Solvents inducing oxidation of hydroxylamines

Article abstract of DOI:10.1248/cpb.50.996

Hydroxylamines gradually undergo oxidation to their oximes on being dissolved in organic solvent (e.g. methanol). This phenomenon was followed by ¹H-NMR and liquid chromatography-mass spectrometry (LC-MS). The oxidation rate was estimated from

Full text of DOI:10.1248/cpb.50.996

996
Notes
Chem. Pharm. Bull. 50(7) 996—1000 (2002)
Vol. 50, No. 7
Solvents Inducing Oxidation of Hydroxylamines
Shizuyo HORIYAMA, ,a Kiyoko SUWA,a Masae YAMAKI,a Hiromi KATAOKA,a Toyoshi KATAGI,a
*
c
Mitsuo TAKAYAMA,b and Takae TAKEUCHI
a Faculty of Pharmaceutical Sciences, Mukogawa Women’s University; 11–68 Koshien Kyuban-cho, Nishinomiya
663–8179, Japan: b Graduate School of Sciences, Yokohama City University; 22–2 Seto, Kanazawa-ku, Yokohama
236–0027, Japan: and c Department of Chemistry, Faculty of Science, Nara Women’s University; Kitauoyanishi-machi,
Nara 630–8506, Japan. Received February 18, 2002; accepted April 2, 2002
Hydroxylamines gradually undergo oxidation to their oximes on being dissolved in organic solvent (e.g.
1
methanol). This phenomenon was followed by H-NMR and liquid chromatography-mass spectrometry (LC-
MS). The oxidation rate was estimated from the peak area observed on the mass chromatogram at the proto-
nated molecule or fragment ion on LC-atmospheric pressure chemical ionization (APCI)-MS. The results
showed that the oxidation rate of hydroxylamines depended on the solvent type.
Key words hydroxylamine; oxidation; LC-MS; NMR
Oxime derivatives are well known as having antiallergic, samples, 4Ј-piperidinoacetophenone oxime (2), 4Ј-morpholi-
anti-inflammatory, antipyretic, fungicidal and parasiticidal noacetophenone oxime (4) and 4Ј-methoxybenzyl methyl ke-
activities.1,2) Recently, Kramer et al. reported that some tone oxime (6).
oximes and hydroxylamines, which were prepared as a wide
range of di-tert-butylphenol containing compounds, were in- Results and Discussion
hibitors of cyclooxygenase (COX) and 5-lipoxygenase (5-
All compounds 1—6 (Fig. 1) involved in this study were
LO).3) We also have synthesized O-acyl oximes, free oximes prepared from the corresponding methylketones in the usual
and hydroxylamines for the purpose of developing a series of manner,11,12) and carefully purified by crystallization. Their
1
nonsteroidal anti-inflammatory drugs having dual inhibitors purities were checked by H-NMR spectrum and LC-MS
of COX4) and 5-LO, and reported their anti-inflammatory ac- chromatogram. Table 1 shows the H-NMR data of com-
1
tivity. We then have found that some O-acyl oximes, depend- pound 1—6 as the standard. Table 2 shows the retention
ing on the measuring conditions of their mass spectra, times of each compound on the liquid chromatogram, and
showed thermal degradation occurring at characteristic sites5) their protonated molecule and characteristic fragment ions in
and reductive degradation by a matrix containing a thiol the mass spectra of each peak on LC-atmospheric pressure
group.6) In the present work,we examined the properties of chemical ionization (APCI)-MS spectra.
hydroxylamines and found that they were unstable in some
organic solvents.
Hydroxylamines 1, 3 and 5 were dissolved in methanol
and chloroform for LC-MS and in methanol-d4 and chloro-
Hydroxylamines have amphetamine-like activity, inhibit form-d for NMR, which were chosen as representative of
monoamine oxidase and other activities (e.g. spontaneous protic and aprotic solvents, respectively. The change on
motor activity and blood pressure effects),7) and also are im- standing was followed by measuring H-NMR and LC-MS
1
1
portant metabolic products of N-oxidation of aliphatic pri- spectra. After 24 h, new signals appeared in the H-NMR
mary and secondary amines, for example, amphetamine and spectra of each methanolic solution, and their intensity grad-
phentamine. In the 1970s, several studies were devoted to ex- ually increased with the elapse of time. In the case of 1, new
amining the instability of hydroxylamines. Beckett et al.8,9) signals appeared at d 2.18 (s), 3.20 (m) and 7.50 (d), corre-
reported that N-hydroxyamphetamine (2-hydroxylamino-1- sponding to 2-CH3, 2Љ,6Љ-CH2 and aromatic protons (2Ј,6Ј-H)
phenylpropane) was readily oxidized to the corresponding of oxime 2, as shown in Fig. 2 (Asterisks indicate new sig-
oxime in nonacidic buffered or non-buffered aqueous solu- nals). In the case of 3, signals appeared at d 2.19 (s), at d
tions by aerial oxygen, with the oxidation being dependent 3.17 (m) and 7.54 (d), matching with those for 2-CH3, 2Љ,6Љ-
on pH and time as well as being enhanced by the presence of CH2 and aromatic protons (2Ј,6Ј-H) of oxime 4. In the case
trace heavy metal ions. They suggested that extraction with of 5, the signals agreed with 3-CH3 and 1-CH2 of oxime 6
organic solvents such as ether, benzene or chloroform is ef-
fective for protecting hydoxylamines from the aqueous envi-
ronment. Lindeke and Anderson.10) reported that the oxida-
tion takes place in aerated toluene, in which, N-hydroxyam-
phetamine gave only the nitrosodimer.
In this paper, we report that some hydroxylamines were
gradually oxidized to the corresponding oximes in some or-
ganic solvents (e.g. methanol) with no aeration. The change
occurring when they were left standing was followed by mea-
suring 1H-NMR and liquid chromatography-mass spectrome-
try (LC-MS) spectra of the mixture of hydroxylamine and the
product. The oxidized products were identified by comparing
1
their H-NMR and LC-MS spectra with those of authentic
Fig. 1. Structures of Compounds Examined in This Study
© 2002 Pharmaceutical Society of Japan
To whom correspondence should be addressed. e-mail: horiyama@mwu.mukogawa-u.ac.jp

