Structural concept of nitroxide as a lipid peroxidation inhibitor

Article abstract of DOI:10.1021/jo200361p

Nitroxides have antioxidative activities toward lipid peroxidation, but the influence of steric factors is not known. We synthesized alkyl-substituted nitroxides at the α-position of the N-O moiety to enhance lipophilicity and the bulk effect. There was good correlation between the IC₅₀ and lipophilicity (log P_o/w) of nitroxides with use of the thiobarbituric acid-reactive substances (TBARS) assay. Furthermore, an inhibitory effect on the TBARS assay was dependent upon the number and length of alkyl groups, though nitroxides had almost identical lipophilicity.

Full text of DOI:10.1021/jo200361p

NOTE
pubs.acs.org/joc
Structural Concept of Nitroxide As a Lipid Peroxidation Inhibitor
Toshihide Yamasaki,†,4 Yuko Ito,†,4 Fumiya Mito,† Kana Kitagawa,† Yuta Matsuoka,†
Mayumi Yamato,‡ and Ken-ichi Yamada†,*
†Department of Bio-functional Science, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi Higashi-ku,
Fukuoka 812-8582, Japan
‡Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka 812-8582, Japan
S Supporting Information
b
ABSTRACT: Nitroxides have antioxidative activities toward lipid peroxi-
dation, but the influence of steric factors is not known. We synthesized alkyl-
substituted nitroxides at the R-position of the NꢀO moiety to enhance
lipophilicity and the bulk effect. There was good correlation between the IC50
and lipophilicity (log Po/w) of nitroxides with use of the thiobarbituric acid-
reactive substances (TBARS) assay. Furthermore, an inhibitory effect on the
TBARS assay was dependent upon the number and length of alkyl groups,
though nitroxides had almost identical lipophilicity.
ipid radicals and peroxyl radicals produced by highly reactive
oxygen species are involved in cell injury. The consecutively
with the rate of nitroxide reduction.16 Conversely, tetraethyl-
substituted nitroxide did not react with reducing agents, which
can be explained by the lower reduction potential. However, this
compound had an inhibitory effect on lipid peroxidation.17 These
results suggest that the feature of the NꢀO group surrounding
the 2,6 position of piperidine nitroxide can be regulated with the
reactivity of the lipid radical without undesirable reduction.
Furthermore, confirmation of this hypothesis would further expand
the application range of nitroxide as not only an antioxidant, but
also as an antifatigue and photoprotecting agent in rubber and
polymers18,19 (which is based on the mechanism of the radicalꢀ
radical coupling reaction). To find a good antioxidant, the
lipophilicity of a compound must be considered. This is because
lipophilicity is a good index for the permeability of the bloodꢀ
brain barrier and as a basic index for drug design.20 Here, the
reaction with lipid-derived radicals would provide stability to
alkyl-substituted piperidine nitroxide via van der Waal’s forces.
Hence, the aim of the present study was to clarify the effect of
lipophilicity and steric factors near the NꢀO moiety of piper-
idine nitroxides to lipid peroxidation to find good candidates for
antioxidants.
