Introduction

Situated in the brush border of the intestine, α-glycosidase (EC 3.2.1.20) is a membrane-bound enzyme responsible for the hydrolysis of complex carbohydrates into D-glucose. This sole monosaccharide can be assimilated from the intestinal lumen1. Regarding its critical function in carbohydrate processing, the inhibition of α-glucosidase can decrease the hydrolysis rate of complex carbohydrates. This mechanism represents a crucial strategy in managing hyperglycemia in type-2 diabetes. Additionally, α-glucosidase inhibitors have also been investigated for their potential therapeutic benefits in treating various diseases, including cancer and viral infections2,3,4. Accordingly, the α-glucosidase inhibitors Acarbose (Glucobay), Miglitol (Glyset), and Voglibose (Volix, Basen), which are readily available on the market, are suggested as the initial pharmacological intervention for the treatment of type-2 diabetes5. These kinds of α-glucosidases inhibitors have sugar-based structures, which need complex multistep processes for their synthesis6. Unfortunately, administrating these medications is associated with undesirable side effects like flatulence, diarrhea, and abdominal discomfort7,8. Because of this, creating new, secure, and effective α-glycosidase inhibitors would be crucial as strong leadership candidates for upcoming antidiabetic drug discovery initiatives9,10.

Nitrogen-containing heterocyclic compounds are known to play crucial roles in medicinal chemistry and exhibit a wide range of pharmacological activities. Among the wide variety of nitrogen-containing heterocyclic compounds, those containing phthalazine and pyrazole moieties are specifically recognized in the fields of medicine and pharmacy as integral components of certain drugs. These compounds are known to demonstrate a broad spectrum of biological activities11.

Pyrazole possesses a diverse range of pharmacological activities, such as anti-inflammatory12, antifungal13, anti-cancer14,15,16, antibacterial17, anti-Alzheimer’s18, antileishmanial19, antihypertensive20, and antitumor21 activities. Furthermore, several pyrazole-containing agents have demonstrated potent antidiabetic7,21,22,23,24,25,26,27 and hypoglycemic28,29,30 properties. Recently, many pyrazole derivatives have been investigated for their antidiabetic properties. For instance, Azimi et al. reported a new series of pyrazole‑benzofuran hybrids as potent α-glucosidase inhibitors (Fig. 1 compound A)27. Yamali et al. discovered the pyrazole-containing derivatives as highly potent and selective carbonic anhydrase inhibitors (Fig. 1 compound B)16. Ren et al. presented novel pyrazole-benzimidazole derivatives as antitumor agents (Fig. 1 compound C)31. Notably, Teneligliptin, an antidiabetic drug containing pyrazole, was approved for treating type II diabetes (Fig. 1)25. According to the success of this scaffold in the chemical class of antidiabetic reagents, significant metabolic stability and pharmacological efficiency of pyrazole-based antidiabetic agents encouraged us to further study pyrazole scaffolds to develop a new agent.

Fig. 1
figure 1

Rationale of this research.

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Phthalazine has been found to exhibit a diverse range of biological activities, including antitumor32,33, antimicrobial18,34,35, antifungal36, anti-inflammatory37,38, vasorelaxant39, cardiotonic40, anticonvulsant41,42, and antioxidative41 activities. Azelastine is one of the commercially available drugs used in the treatment of allergic rhinitis (Fig. 1)41. Furthermore, several phthalazine derivatives have demonstrated α-glucosidase inhibitory activity. For instance, Taslimi et al. have investigated the inhibitory effects of 1H-pyrazolo[1,2-b]phthalazine-5,10-dione derivatives against α-glucosidase through in vitro assays (Fig. 1 compound D)43. Similarly, 2H-indazolo[2,1-b]phthalazine-1,6,11-trione compounds have shown satisfactory inhibitory effects against carbonic anhydrase isoforms I and II, α-glycosidase, and cholinesterases at the nanomolar level (Fig. 1 compound E)44. Novel scaffolds of pyrazolyl-phthalazine-diones have been evaluated for Antiradical activity by Simijonovic et al. (Fig. 1 compound F)41. Chate et al. have reported 2H-indazolo[2,1-b]phthalazine-trione derivatives as potent antimicrobial agents (Fig. 1 compound G)35.

Molecular hybridization is a strategic medicinal chemistry approach that combines pharmacophoric elements or structural features from different bioactive molecules into a single hybrid compound. Hybrid molecules are designed through a rational process that carefully combines the active fragments of parent compounds to achieve the best possible therapeutic outcomes. Molecular hybridization has been widely employed to develop new bioactive compounds possessing special features such as tyrosinase inhibition45,46, anti-Alzheimer47,48,49, and α-glucosidase inhibition27,50 activities. Motivated by the success of this method, we employed a molecular hybridization approach including a humic acid-catalyzed reaction to synthesize novel pyrazole-phthalazine hybrids.

High molecular weight substances are highly beneficial for organic synthesis due to their low toxicity, and straightforward separation. Humic acid is a readily available, cost-effective polymer with impressive catalytic properties found in environments such as peat and soil. Its catalytic capabilities are due to the presence of various functional groups, including carboxyl and phenolic hydroxyl groups51,52. Previous studies such as solvent-free reactions for functionalizing bis(indolyl)methanes and bis(pyrazolyl)methanes53, one-pot, water-based synthesis of 5-substituted 1H-tetrazoles51, and utilizing humic acid as a support for a Pd/Ni bimetallic catalyst in Heck coupling reactions52 showcased the catalytic functions of this material.

All the synthesized conjugates in this work were examined for their possible in vitro α-glucosidase inhibitory actions in accordance with the US-NCI protocol. In the next step, molecular docking simulations were conducted to evaluate the binding mode of the 8l compound.

