Synthesis and tyrosinase inhibitory activity of novel N-hydroxybenzyl-N-nitrosohydroxylamines

Article abstract of DOI:10.1016/S0045-2068(03)00026-9

Several novel N-substituted N-nitrosohydroxylamines were synthesized. They all inhibited mushroom tyrosinase, but the type of inhibition was different depending on the substituent. Some N-(mono- or dihydroxybenzyl)-N-nitrosohydroxylamines exhibited uncompetitive inhibition with respect to L-dopa. Among them, compound 6 was also a competitive inhibitor with respect to oxygen. This observation suggests that another interaction by the meta- or para-hydroxyl group might stabilize the binding of the inhibitor to the enzyme through the oxygen binding site.

Full text of DOI:10.1016/S0045-2068(03)00026-9

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 31 (2003) 129–135
www.elsevier.com/locate/bioorg
Synthesis and tyrosinase inhibitory
activity of novel
N-hydroxybenzyl-N-nitrosohydroxylamines
Mitsuhiro Shiino,a,* Yumi Watanabe,a and Kazuo Umezawab
a
Laboratory of Chemistry, School of Medicine, Keio University, 4-1-1 Hiyoshi, Kohoku-ku,
Yokohama 223-8521, Japan
b
Department of Applied Chemistry, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
Received 16 July 2002
Abstract
Several novel N-substituted N-nitrosohydroxylamines were synthesized. They all inhibited
mushroom tyrosinase, but the type of inhibition was different depending on the substituent.
Some N-(mono- or dihydroxybenzyl)-N-nitrosohydroxylamines exhibited uncompetitive inhi-
bition with respect to L-dopa. Among them, compound 6 was also a competitive inhibitor with
respect to oxygen. This observation suggests that another interaction by the meta- or para-hy-
droxyl group might stabilize the binding of the inhibitor to the enzyme through the oxygen
binding site.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Nitrosohydroxylamine; Tyrosinase; L-dopa; Enzyme inhibition
1. Introduction
Tyrosinase (EC 1.14.18.1) is a copper containing enzyme that catalyzes two differ-
ent reactions, the hydroxylation of monophenolic compounds to o-diphenols and the
oxidation of the o-diphenols to o-quinones. The mammalian tyrosinase is the key
* Corresponding author. Fax: +81-45-566-1310.
E-mail address: mitsuhir@hc.cc.keio.ac.jp (M. Shiino).
0045-2068/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0045-2068(03)00026-9

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M. Shiino et al. / Bioorganic Chemistry 31 (2003) 129–135
Fig. 1. Chemical structures of N-cyclopentyl-N-nitrosohydroxylamine (1), tropolone (2), and cupferron (3).
enzyme in melanin biosynthesis [1], catalyzing the hydroxylation of
L
L-tyrosine to 3,4-
dihydroxy- -phenylalanine ( -dopa) and its subsequent oxidation to dopaquinone.
L
Therefore, inhibitors of tyrosinase should be useful as therapeutic agents for the
treatment of melanin hyperpigmentation [2] and cosmetic materials for whitening af-
ter sunburn [3]. Moreover, tyrosinase, also known as polyphenol oxidase, is known
to cause a browning reaction. Thus, in the food industry, tyrosinase inhibitors are
becoming important to prevent enzymatic browning [4]. In the previous paper, we
reported that N-substituted N-nitrosohydroxylamines strongly inhibit mushroom ty-
rosinase [5]. Among the compounds examined, N-cyclopentyl-N-nitrosohydroxyl-
amine (1, Fig. 1) exhibited the most potent activity, having an IC50 value of
0.6 lM, which is as potent as tropolone (2) [6], the most potent known inhibitor
of tyrosinase. It was suggested that the inhibitory activity was caused by the chela-
tion of the copper in the active site of the enzyme by the N-nitrosohydroxylamino
moiety. The kinetic analysis showed that cupferron (3) was a competitive inhibitor
with respect to L-dopa, but compound 1was not a competitive one. Thus, the mech-
anism of inhibition by N-substituted N-nitrosohydroxylamines might be different de-
pending on the N-substituent. In order to further investigate the effect of the
N-substituent on the mechanism, we synthesized several N-hydroxybenzyl-N-nitro-
sohydroxylamines and related compounds to examine their inhibitory activities.
The results are reported herein.
2. Materials and methods
2.1. Materials
Tyosinase was obtained from Sigma and used without further purification. So-
dium cyanoborohydride was purchased from Aldrich. All other chemical reagents
were supplied by Tokyo Kasei.
2.2. Syntheses
Three N-monohydroxybenzyl derivatives (4, 5, and 6), two N-dihydroxybenzyl
derivatives (7 and 8), 40-chlorobenzyl derivatives (9), 40-methoxybenzyl derivatives
(10), and 40-tert-butylbenzyl derivatives (11) were synthesized by the same method
as described previously [5], starting from the corresponding benzaldehydes, through
the reduction of oximes. All N-substituted N-nitrosohydroxylamines synthesized

