DES‐Promoted Synthesis of 3,4‐Dihydropyrimidinones and Their Antidiabetic and Antioxidant Evaluation Supported With Computational Studies

ABSTRACT

A series of 3,4‐dihydropyrimidinone (DHPM) derivatives was synthesized using a green deep eutectic solvent (DES) system composed of ZnCl₂ and urea, which acted simultaneously as solvent, catalyst, and in situ substrate (urea). The synthesized compounds were evaluated for their antidiabetic potential via α‐glucosidase and α‐amylase inhibition assays, as well as for antioxidant activity using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and nitric oxide (NO) scavenging assays. Among the series, compound 3f exhibited superior inhibitory activity against α‐glucosidase (IC₅₀ = 35.25 µM) and α‐amylase (IC₅₀ = 38.61 µM), being ∼2.3‐ and ∼2.6‐fold more potent, respectively, than the standard drug acarbose. In antioxidant evaluations, compound 3i (IC₅₀ = 35.60 µM) demonstrated ∼2.9‐fold higher activity than gallic acid in the DPPH assay, whereas 3d (IC₅₀ = 30.70 µM) and 3a (IC₅₀ = 44.52 µM) showed ∼3.8‐ and ∼1.5‐fold higher activity in FRAP and NO scavenging assays, respectively. Molecular docking studies revealed key hydrogen bonding interactions of 3f with the active site residues of both enzymes, supported by favorable docking scores. Furthermore, density functional theory (DFT) studies revealed favorable electronic and reactivity profiles, whereas ADME/T predictions indicated good drug‐likeness. Overall, compound 3f shows strong potential as a lead antidiabetic agent for managing postprandial hyperglycemia.

DES‐promoted synthesis and antidiabetic evaluation of 3,4‐dihydropyrimidinones and their complementation with computational studies are reported.

1. Introduction

Over the past few decades, diabetes cases have seen a multifold increase throughout the world [1]. Multiple literature reports have reported that this increment was substantial in the first decade of the 21st century [2], particularly in the middle‐income countries of Asia and Africa [3]. Particularly, Type II diabetes (T2D) is a chronic metabolic disorder caused by insulin resistance, which makes cells unable to respond effectively to insulin. Over time, this leads to progressive beta‐cell dysfunction and a relative insulin deficiency [4, 5, 6, 7]. Additionally, approximately 90% of all diabetic cases are reported to be T2D [8].

According to the International Diabetes Federation, DM poses a significant global health burden, with an estimated 537 million people affected worldwide in 2021, a number projected to rise to 643 million by 2030 and 783 million by 2045 [1, 9, 10, 11, 12]. The condition arises from various genetic, environmental, and lifestyle factors that contribute to impaired glucose regulation [13]. Insulin resistance results in high glucose levels in the blood and can lead to serious complications, such as retinopathy, nephropathy, neuropathy, and cardiovascular disease [14, 15].

The current treatment strategy for T2DM's elevated postprandial glucose levels is impairing the breakdown of dietary carbohydrates, which further delays the absorption of glucose and lowers blood sugar levels [16]. Pancreatic α‐amylase is a key enzyme that breaks complex carbohydrates such as starch into simple monosaccharides, which are further cleaved by α‐glucosidase into glucose, which, on absorption, enters the bloodstream [17, 18, 19]. Currently marketed drugs like acarbose, voglibose, and miglitol inhibit the aforementioned enzymes effectively; however, they are also accompanied by side effects such as bloating, abdominal discomfort, diarrhea, and flatulence [20, 21]. Therefore, it is imperative to identify new compounds that function similarly with no/minimal side effects.

The pharmacological significance of 3,4‐dihydropyrimidinones (DHPMs) (Figure 1) has been widely reported in the literature [22, 23, 24, 25, 26, 27], including more recent ones that discuss their antidiabetic effects [28, 29, 30, 31]. Additionally, their unique structure offers the capacity to adjust or fine‐tune the pharmacophore in three dimensions, increasing the likelihood of discovering new leads [32].

FIGURE 1.

Some potent bioactive compounds containing the DHPM scaffold.

