 88.3 107.6 97.6 26 117.6 123.8 143.8 7 89.1 113.8 94.7 27 86.7 121.9 128.6 8 100.1 102.1 91.2 28 98.1 96.9 104.1 9 117.8 116.2 104.1 29 107.4 106.3 100.3 10 83.6 112.3 93.1 30 114.1 105.5 111.9 11 99.1 114.0 111.9 31 112.2 104.4 104 12 83.3 102.6 100.9 32 98.7 100.2 92.8 13 81.5 108.3 105.1 33 113.8 103.4 109 14 98.9 112.7 107.3 34 100.1 88.0 100.0 15 21.9 48.0 32.4 35 103.6 97.3 105.7 16 98.6 131.9 105.5 36 87.5 92.3 95.2 17 121.5 178.8 126.9 37 95.0 93.7 119.8 18 122.8 192.4 143.4 38 74.5 86.7 116.7 19 118.9 107.4 111.0 39 83.0 89.3 119.0 a N -CF 3 -benzimidazoles derivatives (10 µM) against 786-O, A498 and HepG2 cells. Staurosporine (1 µM) was used as a positive control. Every experiment was repeated independently at least three times. Based on these primary data, we further determined the GI 50 values (defined as the concentration required for 50% tumor cell growth inhibition) of two optimal compounds 15 and 38 , with the calculated results summarized in Table 2. Collectively, the N -CF 3 -benzimidazole analogues displayed moderate micromolar-level cytotoxicity against all three tested cancer cell lines. Table 2 Cytotoxicity of the synthesized compounds 15 and 38 . Cpd. GI 50 (µM) a 786-O A498 HepG2 15 12.45 12.94 10.32 38 12.46 > 20 > 20 a GI 50 values were defined as the compound concentrations that resulted in 50% cell growth inhibition. Every experiment was repeated independently at least three times. 2.4 In Silico Target Prediction. To gain preliminary insight into the potential molecular targets responsible for the antitumor activity of this chemotype, we performed in silico target prediction using the SwissTargetPrediction web server (https://www.swisstargetprediction.ch/). A set of structurally representative N -CF 3 -benzimidazole derivatives, including compounds 5 , 9 , 13 , 16 , 18 , and the most cytotoxic compound 15 , were selected for this analysis based on their distinct substitution patterns and core scaffolds (benzimidazole, naphthyl-containing, substituted phenyl, and diazepine derivatives). Among them, compound 5 , which incorporates a unique naphthalene–thiophene fused system on the benzimidazole core, was predicted to address a pharmacologically attractive target spectrum. The top-ranked targets for 5 were highly enriched in the kinase family, with prominent hits including cyclin-dependent kinases (CDK2, CDK5), glycogen synthase kinase-3 beta (GSK-3β), and the insulin-like growth factor 1 receptor (IGF1R), all of which are intimately linked to tumor cell proliferation, survival, and apoptosis escape. Moreover, a subset of epigenetic regulators, most notably histone deacetylase 6 (HDAC6) and the bromodomain-containing protein BRD4—also surfaced among the predicted targets, albeit with slightly lower probability scores. The coexistence of kinase and epigenetic modulator hits suggests that compound 5 might exert its antiproliferative effects through a concerted polypharmacology mechanism, rather than via a single target. Intriguingly, in the primary viability screen, 5 displayed only modest activity against the three cancer cell lines; this may indicate that the predicted targets are not fully engaged under the tested conditions, or that the compound’s cellular permeability and metabolic stability limit its functional readout. Nevertheless, the remarkable structural novelty of 5 and its in silico target profile provide a highly attractive starting point for targeted structural optimization aimed at improving both potency and target engagement. The predicted target landscapes of the other N -CF 3 -benzimidazole analogues ( 9 , 13 , 16 , 18 ) shared partial overlap with that of 5 , with kinases such as FLT3, EGFR, and Aurora-A appearing recurrently across predictions. The diazepine derivatives 16 and 18 were additionally flagged for potential binding to tubulin and topoisomerase isoforms, suggesting that minor changes in the central ring system can significantly alter the target recognition spectrum. Compound 15 , the most potent derivative in the cell-based assays, was consistently predicted to target multiple receptor tyrosine kinases and phosphodiesterases, hinting at a mechanistic basis for its superior cytotoxicity. However, it must be emphasized that these in silico findings are purely computational predictions. Given that the N -CF 3 -benzimidazole scaffold is quite novel and scarcely investigated in the context of target identification, the current knowledge regarding its true binding partners remains extremely limited. Therefore, these predicted targets require rigorous experimental validation. Further in-depth target deconvolution studies will be pursued in our laboratory to clarify and definitively identify the molecular targets responsible for the antitumor activity of this promising compound class. Conclusions In this study, based on the key screening finding that the benzimidazole moiety is the common active core of antitumor hit compounds from our in-house library, we synthesized 39 structurally diverse N -CF 3 -benzimidazole derivatives and systematically assessed their in vitro antitumor activity via the CCK-8 assay. The results showed that these N -CF 3 -benzimidazole derivatives exerted moderate to significant antiproliferative activity against three human cancer cell lines (786-O, A498 and HepG2) at micromolar concentrations, with compound 15 exhibiting the most potent inhibitory effect on renal clear cell adenocarcinoma 786-O cells. Moreover, in silico target prediction indicated that this compound class may exert antitumor effects through multi-target modulation of cancer-related kinases and epigenetic regulators, warranting future experimental validation. Taken together, this work identifies N -CF 3 -benzimidazoles as a promising class of antitumor scaffolds, providing a solid experimental basis for the development of novel anticancer therapeutic candidates via subsequent structural modification and systematic biological evaluation. Materials and Methods 4.1. General Experiments. All chemical reagents and solvents used for the synthesis of N -CF 3 -benzimidazole derivatives were obtained from reputable commercial vendors, and used directly without additional purification unless otherwise specified. The progression of all chemical reactions was monitored via thin-layer chromatography (TLC) on precoated silica gel GF254 plates, with band visualization under ultraviolet (UV) light at 254 nm. Nuclear magnetic resonance (NMR) spectra, including 1 H NMR and 13 C NMR, were recorded on Bruker Avance III 400 MHz and 500 MHz spectrometers (Bruker BioSpin, Rheinstetten, Germany), with chemical shifts (δ) reported in parts per million (ppm) relative to tetramethylsilane (TMS) as the internal reference. High-resolution mass spectrometry (HRMS) analysis was performed on a Shimadzu LCMS-IT-TOF mass spectrometer. 