Synthesis of novel indole, 1,2,4-triazole derivatives as potential glucosidase inhibitors

2.1 Experimental details

All the analytical grade chemicals used in this experimental work were procured from Merck India and used without further purification. Melting points were recorded by the open capillary method and are uncorrected. The IR spectra were recorded on a Shimadzu FT-IR 157 spectrophotometer. ¹H NMR spectra were recorded using CdCl₃/DMSO‑d₆ as solvent and TMS as an internal standard on a Bruker 400 MHz NMR spectrometer. The mass spectra were recorded on a low-resolution SHIMADZU mass spectrometer operating at 70 eV. The purity of the compounds was checked on completion of the reaction by thin-layer chromatography (TLC) on pre-coated silica gel plates.

2.1.2

2.1.2 Ethyl1-cyclopropyl-5-((5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-yl)methoxy)-2-methyl-1H-indole-3-carboxylate (Compound 5a)

In a 100 mL round bottom flask placed 1 g (0.003 mol) of Compound 4, 0.42 g (0.003 mol) of Anisaldehyde (aromatic aldehyde), 0.47 g (0.006 mol) of ammonium acetate and added 15 mL ethanol, shake the mixture well and stirred the reaction mass at room temperature for 22–24 h. The progress of the reaction monitored by TLC by using the mobile phase (Hexane: Ethyl acetate; ratio 4:1). After 24 h, the reaction was complete, and then the reaction mass was neutralized with aqueous ammonia. A white color compound was precipitated out. The solid was filtered and washed with water, dried at 55–60 °C for 10 h. Yield 78%; Melting point: 191–181 °C. The same process was used for remaining aromatic aldehydes (Table 1; Compounds 5b-5k).

Table 1 List of synthesized compounds.

Code	Structure	Molecular weight	Molecular formula
5a		446.56	C25H26N4O4
5b		450.92	C24H23ClN4O3
5c		477.47	C24H23N5O6
5d		461.47	C24H23N5O5
5e		476.52	C26H28N4O5
5f		485.36	C24H22Cl2N4O3
5g		512.56	C29H28N4O5
5h		476.52	C26H28N4O5
5i		416.47	C24H24N4O3
5j		432.47	C24H24N4O4
5k		521.52	C26H27N5O7

Purity confirmation: After isolation, the product was dried at 55–60 °C for 10 h. Single product spot was observed on TLC plate, and it was confirmed by a clean ¹H NMR spectrum; hence, it was concluded that the product was pure. The derived compounds were sequentially labelled as 5a-5k (the experimental procedures are reported in supplementary file).

2.2 Molecular docking studies with α-glucosidase

The docking simulations were performed using N-terminal human maltase-glucoamylase protein (α-Glucosidase) from the human intestine (PDB ID: 3CTT) obtained from RCSB Protein Data Bank (http://www.rcsb.org/pdb) (Cardona et al., 2009). The docking was performed according to the method described earlier (Chen et al., 2020; Wu et al., 2017). The protein was prepared by adding polar hydrogen atom with Gasteiger-Huckel charges and the removal of water molecules. The enzyme energy minimized using Gasteiger–Huckel charges and Tripos force field in the Sybyl software suite (Clark et al., 1989; Homer et al., 2008). The 3D structures of the ligands 5a-5k were generated by the SKETCH module implemented in the SYBYL-X 2.0 program (Tripos Inc., St. Louis, USA), and its energy-minimized conformation was obtained using the Powel method in SYBYL software. The resultant ligands were subjected to docking run at the ligand-binding site of the α-glucosidase by Surflex dock in SYBYL-X 2.0 program. Finally, Surflex dock module was used to predict and examine the binding modes of the synthesized compounds at the active site of the α-glucosidase enzyme. The general parameter such as starting conformations, max conformations, angstroms to expand search grid and the number of rotatable bonds were set as 0, 20, 6, and 100, respectively. A soft grid treatment and active spin alignment method were performed. The number of spins and density of search per alignment was set at 12 and 3, respectively. From the analysis ligand-receptor complex with the maximum total score was considered as the most stable conformation. The H-bonding interactions between amino acid residues and their bond distances with synthesized compounds were noted and compared to enzyme inhibitor, Casuarine, as a reference molecule (see supplementary file).

