Design, synthesis and biological evaluation of hydroxy- or methoxy-substituted 5-benzylidene(thio) barbiturates as novel tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.04.060

Here a new class of hydroxy- or methoxy-substituted 5-benzylidene(thio) barbiturates were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that several compounds had more potent tyrosinase inhibitory activities than the widely used tyrosinase inhibitor kojic acid (IC₅₀ = 18.25 μM). In particular, 3′,4′-dihydroxylated 1e was found to be the most potent inhibitor with IC₅₀ value of 1.52 μM. The inhibition mechanism analysis revealed that the potential compounds 1e and 2e exhibited such inhibitory effects on tyrosinase by acting as the irreversible inhibitors. Structure-activity relationships' (SARs) analysis also suggested that further development of such compounds might be of interest.

Full text of DOI:10.1016/j.bmc.2014.04.060

Accepted Manuscript
Design, synthesis and biological evaluation of hydroxy- or methoxy-substituted
5-benzylidene(thio) barbiturates as novel tyrosinase inhibitors
Zhiyong Chen, Dachuan Cai, Dehai Mou, Qin Yan, Yifeng Sun, Wenlong Pan,
Yiqian Wan, Huacan Song, Wei Yi
PII:
S0968-0896(14)00334-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.04.060
BMC 11555
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 March 2014
26 April 2014
28 April 2014
Please cite this article as: Chen, Z., Cai, D., Mou, D., Yan, Q., Sun, Y., Pan, W., Wan, Y., Song, H., Yi, W., Design,
synthesis and biological evaluation of hydroxy- or methoxy-substituted 5-benzylidene(thio) barbiturates as novel
tyrosinase inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.04.060
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Design, synthesis and biological evaluation of hydroxy- or
methoxy-substituted 5-benzylidene(thio) barbiturates
as novel tyrosinase inhibitors
Zhiyong Chena, Dachuan Caia, Dehai Moua, Qin Yanb, Yifeng Suna, Wenlong Pana, Yiqian Wanb,
Huacan Song b*, Wei Yib*
aGuangdong Provincial Public Laboratory of Analysis and Testing Technology, China National
Analytical Center (Guangzhou), Building 34, 100 Xianlie Middle Road, Guangzhou, 510070,
P R China
bSchool of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang West Road,
Guangzhou 510275, P R China
*Corresponding Author. Tel :﹢86 20 84110918; Fax :﹢86 20 84112245.
E-mail: yiwei2@mail2.sysu.edu.cn and yjhxhc@mail.sysu.edu.cn
1

Abstract
Here a new class of hydroxy- or methoxy-substituted 5-benzylidene(thio)barbiturates were
designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom
tyrosinase were evaluated. The results showed that several compounds had more potent tyrosinase
inhibitory activities than the widely used tyrosinase inhibitor kojic acid (IC50 = 18.25 μM). In
particular, 3’,4’-dihydroxylated 1e was found to be the most potent inhibitor with IC50 value of
1.52 μM. The inhibition mechanism analysis revealed that the potential compounds 1e and 2e
exhibited such inhibitory effects on tyrosinase by acting as the irreversible inhibitors.
Structure-activity relationships’ (SARs) analysis also suggested that further development of such
compounds might be of interest.
Keywords: 5-Benzylidene(thio)barbiturate; Tyrosinase inhibitor; SARs; Inhibition mechanism.
2

1. Introduction
Tyrosinase (EC 1.14.18.1), also known as polyphenoloxidase (PPO), is a copper-containing
bifunctional enzyme widely distributed in nature. It catalyzes two distinct reactions of melanin
synthesis, the hydroxylation of L-tyrosine by monophenolase action and the oxidation of L-DOPA
to the corresponding o-dopaquinone by diphenolase action.1 Melanin synthesis was responsible for
the undesired enzymatic browning of fruits and vegetables and resulted in a loss of nutritional and
market values.2 The production of abnormal melanin pigmentation (melasma, freckles, ephelide,
sneile lentigines, etc) was a serious esthetic problem in human beings.3 Moreover, tyrosinase was
found to be involved the molting process of insects4 and contributed to the neurodegeneration
associated with Parkinson’s disease.5 Therefore, tyrosinase inhibitors have become increasingly
important in food industry as well as in the medicinal and cosmetic products due to decreasing the
excessive accumulation of pigmentation resulting from the enzyme action.
To date, many efforts have been spent in the search for effective and safe tyrosinase inhibitors,
and a large number of naturally occurring and synthetic tyrosinase inhibitors have been
extensively reported.6-7 However, only few of them are put into practical use due to their weak
individual activities or safety concerns. Undoubtedly, it is still necessary to search and develop
novel tyrosinase inhibitors with better activity and lower side effect.
Among all the known tyrosinase inhibitors, polyphenol derivatives have attracted considerable
attention due to their favorable interaction with the hydrophobic protein pocket surrounding the
binuclear copper active site of tyrosinase,8 such as N-hydroxycinnamoylphenalkyl amides,9
hydroxysubstituted benzaldoximes,10 aurones,11 chalcones,12 flavanones,13 resveratrols and their
analogs,14 benzoate ester derivatives,15 and pyrazole derivatives.16 Recently, our groups also
demonstrated a series of 4-o-substituted 5-benzylidene barbiturate and thiobarbiturate derivatives
as potential tyrosinase inhibitors.17 Inhibition mechanism analysis revealed that such type of
compounds, similar to N-hydroxy-N’-phenylthiourea, N-hydroxy-N’-phenylurea18 and
thiosemicarbazide derivatives,19 were able to efficiently complex the two copper ions in the active
site of tyrosinase.
Taking advantage of above information, we speculated that coupling products of proper
hydroxy-substituted benzaldehydes with barbituric or thiobarbituric acid could not only complex
3

