Design, synthesis, biological evaluation, and in silico characterization of chalcone derivatives as antidiabetic hits

Abstract

A series of 40 chalcone derivatives was synthesized through Claisen–Schmidt condensation and characterized by FT-IR and NMR spectroscopy. The compounds were evaluated for α-amylase inhibitory and DPPH radical-scavenging activities to identify chalcone-based hits with a dual in vitro profile relevant to postprandial glycemic control. The series displayed a broad range of activities, with α-amylase IC₅₀, values spanning 10.41 ± 1.23–1021.64 ± 2.75 µM and radical-scavenging IC₅₀ , values ranging from 31.34 ± 0.20 to 698.34 ± 14.56 µM. Among the evaluated compounds, 4, 17, 19, 31, 33, and 35 emerged as the most active α-amylase inhibitors, with compound 4 being the most potent member of the series (IC₅₀ = 10.41 ± 1.23 µM), outperforming acarbose (IC₅₀ = 73.12 ± 5.04 µM). Structure–activity relationship analysis indicated that hydroxy- and methoxy-substituted aryl motifs were generally favorable, whereas some heteroaryl replacements and heavily halogenated patterns were less well tolerated. To rationalize the experimental profile, an ensemble docking workflow coupled to Naive Bayes rescoring was applied to human pancreatic α-amylase. The predicted binding modes supported productive occupation of the catalytic groove and suggested a key interaction between compound 4 and Glu233, consistent with its superior inhibitory potency. In silico ADMET profiling of the leading compounds indicated acceptable lipophilicity, solubility, intestinal absorption, and low predicted cardiotoxicity, with compound 4 showing the most balanced overall profile. In summary, these findings identify this chalcone series as a promising starting point for hit-to-lead optimization toward multifunctional radical scavenging and α-amylase inhibitors.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-55706-3.

Introduction

Diabetes mellitus remains a major global health burden characterized by chronic hyperglycemia, resulting from impaired insulin secretion, insulin resistance in target tissues, or a combination of these defects. Beyond glycemic dysregulation, sustained hyperglycemia promotes progressive damage to the vasculature, kidney, retina, and heart, thereby contributing to substantial morbidity, mortality, and healthcare expenditure^1–3. Accordingly, identifying small molecules capable of controlling postprandial glucose excursions while exhibiting suitable pharmacological properties remains an important objective in antidiabetic drug discovery^4–6.

Among the validated strategies for postprandial glycemic control, inhibition of digestive enzymes involved in carbohydrate processing is particularly attractive^7–9. In this context, α-Amylase (EC 3.2.1.1) catalyzes the initial hydrolysis of dietary polysaccharides by cleaving α-1,4-glycosidic bonds, producing smaller oligosaccharides that are subsequently converted into absorbable glucose by α-glucosidase. Pharmacological inhibition of α-amylase can therefore attenuate intestinal glucose release and reduce postprandial hyperglycemia^10–12. However, clinically available carbohydrate-hydrolyzing enzyme inhibitors such as acarbose, miglitol, and voglibose are commonly associated with gastrointestinal adverse effects, highlighting the need for new chemotypes with improved potency and a more balanced overall profile^13,14.

In parallel, poorly controlled hyperglycemia and the associated complications of DM dysregulate oxidoreductase enzymes, including xanthine oxidase and lipoxygenase, thereby increasing the generation of reactive oxygen species (ROS), reactive nitrogen species (RNS), and other free radicals during metabolism¹⁵. These highly reactive species can damage biomacromolecules such as proteins, lipids, and DNA, triggering oxidative chain reactions that contribute to cellular dysfunction and tissue injury^16–20. Sustained oxidative and nitrosative stress can ultimately promote cell death and progressive tissue injury, contributing to pathological alterations in vital organs, including the heart, kidneys, liver, and immune system, and thereby favoring the development of inflammatory, cardiovascular, neoplastic, arthritic, and age-related disorders^21–26. In this context, diabetic complications can be managed through controlling ROS and RNS^27–29. From a medicinal chemistry standpoint, this provides a compelling rationale for exploring multifunctional molecules able to combine α-amylase inhibition with radical-scavenging capacity within a single scaffold^30–32.

Chalcones (1,3-diphenylprop-2-en-1-ones) constitute a privileged and synthetically tractable chemotype for this purpose^33–35. Their α,β-unsaturated ketone framework, which links two aromatic or heteroaromatic fragments, enables broad structural diversification (Fig. 1). They provide a safe and straightforward synthetic platform for accessing a wide range of biologically relevant heterocycles, including dihydropyrazole³⁶, azachalcone³⁷, pyrimidine³⁸, cyanopyridine, pyrazoline³⁹, isoxazole, while also being structurally related to natural isoflavonoids, flavonoids, and aurones, which has placed them at the center of considerable medicinal chemistry interest⁴⁰. Moreover, their scaffold readily accommodates hydroxy-, methoxy-, halo-, and heteroaryl substituents, allowing precise modulation of electronic distribution, hydrogen-bonding capacity, and lipophilic balance. This sustained interest is also consistent with the wide range of biological activities reported for chalcone derivatives across different therapeutic contexts^41–59. This translational relevance is further supported by clinically used or investigated chalcone-based agents (Fig. 1), such as metochalcone (i)⁶⁰ and sofalcone (ii)⁶¹, as well as hesperidin methylchalcone (iii) and trimethylchalcone (iv)⁶², which have shown therapeutic utility in gastrointestinal and venous disorders.

Fig. 1.

Representative chalcones, clinically investigated and approved.

Currently, different chalcone scaffold such as metochalcone(i)⁶⁰, and sofalcone(ii)⁶¹, have been investigated in clinical practice and are approved as choleretic and antiulcer drug as shown in Fig. 1. Likewise, hespiridine methylchalcone(iii), and trimethylchalcone(iv), clinical investigations in chronic venous lymphatic insufficiency, trunk and branch varicosis show it more effective in relieving of symptoms and show excellent tolerance level⁶².

The above interesting biological potentials of the chalcone scaffold in drug discovery, and the associated challenges to already in use drugs against DM, have made the situation more interesting and highly pressing to develop more novel and efficient drugs. Here in this work our focus is to use chalcone analogues as the main scaffold in the integral project to develop novel agent having α-amylase inhibition and free radical scavenging features. As part of our research interest in synthesis of biologically relevant compounds^63–77 and keeping in mind the above-mentioned objectives, we have synthesized various analogues of chalcone by using different types of substituted aldehyde and acetophenone, after purification and structural characterization their α-amylase inhibition and free radical scavenging potential were evaluated. Further, the molecular docking, and ADMET studies were conducted.

Materials and methods

Experimental methods

Synthesis and characterization of compounds

All chemicals and reagents were synthetic grade, obtained from commercial sources, and used without further purification unless stated otherwise. Specifically, aromatic aldehydes, acetophenones, sodium hydroxide, and common solvents were purchased from Alfa Aesar. Reaction progress was tracked by thin-layer chromatography (TLC) on Merck silica gel plates. Purification of final products was achieved by recrystallization from absolute ethanol. Infrared spectra were acquired on a Shimadzu Fourier-transform infrared (FT-IR) spectrometer. Nuclear magnetic resonance (NMR) spectra (¹H and ¹³C) were recorded in deuterated DMSO or CDCl₃ using Bruker 400/600 MHz and 100/125 MHz spectrometers, respectively.

General protocol for the synthesis of compounds

For the synthesis of various chalcone derivatives, the Claisen–Schmidt reaction described in the literature was used with minimal modification⁷⁸. An aqueous solution of NaOH (3 M, 3 mL) was added to a solution of aromatic ketone (3 mmol) in EtOH. The resulting solution was stirred at room temperature for a while, then an ethanolic solution of various substituted aldehydes was added dropwise to the reaction mixture. After complete addition of aldehydes, the reaction mixture was stirred at room temperature for 24 h; the reaction progress was monitored by thin-layer chromatography (TLC) at regular intervals. After the reaction was completed, the mixture was cooled and left to stand for 24 h. Then, the residue was collected, and the product was separated, washed with water, and then air-dried. The target compounds were purified by recrystallization from absolute EtOH. The resultant compounds (1–40) were characterized by various spectroscopic techniques. The spectroscopic data for the synthesized compounds are presented below, and their spectra are shown in the supporting information (Figures SS1-SS80).

Spectral data of synthesized compounds

(E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (1): Yield: 78%; white crystalline solid; MP: 118–120 °C (lit. = 120–121 °C)⁷⁹; Rf. value: 0.4 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3027, 1680, 1590,1550,1505,1250,1020. ¹H-NMR(600 MHz, CDCl₃): 7.97(2H, d, J = 8.40), 7.81(1H, d, J = 15.60), 7.61(2H, d, J = 8.50), 7.47(2H, d, J = 8.40), 7.38(1H, d, J = 15.60), 6.95(2H, d, J = 8.50), 3.86(3H, s, -OCH₃). ¹³C-NMR (150 MHz, CDCl₃): 189.00, 162.12, 145.39, 139.27, 136.61, 130.31, 129.82, 128.85, 127.47, 119.20, 114.45, 55.41.

