Life Sciences 93 (2013) 904–911

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Synthesis, angiopreventive activity, and in vivo tumor inhibition of novel benzophenone–benzimidazole analogs V. Lakshmi Ranganatha a, B.R. Vijay Avin b, Prabhu Thirusangu b, T. Prashanth a, B.T. Prabhakar b, Shaukath Ara Khanum a,⁎ a

Department of Chemistry, Yuvaraj's College (Autonomous), University of Mysore, Mysore 570 005, Karnataka, India Molecular Biomedicine Laboratory, Postgraduate Department of Studies and Research in Biotechnology, Sahyadri Science College (Autonomous), Kuvempu University, Shimoga 577203, Karnataka, India

b

a r t i c l e

i n f o

Article history: Received 9 July 2013 Accepted 1 October 2013 Keywords: Benzophenone–benzimidazoles Methoxy group Cytotoxicity Angioprevention

a b s t r a c t Aim: The development of anticancer drugs with specific targets is of prime importance in modern biology. This study investigates the angiopreventive and in vivo tumor inhibition activities of novel synthetic benzophenone– benzimidazole analogs. Main methods: The multistep synthesis of novel benzophenone–benzimidazole analogs (8a–n) allowing substitution with methoxy, methyl and halogen groups at different positions on the identical chemical backbone and the variations in the number of substituents were synthesized and characterized. The newly synthesized compounds were further evaluated for cytotoxic and antiproliferative effects against Ehrlich ascites carcinoma (EAC) cells. The potent lead compounds were further assessed for antiangiogenic effects in a CAM model and a tumor-induced vasculature in vivo model. The effect of angioprevention on tumor growth was verified in a mouse model. Key findings: The cytotoxicity studies revealed that compounds 8f and 8n are strongly cytotoxic. Analyzing the structure–activity relationship, we found that an increase in the number of methyl groups in addition to methoxy substitution at the para position of the benzoyl ring in compound 8n resulted in higher potency compared to 8f. Furthermore, neovessel formation in in vivo systems, such as the chorioallantoic membrane (CAM) and tumor-induced mice peritoneum models, was significantly suppressed and reflected the tumor inhibition observed in mice. Significance: These results suggest the potential clinical application of compound 8n as an antiangiogenic drug for cancer therapy. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved.

Introduction The identification of novel molecular targets for cancer therapy has led to a paradigm shift in drug development, and more emphasis is now placed on molecules that can effectively inhibit the angiogenesis process, in which new blood vessels are formed from preexisting ones to support the growth of tumors and the development of cancer (Folkman, 2007; Albini et al., 2012). Angiogenesis inhibitors are desirable anticancer targets and minimize the side effects of chemotherapy. Specificity in targeting tumor endothelial cells and the formation of neovessels are unique properties that could be incorporated into a potential antiangiogenic drug for cancer therapy (Alicia Chung et al., 2010; Johannessen et al., 2013). Benzophenone derivatives obtained from natural (Henry Jacobs et al., 1999) and synthetic (Karrer et al., 2000) methods are pharmacologically ⁎ Corresponding author at: Department of Chemistry, Yuvaraj's College, University of Mysore, Mysore, India. Tel.: +91 99018 88755; fax: +91 821 2419239. E-mail address: [email protected] (S.A. Khanum).

active molecules (Tzvetomira et al., 2009; Yamazaki et al., 2012) and even display antitumor activity (Sakowski et al., 2001; Hsieh et al., 2003; Balasubramanyam et al., 2004; Prabhakar et al., 2006a, 2006b). Several benzophenones are under study; combretastatin A-4 is known to exhibit antiangiogenic effects and is being studied in clinical trials (Tozer et al., 2008). The presence of a benzimidazole nucleus in numerous categories of therapeutic agents has made it an indispensable anchor for the development of new antiangiogenic therapeutics (Yogita and Silakari, 2012). Previously, we reported the synthesis and antitumor and antiangiogenic properties of (2aroyl-4-methylphenoxy)acetamides 4a–e, which are benzophenone analogs (Prabhakar et al., 2006a, 2006b). More specifically, the benzophenone derivative [2-(4-methoxybenzoyl)-4-methylphenoxy]N-(4-chlorophenyl) acetamide (BP-1, IC50: 42.50 μM) was shown to inhibit angiogenesis, thereby preventing angiogenesis-dependent disorders, such as mammary carcinoma and rheumatoid arthritis, both in vivo and in vitro, where it down-regulated the vascular endothelial growth factor (VEGF) gene expression responsible for angiogenesis (Prabhakar et al., 2006a, 2006b; Shankar et al., 2009). The promising

0024-3205/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.10.001

V.L. Ranganatha et al. / Life Sciences 93 (2013) 904–911

results obtained in that earlier study prompted us to synthesize a new series of analogs, 8a–n, by retaining the methoxy group and varying the position and number of methyl and halogen substituents on the benzophenone ring. In addition, a benzimidazole ring is incorporated in place of the substituted phenyl acetamide group to provide novel benzophenone–benzimidazole analogs 8a–n. In the present study, we have synthesized, characterized and evaluated the biological activity of novel new analogs 8a–n with a special emphasis on their angiopreventive abilities, resulting in tumor inhibition in mice. Among the series of fourteen analogs, compound 8n was identified as the most promising antiangiogenic compound. Experimental

