Synthesis, antiglycation and antioxidant potentials of benzimidazole derivatives

Accepted Manuscript Synthesis, antiglycation and Antioxidant potentials of Benzimidazole Derivatives Muhammad Taha, Ashik Mosaddik, Fazal Rahim, Sajjad Ali, Mohamed Ibrahim, Noor Barak Almandil PII: DOI: Reference:

S1018-3647(18)30473-7 https://doi.org/10.1016/j.jksus.2018.04.003 JKSUS 622

To appear in:

Journal of King Saud University - Science

Received Date: Accepted Date:

14 March 2018 4 April 2018

Please cite this article as: M. Taha, A. Mosaddik, F. Rahim, S. Ali, M. Ibrahim, N.B. Almandil, Synthesis, antiglycation and Antioxidant potentials of Benzimidazole Derivatives, Journal of King Saud University Science (2018), doi: https://doi.org/10.1016/j.jksus.2018.04.003

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Synthesis, antiglycation and Antioxidant potentials of Benzimidazole Derivatives

Muhammad Tahaa, Ashik Mosaddika, Fazal Rahimb, Sajjad Alic, Mohamed Ibrahima, Noor Barak Almandila a

Department of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia. bDepartment of Chemistry, Hazara University Mansehra-21300, Pakistan cDepartment of Chemistry, Karakoram International University, GilgitBaltistan, Gilgit-15100, Pakistan. 

Correspondence and reprints

E-mail: [email protected] and [email protected],

Abstract Benzimidazole derivatives 1-20 were synthesized and evaluated for antiglycation and antioxidant potentials. Among the series some analogs showed antiglycating potential ranging in between 182.30 ± 1.20 to 473.51 ± 2.17 when compared with standard rutin (IC50 value 295.09 ± 1.04 µM) and for antioxidant potential ranging between 22.42 ± 0.26 to 82.30 ±1.33 when compare with standard Propyl gallate (IC50 value 29.20 ± 1.25). Compound 2, 6, 10 and 19 showed potent antioxidant and antiglycation inhibitory potentials. Compounds 7, 11, 13, 15 and 20 showed moderate antiglycating potential with IC50 values 473.51 ± 2.17,325.20 ± 1.70, 440.0 ± 3.60, 370.60 ± 2.80 and 415.20 ± 3.20μM, and these compounds also showed excellent antioxidant potential with IC50 values73.51 ± 1.17, 45.63 ± 0.92, 82.30 ± 1.33, 75.41 ± 1.51, 40.60 ± 0.80

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and 64.92 ± 1.41μM respectively. The remaining compounds 1, 3, 4,5,8, 9, 12, 14, 16, 17and 18 were found inactive. Keywords: Synthesis, Benzimidazole, antiglycating, Antioxidant, SAR. 1. Introduction Discovery of antiglycating agents is a vital tactic to cure diabetic problems, since at present, number of active antiglycating agents is neglected and there is an urgent demand of new compounds to be identified (Ahmed, 2005). As the hostile occurrence of type-2 diabetes is rising, its damaging effects are frequently abetting the production of advanced glycation end products (Brownlee, 1994). These advanced glycation end products (AGEPs) are significant pathogenic intermediaries of approximately all diabetic problems (Peppa et al., 2003). Glycation is a chaotic process which can take place endogenously (Kellow and Savige, 2013) and modifies biomolecules (Misciagna and Michele, 2007). The process is a non-enzymatic reaction of proteins with sugars (Ho et al., 2010). These modifications lead to impaired protein functions (Goodarzi et al., 2010) and perhaps contributes to microvascular diseases that slows down wound healings of diabetic patients, associated complications and ageing-related sicknesses for instance cataracts, retinopathy, renal dysfunction and arteriosclerosis (Rodriguez and Jarvis, 2012; Anguizola et al., 2013; Akash, et al., 2013). Chemical entities that inhibit or slows down the process of glycation play a pivotal role in the treatment of diabetic complications. Exploration for new antiglycating agents is, therefore, still of great scientific interest (Ahmed, 2005). In recent years, certain food stuffs have been identified and attracted attention as having antiglycating and antioxidant properties (Deetae et al., 2012). The most of diseases are linked with amplified oxidative stress due to either the changes in generation of free radicals or the changes antioxidant balanced activity (Tessier et al., 2009). The major methods of antioxidant resistance comprise enzymes CATs, SODs, and GH-PXs. The a number of vitamins and micronutrients are energetic in reducing free-radical species or necessary as cofactors for inhibitions enzymes (Pinzani et al., 2010). Equally defensive and chain contravention antioxidants have a control over the oxidative stress that goes together with disease and aging (Jacomelli et al., 2010). Lately, The concentration in natural product antioxidants such as phenolic compounds and vitamin E (-tocopherol). Mainly phenolic