4.2. Procedures for Synthesis of N -CF 3 benzimidazoles General procedure for synthesis of 2-iodo- N -CF 3 -benzimidazoles 36–39 Under an inert atmosphere, a 10 mL Schlenk tube equipped with a magnetic stir bar was charged with o -diisocyanoarenes (0.1 mmol, 1.0 equiv), silver fluoride (88.7 mg, 0.7 mmol, 7.0 equiv), and N -iodosuccinimide (44.6 mg, 0.2 mmol, 2.0 equiv). DCE (2 mL) was added, and the mixture was stirred at room temperature for 10 min. Upon completion, the reaction mixture was filtered through a short pad of Celite and washed with ethyl acetate (15 mL). The combined filtrates were concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel using petroleum ether/ethyl acetate (PE/EA) as eluent to afford the pure product. Synthesis of compound 28 Under an argon atmosphere, compound 15 (0.05 mmol, 1.0 equiv), benzenethiol (1.0 equiv), NaO t Bu (1.5 equiv) and MeCN (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60°C for 6 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 3:1 v/v) to afford the pure product 28 . Synthesis of compound 29 Under an argon atmosphere, compound 15 (0.05 mmol, 1.0 equiv), EtONa (1.1 equiv), Cs 2 CO 3 (0.4 equiv) and EtOH (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60°C for 6 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product 29 . Synthesis of compound 30 Under an argon atmosphere, compound 15 (0.05 mmol, 1.0 equiv), phenol (3.0 equiv), NaO t Bu (1.5 equiv) and anhydrous MeCN (2 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60°C overnight. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product 30 . Synthesis of compound 31 Under an argon atmosphere, compound 15 (0.05 mmol, 1.0 equiv), morpholine (1.5 equiv) and anhydrous DMF (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60°C overnight. After cooling to room temperature, the reaction mixture was quenched by the addition of a saturated aqueous solution of NaCl and then extracted with ethyl acetate. The combined organic layers were dried over Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 3:1 v/v) to afford the pure product 31 . Synthesis of compound 32 Under an argon atmosphere, compound 15 (0.05 mmol, 1.0 equiv), benzylamine (1.5 equiv) and EtOH (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 130°C for 16. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 3:1 v/v) to afford the pure product 32 . Synthesis of compound 33 Under an argon atmosphere, compound 15 (0.05 mmol, 1.0 equiv), 1-ethyl-4-ethynylbenzene (3.0 equiv), Pd(PPh 3 ) 2 Cl 2 (2.6 mol%), CuI (4.2 mol%) and Et 3 N (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at room temperature for 6 h. After completion, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product 33 . General procedure for synthesis of compounds 34 and 35 Under an argon atmosphere, compound 15 (0.05 mmol, 1.0 equiv), aryl boronic acid ( 3.0 equiv), Pd(PPh 3 ) 2 Cl 2 (10 mol%), SPhos (15 mol%), K 3 PO 4 (1.5 equiv), Ag 2 O (2.5 equiv) and THF (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60°C for 12 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product 34 or 35 . 4.3. Cell culture and MTT assay. Human renal clear cell adenocarcinoma 786-O, renal carcinoma A498, and hepatocellular carcinoma HepG2 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, and maintained at 37°C in a humidified 5% CO 2 incubator. The cytotoxicity of all synthesized N -CF 3 -benzimidazole derivatives was assessed via CCK-8 assay (Beyotime, China), with staurosporine (STS) set as the positive control. Briefly, cells were seeded into 96-well plates at 5.0 × 10 3 cells/well, then treated with DMSO-dissolved compounds for 24 h: either a fixed 10 µM dose for initial activity screening, or a 9-point two-fold serial dilution (0.391–20 µM) for GI 50 determination. After incubation, 10 µL of CCK-8 reagent was added to each well, and absorbance at 450 nm was measured with a microplate reader. GI 50 values were calculated by nonlinear regression using GraphPad Prism 10, with all experiments performed in at least three independent replicates. Declarations AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ; [email protected] . ORCID Yu Zhang: 0000-0003-3179-0538 Qingjiang Li: 0000-0001-5535-6993 Notes The authors declare no competing financial interest. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (22571327 and 22171293), Hainan Provincial Natural Science Foundation of China (825MS158), the Guangdong Basic and Applied Basic Research Foundation (2026A1515011220 and 2024A1515012178), the Hunan Provincial Natural Science Foundation of China (2024JJ6397). Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Molecules 28(7):3012 Crousse B (2023) Recent Advances in the Syntheses of N-CF3 Scaffolds up to Their Valorization. Chem Rec 23(9):e202300011 Wang L, Wang J, Ye S, Jiang B, Guo Z, Mumtaz Y, Yi W (2022) General Access to N-CF 3 Secondary Amines and Their Transformation to N-CF 3 Sulfonamides. Angew Chem Int Ed Engl 61(49):e202212115 Komatsu D, Yamada K, Hanamoto T (2023) Iodine-promoted synthesis of CF 3 -substituted dihydroimidazobenzimidazole and CF 3 -dihydroimidazoindole via C-N bond formation. Org Biomol Chem 21(33):6762–6771 Xu P, Wang XY, Wang Z, Zhao J, Cao XD, Xiong XC, Yuan YC, Zhu S, Guo D, Zhu X (2022) Defluorinative Alkylation of Trifluoromethylbenzimidazoles Enabled by Spin-Center Shift: A Synergistic Photocatalysis/Thiol Catalysis Process with CO 2 •– . Org Lett 24(22):4075–4080 Li JY, Fu JL, Liu HC, Wang H, Li Q (2026) Fluorinative Cyclization of ortho-Diisocyanoarenes: One-Pot Synthesis of N-CF 3 and N-CF 2 H Benzimidazoles. Org Lett 28. 