SYNTHESIS, CHARACTERIZATION, AND TYROSINASE INHIBITORY PROPERTIES OF BENZIMIDAZOLE DERIVATIVES
© 2014 Mert Olgun Karatas", Bulent Alia", Engin £etinkayaA, g dem Bilenc, Nahit Gen^erc, #, and Oktay Arslanc
aInonu University, Faculty of Arts and Sciences, Department of Chemistry 44280, Malatya, Turkey bEge University, Faculty of Science, Department of Chemistry, 35100, Izmir, Turkey cDepartment of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir, 10145, Turkey Received January 8, 2014; in final form, March 3, 2014
1-Alkylbenzimidazole and 1,3-dialkyl benzimidazolium salts were synthesized and characterized by the data of IR, 1H NMR, 13C NMR spectra and elemental analyses. These compounds were investigated as tyrosinase inhibitors. Tyrosinase has been purified from banana by affinity chromatography on a Sepharose 4B gel conjugated with L-tyrosine-p-aminobenzoic acid. All the synthesized compounds inhibited the tyrosinase activity. Among the compounds studied, 1,4-di(1#-benzo[d]imidazol-1-yl)butane was found to be the most active tyrosinase inhibitor (IC50 0.31 mM).
Keywords: benzimidazole, enzymatic browning, tyrosinase inhibitors
DOI: 10.7868/S0132342314040046
INTRODUCTION
Benzimidazole consists of the fusion of benzene and imidazole. Benzimidazole has been widely used as carbon skeleton for synthesis of N-heterocyclic car-benes (NHC). Benzimidazole derivatives and their NHC's are usually used as ligand for transition metal complexes and these complexes are also used as catalyst in various organic synthesis [1]. Besides using on synthesis of NHC's, various biological activities of benzimidazole derivatives were reported [2]. The biologic potential of benzimidazole can be traced back to 1944. Wooley reported that benzimidazole can act similar to purines to give some biological responses [3]. After this study, biological properties of benzimidazole derivatives were investigated intensively. Benzimida-zole bearing bioactive compounds were reported as antihypertensive [4], anti-inflammatory [5], antimicrobial [6], antiviral [7], antioxidant [8], antitumor [9], lipid modulator [10], anticoagulant [11].
Tyrosinase (monophenol or o-diphenol, oxygen oxi-doreductase, EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a copper-containing monooxygenase that is widely distributed in microorganisms, animals, and plants [12]. Tyrosinase could catalyze two distinct reactions involving molecular oxygen in the hydroxylation of monophenols to o-diphenols (monophenolase) and in the oxidation of o-diphenols to o-quinones (dipheno-lase) [13]. Due to the high reactivity, quinines could
# Corresponding author (e-mail: ngencer@balikesir.edu.tr).
polymerize spontaneously to form high molecular weight brown pigments (melanins) or react with amino acids and proteins to enhance brown colour of the pigment produced [14, 15]. Previous reports confirmed that tyrosinase not only was involved in mela-nising in animals, but also was one of the main causes of most fruits and vegetables quality loss during post harvest handling and processing, leading to faster degradation and shorter shelf life [16]. Recently, investigation demonstrated that various dermatological disorders, such as age spots and freckle, were caused by the accumulation of an excessive level of epidermal pigmentation [17, 18]. Tyrosinase has also been linked to Parkinson's and other neurodegenerative diseases [19]. In insects, tyrosinase is uniquely associated with three different biochemical processes, including sclerotiza-tion of cuticle, defensive encapsulation and melanisa-tion of foreign organism, and wound healing [20]. These processes provide potential targets for developing safer and effective tyrosinase inhibitors as insecticides and ultimately for insect control. Thus, the development of safe and effective tyrosinase inhibitors is of great concern in the medical, agricultural, and cosmetic industries. However, only a few such as kojic acid, arbutin, tropolone, and 1-phenyl-2-thiourea are used as therapeutic agents and cosmetic products [18, 21].
In this study we synthesized 1-alkylbenzimidazoles and 1,3-dialkyl benzimidazolium salts and their inhibitory properties on PPO activity were evaluated. Imi-dazolium salts similar to we synthesized in this study were reported as antimicrobial agents [22] but PPO in-
hibitory properties of ionic benzimidazole derivatives were investigated first time in this study.
RESULTS AND DISCUSSION
The synthetic procedures employed to obtain the target compounds (4a—m) are depicted in Scheme. All new synthesized compounds were characterized by IR, 1H-NMR, 13C-NMR spectroscopic methods and elemental analyses. In the IR spectra of compounds (4a—m), it was possible to see the absorption between
1593 and 1553 cm-1 belong to N-C-N bond. In the 1H-NMR it was possible to see characteristic NCflN signals between 9.52-9.01 ppm as singlet. Beside these signals, in anthracene skeleton, at position 10 (Fig. 1), characteristic aromatic proton signals were obtained between 8.95-8.86 ppm as singlet. 13C-NMR signals and number ofpeaks were compatible with structure of synthesized compounds. In 13C-NMR spectra, characteristic NCN signals were obtained between 141.6 and 162.8 ppm. Furthermore, elemental analysis datas were compatible with synthesizes compounds.
