Hydroxy- or methoxy-substituted benzaldoximes and benzaldehyde-O-alkyloximes as tyrosinase inhibitors

Article abstract of DOI:10.1016/S0968-0896(01)00084-0

Several benzaldoximes, benzaldehyde-O-ethyloximes, and acetophenone-oximes were synthesized and evaluated as tyrosinase inhibitors by an assay based on tyrosinase catalyzed L-DOPA oxidation. Whereas benzaldoxime itself is only a weak inhibitor, its deriva

Full text of DOI:10.1016/S0968-0896(01)00084-0

Bioorganic & Medicinal Chemistry 9 (2001) 1879–1885
Hydroxy- or Methoxy-Substituted Benzaldoximes and
Benzaldehyde-O-alkyloximes as Tyrosinase Inhibitors
Jakob P. Ley* and Heinz-Jurgen Bertram
Haarmann & Reimer GmbH, Flavor Division, Research & Development Flavors,
PO Box 1253, D-37601 Holzminden, Germany
Received 14 February 2001; accepted 21 March 2001
Abstract—Several benzaldoximes, benzaldehyde-O-ethyloximes, and acetophenonoximes were synthesized and evaluated as tyr-
osinase inhibitors by an assay based on tyrosinase catalyzed L-DOPA oxidation. Whereas benzaldoxime itself is only a weak inhi-
bitor, its derivatives with one or two hydroxy or methoxy moieties in para and meta positions depress tyrosinase activity.
Acetophenonoximes and trisubstituted benzaldoximes show no inhibitory activity. The IC50 of 3,4-dihydroxybenzaldehyde-O-
ethyloxime (0.3 ꢀ 0.1 mmol Lꢁ1) is of the same magnitude as tropolone (0.13 ꢀ 0.08 mmol Lꢁ1), one of the best tyrosinase inhi-
bitors known so far. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
are either lighter or darker than the surrounding areas.
In addition, many people with dark skin want to lighten
their skin colour for cosmetic reasons.
The two copper atoms containing tyrosinase is widely
distributed in plants, fungi, and animals.1 Tyrosinase
shows two enzymatic activities, ortho-monophenoloxidase
and polyphenoloxidase activity and accepts many phe-
nols and catechols as substrates. Tyrosinase causes the
enzymatic browning of food2 and is the key enzyme for
melanogenesis in mammals.3
Both browning of fruits and skin darkening can be
suppressed, at least partially, by deactivation of tyr-
osinase. The activity of the enzyme in freshly cut fruits
can be supressed by heating, by acidification, by reduc-
tants like ascorbic acid or sulfite,2 or by tyrosinase inhi-
bitors, for example kojic acid,5 kojic acid octanoates,6
oxalic acid,7 salicylhydroxamic acid,8,9 N-acetylcysteine10
or gallic acid esters.11 For the deactivation of the tyr-
osinase of human melanocytes, several inhibitors like
kojic acid and kojic acid derivatives were described and
some like hydroquinone or arbutin are used.4
In particular, the unfavourable darkening of freshly
cutted fruits or vegetables like apples or potatoes is a
severe problem for further processing, for example juice
manufacturing. The enzymatic oxidation of poly-
phenolic compounds (e.g., caffeic acid or its esters such
as chlorogenic acid) to the dark coloured so-called mel-
anoids is mainly catalyzed by tyrosinases.
The best known tyrosinase inhibitor so far is tropolone
In animals l-tyrosine is converted to the red-brown
dopaquinone via l-3,4-dihydroxyphenylalanine (l-
DOPA) by tyrosinase-catalyzed oxidation; dopaquinone
is further oxidized to yield the mostly brown to black
coloured polymeric melanins which are responsible for
the different colours of skin and hair of mammals.3,4
If in the human skin the melanocytes are not distributed
evenly, unfavourable pigmentation spots form which
(1, cf. Scheme 1) showing an IC50 of 0.4 mmol Lꢁ1 12
.
For kojic acid (2, cf. Scheme 1) an IC50 of 23 mmol Lꢁ1
was reported.13 It is assumed that these inhibitors com-
plex the two copper atoms which are presented in the
active site of the enzyme.5 Kubo et al. have described
some benzaldehydes as weak to moderate tyrosinase
inhibitors.14
In our efforts to develop new, low-cost, easy-to-prepare
and highly active tyrosinase inhibitors for skin light-
ening or antibrowning preparations, we investigated
some benzaldoximes because they may be able to com-
plex the two copper atoms in the active site of tyrosinase.
*Corresponding author. Fax: +49-5531-90-3883;
e-mail: jakob.ley.jl@hr-gmbh.de (J.P. Ley).
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00084-0

1880
J. P. Ley, H.-J. Bertram / Bioorg. Med. Chem. 9 (2001) 1879–1885
Scheme 1. Structures of standard inhibitors tropolone (1) and kojic acid (2), the oximes 3–30 (for definition of R1–R6, cf. Table 1), the benzonitriles
31–32 and the benzaldehydes 33–36.
