Studies on the pyrrolinone metabolites derived from the tobacco alkaloid 1-methyl-2-(3-pyridinyl)pyrrole (β-nicotyrine)

Article abstract of DOI:10.1021/tx990019j

Previous studies have established that the tobacco alkaloid 1-methyl-2- (3-pyridyl)pyrrole (β-nicotyrine) is biotransformed by rabbit lung and liver microsomal preparations to an equilibrium mixture of the corresponding 3- and 4-pyrrolin-2-ones. Autoxidation of these pyrrolin-2-ones generates the chemically stable 5-hydroxy-5-(3-pyridinyl)-3-pyrrolin-2-one. This paper summarizes efforts to document more completely the pathway leading to this hydroxypyrrolinone. Chemical and spectroscopic evidence implicates the 2- hydroxy-1-methyl-5-(3-pyridinyl)pyrrole (2-hydroxy-β-nicotyrine) as the key intermediate in this reaction pathway. Of potential toxicological interest is the detection of radical species derived from the autoxidation of this compound.

Full text of DOI:10.1021/tx990019j

508
Chem. Res. Toxicol. 1999, 12, 508-512
Stu d ies on th e P yr r olin on e Meta bolites Der ived fr om th e
Toba cco Alk a loid 1-Meth yl-2-(3-p yr id in yl)p yr r ole
(â-Nicotyr in e)
Xin Liu,† Lunyi Zang,† Cornelis J . Van der Schyf,†,‡ Kazuo Igarashi,§
Kay Castagnoli,† and Neal Castagnoli, J r.*,†
Peters Center for the Study of Parkinson’s Disease, Department of Chemistry,
and Department of Biomedical Sciences and Pathobiology, VA-MD Regional College of Veterinary
Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0212,
and Laboratory of Biochemical Toxicology, Faculty of Pharmaceutical Sciences,
Kobegakuin University, Kobe 651-2180, J apan
Received J anuary 29, 1999
Previous studies have established that the tobacco alkaloid 1-methyl-2-(3-pyridyl)pyrrole
(â-nicotyrine) is biotransformed by rabbit lung and liver microsomal preparations to an
equilibrium mixture of the corresponding 3- and 4-pyrrolin-2-ones. Autoxidation of these
pyrrolin-2-ones generates the chemically stable 5-hydroxy-5-(3-pyridinyl)-3-pyrrolin-2-one. This
paper summarizes efforts to document more completely the pathway leading to this hydroxy-
pyrrolinone. Chemical and spectroscopic evidence implicates the 2-hydroxy-1-methyl-5-(3-
pyridinyl)pyrrole (2-hydroxy-â-nicotyrine) as the key intermediate in this reaction pathway.
Of potential toxicological interest is the detection of radical species derived from the autoxidation
of this compound.
involving a single electron transfer to dioxygen from the
oxyanion 6 of the 2-hydroxypyrrole derivative 5, the enol
In tr od u ction
The pharmacological (1) and toxicological (1-4) prop-
erties of tobacco products have prompted extensive
studies on the metabolic fate of (S)-nicotine (1) (5-9), the
principal pharmacologically active alkaloid present in
tobacco products. A more limited effort, which includes
our studies on 1-methyl-4-(3-pyridinyl)pyrrole [â-nicotyrine
(2)] (10), has focused on the metabolism of some of the
minor tobacco alkaloids (11). Interest in â-nicotyrine is
based in part on reports that structurally related five-
membered heteroarenes may be biotransformed to reac-
tive metabolites that contribute to their toxic properties
(12-16). It has been reported that â-nicotyrine forms
from the autoxidation of nicotine under sunlight (17) and
also is a urinary metabolite of (S)-nicotine in dogs and
rats (18). Results of preliminary studies in which cyto-
chrome P450 rich Clara cells isolated from rabbit lung
were used suggest that â-nicotyrine is bioactivated in an
NADPH-dependent process to pneumotoxic metabolites.1
Previous studies in which NADPH-supplemented rab-
bit liver and lung microsomal preparations were used
and a GC/EIMS assay have led to the characterization
of isomeric pyrrolinones 3 and 4 as metabolites of
â-nicotyrine (Scheme 1) (19). Following their isolation
from the incubation mixtures, these pyrrolinones were
found to undergo autoxidation to yield 5-hydroxy-1-
methyl-5-(3-pyridinyl)-3-pyrrolin-2-one (9). We postulated
a free radical pathway for the conversion of 3 and 4 to 9
tautomer of the pyrrolinones, which yields the resonance-
•-
stabilized radical 7 and superoxide radical anion O2
.