2.3 α-Amylase inhibition assay

The α-amylase inhibitory assay was determined using a method adapted from Kwon et al. with minor modifications (Kwon et al., 2008). The synthesized compounds 5a-5k were dissolved in 1% dimethyl sulfoxide (DMSO) aqueous solution, while standard acarbose was prepared in distilled water. A reaction mixture was prepared to contain 100 μL of standard or compounds (20 to 100 µg/mL), 200 μL of sodium phosphate buffer (20 mM, pH 6.9) and 100 μL of α-amylase (1 U/mL). The mixture was preincubated at 25 °C for 10 min, then 200 μL of 1% starch in the above buffer was added and incubated for another 10 min. The reactions were stopped by adding 1 mL of 3,5-Dinitrosalicylic acid and placing on a boiling water bath for 5 min. The boiled reaction mixture was cooled to room temperature, diluted to 1:5 ratio with water, and absorbance was measured in a UV–visible spectrophotometer at 540 nm. The experiment was done in triplicates, and the percentage of inhibition of enzyme activity was calculated as:$$\%\text{Inhibition}=\left[\left({\text{A}}^{\text{Control}}{\text{- A}}^{\text{Treatment}}\right)/{\text{A}}^{\text{Control}}\right]\times 100$$where A^Control is absorbance of the control and A^Treatment is absorbance upon treatment with various compounds. The percentage of α-amylase inhibitory activity is given in the Table 2.

2.4 α-Glucosidase inhibition assay

The α-Glucosidase inhibitory activity was determined as described earlier by Kim et al. with slight modifications (Kim et al., 2004). A reaction mixture containing 100 μL of synthesized compound 5a-5k or acarbose (20 to 100 µg/mL) was mixed with 50 μL of α-glucosidase (1 U/mL) prepared in 0.1 M phosphate buffer (pH 6.9). The mixture was preincubated at 37 °C for 20 min, then 10 μL of 10 mM p-nitrophenyl-α-D-glucopyranoside (pNPG) in the above buffer was added and incubated for another 30 min. The reactions were stopped by adding 650 μL of 1 M sodium carbonate, and the absorbance was measured in a UV–visible spectrophotometer at 405 nm. DMSO (1%) was used as a solvent control. The experiment was done in triplicates, and the percent inhibition of enzyme activity was calculated as:$$\%\text{Inhibition}=\left[\left({\text{A}}^{\text{Control}}{\text{- A}}^{\text{Treatment}}\right)/{\text{A}}^{\text{Control}}\right]\times 100$$where A^Control is absorbance of the control and A^Treatment is absorbance of treated sample. The percentage of α-glucosidase inhibitory activity is given in the Table 3.

2.5 Statistical analysis

The enzyme assays were performed in triplicates (n = 3), and the results were expressed as mean ± Standard deviation. Un-paired two-tailed t-test performed to compare the mean of each treated group with the mean of the control group using GraphPad prism software (Graph Pad software Inc., San Diego, USA). p values ≤ 0.05 was considered as statistically significant.

3.1 Chemistry

The direction of synthesis of the prepared compounds (5a-5k) was as outlined in the synthesis Scheme 1. These compounds were subjected to various spectroscopic techniques, namely, ¹H NMR and EI-MS to confirm the structures (data shown in supplementary files). The elemental analyses were found to correspond with the calculated values for all the compounds in Scheme 1. The starting material indole hydrazide (compound-4) was prepared by Nenitzescu reaction and used further to synthesize compounds 5a-5k (Eiden and Kucklander, 1971; Gani et al., 2002).

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Scheme 1

Synthesis of Indole fused triazole derivatives (5a-5k).

