Design, synthesis and biological evaluation of novel coumarin-1, 2, 4-triazole derivatives as dual inhibitors of α-glucosidase and PTP1B

2.1. Material and instruments

The α-glu enzyme (G5003-1KU) originating from Saccharomyces cerevisiae was sourced from Sigma-Aldrich in the United States, while the substrate p-NPG (p-Nitrophenyl-α-D-galactopyranoside) was acquired from Macklin, a company based in Shanghai, China. The ¹H NMR and ¹³C NMR were monitored by JNM spectrometer. Besides, the high-resolution mass spectrometry (HRMS) were recorded by the instrument of UHPLC Q-Exactive Plus.

2.2. Chemistry

2.3. Evaluation of α-glu inhibition

In this experiment, all coumarin-1, 2, 4-triazole derivatives were dissolved in DMSO, the reference drug acarbose, substrate p-PNG and α-glu were solubilized in a 100 μM phosphate buffer at pH 6.8. First, 10 μL of the compound with various concentrations was dispensed into a 96-well plate, then mixed with α-glu (0.1 U/mL, 150 μL) and incubated at 37°C for 0.25 h 15 min. After that, 40 μL p-NPG (1.25 mM) was added into the reaction system and incubated for 0.5 h 30 min, the optical density (OD) values were monitored at 405 nm. The values of half-maximal inhibitory concentration were evaluated by GraphPad Prism 8, and all data were repeated three times.

2.4. Assay of PTP1B inhibitory activity

The inhibitory activity of 10n against PTP1B protein was detected [21]. Compound 10n was prepared in a 10% DMSO solution, with a 5 µL aliquot being introduced into a reaction mixture (50 µL) to ensure the final concentration of DMSO was 1%. The assessment of enzyme inhibitory activity was carried out at room temperature for 0.5 h within a 50 µL solution that included 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0), sodium chloride (50 mM), dithiothreitol (DTT) (3 mM), 0.05% Tween 20, 6,8-difluoro-4-methylumbelliferyl phosphate, (DiFMUP) (10 µM), the phosphatase and compound 10n. The fluorescence intensity of PTP1B protein was detected utilizing a plate reader (Tecan Infinite M1000) with excitation was 358 nm and emission was 455 nm. Then, the fluorescence intensity data were dealt with GraphPad Prism 8.

2.5. Enzyme kinetics study of 10n on α-glu

The inhibition mode of active compound 10n on α-glu was identified by the method of enzyme kinetics. Firstly, based on the IC₅₀ value of 10n to set the concentrations of 10n around about half of the IC₅₀ value. Varied concentrations of 10n (0 µM, 4 µM, 8 µM) and 0.05 U/mL α-glu were co-incubated in 96-well plates at 37°C for 0.25 h. After that, p-PNG (1mM, 1.5 mM, 2.0 mM, 2.5 mM) was added to the mixture and the OD values were monitored at 405 nm. Finally, the data were dealt with Origin 64, and the relevant constants were obtained from the Lineweaver-Burk plot.

2.6. Fluorescence quenching

For studying whether 10n could change the α-glu fluorescence intensity, the fluorescence quenching assay was conducted based on the previous method with subtle modification [22]. Compound 10n (0, 2, 4, 6, 8, 10, 12 μM) was mixed with 2 mL α-glu (3.5 U/mL) thoroughly, scanning and recording the fluorescence intensity under the condition of excitation wavelength: 280 nm, emission wavelength: 290-450 nm, slit width: 5 nm.

In this experiment, all outcomes were calculated by the equation: F₀/F = 1+ K_qτ₀ [Q] =1+K_sv [Q]. F₀ stands for the fluorescence intensity of α-glu in the absence of the compound, while F denotes the fluorescence intensity of α-glu with the compound, respectively. K_q stands for the constant of bimolecular quenching rate, K_sv is the constant of fluorescence quenching, [Q] is the concentration of quencher 10n, and τ₀ (10^-8 s) stands for the average lifetime of the biomolecules without the quencher. Besides, Formula log [(F₀-F)/F] = nlog[Q] +logK_a was applied to compute the number of binding sites (n) and the binding constant (K_a).

