2-Hydroxy-4-isopropylbenzaldehyde, a potent partial tyrosinase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2003.11.033

Chamaecin (2-hydroxy-4-isopropylbenzaldehyde) was synthesized and tested for its tyrosinase inhibitory activity. It partially inhibits the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an IC₅₀ of 2.3 μ

Full text of DOI:10.1016/j.bmcl.2003.11.033

Bioorganic & Medicinal Chemistry Letters 14 (2004) 681–683
2-Hydroxy-4-isopropylbenzaldehyde, a potent partial
tyrosinase inhibitor
Ken-ichi Nihei,a Yoshiro Yamagiwa,b Tadao Kamikawab and Isao Kuboa,*
aDepartment of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA
bDepartment of Chemistry, Faculty of Science and Technology, Kinki University, Kowakae, Higashi-Osaka-shi,
Osaka 577-8502, Japan
Received 12 September 2003; revised 11 November 2003; accepted 16 November 2003
In honor of Professor Andrew S. Kende’s 70th birthday.
Abstract—Chamaecin (2-hydroxy-4-isopropylbenzaldehyde) was synthesized and tested for its tyrosinase inhibitory activity. It
partially inhibits the oxidation of l-3,4-dihydroxyphenylalanine (l-DOPA) catalyzed by mushroom tyrosinase with an IC50 of 2.3
mM. The inhibition kinetics analyzed by Dixon plots found that chamaecin is a mixed type inhibitor. This inhibition may come in
part from its ability to form a Schiff base with a primary amino group in the enzyme.
# 2003 Elsevier Ltd. All rights reserved.
Tyrosinase (EC 1.14.18.1), a copper-containing enzyme,
catalyzes two distinct reactions of melanin synthesis, the
hydroxylation of a monophenol and conversion of an
o-diphenol to the corresponding o-quinone.1 The hydro-
xylation of l-tyrosine, the initial step in melanin synth-
esis, is also the initial step in catecholamine synthesis. The
enzymatic oxidation of l-tyrosine to melanin is of con-
siderable importance since melanin has many functions.
Alterations in melanin synthesis occur in many disease
states. Melanin pigments are also found in the mamma-
lian brain. Tyrosinase may play a role in neuromelanin
formation in the human brain and could be central to
dopamine neurotoxicity as well as contribute to the
neurodegeneration associated with Parkinson’s disease.2
Melanoma specific anticarcinogenic activity is also
known to be linked with tyrosinase activity.3
found 2-hydroxy-4-methoxybenzaldehyde to be a mixed
type inhibitor. This inhibition comes from its ability to
form a Schiff base with a primary amino group and to
chelate copper in the enzyme.4 Simultaneously, cumin-
aldehyde (4-isopropylbenzaldehyde) (2) was character-
ized as a tyrosinase inhibitor from the seeds of Cuminum
cyminum, known as cumin and widely used as a food
spice.5 Subsequently, various benzaldehyde derivatives
were tested for their tyrosinase inhibitory activity for com-
parison. Notably, cuminaldehyde is approximately 16-fold
more potent compared to benzaldehyde (3) as far as their
IC50s are compared, though anisaldehyde (4) is only
slightly more potent than benzaldehyde. In contrast,
salicylaldehyde (5) is about 4-fold less potent than benz-
aldehyde. Interestingly, 2-hydroxy-4-methoxybenzaldehyde
is 11-times more potent than anisaldehyde. Hence, intro-
ducing an isopropyl group at the 4-position in salicylalde-
hyde can be expected to enhance the inhibitory activity
(Fig. 1). 2-Hydroxy-4-isopropylbenzaldehyde (6)6 was
previously isolated from various plants. It was first char-
acterized from Eucalyptus cneralifolia7 and Chamaecyparis
taiwanensis, and named as chamaecin.8
In our continuing search for tyrosinase inhibitors from
plants, 2-hydroxy-4-methoxybenzaldehyde (1), was
recently isolated as a potent tyrosinase inhibitor from
African medicinal plants. It inhibits the oxidation of
l-3,4-dihydroxyphenylalanine (l-DOPA) catalyzed by
mushroom tyrosinase with an IC50 of 30 mM.4 The
inhibition kinetics analyzed by Lineweaver–Burk plots
In a previous report,9 Reimer–Tiemann reaction was
described as a convenient method for the preparation of
various salicylaldehyde derivatives from the corres-
ponding phenols as a one step synthesis.10 This reaction
was first applied to commercially available 3-iso-
propylphenol (7). However, the complex mixture was
Keywords: Mushroom tyrosinase; Benzaldehydes; Chamaecin; Vils-
meier reaction; Partial mixed type inhibitor.
* Corresponding author. Tel.:+1-510-643-6303; fax:+1-510-643-0215;
e-mail: ikubo@uclink.berkeley.edu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.033

