Synthesis of N-substituted N-nitrosohydroxylamines as inhibitors of mushroom tyrosinase

Article abstract of DOI:10.1016/S0968-0896(01)00003-7

A series of N-substituted N-nitrosohydroxylamines including six new compounds were synthesized and examined for inhibition of mushroom tyrosinase. Corresponding hydroxylamines were reacted with n-butyl nitrite to give substituted nitrosohydroxylamines as their ammonium salt. The N-substituted hydroxylamines were prepared from the primary amines via the oxaziridine, or from the carbonyl compounds via the oxime. Most of the nitrosohydroxylamines tested inhibited mushroom tyrosinase. Among them, N-cyclopentyl-N-nitrosohydroxylamine exhibited the most potent activity (IC₅₀=0.6 μM), as powerful as that of tropolone, one of the most powerful inhibitors. As removal of nitroso or hydroxyl moiety, the enzyme inhibitory activity was completely diminished. Both N-nitroso group and N-hydroxy group were suggested to be essential for the activity, probably by interacting with the copper ion at the active site of the enzyme. Lineweaver-Burk plotting showed that cupferron was a competitive inhibitor but that N-cyclopentyl-N-nitrosohydroxylamine was not.

Full text of DOI:10.1016/S0968-0896(01)00003-7

Bioorganic & Medicinal Chemistry 9 (2001) 1233±1240
Synthesis of N-substituted N-nitrosohydroxylamines as
Inhibitors of Mushroom Tyrosinase
Mitsuhiro Shiino,a,* Yumi Watanabea and Kazuo Umezawab
aLaboratory of Chemistry, School of Medicine, Keio University, 4-1-1 Hiyoshi, Kohoku-ku, Yokohama 223-0061, Japan
bDepartment of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku,
Yokohama 223-0061, Japan
Received 10 August 2000;accepted 13 December 2000
AbstractÐA series of N-substituted N-nitrosohydroxylamines including six new compounds were synthesized and examined for
inhibition of mushroom tyrosinase. Corresponding hydroxylamines were reacted with n-butyl nitrite to give substituted nitroso-
hydroxylamines as their ammonium salt. The N-substituted hydroxylamines were prepared from the primary amines via the oxaz-
iridine, or from the carbonyl compounds via the oxime. Most of the nitrosohydroxylamines tested inhibited mushroom tyrosinase.
Among them, N-cyclopentyl-N-nitrosohydroxylamine exhibited the most potent activity (IC50=0.6 mM), as powerful as that of
tropolone, one of the most powerful inhibitors. As removal of nitroso or hydroxyl moiety, the enzyme inhibitory activity was
completely diminished. Both N-nitroso group and N-hydroxy group were suggested to be essential for the activity, probably by
interacting with the copper ion at the active site of the enzyme. Lineweaver±Burk plotting showed that cupferron was a competitive
inhibitor but that N-cyclopentyl-N-nitrosohydroxylamine was not. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
we found that dopastin (1) also inhibited mushroom
tyrosinase e€ectively. The inhibitory activity of dopastin
(1) is likely to be derived from the N-nitroso-N-hydroxyl-
amino group, which can interact with the copper ion at
the active site of the enzyme. Therefore, we synthesized
several N-nitroso-N-hydroxylamines and examined their
inhibitory activity on mushroom tyrosinase to study the
structure±activity relationship.
Tyrosinase (E. C.1.14.8.1) is
a
copper-containing
enzyme widely distributed in plants and animals. It cat-
alyses the o-hydroxylation of monophenols and also the
oxidation of o-diphenols to o-quinones. Tyrosinase is
known to be a key enzyme for melanin biosynthesis in
plants and animals. Therefore, tyrosinase inhibitors
should be clinically useful for the treatment of some
dermatological disorders associated with melanin
hyperpigmentation1 and also important in cosmetics for
whitening and depigmentation after sunburn.2,3 In
addition, tyrosinase is known to be involved in the
molting process of insects4 and adhesion of marine
organisms.5
Results and Discussion
Synthesis of N-substituted N-nitrosohydroxylamines
Eleven N-substituted N-nitrosohydroxylamines (4±14)
including six new compounds (5±8, 12 and 13) were
synthesized. The procedure for the synthesis of the N-
substituted N-nitrosohydroxylamines is outlined in
Scheme 1. The N-nitrosohydroxylamines were prepared
by nitrosation of the corresponding hydroxylamine,
which were obtained by procedures A or B in Scheme 1.
