Synthesis, In Vitro α-Glucosidase Inhibitory Activity and Molecular Docking Studies of Novel Benzothiazole-Triazole Derivatives

Benzothiazole-triazole derivatives 6a–6s have been synthesized and characterized by 1H-NMR and 13C-NMR. All synthetic compounds were screened for their in vitro α-glucosidase inhibitory activity by using Baker’s yeast α-glucosidase enzyme. The majority of compounds exhibited a varying degree of α-glucosidase inhibitory activity with IC50 values between 20.7 and 61.1 μM when compared with standard acarbose (IC50 = 817.38 μM). Among the series, compound 6s (IC50 = 20.7 μM) bearing a chlorine group at the 5-position of the benzothiazole ring and a tert-butyl group at the para position of the phenyl ring, was found to be the most active compound. Preliminary structure-activity relationships were established. Molecular docking studies were performed to predict the binding interaction of the compounds in the binding pocket of the enzyme.


Introduction
Type 2 diabetes is the most common type of diabetes, accounting for about 95 percent of diabetes cases. It is characterized by high blood glucose (hyperglycemia), insulin resistance and relative lack of insulin [1]. Hyperglycemia plays an important role in the development of type 2 diabetes and complications associated with diseases such as microvascular and macrovascular diseases [2]. α-Glucosidase catalyzes the hydrolysis of terminal 1,4-linked α-D-glucose residues and releases free α-D-glucose, which is to the main cause of hyperglycemia [3]. Inhibition of α-glucosidase activity is one of the important therapeutic approaches for the treatment of type 2 diabetes by slowing the absorption process of glucose in intestine [4]. Three inhibitors of α-glucosidase enzyme (acarbose, miglitol, and voglibose) are being clinically used for the management of type 2 diabetes, and these are also used as anticancer [5], anti-HIV [6], and anti-hepatitis agents [7].
In continuation of our drug discovery research on potential antidiabetic agents [25][26][27][28], we synthesized a novel series of benzothiazole-triazole derivatives and evaluated for their in vitro αglucosidase inhibitory activity. Furthermore, molecular docking was also performed to predict the binding interaction of the compounds in the binding pocket of the enzyme.

Chemistry
The synthesis of benzothiazole-triazole derivatives 6a-6s is shown in Scheme 1. The reaction of various benzyl chlorides or benzyl bromides 1 with NaN3 in DMF at room temperature for 24 h provided the corresponding (azidomethyl)aryl 2. Treatment of substituted 2-aminobenzenethiol with CS2 in the presence of KOH in reflux EtOH/H2O afforded substituted benzo[d]thiazole-2-thiol 4, which reacted with 3-bromoprop-1-yne to provide the key intermediate 5. In the final step, according to Huisgen, the 1,3-dipolar cycloaddition reaction (click reaction), condensation of intermediate 5 with various (azidomethyl)aryl 2 in the presence of sodium ascorbate and CuSO4·5H2O in DMF solution resulted in the formation of the title products 6a-6s.
The structures of all the title compounds 6a-6s were characterized by 1 H-NMR and 13 C-NMR spectra (Supplementary Materials). For instance, the 1 H-NMR spectrum of 6a shows two singlet signals at δ 4.68 ppm and δ 5.55 ppm, which were assigned to the methylene protons of SCH2 and NCH2, respectively. A doublet of two protons appeared at δ 7.19 ppm (J = 8.4 Hz), which was assigned to the aromatic protons of C2,6-H of the right phenyl ring. The benzothiazole protons appeared as two doublets of one proton each at δ 7.86 and δ 7.99 ppm, and a triplet of one proton at δ 7.37 ppm with a coupling constant of 8.0 Hz and 8.4 Hz, respectively. The rest of the protons of benzothiazole and the right phenyl appeared as a multiplet of three protons between δ 7.45 and 7.51 ppm. The single peak of the C-H of triazole ring was observed at δ 8.17 ppm. The 13 C-NMR spectrum of 6a showed On the other hand, 1,2,3-triazoles are an important class of heterocyclic compounds, and have been widely applied in various fields, including synthetic organic chemistry, biological science, material science and medicinal chemistry [15,16]. Notably, 1,2,3-triazole derivatives have been reported to exhibit various biological activities such as antioxidant [17], antibacterial [18], antitubercular [19] and anticancer [20]. There are some clinical and commercial drugs, such as tazobactam, cefatrizine and carboxyamidotriazole, with 1,2,3-triazole moiety ( Figure 1). In particular, several recent studies have shown that some 1,2,3-triazole derivatives exhibit α-glucosidase inhibitory activity [21][22][23][24][25].
In continuation of our drug discovery research on potential antidiabetic agents [25][26][27][28], we synthesized a novel series of benzothiazole-triazole derivatives and evaluated for their in vitro α-glucosidase inhibitory activity. Furthermore, molecular docking was also performed to predict the binding interaction of the compounds in the binding pocket of the enzyme.

