Synthesis of novel azo compounds containing 5(4H)-oxazolone ring as potent tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.01.014

Six new azo dyes containing of 5(4H)-oxazolone ring were prepared by diazotization of 4-aminohippuric acid and coupling with N,N-dimethylaniline, 1-naphthol and 2-naphthol and condensation with 4-fluoro benzaldehyde or 4-trifluoromethoxy benzaldehyde. The

Full text of DOI:10.1016/j.bmc.2013.01.014

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of novel azo compounds containing 5(4H)-oxazolone ring
as potent tyrosinase inhibitors
a
b
a
c
Hooshang Hamidian a, , Roya Tagizadeh , Samieh Fozooni , Vahid Abbasalipour , Ali Taheri ,
⇑
Mohadeseh Namjou d
a Department of Chemistry, Payame Noor University (PNU), PO Box 19395-3697, Tehran, Iran
b Mining Engineering Department, Zarand High Education Center, Shahid Bahonar University, Kerman, Iran
c School of Advance Medical Science, Golestan University of Medical Science, Gorgan, Iran
d Department of Medical Laboratory, Golestan University of Medical Science, Gorgan, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new azo dyes containing of 5(4H)-oxazolone ring were prepared by diazotization of 4-aminohippuric
acid and coupling with N,N-dimethylaniline, 1-naphthol and 2-naphthol and condensation with 4-fluoro
benzaldehyde or 4-trifluoromethoxy benzaldehyde. The new compounds were fully characterized by
spectroscopic techniques. All synthesized compounds exhibited high tyrosinase inhibitory behavior.
The results of mushroom tyrosinase inhibition assays indicate that the 4-trifluoromethoxy derivatives
have high degrees of inhibition and N,N-dimethylaniline derivatives are better for tyrosinase inhibition
than 1-naphthol and 2-naphthol derivatives. All synthesized azo compounds (4a–4f) showed the most
Received 30 October 2012
Revised 28 December 2012
Accepted 5 January 2013
Available online xxxx
Keywords:
Anti-tyrosinase effect
Diazotization
5(4H)-Oxazolones
Azo compounds
potent mushroom tyrosinase inhibition, comparable to that of Kojic acid and
standard inhibitors.
L-mimosine, as reference
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
oxazolones are important intermediates for the synthesis of fine
chemicals and precursors of several biologically active molecules
such as amino acids and peptides.8
Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase,
EC 1.14.18.1, syn. polyphenol oxidase), also known as polyphenol
oxidase (PPO), is a copper-containing monooxygenase that is
widely distributed in microorganisms, animals, and plants.1 Tyros-
inase inhibitors are clinically useful for the treatment of skin dis-
eases associated with melanin hyperpigmentation and applied in
cosmetics for whitening and depigmentation after sunburn.2 Mel-
anin is a heteropolymer of indole compounds and is produced in-
side melanosomes by the action of the tyrosinase enzyme on the
tyrosinase precursor material in melanocytes. It has recently been
discovered that some other factors such as metal ions and the TRP-
1 and TRP-2 enzymes also contribute to the production of melanin.
However, tyrosinase plays a critical role in the regulation of mela-
nin biosynthesis. Therefore, many tyrosinase inhibitors that sup-
press melanogenesis have been widely studied with the aim of
developing preparations for the treatment of hyperpigmenta-
tion.3–7
On the other hand, oxazolone derivatives are highly versatile
intermediates used for the synthesis of several organic molecules,
including amino acids, peptides, antimicrobial or antitumor com-
pounds, immunomodulators, heterocyclic precursors for biosensors
coupling, and photosensitive composition devices for proteins.9–11
It is well known that azo compounds are the widest class of
industrial synthesized organic dyes due to their versatile applica-
tion in various fields, such as dyeing textile fiber, biological–phar-
macological activities and advanced application in organic
synthesis.12–16
In recent years, the fabrication of azo dyes has been intensively
investigated, due to their unique industrial applications in hyp-
notic drugs,17 in living cells, in detecting cancer18 and having phar-
macological and biological activities.19–22
In this work, we synthesized a number of new azo compounds
containing of oxazolone ring and studied chemical structures. Also
we evaluated inhibitory effect on tyrosinase of new compounds.