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Indole fused with triazole derivatives (5a-5k) were prepared by a condensation reaction between 1-cyclopropyl-3-ethoxycarbonyl-2-methylindole-5-(1-methylethoxy acetic acid hydrazide) with aqueous ammonia, ammonium acetate, ethanol, and different aromatic aldehyde. The reaction mass was stirred for 20–24 h at room temperature. As a result of this novel bis-heterocyclic compounds containing triazole and indole were obtained in moderate to good yield, as shown in Table S1 (supplementary file). The infrared spectral pattern of all compounds 5a-5k is presented in the experimental section in which for all compounds C-3 carbonyl functional group peaks exhibit in the region between 1670 and 1695 cm⁻¹. The band due to √_NH of 1,2,4 triazole ring were observed in the range of 3180–3394 cm⁻¹. The presence of the number of protons in the synthesized compounds was recognized by ¹HNMR spectroscopic technique. The ¹HNMR spectra data were recorded in DMSO‑d₆. All the results have been presented in the experimental section. For all the compounds, a singlet peak at range δ 2.4–2.75 ppm indicates the presence of three protons, which is corresponding to the methyl group present at C-2 position of the indole ring. The spectra showed multiplate at the range between δ 7–8 ppm corresponding to all aromatic protons. δ 11–12 ppm exhibited as a singlet, which is corresponding to NH proton present in the 1,2,4 triazole ring. A singlet at the range between δ 5–5.2 ppm indicates about the presence of methylene protons at C-5 position of the indole ring. A quartet at a range between δ 4.2–4.5 ppm corresponds to the presence of methylene protons at C-3 position of the indole ring. A triplet δ 1.1–1.4 ppm is corresponding to the presence of methyl group at C-3 position of indole moiety. A multiplate δ 4–4.8 is corresponding to the presence of one proton of (N-CH) cyclopropyl ring. A multiplate in the range between at δ 0.9–1.2 ppm corresponding to four protons due to presence of two methylene protons appears in the cyclopropyl ring. Based on all these available information, the structures were determined. The molecular weight of synthesized compounds were recorded on a low-resolution mass spectrometer operating at 70 eV. The molecular weight of all compounds was precisely matching with the calculated molecular mass.

3.2 Molecular docking studies

A virtual screening mechanism using the Consensus-scoring method was applied in order to obtain potential ligands that bind the active site of α-glucosidase. In the beginning, synthesized molecules were built using SKETCH module implemented in the SYBYL program. The X-ray structure of N-terminal human maltase-glucoamylase (α-glucosidase), PDB ID: 3CTT, was obtained from protein data bank and used for docking calculations. The docking was performed with Surflex-Dock program interfaced with Sybyl-X 2.0. The compiled docked view for all the compounds (5a-5k) is shown in Fig. S1 (supplementary file). Based on the analysis, it was found that all the ligands were fitting well into the active site and had the most favorable docking scores, as shown in Table S2 (supplementary file). Among these 5e and 5j had the best Consensus scores. Hence, further docking analysis of binding region and interactions with amino acid residues in the active site were analyzed for these two ligands.

As depicted in Fig. 1, compound 5e makes four hydrogen-bonding interactions at the active site of the enzyme, hydrogen of –NH of triazole ring makes hydrogen bonding interaction with the oxygen of ASP542 (NH–---O-ASP542, 2.53 Å), an oxygen atom of methoxy group makes hydrogen bonding interaction with the hydrogen of ARG526 (O----H-ARG526, 1.88 Å), the hydrogen atom of methyl group present at the second position of indole ring makes hydrogen bonding interaction with the oxygen atom of LEU577 (H----O-LEU577, 2.40 Å). The methoxy group's oxygen atom at the fifth position in the phenyl ring makes hydrogen bonding interaction with the hydrogen of ASP327 (O----H-ASP327, 2.57 Å).

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Fig. 1

(A-B). Docked view of compound 5e at the active site of the enzyme Maltase-glucoamylase (α-glucosidase).

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As shown in Fig. 2, compound 5j makes two hydrogen-bonding interactions at the active site of the enzyme, oxygen atom of the carbonyl group of carboxylate group present at the third position of indole ring makes hydrogen bonding interaction with the hydrogen of THR205 (O----H-GLN603, 2.05 Å) and the hydrogen atom of methyl group present at carboxylate makes hydrogen bonding interaction with the oxygen of GLU300 (H----H-GLU300, 4.98 Å).

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Fig. 2

(A-B). Docked view of compound 5j at the active site of the enzyme Maltase-glucoamylase (α-glucosidase).

