Synthesis of novel compounds containing morpholine and 5(4H)-oxazolone rings as potent tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.09.015

In this study, six new compounds containing morpholine and 5(4H)-oxazolone rings were synthesized. Structures of the new compounds using IR, ¹H NMR, mass spectroscopy and elemental analysis were characterized. All new compounds (4a-4f) have a s

Full text of DOI:10.1016/j.bmc.2015.09.015

Accepted Manuscript
Synthesis of novel compounds containing morpholine and 5(4H)-oxazolone
rings as potent tyrosinase inhibitors
Hooshang Hamidian, Sima Azizi
PII:
S0968-0896(15)30040-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.015
BMC 12562
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 July 2015
7 September 2015
8 September 2015
Please cite this article as: Hamidian, H., Azizi, S., Synthesis of novel compounds containing morpholine and 5(4H)-
oxazolone rings as potent tyrosinase inhibitors, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/
10.1016/j.bmc.2015.09.015
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Synthesis of novel compounds containing morpholine and
5(4H)-oxazolone rings as potent tyrosinase inhibitors
HooshangHamidian*, SimaAzizi
Department of Chemistry, Payame Noor University (PNU),P.O. Box 19395-3697, Tehran, Iran.
*E-mail: h_hamidian@pnu.ac.ir
Abstract
In this study, six new compounds containing morpholine and 5(4H)-oxazolone
rings were synthesized. Structures of the new compounds using IR, 1HNMR, mass
spectroscopy and elemental analysis were characterized. All new compounds (4a-
4f) have a strong inhibitory effect against mushroom tyrosinase. And the inhibitory
effects of these compounds were compared with Kojic acid as standard.
Keywords: Tyrosinase inhibitor, 5(4H)-Oxazolone, Morpholine, Synthesis.
1.
Introduction
Tyrosinase (EC1.14.18.1), also known as polyphenoloxidase (PPO), is a copper
containing enzyme involved in melanin synthesis and widely distributed in nature.
Two reactions involved in melanin biosynthesis pathways catalyzed by tyrosinase
are hydroxylation of monophenols to o-phenols and the oxidation of the o-phenols
to o-quinones.1 Tyrosinase catalyzed the formation of quinones and mediated the
development of brown colour pigment through spontaneous polymerization of
highly reactive quinones.2
In mammals, L-tyrosine serves as a typical monophenol substrate. Numerous
phenolics having monophenol and o-diphenol structures found in plants and insects
*Corresponding author. Tel. & Fax: +98 34 32476624
E-mail addresses: h_hamidian@pnu.ac.ir
1

are similarly oxidized by tyrosinase.3 This enzymatic oxidation is the ratelimiting
step in several phenomena of color development and cuticle sclerotization; the
remainder of the reaction sequence proceeds spontaneously at physiological
conditions.4
Tyrosinase inhibitors are recommended for the treatment of melanin
hyperpigmentation and used in cosmetics for whitening and depigmentation after
sunburn.5Tyrosinasehas an important role in the regulation of melanin
biosynthesis. Therefore, many tyrosinase inhibitors that prevent melanogenesis
have been widely considered with the aim of developing for the treatment of
hyperpigmentation.6
5(4H)-Oxazolones are profitable intermediates for the synthesis of several
biological compounds such as amino acids and peptides.7-8 Also some of the these
compounds represent an extensive range of pharmacological activities including
Anti-inflammatory,9 anticancer,10 antagonistic and antiangiogenic,11-12 tyrosinase
inhibitor13 and etc.
In continuation of our research on 5(4H)-oxazolones as potential compounds in our
13-14
drug discovery program,
we synthesized some of new 5(4H)-oxazolone
derivatives and screened them for their tyrosinase inhibitory properties.
We also briefly studied the structure–activity relationships of these compounds
to find out possible lead compound for the treatment of tyrosinase-related
diseases.
2.
Results and disscusion
2.1. Chemistry
Diazonium salts could react readily with nucleophiles as an aromatic compounds
containing amino or hydroxyl group, which have been extensively researched and
2

