Benzylidene-linked thiohydantoin derivatives as inhibitors of tyrosinase and melanogenesis: Importance of the β-phenyl-α,β-unsaturated carbonyl functionality

Article abstract of DOI:10.1039/c4md00171k

Based on the structural characteristics of the heterocyclic scaffolds of substituted benzylidene-hydantoin, -pyrrolidinedione, and -thiazolidinedione derivatives with potent tyrosinase inhibitory activity, substituted benzylidene derivatives with a 2-thio

Full text of DOI:10.1039/c4md00171k

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Benzylidene-linked thiohydantoin derivatives as
inhibitors of tyrosinase and melanogenesis:
importance of the b-phenyl-a,b-unsaturated
carbonyl functionality
Cite this: Med. Chem. Commun., 2014,
5, 1410
Hye Rim Kim,a Hye Jin Lee,a Yeon Ja Choi,a Yun Jung Park,a Youngwoo Woo,a
a
a
a
b
a
*
Seong Jin Kim, Min Hi Park, Hee Won Lee, Pusoon Chun, Hae Young Chung
a
*
and Hyung Ryong Moon
Based on the structural characteristics of the heterocyclic scaffolds of substituted benzylidene-hydantoin,
-pyrrolidinedione, and -thiazolidinedione derivatives with potent tyrosinase inhibitory activity, substituted
benzylidene derivatives with a 2-thiohydantoin heterocyclic scaffold were synthesized by modified
Knoevenagel condensation between benzaldehydes and 2-thiohydantoin with a view toward producing
more potent, safer tyrosinase inhibitors capable of being utilized in the agricultural, food, cosmetics, and
pharmaceutical industries. Of the twelve compounds synthesized, three compounds, 2c, 2d and 2i,
exhibited even more potent inhibitory activities against mushroom tyrosinase than kojic acid or
resveratrol, which are well-known potent tyrosinase inhibitors. The inhibitory pattern of compounds with
a thiohydantoin template differed from that of compounds with a hydantoin, pyrrolidinedione, or
thiazolidine scaffold, probably because of the loss of the hydrogen bonding ability of the thiocarbonyl
group of thiohydantoin. Considering the high tyrosinase inhibitory activities of 5-(substituted
benzylidene)thiohydantoin derivatives, the thiohydantoin template is considered a near perfect surrogate
for hydantoin, pyrrolidinedione, and thiazolidinedione scaffolds. (Z)-5-(2,4-Dihydroxybenzylidene)-2-
thiohydantoin (2d, IC50 ¼ 1.07 ꢀ 2.30 mM) had 24 times the inhibitory effect of resveratrol (IC50 ¼ 26.63
ꢀ 0.55 mM) and 18 times that of kojic acid (IC50 ¼ 19.69 ꢀ 4.90 mM) against mushroom tyrosinase and
showed anti-melanogenesis through the inhibition of tyrosinase activity in B16 cells with no appreciable
cytotoxicity, which suggests that 2d is a promising candidate for the development of safer and more
potent fruit and food browning preventatives and skin-lightening medicines. This result and our previous
data indicate that it is the “b-phenyl-a,b-unsaturated carbonyl” group that is essential for potent anti-
tyrosinase activity.
Received 16th April 2014
Accepted 25th June 2014
DOI: 10.1039/c4md00171k
www.rsc.org/medchemcomm
Tyrosinase exists widely in bacteria, fungi, plants, insects,
vertebrates, and invertebrates and is the rate-determining
Introduction
enzyme in the biosynthesis of melanin pigments that are
responsible for the coloration of hair, skin, and eyes and the
undesired enzymatic browning of vegetables and fruits.2,3
Tyrosinase has become increasingly important in the medicinal
and cosmetic industries. For example, tyrosinase inhibitors are
used to treat hyperpigmentation, melasma, freckles, and age
spots, and as skin whitening agents, whereas tyrosinase acti-
vators that increase melanogenesis can protect the skin from
UV damage.4 In the food industry, tyrosinase is an important
enzyme in the context of maintaining the quality of fruit and
vegetables under storage because of the damage-associated
browning that occurs during postharvest handling. The devel-
opment of undesirable avors post-harvest also lowers the
values of agricultural products. Quinone compounds are
produced by successive oxidative reactions of tyrosinase during
Over the past couple of decades, tyrosinase (EC 1.14.18.1), a
polyphenol oxidase, has received considerable attention as an
indispensable tool in the search for potent tyrosinase inhibitors
from natural and synthetic sources. Tyrosinase is a copper-
containing multifunctional, glycosylated oxidase that catalyzes
both the hydroxylation of L-tyrosine to L-dopa and the oxidation
of L-dopa to L-dopaquinone, the latter of which is nally con-
verted into melanin through a series of enzymatic and nonen-
zymatic reactions.1
aMolecular Inammation Research Center for Aging Intervention (MRCA), College of
Pharmacy, Pusan National University, Busan 609-735, Republic of Korea. E-mail:
mhr108@pusan.ac.kr; Fax: +82-51-513-6754; Tel: +82-51-510-2815
bCollege of Pharmacy, Inje University, Gimhae, Gyeongnam 621-749, Republic of
Korea. E-mail: hyjung@pusan.ac.kr; Fax: +82-51-518-2821; Tel: +82-51-510-2814
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‘browning’ and are highly reactive species, which are capable of
Compound I was designed by mimicking the chemical
triggering Diels–Alder and Michael addition reactions with the structures of the tyrosinase substrates, L-tyrosine and L-dopa.10
Lys-NH2 (amino) and Cys-SH (sulydryl) groups of proteins.5 Like the primary amines of L-tyrosine and L-dopa, the amide NH
Therefore, tyrosinase inhibitors could possibly prevent and at position 1 of hydantoin I has the ability to form hydrogen
control enzymatic browning reactions by inhibiting quinone bonds with amino acids at the active site of tyrosinase.
