Novel biphenyl ester derivatives as tyrosinase inhibitors: Synthesis, crystallographic, spectral analysis and molecular docking studies

Article abstract of DOI:10.1371/journal.pone.0170117

Biphenyl-based compounds are clinically important for the treatments of hypertension and inflammatory, while many more are under development for pharmaceutical uses. In the present study, a series of 2-([1,1'-biphenyl]-4-yl)-2-oxoethyl benzoates, 2(a-q), and 2-([1,1'- biphenyl]-4-yl)-2-oxoethyl pyridinecarboxylate, 2(r-s) were synthesized by reacting 1-([1,1'- biphenyl]-4-yl)-2-bromoethan-1-one with various carboxylic acids using potassium carbonate in dimethylformamide at ambient temperature. Single-crystal X-ray diffraction studies revealed a more closely packed crystal structure can be produced by introduction of biphenyl moiety. Five of the compounds among the reported series exhibited significant antityrosinase activities, in which 2p, 2r and 2s displayed good inhibitions which are comparable to standard inhibitor kojic acid at concentrations of 100 and 250 μg/mL. The inhibitory effects of these active compounds were further confirmed by computational molecular docking studies and the results revealed the primary binding site is active-site entrance instead of inner copper binding site which acted as the secondary binding site.

Full text of DOI:10.1371/journal.pone.0170117

RESEARCH ARTICLE
Novel biphenyl ester derivatives as tyrosinase
inhibitors: Synthesis, crystallographic, spectral
analysis and molecular docking studies
Huey Chong Kwong1, C. S. Chidan Kumar2*, Siau Hui Mah3, Tze Shyang Chia4, Ching
Kheng Quah4*, Zi Han Loh3, Siddegowda Chandraju5, Gin Keat Lim1
1 School of Chemical Sciences, Universiti Sains Malaysia, Penang, Malaysia, 2 Department of Engineering
Chemistry, Vidya Vikas Institute of Engineering & Technology, Visvesvaraya Technological University,
Alanahalli, Mysuru, Karnataka, India, 3 School of Biosciences, Taylor’s University, Lakeside Campus,
Subang Jaya, Selangor, Malaysia, 4 X-ray Crystallography Unit, School of Physics, Universiti Sains
Malaysia, Penang, Malaysia, 5 Department of Sugar Technology & Chemistry, Sir M.V. PG Center,
University of Mysore, Tubinakere, India
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* ckquah@usm.my (CKQ); chidankumar@gmail.com (CSCK)
Abstract
Biphenyl-based compounds are clinically important for the treatments of hypertension and
inflammatory, while many more are under development for pharmaceutical uses. In the
present study, a series of 2-([1,1’-biphenyl]-4-yl)-2-oxoethyl benzoates, 2(a-q), and 2-([1,1’-
biphenyl]-4-yl)-2-oxoethyl pyridinecarboxylate, 2(r-s) were synthesized by reacting 1-([1,1’-
biphenyl]-4-yl)-2-bromoethan-1-one with various carboxylic acids using potassium carbon-
ate in dimethylformamide at ambient temperature. Single-crystal X-ray diffraction studies
revealed a more closely packed crystal structure can be produced by introduction of biphe-
nyl moiety. Five of the compounds among the reported series exhibited significant anti-
tyrosinase activities, in which 2p, 2r and 2s displayed good inhibitions which are comparable
to standard inhibitor kojic acid at concentrations of 100 and 250 μg/mL. The inhibitory effects
of these active compounds were further confirmed by computational molecular docking
studies and the results revealed the primary binding site is active-site entrance instead of
inner copper binding site which acted as the secondary binding site.
OPEN ACCESS
Citation: Kwong HC, Chidan Kumar CS, Mah SH,
Chia TS, Quah CK, Loh ZH, et al. (2017) Novel
biphenyl ester derivatives as tyrosinase inhibitors:
Synthesis, crystallographic, spectral analysis and
molecular docking studies. PLoS ONE 12(2):
e0170117. doi:10.1371/journal.pone.0170117
Editor: Mohammad Shahid, Aligarh Muslim
University, INDIA
Received: November 2, 2016
Accepted: December 29, 2016
Published: February 27, 2017
Copyright: © 2017 Kwong et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Introduction
Data Availability Statement: All spectral and
tyrosinase assay data are already available in the
manuscript and supporting information files. Three
dimensional crystallography data are deposited in
the Cambridge Crystallographic Data Centre with
CCDC no. 1476974-1476982 and 1477101-
1477107 as supplementary publications. Copies of
available material can be obtained free of charge,
on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK, (Fax: +44-(0)1223-336033 or e-mail:
deposit@ccdc.cam.ac.uk).
Biphenyl are two adjoined benzene rings that attached through their 1,1’-positions. It appeared
as a white crystal with pleasant odor, which served as an important structure analog in various
synthesis. The most widely used biphenyl derivatives is polychlorinated biphenyls (PCBs) in
electrical and chemical industries as dielectric fluids and heat transfer agents [1]. Biphenyl
moiety also served as central building block for basic liquid crystal [2] and fluorescent layers in
OLEDs [3]. As for pharmaceutical uses, to date, there are two simple biphenyl derivatives
which have been applied in clinical usage to treat hypertension [4] and inflammatory [5]; and
many more are in development as potential anti-cholinesterase [6], anti-diabetic [7], anti-
tumor [8], anti-cancer [9] and anti-leukemia agent [10], and as a potential therapeutics for
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Funding: HCK thanks Malaysian Government for
MyBrain15 (MyPhD) scholarship and wishes to
offer his deep gratitude to Ivy Ye Wei Ng, student
from School of Biosciences, Taylor’s University,
Malaysia, for anti-tyrosinase assay data collection.
The authors thank the Malaysian Government and
Universiti Sains Malaysia (USM) for Fundamental
Research Grant Scheme (FRGS) (203.
cardiovascular disease [11] and osteoporosis [12]. The anti-tyrosinase activities of biphenyl-
based compounds were also reported [13–15]. Tyrosinase (EC 1.14.18.1) is a multi-functional
copper-containing enzyme that plays a crucial role in melanin biosynthesis and melanin con-
tributes to skin pigmentation. Therefore, tyrosinase inhibitors were useful in the treatment of
dermatological disorder that associated with melanin hyperpigmentation, in cosmetic for
whitening and in depigmentation after sunburn [16]. The biological activities of biphenyl
derivatives and their use as tyrosinase inhibitor inspired us to work on the synthesis of a series
of new biphenyl esters andto evaluate their anti-tyrosinase activites. In the current project, we
focused on the design and synthesis of new anti-tyrosinase agents with biphenyl-based struc-
ture to reach more active analogs towards inhibition of tyrosinase. Besides, we hope the new
analogs to render minimum side effects. We also investigated in-silico binding mode of the
proposed ligands into tyrosinase enzyme in comparison with kojic acid as reference drug by
docking procedure. In fact, it revealed biphenyl-based derivatives have similar pharmacopho-
ric pattern like kojic acid and are able to bind at the active-site entrance.
PFIZIK.6711563).
Competing interests: The authors have declared
that no competing interests exist.
