European Journal of Medicinal Chemistry 98 (2015) 203e211

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Kinetic and in silico studies of novel hydroxy-based thymol analogues as inhibitors of mushroom tyrosinase Zaman Ashraf a, b, Muhammad Rafiq a, Sung-Yum Seo a, *, Kang Sung Kwon c, Mustafeez Mujtaba Babar d, Najam-us-Sahar Sadaf Zaidi c a

Department of Biology, College of Natural Sciences, Kongju National University, Gongju 314-701, Republic of Korea Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan Department of Chemistry, Chungnam National University Daejeon, 305-764, Republic of Korea d Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, H-12, Kashmir Highway, Islamabad, 44000, Pakistan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 March 2015 Received in revised form 30 April 2015 Accepted 20 May 2015 Available online 21 May 2015

The present studies reports the synthesis of hydoxylated thymol analogues (4aee) and (6aec) as mushroom tyrosinase inhibitors. The title compounds were obtained in good yield and characterized by FTIR, 1H NMR, 13C NMR, Mass spectral data and X-ray crystallography in case of compound (6a). The inhibitory effects on mushroom tyrosinase and DPPH were evaluated and it was observed that 2-[5methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate (6b) showed tyrosinase inhibitory activity (IC50 15.20 mM) comparable to kojic acid (IC50 16.69 mM) while 2-[5-methyl2-(propan-2-yl)phenoxy]-2-oxoethyl 3,4-dihydroxybenzoate (4d) exhibited higher antioxidant potential (IC50 11.30 mM) than standard ascorbic acid (IC50 24.20 mM). The docking studies of synthesized thymol analogues was also performed against tyrosinase protein (PDBID 2ZMX) to compare the binding affinities with IC50 values. The predicted binding affinities are in good agreement with the IC50 values as compound (6b) showed highest binding affinity
7.1 kcal/mol. The kinetic mechanism analyzed by LineweavereBurk plots exhibited that compound (4d) and (6b) inhibit the enzyme by two different pathways displayed mixed-type inhibition. The inhibition constants Ki calculated from Dixon plots for compounds (4d) and (6b) are 34 mM and 25 mM respectively. It was also found from kinetic analysis that derivative (6b) formed reversible enzyme inhibitor complex. It is propose on the basis of our investigation that title compound (6b) may serve as lead structure for the design of more potent tyrosinase inhibitors. © 2015 Published by Elsevier Masson SAS.

Keywords: Thymol analogues Synthesis Antioxidant activity Mushroom tyrosinase inhibitors Kinetic mechanism Molecular docking

1. Introduction Tyrosinase (EC 1.14.18.1) as a binuclear copper-containing metalloenzyme catalyzes two distinct reactions of melanin biosynthesis. It catalyzes L-tyrosine to L-3,4-dihydroxyphenylalanine (LDOPA) and further oxidation of (L-DOPA) to dopaquinone which then transformed through several reactions into brown to black melanin. The distribution patterns of melanin in the surrounding keratinocytes determine the color of human skin [1,2]. Kadekaro et al. and Petit et al. also reported that there are certain other factors such as UV exposure, a-melanocyte-stimulating hormone, melanocortin 1 receptor and agouti-related protein which involved in melanin biosynthesis [3,4]. Abnormal melanin production such

* Corresponding author. . E-mail address: [email protected] (S.-Y. Seo). http://dx.doi.org/10.1016/j.ejmech.2015.05.031 0223-5234/© 2015 Published by Elsevier Masson SAS.

as observed in melasma, freckles, lentigo senilis, and other forms of melanin hyperpigmentation can be a serious aesthetic problem often causing emotional disturbance [5e7]. Taking into account the key role of tyrosinase in melanin production, many tyrosinase inhibitors have found application in cosmetics and pharmaceutical products [8e10]. A large number of tyrosinase inhibitors have been reported but only a few are used because of their limitations with regards to cytotoxicity, selectivity and stability. Thus, it is in great need of developing new tyrosinase inhibitors without causing adverse reactions. A number of cinnamic acid and benzoic acid analogues possessing hydroxy groups at position 3 and 4 of the phenyl ring have been reported as antioxidants and mushroom tyrosinase inhibitors [11,12]. Liu (2003) and Chen (2005) also reported that alkoxy benzoic acids and hydroxy benzoic acids showed potent mushroom tyrosinase inhibitory activity [13,14]. Thymol a naturally occurring monoterpene phenol which is the main constituent of thyme