the binuclear copper active site of tyrosinase but also interact with the hydrophobic enzyme pocket,
and thus, leading to increasing inhibitor-enzyme binding affinity and improving inhibitory effects
on mushroom tyrosinase. Based on these, in our continuing interest to develop new potential
tryosinase inbibitors, here
a
series of new, low-cost and easy-to-prepare
5-benzylidene(thio)barbiturates bearing hydroxyl groups on phenyl ring were designed,
synthesized and their inhibitory effects on the diphenolase activitiy of mushroom tyrosinase were
evaluated. Besides, to verify the importance of hydroxyl group moiety, the corresponding
methoxy-substituted analogues were investigated. Furthermore, their SARs were discussed and the
inhibition mechanism of selected compounds was studied.
2. Experimental section
2.1. General
Melting points were determined on a WRS-1B digital instrument without correction. NMR
spectra were recorded on a Varian Mercury-Plus 300 spectrometers in CDCl3 or DMSO-d6 at 25
oC. All chemical shifts (d) are quoted in parts per million downfield from TMS and coupling
constants (J) are given in Hertz. Mass spectra were obtained from VG ZAB-HS, LCMS-2010A or
LCQ DECA XP spectrometer. Infrared (IR) spectra were measured on VECTOR 22 spectrometer
using a potassium bromide (KBr) disk, scanning from 400 to 4000 cm-1. All reactions were
monitored by TLC and spots were visualized with UV light or iodine. All commercially available
reagents and solvents were used without further purification. Mushroom tyrosinase (specific
activity of the enzyme is 6680 U/mg) and L-DOPA (L-3,4-dhydroxyphenylalanine) were
purchased from Sigma Chemical Co..
2.2. Chemistry
General procedure for the synthesis of 1b-l and 2b-l: To a solution of barbituric acid or
thiobarbituric acid (1.0 mmol) in hot ethanol (20 mL), the corresponding aromatic aldehyde (1.0
mmol) was added. The reaction mixture was heated 2 h at 80 oC and cooled to room temperature.
The precipitated solid was filtered, washed with ethanol and dried under vacuum. The crude
product was recrystallized from N, N-dimethylformamide (DMF) and water. Compound 1a and 2a
have been synthesized and characterized in our previous report.17b
4

2.2.1. 5-(2-Hydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)trione (1b)
o
Yellow powder was obtained (0.20 g, 84%), mp >250 C; IR (KBr, cm-1) υ: 3434, 3209,
3090, 2858, 1701, 1533, 1457, 1364, 1273, 774; 1H NMR (DMSO-d6, 300 MHz) δ: 11.25 (s, 1H,
NH), 11.14 (s, 1H, NH), 10.95 (s, 1H, OH), 7.34-7.05 (m, 5H, CH, ArH); 13C NMR (DMSO-d6,
75 MHz) δ: 164.21, 162.31, 156.06, 157.23, 150.19, 137.43, 133.21, 124.34, 119.09, 117.68,
116.52; FAB-MS m/z (%): 232 (100) (M).
2.2.2. 5-(3-Hydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1c)
o
Yellow powder was obtained (0.20 g, 85%), mp >250 C; IR (KBr, cm-1) υ: 3302, 1736,
1684, 1567, 1404, 1319, 1280, 796; 1H NMR (DMSO-d6, 300 MHz) δ: 11.33 (s, 1H, NH), 11.18 (s,
1H, NH), 9.68 (s, 1H, OH), 8.15 (s, 1H, CH), 7.63 (s, 1H, ArH), 7.43-7.40 (d, J = 7.5Hz, 1H, ArH),
7.26-7.22 (t, J = 7.5Hz, 1H, ArH), 6.94-6.91 (m, 1H, ArH); 13C NMR (DMSO-d6, 75 MHz) δ:
164.21, 162.31, 156.06, 157.23, 150.19, 137.43, 133.21, 124.34, 119.09, 117.68, 116.52; ESI-MS
m/z (%): 231 (75) (M-1).
2.2.3. 5-(2,4-Dihydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1d)
o
Yellow powder was obtained (0.20 g, 81%), mp >250 C; IR (KBr, cm-1) υ: 3455, 3364,
1
1731, 1674, 1556, 1404, 1309, 1289, 795; H NMR (DMSO-d6, 300 MHz) δ: 11.61 (s, 1H, NH),
11.26 (s, 1H, NH), 8.15 (m, 1H, CH), 7.93-7.89 (m, 1H, ArH), 7.00-6.95 (m, 2H, ArH); 13C NMR
(DMSO-d6, 75 MHz) δ: 163.92, 162.16, 157.16, 150.95, 150.29, 145.70, 133.94, 116.45, 113.14,
109.82, 103.27; FAB-MS m/z (%): 248 (100) (M).
2.2.4. 5-(3,4-Dihydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1e)
o
Yellow powder was obtained (0.21 g, 83%), mp >250 C; IR (KBr, cm-1) υ: 3469, 3251,
1
1727, 1663, 1545, 1506, 1403, 1295, 794; H NMR (DMSO-d6, 300 MHz) δ: 11.18 (s, 1H, NH),
11.06 (s, 1H, NH), 8.17 (s, 1H, CH), 8.08 (s, 1H, ArH), 7.62-7.59 (m, 1H, ArH), 6.84-6.81 (m, 1H,
ArH); 13C NMR (DMSO-d6, 75 MHz) δ: 163.90, 162.27, 158.23, 155.15, 152.10, 150.43, 134.57,
125.36, 118.92, 115.33, 112.43; EI-MS m/z (%): 248 (100) (M), 231 (24), 204 (50), 188 (25), 161
(23), 134 (25), 88 (10); HRMS: calculated (248.0433), found (248.0422).
2.2.5. 5-(3-Hydroxyl-4-methoxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1f)
Yellow powder was obtained (0.24 g, 90%), mp >250 oC; IR (KBr, cm-1) υ: 3247, 3090, 1729,
1
1673, 1553, 1508, 1444, 1274, 1147, 795, 520; H NMR (DMSO-d6, 300 MHz) δ: 11.21 (s, 1H,
5