Material and methods
All reagents and solvents were obtained commercially from Sigma Aldrich Corporation with
high purity. Melting points were determined on Stuart (UK) SMP10 apparatus. 1H and 13
C
nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 at 500 MHz and 125
MHz, respectively, using Bruker Avance III 500 spectrometer. Fourier transform infrared
spectroscopy (FTIR) spectra were recorded on Perkin Elmer Frontier FTIR spectrometer
equipped with attenuated total reflection (ATR). The X-ray diffraction analysis were per-
formed using Bruker APEX II DUO CCD diffractometer, employing MoKα radiation (λ =
0.71073 Å) with φ and ω scans. Data reduction and absorption correction were performed
using SAINT and SADABS program [17]. All X-ray structures were solved by using direct
methods and refined by using full-matrix least-squares techniques on F2 through SHELXTL
software package [18]. The C-bound H atoms were calculated geometrically with isotropic dis-
placement parameters set to 1.2times the equivalent isotropic U value of the parent carbon
atoms. N-bound H atoms are located from difference Fourier map and refined freely [N—
H = 0.87 (3)—0.93 (3) Å]. Similar geometry restraint (SAME) was applied to disordered biphe-
nyl moiety of 2n. Crystallographic data for 2b-2e, 2g and 2i-2s were deposited in the Cam-
bridge Crystallographic Data Centre with CCDC no. 1476974–1476982 and 1477101–1477107
as supplementary publications. Copies of available material can be obtained free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-(0)1223-336033 or
e-mail: deposit@ccdc.cam.ac.uk).
Synthesis
Target compounds were synthesized via a two-step reaction (Fig 1). First, 1-([1,1’-biphenyl]-
4-yl)ethan-1-one was refluxed with N-bromosuccinimide and petroleum ether in methanol at
333K for two hours. The resultant precipitate of 1-([1,1’-biphenyl]-4-yl)-2-bromoethan-1-one
(1) was filtered and recrystallized with ethanol. Next, 1 (0.55 g, 0.002 mol) was reacted with
various carboxylic acids (0.003 mol) in the presence of potassium carbonate in DMF (5 ml)
and stirred at room temperature for about four hours. The reaction progress was monitored
by thin layer chromatography (TLC). The reaction mixture was poured into ice-cool water
after the completion of reaction and was stirred for another 10 minutes. The precipitate
obtained was filtered out and washed successively with distilled water [19]. The dried precipi-
tate was purified using silica gel column chromatography, eluting with ethyl acetate/hexane
(2:8). Suitable single crystal specimens were obtained via slow evaporation from various types
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Fig 1. The reaction scheme for the synthesis of 2-([1,1’-biphenyl]-4-yl)-2-oxoethyl benzoates, 2(a-q), and 2-([1,1’-biphenyl]-4-yl)-2-oxoethyl
pyridinecarboxylate, 2r&2s.
doi:10.1371/journal.pone.0170117.g001
of solvents as described below. All target compounds 2(a-s) were synthesized in good yield
and high purity. Their chemical structures were characterized by using NMR and FTIR spec-
troscopy. Crystal structures of all compounds except 2a, 2f and 2h were determined by using
single-crystal X-ray diffraction analysis.
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl benzoate (2a): Yield: 73%; M.P. 442–444 K; FT-IR (ATR
(solid) cm-1): 3063 (Ar C–H, v), 2936 (C–H, ν), 1718, 1696 (C = O, ν), 1599, 1451 (Ar, C–C, ν),
1277, 1234, 1123 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm δ 8.197–8.180 (d, 2H, J = 8.3
Hz, 17–CH, 21–CH), 8.090–8.073 (d, 2H, J = 8.6 Hz, 9–CH, 11–CH), 7.769–7.752 (d, 2H,
J = 8.6 Hz, 8–CH, 12–CH), 7.675–7.661 (d, 2H, J = 7.1 Hz, 1–CH, 5–CH), 7.649–7.620 (t, 1H,
J = 7.4 Hz, 19–CH), 7.530–7.496 (m, 4H, 2–CH, 4–CH, 18–CH, 20–CH), 7.463–7.434 (t, 1H,
J = 7.1 Hz, 3–CH), 5.641 (s, 2H, 14–CH2); 13C NMR (125 MHz, CDCl3): δ 191.77 (C13),
166.11 (C15), 146.62 (C7), 139.68 (C6), 133.39 (C19), 133.01(C10), 130.02 (C9, C11), 129.44
(C16), 129.03 (C2, C4), 128.48 (C17, C21), 128.47 (C18, C20), 127.91 (C3), 127.53 (C8, C12),
127.31 (C1, C5), 66.53 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 2-chlorobenzoate (2b): Solvent for growing crystal: ace-
tone; Yield: 85%; M.P. 392–394 K; FT-IR (ATR (solid) cm-1): 3068 (Ar C–H, v) 2944 (C–H, ν),
1728, 1691 (C = O, ν), 1589, 1470 (Ar, C–C, ν), 1234, 1112, 1029 (C–O, ν), 736 (C–Cl, ν); 1H
NMR (500 MHz, CDCl3): δ ppm 8.111–8.097 (d, 1H, J = 7.3 Hz, 21–CH), 8.085–8.068 (d, 2H,
J = 8.6 Hz, 9–CH, 11–CH), 7.776–7.758 (d, 2H, J = 8.6 Hz, 8–CH, 12–CH), 7.681–7.664 (d, 2H,
J = 8.4 Hz, 1–CH, 5–CH), 7.533–7.503 (m, 3H, 2–CH, 3–CH, 4–CH), 7.495–7.479 (d, 1H,
PLOS ONE | DOI:10.1371/journal.pone.0170117 February 27, 2017
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Novel biphenyl ester derivatives as tyrosinase inhibitors
J = 7.3 Hz, 18–CH), 7.466–7.437 (t, 1H, J = 7.4 Hz, 20–CH), 7.417–7.388 (t, 1H, J = 7.3 Hz, 19–
CH), 5.656 (s, 2H, 14–CH2); 13C NMR (125 MHz, CDCl3): δ ppm 191.43 (C13), 165.27 (C15),
146.76 (C7), 140.26 (C6), 138.46 (C10), 134.14 (C19), 133.04 (C17), 132.88 (C16), 132.00
(C20), 131.24 (C3), 129.10 (C9, C11), 128.49 (C2, C4), 128.27 (C18), 127.59 (C8, C12), 127.35
(C1, C5), 126.78 (C21) 66.55 (C14)
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 3-chlorobenzoate (2c): Solvent for growing crystal: ace-