Nitrogen heterocycles are of special interest because they con-
stitute an important class of natural and nonnatural products,
many of which exhibit useful biological activities. Oxazolone
derivatives are in general well-known five-membered nitrogen-
containing heterocyclic compounds. 4-Arylidene-2-phenyl-5(4)-
2. Results and discussion
2.1. Chemistry
Diazonium salts could react readily with nucleophiles as an aro-
matic compounds containing amino or hydroxyl group, which have
⇑
Corresponding author. Tel./fax: +98 341 3342465.
E-mail address: h_hamidian@pnu.ac.ir (H. Hamidian).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.014
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H. Hamidian et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
O
The absorption maxima of the synthesized dyes changed from
O
NaNO2/HCl
496 to 542 nm. Compounds (4a–4f) are stable solids whose struc-
tures were established by IR, 1H NMR spectroscopy, mass spec-
trometry and elemental analysis.
N
H
N
H
COOH
COOH
-
Cl
+
H2N
N2
(1)
(2)
Scheme 1. Diazotisation of 4-aminohippuric acid.
2.2. Inhibitory activity of tyrosinase
The compounds (4a–4f) demonstrated excellent in vitro tyrosi-
nase inhibitory properties having IC50 values in the range of
4.33 0.52 to 1.44 0.36
l
M, whereas standard inhibitors,
been extensively researched and widely used for the preparation of
molecules with significance for both academic and industrial
applications. 4-Amino hippuric acid is dissolved in a 2.5% sodium
carbonate solution by heating and stirring. In the solution of
4-amino hippuric acid, sodium nitrite is dissolved and 4-amino
hippuric acid was diazotized by slow addition of concd HCl at
0 °C. A yellow precipitate of the diazonium salt (2) was formed
(Scheme 1).23
Coupling components (N,N-dimethylaniline, 1-naphthol and 2-
naphthol) are added to diazonium salt of 4-aminohippuric acid.
Azo dyes (3a–3c) are produced in good yields. Diazonium salt is
coupled to the para-position of the amine group, 2-position of
hydroxyl group in 1-naphthol and 1-position of hydroxyl group
in 2-naphthol (Scheme 2).
Then 4-arylidene-5(4H)-oxazolone azo dyes (4a–4f) are synthe-
sized by classical Erlenmeyer reaction, involving condensation of
compounds (3a–3c) with 4-fluorobenzaldehyde and 4-triflouro-
methoxy benzaldehyde in presence of acetic anhydride and so-
dium acetate under refluxing condition at 100 °C for 3 h
(Scheme 3).
L-mimosine and kojic acid, have IC50 values 3.68 0.02 and
16.67 0.52 lM, respectively (Table 2 and Fig. 1).
Compounds (4a) and (4b) having IC50 values 2.01 0.39 and
1.44 0.36, respectively, were found to be very active members
of the series, even better than both the standard inhibitors. How-
ever, compounds (4c), (4d) and (4e) were found better than the
standard kojic acid but not L-mimosine. 2-(4-{2-[4-(Dimethyl-
amino)phenyl]-1-diazenyl}phenyl)-4-{1-[4-(trifluoromethoxy)
phenyl]methylidene]-1,3-oxazol-5-one (4b) was found to be the
most active one having IC50 = 1.44 0.36
compounds.
Comparing the activities with the structures of compounds, it
turns out that the tyrosinase activity is mainly dependent on the
substituents present at C-2 and C-4 positions of oxazolone ring.
When tyrosinase inhibitory activity of the most active compound
(4b) was compared with other compounds, it was observed that
it has a 4-(trifluoromethoxy) phenyl group on the aliphatic double
bond at C-4 and [4-(dimethylamino)phenyl]-1-diazenylphenyl
group at C-2.
lM among all tested
This shows that extension of conjugation through an aliphatic
double bond could be the prerequisite for activity rather than
extension through an aromatic ring.
A decrease in the activity of compounds (4a), (4c) and (4e) as
compared to compounds (4b), (4d) and (4f) was due to the change
in the substituent in phenyl ring present at C-4 of oxazolone ring.
Generally, variation in color of these dyes results from the alter-
nation in coupling components. Since the synthesized dyes ob-
tained varied in color from red to brown, a convenient method of
measuring the color of the compound was to study the absorption
spectra of their solutions. The visible absorption maxima for the
synthesized dyes were measured in Me2SO at the concentration
of 10ꢀ5 M and are listed in Table 1.
