Design and synthesis of hydroxypyridinone-l-phenylalanine conjugates as potential tyrosinase inhibitors

Article abstract of DOI:10.1021/jf401585f

A range of hydroxypyridinone-l-phenylalanine conjugates were synthesized starting from kojic acid. Their tyrosinase activity was determined, and it was found that one of the compounds ((S)-(5-(benzyloxy)-1-octyl-4-oxo-1,4- dihydropyridin-2-yl)methyl 2-amino-3-phenylpropanoate, 5e) showed potent inhibitory effect against mushroom tyrosinase, the IC₅₀ values for monophenolase and diphenolase activities being 12.6 and 4.0 μM, respectively. It was also demonstrated that these conjugates are mixed-type inhibitors, suggesting they could bind to both the free enzyme and the enzyme-substrate complexes. MTT assay indicated that 5e was nontoxic to three cell lines. This compound may find applications in food preservation and cosmetics.

Full text of DOI:10.1021/jf401585f

Article
pubs.acs.org/JAFC
Design and Synthesis of Hydroxypyridinone‑L‑phenylalanine
Conjugates as Potential Tyrosinase Inhibitors
Dong-Fang Li,† Pan-Pan Hu,† Mu-Song Liu,† Xiao-Le Kong,‡ Jin-Chao Zhang,§ Robert C. Hider,‡
and Tao Zhou*,†
†School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310035, People’s Republic of
China
‡Division of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH,
United Kingdom
§College of Chemistry & Environmental Science, Chemical Biology Key Laboratory of Hebei Province, Hebei University, Baoding 071002,
People’s Republic of China
ABSTRACT: A range of hydroxypyridinone-L-phenylalanine conjugates were synthesized starting from kojic acid. Their
tyrosinase activity was determined, and it was found that one of the compounds ((S)-(5-(benzyloxy)-1-octyl-4-oxo-1,4-
dihydropyridin-2-yl)methyl 2-amino-3-phenylpropanoate, 5e) showed potent inhibitory effect against mushroom tyrosinase, the
IC50 values for monophenolase and diphenolase activities being 12.6 and 4.0 μM, respectively. It was also demonstrated that
these conjugates are mixed-type inhibitors, suggesting they could bind to both the free enzyme and the enzyme−substrate
complexes. MTT assay indicated that 5e was nontoxic to three cell lines. This compound may find applications in food
preservation and cosmetics.
KEYWORDS: hydroxypyridinone, kojic acid, tyrosinase inhibitor
inhibit tyrosinase by chelating copper ion in the active site of
tyrosinase due to the ketone group at position 4 and the hydroxyl
group at position 5, it is assumed that an increase of affinity for
copper may result in a stronger tyrosinase inhibition. As 3-
hydroxypyridin-4-ones are predicted to possess a higher copper
affinity than that of the corresponding hydroxypyranones, we have
synthesized a range of 3-hydroxypyridin-4-one derivatives (5) with
a hydrophobic alkyl group at the N-1 position, and their tyrosinase
inhibitory activities have been investigated.
INTRODUCTION
■
The production of melanin by melanocytes, melanogenesis, is
initiated by tyrosinase upon exposure of the skin to UV
radiation. Tyrosinase (EC 1.14.18.1), a multifunctional copper-
containing enzyme, is widely distributed in plants and animals,1
and plays an important role in the pathway of melanin
biosynthesis from L-tyrosine. It is well-known that tyrosinase
can catalyze the first and rate-limiting step of melanin
formation, the hydroxylation of L-tyrosine to L-3-(3,4-
dihydroxyphenyl)-alanine (L-DOPA) (monophenolase activity)
and also the subsequent oxidation of DOPA to dopaquinone
(diphenolase activity). Dopaquinone is highly reactive and can
polymerize spontaneously to form melanin in a series of
reaction pathways.2 The enzymatic oxidation of L-tyrosine into
melanin is of considerable importance in the coloring of skin,
hair, and eyes and in food browning.3,4 It has also been suggested
that tyrosinase contributes to the neurodegeneration associated
with Parkinson’s disease.5,6 Thus, the development of novel
tyrosinase inhibitors is of interest to agricultural, cosmetic, and
medical fields.7−10 A large number of naturally occurring and
synthetic compounds that are tyrosinase inhibitors have been
reported.11,12 However, only a few of them are sufficiently potent
for practical use and comply with general safety regulations. kojic
acid (1), 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, which is
isolated from metabolic products of many species of Aspergillus
and Penicillium molds, possesses appreciable inhibitory activity
against tyrosinase and also possesses free radical scavenging
activity.13,14 Kojic acid has been used as an ingredient in cosmetics
and as an antibrowning agent in foods that rapidly change
color.15,16 Thus, modification of kojic acid provides a potential
route for superior tyrosinase inhibitors.17−20 As kojic acid can
MATERIALS AND METHODS
■
Instruments. 1H NMR spectra were recorded on a Bruker Avance
400 spectrometer or a Bruker Avance 500 spectrometer (Bruker Corp.,
Germany) with tetramethylsilane as an internal standard. Electrospray
ionization (ESI) mass spectra were obtained by infusing samples
into an LCQ Deca XP ion-trap instrument (ThermoFinnigan, San
Jose, CA). High-resolution mass spectra (HRMS) were obtained on
a QTOFMicro (Waters, U.S.) by direct infusing samples into the ESI
source.
