Design, Synthesis, Molecular Docking, and Kinetic Study of 3-Amino-2,4-Diarylbenzo[4,5]Imidazo[1,2-a]Pyrimidines as Novel, Potent Α-Glucosidase Inhibitor


 In an attempt to find novel, potent α-glucosidase inhibitors, a library of poly-substituted 3-amino-2,4-diarylbenzo[4,5]imidazo[1,2-a]pyrimidines 3a-ag have been synthesized through heating a mixture of 2-aminobenzimidazoles 1 and α-azidochalcone 2 under the mild conditions. This efficient, facile protocol has been resulted into the desirable compounds with a wide substrate scope in good to excellent yields. Afterwards, their α-glucosidase inhibitory activities were investigated. Showing IC50 values ranging from 16.4 ± 0.36 µM to 297.0 ± 1.2 µM confirmed their excellent potency to inhibit α-glucosidase which may provide new drug candidates in the treatment of type II diabetes mellitus. Among various synthesized 3-amino-2,4-diarylbenzo[4,5]imidazo[1,2-a]pyrimidines, compound 3k exhibited the highest potency against α-glucosidase (IC50 = 16.4 ± 0.36 μM) which was 45.7 times more potent than acarbose as standard inhibitor (IC50 = 750.0 ± 1.5 μM). Moreover, the role of amine moiety on the observed activity was studied through substituting with chlorine and hydrogen resulted into a considerable deterioration on the inhibitory activity. Kinetic study and molecular docking study have confirmed the in-vitro results.

The structures of the isolated products (3a-ag, 4a, and 6a) were deduced on the basis of their IR, 1 H-and 13 C-NMR spectroscopy, as well as mass spectrometry. Partial assignments of these resonances are provided in the Experimental Part.
There was the same trend for the activities of compounds 3h-j with their analogs in the rst series (compounds 3a-c). Additionally, results revealed that replacing chlorine at 4-position on 4-aryl ring of compounds 3h and 3i with bromine (compounds 3m and 3n) moderately decreased the α-glucosidase inhibitory activity.

Enzyme kinetic study
To investigate the inhibition mode of synthesized poly-substituted 3-amino-2,4diarylbenzo [4,5]imidazo[1,2-a]pyrimidine 3 against α-glucosidase, kinetic study was performed with standard inhibitor, acarbose, and the most potent derivative 3k. To indicate the type of inhibition and K i , Lineweaver-Burk plots and secondary re-plotting of the mentioned plots were presented ( gure 2). As it was showed in gure 2a, while inhibitor concentration increased, the K m value gradually increased, but V m value remained unchanged. Therefore, it can be implied compound 3k was a competitive inhibitor and competes with acarbose for binding to the enzyme active site. Moreover, plot of K m versus different concentration of compound 3k gave an estimate of the inhibition constant, K i of 16 µM ( gure 2b).

Cytotoxicity studies
Among the potent synthesized 3-amino-2,4-diarylbenzo [4,5]imidazo[1,2-a]pyrimidine 3, the cytotoxicity of some of them including 3a, 3k, 3m and 3ad was evaluated through use of the breast cancer cell lines including MCF-7 and MDA-MB-231, as well as human pancreatic cancer cell lines including HDF and PANC1. The selected compounds did not possess any cytotoxic activity against these cell lines at concentration of 100 µM (IC 50 > 200 µM).

Docking study
Molecular docking study was performed on the compounds 3a, 3k and 3ad to study the mode of their interaction in the active site of the yeast isomaltase from Saccharomyces cerevisiae (Pdb id:3A4A) with 84% similarity to S. cerevisiae α-glucosidase using Auto Dock Tools (version 1.5.6). These compounds showed similar binding modes of interaction with catalytic residues. The superimposed structure of acarbose as a standard inhibitor and the most potent compound 3k in the active site of isomaltase was shown in gure 3. In the most potent compound 3k, benzimidazole and 4-(4Cl-phenyl) ring units created π-π interaction with Phe 303 and Tyr 158, respectively in the active site of the enzyme ( gure 4). The 2-(4Cl-phenyl) ring formed π-anion interaction with the aromatic side chains of Asp352. Moreover, a πcation interaction was observed between pyrimidine moiety and Arg 442.
Compounds 3a and 3k interacted with similar amino acids in the active site of the enzyme. Benzimidazol, pyrimidine, and 4-phenyl ring of compound 3a interacted with Phe303, Arg442, and Tyr158, respectively ( gure 5). Compound 3k had additional π-alkyl interaction between 4-(4Cl-phenyl) ring and Arg315, as well as 2-(4Cl-phenyl) ring and Val 216. Higher observed inhibitory activity of compound 3k could be attributed to the formation of stabilizing interactions with speci c residues like Arg315 and Val 216, which could be resulted from the presence of chlorine atoms led to the electron-de ciency of phenyl rings. Additionally, chlorine atoms in compound 3k could create hydrophobic interactions with Tyr72, His112, Phe178, Arg315 which brought more inhibitory activity in comparison with compound 3a.
In compound 3ad, there was a difference in interaction mode of the 2-(4Cl-phenyl) moiety with the active site of enzyme. Insertion of chlorine in 7 and 8 positions on the benzimidazole moiety led to a signi cant decrease in the inhibitory activity. However, there was not any interaction between 2-(4Cl-phenyl) moiety and Asp352 ( gure 6).
Further studies on the binding energies of selected compounds exhibited that compound 3k had lower free binding energy (−9.63 kcal/mol) as compared to compounds 3ad (−8.89 kcal/mol) and 3a (-9.14 kcal/mol). As observed from the best docking conformations, showed that all three compounds have a lower free binding energy than acarbose (-8.20 kcal/mol). Therefore, the results emphasized that the target compounds bind more easily to the target enzyme (α-glucosidase) than the reference drug, acarbose. These ndings had good agreement with the obtained results through in vitro experiments.