(E)-1-(4-chlorophenyl)-3-(p-tolyl)prop-2-en-1-one (2): Yield: 78%; white crystalline solid; MP: 165 °C (lit. = 163–164 °C)⁸⁰; Rf. value: 0.4 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3065,1665,1605,1545,1515,1260,1020. ¹H-NMR(400 MHz, CDCl₃): 8.08(2H, d, J = 8.60), 7.93(1H, d, J = 15.60), 7.66(2H, d, J = 8.00), 7.59(2H, d, J = 8.60), 7.57(1H, d, J = 15.60), 6.35(2H, d, J = 8.00), 2.50(3H, s, -CH₃). ¹³C-NMR(100 MHz, CDCl₃): 189.38, 145.45, 141.33, 139.07, 136.65, 131.97, 129.88, 129.76, 128.90, 128.56, 120.47, 21.53.

(E)-1-(4-chlorophenyl)-3-phenylprop-2-en-1-one (3): Yield: 80%; off white crystalline solid; MP: 116–118 °C (lit. = 113–117 °C)⁸¹; Rf. value: 0.40 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3018, 1655, 1600, 1575, 1520, 1260, 1015. ¹H-NMR(400 MHz, CDCl₃): 8.05(2H, d, J = 8.30), 7.91(1H, d, J = 15.60), 7.72–7.69(2H, m), 7.59(1H, d, J = 15.60), 7.54(2H, d, J = 8.60), 7.50–7.48(3H, m). ¹³C-NMR(100 MHz, CDCl₃): 189.03, 145.18, 136.35, 134.57, 130.86, 129.93, 129.00, 128.92, 128.55, 121.39.

(E)-1-(4-chlorophenyl)-3-(2-hydroxyphenyl)prop-2-en-1-one (4): Yield: 72%; red color solid; MP: 113–115 °C (lit. = 115–120 °C)⁸²; Rf. value: 0.35 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3450,3013, 1655, 1590, 1570, 1510, 1235, 1005. ¹H-NMR(600 MHz, DMSO-d₆): 10.29 (s, 1H, -OH), 8.13(2H, d, J = 8.60), 8.08(1H, d, J = 15.70), 7.87(1H, d, J = 7.80), 7.85(1H, d, J = 15.70), 7.64(2H, d, J = 8.60), 7.30(1H, t, J = 5.76), 6.96(1H, d, J = 8.16), 6.90(1H, t, J = 7.68). ¹³C-NMR (150 MHz, DMSO-d₆): 188.23, 157.18, 139.91, 137.79, 136.57, 132.12, 128.83, 128.73, 121.29, 120.60, 119.39, 116.25.

(E)-1-(4-chlorophenyl)-3-(furan-2-yl)prop-2-en-1-one (5): Yield: 85%; cream/yellow color solid; MP: 75–76 °C (lit. = 72–74 °C)⁸³; Rf.value:0.5 (Ethyl acetate/n-hexane,1:4); FT-IR(KBr, cm^-1): 3100, 1655, 1595, 1510, 1315. ¹H-NMR(400 MHz, DMSO-d₆): 8.16(2H, d, J = 8.50), 8.0(1H, d, J = 3.30), 7.69(2H, d, J = 8.50), 7.65(1H, d, J = 15.40), 7.56(1H, d, J = 15.40), 7.21(1H, d, J = 3.40), 6.78–7.77(1H, m). ¹³C-NMR(100 MHz, DMSO-d₆): 188.0, 151.51, 146.80, 138.45, 136.39, 131.30, 130.61, 129.35, 118.78, 117.84, 113.64.

(E)-1-(4-chlorophenyl)-3-(3-nitrophenyl)prop-2-en-1-one (6): Yield: 80%; off white amorphous solid; MP: 116–118 °C (lit. = 119 °C)⁸⁴; Rf. value: 0.4 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3095, 1662, 1602, 1510, 1325. ¹H-NMR(400 MHz, CDCl₃): 8.53 (1H, s), 8.29(1H, d, J = 7.70), 8.08(2H, d, J = 9.90), 7.76(1H, d, J = 7.70), 7.87(1H, d, J = 15.70), 7.70(1H, d, J = 15.70), 7.65(2H, d, J = 9.90), 7.58(1H, t, J = 7.80). ¹³C-NMR (100 MHz, DMSO- d₆): 189.47, 148.94, 141.63, 137.77, 137.09, 135.32, 133.74, 130.74, 129.21, 129.10, 125.47, 124.98, 123.47.

(E)-1-(4-chlorophenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one (7): Yield: 82%; deep yellow crystalline solid.; MP: 134–136 °C (lit. = 138–140 °C)⁷⁹; Rf. Value: 0.5 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3040, 1645, 1590, 1560, 1510, 1255, 1015. ¹H-NMR(600 MHz, DMSO-d₆): 8.13(2H, d, J = 8.50), 7.72(2H, d, J = 8.40), 7.69(1H, d, J = 16.0), 7.63(1H, d, J = 16.0), 7.60(2H, d, J = 8.40), 6.76 (2H, d, J = 8.50), 3.01(6H, s, -N(CH₃)₂). ¹³C-NMR (150 MHz, DMSO-d₆): 187.51, 152.20, 145.75, 137.41, 136.98, 130.82, 130.02, 128.76, 121.93, 115.75, 111.64, 39.65.

(E)-1-(4-chlorophenyl)-3-(3H-pyrrol-2-yl)prop-2-en-1-one (8): Yield: 72%; deep yellow crystalline solid; MP: 173–175 °C (lit. = 175 °C)^85,86; Rf. Value: 0.50 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3055, 1635 ,1570, 1545, 1520, 1235, 1025. ¹H-NMR(400 MHz, DMSO-d₆): 11.83 (1H, s, -NH), 8.14(2H, d, J = 8.60), 7.74(1H, d, J = 15.50), 7.71(2H, d, J = 8.60), 7.66(1H, d, J = 15.50), 7.26–7.24(1H, m), 6.86–6.84(1H, m), 6.34–6.32(1H, m). ¹³C-NMR(100 MHz, DMSO-d₆): 189.47, 148.94, 141.63, 137.77, 137.09, 135.32, 133.74, 130.74, 129.21, 129.10, 125.47, 124.98, 123.47.

(E)-1-(4-chlorophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (9): Yield: 73%; light yellow crystalline solid; MP: 105–107 °C (lit. = 105 °C)^87,88; Rf. value:0.43 (Ethylacetate/n-hexane,1:4); FT-IR (KBr, cm^-1):3065,1635,1580,1565,1510,1235,1020. ¹H-NMR(400 MHz, CDCl₃): 8.07(2H, d, J = 8.60), 7.84(1H, d, J = 15.60), 7.59(2H, d, J = 8.60), 7.48(1H, d, J = 15.60), 6.96(1H, s), 4.02(6H, s, -2OCH₃), 4.01(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 189.22, 153.55, 145.69, 139.15, 136.74, 130.11, 129.92, 128.95, 120.84, 105.76, 61.01, 56.37.

(E)-1-(4-chlorophenyl)-3-(thiophen-2-yl)prop-2-en-1-one (10): Yield: 74% ; light yellow crystalline solid; MP: 119–121 °C (lit. = 118–120 °C)⁸³; Rf. value: 0.38 (EtOAc/n-hexane,1:4); FT- IR (KBr, cm ^-1): 3057, 1630, 1560, 1525, 1490, 1225, 1005 ¹H-NMR(400 MHz, CDCl₃): 8.09(1H, d, J = 15.20), 8.05(2H, d, J = 8.70), 7.60(2H, d, J = 8.70), 7.56(1H, d, J = 5.0), 7.49(d. 1H, J = 3.60), 7.41(1H, d, J = 15.20), 7.23–7.21(1H, m). ¹³C-NMR(100 MHz, CDCl₃): 188.65, 140.25, 139.22, 137.73, 136.43, 132.40, 129.82, 129.10, 128.94, 128.42, 120.16.

(E)-1-(4-chlorophenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (11): Yield: 70%; cream/off white crystalline solid; MP: 114–116 °C (lit. = 115–117 °C)⁸⁹; Rf. value:0.42 (EtOAc/nhexane,1:4); FTIR (KBr, cm¹): 3085, 1675, 1610, 1530, 1490, 1275, 1035. ¹H-NMR(600 MHz, DMSO-d₆): 8.28(1H, d, J = 8.50), 8.21(2H, d, J = 8.60), 8.05(1H, d, J = 15.50), 7.99(1H, d, J = 15.50), 7.77(s. 1H), 7.67(2H, d, J = 8.60), 7.58(1H, d, J = 8.50). ¹³C-NMR (150 MHz, DMSO-d₆): 187.87, 138.48, 137.63, 135.79, 135.20, 131.25, 130.57, 129.92, 129.50, 128.95, 127.96, 125.01.

(E)-1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one (12): Yield: 72%; white crystalline solid; MP:106–108 °C (lit. = 104–110 °C)^80,90; Rf. value:0.45(Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3045, 1655–1665,1605, 1550, 1135. ¹H-NMR(400 MHz, CDCl₃): 8.15(2H, d, J = 8.90), 7.92(1H, d, J = 15.60), 7.74–7.71(2H, m), 7.67(1H, d, J = 15.60), 7.51–7.48(3H, m), 7.07(2H, d, J = 8.90), 3.94(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 188.58, 163.38, 143.68, 135.0, 131.06, 130.83, 130.35, 128.93, 128.39, 121.84, 113.85, 55.50.

