Synthesis and tyrosinase inhibition activity of trans-stilbene derivatives

Article abstract of DOI:10.1016/j.bioorg.2016.01.001

Synthesis of a focussed library of trans-stilbene compounds through Wittig and other base catalysed condensation reactions is presented. The synthesized stilbenes were screened for their inhibitory potential against murine tyrosinase activity to explore the structure activity relationship (SAR). Presence of electron withdrawing group (-CN) at the double bond and hydroxyl group or halogen atom especially at para-position on the aromatic rings was found to significantly elevate the inhibitory activity. Among all the compounds screened, compounds 2, 6, 8, 10, 11, 15 and 21 were found to exhibit appreciable inhibitory activity. Compound 21 ((E)-2,3-bis(4-Hydroxyphenyl)acryonitrile) was found to be the most active with an IC₅₀ value of 5.06 μM which is less than half of the value 10.78 μM observed for resveratrol (common standard used in murine tyrosinase activity studies) under similar conditions. The results obtained from the present study reveal structural/functional group sensitivity for the tyrosinase inhibitory activity of stilbenoid moieties and are expected to be very helpful for the design and synthesis of novel, selective and effective tyrosinase inhibitors.

Full text of DOI:10.1016/j.bioorg.2016.01.001

Bioorganic Chemistry 64 (2016) 97–102
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and tyrosinase inhibition activity of trans-stilbene derivatives
Tabasum Ismail a, , Syed Shafi a,b, Jada Srinivas c, Dhiman Sarkar d, Yasrib Qurishi e, Jabeena Khazir a,
⇑
Mohammad Sarwar Alam b, Halmuthur Mahabalarao Sampath Kumar c,
⇑
a Medicinal Chemistry Division, Indian Institute of Integrative Medicine, Jammu 180001, India
b Department of Chemistry, Jamia Hamdard University, New Delhi 110062, India
c Vaccine Immunology Laboratory, NPC Division, Indian Institute of Chemical Technology, Hyderabad 500007, India
d Combi. Chem. Bio Resource Center, Organic Chemistry Division, Dr. HomiBhabha Road, National Chemical Laboratory (NCL), Pune 411008, Maharastra, India
e Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a focussed library of trans-stilbene compounds through Wittig and other base catalysed con-
densation reactions is presented. The synthesized stilbenes were screened for their inhibitory potential
against murine tyrosinase activity to explore the structure activity relationship (SAR). Presence of elec-
tron withdrawing group (–CN) at the double bond and hydroxyl group or halogen atom especially at
para-position on the aromatic rings was found to significantly elevate the inhibitory activity. Among
all the compounds screened, compounds 2, 6, 8, 10, 11, 15 and 21 were found to exhibit appreciable inhi-
bitory activity. Compound 21 ((E)-2,3-bis(4-Hydroxyphenyl)acryonitrile) was found to be the most active
Received 25 October 2015
Revised 30 December 2015
Accepted 4 January 2016
Available online 4 January 2016
Keywords:
Stilbenoids
Tyrosinase inhibitors
Wittig reaction
Electron withdrawing groups
Resveratrol
with an IC50 value of 5.06 lM which is less than half of the value 10.78 lM observed for resveratrol (com-
mon standard used in murine tyrosinase activity studies) under similar conditions. The results obtained
from the present study reveal structural/functional group sensitivity for the tyrosinase inhibitory activity
of stilbenoid moieties and are expected to be very helpful for the design and synthesis of novel, selective
and effective tyrosinase inhibitors.
Murine tyrosinase activity
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
technology and food processing. It is in this context that designing
of novel tyrosinase inhibitors is receiving a considerable attention
Tyrosinase, an enzyme catalysing the rate-limiting step in the
biosynthetic pathway of melanin pigments, is widely distributed
in nature [1–3]. The enzyme participates in several important reac-
tions of host defence, wound healing and sclerotization in insects
and other arthropods [1,4–7]. Tyrosinase has also been found to
be responsible for undesired enzymatic browning of farm products,
such as bruised or cut fruits and vegetables, which subsequently
leads to a significant decrease in their nutritional and market val-
ues [8–10]. In view of these different functions ascribed to tyrosi-
nase, it seems that studies aimed at the design and development of
novel tyrosinase inhibitors and a comprehensive understanding
about their mode of action can prove very useful for understanding
many life processes. Such studies besides providing sufficient
insight into mechanism underlying the regulation of skin pigmen-
tation in mammals are expected to help in a great way in search for
proper cure to many dermatological disorders in mammals, design
of alternative insect control agents and products useful to food
from food and animal scientists [10,11].
