Synthesis of Some Novel Chalcone and 3,5‐Disubstituted Pyrazoline Derivatives: Evaluation of Their α‐Amylase and α‐Glucosidase Inhibitory Activities In Vitro and In Silico

ABSTRACT

This study reports the synthesis of some novel chalcone (C1–C3) and 3,5‐disubstituted pyrazoline derivatives (P1–P3), structurally based on aminophenyl and trifluoromethylphenyl groups. Their potential as antidiabetic agents was evaluated through in vitro inhibition of α‐amylase and α‐glucosidase enzymes and supported by molecular docking studies. Among all the compounds, P2 showed potent dual inhibition, with IC₅₀ values of 9.35 and 2.10 µM against α‐amylase and α‐glucosidase, respectively, comparable to or better than the standard inhibitor acarbose. Kinetic studies revealed that P2 acts via a non‐competitive mechanism for both enzymes. Docking analysis was performed for all of the molecules. These findings suggest that especially P2 has significant potential as a lead compound for further antidiabetic drug development.

A novel pyrazoline derivative (P2) was synthesized and demonstrated potent α‐glucosidase inhibition (IC₅₀ = 2.10 µM), significantly surpassing acarbose and highlighting its promise as an effective oral antidiabetic agent.

1. Introduction

Type 2 diabetes mellitus (T2DM) represents a growing global health burden with a rapidly increasing prevalence in both industrialized and developing countries. According to WHO and IDF estimates, approximately 537 million individuals were affected in 2021, a figure projected to rise to 783 million by 2045 [1, 2]. This increase is especially pronounced in low‐ and middle‐income regions, driven by urbanization, sedentary behavior, and dietary transitions [3, 4].

In T2DM, postprandial hyperglycemia plays a key role in disease progression. Dietary carbohydrates present in grains, legumes, and starchy foods are enzymatically hydrolyzed into glucose, contributing to elevated blood sugar levels. Two enzymes are important in this process. α‐Amylase (EC 3.2.1.1) catalyzes the cleavage of internal α‐1,4 glycosidic bonds in starch, forming oligosaccharides. α‐Glucosidase (EC 3.2.1.3), located in the small intestine, further breaks down oligosaccharides into glucose and facilitates absorption [5, 6]. As these enzymes directly regulate glucose release from complex carbohydrates, they are validated targets in T2DM therapy. However, existing α‐amylase and α‐glucosidase inhibitors (e.g., acarbose and miglitol) often cause gastrointestinal discomfort such as bloating, abdominal pain and diarrhea due to incomplete carbohydrate digestion and subsequent microbial fermentation in the colon [7]. Therefore, it is vital to find new inhibitor molecules that will reduce or eliminate these disturbing side effects.

Chalcones are open‐chain flavonoid compounds (C6–C3–C6) that have two aromatic rings joined by a three‐carbon α,β‐unsaturated carbonyl system (Figure 1).

Figure 1.

General chalcone structure.

Both natural and synthetic chalcones have attracted considerable interest due to their simple structures, ease of synthesis, wide range of derivatives, and diverse biological activities. Researches have demonstrated that chalcone compounds exhibit various pharmacological properties, including antioxidant, anticarcinogenic, antibacterial, anti‐inflammatory, antifungal, anticancer, antihistaminic, analgesic, antituberculosis, and antidiabetic effects [8, 9, 10, 11, 12]. According to published reports, developed drugs and drug candidate compounds contain a significant number of halogenated structures. In particular, halogen‐containing chalcone derivative compounds inhibit the progression of obesity and hyperglycemia by inhibiting protein kinase [13, 14, 15, 16, 17, 18, 19]. The structures of some bioactive halosubstituted chalcones available in the literature are given in Figure 2.

Figure 2.

Structures of some bioactive halosubstituted chalcones.

Beyond their biological activity, chalcones are also important intermediates in the synthesis of new compounds. Their α,β‐unsaturated carbonyl system and substituted aromatic rings render them chemically reactive and suitable for the synthesis of five‐, six‐, and seven‐membered heterocycles. Heterocyclic compounds represent a crucial class of organic molecules, playing a central role in regulating numerous biological processes due to their structural diversity and functional importance in biochemical systems. Among these, 3,5‐disubstituted‐2‐pyrazolines are synthesized via acid‐catalyzed Michael addition of hydrazine hydrate to chalcones. Pyrazolines are five‐membered heterocyclic compounds composed of three carbon atoms, two adjacent nitrogen atoms, and a double bond (Figure 3) [20, 21, 22].

Figure 3.

3,5‐Disubstituted pyrazoline ring system.

Pyrazolines can be considered alkaloids due to their pharmacological activity. Extensive research in medicinal chemistry has demonstrated that pyrazoline derivatives possess a wide range of therapeutic applications. These include activities such as pesticidal, fungicidal, antibacterial, antidiabetic, antimalarial, antitumor, antifungal, antiamoebic, anticonvulsant, antidepressant, anti‐Alzheimer's, antituberculosis, anticancer, anti‐EGFR, anti‐Parkinsonian, monoamine oxidase inhibition, and insecticidal properties [23, 24, 25, 26]. The structures of some bioactive halosubstituted chalcones available in the literature are given in Figure 4.

Figure 4.

Structures of some bioactive halosubstituted 3,5‐disubstituted‐2‐pyrazoline.

Given the high bioactivity of both chalcone and pyrazoline derivatives and the limited number of studies on their antidiabetic effects, particularly in terms of enzyme inhibition, this study aimed to investigate the antidiabetic potential of some newly synthesized chalcone and pyrazoline derivatives substituted with aminophenyl and 2‐/3‐/4‐trifluoromethylphenyl groups. Specifically, the effect of three chalcones (C1, C2, C3) and three pyrazolines (P1, P2, P3) on the activity of α‐amylase and α‐glucosidase were evaluated in vitro and in silico. While C2 and C3 have been previously reported in the literature [27], their inhibitory activity against these enzymes has not been explored. Compounds C1, P1, P2, and P3 were synthesized for the first time as part of this study.

2. Results and Discussion

2.1. Chemistry

In this study, three chalcone compounds and three pyrazoline derivatives originating from these chalcones were synthesized. In the design of both chalcone and pyrazoline compounds, substituents with high biological activity were preferred.

