Synthesis, kinetic mechanism and docking studies of vanillin derivatives as inhibitors of mushroom tyrosinase

Article abstract of DOI:10.1016/j.bmc.2015.06.068

The purpose of the present study was to discover the extent of contribution to antityrosinase activity by adding hydroxy substituted benzoic acid, cinnamic acid and piperazine residues to vanillin. The study showed the transformation of vanillin into este

Full text of DOI:10.1016/j.bmc.2015.06.068

Accepted Manuscript
Synthesis, kinetic mechanism and docking studies of vanillin derivatives as in-
hibitors of mushroom tyrosinase
Zaman Ashraf, Muhammad Rafiq, Sung-Yum Seo, Mustafeez Mujtaba Babar,
Najam-us-Sahar Sadaf Zaidi
PII:
S0968-0896(15)00562-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.068
BMC 12424
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 May 2015
26 June 2015
27 June 2015
Please cite this article as: Ashraf, Z., Rafiq, M., Seo, S-Y., Babar, M.M., Zaidi, N.S., Synthesis, kinetic mechanism
and docking studies of vanillin derivatives as inhibitors of mushroom tyrosinase, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.06.068
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Synthesis, kinetic mechanism and docking studies of vanillin derivatives as inhibitors of
mushroom tyrosinase
Zaman Ashraf1,2, Muhammad Rafiq1, Sung-Yum Seo1,*, Mustafeez Mujtaba Babar3, Najam-us-
Sahar Sadaf Zaidi3
1Department of Biology, College of Natural Sciences, Kongju National University, Gongju 314-
701, Republic of Korea
2Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan
3Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and
Technology, H-12, Kashmir Highway, Islamabad, 44000, Pakistan
Keywords: Vanillin derivatives; Synthesis; Mushroom Tyrosinase Inhibitors; Kinetic
Mechanism, In silico docking
To whom correspondence should be addressed:
Sung-Yum Seo,
Tel.: 0082-41-850-8503; Fax: 0082-41-854-8503;
Email: dnalove@kongju.ac.kr
1

Abstract
The purpose of the present study was to discover the extent of contribution to antityrosinase
activity by adding hydroxy substituted benzoic acid, cinnamic acid and piperazine residues to
vanillin. The study showed the transformation of vanillin into esters as shown in (4a-4d), (6a-6b),
and (8a-8b). In addition, the relationship between structures of these esters and their mushroom
tyrosinase inhibitory activity was explored. The kinetics of inhibition on mushroom tyrosinase
by these esters was also investigated. It was found that hydroxyl substituted benzoic acid
derivatives were weak inhibitors; however hydroxy or chloro substituted cinnamic acid and
piperazine substituted derivatives were able to induce significant tyrosinase inhibition. The
mushroom tyrosinase (PDBID 2ZWE) was docked with synthesized vanillin derivatives and
their calculated binding energies were compared with experimental IC50 values which provided
positive correlation. The most potent derivative 2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl
(2E)-3-(4-hydroxyphenyl)prop-2-enoate (6a) possesses hydroxy substituted cinnamic acid
scaffold having IC50 value 16.13 µM with binding energy of -7.2 Kcal/mol. The tyrosinase
inhibitory activity of (6a) is comparable with standard kojic acid. Kinetic analysis indicated that
compound 6a was mixed-type tyrosinase inhibitor with inhibition constant values Ki (13 µM)
and Ki′ (53 µM) and formed reversible enzyme inhibitor complex. The active vanillin analogue
(6a) was devoid of toxic effects as shown in cytotoxic studies.
2

Introduction
Tyrosinase (EC 1.14.18.1), a membrane bound copper-containing glycoprotein, regulates the
biosynthesis of melanin in melanocytic cells. In mammals L-tyrosine is oxidized by tyrosinase to
dopaquinone through L-3,4-dihydorxyphenylalanine (L-DOPA). Dopaquinone is then
transformed through several reactions into brown to black melanin. Melanin determines the color
of human skin, hair, and eyes. The actual color of skin is determined by the type and amount of
melanin and its distribution pattern in the surrounding keratinocytes.1 There are certain other
environmental, hormonal and genetic factors such as UV exposure, α-melanocyte-stimulating
hormone, melanocortin 1 receptor, and agouti-related protein which are involved in regulation of
melanogenesis.2,3 Melasma and post-inflammatory hyperpigmentation (PIH) are the most
common pigmentation disorders for which patients seek treatment.4 The melanocyte disorders
such as senile lentigo, freckles and pigmented acne scars occur in human of all races worldwide.5
Such disorders may have major influence on a person’s psychological and social well-being
resulting in lower output, overall functioning, and self-confidence.6
Tyrosinase is also responsible for the production of neuromelanins but excessive production
of dopaquinones by oxidation of dopamine results in neuronal damage and cell death. This links
tyrosinase to Parkinson’s and other neurodegenerative diseases.7-9 It has also been reported that
tyrosinase is one of the main causes of most fruit and vegetable damage during postharvest
handling and processing, leading to quicker degradation and lesser shelf life.10-12 A number of
researchers reviewed the importance of mushroom tyrosinase, defined its biochemical
characteristics and inhibition as well as activation by several inhibitors from natural and
synthetic origin for their use in food and cosmetic industry.13-16 Tyrosinase is also linked to the
sclerotization of cuticle, defensive encapsulation and melanization of foreign organism and
wound healing in insects.17 These biochemical processes offer probable targets for the
development of safer and effective tyrosinase inhibitors as insecticides and eventually for insect
control. Thus, the development of safe and effective tyrosinase inhibitors is of great concern in
the medical, agricultural and cosmetic industries.
The pharmacophore modeling and molecular docking has recently developed as a powerful
method complementing traditional high through put screenings. Molecular docking is an
optimization problem that would define the “best-fit” positioning of a compound that binds to a
3