L
produced lipid peroxidation involved in this process is asso-
ciated with the initiation of disorders associated with oxi-
dative stress (e.g., cardiovascular diseases, cancer, chronic
inflammation).1 Moreover, lipid peroxide decomposes into
lipid radicals, peroxyl radicals, and alkoxyl radicals,2 and acts
as a substrate for chain reactions. Once formed, peroxyl radicals
can be rearranged via a cyclization reaction to malondialde-
hyde and 4-hydroxynonenal, which are the major aldehyde
products,3,4 followed by cytotoxic and genotoxic effects.5 The
products of lipid peroxidation also act as signaling molecules6,7
and subsequently undergo secondary reactions as substrates
of various enzymes. Therefore, to minimize and inhibit the
progression of oxidative stress, lipid-derived free radicals
are target molecules because they are the chain-reaction inter-
mediates of lipid peroxidation. Here, nitroxide (>NꢀO•: ni-
troxyl radical or aminoxyl radical) with an unpaired electron
has a direct reaction with a lipid-derived radical (R•) via
radicalꢀradical coupling to produce alkoxyamine derivates
(>NꢀOꢀR)8ꢀ10 as follows:
According to a previously described method,14,16 we used
chain ketone compounds and synthesized alkyl-substituted pi-
peridine nitroxides 2bꢀi (Table 1). However, the yield of the
product was <18%. In the case of cyclic ketones, we reported that
we obtained the 2,2,6,6-tetrasubstituted piperidin-4-one deriva-
tives (precursor compounds of nitroxides (1)) in ∼30% yield.14
The reason for this low yield was thought to be because the chain
>NꢀO• þ R• f >NꢀOꢀR
This process results in termination of the radical chain
reaction.11 This feature of nitroxide would give it the function
of an antioxidant and free-radical tracing agent.12,13 We pre-
viously reported the synthetic method in which the 2,6 position
of the piperidine nitroxide ring was substituted with cyclic ketone
derivatives.14 The synthesized compounds regulated the reactiv-
ity with free radicals and reducing agents.15 For instance, the
calculated Gibbs energy from the difference in redox potential
between ascorbic acid and nitroxide showed a linear relationship
Received: February 18, 2011
Published: April 18, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/jo200361p J. Org. Chem. 2011, 76, 4144–4148
|

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NOTE
Table 1. Synthesized Nitroxides (2bꢀi)a and Their Partition
Coefficients
compd 2
R1
R2
yield (1) log Po/w (2) IC50 (μM) (2)b
c
a
b
c
d
e
f
Me
Et
Me
Et
ꢀ
11
17
4
0.277
1.115
2.158
3.169
0.366
1.133
1.711
2.449
n.d.d
131.4 ( 19.2
86.6 ( 15.4
43.6 ( 4.3
20.2 ( 3.3
59.8 ( 6.5
45.2 ( 5.4
37.7 ( 3.4
11.1 ( 1.5
5.6 ( 0.1
Figure 1. Correlation of the values of IC50 and log Po/w of nitroxides
with use of the thiobarbituric acid-reactive substances assay: 9, 2aꢀd;
b, 2eꢀh.
n-Pr n-Pr
n-Bu n-Bu
Me
Me
Me
Me
Me
Et
8
better protecting effect than 2a, although these two nitroxides
had a similar value of log Po/w. These results suggested that the
protecting effect against lipid peroxidation was affected not only
by the steric effect, but also by the affinity of nitroxides with lipid
radicals.
n-Pr
18
12
15
5
g
h
i
n-Bu
n-C5H11
n-C8H17
a Conditions: (i) NH4Cl, Triton B, DMSO, 60°C; (ii) H2O2, Na2WO4
3
Tetraethyl-substituted piperidine nitroxide bound with lipid-
derived radicals did not decompose the nitroxide and lipid-
derived radical upon heating (100 °C), but the tetramethyl type
did. These observations were confirmed by LC/MS using the
linoleic acid/lipoxygenase system. The alkoxyamine adducts
(>NꢀOꢀR) of nitroxides 2bꢀi were supposed to be stable
because their ESR signals were recovered with only 10ꢀ30%
intensity by heating of the reaction mixture. The reactivity of
these nitroxides in a rat microsomal lipid peroxidation system
was found to be dependent upon the lipophilicity in the present
study. Here, other factors such as steric hindrance and the
stability of >NꢀOꢀR must also be considered. The mono-
alkyl-substituted nitroxides 2eꢀi tended to be more efficient in
inhibiting lipid peroxidation than di-alkyl-substituted nitroxides
2aꢀd. The stability of >NꢀOꢀR was supposed to be almost
identical, but di-alkyl-substituted nitroxides 2bꢀd had slightly
higher stability than mono-alkyl-substituted nitroxides. There-
fore, these alkyl substrate nitroxides are thought to be effective
inhibitors of lipid peroxidation via thermally stabilized alkoxya-
mine derivatives. Nitroxides are also used to identify the structure
of protein-based carbon-centered radicals combined with LC/
MS techniques.21 These results suggest that nitroxides control-
ling the reactivity of the target molecules would be useful not
only for antioxidant and photoprotecting agents, but also as
detection agents of protein- and lipid-derived radicals.
2H2O. b Values are means ( standard deviations (n = 3). c Nitroxide 2a
is commercially available. d n.d.: not determined. The ESR signal in the
water phase was not detected because of high lipophilicity.
ketone compound was not adequate for the aldol reaction
because of intermolecular condensation and hindrance of the
carbonyl group by the alkyl chain. So far, to introduce the alkyl
groups into the R-position of piperidine nitroxide, it has been
synthesized in a stepwise fashion. However, synthetic methods to
substitute the R-position of 1a by alkyl substituents in one step
are lacking. Here, we could readily obtain R-substituted piper-
idin-4-one derivatives substituted with alkyl groups.