The trifluoromethyl (–CF₃) group is widely used in medicinal chemistry because of its significant effect on enzyme inhibition. Its strong electron‐withdrawing nature reduces the electron density of adjacent functional groups, enhancing binding affinity within enzyme active sites by stabilizing interactions such as hydrogen bonding and polar contacts. The –CF₃ group also increases molecular hydrophobicity, promoting more effective binding in hydrophobic pockets and improving overall membrane permeability. The robust carbon–fluorine bond confers metabolic stability, making –CF₃‐containing compounds more resistant to degradation and prolonging their biological activity. Additionally, the steric bulk of the –CF₃ group can modulate or restrict access to enzyme binding sites, contributing to selective inhibition. Collectively, these electronic, steric, and lipophilic properties make –CF₃ substitution a valuable strategy in drug design to enhance the potency and selectivity of enzyme inhibitors. Also, many FDA‐approved drugs contain –CF₃ groups, underlining their importance in modern medicinal chemistry and rational drug design [28, 29, 30, 31, 32].

The amino (–NH₂) group, the other substituent of the synthesized molecules, is a key functional moiety in medicinal chemistry due to its significant impact on enzyme inhibition and biological activity. Its ability to act as both a hydrogen bond donor and acceptor enables –NH₂‐substituted compounds to form strong interactions with amino acid residues within enzyme active sites, thereby enhancing binding affinity and inhibitory potency [33]. The electron‐donating properties of the –NH₂ group can also increase the nucleophilicity of neighboring atoms or stabilize reactive intermediates, further modulating enzyme activity. Additionally, the hydrophilic nature of the –NH₂ group improves aqueous solubility and bioavailability, indirectly supporting enhanced pharmacological effects. Amino groups frequently interact with acidic residues such as aspartate and glutamate in enzyme active sites and can serve as sites for salt formation or prodrug strategies to optimize pharmacokinetics. Notable examples include aminoglycoside antibiotics, where multiple –NH₂ groups bind bacterial ribosomal RNA to inhibit protein synthesis, and amino‐substituted kinase inhibitors, which form key hydrogen bonds in the kinase hinge region, improving potency and selectivity [33, 34]. Collectively, these properties make the –NH₂ group a widely employed functional group in rational drug design and enzyme‐targeted therapies.

For all these reasons, these two functional groups were selected as substituents in the design of the compounds to be synthesized.

As part of this study, three 2”/3”/4”‐trifluoromethyl‐substituted 2'‐aminochalcones (C1–C3) were synthesized via Claisen–Schmidt condensation, and their enzyme inhibition properties were subsequently investigated [23, 35, 36]. In the first step of the reaction, 2'‐aminoacetophenone was deprotonated at the α‐carbon in a basic medium, generating an enolate anion. This enolate then underwent a nucleophilic attack on the carbonyl carbon of 2‐, 3‐, or 4‐(trifluoromethyl)benzaldehyde, which had been added separately to the reaction mixture. This step led to the formation of an alkoxide intermediate. The alkoxide ion subsequently abstracted a proton from water in the reaction medium, yielding a more stable β‐hydroxyketone intermediate. Finally, dehydration of this intermediate resulted in the formation of the α,β‐unsaturated chalcone derivatives C1, C2, and C3.

A literature review of the chalcone compounds obtained in the first stage of the synthesis studies revealed that compound C1 is novel, while compounds C2 and C3 have been previously reported [27]. The structures of these chalcones were elucidated using NMR (¹H, ¹³C (APT)), LC‐QTOF‐MS, and FT‐IR spectroscopic techniques. Additionally, the NMR spectral data were analyzed and supported using the ACD NMR software [37].

Analysis of the ¹H‐NMR spectra of compounds C1, C2, and C3 revealed the characteristic AB spin system of the α,β‐unsaturated carbonyl protons, typically observed in chalcone structures. These appeared in the ranges of 7.54–7.75 ppm (H‐2) and 7.74–8.07 ppm (H‐3), with a coupling constant of J = 16.0 Hz, supporting the formation of the chalcone framework. In the ¹³C‐NMR spectra, the carbonyl carbon (C‐1) resonated at 191.06–191.08 ppm, while the C‐2 and C‐3 carbons appeared in the ranges of 124.75–127.41 ppm and 138.22–140.96 ppm, respectively, further confirming the proposed structures. Upon evaluation of the HRMS spectra of the compounds, the [M + 1]⁺ ions were observed as the base peaks at 292.09498 m/z, 292.09582 m/z, and 292.09533 m/z for compounds C1, C2, and C3, respectively.

The FT‐IR spectra provided additional evidence for the functional groups present in the synthesized chalcones. Asymmetric –NH₂ stretching vibrations were observed in the range of 3507–3384 cm⁻¹, and symmetric –NH₂ stretching vibrations appeared in the 3339–3279 cm^–1 range. C═O stretching vibrations were identified between 1653 and 1648 cm^–1, while C═C stretching vibrations were detected in the 1591–1576 cm^–1 range. The presence of C–F stretching vibrations was confirmed by peaks in the range of 747–745 cm^–1. These spectral features confirm the successful synthesis and structural identity of the chalcone compounds.

Chalcones serve as highly effective intermediates in the synthesis of five‐membered heterocyclic compounds. The α,β‐unsaturated carbonyl group present in their structure plays a crucial role in facilitating cyclization reactions. Accordingly, in the second phase of the synthesis studies, 3,5‐disubstituted‐2‐pyrazoline derivatives (P1, P2, and P3) were synthesized via the Michael addition of hydrazine hydrate to the chalcone compounds C1–C3 under acid‐catalyzed conditions.

The literature search revealed that the three synthesized pyrazoline derivatives are new. The structures of these compounds were elucidated using ¹H‐NMR, ¹³C‐NMR (APT), LC‐QTOF‐MS, and FT‐IR spectroscopic data.

Characteristic splitting patterns were observed in the ¹H‐NMR spectra of compounds P1–P3: two doublet of doublets (dd) in the range of δ 3.09–3.29 ppm (C₄‐H_A) and δ 3.61–3.94 ppm (C₄‐H_B), as well as a triplet (t) in the δ 4.89–5.58 ppm range. These signals correspond to the geminal –CH₂– and methine –CH– protons of the pyrazoline ring, respectively. This pattern indicates the presence of an ABX spin system, resulting from geminal‐vicinal coupling between the diastereotopic protons (H_A and H_B) at C‐4 and the methine proton (H_X) at C‐5 [24, 27].

Further evidence of ring formation is provided by the appearance of a broad singlet corresponding to the –NH proton (H‐1) at δ 6.38, 6.33, and 6.06 ppm for compounds P1, P2, and P3, respectively. In parallel, the disappearance of the H‐2 and H‐3 resonance signals specific to the α,β‐unsaturated system in the starting shells supports the successful cyclization to pyrazoline derivatives.