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compounds are usually separated into flavonoids, stilbenes, tannins, phenolic acids and lignans (Dai and Mumper, 2010). Compounds having benzimidazoles core are general and significant substructures occurring in nature and other bioactive compounds. They do something as glucagon receptor anagonists (Chang et al., 2001), β-Raf kinase (Takle et al., 2006), α-glucosidase (Taha et al., 2016), βglucuronidase (Taha et al., 2015), antiglycation (Taha et al., 2016), CB1 cannabinoid receptor antagonists (Eyers et al., 1998), carbonic anhydrase (Khan et al., 2013a & 2013b). Several imidazoles work as antibacterial (Antolini et al., 1999), antibiotics (Brogden et al., 1978) fungicides (Santo et al., 2005), anti-inflammatory agents (Brimblecombe et al., 1975), antidiabetic and antihypertensive (Pathan et al., 2006; Bellina et al., 2007).

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2.0 Methods and Materials 2.1. Synthesis of benzimidazole Benzimidazole derivatives 1–20 were synthesized by treating 3.12 mmol of 3,5-dichlorobenzene1,2-diamine with 3.16 mmol of arylaldehyde. Then N, N-dimethylformamide (DMF) 10 ml and sodium metabisulphite (Na2S2O5) 3 mmol were mixed in the reaction. The reaction mixture was mixed and refluxed for 6 h. The reaction was observed by Thin layer chromatography (TLC) when reaction completed; the reaction mixture left for cooling 30 min then 30 ml of water was included which brought out precipitation. Finally, the precipitates were separated by filter and the crude product was recrystalized in ethyl acetate to afford pure crystal (Taha et al., 2015).

2.2. Spectroscopic data 2.2.1. 4,6-dichloro-2-o-tolyl-1H-benzo[d]imidazole (1) 1

H NMR (500 MHz, DMSO): 9.45 (1H, NH) 8.37 (d, J = 2.1 Hz, 1H, Ar-H), 8.19 (s, 1H, Ar-

H), 7.78 (dd, J = 2.1 Hz,7.45 Hz, 1H, Ar-H), 7.47–7.41 (m, 3H, Ar-H), 2.61 (s, 3H, CH3);

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C

NMR (125 MHz, DMSO-d6):  151.8 (C-2), 141.4(C-4), 137.4 (C-10), 136.7 (C-11), 136.4 (C5), 131.5 (C-8), 130.2 (C-15), 129.6 (C-14), 129.5 (C-12), 128.6 (C-13), 122.6 (C-7), 122.4 (C6), 114.0 (C-9), 18.5 (CH3); Anal. Cal for C14H10Cl2N2: C = 60.67, H = 3.64, N = 10.11. Found: C = 60.65, H = 3.60, N = 10.07; EI MS m/z (% rel. abund.): 278 (M+ 2, 16), 276 (M+, 52.9), Yield: 78%. 2.2.2. 4-(4,6-dichloro-1H-benzo[d]imidazol-2-yl) benzene-1,3-diol (2) 1

H NMR (500 MHz, DMSO): 10.2 (s, 1H, NH), 9.60 (s, 2H, 2xOH), 7.98 (d, J = 8.4 Hz, 1H,

Ar-H), 7.62 (d, J = 1.4 Hz, 1H, Ar-H), 7.43 (d, J = 1.4 Hz, 1H, Ar-H), 6.47 (dd, J = 2.4, 8.4 Hz, 1H, Ar-H), 6.47 (d, J = 2.4 Hz, 1H, Ar-H); 13C NMR (125 MHz, DMSO):  160.0 (C-13), 156.4 (C-11), 152.9 (C-2), 141.9 (C-4), 136.4 (C-5), 130.6 (C-8), 130.5 (C-15), 123.7 (C-7), 122.1 (C6), 113.7 (C-9), 110.9 (C-10), 109.2 (C-14), 105.8 (C-12); Anal. Cal for C13H8Cl2N2O2: C = 52.91, H = 2.73, N = 9.49. Found: C = 52.88, H = 2.71, N = 9.47; EI MS m/z (% rel. abund.): 297 (M+ 2, 9), 295 (M+, 31), Yield: 83%. 2.2.3. 4,6-dichloro-2-(pyridin-3-yl)-1H-benzo[d]imidazole (3) 1

H NMR (500 MHz, DMSO):  13.61 (s, 1H, NH), 9.37 (s, 1H, Ar-H), 8.74 (dd, J = 1.5, 5.0 Hz,

1H, Ar-H), 8.55 (s, 1H, Ar-H), 7.64 (s, 1H, Ar-H), 7.63 (dd, J = 5.0, 8.2 Hz, 1H, Ar-H), 7.45 (s, 1H, Ar-H); 13C NMR (125 MHz, DMSO):  155.3 (C-11), 152.9 (C-2), 147.7 (C-13), 141.9 (C4