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water to remove potassium carbonate and extracted with diethyl ether (3 × 50 ml). The ether layer was washed with 10% sodium hydroxide solution (3 × 50 ml) followed by water (3 × 30 ml), dried over anhydrous sodium sulfate, and evaporated to dryness to obtain the crude solid, which was recrystallized with ethanol to afford compounds 5a–n. The characterization data for compound 5a are provided as a representative example. 5a Yield 90%. mp 49–52 °C. IR (Nujol, cm−1): 1664 (C_O), 1760 (ester, C_O). 1H NMR (DMSO): δ 1.2 (t, 3H, CH3 of ester), 2.3 (s, 3H, CH3), 4.1 (q, 2H, CH2 of ester), 4.5 (s, 2H, OCH2), 7.1–7.7 (m, 8H, Ar\H). LC–MS m/z 299 (M + 1). Anal. Calcd for C18H18O4: C, 72.47; H, 6.04%. Found: C, 72.46; H, 6.12%.

All solvents and reagents were purchased from Sigma Aldrich Chemicals Pvt Ltd. Melting points were determined on an electrically heated VMP-III melting point apparatus. FT-IR spectra were recorded using KBr disks and Nujol on an FT-IR Jasco 4100 infrared spectrophotometer. 1H NMR spectra were recorded using a Bruker DRX 400 spectrometer at 400 MHz with TMS as an internal standard. Mass spectra were recorded on an LC–MS/MS (API-4000) mass spectrometer. Further elemental analysis of the compounds was performed on a Perkin Elmer 2400 elemental analyzer.

General procedure for the preparation of (4-benzoyl-2-methylphenoxy) acetic acids 6a–n Compound 5a (6.0 mmol) was dissolved in ethanol (15 ml) and treated with a solution of sodium hydroxide (15 mmol) in water (5 ml). The reaction mixture was stirred at reflux for 5–6 h, cooled and acidified with 1 N hydrochloric acid. The precipitate was filtered, washed with water and recrystallized from methanol to afford compound 6a in good yield. Compounds 6b–n were synthesized analogously starting with 5b–n, respectively. The characterization data for compound 6a are provided as a representative example.

Chemistry

6a Yield 75%. mp 130–132 °C. FT-IR (KBr, cm−1): 1675 (C_O), 1730 (acid C_O), 3400–3500 (acid OH). 1H NMR (DMSO): δ 2.3 (s, 3H, CH3), 4.46 (s, 2H, OCH2), 7.2–7.7 (m, 8H, Ar\H), 9.5 (s, 1H, COOH). LC–MS m/z 271 (M + 1). Anal. Calcd for C16H14O4: C, 71.10; H, 5.22%. Found: C, 71.13; H, 5.26%.

General procedure for the preparation of phenylbenzoates 3a–n Substituted benzoates 3a–n were synthesized by benzoylation of substituted phenols 1a–b with benzoyl chlorides 2a–g (1:1) using 10% sodium hydroxide solution. The reaction was stirred for 2–3 h at 0 °C. The reaction was monitored by TLC using a 4:1 n-hexane:ethyl acetate solvent mixture as the eluent. After completion of the reaction, the oily product was extracted with diethyl ether. The ether layer was washed with 10% sodium hydroxide solution (3 × 50 ml) followed by water (3 × 30 ml) and then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to afford compounds 3a–n. The characterization data for compound 3a are provided as a representative example. 3a Yield 90%. Pale yellow liquid. IR (neat): 1715 cm−1 (C_O). 1H NMR (DMSO): δ 2.45 (s, 3H, Ar\CH3), 7.5–8.2 (m, 9H, Ar\H). Anal. Calcd for C14H12O2 (212): C, 79.22; H, 5.70%. Found: C, 79.18; H, 5.76%. General procedure for the preparation of 4-hydroxybenzophenones 4a–n Substituted 4-hydroxy-diarylmethanones, commonly known as hydroxybenzophenones (4a–n), were synthesized via the Fries rearrangement. Compounds 3a–n, (0.001 mol) were treated with anhydrous aluminum chloride (0.002 mol) as a catalyst at 150–170 °C neat for 2–3 h. The reaction mixture was then cooled to room temperature and quenched with 6 N HCl in the presence of ice water. The reaction mixture was stirred for 2–3 h and filters, and the resulting solid was recrystallized with methanol to obtain compounds 4a–n. The characterization data for compound 4a are provided as a representative example. 4a Yield 72%. mp 125–128 °C. IR: (KBr, cm−1) 1640 (C_O), 3510–3600 (O\H); 1H NMR (DMSO): δ 2.35 (s, 3H, CH3), 6.71–7.50 (m, 8H, Ar\H), 12.20 (bs, 1H, OH). LC–MS m/z 212 (M + 1). Anal. Calcd for C14H12O2 (212): C, 79.22; H, 5.70%. Found: C, 79.18; H, 5.69%. General procedure for the preparation of (4-benzoyl-2-methylphenoxy) acetic acid ethyl esters 5a–n Compounds 5a–n were obtained by stirring a mixture of compounds 4a–n (0.013 mol) and ethyl chloroacetate (0.026 mol) in dry acetone (50 ml) with anhydrous potassium carbonate (0.019 mol) at reflux for 8–9 h. The reaction mixture was cooled, and the solvent was removed by distillation. The residual mass was triturated with cold