The partition coefficients of nitroxides between n-octanol and
phosphate buffer (Po/w) were determined by electron spin reso-
nance (ESR) spectrometry (Table 1). The logarithm of Po/w (log
Po/w) was increased along with the length of the alkyl chain at the
2- or 2,6-position of the piperidine ring. The inhibitory effect on
lipid peroxidation was then measured by the thiobarbituric acid-
reactive substances (TBARS) assay. The half-maximal inhibitory
concentration (IC50) was calculated (i.e., the nitroxide concen-
tration at 50% inhibition of lipid peroxidation (Table 1)). IC50
values were dependent upon the structures of the substituent.
The nitroxides 2h and 2i, which had n-pentyl and n-octyl groups,
respectively, had the highest inhibitory effects.
The correlation of IC50 and log Po/w is shown in Figure 1. IC50
values decreased with an increase in the log Po/w. Therefore,
nitroxides which had a large value of log Po/w had a good
protecting effect upon lipid peroxidation. In addition, the IC50
values of nitroxides 2eꢀh (which had three methyl and one alkyl
groups at the 2,6-position) were relatively smaller than those of
nitroxides 2aꢀd (which had two methyl and two alkyl groups at
the same position). These results suggested that the bulky alkyl
substituent that approximated to the NꢀO moiety induced
resistance against lipid radicals. With respect to nitroxides 2b
and 2f, although these compounds had the same number of
carbon atoms and similar lipophilicity, the IC50 value of nitroxide
2f was lower than that of 2b. This indicated that the protecting
effect was involved with the steric hindrance surrounding the
NꢀO moiety. Conversely, nitroxide 2e (which substituted the
one methyl group of nitroxide 2a with an ethyl group) had a
’ EXPERIMENTAL SECTION
Reagents and solvents from commercial suppliers were used without
further purification. Silica gel (100ꢀ200 mesh) was used for column
chromatography. Compound 2b was synthesized as previously des-
cribed.16 TLC was detected with iodine vapor.
2,2-Diethyl-6,6-dimethylpiperidin-4-one (1b). NH4Cl (5.35 g,
100 mmol) and Triton B (4 mmol) were added portion-wise to
a stirred solution of 1,2,2,6,6-pentamethylpiperidin-4-one (3.38 g,
20 mmol) and 3-pentanone (5.17 g, 60 mmol) in DMSO (32 mL) at
room temperature. The mixture was then heated for 4 h at 50 °C. It was
diluted with H2O, acidified with 7% aq HCl, and extracted with ether
(3ꢁ) to remove the neutral fraction. The reaction mixture was adjusted
to pH 9 with use of K2CO3 and then extracted with AcOEt (3ꢁ). The
AcOEt extract was dried over anhydrous sodium sulfate and concen-
trated in vacuo. The residue was separated by column chromatography
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NOTE
with hexane/AcOEt (80:20) to afford 1b (413 mg, 11%). The analytical
data were in agreement with a previous study.16
2,2-Diethyl-6,6-dimethylpiperidin-4-one-1-oxyl (2b). Com-
pound 2b was synthesized from compound 1b according to a previously
described method.16
concentrated in vacuo. The residue was separated by column chroma-
tography with hexane/AcOEt (8:2) to afford 2-ethyl-2,6,6-trimethylpi-
peridin-4-one (1e) (400 mg, 8%) as an orange oil: MS (FABþ) 170.2
1
(Mþ þ 1); νmax/cmꢀ1 1704 (CdO); H NMR (400 MHz; CDCl3)
δ (ppm) 0.912 (3H, t, J = 7.6 Hz), 1.157 (3H, s), 1.233 (3H, s), 1.237 (3H,
s), 1.40ꢀ1.59 (2H, m), 2.229 (1H, s), 2.247 (1H, s), 2.268 (1H, s), 2.278
(1H, s); 13C NMR (125 MHz; CDCl3) δ (ppm) 8.1, 28.4, 31.7, 32.4, 36.8,
52.3, 54.3, 54.9, 57.4, 211.0; HRMS (ESIþ) calcd for C10H20NO [M þ
H]þ 170.1545, found 170.1562. Anal. Calcd for C10H19NO: C, 70.96; H,
11.31; N, 8.28. Found: C, 70.85; H, 11.11; N, 8.14.