formation and thus improve the quality and nutritional value of Furthermore, the imido group of I could serve as an acid like the
agricultural products.
carboxylic acids of tyrosinase substrates. Compounds I, II, and
Furthermore, recent studies have demonstrated that tyrosi- III exhibited more potent tyrosinase inhibition than kojic acid
nase inhibitors can be used to control insect pests by inhibiting the positive control (according to reports, kojic acid is three fold
their developmental and defensive functions,6–8 and thus, the or more potent than arbutin), which prompted us to synthesize
development of suitable novel tyrosinase inhibitors could also benzylidene-heterocyclic compounds with another surrogate of
improve the output and quality of agricultural products by carboxylic acid. During our continued efforts to identify more
reducing the impact of insect pests.
potent, safer tyrosinase inhibitors, 5-(hydroxyl- or alkoxy-
Over the last few decades, a large number of naturally substituted benzylidene)thiohydantoin analogs 2a–2l with a
occurring and synthetic tyrosinase inhibitors have been repor- monothioimido group as a carboxylic acid surrogate were
ted. Currently, arbutin, a hydroquinone glycoside extracted designed, synthesized, and evaluated with respect to their
from the bearberry plant of the genus Arctostaphylos is used in tyrosinase inhibitory and anti-melanogenic activities. Currently,
the cosmetic industry as a whitening agent due to its tyrosinase hydantoin analogs, including phenytoin and fosphenytoin, are
inhibitory activity. However, it has also been reported that used for the treatment of epilepsy.14 Therefore, we considered
arbutin is decomposed by temperature (10% decomposition at that replacement of the carbonyl group of hydantoin analogs I
20 ꢁC for 15 days), enzymes, such as b-glycosidase, and by by a thiocarbonyl group might diminish the potential anticon-
intestinal bacteria to glucose and hydroquinone, the latter of vulsant effect and increase tyrosinase inhibitory activity and
which could cause immunotoxicity or possibly cancer.9 More- bioavailability by enhancing lipophilicity. Benzylidene-thio-
over, several reportedly active agents, such as arbutin, kojic hydantoin derivatives have been reported to show potent
acid, and tropolone, have not been demonstrated to be clini- fungicidal, aldose reductase inhibitory, anti-tuberculosis, and
cally efficacious when critically analyzed in carefully controlled selective COX-2 inhibitory activities.15–19 However, to the best of
studies. Therefore, there is a continuous need for tyrosinase our knowledge, the anti-tyrosinase effects of benzylidene-thio-
inhibitors that are potent enough to be of practical use and yet hydantoin analogs have not been previously reported.
safe enough to be used in pharmaceutical, cosmetic, food, and
agriculture elds.
The target compounds 2a–2l were prepared by modied
Knoevenagel condensation between benzaldehydes with
various substituents and 2-thioxoimidazolidin-4-one, also
known as 2-thiohydantoin. Reuxing 2-thiohydantoin and
benzaldehydes with hydroxyl, methoxyl, and/or ethoxyl
substituents at different positions in the presence of sodium
acetate and acetic acid afforded benzylidene-thiohydantoin
derivatives 2a–2l, as shown in Scheme 1 and Table 1. Twelve 5-
(substituted benzylidene)-2-thiohydantoin analogs were
obtained in moderate yields. In all reactions, (Z)-isomers were
produced predominately. The products were characterized by1H
and 13C NMR and mass spectroscopy and all spectral data were
identical to those of authentic compounds.
Results and discussion
Recently, we synthesized benzylidenehydantoin I, benzylidene-
pyrrolidinedione II, and benzylidenethiazolidine-2,4-dione III
derivatives as potential tyrosinase inhibitors (Fig. 1) and
showed that these compounds are promising anti-browning
agent candidates in food and agricultural products and prom-
ising active ingredients in skin-lighting cosmetics and in
medicines for the treatment of diseases associated with
hyperpigmentation.10–13
Fig. 1 The rationale for the design of benzylidene-linked thiohydantoin derivatives.
