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Article

In Vitro and In Vivo Biological Evaluation of Indole-thiazolidine-2,4-dione Derivatives as Tyrosinase Inhibitors

by
Li Lu
1,†,
Chunmei Hu
1,†,
Xiaofeng Min
1,
Zhong Liu
2,
Xuetao Xu
1,* and
Lishe Gan
1,*
1
Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China
2
College of Life Science and Technology, Jinan University, Guangzhou 510632, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2023, 28(22), 7470; https://doi.org/10.3390/molecules28227470
Submission received: 16 October 2023 / Revised: 30 October 2023 / Accepted: 2 November 2023 / Published: 7 November 2023
(This article belongs to the Section Natural Products Chemistry)
Browse Figures
Versions Notes

Abstract

:
Tyrosinase is an important rate-limiting enzyme in melanin biosynthesis. To find potential tyrosinase inhibitors with anti-melanogenic activity, a series of indole-thiazolidine-2,4-dione derivatives 5a~5z were synthesized by incorporating indole with thiazolidine-2,4-dione into one compound and assayed for their biological activities. All compounds displayed tyrosinase inhibitory activities and 5w had the highest anti-tyrosinase inhibitory activity with an IC50 value of 11.2 μM. Inhibition kinetics revealed 5w as a mixed-type tyrosinase inhibitor. Fluorescence quenching results indicated that 5w quenched tyrosinase fluorescence in a static process. CD spectra and 3D fluorescence spectra results suggested that the binding of 5w with tyrosinase could change the conformation and microenvironment of tyrosinase. Molecular docking also represented the binding between 5w and tyrosinase. Moreover, 5w could inhibit tyrosinase activity and melanogenesis both in B16F10 cells and the zebrafish model. Therefore, compound 5w could serve as a tyrosinase inhibitor with anti-melanogenic activity.

1. Introduction

Melanin, as a crucial biopolymer, is produced by melanocytes and is responsible for eye, skin, and hair color [1,2,3]. One important role of melanin is to protect the skin against ultraviolet damage [4,5,6]. Excessive ultraviolet exposure typically leads to abnormal melanin production and deposition, which generally results in several pigmentation skin diseases, including lentigo, freckles, and melisma [7,8,9].
Tyrosinase (EC 1.14.18.1) is one important rate-limiting enzyme for melanin production and is responsible for regulating the synthetic pathway of melanin production [10,11]. Tyrosinase is also a metalloenzyme, containing two bifunctional copper ions in its catalytic site [12,13]. In the process of melanin production, tyrosinase modulates the first two steps by the catalytic oxidation of L-tyrosine to dopaquinone [14]. In the first step, tyrosinase catalyzes L-tyrosine to convert it into 3,4-dihydroxyphenylalanine (L-DOPA), and in the second step, tyrosinase further oxidizes L-DOPA to dopaquinone, which is the important intermediate in melanin biosynthesis and is converted to melanin pigments by subsequent related chemical reactions [15,16,17]. Therefore, inhibiting tyrosinase activity is essential for the reduction in melanin synthesis [18]. Apart from their application in pigmentation disorders, tyrosinase inhibitors play important roles in the food industry. Tyrosinase can also catalyze the oxidation of phenolic molecules in fruit and vegetables into quinones, leading to enzymatic browning. Therefore, controlling tyrosinase activity is important for the quality of fruit and vegetables. Multitudinous natural and synthetic tyrosinase inhibitors displaying wide structural diversity have been identified [19,20,21]. But only a few tyrosinase inhibitors are used as efficacy agents in the pharmaceutical, cosmetic, and food industries, such as arbutin, paeonol, glabridin, and kojic acid. However, they still present some drawbacks like poor efficacy, skin irritation, and low stability. Therefore, it is urgent to develop safer and more effective tyrosinase inhibitors.
Indole scaffold with a structure of pyrrole in parallel with benzene has been one of the key structural nuclei in drug development due to its rich pharmacological activity and specific property of imitating peptide structure to bind with enzymes [22,23,24,25,26]. Previous studies have reported some indole derivatives as potential tyrosinase inhibitors. For example, the Mirfazli group introduced carbohydrazide into an indole scaffold to obtain substituted indole-carbohydrazides (Figure 1A), which presented excellent tyrosinase inhibitory activity [24]. The Abbasi group also synthesized the indole-N-ethyltriazole hybrids amalgamated with N-arylated ethanamides (Figure 1B) as tyrosinase inhibitors [25]. In another study, the Hoang group found 3-aminoalkylated indoles (Figure 1C) presented potential tyrosinase inhibitory activities [26]. Moreover, indole and its homologues widely exist in nature, showing good safety. The above investigations indicate that indole can serve as a core scaffold for developing potential tyrosinase inhibitors.
On the other hand, thiazolidine-2,4-diones (TZDs) are an important kind of antidiabetic agent and have been approved by the FDA to treat type 2 diabetes [27]. Their therapeutic target is peroxisome proliferator-activated receptor-γ (PPARγ). Recently, TZD derivatives have been reported to be potent inhibitors against tyrosinase, such as benzylidene-TZD derivatives (Figure 1D) [28] and 2,4-dihydroxybenzylidene-TZD (Figure 1E) [29]. The above analysis suggests that TZD can also serve as a core framework for designing potential tyrosinase inhibitors.
Drug design based on molecular hybridization involves combining two or more active molecules into a completely new molecule, which can inherit the advantageous structure of the parent molecule. This strategy can enhance the affinity or other performance of the molecule. Hence, to continue our efforts in the development of potential anti-tyrosinase agents [30,31,32], we report herein the synthesis of indole-thiazolidine-2,4-dione derivatives by incorporating indole with thiazolidine-2,4-dione to construct target compounds (Figure 1F). After determining their inhibitory activities against tyrosinase and the subsequent inhibitory mechanism, the effects on tyrosinase activity and melanogenesis in B16F10 cells and the zebrafish model were gradually evaluated.