Results and discussion

Designing consideration

This study leverages the established biological activities of two key pharmacophores: the pyrazole and phthalazine scaffolds. Both of these scaffolds have demonstrated diverse biological effects, including the inhibition of various targets, with particular relevance to the alpha-glucosidase enzyme (Fig. 1). For example, Taslimi et al. reported the synthesis of a phthalazine derivative, compound E, which exhibited a 20-fold greater alpha-glucosidase inhibitory activity than Acarbose, a standard alpha-glucosidase inhibitor44. Similarly, Azimi et al. synthesized the pyrazole derivative A, demonstrating an 18.5-fold increase in inhibitory activity against alpha-glucosidase compared to Acarbose54. Given the demonstrated potency of these two scaffolds as alpha-glucosidase inhibitors, this project aimed to exploit a synergistic effect by combining these pharmacophores into a hybrid molecule. This strategy led to identifying compound 8l, which exhibited a 52-fold increase in inhibitory activity relative to Acarbose.

Chemistry

The synthetic route for the synthesis of pyrazole-phthalazine hybrids 8a–n is depicted in Fig. 2. The hydrazones 8a–g were prepared through the reaction of acetophenone derivatives (1) with phenylhydrazine (2) in absolute ethanol, using a catalytic amount of sulfuric acid under refluxed conditions for 12 h similar to previously reported approaches for synthesizing Schiff base compounds in acidic media55,56,57,58. During the reaction, the progress was monitored by thin-layer chromatography (TLC), where the formation of hydrazone derivatives was confirmed.

Fig. 2
figure 2

Synthetic route for pyrazole‑phthalazine derivatives.

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The hydrazone derivatives (3) a–g were subjected to a Vilsmeier-Haack reaction to form 4-formyl pyrazole derivatives (4a–g). This reaction was carried out with POCl3 and DMF at 60–70 °C for 5 h. The reaction was monitored by TLC to ensure the complete conversion of the hydrazone to the desired formyl pyrazole derivatives59. POCl3 is a reactive reagent that must be handled in a well-ventilated fume hood.

Phthalhydrazide (6) was prepared by reacting phthalic anhydride (5) with hydrazine monohydrate in acetic acid under reflux for 4 h. The reaction progress was monitored by TLC, and once complete, the products were isolated and purified through recrystallization using ethanol60. Finally, the desired pyrazole-phthalazine hybrids (8a-n) were obtained through a multicomponent reaction of the synthesized aldehydes (4a–g), phthalhydrazide (6), and dimedones (7a or 7b) in the presence of humic acid in ethanol. The reaction was carried out under reflux for 12–18 h, with regular monitoring by TLC to track the formation of the final products. Once the reaction was complete, the products were purified by column chromatography. The structures of the newly synthesized derivatives were characterized by IR, 1H NMR, 13C NMR spectra, and elemental analysis, confirming their purity and structure.

The plausible mechanism for the synthesis of pyrazole‑phthalazine derivatives is shown in Fig. 3.

Fig. 3
figure 3

The plausible mechanism for the synthesis of pyrazole‑phthalazine hybrids .

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First, the humic acid catalyst activates the aldehydes (4a–g), through its carboxylic acid moieties (–COOH) to enhance the electrophilicity of the carbonyl functional group for facilitating the subsequent nucleophilic attack by enolized diketones 7a or 7b during Knoevenagel condensation. in the next steps, Michael-type addition of phthalhydrazide to the constructed heterodienes, and subsequent cyclization and dehydration afford the corresponding compounds 8 a-g or 8 h-n.

In vitro α-glucosidase inhibitory activity

All synthesized compounds 8 a-n were evaluated for their in-vitro inhibitory activity against α-glucosidase (Saccharomyces cerevisiae) in comparison to Acarbose as the standard drug (Table 1). The choice of substituents (H, 4–Cl, 4–CH3, 4–NO2, 4–OMe, 4–OH, and 4–Br) in the synthesized hybrid compounds was deliberate, aimed at exploring the structure–activity relationship (SAR) of these molecules concerning α-glucosidase inhibition. These substituents were selected to represent a range of electronic and steric properties, allowing us to investigate how these characteristics influence the interaction of the compounds with the enzyme. Specifically, electron-donating groups like methoxy (4–OMe) and methyl (4–CH3) were included to assess the impact of increased electron density, while electron-withdrawing groups such as nitro (4–NO2), Chloro (4–Cl), and Bromo (4–Br) were chosen to evaluate the effects of reduced electron density. The hydroxyl group (4–OH) was used to explore the potential for hydrogen bonding interactions. The unsubstituted phenyl (H) served as a baseline to understand the impact of the different substitution patterns. By systematically varying these substituents, we aimed to identify key structural features crucial for potent α-glucosidase inhibitory activity and optimize the lead compounds.

Table 1 In vitro α-glucosidase inhibitory activities of pyrazole‑phthalazine hybrids.
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As can be seen in Table 1, synthesized compounds can be divided into two groups: derivatives with 5,5-dimethylcyclohexane-1,3-dione (7a) in their synthetic route were named group 1 (compounds 8 a-g) and derivatives with acetylacetone in their synthetic route (7b) were named group 2 (compounds 8 h-n).

The obtained IC50 values demonstrated that all the target compounds showed excellent inhibition against yeast α-glucosidase (ranging from 13.66 ± 0.009 µM to 494 ± 0.006 µM) while Acarbose exhibited IC50 value = 720.18 ± 0.008 µM. Among them, compound 8l (IC50 = 13.66 ± 0.009), having a methoxy group in R position, was found to be the most potent inhibitor. This is approximately 52-fold more potent than the standard Acarbose.