2.7. Synchronous fluorescence

Setting the parameters of the steady-state/transient fluorescence spectrometer (FLS 1000, Edinburgh) as follows: wavelength range of 240 nm to 350 nm, both slit widths set to 5 nm, Δλ = 15 nm, and Δλ = 60 nm. The fluorescence spectra of α-glu without compound 10n and with different concentrations of 10n were scanned under Δλ = 15 nm and Δλ = 60 nm, respectively. Before scanning, the solvent was scanned to subtract the background. The relative synchronous fluorescence quenching ratio (RSFQ) was calculated using the formula: RSFQ = 1 - F/F₀, where F and F₀ were as mentioned in the part of fluorescence quenching.

2.8. Circular dichroism spectra

The method was performed according to our reported work [1]. Briefly, before scanning, the solvent should be scanned to subtract the effect of the background. Then, 2 mL of α-glu (3 U/mL) was prepared, and compound 10n of 0, 1, 3, 5, 7, 9 μM were added to the enzyme system. Then, circular dichroism (CD) was monitored from 200-260 nm using a CD spectrometer Chirascan Plus (Applied Photophysics, Leatherhad, UK). The data were processed by Origin 64 software, and the results were analyzed by CDNN.

2.9. Three-dimensional (3D) fluorescence spectra

The 3D fluorescence spectra of α-glu and the compound-enzyme mixture were acquired based on the previously reported method [5]. Both the wavelength of emission and excitation were set to 200-500 nm, and the slit width was 5 nm. Firstly, the absorption of solvent was scanned to subtract the background. After that, the spectra of α-glu (1 U/mL) were recorded, then adding 5 μM compound 10n into the enzyme system rapidly and mixing well, scanning under the same conditions.

2.10. Cytotoxicity assay in vitro

HEK-293 cells were selected to test the effect of compound 10n on normal cytotoxicity as described as reported work previously [3,9,16]. Before the addition of various concentrations of 10n, cells need to be placed into 96-well plates and allowed to incubate for a period of 24 h. After cells were co-cultured with 10n for 24 h, 15 μL MTT (5mg/mL) was added into the plates and cultured for 1-4 h, discarding the supernatant and adding 150μL DMSO, shaking the mixture and measuring the OD values at 490 nm.

2.11. Molecular docking

Docking modes of 10n with both enzymes were performed by the software of Autodock vina 1.1.2 [23]. Besides, the search grid for α-glu featured center coordinates x: -19.676, y: -7.243, z: -21.469, and dimensions size_x: 17, size_y: 17, size_z: 17, and the search grid of PTP1B was the same as in previous work [16]. Between these two proteins, the exhaustiveness was 16. If not mentioned, use the default parameters. Selecting the best pose evaluated by Vina docking scores and performing a visual analysis using PyMOL 1.7.6.

2.12. The sucrose-loading test in vivo

The in vivo sucrose-loading test was carried out to detect whether compound 10n could exert an effect on the postprandial blood glucose levels, mainly based on the method reported previously [2]. The normal male Kunming mice (7-8 weeks, 30-35 g, SCXK (2019-0010)) were purchased from SPF (Beijing) Biotechnology Co., (Beijing, China). These experiments were approved by the Laboratory Animal Management and Ethics Committee of Guizhou Medical University (Ethics Code: 2402935). After the experimental mice were adapted to culture for 7 days, all mice were fasted but drank water normally for 12 h. All animals were divided into four groups (n = 4) randomly, which included the blank group, positive acarbose group, control and treated group with 10n. Before the experiment, compound 10n (4 mg/mL), sucrose (0.7 g/mL) and acarbose (2 mg/mL) were homogeneously suspended in 0.5% CMC-Na aqueous solution. After that, each mouse in each group was numbered and weighed. Subsequently, both the compound group and acarbose group were gavaged with a dosage of 20 milligrams per kilogram (20 mg/kg). After 40 min, the control and blank groups were gavaged with the pre-prepared sucrose solution (2.5 g/kg) and 0.5% CMC-Na aqueous solution, respectively. Immediately thereafter, using the Roche Accu-Chek Instant blood glucose meter to monitor the concentrations of blood glucose in the tail tip of the mice at 0, 15, 30, 60, 90, and 120 min after the administration of sugar. The outcomes of the experiment were analyzed using Origin 64.