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K. Nihei et al. / Bioorg. Med. Chem. Lett. 14 (2004) 681–683
Figure 2. Dose–response plots by chamaecin on tyrosinase oxidation
of l-DOPA. Plots in inset was converted from dose–response plots.
Figure 1. Structure of chamaecin and related compounds. Number in
parenthesis is IC50 value.
chamaecin acts as a partial inhibitor on diphenolase
activity by tyrosinase.
The kinetic behavior of the oxidation of l-DOPA cata-
lyzed by tyrosinase at different concentrations of cha-
maecin was studied. The inhibition kinetics analyzed by
Dixon plots showed that the plots of 1/v versus [I] give a
family of hyperbolic curves,17 but intersected one
another near the x-axis, indicating that chamaecin is a
mixed (competitive and noncompetitive mixed) type
inhibitor as shown in Figure 3. This observed behavior
indicates that inhibitor can bind not only with free
enzyme, but also with the enzyme–substrate complex,
and their equilibrium constants are different. The mixed
inhibition exerted by chamaecin comes from its ability
to form a Schiff base with a primary amino group and
to chelate copper in the active site, although its direct
effect to the active site contributes more to the inhibi-
tion since the dissociation equilibrium constant, Ki (1.1
Scheme 1. Synthesis of chamaecin.
observed on TLC and their separation was not easily
performed. Hence, to introduce a carbonyl group at the
ortho position of the phenolic group in 7, Vilsmeier
reaction11,12 was selected. The efficient total synthesis of
chamaecin was carried out as follows (Scheme 1).
3-Isopropylphenol was converted to 3-isopropylanisole
(8) using iodomethane and K2CO3. Applying Vilsmeier
reaction to 8 with N-methylformanilide (MFA) and
P(O)Cl3 afforded 9 in excellent yield.12 In this step,
2-alkylbenzaldehyde could not be furnished, at least not
as a major product because of the steric hindrance of
the adjacent isopropyl group. Finally the removal of the
methyl group from 9 by reflux with LiCl in DMF was
achieved and chamaecin was obtained in high yield.13,14
The overall yield from 7 was 65%.
Dose–response curve of tyrosinase inhibition by cha-
maecin was shown in Figure 2.15 The IC50 was esti-
mated to be 2.3 mM which is about 350-fold more
potent than that of benzaldehyde. It should be noted
that as the concentrations of chamaecin increased, the
remaining enzyme activity was decreased but not
completely suppressed. The remaining enzyme activity
was about 6.7% when the concentration of chamaecin
reached to 1.0mM. In order to get more diagnostic
signature, dose-response plots obtained were trans-
formed to the plots of 1/v versus [I] (inset in Fig. 2).16
It displays an apparent hyperbolic nature so that
Figure 3. Dixon plots for inhibition of chamaecin on mushroom tyr-
osinase for the catalysis of l-DOPA. The inhibitor concentrations
used were 0.24, 0.49, 0.98, 1.95, 3.91, and 7.81 mM. The concentration
of l-DOPA for curves 1–3 was 0.25, 0.50, and 0.75 mM, respectively.

