Synthesis of novel azo-resveratrol, azo-oxyresveratrol and their derivatives as potent tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.10.050

Ten azo compounds including azo-resveratrol (5) and azo-oxyresveratrol (9) were synthesized using a modified Curtius rearrangement and diazotization followed by coupling reactions with various phenolic analogs. All synthesized compounds were evaluated for their mushroom tyrosinase inhibitory activity. Compounds 4 and 5 exhibited high tyrosinase inhibitory activity (56.25% and 72.75% at 50 μM, respectively). The results of mushroom tyrosinase inhibition assays indicate that the 4-hydroxyphenyl moiety is essential for high inhibition and that 3,5-dihydroxyphenyl and 3,5-dimethoxyphenyl derivatives are better for tyrosinase inhibition than 2,5-dimethoxyphenyl derivatives. Particularly, introduction of hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mushroom tryosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (5) showed the most potent mushroom tyrosinase inhibition with an IC₅₀ value of IC₅₀ = 36.28 ± 0.72 μM, comparable to that of resveratrol, a well-known tyrosinase inhibitor.

Full text of DOI:10.1016/j.bmcl.2012.10.050

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7451–7455
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel azo-resveratrol, azo-oxyresveratrol and their derivatives
as potent tyrosinase inhibitors
Yu Min Song a, , Young Mi Ha a, , Jin-Ah Kim a, , Ki Wung Chung a, Yohei Uehara a, Kyung Jin Lee a,
a,
Pusoon Chun b, Youngjoo Byun c, Hae Young Chung a, , Hyung Ryong Moon
⇑
⇑
a Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
b College of Pharmacy, Inje University, Gimhae, Gyeongnam 621-749, Republic of Korea
c College of Pharmacy, Korea University, Chungnam 339-700, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Ten azo compounds including azo-resveratrol (5) and azo-oxyresveratrol (9) were synthesized using a
modified Curtius rearrangement and diazotization followed by coupling reactions with various phenolic
analogs. All synthesized compounds were evaluated for their mushroom tyrosinase inhibitory activity.
Received 3 August 2012
Revised 8 October 2012
Accepted 10 October 2012
Available online 17 October 2012
Compounds 4 and 5 exhibited high tyrosinase inhibitory activity (56.25% and 72.75% at 50 lM, respec-
tively). The results of mushroom tyrosinase inhibition assays indicate that the 4-hydroxyphenyl moiety
is essential for high inhibition and that 3,5-dihydroxyphenyl and 3,5-dimethoxyphenyl derivatives are
better for tyrosinase inhibition than 2,5-dimethoxyphenyl derivatives. Particularly, introduction of
hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mush-
room tryosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (5) showed the most
Keywords:
Azo-resveratrol
Azo-oxyresveratrol
Curtius rearrangement
Anti-tyrosinase effect
Diazotization
potent mushroom tyrosinase inhibition with an IC50 value of IC50 = 36.28 0.72
of resveratrol, a well-known tyrosinase inhibitor.
lM, comparable to that
Ó 2012 Elsevier Ltd. All rights reserved.
Resveratrol (Fig. 1) is a naturally occurring phytoalexin with a
stilbene structure that is found in grapes, berries, peanuts, and
red wine1 and has attracted much attention for many years due
to its diverse biological effects. There have been many reports
proving its effects such as antioxidant, anti-aging, anti-inflamma-
tion, anticancer, cardio-protection and anti-tyrosinase activities.2
Resveratrol is well-known as one of the most potent antioxidants
against ROS and oxidative stress. Some of the biological activities
of resveratrol, such as neuroprotective, cardioprotective, and anti-
cancer effects, are attributed to its anti-oxidative property.3–5 Its
anti-oxidative action is reported to be exerted via hydrogen trans-
fer from its hydroxyl group to reactive free radicals, converting it
into a long-lived and less reactive ArOÅ radical.6 Recently, it was
shown that resveratrol can have anti-hypertensive properties
through both endothelium-independent and endothelium-medi-
ated vasorelaxing effects.7–9 All these beneficial effects on human
health have prompted the use of resveratrol as a therapeutic agent.
Therefore, it is of interest to develop a series of resveratrol analogs
that show resveratrol-like biological activities.
