Design, synthesis and antitumor activity evaluation of Benzimidazole derivatives with potent HDAC inhibitory activity

Design, synthesis and antitumor activity evaluation of Benzimidazole derivatives with potent HDAC inhibitory activity | 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 Design, synthesis and antitumor activity evaluation of Benzimidazole derivatives with potent HDAC inhibitory activity Jiantao Ping, Hongrui Chu, Yisheng Zhao, Chen Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4988136/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Medicinal Chemistry Research → Version 1 posted 4 You are reading this latest preprint version Abstract This study aims to design and synthesize novel benzimidazole HDAC inhibitors to explore their potential applications in the treatment of cancer and other related diseases. By comparing the structures of our reported benzimidazole HDAC inhibitors, we designed a series of compounds accordingly. We then used experimentally verified their inhibitory activity against HDAC enzymes. The results showed that several of the newly synthesized compounds showed good HDAC inhibition and anti-proliferative activity. Therefore, we conclude that these novel HDAC inhibitors have potential as drug candidates for the treatment of cancer. Benzimidazole derivatives HDAC inhibitors Anti-tumor Figures Figure 1 Figure 2 Figure 3 1. Introduction Histone deacetylases (HDACs) are enzymes that remove acetyl groups from histones, leading to chromatin condensation and repression of gene transcription 1 . By inhibiting the activity of HDACs, HDAC inhibitors promote hyperacetylation of histones and alter the structure of chromatin, resulting in increased accessibility of DNA to transcription factors and ultimately leading to changes in gene expression patterns 2 , 3 . This can lead to the activation or suppression of genes involved in cell cycle regulation, apoptosis, differentiation, and other cellular processes relevant to cancer development 4 , 5 . HDAC inhibitors, also known as histone deacetylase inhibitors, are a class of compounds that have gained significant attention in the field of cancer research and treatment 6 . These compounds work by inhibiting the activity of histone deacetylases, which play a role in regulating gene expression 7 . The dysregulation of gene expression is often observed in cancer cells, making HDAC inhibitors an attractive target for therapeutic intervention 8 . Benzimidazole derivatives have garnered significant attention in cancer research as HDAC inhibitors, due to their potential as anti-cancer agents 9 . In our study, we have reported a series of Benzimidazole derivatives designed to target both the DNA minor groove and histone deacetylases 10 . The most active compounds not only demonstrated enhanced HDAC inhibitory effects but also exhibited potent inhibition activities against tumors by eliciting anti-tumor immunity. Building on our previous experience in designing benzimidazole HDAC inhibitors, this article presents the synthesis and evaluation of a new series of benzimidazole-hydroxamate derivatives for their biological activities. The compounds were subjected to biological characterizations, including assessment of HeLa nuclear extract activity, HDAC isozymes activity, and pro-apoptotic activity. Among the compounds tested, 8f emerged as the most potent compound. The findings regarding the synthesis and biological activities of these derivatives are detailed in this article. 2. Results and discussion 2.1. Chemistry The synthesis methods of all target compounds are described in Schemes 1 and 2 . As shown in Schemes 1 , compound A couple with o-Phenylenediamine in the presence of Na 2 S 2 O 5 to yield benzimidazole derivative C . Compound C do coupling reaction with different carbamate hydrochlorides to get 4a - f , which were next converted to hydroxamic acids 5a - f by treating with hydroxylamine. The synthesis methods in Schemes 2 were similar to Schemes 1 . 2.2. Inhibitory activity of HeLa cell extract Unlike previous our reported benzimidazole HDAC inhibitors, we use amide bonds instead of oxygen to obtain a new series of compounds 5a - 5f . Then HDAC enzymes inhibition assay was conducted to verify their inhibitory activity. Unfortunately, their inhibitory activity improved little, and the most active compound 5f only showed efficacy comparable to SAHA. Subsequently, 6-substituted benzimidazole derivatives were designed and synthesized. Compared to 5f and SAHA, the activity of 8e and 8f has increased significantly, up to 10 nM. Table.1. The chemical structures and HDACs inhibitory activities of hydroxamate derivatives. Cpd. n IC 50 (nM) Cpd. n IC 50 (nM) 5a 1 >1000 8a 1 >1000 5b 2 >1000 8b 2 >1000 5c 3 >1000 8c 3 >1000 5d 4 503.2 ± 78.4 5057-75(25) 8d 4 237.4 ± 22 5e 5 306.5 ± 44.1 8e 5 18.0 ± 2.0 5f 6 89.8 ± 11.2 8f 6 15.2 ± 0.3 SAHA 102.3 ± 9.6 2.3. HDAC isoform selectivity Compounds 8e and 8f were chosen based on their in vitro inhibitory activities against different HDAC isoforms. As shown in Table 2, the two compounds demonstrated superior activities against HDAC1, 2, and 6 (IC 50 1000 nM). In comparison to SAHA, the two compounds displayed enhanced inhibitory activities against HDAC1 and HDAC2. Notably, compound 8e exhibited a significantly improved HDAC1 inhibitory activity (IC 50 = 9.4 nM), which is eight times better than that of SAHA (IC 50 = 72 nM). Table.2. HDAC isoforms inhibitory activity of compounds 4k , 4l, 8e, 8f and SAHA Cpd. IC 50 a (nM) HDAC1 HDAC2 HDAC6 HDAC8 8e 9.4 ± 0.3 12.5 ± 1.0 39.3 ± 3.2 16776 ± 1036 8f 10.1 ± 2.1 23.0 ± 1.63 45.1 ± 2.7 3829 ± 10 SAHA 72 ± 2 61 ± 11 22 ± 3 17879 ± 93 2.4. In vitro antiproliferative assay Compounds 8e and 8f were chosen for evaluation of their in vitro anti-proliferative activities due to their strong inhibition of HDACs. SAHA was utilized as the positive control. However, these two compounds exhibited weak inhibitory activity on the four selected cell lines. Only compound 8f demonstrated a 2 µM inhibition activity on HEL cells. Table 3 Anti-proliferative activities of compounds 4k , 4l , 8e , 8f and SAHA Cpd IC 50 a (µM) HEL KG1 K562 PC-3 8e 7.22 ± 0.42 44.49 ± 0.58 32.18 ± 2.49 > 20 8f 2.62 ± 0.35 13.45 ± 1.65 7.17 ± 0.11 > 20 SAHA 0.36 ± 0.05 1.38 ± 0.14 0.99 ± 0.07 7.9 ± 0.54 2.5. Apoptotic assay Annexin VFTIC/propidium iodide (PI) assay was performed to investigate the apoptosis-inducing ability of compound 8f . HEL cells were treated with compounds 8f at 1.25, 2. 5 and 5 µM for 24 h. As shown in Fig. 2 , compounds 8f could significantly induce apoptosis in a dose-dependent manner. Notably, the apoptosis-inducing ability of 8f were much stronger than those of SAHA at the concentration of 5 µM, confirming its notable cellular potencies. The Annexin V/propidium iodide (PI) assay was conducted to investigate the apoptosis-inducing potential of compound 8f . HEL cells were exposed to varying concentrations of compound 8f (1.25, 2.5, and 5 µM) for a duration of 24 hours. As depicted in Fig. 5, compound 8f demonstrated a significant dose-dependent induction of apoptosis. Notably, the apoptotic effects induced by compound 8f were considerably stronger than those observed with SAHA at a concentration of 5 µM, thus confirming its remarkable cellular potency. 2.6. Western Blot Analysis Figure 3 shows the results of western blot analysis to determine the cellular potency of compound 8f . The results showed that compound 8f dose-dependently increased the acetylation level of H3 and tubulin. 3. Conclusion In this study, a series of novel benzimidazole-hydroxamate analogues were designed and synthesized as HDAC inhibitors. Compound 8f exhibited potent enzyme inhibition and demonstrated inhibitory effects on tumor cells through screening. Flow cytometry assay indicated that compound 8f effectively induced apoptosis. Furthermore, western blot assay revealed that compound 8f had the capacity to induce histone h3 and tubulin acetylation. These results suggest that benzimidazole-hydroxamate derivative 8f may serve as a potential candidate for further structural optimization research in anti-tumor molecule development. 