10.1021/acs.orglett.6c01137 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Scheme1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 May, 2026 Reviews received at journal 07 May, 2026 Reviews received at journal 06 May, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 28 Apr, 2026 Editor assigned by journal 25 Apr, 2026 Submission checks completed at journal 25 Apr, 2026 First submitted to journal 24 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9514025","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630867258,"identity":"30181da9-5f6f-47f7-8c52-a12073b69947","order_by":0,"name":"Qigang Sun","email":"","orcid":"","institution":"Hainan Affiliated Hospital of Hainan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qigang","middleName":"","lastName":"Sun","suffix":""},{"id":630867262,"identity":"942bfd81-23c1-4fdd-a389-3b1f560ca7d2","order_by":1,"name":"Jia-Yi Li","email":"","orcid":"","institution":"Sun Yat-sen 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a benzene ring with an imidazole ring, has emerged as a pivotal pharmacophore in the fields of medicinal chemistry and natural product research, owing to its distinctive structural features and physicochemical properties.\u003csup\u003e1–3\u003c/sup\u003e In drug discovery, the benzimidazole core exhibits remarkable druggability, serving as the central scaffold for numerous clinically approved drugs such as omeprazole (proton pump inhibitor), albendazole (antiparasitic agent), and maribavir (antiviral drug), which are widely utilized in the treatment of gastrointestinal disorders, infectious diseases, and cancers.\u003csup\u003e1,4\u003c/sup\u003e Furthermore, this scaffold has been demonstrated to possess broad-spectrum antimicrobial, antifungal, antiviral, and antitubercular activities, positioning it as a valuable lead structure for the development of novel agents.\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe trifluoromethyl (CF\u003csub\u003e3\u003c/sub\u003e) group represents one of the most important fluorine-containing modification units in modern medicinal chemistry, with its widespread application in drug molecule design stemming from its unique physicochemical properties. Incorporation of the CF\u003csub\u003e3\u003c/sub\u003e group can significantly modulate the lipophilicity, metabolic stability, and membrane permeability of small molecules, thereby enhancing their pharmacokinetic profiles and bioavailability.\u003csup\u003e5–8\u003c/sup\u003e Notably, 14 out of 29 (approximately 48%) of FDA-approved small-molecule drugs in 2025 contain fluorine atoms, underscoring the critical role of fluorine modification in drug development.\u003csup\u003e9\u003c/sup\u003e When the CF\u003csub\u003e3\u003c/sub\u003e group is directly attached to a nitrogen atom to form the \u003cem\u003eN\u003c/em\u003e-trifluoromethyl (\u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e) moiety, the resulting \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e compounds exhibit higher lipophilicity, enhanced metabolic stability, and reduced basicity compared to their traditional N-methyl (\u003cem\u003eN\u003c/em\u003e-CH\u003csub\u003e3\u003c/sub\u003e) counterparts, rendering this moiety an important strategy for drug structural optimization.\u003csup\u003e10,11\u003c/sup\u003e Nevertheless, the synthesis of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e compounds has long faced significant challenges: when CF\u003csub\u003e3\u003c/sub\u003e is directly linked to a nitrogen atom bearing a lone pair of electrons, the corresponding intermediates and products tend to exhibit poor stability and are prone to defluorination, rendering conventional synthetic methods largely inapplicabl.\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAgainst this backdrop, the incorporation of the N-trifluoromethyl moiety into the benzimidazole framework to construct \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives holds promise for further enhancing the druggability of the parent core and expanding its application potential in medicinal chemistry. In recent years, the significance of this unique scaffold has garnered increasing attention; however, existing research has primarily focused on synthetic methodology, while systematic investigations into its pharmacological activities remain scarce, with its potential antitumor, antimicrobial, and other medicinal values yet to be fully explored.\u003csup\u003e13,14\u003c/sup\u003e Leveraging an efficient synthetic strategy previously established by our research group,\u003csup\u003e15\u003c/sup\u003e we have successfully constructed a series of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-substituted benzimidazole derivatives and conducted evaluations of their antitumor activities. Building upon these findings, we aim to further advance the synthesis and biological activity studies of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole compounds, exploring their application prospects in antitumor drug development and laying a foundation for the in-depth investigation and potential utilization of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e2.1 Hit Discovery for Potential Anti-Cancer Agents\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo identify novel anticancer therapeutic candidates, we conducted phenotypic screening of our in-house library comprising natural products and synthetic derivatives via the Cell Counting Kit-8 (CCK-8) cell viability assay. The screening outputs demonstrated that a panel of structurally diverse chemotypes exerted measurable antiproliferative activity against tumor cells. Notably, all active hit scaffolds were found to harbor a conserved benzimidazole core motif. Building on our well-established synthetic methodology for \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles from prior work, we therefore set out to systematically characterize the antitumor potency of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles, as well as to comprehensively dissect their pharmacological profiles and translational application potential in cancer therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Chemistry.