Generally, resveratrol and its analogs contain a double bond that
may undergo E/Z-isomerization and two phenolic rings located on
each end of the double bond (Fig. 1).10,11 In previous studies, resve-
ratrol analogs were mainly synthesized by modifying the substitu-
ents on both phenyl rings, not the double bond of the stilbene
scaffold.5,12 Benzene-fusioned (I) and heterocycle-fusioned (II and
III) analogs of resveratrol were also recently synthesized and found
to have antitumor, vasorelaxing or anti-tyrosinase activity (Fig. 1).
The structural feature of these analogues is that their benzene, furan
and thiazole rings serve as a double bond connecting two aromatic
rings.9 Oxyresveratrol, which is a resveratrol analog with an addi-
tional hydroxyl group, also showed comparable anti-tyrosinase
activity to resveratrol, a well-known tyrosinase inhibitor. Espe-
cially, benzene-fusioned analogs, including compound I developed
by our laboratory exhibited even more potent anti-tyrosinase activ-
ity than resveratrol.13 Tyrosinase exists widely in bacteria, fungi,
plants, insects, vertebrates and invertebrates14 and is the rate limit-
ing enzyme in the biosynthesis of melanin pigments responsible for
coloration of hair, skin and eyes, the undesired enzymatic browning
of fruits and vegetables and the developmental and defensive func-
tions of insects.15 Therefore, tyrosinase inhibitors have become
increasingly important in the agricultural (for insecticides and
browning inhibitors of vegetables and fruits), medicinal (for treat-
ment of hyperpigmentation diseases) and cosmetic (for whitening
agents) industries.
⇑
Corresponding authors. Tel.: +82 51 510 2815; fax: +82 51 513 6754 (H.R.M.);
tel.: +82 51 510 2814; fax: +82 51 518 2821 (H.Y.C.).
E-mail addresses: hyjung@pusan.ac.kr (H.Y. Chung), mhr108@pusan.ac.kr (H.R.
Moon).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.050

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Y. M. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7451–7455
Figure 1. Chemical structures of resveratrol and its derivatives.
Aromatic azo compounds are widely used in various fields as
followed by hydrolysis with 1 N-NaOH afforded 3,5-dihydroxyben-
organic dyes, radical initiators, therapeutics and drug delivery
agents.16–18 Moreover, azo compounds are also involved in a
number of biological reactions such as inhibition of DNA, RNA
and protein synthesis, carcinogenesis and nitrogen fixation.19,20
In this work, modification to convert an ethylene linker
connecting two phenyl rings of resveratrol into an azo linker was
attempted (Fig. 2). More varied and increased effects on anti-
tyrosinase, as well as antioxidant and other activities, can be
expected by azo-resveratrol or azo-oxyresveratrol due to the
combination of the chemical properties of azo compounds and
resveratrol or oxyresveratrol.
The double bond of resveratrol, oxyresveratrol or their deriva-
tives was converted into an azo linkage but, in most compounds,
the para-hydroxyl group on their phenyl ring was maintained be-
cause the para-hydroxyl group is more responsible for the radi-
cal-scavenging activity than the meta-hydroxyl group in the
antioxidant reaction.6,11,21
In this work, ten non-symmetric azo compounds including azo-
resveratrol and azo-oxyresveratrol were synthesized. Generally,
symmetric aromatic azo compounds are prepared by the oxidation
of aromatic amines using transition metals or by the reduction of
nitro aromatics using metals.22,23 In order to prepare non-symmet-
ric aromatic azo compounds, a coupling reaction of aryl diazonium
salts with electron-rich aromatics such as phenolic compounds
was employed.24 The synthesis of azo-resveratrol (5), azo-oxyres-
veratrol (9) and their O-methyl analogs is depicted in Scheme 1.
For the preparation of azo-resveratrol (5) and its analogs,
3,5-dimethoxyaniline (3) was synthesized from methyl
3,5-dihydroxybenzoate. O-methylation of the phenolic hydroxyl
groups of methyl 3,5-dihydroxybenzoate with dimethyl sulfate
zoic acid (2). A modified Curtius rearrangement of the resulting
acid
2
with diphenylphosphoryl azide (DPPA)25 in refluxing
1,2-dichloroethane gave a rather stable isocyanate intermediate
that required further hydrolysis with sodium hydroxide to gener-
ate the corresponding amine 3, a key material for preparation of
the diazonium salt. Treatment of 3 with sodium nitrite in an
aqueous HCl solution generated arenediazonium salt,26 which
was reacted with various electron-rich phenols such as phenol,
3-methoxyphenol, 2-methoxyphenol, and resorcinol (1,3-dihy-
droxybenzene) to give non-symmetric azo aromatic derivatives
4–8, respectively. Coupling between aryl diazonium salt and phe-
nols takes place almost exclusively at the para position if it is open.
