Synthesis and biological evaluation of polyhydroxy benzophenone as mushroom tyrosinase inhibitors

Article abstract of DOI:10.3109/14756366.2010.521745

A series of polyhydroxy benzophenone were synthesized and evaluated as mushroom tyrosinase inhibitors. The results demonstrated that most of the target compounds had remarkable inhibitory activities on mushroom tyrosinase. Among all these compounds, 2,3,4,3′,4′,5′-hexahydroxy-diphenylketone 10 was found to be the most potent tyrosinase inhibitor with IC50 value of 1.4 μM. In addition, the inhibition kinetics analyzed by Lineweaver-Burk plots revealed that such compounds were competitive inhibitors. These results suggested that such compounds might be utilized for the development of new candidate for treatment of dermatological disorders.

Full text of DOI:10.3109/14756366.2010.521745

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(3): 449–452
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.521745
SHORT COMMUNICATION
Synthesis and biological evaluation of polyhydroxy
benzophenone as mushroom tyrosinase inhibitors
Jianlong Wu, Xuesen Hu, and Lin Ma
School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, P. R. China
Abstract
A series of polyhydroxy benzophenone were synthesized and evaluated as mushroom tyrosinase inhibitors.
The results demonstrated that most of the target compounds had remarkable inhibitory activities on mushroom
tyrosinase. Among all these compounds, 2,3,4,3',4’,5’-hexahydroxy-diphenylketone 10 was found to be the most
potent tyrosinase inhibitor with IC value of 1.4 μM. In addition, the inhibition kinetics analyzed by Lineweaver–Burk
plots revealed that such compou5n0ds were competitive inhibitors. These results suggested that such compounds
might be utilized for the development of new candidate for treatment of dermatological disorders.
Keywords: Polyhydroxy benzophenone, tyrosinase, inhibitors
Tyrosinase (EC 1.14.18.1; polyphenol oxidase, PPO), a
multifunctional copper-containing enzyme, catalyzed
two distinct reaction of melanin synthesis: the hydroxyla-
tion of monophenols and the oxidation of the o-phenols.1,2
ereby, the enzyme converts tyrosine to 3,4-dihydroxy-
phenylalanine (l-dopa) and oxidizes l-dopa to form dop-
aquinone, which plays important roles in the process of
melanin biosynthesis.3 Tyrosinase is widespread in plants
and animals. erefore, tyrosinase inhibitors have become
increasingly important in agriculture,2,4 cosmetic industry,5
and medication6 due to decreasing the excessive accumula-
tion of pigmentation resulting from the enzyme action.7–12
Presently, tyrosinase inhibitors have been established
as important constituents of cosmetic materials, food
preservative, and depigmenting agents for hyperpigmen-
tation. Many tyrosinase inhibitors have been reported, for
example, hydroquinone,13–16 kojic acid,17 azelaic acid,18,19