Realization about the importance of tyrosinase inhibitors for
their impact on many physiological aspects especially in mammals
and insects has stimulated an intense research activity aimed at
designing of novel tyrosinase inhibitors and exploration of their
mode of action. As an outcome of these studies, a huge library of
tyrosinase inhibitors has been discovered from natural sources or
synthesized in the laboratories [12–15]. However, various limita-
tions have been reported about the use of these currently known
tyrosinase inhibitors. While potentially active agents, such as kojic
acid and arbutin, are yet to be demonstrated clinically efficient,
others are associated with disadvantages like high cytotoxicity,
insufficient penetrating power, low activity and low stability
[16,17]. It is in this context that the use of traditional skin whitening
products like hydroquinone, corticosteroids and mercury contain-
ing products has been prohibited, because these compounds have
been found to be potentially mitogenic on account of their cytotox-
icity to melanocytes [16,18–22]. Therefore, many alternative tyrosi-
nase inhibitors with potential to suppress melanogenesis are being
actively studied with the aim of developing safer preparations for
⇑
Corresponding authors.
E-mail addresses: tabsismail@gmail.com (T. Ismail), hmskumar@gmail.com
(H.M.S. Kumar).
http://dx.doi.org/10.1016/j.bioorg.2016.01.001
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

98
T. Ismail et al. / Bioorganic Chemistry 64 (2016) 97–102
Table 1
the treatment of hyperpigmentation [23–25]. In light of these
reports, currently there is a great demand for the development of
natural product inspired skin whitening agents, which are free from
harmful side effects. In this regard, phenolic compounds whose
activity has been attributed to their structural resemblance to
L-DOPA and tyrosine (the natural substrates of tyrosinase) [26,27],
seem to be very potent agents. The present work is an outcome of
our investigations aimed at designing of safe to use, natural product
inspired potent tyrosinase inhibitors. Our literature survey exercise
in this regard, motivated us for the synthesis and tyrosinase inhibi-
tion activity studies of stilbene based inhibitors which are presaged
to be very promising but the mechanism behind their activity is yet
to be fully understood [12,28].
Stilbenes synthesized using Wittig reaction.
Compound
R1
R2
Yield (%)a
1
2
3
4
5
6
7
8
9
H
4-F
4-Cl
4-Br
4-NO2
4-CN
4-OCOCH3
4-OH
4-OCH3
4-F
H
H
H
H
H
H
H
H
89
88
88
84
81
86
81
82
86
82
86
H
10
11
40-OCH3
H
3-OCH3,
4-Br
For a molecular level understanding of the inhibitory action of
stilbenoid moieties, we explored the structure-murine tyrosinase
inhibition activity relation for variedly substituted stilbene com-
pounds using resveratrol as a positive control. Since the already
established SARs for this class of inhibitors indicate that the
trans-olefin structure in the parent stilbene skeleton is essential
for tyrosinase inhibition [29,30], we restricted our studies to only
trans derivatives of stilbene compounds with a diverse substitution
pattern on and around aromatic rings and olefinic bond.
12
13
14
15
3,5-F
4-OCH3
2,3,4,5,6-F
–
H
85
85
84
86
30, 40, 50-OCH3
H
One benzene ring replaced
by anthracene
a
Isolated yields after chromatographic purification.
and phenyl methylacetate in presence of pyridine/acetic anhydride
or sodium methoxide/methanol were used to achieve the substitu-
tion across olefinic bond; the reaction exclusively yields the E-
isomer [33,34].
2. Material and methods
Base (piperidine/pyridine) catalysed condensation of 4-
hydroxyphenylacetonitrile with 4-hydroxybenzaldehyde and
nicotinaldehyde respectively at 115 °C (Scheme 3), was followed
for the synthesis of 21 and 22 in E-form (Table 3) [34,35]. The forma-
tion of E-isomers in all cases was established from the NMR spectra
of the final products.
2.1. Chemistry
All solvents were dried and freshly distilled prior to use. 1H was
recorded on a Bruker DPX 200 instrument in CDCl3 using TMS as
internal standard for protons. Mass spectra were recorded on
ESI-esquire 3000 Bruker Daltonics instrument. Elemental analysis
was carried out using Elemental Vario EL III elemental analyser.
Chemical shift values are mentioned in d (ppm) and coupling con-
stants (J) are given in Hz. Mass-spectrometric (MS) data is reported
in m/z. Elemental analysis data is reported in % standard. The pro-
gress of all reactions was monitored by TLC on 2 cm ꢀ 5 cm pre-
coated silica gel 60 F254 plates of thickness 0.25 mm (Merck).
The chromatograms were visualized under UV 254–366 nm and
iodine. Melting points were determined on Buchi B-542 apparatus
by an open capillary method and are uncorrected. Chemicals were
purchased from M/s Aldrich Chemicals, Mumbai. Reagents and
solvents used were mostly of LR grade.
