Design, synthesis, and molecular dynamics simulation studies of some novel kojic acid fused 2-amino-3-cyano-4H-pyran derivatives as tyrosinase inhibitors

Abstract

A novel series of kojic acid fused 2-amino-3-cyano-4H-pyran derivatives were synthesized via a multicomponent reaction involving kojic acid, benzyloxy benzaldehyde, and malonitrile as tyrosinase inhibitors. Subsequently, the structures of the compounds were characterized using FT-IR, ¹H-, and ¹³C-NMR spectroscopic analyses. The designed compounds fall into three series: (1) 4-benzyloxy-phenyl kojopyran 6a–e, (2) 3-benzyloxy- phenyl kojopyran derivatives 6f–j, and (3) 4-benzyloxy-3-methoxy-phenyl kojopyran derivative 6 k–o. The assessment of tyrosinase inhibition activity was conducted using L-Dopa as the substrate. Among synthesized compounds, 2-amino-4-(4-((4-fluorobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6b) demonstrated the highest antityrosinase activity with a competitive inhibition pattern (IC₅₀ = 7.69 ± 1.99 μM) as compared to the control agent kojic acid (IC₅₀ = 23.64 ± 2.56 µM). Since compound 6b was synthesized as a racemic mixture, in silico studies were performed for both R and S enantiomers. The R- enantiomer showed critical interactions compared with the S-enantiomer. Specifically, it established hydrogen bonds and hydrophobic interactions with crucial and highly conserved amino acids within the enzyme's binding site in the target protein. Moreover, the molecular dynamics simulations revealed that compound 6b demonstrated significant interactions with essential residues of the binding site, resulting in a stable complex throughout the entire simulation run. The drug-like and ADMET properties predictions showed an acceptable profile for compound 6b. Thus, it can serve as a drug candidate to develop more potent antityrosinase agents due to its low toxicity and its high inhibition activity.

Introduction

Melanogenesis is a physiological process that produces a natural pigment called melanin which plays an important role in preventing sun-induced skin injuries in all organisms [1]. Modulating melanogenesis is a widely employed approach for addressing irregular skin pigmentation using both pharmaceutical and cosmetic interventions [2].

Tyrosinase enzyme plays a key role in the early stage of melanogenesis by catalyzing the oxidation of phenol to o-quinone observed. Enzyme activity may cause disturbances in pigment production [3]. Excessive production and hyperpigmentation of melanin are seen in various skin disorders such as freckles, age spots, pregnancy spots, melisma, pigmented scars caused by acne, and melanoma as one of the deadly types of cancer [4].

Browning in plants and fruits such as mushrooms, apples, pears, and bananas results from overactivity of the tyrosinase enzyme. It leads to a decrease in these products' quality and commercial value [5]. On the other hand, the defense mechanisms, such as skin shedding and wound healing in insects, are accomplished by tyrosinase enzyme. As a result, the inhibition of tyrosinase enzyme in plants and insects can have great importance in agriculture industries [6].

Tyrosinase is also related to neurodegenerative diseases such as Parkinson’s by oxidizing excess dopamine to produce dopamine quinone. These highly reactive species induce neural damage and cell death, and the production of a pigment called neuromelanin in the human brain and some neurons. An imbalance in dopamine quinones and neuromelanin levels is associated with the development of neurodegenerative disorders [7, 8].

Kojic acid isolated from the fungus Aspergillus oryzae is a well-known competitive inhibitor against antityrosinase and free radical scavenger agent [9]. As a result, the development of new kojic acid Although using products containing kojic acid may be considered safe for most people, there are some risks and possible side effects such as sunburn and contact dermatitis in some people, especially those with sensitive skin. As a result, the development of new kojic acid-related compounds that have fewer side effects is considered by many scientists [10, 11]. On another hand, pyran-fused compounds have long captivated the attention of synthetic and biological researchers due to their diverse range of chemical and biological properties [12].

Therefore, medicinal chemists have used kojic acid and pyran core (compounds a, b, d, c, Fig. 1) as effective pharmacophores in designing the new tyrosinase inhibitors [12,13,14,15,16,17]. Based on the biological importance of kojic acid compounds and in continuation of our research programs [18, 19], some novel kojic acid fused 2-amino-3-cyano-4H-pyran derivatives were designed, synthesized, and evaluated as tyrosinase inhibitors. Afterward, kinetic and in silico studies including ADMET, molecular docking, and molecular dynamics simulation, were conducted to predict the potential of potent derivatives as ideal drug candidates for further studies.

Fig. 1

Kojic acid and 4H-pyran related structures as tyrosinase inhibitors and newly designed hybrid

Results and discussion

Chemistry

The synthetic routes for the synthesis of kojic acid fused 2-amino-3-cyano-4H-pyran derivatives 6a–o were shown in Fig. 2. For this purpose, in the first step, various benzyloxy-benzaldehydes 3a–o were obtained by the nucleophilic reaction between 4-hydroxyl-benzaldehydes or 3-hydroxyl-benzaldehydes or vanillin (1a–c) and benzyl halide derivatives 2a–e in the presence of potassium carbonate in DMF at the room temperature [20]. In the second step, the multicomponent reaction between benzyloxy-benzaldehydes 3a–o, malonitrile 4 and kojic acid 5 was conducted in ethanol (5 ml) in reflux condition to produce the target compounds 6a–o in good to acceptable yields [21]. The structure of all new kojic acid derivatives was confirmed by ¹H, ¹³C NMR, IR spectroscopy, and elemental analysis.