3.1. Chemistry

The synthetic route of coumarin-1, 2, 4-triazole derivatives 10a-10n was demonstrated in Figure 2. At first, treatment of 4-hydroxycoumarin with epichlorohydrin and KOH in EtOH yields key intermediate 4-(oxiran-2-ylmethoxy)-2H-chromen-2-one (3). Secondly, phenol reacted with chloroacetic acid to yield the intermediate 2-phenoxyacetic acid (5). Subsequently, intermediate 5 undergoes a cyclization reaction with thiosemicarbazide 6 under high temperature to form intermediate 4-amino-5-(phenoxymethyl)-4H-1,2,4-triazole-3-thiol (7) [9]. Moreover, substituted benzaldehydes (8a-8n) reacted with intermediate 7 to obtain intermediates substituted (E)-4-((benzylidene)amino)-5-(phenoxymethyl)-4H-1,2,4-triazole-3-thiol (9a-9n). Finally, the intermediate 3 yielded in the first step was reacted with 9a-9n to get the final products coumarin-1, 2, 4-triazole derivatives 10a-10n. During the synthesis of the compounds, our primary objective was to obtain the target compounds for the evaluation of their biological activities; thus, we did not dedicate excessive research efforts to optimizing compound yields and synthetic conditions. In addition, a chiral center is generated in the final reaction step, which makes the target compound a racemate. In this study, we did not perform resolution, and the subsequent studies were conducted in the form of the racemate. All title compounds were confirmed by the method of ¹H NMR, ¹³C NMR, and HRMS (Supplementary materials).

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Figure 2.

The synthetic route of title compounds 10a-10n. (a) NaOH, EtOH, 80°C, 5h; (b) NaOH, EtOH, 80°C, 5h; (c) 160°C, 1 h; (d) AcOH, 120°C, 4h; (e) EtOH, 80°C, 5h.

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For example, the ¹H NMR spectrum of compound 10d (with R = 4-CH₃) showed a singlet signal for three protons at 2.32 ppm, which was assigned to the methyl (CH₃) substituent. According to the literature [19], 3.32-3.70 (m, 1H), 3.48 (dd, 1H, J = 13.2 Hz, 4.4 Hz), and 4.15-4.23 (m, 3H) are assigned to the C-H signals of the linker between coumarin and 1,2,4-triazole. For the other compounds in this series, the peaks at 3.32-3.37 ppm are partially obscured by the water peak in the deuterated solvent DMSO and thus not clearly observed, because their chemical shift is very close to that of the water peak. The hydroxyl group (-OH) on the linker appears as a doublet with a coupling constant J = 4.0 Hz at 5.76 ppm. The singlet at 5.24 ppm with an integration of two is assigned to the methylene group (-CH₂OPh) of the phenoxymethylene side chain. The singlet at 5.82 ppm is the signal for the C-3 hydrogen of coumarin. The remaining hydrogen signals of the coumarin ring are observed at 7.38-7.32 ppm (m, 2H), 7.60-7.64 ppm (m, 1H), and 7.87 ppm (d, J = 8.0 Hz, 1H) [24]. The phenyl ring peaks of the phenoxy side chain are observed at 6.90-6.94 ppm (m, 1H) and 7.22-7.29 ppm (m, 4H). Two double peaks at 6.97 and 7.66 ppm with a coupling constant of 8.0 Hz were attributed to the aromatic protons of C3,5-H and C2,6-H at the 4-methylphenyl moiety, respectively. For the other compounds in this series, different aromatic hydrogen signals will be exhibited in the range of 6.5-8.5 ppm due to differences in substituents. The singlet peak of the CH proton of the hydrazone moiety was observed at 8.82 ppm.

In the ¹³C NMR spectra of compound 10d, the five carbon signals in the range of 0-75 ppm are assigned to aliphatic carbons, including methyl (CH₃) substituent, methylene group (-CH₂OPh), and linker (-OCH₂CHOHCH₂S-). The peak at 91.1 ppm is the signal for the C-3 carbon of coumarin. The seven peaks in the downfield region above 145 ppm are assigned to carbons directly bonded to heteroatoms (O, N, or S). The signals in the range of 110-145 ppm are the remaining aromatic ring carbon signals. Furthermore, the HRMS analysis of all compounds 10a-10n was consistent with their respective molecular formulas. Hence, the characterizations by ¹H NMR, ¹³C NMR, and HRMS clearly confirmed the structural correctness of the target compounds in this study.