2.1.1.1. Synthesis of 1–15: representative procedure for synthesis of
Trans-1,2-Diphenylethene (1). To a solution of benzyl chloride
(0.126 g, 1.00 mmol) in dry toluene (7 mL), was added triph-
enylphosphine (0.313 g, 1.20 mmol) at ambient temperature. The
reaction mixture was allowed to reflux for 18 h under N2 atmo-
sphere; during this time the Wittig salt precipitated out, it was fil-
tered, washed and used for further reaction. To a solution of Wittig
salt (0.08 g, 0.20 mmol) in CH2Cl2, was added NaOH solution
(0.01 g, 0.25 mmol, 3 mL water) at ambient temperature. The solu-
tion turned orange red indicating the formation of Wittig ylide. To
this solution was added benzaldehyde (0.018 g, 0.16 mmol) and
the reaction mixture allowed to stir at ambient temperature for
3 h. The crude product formed was extracted with CH2Cl2, washed
with brine and evaporated to dryness. After usual column chro-
matography (hexane: EtOAc; 9:1), the product obtained was a mix-
ture of cis and trans-isomer, isolated in 87% yield. The above
mixture was dissolved in hexane containing catalytic amount of
iodine and allowed to reflux for 1 h. The reaction mixture was
cooled down to ambient temperature, washed with Na2S2O5 solu-
tion and the organic layer evaporated to give pure trans-isomer (1)
in quantitative yield (89%).
2.1.1. Synthesis of stilbene derivatives
A library of twenty-two stilbene derivatives was synthesized in
the present study. Wittig reaction between substituted aromatic
aldehydes and Wittig-salts derived from simple/substituted benzyl
chlorides (Scheme 1) was employed for the synthesis of com-
pounds 1–15 (Table 1). Trans stilbenes were obtained from a mix-
ture of cis- and trans-isomers by following reported procedures
[31,32].
Base catalysed condensation reaction of substituted benzalde-
hydes with phenyl acetic acid (Scheme 2), was used for the
synthesis of compounds 16–20 (Table 2). Phenyl acetaldehyde
Cl-
+
P(C6H5)3
CHO
(cis)
+
I2 / Hexane
R2
NaOH/H2O
DCM
R1
+
R2
R2
R1
R1
R2
(trans)
(trans)
R1
Scheme 1. Synthesis of stilbenes (1–15) using Wittig reaction.

T. Ismail et al. / Bioorganic Chemistry 64 (2016) 97–102
99
2.1.1.5. Trans-1-Nitro-4-styrylbenzene (5). Yellow solid, mp: 156–
157 °C [38], 1H NMR (200 MHz, CDCl3):
7.14 (d, 1H,
d
J = 16.40 Hz), 7.28 (d, 1H, J = 14.41 Hz), 7.32–7.43 (m, 3H), 7.56
(d, 2H, J = 7.21 Hz), 7.64 (d, 2H, J = 8.80 Hz), 8.23 (d, 2H,
J = 8.82 Hz), ESI-MS: 224.7 (M+ꢁH), C, H,
N analysis for
14H11NO2: Calculated C, 74.65; H, 4.92; N, 6.22. Found: C, 74.68;
C
H, 4.96; N, 6.26.
Scheme 2. Base catalysed condensation for synthesis of stilbenes (16–20).
2.1.1.6. Trans-4-Styrylbenzonitrile (6). White solid, mp: 116–117 °C
[37], 1H NMR (200 MHz, CDCl3): d 7.26 (d, 1H, J = 16.16 Hz), 7.29 (d,
1H, J = 16.44 Hz), 7.31–7.39 (m, 3H), 7.52–7.67 (m, 6H), ESI-MS:
228 (M++Na), C, H, N analysis for C15H11N: Calculated C, 87.77; H,
5.40; N, 6.82. Found: C, 87.72; H, 5.48; N, 6.83.
Table 2
Stilbenes synthesized through base catalysed condensation.
Compound
R1
R2
R3
Yield (%)a
16
17
18
19
20
4-OCH3
4-OCH3
4-OCH3
4-OCH3
H
30, 40-OCH3
40-OCOCH3
COOH
COOH
COOH
COOCH3
CHO
35
37
46
44
43
2.1.1.7. Trans-Acetic acid 4-styryl-phenyl ester (7). White solid, mp:
149–150 °C [39], 1H NMR (200 MHz, CDCl3): d 2.31 (s, 3H), 7.09–
7.14 (m, 4H), 7.33–7.38 (m, 3H), 7.52–7.57 (m, 4H), ESI-MS: 261
(M++Na), C, H, N analysis for C16H14O2: Calculated C, 80.65; H,
5.92. Found: C, 80.62; H, 5.96.
30-OCH3, 40-OH
30-OCH3, 40-OH
40-OCH3
a
Isolated yields after chromatographic purification.