tone; Yield: 80%; M.P. 427–429 K; FT-IR (ATR (solid) cm-1): 3076 (Ar C-H, v), 2941 (C–H, ν),
1727, 1696 (C = O, ν), 1600, 1412 (Ar, C–C, ν), 1295, 1232, 1131 (C–O, ν), 744 (C–Cl, ν); 1H
NMR (500 MHz, CDCl3): δ ppm 8.169 (s, 1H, 17–CH), 8.080–8.063 (d, 3H, J = 8.6 Hz, 9–CH,
11–CH, 21–CH), 7.774–7.757 (d, 2H, J = 8.6 Hz, 8–CH, 12–CH), 7.676–7.662 (d, 2H, J = 7.1
Hz, 1–CH, 5–CH), 7.618–7.600 (d, 1H, J = 9.1 Hz, 19–CH), 7.534–7.504 (t, 2H, J = 7.1 Hz,
2–CH, 4–CH), 7.473–7.441 (m, 2H, 3–CH, 20–CH), 5.651 (s, 2H, 14–CH2); 13C NMR (125
MHz, CDCl3): δ ppm 191.31 (C13), 164.94 (C15), 146.76 (C7), 139.62 (C6), 134.66 (C10),
133.44 (C19), 132.81 (C10), 131.17 (C16), 130.08 (C20), 129.82 (C17), 129.05 (C9, C11), 128.50
(C21), 128.44 (C2, C4), 128.15 (C3), 127.58 (C8, C12), 127.32 (C1, C5) 66.72 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 4-chlorobenzoate (2d): Solvent for growing crystal: ace-
tone, acetonitrile (1:1 v/v); Yield: 84%; M.P. 435–437 K; FT-IR (ATR (solid) cm-1): 3066 (Ar
C-H, v), 2944 (C–H, ν), 1719, 1690 (C = O, ν), 1598, 1425 (Ar, C–C, ν), 1274, 1232, 1107 (C–
O, ν), 760 (C–Cl, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.134–8.116 (d, 2H, J = 8.8 Hz, 17–
CH, 21–CH), 8.080–8.063 (d, 2H, J = 8.6 Hz, 9–CH, 11–CH), 7.772–7.755 (d, 2H, J = 8.6 Hz,
8–CH, 12–CH), 7.675–7.661 (d, 2H, J = 7.1 Hz, 1–CH, 5–CH), 7.533–7.503 (t, 2H, J = 7.1 Hz,
2–CH, 4–CH), 7.498–7.480 (d, 2H, J = 8.8 Hz, 18–CH, 20–CH), 7.467–7.437 (t, 1H, J = 7.1 Hz,
3–CH), 5.639 (s, 2H, 14–CH2); 13C NMR (125 MHz, CDCl3): δ ppm 191.52 (C13), 16532
(C15), 146.77 (C7), 139.95 (C6), 139.66 (C19), 132.93 (C10), 131.44 (C9, C11), 129.05 (C17,
C21), 128.86 (C18, C20), 128.50 (C16), 128.44 (C2, C4), 127.89 (C3), 127.57 (C8, C12), 127.32
(C1, C5), 66.61 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 2,4-dichlorobenzoate (2e): Solvent for growing crystal:
chloroform; Yield: 79%; M.P. 383–385 K; FT-IR (ATR (solid) cm-1): 3092 (Ar C-H, v), 2933
(C–H, ν), 1735, 1693 (C = O, ν), 1603, 1415 (Ar, C–C, ν), 1229, 1134, 1105 (C–O, ν), 763 (C–
Cl, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.078–8.052 (m, 3H, 9–CH, 11–CH, 17–CH),
7.769–7.752 (d, 2H, J = 8.5 Hz, 8–CH, 12–CH), 7.672–7.658 (d, 2H, J = 7.1 Hz, 1–CH, 5–CH),
7.546 (s, 1H, 20–CH), 7.530–7.500 (t, 2H, J = 7.1 Hz, 2–CH, 4–CH), 7.465–7.436 (t, 1H, J = 7.1
Hz, 3–CH), 7.392–7.375 (d, 1H, J = 8.5 Hz, 18–CH), 5.644 (s, 2H, 14–CH2); 13C NMR (125
MHz, CDCl3): δ ppm 191.17 (C13), 164.05 (C15), 146.79 (C7), 139.58 (C6), 138.84 (C19),
135.38 (C10), 133.09 (C21), 132.75 (C17), 131.11 (C18), 139.05 (C9, C11), 128.52 (C20), 128.43
(C2, C4), 127.57 (C8, C12), 127.50 (C16), 127.31 (C1, C5), 127.14 (C3), 66.74 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 2-methylbenzoate (2f): Yield: 68%; M.P. 368–370 K;
FT-IR (ATR (solid) cm-1): 3063 (Ar C-H, v), 2931 (C–H, ν), 1723, 1696 (C = O, ν), 1605, 1419
(Ar, C–C, ν), 1263, 1234, 1099 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.126–8.112 (d,
1H, J = 7.3 Hz, 21–CH), 8.091–8.073 (d, 2H, J = 8.6 Hz, 9–CH, 11–CH), 7.770–7.753 (d, 2H,
J = 8.6 Hz, 8–CH, 12–CH), 7.677–7.663 (d, 2H, J = 7.1 Hz, 1–CH, 5–CH), 7.530–7.500 (t, 2H,
J = 7.1 Hz, 2–CH, 4–CH), 7.485–7.433 (m, 3H, 3–CH, 18–CH), 7.331–7.301 (t, 2H, J = 7.3 Hz,
19–CH, 20–CH), 5.621 (s, 2H, 14–CH2), 2.679 (s, 3H, 22–CH3); 13C NMR (125 MHz, CDCl3):
δ ppm 189.12 (C13), 164.05 (C15), 146.67 (C7), 140.69 (C6), 139.55 (C17), 133.05 (C10),
132.40 (C19), 131.72 (C18), 131.03 (C21), 129.03 (C9, C11), 128.85 (C16), 128.45 (C2, C4),
127.71 (C3), 127.53 (C8, C12), 127.31 (C1, C5), 125.81 (C20), 66.28 (C14), 21.71 (C22).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 3-methylbenzoate (2g): Solvent for growing crystal: ace-
tone, ethanol and acetonitrile (1:1:1 v/v/v); Yield: 79%; M.P. 413–415 K; FT-IR (ATR (solid)
cm-1): 3033 (Ar C-C, v), 2942 (C–H, ν), 1712, 1696 (C = O, ν), 1602, 1416, (Ar, C–H, ν), 1279,
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Novel biphenyl ester derivatives as tyrosinase inhibitors
1196, 1118 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm δ 8.090–8.073 (d, 2H, J = 8.6 Hz,
9–CH, 11–CH), 8.008 (s, 1H, 17–CH), 7.997–7.981 (d, 1H, J = 7.8 Hz, 21–CH), 7.768–7.751 (d,
2H, J = 8.6 Hz, 8–CH, 12–CH), 7.676–7.660 (d, 2H, J = 7.5 Hz, 1–CH, 5–CH), 7.531–7.501 (t,
2H, J = 7.5 Hz, 2–CH, 4–CH), 7.464–7.434 (m, 2H, 19–CH, 20–CH), 7.410–7.380 (t, 1H,
J = 7.5 Hz, 3–CH), 5.633 (s, 2H, 14–CH2), 2.454 (s, 3H, 22–CH3); 13C NMR (125 MHz,
CDCl3): δ ppm 191.81 (C13), 166.28 (C15), 146.62 (C7), 139.68 (C6), 138.29 (C18), 134.17
(C19), 133.02 (C10), 130.52 (C21), 129.33 (C16), 129.03 (C9, C11), 128.47 (C2, C4), 128.45
(C17), 128.39 (C20), 127.53 (C8, C12), 127.32 (C1, C5), 127.17 (C3), 66.45 (C14), 21.30 (C22).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 4-methylbenzoate (2h): Yield: 71%; M.P. 438–440 K;
FT-IR (ATR (solid) cm-1): 3038 (Ar C-H, v), 2928 (C–H, ν), 1715, 1696 (C = O, ν), 1602, 1411,
(Ar, C–C, ν), 1274, 1234, 1129 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.086–8.065 (m,
4H, 9–CH, 11–CH, 17–CH, 21–CH), 7.763–7.746 (d, 2H, J = 8.6 Hz, 8–CH, 12–CH), 7.673–
7.659 (d, 2H, J = 7.2 Hz, 1–CH, 5–CH), 7.528–7.498 (t, 2H, J = 7.2 Hz, 2–CH, 4–CH), 7.460–