General procedure for the preparation of N-(2-aminophenyl)-2-(4-benzoyl2-methylphenoxy) acetamides 7a–n To compounds 6a–n (0.0037 mol) in dry dichloromethane (15 ml), lutidine (1.2 vol.) was added at 25–30 °C, followed by the addition of 1,2-diaminobenzene (0.0037 mol). The mixture was stirred at 25–30 °C for 30 min. The reaction was cooled to 0–5 °C, and TBTU (0.0037 mol) was added over a period of 30 min while maintaining the temperature below 5 °C. The reaction was stirred overnight and monitored by TLC using chloroform:methanol (9:1). The reaction mixture was diluted with 20 ml of dichloromethane and treated with 1.5N HCL solution (20 ml). The organic layer was washed with water (25 ml), dried over anhydrous sodium sulfate, concentrated to a syrupy liquid and recrystallized twice from diethyl ether to afford compounds 7a–n. The characterization data for compound 7a are provided as a representative example. 7a Yield 95%. mp 190–192 °C. FT-IR (KBr, cm−1): 1675 (C_O), 1730 (amide C_O), 3120–3220 (amide CO\NH). 1H NMR (DMSO): δ 2.2 (s, 3H, CH3), 4.56 (s, 2H, OCH2), 7.1–7.6 (m, 12H, Ar\H), 9.8 (s, 1H, NH), 11.5 (d, 2H, NH2). LC–MS m/z 361 (M + 1). Anal. Calcd for C22H20N2O3: C, 73.32; H, 5.59; N, 7.77%. Found: C, 73.37; H, 5.66; N, 7.80%. General procedure for the preparation of [4-(1H-benzimidazol-2ylmethoxy)-3-methylphenyl]-phenyl-methanones 8a–n Compounds 8a–n were synthesized via cyclization of compounds 7a–n using neat acetic acid as the cyclizing agent under refluxing conditions for 5–6 h. The reaction was monitored by TLC using n-hexane:dichloromethane:acetone (5:3:2). After completion of the reaction, the reaction mixture was cooled to room temperature, poured into crushed ice and neutralized with a 10% sodium bicarbonate solution. After filtration, the solid was recrystallized from acetonitrile. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-phenylmethanone 8a. Yield 80%. mp 205–207 °C. FT-IR (KBr, cm−1): 1698 (C_N), 3176 (N\H), 1H NMR (DMSO): δ 2.4 (s, 3H, CH3), 4.50 (s, 2H, OCH2),

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7.3–8.1 (m, 12H, Ar\H), 11.89 (s, 1H, NH). LC–MS m/z 343 (M + 1). Anal. Calcd for C22H18N2O2: C, 77.17; H, 5.30; N, 8.18%. Found: C, 77.24; H, 5.35; N, 8.22%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-(2-fluorophenyl) methanone 8b. Yield 82%. mp 200–202 °C. FT-IR (KBr, cm−1): 1690 (C_N), 3186 (N\H), 1H NMR (DMSO): δ 2.2 (s, 3H, CH3), 4.81 (s, 2H, OCH2), 7.00–8.05 (m, 11H, Ar\H), 11.86 (s, 1H, NH). LC–MS m/z 361 (M + 1). Anal. Calcd for C22H17FN2O2: C, 73.32; H, 4.75; N, 7.77%. Found: C, 73.34; H, 4.85; N, 7.72%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-(4-fluorophenyl) methanone 8c. Yield 70%. mp 204–206 °C. FT-IR (KBr, cm−1): 1678 (C_N), 3196 (N\H), 1H NMR (DMSO): δ 2.15 (s, 3H, CH3), 4.30 (s, 2H, OCH2), 7.2–8.0 (m, 11H, Ar\H), 11.81 (s, 1H, NH). LC–MS m/z 361 (M + 1). Anal. Calcd for C22H17FN2O2: C, 73.32; H, 4.75; N, 7.77%. Found: C, 73.38; H, 4.79; N, 7.82%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-(3-bromophenyl) methanone 8d. Yield 78%. mp 185–187 °C. FT-IR (KBr, cm−1): 1688 (C_N), 3172 (N\H). 1H NMR (DMSO): δ 2.31 (s, 3H, CH3), 4.80 (s, 2H, OCH2), 7.1–8.0 (m, 11H, Ar\H), 11.89 (s, 1H, NH). LC–MS m/z 422 (M + 1). Anal. Calcd for C22H17N2O2: C, 62.72; H, 4.07; N, 6.65. Found: C, 62.76; H, 4.10; N, 6.69%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-(3-fluorophenyl) methanone 8e. Yield 83%. mp 210–211 °C. FT-IR (KBr, cm−1): 1692 (C_N), 3188 (N\H), 1H NMR (DMSO): δ 2.15 (s, 3H, CH3), 4.88 (s, 2H, OCH2), 7.10–8.10 (m, 11H, Ar\H), 11.81 (s, 1H, NH). LC–MS m/z 361 (M + 1). Anal. Calcd for C22H17FN2O2: C, 73.32; H, 4.75; N, 7.77. Found: C, 73.34; H, 4.85; N, 7.72%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-(4-methoxyphenyl) methanone 8f. Yield 88%. mp 160–163 °C. FT-IR (KBr, cm−1): 1698 (C_N), 3176 (N\H). 1H NMR (DMSO): δ 2.25 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 4.55 (s, 2H, OCH2), 7.2–8.2 (m, 11H, Ar\H), 11.77 (s, 1H, NH). LC–MS m/z 373 (M + 1). Anal. Calcd for C23H20N2O3: C, 74.18; H, 5.41; N, 7.52%. Found: C, 74.24; H, 5.45; N, 7.56%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-(2-chlorophenyl) methanone 8g. Yield 74%. mp 192–195 °C. FT-IR (KBr, cm−1): 1698 (C_N), 3176 (N\H). 1H NMR (DMSO): δ 2.18 (s, 3H, CH3), 4.40 (s, 2H, OCH2), 7.15–8.10 (m, 11H, Ar\H), 11.87 (s, 1H, NH). LC–MS m/z 377 (M + 1). Anal. Calcd for C22H17ClN2O2: C, 70.12; H, 4.55; N, 7.43%. Found: C, 70.17; H, 4.59; N, 7.48%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-o-tolylmethanone 8h. Yield 83%. mp 163–164 °C. FT-IR (KBr, cm−1): 1688 (C_N), 3179 (N\H), 1H NMR (DMSO): δ 2.38 (s, 3H, CH3), 2.22 (s, 3H, CH3), 4.30 (s, 2H, OCH2), 7.05–8.10 (m, 11H, Ar\H), 11.25 (s, 1H, NH). LC–MS m/z 357 (M + 1). Anal. Calcd for C23H20N2O2: C, 77.51; H, 5.66; N, 7.86. Found: C, 77.55; H, 5.69; N, 7.90%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-m-tolylmethanone 8i. Yield 76%. mp 169–171 °C. FT-IR (KBr, cm−1): 1698 (C_N), 3176 (N\H), 1H NMR (DMSO): δ 2.38 (s, 3H, CH3), 2.22 (s, 3H, CH3), 4.30 (s, 2H, OCH2), 7.03–8.20 (m, 11H, Ar\H), 11.25 (s, 1H, NH). LC–MS m/z 357 (M + 1). Anal. Calcd for C23H20N2O2: C, 77.51; H, 5.66; N, 7.86. Found: C, 77.55; H, 5.69; N, 7.90%. [4-(1H-benzimidazol-2-ylmethoxy)-3-methylphenyl]-(4-bromophenyl) methanone 8j. Yield 81%. mp 176–177 °C. FT-IR (KBr, cm−1): 1680 (C_N), 3178 (N\H), 1H NMR (DMSO): δ 2.31 (s, 3H, CH3), 4.80 (s, 2H, OCH2), 7.1–8.0 (m, 11H, Ar\H), 11.88 (s, 1H, NH). LC–MS m/z 422 (M + 1). Anal. Calcd for C22H17N2O2: C, 62.72; H, 4.07; N, 6.65%. Found: C, 62.76; H, 4.10; N, 6.69%.

