Carvacrol derivatives as mushroom tyrosinase inhibitors; synthesis, kinetics mechanism and molecular docking studies

The present work describesthe development of highly potent mushroom tyrosinase inhibitor better than the standard kojic acid. Carvacrol derivatives 4a-f and 6a-d having substituted benzoic acid and cinnamic acidresidues were synthesized with the aim to possess potent tyrosinase inhibitory activity.The structures of the synthesized compounds were ascertained by their spectroscopic data (FTIR, 1HNMR, 13CNMR and Mass Spectroscopy).Mushroom tyrosinase inhibitory activity of synthesized compounds was determined and it was found that one of the derivative 6c possess higher activity (IC50 0.0167μM) than standard kojic acid (IC50 16.69μM). The derivatives 4c and 6b also showed good tyrosinase inhibitory activity with (IC50 16.69μM) and (IC50 16.69μM) respectively.Lineweaver—Burk and Dixon plots were used for the determination of kinetic mechanism of the compounds 4c and 6b and 6c. The kinetic analysis revealed that compounds 4c and 6b showed mixed-type inhibition while 6c is a non-competitive inhibitor having Ki values19 μM, 10 μM, and 0.05 μMrespectively. The enzyme inhibitory kinetics further showed thatcompounds 6b and 6c formed irreversible enzyme inhibitor complex while 4c bind reversibly with mushroom tyrosinase.The docking studies showed that compound 6c have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (-7.90 kcal/mol) as compared to others.The 2-hydroxy group in compound 6c interacts with amino acid HIS85 which is present in active binding site. The wet lab results are in good agreement with the dry lab findings.Based upon our investigation we may propose that the compound 6c is promising candidate for the development of safe cosmetic agent.


Introduction
Tyrosinase a copper containing metalloenzymemediating the o-hydroxylation of monophenols to catechols and the subsequent two-electron oxidation to quinines [1]. The physiological function of tyrosinase is to convert tyrosine into dopaquinone, which represents the first step of melaninbiosynthesis in melanosomes [2][3].The key starting material for melanin biosynthesis is aromatic amino acid L-tyrosine [4].The L-tyrosine and L-3,4-dihydroxyphenylalanin (L-DOPA) play vital role in regulation of the melanin synthesis [5][6][7]. The color of human skin is determined by the presence of melanin in the surrounding keratinocytes [8][9]. A number of other factors such as UV exposure, α-melanocyte-stimulating hormone, melanocortin 1 receptor and agouti-related protein are also involved in melanogenesis [10][11].The melanogenesiscorrespondsto a possible cellular danger and is confined to special melanosomes in melanocytes, which synthesize pigments and transfer them to recipient cells [12].The abnormal proliferation of melanocytes produced melanoma a type of skin cancer [13][14].
The abnormal accumulation of melaninin keratinocytes caused melasma and post-inflammatory disorders for which patients pursue treatment [15].A number of other melanocytedisorders like senile lentigo, freckles and pigmented acne scars occur in human of all races worldwide [16].Hyperpigmetationdisorders adversely affect person's psychological and social well-beingwhich results in lower output, overall performance, and self-confidence [17].Tyrosinasecan also be linked to neurodegenerative diseases owing to excessive production ofdopaquinonesbyoxidation of dopamine results in neuronal damage and cell death [18][19][20].It has also been reported that tyrosinase is responsible for quicker degradation and lesser shelf life of fruits and vegetables during postharvest handling and processing [21][22][23].The therapeutics used currently to treat hyperpigmentation disorders generally associated with side effects. Thus synthesis of potent tyrosinase inhibitors with minimal side effects is of great interest in the medical, agricultural and cosmetic industries.
The antioxidant and tyrosinase inhibitory activities of substituted benzoic and cinnamic acids have been reported previously [24][25]. Carvacrol is a naturally occurring monoterpene phenol present in thyme along with thymol. Carvacrol besides its odoriferous functions exhibited antimicrobial activities [26][27][28]. Thus, carvacrol and thymol are employed as meat preservatives or flavoring agents in the food industry. The antioxidant activity of thyme essential oils was previously reported, and it has been identified to be due to the presence of carvacrol and thymol [29][30][31]. Carvacrol as antioxidant protects food qualities and organisms from damage induced by oxidative stress. In contrast to these well-studied biological effects the tyrosinase inhibitory potential of carvacrol is poorly understood. The present work describes the synthesis, tyrosinase inhibitory kinetics and computation studies of carvacrol derivatives. The title compounds were synthesized by incorporating the substituted benzoic and cinnamic acids. The enzyme inhibitory kinetics of the most potent derivatives was also determined. Molecular docking studies were also carried to compare the wet lab results with the computational results.