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Table 1. 1H-NMR Spectral Data of Hydroxylamines 1, 3 and 5, and Oximes 2, 4 and 6
H
1
2
3
4
H
5
6
1
2
3.96 q (6.41)
1.33 d (6.41)
7.23 d (8.55)
6.94 d (8.55)
3.10 m
1.58 m
1.71 m
N.D.
N.D.
—
2.18 s
3.97 q (6.59)
1.33 d (6.59)
7.24 d (8.42)
6.93 d (8.79)
3.11 m
—
3.82 m
N.D.
N.D.
—
2.19 s
1
2.45 dd (13.55, 7.69)
2.85 dd (13.55, 5.86)
3.07 m
1.00 d (6.22)
7.11 d (8.42)
6.84 d (8.79)
3.76 s
3.39 s
2Ј,6Ј
3Ј,5Ј
2Љ,6Љ
4Љ
3Љ,5Љ
OH
NH
7.50 d (8.98)
6.93 d (8.98)
3.20 m
1.61 m
1.70 m
N.D.
7.54 d (8.98)
6.94 d (8.98)
3.17 m
—
3.82 m
N.D.
2
3
—
1.72 s
7.12 d (8.55)
6.85 d (8.55)
3.77 s
2Ј,6Ј
3Ј,5Ј
OCH3
OH
NH
N.D.
N.D.
N.D.
—
—
—
* Coupling constants (J in Hz) are given in parentheses. N.D.; not detected.
Table 2. Retention Time (tR), [MϩH]ϩ and Fragment Ion of Hydroxylamines 1, 3 and 5, Oximes 2, 4 and 6 on LC-MS
tR (min)
[MϩH]ϩ
[MϪ17]ϩ
[MϪ15]ϩ
[MϪ33]ϩ
1
2
3
4
5
6
5.4
6.3
4.2
4.8
5.9
5.5
221 (38)
219 (100)
223 (4)
221 (100)
182 (100)
180 (100)
203 (100)
—
205 (100)
—
164 (11)
—
—
203 (11)
—
205 (26)
—
188 (88)
—
190 (92)
—
149 (9)
—
164 (4)
* % Intensity is given in parentheses.
Fig. 2. 1H-NMR Spectra of 1 at (a) Soon After and (b) After 24 h Dissolution in Methanol-d4
the presence of syn-isomer from [MϩH]ϩ m/z 180 in its
mass spectrum, agreeing with the result observed for the 1H-
NMR spectrum. These findings, confirmed that hydroxyl-
amines 1, 3 and 5 gradually changed to oxime 2, 4 and 6 only
on being dissolved in methanol; no change was seen in chlo-
roform solution.
Next, we tried to calculate the oxidation rates from mass
chromatograms, using the peak area of [MϩH]ϩ m/z 221 for
1, while fragment ion m/z 190 was used for 3 and m/z 149 for
5 because the retention time of each oxidizing compound
was very close to the original peak. The peak area of
being observed at d 1.72 (s), 3.38 (s). Also, slightly smaller
signals appeared at d 1.71 (s, 3-CH3) and 3.65 (s, 1-CH2),
1
suggesting the presence of a syn-isomer.13) In the H-NMR
spectra of each chloroform-d solution, no changes were ob-
served.
LC-MS spectra of each methanolic solution showed a new
peak after 24 h, with the peak area increaseing with time. The
retention time of the new peak was 6.3 min for 1, 4.8 for 3
and 5.5 min for 5, which agreed with the retention time of the
corresponding oximes, respectively. In spectra of 5, a small
peak appeared at 5.3 min, which seemed to be dependent on