The least activity of compound (4c) (IC50 4.33 0.52 lM) may be
O
O
-
+
NaOH
Na
O
N
H
N
H
COOH
Ar
-
Cl
+
N
+
Ar
N
N2
O
H3C
CH3
(3a-3c)
N
OH
OH
,
,
Ar=
Scheme 2. Coupling of diazonium salt with aromatic compounds.
O
CHO
O
X
N
O
-
+
Ac2O/NaOAc
Na
N
COO
N
+
N
H
Y
Y
N
N
z
(4a-4f)
Y= -F, -OCF3
H3C
CH3
N
OCOCH3
OCOCH3
Z=
,
,
Scheme 3. Synthesis of fluoro 5(4H)-oxazolone azo dyes (4a–4f).
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H. Hamidian et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Table 1
Structure, yields and kmax of new 5(4H)-oxazolone azo dyes (4a–4f)
Entry
R
Product
kmax (Me2SO)
Yielda (%)
42
H3C
CH3
CH3
O
O
O
N
N
N
F
CH3
CH3
N
N
1
501
N
4a
H3C
CF3
O
O
N
N
CH3
CH3
2
3
542
534
47
56
4b
N
N
OH
H3C
O
O
O
O
N
F
4c
N
N
OH
H3C
O
O
CF3
O
O
O
N
4
5
517
515
61
73
4d
N
N
OH
OH
O
O
N
F
N
N
4e
O
O
H3C
CF3
O
O
O
N
N
N
4f
6
510
69
O
H3C
O
a
Isolated yields.
Table 2
Tyrosinase inhibitory activities of the compounds (4a–4f), as compared to the
standard inhibitors
Entry
Compound
IC50 SEMa
(lM)
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
2.01 0.39
1.44 0.36
4.33 0.52
3.98 0.16
3.91 0.24
3.04 0.31
16.67 0.52
3.68 0.02
Kojic acidb
L
-Mimosineb
a
SEM is the standard error of the mean.
Standard inhibitors of the enzyme tyrosinase.
b
Figure 1. Comparative graphical presentation of the tyrosinase inhibitory poten-
tials of the compounds (4a–af).
due to changing the substituent in phenyl rings present at C-4 and
aromatic ring at C-2. Compound (4b) (IC50 1.44 0.36 M) was
compounds (4c–4f) having IC50 values 4.33 0.52, 3.98 0.16,
3.91 0.24 and 3.04 0.31 lM, respectively, showed good activity.
l
found to be highly active member of the present series of azo com-
pounds. Its excellent activity may be due to the presence of
dimethylamino group in phenyl ring at C-2 and the presence of a
trifluoromethoxy group in phenyl ring at C-4, which meets the cri-
teria for achieving extension of conjugation. Compound (4a) (IC50
The activities may be due to the presence of electron-withdrawing
substituent on phenyl ring at C-4 and electron-donating substitu-
ent on aromatic ring at C-2.
Effect of the compound 4b on tyrosinase tertiary structure was
considered by measurements of intrinsic fluorescence. We found
that 4b had a quenching effect on the intrinsic fluorescence, which
gradually occurred with increasing concentration.
2.01 0.39
lM) is structurally similar to compound (4b) except
where trifluoromethoxy group is replaced by fluoro. Interestingly,
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4
H. Hamidian et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Table 3
was added to suspension of diazotized hippuric acid, with stirring,
Melanin production and cytotoxicity
and acid-stable form of the dye was separated. A stiff paste was
formed in 5–10 min and then sodium hydroxide (5 g) was added
to produce the orange sodium salt. The product was collected using
saturated sodium chloride solution. The crude product was crystal-
lized from water. Orange powder, decomposed >270 yield is 81%.
Entry Compound Melanin production
inhibition (%)
Cytotoxicity cell
viability (%)
1
2
3
4
5
6
7
4a
4b
4c
4d
4e
4f
35.70 4.09
34.33 3.21
31.31 1.87
33.42 1.48
32.50 1.39
35.63 0.74
71.90 2.87
67.26 3.22
72.13 1.33
73.52 2.44
66.66 5.31
68.71 2.73
81.04 1.23b
IR (KBr):
t .
= 3354, 1716 cmꢀ1 1H NMR (400 MHz, DMSO-d6):
3.07 (s, 6H, 2CH3), 3.61 (d, 2H, J 4.4 Hz, CH2), 6.84 (d, 2H, J
8.9 Hz, ArH), 7.80–7.82 (m, 4H, ArH), 7.92 (br, 1H, NH), 7.98 (d,
2H, J 8.5, ArH) ppm. C17H17O3N4Na (348) Calcd C 58.65, H 4.88, N
16.08. Found: C 58.54, H 4.92, N 16.18.19
Kojic acida 17.20 1.22b
a
Standard inhibitors of the enzyme tyrosinase.