Para-products were exclusively generated in all coupling reactions.
O-demethylation of 4 was performed using boron tribromide at
room temperature to afford azo-resveratrol (5) contaminated with
borate salts. Crude azo-resveratrol was acetylated with acetic
anhydride to remove the borate salts, and finally the corresponding
acetylated azo-resveratrol was deacetylated using sodium methox-
ide to generate pure azo-resveratrol (5). Azo-oxyresveratrol (9)
was also prepared from O-demethylation of 8 using BBr3 followed
by the same purification processes used in the purification of
azo-oxyresveratrol (5).
As shown in Scheme 2 and 2,5-dimethoxyphenyl azo compounds
13–16 were also synthesized from methyl 2,5-dihydroxybenzoate
using the same modified Curtius rearrangement and diazotization
followed by a coupling reaction with phenols. It is interesting to
note that the coupling reaction of 2,5-dimethoxyphenyl diazonium
ion with 3-methoxyphenol did not give the corresponding para-
product exclusively. Instead, the para- and ortho-products were ob-
tained as an inseparable mixture. According to the 1H NMR experi-
ments, the ratio of the para- and ortho-products was 2:1. Acetylation
of the mixture with acetic anhydride in the absence of tertiary
amine produced a less polar spot as a sole product on TLC along with
the unreacted starting material. The less polar spot turned out to be
an acetylated mixture of 14 and 15 by NMR analysis; notably, the
unreacted starting material was compound 15. This result suggests
that the phenolic hydroxyl group of 14 is more favorable to acetyla-
tion than that of 15, probably due to an intramolecular hydrogen
bond between the hydroxyl group of 15 and the neighboring
Figure 2. Chemical structure of stilbene with an azo linker designed from
resveratrol.

Y. M. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7451–7455
7453
Scheme 1. Synthesis of azo-resveratrol, azo-oxyresveratrol and their analogs. Reagents and conditions: (a) (CH3)2SO4, K2CO3, CH3CN, rt, 88%; (b) 1 N-NaOH, 1,4-dioxane, rt,
98%; (c) (i) DPPA, Et3N, 1,2-dichloroethane, reflux, (ii) 2 N-NaOH, THF, rt, 87%; (d) (i) NaNO2, 1 N-HCl, THF, 0–5 °C, (ii) phenols, 1 N-NaOH, 0–5 °C, 80% for 4, 72% for 6, 73% for
7, 66% for 8; (e) (i) BBr3, CH2Cl2, 0 °C to rt, (ii) Ac2O, pyridine, rt, (iii) 1 N-NaOMe, MeOH, rt, 54% for 5, 61% for 9.
Scheme 2. Synthesis of 2,5-dimethoxyphenyl azo compounds. Reagents and conditions: (a) (CH3)2SO4, K2CO3, CH3CN, rt, 81%; (b) 1 N-NaOH, 1,4-dioxane, rt, 92%; (c) (i) DPPA,
Et3N, 1,2-dichloroethane, reflux, (ii) 2 N-NaOH, THF, rt, 76%; (d) (i) NaNO2, 1 N-HCl, THF, 0–5 °C, (ii) resorcinol, 1 N-NaOH, 0–5 °C, (iii) Ac2O, pyridine, rt, (iv) 1 N-NaOMe,
MeOH, rt, 49%; (e) (i) NaNO2, 1 N-HCl, THF, 0–5 °C, (ii) 3-methoxyphenol, 1 N-NaOH, 0–5 °C, (iii) Ac2O, CH2Cl2, rt, 11% for 15; (f) (i) NaNO2, 1 N-HCl, THF, 0–5 °C, (ii) guiacol,
1 N-NaOH, 0–5 °C, 18%.
Table 1
Tyrosinase inhibition and substitution pattern of the substituted azo analogs 4–9, 13, 15 and 16
R4
R1
N
N
R5
R6
R3
R2
Compound
R1
R2
R3
R4
R5
R6
Tyrosinase inhibitiona (%)
4
5
6
7
8
OMe
OH
OMe
OMe
OMe
OH
OMe
OMe
OMe
OMe
OH
OMe
OMe
OMe
OH
H
H
H
H
H
H
H
OMe
OMe
OMe
OH
OH
OH
OH
OH
OH
OH
OMe
OH
H
H
H
OMe
H
H
H
H
OMe
H
H
OMe
H
OH
OH
OH
OH
H
56.25 0.45
72.75 1.60
8.90 1.87
1.03 9.08
48.50 3.47
41.46 2.17
27.80 1.14
6.38 2.79
0.44 1.65
9
13
15
16
H
H
Values represent means S.E. of three experiments.