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surprising that 4-methoxybenzylidene and 2,4-dimethoxy-
benzylidene analogs 2c and 2i with a thiohydantoin scaffold
displayed impressive mushroom tyrosinase inhibitory activities
(72.62 and 58.71%, respectively). Although most 3,5-dihydroxy-
benzylidene analogs synthesized previously by our laboratory
exhibited only weak or no inhibition against mushroom tyros-
Scheme
1 Preparation of (Z)-5-(substituted benzylidene)-2-thio-
hydantoin derivatives. Reagents and conditions: (a) NaOAc, AcOH, inase, their congener 2f with a thiohydantoin scaffold inter-
reflux, 4–24 h, 15.1–84.6%.
estingly exhibited moderate inhibitory activities with 46.22%
inhibition at 50 mM. It was also interesting to note that 4-
hydroxybenzylidene derivatives constructed on templates, such
Mushroom tyrosinase is commonly used for screening and
characterizing potential tyrosinase inhibitors, because it is
commercially available, relatively inexpensive, and provides
reliable results. Accordingly, it was used in the present study to
evaluate the tyrosinase inhibitory abilities of the synthesized
thiohydantoin compounds. The mushroom tyrosinase inhibi-
tory activities of the synthesized compounds were examined as
described previously, with minor modications20 and are shown
in Table 1. All tyrosinase inhibitions were measured at
concentrations of 25 or 50 mM. 2,4-Dihydroxybenzylidene-2-
thiohydantoin 2d (96.54% inhibition at 25 mM) inhibited
mushroom tyrosinase activity more than the other eleven thio-
hydantoins synthesized. The 2,4-dihydroxyl functional group on
the benzylidene remarkably increased the activity. This
tendency was also previously observed for benzylidene-pyrroli-
dinedione and benzylidene-thiazolidinedione derivatives
reported by our laboratory.11–13 Mono- and di-methox-
ybenzylidene analogs with a hydantoin, pyrrolidinedione, or
thiazolidinedione scaffold showed only low inhibitory activities
against mushroom tyrosinase.10–13 Therefore, it was somewhat
as hydantoin, pyrrolidinedione, or thiazolidinediones, showed
moderate to high inhibitory inhibition against mushroom
tyrosinase10,11 whereas the 4-hydroxybenzylidene analog 2a
constructed on a thiohydantoin template showed almost none.
This dramatic change in heterocyclic scaffold-dependent
tyrosinase inhibitory activity could be due to the hydrogen
bonding abilities of scaffolds with tyrosinase. For 4-hydroxy-
benzylidene-hydantoin, -pyrrolidinedione, and -thiazolidine-
dione compounds, the carbonyl group of the heterocyclic
templates could play an important role in interactions with
tyrosinase, probably via hydrogen bonding. However, conver-
sion of the heterocyclic template into thiohydantoin provides a
thiocarbonyl group, instead of a carbonyl group, and this might
prevent this hydrogen bonding and result in a loss of inhibitory
activity.
We further investigated the IC50 values of 2c, 2d, and 2i
because these three compounds exhibited strong inhibitory
activity. The IC50 values of 2c, 2d, 2i, resveratrol, and kojic acid
for the inhibition on mushroom tyrosinase are shown in Table
2. The inhibitory effects of 2c, 2d, 2i, resveratrol, and kojic acid
Table 1 Reaction yields and tyrosinase inhibitions of the synthesized (Z)-5-(benzylidene)-2-thioxoimidazolidin-4-ones 2a–2la
Compound
R1
R2
R3
R4
Yield (%)
Tyrosinase inhibitionb (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
H
OH
H
OH
H
H
H
H
OMe
H
H
H
H
H
H
OH
OH
OMe
OEt
H
OMe
OMe
OMe
OH
H
OMe
OH
OH
H
OH
OH
OMe
OMe
OH
OMe
H
H
H
H
H
OH
H
H
H
45.0
61.4
64.0
58.1
76.6
52.6
59.4
15.1
80.3
71.7
84.6
52.8
3.78 ꢀ 3.62
42.60 ꢀ 5.09
72.62 ꢀ 0.58
96.54 ꢀ 1.80c
23.73 ꢀ 4.76
46.22 ꢀ 4.95
10.43 ꢀ 3.58
20.45 ꢀ 1.09
58.71 ꢀ 0.35
33.97 ꢀ 3.48
5.47 ꢀ 7.54
19.27 ꢀ 4.75
78.26 ꢀ 2.31
H
OMe
OMe
H
Kojic acid
a
b
Inhibition values represent means ꢀ SEs of three independent experiments. Tyrosinase inhibition was measured using L-tyrosinase as a
substrate at 50 mM. c Tyrosinase inhibition was measured using L-tyrosinase at 25 mM.
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Table 2 IC50 values for tyrosinase inhibition by 2c, 2d, 2i, resveratrol,
and kojic acid
treated cells (186.25%) and untreated controls (100%). In view
of our cytotoxicity results (Fig. 2(a)), the inhibition of melano-
genesis in B16 cells by compound 2d did not appear to be due to
the cytotoxic effect or cell growth inhibition, which suggested
that it could have been caused by the inhibition of murine-
derived tyrosinase by 2d. To identify the mechanism involved,
we examined the inhibitory effect of 2d on cellular tyrosinase
activity using murine-derived tyrosinase. Aer incubation for 24
h with compound 2d, murine-derived tyrosinase activities
decreased dose-dependently, showing 379.82% at 5.0 mM and
332.11% at 20 mM (Fig. 2(c)), compared with 100 nM a-MSH-
only-treated cells (466.06%) and untreated controls (100%),
indicating that the anti-melanogenic effect of 2d was due to its
inhibition of murine-derived tyrosinase.
These results suggest that the heterocyclic thiohydantoin
serves as an effective scaffold for novel tyrosinase inhibitors,
and that 2d is a promising preventative of vegetable and fruit
browning, a pesticide, and a promising skin-whitening agent.
