Acetylcholine Esterase Inhibitory Potential of Some Benzimidazole Derivatives

This study was undertaken to evaluate anticholinesterase activity of some benzimidazole derivatives due to their biological importance. Previously synthesized 14 benzimidazole compounds were used in biological studies. Inhibitory activities of related compounds on Acetylcholine esterase (AChE, E.C.3.1.1.7 from Electric Eel) enzyme have been determined by a modified Ellman’s colorimetric method. Compound 4f was found as the most active derivative with 69.26%, 51.23% and 38.22% inhibition profiles at 1 mM, 0.1 mM and 0.01 mM concentrations, respectively. It was determined that the substituents increasing liphophilicty of the compounds have an influence on biological activity. Keywods: Benzimidazole, anticholinesterase activity, liphophilicity INTRODUCTION Alzheimer's disease (AD) is the most common reason of dementia among the elderly. As average duration of life increases, the number of AD cases worldwide is expected to rise from 35 million today to more than 115 million by 2050. Molecular studies of the brain at the initial phases of memory loss related with AD have been problematic in humans (Ashe and Zahs, 2010). European International Journal of Science and Technology ISSN: 2304-9693 www.eijst.org.uk 116 AD patients display episodic memory deficiencies, judgment insufficiencies, confusion, altered mood, and motor impairments at later periods (McKhann et al.,1984). Although no cure exists for AD, there are two types of drugs currently being used for treatment of AD. The NMDA receptor antagonist memantine prevents glutamate excitotoxicity caused by over activation of the NMDA receptor (Chen et al., 1992). Although memantine can slow the cognitive failure in patients with moderate to severe AD, it neither reverses nor prevents cognitive deficiencies and offers no apparent advantage to patients with mild to moderate AD. The second class of AD therapeutics are the cholinesterase inhibitors (ChEIs), which elevate acetylcholine (ACh) levels (Cuadra et al., 1994), which partially offsets the loss of cholinergic input to the cerebral cortex that occurs in AD (Whitehouse et al., 1981). ChEIs are effective for mild to moderate AD, unlike memantine, with clinical trials reporting slower rates of cognitive failure, as well as functional improvements (Rogers et al., 1998; Rösler et al., 1999; Raskind et al., 2000). Hence, ChEIs are the first choice for treatment of cognitive, functional and behavioral symptoms of AD. Benzimidazole compounds have an essential role in medical area due to various pharmacological effects as antimicrobial, antiviral, antidiabetic, anticancer, amti-HIV, antihypertensive, antiulcerative, antioxidant and anticonvulsant (Walia et al., 2011). Furthermore, some studies about antichlolinesterase effects of benzimidazole derivatives have been reported (Bacharaju et al., 2012; Alpan et al., 2013;Yoon et al., 2013a; Yoon et al., 2013b). Prompted from above observations, we performed an activity study by using previously synthesized benzimidazole compounds to investigate their Acetylcholine esterase inhibitory potencies (Özkay et al., 2011) MATERIALS AND METHODS Chemistry Test compounds were synthesized at four steps. Synthetic pathway was outlined in Scheme 1. In first reaction step, Schiff base of 2-aminobenzimidazole and 4-formylbenzoic acid methyl ester was prepared (1). In second step, compound 1 was reduced by NaBH4 to obtain compound 2, which was reacted with hydrazine hydrate to gain hydrazide compound (3). In final step, reaction between compound 3 and corresponding benzaldehyde derivatives gave target benzimidazole derivatives (4a-4n). Detailed synthesis procedures and structure confirmations of the benzimidazole derivatives were reported in our previous study (Özkay et al., 2011). Some physicochemical properties of the compounds were presented in Table 1. Pharmacology AChE Inhibition AChE inhibition potency of the compounds 4a-4n were investigated by modified method of Ellman (Ellman et al., 1961). This spectrophotometric method is based on the reaction of released