NDMS and 2-BS compounds were synthesized with alloxan monohydrate 98% (Chai 10G, Sigma, CAS 2244-11-3), acarbose (Alfa Aesar), yeast α-glucosidase (CAS 9001-42-7), porcine α-amylase (CAS 9000-90-2), Tween 80 (CAS 9005-65-6) (Sigma Aldrich), normal saline (Medipak Limited), creatinine Jafee (Kit Lot CF 11231JB-CentronicGmbH/Germany), GPT/ALT-LQ- (Lot-LIQ-1259 LABKIT, China), ACCU-CHEK glucose measuring kits (Roche), ethanol strips, and pricking needles. All chemicals and solvents were of analytical grade and used without further purification.

Healthy, 8–12 weeks Swiss albino mice were purchased from the Directorate of Veterinary Research Institute, Peshawar, and housed in sanitized polypropylene cages. Mice were housed in the animal house of the Department of Pharmacy, University of Swabi, Pakistan, and nourished under standard conditions (12 h light/dark) at 25 ± 2°C temperature 34 and 50 ± 15% relative humidity and were fed standard food (pellet diet) and water ad libitum. Upon reaching an average body weight of 35 g, the animals were inspected for in vivo experimentation. The randomized animals were adapted to the laboratory environment for ten days prior to the start of the study. The experimental protocol was approved by the Ethical Committee (UOS/Pharm11), Department of Pharmacy, University of Swabi, Khyber Pakhtunkhwa, Pakistan. The Advance Academic and Research Board (ASRB), University of Swabi, approved this study (study permission no. F. NO. DA/UOS/2023/1766 dated August 01, 2023). Following the experiment, the animals were euthanized in compliance with AVMA regulations³⁵.

NDMS and 2-BS were synthesized by M.S. Jan et al. (2020) and further evaluated for their antidiabetic potential. The synthesis of the N-benzene sulfonamide derivative of pyrrolidine-2,5-dione shown in Scheme 2 (see S. Figure 1) ³⁶. The analyzed compounds were synthesized by Michael's addition of ketones to N-substituted maleimides. A mixture of ethyl-2-oxy-cyclohexanecarboxylate (2.0 equivalents) and a catalyst (20 mol% of either 8-hydroxyquinoline or a combination of creatinine and KOH) was mixed in 1 M dichloromethane (DCM) and magnetically agitated for 3–4 min, followed by N-cyclohexylmaleimide additions to synthesize the desired molecules. Thin-layer chromatography (TLC) plates were used to track the reaction progress (see S. Figure 2). After the reaction, the mixture was cooled and diluted with 15 mL of water. Dichloromethane (3 × 15 mL) was then used to extract the material, which was filtered and concentrated using a low-pressure rotary evaporator. Column chromatography was used to purify the crude mixture.

The in-depth summary begins with the combination of N-substituted maleimides with different cyclic alkyl/alkyl ketones to create ketone derivatives of pyrrolidine-2,5-dione. All reactions were straightforward, single-step processes that were completed quickly and produced good isolated yields. To the best of our knowledge, this is the first report of a tricomponent non-covalent organocatalytic system for these reactions. N-sulfamoyl-phenylmaleic acid ([(4-sulfamoylphenyl) carbamoyl] prop-2-enoic acid) intermediate was produced in the first stage by reacting maleic anhydride with sulfanilamide in diethyl ether to form an N-substituted sulfonamide derivative. The sulfanilamide derivative was obtained by cyclizing the intermediate in acetic anhydride with sodium acetate. After reacting with the N-sulfonamide derivative, cyclohexanone and cycloheptanone were produced in the reaction mixture. Biologically screened compounds were > 95% pure, as determined by HPLC³⁶. The physical and chemical properties of NDMS include the following: Molecular Formula: C₁₂H₁₄N₂O₄S, Molecular Weight, 282.32 g/mol; Melting Point, 160°C; Solubility: Soluble in methanol, ethanol, and DMSO Low solubility in hexane and chloroform; moderate water solubility; Appearance: White to off-white solid; and Boiling Point, 360°C. 2-Benzylsuccinimide molecular formula of C₁₁H₁₁NO₂, a Molecular Weight of 189.21 g/mol, and a Melting Point of 102°C. solubility in water is likely low, as is common for succinimide derivatives, owing to their relatively nonpolar nature. 2-BS is more soluble in polar organic solvents such as acetone, ethanol, methanol, and dimethyl sulfoxide (DMSO) because of the ability of the succinimide and benzyl groups to interact with these solvents. Appearance: white or off-white crystalline solid; boiling point: 325°C.