7.431 (t, 1H, J = 7.2 Hz, 3–CH), 7.313–7.297 (d, 2H, J = 7.9 Hz, 18–CH, 20–CH), 5.616 (s, 2H,
14–CH2), 2.460 (s, 3H, 22–CH3); 13C NMR (125 MHz, CDCl3): δ ppm 191.94 (C13), 166.15
(C15), 146.58 (C7), 144.14 (C19), 139.69 (C6), 133.05 (C10), 130.05 (C9, C11), 129.20 (C17,
C21), 129.02 (C18, C20), 128.47 (C2, C4), 128.44 (C3), 127.52 (C8, C12), 127.31 (C1, C5),
126.67 (C16), 66.38 (C14), 21.35 (C22).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 2-methoxybenzoate (2i): Solvent for growing crystal: ace-
tone; Yield: 85%; M.P. 400–402 K; FT-IR (ATR (solid) cm-1): 3073 (Ar C-H, v), 2998, 2936,
2843 (C–H, ν), 1731, 1699 (C = O, ν), 1599, 1411 (Ar, C–H, ν), 1244, 1225, 1102, 1016 (C–O,
ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.088–8.072 (d, 2H, J = 8.3 Hz, 9–CH, 11–CH), 8.059–
8.044 (d, 1H, J = 7.6 Hz, 17–CH), 7.762–7.745 (d, 2H, J = 8.3 Hz, 8–CH, 12–CH), 7.674–7.660
(d, 2H, J = 7.2 Hz, 1–CH, 5–CH), 7.565–7.550 (d, 1H, J = 7.6 Hz, 20–CH), 7.529–7.498 (t, 2H,
J = 7.2 Hz, 2–CH, 4–CH), 7.460–7.431 (t, 1H, J = 7.2 Hz, 3–CH), 7.070–7.031 (m, 2H, 18–CH,
19–CH), 5.603 (s, 2H, 14–CH2), 3.957 (s, 3H, 22–CH3); 13C NMR (125 MHz, CDCl3): δ ppm
191.05 (C13), 165.30 (C15), 159.63 (C17), 146.50 (C7), 139.74 (C6), 134.10 (C19), 133.17
(C10), 132.30 (C21), 129.04 (C9, C11), 128.52 (C2, C4), 128.42 (C3), 127.48 (C8, C12), 127.32
(C1, C5), 120.26 (C20), 119.04 (C16), 112.09 (C18), 66.29 (C14), 56.08 (C22).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 3-methoxybenzoate (2j): Solvent for growing crystal: ace-
tone; Yield: 84%; M.P. 397–399 K; FT-IR (ATR (solid) cm-1): 3092 (Ar C-H, v), 2933, 2838 (C–
H, ν), 1719, 1704 (C = O, ν), 1603, 1417 (Ar, C–C, ν), 1288, 1110, 1030 (C–O, ν); 1H NMR
(500 MHz, CDCl3): δ ppm 8.088–8.071 (d, 2H, J = 8.6 Hz, 9–CH, 11–CH), 7.802–7.787 (d, 1H,
J = 7.5 Hz, 21–CH), 7.769–7.752 (d, 2H, J = 8.6 Hz, 8–CH, 12–CH), 7.696 (s, 1H, 17–CH),
7.676–7.659 (d, 2H, J = 7.9 Hz, 1–CH, 5–CH), 7.531–7.501 (t, 2H, J = 7.9 Hz, 2–CH, 4–CH),
7.464–7.435 (t, 1H, J = 7.5 Hz, 20–CH), 7.431–7.399 (t, 1H, J = 7.9 Hz, 3–CH), 7.189–7.174 (d,
1H, J = 7.5 Hz, 19–CH), 5.634 (s, 2H, 14–CH2), 3.898 (s, 3H, 22–CH3); 13C NMR (125 MHz,
CDCl3): δ ppm 191.69 (C13), 165.99 (C15), 159.62 (C17), 146.64 (C7), 139.66 (C6), 132.98
(C10), 130.68 (C16), 129.52 (C20), 129.04 (C9, C11), 128.47 (C2, C4), 127.54 (C8, C12), 127.53
(C3), 127.32 (C1, C5), 122.49 (C19), 120.15 (C21), 114.22 (C17), 66.59 (C14), 55.49 (C22).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 4-methoxybenzoate (2k): Solvent for growing crystal: ace-
tone and acetonitrile (1:1 v/v); Yield: 79%; M.P. 437–439 K; FT-IR (ATR (solid) cm-1): 3005
(Ar C-H, v), 2976, 2931, 2841 (C–H, ν), 1714, 1698 (C = O, ν), 1603, 1420 (Ar, C–C, ν), 1256,
1168, 1126, 1028 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.150–8.133 (d, 2H, J = 9.0 Hz,
17–CH, 21–CH), 8.087–9.070 (d, 2H, J = 8.6 Hz, 9–CH, 11–CH), 7.763–7.746 (d, 2H, J = 8.6
Hz, 8–CH, 12–CH), 7.674–7.659 (d, 2H, J = 7.5 Hz, 1–CH, 5–CH), 7.529–7.499 (t, 2H, J = 7.5
Hz, 2–CH, 4–CH), 7.461–7.432 (t, 1H, J = 7.5 Hz, 3–CH), 6.994–6.976 (d, 2H, J = 9.0 Hz, 18–
CH, 20–CH), 5.605 (s, 2H, 14–CH2), 3.910 (s, 3H, 22–CH3); 13C NMR (125 MHz, CDCl3):
δ ppm 192.07 (C13), 165.79 (C15), 163.74 (C19), 146.56 (C7), 139.70 (C6), 133.07 (C10),
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Novel biphenyl ester derivatives as tyrosinase inhibitors
132.11 (C17, C21), 129.02 (C9, C11), 128.47 (C2, C4), 128.43 (C3), 127.51 (C8, C12), 127.31
(C1, C5), 121.80 (C16), 113.75 (C18, C20), 66.30 (C14), 55.49 (C22).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 2-nitrobenzoate (2l): Solvent for growing crystal: acetone,
acetonitrile (1:1 v/v); Yield: 80%; M.P. 413–415 K; FT-IR (ATR (solid) cm-1): 3092 (Ar C-H,
v), 2941, 2864 (C–H, ν), 1743, 1690 (C = O, ν), 1603, 1423 (Ar, C–C, ν), 1529, 1343 (N–O, ν),
1290, 1240, 1123, 1078 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.068–8.051 (d, 2H,
J = 8.6 Hz, 9–CH, 11–CH), 8.039–8.011 (m, 2H, 18–CH, 19–CH), 7.797–7.762 (m, 3H, 8–CH,
12–CH, 21–CH), 7.723–7.692 (t, 1H, J = 7.9 Hz, 20–CH), 7.681–7.660 (d, 2H, J = 7.5 Hz,
1–CH, 5–CH), 7.534–7.504 (t, 2H, J = 7.5 Hz, 2–CH, 4–CH), 7.468–7.439 (t, 1H, J = 7.5 Hz,
3–CH), 5.664 (s, 2H, 14–CH2); 13C NMR (125 MHz, CDCl3): δ ppm 190.92 (C13), 165.10
(C15), 147.79 (C7), 146.88 (C17), 139.57 (C6), 133.28 (C20), 132.62 (C10), 131.94 (C19),
130.40 (C21), 129.05 (C9, C11), 128.53 (C3), 128.45 (C2, C4), 127.61 (C8, C12), 127.37 (C16),
127.32 (C1, C5), 124.05 (C18), 67.36 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 3-nitrobenzoate (2m): Solvent for growing crystal: ace-
tone, acetonitrile (1:1 v/v); Yield: 82%; M.P. 426–428 K; FT-IR (ATR (solid) cm-1): 3092 (Ar,
C–H, ν), 2925, 2880 (C–H, ν), 1738, 1690 (C = O, ν), 1603, 1441 (Ar, C–C, ν), 1537, 1348 (N–
O, ν), 1229, 1136 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 9.027 (s, 1H, 21–CH), 8.525–
8.492 (t, 2H, J = 8.0 Hz, 18–CH, 19–CH), 8.085–8.068 (d, 2H, J = 8.6 Hz, 9–CH, 11–CH),
7.787–7.769 (d, 2H, J = 8.6 Hz, 8–CH, 12–CH), 7.751–7.719 (t, 1H, J = 8.0 Hz, 17–CH), 7.680–
7.666 (d, 2H, J = 7.2 Hz, 1–CH, 5–CH), 7.538–7.508 (t, 2H, J = 7.2 Hz, 2–CH, 4–CH), 7.473–
7.443 (t, 1H, J = 7.2 Hz, 3–CH), 5.715 (s, 2H, 14–CH2); 13C NMR (125 MHz, CDCl3): δ ppm