3.2. α-Glu inhibitory activity

All target compounds 10a-10n were tested for their α-Glu inhibitory activity. As depicted in Table 1, compounds 10a (28.62 ± 0.54 μM), 10b (31.07 ± 1.10 μM), 10e (24.95 ± 1.57 μM), 10g (16.68 ± 0.63 μM), 10h (21.22 ± 0.65 μM), 10i (37.31 ± 1.00 μM), and 10n (9.71 ± 0.28 μM) displayed excellent inhibitory activity more than the positive control acarbose (IC₅₀ = 309.83 ± 8.74 μM). Compounds 10c, 10f, 10l, 10m exhibited moderate α-glu inhibitory activity with IC₅₀ values of 160.67 ± 5.10 μM, 125.50 ± 0.92 μM, 39.40 ± 0.92 μM, 65.82 ± 0.39 μM, respectively. Other compounds 10d (4-CH₃), 10j (4-Cl) and 10k (4-Br) showed low inhibitory activity with IC₅₀ values more than 200 μM. Amongst them, compound 10n demonstrated the strongest inhibition with an IC₅₀ value was 9.71 ± 0.28 μM, which was approximately 32-fold that of acarbose. Since compounds 10a-10n share the same chemical scaffold and 10n is the most active compound in this series, 10n will be selected subsequently as the representative compound of this series to conduct studies on its mechanism of action and activity evaluation.

3.3. PTP1B inhibitory activity

On the basis of the results of α-glu inhibition, derivative 10n with the strongest α-glu inhibitory activity was selected to detect its PTP1B inhibitory effect. In this experiment, ursolic acid was chosen as the reference compound according to the previous literature [25]. Results displayed that the activity of PTP1B could be decreased after being treated with compound 10n with different concentrations, and the downward trend of PTP1B activity in the 10n treatment group was similar to the positive ursolic acid [16]. The results revealed that 10n exhibited excellent PTP1B inhibition with an IC₅₀ value was 7.31 ± 1.55 μM, as compared to the positive ursolic acid (4.55 ± 1.08 μM) (Figure 3).

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Figure 3.

PTP1B inhibitory activity of compound 10n and ursolic acid.

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3.4. Analysis of SAR

A total of fourteen compounds (10a-10n) were synthesized, and their α-glu inhibitory activity was tested, but compounds with different substituents displayed varied α-glu inhibition. Therefore, for the purpose of discussing the relationship between the difference of R groups and α-glu inhibition, the structure-activity relationships (SAR) were analyzed. Through SAR discussion of these compounds, it was found that compounds containing -OH (10a, 10b, 10e, 10g and 10h) were beneficial for improving the inhibitory activity of α-glu. Especially, by comparing compound 10e (R: 3-Cl, 4-OH) and 10g (R: 2-F, 4-OH) that introducing the halogen groups could enhance the inhibition. However, when the substitutes were only halogen, the enzyme inhibition could be reduced (10f and 10l) or even inactive (10i and 10k), which depended on the position of the halogen. These results suggest that introducing an electron-donating substituent (OH) at the para position of the benzene ring enhances inhibitory activity, while introducing electron-withdrawing substituents (F, Cl, Br) at the para position results in decreased activity. However, if the electron-withdrawing group (NO₂) is at the meta position, it has little effect on activity. This suggests that the electronic effect exerts a significant influence on activity, and the effect of substituents at the para position on the compound’s electron cloud is much greater than that at the meta position. Additionally, the existence of electron-donating groups like -CH₃ had a great influence on α-glu inhibition. For example, comparing compounds 10c (R: 3,5-Me₂, 4-OH) and 10h (R: 2,6-Me₂, 4-OH), despite their identical substituents, but there was a large difference in inhibiting α-glu for changing the positions of methyl. Changing the 2,6-dimethyl substitution to 3,5-dimethyl substituents resulted in a decrease in inhibitory activity by approximately 8-fold. The introduction of a phenyl at the para position of benzene ring (10n) significantly enhanced the α-glu inhibitory activity, indicating that the larger steric hindrance of the compound may be favorable for enhancing activity (Figure 4).