4. Experiment sections 4.1. General synthesis of compounds General procedure for the synthesis of 4a - f , 7a - f Compound C (1 mmol, 0.32 g, 1 equiv) and amino acid esters (1 mmol, 1.1 equiv) were dissolved in DMF. TBTU (1.2 g, 0.31 mmol, 1.2 equiv) and DIEA (0.39 g, 3 mmol, 3 equiv) were added to the solution. The mixture was stirred for 4 h before being poured into water and extracted with ethyl acetate (3 × 25 mL). The organic layers were combined and washed successively with a solution of 1 M citric acid, saturated NaHCO 3 solution, and brine; then dried over anhydrous MgSO 4 before being filtered and evaporated to yield the crude product. The crude product was subsequently purified by column chromatography using petroleum ether/EtOAc as eluent to afford 0.34 g of compound 4a as a light yellow solid. methyl (4-(1H-benzo[d]imidazol-2-yl)benzoyl)glycinate ( 4a ) Yield: 73%. 1 H NMR (400 MHz, DMSO) δ 8.25 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.76 – 7.52 (m, 2H), 7.46 (m, 2H), 3.59 (s, 3H), 3.41 (m, 2H). methyl 3-(4-(1H-benzo[d]imidazol-2-yl)benzamido)propanoate ( 4b ) Yield: 54%. 1 H NMR (400 MHz, DMSO) δ 8.38 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 1.6 Hz, 2H), 7.64 – 7.56 (m, 1H), 3.69 (s, 3H), 3.50 (d, J = 5.9 Hz, 2H), 2.31 (t, J = 7.2 Hz, 2H). ethyl 4-(4-(1H-benzo[d]imidazol-2-yl)benzamido)butanoate ( 4c ) Yield: 64%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.62 (t, J = 5.6 Hz, 1H), 8.30 – 8.22 (m, 2H), 8.04 – 7.99 (m, 2H), 7.72 – 7.66 (m, 1H), 7.59 – 7.53 (m, 1H), 7.30 – 7.17 (m, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.32 (d, J = 6.2 Hz, 2H), 2.38 (t, J = 7.4 Hz, 2H), 1.81 (p, J = 7.2 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H). methyl 5-(4-(1H-benzo[d]imidazol-2-yl)benzamido)pentanoate ( 4d ) Yield: 56%. 1 H NMR (400 MHz, DMSO) δ 13.06 (s, 1H), 8.60 (s, 1H), 8.25 (d, J = 6.7 Hz, 2H), 8.01 (d, J = 6.8 Hz, 2H), 7.62 (d, J = 50.8 Hz, 2H), 7.23 (s, 2H), 3.59 (s, 3H), 3.30 (t, 2H), 2.37 (t, 2H), 1.51-1.57 (m, 4H). methyl 6-(4-(1H-benzo[d]imidazol-2-yl)benzamido)hexanoate ( 4e ) Yield: 47%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.57 (t, J = 5.6 Hz, 1H), 8.28 – 8.22 (m, 2H), 8.03 – 7.97 (m, 2H), 7.62 (s, 2H), 7.23 (dq, J = 7.1, 3.7 Hz, 2H), 3.58 (s, 3H), 3.28 (q, J = 6.9 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.61 – 1.48 (m, 4H), 1.34 (dtd, J = 8.6, 6.2, 2.1 Hz, 2H). ethyl 7-(4-(1H-benzo[d]imidazol-2-yl)benzamido)heptanoate ( 4f ) Yield: 73%. 1 H NMR (400 MHz, Chloroform- d ) δ 8.01 (d, J = 7.9 Hz, 2H), 7.66 (d, J = 8.2 Hz, 4H), 7.28 (d, J = 3.5 Hz, 2H), 6.74 (t, J = 5.7 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.43 (q, J = 6.7 Hz, 2H), 2.27 (t, J = 7.5 Hz, 2H), 1.60 (t, J = 7.2 Hz, 4H), 1.34 (dt, J = 7.3, 4.1 Hz, 4H), 1.24 (t, J = 7.1 Hz, 3H). methyl (2-phenyl-1H-benzo[d]imidazole-6-carbonyl)glycinate ( 7a ) Yield: 71%. 1 H NMR (400 MHz, DMSO) δ 13.19 (s, 1H), 8.98 (s, 1H), 8.21 (d, J = 7.2 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H), 7.64 – 7.50 (m, 4H), 4.06 (d, J = 5.7 Hz, 2H), 3.68 (s, 3H). methyl 3-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)propanoate ( 7b ) Yield: 79%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.22 (d, J = 4.7 Hz, 1H), 8.18 (d, J = 2.4 Hz, 1H), 7.64 (s, 1H), 7.45 (t, J = 6.0 Hz, 1H), 7.11 (t, J = 5.7 Hz, 1H), 3.80 (s, 1H), 3.71 (d, J = 2.1 Hz, 1H), 2.68 (dd, J = 10.5, 4.4 Hz, 1H). methyl 4-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)butanoate ( 7c ) Yield: 81%. 1 H NMR (400 MHz, DMSO) δ 8.58 – 8.44 (m, 1H), 8.20 (d, J = 7.6 Hz, 2H), 8.02 (s, 1H), 7.71 (d, J = 3.9 Hz, 1H), 7.56 (dt, J = 11.8, 5.0 Hz, 4H), 3.59 (s, 3H), 3.34 – 3.27 (m, 2H), 2.40 (t, J = 7.4 Hz, 2H), 1.88 – 1.77 (m, 2H). methyl 5-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)pentanoate ( 7d ) Yield: 44%. 1 H NMR (400 MHz, DMSO) δ 8.48 (d, J = 19.6 Hz, 1H), 8.20 (d, J = 7.3 Hz, 2H), 7.75 (dd, J = 17.1, 8.6 Hz, 2H), 7.55 (dq, J = 14.3, 7.0 Hz, 4H), 3.59 (s, 3H), 3.30 (d, J = 5.8 Hz, 2H), 2.37 (t, J = 6.9 Hz, 2H), 1.67 – 1.50 (m, 4H). methyl 6-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)hexanoate ( 7e ) Yield: 59%. 1 H NMR (400 MHz, DMSO) δ 8.46 (d, J = 22.5 Hz, 1H), 8.22 – 8.18 (m, 2H), 7.76 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 3.3 Hz, 1H), 7.62 – 7.50 (m, 4H), 3.58 (s, 3H), 3.28 (dd, J = 12.8, 6.5 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.65 – 1.49 (m, 4H), 1.34 (dd, J = 15.3, 8.2 Hz, 2H). methyl 7-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)heptanoate ( 7f ) Yield: 61%. 1 H NMR (400 MHz, DMSO) δ 8.44 (s, 1H), 8.21 (d, J = 7.4 Hz, 1H), 8.13 (s, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.67 – 7.50 (m, 2H), 3.58 (s, 2H), 3.29 (d, J = 6.3 Hz, 1H), 2.30 (t, J = 7.4 Hz, 1H), 1.55 (d, J = 6.5 Hz, 2H), 1.33 (d, J = 3.5 Hz, 2H). General procedure for the synthesis of 5a - f , 8a - f The synthesis route was similar to previous publication 11 . 4-(1H-benzo[d]imidazol-2-yl)-N-(2-(hydroxyamino)-2-oxoethyl)benzamide ( 5a ) Yield: 43%. 1 H NMR (400 MHz, DMSO) δ 10.66 (s, 1H), 8.92 (t, J = 5.7 Hz, 1H), 8.30 (d, J = 8.3 Hz, 2H), 8.08 (d, J = 8.3 Hz, 2H), 7.67 (dd, J = 6.0, 3.1 Hz, 2H), 7.30 (dd, J = 6.0, 3.1 Hz, 2H), 3.84 (d, J = 5.8 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 166.30, 150.47, 135.82, 131.89, 128.59, 126.97, 123.53, 115.59, 41.17. HRMS (ESI) m/z Calcd [M+H] + 311.1138, found: 311.1136. 4-(1H-benzo[d]imidazol-2-yl)-N-(3-(hydroxyamino)-3-oxopropyl)benzamide ( 5b ) Yield: 64%. 1 H NMR (400 MHz, DMSO) δ 10.49 (s, 1H), 8.78 (s, 1H), 8.68 (t, J = 5.4 Hz, 1H), 8.26 (d, J = 8.3 Hz, 2H), 8.01 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 6.5 Hz, 1H), 7.56 (d, J = 6.6 Hz, 1H), 7.23 (s, 2H), 3.49 (dd, J = 12.9, 6.8 Hz, 2H), 2.30 (t, J = 7.2 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 167.77, 166.07, 150.85, 144.28, 135.70, 132.93, 128.29, 126.68, 123.39, 122.38, 119.54, 111.97, 36.60, 32.89. HRMS (ESI) m/z Calcd [M+H] + 325.1295, found: 325.1292. 4-(1H-benzo[d]imidazol-2-yl)-N-(4-(hydroxyamino)-4-oxobutyl)benzamide ( 5c ) Yield: 73%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.84 (t, J = 5.5 Hz, 1H), 8.46 (d, J = 8.2 Hz, 2H), 8.15 (d, J = 8.2 Hz, 2H), 7.85 (dd, J = 6.1, 3.1 Hz, 2H), 7.55 (dd, J = 6.1, 3.2 Hz, 2H), 3.31 (p, J = 6.5, 6.1 Hz, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.80 (q, J = 7.2 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 174.98, 167.03, 153.43, 130.66, 130.32, 129.47, 129.33, 127.13, 33.94, 29.24, 22.59. HRMS (ESI) m/z Calcd [M+H] + 337.1311, found: 337.1306. 4-(1H-benzo[d]imidazol-2-yl)-N-(5-(hydroxyamino)-5-oxopentyl)benzamide ( 5d ) Yield: 66%. 1 H NMR (400 MHz, DMSO) δ 8.79 (t, J = 5.5 Hz, 1H), 8.45 (d, J = 8.5 Hz, 2H), 8.14 (d, J = 8.5 Hz, 2H), 7.85 (dt, J = 6.6, 3.3 Hz, 2H), 7.61 – 7.52 (m, 2H), 3.33 – 3.26 (m, 2H), 2.01 (t, J = 6.7 Hz, 2H), 1.66 – 1.48 (m, 4H). 13 C NMR (101 MHz, DMSO) δ 169.58, 166.97, 153.36, 130.72, 130.18, 129.47, 127.18, 122.17, 32.75, 29.48, 26.66, 25.44. HRMS (ESI) m/z Calcd [M+H] + 353.1535, found: 353.1539. 4-(1H-benzo[d]imidazol-2-yl)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide ( 5e ) Yield: 83%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.72 (t, J = 5.5 Hz, 1H), 8.35 (d, J = 8.1 Hz, 2H), 8.11 (d, J = 8.2 Hz, 2H), 7.81 (dd, J = 6.1, 3.2 Hz, 2H), 7.50 (dt, J = 6.0, 3.6 Hz, 2H), 3.29 (q, J = 6.7 Hz, 3H), 1.96 (t, J = 7.4 Hz, 2H), 1.54 (q, J = 7.4 Hz, 4H), 1.31 (q, J = 7.8 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 167.45, 166.72, 150.77, 130.72, 129.50, 127.38, 125.93, 122.20, 38.88, 33.45, 29.64, 26.39, 24.25. HRMS (ESI) m/z Calcd [M+H] + 366.1813, found: 366.1812. 4-(1H-benzo[d]imidazol-2-yl)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide ( 5f ) Yield: 40%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.37 (s, 1H), 8.73 (t, J = 5.6 Hz, 1H), 8.43 – 8.36 (m, 2H), 8.16 – 8.09 (m, 2H), 7.84 (dd, J = 6.1, 3.1 Hz, 2H), 7.57 – 7.51 (m, 2H), 3.29 (q, J = 6.6 Hz, 2H), 1.95 (t, J = 7.4 Hz, 2H), 1.60 – 1.45 (m, 4H), 1.37 – 1.21 (m, 4H). 13 C NMR (101 MHz, DMSO) δ 174.95, 166.06, 151.59, 133.03, 131.85, 129.93, 128.31, 124.50, 114.33, 114.13, 34.12, 29.46, 28.80, 26.75, 24.95. HRMS (ESI) m/z Calcd [M+H] + 381.1921, found: 381.1913. N-(2-(hydroxyamino)-2-oxoethyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide ( 8a ) Yield: 61%. 1 H NMR (400 MHz, DMSO) δ 10.62 (s, 1H), 8.81 (s, 1H), 8.74 (d, J = 21.0 Hz, 1H), 8.22 (d, J = 6.9 Hz, 2H), 7.84 – 7.68 (m, 2H), 7.59 – 7.51 (m, 3H), 3.83 (d, J = 5.7 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 167.40, 166.70, 153.56, 130.73, 130.25, 129.49, 128.64, 127.16, 41.23. HRMS (ESI) m/z Calcd [M+H] + 311.1006, found: 311.1002. N-(3-(hydroxyamino)-3-oxopropyl)-2-phenyl-1H-benzo[d]imidazole-6 carboxamide(8b) Yield: 61%. 1 H NMR (400 MHz, DMSO) δ 10.55 (s, 1H), 8.85 (t, J = 5.3 Hz, 1H), 8.55 – 8.42 (m, 2H), 8.28 (s, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.76 – 7.66 (m, 3H), 7.41 (t, J = 50.8 Hz, 1H), 3.51 (dd, J = 12.7, 6.8 Hz, 2H), 2.33 (t, J = 7.1 Hz, 2H). 