paper 2 published

Design, synthesis and biological evaluation of hydroxy substituted
amino chalcone compounds for antityrosinase activity in B16 cells
Sini Radhakrishnan ⇑
, Ronald Shimmon, Costa Conn, Anthony Baker
School of Chemistry and Forensic Science, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia
a r t i c l e i n f o
Article history:
Received 22 July 2015
Revised 21 August 2015
Accepted 24 August 2015
Available online 24 August 2015
Keywords:
Hyperpigmentation
Melanin
Cytotoxic
Tyrosinase inhibitor
Docking
a b s t r a c t
A series of hydroxy substituted amino chalcone compounds have been synthesized. These compounds
were then evaluated for their inhibitory activities on tyrosinase and melanogenesis in murine B16F10
melanoma cell lines. The structures of the compounds synthesized were confirmed by 1
H NMR, 13
C
NMR, FTIR and HRMS. Two novel amino chalcone compounds exhibited higher tyrosinase inhibitory
activities (IC50 values of 9.75 lM and 7.82 lM respectively) than the control kojic acid (IC50:
22.83 lM). Kinetic studies revealed them to act as competitive tyrosinase inhibitors with their Ki values
of 4.82 lM and 1.89 lM respectively. Both the compounds inhibited melanin production and tyrosinase
activity in B16 cells. Docking results confirm that the active inhibitors strongly interact with mushroom
tyrosinase residues. This study suggests that the depigmenting effect of novel amino chalcone
compounds might be attributable to inhibition of tyrosinase activity, suggesting amino chalcones to be
a promising candidate for use as depigmentation agents or as anti-browning food additives.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
Melanogenesis is considered as a major defense mechanism of
human skin against UV light. However, an excessive accumulation
of the pigment, melanin could lead to serious aesthetic issues.
Tyrosinase [EC 1.14.18.1], also known as polyphenol oxidase is
responsible for melanization in animals, and also plays an impor-
tant role in cuticle formation in insects. Alterations in tyrosinase
dysfunction could culminate with serious maladies including
Café au lait macules, ephelides (freckles), solar lentigo (age spots),
and melasma [1–5]. Tyrosinase is a key enzyme in the melanogenic
pathway responsible for the hydroxylation of L-tyrosine to 3,4-
dihydroxy phenylalanine (L-DOPA) and oxidation of L-DOPA to
dopaquinone [6]. Therefore, tyrosinase inhibitors should be clini-
cally useful for the treatment of some dermatological disorders
associated with melanin hyperpigmentation and also important
in cosmetics for whitening and depigmentation after sunburn. In
addition, tyrosinase is responsible for undesired enzymatic brown-
ing of fruits and vegetables that take place during senescence or
damage in post-harvest handling. This could culminate with a
decline in the functional and organoleptic qualities, such as dark-
ening, softening and off-favour development including a loss in
the nutritional value of foods which makes the identification of
novel tyrosinase inhibitors extremely important [7,8].
From a structural perspective, tyrosinase has two copper ions in
its active site which play a vital role in its catalytic activity. At the
active site of tyrosinase, a dioxygen molecule binds in a side-on
coordination between two copper ions. Each of the copper ions is
coordinated by three histidines in the protein matrix [9]. The cop-
per atoms participate directly in hydroxylation of monophenols to
diphenols (cresolase activity) and in the oxidation of o-diphenols
to o-quinones (catechol oxidase activity) that enhance the produc-
tion of the brown color [10].
Previously, we have reported the synthesis of hydroxy
substituted azachalcone compounds with potential inhibitory
effects on mushroom tyrosinase activity [11]. Presence of the
20
-hydroxyl group on ring A of the chalcone was considered impor-
tant as it was involved in binding to the Cu atoms in the tyrosinase
active site. Studies have shown the presence of an electron-
donating group at para position to increase the mushroom tyrosi-
nase inhibitory activity [12]. It was interesting to note that the
scaffold of the amino chalcone was structurally quite similar to
the substrate L-DOPA. In addition, amino chalcones have been
reported to have promising biological activities [13,14]. Also,
naphthalene derivatives of oxyresveratrol have served to be a
favourable scaffold with potent tyrosinase inhibition [15]. On the
basis of these findings, we considered that it might be interesting
to synthesize a series of hydroxyphenyl and hydroxynaphthyl
http://dx.doi.org/10.1016/j.bioorg.2015.08.005
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding author.
E-mail address: Sini.KaranayilRadhakrishnan@student.uts.edu.au (S. Radhakrishnan).
Bioorganic Chemistry 62 (2015) 117–123
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

substituted amino chalcone compounds for use as depigmentation
agents and as anti-browning food additives.
In the first step, nitro chalcones were synthesized by the base-
catalyzed Claisen–Schmidt condensation of an aldehyde and an
appropriate ketone in a polar solvent like methanol. These nitro
chalcones were then successfully reduced to their corresponding
primary amines in the presence of palladium/carbon, using ammo-
nium formate as the hydrogen source (Scheme 1). The reaction was
based on a facile mechanism of catalytic hydrogen transfer
hydrogenation, an extension of the ‘Leuckart reaction’ [16]. The
structures of the compounds synthesized were confirmed by 1
H
NMR, 13
C NMR, FTIR and HRMS. Assays were performed with
L-DOPA as the substrate, using kojic acid, a well-known strong
tyrosinase inhibitor as the positive control. For the most promising
inhibitors, Lineweaver–Burk kinetic analysis was performed to
determine the mechanism of inhibition. Further, we have inte-
grated our experimental results with computational simulation
methods to obtain a relative insight into the molecular mecha-
nisms governing the mode of inhibition. Previous studies have
indicated the use of chalcones as anticancer agents [17]. This
prompted us to further investigate the effect of aminochalcone
compounds on melanin formation in murine B16F10 cells.
2. Materials and methods
2.1. Chemical reagents and instruments
Melting points (Mp) were determined with WRS-1B melting
point apparatus and the thermometer was uncorrected. NMR spec-
tra were recorded on Agilent 500 spectrometer at 25 °C in CDCl3 or
DMSO-d6. All chemical shifts (o) are quoted in parts per million
downfield from TMS and coupling constants (J) are given in Hz.
Abbreviations used in the splitting pattern were as follows:
s = singlet, d = doublet, t = triplet, m = multiplet. HRMS spectra
were recorded using the Agilent Technologies 6520 LC/MS-QTOF.