Procedure A included condensation of the primary amine
with benzaldehyde and oxidation of the resulting imine
with m-chloroperbenzoic acid followed by hydrolysis of
the oxaziridine.9,10 Procedure B included condensation
of the aldehyde or ketone with hydroxylamine and
reduction of the resulting oxime.11 Nitrosation of all of
Several N-substituted N-nitrosohydroxylamines are
known to inhibit various types of enzymes;e.g., dopastin
(1) is an inhibitor of dopamine-b-monohydroxylase (E.
C.1.14.2.1),6 nitrosoxacins inhibit 5-lipoxygenase (E.
C.1.13.11.12),7 and cupferron (2) and neocupferron (3),
which are metal-chelating agents, inhibit superoxide
dismutase (E. C.1.15.1.1)8 (Fig. 1). In the present paper,
*Corresponding author. Tel.: +81-45-566-1311;fax: +81-45-566-1310;
e-mail: mitsuhir@hc.cc.keio.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00003-7

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M. Shiino et al. / Bioorg. Med. Chem. 9 (2001) 1233±1240
It has been reported that the inhibitory activities of
cupferron (2) on superoxide dismutase and dopastin (1)
on dopamine-b-monohydroxylase were due to the che-
lation between the nitrosohydroxylamino group and the
copper ion in the enzyme.6,8 It has also been reported
that some N-substituted N-nitrosohydroxylamines were
copper chelating compounds.19,20 Therefore, we also
examined the chelating activity of N-substituted N-nitro-
sohydroxylamines. The addition of each N-substituted
N-nitrosohydroxylamine to a copper carbonate solution
gave the corresponding chelate compound as the pre-
cipitate, indicating the ability of each compound to
chelate Cu2+. Besides, the addition of copper sulfate to
the reaction medium in the inhibitory assay recovered
the enzyme activity inhibited by N-cyclopentyl-N-nitroso-
hydroxylamine (9), as shown in Figure 2. Based on these
results, we propose that the synthesized or desigaed inhi-
bitors might bind to the copper ion at the active site of
the enzyme.
Figure 1. Structure of dopastin and related N-nitrosohydroxylamines.
the hydroxylamines obtained was achieved by use of
n-butyl nitrite in etherial solution saturated with
ammonia, and N-nitroso-N-hydroxylamines were isolated
as ammonium salts. All compounds synthesized were
characterized by chemical and spectral methods.
Kinetic analysis for o-dihydroxyphenolase inhibition
with respect to l-dopa and oxygen was carried out with
cupferron (2) and compound 9, as shown in Figure 3.
The o-dihydroxyphenolase reaction is an orderded bi-bi
reaction. Thus, cupferron was competitive with l-dopa
(Ki=0.3 mM) and noncompetitive with oxygen. On the
other hand, compound 9 did not show competitive
inhibition. Therefore, these two compounds a€ect the
enzyme in di€erent ways. To look into the mechanism
of inhibition, we tested the e€ect of pre-incubation of the
enzyme with the inhibitor prior to the addition of l-dopa.
Pre-incubation of the enzyme in the presence of cupferron
or compound 9 and in the absence of the substrate
decreased the enzyme activity signi®cantly. The inhibi-
tory activity at an equivalent amount of IC50 of either
compound was increased 1.6 times by the pre-incubation.
As the enzyme pre-incubated is mostly met tyrosinase,
known as the resting form of the enzyme,21,22 the above
results indicate that both cupferron and compound 9
can react with the met-form of tyrosinase before oxygen
binds.
