N-dealkylation of an N-cyclopropylamine by horseradish peroxidase. Fate of the cyclopropyl group

Article abstract of DOI:10.1021/ja0111479

Cyclopropylamines inactivate cytochrome P450 enzymes which catalyze their oxidative N-dealkylation. A key intermediate in both processes is postulated to be a highly reactive aminium cation radical formed by single electron transfer (SET) oxidation of the nitrogen center, but direct evidence for this has remained elusive. To address this deficiency and identify the fate of the cyclopropyl group lost upon N-dealkylation, we have investigated the oxidation of N-cyclopropyl-N-methylaniline (3) by horseradish peroxidase, a well-known SET enzyme. For comparison, similar studies were carried out in parallel with N-isopropyl-N-methylaniline (9) and N,N-dimethylaniline (8). Under standard peroxidatic conditions (HRP, H₂O₂, air), HRP oxidizes 8 completely to N-methylaniline (4) plus formaldehyde within 15-30 min, whereas 9 is oxidized more slowly (<10% in 60 min) to produce only N-isopropylaniline (10) and formaldehyde (acetone and 4 are not formed). In contrast to results with 9, oxidation of 3 is complete in <60 min and affords 4 (20% yield) plus traces of aniline. By using [1′-¹⁴C]-3, [1′-¹³C]-3, and [2′,3′-¹³C]-3 as substrates, radiochemical and NMR analyses of incubation mixtures revealed that the complete oxidation of 3 by HRP yields 4 (0.2 mol), β-hydroxypropionic acid (17, 0.2 mol), and N-methylquinolinium (16, 0.8 mol). In buffer purged with pure O₂, the complete oxidation of 3 yields 4 (0.7 mol), 17 (0.7 mol), and 16 (0.3 mol), while under anaerobic conditions, 16 is formed quantitatively from 3. These results indicate that the aminium ion formed by SET oxidation of 3 undergoes cyclopropyl ring fragmentation exclusively to generate a distonic cation radical (14^+?) which then partitions between unimolecular cyclization (leading, after further oxidation, to 16) and bimolecular reaction with dissolved oxygen (leading to 4 and 17 in a 1:1 ratio). Neither β-hydroxypropionaldehyde, acrolein, nor cyclopropanone hydrate are formed as SET metabolites of 3. The synthetic and analytical methods developed in the course of these studies should facilitate the application of cyclopropylamine-containing probes to reactions catalyzed by cytochrome P450 enzymes.

Full text of DOI:10.1021/ja0111479

8502
J. Am. Chem. Soc. 2001, 123, 8502-8508
N-Dealkylation of an N-Cyclopropylamine by Horseradish Peroxidase.
Fate of the Cyclopropyl Group
Christopher L. Shaffer, Martha D. Morton, and Robert P. Hanzlik*
Contribution from the Department of Medicinal Chemistry, UniVersity of Kansas,
Lawrence, Kansas 66045-7582
ReceiVed May 8, 2001
Abstract: Cyclopropylamines inactivate cytochrome P450 enzymes which catalyze their oxidative N-
dealkylation. A key intermediate in both processes is postulated to be a highly reactive aminium cation radical
formed by single electron transfer (SET) oxidation of the nitrogen center, but direct evidence for this has
remained elusive. To address this deficiency and identify the fate of the cyclopropyl group lost upon
N-dealkylation, we have investigated the oxidation of N-cyclopropyl-N-methylaniline (3) by horseradish
peroxidase, a well-known SET enzyme. For comparison, similar studies were carried out in parallel with
N-isopropyl-N-methylaniline (9) and N,N-dimethylaniline (8). Under standard peroxidatic conditions (HRP,
H2O2, air), HRP oxidizes 8 completely to N-methylaniline (4) plus formaldehyde within 15-30 min, whereas
9 is oxidized more slowly (<10% in 60 min) to produce only N-isopropylaniline (10) and formaldehyde (acetone
and 4 are not formed). In contrast to results with 9, oxidation of 3 is complete in <60 min and affords 4 (20%
yield) plus traces of aniline. By using [1′-14C]-3, [1′-13C]-3, and [2′,3′-13C]-3 as substrates, radiochemical and
NMR analyses of incubation mixtures revealed that the complete oxidation of 3 by HRP yields 4 (0.2 mol),
â-hydroxypropionic acid (17, 0.2 mol), and N-methylquinolinium (16, 0.8 mol). In buffer purged with pure
O2, the complete oxidation of 3 yields 4 (0.7 mol), 17 (0.7 mol), and 16 (0.3 mol), while under anaerobic
conditions, 16 is formed quantitatively from 3. These results indicate that the aminium ion formed by SET
oxidation of 3 undergoes cyclopropyl ring fragmentation exclusively to generate a distonic cation radical (14+•)
which then partitions between unimolecular cyclization (leading, after further oxidation, to 16) and bimolecular
reaction with dissolved oxygen (leading to 4 and 17 in a 1:1 ratio). Neither â-hydroxypropionaldehyde, acrolein,
nor cyclopropanone hydrate are formed as SET metabolites of 3. The synthetic and analytical methods developed
in the course of these studies should facilitate the application of cyclopropylamine-containing probes to reactions
catalyzed by cytochrome P450 enzymes.