Tested at 200 lg/ml.
b
3.4. Sodium 2-({4-[2-(1-hydroxy-2-naphthyl)-1-
diazenyl]benzoyl}amino) acetate (3b)
2.3. Melanin production inhibition and cytotoxicity
1-Naphthol (0.01 mol) was dissolved in 5% sodium hydroxide
solution (30 mL). The solution of 2-naphthol was added to suspen-
sion of diazotized hippuric acid, with stirring, and base-stable form
of the dye was separated. A stiff paste was formed in 5–10 min and
then 10 mL acetic acid 10% was added to produce the red sodium
salt. The product was collected using saturated sodium chloride
solution. The crude product was crystallized from water. The crude
product was crystallized from water. Red powder, decomposed
The inhibitory of the compounds (4a–4f) were also tested on
melanin production and their cytotoxicity on B16F10 mouse mela-
noma cells at concentrations of 20 lg/ml. The results of melanin
production inhibition and cytotoxicity by the compounds (4a–4f)
are showed in Table 3. Compounds 4a–4f prevented melanin pro-
duction by 35.7%, 34.3%, 31.3%, 33.4%, 32.5% and 35.6%, respectively,
at concentrations of 20 lg/ml. On the other hand, compounds (4a–
>236 yield is 81%. IR (KBr):
t .
= 3469, 3364, 1714 cmꢀ1 1H NMR
4f) have shown moderate inhibition of melanin production. Cyto-
toxicity of new compounds (4a–4f) was evaluated and was defined
that all compounds were relatively less toxic (Table 3).
(400 MHz, DMSO-d6): 3.61 (d, 2H, J 4.4 Hz, CH2), 6.88–8.63 (m,
12H, ArH, NH, OH) ppm. C19H14N3O4Na (371) Calcd C 61.46, H
3.77, N 11.32. Found: C 61.73, H 3.66, N 11.09.
3. Experimental
3.1. General
3.5. Sodium 2-({4-[2-(2-hydroxy-1-naphthyl)-1-
diazenyl]benzoyl}amino) acetate (3c)
All the chemicals were obtained from Merck, Fluka, Sigma and
Aldrich Companies and used without further purification. Melting
points were measured using Thermo Fisher Scientific. IR spectra
were recorded Bruker tensor 27, FT-IR Spectrophotometer. All 1H
NMR spectra were recorded on a Bruker 400 MHz Spectrophotom-
eter. Chemical shifts are reported in parts per million (ppm) using
tetramethylsilane (TMS) as an internal standard. Ultraviolet–visi-
ble (UV–vis) absorption spectra were recorded on an Perkin–Elmer
spectrophotometer at the wavelength of maximum absorption
(kmax) in a range of DMSO at same concentrations (1 ꢁ 10ꢀ6M).
The mass Spectra were run on a Shimadzu Qp 5050 Ex Spectrom-
eter. The microanalyses for C, H and N were performed on Per-
kin–Elmer elemental analyzer.
2-Naphthol (0.01 mol) was dissolved in 5% sodium hydroxide
solution (30 mL). The solution of 2-naphthol was added to suspen-
sion of diazotized hippuric acid, with stirring, and base-stable form
of the dye was separated. A stiff paste was formed in 5–10 min and
then 10 mL acetic acid 10% was added to produce the red sodium
salt. The product was collected using saturated sodium chloride
solution. The crude product was crystallized from water. Red pow-
der, decomposed >259 yield is 81%. IR (KBr):
t = 3477, 3355,
1710 cmꢀ1 1H NMR (400 MHz, DMSO-d6): 3.64 (d, 2H, J 4.4 Hz,
.
CH2), 6.90–8.89 (m, 12H, ArH, NH, OH) ppm. C19H14N3O4Na (371)
Calcd C 61.46, H 3.77, N 11.32. Found: C 61.25, H 4.02, N 11.18.
3.6. General procedure for synthesis of compounds (4a–4f)
The fluorescence emission spectra were determined with a RF-
5301PC Shimadzu spectrofluorometer using a cuvette with a 1 cm
path length. An excitation wave length of 280 nm was used for the
tryptophan fluorescence measurements, and the emission wave-
length ranged between 300 and 420 nm.