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecently, we developed a mild and efficient one-pot method for the synthesis of 2-iodo-\u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e benzimidazoles via fluorinative cyclization of readily accessible \u003cem\u003eortho\u003c/em\u003e-diisocyanoarenes.\u003csup\u003e15\u003c/sup\u003e In the present study, the tested compounds \u003cstrong\u003e1\u003c/strong\u003e–\u003cstrong\u003e27\u003c/strong\u003e were selected from our established library of 2-iodo-\u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e benzimidazole, with their structures illustrated in Scheme 1A. Four new 2-iodo-\u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e benzimidazole derivatives (\u003cstrong\u003e36\u003c/strong\u003e–\u003cstrong\u003e39\u003c/strong\u003e) were additionally synthesized using the same protocol (Scheme 1C). A broad range of substituents on the benzene ring of the benzimidazole core were investigated, including methyl (\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e19\u003c/strong\u003e–\u003cstrong\u003e22\u003c/strong\u003e), chloro (\u003cstrong\u003e3\u003c/strong\u003e), bromo (\u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e21\u003c/strong\u003e–\u003cstrong\u003e22\u003c/strong\u003e, \u003cstrong\u003e25\u003c/strong\u003e–\u003cstrong\u003e26\u003c/strong\u003e), phenyl (\u003cstrong\u003e6, 12\u003c/strong\u003e), substituted phenyl groups (\u003cstrong\u003e7\u003c/strong\u003e–\u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e13\u003c/strong\u003e–\u003cstrong\u003e15\u003c/strong\u003e, \u003cstrong\u003e25\u003c/strong\u003e–\u003cstrong\u003e27\u003c/strong\u003e, \u003cstrong\u003e36\u003c/strong\u003e–\u003cstrong\u003e39\u003c/strong\u003e), and methoxy (\u003cstrong\u003e23\u003c/strong\u003e–\u003cstrong\u003e24\u003c/strong\u003e). Furthermore, two benzimidazoles incorporating naphthalene and thiophene moieties (\u003cstrong\u003e5\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e) were evaluated. An \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e dihydroimidazole derivative (\u003cstrong\u003e11\u003c/strong\u003e) and three diazepine analogs (\u003cstrong\u003e16\u003c/strong\u003e–\u003cstrong\u003e18\u003c/strong\u003e) were also included in the screening panel. Finally, a series of C2-substituted \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e benzimidazole derivatives (\u003cstrong\u003e28\u003c/strong\u003e–\u003cstrong\u003e35\u003c/strong\u003e) were examined. These compounds were prepared via further functionalization of the C-I bond in compound \u003cstrong\u003e15\u003c/strong\u003e through nucleophilic aromatic substitution (S\u003csub\u003eN\u003c/sub\u003eAr) and palladium-catalyzed cross-coupling reactions (Scheme 1B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Cytotoxicity Evaluation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe in \u003cem\u003evitro\u003c/em\u003e cytotoxicity of all synthesized \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives was determined via the CCK-8 assay against three human cancer cell lines: renal carcinoma 786-O, A498, and hepatocellular carcinoma HepG2, with staurosporine (STS) serving as the positive control. We first assessed the tumor cell growth inhibition rate of each derivative at 10 µM (Table 1), and the results showed that the tested \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles exhibited varying levels of anti-proliferative activity against the tested cancer cell lines.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe cell viability of the \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eCpd.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eCell Viability (100%)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eCpd.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eCell Viability (100%)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e786-O\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA498\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHepG2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e786-O\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA498\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHepG2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSTS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e53.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e114.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e116.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e111.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e115.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e113.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e102.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e114.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e106.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e101.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e129.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e113.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e103.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e104.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e83.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e107.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e117.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e123.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e143.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e89.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e113.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e27\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e121.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e128.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e102.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e104.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e117.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e116.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e104.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e107.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e106.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e112.