Deep eutectic solvents (DESs) are a subclass of ionic liquids, containing a mixture of a hydrogen bond donor and a hydrogen bond acceptor. In the past decades, it was utilized as a green solvent as well as a catalyst in organic transformations [33]. DESs have shown remarkable properties such as low volatility, cost‐effectiveness, biodegradability, and chemical and thermal stability [34, 35, 36, 37]. Due to the aforementioned beneficial properties, DESs have been utilized in various organic reactions, such as Paal–Knorr, Diels–Alder, Perkin, Biginelli, Hantzsch, and Henry reactions [38].

In the present study, ZnCl₂/urea DES was utilized for the green synthesis of DHPM. The present protocols offered significant advantages, such as short reaction time, green solvent and catalyst, and high‐to‐excellent product yield. The synthesized DHPM analogs were subsequently evaluated for their antidiabetic potential through α‐amylase and α‐glucosidase inhibition as well as antioxidant activity using established assays (2,2‐diphenyl‐1‐picrylhydrazyl [DPPH], ferric reducing antioxidant power [FRAP], and nitric oxide [NO]). The computational studies were also performed to complement the experimental results.

2. Result and Discussion

Mahdipour et al. reported the synthesis of DHPMs using α‐ketoester (ethylacetoacetate or methylacetoacetate) and aldehydes as starting materials in ZnCl₂/urea DES (Scheme S1) [39]. To expand the scope of the catalytic efficacy of ZnCl₂/urea DES, we employed acetylacetone as an active methylene component and engaged this in a condensation reaction and various aldehydes to generate the corresponding DHPM analogues. Acetylacetone reacted rapidly and efficiently with aldehydes, affording the target compounds in excellent yields (90%–98%) within 10 min (Scheme 1). The results summarized in Scheme S2 demonstrated that aromatic aldehydes with electron‐withdrawing groups (EWGs) reacted rapidly and provided a better yield than aldehydes with electron‐donating groups (EDG).

SCHEME 1.

Illustration of the synthesis of DHPMs using DES.

The synthesized compounds were characterized by using spectroscopic techniques, including ¹H NMR, ¹³C NMR, and HRMS. For example, compound 3d (Figures 2 and 3), in its ¹H spectrum, displayed two singlets at δ _H 2.07 and 2.27 ppm, which correspond to H‐8 and H‐9, respectively. The ─OCH₃ protons (H‐17) exhibited their resonance at δ _H 3.72 ppm. The C─H proton (H‐4) coupled with H‐3 (NH) to form a doublet at δ _H 5.20 ppm, which confirmed that the cyclization had occurred. However, the most downfield signal at δ _H 9.15 ppm corresponded to the H‐1 proton. Furthermore, 3d showed the expected peaks in its ¹³C APT (attached proton test) NMR spectrum, for example, C‐8 at δ 19.28 ppm, C‐9 at δ 30.60 ppm, C‐4 at δ 53.82 ppm, C‐17 at δ 55.55 ppm, and C‐7 at δ 194.83 ppm. The HRMS finally showed a molecular ion peak at m/z 283.1048, confirming the assigned structure of compound 3d.

FIGURE 2.

¹HNMR of compound 3d.

FIGURE 3.

¹³C (APT) NMR of compound 3d.

Figure 4 demonstrates that aromatic protons of all five compounds resonate in the aromatic region. The four‐substituted analogues (3a, 3d, and 3f), as expected, exhibited two well‐resolved, split signals, except for 3b (4‐CH₃ analogue), which displayed an unresolved broad singlet in the aromatic region. The N─H (N1) and N─H (N3) protons, for example, in compounds (3f and 3h) bearing EWGs on the phenyl ring appeared relatively more downfield, attributed to their inductive effect. However, the same protons in compounds (3b and 3d) bearing EDG appeared relatively in the upfield region when compared to their EWG counterparts.

FIGURE 4.

Expanded overlay of ¹H NMR spectra of 3a, 3b, 3d, 3f, and 3h; however, the full spectra can be viewed in supplementary data.

3. Biological Studies

3.1. α‐Glucosidase and α‐Amylase Inhibition

The in vitro antidiabetic activity of compounds 3a–l was evaluated by measuring their IC₅₀ values against α‐glucosidase and α‐amylase enzymes, using acarbose as a standard, as shown in Table 1. Generally, the synthesized compounds demonstrated promising inhibition activity against α‐amylase as compared to α‐glucosidase, with IC₅₀ values ranging between 38.61–282.00 and 35.25–375.10 µM, respectively.