Chemistry
The synthesis of benzothiazole-triazole derivatives 6a-6s is shown in Scheme 1. The reaction of various benzyl chlorides or benzyl bromides 1 with NaN 3 in DMF at room temperature for 24 h provided the corresponding (azidomethyl)aryl 2. Treatment of substituted 2-aminobenzenethiol with CS 2 in the presence of KOH in reflux EtOH/H 2 O afforded substituted benzo[d]thiazole-2-thiol 4, which reacted with 3-bromoprop-1-yne to provide the key intermediate 5. In the final step, according to Huisgen, the 1,3-dipolar cycloaddition reaction (click reaction), condensation of intermediate 5 with various (azidomethyl)aryl 2 in the presence of sodium ascorbate and CuSO 4 ·5H 2 O in DMF solution resulted in the formation of the title products 6a-6s.
The structures of all the title compounds 6a-6s were characterized by 1 H-NMR and 13 C-NMR spectra (Supplementary Materials). For instance, the 1 H-NMR spectrum of 6a shows two singlet signals at δ 4.68 ppm and δ 5.55 ppm, which were assigned to the methylene protons of SCH 2 and NCH 2 , respectively. A doublet of two protons appeared at δ 7.19 ppm (J = 8.4 Hz), which was assigned to the aromatic protons of C2,6-H of the right phenyl ring. The benzothiazole protons appeared as two doublets of one proton each at δ 7.86 and δ 7.99 ppm, and a triplet of one proton at δ 7.37 ppm with a coupling constant of 8.0 Hz and 8.4 Hz, respectively. The rest of the protons of benzothiazole and the right phenyl appeared as a multiplet of three protons between δ 7.45 and 7.51 ppm. The single peak of the C-H of triazole ring was observed at δ 8.17 ppm. The 13 C-NMR spectrum of 6a showed two characteristic peaks for methylene carbon at δ 27.9 ppm and δ 52.5 ppm. The remaining thirteen signals were assigned to benzothiazole, triazole and phenyl carbon in the compound 6a. Therefore, the data of 1 H-NMR and 13 C-NMR is in agreement with the structure of compound 6a.
Molecules 2017, 22, 1555 3 of 10 two characteristic peaks for methylene carbon at δ 27.9 ppm and δ 52.5 ppm. The remaining thirteen signals were assigned to benzothiazole, triazole and phenyl carbon in the compound 6a. Therefore, the data of 1 H-NMR and 13 C-NMR is in agreement with the structure of compound 6a.

α-Glucosidase Inhibition Assay
All the synthetic benzothiazole-triazole derivatives (6a-6s) were screened to evaluate their in vitro α-glucosidase inhibitory activity by using Baker's yeast α-glucosidase enzyme. The results are summarized in Table 1

α-Glucosidase Inhibition Assay
All the synthetic benzothiazole-triazole derivatives (6a-6s) were screened to evaluate their in vitro α-glucosidase inhibitory activity by using Baker's yeast α-glucosidase enzyme. The results are summarized in Table 1

Structure-Activity Relationship
The structure-activity relationship of this class of compounds has been summarized. The introduction of electron withdrawing groups such as fluorine, chlorine, and bromine into the phenyl ring results in an increase in inhibitory activity. Furthermore, shifting these groups from the para to the ortho position decreased inhibitory activity. Introduction of the electron-withdrawing group chlorine into the benzothiazole ring results in a significant increase in inhibitory activity. It is interesting to point out that 6i and 6s containing the tert-butyl group at the para position of the phenyl ring exhibited potent α-glucosidase inhibitory activity, with IC 50 values of 29.4 and 20.7 µM, respectively. In particular, compound 6s with a chlorine group at the 5-position of the benzothiazole ring and a tert-butyl group at the para position of the phenyl ring, was found to be the most active compound in this series. In order to illustrate the binding interactions of molecules into the active site of α-glucosidase, molecular docking studies were conducted.

