Enhanced substituted resorcinol hydrophobicity augments tyrosinase inhibition potency

Article abstract of DOI:10.1021/jm061361d

The objective of the present study was to investigate to what extent the addition of hydrophobic residues to a 2,4-resorcinol derivative would contribute to their tyrosinase inhibitory potency. Hence, 3-(2,4-dihydroxyphenyl)propionic acid, isolated from F

Full text of DOI:10.1021/jm061361d

2676
J. Med. Chem. 2007, 50, 2676-2681
Enhanced Substituted Resorcinol Hydrophobicity Augments Tyrosinase Inhibition Potency
Soliman Khatib,†,‡ Ohad Nerya,† Ramadan Musa,† Snait Tamir,†,‡ Tal Peter,‡ and Jacob Vaya†,‡,
*
The Laboratory of Natural Medicinal Compounds, MIGAL - Galilee Technology Center, P.O. Box 831, Kiryat Shmona 11016, Israel, and
Department of Biotechnology, Tel-Hai Academic College, Israel
ReceiVed NoVember 27, 2006
The objective of the present study was to investigate to what extent the addition of hydrophobic residues to
a 2,4-resorcinol derivative would contribute to their tyrosinase inhibitory potency. Hence, 3-(2,4-
dihydroxyphenyl)propionic acid, isolated from Ficus carica, was transformed into esters, and the relationship
between the structure of these esters to their mushroom tyrosinase inhibition activity was explored. The
enzyme crystallographic structure, published recently (Matoba, Y. et al. J. Biol. Chem. 2006, 281, 8981-
8990) was docked with the new esters, and their calculated free energy (FE) and docking energy (DE)
were compared with the experimental IC50 values, providing good correlations. The observed IC50 of the
isopropyl ester was 0.07 µM, and its interaction with the enzyme binding site appears to be composed of
four hydrogen bonds and two hydrophobic interactions. It may be concluded that the addition of a hydrophobic
moiety to 2,4-resorcinol derivatives augments tyrosinase inhibitory potency as was predicted from the
modeling study.
Introduction
In reports on the inhibitory effect of dodecyl gallate, it has been
debated that its enzyme inhibition is derived, in part, from the
interaction of the dodecyl hydrophobic moiety with the hydro-
phobic domain of the enzyme.15 This finding emphasized the
possibility that a hydrophobic moiety may also contribute to
the inhibition.
Tyrosinase is a copper-containing enzyme, widely distributed
in nature. It has a diverse role in agriculture, the cosmetic
industry, and medicine, is involved in the browning of fruits
and vegetables2 and in the formation of brown pigments
(melanins), and is responsible for mammalian skin and hair
color.3 Tyrosinase is also linked to Parkinson’s and other
neurodegenerative diseases,4,5 oxidizing excess dopamine to
produce DOPA quinones, highly reactive compounds that induce
neuronal damage and cell death.
As part of our study in search of new biologically active
agents from plants and fungi, we have isolated a tyrosinase
inhibitor from fig leaves and fruits. The structure of the active
compound has been elucidated as 3-(2,4-dihydroxyphenyl)-
propionic acid (DPPAa). Some of the biochemical properties
of this compound have been characterized previously,16,17
including its tyrosinase inhibition. Apparently, DPPA is a
competitive inhibitor of tyrosinase, with reported IC50 of 3.2
µM. In a recent study, aliquots of DPPA (1 µg/mL) reduced
the browning of slide mushrooms by 50%.2 The new tyrosinase
three-dimensional structure revealed the presence of a hydro-
phobic protein pocket adjoining the binuclear copper active site.
On the basis of data from literature7 and our lab, including a
modeling study,8 we hypothesize that compounds of 2,4-
resorcinol structure can fit the binuclear copper active site well,
and that in the presence of a hydrophobic subunit connected to
the resorcinol aromatic ring, may interact with the hydrophobic
enzyme pocket, leading to increased inhibitor-enzyme binding
affinity, and thus to an improved compound activity. In the
present study, on the basis of the new tyrosinase three-
dimensional structure,1 we have aimed to form DPPA esters
by reactions of DPPA with hydrophobic alcohols of various
structures and to examine the above hypothesis.