(E)-3-(2-hydroxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (13): Yield: 70%; dark color solid; MP: 152–154 °C (lit. = 151–153 °C)⁹⁰; Rf. value. 0.40 (Ethyl acetate/n-hexane,1:4);FT-IR (KBr, cm ^-1): 3242, 3010, 1650, 1580, 1550, 1505, 1230, 1150. ¹H-NMR(400 MHz, DMSO-d₆): 8.16(2H, d, J = 8.90), 8.11(1H, d, J = 15.40), 8.07(1H, d, J = 15.40), 7.63(1H, d, J = 7.80), 7.17(2H, d, J = 8.90), 7.13 (1H,t, J = 7.0), 6.85(1H, d, J = 7.70), 6.56(1H, t, J = 6.90), 3.94(3H, s, -OCH₃). ¹³C-NMR (100 MHz, DMSO-d₆): 188.56, 163.10, 143.35, 132.16, 132.08, 130.74, 130.47, 122.39, 119.60, 117.78, 114.30, 55.92.

(E)-3-(furan-2-yl)-1-(4-methoxyphenyl)prop-2-en-1-one (14): Yield: 80%; cream color crystalline solid; MP: 77–79 °C (lit. = 76–78 °C)⁸³; Rf. Value. 0.40 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3110, 1650, 1585, 1505, 1310. ¹H-NMR(400 MHz, CDCl₃): 8.16(2H, d, J = 9.0), 7.71(1H, d, J = 15.30), 8.62(1H, d, J = 2.0), 7.59(1H, d, J = 15.30), 7.09(2H, d, J = 9.0), 6.81 (1H,d, J = 3.45), 6.62–6.61(1H, m), 3.99(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 188.29, 163.65, 151.76, 131.05, 130.61, 130.0, 119.21, 115.77, 113.77, 112.59, 55.92.

(E)-1-(4-methoxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one (15): Yield: 70%; white solid; MP:152–154 °C (lit. = 151–152 °C)⁹¹; Rf. Value:0.40(Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3090, 1660, 1595, 1505, 1320. ¹H-NMR(600 MHz, CDCl₃): 8.52 (1H, s), 8.28(1H, d, J = 7.70), 8.09(2H, d, J = 8.80), 7.94(1H, d, J = 7.70), 7.85(1H, d, J = 15.60), 7.70(1H, d, J = 15.60), 7.64(1H, t, J = 7.90), 7.03(2H, d, J = 8.80), 3.92 (3H, s, -OCH₃). ¹³C-NMR (150 MHz, CDCl₃): 187.87, 163.89, 148.65, 140.74, 136.94, 134.45, 131.01, 130.50, 130.03, 124.49, 122.21, 114.04, 55.66.

(E)-3-(4-(dimethylamino)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (16): Yield: 65%; deep yellow crystalline solid; MP: 129–130 °C (lit. = 129–131 °C)^90,92; Rf.value:0.40(Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3030, 1640, 1590, 1560, 1510, 1150,1320. ¹H-NMR(400 MHz, CDCl₃): 8.15(2H, d, J = 8.80), 7.921(1H, d, J = 15.50), 8.65(2H, d, J = 8.80), 7.48(1H, d, J = 15.50), 7.08(2H, d, J = 8.80), 6.78 (2H,d, J = 8.80), 3.95(3H, s, -OCH₃), 3.10(6H, s, -N(CH₃)₂. ¹³C-NMR (100 MHz, CDCl₃): 188.56, 162.82, 152.04, 144.53, 131.85, 130.53, 130.29, 122.76, 116.55, 113.69, 111.83, 55.59, 39.89.

(E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one (17): Yield: 70%; white crystalline solid; MP: 99–102 °C (lit. = 100–103 °C)⁸⁰; Rf. value: 0.4 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3050, 1655 ,1575, 1555, 1510, 1240, 1150. ¹H-NMR(400 MHz, CDCl₃): 8.15(2H, d, J = 8.90), 7.90(1H, d, J = 15.60), 7.1(2H, d, J = 8.70), 7.55(1H, d, J = 15.90), 7.09(2H, d, J = 8.90), 7.04 (2H,d, J = 8.70), 3.98(3H, s, -OCH₃), 3.94(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 188.56, 163.38, 161.64, 143.70, 131.34, 130.69, 130.06, 127.79, 119.53, 114.38, 113.79, 55.54, 39.89.

(E)-1-(4-methoxyphenyl)-3-(p-tolyl)prop-2-en-1-one (18): Yield: 75%; white crystalline solid; MP: 124–126 °C (lit. = 127–127.5 °C)⁸⁰; Rf. Value. 0.4(Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3058, 1646 ,1580, 1570, 1515, 1265, 1025 ¹H-NMR(400 MHz, CDCl₃): 8.03(2H, d, J = 8.90), 7.79(1H, d, J = 15.60), 7.53(2H, d, J = 8.40), 7.47(1H, d, J = 15.60), 7.21(2H, d, J = 8.40), 6.97 (2H,d, J = 8.90), 3.85(3H, s, -OCH₃), 2.36(3H, s, -CH₃). ¹³C-NMR(100 MHz, CDCl₃): 188.58, 163.35, 143.97, 140.77, 132.34, 130.77, 129.68, 128.40, 120.85, 113.71, 55.54, 31.32.

(E)-1-(4-methoxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one (19): Yield: 70%; yellow crystalline solid; MP: 126–128 °C (lit. = 128–130 °C)⁹³; Rf. value: 0.45(Ethyl acetate/n-hexane,1:5); FT-IR (KBr, cm ^-1): 3365, 3050, 1645, 1580, 1555, 1510, 1250, 1010. ¹H-NMR(600 MHz,DMSO-d₆): 8.09(1H, d, J = 15.50), 8.06(2H, d, J = 7.90), 7.85(1H, d, J = 15.50), 7.32(1H, d, J = 7.70), 7.07(2H, d, J = 7.90), 6.87(1H, d, J = 7.70), 6.58 (1H,s), 3.85(3H, s, -OCH₃), 3.75(3H, s, -OCH₃). ¹³C-NMR (150 MHz, DMSO-d₆): 187.75, 162.75, 149.54, 140.53, 131.27, 130.40, 121.27, 118.62, 115.53, 113.77,112.91, 55.68, 55.44.

(E)-1-(4-methoxyphenyl)-3-(3H-pyrrol-2-yl)prop-2-en-1-one (20): Yield: 72%; deep yellow crystalline solid; MP: 168–170 °C (lit. = 168 °C)⁸⁵; Rf. value. 0.50(Ethyl acetate/n-hexane,1:4); FT-IR(KBr, cm ^-1): 3045, 1635 ,1595, 1565, 1520, 1235, 1025 ¹H-NMR(400 MHz, CDCl₃): 8.13(2H, d, J = 8.90), 7.86(1H, d, J = 15.50), 7.28(1H, d, J = 15.50),7.10–7.09(1H, m), 7.07(2H, d, J = 8.90), 6.83–6.81(1H, m), 6.46–6.43(1H, m), 4.0 (3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 188.57, 163.11, 133.73, 131.50, 130.51, 129.45, 122.72, 115.56, 114.80, 113.76, 111.40, 55.71.

(E)-1-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (21): Yield: 70% ; off white crystalline solid; MP: 129–130 °C (lit. = 131.1–132.5 & 129 °C)^84,87,94; Rf. value: 0.50 (EtOAc/n-hexane,1:4); FT-IR(KBr, cm ^-1): 3060, 1665, 1595, 1580, 1530, 1235, 1005. ¹H-NMR(400 MHz, CDCl₃): 8.04(2H, d, J = 8.80), 7.72(1H, d, J = 15.50), 7.44(1H, d, J = 15.50), 6.98(2H, d, J = 8.80), 6.86(2H, s), 3.91(6H, s, -2OCH₃), 3.89(3H, s, -OCH₃), 3.87(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 188.57, 163.38, 153.34, 144.10, 140.23, 131.08, 130.78, 130.56, 121.19, 113.82, 105.57, 60.97, 56.20, 55.47.

(E)-1-(4-methoxyphenyl)-3-(thiophen-2-yl)prop-2-en-1-one (22): Yield: 70%; off white crystalline solid; MP: 107–109 °C (lit. = 104–106 °C)⁸³; Rf. value: 0.43 (EtOAc/n-hexane,1:4), FT-IR (KBr, cm ^-1): 3025, 1625, 1560, 1530, 1490, 1245, 1025. ¹H-NMR(400 MHz, CDCl₃): 8.13(2H, d, J = 8.90), 8.05(1H, d, J = 15.30), 7.50(1H, d, J = 5.0), 7.46(1H, d, J = 15.30), 7.42(d. 1H, J = 4.0), 7.18–7.16(1H, m), 7.08(2H, d, J = 8.90), 3.96 (3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 188.01, 163.38, 140.49, 136.37, 131.74, 130.98, 130.71, 128.48, 128.31, 120.62, 113.85, 55.48.