2. Results and Discussion

2.1. Chemistry

Indole-thiazolidine-2,4-dione derivatives 5a~5z were synthesized using a convergent approach according to Scheme 1. Tryptamine (1) underwent an amidation reaction with bromoacetyl bromide in the presence of potassium carbonate in DCM solution to produce intermediate (2). Thiazolidine-2,4-dione (3) reacted with substituted benzaldehyde in the presence of sodium hydroxide in EtOH solution to give intermediate (4). Then, compounds (2) and (4) underwent substitution reactions in the presence of tetrabutylammonium bromide and potassium carbonate in DMF solution to provide target products 5a~5z.
The detailed synthesis steps and NMR and HRMS data are shown in the Supplementary Materials. For example, the 1H NMR spectrum of compound 5a exhibited a single peak at 10.85 ppm due to the imine proton of the indole ring; triple peaks at 8.43 ppm due to the imine proton of amide group; a single peak at 7.98 ppm attributed to vinyl protons; a single peak at 7.67 ppm; double peaks at 7.66 ppm (J = 1.1 Hz); double peaks at 7.58 ppm (J = 7.0 Hz); double peaks at 7.56 ppm (J = 5.9 Hz); double peaks at 7.53 ppm (J = 1.5 Hz); double peaks at 7.51 ppm (J = 7.5 Hz); multiple peaks at 7.36–7.31 ppm; a single peak at 7.16 ppm; multiple peaks at 7.09–7.04 ppm (J = 7.5 Hz); and multiple peaks at 7.01–6.96 ppm, all attributed to the aromatic protons of the benzene ring, respectively; and a single peak at 4.28 ppm; multiple peaks at 3.38–3.33 ppm; and triple peaks at 2.83 ppm (J = 7.4 Hz), all due to the methylene, respectively. The 13C NMR spectrum of 5a showed two characteristic peaks at 167.63 and 165.83 ppm due to two carbonyl carbons of TZD; a characteristic peak at 165.34 ppm attributed to the carbonyl carbon of the amide group; a characteristic peak at 136.70 ppm due to the vinyl carbon; characteristic peaks at 133.77, 133.40, 131.25, 130.66, 129.92, 127.65, 123.24, 121.72, 121.41, 118.76, 118.69, and 111.86 ppm attributed to the aromatic carbon of the benzene ring; a characteristic peak at 112.01 ppm due to the carbon atom of TZD; characteristic peaks at 44.00 and 25.47 ppm attributed to methylene carbons. For the HRMS of compound 5a, its molecular ion peak appeared at m/z 406.1224 as [M + H]+.

2.2. Tyrosinase Inhibitory Activity Assay

Based on the preliminary experimental results of the positive control, kojic acid, the anti-tyrosinase inhibitory activities of all compounds 5a~5z were first screened at the initial concentration of 32 μM (Table 1). Among them, compounds 5l and 5w displayed the strongest tyrosinase inhibitory activities with inhibition ratios higher than 70%, while those of other compounds were unsatisfactory and below 50%. Their 50% inhibitory concentration (IC50) values were not further assayed, while compounds 5l and 5w were further detected for IC50 values of 13.3 and 11.2 μM, respectively, which were better than that of kojic acid (15.6 μM). That was to say that the hybridization strategy of indole with thiazolidine-2,4-dione into one compound would be an effective method for obtaining potential tyrosinase inhibitors.
The preliminary structure–activity relationship was analyzed based on the tyrosinase inhibitory activities of all compounds. It could be observed that substitution groups on the benzene ring resulted in differences in tyrosinase inhibitory activity. Compound 5a, with no substituent group on the benzene ring, was selected as the parent molecule. The introduction of substituent groups (like methyl, fluorine, chlorine, bromine, hydroxyl, cyano, and nitro) would enhance the inhibitory activities, respectively, while the introduction of substituent groups, i.e., trifluoromethyl and methoxy groups would reduce the inhibitory activities. For the same substituent groups at different positions of the benzene ring, the meta-position contributes more to the inhibitory activity than the ortho- and para-position. In addition, for all substituent groups at the meta-position of the benzene ring, the contribution order of substituent groups on inhibitory activity is cyanide > bromine > chlorine > hydroxyl > methyl > fluorine > nitro > hydrogen > trifluoromethyl > methoxy. Cyanide groups contributed most to the tyrosinase inhibitory activity. These results will guide our further research works on the development of tyrosinase inhibitors.

2.3. Inhibition Kinetics

To understand the inhibition mechanism of this kind of compound on tyrosinase, the inhibition kinetics of the preferable compound 5w were investigated. As displayed in Figure 2A, the Lineweaver–Burk plots of 1/v vs. 1/[S] produced a group of straight lines, which intersect in the second quadrant. With the increasing concentrations of 5w, the Y-intercept of corresponding lines increased and the X-intercept decreased, meaning the decrease of Vmax and Km. Compound 5w could then be identified as a mixed-type inhibitor. This result also suggested that compound 5w could bind with both free enzyme and enzyme–substrate complex. In addition, the inhibition constant (Ki) of compound 5w to tyrosinase was determined to be 11.73 μM from the plots of the slope vs. inhibitor concentration (Figure 2B).