2.1.1.8. Trans-4-Styryl-phenol (8). White solid, mp: 189–190 °C
[37], 1H NMR (200 MHz, CDCl3): d 6.86 (d, 2H, J = 7.32 Hz), 7.04
(d, 2H, J = 16.16 Hz), 7.34–7.54 (m, 7H), ESI-MS: 194.7 (M+ꢁH), C,
H, N analysis for C14H12O: Calculated C, 85.68; H, 6.16. Found: C,
85.70; H, 6.19.
2.1.1.9. Trans-1-Methoxy-4-styryl-benzene (9). White solid, mp:
135–136 °C [36], 1H NMR (200 MHz, CDCl3): d 3.84 (s, 3H), 6.91
(d, 1H, J = 9.06 Hz), 7.03 (dd, 2H, J1 = 15.86 Hz, J2 = 16.14 Hz), 7.18
(t, 1H, J = 7.55 Hz), 7.31–7.36 (m, 3H), 7.45–7.51 (m, 4H), ESI-MS:
210.9 (M++H), C, H, N analysis for C15H14O: Calculated C, 85.68;
H, 6.71. Found: C, 85.65; H, 6.76.
Scheme 3. Base catalysed condensation for synthesis of stilbenes (21–22).
Table 3
Stilbenes synthesized through base (piperidine/pyridine) catalysed condensation.
2.1.1.10. Trans-1-fluoro-4-(4-methoxystyryl)benzene (10). White
solid, mp: 147–148 °C [40], 1H NMR (200 MHz, CDCl3): d 3.84 (s,
3H), 6.99–7.04 (m, 6H), 7.46–7.52 (m, 4H), ESI-MS: 228 (M+), C,
H, N analysis for C15H13FO: Calculated C, 78.93; H, 5.74. Found:
C, 78.97; H, 5.75.
Compound
X
R
Yield (%)a
21
22
CH
N
OH
H
35
37
a
Isolated yields after chromatographic purification.
White solid, mp: 122–124 °C [36], 1H NMR (200 MHz, CDCl3): d
2.1.1.11. Trans-2-Bromo-1-methoxy-4-styryl-benzene (11). Brown
solid, mp: 135–136 °C, 1H NMR (200 MHz, CDCl3): d 3.95 (s, 3H),
6.92 (d, 1H, J = 8.54 Hz), 7.02 (m, 2H), 7.29–7.44 (m, 5H), 7.78 (d,
1H, J = 7.07 Hz), 8.43 (s, 1H), ESI-MS: 289.17 (M+), C, H, N analysis
for C15H13BrO: Calculated C, 62.30; H, 4.53. Found: C, 62.35; H,
4.52.
7.12 (s, 2H), 7.30–7.45 (m, 6H), 7.72 (m, 4H), ESI-MS: 180.9 (M++H),
C, H, N analysis for C14H12: Calculated C, 93.29; H, 6.71. Found: C,
93.27; H, 6.76.
All the remaining reactions were carried out using this general
procedure with different substituted benzaldehydes.
2.1.1.12. Trans-1,3-Difluoro-5-styrylbenzene (12). White solid, mp:
132–133 °C, 1H NMR (200 MHz, CDCl3): d 7.45–7.53 (m, 5H),
2.1.1.2. Trans-1-Fluoro-4-styryl-benzene (2). White solid, mp: 120–
121 °C [37], 1H NMR (200 MHz, CDCl3): d 7.03–7.09 (m, 4H),
7.26–7.37 (m, 3H), 7.46–7.52 (m, 4H), ESI-MS: 198 (M+), C, H, N
analysis for C14H11F: Calculated C, 84.82; H, 5.59. Found: C,
84.88; H, 5.57.
7.62–7.72 (m, 5H), ESI-MS: 216 (M+), C, H,
N analysis for
C14H10F2: Calculated C, 77.77; H, 4.66. Found: C, 77.72; H, 4.68.
2.1.1.13. Trans-1,2,3-Trimethoxy-5-[2-(4-methoxyphenyl)-vinyl]-ben-
zene (13). White solid, mp: 140–142 °C [41], 1H NMR (200 MHz,
CDCl3): d 3.84 (s, 3H), 3.91 (s, 9H), 6.72 (s, 2H), 6.90–6.95 (m,
4H), 7.45 (d, 2H, J = 8.70 Hz), ESI-MS: 301 (M++H), C, H, N analysis
for C18H20O4: Calculated C, 71.98; H, 6.71. Found: C, 71.92; H, 6.73.