Introduction
Scheme 1
The cyclopropylamine substructure is found in a number of
drugs and drug candidates and is known to undergo biotrans-
formation by cytochrome P450-catalyzed oxidative N-de-
alkylation reactions with loss of the cyclopropyl moiety. For
ordinary amines not containing an N-cyclopropyl group, alkyl
groups lost during N-dealkylation are converted to carbonyl
metabolites formed by hydrolysis of a carbinolamine precursor.
Thus, N-dealkylation was originally viewed as occurring via a
hydrogen atom transfer (HAT) process analogous to ordinary
aliphatic hydroxylation but R to the nitrogen atom.
For cyclopropylamines, metabolic loss of the cyclopropyl
group could, in principle, involve a similar mechanism (viz.
Scheme 1), but the unique electronic structure of the cyclopropyl
group compared to ordinary aliphatic groups,1 and the unique
ability of cyclopropylamines (e.g., 12,3) to act as suicide
substrates for cytochrome P450 enzymes, suggest that other
mechanisms may be involved. Indeed, the fact that cyclopro-
pylamine 2 inactivates P450 as efficiently as 12,3 shows that a
C-1′ hydrogen is not required in the P450 inactivation process
and rules out enzyme inactivation occurring as a side branch in
the classical HAT pathway of N-dealkylation (Scheme 1).
* Address correspondence to this author. Tel. 785-864-3750. Fax 785-
864-5326. rhanzlik@ukans.edu.
(1) Patai, S. The Chemistry of the Cyclopropyl Group Part 2; John Wiley
& Sons: New York, 1987; Vol. 1.
(2) Hanzlik, R. P.; Tullman, R. H. J. Am. Chem. Soc. 1982, 104, 4, 2048-
2050.
To account for the inactivation of P450 by cyclopropylamines,
a mechanism involving single electron transfer (SET) as an
(3) Macdonald, T. L.; Zirvi, K.; Burka, L. T.; Peyman, P.; Guengerich,
F. P. J. Am. Chem. Soc. 1982, 104, 2050-2052.
10.1021/ja0111479 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/10/2001

N-Dealkylation of an N-Cyclopropylamine
J. Am. Chem. Soc., Vol. 123, No. 35, 2001 8503
Scheme 2
Scheme 3
initiating event has been proposed (Scheme 2).2,3 Such a
mechanism could also account for N-dealkylation of both
cyclopropyl and ordinary aliphatic amines by cytochrome P450
enzymes and has been used to explain other features of the
N-dealkylation process that are not well accounted for by a HAT
mechanism like that shown in Scheme 1, such as the occurrence
of very low KDIEs for N-dealkylation vs aliphatic hydroxyla-
tion,4,5 negative Hammett F values for dealkylation of N,N-
dialkylanilines,6 and the release of alkyl radicals during oxidative
aromatization of 4-alkyl-1,4-dihydropyridines.7
The one-electron oxidation of an N-cyclopropylamine leads
to a highly unstable8 cation radical that undergoes rapid
cyclopropyl ring-opening.9-11 This is consistent with the
reported formation of acyclic metabolites during the oxidation
of cyclopropylamines by non-P450 enzymes that are proposed
utilize a SET mechanism. For example, oxidation of 1 by the
flavoprotein monoamine oxidase (MAO) leads to formation of
acrolein and acrolein-inactivated enzyme.12 Oxidation of tra-
nylcypromine (trans-2-phenylcyclopropylamine) by horseradish
peroxidase (HRP), a hemoprotein whose redox cycle involves
a ferryl intermediate similar to one postulated in P450 turnover13
(cf. Scheme 2), leads to formation of cinnamaldehyde.14
Unfortunately, in no case has the three-carbon metabolite arising
from the P450-catalyzed N-dealkylation of a cyclopropylamine
been determined.15,16 Extensive data already support the hy-
pothesis that P450, like MAO and HRP, can oxidize at least
some of its substrates by an initial one-electron oxidation to
generate a radical-cation.13,17-20 However, to reach this conclu-
sion for cytochrome P450-catalyzed N-dealkylations will require
elucidating the fate of the cyclopropyl group that is lost.