2.4. Fluorescence Quenching

Fluorescence quenching, an effective method for monitoring the binding of a compound to its target protein, was operated to examine the binding of compound 5w with tyrosinase. As presented in Figure 3A–C, tyrosinase demonstrated a fluorescence characteristic peak (332 nm) at 298, 303, and 308 K, respectively, due to the chromophore of tyrosinase. When compound 5w was added by titration, the fluorescence spectra of tyrosinase gradually decreased. The results indicated that the binding between compound 5w and tyrosinase occurred. Moreover, the fluorescence characteristic peak of tyrosinase slightly red-shifted with the titration addition of compound 5w, which suggested there were microenvironmental changes in fluorescent chromophores.
For a further understanding of the binding process of compound 5w with tyrosinase, the fluorescence quenching manner was assayed using the Stern–Volmer equation. In the Stern–Volmer plots of compound 5w (Figure 3D), the lines at different temperatures presented a good linear, which illustrated only one quenching manner in their binding process. Although the quenching constant (Ksv) values were slightly proportional to temperature, the quench rate constant (Kq) values at different temperatures were all higher than 2 × 1010 L mol−1 S−1, which is the maximum scattering collision quenching constant (Table 2). The fluorescence quenching process of compound 5w to tyrosinase was then a static process. In addition, the number of binding sites (n) was obtained to be about one, meaning there was only one binding site between compound 5w and tyrosinase (Table 2).

2.5. CD Spectra

CD spectra, one widely used method for determining the secondary structure of protein, were applied to assay the effects of 5w on the conformation change of tyrosinase. The CD spectra of the tyrosinase–compound 5w mixture were monitored and two negative characteristic bands around 210 and 222 nm appeared (Figure 4), which represented typical features of α-helixes. The addition of compound 5w caused a significant increase in negative peak intensity and changes in characteristic bands, which suggested the conformation change of tyrosinase by binding with compound 5w. The secondary structure contents of tyrosinase were then obtained from the CD spectra data and are listed in Table 3. The results indicated that treatment of compound 5w could result in content changes in the secondary structure of tyrosinase. Compound 5w especially (molar ratio of 3:1) could cause the content increase in the α-helix (from 8.7 to 9.0%); β-turn (from 19.8 to 20.1%); and random coil (from 30.6 to 35.5%); and the content decrease in the β-Sheet (from 34.0 to 32.7%), respectively.

2.6. Three-Dimensional Fluorescence Spectra

Three-dimensional fluorescence spectra were employed to monitor the effects of compound 5w on the conformation and microenvironment changes in tyrosinase. In the 3D fluorescence spectra of tyrosinase (Figure 5A), two characteristic peaks 1 and 2 appeared, which corresponded to Tyr and Trp residues and polypeptide strand transition, respectively. After being treated with compound 5w (8.5 μM), the characteristic peaks 1 and 2 of tyrosinase reduced significantly accompanied by a slight shift, which indicated that the binding of 5w to tyrosinase would result in conformation and microenvironment changes in tyrosinase. These results were inconsistent with the results of fluorescence quenching and CD spectra.

2.7. Molecular Docking

The binding information between compound 5w and tyrosinase (PDB: 2Y9X) was investigated using SYBYL software (2.0). After compound 5w and tyrosinase were prepared using the self-taking program, molecular docking was conducted, and the results are shown in Figure 6. From the docking results (Figure 6A–C), compound 5w closely bound itself to the active site, especially with the indole ring section located in the copper ion catalytic region and the benzene ring section with a cyanide group located outside the active site. Then, the specific binding information was analyzed (Figure 6D) and it was found that the nitrogen hydrogen group in the indole ring formed one hydrogen bond with Met280 (2.3 Å) and the cyanide group in the benzene ring formed another hydrogen bond with Asn81 (2.5 Å). Moreover, compound 5w made hydrophobic interactions with His244, His263, Val283, and Ala286. The existence of the above interactions between compound 5w and tyrosinase affected the structure of tyrosinase active sites, leading to a decrease in its enzyme activity.

2.8. Tyrosinase Activity and Melanogenesis Assay in B16F10 Cells

Although the synthesized compounds presented good activities against mushroom tyrosinase, due to the differences between mushroom tyrosinase and human tyrosinase [33], their effects on tyrosinase activity and melanogenesis both in B16F10 cells and zebrafish were assayed. The cytotoxicities of 5w and kojic acid on B16F10 cells were assayed and the results showed that 5w and kojic acid both had obvious effects on the cells’ viability up to 32 μM (Supplementary Materials). Therefore, the effects of 5w and kojic acid on cellular tyrosinase activity and melanogenesis in B16F10 cells were detected at concentrations of 16 and 32 μM. As displayed in Figure 7A,B, 5w and kojic acid could inhibit both cellular tyrosinase activity and melanogenesis in B16F10 cells, and 5w presented obvious higher cellular anti-tyrosinase activity and anti-melanogenesis activity than kojic acid at the test concentrations.

2.9. Tyrosinase Activity and Melanogenesis Assay in Zebrafish

Finally, the effects of 5w on tyrosinase activity and the melanogenesis assay in zebrafish were investigated. The acute toxicity results showed that compound 5w and kojic acid had an obvious toxicity to the zebrafish embryos up to 20 μM (Supplementary Materials). Then, the tyrosinase activity and melanogenesis in zebrafish treated with 5w and kojic acid were assayed, and the results are displayed in Figure 8. It was observed that 5w and kojic acid treatment could reduce the tyrosinase activity in zebrafish, and the compound 5w had higher anti-tyrosinase activity than kojic acid at a concentration of 10 μM. From the embryo photos (Figure 8B), treatment with 5w and kojic acid results in a pigmentation decrease in the embryo’s eyes, yolk sac, and notum compared to the control embryo. The grayscale values of whole embryos were determined using Image J software (1.8.0) (Figure 8C), and it was found that 5w and kojic acid treatment also led to a decrease in grayscale values compared to the control embryo, and 5w displayed a higher decrease. All the above results show that 5w could inhibit the tyrosinase activity in vitro and in vivo, therefore reducing melanogenesis both in B16F10 cells and zebrafish.

3. Materials and Methods

3.1. Chemistry

Mushroom tyrosinase and L-DOPA were supplied by Sigma-Aldrich (Shanghai, China). All other reagents were commercially available. NMR spectra were recorded on a Bruker (500 MHz) NMR spectrometer. HRMS data were measured on Apex II by means of the ESI technique.