TABLE 1.

IC₅₀ (µM) values for the in vitro evaluation of antidiabetic activity of compounds 3a–l.

Compound	α‐Glucosidase	α‐Amylase
	125.00	99.88
	252.60	145.50
	377.10	66.99
	128.20	141.40
	166.30	132.00
	35.25	38.61
	118.30	50.46
	495.80	40.53
	211.00	282.00
	146.80	148.70
	113.40	97.93
	223.70	70.98
Acarbose	84.21	102.72

Compound numbers highlighted in bold exhibited more potency than the standard drug (Acarbose).

As shown in Table 1, compound 3f showed the highest inhibitory effects on both α‐glucosidase (IC₅₀ = 35.25 µM) and α‐amylase (IC₅₀ = 38.61 µM) and was found to be $\sim$ 2.3‐fold superior against α‐glucosidase and $\sim$ 2.6‐fold superior against α‐amylase when compared to the standard acarbose (IC₅₀ = 84.21 and 102.72 µM). The remaining compounds, however, displayed lower inhibition of α‐glucosidase with respect to acarbose.

For α‐amylase, several compounds, such as 3a, 3c, 3f–3h, 3k, and 3l, displayed superior inhibition of the α‐amylase enzyme as compared to acarbose. Compound 3h emerged as the strongest inhibitor (IC₅₀ = 40.53 µM) of α‐amylase, displaying $\sim$ 2.5‐fold superior activity when compared to acarbose. These results suggest that the synthesized compounds have promising antidiabetic potential, particularly as α‐amylase inhibitors.

The structure–activity relationship (SAR) analysis for α‐glucosidase inhibition highlights the significance of functional groups on the phenyl ring (Figure 5). Generally, the placement of substituents, regardless of their electronic nature and positioning on the phenyl ring, decreased their activity. Particularly, the EDG (CH₃ and OCH₃) offered less active compounds (3b–3e). Halogen substituents, particularly chlorine at para (3f, IC₅₀ = 35.25 µM) and meta (3g, IC₅₀ = 118.30 µM) positions, noticeably improved the activity, with the former emerging as the most active compound of the series. The positioning of strong EWGs such as fluorine (3h) and NO₂ (3l) offered much lower inhibition when compared to their unsubstituted analogues and acarbose.

FIGURE 5.

Summarized SAR of the tested compounds for their antidiabetic activity.

For α‐amylase inhibition, the presence of EDG on the phenyl ring offered weak inhibitors (3b–e, IC₅₀ = 132.00–145.50 µM), except for the methyl group at the meta‐position (3c, IC₅₀ = 66.99 µM). Their mono‐halogenated derivatives (3f–3h, 3k) demonstrated good activity with the IC₅₀ values ranging between 38.61 and 97.93 µM and were more active than acarbose. In contrast to α‐glucosidase, the nitro group placement at para‐position (3l) generated a stronger inhibitor of α‐amylase with superior potency to acarbose. Overall, the results indicated that EWGs at the para‐position favored the amylase inhibitory activity of these compounds.

3.2. Antioxidant Activity Profiling

3.2.1. DPPH Radical Scavenging Activity

The DPPH radical scavenging assay measures a compound's ability to donate electrons and neutralize free radicals, indicating its potential as an antioxidant [40]. This assay is crucial for evaluating the effectiveness of compounds in protecting cells from oxidative damage. In this context, the relative antioxidant potency of compounds 3a–l was evaluated under in vitro conditions, and the results are listed in Table 2.

TABLE 2.

IC₅₀ (µM) values for the in vitro evaluation of antioxidant activity of compounds 3a–l.

Compound	DPPH	FRAP	NO
	234.20	78.81	44.52
	214.10	40.00	268.50
	133.50	221.10	203.20
	53.14	30.70	96.42
	86.42	216.80	259.40
	40.71	144.40	139.00
	272.60	280.40	282.80
	49.39	201.20	232.90
	35.60	116.10	140.00
	149.30	261.80	112.10
	232.90	107.40	115.50
	135.70	282.80	265.00
Gallic acid	105.10	116.60	78.50

Abbreviations: DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl; FRAP, ferric reducing antioxidant power; NO, nitric oxide.