Molecular Docking
The theoretical binding mode between 6i and Saccharomyces cerevisiae α-glucosidase is shown in Figure 2. Compound 6i adopted an "L-shaped" conformation in the pocket of the α-glucosidase. The benzothiazole group of 6i was located at the hydrophobic pocket, surrounded by the residues Phe-157, Phe-310 and Phe-311, while the 4-(tert-butyl)phenyl group of 6i stretched into the hydrophobic pocket consisting of Phe-157, Phe-177 and Phe-300, forming a stable hydrophobic binding. Detailed analysis showed that the triazole group in the middle of 6i formed CH-π interaction with the residue Phe-300. In addition, cation-π interactions were observed between 6i and the residues Lys-155, Arg-312 and Arg-439. Furthermore, 6i formed anion-π interactions with the residues Glu-276 and Asp-349, respectively. All these interactions helped 6i to anchor in the binding site of the α-glucosidase.

Structure-Activity Relationship
The structure-activity relationship of this class of compounds has been summarized. The introduction of electron withdrawing groups such as fluorine, chlorine, and bromine into the phenyl ring results in an increase in inhibitory activity. Furthermore, shifting these groups from the para to the ortho position decreased inhibitory activity. Introduction of the electron-withdrawing group chlorine into the benzothiazole ring results in a significant increase in inhibitory activity. It is interesting to point out that 6i and 6s containing the tert-butyl group at the para position of the phenyl ring exhibited potent α-glucosidase inhibitory activity, with IC50 values of 29.4 and 20.7 μM, respectively. In particular, compound 6s with a chlorine group at the 5-position of the benzothiazole ring and a tert-butyl group at the para position of the phenyl ring, was found to be the most active compound in this series. In order to illustrate the binding interactions of molecules into the active site of α-glucosidase, molecular docking studies were conducted.

Molecular Docking
The theoretical binding mode between 6i and Saccharomyces cerevisiae α-glucosidase is shown in Figure 2. Compound 6i adopted an "L-shaped" conformation in the pocket of the α-glucosidase. The benzothiazole group of 6i was located at the hydrophobic pocket, surrounded by the residues Phe-157, Phe-310 and Phe-311, while the 4-(tert-butyl)phenyl group of 6i stretched into the hydrophobic pocket consisting of Phe-157, Phe-177 and Phe-300, forming a stable hydrophobic binding. Detailed analysis showed that the triazole group in the middle of 6i formed CH-π interaction with the residue Phe-300. In addition, cation-π interactions were observed between 6i and the residues Lys-155, Arg-312 and Arg-439. Furthermore, 6i formed anion-π interactions with the residues Glu-276 and Asp-349, respectively. All these interactions helped 6i to anchor in the binding site of the α-glucosidase. To explain the activity order of 6i and 6s against α-glucosidase, 6s was then docked to the binding site of α-glucosidase; the theoretical binding mode between 6s and α-glucosidase is shown in Figure 3A. The interaction between 6s and α-glucosidase was almost the same as the precursor 6i. The only difference was that the benzothiazole group of 6s formed π-π stacking interactions with the residues His-239 and Phe-157, which were not formed by 6i, making 6s more active than 6i against α-glucosidase ( Figure 3B). In addition, the estimated binding energies were −8.6 kcal·mol −1 for 6i and To explain the activity order of 6i and 6s against α-glucosidase, 6s was then docked to the binding site of α-glucosidase; the theoretical binding mode between 6s and α-glucosidase is shown in Figure 3A. The interaction between 6s and α-glucosidase was almost the same as the precursor 6i. The only difference was that the benzothiazole group of 6s formed π-π stacking interactions with the residues His-239 and Phe-157, which were not formed by 6i, making 6s more active than 6i against α-glucosidase ( Figure 3B). In addition, the estimated binding energies were −8.6 kcal·mol −1 for 6i and −8.9 kcal·mol −1 for 6s, respectively, which was consistent with the results of the in vitro anti-α-glucosidase assay. In summary, the above molecular simulations give us rational explanation of the interactions between 6i, 6s and α-glucosidase, which provided valuable information for further development of α-glucosidase inhibitors.
Molecules 2017, 22, 1555 5 of 10 −8.9 kcal·mol −1 for 6s, respectively, which was consistent with the results of the in vitro anti-αglucosidase assay. In summary, the above molecular simulations give us rational explanation of the interactions between 6i, 6s and α-glucosidase, which provided valuable information for further development of α-glucosidase inhibitors.

General
All starting materials and reagents were purchased from commercial suppliers. Nuclear magnetic resonance spectra (NMR) were recorded on a Bruker spectrometer (400 MHz) with TMS as an external reference and reported in parts per million.