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 conc. HCl at 0°C.A yellow precipitate of the diazonium salt (2) was
formed (Scheme 1).15
O
O
NaNO2/HCl
N
N
COOH
COOH
-
Cl
H
+
N2
H
H2N
(1)
(2)
Scheme 1. Diazotisation of 4-aminohippuric acid.
N- phenylmorpholine (Coupling component) is added to diazonium salt of 4-
aminohippuric acid. Azo dye (3) is produced in good yields. Diazonium salt is
coupled to the para-position of the morpholino group (Scheme 2).
O
N
O
COOH
O
N
H
N
COOH
EtOH
H
-
Cl
+
2
+
N
N
N
N
O
(3)
Scheme 2. Coupling of diazonium salt with aromatic compounds.
3

Then 4-arylidene-5(4H)-oxazoloneazo dyes (4a–4f) are synthesized by classical
Erlenmeyer reaction, involving condensation of compound (3) with 4-fluoro
benzaldehyde, benzaldehyde, 2-chloro-4-fluoro benzaldehyde, 4-chloro
benzaldehyde, 2-nitro benzaldehyde and thiophen 2- carbaldehyde in presence of
acetic anhydride and sodium acetate under refluxing condition at 100 C for 4 h
(Scheme 3).
COOH
O
O
N
Ar
H
O
N
N
N
Ac2O/NaOAc
N
N
ArCHO
+
N
N
O
(4a-4f)
O
Cl
NO2
,
Ar:
,
,
,
,
S
F
F
Cl
Scheme 3. Synthesis of new 5(4H)-oxazolone compounds (4a-4f).
The synthesized compounds obtained varied in color from red to brown, a suitable
method of measuring the color of the compound was to study the absorption
spectra of their solutions. The visible absorption maximums for the synthesized
4

compounds were measured in DMSO at the concentration of 10-6 M and are
listed in Table 1. The absorption maximum of the synthesized dyes changed from
1
340 to 653 nm. Structures of synthesized compounds were determined by H
NMR, mass spectroscopic and elemental analysis.
Table 1. Structure, yields and max of new azo dyes (4a-4f)
max
Entry
1
ArCHO
Product
Yield(%)a
60
(DMSO)
O
H
O
O
N
N
653
4a
4b
O
N
N
N
O
H
O
O
F
F
2
644
64
O
O
N
N
N
N
N
F
O
H
O
O
N
N
Cl
3
4
624
641
73
70
4c
Cl
F
O
H
O
O
N
Cl
N
4d
O
N
N
Cl
H
O
O
O
N
N
N
O
O
N
NO2
N
N
5
6
340
351
65
55
4e
4f
NO2
O
O
O
N
S
N
S
H
a Isolated yields
2.2. Inhibitory activity of tyrosinase
IC50 value, a concentration giving 50% inhibition of tyrosinase activity, was
determined by interpolation of the dose–response curves (Figure 1).
5

4a
4b
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
50
100
150
0
50
100
150
Concentration (μM)
Concentration (μM)
4c
4d
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
50
100
150
0
50
100
150
Concentration (μM)
Cocentration (μM)
4e
4f
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
50
100
150
0
50
100
150
Concentration (µM)
Concentration (µM)
Figure 1. Dose-Dependent curves of compounds (4a-4f) on tyrosinase activity.
6