2-Ethyl-2,6,6-trimethylpiperidin-4-one-1-oxyl (2e). Com-
pound 1e (400 mg, 2.36 mmol) was oxidized according to the method
for compound 2c. The crude product was separated by column
chromatography with CHCl3/MeOH to afford compound 2e (173.2
mg, 40%) as an orange oil: MS (FABþ) 184.2 (Mþ); νmax/cmꢀ1 1719
(CdO); HRMS (ESIþ) calcd for C10H18NNaO2 [M þ Na]þ 207.1235,
found 207.1247; AN = 1.513 mT. Anal. Calcd for C10H18NO2: C, 65.19;
H, 9.85; N, 7.60. Found: C, 64.97; H, 9.61; N, 7.42.
2,2,6-Trimethyl-6-propylpiperidin-4-one (1f). 2-Pentanone
(5.17 g, 60 mmol) was used in place of 3-pentanone according to the
method for compound 1b. The product was separated by column
chromatography with hexane/AcOEt (8:2) to afford 1f (700 mg,
18%) as a pale-yellow oil: MS (FABþ) 184.2 (Mþ þ 1); νmax/cmꢀ1
1703 (CdO); 1H NMR (400 MHz; CDCl3) δ (ppm) 0.910 (3H, t, J =
6.8 Hz), 1.170 (3H, s), 1.229 (6H, br s), 1.29ꢀ1.52 (4H, m), 2.231 (1H,
s), 2.256 (1H, s), 2.266 (1H, s), 2.273 (1H, s); 13C NMR (125 MHz;
CDCl3) δ (ppm) 14.4, 17.0, 28.9, 31.8, 32.4, 46.9, 52.8, 54.3, 54.9, 57.4,
211.0; HRMS (ESIþ) calcd for C11H22NO [M þ H]þ 184.1701, found
184.1696. Anal. Calcd for C11H21NO: C, 72.08; H, 11.55; N, 7.64.
Found: C, 71.73; H, 11.32; N, 7.58.
2,2,6-Trimethyl-6-propylpiperidin-4-one-1-oxyl (2f). Com-
pound 1f (500 mg, 2.53 mmol) was oxidized according to the method
for compound 2c. The crude product was separated by column
chromatography with hexane/AcOEt (98:2) to afford compound 2f
(253 mg, 50%) as an orange oil: MS (FABþ) 198.2 (Mþ); νmax/cmꢀ1
1720 (CdO); HRMS (ESIþ) calcd for C11H20NNaO2 [M þ Na]þ
221.1392, found 221.1409; AN = 1.589 mT. Anal. Calcd for C11H20NO2:
C, 66.63; H, 10.17; N, 7.06. Found: C, 66.30; H, 9.88; N, 6.96.
2-Butyl-2,6,6-trimethylpiperidin-4-one (1g). 2-Hexanone
(3.00 g, 30 mmol) was used in place of 4-heptanone according to the
method for compound 1c. The product was separated by column
chromatography with hexane/AcOEt (9:1) to afford 1g (239 mg, 12%)
as a pale-yellow oil: MS (FABþ) 198.3 (Mþ þ 1); νmax/cmꢀ1 1703
(CdO); 1H NMR (400 MHz; CDCl3) δ (ppm) 0.909 (3H, t, J = 6.4 Hz),
1.170 (3H, s), 1.231 (6H, br s), 1.24ꢀ1.54 (6H, m), 2.230 (1H, s), 2.255
(1H, s), 2.265 (1H, s), 2.274 (1H, s); 13C NMR (125 MHz; CDCl3) δ
(ppm) 14.0, 23.0, 26.0, 29.0, 31.8, 32.5, 44.3, 52.8, 54.3, 54.9, 57.4, 211.0;
HRMS (ESIþ) calcd for C12H24NO [M þ H]þ 198.1858, found
198.1845. Anal. Calcd for C12H23NO: C, 73.04; H, 11.75; N, 7.10. Found:
C, 72.83; H, 11.63; N, 7.02.