specific protein of interest.18-19 The computational chemistry and chemoinformatics play an
important role in preliminary drug research.
A number of aromatic aldehydes possessing electron donating or electron withdrawing
groups at 3 or 4 position of benzene ring have been reported for their potential for inhibition of
tyrosinase.20-23 Nihei and coworkers reported that 2-hydroxy-4-isopropyl benzaldehyde, known
as chamaecin, showed potent tyrosinase inhibition activity with IC50 2.3 µM.24 Vanillin (4-
hydroxy-3-methoxybenzaldehyde) is a well-known natural product and is extensively used as
flavoring agent in food and cosmetics with an estimated yearly universal consumption of
synthetic vanillin more than 200 tons. Additionally, vanillin displays antimicrobial, anticancer
and antioxidant activities. Also, it has been used as a potential starting material for the
production of fine chemicals cationic surfactants.25-26 It was also reported that vanillin and
vanillic acid isolated from Origanum vulgare may serve as agents for antimelanogenesis.27 Most
of the clinically used tyrosinase inhibitors such as kojic acid, arbutine, oxyresveratrol, p-
coumaric acid, kaempferol, glabridin etc., all possess free phenolic hydroxyl moiety which play
important role in tyrosinase inhibitory activity.28-29 The presence of these structural features may
help in the designing of more potent tyrosinase inhibitors. Hence, vanillin was selected as a core
structure for the concise and cost-effective synthesis of ester derivatives having hydroxy
substituted aryl moiety for their improved tyrosinase inhibitory activity. Vanillin derivatives
have also been synthesized by incorporating heterocyclic piperazine ring to explore its role in
tyrosinase inhibitory activity.
Material and Methods
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 FT-IR spectra were recorded with Shimadzu FTIR–8400S
spectrometer (Kyoto, Japan, υ, cm-1). The 1H NMR and 13C NMR spectra (CDCl3) and (DMSO-
d6) 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. The procedure for the synthesis of the desired compounds is depicted in
Scheme I, II and III.
4

Reagents
Mushroom tyrosinase was purchased from Sigma (USA); L-DOPA and vanillin were
purchased from Sigma (USA). Stock solutions of the reducing substrates were prepared in
phosphate buffer (20 mM, pH 6.8).
Synthesis of vanillin chloroacetyl derivative (2)
A mixture of vanillin (1) (0.01 mol), triethylamine (0.01 mol) in anhydrous dichloromethane
o
(25 mL) was cooled in an ice salt mixture to 0 to -5 C. To this reaction mixture, chloroacetyl
chloride (0.01 mol) 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 5h, 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 product was
purified by silica gel column to afford the corresponding vanillin chloroacetyl derivative (2) as
white solid.
4-formyl-2-methoxyphenyl chloroacetate (2) White solid; reaction time, 5 h; yield, 82 %;
melting point, 63-65 °C; Rf 0.64 (n-hexane:ethyl acetate 2:1), FTIR νmax cm-1: 3016 (sp2 C-H),
1
2960 (sp3 C-H), 1761 (C=O ester), 1689 (C=O Aldehyde), 1598 (C=C aromatic); H NMR
(DMSO-d6, δ ppm): 9.99 (s, 1H, CHO), 7.65 (d, J=1.6 Hz, 1H, H-2), 7.62 (dd, J=6.4, 2.0 Hz,
1H, H-6), 7.43 (d, J=8.0 Hz, 1H, H-5), 4.76 (s, 2H, -CH2), 3.89 (s, 3H, -OCH3); 13C NMR
(DMSO-d6, δ ppm); 192.5 (C=O, Aldehyde), 165.8 (C=O ester), 151.8 (C-3), 144.0 (C-4), 135.9
(C-1), 124.0 (C-5), 123.9 (C-6), 112.7 (C-2), 59.8 (OCH3), 56.0 (CH2).
Synthesis of vanillin analogues (4a-4d) and (6a-6b)
A mixture of vanillin chloroacetyl derivative (2) (0.01 mol), hydroxy substituted benzoic
acids (3a-g) (0.01mole), triethyl amine (0.01 mol), potassium iodide (0.01 mol) in dimethyl
formamide (25 mL) was stirred overnight at room temperature (Scheme I). The reaction mixture
was poured into finely crushed ice with stirring and extracted with ethyl acetate (4x25 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-d).
5