The crystallographic structure of tyrosinase has been estab-
lished recently,1 enabling a close look at its three-dimensional
structure and a better understanding of its mechanism of action.
The enzyme catalyzes two reactions in the melanin biosynthesis
pathway involving molecular oxygen: the hydroxylation of
monophenols to o-phenols, and the oxidation of the o-phenols
to o-quinones, which further polymerize spontaneously into
melanin.
Tyrosinase inhibitors have become increasingly important,
and a number of naturally occurring ones have been described.
Most consist of a polyphenol structure with poor (IC50 > 100
µM)6 to moderate (IC50 ) 10-100 µM) activity7 and a few
with high inhibition potency (IC50 < 10 µM).8 Reports on
the relation between the structure of such polyphenol deriva-
tives and their enzyme inhibition have emphasized the contribu-
tion of the 2,4-substituted resorcinol structure and the signifi-
cance of the location of their hydroxyl groups on both aromatic
rings, with a preference for a 4-substituted B ring rather than
a substituted A ring in regard to the potency.8-10 Resorcinol
residues, such as in flavonoids, in 4-alkyl-substituted
resorcinols,7,11-13 in stilbenes3,6,14 and in chalcones8 have been
investigated elsewhere as tyrosinase inhibitors. Among the
various 4-alkyl-substituted resorcinols, the 4-hexyl derivative
has been found to be a superior inhibitor to the 4-dodecyl- or
4-ethylresorcinol, and slightly less effective than kojic acid.11
Material and Methods
Chemicals and Reagents. Tyrosinase (EC 1.14.18.1, with an
activity of 6680 units/mg, T7755) and the DPPA standard were
purchased from Sigma. 3-(2,4-Dihydroxyphenyl)propionic acid
a Abbreviations: FE, free energy; DE, docking energy; DPPA, 3-(2,4-
dihydroxyphenyl)propionic acid; EtOH, ethanol; THF, tetrahydrofuran;
ADT, AutoDock Tools program; RP, reverse phase; ES, electrospray
ionization; Rij, equilibrium separation between two nucleus; epsij,
pairwise potential energy; GA, genetic algorithm; Ki, inhibition constant;
MOPAC, semiempirical molecular orbital package; PM3, semiempirical
Hamiltonian.
* Corresponding author. Tel: 972-4-6953512. Fax: 972-4-6944980.
E-mail: vaya@migal.org.il.
† MIGAL - Galilee Technology Center.
‡ Tel-Hai Academic College.
10.1021/jm061361d CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/21/2007

Tyrosinase Inhibition Potency of Resorcinol DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2677
1H NMR Data. DPPA ethyl ester [3-(2,4-dihydroxyphenyl)-
propionic acid ethyl ester, (compound 2] (acetone-d6): δ 6.9 (1H,
d, J ) 8.20 Hz); 6.38 (1H, d, J ) 2.60 Hz); 6.27 (1H, dd, J )
8.18, 2.20 Hz); 3.58 (2H, q, J ) 6.70 Hz); 2.79 (2H, t, J ) 7.80
Hz), 2.56 (2H, t, J ) 7.40 Hz); 1.12 (3H, t, J ) 6.70 Hz).
DPPA isopropyl ester [3-(2,4-dihydroxyphenyl)propionic acid
isopropyl ester, compound 3] (acetone-d6): δ 6.9 (1H, dd, J ) 8.18,
6.30 Hz), 6.37 (1H, d, J ) 2.23 Hz), 6.28 (1H, dt, J ) 8.56, 2.20
Hz), 4.9 (1H, sept., J ) 6.33 Hz), 2.79 (2H, t, J ) 7.80 Hz), 2.55
(2H, t, J ) 7.44 Hz), 1.19 (6H, d, J ) 6.33 Hz).