General Synthesis. All chemicals were of AR grade and used
without any further purification.
Synthesis of 5-(Benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one
(2). To a solution of kojic acid (71 g, 0.5 mol) in methanol (150 mL)
was added NaOH solution (22 g in 50 mL of H2O). The mixture was
heated to reflux, BnCl (75.9g, 0.6 mol) was added dropwise over 1.5 h,
and the reflux was continued for 6 h. The reaction mixture was cooled to
room temperature, filtered to remove the inorganic salt, and the filtrate
was cooled. White solid was precipitated and filtered. The collected
Received: April 10, 2013
Revised: June 13, 2013
Accepted: June 13, 2013
Published: June 13, 2013
© 2013 American Chemical Society
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product was washed with cold methanol and obtained as a white solid 2
(83 g, 72% yield).
(m, 2H, CH2), 3.06 (d, J = 6.4 Hz, 2H, CH2), 3.50 (m, 2H, CH2), 4.60
(q, J = 7.2 Hz, 1H, CH), 4.88 (m, 2H, CH2), 5.06 (s, 2H, CH2), 5.19
(s, 2H, CH2), 6.44 (s, 1H, C6−H in pyridinone ring), 6.84 (s, 1H,
C3−H in pyridinone ring), 7.02 (m, 2H, Ph), 7.18 (m, 2H, Ph), 7.27−
7.39 (m, 12H, 11 from Ph, 1 from NH). ESI−MS: m/z 569 (MH+).
(S)-(5-(Benzyloxy)-1-hexyl-4-oxo-1,4-dihydropyridin-2-yl)methyl
2-(Benzyloxycarbonyl)-3-phenylpropanoate (4d). 1H NMR (400 MHz,
CDCl3): δ 0.86 (t, J = 7.2 Hz, 3H, CH3), 1.13 (m, 2H, CH2), 1.25
(m, 4H, CH2), 1.43 (m, 2H, CH2), 2.96 (d, J = 6.4 Hz, 2H, CH2), 3.49
(m, 2H, CH2), 4.55 (q, J = 6.8 Hz, 1H, CH), 4.86 (m, 2H, CH2), 5.04
(s, 2H, CH2), 5.19 (s, 2H, CH2), 6.45 (s, 1H, C6−H in pyridinone),
6.85 (s, 1H, C3−H in pyridinone ring), 7.01 (m, 2H, Ph), 7.18
(m, 2H, Ph), 7.27−7.39 (m, 12H, 11 from Ph, 1 from NH). ESI−MS:
m/z 597 (MH+).
(S)-(5-(Benzyloxy)-1-octyl-4-oxo-1,4-dihydropyridin-2-yl)methyl
2-(Benzyloxycarbonyl)-3-phenylpropanoate (4e). 1H NMR (400 MHz,
CDCl3): δ 0.89 (t, J = 7.2 Hz, 3H, CH3), 1.13 (m, 2H, CH2), 1.22
(m, 8H, CH2), 1.49 (m, 2H, CH2), 3.06 (d, J = 6.4 Hz, 2H, CH2), 3.51
(m, 2H, CH2), 4.61 (q, J = 6.8 Hz, 1H, CH), 4.88 (m, 2H, CH2), 5.07
(s, 2H, CH2), 5.19 (s, 2H, CH2), 6.46 (s, 1H, C6−H in pyridinone
ring), 6.86 (s, 1H, C3−H in pyridinone ring), 7.03 (m, 2H, Ph), 7.18
(m, 2H, Ph), 7.28−7.40 (m, 12H, 11 from Ph, 1 from NH). ESI−MS:
m/z 625 (MH+).
(S)-(5-(Benzyloxy)-1-decyl-4-oxo-1,4-dihydropyridin-2-yl)methyl
2-(Benzyloxycarbonyl)-3-phenylpropanoate (4f). 1H NMR (400 MHz,
CDCl3): δ 0.84 (t, J = 6.8 Hz, 3H, CH3), 1.21 (m, 14H, CH2), 1.61
(m, 2H, CH2), 2.91 and 3.04 (m, 2H, CH2), 3.76 (m, 2H, CH2), 4.32 (m,
1H, CH), 4.93−5.08 (m, 6H, CH2), 6.27 (s, 1H, C6−H in pyridinone
ring), 7.19−7.41 (m, 15H, Ph), 7.59 (s, 1H, C3−H in pyridinone ring),
7.93 (d, J = 8.0 Hz, 1H, NH). ESI−MS: m/z 653 (MH+).
