Evaluation of dihydropyrimidin-(2H)-one analogues and rhodanine derivatives as tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.02.076

A series of dihydropyrimidin-(2H)-one analogues and rhodanine derivatives were synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that some of the synthesized compounds exhibited

Full text of DOI:10.1016/j.bmcl.2011.02.076

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2376–2379
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of dihydropyrimidin-(2H)-one analogues and rhodanine
derivatives as tyrosinase inhibitors
Jinbing Liu a, , Fengyan Wu a, Lingjuan Chen b, Jianming Hu a, Liangzhong Zhao a,
⇑
Changhong Chen a, Liwang Peng a
a Department of Biology and Chemical Engineering, Shaoyang University, Shao Shui Xi Road, Shaoyang 422100, People’s Republic of China
b College of Food Science and Engineering, Central South University of Forestry and Technology, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dihydropyrimidin-(2H)-one analogues and rhodanine derivatives were synthesized and their
inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results
showed that some of the synthesized compounds exhibited significant inhibitory activities. Especially,
compound 15 bearing a hydroxyethoxyl group at position-4 of phenyl ring exhibited most potent tyros-
inase inhibitory activity with IC50 value of 0.56 mM. The inhibition mechanism analysis of compound 15
demonstrated that the inhibitory effect of the compound on the tyrosinase was irreversible. These results
suggested that such compounds might be served as lead compounds for further designing new potential
tyrosinase inhibitors.
Received 9 December 2010
Revised 16 February 2011
Accepted 21 February 2011
Available online 26 February 2011
Keywords:
Dihydropyrimidin-(2H)-one
Rhodanine
Tyrosinase inhibitors
Inhibition mechanism
Ó 2011 Elsevier Ltd. All rights reserved.
Dihydropyrimidinones (DHPMs) and their derivatives have at-
tracted interest in medicinal chemistry, exhibiting pharmacologi-
cal and therapeutic properties. In the past decades, a broad
range of biological effects including antiviral, antitumor, antibac-
terial and anti-inflammatory activities have been described for
these compounds.1,2 Compounds containing the 2-thioxothiazoli-
din-4-one ring have showed a wide range of pharmacological
activities, which includes antimicrobial, antiviral, and anticonvul-
sant effects.3 Prominent amongst the resultant hits was a series of
S
S
NH
O
Figure 1. The substructure of 5-benzylidenerhodanine.
nones (diphenolase).6 Due to the high reactivity, quinines could
polymerize spontaneously to form high molecular weight brown-
pigments (melanins) or react with amino acids and proteins to en-
hance brown color of the pigment produced.7,8 Previous reports
confirmed that tyrosinase not only was involved in melanizing in
animals, but also was one of the main causes of most fruits and
vegetables quality loss during post harvest handling and process-
ing, leading to faster degradation and shorter shelf life.9 Recently,
investigation demonstrated that various dermatological disorders,
such as age spots and freckle, were caused by the accumulation of
an excessive level of epidermal pigmentation.10,11 Tyrosinase has
also been linked to Parkinson’s and other neurodegenerative dis-
eases.12 In insects, tyrosinase is uniquely associated with three dif-
ferent biochemical processes, including sclerotization of cuticle,
defensive encapsulation and melanization of foreign organism,
and wound healing.13 These processes provide potential targets
for developing safer and effective tyrosinase inhibitors as insecti-
cides and ultimately for insect control. Thus, the development of
safe and effective tyrosinase inhibitors is of great concern in the
medical, agricultural, and cosmetic industries. However, only a
few such as kojic acid, arbutin, tropolone, and 1-phenyl-2-thiourea
derivatives containing
a 5-benzylidenerhodanine substructure
(Fig. 1). Benzylidenerhodanines have been reported as small mol-
ecule inhibitors of numerous targets such as cyclooxygenase and
5-lipoxygenase, b-lactamase, cathepsin D, HCV NS3 protease, al-
dose reductase, protein mannosyl transferase and JNK-stimulating
phosphatase.4 Although, during the last years extensive studies on
the pharmacology of DHPMs and rhodanine derivatives have been
reported, the tyrosinase inhibitor activities of this kind of
compounds have never appeared in the literature.
Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase,
EC 1.14.18.1, syn. polyphenol oxidase), also known as polyphenol
oxidase (PPO), is a copper-containing monooxygenase that is
widely distributed in microorganisms, animals, and plants.5 Tyros-
inase could catalyze two distinct reactions involving molecular
oxygen in the hydroxylation of monophenols to o-diphenols
(monophenolase) and in the oxidation of o-diphenols to o-qui-
⇑
Corresponding author. Tel.: +86 739 5431768; fax: +86 739 5431768.