electron-rich phenols,20 corticosteroids,21,22 resinoids,23,24
resveratrol,25 oxyresveratrol,26 and arbutin have been uti-
lized as cosmetic agents. Most of these compounds have
the structures of benzene rings and hydroxyl radicals.
In the last decades, polyhydroxy benzophenones have
been widely utilized for the production and develop-
ment of plastic, resin, coating materials, synthetic rubber,
light-sensitive materials, and cosmetic materials. However,
its other properties such as antioxidant activity and
tyrosinase inhibitory activity have not been reported yet.
Since polyhydroxy benzophenones have the similar
structures to the tyrosinase inhibitors mentioned earlier,
we speculated that polyhydroxy benzophenones might
exhibit potent tyrosinase inhibitory activity. erefore,
our team synthesized a series of polyhydroxy benzo-
phenones and evaluated their inhibitory effects on the
diphenolase activity of mushroom tyrosinase. To the best
of our knowledge, this is the first time to report the inhib-
itory effects on the diphenolase activity of mushroom
tyrosinase of polyhydroxy benzophenones.
e synthetic routes of compounds 1–181 were out-
lined in Scheme 1, and the chemical structures of poly-
hydroxy benzophenones 1–18 were given in Table 1.
Tyrosinase inhibition assays were performed accord-
ing to the developed method described earlier by
1e synthesis of compounds 1–18: To the mixture of appropriate phenol
(50 mmol) and polyhydroxybenzoic acid (55.6 mmol), anhydrous zinc
chloride (27.2 g) and phosphorus oxychloride (25 mL) were added.
e reaction mixture was refluxed for 2.5 h in the water bath of 70°C
with stirring. en add the mixture to appropriate ice and cool to 4°C
for 24 h. e precipitate solid was filtered, washed with 3% sodium
bicarbonate twice, and purified by recrystallization from boiling
water to afford compounds 1–18 (Figure 1). e synthetic route of
compounds 1–18 is shown in Scheme 1. Compound 10: 2,3,4,3',4’,5’-
hexahydroxydiphenylketone (10). Yield: 90%; yellow powders; mp
272–273°C; 1H NMR (dimethyl sulfoxide-d6, 300 MHz) δ (ppm): 6.4
(1H, d, J= 6.9 Hz, ArH), 6.6 (2H, s, ArH), 7.0 (1H, d, J= 4.2 Hz, ArH), 8.6
(1H, s, OH-R3), 8.9 (1H, s, OH-R2), 9.3 (2H, s, OH-R6, OH-R8), 10.0 (1H, s,
OH-R7), 12.1 (1H, s, OH-R1). 13C NMR(dimethyl sulfoxide-d6, 300 MHz)
δ: 13C NMR(dimethyl sulfoxide-d6, 300 MHz) δ: 198.60(Ccarbonyl), 175.94
(Cphenyl-4), 152.43 (Cphenyl-3′), 145.96 (Cphenyl-3,5), 138.27 (Cphenyl-4′),
133.45 (Cphenyl-2′), 128.61 (Cphenyl-5′), 125.31 (Cphenyl-6′), 113.76 (Cphenyl-1′),
109.64 (Cphenyl-2,6), 107.83 (Cphenyl-1).
Address for Correspondence: Lin Ma, School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang Xi Road,
Guangzhou 510275, P. R. China. E-mail: cesmal@mail.sysu.edu.cn
(Received 07 July 2010; revised 01 September 2010; accepted 02 September 2010)
449