Toward this end, we have investigated the oxidation of
N-cyclopropyl-N-methylaniline (3) by HRP, a well-known SET
enzyme.20-22 We used 3 rather than 1 as a probe because HRP
has a lower oxidation potential than P450 (∼0.75 V23 vs 1.7-
2.0 V,24 respectively) and is therefore unable to oxidize amines
25
such as 1 because of their higher E1/2 and pKa values14
compared to anilines such as 3. We previously reported26 that
the HRP oxidation of [1′-14C]-3 yields N-methylaniline (4), but
the anticipated three-carbon metabolites acrolein (5) and 3-hy-
droxypropanal (6) were not formed (see Scheme 3). Instead, a
polar unreactive 14C-containing metabolite eluted in the solvent
front on reverse phase HPLC, but was refractory to isolation
and characterization. In an attempt to trap an intermediate in
the formation of this solvent front metabolite, we added cyanide
ion to the incubations. This substantially decreased the amount
of 14C in the solvent front peak and resulted in the formation
of a new extractable metabolite, which we identified as
cyanotetrahydroquinoline 7.26
We have now extended this work and report herein the
complete fate of the cyclopropyl moiety during HRP-catalyzed
SET oxidation of 3 in the presence and absence of cyanide.
The results demonstrate that after one-electron oxidation the
cyclopropyl group in 3 undergoes rapid quantitative ring-opening
to an acyclic, distonic radical cation, and that the latter partitions
between (a) reacting with molecular oxygen leading to 4 and
3-hydroxypropionic acid in a 1:1 ratio, and (b) intramolecular
cyclization leading to quinoline-derived products. This work also
demonstrates and validates methodology appropriate for eluci-
dating the fate of the cyclopropyl moiety of cyclopropylamines
undergoing P450-catalyzed N-dealkylation reactions; such stud-
ies are currently under way in our laboratory.
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Results and Discussion
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Product Identification. To provide context for our study of
the SET oxidation of cyclopropylaniline 3 by HRP, we first
investigated the HRP oxidation of the related compounds N,N-
dimethylaniline (8) and N-isopropyl-N-methylaniline (9). HRP
oxidizes 8 completely within 15-30 min producing 4 plus
formaldehyde. Substrate 9 is oxidized very slowly (<10%
consumption after 60 min) to N-isopropylaniline (10) and
(10) Ha, J. D.; Lee, J.; Blascktock, S. C.; Cha, J. K. J. Org. Chem. 1998,
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Dihydropyridines, Cycloalkylamines, and N, N.-Dimethylanilines by Cyto-
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2001, 123, 349-350.

8504 J. Am. Chem. Soc., Vol. 123, No. 35, 2001
Shaffer et al.
formaldehyde; neither 4 nor acetone are formed. The slow,
minimal oxidation of 9 by HRP may be due to unfavorable steric
interactions between the isopropyl group and the HRP apopro-
tein,13,27 but the preference for HRP-catalyzed N-demethylation
over N-dealkylation has been reported previously for other
N-alkyl-N-methylanilines.28
Incubation of 3 with HRP results in complete oxidation of
the substrate within 30-60 min. However, in contrast to the
oxidation of 9 to 10, the only observed metabolite of 3 is 4;
neither N-cyclopropylaniline (11) nor formaldehyde are formed,
nor is there any inactivation of HRP. Control experiments
demonstrated that 11 is susceptible to oxidation by HRP, but
not so fast as to preclude its detection if it had actually formed
in the incubation. On the other hand, mass balance studies by
HPLC (A254) showed that the amount of 4 detected accounts
for only 20-25% of the amount of substrate 3 consumed.