2.1.1.3. Trans-1-Chloro-4-styrylbenzene (3). White solid, mp: 129–
130 °C [36], 1H NMR (200 MHz, CDCl3): d 7.08–7.13 (m, 2H),
7.48–7.53 (m, 9H), ESI-MS: 214.6 (M+), C, H, N analysis for
2.1.1.14. Trans-1,2,3,4,5-Pentafluoro-6-styryl-benzene (14). White
solid, mp: 139–140 °C, 1H NMR (200 MHz, CDCl3): d 6.99–7.04
(m, 2H), 7.40–7.46 (m, 3H), 7.50–7.57 (m, 2H), ESI-MS: 292.7
(M++Na), C, H, N analysis for C14H7F5: Calculated C, 62.23; H,
2.61. Found: C, 62.21; H, 2.63.
C
14H11Cl: Calculated C, 78.32; H, 5.16. Found: C, 78.30; H, 5.18.
2.1.1.4. Trans-1-Bromo-4-styrylbenzene (4). Brown solid, mp: 139–
140 °C [38], 1H NMR (200 MHz, CDCl3):
7.04 (d, 1H,
d
J = 13.96 Hz), 7.13 (d, 1H, J = 16.22 Hz), 7.24–7.31 (m, 3H), 7.37
(d, 2H, J = 7.80 Hz), 7.50–7.54 (m, 4H), ESI-MS: 260 (M++H), C, H,
N analysis for C14H11Br: Calculated C, 64.89; H, 4.28. Found: C,
64.85; H, 4.23.
2.1.1.15. Trans-9-Styryl-anthracene (15). White solid, mp: 132–
133 °C [42]; 1H NMR (200 MHz, CDCl3):
d
6.96 (d, 1H,
J = 16.17 Hz), 7.02 (d, 1H, J = 16.22 Hz), 7.43–7.52 (m, 6H),

100
T. Ismail et al. / Bioorganic Chemistry 64 (2016) 97–102
7.69–7.74 (m, 2H), 7.96–8.01 (m, 3H), 8.39–8.44 (m, 3H), ESI-MS:
280.36 (M+), C, H, N analysis for C22H16: Calculated C, 94.25; H,
5.75. Found: C, 94.22; H, 5.78.
White solid, mp: 248–249 °C, 1H NMR (200 MHz, CDCl3): d 5.32
(broad s, 2 ꢀ OH), 6.76 (d, 2H, J = 7.07 Hz), 6.83 (d, 2H, J = 7.02 Hz),
7.57–7.75 (m, 4H), 8.04 (s, 1H), ESI-MS: 260.1 (M++Na), C, H, N
analysis for C15H11NO2: Calculated C, 75.94; H, 4.67; N, 5.90.
Found: C, 75.95; H, 4.65; N, 5.94.
2.1.1.16. (E)-3-(3,4-Dimethoxyphenyl)-2-(4-methoxyphenyl)acrylic
acid (16). A mixture of 4-methoxyphenylacetic acid (0.2 g,
12.00 mmol), 3,4-dimethoxybenzaldehyde (0.2 g, 12.00 mmol)
and Et3N (1 mL) in Ac2O (5 mL), was heated at 140 °C for 12 h. After
completion of the reaction (monitored by TLC), the reaction mix-
ture was brought to ambient temperature and evaporated to dry-
ness. The residue was diluted with aqueous NaOH for
saponification. The solution was then acidified with AcOH and
extracted with CH2Cl2 (2 ꢀ 50 mL). The combined organic layers
were dried over Na2SO4 and evaporated under vacuum. The crude
product was purified by column chromatography (hexane: EtOAc;
6:4) to yield compound 16 (35%).
2.1.1.22.
(E)-2-(4-Hydroxyphenyl)-3-(pyridin-3-yl)acrylonitrile
(22). White solid, mp: 68–69 °C, 1H NMR (200 MHz, CDCl3): d
5.32 (broad s, OH), 6.90 (m, 3H), 7.46 (s, 1H), 7.49 (d, 2H,
J = 8.71 Hz), 7.75 (d, 2H, J = 8.71 Hz), 8.26 (s, 1H), ESI-MS: 246
(M++Na), C, H, N analysis for C14H10N2O: Calculated C, 75.66; H,
4.54; N, 12.60. Found: C, 75.67; H, 4.52; N, 12.62.
2.2. Biology
L-3,4-Dihydroxyphenylalanine (L-DOPA), resveratrol (3,5,40-
Trihydroxy-trans-stilbene) and other chemical reagents were pur-
chased from Aldrich Chemical Co.
Yellow solid, mp: 218–219 °C [43], 1H NMR (200 MHz, CDCl3): d
3.81 (s, 3H), 3.85 (s, 3H), 3.91 (s, 3H), 6.35 (d, 1H, J = 7.75 Hz), 6.67–
7.05 (m, 3H), 7.15 (d, 2H, J = 8.48 Hz), 7.46 (d, 1H, J = 8.56 Hz), 7.85
(s, 1H), ESI-MS: 337 (M++Na), C, H, N analysis for C18H18O5: Calcu-
lated C, 68.78; H, 5.77. Found: C, 68.77; H, 5.78.