¹³C‐NMR (APT) spectra further confirm the structural assignment. The quaternary carbon signals at 153.07, 153.68, and 155.67 ppm for P1, P2, and P3, respectively, are attributed to the newly formed C‐3 carbon in the pyrazoline ring. Resonance signals for C‐4 (–CH₂) in the range of 43.09–43.63 ppm and for C‐5 (–CH) in the range of 57.91–62.47 ppm are additional indicators of the pyrazoline scaffold. In addition, the disappearance of the carbonyl carbon signals (previously observed at ~191.06–191.08 ppm in the chalcones) is further confirmation of the successful ring closure.

Upon evaluation of the HRMS spectra of the compounds, the [M + 1]⁺ ions were observed as the base peaks at 306.12190 m/z, 306.12196 m/z, and 306.12226 m/z for compounds P1, P2, and P3, respectively.

The FT‐IR spectra of P1–P3 show sharp double bands corresponding to the asymmetric –NH₂ stretching at 3461, 3460, and 3463 cm⁻¹, and the symmetric –NH₂ stretching at 3324, 3322, and 3318 cm⁻¹, respectively. Crucial vibrations confirming the pyrazoline core, namely the N–H, C═N, and C–N stretching bands, were observed in the ranges of 3324–3318 cm⁻¹, 1611–1608 cm⁻¹, and 1209–1208 cm⁻¹, respectively, corresponding to the expected values.

Spectra of the compounds are given in the Supporting Information.

2.2. Characterization of the Compounds by HPLC‐DAD Evaluation

The new compounds were characterized with respect to purity, hydrophobicity, UV‐Vis spectra, and λ_max values by using HPLC‐DAD and molecular weight data. The purity of the compound samples analyzed in all experiments was tested with area normalization method in HPLC‐DAD using the chromatograms at 280 nm and also by using the peak purity check function in Chemstation software, which compares multiple spectra of the peak (Supporting Information S1: Figures S25–S30). The results show a high level of purity of above 99.5% (Table 1). The impurities with a percentage of less than 0.5% may be a result of carry‐over or other sources in the HPLC system, as NMR data and other characterization tools do not show any impurity.

Table 1.

Purity levels of compounds based on HPLC‐DAD chromatograms at 280 nm.

Compound	Purity (%)		Compound	Purity (%)
C1	99.54		P1	99.84
C2	99.95		P2	99.69
C3	99.85		P3	99.52

λ_max values of the compounds were obtained from HPLC‐DAD data of acetonitrile (ACN) solutions of the samples of 5.0 × 10^–4 M concentration. The mobile phase composition at the time of peaks and λ_max values are reported below:

UV–Vis λ_max values of compounds C1–C3; mobile phase composition 77% ACN and 0.1% HAc: λ_max = 294, 402 nm; additional strong absorption in the 200–250 nm region (Supporting Information S1: Figures S25–S27). UV–Vis λ_max values of compounds P1–P3; mobile phase composition 68% ACN and 0.1% HAc (acetic acid): λ_max = 283, 350 nm; additional strong absorption in the 200–250 nm region (Supporting Information S1: Figures S28–S30).

The hydrophobicity evaluation was done by utilizing log k' values, the values that are widely used for hydrophobicity comparisons. log k' values are reported in Table 2. These values are in inverse relation with polarity of the compounds, larger log k' values for later eluting compounds with lower polarity in reverse phase HPLC. The data showed the C1–C3 series are less polar eluting later at around 19.5 min with larger log k' values as compared with P1–P3 series with peaks around 17.1 min and smaller log k' values.

Table 2.

Hydrophobicity index (log k’) of the compounds.

Compound	RT (min)	k’	log k’	Compound	RT (min)	k’	log k’
C1	19.190	3.569	0.553	P1	17,177	3.090	0.490
C2	19.530	3.650	0.562	P2	17,007	3.049	0.484
C3	19.749	3.702	0.568	P3	17.113	3.075	0.488

Note: Chromatograms of compounds are given in the Supporting Information.

2.3. α‐Amylase Inhibitory Activity of the Chalcones and 3,5‐Disubstituted Pyrazolines In Vitro

As a result of the inhibition studies, it was determined that all molecules except for the C2 molecule caused α‐amylase inhibition. The C2 molecule did not exhibit any inhibitory effect up to a concentration of 130 µM in the reaction medium. Due to the precipitation of the molecule in the reaction medium, it was not possible to exceed this concentration. Based on the %Relative Activity graphs obtained against the concentrations of the tested molecules, the IC₅₀ values were calculated. The results were given in Table 3.

Table 3.

IC₅₀ values for the inhibition of α‐amylase by the chalcones and 3,5‐disubstituted pyrazolines.

Molecule	IC50 (µM)a
C1	93.11 ± 6.18D
C2	ND
C3	75.22 ± 8.34C
P1	41.08 ± 2.49B
P2	9.35 ± 1.16A
P3	45.05 ± 3.64B
Acarbose	1.65 ± 0.36A

^aIC₅₀ values (mean ± standard deviation) labeled with different superscript letters are significantly different at p < 0.05. “ND” denotes values that could not be determined.

It was observed that the P2 molecule exhibited statistically the same IC₅₀ value as the standard inhibitor acarbose. In contrast, the other molecules displayed significantly different and higher IC₅₀ values compared with acarbose. To determine the inhibition mechanism of P2, α‐amylase activities were measured at various starch concentrations both in the absence and presence of two different concentrations of the inhibitor (4.35 and 8.70 µM). The enzyme activity values obtained were used to construct a Lineweaver–Burk (LB) plot (Figure 5).

Figure 5.

LB plot of α‐amylase in the absence and at two concentrations of P2.

As seen in Figure 5, P2 molecule inhibits α‐amylase via a non‐competitive mechanism. The inhibitor binding constant (K _i) of the molecule was also calculated as 5.34 µM.

2.4. α‐Glucosidase Inhibitory Activity of the Chalcones and 3,5‐Disubstituted Pyrazolines In Vitro

As a result of the α‐glucosidase inhibition studies, approximately 7%, 3%, and 22% inhibition were observed for the C1, C2, and C3 molecules, respectively, when their concentrations in the reaction medium were increased up to 50, 80, and 85 µM. At higher concentrations, precipitation occurred in the reaction medium, which prevented further testing. Therefore, IC₅₀ values could not be determined for these molecules.

On the other hand, P1, P2, and P3 molecules significantly inhibited α‐glucosidase activity. From the %Relative Activity graphs obtained against the concentrations of the tested molecules, the IC₅₀ values were calculated. The results were given in Table 4.

Table 4.

IC₅₀ values for the inhibition of α‐glucosidase by the chalcones and 3,5‐disubstituted pyrazolines.