3.2. Tyrosinase Inhibition and Kinetics Study

The tyrosinase inhibitory activities of compounds 5a~5z were determined according to previous reports [34,35]. In brief, 10 μL of compound was added into 140 μL of tyrosinase, incubated for 5 min at room temperature. Then, 50 μL of L-DOPA was added, followed by the measurement of absorbance change at 475 nm. The inhibition rate at different concentrations was calculated and compared to the blank control.
The kinetics studies of 5w were conducted using the above experimental procedure. The absorbance changes in the mixture containing 5w (0, 4, 8, and 16 μM), tyrosinase (66.7 U/mL), and L-DOPA (2, 4, 6, and 8 mM) were recorded, respectively. The data were analyzed using Lineweaver–Burk plots [36].

3.3. Fluorescence Quenching

According to a previous report [37], the fluorescence quenching titration was run. In total, 1 μL of 5w solution was progressively titrated into 2 mL of tyrosinase (0.17 mg/mL), followed by fluorescence spectra determination (λex = 280 nm) at temperatures of 298, 303, and 308 K, respectively. The quenching parameters were obtained using the Stern–Volmer equation.

3.4. CD Spectra

The CD spectra of tyrosinase (33 μM) and tyrosinase–compound 5w mixture were measured at room temperature, respectively, based on previous reports [38]. The test wavelengths were 195~250 nm.

3.5. Three-Dimensional Fluorescence Spectra

Based on a previous report [39], the 3D fluorescence spectra of tyrosinase (0.17 mg/mL) and tyrosinase–compound 5w mixture were measured, respectively, at room temperature. The test wavelengths were 200–400 nm.

3.6. Molecular Docking

The molecular docking of compound 5w with tyrosinase was conducted using SYBYL software based on previous reports [40,41]. Compound 5w was treated with energy minimization accompanied by charge addition with the Gasteiger–Huckel model. The crystal structure of Agaricus bisporus tyrosinase (PDBID: 2Y9X) was obtained from the RCSB. Tyrosinase protein was then treated by removing the ligand and water, adding hydrogen atoms and charge, and repairing end residues, followed by the generation of the active pocket. Finally, the docking of compound 5w with tyrosinase was run.

3.7. Cell Assay

3.7.1. Cell Cytotoxicity

The effects of compound 5w and kojic acid on the cells’ viability were determined using the MTT method. B16F10 cells (5000 cells/well) were seeded into the 96-well plate for 24 h, followed by the treatment of compound 5w or kojic acid (4~32 μM) for another 48 h. Then, MTT solution (0.5 mg/mL) was added and incubated for 4 h. DMSO (100 μL) was used to dissolve the violet precipitates, and then the mixture was measured for its absorbance at 570 nm.

3.7.2. Tyrosinase Activity in B16F10 Cells

The tyrosinase inhibitory activity of 5w in B16F10 cells was determined. B16F10 cells (1 × 105 cells/well) were seeded into the 6-well plate for 24 h and subsequently treated with compound 5w or kojic acid for another 48 h. Then, the harvested cells were suspended into the appropriate amount of Triton X-100 (1%) and frozen for 1 h at −80 °C. After it had thawed, the mixture was centrifuged to collect the supernatant. Then, its protein concentration was determined using a BCA Kit. After 20 μL of L-DOPA (10 mM) was added into 80 μL of protein solution, the absorbance change at 475 nm was measured.

3.7.3. Melanin Content in B16F10 Cells

B16F10 cell treatment was consistent with the above method. The harvested cells were suspended into NaOH (1 M, 200 μL). After boiling for 1 h, the mixture was measured to determine the absorbance change at wavelength of 405 nm.

3.8. Zebrafish Assay

3.8.1. Acute Toxicity

In this experiment, 6~8 hpf embryos (1 embryo per well) were seeded in 24-well plates, then the compound 5w or kojic acid (0~40 μM) was added, followed by observation of the survival rate at 72 hpf.

3.8.2. Tyrosinase Activity in Zebrafish

The embryos treated with compound 5w or kojic acid were lysed in PBS, followed by centrifugation to obtain the supernatant. The appropriate amount of supernatant, equal L-DOPA (5 mg/mL), was added and the absorbance at 475 nm was determined.

3.8.3. Melanin Content in Zebrafish

The embryos treated with compound 5w or kojic acid were observed under a microscope and the melanin content was assayed using Image J software.

4. Conclusions

In summary, a series of indole-thiazolidine-2,4-dione derivatives 5a~5z were synthesized by incorporating indole with thiazolidine-2,4-dione into the target compounds, and their potential anti-tyrosinase and anti-melanogenic activities were evaluated. All compounds displayed tyrosinase inhibitory activities, and 5w had the highest anti-tyrosinase inhibitory activity with an IC50 value of 11.2 μM compared to the positive control kojic acid (IC50 = 15.6 μM). Inhibition kinetics indicated 5w as a mixed-type inhibitor of tyrosinase. Fluorescence quenching results illustrated that the binding of 5w to tyrosinase could quench the fluorescence of tyrosinase with a static quenching process. CD spectra and 3D fluorescence spectra suggested that the binding of 5w to tyrosinase could change the conformation and microenvironment of tyrosinase. Molecular docking revealed the detailed binding between 5w and tyrosinase. Moreover, compound 5w could inhibit tyrosinase activity and melanogenesis both in B16F10 cells and the zebrafish model. Therefore, compound 5w could serve as a tyrosinase inhibitor with anti-melanogenic activity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28227470/s1, Synthesis of indole-thiazolidine-2,4-dione derivatives 5a~5z; 1H NMR and 13C NMR of compounds 5a~5z; HRMS of compounds 5a~5z; Cytotoxicity of 5w on B16F10 cells and zebrafish model [42,43,44].