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possesses tyrosinase inhibitory activity [15] along with other numerous pharmacological activities. As phenolic antioxidants, thymol protects food qualities and organisms from damage induced by oxidative stress. Thymol is used as meat preservatives or flavoring agent in the food industry. It exhibited anti-inflammatory effect in human neutrophils incubated [16] and also inhibits the formation of lethal products through reactive nitrogen species [17]. Thymol displays antimicrobial [18e20] and wound-healing activity [21] and is able to rise the levels of macrophage migration inhibitory factor (MIF) in central nervous system [22]. Thymol increased the in vitro fibroblast growth [23], effectively inhibited COX-1 [24] and prevented inducible lymphocyte proliferation [25]. Keeping in view the importance of these structural features we have synthesized the hydroxylated thymol analogues having benzoic acid and cinnamic acid moieties in order to discover their tyrosinase inhibitory potential to offer a source for the development of new effective tyrosinase inhibitors. The antioxidant activity was also carried out as most of the clinically used tyrosinase inhibitors kojic acid, arbutin, kaempferol, hydroquinone, etc. all possess antioxidant activity. In addition to evaluate the tyrosinase inhibitory potential and antioxidant activity of synthesized hydroxylated thymol derivatives molecular docking was carried out to predict the position of the synthesized compounds in the active site of the 3D structure of tyrosinase (PDB ID 2ZMX). The investigation of the binding interactions during docking analysis between ligandprotein functionalities is important to elucidate the possible molecular mechanism [26,27]. 2. Results and discussion 2.1. Chemistry The title compounds (4aee) and (6aec) were synthesized by following the already reported method [28] with some modification shown in Schemes 1 and 2. The thymol chlroacetyl derivative (2) was synthesized by esterification of phenolic eOH group of thymol with chloroacetyl chloride in the presence of (C2H5)3N and anhydrous methylene chloride as solvent. The presence of ester carbonyl stretching at 1723 cm
1 and disappearance of the eOH stretching in FTIR spectra confirmed the formation intermediate (2). The final products (4aee) and (6aec) were prepared by simple

nucleophilic substitution at intermediate (2) with hydroxy substituted benzoic acids (3aee) and substituted and unsubstituted cinnamic acids (5aec) respectively. All of the synthesized compounds have been characterized by FTIR, 1HNMR, 13CNMR and Mass spectroscopic data. Compound (6a) yielded single crystals, suitable for X-ray diffraction studies. The X-ray diffraction analysis also confirmed the formation of the desired product and this is an ambiguous evidence of the structure. 2.2. Bioassay for tyrosinase inhibitory activity Hydroxylated thymol analogues have been designed to evaluate their inhibitory effects on mushroom tyrosinase activity. Kojic acid a competitive tyrosinase inhibitor was used as standard for comparison purpose. The hydroxylated tyrosinase inhibitors as described in preceding section are of special interest because of their high activity (IC50 < 10 mM). Novel thymol analogues (4aee) and (6aec) have been synthesized by incorporation of hydroxylated benzoic acids and cinnamic acids. The synthesis of mono and di-hydroxylated derivatives with different position of eOH at phenyl ring was carried out to explore the role of multiple hydroxyl groups in tyrosinase inhibition. It has been exposed from our bioassay results (Table 1) that the major determining factor of inhibitory activity is the position and not the number of the hydroxyl groups. Interestingly, compound (4d) bearing 3,4-dihydroxy substituted benzoic acid moiety showed higher activity (IC50 45.0 mM) than (4c) and (4e) having IC50 56.1 and 220.9 mM respectively. The derivatives (4c) and (4e) possess 2,4- and 3,5dihydroxy substituted benzoic acid residues respectively. In case of compound (4d) two hydroxy groups are present on adjacent positions of phenyl ring; this impedes the molecule to interact well with enzyme. This structural feature can be well correlated with LDOPA which is used as substrate for tyrosinase enzyme during bioassay. Thus compound (4d) because of close structural similarities with L-DOPA is more active among the dihydroxylated thymol analogues. Table 1 presented the IC50 values of the synthesized thymol analogues and it was observed that kojic acid is more active than all of the synthesized thymol analogues except compound (6b). Compound (6b) showed excellent tyrosinase inhibitory activity with IC50 15.2 mM and is so inhibitor as kojic acid (IC50 16.69 mM). The synthesized thymol derivatives (6a), (6b) and (6c)

CH3

CH3 O

+

HO H3C

Cl

(C2H5)3N/CH2Cl2

O

0 to -5°C

Cl

O Cl

CH3

H3C

(1)

(2)

CH3

Cl

O

CH3 OH

+

O

(C2H5)3N/KI

O

DMF

R H3C

CH3

(2)

CH3

O O

R O

(3a-3e)

R= 3a = 3-OH 3c = 2,4-di-OH 3e = 3,5-di-OH

(4a-4e) 3b = 4-OH 3d = 3,4-di-OH

O H3C

R is same as in (3a-3e)

Scheme 1. Synthesis of thymol analogues (4aee).

CH3

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205

Table 1 Tyrosinase inhibitory activity and free radical scavenging activity of the synthesized thymol analogues (4aee) and (6aec). Codes

4a 4b 4c 4d 4e 6a 6b 6c Kojic acid Ascorbic acid a

Tyrosinase inhibition Activity

DPPH Activity

% Inhibition (25 mg/mL)

IC50 ± SEM mM

% Inhibition (500 mg/mL)

IC50 ± SEM mM

48 ± 1 33 ± 2 68 ± 2 55 ± 3 5±2 42 ± 3 93 ± 4 77 ± 2 100

79.3 ± 5.3 91.5 ± 9.4 56.1 ± 5.9 45.0 ± 1.5 220.9 ± 11.6 100.8 ± 14.3 15.2 ± 0.42 45.2 ± 4.3 16.69 ± 2.8

3±1 2±1 3±1

a

9±2 8±3 5±2 50 ± 6

1344.0 ± 18.76

a

a

97 ± 1

24.20 ± 1.93

a

a

a a

11.30 ± 2.09 a a a

a

Not determined.