K. Nihei et al. / Bioorg. Med. Chem. Lett. 14 (2004) 681–683
683
mM) is lower than ꢀKi (6.4 mM). On the other hand, the
kinetics experiments by chamaecin provided burst ampli-
tude (b-value) (0.43) that is between 0 and 1. Hence, this
molecule dose not completely block the ability of the
enzyme to turnover when bounded to the inhibitor.
2. Xu, Y.; Stokes, A. H.; Freeman, W. M.; Kumer, S. C.;
Vogt, B. A.; Vrana, K. E. Mol. Brain Res. 1997, 45, 159.
3. Prezioso, J. A.; Epperly, M. W.; Wang, N.; Bloomer,
W. D. Cancer Lett. 1992, 63, 73.
4. Kubo, I.; Kinst-Hori, I. Planta Med. 1999, 65, 19.
5. Kubo, I., Kinst-Hori, I. J. Agric. Food Chem. 1998. 46,
5338. For recent papers concerned about benzaldehyde
derivatives as a tyrosinase inhibitor, see: Kubo, I.; Kinst-
Hori, I. J. Agric. Food Chem. 1999, 47, 4574. Lee, H. S. J.
Agric. Food Chem. 2002. 50, 1400, and Jimenez, M.;
Chazarra, S.; Escribano, J.; Cabanes, J.; Garcıa-Car-
mona, F. J. Agric. Food Chem. 2001. 49, 4060.
6. Cytotoxic activity of chamaecin against melanoma carci-
noma cells from both human HTB-140and mouse B-16
was found to be greater than 100 mg/mL. Hence, the
depigmenting activity of the cells may be expected at dose
levels causing no cytotoxicity.
Although the precise explanation how chamaecin inter-
acts with the enzyme on a molecular basis is still
unknown, the ability to form a Schiff base with a pri-
mary amino group in the enzyme is more likely. The
2-hydroxy-4-methoxybenzaldehyde that form more
stable Shiff base adducts with a primary amino group
showed more potent activity.4 The Schiff base is expec-
ted to be largely governed by those factors affecting the
stability of the carbon–nitrogen double bond. Since
2-methoxy-4-isopropylbenzaldehyde (9) did not show
any inhibitory activity up to 0.5 mM, the salicylalde-
hyde moiety was requisite to interact with enzyme. In
addition, the partial inhibition kinetics of chamaecin
was probably explained by the fact that the Schiff base
adduct generated is too small to prevent complete
adsorption of the substrate in the active pocket.18
7. Reuter, F. H. Roy. Australian Chem. Inst. J. Proc. 1950,
17, 33.
8. Lin, Y. T.; Wang; K. T.; Chang, L. H. J. Chinese Chem.
Soc. 1963, 10, 139. 2-Hydroxy-4-isopropylbenzaldehyde
was also characterized from E. cneralifolia and designated
as macropone.7,9
.
9. Birch, A. J.; Elliott, P. Aust. J. Chem. 1953, 6, 369.
10. Thoer, A.; Denis, M.; Delmas, M.; Gaset, A. Synth.
Commun. 1988, 18, 2095.
The primary amino group very likely plays an impor-
tant role in the tertiary structure of tyrosinase. For
instance, the amino group in the enzyme may be involved
with hydrogen bonding which is essential to maintain the
tertiary structure of the enzyme. This may be supported
by the previous report that the hydrogen-bonding inter-
actions are known to stabilize the oxy-form of Strepto-
myces glaucescens tyrosinase.19 Chamaecin, a relatively
nonpolar molecule, may form a Schiff base with an
amino group possibly located nearby hydrophobic
region of the enzyme. However, native proteins form a
sort of intramolecular micelle in which the nonpolar
portion is likely to be out of contact in the water based-
test solution. Hence, some substrates first approach the
binuclear active site and form the enzyme-substrate
complex, and then chamaecin forms a Schiff base with a
free primary amino group of the enzyme. The low con-
formational stabilities of native proteins make them
easily susceptible to denaturation by altering the bal-
ance of the weak nonbonding forces that maintain the
native conformation. It appears that chamaecin indir-
ectly disrupts the tertiary structure of the enzyme.
However, the conclusive interpretation remains to be
clarified since the structure of tyrosinase used for this
study has not yet been established.
11. Singh, I. P.; Shukla, V. K.; Dwivedi, A. K.; Khanna,
N. M. Indian J. Chem. 1989, 28B, 692.
12. Iwata, M.; Emoto, S. Bull. Chem. Soc. Jpn. 1974, 47,
1687.
13. Berbard, A. M.; Ghiani, M. R.; Piras, P. P.; Rivoldini, A.
Synthesis 1989, 287.
14. Chamaecin; IR (film) 3140, 2950, 1650, 1620, 1565, 1500,
1320, 1195, 940 cmÀ1. 1H NMR (CDCl3, 50 0 MHzd, ): 1.26
(d, J=7.0Hz, 6H), 2.92 (sep, J=7.0=Hz, 1H), 6.87 (d,
J=1.5 Hz, 1H), 6.90(dd, J=1.5, 7.5 Hz, 1H), 7.45 (d, J=7.5
Hz, 1H), 9.84 (s, 1H), 11.05 (s, 1H). HRMS-EI [M]+ calcd
1
for C10H12O2 164.0837, found 164.0824. IR and H NMR
spectra are consisted with those reported by Lin, Y. T et al.8
15. The assay was performed as previously reported4 with
slight modifications.20 The commercial mushroom tyro-
sinase purchased from Sigma was purified by the proce-
dure as previously reported.21 All data show that the
mean of three separate experiments and were reproducible
to within 15% of average. The IC50s were obtained by
fitting experimental data to the logistic curve by Sigma
Plot (SPSS Inc, Chicago, IL).
16. Copeland, R. A. Enzymes: A Practical Introduction to
Structure, Mechanism, and Data Analysis; Wiley: New
York, 2000; pp. 266–304.
17. The partial inhibition curve was fitted to the equation that
was identical to Segel’s mathematical model, see: Segel, I.
Enzyme Kinetics; Wiley: New York, 1975; pp 161–226.
18. Cleland, W. W. In The Enzyme, vol. 2; Boyer, P. D., Ed.;
Academic Press: New York, 1970; p 1–65.
References and notes
19. Jackman, M. P.; Huber, M.; Hajnal, A.; Lerch, K. Bio-
chem. J. 1992, 282, 915.
1. Sanchez-Ferrer, A.; Rodrıguez-Lopez, J. N.; Garcia-
Canovas, F.; Garcıa-Carmona, F. Biochem. Biophys. Acta
1995, 1247, 1.
20. Nihei, K.; Kubo, I. Bioorg. Med. Chem. Lett. 2003, 13, 2409.
21. Espın, J. C.; Wichers, H. J. J. Agric. Food Chem. 1999, 47,
2638.