Article Potent Tyrosinase Inhibitory Activity of Curcuminoid Analogues and Inhibition Kinetics Studies

Anan Athipornchai 1 , Nattisa Niyomtham 2, Wachirachai Pabuprapap 3 , Vachiraporn Ajavakom 3, Maria Duca 4, Stéphane Azoulay 4,* and Apichart Suksamrarn 3,*

1 Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131, Thailand; [email protected] 2 International College of Dentistry, Walailak University, Nakhon Si Thammarat 80161, Thailand; [email protected] 3 Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkapi, Bangkok 10240, Thailand; [email protected] (W.P.); [email protected] (V.A.) 4 Université Côte d’Azur, CNRS, ICN, 28 Avenue Valrose, CEDEX 2, 06108 Nice, France; [email protected] * Correspondence: [email protected] (S.A.); [email protected] or [email protected] (A.S.); Tel.: +33-4-89-15-10-29 (S.A.); +66-81849-8420 (A.S.)

Abstract: Natural tyrosinase inhibitors from herbal plants are promising therapeutic agents for skincare and cosmetic products. Natural curcuminoids exhibit weak antityrosinase properties. The structural modification of , the major curcuminoid from Curcuma longa, gave 14 analogues.   The tyrosinase inhibitory activity of the natural curcuminoids and the modified analogues on both L-tyrosine and DOPA substrates were evaluated. The inhibition kinetics were also undertaken. Citation: Athipornchai, A.; For analogues with potent activity on the L-tyrosine substrate, the isoxazole analogue 12 and two Niyomtham, N.; Pabuprapap, W.; reduced analogues, hexahydrocurcumin (16) and the α,β-unsaturated analogue 17, showed IC Ajavakom, V.; Duca, M.; Azoulay, S.; 50 Suksamrarn, A. Potent Tyrosinase values of 8.3, 14.6 and 9.4 µM, and were 20.9-, 11.9- and 18.4-fold more active, respectively, than kojic Inhibitory Activity of Curcuminoid acid, the reference compound. For the analogues with potent antityrosinase on DOPA substrate, the Analogues and Inhibition Kinetics dimethylated analogue 5 exhibited the strongest antityrosinase activity against the DOPA substrate, Studies. Cosmetics 2021, 8, 35. with the IC50 value of 8.0 µM, which was 16.6-fold more active than kojic acid. The inhibition kinetics https://doi.org/10.3390/ revealed that curcuminoid 5 could bind with both free enzyme and with the enzyme–substrate cosmetics8020035 complex. It acted as a competitive–uncompetitive mixed-II type inhibitor. Curcuminoid 17 could bind with both free enzyme and the enzyme–substrate complex. The results indicated that 17 acted Academic Editor: Enzo Berardesca as a competitive–uncompetitive mixed-I type inhibitor, while curcuminoid 12 was a noncompetitive inhibitor which bound with both free enzymes and the enzyme–substrate complex. These potent Received: 9 April 2021 analogues might serve as new potential tyrosinase inhibitors for the prevention and treatment of skin Accepted: 28 April 2021 pigmentation disorders. Published: 4 May 2021

Keywords: curcuminoid analogues; antityrosinase activity; inhibition kinetics; skin pigmentation Publisher’s Note: MDPI stays neutral disorders with regard to jurisdictional claims in published maps and institutional affil- iations.

1. Introduction Increasing attention has been focused on the use of natural ingredients from herbal plants for developing into modern medicine and cosmetic products because of their rel- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. atively few side effects. Moreover, the pursuit of white skin and healthiness is the latest This article is an open access article trend amongst most of the Asian populations, as they want to look younger than their distributed under the terms and age. Human skin color is determined by the amount of natural pigment, melanin, which is conditions of the Creative Commons synthesized by the tyrosinase enzyme catalyst. Attribution (CC BY) license (https:// Tyrosinase, also known as oxidase, is a copper-containing monooxygenase creativecommons.org/licenses/by/ enzyme which catalyzes two distinct reactions of melanin biosynthesis, including the 4.0/). ortho-hydroxylation of the amino acid, tyrosine, to 3,4-dihydroxyphenylalanine (DOPA)

Cosmetics 2021, 8, 35. https://doi.org/10.3390/cosmetics8020035 https://www.mdpi.com/journal/cosmetics Cosmetics 2021, 8, 35 2 of 10

(cresolase or monophenolase activity), and the oxidation of DOPA to ortho-quinone (cat- echolase or diphenolase activity). The ortho-quinone was also synthesized to melanin pigment by various reactions [1]. Many researchers have been searching for natural tyrosi- nase inhibitors from herbal plants for skincare and cosmetic products. Curcuminoids are natural, strong which are isolated from the rhizomes of Zingiberaceae plants such as Curcuma longa L. The major curcuminoid constituent of this plant is curcumin (1)[2], which exhibits several biological activities such as antioxidant [3], anti-inflammatory [4], anticancer [5], antibacterial [6], antimicrobial [7] and antiviral [8] activities. Recently, several curcuminoids and their analogues have been developed and used as oral supplements for various medical conditions [9]. The safety of curcumin is evident from its consumption for centuries at levels of up to 100 mg/day by people in many countries. In addition, there is much evidence to confirm that curcumin and its derivatives are safe for topical application, and are used in commercially available cosmetic products and for wound healing [10,11]. For this reason, the beneficial properties of curcuminoids and their analogues have attracted numerous efforts for development in cosmetics for use as a safe therapeutic agent. The carbocyclic diarylpentanoids, a group of polyphenolic curcumin analogues, have been synthesized and their antityrosinase and antioxidant activities have been evaluated [4,12]. Symmetrical and unsymmetrical carbo- cyclic curcumin analogues have also been synthesized and their antityrosinase activities have also been determined [13–15]. However, there are few reports on antityrosinase activity of the linear curcumin analogues [16,17]. In this study, aiming the critical roles of the linear curcuminoid analogues as tyrosinase inhibitors, a series of curcumin analogues were synthesized and their tyrosinase inhibitory activity were evaluated. In addition, the inhibition mechanism and structure−activity relationships for the antityrosinase properties of linear curcuminoid analogues were also investigated.