13 C NMR (101 MHz, DMSO) δ 167.81, 166.08, 151.21, 133.38, 129.96, 128.59, 124.83, 36.75, 32.86. HRMS (ESI) m/z Calcd [M+H] + 325.1222, found: 325.1226. N-(4-(hydroxyamino)-4-oxobutyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide ( 8c ) Yield: 49%. 1 H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 8.71 (s, 1H), 8.50 (d, J = 22.2 Hz, 1H), 8.21 (d, J = 7.9 Hz, 2H), 7.79 – 7.66 (m, 2H), 7.61 – 7.49 (m, 4H), 3.32 – 3.25 (m, 2H), 2.05 (t, J = 7.3 Hz, 2H), 1.83 – 1.73 (m, 2H). 13 C NMR (101 MHz, DMSO) δ 169.46, 167.12, 153.44, 130.71, 130.24, 129.49, 129.23, 127.14, 122.24, 30.56, 25.90. HRMS (ESI) m/z Calcd [M+H] + 339.1379, found: 339.1382. N-(5-(hydroxyamino)-5-oxopentyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide ( 8d ) Yield: 33%. 1 H NMR (400 MHz, DMSO) δ 8.69 (s, 1H), 8.47 (d, J = 6.2 Hz, 1H), 8.24 (d, J = 7.9 Hz, 2H), 7.71 – 7.61 (m, 2H), 7.51 – 7.42 (m, 4H), 3.43 – 3.36 (m, 2H), 2.07 (t, J = 5.7 Hz, 2H), 1.66 – 1.51 (m, 4H). 13 C NMR (101 MHz, DMSO) δ 169.55, 167.00, 130.69, 130.28, 129.49, 127.12, 32.54, 29.37, 23.29. HRMS (ESI) m/z Calcd [M+H] + 353.1535, found: 353.1539. N-(6-(hydroxyamino)-6-oxohexyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide ( 8e ) Yield: 42%. 1 H NMR (400 MHz, DMSO) δ 10.34 (s, 1H), 8.66 (s, 1H), 8.45 (s, 1H), 8.20 (d, J = 7.1 Hz, 2H), 7.74 (d, J = 8.0 Hz, 1H), 7.65 – 7.45 (m, 4H), 3.30 – 3.23 (m, 2H), 1.96 (t, J = 7.3 Hz, 2H), 1.61 – 1.49 (m, 4H), 1.36 – 1.26 (m, 2H). 13 C NMR (101 MHz, DMSO) δ 169.58, 166.97, 153.39, 130.72, 130.21, 129.50, 129.38, 127.13, 122.20, 39.68, 32.75, 29.49, 26.66, 25.45. HRMS (ESI) m/z Calcd [M+H] + 367.1692, found: 367.1695. N-(7-(hydroxyamino)-7-oxoheptyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide ( 8f ) Yield: 81%. 1 H NMR (400 MHz, DMSO) δ 10.34 (s, 1H), 8.65 (s, 1H), 8.44 (d, J = 5.3 Hz, 1H), 8.21 (d, J = 7.1 Hz, 2H), 8.12 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.66 – 7.50 (m, 4H), 3.28 (dd, J = 6.4 Hz, 2H), 1.95 (t, J = 7.3 Hz, 2H), 1.58 – 1.47 (m, 4H), 1.37 – 1.25 (m, 4H). 13 C NMR (101 MHz, DMSO) δ 169.62, 166.94, 153.28, 130.85, 129.94, 129.51, 127.22, 122.33, 32.73, 29.61, 28.87, 26.77, 25.62. HRMS (ESI) m/z Calcd [M+H] + 381.1848, found: 381.1891. 4.2. HeLa nuclear extract and HDAC enzymatic assay The mixed solution containing the enzyme solution (HeLa nuclear extract, HDAC1, HDAC2, HDAC6 or HDAC8), the tested compound solution, and the fluorogenic substrate solution [Boc-Lys (acetyl)-AMC or Boc-Lys (trifluoroacetyl)-AMC] was plated onto an enzyme-labeled plate. After a 30-minute incubation at 37 °C, the reaction was stopped by trypsin and trichostatin A. Fluorescence analysis was performed using a Microplate reader (Tecan) with excitation/emission wavelengths of 360/460 nm. 4.3 In vitro antiproliferative activity Cancer cells were seeded into 96-well plates and then treated with various concentrations of compounds. After co-incubation for 48 hours, 20μl of CCK8 solution was added to each well and incubated for 2 hours at 37°C. A microplate reader (Tecan) was used to measure the absorbance at 450 nm for each well. 4.4 Flow cytometric assay HEL cells were treated with compounds for 24 hours. Then the cells were collected pellets for annexin V-FITC/PI staining. The stained cells were analyzed by a flow cytometer. 4.5. Werstern blot assay Cancer cells were treated with compounds for 24 hours. Subsequently, the cells were collected and pellets were obtained for protein extraction. The protein content was determined using the BCA protein determination assay. Equal amounts of total protein were measured for Western blot analysis, including Ac-HH3 and Ac-Tubulin. Declarations The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Jiantao Ping: Investigation, Visualization, Funding acquisition, Project administration.Hongrui Chu: Investigation, Methodology, Writing-original draft.Yisheng Zhao: Investigation, Visualization,Funding acquisition.Chen Chen: Conceptualization, Investigation,Supervision, Formal analysis, Writing – review & editing. Acknowledgments This study was funded by the Education and Industry Integration Pilot Project of Qilu University of Technology (2022PY036), the National Natural Science Foundation of China (No. 62105175), Natural Science Foundation of Shandong Province (ZR2021QF058, ZR2021QB174) References Ho, T. C. S.; Chan, A. H. Y.; Ganesan, A. Thirty Years of HDAC Inhibitors: 2020 Insight and Hindsight. Journal of Medicinal Chemistry 2020, 63, 12460–12484. Cheng, B.; Pan, W.; Xiao, Y.; Ding, Z.; Zhou, Y.; Fei, X.; Liu, J.; Su, Z.; Peng, X.; Chen, J. HDAC-targeting epigenetic modulators for cancer immunotherapy. European Journal of Medicinal Chemistry 2024, 265, 116129. Chen, I. C.; Sethy, B.; Liou, J. P. Recent Update of HDAC Inhibitors in Lymphoma. Frontiers in Cell and Developmental Biology 2020, 8. Wang, X.; Waschke, B. C.; Woolaver, R. A.; Chen, S. M. Y.; Chen, Z.; Wang, J. H. HDAC inhibitors overcome immunotherapy resistance in B-cell lymphoma. Protein & Cell 2020, 11, 472–482. Chen, C.; Chu, H. R.; Wang, A. Y.; Yin, H. H.; Gao, Y. Q.; Liu, S. H.; Li, W.; Han, L. Q. Discovery of 2,5-diphenyl-1,3,4-thiadiazole derivatives as HDAC inhibitors with DNA binding affinity. European Journal of Medicinal Chemistry 2022, 241. Zhang, W.-X.; Huang, J.; Tian, X.-Y.; Liu, Y.-H.; Jia, M.-Q.; Wang, W.; Jin, C.-Y.; Song, J.; Zhang, S.-Y. A review of progress in o-aminobenzamide-based HDAC inhibitors with dual targeting capabilities for cancer therapy. European Journal of Medicinal Chemistry 2023, 259, 115673. Biersack, B.; Polat, S.; Höpfner, M. Anticancer properties of chimeric HDAC and kinase inhibitors. Seminars in Cancer Biology 2022, 83, 472–486. Sun, L.; Han, L.; Zhang, L.; Chen, C.; Zheng, C. Design, synthesis, and antitumor activity evaluation of carbazole derivatives with potent HDAC inhibitory activity. Medicinal Chemistry Research 2023, 32, 1677–1689. Lee, Y. T.; Tan, Y. J.; Oon, C. E. Benzimidazole and its derivatives as cancer therapeutics: The potential role from traditional to precision medicine. Acta Pharmaceutica Sinica B 2023, 13, 478–497. Chen, C.; Li, X.; Zhao, H.; Liu, M.; Du, J.; Zhang, J.; Yang, X.; Hou, X.; Fang, H. Discovery of DNA-Targeting HDAC Inhibitors with Potent Antitumor Efficacy In Vivo That Trigger Antitumor Immunity. Journal of Medicinal Chemistry 2022, 65, 3667–3683. Chen, C.; Hou, X.; Wang, G.; Pan, W.; Yang, X.; Zhang, Y.; Fang, H. Design, synthesis and biological evaluation of quinoline derivatives as HDAC class I inhibitors. European Journal of Medicinal Chemistry 2017, 133, 11–23. Scheme Schemes 1 and 2 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files 1.jpg Scheme 1. Synthesis of target compounds 5a-f. Reagents and conditions: (a) Na 2 S 2 O 5, EtOH, H 2 O, reflux, 4h; (b) EDCI, HOBT, DCM, rt, 6h; (c)NH 2 OH•HCl, KOH, MeOH, rt, 1-2 h. 2.jpg Scheme 2. Synthesis of target compounds 8a-f. Reagents and conditions: (a) Benzaldehyde, Na 2 S 2 O 5, EtOH, H 2 O, 80℃, 8h; (b) TBTU, DCM, rt, 6h; (c) NH 2 OH•HCl, KOH, MeOH, rt, 4 h. supportinginformation.docx Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Medicinal Chemistry Research → Version 1 posted Editorial decision: Revision requested 28 Aug, 2024 Editor assigned by journal 28 Aug, 2024 Submission checks completed at journal 28 Aug, 2024 First submitted to journal 28 Aug, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4988136","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":348162843,"identity":"a393b985-bc9d-49a8-9197-8ca90b4bbdcd","order_by":0,"name":"Jiantao Ping","email":"","orcid":"","institution":"Qilu University of Technology (Shandong Academy of Sciences)","correspondingAuthor":false,"prefix":"","firstName":"Jiantao","middleName":"","lastName":"Ping","suffix":""},{"id":348162845,"identity":"009d864a-b1a4-4b4c-98c9-e5d50ad678b4","order_by":1,"name":"Hongrui Chu","email":"","orcid":"","institution":"Qilu University of Technology (Shandong Academy of Sciences)","correspondingAuthor":false,"prefix":"","firstName":"Hongrui","middleName":"","lastName":"Chu","suffix":""},{"id":348162852,"identity":"04034721-5f1b-4476-9018-ce68e9cfb58b","order_by":2,"name":"Yisheng Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYDACZgY2CEMCiD8wWICYBsRrYZwB1khICwOSFmYeYrQYHGd/9uDjjloG+dnNDx/blEkkNrA3b5NgqLmDW8thHnPDmWeOMzDOOWZsnHMOqIXnWJkEw7Fn+LSwSfO2HWNglkgwk85tA2qRyDGTYGw4jEcL+zPpv0AtbBLp36QtQVrk3xDSwmAmzdhWw8ADNBzIANnCg1+L5GEeM8netgMMEhI5xYY95ySM23jSii0SjuHWwnf++DOJn211DPIz0jc++FFmI9vPfnjjjQ81uLUoHABTh+sbwDQbNJoScGpgYJCHKK2DctnwKB0Fo2AUjIIRCwA4A014GczBcgAAAABJRU5ErkJggg==","orcid":"","institution":"Qilu University of Technology (Shandong