Subsequent radical recombination of 7b with HOO• (the
conjugate acid of O2•-) and hydrolysis of the resulting
hydroperoxide 8 would yield 9 (Scheme 1). Mass spectral
evidence also suggested the presence of an isomeric
hydroxypyrrolinone (possibly 11) that could be derived
from the isomeric radical recombination product 10 (19).
This paper describes the results of studies designed to
evaluate the pathway leading from the pyrrolinones 3
and 4 to the 5-hydroxypyrrolinone 9.
Ma ter ia ls a n d Meth od s
Ch em istr y. Syntheses were carried out under a nitrogen
atmosphere. All starting materials and the spin trapping
reagents N-tert-butyl-R-phenylnitrone [PBN2 (20)] and 5,5-
dimethyl-1-pyrroline N-oxide [DMPO (21)] were purchased from
Aldrich Chemical Co. (Milwaukee, WI), while TRIS, HEPES,
potassium chloride, EGTA, acetonitrile, triethylamine, and
acetic acid were obtained from Fisher Chemical Co. (Pittsburgh,
PA). NADP+, magnesium chloride, D-glucose 6-phosphate, glucose-
6-phosphate dehydrogenase, and mono-, di-, and tribasic phos-
phates were purchased from Sigma Chemical Co. (St. Louis,
MO). The tartrate salt of â-nicotyrine (2) (10), 3,3-dibromoco-
tinine (20), 5-hydroxycotinine (16) (21) (improved synthesis
described later in the text), and 5-methoxy-1-methyl-5-(3-
pyridyl)-3-pyrrolin-2-one (13) (21) were synthesized as described
previously.
* To whom correspondence should be addressed.
† Department of Chemistry.
Abbreviations: aN, R nitrogen hyperfine splitting constant; aâH, â
2
‡ Department of Biomedical Sciences and Pathobiology.
hydrogen hyperfine splitting constant; DMPO, 5,5-dimethyl-1-pyrroline
N-oxide; DMSO, dimethyl sulfoxide; ESR, electron spin resonance; GC/
MS, gas chromatography/mass spectrometry; NADP+, nicotinamide
adenine dinucleotide phosphate; NADPH, 1,4-dihydronicotinamide
adenine dinucleotide phosphate; PBN, N-tert-butyl-R-phenylnitrone.
§ Kobegakuin University.
1
W. K. Nichols, M. O. Covington, G. S. Yost, X. Liu, and N.
Castagnoli, J r. (1990) Bioactiviation of the tobacco alkaloid â-nicotyrine
in isolated Clara cells (unpublished data).
10.1021/tx990019j CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/08/1999

Metabolic Studies on â-Nicotyine
Chem. Res. Toxicol., Vol. 12, No. 6, 1999 509
Sch em e 1. In Vitr o Meta bolic F a te of â-Nicotyr in e
Proton NMR spectra were recorded on a Bruker WP 270 MHz
spectrometer linked to an Aspect 2000 computer. Chemical
shifts are reported in parts per million relative to Me4Si in
CDCl3 or to 3-(trimethylsilyl)propionic-2,2,3,3-acid-d4, sodium
salt (TSP), in D2O and CD3OD. Spin multiplicities are given as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or
b (broad). ESR spectra were performed on a Bruker ER 200 D
instrument. UV spectra were recorded on a Beckman DU 50
spectrophotometer. GC/EI mass spectral analyses were per-
formed on an HP 5970 mass selective detector interfaced with
an HP 5890 gas chromatograph equipped with an HP-1 100%
dimethylpolysiloxane capillary column (12.5 m × 0.2 mm × 0.33
µm). The analyses were performed using a temperature program
starting at 100 °C and, following a 1 min hold, increasing the
temperature to 275 °C at a rate of 25 °C/min. HPLC/diode array
analyses were performed with a dual (Beckman 114M) pump
system at a flow rate of 1 mL/min, a Beckman 421A controller,
a C8 Alltech Econosil (25 cm × 4.6 mm × 10 µm) column, and
an HP 1040A diode array detector. Elution proceeded first with
100% mobile phase A [90:10:0.5:0.1 (v/v) water/acetonitrile/acetic
acid/triethylamine] for 10 min employing a 10 min ramp and
ended with 40% mobile phase B (100% acetonitrile). After
elution for 10 min with 40% B, the mobile phase was ramped
back to 100% A over a 5 min period, after which the elution
was continued for another 10 min, allowing the column to
equilibrate. The total eluting time was 45 min. The effluent was
monitored simultaneously at 260 and 282 nm. Typical retention
times were as follows: â-nicotyrine, 23.5 min; pyrrolinone 3,
21.5 min; pyrrolinone 4, 20.5 min; 5-hydroxypyrrolinone 9, 13.5
min; and 5-hydroxycotinine (16), 11 min. Nicotinamide (8.5 min)
was observed in both control and experimental incubations.