All the synthesized compounds were screened for their tyrosi-
nase inhibitory activity using resveratrol as the positive control.
The inhibitory effect of all synthesised stilbenoid derivatives on
murine tyrosinase activity was evaluated using L-DOPA as sub-
strate. Tyrosinase was prepared from murine B16 melanoma cells
(Riken Cell Bank, Tsukuba, Japan). The cells were lysed by incubat-
ing at 4 °C for 1 h in lysis buffer (10 mM Tris HCl, pH 7.5, 1% NP-40,
0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA,
0.5 mM 4-(2-aminoethyl)-benzenesulfonylfluride hydrochloride,
2.1.1.17. (E)-3-(4-Acetoxyphenyl)-2-(4-methoxyphenyl)acrylic acid
(17). White solid, mp: 149–150 °C, 1H NMR (200 MHz, CDCl3): d
2.24 (s, 3H), 3.83 (s, 3H), 6.83–6.98 (m, 5H), 7.14–7.21 (m, 3H),
7.85 (s, 1H), ESI-MS: 335 (M++Na), C, H, N analysis for C18H16O5:
Calculated C, 69.22; H, 5.16. Found: C, 69.21; H, 5.18.
and 10 lg/mL leupeptin). The lysates were centrifuged at 50,000g
2.1.1.18. (E)-3-(4-Hydroxy-3-methoxyphenyl)-2-(4-methoxyphenyl)
acrylic acid (18). A mixture of 4-methoxyphenylacetic acid (0.2 g,
1.20 mmol) and 4-hydroxy-3-methoxybenzaldehyde (0.915 g,
6.00 mmol) was charged in THF (15 mL). A solution of NaOMe
(0.648 g, 12 mmol) in MeOH (25 mL) was added to the above mix-
ture. The reaction mixture was allowed to stir at ambient temper-
ature for 6 h. After completion of the reaction (monitored by TLC),
the reaction mixture was neutralized with 0.1 N HCl, extracted
with EtOAc (2 ꢀ 50 mL), dried over Na2SO4 and evaporated under
vacuum. Finally the product was purified by column chromatogra-
phy (hexane: EtOAc; 7:3) to yield compound 18 (46%).
for 30 min to obtain the supernatant as a source of tyrosinase.
The reaction mixture contained 50 mM phosphate buffer, pH 6.8,
0.05% L-DOPA and the supernatant (tyrosinase). After incubation
in the absence or presence of various stilbene derivatives (with
varying concentrations ranging from 1 lg/mL to 100 lg/mL) at
37 °C for 20 min, dopachrome was monitored by measuring absor-
bance at wavelength 492 nm by using a Molecular Devices micro-
plate reader. Finally the IC50 values were calculated using
Microsoft Office Excel.
White solid, mp: 82–83 °C, 1H NMR (200 MHz, CDCl3): d 3.83 (s,
3H), 3.95 (s, 3H), 6.89 (m, 3H), 7.00 (d, 2H, J = 8.13 Hz), 7.43 (d, 2H,
J = 8.66 Hz), 7.87 (s, 1H), ESI-MS: 301 (M++H), C, H, N analysis for
C17H16O5: Calculated C, 67.99; H, 5.37. Found: C, 67.98; H, 5.36.
3. Results and discussion
The stilbene derivatives were screened for their murine tyrosi-
nase inhibitory activity at varying concentrations (10, 30 and
100 lg/mL) and the results are presented in Table 4. As is evident
2.1.1.19. (E)-Methyl-3-(4-hydroxy-3-methoxyphenyl)-2-(4-methoxy-
phenyl)acrylate (19). White solid, mp: 80–81 °C, 1H NMR
(200 MHz, CDCl3): d 3.49 (s, 3H), 3.97 (s, 6H), 7.05 (d, 2H,
J = 7.48 Hz), 7.43–7.49 (m, 6H), ESI-MS: 314 (M+), C, H, N analysis
for C18H18O5: Calculated C, 68.78; H, 5.77. Found: C, 68.74; H, 5.78.
from the data, the screened compounds especially 2, 6, 8, 10, 11, 15
and 21 showed remarkably significant tyrosinase inhibitory activ-
ity at investigated range of concentrations.
Since the % inhibition by resveratrol, 6, 8 and 21 was more than
50% at 10
dosage their tyrosinase inhibition activity was also explored at
g/mL and the recorded observations are presented as Table 5.