Treatment of quenched incubations with DNP reagent29 and
subsequent HPLC and GC/EIMS analysis revealed the absence
of DNP derivatives of 5 and 6, the acyclic carbonyl metabolites
expected to arise from the N-dealkylation of 3 (viz. Scheme 3),
although control experiments showed these compounds to be
readily detectable at levels corresponding to as little as 5%
conversion of substrate. Unfortunately, cyclopropanone hydrate
(12), which could conceivably arise via carbinolamine 13 if C-1′
deprotonation of 3+• occurred instead of ring-opening to 14+•,
does not react with DNPH.26 Because 12 is a well-known
irreversible inactivator of HRP,30 it would be expected to
inactivate HRP at least partially if it were formed during the
oxidation of 3, causing deviations from the strict first-order
kinetics observed for loss of 3 under standard incubation
conditions. To explore this possiblity we synthesized 12 and
added it to incubations with HRP to observe its effect on enzyme
activity. In this control experiment, HRP was incubated with
12 (250 µM, half the normal concentration used for 3 and related
aniline-type substrates) under otherwise standard conditions for
5 min, after which dimethylaniline (8, 500 µM) was added and
the incubation monitored visually and by HPLC. Visually, the
reaction remained colorless for 60 min, and HPLC showed that
no loss of 8 took place. Under identical conditions without
addition of 12, HRP incubations of 8 become quite colorful as
cation-radical intermediates and quinoid reaction products are
generated, and HPLC shows complete consumption of 8 within
ca. 15 min. Since incubations with 3 follow strict first-order
kinetics up to >90% consumption of 3, no enzyme inactivation
is occurring, which suggests that 12 is not forming as a
significant metabolite from 3.
Figure 1. HPLC chromatograms (A254 and radioactivity) of a 15 min
incubation of 3 with HRP/H2O2 in the absence of cyanide (Panel A)
and a 45 min incubation of 3 with HRP/H2O2 in the presence of 1 mM
cyanide (Panel B).
lyophilization, not extractable from aqueous solution by organic
solvents, and unaffected by refluxing HCl (1.0 M). The latter
two observations in particular rule out the possibility that this
material was N-methylpropionanilide, formed by acid catalyzed
rearrangement of carbinolamine 13.31
Because oxidative N-dealkylation reactions often involve
iminium ion intermediates that can be trapped by cyanide,32-34
HRP incubations with 3 (0.5 mM) were conducted with 1 mM
KCN added. The presence of dilute cyanide slows the oxidation
of 3 slightly (∼60-90 min to attain complete oxidation) and
gives rise to a major new UV-absorbing radioactive peak which
was extracted and identified as the tetrahydroquinoline derivative
7 (Figure 1, Panel B).26 Throughout the incubation, 14C only
eluted in the solvent front peak and under the peaks corre-
sponding to unmetabolized 3 and cyanide adduct 7, the ratio of
7 to the solvent front metabolite was constant at 60:40, and the
total recovery of injected radioactivity was quantitative. The
radioactive material in the solvent front displayed the same
physical and chemical properties as that formed in the cyanide-
free incubations.
That the cyanide-trapped intermediate formed in the HRP
oxidation of 3 is a tetrahydroquinoline derivative suggests that
the ring-opening of 3+• is substantially faster9,11 than its
deprotonation35 on either the methyl group or the C-1′ position
of the cyclopropyl group (Scheme 3). Once the cyclopropyl ring
of 3+• opens to form 14+•, intramolecular cyclization followed
by deprotonation and a second one-electron oxidation would
form enamine 15, a two-electron oxidation product of 3, whose
conjugate acid form 15H+ could readily be trapped by cyanide36
to form 7 (Scheme 4). The fact that cyanide substantially
decreases the amount of 14C in the solvent front peak suggests
that cyanide is indeed trapping and stabilizing a precursor to
the solvent front metabolite. In the presence of cyanide, 4 is
Since DNP-trapping and HPLC with UV detection failed to
reveal any metabolites derived from the cyclopropyl moiety of
3, we resorted to radiolabeling it with 14C. HPLC analyses of
incubations showed that HRP oxidation of [1′-14C]-3 generates
one weakly UV-absorbing radioactive metabolite that elutes
within the solvent front (Figure 1, Panel A). Throughout the
entire incubation period, 14C eluted only in the solvent front
peak and under the peak corresponding to unmetabolized 3, and
total recovery of injected radioactivity was quantitative. As
shown by HPLC with both UV and 14C detection, the solvent
front metabolite is inert to DNPH reagent, not volatile upon
(31) Turro, N. J.; Hammond, W. B. Tetrahedron 1968, 24, 6029-6035.
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1376.

N-Dealkylation of an N-Cyclopropylamine
J. Am. Chem. Soc., Vol. 123, No. 35, 2001 8505
Scheme 4
identified the major metabolite of 3 as 1-methylquinolinium (16)
and the minor metabolite as 3-hydroxypropionic acid (17).