2. Materials and Methods 2.1. Chemical Reagents and General Experimental Procedures All chemicals used for the synthesis of the curcuminoid analogues were supplied by Merck (Damstadt, Germany), Fluka (Buchs, Switzerland), or Sigma-Aldrich (St. Louis, MO, USA). Tyrosinase from mushroom (EC 1.14.18.1), L-tyrosine, 3,4-dihydroxy-L-phenylalanine (DOPA), kojic acid and (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Melting points were determined using an electrothermal melt- ing point apparatus and were uncorrected. IR spectra were recorded in the ATR mode on a Perkin-Elmer FT-IR Spectrum 400 spectrophotometer (PerkinElmer, Waltham, MA, USA). 1H and 13C NMR spectra were recorded on a Bruker AVANCE 400 spectrometer (Bruker, Faellanden, Switzerland) operating at 400 and 100 MHz, respectively. Mass spectra were obtained using a Finnigan LC-Q mass spectrometer (Finnigan, Bremen, Germany). High-resolution mass spectra were obtained using a Bruker micrOTOF mass spectrometer (Bruker, Bremen, Germany). Unless indicated otherwise, column chromatography was carried out using Merck silica gel 60 (finer than 0.063 mm). For TLC, Merck precoated silica gel 60 F254 plates were used. Spots on TLC were detected under UV light and by spraying with anisaldehyde-H2SO4 reagent followed by heating. Untreated 96-well plates were obtained from Thermo Scientific Nunc (Illkirch, France). Adhesive films (Greiner Bio-One, Les Ulis, France) were used to cover the plates during incubation. In vitro biologi- cal tests were realized in triplicate with the automated system epMotion 5075 (Eppendorf, Montesson, France) and absorbance measurements were carried out using a Spectramax Plus 384 spectrophotometer (Molecular Devices, Wokingham, UK).

2.2. Chemical Modification of Curcuminoids The natural curcuminoids 1–3 were isolated from Curcuma longa rhizomes by the method described previously [18] and were used in chemical modifications to the analogues 4–17 for antityrosinase evaluations. The diacetate 4, dimethyl ether 5, the hydroxyethyl ether 6, the didemethyl ether 7 and the reduced curcuminoid analogues 15–17 were synthe- Cosmetics 2021, 8, 35 3 of 10

sized by the literature procedures [18]. The pyrazoles 8–10 and the isoxazoles 11–14 were modified from the corresponding curcuminoids 1–3 [19]. The 1H and 13C NMR spectra of all compounds are in the Supplementary Materials. (Figures S1–S34)

2.3. Tyrosinase Inhibition Assay Tyrosinase inhibitory activity was determined according to the method as previously described [20] with modifications by measuring the DOPA chrome formed due to the action of tyrosinase enzyme on L-tyrosine and DOPA substrates. Briefly, the assays were performed in 96-well plates as follows. First, 150 µL of a of mushroom tyrosinase (171.66 U mL−1 for tyrosine assays and 85.83 U mL−1 for DOPA assays) in phosphate buffer (50 mmol L−1, pH 6.8) was distributed in each well (enzyme final per well: 100 U mL−1), together with 7.5 µL of samples diluted at a concentration of 3.433 mg mL−1 in DMSO (sample final concentration per well: 100 µg mL−1). Kojic acid (3.433 mmol L−1 in DMSO) was used as the positive control and DMSO alone was used as the negative control (OD control). The plate was filmed and incubated at room temperature for 20 min. A first reading was performed at 480 nm to obtain ODs from the blank. Then, 100 µL of a solution of L-tyrosine 1 mM in phosphate buffer (pH = 6.8; final L-tyrosine concentration per well: 0.388 mM) were distributed in each well. After 20 min of incubation, OD reading was performed at 480 nm to assess the percentage of inhibition using the equation 1 below:

The percentage of tyrosinase inhibition (%) = {[(A - B) - (C - D)]/(A - B)} × 100 (1)

where A is the absorbance of the control with the enzyme, B is the absorbance of the control without the enzyme, C is the absorbance of the test sample with the enzyme and D is the absorbance of the test sample without the enzyme.

2.4. Kinetic Analysis of Tyrosinase Inhibition Assay The kinetic properties of the highly active curcuminoids (5, 12 and 17) were found using the procedure described above but varying the concentration of L-tyrosine or DOPA as substrates (0.125, 0.25, 0.50 and 1.00 mM) and doing a reading every 30 s after addition of the substrate. The Lineweaver−Burk plot was applied to determine the inhibition mode. The inhibition constant (KI and KIS) was determined by the secondary plots of the apparent slope or intercept versus different of inhibitor [21].

2.5. Statistical Analysis All assays were performed at least three times and in triplicate. The statistical analyses were done using the software GraphPad Prism for Windows, Version 5.03 (GraphPad Software, La Jolla, CA, USA).

3. Results and Discussion 3.1. Chemistry Natural curcuminoids 1–3 were isolated from the rhizomes of C. longa which were subjected to chemical modifications following our published procedures [18,19] to give 14 synthetic analogues (4–17) for antityrosinase evaluations.