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The binding simulation of ligand, Casuarine, a well-known inhibitor with α-glucosidase protein active site, was used as reference ligand. As reported earlier and also analyzed by us, casuarine showed eight bonding interactions. The docked view of the same is shown in Fig. S2 (supplementary file) (Cardona et al., 2009). The docking results revealed that the synthesized compounds are well accommodated in the same binding pocket of α-glucosidase. Moreover, the synthesized compounds bind similarly to the protein as that of the Casuarine-enzyme complex.

3.3 In vitro α-amylase inhibitory potential

We have synthesized eleven indole derivatives containing 1, 2, 4-triazole moiety, and screened for α-amylase inhibitory potential. Tested compounds showed a variable degree of α-amylase inhibition ranging from 17.30 ± 0.68 to 71.29 ± 0.75 at a concentration range from 20 to 100 μg/mL. While at similar concentrations, standard acarbose showed 40.10 ± 0.75 to 94.02 ± 0.54 respectively. Among the compounds, 5e showed good α-amylase inhibition potential (30.05 ± 0.51, 39.04 ± 0.78, 48.05 ± 0.61, 54.30 ± 0.45, 63.63 ± 0.75 at concentration gradient 20 to 100 μg/mL respectively). Likewise compound 5j showed similar α-amylase inhibitory potential (30.08 ± 0.59, 40.11 ± 0.76, 54.05 ± 0.56, 62.09 ± 0.85, 71.29 ± 0.75 at concentration 20 to 100 μg/mL respectively). Remaining compounds including the parent molecule (compound-4) showed moderate to low inhibitory potential when compared to standard acarbose (Table 2). The DMSO (1%) did not affect the enzyme activity (data not shown). α-Amylase facilitates the digestion of complex carbohydrates by breaking 1,4-glycosidic linkages of polysaccharides to disaccharides. Hence inhibition of α-amylase is an important step in controlling hyperglycemia in humans. In this study, compounds 5e, and 5j retained 69–75% inhibitory activity compared to standard acarbose.

3.4 In vitro α-glucosidase inhibitory potential

α-Glucosidase is a carbohydrate hydrolyzing enzyme responsible for the conversion of disaccharides to monosaccharides. This enzyme's inhibition has been correlated with improved postprandial hyperglycemia and decreased insulin requirements in diabetic patients (Hanefeld et al., 2004). In our study, tested compounds showed a variable degree of α-glucosidases inhibition, ranging from 18.19 ± 0.68 to 71.02 ± 0.69 at a concentration gradient 20 to 100 μg/mL respectively. Compound 5e showed good α-glucosidase inhibition potential (30.07 ± 0.41 to 70.24 ± 0.86). Similarly, compound 5j had inhibitory potential between 30.04 ± 0.69 to 71.02 ± 0.69 respectively. Remaining compounds showed lower α-glucosidase inhibitory potential (Table 3). The DMSO (1%) did not affect the enzyme activity (data not shown). Thus it can be concluded that compounds 5e and 5j confer inhibitory effects on α-glucosidase; however, at moderate levels (∼75%) compared to standard acarbose.

All structural features such as, 5-substituted indole moiety, aryl substituted 1, 2 4 triazole play an important role in the α-glucosidase and α-amylase inhibition. Addition of 2,5 dimethoxy substituent (2,5-dimethoxybenzaldehyde) at ortho and meta position in compound 5e and hydroxy, methoxy substituents (6-methoxy-2-naphthol aldehyde) at ortho and meta position in compound 5j have exhibited good inhibitory activities against α-glucosidase enzymes. The remaining compounds had low to moderate α-glucosidase inhibitory potential. Earlier studies had shown similar observation, where Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, derived from the marine algae, had good α-glucosidase inhibitory activity (Liu et al., 2011). Also, it is shown that methoxy-substituted benzohydrazide derivatives had good antioxidant and α-glucosidase inhibitory activity attributed to the methoxy group at the meta and ortho position on the benzene ring (Prachumrat et al., 2018). These reports hence substantiate our observation on 5e and 5j being better ligands among the synthesized compounds.