Chemistry
The carvacrol derivatives 4a-f and 6a-d were obtained by following the previouslydescribed method [32] with slight modification shown insupporting informations (S1 and S2 Figs). The carvacrol in the first step is converted in tointermediate 2 by esterification reaction with chloroacetyl chloride in the presence of (C 2 H 5 ) 3 N and anhydrous methylene chloride as solvent. The formation of the intermediate 2 was ascertained by the presence of ester carbonyl stretching at 1731cm -1 and disappearance of the-OH stretching in FTIR spectra. The title carvacrol derivatives 4a-f and 6a-d were synthesized by simple nucleophilic replacement of chloro group from intermediate (2) by carboxylic group (-COOH) of substituted benzoic acids 3a-f and cinnamic acids 5a-d respectively. The structures of the final products were confirmed by FTIR, 1 HNMR, 13 CNMR and Mass spectroscopic data.

Bioassay for tyrosinase inhibitory activity
The substituted thymol and vanillin based tyrosinase inhibitors have already been reported by the author and got comparable IC 50 values with the standard kojic acid repeated. We have evaluated the role of alkyl, methoxy and aldehyde functional groups in tyrosinase inhibitory activity. In this paper carvacrol derivatives 4a-f and 6a-d were designed based upon our previous investigations. This time 2,4-dihydroxy cinnamic acid moiety is also attached with the carvacrol and we got excellent activity compared to standard kojic acid. The hydroxy substituted benzoic acids and cinnamic acids were selected to synthesize derivatives 4a-f and 6a-d respectively. Thehydroxy substituted tyrosinase inhibitors with IC 50 value less than 10μM are of special interest. The well known clinically used tyrosinase inhibitor kojic acid was used as reference drug for comparison purpose. The bioassay results showed that hydroxy substituted cinnamic acid moiety play major role in tyrosinase inhibitory activity. The substitution pattern of hydroxyl groups at phenyl ring is the decisive factor of inhibitory activity. Thecompound 6c bearing 2,4-dihydroxy substituted cinnamic acid residue exhibited excellent tyrosinase inhibitory activity (IC 50 0.0167μM).The compounds which possess hydroxy substituted cinnamic acid residue are in general are more active than those having hydroxy substituted benzoic acid moiety ( Table 1). The derivatives 4a and 4b having mono hydroxy substituted benzoic acid ring showed comparable activity (IC 50 14.9μM) to that ofkojic acid (IC 50 16.69μM).On the other hand the compound 4c having 2,4-dihydroxy substituted benzoic acid residue exhibited higher tyrosinase inhibitory activity (IC 50 6.7μM) than kojic acid. The compounds 6b and 6d possessing substituted cinnamic acid functionalities showed IC 50 values 6.5 and 6.7 μM respectively better than kojic acid. Interestingly, carvacrol derivative 6c bearing 2,4-dihydroxy substituted cinnamic acid moiety showed most potent activity than all of the synthesized compounds and kojicacid.We may propose that the hydroxy substitution pattern on phenyl ring in case of compound 6c impedes the molecule to interact well with the active sites of enzyme. The kinetic analysis and docking studies helps us to explore the mode of binding and residual interactions between enzyme and synthesized inhibitors. Kinetic studies. The mode of inhibition of synthesized carvacrol derivatives 4c, 6b and 6c on the diphenolase activity, during the oxidation of L-DOPA, was determined from Lineweaver-Burk and Dixon plots. In the presence of compounds 6b, 6c and 4c the kinetics of the enzyme was shown in Figs 1(a)-3(a). The plots of 1/V versus 1/ [S] gave a family of straight lines with different slopes. The analysis showed that V max decreased with changing K m in the  presence of increasing concentrations of compound 6b and 4c which indicated that both of thesecompoundsinhibit tyrosinase by two different pathways, and show mixed type inhibition. This result showed that compounds 6b and 4c can bind, not only with free enzyme, but also with the enzyme-substrate complex [33]. The kinetic study of most active compound 6c showed that it is a non-competitive inhibitor of mushroom tyrosinase Fig 2(a). The value of 1/ V max is increased to a new value while that of K m remains same which indicated that the compound 6c simply lowers the concentration of functional enzyme by non-competitively binding mode at enzyme [34]. On the other hand in Dixon plot, slope obtained from the plots for uninhibited enzyme and with different concentrations of inhibitors 6b, 6c and 4c was consistent with the characteristic patterns of inhibition of compounds 6b, 6c and 4c with Ki value 10 μM, 0.05 μM and 19 μM as shown in Figs 1(b)-3(b), respectively.The inhibition constant Ki for compound 6c was also calculated by secondary replot of slope from Lineweaver-Bruk plot versus inhibitor concentrations as shown in Fig 2( The behavior of compound 6b indicated that it inhibits tyrosinase by two different pathways; competitively forming enzyme-inhibitor (EI) complex and interrupting enzyme-substrate-inhibitor (ESI) complex in non-competitive manner. Binding affinities of EI and ESI complexes were determined to gain insightful pathway in which compounds 6b preferentially preceded, the secondary replots of slope versus concentration of compounds 6b showed EI dissociation constant Fig 1(c), while ESI dissociation constant was shown by secondary replots of intercept versus concentration of compounds 6b Fig 1(d). A lower dissociation constant of Kipointed out stronger binding between enzyme and compounds 6b which suggested preferred competitive over noncompetitive manners [35].
The inhibitory effect of compounds (6b), (6c) and (4c) on diphenolase activity of tyrosinase. The inhibitory mechanism of mushroom tyrosinase by compounds 6b, 6c and 4c for the oxidation of L-DOPAwas studied as shown in Fig 4(a), 4(b) and 4(c), respectively. The plots of the remaining enzyme activity versus the concentration of enzyme (2, 4, 6, 8 and 10 μg/mL) in the presence of different concentrations of compound 6b, 6c and 4c for the catalysis of L-DOPA gave a series of straight lines.In this study, we found the parallel straight lines with the same slopes, indicating that the inhibitory effect of compounds 6b and 6c was irreversible [36][37]to the enzyme Fig 4(a) and 4(b). These results suggested that both the compounds 6b and 6c effectively inhibited the enzyme by binding to its binuclear active site irreversibly. In case of compound 4c all the lines intersected at the same point on X-axis. Increasing the inhibitor concentration resulted in a decrease in the slope of the lines, which indicated that the compound 4c was a reversible inhibitor of tyrosinase [38] (Fig 4(c).