[4-(1H-benzimidazol-2-ylmethoxy)-3,5-dimethylphenyl]-phenylmethanone 8k. Yield 80%. mp 187–188 °C. FT-IR (KBr, cm−1): 1672 (C_N), 3188 (N\H), 1H NMR (DMSO): δ 2.30–2.45 (s, 6H, 2CH3), 4.85 (s, 2H, OCH2), 7.18–8.14 (m, 11H, Ar\H), 11.44 (s, 1H, NH). LC–MS m/z 357 (M + 1). Anal. Calcd for C23H20N2O2: C, 77.51; H, 5.66; N, 7.86%. Found: C, 77.56; H, 5.69; N, 7.88%. [4-(1H-benzimidazol-2-ylmethoxy)-3,5-dimethylphenyl]-p-tolylmethanone 8l. Yield 70%. mp 169–171 °C. FT-IR (KBr, cm−1): 1698 (C_N), 3176 (N\H), 1H NMR (DMSO): δ 2.12–2.34 (s, 6H, 2CH3), 2.42 (s, 3H, CH3), 4.80 (s, 2H, OCH2), 7.3–8.1 (m, 10H, Ar\H), 10.8 (s, 1H, NH). LC–MS m/z 371 (M + 1). Anal. Calcd for C24H22N2O2: C, 77.81; H, 5.99; N, 7.56%. Found: C, 77.85; H, 5.96; N, 7.59%. [4-(1H-benzimidazol-2-ylmethoxy)-3,5-dimethylphenyl]-(4-fluorophenyl)methanone 8m. Yield 76%. mp 181–183 °C. FT-IR (KBr, cm− 1): 1698 (C_N), 3176 (N\H), 1H NMR (DMSO): δ 2.22–2.40 (s, 6H, 2CH3), 4.80 (s, 2H, OCH2), 7.3–8.1 (m, 10H, Ar\H), 11.55 (s, 1H, NH). LC–MS m/z 375 (M + 1). Anal. Calcd for C23H19FN2O2: C, 73.78; H, 5.11; N, 7.48%. Found: C, 73.79; H, 5.14; N, 7.51%. [4-(1H-benzimidazol-2-ylmethoxy)-3,5-dimethylphenyl]-(4-methoxyphenyl)methanone 8n. Yield 88%. mp 172–174 °C. FT-IR (KBr, cm−1): 1686 (C_N), 3179 (N\H), 1H NMR (DMSO): δ 2.22–2.35 (s, 6H, CH3), 3.44 (s, 3H, OCH3), 4.62 (s, 2H, OCH2), 7.20–8.13 (m, 10H, Ar\H), 10.8 (s, 1H, NH). LC–MS m/z 387 (M + 1). Anal. Calcd for C24H22N2O3: C, 74.59; H, 5.74; N, 7.25. Found: C, 74.63; H, 5.77; N, 7.29%. Biology Cell culture and in vitro compound treatment EAC cells were used for the present study and were cultured as reported previously (Prabhakar et al., 2006a, 2006b). The cells were treated with varying concentrations of compounds 8a–n (0, 10, 20, 50, and 100 μM in DMSO) for various time intervals (0–48 h) and utilized in further experiments. An appropriate vehicle control was used, and each experiment was repeated a minimum of three times independently. Trypan blue dye exclusion assay The effect of compounds 8a–n on the cell viability of EAC cells was determined using a trypan blue dye exclusion assay (Prabhakar et al., 2006a, 2006b). EAC cells treated with or without the compounds were harvested and resuspended in 0.4% trypan blue, and the viable cells were counted using a hemocytometer. The inhibitory concentration (IC50) value was estimated after 48 h of treatment. MTT assay The effect of compounds 8a–n on cell proliferation of EAC cells was determined by MTT assay, as described previously (Prabhakar et al., 2006a, 2006b). Cells treated with or without compounds were incubated for 48 h. MTT reagent (5 mg/mL) was added, and the color change due to the proliferating cells was estimated. LDH release assay A lactate dehydrogenase (LDH) assay was performed to assess LDH release following the treatment of EAC cells with compounds 8a–n (0, 10, 20, 50, and 100 μM) after 48 h of incubation, as described previously (Kavitha et al., 2009). Briefly, the cells were lysed using 0.1% Triton X-100 in PBS. The amount of LDH released in both the culture media and the cell lysate was measured at 490 nm using an ELISA reader (Robotronics, India). The percentage of LDH release was calculated as LDH release in media / (LDH release in media + intracellular LDH release) × 100.