residue was treated with saturated sodium bicarbonate and the
neutralized solution extracted with chloroform. The extract was
washed with aqueous solutions of sodium sulfite and dried over
anhydrous Na2SO4. The solvent was removed under vacuum to
leave a light brown residue which, after crystallization from
diethyl ether, provided pure 5-hydroxycotinine (0.87 g, 4.5 mmol,
90%) which was identical in all respects to the previously
reported product which was obtained by catalytic reduction of
the corresponding hydroxypyrrolinone 9 (21).
5-Hyd r oxy-1-m eth yl-5-(3-p yr id yl)-3-p yr r olin -2-on e (9).
Following the procedure for the O-demethylation of 5-methoxy-
cotinine (14), 5-hydroxy-1-methyl-5-(3-pyridyl)-3-pyrrolin-2-one
(9) was obtained from the corresponding methyl ether 13 as
yellow crystals in 80% yield. This product was identical in every
respect to that obtained by treatment of 13 with HBr.
Mixtu r e of 1-Meth yl-5-(3-p yr id yl)-4-p yr r olin -2-on e (3)
a n d 1-Meth yl-5-(3-p yr id yl)-3-p yr r olin -2-on e (4). 5-Hydroxy-
cotinine (16, 87.2 mg, 0.454 mmol) was placed in a flask
connected to a 50 cm long pyrolysis tube wrapped with electro-
thermal wire that was connected to a cold trap cooled in a dry
ice/acetone bath. The whole system was purged with nitrogen.
The flask containing the 5-hydroxycotinine was heated slowly
in an oil bath to a final temperature of 135 °C, while the
pyrolysis tube was kept at 280 °C. The process was carried out
under vacuum (0.12-0.14 mmHg) for 1.5 h and resulted in a
dark purple-colored condensate containing a mixture of the two
pyrrolinones 3 and 4 in a ratio of 3:1 as determined by GC/MS
(9 mg, 11.4%): 1H NMR (CDCl3) of 1-methyl-5-(3-pyridyl)-4-
pyrrolin-2-one (2) δ 8.67 (m, 2H, C2′- and C6′-H), 7.69 (d, 1H,
C4′-H), 7.35 (dt, 1H, C5′-H), 5.38 (t, 1H, C4-H), 3.22 (d, 2H, C3-
H), 3.02 (s, 3H, N-Me); 1H NMR (CDCl3) of 1-methyl-5-(3-
pyridyl)-3-pyrrolin-2-one (4) δ 8.50 (m, 2H, C2′- and C6′-H), 7.65
(dt, 1H, C4′-H), 7.30 (m, 1H, C5′-H), 7.03 (dd, 1H, C3-H), 6.30
(dd, 1H, C4-H), 5.01 (t, 1H, C5-H), 2.83 (s, 3H, N-Me). Direct
insertion probe high-resolution EI-MS (M•+) calcd for C10H10N2O
174.0793131, found 174.078354.
1-Met h yl-5-m et h oxy-5-(3-p yr id yl)-2-p yr r olid in on e [5-
Meth oxycotin in e (14)]. A solution of 1-methyl-5-methoxy-5-
(3-pyridyl)-3-pyrrolin-2-one (13, 10.2 g, 100 mmol) in 300 mL
of methanol containing 2 g of 10% Pd/C was stirred vigorously
under hydrogen at atmospheric pressure and room temperature
for 24 h. After filtration of the catalyst and removal of the
solvent by evaporation under vacuum, 10.3 g (50 mmol, 100%)
of a pale yellow oil was obtained. The product solidified upon
storage in the refrigerator. Recrystallization from ethyl acetate
yielded cubic colorless crystals that were suitable for analyses:
mp 75-76 °C; GC/EIMS (tR ) 3.7 min; relative intensity) m/z
206 (5%), 175 (100%), 149 (10%), 128 (10%), 100 (18%), 78 (30%);
1H NMR (CDCl3) δ 8.67 (1H, d, C2′-H), 8.59 (1H, d of d, C6′-H),
7.70 (1H, t of d, C4′-H), 7.32 (1H, m, C5′-H), 3.27 (3H, s, O-CH3),
2.69-2.60 (2H, m, C3-H), 2.58 (3H, s, N-CH3), 2.52-2.41 (1H,
m, C4-H), 2.32-2.15 (1H, m, C4-H); UV (in CH3OH) λmax 216 (ꢀ
) 7300), 260 nm (ꢀ ) 4500). Anal. Calcd for C11H14N2O2: C,
64.06; H, 6.84; N, 13.58. Found: C, 64.16; H, 6.86; N, 13.64.