Results and Discussion
Synthesis
The benzaldoximes 5–15 and the acetophenonoximes
28–30 were prepared starting from unprotected benzal-
dehydes or acetophenones, respectively, by reaction
with hydroxylamine in aqueous solution with the aid of
sodium acetate in moderate to good yields (cf. Scheme 2
and Table 1). The oxime ethers 16–25 were synthesized
in a similiar manner by reaction with O-substituted
Scheme 2. Synthesis of benzaldoximes 5–15, benzaldehyde-O-ethylox-
imes 16–27 and acetophenone oximes 28–30. For definition of R1–R6,
cf. Table 1.
hydroxylamines. If necessary, the oximes were purified
by simple recrystallization. Analytical data of the
known compounds corresponded to the expected
values. All new compounds (16–23, 25–27 and 29–30)
Several free oximes (3–16), oximeethers (17–27 and 30),
trisubstituted oximes (8, 11, 12, 13, 20, 23, 27 and 29),
some acetophenones (28–30) with different substitution
pattern (hydroxy, methyl, methoxy) have been eval-
uated for structure–activity relationships (cf. Table 1).
1
were analyzed by H, 13CNMR, and MS and showed
satisfying high resolution MS (HR-MS) data. The che-
mical shifts for carbonyl hydrogens of benzaldoximes 5–
15 are in the typical range (7.81 – 8.26 ppm) for meth-
oxy- or hydroxysubstituted Z-benzaldoximes.15
Table 1. Structures of oximes 3–30a
R1
R2
R3
R4
R5
R6
Tyrosinase experiments
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
For evaluating the tyrosinase inhibitory activity, a typi-
cal assay protocol with l-DOPA as substrate was cho-
sen.5,16 The commercial tyrosinase from mushrooms
was used without further purification after determining
the monophenolase activity by the standard protocol
with l-tyrosine as substrate. The assay was conducted
in a 96 multiwell plate using a photometer with a fixed
wavelength of 495 nm. A series of dilutions of appro-
piate test compounds and a blank control solution were
pre-incubated with tyrosinase for 10min at 37 ꢂC.
l-DOPA was addded and the absorptions were imme-
diately recorded for at least 10min. For the first min-
utes, absorptions increased mostly linear and
subsequently they showed a typical saturation beha-
OH
OH
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OMe
OEt
OMe
Me
t-Bu
H
OH
OMe
OH
OH
OH
OH
OH
OH
OH
OMe
H
OH
OH
OMe
OH
OH
OH
OH
OH
OMe
OH
OH
OH
OH
OH
10
11
12
13
14
15
16b
17b
18b
19b
20b
21b
22b
23b
24
25
26b
27b
28
29b
30b
H
OMe
Me
t-Bu
H
H
H
H
H
H
OH
H
H
OMe
H
H
H
Me
H
OMe
H
H
H
OH
OH
OH
OH
OH
OMe
OEt
OMe
H
Et
Et
pMeBn
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
viour. With increasing inhibitor concentration,
a
decreasing maximum absorption was reached which
shows no approximation to the absorption of the blank
control during the experiment.
H
Me
Me
OH
OMe
OH
The relative tyrosinase activities against blank control
at 3 min for the different samples were plotted against
the logarithm of sample concentration. From these
plots, the IC50 values were calculated. The alternative
calculation from the initial slopes of the time/absorption
plots resulted in nearly identical inhibition data.
H
Et
H
aFor structural formula and preparation, cf. Schemes 1 and 2.
bNot listed in Beilstein handbook or in Chemical Abstracts.

J. P. Ley, H.-J. Bertram / Bioorg. Med. Chem. 9 (2001) 1879–1885
1881
The parent compound benzaldoxime (3) shows a very
weak inhibition activity against tyrosinase, but when a
hydroxy or methoxy moiety is introduced the activity
sharply increased. One could expect that the salicyl-
aldoxime (4) inhibits tyrosinase better than 4-hydroxy-
benzaldoxime (14) because of the higher complexation
capability. Surprisingly, 14 shows double inhibition
potential compared to 4. Furthermore, the 2,3-dihy-
droxybenzaldoxime (5) is completely inactive and 3,4-
dihydroxybenzaldoxime (6) shows a very potent IC50 of
18 mM. The most active compound tested so far is the
3,4-dihydroxybenzaldehyde-O-ethyloxime (17), showing
an IC50 in the nanomolar range (300 nmol Lꢁ1) com-
parable to tropolone (1).12 2-Hydroxy moieties are not
necessary for good inhibition, but 4-hydroxy or meth-
oxy are. All acetophenone oxime derivatives are com-
pletely inactive irrespective of the substitution pattern of
the phenyl ring and the oxime group. These data show
that, in fact, an inhibition occurs because the reduction
potentials of benzaldoximes and ketoximes are very
similiar. With exception of the moderate inhibitors
3,4,5-trihydroxybenzaldoxime (8) and 3,4,5-trihydrox-
ybenzaldehyde-O-ethyloxime (20) trisubstituted oximes
show no reduction of tyrosinase activity. It may be
possible that the third substituent hinders the correct
docking of the inhibitor to the active site of tyrosinase,
which is optimized for 4-hydroxy- or 3,4-dihydroxy-
phenyl moieties. Some of the oxime O-ethylethers are
more potent compared to their free counterparts, but
not all.