Compounds 8h (IC50 = 15.44 ± 0.024 µM), 8l (IC50 = 13.66 ± 0.009 µM), and 8n (IC50 = 18.63 ± 0.041 µM) from group 2 containing phenyl, 4-methoxyphenyl, and 4-bromophenyl moieties, respectively, demonstrated highest activities. Also, compound 8a with phenyl group with IC50 of 74.22 ± 0.012 demonstrated the best activity in group 1 and stood in the fourth rank of the highest activities. Interestingly, the fifth and seventh ranks of the best activities also belong to the second group (i.e., 8k and 8i with IC50 of 86.68 ± 0.012 and 358.57 ± 0.008, respectively). This shows that the second group may have a better main skeleton against alpha-glucosidase.

However, the best compound in group 1, 8a, with the basic phenyl group, is fourth-ranked among the screened compounds, which has about sixfold lower activity than the best compound, but it shows a tenfold better activity in comparison with the Acarbose. The results showed that the basic phenyl group has good inhibitory activity in both group 1 and group 2. The 8m compound, with the lowest inhibitory activity (IC50 = 494 ± 0.006), shows 31 percent higher activity in comparison with the Acarbose.

The α-glucosidase inhibitory activity of the second group containing 3-phenyl (8h-n) demonstrated that compound 8l, containing a 4-methoxyphenyl moiety, was the most potent compound. Changing from the bromine to nitro in group 2 (8n to 8k) resulted in a dramatic decrease in the IC50 values of 18.63 ± 0.041 to 86.68 ± 0.012 µM, respectively. In both groups, the introduction of other electron-withdrawing groups, such as halogen atoms (Br and Cl) and nitro group, led to lower activity than compounds 8a and 8h. In the second group, halogenated cou/nterparts of compound 8h exhibited inhibitory activity generally depending on the size of halogen substitution. Compound 8n (IC50 = 18.63 ± 0.041) bearing bromo substituent was found to be the third most active analog in this category and 38-fold more potent than standard Acarbose. Compounds 8i, having a relatively smaller chlorine substituent, showed dramatically lower activity with an IC50 value of 358.57 ± 0.008. This trend was not consistent with the same compounds in the first group.

In both categories, the replacement of the methoxy group with methyl resulted in a decrease in the biological activity (group 1: 8e with IC50 of 228.96 ± 0.016 μM to 8c with IC50 of 281.92 ± 0.012 μM and group 2: 8l with IC50 of 13.66 ± 0.009 μM to 8j with IC50 of 167.24 ± 0.052 μM). This enhancement could be due to the electron-donating nature and steric effects of the methoxy group, which might facilitate better interactions with the biological target, thus improving efficacy.

Enzyme kinetic study

To investigate the mechanism of inhibition of synthesized compounds against α-glucosidase, a kinetic study was performed with the most potent compound 8l through the varying concentrations of p-nitrophenyl α-D-glucopyranoside (PNPG) as substrate in the absence and presence of compound 8l at different concentrations.

Type of inhibition and Ki were indicated by Lineweaver– Burk plots and the secondary re-plotting of these plots was presented in Fig. 4. The Michaelis–Menten constants (Km) values were determined. As it was illustrated in Fig. 4a, while inhibitor concentration increased, the Km value gradually increased, but the Vmax value remained unchanged. Therefore, this finding revealed that compound 8l is a competitive inhibitor for α-glucosidase. Moreover, the plot of Km versus different concentrations of compound 8l resulted in the inhibition constant, Ki, of 34.75 µM, which is shown in Fig. 4b.

Fig. 4
figure 4

(a) The Lineweaver–Burk plot in the absence and presence of various concentrations of compound 8l. (b) The secondary plot between Km and various concentrations of compound 8l.

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Molecular docking study

Several studies have successfully utilized molecular docking to elucidate the interactions between small molecules and their target proteins, providing valuable insights into their binding mechanisms58,61,62. In this work, molecular docking studies were performed to rationalize the results of biological assays and gain structural insight into the binding of the synthetic derivatives against α-glucosidase. Following the in vitro enzymatic assays, compound 8l, which exhibited notable inhibitory activity against α-glucosidase, was identified for further investigation through a docking study. The primary sequence of MAL12 alpha-glucosidase was retrieved and downloaded from the UniProt database with the access code P53341. As far as the crystal structure of the mentioned protein, has not been experimentally resolved, the AlphaFold 2 Protein Structure Database was employed to predict the three-dimensional (3D) structure63. The PROCHECK program64 was applied to assess the stereochemical quality of the model65. The phi/psi Ramachandran plot distributions indicated that 99.2% of residues are in the favored and allowed regions, and only 0.8% of residues lie in the outlier region (Fig. 5).

Fig. 5
figure 5

Ramachandran plot of the modeled a-glucosidase enzyme.

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The docking studies involved the superposition of the structure of Acarbose (a standard inhibitor) and the most potent compound, 8l, within the active site of α-glucosidase. The comparative conformation, depicted in Fig. 6, confirms that both compounds, 8l and Acarbose, share the same binding region within the protein. This overlap in structural orientation substantiates the validity of this conformation.

Fig. 6
figure 6

Acarbose (pink) and most potent compound 8l (blue).

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The detailed binding mode of Acarbose illustrates hydrogen bonding interactions with residues ARG312, HIS279, THR307, SER308, and GLU304, in addition to one electrostatic interaction with GLU304 (Fig. 7).

Fig. 7
figure 7

Results of molecular ducking of Acarbose and Alpha Glucosidase (A) 3D interactions and (B) 2D interactions.

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Figure 8 illustrates the binding mode of the most potent compound, 8l. Analysis of the binding mode revealed several significant interactions: 1- The carbonyl of the acetyl and Oxygen atom of the methoxy groups form hydrogen bonds with LYS425 and ASN314, respectively. 2- The pyrazole ring and the phenyl group attached to the Nitrogen atom of the pyrazole ring exhibit a Pi-Anion electrostatic interaction with ASP429 and LYS432, respectively. 3- The phenyl group attached to the Nitrogen atom of the pyrazole ring also demonstrates a Pi-Alkyl hydrophobic interaction with ILE315. 4-The carbonyl group of the phthalazide moiety exhibits carbon hydrogen bond with PRO317. 5- There is a Pi-sigma interaction between benzene ring of the phthalazide and VAL316.

