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2
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1
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0.06
[I] (mM)
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2
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17. 5,50-Methylene-bis-salicylaldehyde (2). Purified by chromatography using 6:1
dichloromethane/petroleum ether as the eluent. Mp. 141–142 °C (Ref. 14 142–
143 °C). 1H NMR (CDCl3) d (ppm) 3.96 (s, 2H, –CH2), 6.95 (d, 2H, aromatic), 7.26
0
-20
-15
-10
-5
0
5
10
15
1/[S] (mM-1)
(s, 2H, aromatic), 7.34 (d, 2H, aromatic), 9.85 (s, 2H, CHO), 10.91 (s, 2H, OH). 13
C
Figure 2. Lineweaver–Burk plots for inhibition of compound
tyrosinase for catalysis of -DOPA. Concentrations of 2 were 0, 0.005 mM, 0.02 mM,
0.05 mM, respectively. The inset represent the secondary plot of 1/Vmax versus
concentration of compound 2, to determine the inhibition constant (Ki).
2 on mushroom
NMR (CDCl3) d (ppm): 39.35, 117.92, 120.41, 131.90, 133.15, 137.52, 160.14,
196.34. MS m/z (%): 256 (100), 227 (67), 209 (17), 199 (25), 181 (75), 152 (12),
135 (22), 107 (15), 77 (22).
L
18. Brahmbhalt, D. I.; Jayabalan, L.; Hirani, B. R.; Singh, S. Indian J. Chem., Sect. B
1998, 37, 683.
19. 3,30-Dimethoxy-5,50-methylene-bis-salicylaldehyde (3). Purified by chromato-
graphy using 6:1 dichloromethane/petroleum ether as the eluent. Mp. 157–
158 °C (Ref. 16156–158 °C). 1H NMR (CDCl3) d (ppm): 3.81 (s, 6H, –OCH3), 3.96 (s,
2H, –CH2), 6.85 (s, 2H, aromatic), 6.87 (s, 2H, aromatic), 9.81 (s, 2H, –CHO), 10.93
(s, 2H, –OH). 13C NMR (CDCl3) d (ppm): 40.44, 56.37, 113.32, 123.84, 127,58,
133.02, 150, 40, 196.38. MS m/z (%): 316 (100), 288 (12), 270 (5), 165 (8).
20. 4,40-Dimethoxy-5,50-methylene-bis-salicylaldehyde (4): This compound was
that bis-salicylaldehyde compounds act as better inhibitors of
mushroom tyrosinase than salicylaldehyde. Moreover, we deter-
mined that compound (2), the most effective inhibitor, acts as a
non-competitive inhibitor. We determined the effects of different
chemical groups on the inhibitory activity of these compounds.
First, the joining of two benzaldehyde rings facilitated the inhibi-
tory effects on the diphenolase activity of mushroom tyrosinase.
Second, the position of substitutions on the phenyl rings and the
type of substitutions critically affected the inhibitory activity. In-
deed, the substituents introduced in the meta position on the phe-
nyl rings may have hindered docking of the inhibitor to tyrosinase,
whereas substitutions in the para position did not affect the en-
zyme–inhibitor interaction. Furthermore, when the –OH groups
were in the ortho position of each benzaldehyde ring each com-
pound exhibited remarkable tyrosinase inhibition.
obtained with a yield of 58%: Mp.: 185–187 °C. IR (KBr): 1670 (–C@O) cmꢁ1
.
1H NMR (CDCl3) d (ppm): 3.81 (s, 6H, –OCH3), 3.99 (s, 2H, –CH2), 7.50 (d, 2H,
J = 8.7), 7.30 (d, 2H, J = 8.7), 9.67 (s, 2H, CHO), 11.44 (s, 2H, OH). 13C NMR
(CDCl3) d (ppm): 29.70, 56.04, 103.22, 132.14, 133.7, 134.5, 161.49, 164.77,
194.75. MS m/z (%): 316 (100), 288 (9), 270 (5), 165 (80), 151 (17), 137 (12), 77
(2). Anal. Calcd for C17H16O6: C, 64.55; H, 5.10. Found: C, 64.7; H, 5.22.
21. Begala, M.; Delogu, G.; Maccioni, E.; Podda, G.; Tocco, G.; Quezada, E.; Uriarte,
E.; Fedrigo, M. A.; Favretto, D.; Traldi, P. Rapid Commun. Mass Spectrom. 2001,
15, 1000.
22. Nechifor, M. React. Funct. Polym. 2009, 69, 27.
23. 3,30-Dihydroxy-5,50-methylene-bis-salicylaldehyde (5). It was obtained with a
yield of 51%: mp: 190–192 °C (Ref.21 188–189 °C, yield 41%).
24. 4,40-Dihydroxy-5,50-methylene-bis-salicylaldehyde (6): This compound was
obtained with a yield of 46%: Mp: 193–195 °C. IR (KBr): 1650 (–C@O) cmꢁ1
.
These results suggest that these novel compounds may serve as
structural templates for the design and development of novel
tyrosinase inhibitors.
1H NMR (DMSO-d6) d (ppm): 3.92 (s, 2H, –CH2), 7.30 (d, 2H, J = 9.0), 7.65 (d, 2H,
J = 9.0), 9.81 (s, 2H, CHO), 10.81 (s, 2H, OH), 11.63 (s, 2H,OH). 13C NMR (DMSO-
d6) d (ppm): 43.0, 120.0, 122.4, 135.0, 137.5, 140.6, 163.2, 198.0. MS m/z (%):
288 (100), 260 (17), 151 (91), 137 (32), 77 (5). Anal. Calcd for C15H12O6: C,
62.50; H, 4.20. Found: C, 62.54; H, 4.22.
Acknowledgments
25. Pre-incubation with the enzyme consisted of a 1/15 M phosphoric acid buffer
solution (pH 6.8, 1.8 mL), an aqueous solution of mushroom tyrosinase (1000
U/mL, Sigma Chemical Co., 0.1 mL) and DMSO (0.1 mL) with or without the
The authors acknowledge the University of Cagliari (project
grant ex 60% 2007), Fondazione Banco Sardegna 2007 and MIUR
2008 for their financial support.
sample. The mixture was incubated at 25 °C for 10 min. Then, a 1.05 mM of L-
3,4-dihydroxyphenylalanine (DOPA) solution (1 mL) was added and the
reaction was monitored at 475 nm for 5 min. The percent inhibition of
tyrosinase activity was calculated as: inhibition (%) = (A ꢁ B)/A ꢀ 100, where
A represents the difference in the absorbance of control sample between 0.5
and 1.0 min, and B represents the difference in absorbance of the test sample
between 0.5 and 1.0 min. The IC50 value, a concentration giving 50% inhibition
of tyrosinase activity, was determined by interpolation of dose–response
curves. The activity of mushroom tyrosinase was determinate by
spectrophotometric techniques (Varian Cary 50). Kojic acid was used as a
reference tyrosinase inhibitor.
References and notes
1. Lee, S. E.; Kim, M. K.; Lee, S. G.; Ahn, Y. J.; Lee, H. S. Food Sci. Biotechnol. 2000, 9,
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2. Kubo, I.; Kinst-Hori, I. J. Agric. Food. Chem. 1999, 47, 4574.
3. Robb, D. A.. In Lontie, R., Ed.; Copper Proteins and Copper Enzyme; CRC Press:
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