Synthesis and biological evaluation of novel 4-hydroxybenzaldehyde derivatives as tyrosinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.11.007

A series of novel 4-hydroxybenzaldehyde derivatives were synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were investigated. Most of target compounds had more potent inhibitory activities than the parent compound 4-hydroxybenzaldehyde (IC₅₀ = 1.22 mM). Interestingly, compound 3c bearing a dimethoxyl phosphate was found to be the most potent inhibitor with IC₅₀ value of 0.059 mM. The inhibition kinetics analyzed by Lineweaver-Burk plots revealed that compound 3c was a non-competitive inhibitor (K_I = 0.0368 mM). In particular, compound 3c showed no side effects at dose of 1600 mg/kg in mice. These results suggested that such compounds might be served as lead compounds for further designing new potential tyrosinase inhibitors.

Full text of DOI:10.1016/j.ejmech.2009.11.007

European Journal of Medicinal Chemistry 45 (2010) 639–646
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel 4-hydroxybenzaldehyde derivatives
as tyrosinase inhibitors
,
b
a
a
a
a
a,
*
Wei Yi a, Rihui Cao a , Wenlie Peng , Huan Wen , Qin Yan , Binhua Zhou , Lin Ma , Huacan Song
*
a School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
b State Key Laboratory of Biocontrol, School of Life Science, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275, PR 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 novel 4-hydroxybenzaldehyde derivatives were synthesized and their inhibitory effects on the
diphenolase activity of mushroom tyrosinase were investigated. Most of target compounds had more
potent inhibitory activities than the parent compound 4-hydroxybenzaldehyde (IC50 ¼ 1.22 mM). Inter-
estingly, compound 3c bearing a dimethoxyl phosphate was found to be the most potent inhibitor with
IC50 value of 0.059 mM. The inhibition kinetics analyzed by Lineweaver–Burk plots revealed that
compound 3c was a non-competitive inhibitor (KI ¼ 0.0368 mM). In particular, compound 3c showed no
side effects at dose of 1600 mg/kg in mice. These results suggested that such compounds might be served
as lead compounds for further designing new potential tyrosinase inhibitors.
Received 19 May 2008
Received in revised form
21 October 2009
Accepted 2 November 2009
Available online 10 November 2009
Keywords:
Synthesis
4-Hydroxybenzaldehyde derivatives
Tyrosinase inhibitors
Inhibition mechanism
Acute toxicity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
sources, such as 4-hydroxybenzaldehyde [4], anisaldehyde [4],
cuminaldehyde [11] and 4-methoxysalicylaldehyde [12] (Fig. 1).
Tyrosinase is
a
multifunctional copper-containing enzyme
Unfortunately, most of 4-substituted benzaldehyde derivatives
cannot be considered of practical use because of their lower
activities or serious side effects. Therefore, it is still necessary to
search and discover novel tyrosinase inhibitors with higher activ-
ities and lower side effects.
widely distributed in microorganisms, plants and animals [1]. It is
well known that tyrosinase can catalyzes two distinct reactions of
melanin synthesis, the hydroxylation of monophenols to o-diphe-
nols (monophenolase activity) and the oxidation of o-diphenols to
o-quinones (diphenolase activity) [2]. Previous reports confirmed
that tyrosinase was involved, not only in melanizing in animals, but
also in browning in plants. 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 [3]. Therefore, the control of tyrosinase activity is of
great importance in preventing the synthesis of melanin in the
browning of fruits and vegetables and the accumulation of an
excessive level of epidermal pigmentation in animals.
Tyrosinase inhibitors have become increasingly important in
medication [4], cosmetic industry [5] and food industry due to
decreasing the excessive accumulation of pigmentation resulting
from the enzyme action [6–10]. So far, numerous 4-substituted
benzaldehyde derivatives and their analogues as potential tyrosi-
nase inhibitors have been discovered from natural or synthetic
Previous literatures [4,12] reported that the 4-substituted
benzaldehyde derivatives exhibited the tyrosinase inhibitory
activity by the formation of a Schiff base between the aldehyde
group and a primary amino group of the enzyme, and the inductive
effect of the electron-donating group at 4-position are necessary for
the actions [11]. However, the inductive effect cannot be the only
explanation for the binding stability of the enzyme and the inhibitor
[1]. In addition, it is well known that phosphates and amino acids
are involved in the metabolism of organism and exhibit various
biochemical and pharmacological functions. More recently, our
groups reported that condensation products of 4-formylphenoxy-
acetic acid and the appropriate amino acid methyl esters displayed
potent acetylcholinesterase inhibitory activities [13].
Taking advantage of previously developed structure–activitiy
relationships (SARs) of 4-substituted benzaldehydes as tyrosinase
inhibitors, in the present investigation,
a series of novel
4-hydroxybenzaldehyde derivatives were synthesized and their
inhibitory effects on the diphenolase activity of mushroom tyrosi-
nase were evaluated. Meanwhile, the inhibition mechanism and
* Corresponding authors. Tel.: þ86 20 84110918; fax: þ86 20 84112245.
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), yjhxhc@mail.sysu.edu.cn
(H. Song).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.007

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W. Yi et al. / European Journal of Medicinal Chemistry 45 (2010) 639–646
preliminary acute toxicity of the most potent compound were also
H3CO
CHO
investigated. The purpose of this study was to investigate effects of
substituents at postion-4 of benzene on tyrosinase inhibitory
activity, with the ultimate aim of developing novel tyrosinase
inhibitors with potent activities, together with lower side effects.