Table 2. Results of tyrosinase assay with the substrate l-DOPA in
buffer pH 6.8 for tropolone (1) and kojic acid (2), the oximes 3–30, the
benzonitriles 31–32, and the benzaldehydes 33–36a
IC50 (mmol Lꢁ1
)
IC50 (mmol Lꢁ1
)
1
2
3
4
5
6
7
8
0.13ꢀ0.08
22ꢀ5
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
18ꢀ3
380ꢀ70
4.2ꢀ1.5
14ꢀ4
2200ꢀ770
64ꢀ11
>4000
18ꢀ5
>4000
43ꢀ16
>4000
124ꢀ18
500ꢀ240
>4000
>4000
>4000
45ꢀ14
>4000
>4000
620ꢀ70
>4000
>4000
4.6ꢀ0.8
20.2ꢀ6.6
2.3ꢀ0.7
3.5ꢀ0.4
>4000
>4000
>4000
25ꢀ3
9
10
11
12
13
14
15
16
17
18
56ꢀ9
>4000
0.3ꢀ0.1
3ꢀ3
aEach IC50 was calculated from time/absorption plot for six different
concentrations of test compounds 3 min after addition of l-DOPA
(T=37 ꢂC, pre-incubation time 10min).
reducing agents but as real inhibitors. In addition to this
specificity, the time plots suggest that the aromatic ring
is bound to the active site or to the cofactor binding
site3 of tyrosinase and the enzyme is blocked.
To exclude artefacts, the tyrosinase inhibiting activities
of 3,4-dihydroxybenzonitrile (31, cf. Scheme 1) and
4-hydroxy-3-methoxybenzonitrile (32) were tested,
which are oxidation products of the corresponding oxi-
mes. 4-Hydroxy-3-methoxy-benzonitrile (32) shows no
activity and 3,4-dihydroxybenzonitrile (31) yields an
Therefore, we have performed a kinetic experiment on
vanillinoxime (9). For different inhibitor concentrations
(0, 1.5, 3 mmol Lꢁ1) the l-DOPA concentration was
varied. The plot of initial velocity against substrate
concentration shows decreasing Vmax values for
increasing inhibitor concentration. In addition, the
Lineweaver–Burk plot shown in Figure 1 suggests that
vanillinoxime (9) inhibits tyrosinase by non-competitive
type of kinetics. As control the apparent Km value for
the l-DOPA/mushroom tyrosinase system was calcu-
lated (0.39 mmol Lꢁ1), which is similiar to that deter-
mined by Son et al.7 (0.64 mmol Lꢁ1) for a catechol/
mushroom tyrosinase system. Considering these data,
we propose that vanillinoxime (9) does not block the
active site itself but binds to an other essential domain
of tyrosinase.
IC50 of 45 mmol
corresponding oxime 6.
L
ꢁ1, 2.5 times less active than the
Some benzaldehydes like 2-hydroxy-4-methoxy-
benzaldehyde have been reported to show an IC50 of
about 30 mmol Lꢁ1 14
.
Some other hydroxy-
benzaldehydes were shown to be much worse inhibitors;
for example vanillin shows an IC50 of about 70,000
mmol Lꢁ1. In Table 2, the IC50 of aldehydes 33–36 are
shown (for structures, cf. Scheme 1). They are either not
or only very weak inhibitors, especially when they are
compared with their corresponding oximes.
Conclusions
From these facts, we conclude that the inhibition activ-
ity of the oximes is caused by the compounds themselves
and not by their degradation products.
In conclusion, mono- or disubstituted benzaldoximes
and benzaldehyde-O-ethyloximes containing a 4-hy-
droxy- or methoxy-moiety show potent tyrosinase inhi-
bition activities. They directly interact with the enzyme
and do not simply reduce the oxidation products of l-
DOPA as postulated for ascorbic acid and hydro-
quinone. Vanillinoxime shows noncompetitive inhibi-
tion kinetics. The benzaldoximes are easy to prepare
and to purify from simple chemicals. Further studies
regarding in vitro assays on melanocytes and in vivo
tests are required for the evaluation as skin lightening
agents, and will be performed in the near future.
Only oxime O-ethylethers 20 and 22 show some sub-
strate activity, as can be seen by the increase in absor-
bance at 490nm for the mixture of tyrosinase and the
test compound without l-DOPA but, in combination
with l-DOPA, oxime 22 shows inhibitor activity with
an IC50 of 14 mmol Lꢁ1
.
Because of the high specificity of inhibitor activity, we
conclude that the oximes act not as pure chemical

1882
J. P. Ley, H.-J. Bertram / Bioorg. Med. Chem. 9 (2001) 1879–1885
Figure 1. Lineweaver–Burk plot for determination of inhibition kinetics of vanillinoxime (9) on l-DOPA/tyrosinase model system. v=1/v: (Auꢁ1
min), 1/c(DOPA): (mmolꢁ1 L); c(vanillinoxime)=(^) 0 mmol Lꢁ1, (ꢃ) 1.5 mmol Lꢁ1, (o) 3.0 mmol Lꢁ1. The apparent Km value for the DOPA/
tyrosinase system was calculated as 0.39 mmol Lꢁ1. Determination was performed in triplicate.
Experimental
(17%), 135 (54%), 108 (19%), 107 (44%), 80 (25%), 79
(35%), 53 (21%), 52 (41%).
All chemicals were obtained from Sigma-Aldrich (Dei-
senhofen, Germany) or Lancaster Synthesis (Mulheim,
Germany). Tyrosinase (from mushrooms) was delivered
by Sigma and the monophenoloxidase activity was
determined using the usual method described in the
catalogue. NMR spectra were recorded using Varian
VXR400S (1H: 400 MHz) or Gemini 2000 (1H:
200 MHz) spectrometers (Varian, Darmstadt, Germany)
at 25 ꢂC using tetramethylsilane as internal standard.