Inhibition of tyrosinase
Most of the N-nitroso-N-hydroxylamines tested inhibited
the mushroom tyrosinase, and the IC50 values ranged
widely from 0.6 to 273 mM, as summarized in Table 1.
Tropolone,12,13 kojic acid14À16 and mimosine,12,17,18 are
known to be the most powerful inhibitors of tyrosinase.
Among the compounds tested, cupferron (2;
IC50=1.1 mM), N-cycloalkyl derivatives (9±11;0.6±
1.2 mM), and N-n-alkyl derivatives (12 and 13;1.5 and
2.2 mM, respectively) exhibited the inhibitory activity of
almost the same order with the reported inhibitors.
Interestingly, activities of N-n-alkyl derivatives were not
a€ected by the length of their alkyl chain (12 and 13).
On the other hand, the inhibitory activity dramatically
decreased with an increase in the length of the alkyl
chain (4, 6 and 7;3.0±273 mM) in the compounds having
the phenyl substituent at the a-carbon of the N-nitroso-
N-hydroxylamino group. Compound 8, having two
phenyl groups at the a-carbon, exhibited the activity two
orders lower than that of the corresponding mono-
phenyl derivative (4). The activity of the N-tert-butyl
derivative (14;19.3 mM) was one order lower than that
of N-n-alkyl derivatives (12 and 13). Therefore, the
inhibitory activity was a€ected signi®cantly with either
the phenyl group or alkyl group as a substituent on the
a-carbon of the N-nitroso-N-hydroxylamino group, but
not by subsituents at other positions. These observations
suggest that the phenyl or alkyl substituent at the a-
carbon of the N-nitroso-N-hydroxylamino group may
cause steric hindrance for the approach of the inhibitors
to the active site of the enzyme.
Since the most potent inhibitors such as kojic acid,
mimosine, and toropolone resemble the structure of the
substrate in part (Fig. 4A),12,23,24 this structural similarity
may explain the inhibitory activity. Though N-nitroso-
hydroxylamines seem to have no structural similarity,
some of them exhibited inhibitory activity of the same
order. Recently, N-nitrosohydroxylamines were reported
to exist mainly as a tautomeric hydroxydiazenium N-oxide
form, being a planar structure (Fig. 4B).25 As a planar
structure, the distance between the two oxygens of the
nitrosohydroxylamino group can be estimated from a
molecular model, and it was nearly the same as that of the
two hydroxy groups of catechols. Therefore, the N-nitroso-
hydroxylamino group may interact with binuclear copper
at the active site of the enzyme, as a bridge structure similar
to the substrate (Fig. 4C). There are two separate substrate-
binding sites in the binuclear copper active center of the
oxy-form of tyrosinase, one for catechol and the other
for oxygen.26 Being a competitive inhibitor, cupferron
only interacts with the catechol-binding site. On the
other hand, compound 9 may interact not only with the
The related hydroxylamines and N-nitrosoamines did
not inhibit the enzyme, as shown in Table 2. Therefore,
both N-nitroso group and N-hydroxyl group are essential
for inhibition of the enzyme.

M. Shiino et al. / Bioorg. Med. Chem. 9 (2001) 1233±1240
1235
Scheme 1. Routes for synthesis of N-nitrosohydroxylamines: (a) CH2Cl2, MgSO4, rt, 2 h;(b) m-chloroperbenzoic acid, CH2Cl2, 4 ꢀC, 4 h;(c) H 2SO4/
MeOH/H2O, 4 ꢀC, 18 h;(d) n-butyl nitrite, ether, NH3, 0 ꢀC, 15 min;(e) NH 2OHꢁ HCl, NaOH, MeOH, rt, 2 h;(f) NaBH 3CN, HCl, MeOH, rt, 16 h.
catechol-binding site but also with the oxygen-binding
site in the oxy-form of tyrosinase.
cause steric hindrance for the approach of inhibitors to the
active site of the enzyme. As a mechanism of inhibition, we
suggested that both N-nitroso and N-hydroxyl groups were
essential to inhibit the enzyme, interacting with the copper
ion at the active site of the enzyme. Furthermore, kinetic
analysis of the enzyme indicated that N-cyclopentyl-N-
nitrosohydroxylamine might interact to the copper ion at
the active site of the enzyme not only from the substrate-
binding site but also from the oxygen-binding site.