HO
CHO
Anisaldehyde
4-Hydroxybenzaldehyde
OH
CHO
4-methoxysalicylaldehyde
2. Chemistry
H3CO
CHO
The synthesis of 4-hydroxybenzaldehyde derivatives was
summarized in Scheme 1–3. The reaction of 4-hydroxy-
benzaldehyde with 2-chloroethanol and epichlorohydrin, respec-
tively, in 1,4-dioxane and water gave the expected derivatives 3a
and 2e in good yield [14,15]. The intermediate 2-butoxyethyl-4-
methylbenzene sulfonate was prepared from the commercially
available 2-butoxyethanol and TsCl according to already published
methods [16]. Then, 2-butoxyethyl-4-methylbenzene sulfonate was
reacted with 4-hydroxybenzaldehyde in anhydrous THF in the
presence of anhydrous potassium carbonate to afford the derivative
2a. Following the similar procedure, compounds 2b–d could be
carried out smoothly in good yield. Compound 2e was hydrolyzed
in sodium hydroxide solution to afford compound 2f. Esterification
of compound 2e with sodium methoxide in methanol provided
compound 2g. The intermediate 2-(4-formyl-phenoxy)-methox-
yethyl acetate was prepared from the starting materials 2-oxo-1,4-
butanediol diacetate ester, 4-methylbenzenesulfonic acid and
4-hydroxybenzaldehyde according to the usual methods [17,18],
followed by hydrolyzation in NaOH solution to provide the target
compound 2h. Compound 2i was given according to the same
procedure described for compound 2h (Scheme 1).
Cuminaldehyde
Fig. 1. Chemical structures of known tyrosinase inhibitors.
i
OR1
OHC
OHC
OH
2a R1=CH2CH2OCH2CH2CH2CH3
2b R1=CH2CH2OCH2CH2OCH3
2c R1=CH2CH2CH2CH2OCH3
2d R1=CH2CH2OCH3
1
ii
iv
OHC
OCH2
O
2e
OR1
OHC
iii
2h R1 =CH2OCH2CH2OH
2i R1 =CH2OCH(CH2OH)2
OR1
OHC
2f R1 =CH2CHOHCH2OH
2g R1 =CH2CHOHCH2OCH3
Compound 3a reacted with phosphorous oxychloride in anhy-
drous dichloromethane, followed by hydrolyzation in sodium
hydroxide solution to provide compound 3b, and followed by
esterification with anhydrous methanol in the presence of sodium
methoxide and with anhydrous ethanol in the presence of sodium
ethoxide to provide compound 3c and 3d, respectively. Esterifica-
tion of compound 2a with succinic anhydride in anhydrous
dichloromethane gave compound 3e (Scheme 2).
Scheme 1. Synthesis of compounds 2a–i. Reagents and conditions: (i) 2-butoxyethanol
or 2-(2-methoxyethoxy)ethanol or 4-methoxybutan-1-ol or 2-methoxyethanol, NaOH,
TsCl, 0 ꢀC, 5 h; K2CO3, THF, reflux, 16 h (ii) epichlorohydrin, 1,4-dioxane, NaOH, H2O,
reflux, 2 h (iii) NaOH, H2O, reflux, 3 h; methanol, sodium methoxide, reflux, 24 h (iv)
2-oxo-1,4-butanediol diacetate ester or 2-[(propionyloxy)methyl-hydrogen]-1,3-pro-
panediol diacetate, toluene, 4-methylbenzenesulfonic acid, 100 ꢀC, 0.5 h, then reflux,
10 h; methanol, sodium methoxide, rt, 1 h.
i
OHC
OCH2CH2OH
OH
OHC
1
3a
iv
ii
O
O
OHC
OCH2CH2OP OH
OH
OHC
OCH2CH2OCCH2CH2COOH
3b
3e
iii
O
OCH2CH2OP OR2
OR2
OHC
3c R2=CH3
3d R2=CH2CH3
Scheme 2. Synthesis of compounds 3a–e. Reagents and conditions: (i) 2-chloroethanol, 1,4-dioxane, NaOH, H2O, reflux, 2 h (ii) Et3N, CH2Cl2, POCl3, 0 ꢀC, 1.5 h; NaOH, rt, 5 h
(iii) methanol/sodium methoxide or ethanol/sodium ethoxide, reflux, 2 h (iv) Et3N, CH2Cl2, succinic anhydride, 100 ꢀC, 0.5 h; DMAP, reflux, 4 h.

W. Yi et al. / European Journal of Medicinal Chemistry 45 (2010) 639–646
641
H
N
OH
O
O
COOCH3
COOH
COOCH3
O
R3
O
ii
i
iii
CHO
1
CHO
5
CHO
4
CHO
6a-i
6d R3= CH(CH3)C2H5
6c R3 = CH 2CH2SCH3
6a R3 = H
6b R3= CH3
6e R3 = CH2CH(CH3)2
6f R3 =
CH2Ph
N
O
H2
C
O
6h R3 = CH2Ph(p-OH)
C
6g R3 =
6i
O
OCH3
N
H
CHO
Scheme 3. Synthesis of compounds 6a–i. Reagents and conditions: (i) ClCH2COOCH3, K2CO3, acetone, 60 ꢀC, 5 h (ii) NaOH, H2O; Hþ, H2O. (iii) DCC, DAMP, CHCl3, rt, overnight.
The reaction of compound 1 with methyl chloroacetate gave
compound 4, followed by hydrolyzation in sodium hydroxide to
provide 4-formylphenoxyacetic acid 5. The condensation of various
amino acid methyl esters with compound 5 in the presence of N,N0-
dicyclohexyl-carbodiimide (DCC) and 4-(dimethylamino)pyridine
(DMAP) produced compound 6a–i [13] (Scheme 3).
And compounds 2b–e, 2g–f, 3b–e, 6a–d and 6f–i displayed inhib-
itory effects on the diphenolase activity of mushroom tyrosinase in
the sub-millimolar ranges. Interestingly, compound 3c bearing
a dimethoxyl phosphate substituent was found to be the most
potent inhibitor with IC50 value of 0.059 mM. Replacement of
methoxy group of compound 3c with ethoxy group led to
a dramatic decline in tyrosinase inhibitory activity (compound 3d,
IC50 ¼ 0.55 mM). This might be due to molecular dimension of the
ethoxy substitution to impede this molecule to interact well with
the enzyme. In addition, compound 3b (IC50 ¼ 0.82 mM) bearing
a free phosphate group and compound 3e (IC50 ¼ 0.62 mM) having
a free carboxyl substituent showed moderate tyrosinase inhibitory
activities. These results suggested that introducing appropriate
hydrophobic subunits into position-4 of benzaldehyde might be
facilitated their inhibitory effects on the diphenolase activity of
mushroom tyrosinase, and dimethoxyl phosphate group repre-
sented the most optimal structure for these compounds to exhibit
remarkable tyrosinase inhibitory effects.