all possess cinnamic acid moiety but varied in substitution pattern on phenyl ring. The derivative (6a) have unsubstituted cinnamic acid residue while compound (6b) and (6c) possess 4-hydroxyl and 4-chloro cinnamic acid moiety respectively. Interestingly, presence of 4-hydroxyl group on cinnamic acid phenyl ring in compound (6b) led to a dramatic increase in tyrosinase inhibitory activity. The compound (6a) (IC50 100.8 mM) is 6.6 times less active while (6c) (IC50 45.2 mM) is 2.97 times less active than the most potent derivative (6b). Based upon our results we propose that thymol analogue (6b) may serve as a lead structure for the design of more potent tyrosinase inhibitors. 2.3. Kinetic mechanism The inhibitory mechanism of the most active compounds (6b) and (4d) on mushroom tyrosinase for the oxidation of L-DOPA was studied. Kinetic studies showed a concentration dependent inhibition of mushroom tyrosinase by thymol derivatives (4d) and (6b). Continuous monitoring of the reaction showed a marked decrease in reaction rate in the presence of the inhibitors, which is ultimately, indicated the decrease in the final absorbance when compared with controls containing no inhibitor. The potency of inhibition exhibited by these compounds varied depending on the presence of type and position of the functionalities on the aromatic rings [29]. Inhibition kinetics was analyzed by Lineweaver-Burk plot and Dixon plots to determine the type of inhibition and inhibition constant (Ki). The results of Figs. 1e2(a) showed that in case of compounds (4d) and (6b) by increasing the concentration of substrate (L-DOPA) gave family of straight lines, which intersected within the second quadrant. The analysis showed that Vmax decreased with changing Km in the presence of increasing

concentrations of compounds. This behavior of compounds (4d) and (6b) indicated that it inhibits tyrosinase by two different pathways and show mixed type inhibition. On the other hand in Dixon plot, slope obtained from the plots for uninhibited enzyme and with different concentrations of inhibitors (4d) and (6b) was consistent with the characteristic patterns of mixed-type inhibition with Ki value 34 mM and 25 mM as shown in Figs. 1e2(b) respectively. 2.4. Reversible inhibition The most potent derivative (6b) was selected to ascertain the formation of reversible enzyme-Inhibitor complex between tyrosinase and inhibitor (6b). The plots of initial rate verses enzyme concentrations (4, 6, 8, 10, 15 and 20 mg/mL) for compound (6b) all consisted of straight lines passing through the origin. Increasing the inhibitor concentration resulted in a decrease in the slope of the lines, which indicate that the enzyme undergoes a reversible inhibition [30] (Fig. 3). 2.5. DPPH radical scavenging assay Thymol derivatives were evaluated for the DPPH free radical scavenging ability as excessive melanogenesis generates free radicals which start unwanted reactions. The thymol derivative (4d) exhibited excellent radical scavenging activity when compared with the standard ascorbic acid. The IC50 value of compound (4d) is 11.30 mM while IC50 value of ascorbic acid is 24.20 mM. The presence of ortho disubstituted phenyl ring in compound (4d) play significant role in radical scavenging activity as it showed more potent radical scavenging activity than ascorbic acid. All of the other

Fig. 1. a) Lineweaver-Burk plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (4d) in the presence of different concentrations of L-DOPA. b) Dixon plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (4d) in the presence of different concentrations of L-DOPA (0.062, 0.125, 0.25, 0.5 and 1 mM, respectively).

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Fig. 2. a) Lineweaver-Burk plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (6b) in the presence of different concentrations of L-DOPA. b) Dixon plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (6b) in the presence of different concentrations of L-DOPA (0.062, 0.125, 0.25, 0.5 and 1 mM, respectively).

Fig. 3. Effects of concentrations of mushroom tyrosinase on its activity for the catalysis of L-DOPA at different concentrations of compound (6b).

thymol derivatives showed little activity even at high concentration (500 mg/mL) Table 1. 2.6. Docking studies The in silico docking of the synthesized thymol derivatives was performed against crystal structure of tyrosinase protein (PDBID 2ZMX) to compare their experimental IC50 with binding affinities. Matoba et al. published recently the crystallographic structure of tyrosinase (PDBID 2ZMX) which enables us to predict the binding affinities of thymol derivatives. The compound (6b) showed highest binding affinity with the target protein having binding energy value
7.1 kcal/mol. The experimentally determined tyrosinase inhibitory activity results are in good agreement with the predicted binding affinities by in silico docking. The 4ehydroxy group of cinnamic acid phenyl ring in compound (6b) formed hydrogen bonds with GLU67 having distance 1.99 Å. The p-p stacking also present in the same compound between phenyl ring of thymol moiety and PHE34 having distance 3.81 Å (Fig. 4). The phenolic hydroxyls present at positions 3 and 4 in compound (4d) formed hydrogen bonds with SER96 having bond lengths 2.36 Å and 2.89 Å respectively (Fig. 5) The hydrogen of 3hydroxyl in the same compound also interact with VAL95 having distance 2.40 Å between two interacting groups. The ester carbonyl oxygen in compound (4d) also formed hydrogen bond with ARG140 having bond length 2.40 Å. Table 2 presented the calculated binding energies of the synthesized thymol analogues and it determines