Conclusion
We found that N-substituted N-nitrosohydroxylamines
inhibited mushroom tyrosinase. Their potency was a€ected
by the structure of N-substitutent. N-Substitution might

1236
M. Shiino et al. / Bioorg. Med. Chem. 9 (2001) 1233±1240
Table 1. Inhibitory activity of N-substituted N-nitrosohydroxyl-
amines on mushroom tyrosinase
Compd
R
IC50 (mM)
1
20
2
3
1.1
Figure 2. E€ect of Cu2+ ion on the inhibitory activity of N-cyclo-
pentyl-N-Nitrosohydroxylamine. The reaction was carried out with
0.5 mM l-dopa, 67 mM phosphate bu€er (pH 6.8), 80 units of mush-
room tyrosinase, CuSO4 and 1.0 mM (*) or 2.0 mM (&) of N-cyclo-
pentyl-N-nitrosohysroxylamine. Percentage of recovered activity was
calculated in reference to the control value.
46.0
4
5
3.0
1.3
Experimental
Materials
6
7
23.5
273
Cupferron (2) and neocupferron (3) were purchased
from Tokyo Kashei. Dopastine (1) was kindly supplied
by Dr. H. Iinuma, Institute of Microbial Chemistry,
Tokyo. Tyrosinase was obtained from Sigma.
Syntheses
8
139
1
The structures were determined by 60 MHz H NMR
(Hitachi R-24), IR (Jasco A-100, Bio-Rad FTS-60A
(FTIR)), HRMS (JEOL JMS-700 MSstaition) and the
elemental analysis. The typical procedure for the pre-
paration of N-nitrosohydroxylamines 5 and 8 (procedure
A) is described for 5.
9
0.6
1.2
1.1
10
11
N-(3-Phenylpropyl)hydroxylamine. N-(3-Phenylpropyl)
amine (6.76 g, 0.05 M) in dichloromethane (75 mL),
benzaldehyde (10 g, 0.05 M) in dichloromethane (25 mL),
and anhydrous MgSO4 (8.7 g, 0.05 M) were mixed, and
stirred for 2 h at room temperature. After removal of
MgSO4 by ®ltration, the ®ltrate was evaporated in vacuo.
The residue was dissolved in 100 mL of dichloromethane
and added dropwise by 12.1 g of m-chloroperbenzoic
acid in 150 mL of dichloromethane, and the mixture was
stirred at 4 ꢀC for 4 h. After ®ltration, the ®ltrate was
washed with 10% K2CO3 aq solution, dried, and con-
centrated in vacuo. The residue was dissolved in 50 mL of
methanol and then H2SO4:CH3OH:H2O (32:75:93 mL)
was added dropwise at 4 ꢀC, and then the mixture was
stirred for 18 h. After concentration, the acidic solution
was neutralized with 6 M NaOH aq solution, and the pH
of the solution was adjusted to above 10 with saturated
NaHCO3 aq solution. The alkaline solution was extracted
with 300 mL of ethyl acetate, and the extract was dried
over Na2SO4 and evaporated to dryness. The residue
was crystallized from n-hexane and benzene to give
0.85 g of N-(3-phenylpropyl)hydroxylamine with a total
yield of 11.2%: mp 41.8±43.4 ꢀ;IR (KBr): 3245, 3140,
12
13
1.5
2.2
14
19.3
Table 2. E€ect of N-hydroxyl and N-nitroso amines on tyrosinase
IC50 (mM)
R
R-NHOH
R-N(NO)-R0
Phenyl (2)a
Benzyl (4)a
Cyclohexyl (10)a
Cyclooctyl (11)a
tert-butyl (14)a
N.T.