3.2. Inhibitory Effects on Tyrosinase The natural curcuminoids 1, 2 and 3 and their synthetic analogues 4–17 were sub- jected to tyrosinase inhibitory assay by measuring the hydroxylation of L-tyrosine and the oxidation of DOPA substrates to assess monophenolase and diphenolase activities, respectively. Based on the data shown in Table1, it was found that the natural curcumi- noids 1–3 showed moderate to good inhibitory activities against tyrosinase enzyme on both pathways. Against the monophenolase activity, curcumin (1) showed moderate inhibitory effect with IC50 of 326.5 µM and the action of demethoxycurcumin (2) was comparable to that of the curcuminoid 1, whereas (3) (IC50 46.5 µM) was more active than 1 and 2; it was 10.1-fold more active than compound 2 and was 3.7-fold more Cosmetics 20212021,, 88,, xx FORFOR PEERPEER REVIEWREVIEW 55 ofof 1010

value of 9.4 µM, which was 34.7- and 18.4-fold more active than the parent compound 1 Cosmetics 2021, 8, x FOR PEER REVIEWvalue of 9.4 µM, which was 34.7- and 18.4-fold more active than the parent compound5 of 10 1 and kojic acid, respectively. However, for the DOPA substrate, compound 17 waswas onlyonly slightlyslightly moremore activeactive thanthan itsits parentparent compoundcompound 1 andand kojickojic acid.acid. FromFrom thesethese results,results, itit waswas concludedconcluded thatthat reducedreduced curcuminoidcurcuminoid analoguesanalogues 15–17 contributedcontributed toto highhigh tyrosinasetyrosinase in-in-

hibiting activities on L-tyrosine substrate which could be explained by more adaptative hibitingvalue ofactivities 9.4 µM, onwhich L-tyrosine was 34.7- substrate and 18.4-fold which more could active be than explained the parent by compound more adaptative 1 flexibility.flexibility. TheThe structurestructure−activity relationship (SAR) analysis of curcuminoids and their flexibility.flexibility.and kojic TheThe acid, structurestructure respectively.−activity However, relationship for the DOPA (SAR) substrate, analysis compound of curcuminoids 17 was onlyand their analogues is summarized in Figure 1. analoguesslightly moreis summarized active than itsin parentFigure compound 1. 1 and kojic acid. From these results, it was concluded that reduced curcuminoid analogues 15–17 contributed to high tyrosinase in- hibiting activities on L-tyrosine substrate which could be explained by more adaptative flexibility. The structure−activity relationship (SAR) analysis of curcuminoids and their analogues is summarized in Figure 1.

Cosmetics 2021, 8, 35 4 of 10

active than the standard kojic acid (IC50 132.8 µM). Against the diphenolase activity, com- pound 3 (IC50 34.8 µM) was more active than 1 and 2; it was 3.8-fold more active than kojic acid. The results indicate that the absence of the methoxy group increases tyrosinase in- hibitory activity of curcuminoids especially for the monophenolase step, probably due the

ability of hydroxyl groups to create a metal-ligand binding interaction with the dicopper nucleus [22]. Figure 1. StructureStructure−−activityactivity relationshiprelationship (SAR)(SAR) analysisanalysis of curcuminoids and their analogues.

Table 1. Tyrosinase inhibitory activity of natural curcuminoids (1–3) and synthetic analogues (4–17) Table 1. Tyrosinase inhibitory activity ofof naturalnatural curcuminoidscurcuminoids ((11––33)) andand syntheticsynthetic analoguesanalogues ((44––1717)) onon L-tyrosineL-tyrosine andand DOPADOPA sub-sub- Figure 1. Structure−activity relationship (SAR) analysis of curcuminoids and their analogues. strates.strates. on L-tyrosine and DOPA substrates.

1 1 Table 1. Tyrosinase inhibitory activity of natural curcuminoids (1–3) andTyrosinase synthetic analogues InhibitoryTyrosinase ( Activity4–17) Inhibitoryon (ICL-tyrosine; µM) Activityand DOPA (IC sub-50; µM) 11 Tyrosinase Inhibitory50 Activity (IC5050;; µM)µM) 1 Compoundstrates. Compound StructureStructure L-tyrosineL-tyrosine DOPA DOPA O O 1 OO OO Tyrosinase Inhibitory Activity (IC50; µM) Compound MeO Structure OMe MeO OMeOMe 1 1 326.5L-tyrosine326.5 94.8 DOPA94.8 HO O O OH HOHO OHOH MeO OMe 1 O O 326.5 94.8 OO OO MeO HO OH 2 2 MeO 470.0470.0 41.1 41.1 2 O O 470.0 41.1 HO OH HOHOMeO OHOH 2 470.0 41.1 OO OO HO O O OH 3 3 O O 46.546.5 34.8 34.8 HO OH 3 HOHO OHOH 46.5 34.8 O O HO OO OO OH MeO OMe MeO OMeOMe 2 2 4 O O 2 InactiveInactive 222 2 InactiveInactive 22 4 4 MeO OMe Inactive InactiveInactive InactiveInactive 4 AcO OAc Inactive 2 Inactive 2 AcOAcO OAcOAc AcO O O OAc OO OO MeO OMe MeO O O OMeOMe 5 5 MeO OMe 86.286.2 8.0 8.0 5 MeO OMe 86.2 8.0 MeO OMeOMe MeO OMe OO OO O O O O MeO OMeOMe MeOMeO OMe 22 22 6 2 InactiveInactive2 22 2 InactiveInactive2 22 6 6 6 HO OH Inactive InactiveInactiveInactive Inactive InactiveInactiveInactive HOHO HO O O OHOHOH OO O OO O O OO O OOO HO OH HOHO HO OHOH 7 7 7 66.966.966.9 54.6 54.6 54.6 Cosmetics 2021,, 8,, xx FORFOR PEERPEER REVIEWREVIEW HOHO HO OHOH 6 of 10 HO OH N N NHNH NN NHNH MeOMeO OMe 8 MeO OMe 677.1 21.9 8 8 MeO OMe 677.1677.1 21.9 21.9 8 HO OH 677.1 21.9 HO OH HOHO OHOH N NH MeO 9 9 154.4154.4 19.7 19.7 HO OH N NH N NH 10 10 129.1129.1 19.2 19.2

HO OH N O MeO OMe 11 11 24.324.3 330.9 330.9 HO OH N O 12 8.3 86.2 HO OH N O MeO OMe 13 Inactive 2 InactiveInactive 2 MeO OMe N O MeO OMe 14 Inactive 2 InactiveInactive 2 AcO OAc O O MeO OMe 15 51.6 283.1 HO OH O OH MeO OMe 16 14.6 25.4 HO OH O MeO OMe 17 9.4 63.5 HO OH O OH 33 Kojic acid HO 173.2 132.8 O 1 2 3 11 StandardStandard deviationsdeviations areare notnot shown,shown, butbut allall areare withinwithin 10%10% ofof thethe ICIC5050 value.value. 22 Inactive at 1000 µM. 33 Kojic acid was used as the posi- tive control.