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Figure 4.

Analysis of SAR between 10a-10n and α-glu inhibition.

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3.5. Inhibition kinetics

For further understanding of the inhibition mode of the most active compound 10n on α-glu, the enzyme inhibition kinetics test was carried out. The Lineweaver-Burk plot was used to describe the relationship between concentrations of substrate (p-NPG) and activity of residual enzyme. According to Figure 5(a), a group of straight lines intersecting in the second quadrant was detected, which suggests compound 10n was a mix-type inhibitor. This type of inhibition indicates that 10n could both inhibit free α-glu enzyme and the complex of α-glu and substrate. In addition, after quadratic plotting of the double reciprocal plot, the constant of inhibitor binding to the free enzyme (K_i = 21.12 μM) was calculated to be greater than the constant of inhibitor binding to the enzyme-substrate complex (K_is = 4.96 μM) (Figures 5b and c), demonstrating that compound 10n was more inclined to bind to the enzyme-substrate complex [26]. Simultaneously, the result revealed that as the concentration of compound 10n increased, the Km value progressively climbed, whereas the Vmax value steadily declined.

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Figure 5.

Analysis of enzyme inhibition kinetics of compound 10n against α-glu. (a) Lineweaver-Burk plot for 10n; (b) The plot between slope and concentrations of 10n; (c) The plot between Y-intercept and concentrations of 10n.

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3.6. Fluorescence quenching

Fluorescence spectroscopy is currently the most popular tool for detecting ligand-protein interactions because it is easy to operate and the experimental results are accurate [27]. As shown in Figure 6(a), the distinctive fluorescence peak of α-glu was found at 331 nm. Besides, the addition of 10n could decrease the fluorescence intensity of this enzyme from 1.62×10⁶ to 8.51×10⁵ (a decrease of 47.5%), demonstrating that 10n could inhibit the activity of α-glu by quenching the fluorescence intensity of α-glu.

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Figure 6.

(a) The fluorescence spectra of α-glu treated with various concentrations of compound 10n. (b) The Stern-Volmer plot of α-glu enzyme. (c) The double log plots of α-glu. (d) Synchronous fluorescence spectra of α-glu at ∆λ = 15 nm. (e) Synchronous fluorescence spectra of α-glu at ∆λ = 60 nm. (f) The fluorescence-quenching ratio of Tyr and Trp. (g) The CD spectra of α-glu after being treated with compound 10n (0, 1, 3, 5, 7, 9 μM).

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The mechanism of fluorescence quenching contains static quenching and dynamic quenching. These two mechanisms are led by a complex combination of enzymes and compounds and the diffusion and collision of molecules, respectively. For exploring the fluorescence quenching mechanism of compound 10n on α-glu, the data were analyzed utilizing the Stern-Volmer equation [4]. As depicted in the Stern-Volmer plot (Figures 6b and c), the linear relationship between the fluorescence intensity ratio (F₀/F) and the concentration of the quencher within the specified range indicated that the quenching of α-glu by 10n was governed by a single quenching mechanism. K_sv (0.75×10⁵ L∙mol^-1) represented the quenching constant of fluorescence, and K_q (0.75×10¹³ L∙mol⁻¹∙S⁻¹) was the bimolecular quenching rate constant. As K_q (0.75×10¹³ L∙mol⁻¹∙S⁻¹) surpassed the maximum diffusion constant for collision quenching (2×10¹⁰ L∙mol⁻¹∙S⁻¹), it confirmed that 10n quenched α-glu via a static quenching mechanism. The K_sv and K_q values calculated in this study are similar to those reported in the literature, and both fall within the same order of magnitude [28]. By calculating the binding constant (K_a = 7.53×10³ L∙mol⁻¹) and the number of binding sites (n = 0.98), which was close to one, it was further demonstrated that 10n and α-glu had only one binding site.