Before the administration of the compounds to lab animals, 5-2000 mg/kg/b. w., comprising 8 doses, was determined by the results of preliminary range-finding experiments from LD0-100. For every eight doses, three replicates were examined using both the positive and negative controls. Oral routes were followed for each group of five mice, and after implementing the predetermined operations, the animals were closely monitored for allergic reactions, behavioral changes, and mortality rates for up to three days. The comprehensive dosage schedules and animal requirements are provided in Tables^{36, 37}.

Diabetes was induced by administering intraperitoneal alloxan at 120 mg/kg/bw. w. to mice ⁴¹. Not only was the symptomatic assessment performed for polyuria and polydipsia, but the model was also evaluated by fasting blood glucose levels above 200 mg/dL for diabetes induction. The animals were grouped into normal control (nondiabetic, saline nourished), diabetic control (alloxan administered), group 3 was standardly treated with glibenclamide (0.5 mg/kg), and groups 4 and 5 were treated with oral NDMS at doses of 5 and 10 mg/kg, and 2-BS at the same dose of NDMS was administered orally to groups 6 and 7. Glucose levels were recorded regularly for up to 15 days using an Accu-Check glucometer.

After 15 days, blood samples were collected in heparin-coated sample tubes. Serum was isolated and processed for determination of total cholesterol, LDL, HDL, and TGs ^{17, 42} before centrifugation at room temperature for 30 minutes at 15000 rpm. For triglyceride (TG), the separated serum was analyzed using a calorimetric machine (MicroLab 300; Q-Line Biotech Pvt. Ltd.). Yellow gel tubes (clot activators) were used for TG quantification. Approximately 1 ml of TG reagent was added to the tube and mixed with 10 µL of serum, followed by incubation for 10 min at 37°C. The tube was then processed using a machine. Similarly, for total cholesterol, the same process was repeated, but the serum was mixed with a cholesterol reagent and placed in a water bath for 10 min at 37°C. The prepared tube was placed in a MicroLab 300 (Q-Line Biotech Pvt. Ltd.) for analysis, and the readings were recorded. The levels of total cholesterol, total triglycerides, low-density lipoproteins, and high-density lipoproteins in the plasma were measured.

The same isolated plasma was used for the analysis of liver enzymes (SGPT, SGOT, and ALP). Briefly, for SGPT, SGOT, and ALP profiling, the previously separated serum (10–50 µL) was mixed in a tube with an ALT reagent (Thermo Fisher Scientific Company) and subjected to calorimetric analysis. For creatinine analysis, mice blood samples (1.5 ml) in a heparin-coated sample tube already placed in the tube rack were used using the standard kit method. A blank solution was tested to check the machine function, followed by validation that the creatinine reagent fell within the acceptable reading range of ≤ 2 mg/dL. The desired blood sample was then aspirated after mixing with the reagent and waiting for 10 s using a stopwatch, followed by machine readings for further statistical analysis.

To check the recovery of the pancreas, approximately 3–4 pancreas samples from each group were collected from the sacrificed mice for histopathological examination of the pancreas at the end of the anti-diabetic study. The study was continued for up to 4 weeks after the administration of various dosages to the respective groups. A standard histological protocol was used (IETIE). Briefly, pancreatic sections were preserved in formalin saline. Subsequently, ethanol was used to dehydrate the tissues, followed by xylene tissue cleansing at increasing concentrations. Before embedding in paraffin blocks, the tissues were impregnated with molten paraffin. Fine sectioning was followed by hematoxylin and eosin (H and E) staining, and the samples were examined under a light microscope. For histological examination of the liver, three to four mice from each group that had the greatest change in blood sugar levels were sacrificed. This was performed using a standard histological method. The liver was immediately preserved in 10% formalin for 24 h at room temperature after euthanasia. The tissues were subsequently divided into sections, placed on glass microscope slides, and fixed in paraffin. The slices (4 mm) were stained with hematoxylin and eosin and viewed under a light microscope.