The DPPH assay results show most compounds demonstrated activity within the range 35.60–86.42 µM and were multiple‐fold more active than the standard gallic acid (105.10 µM). Compound 3i, with an IC₅₀ of 35.6 µM, exhibited $\sim$ 3‐fold stronger radical scavenging activity than the standard. Other notable compounds included 3f showing $\sim$ 2.5‐fold (IC₅₀ = 40.71 µM), 3h showing $\sim$ 2‐fold (IC₅₀ = 49.39 µM), and 3d showing $\sim$ 2‐fold (IC₅₀ = 53.14 µM) superior activity to gallic acid, highlighting their significant antioxidant potential.

3.2.2. Ferric Reducing Antioxidant Power

The FRAP assay assesses the ability of a compound to reduce ferric ions (Fe³⁺) to ferrous ions (Fe²⁺), reflecting its overall antioxidant power [41]. This assay is important for determining a compound's capacity to act as an electron donor and reduce oxidative stress [42]. In the FRAP assay, compound 3d demonstrated the $\sim$ 3.7‐fold highest reducing power with an IC₅₀ of 30.70 µM, followed by 3b $\sim$ 2.9‐fold (IC₅₀ = 40.00 µM) and 3a $\sim$ 1.5‐fold (IC₅₀ = 78.81 µM). These results suggest that these compounds have strong electron‐donating abilities, contributing to their antioxidant properties.

3.2.3. NO Activity

The NO scavenging assay evaluates a compound's ability to neutralize NO radicals, which are involved in various physiological processes and oxidative stress [43]. Effective NO scavenging indicates potential anti‐inflammatory and antioxidant benefits. For NO scavenging, compound 3a displayed an IC₅₀ of 44.52 µM, significantly outperforming gallic acid (IC₅₀ = 78.50 µM). Other compounds, such as 3j (IC₅₀ = 112.1 µM) and 3k (IC₅₀ = 115.5 µM), also showed moderate NO inhibition, indicating their role in reducing oxidative stress.

The SAR analysis of the synthesized DHPM derivatives revealed important insights into antioxidant activities (Figure 6). In antioxidant assays, varying trends were observed depending on the type of assay. For DPPH radical scavenging activity, the compound 3a without any substituent displayed the second‐highest IC₅₀ value in the series. The methoxy group at meta‐ and para‐positions (3d and 3e) demonstrated a good antioxidant activity as compared to the methyl group (3b and 3c) but did not show higher activity than the standard. In compounds bearing electron‐withdrawing halogen groups (─F, ─Cl, ─Br), the para‐substituted fluoro (3h) and chloro (3f) derivatives exhibited excellent DPPH radical scavenging activity. Noticeably, the disubstituted halogen (3i, ‐2,4‐DiF) exhibited excellent DPPH activity among all other compounds. The stronger EWG (3l, ‐p‐NO₂) at the para‐position diminished the activity. It was observed that EDG displayed good activity at the meta‐position as compared to EDG.

FIGURE 6.

Summarized SAR of the tested compounds for their antioxidant activity. DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl; FRAP, ferric reducing antioxidant power; NO, nitric oxide.

For the FRAP assay, compounds (3b and 3d) with EDG at the para‐position demonstrated the highest FRAP activity than their meta analogues (3c and 3e). The compound (3a) without any substituent also displayed good FRAP activity. The compounds with EWG displayed lower activity than EDG.

For the NO assay, only one compound exhibited excellent NO activity when compared to the substituted compounds with EDG and EWG.

4. Computational Studies

4.1. Molecular Docking

To understand how these compounds interact with the α‐glucosidase and human pancreatic α‐amylase (HPA), we performed the molecular docking of the most active compound (3f) with the active site of α‐glucosidase (previously built model) [9] and α‐amylase (PDB: 2QV4). The docking experiments were performed using Maestro 13.1. The standard drug acarbose was used as a reference for α‐glucosidase and α‐amylase enzyme inhibition.