Fig. 8
figure 8

(A) 3D and (B) 2D docked conformation of compounds 8l.

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Molecular dynamics investigation

To evaluate the stability of the protein–ligand complex, the root mean square deviation (RMSD) changes of the backbone of the α-glycosidase were measured and analyzed over 20 ns MD simulation (Fig. 9). The RMSD values were 0.13 ± 0.024 and 0.2543 ± 0.04 nm, for protein–ligand complex and the α-glycosidase respectively. The low amount of these changes over the simulation time indicates the stability of the ligand–protein complex and the strong connection of the ligands to the connection position. The stability of this connection has allowed the ligands to remain in the junction. Moreover, the lower amount of RMSD for the protein–ligand complex indicates that the protein is more stable in the presence of the ligand, which indicates a high affinity between the α-glycosidase and compound 8l.

Fig. 9
figure 9

RMSD of the α-glycosidase complexed with compound 8l (in red) and the Apo α-glycosidase (in yellow) for over 20 ns MD simulation time.

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The root mean square fluctuation (RMSF) refers to the fluctuation of the Cα atom from its average position throughout the simulation time. To assess the effect of ligand binding on the flexibility of the α-glycosidase protein residues, the RMSF plot was calculated and analyzed. Figure 10 compares the RMSF values of apo α-glycosidase, the α-glycosidase, and compound 8l complex. Generally, the apoenzyme (yellow color line in Fig. 10) had higher RMSF fluctuations compared to the complex (red-colored lines in Fig. 10). This observation occurs upon ligand binding to the enzyme, in which residue movement decreases due to non-bonding interaction between the ligand and the enzyme.

Fig. 10
figure 10

RMSF plot of the α-glycosidase and compound 8l complex (in red) and the Apo α-glycosidase (in yellow) for over 20 ns MD simulation time.

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The results show that in the free α-glycosidase structure, specific residues such as residues 2, 4, 38, 66, 85, 98, 233,315, 453, 462, 471, 473, and 483 have higher fluctuations, indicating structural flexibility and no restriction of movement in these regions. With the binding of compound 8l, a decrease in the fluctuations in these regions is observed.

ADME analysis

The study of toxicity, physicochemical, and pharmacokinetic properties plays crucial roles in the rational development of drug discovery. ADME consists of a foundational framework in pharmacokinetics that plays an essential role in the drug design and development process. The primary key view of ADMET helps to predict and understand the efficacy, mechanical stability, and toxicological risk of a drug. Interaction with the target biological macromolecules might produce desirable or undesirable pharmacological effects. The bioavailability of a drug depends on its safety and efficacy, which mainly depend on the ADME properties66. Pharmacokinetic parameters like Lipinski’s rule of five (molecular weight (MW), number of hydrogen bond acceptors (HBA), number of hydrogen bond donors (HBD), lipophilicity (clogP), and number of rotatable bonds (NROTB)), and Veber’s rule (topological polar surface area (TPSA) and solubility) are involved in the assessment of ADME.

Here, we evaluate the ADME properties compounds 8a-n by using an in silico SwissADME server67 to see the pharmacokinetic properties. Absorption determines how effectively a drug enters systemic circulation; distribution assesses how it disperses throughout various tissues; metabolism examines the chemical alterations within the body; and excretion focuses on the elimination pathways of the drug and its metabolites. The ADME properties of compounds 8 a-n are shown in Table 2.

Table 2 Predicted pharmacokinetic properties of compounds 8 a-n ADME.
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Conclusion

With the aim of developing novel α-glucosidase inhibitors, new types of pyrazole‑phthalazine derivatives were designed, synthesized, and evaluated for their α-glucosidase inhibition. All screened compounds displayed enhanced inhibitory strength in the range of 13.66 ± 0.009 to 494 ± 0.006 μM when compared to Acarbose (IC50 = 720.18 ± 0.008 μM). Among them, compound 8l was found to be the most potent inhibitor. This compound was demonstrated to be approximately 53-fold more potent than the standard Acarbose. Binding mode analysis showed that almost all structural features, such as the pyrazole ring, phenyl group attached to the pyrazole moiety, carbonyl groups, methoxy, and methyl group are contributed to binding through hydrogen bonding, hydrophobic and electrostatic interactions.

In summary, the study highlights the potential of pyrazole-phthalazine hybrids as promising α-glucosidase inhibitors with significantly higher potency than the available standard drug. Beyond their enhanced inhibitory profiles, these compounds can provide a foundation for further optimization to improve pharmacokinetic properties, selectivity, and safety profiles. Future studies could involve in-depth in vivo validations, toxicity profiling, and structural modifications to yield derivatives with improved drug-like properties for clinical applications.

Experimental

All materials were purchased from Sigma-Aldrich. Tin-layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (F254). Melting points of synthesized materials 8a–n were measured on a Kofer apparatus. 1H NMR and 13C NMR spectra were carried out on a Bruker FT-500 spectrometer in DMSO-d6 and TMS as an internal standard at room temperature. FTIR spectra were recorded on S-8400 shimadzu in KBr pellets. Elemental analysis was conducted using an Elemental Analyzer system GmbH VarioEL CHNS mode.

General procedure for the synthesis of 1,3-disubstituted-4-pyrazole carbaldehyde derivatives 4 a-g.