Molecule	IC50 (µM)a
C1	ND
C2	ND
C3	ND
P1	9.83 ± 0.44A
P2	2.10 ± 0.28A
P3	7.16 ± 0.24A
Acarbose	545.39 ± 9.86B

^aIC₅₀ values (mean ± standard deviation) labeled with different superscript letters are significantly different at p < 0.05. “ND” denotes values that could not be determined.

When the data in Table 4 were statistically analyzed, it was found that all of the 3,5‐disubstituted pyrazoline molecules exhibited lower IC₅₀ values than the standard inhibitor acarbose. However, no statistically significant differences were observed among the IC₅₀ values of the pyrazoline molecules themselves. As a result, all of the molecules demonstrated stronger α‐glucosidase inhibition compared with acarbose.

To determine the inhibition mechanism of P2, with the lowest IC₅₀, α‐glucosidase activities were measured at various p‐NPG concentrations both in the absence and presence of two different concentrations of the inhibitor (2.90 and 5.80 µM). The enzyme activity values obtained were used to construct a Lineweaver–Burk (LB) plot (Figure 6).

Figure 6.

LB plot of α‐glucosidase in the absence and at two concentrations of P2.

As seen in Figure 6, P2 molecule inhibits α‐glucosidase via a non‐competitive mechanism. The inhibitor binding constant (K _i) of the molecule was calculated as 1.14 µM.

Although the number of studies investigating the antidiabetic potential of chalcone and pyrazoline derivatives is limited, several findings indicate promising bioactivity. In a study, a comprehensive library of 1,3,5‐triaryl‐2‐pyrazolines was designed and synthesized. These newly developed compounds were screened for their α‐glucosidase inhibitory activity, and all demonstrated enzyme inhibition to varying degrees. Among them, four compounds exhibited stronger inhibitory effects than acarbose (IC₅₀ = 375.82  ±  1.76 μM) [38]. In another study, a series of pyrazolinyl‐acyl thiourea derivatives was evaluated for their inhibitory effects on α‐amylase and α‐glucosidase. Most of the compounds showed moderate α‐amylase inhibition, while a few exhibited relatively stronger activities with IC₅₀ values around 90–100 μM. The remaining derivatives displayed lower potency, with IC₅₀ values in the range of 103–131 μM, compared with acarbose (IC₅₀ = 10.2 μM). In contrast, all compounds demonstrated moderate to strong α‐glucosidase inhibition. The most active members of the series showed IC₅₀ values of approximately 68–70 μM, whereas acarbose exhibited an IC₅₀ of 9.5 μM [26]. Uğraş et al. (2024) also investigated a newly synthesized series of pyrazoline derivatives for their in vitro α‐amylase and α‐glucosidase inhibitory activities. The majority of the synthesized pyrazoline derivatives demonstrated stronger inhibition of both α‐amylase and α‐glucosidase enzymes than the reference drug acarbose [39].

In the current study, the P2 molecule showed an IC₅₀ value for α‐amylase that was statistically comparable to that of acarbose, indicating similar inhibitory efficiency. Besides this, in the case of α‐glucosidase, compounds P1, P2, and P3 were found to be 55–250 times more potent than acarbose. These results strongly indicate that P2 possesses highly effective in vitro antidiabetic activity, particularly as an α‐glucosidase inhibitor.

In the synthesized compounds, the amino group is kept constant, while the position of the trifluoromethyl substituent varies. The differences observed in the activity of the pyrazoline derivatives are considered to arise predominantly from the positional effects of the –CF₃ group. The obtained results indicate that compound P2, which contains the –CF₃ substituent at the meta position, exhibits the strongest α‐amylase inhibitory activity. The meta position enhances the potential of the fluorine atoms within the substituent to form hydrogen bonds with the enzyme, thereby strengthening the interaction. In contrast, intramolecular hydrogen bonding is favored at the ortho position (P1), while intermolecular hydrogen bonding is more likely at the para position (P3). Both situations are predicted to reduce the likelihood of forming enzyme–ligand hydrogen bonds, consequently decreasing the inhibition potential.

Conversion of chalcones (C1–C3) into pyrazolines (P1–P3) results in increased α‐glucosidase and α‐amylase inhibitory activities. Unlike chalcones, the pyrazoline ring system contains nitrogen heteroatoms, which enhances their ability to form hydrogen bonds with enzymes. The presence of nitrogen atoms and their associated hydrogen atoms in pyrazolines increases the interaction potential, which is considered to contribute significantly to the observed enzyme inhibition.

2.5. Molecular Docking

The binding energies indicating the binding affinities of the chalcones and 3,5‐disubstituted pyrazolines to the active sites of the α‐amylase and α‐glucosidase enzymes are presented in Table 5.

Table 5.

Binding energies (ΔG) of the chalcones and 3,5‐disubstituted pyrazolines.

Molecule	α‐Amylase (ΔG, kcal/mol)	α‐Glucosidase (ΔG, kcal/mol)
C1	–9.0	–7.0
C2	–8.7	–7.6
C3	–8.5	–7.5
P1	–8.8	–7.6
P2	–8.2	–7.1
P3	–8.9	–8.0

According to the results, all studied molecules were found to bind to the active sites of the enzymes. The calculated theoretical binding energies ranged from −8.2 to −9.0 kcal/mol for α‐amylase and from −7.0 to −8.0 kcal/mol for α‐glucosidase. Among these, compound C1 exhibited the highest theoretical binding affinity for α‐amylase. However, under in vitro conditions, the lowest IC₅₀ value for α‐amylase inhibition was observed with compound P2. Moreover, P3 showed the highest theoretical binding affinity for α‐glucosidase. Statistically, all pyrazoline derivatives inhibited α‐glucosidase to a similar extent, but compound P2 again demonstrated the lowest IC₅₀ value. These findings indicate a discrepancy between the theoretical and experimental results, especially in the case of α‐amylase. There may be several reasons for this. First, due to solubility problems with the chalcones, it was not possible to determine their IC₅₀ values experimentally. Additionally, molecular docking studies are designed to model competitive inhibition by simulating binding to the active site. However, in vitro studies have shown that the P2 molecule inhibited both α‐amylase and α‐glucosidase through non‐competitive mechanism. This difference in inhibition type may contribute to the inconsistency observed between the computational and experimental findings.

Interaction of the P2 with the active site of α‐amylase and interaction types and interaction distances are given in Figure 7 and Table 6, respectively. The binding of the P2 molecule to the active site of α‐amylase indicates a strong and specific molecular affinity (ΔG = –8.2 kcal/mol). Key interactions in this binding include hydrogen bonds primarily with GLU233, ALA198, SER199, LYS200, and ILE235. Also π‐Interactions are involved in HIS201, ILE235, ALA198, LYS200 with π‐cation, π‐sigma, π‐π T‐shaped, and π‐alkyl types. Particularly, residues GLU233, HIS201, and LYS200 appear critical in mediating stable ligand binding.

