GREEN SYNTHESIS OF PYRANO [2, 3-D] PYRAZOLE DERIVATIVES USING SPINACH (SPINACIA OLERACEA) LEAF EXTRACT AS A NATURAL CATALYST: SYNTHESIS, CHARACTERIZATION, AND EVALUATION OF ANTI-DIABETIC ACTIVITY

A highly efficient, eco-friendly, and straightforward multi-component synthesis of biologically potent pyrano [2, 3-d] pyrazole derivatives (4a–4j) has been developed using an aqueous extract of fresh spinach leaves (Spinacia oleracea) as a natural, recyclable, and biodegradable catalyst. The reaction involves a one-pot, three-component condensation of substituted aromatic aldehydes, malononitrile, and ethyl acetoacetate in ethanol at 60°C. Under the optimized conditions (10 mol% spinach extract, EtOH, 60°C), all target compounds were obtained in excellent yields (83–94%) within 25–35 minutes. The catalytic efficiency of spinach extract originates from its rich phytochemical profile — particularly chlorophyll, flavonoids, phenolic acids, and Mg²⁺ ions — which collectively facilitate Knoevenagel condensation, Michael addition, and intramolecular cyclization steps through hydrogen-bond-donor/acceptor interactions and mild base catalysis. All synthesized compounds were thoroughly characterized by IR, ¹H NMR, ¹³C NMR spectroscopy, and HRMS analysis. The anti-diabetic activity was assessed in vitro through α- and β-glucosidase inhibition assays. Compound 4e emerged as the most potent inhibitor, exhibiting IC₅₀ values of 15.8 μg/mL and 17.9 μg/mL for α- and β-glucosidase, respectively — values closely comparable to, and in some cases surpassing, the standard drug acarbose (IC₅₀ = 14.2 and 12.8 μg/mL). Molecular docking studies further validated the binding interactions of the active compounds within the active sites of both glucosidase enzymes. This work demonstrates the remarkable potential of plant-derived catalysts as sustainable alternatives to conventional chemical catalysts in multi-component heterocyclic synthesis.