A stirring solution of phenylhydrazine 2 (6 mmol) in ethanol (7.5 mL), acetophenone 1 (5 mmol), and 0.25 ml acetic acid was heated at 70 °C for 12 h. After completion of the reaction (monitored by TLC), the mixture was cooled to room temperature and subsequently poured on crushed ice and water mixture to afford precipitations of hydrazone intermediate 3 a–g. The resulting product was filtered off and washed thoroughly wit distilled water. In a round bottom flask, a solution of hydrazone 3 a–g (5 mmol) in DMF (5 mL) was added drop-wise to an ice-cold solution of DMF (5 mL) and POCl3 (15 mmol) and the resulting mixture was refluxed at 60–70 °C for 5 h. After completion of the reaction, the reaction mixture was allowed to cool to room temperature, poured into a mixture of ice and water following by neutralizing with a saturated solution of sodium hydroxide. finally, the precipitations was filtered off, washed with a few cold water and recrystallized from ethanol to obtain pure aldehydes 4 a–g.

Procedure for the preparation of phthalhydrazide 6

A mixture of hydrazine hydrate (11 mmol), was added to a stirring solution of phthalic anhydride (10 mmol) in acetic acid (22 ml) and heated the mixture and was refluxed for 4 h. After completion of the reaction, the precipitated white solids were collected, filtered, and washed with water. The air-dried powder was used in the next step without further purification.

Typical procedure for the preparation of pyrazole‑phthalazine derivatives (8 a-g)

A mixture of dimedone 7 (0.5 mmol), phthalhydrazide 6 (0.5 mmol), aldehyde 4 (0.5 mmol), and humic acid (0.13 gr) in 12.5 ml ethanol was heated at 80 °C overnight. After cooling, the filtrate was concentrated and the formed crystals further recrystallized from ethanol to afford the pure products 8 a-g. (see related spectra in Supplementary data).

13-(1,3-diphenyl-1H-pyrazol-4-yl)-3,3-dimethyl-2,3,4,13-tetrahydro-1H-indazolo[1,2-b]phthalazine-1,6,11-trione (8a) Yellow solid; isolated yield: 89%; mp 204–206 °C; IR (KBr, υ): 3024, 2957, 1659, 1590 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H, H5-pyr), 8.12 (dd, J = 8.0, 1.5 Hz, 1H), 7.91–7.83 (m, 1H), 7.60–7.56 (m, 3H), 7.55–7.49 (m, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.35–7.22 (m, 7H), 6.44 (1H, s, CHN), 3.20–3.36 (2H, AB system, CHaHb), 2.34 (2H, s, CH2), 1.23 (6H, s, 2Me); 13C NMR (101 MHz, DMSO-d6) δ 192.26, 158.17, 157.18, 155.65, 139.33, 136.78, 133.80, 133.56, 131.69, 130.49, 129.82, 129.17, 128.95, 128.20, 128.09, 127.66, 127.02, 126.52, 122.46, 119.68, 118.84, 118.63, 64.36, 50.66, 38.40, 34.46, 28.83, 28.28; Anal Calcd for C32H26N4O3, C, 74.69; H, 5.09; N, 10.89 found: C, 74.71; H, 5.09; N, 10.80.

13-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3,3-dimethyl-2,3,4,13-tetrahydro-1H-indazolo[1,2-b]phthalazine-1,6,11-trione (8b) Yellow solid; isolated yield: 91%; mp 181–183 °C; IR (KBr, υ): 3026, 2959, 1659, 1595 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H, H5-pyr), 8.12 (dd, J = 7.9, 1.2 Hz, 1H), 7.91–7.83 (m, 1H), 7.61–7.57 (m, 3H), 7.56–7.49 (m, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.40–7.34 (m, 2H), 7.35–7.31 (m, 1H), 7.31–7.26 (m, 3H), 6.48 (1H, s, CHN), 3.20–3.35 (2H, AB system, CHaHb), 2.38 (2H, s, CH2), 1.23 (6H, s, 2Me); 13C NMR (101 MHz, DMSO-d6) δ 192.68, 159.65, 155.67, 154.14, 140.08, 136.86, 135.40, 135.18, 132.94, 130.10, 129.83, 129.15, 128.82, 128.66, 128.51, 128.04, 126.45, 126.31, 123.03, 120.11, 119.54, 118.91, 65.05, 51.44, 38.19, 34.48, 28.97, 28.62; Anal Calcd for C32H25ClN4O3, C, 70.01; H, 4.59; N, 10.20 found: C, 70.07; H, 4.66; N, 10.16.

3,3-dimethyl-13-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-2,3,4,13-tetrahydro-1H-indazolo[1,2-b]phthalazine-1,6,11-trione (8c) Yellow solid; isolated yield: 85%; mp 223–225 °C; IR (KBr, υ): 3023, 2959, 1663, 1588 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H, H5-pyr), 8.12 (dd, J = 7.9, 1.2 Hz, 1H), 7.83–7.92 (m, 1H), 7.73–7.78 (m, 1H), 7.70–7.62 (m, 2H), 7.58–7.51 (m, 1H), 7.48–7.35 (m, 3H), 7.22–7.16 (m, 2H), 7.05 (d, J = 8.7 Hz, 2H), 6.44 (1H, s, CHN), 3.18–3.33 (2H, AB system, CHaHb), 2.37 (3H, s, Me), 2.30 (2H, s, CH2), 1.22 (6H, s, 2Me); 13C NMR (101 MHz, DMSO-d6) δ 192.17, 157.89, 156.27, 155.25, 139.70, 137.51, 134.82, 133.18, 131.90, 131.79, 131.75, 130.65, 130.06, 129.35, 129.24, 128.47, 127.02, 126.78, 123.11, 121.44, 119.47, 119.17, 65.37, 50.39, 37.05, 35.01, 29.94, 28.86, 21.23; Anal Calcd for C33H28N4O3, C, 74.98; H, 5.34; N, 10.60 found: C, 74.93; H, 5.35; N, 10.51.