From this point of time, we strongly suggest that considering
these results and our previous experimental data reported, the
“b-phenyl-a,b-unsaturated carbonyl” group might serve as a key
pharmacophore for high tyrosinase inhibition.
Compound Conc. (mM) Tyrosinase inhibitiona (%) IC50b (mM)
2c
1.0
5.0
10.0
25.0
50.0
1.0
31.12 ꢀ 3.05
50.43 ꢀ 3.05
61.67 ꢀ 2.75
70.32 ꢀ 1.04
72.62 ꢀ 0.58
41.79 ꢀ 4.07
62.25 ꢀ 4.53
73.49 ꢀ 2.92
96.54 ꢀ 1.80
39.48 ꢀ 2.59
44.96 ꢀ 4.53
49.28 ꢀ 5.38
53.89 ꢀ 2.25
68.59 ꢀ 5.29
13.67 ꢀ 4.24
16.26 ꢀ 0.75
27.09 ꢀ 7.79
38.05 ꢀ 1.84
47.29 ꢀ 1.07
47.78 ꢀ 1.30
11.70 ꢀ 4.07
44.47 ꢀ 4.07
63.30 ꢀ 4.07
68.30 ꢀ 4.07
74.47 ꢀ 4.07
7.36 ꢀ 1.79
2d
2i
1.07 ꢀ 2.30
5.0
10.0
25.0
1.0
14.12 ꢀ 3.49
5.0
10.0
25.0
50.0
0.5
1.0
5.0
10.0
20.0
30.0
2.0
10.0
20.0
30.0
40.0
Resveratrol
26.63 ꢀ 0.55
14.27 ꢀ 4.07
Kojic acid
Materials and methods
General
a
Values represent means ꢀ SEs of three independent experiments.
1
Melting points are uncorrected. H and 13C NMR spectra were
b
50% inhibitory concentration.
recorded on Varian Unity INOVA 400 and Varian Unity AS 500
instruments. Chemical shis are reported with reference to the
respective residual solvent or deuterated peaks (dH 3.30 and dC
49.0 for CD3OD, dH 7.27 and dC 77.0 for CDCl3). Coupling
constants are reported in hertz. The abbreviations used are as
follows: s (singlet), d (doublet), t (triplet), q (quartet), dd
(doublet of doublets), and brs (broad singlet). All the reactions
described below were performed under argon or nitrogen
atmosphere and monitored by TLC. All anhydrous solvents were
distilled over CaH2 or Na/benzophenone prior to use.
on mushroom tyrosinase were determined using L-tyrosine as a
substrate; all compounds inhibited mushroom tyrosinase in a
concentration-dependent manner. Compounds 2c (IC50 ¼ 7.36
ꢀ 1.79 mM) and 2i (IC50 ¼ 14.12 ꢀ 3.49 mM) were found to be 3.6-
and 1.9-fold more potent, respectively, than resveratrol (IC50
¼
26.63 ꢀ 0.55 mM), a well-known tyrosinase inhibitor, and 2.7-
and 1.4-fold, respectively, more potent than kojic acid (IC50
¼
19.69 ꢀ 4.90 mM), the compound most frequently used as a
General procedure for the synthesis of (Z)-5-(substituted
benzylidene)thiohydantoin analogues (2a–2l). A mixture of
substituted benzaldehydes (1.53–2.46 mmol), 2-thiohydantoin
(1.1 eq.) and sodium acetate (3.0 eq.) in acetic acid (4 mL/1.0 g of
sodium acetate) was reuxed for 4–24 h. Aer cooling, water was
added and the precipitates generated were ltered and washed
with water and ethyl acetate and/or methylene chloride,
depending on the physical properties of the remaining starting
materials, to give (Z)-5-(substituted benzylidene)-2-thio-
xoimidazolidin-4-one derivatives as solids in 15.1–85.2% yields.
(Z)-5-(4-Hydroxybenzylidene)-2-thioxoimidazolidin-4-one
(2a). Green solid; reaction time, 4 h; yield, 45.0%; melting point,
>300 ꢁC; 1H NMR (500 MHz, DMSO-d6) d 12.23 (s, 1H, NH), 11.96
(s, 1H, NH), 10.02 (s, 1H, OH), 7.61 (d, 2H, J ¼ 8.0 Hz, 20-H, 60-H),
6.79 (d, 2H, J ¼ 8.5 Hz, 30-H, 50-H), 6.41 (s, 1H, vinylic H); 13C
NMR (100 MHz, DMSO-d6) d 178.9 (C2), 166.5 (C4), 159.7 (C40),
133.1 (C20, C60), 125.8 (C5), 124.0 (C10), 116.5 (C30, C50), 113.5
(benzylic C); LRMS(ESI) m/z 219 (M ꢂ H)ꢂ.
reference for the evaluation of tyrosinase inhibitors. More
interestingly,
(Z)-5-(2,4-dihydroxybenzylidene)thiohydantoin
(2d, IC50 ¼ 1.07 ꢀ 2.30 mM) was found to be 24- and 18-fold
more potent than resveratrol and kojic acid, respectively.
To evaluate the cytotoxic effects of compound 2d, B16F10
melanoma cells (B16 cells, murine melanoma cells) were used.