4.4
>0.28
0.22
5.5
12.8 (R0ˆCH3)
1.0 (R0ˆCH3)
N.T.
N.T.
>78 (R0ˆC2H5)
aCompound number of related chemical in Table 1.
N.T.: not tested.
3025, 2930, 2850, 1600, 1490, 1450, 1060, 740, 685 cmÀ1
;

M. Shiino et al. / Bioorg. Med. Chem. 9 (2001) 1233±1240
1237
Figure 3. Lineweaver±Burk plots of the mushroom tyrosinase reaction with (A) cupferron and (B) N-cyclopentyl-N-nitrosohydroxylamine: (a)
respect to l-dopa;(b) respect to oxygen.
1H NMR (CDCl3)d1.92 (2H, m), 2.68 ( 2H, t, J=7 Hz),
2.94 (2H, t, J=7 Hz), 6.12 (2H, s), 7.18 (5H, s). Anal.
calcd for C9H13NO: C, 71.49;H, 8.67;N, 9.26. Found:
C, 71.41;H, 8.60;N, 9.18.
of the supernatant, the precipitate was washed three
times sequentially with dried ether and n-hexane to give
the white crystal, which was dried with nitrogen gas.
Yield: 181 mg (0.92 mM, 92%);mp 86.8 ꢀC;IR (KBr):
3150 (broad), 3028, 2932, 1500, 1450, 1402, 1075, 937,
1
N-Nitroso-N-(3-phenylpropyl)hydroxylamine ammonium
salt (5). (3-Phenylpropyl)hydroxylamine (150 mg,
1 mM) dissolved in ether (8 mL) saturated ammonia at
0 ꢀC. After 15 min of stirring, the reaction mixture was
centrifuged at 500 g for 10 min at 4 ꢀC. After decantation
742, 698 cmÀ1; H NMR (DMSO-d6) d: 2.04 (2H, m),
2.61 (2H, t, J=7 Hz), 3.88 (2H, t, J=6 Hz), 6.92 (4H, s,
broad), 7.20 (5H, s);HRMS(FAB) calcd for C 9H11N2O2
(MÀNH4)À 179.0820, found 179.0835. Anal. calcd for
C9H15N3O2: C, 54.80;H, 7.67;N, 21.31. Found: C,

1238
M. Shiino et al. / Bioorg. Med. Chem. 9 (2001) 1233±1240
Figure 4. (A) The known potent inhibitors. (B) A possible planar structure of the tautomeric hydroxydiazenium N-oxide. (C) Binding models to the
binuclear copper active center in the oxy-form of tyrosinase. N-nitrosohydroxylamines (right) may also interact with the binuclear copper as shown
for catechol (left, ref 22).
54.31;H, 7.92;N, 20.85. Both Liebermann and Griess'
reactions for the nitroso group were positive. Compound 8
was obtained in a similar way from the appropriate amine.
containing a trace of methyl orange, NaBH3CN (1.5 g,
0.024 M) was added;and then 5 M HCl-MeOH was
added dropwise with stirring to maintain the red color
of the solution for 30 min. The reaction mixture was
stirred overnight, and methanol was removed in vacuo.
The residue was suspended in water and brought to pH
10 using 6 M NaOHaq solution and saturated NaHCO3
aq solution, and extracted with ethyl acetate. The
extract was dried over Na2SO4 and concentrated in
vacuo to dryness. Recrystallization of the crude product
from n-hexane gave 1.3 g of N-hexylhydroxylamine with
a yield of 29% from n-hexylaldehyde: mp 65.0±65.8 ꢀC;
N-Nitroso-N-(diphenylmethyl)hydroxylamine ammonium
salt (8). Overall yield 12.3%;mp 114.2±117.6 ꢀC;IR
(KBr): 3211 (broad), 1485, 1457, 1385, 1072, 934, 750,
;
698 cmÀ1 1H NMR (DMSO-d6) d 6.48 (1H, s), 6.65
(4H,broad), 7.12 (10H, s);HRMS(FAB) calcd for
C13H11N2O2 (MÀNH4)À 227.0821, found 227.0838;
Both Liebermann and Griess' reactions were positive.