3,3-dimethyl-13-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-2,3,4,13-tetrahydro-1H-indazolo[1,2-b]phthalazine-1,6,11-trione (8d) Yellow solid; isolated yield: 88%; mp 173–175 °C; IR (KBr, υ): 3021, 2961, 1660, 1594 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H, H5-pyr), 8.11 (dd, J = 7.9, 1.2 Hz, 1H), 7.89–7.83 (m, 1H), 7.71 (d, 1H, J = 8.4 Hz), 7.61–7.56 (m, 3H), 7.54–7.49 (m, 1H), 7.38–7.34 (m, 2H), 7.31–7.27 (m, 4H), 6.47 (1H, s, CHN), 3.21–3.36 (2H, AB system, CHaHb), 2.34 (2H, s, CH2), 1.22 (6H, s, 2Me); 13C NMR (101 MHz, DMSO-d6) δ 192.96, 155.65, 154.92, 154.35, 150.42, 140.06, 136.85, 135.38, 134.65, 133.67, 131.66, 130.08, 129.85, 129.13, 127.96, 127.12, 126.70, 126.44, 122.94, 119.53, 119.41, 118.61, 65.17, 50.86, 37.21, 34.03, 28.95, 28.38; Anal Calcd for C32H25N5O5, C, 68.69; H, 4.50; N, 12.52 found: C, 68.71; H, 4.47; N, 12.54.

13-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3,3-dimethyl-2,3,4,13-tetrahydro-1H-indazolo[1,2-b]phthalazine-1,6,11-trione (8e) Yellow solid; isolated yield: 90%; mp 216–218 °C; IR (KBr, υ): 3025, 2961, 1655, 1591 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H, H5-pyr), 8.17–8.14 (m, 1H), 7.90–7.84 (m, 1H), 7.61–7.57 (m, 2H), 7.46 (d, J = 8.0 Hz, 1H), 7.24–7.20 (m, 4H), 7.03–7.00 (m, 2H), 6.78–6.75 (m, 2H), 6.40 (1H, s, CHN), 3.74 (3H, s, OMe), 3.16–3.34 (2H, AB system, CHaHb), 2.38 (2H, s, CH2), 1.26 (6H, s, 2Me); 13C NMR (101 MHz, DMSO-d6) δ 192.40, 160.20, 157.80, 155.64, 155.26, 139.37, 136.56, 136.45, 131.94, 130.78, 129.76, 128.89, 128.83, 128.45, 127.95, 126.50, 126.42, 122.64, 119.71, 118.49, 118.21, 114.43, 64.37, 55.49, 50.83, 38.48, 35.36, 28.92, 28.34; Anal Calcd for C33H28N4O4, C, 72.78; H, 5.18; N, 10.29 found: C, 72.76; H, 5.14; N, 10.21.

13-(3-(4-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3,3-dimethyl-2,3,4,13-tetrahydro-1H-indazolo[1,2-b]phthalazine-1,6,11-trione (8f.) Yellow solid; isolated yield: 84%; mp 171–173 °C; IR (KBr, υ): 3022, 2959, 1663, 1588 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H, OH), 8.53 (s, 1H, H5-pyr), 8.10 (dd, J = 7.9, 1.2 Hz, 1H), 7.90–7.82 (m, 1H), 7.69–7.62 (m, 2H), 7.56–7.49 (m, 1H), 7.47–7.39 (m, 3H), 7.35–7.27 (m, 3H), 7.24–7.18 (m, 2H), 6.47 (s, 1H, CHN), 3.18–3.33 (2H, AB system, CHaHb), 2.34 (2H, s, CH2), 1.23 (6H, s, 2Me); 13C NMR (101 MHz, DMSO-d6) δ 192.62, 158.30, 156.79, 155.29, 154.68, 139.11, 136.88, 134.84, 134.09, 130.47, 130.12, 129.16, 128.95, 128.79, 127.86, 127.07, 126.48, 122.37, 119.53, 118.81, 118.24, 114.82, 64.61, 50.75, 37.13, 34.51, 28.69, 28.30; Anal Calcd for C32H26N4O4, C, 72.44; H, 4.94; N, 10.56 found: C, 72.48; H, 4.94; N, 10.53.

13-(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3,3-dimethyl-2,3,4,13-tetrahydro-1H-indazolo[1,2-b]phthalazine-1,6,11-trione (8 g) Yellow solid; isolated yield: 89%; mp 179–181 °C; IR (KBr, υ): 3022, 2957, 1655, 1591 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H, H5-pyr), 8.13–8.05 (m, 1H), 7.88–7.79 (m, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.34–7.20 (m, 7H), 7.19–7.13 (m, 2H), 6.41 (s, 1H, CHN), 3.17–3.33 (2H, AB system, CHaHb), 2.33 (2H, s, CH2), 1.24 (6H, s, 2Me); 13C NMR (101 MHz, DMSO-d6) δ 192.39, 156.34, 155.77, 154.79, 139.29, 136.77, 134.30, 133.02, 132.10, 131.02, 130.75, 129.58, 129.17, 128.24, 128.09, 127.69, 127.02, 126.48, 122.87, 120.13, 119.68, 118.88, 64.00, 51.31, 36.79, 34.37, 28.84, 28.13; Anal Calcd for C32H25BrN4O3, C, 64.76; H, 4.25; N, 9.44 found: C, 64.71; H, 4.23; N, 9.48.

2-acetyl-1-(1,3-diphenyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8 h) Yellow solid; isolated yield: 90%; mp 219–221 °C; IR (KBr, υ): 3025, 2959, 1654, 1579 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, H5-pyr), 8.12 (dd, J = 7.9, 1.2 Hz, 1H), 7.91–7.84 (m, 1H), 7.62–7.55 (m, 3H), 7.55–7.47 (m, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.30–7.20 (m, 5H), 7.19–7.13 (m, 2H), 6.47 (s, 1H, CHN), 3.09 (3H, s, Me), 2.07 (3H, s, Me); 13C NMR (101 MHz, DMSO-d6) δ 193.19, 157.81, 156.65, 154.49, 145.84, 139.02, 134.87, 130.96, 130.50, 129.99, 129.87, 129.58, 128.37, 128.21, 127.95, 127.04, 126.53, 122.09, 120.16, 119.69, 119.53, 116.73, 66.92, 30.80, 14.58; Anal Calcd for C29H22N4O3, C, 73.40; H, 4.67; N, 11.81 found: C, 73.37; H, 4.61; N, 11.86.