V.L. Ranganatha et al. / Life Sciences 93 (2013) 904–911

Mayo Lynx Reg microscope, as previously reported (Prabhakar et al., 2006a, 2006b).

Chorioallantoic membrane (CAM) assay The inhibition of neovessels induced by rVEGF165 was analyzed following the treatment of fertilized egg CAM with compounds 8f and 8n (10 μM) (Prabhakar et al., 2006a, 2006b), and changes in the microvessel density (MVD) were photographed using a Nikon D3200 camera.

Statistical analysis Values are expressed as the means + SEM for the control and experimental samples, and statistical analysis was performed using student's t-test. The values were considered statistically significant if the p value was less than 0.05.

In vivo tumor growth and treatment with compounds EAC cells were cultured in vivo (n = 10), and the compounds 8f and 8n were partially dissolved in DMSO and diluted with sterile PBS. Three doses were injected intraperitoneally (i.p.) at a concentration of 100mg/kg body weight using a 26 gauge needle every other day starting on the fourth day of tumor implantation. An appropriate vehicle control was maintained. The survivability of the animals (n = 10) was also determined (Prabhakar et al., 2006a, 2006b).

Results and discussion Chemistry The synthesis of compounds 8a–n is outlined in Scheme 1. Substituted phenols 1a–b reacted with substituted benzoyl chlorides 2a–j in the presence of 10% sodium hydroxide to give phenyl benzoates 3a–n. Compounds 3a–n underwent Fries rearrangement to afford substituted diaryl methanones, commonly known as hydroxyl benzophenones (4a–n). Compounds 4a–n were reacted with ethyl chloroacetate to give ethyl(2-aroyl-4-methylphenoxy)

Peritoneal angiogenesis assay and H&E staining for MVD The suppression of angiogenesis in the peritoneum of tumorbearing mice treated with or without compounds 8f and 8n was photographed, and the formaldehyde-fixed peritoneum was processed for H&E staining for the measurement of MVD using a Lawrence and COCl R2

OH R1

R

+ 1(a-b)

R 10% NaOH 2-3 h stirring

R3 2(a-j)

O

R2 R3 R4

3(a-n)

R Anhydrous AlCl3

O C

R1

R4 a; R2=R3=R4=H b; R2=R3=H, R4=CH3 c; R3= R2=H, R4=F d; R2=R4=H, R3=Br e; R3=R4=H, R2=F f; R2=R3=H, R4=OCH3 g; R3=R4=H, R2=Cl h; R3=R4=H, R2=CH3 i; R2=R4=H, R3=CH3 j; R2=R3=H, R4=Br

a; R=CH3, R1=H b; R=R1=CH3

907

R3

HO

R2

R1

C

R4

ClCH 2COOC 2H5

0

150-170 C

K2CO3/Acetone

O 4(a-n) R

O O

O

R2

R1

C 5(a-n)

R4

NaOH/H2O

O

C2H5OH

R3

O

R2

R1

C 6(a-n) O

O

R4

NH 2 TBTU/Lutidine DCM/STIRRING

N N H

R

OH

R3

R1 O

R2

R

C

R4 ACETIC ACID H 2N REFLUX

8(a-n) O a; R=CH3, R1=R2=R3=R4=H b; R=CH 3, R2=F, R1=R3=R4= H 3 c; R=CH 3, R1=R2=R3=H, R4=F d; R=CH 3, R1= R2=R4=H, R3=Br e; R=CH3, R1= R3=R4=H, R2=F f; R=CH3, R1=R2=R3=H, R4=OCH 3 g; R=CH3, R1=R3=R4=H, R2=Cl

HN

R3

R

O

R3

NH 2

O

R2

R1

C O

R4

7(a-n)

h; R=CH3, R1= R3=R4=H, R2=CH3 i; R=CH3, R1=R2=R4=H, R3=CH3 j; R=CH3, R1= R2=R3=H, R4=Br k; R=R1=CH3, R2=R3=R4=H l; R=R1=CH3, R2=R3=H, R4=CH3 m; R=R1=CH3, R2=R3=H, R4=F n; R=R1=CH3, R2=R3=H, R4=OCH3

Scheme 1. Synthesis of benzophenone-integrated benzimidazole analogs.