All reactions were monitored by TLC (Merck Kieselgel 60 F254)
and the spots were visualized under UV light. Infrared (IR) spectra
were recorded on Thermo Scientific NICOLET 6700 FT-IR spectrom-
eter. Tyrosinase, L-3,4-dihydroxyphenylalanine (L-DOPA), kojic
acid and a-MSH (alpha-melanocyte stimulating hormone) were
purchased from Sigma–Aldrich Chemical Co.
2.2. General method for the synthesis of nitro chalcone derivatives
(1a–1f)
To a stirred solution of the appropriate ketone (1 mM) and a
substituted aldehyde (1 mM) in 25 ml methanol, was added
pulverized NaOH (2 mM) and the mixture was stirred at room
temperature for 24–36 h. The reaction was monitored by TLC using
n-hexane: ethyl acetate (7:3) as mobile phase. The reaction
mixture was cooled to 0 °C (ice-water bath) and acidified with
HCl (10% v/v aqueous solution) to afford total precipitation of the
compounds. In most cases, a yellow precipitate was formed, which
was filtered and washed with 10% aqueous HCl solution. In the
cases where an orange oil was formed, the mixture was extracted
with CH2Cl2, the extracts were dried (Na2SO4) and the solvent was
evaporated to give the respective chalcone (1a–1f).
(1a). (2E)-1-(2-hydroxyphenyl)-3-(4-methoxy-3-nitrophenyl)
prop-2-en-1-one Mp: 110–112 °C; 1
H NMR (500 MHz, CDCl3): o
12.47 (s, 1H, OH), 8.10 (s, 1H), 7.86 (d, 1H, J = 12.5, Hb), 7.74
(d, 1H, J = 10.0, H-60
), 7.72 (d, IH, J = 12.5, Ha), 7.65 (d, 1H, J = 9.5,
H-5), 7.54 (d, 1H, J = 8.0, H-6), 7.49 (t, 1H, J = 8.5, H-40
), 6.92 (dd,
1H, J = 9.5, H-30
), 6.90 (dd, 1H, J = 9.0, H-50
), 3.86 (s, 3H); 13
C NMR
(125 MHz, DMSO-d6) o 192.22 (C@O), 130.52 (C60
), 119.25 (C10
),
118.27 (C50
), 136.77 (C40
), 118.28 (C30
), 161.92 (C20
), 126.90 (C1),
120.54 (C2), 137.22 (C3), 147.50 (C4), 112.42 (C5), 114.23 (C6),
56.67 (Me), 122.40 (vinylic), 141.83 (vinylic); IR (KBr) m (cmÀ1
):
3200, 3070, 2914, 2864, 2720, 1686, 1578, 1550, 1468, 1349,
970, 720, 580; MS (ESI): 270.1 ([M + H])+
.
(1b). (2E)-1-(2-hydroxyphenyl)-3-(2-methoxy-4-nitrophenyl)
prop-2-en-1-one Mp: 123–125 °C; 1
12.22 (s, 1H, OH), 8.22 (s, 1H), 7.95 (d, 1H, J = 14.0, Hb), 7.86 (m,
1H, J = 9.5, H-6), 7.80 (d, IH, J = 14.0, Ha), 7.75 (d, 1H, J = 10.5,
O CH3
HO
+
O
H
O
OH
O
OH
R = 4-O -CH3, R1 = 3-NO2; 1a
R = 2-O -CH3, R1 = 4-NO2; 1b
R R1
R
R1
R
R2
a b
R = 4-O -CH3, R1 = 3-NH2; 2a
R = 2-O -CH3, R1 = 4-NH2; 2b
O
CH3
OH
+
O
H
O
OH
O
OH
R RR1
R1
R R2
a b
R = H, R1 = 3-NO2; 1c
R = H, R1 = 4-NO2; 1d
R = 4-O -CH3, R1 = 3-NO2; 1e
R = 2-O -CH3, R1 = 4-NO2; 1f
R = H, R1 = 3-NH2; 2c
R = H, R1 = 4-NH2; 2d
R = 4-O -CH3, R1 = 3-NH2; 2e
R = 2-O -CH3, R1 = 4-NH2; 2f
Scheme 1. (General method for the synthesis of nitro chalcones (1a–1f) and amino chalcones (2a–2f). Reagents and conditions: (a) MeOH, NaOH, 0 °C, 24 hrs; (b) ammonium
formate, palladium on carbon, MeOH, RT.
118 S. Radhakrishnan et al. / Bioorganic Chemistry 62 (2015) 117–123

H-60
), 7.69 (d, 1H, J = 8.5, H-5), 7.59 (dd, 1H, J = 8.5, H-40
), 7.02 (dd,
1H, J = 9.0, H-30
), 6.97 (m, 1H, J = 10.0, H-50
), 3.82 (s, 3H); 13
C NMR
(125 MHz, DMSO-d6) o 194.25 (C@O), 130.24 (C60
), 119.33 (C10
),
118.20 (C50
), 137.07 (C40
), 118.48 (C30
), 162.90 (C20
), 128.22 (C1),
115.24 (C2), 112.22 (C3), 137.50 (C4), 122.47 (C5), 116.55 (C6),
125.50 (vinylic), 140.87 (vinylic); IR (KBr) m (cmÀ1
): 3250, 3345,
3015, 2910, 2865, 2700, 1702, 1682, 1552, 1465, 1305, 979, 735,
680, 575; MS (ESI): 270.1 ([M + H])+
.
(1c). (2E)-1-(3-hydroxynaphthalen-2-yl)-3-(3-nitrophenyl)
prop-2-en-1-one Mp: 125–127 °C; 1
11.97 (s, 1H, OH), 8.72 (s, 1H), 8.49 (dd, 1H, J = 10.5, H-4), 8.25
(d, 1H, J = 8.0, H-6), 8.10 (dd, 1H, J = 9.5, H-100
), 8.05 (dd, 1H,
J = 9.0, H-50
), 7.85 (d, 1H, J = 11.5, Hb), 7.80 (dd, 1H, J = 9.5, H-5),
7.75 (dd, 1H, J = 9.0, H-80
), 7.54 (t, 1H, J = 8.5, H-60
), 7.52 (d, IH,
J = 11.5, Ha), 7.37 (m, 1H, J = 10.0, H-70
), 7.11 (dd, 1H, J = 7.5, H-
30
); 13
C NMR (125 MHz, DMSO-d6) o 202.44 (C@O), 136.45 (C1),
124.42 (C2), 149.89 (C3), 124.17 (C4), 130.42 (C5), 128.95 (C6),
115.25 (C10
), 165.04 (C20
), 119.99 (C30
), 131.12 (C40
), 124.20 (C50
),
128.12 (C60
), 122.92 (C70
), 129.44 (C80
), 128.51 (C90
), 139.22
(C100
), 127.66 (vinylic), 143.27 (vinylic); IR (KBr) m (cmÀ1
): 3080,
1710, 1561, 1526, 1426, 1340, 1291, 1253, 1158, 988, 973, 827,
728; MS (ESI): 320.1 ([M + H])+
.