NH), 11.08 (s, 1H, NH), 9.36 (s, 1H, OH), 8.12-8.08 (m, 2H, CH, ArH), 7.72-7.68 (m, 1H, ArH),
7.04-7.02 (m, 1H, ArH), 3.87 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75 MHz) δ: 164.46, 162.57,
156.06, 153.38, 150.67, 146.26, 130.73, 125.88, 120.93, 115.52, 111.77, 56.26; ESI-MS m/z (%):
261 (20) (M-1).
2.2.6. 5-(3-Methoxyl-4-hydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1g)
Yellow powder was obtained (0.21 g, 78%), mp >250 oC; IR (KBr, cm-1) υ: 3272, 2863, 1746,
1663, 1540, 1499, 1407, 1309, 1281, 1176, 825, 523; 1H NMR (DMSO-d6, 300 MHz) δ: 11.20 (s,
1H, NH), 11.08 (s, 1H, NH), 10.48 (s, 1H, OH), 8.43 (s, 1H, CH), 8.20 (s, 1H, ArH), 7.80-7.76 (d,
J = 10.8Hz, 1H, ArH), 6.90-6.86 (d, J = 10.8Hz, 1H, ArH), 3.82 (s, 3H, OCH3); 13C NMR
(DMSO-d6, 75 MHz) δ: 164.45, 162.58, 156.02, 152.50, 151.66, 146.29, 130.66, 125.95, 120.92,
115.58, 112.50, 56.43; ESI-MS m/z (%): 261 (100) (M-1).
2.2.7. 5-(3,4,5-Trimethoxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1h)
Yellow powder was obtained (0.23 g, 76%), mp >250 oC; IR (KBr, cm-1) υ: 3265, 2894, 1730,
1
1667, 1544, 1505, 1355, 1278, 1169, 792; H NMR (DMSO-d6, 300 MHz) δ: 11.35 (s, 1H, NH),
11.23 (s, 1H, NH), 8.24 (s, 1H, CH), 7.82 (s, 2H, ArH), 3.81 (s, 6H, OCH3), 3.77 (s, 3H, OCH3);
13C NMR (DMSO-d6, 75 MHz) δ: 164.31, 162.69, 155.78, 152.54(2C), 150.74, 142.54, 128.17,
117.92, 113.26 (2C), 61.04, 56.76 (2C); ESI-MS m/z (%): 337 (67) (M+CH3OH).
2.2.8. 5-(4-Methoxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1i)
Yellow powder was obtained (0.23 g, 93%), mp >250 oC; IR (KBr, cm-1) υ: 3251, 3066, 2895,
1738, 1695, 1651, 1601, 1562, 1522, 1431, 1269, 869; 1H NMR (DMSO-d6, 300 MHz) δ: 11.29 (s,
1H, NH), 11.17 (s, 1H, NH), 8.37-8.33 (d, J = 9.0Hz, 2H, ArH), 8.22 (s, 1H, CH), 7.06-7.03 (d, J
= 9.0Hz, 2H, ArH), 3.86 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75 MHz) δ: 164.47, 164.00, 162.72,
155.59, 150.80, 138.11 (2C), 125.78, 116.12, 114.57 (2C), 56.44; ESI-MS m/z (%): 277 (27)
(M+CH3OH).
2.2.9. 5-(3,4,5-Trihydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1j)
Yellow powder was obtained (0.18 g, 68%), mp >250 oC; IR (KBr, cm-1) υ: 3215, 1687, 1661,
1524, 1503, 1218, 792; 1H NMR (DMSO-d6, 300 MHz) δ: 11.16 (s, 1H, NH), 11.03 (s, 1H, NH),
7.98 (s, 1H, CH), 7.51 (s, 2H, ArH); 13C NMR (DMSO-d6, 75 MHz) δ: 164.81, 162.72, 156.99,
150.83, 145.80 (2C), 141.67, 123.54, 116.27 (2C), 114.34; ESI-MS m/z (%): 263 (47) (M-1).
6