Fig. 2

General procedure for the synthesis of kojic acid fused 2-amino-3-cyano-4H-pyran derivatives 6a–o

Tyrosinase inhibitory activity

The inhibitory activities of the kojic acid fused 2-amino-3-cyano-4H-pyran derivatives 6a-o on mushroom tyrosinase were investigated using kojic acid as a positive control by evaluation of their potential to inhibit enzymatic oxidation of L-Dopa. All results are presented as mean ± SE in Table 1. The synthesized products shown in Fig. 2 can be divided into three series based on starting aldehyde: (1) 4-benzyloxy-phenyl kojopyran 6a–e (4-hydroxy-benzaldehyde derived compounds), (2) 3-benzyloxy- phenyl kojopyran derivatives 6f–j (3-hydroxy-benzaldehyde derived compounds), and (3) 4-benzyloxy-3-methoxy-phenyl kojopyran derivative 6 k–o (vanillin derived compounds).

Table 1 Tyrosinase inhibitory activities of compounds 6a-o

Among synthesized compounds 6a–o, compound 6b exhibited the most inhibitory activity against tyrosinase enzyme with an IC₅₀ value of 7.69 ± 1.99 µM, which was better than kojic acid inhibitory activity (IC₅₀ = 23.64 ± 2.56 µM). As seen in Table 1, the first and second groups, compounds 6a–j, indicated better inhibitory activities than the third group 6k–o. It seems that the methoxy group and the phenyl ring diminished antityrosinase activity. Both electronic and spatial effects can reduce the third group's activity, although spatial effects play a more important role. In the first series, compound 6a, without substituting the benzyl moiety, was inactive against the tyrosinase enzyme. The introduction of electron-withdrawing groups (EWGs), including 4-F, 4-Cl, and 4-Br on the benzyl pendant group were, generated compounds 6b, 6c, and 6d with IC₅₀ values of 7.69, 79.25, and 33.96 µM, respectively. Notably, electron-withdrawing effect orders are F > Cl > Br. The compound 6b with fluorine at benzyl moiety's 4th position illustrated better inhibitory activity than chlorine and bromine atoms. Also, a comparison between compound 6c having 4-chlorine and 6e having 4-methyl as electron donating group (EDG) at the benzyl moiety with the same size showed that electron-withdrawing groups could produce more potent agents than the electron donating group. The second series of the synthesized compounds 6f–j were structurally different from the first series since pendant benzyl moiety connected to phenyl at the 3rd position. The compound 6f exhibited no inhibitory activity towards tyrosinase.

Similar to the first series introduction of EWGs, including 4-F, 4-Cl, and 4-Br led to improve antityrosinase activities in the compounds 6g, 6h, and 6i with IC₅₀ values of 9.72, 10.66, and 15.34 µM. It also introduced the 4-methyl group as an EDG at the benzyl pendant moiety that created compound 6j without any activity. Fluorine as a small EWG apparently produced more effective ligand-enzyme interaction than chlorine and bromine atoms as large EWGs.

Kinetic studies

Mechanism and type of inhibition were elucidated through kinetic investigations. Lineweaver utilized Burk plot analysis to determine the enzyme inhibition mode, employing the most potent derivative 6b (see Fig. 3A). Lineweaver–Burk plots, depicting 1/V against 1/[S], were constructed to illustrate the inhibition of tyrosinase by 6b across varying concentrations of both 6b and L-Dopa, the substrate. In this context, K_m represents the Michaelis–Menten constant, while V_max signifies the maximum reaction velocity.

Fig. 3

a Lineweaver − Burk plot of tyrosinase inhibition by different concentrations of 6b in the presence of L-DOPA. b The secondary Lineweaver–Burk plot of compound 6b

Upon analyzing the data, it was observed that the enzyme's V_max remained consistent, while the K_m values exhibited a rise when subjected to increasing concentrations of compound 6b (depicted in Fig. 3A). Based on these findings, it can be inferred that the inhibitory effect of compound 6b is competitive. This deduction is further supported by the secondary graph (Fig. 3B) portraying the relationship between Km and varied concentrations of compound 6b, facilitating the estimation of inhibition constants (K_i = 7.57 µM).

Drug-likeness prediction

To remove molecules with unfavorable properties from screening libraries and increase the chance of drug discovery, the drug-likeness concept was first proposed by Lipinski known as the rule of five to define the physicochemical ranges of an oral drug. This rule includes the molecular weight (MW ≤ 500), the number of hydrogen bonding acceptors (HBA ≤ 10), the number of hydrogen bonding acceptors (HBD ≤ 5), the lipophilicity index (logP ≤ 5), rotatable bond count (RBC ≤ 10) and polar surface area (PSA ≤ 140) of compounds. Two online servers, including pkCSM and SwissADME, were used to calculate these parameters [22, 23]. Some compounds (6b, 6d, 6g, 6h, and 6i) with the best anti-tyrosinase activities were selected to evaluate physicochemical properties. Data in Table 2 confirm that all selected compounds successfully passed Lipinski's rule.