We observed that compound 21 exhibits maximum inhibitory
effects, with 40.46% inhibition at 1 g/mL, 90.94% at 10 g/mL,
94.33% at 30 g/mL and 94.43% at 100 g/mL and an IC50 value
of 1.20 g/mL (5.06 M) on murine tyrosinase activity (Fig. 1).
lg/mL, to test the efficacy of these compounds at low
2.1.1.20. (E)-3-(4-Methoxyphenyl)-2-phenylpropenal (20). White
solid, mp: 114–115 °C, 1H NMR (200 MHz, CDCl3): d 3.79 (s, 3H),
6.75 (d, 2H, J = 8.84 Hz), 7.15–7.44 (m, 8H), 9.73 (s, 1H), ESI-MS:
261 (M++Na), C, H, N analysis for C16H14O2: Calculated C, 80.65;
H, 5.92. Found: C, 80.68; H, 5.96.
1 l
l
l
l
l
l
l
Resveratrol, the commonly used standard in tyrosinase inhibition
2.1.1.21. (E)-2,3-bis(4-Hydroxyphenyl)acryonitrile (21). A mixture of
4-hydroxyphenylacetonitrile (0.3 g, 2.20 mmol), 4-hydroxybenzal-
dehyde (0.274 g, 2.20 mmol) and catalytic amount of dry piperi-
dine, in 5 mL of dry pyridine, was allowed to reflux at 120 °C for
48 h. After completion of the reaction (monitored by TLC), the reac-
tion mixture was neutralized with 2 N HCl, poured into ice cold
water and extracted with EtOAc (2 ꢀ 50 mL), dried over Na2SO4
and evaporated under vacuum. Finally column chromatography
was performed to purify compound 21 (hexane: EtOAc; 6:4) giving
45% yield.
studies showed similar levels of inhibitory effects on the enzyme
activity with 41.49% inhibition at 1
88.68% at 30 g/mL and 94.02% at 100
of 2.46 g/mL = 10.78 M (Fig. 1).
l
g/mL, 80.74% at 10
lg/mL,
l
lg/mL and an IC50 value
l
l
In the investigated series of compounds, 2, 6, 8, 10, 11 and 15
were less active than 21, while 3, 4, 5, 12, 13, 14, 20 and 22 were
found to be least active. Compounds 1, 7, 9, 16–19 exerted little
or no inhibitory effect compared to the impact of rest of the inves-
tigated compounds on tyrosinase activity. Comparison of the
observed IC50 values suggest that compound 21 exhibits a 2-fold

T. Ismail et al. / Bioorganic Chemistry 64 (2016) 97–102
101
Table 4
Results from biological studies of compounds 2, 10, 12 and 14
indicate that fluorination of compound 1 increases its tyrosinase
inhibition activity and the extent of increase depends upon the
extent and position of fluorination. It is pertinent to mention that
in pharmaceutical research fluorination of pharamcaphores is fre-
quently employed to increase the efficacy and bioavailability of
drugs [44,45]. Fluorination has been observed to increase the
binding affinity and metabolic stability of many drugs which is
mainly attributed to its impact on the lipophilicity, basicity,
molecular conformation and polar interactions of the fluorinated
drug [46–48]. Comparison of the IC50 values of fluoro-
substituted compounds with other halo-substituted stilbenes
and the unsubstituted compound (2 < 4 < 3 < 1), does not indicate
a gradual increase with increase in the molecular mass. This sug-
gests that the impact of the fluorination on the activity of stilbene
derivatives in present study cannot be attributed of its impact on
the lipophilicity, rather it is probably due to increased favourable
and specific dipolar interactions [49]. This hypothesis is further
supported by the IC50 values of stilbenes substituted with nitro,
cyano, acetoxy, hydroxyl, and methoxy groups (5, 6, 7, 8, 9). Pres-
ence of nitro, cyano and hydroxyl groups at position 4 of the aro-
matic ring was found to be very effective in enhancing the
efficacy of stilbenes towards tyrosinase inhibition. Attachment
of methoxy groups to the basic skeleton has been reported to
reduce the inhibitory activity [20]. This is well attested by our
Effect of stilbenoid derivatives on murine tyrosinase activity (SD < 5%).
Compound
% inhibition for tyrosinase in
mL concentration
l
g/
IC50 value lM
10
6.49
30
100
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
12.48
67.15
27.34
26.65
23.75
62.28
6.82
63.02
15.09
55.01
71.33
22.41
5.69
28.66
99.28
65.96
63.81
60.47
81.36
11.12
63.60
55.51
68.43
>500
41.00
16.64
13.08
14.94
49.04
10.53
57.27
4.64
50.05
38.83
9.83
2.12
5.94
79.44
319.75
276.56
360.70
49.95
>500
17.42
432.97
50.04
56.16
393.99
>500
100
56.76
52.69
29.24
100.00
0
8.35
28.64
1.95
>500
17.07
10.20
4.04
166.21
NTb
17
18
19
20
21
22
0.23
0
0
NTb
2.24
0.69
NTb
1.44
1.48
10.64
76.83
94.43
57.50
94.02
>500
276.05
5.06
270.22
10.78
16.55
90.94
34.06
80.74
21.68
94.33
40.18
88.68
Resveratrolc
The bold entries are to highlight the significant inhibition activity results from the
presented study.