The title compounds (4a-d) were purified by silica gel column chromatography. The same
procedure was used for the preparation of compounds (6a and 6b) Scheme II.
2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl 4-hydroxybenzoate (4a) solid; reaction time, 24 h;
yield, 76 %; melting point, 111-113 °C; Rf 0.46 (n-hexane:ethyl acetate 2:1),FTIR νmax cm-1:
3126 (O-H), 2940 (sp2 C-H), 2820(sp3 C-H), 1723 (C=O ester), 1690 (C=O Aldehyde), 1588
(C=C aromatic), 1143 (C-O, ester); 1H NMR (DMSO-d6, δ ppm): 9.78 (s, 1H, -CHO), 8.12 (d,
J=8.0 Hz, 1H, H-6’), 7.90 (dd, J=6.8, 2.0 Hz, 2H, H-2, H-6), 7.64 (dd, J=6.6, 2.0 Hz, 1H, H-5’),
7.59 (d, J=2.0 Hz, 1H, H-3’), 6.89 (dd, J=5.2, 2.8 Hz, 2H, H-3, H-5), 5.20 (s, 2H, -CH2), 3.88 (s,
13
3H, -OCH3), 2.50 (s, 1H, -OH); C NMR (DMSO-d6, δ ppm); 191.4 (C=O Aldehyde), 166.2
(C=O Aliphatic ester), 164.8 (C=O Aromatic ester), 163.0 (C-4), 153.5 (C-1’), 148.6 (C-2’),
135.8 (C-4’), 132.3 (C-2, C-6), 129.1 (C-1), 123.9 (C-5’), 122.5 (C-6’), 119.5 (C-3’), 115.8 (C-3,
C-5), 60.9 (CH2), 56.6 (-OCH3).
2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl 2,4-dihydroxybenzoate (4b) solid; reaction time, 24
h; yield, 70 %; melting point, 141-143 °C; Rf 0.42 (n-hexane:ethyl acetate 2:1), FTIR νmax cm-1:
3320 (O-H), 2943 (sp2 C-H), 2867 (sp3 C-H), 1741 (C=O ester), 1687 (C=O Aldehyde), 1604
(C=C aromatic), 1123 (C-O, ester); 1H NMR (DMSO-d6, δ ppm): 9.77 (s, 1H, -CHO), 7.73 (d,
J=8.8 Hz, 1H, H-6), 7.64 (d, J=1.6 Hz, 1H, H-3’), 7.61 (dd, J=6.0, 2.0 Hz, 1H, H-5’), 7.43 (d,
J=4.4, 1H, H-6’), 6.43 (dd, J=6.4, 2.4 Hz, 1H, H-5), 6.35 (d, J=2.4 Hz, 1H, H-3), 5.28 (s, 2H, -
CH2), 3.88 (s, 3H, -OCH3), 3.36 (s, 2H, -OH); 13C NMR (DMSO-d6, δ ppm); 192.4 (C=O
Aldehyde), 168.3 (C=O Aliphatic ester), 166.2 (C=O Aromatic ester), 165.1 (C-2), 163.2 (C-4),
151.8 (C-1’), 143.7 (C-2’), 135.8 (C-4’), 132.6 (C-6), 129.1 (C-3’), 124.1 (C-5’), 112.7 (C-6’),
110.9 (C-5), 109.1 (C-3), 103.9 (C-1), 61.4 (CH2), 56.5 (-OCH3).
2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl 3,4-dihydroxybenzoate (4c) solid; reaction time, 24
h; yield, 76 %; melting point, 144-145 °C; Rf 0.40 (n-hexane:ethyl acetate 2:1), FTIR νmax cm-1:
3290 (O-H), 2976 (sp2 C-H), 2856 (sp3 C-H), 1726 (C=O ester), 1692 (C=O Aldehyde), 1594
(C=C aromatic), 1178 (C-O, ester); 1H NMR (DMSO-d6, δ ppm): 9.78 (s, 1H, -CHO), 7.75 (d,
J=8.8 Hz, 1H, H-6’), 7.64 (d, J=1.2 Hz, 1H, H-3’), 7.62 (dd, J=6.4, 1.2 Hz, 1H, H-5’), 7.42 (d,
J=8.0 Hz, 1H, H-5), 6.43 (dd, J=6.4, 2.4 Hz, 1H, H-6), 6.34 (d, J=2.4 Hz, 1H, H-2), 5.24 (s, 2H,
-CH2), 3.88(s, 3H, -OCH3), 3.35 (s, 2H, -OH); 13C NMR (DMSO-d6, δ ppm);192.5 (C=O
Aldehyde), 168.2 (C=O Aliphatic ester), 166.2 (C=O Aromatic ester), 165.1 (C-4), 163.2 (C-3),
6

151.8 (C-1’), 143.7 (C-2’), 135.8 (C-4’), 132.5 (C-6), 124.0 (C-3’), 115.1 (C-5’), 112.7 (C-6’),
109.1 (C-2), 103.9 (C-5), 103.1 (C-1), 61.1 (CH2), 56.5 (-OCH3).
2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl 3,5-dihydroxybenzoate (4d) solid; reaction time, 24
h; yield, 81 %; melting point, 138-139 °C; Rf 0.38 (n-hexane:ethyl acetate 2:1), FTIR νmax cm-1:
3354 (O-H), 2971 (sp2 C-H), 2887 (sp3 C-H), 1735 (C=O ester), 1676 (C=O Aldehyde), 1601
(C=C aromatic), 1154 (C-O, ester); 1H NMR (DMSO-d6, δ ppm): 9.78 (s, 1H, -CHO), 7.43 (dd,
J=6.4, 1.6 Hz, 1H, H-5’), 7.39 (d, J=2.0 Hz, 1H, H-3’), 6.96 (d, J=8.0 Hz, 1H, H-6’), 6.89 (dd,
J=2.4, 2.4 Hz, 2H, H-2, H-6), 6.50 (dd, J=2.4, 2.4 Hz, 1H, H-4), 4.87 (s, 2H, -CH2), 3.71 (s, 3H,
-OCH3), 3.32 (s, 2H, -OH); 13C NMR (DMSO-d6, δ ppm); 191.4 (C=O Aldehyde), 168.5 (C=O
Aliphatic ester), 167.1 (C=O Aromatic ester), 159.1 (C-3, C-5), 153.5 (C-1’), 148.6 (C-2’), 130.8
(C-4’), 129.1 (C-2, C-6), 126.5 (C-3’), 115.8 (C-5’), 111.1 (C-6’), 108.2 (C-1), 107.7 (C-4), 61.5
(CH2), 56.0 (-OCH3).
2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate (6a) solid;
reaction time, 24 h; yield, 72 %; melting point, 145-147 °C; Rf 0.48 (n-hexane:ethyl acetate 2:1),
FTIR νmax cm-1: 3410 (O-H), 2939 (sp2 C-H), 2851 (sp3 C-H), 1715 (C=O ester), 1702 (C=O
ester), 1680 (C=O aldehyde), 1600 (C=C aliphatic) 1588 (C=C aromatic), 1167 (C-O, ester); 1H
NMR (DMSO-d6, δ ppm): 9.77 (s, 1H, -CHO), 7.87 (d, J=8.8 Hz, 1H, H-6’’), 7.82 (d, J=16.0
Hz, 1H, H-3), 7.64 (dd, J=6.8, 1.6 Hz, 2H, H-2’, H-6’), 7.42 (dd, J=8.0, 1.6 Hz, 1H, H-5’’), 7.25
(dd, J=9.4, 1.6 Hz, 2H, H-3’, 5’), 6.76 (d, J=2.0 Hz, 1H, H-3’’), 6.55 (d, J=16.0 Hz, 1H, H-2),
5.15 (s,2H, -CH2), 3.89 (s, 3H, -OCH3), 1.99 (s, 1H, -OH); 13C NMR (DMSO-d6, δ ppm); 192.5
(C=O Aldehyde), 166.3 (C=O ester), 165.9 (C=O ester), 160.5 (C-4’), 151.8 (C-2’’), 146.8 (C-
3), 145.4 (C-4’’), 135.8 (C-1’), 132.3 (C-1’’), 131.1 (C-2’,6’), 130.5 (C-3’,5’), 125.3 (C-2),
124.0 (C-3’’), 116.2 (C-5’’), 113.2 (C-6’’), 60.9 (-OCH3), 56.6 (-CH2).
2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl (2E)-3-(4-chlorophenyl)prop-2-enoate (6b) solid;
reaction time, 24 h; yield, 88 %; melting point, 134-136 °C; Rf 0.51 (n-hexane:ethyl acetate 2:1),
FTIR νmax cm-1: 3018 (sp2 C-H), 2940 (sp3 C-H), 1780 (C=O ester), 1723 (C=O), 1690 (C=O
aldehyde), 1602 (C=C aliphatic), 1588 (C=C aromatic), 1108 (C-O, ester); 1H NMR (DMSO-d6,
δ ppm): 9.98 (s, 1H, -CHO), 7.84 (dd, J=9.6, 1.6 Hz, 2H, H-2’,6’), 7.81 (d, J=2.0 Hz, 1H, H-
3’’), 7.80 (d, J=16.4 Hz, 1H, H-3), 7.64 (dd, J=3.6, 2.0 Hz, 1H, H-5’’),7.52 (dd, J=9.2, 1.6 Hz,
2H, H-3’, 5’), 7.42 (d, J=8.0 Hz, 1H, H-6’’), 6.79 (d, J=16.0 Hz, 1H, H-2), 5.15 (s,2H, -CH2),
7

