J. Xu et al. / Food Chemistry xxx (2016) xxx–xxx
3
7.53 (s, 1H, CH), 7.45 (s, 1H, thiophene-H), 7.35 (s, 1H, thiophene-
H), 4.35 (s, H, OH), 3.43 (m, 2H, CH2), 1.1(t, 3H, CH3). 13C NMR
(400 MHz, DMSO-d6) d 177.89(1C, C = S), 140.79(1C, C@N), 139.21
H), 2.50 (s, 3H, CH3). 13C NMR (400 MHz, DMSO-d6) d 177.70(1C,
C@S), 143.22(1C, C@N), 138.24(1C, thiophene-C), 136.81(1C,
thiophene-C), 131.48(1C, thiophene-C), 126.84(1C, thiophene-C),
15.80(1C, CH3). MS (ESI): m/z 200.0 [M + H]+.
(1C,
thiophene-C),
137.89(1C,
thiophene-C),
130.97(1C,
thiophene-C), 126.13(1C, thiophene-C), 67.28(1C, CH2), 65.34(1C,
5-Nitro-thiophene-2-carbaldehyde thiosemicarbazone (11):
yellow powder, yield 84.2%, IR(KBr) 3470, 1608, 1540,
CH2), 57.84(1C, CH3). MS (ESI): m/z 242.1 [M-H]-.
4-(2-Hydroxy-ethoxymethyl)-thiophene-2-carbaldehyde
thiosemicarbazone (4): pale yellow crystals, yield 84.1%, IR(KBr)
1335 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.77 (s, 1H, NH),
;
8.41, 8.17 (d, 2H, NH2), 8.06 (d,1H, thiophene-H), 8.00(s, 1H, CH),
7.51 (d,1H, thiophene-H). 13C NMR (400 MHz, DMSO-d6) d 178.55
(1C, C@S), 151.50(1C, C@N), 147.19(1C, thiophene-C), 135.67(1C,
thiophene-C), 130.88(1C, thiophene-C), 129.66(1C, thiophene-C).
MS (ESI): m/z 229.1 [M-H]-.
3441, 3178, 1608, 1528 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d
;
11.39 (s, 1H, NH), 8.17 (s, 2H, NH2), 7.52 (s, 1H, CH), 7.47 (s,1H,
thiophene-H), 7.37 (s,1H, thiophene-H), 4.60 (s, H, OH), 4.40 (s,
2H, CH2), 3.48(t, 2H, CH2), 3.42(t, 2H, CH2). 13C NMR (400 MHz,
DMSO-d6)
d
177.88(1C, C@S), 140.76(1C, C@N), 139.17(1C,
5-Phenyl-thiophene-2-carbaldehyde thiosemicarbazone (12):
thiophene-C), 137.90(1C, thiophene-C), 131.07(1C, thiophene-C),
126.18(1C, thiophene-C), 72.03(1C, CH2), 67.71(1C, CH2), 60.62
(1C, CH2). MS (ESI): m/z 258.1 [M-H]-.
5-Hydroxymethyl-thiophene-2-carbaldehyde
bazone (5): pale yellow acicular crystal, yield 74.9%, IR(KBr)
3439, 3166, 1589, 1549 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d
11.36 (s, 1H, NH), 8.13 (d, 2H, NH2), 7.46 (s, 1H, CH), 7.25 (d, 1H,
thiophene-H), 6.90 (d, 1H, thiophene-H), 5.53 (s, H, OH), 4.59 (s,
2H, CH2). 13C NMR (400 MHz, DMSO-d6) d 177.77(1C, C@S),
150.21(1C, C@N), 138.25(1C, thiophene-C), 137.62(1C, thiophene-
C), 130.89(1C, thiophene-C), 124.74(1C, thiophene-C), 58.98(1C,
CH2). MS (ESI): m/z 214.2 [M-H]-.
yellow powder, yield 83.7%, IR(KBr) 3164, 1589, 1521 cmꢀ1 1H
;
NMR (400 MHz, DMSO-d6) d 11.47 (s, 1H, NH), 8.19 (s, 2H, NH2),
7.67 (d,2H, Ph-H), 7.55(s, 1H, CH), 7.49 (d,1H, thiophene-H), 7.43–
7.39 (t, 2H, Ph-H; d, H, thiophene-H), 7.32 (t, H, Ph-H). 13C NMR
(400 MHz, DMSO-d6) d 177.86(1C, C@S), 145.74(1C, C@N), 138.39
(1C, thiophene-C), 137.76(1C, thiophene-C), 133.65(1C, Ph-C),
132.35(1C,Ph-C), 129.68(2C,Ph-C), 128.74(1C, thiophene-C), 125.88
(2C,Ph-C), 124.84(1C, thiophene-C). MS (ESI): m/z 260.1 [M-H]-.
thiosemicar-
;
2.3. Determination of water solubility and lipid solubility
The experiment was performed as reference (Bai, Yan, Hu,
Zhang, & Huang, 2006) described with minor modifications to
determine the water/lipid solubility of selected compounds
preliminarily. An excess of sample was dissolved in water/ethyl
acetate, sonic disruption for 1 h, and oscillation incubation for
6 h in room temperature, after centrifugation for 10 min, the upper
clear liquid was retained by filtration. 1 mL Filtrate was then
diluted with water/ethyl acetate into five concentrations from
saturated to low, then the absorbance values were recorded using
a Shimadzu UV-2450 spectrophotometer (Japan) at its maximum
absorption wavelength, and standard curve was determined.
Known concentration of the sample solution(dissolved in water/
ethyl acetate) was configured, recorded the absorbance value using
a Shimadzu UV-2450 spectrophotometer (Japan) at its maximum
absorption wavelength. Since dilute solution conformed to
Lambert-Beer’s law, according to the obtained standard curve
and the absorbance value of known concentration, the sample’s
water solubility and lipid solubility can be calculated.