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e93.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e114.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e114.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e111.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e112.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e104.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e104\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e102.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e108.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e113.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e103.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e109\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e112.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e107.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e103.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e131.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e105.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e36\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e92.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e121.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e178.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e126.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e37\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e93.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e122.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e192.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e143.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e118.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e107.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e111.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e39\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e89.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles derivatives (10 µM) against 786-O, A498 and HepG2 cells. Staurosporine (1 µM) was used as a positive control. Every experiment was repeated independently at least three times.\u003c/p\u003e\n\u003cp\u003eBased on these primary data, we further determined the GI\u003csub\u003e50\u003c/sub\u003e values (defined as the concentration required for 50% tumor cell growth inhibition) of two optimal compounds \u003cstrong\u003e15\u003c/strong\u003e and \u003cstrong\u003e38\u003c/strong\u003e, with the calculated results summarized in Table 2. Collectively, the \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole analogues displayed moderate micromolar-level cytotoxicity against all three tested cancer cell lines.\u003c/p\u003e\n\u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eCytotoxicity of the synthesized compounds \u003cstrong\u003e15\u003c/strong\u003e and \u003cstrong\u003e38\u003c/strong\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003eCpd.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003eGI\u003csub\u003e50\u003c/sub\u003e (µM)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e786-O\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA498\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHepG2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e38\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt; 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt; 20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eGI\u003csub\u003e50\u003c/sub\u003e values were defined as the compound concentrations that resulted in 50% cell growth inhibition. Every experiment was repeated independently at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 In Silico Target Prediction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain preliminary insight into the potential molecular targets responsible for the antitumor activity of this chemotype, we performed in silico target prediction using the SwissTargetPrediction web server (https://www.swisstargetprediction.ch/). A set of structurally representative \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives, including compounds \u003cstrong\u003e5\u003c/strong\u003e, \u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e13\u003c/strong\u003e, \u003cstrong\u003e16\u003c/strong\u003e, \u003cstrong\u003e18\u003c/strong\u003e, and the most cytotoxic compound \u003cstrong\u003e15\u003c/strong\u003e, were selected for this analysis based on their distinct substitution patterns and core scaffolds (benzimidazole, naphthyl-containing, substituted phenyl, and diazepine derivatives).\u003c/p\u003e\n\u003cp\u003eAmong them, compound \u003cstrong\u003e5\u003c/strong\u003e, which incorporates a unique naphthalene–thiophene fused system on the benzimidazole core, was predicted to address a pharmacologically attractive target spectrum. The top-ranked targets for \u003cstrong\u003e5\u003c/strong\u003e were highly enriched in the kinase family, with prominent hits including cyclin-dependent kinases (CDK2, CDK5), glycogen synthase kinase-3 beta (GSK-3β), and the insulin-like growth factor 1 receptor (IGF1R), all of which are intimately linked to tumor cell proliferation, survival, and apoptosis escape. Moreover, a subset of epigenetic regulators, most notably histone deacetylase 6 (HDAC6) and the bromodomain-containing protein BRD4—also surfaced among the predicted targets, albeit with slightly lower probability scores. The coexistence of kinase and epigenetic modulator hits suggests that compound \u003cstrong\u003e5\u003c/strong\u003e might exert its antiproliferative effects through a concerted polypharmacology mechanism, rather than via a single target. Intriguingly, in the primary viability screen, \u003cstrong\u003e5\u003c/strong\u003e displayed only modest activity against the three cancer cell lines; this may indicate that the predicted targets are not fully engaged under the tested conditions, or that the compound’s cellular permeability and metabolic stability limit its functional readout. Nevertheless, the remarkable structural novelty of \u003cstrong\u003e5\u003c/strong\u003e and its in silico target profile provide a highly attractive starting point for targeted structural optimization aimed at improving both potency and target engagement.