The compounds (4a–4f) exhibited excellent in vitro tyrosinase inhibitory properties
having IC50 values in the range of 20.33 ± 0.47 to 13.65 ± 0.22 µM, since standard
inhibitor, Kojic acid, have IC50 value 16.01 ± 0.56 µM, respectively (Table 2).
Table 2.Tyrosinase inhibitory activities of the compounds (4a-4f), as compared to the standard
inhibitor
Entry
Compound
IC50 ± SEMa (µM)
17.22 ± 0.41
15.78 ± 0.24
16.02 ± 032
18.91 ± 0.15
20.33 ± 0.47
13.65 ± 0.22
16.01± 0.56
1
2
3
4
5
6
7
4a
4b
4c
4d
4e
4f
Kojic acidb
a SEM is the standard error of the mean.
b Standard inhibitor of the enzyme tyrosinase.
Compound (4f) having IC50 value 13.65 ± 0.22 µM, was found to be very active
members of the series, even better than the standard inhibitor. 2-{4-[2-(4-
morpholinophenyl)-1-diazenyl]phenyl}-4-[1-(2-thianyl) methylidene)]-1,3-oxazol-
5(4H)-one(4f) was found to be the most active one having IC50 = 13.65 ± 0.22 µM
among all tested compounds.
Comparing the activities with the structures of compounds, it clear that the
tyrosinase activity is mainly relevant on the substituents present at C-4 position of
oxazolone ring. When tyrosinase inhibitory activity of the most active compound
(4f) was compared with other compounds, it was observed that it has a thiophen
ring on the aliphatic double bond at C-4.
This shows that expansion of conjugation through an aliphatic double bond could
be the prerequisite for activity rather than expansion through an aromatic ring.
The minimum activity of compound (4e) (IC50 20.33 ± 0.47 µM) may be due to
changing the substituent in phenyl rings present at C-4.
7

Effect of the compound (4f) on tyrosinase tertiary structure was considered by
measurements of intrinsic fluorescence. We found that (4f) had a quenching effect
on the intrinsic fluorescence, which gradually occurred with increasing
concentration. Consequently, compound (4f) effects on tyrosinase activity by
destruction of tertiary structure it.
2.3. Melanin production inhibition and cytotoxicity
The inhibitory of the compounds (4a-4f) were also tested on melanin production
and their cytotoxicity on B16F10 mouse melanoma cells at concentrations of 20
µg/ml. The results of melanin production inhibition and cytotoxicity by the
compounds(4a-4f) are showed in Table 3.Compounds 4a–4f prevented melanin
production by 40.15%, 38.83%, 32.40%, 34.37%, 30.68% and 31.17%,
respectively, at concentrations of 20µg/ml. On the other hand, compounds (4a–4f)
have shown mediate inhibition of melanin production. Cytotoxicity of new
compounds (4a–4f) was appraised and was explained that all compounds were the
more toxic than kojic acid (Table 3).
Table 3. Melanin production and cytotoxicity
Entry
Compound
Melanin production Inhibition (%) Cytotoxicity Cell viability (%)
1
2
3
4
5
6
7
4a
4b
4c
4d
40.15 ± 4.72
38.83 ± 1.50
32.40 ± 1.13
34.37 ± 2.69
30.68 ± 0.56
31.17 ± 1.25
16.90 ± 3.71b
62.87 ± 4.11
57.24 ± 1.42
60.66 ± 3.33
61.87 ± 1.96
58.11 ± 6.32
56.71 ± 2.91
80 ± 3.26b
4e
4f
Kojic acida
a Standard inhibitors of the enzyme tyrosinase.
b Tested at 200 µg/ml.
3.
Experimental
8

3.1. General
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
1
Spectrophotometer. All H NMR spectra were recorded on a Bruker 500 MHZ
Spectrophotometer. Chemical shifts are reported in parts per million (ppm) using
tetramethylsilane (TMS) as an internal standard. Ultraviolet-visible (UV-vis)
absorption spectra were recorded on a Perkin-Elmer spectrophotometer at the
wavelength of maximum absorption (λmax) in a range of DMSO at same
concentrations (1× 10-6M). The mass Spectra were run on a Shimadzu Qp 5050 Ex
Spectrometer. The microanalyses for C, H and N were performed on perkin-elmer
elemental analyzer.
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 wavelength ranged between 300 and 420nm.
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 nitrite (0.01mol) was added, with stirring, until it was
dissolved. The solution was cooled by placing it in an ice bath, and then
concentrated hydrochloric acid (2 mL) and water (3 mL) were added. By
acidifying the solution, a powdery yellow precipitate of the diazonium salt was
separated. 15
9