4.1.1. α‐Glucosidase

Interpretation of the α‐glucosidase‐compound 3f complex revealed that compound 3f exhibits three hydrogen bonds and one hydrophobic interaction ($\pi -\pi$ interaction) as presented in Figure 7a. The nitrogen atom (N1) formed an H‐bond with Ser339 (d = 1.99 Å), another nitrogen atom (N3) displayed an H‐bond with Val294 (d = 2.11 Å), and the carbonyl group of the pyrimidine ring also showed H‐bonding with Trp340 (d = 2.28 Å). The aromatic ring demonstrated a hydrophobic interaction with Tyr286 (d = 4.91 Å) amino acid residue. The complex was characterized by a docking score of −10.62.

FIGURE 7.

(a) 2D (left side) and 3D (right side) ligand–receptor complex of α‐glucosidase–3f; (b) 2D (left side) and 3D (right side) ligand–receptor complex of α‐amylase–3f.

4.1.2. α‐Amylase

The molecular docking result for α‐amylase–3f complex showed that the compound interacted with the protein via two hydrogen bond interactions (Figure 7b)—one H‐bond between the nitrogen atom (N1) and Gly249 amino residue (d = 1.87 Å) and the second hydrogen bond (d = 2.17 Å) between the N2 atom and Asp212 amino residue. The complex displayed a docking score of −6.67.

4.2. Absorption, Distribution, Metabolism, and Excretion

ADME stands for absorption, distribution, metabolism, and excretion. It is crucial in drug development, as it determines how a drug is absorbed into the bloodstream, distributed throughout the body, metabolized into different forms, excreted, and its potential toxicity. Understanding ADME ensures effective and safe drug design, optimizing therapeutic efficacy while minimizing adverse effects.

4.2.1. Physicochemical Properties

The physicochemical properties of the most active compound 3f were computed and are summarized in Table 3. It can be observed that the compound showed compliance with all these parameters. The boiled egg and radar diagram (Figure 8) also further demonstrated that 3f is a safe candidate for drug development.

TABLE 3.

The physicochemical properties of compound 3f.

Compound	MF	Physicochemical properties
		MW	HBA	HBD	rotBOND	tPSA (Å2)
3f	C13H13ClN2O2	264.07	2	2	2	58.20
Standard range	—	<500 [9, 44]	0–20 [44]	0–6 [44]	0–11 [45]	0–140 [44]

Abbreviations: HBA, no. of hydrogen bond acceptor; HBD, no. of hydrogen bond donor; MF, molecular formula; MW, molecular weight; ^rotBOND, no. of rotatable bond; ^tPSA, total polar surface area.

FIGURE 8.

The boiled egg and radar diagram of compound 3f.

4.2.2. In Silico Rules and Bioavailability

Table 4 presents the bioavailability and adherence to various in silico rules, such as Lipinski, Muegge, Ghose, Veber, and Egan, for the most active compound 3f. The results indicated that compound 3f meets the criteria for in silico bioavailability according to all five rule sets, as denoted by “Yes” in the “In silico bioavailability” column.

TABLE 4.

In silico rules for bioavailability and bioavailability score for compound 3f.

Compound	In silico bioavailability					Bioavailability score
	Lipinski	Muegge	Ghose	Veber	Egan
3f	Yes	Yes	Yes	Yes	Yes	0.55

4.2.3. In Silico Toxicity Prediction

The in silico toxicity prediction was conducted for the most active compound (3f) using an online web portal (https://biosig.lab.uq.edu.au/pkcsm/), and the results are summarized in Table 5. The results indicated that compound 3f displays no toxicity for the tested parameters.

TABLE 5.

The toxicity prediction of compound 3f.

S. no.	Model	3f
1	AMES toxicity	No
2	hERG I inhibitor	No
3	hERG II inhibitor	No
4	Hepatotoxicity	No
5	Skin sensitization	No

4.3. Density Functional Theory (DFT) Analysis

Frontier molecular orbitals (FMOs) play a crucial role in analyzing the chemical stability and electronic and optical properties of molecules. FMOs consist of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). The HOMO, being the highest energy occupied orbital, exhibits electron‐donating or nucleophilic characteristics. Conversely, the LUMO, the lowest energy unoccupied orbital, is available for electron acceptance and demonstrates electrophilic behavior. FMO theory leverages these orbitals to analyze molecular structure and reactivity [46, 47].