LC-MS spectra were recorded using the LCQ-HPLC
system Finnigan MAT HP1100 (Finnigan MAT, Egels-
bach, Germany). Abbreviations used for MS: APCI
atmospheric pressure chemical ionisation, ESI electro
spray ionisation, EI electron impact.
3,4-Dihydroxybenzaldoxime (6). Yield 88% (purity
>97%, GC; >98%, HPLC); mp 143 ꢂC (dec.); 1H
NMR (200 MHz, CD3COCD3): d 9.93 (1H, bs, OH),
8.15 (1H, bs, OH), 7.95 (1H, s, HCO), 7.16 (1H, d,
6
J=1.9 Hz, H2), 6,92 (1H, dd, J=8.2, 2.0Hz, H ), 6.81
(1H, d, J=8.3 Hz, H5), 2.88 (1H, bs, OH) ppm; 13C
NMR (50MHz, CD 3COCD3): 149.2 (CH, CCO), 147.5
(C, C3 or C4), 146.1 (C, C4 or C3), 126.3 (C, C1), 120.6
(CH, C6), 115.9 (CH, C2 or C5), 113.4 (CH, C5 or C2)
ppm; MS (ESIꢁ): m/z 305.0 ([2MꢁH]ꢁ, 100%), 152.2
([MꢁH]ꢁ, 80%).
3-Hydroxy-4-methoxybenzaldoxime (7). Yield 69%;
(purity 92%, GC) mp 142.4 ꢂC; MS (EI): m/z 167 (M+,
100%), 152 (31%), 134 (14%), 125 (14%), 124 (52%),
109 (15%), 106 (16%), 79 (20%), 52 (19%), 51 (21%).
The multiwell plates (96 well) used were of polystyrene
and were read out using a Ceres UV 900 C photometer
(Bio-Tek Inc., USA).
3,4,5-Trihydroxybenzaldoxime (8). Yield 86% (purity
>98%, GC); mp 177 ꢂC (dec.); 1H NMR (200 MHz,
(CD3)2CO): d 7.89 (1H, s, HCO), 6.70(2H, s, H 2 and H6)
ppm; 13C NMR (50MHz, (CD 3)2CO): d 149.5 (CH,
Synthesis of oximes
The appropriate benzaldehyde or acetophenone (87
mmol) was dissolved in water (45 mL) at 40 ꢂC. A solu-
tion of the corresponding hydroxylamine hydrochloride
(90mmol) and sodium acetate (87 mmol) in water (25
mL) was added, and the reaction mixture was stirred at
about 80 ꢂC under nitrogen for 2 h. The mixture was
cooled and extracted with tert-butyl methyl ether (200
mL), the organic phase was washed with saturated
NaCl solution, dried over Na2SO4, filtered, and the fil-
trate was evaporated to dryness in vacuo. If necessary,
the residue was recrystallized.
C
CO), 146.6 (2 C, C3 and C5), 135.4 (C, C4), 125.4 (C,
C1), 106.8 (2 CH, C2 and C6) ppm; MS (APCI+): m/z
170.0 ([M+H]+, 100%), 154.3 (11%).
4-Hydroxy-3-methoxybenzaldoxime (9). Yield 91%
(purity 94%, GC, 98%, HPLC); mp 118.2 ꢂC; 1H NMR
(400 MHz, (CD3)2SO): d 7.99 (1H, s, HCO), 7.16 (1H, d,
J=2 Hz, H2), 6.97 (1H, dd, J=8.1, 2 Hz, H5), 6.78 (1H,
d, J=8.1 Hz, H6), 3.78 (3H, s, HArOMe) ppm; 13C NMR
4
(100 MHz, (CD3)2SO): d 148.0(C, C or C3), 147.96
2,3-Dihydroxybenzaldoxime (5). Yield 44% (purity
>98%, GC); MS (EI): m/z 153 (M+, 100%), 136
(CH, CCO), 147.8 (CH, C3 or C4), 124.32 (C, C1), 120.4
(CH, C6), 115.4 (CH, C5), 109.2 (CH, C2), 55.4 (CH3,

J. P. Ley, H.-J. Bertram / Bioorg. Med. Chem. 9 (2001) 1879–1885
1883
CArOMe) ppm; MS (EI): m/z 167 (M+, 100%), 152
(13%), 134 (22%), 125 (21%), 124 (61%), 109 (18%),
106 (19%), 79 (15%), 52 (15%), 51 (16%).
mp < 23 ꢂC; 1H NMR (400 MHz, CD3OD): d 7.91 (1H,
s, HCO), 7.08 (1H, d, J=2 Hz, H2), 6.66 (1H, ddd,
J=8.2, 2, 0.5 Hz, H6), 6.75 (1H, d, J=8.2 Hz, H5), 4.12
(2H, q, J=7 Hz, HNOEt,1), 1.27 (3H, t, J=7 Hz,
3-Ethoxy-4-hydroxybenzaldoxime (10). Yield 24%
(recryst., purity 90%, GC); mp 189 ꢂC (dec.); MS (EI):
m/z 181 (M+, 90%), 153 (28 %), 152 (17%), 136 (26%),
135 (38%), 126 (21%), 110 (100%), 52 (19%), 51 (18%),
29 (16%).