DPPA octyl ester [3-(2,4-dihydroxyphenyl)propionic acid octyl
ester, compound 4] (acetone-d6): δ 7.02 (1H, d, J ) 7.80 Hz); 6.5
(1H, d, J ) 2.23 Hz); 6.4 (1H, dd, J ) 8.18, 2.23 Hz); 3.72 (2H,
t, J ) 7.07 Hz); 2.92 (2H, t, J ) 7.80 Hz); 2.69 (2H, t, J ) 7.80
Hz); 1-1.7 (15H, m).
DPPA tert-butyl ester [3-(2,4-dihydroxyphenyl)propionic acid
tert-butyl ester, compound 5] (acetone-d6): δ 6.89 (1H, d, J ) 8.18
Hz); 6.38 (1H, d, J ) 2.23 Hz); 6.28 (1H, dd, J ) 8.10, 2.23 Hz);
2.77 (2H, t, J ) 8.20 Hz); 2.54 (2H, t, J ) 7.815 Hz); 1.5 (9H, s).
DPPA 2,2-dimethylpropyl ester [3-(2,4-dihydroxyphenyl)-
propionic acid 2,2-dimethylpropyl ester, compound 6] (acetone-
d6): δ 6.89 (1H, d, J ) 8.18 Hz); 6.38 (1H, d, J ) 2.23 Hz); 6.28
(1H, dd, J ) 8.10, 2.23 Hz); 3.74 (2H, s); 2.82 (2H, t, J ) 8.20
Hz); 2.59 (2H, t, J ) 7.815 Hz); 0.9 (9H, s).
3,3,5,5-Tetramethyl-4-heptyl ester [3-(2,4-dihydroxyphenyl)-
propionic acid 3,3,5,5-tetramethyl-4-heptyl ester, compound 7]
(CDCl3): δ 6.87 (1H, d, J ) 8.10 Hz); 6.34 (1H, d, J ) 2.40 Hz);
6.32 (1H, dd, J ) 8.10, 2.70 Hz); 3.74 (1H, s); 2.9 (2H, t, J ) 8.00
Hz); 2.66 (2H, t, J ) 8.00 Hz); 1.2 (4H, q, J ) 7.20 Hz); 0.77
(12H, s); 0.735 (6H, t, J ) 7.50 Hz).
Tyrosinase Inhibitory Effect of DPPA Derivatives. Potassium
phosphate buffer (70 µL, 50 mM) at pH 6.5, 30 µL of tyrosinase
(final concentration of 50 units/mL), and 3 µL of the tested
compounds dissolved in absolute ethanol (final concentration of
5-100 µM) were added to wells of 96-well plates. After 10 min
incubation at 25 °C, L-tyrosine (final concentration of 1 mM) was
added. The optical density (450 nm) of the samples was measured
(ELISA SLT Lab Instruments Co. A-5082) every 5 min, relative
to the control, which contained ethanol (2 µL and without inhibitor),
demonstrating a linear color change with time. The IC50 was
calculated at the linear zone of the curve (20 min with L-tyrosine,
as the substrate).
Preincubation of Compounds 6, 7, and DPPA with Tyrosi-
nase. Either compouns 5 or 7 or DPPA was tested, with and without
preincubation, with the enzyme. Each of these compounds was
added separately to the enzymatic reaction, as described above, at
concentrations that inhibit 80% of the enzyme activity (0.5, 0.3,
and 10 µM for compounds 5, 7, and DPPA, respectively). After
10 min of incubation, the substrate (L-tyrosine, 1.0-3.75 mM) was
added. The formation of DOPA-quinone was followed by an
absorption measurement at 450 nm, 20 min after the addition of
L-tyrosine.
Sample Dialysis. DPPA (10 µM) was incubated with tyrosinase
for 20 min, followed by dialysis (dialysis cassette, 10000 MWCO,
Pierce) overnight. The activity of the enzyme was measured after
dialysis and compared to that of the enzyme under the same
conditions before dialysis. After dialysis, the presence of free
DPPA, was also tested by LC-MS at the ES- mode (LC- Waters
2790 HPLC, with the Waters Photodiode Array Detector 996,
attached to the Micromass Quattro Ultima MS). The protein
quantity was calculated by Bradford analysis, before and after the
dialysis.