2-Acetoxy-1-m eth yl-5-(3-p yr id yl)p yr r ole (12). A solution
of 5-methoxycotinine (14, 1.03 g, 5 mmol) in 5 mL (100 mmol)
of acetic anhydride was heated to 80 °C in an oil bath for 6 h
and then allowed to remain at room temperature overnight. The
reaction was monitored by GC/EIMS. When the starting mate-
rial (retention time of 3.7 min) had been fully consumed, the
GC/EIMS showed only one peak at m/z 216 (retention time of
4.4 min). After removal of the remaining acetic anhydride, the
brown oily residue was purified first by passage through a
neutral alumina column with CHCl3 followed by chromatogra-
phy on a silica gel column (1:20 w/w), again eluting with CHCl3.
The first fraction gave 0.83 g (76.5%) of 2-acetoxy-1-methyl-5-
(3-pyridyl)pyrrole (12) as a pale yellow oil: GC/EIMS (tR ) 4.5
min; relative intensity) m/z 216 (10%), 174 (100%), 145 (10%),
131 (10%), 104 (10%), 78 (10%); 1H NMR (CDCl3) δ 8.66 (1H, d,
C2′-H), 8.52 (1H, q, C6′-H), 7.68 (1H, q, C4′-H), 7.32 (1H, q, C5′-
H), 6.21 (1H, d, C3-H), 5.95 (1H, d, C4-H), 3.45 (3H, s, NCH3),
2.35 (3H, s, COCH3); UV (in the HPLC mobile phase of 50:50:
0.1:0.5 v/v water/acetonitrile/triethylamine/acetic acid at pH 3.8)
5-Hydr oxy-1-m eth yl-5-(3-pyr idyl)-2-pyr r olidin on e [5-Hy-
d r oxycotin in e (16)]. A solution of 5-methoxycotinine (14, 1.03
g, 5 mmol) and trimethylsilyl iodide (0.925 mL or 1.3 g, 6.5
mmol) in 2 mL of chloroform was stirred at room temperature
for 24 h (22). After the addition of 0.82 mL of methanol (0.64 g,
20 mmol), the solvent was removed under vacuum at 50 °C. The

510 Chem. Res. Toxicol., Vol. 12, No. 6, 1999
Liu et al.
Sch em e 2. Syn th etic Rou tes to Com p ou n d s
Discu ssed in th e Text
4 (19, 23). The overall yield, however, was very low, and
our attempts to prepare useful quantities of both 3 and
4 by this approach were unsuccessful. A mixture of 3 and
4 could be obtained by vacuum tube pyrolysis of 5-hy-
droxycotinine (16). GC/EIMS and 1H NMR analyses of
the distillate gave spectra which were in agreement with
this earlier report. Solutions of this mixture in anhy-
drous, argon-purged CHCl3 slowly turned a deep purple
color which disappeared within hours upon storage due
to decomposition. The chemical instability of these pyr-
rolinones has been taken as evidence of their equilibra-
tion with the highly electron rich and, presumably,
unstable 5-hydroxypyrrole derivative 5 as proposed in
Scheme 1.
An alternative approach to the preparation of 3 that
was examined involved reaction of 5-hydroxycotinine (16)
with acetic anhydride with the hope that the expected
5-acetoxy species 21, containing a better leaving group,
would undergo elimination to form 3. GC/EI mass
spectral analysis of the principal product isolated from
this reaction, however, displayed a parent ion at m/z 216
rather than at m/z 174 as expected for 3. Proton NMR
and elemental analyses (see Materials and Methods) led
to the characterization of this product as 2-acetoxy-1-
methyl-5-(3-pyridinyl)pyrrole (12). Since 5-methoxycoti-
nine (14) was an even better substrate for this reaction,
it seems likely that the pathways to 12 proceed through
pyrrolinyl acetate 18 or 19 (Scheme 2) followed by loss
of water (from 18) or methanol (from 19).