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acetates 5a–n in excellent yield; 5a–n provided (4-aroyl-phenoxy) ethanoic acids 6a–n upon alkaline hydrolysis. N-(2-Aminophenyl)2-(4-benzoylphenoxy)acetamides 7a–n were obtained by treating compounds 6a–n with 1,2-diaminobenzene in the presence of N,N,N′,N′-tetramethyl-o-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU) and 2,6-dimethylpyridine (lutidine) as coupling agents. Finally, the title compounds 8a–n were obtained in excellent yield by treating compounds 7a–n with glacial acetic acid as a cyclizing agent. Pharmacology Ehrlich ascites carcinoma (EAC) cells were used for preliminary anticancer screening of the newly synthesized compounds 8a–n. To assess their cytotoxicity and antiproliferative effects, three independent assays, trypan blue dye exclusion, MTT assay and LDH release assay, were performed against EAC cells growing in the log phase by treatment with varying concentrations (0–100 μM) of compounds 8a–n. The more potent analogs were further assayed for angiopreventive activity in the in vivo CAM model, in which angiogenesis was induced by rVEGF165 or by tumors in the mouse peritoneum. In addition, the effect of the compounds on tumor growth was evaluated. 8f and 8n as lead compounds In the present study, the cytotoxic and antiproliferative effects of compounds 8a–n on EAC cells were investigated following 48 h of exposure using three independent assay systems (trypan blue, MTT and LDH release). This method is a preliminary screening method to eliminate those analogs that do not show any cytotoxic effects. The EAC cells were treated with increasing concentrations of the compounds (0, 10, 20, 50 and 100 μM in DMSO), and cell viability was determined using a trypan blue assay. DMSO was used as the solvent to dissolve the compounds, and DMSO alone served as a vehicle control. The results showed that the levels of sensitivity differed drastically among the compounds. Compound 8f, with one methyl group on the phenyl ring and one methoxy group on the benzoyl ring, and 8n, with two methyl groups on the phenyl ring and a methoxy group on the benzoyl ring, showed potent activity by inducing minimum viability at inhibitory concentrations of 17 μM and 12 μM, respectively. Compounds 8a, 8b, 8e, 8g, 8i, 8k and 8m showed moderate activity, whereas compounds 8c, 8d, 8h, 8j and 8l showed less sensitivity. Importantly, these compounds do not contain a methoxy group. The IC50 values of each compound were calculated and are summarized in Table 1. The cytotoxic effects of compounds 8a–n on cell proliferation were further tested using MTT assay. The results observed agreed with Table 1 IC50 values of compounds 8a–n calculated based on trypan blue, MTT and LDH release assay at 48 h in EAC cells. Based on the IC50 values, compounds 8f and 8n were chosen as lead compounds.

Control 8a 8b 8c 8d 8e 8f 8g 8h 8i 8j 8k 8l 8m 8n

Trypan blue assay IC50 value (μM)

MTT assay IC50 values (μM)

LDH release assay IC50 values (μM)