2,2-Dimethyl-6,6-dipropylpiperidin-4-one (1c). NH4Cl
(2.68 g, 50 mmol) and Triton B (40 wt % soln in MeOH, 2 mL) were
added portion-wise to a stirred solution of 1,2,2,6,6-pentamethylpiper-
idin-4-one (1.69 g, 10 mmol) and 4-heptanone (3.43 g, 30 mmol) in
DMSO (16 mL) at room temperature. The mixture was then heated for
4.5 h at 50 °C. It was diluted with H2O, acidified with 7% aq HCl, and
extracted with ether (3ꢁ) to remove the neutral fraction. The reaction
mixture was adjusted to pH 12 with K2CO3 and then extracted with
AcOEt (3ꢁ). The AcOEt extract was dried over anhydrous sodium
sulfate, and concentrated in vacuo. The residue was separated by column
chromatography with hexane/AcOEt to afford 2,2-dimethyl-6,6-dipro-
pylpiperidin-4-one (1c) (368 mg, 17%) as a pale-yellow needle after
recrystallization: mp 53.4ꢀ55.0 °C (hexane); MS (FABþ):212.2
1
(Mþ þ 1); νmax/cmꢀ1 1690 (CdO); H NMR (400 MHz; CDCl3)
δ (ppm) 0.886 (6H, t, J = 6.8 Hz), 1.218 (6H, s), 1.23ꢀ1.49 (8H, m),
2.235 (2H, s), 2.264 (2H, s); 13C NMR (125 MHz; CDCl3) δ (ppm)
14.4, 16.8, 32.2, 42.9, 51.3, 54.4, 54.5, 59.6, 211.3; HRMS (ESIþ) calcd
for C13H26NO [M þ H]þ 212.2014, found 212.2030. Anal. Calcd for
C13H25NO: C, 73.88; H, 11.92; N, 6.63. Found: C, 73.74; H, 11.88;
N, 6.67.
2,2-Dimethyl-6,6-dipropylpiperidin-4-one-1-oxyl (2c). Com-
pound 1c (59 mg, 0.28 mmol) and Na2WO4 2H2O (50 mg, 0.15 mmol)
3
were added in methanol (5 mL); H2O2 (30%, 1 mL) was slowly added to
the solution. The mixture was stirred at room temperature, and H2O2
added (monitoring by TLC). After stirring, the solution was saturated with
K2CO3 and extracted with chloroform. The chloroform extracts were dried
over anhydrous sodium sulfate and evaporated. The residue was separated
by column chromatography with hexane/AcOEt to afford compound 2c
(38 mg, 60%) as an orange oil: MS (FABþ) 226.3 (Mþ); νmax/cmꢀ1 1720
(CdO); AN = 1.568 mT. Anal. Calcd for C13H24NO2: C, 68.99; H, 10.69;
N, 6.19. Found: C, 68.95; H, 10.76; N, 6.28.
2,2-Dibutyl-6,6-dimethylpiperidin-4-one (1d). 5-Nonanone
(4.27 g, 30 mmol) was used in place of 4-heptanone according to the
method for compound 1c. The mixture was stirred for 4 h at 60 °C. The
product was separated by column chromatography with hexane/AcOEt
(85:15) to afford compound 1d (96 mg, 4%) as a pale-yellow oil: MS
1
(FABþ) 240.3 (Mþ þ 1); νmax/cmꢀ1 1705 (CdO); H NMR (400
MHz; CDCl3) δ (ppm) 0.900 (6H, t, J = 6.8 Hz), 1.223 (6H, s), 1.15ꢀ
1.52 (12H, m), 2.239 (2H, s), 2.267 (2H, s); 13C NMR (125 MHz;
CDCl3) δ (ppm) 14.0, 23.0, 25.7, 32.2, 40.1, 51.4, 54.4, 54.6, 59.5, 211.3;
HRMS (ESIþ) calcd for C15H30NO [M þ H]þ 240.2327, found
240.2314. Anal. Calcd for C15H29NO: C, 75.26; H, 12.21; N, 5.85.
Found: C, 75.15; H, 12.11; N, 5.75.