2-acetyl-1-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8i) Yellow solid; isolated yield: 87%; mp 194–196 °C; IR (KBr, υ): 3026, 2958, 1655, 1578 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H, H5-pyr), 8.09 (d, J = 7.5 Hz, 1H), 7.85 (t, J = 7.2 Hz, 1H), 7.69–7.61 (m, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.45–7.38 (m, 3H), 7.32–7.22 (m, 5H), 6.47 (s, 1H, CHN), 3.07 (3H, s, Me), 2.00 (3H, s, Me); 13C NMR (101 MHz, DMSO-d6) δ 193.13, 156.49, 156.20, 154.70, 146.37, 142.76, 134.08, 130.10, 129.46, 129.18, 128.60, 128.26, 128.10, 127.34, 127.01, 126.75, 126.53, 122.62, 119.67, 119.29, 118.96, 116.99, 66.27, 29.38, 14.71; Anal Calcd for C29H21ClN4O3, C, 68.44; H, 4.16; N, 11.01 found: C, 68.46; H, 4.11; N, 11.06.

2-acetyl-3-methyl-1-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8j) Yellow solid; isolated yield: 88%; mp 195–197 °C; IR (KBr, υ): 3023, 2960, 1652, 1580 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H, H5-pyr), 8.13 (dd, J = 7.9, 1.2 Hz, 1H), 7.91–7.82 (m, 1H), 7.62–7.51 (m, 4H), 7.46 (d, J = 8.0 Hz, 1H), 7.37–7.27 (m, 2H), 7.25–7.15 (m, 2H), 7.04 (d, J = 8.7 Hz, 2H), 6.54 (s, 1H, CHN), 3.15 (3H, s, Me), 2.37 (3H, s, Me), 2.06 (3H, s, Me); 13C NMR (101 MHz, DMSO-d6) δ 193.22, 157.43, 156.64, 154.60, 147.35, 139.72, 135.39, 133.15, 130.49, 129.42, 129.22, 128.44, 128.28, 128.26, 126.81, 126.72, 126.49, 122.75, 119.93, 119.71, 118.66, 116.46, 66.31, 31.41, 21.82, 14.22; Anal Calcd for C30H24N4O3, C, 73.76; H, 4.95; N, 11.47 found: C, 73.72 H, 4.91; N, 11.51.

2-acetyl-3-methyl-1-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8 k) Yellow solid; isolated yield: 87%; mp 201–203 °C; IR (KBr, υ): 3021, 2955, 1651, 1577 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H, H5-pyr), 8.13 (dd, J = 8.0, 1.2 Hz, 1H), 7.85–7.76 (m, 1H), 7.53–7.47 (m, 1H), 7.34–7.20 (m, 7H), 7.20–7.15 (m, 3H), 6.46 (s, 1H, CHN), 3.03 (3H, s, Me), 2.00 (3H, s, Me); 13C NMR (101 MHz, DMSO-d6) δ 193.92, 157.66, 155.08, 154.13, 149.22, 146.87, 139.96, 135.38, 130.05, 129.53, 129.08, 128.43, 128.10, 128.07, 127.49, 127.12, 126.85, 122.72, 120.60, 119.27, 118.28, 116.58, 66.71, 31.04, 15.06; Anal Calcd for C29H21N5O5, C, 67.05; H, 4.07; N, 13.48 found: C, 67.04; H, 4.09; N, 13.51.

2-acetyl-1-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8 l) Yellow solid; isolated yield: 89%; mp 188–190 °C; IR (KBr, υ): 3021, 2953, 1651, 1575 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H, H5-pyr), 8.14 (dd, J = 7.9, 1.2 Hz, 1H), 7.92–7.83 (m, 1H), 7.61–7.56 (m, 3H), 7.50–7.44 (m, 1H), 7.33–7.28 (m, 1H), 7.26–7.16 (m, 2H), 7.05 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 8.7 Hz, 2H), 6.55 (s, 1H, CHN), 3.76 (3H, s, Me), 3.12 (3H, s, Me), 2.03 (3H, s, Me); 13C NMR (101 MHz, DMSO-d6) δ 194.03, 160.54, 157.56, 156.18, 154.71, 145.39, 139.37, 135.38, 133.88, 133.15, 132.16, 131.32, 130.49, 129.93, 127.02, 126.48, 123.54, 120.11, 119.71, 117.89, 115.68, 114.42, 64.64, 54.69, 30.01, 13.15; Anal Calcd for C30H24N4O4, C, 71.42; H, 4.79; N, 11.10 found: C, 71.40; H, 4.81; N, 11.16.

2-acetyl-1-(3-(4-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8 m) Yellow solid; isolated yield: 88%; mp 183–185 °C; IR (KBr, υ): 3021, 2959, 1653, 1580 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H, OH), 8.66 (s, 1H, H5-pyr), 8.15 (dd, J = 8.0, 1.2 Hz, 1H), 7.87–7.77 (m, 1H), 7.58–7.48 (m, 1H), 7.36 (d, J = 7.9 Hz, 1H), 7.33–7.25 (m, 2H), 7.23–7.16 (m, 3H), 7.08–6.98 (m, 2H), 6.79–6.70 (m, 2H), 6.53 (s, 1H, CHN), 3.05 (3H, s, Me), 2.13 (3H, s, Me); 13C NMR (101 MHz, DMSO-d6) δ 193.60, 158.14, 156.81, 156.45, 154.20, 145.31, 139.59, 134.67, 133.00, 130.65, 129.10, 128.55, 128.48, 127.45, 127.08, 126.40, 123.29, 122.82, 119.70, 119.29, 116.02, 115.73, 66.38, 29.76, 14.59; Anal Calcd for C29H22N4O4, C, 71.01; H, 4.52; N, 11.42 found: C, 71.05; H, 4.57; N, 11.46.