Academy of Sciences)","correspondingAuthor":true,"prefix":"","firstName":"Yisheng","middleName":"","lastName":"Zhao","suffix":""},{"id":348162859,"identity":"e40ef35b-a4ba-41b7-a7e7-abc1f492ed7f","order_by":3,"name":"Chen Chen","email":"","orcid":"","institution":"Shandong Chengchuang Blue Sea Pharmaceutical Technology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-08-28 04:55:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4988136/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4988136/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00044-024-03349-2","type":"published","date":"2024-11-21T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65385374,"identity":"1ec9783a-a163-4257-989b-292014ad5494","added_by":"auto","created_at":"2024-09-26 19:37:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27947,"visible":true,"origin":"","legend":"\u003cp\u003eDesign strategy of target compounds\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4988136/v1/d42d2e45e463f7c00efdfbdc.jpg"},{"id":65385712,"identity":"9c4f3df8-e35f-412b-90b5-2bd5fe7c9396","added_by":"auto","created_at":"2024-09-26 19:45:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":152617,"visible":true,"origin":"","legend":"\u003cp\u003eInducing apoptosis of HEL cells by \u003cstrong\u003e8f\u003c/strong\u003e at 1.25, 2.5 and 5 μM after 24 h.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4988136/v1/fa58f0215cd78e9dfeb5d651.jpg"},{"id":65385377,"identity":"17f176d5-24e8-43c6-887d-f16dcf048631","added_by":"auto","created_at":"2024-09-26 19:37:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25034,"visible":true,"origin":"","legend":"\u003cp\u003eHEL cells were treated with DMSO, \u003cstrong\u003e8f \u003c/strong\u003eor SAHA for 24 h.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4988136/v1/e5b593bc7c1d68b62377533a.jpg"},{"id":69835160,"identity":"948e01cd-d57f-4455-b876-c6f5609bfe0a","added_by":"auto","created_at":"2024-11-25 16:12:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1169426,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4988136/v1/3069f714-1b6a-4d40-87f9-07c1a18107df.pdf"},{"id":65385375,"identity":"1c3c82da-8229-410a-b1ea-2d73f6ddd82a","added_by":"auto","created_at":"2024-09-26 19:37:01","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Synthesis of target compounds \u003cstrong\u003e5a-f\u003c/strong\u003e. Reagents and conditions: (a) Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5, \u003c/sub\u003eEtOH, H\u003csub\u003e2\u003c/sub\u003eO, reflux, 4h; (b) EDCI, HOBT, DCM, rt, 6h; (c)NH\u003csub\u003e2\u003c/sub\u003eOH•HCl, KOH, MeOH, rt, 1-2 h.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4988136/v1/39b50e5ee8ea309c8136d4dc.jpg"},{"id":65385713,"identity":"3dd3ed31-bd92-4a79-a81b-1db995e6a6b6","added_by":"auto","created_at":"2024-09-26 19:45:01","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 2.\u003c/strong\u003e Synthesis of target compounds \u003cstrong\u003e8a-f\u003c/strong\u003e. Reagents and conditions: (a) Benzaldehyde, Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5, \u003c/sub\u003eEtOH, H\u003csub\u003e2\u003c/sub\u003eO, 80℃, 8h; (b) TBTU, DCM, rt, 6h; (c) NH\u003csub\u003e2\u003c/sub\u003eOH•HCl, KOH, MeOH, rt, 4 h.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4988136/v1/bc4bc798a0a59d3ac722bd86.jpg"},{"id":65385379,"identity":"f3cc400f-0209-4f74-b7b8-a8a15b59085c","added_by":"auto","created_at":"2024-09-26 19:37:01","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1023197,"visible":true,"origin":"","legend":"","description":"","filename":"supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4988136/v1/7cd0685417aac2cd32389e87.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Design, synthesis and antitumor activity evaluation of Benzimidazole derivatives with potent HDAC inhibitory activity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHistone deacetylases (HDACs) are enzymes that remove acetyl groups from histones, leading to chromatin condensation and repression of gene transcription\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. By inhibiting the activity of HDACs, HDAC inhibitors promote hyperacetylation of histones and alter the structure of chromatin, resulting in increased accessibility of DNA to transcription factors and ultimately leading to changes in gene expression patterns\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This can lead to the activation or suppression of genes involved in cell cycle regulation, apoptosis, differentiation, and other cellular processes relevant to cancer development\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHDAC inhibitors, also known as histone deacetylase inhibitors, are a class of compounds that have gained significant attention in the field of cancer research and treatment\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These compounds work by inhibiting the activity of histone deacetylases, which play a role in regulating gene expression\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The dysregulation of gene expression is often observed in cancer cells, making HDAC inhibitors an attractive target for therapeutic intervention\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBenzimidazole derivatives have garnered significant attention in cancer research as HDAC inhibitors, due to their potential as anti-cancer agents\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In our study, we have reported a series of Benzimidazole derivatives designed to target both the DNA minor groove and histone deacetylases\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The most active compounds not only demonstrated enhanced HDAC inhibitory effects but also exhibited potent inhibition activities against tumors by eliciting anti-tumor immunity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBuilding on our previous experience in designing benzimidazole HDAC inhibitors, this article presents the synthesis and evaluation of a new series of benzimidazole-hydroxamate derivatives for their biological activities. The compounds were subjected to biological characterizations, including assessment of HeLa nuclear extract activity, HDAC isozymes activity, and pro-apoptotic activity. Among the compounds tested, \u003cb\u003e8f\u003c/b\u003e emerged as the most potent compound. The findings regarding the synthesis and biological activities of these derivatives are detailed in this article.\u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Chemistry\u003c/h2\u003e\n \u003cp\u003eThe synthesis methods of all target compounds are described in Schemes \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. As shown in Schemes \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, compound \u003cstrong\u003eA\u003c/strong\u003e couple with o-Phenylenediamine in the presence of Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e to yield benzimidazole derivative \u003cstrong\u003eC\u003c/strong\u003e. Compound \u003cstrong\u003eC\u003c/strong\u003e do coupling reaction with different carbamate hydrochlorides to get \u003cstrong\u003e4a\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e, which were next converted to hydroxamic acids \u003cstrong\u003e5a\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e by treating with hydroxylamine. The synthesis methods in Schemes \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e were similar to Schemes \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Inhibitory activity of HeLa cell extract\u003c/h2\u003e\n \u003cp\u003eUnlike previous our reported benzimidazole HDAC inhibitors, we use amide bonds instead of oxygen to obtain a new series of compounds \u003cstrong\u003e5a\u003c/strong\u003e-\u003cstrong\u003e5f\u003c/strong\u003e. Then HDAC enzymes inhibition assay was conducted to verify their inhibitory activity. Unfortunately, their inhibitory activity improved little, and the most active compound \u003cstrong\u003e5f\u003c/strong\u003e only showed efficacy comparable to SAHA. Subsequently, 6-substituted benzimidazole derivatives were designed and synthesized. Compared to \u003cstrong\u003e5f\u003c/strong\u003e and SAHA, the activity of \u003cstrong\u003e8e\u003c/strong\u003e and \u003cstrong\u003e8f\u003c/strong\u003e has increased significantly, up to 10 nM.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable.1.\u003c/strong\u003e The chemical structures and HDACs inhibitory activities of hydroxamate derivatives.