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Vol. 50, No. 7
Fig. 3. LC-APCI-MS Chromatograms (a, c, e) and Spectra (b, d, f) of 1 and 2 in Methanol
(a), (b): Immediately after dissolution of 1. (c), (d): 24 h after dissolution of 1. (e), (f): 2.
Fig. 4. Calibration Curve of (a) 1 (m/z 221), (b) 3 (m/z 190) and (c) 5 (m/z 149)

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Fig. 5. First-order Plots for the Oxidation of (a) 1, (b) 3 and (c) 5 in Methanol
Fig. 6. First-order Plots for the Oxidation of (a) 1, (b) 3 and (c) 5 in Chloroform
[MϩH]ϩ may be affected by the isotope peak of [MϩH]ϩ vent.
from oxidizing compounds. The calibration graphs (Fig. 4)
We also examined the stability of hydroxylamine 1 in vari-
for 1, 3 and 5 were generated from the peak areas at m/z 221, ous solvents, ethanol, pyridine, methylamine, benzene and
m/z 190 and m/z 149 of increasing amounts of standard sam- toluene, and calculated their k-values, which were 0.009,
ple of 1, 3 and 5 and the external standard was 2.5 mM 0.017, 0.401, 0.002 and 0.003, respectively. These results
methanol solution of 2. A linear calibration curve was con- showed that the oxidation proceeded very rapidly in methyl-
structed using the least-squares method of quantities versus amine, slowly in the ethanol and pyridine, and almost not at
peak area. The linearity was good in the range of 0.26 to all in benzene and toluene. It seems that the oxidation pro-
6.59 mM (r2ϭ0.9969) for 1, of 0.02 to 2.35 mM (r2ϭ0.9985) ceeded in protic solvents more than in aprotic solvents, but
for 3 and 0.13 to 10.07 mM (r2ϭ0.9981) for 5. Using the re- the data did not indicate a dependence on the basicity (pKa)14)
maining percentages of these peak areas, we determined the of the solvents, which was 15.5 for methanol, 16.0 for
pseudo first-order rate constants (Ϫk) for the oxidation of hy- ethanol, 5.1 for pyridine and 10.6 for methylamine.
droxylamines. Figures 5 and 6 show pseudo first-order plots
Beckett and Lindeke et al. reported that aerial oxygen,
for the oxidation of 1, 3 and 5 in methanol and chloroform, heavy metal and pH cause the oxidation of hydroxylamine,
with the percentage of log(At/A0) being plotted against time but in our case, the oxidation occurred only on dissolution in
(h). k-Values (Ϫh) of 1, 3 and 5 in methanol were 0.018, solvent. We confirmed the oxidation phenomena of hydroxy-
0.013 and 0.018 and in chloroform were 3ϫ10Ϫ5, 2ϫ10Ϫ4 lamines 1, 3 and 5 to oxime 2, 4 and 6 in methanol solution
1
and 3ϫ10Ϫ4, respectively. These results indicated that the ox- by using H-NMR and LC-APCI-MS techniques. Further
idation phenomenon of hydroxylamines depended on the sol- study is needed to clarify the oxidation mechanism.