The energy gap ($\mathrm{\Delta }$ E), calculated by using E _HOMO and E _LUMO, is crucial for assessing chemical reactivity [48]. A larger gap suggests greater stability, whereas a smaller energy gap indicates higher reactivity [49]. Table 6 indicates that for compound 3f, the calculated E _HOMO and E _LUMO energies are −6.67 and −1.93 eV, respectively, resulting in an energy gap of 4.73 eV. Figure 9a illustrates the HOMO and LUMO distributions and their energy levels using the B3LYP/6‐31G++dp basis set. Figure 9a shows HOMO and LUMO charge densities across the entire molecule, with green and red representing positive and negative phases. The observed energy gap and electron density distribution provide insights into the chemical stability of compound 3f.

TABLE 6.

The frontier molecular orbital of compound 3f using B3LYP/6‐31G++dp basis set.

Compound	E HOMO	E LUMO	$\mathrm{\Delta }$ E
3f	−6.67	−1.93	4.73

FIGURE 9.

(a) The FMOs of compound 3f. Part (b) illustrates the MEP map of compound 3f, where the predominant blue coloration across the molecule highlights its susceptibility to nucleophilic attack.

The E _HOMO and E _LUMO and $\mathrm{\Delta }$ E further utilized for the calculation of various global reactivity parameters [48], including ionization potential (I = −E _HOMO), electron affinity (A = −E _LUMO), electronegativity (χ = ((I + A)/2)) chemical potential (μ = −((I + A)/2)), hardness (η = ((I − A)/2)), softness (S = 1/η), and electrophilicity index (ω = ((E _HOMO + E _LUMO)²/4(E _LUMO − E _HOMO)) [4, 11, 35, 50]. The energy of the HOMO is directly related to the ionization potential (I), which helps in assessing a molecule's reactivity and its electron‐donating ability. A higher ionization potential indicates lower reactivity, whereas a lower ionization potential suggests higher reactivity [51]. Similarly, the LUMO energy correlates with electron affinity, where a high electron affinity signifies a molecule's strong capacity to accept electrons.

Electronegativity (χ) describes an atom's ability to attract electrons. Chemical potential (μ) is related to electronegativity and measures the tendency of electrons within a molecule to be drawn toward each other. Global hardness (η) quantifies a molecule's resistance to charge transfer, whereas global softness (S) measures its capacity for charge transfer. Ideally, a molecule should have lower global hardness and higher global softness to enhance charge transfer interactions with nearby biomolecules [52].

The electrophilicity index (ω) assesses a molecule's ability to attract electrons, indicating the energy gained when the molecule interacts with nearby electrons from its environment. The parameters for compound 3f were computed using Gaussian software; results are summarized in Table 7.

TABLE 7.

The global reactivity parameter of compound 3f.

Compound	Global reactivity parameters
	I	A	H	S	χ	μ	ω
3f	6.67	1.93	2.36	0.15	3.37	−3.37	2.40

4.4. Molecular Electrostatic Potential (MEP)

MEP represents the electrostatic field around a molecule, reflecting the net effect of its charge distribution, which includes contributions from both electrons and nuclei. This spatial distribution is closely related to the molecule's dipole moments, partial charges, electronegativity, and overall chemical reactivity. The MEP is visualized using a color scale to represent different levels of electrostatic potential: blue indicates a positive potential, green represents zero potential, and yellow to red shows increasingly negative potential.

On the MEP surface, green areas correspond to regions of zero electrostatic potential, red and yellow areas indicate negative electrostatic potentials associated with electrophilic reactivity, and blue areas signify positive electrostatic potentials linked to nucleophilic reactivity. The MEP of compound 3f is illustrated in Figure 9b and indicates that the nitrogen atoms N1 and N3 of compound 3f both show a nucleophilic character (shown in blue).

5. Conclusion

The present study reported the synthesis of a series of 3,4‐diydropyrimidinones in the presence of DES that played a dual role as solvent as well as catalyst. Their antidiabetic testing revealed them to be inhibitors of both α‐glucosidase and α‐amylase enzymes, with the representative compound 3f exhibiting a significant dual inhibition. Moreover, compounds 3i, 3d, and 3a demonstrated robust DPPH, FRAP, and NO scavenging capabilities, respectively, compared to gallic acid. The SAR analysis revealed that the para‐substitution of the chloro group on the phenyl ring is important in their α‐glucosidase and α‐amylase inhibition and could be explored further in future investigations. The molecular‐docking studies revealed that 3f predominantly formed hydrogen bonding with the enzymes. Finally, DFT studies explored the FMOs and global reactivity parameters of 3f. This study highlights the potential of DHPMs in developing new antidiabetic agents in the future.