Meta bolism . Consistent with earlier results, â-nico-
tyrine was metabolized efficiently in NADPH-supple-
mented rabbit liver microsomal preparations with es-
sentially 100% of the substrate being consumed within
75 min. HPLC/diode array analysis of the incubation
mixture and synthetic standards established the two
pyrrolinones 3 and 4 as major metabolites. Two ad-
ditional minor peaks were shown to be due to the
hydroxypyrrolinone 9 and 5-hydroxycotinine (16). As will
become evident from the discussion below, compounds 9
and 16 are likely to be derived from the pyrrolinones 3
and 4. No metabolite was formed in the absence of
microsomes or NADPH.
λmax 230 (ꢀ ) 8600), 290 nm (ꢀ ) 15 000). Anal. Calcd for
C
12H12N2O2‚0.15H2O: C, 65.83; H, 5.59; N, 12.79. Found: C,
65.62; H, 5.53; N, 12.81.
Hyd r olysis of 2-Acetoxy-1-m eth yl-5-(3-p yr id yl)p yr r ole
(12). Solutions of 12 (10 mg in 0.5 mL) were prepared in 10%
DCl in D2O and 0.1 M phosphate buffer in D2O (pH 7.4). The
1
reaction mixtures were monitored by H NMR at room temper-
ature and the products analyzed by GC/MS.
Meta bolism . Rabbit liver microsomes were prepared from
male New Zealand white rabbits as described previously (20).
Incubation mixtures contained rabbit lung (4 mg of protein/mL)
or liver microsomes (5 mg of protein/mL), â-nicotyrine tartrate
(154 mg/mL, 0.5 mM), and an NADPH-regenerating system (0.5
mM NADP+, 8 mM glucose 6-phosphate, 1 unit/mL glucose-6-
phosphate dehydrogenase, and 4 mM MgCl2) in a total of 4 mL
of a 0.1 M Tris-HCl buffer (pH 7.4) containing 0.1 mM EGTA.
Incubations were carried out at 37 °C for 75 min in a metabolic
shaker at 60 rpm. At the end of the specified incubation period,
the mixtures were added to an equal volume of ice-cold aceto-
nitrile. The quenched mixtures were centrifuged at 16000g for
5 min, and the resulting supernatants were centrifuged at
16000g for an additional 5 min and filtered to remove the
precipitated protein fraction. The final supernatants were
analyzed by the HPLC method described above.
ESR Stu d ies. All studies were carried out at room temper-
ature. Solutions of the acetoxypyrrole 12 (6 mM in 0.1 mM
phosphate buffer at pH 7.0) contained 0.3 mM purified DMPO
in water or saturated PBN in water (both at 1:1 v/v) were used
in these studies. Control studies were carried out in which the
trapping reagents or 12 was omitted. Additional controls
included incubations in which 12 was replaced with 13 mM
â-nicotyrine (2). Some incubations also contained 2 mM ethanol,
a hydroxyl radical scavenger. Each sample was siphoned into 2
in. long capillary tubes (inside diameter of 1 mm), and the tubes
were sealed. Unless stated otherwise, the ESR parameters were
set as follows: X-band, 9.3 GHz, 100 kHz modulation frequency;
microwave power of 20 mW; modulation amplitude of 2.0 G; time
constant of 1.25 s; scan time of 500 s; receiver gain of 8.0 × 105;
central field of 3483 G; and scan width of 100 G. The line width
of the ESR was measured with an E-500 gauss meter.
The availability of 12 provided an opportunity to
examine the chemical behavior of the putative hydroxy-
pyrrole metabolite 5 (Scheme 1) which should be formed
following hydrolysis of the ester group. We monitored the
1
fate of 12 in deuterated phosphate buffer (pH 7.4) by H
NMR spectroscopy. The unique chemical shift values for
the signals for the N-methyl groups for the compounds
of interest [δ 3.52 (12), 3.06 (3), 2.88 (4), 2.74 (9), and
2.65 ppm (16)] provided an opportunity to make rough
estimates of the concentrations of reactant and products
over the time course of the incubations. Under neutral
conditions, the hydrolysis of 12 was very slow, perhaps
due to its limited water solubility. Signals corresponding
to the hydroxypyrrole 5 were not observed at any time.
Instead, the principal products formed early in the
reaction were the two pyrrolinones 3 and 4 which were
present in approximately equal amounts during the
Resu lts a n d Discu ssion
Ch em istr y. Synthetic standards of compounds 3, 4,
1
9, and 16 were required for HPLC and H NMR studies.