observations for compounds
9
(IC50 = 432.97 lM) and 13
(IC50 > 500 M) carrying one and four methoxy groups respec-
l
a Measurement of tyrosinase activity was performed as described in materials and
tively. To ascertain the role of such groupings, we introduced
electron withdrawing groups to methoxy/acetoxy substituted stil-
benes and evaluated their inhibitory potential. As is clear from
data related to compounds 16–19, COOH/COOCH3 groups showed
no effect over the inhibitory effect of stilbenes. Even the introduc-
tion of hydroxyl group to these moieties (compounds 18 and 19)
was observed to have no significant effect on their inhibitory
potential.
Since the IC50 values were lower for hydroxyl (OH) and cyano
(CN) group, we synthesized compound 21 (containing both groups)
and evaluated its tyrosinase inhibitory activity. In our investiga-
tions, we found that compound 21 shows a stronger suppressive
methods.
b
Less than 10% inhibition at the concentration of 100
Resveratrol was used as a positive control.
lg/mL. NT, not tested.
c
Table 5
Effect of stilbenoid derivatives at lower concentrations on murine tyrosinase Activity.
Compound
% inhibition for tyrosinase at 1 lg/mL concentration
Resveratrol
6
8
21
41.49
18.17
24.34
40.46
action on the enzyme tyrosinase with IC50 value of 1.20
(5.06 M), in comparison to the standard for which IC50 value of
2.46 g/mL (10.78 M) was observed under similar conditions.
lg/mL
l
l
l
The observed efficacy of compound 21 against other tyrosinase
inhibitors seems very promising in light of the reported IC50 values
of well-known tyrosinase inhibitors viz., kojic acid (IC50 = 139
[50] oxyresveratrol (IC50 = 52.7 M) [1] and gnetol (IC50 = 4.5
l
l
M)
M)
l
[50] which are often used as standard inhibitors for murine tyrosi-
nase activity. The tyrosinase inhibitory effect observed for 21 is sig-
nificantly stronger than that observed for resveratrol, kojic acid
and oxyresveratrol and almost equivalent to what has been
reported for gnetol. This proves that compared to three hydroxyl
groups in stilbene skeleton (as in resveratrol), a cyano group
together with two hydroxyl groups lead to better tyrosinase inhi-
bition (as observed for compound 21).
Activity studies for compound 22 indicate that pyridine ring
reduces the inhibitory power to a great extent. This is evident from
IC50 value of this compound, which is equal to 270.22
lM. The
results for compound 15 (IC50 166.42 M) indicate that increase
in the aromatic character of the stilbene moiety increase its inhibi-
tory potential.
Overall it seems that the presence of electron withdrawing
groups enhances the tyrosinase inhibition activity of trans-
stilbene moieties, this is in agreement with observations reported
for the case of biphenyl based tyrosinase inhibitors [51]. A compar-
ison of the IC50 values of the stilbenes with electron withdrawing
groups indicates some interesting patterns.
l
Fig. 1. Dose-dependent inhibitory effects on murine tyrosinase by Resveratrol and
compound 21. Effect of tyrosinase activity by the samples as function of
concentration. Data are Mean SD (n = 7) of three similar experiments carried out
in triplicate at different concentrations. Comparisons were made between control
and treated samples using student’s t-test. ⁄P 6 0.05, ⁄⁄P 6 0.001 and ⁄⁄⁄P 6 0.001
for each analysis vs control.
a
stronger inhibitory effect on murine tyrosinase activity than the
standard (Table 4).

102
T. Ismail et al. / Bioorganic Chemistry 64 (2016) 97–102
(1) A comparison of IC50 values of 2, 4, 10 and 11 suggests that
Acknowledgment
two substituents can exhibit synergism for their overall
impact on the efficacy of the stilbene and the extent being
very sensitive to the relative position and the steric aspects
of the substituents. IC50 values of 10 and 11 suggest that
presence of halogens in vicinity of an oxygen containing
group proves very effective probably on account of its ability
to increase the overall polarity of the molecule.
The authors thank CSIR/UGC-New Delhi for the award of
fellowship.
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The impact of substitution on the basic stilbene skeleton over
their murine tyrosinase inhibitory activity has been explored. The
presence of hydroxyl group on phenyl rings was observed to be
very important for their tyrosinase inhibition activity and presence
of electron withdrawing groups was found to significantly enhance
the tyrosinase inhibitory activity of stilbenes. A cyano group
together with two hydroxyl groups (compound 21) leads to better
tyrosinase inhibition than three hydroxyl groups in stilbene skele-
ton (as in resveratrol). The tyrosinase inhibition activity of com-
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design and development of new, safe and effective tyrosinase
inhibitors.