Structural assignments of these metabolites were confirmed by
spiking incubation samples with authentic standards and re-
measuring the NMR spectra; this approach avoided small pH-
dependent variations in the chemical shifts of various signals,
which were otherwise often observed. Thus, complete oxidation
of 3 by HRP under standard peroxidatic conditions yields 4 and
17 (0.2 mol each) and 16 (0.8 mol). Complete oxidation of 3
by HRP in the presence of 1 mM cyanide yields 4 and 17 (0.08
mol each), 16 (0.32 mol), and 7 (0.6 mol). With the elucidation
of 16 and 17 as solvent front metabolites of 3, it became obvious
why our earlier HPLC-based mass balance study accounted for
only 20-25% of starting material consumed; compound 17 has
no UV activity at 254 nm, and, ironically, compound 16 has a
UV absorption minimun at 254 nm.37
also formed in lower amounts; however, with or without
cyanide, much more 14C is found in the solvent front than would
be expected if the solvent front metabolite were only being
formed in a process stoichiometric with the formation of 4.
In an attempt to identify the highly polar, nonderivatizable
solvent front metabolite, we next resorted to 13C-labeling of the
cyclopropyl moiety of 3, which we hoped would allow us to
structurally elucidate the metabolite by NMR in situ. Thus, we
synthesized two different 13C-enriched forms of 3, namely [1′-
13C]-3 and [2′,3′-13C2]-3. Each 13C-enriched probe (with [1′-
14C]-3 added as a tracer for quantitation) was incubated
separately with HRP under standard peroxidatic conditions.
After complete oxidation of 3, the incubations were quenched,
and the solvent front metabolite peaks were isolated by
preparative HPLC and, following concentration in vacuo,
analyzed by 1H-decoupled 13C NMR. To our surprise, two
metabolites were detected in the same ratio in each isolated
solvent front fraction. The 13C spectrum of the solvent front
fraction from [1′-13C]-3 (Figure 2, Spectrum A) showed a major
metabolite as a singlet at δ149.1 and a minor metabolite as a
singlet at δ178.7. In the 13C spectrum of the solvent front
fraction from [2′,3′-13C]-3 (Figure 2, Spectrum B), the major
metabolite appeared as two doublets at δ147.1 (1JC-C ) 56 Hz)
and δ121.4 (1JC-C ) 56 Hz) while the minor metabolite
appeared as two doublets at δ 58.5 (1JC-C ) 37 Hz) and δ 39.5
(1JC-C ) 37 Hz). The observation of carbon-carbon couplings
in both of the solvent front metabolites from [2′,3′-13C]-3 shows
that the two enriched carbons remain contiguous in both of the
Mechanistic Considerations. The identification of 16 as the
major HRP metabolite of 3 is consistent with our previous
suggestion1 that enamine 15, a 2-electron oxidation product from
3, is an intermediate en route to cyanide adduct 7. However,
formation of 17 from 3 requires a 4-electron oxidation process.
To determine if 17 arose via oxidation of either 5 or 6, which
were anticipated but never observed as metabolites of 3, each
of these compounds was incubated separately with HRP and
H2O2 but neither was affected. Carbon-centered radicals gener-
ated from amines and other substrates undergoing HRP-
oxidation have been reported to react avidly with molecular
oxygen.38-40 We therefore conducted incubations of 3 with HRP/
H2O2 under anaerobic conditions to determine if molecular
oxygen was involved in the formation of 17 (and 4). Under
these conditions we found that 3 was converted quantitatively
to 16 and that this conversion was twice as fast under nitrogen
as under air. Anaerobic incubation of 3 in the presence of
cyanide yields both 16 (65%) and 7 (35%). In neither anaerobic
reaction is either 4 or 17 formed. Incubations were next
conducted under an atmosphere of 100% O2. Upon complete
consumption of 3, the products formed included 16 (0.3 mol)
and 4 and 17 (0.7 mol each). These observations show that
mechanistic branching occurs during the HRP oxidation of 3
and that the metabolites formed are dependent on the interaction
of 14+• with O2. In other words, if 14+• is not trapped by
molecular oxygen it undergoes intramolecular cyclization lead-
ing to 16 (Scheme 5, Path A), while if 14+• is trapped by oxygen,
intramolecular cyclization is prevented and 4 and 17 are formed
in equimolar amounts (Scheme 5, Path B).