4), 136.6 (C-5), 135.6 (C-15), 132.7 (C-10), 130.6 (C-8), 124.2 (C-14), 123.7 (C-7), 122.1 (C-6), 113.9 (C-9); Anal. Cal for C12H7Cl2N3: C = 54.57, H = 2.67, N = 15.91. Found: C = 54.55, H = 2.67, N = 15.88; EI MS m/z (% rel. abund.): 265 (M+ 2, 26), 263 (M+, 73), Yield: 74%. 2.2.4. 4,6-dichloro-2-(4-fluorophenyl)-1H-benzo[d]imidazole (4) 1

H NMR (500 MHz, DMSO):  13.24 (s, 1H, NH), 8.25 (s, 1H, Ar-H), 7.47 (d, J = 2.4 Hz, 1H,

Ar-H), 7.66 (s, 1H, Ar-H), 7.45 (s, 1H, Ar-H), 6.67 (dd, J = 2.4, 8.4 Hz, 1H, Ar-H) 6.61 (d, J = 2.4 Hz, 1H, Ar-H);

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C NMR (1254 MHz, DMSO):  163.2 (C-13), 152.6 (C-2), 141.6 (C-4),

136.4 (C-5), 130.8 (C-8), 130.3 (C-10), 129.1 (C-11), 129.3 (C-15), 123.9 (C-7), 122.3 (C-6), 116.2 (C-12), 116.0 (C-14), 113.9 (C-9); Anal. Cal for C13H7Cl2FN2: C = 55.54, H = 2.51, N = 9.97. Found: C = 55.52, H = 2.51, N = 9.96; EI MS m/z (% rel. abund.): 282 (M + 2, 24), 280 (M+, 80), Yield: 77%. 2.2.5. 4,6-dichloro-2-(2-chlorophenyl)-1H-benzo[d]imidazole (5) 1

H NMR (500 MHz, DMSO):  8.22 (s, 1H, NH), 7.76 (d, J = 7.74 Hz, 1H, Ar-H), 7.58 (d, J =

7.43 Hz, 1H, Ar-H), 7.37 (t, 1H, Ar-H), 7.41 (t, 1H, Ar-H), 7.31 (s, 2H, Ar-H); 13C NMR (125 MHz, DMSO):  153.2 (C-7), 142.1 (C-1), 138.7 (C-8), 136.6 (C-2), 132.5 (C-13), 130.7 (C-5), 130.4 (C-11), 129.7 (C-12), 129.5 (C-9), 127.7 (C-10), 124.2 (C-4), 122.3 (C-3), 114.4 (C-6); Anal. Cal for C13H7Cl3N2: C = 52.49, H = 2.34, N = 9.43. Found: C = 52.47, H = 2.32, N = 9.41; EI MS m/z (% rel. abund.): 296 (M + 2, 12), 298 (M+, 34), Yield: 79%. 2.2.6. 4-(4,6-dichloro-1H-benzo[d]imidazol-2-yl) benzene-1,2-diol (6) 1

H NMR (500 MHz, DMSO):  9.58 (s, 1H, NH), 9.32 (s,1H, OH), 9.10 (s, 1H, OH), 7.64 (d, J

= 2.1 Hz, 1H, Ar-H), 7.52–7.48 (m, 2H, Ar-H), 7.34 (d, J = 2.1 Hz, 1H, Ar-H), 6.88 (d, J = 8.1 Hz, 1H, Ar-H); 13C NMR (125 MHz, DMSO):  152.7 (C-2), 147.1 (C-13), 145.7 (C-12), 141.7 (C-4), 136.6 (C-5), 130.6 (C-8), 124.4 (C-10), 123.5 (C-7), 123.3 (C-15), 122.3 (C-6), 116.2 (C14), 114.5 (C-11), 114.1 (C-9); Anal. Cal for C13H8Cl2N2O2: C = 52.91, H = 2.73, N = 9.49. Found: C = 52.91, H = 2.72, N = 9.48; EI MS m/z (% rel. abund.): 297 (M + 2, 6), 295 (M+, 22), Yield: 81%.

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2.2.7. 5-(4,6-dichloro-1H-benzo[d]imidazol-2-yl)-2-methoxyphenol (7) 1

H NMR (500 MHz, DMSO):  11.50 (s, 1H, NH), 9.80 (s,1H, OH), 7.67 (d, J = 2.1 Hz, 1H, Ar-

H), 7.66 (dd, J = 2.1, 8.2 Hz, 1H, Ar-H), 7.57 (d, J = 2.1 Hz, 1H, Ar-H), 7.37 (d, J = 2.2 Hz, 1H, Ar-H), 7.12 (d, J = 8.3 Hz, 1H, Ar-H), 3.81 (s, 3H, OCH3);

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C NMR (125 MHz, DMSO): 