1
The typical procedure for the preparation of N-nitroso-
hydroxylamines 6, 7, 12 and 13 (procedure B) is described
for 12.
IR (KBr): 3266, 3162, 2924, 2857, 1460, 1378 cmÀ1; H
NMR (DMSO-d6) d 0.83 (3H, t, J=4 Hz), 1.05±1.55
(8H, m), 2.88 (2H, t, J=6 Hz), 6.25 (2H, s);
HRMS(FAB) calcd for C6H16NO (M+H)+ 118.1232,
found 118.1253. Anal. calcd for C6H15NO: C, 61.49;H,
12.90;N, 11.95. Found: C, 61.78;H, 13.09;N, 11.88.
N-Hexylhydroxylamine. To a mixture of hexylaldehyde
(4.0 g, 0.04 M) in methanol (100 mL) and hydroxyl-
amine hydrochloride (4.1 g, 0.06 M) in water (10 mL), 6
M NaOH aq solution (18 mL) was added. After 2 h of
stirring, the mixture was concentrated;and the residue
was then extracted with ethyl acetate. The extract was
dried over Na2SO4 and evaporated to give crude oxime.
To a solution of the crude oxime in methanol (60 mL)
N-Nitroso-N-hexylhydroxylamine ammonium salt (12).
Nitrosation of N-hexylhydroxylamine was accom-
plished by the same method described above. Yield of
82%;IR (KBr): 3181(broad), 2930, 2860, 1460, 1066,
1
941 cmÀ1; H NMR (D2O)d 0.68 (3H, t, J=4 Hz), 1.08

M. Shiino et al. / Bioorg. Med. Chem. 9 (2001) 1233±1240
1239
(6H, m), 1.61 (2H, m), 3.78 (2H, t, J=4 Hz);
HRMS(FAB) calcd for C6H13N2O2 (MÀNH4)À
145.0977, found 145.0969. Anal. calcd for C6H17N3O2:
C, 44.15;H, 10.50;N, 25.75. Found: C, 43.36;H, 10.51;
N, 25.02. Both Liebermann and Griess' reactions were
positive. Compounds 6, 7 and 13 were obtained in a
similar way from the appropriate aldehyde or ketone.
reaction mixture (3.2 mL of ®nal volume) containing
0.54 mM l-dopa, 67 mM phosphate bu€er (pH 6.8), 80
units of mushroom tyrosinase, and the test sample was
incubated at 30 ꢀC for 2 min. The formation of dopa-
chrome was monitored spectrophotometrically by absorp-
tion at 475 nm with a Shimadzu UV-160A spectrophoto-
meter.
N-Nitroso-N-(ꢀ-metylbenzyl)hydroxylamine ammonium
salt (6). Overall yield 20.6%;mp 71.3±78.0 ꢀC;IR
Tyrosinase assay by oxygen monitor
(KBr): 3176 (broad), 1447, 1063, 931, 703 cmÀ1
;
1H
The enzyme activity was measured for oxygen con-
sumption by a modi®ed method.31 The reaction mixture
(0.92 mL of ®nal volume) containing 0.5 mM l-dopa,
67mM phosphate butter (pH 6.8), 80 units of mushroom
tyrosinase, and the test sample was incubated at 30 ꢀC
for 5 min. Before addition of the enzyme, the initial
oxygen concentration was controlled by passing nitrogen
into the reaction mixture. Oxygen consumption rates
were determined with a Central Oxygraph-9, a Clark-type
oxygen electrode.