3.3. Preparetion
of
Sodium2-({4-[2-(4-morpholinophenyl)
-1-
diazenyl]benzoyl}amino)acetate (3)
N-phenylmorpholin (0.01mol) in 96% ethanol (20 ml) was dissolved by heating.
The solution N-phenylmorpholine was added to suspension of diazotized hippuric
acid, with stirring, and acid-stable form of the dye was separated.
Red powder; decomposed >256 °C. Yield: 88%. IR (KBr) υ: 3361, 1712, 1674cm-1.
1H NMR (500 MHz, DMSO-d6): 3.22 (t, 4H), 3.64 (d, 2H), 3.80 (t, 4H), 6.84 (d,
2H, J 8.9 Hz, ArH), 6.93-8.82 (m, 9H, ArH, NH) ppm. Anal.Calcd.for
C19H19N4O4Na: C, 58.46; H, 4.87; N, 14.36. Found: C, 58.65; H, 5.05; N, 14.48.
3.4. General procedure for synthesis of compounds (4a-4f):
A mixture of anhydrous sodiumacetate (0.001 mol), 4-fluoro benzaldehyde ,
benzaldehyde, 2-chloro-4-fluoro benzaldehyde, 4-chloro benzaldehyde, 2-nitro
benzaldehyde and thiophen 2- carbaldehyde (0.001 mol), sodium salt of azo dye
(3) (0.001 mol) and acetic anhydride (10 mL) was heated with stirring until the
mixture is transformed from an orange semi-solid mass to a deep red liquid (4 h).
After cooling, the precipitated product was filtered and recrystallized in ethanol.
3.4.1.
2-{4-[2-(4-morpholinophenyl)
-1-diazenyl]phenyl}-4-[1-
phenylmethylidene]-1,3-oxazol-5(4H)-one (4a):
1
Red powder; mp: 180-182 C. IR (KBr) : 1787, 1650 cm-1. H NMR (DMSO-d6,
500 MHz) δ: 3.27 (t, 4H), 3.78 (t, 4H), 6.88–8.22 (m, 14H). MS (EI) m/z (%): 438
(M+, 66), 294(43), 248(2),190(8), 162(100), 104(81), 77(29).Anal.Calcd.for
C26H22N4O3: C, 71.23; H, 5.02; N, 12.78. Found: C, 71.12; H, 4.89; N, 12.97.
10

3.4.2
4-[1-(4-flourophenyl)methylidene]-2-{4-[2-(4-morpholinophenyl)-1-
diazenyl] phenyl}-1,3-oxazol-5(4H)-one (4b):
1
Light brown powder; mp: 200-201 C. IR (KBr) : 1790, 1651 cm-1. H NMR
(DMSO-d6, 500 MHz) δ: 3.20 (t, 4H), 3.91 (t, 4H), 7.16–8.31 (m, 13H). MS (EI)
m/z (%): 456(M+, 27), 294(34), 236(30), 162(100), 104(89), 120(27), 77(40). Anal.
Calcd for C26H21N4O3F: C, 68.42; H, 4.61; N, 12.28. Found: C, 68.57; H, 4.44; N,
12.08.
3.4.3.
4-[1-(2-chloro-4-flourophenyl)methylidene]-2-{4-[2-(4-
morpholinophenyl)-1-diazenyl] phenyl}1,3-oxazol-5(4H)-one (4c):
1
Brown powder; mp: 230-232 C. IR (KBr) : 1796, 1600 cm-1. H NMR (DMSO-
d6, 500 MHz) δ: 3.36 (t, 4H), 3.88 (t, 4H), 7.02–8.18 (m, 12H). MS (EI) m/z
(%):492(M+2, 2), 490(M+, 6), 311(82), 162(100), 104(74), 120(53), 77(42). Anal.
Calcd. for C26H20N4O3ClF: C, 63.61; H, 4.08; N, 11.42. Found: C, 63.43; H, 3.93;
N, 11.39.
3.4.4.
4-[1-(4-chlorophenyl)methylidene]-2-{4-[2-(4-morpholinophenyl)-1-
diazenyl]phenyl}-1,3-oxazol-5(4H)-one (4d):
1
Dark brown powder; mp: 220-222 C. IR (KBr) : 1792, 1647 cm-1. H NMR
(DMSO-d6, 500 MHz) δ: 3.49 (t, 4H), 3.94 (t, 4H), 7.06–8.27 (m, 13H). MS (EI)
m/z (%): 474(M+2, 12), 472(M+, 36), 249(27), 162(100), 120(92), 104(52), 77(23).
Anal. Calcd for C26H21N4O3Cl: C, 66.03; H, 4.44; N, 11.85. Found: C, 65.90; H,
4.53; N, 12.09.
3.4.5.
2-{4-[2-(4-morpholinophenyl)-1-diazenyl]phenyl}-4-[1-(2-
nitrophenyl)methylidene]-1,3-oxazol-5(4H)-one (4e):
11