thiocholine to give a colored product with a chromogenic reagent 5,5-dithio-bis(2-nitrobenzoic)acid (DTNB). AChE, (E.C.3.1.1.7 from Electric Eel, 500 units), and Donepezil hydrochloride were purchased from Sigma– Aldrich (Steinheim, Germany). Potassium dihydrogen phosphate, DTNB, potassium hydroxide, sodium hydrogen carbonate, gelatine and acetylthiocholine iodide (ATC) were obtained from Fluka (Buchs, Switzerland). Spectrophotometric measurements were performed on a Thermo microplate spectrophotometer (Multiskan GO, Massachusetts, USA). Cholinesterase activity of the compounds (4a-4n) was measured in 100 mM phosphate buffer (pH 8.0) at 25 °C, using ATC as substrates, respectively. DTNB (10 mM) was used in order to observe absorbance changes at 412 nm. Donepezil hydrochloride was used as a positive control. European International Journal of Science and Technology Vol. 3 No. 7 September, 2014 117 Enzymatic assay AChE inhibition of the compounds 4a-4n were investigated by modified Ellman’s test (Ellman et al., 1961; Perry et al., 2000). Donepezil hydrochloride was used as a positive control (Table 2). Enzyme solutions were prepared in gelatin solution (1%), at a concentration of 2.5 units/mL. AChE and compound solution (50 μL) which is prepared in 2% DMSO at 0.1 and 1 mM concentrations were added to 3.0 mL phosphate buffer (pH 8±0.1) and incubated at 25 oC for 5 min. The reaction was started by adding DTNB) (50 μL) and ATC (10 μL) to the enzyme-inhibitor mixture. The production of the yellow anion was recorded for 10 min at 412 nm. As a control, an identical solution of the enzyme without the inhibitor is processed following the same protocol. The blank reading contained 3.0 mL buffer, 50 μL 2% DMSO, 50 μL DTNB and 10 μL substrate. All processes were assayed in triplicate. The inhibition rate (%) was calculated by the following equation: Inhibition % = [(AC-AB) – (AI-AB)] / (AC-AB) x 100 Where AI is the absorbance in the presence of the inhibitor, AC is the absorbance of the control and AB is the absorbance of blank reading. Both of the values are corrected with blank-reading value. SPSS for Windows 15.0 was used for statistical analysis. Student's ttest was used for all statistical calculations. Data were expressed as Mean ± SD inactive in culture medium. RESULTS AND DISCUSSION In the present study, anticholinesterase activity of previously synthesized benzimidazole derivatives was investigated (Özkay et al., 2011). The anticholinesterase effects of the compounds (4a-4n) were determined by modified Ellman’s colorimetric assay (Ellman et al., 1961) (Table 2). In the series, compound 4f that bear methoxy substituent was determined as the most active compound with 69.26%, 51.23% and 38.22% inhibition potencies at 1 mM, 0.1 mM and 0.01 mM concentrations, respectively. In addition compound 4g carrying ethoxy substituent showed 64.70% and 49.42% inhibition at 1 mM and 0.1 mM concentrations. The IC50 values were calculated as 0.091 mM and 0.134 mM for the compounds 4f and 4g. The inhibition percentages of the compounds 4a-4e, 4h-4n were lower than 50% at 1 mM concentration and thus enzyme inhibitory potency of these compounds were not determined in further concentrations. Standard drug Donepezil was studied at lower concentrations in order to determine IC50 value and it was calculated as 0.054 μM. None of the compounds showed comparable activity with reference drug. CONCLUSION Consuquently, a series of benzimidazole derivatives were observed for their AChE inhibitory potency. In respect of the activity test results, compound 4f displayed the best inhibitory activity with a value of IC50=0.091 mM. Compound 4g (IC50=0.134 mM) displayed moderate enzyme inhibition. The other compounds in the series did not show adequate biological activity. None of the tested compounds displayed biological activity as much as standard drug Donepezil. It was determined that the substituents as methoxy and ethoxy, which increase liphophilicty of the compounds have an influence on biological activity. European International Journal of Science and Technology ISSN: 2304-9693 www.eijst.org.uk