Structural assessment of mushroom tyrosinase
Mushroom tyrosinase (Agaricusbisporus) is a class of oxidoreductase copper containing protein comprises 391 amino acids. The structure architecture of mushroom tyrosinase showed that, it consists of 39% helial (154 residues) and 14% β sheet (57 residues) and 46% coil (180 residues). The X-Ray diffraction study confirmed its resolution 2.78Å, R-value 0.238 and unit cell length values were observed for a = 103.84, b = 104.82 and c = 119.36 with angles 90˚, 110.45˚and 90˚for all α, β and γ dimensions respectively. The Ramachandran plots and values indicated that 95.90% of residues were in favored regions and 100.0% residues were lies in allowed regions. This selected Ramachandran graph values showed the good accuracy of phi (φ) and psi (ψ) angles among the coordinates of receptor molecules and most of residues plummeted in acceptable region.

Molecular docking analysis
The molecular docking studies of the synthesized compounds 4a-f and 6a-d were performed against mushroom tyrosinase (PDBID: 2Y9X) to determine their binding affinities. The 2,4-dihydroxy substituted derivatives showed best affinity for the target protein compared to monosubstituted, 3,4-and 3,5-dihydroxy substituted ones. The docking results displayed that derivatives having hydroxy substituted cinnamic acids functionality exhibited highest affinity than all other synthesized compounds Table 2.
The compound 6c having 2,4-dihydroxy substitution showed maximum binding affinity with binding energy value (-7.90 kcal/mol) as compared to others.The 2-hydroxy group of cinnamic acid in 6c interacts with amino acid HIS85 of the target protein forming two hydrogen bonds with bonding distance 2.07 and 2.12Å. The ester carbonyl oxygen in the same compound also interacts with HIS284 and GLU256 at a bonding distance 2.10 and 2.97Å respectively ( Fig  5). It has been reported that these amino acids are located in active binding site and are significant in the downstream signaling pathways [39].The compound 4c having 2,4-dihydroxy substituted benzoic acid residue showed good binding affinity with binding energy value(-7.80 kcal/ mol). Interestingly 2-hydroxy group of benzoic acid moiety in compound 4c interacts with-HIS85 and 4-hydroxy interacts with ASN81having bond length 2.18 and 2.35Å respectively ( Fig  6). The