The synthesis of the 5-hydroxypyrrolinone 9 was achieved
in good yield by treatment of the readily available
methoxypyrrolinone 13 (21) with trimethylsilyl iodide to
cleave the methyl ether (Scheme 2). Catalytic hydrogena-
tion of 13 followed by ether cleavage of the resulting
5-methoxycotinine 14 with trimethylsilyl iodide, rather
than with HBr, provided an improved route to 5-hy-
droxycotinine (16).
We have earlier reported the synthesis of the 4-pyr-
rolinone 3 via 3-phenylselenylation of cotinine followed
by elimination of the corresponding phenylselenyl oxide
and rearrangement of the initially formed 3-pyrrolinone
1
course of the 8 day incubation. The H NMR signals for
pyrrolinones were slowly replaced with signals corre-
sponding to the hydroxypyrrolinone 9 (20% yield) and
5-hydroxycotinine [16 (65% yield)].
The rate of hydrolysis of 12 was dramatically enhanced
at pH 1 (5% HCl/H2O), conditions under which 12 was
1
soluble. H NMR signals for the starting material could

Metabolic Studies on â-Nicotyine
Chem. Res. Toxicol., Vol. 12, No. 6, 1999 511
Sch em e 3. P r op osed Str u ctu r es of th e Tr a p p ed Ra d ica ls Der ived fr om 5-Hyd r oxy-â-n icotyr in e
not be detected after 10 min. At this time, 3-pyrrolinone
4 (2%), 4-pyrrolinone 3 (37%), and 5-hydroxycotinine [16
(59%)] were present; no signals reflecting the formation
of the hydroxypyrrolinone 9 were observed. By 70 min,
5-hydroxycotinine accounted for all of the starting mate-
rial. The conversion of the pyrrolinones to 5-hydroxyco-
tinine presumably proceeds via the protonation of the
amidoiminium intermediate 15 (Scheme 1).
The results from these studies suggest that the puta-
tive 5-hydroxypyrrole metabolite 5 (derived chemically
from the corresponding acetoxy compound 12 and meta-
bolically from â-nicotyrine) exists predominantly as the
equilibrium mixture of pyrrolinones 3 and 4 in aqueous
solution. These pyrrolinones undergo acid-catalyzed hy-
dration, to yield 5-hydroxycotinine 16, and autoxidation,
to yield the 5-hydroxypyrrolinone 9.
The autoxidative pathway proposed to account for the
conversion of the pyrrolinones 3 and 4 to the hydroxy-
pyrrolinone 9 (Scheme 1) leads to O2•- and the free radical
species 7. Evidence to support this proposal was sought
with the aid of the acetoxypyrrole 12 which was incu-
bated at pH 7.0 and room temperature in the presence
of the spin trapping reagent PBN (20) or DMPO (21). No
ESR signals were observed in several control incubations
performed in the absence of the spin trapping reagents
or 12. Furthermore, even after a 5 day incubation period,
no ESR signals were observed when â-nicotyrine replaced
12. Incubation mixtures containing PBN and 12 slowly
developed a six-line ESR pattern after 24 h. This pattern
is that expected for a nitroxyl radical in which the
unpaired electron is split into a triplet by the R-nitrogen
atom (hyperfine splitting constant aN ) 15.2 G) with each
line of the triplet being split further by the â-hydrogen
(aâH ) 2.8 G). Although these spectral data are not
definitive, they are consistent with the hydroxyl radical
adduct 22 (aN ) 15.2 G, and aâH ) 2.75 G) (24). An
alternative interpretation is an adduct 23 (Scheme 3),
resulting from trapping of a carbon-centered radical
(reported aN ) 14-16.2 G, and aâH ) 2.64-4.35 G) (24).
Favoring the carbon radical adduct is the chemical
instability of 22 (25). The absence of evidence of further
coupling with the OH proton (agH ) 0.21 G) (26) present
in 22 also is support for the carbon adduct. A similar
experiment carried out with DMPO (21) gave a weak 10-
line spectrum after 2 h. The spectrum may be interpreted
as a mixture of a four-line pattern (aN ) 15 G, and aHâ
)
15 G), expected for the hydroxyl radical adduct 24, with
a six-line pattern (aN ) 15.9 G, and aâH ) 23.0 G) (24),
expected for a carbon radical adduct (25). The absence
of adducts resulting from trapping of the hydroperoxyl
radical (HOO•) is expected because of the short half-life
and ease with which HOO• is reduced to HO• (27, 28).