Table 2 Physicochemical properties of the selected synthesized compounds (6b, 6d, 6g, 6h, and 6i)

Prediction of ADMET properties

ADMET describes the Absorption, Distribution, Metabolism, Excretion, and Toxicity of a pharmaceutical compound within an organism. ADMET properties enable drug developers to investigate the safety and efficacy of drugs for preclinical and clinical development. Table 3 shows ADMET prediction for the selected compounds (6b, 6d, 6g, 6h, and 6i) calculated by pkCSM and SwissADME online servers [22, 23]. The estimated Human Intestinal Absorption (HIA) of the selected compounds is high, which illustrates high absorption by gastrointestinal tracts. All compounds are predicted to have acceptable skin permeability. The steady-state volume of distribution (VDss) refers to the volume where the total medication content is distributed to attain a consistent concentration in the blood plasma. The acceptable value for VDss is 0.45 > LogVDss > − 0.15. All synthesized compounds are within the defined range. Regarding metabolism, all compounds were predicted not to be inhibitors or substrates of CYP450 2D6 while expected to be CYP450 3A4 and 2C9 inhibitors. For the excretion, none of the compounds is expected to be substrates for the Renal Organic Cation Transporter 2 (OCT2). Finally, all products are not considered toxic regarding AMES, skin sensitization and hERG I channel inhibition. Also, the calculated skin permeability for all compounds was sited in the allowed range. According to these data, it could be predicted that the selected potent compounds (6b, 6d, 6g, 6h, and 6i) can have an acceptable pharmacokinetic profile.

Table 3 ADMET^a prediction of the synthesized compounds 6b, 6d, 6g, 6h, and 6i

Molecular docking study

This study employed the induced fit docking method to analyze the most potent compound 6b binding mode within the tyrosinase active site. To ensure the reliability of the docking protocol, re-docking experiment was performed with the crystalographic inhibitor tropolone into the enzyme’s active site. The resulting superimposed structures of the docked and crystallized tropolone showed good agreement, with a record RMSD value of less than 2 Å (Fig. 4).

Fig. 4

The superimpose structure of crystalographic tropolone (yellow) vs docked tropolone (blue)

Next, induced fit docking was applied to 6b as the most potent analog. It should be noted that compound 6b was produced as a racemic mixture including R- and S- enantiomers; as a result, comprehensive molecular docking studies were undertaken to evaluate both enantiomers in the active site of tyrosinase.

The results of S- enantiomer of compound 6b within the tyrosinase binding site are depicted in Fig. 5, which recorded the binding energy of − 6.304 kcal/mol. The OH group of S- enantiomer 6b exhibited H-bound interaction with Met280 (1.76 Å), and the other H-bound interaction is recorded between NH₂ and Glu256 residue (1.95 Å). Kojopyran moiety also participated in pi-pi stacking interaction with His263 (3.96 Å).

Fig. 5

3D and 2D interaction of S-enantiomer of compound 6b in complex with tyrosinase

The interaction between tyrosinase and R- enantiomer of compound 6b is depicted in Fig. 6 with the binding energy of − 6.825 kcal/mol. In this complex, the kojopyran moiety engaged in a hydrogen bonding interaction with Gly281 (2.52 Å). In contrast, the NH group of kojopyran participated in a pi-pi stacking interaction with Phe264 (5.18 Å). Furthermore, the OH group of R- enantiomer 6b exhibited metal chelation with Cu401 (2.44 Å) involving residues categorized as critical amino acids for the binding. Additionally, the 4-fluoro-benzyl terminal of the compound formed another important pi-pi stacking interaction with His244 (5.14 Å), further contributing to the compound's binding affinity.

Fig. 6

3D and 2D interaction of R-enantiomer of compound 6b in complex with tyrosinase

Molecular dynamics simulations

It is worth noting that the molecular docking study revealed no significant disparities in the binding energies between the S- and R- enantiomers. Consequently, molecular dynamics simulations were accompanied to extract the optimal enantiomeric conformation with better interaction and stability with the enzyme.

An analysis of the root mean square deviation (RMSD) data throughout the molecular dynamics run indicated that during the initial three nanoseconds (ns) R-enantiomer 6b, there was a significant increase in RMSD, reaching a value of 2 Å (Fig. 7). However, after this initial phase, a gradual decrease in RMSD occurred, indicating a convergence towards a more stable configuration. Subsequently, the RMSD values recorded steady fluctuations around 1.5 Å. This behavior suggested that the simulation had reached equilibrium, and the protein–ligand complex remained relatively stable during the molecular dynamics run. Such stability is crucial for potent enzyme inhibitor, as it demonstrates a consistent and prolonged inhibition of the enzyme activity. In contrast, S-6b exhibited less stability than the R- 6b isomer with an average value of 3.5 Å.

Fig. 7

RMSD of the tyrosinase backbone in complexed R- enantiomer 6b (in blue) S- enantiomer 6b (in orange) for over 30 ns molecular dynamics simulation time

The root mean square fluctuation (RMSF) plot of R-6b and S-6b, shown in Fig. 8, illustrates the flexibility of the enzyme structure concerning its secondary structure elements. It can be observed that the N- and C-terminal ends of the enzyme and the unstructured regions exhibited higher fluctuations compared to the secondary structure elements. This is common as terminal regions and unstructured parts are generally more dynamic. The results revealed that a reduction in residue motion was observed upon binding the ligand to tyrosinase. This reduction can be attributed to the non-bonding interactions between the ligand and the enzyme. The most significant disparities in RMSF between the two systems were found at residue indices that directly interacted with the ligand. These residues experienced reduced fluctuations, indicating that the ligand binding stabilized these regions by restricting their motion Overall, the RMSF analysis demonstrated that the enzyme- 6b (R) complex exhibited lower RMSF values than the apoenzyme. More stability was further corroborated by the RMSF analysis of R-6b (highlighted in green), which demonstrated fewer fluctuations in the ligand’s atoms vs of S- 6b. This suggests an increased propensity for interactions with the enzyme’s binding site.