13
3.89 (s, 3H, -OCH3); C NMR (DMSO-d6, δ ppm);192.4 (-CHO), 166.3 (C=O ester), 165.9
(C=O, ester),151.8 (C-2’’), 145.1 (C-3), 143.8 (C-4’’), 135.8 (C-1’), 134.9 (C-1’’), 133.2 (C-
2’,6’), 130.9 (C-4’), 130.8 (C-3’,5’), 129.4 (C-2), 123.9 (C-3’’), 118.0 (C-5’’), 112.7 (C-6’’),
60.9 (-OCH3), 56.6 (-CH2).
Synthesis of vanillin analogues (8a-8b)
A solution of N-substituted piperazines (7a-7b) (0.01mol) and vanillin chloroacetyl
derivative (2) (0.011mol) in anhydrous dichloromethane (25 mL) was mixed with triethyl amine
(0.03mol) at ambient temperature. The mixture was stirred for two days, then CH2Cl2 (50 mL)
was added. The products were sequentially washed with saturated Na2CO3 (30 mL x 2), with
saturated sodium chloride and dried over anhydrous magnesium sulphate (Scheme III). After
filtration and removal of the solvent under reduced pressure, the crude products were purified by
silica gel column chromatography to afford the title compounds (8a-8b).
4-formyl-2-methoxyphenyl (4-methylpiperazin-1-yl)acetate (8a) solid; reaction time, 24h; yield,
81 %; melting point, 66-68 °C; Rf 0.58 (n-hexane:ethyl acetate 2:1), FTIR νmax cm-1: 2987 (sp2
C-H), 2820 (sp3 C-H), 1725 (C=O ester), 1656 (C=O Aldehyde), 1597 (C=C aromatic), 1348 (C-
N); 1H NMR (DMSO-d6, δ ppm): 9.84 (s, 1H, -CHO), 7.33 (d, J=2.0 Hz, 1H, H-3’), 7.29 (dd,
J=5.2, 2.0 Hz, 1H, H-5’), 6.96 (d, J=6.4 Hz, 1H, H-6’), 4.92 (s, 2H -CH2), 4.15 (s, 3H, -OCH3),
13
3.70 (m, 4H, H-3, H-5), 3.20 (m, 4H, H-2, H-6), 1.27 (s, 3H, -CH3); C NMR (DMSO-d6, δ
ppm); 190.8 (-CHO), 164.2 (C=O ester), 140.2 (C-2’), 138.2 (C-1’), 122.3 (C-5’), 115.4 (C-3’),
114.4 (C-4’), 108.8 (C-6’), 56.1 (-OCH3), 43.2 (CH2), 31.9 (C-2, C-6), 29.6 (C-3, C-5), 14.1
(CH3).
4-formyl-2-methoxyphenyl (4-phenylpiperazin-1-yl)acetate (8b) solid; reaction time, 24 h;
yield, 84 %; melting point, 70-72 °C; Rf 0.52 (n-hexane:ethyl acetate 2:1), FTIR νmax cm-1: 2967
(sp2 C-H), 2865 (sp3 C-H), 1743 (C=O ester), 1687 (C=O Aldehyde), 1590 (C=C aromatic),
1365 (C-N); 1H NMR (DMSO-d6, δ ppm): 9.84 (s, 1H, -CHO), 7.33 (dd, J=5.2, 2.0 Hz, 1H, H-
5’), 7.29 (d, J=2.0 Hz, 1H, H-3’), 6.96 (d, J=6.4 Hz, 1H, H-6’), 6.92-6.94 (m, 5H, 4-phenyl),
13
4.92 (s, 2H -CH2), 4.13 (s, 3H, -OCH3), 3.69 (m, 4H, H-3, H-5), 3.23 (m, 4H, H-2, H-6); C
NMR (DMSO-d6, δ ppm); 192.4 (-CHO), 165.1 (C=O ester), 150.9 (C-2’’), 150.7 (C-1’’), 149.1
8