3.3. Inhibition Mechanism on the Mushroom Tyrosinase The inhibition mechanism on the mushroom tyrosinase was investigated for the best inhibitor through kinetic analysis. The inhibitory types of curcuminoid 5 onon mushroommushroom tyrosinase were determined during the oxidation of DOPA from Lineweaver−Burk plots [21], the results of which are summarized in Table 2.

Table 2. TheThe inhibitioninhibition typetype andand constantsconstants ofof actiactive curcuminoids on mushroom tyrosinase.

Inhibition Constants (µM) Compound Inhibition Type KII KKISIS 5 Mixed-II type 4.64 3.44 12 Non-competitive 0.12 0.08 17 Mixed-I type 0.01 0.05

Amongst them, the double-reciprocal plots of the enzyme inhibited by curcuminoid 5 areare shownshown inin FigureFigure 2.2. TheThe equilibriumequilibrium constantsconstants forfor thethe bindingbinding ofof 5 withwith freefree enzyme,enzyme, KII,, andand withwith thethe enzyme–substrateenzyme–substrate complex,complex, KKISIS,, werewere obtainedobtained fromfrom thethe secondsecond plotsplots ofof Km/Vm (slope)(slope) andand 1/V1/Vm (intercept)(intercept) versusversus thethe concentrationsconcentrations ofof thethe inhibitors.inhibitors. FromFrom thethe data, we found that curcuminoid 5 couldcould bind,bind, notnot onlyonly withwith thethe freefree enzyme,enzyme, butbut alsoalso with the enzyme–substrate complex. The KISIS valuesvalues werewere lowerlower thanthan KKII,, indicatingindicating thatthat

CosmeticsCosmetics 20212021,,,, 88,,,, xxxx FORFORFORFOR PEERPEERPEERPEER REVIEWREVIEWREVIEWREVIEW 66 ofof 1010 CosmeticsCosmetics Cosmetics 2021 20212021,, , 8 88,, , x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 666 of ofof 10 1010

NN NHNH NNN NHNHNH MeOMeO 99 MeOMeOMeO 154.4154.4 19.719.7 Cosmetics 2021, 8, 35 999 154.4154.4154.4 5 of 10 19.719.719.7 HOHO OHOH HOHOHO OHOHOH NN NHNH NNN NHNHNH 101010 129.1129.1129.1 19.219.219.2 HOHO OHOH Table 1. Cont. HOHOHO OHOHOH NN OO NNN OOO MeOMeO OMeOMe 1 11 MeOMeOMeO OMeOMeOMe Tyrosinase Inhibitory Activity24.3 (IC 50; µM) 330.9 111111 Compound Structure 24.324.324.3 330.9330.9330.9 HOHO OHOH HOHOHO OHOHOH L-tyrosine DOPA NN OO NNN OOO 12 8.3 86.2 121212 12 8.38.38.38.3 86.2 86.286.286.2 HOHO OHOH HOHOHO OHOHOH NN OO NNN OOO MeO OMe MeOMeO OMeOMe 2 2 13 MeOMeO OMeOMe Inactive 22 InactiveInactive 22 131313 13 Inactive 2 InactiveInactiveInactiveInactive 2 2 2 InactiveInactiveInactive 2 2 MeOMeO OMeOMe MeOMeOMeO OMeOMeOMe NN OO NNN OOO MeO OMe MeOMeO OMeOMe 2 2 14 MeOMeO OMeOMe Inactive 22 Inactive 22 141414 14 Inactive 2 InactiveInactiveInactiveInactive 2 2 2 InactiveInactiveInactiveInactive 2 2 AcOAcO OAcOAc AcOAcOAcO OAcOAcOAc OO OO OOO OOO MeOMeO OMeOMe MeOMeOMeO OMeOMeOMe 151515 15 51.651.651.651.6 283.1 283.1283.1283.1 HOHO OHOH HOHOHO OHOHOH OO OHOH OOO OHOHOH MeOMeO OMeOMe MeOMeOMeO OMeOMeOMe 161616 16 14.614.614.614.6 25.4 25.425.425.4 HOHO OHOH HOHOHO OHOHOH OO OOO MeOMeO OMeOMe MeOMeOMeO OMeOMeOMe 171717 17 9.49.49.49.4 63.5 63.563.563.5 HOHO OHOH HOHOHO OHOHOH OO OOO OHOH OHOHOH Kojic acid 3 333 Kojic acid 3 173.2173.2 132.8 132.8 KojicKojic acidacid 3 33 HOHO 173.2173.2 132.8132.8 KojicKojicKojic acid acidacid HOHOHO 173.2173.2173.2 132.8132.8132.8 OO OOO 11 1 22 2 33 3 11 StandardStandard deviationsdeviationsStandard areare deviations notnot shown,shown, are not butbut shown, allall areare but withinwithin all are 10%10% within ofof thethe 10% ICIC of505050 the value.value. IC value. 22 InactiveInactive at 1000 at 1000µM.µ 33M. KojicKojic acid acid was used as the posi- 11 Standard StandardStandard deviations deviationsdeviations are areare not notnot shown, shown,shown, but butbut all allall are areare within withinwithin 10% 10%10% of ofof the thethe IC ICIC505050 value. value.value.50 2 2 Inactive InactiveInactive at atat 1000 10001000 µM. µM.µM. 3 3 Kojic KojicKojic acid acidacid was waswas used usedused as asas the thethe posi- posi-posi- tive control. was used as the positive control. tivetivetive control. control.control.