190.88 (C13), 164.07 (C15), 148.35 (C18), 146.93 (C7), 139.56 (C6), 135.67 (C21), 132.63
(C10), 131.25 (C16), 129.77 (C20), 129.07 (C9, C11), 128.56 (C3), 128.44 (C2, C4), 127.82
(C19), 127.63 (C8, C12), 127.32 (C1, C5), 126.06 (C17), 67.05 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 4-nitrobenzoate (2n): Solvent for growing crystal: acetone,
ethanol and acetonitrile (1:1:1 v/v/v); Yield: 75%; M.P. 459–461 K; FT-IR (ATR (solid) cm-1):
3116 (Ar, C–H, ν), 2931, 2859 (C–H, ν), 1733, 1696 (C = O, ν), 1603, 1420 (Ar, C–C, ν), 1518,
1348 (N–O, ν), 1282, 1237, 1120, 1105 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.361 (s,
4H, 17–CH, 18–CH, 20–CH, 21–CH), 8.080–8.063 (s, 2H, J = 8.6 Hz, 9–CH, 11–CH), 7.784–
7.767 (d, 2H, J = 8.6 Hz, 8–CH, 12–CH), 7.677–7.663 (d, 2H, J = 7.2 Hz, 1–CH, 5–CH), 7.536–
7.506 (t, 2H, J = 7.2 Hz, 2–CH, 4–CH), 7.472–7.443 (t, 1H, J = 7.2 Hz, 3–CH), 5.701 (s, 2H, 14–
CH2); 13C NMR (125 MHz, CDCl3): δ ppm 190.87(C13), 164.28 (C15), 150.81 (C19), 146.95
(C7), 139.53 (C6), 134.86 (C16), 132.63 (C10), 131.15 (C17, C21), 129.07 (C9, C11), 128.57
(C3), 128.43 (C2, C4), 127.63 (C8, C12), 127.31 (C1, C5), 123.65 (C18, C20), 67.05 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 2-aminobenzoate (2o): Solvent for growing crystal: ace-
tone, ethanol and acetonitrile (1:1:1 v/v); Yield: 73%; M.P. 445–447 K; FT-IR (ATR (solid)
cm-1): 3479, 3368 (N–H, ν), 3058 (Ar C-H, v), 2933 (C–H, ν), 1688 (C = O, ν), 1619, 1423 (Ar,
C–C, ν), 1603 (N–H, δ), 1232, 1145 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ 8.093–8.076 (d,
2H, J = 8.6 Hz, 9–CH, 11–CH), 8.065–8.048 (d, 1H, J = 8.4 Hz, 21–CH), 7.767–7.750 (d, 2H,
J = 8.6 Hz, 8–CH, 12–CH), 7.678–7.663 (d, 2H, J = 7.3 Hz, 1–CH, 5–CH), 7.530–7.500 (t, 2H,
J = 7.3 Hz, 2–CH, 4–CH), 7.462–7.433 (t, 1H, J = 7.3 Hz, 3–CH), 7.350–7.316 (t, 1H, J = 8.4
Hz, 19–CH), 6.735–6.706 (m, 2H, 18–CH, 20–CH), 5.592 (s, 2H, 14–CH2); 13C NMR (125
MHz, CDCl3): δ ppm 192.01 (C13), 167.36 (C15), 150.67 (C17), 146.61 (C7), 139.67 (C6),
134.59 (C19), 133.00 (C10), 131.67 (C21), 129.03 (C9, C11), 128.49 (C2, C4), 128.45 (C3),
127.53 (C8, C12), 127.32 (C1, C5), 116.76 (C18), 116.51 (C20), 110.10 (C16), 66.08 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 3-aminobenzoate (2p): Solvent for growing crystal: ace-
tone, ethanol (1:1 v/v); Yield: 78%; M.P. 424–426 K; FT-IR (ATR (solid) cm-1): 3458, 3356 (N–
H, ν), 3038 (Ar C-H, v), 2939 (C–H, ν), 1707, 1685 (C = O, ν), 1632, 1403 (Ar, C–C, ν), 1602
(N–H, δ), 1303, 1223, 1110 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.083–8.066 (d, 2H,
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Novel biphenyl ester derivatives as tyrosinase inhibitors
J = 8.6 Hz, 9–CH, 11–CH), 7.764–7.747 (d, 2H, J = 8.6 Hz, 8–CH, 12–CH), 7.673–7.657 (d, 2H,
J = 7.4 Hz, 1–CH, 5–CH), 7.581–7.566 (d, 1H, J = 7.8 Hz, 21–CH), 7.528–7.498 (t, 1H, J = 7.4
Hz, 2–CH, 4–CH), 7.486 (s, 1H, 17–CH), 7.460–7.431 (t, 1H, J = 7.4 Hz, 3–CH), 7.297–7.265
(t, 1H, J = 7.8 Hz, 20–CH), 6.936–6.920 (d, 1H, J = 7.8 Hz, 19–CH), 5.608 (s, 2H, 14–CH2);
13C NMR (125 MHz, CDCl3): δ ppm 191.88 (C13), 165.29 (C15), 152.04 (C18), 146.01 (C7),
139.74 (C6), 135.75 (C10), 130.40 (C20), 129.41 (C16), 129.06 (C9, C11), 128.58 (C2, C4),
128.48 (C3), 127.54 (C8, C12), 127.33 (C1, C5), 120.21 (C19), 119.89 (C21), 116.12 (C16),
66.45 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl 4-aminobenzoate (2q): Solvent for growing crystal: ace-
tone Yield: 83%; M.P. 477–479 K; FT-IR (ATR (solid) cm-1): 3437, 3342, 3219 (N–H, ν), 3028
(Ar C-H, v), 2931 (C–H, ν), 1683 (C = O, ν), 1629, 1419 (Ar, C–C, ν), 1594 (N–H, δ), 1282,
1236, 1169, 1126 (C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.082–8.065 (d, 2H, J = 8.5 Hz,
9–CH, 11–CH), 7.999–7.981 (d, 2H, J = 8.8 Hz, 17–CH, 21–CH), 7.754–7.737 (d, 2H, J = 8.5
Hz, 8–CH, 12–CH), 7.669–7.655 (d, 2H, J = 7.2 Hz, 1–CH, 5–CH), 7.524–7.494 (t, 2H, J = 7.2
Hz, 2–CH, 4–CH), 7.455–7.426 (t, 1H, J = 7.2 Hz, 3–CH), 6.705–6.688 (d, 2H, J = 8.8 Hz,
18–CH, 20–CH), 5.568 (s, 2H, 14–CH2); 13C NMR (125 MHz, CDCl3): δ ppm 191.66 (C13),
166.18 (C15), 151.22 (C18), 146.01 (C7), 139.74 (C6), 133.00 (C10), 132.16 (C17, C21), 129.02
(C9, C11), 128.49 (C2, C4), 128.40 (C3), 127.49 (C8, C12), 127.32 (C1, C5), 118.90 (C16),
113.85 (C18, C20), 66.21 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl picolinate (2r): Solvent for growing crystal: acetone, ace-
tonitrile (1:1 v/v); Yield: 77%; M.P. 389–391 K; FT-IR (ATR (solid) cm-1): 3061 (Ar C-H, v),
2931 (C–H, ν), 1741, 1717, 1693 (C = O, ν), 1603, 1404 (Ar, C–C, ν), 1309 (C–N, ν) 1234, 1131
(C–O, ν); 1H NMR (500 MHz, CDCl3): δ ppm 8.851–8.837 (d, 1H, J = 7.5 Hz, 17–CH), 8.277–
8.262 (d, 1H, J = 7.5 Hz, 20–CH), 8.085–8.068 (d, 2H, J = 8.5 Hz, 9–CH, 11–CH), 7.935–7.904
(t, 1H, J = 7.5 Hz, 19–CH), 7.766–7.748 (d, 2H, J = 8.5 Hz, 8–CH, 12–CH), 7.669–7.655 (d, 2H,
J = 7.2 Hz, 1–CH, 5–CH), 7.571–7.547 (t, 1H, J = 7.5 Hz, 18–CH), 7.526–7.496 (t, 2H, J = 7.2
Hz, 2–CH, 4–CH), 7.459–7.430 (t, 1H, J = 7.2 Hz, 3–CH) 5.735 (s, 2H, 14–CH2); 13C NMR
(125 MHz, CDCl3): δ ppm 190.92 (C13), 164.64 (C15), 149.99 (C16), 147.41 (C7), 146.71
(C20), 139.65 (C6), 137.13 (C18), 132.83 (C10), 129.03 (C9, C11), 128.51 (C2, C4), 128.45
(C3), 127.55 (C8, C12), 127.31 (C1, C5), 127.28 (C19), 125.69 (C17), 67.18 (C14).