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\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\u003eCpd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCpd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e503.2\u0026thinsp;\u0026plusmn;\u0026thinsp;78.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5057-75(25)\u003cstrong\u003e8d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e237.4\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e306.5\u0026thinsp;\u0026plusmn;\u0026thinsp;44.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e5f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89.8\u0026thinsp;\u0026plusmn;\u0026thinsp;11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSAHA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. HDAC isoform selectivity\u003c/h2\u003e\n \u003cp\u003eCompounds \u003cstrong\u003e8e\u003c/strong\u003e and \u003cstrong\u003e8f\u003c/strong\u003e were chosen based on their in vitro inhibitory activities against different HDAC isoforms. As shown in Table 2, the two compounds demonstrated superior activities against HDAC1, 2, and 6 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;100nM) compared to HDAC8 (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;1000 nM). In comparison to SAHA, the two compounds displayed enhanced inhibitory activities against HDAC1 and HDAC2. Notably, compound \u003cstrong\u003e8e\u003c/strong\u003e exhibited a significantly improved HDAC1 inhibitory activity (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.4 nM), which is eight times better than that of SAHA (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;72 nM).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable.2.\u003c/strong\u003e HDAC isoforms inhibitory activity of compounds \u003cstrong\u003e4k\u003c/strong\u003e, \u003cstrong\u003e4l, 8e, 8f\u003c/strong\u003e and \u003cstrong\u003eSAHA\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tabb\" border=\"1\"\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCpd.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e(nM)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHDAC1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHDAC2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHDAC6\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHDAC8\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\u003e8e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16776\u0026thinsp;\u0026plusmn;\u0026thinsp;1036\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3829\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSAHA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e72\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61\u0026thinsp;\u0026plusmn;\u0026thinsp;11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17879\u0026thinsp;\u0026plusmn;\u0026thinsp;93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. In vitro antiproliferative assay\u003c/h2\u003e\n \u003cp\u003eCompounds \u003cstrong\u003e8e\u003c/strong\u003e and \u003cstrong\u003e8f\u003c/strong\u003e were chosen for evaluation of their in vitro anti-proliferative activities due to their strong inhibition of HDACs. SAHA was utilized as the positive control. However, these two compounds exhibited weak inhibitory activity on the four selected cell lines. Only compound\u0026nbsp;\u003cstrong\u003e8f\u003c/strong\u003e demonstrated a 2 \u0026micro;M inhibition activity on HEL cells.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAnti-proliferative activities of compounds \u003cstrong\u003e4k\u003c/strong\u003e, \u003cstrong\u003e4l\u003c/strong\u003e, \u003cstrong\u003e8e\u003c/strong\u003e, \u003cstrong\u003e8f\u003c/strong\u003e and \u003cstrong\u003eSAHA\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCpd\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003csup\u003ea\u003c/sup\u003e(\u0026micro;M)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHEL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eKG1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eK562\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePC-3\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\u003e8e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSAHA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Apoptotic assay\u003c/h2\u003e\n \u003cp\u003eAnnexin VFTIC/propidium iodide (PI) assay was performed to investigate the apoptosis-inducing ability of compound \u003cstrong\u003e8f\u003c/strong\u003e. HEL cells were treated with compounds \u003cstrong\u003e8f\u003c/strong\u003e at 1.25, 2. 5 and 5 \u0026micro;M for 24 h. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, compounds \u003cstrong\u003e8f\u003c/strong\u003e could significantly induce apoptosis in a dose-dependent manner. Notably, the apoptosis-inducing ability of \u003cstrong\u003e8f\u003c/strong\u003e were much stronger than those of SAHA at the concentration of 5 \u0026micro;M, confirming its notable cellular potencies.\u003c/p\u003e\n \u003cp\u003eThe Annexin V/propidium iodide (PI) assay was conducted to investigate the apoptosis-inducing potential of compound \u003cstrong\u003e8f\u003c/strong\u003e. HEL cells were exposed to varying concentrations of compound \u003cstrong\u003e8f\u003c/strong\u003e (1.25, 2.5, and 5 \u0026micro;M) for a duration of 24 hours. As depicted in Fig. 5, compound \u003cstrong\u003e8f\u003c/strong\u003e demonstrated a significant dose-dependent induction of apoptosis. Notably, the apoptotic effects induced by compound \u003cstrong\u003e8f\u003c/strong\u003e were considerably stronger than those observed with SAHA at a concentration of 5 \u0026micro;M, thus confirming its remarkable cellular potency.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Western Blot Analysis\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the results of western blot analysis to determine the cellular potency of compound \u003cstrong\u003e8f\u003c/strong\u003e. The results showed that compound \u003cstrong\u003e8f\u003c/strong\u003e dose-dependently increased the acetylation level of H3 and tubulin.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn this study, a series of novel benzimidazole-hydroxamate analogues were designed and synthesized as HDAC inhibitors. Compound \u003cb\u003e8f\u003c/b\u003e exhibited potent enzyme inhibition and demonstrated inhibitory effects on tumor cells through screening. Flow cytometry assay indicated that compound \u003cb\u003e8f\u003c/b\u003e effectively induced apoptosis. Furthermore, western blot assay revealed that compound 8f had the capacity to induce histone h3 and tubulin acetylation. These results suggest that benzimidazole-hydroxamate derivative \u003cb\u003e8f\u003c/b\u003e may serve as a potential candidate for further structural optimization research in anti-tumor molecule development.\u003c/p\u003e"},{"header":"4. Experiment sections","content":"\u003cp\u003e4.1. General synthesis of compounds\u003c/p\u003e\n\u003cp\u003eGeneral procedure for the synthesis of\u0026nbsp;\u003cstrong\u003e4a\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003e7a\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003eC\u003c/strong\u003e (1 mmol, 0.32 g, 1 equiv) and amino acid esters (1 mmol, 1.1 equiv) were dissolved in DMF. TBTU (1.2 g, 0.31 mmol, 1.2 equiv) and DIEA (0.39 g, 3 mmol, 3 equiv) were added to the solution. The mixture was stirred for 4 h before being poured into water and extracted with ethyl acetate (3 \u0026times; 25 mL). The organic layers were combined and washed successively with a solution of 1 M citric acid, saturated NaHCO\u003csub\u003e3\u003c/sub\u003e solution, and brine; then dried over anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e before being filtered and evaporated to yield the crude product. The crude product was subsequently purified by column chromatography using petroleum ether/EtOAc as eluent to afford 0.34 g of compound \u003cstrong\u003e4a\u003c/strong\u003e as a light yellow solid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl (4-(1H-benzo[d]imidazol-2-yl)benzoyl)glycinate\u003c/strong\u003e (\u003cstrong\u003e4a\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;73%.