The effects of3.3.3.3.3.3. the Inhibition InhibitionInhibition free hydroxyl Mechanism MechanismMechanism group on onon of the thethe the Mushroom MushroomMushroom function Tyrosinase TyrosinaseTyrosinase at the aromatic ring on the tyrosinase inhibition were then investigated. The diacetylated curcuminoid analogue TheTheThe inhibition inhibitioninhibition mechanism mechanismmechanism on onon the thethe mushroom mushroommushroom tyrosinase tyrosinasetyrosinase was waswas investigated investigatedinvestigated for forfor the thethe best bestbest 4 and the dihydroxyethyl ether analogue 6 showed no inhibition on tyrosinase on both inhibitorinhibitorinhibitor throughthroughthrough kinetickinetickinetic analysis.analysis.analysis. TheTheThe inhibitoryinhibitoryinhibitory typestypestypes ofofof curcuminoidcurcuminoidcurcuminoid 555 on onononon mushroom mushroommushroommushroommushroom substrates, indicating that acetylation and hydroxyethylation of the free hydroxyl groups tyrosinasetyrosinasetyrosinase were werewere determined determineddetermined during duringduring the thethe oxidation oxidationoxidation of ofof DOPA DOPADOPA from fromfrom Lineweaver LineweaverLineweaver−−−BurkBurkBurk plots plotsplots resulted in a loss of tyrosinase inhibition. In contrast, the methylation of compound 1 [21],[21],[21], the thethe results resultsresults of ofof which whichwhich are areare summarized summarizedsummarized in inin Table TableTable 2. 2.2. to the dimethoxylated analogue 5 gave rise to more potent inhibitory activities against tyrosinase on bothTable substrates, 2. The inhibition especially type the DOPAand constants substrate, of acti whichve curcuminoids exhibited highly on mushroom potent tyrosinase. TableTableTable 2. 2.2. The TheTheThe inhibition inhibitioninhibitioninhibition type typetypetype and andandand constants constantsconstantsconstants of ofofof acti actiactiactiveveve curcuminoids curcuminoidscurcuminoids on onon mushroom mushroommushroom tyrosinase. tyrosinase.tyrosinase. inhibitory activity on the DOPA substrate at the IC50 value of 8.0 µM, 11.9-fold more active than the parent 1. It was 16.6-fold more active than kojic acid. The resultsInhibitionInhibitionInhibition have indicated Constants ConstantsConstants (µM) (µM)(µM) CompoundCompound InhibitionInhibition TypeType CompoundCompound InhibitionInhibition TypeType KIII KKISISIS that methylation of the hydroxyl group increased tyrosinase inhibition. InKK addition,KII I the KKKISISIS didemethylated curcumin555 7 was also moreMixed-IIMixed-IIMixed-II active than type typetype the parent 1. However, 4.64 4.64 4.64 it was 3.44 3.44 3.44 5 less active than the dimethoxylated121212 analogueNon-competitiveNon-competitiveNon-competitiveon DOPA substrate and was0.120.120.12 as active as 0.08 0.08 0.08 compound 5 on L-tyrosine substrate. 171717 Mixed-IMixed-IMixed-I type typetype 0.01 0.01 0.01 0.05 0.05 0.05 In order to see the influence of the diketo function on tyrosinase activities, the pyra- zole (8–10) and the isoxazole (11–14) analogues were synthesized for the evaluation of AmongstAmongstAmongst them, them,them, the thethe double-reciprocal double-reciprocaldouble-reciprocal plots plotsplots of ofof the thethe enzyme enzymeenzyme inhibited inhibitedinhibited by byby curcuminoid curcuminoidcurcuminoid antityrosinase activities. For the L-tyrosine substrate, most of the pyrazole analogues were 555 are areareareare shown shownshownshownshown in inininin Figure FigureFigureFigureFigure 2. 2.2.2.2. The TheTheTheThe equilibrium equilibriumequilibriumequilibriumequilibrium constants constantsconstantsconstantsconstants for forforforfor the thethethethe binding bindingbindingbindingbinding of ofofofof 5 55 with withwithwithwith free freefreefreefree enzyme, enzyme,enzyme,enzyme,enzyme, 1 3 less active than theKIII, corresponding, andand withwith thethe enzyme–substrateenzyme–substrate parent natural curcuminoids complex,complex, KKISISIS–,, werewere. The obtainedobtained results indicated fromfrom thethe secondsecond plotsplots ofof KKKII,I, , and andand with withwith the thethe enzyme–substrate enzyme–substrateenzyme–substrate complex, complex,complex, K KKISISIS,, , were werewere obtained obtainedobtained from fromfrom the thethe second secondsecond plots plotsplots of ofof that the replacement of the β-diketo function in curcuminoid caused a marked decrease in Kmm/Vmm (slope)(slope) andand 1/V1/Vmm (intercept)(intercept) versusversus thethe concentrationsconcentrations ofof thethe inhibitors.inhibitors. FromFrom thethe KKKmmm/V/V/Vmmm (slope) (slope)(slope) and andand 1/V 1/V1/Vmmm (intercept) (intercept)(intercept) versus versusversus the thethe concentrations concentrationsconcentrations of ofof the thethe inhibitors. inhibitors.inhibitors. From FromFrom the thethe tyrosinase inhibitory activity on the L-tyrosine substrate. However, for the DOPA substrate, data,data, wewe foundfound thatthat curcuminoidcurcuminoid 55 couldcouldcouldcould bind,bind,bind,bind, notnotnotnot onlyonlyonlyonly withwithwithwith thethethethe freefreefreefree enzyme,enzyme,enzyme,enzyme, butbutbutbut alsoalsoalsoalso they were more activedata, we than found the curcuminoidsthat curcuminoid1–3 and5 could the mostbind, activenot only analogue with the was free the enzyme, but also with the enzyme–substrate complex. The KISISIS valuesvalues werewere lowerlower thanthan KKIII,, indicatingindicating thatthat withwithwith the thethe enzyme–substrate enzyme–substrateenzyme–substrate complex. complex.complex. The TheThe K KKISISIS values valuesvalues were werewere lower lowerlower than thanthan K KKII,I, , indicating indicatingindicating that thatthat pyrazole 10; its IC50 was 19.2 µM, which was 6.9-fold more active than kojic acid. On the other hand, the isoxazoles 11 and 12 were more strongly active than the curcuminoids 1 and 3 on L-tyrosine substrate, with the IC of 24.3 and 8.3 µM, respectively. They were 50 13.4- and 5.6-fold more active than the curcuminoids 1 and 3, respectively. However, compounds 11 and 12 were considerably less active than the parent natural curcuminoids 1–3 on DOPA substrate. Moreover, the dimethoxylated isoxazole 13 and the diacetylated Cosmetics 2021, 8, 35 6 of 10