2-([1,1'-biphenyl]-4-yl)-2-oxoethyl nicotinate (2s): Solvent for growing crystal: acetone, etha-
nol and acetonitrile (1:1:1 v/v/v); Yield: 72%; M.P. 389–391 K;FT-IR (ATR (solid) cm-1):3034
(Ar C-H, v), 2928 (C–H, ν), 1735, 1722, 1696 (C = O, ν), 1595, 1417 (Ar, C–H, ν), 1327 (C–N,
ν) 1285, 1134 (C–O, ν);1H NMR (500 MHz, CDCl3): δ ppm 9.483 (s, 1H, 20–CH), 8.989–8.978
(d, 1H, J = 5.4 Hz, 19–CH), 8.956–8.940 (d, 1H, J = 5.4 Hz, 17–CH), 8.066–8.049 (d, 2H, J = 8.6
Hz, 9–CH, 11–CH), 7.978–7.955 (t, 1H, J = 5.4 Hz, 18–CH), 7.795–7.778 (d, 2H, J = 8.6 Hz,
8–CH, 12–CH), 7.679–7.665 (d, 2H, J = 7.4 Hz, 1–CH, 5–CH), 7.540–7.510 (t, 2H, J = 7.4 Hz,
2–CH, 4–CH), 7.477–7.448 (t, 1H, J = 7.4 Hz, 3–CH) 5.759 (s, 2H, 14–CH2); 13C NMR (125
MHz, CDCl3): δ ppm 189.96 (C13), 165.89 (C15), 147.26 (C7), 143.37 (C6), 139.55 (C19),
138.62 (C20), 133.22 (C10), 132.28 (C17), 129.08 (C9, C11), 128.95 (C18), 128.66 (C3), 128.43
(C2, C4), 127.71 (C8, C12), 127.32 (C1, C5) 126.14 (C16), 67.78 (C14).
Anti-tyrosinase assay
The evaluations of anti-tyrosinase activities of biphenyl esters 2(a-s) were carried out accord-
ing to the methods reported by Nithitanakool et al. (2009) with some modifications [20].
Briefly, the biphenyl esters with concentrations of 50, 100 and 250 μg/mL were diluted with
40 μL of acetone and mixed with 80 μL of mushroom tyrosinase (100 U/mL) in 0.1 M PBS (pH
6.8) solution. A similar volume of acetone with tyrosinase was used as control. Each biphenyl
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Novel biphenyl ester derivatives as tyrosinase inhibitors
ester and control was prepared in triplicate. The mixtures were incubated at 37˚C for 10 min.
Then, 40 μL of 0.01 M L-DOPA solution was added and further incubated at 37˚C for 25 min.
The absorbance was measured at 475 nm using a microplate reader. Kojic acid was used as the
standard drug. The percentage of inhibition of tyrosinase enzyme was calculated by using the
following formula:
Absorbance of control À Absorbance of sample
% Inhibition ¼
x 100
Absorbance of control
Statistical analysis
The results of anti-tyrosinase assay were expressed as mean standard deviation (SD) and
were labeled if p < 0.05 by using ANOVA of IBM SPSS Statistics for Windows, Version 23.0
(IBM, New York).
Docking protocol
The crystal structures of tyrosinase from Bacillus megaterium (TyrBm) in complex with inhibi-
tor kojic acid (PDB entry: 3NQ1) [21] and biphenyl esters (2i, 2o, 2p, 2r and 2s) were used as
target and ligands, respectively, for molecular docking using Genetic Optimization for Ligand
Docking (GOLD) package 5.4.1 [22–24]. Genetic algorithm (GA) was used to explore the
ligand-protein binding space and the conformational flexibility of ligand inside the protein. A
spherical binding site with a radius of 6 Å was used across residues Phe197, Pro201, Asn205
and Arg209 in the active-site entrance. 100 GA runs were carried out and the top 100 ranked
docking poses were scored using the Piecewise Linear Potential (PLP) scoring function.
Default values were used for all other parameters. The intermolecular interaction of the best
scored pose of each ligand was analyzed and illustrated using the Discovery Studio 4.5 software
[25].
All spectral, crystallography data, crystal packing and tyrosinase assay data are described in
detail in the S1 Dataset.
Results and discussion
Spectroscopic analysis
The IR spectra of biphenyl esters 2(a-s) showed absorption bands above 3000 cm-1, indicating
the presence of unsaturated C–H (benzene and biphenyl) groups, whereas the aromatic
v(C = C) were shown near 1600 cm-1 and 1410 cm-1. The methyl (–CH3) and methylene
(–CH2–) group’s C–H stretching were observed around 2970 and 2940 cm-1. In addition, dis-
tinct v(C = O) and v(C–O) bands were found in the range of 1743–1683 cm-1 and 1300–1028
cm-1. Absorption band for aryl halides were revealed at far right of the spectra, near 750 cm-1.
The N = O stretching (2l-2m), N–H stretching in (2o-2q) and C–N stretching in (2r-2s) were
observed at ~1530 cm−1, ~3450 cm−1 and ~1310 cm−1, respectively [26, 27]. The 1H NMR
spectra showed presences of –CH2– protons centering around δꢀ 5.65 ppm and revealed two
well-resolved sets of doublet centering around δꢀ 8.09 and 7.77 ppm with the integration
values of 2:2, ascribed to the -CH- protons of second phenyl ring. The first phenyl ring was
shown as a doublet and two triplet near δꢀ7.67, 7.53 and 7.47 ppm with the integration values
of 2:2:1. Furthermore, the benzene protons were revealed at down-field region in the 1H NMR
spectra with different set of multiplicity and integration values due to different position of sub-
stituent. Biphenyl and benzene rings can be distinguished by their identical J-coupling values.