the stability of ligandetarget complex. There is hydrogen bond between 4-hydroxy group of derivative (4c) and amino acid THY37 of the target protein with bond length 1.84 Å (Fig. 6). The binding energy of thymol analogue (4c) is
6.5 kcal/mo. The presence of phenolic hydroxyl on acid moiety in the synthesized compounds is the determining factor of binding energies. Another important structural feature is the cinnamic acid moiety. It was observed that when phenolic hydroxyl is present on para position of the cinnamic acid residue then ligand has maximum binding affinity with the receptor protein. The experimentally determined IC50 values also confirmed the docking results as compound (6b) showed the maximum inhibition of the mushroom tyrosinase activity. The partition coefficient values (log P) of the synthesized compounds were calculated by ChemSketch ACD labs 2012 to verify the druglikeness properties of the compounds. The most potent derivatives (6b) and (4d) have partition coefficient values 4.92 ± 0.33 and 4.97 ± 0.32 respectively which are not beyond the limits of (log P) values. 3. Conclusion The hydroxylated thymol analogues (4aee) and (6aec) having substituted benzoic acids and cinnamic acids moieties have been synthesized and fully characterized. The computational studies investigation revealed that thymol analogue (6b) possesses maximum binding affinity (
7.1 kcal/mol) with the target protein (PDBID 2ZMX). The wet lab results are in good agreement with the calculated docking scores. The most potent activity against mushroom tyrosinase was exhibited by the compound (6b) having IC50 15.2 mM. The presence of phenolic eOH at para-position on cinnamic acid phenyl ring play very significant role in tyrosinase inhibition activity. The kinetic analysis of compound (6b) shown that it is mixed type inhibitor with Ki value 25 mM and formed reversible drugereceptor complex with mushroom tyrosinase. It was concluded from our results that the title compound (6b) may serve as lead structure for the design of more potent tyrosinase inhibitors. 4. 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 FTIRe8400S spectrometer (Kyoto, Japan, y, cm
1). Mass spectra were performed on an Agilent 6460 Series Triple

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207

Fig. 4. The interactions of compound (6b) with the active site of mushroom tyrosinase (PDBID 2ZMX) generated by using Discovery Studio 4.1.0. The a) shows the two dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein. The b) shows the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate bond distances between interacting functionalities of the ligand and receptor.

Fig. 5. The interactions of compound (4d) with the active site of mushroom tyrosinase (PDBID 2ZMX) generated by using Discovery Studio 4.1.0. The a) shows the two dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein. The b) shows the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate bond distances between interacting functionalities of the ligand and receptor.

Table 2 Average binding affinity and Log P values of the synthesized thymol analogues (4aed) and (6aec). Compound

Average binding affinity (kcal/mol)

Log pa

4a 4b 4c 4d 4e 6a 6b 6c

5.8 6.4 6.5 6.6 6.5 5.6 7.1 5.7

5.17 5.14 5.20 4.97 4.88 5.45 4.92 5.98

a

± ± ± ± ± ± ± ±

0.30 0.30 0.33 0.32 0.32 0.30 0.33 0.33

was 350  C. Nitrogen was used as a desolvation gas (flow 600 L/h). Elemental Analysis (C, H) were carried out on a Flash 2000 series elenatal analyzer with TCD detector system and results are with ±0.3%. The 1H NMR and 13C NMR spectra (CDCl3) and (DMSO-d6) were recorded using a Bruker 400 MHz spectrometer. Chemical shifts (d) 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 nhexane and ethyl acetate as mobile phase. The procedure for the synthesis of the desired compounds is depicted in Schemes 1 and 2.

Log p calculated from ChemSketch ACD labs 2012.

4.1. Reagents Quadrupole instrument (Agilent). The ionization was achieved by electrospray ionization in the positive ion mode (ESIþ) and negative ion mode (ESI-). The capillary voltage was set to 4.0 kV. The source temperature was 120  C, and the desolvation temperature

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).

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Fig. 6. The interactions of compound (4c) with the active site of mushroom tyrosinase (PDBID 2ZMX) generated by using Discovery Studio 4.1.0. The a) shows the two dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein. The b) shows the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate bond distances between interacting functionalities of the ligand and receptor.

CH3

O

CH3

+

O Cl

OH

H3C

(2)

CH3

O

(C2H5)3N/KI

R

O

R

(5a-5c)

O

DMF O

R= 5a = -H 5c = -Cl

(6a-6c)

5b = -OH

O H3C

CH3

R is same as in (5a-5c) Scheme 2. Synthesis of thymol analogues (6aec).

4.1.1. Synthesis of thymol chloroacetyl derivative (2) A mixture of thymol (1) (0.01 mol), triethylamine (0.01 mol) in anhydrous dichloromethane (25 mL) was cooled in an ice salt mixture to 0 to
5  C. To this reaction mixture chloroacetyl chloride (0.01mol) in dry dichloromethane was added drop wise with constant stirring over a period of 1 h maintaining the temperature constant. The reaction mixture was then stirred at room temperature for further 5 h, washed with 5% HCl, and 5% sodium hydroxide solution. The organic layer was washed with saturated aqueous NaCl, dried over anhydrous magnesium sulfate, filtered and solvent was removed under reduced pressure. The crude product was purified by silica gel column to afford the corresponding thymol chloroacetyl derivative (2). Colorless oil; reaction time, 6 h; yield, 84%; Rf 0.58 (n-hexane: ethyl acetate 3:1), FTIR nmax cm
1: 2923 (sp2 CeH), 2843 (sp3 CeH), 1723 (C]O ester), 1599 (C]C aromatic), 1148 (CeO, ester). 4.1.2. Synthesis of thymol analogues (4aee) and (6aec) A mixture of thymol chloroacetyl derivative (2) (0.01 mol), hydroxy substituted benzoic acids (3aee) (0.01 mol), triethyl amine (0.01 mol), potassium iodide (0.01 mol) in dimethyl formamide (25 mL) was stirred overnight at room temperature (Scheme 1). The reaction mixture was poured into finely crushed ice with stirring 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 sulfate, filtered and the solvent was removed under reduced pressure to afford the crude products (4aee). The title compounds (4aee) were purified by silica gel