Author Contributions

Conceptualization, X.X., L.G. and Z.L.; methodology, software, validation, formal analysis, investigation, resources, data curation, and visualization, L.L., C.H. and X.M.; writing—original draft preparation, X.X.; writing—review and editing, L.G. and Z.L.; supervision, X.X.; project administration, X.X., L.G. and Z.L.; funding acquisition, X.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Department of Education of Guangdong Province (2021KCXTD044) and the Science and Technology Planning Project of Guangdong Province (2021B1212040016).

Institutional Review Board Statement

The animal study protocol was approved by the Research Ethics Committee of Jinan University (No: 20230310-0001).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Romagnoli, R.; Oliva, P.; Prencipe, F.; Manfredini, S.; Germanò, M.P.; Luca, L.D.; Ricci, F.; Corallo, D.; Aveic, S.; Mariotto, E. Cinnamic acid derivatives linked to arylpiperazines as novel potent inhibitors of tyrosinase activity and melanin synthesis. Eur. J. Med. Chem. 2022, 231, 114147. [Google Scholar] [CrossRef] [PubMed]
  2. Moreiras, H.; Pereira, F.J.C.; Neto, M.V.; Seabra, M.C.; Barral, D.C. The exocyst is required for melanin exocytosis from melanocytes and transfer to keratinocytes. Pigm. Cell. Melanoma R. 2020, 33, 366–371. [Google Scholar] [CrossRef] [PubMed]
  3. Su, J.; Li, D.; Hu, Y.; You, X.; Guo, X.; Li, X.; Chen, B. A novel C6-sulfonated celastrol analog as a tyrosinase and melanin inhibitor: Design, synthesis, biological evaluation and molecular simulation. J. Mol. Struc. 2023, 1283, 135288. [Google Scholar] [CrossRef]
  4. Nasab, N.H.; Raza, H.; Eom, Y.S.; Hassan, M.; Kloczkowski, A.; Kim, S.J. Synthesis and discovery of potential tyrosinase inhibitor of new coumarin-based thiophenyl-pyrazolylthiazole nuclei: In-vitro evaluation, cytotoxicity, kinetic and computational studies. Chem. Biol. Drug. Des. 2023, 101, 13–88. [Google Scholar]
  5. Pillaiyar, T.; Namasivayam, V.; Manickam, M.; Jung, S.H. Inhibitors of melanogenesis: An updated review. J. Med. Chem. 2018, 61, 7395–7418. [Google Scholar] [CrossRef] [PubMed]
  6. Najafi, Z.; Rafiei, F.; Ghafouri-Khosrowshahi, A.; Mahdavi, M.; Dianatpour, M.; Iraji, A. Design, synthesis, and molecular dynamics simulation studies of new chalcone-based 2-arylidene-1,3-indandiones as tyrosinase inhibitors. Chemistryselect 2023, 33, e202302192. [Google Scholar] [CrossRef]
  7. Ielo, L.; Deri, B.; Germano, M.P.; Vittorio, S.; Mirabile, S.; Gitto, R.; Rapisarda, A.; Ronsisvalle, S.; Floris, S.; Pazy, Y. Exploiting the 1-(4-fluorobenzyl) piperazine fragment for the development of novel tyrosinase inhibitors as anti-melanogenic agents: Design, synthesis, structural insights and biological profile. Eur. J. Med. Chem. 2019, 178, 380–389. [Google Scholar] [CrossRef]
  8. Roberts, N.B.; Curtis, S.A.; Milan, A.M.; Ranganath, L.R. The pigment in alkaptonuria relationship to melanin and other coloured substances: A review of metabolism, composition and chemical analysis. JIMD Rep. 2015, 24, 51–66. [Google Scholar]
  9. Chortani, S.; Hajlaoui, A.; Jlizi, S.; Harrath, A.H.; Jannet, H.B.; Romdhane, A. Access to new phosphonate-and imidazolidine-benzopyrimidinone derivatives as antityrosinase and anti-acetylcholinesterase agents: Design, synthesis and molecular docking. J. Mol. Struct. 2022, 1268, 133693. [Google Scholar] [CrossRef]
  10. Hassan, M.; Shahzadi, S.; Kloczkowski, A. Tyrosinase inhibitors naturally present in plants and synthetic modifications of these natural products as anti-agents: A review. Molecules 2023, 28, 378. [Google Scholar] [CrossRef]
  11. Li, J.; Feng, L.; Liu, L.; Wang, F.; Ouyang, L.; Zhang, L.; Hu, X.; Wang, G. Recent advances in the design and discovery of synthetic tyrosinase inhibitors. Eur. J. Med. Chem. 2021, 224, 113744. [Google Scholar] [CrossRef] [PubMed]
  12. Wang, G.; He, M.; Huang, Y.; Peng, Z. Synthesis and biological evaluation of new kojic acid-1, 3, 4-oxadiazole hybrids as tyrosinase inhibitors and their application in the anti-browning of fresh-cut mushrooms. Food Chem. 2023, 409, 135275. [Google Scholar] [CrossRef] [PubMed]
  13. He, M.; Fan, M.; Yang, W.; Peng, Z.; Wang, G. Novel kojic acid-1, 2, 4-triazine hybrids as anti-tyrosinase agents: Synthesis, biological evaluation, mode of action, and anti-browning studies. Food Chem. 2023, 419, 136047. [Google Scholar] [CrossRef] [PubMed]
  14. Xue, S.; Li, Z.; Ze, X.; Wu, X.; He, C.; Shuai, W.; Marlow, M.; Chen, J.; Scurr, D.; Zhu, Z. Design, synthesis, and biological evaluation of novel hybrids containing dihydrochalcone as tyrosinase inhibitors to treat skin hyperpigmentation. J. Med. Chem. 2023, 66, 5099–5117. [Google Scholar] [CrossRef]