2.2.10. 5-(2,4,6-Trihydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1k)
Yellow powder was obtained (0.16 g, 61%), mp >250 oC; IR (KBr, cm-1) υ: 3447, 3148, 1681,
1656, 1531, 1496, 1220, 811; 1H NMR (DMSO-d6, 300 MHz) δ: 11.14 (s, 1H, NH), 11.05 (s, 1H,
NH), 8.10 (s, 1H, CH), 7.56 (s, 2H, ArH); 13C NMR (DMSO-d6, 75 MHz) δ: 164.82, 162.90,
157.04, 150.79 (2C), 145.71, 142.33, 118.45, 115.53, 111.06 (2C); ESI-MS m/z (%): 263 (24)
(M-1).
2.2.11. 5-(3,4,5-Trihydroxybenzyliden)pyrimidine-2,4,6(1H, 3H, 5H)-trione (1l)
Yellow powder was obtained (0.19 g, 70%), mp >250 oC; IR (KBr, cm-1) υ: 3450, 3220, 1677,
1645, 1520, 1499, 1231, 1108, 687; 1H NMR (DMSO-d6, 300 MHz) δ: 11.23 (s, 1H, NH), 11.10 (s,
1H, NH), 10.95 (s, 1H, OH), 10.60 (s, 1H, OH), 9.83 (s, 1H, OH), 8.77 (s, 1H, CH), 8.32-8.28 (d,
J = 6.0Hz, 1H, ArH), 7.46-7.42 (d, J = 9.0Hz, 1H, ArH), 6.39-6.35 (d, J = 9.0Hz, 1H, ArH); 13C
NMR (DMSO-d6, 75 MHz) δ: 165.21, 163.05, 153.96, 152.20, 151.05, 150.93, 132.53, 126.87,
113.92, 112.52, 108.07; ESI-MS m/z (%): 263 (53) (M-1).
2.2.12. 5-(2-Hydroxybenzyliden)-2-thioxo-dihyropyrimidine-4,6(1H, 5H)-dione (2b)
Yellow powder was obtained (0.20 g, 83%), mp >250 oC; IR (KBr, cm-1) υ: 3427, 3134, 1655,
1558, 1457, 1382, 1276, 1134, 761; 1H NMR (DMSO-d6, 300 MHz) δ: 13.28 (s, 1H, NH), 12.27 (s,
1H, NH), 7.19 (m, 2H, CH, ArH), 7.09 (m, 2H, ArH), 6.98-6.96 (m, 1H, ArH); 13C NMR
(DMSO-d6, 75 MHz) δ: 178.57, 162.41, 160.11, 158.41, 150.39, 133.40, 131.72, 122.33, 119.59,
117.86, 115.25; ESI-MS m/z (%): 247 (78) (M-1).
2.2.13. 5-(3-Hydroxybenzyliden)-2-thioxo-dihyropyrimidine-4,6(1H, 5H)-dione (2c)
Yellow powder was obtained (0.24 g, 87%), mp >250 oC; IR (KBr, cm-1) υ: 3235, 1664, 1561,
1458, 1377, 1280, 812; 1H NMR (DMSO-d6, 300 MHz) δ: 12.42 (s, 1H, NH), 12.31 (s, 1H, NH),
9.75 (s, 1H, OH), 8.15 (s, 1H, CH), 7.68 (s, 1H, ArH), 7.48-7.44 (d, J = 7.8Hz, 1H, ArH),
7.29-7.24 (t, J = 7.8Hz, 1H, ArH), 6.97-6.93 (dd, J1 = 7.8Hz, J2 = 2.4Hz, 1H, ArH); 13C NMR
(DMSO-d6, 75 MHz) δ: 179.53, 162.43, 160.02, 157.55, 156.30, 134.30, 129.86, 126.04, 120.09,
117.13, 113.21; ESI-MS m/z (%): 247 (100) (M-1).
2.2.14. 5-(2,4-Dihydroxybenzyliden)-2-thioxo-dihyropyrimidine-4,6(1H, 5H)-trione (2d)
Yellow powder was obtained (0.19 g, 73%), mp >250 oC; IR (KBr, cm-1) υ: 3037, 1710, 1673,
1
1594, 1466, 1384, 1261, 1125, 851, 793; H NMR (DMSO-d6, 300 MHz) δ: 11.58 (s, 1H, NH),
7