3. Results and discussion
3.1. Inhibitory effects on the diphenolase activity of mushroom
tyrosinase
Taking compound 2a–i, 3a–e and 6a–i as the effectors, we
investigated the inhibitory effects of 4-hydroxybenzaldehyde
derivatives on the diphenolase activity of mushroom tyrosinase for
the oxidation of L-DOPA. Fig. 2 showed that the remaining enzyme
activity rapidly decreased with the increasing concentrations of
compound 3c. The IC50 value of all compounds investigated was
summarized in Table 1.
As shown in Table 1, all compounds exhibited tyrosinase
inhibitory effects with IC50 values ranged from 0.059 to 3.26 mM.
Compound 3a bearing a hydroxyethoxyl group at position-4 of
phenyl ring exhibited weak tyrosinase inhibitory activity with IC50
value of 2.55 mM. Interestingly, replacement of the hydroxyl group
of compound 3a with methoxy (compound 2d, IC50 ¼ 0.66 mM)
and methoxyethoxy (compound 2b, IC50 ¼ 0.9 mM), respectively,
100
80
60
40
20
0
Table 1
Inhibitory effects on mushroom tyrosinase of 4-substituted benzaldehyde
derivatives.
Compound
IC50(mmol/L)a
Compound
IC50(mmol/L)a
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3.26
0.90
0.62
0.66
0.59
1.26
0.75
0.37
0.27
2.55
0.82
0.059
0.55
3e
6a
6b
6c
6d
6e
6f
6g
0.62
0.17
0.16
0.40
0.46
1.65
0.48
0.11
0.22
0.76
1.22b
0.41c
6h
6i
1
4-methoxyinnamic acid
0.0
0.2
0.4
0.6
0.8
1.0
a
Values were determined from logarithmic concentration–inhibition curves (at
least eight points) and are given as means of three experiments.
[I] (mM)
b
Fig. 2. Effect of compound 3c on the diphenolase activity of mushroom tyrosinase for
the catalysis of L-DOPA at 25 ꢀC.
Values in the literature [4] is 1.20 mM.
Values in the literature [20] is 0.42 mM.
c

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W. Yi et al. / European Journal of Medicinal Chemistry 45 (2010) 639–646
Fig. 3. Lineweaver–Burk plots for inhibition of selected compounds 3c, 6a, 6b and 6g on mushroom tyrosinase for the catalysis of L-DOPA. Concentrations of 3c, 6a, 6b and 6g for
curves 1–3 were 0, 12.94 M, 25.88 M; 0, 0.05 mM, 0.10 mM; 0, 0.055 mM, 0.11 mM; 0, 0.0503 mM, 0.106 mM, respectively. The inset represents the secondary plot of 1/Vmax
versus concentration of 3c, 6a, 6b and 6g, respectively, to determine the inhibition constants (KI).
m
m
led to
a
dramatic increase in inhibitory activities. Whereas
indicated that the additional oxygen in the side chain might play an
important role in determining their inhibitory effects on the
diphenlosase activity of mushroom tyrosinase.
compound 2a (IC50 ¼ 3.26 mM) having a butoxyethoxy substituent
was 4.9 times less active than compound 2d (IC50 ¼ 0.66 mM).
These results indicated that the terminal methoxy group contrib-
uted to tyrosinase inhibitory activities, but the elongation of the
alkyl chain might retard the binding of inhibitors with the active
site of tyrosinase, leading to a decrease of tyrosinase inhibition
activity.
Compound 2e (IC50 ¼ 0.59 mM) bearing ethylene oxide moiety
in the side chain showed potent tyrosinase inhibitory activities,
whereas its opened-ring congener 2f (IC50 ¼ 1.26 mM) exhibited
weaker inhibitory effect. Interestingly, replacement of terminal
hydroxyl group of compound 2f with a methoxy substituent to give
compound 2g (IC50 ¼ 0.75 mM) demonstrated more potent tyrosi-
nase inhibitory activities. These results suggested that the opened-
ring of ethylene oxide might be detrimental to tyrosinase inhibitory
activity.
Our previous report [13] described that the condensation
products of 4-formylphenxoyacetic acid and various amino acid
methyl esters exhibited potent acetylcholinesterase inhibitory
activities. In this investigation, these compounds were subjected
for tyrosinase inhibition assays. As shown in Table 1, all compounds
(except 6e) exhibited more potent inhibitory activities than the
parent compound 4-hydroxybenzadehyde (IC50 ¼ 1.22 mM). Espe-
cially, compounds 6a, 6b and 6g displayed significant tyrosinase
inhibitory activities with IC50 value of 0.17, 0.16 and 0.11 mM,
respectively. These results suggested that the introduction of
appropriate amino acid moieties into position-4 of benzaldehyde
might be enhanced their inhibitory effects on the diphenolase
activities of mushroom tyrosinase, and
L-glycine (6a), L-alanine (6b)
and -tryptophan (6g) was more favorable.
L
Unlike compound 3a having a hydroxyethoxyl group at posi-
tion-4 of phenyl ring, compound 2h (IC50 ¼ 0.37 mM) bearing an
additional oxygen in the side chain attached to phenyl ring
exhibited potent tyrosinase inhibitory activity. Similarly, the
inhibitory activity of compound 2i (IC50 ¼ 0.27 mM) was 4.5 times
than that of compound 2f (IC50 ¼ 1.26 mM). These resulted
3.2. Inhibition mechanism of the selected compounds on mushroom
tyrosinase
The inhibitory mechanism of the selected compounds 3c, 6a, 6b
and 6g on mushroom tyrosinase for the oxidation of L-DOPA was

W. Yi et al. / European Journal of Medicinal Chemistry 45 (2010) 639–646
643
Table 2
affected tyrosinase activity; (2) the elongation of the alkyl chain
might retard the binding of inhibitors with the active site of
tyrosinase, leading to a decrease of tyrosinase inhibition activities;
(3) the molecular symmetry might be play a very important role in
determining enzyme activities; (4) the introduction of the appro-
Kinetics and inhibition constants of compounds 3c, 6a, 6b and 6g on the activity of
mushroom tyrosinase.