4-Methoxyacetyl-thiophene-2-carbaldehyde
bazone (6): pale yellow flaky crystals, yield 73.1%, IR(KBr) 3155,
1742, 1601, 1531 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.40 (s,
1H, NH), 8.16 (s, 2H, NH2), 7.57 (s,1H, thiophene-H), 7.53 (s, 1H,
CH), 7.38 (s,1H, thiophene-H), 4.40 (s, 2H, CH2), 2.03(s, 3H, CH3).
13C NMR (400 MHz, DMSO-d6) d 177.99(1C, C@S), 170.64(1C,
C@O), 139.57(1C, C@N), 137.98(1C, thiophene-C), 137.69(1C,
thiophene-C), 131.04(1C, thiophene-C), 127.64(1C, thiophene-C),
61.04(1C, CH2), 21.13(1C, CH3). MS (ESI): m/z 256.0 [M-H]-.
thiosemicar-
;
5-Methoxymethyl-thiophene-2-carbaldehyde
bazone (7): yellow powder, yield 80.3%, IR(KBr) 3144, 1601,
1522 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d11.39 (s, 1H, NH),
thiosemicar-
;
8.15 (s, 2H, NH2), 7.50 (s, 1H, CH), 7.27 (d, 1H, thiophene-H), 6.98
(d, 1H, thiophene-H), 4.53 (s, 2H, CH2), 3.25(s, 3H, CH3). 13C NMR
(400 MHz, DMSO-d6) d 177.87(1C, C@S), 144.44(1C, C@N), 139.00
(1C,
thiophene-C),
137.93(1C,
thiophene-C),
130.67(1C,
thiophene-C), 127.23(1C, thiophene-C), 68.82(1C, CH2), 57.78(1C,
CH3). MS (ESI): m/z 228.0 [M-H]-.
5-(2-Hydroxy-ethoxymethyl)-thiophene-2-carbaldehyde
thiosemicarbazone (8): pale yellow powder, yield 72.2%, IR(KBr)
2.4. Tyrosinase activity assay
3162, 1628, 1545 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.39 (s,
;
The assay of inhibition of target compounds on the diphenolase
activity of mushroom tyrosinase was performed by our reported
procedure with minor modifications (Xie et al., 2016). L-DOPA
was used as substrate for the assay. The reaction media (3 mL)
for activity assay contained 2.8 mL 0.5 mM l-DOPA in 50 mM Na2
HPO4-NaH2PO4 buffer (pH = 6.8) and 0.1 mL of different concentra-
tions of inhibitor (dissolved by DMSO previously), and then 0.1 mL
1H, NH), 8.16 (s, 2H, NH2), 7.50(s, 1H, CH), 7.27 (d,1H, thiophene-
H), 6.99 (d,1H, thiophene-H), 4.62 (s, 2H, CH2), 3.49(t, 2H, CH2),
3.45(t, 2H, CH2). 13C NMR (400 MHz, DMSO-d6) d 177.72(1C,
C@S), 145.04(1C, C@N), 138.90(1C, thiophene-C), 137.99(1C,
thiophene-C), 130.68(1C, thiophene-C), 127.10(1C, thiophene-C),
71.91(1C, CH2), 67.42(1C, CH2), 60.58(1C, CH2). MS (ESI): m/z
258.1 [M-H]-.
of the aqueous solution of mushroom tyrosinase(166.5 lg/mL) was
5-Methoxyacetyl-thiophene-2-carbaldehyde
bazone (9): pale yellow crystal, yield 81.1%, IR(KBr) 3174, 1723,
1611, 1536 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 11.43 (s, 1H,
thiosemicar-
added to the mixture. The solution was immediately monitored by
measuring the linear increase in optical density at 475 nm of for-
mation of the DOPA chrome for 150 s using a Shimadzu UV-2450
spectrophotometer (Japan). The extent of inhibition by the addi-
tion of the sample was expressed as the percentage necessary for
50% inhibition (IC50), calculated by SPSS19. The value of inhibition
ratio of compounds on tyrosinase can be calculated by the follow-
ing equation:
;
NH), 8.16 (s, 2H, NH2), 7.54(s, 1H, CH), 7.29 (d,1H, thiophene-H),
7.10 (d,1H, thiophene-H), 5.20 (s, 2H, CH2), 2.03(s, 3H, CH3). 13C
NMR (400 MHz, DMSO-d6) d 177.95(1C, C@S), 170.13(1C, C@O),
141.07(1C, C@N), 140.11(1C, thiophene-C), 137.74(1C, thiophene-
C), 130.57(1C, thiophene-C), 129.25(1C, thiophene-C), 60.59(1C,
CH2), 21.06(1C, CH3). MS (ESI): m/z 258.0 [M + H]+.
5-Methyl-thiophene-2-carbaldehyde thiosemicarbazone (10):
Inhibition ratioð%Þ ¼ ð1 ꢀ OD1=OD2Þ ꢁ 100%
yellow powder, yield 86.6%, IR(KBr) 3148, 1590, 1524 cmꢀ1 1H
;
NMR (400 MHz, DMSO-d6) d 11.37 (s, 1H, NH), 8.14 (s, 2H, NH2),
7.44(s, 1H, CH), 7.23 (d,1H, thiophene-H), 6.81 (d,1H, thiophene-
where OD1 is the slope of reaction kinetics equation obtained from
reaction with inhibitor; OD2 is the slope of reaction kinetics
Please cite this article in press as: Xu, J., et al. Novel inhibitors of tyrosinase produced by the 4-substitution of TCT. Food Chemistry (2016), http://dx.doi.org/