(1d). (2E)-1-(3-hydroxynaphthalen-2-yl)-3-(4-nitrophenyl)
prop-2-en-1-one Mp: 114–116 °C; 1
12.17 (s, 1H, OH), 8.32 (dd, 1H, J = 10.0, H-50
), 8.19 (dd, 2H,
J = 9.0, H-2 & H-6), 8.15 (dd, 1H, J = 8.0, H-100
), 7.92 (d, 1H,
J = 12.5, Hb), 7.75 (dd, 1H, J = 9.5, H-80
), 7.59 (d, IH, J = 12.5, Ha),
7.57 (dd, 1H, J = 9.5, H-60
), 7.42 (m, 1H, J = 10.5, H-70
), 7.29 (dd,
1H, J = 8.5, H-30
), 6.92 (dd, 2H, J = 11.5, H-3 & H-5); 13
C NMR
(125 MHz, DMSO-d6) o 197.47 (C@O), 139.49 (C1), 134.45 (C2 &
C6), 129.82 (C3 & C5), 150.02 (C4), 119.27 (C10
), 160.21 (C20
),
119.92 (C30
), 130.92 (C40
), 126.27 (C50
), 129.12 (C60
), 124.90 (C70
),
130.45 (C80
), 129.22 (C90
), 140.20 (C100
), 129.62 (vinylic), 146.20
(vinylic); IR (KBr) m (cmÀ1
): 3075, 1685, 1652, 1586, 1439,
13450, 1285, 1249, 1155, 1025, 970, 835, 732; MS (ESI): 320.1
([M + H])+
.
(1e). (2E)-1-(3-hydroxynaphthalen-2-yl)-3-(4-methoxy-3-nitro-
phenyl) prop-2-en-1-one Mp: 132–134 °C; 1
H NMR (500 MHz,
CDCl3): o 12.29 (s, 1H, OH), 8.15 (s, 1H), 8.12 (dd, 1H, J = 8.0,
H-100
), 8.07 (dd, 1H, J = 10.5, H-50
), 7.90 (dd, 1H, J = 10.5, H-80
),
7.79 (d, 1H, J = 13.5, Hb), 7.65 (d, 1H, J = 9.0, H-5), 7.62 (d, IH,
J = 13.5, Ha), 7.50 (dd, 1H, J = 9.0, H-60
), 7.42 (d, 1H, J = 8.5, H-6),
7.39 (m, 1H, J = 7.5, H-70
), 7.24 (dd, 1H, J = 9.5, H-30
), 3.72 (s, 3H);
13
C NMR (125 MHz, DMSO-d6) o 195.40 (C@O), 120.85 (C10
),
162.44 (C20
), 119.40 (C30
), 130.22 (C40
), 125.87 (C50
), 130.10 (C60
),
124.40 (C70
), 130.49 (C80
), 130.20 (C90
), 142.25 (C100
), 128.99
(vinylic), 147.25 (vinylic), 127.55 (C1), 122.50(C2), 139.22 (C3),
147.42 (C4), 114.22 (C5), 114.20 (C6), 59.30 (Me); IR (KBr) m
(cmÀ1
): 3255, 3065, 2924, 2847, 2720, 2650, 16,702, 1570, 1550,
1425, 1315, 968, 725, 580; HRMS m/z: 350.1012 ([M + H])+
; Calcd:
350.1022.
(1f). (2E)-1-(3-hydroxynaphthalen-2-yl)-3-(2-methoxy-4-nitro-
phenyl) prop-2-en-1-one Mp: 115–117 °C; 1
H NMR (500 MHz,
CDCl3): o 12.69 (s, 1H, OH), 8.37 (dd, 1H, J = 10.5, H-50
), 8.20 (dd,
1H, J = 9.0, H-100
), 7.68 (dd, 1H, J = 9.5, H-80
), 7.60 (dd, 1H, J = 8.5,
H-60
), 7.57 (d, 1H, J = 12.0, Hb), 7.52 (d, IH, J = 12.0, Ha), 7.40 (m,
1H, J = 10.0, H-70
), 7.25 (m, 1H, J = 8.5, H-30
); 13
C NMR (125 MHz,
DMSO-d6) o 194.50 (C@O), 125.04 (C10
), 159.84 (C20
), 119.22 (C30
),
130.27 (C40
), 125.44 (C50
), 132.55 (C60
), 126.42 (C70
), 132.40 (C80
),
130.27 (C90
), 144.20 (C100
), 130.25 (vinylic), 149.85 (vinylic), 55.62
(Me), 130.29 (C1), 119.87 (C2), 117.44 (C3), 130.49 (C4), 124.72
(C5), 119.50 (C6); IR (KBr) m (cmÀ1
): 3350, 3249, 3017, 2850, 2650,
1685, 1605, 1552, 1450, 1325, 975, 728, 687, 568; HRMS m/z:
350.1009 ([M + H])+
; Calcd: 350.1022.
2.3. General method for the synthesis of amino chalcones (2a–2f)
To a stirred suspension of the appropriate nitro chalcones 1a–1f
(5 mmol), 0.25 g 10% palladium on charcoal in 10 cm3
dry metha-
nol at room temperature, was added anhydrous ammonium for-
mate (23 mmol), in a single portion under N2. The resulting
mixture was stirred at room temperature for 3 h. The catalyst
was removed by ﬁltration through Celite and washed with
2 Â 10 cm3
methanol. The ﬁltrate was evaporated under reduced
pressure and the residue was taken up in CHCl3 and washed with
3 Â 25 cm3
H2O. The organic layer was dried (Na2SO4) and evapo-
rated to dryness to give the products 2a–2f.