Compound
Inhibition type
Inhibition constant (KI) (mM)
3c
6a
6b
6g
Non-competitive
Non-competitive
Non-competitive
Non-competitive
0.0368
0.117
0.125
0.054
priate amino acid moieties such as L-glycine, L-alanine, L-trypto-
phan enhanced inhibitory activities. Interestingly, compound 3c
bearing a dimethoxyl phosphate was found to be the most potent
inhibitor with IC50 value of 0.059 mM. The inhibition kinetics
analyzed by Lineweaver–Burk plots revealed that compound 3c
determined from Lineweaver–Burk double reciprocal plots. Fig. 3
showed the double-reciprocal plots of the enzyme inhibited by the
above-mentioned compounds. The results displayed that the plots
of 1/V versus 1/[S] gave three straight lines with different slopes,
but intersected on the horizontal axis. Accompanying the increase
of the concentration of compound, the value of Vmax descended but
the values of Km remained the same, which suggested that these
compounds was a non-competitive inhibitor of the tyrosinase. The
behaviour indicated that these compounds could bind with both
the free enzyme and the enzyme–substrate complex, and the
equilibrium constants are the same [19]. The inhibition constants
for compounds 3c, 6a, 6b and 6g binding with the free enzyme (KI)
were determined by the plot of the values of intercept versus the
concentration of the corresponding compound as shown in Fig. 3.
The values obtained are summarized in Table 2. As shown in Table
2, the inhibition constant (KI) of compound 3c was less than that of
compounds 6a, 6b and 6g, suggesting that compound 3c had most
potent inhibitory effect.
The reason for the exceptionally potent inhibition activity of
compound 3c might be that, to a certain extent, intermolecular
hydrogen bond formed between the dimethoxyl phosphate group
of compound 3c and the sulfhydryl, amino, carboxyl or hydroxyl in
tyrosinase, and the interaction increased the chance for aldehyde
group of compound 3c to form Schiff base with amino group
locating in the outside of enzyme active center [20]. Undoubtedly,
the more the numbers of hydrogen bond formed between
compound 3c and tyrosinase, the tighter and the more stable the
interaction between 3c and tyrosinase. This assumption is consis-
tent with the deduction in the previous report that the hydrogen-
bonding interactions could stabilize the oxy-form of Streptomyces
glaucescens tyrosinase [21]. However, the conclusive interpretation
remains to be clarified since the structure of tyrosinase used for this
study has not yet been understood completely.
was
a
non-competitive inhibitor (KI ¼ 0.0368 mM). Safety is
a primary consideration for tyrosinase inhibitors, especially for
those materials used in food and cosmetic products. It is worth
noting that compound 3c did not showed any side effects at dose of
1600 mg/kg in mice. All these data suggested that such compounds
might serve as candidates for the treatment of tyrosinase based
disorders and as lead compounds for further designing new
potential tyrosinase inhibitors.
5. Experimental protocols
5.1. Reagents and general procedures
Melting points were determined on a WRS-1B digital instru-
ment without correction. 1H and 13C NMR spectra were recorded on
a Varian Mercury-Plus 300 NMR instrument (1H 300 MHz; 13C
75 MHz) in either CDCl3 or DMSO-d6. Abbreviations for data quoted
are s, singlet; br s, broad singlet; d, doublet; t, triplet; dd, doublet of
doublets; m, multiplet. Mass spectra were recorded on a Thermo
Finnigan LCQ DECAXP ion trap mass spectrometer or VS ZAB-HS
spectrometer. IR spectra were recorded as potassium bromide
pellets on a Bruker Equinox 55 FT/IR spectrometer. Elemental
analyses (C, H, N) were carried out on an Elementary Vario EL series
elemental analyzer and the results were within ꢁ0.4%. Thin-layer
chromatographies were done on pre-coated silica gel 60 F254
plates (Merck).
Tyrosinase and L-3, 4-dhydroxyphenylalanine (L-DOPA) were
purchased from Sigma–Aldrich Chemical Co. Other chemicals were
purchased from commercial suppliers and were dried and purified
when necessary. The water used was re-distillated and ion-free.
Compounds 4, 5 and 6a–i were synthesized, purified and charac-
terized as previously described [13].
5.2. Synthesis
3.3. Acute toxicity of compound 3c in mice
5.2.1. 4-(2-Butoxyethoxy)benzaldehyde (2a)
Compound 3c, the most potent inhibitor of this investigation,
was selected to evaluate acute toxicity in mice. Clinical symptom
was measured for 20 days after the oral single gavage administra-
tion of 1600 mg/kg. The results showed that all of the mice after
administration at dose of 1600 mg/kg body weight/day did not
show any mortality, and autopsy of the animals at the end of the
experimental period (20 days) revealed no apparent changes in any
organs. The results indicated that the compound 3c was safe at dose
of 1600 mg/kg in mice.
A
mixture of 2-butoxyethanol (6.0 g, 50 mmol), sodium
hydroxide (4.0 g, 100 mmol) and water (10 ml) was cooled to 0 ꢀC,
and then TsCl (20.96 g, 11 mmol) in anhydrous THF was added
dropwise and stirred for 5 h. The resulting mixture was treated
with sodium hydroxide (1.6 g, 40 mmol) and extracted with chlo-
roform. The organic layer was dried over anhydrous magnesium
sulfate, filtered and evaporated. The crude product was purified by
silica gel column chromatography (ethyl acetate: petroleum
ether ¼ 1:5) to afford 2-butoxyethyl-4-methylbenzene sulfonate
(11.88 g, 92.0%) as a liquid. 1H NMR (CDCl3, 300 MHz)
d: 7.79 (d,
4. Conclusion
J ¼ 8.4 Hz, 2H, ArH), 7.32 (d, J ¼ 8.4 Hz, 2H, ArH), 4.60 (t, J ¼ 5.3 Hz,
2H, CH2), 4.15 (t, J ¼ 5.3 Hz, 2H, CH2), 3.40 (t, J ¼ 4.6 Hz, 2H, CH2),
2.44 (s, 3H, CH3), 1.46 (m, 2H, CH2), 1.31 (m, 2H, CH2), 0.89 (t,
J ¼ 4.6 Hz, 3H, CH3).