a
Tyrosinase inhibition was measured using L-tyrosinase as the substrate at 50 lM.
nitrogen, as well as steric hindrance. In the coupling reaction of dia-
zonium salt with resorcinol and 2-methoxyphenol, the para-prod-
ucts 13 and 16 were formed exclusively. All synthesized
compounds were characterized by melting point, 1H and 13C NMR
and low/high resolution mass experiments.
Tyrosinase inhibition by the synthesized azo-phenols 4–9, 13,
15 and 16 was examined with an expectation that the compounds
would show good anti-tyrosinase activity. The inhibitory activities
were examined using mushroom tyrosinase as described previ-
ously with minor modification.27 The inhibition of mushroom
tyrosinase using the synthesized azo-phenol derivatives is summa-
rized in Table 1.28 Whereas compounds 6, 7, 15 and 16 exhibited
weak or no inhibition, compounds 4, 8 and 13 showed moderate
inhibitory activity against mushroom tyrosinase, with 56.25%,
48.50% and 27.80% inhibition at 50
lM, respectively. Azo-oxyres-
veratrol (9) also showed moderate inhibitory potency with

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Y. M. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7451–7455
Table 2
inhibition by the synthesized compounds was examined.
4-Hydroxyphenyl derivatives suppressed mushroom tyrosinase
activity more effectively than the other derivatives including 2,4-
dihydroxyphenyl, 2-methoxy-4-hydroxyphenyl and 2-hydroxy-4-
methoxyphenyl. 4-Hydroxy-3-methoxyphenylanalogssignificantly
reduced the antityrosinase effect. Generally, 3,5-dihydroxyphenyl
and 3,5-dimethoxyphenyl analogs were more potent tyrosinase
inhibitors than 2,5-dimethoxyphenyl analogs. Azo-resveratrol (5)
exhibited the most potent mushroom tyrosinase inhibition. The
IC50 value of azo-resveratrol was comparable to that of resveratrol.
Higher LogP value of azo-resveratrol compared with resveratrol
might provide merit superior to resveratrol as a lead compound for
the development of whitening agents and pharmaceutical drugs to
treat hyperpigmentation. Synthetic methodology and structure-
activity relationships of azo-resveratrol (5), azo-oxyresveratrol (9)
and their analogs will be extensively utilized in designing and syn-
thesizing potent tyrosinase inhibitors.
Inhibitory effects of compound 4, azo-resveratrol (5) and resveratrol on mushroom
tyrosinase activity
a
b
Compound
Conc.
(lM) Tyrosinase inhibition (%) IC50 (lM)
4
5.0
20.0
50.0
80.0
8.40 0.70
46.04 0.82
56.31 0.51
61.41 0.72
7.60 1.09
44.29 1.13
72.75 1.60
13.67 4.24
16.26 0.75
27.09 7.79
38.05 1.84
47.29 1.07
47.78 1.30
50.2 1.01
Azo-resveratrol (5) 15.0
36.28 0.72
26.63 0.55
30.0
50.0
0.5
1.0
Resveratrol
5.0
10.0
20.0
30.0
a
Values represent means S.E. of three experiments.
50% inhibitory concentration.
b
Acknowledgment
41.46% inhibition at 50 lM. Azo-resveratrol (5) exerted the most
This work was supported by a National Research Foundation of
Korea (NRF) Grant funded by the Korea government (MEST) (No.