(2a). (2E)-3-(3-amino-4-methoxyphenyl)-1-(2-hydroxyphenyl)
prop-2-en-1-one Mp: 202–204 °C; 1
11.97 (s, 1H, OH), 8.27 (s, 1H), 7.55 (d, 1H, J = 9.0, H-5), 7.92 (d,
1H, J = 14.5, Hb), 7.72 (d, 1H, J = 9.5, H-60
), 7.49 (d, 1H, J = 8.0, H-
6), 7.40 (t, 1H, J = 8.5, H-40
), 7.32 (d, IH, J = 14.5, Ha), 6.87 (dd, 1H,
J = 8.5, H-50
), 6.85 (dd, 1H, J = 7.5, H-30
), 5.52 (s, 2H), 3.89 (s, 3H);
13
),
119.20 (C10
), 117.67 (C50
), 136.70 (C40
), 118.22 (C30
), 158.94 (C20
),
126.68 (C1), 119.54 (C2), 145.89 (C3), 147.52 (C4), 112.48 (C5),
114.22 (C6), 123.02 (vinylic), 140.87 (vinylic); IR (KBr) m (cmÀ1
):
3225, 3100, 2955, 2860, 2680, 2015, 1690, 1580, 1550, 1440,
1350, 972, 727, 582; HRMS m/z: 270.1118 ([M + H])+
; Calcd:
270.1124.
(2b). (2E)-3-(4-amino-2-methoxyphenyl)-1-(2-hydroxyphenyl)
prop-2-en-1-one Mp: 235–237 °C; 1
11.95 (s, 1H, OH), 8.27 (s, 1H), 8.05 (d, 1H, J = 12.0, Hb), 7.95 (d,
1H, J = 9.5, H-60
), 7.92 (d, IH, J = 12.0, Ha), 7.89 (m, 1H, J = 9.5, H-
6), 7.75 (d, 1H, J = 9.0, H-5), 7.62 (dd, 1H, J = 8.0, H-40
), 7.20 (dd,
1H, J = 9.5, H-30
), 6.99 (dd, 1H, J = 10.5, H-50
), 5.68 (s, 2H), 3.69 (s,
3H); 13
),
120.30 (C10
), 119.25 (C50
), 139.10 (C40
), 118.42 (C30
), 160.90 (C20
),
129.25 (C1), 117.64(C2), 112.20 (C3), 149.50 (C4), 125.40 (C5),
119.45 (C6), 126.40 (vinylic), 145.27 (vinylic); IR (KBr) m (cmÀ1
):
3350, 3325, 3224, 3019, 2922, 2850, 2685, 1700, 1656, 1550,
1425, 1329, 1015, 970, 728, 572; HRMS m/z: 270.1114 ([M + H])+
;
Calcd: 270.1124.
(2c). (2E)-3-(3-aminophenyl)-1-(3-hydroxynaphthalen-2-yl)
prop-2-en-1-one. Mp: 228–230 °C; 1
12.25 (s, 1H, OH), 8.69 (s, 1H), 8.57 (dd, 1H, J = 9.5, H-4), 8.27 (d,
1H, J = 8.5, H-6), 8.22 (dd, 1H, J = 9.5, H-50
), 8.17 (dd, 1H, J = 9.0,
H-100
), 7.91 (d, 1H, J = 10.0, Hb), 7.89 (dd, 1H, J = 9.5, H-5), 7.82
(d, IH, J = 10.0, Ha), 7.79 (dd, 1H, J = 9.0, H-80
), 7.57 (dd, 1H,
J = 9.5, H-60
), 7.40 (m, 1H, J = 11.5, H-70
), 7.20 (dd, 1H, J = 8.5, H-
30
), 5.98 (s, 2H); 13
C NMR (125 MHz, DMSO-d6) o 198.98 (C@O),
134.25 (C1), 125.26 (C2), 152.69 (C3), 125.20 (C4), 130.22 (C5),
127.62 (C6), 116.20 (C10
), 162.54 (C20
), 120.10 (C30
), 134.32 (C40
),
124.20 (C50
), 128.75 (C60
), 122.52 (C70
), 129.02 (C80
), 128.50 (C90
),
140.50 (C100
), 129.25 (vinylic), 145.20 (vinylic); IR (KBr) m
(cmÀ1
): 3345, 3328, 3060, 2600, 1717, 1650, 1567, 1535, 1420,
1335,1228, 980, 935, 820, 752; HRMS m/z: 320.1149 ([M + H])+
;
Calcd: 320.1175.
(2d). (2E)-3-(4-aminophenyl)-1-(3-hydroxynaphthalen-2-yl)
prop-2-en-1-one. Mp: 240–242 °C; 1
12.25 (s, 1H, OH), 8.27 (dd, 1H, J = 9.0, H-50
), 8.07 (d, 1H, J = 9.5,
Hb), 8.05 (dd, 1H, J = 8.0, H-100
), 7.85 (d, IH, J = 9.5, Ha), 7.80 (m,
1H, J = 10.5, H-80
), 7.75 (dd, 2H, J = 10.0, H-2 & H-6), 7.55 (dd, 1H,
J = 9.0, H-60
), 7.47 (dd, 1H, J = 10.5, H-70
), 7.35 (dd, 1H, J = 8.0, H-
30
), 6.87 (dd, 2H, J = 15.0, H-3 & H-5), 5.97 (s, 2H); 13
C NMR
(125 MHz, DMSO-d6) o 192.45 (C@O), 139.25 (C1), 125.68 (C3 &
C5), 155.25 (C4), 132.23 (C2 & C6), 120.20 (C10
), 164.66 (C20
),
119.28 (C30
), 133.87 (C40
), 122.90 (C50
), 128.65 (C60
), 122.48 (C70
),
130.15 (C80
), 129.20 (C90
), 140.25 (C100
), 132.15 (vinylic), 144.25
S. Radhakrishnan et al. / Bioorganic Chemistry 62 (2015) 117–123 119

(vinylic); IR (KBr) m (cmÀ1
): 3425, 3345, 3312, 3026, 2559, 1712,
1648, 1525, 1508, 1415, 1329, 1209, 905, 817, 762; HRMS m/z:
320.1155 ([M + H])+
; Calcd: 320.1175.