H
NOEt,2) ppm; 13C NMR (100 MHz, CD3OD): d 149.9
(CH, CCO), 148.8 (C, C3 or C4), 146.8 (C, C4 or C3),
125.7 (C, 1), 121.3 (CH, C6), 116.3 (CH, C5 or C2), 113.9
(CH, C2 or C5), 70.2 (CH2, CNOEt,1), 15.0(CH
C
,
3
NOEt,2) ppm; MS (EI): m/z 181 (M+, 100%), 153
(20%), 152 (19%), 136 (28%), 126 (26%), 110 (47%),
109 (30%), 81 (18%), 53 (14%), 29 (16%). HR-MS m/z
(M+): Calcd. for C9H11NO3: 181.0739. Found
181.0747.
3,5-Dimethoxy-4-hydroxybenzaldoxime (11). Yield 89%
(purity 97%, GC); 1H NMR (200MHz, CD3COCD3): d
8.02 (1H, s, HCO), 6.93 (1H, s, H2 and H6), 3.84 (6H, s,
H
ArOMe), 2.97 (bs, OH) ppm; 13C NMR (50MHz,
CD3COCD3): d 149.3 (CH, CCO), 148.6 (2C, C3 and
C5), 138.5 (C, C4), 124.6 (C, C1), 104.8 (2CH, C2 and
C6), 56.4 (2CH3-, CArOMe) ppm; MS (EI): m/z 197 (M+,
100%), 155 (15%), 154 (57%), 139 (12%), 67 (12%), 66
(11%), 65 (10%), 53 (14%), 39 (11%).
3,4-Dihydroxybenzaldehyde-O-(4-methylbenzyl)-oxime
(18). Yield 8% (purity 98%, GC); mp 85–86 ꢂC; 1H
NMR (200 MHz, CD3OD): d 7.95 0 (1H, s, HCO), 7.26
0
(2H, BB0, HAr ), 7.14 (2H, AA0, HAr ), 7.08 (1H, d, J=2
Hz, H2), 6.86 (1H, dd, J=8.5 Hz, 2 Hz, H6), 6.74 (1H,
0
d, J=8.5 Hz, H5), 5.05 (2H, s, HꢁA2 r ꢁCH), 2.34 (3H, s,
0
3,5-Dimethyl-4-hydroxybenzaldoxime (12). Yield 48%
(recryst., purity 97%, GC); MS (EI): m/z 165 (M+,
100%), 149 (24%), 148 (38%), 147 (36%), 132 (35%),
122 (85%), 107 (28%), 91 (55%), 77 (43%), 39 (24%).
H
Ar ꢁMe) ppm; 13C NMR (50MHz, CD 3OD): d 150.3
(CH, CCO), 148.7 (C, C3 o0r C4), 146.6 (C, C30 or C40),
0
138.5 (C, C1 ), 136.2 (C, C4 ), 129.8 (2 CH, C2 or C3 ),
0
0
129.4 (2 CH, C3 or C2 ), 125.4 (C, C1), 121.4 (CH, C6),
0
116.1 (CH, C5),0 113.8 (CH, C2), 76.8 (CH2, CꢁA2 r ꢁCH),
3,5-Di-tert-butyl-4-hydroxybenzaldoxime (13). Yield
94% (purity 98%, GC); 1H NMR (200 MHz, CDCl3): d
21.2 (CH3, CAr ꢁMe) ppm; MS (APCIꢁ): m/z 256.3
([MꢁH]ꢁ, 100%). HR-MS m/z (M+): calcd for
C15H15NO3: 257.1052. Found 257.1059.
2
8.07 (1H, s, HCO) 7.40(2H, s, H and H6), 7.0(1H, bs,
OH), 5.44 (1H, s, OH), 1.45 (18H, s, HtBu,2) ppm; 13C
NMR (50MHz, CD 3COCD3): d 159.3 (C, C4), 155.6
(C), 151.1 (CH, CCO 136.3 (2C, C3 and C5), 124.1 (2CH,
C2 and C6), 34.3 (2C, CtBu,1), 30.2 (6CH3, CtBu,2) ppm.
MS (EI): m/z 249 (M+, 33%), 235 (15%), 234 (100%),
231 (16%), 218 (50%), 216 (84%), 188 (30%), 115
(15%), 57 (25%), 41 (26%).
3-Hydroxy-4-methoxybenzaldehyde-O-ethyloxime (19).
Yield 98% (purity >97%, GC); mp 61.4 ꢂC; 1H NMR
(200 MHz, CD3OD): d 7.93 (1H, s, HCO), 7.10(1H, d,
J=2 Hz, H2), 6.97 (1H, dd, J=8, 2 Hz, H6), 6.90(1H,
d, J=8 Hz, H5), 4.12 (2H, q, J=7 Hz, HNOEt,1), 3.87
(3H, s, HArOMe), 1.27 (3H, t, J=7 Hz, HNOEt,2) ppm;
13C NMR (50MHz, CD 3OD): d 150.6 (C, C4), 149.3
(CH, CCO), 147.7 (C, C3), 126.8 (C, C1), 121.0(CH, C 6),
113.4 (CH, C2), 112.2 (CH, C5), 70.2 (CH2, CNOEt,1),
56.2 (CH3, CNOEt,2), 14.9 (CH3, CArOMe) ppm; MS (EI):
m/z 195 (M+, 100%), 140 (20%), 124 (36%), 123
(27%), 106 (21%), 79 (22%), 65 (22%), 52 (25%), 51
(22%), 29 (27%). HR-MS m/z (M+): calcd for
C10H13NO3: 195.0895. Found 195.0918.