The effect of compound 2d on B16 cell viability is presented in
Fig. 2(a). No signicant cytotoxic effect was found up to a
concentration of 20 mM.
Next, effects of 2d on cellular tyrosinase activity and mela-
nogenesis were investigated using B16 cells within 20 mM
concentration at which 2d showed no appreciable cytotoxicity.
To assess the inhibitory effect of compound 2d on melano-
genesis, we quantied the melanin contents of B16 cells treated
with compound 2d (Fig. 2(b)). The melanin content of B16 cells
treated with compound 2d in the presence of a 100 nM
a-melanocyte-stimulating hormone (a-MSH) decreased as the
concentration of 2d was increased, that is, 160.50% at 5.0 mM
and 138.25% at 20 mM, as compared with 100 nM a-MSH-only-
(Z)-5-(2-Hydroxybenzylidene)-2-thioxoimidazolidin-4-one
(2b). Dark greenish yellow solid; reaction time, 4 h; yield, 61.4%;
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Fig. 2 Effects of 2d on cytotoxicity, melanin production and tyrosinase activity in B16 cells. (a) The effect of 2d on the viability of B16 cells. Cells
were treated with various doses of 2d (0–20 mM) and its effect on cell viability was examined by performing MTT assays. Data are expressed as
percentages of non-treated controls. (b) The ability of 2d to inhibit melanogenesis in the presence of 100 nM a-MSH in B16 cells. Melanin levels
were measured at 405 nm. Values represent the means ꢀ SEs of three independent experiments. Data are expressed as percentages of non-
treated controls. ###p < 0.001 vs. untreated controls, *p < 0.05 and ***p < 0.001 vs. cells treated with 100 nM a-MSH. (c) Inhibition of tyrosinase
in B16 cells by 2d. B16 cells were co-treated with 5 or 20 mM of 2d and 100 nM a-MSH for 24 h. Results are expressed as percentages of non-
treated controls, and columns represent the means ꢀ SEs of three independent experiments. ###p < 0.001 vs. non-treated control, *p < 0.05
and ***p < 0.001 vs. cells treated with 100 nM a-MSH.
1
ꢁ
melting point, 283.2–285.1 C; H NMR (500 MHz, DMSO-d6) d 132.6 (C60), 124.7 (C5), 111.8 (C10), 109.3 (bꢂenzylic C), 108.5
12.28 (s, 1H, NH), 11.84 (s, 1H, NH), 10.30 (s, 1H, OH), 7.66 (d, (C50), 102.9 (C30); LRMS(ESI) m/z 235 (M ꢂ H) .
1H, J ¼ 7.5 Hz, 60-H), 7.19 (t, 1H, J ¼ 8.0 Hz, 40-H), 6.87 (d, 1H, J ¼
(Z)-5-(3,4-Dihydroxybenzylidene)-2-thioxoimidazolidin-4-
8.0 Hz, 30-H), 6.82 (t, 1H, J ¼ 7.5 Hz, 50-H), 6.70 (s, 1H, vinylic H); one (2e). Greenish yellow solid; reaction time, 5 h; yield, 76.6%;
13C NMR (100 MHz, DMSO-d6) d 179.0 (C2), 166.4 (C4), 157.0 melting point, >300 ꢁC; 1H NMR (500 MHz, DMSO-d6) d 12.20 (s,
(C20), 131.7 (C40), 131.1 (C60), 127.6 (C5), 120.1 (C10), 120.1 (C50), 1H, NH), 11.93 (s, 1H, NH), 9.63 (s, 1H, OH), 9.04 (s, 1H, OH),
116.2 (benzylic C), 107.9 (C30); LRMS(ESI) m/z 219 (M ꢂ H)ꢂ.
7.09 (dd, 1H, J ¼ 2.0, 8.0 Hz, 60-H), 7.07 (d, 1H, J ¼ 1.5 Hz, 20-H),
(Z)-5-(4-Methoxybenzylidene)-2-thioxoimidazolidin-4-one 6.75 (d, 1H, J ¼ 8.0 Hz, 50-H), 6.32 (s, 1H, vinylic H); 13C NMR
(2c). Green solid; reaction time, 4 h; yield, 64.0%; melting point, (100 MHz, DMSO-d6) d 178.9 (C2), 166.5 (C4), 148.3 (C40), 146.1
1
266.9–267.5 ꢁC; H NMR (400 MHz, DMSO-d6) d 12.26 (s, 1H, (C30), 126.0 (C5), 124.4 (C10), 123.6 (C60), 118.5 (C50), 116.5 (C20),
NH), 12.03 (s, 1H, NH), 7.70 (d, 2H, J ¼ 8.8 Hz, 20-H, 60-H), 6.94 114.1 (benzylic C); LRMS(ESI) m/z 235 (M ꢂ H)ꢂ.