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Fig 2. General scheme of biphenyl esters with torsion angles τ1, τ2, τ3 and τ4.
doi:10.1371/journal.pone.0170117.g002
In addition, protons of –CH3 and –OCH3 substitutions in compounds 2(f-k) were revealed at
the up-field region near δꢀ2.50 and 3.90 ppm. Based on the integration values, numbers of
protons are in agreement with the proposed values. 13C NMR spectra of 2(a-s) showed three
distinct sets of carbonyl carbon, aromatic carbon and saturated carbon signals. In the down-
field region, both δ(C = O) and δ(COO) carbonyl signals are centering around δꢀ191 and
165 ppm, respectively, whereas the –CH2– saturated carbon signals are at the up-field region
located around δꢀ66 ppm. The aromatic carbon signals of biphenyl and benzene groups were
found in the range of δꢀ152 to 110 ppm. The –CH3 and –OCH3 carbon signals of compounds
2(f-k) were located in the up-field region centering at δꢀ21 and 56 ppm, respectively [28, 29].
Single crystal structure commentary
The asymmetric unit (Z’) of all studied compounds consists of a crystallographic independent
molecule except 2m, 2r, and 2s, which each consists of two crystallographic independent mole-
cules (denoted as molecules A and B, respectively). The molecular conformation of biphenyl
esters (Fig 2, Table 1) can be characterized by four degree-of-freedom, which are torsion angles
C5—C6—C7—C12 (τ1), C9—C10—C13—C14 (τ2), C13—C14—O1—C15 (τ3) and O1—C15
—C16—C17 (τ4).
From the classical point of view, the planarity of biphenyl ring is distorted by the non-
bonded steric repulsion force between two ortho-hydrogen atoms [30]. However, molecular
X-ray structure showed τ1 varied from almost perpendicular (τ1 = -80.25˚ and -109.38˚ in
2m) to almost planar (τ1 = 178.39˚ in 2k) which opposed to the classical view. Instead of steric
repulsion force between the ortho-hydrogen atoms, the planarity of τ1 in compounds 2d, 2i,
2j, 2m and 2p might be influenced by C—HÁÁÁπ interaction in the crystal packing. The torsion
angle between biphenyl moiety and adjacent carbonyl group, C9—C10—C13—C14 (τ2), is
nearly planar for most of the compounds and the largest deviation from planarity is observed
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Table 1. Summary of torsion angles*.
Compound
Substituent
τ1
-176.57
τ2
-160.15
172.23
173.43
-175.31
2.23
τ3
70.26
τ4
172.44
2b
2c
2d
2e
2g
2i
2-chlorobenzene
3-chlorobenzene
4-chlorobenzene
2,4-dichlorobenzene
3-methylbenzene
2-methoxybenzene
3-methoxybenzene
4-methoxybenzene
2-nitrobenzene
3-nitrobenzene
4-nitrobenzene
2-aminobenzene
3-aminobenzene
4-aminobenzene
2-pyridine
-27.21
76.16
8.26
149.13
73.32
154.95
-151.14
77.09
174.74
-176.21
76.32
0.04
-127.41
-170.83
7.15
77.17
-148.66
-6.36
2j
149.09
-75.65
2k
2l
178.39
-6.54
-72.21
-178.03
-101.32
173.27, 173.28
-158.17
169.15
-177.09
6.76
76.59
2m
2n
2o
2p
2q
2r
-80.25, -109.38
-178.13
5.94, 164.3
-5.47
-79.04, 78.89
83.95
149.63
-14.78
77.31
-156.75
170.24
12.88
89.69
1.4
161.03
71.34
-5.13
-164.34, -178.05
141.69, 145.88
-176.66, -33.91
5.35, 26.92
-78.65, -79.57
74.9, -77.11
177.08, -16.00
16.14, 1.67
2s
3-pyridine
* τ1 = Torsion angle of C5—C6—C7—C12; τ2 = Torsion angle of C9—C10—C13—C14; τ3 = Torsion angle of C13—C14—O1—C15; τ4 = Torsion angle of
O1—C15—C16—C17.
doi:10.1371/journal.pone.0170117.t001
in molecule B of compound 2r (τ2 = -33.91˚). Torsion angle τ3, which interconnecting two
carbonyl groups, in phenacyl benzoates tends to adopt two types of conformations, either syn-
clinal or periplanar [19]. However, τ3 for all crystals in this report only adopts synclinal confor-
mation, ranging from 70.26˚ to 89.69˚, which is similar to adamantyl-based ester derivatives
[31]. The torsion angle between carboxylate group and the attached phenyl ring, O1—C15—
C16—C17 (τ4), is observed in the ranges from 0.04˚ to 16.14˚ and 101.32˚ to 178.03˚. The tor-
sion angle τ4 of compound with methoxy– (2i) or nitro—substituted (2l) at ortho–position is
largely twisted due to the steric repulsion between ortho–substituent and adjacent carbonyl
oxygen atom. The benzoate groups of 2b (2-chlorobenzene), 2d (2,4-dichlorobenzene) and 2o
(2-aminobenzene) are nearly planar with angle τ4 of 172.44˚, 174.74˚ and 169.15˚, respectively.
The ortho-amino substituent in 2o forms a strong intramolecular N–HÁÁÁO hydrogen bond
with the adjacent carbonyl group, featuring a S(6) ring motif.
Crystal packing similarity and structural occupancy
A Cambridge Structure Database (CSD) search using phenacyl benzoate was performed to
locate previously reported phenacyl benzoate and adamantyl benzoate derivatives and 58 simi-
lar structures were found. In order to identify the effect of the replacement of phenyl ring with
relatively more electron rich biphenyl rings on the crystal packing similarity and structure
occupancy, sixteen of the present biphenyl benzoate derivatives were compared with 42
reported phenacyl benzoate derivatives and sixteen adamantyl benzoate derivatives. In con-
trast to the high occurrence of isostructures in adamantanyl benzoate derivatives [31], there
is only a pair of isostructural crystals (2d and 2o) (Fig 3) is observed in the present work.
The comparison of crystal structure occupancy between the search results and the present
compounds are summarized in Table 2. The introduction of adamantane moiety in phenacyl
benzoate had reduced the occurrence of πÁÁÁπ interactions as compared to phenacyl benzoates
which consist of two terminal phenyl rings. In this study, the replacement of adamantane
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Fig 3. Partial crystal packing of 2d and 2o and their overlay diagram.
doi:10.1371/journal.pone.0170117.g003
moiety with biphenyl moiety encouraged the formation of weak intermolecular πÁÁÁπ and C–
HÁÁÁπ interactions in crystal packing. Thus, the packing coefficient of most of the present com-
pounds (12 out of 16) laid above 64% and some are even higher than phenacyl benzoates (2c =
71%, 2m = 75% and 2r = 77%) (Fig 4).
Anti-tyrosinase activities
In the present study, all synthesized compounds were screened for tyrosine inhibitory activity.
Five out of nineteen compounds (2i, 2o, 2p, 2r and 2s) with electron-donating substituents
(–methoxy &–amino) and pyridine ring showed positive results. The anti-tyrosinase effects
of biphenyl esters were evaluated at concentrations of 50, 100 and 250 μg/mL. The percentage
of inhibition against tyrosinase enzyme of 2i, 2o, 2p, 2r and 2s are presented in Fig 5. At the
concentration of 250 μg/mL, compounds 2p (3-amino), 2r (2-pyridine) and 2s (3-pyridine)
showed strong activities with inhibition percentage of 57.33%, 58.90% and 60.34%, respec-
tively, which are comparable to standard drug, kojic acid (57.22%), with no statistical differ-
ence. On the other hand, all five active compounds showed an average inhibition percentage
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Table 2. Summary of structural occupancy of present and reported compounds.