\u003csup\u003e\u0026nbsp;1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.25 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 8.01 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 7.76 \u0026ndash; 7.52 (m, 2H), 7.46 (m, 2H), 3.59 (s, 3H), 3.41 (m, 2H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 3-(4-(1H-benzo[d]imidazol-2-yl)benzamido)propanoate\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e4b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;54%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.38 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 8.09 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 2H), 7.96 (d, \u003cem\u003eJ\u003c/em\u003e = 1.6 Hz, 2H), 7.64 \u0026ndash; 7.56 (m, 1H), 3.69 (s, 3H), 3.50 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H), 2.31 (t, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eethyl 4-(4-(1H-benzo[d]imidazol-2-yl)benzamido)butanoate\u003c/strong\u003e (\u003cstrong\u003e4c\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield: 64%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 8.62 (t, \u003cem\u003eJ\u003c/em\u003e = 5.6 Hz, 1H), 8.30 \u0026ndash; 8.22 (m, 2H), 8.04 \u0026ndash; 7.99 (m, 2H), 7.72 \u0026ndash; 7.66 (m, 1H), 7.59 \u0026ndash; 7.53 (m, 1H), 7.30 \u0026ndash; 7.17 (m, 2H), 4.06 (q, \u003cem\u003eJ\u003c/em\u003e = 7.1 Hz, 2H), 3.32 (d, \u003cem\u003eJ\u003c/em\u003e = 6.2 Hz, 2H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.81 (p, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H), 1.19 (t, \u003cem\u003eJ\u003c/em\u003e = 7.1 Hz, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 5-(4-(1H-benzo[d]imidazol-2-yl)benzamido)pentanoate\u003c/strong\u003e (\u003cstrong\u003e4d\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield: 56%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 13.06 (s, 1H), 8.60 (s, 1H), 8.25 (d, \u003cem\u003eJ\u003c/em\u003e = 6.7 Hz, 2H), 8.01 (d, \u003cem\u003eJ\u003c/em\u003e = 6.8 Hz, 2H), 7.62 (d, \u003cem\u003eJ\u003c/em\u003e = 50.8 Hz, 2H), 7.23 (s, 2H), 3.59 (s, 3H), 3.30 (t, 2H), 2.37 (t, 2H), 1.51-1.57 (m, 4H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 6-(4-(1H-benzo[d]imidazol-2-yl)benzamido)hexanoate\u003c/strong\u003e (\u003cstrong\u003e4e\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield: 47%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 8.57 (t, \u003cem\u003eJ\u003c/em\u003e = 5.6 Hz, 1H), 8.28 \u0026ndash; 8.22 (m, 2H), 8.03 \u0026ndash; 7.97 (m, 2H), 7.62 (s, 2H), 7.23 (dq, \u003cem\u003eJ\u003c/em\u003e = 7.1, 3.7 Hz, 2H), 3.58 (s, 3H), 3.28 (q, \u003cem\u003eJ\u003c/em\u003e = 6.9 Hz, 2H), 2.32 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.61 \u0026ndash; 1.48 (m, 4H), 1.34 (dtd, \u003cem\u003eJ\u003c/em\u003e = 8.6, 6.2, 2.1 Hz, 2H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eethyl 7-(4-(1H-benzo[d]imidazol-2-yl)benzamido)heptanoate\u003c/strong\u003e (\u003cstrong\u003e4f\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield: 73%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) \u0026delta; 8.01 (d, \u003cem\u003eJ\u003c/em\u003e = 7.9 Hz, 2H), 7.66 (d, \u003cem\u003eJ\u003c/em\u003e = 8.2 Hz, 4H), 7.28 (d, \u003cem\u003eJ\u003c/em\u003e = 3.5 Hz, 2H), 6.74 (t, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 1H), 4.11 (q, \u003cem\u003eJ\u003c/em\u003e = 7.1 Hz, 2H), 3.43 (q, \u003cem\u003eJ\u003c/em\u003e = 6.7 Hz, 2H), 2.27 (t, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 1.60 (t, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 4H), 1.34 (dt, \u003cem\u003eJ\u003c/em\u003e = 7.3, 4.1 Hz, 4H), 1.24 (t, \u003cem\u003eJ\u003c/em\u003e = 7.1 Hz, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl (2-phenyl-1H-benzo[d]imidazole-6-carbonyl)glycinate\u003c/strong\u003e (\u003cstrong\u003e7a\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield: 71%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 13.19 (s, 1H), 8.98 (s, 1H), 8.21 (d, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H), 7.79 (d, \u003cem\u003eJ\u003c/em\u003e = 8.0 Hz, 1H), 7.64 \u0026ndash; 7.50 (m, 4H), 4.06 (d, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 2H), 3.68 (s, 3H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 3-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)propanoate\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e7b\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield: 79%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u0026delta; 8.22 (d, \u003cem\u003eJ\u003c/em\u003e = 4.7 Hz, 1H), 8.18 (d, \u003cem\u003eJ\u003c/em\u003e = 2.4 Hz, 1H), 7.64 (s, 1H), 7.45 (t, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 1H), 7.11 (t, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 1H), 3.80 (s, 1H), 3.71 (d, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 1H), 2.68 (dd, \u003cem\u003eJ\u003c/em\u003e = 10.5, 4.4 Hz, 1H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 4-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)butanoate\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e7c\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield: 81%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.58 \u0026ndash; 8.44 (m, 1H), 8.20 (d, \u003cem\u003eJ\u003c/em\u003e = 7.6 Hz, 2H), 8.02 (s, 1H), 7.71 (d, \u003cem\u003eJ\u003c/em\u003e = 3.9 Hz, 1H), 7.56 (dt, \u003cem\u003eJ\u003c/em\u003e = 11.8, 5.0 Hz, 4H), 3.59 (s, 3H), 3.34 \u0026ndash; 3.27 (m, 2H), 2.40 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.88 \u0026ndash; 1.77 (m, 2H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 5-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)pentanoate\u003c/strong\u003e (\u003cstrong\u003e7d\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield: 44%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.48 (d, \u003cem\u003eJ\u003c/em\u003e = 19.6 Hz, 1H), 8.20 (d, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 7.75 (dd, \u003cem\u003eJ\u003c/em\u003e = 17.1, 8.6 Hz, 2H), 7.55 (dq, \u003cem\u003eJ\u003c/em\u003e = 14.3, 7.0 Hz, 4H), 3.59 (s, 3H), 3.30 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H), 2.37 (t, \u003cem\u003eJ\u003c/em\u003e = 6.9 Hz, 2H), 1.67 \u0026ndash; 1.50 (m, 4H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 6-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)hexanoate\u003c/strong\u003e (\u003cstrong\u003e7e\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield: 59%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.46 (d, \u003cem\u003eJ\u003c/em\u003e = 22.5 Hz, 1H), 8.22 \u0026ndash; 8.18 (m, 2H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 1H), 7.70 (d, \u003cem\u003eJ\u003c/em\u003e = 3.3 Hz, 1H), 7.62 \u0026ndash; 7.50 (m, 4H), 3.58 (s, 3H), 3.28 (dd, \u003cem\u003eJ\u003c/em\u003e = 12.8, 6.5 Hz, 2H), 2.32 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.65 \u0026ndash; 1.49 (m, 4H), 1.34 (dd, \u003cem\u003eJ\u003c/em\u003e = 15.3, 8.2 Hz, 2H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emethyl 7-(2-phenyl-1H-benzo[d]imidazole-6-carboxamido)heptanoate\u003c/strong\u003e (\u003cstrong\u003e7f\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield: 61%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.44 (s, 1H), 8.21 (d, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 1H), 8.13 (s, 1H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 1H), 7.67 \u0026ndash; 7.50 (m, 2H), 3.58 (s, 2H), 3.29 (d, \u003cem\u003eJ\u003c/em\u003e = 6.3 Hz, 1H), 2.30 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 1H), 1.55 (d, \u003cem\u003eJ\u003c/em\u003e = 6.5 Hz, 2H), 1.33 (d, \u003cem\u003eJ\u003c/em\u003e = 3.5 Hz, 2H).\u003c/p\u003e\n\u003cp\u003eGeneral procedure for the synthesis of\u0026nbsp;\u003cstrong\u003e5a\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e,\u0026nbsp;\u003cstrong\u003e8a\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis route was similar to previous publication\u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(1H-benzo[d]imidazol-2-yl)-N-(2-(hydroxyamino)-2-oxoethyl)benzamide\u003c/strong\u003e (\u003cstrong\u003e5a\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;43%.\u003csup\u003e\u0026nbsp;1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 10.66 (s, 1H), 8.92 (t, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 1H), 8.30 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 2H), 8.08 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 2H), 7.67 (dd, \u003cem\u003eJ\u003c/em\u003e = 6.0, 3.1 Hz, 2H), 7.30 (dd, \u003cem\u003eJ\u003c/em\u003e = 6.0, 3.1 Hz, 2H), 3.84 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H).\u0026nbsp;\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 166.30, 150.47, 135.82, 131.89, 128.59, 126.97, 123.53, 115.59, 41.17.\u0026nbsp;HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 311.1138, found: 311.1136.