(d, 2H, J ¼ 8.8 Hz, 30-H, 50-H), 6.43 (s, 1H, vinylic H), 3.77 (s, 3H,
(Z)-5-(3,5-Dihydroxybenzylidene)-2-thioxoimidazolidin-4-
40-OCH3); 13C NMR (100 MHz, DMSO-d6) d 179.2 (C2), 166.5 one (2f). Dark brown solid; reaction time, 6 h; yield, 52.6%;
(C4), 160.9 (C40), 132.8 (C20, C60), 126.5 (C5), 125.5 (C10), 115.1 melting point, >300 ꢁC; 1H NMR (500 MHz, DMSO-d6) d 12.29 (s,
(C30, C50), 112.8 (benzylic C), 56.0 (40-OCH3); LRMS(ESI) m/z 233 1H, NH), 12.03 (s, 1H, NH), 9.38 (s, 2H, OH), 6.52 (s, 2H, 20-H, 60-
(M ꢂ H)ꢂ.
H), 6.27 (s, 2H, 40-H, vinylic H); 13C NMR (100 MHz, DMSO-d6) d
(Z)-5-(2,4-Dihydroxybenzylidene)-2-thioxoimidazolidin-4- 179.8 (C2), 166.4 (C4), 159.1 (C30, C50), 134.5 (C10), 128.5 (C5),
one (2d). Yellowish green solid; reaction time, 5 h; yield, 58.1%; 113.1 (benzylic C), 108.9 (C20, C60), 104.7 (C40); LRMS(ESI) m/z
melting point, >300 ꢁC; 1H NMR (500 MHz, DMSO-d6) d 12.13 (s, 235 (M ꢂ H)ꢂ.
1H, NH), 11.65 (s, 1H, NH), 10.30 (s, 1H, OH), 9.92 (s, 1H, OH),
(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-2-thioxoimidazo-
7.56 (d, 1H, J ¼ 8.5 Hz, 60-H), 6.68 (s, 1H, vinylic H), 6.34 (d, 1H, J lidin-4-one (2g). Dark yellow solid; reaction time, 4 h; yield,
¼ 2.0 Hz, 30-H), 6.27 (dd, 1H, J ¼ 2.0, 8.5 Hz, 50-H); 13C NMR (100 59.4%; melting point, ꢁC; 1H NMR (500 MHz, DMSO-d6) d 12.13
MHz, DMSO-d6) d 177.7 (C2), 166.5 (C4), 161.4 (C40), 158.9 (C20), (br s, 2H, 2 ꢃ NH), 9.15 (br s, 1H, OH), 7.27 (s, 1H, 20-H), 7.25 (d,
1H, J ¼ 8.5 Hz, 60-H), 6.79 (d, 1H, J ¼ 8.0 Hz, 50-H), 6.37 (s, 1H,
1414 | Med. Chem. Commun., 2014, 5, 1410–1417
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vinylic H), 3.83 (s, 3H, 30-OCH3); 13C NMR (100 MHz, DMSO-d6) d acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] and a-MSH
179.1 (C2), 167.2 (C4), 149.1 (C30), 148.4 (C40), 127.3 (C5), 125.4 (alpha-melanocyte stimulating hormone) were purchased from
(C60), 124.8 (C10), 116.4 (C50), 114.7 (C20), 113.6 (benzylic C), 56.6 Sigma Chemical Co (St. Louis, MO, U.S.A.). Solvents used for
(30-OCH3); LRMS(ESI) m/z 249 (M ꢂ H)ꢂ.
organic syntheses were redistilled. All other chemicals and
(Z)-5-(3-Ethoxy-4-hydroxybenzylidene)-2-thioxoimidazolidin- solvents were of analytical grade and used without further
4-one (2h). Yellow solid; reaction time, 24 h; yield, 15.1%; purication.
1
ꢁ
melting point, 175.4–177.2 C; H NMR (400 MHz, DMSO-d6) d
12.22 (br s, 1H, NH), 12.01 (br s, 1H, NH), 9.54 (s, 1H, OH), 7.20 Cell culture
(d, 1H, J ¼ 8.8 Hz, 60-H), 7.20 (s, 1H, 20-H), 6.79 (d, 1H, J ¼ 8.8 Hz,
B16 cells (obtained from the Korean Cell Line Bank) were
cultured in DMEM with 10% fetal bovine serum (FBS; Gibco)
50-H), 6.39 (s, 1H, vinylic H), 4.08 (q, 2H, J ¼ 6.8 Hz, 30-OCH2),
1.31 (t, 3H, J ¼ 6.8 Hz, CH2CH3); 13C NMR (100 MHz, DMSO-d6)
and penicillin/streptomycin (100 IU/50 mg mLꢂ1) in a humidi-
d 179.0 (C2), 166.5 (C4), 149.6 (C30), 147.6 (C40), 125.8 (C5), 125.5
ꢁ
ed atmosphere containing 5% CO2 in air at 37 C. B16 cells
(C60), 124.4 (C10), 116.5 (C50), 116.0 (C20), 114.1 (benzylic C), 64.8
were cultured in 24-well plates for melanin quantication and
enzyme activity assays.
(30-OCH2), 15.3 (CH2CH3); LRMS(ESI) m/z 263 (M ꢂ H)ꢂ.