152.8 (C-2), 147.3 (C-13), 147.4 (C-12), 141.7 (C-4), 136.5 (C-5), 130.4 (C-8), 124.1 (C-10), 123.7 (C-7), 122.7 (C-15), 122.1 (C-6), 113.7 (C-9), 113.7 (C-11), 111.6 (C-14), 56.3 (OCH3); Anal. Cal forC14H10Cl2N2O2: C = 54.39, H = 3.26, N = 9.06. Found: C = 54.39, H = 3.24, N = 9.08; EI MS m/z (% rel. abund.): 310 (M + 2, 10), 308 (M+, 36), Yield: 76%. 2.2.8. 4,6-dichloro-2-m-tolyl-1H-benzo[d]imidazole (8) 1

H NMR (500 MHz, DMSO):  13.38 (s, 1H, NH), 8.05 (s, 1H, Ar-H), 7.97 (d, J = 8.2 Hz, 1H,

Ar-H), 7.55 (d, J = 1.4 Hz, 1H, Ar-H), 7.49 (t, J = 7.4 Hz, 1H, Ar-H), 7.39 (d, J = 1.9 Hz, 1H, Ar-H), 7.35(s, 1H, Ar-H); 2.51 (s, 3H, CH3); 13C NMR (125 MHz, DMSO):  152.7 (C-2), 141.5 (C-4), 137.7 (C-10), 137.3 (C-11), 136.4 (C-5), 131.1 (C-12), 130.6 (C-8), 129.1 (C-14), 129.2 (C-13), 128.7 (C-15), 123.7 (C-7), 122.1 (C-6), 113.9 (C-9), 21.6 (CH3); Anal. Cal for C14H10Cl2N2: C = 60.67, H = 3.64, N = 10.11. Found: C = 60.63, H = 3.63, N = 10.09; EI MS m/z (% rel. abund.): 278 (M + 2, 21), 276 (M+, 67), Yield: 80%. 2.2.9. 4,6-dichloro-2-p-tolyl-1H-benzo[d]imidazole (9) 1

H NMR (500 MHz, DMSO):  13.33 (s, 1H, NH), 8.08 (d, J = 8.4 Hz, 2H, Ar-H), 7.54 (s, 1H,

Ar-H), 7.41 (d, J = 8.4 Hz, 2H, Ar-H) 7.38 (s, 1H, Ar-H), 2.52 (s, 3H, CH3);

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C NMR (125

MHz, DMSO):  153.1 (C-2), 141.7 (C-4), 136.6 (C-5), 131.5 (C-10), 131.7 (C-13), 130.6 (C-8), 129.5 (C-12), 129.6 (C-14), 128.7 (C-11), 128.9 (C-15), 123.7 (C-7), 122.1 (C-6), 113.8 (C-9), 21.3 (CH3); Anal. Cal for C14H10Cl2N2: C = 60.67, H = 3.64, N = 10.11. Found: C = 60.65, H = 3.61, N = 10.07; EI MS m/z (% rel. abund.): 278 (M + 2, 11), 276 (M+, 39). Yield: 86%. 2.2.10. 2-(4,6-dichloro-1H-benzo[d]imidazol-2-yl) benzene-1,4-diol (10) 1

H NMR (500 MHz, DMSO):  13.24 (1H, NH), 11.48 (1H, OH), 9.15 (1H, OH), 7.66 (s, 1H,

Ar-H), 7.54 (s, 1H, Ar-H), 7.43 (d, J = 1.4 Hz, 1H, Ar-H), 6.92 (d, J = 9.1 Hz, 1H, Ar-H), 6.87 (dd, J = 6.4, 9.1 Hz, 1H, Ar-H);

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C NMR (125 MHz, DMSO):  153.1 (C-2), 150.1 (C-14),

146.8 (C-11), 141.7 (C-4), 136.4 (C-5), 130.6 (C-8), 123.7 (C-7), 122.1 (C-6), 119.7 (C-10), 117.8 (C-12), 117.1 (C-13), 114.3 (C-15), 113.7 (C-9); Anal. Cal for C13H8Cl2N2O2: C = 52.91,

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H = 2.73, N = 9.49. Found: C = 52.88, H = 2.71, N = 9.47; EI MS m/z (% rel. abund.): 297 (M + 2, 23), 295 (M+, 73), Yield: 84%. 2.2.11. 2-(4,6-dichloro-1H-benzo[d]imidazol-2-yl)-5-methoxyphenol (11) 1