450 J. Wu et al.
O
R5
R1
R5
R1
R6
R7
R6
R7
COOH
R2
R3
R2
ZnCl2, POCl3
+
R4
R3
R4
R8
R8
Scheme 1. Synthetic route of polyhydroxy benzophenones 1–18.
Table 1. Chemical structure of polyhydroxy benzophenones 1–18 and inhibitory effects on mushroom tyrosinase of polyhydroxy
benzophenones and kojic acid as reference inhibitor.
O
R5
R1
R6
R7
R2
R3
R4
R8
Compound R1
R2
R3
R4
H
R5
H
R6
R7
R8
H
IC50 (μmol/L)
NTa
1
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
2
3
OH
H
H
H
H
H
H
NT
H
H
H
OCH3
H
H
NT
4
5
6
7
8
9
10
11
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
OH
H
H
≥200
≥200
17.4
≥100
10.1
6.4
H
H
OCH3
OH
H
H
H
H
H
OH
H
OH
OH
OH
OH
H
H
H
H
OH
H
H
H
H
OCH3
OH
H
H
H
H
OH
H
1.4
OH
H
OH
H
≥200
≥200
7.3
12
13
14
H
OH
H
H
H
H
OH
H
H
H
H
H
OH
H
OH
H
OH
H
98.3
8.8
15
16
17
18
OH
OH
OH
OH
—
OH
H
H
OH
H
OH
H
H
H
H
≥200
10.5
≥200
19.0
OH
OH
—
H
H
H
H
H
H
H
H
H
Kojic acid30
—
—
—
—
—
—
aNT: not tested.
Hearing27 with some modification.2 e IC50 values of all
compounds investigated were summarized in Table 1
and Figure 1. As the results reported in Table 1, com-
pounds 6, 8, 9, 10, 13, 15, and 17 exhibited more potent
tyrosinase inhibitory activities than kojic acid, especially
compound 10 (IC50 = 1.4 μmol/L), which demonstrated
excellent in vivo activity.
Of all polyhydroxy benzophenones 1–18, compounds
1, 18, and 3, which are dihydroxy-substituted diphe-
nylketones, display none or very weak inhibitory effect
on tyrosinase, whereas trihydroxy-, tetrahydroxy-, penta-
hydroxy-, and hexahydroxy-substituted diphenylketones
exhibited a significant increase inhibitory activity on
tyrosinase along the number of hydroxyl group grows.
It seems to be a tendency that the inhibitory activity of
polyhydroxy benzophenones on tyrosinase increase
along the number of hydroxyl group grows.
2Tyrosinase Inhibition Assay: In brief, a 10 μL sample was added to an
assay mixture containing with 10 μL tyrosinase solution (0.5 mg/mL)
and 900 μL phosphate buffer (pH 6.8) to a total volume assay mixture
of 920 μL, for 20 min at 25°C. en 80 μL of l-dopa (1.50 mg/mL) was
added to the reaction mixture and the enzyme reaction was monitored
by measuring the change in absorbance at 475 nm of the l-dopa for
1 min. IC50 value, a concentration giving 50% inhibition of tyrosi-
nase activity, was determined by interpolation of the dose–response
curves. Here, kojic acid (IC50 = 19.0 μmol/L) was used as the reference
inhibitors.
Interestingly, most of the compounds that display
potent tyrosinase inhibitory activities are substituted
by hydroxyl group on positions R1, R2, and R3. e result
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and biological evaluation of polyhydroxy benzophenone 451
80
70
60
50
40
30
20
10
0
30
25
6
20
8
9
20 µM
10
13
14
15
17
15
0 µM
10
5
0
20 40 60 80 100 120 140 160 180 200
0
c/µmol L−1
0.0 0.5 1.0 1.5 2.0 2.5
−2.5 −2.0 −1.5 −1.0 −0.5
1/[s](mM−1
)
Figure 1. Dose-dependent inhibition of mushroom tyrosinase by
polyhydroxy benzophenones.
Figure 2. Lineweaver–Burk plots for inhibition of 2,3′,4,4′,5′-
pentahydroxydiphenylketone on mushroom tyrosinase for the
catalysis of dopa at 25°C, pH 6.8. Concentrations of 2,3′,4,4′,5′-
pentahydroxydiphenylketone (compound 6) for curves were
0 and 20 μmol/L, respectively.
suggests that the introduction of hydroxyl group on posi-
tions R1, R2, and R3 strengthens the tyrosinase inhibitory
activity. Compared with compounds 2and 12, compound
13 (IC50 = 7.3 μmol/L) bearing a hydroxyl substituent at
position R5 displays potent inhibitory activity. When
position R7 is replaced by hydroxyl group, such as com-
pounds 4 (IC50 ≥ 200 μmol/L) and 7 (IC50 ≥ 100 μmol/L), a
significant decrease in inhibition potency was observed,
whereas the IC50 values of compounds 13 and 17 are
7.3 μmol/L and 10.5 μmol/L, respectively.
both the number and position of hydroxyl groups in
diphenylketone seem to play a critical role in exerting
the inhibitory effect on dopa oxidase activity of tyrosi-
nase. In addition, the inhibition kinetics analyzed by
Lineweaver–Burk plots revealed that such compounds
were competitive inhibitors. ese results suggested
that such compounds might be utilized for the develop-
ment of new candidate for treatment of dermatological
disorders.
Inaddition,compoundssubstitutedbymethoxylgroup
exhibited more potent tyrosinase inhibition potencies
than unsubstituted compounds with similar structure,
such as compounds 9 and 7 or compounds 2 and 5.
As the compounds that showed potent inhibitory
activities on tyrosinase have similar structures to each
other, we can get the conclusion that they have the same
inhibition type on mushroom tyrosinase. erefore,
2,3′,4,4′,5′-pentahydroxy-diphenylketone (compound
6) had been chosen to investigate the kinetic behav-
ior of mushroom tyrosinase during the oxidation of
Declaration of interest
is work was supported by the Undergraduate Research
Programmes of Sun Yat-sen University, China (2009-73).
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