isoxazole 14 showed no inhibitory activities against tyrosinase on both substrates. The findings indicated that the free hydroxyl groups on the benzene rings are essential for high activity on L-tyrosine substrate. From these results, it was concluded that the presence of the pyrazole ring contributed to high inhibitory activity against tyrosinase on DOPA substrate whereas the presence of the isoxazole ring contributed to high inhibitory activity against tyrosinase on L-tyrosine substrate. In order to see the influence of the alkene function of the alkyl side chain on anti- tyrosinase activities, the reduced curcuminoid analogues 15–17 were prepared from the curcuminoid 1. The antityrosinase evaluation of these reduced curcuminoids revealed that tetrahydrocurcumin 15 was 6.3-fold more active than the parent curcuminoid 1 on L-tyrosine substrate, but it was less active than compound 1 on the DOPA substrate. In Cosmetics 2021, 8, x FOR PEER REVIEW 5 of 10 addition, hexahydrocurcumin (16) was 22.3- and 3.7-fold more active than curcumin (1) on L-tyrosine and DOPA substrates, respectively. The α,β-unsaturated ketone 17 showed highly potent inhibitory activity against tyrosinase on L-tyrosine substrate, with an IC50 value of 9.4 µM, which was 34.7- and 18.4-fold more active than the parent compound 1 valueand kojic of 9.4 acid, µM, which respectively. was 34.7- However, and 18.4-fold for more the DOPA active than substrate, the parent compound compound17 1was only andslightly kojic moreacid, respectively. active than However, its parent for compound the DOPA 1substrate,and kojic compound acid. From 17 was these only results, it wasslightly concluded more active that than reduced its parent curcuminoid compound analogues1 and kojic acid.15–17 Fromcontributed these results, to high it was tyrosinase concluded that reduced curcuminoid analogues 15–17 contributed to high tyrosinase in- inhibiting activities on L-tyrosine substrate which could be explained by more adaptative hibiting activities on L-tyrosine substrate which could be explained by more adaptative flexibility. The structure−activity relationship (SAR) analysis of curcuminoids and their flexibility. The structure−activity relationship (SAR) analysis of curcuminoids and their analogues is is summarized summarized in Figure in Figure 1. 1.

Figure 1. Structure−activity relationship (SAR) analysis of curcuminoids and their analogues. Figure 1. Structure−activity relationship (SAR) analysis of curcuminoids and their analogues. 3.3. Inhibition Mechanism on the Mushroom Tyrosinase Table 1. Tyrosinase inhibitory activity of natural curcuminoids (1–3) and synthetic analogues (4–17) on L-tyrosine and DOPA sub- strates. The inhibition mechanism on the mushroom tyrosinase was investigated for the best inhibitor through kinetic analysis. The inhibitory types of curcuminoid 5 on mushroom ty- Tyrosinase Inhibitory Activity (IC50; µM) 1 Compound rosinase wereStructure determined during the oxidation of DOPA from Lineweaver−Burk plots [21], L-tyrosine DOPA the results of which are summarized in Table2. O O MeO OMe 1 Table 2. The inhibition type and constants of active curcuminoids326.5 on mushroom94.8 tyrosinase. HO OH O O Inhibition Constants (µM) MeO 2 Compound Inhibition Type 470.0 41.1 K K HO OH I IS 5 O O Mixed-II type 4.64 3.44 3 12 Non-competitive46.5 0.12 34.8 0.08 HO 17 OH Mixed-I type 0.01 0.05 O O MeO OMe 4 Amongst them, the double-reciprocal plots ofInactive the enzyme 2 inhibitedInactive by curcuminoid 2 5 AcO OAc are shown in Figure2. The equilibrium constants for the binding of 5 with free enzyme, O O K ,MeO and with the enzyme–substrateOMe complex, K , were obtained from the second plots of 5 I IS 86.2 8.0 K /V (slope) and 1/V (intercept) versus the concentrations of the inhibitors. From mMeO m OMem the data, weO foundO that curcuminoid 5 could bind, not only with the free enzyme, but MeO OMe 6 also with the enzyme–substrate complex. The KInactiveIS values 2 were lowerInactive than K2 I, indicating HO OH O O O O HO OH 7 66.9 54.6 HO OH N NH MeO OMe 8 677.1 21.9 HO OH

CosmeticsCosmetics 2021 2021, 8,, 8x, FORx FOR PEER PEER REVIEW REVIEW 7 of7 10 of 10

Cosmetics 2021, 8, 35 7 of 10

curcuminoidcurcuminoid 5 acts5 acts as as a competitive–uncompetitivea competitive–uncompetitive mi mixed-IIxed-II type type inhibitor inhibitor [21,23]. [21,23]. These These datadata indicatedthat indicated curcuminoid that that the the affinity affinity5 acts of as ofthe athe competitive–uncompetitiveinhibitor inhibitor for for the the enzyme–substrate enzyme–substrate mixed-II complex complex type was inhibitor was [21,23]. strongerstrongerThese than thandata that that for indicated for the the free free thatenzyme. enzyme. the affinity of the inhibitor for the enzyme–substrate complex was stronger than that for the free enzyme.