The present investigation reported the effects of 4-hydroxy-
benzaldehyde derivatives on the diphenolase activity of mushroom
tyrosinase for the oxidation of
that most of target compounds had more potent inhibitory activi-
ties than the parent compound 4-hydroxybenzaldehyde. Prelimi-
nary SARs analysis indicated that: (1) the numbers of oxygen atoms
contained in the chain attached position-4 of benzaldehyde
L
-DOPA. The results demonstrated
A mixture of 2-butoxyethyl-4-methylbenzene sulfonate (2.74 g,
10 mmol), 4-hydroxybenzaldehyde (1.22 g, 10 mmol), anhydrous
potassium carbonate (2.07 g, 15 mmol) and anhydrous THF (25 ml)
was refluxed for 16 h. The reaction mixture was poured into ice
water and extracted with chloroform. The organic layer was dried

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W. Yi et al. / European Journal of Medicinal Chemistry 45 (2010) 639–646
over anhydrous magnesium sulfate, filtered and evaporated. The
crude product was purified by silica gel column chromatography
(ethyl acetate:petroleum ether ¼ 1:5) to give pale yellow oil (1.63 g,
5.2.6. 4-(2,3-Dihydroxypropoxy)benzaldehyde (2f)
A mixture of compound 2e (1.78 g, 10 mmol) and 1 M NaOH
solution (10 mL) was refluxed for 3 h. The solvent was removed
under reduced pressure and the residue was extracted with ethyl
acetate. The organic phase was dried over anhydrous sodium
sulfate, filtered and evaporated. The crude product was purified by
silica gel column chromatography (ethyl acetate: petroleum
ether ¼ 1:4) to afford compound 2f (1.17 g, 57%) as yellow oil. ESI-
74%). ESI-MS: m/z 223 (M þ 1). IR (KBr, cmꢂ1
) n: 2958(C–H), 1693
(CHO),1601 (Ph),1509 (Ph), 1259 (C–O), 1161 (C–O), 1126 (C–O), 833
(Ph); 1H NMR (CDCl3, 300 MHz)
d: 9.88 (s, 1H, CHO), 7.82(d,
J ¼ 8.3 Hz, 2H, ArH), 7.02 (d, J ¼ 8.3 Hz, 2H, ArH), 4.20 (t, J ¼ 5.5 Hz,
2H, CH2), 3.80 (t, J ¼ 5.5 Hz, 2H, CH2), 3.54 (t, J ¼ 4.8 Hz, 2H, CH2),
1.59 (m, 2H, CH2), 1.35 (m, 2H, CH2), 0.93 (t, J ¼ 4.5 Hz, 3H, CH3); 13
C
MS: m/z 195 m/z (M ꢂ 1). IR (KBr, cmꢂ1
)
n
: 3370, 1684, 1600, 150,
NMR (CDCl3, 75 MHz)
d
: 190.4, 163.6, 131.6, 129.7, 114.6, 71.2, 68.7,
1309,1164,1112,1034, 839; 1H NMR (CDCl3, 300 MHz)
d: 9.82 (s, 1H,
67.6, 31.5, 19.1, 13.8; Anal. Calcd for C13H18O3: C, 70.27; H, 8.11;
found: C, 70.09; H, 8.34.
CHO), 7.78 (d, J ¼ 8.8 Hz, 2H, ArH), 6.97 (d, J ¼ 8.8 Hz, 2H, ArH), 4.26
(m, 1H, CH), 4.19 (m, 2H, CH2), 3.78 (m, 2H, CH2); 13C NMR (CDCl3,
75 MHz) d: 190.3, 163.0, 131.6, 130.0, 114.6, 72.5, 70.2, 68.8; Anal.
5.2.2. 4-[2-(2-Methoxyethoxy)ethoxy]benzaldehyde (2b)
Calcd for C10H12O4: C, 61.22; H, 6.12; found: C, 62.45; H, 5.98.
Compound 2b (1.62 g, 73%) was obtained as pale yellow solid by
the same procedure described for 2a from 4-hydroxybenzaldehyde
(1.22 g, 10 mmol) and 2-(2-methoxyethoxy)ethanol (ꢂ61.0 g,
5.2.7. 4-(2-Hydroxy-3-methoxypropoxy)benzaldehyde (2g)
A mixture of compound 2e (1.78 g, 10 mmol) and sodium
methoxide in methanol (0.2 M, 20 ml) was refluxed for 24 h. The
solvent was removed under reduced pressure and the residue was
extracted with ethyl acetate. The organic phase was dried over
anhydrous sodium sulfate, filtered and evaporated. The crude
product was purified by silica gel column chromatography (ethyl
acetate: petroleum ether ¼ 1:4) to afford compound 2g (1.17 g, 57%)
as pale yellow oil. ESI-MS: m/z 211 (M þ 1). 1H NMR (CDCl3,
50 mmol). Mp 44–45 ꢀC. ESI-MS: m/z 225 (M þ 1). IR (KBr, cm
) n:
2891, 1689, 1601, 1509, 1129, 1104, 1034, 836; 1H NMR (CDCl3,
300 MHz)
d
: 9.89 (s, 1H, CHO), 7.81 (d, J ¼ 8.7 Hz, 2H, ArH), 7.02 (d,
J ¼ 8.7 Hz, 2H, ArH), 4.23 (t, J ¼ 4.8 Hz, 2H, CH2), 3.90 (t, J ¼ 4.8 Hz,
2H, CH2), 3.73 (dd, J ¼ 2.0 Hz, 4.8 Hz, 2H, CH2), 3.59 (dd, 2H,
J ¼ 2.0 Hz, 4.8 Hz, CH2), 3.34 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz)
d: 190.4, 163.2, 131.6, 129.7, 114.6, 71.7, 70.6, 69.3, 67.2, 58.9; Anal.
Calcd for C12H16O4: C, 64.27, H, 7.19; found: C, 64.35; H, 7.12.