(C-1’), 129.3 (C-3’, C-5’), 120.7 (C-2’, C-6’), 117.1 (C-4’), 116.8 (C-5’’), 114.4 (C-3’’), 112.5
(C-4’’), 108.8 (C-6’’), 56.0 (-OCH3), 49.9 (C-2, C-6), 49.2 (C-3, C-5), 46.2 (CH2).
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.30-31 Briefly, 140 µL of phosphate
buffer (20 mM, pH 6.8), 20 µL of mushroom tyrosinase (30 U/mL) and 20 µL 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 µL of L-DOPA (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 phosphate buffer was used instead of the inhibitor solution for negative
control. 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.
Free radical scavenging assay
Radical scavenging activity was determined by modifying already reported method32-33 by
2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The assay solution consisted of 100 µL of DPPH
(150 µM), 20 µL of increasing concentration of test compounds and the volume was adjusted to
200 µL in each well with DMSO. The reaction mixture was then incubated for 30 minutes 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 (OPTIMax, Tunable) at 517 nm. The
reaction rates were compared and the percent inhibition caused by the presence of tested
inhibitors was calculated. Each concentration was analyzed in three independent experiments run
in triplicate.
Kinetic analysis of the inhibition of tyrosinase
A series of experiments were performed to determine the inhibition kinetics of derivatives
4b, 6a, 6b, and 8a by following the already reported method.34-35 The inhibitor concentrations
are: 0, 72.2, 144.5 and 289 µM for 4b; 0, 8.75 and 17.5 µM for 6a; 0, 8.3, 16.6 and 33.3 µM for
9

6b; 0, 10.68, 21.37, 42.75, 85.5 and 342 µM for 8a. Substrate L-DOPA concentration was
between 0.0625 to 2 mM in all kinetic studies. Pre-incubation and measurement time was the
same as discussed in mushroom tyrosinase inhibition assay protocol. Maximal initial velocity
was determined from initial linear portion of absorbance up to five minutes after addition of
enzyme at a 15s interval. 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. The EI
dissociation constant Ki was determined by secondary plot of 1/V versus inhibitors
concentrations while ESI dissociation constant Kiʹ was determined by intercept versus inhibitors
concentrations. The reversible kinetics of the enzyme inhibitor complex was also determined for
different concentration of derivatives 6a and 8a versus the enzyme concentration (4, 6, 8, 10, 15
and 20 µg/mL).
Molecular Modeling
Protein preparation and Grid Selection
Molecular docking analysis of the synthesized compounds was performed to determine
the relative affinity with tyrosinase enzyme using the AutoDock Vina (1.1.2).36 The program
helps in determining the binding affinity of the ligand molecules with the tertiary structure of the
protein. Moreover, by default 9 different binding conformations are generated for each molecular
docking analysis. For the analysis, the tertiary structure of tyrosinase was retrieved from the
RCSB Protein Databank using the PDBID 2ZWE.37-38 The tertiary structure is based on sequence
homology modeling. The model contains a complex of two proteins; tyrosinase enzyme and a
caddie protein melC/ORF378. Moreover, copper ions, nitrate ions and solvent molecules have
also been represented in the crystal structure. In order to modify the protein for docking analysis,
Discovery Studio (4.1.0) was used.39 The caddie protein, bound to the active site of the
tyrosinase, was removed. Moreover, the solvent molecules and nitrate ions were also removed.
The active site of the molecule comprises the amino acid residues surrounding the two copper
ions. After modifying the enzyme and confirming that there were no chemical entities that could
hinder the ligand-protein binding, the protein structure was saved in pdb format. Using
AutoDock Tools (1.5.6), the crystal structure was then prepared for docking. For the purpose, the
molecule was protonated and energy was minimized. Moreover, the search space coordinates for
the binding of the ligand to the protein molecule were also defined. The search space grid box
10

was set in a manner that it ensured that the active site was fully enclosed. Therefore, the spacing
was kept at 1Å with 40x40x40 as the size for x, y and z coordinates. The center of x, y and z
axes were kept at -11,-13 and 12. Default values were retained for the rest of the parameters. The
protein was then saved in pdbqt format that could be read by AutoDock Vina for determining
binding potential of the ligands with the molecule.
Ligand preparation
The ligand molecules were sketched in ChemSketch software (11.02) and subjected to 3-
D auto-optimization. The structures were saved in mol format and the files were converted to
pdb format using ArgusLab (v.4.0.1).40 The files were retrieved in the AutoDock Tools to
determine and optimize the ligand geometry and bond flexibility based upon semi-empirical
method. The ligand structures were then saved in pdbqt format.
Docking analysis and visualization
After the protein and ligand files had been prepared, docking analysis was performed
using AutoDock Vina. The program employs an iterated local search algorithm using the user-
provided search parameters. The docking for each ligand was carried out 10 times on 3 different
computer platforms in order to remove machine based bias. Output was obtained in the form of
binding energy (kcal/mol) and binding pose file for each ligand-protein binding conformation.
The best ligand binding pose was selected on the basis of binding energy, number and nature of
amino acid residues involved in the interaction, type of interaction and intermolecular distance
between the ligand atoms and the amino acid residues. The selected pose was visualized in
Discovery Studio. Both 2-D and 3-D ligand interaction maps were generated in order to
determine the amino acids involved in binding of the ligand to the crystal structure of the protein.
Cell viability assay
The cell viability experiment using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Bromide (MTT) assay was performed to investigate cytotoxicity of compound 6a. The mouse
skin fibroblast (L929) and human keratinocyte (HaCaT) were cultured in 48 well plates (Crystal
grade polystyrene, gamma sterilized, SPL Korea) using complete DMEM medium containing 1
% antibiotics and 10 % fetal bovine serum. The cells (5x104 cells/well) were incubated 24 hours
before exposing to the various concentrations of test compound. The vanillin analogue 6a was
11