General Procedure for the Synthesis of 5. To a suspension of 4
(2 mmol) and benzyl chloride (5 mmol) in MeOH/EtOAc (15 mL/
15 mL) was added 5% Pd/C (10% weight of 4). Hydrogenation was
carried out at 30 psi H2 for 5−6 h at room temperature. After filtration
to remove the catalyst, the filtrate was concentrated to dryness. The
residue was redissolved in a small amount of methanol, and precipitated
by the addition of diethyl ether. After removal of the supernatant, the
residue was dried at 40 °C under vacuum, providing hydrochlorides of 5
as pale brown solids.
General Procedure for the Synthesis of 3. A mixture of 2 (5 mmol),
NaOH (1.0 g), and amine RNH2 (5.5 mmol) in MeOH/H2O
(20 mL/10 mL) was refluxed for 2−3 h. The reaction was monitored
by TLC. After completion of the reaction, the mixture was concentrated
to about half volume, and extracted with CH2Cl2 (3 × 50 mL). The
combined organic layer was washed with brine, and dried over anhydrous
Na2SO4. After removal of the solvent, the crude product 3 was obtained,
which was used in the next reaction without further purification.
5-(Benzyloxy)-2-(hydroxymethyl)-1-methylpyridin-4(1H)-one
1
(3a). H NMR (400 MHz, DMSO-d6): δ 3.58 (s, 3H, CH3), 4.36
(s, 2H, CH2), 4.98 (s, 2H, CH2), 6.22 (s, 1H, C6−H in pyridinone
ring), 7.31−7.41 (m, 5H, Ph), 7.55 (s, 1H, C3−H in pyridinone ring).
ESI−MS: m/z 246 (MH+).
5-(Benzyloxy)-2-(hydroxymethyl)-1-ethylpyridin-4(1H)-one (3b).
1H NMR (400 MHz, CDCl3): δ 1.29 (t, J = 7.2 Hz, 3H, CH3),
3.96 (q, J = 7.2 Hz, 2H, CH2), 4.44 (s, 2H, CH2), 5.11 (s, 2H, CH2),
6.32 (s, 1H, C6−H in pyridinone ring), 6.94 (s, 1H, C3−H in
pyridinone ring), 7.27−7.38 (m, 5H, Ph). ESI−MS: m/z 260 (MH+).
5-(Benzyloxy)-2-(hydroxymethyl)-1-butylpyridin-4(1H)-one (3c).
1H NMR (400 MHz, CDCl3): δ 0.85 (t, J = 7.2 Hz, 3H, CH3),
1.14 (m, 2H, CH2), 1.47 (m, 2H, CH2), 3.92 (q, J = 7.2 Hz, 2H, CH2),
4.43 (s, 2H, CH2), 5.09 (s, 2H, CH2), 6.32 (s, 1H, C6−H in
pyridinone ring), 6.92 (s, 1H, C3−H in pyridinone ring), 7.27−7.38
(m, 5H, Ph). ESI−MS: m/z 288 (MH+).
5-(Benzyloxy)-2-(hydroxymethyl)-1-hexylpyridin-4(1H)-one (3d).
1H NMR (400 MHz, CDCl3): δ 0.87 (t, J = 6.8 Hz, 3H, CH3),
1.21 (m, 6H, CH2), 1.58 (m, 2H, CH2), 3.87 (q, J = 7.6 Hz, 2H, CH2),
4.43 (s, 2H, CH2), 5.10 (s, 2H, CH2), 6.34 (s, 1H, C6−H in
pyridinone ring), 6.91 (s, 1H, C3−H in pyridinone ring), 7.27−7.39
(m, 5H, Ph). ESI−MS: m/z 316 (MH+).
5-(Benzyloxy)-2-(hydroxymethyl)-1-octylpyridin-4(1H)-one (3e).
1H NMR (400 MHz, CDCl3): δ 0.89 (t, J = 7.2 Hz, 3H, CH3),
1.15 (m, 2H, CH2), 1.23 (m, 8H, CH2), 1.51 (m, 2H, CH2), 3.88 (q,
J = 7.2 Hz, 2H, CH2), 4.42 (s, 2H, CH2), 5.10 (s, 2H, CH2), 6.31
(s, 1H, C6−H in pyridinone ring), 6.89 (s, 1H, C3−H in pyridinone
ring), 7.27−7.38 (m, 5H, Ph). ESI−MS: m/z 344 (MH+).
5-(Benzyloxy)-2-(hydroxymethyl)-1-decylpyridin-4(1H)-one (3f).
1H NMR (400 MHz, CDCl3): δ 0.85 (t, J = 6.8 Hz, 3H, CH3),
1.22 (m, 14H, CH2), 1.60 (m, 2H, CH2), 3.86 (q, J = 7.2 Hz, 2H,
CH2), 4.42 (s, 2H, CH2), 5.09 (s, 2H, CH2), 6.33 (s, 1H, C6−H in
pyridinone ring), 6.92 (s, 1H, C3−H in pyridinone ring), 7.28−7.39
(m, 5H, Ph). ESI−MS: m/z 372 (MH+).