Conclusion
The carvacrol derivatives 4a-4f and 6a-6d were synthesized by incorporation of the substituted benzoic acids and cinnamic acids to discover the highly potent mushroom tyrosinase inhibitor.The hydroxy substituted derivatives showed good to excellent tyrosinase inhibitory activity. The compound 6c bearing 2,4-dihydroxy substituted cinnamic acid residue exhibited excellent tyrosinase inhibitory activity (IC 50 0.0167μM).The derivatives with hydroxy substituted cinnamic acid residue possess greater tyrosinase inhibitory potential as compared to benzoic acids.The kinetic study of most active compound 6c showed that it is a non-competitive inhibitor with Ki value 0.05 μM.The kinetic results further revealed that derivative 6c effectively inhibited the enzyme by binding to its binuclear active site irreversibly.The docking studies showed that compound 6c have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (-7.90 kcal/mol) as compared to others.The 2-hydroxy group in compound 6c interacts with amino acid HIS85 which is present in active binding site. Based upon our investigation it is proposed that carvacrol derivative 6c may have potential to design new cosmetic agents.

Experimental
All chemicals used for the synthesis of compounds were purchased from Sigma Chemical Co. Melting points were determined using a Digimelt MPA 160, USA melting point apparatus and are reported uncorrected. The FTIR spectra were recorded with Shimadzu FTIR-8400S spectrometer (Kyoto, Japan, υ, cm -1 ). Mass spectra were performed on an Agilent 6460 Series Triple Quadrupole instrument (Agilent). Electrospray ionization both in the positive (ESI+) and negative ion mode (ESI-) was employed for ionization. Elemental Analysis (C, H) were carried out on a Flash 2000 series elemental analyzer with TCD detector system and results are with ±0.3%. The 1 H NMR and 13 C NMR spectra (DMSO-d 6 ) were recorded using a Bruker 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm downfield from the internal standard tetramethylsilane (TMS). The purity of the compounds was checked by thin layer chromatography (TLC) on silica gel plate using n-hexane and ethyl acetate as mobile phase.

Reagents
Mushroom tyrosinase was purchased from Sigma (USA); L-DOPA and thymol were purchased from Sigma (USA). Stock solutions of the reducing substrates were prepared in phosphate buffer (20 mM, pH 6.8).

Synthesis of intermediate (2)
Carvacrol 1 (0.01 mol), triethylamine (0.01 mol) were mixed in dry dichloromethane (25mL) at 0 to -5˚C. The reaction mixture was treated withchloroacetyl chloride (0.01mol) in dry dichloromethane drop wise with constant stirring over a period of 1h maintaining the temperature constant. The reaction mixture was then stirred at room temperature for further 5hand progress of the reaction was determined by thin layer chromatography. After the completion of reaction the mixture was washed with 5% HCl, and 5% sodium hydroxide solution. The organic layer was washed with saturated aqueous NaCl, dried over anhydrous magnesium sulphate, filtered and solvent was removed under reduced pressure. The crude  and extracted with ethyl acetate (4×25 mL). The combined organic layer was washed with 5% HCl, 5% sodium hydroxide and finally with aqueous NaCl solution. The organic layer was dried over anhydrous magnesium sulphate, filtered and the solvent was removed under reduced pressure to afford the crude products 4a-4f. The title compounds 4a-f were purified by silica gel column chromatography (n-hexane: ethyl acetate 4:1). The same procedure was used for the preparation of compounds 6a-d.