\u003c/p\u003e\n\u003cp\u003eThe predicted target landscapes of the other \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole analogues (\u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e13\u003c/strong\u003e, \u003cstrong\u003e16\u003c/strong\u003e, \u003cstrong\u003e18\u003c/strong\u003e) shared partial overlap with that of \u003cstrong\u003e5\u003c/strong\u003e, with kinases such as FLT3, EGFR, and Aurora-A appearing recurrently across predictions. The diazepine derivatives \u003cstrong\u003e16\u003c/strong\u003e and \u003cstrong\u003e18\u003c/strong\u003e were additionally flagged for potential binding to tubulin and topoisomerase isoforms, suggesting that minor changes in the central ring system can significantly alter the target recognition spectrum. Compound \u003cstrong\u003e15\u003c/strong\u003e, the most potent derivative in the cell-based assays, was consistently predicted to target multiple receptor tyrosine kinases and phosphodiesterases, hinting at a mechanistic basis for its superior cytotoxicity. However, it must be emphasized that these in silico findings are purely computational predictions. Given that the \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole scaffold is quite novel and scarcely investigated in the context of target identification, the current knowledge regarding its true binding partners remains extremely limited. Therefore, these predicted targets require rigorous experimental validation. Further in-depth target deconvolution studies will be pursued in our laboratory to clarify and definitively identify the molecular targets responsible for the antitumor activity of this promising compound class.\u003c/p\u003e\n\n"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, based on the key screening finding that the benzimidazole moiety is the common active core of antitumor hit compounds from our in-house library, we synthesized 39 structurally diverse \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives and systematically assessed their in \u003cem\u003evitro\u003c/em\u003e antitumor activity via the CCK-8 assay. The results showed that these \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives exerted moderate to significant antiproliferative activity against three human cancer cell lines (786-O, A498 and HepG2) at micromolar concentrations, with compound \u003cstrong\u003e15\u003c/strong\u003e exhibiting the most potent inhibitory effect on renal clear cell adenocarcinoma 786-O cells. Moreover, in silico target prediction indicated that this compound class may exert antitumor effects through multi-target modulation of cancer-related kinases and epigenetic regulators, warranting future experimental validation. Taken together, this work identifies \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles as a promising class of antitumor scaffolds, providing a solid experimental basis for the development of novel anticancer therapeutic candidates via subsequent structural modification and systematic biological evaluation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e4.1. General Experiments.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemical reagents and solvents used for the synthesis of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives were obtained from reputable commercial vendors, and used directly without additional purification unless otherwise specified. The progression of all chemical reactions was monitored via thin-layer chromatography (TLC) on precoated silica gel GF254 plates, with band visualization under ultraviolet (UV) light at 254 nm. Nuclear magnetic resonance (NMR) spectra, including \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR, were recorded on Bruker Avance III 400 MHz and 500 MHz spectrometers (Bruker BioSpin, Rheinstetten, Germany), with chemical shifts (\u0026delta;) reported in parts per million (ppm) relative to tetramethylsilane (TMS) as the internal reference. High-resolution mass spectrometry (HRMS) analysis was performed on a Shimadzu LCMS-IT-TOF mass spectrometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Procedures for Synthesis of\u003c/strong\u003e \u003cstrong\u003eN\u003c/strong\u003e\u003cstrong\u003e-CF\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003ebenzimidazoles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral procedure for synthesis of 2-iodo-\u003c/strong\u003e \u003cstrong\u003eN\u003c/strong\u003e \u003cstrong\u003e-CF\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003e3\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e \u003cstrong\u003e-benzimidazoles 36\u0026ndash;39\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder an inert atmosphere, a 10 mL Schlenk tube equipped with a magnetic stir bar was charged with \u003cem\u003eo\u003c/em\u003e-diisocyanoarenes (0.1 mmol, 1.0 equiv), silver fluoride (88.7 mg, 0.7 mmol, 7.0 equiv), and \u003cem\u003eN\u003c/em\u003e-iodosuccinimide (44.6 mg, 0.2 mmol, 2.0 equiv). DCE (2 mL) was added, and the mixture was stirred at room temperature for 10 min. Upon completion, the reaction mixture was filtered through a short pad of Celite and washed with ethyl acetate (15 mL). The combined filtrates were concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel using petroleum ether/ethyl acetate (PE/EA) as eluent to afford the pure product.\u003c/p\u003e\n\u003ch3\u003eSynthesis of compound 28\u003c/h3\u003e\n\u003cp\u003eUnder an argon atmosphere, compound \u003cstrong\u003e15\u003c/strong\u003e (0.05 mmol, 1.0 equiv), benzenethiol (1.0 equiv), NaO\u003csup\u003e\u003cem\u003et\u003c/em\u003e\u003c/sup\u003eBu (1.5 equiv) and MeCN (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60\u0026deg;C for 6 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 3:1 v/v) to afford the pure product \u003cstrong\u003e28\u003c/strong\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eSynthesis of compound 29\u003c/h2\u003e\n \u003cp\u003eUnder an argon atmosphere, compound \u003cstrong\u003e15\u003c/strong\u003e (0.05 mmol, 1.0 equiv), EtONa (1.1 equiv), Cs\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (0.4 equiv) and EtOH (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60\u0026deg;C for 6 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product \u003cstrong\u003e29\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSynthesis of compound 30\u003c/h3\u003e\n\u003cp\u003eUnder an argon atmosphere, compound \u003cstrong\u003e15\u003c/strong\u003e (0.05 mmol, 1.0 equiv), phenol (3.0 equiv), NaO\u003csup\u003e\u003cem\u003et\u003c/em\u003e\u003c/sup\u003eBu (1.5 equiv) and anhydrous MeCN (2 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60\u0026deg;C overnight. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product \u003cstrong\u003e30\u003c/strong\u003e.\u003c/p\u003e\n\u003ch3\u003eSynthesis of compound 31\u003c/h3\u003e\n\u003cp\u003eUnder an argon atmosphere, compound \u003cstrong\u003e15\u003c/strong\u003e (0.05 mmol, 1.0 equiv), morpholine (1.5 equiv) and anhydrous DMF (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60\u0026deg;C overnight. After cooling to room temperature, the reaction mixture was quenched by the addition of a saturated aqueous solution of NaCl and then extracted with ethyl acetate. The combined organic layers were dried over Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 3:1 v/v) to afford the pure product \u003cstrong\u003e31\u003c/strong\u003e.\u003c/p\u003e\n\u003ch3\u003eSynthesis of compound 32\u003c/h3\u003e\n\u003cp\u003eUnder an argon atmosphere, compound \u003cstrong\u003e15\u003c/strong\u003e (0.05 mmol, 1.0 equiv), benzylamine (1.5 equiv) and EtOH (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 130\u0026deg;C for 16. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 3:1 v/v) to afford the pure product \u003cstrong\u003e32\u003c/strong\u003e.\u003c/p\u003e\n\u003ch3\u003eSynthesis of compound 33\u003c/h3\u003e\n\u003cp\u003eUnder an argon atmosphere, compound \u003cstrong\u003e15\u003c/strong\u003e (0.05 mmol, 1.0 equiv), 1-ethyl-4-ethynylbenzene (3.0 equiv), Pd(PPh\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (2.6 mol%), CuI (4.2 mol%) and Et\u003csub\u003e3\u003c/sub\u003eN (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at room temperature for 6 h. After completion, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product \u003cstrong\u003e33\u003c/strong\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003eGeneral procedure for synthesis of compounds 34 and 35\u003c/h2\u003e\n \u003cp\u003eUnder an argon atmosphere, compound \u003cstrong\u003e15\u003c/strong\u003e (0.05 mmol, 1.0 equiv), aryl boronic acid ( 3.0 equiv), Pd(PPh\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (10 mol%), SPhos (15 mol%), K\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e(1.5 equiv), Ag\u003csub\u003e2\u003c/sub\u003eO (2.5 equiv) and THF (1 mL) were added to a 15-mL Schlenk tube equipped with a magnetic stir bar. The mixture was stirred at 60\u0026deg;C for 12 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel with eluent (petroleum ether/ethyl acetate 10:1 v/v) to afford the pure product \u003cstrong\u003e34\u003c/strong\u003e or \u003cstrong\u003e35\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4.3. Cell culture and MTT assay.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eHuman renal clear cell adenocarcinoma 786-O, renal carcinoma A498, and hepatocellular carcinoma HepG2 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, and maintained at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The cytotoxicity of all synthesized \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives was assessed via CCK-8 assay (Beyotime, China), with staurosporine (STS) set as the positive control. Briefly, cells were seeded into 96-well plates at 5.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well, then treated with DMSO-dissolved compounds for 24 h: either a fixed 10 \u0026micro;M dose for initial activity screening, or a 9-point two-fold serial dilution (0.391\u0026ndash;20 \u0026micro;M) for GI\u003csub\u003e50\u003c/sub\u003e determination. After incubation, 10 \u0026micro;L of CCK-8 reagent was added to each well, and absorbance at 450 nm was measured with a microplate reader. GI\u003csub\u003e50\u003c/sub\u003e values were calculated by nonlinear regression using GraphPad Prism 10, with all experiments performed in at least three independent replicates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAUTHOR INFORMATION\u003c/p\u003e\n\u003cp\u003eCorresponding Author\u003c/p\u003e\n\u003cp\u003e*E-mail: [email protected]; [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYu Zhang: 0000-0003-3179-0538\u003c/p\u003e\n\u003cp\u003eQingjiang Li: 0000-0001-5535-6993\u003c/p\u003e\n\u003cp\u003eNotes\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (22571327 and\u0026nbsp;22171293), Hainan Provincial Natural Science Foundation of China (825MS158), the Guangdong Basic and Applied Basic Research Foundation (2026A1515011220 and\u0026nbsp;2024A1515012178), the Hunan Provincial Natural Science Foundation of China (2024JJ6397).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec11\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAroua LM, Alminderej FM, Almuhaylan HR, Alosaimi AH, Medini F, Mohammed HA, Almahmoud SA, Khan RA, Mekni NH (2025) Benzimidazole(s): synthons, bioactive lead structures, total synthesis, and the profiling of major bioactive categories. RSC Adv 15(10):7571\u0026ndash;7608\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadawy MAS, Br\u0026auml;se S, Ali TFS, Abdel-Aziz M, Abdel-Rahman HM (2025) Biologically Active Benzimidazole Hybrids as Cancer Therapeutics: Recent Advances. Pharmaceuticals (Basel) 18(10):1454\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar R, Marianesan AB, Pathak S (2024) Benzimidazole as a Privileged Scaffold in Drug Design and Discovery. Curr Top Med Chem 24(17):1504\u0026ndash;1528\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTarek A, Jaballah MY, Elrazaz EZ, Samir N (2025) The recent advances in benzimidazole\u0026ndash;based antimicrobials and antitubercular agents. Futur J Pharm Sci 11:109\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Wang J, Gu Z, Wang S, Zhu W, Ace\u0026ntilde;a JL, Soloshonok VA, Izawa K, Liu H (2016) Next Generation of Fluorine-Containing Pharmaceuticals, Compounds Currently in Phase II-III Clinical Trials of Major Pharmaceutical