11.24 (s, 1H, NH), 8.80 (m, 1H, CH), 7.92-7.88 (m, 1H, ArH), 6.95 (m, 2H, ArH); 13C NMR
(DMSO-d6, 75 MHz) δ: 179.14, 162.37, 160.17, 159.89, 159.75, 152.34, 131.12, 120.60, 111.03,
109.42, 105.20; FAB-MS m/z (%): 264 (100) (M).
2.2.15. 5-(3,4-Dihydroxybenzyliden)-2-thioxo-dihyropyrimidine-4,6(1H, 5H)-dione (2e)
Yellow powder was obtained (0.21 g, 80%), mp >250 oC; IR (KBr, cm-1) υ: 3447, 3120, 2908,
1650, 1575, 1505, 1443, 1279, 1144, 1003, 695; 1H NMR (DMSO-d6, 300 MHz) δ: 12.26 (s, 1H,
NH), 12.18 (s, 1H, NH), 8.27-8.26 (d, J1 = 2.1Hz, 1H, ArH), 8.12 (s, 1H, CH), 7.66-7.62 (dd, J1 =
2.1Hz, J2=8.7Hz, 1H, ArH), 6.86-6.83 (d, J2 = 8.7Hz, 1H, ArH); 13C NMR (DMSO-d6, 75 MHz) δ:
178.62, 163.10, 160.59, 157.74, 153.85, 145.60, 132.96, 125.16, 122.07, 116.22, 114.32; EI-MS
m/z (%): 264 (100) (M), 247 (30), 204 (25), 166 (55), 134 (33); HRMS: calculated (2464.0422),
found (264.0196).
2.2.16. 5-(3-Dydroxyl-4-methoxybenzyliden)-2-thioxodihyropyrimidine-4,6(1H, 5H)-dione
(2f)
Yellow powder was obtained (0.23 g, 88%), mp >250 oC; IR (KBr, cm-1) υ: 3447, 3120, 1650,
1575, 1505, 1442, 1317, 1279, 1188, 1144, 1002, 482; 1H NMR (DMSO-d6, 300 MHz) δ: 12.29 (s,
1H, NH), 12.20 (s, 1H, NH), 8.15 (m, 2H, CH, ArH), 7.76 (m, 1H, ArH), 7.08-7.04 (m, 1H, ArH),
3.88 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75 MHz) δ: 178.41, 162.20, 160.36, 153.38, 150.58,
148.65, 130.98, 125.01, 120.93, 118.20, 111.55, 56.70; EI-MS m/z (%): 278 (100) (M), 261 (20),
180 (35), 161(23), 133 (25), 105 (24).
2.2.17. 5-(3-Methoxyl-4-hydroxybenzyliden)-2-thioxodihyropyrimidine-4,6(1H, 5H) -dione
(2g)
Yellow powder was obtained (0.20 g, 74%), mp >250 oC; IR (KBr, cm-1) υ: 3519, 3116, 2910,
1652, 1503, 1430, 1395, 1283, 1170, 1026, 960, 871, 786, 525; 1H NMR (DMSO-d6, 300 MHz) δ:
12.29 (s, 1H, NH), 12.19 (s, 1H, NH), 10.67 (s, 1H, OH), 8.46 (s, 1H, CH), 8.22 (s, 1H, ArH),
7.86 (m, 1H, ArH), 6.89 (m, 1H, ArH), 3.82 (s, 3H, OCH3); 13C NMR (DMSO-d6, 75 MHz) δ:
178.27, 162.19, 160.86, 155.31, 150.44, 146.94, 130.88, 123.12, 120.52, 118.86, 112.70, 56.53;
EI-MS m/z (%): 278 (100) (M), 261 (20), 218 (17), 180 (35), 105 (16).
2.2.18. 5-(3,4,5-Trimethoxybenzyliden)-2-thioxodihydropyrimidine-4,6(1H, 5H)-dione (2h)
Yellow powder was obtained (0.23 g, 71%), mp >250 oC; IR (KBr, cm-1) υ: 3118, 2907, 1655,
8