Molecular Docking: Preparation of the Enzyme. The crystal
structure of the tyrosinase oxy form, complexed with a Caddie
Protein and prepared by the addition of hydrogen peroxide, was
retrieved from the Protein Data Bank.1 The enzyme was prepared
for docking by the ADT program (AutoDock Tools program), an
accessory program that allows the user to interact with AutoDock18-20
from a graphic user interface. Water, heteroatoms, and the Caddie
Protein were removed from the protein PDB file. Polar hydrogen
Table 1. Docking and Free Energies Calculated by Using AutoDock
Program. A Comparison between Experimental and Calculated IC50
Values
IC50 (µM)
docking
energy
free
energy
R
(kcal/mol) (kcal/mol) experimental calculateda
1
2
3
4
5
6
7
H
ethyl
isopropyl
octyl
-6.21
-7.41
-8.01
-7.98
-7.59
-8.19
-6.17
-5.43
-5.88
-6.45
-4.29
-6.9
-6.27
-6.73
1.85
0.22
0.07
0.70
14.87
0.18
0.15
0.40
0.27
0.12
0.70
0.0025
0.17
0.05
tert-butyl
2,2-dimethylpropyl
3,3,5,5-tetramethyl-
4-heptyl
a Calculated IC50 values were generated from free energy values by the
following equation, FE ) 3.738 × (IC50) - 6.909 (generated from a
regression curve relating free energy to experimental IC50).
(DPPA, 1) was isolated from fig leaves, as described below.
Solvents used were of HPLC grade.
Isolation and Structure Determination of DPPA from Fig
Leaves and Fruits. Dry leaves (380 g) from fig trees (Ficus carica
L.) were extracted twice, each time with 760 mL of 70% ethanol
in water. The crude extract (28.12 g, 7.4% yield) exhibited 80%
inhibition of the mushroom tyrosinase activity, at a concentration
of 0.032 mg/mL.
The dry extract was dissolved in 100 mL of 20% methanol in
water and centrifuged (12000 rpm, 4 °C) to remove insoluble
materials, and the supernatant was collected. The filtrate was
chromatographed on a silica gel column (silica gel S, 0.032-0063)
and eluted with a solvent mixture gradient (0% to 100% methanol/
dichloromethane). A sample from each fraction was examined for
tyrosinase inhibition, and the active fractions were collected and
rechromatographed on a similar form of silica gel. Fractions with
active compounds were then separated on an HPLC preparative
RP-18 column, using a gradient of eluents (5% to 30% acetonitrile/
water), followed by an additional separation on sephadex LH-25,
with methanol as an eluent, to afford a pure active compound (28
mg). The structure of the pure isolated active compound was
elucidated as 3-(2,4-dihydroxyphenyl)- propionic acid (DPPA) by
a conventional analytical method. UV-vis spectra (EtOH three
bands at λmax ) 210, 225, 280 nm); IR(KBr) ν ) 3387, 2933, 1701,
1605, 1515; NMR 6.94 (1H, d, J ) 8.18 Hz), 6.38 (1H, d, J )
2.60 Hz), 6.27 (1H, dd, J ) 8.18, 2.60 Hz), 2.79 (2H, t, J ) 7.44
Hz), 2.56 (2H, t, J ) 7.80 Hz).
Synthesis of DPPA Esters. DPPA ethyl (2), isopropyl (3), octyl
(4), tert-butyl (5), 2,2-dimethylpropyl (6), and 3,3,5,5-tetramethyl-
4-heptyl (7) esters (Table 1) were synthesized by conventional
procedures. Briefly, 20 mg (0.11mmol) of DPPA were dissolved
in 5 mL of the respective alcohol (for esters 2-6), 100 µL H2SO4
(98%) was added, and the resulting solution was heated to either
reflux or 100 °C for 2 h. A slight modification was employed for
the synthesis of ester 7. Briefly, 20 mg (0.11mmole) of DPPA was
reacted with 20 mg (0.22 mmol) of 3,3,5,5-tetramethyl-4-heptanol
in 1 mL of dry THF, followed by the addition of 50 µL of H2SO4.