(E)-3-(2-chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (23): Yield: 64%; white crystalline solid; MP: 131–134 °C (lit. = 135–138 °C)⁹⁵; Rf. value: 0.38(EtOAc/n-hexane,1:4), FT-IR(KBr,cm^-1): 3058,1685,1580,1540,1500,1125,840. ¹H-NMR(600 MHz, DMSO-d₆): 8.21–8.20 (m, 1H), 8.19(2H, d, J = 8.90), 8.04(1H, d, J = 15.50), 7.99(1H, d, J = 15.50), 7.56–7.44(3H, m), 7.10(2H, d, J = 8.90), 3.80(s, 3H, -OCH₃). ¹³C-NMR (150 MHz, DMSO-d₆): 187.04, 163.64, 137.70, 134.22, 132.44, 131.72, 140.44, 130.14, 129.94, 128.51, 127.60, 124.78, 114.06, 55.70.

(E)-3-(2,4-dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (24): Yield: 65%; white crystalline solid; MP: 152–154 °C (lit. = 155–158 °C)⁹⁵; Rf.value:0.50 (EtOAc/n-hexane,1:4). FT-IR (KBr, cm ^-1): 3090,1695,1650,1550,1520,1230,1025. ¹H-NMR(400 MHz, DMSO-d₆): 8.26(1H, d, J = 8.60), 8.19(2H, d, J = 8.90), 8.05(1H, d, J = 15.50), 7.95(1H, d, J = 15.50), 7.74(s. 1H), 7.67(2H, d, J = 8.60), 7.55(1H, d, J = 8.50), 7.11(2H, d, J = 8.90), 3.88(s, 3H, -OCH₃). ¹³C-NMR (100 MHz, DMSO-d₆): 187.46, 163.94, 136.92, 135.47, 131.95, 130.46, 130.24, 129.90, 128.36, 125.76, 114.60, 56.08.

(E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (25): Yield: 75%; yellow crystalline solid; MP: 118–120 °C (lit. = 120–121 °C)⁹⁰; Rf. Value. 0.50 (Ethyl acetate/n hexane,1:4); FT-IR (KBr, cm^-1): 3075, 1660, 1600, 3380. ¹H-NMR(400 MHz, DMSO-d₆): 8.11(2H, d, J = 8.80), 8.01(1H, d, J = 15.60), 7.94–7.92(m, 2H), 7.75(1H, d, J = 15.60), 7.56–7.51(3H, m), 6.88(2H, d, J = 8.80). ¹³C-NMR (100 MHz, DMSO-d₆): 186.84, 167.13, 142.24, 135.55, 131.83, 131.21, 130.50, 129.32, 128.99, 122.90, 116.83.

(E)-3-(furan-2-yl)-1-(4-hydroxyphenyl)prop-2-en-1-one (26): Yield: 80%; deep yellow crystalline solid; MP: 73–75 °C (lit. = 69–71 °C)⁸³; Rf. value. 0.55(Ethyl acetate/n-hexane,1:4); FT-IR(KBr, cm^-1): 3385, 3050, 1655, 1620, 1550, 1440 ¹H-NMR(600 MHz, CDCl₃): 8.06(2H, d, J = 8.60), 7.63(1H, d, J = 15.30), 7.55(m, 1H), 7.50(1H, d, J = 15.30), 6.97(2H, d, J = 8.60), 6.73(1H, d, J = 3.40), 6.54–6.53(1H, m). ¹³C-NMR(150 MHz, CDCl₃): 188.62, 160.29, 151.74, 144.87, 131.14, 130.29, 119.09, 116.10, 115.49, 112.67.

(E)-1-(4-hydroxyphenyl)-3-(3-nitrophenyl)prop-2-en-1-one (27): Yield: 65%; orange crystalline solid; MP: 181–182 °C (lit. = 180–182 °C)⁹⁶; Rf. Value. 0.35 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm ^-1): 3370,3080,1655, 1590, 1503, 1315. ¹H-NMR(600 MHz, DMSO-d6): 10.48((1H, s, -OH), 8.77 (1H, s), 8.33(1H, d, J = 6.20), 8.27(1H, d, J = 8.30), 8.16(1H, d, J = 14.30), 8.14(2H, d, J = 7.0), 7.81(1H, d, J = 14.30), 7.76(1H, t, J = 6.40), 6.92(2H, d, J = 7.0). ¹³C-NMR (150 MHz, DMSO-d₆): 186.84, 167.13, 142.24, 135.55, 131.83, 131.21, 130.50, 129.32, 128.99, 122.90, 116.83.

(E)-1-(4-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (28): Yield: 75%; yellow crystalline solid; MP: 182 °C (lit. = 179–181 °C)⁹⁰; Rf. value: 0.45 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr,cm^-1): 3380,3025, 1650, 1580, 1550,3378,1160. ¹H-NMR(600 MHz, CDCl₃): 10.45(s, 1H, -OH), 8.10(2H, d, J = 8.0), 7.83(2H, d, J = 8.20), 7.78(1H, d, J = 15.40),7.67(2H, d, J = 15.40), 7.0(2H, d, J = 8.0), 6.93(2H, d, J = 8.20), 3.79(3H, s, -OCH₃). ¹³C-NMR (150 MHz, CDCl₃): 187.07, 162.01, 161.06, 142.68, 131.02, 130.49, 129.32, 127.47, 119.48, 115.31, 114.30, 54.89.

(E)-1-(4-hydroxyphenyl)-3-(pyridin-2-yl)prop-2-en-1-one (29): Yield: 70%; cream color crystalline solid; MP: 90–92 °C (lit. = 93 °C)⁸⁶; Rf. value: 0.38(EtOAc/n hexane,1:4); FT-IR (KBr, cm ^-1): 3240, 3054, 1642, 1550, 1530, 1505, 1235, 1025. ¹H-NMR(600 MHz, DMSO-d₆): 8.66(2H, d, J = 8.60), 8.13(1H, d, J = 15.40), 7.88–7.85(3H ,m), 7.59(1H, d, J = 15.40), 7.39–7.37(1H, m), 6.60(2H, d, J = 8.60). ¹³C-NMR (150 MHz, DMSO-d₆): 185.28, 171.36, 153.63, 149.83, 139.81, 136.91, 131.44, 126.61, 124.07, 117.52, 116.83.

(E)-1-(4-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (30): Yield: 70%; light yellow crystalline solid; MP: 183–185 °C (lit. = 180–182 °C)⁸⁷; Rf. value:0.35 (EtOAc/n-hexane); FT-IR (KBr,cm^-1): 3260, 3024, 1637, 1575, 1555, 1520, 1257, 1025. ¹H-NMR(400 MHz, CDCl₃): 8.01(2H, d, J = 8.50), 7.69(1H, d, J = 15.50), 7.40(1H, d, J = 15.50), 6.95(2H, d, J = 8.50), 6.81(2H, s), 3.87(6H, s, -(OCH₃)₂), 3.85(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 188.59, 163.39, 153.50, 144.26, 130.89, 121.08, 105.08, 61.05, 56.25, 55.52.

(E)-1-(4-hydroxyphenyl)-3-(thiophen-2-yl)prop-2-en-1-one (31): Yield: 67%; deep yellow crystalline solid; MP: 150–152 °C (lit. = 154–156 °C)⁸³; Rf. value: 0.35 (EtOAc/nhexane,1:4); FT-IR (KBr, cm ^-1): 3024, 1705,1690,1460,1410,1355, 1045. ¹H-NMR(600 MHz, DMSO-d₆): 8.0(2H, d, J = 8.70), 7.85(1H, d, J = 15.20), 7.72(1H, d, J = 5.0), 7.62(1H, d, J = 3.40), 7.54(1H, d, J = 15.20), 7.17–7.16(1H, m), 6.90(2H, d, J = 8.70). ¹³C-NMR (150 MHz, DMSO-d₆): 186.78, 161.89, 139.92, 135.56, 132.32, 131.02, 129.91, 128.86, 128.69, 120.24,115.14.

(E)-3-(2,4-dichlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (32): Yield: 62%; deep yellow crystalline solid; MP: 116–117 °C (lit. = 120–121 °C)⁸⁹; Rf. Value. 0.42 (EtOAc/n-hexane,1:4); FT IR (KBr, cm¹):3175,3065,1695,1560,1530,1480,1235,1045. ¹H-NMR(600 MHz, CDCl₃): 7.94 (1H, d, J = 15.7 Hz), 7.84 (2H, d, J = 8.3 Hz), 7.60 (1H, d, J = 8.4 Hz), 7.40 (1H, d, J = 15.7 Hz), 7.35 (1H. s), 7.19 (1H, d, J = 8.3 Hz), 6.80 (2H, d, J = 8.2 Hz).¹³C-NMR(150 MHz, CDCl₃): 188.96, 162.30, 138.36, 136.21, 135.82, 131.94, 131.27, 129.95, 129.41, 128.52, 127.49, 125.03, 115.50.

(E)-3-(4-hydroxy-3-methoxyphenyl)-1-phenylprop-2-en-1-one (33): Yield: 70%; dark brown crystalline solid; MP: 117–118 °C (lit. = liquid)³³; Rf. value: 0.45 (Ethyl acetate/n-hexane,1:4); IR (KBr, cm ^-1): 3023, 1655, 1595, 1570, 1515, 1265, 1020. ¹H-NMR(600 MHz, DMSO-d₆): 8.07(1H, d, J = 14.90), 7.94(2H, d, J = 7.80), 7.78(1H, d, J = 14.90), 7.55–7.44(3H, m), 6.87(1H, d, J = 7.80), 6.42(1H, d, J = 6.72), 5.85(1H, s), 3.62(3H, s, -OCH₃). ¹³C-NMR (150 MHz, DMSO-d₆): 189.08, 153.38, 146.62, 140.55, 131.12, 128.25, 127.56, 120.97, 111.44, 106.25, 55.06.