2,2-Dibutyl-6,6-dimethylpiperidin-4-one-1-oxyl (2d). Com-
pound 1d (100 mg, 0.42 mmol) was oxidized according to the method
for compound 2c. The crude product was separated by column chroma-
tography with hexane/AcOEt to afford compound 2d (53 mg, 50%) as an
2-Butyl-2,6,6-trimethylpiperidin-4-one-1-oxyl (2g). Compound
1g (199 mg, 1.01 mmol) was oxidized according to the method for
compound 2c. The crude product was separated by column chromatography
with hexane/AcOEt (95:5) to afford compound 2g (68 mg, 32%) as an
orange oil: MS (FABþ) 212.3 (Mþ); νmax/cmꢀ1 1720 (CdO); AN = 1.598
mT. Anal. Calcd for C12H22NO2: C, 67.89; H, 10.44; N, 6.60. Found: C,
68.01; H, 10.44; N, 6.53.
orange oil: MS (FABþ) 254.3 (Mþ); νmax/cmꢀ1 1720 (CdO); AN
=
1.575 mT. Anal. Calcd for C15H28NO2: C, 70.82; H, 11.09; N, 5.51.
Found: C, 70.55; H, 11.05; N, 5.24.
2-Ethyl-2,6,6-trimethylpiperidin-4-one (1e). NH4Cl (8.02 g,
150 mmol) and Triton B (40 wt % soln in MeOH, 15 mL) were added
portion-wise to a stirred solution of 1,2,2,6,6-pentamethylpiperidin-4-
one (5.08 g, 30 mmol) and 2-butanone (6.48 g, 90 mmol) in DMSO
(25 mL) at room temperature. The mixture was then heated for 4.5 h at
50 °C. It was diluted with H2O, acidified with 7% aq HCl, and extracted
with ether (4ꢁ) to remove the neutral fraction. The reaction mixture
was adjusted to pH 12 with K2CO3 and then extracted with AcOEt
(7ꢁ). The AcOEt extract was dried over anhydrous sodium sulfate and
2,2,6-Trimethyl-6-pentylpiperidin-4-one (1h). 2-Heptanone
(3.43 g, 30 mmol) was used in place of 4-heptanone according to the
method for compound 1c. The product was separated by column
chromatography with hexane/AcOEt to afford 2,2,6-trimethyl-6-pen-
tylpiperidin-4-one (1h) (316 mg, 15%) as an orange oil: MS (FABþ)
1
212.3 (Mþ þ 1); νmax/cmꢀ1 1690 (CdO); H NMR (400 MHz;
CDCl3) δ (ppm) 0.888 (3H, t, J = 6.8 Hz), 1.170 (3H, s), 1.229 (3H, s),
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NOTE
1.232 (3H, s), 1.24ꢀ1.53 (8H, m), 2.230 (1H, s), 2.253 (1H, s), 2.266
(1H, s), 2.273 (1H, s); 13C NMR (125 MHz; CDCl3) δ (ppm) 14.0,
22.5, 23.4, 29.0, 31.8, 32.2, 32.4, 44.5, 52.8, 54.3, 54.9, 57.4, 211.0;
HRMS (ESIþ) calcd for C13H26NO [M þ H]þ 212.2014, found
212.2005. Anal. Calcd for C13H25NO: C, 73.88; H, 11.92; N, 6.63.
Found: C, 73.61; H, 11.70; N, 6.56.
2,2,6-Trimethyl-6-pentylpiperidin-4-one-1-oxyl (2h). Com-
pound 1h (316 mg, 1.5 mmol) was oxidized according to the method
for compound 2c. The crude product was separated by column chroma-
tography with hexane/AcOEt to afford compound 2h (226 mg, 70%) as an
orange oil: MS (FABþ) 226.3 (Mþ); νmax/cmꢀ1 1720 (CdO); HRMS
(ESIþ) calcd for C13H24NNaO2 [M þ Na]þ 249.1705, found 249.1720;
AN = 1.498 mT. Anal. Calcd for C13H24NO2: C, 68.99; H, 10.69; N, 6.19.
Found: C, 68.75; H, 10.47; N, 6.10.
d. Partition Coefficients between n-Octanol and Phos-
phate Buffer (PB). The nitroxide solution was prepared in 10 mM
phosphate buffer (PB) (pH 7.4; treated with Chelex100). A 50-μL
aliquot of the nitroxide solution was vigorously mixed with 500 μL of n-
octanol, and the mixture was centrifuged at 3000 rpm for 5 min. The
ESR spectra of n-octanol and PB were measured with an X-band ESR
spectrometer (JES-FA100; JEOL, Tokyo, Japan). The amount of
nitroxide was calculated from the double-integrated ESR signal intensity
by using Mn2þ as an external standard for correcting the sensitivities of
n-octanol and PB. The conditions for the ESR measurements were as
follows: power, 10 mW; frequency, 9.4 GHz; magnetic field, 337 mT;
modulation amplitude, one-third line width or less; and time con-
stant, 0.3 s.