  15. Barros, M.R.; Menezes, T.M.; Garcia, Y.S.; Neves, J.L. Inhibitory effects of iron-based carbonaceous nanocomposites on mushroom tyrosinase activity: Molecular aspects and mechanistic insights. New J. Chem. 2023, 47, 9134–9142. [Google Scholar] [CrossRef]
  16. Najafi, Z.; Ebadi, A.; Chehardoli, G.; Ziaei, M.; Akbarzadeh, T.; Saeedi, M.; Gholamhoseini, P.; Mahdavi, M. Design, synthesis, in vitro, and in silico studies of novel benzylidene 6-methoxy-1-tetralone linked to benzyloxy and benzyl-1, 2, 3-triazole rings as potential tyrosinase inhibitors. J. Mol. Struct. 2023, 1271, 134018. [Google Scholar] [CrossRef]
  17. Dong, H.H.; Wang, X.Y.; Li, G.; Zhao, L.X.; Zhao, C.H. Synthesis, characterization, antioxidant and tyrosinase inhibitory activities of hesperetin derivatives. Synth. Commun. 2023, 53, 1579–1587. [Google Scholar] [CrossRef]
  18. Roulier, B.; Rush, I.; Lazinski, L.M.; Pérès, B.; Olleik, H.; Royal, G.; Fishman, A.; Maresca, M.; Haudecoeur, R. Resorcinol-based hemiindigoid derivatives as human tyrosinase inhibitors and melanogenesis suppressors in human melanoma cells. Eur. J. Med. Chem. 2023, 246, 114972. [Google Scholar] [CrossRef]
  19. Mermer, A.; Demirci, S. Recent advances in triazoles as tyrosinase inhibitors. Eur. J. Med. Chem. 2023, 259, 115655. [Google Scholar] [CrossRef]
  20. Vittorio, S.; Dank, C.; Ielo, L. Heterocyclic compounds as synthetic tyrosinase inhibitors: Recent advances. Int. J. Mol. Sci. 2023, 24, 9097. [Google Scholar] [CrossRef]
  21. Obaid, R.J.; Mughal, E.U.; Naeem, N.; Sadiq, A.; Alsantali, R.I.; Jassas, R.S.; Moussa, Z.; Ahmed, S.A. Natural and synthetic flavonoid derivatives as new potential tyrosinase inhibitors: A systematic review. RSC Adv. 2021, 11, 22159–22198. [Google Scholar] [CrossRef] [PubMed]
  22. Ubeid, A.A.; Do, S.; Nye, C.; Hantash, B.M. Potent low toxicity inhibition of human melanogenesis by novel indole-containing octapeptides. BBA-Gen. Subj. 2012, 1820, 1481–1489. [Google Scholar] [CrossRef] [PubMed]
  23. Kaushik, N.K.; Kaushik, N.; Attri, P.; Kumar, N.; Kim, C.H.; Verma, A.K.; Choi, E.H. Biomedical importance of indoles. Molecules 2013, 18, 6620–6662. [Google Scholar] [CrossRef] [PubMed]
  24. Kudličková, Z.; Michalková, R.; Salayová, A.; Ksiažek, M.; Vilková, M.; Bekešová, S.; Mojžiš, J. Design, synthesis, and evaluation of novel indole hybrid chalcones and their antiproliferative and antioxidant activity. Molecules 2023, 28, 6583. [Google Scholar] [CrossRef]
  25. Citarella, A.; Moi, D.; Pedrini, M.; Pérez-Peña, H.; Pieraccini, S.; Dimasi, A.; Stagno, C.; Micale, N.; Schirmeister, T.; Sibille, G.; et al. Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses. Org. Biomol. Chem. 2023, 21, 3811. [Google Scholar] [CrossRef]
  26. Garrepalli, S.; Gudipati, R.; Kapavarapu, R.; Ravindhranath, K.; Pal, M. Synthesis and characterization of two known and one new impurities of dolutegravir: In silico evaluation of certain intermediates against SARS-CoV-2 O-ribose methyltransferase (OMTase). J. Mol. Struct. 2023, 1271, 133992. [Google Scholar] [CrossRef]
  27. Iraji, A.; Sheikhi, N.; Attarroshan, M.; Ardani, G.R.S.; Kabiri, M.; Bafghi, A.N.; Kobarfard, F.; Rezaei, Z.; Khoshneviszadeh, M.; Foroumadi, A.; et al. Design, synthesis, spectroscopic characterization, in vitro tyrosinase inhibition, antioxidant evaluation, in silico and kinetic studies of substituted indole-carbohydrazides. Bioorg. Chem. 2022, 129, 106140. [Google Scholar] [CrossRef]
  28. Shakila; Abbasi, M.A.; Aziz-ur-Rehman; Siddiqui, S.Z.; Nazir, M.; Raza, H.; Zafar, A.; Shah, S.A.A.; Shahid, M.; Seo, S. Multi-step synthesis of indole-N -ethyltriazole hybrids amalgamated with N-arylated ethanamides: Structure-activity relationship and mechanistic explorations through tyrosinase inhibition, kinetics and computational ascriptions. J. Mol. Struct. 2022, 126, 1132953. [Google Scholar] [CrossRef]
  29. Huynh, T.; Ngo, K.; Nguyen, H.; Dang, H.; Phung, V.; Thaie, K.; Hoang, T. Catalyst-free and multicomponent synthesis of 3-aminoalkylated indoles via a Mannich-type reaction: Multitargeted anticancer, tyrosinase and a-glucosidase inhibitory activities. New J. Chem. 2021, 45, 18183. [Google Scholar] [CrossRef]
  30. Asati, V.; Mahapatra, D.K.; Bharti, S.K. Thiazolidine-2,4-diones as multi-targeted scaffold in medicinal chemistry: Potential anticancer agents. Eur. J. Med. Chem. 2014, 87, 814–833. [Google Scholar] [CrossRef]