H NMR (500 MHz, DMSO):  12.56 (1H, NH), 10.40 (1H, OH), 8.06 (d, J = 9.1 Hz, 1H, Ar-

H), 7.66 (s, 1H, Ar-H), 7.45 (d, J = 2.1 Hz, 1H, Ar-H), 6.65 (dd, J = 2.4, 8.6 Hz, 1H, Ar-H), 6.63 (d, J = 2.4 Hz, 1H, Ar-H), 3.84 (s, 3H, OCH3); 13C NMR (125 MHz, DMSO):  162.2 (C-13), 156.2 (C-15), 152.7 (C-2), 141.5 (C-4), 136.6 (C-5), 130.6 (C-8), 129.7 (C-11), 123.7 (C-7), 122.1 (C-6), 113.7 (C-9), 110.6 (C-10), 107.2 (C-12), 104.2 (C-14), 55.8 (OCH3); Anal. Cal for C14H10Cl2N2O2: C = 54.39, H = 3.26, N = 9.06. Found: C = 54.37, H = 3.26, N = 9.06; EI MS m/z (% rel. abund.): 310 (M + 2, 15), 308 (M+, 48), Yield: 91%. 2.2.12. 4,6-dichloro-2-(3-chlorophenyl)-1H-benzo[d]imidazole (12) 1

H NMR (500 MHz, DMSO):  8.26 (s, 1H, NH), 8.17 (s, 1H, Ar-H), 7.67 (s,1H, Ar-H), 7.61–

7.58 (m, 3H, Ar-H), 7.41 (d, J = 2.1 Hz, 1H, Ar-H); 13C NMR (125 MHz, DMSO):  152.7 (C2), 141.5 (C-4), 139.4 (C-10), 136.2 (C-5), 134.6 (C-12), 130.4 (C-8), 130.3 (C-11), 130.2 (C15), 129.5(C-14), 128.6 (C-13), 123.5 (C-7), 122.1 (C-6), 113.7 (C-9); Anal. Cal for C13H7Cl3N2: C = 52.47, H = 2.37, N = 9.41. Found: C = 52.46, H = 2.36, N = 9.42; EI MS m/z (% rel. abund.): 298 (M + 2, 17), 296 (M+, 52), Yield: 82%. 2.2.13. 4-(4,6-dichloro-1H-benzo[d]imidazol-2-yl) phenol (13) 1

H NMR (500 MHz, DMSO):  10.18 (s, 1H, NH), 9.20 (1H, OH), 8.05 (d, J = 8.4, 1H, Ar-H),

7.56 (d, J = 1.1 Hz, 1H, Ar-H), 7.8 (d, J = 1.1 Hz, 1H, Ar-H), 6.95 (d, J = 8.4, 2H, Ar-H); 13C NMR (125 MHz, DMSO):  158.3 (C-13), 152.7 (C-2), 141.5 (C-4), 136.2 (C-5), 130.7 (C-11), 130.6 (C-15), 130.4 (C-8), 123.7 (C-7), 122.1 (C-6), 116.4 (C-12), 116.3 (C-14), 113.7 (C-9), 113.3 (C-10); Anal. Cal for C13H8Cl2N2: C = 55.94, H = 2.89, N = 10.04; O, 5.73 Found: C = 55.92, H = 2.87, N = 10.05; EI MSm/z (% rel. abund.): 280 (M + 2, 23), 278 (M+, 67), Yield: 85%. 2.2.14. 4,6-Dichloro-2-(4-chlorophenyl)-1H-benzo[d]imidazole (14) 1

H NMR (500 MHz, DMSO):  10.26 (s, 1H, NH), 8.22 (s, 1H, Ar-H), 7.65 (d, J = 8.5 Hz, 2H,

Ar-H), 7.58 (s, 1H, Ar-H), 7.42 (J = 8.5 Hz, 2H, Ar-H); 13C NMR (125 MHz, DMSO):  152.7 (C-2), 141.5 (C-4), 136.4 (C-5), 134.3 (C-13), 132.6 (C-10), 130.4 (C-8), 129.3 (C-12), 129.5 (C14), 128.7 (C-11), 128.9(C-15), 123.7 (C-7), 122.3 (C-6), 113.7 (C-9); Anal. Cal for C13H7Cl3N2:

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C = 52.47, H = 2.37, N = 9.41 Found: C = 52.47, H = 2.37, N = 9.42; EI MS m/z (% rel. abund.): 298 (M + 2, 12), 296 (M+, 32), Yield: 85%.

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2.2.15. 2-(4,6-dichloro-1H-benzo[d]imidazol-2-yl) phenol (15) 1

H NMR (500 MHz, DMSO):  11.66 (s, 1H, NH), 9.89 (1H, OH), 8.11 (d, J = 9.1 Hz, 1H, Ar-

H), 7.64 (s, 1H, Ar-H), 7.45 (d, J = 2.0 Hz, 1H, Ar-H), 7.41 (d, J = 2.0 Hz, 1H, Ar-H), 7.08 (d, J = 8.0 Hz, 1H, Ar-H), 7.02 (t, J = 7.1 Hz, 1H, Ar-H); 13C NMR (125 MHz, DMSO):  154.1 (C11), 152.7 (C-2), 141.7 (C-4), 136.2 (C-5), 131.7 (C-15), 130.4 (C-8), 130.1 (C-13), 123.5 (C-7), 122.1 (C-6), 121.6 (C-14), 118.3 (C-10), 117.6 (C-12), 113.7 (C-9); Anal. Cal for C13H8Cl2N2O: C = 55.94, H = 2.89, N = 10.04. Found: C = 55.92, H = 2.88, N = 10.04; EI MS m/z (% rel. abund.): 280 (M + 2, 24), 278 (M+, 78), Yield: 81%. 2.2.16. 4,6-dichloro-2-(2,6-dimethoxyphenyl)-1H-benzo[d]imidazole (16) 1