300 MHz)
J ¼ 8.6 Hz, 2H, ArH), 4.18 (m, 1H, CH), 4.11 (m, 2H, CH2), 3.58 (m, 2H,
CH2); 3.43 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz)
: 190.5, 163.3,
d
: 9.87 (s, 1H, CHO), 7.82 (d, J ¼ 8.6 Hz, 2H, ArH), 7.02 (d,
5.2.3. 4-(4-Methoxybutoxy)benzaldehyde (2c)
d
Compound 2c (1.43 g, 69.0%) was obtained as yellow oil by the
same procedure described for 2a from 4-hydroxybenzaldehyde
(1.22 g, 10 mmol) and 4-methoxybutan-1-ol (5.21 g, 50 mmol). ESI-
131.8, 130.0, 114.6, 73.0, 68.9, 68.6, 59.1; Anal. Calcd for C11H11O4: C,
62.85; H, 6.71; found: C, 63.11; H, 6.58.
MS: m/z 209 (M þ 1). IR (KBr, cmꢂ1
)
n
: 2950, 1691, 1600, 1509, 1253,
: 9.90 (s, 1H, CHO), 7.76
5.2.8. 4-(2-Hydroxyethoxymethoxyl)benzaldehyde (2h)
1160, 1125, 834; 1H NMR (CDCl3, 300 MHz)
d
2-Oxo-1,4-butanediol diacetate ester (4.4 g, 25 mmol) in
toluene (50 ml) was added to 4-methylbenzenesulfonic acid
(0.35 g,1.5 mmol). The mixture was stirred at 100 ꢀC for 0.5 h. Then,
4-hydroxybenzaldehyde (3.05 g, 25 mmol) was added and the
solution was refluxed for 10 h. The resulting mixture was evapo-
rated under reduced pressure to remove the toluene and extracted
with chloroform. The organic layer was washed with water and
sodium hydroxide (1 M), then dried over anhydrous magnesium
sulfate, filtered and evaporated. The crude product was purified by
silica gel column chromatography, and the intermediate 2-(4-for-
mylphenoxy)methoxyethyl acetate (3.37 g, 57%) was obtained as
(d, J ¼ 8.4 Hz, 2H, ArH), 7.01(d, J ¼ 8.4 Hz, 2H, ArH), 4.15 (t,
J ¼ 4.5 Hz, 2H, CH2), 3.76 (t, J ¼ 6.3 Hz, CH2, 2H,), 3.37 (s, 3H, OCH3),
1.73 (m, 2H, CH2), 1.65 (m, 2H, CH2); 13C NMR (CDCl3, 75 MHz)
d:
190.4,163.9,131.7,129.6,114.5, 72.1, 68.1, 58.4, 26.1, 25.9; Anal. Calcd
for C12H16O3: C, 69.21; H, 7.74; found: C, 69.12; H, 7.88.
5.2.4. 4-(2-Methoxyethoxy)benzaldehyde (2d)
Compound 2d (1.48 g, 82%) was obtained as yellow oil by the
same procedure described for 2a using 4-hydroxybenzaldehyde
(1.22 g, 10 mmol) and 2-methoxyethanol (3.83 g, 50 mmol) as
starting materials. EI-MS: m/z 180 (M þ 1). IR (KBr, cmꢂ1
)
n
: 2925,
colorless oil. 1H NMR (CDCl3, 300 MHz)
d: 9.88 (s, 1H, CHO), 7.82 (d,
1690, 1600, 1508, 1258, 1159, 1124, 833; 1H NMR (CDCl3, 300 MHz)
J ¼ 8.7 Hz, 2H, ArH), 7.15 (d, J ¼ 8.7 Hz, 2H, ArH), 5.32 (s, 2H, CH2),
d
: 9.90 (s, 1H, CHO), 7.79 (d, J ¼ 8.4 Hz, 2H, ArH), 7.02 (d, J ¼ 8.4 Hz,
2H, ArH), 4.19 (t, J ¼ 5.1 Hz, 2H, CH2), 3.79 (t, J ¼ 5.1 Hz, 2H, CH2),
3.47 (s, 3H, CH3); 13C NMR (CDCl3, 75 MHz)
: 190.3, 163.4, 131.5,
4.23 (m, 2H, CH2), 3.89 (m, 2H, CH2), 1.97 (s, 3H, CH3).
Then, the intermediate 2-(4-formylphenoxy)methoxyethyl
acetate (2.0 g, 8.4 mmol) was added to a solution of sodium
methoxide-methanol (0.2 M, 20 ml). The reaction mixture was
stirred for 1 h at room temperature, and methanol was evaporated
under reduced pressure. The crude product was purified by silica
gel column chromatography (ethyl acetate:petroleum ether ¼ 3:7).
Compound 2h (1.60 g, 97.0%) was obtained as a liquid. ESI-MS: m/z
d
129.7, 114.5, 70.4, 67.4, 58.9; Anal. Calcd for C10H12O3: C, 66.67; H,
6.67; found: C, 66.42; H, 6.85.
5.2.5. 4-(2-Oxiranylmethoxy)benzaldehyde (2e)
A
mixture of 4-hydroxybenzaldehyde (1.22 g, 10 mmol),
epichlorohydrin (3 ml), 1,4-dioxane (15 ml), water (5 ml) and
sodium hydroxide (4.4 g, 110 mmol) was refluxed for 2 h. The
solvent was removed under reduced pressure and the residue was
extracted with ethyl acetate. The organic phase was dried over
anhydrous sodium sulfate, filtered and evaporated. The crude
product was purified by silica gel column chromatography (ethyl
acetate: petroleum ether ¼ 1:4) to afford compound 2e (1.49 g,
83%) as a yellow oil. ESI-MS: m/z 179 (M þ 1). 1H NMR (CDCl3,
195 (M ꢂ 1). IR (KBr, cmꢂ1
)
n
: 3420, 2925, 1686, 1601, 1508, 1231,
: 9.87 (s, 1H, CHO),
1162,1068, 983, 835; 1H NMR (CDCl3, 300 MHz)
d
7.83 (d, J ¼ 8.7 Hz, 2H, ArH), 7.14 (d, J ¼ 8.7 Hz, 2H, ArH), 5.35 (s, 2H,
CH2), 3.83 (m, 2H, CH2), 3.76 (m, 2H, CH2); 13C NMR (CDCl3,
75 MHz) d: 190.8, 161.8, 131.7, 130.5, 116.1, 93.0, 70.5, 61.5; Anal.
Calcd for C10H12O4: C, 61.22; H, 6.12; found: C, 61.08; H, 6.41.