  31. Ha, Y.M.; Park, Y.J.; Kim, J.A.; Park, D.; Park, J.Y.; Lee, H.J.; Lee, J.Y.; Moon, H.R.; Chung, H.Y. Design and synthesis of 5-(substituted benzylidene) thiazolidine-2,4-dione derivatives as novel tyrosinase inhibitors. Eur. J. Med. Chem. 2012, 49, 245–252. [Google Scholar] [CrossRef] [PubMed]
  32. Kim, S.H.; Ha, Y.M.; Moon, K.M.; Choi, Y.J.; Park, Y.J.; Jeong, H.O.; Chung, K.W.; Lee, H.J.; Chun, P.; Moon, H.R.; et al. Anti-melanogenic effect of (Z)-5-(2,4-dihydroxybenzylidene) thiazolidine-2,4-dione, a novel tyrosinase inhibitor. Arch. Pharm. Res. 2013, 36, 1189–1197. [Google Scholar] [CrossRef] [PubMed]
  33. Roulier, B.; Pérès, B.; Haudecoeur, R. Advances in the Design of Genuine Human Tyrosinase Inhibitors for Targeting Melanogenesis and Related Pigmentations. J. Med. Chem. 2020, 63, 13428–13443. [Google Scholar] [CrossRef] [PubMed]
  34. Lu, L.; Zhang, X.; Kang, Y.; Xiong, Z.; Zhang, K.; Xu, X.T.; Bai, L.P.; Li, H.G. Novel coumarin derivatives as potential tyrosinase inhibitors: Synthesis, binding analysis and biological evaluation. Arab. J. Chem. 2023, 16, 104724. [Google Scholar] [CrossRef]
  35. Li, J.; Min, X.; Zheng, X.; Wang, S.; Xu, X.; Peng, J. Synthesis, anti-tyrosinase activity, and spectroscopic inhibition mechanism of cinnamic acid-eugenol esters. Molecules 2023, 28, 5969. [Google Scholar] [CrossRef]
  36. Zargaham, M.K.; Ahmed, M.; Akhtar, N.; Ashraf, Z.; Abdel-Maksoud, M.A.; Aufy, M.; Nadeem, H. Synthesis, in silico studies, and antioxidant and tyrosinase inhibitory potential of 2-(substituted phenyl) thiazolidine-4-carboxamide derivatives. Pharmaceuticals 2023, 16, 835. [Google Scholar] [CrossRef]
  37. Nunes, J.A.; de Araújo, R.S.A.; da Silva, F.N.; Cytarska, J.; Łączkowski, K.Z.; Cardoso, S.H.; Mendonça-Júnior, F.J.B.; da Silva-Júnior, E.F. Coumarin-based compounds as inhibitors of tyrosinase/tyrosine hydroxylase: Synthesis, kinetic studies, and in silico approaches. Int. J. Mol. Sci. 2023, 24, 5216. [Google Scholar] [CrossRef]
  38. Peng, Z.; Wang, G.; Zeng, Q.; Li, Y.; Wu, Y.; Liu, H.; Wang, J.; Zhao, Y. Synthesis, antioxidant and anti-tyrosinase activity of 1,2,4-triazole hydrazones as antibrowning agents. Food Chem. 2021, 34, 1128265. [Google Scholar] [CrossRef]
  39. Xiao, D.; Lu, L.; Liang, B.; Xiong, Z.; Xu, X.T.; Chen, W.H. Identification of 1,3,4-oxadiazolyl-containing β-carboline derivatives as novel α-glucosidase inhibitors with antidiabetic activity. Eur. J. Med. Chem. 2023, 261, 115795. [Google Scholar] [CrossRef]
  40. Lin, J.; Xiao, D.; Lu, L.; Liang, B.W.; Xiong, Z.; Xu, X.T. New β-carboline derivatives as potential α-glucosidase inhibitor: Synthesis and biological activity evaluation. J. Mol. Struct. 2023, 1283, 135279. [Google Scholar] [CrossRef]
  41. Wu, X.Z.; Zhu, W.J.; Lu, L.; Hu, C.M.; Zheng, Y.Y.; Zhang, X.; Lin, J.; Wu, J.Y.; Xiong, Z.; Zhang, K.; et al. Synthesis and anti- α-glucosidase activity evaluation of be-tulinic acid derivatives. Arab. J. Chem. 2023, 16, 104659. [Google Scholar] [CrossRef]
  42. Pakhare, D.; Radhika, K. Application of Horner-Wadsworth-Emmons olefination for the synthesis of granulatamide A, its E isomer and other amides of tryptamine. New J. Chem. 2016, 40, 5428–5431. [Google Scholar] [CrossRef]
  43. Touaibia, M.; St-Coeur, P.; Duff, P.; Faye, D.C.; Pichaud, N. 5-Benzylidene, 5-benzyl, and 3-benzylthiazolidine-2,4-diones as potential inhibitors of the mitochondrial pyruvate carrier: Effects on mitochondrial functions and survival in Drosophila melanogaster. Eur. J. Pharmacol. 2021, 913, 174627. [Google Scholar] [CrossRef] [PubMed]
  44. Shaikh, F.M.; Patel, N.B.; Sanna, G.; Busonera, B.; Colla, P.L.; Rajani, D.P. Synthesis of some new 2-amino-6-thiocyanato benzothiazole derivatives bearing 2,4-thiazolidinediones and screening of their in vitro antimicrobial, antitubercular and antiviral activities. Med. Chem. Res. 2015, 24, 3129–3142. [Google Scholar] [CrossRef]
Molecules 28 07470 g001
Figure 1. The reported indole or thiazolidine-2,4-dione based tyrosinase inhibitors, and the target indole-thiazolidine-2,4-dione derivatives in this work.
Figure 1. The reported indole or thiazolidine-2,4-dione based tyrosinase inhibitors, and the target indole-thiazolidine-2,4-dione derivatives in this work.
Molecules 28 07470 g001
Molecules 28 07470 sch001
Scheme 1. Synthetic route of compounds 5a~5z. Reagents and conditions: (a) K2CO3, DCM, r.t., 1 h, yield 70%; (b) NaOH, EtOH, r.t., 6 h, yield 65%; (c) K2CO3, TBAB, DMF, r.t., 2 h, yield 28~76%.