H NMR (500 MHz, DMSO):  10.60 (s, 1H, NH), 8.06 (d, J = 9.1 Hz, 1H, Ar-H), 7.63 (s, 1H,

Ar-H), 7.43 (d, J = 2.1 Hz, 1H, Ar-H), 6.67 (dd, J = 2.4, 8.4 Hz, 1H, Ar-H), 6.62 (d, J = 2.4 Hz, 2H, Ar-H), 3.82 (s, 6H, 2xOCH3), 13C NMR (125 MHz, DMSO):  162.2 (C-13), 156.2 (C-15), 152.7 (C-2), 141.5(C-4), 136.4 (C-5), 130.6(C-8), 129.7 (C-11), 123.5 (C-7), 122.1 (C-6), 113.7 (C-9), 110.6 (C-10), 107.2 (C-12), 104.2 (C-14), 55.8 (OCH3), 55.5 (OCH3); Anal. Cal for C15H12Cl2N2O2: C = 55.75, H = 3.74, N = 8.67. Found: C = 55.60, H = 3.264, N = 8.59; EI MS m/z (% rel. abund.): 310 (M + 2, 15), 308 (M+, 48), Yield: 91%.

2.2.17. 4,6-dichloro-2-(3-methoxyphenyl)-1H-benzo[d]imidazole (17) 1

H NMR (500 MHz, DMSO):  11.60 (s, 1H, NH), 7.82 (d, J = 7.6 Hz, 1H, Ar-H), 7.76 (d, J =

1.6 Hz, 1H, Ar-H), 7.62 (d, J = 1.6 Hz, 1H, Ar-H), 7.52 (t, J = 8.1 Hz, 1H, Ar-H), 7.42 (d, J = 1.6 Hz, 1H, Ar-H), 7.12 (dd, J = 8.2, 2.1 Hz, 1H, Ar-H), 3.87 (s, 3H, OCH3);

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C NMR (125

MHz, DMSO):  161.1 (C-12), 152.7 (C-2), 141.7 (C-4), 136.4 (C-5), 131.6 (C-10), 130.6 (C-8), 130.2 (C-14), 123.5 (C-7), 122.1 (C-6), 120.0 (C-15), 114.1 (C-13), 114.1 (C-9), 111.3 (C-11), 55.8 (OCH3); Anal. Cal for C14H10Cl2N2O: C = 57.36, H = 3.44, N = 9.56. Found: C = 57.35, H = 3.41, N = 9.56; EI MS m/z (% rel. abund.): 294 (M + 2, 15), 292 (M+, 34), Yield: 88%. 2.2.18. 4,6-dichloro-2-(pyridin-2-yl)-1H-benzo[d]imidazole (18) 1

H NMR (500 MHz, DMSO):  13.55 (s, 1H, NH), 8.76 (d, J = 2.1 Hz, 1H, Ar-H), 8.35 (d, J =

7.6 Hz, 1H, Ar-H), 8.06 (t, J = 8.1 Hz, 1H, Ar-H), 7.61(t, J = 5.1 Hz, 1H, Ar-H), 7.55 (s, 1H, ArH), 7.43 (d, J = 2.1 Hz, 1H, Ar-H); 13C NMR (125 MHz, DMSO):  155.2 (C-10), 152.7 (C-2), 149.2 (C-12), 141.7 (C-4), 137.2 (C-14), 136.4 (C-5), 130.4 (C-8), 124.2 (C-15), 123.7 (C-7), 123.6 (C-13), 122.1 (C-6), 113.7 (C-9); Anal. Cal for C12H7Cl2N3: C = 54.57, H = 2.67, N = 9

15.91. Found: C = 54.54, H = 2.65, N = 15.92; EI MS m/z (% rel. abund.): 265 (M + 2, 12), 263 (M+, 38.4), Yield: 88%. 2.2.19. 4-(4,6-dichloro-1H-benzo[d]imidazol-2-yl) phenol (19) 1