General Procedure for the Synthesis of 2
A mixture of substituted benzyl chlorides or benzyl bromides 1 (1.0 mmol) and NaN3 (1.2 mmol) in 10 mL DMF was stirred at room temperature for 24 h. After the completion of the reaction (monitored by TLC), the mixture was poured into water and extracted 3 times for ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated under vacuum. The residue was purified by chromatography to give 2.

General Procedure for the Synthesis of 4
To a solution of substituted 2-aminobenzenethiol (2 mmol) in EtOH (20 mL) was added CS2 (4 mmol) and KOH (4 mmol), and the mixture was stirred at 80 °C for 12 h. After cooling to the room temperature, the mixture was treated with ice-water (100 mL) and 10% HCl was added to adjust pH to 2-3. The solid precipitate was collected by filtration to obtain 4, which was used in the next step without further purification.

General Procedure for the Synthesis of 5
A mixture of 4 (1 mmol), 3-bromoprop-1-yne (1.5 mmol) and K2CO3 (3 mmol) in acetone (20 mL) was stirred at reflux for 5 h. The solvent was evaporated under reduced pressure and the residue was purified by chromatography to give 5.

General
All starting materials and reagents were purchased from commercial suppliers. Nuclear magnetic resonance spectra (NMR) were recorded on a Bruker spectrometer (400 MHz) with TMS as an external reference and reported in parts per million.

General Procedure for the Synthesis of 2
A mixture of substituted benzyl chlorides or benzyl bromides 1 (1.0 mmol) and NaN 3 (1.2 mmol) in 10 mL DMF was stirred at room temperature for 24 h. After the completion of the reaction (monitored by TLC), the mixture was poured into water and extracted 3 times for ethyl acetate. The combined organic layers were dried over Na 2 SO 4 and concentrated under vacuum. The residue was purified by chromatography to give 2.

General Procedure for the Synthesis of 4
To a solution of substituted 2-aminobenzenethiol (2 mmol) in EtOH (20 mL) was added CS 2 (4 mmol) and KOH (4 mmol), and the mixture was stirred at 80 • C for 12 h. After cooling to the room temperature, the mixture was treated with ice-water (100 mL) and 10% HCl was added to adjust pH to 2-3. The solid precipitate was collected by filtration to obtain 4, which was used in the next step without further purification.

General Procedure for the Synthesis of 5
A mixture of 4 (1 mmol), 3-bromoprop-1-yne (1.5 mmol) and K 2 CO 3 (3 mmol) in acetone (20 mL) was stirred at reflux for 5 h. The solvent was evaporated under reduced pressure and the residue was purified by chromatography to give 5.

General Procedure for the Synthesis of Benzothiazole-Triazole Derivatives (6a-6s)
A mixture of 5 (1.0 mmol), 2 (1.0 mmol), CuSO 4 ·5H 2 O (0.025 g; 0.1 mmol) and sodium ascorbate (0.10 g, 0.5 mmol) in DMF (10 mL) was stirred at room temperature for 4 h. After completion of reaction, the content was poured into 50 mL ice cold water and extracted with ethylacetate (50 mL × 3). The combined organic extracts were dried over Na 2 SO 4 , filtered, and concentrated. The residue was purified by chromatography on silica gel with EtOAc/petroleum ether to give the title products 6a-6s.

Molecular Docking
Molecular docking studies were performed to investigate the binding mode between 6i, 6s and α-glucosidase using Autodock vina 1.1.2. The 3D structure of α-glucosidase of Saccharomyces cerevisiae have been predicted using homology modeling in our previous report [25]. The 3D structure of the compounds were obtained by ChemBioDraw Ultra 14.0 and ChemBio3D Ultra 14.0 softwares. The AutoDockTools 1.5.6 package was employed to generate the docking input files. The search grid of α-glucosidase was identified as center_x: −19.676, center_y: −7.243, and center_z: −21.469, with dimensions size_x: 15, size_y: 15, and size_z: 15. The value of exhaustiveness was set to 20. For Vina docking, the default parameters were used if it was not mentioned. The best-scoring pose as judged by the Vina docking score was chosen and visually analyzed using PyMoL 1.7.6 software (http://www.pymol.org/).

Conclusions
In summary, a novel series of benzothiazole-triazole derivatives 6a-6s have been synthesized and evaluated for their α-glucosidase inhibitory activity. The majority of compounds exhibited superior α-glucosidase inhibitory activity in the range of IC 50 = 20.7 and 61.1 µM as compared to standard acarbose (IC 50 = 817.38 µM). Molecular docking study was carried out to understand the molecular interaction of compounds with the active site of α-glucosidase. This study has identified a novel series of lead compounds which can be used for the development of new α-glucosidase inhibitors.