column chromatography (n-hexane: ethyl acetate 4:1). The same procedure was used for the preparation of compounds (6aec) Scheme 2. 4.1.2.1. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3hydroxybenzoate (4a). solid; reaction time, 24 h; yield, 82%; melting point, 74e76  C; Rf 0.52 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3079 (OeH), 3025 (sp2 CeH), 2975 (sp3 CeH), 1728 (C] O ester), 1588 (C]C aromatic), 1158 (CeO, ester); ESI-MS: m/z 351 [M þ 23] (M þ Na)þ; 1H NMR (CDCl3, d ppm): 7.72 (d, J ¼ 8.0 Hz, 1H, H-6), 7.58 (s, 1H, H-2), 7.34 (dd, J ¼ 8.4, 7.6 Hz, 1H, H-5), 7.23 (d, J ¼ 7.6 Hz, 1H, H-4), 7.08 (d, J ¼ 2.8 Hz, 1H, H-30 ), 7.05 (d, J ¼ 7.6 Hz, 1H, H-40 ), 6.90 (s, 1H, H-60 ), 5.13 (s, 2H, -CH2), 3.03 (sept, 1H, J ¼ 6.8 Hz, H-100 ), 2.33 (s, 3H, H-300 ), 1.37 (s, 1H, -OH), 1.21 (d, J ¼ 6.8 Hz, 6H, H-200 ); 13C NMR (CDCl3, d ppm); 166.7 (C]O ester), 165.7 (C]O, ester), 155.7 (C-3), 147.2 (C-10 ), 136.9 (C-20 ), 136.7 (C50 ), 130.3 (C-6), 129.8 (C-2), 127.5 (C-1), 126.5 (C-30 ), 122.4 (C-40 ), 122.3 (C-60 ), 120.7 (C-4), 116.5 (C-5), 61.2 (eCH2), 29.7 (C-100 ), 27.0 (C-300 ), 23.0 (C-200 ); Anal Calcd For C19H20O5: C, 69.51; H, 6.10; Found C, 69.48; H, 6.17. 4.1.2.2. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 4hydroxybenzoate (4b). Solid; reaction time, 24 h; yield, 80%; melting point, 83e85  C; Rf 0.50 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3126 (OeH), 2913 (sp2 CeH), 2852 (sp3 CeH), 1705 (C] O ester), 1589 (C]C aromatic), 1148 (CeO, ester); ESI-MS: m/z 351 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6, d ppm): 7.99 (dd, J ¼ 6.0, 2.4 Hz, 2H, H-2, H-6), 7.28 (d, J ¼ 2.8 Hz, 1H, H-20 ), 7.22 (dd, J ¼ 5.2, 2.4 Hz, 1H, H-40 ), 7.05 (d, J ¼ 7.6, 1H, H-60 ), 6.85 (dd, J ¼ 6.0, 2.4 Hz,

Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211

2H, H-3, H-5), 5.11 (s, 2H, -CH2), 3.57 (s, 1H, -OH), 3.04 (sept, J ¼ 6.8 Hz, 1H, H-100 ), 2.32 (s, 3H, -H-300 )), 1.21 (d, J ¼ 3.6 Hz, 6H, H200 ); 13C NMR (DMSO-d6, d ppm); 167.2 (C]O ester), 165.7 (C]O, ester), 160.5 (C-4), 147.2 (C-10 ), 136.9 (C-20 ), 136.7 (C-50 ), 132.3 (C-3, C-5), 127.5 (C-1), 126.5 (C-30 ), 122.4 (C-40 ), 121.2 (C-60 ), 115.3 (C-2,C6), 60.9 (eCH2), 29.7 (C-100 ), 27.0 (C-300 ), 23.0 (C-200 ); Anal Calcd For C19H20O5: C, 69.51; H, 6.10; Found C, 69.57; H, 6.02. 4.1.2.3. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 2,4dihydroxybenzoate (4c). Solid; reaction time, 24 h; yield, 78%; melting point, 103e105  C; Rf 0.46 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3093 (OeH), 2924 (sp2 CeH), 2852 (sp3 CeH), 1710 (C]O ester), 1602 (C]C aromatic), 1159 (CeO, ester); ESI-MS: m/z 367 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6, d ppm): 7.82 (d, J ¼ 8.0 Hz, 1H, H-6), 7.23 (d, J ¼ 8.0 Hz, 1H, H-5), 7.08 (d, J ¼ 0.8 Hz, 1H, H-60 ), 6.89 (s, 1H, H-3), 6.38 (d, J ¼ 2.4 Hz, 1H, H-30 ), 6.36 (dd, J ¼ 6.4, 2.4 Hz, 1H, H-40 ), 5.12 (s, 2H, -CH2), 3.02 (sept, J ¼ 7.2 Hz, 1H, H-100 ), 2.34 (s, 3H, H-300 ), 1.32 (s, 2H, -OH), 1.22 (d, J ¼ 6.8 Hz, 6H, H200 ); 13C NMR (DMSO-d6, d ppm); 169.0 (C]O ester), 166.8 (C]O ester), 163.9 (C-2), 162.7 (C-4), 147.1 (C-10 ), 136.9 (C-20 ), 136.8 (C-50 ), 132.1 (C-6), 127.6 (C-40 ), 126.6 (C-30 ), 122.3 (C-60 ), 108.2 (C-3), 104.8 (C-5), 103.1 (C-1), 60.9 (eCH2), 29.7 (C-100 ), 27.0 (C-300 ), 23.0 (C-200 ); Anal Calcd For C19H20O6: C, 66.28; H, 5.81; Found C, 66.16; H, 5.73. 4.1.2.4. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3,4dihydroxybenzoate (4d). Solid; reaction time, 24 h; yield, 75%; melting point, 159e161  C; Rf 0.42 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3126 (OeH), 2965 (sp2 CeH), 2827 (sp3 CeH), 1712 (C]O ester), 1604 (C]C aromatic), 1128 (CeO, ester); ESI-MS: m/z 367 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6, d ppm): 7.43 (d, J ¼ 2.4 Hz, 1H, H-2), 7.39 (dd, J ¼ 6.0, 2.4 Hz, 1H, H-6), 7.25 (d, J ¼ 8.0 Hz, 1H, H-5), 7.07 (d, J ¼ 0.8 Hz, 1H, H-50 ), 6.96 (d, J ¼ 7.6 Hz, 1H, H-30 ), 6.87 (dd, J ¼ 8.4, 0.8 Hz, 1H, H-40 ), 5.14 (s, 2H, -CH2), 3.36 (s, 2H, -OH), 2.98 (sept, J ¼ 6.8 Hz, 1H, H-100 ), 2.26 (s, 3H, H-300 ), 1.11 (d, J ¼ 6.8 Hz, 6H, H-200 ); 13C NMR (DMSO-d6, d ppm); 167.6 (C]O ester), 165.7 (C]O ester), 151.5 (C-3), 147.4 (C-4), 145.6 (C-10 ), 137.2 (C-20 ), 136.7 (C-50 ), 127.7 (C-6), 126.9 (C-40 ), 122.8 (C-30 ), 122.7 (C60 ), 119.8 (C-2), 116.9 (C-5), 115.9 (C-1), 61.4 (eCH2), 26.7 (C-100 ), 23.3 (C-300 ), 20.7 (C-200 ); Anal Calcd For C19H20O6: C, 66.28; H, 5.81; Found C, 66.39; H, 5.89. 4.1.2.5. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3,5dihydroxybenzoate (4e). Solid; reaction time, 24 h; yield, 82%; melting point, 110e112  C; Rf 0.46 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3125 (OeH), 2924 (sp2 CeH), 2852 (sp3 CeH), 1709 (C]O ester), 1600 (C]C aliphatic) 1603 (C]C aromatic), 1132 (CeO, ester); ESI-MS: m/z 367 [M þ 23] (M þ Na)þ; 1H NMR (DMSOd6, d ppm): 7.26 (d, J ¼ 8.0 Hz, 1H, H-30 ), 7.06 (d, J ¼ 8.0 Hz, 1H, H-40 ), 6.89 (m, 3H, H-2, H-4, H-6), 6.48 (s, 1H, H-60 ), 5.17 (s, 2H, -CH2), 3.47 (s, 2H, -OH), 2.99 (sept, J ¼ 6.8 Hz, 1H, H-100 ), 2.27 (s, 3H, H-300 ), 1.10 (d, J ¼ 6.8 Hz, 6H, H-200 ); 13C NMR (DMSO-d6, d ppm); 169.0 (C]O ester), 166.9 (C]O ester), 163.9 (C-3, C-5), 162.8 (C-10 ), 147.1 (C-20 ), 136.9 (C-50 ), 132.1 (C-2, C-6), 127.7 (C-40 ), 126.6 (C-30 ), 122.2 (C-60 ), 108.2 (C-4), 104.7 (C-1), 60.9 (eCH2), 29.7 (C-100 ), 27.0 (C-300 ), 23.0 (C-200 ); Anal Calcd For C19H20O6: C, 66.28; H, 5.81; Found C, 66.12; H, 5.91. 4.1.2.6. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3phenylprop-2-enoate (6a). Solid; reaction time, 24 h; yield, 86%; melting point, 94e96  C; Rf 0.54 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3918 (sp2 CeH), 2820 (sp3 CeH), 1723 (C]O ester), 1593 (C]C aromatic), 1146 (CeO, ester); ESI-MS: m/z 361 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6, d ppm): 7.80 (d, J ¼ 16.0 Hz, 1H, H2), 7.50 (dd, J ¼ 4.8, 2.0 Hz, 2H, H-20 , 60 ), 7.39e7.41 (m, 3H, H-30 , H40 , H-50 ), 7.25 (d, J ¼ 4.8 Hz, 1H, H-300 ), 7.09 (d, J ¼ 6.4 Hz, 1H, H-400 ),

209

6.89 (s, 1H, H-600 ), 6.56 (d, J ¼ 16.0 Hz, 1H, H-1), 5.02 (s, 2H, -CH2), 3.03 (sept, J ¼ 6.8 Hz, 1H, H-1000 ), 2.34 (s, 3H, H-3000 ), 1.19 (d, J ¼ 6.8 Hz, 6H, H-2000 ); 13C NMR (DMSO-d6, d ppm); 166.7 (C]O ester), 166.1 (C]O, ester), 147.3 (C-100 ), 146.4 (C-2), 136.9 (C-200 ), 134.1 (C-500 ), 130.6 (C-20 ,C-60 ), 128.9 (C-30 , C-50 ), 128.2 (C-40 ), 127.5 (C-10 ), 126.5 (C-300 ), 122.4 (C-400 ), 116.6 (C-600 ), 60.6 (eCH2), 27.1 (C1000 ), 23.0 (C-3000 ), 20.7 (C-2000 ); Anal Calcd For C21H22O4: C, 74.56; H, 6.51; Found C, 74.45; H, 6.69.