Conclusion
In conclusion, we introduced a novel, potent series of α-glucosidase inhibitors. Poly-substituted 3-amino-2,4-diarylbenzo [4,5]imidazo [1,2-a]pyrimidines were synthesized through an e cient, short-time, high-yield Michael addition-cyclization between 2-aminobenzimidazoles and α-azidochalcones under the mild conditions. No need to column chromatography led us to obtain a large scope of substrates, all of which exhibited good to excellent inhibitory activity. Among them, compound 3k showed the best inhibitory potency having IC 50 value of 16.4 ± 0.36 μM which was 45.7 times more potent than acarbose as standard inhibitor (IC 50 = 750.0 ± 1.5 μM). The kinetic study for this compound showed there was a competitive mechanism. Moreover, docking studies revealed that 3-amino-2,4diarylbenzo [4,5]imidazo [1,2-a]pyrimidines could interact with important amino acids in the active site of α-glucosidase.

Methods
All chemicals were purchased from Merck (Germany) and were used without further puri cation. Melting points were measured on an Electrothermal 9100 apparatus and were not corrected. Mass spectra were recorded on an Agilent Technologies (HP) 5973 mass spectrometer operating at an ionization potential of 20 eV. IR spectra were recorded on a Shimadzu IR-460 spectrometer. 1 H and 13 C NMR spectra were measured (DMSO-d 6 solution) with Bruker DRX-300 AVANCE (at 300.1 and 75.1 MHz) spectrometer with TMS as an internal standard. α-Azido chalcones 2 were obtained from the corresponding benzylidene acetophenones in two steps following the literature procedure 15 .
General procedure for the preparation of 3-chloro-2,4-diphenylbenzo [4,5]imidazo[1,2-a]pyrimidine 4a: To a stirring solution of concentrated sulfuric acid (1.6 mmol), sodium nitrite (2.2 mmol) was added gradually over 10-15 min. After addition was completed, the temperature was raised to 70 °C, and the mixture was stirred until sodium nitrite dissolved thoroughly. Then, the mixture is cooled to 25 °C with an ice bath, and a solution of 3-amino-2,4-diphenylbenzo [4,5]imidazo[1,2-a]pyrimidin 3a (2.0 mmol) in glacial acetic acid (4.0 ml) was added slowly with stirring, at such a rate that temperature remains below 40 °C. After 30 min, TLC monitoring con rmed compound 3a was completely converted to corresponding diazonium salt. The obtained mixture was added at 10 °C in portions to a solution of CuCl (4.4 mmol) in concentrated hydrochloric acid (4.0 mmol) over a period of about 5 minutes. Afterward, temperature was raised to 80 °C and the reaction mixture was heated for almost 5h. After completion of the reaction which was monitored by TLC, mixture was quenched by iced water. The precipitate was ltered and recrystallized in EtOH to afford the pure product 4a.
Docking studies were performed using Auto Dock Tools (version1.5.6), and the pdb structure of 3A4A was taken from theBrookhaven protein database (http://www.rcsb.org) as a complex bound with α-D-glucose. The 3D structures of the selected compounds were created by MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and converted to pdbqt coordinate using Auto dock Tools.. Before preparation of auto dock format of protein, the water molecules and the inhibitors were removed from it. Then, using Auto Dock Tools, polar hydrogen atoms were added, Kollman charges were assigned, and the obtained enzyme structure was used as an input le for the AUTOGRID program. In AUTOGRID for each atom type in the ligand, maps were calculated with 0.375 A spacing between grid points, and the center of the grid box was placed at x = 22.625, y = -8.069, and z = 24.158. The dimensions of the active site box were set at 50 × 50 × 50 A. Each docked system was carried out by 50 runs of the AUTODOCK search by the Lamarckian genetic algorithm. The best pose of each ligand was selected for analyzing the interactions between α-glucosidase and the inhibitor. The results were visualized using Discovery Studio 4.0 Client and LigPlot (Figure 3-6).