1
Light red powder; mp: 256-257 C. IR (KBr) : 1794, 1642, 1599, 1339 cm-1. H
NMR (DMSO-d6, 500 MHz) δ: 3.40 (t, 4H), 3.96 (t, 4H), 7.00–8.72 (m, 13H). MS
(EI) m/z (%): 483(M+, 7), 162(94), 120(47), 104(100), 92(48), 76(58), 65(41).
Anal. Calcd for C26H21N5O5: C, 64.60; H, 4.35; N, 14.49. Found: C, 64.75; H, 4.21;
N, 14.28.
3.4.6.
2-{4-[2-(4-morpholinophenyl)-1-diazenyl]phenyl}-4-[1-(2-
thianyl)methylidene)]-1,3-oxazol-5(4H)-one (4f):
1
Purple powder; mp: 230-231 C. IR (KBr) : 1786, 1643 cm-1. H NMR (DMSO-
d6, 500 MHz) δ: 3.35 (t, 4H), 3.88 (t, 4H), 7.02–8.18 (m, 12H). MS (EI) m/z (%):
444(M+, 13), 294(19), 178(11), 162(100), 120(90), 104(90), 77(37). Anal. Calcd
for C24H20N4O3S: C, 64.86; H, 4.50; N, 12.61. Found: C, 65.02; H, 4.67; N, 12.89.
3.5. Biology
3.5.1. Tyrosinase inhibition assay
Tyrosinase inhibition was assayed according to the method of Ref.16 Briefly, all the
synthesized compounds were sieved for the diphenolase inhibitory activity of
tyrosinase using L-DOPA as substrate. All the compounds were dissolved in
DMSO. The final concentration ofDMSOin 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
LDOPA (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 measurement was performed in triplicate for each
concentration and averaged before further calculation. The percent of inhibition of
tyrosinase reaction was calculated as following:
12

Inhibition (%) = [B – S/B] × 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 acid was used as reference standard
inhibitors for comparison.
3.5.2. Inhibition of melanin production
Melanin production inhibition was confirmed by method of Wang et al.17 A total of
8 × 104 cells were added to 60 mm plates, and were incubated at 37 C in a CO2
incubator then 10 µl test samples in M were added to plates and were
incubated for 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 µl portions of raw cell extracts were
moved to 96-well plates. Melanin production inhibition was determined by
recording absorbance at 475 nm. The effects of test samples on melanin contents
are explained as percent inhibitions of the value obtained in B16F10 mouse
melanoma cells which were cultured with DMSO alone.
3.5.3. Cytotoxicity assay
Cytotoxicity assays were implemented using a micro-culture MTT method
described by Han et al.18 A B16F10 mouse melanoma cell suspensionwas poured
into a 96-well plate (103 cells well) and cells were allowed to completely stic to
each other overnight. est samples were then added to the plate and were incubated
at 37 C for 72 h in a CO2 incubator. 20 µl of MTT solution (2 mg/ml) was then
added per well and incubated for 4 h. Supernatant was then removed and formazan
was soluiblized by adding 150 µl DMSO to each well with mild shaking.
Absorbance at 490 nm was recorded using an ELISA plate reader.
13

Acknowledgements
The authors thank Payame Noor University (PNU) of Kerman for the financial
support.
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Graphical abstract
16