General Procedure for the Synthesis of 4. A mixture of compound
3 (5 mmol), N-Cbz-L-phenylalanine (5.5 mmol), EDC·HCl (5.5 mmol),
and DMAP (0.2 g) in DMF (20 mL) was stirred at room temperature.
After removal of the solvent, the residue was dissolved in dichloromethane
(100 mL), washed with brine (2 × 50 mL), and dried over anhydrous
Na2SO4. After filtration, the filtrate was concentrated, and the residue was
purified by silica gel column chromatography (CH2Cl2/CH3OH, 10:1),
giving the product 4 as a pale yellow powder.
Hydrochloride Salt of (S)-(5-(Benzyloxy)-1-methyl-4-oxo-1,4-
1
dihydropyridin-2-yl)methyl 2-Amino-3-phenylpropanoate (5a). H
NMR (400 MHz, DMSO-d6): δ 3.25 (m, 2H, CH2), 3.87 (t, J = 7.2
Hz, 3H, CH3), 4.12 (m, 1H, CH), 5.29 (m, 2H, CH2), 7.21 (m, 5H,
Ph), 7.40 (s, 1H, C6−H in pyridinone ring), 8.39 (s, 1H, C3−H in
+
pyridinone ring), 8.85 (br, 3H, NH3 ). ESI−MS: m/z 303 (MH+).
HR−MS: m/z calcd for C16H19N2O4 (MH+) 303.1345, found
303.1341.
Hydrochloride Salt of (S)-(5-(Benzyloxy)-1-ethyl-4-oxo-1,4-dihy-
dropyridin-2-yl)methyl 2-Amino-3-phenylpropanoate (5b). 1H
NMR (400 MHz, DMSO-d6): δ 1.33 (t, J = 7.2 Hz, 3H, CH3), 3.11
and 3.25 (m, 2H, CH2), 4.16 (m, 2H, CH2), 4.32 (m, 1H, CH), 5.31
(m, 2H, CH2), 7.21 (m, 5H, Ph), 7.39 (s, 1H, C6−H in pyridinone
(S)-(5-(Benzyloxy)-1-methyl-4-oxo-1,4-dihydropyridin-2-yl)-
+
ring), 8.37 (s, 1H, C3−H in pyridinone ring), 8.87 (br, 3H, NH3 ).
1
methyl 2-(Benzyloxycarbonyl)-3-phenylpropanoate (4a). H NMR
ESI−MS: m/z 317 (MH+). HR-MS: m/z calcd for C17H21N2O4
(400 MHz, CDCl3): δ 3.06 (m, 2H, CH2), 3.26 (s, 3H, CH3), 4.61
(q, J = 7.2 Hz, 1H, CH), 4.87 (m, 2H, CH2), 5.07 (s, 2H, CH2), 5.18
(s, 2H, CH2), 6.48 (s, 1H, C6−H in pyridinone ring), 6.86 (s, 1H,
C3−H in pyridinone ring), 7.04 (m, 2H, Ph), 7.16 (m, 2H, Ph), 7.29−
7.42 (m, 12H, 11 from Ph, 1 from NH). ESI−MS: m/z 527 (MH+).
(S)-(5-(Benzyloxy)-1-ethyl-4-oxo-1,4-dihydropyridin-2-yl)methyl
2-(Benzyloxycarbonyl)-3-phenylpropanoate (4b). 1H NMR (400
MHz, CDCl3): δ 1.17 (t, J = 7.2 Hz, 3H, CH3), 3.07 (m, 2H, CH2),
3.55 (m, 2H, CH2), 4.62 (m, 1H, CH), 4.88 (m, 2H, CH2), 5.07 (s,
2H, CH2), 5.19 (s, 2H, CH2), 6.46 (s, 1H, C6−H in pyridinone ring),
6.87 (s, 1H, C3−H in pyridinone ring), 7.03 (m, 2H, Ph), 7.18 (m,
2H, Ph), 7.29−7.41 (m, 12H, 11 from Ph, 1 from NH). ESI−MS: m/z
541 (MH+).
(MH+) 317.1501, found 317.1496.
Hydrochloride Salt of (S)-(5-(Benzyloxy)-1-butyl-4-oxo-1,4-dihy-
dropyridin-2-yl)methyl 2-Amino-3-phenylpropanoate (5c). 1H
NMR (400 MHz, DMSO-d6): δ 0.89 (t, J = 7.2 Hz, 3H, CH3), 1.29
(m, 2H, CH2), 1.66 (m, 2H, CH2), 3.10 and 3.24 (m, 2H, CH2), 4.14
(m, 2H, CH2), 4.32 (m, 1H, CH), 5.31 (s, 2H, CH2), 7.21 (m, 5H, Ph),
7.38 (s, 1H, C6−H in pyridinone ring), 8.35 (s, 1H, C3−H in pyridinone
ring), 8.86 (br, 3H, NH3+). ESI−MS: m/z 345 (MH+). HR−MS: m/z
calcd for C19H25N2O4 (MH+) 345.1814, found 345.1805.