A mixture of anhydrous sodium acetate (0.01 mol), 4-fluoro benz-
aldehyde or 4-trifluoromethoxy benzaldehyde (0.01 mol), sodium
salt of azo dye (3a–3c) (0.01 mol) and acetic anhydride (40 mL)
was heated with stirring until the mixture is transformed from an or-
ange semi-solid mass to a deep red liquid (2–4 h). After cooling, the
precipitated product was filtered and recrystallized in toluene.
3.2. Preparetion of diazonium salt of 4-aminohippuric acid (2)
In a 125-mL Erlenmeyer flask, 4-aminohippuric acid (0.01 mol)
was added to 2% sodium carbonate solution (30 mL) until it was
dissolved by boiling. The solution was then cooled and sodium ni-
trite (0.01 mol) was added, with stirring, until it was dissolved. The
solution was cooled by placing it in an ice bath, and then concen-
trated hydrochloric acid (2 mL) and water (3 mL) were added. By
acidifying the solution, a powdery yellow precipitate of the diazo-
nium salt was separated.23
3.6.1. 2-(4-{2-[4-(Dimethylamino)phenyl]-1-diazenyl}phenyl)-
4-[1-(4-fluorophenyl)methylidene]-1,3-oxazol-5-one (4a)
Dark red powder; mp: 264–266 °C. IR (KBr) m .
: 1789, 1657 cmꢀ1
1H NMR (DMSO-d6, 400 MHz): d 3.15 (s, 6H), 6.78–8.30 (m, 13H).
MS (EI) m/z (%): 42(100), 76(7), 104(2), 120(6), 171(4), 247(10),
275(6). Anal. Calcd for C24H19N4O2F: C, 69.57; H, 4.59; N, 13.53.
Found: C, 69.66; H, 4.39; N, 13.71.
3.6.2. 2-(4-{2-[4-(Dimethylamino)phenyl]-1-diazenyl}phenyl)-
4-{1-[4-(trifluoromethoxy)phenyl]methylidene]-1,3-oxazol-5-
one (4b)
3.3. Sodium 2-[4-{2-[4-(dimethylamino)phenyl]-1-diazenyl}
benzoyl amino] acetate (3a)
Brown powder; mp: 248–250 °C. IR (KBr) m .
: 1794, 1655 cmꢀ1
N,N-Dimethylaniline (0.01 mol) and glacial acetic acid
(0.01 mol) was mixed. The solution of N,N-dimethylaniline acetate
1H NMR (DMSO-d6, 400 MHz) d: 3.14 (s, 6H), 6.77–8.30 (m, 13H).
MS (EI) m/z (%): 42(3), 76(17), 104(9), 119(100), 148(17), 224(3),
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H. Hamidian et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
252(9), 480(M+, 3). Anal. Calcd for C25H19N4O3F3: C, 62.50; H, 3.96;
N, 11.67. Found: C, 62.67; H, 4.11; N, 11.51.
acid and L-mimosine was used as reference standard inhibitors for
comparison.
3.6.3. 2-[2-(4-{4-[1-(4-Fluorophenyl)methylidene]-5-oxo-4,5-
dihydro-1,3-oxazol-2-yl}phenyl)-1-diazenyl]-1-naphthyl
acetate (4c)
3.7.2. Inhibition of melanin production
Melanin production inhibition was ascertained by method of
Wang et al.24 A total of 8 ꢁ 104 cells were added to 60 mm plates,
Brown powder; mp: 246–248 °C. IR (KBr)
v
: 1800, 1726,
and were incubated at 37 °C in a CO2 incubator then 10 ll test
1654 cmꢀ1 1H NMR (DMSO-d6, 400 MHz) d: 2.53 (s, 3H), 7.19–
.
samples in DMSO were added to plates and were incubated for
9.04 (m, 15H). MS (EI) m/z (%): 42(100), 76(11), 91(9), 104(10),
143(51), 171(5), 247(3), 275(7). Anal. Calcd for C28H18N3O4F: C,
70.15; H, 3.76; N, 8.77. Found: C, 70.23; H, 3.59; N, 9.02.
72 h at 37 °C in a CO2 incubator. After washing with PBS, cells were
destroyed with 1 ml of 1 N NaOH, and 200 ll portions of raw cell
extracts were moved to 96-well plates. Melanin production inhibi-
tion was determined by recording absorbance at 475 nm. The ef-
fects of test samples on melanin contents are stated as percent
inhibitions of the value obtained in B16F10 mouse melanoma cells
which were cultured with DMSO alone.