(E)-1-phenyl-3-(3H-pyrrol-2-yl)prop-2-en-1-one (34): Yield:73%; deep yellow crystalline solid; MP: 131–133 °C (lit. = 130 °C)⁸⁵; Rf. value: 0.50(Ethylacetate/n-hexane,1:4);FT-IR(KBr,cm^-1):3056,1642,1595,1565,1525,1260,1020 ¹H-NMR(400 MHz, DMSO-d₆): 11.81(1H, s), 8.13–8.11(2H, m), 7.73–7.64(5H, m), 7.25–7,23(1H, m), 6.84–6.82(1H, m), 6.33–6.31( 1H, m). ¹³C-NMR (100 MHz, DMSO-d₆): 188.83, 138.81, 134.76, 133.16, 129.58, 128.38, 124.78, 116.88, 115.05, 111.14.

(E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (35): Yield: 72%; off white crystalline solid; MP: 118–120 °C (lit. = 120–122 °C)⁸⁷; Rf. value:0.40 (Ethylacetate/n-hexane,1:4); FT-IR (KBr,cm^-1): 3034, 1635, 1570, 1550, 1525, 1275, 1025. ¹H-NMR(600 MHz, DMSO-d₆): 8.04–8.02(2H, m), 7.75(1H, d, J = 15.60), 7.62–7.52(3H, m), 7.43(1H, d, J = 15.60), 6.89(2H, s), 3.44(6H, s, 2-OCH₃), 3.43(3H, s, -OCH₃). ¹³C-NMR (150 MHz, DMSO-d₆): 190.76, 153.62, 144.83, 140.68, 138.35, 132.82, 130.50, 128.39, 121.71, 105.57, 60.83, 56.40.

(E)-3-(4-(dimethylamino)phenyl)-1-phenylprop-2-en-1-one (36): Yield: 78%; orange/deep yellow color crystalline solid; MP: 110–112 °C (lit. = 111–113 °C⁹⁶; Rf. Value: 0.50(Ethylacetate/nhexane,1:4); FTIR (KBr, cm¹): 3015, 1667, 1560, 1330. ¹H-NMR(400 MHz, CDCl₃): 8.12(2H, d, J = 5.60), 7.93(1H, d, J = 15.0), 7.65–7.60(5H, m), 7.47(1H, d, J = 15.0), 6.81(2H, d, J = 5.60), 3.14(6H, s, -N(CH₃)₂. ¹³C-NMR (100 MHz, CDCl₃): 190.93, 152.06, 145.71, 129.03, 132.35, 130.62, 128.32, 122.82, 116.98, 112.03, 40.45.

(E)-1-phenyl-3-(p-tolyl)prop-2-en-1-one (37): Yield: 75%; white crystalline solid; MP: 92–94 °C (lit. = 94–96 °C)⁸⁰; Rf. Value: 0.5 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3040, 1650,1580, 1550, 1520, 1265, 1020. ¹H-NMR(40 MHz, CDCl₃): 8.15(2H, d, J = 7.0), 7.94(1H, d, J = 15.70), 7.67–7.59(6H, m), 7.34(2H, d, J = 7.70), 2.50(3H, s, -CH₃). ¹³C-NMR(100 MHz, CDCl₃): 190.65, 145.08, 141.05, 138.48, 132.70, 132.20, 129.76, 128.61, 128.52, 128.49,121.08, 21.56.

(E)-1-(thiophen-2-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (38): Yield: 88%; white crystalline solid; MP: 146–148 °C (lit. = 147–148 °C)⁹⁷; Rf. Value: 0.5 (Ethyl acetate/n-hexane,1:5); FT-IR (KBr, cm^-1): 3038, 1651,1578, 1548, 1521, 1266, 1022. ¹H-NMR(400 MHz, CDCl₃): 7.89–7.87(1H, m), 7.78(1H, d, J = 15.50), 7.69–7.67(1H, m), 7.32(1H, d, J = 15.50), 7.19–7.17(1H, m), 6.86(2H, s), 3.92(6H, s, -2OCH₃). 3.90(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 190.65, 145.08, 141.05, 138.48, 132.70, 132.20, 129.76, 128.61, 128.52, 128.49,121.08, 21.56.

(E)-3-(4-methoxyphenyl)-1-(thiophen-2-yl)prop-2-en-1-one (39): Yield: 90%; white crystalline solid; MP: 69–69 °C (lit. = 68 °C)⁹⁸; Rf. Value: 0.50 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3040, 1652,1576, 1547, 1520, 1265, 1021. ¹H-NMR(400 MHz, CDCl₃): 7.85–7.83(1H, m), 7.65–7.64(1H, m), 7.79(1H, d, J = 15.50), 7.60(2H, d, J = 8.50), 7.32(1H, d, J = 15.50), 7.17–7.15(1H, m), 6.93(2H, d, J = 8.50), 3.84(3H, s, -OCH₃). ¹³C-NMR (100 MHz, CDCl₃): 181.95, 161.92, 145.98, 143.97, 133.83, 131.54, 130.28, 128.16, 127.46, 119.29,114.44, 55.65.

(E)-3-(4-chlorophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (40): Yield: 90%; white crystalline solid; MP: 126–127 °C (lit. = 128 °C)⁹⁸; Rf. Value: 0.50 (Ethyl acetate/n-hexane,1:4); FT-IR (KBr, cm^-1): 3040, 1650, 1570, 1545, 1525, 1260, 1020. ¹H-NMR(400 MHz, CDCl₃): 7.86–7.85(1H, m), 7.79(1H, d, J = 15.60), 7.68–7.67(1H, m), 7.56(2H, d, J = 8.48), 7.39(1H, d, J = 15.60), 7.36(1H, d, J = 15.60), 7.18–7.16(1H, m), 6.93(2H, d, J = 8.48). ¹³C-NMR (100 MHz, CDCl₃): 181.98, 145.05, 142.54, 136.49, 133.17, 131.92, 129.64, 129.25, 128.33, 122.05.

In vitro α-amylase inhibition protocol

The structurally characterized chalcones (1–40) were investigated against α-amylase enzyme via a previously reported protocol with little modification⁹⁹, which involves the addition of 20 μl of dimethyl sulfoxide (DMSO) to each well, then twenty microliters of each sample was added, subsequently 50 μl of PBS (PH = 6.8) was added to each respective well. Afterward, 20 µL of starch was added to each well, and the mixture was incubated for 20 min at 37 °C. In the next step, 10 µL of the α-amylase enzyme was introduced into a separate well. At the end, 100 µL of dinitrosalicylic acid (DNS) was added to a separate well and incubated for 30 min at 37 °C. Finally, absorbance was measured at 540 nm by using a Biotech microplate reader, and acarbose was used as a standard at different concentrations. All the experiments were conducted in triplicate⁹⁹. At the end, the obtained results were expressed in IC₅₀ by using GraphPad Prism software.

In vitro DPPH protocol

The antioxidant activity of the synthesized chalcone series was measured using the DPPH free radical scavenging assay, as previously reported with minor modifications^100,101. The investigation was conducted by adding 100 µL of each tested sample, dissolved in dimethyl sulfoxide (DMSO), to a 96-well plate. Afterward, an equal volume of DPPH (100 mM) dissolved in methanol was charged to the separate sample. Then, incubated for 30 min at ambient temperature in the absence of light, and ascorbic acid was applied as a standard. All the experiments were conducted in triplicate¹⁰⁰. Lastly, the investigation was conducted by measuring their IC₅₀ values via absorbance at 517 nm through a Biotech microplate reader.

Computational methods

Protein preparation

The X-ray structure of human pancreatic α-amylase (PDB ID: 1B2Y)¹⁰² was imported into the Maestro workspace v.9.3 (Schrödinger, LCC, New York, 2012) and prepared using Protein Preparation Wizard. In this step, hydrogen atoms were added, and bond orders and formal charges were adjusted accordingly. The Epik¹⁰³ was employed to predict the protonation states (pK_a) of polar amino acids at pH 7.4 ± 0.5, whereas PROPKA v.3.1¹⁰⁴ was used to optimize hydrogen orientations. Missing loop regions were reconstructed using Prime’s loop refinement under the OPLS2005 force field¹⁰⁵.

Conformer generation

To account for protein flexibility, a pool of conformations was generated using CABS-flex 3.0, a coarse-grained molecular dynamics simulation tool¹⁰⁶. It allows efficient exploration of conformational space while maintaining computational efficiency. Simulations were performed using default parameters, with particular attention to the flexible loops surrounding the catalytic site. Four representative conformations were selected by aligning all generated models to the crystallographic reference. Special attention was given to selecting conformations that displayed significant differences in active-site geometry, ensuring adequate sampling of the conformational landscape of the substrate-binding groove.