After the solution had been stirred under argon for 24 h at 37 °C,
5 mL of water was added and each ester was extracted three times
with 10 mL of chloroform. The chloroform was evaporated under
reduced pressure, and the product was purified by flash chroma-
tography (silica gel, hexane:ethyl acetate, 70:30 as solvents),
affording purified esters, with a 75-85% yield. The products were
injected into the LC-MS under the electrospray ionization mode
(ES-), resulting in the expected molecular ion at m/z ) 181, 209,
223, 293, 237, 251, and 335 (M - 1) for compounds 1-7,
respectively.

2678 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Khatib et al.
tively, as compared to the IC50 of DPPA, 1.9 µM. These results
may indicate that increased inhibitory potency does not neces-
sarily correlate with the increase in the carbon chain length,
but rather may be related to greater ester bulkiness.
At this point, we questioned if such a performance could be
predicted theoretically, prior to inhibitor synthesis, by calculating
parameters related to interactions between the inhibitor and the
enzyme’s amino acids, within the enzyme core. Thus, parameters
such as docking and free energy were calculated using the
AutoDock program, as detailed in Materials and Methods. The
crystallographic structure of the enzyme was acquired from
newly available data.1 The structure of each potential inhibitor,
requiring minimum energy, as determined by the MOPAC
program,21 and according to the energy calculations of the
enzyme-inhibitor complexes, was used. A summary of the
results, which is shown in Table 1, implies that both parameters,
the docking and free energy, were useful predictors of the
expected inhibitory potency. The values of the energy level of
both parameters decreased with the elevation of the observed
inhibitor activity (experimental IC50) and correlated with the
expected IC50. On the basis of the above data of the free energy,
three additional potential inhibitors, with a variety of bulky
esters, were docked. The predicted FE values of the compounds,
5 (tert-butyl ester of DPPA), 6 (2,2-dimethylpropyl ester), and
7 (1-(1,1-dimethylpropyl)-2,2-dimethylbutyl ester), were -6.9,
-6.27, and -6.73 kcal/mol, respectively (Table 1) and from
ploting a regression curve of free energy and docking energy
versus the experimental IC50, the expected IC50 for compounds
5, 6, and 7 were calculated to be 0.0025, 0.17, and 0.05 µM,
respectively. These three compounds, 5, 6, and 7, were
synthesized and their observed IC50 values found to be 14.90,
0.18, and 0.15, respectively. A new regression curve was
calculated relating the above energies values to the experimental
IC50 of all esters synthesized including those of compounds 5,
6, and 7, excluding the tert-butyl ester of the DPPA, afforded
an R2 of 0.94 and 0.76, respectively, with P values of 0.0063
and 0.0525, respectively.
Figure 1. Inhibition of mushroom tyrosinase by three natural
compounds, each at concentration at or above 50% inhibition: (9) 2
µM 3-(2,4-dihydroxyphenyl)propionic acid (DPPA), (b) 7 µM glabrene,
and (2) 0.07 µM glabridin.
atoms were added and Kollman united atomic charges assigned.
The solvation parameters were added by the Addsol program (part
of the ADT program). The two copper ions and the oxygen atoms
of the enzyme active sites were returned to their original sites. The
“C” letter of the copper ions was changed to “M”, and the solvation
parameters of the copper ions and peroxide oxygen were fixed to
zero.
Preparation of Ligands. The ligands were constructed by “CS
Chemdraw Pro” and “CS ChemBats3D Pro” and optimized to their
minimum energy with the MOPAC program and the PM3 semiem-
pirical Hamiltonian.21 Rotatable bonds and charges in the ligands
were assigned by the ADT program.
Grid Parameters. The grid points were set to the catalytic site of
the enzyme. The number of grid points in xyz was set to 40, 40,
40, the spacing value equivalent to 0.375, and the grid center to
-14.041, -13.964, 21.713.
The copper van der Waals parameters, which were obtained from
the “Quanta 3.0 Parameters Handbook” are atom type M equilib-
rium separation between the nucleus of the two copper ions, Rij )
1.27 A0, and pairwise potential energy, epsij ) 0.06.
Docking. Ligand docking was carried out with the AutoDock 3.0.5
Lamarckian Genetic Algorithm (GA). The approximate binding free
energies, calculated by this program, are based on an empirical
function, derived by linear regression analysis of protein-ligand
complexes with known binding constants. This function includes
terms for changes in energy due to van der Waals, hydrogen bonds,
and electrostatic forces, as well as ligand torsion and desolvation.