Hydrochloride Salt of (S)-(5-(Benzyloxy)-1-hexyl-4-oxo-1,4-dihy-
dropyridin-2-yl)methyl 2-Amino-3-phenylpropanoate (5d). 1H
NMR (500 MHz, MeOD): δ 0.91 (t, J = 7.0 Hz, 3H, CH3), 1.35
(m, 4H, CH2), 1.41 (m, 2H, CH2), 1.87 (m, 2H, CH2), 3.21 and 3.30
(m, 2H, CH2), 4.23 (m, 1H, CH), 4.27 (t, J = 7.0 Hz, 2H, CH2), 4.75
(s, 2H, CH2), 7.31 (m, 6H, Ph and C6−H in pyridinone ring), 8.12
(S)-(5-(Benzyloxy)-1-butyl-4-oxo-1,4-dihydropyridin-2-yl)methyl
2-(Benzyloxycarbonyl)-3-phenylpropanoate (4c). 1H NMR (400 MHz,
CDCl3): δ 0.85 (t, J = 7.2 Hz, 3H, CH3), 1.14 (m, 2H, CH2), 1.47
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a
Scheme 1
a
Reagents and conditions: (a) BnCl, MeOH/H2O, 70 °C, 6 h; (b) RNH2, EtOH/H2O, NaOH, reflux, 3 h, 72−85% yield; (c) EDC, DMAP; (d) H2
(30 psi), Pd/C, EtOAc/MeOH (1:1), room temperature, 5 h, 88−93% yield.
(s, 1H, C3−H in pyridinone ring). ESI−MS: m/z 373 (MH+). HR−
MS: m/z calcd for C21H29N2O4 (MH+) 373.2127, found 373.2132.
Hydrochloride Salt of (S)-(5-(Benzyloxy)-1-octyl-4-oxo-1,4-dihy-
dropyridin-2-yl)methyl 2-Amino-3-phenylpropanoate (5e). 88%
yield. 1H NMR (500 MHz, MeOD): δ 0.91 (t, J = 7.0 Hz, 3H,
CH3), 1.32 (m, 10H, CH2), 1.86 (m, 2H, CH2), 3.2 and 3.33 (m, 2H,
CH2), 4.25−4.32 (m, 3H, CH and CH2), 4.78 (s, 2H, CH2), 7.33 (m,
6H, Ph and C6−H in pyridinone ring), 8.18 (s, 1H, C3−H in
pyridinone ring). ESI−MS: m/z 401 (MH+). HR−MS: m/z calcd for
C23H33N2O4 (MH+) 401.2440, found 401.2447.
25 0.1 °C by using a Techne TE-8J temperature controller. The
solution under investigation was stirred vigorously during the experiment.
A Gilson Mini-plus#3 pump with speed capability (20 mL/min) was used
to circulate the test solution through a Hellem quartz flow cuvette. For
the stability constant determinations, a 50 mm path length cuvette was
used, and for pKa determinations, a cuvette path length of 10 mm was
used. The flow cuvette was mounted on an HP 8453 UV−visible
spectrophotometer. All instruments were interfaced to a computer and
controlled by a Visual Basic program. Automatic titration and spectral
scans adopted the following strategy: the pH of a solution was increased
by 0.1 pH unit by the addition of KOH from the autoburet; when pH
readings varied by <0.001 pH unit over a 3 s period, an incubation period
was activated. For pKa determinations, a period of 1 min was adopted; for
stability constant determinations, a period of 5 min was adopted. At the
end of the equilibrium period, the spectrum of the solution was then
recorded. The cycle was repeated automatically until the defined end
point pH value was achieved. All of the titration data were analyzed with
the pHab program.22 The species plot was calculated with the HYSS
program.23 Analytical grade reagent materials were used in the preparation
of all solutions.
Determination of Cell Survival. Cell Culture. Three different
human carcinoma cell lines, Hela, A549, and MCF-7, were cultured in
DMEM medium supplemented with 10% fetal bovine serum, 100
units/mL of penicillin, and 100 mg/mL of streptomycin. Cells were
maintained at 37 °C in a humidified atmosphere of 5% CO2 in air.
Samples were dissolved in DMSO at a concentration of 10 mM as
stock solution, and diluted with PBS buffer to concentrations of 100, 10,
and 1 μM, then diluted with DMEM medium to final concentrations of
10, 1, and 0.1 μM as working solutions. To avoid DMSO toxicity, the
concentration of DMSO was less than 0.1% (v/v) in all experiments.
Cytotoxicity Analysis. The cells harvested from exponential phase
(2 × 104 cells/mL, 100 μL) were seeded into a 96-well plate. Cells
were allowed to completely adhere overnight, and then the
supernatant was removed. Fresh DMEM medium (90 μL) was
complemented, and then the samples (10 μL) were added to the wells.