E-mail address: xtliujb@yahoo.com.cn (J. Liu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.076

J. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2376–2379
2377
O
O
OH
OH
OH
S
O
O
OH
N
H
NH2
HO
HO
HO
O
OH
arbutin
1-phenylthiourea
tropolone
kojic acid
Figure 2. Chemical structure of known tyrosinase inhibitors.
(PTU) (Fig. 2) are used as therapeutic agents and cosmetic
products.11,14
substances. Figure 3 showed that the remaining enzyme activity
rapidly decreased with the increasing concentrations of compound
15. The IC50 values of dihydropyrimidin-(2H)-one analogues and
rhodanine derivatives against tyrosinase were summarized in
Table 1, and IC50 values of all these compounds were determined
from logarithmic concentration–inhibition curves (at least eight
points) and are given as means of three experiments .
Our results showed that compounds 7–16 exhibited potent
inhibitory on mushroom tyrosinase with IC50 values ranged from
0.56 to 25.11 mM. Some of the synthesized compounds demon-
strated more potent inhibitory activities than the reference stan-
dard inhibitor arbutin. Compounds 1–6 and 17 did not show any
significant activities at 50 mM concentration. The result may be re-
lated to the structure of tyrosinase contained a type-3 copper cen-
ter with a coupled dinuclear copper active site in the catalytic core.
Tyrosinase inhibition of compounds 7–16 depended on the compe-
tency of the sulfur atom to chelate with the dicopper nucleus in the
active site, and tyrosinase would lose its catalyzing ability after
forming complex.
Comparing to the tyrosinase inhibitory activities of 3,4-dihydro-
pyrimidin-2-(1H)-thione analogs, compounds with hydroxyl group
in the benzene ring exhibited more potent inhibitory activities.
This suggested that hydroxyl group might play an important role
in determining their inhibitory activities. The result was consistent
with the Kim’s propose that the hydroxyl groups might inhibit the
tyrosinase enzyme by participating in a metal–ligand binding
interaction with the dicopper nucleus.27 The introduction of a hy-
droxy group in the para position led to appreciably better inhibi-
tory activity than the hydroxyl group in the ortho position, when
the substituents introduced in the meta position on the phenyl
rings may have hindered docking of the inhibitor to tyrosinase,
and led to the decrease in inhibitory activities. Interestingly, com-
pound 10 was found to be the most potent inhibitor with an IC50
value of 10.67 mM. These results suggested that benzene ring
might cause stereohindrance for the inhibitors approaching the ac-
tive site of the enzyme.
On the other hand, it was reported that phenyl thioureas and al-
kyl thioureas could exhibit weak to moderate depigmenting activ-
ity15 and Ley and Bertram reported that benzaldoximes and
benzaldehyde-o-alkyloximes possess higher tyrosinase inhibitory
ability.16 Tyrosinase belongs with catechol oxidase to the type-3
copper protein group. Crystal structure of phenyl thioureas bound
catechol oxidase was reported. The sulfur atom of this compound
binds to both copper ions in the active site of the enzyme.8 More
recently, our investigation also demonstrated that condensation
products of aldehyde or ketone and thiosemicarbazide derivatives
exhibited potent inhibitory activities against mushroom tyrosi-
nase.17,18 Stimulated by these results, in the present investigation,
we synthesized a series of dihydropyrimidin-(2H)-ones analogues
and rhodanine derivatives, and their inhibitory effects on the
diphenolase activity of mushroom tyrosinase were investigated.
According to the general procedure shown in Scheme 1,19,20 we
carried out the preparation of 3,4-dihydropyrimidin-2-(1H)-ones
(DHPMs) and thione analogs from the corresponding aldehydes,
pentane-2,4-dione and urea or thiourea in the presence of magne-
sium bromide under solvent-free conditions. The synthesis is usu-
ally complete within 0.5–3 h at 50 °C or 100 °C, and the target
compounds could be purified by recrystallization from 95% alcohol.
The yields for these reactions are from moderate to good, and all the
target compounds were characterized by IR, NMR and MS. The Schiff
base of 3,4-dihydropyrimidin-2(1H)-one was prepared according to
Scheme 2.21 Rhodanine derivatives were synthesised for this study
by means of the Knoevenagel condensation ofthe suitable aldehydes
with 2-thioxo-4-thiazolidinone, respectively, in refluxing acetic acid
in the presence of sodium acetate (Scheme 3).22,23 The target com-
pounds were also characterized by IR, NMR and MS.