H NMR (500 MHz, DMSO):  10.21 (s, 1H, NH), 9.50 (s, IH, OH), 8.07 (d, J = 8.6, 2H, Ar-H),

7.58 (d, J = 1.1 Hz, 1H, Ar-H), 7.38 (d, J = 1.1 Hz, 1H, Ar-H), 6.95 (d, J = 8.6, 2H, Ar-H); 13C NMR (125 MHz, DMSO):  158.3 (C-13), 152.7 (C-2), 141.5 (C-4), 136.2 (C-5), 130.7 (C-11), 130.9 (C-15), 130.4 (C-8), 123.7 (C-7), 122.1 (C-6), 116.4 (C-12), 116.4 (C-14), 113.7 (C-9), 113.3 (C-10); Anal. Cal for C13H8Cl2N2: C = 55.94, H = 2.89, N = 10.04; O, 5.73 Found: C = 55.94, H = 2.87, N = 10.04; EI MS m/z (% rel. abund.): 280 (M + 2, 23), 278 (M+, 67), Yield: 85%. 2.2.20. 2-(4,6-dichloro-1H-benzo[d]imidazol-2-yl)-5-methoxyphenol (20) 1

H NMR (500 MHz, DMSO):  10.60 (s, 1H, NH), 9.30 (s, IH, OH), 8.06 (d, J = 9.1 Hz, 1H, Ar-

H), 7.65 (s, 1H, Ar-H), 7.45 (d, J = 2.1 Hz, 1H, Ar-H), 6.67 (dd, J = 2.4, 8.6 Hz, 1H, Ar-H), 6.62 (d, J = 2.4 Hz, 1H, Ar-H), 3.84 (s, 3H, OCH3); 13C NMR (125 MHz, DMSO):  162.2 (C-13), 156.2 (C-15), 152.7 (C-2), 141.5 (C-4), 136.6 (C-5), 130.6(C-8), 129.7 (C-11), 123.5 (C-7), 122.1 (C-6), 113.7 (C-9), 110.6 (C-10), 107.2 (C-12), 104.2 (C-14), 55.8 (OCH3); Anal. Cal for C14H10Cl2N2O2: C = 54.39, H = 3.26, N = 9.06. Found: C = 54.37, H = 3.24 N = 9.07; EI MS m/z (% rel. abund.): 310 (M + 2, 15), 308 (M+, 48), Yield: 91%.

2.3. Biological evaluation 2.3.1. Antiglycation Assay

Bovine Serum Albumin (BSA) was purchased from Merck Marker Pvt. Ltd. (Germany), rutin and methylglyoxal (MG) (40% aqueous solution) were from Sigma Aldrich (Japan), sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4) and sodium azide (NaN3) were purchased from Scharlau Chemie, S. A. (Spain), while dimethyl sulphoxide (DMSO) was purchased from Fischer Scientific (UK). Bovine Serum Albumin (10 mg/mL), methyl glyoxal (14 mM), various concentrations of the compounds (prepared in DMSO, 10% final concentration), and 0.1 M phosphate buffer (pH 7.4) containing sodium azide (30 mM) was incubated under aseptic conditions at 37 °C for 9 days. After 9 days, each sample was examined for the development of specific fluorescence (excitation, 330 nm; emission, 440 nm) against 10

sample blank (Khan et al., 2011; Khan et al., 2013a; Taha et al., 2014). Rutin was used as a positive control. The percent inhibition of AGE formation in the test sample versus control was calculated for each inhibitor compound by using the following formula: % inhibition= (1fluorescence of test sample/ Fluorescence of the control group) × 100. 2.3.2 DPPH (1, 1-Diphenyl-2-picryl hydrazyl) free radical scavenging activity: The free radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazil (DPPH) using literature protocols. Reaction mixture contains 5 μL of test sample (1 mM in DMSO) and 95 μL of DPPH (Sigma, 300 μM) in ethanol. The reaction mixture was taken into a 96-well microtiter plate and incubated at 37º C for 30 min. The absorbance was measured at 515 nm on microtiter plate reader (Molecular Devices, CA, USA). Percent radical scavenging activity was determined in comparison with a DMSO containing control. IC50 values represent the concentration of compounds to scavenge 50% of DPPH radicals. Propyl gallate was used as a positive control. All the chemicals used were of analytical grade (Sigma, USA) (Anouar et al., 2013; Khan et al., 2012).

3.Results and discussion 3.1. Chemical synthesis Benzimidazole derivatives 1–20 were synthesized by treating of 3,5-dichlorobenzene-1,2diamine with of arylaldehyde. Finally the precipitates were separated by filter and the crude product was recrystalized in ethyl acetate to afford pure crystal and characterized by different spectroscopic methods.

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Scheme: Synthesis of Benzimidazole derivatives (1-20) Benzimidazole derivatives 1–20 (Table 1) were subjected to various spectroscopic analysis and their data partially matched with the similar type of derivatives previously published (Alkahtani et al., 2015; Tunçbilek et al., 2009).

Table 1. Structures of Benzimidazole derivatives (1-20) No.

Structure

No.