M. Shiino et al. / Bioorganic Chemistry 31 (2003) 129–135
131
were isolated as ammonium salts, which were characterized by chemical and spectral
1
methods. H NMR spectra were recorded on a Hitachi R-24 60 MHz spectrometer.
FTIR spectra were recorded on a Bio-Rad FTS-60A spectrometer. HRMS data were
obtained on a JEOL JMS-700 ms station spectrometer. The general procedure for
the preparation of N-hydroxybenzyl-N-nitrosohydroxylamines is described for
compound 4.
N-(20 -hydroxybenzyl)-N-nitrosohydroxylamine, ammonium salt (4). To a mix-
ture of salicylaldehyde (3.0 g, 25 mM) in methanol (150 mL) and hydroxylamine
hydrochloride (2.6 g, 37 mM) in water (5 mL) was added aqueous solution of
6 M NaOH (7.5 mL). After 2 h of stirring, the mixture was concentrated in vacuo.
The residue was extracted with ethyl acetate. The extract was dried over Na2SO4
and evaporated to give the crude oxime. To a solution of the crude oxime in
methanol (50 mL), containing a trace of methyl orange, NaBH3 CN (1.6 g,
25 mM) was added. Subsequently, 5 M HCl–MeOH was added dropwise with stir-
ring to maintain the red color of the solution for 30 min. The reaction mixture
was stirred overnight, and methanol was removed in cacuo. The residue was sus-
pended in water and brought to pH 10 using aqueous solutions of 6 M NaOH
and saturated NaHCO3, and extracted with ethyl acetate. The extract was dried
over Na2SO4 and concentrated in cacuo to dryness. Recrystallization of the crude
product from ethyl acetate gave 1.4 g of N-(20-hydroxybenzyl)hydroxylamine with
a yield of 37% from salicylaldehyde. N-(20-hydroxybenzyl)hydroxylamine (100 mg,
0.6 mM) was dissolved in 1mL of ethyl acetate and added to 8 mL of ether. Sub-
sequently, n-butyl nitrite (0.3 mL) was added and then ammonia gas was bubbled
through the mixture at 0 °C for 10 min. After 30 min of stirring, the reaction mix-
ture was centrifuged at 500g for 10 min at 4 °C. After decantation of the super-
natant, the precipitate was washed three times sequentially with dried ether
followed by n-hexane to give white crystals, which were dried using nitrogen
gas. The yield of N-(20-hydroxybenzyl)-N-nitrosohydroxylamine, ammonium salt
was 80 mg (72%). mp 137.5–142.2 °C; IR (KBr): 3423 (broad), 1604, 1463, 927,
749, 698 cmÀ1
;
1H NMR (D2OÞd: 5.02 (2H, s), 6.68–7.15 (4H, m); HRMS
À
(FAB) calcd. for C7H7N2O3 [M–HŠ 167.046, found 167.046. Both Liebermann
[7] and GriessÕ [8] reactions were positive indicating the presence of the nitroso
group.
N-(30 -hydroxybenzyl)-N-nitrosohydroxylamine, ammonium salt (5). The overall
yield from 3-hydroxybenzaldehyde was 30.6%. mp 126.6–130.1 °C; IR (KBr): 3422
(broad), 1605, 1466, 929, 740, 694 cmÀ1; H NMR (D2OÞd: 4.90 (2H, s), 6.65–7.10
1
À
(4H, m); HRMS(FAB) calcd. for C7H7N2O3 [M–HŠ 167.046, found 167.046. Both
Liebermann and GriessÕ [7,8] reactions were positive indicating the presence of the
nitroso group.
N-(40-hydroxybenzyl)-N-nitrosohydroxylamine, ammonium salt (6). The overall
yield from 4-hydroxybenzaldehyde was 22.4%. mp 101.4–103.4 °C; IR (KBr): 3427
1
(broad), 1602, 1436, 945, 748, 696 cmÀ1; H NMR (D2OÞd: 4.85 (2H, s), 6.65 (2H,
d, J ¼À8:0 Hz), 7.05 (2H, d, J ¼ 8:0 Hz); HRMS(FAB) calcd. for C7H7N2O3
[M–HŠ 167.046, found 167.045. Both Liebermann and GriessÕ reactions were
positive indicating the presence of the nitroso group [7,8].