dissolved in DMSO and diluted with DMEM medium to achieve final concentrations; 0, 8.70,
17.4 and 35.1µM. Then cells in the presence of compound 6a were again incubated for 24 hours at
37 °C and 5 % CO2. Then MTT assay was conducted, absorbance was measured at 570 nm by
using ELISA reader and all determinations were performed in triplicate. All data were expressed
as a mean standard deviation and significance was analyzed using Student’s t-tests. Statistical
significance was considered as (p<0.005).
Results and discussion
Synthesis
The vanillin derivatives (4a-4d), (6a-6b), and (8a-8b) were synthesized by following the
already reported method41 with some modification shown in Scheme I, II and III. The vanillin
chlroacetyl derivative (2) was synthesized by esterification of phenolic -OH group of vanillin
with chloroacetyl chloride in the presence of (C2H5)3N and anhydrous methylene chloride as
1
solvent. The presence of a singlet at 4.76ppm for methylene protons in HNMR and a signal at
13
56.0ppm for methylene carbon in CNMR spectra confirm the formation of precursor (2). The
final products (4a-4d) and (6a-6b) were prepared by simple nucleophilic substitution at (2) with
hydroxy substituted benzoic acids (3a-3d) and para substituted cinnamic acids (5a-5b)
respectively. The title compounds (8a-8b) were prepared by incorporation of the N-methyl and
N-phenyl substituted piperazine moiety. All of the synthesized compounds have been
characterized by FTIR, 1H NMR and 13C NMR spectroscopic data.
H3CO
CHO
O
CHO
H3CO
(C2H5)N/CH2Cl2
0 to -5°C
+
Cl
Cl
O
HO
Cl
(1)
(2)
O
O
H3CO
CHO
H3CO
CHO
OH
(C2H5)3N/KI
DMF
+
O
O
O
R
O
Cl
(2)
(3a-d)
O
O
(4a-d)
R is same as in (3a-3d)
R=
3a = 4-OH
3c = 3,4-di-OH 3d = 3,5-di-OH
R
3b = 2,4-di-OH
Scheme I Synthesis of vanillin analogues (4a-4d)
12

O
H3CO
O
CHO
H3CO
O
CHO
OH
O
+
R
(C2H5)3N/KI
O
O
DMF
(5a-b)
Cl
(2)
O
(6a-6b)
R
R=
5a = -OH
5b = -Cl
R is same as in (5a-5b)
Scheme II Synthesis of vanillin analogues (6a-6b)
H3CO
O
CHO
NH
R
CHO
N
O
(C2H5)3N
CH2Cl2
+
R/Ar
N
N
O
(7a-b)
Cl
(2)
OCH 3
O
(8a-8b)
R/Ar=
R is same as in (7a-7b)
7a = -CH3
7b = -C6H5
Scheme III Synthesis of vanillin analogues (8a-8b)
Bioassay for tyrosinase inhibitory activity
Inhibitory effects of the synthesized vanillin derivatives on mushroom tyrosinase activity
have been evaluated. Kojic acid, a competitive tyrosinase inhibitor, was used as standard in this
study for comparison purposes. The synthesized compounds 4b, 6a, 6b, and 8a showed good to
excellent inhibition of mushroom tyrosinase with IC50 ranged from 16.13 µM to 42.60 µM while
rest of the vanillin analogues displayed lesser activities compared to standard kojic acid. Table 1
presented the IC50 values of the synthesized vanillin derivatives and it was observed that
compound 6a exhibited the most potent tyrosinase inhibitory activity with IC50 16.13 µM. The
presence of the hydroxy substituted cinnamic acid moiety in compound 6a plays very important
role in the tyrosinase inhibitory activity as it showed same enzyme inhibition response at a lower
dose than the standard kojic acid. The role of chlorine in tyrosinase inhibitory activity was also
investigated in case of compound 6b. The derivative 6b possesses chlorine at position 4 of the
phenyl ring of cinnamic acid having IC50 30.85 µM and showed activity lower than compound
6a. It showed higher activity than derivatives with mono- and dihydroxy substituted benzoic
acids scaffold. The compound 4b possesses a 2,4-dihydroxy substituted benzoic acid moiety and
exhibited higher tyrosinase inhibition potential with IC50 42.60 µM than the derivatives which
13

possess mono substituted or 3,4-dihydroxy or 3,5-dihydroxy substituted benzoic acid
functionality. The compound 8a showed excellent tyrosinase inhibitory activity with IC50 21.60
µM more active than all other derivatives except compound 6a. It possesses N-methyl
substituted piperazine ring which plays vital role in inhibition of tyrosinase activity. The IC50
value of vanillin reported in literature19 is 70.0 mM; all of the reported vanillin analogues have
higher potential to inhibit the activity of tyrosinase than vanillin. It is worth noting that
antityrosinase activity was increased when chloroacetyl vanillin derivative 2 was esterified with
hydroxy substituted cinnamic acid rather than monohydroxy or dihydroxy substituted benzoic
acids. On the basis of the results we propose that vanillin derivative 6a may serve as a structural
model for the design and development of novel tyrosinase inhibitors.
Table 1 The inhibitory effects of vanillin derivatives on mushroom tyrosinase activity: (substrate: L-
DOPA).
DPPH Assay
% Inhibition SEM (100
µg/mL)b
Tyrosinase Activity
IC50 SEMa µM
Compounds
78.9 5.87
201.15 21.9
42.60 14.5
156.87 25.1
59.76 2.02
16.13 0.94
30.85 11.8
21.60 2.43
101.38 18.5
16.69 2.8
---
18
10
13
17
16
1
1
1
2
1
2
2
4a
4b
4c
4d
1
6a
6b
7
1
1
1
1
8a
8b
4
---
Kojic acid
Vitamin C
97
1
aSEM=Standard error of the mean; values are expressed in mean SEM for three parallel measurements
bAll inhibitors and vitamin C concentrations are 100 µg/mL
Kinetic Mechanism
Based upon our results we selected the most potent vanillin derivatives 4b, 6a, 6b and 8a to
determine their inhibition type and inhibition constants on mushroom tyrosinase activity. The
potential of these derivatives to inhibit the free enzyme and enzyme-substrate complex was
determined in terms of EI and ESI constants respectively. The kinetic studies of the enzyme by
the Lineweaver–Burk plot of 1/V versus 1/[S] in the presence of different inhibitors
concentrations gave a series of straight lines as shown in Fig 1-4(a). The results of Fig 1-3(a)
showed that compounds 4b, 6a, and 6b intersected within the second quadrant. The analysis
14