Benchmark dataset

A curated dataset of 189 known α-amylase inhibitors (IC₅₀ ≤ 20 µM) was extracted from the ChEMBL database (CHEMBL6066863). For each active compound, 36 property-matched decoys (6,804 compounds) were generated using the LUDe (Ligand Unbiased Decoys) server¹⁰⁷, that maintain similar physicochemical properties while ensuring topological dissimilarity. All actives and decoys were imported into the Maestro workspace v.9.3 and prepared using LigPrep v.2.5 (Schrödinger, LLC) at a pH of 7.4 ± 0.5, followed by geometric optimization using OPLS2005.

Molecular docking protocol

A receptor grid was generated for each protein conformation using the Receptor Grid Generation panel in Glide v.5.8. The grid center was defined based on the acarbose binding site in the co-crystal structure (coordinates: x = 37.64 Å, y = -25.58 Å, z = -49.06 Å). The outer box was set to 20 Å to encompass the entire substrate-binding groove spanning pockets -3 to + 2. The inner box, defining the ligand center-of-mass sampling region, was set to 10 Å. Molecular docking calculations were performed using Glide v.5.8 in Standard Precision (SP) mode, with flexible ligand sampling and Epik state penalties applied. Poses were scored using the Docking Score empirical function, which integrates Van der Waals and electrostatic interaction terms, hydrogen-bond rewards, lipophilic complementarity, and Epik-derived ionization-state penalties. Each compound in the benchmark dataset was docked independently against all five protein conformations, yielding a comprehensive ensemble protocol. The docking results were then aggregated by extracting 18 intermolecular descriptors (i.e., scores, energy terms) per conformation. Additionally, to assess pose reliability, redocking experiments were performed using co-crystallized acarbose. Root-mean-square deviation (RMSD) was calculated for heavy atoms to assess pose reproduction accuracy, with RMSD < 2.0 Å considered successful.

Machine learning re-scoring

To improve the discrimination between actives and decoys beyond conventional Docking Scores, the aggregated ensemble docking dataset was analyzed in KNIME v.5.2.5 to derive a Bayesian scoring function. In this context, docking-derived energy terms and scoring functions were used as intermolecular descriptors. The most informative variables were then selected using a genetic algorithm, and a Naive Bayes classifier was trained under fivefold cross-validation. Model performance was evaluated using Enrichment Factor at 1% (EF₁_%), Boltzmann-Enhanced Discrimination of Receiver Operating Characteristic at 1% (BEDROC₁_%, α = 20), and Area Under the ROC curve (AUROC).

In silico ADMET analysis

ADMETLab v.3.0¹⁰⁸ was used to predict key drug-like properties and the ADMET profile of the top synthesized hits. The analysis included physicochemical descriptors, namely logS, logD, and logP, which inform aqueous solubility, pH-dependent distribution behavior, and intrinsic lipophilicity, respectively. Absorption-related parameters were also evaluated, including Caco-2 permeability and the parallel artificial membrane permeability assay (PAMPA) for blood–brain barrier permeation. Metabolism-related properties were also examined, including human liver microsome (HLM) stability. Elimination markers included plasma clearance (CLplasma). Toxicity endpoints included human ether-à-go-go-related gene (hERG) channel blockade, drug-induced liver injury (DILI) hepatotoxicity, and Ames mutagenicity.

Results and discussion

Synthesis and structural characterization

In this work, we synthesized a series of 40 chalcone analogs (compounds 1–40, Table 1) through a Claisen–Schmidt condensation. The compounds were obtained by base-promoted coupling of appropriately substituted aromatic aldehydes with acetophenone derivatives at room temperature, as outlined in Fig. 2. The substitution pattern was designed to sample electron-donating, electron-withdrawing, hydrogen-bonding, and heteroaryl environments, thereby enabling an initial assessment of how electronic and pharmacophoric features influence the biological profile of the chalcone scaffold. For clarity, the synthesized chalcones were organized into five series according to the substitution pattern of ring A. In the first series (1–11), second (12–24), third (25–32), fourth (33–37), and fifth (38–40) series, p-chlorophenyl, p-methoxyphenyl, p-hydroxyphenyl, phenyl, and thiophene were used as ring A, respectively, while different substituted benzene were used as ring B. Reaction progress was monitored by TLC, and the crude products were purified through recrystallization from ethanol, yielding amounts between 64 and 90%.

Table 1.

Synthetic Chalcone derivatives (1–40).

Code of compd	Ring A	Ring B	α-Amylase IC50 (µM ± SD)	DPPH IC50 (µM ± SD)
1			48.03 ± 2.78	75.97 ± 0.90
2			37.15 ± 0.30	211.23 ± 0.68
3			49.03 ± 0.89	264.07 ± 0.11
4			10.41 ± 1.23	237.06 ± 0.07
5			21.24 ± 0.63	135.51 ± 2.70
6			92.21 ± 3.57	117.62 ± 1.40
7			32.34 ± 2.95	47.34 ± 1.18
8			587.44 ± 6.71	365.63 ± 0.43
9			38.11 ± 2.90	120.57 ± 0.46
10			393.51 ± 5.86	698.34 ± 14.56
11			194.10 ± 6.61	158.79 ± 4.24
12			174.21 ± 0.64	180.54 ± 0.18
13			117.21 ± 4.45	199.81 ± 0.07
14			104.14 ± 0.73	113.87 ± 0.72
15			51.32 ± 1.97	229.66 ± 0.31
16			32.13 ± 3.38	56.33 ± 0.44
17			23.07 ± 0.55	99.17 ± 0.40
18			26.60 ± 0.98	56.87 ± 0.36
19			15.44 ± 0.29	202.80 ± 0.64
20			187.14 ± 0.95	340.79 ± 0.13
21			86.49 ± 0.55	92.45 ± 2.00
22			57.67 ± 0.30	207.60 ± 14.38
23			277.11 ± 3.18	359.25 ± 1.75
24			365.27 ± 3.74	261.28 ± 1.14
25			64.83 ± 0.28	198.30 ± 0.23
26			26.19 ± 1.72	63.44 ± 0.29
27			27.33 ± 4.67	266.51 ± 1.19
28			65.12 ± 2.68	240.79 ± 0.99
29			374.08 ± 1.91	299.36 ± 1.16
30			39.03 ± 0.50	49.12 ± 0.53
31			19.46 ± 0.03	99.70 ± 0.43
32			218.35 ± 10.09	290.98 ± 6.70
33			16.54 ± 0.25	227.46 ± 0.02
34			1021.64 ± 2.75	316.78 ± 0.70
35			31.97 ± 1.05	31.34 ± 0.20
36			61.27 ± 1.37	219.95 ± 1.34
37			152.67 ± 2.92	192.96 ± 0.10
38			87.24 ± 2.67	118.52 ± 3.39
39			65.41 ± 1.33	201.50 ± 10.39
40			380.14 ± 6.71	675.12 ± 10.46
Standard	Acarbose		73.12 ± 5.04	–––––-
Standard	Ascorbic acid		–––––––	287.30 ± 4.00

Fig. 2.

Representative scheme for synthesizing chalcone analogs.

The structural characterization of the synthesized analogs was then performed by FT-IR and ¹H/¹³C NMR spectroscopy. In the FT-IR spectra, the chalcone derivatives displayed the expected absorptions associated with aromatic C–H stretching (3090–3010 cm^-1), olefinic C–H (1600–1650 cm^-1), and conjugated enone carbonyl group (1690–1650 cm^-1). Furthermore, the ¹H-NMR spectra of the synthesized compounds were recorded in deuterated solvents such as DMSO and chloroform on 600- and 400-MHz NMR spectrometers. Interestingly, all characterized compounds exhibit characteristic peaks of the trans-chalcone scaffold which lies in the range of 8.00 to 7.00 ppm, with large coupling constants of around 15–19 Hz.

In addition, peaks for various substituted benzene rings were found in the aromatic range. Similarly, singlet peaks for different substituents, such as methoxy, methyl, and dimethylamino groups, were observed at 3.80–3.70 ppm, 2.80–2.70 ppm, and around 3.00 ppm, respectively. Likewise, ¹³C-NMR recorded spectra show that the peaks in the range such as 190–188 ppm is due to carbonyl carbon, whereas various peaks in the range of 140–110 ppm are due to various aryl carbons, while peaks in the range of 80–50 ppm, are because of carbon in various substituents such as methoxy-, dimethylamino-, methyl- attached to aryl groups in chalcone derivatives. In short, all these spectroscopic data collectively confirm the presence of the said nucleus-containing compounds and are in good agreement with the literature.

α-amylase inhibition

All synthesized analogs (1–40) were evaluated for their α-amylase inhibitory activity, with IC₅₀ values ranging from 10.41 ± 1.23 to 1021.64 ± 2.75 µM. Among them, compounds 4 (IC₅₀ = 10.41 ± 1.23 µM), 19 (IC₅₀ = 15.44 ± 0.29 µM), 33 (IC₅₀ = 16.54 ± 0.25 µM), 31 (IC₅₀ = 19.46 ± 0.03 µM), and 5 (IC₅₀ = 21.24 ± 0.63 µM) emerged as the most potent members of the series, showing markedly stronger inhibition than the reference drug acarbose (IC₅₀ = 73.12 ± 5.04 µM). Additional highly active analogs, including 17 (IC₅₀ = 23.07 ± 0.55 µM), 26 (IC₅₀ = 26.19 ± 1.72 µM), 18 (IC₅₀ = 26.60 ± 0.98 µM), and 27 (IC₅₀ = 27.33 ± 4.67 µM), further defined a consistent cluster of sub-30 µM inhibitors, highlighting the chalcone scaffold as a promising chemotype for α-amylase inhibition.