SigmaPlot@17 was used for all graphical and statistical analyses. A P-value < 0.05 was considered to indicate a significant statistical difference.

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NDMS and 2-BS directly blocked α-amylase and α-glucosidase enzymes via an enzyme inhibition assay experiment using acarbose as the reference drug (Fig. 1). NDMS showed significant inhibition of α-amylase in a dose-dependent manner of 85.49 ± 0.60, 76.28 ± 1.94, 71.08 ± 1.04, 61.37 ± 0.56, and 54.12 ± 0.52 at a concentration range of 62.5–1000 µg/mL, with an IC50 value of 49.05 µM. The 2-BS displayed percent inhibition of 77.58 ± 0.63, 716.61 ± 0.43, 60.93 ± 0.67, 53.65 ± 0.91, and 45.12 ± 0.12 at the same concentration, with an IC50 value of 95.05 µM. In comparison, acarbose had an IC50 value of 22.61 µM (Fig. 1B) ^46–48. The reduction in the activity of α-glucosidase by NDMS was 87.30 ± 1.42, 82.80 ± 0.41, 69.51 ± 0.59, 65.90 ± 0.32, and 61.56 ± 0.52 at a concentration range of 62.5–1000 µg/ml with an IC50 value of 30.93 µM, whereas 2-BS displayed an inhibition of 76.32 ± 0.87, 69.33 ± 0.66, 56.40 ± 0.82, 51.90 ± 0.45, and 42.34 ± 0.42 at the same dose, respectively, with an IC50 value of 120.3 µM (Fig. 1C). In comparison, acarbose has an IC50 of 23.78 µM ⁴⁹.

The body weights of treated and nontreated mice were measured at different time intervals ranging from days 1 to 15, and the percent change in body weight was calculated, as shown in Fig. 2A. The data demonstrated that weight gain in diabetic mice (23.59%) was less than that in normal control mice (41.7%). Furthermore, diabetic mice treated with glibenclamide (0.5 mg/kg/b. w.) resulted in almost equivalent weight gain to that of normal control mice (47.8%). The NDMS (5 and 10 mg/kg/b.w.)-treated diabetic mice have shown a good weight gain of 50–52.3% compared to glibenclamide ^{41, 50}. While the 2-BS (5 and 10 mg/kg/b. w.)-treated animals had a minimum effect on controlling weight loss of approximately 29–36% in diabetic mice. Thus, 2-BS is less effective in controlling weight loss in diabetic models than NDMS and glibenclamide (Fig. 2A).

Similarly, the antihyperglycemic effects of NDMS and 2-BS (5 and 10 mg/kg/b. w.) with the reference drug, glibenclamide (0.5 mg/kg/b.w.), via oral administration considerably reduced fasting blood glucose levels from days 1–15. The NDMS (5 and 10 mg/kg/b.w.)-treated Blood glucose levels (BGL) of C. sinensis-treated mice were noted as 247.9 ± 0.9, 236.1 ± 1.7, 208 ± 1.9, 187 ± 1.4, and 178 ± 1.3, respectively. While the 2-BS (5 and 10 mg/kg/b. w.)-treated mice exhibited a decreased BGL of 251.6 ± 1.4, 232.4 ± 1.5, 223 ± 1.1, 208 ± 1.7, and 199 ± 1.6 mg/dL, respectively. The noted BGL decline was significantly less than that in the diabetic control but was still higher than that in glibenclamide-treated mice (Fig. 2B).