(Z)-5-(2,4-Dimethoxybenzylidene)-2-thioxoimidazolidin-4-
one (2i). Green solid; reaction time, 4 h; yield, 80.3%; melting
Cell viability
point, 237.1–238.6 ꢁC; 1H NMR (500 MHz, DMSO-d6) d 12.23 (br
Cell survival was quantied by a colorimetric MTT assay that
s, 1H, NH), 11.93 (br s, 1H, NH), 7.73 (d, 1H, J ¼ 8.5 Hz, 60-H),
measures mitochondrial activity in viable cells. This method is
based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-
6.68 (d, 1H, J ¼ 1.5 Hz, 30-H), 6.60 (s, 1H, vinylic H), 6.57 (dd, 1H,
J ¼ 2.0, 8.5 Hz, 50-H), 3.85 (s, 3H, OCH3), 3.82 (s, 3H, OCH3); 13C
diphenyltetrazolium bromide (MTT, Sigma) to MTT-formazan
NMR (100 MHz, DMSO-d6) d 178.8 (C2), 166.5 (C4), 162.8 (C40),
crystal by mitochondrial enzymes as previously described.21
159.9 (C20), 132.0 (C60), 126.4 (C5), 114.3 (C10), 107.2 (benzylic
Briey, cells seeded at a density of 3 ꢃ 104 per cell in a Corning
C), 106.6 (C50), 98.8 (C30), 56.5 (20-OCH3), 56.2 (40-OCH3);
LRMS(ESI) m/z 263 (M ꢂ H)ꢂ.
48-well plate (Corning, NY, USA), were allowed to adhere over-
night; the culture medium was then replaced with fresh serum
(Z)-5-(3,4-Dimethoxybenzylidene)-2-thioxoimidazolidin-4-
free DMEM. MTT was freshly prepared at 5 mg mLꢂ1 in phos-
one (2j). Greenish yellow solid; reaction time, 4 h; yield, 71.7%;
1
phate-buffered saline (PBS). Aliquots of 500 mL of MTT stock
solution were added to each well, and the plate was incubated at
37 ꢁC for 4 hours in a humidied 5% CO2 incubator. Aer 4
hours, the medium was removed. To each well, 500 mL of EtOH–
DMSO (1 : 1 mixture solution) was added to dissolve formazan.
Aer 10 min, the optical density of each well was measured
spectrophotometrically with a 560 nm lter. Results from three
experiments are shown.
ꢁ
melting point, 236.2–238.0 C; H NMR (500 MHz, DMSO-d6) d
12.30 (br s, 1H, NH), 12.14 (br s, 1H, NH), 7.35 (dd, 1H, J ¼ 2.0,
9.0 Hz, 60-H), 7.23 (d, 1H, J ¼ 2.0 Hz, 20-H), 6.98 (d, 1H, J ¼ 8.5
Hz, 50-H), 6.45 (s, 1H, vinylic H), 3.83 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3); 13C NMR (100 MHz, DMSO-d6) d 179.3 (C2), 166.5 (C4),
150.9 (C30), 149.5 (C40), 126.5 (C5), 125.7 (C10), 125.0, (C60) 114.0
(benzylic C), 113.4 (C50), 112.4 (C20), 56.5 (OCH3), 56.2 (OCH3);
LRMS(ESI) m/z 263 (M ꢂ H)ꢂ.
(Z)-5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2-thioxoimidazo-
lidin-4-one (2k). Yellowish green solid; reaction time, 4 h; yield,
84.6%; melting point, 240.1–242.6 ꢁC; 1H NMR (500 MHz,
Assay to measure inhibitory effects of compounds on
mushroom tyrosinase activity
DMSO-d6) d 12.15 (br s, 1H, NH), 11.98 (br s, 1H, NH), 9.10 (br s, Mushroom-derived tyrosinase was used as the source of the
1H, OH), 7.57 (s, 2H, 20-H, 60-H), 6.50 (s, 1H, vinylic H), 3.76 (s, enzyme for the entire study. Tyrosinase activity was determined
6H, 30-OCH3, 50-OCH3); 13C NMR (100 MHz, DMSO-d6) d 174.9 as described previously with minor modication.20 Briey, 20 mL
(C2), 164.2 (C4), 148.1 (C30, C50), 138.8 (C40), 128.5 (C5), 123.7 of an aqueous solution of mushroom tyrosinase (1000 units)
(C10), 121.9 (benzylic C), 109.5 (C20, C60), 56.6 (30-OCH3); was added to a 96-well microplate (Nunc, Denmark), in a total
LRMS(ESI) m/z 279 (M ꢂ H)ꢂ.
assay mixture volume of 200 mL containing 1 mM L-tyrosine
solution, and 50 mM phosphate buffer (pH 6.5). The assay
(Z)-2-Thioxo-5-(3,4,5-trimethoxybenzylidene)imidazolidin-4-
one (2l). Pale brown solid; reaction time, 4 h; yield, 52.8%; mixture was incubated at 25 ꢁC for 30 minutes. Following
1
ꢁ
melting point, 264.0–266.6 C; H NMR (500 MHz, DMSO-d6) d incubation, the amount of dopachrome produced in the reac-
12.25 (br s, 1H, NH), 12.08 (br s, 1H, NH), 7.54 (s, 2H, 20-H, 60-H), tion mixture was determined spectrophotometrically at 492 nm
6.52 (s, 1H, vinylic H), 3.79 (s, 6H, 30-OCH3, 50-OCH3), 3.69 (s, (OD492) using a microplate reader (Hewlett Packard). IC50
,
3H, 40-OCH3); 13C NMR (100 MHz, DMSO-d6) d 175.9 (C2), 164.2 inhibitory concentration-50, is the concentration of a drug that
(C4), 153.1 (C30, C50), 139.6 (C40), 130.1 (C10), 128.7 (C5), 120.3 inhibits a standard 50% response. IC50 is a value derived from
(benzylic C), 109.0 (C20, C60), 60.8 (40-OCH3), 56.5 (30-OCH3, 50- the X-axis and is determined from the alignment of the dose–
OCH3); LRMS(ESI) m/z 293 (M ꢂ H)ꢂ.