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M. Shiino et al. / Bioorganic Chemistry 31 (2003) 129–135
2.3. Kinetics
Tyrosinase activity was measured by a spectrophotometric method using a Shima-
dzu UV-160A spectrophotometer or by a polarographic method using a Central
Oxygraph-9 equipped with a Clark-type oxygen electrode [5].
The IC50 values were obtained from the spectrophotometric assay. Thus, 3 mL of
0.5 mM L-dopa in 67 mM phosphate buffer (pH 6.8) and 0.1mL of the test sample
solution were mixed at 30 °C. Subsequently, 0.1mL of the aqueous solution of mush-
room tyrosinase (80 units) was added to the mixture to measure immediately,
the initial rate of linear increase in optical density at 475 nm, corresponding to the
formation of dopachrome. The inhibitory effect of the sample was represented as
percentage of inhibition compared with the control.
The effect of copper sulfate on the inhibition of mushroom tyrosinase by com-
pound 6 was performed by the spectrophotometric assay. Accordingly, the mixture
contained, in a total volume of 3.2 mL, 67 mM phosphate buffer (pH 6.8), 0.5 mM
L
-dopa, 13.7 lM of compound 6 (equivalent amount of IC50), and 50 lM copper
sulfate. The mixture was incubated at 30 °C for 3 min. Subsequently, 0.1mL of the
aqueous solution of mushroom tyrosinase (80 units) was added to the mixture.
The reaction was monitored at 475 nm.
3. Results and discussion
The inhibitory activity of the newly synthesized compounds on mushroom tyros-
inase and the type of inhibition obtained from Lineweaver–Burk plots are listed in
Table 1. The synthesized compounds show activities with IC50 values ranging from
1 0 to 24lM, which are nearly comparable with that of the well-known potent inhib-
itor, kojic acid (12, Fig. 2) [9]. Since these compounds have IC50 values less than the
parent compound, N-benzyl-N-nitrosohydroxylamine (IC50 ¼ 3:0lM), the substitu-
ents on the benzene ring slightly decrease the inhibition activities, regardless of the
nature of the substituent or its position. Compound 6 showed an IC50 value that
is two orders of magnitude greater than that of the corresponding hydroxylamine
(IC50 ¼ 1.1 mM), confirming that the activity is caused by the N-nitrosohydroxyla-
mino group as reported in our previous paper [5].
The inhibition data obtained at pH 6.8, were plotted using Lineweaver–Burk plots
(Fig. 3) and analyzed. Compounds 4, 9, 10, and 11 showed mixed type of inhibition
with respect to
hydroxyl group at the meta- or para-position, inhibited uncompetitively with respect
to -dopa. Moreover, compound 6 exhibited competitive inhibition with respect to
L-dopa. On the other hand, compounds 5–8, which have a phenolic
L
oxygen (Fig. 3).
To confirm the mechanism of inhibition by compound 6, the effect of copper sul-
fate on the inhibitory activity was examined. The results showed that the activity of
compound 6 decreased from 50% of inhibition to 41% by the addition of copper sul-
fate, similar to that observed with cupferron in the presence of copper sulfate (38%).
These observations suggest that the mechanism of inhibition by compound 6 may be