3.6.4. 2-{2-[4-(5-Oxo-4-{1-[4-
(trifluoromethoxy)phenyl]methylidene}-4,5-dihydro-1,3-
oxazol-2-yl)phenyl]-1-diazenyl}-1-naphthyl acetate (4d)
Red powder; mp: 214–216 °C. IR (KBr)
v: 1796, 1762,
1656 cmꢀ1 1H NMR (DMSO-d6, 400 MHz) d: 2.53 (s, 3H), 7.19–
.
3.7.3. Cytotoxicity assay
9.03 (m, 15H). MS (EI) m/z (%): 42(100), 91(75), 143(59), 171(11),
247(4), 275(17). Anal. Calcd for C29H18N3O5F3: C, 63.85; H, 3.30;
N, 12.07. Found: C, 63.92; H, 3.13; N, 12.33.
Cytotoxicity assays were performed using a micro-culture MTT
method described by Han et al.25 A B16F10 mouse melanoma cell
suspensionwas poured into a 96-well plate (103 cells/well) and
cells were allowed to completely stick to each other overnight. Test
samples were then added to the plate and were incubated at 37 °C
3.6.5. 1-[2-(4-{4-[1-(4-Fluorophenyl)methylidene]-5-oxo-4,5-
dihydro-1,3-oxazol-2-yl}phenyl)-1-diazenyl]-2-naphthyl
acetate (4e)
for 72 h in a CO2 incubator. 20
then added per well and incubated for 4 h. Supernatant was then
removed and formazan was solubilized by adding 150 l DMSO
ll of MTT solution (2 mg/ml) was
Dark red powder; mp: 280–282 °C. IR (KBr)
v
: 1790, 1763,
l
1652 cmꢀ1 1H NMR (DMSO-d6, 400 MHz) d: 2.37 (s, 3H), 7.19–
.
to each well with mild shaking. Absorbance at 490 nm was re-
8.76 (m, 15H). MS (EI) m/z (%): 42(100), 91(14), 104(4), 120(8),
143(44), 171(9), 274(7), 275(9). Anal. Calcd for C28H18N3O4F: C,
70.15; H, 3.76; N, 8.77. Found: C, 69.98; H, 3.96; N, 8.88.
corded using an ELISA plate reader.
Acknowledgment
3.6.6. 1-{2-[4-(5-Oxo-4-{1-[4-
(trifluoromethoxy)phenyl]methylidene}-4,5-dihydro-1,3-
oxazol-2-yl)phenyl]-1-diazenyl}-2-naphthyl acetate (4f)
The authors thank Payame Noor University (PNU) of Kerman for
the financial support.
Red powder; mp: 206–208 °C. IR (KBr)
v
:
1797, 1767,
References and notes
1660 cmꢀ1 1H NMR (DMSO-d6, 400 MHz) d: 2.37 (s, 3H), 7.16–
.
1. Song, K. K.; Huang, H.; Han, P.; Zhang, C. L.; Shi, Y.; Chen, Q. X. Biochem. Biophys.
Res. Commun. 2006, 342, 1147.
2. Lee, H. S. J. Agric. Food Chem. 2002, 50, 1400.
3. Masamoto, Y.; Ando, H.; Murata, Y.; Shimoishi, Y.; Tada, M.; Takahata, K. Biosci.
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3.7. Biology
3.7.1. Tyrosinase inhibition assay
The spectrophotometric assay for tyrosinase was performed
according to the method Ref. 20. Briefly, all the synthesized com-
pounds were screened for the diphenolase inhibitory activity of
tyrosinase using L-DOPA as substrate. All the compounds were dis-
solved in DMSO. 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 tyrosi-
nase (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 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 L-DOPA chrome for 10 min. The mea-
surement was performed in triplicate for each concentration and
averaged before further calculation. IC50 value, a concentration giv-
ing 50% inhibition of tyrosinase activity, was determined by interpo-
lation of the dose–response curves. The percent of inhibition of
tyrosinase reaction was calculated as following:
Inhibition ð%Þ ¼ ½B ꢀ S=Bꢂ ꢁ 100
Here, the B and S are the absorbances for the blank and samples.
All the experiments were carried out at least in triplicate and the
results represent means SEM (standard error of the mean). Kojic
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Please cite this article in press as: Hamidian, H.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.01.014