Fig. 8

a RMSF of the R- enantiomer 6b (in blue) S- enantiomer 6b (in orange) for over 30 ns molecular dynamics simulation time

Figure 9 shows RMSF values for the heavy atoms of ligand R-6b bound to tyrosinase. All R- enantiomer 6b atoms, except those in the 4-fluoro benzyl substituted regions, exhibit RMSF values below 2 Å. This low fluctuation indicates that these atoms form a stable complex with tyrosinase due to strong intermolecular interactions, limiting their movements during the molecular dynamics simulation. However, RMSF values for the heavy atoms of ligand S-6b (Fig. 10) demonstrated higher RMSF value, particularly evidenced by the RMSD value exceeding 4 Å for the para-fluorobenzyl moiety. These results suggest that ligand R-6b has the potential to be an effective inhibitor and more stable in the tyrosinase active site.

Fig. 9

RMSF graph of the heavy atoms of R- enantiomer 6b in complex with tyrosinase. The structure of R- enantiomer 6b is depicted, exhibiting the regions of the molecule with the highest fluctuations

Fig. 10

RMSF graph of the heavy atoms of S- enantiomer 6b in complex with tyrosinase. The structure of S- enantiomer 6b is depicted, exhibiting the regions of the molecule with the highest fluctuations

Figure 11a presents a detailed timeline of interactions during the molecular dynamics simulation. The enzyme’s interactions with the ligand were continuously monitored throughout the simulation, and these interactions have been categorized and summarized in Fig. 8b. The interactions of R- enantiomer 6b with the active site pocket of the enzyme were observed to occur for more than 50% of the simulation duration. These interactions can be summarized as follows (Fig. 8c):

Fig. 11

a A timeline representation of the interactions and contacts, b Protein- R- enantiomer 6b contacts based on the type of interactions, c Protein residues interactions with R- enantiomer atom

The OH group of the kojopyran ring effectively participated in a critical metal chelation interaction with the Cu cofactor, persisting throughout 99% of the molecular dynamics run. This metal chelation interaction also affected the interactions with other amino acids, including His256, His263, His296, and Glu256.

The NH group of kojopyran demonstrated hydrogen bonding interaction with Gly281, which occurred approximately 55% of the simulation time.

Additionally, the mentioned ring structure of the ligand was involved in a pi-pi stacking interaction with Phe264. Interestingly, there is a high similarity between the docking interaction of R- enantiomer 6b and the results of molecular dynamics. A similar interaction is observed in molecular dynamics simulation vs molecular docking, so the OH group of the kojopyran ring participated in a critical metal chelation interaction with the Cu cofactor, and NH of the kojopyran ring showed hydrogen bonding interaction with Gly281. Similarly, phe264 exhibited pi-pi stacking interaction with kojopyran.

The timeline of interactions during the molecular dynamics simulation of S- enantiomer of 6b is demonstrated in Fig. 12a. Also, the enzyme's interactions with the ligand were continuously monitored throughout the simulation, summarized in Fig. 12b. Importantly, the S- enantiomer displayed no interactions with the enzyme's binding site throughout the simulation in 30% run time, and just one interaction with Met290 mediated with water is presented in 25% of simulation run (12c). The interaction with this residue was also recorded in molecular docking study.

Fig. 12

a A timeline representation of the interactions and contacts, b Protein- S- enantiomer of 6b contacts based on the type of interactions, c Protein residues interactions with S- enantiomer of 6b atom

Consequently, molecular dynamics evaluations of R and S- enantiomer of 6b provided valuable insights into the binding interactions between 6b enantiomers and the tyrosinase active site, supporting the role of R- enantiomer of 6b as a promising inhibitor.

Conclusions

As a result, a new series of some novel kojic acid fused 2-amino-3-cyano-4H-pyran derivatives as tyrosinase inhibitors were designed, synthesized, and asse R- enantiomer assessed against tyrosinase enzyme. Among synthesized derivatives, compound 6b showed the most potent antityrosinase effect with an IC₅₀ value of 7.69 ± 1.99 μM and a competitive mode of kinetic inhibition as compared to kojic acid as the control agent (23.64 ± 2.56 µM).

The in silico study R- enantiomer of compound 6b within the enzyme’s binding site demonstrated significant interactions with critical and highly conserved amino acids, including hydrogen bonds and hydrophobic interactions. Additionally, molecular dynamics simulations of compound 6b (R) revealed strong and stable interactions of the kojopyran moiety with essential residues and the Cu cofactor in the binding site. Furthermore, during the simulation run, the tyrosinase-6b complex was stable. The drug-like and ADMET properties predictions showed an acceptable profile for these agents. Our results proposed that compound 6b can serve as a drug candidate to develop more potent antityrosinase agents.