Scheme 1. Synthetic route of compounds 5a~5z. Reagents and conditions: (a) K2CO3, DCM, r.t., 1 h, yield 70%; (b) NaOH, EtOH, r.t., 6 h, yield 65%; (c) K2CO3, TBAB, DMF, r.t., 2 h, yield 28~76%.
Molecules 28 07470 sch001
Molecules 28 07470 g002
Figure 2. (A) The Lineweaver–Burk plots of 5w against tyrosinase; (B) The plots of the slope versus 5w concentration.
Figure 2. (A) The Lineweaver–Burk plots of 5w against tyrosinase; (B) The plots of the slope versus 5w concentration.
Molecules 28 07470 g002
Molecules 28 07470 g003
Figure 3. (A–C) The fluorescence quenching of 5w against tyrosinase; curves a to k stood for 5w concentrations of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 μM, respectively; (D) the Stern–Volmer plots of 5w.
Figure 3. (A–C) The fluorescence quenching of 5w against tyrosinase; curves a to k stood for 5w concentrations of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 μM, respectively; (D) the Stern–Volmer plots of 5w.
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Molecules 28 07470 g004
Figure 4. CD spectra of tyrosinase–compound 5w complex.
Figure 4. CD spectra of tyrosinase–compound 5w complex.
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Molecules 28 07470 g005
Figure 5. The 3D fluorescence spectra of tyrosinase (A) and tyrosinase–5w mixture (B).
Figure 5. The 3D fluorescence spectra of tyrosinase (A) and tyrosinase–5w mixture (B).
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Molecules 28 07470 g006
Figure 6. The molecular docking between compound 5w and tyrosinase. (A–C) Binding pose of 5w in the active site; (D) binding details of 5w.
Figure 6. The molecular docking between compound 5w and tyrosinase. (A–C) Binding pose of 5w in the active site; (D) binding details of 5w.
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Molecules 28 07470 g007
Figure 7. (A) The effects of 5w on tyrosinase activity in B16F10 cells; (B) the effects of 5w on melanogenesis in B16F10 cells. * p < 0.05, *** p < 0.001.
Figure 7. (A) The effects of 5w on tyrosinase activity in B16F10 cells; (B) the effects of 5w on melanogenesis in B16F10 cells. * p < 0.05, *** p < 0.001.
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Molecules 28 07470 g008
Figure 8. (A) The effects of 5w on tyrosinase activity in zebrafish; (B) the embryo photos of each group; (C) the effects of 5w on melanogenesis in zebrafish. “ns” means no significant difference (p > 0.05); *** p < 0.001, **** p < 0.0001.
Figure 8. (A) The effects of 5w on tyrosinase activity in zebrafish; (B) the embryo photos of each group; (C) the effects of 5w on melanogenesis in zebrafish. “ns” means no significant difference (p > 0.05); *** p < 0.001, **** p < 0.0001.
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Table 1. Tyrosinase inhibitory activities of compounds 5a~5z.
Table 1. Tyrosinase inhibitory activities of compounds 5a~5z.
Molecules 28 07470 i001
No.RInhibition (%)IC50
(μM)		No.RInhibition
(%)		IC50
(μM)
5aH14.2-5n2-CF33.8-
5b2-Me19.3-5o3-CF34.5-
5c3-Me33.2-5p4-CF33.0-
5d4-Me15.1-5q2-OMe1-
5e2-F16.5-5r3-OMe2-
5f3-F32.6-5s4-OMe1-
5g4-F27.4-5t2-OH26.9-
5h2-Cl16.8-5u3-OH36.2-
5i3-Cl37.6-5v4-OH24.6-
5j4-Cl36.6-5w3-CN71.511.2
5k2-Br31.7-5x4-CN24.6-
5l3-Br70.313.35y3-NO219.8-
5m4-Br19.5-5z4-NO219.1-
Kojic acid 15.6
“-” means not determined.
Table 2. Quenching parameters between compound 5w and tyrosinase.
Table 2. Quenching parameters between compound 5w and tyrosinase.
TKSV (×104 Lmol−1)Kq (×1012 Lmol−1)n
2983.773.770.72
3033.843.840.74
3084.474.470.82
Table 3. The secondary structure content of tyrosinase.
Table 3. The secondary structure content of tyrosinase.
[Enzyme]: [5w]α-Helix (%)β-Sheet (%)β-Turn (%)Random Coil (%)
1: 08.734.019.830.6
1: 18.833.619.932.5
1: 28.933.120.034.5
1: 39.032.720.135.5
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Lu, L.; Hu, C.; Min, X.; Liu, Z.; Xu, X.; Gan, L. In Vitro and In Vivo Biological Evaluation of Indole-thiazolidine-2,4-dione Derivatives as Tyrosinase Inhibitors. Molecules 2023, 28, 7470. https://doi.org/10.3390/molecules28227470

AMA Style

Lu L, Hu C, Min X, Liu Z, Xu X, Gan L. In Vitro and In Vivo Biological Evaluation of Indole-thiazolidine-2,4-dione Derivatives as Tyrosinase Inhibitors. Molecules. 2023; 28(22):7470. https://doi.org/10.3390/molecules28227470

Chicago/Turabian Style

Lu, Li, Chunmei Hu, Xiaofeng Min, Zhong Liu, Xuetao Xu, and Lishe Gan. 2023. "In Vitro and In Vivo Biological Evaluation of Indole-thiazolidine-2,4-dione Derivatives as Tyrosinase Inhibitors" Molecules 28, no. 22: 7470. https://doi.org/10.3390/molecules28227470

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