M. Miura et al. / Bioorg. Med. Chem. 19 (2011) 5693–5697
5697
H2O2 (34 mL, 100 mmol). NO gas (250 mL, 100 mmol) was intro-
duced to the reaction for 1 h under nitrogen atmosphere, and incu-
bated for 2 h at 37 °C. The reaction mixture was extracted twice
with hexane 100 mL, twice with dichloromethane 100 mL, twice
with ethyl acetate 100 mL, and twice with ethyl acetate 100 mL
with salting out. Each organic phase was dried over Na2SO4, fil-
tered, and evaporated in vacuo to produce a yellow oil. The CH2Cl2
extract showed the highest mutagenicity among the organic ex-
tracts. The crude product was purified by column chromatography
twice (silica gel 60, hexane:CH2Cl2/Et2O = 2:2:1, UV 254 nm), and
temperature. After 12 h, the reaction mixture was refluxed and
dimethyl sulfoxide 1 mL was added to remove the excess MCPBA.
The reaction mixture was washed twice with water 10 mL and
brine 5 mL, dried over Na2SO4, filtered, and evaporated in vacuo
to obtain a residue. The crude product was purified by column
chromatography (Silica Gel 60, hexane/CH2Cl2/Et2O = 2:2:1, UV
254 nm, diphenylamine, anisaldehyde/sulphuric acid), by a pre-
parative HPLC (Lichrosorb Si 60, hexane: CH2Cl2/EtOH = 8:1:0.4,
UV 254 nm), and by a preparative TLC (silica gel, hexane/CH2Cl2/
Et2O = 4:4:1, UV 254 nm) to produce a colorless oil (yield 1%). 1H
NMR (CDCl3, 500 MHz): d 1.06 (3H, t, J = 7.4 Hz, –CH2CH3), 2.50
(1H, dq, J = 14.9, 7.4 Hz, –CH2CH3), 2.57 (1H, m, –CH2CH3), 2.60–
2.68 (1H, m, H-4), 2.92–2.98 (1H, m, H-4), 4.21–4.35 (2H, m, H-3).
by preparative HPLC (Lichrosorb Si60 7.5 ꢁ 250 mm 10
lm, hex-
ane: CH2Cl2/EtOH = 8:1:0.2, 2.0 mL/min, UV 254 nm) to produce a
colorless oil. The oil was crystallized by cooling, followed by wash-
ing with CCl4 (mp 37.5–38.5 °C).
Acknowledgment
4.7. X-ray data for the direct-acting mutagen
This work was supported in part by a Grant-in-Aid for the Min-
istry of Education, Culture, Sports, Sciences and Technology of
Japan.
A
colorless prismatic crystal with dimensions of
0.48 ꢁ 0.20 ꢁ 0.07 mm was obtained by cooling, followed by wash-
ing with CCl4. Diffraction data were collected on a crystal of the
direct-acting mutagen at 173 K on a Bruker/AXS Smart 1000
References and notes
diffractometer with graphite monochromated Mo-K
a
radiation
0
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0
0
a = 6.5597(4) ÅA, b = 9.9753(6) ÅA, c = 10.9713(8) ÅA. The structure
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expanded using Fourier techniques. The non-hydrogen atoms were
refined anistropically by full matrix least-squares calculations
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CCDC-821046 (the direct-acting mutagen) contains the
supplementary crystallographic data for this paper. These data
Center, 12 Union Road Cambridge CB2 1EZ, UK; fax: +44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk).
4.8. Synthesis of 3-ethyl-3-nitro-1-pyrazoline
Diazomethane was generated from N-methyl-N-nitroso-p-
toluenesulfonamide (8.45 g, 0.039 mol) with KOH.39 2-Nitrobutene
(2.03 g) in diethyl ether 10 mL was slowly added to a solution of
diazomethane in diethyl ether at ꢂ78 °C, and the mixture was stir-
red. After 30 min, the reaction was allowed to warm to room tem-
perature. After 3 h, the yellow color disappeared. The reaction
mixture was washed twice with water 50 mL and brine 20 mL.
The ether phase was dried over Na2SO4, filtered, and evaporated
in vacuo to produce a yellow oil. The oil was purified chromato-
graphically [Silica Gel 60, hexane/CH2Cl2/Et2O = 4:2:1, UV 254 nm
or diphenylamine] to obtain 3-ethyl-3-nitro-1-pyrazoline (yield
42%). UV (nm, CH3CN) k = 200 (
e = 4930), 249 (e = 440), 325
(e
= 75). 1H NMR (CDCl3, 500 MHz): d 1.00 (3H, t, J = 7.4 Hz,
–CH2CH3), 1.86 (1H, ddd, J = 14.5, 8.6, 6.0 Hz, H-4), 2.36 (1H, dq,
J = 14.6, 7.4 Hz, –CH2CH3), 2.39 (1H, ddd, J = 14.3, 8.9, 5.4 Hz,H-4),
2.58 (1H, dq, J = 14.6, 7.4 Hz, –CH2CH3), 4.72–4.88 (2H, m, H-5).
13C NMR (CDCl3, 100 MHz): 7.82 (–CH2CH3), 26.0 (–CH2CH3), 28.7
(C-4), 78.4 (C-5), 127.8 (C-3). IR (neat) cmꢂ1: 1544, 1352 (NO2).
Anal. Calcd for C5H9N3O2: C, 41.95; H, 6.34; N, 29.36; O, 22.35.
Found: C, 42.17; H, 6.30; N, 29.04; O, 22.49.
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4.9. Synthesis of 5-ethyl-5-nitro-1-pyrazoline 1-oxide
Small portions of MCPBA 860 mg were added to a solution of
3-ethyl-3-nitro-1-pyrazoline 333 mg in CH2Cl2 6 mL at room