41 25 N100 N100 33 17 28 91 23 82 35 N100 24 12

38 22 98 N100 30 16 26 87 19 78 29 N100 23 10

39 23 99 98 32 17 26 90 24 81 34 99 26 10

those of the trypan blue assay (Table 1). Compound 8n showed the highest activity, with an IC50 value of ~10 μM, followed by compound 8f, ~16 μM. The obtained cytotoxic data were reconfirmed by an LDH release assay to determine the cellular integrity following treatment with compounds 8a–n (0, 10, 20, 50, and 100 μM). The results showed a concentration-dependent increase in LDH release upon treatment of EAC cells with compounds 8a–n (Table 1). Thus, our studies using three independent cytotoxic assay systems confirmed the cytotoxic nature of compounds 8f and 8n, and these two compounds were further investigated as lead compounds. Structure activity relationship of compounds 8f and 8n Benzophenone derivatives are known to be pharmacologically active molecules against various pathological conditions including cancer (Kumazawa et al., 1997; Hsieh et al., 2003). Earlier, our group reported the antiinflammatory (Khanum et al., 2004), antimicrobial (Khanum et al., 2005), antitumor (Prabhakar et al., 2006a, 2006b) and antiangiogenic (Prabhakar et al., 2006a, 2006b) properties of benzophenone derivatives. The molecular mechanism underlying the antiangiogenic (Prabhakar et al., 2006a, 2006b) effect of BP-1 has been studied extensively Prabhakar et al., 2006a, 2006b; Shankar et al., 2009). Structurally, BP-1 has a methoxy group in the para position of the benzoyl ring and a methyl group at the para position of the phenyl ring (Table 2). BP-1 has an IC50 value of 42.5 μM. Encouraged by the potent activity of BP-1, in the present study, we synthesized analogs of benzophenone (8a–n) that retain the methoxy group and vary the number and position of the methyl and halogen groups on the benzophenone moiety. In addition, we modified the acetamide group and the terminally substituted aromatic rings by replacing them with a benzimidazole ring system. Surprisingly, the compounds with a methoxy group in the para position of the benzoyl ring and a methyl group at the ortho position of the phenyl ring showed a significant cytotoxic effect, with enhancement in the IC50 values, as verified by an MTT assay (Table 2). There was an approximately 3-fold increase in the IC50 value of the newly synthesized compound 8n (~10 μM), with one methoxy and two methyl groups on the benzophenone ring and the newly incorporated benzimidazole ring, compared to the previously reported BP-1 (Prabhakar et al., 2006a, 2006b). Interestingly, 8f has a structure similar to 8n but lacking one methyl group and showed a slightly decreased IC50 (~16 μM). In general, the methoxy and the number of methyl groups at the ortho position played an important role in the biological activity of the compounds. Supporting this, compound 8m, without a methoxy group but with a fluoro and two methyl groups, showed decreased activity compared to 8f and 8n (Table 1). Thus, the methoxy group at the para position and the two methyl groups at the ortho position are important for the biological activity. No other substituents resulted in any significant cytotoxicity (Table 1). Compounds 8f and 8n were selected, based on their significant structure–activity relationships and their increased IC50 values, as lead compounds for further evaluation of their angiopreventive activity. Compounds 8f and 8n inhibit neovascularization and tumor growth in vivo Neovascularization, or microvessel density (MVD), is a widely used surrogate measure in pathological specimens and tumor models to assess disease progression. Increased MVD has a direct correlation with tumor progression, and the inhibition of blood vessels results in regression of tumor growth (Alicia Chung et al., 2010; Shivakumar et al., 2008). Earlier, we showed that benzophenone derivatives are potent antiangiogenic compounds that inhibit neovascularization and tumor growth in mice (Prabhakar et al., 2006a, 2006b). In the present study, inhibition of neovascularization was studied with new benzophenone-benzimidazole derivatives 8f and 8n. In the CAM assay model, we used rVEGF165 (10 ng/embryo) as a potent angiogenic factor (Lee Ellis and Daniel, 2008) to induce neovascularization. Simultaneous

V.L. Ranganatha et al. / Life Sciences 93 (2013) 904–911

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Table 2 Structure–activity relationships of compounds. Compounds

Structure

Methoxy group position

Methyl group position

MTT assay IC50 values (μM)

A* BP-1

Para position of benzoyl ring

(1CH3) Para position of the phenyl ring

42.5

8f

Para position of benzoyl ring

(1CH3) Ortho position of phenyl ring

16

8n

Para position of benzoyl ring

(2CH3) Ortho position of phenyl ring

10

treatment with 8f and 8n was carried out to determine the effects. Compounds 8f and 8n induced a vascular zone in the developing embryos. Notably, newly formed microvessels regressed around the area of the implanted disk (Fig. 1A). Angiogenesis is evident in the inner peritoneal lining of EAC-bearing mice; thus, mouse peritoneum is a reliable model for angiogenesis (Shibuya et al., 1999). Hence, the peritoneal lining of mice treated

with compounds 8f and 8n was examined to gauge the effects on peritoneal angiogenesis. Tumor-bearing mice treated with compounds 8f and 8n showed decreased peritoneal angiogenesis compared to the massive angiogenesis in untreated mice (Fig. 1B). Further H & E staining of the peritoneum sections of the control group appeared well– vascularized, in contrast to the peritoneum sections treated with 8f and 8n, which were characterized by a pronounced decrease in MVD

Fig. 1. Angiopreventive effect of compounds 8f and 8n (A) CAM photos illustrate the formation of blood vessels in the normal, control (VEGF induced) and treated (VEGF + compounds 8f and 8n) groups. Inhibition of angiogenesis is evident. (B) Compounds 8f and 8n suppress EAC-induced neovascularization in the peritoneum of mice. The peritoneum lining of the mice was observed and photographed. (C) Representative photograph of peritoneum sections stained with hematoxylin and eosin showing varying numbers of MVD in normal, control and compound 8f- and 8n-treated mice. (D) Decrease in microvessel density in compound 8f- and 8n-treated mice compared to control mice.

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and the caliber of detectable vascular channels (Fig. 1C). The control peritoneum sections showed 20.4 ± 1.4 vessels/high power field (V/HPF), whereas after treatment with compounds 8f and 8n, the sections showed 9.9 ± 1.9 and 8.2 ± 1.2 V/HPF, respectively (Fig. 1D). This indicates that these compounds primarily act on endothelial cells and thus inhibit angiogenesis. Because angiogenesis plays an important role in tumor growth (Folkman, 1990; Hanahan and Folkman, 1996) and our compounds inhibit angiogenesis, they also inhibit tumor growth as well as the accumulation of ascites fluid in mice, which is angiogenesis dependent (Fig. 2A). Ascites fluid is a direct nutritional source for tumor cells, and a rapid increase in ascites fluid would be a means to meet the nutritional requirements of growing tumor cells [Gupta et al., 2004]. Hence, a decrease in ascites fluid accounts for suppression of tumor growth. Our data showed that the effects of compounds 8f and 8n after i.p. injection was a reduction in the formation of ascites, which directly impacts the growth of the tumor (Fig. 2B and C). The mice bearing Ehrlich ascites carcinoma (EAC) cells survive for only 10 days after implantation of the tumor cells. In the present study, the compounds were administered on the fourth, sixth and eighth day of tumor growth. The control mice died exactly on the tenth day. No treatment was continued for the test animals, and they were kept for survival studies. The results revealed prolonged survivability for the 8f- and 8n-treated mice until the 23rd and 26th day, respectively (Fig. 2D). Compounds 8f and 8n induced tumor inhibitions of ~70% and ~90%, respectively. The body weight of the treated animals was monitored regularly, and a slight increase (~10%) was observed. The measurement of the ascites volume and the tumor cells after the death of the animals indicated a moderate increase, suggesting re-occurrence of the tumor (Fig. 1 supplementary). Hence, compounds 8f and 8n prolonged the lifespan of the animals. The death of the animals may be due to reappearance of the tumor and not the treatment. Surgical pathology studies of dissected organs, such as the liver and spleen, of the treated animals after the 10th and 23rd day were comparable with those of normal animals, confirming