1
1505, 1442, 1385, 1278, 1169, 1009, 783; H NMR (DMSO-d6, 300 MHz) δ: 12.43 (s, 1H, NH),
12.32 (s, 1H, NH), 8.25 (s, 1H, CH), 7.87 (s, 2H, ArH), 3.82 (s, 6H, OCH3), 3.79 (s, 3H, OCH3);
13C NMR (DMSO-d6, 75 MHz) δ: 178.81, 162.61, 160.48, 152.55 (2C), 143.10, 128.22, 117.93,
113.62 (2C), 109.98, 61.12 (2C), 56.82; ESI-MS m/z (%): 353 (100) (M+Na).
2.2.19. 5-(4-Methoxybenzyliden)-2-thioxo-dihydropyrimidine-4,6(1H, 5H)-dione (2i)
Yellow powder was obtained (0.25 g, 94%), mp >250 oC; IR (KBr, cm-1) υ: 3187, 2913, 1665,
1502, 1443, 1274, 870, 629; 1H NMR (DMSO-d6, 300 MHz) δ: 12.37 (s, 1H, NH), 12.27 (s, 1H,
NH), 8.42-8.38 (d, J = 9.0Hz, 2H, ArH), 8.24 (s, 1H, CH), 7.08-7.04 (d, J = 9.0Hz, 2H, ArH), 3.87
(s, 3H, OCH3); 13C NMR (DMSO-d6, 75 MHz) δ: 178.81, 164.54, 162.80, 160.55, 156.56, 138.60
(2C), 125.92, 116.25, 114.77 (2C), 56.57; ESI-MS m/z (%): 293 (30) (M+CH3OH).
2.2.20. 5-(3,4,5-Trihydroxybenzyliden)-2-thioxodihydropyrimidine-4,6(1H, 5H)-dione (2j)
Yellow powder was obtained (0.17 g, 62%), mp >250 oC; IR (KBr, cm-1) υ: 3457, 3164, 1663,
1503, 1445, 1278, 804; 1H NMR (DMSO-d6, 300 MHz) δ: 12.25 (m, 2H, NH), 9.91 (s, 1H, OH),
9.53 (s, 2H, OH), 7.98 (s, 1H, CH), 7.58 (s, 2H, ArH); 13C NMR (DMSO-d6, 75 MHz) δ: 178.58,
162.83, 160.67, 150.81, 145.94 (2C), 142.77, 123.79, 116.77 (2C), 114.32; ESI-MS m/z (%): 279
(100) (M-1).
2.2.21. 5-(2,4,6-Trihydroxybenzyliden)-2-thioxodihydropyrimidine-4,6(1H, 5H)-dione (2k)
Yellow powder was obtained (0.15 g, 57%), mp >250 oC; IR (KBr, cm-1) υ: 3449, 3124, 1656,
1
1505, 1446, 1280, 798; H NMR (DMSO-d6, 300 MHz) δ: 12.24 (m, 2H, NH), 8.13 (s, 1H, CH),
7.58 (s, 2H, ArH); 13C NMR (DMSO-d6, 75 MHz) δ: 178.46, 162.50, 160.39 151.04 (2C), 144.69,
143.54, 119.78, 115.87, 113.64 (2C); ESI-MS m/z (%): 279 (65) (M-1).
2.2.22. 5-(3,4,5-Trihydroxybenzyliden)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (2l)
Yellow powder was obtained (0.16 g, 63%), mp >250 oC; IR (KBr, cm-1) υ: 3451, 3143, 1649,
1
1507, 1457, 1291, 1121, 761, 687; H NMR (DMSO-d6, 300 MHz) δ: 12.52 (m, 2H, NH), 10.12
(m, 1H, OH), 8.89 (m, 2H, CH, ArH), 7.52-7.48 (d, J = 8.4Hz, 1H, ArH), 7.07-7.04 (d, J = 8.4Hz,
1H, ArH), 6.39-6.35 (d, J = 9.0Hz, 1H, ArH); 13C NMR (DMSO-d6, 75 MHz) δ: 178.61, 163.19,
161.22, 159.86, 153.44, 144.21, 133.57, 124.32, 116.49, 115.09, 110.27; ESI-MS m/z (%): 279
(31) (M-1).
2.3. Biology
9

Tyrosinase inhibition assay was performed as described by our groups17 with some slight
modifications. Briefly, all the synthesized compounds were screened for the diphenolase
inhibitory activity of tyrosinase using L-DOPA as substrate. 5-Benzylidene(thio)barbiturates were
first dissolved in DMSO, and the final concentration of DMSO in the test solution was 1.0%.
Phosphate buffer, pH 6.8, was used to dilute the DMSO stock solution of test compounds. Thirty
units of mushroom tyrosinase (0.5 mg/ml) was first pre-incubated with the compounds, in 50 mM
o
phosphate buffer (pH = 6.8), for 10 min at 25 C. Then the L-DOPA (0.5 mM) was added to the
reaction mixture and the enzyme reaction was monitored by measuring the change in absorbance
at 475 nm of formation of the DOPAchrome for 1 min. The measurement was performed in
triplicate for each concentration and averaged before further calculation. IC50 value, a
concentration giving 50% inhibition of tyrosinase activity, was determined by interpolation of the
dose-response curves. As a control, the IC50 of kojic acid was also measured.
3. Results and Discussion
3.1. Synthesis
5-Benzylidene(thio)barbiturates were prepared through the well-known Knoevenegal
condensation of the corresponding benzaldehydes and barbituric or thiobarbituric acid as
described previously.17,20 Briefly, a number of commercially available benzaldehydes were reacted
with barbituric or thiobarbituric acid in hot ethanol for 2 h to afford 5-benzylidenebarbiturate 1a-l
and 5-benzylidenethiobarbiturate 2a-l in good yields (Scheme 1).
3.2. Biological activity
3.2.1. Inhibitory effects on the diphenolase activity of mushroom tyrosinase
Our previous work have showed that such coupling products 1a and 2a bearing a hydroxyl
substituent at position-4’ of A-ring had excellent inhibitory activities on mushroom tyrosinase
with IC50 values of 13.98 μM and 14.49 μM, respectively.17b Inspired by this, we sought to probe
the position and number of hydroxyl substituent for the influence of tyrosinase inhibitory activity.
It was noteworthy that such expansion of 5-benzylidene(thio)barbiturates has not been reported so
far. Therefore, here the inhibition of our synthetic hydroxy- or methoxyl-substituted
5-benzylidene(thio)barbiturates on the mushroom tyrosinase was investigated and compared with
10

