Thiazolidine-2,4-dione hybrids as dual alpha-amylase and alpha-glucosidase inhibitors: design, synthesis, in vitro and in vivo anti-diabetic evaluation

Abstract

Twelve 3,5-disubstituted-thiazolidine-2,4-dione (TZD) hybrids were synthesized using solution phase chemistry. Continuing our previous work, nine O-modified ethyl vanillin (8a-i) derivatives were synthesized and reacted with the TZD core via Knoevenagel condensation under primary reaction conditions to obtain final derivatives 9a-i. Additionally, three isatin-TZD hybrids (11a-c) were synthesized. The intermediates and final derivatives were characterized using ¹H and ¹³C NMR spectroscopy, and the observed chemical shifts agreed with the proposed structures. The in vitro alpha-amylase and alpha-glucosidase inhibitory evaluation of newly synthesized derivatives revealed compounds 9F and 9G as the best dual inhibitors, with IC₅₀ values of 9.8 ± 0.047 μM for alpha-glucosidase (9F) and 5.15 ± 0.0017 μM for alpha-glucosidase (9G), 17.10 ± 0.015 μM for alpha-amylase (9F), and 9.2 ± 0.092 μM for alpha-amylase (9G). The docking analysis of synthesized compounds indicated that compounds have a higher binding affinity for alpha-glucosidase as compared to alpha-amylase, as seen from docking scores ranging from -1.202 to -5.467 (for alpha-amylase) and -4.373 to -7.300 (for alpha-glucosidase). Further, the molecules possess a high LD₅₀ value, typically ranging from 1000 to 1600 mg kg^-1 of body weight, and exhibit non-toxic properties. The in vitro cytotoxicity assay results on PANC-1 and INS-1 cells demonstrated that the compounds were devoid of significant toxicity against the tested cells. Compounds 9F and 9G showed high oral absorption, i.e., oral absorption >96%, and their molecular dynamics simulation yielded results closely aligned with the observed docking outcomes. Finally, compounds 9F and 9G were evaluated for in vivo antidiabetic assessment by the induction of diabetes in Wistar rats using streptozotocin. Molecule 9G has been identified as the most effective anti-diabetic molecule due to its ability to modulate several biochemical markers in blood plasma and tissue homogenates. The results were further confirmed by histology investigations conducted on isolated pancreas, liver, and kidney.

Fig. 1. Rationale for designing of proposed molecules.

Scheme 1. Synthesis of thiazolidinedione.

Scheme 2. Synthesis of 3-nitrobenzyl thiazolidinediones.

Scheme 3. Synthesis of O-substituted ethyl vanillin.

Scheme 4. Synthesis of title compounds 9a–i.

Scheme 5. Synthesis of ISATIN–TZD hybrids.

Fig. 2. Docking analysis of 9f and 9g within the active site of human pancreatic alpha-glucosidase. For the rest of the poses (see ESI 1).

Fig. 3. 2-D interaction pose of 9f and 9g within the active site of 1B2Y.

Fig. 4. MD simulation of 9G within the active site of human pancreatic alpha-glucosidase (UniProt id: P00689). 4a: RMSD variation during the simulation 4b: RMSF variation of atoms in the ligand.

Fig. 5. MD simulation of 9G within the active site of human pancreatic alpha-amylase (PDB id: 1B2Y). 5a: RMSD variation during the simulation 5b: RMSF variation of atoms in the ligand.

Fig. 6. A and B: Cytotoxicity evaluation of compounds 9F and 9G.

Fig. 7. In vitro antioxidant evaluation using ROS assay of synthesized compounds.

Fig. 8. Modulation of ROS production in animal pancreas by test compounds 9F and 9G.

Fig. 9. Modulation of alpha-amylase and alpha-glucosidase activity by 9F and 9G in pancreatic cells.

Fig. 10. Modulation of alpha-amylase and alpha-glucosidase activity by 9F and 9G in animal pancreas.

Fig. 11. Structure–activity relationship of synthesized compounds. Compounds were more active against α-glucosidase than α-amylase. Aliphatic groups showed higher activity against both of the tested enzymes. The molecule binds more tightly within the active site of α-glucosidase than α-amylase. Among substituted benzyls, the 4-Me benzyl group displayed the highest activity against α-glucosidase. Electron withdrawing groups showed lower activity than electron donating groups. Isatin derivatives showed weaker inhibition of α-glucosidase than α-amylase. Among isatin, 5-Cl displayed the highest activity against the tested enzymes.

Fig. 12. Oral glucose tolerance test. The data were reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a two-way analysis of variance (ANOVA) with post hoc Bonferroni's test (n = 6). A, v/s normal control. B, v/s STZ + 9F. c, v/s STZ + 9F, STZ + 9G.

Fig. 13. Effect of the test compounds on body weight in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a two-way analysis of variance (ANOVA) with post hoc Bonferroni's test (n = 6). a v/s normal control; vehicle control. b v/s STZ. c v/s STZ + 9F. d v/s STZ + 9G, STZ + 9F.

Fig. 14. Effect of the test compounds on modulation of blood glucose level in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a two-way analysis of variance (ANOVA) with post hoc Bonferroni's test (n = 6). a, v/s normal control; vehicle control. b, v/s STZ. c, v/s STZ + 9F. d, v/s STZ + 9G, STZ + 9F.

Fig. 15. Effect of the test compounds on PPAR-gamma levels in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 16. Modulation of serum alpha-amylase levels. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 17. Effect of the synthesized compounds on the lipid profile in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 18. Effect of synthesized compounds on kidney parameters in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 19. Effect of synthesized compounds on liver parameters in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 20. Effect of synthesized compounds on oxidative stress markers in pancreatic tissue in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 21. Effect of synthesized compounds on oxidative stress markers in the blood serum in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 22. Effect of synthesized compounds on TNF-alpha levels in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 23. Effect of synthesized compounds on IL-1beta levels in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD); p < 0.001 and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, v/s normal control, vehicle control; b, v/s STZ; c, v/s STZ + 9F. d, v/s STZ + 9F, STZ + 9G.

Fig. 24. Effect of synthesized compounds on complete blood count (CBC) parameters in STZ-induced diabetic rats. The data are reported as mean ± standard deviation (SD) and were evaluated using a one-way analysis of variance (ANOVA) with post hoc Tukey's test (n = 6). a, (p < 0.001) v/s normal control, vehicle control; b, (p < 0.001) STZ; c, (p < 0.001) STZ + 9F, STZ; d (p < 0.001) STZ + 9F, STZ + 9G, STZ.

Fig. 25. Effect of synthesized compounds on histopathology of pancreas in STZ-induced diabetic rats (I – normal control, II – vehicle control, III – STZ, IV – STZ + 9F, V – STZ + 9G and VI – STZ + PGZ).

Fig. 26. Effect of synthesized compounds on the histopathology of the liver in STZ-induced diabetic rats (I – normal control, II – vehicle control, III – STZ, IV – STZ + 9F, V – STZ + 9G, and VI – STZ + PGZ).

Fig. 27. Effect of synthesized compounds on histopathology of kidney in STZ-induced diabetic rats (I – normal control, II – vehicle control, III – STZ, IV – STZ + 9F, V – STZ + 9G, and VI – STZ + PGZ).