5.2.9. 2-(4-Formylphenoxy)methoxypropane-1,3-diol (2i)
300 MHz)
d
: 9.86 (s, 1H, CHO), 7.81 (d, J ¼ 8.8 Hz, 2H, ArH), 7.01 (d,
Compound 2i (1.17 g, 26%) was obtained as colorless oil by the
same procedure described for 2h using 4-hydroxybenzaldehyde
(3.0 g, 25 mmol) and 2-[(propionyloxy)methyl-hydrogen]-1,3-pro-
panediol diacetate (6.5 g, 25 mmol) as reaction agents. ESI-MS: m/z
J ¼ 8.8 Hz, 2H, ArH), 4.34 (m, 1H, CH2), 4.01 (m, 1H, CH2), 3.38 (m,
1H, CH); 2.93 (m, 1H, CH2), 2.71 (m, 1H, CH2); 13C NMR (CDCl3,
75 MHz)
d: 190.3, 162.9, 131.5, 129.8, 114.5, 68.7, 49.6, 44.2; Anal.
Calcd for C10H10O3: C, 67.41; H, 5.66; found: C, 67.68; H, 6.45.
225 (M ꢂ 1). IR (KBr, cmꢂ1
) n: 3425, 2935, 1687, 1605, 1509, 1229,

W. Yi et al. / European Journal of Medicinal Chemistry 45 (2010) 639–646
645
1162, 1068, 825; 1H NMR (CDCl3, 300 MHz)
d
: 9.75 (s, 1H, CHO), 7.73
dropwise into succinic anhydride (1.1 g, 11 mmol) in anhydrous
dichloromethane (50 ml). The solution was stirred at 100 ꢀC for
0.5 h and then treated with 4-dimethylaminopyridine and refluxed
for 4 h. The solvent was removed under reduced pressure and the
crude product was purified by silica gel column chromatography
(ethyl acetate:petroleum ether ¼ 1:6) to yield yellow solid (1.3 g,
(d, 2H, J ¼ 8.8 Hz, ArH), 7.08 (d, 2H, J ¼ 8.8 Hz, ArH), 5.24 (s, 2H, CH2),
3.82 (m, 1H, CH), 3.65 (m, 2H, CH2), 3.53 (m, 2H, CH2); 13C NMR
(CDCl3, 75 MHz) d: 190.6, 162.2, 131.9, 130.5, 116.1, 92.7, 80.7, 62.3;
Anal. Calcd for C11H14O5: C, 58.41; H, 6.19; found: C, 58.58; H, 6.31.
5.2.10. 4-(2-Hydroxyethoxy)benzaldehyde (3a)
50%). mp 97–99 ꢀC. ESI-MS: m/z 265 (M ꢂ 1). IR (KBr, cmꢂ1
) n: 3452,
A mixture of 4-hydroxybenzaldehyde (1.22 g, 10 mmol), 2-
chloroethanol (3 ml, 15 mmol), 1,4-dioxane (15 ml), sodium
hydroxide (4.4 g, 110 mmol) and water (5 ml) was refluxed for 3 h.
The solvent was removed under reduced pressure and the residue
was extracted with ethyl acetate. The organic phase was dried over
anhydrous sodium sulfate, filtered and evaporated. The crude
product was purified by silica gel column chromatography (ethyl
acetate: petroleum ether ¼ 1:3) to afford compound 3a (1.35 g, 81%)
2963, 1731, 1666, 1595, 1509, 1407, 1347, 1315, 1263, 1211, 1162, 834;
1H NMR (CDCl3, 300 MHz)
d
: 9.88 (s, 1H, CHO), 7.85 (d, J ¼ 8.7 Hz,
2H, ArH), 7.03 (d, J ¼ 8.7 Hz, 2H, ArH), 4.50 (t, J ¼ 4.5 Hz, 2H, CH2);
4.26 (t, J ¼ 4.5 Hz, 2H, CH2), 2.70 (s, 4H, 2 ꢃ CH2); 13C NMR (CDCl3,
75 MHz) d: 191.1, 177.7, 172.2, 163.6, 132.3, 130.4, 115.3, 115.1, 66.4,
63.0, 29.2, 29.1; Anal. Calcd for C13H14O6: C, 58.65; H 5.26; found: C,
58.32; H, 5.55.
as a yellow oil. ESI-MS: m/z 167(M þ 1). IR (KBr, cmꢂ1
)
n: 3370,1684,
5.3. Tyrosinase assay
1600,1509,1309,1164,1112,1034, 839; 1H NMR (CDCl3, 300 MHz)
d:
9.89 (s, 1H, CHO), 7.79(d, J ¼ 8.4 Hz, 2H, ArH), 7.32 (d, J ¼ 8.4 Hz, 2H,
The spectrophotometric assay for tyrosinase was performed
according to the method reported by our groups [22,23] with some
slight modifications. Briefly, all the synthesized compounds were
screened for the diphenolase inhibitory activity of tyrosinase using
ArH), 4.75 (t, J ¼ 4.8 Hz, 2H, CH2), 4.42 (t, J ¼ 4.8 Hz, 2H, CH2); 13C
NMR (CDCl3, 75 MHz) d: 190.2, 163.3, 131.5, 130.1, 114.4, 70.1, 61.1;
Anal. Calcd for C9H10O3: C, 65.06; H, 6.02; found: C, 65.18; H, 6.10.
L-DOPA as substrate. All the compounds were dissolved in DMSO.
5.2.11. 2-(4-Formylphenoxy)ethyl dihydrogen phosphate (3b)
The final concentration of DMSO in the test solution was 2.0%.