(2e). (2E)-3-(3-amino-4-methoxyphenyl)-1-(3-hydroxynaph-
thalen-2-yl) prop-2-en-1-one. Mp: 255–257 °C; 1
H NMR
(500 MHz, CDCl3): o 12.45 (s, 1H, OH), 8.22 (dd, 1H, J = 9.5, H-50
),
8.19 (s, 1H), 8.02 (d, 1H, J = 11.5, Hb), 7.95 (dd, 1H, J = 8.0, H-100
),
7.89 (d, 1H, J = 7.5, H-6), 7.85 (dd, 1H, J = 10.5, H-80
), 7.79 (d, IH,
J = 11.5, 7.60 (dd, 1H, J = 8.5, H-60
), 7.47 (dd, 1H, J = 9.5, H-70
),
Ha), 7.41 (d, 1H, J = 8.0, H-5), 7.39 (dd, 1H, J = 8.0, H-30
), 5.85 (s,
2H), 3.72 (s, 3H), 3.68 (s, 3H); 13
C NMR(125 MHz, DMSO-d6) o
118.89 (C10
), 165.20 (C20
), 119.27 (C30
), 130.85 (C40
), 120.94 (C50
),
126.46 (C60
), 122.44 (C70
), 130.29 (C80
), 130.09 (C90
), 142.75
(C100
), 134.28 (vinylic), 142.77 (vinylic), 192.85 (C@O), 124.66
(C1), 120.05 (C2), 143.19 (C3), 145.22 (C4), 112.01 (C5), 114.20
(C6), 55.03 (Me); IR (KBr) m (cmÀ1
): 3449, 3250, 3124, 2948,
2715, 1702, 1550, 1465, 1352, 967, 735, 584; HRMS m/z:
320.1254 ([M + H])+
; Calcd: 320.1287.
(2f). (2E)-3-(4-amino-2-methoxyphenyl)-1-(3-hydroxynaphtha-
len-2-yl) prop-2-en-1-one. Mp: 262–264 °C; 1
H NMR (500 MHz,
CDCl3): o 13.05 (s, 1H, OH), 8.17 (s, 1H), 8.14 (d, 1H, J = 13.5, Hb),
8.09 (dd, 1H, J = 9.0, H-50
), 8.02 (dd, 1H, J = 8.0, H-100
), 7.95 (d, 1H,
J = 9.0, H-60
), 7.92 (m, 1H, J = 10.0, H-80
), 7.87 (m, 1H, J = 9.0, H-6),
7.82 (d, IH, J = 13.5 Ha), 7.72 (d, 1H, J = 9.0, H-5), 7.62 (dd, 1H,
J = 9.5, H-60
), 7.44 (dd, 1H, J = 10.5, H-70
), 7.40 (dd, 1H, J = 8.5,
H-30
), 5.48 (s, 2H), 3.58 (s, 3H); 13
C NMR (125 MHz, DMSO-d6) o
119.88 (C10
), 162.46 (C20
), 120.08 (C30
), 130.95 (C40
), 122.40 (C50
),
129.02 (C60
), 122.11 (C70
), 130.12 (C80
), 130.20 (C90
), 140.55 (C100
),
131.20 (vinylic), 143.04 (vinylic), 193.02 (C@O), 129.14 (C1),
116.88 (C2), 112.45 (C3), 150.05 (C4), 127.40 (C5), 119.47 (C6),
55.89 (Me); IR (KBr) m (cmÀ1
): 3462, 3248, 3016, 2970, 2862,
1716, 1680, 1555, 1465, 1355, 979, 727, 657, 582; HRMS m/z:
320.1246 ([M + H])+
; Calcd: 320.1287.
2.4. Tyrosinase inhibition assay
The mushroom tyrosinase inhibition activity of all tested com-
pounds was measured using L-DOPA as substrate [18]. Mushroom
tyrosinase, L-DOPA and tested samples were prepared by dissolv-
ing in 50 mM Na2HPO4–NaH2PO4 buffer (pH 6.8). Reaction mix-
tures containing 50 lL of 2 mmol LÀ1
of L-DOPA, 50 lL of
phosphate buffer and 50 lL of different concentrations (0.5 lM,
1.0 lM, 2.5 lM, 5.0 lM and 10.0 lM) of tested compounds were
added in 96 well microtiter plates, followed by the addition of
50 lL of 0.2 mg mLÀ1
of mushroom tyrosinase. The assay mixture
was incubated at 25 °C for 10 min. Then the enzyme reaction
was monitored by measuring the change in absorbance at
492 nm of formation of the DOPA chrome for 1 min. We deter-
mined the inhibition at 20 lM for all the synthesized compounds.
In addition, we determined the IC50, that is, the concentration of
compound that causes 50% inhibition for kojic acid and the active
inhibitors 2a and 2b. In the present study, dose-dependent inhibi-
tion experiments were performed in triplicate to determine the
IC50 of test compounds. The average results from three experi-
ments are shown.
2.4.1. Determination of the inhibition type of compounds 2a and 2b on
mushroom tyrosinase
Inhibitors were first dissolved in DMSO and used for the test
after a 30-fold dilution. The final concentration of DMSO in the test
solution was 3.33%. Mushroom tyrosinase (50 lL; 0.2 mg mLÀ1
)
was incubated with 50 lL of various concentrations of enzyme
substrate (0.2–0.6 mM) and 50 lL of phosphate buffer, and then
50 lL of different concentrations of tested samples (0, 1.25, 5.0
and 20.0 lM) were simultaneously added to the reaction mixtures.
Pre-incubation and measurement time was the same as before. The
measurement was performed in triplicate for each concentration
and averaged before further calculation. The absorbance variations
from these studies were used to generate Lineweaver–Burk plots to
determine the inhibition type. The kinetic parameter (Km) of the
tyrosinase activity was calculated by linear regression from Line-
weaver–Burk plots [19]. For the type of enzyme inhibition and
the inhibition constant (Ki) for an enzyme–inhibitor complex, the
mechanisms were analyzed by Dixon plot, which is a graphical
method [plot of 1/enzyme velocity (1/V) versus inhibitor concen-
tration (I)] with varying concentrations of the substrate.
2.5. In silico docking between tyrosinase and target compounds
To further understand the binding modes of the most active
compounds with mushroom tyrosinase, molecular docking studies
of compounds 2a & 2b were performed using Discovery Studio 4.5
(Accelrys, San Diego, CA, USA). To model the tyrosinase structure,
we used the crystal structure of Agaricus bisporus tyrosinase (PDB
ID: 2Y9X), A chain. Hot spot identification revealed the natural
ligand tropolone binding at the same site as that of the inhibitor
compounds. Tropolone was removed and docking was done for
the inhibitor compounds 2a and 2b. From the docking results, we
checked for possible hydrogen-bonding and non-bonding interac-
tions with the amino acid residues.