During the aerobic oxidation of 3 by HRP we observed by
HPLC a minor transient peak at early times in the oxidation
reaction. This material is formed in larger amounts in anaerobic
oxidations and is not seen in incubations conducted under a
pure oxygen atmosphere. By means of GC/EIMS examination
of extracts of reaction mixtures, and GC/EIMS and HPLC
comparisons to an authentic standard, this transient intermediate
was identified as N-methyl-1,2,3,4-tetrahydroquinoline (18). The
transient appearance of 18 during the oxidation of 3 by HRP is
somewhat surprising, because it is an isomer of 3 rather than
an oxidation product. Nevertheless, control studies demonstrated
that HRP and H2O2 are both required for the conversion of 3 to
1
1
solvent front metabolites. Extensive H, 13C, H-1H COSY,
HMQC, and HMBC studies (see Supporting Information)
(37) For 16, λmax ) 315 nm, ꢀ315 ) 5,990 M-1 cm-1 (ꢀ254 ) 306 M-1
cm-1, ꢀ320 ) 5,390 M-1 cm-1); for 4, λmax ) 246 nm, ꢀ246 ) 12,720 M-1
cm-1 (ꢀ254 ) 9,093 M-1 cm-1, ꢀ320 ) 258 M-1 cm-1).
(38) Ashley, P. L.; Davis, D. K.; Griffin, B. W. Biochem. Biophys. Res.
Commun. 1980, 97, 660-666.
Figure 2. 1H-Decoupled 13C NMR spectra (125.8 MHz, NS ) 57730)
of the HPLC-isolated solvent front metabolites from [1′-13C]-3 (Spec-
trum A) and [2′,3′-13C]-3 (Spectrum B). Asterisks denote the sites of
13C-enrichment.
(39) Kedderis, G. L.; Rickert, D. E.; Pandey, R. N.; Hollenberg, P. F. J.
Biol. Chem. 1986, 261, 15910-15914.
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Chem. 1989, 264, 19828-19836.

8506 J. Am. Chem. Soc., Vol. 123, No. 35, 2001
Shaffer et al.
Scheme 5
should facilitate the application of cyclopropylamine-containing
mechanistic probes to reactions catalyzed by cytochrome P450
enzymes.
Experimental Section
Materials. Horseradish peroxidase (EC 1.11.1.7, RZ ) 0.5) was
obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in
0.4 M potassium phosphate buffer (pH 5.5) at a concentration of 102
µg/mL; this working stock solution was stable for months at 4 °C.
Sodium [14C]-formate (0.5 mCi, 55 mCi/mmol) was purchased from
Moravek Biochemicals, Inc. (Brea, CA). Sodium [13C]-formate (99%
enrichment) and [1,2-13C2]-ethyl iodide (99% enrichment) were procured
from Cambridge Isotope Labs (Cambridge, MA). Chemicals and
solvents of reagent or HPLC grade were supplied by Aldrich Fine
Chemical Co. (Milwaukee, WI), TCI America (Portland, OR) and Fisher
Scientific (Pittsburgh, PA). Water for incubation reagents and chro-
matography was distilled, followed by passage through a Millipore
Milli-Q Water System. Tetrahydrofuran (THF) was distilled under
nitrogen from sodium and benzophenone, and MeCN and Et2O were
distilled under nitrogen from CaH2 prior to use. Unless otherwise noted,
all air and moisture sensitive reactions were performed under a nitrogen
environment. Analytical thin-layer chromatography (TLC) was con-
ducted on Analtech Uniplate 250 µm silica gel plates with detection
by UV light and I2. For flash chromatography, Selecto Scientific 63-
200 mesh silica gel was employed. 1H NMR spectra were obtained at
either 400 or 500 MHz with Bruker DRX 400 and DRX 500
spectrometers, while 1H-decoupled 13C NMR spectra were obtained at
100.6 MHz on a Bruker 400 spectrometer. Proton chemical shifts are
reported in ppm (δ) relative to tetramethylsilane as inferred from shifts
of residual protons in the deuterated solvents (i.e., 7.26 ppm for CDCl3,
4.80 ppm for D2O, 2.50 ppm for d6-DMSO, and 3.31 ppm for CD3-
OD); 13C chemical shifts are in ppm relative to internal CDCl3 (77.23
ppm).