4-Hydroxybenzaldoxime (14). Yield 88% (purity 97%,
GC); mp 86.3 ꢂC; MS (EI): m/z 137 (M+, 100%), 120
(14%), 119 (16%), 94 (71%), 93 (21%), 65 (39%), 64
(12%), 63 (15%), 53 (10%), 39 (21%).
4-Methoxybenzaldoxime (15). Yield 78% (purity 98%,
GC); MS (EI): m/z 151 (M+, 100%), 135 (19%), 134
(33%), 133 (34%), 108 (67%), 92 (24%), 90 (22%), 77
(36%), 64 (23%), 63 (26%).
3,4,5-Trihydroxybenzaldehyde-O-ethyloxime (20). Oily
residue, which was not purified furthermore; quant yield
1
2,3-Dihydroxy-benzaldehyde-O-ethyloxime (16). Yield
96% (purity >98%, GC); 1H NMR (200 MHz,
CD3OD): d 8.26 (1H, s, HCO), 6.94–6.73 (3H, m, H4, H5
and H6), 4.23 (2H, q, J=7 Hz, HNOEt,1), 1.33 (3H, t,
J=7 Hz, HNOEt,2) ppm; 13C NMR (50MHz, CD 3OD):
d 151.7 (CH, CCO), 146.4 (C, C3), 146.2 (C, C2), 121.9
(purity 98.6%, GC); H NMR (200 MHz, CD3OD): d
7.81 (1H, s, HCO), 6.59 (2H, s, H2 and H6), 4.09 (2H, q,
J=7.0Hz, H NOEt,1), 1.26 (3H, t, J=7 Hz, HNOEt,2
)
ppm; 13C NMR (50MHz, CD 3OD): d 149.9 (CH, CCO),
146.8 (2C, C3 and C5), 136.3 (C, C4), 124.5 (C, C1),
107.2 (2CH, C2 and C6), 70.1 (CH2, CNOEt,1), 14.9
(CH3, CNOEt,2) ppm; MS (EI): m/z 197 (M+, 100%),
153 (17%), 152 (29%), 142 (31%), 126 (47%), 125
(33%), 96 (18%), 79 (27%), 51 (17%), 29 (24%). HR-
MS m/z (M+): calcd for C9H11NO4: 197.0688. Found
197.0668.
(CH, C4 or C5 or C6), 120.7 (CH, C4 or C5 or C6), 118.3
4
(C, C1), 118.0(CH, C
C
or C5 or C6), 71.1 (CH2ꢁ
,
NOEt,1), 14.7 (CH3, CNOEt,2) ppm; MS (EI): m/z 181
(M+, 89%), 136 (22%), 135 (100%), 108 (26.6%), 107
(61%), 80(24%), 79 (33%), 53 (18%), 52 (28%), 29
(16%). HR-MS m/z (M+): calcd for C9H11NO3:
181.0739. Found 181.0827.
4-Hydroxy-3-methoxybenzaldehyde-O-ethyloxime (21).
Yield 75% (purity 99%, GC); mp 31.7 ꢂC; H NMR
1
3,4-Dihydroxy-benzaldehyde-O-ethyloxime (17). Oily
residue, which crystallized in a refrigerator and was not
purified furthermore; quant. yield (purity 99.8%, GC);
(200 MHz, CD3OD): d 7.98 (1H, s, HCO), 7.23 (1H, d,
J=2 Hz, H2), 6.98 (1H, dd, J=8, 2 Hz, H6), 6.78 (1H,
d, J=8 Hz, H5), 4.15 (2 H, q, J=6.8 Hz, HNOEt,1), 3.85

1884
J. P. Ley, H.-J. Bertram / Bioorg. Med. Chem. 9 (2001) 1879–1885
(3H, s, HArOMe), 1.27 (3H, t, J=6.8 Hz, HNOEt,2) ppm;
13C NMR (50MHz, CD 3OD): d 149.7 (CH, CCO), 149.5
(C, C4 or C3), 149.1 (C, C3 or C4), 125.4 (C, C1),
3,5-Dimethyl-4-hydroxybenzaldehyde-O-ethyloxime (27).