The docked energy also includes the ligand internal energy or the
intramolecular interaction energy of the ligand. All of the parameters
were assigned the default values, implemented by the program.
Analysis of Results. All of the docking results were visualized
and analyzed with ADT and Chimera software. The lowest docked
energy was taken as the best-docked conformation of the compound
for the macromolecule.
The effect of Preincubation of Each of the Compounds,
6, 7, and DPPA with Tyrosinase. The tyrosinase inhibitory
effect of the two synthetic DPPA esters, compounds 6 and 7
(share a resorcinol aromatic ring), were tested with and without
preincubation in the presence of the enzyme. Thus, DPPA 1
(at 10 µM) or compound 6 (at 0.5 µM) or 7 (at 0.3 µM) were
each incubated for 10 min with the enzyme alone, before the
addition of tyrosine. The formation of DOPA-quinone was
recorded after 20 min. In order to assess the effect of competition
between the inhibitors and tyrosine, these experiments were
repeated with increasing tyrosine concentrations from 1 to 3.75
mM, and the inhibition level of the enzyme-inhibitor complex
tested. Results summarized in Figure 2 I-III show that without
preincubation with the enzyme, the inhibitory levels of the
compounds, 6 (Figure 2II) and 7 (Figure 2III), decreased with
the increase in tyrosine concentration. Similarly, the inhibitory
effect of DPPA (Figure 2I) dropped in the presence of 1, 2,
and 2.5 mM tyrosine, but increased again at 3 and 3.75 mM
tyrosine. When these inhibitors were preincubated with the
enzyme for 10 min prior to the addition of tyrosine, the
inhibitory potency of ester 6 (0.5 µM) remained the same even
in the presence of 3.75 mM tyrosine, and that of ester 7 (0.3
µM) diminished slightly with the rise in tyrosine concentration,
from 97% inhibition with 1 mM tyrosine to 86% inhibition with
3.75 mM tyrosine. Such a preincubation of DPPA with
tyrosinase only slightly affected its inhibitory behavior.
Results
Isolation of DPPA. A 70% ethanolic extract from dry fig
leaves exhibited potent inhibition of the mushroom tyrosinase.
The active compound from the extract was identified as DPPA,
which was identical to the authentic sample of this compound.
The characteristic DPPA inhibitory effect on mushroom tyro-
sinase, in comparison to other two known inhibitors isolated
from licorice root,13 is shown in Figure 1. The results demon-
strate that while the DPPA inhibitory level remained stable
during the first hour of incubation those of the other two
inhibitors declined rapidly.
Tyrosinase Inhibitory Level of DPPA Esters. In order to
assess the effect of the increasing hydrophobicity of DPPA
derivatives on enzyme inhibition, DPPA ethyl (2), isopropyl
(3) and octyl (4) esters were synthesized (Table 1) and their
activities determined, 0.22 µM, 0.07 µM, and 0.7 µM, respec-

Tyrosinase Inhibition Potency of Resorcinol DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11 2679
Discussion
Among the various tyrosinase inhibitors reported in literature,
those containing a resorcinol subunit are of special interest, since
they are usually highly active (IC50 < 10 µM). The 2,4-
resorcinol derivatives are distributed among the stilbene, fla-
vonoid, and chalcone families and are significantly more potent
as inhibitors than the 3,5-substituted resorcinol derivatives. For
example, among the stilbene family, oxyresveratrol (2′,4′,3,5-
tetrahydroxy-trans-stilbene) has an IC50 of 1.2 µM, whereas
resveratrol (4′,3,5-trihydroxy-trans-stilbene) has an IC50 of 54.6
µM.22 These differences in the IC50 values are not necessarily
a result of the additional hydroxyl group found in oxyresveratrol,
as compared to resveratrol. It has been reported by others that
in the case of polyphenols, such as N-benzylbenzamides9 and
chalcones,10 the major determining factor of their inhibitory
efficacy is the location and not the number of the hydroxyl
groups.