For evaluating the tyrosinase inhibitory activity, all the synthe-
sized compounds were subjected to tyrosinase inhibition assay
with L-DOPA as substrate, according to the method reported by
our groups with some slight modifications.9,18 The tyrosinase
inhibitory activities of arbutin and 4-methoxycinnamic acid
were ever reported, therefore, they were selected as comparing
As shown in Table 1, rhodanine derivatives exhibited more
potent inhibitor activities than dihydropyrimidin-(2H)-ones
O
R
X
O
O
O
CH
MgBr2
NH
+
+
H2N
NH2
R
N
H
X
1. R = Ph, X = O
2. R = 4-OHPh, X = O
4. R = 2-OHPh, X = O
6. R = Et, X = O
3. R = 4-MeOPh, X = O
5. R = 4-OH-3-MeOPh, X = O
7. R = 2-OHPh, X = S
8 .R = 4-OH-3-MeOPh, X = S
10. R = Et, X = S
9. R = 4-MeOPh, X = S
11. R = 4-OHPh, X = S
Scheme 1. Synthesis of 3,4-dihydropyrimidin-2-(1H)-ones (DHPMs) and thione analogs. Reagents and conditions: MgBr2, 50 °C or 100 °C, 0.5–3 h.

2378
J. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2376–2379
O
O
O
O
MgBr2
NH
+
+
O
H2N
NH2
i
N
H
O
O
N
CH3CH2ONH2
ii
NH
N
H
O
Scheme 2. Synthesis of the Schiff base of 3,4-dihydropyrimidin-2-(1H)-ones (DHPMs). Reagents and conditions: (i) MgBr2, 50 °C, 3 h. (ii) anhydrous ethanol, rt, 24 h.
O
O
NH
O
NH
S
S
R
S
+
S
R
12. 4-OH;
13. 3,4-(HOCH2CH2O)2;
14. 2-OH;
15. 4-HOCH2CH2O; 16. 4-OH-3-Me
Scheme 3. Synthesis of Rhodanine derivatives. Reagents: acetic acid, sodium acetate, reflux.
Table 1
Tyrosinase inhibitory activities and yields of the synthesized compounds
a
Compounds
Yield (%)
IC50 (mM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
58.5
48.9
55.0
53.7
87.2
57.0
51.2
52.6
58.4
47.2
70.1
33.2
35.8
23.6
35.3
37.2
77.7
NAb
NAb
NAb
NAb
NAb
NAb
16.25
20.12
25.11
10.67
12.25
10.06
5.69
9.55
0.56
11.08
17
NAb
4-Methoxycinnamic acid
Arbutin
0.41 (mM)c
10.40 (mM)d
Figure 3. Effect of compound 15 on the diphenolase activity of mushroom
tyrosinase for the catalysis of L-DOPA at 25 °C.
a
Values were determined from logarithmic concentration–inhibition curves (at
least eight points) and are given as means of three experiments
b
Not active at 50 mM concentration.
analogues, which indicated that the smaller size of the ring in vol-
ume would be beneficial to the molecules to approach the active
centre of the enzyme. Compound 15 bearing a hydroxyethoxyl
group at position-4 of phenyl ring exhibited most potent tyrosi-
nase inhibitory activity with IC50 value of 0.56 mM. These results
suggested that introducing appropriate hydrophobic subunits into
position-4 of benzaldehyde might be facilitated their inhibitory ef-
fects on the diphenolase activity of mushroom tyrosinase. Interest-
ingly, hydroxyethoxyl group introduced to the position-3,4 of
phenyl ring (compound 13, IC50 = 5.69 mM), led to a dramatic de-
crease in inhibitory activities. These results further confirmed that
the substituents introduced in the meta position on the phenyl
rings may hinder the complex formation between inhibitors unit
and the metallic center of the active site in enzyme due to the ste-
ric hindrance.
c
IC50 values in the literature is 0.34–0.43 mM.24,25
d
The concentration of 10.4 mM corresponding to inhibition percentage, deter-
mined in this work, is 30%. The reported IC50 value of arbutin was more than
30 mM.26
concentration in the presence of different concentration of the
above-mentioned compound. The results displayed that the plots
of V versus [E] gave a family of parallel straight lines with the same
slopes. These results demonstrated that the inhibitory effect of
compound 15 on the tyrosinase was irreversible, suggesting that
2-thioxothiazolidin-4-one moiety of 5-(4-(2-hydroxyethoxy)-
benzylidene)-2-thioxothiazolidin-4-one effectively inhibited the
enzyme by binding to its binuclear active site irreversibly. The re-
sult may be related to the structure of tyrosinase, within the struc-
ture, there are two copper ions in the active centre of tyrosinase
and a lipophilic long-narrow gorge near to the active centre.