Scheme 6
Incubation Procedures. Incubations were generally conducted on
a 1.0 mL scale in 1.5 dram (ca. 15 × 50 mm) screw-cap vials and
contained, in order of addition, 962 µL of buffer (0.4 M potasium
phosphate, pH 5.5), 8 µL of HRP stock solution (0.021 nmol HRP),
20 µL of substrate stock solution (25 mM in MeCN) and, to initiate
reaction, 10 µL of 0.1 M aqueous hydrogen peroxide. Reactions were
stirred magnetically at room temperature (22-23 °C) under air and
stopped by addition of 670 µL of MeCN. Larger scale incubations used
the same proportions and procedure. Anaerobic incubations were
conducted similarly except that buffer, substrate, and HRP were
combined in a tube closed by a septum penetrated by a long syringe
needle (nitrogen inlet) and a short needle (nitrogen outlet). The solution
was purged for 10-12 min by a moderate stream of nitrogen gas so as
not to cause foaming or evaporative loss of substrate, and reaction was
initated by addition of nitrogen-purged hydrogen peroxide solution. The
nitrogen purge was maintained at a slow speed throughout the reaction.
At the appropriate time nitrogen-purged MeCN was added to stop the
reaction. Reactions under oxygen were conducted following the same
protocol using oxygen instead of nitrogen.
18. In separate experiments, HRP was shown to oxidize 18 to
16, whereas in the presence of cyanide both 16 and 7 were
formed from 18. To account for this aspect of the reaction we
propose the 14+• undergoes cyclization and deprotonation to
generate radical 19 as shown in Scheme 6; bimolecular
disproportionation of 19 would then yield 18 and 15, both of
which are ultimately oxidized to quinolinium ion 16.
Conclusions
Results presented above indicate that the cyclopropylaminium
ion generated by HRP oxidation of 3 reacts exclusively by ring-
opening fragmentation to the acyclic distonic radical 14+•. This
is in sharp contrast to the reactivity of the related aminium ions
8
+• and 9+•, both of which undergo exclusive deprotonation on
Metabolite Analysis by HPLC and GC/EIMS. Procedures for
direct analysis of incubations by HPLC, for derivatization of carbonyl
metabolites with dinitrophenylhydrazine, and for analysis of incubation
extracts by GC/EIMS have been described previously.26 Standard
solutions of potential carbonyl metabolites were prepared and analyzed
similarly. Thus, formaldehyde (380 µL, 5.1 mmol, 37% (w/v) solution)
was added dropwise with stirring to 0.15 M DNPH trapping solution
(35 mL, 5.3 mmol); a yellow precipitate formed immediately. After
15 min the yellow crystals were isolated by suction filtration, washed
with H2O (100 mL), and recrystallized from absolute EtOH to afford
N-(2,4-dinitrophenyl)-N′-methylene-hydrazine (978 mg, 92%) as yel-
low-orange crystals. Mp 166 °C, lit. mp 167 °C.29 Similarly, acetone
(370 µL, 5.0 mmol) was used to produce N-(2,4-dinitrophenyl)-N′-
isopropylidene-hydrazine (869 mg, 73%) as yellow crystals. Mp 125
°C, lit. mp 128 °C.29 Acrolein (340 µL, 5.1 mmol) afforded a cyclic
DNP derivative 1-(2,4-dinitrophenyl)-4,5-dihydro-1H-pyrazole, 710 mg,
59%) as dark orange crystals. Mp 165 °C, lit. mp 165 °C.29 3,3-
Diethoxy-1-propanol (500 µL, 3.2 mmol) was used as described above
their methyl group, and no doubt reflects the kinetic9-11 and
thermodynamic8 instability of 3+• vs that of its isomer 14+•.