The absence of evidence for the formation of an adduct
between the form of 7 in which the radical is centered
on oxygen (7a ) also would be expected because the
greater electronegativity of oxygen versus that of carbon
makes alkoxyl radicals less stable than carbon radicals.
In an effort to provide further information on the
putative hydroxyl radical pathway, ethanol, a specific
hydroxyl radical scavenger (29), was added to the reaction
mixtures containing 12 and either spin trap. The DMPO-
containing system gave a six-line ESR signal (aN ) 15.7,
and aâH ) 22.8), while the PBN-containing system gave
another six-line ESR signal (aN ) 15.5, and aâH ) 3.6).
Comparison with literature values (29) led us to conclude
that these ESR signals correspond to adducts 26 and 27
formed between the hydroxyl radical-generated methyl
hydroxyl carbinyl radical (28) and the spin traps DMPO
and PBN, respectively. Again, no ESR signals were
observed in the control samples which did not contain
12.
Su m m a r y. The results of the studies reported here
confirm previous observations documenting the efficient

512 Chem. Res. Toxicol., Vol. 12, No. 6, 1999
Liu et al.
(11) Liu, X., J acob, P., III, and Castagnoli, N., J r. (1993) Metabolism
of minor tobacco alkaloids. In Absorption, distribution, metabolism
& excretion of nicotine and related alkaloids (Gorrod, J . W., and
Wahrens, J ., Eds.) Chapman & Hall, London.
(12) Gordon, S. M. (1990) Identification of exposure markers in
smokers’ breath. J . Chromatogr. 511, 291-302.
(13) Fransson-Steen, R., Goldsworthy, T. L., Kedderis, G. L., and
Maronpot, R. R. (1997) Furan-induced liver cell prolferation and
apopotosis in female B6C3F1 mice. Toxicology 118, 195-204.
(14) Wolson, D. M., Goldsworthy, T. L., Popp, J . A., and Butterworth,
B. E. (1992) Evaluation of genotoxicity, pathological lesions, and
cell proliferation in livers of rats and mice treated with furan.
Environ. Mol. Mutagen. 19, 209-222.
(15) Wilcox, A. L., Bao, Y. T., and Loeppky, R. N. (1991) Pyrroles as
effective agents for blocking amine nitrosation. Chem. Res.
Toxicol. 4, 373-381.
NADPH-dependent oxidation of â-nicotyrine by rabbit
liver and lung microsomal preparations in forming a
mixture of the chemically labile pyrrolinones 3 and 4.
These pyrrolinones may undergo hydration, in an acid-
dependent reaction, to form the known (S)-nicotine
metabolite 5-hydroxycotinine (16), which has been re-
ported to be a urinary metabolite of (S)-nicotine, or
autoxidation to form the hydroxypyrrolinone 9. Evidence
obtained with synthetic 5-acetoxy-1-methyl-2-(3-pyridi-
nyl)pyrrole (12) suggests that the pyrrolinones 3 and 4
are derived from the corresponding 5-hydroxypyrrole
derivative 5. The autoxidation leading to the hydroxy-
pyrrolinone 9 is likely to proceed via the conjugate base
6 derived from 5 since assignable ESR signals were
detected when 12 underwent hydrolysis in the presence
of radical spin trapping reagents. Studies on the in vivo
metabolic fate of â-nicotyrine are underway.
(16) Sta˜lhandske, T., and Slanina, P. (1982) Nicotyrine inhibits in vivo
metabolism of nicotine without increasing its toxicity. Toxicol.
Appl. Pharmacol. 65, 366-372.
(17) Wada, E., Kisaki, T., and Saito, K. (1959) Autoxidation of nicotine.
Arch. Biochem. Biophys. 79, 124-130.
(18) Werle, E., and Meyer, A. (1950) U¨ ber den Abbau des Nicotins
durch tierisches Gewebe. Biochem. Z. 321, 221-235.
(19) Shigenaga, M. K., Kim, B. H., Caldera-Munoz, P., Cairns, T.,
J acob, P., III, Trevor, A. J ., and Castagnoli, N., J r. (1989) Liver
and lung microsomal metabolism of the tobacco alkaloid
â-nicotyrine. Chem. Res. Toxicol. 2, 282-287.
(20) Duffield, A. M., Budzikiewiz, H., and Djerassi, C. (1965) Mass
spectrometry in structure and stereochemical problems. LXXII.
A study of the fragmentation processes of some tobacco alkaloids.