3. Cell culture
B16 F10 cells (obtained from National Centre for Cell Sciences
(NCCS), Pune, India) were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; Sigma Aldrich Co., St Louis, USA) with
10% fetal bovine serum (FBS) and penicillin/streptomycin
(100 IU.50 lg mlÀ1
) in a humidified atmosphere of 5% CO2 at
37 °C. B16 cells were cultured in 24-well plates for melanin quan-
tification and enzyme activity assays.
3.1. Cell viability
Cell survival was quantified by a colorimetric assay that used
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) and that measured mitochondrial activity in viable cells.
This method is based on the conversion of MTT (Sigma) to
MTT-formazan crystals by a mitochondrial enzyme, as previously
described [20]. Briefly, cells seeded at a density of 3 Â 104
/cell in
a 48-well plate, were allowed to adhere overnight; the culture
medium was then replaced with fresh serum-free DMEM. MTT
was freshly prepared at 5 mg/ml in phosphate-buffered saline
(PBS). Aliquots of 500 ll of MTT stock solution were added to each
well, and the plate was incubated at 37 °C for 4 h in a humidified
5% CO2 incubator. After 4 h, the medium was removed. To each
well, 500 ll of EtOH–DMSO (solution of a 1:1 mixture) was added
to dissolve the formazan. After 10 min, the optical density of each
well was measured spectrophotometric ally using a 560 nm filter.
The results from three experiments are shown.
3.2. Assay of murine tyrosinase activity
Tyrosinase activity was estimated by measuring the rate of
L-DOPA oxidation [21]. Cells were plated in 24-well dishes at a
density of 5 Â 104
cells mlÀ1
. B16 cells were incubated in the pres-
ence or absence of 100 nM a-MSH, and then treated for 24 h with
various concentrations of 2a or 2b (0–10 lM). Cells were washed
and lysed in 100 ll of 50 mM sodium phosphate buffer (pH 6.5)
containing 1% Triton X-100 (Sigma) and 0.1 mM PMSF (phenyl-
methylsulfonyl fluoride), and then frozen at À80 °C for 30 min.
After thawing and mixing, cellular extracts were clarified by

centrifugation at 12,000 rpm for 30 min at 4 °C. An 8 ll sample of
the supernatant and 20 ll of L-DOPA (2 mg mlÀ1
) were placed in
a 96-well plate, and the absorbance at 492 nm was read every
10 min for 1 h at 37 °C using an ELISA plate reader. The final activ-
ity is expressed as D O.D. minÀ1
for each condition. The results
from three experiments are shown.
3.3. Determination of melanogenesis in B16 cells
Determination of melanin content of cells was done using a
modification of the method of Bilodeau et al. [22]. In the present
study, the amount of melanin was used as an index of melanogen-
esis. B16 F10 cells (5 Â 104
) were transferred to 24-well dishes and
incubated in the presence or absence of 100 nM a-MSH. Cells were
then incubated for 24 h with various concentrations of 2a and 2b
(0–10 lM). After washing twice with PBS, samples were dissolved
in 100 ll of 1 N NaOH. The samples were then incubated at 60 °C
for 1 h and mixed to solubilize the melanin. Absorbance at
405 nm was compared with a standard curve of synthetic melanin.
The results from three experiments are shown.
4. Results and discussion
4.1. Chemistry
In terms of the structure-activity relationships, compound 2b
(2E)-3-(4-amino-2-methoxyphenyl)-1-(2-hydroxyphenyl) prop-2-
en-1-one) exhibited the most potent tyrosinase inhibitory activity
with inhibition of 75.51% (Table 1). This could be accounted for the
presence of strong ortho–para activating substituents on ring B.
Presence of strong electron releasing groups in the ortho position
(AOMe) and a strongly activating para amino substituent modu-
lates the electronic structure of ring B significantly. This is consis-
tent with the docking results where the electron-donating groups
increase the electron density of ring B through a resonance donat-
ing effect and higher electron density binds copper ions more
effectively in the active site of enzyme. Among all the investigated
compounds, the amino chalcone compound 2b showed better inhi-
bitory potential (IC50: 7.82 lM) than kojic acid (IC50: 22.83 lM).
Removal of electron donating substituents on ring B brought about
a slight decrease in tyrosinase inhibition as seen with compound
2a that had an IC50 of 9.75 lM. The fact that the activity is mark-
edly affected by altering the substituents on the aminophenyl ring,
suggests that this aromatic ring makes a specific contribution to
the binding via an aromatic ring orientation. Replacement of the
phenyl group with a naphthyl group led to a slight decline in inhi-
bitory activities (compounds 2c and 2d) indicating that the bulky
naphthyl group in ring A might cause stereo-hindrance for the
inhibitors approaching the active site. However, substituting the
naphthyl amino chalcone with a para activating group on ring B
gave compound 2e with an inhibitory potential (58.5 ± 1.62%) that
was still more effective than kojic acid (42.6 ± 0.32%) while substi-
tution with an ortho directing methoxy group gave compound 2f
that showed a considerable decline in tyrosinase inhibition
(39.6 ± 0.66%).
4.2. Kinetics
We investigated in greater detail the bioactivities of compounds
2a and 2b, which had more potent activity than kojic acid. Com-
pounds 2a, 2b and kojic acid were found to inhibit mushroom
tyrosinase activity in a concentration-dependent manner (Table 2).
We measured the reaction rates in the presence of active inhibitors
with various concentrations of L-DOPA as a substrate. As the con-
centrations of active inhibitors 2a and 2b increased, Km values
gradually increased, but Vmax values did not change, thereby indi-
cating that the inhibitors act as competitive inhibitors of mush-
room tyrosinase (Fig. 1). The inhibition kinetics were illustrated
by Dixon plots, which were obtained by plotting 1/V versus [I] with
varying concentrations of substrate. Dixon plots gave a family of
straight lines passing through the same point at the second quad-
rant, giving the inhibition constant (Ki) (Fig. 2). The Ki value esti-
mated from this Dixon plot was 6.75 lM and 5.80 lM for the
compounds 2a and 2b respectively. A comparison of the Km and
Ki values of the compounds with that of kojic acid revealed that
they possess much higher affinity to tyrosinase than kojic acid.