2,2,6-Trimethyl-6-octylpiperidin-4-one (1i). 2-Decanone (4.69 g,
30 mmol) was used in place of 4-heptanone according to the method for
compound 1c. The mixture was heated for 4 h at 50 °C. The product was
separated by column chromatography with hexane/AcOEt to afford com-
pound 1i (127 mg, 5%) as a pale-yellow oil: MS (FABþ) 254.3 (Mþ þ 1);
νmax/cmꢀ1 1708 (CdO); 1H NMR (400 MHz; CDCl3) δ (ppm) 0.879
(3H, t, J = 6.8 Hz), 1.168 (3H, s), 1.230 (6H, br s), 1.24ꢀ1.53 (14H, m),
2.228 (1H, s), 2.251 (1H, s), 2.265 (1H, s), 2.272 (1H, s); 13C NMR (125
MHz; CDCl3) δ (ppm) 14.0, 22.6, 23.8, 29.0, 29.2, 29.5, 30.0, 31.8, 32.5,
44.6, 52.8, 54.3, 55.0, 57.4, 211.0; HRMS (ESIþ) calcd for C16H32NO [M þ
H]þ 254.2484, found 254.2489. Anal. Calcd for C16H31NO: C, 75.83; H,
12.33; N, 5.53. Found: C, 75.77; H, 11.99; N, 5.54.
2,2,6-Trimethyl-6-octylpiperidin-4-one-1-oxyl (2i). Compound
1i (100 mg, 0.39 mmol) was oxidized according to the method for
compound 2c. The crude product was separated by column chromatogra-
phy with hexane/AcOEt to afford compound 2i (78 mg, 74%) as an orange
oil: MS (FABþ): 268.3 (Mþ); νmax/cmꢀ1 1721 (CdO); AN = 1.596 mT.
Anal. Calcd for C16H30NO2: C, 71.59; H, 11.27; N, 5.22. Found: C, 71.45;
H, 11.13; N, 5.10.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra. This
S
b
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: yamada@pch.phar.kyushu-u.ac.jp.
Author Contributions
4These authors contributed equally.
’ ACKNOWLEDGMENT
This study was partially supported by a Grant-in-Aid for
Young Scientists (B) from the Japan Society for the Promotion
of Science, and the Development of Systems and Technology for
Advanced Measurement and Analysis of the Japan Science and
Technology Agency. We appreciate the technical support pro-
vided by the Research Support Center, Graduate School of
Medical Sciences, Kyushu University.
Thiobarbituric Acid-Reactive Substances Assay
a. Animals. Animal experiments and all procedures were approved
by the Committee on the Ethics of Animal Experiments, Faculty of
Pharmaceutical Sciences, Kyushu University (Kyushu, Japan). Male
Wistar rats were purchased from Kyudo Company Limited (Saga,
Japan). Rats were allowed free access to water and food (MF, Oriental
Yeast Company, Tokyo, Japan).
’ REFERENCES
b. Preparation of Microsomal Suspensions. Liver samples
from male Wistar rats were obtained after killing under deep anesthesia.
They were washed with chilled 0.9% NaCl and homogenized with a
Potter-type homogenizer in three volumes (w/v) of chilled phosphate-
buffered saline (PBS). The homogenate from each sample was centrifuged
at 700 ꢁ g for 10 min at 4 °C, and the supernatant centrifuged at 7 000 ꢁ g
for 10 min at 4 °C. The resulting supernatant was centrifuged at 105 000 ꢁ g
for 60 min at 4 °C. Protein concentrations of microsomes were determined
with use of a BCA protein assay kit (Pierce, Rockford, IL, USA).
c. Protective Effect of Nitroxides on Lipid Peroxidation.
Peroxidation of a microsomal suspension (0.5 mg/mL) was initiated by
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tion of lipid peroxidation is achieved, was calculated with use of Sigma
Plot 11.2 software (Systat Software Incorporated, San Jose, CA) with the
4ꢀ8 parameter exponential function standard curve analysis for dose
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