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) were first pre-incubated with the compounds, in
50 mM phosphate buffer (pH 6.8), for 10 min at 25 ꢀC. Then the
A mixture of compound 3a (1.66 g, 10 mmol), triethylamine
(3.03 g, 30 mmol) and dichloroethane (40 ml) was cooled to 0 ꢀC and
treated dropwise with a solution of phosphorus oxychloride (3.04 g,
20 mmol) in dichloroethane and then stirred for 1.5 h. After the
solvent evaporating, 1 M NaOH solution (20 mL) was added drop-
wise and stirred at room temperature for 5 h. The resulting mixture
was evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (ethyl acetate: petroleum
ether ¼ 1:5) to afford compound 3b (1.25 g, 51%) as yellow oil. ESI-
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 concentra-
tion and averaged before further calculation. IC50 value, a concen-
tration giving 50% inhibition of tyrosinase activity, was determined
by interpolation of the dose–response curves. 4-Methoxycinnamic
acid and 4-hydroxybenzaldehyde were used as standard inhibitors
for the tyrosinase.
MS: m/z 247 (M þ 1). 1H NMR (CDCl3, 300 MHz)
d: 9.88 (s, 1H, CHO),
7.85 (d, J ¼ 8.9 Hz, 2H, ArH), 7.02 (d, J ¼ 8.9 Hz, 2H, ArH), 4.32 (t,
J ¼ 5.3 Hz, 2H, CH2), 3.86 (t, J ¼ 5.3 Hz, 2H, CH2),1.26 (s, OH); 13C NMR
(CDCl3, 75 MHz) d: 190.5, 163.1, 131.9, 130.4, 114.8, 69.9, 65.4; Anal.
Calcd for C9H11O6 P: C, 43.91; H, 4.50; found: C, 43.88; H, 4.32.
5.4. Inhibition kinetics of compound 3c
5.2.12. 2-(4-Formylphenoxy)ethyl dimethyl phosphate (3c)
According to the same procedure described for 3b, compound 3c
(1.10 g, 40%) was obtained as yellow oil by the esterification with
anhydrous methanol (50 ml) and sodium methoxide (20 ml, 0.2 M).
The determination of inhibitor kinetics was performed by
modification of the above-mentioned method: for each of three
different inhibitor concentrations (0, 12.94
respectively.) -DOPA concentration was varied (0, 5, 10, 15, 20 and
25 L). Pre-incubation and measurement time was the same as
above. Maximal initial velocity v ¼ A/ t was determined from
mM and 25.88 mM,
ESI-MS: m/z 297 (M þ Na). IR (KBr, cmꢂ1
)
n
: 3475, 2958, 2856, 2742,
L
1688, 1600, 1510, 1457, 1262, 1168, 1039, 983, 847; 1H NMR (CDCl3,
m
300 MHz):
J ¼ 8.5 Hz, 2H, ArH), 4.42 (m, 2H, CH2), 4.28 (m, 2H, CH2), 3.81 (s, 3H,
OCH3), 3.77 (s, 3H, OCH3); 13C NMR (CDCl3, 75 MHz)
: 190.3, 163.2,
132.4, 130.6, 114.2, 70.1, 65.5, 55.6, 55.3; 31P NMR [a solution
: 2.32 ppm. Anal.
d
9.88 (s, 1H, CHO), 7.85(d, J ¼ 8.5 Hz, 2H, ArH), 7.03 (d,
d d
initial linear portion of absorbance between 0 and 60 s after addi-
tion of mushroom tyrosinase. The inhibition type on the enzyme
was assayed by Lineweaver–Burk plots, and the inhibition constant
was determined by the second plots of the apparent Km/Vm or 1/Vm
versus the concentration of compound.
d
(H3PO4:H2O ¼ 1:10) as an external standard]
d
Calcd for C11H15O6P: C, 48.18; H, 5.47; found: C, 48.01; H, 5.65.
5.2.13. 2-(4-Formylphenoxy)ethyl diethoxyl phosphate (3d)
5.5. Preliminary acute toxicity assays in mice
According to the same procedure described for 3b, compound
3d (1.27 g, 42%) was obtained as yellow oil by the esterification with
anhydrous ethanol (50 ml) and sodium ethoxide (20 ml, 0.2 M).
Acute toxicity assay was performed according to the method
described by Shim et al. [24] with some slight modifications. Briefly,
ten mice (specific pathogen free, 4 weeks of age) were acclimated
for a week in the housing: Temperature was maintained at
20 ꢁ 2 ꢀC and relative humidity was 50 ꢁ 10%. Body weight of the
mice at study beginning was 20 ꢁ 2 g. Mice were fasted for 4 h prior
to oral administration. Compound 3c was dissolved in CMC (car-
boxymethyl cellulose). Constant volume (10 ml/kg body weight)
containing the concentration of compound 3c (160 mg/ml) was
orally administered to the mice to achieve the dose of 1600 mg/kg.
CMC was used as negative control. Feeds and water were provided
after 4 h of oral administration. Clinical symptom was observed
ESI-MS: m/z 303 (M þ 1). 1H NMR (CDCl3, 300 MHz)
d: 9.86 (s, 1H,
CHO), 7.83 (d, J ¼ 8.9 Hz, 2H, ArH), 7.01 (d, J ¼ 8.9 Hz, 2H, ArH), 4.30
(t, J ¼ 5.4 Hz, 2H, CH2), 4.11 (m, 4H, CH2), 3.85 (t, J ¼ 5.4 Hz, 2H,
CH2); 1.27 (m, 6H, CH3); 13C NMR (CDCl3, 75 MHz)
d: 190.6, 163.3,
131.6, 130.0, 114.6, 70.3, 64.8, 63.5, 63.2, 15.1, 14.9; Anal. Calcd for
C13H19O6P: C, 51.66; H, 6.34; found: C, 51.82; H, 6.28.
5.2.14. 3-[(4-Formylphenoxy)ethoxycarbonyl]propanoic acid (3e)
A
mixture of triethylamine (2.0 ml), anhydrous dichloro-
methane (20 ml) and compound 3a (1.66 g, 10 mmol) was added

646
W. Yi et al. / European Journal of Medicinal Chemistry 45 (2010) 639–646
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