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(1H-benzo[d]imidazol-2-yl)-N-(3-(hydroxyamino)-3-oxopropyl)benzamide\u003c/strong\u003e (\u003cstrong\u003e5b\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;64%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 10.49 (s, 1H), 8.78 (s, 1H), 8.68 (t, \u003cem\u003eJ\u003c/em\u003e = 5.4 Hz, 1H), 8.26 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 2H), 8.01 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 2H), 7.69 (d, \u003cem\u003eJ\u003c/em\u003e = 6.5 Hz, 1H), 7.56 (d, \u003cem\u003eJ\u003c/em\u003e = 6.6 Hz, 1H), 7.23 (s, 2H), 3.49 (dd, \u003cem\u003eJ\u003c/em\u003e = 12.9, 6.8 Hz, 2H), 2.30 (t, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 167.77, 166.07, 150.85, 144.28, 135.70, 132.93, 128.29, 126.68, 123.39, 122.38, 119.54, 111.97, 36.60, 32.89. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 325.1295, found: 325.1292.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(1H-benzo[d]imidazol-2-yl)-N-(4-(hydroxyamino)-4-oxobutyl)benzamide\u003c/strong\u003e (\u003cstrong\u003e5c\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;73%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 8.84 (t, \u003cem\u003eJ\u003c/em\u003e = 5.5 Hz, 1H), 8.46 (d, \u003cem\u003eJ\u003c/em\u003e = 8.2 Hz, 2H), 8.15 (d, \u003cem\u003eJ\u003c/em\u003e = 8.2 Hz, 2H), 7.85 (dd, \u003cem\u003eJ\u003c/em\u003e = 6.1, 3.1 Hz, 2H), 7.55 (dd, \u003cem\u003eJ\u003c/em\u003e = 6.1, 3.2 Hz, 2H), 3.31 (p, \u003cem\u003eJ\u003c/em\u003e = 6.5, 6.1 Hz, 2H), 2.06 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.80 (q, \u003cem\u003eJ\u003c/em\u003e = 7.2 Hz, 2H).\u0026nbsp;\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 174.98, 167.03, 153.43, 130.66, 130.32, 129.47, 129.33, 127.13, 33.94, 29.24, 22.59. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 337.1311, found: 337.1306.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(1H-benzo[d]imidazol-2-yl)-N-(5-(hydroxyamino)-5-oxopentyl)benzamide\u003c/strong\u003e (\u003cstrong\u003e5d\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;66%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.79 (t, \u003cem\u003eJ\u003c/em\u003e = 5.5 Hz, 1H), 8.45 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 2H), 8.14 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 2H), 7.85 (dt, \u003cem\u003eJ\u003c/em\u003e = 6.6, 3.3 Hz, 2H), 7.61 \u0026ndash; 7.52 (m, 2H), 3.33 \u0026ndash; 3.26 (m, 2H), 2.01 (t, \u003cem\u003eJ\u003c/em\u003e = 6.7 Hz, 2H), 1.66 \u0026ndash; 1.48 (m, 4H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 169.58, 166.97, 153.36, 130.72, 130.18, 129.47, 127.18, 122.17, 32.75, 29.48, 26.66, 25.44.\u0026nbsp;HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 353.1535, found: 353.1539.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(1H-benzo[d]imidazol-2-yl)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide\u003c/strong\u003e (\u003cstrong\u003e5e\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;83%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 8.72 (t, \u003cem\u003eJ\u003c/em\u003e = 5.5 Hz, 1H), 8.35 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 8.11 (d, \u003cem\u003eJ\u003c/em\u003e = 8.2 Hz, 2H), 7.81 (dd, \u003cem\u003eJ\u003c/em\u003e = 6.1, 3.2 Hz, 2H), 7.50 (dt, \u003cem\u003eJ\u003c/em\u003e = 6.0, 3.6 Hz, 2H), 3.29 (q, \u003cem\u003eJ\u003c/em\u003e = 6.7 Hz, 3H), 1.96 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.54 (q, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 4H), 1.31 (q, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 2H).\u0026nbsp;\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 167.45, 166.72, 150.77, 130.72, 129.50, 127.38, 125.93, 122.20, 38.88, 33.45, 29.64, 26.39, 24.25. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 366.1813, found: 366.1812.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(1H-benzo[d]imidazol-2-yl)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide\u003c/strong\u003e (\u003cstrong\u003e5f\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield: 40%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 10.37 (s, 1H), 8.73 (t, \u003cem\u003eJ\u003c/em\u003e = 5.6 Hz, 1H), 8.43 \u0026ndash; 8.36 (m, 2H), 8.16 \u0026ndash; 8.09 (m, 2H), 7.84 (dd, \u003cem\u003eJ\u003c/em\u003e = 6.1, 3.1 Hz, 2H), 7.57 \u0026ndash; 7.51 (m, 2H), 3.29 (q, \u003cem\u003eJ\u003c/em\u003e = 6.6 Hz, 2H), 1.95 (t, \u003cem\u003eJ\u003c/em\u003e = 7.4 Hz, 2H), 1.60 \u0026ndash; 1.45 (m, 4H), 1.37 \u0026ndash; 1.21 (m, 4H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 174.95, 166.06, 151.59, 133.03, 131.85, 129.93, 128.31, 124.50, 114.33, 114.13, 34.12, 29.46, 28.80, 26.75, 24.95. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 381.1921, found: 381.1913.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN-(2-(hydroxyamino)-2-oxoethyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e8a\u003c/strong\u003e) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;61%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 10.62 (s, 1H), 8.81 (s, 1H), 8.74 (d, \u003cem\u003eJ\u003c/em\u003e = 21.0 Hz, 1H), 8.22 (d, \u003cem\u003eJ\u003c/em\u003e = 6.9 Hz, 2H), 7.84 \u0026ndash; 7.68 (m, 2H), 7.59 \u0026ndash; 7.51 (m, 3H), 3.83 (d, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 167.40, 166.70, 153.56, 130.73, 130.25, 129.49, 128.64, 127.16, 41.23. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 311.1006, found: 311.1002.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN-(3-(hydroxyamino)-3-oxopropyl)-2-phenyl-1H-benzo[d]imidazole-6 carboxamide(8b)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;61%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 10.55 (s, 1H), 8.85 (t, \u003cem\u003eJ\u003c/em\u003e = 5.3 Hz, 1H), 8.55 \u0026ndash; 8.42 (m, 2H), 8.28 (s, 1H), 7.99 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 1H), 7.83 (d, \u003cem\u003eJ\u003c/em\u003e = 8.5 Hz, 1H), 7.76 \u0026ndash; 7.66 (m, 3H), 7.41 (t, \u003cem\u003eJ\u003c/em\u003e = 50.8 Hz, 1H), 3.51 (dd, \u003cem\u003eJ\u003c/em\u003e = 12.7, 6.8 Hz, 2H), 2.33 (t, \u003cem\u003eJ\u003c/em\u003e = 7.1 Hz, 2H).\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 167.81, 166.08, 151.21, 133.38, 129.96, 128.59, 124.83, 36.75, 32.86. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 325.1222, found: 325.1226.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN-(4-(hydroxyamino)-4-oxobutyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide\u003c/strong\u003e(\u003cstrong\u003e8c\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;49%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 10.41 (s, 1H), 8.71 (s, 1H), 8.50 (d, \u003cem\u003eJ\u003c/em\u003e = 22.2 Hz, 1H), 8.21 (d, \u003cem\u003eJ\u003c/em\u003e = 7.9 Hz, 2H), 7.79 \u0026ndash; 7.66 (m, 2H), 7.61 \u0026ndash; 7.49 (m, 4H), 3.32 \u0026ndash; 3.25 (m, 2H), 2.05 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.83 \u0026ndash; 1.73 (m, 2H).\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 169.46, 167.12, 153.44, 130.71, 130.24, 129.49, 129.23, 127.14, 122.24, 30.56, 25.90. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 339.1379, found: 339.1382.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN-(5-(hydroxyamino)-5-oxopentyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide\u0026nbsp;\u003c/strong\u003e(\u003cstrong\u003e8d\u003c/strong\u003e)\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;33%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 8.69 (s, 1H), 8.47 (d, \u003cem\u003eJ\u003c/em\u003e = 6.2 Hz, 1H), 8.24 (d, \u003cem\u003eJ\u003c/em\u003e = 7.9 Hz, 2H), 7.71 \u0026ndash; 7.61 (m, 2H), 7.51 \u0026ndash; 7.42 (m, 4H), 3.43 \u0026ndash; 3.36 (m, 2H), 2.07 (t, J = 5.7 Hz, 2H), 1.66 \u0026ndash; 1.51 (m, 4H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 169.55, 167.00, 130.69, 130.28, 129.49, 127.12, 32.54, 29.37, 23.29. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 353.1535, found: 353.1539.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN-(6-(hydroxyamino)-6-oxohexyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide\u003c/strong\u003e(\u003cstrong\u003e8e\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield:\u0026nbsp;42%.\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 10.34 (s, 1H), 8.66 (s, 1H), 8.45 (s, 1H), 8.20 (d, \u003cem\u003eJ\u003c/em\u003e = 7.1 Hz, 2H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e = 8.0 Hz, 1H), 7.65 \u0026ndash; 7.45 (m, 4H), 3.30 \u0026ndash; 3.23 (m, 2H), 1.96 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.61 \u0026ndash; 1.49 (m, 4H), 1.36 \u0026ndash; 1.26 (m, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 169.58, 166.97, 153.39, 130.72, 130.21, 129.50, 129.38, 127.13, 122.20, 39.68, 32.75, 29.49, 26.66, 25.45. HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 367.1692, found: 367.1695.