Compound
Packing coefficient (%)
64.59
Compound
BUTPOX[31]
BUTPUD[31]
BUTQAK[31]
BUTQEO[31]
BUTQIS[31]
BUTQOY[31]
BUTQUE[31]
AZULUD[39]
BOQXOW [41]
CIQNEW[43]
CIXVUC[19]
CIXWAJ[19]
CIXWEN[19]
CIXWIR[19]
CIYCAQ[19]
CIYCEU[19]
CIYCIY[19]
Packing coefficient (%)
61.08
Compound
EVEGOC[32]
EVEVEH[33]
GARCEJ[34]
GITHUN[35]
IDIWID [36]
Packing coefficient (%)
63.22
2b
2c
71.73
61.07
63.04
2d
65.37
61.85
65.80
2e
63.11
63.05
64.40
2g
63.28
61.24
65.26
2i
65.07
61.81
KULLIO [37]
MANGIR[38]
OBOYIP[40]
OCAKUA [42]
OCAQUG[44]
OCEFEJ[45]
PAXCOI [46]
PECZAA[47]
PODQIK[48]
PODRAD [49]
USIWID[50]
USIWID01 [51]
USIWOJ[52]
VOBYUI[53]
WIGTUD [54]
YAFWEJ[56]
YAFZAI[58]
YAHGUL[60]
YAHYOX[62]
62.07
2j
66.93
60.61
61.06
2k
63.76
63.85
67.22
2l
65.74
66.55
63.92
2m
75.69
64.07
66.98
2n
64.99
63.94
68.55
2o
62.42
64.33
66.18
2p
67.17
62.08
64.37
2q
68.61
63.98
60.66
2r
77.33
67.27
63.77
2s
64.42
65.07
62.53
BUVCIG[31]
BUVCOM[31]
BUVCUS[31]
BUVDAZ[31]
BUVDED[31]
BUVDIH [31]
BUVDON[31]
BUVDUT[31]
BUVFAB[31]
61.11
68.83
62.37
61.32
CIYCOE[19]
CIYFUN[19]
CIYGAU[19]
EVAFOX[55]
EVAJAN[57]
EVAJIV[59]
EVAZEH[61]
EVEGIW[63]
62.96
66.04
62.33
62.38
63.97
62.53
64.89
64.99
60.79
68.00
66.25
60.97
65.64
68.65
61.68
64.03
63.55
61.02
63.03
63.37
61.58
63.25
doi:10.1371/journal.pone.0170117.t002
of 50% at 100 μg/mL. All compounds showed weaker effect than kojic acid at 50 μg/mL, except
2s, which is the best inhibitor in this study, able to inhibit 46.24% of tyrosinase enzyme. In
summary, biphenyl compounds which consist of ortho-/meta-amino group and pyridine ring
showed significant response towards tyrosinase enzyme inhibition.
Fig 4. Structural occupancy comparison of biphenyl benzoates, phenacyl benzoates and adamantyl benzoates.
doi:10.1371/journal.pone.0170117.g004
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Fig 5. The percentage of tyrosinse inhibition (mean±SD) with n = 3. # and * indicate p<0.05.
doi:10.1371/journal.pone.0170117.g005
Docking and structure-activity relationship
In the protein crystal structure of 3NQ1, inhibitor kojic acid was bound to the active-site
entrance of protein TyrBm with hydrogen bond and C–HÁÁÁπ interaction, involving residues
Gly200, Arg209 and Pro201 (Fig 6a). The docking models of active compounds 2i, 2o, 2p,
2r and 2s are illustrated in Fig 6b and 6f. The first phenyl ring in biphenyl moiety was
bound to the copper binding site via C–HÁÁÁπ and πÁÁÁπ interactions with residues His208
and Val218 (additional C–HÁÁÁπ interaction with residue Ala221 for ligand 2s), while the
second phenyl ring in biphenyl moiety was bound to residue Arg209 through C-HÁÁÁπ inter-
action. For compounds 2i and 2o, their carbonyl moiety was bound with residue Gly200 but
the substituted phenyl ring failed to bind with other residues at the active-site entrance
(except Met184 for compound 2i) and this leads to a decrease in enzyme inhibition activi-
ties. For compound 2p, its amino benzene moiety was bound with two residues (Met184
and Phe197) via C–HÁÁÁπ interaction, thus exhibiting stronger inhibition effects than 2i and
2o. Similar to kojic acid, the compounds 2r and 2s, which showed the strongest activities,
contain electron donating pyridine ring that are able to bind with both Pro201 and Gly200
residues at the active-site entrance. The docking results showed that the compound with
biphenyl moiety are able to penetrate the active-site entrance and bind to the copper bind-
ing site (Ala221, His208 and Val218) by using the advantage of electron mobilized benzene
ring, in succession suggesting that the key for strong tyrosinase inhibition effects are the
attached heterocyclic ring which are able to shield the active-site entrance by binding itself
with residue Pro201 and Gly200.
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Fig 6. (a) Kojic acic binding mode in the crystal structure of TyrBm. Putative binding mode inside the binding gorge of TyrBm of
ligands: (b) 2i, (c) 2o, (d) 2p, (e)2r and (f) 2s.
doi:10.1371/journal.pone.0170117.g006
Conclusion
A series of 2-([1,1’-biphenyl]-4-yl)-2-oxoethyl benzoates, 2(a-q), and 2-([1,1’-biphenyl]-4-yl)-
2-oxoethyl pyridinecarboxylate, 2(r-s), were synthesized and characterized by FTIR, 1H and
13C NMR spectroscopic analysis, its 3D structure was further confirmed by single-crystal X-
ray diffraction studies. Introduction of biphenyl moiety into the synthesis of 2-oxopropyl ben-
zoate derivatives produced crystal structure with higher structural occupancy by augment of
the weak πÁÁÁπ and C–HÁÁÁπ interactions. Five compounds showed tyrosine inhibitory activi-
ties, while at 250 μg/mL, 2p, 2r, and 2sexhibited high inhibition comparable to the standard
drug, kojic acid. In addition, the computational molecular docking results suggested pyridine
ring has a better binding affinity toward TyrBm. Thus, further modification of biphenyl com-
pounds substituted with heterocyclic ring can potentially produce promising anti-tyrosinase
agents for clinical use in the future.
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Novel biphenyl ester derivatives as tyrosinase inhibitors
Supporting information
S1 Dataset.
(DOCX)
Acknowledgments
HCK thanks Malaysian Government for MyBrain15 (MyPhD) scholarship and wish to offer
his deep gratitude to Ivy Ye Wei Ng, student from School of Biosciences, Taylor’s University,
Malaysia for anti-tyrosinase assay data collection. The authors thank the Malaysian Govern-
ment and Universiti Sains Malaysia (USM) for the Fundamental Research Grant Scheme
(FRGS) (203.PFIZIK.6711563).
Author Contributions
Conceptualization: HCK CSCK.
Data curation: HCK ZHL.
Formal analysis: HCK TSC SC.
Funding acquisition: CKQ.
Investigation: CSCK SHM TSC.
Methodology: HCK CSCK.
Project administration: CKQ.
Resources: CKQ SHM GKL.
Software: CKQ.
Supervision: CKQ GKL.
Validation: HCK TSC.
Visualization: HCK TSC.
Writing – original draft: HCK SHM.
Writing – review & editing: HCK CSCK TSC.
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