Figure 2. (A) Lineweaver−Burk plots for the inhibition of 5 on the mushroom tyrosinase for the Figure 2. (A) Lineweaver−Burk plots for the inhibition of 5 on the mushroom tyrosinase for the Figurecatalysis 2. (A) Lineweaver of DOPA− atBurk 28 plots◦C, pH for 6.8.the inhibition Concentrations of 5 on the of mushroom5 for curves tyrosinase 1–5 were for 0.0,the 2.5, 5.0, 7.5 and catalysiscatalysis of ofDOPA DOPA at at28 28 °C, °C, pH pH 6.8. 6.8. Concentrations Concentrations of 5of for 5 for curves curves 1–5 1–5 were were 0.0, 0.0, 2.5, 2.5, 5.0, 5.0, 7.5 7.5and and µ 10.010.0 µΜ 10.0µ,Μ respectively., respectively.M, respectively. The The insets insets The(B ,(CB) insets, Crepresent) represent (B,C the) the represent secondary secondary theplot plotsecondary of theof the slopes slopes plot and and ofintercepts theintercepts slopes and intercepts versusversus versusconcentrations concentrations concentrations of of5 to 5 todetermine ofdetermine5 to determine the the inhibition inhibition the constants. inhibition constants. constants.

InIn addition, addition,In addition, the the inhibitory inhibitory the inhibitory type type of ofcurcuminoid curcuminoid type of curcuminoid 17 17on on mushroom mushroom17 on tyrosinase tyrosinase mushroom using usingtyrosinase L- L- using tyrosinetyrosineL-tyrosine substrate substrate substratewas was determined determined was determined by by the the same same bymethods. methods. the same Amongst Amongst methods. them, them, Amongstthe the double-re- double-re- them, the double- ciprocalciprocalreciprocal plots plots of of the plots the enzyme enzyme of the inhibited inhibited enzyme by inhibitedby curcuminoid curcuminoid by curcuminoid17 17are are shown shown in17 inFigureare Figure shown 3. The3. The re- in re- Figure3. The sultssults showedresults showed that showed that curcuminoid curcuminoid that curcuminoid 17 17 could could bind, 17bind,could not not only bind,only with with not free onlyfree enzyme, enzyme, with freebut but also enzyme, also with with but also with the enzyme−substrate complex. The values of both Km and Vmax decreased with increasing the enzymethe enzyme−substrate−substrate complex. complex. The values The of values both K ofm and both V Kmaxm decreasedand Vmax withdecreased increasing with increasing inhibitor concentrations and the KI values were lower than KIS, indicating that 17 acts as a inhibitorinhibitor concentrations concentrations and the and KI values the K werevalues lower were than lower KIS, indicating than K ,that indicating 17 acts as that a 17 acts as competitive–uncompetitive mixed-I type inhiI bitor [23,24]. The competitiveIS effect was competitive–uncompetitivea competitive–uncompetitive mixed-I type mixed-I inhibitor type inhibitor[23,24]. The [23 co,24mpetitive]. The competitive effect was effect was stronger than the uncompetitive effect, meaning that curcuminoid 17 inhibited the free strongerstronger than thanthe uncompetitive the uncompetitive effect, meaning effect, meaning that curcuminoid that curcuminoid 17 inhibited17 theinhibited free the free enzymeenzyme more more than than the the en enzyme–substratezyme–substrate complex. complex. enzyme more than the enzyme–substrate complex.

Figure 3. (A) Lineweaver−Burk plots for the inhibition of 17 on the mushroom tyrosinase for the catalysis of L-tyrosine at 28 ◦C, pH 6.8. Concentrations of 17 for curves 1–5 were 0.0, 5.0, 10.0, 15.0 µ and 20.0 M, respectively. The insets (B,C) represent the secondary plot of the slopes and intercepts versus concentrations of 17 to determine the inhibition constants.

Moreover, the inhibitory mechanisms of compound 12 on mushroom tyrosinase using L-tyrosine substrate were also studied from Lineweaver−Burk plots [21]. Amongst them, Cosmetics 2021, 8, x FOR PEER REVIEW 8 of 10

Figure 3. (A) Lineweaver−Burk plots for the inhibition of 17 on the mushroom tyrosinase for the catalysis of L-tyrosine at 28 °C, pH 6.8. Concentrations of 17 for curves 1–5 were 0.0, 5.0, 10.0, 15.0 and 20.0 µΜ, respectively. The insets (B,C) represent the secondary plot of the slopes and inter- cepts versus concentrations of 17 to determine the inhibition constants. Cosmetics 2021, 8, 35 8 of 10 Moreover, the inhibitory mechanisms of compound 12 on mushroom tyrosinase us- ing L-tyrosine substrate were also studied from Lineweaver−Burk plots [21]. Amongst them, the double-reciprocal plots of the enzyme inhibited by curcuminoid 12 are shown in Figurethe double-reciprocal 4. From these data, plots the enhancemen of the enzymet of inhibited inhibitor byconcentration curcuminoid could12 aredecrease shown in the valuesFigure of4 .V Frommax, but these the data,values the of enhancementKm remained the of inhibitor same, whic concentrationh indicated couldthat curcumi- decrease the noid values12 was of a Vnoncompetitivemax, but the values inhi ofbitor Km forremained mushroom the same, tyrosinase. which This indicated behavior that curcuminoidshowed that 1212 couldwas a bind noncompetitive with both free inhibitor enzyme for and mushroom enzyme–substrate tyrosinase. Thiscomplex behavior and their showed equi- that 12 could bind with both free enzyme and enzyme–substrate complex and their equilibrium librium constants were the same [23,25]. In all cases, the Ki value was considerably lower than constantsthat recently were reported the same for [23 kojic,25]. Inacid all (9.23 cases, µM) the K[26],i value which was demonstrates considerably lowerthat com- than that µ poundsrecently 5, 12 and reported 17 are for potent kojic tyrosinase acid (9.23 inhibitors.M) [26], which demonstrates that compounds 5, 12 and 17 are potent tyrosinase inhibitors.