The incubation was continued for 48 h. Control wells were prepared
by addition of culture medium. Wells containing culture medium
without cells were used as blanks. All experiments were performed in
quintuplicate. The MTT assay was performed as described by
Mosmann.24 Upon completion of the incubation, stock MTT dye
solution (10 μL, 5 mg/mL) was added to each well. After 4 h incubation,
the supernatant was removed, and DMSO (100 μL) was added to
dissolve the MTT formazan. The OD of each well was measured on a
microplate spectrophotometer at a wavelength of 570 nm.
Hydrochloride Salt of (S)-(5-(Benzyloxy)-1-decyl-4-oxo-1,4-dihy-
dropyridin-2-yl)methyl 2-Amino-3-phenylpropanoate (5f). 1H NMR
(400 MHz, DMSO-d6): δ 0.86 (t, J = 6.8 Hz, 3H, CH3), 1.24 (m, 14H,
CH2), 1.70 (m, 2H, CH2), 3.12 and 3.25 (m, 2H, CH2), 4.16 (m, 2H,
CH2), 4.31 (m, 1H, CH), 5.33 (s, 2H, CH2), 7.21 (m, 5H, Ph), 7.47
(s, 1H, C6−H in pyridinone ring), 8.42 (s, 1H, C3−H in pyridinone
+
ring), 8.89 (br, 3H, NH3 ). ESI−MS: m/z 429 (MH+). HR−MS: m/z
calcd for C25H37N2O4 (MH+) 429.2753, found 429.2762.
Tyrosinase Inhibition Assay. Mushroom tyrosinase (lyophilized
powder, ≥1000 unit/mg solid) was purchased from Sigma. One unit =
ΔA280 of 0.001 per min at pH 6.5 at 25 °C in 3 mL of reaction mixture
containing L-tyrosine.
Prepration of Tyrosinase Solution. Ten milligrams of mushroom
tyrosinase was dissolved in distilled water and diluted to a final
concentration of 100 unit/mL.
Inhibitory Effect of Hydroxypyridinone-L-phenylalanine Conju-
gates on the Tyrosinase Activity. In this investigation, L-tyrosine was
used as the substrate for the monophenolase activity assay, and L-
DOPA was used as the substrate for the diphenolase activity assay. The
reaction system (3 mL) containing different concentrations of 0.1 mL
inhibitors was first mixed with 1 mL of substrate solution containing
1.8 mL of 50 mM phosphate buffer (pH 6.8), then a portion of 0.1 mL
of enzyme solution was added to this blend, and the reaction was
carried out at 30 °C for 10 min. Absorption and kinetic measurements
were carried out on a UV-2100 spectrophotometer at 475 nm.
Controls, without inhibitors, were routinely carried out. The inhibitory
activity of the sample was expressed as the concentration that inhibits
50% of the enzyme activity (IC50).
Determination of the Inhibition Type and Inhibition Constant.
The inhibition type was assayed by the Lineweaver−Burk plot based
on the results of inhibitory effect on the diphenolase activity of
tyrosinase, and the inhibition constant was determined by the
secondary plots of the apparent Km/Vm or 1/Vm versus the
concentration of the inhibitor, as described by Chen et al.21
pKa and Copper(II) Stability Constants’ Determination. The
automatic titration system used in this study was comprised of an
autoburet (Metrohm Dosimat 765 L mL syringe), Mettler Toledo
MP230 pH meter with Metrohm pH electrode (6.0133.100), and a
reference electrode (6.0733.100). 0.1 M KCl electrolyte solution was
used to maintain the ionic strength. The temperature of the test
solutions was maintained in a thermostatic jacketed titration vessel at
Statistical Analyses. All experiments were performed in triplicate.
The data were statistically analyzed using analysis of variance test
(SPSS version 13.0). Data in the tables and figure were given as mean
SD and analyzed by one-way ANOVA. Significant differences
between the treatments were examined by Duncan’s multiple range
test. P < 0.05 was considered as statistically significant.
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hydrophobic compound with the highest C log P value25 of 4.707
(Table 1), was found to possess a weak inhibitory activity. This is
probably due to the fact that 5f has a poor water solubility. Thus,
for the hydroxypyridinone derivatives to exert a good tyrosinase
inhibitory effect, it is essential for the compounds to possess a
suitable hydrophobicity.
Although some reported kojic acid derivatives possess higher
tyrosinase inhibitory activity, for example, Kim et al. reported
the synthesis of a kojic acid derivative with a potent tyrosinase
inhibitory activity (IC50 = 3.63 μM),26 it needs longer synthetic
procedure. As compared to pyranone ring in kojic acid deriva-
tives, pyridinone ring in hydroxypyridinone derivatives is more
stable because it possesses aromaticity due to the existence of
tautomerism 7 (Scheme 2). In comparison with the reported
RESULTS AND DISCUSSION
■
Synthesis of Hydroxypyridinone-L-phenylalanine
Conjugates (5). Compounds 5 were synthesized starting
from kojic acid (1) (Scheme 1). Benzylation of 5-hydroxy group
in kojic acid was achieved by the reaction of kojic acid and benzyl
chloride under basic condition to provide 2 in good yield.