3.7. Synchronous fluorescence

Synchronous fluorescence spectroscopy, as a means of studying changes in the microenvironment near functional fluorescent groups in enzymes, is often applied to the mechanistic study of various enzymes. ∆λ refers to the difference between the wavelength of excitation and emission. For tyrosine residues (Tyr), the wavelength difference (∆λ) is 15 nm, whereas for tryptophan residues (Trp), the wavelength difference (∆λ) is 60 nm. As shown in Figures 6 (d and e), as the concentrations of 10n increased, the fluorescence intensity decreased progressively at Δλ = 15 nm and Δλ = 60 nm, which was consistent with the outcomes of fluorescence quenching. The decrease in fluorescence intensity indicated that 10n bound to the α-glu. The results in Figure 6(f) exhibited that under identical concentration conditions, the quenching ratio (RSFQ%) of tryptophan residues exceeded that of tyrosine residues, suggesting that the binding site of 10n was closer to the tryptophan residues, which led to faster quenching of this amino acid residue.

3.8. Thermodynamics analysis

Non-covalent interactions are the primary contributors to the stability of enzyme-small molecule complexes, including hydrophobic forces, hydrogen bonds, Van der Waals forces, and electrostatic forces. All these forces are critical to the thermodynamic parameters of the complex. To explore the binding mechanism between compound 10n and α-glu, the corresponding thermodynamic parameters (ΔG, ΔH, and ΔS) were derived from the van’t Hoff equation (Table S1). Negative ΔG values suggested that the binding of compound 10n to α-glu was a spontaneous process. Additionally, the results showed ΔH<0 and ΔS>0, which indicated that both hydrogen bonds and the hydrophobic interactions served as the dominant driving force for this binding [29].

3.9. Circular dichroism

Circular dichroism (CD) was an effective tool to detect the effect of inhibitors on the conformational changes of the enzyme. Therefore, the CD spectra were recorded to further elucidate how the compound 10n affected the protein conformation [30]. From Figure 6 (g), two distinct negative peaks at 209 nm and 220 nm were observed, which were typical peaks of α-helixes of α-glu. With an increase in the concentrations of 10n, these two characteristic negative peaks gradually flattened, indicating that the addition of compound 10n could change the secondary structure of α-glu. Besides, this phenomenon also indicates that the inhibitor induces folding or unfolding of the polypeptide chains composing the enzyme, resulting in a decrease in the content of α-helixes. As shown in the calculations in Table 2, it was evident that with the concentrations of 10n increased, the α-helix content decreased from 36.8% to 26.9%, the β-sheets content and β-turns respectively increased from 15.5% to 21.4% and 16.3% to 18.1%, respectively. The proportion of random coils also rose from 29.4% to 37.3%, suggesting that the α-glu secondary structure became more unstable and looser after interacting with 10n. The reduction in enzymatic activity was primarily attributed to alterations in α-glu’s secondary structure and microenvironment. The alterations of the secondary structure and microenvironment may affect the conformation of the α-glu active center, modify its binding affinity and catalytic efficiency towards substrates, and thereby influence its enzymatic activity [16,31].

3.10. 3D fluorescence

The function and activity of a protein could be affected by the interaction of a ligand through changing the conformation and structure of the target protein [32]. Thus, 3D fluorescence spectroscopy was employed to assess the influences of 10n on the conformation and structural integrity of α-glu. The data presented in Figure 7 shows that there were two distinctive peaks in the spectra of fluorescence in 3D of α-glu. Peak 1 (λex/λem: 310/380 nm) and peak 2 (λex/λem: 230/401 nm) represented the absorption peaks of tryptophan and tyrosine residues, as well as the fluorescence characterization of peptide backbone structure, respectively. As Figures 7(a,c) displayed that the addition of 10n could reduce the fluorescence intensity of these two characteristic peaks, demonstrating that compound 10n could destroy the three-dimensional structure of α-glu. All these results were similar to the reported literature [33].

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Figure 7.

3D fluorescence spectroscopy of α-glu with compound 10n. (a) 3D fluorescence spectra of α-glu. (b) Contour maps of α-glu. (c) 3D fluorescence spectra of α-glu with 10n. (d) Contour maps of α-glu with 10n.

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3.11. HEK-293 cytotoxicity

To investigate whether the compound 10n had significant toxicity to normal human cells, an MTT assay was performed to examine the impact of 10n at concentrations of 300, 150, 75, 37.5, 18.75, and 9.375 μM on the survival rates of human embryonic kidney cells (HEK-293). The experiment found that at the maximum concentration of 10n (300 μM), the cell survival rate reached 96.34%, demonstrating that 10n was almost non-toxic to normal human cells. Other more in-depth safety evaluations will be conducted in future research.