From a structure–activity relationship (SAR) perspective, α-amylase inhibition was strongly modulated by the substitution pattern on both aromatic termini of the chalcone scaffold. Within the 4-chlorophenyl series (1–11), the most pronounced gain in potency was observed for the 2-hydroxyphenyl analog 4, indicating that introduction of an ortho-hydroxyl group on ring B is particularly favorable in this background. Good activity was also retained for the furan-2-yl derivative 5, as well as for electron-rich aryl substituents such as 4-dimethylaminophenyl, 7 (IC₅₀ = 32.34 ± 2.95 µM), 4-methoxyphenyl 1 (IC₅₀ = 48.03 ± 2.78 µM), p-tolyl, 2 (IC₅₀ = 37.15 ± 0.30 µM), and 3,4,5-trimethoxyphenyl, 9 (IC₅₀ = 38.11 ± 2.90 µM), whereas potency dropped markedly for the 3-nitrophenyl, 6 (IC₅₀ = 92.21 ± 3.57 µM), 2,4-dichlorophenyl, 11 (IC₅₀ = 194.10 ± 6.61 µM), pyrrolyl 8 (IC₅₀ = 587.44 ± 6.71 µM), and thiophenyl, 10 (IC₅₀ = 393.51 ± 5.86 µM) congeners.

In the 4-methoxyphenyl subseries (12–24), the most favorable profiles were associated with electron-rich ring B motifs, particularly 3-hydroxy-4-methoxyphenyl, 19(C₅₀ = 15.44 ± 0.29 µM), 4-methoxyphenyl,17 (IC₅₀ = 23.07 ± 0.55 µM), p-tolyl, 18 (IC₅₀ = 26.60 ± 0.98 µM), and 4-dimethylaminophenyl, 16 (IC₅₀ = 32.13 ± 3.38 µM), whereas halogenated aryl groups such as 2-chlorophenyl, 23(IC₅₀ = 277.11 ± 3.18 µM) and 2,4-dichlorophenyl,24(IC₅₀ = 365.27 ± 3.74 µM) were clearly detrimental.

A distinct but still favorable pattern emerged in the 4-hydroxyphenyl series (25–32), where heteroaryl replacements became more productive, as illustrated by the potent furan-2-yl, 26 (IC₅₀ = 26.19 ± 1.72 µM) and thiophen-2-yl, 31 (IC₅₀ = 19.46 ± 0.03 µM) analogs, while the 3-nitrophenyl derivative, 27 (IC₅₀ = 27.33 ± 4.67 µM) also remained highly active in this context. In the unsubstituted phenyl series (33–37), the best activities were again associated with oxygenated ring B substituents, with 3-hydroxy-4-methoxyphenyl, 33 (IC₅₀ = 16.54 ± 0.25 µM) and 3,4,5-trimethoxyphenyl,35(IC₅₀ = 31.97 ± 1.05 uM) standing out, whereas the pyrrolyl analog 34 (IC₅₀ = 1021.64 ± 2.75 µM) showed a dramatic loss of potency.

These data suggest that potent α-amylase inhibition in this series is favored by a balanced combination of aromatic complementarity and well-positioned hydrogen-bonding functionality on ring B, particularly ortho-hydroxyl and methoxy/hydroxyl-rich aryl motifs, whereas excessive halogenation and some heteroaryl replacements are generally less tolerated.

Antioxidant potential

Chalcone analogs were further evaluated for their DPPH radical-scavenging activity, revealing a broad range of activity, with IC₅₀ values spanning 31.34 ± 0.20–698.34 ± 14.56 µM. The most potent compound in the series was 35 (IC₅₀ = 31.34 ± 0.20 µM), followed by 7 (IC₅₀ = 47.34 ± 1.18 µM), 30 (IC₅₀ = 49.12 ± 0.53 µM), 16 (IC₅₀ = 56.33 ± 0.44 µM), 18 (IC₅₀ = 56.87 ± 0.36 µM), and 26 (IC₅₀ = 63.44 ± 0.29 µM). Additional active analogs, including 1 (IC₅₀ = 75.97 ± 0.90 µM), 21 (IC₅₀ = 92.45 ± 2.00 µM), 17 (IC₅₀ = 99.17 ± 0.40 µM), and 31 (IC₅₀ = 99.70 ± 0.43 µM), further support the ability of this chalcone series to deliver consistent radical-scavenging activity. Notably, 80% derivatives displayed stronger activity than the reference antioxidant ascorbic acid (IC₅₀ = 287.30 ± 4.00 µM).

From a SAR standpoint, the DPPH data indicate that radical-scavenging activity is generally favored by electron-rich substitution patterns, particularly when ring B bears dimethylamino- or methoxy-substituted aryl groups. In the 4-chlorophenyl series (1–11), the 4-dimethylaminophenyl derivative, 7(IC₅₀ = 47.34 ± 1.18 µM) emerged as the most active member, while 4-methoxyphenyl,1(IC₅₀ = 75.97 ± 0.90 µM) and 3,4,5-trimethoxyphenyl,9(IC₅₀ = 120.57 ± 0.46 µM) also retained good activity. By contrast, replacement with pyrrolyl, 8 (IC₅₀ = 365.63 ± 0.43 µM) or thiophen-2-yl, 10 (IC₅₀ = 698.34 ± 14.56 µM) resulted in a pronounced loss of potency.

A similar trend was observed in the 4-methoxyphenyl subseries (12–24), in which the most favorable profiles were associated with 4-dimethylaminophenyl, 16 (IC₅₀ = 56.33 ± 0.44 µM), p-tolyl, 18 (IC₅₀ = 56.87 ± 0.36 µM), 4-methoxyphenyl, 17 (IC₅₀ = 99.17 ± 0.40 µM), and 3,4,5-trimethoxyphenyl, 21 (IC₅₀ = 92.45 ± 2.00 µM) motifs, whereas halogenated aryl substituents, such as 2-chlorophenyl,23(IC₅₀ = 359.25 ± 1.75 µM) and 2,4-dichlorophenyl, 24 (IC₅₀ = 261.28 ± 1.14 µM) were clearly detrimental. In the 4-hydroxyphenyl series (25–32), the best activities were observed for the 3,4,5-trimethoxyphenyl analog 30 (IC₅₀ = 49.12 ± 0.53 µM) and the furan-2-yl derivative 26 (IC₅₀ = 63.44 ± 0.29 µM), while 3-nitrophenyl, 27 (IC₅₀ = 266.51 ± 1.19 µM), 2-pyridyl, 29 (IC₅₀ = 299.36 ± 1.16 µM), and 2,4-dichlorophenyl, 32 (IC₅₀ = 290.98 ± 6.70 µM) were less favorable.

In the unsubstituted phenyl series (33–37), the superior activity of 35 (IC₅₀ = 31.34 ± 0.20 µM) highlights the strong beneficial effect of a trimethoxy-substituted ring B, whereas the pyrrolyl analog 34 (IC₅₀ = 316.78 ± 0.70 µM) was among the least active compounds in the entire set. Overall, these results indicate that DPPH radical scavenging in this series is primarily driven by electron-donating substituents that stabilize the radical. In addition, the structural requirements for antioxidant activity partially overlap with those for α-amylase inhibition.

Molecular docking

To enhance our understanding of the experimental SAR trends, we conducted molecular docking simulations in the α-amylase catalytic site. Initially, molecular docking calculations were performed against the crystallographic structure of α-amylase (PDB ID: 1B2Y)¹⁰² using the standard Docking Score function. To assess the prospective utility of this protocol for ligand prioritization, its enrichment performance was evaluated on a benchmarking set comprising 189 known α-amylase inhibitors and 6,804 property-matched decoys. Under these conditions, the docking workflow showed poor discriminatory power, yielding an AUROC of 0.40, EF₁% of 1.15, and BEDROC₁% of 0.04, indicating limited ability to distinguish active ligands from decoys (Fig. 3a). This result reflects a common limitation of conventional docking, where empirical scoring functions often poorly correlate with experimental binding affinity¹⁰⁹.

Fig. 3.

Validation of the docking protocol and predicted binding modes of the most active chalcone derivatives in α-amylase. (a) ROC curve for standard docking using the Docking Score function. (b) ROC curve for Naive Bayes rescoring based on genetic algorithm-selected docking descriptors. (c) ROC curve for ensemble docking combined with Naive Bayes rescoring after incorporation of four representative protein conformations generated by CABS-flex. (d) Superposition of the crystallographic and re-docked poses of acarbose (RMSD = 1.3 Å). (e) Crystallographic binding mode of acarbose in the α-amylase active site. (f–i) Predicted binding modes of compounds 4, 19, 31, and 33, respectively. Hydrogen bonds are shown in yellow, π–π interactions in green, and the protein surface in gray.