Figure 4. (A) Lineweaver−Burk plots for the inhibition of 12 on the mushroom tyrosinase for the Figure 4. (A) Lineweaver−Burk plots◦ for the inhibition of 12 on the mushroom tyrosinase for the catalysiscatalysis of L-tyrosine of L-tyrosine at 28 at°C, 28 pHC, 6.8. pH Concentrations 6.8. Concentrations of 12 offor12 curvesfor curves 1–5 were 1–5 were 0.0, 0.5, 0.0, 1.0, 0.5, 2.0 1.0, 2.0 and and 3.03.0 µµΜM,, respectively. respectively. The The insets insets ( B,,C) represent thethe secondarysecondary plotplot ofof the the slopes slopes and and intercepts intercepts versus versusconcentrations concentrations of of12 12to to determine determine the the inhibition inhibition constants. constants. 4. Conclusions 4. Conclusions In this study, we have identified a number of chemically modified curcuminoid ana- In this study, we have identified a number of chemically modified curcuminoid ana- logues with potent inhibitory activities against tyrosinase enzyme on L-tyrosine and DOPA logues with potent inhibitory activities against tyrosinase enzyme on L-tyrosine and substrates. For the analogues with potent antityrosinase on the L-tyrosine substrate, the DOPA substrates. For the analogues with potent antityrosinase on the L-tyrosine sub- isoxazole analogue 12 and two reduced curcuminoid analogues, hexahydrocurcumin (16) strate, the isoxazole analogue 12 and two reduced curcuminoid analogues, hexahydrocur- and the α, β-unsaturated curcuminoid analogue 17, showed IC50 values of 8.3, 14.6 and 9.4 cumin (16) and the α, β-unsaturated curcuminoid analogue 17, showed IC50 values of 8.3, µM, or 20.9-, 11.9- and 18.4-fold, respectively, more active than the reference kojic acid. For 14.6 and 9.4 µM, or 20.9-, 11.9- and 18.4-fold, respectively, more active than the reference the analogues with potent antityrosinase on DOPA substrate, the dimethylated analogue 5 kojic acid. For the analogues with potent antityrosinase on DOPA substrate, the dimethyl- exhibited the strongest activity, with the IC50 value of 8.0 µM, followed by the pyrazoles ated analogue 5 exhibited the strongest activity, with the IC50 value of 8.0 µM, followed 9 and 10, with the IC50 values of 19.7 and 19.2 µM. These three compounds were 16.6-, by the6.7- pyrazoles and 6.9-fold 9 and more 10, activewith the than IC50 kojic values acid. of It19.7 is noteworthy and 19.2 µM. that These hexahydrocurcumin three com- pounds(16 )were may 16.6-, be regarded 6.7- and as 6.9-fold an analogue more active with antityrosinasethan kojic acid. activity It is noteworthy on both L-tyrosine that hex- sub- ahydrocurcuminstrates. The inhibitory(16) may be types regarded of curcuminoid as an analogue5 on with mushroom antityrosinase tyrosinase activity were on determined both L-tyrosineduring substrates. the oxidation The ofinhi DOPAbitory from types the of Lineweaver curcuminoid−Burk 5 onplots mushroom and it wastyrosinase found that werecompound determined5 duringcould bindthe oxidation with the of free DOPA enzyme from and the theLineweaver enzyme–substrate−Burk plots complex. and it It was foundacted asthat a competitive–uncompetitivecompound 5 could bind with mixed-II the free type enzyme inhibitor. and the These enzyme–substrate data indicated that the affinity of the inhibitor for the enzyme–substrate complex was stronger than that for the free enzyme. For the curcuminoid 17, the inhibitory type showed that it could bind, not only with the free enzyme, but also with the enzyme-substrate complex. The results indicated that 17 acted as a competitive–uncompetitive mixed-I type inhibitor; it inhibited the free enzyme more than the enzyme–substrate complex. These potent analogues might serve as lead compounds for further design of new potential tyrosinase inhibitors and play a potential role in the prevention and treatment of skin pigmentation disorders. Cosmetics 2021, 8, 35 9 of 10

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/cosmetics8020035/s1, Figures S1–S34: The 1H and 13C NMR spectra of compounds 1–17. Author Contributions: Conceptualization, A.S. and S.A.; methodology, A.A., N.N., W.P. and V.A.; software, S.A.; validation, S.A. and M.D.; formal analysis, S.A. and A.S.; investigation, A.A., N.N. and W.P.; resources, A.S. and S.A.; data curation, S.A., M.D. and A.S.; writing—original draft preparation, A.A.; writing—review and editing, A.S., W.P., V.A. and S.A..; visualization, N.N.; supervision, A.S. and S.A.; project administration, A.S. and S.A.; funding acquisition, A.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by The Thailand Research Fund, grant number DBG6180030. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: This work was supported by the Junior Research Fellowship Program, French Embassy, Thailand, the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research and Innovation, and The Research Unit in Synthetic Compounds and Synthetic Analogues from Natural Product for Drug Discovery (RSND), Burapha University. Supports from Ramkhamhaeng University and Burapha University are gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest.

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