kojic acid, which is a widely used skin-whitening material in cosmetic industry. As shown in
Table 1, parent compounds barbituric acid, thiobarbituric acid and 3,4-dihydroxybenzaldehyde
showed no activities at the concentration of 100 μM. As predicted, many condensation products of
barbituric acid or thiobarbituric acid and hydroxybenzaldehydes displayed potent tyrosinase
inhibitory activities. Especially, the obtained compounds 1d (IC50 = 17.15 μM), 1e (IC50 = 1.52
μM), 1g (IC50 = 5.50 μM), 1j (IC50 = 5.51 μM), 2e (IC50 = 6.10 μM), 2g (IC50 = 7.80 μM) and 2j
(IC50 = 7.14 μM) exhibited more potent tyrosinase inhibitory activities than the reference inhibitor
kojic acid (IC50 = 18.25 μM). These results showed that the introduction of 5-benzylidene
substructures was crucial for presenting the inhibitory effect on tyrosinase.
Compared with compounds 1a and 2a, unfortunately, the results from Table 1 showed that our
synthesized compounds 1b and 2b having a hydroxyl group at position-2’ and 1c and 2c having a
hydroxyl group at position-3’ of A-ring displayed a loss of activity (less than 20% inhibition at a
concentration of 100 μM). Interestingly, incorporation of additional hydroxyl group onto
position-3’ of 1a to afford the most potent compound 1e with IC50 value of 1.52 μM, and its
thiobarbiturate analogue 2e (IC50 = 6.10 μM) also showed significant inhibitory activity.
Compared with 1a, compound 1d bearing an additional hydroxyl group at positon-2’ of A-ring
exhibited comparable activity, whereas its thiobarbiturate analogue 2d showed no inhibitiory
activity on tyrosinase. Replacement of hydroxyl group at position-3’ of 1e and 2e with a methoxy
group to give 1g (IC50 = 5.50 μM) and 2g (IC50 = 7.80 μM) led to a reduction in potency. Similar
findings were also observed at position-4’ of A-ring (1a vs 1i, 2a vs 2i, 1e vs 1f and 2e vs 2f).
These results suggested that 4’-hydroxyl group might be absolutely necessarily and 3’-hydroxyl
group facilitated their inhibitory activities while 3’ or 4’-methoxy group was unfavorable.
To further investigate the effect of the number and position of hydroxyl substituents on A-ring,
3’, 4’, 5’-trihydroxlated 1j and 2j, 2’, 4’, 6’-trihydroxlated 1k and 2k, 2’,3’,4’ -trihydroxlated 1l
and 2l were examined for the tyrosinase inhibitory activity. As shown in Table 1, the inhibitory
effects of 1j (IC50 = 5.51 μM) and 2j (IC50 = 7.14 μM) were slightly lower than that of
3’,4’-dihydroxylated 1e and 2e. Compound 2l (IC50 = 95.83 μM) showed weak inhibitory activity,
whereas 1k, 2k and 1l exhibited a completely loss of activity. Replacement of hydroxyl groups at
position-3’, 4’, 5’ of A-ring with methoxy substituents to afford 1h and 2h also showed no activity.
11

These data further revealed that 4’-hydroxyl group might played a very vital role in determining
inhibitory effects on mushroom tyrosinase and methoxy group was not advantageous for the
inhibitory potency.
Furthermore, 3’, 4’-dihydroxylated 1e and 2e showed more potent activities than 3’, 4’,
5’-trihydroxylated 1j and 2j, 2’, 4’, 6’-trihydroxylated 1k and 2k, and 2’, 3’, 4’-trihydroxylated 1l
and 2l, suggesting that the third substituent might hinder the correct docking of the inhibitor to the
active site of tyrosinase.
3.2.2. Inhibitory mechanism of compound 1e and 2e on mushroom tyrosinase
The inhibition mechanism on mushroom tyrosinase by 1e for the oxidation of L-DOPA was
investigated. Fig 1A showed the relationship between enzyme activity and its concentration in the
presence of 1e. The plots of the remaining enzyme activity (V) versus the concentrations of
enzyme at different inhibitor concentrations ([E]) gave a family of parallel straight lines with the
same slopes, indicating that the inhibitory effect of 1e on the tyrosinase was irreversible. Identical
research was carried out on 2e, and same conclusion was drawn that 2e was irreversible inhibitor
(Fig. 1B). These results suggested that 5-benzylidene(thio)barbiturates effectively inhibited the
enzyme by binding to its binuclear active site irreversibly.
On the basis of the crystallographic structure of tyrosinase and our obtained results, a possible
binding mode was postulated (Fig. 2). As shown in Fig. 2, the barbiturate moiety could fit the
binuclear copper active site well because the oxygen and/or sulfur atom formed strong chelation
with the binuclear copper in the active center of tyrosinase. Such interaction acted like a bridge to
link 5-benzylidene moiety bearing the hydroxyl or methoxyl group and the hydrophobic protein
pocket in tyrosinase, and thus facilitating them to interact. Moreover, the additional hydrogen
bonding interaction might occur between hydroxyl groups and some key bioactive amino acids
existed in the hydrophobic domain,8,21 since the above SARs analysis have disclosed that
5-benzylidene(thio)barbiturates bearing the hydroxyl substituent showed more potent tyrosinase
inhibitory activities than those bearing the methoxyl substituent.
4. Conclusion
In summary, here we have developed a series of hydroxy- or methoxyl-substituted
5-benzylidene(thio)barbiturates from simple chemicals as novel tyrosinase inhibitors. The results
12