4.1.2.7. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4hydroxyphenyl)prop-2-enoate (6b). Solid; reaction time, 24 h; yield, 80%; melting point, 117e119  C; Rf 0.48 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3103 (eOH), 2945 (sp2 CeH), 2864 (sp3 CeH), 1726 (C]O), 1601 (C]C aromatic), 1122 (CeO, ester); ESIMS: m/z 377 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6, d ppm): 7.83 (d, J ¼ 16.0 Hz, 1H, H-2), 7.57 (dd, J ¼ 4.0, 2.0 Hz, 2H, H-20 , 60 ), 7.42 (dd, J ¼ 4.0, 2.4 Hz, 2H, H-30 , 50 ), 7.24 (d, J ¼ 6.8 Hz, 1H, H-300 ), 7.07 (d, J ¼ 8.0 Hz, 1H, H-400 ), 6.90 (s, 1H, H-600 ), 6.57 (d, J ¼ 16.0 Hz, 1H, H-1), 5.03 (s, 2H, -CH2), 3.04 (sept, J ¼ 6.8 Hz, 1H, H-1000 ), 2.34 (s, 3H, H-3000 ), 1.23 (d, J ¼ 6.8 Hz, 6H, H-2000 ); 13C NMR (DMSO-d6, d ppm); 167.3 (C]O ester), 166.6 (C]O, ester), 158.1 (C-100 ), 147.2 (C-2), 146.3 (C-200 ), 136.9 (C-500 ), 130.2 (C-30 , C-50 ), 127.5 (C-40 ), 126.8 (C-10 ), 126.5 (C-300 ), 122.4 (C-400 ), 115.9 (C-20 ,C-60 ), 113.7 (C-600 ), 56.6 (eCH2), 29.1 (C-1000 ), 27.1 (C-3000 ), 23.0 (C-2000 ); Anal Calcd For C21H22O5: C, 71.19; H, 6.21; Found C, 71.28; H, 6.13.

4.1.2.8. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4chlorophenyl)prop-2-enoate (6c). solid; reaction time, 24 h; yield, 84%; melting point, 82e84  C; Rf 0.52 (n-hexane:ethyl acetate 2:1), FTIR nmax cm
1: 3923 (sp2 CeH), 2854 (sp3 CeH), 1728 (C]O ester), 1627 (C]C aromatic), 1162 (CeO, ester); ESI-MS: m/z 395 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6, d ppm): 7.75 (d, J ¼ 16.0 Hz, 1H, H-2), 7.42 (d, J ¼ 8.8 Hz, 2H, H-20 , 60 ), 7.24 (d, J ¼ 8.0 Hz, 1H, H-300 ), 7.09 (d, J ¼ 7.2 Hz, 1H, H-400 ), 6.89 (s, 1H, H-600 ), 6.83 (d, J ¼ 8.8 Hz, 2H, H-30 , H-50 ), 6.38 (d, J ¼ 16.0 Hz, 1H, H-1), 5.03 (s, 2H, -CH2), 3.03 (sept, J ¼ 6.8 Hz, 1H, H-1000 ), 2.33 (s, 3H, H-3000 ), 1.19 (d, J ¼ 6.8 Hz, 6H, H-2000 ); 13C NMR (CDCl3, d ppm); 166.6 (C]O ester), 165.9 (C]O, ester), 147.2 (C-100 ), 144.9 (C-2), 136.9 (C-200 ), 136.6 (C-500 ), 132.6 (C-20 ,C-60 ), 129.4 (C-30 , C-50 ), 129.2 (C-40 ), 127.5 (C-10 ), 126.5 (C-300 ), 122.4 (C-400 ), 117.2 (C-600 ), 60.8 (eCH2), 27.0 (C1000 ), 23.0 (C-3000 ), 20.7 (C-2000 ); Anal Calcd For C21H21O4Cl: C, 67.65; H, 5.64; Found C, 67.51; H, 5.73.

4.2. Mushroom tyrosinase inhibition assay The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was used for in vitro bioassays as described previously with some modifications [31,32]. Briefly, 140 mL of phosphate buffer (20 mM, pH 6.8), 20 mL of mushroom tyrosinase (30 U/mL) and 20 mL of the inhibitor solution were placed in the wells of a 96-well micro plate. After pre-incubation for 10 min at room temperature, 20 mL of LDOPA (3,4-dihydroxyphenylalanine) (0.85 mM) was added and the plate was further incubated at 25  C for 20 min. Subsequently the absorbance of dopachrome was measured at 475 nm 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 test compounds was expressed as the percentage of concentration necessary to achieve 50% inhibition (IC50). Each concentration was analyzed in three independent experiments. The IC50 values were determined by the data analysis and graphing software Origin 8.6, 64-bit.