Natural and synthetic substituted 2,4-resorcinol compounds,
such as artocarbene and chlorophorin (trans-stilbene), norarto-
carpanone (flavanone),7,23 and chalcones8 are all constructed
from a 4-substituted resorcinol skeleton and have IC50 values
of <10 µM. In the present study, DPPA, with a 2, 4-dihydroxy
resorcinol skeleton demonstrated a high tyrosinase inhibitory
potency (1.9 µM), which decayed very slowly with time (Figure
1). We questioned if the addition of a hydrophobic moiety to
the basic structure of the DPPA would increase enzyme-binding
affinity. A new 2,4-dihydroxystilbene, isolated from Chloro-
phora excelsa by Shimizu et al.,24 and chlorophorin contain an
additional substituted octyl side chain at position 3 of the
aromatic ring, unlike the new stilbene, which contains, in
addition, a hydroxyl group on carbon 7′′ of the octyl residue.
Both compounds were inhibitors of tyrosinase with an IC50 of
96 µM in the case of the latter, and 1.9 µM for the more
hydrophobic compound (chlorophorin). In another study, the
inhibitory effect of the 4-substituted resorcinol, e.g., 4-hexyl-
resorcinol, was found to be more active in preventing L-DOPA
oxidation to DOPA-quinone than was 4-ethylresorcinol,11 with
the less hydrophobic side chain. Similarly, when studying the
inhibitory effect of p-alkylbenzoic acids, it was found that with
the increase in the length of the alkyl side chain, the tyrosinase
inhibitory effect improved.25 In the present study, the docked
2,4-substituted resorcinol derivative (compound 3) appeared to
fit well in the enzyme active site (Figure 3), close to the copper
ion couple. Figure 3 demonstrates that the two resorcinol
hydroxyl groups seem to form three hydrogen bonds, two with
the nitrogen atoms of the amino acids, Arg 55 and Trp 184, on
the side chains, and one with the carboxylic group of the Glu
(182). An additional hydrogen bond is located between the
carbonyl oxygen of DPPA and the imidazole hydrogen of His
190. Furthermore, hydrophobic interactions of compound 3 are
also possible between its resorcinol aromatic ring and Ile 42,
as well as between the isopropyl ester and Ala 202. Other
hydrophobic interactions may occur if one of the two methyl
groups of the isopropyl ester is replaced by a long chain, which
may interact with Ile 42 and Met 43 in the enzyme hydrophobic
pocket (Figure 3B); such a compound will be considered for
future synthesis.
Figure 2. The effect of preincubation of enzyme inhibitors with
tyrosinase. Tyrosinase was incubated either with 10 µM DPPA (I),
with 0.5 µM compound 6 (II), or with 0.3 µM compound 7 (III) (each
at concentration exhibiting 80% inhibition) for 10 min, before the
addition of tyrosine (1-3.75 mM). Twenty minutes after the addition
of tyrosine, the level of DOPA-quinone formation was monitored
spectrophotometrically at 450 nm. Letters represent significant differ-
ences (p < 0.05, according to Duncan’s multiple range test) among
the results of % enzyme inhibition obtained at the various concen-
trations of tyrosine (uppercase, with preincubation with enzyme;
lower case, without preincubation with enzyme). *Significance of
p < 0.05 according to t-test between the results obtained with
preincubation and without preincubation with the enzyme at the same
concentration.
bulky esters are 6-fold better inhibitors of tyrosinase than
dodecyl gallate with the linear chain. This is assuming that this
preferential inhibitory effect of the bulky portion is apparently
due to a better match of the hydrophobic protein pocket
surrounding the binuclear copper active site. The present study
further elucidated the effect of the increased bulkiness of the
hydrophobic group. We questioned if such an effect could be
predicted from the calculated parameters related to the energy
of the interaction between the potential inhibitor and the enzyme.