1

11

2

12

3

13

4

14

5

15

6

16

7

17

Structure

12

8

18

9

19

10

20

3.2. Biological activity 3.2.1. Antiglycation activity All synthesized benzimidazole derivatives (1-20) were evaluated for their antiglycation potential. Among the series, compounds 2 (IC50 = 240.50 ± 1.30µM), 10 (IC50 = 182.30 ± 1.20µM), and 19 (IC50 = 288.60 ± 1.30µM) showed most potent antiglycation activity if compared with the standard rutin (IC50 = 295.09 ± 1.04 µM) (Table 2). Structure activity relationships were mainly based on substitution pattern on aldehyde phenyl ring. Compound 10, a 2,5-dihydroxy analog was found to be the most active among the series. If we compare compound 10 with compound 2, a 2,4-dihydroxy analog, and compound 6, a 3,4-dihydroxy, compound 10 is superior. The difference in their potential is mainly due to the position difference of substituents. The greater potential shown by these compounds might be due to the acetal formation with fructose carbonyl group. Similarly compound 19, a para hydroxyl analog was found to the third most active compound among the series. The decline in the potential of this compound might be due to less number of hydroxyl group on phenyl ring if we compare it with compound 10 and 2. By comparing compound 19 with other monohydroxy analogs like 13, a meta hydroxyl analog and 15, a ortho hydroxyl analog, compound 19 is superior. The difference in their potential is mainly due the position difference of hydroxyl group on phenyl ring. By bringing about other substituents like chloro, flouro or methyl group on phenyl ring makes it completely inactive. So, it was concluded that only those analogs showed antiglycation potential among these synthesized scaffolds which have hydroxyl group on phenyl ring. The number and position of hydroxyl further effect their potential. 3.2.2. Antioxidant Activity 13

Benzimidazole derivatives 1-20 were also evaluated for their antioxidant potential. Among the series, compounds 6 (IC50 = 29.14± 0.47µM) and 10 (IC50 = 22.42 ±0.26µM) showed potent antioxidant activity as compared to standard propyl gallate (IC50 = 29.20 ± 1.25µM). The greater potential of these compounds i.e. 6, a 3,4-dihydroxyanalogand 10, a 2,5-dihydroxyanalogmight be due to the stabilization of phenoxy radical formed during this assay. The other dihydroxy analog like Compound 2 (IC50 = 31.71 ± 0.42µM) also showed good potential. The small difference in the activities of these analogs might be due to the position difference of substituents. Other analogs like7 (IC50 = 73.51±1.17µM),11(IC50 = 45.63 ± 0.92µM), 13 (IC50 = 82.30 ± 1.33µM), 15 (IC50 = 75.41±1.51µM), 19 (IC50 = 40.60 ± 0.80µM) and 20 (IC50 = 64.92±1.41µM) having hydroxyl group also displayed better antioxidant activity. The difference in the activities of these compounds were mainly affected by the number and position of substituents. The remaining compounds were found inactive.

Table 2. Antiglycation inhibition activity of benzimidazole derivatives 1-20 No.

1 2

Antioxidant

Antiglycation

(IC50± SEMµM)

(IC50 + SEMµM)

N.A.

N.A.

31.71 ± 0.42

No.

Antioxidant

Antiglycation

(IC50± SEMµM)

(IC50± SEMµM)

11

45.63±0.92

325.20 ± 1.70

240.50 ± 1.30

12

N.A.

N.A.

3

N.A.

N.A.

13

82.30±1.33

440.0 ± 3.60

4

N.A.

N.A.

14

N.A.

N.A.

5

N.A.

N.A.

15

75.41±1.51

370.60 ± 2.80

6

29.14 ± 0.47

310.20 ± 1.60

16

N.A.

N.A.

7

73.51±1.17

473.51 ± 2.17

17

N.A.

N.A.

8

N.A.

N.A.

18

N.A.

N.A.

9

N.A.

N.A.

19

40.60 ± 0.80

288.60 ± 1.30

10

22.42±0.26

182.30 ± 1.20

20

64.92±1.41

415.20 ± 3.20

(Propyl gallate)

(Rutin)

(Propyl gallate)

(Rutin)

29.20 ± 1.25

296.20 ± 1.10

29.20 ± 1.25

296.20 ± 1.10

SEMa is the standard error of the mean, NAb Not active, Rutin,c standard inhibitor for glycation activity.

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4. Conclusion In this study, benzimidazole derivatives 2 and 10 have been identified as potent inhibitors of both glycation of proteins and oxidative damages. It was found that most of the active compounds possess hydroxyl substitutions. It was also observed that substituents such as methyl, methoxy and halides do not play any significant role in inhibiting glycation and oxidative potentials. But the halides, methoxy and methyl moiety has not role to inhibit the glycation and oxidation process. These results suggest benzimidazole class of compounds is an effective class of lead molecules that may act as dual inhibitors of both glycation of proteins and the oxidative damages caused by free radicals. These finding suggest that benzimidazole is an effective class of compounds or a lead molecule that may act as dual inhibitors of both glycation of proteins and the oxidative damages caused by free radicals

Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.

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Graphical Abstract:

19