Oily product, which was not further purified; quant.
yield (purity 96%, GC); 1H NMR (200 MHz, CDCl3): d
7.96 (1H, s, HCO), 7.22 (2H, bs, H2 and H6), 4.7–4.6
(1H, m, OH), 4.19 (2H, q, J=7.0Hz, H NOEt,1), 2.25
(6H, s, HArMe), 1.32 (3H, t, J=7.2 Hz, HNOEt,2) ppm;
13C NMR (50MHz, CDCl 3): d 153.5 (C, C4), 148.2
(CH, CCO), 127.3 (2 CH, C2 and C6), 124.3 (C, C1),
123.2 (2 C, C3 and C5), 69.4 (CH2, CNOEt,1), 15.9 (2
CH3, CArMe), 14.6 (CH3, CNOEt,1) ppm; MS (EI): m/z
193 (M+, 100%), 165 (28%), 164 (45%), 150 (20%),
148 (25%), 122 (35%), 121 (19%), 91 (44%), 77 (33%),
29 (14%). HR-MS m/z (M+): calcd for C11H15NO2:
193.1103. Found 193.1113.
5
122.6 (CH, C6), 116.0(CH, C ), 109.6 (CH, C2), 70.2
(CH2, CNOEt,1), 56.2 (CH3, CArOMe), 14.9 (CH3,
C
NOEt,2) ppm; MS (EI): m/z 195 (M+, 100%), 140
(19%), 124 (36%), 123 (27%), 106 (20%), 79 (22%), 65
(22%), 52 (24%), 51 (21%), 29 (26%). HR-MS m/z
(M+): calcd for C10H13NO3: 195.0895. Found
195.0902.
3-Ethoxy-4-hydroxybenzaldehyde-O-ethyloxime
(22).
Oily residue, which was not purified furthermore;
quant yield (purity 94%, GC); mp 43 ꢂC; 1H NMR
(200 MHz, CD3OD): d 7.97 (1H, s, HCO), 7.21 (1H, d,
J=2 Hz, H2), 6.97 (1H, dd, J=8, 2 Hz, H6), 6.79 (1H,
d, J=8 Hz, H5), 4.14 (2H, q, J=6.8 Hz, HOEt,1 or
3,4-Dihydroxyacetophenone oxime (28). Yield 90%
1
(purity 97%, GC, 98%, HPLC); H NMR (200 MHz,
H
NOEt,1), 4.11 (2H, q, J=6.8 Hz, HNOEt,1 or HOEt,1),
1.42 (3H, t, J=6.8 Hz, HOEt,2), 1.27 (3H, t, J=6.8 Hz,
(CD3)2SO): d 10.81 (1H, bs, OH), 9.09 (1H, bs, OH),
8.98 (1H, bs, OH), 7.10(1H, d, J=2.2 Hz, H2), 6.90
(1H, dd, J=8.3 Hz, 2.2 Hz, H6), 6.71 (1H, d, J=8.3 Hz,
H5), 2.05 (3H, s, HCOMe) ppm; 13C NMR (50MHz,
(CD3)2SO): d 152.4 (C, CCO), 146.1 (C, C3 or C4),
144.9 (C, C4 or C3), 128.2 (C, C1), 117.1 (CH, C6),
H
NOEt,2) ppm; 13C NMR (50MHz, CD 3OD): d 149.8
(C, C3 or C4), 149.7 (CH, CCO), 148.3 (C, C3 or C4),
125.4 (C, C1), 122.5 (CH, C6), 116.1 (CH, C5), 111.0
(C2), 70.2 (CH2, CNOEt,1), 65.4 (CH2, COEt,1), 15.0
(CH3, COEt,2 or CNOEt,2), 14.9 (CH3, CNOEt,2 or COEt,2
)
115.0(CH, C ), 112.5 (CH, C2), 11.3 (CH3, CCOMe); MS
5
ppm; MS (EI): m/z 209 (M+, 100%), 153 (39%), 152
(25%), 136 (26%), 126 (28%), 110(36%), 52 (23%), 51
(20%), 29 (40%), 27 (26%). HR-MS m/z (M+): calcd
for C11H15NO3: 209.1052. Found 209.1043.
(APCIꢁ): m/z 332.70([2M ꢁH]ꢁ, 11%), 166.44 ([MꢁH]ꢁ,
100%).
3,5-Dimethoxy-4-hydroxyacetophenone
1
oxime
(29).
Yield 47% (purity > 91%, GC); H NMR (200 MHz,
3,5-Dimethoxy-4-hydroxybenzaldehyde-O-ethyloxime (23).
Yield 60% (purity 97%, GC); 1H NMR (200 MHz,
CDCl3): d 7.98 (1H, s, HCO), 6.83 (2H, s, H2 and H6),
5.67 (1H, s, OH), 4.28 (2H, q, J=7.1 Hz, HNOEt,1), 3,93
(6H, s, HArOMe), 1.32 (3H, t, J=7.2 Hz, HNOEt,2) ppm;
MS (EI): m/z 225 (M+, 100%), 154 (23%), 153 (30%),
123 (15%), 122 (16%), 108 (13%), 67 (13%), 65 (13%),
29 (15%). HR-MS m/z (M+): calcd for C11H15NO4:
225.1001. Found 225.1002.
CD3OD): d 6.94 (2H, s, H2 and H6), 3.83 (6H, s,
H
ArOMe), 2.21 (3H, s, HCOMe) ppm; 13C NMR (50MHz,
CD3OD): d 156.2 (C, CCO), 148.9 (2C, C3 and C5), 137.7
(C, C4), 129.1 (C, C1), 104.6 (2 CH, C2 and C6), 56.7 (2
CH3, CArOMe), 12.2 (CH3, CCOMe) ppm; MS (EI): m/z
211 (M+, 100%), 194 (14%), 164 (12%), 155 (20%),
154 (41%), 153 (13%), 136 (12%), 123 (12%), 108
(12%). HRMS m/z (M+): calcd for C10H13NO4:
211.0845. Found 211.0822.
4-Hydroxybenzaldehyde-O-ethyloxime (24). Yield 73%
(purity 99%, GC); MS (EI): m/z 165 (M+, 100%), 137
(50%), 136 (73%), 120 (38%), 94 (61%), 93 (32%), 65
(50%), 29 (31%), 27 (20%).