NMR (D2O)d 1.65 (3H, d, J=7.0 Hz), 5.36 (1H, q,
J=7.0 Hz), 7.25 (5H, s);HRMS(FAB) calcd for
C8H9N2O2 (MÀNH4)À 165.0664, found 165.0659. Anal.
calcd for C8H13N3O2: C, 52.44;H, 7.15;N, 22.94.
Found: C, 51.93;H, 7.42;N, 22.40;Both Liebermann
and Griess' reactions were positive.
N-(ꢀ-Propylbenzyl)hydroxylamine. Mp 65.3±66.7 ꢀC;IR
1
(KBr): 3261, 3148, 1635, 1496, 1027, 752, 694 cmÀ1; H
NMR(CDCl3) d 0.83 (3H, t, J=5.0 Hz), 1.12 (2H, m),
1.65 (2H, m), 3.85 (1H, dd, J=8.0 Hz), 5.58 (2H, s), 7.21
(5H, s);HRMS(FAB) calcd for C 10H16NO (M+H)+
166.1232, found 166.1215. Anal. calcd for C10H15NO:
C, 72.69;H, 9.15;N, 8.48. Found: C, 72.88;H, 9.00;N,
8.62.
E€ect of pre-incubation of mushroom tyrosinase with
inhibitor
Pre-incubation mixtures consisted of 0.1 mL of the
sample solution (equivalent amount of IC50) and 0.1 mL
of the aqueous solution of the tyrosinase (80 units). The
mixture was pre-incubated at 0 ꢀC for 5 min. Then,
3.0 mL of 0.54 mM l-dopa was added, and the reaction
monitored at 475 nm for 15 min.
N-Nitroso-N-(ꢀ-propylbenzyl)hydroxylamine ammonium
salt (7). Overall yield 47.1%;mp 103.9±108.9 ꢀC;IR
(KBr): 3150 (broad), 1456, 1061, 943, 748, 700 cmÀ1; 1H
NMR (D2O) d 0.98 (3H, t, J=5.0 Hz), 1.35 (2H, m),
2.10 (2H, m), 5.35 (1H, m), 7.40 (5H, s). HRMS(FAB)
calcd for C10H13N2O2 (MÀNH4)À 193.0977, found
193.0977;Both Liebermann and Griess' reactions were
positive.
Acknowledgements
The authors wish to thank Dr. H. Iinuma, Institute of
Microbial Chemistry, for the gift of dopasitin and valuable
suggestions.
N-Dodecylhydroxylamine. Mp 88.6±90.7 ꢀC;IR (KBr):
;
3263, 3156, 2918, 2851, 1462, 1378±1061 cmÀ1 1H
NMR (CDCl3)d 0.90 (3H, t, J=5.0 Hz), 1.28 (20H, s),
2.94 (2H, t, J=5.0 Hz), 5.10 (2H, broad);HRMS(FAB)
calcd for C12H28NO (M+H)+ 202.2171, found
202.2165. Anal. calcd for C12H27NO: C, 71.58;H, 13.52;
N, 6.96. Found: C, 71.61;H, 13.82;N, 6.95.
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N-Nitroso-N-dodecylhydroxylamine ammonium salt (13).
Overall yield 31.8%;mp 62.3±64.2 ꢀC;IR (KBr): 3157
(broad), 2920, 2850, 1462, 949 cmÀ1; 1H NMR (DMSO-
d6) d 0.63 (3H, s), 1.02 (20H, s), 2.96 (4H, s), 3.70 (2H, t,
J=7.0 Hz). HRMS(FAB) calcd for C12H25N2O2
(MÀNH4)À 229.1916, found 229.1922;Both Lie-
bermann and Griess' reactions were positive.
The other N-substituted N-nitrosohydroxylamines were
synthesized in the same way. Compound 427 and 1428
were obtained by the procedure A with total yield of
12.8 and 9.9%, respectively. Compound 9,29 1028 and
1130 were obtained by the procedure B with total yield
of 26.3%, 21.9% and 25.1%, respectively.
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