Companies: New Structural Trends and Therapeutic Areas. Chem Rev 116(2):422\u0026ndash;518\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue M, Sumii Y, Shibata N (2020) Contribution of Organofluorine Compounds to Pharmaceuticals. ACS Omega 5(19):10633\u0026ndash;10640\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeanwell NA (2018) Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug Design. J Med Chem 61(14):5822\u0026ndash;5880\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSwallow S (2015) Fluorine in medicinal chemistry. Prog Med Chem 54:65\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra S, Jadala C, Cardoza S, Potuganti GR, Velma GR (2026) Fluorine-containing FDA-approved small-molecule drugs in 2025: significance, synthetic insights, and therapeutic applications. RSC Adv 16(15):13830\u0026ndash;13850\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei Z, Chang W, Guo H, Feng J, Zhang Z (2023) A Brief Review on the Synthesis of the N-CF\u003csub\u003e3\u003c/sub\u003e Motif in Heterocycles. Molecules 28(7):3012\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrousse B (2023) Recent Advances in the Syntheses of N-CF3 Scaffolds up to Their Valorization. Chem Rec 23(9):e202300011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Wang J, Ye S, Jiang B, Guo Z, Mumtaz Y, Yi W (2022) General Access to N-CF\u003csub\u003e3\u003c/sub\u003e Secondary Amines and Their Transformation to N-CF\u003csub\u003e3\u003c/sub\u003e Sulfonamides. Angew Chem Int Ed Engl 61(49):e202212115\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomatsu D, Yamada K, Hanamoto T (2023) Iodine-promoted synthesis of CF\u003csub\u003e3\u003c/sub\u003e-substituted dihydroimidazobenzimidazole and CF\u003csub\u003e3\u003c/sub\u003e-dihydroimidazoindole via C-N bond formation. Org Biomol Chem 21(33):6762\u0026ndash;6771\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu P, Wang XY, Wang Z, Zhao J, Cao XD, Xiong XC, Yuan YC, Zhu S, Guo D, Zhu X (2022) Defluorinative Alkylation of Trifluoromethylbenzimidazoles Enabled by Spin-Center Shift: A Synergistic Photocatalysis/Thiol Catalysis Process with CO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026ndash;\u003c/sup\u003e. Org Lett 24(22):4075\u0026ndash;4080\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi JY, Fu JL, Liu HC, Wang H, Li Q (2026) Fluorinative Cyclization of ortho-Diisocyanoarenes: One-Pot Synthesis of N-CF\u003csub\u003e3\u003c/sub\u003e and N-CF\u003csub\u003e2\u003c/sub\u003eH Benzimidazoles. Org Lett 28. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.orglett.6c01137\u003c/span\u003e\u003cspan address=\"10.1021/acs.orglett.6c01137\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"N-CF3 moiety, benzimidazole, anti-cancer activity","lastPublishedDoi":"10.21203/rs.3.rs-9514025/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9514025/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBenzimidazole is a privileged heterocyclic pharmacophore widely used in medicinal chemistry, while the \u003cem\u003eN\u003c/em\u003e-trifluoromethyl (\u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e) moiety represents a valuable modification strategy to optimize the druggability of small molecules. However, systematic studies on the antitumor activity of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles remain scarce to date. In this study, the \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-substituted benzimidazole scaffold was identified as a core active antitumor motif through phenotypic screening of our in-house compound library. Leveraging our established synthetic methodology, a library of 39 structurally diverse \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazole derivatives was prepared and systematically evaluated for their in \u003cem\u003evitro\u003c/em\u003e antiproliferative activity against three human cancer cell lines (786-O, A498, HepG2) using the CCK-8 assay. The results showed that most compounds exhibited varying degrees of antiproliferative activity at 10 \u0026micro;M. Notably, the lead compound \u003cb\u003e15\u003c/b\u003e displayed micromolar GI\u003csub\u003e50\u003c/sub\u003e values and showed the most potent activity against 786-O cells. Furthermore, in silico target prediction of representative derivatives suggested potential interactions with a panel of kinases and epigenetic modulators, providing preliminary insights into their antitumor mechanism. This work characterizes the antitumor potential of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-benzimidazoles, offering a critical underpinning for the development of novel antitumor drug candidates.\u003c/p\u003e","manuscriptTitle":"Discovery, synthesis, and anti-cancer activity of N-CF 3 -benzimidazoles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 11:28:49","doi":"10.21203/rs.3.rs-9514025/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-08T05:47:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T13:58:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T13:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171796668893452483322209832889697006420","date":"2026-04-28T07:58:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286028078468146295107014595918740554092","date":"2026-04-28T06:15:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-28T05:53:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-25T17:03:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-25T16:48:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Diversity","date":"2026-04-24T07:46:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8ed762ff-6059-4590-932b-96f3683642c2","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-08T05:47:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T13:58:09+00:00","index":11,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T13:49:57+00:00","index":10,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T10:38:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 11:28:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9514025","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9514025","identity":"rs-9514025","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}