Hydrophobic subunit and sulfur atom exist in the structure of
The inhibitory mechanism of the selected compound 15 on
mushroom tyrosinase for the oxidation of L-DOPA was determined.
Figure 4 showed the relationship between enzyme activity and

J. Liu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2376–2379
2379
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.076.
References and notes
1. Kappe, C. O. Eur. J. Med. Chem. 2000, 35, 1043.
2. Liang, B.; Wang, X.; Wang, J. X. Tetrahedron 2007, 63, 1981.
3. Sortino, M.; Delgado, P. Bioorg. Med. Chem. 2007, 15, 484.
4. Irvine, M. W.; Patrick, G. L. Bioorg. Med. Chem. Lett. 2008, 18, 2032.
5. Song, K. K.; Huang, H.; Han, P.; Zhang, C. L.; Shi, Y.; Chen, Q. X. Biochem. Biophys.
Res. Commun. 2006, 342, 1147.
6. Chen, Q. X.; Liu, X. D.; Huang, H. Biochemistry (Moscow) 2003, 68, 644.
7. Kubo, I.; Nihei, K. I.; Shimizu, K. Bioorg. Med. Chem. Lett. 2004, 12, 5343.
8. Matsuura, R.; Ukeda, H.; Sawamura, M. J. Agric. Food. Chem. 2006, 54, 2309.
9. Yi, W.; Cao, R. Eur. J. Med. Chem. 2010, 45, 639.
10. Gupta, A. K.; Gover, M. D.; Nouri, K. N.; Taylor, S. J. Am. Acad. Dermatol. 2006, 55,
1048.
11. Thanigaimalai, P.; Hoang, T. A. L. Bioorg. Med. Chem. Lett. 2010, 20, 2991.
12. Zhu, Y. J.; Zhou, H. T.; Hu, Y. H. Food Chem. 2011, 124, 298.
13. Ashida, M.; Brey, P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10698.
14. Battaini, G.; Monzani, E.; Casella, L.; Santagostini, L.; Pagliarin, R. J. Biol. Inorg.
Chem. 2000, 5, 262.
Figure 4. The effect of concentrations of tyrosinase on its activity for the catalysis
of -DOPA at different concentration of compound 15. The concentrations of
L
15. Klabunde, T.; Eicken, C. Nat. Struct. Biol. 1998, 5, 1084.
16. Ley, J. P.; Bertram, H. J. Bioorg. Med. Chem. 2001, 9, 1879.
17. Liu, J. B.; Yi, W.; Wan, Y. Q.; Ma, L.; Song, H. C. Bioorg. Med. Chem. 2008, 14,
1096.
compound 15 for curves 1–4 are 0, 0.1, 0.2 and.3 0 mmol/L, respectively.
18. Liu, J. B.; Cao, R. H.; Yi, W.; Ma, C. M.; Wan, Y. Q.; Zhou, B. H.; Ma, L.; Song, H. C.
Eur. J. Med. Chem. 2009, 44, 1773.
compound 15, the compound could exhibit strong affinity for cop-
per ions in the active centre and form a reversible non-covalent
complex with the tyrosinase, and this then reacts to produce the
covalently modified ‘dead-end complex’.
19. Ahmed, N.; Lier, J. E. Tetrahedron Lett. 2007, 48, 5407.
20. General procedures for the synthesis of 3,4-dihydropyrimidin-2-(1H)-ones
(DHPMs) compounds: To a 50 mL flame dried round-bottom flask were added
20 mmol of benzaldehyde, 20 mmol of acetophenone, 20 mmol of urea or
thiourea and 3.5 mmol of MgBr2. The resulting mixture was heated to 100 °C.