showed that Vmax decreased with increasing Km in the presence of increasing concentrations of
compounds 4b, 6a, and 6b, respectively. This behavior of vanillin derivatives 4b, 6a, and 6b
indicated that it inhibits tyrosinase by two different pathways42; competitively forming enzyme-
inhibitor (EI) complex and interrupting enzyme-substrate-inhibitor (ESI) complex in non-
competitive manner. The secondary plots of slope versus concentration of compounds 4b, 6a,
and 6b showed EI dissociation constants Ki Fig. 1-3(b), while ESI dissociation constants Ki′
were shown by secondary plots of intercept versus concentration of compounds 4b, 6a, and 6b,
Fig. 1-3(c). A lower value of Ki than Ki′ pointed out stronger binding between enzyme and
compounds 4b, 6a and 6b which suggested preferred competitive over noncompetitive manners
(Table 2). While in case of compound 8a, Lineweaver–Burk plot gave family of straight lines, all
of which intersected at the same point on the x-axis Fig. 4(a).The analysis showed that 1/Vmax
increased to a new value while that of Km remains the same as a result of increase in the
concentrations of compound 8a. This behavior indicated that compound 8a inhibits tyrosinase
non-competitively to form enzyme inhibitor (EI) complex.33 Secondary plot of slope against
concentration of 8a showed EI dissociation constant (Ki) Fig. 4(b). The results of kinetic
constants and inhibition constants are summarized in Table 2.
FIGURE 1 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of Compound 4b. (a)
Concentrations of 4b were 0, 72.2, 144.5 and 289 µM, respectively. Substrate L-DOPA Concentrations
were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (b) The insets represent the plot of the slope or (c) of the
vertical intercepts versus inhibitor 4b concentrations to determine inhibition constants. The lines were
drawn using linear least squares fit.
15

FIGURE 2 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of Compound 6a. (a)
Concentrations of 6a were 0, 8.75 and 17.5 µM, respectively. Substrate L-DOPA Concentrations were
0.25, 0.5, 1 and 2 mM, respectively. (b) The insets represent the plot of the slope or (c) the vertical
intercepts versus inhibitor 6a concentrations to determine inhibition constants. The lines were drawn
using linear least squares fit.
FIGURE 3 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of Compound 6b. (a)
Concentrations of 6b were 0, 8.3, 16.6 and 33.3 µM, respectively. Substrate L-DOPA Concentrations
were 0.25, 0.5, 1 and 2 mM, respectively. (b) The insets represent the plot of the slope or (c) of the
vertical intercepts versus inhibitor 6b concentrations to determine inhibition constants. The lines were
drawn using linear least squares fit.
16

FIGURE 4 Lineweaver–Burk plots for inhibition of tyrosinase in the presence of Compound 8a. (a)
Concentrations of 8a were 0, 10.68, 21.37, 42.75, 85.5 and 342 µM, respectively. Substrate L-DOPA
Concentrations were 0.062, 0.125, 0.5, 1 and 2 mM, respectively. (b) The insets represent the plot of the
slope versus inhibitor (8a) concentrations to determine inhibition constant. The lines were drawn using
linear least squares fit.
Table 2 Kinetic parameters of the mushroom tyrosinase for L-DOPA activity in the presence of different
concentration of vanillin derivatives 4b, 6a, 6b and 8a.
Code Dose Vmax
(µM) (∆A /Sec)
Km
(mM) Type
Inhibition
Inhibition
Ki
(µM) (µM)
Kiʹ
0
2.44 × 10 -4
0.62
72.2 2.00 × 10 -4
144.5 1.53 × 10 -4
289.0 1.05 × 10 -4
0.83
1.00
1.25
0.83
1.33
1.53
0.74
0.90
1.00
1.05
0.4
Mixed-
inhibition
---
65
205
4b
0
2.66 × 10 -4
8.75 2.22 × 10 -4
17.5 2.00 × 10 -4
Mixed-
inhibition
Reversible
---
13
41
53
6a
6b
0
8.3
2.50 × 10 -4
2.22 × 10 -4
Mixed-
inhibition
100
16.6 1.96 × 10 -4
33.3 1.85 × 10 -4
0
2.0 × 10 -4
10.68 1.66 × 10 -4
21.37 1.42 × 10 -4
42.75 1.17 × 10 -4
85.5 7.14 × 10 -5
0.4
0.4
Non-
Reversible
60
---
8a
0.4
competitive
0.4
0.4
342
2.85 × 10 -5
Vmax is the reaction velocity
17

Km is the Michaelis-Menten constant
Ki is the EI dissociation constant
Kiʹ is the ESI dissociation constant
--- Not determined
The reversibility of the enzyme inhibitor complex was also studied for compounds 6a and 8a.
Fig. 5 and Fig. 6 showed the remaining enzyme activity versus the concentration of enzyme (4,
6, 8, 10, 15 and 20 µg/mL) in the presence of different concentrations of compounds 6a and 8a
for the catalysis of L-DOPA respectively. Increasing the inhibitor concentration resulted in a
decrease in the slope of the lines, which indicate that the enzyme undergoes a reversible
inhibition.
FIGURE 5 Effects of concentrations of mushroom tyrosinase on its activity for the catalysis of L-DOPA
at different concentrations of inhibitor 6a.
18

FIGURE 6 Effects of concentrations of mushroom tyrosinase on its activity for the catalysis of L-DOPA
at different concentrations of inhibitor 8a.
DPPH radical scavenging assay
All of the synthesized vanillin derivatives were evaluated for the DPPH free radical
scavenging ability. The synthesized compounds showed no radical scavenging potential even at
high concentration (100µg/mL).
Docking studies
In an attempt to theoretically describe the difference found in the antityrosinase activity of
the synthesized vanillin derivatives, docking studies using tyrosinase enzyme (2ZWE) were
performed. The molecular construction and the docking analysis were accomplished as described
in the subsequent section. All of the computationally predicted lowest energy complexes are
stabilized by the intermolecular hydrogen bonds and stacking interactions. The steric bulk and
type of functional groups determine the interactions in these complexes. The binding affinities of
the enzyme-inhibitor complexes during docking were calculated and presented in Table 3. The
lower value showed the more stable complex formed between the ligand and target protein. The
vanillin derivative 6a showed the lowest binding energy calculated during docking studies (-7.2
kcal/mol), whereas the binding energies of compounds 4b, 6b and 8a were -7.0, -6.7, -6.5
kcal/mol, respectively. The values of binding energies proved that compound 6a formed the most
19

stable drug receptor complex with target protein. The possible enzyme-inhibitor interactions of
derivatives 4b, 6a, 6b and 8a with the active site of mushroom tyrosinase are shown in Fig.7 to
Fig.10, respectively.
FIGURE 7 The ligand-protein interactions of compound 4b with the active site of mushroom tyrosinase
(2ZWE) generated by using Discovery Studio 4.0. 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 the interactions between the ligand and the amino acids of the receptor.
20