Furthermore, liver function analysis was performed by assessing the key enzymes, including serum pyruvic transaminase (SGPT; 61.4 ± 1.5), serum glutamic oxaloacetic transaminase (SGOT; 46.9 ± 0.8), alkaline phosphatase (ALP; 275 ± 1.1), and serum creatinine (2.8 ± 0.7) in diabetic mice. The reference drug, glibenclamide treatment, halted the liver enzymes SGPT (25.8 ± 1.8), SGOT (23.9 ± 1.5), ALP (188 + 2.5), and serum creatinine (0.5 ± 0.8), respectively. The NDMS (5 and 10 mg/kg/b.w.)-treated diabetic mice reduced the same markers up to SGPT (24–26.8 ± 1.4), SGOT (23–27.8 ± 1.5), ALP (193–182 + 1.8), and serum creatinine (0.7–0.9 ± 0.2), respectively. The reduction was significant at both doses compared to that in the diabetic control group. Similarly, 2-BS (5 and 10 mg/kg/b. w.)-treated diabetic mice reduced the SGPT (22-25.8 ± 1.2), SGOT (21-22.9 ± 1.4), ALP (187-191.4 + 1.17), and serum creatinine (0.6–0.8 ± 0.15) by respectively ^{47, 51–53}. The reduction was remarkable at both doses compared to that in the diabetic control group (Fig. 2C and E).

Lipid profiling studies on mice treated with NDMS and 2-BS for 15 days showed considerably reduced levels of total cholesterol, triglycerides (TGs), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). This outcome was proportionate to that obtained with the recommended dosage of the standard drug glibenclamide (0.5 mg/kg/b.w.) ^{3, 7, 8, 16}. HDL cholesterol levels were also remarkably higher in mice treated with NDMS and 2-BS (Fig. 2D).

The effects of various combinations of treatments on the structure of pancreatic β-cells were compared with those of the alloxan-induced diabetic model (control) and reference drug (positive control) groups. The cell morphology of islets in alloxan-induced diabetic control mice was altered by healing and fibrosis, which showed β-cell destruction, causing the cells to contract and deviate from their normal histological appearance (Fig. 3Ac). In the NMDS-treated (5 and 10 mg/kg/b. w.) group, a good trend of pancreatic β-cell recovery was noted compared to the reference molecules, as indicated by the red arrow in Fig. 3A1. Similarly, the diabetic model group treated with 2-BS (5 and 10 mg/kg/b. w.) also shows a better trend of pancreatic β-cell recovery, as depicted in Fig. 3A2 ¹⁶. The NDMS- and 2-BS-treated groups showed significantly improved cell mass, islet cell shape, and total islet cell volume compared to the reference group (Fig. 3A).

The livers of diabetic animals after receiving alloxan injections displayed several abnormalities, including hazy edema, lymphocyte infiltration, severe congestion, necrotic foci, and hydropic changes (Fig. 3B). The hepatic portal vein was also congested in the alloxan (Diabetic Control) section from the liver of a diabetic mouse, showing hydropic degeneration (HD) and severe congestion (CO) (Fig. 3Bc) ^{8, 50}. The NMDS and 2-BS-treated diabetic mice livers (5 and 10 mg/kg/b. w.) show a nearly similar morphology of hepatic architecture with an apparently normal appearance and good recovery (Fig. 3B1-2).

To investigate the specific sites of interaction responsible for these biological activities, a docking study was performed on these keys’ pharmacophores. Docking analyses of NDMS and 2-BS revealed several possible binding interactions with the target enzymes. NDMS established a significant interaction with the binding site residues of amylase, with a dock score of 5.6607 kcal/mol, while 2-BS had a lower amylase binding affinity, with a docking score of -4.3213 kcal/mol. The docking scores of NDMS and 2-BS to α-glucosidase were 5.8803 and − 4.9448 kcal/mol ^{52, 54}. Overall, the docking simulations of the synthetic compounds demonstrated the significant inhibitory activities of α-amylase (Table 1) and α-glucosidase (Table 2), both of which showed significant interactions with varying binding affinities toward the target proteins (Fig. 4AB).