Condensation of 2 with amine generated 3, which was then
coupled with Cbz-L-phenylalanine by ester bond formation in the
presence of EDC and DMAP in N,N-dimethylformamide (DMF)
at room temperature, yielding product 4. Deprotection of the
benzyl and Cbz groups on 4 was achieved by hydrogenation in the
presence of palladium/charcoal, generating hydroxypyridinone-L-
phenylalanine conjugates 5. All of the compounds have been fully
1
characterized by H NMR, MS, and HRMS.
Inhibitory Effect of Compounds 5 on Monophenolase
Activity of Tyrosinase. Tyrosinase inhibitory activities of the
hydroxypyridinone-phenylalanine conjugates were evaluated by
means of the mushroom tyrosinase inhibition assay method.
Using L-tyrosine as substrate, the inhibitory effects on
tyrosinase increased with the increase of concentrations of 5
(Figure 1). Statistical analysis of the results indicated that a
Scheme 2. Tautomerization of Hydroxypyridinone Derivatives
hydroxypyridinone derivatives (the lowest IC50 value is 0.37 mM),27
5e exhibited stronger tyrosinase inhibitory activity.
Inhibitory Effect of 5d and 5e on Diphenolase Activity
of Tyrosinase. The inhibitory activity of 5e and 5d on the
diphenolase activity of mushroom tyrosinase was investigated
using L-DOPA as a substrate. The IC50 values of 5d and 5e on
the diphenolase activity of the enzyme were determined as 42.8
and 4.0 μM, respectively (Table 1). Thus, 5e also exhibited a
potent inhibitory effect on the diphenolase activity of mushroom
tyrosinase greater than kojic acid (IC50 = 20 μM).28 The kinetic
data of the inhibition of L-DOPA oxidation by 5e and 5d were
expressed in a nonlinear Michaelis−Menten plot (Figure 2A-I, B-I)
and Lineweaver−Burk double-reciprocal plots (Figure 2A-II, B-II).
As can be seen in the nonlinear Michaelis−Menten plot, Vmax
decreased with the increase of inhibitor concentration, whereas Km
increased with the increase of inhibitor concentration, indicating
that 5d and 5e can bind not only with free enzyme but also with
the enzyme−substrate complex. This result can also be obtained
from Lineweaver−Burk double-reciprocal plots. As is shown in
Figure 2A-II and B-II, the intersection of the lines in the second
quadrant indicated that both 5e and 5d were competitive−
uncompetitive mixed-type inhibitors, suggesting that both com-
pounds could bind with both free enzyme and the enzyme−
substrate complexes (ES). The equilibrium constant for inhibitor
binding with free enzyme (KI) was obtained from a plot of slope
(Km/Vm) versus the concentration of the inhibitor, and with ES
(KIS) was obtained from a plot of the vertical intercept (1/Vm)
Figure 1. Inhibitory effects of hydroxypyridinone-phenylalanine
conjugates on monophenolase activity of mushroom tyrosinase. The
assays were carried out in triplicates under the following reaction
conditions: pH 6.8, 30 °C, 10 min.
significant difference was observed between the uses of different
inhibitors (P < 0.05). For each inhibitor, the significant
difference in inhibitory effect also occurred between the each
tested concentration (P < 0.05). IC50 values were summarized
in Table 1. Compounds 5e was found to exhibit potent inhibitory
activity against tyrosinase with an IC50 value of 12.6 μM, which
was lower than that of kojic acid (IC50 = 26.8 μM). From
compounds 5a to 5e, the inhibitory effect increased with the
increase of hydrophobicity of the compound, which is in good
agreement with previously published data.6 However, 5f, the most
Table 1. Inhibitory Effect of Hydroxypyridinone-L-phenylalanine Conjugates (5) against Monophenolase and Diphenolase
Activity of Mushroom Tyrosinase
compounds
C log P
IC50 (μM) (against monophenolase activity)
IC50 (μM) (against diphenolase activity)
KI (μM)
KIS (μM)
5a
0.238
0.614
1.676
2.686
3.697
4.707
−0.888
2590 55.8
1150 31.5
190 6.4
5b
5c
5d
19.2 0.8
12.6 0.3
1650 41.3
26.8 1.2
42.8 2.3
4.0 0.1
55.5 1.5
4.0 0.03
193.5 12.5
8.36 0.53
5e
5f
kojic acid
20
13.0
100.0
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Figure 2. Nonlinear Michaelis−Menten plot (A-I and B-I) and Lineweaver−Burk plots (A-II and B-II) of mushroom tyrosinase with L-DOPA as a
substrate in the presence of 5d (A) and 5e (B). A-I and A-II: The concentrations of 5d for curves 1−4 were 0, 20, 50, and 150 μM, respectively. B-I
and B-II: The concentrations of 5e for curves 1−4 were 0, 2, 5, and 15 μM, respectively. A-III and B-III represent the plot of slope versus the
concentration of 5d and 5e for determining the inhibition constants KI. A-IV and B-IV represent the plot of intercept versus the concentration of 5d
and 5e for determining the inhibition constants KIS.