Experimental Section

Chemistry

All chemical reagents were sourced from Merck and Sigma and employed without additional purification steps. Melting points were determined using a Stuart melting point smp3 apparatus. The structural integrity of all synthesized compounds was validated by utilizing IR spectra, ¹H-NMR, ¹³C-NMR, and MS spectroscopy. The NMR data (¹H and ¹³C) and IR spectra were acquired using a Bruker 400-NMR and ALPHA FT-IR spectrometer on KBr disks. Chemical shifts (δ) and coupling constants (J) were expressed in parts per million (ppm) and Hertz, respectively. The atom numbering of the target compounds, derived from their IUPAC nomenclature, was employed for the assignment of ¹H-NMR data. The original spectra of the investigated compounds are provided as Additional file 1.

General procedure for the synthesis of Benzyloxy-benzaldehyde derivatives 3a-o

In a 25 ml round-bottom flask, a mixture of 3-hydroxybenzaldehyde or 4-hydroxybenzaldehyde or vanillin 1a–c (1 mmol), various benzyl halides 2a–e (1.1 mmol), and potassium carbonate (1.1 mmol) in dimethylformamide solvent 5 ml (DMF) were stirred at room temperature until the reaction was complete. The progress of the reaction was monitored using thin-layer chromatography (TLC). After the completion of reaction, ice was added to the mixture and stirred for 10 min. Then, the precipitate 3a–o were filtered and washed with cold water. They were used for the next step without further purification [20].

General procedure for the synthesis of dihydropyrano [3, 2-b] pyran-3-carbonitrile 6a-o

In a 25 ml round-bottom flask, a three-component reaction including benzyloxy aldehyde derivatives 3a–o (1.2 mmol), kojic acid 4 (1 mmol), and malononitrile 5 (1.2 mmol) was refluxed in the presence of ammonium acetate as the catalyst in ethanol. TLC was used to follow the reaction progress. Following the completion of the reaction, the products 6a–o were isolated through filtration and subjected to a wash with an ethanol/water mixture [21].

2-Amino-4-(4-(benzyloxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6a).

White solid; Mp: 221–224 °C; IR (KBr): υ (cm⁻¹) = 3400, 3327, 3207, 2874, 2199, 1677. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.10 (m, 2H, -CH₂), 4.74 (s, 1H, -CH), 5.09 (s, 2H, -CH₂), 5.70 (m, 1H, -CH₂), 6.63 (s, 1H, -OH), 7.03 (d, 2H, -NH₂), 7.20 (dd, J = 8.8 Hz, 2H, Aromatic), 7.36 (m, 5H, Aromatic).¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 165.6, 168.2, 159.1, 157.9, 149.2, 136.9, 136.1, 133.0, 128.9, 128.4, 127.9, 127.7, 119.3, 115.0, 111.3, 69.2, 59.1 and 55.9. Anal.Calcd for C₂₃H₁₈N₂O₅: C, 68.65; H, 4.51; N, 6.96. Found: C, 68.43; H, 4.75; N, 6.90.

2-Amino-4-(4-((4-fluorobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6b).

White solid; Mp: 227–230 °C; IR (KBr): υ (cm⁻¹) = 3271, 2207, 1645, 1598. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.32 (s, 3H, -CH₃), 4.11 (m, 2H, -CH₂), 4.72 (s, 1H, -CH), 5.08 (s, 2H, -CH₂), 5.70 (t, 1H, -CH₂), 6.35 (s, 1H, -OH), 6.87 (d, 2H, -NH₂), 6.89 (m, 1H, Aromatic), 7.00 (m, 4H, Aromatic), 7.24 (m, 1H, Aromatic), 7.54 (m, 2H, Aromatic).¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.5, 168.1, 162.9, 160.5, 159.3, 158.6, 148.8, 142.3, 136.4, 133.1, 120.1, 119. 2, 115.3, 114.4, 113.8, 111.4, 68.5, 59.1 and 55.5. Anal.Calcd for C₂₃H₁₇FN₂O₅: C, 65.71; H, 4.08; N, 4.52. Found: C, 65.82; H, 4.15; N, 4.61.

2-Amino-4-(4-((4-chlorobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8- dihydropyrano[3,2-b]pyran-3-carbonitrile (6c).

White solid; Mp: 236–239 °C; IR (KBr): υ (cm⁻¹) = 3175, 2197, 1638, 1509. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.10 (m, 2H, -CH₂), 4.74 (s, 1H, -CH), 5.09 (s, 2H, -CH₂), 5.70 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 7.01 (d, 2H, -NH₂), 7.22 (m, 4H, Aromatic), 7.48 (td, 4H, Aromatic).¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.5, 168.1, 159.1, 157.8, 149.2, 136.4, 136.1, 133.0, 132.5, 130.1, 129.5, 128.9, 120.9, 119.3, 115.9, 111.3, 68.4, 59.1 and 55.8. Anal.Calcd for C₂₃H₁₇ClN₂O₅: C, 65.71; H, 4.08; N, 4.52. Found: C, 65.82; H, 4.15; N, 4.61.

2-Amino-4-(4-((4-bromobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8 dihydropyrano[3,2-b]pyran-3-carbonitrile (6d).