that administration of 8f and 8n did not lead to visible changes (Fig. 2 supplementary). This suggests that the target-specific action of compounds 8f and 8n corresponds to the previously reported angiogenic inhibition, similar to combretastatin A4, curcumin and 2methoxystradiol. The potency of these previously reported angiogenic inhibitors is also due to the presence of a methoxy group (Tozer et al., 2008; Gururaj et al., 2002; Rschin et al., 2003). However, our studies are novel in that the presence of the additional methyl group in compound 8n increased the angiopreventive effect and thus the tumor growth.

Conclusion The regulation of angiogenesis is crucial in cancerous conditions. In the present study, we synthesized [4-(1H-benzimidazol-2-ylmethoxy)3-methylphenyl]-phenylmethanones (8a–n) and studied their cytotoxicity and effects on tumor cell viability. By analyzing the structure– activity relationships, it was determined that compounds with methoxy and methyl groups showed very good activity (8f and 8n). An increase in the number of methyl groups while retaining the methoxy group increased the cytotoxic effects, as shown by compound 8n. There was a direct structure–activity relationship observed with the previously reported molecule BP-1, which also displays angiopreventive activity. We selected both 8f and 8n as lead compounds and further investigated their biological activity. The results observed in the CAM and mouse peritoneum models for the inhibition of neovascularization and tumor growth strongly supported our hypothesis. Therefore, we conclude that 8n (BP-1b) has a strong angiopreventive effect and could be developed further for applications in cancer therapeutics.

Conflict of interest statement The authors declare that there are no conflicts of interest.

Fig. 2. Tumor inhibition effect of compounds 8f and 8n on EAC mice. (A) Decrease in body weight of mice treated with compounds 8f and 8n compared with the control mice. (B) Percentage of tumor growth with and without compounds 8f and 8n. (C) Decrease in ascites secretion after treatment with compounds 8f and 8n. (D) Cumulative survivability curves of EAC-bearing mice treated with and without compounds 8f and 8n.

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Acknowledgments V Lakshmi Ranganatha gratefully acknowledges the financial support provided by the DST, New Delhi, under the INSPIRE Fellowship Scheme [IF110555]. Dr. Shaukath Ara Khanum is grateful to UGC New Delhi for the grant of MRP [UGC No. F.39-737/2010 (SR) dated 06/01/2010]. Dr. B. T. Prabhakar is grateful for the grants provided by VGST (VGST/P-9/ SMYSR/2011-12/1171), SERB-DST (SR/FT/LS-25/2011) and UGC (F. No. 41-507/2012 (SR)). We express our sincere thanks to the National College of Pharmacy, Shimoga, India, for the ethical clearance certificate for the animal experiments. We also extend our thanks to Dr. R. G Nayak, Nanjappa Hospital, Shimoga, for the histopathological experiments. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2013.10.001. References Albini A, Tosetti F, Li VW, Noonan DM, Li WW. Cancer prevention by targeting angiogenesis. Nat Rev Clin Oncol 2012;9(9):498–509. Alicia Chung S, Lee Jhon, Nepoleon F. Targeting the tumor vasculature: insights from physiological angiogenesis. Nat Rev Cancer 2010;10:505–14. Balasubramanyam K, Altaf M, Radhika AV, Swaminathan V, Aarthi R, Parag P, et al. Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression. J Biol Chem 2004;279:33716–26. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:4–6. Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 2007;6(4):273–86. Gupta M, Mazumder UK, Kumar RS, Sivakumar T, Vamsi MLM. Antitumor activity and antioxidant status of Caesalpinia bonducella against Ehrlich ascites carcinoma in Swiss albino mice. J Pharmacol Sci 2004;94:177–84. Gururaj AE, Belakavadi M, Venkatesh DA, Marmé D, Salimath BP. Molecular mechanisms of anti-angiogenic effect of curcumin. Biochem Biophys Res Commun 2002;297(4):934–42. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86(3):353–64. Henry Jacobs GE, Carrington CMS, McLean S, Freeholds W. Prenylated benzophenone derivatives from Caribbean Clusia species (Guttiferae). Plukenetiones B-G and xerophenone A. Tetrahedron 1999;55:1581–96.

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Synthesis, angiopreventive activity, and in vivo tumor inhibition of novel benzophenone-benzimidazole analogs.

The development of anticancer drugs with specific targets is of prime importance in modern biology. This study investigates the angiopreventive and in...
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