M. Shiino et al. / Bioorganic Chemistry 31 (2003) 129–135
133
Table 1
Inhibition of tyrosinase by N-substituted N-nitrosohydroxylamines
Compound
IC50 ðlMÞ
Type of inhibition with
respect to
L-dopa
4
5
19.2
20.3
Mixed
Uncompetitive
6
7
13.7
11.6
Uncompetitive
Uncompetitive
8
23.8
Uncompetitive
9
13.1
9.7
Mixed
Mixed
10
11
15.8
Mixed
Fig. 2. Chemical structures of kojic acid (12) and barbarin (13).
due to its chelation of copper ion in the active site of the enzyme as has been shown
for cupferron, even though their type of inhibition is different. In addition, when the
enzyme was pre-incubated with cupferron without L-dopa, the enzyme activity was
decreased [5]. However, pre-incubation of the enzyme with compound 6 did not
change activity. Since the resting form of the enzyme is mostly met-tyrosinase [10],
it appears that compound 6 interacts with only oxy-tyrosinase, but not with met-ty-
rosinase, in contrast to cupferron.
Among the N-monohydroxybenzyl derivatives (4–6), the ortho-hydroxyl deriva-
tive (4) showed mixed type of inhibition while both N-dihydroxybenzyl derivatives

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M. Shiino et al. / Bioorganic Chemistry 31 (2003) 129–135
Fig. 3. Lineweaver–Burk plots of the mushroom tyrosinase reaction with N-(40-hydroxybenzyl)-N-nitro-
sohydroxylamine (6) at pH 6.8: (A) with respect to -dopa and (B) with respect to oxygen.
L
(7 and 8) exhibited uncompetitive inhibition, as noted above. Therefore, the meta- or
para-hydroxyl group on the benzyl residue would play a role in this mechanism. One
possibility is that the meta- or para-hydroxyl group strengthens the interaction be-
tween the inhibitors and the copper in the active site of the oxy-tyrosinase at the ox-
ygen binding site, by forming a hydrogen bond to the amino nitrogen near to the
active site of the enzyme (Fig. 4). This idea was supported by the results of inhibition
under weakly acidic conditions. At pH 5.8, compound 6 exhibited mixed type of in-
hibition with respect to both L-dopa and oxygen, in contrast to uncompetitive inhi-
Fig. 4. Proposed binding models for the active site of the oxy-tyrosinase. (A) A model of
substrate [13]; (B) a model of cupferron as a competitive inhibitor [5]; (C) a model of compound 6 as
an uncompetitive inhibitor with -dopa as a substrate.
L-dopa as a
L

M. Shiino et al. / Bioorganic Chemistry 31 (2003) 129–135
135
bition with respect to L-dopa and competitive inhibition with respect to oxygen at
pH 6.8. Under acidic conditions, hydrogen bonding between phenolic hydroxyl
group of compound 6 and the nitrogen might weaken so that the interaction at
the oxygen binding site might decrease. In case of compound 4, hydrogen bonding
of the ortho-hydroxyl group may be weakened by steric hindrance, making it difficult
to show uncompetitive inhibition.
Most tyrosinase inhibitors that chelate the copper in the active site of the enzyme
show competitive inhibition such as tropolone (2) and kojic acid (12) (Figs. 1and 2).
Here, we have shown that N-substituted N-nitrosohydroxylamine derivatives, which
have the ability to chelate the copper, exhibit a different type of inhibition depending
on the substituent. The different type of inhibition might be due to the differences in
the binding ability of the inhibitor to the copper in the active site of enzyme as well as
the differences in the interaction of the N-substituent with the enzyme. Among tyros-
inase inhibitors, uncompetitive inhibitor with respect to
Fig. 2) has been known as an uncompetitve tyrosinase inhibitor with
L
-dopa is rare. Barbarin (13,
-dopa, but its
L
mechanism of inhibition has not been elucidated yet [11]. Although cyanide is a com-
petitive inhibitor of oxygen, it exhibited noncompetitive inhibition with respect to
L
-dopa [12]. In this paper, we showed that N-ð40-hydroxybenzyl)-N-nitrosohydroxyl-
amine (6) is a tyrosinase inhibitor, which is uncompetitive with L-dopa and compet-
itive with oxygen, and acts through a chelation mechanism.
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