Figure 7.

2D and 3D representation of the interaction of the P2 with the active site of α‐amylase.

Table 6.

Interactions of P2 in the active site of α‐amylase.

ΔG (kcal/mol)	Residue	Interaction distance (Å)	Interaction type
–8.2	A:ALA198:HN ‐ A:GLU233:OE1	1.77958	conventional hydrogen bond
	A:SER199:HN ‐ A:GLU233:O	2.15607	conventional hydrogen bond
	A:LYS200:HN ‐ A:GLU233:O	2.62535	conventional hydrogen bond
	A:LYS200:HZ1 ‐ A:ILE235:O	1.81727	conventional hydrogen bond
	A:HIS201:HN ‐ A:ALA198:O	2.17495	conventional hydrogen bond
	:UNK1:H ‐ A:GLU233:O	2.88991	conventional hydrogen bond
	:UNK1:H ‐ A:GLU233:OE1	2.26213	conventional hydrogen bond
	A:HIS201:HE2 ‐:UNK1	2.49096	pi‐cation; pi‐donor hydrogen bond
	A:ILE235:CD1 ‐:UNK1	3.51674	pi‐sigma
	A:HIS201 ‐:UNK1	4.45245	pi‐pi T‐shaped
	A:HIS201 ‐ A:ALA198	5.0245	pi‐alkyl
	:UNK1 ‐ A:LYS200	4.93361	pi‐alkyl

Interaction of the P2 with the active site of α‐glucosidase and interaction types and interaction distances are given in Figure 8 and Table 7, respectively. The binding mode of P2 in α‐glucosidase is particularly dominated by hydrophobic (π‐alkyl and alkyl) interactions. A single conventional hydrogen bond is formed between the ligand (:UNK1:H) and A:THR235:O with a distance of 2.31 Å. Residues THR235, PHE90, VAL236, and PRO238 appear to be key players in the stabilization.

Figure 8.

2D and 3D representation of the interaction of the P2 with the active site of α‐glucosidase.

Table 7.

Interactions of P2 in the active site of α‐glucosidase.

ΔG (kcal/mol)	Residue	Interaction distance (Å)	Interaction type
−7.1	:UNK1:H ‐ A:THR235:O	2.30588	conventional hydrogen bond
	A:SER325:CB ‐ A:SER88:OG	3.5875	carbon hydrogen bond
	A:ALA327 ‐:UNK1:CL	4.2187	alkyl
	:UNK1:CL ‐ A:VAL236	3.73342	alkyl
	A:PHE90 ‐ A:VAL236	5.17761	pi‐alkyl
	A:PHE90 ‐ A:ALA327	5.05417	pi‐alkyl
	A:PHE90 ‐:UNK1:CL	4.7875	pi‐alkyl
	:UNK1 ‐ A:PRO131	5.19531	pi‐alkyl
	:UNK1 ‐ A:PRO238	5.08008	pi‐alkyl
	:UNK1 ‐ A:VAL236	4.77541	pi‐alkyl

3. Conclusion

This study reports the successful synthesis of novel chalcone (C1–C3) and 3,5‐disubstituted pyrazoline derivatives (P1–P3) bearing aminophenyl and trifluoromethylphenyl groups. Their in vitro and in silico evaluation revealed strong α‐amylase and α‐glucosidase inhibitory activities, particularly for compound P2, which showed IC₅₀ values comparable to or better than the standard drug acarbose. Kinetic studies demonstrated a non‐competitive inhibition mechanism. These findings suggest that P2 is a promising lead compound for the development of new antidiabetic agents with improved efficacy and pharmacokinetics.

4. Materials and Methods

4.1. Chemistry

4.1.1. Materials and Instrumentation

All reagents and solvents used in the synthesis were of analytical grade. 2'‐Aminoacetophenone, 2‐, 3‐, and 4‐(trifluoromethyl)benzaldehyde, and hydrazine monohydrate were obtained from Merck and Sigma‐Aldrich. Sodium hydroxide and ethanol were purchased from Acros Organics. Glacial acetic acid, hexane, diethyl ether, chloroform, and ethyl acetate were supplied by Isolab. The structures of the synthesized compounds were characterized using a Bruker 400 MHz NMR spectrometer for ¹H‐ and ¹³C (APT)‐ NMR analyses, and a Perkin‐Elmer 1600 FT‐IR spectrophotometer (4000–400 cm⁻¹ range) for FT‐IR spectroscopy. HRMS spectra were recorded on an Agilent 6530 Accurate mass spectrometer. The melting points of the synthesized compounds were measured with a Stuart SMP10 melting point apparatus, without correction. Compound purification was performed using column chromatography (CC) and monitored through thin‐layer chromatography (TLC) methods.

The enzymes, p‐nitrophenyl‐α‐d‐glucopyranoside (p‐NPG), acarbose, and soluble starch were obtained from Sigma‐Aldrich. Absorbance measurements were performed using a UV‐visible spectrophotometer (Lambda 25, PerkinElmer).

4.1.2. Synthesis of Chalcones (C1, C2, C3)

Claisen–Schmidt condensation was used for the synthesis of chalcone derivatives (C1, C2, C3) as described in the literature. Sodium hydroxide (5 g, 12.5 mmol) was dissolved in a 30 mL water–ethanol mixture (1:2, v/v) in a beaker and stirred at room temperature using a magnetic stirrer. Separately, 2'‐aminoacetophenone (1.35 g, 10 mmol) was dissolved in 10 mL of ethanol and added to the NaOH solution. The mixture was stirred until the substrate was completely dissolved. Subsequently, 2‐, 3‐, or 4‐(trifluoromethyl)benzaldehyde (1.35 mL, 1.38 mL, or 1.39 mL; 10 mmol, respectively) dissolved in 10 mL of ethanol was added dropwise to the reaction mixture over a period of 30 min. The reaction was stirred at room temperature for 12 h. The progress of the reaction was monitored by TLC. After completion, water was added to the reaction mixture to facilitate precipitation. The resulting solid was collected by vacuum filtration using a crucible, washed with cold water, and dried using a lyophilizer to yield yellow crystalline chalcone derivatives (C1–C3). The purity of the products was assessed by TLC, and structural characterization of the compounds was performed using spectroscopic techniques: ¹H‐NMR, ¹³C‐NMR (APT), and FT‐IR [9, 10, 35]. The synthesis equation and the structures of chalcones are given in Schemes 1 and 2, respectively.