response curve on the dependent Y-axis. In the present study,
dose-dependent inhibition experiments were performed in
triplicate to determine the IC50 of a drug. According to the
inhibition percentage of three doses in each experiment, log-
Materials
Mushroom tyrosinase, L-tyrosine [3-(4-hydroxyphenyl)]-L- linear curves and their equations were determined. Then, the
alanine, (S)-2-amino-3-(4-hydroxyphenyl)propionic acid, kojic individual IC50 was calculated as the concentration at which the
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Y-axis value equaled 50% inhibition. Results are shown from and 2-thiohydantoin. The inhibitory effects of the synthesized
three experiments.
thiohydantoin analogs on mushroom tyrosinase were assessed
using kojic acid and resveratrol as positive reference
compounds. Compounds with a thiohydantoin template were
found to have a tyrosinase inhibitory pattern that differed from
that of compounds with a hydantoin, pyrrolidinedione, or
thiazolidine scaffold, presumably due to the inability of the
thiocarbonyl group of thiohydantoin to form a hydrogen bond.
Of the twelve compounds synthesized, compounds 2c, 2d, and
2i were found to have greater inhibitory effects than kojic acid
or resveratrol. In particular, (Z)-5-(2,4-dihydroxybenzylidene)-2-
thiohydantoin (2d) was found to be a 24- and 18-fold more
potent tyrosinase inhibitor than resveratrol or kojic acid,
respectively, and to inhibit melanin production through the
inhibition of tyrosinase activity in B16 cells without exhibiting a
cytotoxic effect. These results suggest that 2d should be regar-
ded a lead compound for the development of safer, more potent
vegetable and food browning preventatives and skin-lightening
medicines. Considering the tyrosinase inhibitory activities of 5-
Assay of murine tyrosinase activity
Murine tyrosinase activity was estimated by measuring the rate
of oxidation of L-DOPA.22 Cells were plated in 24-well dishes at a
density of 5ꢃ104 cells per mL. B16 cells were incubated in the
presence or absence of 100 nM a-MSH and then treated for 24
hours at various concentrations of (Z)-5-(2,4-dihydroxy-
benzylidene)-2-thioxoimidazolidin-4-one (2d) (0–20 mM). Cells
were washed and lysed in 100 mL of 50 mM sodium phosphate
buffer (pH 6.5) containing 1% Triton X-100 (sigma) and 0.1 mM
PMSF (phenylmethylsulfonyl uoride) and then frozen at
ꢁ
ꢂ80 C for 30 min. Aer thawing and mixing, cellular extracts
were claried by centrifugation at 12 000 rpm for 30 min at 4 ꢁC.
Eight mL of the supernatant and 20 mL of L-DOPA (2 mg mLꢂ1
)
were put in a 96-well plate, and the absorbance at 492 nm was
read every 10 min for 1 hour at 37 ꢁC using an ELISA plate
reader. The nal activity was expressed as DO.D. per min for
each condition. Results from three experiments are shown.
(substituted
benzylidene)thiohydantoin
derivatives,
we
consider the thiohydantoin template a near perfect surrogate
for hydantoin, pyrrolidinedione, and thiazolidinedione scaf-
folds in the context of inhibiting tyrosinase activity. In addition,
these results proved in part that the “b-phenyl-a,b-unsaturated
carbonyl” group might be an essential template for great
tyrosinase inhibition.
Determination of melanogenesis in B16 cells
Determination of the melanin content was done using a
modication of the method reported by Bilodeau et al.23 The
amount of melanin was used as an index of melanogenesis. B16
cells (5 ꢃ 104) were plated on 24-well dishes and incubated in
the presence or absence of 100 nM a-MSH. Cells were then
incubated for 24 hours with various concentrations of (Z)-5-(2,4-
dihydroxybenzylidene)-2-thioxoimidazolidin-4-one (2d) (0–20
mM). Aer washing twice with PBS, samples were dissolved in
100 mL of 1 N NaOH. The samples were incubated at 60 ꢁC for 1
hour and mixed to solubilize the melanin. The absorbance at
405 nm was compared with a standard curve of synthetic
melanin. Results from three experiments are shown.
Acknowledgements
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korean government (MSIP)
(Grant no. 2009-0083538) and by the Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Tech-
nology (Grant no. NRF-2013R1A1A2009949).
Statistical analysis
The inhibition of tyrosinase activity is expressed as a percentage
of inhibition based on: 100 ꢂ [(A ꢃ 100)/B], where A¼ OD492
with a test sample and B ¼ OD492 without a test sample. Data
collected are presented as means ꢀ standard errors (n ¼ 3). The
statistical signicance of differences between groups was
determined by one-factor analysis of variance (ANOVA) followed
by Fischer's protected least signicant difference post hoc test.
Values of *p < 0.05 were considered statistically signicant.
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