The development of TYR inhibitors remains a focal point in both scientific research and industrial applications due to their potential in treating hyperpigmentation disorders and their utility in cosmetic formulations [55,56]. Concurrently, there is a growing interest in the synthesis of novel cannabinoid derivatives to evaluate their diverse biological activities. CBD, in particular, has garnered significant attention for its therapeutic potential, prompting medicinal chemists to explore its chemical framework for drug development. Recent studies have investigated the anti-melanogenic and anti-TYR properties of CBD and minor phytocannabinoids, expanding the potential applications of these compounds in skin-related treatments [9,25,26].

As a part of our research, in this work, we synthesized and characterized new thiosemicarbazone–CBD–aldehyde derivatives (compounds 6–9), building upon our previously synthesized compounds 1–5. These novel derivatives were obtained through standard methods under specific reaction conditions, yielding moderate-to-good yields. The chemical structures of the synthesized compounds were confirmed using various spectroscopic techniques, including NMR, FTIR, and mass spectrometry. Our findings contribute to expanding the research on CBD derivatives and their potential biological activities.

The antioxidant properties of the newly synthesized thiosemicarbazone–CBD–aldehyde derivatives were evaluated through antiradical and ferric-reducing assays, with trolox serving as the standard. The results demonstrated that these compounds exhibit significant antioxidant activity. The scavenging effect is attributed to the N–H group in the thiosemicarbazone moiety and the O–H groups of the CBD skeleton, which can donate hydrogen atoms to free radicals. Upon donating a hydrogen atom, the compounds form a radical that delocalizes onto the benzene ring of the CBD, resulting in a stable resonance hybrid [57]. Among the derivatives, compound 5 exhibited the highest antioxidant potential. The IC₅₀ values for compounds 6 and 7 were 63.69 and 115.88 μM, respectively, compared to 5 with an IC₅₀ of 53.51 μM. These findings suggest that substituting the NH₂ group with electron-donating groups (such as methyl in 6 or dimethyl in 7) reduces the scavenging ability of the DPPH free radical. In contrast, substitution with a phenyl group resulted in a similar activity to 5 (IC₅₀ of 57.58 μM for compound 8), likely due to the phenyl group enhancing radical stability compared to methyl or dimethyl groups [58]. Compound 9 displayed lower scavenging activity than 5–8, probably due to substituting the OH groups with dimethyl ether groups. Collectively, these studies suggest that incorporating thiosemicarbazone moieties into the CBD framework enhances its antioxidant properties, making these derivatives promising candidates for therapeutic applications targeting oxidative-stress-related conditions. On the other hand, the FRAP assay showed similar activity for compounds 5–9, indicating significantly better ferric-reducing activity than CBD. This suggests that the presence of the thiosemicarbazone moiety in the CBD framework plays a crucial role in enhancing antioxidant activity.

To evaluate the TYR-inhibitory properties of our compounds, we utilized mushroom TYR (Agaricus bisporus) as a model system, as it shares a 23% amino acid identity with human TYR and is widely used for studying mammalian TYR due to its high homology [59]. The anti-TYR activity of compounds 1–9 was tested, with compounds 1–4 showing a low inhibitory effect on TYR activity in mushroom TYR’s monophenolase and diphenolase activities. As can be observed in Table 1, the semicarbazone and aminoguanidyl hydrazone derivatives (compounds 3 and 4, respectively) did not exhibit any inhibitory activity, suggesting that the thiosemicarbazone moiety is crucial for the activity. Inhibition likely occurs through the coordination of copper ions present in the active site of TYR by the C=S group. Some studies have indicated that TYR inhibitor activity is reduced when sulfur is substituted with an oxygen atom [60,61]. The sulfur atom in thiosemicarbazones interacts more effectively with the copper ion in the TYR active site than the oxygen and nitrogen atoms in analogous derivatives.

In contrast, compounds 5–9 were found to be more effective or similar to that for KA. SAR analysis for compounds 5–9 in diphenolase activity revealed that substituting the hydrogen of the NH₂ group with methyl, dimethyl, or phenyl groups reduces TYR inhibition. While the methyl and dimethyl substitutions (compounds 6 and 7, respectively) only slightly decrease the inhibitory effect, the phenyl group reduces the inhibitory effect by about two-fold, most likely due to steric hindrance. However, compound 9 exhibited a better inhibitory effect than 5 and KA. All these considerations suggest that, among the structural modifications made in the studied compounds, introducing sulfur and NH₂ groups in the thiosemicarbazone moiety could be a promising strategy to improve the efficacy of tyrosinase inhibitors. When the monophenolase activity of TYR, using L-tyrosine as the substrate, was tested with thiosemicarbazone derivatives (5–9), an extension of the lag phase was observed. Compound 5 notably increased the lag phase approximately two-fold. This observation suggests that the thiosemicarbazone derivatives may interact with the enzyme in a way that influences the enzyme’s activity, potentially through a reducing effect on the copper ions at the active site. The extension of the lag phase indicates a delay in the enzyme’s catalytic action, possibly due to the binding or modification of the enzyme’s active site by the derivatives. More detailed studies would be needed to elucidate the precise mechanism behind this effect.