4.3. Docking studies
We simulated binding between the active site of mushroom
tyrosinase and the active inhibitors 2a and 2b using Accelrys Dis-
covery studio 4.5 suite. Compounds 2a and 2b exhibited hydrogen
bonding interactions with His259, His85 and His61. Both the active
compounds showed hydrophobic p–p stacking interactions with
His263 and T-shaped edge to face aryl–aryl interactions with
Phe264 and His244 (Fig. 3). Docking results show that compound
2b (À25.75 kcal molÀ1
) combines with mushroom tyrosinase more
Table 1
Docking results and tyrosinase inhibition effects of amino chalcones (2a–2f).
Compounds Yield
(%)
CDOCKER energy
(kcal/mol)
Tyrosinase inhibitiona
(%)
2a 62.35 À24.86 63.2 ± 0.12
2b 55.65 25.75 75.5 ± 1.12
2c 72.20 13.90 49.3 ± 0.45
2d 78.60 À11.39 54.3 ± 2.01
2e 42.55 À12.13 58.5 ± 1.62
2f 68.75 À7.67 39.6 ± 0.66
Kojic acid – À10.59 48.4 ± 0.32
a
Values indicate means ± SE for three determinations.
Table 2
Inhibitory effects of kojic acid, 2b and 2a on mushroom tyrosinase activity.
Sample Concentration (lM) Inhibition (%) Average of inhibition (%) IC50 (lM)a
Ki (lM)#
Kojic acid 1.25 3.62 3.60 3.65 3.62 22.83 ± 0.66 9.23
5.00 16.45 16.52 16.42 16.46
20.00 49.07 48.05 48.25 48.45
2b 1.25 23.55 25.20 21.25 23.25 7.82 ± 0.42 1.89
5.00 44.60 42.20 40.25 42.35
20.00 77.60 73.75 75.20 75.51
2a 1.25 39.36 34.25 35.50 22.95 9.75 ± 1.22 4.82
5.00 46.45 44.25 44.65 45.11
20.00 72.50 70.23 73.65 63.25
a
50% inhibitory concentration (IC50).
#
Values were measured at 5 lM of active compounds and Ki is the (inhibitor constant).

strongly than compound 2a (À24.86 kcal molÀ1
) (Table 1). The
lower tyrosinase inhibition of compound 2a could be accounted
for the formation of a strong intramolecular hydrogen bond
(1.95 Å) formed between the 20
-hydroxyl hydrogen with the car-
bonyl oxygen. Copper ion (Cu400) was strongly bound by the
hydroxyl group (20
) of compound 2b at a distance of 1.95 Å. There
was also a coordination between Cu401 and the hydroxy oxygen
(20
) of the ligand 2b at a distance of 3.15 Å. Formation of a complex
between a ligand and the copper ion in the active site of mushroom
tyrosinase could prevent electron transfer by the metal ion. More-
over, the binding of the inhibitor via a coordinate bond will ensure
that access to the active site by the substrate is effectively blocked.
This could curb the enzymes ability to oxidize the substrates
subsequently leading to an inhibition in mushroom tyrosinase.
4.4. Effect on melanogenesis
We then explored whether compounds 2a and 2b that showed
good tyrosinase inhibition activity were cytotoxic to B16F10
melanoma cells. The cytotoxicity of these active compounds was
estimated by using the MTT assay and the results implied that
these compounds were not cytotoxic up to 10 lM but showed little
cytotoxicity at 100 lM (Fig. 4). The melanin content of B16
cells after treatment with compound 2a in the presence of
100 nM a-melanocyte-stimulating hormone (a-MSH) decreased
-200
-100
0
100
200
300
400
500
-4 -2 0 2 4 6
1/V(µM/min)-1
1/S (mM/L)
2a
-200
-100
0
100
200
300
400
500
-4 -2 0 2 4 6
1/V(µM/min)-1
1/S (mM/L)
2b
Fig. 1. Lineweaver Burk plot for inhibition of compounds 2a and 2b on mushroom
tyrosinase. Data were obtained as mean values of 1/V, the inverse of the absorbance
increase at a wavelength of 492 nm per min of three independent tests with
different concentrations of L-DOPA as a substrate. The concentration of compounds
2a and 2b from top to bottom is 20 lM, 5 lM, 1.25 lM and 0 lM respectively.
-150
-100
-50
0
50
100
150
200
250
300
350
400
-5 0 5 10
1/V(µM/min)-1
[Inhibitor] µM
2b
-5
0
5
10
15
20
25
30
35
40
-20 -10 0 10 20 30
1/v(µM/min)-1
[inhibitor] µM
2a
Fig. 2. Dixon plot for the inhibitory effect of compounds 2b and 2a on L-DOPA
oxidation catalyzed by mushroom tyrosinase. The inhibitor concentrations were 0,
10 lM and 20 lM respectively. The L-DOPA concentrations were 200, 400 and
600 lM.
Fig. 3. Docking result of compounds 2a and 2b in the tyrosinase catalytic pocket.
Ligands 2a and 2b are displayed as ball and stick while the core amino acid residues
are displayed as stick. The green dotted lines show the hydrogen bond interactions
and the purple lines show the non-bonding interactions. The ochre balls represent
the copper ions.

dose-dependently, showing 215.22% at 1.0 lM, 182.56% at 5.0 lM
and 123.55% at 10.0 lM. Similarly, cells treated with compound 2b,
exhibited melanin contents of 270.18% at 5.0 lM, 244.11% at
5.0 lM and 200.05% at 10.0 lM (Fig. 5) as compared with
100 nM a-MSH-only-treated group (280.24%) and the control
group (100%).
Finally, to examine the mechanisms by which compounds 2a
and 2b inhibit melanin production, the effect of these compounds
on cellular tyrosinase activity in B16F10 melanoma cells treated
with 100 nM a-MSH was examined. These compounds effectively
diminished tyrosinase activity in a dose-dependent pattern com-
pared to the control (Fig. 6). These results support the hypothesis
that the inhibitory effect of compounds 2a and 2b on melanin
biosynthesis should be attributed to inhibition of tyrosinase
activity.