2009-0083538).
potent inhibitory activity against mushroom tyrosinase, with
72.75% inhibition at the same concentration. The inhibitory
activities decreased in the order of 5 > 4 > 8 > 9 > 13 > 6, 15 > 7,
16. These results suggest that 3,5-dimethoxyphenyl and 3,5-
dihydroxyphenyl derivatives are superior to 2,5-dimethoxy
derivatives in the inhibition of tyrosinase (4, 5, 8 and 9 vs 13, 15
and 16) and that 4-hydroxyphenyl is an essential group for potent
inhibitory activity (4 and 5). It is worthy to note that introduction
of the second substituent into the 4-hydroxyphenyl moiety
decreases mushroom tyrosinase inhibition, depending on the
substituent and position. Insertion of a methoxy group into the
3-position dramatically diminished the tyrosinase inhibitory effect
(7 and 16). Introduction of a methoxy group into the 2-position (6)
lagged behind introduction into the 3-position in decreasing order
of inhibitory potency. It is also notable that 4-hydroxyphenyl azo
compounds exhibited more significant inhibitory activity against
mushroom tyrosinase than 2,4-dihydroxyphenyl azo compounds
(5 vs 9 and 4 vs 8). Interestingly, this result is contrary to that
observed in phenyl-benzo[d]thiazole, phenyl-thiazolidine-4-car-
boxylic acid and phenyl-pyrrolidine-2,5-dione analogs, in which
2,4-dihydroxyphenyl derivatives exhibited higher mushroom
tyrosinase inhibition than 4-hydroxyphenyl derivatives.29 The
bioactivities of compounds 4 and 5 were investigated in greater de-
tail. As depicted in Table 2, resveratrol, a positive control, inhibited
mushroom tyrosinase activity in a concentration-dependent man-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.050.
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M.; Kim, J.-A.; Park, J. Y.; Lee, H. J.; Moon, H. R.; Chung, H. Y. Biochimie 2012, 94,
533.
28. Assay to measure inhibitory effects on mushroom tyrosinase: Mushroom
tyrosinase was used to measure tyrosinase activity for the entire study.
Tyrosinase activity was determined as described previously with minor
modification.27 Briefly, 20
lL
of aqueous mushroom tyrosinase solution
(1000 units) was added to a 96-well microplate (Nunc, Denmark) in a 200
l
L
30. (E)-5-((4-Hydroxyphenyl)diazenyl)benzene-1,3-diol (azo-resveratrol, 5): Yellow
solid; melting point, 179–181 °C; 1H NMR (500 MHz, CD3OD) d 7.77 (d, 2H,
J = 9.0 Hz), 6.90 (d, 2H, J = 9.0 Hz), 6.80 (d, 2H, J = 2.0 Hz), 6.37 (t, 1H, J = 2.0 Hz);
13C NMR (100 MHz, CD3OD) d 160.8, 158.8, 155.0, 146.2, 124.6, 115.5, 104.3,
101.0; HRMS (ESI) m/z C12H9N2O3 (MÀH)À Calcd 229.0613, obsd 229.0620; (E)-
assay mixture containing 1 mM l-tyrosine solution and 50 mM phosphate
buffer (pH 6.5). The assay mixture was incubated at 25 °C for 30 min. Following
incubation, the amount of dopachrome produced was determined
spectrophotometrically at 492 nm (OD492) using a microplate reader
(Hewlett Packard, Palo Alto, CA, USA). IC50, or inhibitory concentration 50, is
the concentration of a compound that inhibits the maximal enzyme velocity by
half. IC50 is derived from the X-axis on an inhibitor concentration versus
product formation curve and is determined from the alignment of a dose-
response curve on the dependent Y-axis. In the present study, dose-dependent
inhibition experiments were performed in triplicate to determine the IC50 of
compounds. According to the inhibition percentage of three doses in each
4-((3,5-Dihydroxyphenyl)diazenyl)benzene-1,3-diol
(azo-oxyresveratrol,
9):
Orange-colored solid; melting point, 197–199 °C; 1H NMR (500 MHz, CD3OD)
d 7.63 (d, 1H, J = 8.5 Hz), 6.73 (d, 2H, J = 2.0 Hz), 6.50 (dd, 1H, J = 2.0, 8.5 Hz),
6.34 (t, 1H, J = 2.0 Hz), 6.30 (d, 1H, J = 2.0 Hz); 13C NMR (100 MHz, CD3OD) d
163.0, 159.2, 157.1, 152.3, 134.4, 132.4, 109.0, 104.1, 102.9, 99.8; LRMS (ESI) m/
z (MÀH)À 245; HRMS (ESI) m/z C12H9N2O4 (MÀH)À Calcd 245.0562, obsd
245.0578.