R
R
CH3
CH2CH=CH2 -CH2CH2CH2CH3 CH2CH2OCH3
CH2C6H5
CH2C6H4(CH3)-2 CH2C6H4(CH3)-4 -CH2C6(CH3)5-2,3,4,5,6 CH2C6H2(OCH3)3-3,4,5 CH9C1AH7-2
Compound (2a), (4a) (2b), (4b) (2c), (4c) (2d), (4d) (2e), (4e) (2f), (4f) (2g), (4g) (2h), (4h) (2i), (4i) (2j), (4j)
n Compound \_j N '
4 (2k), (4k) /^K
5 (21), (4l) \__y
(2k, l)
R
N
+>ci-
N
(2m)
(4k, l)
(4m)
Scheme. Synthesized Compounds. Reagents and Conditions: (i) Ethanol, KOH, RX, 8h, reflux (ii) compound (3), 90°C, DMF, 3 days. (iii) 9-(Chlormethyl)anthracene (3), 90°C, DMF, 5 days.
For evaluating the tyrosinase inhibitory activity, all the synthesized compounds were subjected to tyrosinase inhibition assay with catechol as substrate. The result showed that all the synthesized compounds (4a-m) inhibited the tyrosinase enzyme activity. IC50 values were calculated from inhibition curves obtained
with benzimidazole derivatives. The inhibition values of analogues (4a—m) against PPO were summarized in table. We have determined the IC50 values of 0.31— 13.14 mM for the inhibition of banana PPO. According to IC50 values, compound (2k) was the most effective inhibitor for BPPO (0.31 mM). Except compound
SYNTHESIS, CHARACTERIZATION, AND TYROSINASE INHIBITORY PROPERTIES
499
5 10 4 Fig. 1. Anthracene skeleton.
(5) and compounds (2a, 2b, 2c, 2d, 4a), IC50 values for 23 compounds were in the range of 0.31—2.56 mM. These results show that aliphatic R groups at position of benzimidazole skeleton decreased PPO inhibitory activity. Inhibition of PPO was investigated using gallic acid as a standard inhibitor, which showed the strongest inhibitory activity with IC50 value of 0.03 mM (Fig. 2). Gallic acid (3,4,5-trihydroxybenzoate) has been isolated and identified as a tyrosinase inhibitor from many plants, and its inhibitory mechanism together with those of its ester derivatives has been well studied by Kubo et al. [23—25]. They found that gallic acid inhibited diphenolase activity of mushroom tyrosinase is 100-fold lower than that of kojic acid. The enzymatic browning by a specific inhibitor may involve a single mechanism or may be the result of interplay of two or more mechanisms of inhibitor action.
Enzymatic browning of plants may be delayed or eliminated by removing the reactants, such as oxygen and phenolic compounds, or by using PPO inhibitors. Complete elimination of oxygen from plants during drying is difficult because oxygen is ubiquitous [26]. There are a number of inhibitors, such as sodium met-abisulphite [27], ascorbic acid [28], glutathione [29], tropolone [30] decreasing the activity of PPO. Acidu-lants, such as citric acid can inhibit PPO activity by reducing pH and/or chelating Cu in a food product [31, 32]. Ascorbic acid can also be considered as an effective compound at higher concentrations. The mechanism of ascorbic acid inhibition involves the reduction of quinones generated by PPO [33]. The goal of these studies is determine to the best inhibitor for decreasing the enzymatic browning.
EXPERIMENTAL
All reactions for preparation of benzimidazolium salts were carried out in standard Schlenk-type flasks. Chemicals were purchased from Sigma Aldrich. DMF used as a solvent in the synthesis of benzimidazolium salt was dried by P2O5. 9-(Chloromethyl) anthracene (3) was used without further purification. Melting points were determined using an Electrothermal-9200 melting point apparatus. FT-IR spectra were recorded on an ATR unit in the range of400—4000 cm-1 using a Perkin Elmer Spectrum 100 Spectrophotometer. 1H-NMR and 13C NMR were recorded in DMSO-d6 using a Bruker AC300P FT spectrometer operating at 300.13 MHz
Activity, %
Gallic Acid, mM
Fig. 2. Inhibitory activity of gallic acid on BPPO.
(1H), 75.47 MHz (13C). Chemical shifts (S) are given in ppm relatively TMS and coupling constants (J) are given in Hz. Elementary analysis were done by IBTAM (Inonu University Scientific and Technological Research Central).
Synthesis of 1-alkylbenzimidazole and 1,1'-bisbenz-imidazole compounds (2a—m). 1-Alkylbenzimidazole and bisbenzimidazole compounds were synthesized according to the procedure of Ozdemir et al. [34]. Potassium hydroxide (1 mmol) was added to a solution of benzimidazole (1 mmol) in ethanol (20 mL), the mixture was stirred for 1 h at room temperature, and the corresponding alkyl halides was added dropwise and heated for 8 h at 76°C. The mixture was diluted with 30 mL of water and extracted with chloroform (3 x 10 mL). The compounds (2a—d) were distilled under reduced pressure
IC50 values for the benzimidazole derivatives tested as tyrosinase inhibitors
Compound IC50 (mM) Compound
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