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Vol. 50, No. 7
1
7.73. Found: C, 66.18; H, 8.63; N, 7.77. APCI-MS: m/z 182 [MϩH]ϩ. H-
Experimental
NMR (methanol-d4): Table 1.
General All melting points were measured with a Yanaco MP-S3 appa-
ratus and are uncorrected. Nuclear magnetic resonance spectra were ob-
tained at 500 MHz by means of a JEOL GSX-500 instrument (JEOL, Tokyo,
Japan). Solutions were prepared in chloroform-d or methanol-d4 and tetra-
methylsilane (TMS) serves as the internal reference. Mass spectrometric ex-
periments were performed using a TSQ-7000 triple stage quadruple mass
spectrometer (Thermo Quest, San Jose, CA, U.S.A.) operating with APCI,
connected with an HP series 1050 HPCL (Hewlett-Packard, Palo Alto, CA,
U.S.A.). Chromatography was conducted at ambient temperature on an Ex-
celpack SIL-C18/5 reverse-phase C18 column (5 mm, 5ϫ150 mm) (Yoko-
gawa, Tokyo, Japan) and Capcell Pack C18 reverse-phase C18 column (5 mm,
4.6ϫ150 mm) (Shiseido, Tokyo, Japan) operated at flow-rate of 0.6 ml/min
with eluent CH3OH–H2O (80 : 20) for 1 and CH3OH–H2O (70 : 30) for 3 and
5. The mass spectrometer was operated in the positive ion mode with the va-
porizer at 500 °C and the capillary at 175 °C. Nitrogen was used as the
sheath gas (70 psi) to assist neblization. The electron multiplier was set at
1000 V.
References
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Calibration 1, 3 and 5 were dissolved in chloroform to 10 mM (stock
solution) and further calibration levels were prepared by diluting the stock
solution.
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Sample Preparation for LC-MS 1, 3 and 5 dissolved in He-degassed
methanol, chloroform, ethanol, pyridine, methylamine, benzene and toluene
to 1 mM, and 3 ml was injected at set intervals of about 24 h or more.
Synthesis Hydroxylamines 1,6) 3 and 5 were prepared from 2, 4 and 6
by modification of Borch’s11) method using NaBH3CN. Oximes 2, 4 and 6
were prepared from the corresponding methylketone with hydroxylamine in
the usual way.12)
1-Hydroxylamino-1-(4Ј-piperidinophenyl) Ethane (1)6): 1H-NMR
(methanol-d4): Table 1.
12) Buck J. S., Ide W. S., “Organic Syntheses,” Coll. Vol. II, ed. by Blatt
A. H., John Wiley and Sons, Inc., New York, 1943, p. 622.
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Suec., 10, 493—506 (1973).
1-Hydroxylamino-1-(4Ј-morpholinophenyl) Ethane (2): mp 111.5—
113 °C (from ethyl acetate). Anal. Calcd for C12H18N2O2: C, 64.84; H, 8.16;
N, 12.60. Found: C, 64.69; H, 8.41; N, 12.59. APCI-MS: m/z 223 [MϩH]ϩ.
1H-NMR (methanol-d4): Table 1.
14) McMurry J., “Organic Chemistry,” Brooks/Cole, U.S.A., 1999, p. 660
and p. 985.
2-Hydroxylamino-1-(4Ј-mothoxyphenyl) Propane (3): mp 65—67 °C
(from petroleum ether). Anal. Calcd for C10H15NO2: C, 66.27; H, 8.34; N,