Several DPPA esters were selected, their structures were drawn,
and the conformation with the minimum energy was chosen,
using the MOPAC program. Each of these esters was allowed
to interact with the three-dimensional structure of the enzyme1
by means of the docking program. The interactions with the
minimum energy were selected. According to these calculations,
Docking calculations revealed that the inhibitory effect of
DPPA esters somewhat correlated with increasing bulkiness of
the hydrophobic side chain. The DPPA isopropyl ester was
discovered to be a more potent inhibitor than the ethyl or octyl
esters. This finding is in agreement with that of Kubo,15 who
studied the inhibitory effect of the gallic acid ester, and showed
that geranyl gallate and decahydro-2-naphthyl gallate with their

2680 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 11
Khatib et al.
Figure 3. Isopropyl 3-(2,4-dihydroxyphenyl)propionate (compound 3) interaction with the tridimensional tyrosinase binding site (Matoba, Y.
et al. J. Biol. Chem. 2006, 281, 8981). (A) Lowest energy AutoDock of compound 3 in the tyrosinase active site. The hydrophilic and
hydrophobic interactions of the inhibitor with the enzyme’s amino acids at the enzyme binding site are shown together with their distance;
red ) oxygen, blue ) nitrogen, and gray ) carbon. (B) Surface representation of tyrosinase with the enzyme; hydrophilic (red) and hydrophobic
(blue) zones docked with the DPPA isopropyl ester are shown. The cleft to which compound 3 is bound is the enzyme-binding site.
three potentially promising esters of DPPA were chosen:
compound 5 (containing a tert-butyl ester), compound 6 (with
a 1-[1,1-dimethylpropyl]-2,2-dimethylbutyl ester), and com-
pound 7 (with a 3,3,5,5-tetramethyl-4-heptyl ester). These esters
were synthesized and their tyrosinase inhibitory effects (ob-
served IC50) tested and compared with the predicted IC50. All
three compounds were expected to demonstrate an IC50 < 1.0
µM, and compounds 6 and 7 indeed inhibited the enzyme at
the IC50 values of 0.18 and 0.15 µM, respectively. However,
compound 5 had an IC50 of 14.9 µM, much higher than the
predicted value; possibly such predictions are allowed only with
primary or secondary alcohols as in the case of compounds 2-4,
6, and 7, but not with tertiary alcohols, as in the case of the
DPPA tert-butyl ester (5). The correlation between the observed
IC50 values and the predicted values afforded a regression curve
with the R2 of 0.94 and P value <0.01, based on the free energy
values.
The incubation of DPPA (10 µM) or DPPA esters (each at
concentrations inhibiting 80% of the enzyme activity [0.5 and
0.3 µM for 6 and 7, respectively]), with the enzyme and with
increasing tyrosine concentrations (1-3.75 mM), resulted, as
expected, in an inverse relationship, i.e., the inhibition decreased
with an elevation in tyrosine concentration. When the enzyme
was first preincubated with either compound, 6 or 7, for 10
min, different results were anticipated (Figure 2). The prein-
cubation of the enzyme with the ester almost entirely prevented
the entrance of the tyrosine, supplemented in increasing doses,
into the enzyme active site. This could be explained by the
additional contribution of the hydrophobic bulky group to the
inhibition constant (Ki) by increasing van der Waals forces with
the enzyme hydrophobic core and thus preventing effective
competition with tyrosine. Compounds 6 and 7 were discovered
to be more potent than DPPA, with and without preincubation
with the enzyme, at all tyrosine concentration ranges tested
(Figure 2).
the inhibitors. Such a lipophilic unit, that contains a minor bulky
group, was a preferred inhibitor over a long chain or highly
bulky functional moiety, as it may interact with the enzyme
hydrophobic pocket and augment binding affinity. The lowest
energy AutoDock of compound 3 at the tyrosinase active site
revealed that the 2,4-resorcinol unit fits the enzyme-binding site
well, forming four hydrogen bonds and one lipophilic interac-
tion. By means of such docking methodology, one may design
new inhibitors with additional van der Waals forces (with Met
43 and Thr 203) and hydrogen bonds (toward His 190) and
thus enhanced binding affinity. Further study is required to
synthesize such inhibitors and elucidate the type of inhibition
that occurs.
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