Subsequently, a genetic algorithm was used to select the most informative descriptors from the raw docking scores and individual energy terms, which were then used to build a Naive Bayes-based rescoring function. This machine learning approach substantially improved discrimination, achieving an AUROC of 0.75, an EF₁% of 19.25, and a BEDROC₁_% of 0.71 (Fig. 3b). Additional gains were achieved after the incorporation of four representative α-amylase conformations generated by CABS-flex¹⁰⁶ into an ensemble docking scheme. These conformations were selected to represent structurally diverse states of the binding site, thereby broadening the conformational space sampled during docking. Under this refined protocol, the enrichment metrics further improved to an AUROC of 0.80, an EF₁% of 22.13, and a BEDROC₁% of 0.78 (Fig. 3b), indicating that explicit treatment of protein flexibility enhanced active–decoy discrimination. Notably, the genetic algorithm selected the Coulomb energy term, ligand efficiency, and rotatable-bond penalty among the docking-derived descriptors used for Naive Bayes training. This pattern suggests that α-amylase recognition is governed by electrostatic complementarity, size-efficient binding, and limited conformational strain. Accordingly, the ensemble docking framework was selected for the subsequent binding-mode analysis of the most active chalcone analogs.

The docking protocol was further validated by re-docking acarbose, a well-characterized α-amylase inhibitor. The predicted binding pose showed excellent agreement with the crystallographic structure, with an RMSD of 1.3 Å and preservation of key interactions within the enzyme’s pockets (Fig. 3d). Acarbose occupied the -3 to + 2 pockets of the active site, establishing critical hydrogen bonds with catalytic residues (Asp197, Glu233, and Asp300) that serve as pharmacophoric anchors for inhibitor design (Fig. 3e).

Structural insights for α-amylase inhibition

After confirming the reliability of the docking setup, detailed inspection of the predicted binding poses was performed to characterize how the four most potent chalcone derivatives, 4 (IC₅₀ = 10.41 ± 1.23 µM); 19 (C₅₀ = 15.44 ± 0.29 µM); 33 (IC₅₀ = 16.54 ± 0.25 µM); and 31 (IC₅₀ = 19.46 ± 0.03 µM), interact with key residues in the α-amylase catalytic pocket. As shown in Fig. 3d, 4 adopted a favorable orientation spanning the -1 and + 1 pockets, with the 2’-hydroxyl performing a hydrogen bond to Glu233, while the α,β-unsaturated carbonyl forms a hydrogen bond with Arg195. These interactions with catalytic residue Glu233 suggest direct interference with the enzyme’s hydrolytic mechanism.

Compound 19 also adopted a favorable orientation spanning the − 1 and + 1 pockets (Fig. 3g), closely resembling the binding mode observed for 4. In this pose, the 2′-hydroxyl forms a hydrogen bond with Glu233, whereas the α,β-unsaturated carbonyl is anchored by a hydrogen bond with Arg195, supporting productive recognition of the catalytic groove. Similarly, compound 31 demonstrated π-π stacking interactions with both His201 and Tyr62, complemented by a hydrogen bond with Lys200 (Fig. 3h). The engagement of multiple aromatic residues suggests enhanced binding affinity through cooperative hydrophobic and π-electron interactions. Compound 33 maintained the conserved π-π stacking interaction with His201 while its carbonyl forms a hydrogen bond with Arg195 (Fig. 3i).

These binding mode analyses reveal that the chalcone derivatives exploit both the catalytic machinery and the extended substrate-binding groove of α-amylase, with the aromatic stacking interactions with His201 emerging as a conserved pharmacophoric element. Critically, compound 4 distinguishes itself by directly engaging Glu233, the key acid/base catalyst within α-amylase’s catalytic site (comprising the catalytic residues Asp197, Glu233, and Asp300). This strategic interaction with the catalytic machinery, rather than the peripheral binding sites favored by compounds 19, 31, and 33, provides a compelling mechanistic explanation for compound 4’s superior inhibitory profile.

In silico ADMET analyses

In silico ADMET profiling was performed with ADMETLab v.3.0¹⁰⁸ to assess the developability of the prioritized chalcone derivatives. All four compounds satisfied Lipinski’s Rule of Five and Veber’s oral bioavailability criteria, supporting their overall drug-like character. The predicted physicochemical profiles were also favorable, with adequate aqueous solubility (logS =  − 4.41 to − 2.61) and balanced lipophilicity (logD = 2.00–3.42; logP = 2.21–3.75) (Table 2). Compounds 4, 31, and 33 showed good Caco-2 permeability (> − 5.15 log cm/s), whereas 19 displayed a borderline value. Overall, the set remained compatible with acceptable membrane permeation. In addition, all four compounds showed moderate predicted human liver microsomal stability (instability probability = 0.30–0.61) and intermediate plasma clearance (7.53–11.04 mL/min/kg).

Table 2.

In silico ADMET profile for top compounds.

Cpd	logS	logD	logP	Caco-2	PAMPA	HLM	CLplasma	hERG	DILI	Ames
4	-4.41	3.42	3.75	(-4.63)	(0.06)	(0.3)	(7.79)	(0.25)	(0.64)	(0.15)
19	-2.61	2.0	2.21	(-5.15)	(0.11)	(0.31)	(7.53)	(0.09)	(0.37)	(0.56)
31	-3.31	3.12	2.95	(-4.75)	(0.16)	(0.61)	(11.04)	(0.23)	(0.97)	(0.49)
33	-3.52	3.14	3.01	(-4.84)	(0.01)	(0.37)	(10.15)	(0.21)	(0.49)	(0.32)

Color coding indicates favorable (green), intermediate (yellow), and unfavorable (red) predicted profiles, with numerical values shown in parentheses. Reference criteria were: logS, − 4 to 0.5; logD, 1–3; logP, 0–3; Caco-2, >  − 5.15 log cm/s; CLplasma, < 5 low, 5–15 moderate, and > 15 high; and, for all probability-based endpoints (PAMPA, HLM instability, hERG, DILI, and AMES), 0–0.3 favorable, 0.3–0.7 intermediate, and 0.7–1.0 unfavorable.

The toxicity predictions differentiated the series more clearly. All compounds showed low predicted cardiotoxicity liability, with minimal hERG-blocking potential (probability = 0.09–0.25). In contrast, compound 31 displayed a high predicted DILI risk (0.97), plausibly associated with the thiophene ring, a known structural alert for bioactivation. Compounds 4, 19, and 33 showed moderate hepatotoxicity risk (0.37–0.64), which may be related to the intrinsic Michael acceptor character of the chalcone enone system. Predicted AMES mutagenicity was lowest for compound 4 (0.15), whereas 19, 31, and 33 showed intermediate probabilities (0.32–0.56), supporting the need for experimental genotoxicity assessment during prospective studies. In summary, these results confirm compound 4 as the most balanced ADMET hit for prospective hit-to-lead investigations.

Conclusions

In this work, a combined synthetic, biological, and computational workflow identified chalcone derivatives with α-amylase inhibitory activity and DPPH radical-scavenging properties. A library of 40 chalcone analogs was synthesized, characterized, and evaluated in vitro, leading to the identification of compound 4 as the most promising hit in the series. Structure-based analysis with a validated ensemble and Naive Bayes scoring suggested that compounds 4, 19, 31, and 33 binds effectively within the α-amylase catalytic groove, with an interaction with Glu233 directly interfering with the enzyme’s hydrolytic mechanism, explaining its increased inhibitory potency. In silico ADMET profiling further indicated acceptable developability features for the hit compounds, including suitable solubility, intestinal permeability, and low predicted cardiotoxicity, with compound 4 showing the most balanced overall profile. In summary, these findings identify this chalcone series as a promising starting point for hit-to-lead optimization toward multifunctional radical-scavenging and α-amylase-inhibitory activities.

Author contributions

Conceptualization: M.I.A.S., J.R., and S.S.; Methodology: A.A., F.B.O.D., G.R.R., and Z.H.; Validation: A.A., F.B.O.D., G.R.R., and Z.H.,; Formal analysis: A.Z., G.S.S.F., T.E.A.F., F.A.O., A.C., M.N., B.J.N., and M.I.A.S.; Investigation: .A., F.B.O.D., G.R.R., G.S.S.F. and B.J.N.; Resources: M.I.A.S., S.S., and J.R.; Data curation: A.A., F.B.O.D., G.R.R., Z.H. and T.E.A.F.; Writing—Original draft: M.I.A.S., J.R., and S.S.; Writing—Review & Editing: B.J.N., M.I.A.S., J.R., and S.S.,; Visualization: M.I.A.S., J.R., and J.R.; Supervision: M.I.A.S., J.R., and S.S.; Project administration: M.I.A.S., J.R., and S.S.; Funding acquisition: J.R., and S.S.

All authors read and approved the final draft of the manuscript.

Funding

We gratefully acknowledge CNPq, CAPES (001), and HEC (Pakistan) for financial support. J.R., and S.S., would like to acknowledge CNPq (308875/2026–5, 316687/2023–5, 309975/2022–0, 401355/2025–0, 404172/2023–7, and 405655/2023–1) for the Scholarships. B.J.N. was supported by CNPq (311100/2023–6). M.I.A.S. would like to acknowledge NRPU Project No. 17234. S.S. and J.R. also acknowledge the following FAPEG public calls: Chamada Pública FAPEG/SES Nº 18/2025 (ARB2025191000003), Chamada Pública FAPEG Nº 05/2025 (PVE2025041000055), and Chamada Pública FAPEG Nº 21/2025 (PEE2025331000083 and PEE2025331000108).

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information file.

Competing interests

The authors declare no competing interests.