3,4-Dihydroxyacetophenone-O-ethyloxime (30). Yield
60% (purity 98%, HPLC); 1H NMR (200 MHz,
CD3OD): d 7.14 (1H, d, J=2 Hz, H2), 6.99 (1H, dd,
J=8, 2 Hz, H6), 6.76 (1H, d, J=8 Hz, H5), 4.12 (2H, q,
J=7 Hz, HNOEt,1), 2.14 (3H, s, HCOMe), 1.28 (3H, t,
J=7 Hz, HNOEt,1) ppm; MS (ESI +): m/z 196.05
(100%, [M+H]+). HR-MS m/z (M+): calcd for
C10H13NO3: 195.0895. Found 195.0887.
4-Methoxybenzaldehyde-O-ethyloxime (25). Yield 84%
(purity 99%, GC); MS (EI): m/z 179 (M+, 100%), 151
(57%), 150 (75%), 134 (33%), 108 (40%), 92 (30%), 91
(26%), 77 (50%), 63 (24%), 29 (28%).
Tyrosinase assay
4-Hydroxy-3-methylbenzaldehyde-O-ethyloxime
(26).
1
Yield 69% (purity >99%, GC); H NMR (200 MHz,
CD3OD): d 7.96 (1H, s, HCO), 7.34 (1H, m, H2), 7.25
(1H, dd, J=8.5, 2 Hz, H6), 6.74 (1H, d, J=8.5 Hz, H5),
4.09 (2H, q, J=7.0Hz, H NOEt,1), 2.17 (3H, s, HArMe),
1.26 (3H, t, J=7.0Hz, H NOEt,1) ppm; 13C NMR
(50MHz, CD 3OD): d 158.3 (C, C4), 149.8 (CH, CCO),
130.4 (CH, C2), 127.0(CH, C 6), 125.9 (C, C3), 124.8 (C,
C1), 115.5 (CH, C5), 70.0 (CH2, CNOEt,1), 16.2 (CH3,
Tyrosinase (2000 Units/mg) was dissolved in phosphate
buffer (pH 6.8, c 0.067 mol Lꢁ1) to a concentration of
120U mL ꢁ1, and in each case 100 mL of this tyrosinase
solution were pipetted into a cavity of a 96-well multi-
well plate of clear polystyrene. Phosphate buffer pH 6.8
(25 mL) and stepwise diluted test compound or standard
(75 mL) were added. Phosphate buffer was used to dilute
the DMSO stock solution of test compound. The con-
trol used was phosphate buffer.
C
ArMe), 14.9 (CH3, CNOEt,2) ppm; MS (EI): m/z 179
(M+, 100%), 151 (31%), 150 (56%), 134 (27%), 108
(36%), 107 (21%), 91 (15%), 77 (34%), 29 (13%).
HRMS m/z (M+): calcd for C10H13NO2: 179.0946.
Found 179.0969.
The resulting mixtures in the microtiter plate were
incubated at 37 ꢂC for 10min. A solution of l-DOPA in
phosphate buffer pH 6.8 (c0.03%) (100 mL) was added

J. P. Ley, H.-J. Bertram / Bioorg. Med. Chem. 9 (2001) 1879–1885
1885
and the absorption (A) was recorded at 495 nm for at
Acknowledgements
least 10min. The measurement was performed in tripli-
cate for each concentration and averaged before further
calculation. The residual tyrosinase activities after 3 min
incubation in the presence of test compounds were cal-
culated in accordance with the following equation:
N. Schlichter and C. Strempel are gratefully thanked
for their excellent technical assistance and G. Kram-
mer, C.-O. Schmidt and H. Sommer for the analysis of
the compounds.
References and Notes
Residual tyrosinase activity ð%Þ
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3. Prota, G. Melanins and Melanogenesis; Academic: New
York, 1992.
À
Á
¼ ATest compound=AControl ꢀ 100
ð1Þ
4. Prota, G. Cosmet. Toiletries 1996, 111, 43.
5. Kahn, V.; Lindner, P.; Zaka, V. J. Food Biochem. 1995, 18,
253.
The IC50 was calculated from the residual tyrosinase
activities (%) in a series of dilutions of test compounds.
This is the concentration of a test compound in which
the tyrosinase is inhibited to an extent of 50%.
6. Kaatz, H.; Wollenberger, U.; Peter, M. G.; Streffer, K.
Zeitschrift fur Naturforschung C — J. of Biosci. 1999, 54, 70 .
¨
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The determination of inhibitor kinetics was performed
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each of three different inhibitor concentrations (0, 1.5
and 3 mmol Lꢁ1) l-DOPA concentration was varied (0,
0.1, 0.2, 0.4, 0.5, 0.75 and 1 mmol Lꢁ1). Pre-incubation
and measurement time was the same as above. Maximal
initial velocity v=ꢀA/ꢀt was determined from initial
linear portion of absorbance between 20and 60s after
addition of l-DOPA. Kinetic parameters (Km, appar-
ent, and Vmax) were determined using double-reciprocal
plots (Lineweaver–Burk) of enzyme activity against
l-DOPA concentration.
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