3.12. Molecular docking between α-glu and compound 10n

Molecular docking of 10n to the active pocket of α-glu was illustrated in Figure 8 (a,b), where 10n was well wrapped within the interaction site of α-glu. The evaluated binding energy between the α-glucosidase and 10n was -11.7 kcal•mol^-1. Derivative 10n was found to be situated in a hydrophobic pocket, flanked by residues Phe157, Leu176, Phe177, Leu218, Pro240, Ala278, Phe300, Val303, and Phe311, which together established robust hydrophobic contacts (Figure 8 c and d). Besides, the coumarin moiety of compound 10n engaged in a π-π stacking association with the phenylalanine residue at position 157, and the biphenyl group formed a π-π stacking interaction with Phe300. Notably, a significant hydrogen bond was detected between 10n and the amino acid residue Arg312 (bond length: 2.6 Å). These interactions facilitated the strong binding of compound 10n to the active site of α-glu, thereby exerting its inhibitory activity.

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Figure 8.

Molecular docking of 10n in α-glu. (a) Compound 10n. (b) The overall structure of compound 10n binding to α-glu. (c) Binding posture of 10n at the active pocket of α-glu. (d) Binding posture of 10n in the surface of the binding of α-glu.

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3.13. Molecular docking between PTP1B and compound 10n

There were two active sites in PTP1B, containing the catalytic active site and the second aromatic active site. The former is composed of amino acid residues Cys215-Arg221, and Cys215 being the catalytic center, and the latter consists of six amino acid residues: Arg24, Arg254, Met258, Gly259, Gln262, and Gln266 [34]. Importantly, it is significant to introduce aromatic structures into PTP1B inhibitors, which could enhance PTP1B inhibition. Therefore, in order to observe the binding mode between PTP1B protein and the active compound 10n, the molecular docking was carried out, and docking outcomes of compound 10n with PTP1B protein were shown in Figure 9 (a, b). The evaluated binding energy between the PTP1B and 10n was -8.3 kcal•mol^-1. The 2H-chromene-2-one framework of 10n was positioned at the catalytic active site, surrounded by residues Phe182, Ala217, and Ile219, forming a strong hydrophobic interaction. The phenyl group of 10n could extend to the secondary aromatic binding pocket of PTP1B and form van der Waals forces with it (Figure 9 c and d). Additionally, the 2H-chromene-2-one framework of compound 10n established a π-π stacking interaction with the phe182 and a CH-π interaction with the tyrosine residue at position 46. Obviously, a significant hydrogen bond was identified between 10n and the Gln262, with a bond length of 2.3 Å. These combined interactions effectively secured 10n within the active pocket of PTP1B.

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Figure 9.

Molecular docking of 10n in PTP1B. (a) The structure of compound 10n. (b) Compound 10n at the binding site. (c) Compound 10n is in the surface of the binding pocket.

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3.14. Sucrose-loading test in vivo

To ascertain whether compound 10n could lower blood sugar levels after meals, a sucrose-loading test was done following previously reported protocols [3,5,16]. As depicted in Figure 10, during the test, the blood sugar levels of the blank group remained stable. After the administration of sucrose (2.5 g/kg), there was a rapid increase in the control group, reaching a peak at 15 min. Compared to the control group, both acarbose and compound 10n could quickly decrease the concentrations of postprandial blood sugar at 15 min after administration. From Figure 10(a), it could be found that although the effect of compound 10n on reducing postprandial blood sugar was lower than acarbose, it still had a good blood sugar-lowering effect compared to the blank group. As illustrated in Figure 10(b), the area under the curve (AUC) administered with acarbose and 10n was remarkably lower in comparison with the blank group (p<0.05), demonstrating compound 10n has the capacity to decrease postprandial blood sugar levels in mice, and the results were similar to the reported literature [16]. In our future research, we plan to use normal mouse models and diabetic mouse models, respectively. By investigating long-term administration and different administration doses, we aim to further clarify the therapeutic effect of compound 10n on diabetes.

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Figure 10.

Sucrose-loading test of 10n. (a) Postprandial blood glucose in mice (n = 8). (b) Blood glucose AUC in mice at 150 min post-administration. (***p < 0.001, ****p < 0.0001 vs. the control)

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