6. Experimental Section

6.1. General Information

All chemicals and solvents were purchased from Sigma Aldrich and Merck and were used without any further purification. NMR analysis was recorded on a Bruker AVANCE III spectrometer (600 MHz for ¹H and 151 MHz for ¹³C). Chemical shifts (δ) were reported in parts per million (ppm). The chemical shifts for ¹H and ¹³C are referenced to DMSO‐d₆ at 2.50 and 39.52 ppm, respectively. Spin multiplicities are abbreviated as follows: singlet (s), doublet (d), doublet of doublet of doublets (ddd), doublet of doublets (dd), and triplet (t).

6.2. Preparation of DES

The DES was prepared as per the reported document [39]. ZnCl₂ (1.0 eq.) and urea (3.50 eq.) were heated at 100°C until the solid converted into a liquid phase and used further without any purification.

6.3. Synthesis of DHPMs (3a–3l)

A mixture of aldehyde 1a–1l (1.0 eq.) and acetylacetone 2 (1.0 eq.) was taken in a test tube containing the previously prepared DES (ZnCl₂/urea) and heated up to 110°C for 5–10 min. The progress of the reaction was monitored by using TLC. Water was then added to the reaction mixture, and the precipitates formed were filtered and recrystallized from ethanol to afford the target compounds.

6.4. α‐Glucosidase Inhibitory Activity

The inhibitory activity was studied against α‐glucosidase [53] with slight modifications to the reported procedure. Briefly, acarbose (25–400 µM) or a 100 µL aliquot of each compound was added to α‐glucosidase (1.0 U/mL) solution in 100 mM sodium phosphate buffer (pH 6.8). Further, the reaction was incubated for 15 min at 37°C, and then 50 µL of para‐nitrophenyl β‐d‐glucopyranoside solution (5 mM) was added. Next, the reaction was incubated for another 30 min at 37°C, and the absorbance of the resulting solution was measured at 405 nm. By using the following expression, the inhibitory activity of the compounds was calculated:

$$\%\mathrm{Inhibition}=\left(1-\frac{\mathrm{Absorbance}\mathrm{of}\mathrm{compound}}{\mathrm{Absorbance}\mathrm{of}\mathrm{control}}\right)\times 100$$

6.5. α‐Amylase Inhibitory Activity

α‐Amylase inhibitory activity was determined according to the reported method [54] with slight modifications. Briefly, 200 µL of the compounds or acarbose (75–600 µM) was incubated at 25°C for 10 min with 200 µL of a solution of porcine pancreatic amylase (0.5 mg/mL) prepared in 200 mM sodium phosphate buffer (pH 6.8). Then, 500 µL of 1% starch solution was added before further incubation at 25°C for 15 min. The reaction in the mixture was terminated with a 1 mL dinitrosalicylate reagent before boiling for 10 min. The cooled mixture was diluted with 5 mL of distilled water, and the absorbance was read at 540 nm. The inhibitory activity was calculated using the following formula:

$$\%\mathrm{Inhibition}=\left(1-\frac{\mathrm{absorbance}\mathrm{of}\mathrm{compound}}{\mathrm{absorbance}\mathrm{of}\mathrm{control}}\right)\times 100$$

6.6. DPPH Activity

The compounds were evaluated for their ability to scavenge stable DPPH radicals by adopting the reported method [55] with slight modifications. Varied concentrations of 2 mL (250–2000 mM) of the compounds or Trolox were added to 2 mL of 0.3 mM DPPH prepared in methanol. After thorough mixing, the solution was kept in the dark chamber at room temperature (25°C) for 30 min. Then, the absorbance was measured at 517 nm, and the DPPH radical mopping activity was calculated as follows:

$$\begin{array}{ccc}& & \mathrm{DPPH}\mathrm{scavenging}\mathrm{activity}\left(\%\right)\hfill \\ & & =\left(1-\frac{\mathrm{absorbance}\mathrm{of}\mathrm{compound}}{\mathrm{absorbance}\mathrm{of}\mathrm{control}}\right)\times 100\hfill \end{array}$$