2-acetyl-1-(3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-3-methyl-1H-pyrazolo[1,2-b]phthalazine-5,10-dione (8n) Yellow solid; isolated yield: 88%; mp 213–215 °C; IR (KBr, υ): 3022, 2957, 1650, 1576 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H, H5-pyr), 8.12 (d, J = 7.9 Hz, 1H), 7.90–7.82 (m, 1H), 7.60–7.39 (m, 3H), 7.36–7.31 (m, 2H), 7.28–7.20 (m, 4H), 7.14–7.07 (m, 2H), 6.48 (s, 1H, CHN), 3.11 (3H, s, Me), 2.14 (3H, s, Me); 13C NMR (101 MHz, DMSO-d6) δ 193.53, 156.51, 156.37, 154.31, 145.85, 140.09, 135.31, 133.99, 129.77, 129.68, 129.15, 128.92, 128.52, 128.41, 128.27, 127.69, 126.42, 122.44, 119.71, 119.54, 118.38, 117.30, 66.47, 31.23, 14.61; Anal Calcd for C29H21BrN4O3, C, 62.94; H, 3.82; N, 10.12 found: C, 62.90; H, 3.76; N, 10.18.

Saccharomyces cerevisiae α-glucosidase inhibition assay

An α-Glucosidase enzyme ((EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg), Acarbose, and p-nitrophenyl glucopyranoside as substrate were purchased from Sigma-Aldrich. Phosphate buffer saline (135 µL, pH 6.8, 50 mM), a test compound (20 µL) dissolved in DMSO (10% final concentration), and a solution of α-glucosidase (20 µL) were added to the 96-well plate and incubated at 37 °C for 10 min. Then 25 µL of p-nitrophenyl-α-D-glucopyranoside (4 mM) as substrate was added to each well and the absorbance change was recorded before and after 20 min incubation at 37 °C at 405 nm. DMSO was used as the control. Acarbose was used as the standard inhibitor. The percentage of enzyme inhibition was calculated by using the following formula: %Inhibition = [(Abs. control–Abs. test sample)/Abs. control] × 100. Then IC50 values of test compounds were obtained from the nonlinear regression curve in triplicate.

Kinetic studies

The kinetic study was carried out to investigate the inhibition mode of the most potent compound 8l under the mentioned reaction condition. The 20 mL of enzyme solution (1 U/mL) was incubated with different concentrations (0, 63, 125, and 250 μM) of compound 8l for 10 min at 37 °C. Then different concentrations of p-nitrophenyl glucopyranoside (4, 6, 8 & 10 μM) as substrate was added, and the change in absorbance was measured for 20 min at 37 °C at 405 nm using a spectrophotometer (Gen5, Power wave xs2, BioTek, America).

Docking study

Due to the lack of alpha-glucosidase crystal structure, the AlphaFold 2 Protein Structure Database (https://alphafold.com/) was utilized to predict its three-dimensional (3D) structure63. The accuracy of the AlphaFold-generated structure was validated using the Ramachandran plot65, integrated into the PROCHECK online program (https://saves.mbi.ucla.edu/)64. The ligands 8l and Acarbose were modeled using ChemBio3D Ultra 12.0 (https://www.chemdraw.com.cn/) and subjected to energy minimization with Molecular Mechanics (MM2) until reaching a minimum route-mean-square (RMS) gradient of 0.100. To identify the enzyme’s binding site, the 3D structure obtained from AlphaFold was submitted to the CastP tool22.

Molecular docking was performed on the predicted structures using PyRx (AutoDock Vina) software22, based on scoring functions for the structure of Acarbose and the conformation of the most potent compound 8l in the active site of α-glucosidase (shown in Fig. 6). According to the evaluated data from the CastP, the grid box was set as 54 Å × 31 Å × 36.5 Å, and centered at X = 15.5, Y = 2.75, Z = -1.5. The critical parameters, such as grid number and algorithm, were set to default values during docking. The docking analysis was performed using Discovery Studio.

Molecular dynamics

Molecular dynamic simulation was performed to investigate the effect of the most potent compound, 8l, on the active site of α-glycosidase. In this regard, the best docking mode for the high-scoring ligand molecule compound (8l) was selected as the initial atom coordination for molecular dynamics (MD) simulations. The GROMACS molecular dynamics package was used to conduct MD simulations68 employing the AMBER03 force field and the TIP3P water model. Simulations were executed under periodic boundary conditions (PBC) using a dodecahedral simulation box, ensuring a minimum distance of 1.0 Å between the protein and the box’s edges.

The simulation box contained the protein structure of the apoenzyme, as well as the protein structure and heteroatoms of ligand 8l and α-glycosidase. It was filled with TIP3P water molecules, and a physiological concentration of NaCl (0.15 mM) was added to neutralize the system. Energy minimization of all atoms was performed using the steepest descent algorithm. Subsequently, each system underwent equilibration with 2000 ps of NVT followed by 2000 ps of NPT. Finally, a 20 ns MD simulation was carried out with a two fs time step under constant pressure (1 atm) and temperature (310 K).

ADME study

The ADME (Absorption, Distribution, Metabolism, and Excretion) properties of compounds 8a–8n have been evaluated using the free SwissADME (http://www.swissadme.ch/index.php) in silico online toolkit 67. These properties are key determinants of the drug-likeness and oral bioavailability of compounds as a part of preclinical research to recognize potential drug candidates.