5. Conclusion
Among substituted amino chalcone compounds, compounds 2a
[(2E)-3-(3-amino-4-methoxyphenyl)-1-(2-hydroxyphenyl) prop-
2-en-1-one] and 2b [(2E)-3-(4-amino-2-methoxyphenyl)-1-(2-hy-
droxyphenyl) prop-2-en-1-one] were found to be the most active
tyrosinase inhibitors with their IC50 values of 9.75 ± 1.22 lM and
7.82 ± 0.42 lM, respectively indicating them to be more potent
than the reference compound, kojic acid (22.83 ± 0.66 lM). Both
2a and 2b were identified as competitive inhibitors of mushroom
tyrosinase in a kinetic study. Docking simulation identified the
ligand binding residues that could act possibly as the key determi-
nants to enhance the binding affinity between the inhibitor com-
pounds and the enzyme tyrosinase. In cell based experiments
both the compounds 2a and 2b showed very effective inhibitions
of both melanin production and tyrosinase activity, suggesting
amino chalcones to be a promising candidate for use as depigmen-
tation agents in the field of cosmetics or as anti-browning food
additives in the field of agriculture.
Declaration of interest
The authors declare no conflict of interest.
References
[1] H. Ando, H. Kondoh, M. Ichihashi, V.J. Hearing, J. Invest. Dermatol. 127 (2007)
751–761.
[2] K. Iozumi, G.E. Hoganson, R. Pennella, M.A. Everett, B.B. Fuller, J. Invest.
Dermatol. 100 (1993) 806–811.
[3] Q. Wang, J. Yan, J. He, K. Bai, H. Li, J. Lumin. 138 (2013) 1–7.
[4] S. Briganti, E. Camera, M. Picardo, Pigment Cell Res. 16 (2003) 101–110.
[5] Y.X. Si, S.J. Yin, D. Park, H.Y. Chung, L. Yan, Z.R. Lϋ, H.M. Zhou, J.M. Yang, G.Y.
Qian, Y.D. Park, Int. J. Biol. Macromol. 48 (2011) 700–704.
[6] A.B. Lerner, T.B. Fitzpatrick, E. Calkins, W.H. Summerson, J. Biol. Chem. 187
(1950) 793–802.
[7] M.V. Martinez, J.R. Whitaker, Trends Food Sci. Tech. 6 (1995) 195–200.
[8] M. Jiménez-Atiénzar, M. Atienzar, J. Escribano, J. Cabanes, F. Gandıa-Herrero,
Garcıa-Carmona, Plant Physiol. Biochem. 43 (2005) 866–873.
[9] Y. Matoba, T. Kumagai, A. Yamamoto, H. Yoshitsu, M. Sugiyama, J. Biol. Chem.
281 (2006) 8981–8990.
[10] H. Decker, F. Tuczek, Trends Food Sci. Tech. 25 (2000) 392–397.
[11] S.K. Radhakrishnan, R. Shimmon, C. Conn, A.T. Baker, Bioorg. Med. Chem. Lett.
25 (2015) 1753–1756.
[12] I. Kubo, I. Kinst Hori, J. Agr. Food Chem. 47 (1999) 4121–4125.
[13] Y. Xia, Z.Y. Yang, P. Xia, K.F. Bastow, Y. Nakanishi, K.H. Lee, Bioorg. Med. Chem.
Lett. 10 (2000) 699–701.
[14] Y.R. Prasad, A.S. Rao, R. Rambabu, Asian J. Chem. 21 (2009) 907–914.
[15] S. Song, H.J. Lee, Y. Jin, Y.M. Ha, S.J. Bae, H.Y. Chung, H. Suh, Bioorg. Med. Chem.
Lett. 17 (2007) 461–464.
[16] A.I.R.N.A. Barros, A.M.S. Silva, Tetrahedron Lett. 44 (2003) 5893–5896.
[17] T.W. Yan, J.Q. Ya, L.Z. Ya, J.L. Yu, R. Bing, Q.Z. Yan, R.Y. Meng, Q.J. Ai, L.Q. Jin, L.Z.
Hai, RSC Adv. 61 (2014) 32263–32275.
[18] Y.F. Lin, Y.H. Hu, Y.L. Jia, Z.C. Li, Y.J. Guo, Q.X. Chen, H.T. Lin, Int. J. Biol.
Macromol. 51 (2012) 32–36.
[19] S.J. Cho, J.S. Roh, W.S. Sun, S.H. Kim, K.D. Park, Bioorg. Med. Chem. Lett. 16
(2006) 2683–2684.
[20] H. Tada, O. Shiho, K. Kuroshima, M. Koyama, K. Tsukamoto, J. Immunol.
Methods 93 (1986) 157–165.
[21] D.S. Kim, S.Y. Kim, J.H. Chung, K.H. Kim, H.C. Eun, K.C. Park, Cell. Signal. 14
(2002) 779–785.
[22] M.L. Bilodeau, J.D. Greulich, R.L. Hullinger, C. Bertolotto, R. Ballotti, O.M.
Andrisani, Pigment Cell Res. 14 (2001) 328–336.
Fig. 4. Effect of compounds 2a and 2b on cell viability. Data are expressed as a
percentage of the control.
0
50
100
150
200
250
300
350
400
control
α-MSH
α-MSH+1.0μM
α-MSH+5.0μM
α-MSH+10.0μM
control
α-MSH
α-MSH+1.0μM
α-MSH+5.0μM
α-MSH+10.0μM
2b2a
melanincontents(%ofcontrol)
***
***
***
***
***
***
***
###
Fig. 5. Inhibitory effect of compounds 2a and 2b after treatment with 100 nM a-
MSH in B16 cells. Melanin contents were measured at 405 nm. Values represent the
mean ± S.E. of three experiments. Data are expressed as a percentage of the control.
⁄⁄⁄
p < 0.001 compared to the group treated with 100 nm a-MSH and ###
p < 0.001,
compared with the untreated control.
0
20
40
60
80
100
120
140
control
α-MSH
α-MSH+1.0μM
α-MSH+5.0μM
α-MSH+10.0μM
control
α-MSH
α-MSH+1.0μM
α-MSH+5.0μM
α-MSH+10.0μM
2b2a
Tyrosinaseactivity(%ofcontrol)
###
***
***
***
***
*** ***
***
***
Fig. 6. Inhibitory effect of compounds 2a and 2b on B16 cells tyrosinase. Values
represent the mean ± S.E. of three experiments. Data are expressed as a percentage
of the control. ⁄⁄⁄
p < 0.001 compared to the group treated with 100 nm a-MSH and
###
p < 0.001, compared with the untreated control.

paper 2 published

More Related Content

What's hot (20)

Viewers also liked (20)

Similar to paper 2 published (20)

paper 2 published