Kinetic analyses of the diphenolase activity of TYR demonstrated that compound 5 is a reversible, mixed-type inhibitor of mushroom TYR (Figure 4). The Lineweaver–Burk plot revealed straight lines with varying slopes that intersected in the second quadrant. As the concentration of compound 5 increased, the v_max value decreased while the k_m value increased. This behavior confirms that 5 is a mixed-type inhibitor, capable of binding not only to the free enzyme but also to the enzyme–substrate complex, thereby affecting the TYR-L-DOPA affinity upon inhibitor binding. When compounds 5–9 were incubated with TYR for 3 h and subsequently dialyzed, the enzyme’s activity was restored to levels similar to the control enzyme that had not been exposed to the inhibitors. Additionally, ESI-MS analysis of the inhibitors revealed no changes in their mass during the incubation period, and no oxidation was observed. These results suggest that the inhibitors suppress TYR activity by chelating the copper ions at the enzyme’s active site without undergoing structural changes themselves.

Moreover, fluorescence quenching experiments were conducted to analyze the interactions between compounds 5–9 and TYR (Figure 5 and Figure S5). As shown in Figure 5, the fluorescence emission spectra of TYR at 310 K decreased significantly in a dose-dependent manner with the gradual addition of 5. However, the maximum emission peak remained unchanged, indicating that 5 interacts with Trp residues or nearby amino acids in mushroom TYR without significantly affecting the polarity of the microenvironment around Trp. The Stern–Volmer plots (Figure 5B) derived from the fluorescence quenching data exhibited good linearity, indicating a single quenching mechanism. The quenching constants were determined to elucidate the quenching process (Table 3). Fluorescence quenching in proteins can be categorized as either dynamic or static depending on the type of interaction between the quencher and the protein [62]. It was reported that the limiting fluorescence quenching rate constant of biological macromolecules with various quenchers in an aqueous medium via diffusion collision is about 2 × 10¹⁰ M⁻¹·s⁻¹. If k_q is greater than this value, the quenching process is predominantly static, involving the formation of a stable complex. As shown in Table 3, the k_q values for compounds 5–9 binding to TYR exceeded 2 × 10¹⁰ M⁻¹·s⁻¹, indicating that static quenching is the dominant mechanism.

The K_a values for the binding of thiosemicarbazone–CBD derivatives with TYR ranged from 0.52 × 10⁴ to 2.55 × 10⁴ M⁻¹. Notably, the K_a values of compounds 5 and 9 were similar, both exhibiting high inhibitory potency against TYR (IC₅₀ = 27.72 and 22.41 μM for 5 and 9, respectively), suggesting that the NH₂ group plays a crucial role in TYR inhibition. In contrast, compound 8 displayed the highest K_a value but the lowest inhibitory potency (IC₅₀ = 42.16 μM). These observations suggest that the presence of a phenyl group may enhance hydrophobic or π-π stacking interactions with the TYR environment, stabilizing the TYR–quencher complex. However, high K_a values do not always correlate with effective inhibition, potentially because binding alone may not sufficiently disrupt the enzyme’s catalytic function [63,64].

The interactions between the inhibitors and copper ions were evaluated using UV-Vis spectroscopy at pH 6.8. The formation of complexes between the tested compounds and copper ions led to a bathochromic (red) shift in the UV-Vis spectrum, with a new peak appearing around 400 nm (Figure 5A and Figure S6). This shift indicates the donation of lone pairs from the nitrogen atoms of the azomethine group in the Schiff base to the copper ions, suggesting an LMCT. Notably, compound 9 displayed a distinct UV-Vis profile compared to compounds 5–8, highlighting the critical role of the hydroxyl group (OH) of the phenol in contributing to the coordination with copper. It is suggested that the copper complex adopts a tridentate SNO coordination mode with the thiosemicarbazone derivatives, leading to the formation of thermodynamically stable five- and six-membered conjugate rings (Figure 8). Upon the addition of 100 μM ethylenediaminetetraacetic acid (EDTA), the spectrum reverted to that of the free ligand, confirming the displacement of the compounds from the copper ions by EDTA. When compound 5 was incubated with TYR, a change in its UV-Vis spectrum was observed, suggesting an interaction between the copper ion in TYR’s active site and the thiosemicarbazone moiety of 5 (Figure 7). To gain a deeper understanding of this interaction, further confirmation through ongoing docking studies is being conducted, with results to be published in the near future. Moreover, the ESI-MS analysis of the copper complex formed with 5 revealed a peak at m/z 477, corresponding to the chelation of copper (Figure 6B). The observed isotopic pattern further confirmed the formation of the copper complex, as it matched the characteristic distribution expected for copper-containing species. This isotopic signature is a reliable indicator of metal–ligand complexation in mass spectrometric studies. All the thiosemicarbazone–CBD–aldehyde derivatives (6–9) also exhibited new peaks, with masses and patterns consistent with copper chelation, as well as characteristic absorption changes in UV-Vis spectra (Figure S7).