White solid; Mp: 214–216 °C; IR (KBr): υ (cm⁻¹) = 3406, 3273, 2191, 1636, 1488. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.10 (m, 2H, -CH₂), 5.08 (s, 1H, -CH), 5.09 (s, 2H, -CH₂), 5.70 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 7.02 (d, 2H, -NH₂), 7.22 (m, 4H, Aromatic), 7.41 (td, 2H, Aromatic), 7.59 (t, 2H, Aromatic) .¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.5, 168.1, 160.4, 159.1, 157.8, 149.2, 136.4, 136.1, 133.2, 131.5, 130.1, 129.8, 128.9, 120.9, 119.3, 115.9, 111.3, 68.4, 59.1 and 55.8. Anal.Calcd for C₂₃H₁₇BrN₂O₅: C, 57.40; H, 3.56; N, 5.82. Found: C, 57.58; H, 3.45; N, 5.71.

2-Amino-6-(hydroxymethyl)-4-(4-((4-methylbenzyl)oxy)phenyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6e).

Yellow solid; Mp: 215–218 °C; IR (KBr): υ (cm⁻¹) = 3417, 2193, 1635, 1512.¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.32 (s, 3H, -CH₃), 4.10 (m, 2H, -CH₂), 4.73 (s, 1H, -CH), 5.04 (s, 2H, -CH₂), 5.70 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 7.01 (d, 2H, -NH₂), 7.19 (m, 6H, Aromatic), 7.35 (m, 2H, Aromatic).¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.5, 168.1, 159.1, 157.9, 149.2, 137.1, 136.1, 133.9, 132.9, 129.1, 128.9, 127.8, 119.3, 115.9, 115.0, 111.3, 69.1, 59.1, 55.9 and 20.8. Anal.Calcd for C₂₄H₂₀N₂O₅: C, 69.22; H, 4.84; N, 6.73. Found: C, 69.28; H, 4.93; N, 6.65.

2-Amino-4-(3-(benzyloxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6f).

White solid; Mp: 235–238 °C; IR (KBr): υ (cm⁻¹) = 3271, 2191, 1637, 1487.

¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.10 (m, 2H, -CH₂), 4.24 (s, 1H, -CH), 5.10 (s, 2H, -CH₂), 5.69 (t, 1H, -CH₂), 6.86 (s, 1H, -OH), 6.86 (m, 1H, Aromatic), 6.98 (s, 1H, Aromatic), 7.31 (d, 2H, -NH₂), 7.34 (m, 4H, Aromatic), 7.45 (m, 2H, Aromatic).¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.5, 168.2, 159.2, 158.7, 142.4, 136.8, 136.4, 130.1, 128.4, 127.9, 127.8, 120.1, 119.2, 114.4, 113.8, 111.4, 69.2, 59.1 and 55.5. Anal.Calcd for C₂₃H₁₈N₂O₅: C, 68.65; H, 4.51; N, 6.96. Found: C, 68.77; H, 4.39; N, 6.88.

2-Amino-4-(3-((4-fluorobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6g).

White solid; Mp: 239–242 °C; IR (KBr): υ (cm⁻¹) = 3192, 2194, 1634, 1508. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.10 (m, 2H, -CH₂), 4.75 (s, 1H, -CH), 5.08 (s, 2H, -CH₂), 5.69 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 7.02 (d, 2H, Aromatic), 7.22 ( m, 6H, Aromatic), 7.52 (d, 2H, -NH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.6, 168.1, 162.9, 160.5 159.1, 157.9, 149.2, 136.1, 133.2, 130.1, 129.9, 128.9, 119.3, 115.3, 111.3, 68.5, 59.1 and 55.8. Anal.Calcd for C₂₃H₁₇FN₂O₅: C, 65.71; H, 4.08; N, 6.66. Found: C, 65.67; H, 4.15; N, 6.16.

2-Amino-4-(3-((4-chlorobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6h).

White solid; Mp: 232–236 °C; IR (KBr): υ (cm⁻¹) = 3406, 3273, 2191, 1636. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.10 (m, 2H, -CH₂), 4.77 (s, 1H, -CH), 5.10 (s, 2H, -CH₂), 5.70 (m, 1H, -CH₂), 6.89 (s, 1H, -OH), 6.97 (d, 2H, Aromatic), 6.99 (d, 1H, Aromatic), 7.30 (d, 2H, -NH₂), 7.31 (m, 1H, Aromatic), 7.33 (m, 4H, Aromatic). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.5, 168.1, 159.2, 158.5, 148.8, 142.5, 136.4, 135.9, 132.4, 130.1, 129.6, 128.4, 120.2, 119.2, 114.4, 113.8, 111.4, 68.3, 59.1, and 55.5. Anal.Calcd for C₂₃H₁₇ClN₂O₅: C, 63.24; H, 3.92; N, 6.41. Found: C, 63.17; H, 4.05; N, 6.38.

2-Amino-4-(3-((4-bromobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6i).

White solid; Mp: 236–238 °C; IR (KBr): υ (cm⁻¹) = 3302, 2193, 1639, 1510. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.10 (m, 2H, -CH₂), 4.77 (s, 1H, -CH), 5.09 (s, 2H, -CH₂), 5.69 (m, 1H, -CH₂), 6.34 (s, 1H, -OH), 6.87 (d, 2H, Aromatic), 6.91 (d, 1H, Aromatic), 7.26 (d, 2H, -NH₂), 6.99 (m, 1H, Aromatic), 7.35 (dd, 2H, Aromatic), 7.60 (m, 2H, Aromatic). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.5, 168.1, 159.2, 158.5, 148.8, 142.5, 136.4, 135.9, 132.4, 130.1, 129.6, 128.4, 120.2, 119.2, 114.4, 113.8, 111.4, 68.3, 59.1, and 55.5. Anal.Calcd for C₂₃H₁₇BrN₂O₅: C, 57.40; H, 3.56; N, 5.82. Found: C, 57.47; H, 3.63; N, 5.88.