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN-(7-(hydroxyamino)-7-oxoheptyl)-2-phenyl-1H-benzo[d]imidazole-6-carboxamide\u003c/strong\u003e(\u003cstrong\u003e8f\u003c/strong\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYield: 81%. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) \u0026delta; 10.34 (s, 1H), 8.65 (s, 1H), 8.44 (d, \u003cem\u003eJ\u003c/em\u003e = 5.3 Hz, 1H), 8.21 (d, \u003cem\u003eJ\u003c/em\u003e = 7.1 Hz, 2H), 8.12 (s, 1H), 7.75 (d, \u003cem\u003eJ\u003c/em\u003e = 8.4 Hz, 1H), 7.66 \u0026ndash; 7.50 (m, 4H), 3.28 (dd, \u003cem\u003eJ\u003c/em\u003e = 6.4 Hz, 2H), 1.95 (t, \u003cem\u003eJ\u003c/em\u003e = 7.3 Hz, 2H), 1.58 \u0026ndash; 1.47 (m, 4H), 1.37 \u0026ndash; 1.25 (m, 4H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) \u0026delta; 169.62, 166.94, 153.28, 130.85, 129.94, 129.51, 127.22, 122.33, 32.73, 29.61, 28.87, 26.77, 25.62.\u0026nbsp;HRMS (ESI) m/z Calcd [M+H]\u003csup\u003e+\u003c/sup\u003e 381.1848, found: 381.1891.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. HeLa nuclear extract and HDAC enzymatic assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mixed solution containing the enzyme solution (HeLa nuclear extract, HDAC1, HDAC2, HDAC6 or HDAC8), the tested compound solution, and the fluorogenic substrate solution [Boc-Lys (acetyl)-AMC or Boc-Lys (trifluoroacetyl)-AMC] was plated onto an enzyme-labeled plate. After a 30-minute incubation at 37 \u0026deg;C, the reaction was stopped by trypsin and trichostatin A. Fluorescence analysis was performed using a Microplate reader (Tecan) with excitation/emission wavelengths of 360/460 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 In vitro antiproliferative activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCancer cells were seeded into 96-well plates and then\u0026nbsp;treated with various concentrations of compounds. After co-incubation for 48 hours, 20\u0026mu;l of CCK8 solution was added to each well and incubated for 2 hours at 37\u0026deg;C. A microplate reader (Tecan) was used to measure the absorbance at 450 nm for each well.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Flow cytometric assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEL cells were treated with compounds for 24 hours. Then the cells were collected pellets for annexin V-FITC/PI staining. The stained cells were analyzed by a flow cytometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5. Werstern blot assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCancer cells were treated with compounds for 24 hours. Subsequently, the cells were collected and pellets were obtained for protein extraction. The protein content was determined using the BCA protein determination assay. Equal amounts of total protein were measured for Western blot analysis, including Ac-HH3 and Ac-Tubulin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJiantao Ping: Investigation, Visualization, Funding acquisition, Project administration.Hongrui Chu: Investigation, Methodology, Writing-original draft.Yisheng Zhao: Investigation, Visualization,Funding acquisition.Chen Chen: Conceptualization, Investigation,Supervision, Formal analysis, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was funded by the Education and Industry Integration Pilot Project of Qilu University of Technology (2022PY036), the National Natural Science Foundation of China (No. 62105175), Natural Science Foundation of Shandong Province (ZR2021QF058, ZR2021QB174)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHo, T. C. S.; Chan, A. H. Y.; Ganesan, A. Thirty Years of HDAC Inhibitors: 2020 Insight and Hindsight. Journal of Medicinal Chemistry 2020, 63, 12460\u0026ndash;12484.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng, B.; Pan, W.; Xiao, Y.; Ding, Z.; Zhou, Y.; Fei, X.; Liu, J.; Su, Z.; Peng, X.; Chen, J. HDAC-targeting epigenetic modulators for cancer immunotherapy. European Journal of Medicinal Chemistry 2024, 265, 116129.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, I. C.; Sethy, B.; Liou, J. P. Recent Update of HDAC Inhibitors in Lymphoma. Frontiers in Cell and Developmental Biology 2020, 8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X.; Waschke, B. C.; Woolaver, R. A.; Chen, S. M. Y.; Chen, Z.; Wang, J. H. HDAC inhibitors overcome immunotherapy resistance in B-cell lymphoma. Protein \u0026amp; Cell 2020, 11, 472\u0026ndash;482.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, C.; Chu, H. R.; Wang, A. Y.; Yin, H. H.; Gao, Y. Q.; Liu, S. H.; Li, W.; Han, L. Q. Discovery of 2,5-diphenyl-1,3,4-thiadiazole derivatives as HDAC inhibitors with DNA binding affinity. European Journal of Medicinal Chemistry 2022, 241.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, W.-X.; Huang, J.; Tian, X.-Y.; Liu, Y.-H.; Jia, M.-Q.; Wang, W.; Jin, C.-Y.; Song, J.; Zhang, S.-Y. A review of progress in o-aminobenzamide-based HDAC inhibitors with dual targeting capabilities for cancer therapy. European Journal of Medicinal Chemistry 2023, 259, 115673.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiersack, B.; Polat, S.; H\u0026ouml;pfner, M. Anticancer properties of chimeric HDAC and kinase inhibitors. Seminars in Cancer Biology 2022, 83, 472\u0026ndash;486.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, L.; Han, L.; Zhang, L.; Chen, C.; Zheng, C. Design, synthesis, and antitumor activity evaluation of carbazole derivatives with potent HDAC inhibitory activity. Medicinal Chemistry Research 2023, 32, 1677\u0026ndash;1689.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, Y. T.; Tan, Y. J.; Oon, C. E. Benzimidazole and its derivatives as cancer therapeutics: The potential role from traditional to precision medicine. Acta Pharmaceutica Sinica B 2023, 13, 478\u0026ndash;497.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, C.; Li, X.; Zhao, H.; Liu, M.; Du, J.; Zhang, J.; Yang, X.; Hou, X.; Fang, H. Discovery of DNA-Targeting HDAC Inhibitors with Potent Antitumor Efficacy In Vivo That Trigger Antitumor Immunity. Journal of Medicinal Chemistry 2022, 65, 3667\u0026ndash;3683.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, C.; Hou, X.; Wang, G.; Pan, W.; Yang, X.; Zhang, Y.; Fang, H. Design, synthesis and biological evaluation of quinoline derivatives as HDAC class I inhibitors. European Journal of Medicinal Chemistry 2017, 133, 11\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Benzimidazole derivatives, HDAC inhibitors, Anti-tumor","lastPublishedDoi":"10.21203/rs.3.rs-4988136/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4988136/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aims to design and synthesize novel benzimidazole HDAC inhibitors to explore their potential applications in the treatment of cancer and other related diseases. By comparing the structures of our reported benzimidazole HDAC inhibitors, we designed a series of compounds accordingly. We then used experimentally verified their inhibitory activity against HDAC enzymes. The results showed that several of the newly synthesized compounds showed good HDAC inhibition and anti-proliferative activity. Therefore, we conclude that these novel HDAC inhibitors have potential as drug candidates for the treatment of cancer.\u003c/p\u003e","manuscriptTitle":"Design, synthesis and antitumor activity evaluation of Benzimidazole derivatives with potent HDAC inhibitory activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-26 19:36:57","doi":"10.21203/rs.3.rs-4988136/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-28T14:46:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-28T07:36:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-28T07:36:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medicinal Chemistry Research","date":"2024-08-28T04:53:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9fc19076-e492-4391-ad30-878c9b96b5dd","owner":[],"postedDate":"September 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:06:10+00:00","versionOfRecord":{"articleIdentity":"rs-4988136","link":"https://doi.org/10.1007/s00044-024-03349-2","journal":{"identity":"medicinal-chemistry-research","isVorOnly":false,"title":"Medicinal Chemistry Research"},"publishedOn":"2024-11-21 15:57:23","publishedOnDateReadable":"November 21st, 2024"},"versionCreatedAt":"2024-09-26 19:36:57","video":"","vorDoi":"10.1007/s00044-024-03349-2","vorDoiUrl":"https://doi.org/10.1007/s00044-024-03349-2","workflowStages":[]},"version":"v1","identity":"rs-4988136","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4988136","identity":"rs-4988136","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}