Anti-tyrosinase activity
The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was used for in vitro bioevaluation as described previously with some modifications [40][41]. Briefly, 140μL of phosphate buffer (20mM, pH 6.8), 20μL of mushroom tyrosinase (30U/mL) and 20μL of the inhibitor solution were placed in the wells of a 96-well micro plate. After pre-incubation for 10 min at room temperature, 20μL of L-DOPA (3,4-dihydroxyphenylalanine) (0.85mM) was added and the plate was further incubated at 25˚C for 20min. Subsequently the absorbance of dopachrome was measured at 492nm using a micro plate reader (OPTI Max , Tunable). Kojic acid was used as a reference inhibitor and for negative tyrosinase inhibitor phosphate buffer was used instead of the inhibitor solution. The extent of inhibition by the synthesized carvacrol derivatives 4a-f and 6a-d was expressed as the percentage of concentration necessary to achieve 50% inhibition (IC 50 ). Each concentration was analyzed in three independent experiments run in triplicate. The IC 50 values were determined by the data analysis and graphing software Origin 8.6, 64-bit.
Kinetic analysis of tyrosinase inhibition activity. The carvacrol derivatives 4c, 6b and 6c were selected to determine their kinetic mechanism of enzyme inhibition.A series of experiments were performed to determine the inhibition kinetics by following the already reported method [42][43]. Inhibitor 6b with concentrations 0.0, 7.0, 28 μM, in case of Lineweaver-Burk plot and with concentrations0.0, 1.75, 7.0, 14.0, 28 μM in case of Dixon plot was used respectively. Compound 6c with concentrations0.0, 0.01, 0.02, 0.04μMand 4c with concentrations 0.0, 1.68, 3.37, 6.75, 13.5 μM,respectively were usedin case of Lineweaver-Burk plot as well as in Dixon plot. Substrate L-DOPA concentration was among 0.0625 to 2 mM in all kinetic study. Pre-incubation and measurement time was the same as discussed in mushroom tyrosinase inhibition assay protocol. Formation of DOPAchrome was continuously monitored at 475nm for 5min at a 30s interval in the microplate reader after addition of enzyme. The inhibition type on the enzyme was assayed by Lineweaver-Burk plots of inverse of velocities 1/ V versus inverse of substrate concentration 1/[S] mM -1 , and the inhibition constant Ki was determined by Dixon plot of 1/V versus inhibitor concentrations.
The effect of concentrations of compounds 4c, 6b and 6c on the activity of mushroom tyrosinase for the catalysis of DOPA at 37˚C was also studied. Assay conditions were same as described in assay protocolexceptthat the final concentration of enzyme was varied (2 to 10 μg/mL).

Molecular docking studies
Retrieval of target structure. The crystal structure of mushroom tyrosinase (PDBID: 2Y9X) was retrieved from Protein Data Bank (PDB) (http://www.rcsb.org). The energy minimizations target protein was carried out by employing conjugate gradient algorithm and Amber force field in UCSF Chimera 1.10.1 [44]. The stereo-chemical properties, Ramachandran graph and values [45] of mushroom tyrosinase structure were assessed by Molprobity server [46], while the hydrophobicity graph was generated by Discovery Studio 4.1 Client [47]. The protein architecture and statistical percentage values of helices, beta-sheets, coils and turn were accessed by using online tool VADAR 1.8 [48]. Candidate structures. The synthesized candidate molecules 4a-f and 6a-d were sketched in drawing ACD/ChemSketch tool. The designed ligand molecules were further visualized and minimized by UCSF Chimera 1.10.1. Multiple online drug assessment computational tools like Molinspiration(http://www.molinspiration.com/) and Molsoft (http://www.molsoft.com/) were used to predict the drug-likeness and biological properties of these designed candidate molecules. The Osiris Property Explorer was used to evaluate their possible tumorigenic or mutagenic risks and also to calculate the drug-likeness values. Lipinski's rule of five was analyzed using Molsoft and Molinspiraion tools. Furthermore, their predicted Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties were evaluated by pkCSM online tool [49]. Similarly, different molecular properties like Molar Refractivity, Density, Surface tension and Polarizability were accessed by ChemSketch.
Molecular docking. Molecular docking of all the synthesized ligand molecules 4a-f and 6a-d against mushroom tyrosinase were carried out using diverse AutoDock 4.2 tool according to the specified instructions [50]. In brief, for receptor protein the polar hydrogen atoms and Kollman charges were apportioned and for ligand, Gasteiger partial charges were designated and non-polar hydrogen atoms were merged. All the torsion angles for all the ligands were set free to rotate through docking experiments. The docking experiments were carried out by considering receptor as a rigid while ligands as flexible molecules. A grid map of 80 Åx80 Åx80 Å was set on the whole protein structure to generate the grid map. The number of runs for each docking experiment was set to 100. The Lamarckian genetic algorithm (LGA) and empirical free energy function were applied by taking docking parameters default. The docked complexes were further evaluated on lowest binding energy (Kcal/mol) values and hydrogen bond analysis using Discovery Studio (4.1) and UCSF Chimera 1.10.1.