2-Amino-4-(3-((para-tolyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6j).

Brown solid; Mp: 245–247 °C; IR (KBr): υ (cm⁻¹) = 3431, 2200, 1634.

¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.31 (s, 3H, -CH₃), 4.10 (m, 2H, -CH₂), 4.73 (s, 1H, -CH), 5.09 (s, 2H, -CH₂), 5.69 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 7.01 (d, 2H, Aromatic), 7.19 (m, 6H, Aromatic), 7.35 (d, 2H, -NH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.6, 168.2, 159.1, 157.9, 149.2, 137.1, 136.1, 133.9, 132.9, 128.8, 127.8, 119.3, 115.0, 111.3, 69.1, 59.1, 55.9 and 20.8. Anal.Calcd for C₂₃H₁₈N₂O₅: C, 68.65; H, 4.51; N, 6.96. Found: C, 68.76; H, 4.67; N, 6.98.

2-Amino-4-(4-(benzyloxy)-3-methoxyphenyl)-6-(hydroxymethyl)-8-oxo-4,8 dihydropyrano[3,2-b]pyran-3-carbonitrile (6k).

Yellow solid; Mp: 234–237 °C; IR (KBr): υ (cm⁻¹) = 3336, 2196, 1652, 1511. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.13 (s, 3H, -CH₃), 4.10 (m, 2H, -CH₂), 4.75 (s, 1H, -CH), 5.07 (s, 2H, -CH₂), 5.69 (m, 1H, -CH₂), 6.34 (s, 1H, -OH), 6.77 (d, 1H, Aromatic), 6.77 ( s, 1H, Aromatic), 6.90 (d, 1H, Aromatic), 7.40 (d, 2H, -NH₂), 7.43 (m, 5H, Aromatic). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.6, 168.1, 159.2, 149.1, 147.5, 137.0, 136.1, 133.5, 128.4, 127.9, 119.7, 113.5, 111.6, 69.9, 59.1, and 55.6. Anal.Calcd for C₂₄H₂₀N₂O₅: C, 66.66; H, 4.66; N, 6.48. Found: C, 66.73; H, 4.57; N, 6.41.

2-Amino-4-(4-((4-fluorobenzyl)oxy)-3-methoxyphenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6l).

Brown solid; Mp: 232–235 °C; IR (KBr): υ (cm⁻¹) = 3320, 2200, 1658, 1590.¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.77 (s, 3H, -CH₃), 4.19 (s, 2H, -CH₃), 4.25 (s, 1H, -CH), 4.75 (s, 2H, -CH₂), 5.69 (m, 1H, -CH₂), 6.34 (s, 1H, -OH), 6.77 (d, 1H, Aromatic), 6.79 ( s, 1H, Aromatic), 6.94 (d, 1H, Aromatic), 7.43 (m, 4H, Aromatic), 7.52 (d, 2H, -NH₂). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.8, 168.1, 162.9, 160.5, 159.2, 149.1, 147.3, 136.1, 133.6, 130.1, 119.7, 115.3, 113.5, 111.6, 69.1, 59.1, 55.6, and 55.5. Anal.Calcd for C₂₄H₁₉FN₂O₅: C, 64.00; H, 4.25; N, 6.22. Found: C, 63.98; H, 4.27; N, 6.18.

2-Amino-4-(4-((4-cholorobenzyl)oxy)-3-methoxyphenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6m).

White solid; Mp: 210–213 °C; IR (KBr): υ (cm⁻¹) = 3331, 2221, 1631, 1518. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.77 (s, 3H, -CH₃), 4.18 (s, 2H, -CH₃), 4.75 (s, 1H, -CH), 5.07 (s, 2H, -CH₂), 5.68 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 6.76 (d, 1H, Aromatic), 6.79 ( s, 1H, Aromatic), 6.91 (d, 1H, Aromatic), 7.06 (s, 2H, -NH₂), 7.48 (s, 4H, Aromatic). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.6, 168.1, 159.2, 149.1, 147.2, 136.1, 133.6, 132.4, 129.6, 128.4, 119.7, 113.6, 116.7, 111.3, 69.0, 59.1, 55.6 and 55.5. Anal.Calcd for C₂₄H₁₉ClN₂O₅: C, 61.74; H, 4.10; N, 6.00. Found: C, 61.65; H, 4.17; N, 6.10.

2-Amino-4-(4-((4-boromobenzyl)oxy)-3-methoxyphenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6n).

Yellow solid; Mp: 202–206 °C; IR (KBr): υ (cm⁻¹) = 3331, 2221, 1631, 1518.¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.77 (s, 3H, -CH₃), 4.13 (s, 2H, -CH₃), 4.23 (s, 1H, -CH), 4.75 (s, 2H, -CH₂), 5.69 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 6.76 (d, 1H, Aromatic), 6.79 ( s, 1H, Aromatic), 6.90 (d, 1H, Aromatic), 7.05 (s, 2H, -NH₂), 7.42 (d, 2H, Aromatic), 7.60 (d, 2H, Aromatic). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.6, 168.1, 159.2, 149.1, 147.2, 136.5, 133.7, 131.3, 129.9, 120.9, 120.9, 119.7, 113.6, 111.6, 69.1, 59.1, and 55.6. Anal.Calcd for C₂₄H₁₉BrN₂O₅: C, 61.74; H, 4.10; N, 6.00. Found: C, 61.65; H, 4.17; N, 6.10.