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4.3. Free radical scavenging assay

5.3. Grid parameters

Radical scavenging activities was determined by modifying method of [33] by 2,2-diphenyl-1- picrylhydrazyl (DPPH) assay. The assay solution consisted of 100 mL of (150 mM) 2,2-diphenyl-1picrylhydrazyl (DPPH), 20 mL of increasing concentration of test compounds and the volume was adjusted to 200 mL in each well with DMSO. This reaction mixture was then incubated for 30 min at room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The assay measurements were carried out by using a micro plate reader (OPTI Max, Tunable) at 517 nm. The reaction rates were compared and the percent inhibition due to the presence of tested inhibitors was calculated. Each concentration was analyzed in three independent experiments run in triplicate. The concentration required for 50% decrease in the absorbance of a control solution of DPPH was expressed as IC50. The IC50 values were determined by the Data analysis and graphing software Origin 8.6, 64-bit.

For the docking of ligand molecules to the tyrosinase protein structure, search space coordinates were provided to the AutoDock Vina using AutoDock Tools. The dimensions of the grid box were set in a manner to ensure that the ligand could bind to all the potential binding sites of the protein and, hence, provide the best binding conformation. Default values provided by the program were retained for the rest of the parameters. The number of grid points in xyz was set to the spacing value equivalent to 1.0 Å and the grid center to
5,
15 and 18.

4.4. Kinetic analysis of the inhibition of tyrosinase A series of experiments were performed to determine the inhibition kinetics by following method [34]. Inhibitor (4d) with concentrations 0, 9, 18, 36.2, 72.5 mM, and (6b) with concentrations 0, 7, 14 and 28 mM, respectively were used. Substrate L-DOPA concentration was among 0.0625e2 mM in all kinetic study. Preincubation and measurement time was the same as discussed in mushroom tyrosinase inhibition assay protocol. Formation of DOPAchrome was continuously monitored at 475 nm for 5 min at a 30s interval in the microplate reader after addition of enzyme. The inhibition type on the enzyme was assayed by LineweavereBurk 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 reversible kinetics of the enzyme inhibitor complex was also determined for different concentration (0.0, 2.2, 4.4, 8.8 and 17.5 mM) of derivatives (6b) versus the enzyme concentration (4, 6, 8, 10, 15 and 20 mg/mL). 5. Molecular docking 5.1. Preparation of target protein Docking analysis of the thymol analogues (4aee) and (6aec) against the tyrosinase enzyme was performed in order to compare the relative affinity of the chemicals against the enzyme. For this purpose, AutoDock Vina (1.1.2) was utilized which provides an accurate and efficient means to determine the protein-ligand binding mode predictions [35]. The tertiary structure of the protein (PDB ID 2ZMX) was retrieved from RCSB Protein DataBank Site [36,37]. Bound ligand and solvent molecules were removed from the protein structure using the Chimera Visualization software (1.8) [38]. The protein was saved in the pdb format. AutoDock Tools (1.5.6) was then used to protonate the protein tertiary structure and finally save it in the dockable file format (pdbqt). 5.2. Ligand preparation Thymol analogues were drawn in the ChemSketch software (11.02). After the 3-D optimization, the ligand structures were saved in the mol format. For file format conversion, ArgusLab (4.0.1) was used and the ligands were saved in the pdb format [39]. These files were then retrieved in the AutoDock Tools which was used to observe the ligand geometry and bond flexibility. The ligands were then saved in the pdbqt format.

5.4. Docking analysis and visulaization of binding conformations Docking analysis was carried out using AutoDock Vina which uses an iterated local search algorithm by employing the search space parameters provided by the user. The output was obtained in the form of binding energies (Kcal/mol) and the best binding conformations. The binding conformations with the lowest energy coefficients were selected and visualized in Discovery Studio (4.1.0) [40]. Spatial (3D) and linear (2D) interaction maps were studied in order to determine the amino acids involved in the ligand-protein interactions. 5.5. Crystallographic data collection for (6a) Single crystal X-ray diffraction data set for (6a) was collected at room temperature up to a max 2.04 q of ca. 28.22 on a Bruker SMART CCD area-detector diffractometer using monochromatic MoKa radiation, l ¼ 0.71069 Å. The structure was solved by direct method [41] and refined by full matrix least squares on F2 with anisotropic displacement parameters for non-H atoms using SHELXL-2013 [42]. All hydrogen atoms were located from difference Fourier maps and refined as riding on their parent atoms. 5.6. Crystal structure description The colorless single crystals of compound (6a) suitable for X-ray diffraction were grown from ethyl acetate using the slow evaporation technique. Diffraction intensity data were collected with FR590 MACH3 single crystal diffractometer using Moka monochromatic radiation (l ¼ 0.71073 Å) at room temperature (296 K). The compound (6a) crystallizes in orthorhombic crystal system with Pna21 space group and detailed structure refinement data and crystallographic data are presented in (Table S1) as supplementary material. The bond length of carbonyl p-bond between C(5)-O(19) is less than carbonyl p-bond between C(2)-O(18) due to p-electrons delocalization in C(5)-O(19) p-bond. As these p-electrons are in conjugation with the p-electrons of cinnamic acid double bond between C(6)-C(7). The detailed bond lengths and bond angle of atomic coordinates for all atoms are tabulated as (Table S2 & S3). The additional data for the molecule (6a) are alternatively available from the Cambridge Crystallographic Data Centre as CCDC 1054911. An ORTEP drawing of the compound (6a) with the atomic numbering scheme is shown in Figure S1 (Supplementary material). Acknowledgements This work (Grants No.C0036335) was supported by Business for Cooperative R & D between Industry, Academy, and Research Institute and funded by Korea Small and Medium Business Administration in 2012.

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