After completion of the reaction as indicated by TLC, and then the mixture was
cooled to room temperature and poured into 50 mL ice-water. The solid
product was filtered, washed with ice-water and ethanol (95%), dried, and
recrystallized from 95% ethanol. Compound 11: Yellow solid power, mp 279.6–
The present investigation reported that dihydropyrimidin-
(2H)-one analogues and rhodanine derivatives had potent inhibi-
tory effects on the diphenolase activity of mushroom tyrosinase.
Interestingly, compound 15 was found to be the most potent inhib-
itor with IC50 value of 0.56 mM. Preliminary structure–activity
relationships (SARs) analysis indicated that (1) hydroxyl group in
the benzene ring might play an important role in determining their
inhibitory activities, big groups might cause stereohindrance for
the inhibitors approaching the active site of the enzyme resulting
in the decrease of inhibitory activities, the substituents introduced
in the meta position of the phenyl rings may have hindered docking
of the inhibitor to tyrosinase; (2) the smaller size of the ring in vol-
ume would be beneficial to the molecules to approach the active
centre of the enzyme; (3) rhodanine derivatives bearing a hydroxy-
ethoxyl group at position-4 of phenyl ring might be beneficial to
increase the inhibitory activities. The inhibition mechanism analy-
sis of compound 15 demonstrated that the inhibitory effect of the
compound on the tyrosinase was irreversible.
281.3; IR (KBr, cmÀ1
) m: 3267, 2998, 2889, 1608, 1566, 1505, 1463, 1377, 1329,
1252, 1191, 832; 1H NMR (300 MHz, DMSO-d6): d 10.54(bs, 1H, NH), 9.38 (br s,
1H, NH), 9.16 (s, 1H, OH), 7.10 (d, J = 8.4 Hz, 2H, Ph), 6.71 (d, J = 8.4 Hz, 2H, Ph),
5.19(d, J = 2.7 Hz, 1H, CH), 2.31 (s, 3H, CH3), 2.24 (s, 3H, CH3); 13C NMR
(75 MHz, DMSO-d6): d 196.5, 175.1, 158.9, 149.2, 135.8, 128.5, 115.8, 108.1,
55.8, 31.2, 19.1; MS (ESI): m/z (100%) 263 (M+1).
21. Liu, J. B.; Cao, R. H.; Wang, Z. H.; Peng, W. L.; Song, H. C. Eur. J. Med. Chem. 2009,
44, 1737.
22. Irvine, M. W.; Patrick, G. L.; Kewney, J. Bioorg. Med. Chem. Lett. 2008, 18, 2032.
23. General procedures for the synthesis of Rhodanine derivatives: The mixture of 2-
thioxo-4-thiazolidinone (2.48 mmol), suitable aldehydes (2.54 mmol) and
sodium acetate (7.32 mmol) in acetic acid (30 mL) was heated to reflux for
3–6 h. The reaction was monitored by TLC. After completion of the reaction,
upon cooling of the mixture to room temperature, a precipitate formed which
was collected by filtration, washed with cold ethanol, and dried under vacuum
to provide the target compound. Compound 15: Yellow solid power, mp 197.7–
198.4; IR (KBr, cmÀ1
) m: 3068, 2837, 1726, 1684, 1579, 1502, 1438, 1230, 1201,
1172, 1060, 800; 1H NMR (300 MHz, CDCl3): d 7.63(s, 1H, @CH), 7.46 (d,
J = 8.7 Hz, 2H, Ph), 7.03 (d, J = 8.7 Hz, 2H, Ph), 4.75 (t, J = 4.5 Hz, 2H, OCH2), 4.26
(t, J = 4.5 Hz, 2H, OCH2); 13C NMR (75 MHz, CDCl3): d 196.2, 170.0, 161.8, 143.7,
129.6, 126.4, 117.0, 114.8, 70.3, 60.8; MS (ESI): m/z (100%) 282 (M+1).
24. Shi, Y.; Chen, Q. X.; Wang, Q.; Song, K. K.; Qiu, L. Food Chem. 2005, 92, 707.
25. Lee, H. S. J. Agric. Food. Chem. 2002, 50, 1400.
Acknowledgments
26. Kazuhisa, S.; Koji, N.; Takahisa, N.; Taro, K.; Kenji, S. J. Biosci. Bioeng. 2005, 99,
272.
27. Kim, D.; Park, J.; Kim, J.; Han, C.; Yoon, J.; Kim, N.; Seo, J.; Lee, C. J. Agric. Food.
Chem. 2006, 54, 935.
This work was financially supported by the Foundation of
Education Department of Hunan Province, China (09B091), and
the Key Subject Foundation of Shaoyang University (2008XK201).