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versus the concentration of the inhibitor. The KI and KIS values of
5d were determined to be 55.5 and 193.5 μM, respectively. The
inhibitor constants (KI and KIS) of 5e were measured as 4.0 and
8.36 μM, respectively, which are lower than those of kojic acid
(KI = 13.0 μM, KIS = 100.0 μM).28 In both cases, the KIS value is
larger than the KI value, indicating that the affinity of the inhibitors for
free enzyme is greater than that for the enzyme−substrate complex.
pKa Values and Copper Affinity of 5e. To explore the
inhibitory mechanism of 5e on tyrosinase, the pKa values and the
affinity for copper were evaluated. The pH dependence UV spectra
of 5e (Figure 3) was recorded between 250 and 350 nm over the
complexes, CuL+ and CuL2. The stability constants of these two
metal−ligand complexes can be expressed as follows:
+
[CuL ]
−
+
Cu2+ + L ⇌ CuL β1 =
[Cu2+][L ]
−
(1)
(2)
[CuL2]
−
Cu2+ + 2L ⇌ CuL2 β2 =
− 2
[Cu2+][L ]
where L− represents ligand.
Employing spectrophotometric titration against the hydroxyl
anion, the log stability constants of the two complexes (CuL+ and
CuL2), log β1 and log β2, were determined to be 9.294 0.09 and
15.719 0.11, respectively. A series of UV spectra of 5e in the
presence of copper at different pH values is shown in Figure 4.
As the pH increased from 2, the absorbance at 700 nm gradually
rose, and then the peak shifted to 650 nm. This indicates
the formation of the copper complexes CuL+ and CuL2
correspondingly.
Other synthesized compounds (5a−5d and 5f) were
assumed to possess similar affinities for copper, which is higher
than that of kojic acid (log β1 = 6.6, log β2 = 11.7).29 However,
with the exception of 5e and 5d, other compounds exhibited a
lower activity than kojic acid. It is possible that the hydrophobic
alkyl chain at the N-1 position plays an important role in the
inhibitory activity of 5e and 5d. Hydrophobic interaction of the
hydrophobic alkyl chain with the hydrophobic protein pocket
surrounding the binuclear copper active site in the enzyme may
disrupt tyrosinase’s quaternary structure.30
Figure 3. UV spectra of 5e. [5e] = 870 μM (in 20.120 mL of 0.1 M KCl),
pH was changed from 1.884 to 11.110 by the addition of KOH at 25 °C.
The cytotoxicity of 5e and kojic acid on Hela, A549, and MCF-
7 cell lines was examined using a MTT assay. From the assay
results, it was found that 5e was slightly less toxic than kojic acid
overall, lacking obvious toxicity up to 10 μM (Table 2).
In conclusion, a range of hydroxypyridinone-L-phenylalanine
conjugates were synthesized. Among them, 5e exhibited higher
tyrosinase inhibitory activity than did kojic acid. Moreover, 5e was
demonstrated to be nontoxic to three cell lines, and also could find
application in the cosmetic and food preserving areas.
AUTHOR INFORMATION
Corresponding Author
*Tel.: (+86) 571 88071024-7587. Fax: (+86) 571 88905733. E-
mail: taozhou@zjgsu.edu.cn.
■
Figure 4. UV spectra of 5e in the presence of copper. [5e] = 814.6 μM,
[Cu2+] = 237 μM (in 20.414 mL of 0.1 M KCl), pH was changed from
2.078 to pH 6.052 by the addition of KOH at 25 °C.
Funding
This research work was financially supported by Zhejiang Provincial
National Natural Science Foundation of China (no. LY12B02014) and
the National Natural Science Foundation of China (no. 20972138).
pH range 1.88−11.11 for the free ligand. The speciation spectra
demonstrate a clear shift in λmax from 280 to 310 nm, which reflects
the pH dependence of the ligand ionization equilibrium. Using the
spectrophotometric titration method, the pKa values of 5e obtained
from nonlinear least-squares regression analysis were found to be
3.191 0.06, 8.776 0.07, and 10.34 0.05, which correspond to
the 4-oxo function, the 5-hydroxyl function, and the amino group,
respectively. The stability constant of a metal−ligand complex is
one of the key parameters related to the chelation efficacy of a
ligand. As a bidentate, 5e can form two species of copper
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
DMAP, 4-dimethylaminopyridine; EDC, N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride; DMF, N,N-dime-
thylformamide; ES, enzyme−substrate complexes; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Table 2. Cell Survival Results Based on MTT Assay (%)
5e (μM)
kojic acid (μM)
cell lines
0.1
1.0
10
0.1
1.0
10
Hela
104.2 2.7
97.7 3.1
96.7 1.9
104.8 3.6
97.0 2.2
102.0 2.4
101.4 3.7
100.1 2.8
99.6 2.2
102.7 2.2
99.3 2.5
97.8 3.1
104.6 3.9
99.7 2.6
96.5 2.7
102.2 3.4
98.9 3.2
92.3 2.6
A549
MCF-7
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