J . Am. Chem. Soc. 87, 2926-2932.
Ack n ow led gm en t. This work was supported by a
grant from NIDA (1RO1DA11089-01A1) and the Harvey
W. Peters Center for the Study of Parkinson’s Disease,
Department of Chemistry, Virginia Polytechnic Institute
and State University.
Refer en ces
(21) Nguyen, T., Dagne, E., Gruenke, L., Bhargava, H., and Castagnoli,
N., J r. (1981) The tautomeric structure of 5-hydroxycotinine, a
secondary mammalian metabolite of nicotine. J . Org. Chem. 46,
758-760.
(22) J ung, M. E., and Lyster, M. A. (1977) Quantitative dealkylation
of alkyl ethers via treatment with trimethylsilyl iodide. A new
method for ether hydrolysis. J . Org. Chem. 42, 3761-3764.
(23) Williams, D. E., Shigenaga, M. K., and Castagnoli, N., J r. (1990)
The role of cytochromes P-450 and flavin-containing monooxyge-
nase in the metabolism of (S)-nicotine by rabbit lung. Drug Metab.
Dispos. 18, 418-428.
(1) Crooks, P. A., and Dwoskin, L. P. (1997) Contribution of CNS
nicotine metabolites to the neuropharmacological effects of nico-
tine and tobacco smoking. Biochem. Pharmacol. 54, 743-753.
(2) U.S. Department of Health and Human Services (1984) The
Health consequence of smoking: Cardiovascular disease, a report
of the surgeons general 1984. U.S. Department of Health and
Human Services DHHS Publication No. (PHS) 84-50204, p 384,
U.S. Government Printing Office, Washington, DC.
(3) Nair, J ., Oshima, H., Nair, U. J ., and Bartsch, H. (1996)
Endogenous formation of nitrosamines and oxidative DNA-
damaging agents in tobacco users. Crit. Rev. Toxicol. 26, 149-
161.
(4) Silbergeld, E. K. (1995) The hazards of synthetic (anthropogenic)
chemicals. Toxicol. Lett. 82/83, 935-941.
(5) Castagnoli, N., J r., Rimoldi, J . M., Bloomquist, J ., and Castagnoli,
K. P. (1997) Potential metabolic bioactivation pathways involving
cyclic tertiary amines and azaarenes. Chem. Res. Toxicol. 10,
924-940.
(24) Buettner, G. R. (1987) Spin trapping: ESR parameters of spin
adducts. Free Radical Biol. Med. 3, 259-303.
(25) J anzen, E. G. (1992) Stabilities of hydroxyl radical spin adducts
of PBN-type spin traps. Free Radical Biol. Med. 12, 1257-1260.
(26) Buettner, G. R. (1985) Spin trapping of hydroxyl radicals. In CRC
Handbook of Methods of Oxygen Radical Research (Greenwald,
R. A., Ed.) pp 151-155, CRC Press, Boca Raton, FL.
(27) Thornalley, P. J ., and Bannister, J . V. (1985) The spin trapping
of superoxide radicals. In CRC Handbook of Methods for Oxygen
Radical Research (Greenwald, R. A., Ed.) pp 133-136, CRC Press,
Boca Raton, FL.
(6) Kyerematen, G. A., and Vesell, E. S. (1991) Metabolism of nicotine.
Drug Metab. Rev. 23, 3-41.
(7) Nakayama, H. (1988) Nicotine metabolism in mammals. Drug
Metab. Drug Interact. 6, 95-122.
(8) Svensson, C. K. (1987) Clinical pharmacokinetics of nicotine. Clin.
Pharmacokinet. 12, 30-40.
(9) Gorrod, J . W., and J enner, P. (1985) The metabolism of tobacco
alkaloids. In Essays in Toxicology (Hayes, W. J ., J r., Ed.) Vol. 6,
pp 35-78, Academic Press, New York.
(10) Shigenaga, M. K. (1989) Study on the metabolism and bioacti-
vation of (S)-nicotine and â-nicotyrine. Ph.D. Thesis, University
of California, San Francisco, CA.
(28) Harbour, J . R., and Hair, M. L. (1978) Detection of superoxide
ions in nonaqueous media. Generation by photolysis op pigment
dispersions. J . Phys. Chem. 82, 1397-1399.
(29) Lai, C.-S., and Piette, L. H. (1978) Spin-trapping studies of
hydroxyl radical production involved in lipid peroxidation. Arch.
Biochem. Biophys. 190, 27-38.
TX990019J