2-Amino-6-(hydroxymethyl)-4-(3-methoxy-4-((4-methylbenzyl)oxy)phenyl)-8-oxo-4,8 dihydropyrano[3,2-b]pyran-3-carbonitrile (6o).

Brown solid; Mp: 241–245 °C; IR (KBr): υ (cm⁻¹) = 3345, 2200, 1643, 1500. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 2.33 (s, 3H, -CH₃), 3.76 (s, 3H, -CH₃), 4.18 (s, 2H, -CH₃), 4.75 (s, 1H, -CH), 5.01 (s, 2H, -CH₂), 5.73 (m, 1H, -CH₂), 6.33 (s, 1H, -OH), 6.76 (d, 1H, Aromatic), 6.79 ( s, 1H, Aromatic), 6.91 (d, 1H, Aromatic), 7.06 (s, 2H, -NH₂), 7.48 (s, 4H, Aromatic). ¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.6, 168.1, 159.2, 149.1, 147.4, 137.1, 136.1, 133.9, 128.9, 127.9, 119.6, 113.4, 111.6, 111.3, 69.7, 59.1, 55.6, 20.8. Anal.Calcd for C₂₅H₂₂N₂O₅: C, 67.26; H, 4.97; N, 6.27. Found: C, 67.31; H, 5.07; N, 6.33.

Tyrosinase inhibition assay

We conducted the assay of mushroom tyrosinase (EC 1.14.18.1) utilizing L-Dopa as the substrate, as previously described, with minor adaptations [24]. We employed a spectrophotometric technique to assess the enzyme's diphenolase activity, observing dopachrome formation at 490 nm. All compounds being tested and kojic acid were initially dissolved in DMSO and diluted to the desired levels. In a 96-well microplate, 10 µl of the test samples were combined with 140 µl of 50 mM phosphate buffer (pH = 6.8). Subsequently, 20 µl of tyrosinase (273 U mL⁻¹) was introduced. Following a pre-incubation period of 20 min at 28 °C, 20 µl of L-Dopa solution (resulting in a final concentration of 0.7 mM) was added to the mixture. Following a 10-min incubation, the absorbance of the samples was measured. The inhibitory activity of the tested compounds was stated as IC₅₀. The ratio percentage of inhibition was calculated according to the equation below:

$${\text{Inhibition }}\left( \% \right) \, = { 1}00 \, \left( {{\text{Abs}}_{{{\text{control}}}} - {\text{ Abs}}_{{{\text{compound}}}} } \right)/{\text{Abs}}_{{{\text{control}}}}$$

Determination of the inhibition type

For the purpose of kinetic analysis, we focused on 6b, the most potent derivative. The inhibitor concentrations employed ranged from 2 to 20 μM, while the substrate (L-Dopa) concentrations in the rane of 0.1 mM to 1.5 mM through all kinetic experiments. The pre-incubation and measurement durations followed the protocol outlined in the mushroom tyrosinase inhibition assay [24]. The maximum initial velocity was ascertained from the initial linear segment of absorbance within a 10-min window following the introduction of L-Dopa, with measurements taken at 1-min intervals. Utilizing the Lineweaver–Burk plot technique, we calculated the Michaelis constant (K_m) and the tyrosinase activity's maximal velocity (V_max). This plot involved various L-Dopa concentrations as the substrate. For assessing the enzyme's inhibition type, we employed Lineweaver–Burk plots featuring the reciprocal of velocities (1/V) plotted against the reciprocal of substrate concentrations (1/[S] mM⁻¹) [24].

Molecular docking study

According to previously reported procedures, an induced fit docking study (IFD) was performed using Maestro Molecular Modeling platform to understand the interaction mode of the most potent compound over tyrosinase enzyme with PDB ID of 2Y9X with the grid center of X = − 8.06, Y = − 25.78, and Z = − 39.39 [25, 26].

Molecular dynamics simulations

Molecular dynamics simulation was performed using the Desmond v5.3 module implemented in the Maestro interface (from Schrödinger 2018‐4 suite). The appropriate pose for the molecular dynamics simulation procedure of compound 6b was achieved by the IFD method.

An molecular dynamics simulation of compound 6b was performed using the Desmond v5.3 module in the Maestro interface. The system was prepared by solvating the protein–ligand complex with explicit water molecules and adding counter-ions and NaCl to achieve cellular ionic concentrations. The molecular dynamics protocol involved minimization, pre-production, and production molecular dynamics steps. The simulations were carried out in the NPT ensemble with constant pressure and temperature. The system underwent 30 ns of molecular dynamics simulation, and the dynamic behavior and structural changes were analyzed using the RMSD. The protein–ligand interactions were investigated using the energy-minimized structure from the equilibrated trajectory [25, 26].

In silico prediction of drug-likeness and ADMET properties

The number of hydrogen bond acceptor (HBA) and donor (HBD), logP values, the topological polar surface area (tPSA), and the rotatable bond count (RBCs) of the selected compounds were calculated using the SwissADME and pkCSM online softwares. ADMET prediction of the selected compounds was performed by online software pkCSM [22, 23].