6.7. Ferric Reducing Antioxidant Power

The ferric reducing antioxidant power of the chemical compounds was evaluated using the modified method [56]. Varying concentrations of 500 µL of the compounds or Trolox (250–2000 mM) were added to 250 µL of distilled water, 100 µL of 200 mM phosphate buffer (pH = 6.6), and 100 µL of 1% potassium ferricyanide [K₃Fe (CN)₆]. The mixture was incubated at 50°C for 20 min, followed by acidification with 100 µL trichloroacetic acid (10%). After centrifugation at 3500 rpm for 10 min, 200 µL of the supernatant was transferred into another test tube containing 200 µL of distilled water and 0.8 mL of FeCl₃ (0.1%). Finally, the absorbance was read at 700 nm, and the total reductive antioxidant power was calculated thus:

$$\mathrm{FRAP}\left(\%\right)=\frac{\mathrm{absorbance}\mathrm{of}\mathrm{compound}}{\mathrm{absorbance}\mathrm{of}2000\mathrm{mM}\mathrm{Trolox}}\times 100$$

6.8. NO Activity

The antioxidant capacity of the compounds to mop up NO radicals was determined with the modified protocol [57]. Briefly, 250 µL of sodium nitroprusside (10 mM) prepared in sodium phosphate buffer (pH 7.4) was added to 500 µL of the compounds (250–2000 mM) solution or distilled water (control). The resulting solution was incubated at 37°C for 2 h. Then, 250 µL of Griess reagent was added to the reaction mixture before the absorbance was measured at 540 nm. The % NO scavenging activity of the compounds was calculated with the formula:

$$\mathrm{NO}\mathrm{scavenging}\left(\%\right)=\left(1-\frac{\mathrm{absorbance}\mathrm{of}\mathrm{compound}}{\mathrm{absorbance}\mathrm{of}\mathrm{control}}\right)\times 100$$

6.9. Molecular Docking

The selected compound was saved as a ChemDraw file and imported into Schrödinger Maestro 13.3 software. 3D conformation of the ligand was then generated using the LigPrep module (Schrödinger Release 2021‐2: LigPrep [58, 59], S.; LLC: New York, NY, USA, 2021), assigning their ionization states with Epik (Schrödinger Release 2021‐2: Epik, S.; LLC: New York, NY, USA, 2021). HPA enzyme (PDB ID: 2QV4) was retrieved from the protein data bank and prepared using Protein Preparation Wizard (Schrödinger Release 2021‐2: Protein Preparation Wizard, Epik, S.; LLC: New York, NY, USA, 2021) [60] by adding hydrogens, filling in missing loops, and generating heteroatoms and tautomers with Epik at pH 7.2 ± 0.2. The prepared ligand was then docked into the allosteric site using the Induced Fit Docking protocol (Schrödinger Release 2021‐2: Induced Fit Docking protocol; Glide, Schrödinger, LLC, New York, NY, 2021; Prime, Schrödinger, LLC, New York, NY, 2021) [61] following default settings. The ligand grid box was set as the centroid of Sitemap amino acid residues with a size of ≤14 Å.

6.10. Absorption, Distribution, Metabolism, and Excretion

The physicochemical properties, in silico rules, and bioavailability were analyzed using the online SwissADME server (http://www.swissadme.ch/index.php).

6.11. DFT Calculations

Compound 9, saved as an .sdf file using ChemDraw, was imported into Gaussian 16w software [62]. Geometry optimization and frequency calculations were carried out at the B3LYP/6‐31G++dp theory level. FMOs and MEP maps were acquired at the same theory level and were visualized using GaussView 6.0 software package.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv70439‐sup‐0001‐SuppMat.pdf

Kumar G., Mishra S., Seboletswe P., et al. “DES‐Promoted Synthesis of 3,4‐Dihydropyrimidinones and Their Antidiabetic and Antioxidant Evaluation Supported With Computational Studies.” Chemistry & Biodiversity 22, no. 12 (2025): e00969. 10.1002/cbdv.202500969

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.