Scheme 1.

Synthesis of target compounds. Reaction conditions: (a) NaOH, H₂O/EtOH, RT.

Scheme 2.

Structures of chalcones.

(2E)‐1‐(2‐Aminophenyl)‐3‐[2‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (C1): Yield (%): 85; m.p. (°C): 104–106; Rf: 0.80 (hexane/diethyl ether: 1:2). FT‐IR (cm^–1): 3507, 3339, 1652, 1571, 747.¹H‐NMR (400 MHz, CDCl₃, ppm): δ = 7.54 (AB, J = 16.0 Hz, 1H, H‐2); δ = 8.07 (AB, J = 16.0 Hz, 1H, H‐3); δ = 6.70 (d, J = 8.0 Hz, 1H, H‐3′); δ = 7.30 (t, J = 8.0 Hz, 1H, H‐4′); δ = 6.69 (t, J = 8.0 Hz, 1H, H‐5′); δ = 7.78 (t, J = 8.0 Hz, 1H, H‐6′); δ = 7.83 (d, J = 8.0 Hz, 1H, H‐3″); δ = 7.59 (t, J = 8.0 Hz, 1H, H‐4″); δ = 7.48 (t, J = 8.0 Hz, 1H, H‐5″); δ = 7.81 (d, J = 8.0 Hz, 1H, H‐6″); δ = 6.39 (bs, 2H, ‐NH₂).¹³C‐NMR (100 MHz, CDCl₃, ppm): 191.08 (C‐1), 127.41 (C‐2), 138.22 (C‐3), 118.53 (C‐1'), 151.27 (C‐2′), 115.88 (C‐3′), 134.67 (C‐4′), 117.39 (C‐5′) 131.18 (C‐6′), 134.47 (C‐1″), 129.51, 129.19, 128.89, 128.57 (q, J = 32 Hz, C‐2″), 126.31, 126.25, 126.19, 126.13 (q, J = 6 Hz, C‐3″), 129.33 (C‐4″), 132.08 (C‐5″), 127.94 (C‐6″), 128.16, 125.43, 122.70, 119.97 (q, J = 273 Hz, CF₃). LC‐QTOF‐MS m/z calculated for C₁₆H₁₂F₃NO [M + H]⁺: 292.09492, Found:292.09498.

(2E)‐1‐(2‐Aminophenyl)‐3‐[3‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (C2): Yield (%): 80; m.p. (°C): 129–131, Rf: 0.75 (hexane/diethyl ether: 1:2). FT‐IR (cm^–1): 3384, 3389, 1648, 15780, 745. ¹H‐NMR (400 MHz, CDCl₃, ppm): δ = 7.67 (AB, J = 16.0 Hz, 1H, H‐2); δ = 7.74 (AB, J = 16.0 Hz, 1H, H‐3); δ = 6.71 (d, J = 8.0 Hz, 1H, H‐3'); δ = 7.31 (t, J = 8.0 Hz, 1H, H‐4′); δ = 6.72 (t, J = 8.0 Hz, 1H, H‐5′); δ = 7.78 (d, J = 8.0 Hz, 1H, H‐6′); δ = 7.87 (s, 1H, H‐2″); δ = 7.86 (d, J = 8.0 Hz, 1H, H‐4″); δ = 7.57 (t, J = 8.0 Hz, 1H, H‐5″); δ = 7.64 (d, J = 8.0 Hz, 1H, H‐6″); δ = 6.38 (bs, 2H, ‐NH₂). ¹³C‐NMR (100 MHz, CDCl₃, ppm): 191.06 (C‐1), 124.75 (C‐2), 140.96 (C‐3), 118.67 (C‐1′), 151.15 (C‐2′), 115.96 (C‐3′), 134.64 (C‐4′), 117.37 (C‐5′) 131.04 (C‐6′), 136.06 (C‐1″), 124.58, 124.52, 124.48, 124.44 (q, J = 4 Hz, C‐2″), 131.86, 131.54, 131.22, 130.90 (q, J = 32 Hz, C‐3″), 126.45, 126.41, 126.38, 126.34 (q, J = 4 Hz, C‐4″), 131.44 (C‐5″), 129.44 (C‐6″), 127.94, 125.23, 122.52, 119.81 (q, J = 271 Hz, CF₃). LC‐QTOF‐MS m/z calculated for C₁₆H₁₂F₃NO [M + H]⁺: 292.09492, Found:292.09582.

(2E)‐1‐(2‐Aminophenyl)‐3‐[4‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (C3): Yield (%): 81; m.p. (°C): 90–92, Rf:0.80 (hexane‐diethyl ether:1:2). FT‐IR (cm^–1): 3385, 3279, 1650, 1576, 746. ¹H‐NMR (400 MHz, CDCl₃, ppm): δ = 7.75–7.66 (m, 6H, H‐2/3/2″/3″/4″/5″/6″); δ = 6.71 (d, J = 8.0 Hz, 1H, H‐3′); δ = 7.33 (t, J = 8.0 Hz, 1H, H‐4′); δ = 6.73 (t, J = 8.0 Hz, 1H, H‐5′); δ = 7.86 (d, J = 8.0 Hz, 1H, H‐6′); δ = 6.89 (bs, 2H, –NH₂). ¹³C‐NMR (100 MHz, CDCl₃, ppm): 191.08 (C‐1), 125.37 (C‐2), 140.87 (C‐3), 118.64 (C‐1′), 151.18 (C‐2′), 115.94 (C‐3′), 134.68 (C‐4′), 117.39 (C‐5′) 131.01 (C‐6′), 138.66 (C‐1″), 128.30 (C‐2″/6″), 125.90, 125.86, 125.82, 125.78 (q, J = 4 Hz, C‐3″/5″), 131.94, 131.62, 131.29, 130.97 (q, J = 32 Hz, C‐4″), 127.96, 125.25, 122.55, 119.84 (q, J = 271 Hz, CF₃). LC‐QTOF‐MS m/z calculated for C₁₆H₁₂F₃NO [M + H]⁺: 292.09492, Found:292.09533.