FIGURE 8 The ligand-protein interactions of compound 6a with the active site of mushroom tyrosinase
(2ZWE) generated by using Discovery Studio 4.0. 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 the interactions between the ligand and the amino acids of the receptor.
Table 3 The average binding energy was calculated after docking of synthesized vanillin derivatives
Average Binding
Energy kcal/mol
LogPa
Compounds
-6.7
-6.9
-7.0
-6.5
-7.2
-6.7
-6.5
-5.7
2.89 0.37
2.94 0.41
2.62 0.41
2.72 0.41
2.66 0.40
3.73 0.40
0.75 0.45
3.04 0.50
4a
4b
4c
4d
6a
6b
8a
8b
aLogP calculated from ChemSketch ACD labs 2012
There is a hydrogen bond between phenolic hydroxyl in case of compound 6a and amino
acid SER206 of the enzyme with bond length 2.27Å (Fig. 8). The methoxy of vanillin ring in the
same compound formed a hydrogen bond with ASN188 having bond distance 3.03Å and ester
oxygen is linked with ASN191 with bond length 3.15Å of the target enzyme. There is also π-π
stacking present between cinnamic acid phenyl ring and HIS194 residue of the tyrosinase having
bond distance 3.93Å. Figure 7 depicted different types of interactions present between
21

compound 4b and amino acid residues of the target enzyme. The aldehyde group in compound
4b interacts with ARG55 of target protein by hydrogen bonds having bond length 2.87Å. There
is also π-π stacking present between vanillin phenyl ring and ARG55 while the phenolic
hydroxyl at 4-position of phenyl ring in compound 4b forms hydrogen bond with SER206
residue of the active site of tyrosinase. For compound 6b the carbonyl oxygen of ester
functionality interacts with HIS194 through hydrogen bonding while GLY204 amino acid
residue of target protein forms hydrogen bond with methoxy oxygen of vanillin ring having bond
distance 3.14Å (Fig. 9). There are also π-π stacks interactions between phenyl ring of cinnamic
acid moiety and ARG55 with bond distance 3.63Å. The aldehyde oxygen in compound 8a
interacts through hydrogen bonding with ARG55 residue of the enzyme having bond distance
3.21Å (Fig. 10). The calculated binding energies of derivatives 6a and 6b are -7.2 and -6.7
kcal/mol, respectively, the former possesses hydroxyl substituted cinnamic acid while the later
has chloro substituted cinnamic acid scaffold. The docking results are in good agreement of the
experimental calculated IC50 values. These results further confirmed that hydroxyl groups in the
phenyl ring of cinnamic acid play a significant role in the formation of stable ligand-target
complex as well as in mushroom tyrosinase inhibitory activity.
FIGURE 9 The ligand-protein interactions of compound 6b with the active site of mushroom tyrosinase
(2ZWE) generated by using Discovery Studio 4.0. 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 the interactions between the ligand and the amino acids of the receptor.
22

FIGURE 10 The ligand-protein interactions of compound 8a with the active site of mushroom tyrosinase
(2ZWE) generated by using Discovery Studio 4.0. 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) show the three-dimensional docking of the compound in the binding
pocket. Dashed lines indicate the interactions between the ligand and the amino acids of the receptor.
Cytotoxicity studies
According to our results, the vanillin derivative 6a exhibited most potent tyrosinase
inhibitory activity and highest binding affinity with tyrosinase enzyme. The viability assay of
derivative 6a on mouse skin fibroblast (L929) and human keratinocyte (HaCaT) was carried out.
The results showed that compound 6a exert cytotoxic effects in a dose dependent manner Fig.
11. The cytotoxic effects exerted by compound 6a are minor in case of human keratinocyte
(HaCaT) at higher concentration (35.1µM). On the other hand in case of mouse skin fibroblast
(L929) cytotoxic effects are smaller at lower concentration (8.7µM) and significant at higher
concentration (35.1µM).
23

FIGURE 11 The effects of vanillin derivative 6a on cell viability of the mouse skin fibroblast (L929) and
human keratinocyte (HaCaT) cells. The cells were treated with various concentrations of compound 6a (0,
8.70, 17.4 and 35.1 µM). All data were expressed as a mean standard deviation and significance
was analyzed using Student’s t-tests. Statistical significance was considered as p<0.05 (*p<0.05;
**p<0.005 vs. control).
Conclusion
The vanillin derivatives with various homocyclic or heterocyclic and hydrophobic or
hydrophilic moieties have been synthesized to validate their role in tyrosinase inhibitory activity.
The vanillin analogue 6a showed comparable tyrosinase inhibitory activity (IC50 16.13 µM) with
standard kojic acid. The kinetic analysis showed that compound 6a was a mixed-type tyrosinase
inhibitor and formed reversible enzyme inhibitor complex exhibited no cytotoxicity on mouse
skin fibroblast (L929) and human keratinocyte (HaCaT) cells. The lowest energy AutoDock of
the most potent analogue 6a at the tyrosinase (2ZWE) active site revealed that hydroxy
substituted cinnamic acid unit fits the enzyme-binding site well. It was confirmed that hydroxyl
groups in the phenyl ring of cinnamic acid play a significant role in the formation of stable
ligand-target complex as well as in mushroom tyrosinase inhibitory activity. Based on our results
we propose that compound 6a may serve as a structural model for the design and development of
novel tyrosinase inhibitors.
24

Declaration of interest or Acknowledgement
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.
25

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27

Synthesis, kinetic mechanism and docking studies of vanillin derivatives as inhibitors of mushroom
tyrosinase
Graphical Abstract
28