showed that several compounds had more potent tyrosinase inhibitory activities than the widely
used tyrosinase inhibitor kojic acid. Especially, compound 1e exhibited the most potent tyrosinase
inhibitory activity with IC50 value of 1.52 μM. SARs analysis indicated (1) the introduction of
5-benzylidene substructures was crucial for the inhibition of tyrosinase activity; (2) the number
and position of hydroxyl substituent on the phenyl ring played a vital role in determining
inhibitory effects; (3) the 4-hydroxyl group of phenyl ring might be absolutely necessarily. The
inhibition mechanism analysis revealed that 5-benzylidene(thio)barbiturates exhibited such
inhibitory effects on tyrosinase by acting as the irreversible inhibitors. All the results suggested
that these molecules can serve as the interesting candidates for the treatment of tyrosinase-related
disorders and as the lead for the development of new and potent tyrosinase inhibitors. However, it
should be noted that, from a clinical point of view, this model using mushroom tyrosinase was not
sufficient for evaluating active molecules destined for human use.6,11a Therefore, further
investigations of these potent compounds using human tyrosinase and a human melanoma cell line
are underway in our laboratory, and the research results will be reported in due course.
Acknowledgments
This work was supported by the Science and Technology Project of Guangdong Province,
China (2009B060700048) and the Fundamental Research Funds for the Central Universities.
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15

Table 1 Structures and inhibitory activity against mushroom tyrosinase (diphenolase) of
5-benzylidene(thio)barbiturates 1a-l and 2a-l.
R3 2'R2
O
3
3'
4
NH
2
1'
5
R4
X
4'
6'
6
NH
5'
1
R5
R6
O
Compd
R2’
R3’
R4’
R5’
R6’
X
IC50 (μM)a
H
H
OH
H
H
H
O
13.98±1.65b
1a
OH
H
H
H
O
NIc
1b
H
OH
H
OH
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
S
NI
17.15±1.56
1.52±0.25
NI
1c
OH
OH
OMe
OH
OMe
OMe
OH
OH
OH
OH
H
1d
OH
OH
OMe
OMe
H
H
H
1e
H
H
H
1f
H
H
H
5.50±0.64
NI
1g
H
OMe
H
H
1h
H
H
100.0±5.5
5.51±0.48
NI
1i
H
OH
H
OH
H
H
1j
OH
OH
H
OH
H
1k
OH
H
H
NI
1l
H
H
14.49±1.21b
2a
OH
H
H
H
H
S
NI
2b
OH
H
H
H
H
S
NI
2c
OH
H
OH
OH
OMe
OH
OMe
OMe
OH
OH
OH
H
H
S
NI
2d
OH
OH
OMe
OMe
H
H
H
S
6.10±0.76
NI
2e
H
H
H
S
2f
H
H
H
S
7.80±0.83
NI
2g
H
OMe
H
H
S
2h
H
H
S
70.12±1.92
7.14
2i
H
OH
H
OH
H
H
S
2j
OH
OH
OH
H
S
NI
2k
OH
H
S
95.83±2.25
NI
2l
Barbituric acid
Thiobarbituric acid
NI
3,4-Dihydroxybenzaldehyde
Kojic acid
NI
18.25±1.85
a Assay performed using mushroom tyrosinase. Values are means of three different experiments. IC50 = mean ±
S.E.M., S.E.M.: standard error of mean.
b The data have been reported in reference 17b.
c No obvious inhibition at a concentration of 100 μM (<20%).
16

Fig. 1 The inhibitory effects of 1e and 2e on mushroom tyrosinase for the catalysis of L-DOPA
(A and B). The concentrations of compound 1e for curves 1-4 were 0, 1.0, 1.5 and 2.0 μM,
respectively. The concentrations of compound 2e for curves 1-3 were 0, 5.0 and 10.0 μM,
respectively.
1
A
0.25
2
0.20
3
0.15
4
0.10
0.05
0.00
4
6
8
10
E (μg/mL)
0.25
0.20
0.15
0.10
0.05
0.00
1
B
2
3
4
5
6
7
8
9
10
E (μg/mL)
17

Fig. 2 Postulated binding mode.
interact with the hydrophobic
protein pocket in tyrosinase
O
NH
OH or OMe
O
N
H
X
irreversibly complex the binuclear
copper in the active center of tyrosinase
X= O or S
18

Scheme 1 Synthesis of compounds 1a-l and 2a-l.
O
O
CHO
NH
NH
NH
EtOH
80oC, 2h
+
X
R
R
O
N
H
X
O
A: X=O
B: X=S
1a~l: X=O
2a~l: X=S
a~l
19

Graphical Abstract
O
O
HO
HO
NH
NH
HO or MeO
O
N
X
O
N
H
O
H
1e
IC50 = 1.52 μM
X=O, 1a-l
X=S, 2a-l
A series of hydroxy- or methoxy-substituted 5-benzylidene (thio)barbiturate derivatives were
designed, synthesized, and evaluated as novel tyrosinase inhibitors. Compounds 1e were found to
be the most potent inhibitor. SARs were discussed and the inhibition mechanism of selected
compounds 1e and 2e was also investigated.