4.1.3. Synthesis of 3,5‐Disubstituted‐2‐Pyrazoline Derivatives (P1, P2, P3)

The Michael addition reaction of hydrazine monohydrate to chalcones in an acid‐catalyzed medium was used to synthesize 3,5‐disubstituted‐2‐pyrazoline compounds (P1, P2, and P3) [19, 23, 24]. Chalcones (1.45 g, 5 mmol) were taken separately into 50 mL round‐bottomed flasks, and 20 mL dry ethanol was added to synthesize pyrazoline derivatives. The resultant solutions were mixed with 0.5 mL of hydrazine monohydrate (10 mmol) and 2 mL of glacial acetic acid. The reaction mixtures were then stirred for 6 h at 80°C under reflux. The reactions were stopped by using TLC to monitor their progress, and the cooled reaction contents were then poured onto cold water that had been salted and precipitated. After being cleaned with pure water to prevent air contact, the white solids were filtered out of the crucible and allowed to dry in a lyophilizer. The compounds were examined for purity using TLC, and spectroscopic techniques (¹H‐NMR, ¹³C‐NMR (APT), FT‐IR) were used to determine their structures. Schemes 3 and 4 show the synthesis equation and structures of pyrazolines.

Scheme 3.

Synthesis of target compounds. Reaction conditions: (a) Dry EtOH, AcOH, Reflux (80°C, 6 h).

Scheme 4.

Structures of pyrazolines.

2‐{5‐[2‐(Trifluoromethyl)phenyl]‐4,5‐dihydro‐1H‐pyrazol‐3‐yl}phenyl)amine (P1): Yield (%): 63; m.p. (°C): 59–61; Rf:0.60 (hexane/diethyl ether 1:2). FT‐IR (cm^–1): 3461, 3325, 1611, 1496, 1452, 745. ¹H‐NMR (400 MHz, CDCl₃, ppm): δ = 3.21–3.15 (dd, J _AB  = 16 Hz, J _AX  = 8.0 Hz, 1H, H‐4a); δ = 3.95‐3.87 (dd, J _BA  = 20.0 Hz, J _BX  = 12.0 Hz, 1H, H‐4b); δ = 5.93–5.89 (dd, J _5/4a = 4 Hz, J _5/4b = 12 Hz, 1H, H‐5); δ = 6.75 (m, 1H, H‐3′); δ = 7.23 (t, J = 8.0 Hz, 1H, H‐4′); δ = 6.75 (t, J = 8.0 Hz, 1H, H‐5′); δ = 7.17 (d, J = 8.0 Hz, 1H, H‐6′); δ = 7.25 (d, J = 8.0 Hz, 1H, H‐3″); δ = 7.50 (t, J = 8.0 Hz, 1H, H‐4″); δ = 7.38 (t, J = 8.0 Hz, 1H, H‐5″); δ = 7.71 (d, J = 8.0 Hz, 1H, H‐6″); The –NH₂ and –NH protons have undergone deuterium exchange, and their presence has been confirmed by FT‐IR. ¹³C‐NMR (100 MHz, CDCl₃, ppm): 155.49 (C‐3), 44.16 (C‐4), 54.93 (C‐5), 112.91(C‐1′), 146.90 (C‐2′), 116.10 (C‐3′), 129.58 (C‐4′), 116.89 (C‐5′), 127.56 (C‐6′), 140.51 (C‐1″), 127.05, 126.74, 126.44, 126.13(q, J = 31 Hz, C‐2″), 126.41,126.35, 126.30, 126.24 (q, J = 6 Hz, C‐3″), 125.25 (H‐4″), 132.83 (C‐5″), 131.26 (C‐6″), 128.42, 125.70, 122.97, 120.25 (q, J = 272 Hz, CF₃). LC‐QTOF‐MS m/z calculated for C₁₆H₁₄F₃N₃ [M + H]⁺: 306.12181 Found:306.12190.

2‐{5‐[3‐(Trifluoromethyl)phenyl]‐4,5‐dihydro‐1H‐pyrazol‐3‐yl}phenyl)amine (P2): Yield (%): 57; m.p. (°C): Oil; Rf: 0.60 (hexane/diethyl ether 1:2). FT‐IR (cm^–1): 3460, 3322, 1611, 1496, 1451, 747. ¹H‐NMR (400 MHz, CDCl₃, ppm): δ = 33.32–3.27 (dd, J _AB  = 16.0 Hz, J _AX  = 4.0 Hz, 1H, H‐4a); δ = 3.96–3.89 (dd, J _BA  = 16.0 Hz, J _BX  = 12.0 Hz, 1H, H‐4b); δ = 5.60–5.56 (dd, J _5/4a = 4 Hz, J _5/4b = 12 Hz, 1H, H‐5); δ = 6.84 (d, J = 8.0 Hz, 1H, H‐3′); δ = 7.25 (t, J = 8.0 Hz, 1H, H‐4′); δ = 6.76 (t, J = 8.0 Hz, 1H, H‐5′); δ = 7.20(d, J = 8.0 Hz, 1H, H‐6′); δ = 7.52 (s, 1H, H‐2″); δ = 7.54 (d, J = 8.0 Hz, 1H, H‐4″); δ = 7.48 (t, J = 8.0 Hz, 1H, H‐5″); δ = 7.45 (d, J = 8.0 Hz, 1H, H‐6″); The –NH₂ and –NH protons have undergone deuterium exchange, and their presence has been confirmed by FT‐IR. ¹³C‐NMR (100 MHz, CDCl₃, ppm): 153.65 (C‐3), 43.03 (C‐4), 62.47 (C‐5), 114.65 (C‐1′), 146.73 (C‐2′), 115.78 (C‐3′), 129.64 (C‐4′), 116.48 (C‐5′), 128.90 (C‐6′), 143.65 (C‐1″), 124.64, 124.60, 124.56, 124.53 (q, J = 4 Hz, C‐2″), 131.55, 131.23, 130.91, 130.59 (q, J = 32 Hz, C‐3″), 123.63, 123.59, 123.55, 123.51 (q, J = 4 Hz, H‐4″), 129.38 (C‐5″), 129.95 (C‐6″), 128.14, 125.43, 122.72, 120.01 (q, J = 271 Hz, CF₃). LC‐QTOF‐MS m/z calculated for C₁₆H₁₄F₃N₃ [M + H]⁺: 306.12181 Found: 306.12196.

2‐{5‐[4‐(Trifluoromethyl)phenyl]‐4,5‐dihydro‐1H‐pyrazol‐3‐yl}phenyl)amine (P3): Yield (%): 66; m.p. (°C): 167‐169; Rf:0.70 (hexane/diethyl ether 1:3). FT‐IR (cm^–1): 3464, 3319, 1608, 1496, 1453, 754. ¹H‐NMR (400 MHz, CDCl₃, ppm): δ = 3.32‐3.26 (dd, J _AB  = 16 Hz, J _AX  = 4.0 Hz, 1H, H‐4a); δ = 3.95–3.88 (dd, J _BA  = 16.0 Hz, J _BX  = 12.0 Hz, 1H, H‐4b); δ = 5.60–5.56 (dd, J _5/4a = 4 Hz, J _5/4b = 12 Hz, 1H, H‐5); δ = 6.87 (d, J = 8.0 Hz, 1H, H‐3′); δ = 7.26 (t, J = 8.0 Hz, 1H, H‐4′); δ = 6.77 (t, J = 8.0 Hz, 1H, H‐5′); δ = 7.20 (d, J = 8.0 Hz, 1H, H‐6′); δ = 7.61 (d, J = 8.0 Hz, 2H, H‐2″/6″); δ = 7.39 (d, J = 8.0 Hz, 2H, H‐3″/5″); The –NH₂ and –NH protons have undergone deuterium exchange, and their presence has been confirmed by FT‐IR. ¹³C‐NMR (100 MHz, CDCl₃, ppm): 155.67 (C‐3), 43.53 (C‐4), 57.93 (C‐5), 112.77 (C‐1′), 146.94 (C‐2′), 116.18 (C‐3′), 131.37 (C‐4′), 116.93 (C‐5′), 129.52 (C‐6′), 145.62 (C‐1″), 126.14 (C‐2″/6″), 126.06, 126.03, 125.99, 125.95 (q, J = 4 Hz, C‐3″/5″), 129.81 (C‐4″), 130.13, 127.42, 125.35, 122.64 (q, J = 271 Hz, CF₃). LC‐QTOF‐MS m/z calculated for C₁₆H₁₄F₃N₃ [M + H]⁺: 306.12181 Found: 306.12226.

4.2. HPLC‐DAD Evaluation

The new compounds were evaluated for purity check, hydrophobicity index (log k´) evaluation, and obtaining UV‐Vis spectra, with λ _max determination, by using an HPLC‐DAD system (Agilent). The purity of the compounds was expressed in terms of percentage area upon area normalization from the chromatograms obtained at 280 nm. Hydrophobicity index was calculated taking the log of the capacity factor k´, which was calculated as k´ = (t _R–t ₀)/t ₀, using a 280 nm chromatogram. λ_max values were obtained from the UV‐Vis spectra from DAD data.

4.3. α‐Amylase Inhibitory Activity of the Chalcones and 3,5‐Disubstituted Pyrazolines In Vitro

α‐Amylase inhibitory activity of chalcone and 3,5‐disubstituted pyrazoline molecules was evaluated using the dinitrosalicylic acid (DNS) method. A specified volume of the test compound solution (dissolved in DMSO) was added to a reaction mixture consisting of 20 mM phosphate buffer (containing 6 mM NaCl, pH 7.0) and 1% soluble starch solution (w/v). Following the addition of α‐amylase from porcine pancreas (Sigma, 1 U), the reaction was incubated at 37°C for 10 min. The enzymatic reaction was then stopped by the addition of DNS reagent, and the mixture was incubated in a boiling water bath for 10 min. After cooling, the absorbance was measured at 540 nm. A control reaction mixture prepared with enzyme and without inhibitor was prepared in the same way, and its activity was taken as 100%. The IC₅₀ value was calculated. Acarbose was used as the positive control due to its well‐established inhibitory activity on α‐amylase. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar (as maltose equivalent) per minute under the specified assay conditions [40, 41, 42, 43, 44, 45]. The enzyme reaction rates were determined with two different concentrations of the organic molecule with varying concentrations of soluble starch. The maximum velocity (V _max) of α‐amylase and the Michaelis–Menten constant (K _m) values were calculated from the Lineweaver–Burk plots [46]. The inhibition constant (Kᵢ) was also determined. The DMSO concentration in the reaction medium was kept below 2% during the study.

4.4. α‐Glucosidase Inhibitory Activity of the Chalcones and 3,5‐Disubstituted Pyrazolines In Vitro

The α‐glucosidase inhibitory activity was assessed following a previously described method with minor modifications [42, 43, 44, 45, 47, 48]. Briefly, α‐glucosidase from Saccharomyces cerevisiae (Sigma, 1 U) was incubated with a reaction mixture containing the test compound (dissolved in DMSO), 100 mM phosphate buffer (pH 6.8), and an appropriate volume of p‐NPG. The mixture was incubated at 37°C for 10 min, and the reaction was terminated by the addition of 0.1 M Na₂CO₃. The absorbance of the released p‐nitrophenol was measured at 405 nm. A control reaction mixture prepared with enzyme and without inhibitor was prepared in the same way, and its activity was taken as 100%. The IC₅₀ value, representing the concentration required to inhibit 50% of enzyme activity, was determined for each test compound. Acarbose was used as a reference standard inhibitor. One unit (U) of α‐glucosidase activity was defined as the amount of enzyme that liberates 1 μmol of p‐nitrophenol per minute under the assay conditions. To evaluate the inhibition kinetics, enzyme reactions were performed using two fixed concentrations of the test compound with varying concentrations of p‐NPG. The Michaelis–Menten constant (Kₘ) and maximum reaction velocity (Vₘₐₓ) were calculated using Lineweaver–Burk double reciprocal plots [49]. Furthermore, the inhibition constant (Kᵢ) was determined. The DMSO concentration in the reaction medium was kept below 2% during the study.

4.5. Molecular Docking

The optimization studies of the organic molecules were performed using Spartan 18 V1.3.0 software. The selected optimization protocol involved the following methods: Conformer distribution with molecular mechanics/MMFF, equilibrium geometry at ground state in gas phase using semi‐empirical PM6 and density functional/M06‐2X/6‐31 G**. The most stable conformers of the compounds were identified based on this protocol.

The crystal structures of α‐amylase (PDB ID: 2QV4; human pancreatic α‐amylase complexed with nitrite and acarbose) and α‐glucosidase (PDB ID: 5NN8; crystal structure of human lysosomal acid‐alpha‐glucosidase, GAA, in complex with acarbose) were retrieved from the Protein Data Bank (PDB) for use in molecular docking studies. Before docking, both enzyme structures were prepared using AutoDock Tools‐1.5.6, during which all water molecules, heteroatoms, and co‐crystallized ligands were removed to ensure proper receptor preparation [50]. The interactions between the optimized ligands and the active sites of the enzymes were visualized and analyzed using the Discovery Studio Visualizer 4.5 software [51, 52, 53].

4.6. Statistical Analysis

All experiments were conducted in triplicate (n = 3), and the results are presented as mean ± standard deviation (SD). The data were analyzed using one‐way analysis of variance (ANOVA) followed by Duncan's multiple range test for post hoc comparisons. Statistical analysis was performed using SPSS software (version 26.0, SPSS Inc., Chicago, IL, USA).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

SUPPLEMENTARY FILE‐R1.

ArchPharm SupplMat InChI.

Data Availability Statement

Data will be made available on request.