Once formed, 14+• reacts via two competing pathways, one
unimolecular and one bimolecular and oxygen-dependent (Scheme
5). Intramolecular cyclization of 14+• into its phenyl ring,
followed by deprotonation and a second one-electron oxidation,
generates the iminium ion 15H+ and its conjugate base 15. In
incubations with cyanide present, 15H+ is trapped as 7;
otherwise 15 is further oxidized by HRP to quinolinium ion
16, which is the exclusive metabolite of 3 in the absence of
both cyanide and oxygen. In competition with intramolecular
cyclization, cation radical 14+• can also react with dissolved
oxygen via C-O bond formation leading ultimately to formation
of the N-dealkylated metabolite 4 plus 3-hydroxypropionic acid
(17) derived from the cyclopropyl moiety of 3. The synthetic
and analytical methods developed in the course of these studies

N-Dealkylation of an N-Cyclopropylamine
J. Am. Chem. Soc., Vol. 123, No. 35, 2001 8507
to produce N-(2,4-dinitrophenyl)-N′-(3-hydroxypropylidene)-hydrazine,
the DNP derivative of 3-hydroxypropanal, (138 mg, 17%) obtained as
gold crystals after flash chromatography (5% MeOH in CH2Cl2). Mp
123-124 °C; TLC (CH2Cl2:MeOH, 9.5:0.5) Rf ) 0.34; 1H NMR (400
MHz, CD3OD) δ 2.65 (q, J ) 6.0 Hz, 2H), 3.87 (t, J ) 6.3 Hz, 2H),
7.79 (t, J ) 5.3 Hz, 1H), 8.01 (d, J ) 9.6 Hz, 1H), 8.36 (dd, J ) 9.6,
2.6 Hz, 1H), 9.06 (d, J ) 2.6 Hz, 1H); GC tR ) 23.7 min; EIMS: [M
- 18]+ ) 236; Elemental analysis calcd for C9H10N4O5 C: 42.53, H:
3.97, N: 22.04; found, C: 42.75, H: 3.68, N: 21.60.
(33% Et2O in pentane) to yield N-methyl-[14C]-formanilide (195 µCi,
69%) as a pale yellow oil which was stored in 6 mL of solvent (pentane:
Et2O:EtOAc, 4:1:1) at 0 °C. TLC (pentane:Et2O, 2:1) Rf ) 0.22. Using
the same procedure as described for the synthesis of 3, N-methyl-[14C]-
formanilide (195 µCi, 0.39 mmol), titanium isopropoxide (250 µL, 0.85
mmol), ethylmagnesium bromide (3.0 M in Et2O, 600 µL, 1.8 mmol),
and THF (5 mL) afforded [1′-14C]-3 (56 µCi, 29%) as a colorless oil,
which was stored in 5 mL of pentane at -4 °C. TLC (pentane:Et2O,
40:1) Rf ) 0.70. Prior to its use in incubations, [1′-14C]-3 was purified
(>99%) by preparative HPLC using an Alltech Silica column (10 µµ,
4.6 × 250 mm), eluting with 0.1% EtOAc in hexanes at a flow rate of
1 mL/min. This step was necessary to remove small amounts of
N-methyl-N-propylaniline and N-methyl-N-(3-pentyl)aniline formed as
minor byproducts in the cyclopropanation reaction.
NMR Analysis of Metabolites. Incubations were conducted as
described above and contained 33 µg (0.87 nmol) of HRP, 10 µmol of
13C-enriched 3 and 20 µmol of H2O2 in a final volume of 10 mL of
potassium phosphate buffer (0.4 M, pH 5.5). After complete oxidation
of 3 (as determined by HPLC), the incubation was quenched with
MeCN (6.7 mL) and concentrated in vacuo to ca. 2 mL. The solvent
front metabolite peak (tR ) 2.7-3.9 min) was isolated by preparative
HPLC (as described for aniline HPLC analysis) by collecting the column
effluent of seven injections. The isolated effluent was concentrated in
vacuo to ca. 500 µL and added to a 5 mm NMR tube containing 50 µL
D2O, and the 1H-decoupled 13C NMR spectrum was obtained by a
Bruker 500 MHz NMR at a spectral frequency of 125.8 MHz (NS )
57730). Spectra of authentic standards of 16 and 17 were obtained
similarly: 16 1H NMR (400 MHz, D2O) δ 4.58 (s, 3H), 7.94 (m, 2H),
8.17 (t, J ) 7.4 Hz, 1H), 8.27 (m, 2H), 9.02 (d, J ) 8.4 Hz, 1H), 9.14
(d, J ) 5.7 Hz, 1H); 13C NMR (100.6 MHz, D2O) δ 45.18, 118.01,
121.35, 129.39, 129.91, 130.24, 135.69, 138.47, 147.36, 149.00; 17
1H NMR (400 MHz, D2O) δ 2.43 (t, J ) 6.0 Hz, 2H), 3.72 (t, J ) 5.9
Hz, 2H); 13C NMR (100.6 MHz, D2O) δ 38.64, 58.46, 178.87.
[1′-13C]-3 was prepared from sodium [13C]-formate using the
methods described above. Sodium [13C]-formate (415 mg, 6.0 mmol),
18-crown-6 (3.41 g, 12.9 mmol), THF (15 mL), and benzyl bromide
(900 µL, 7.6 mmol) afforded benzyl [13C]-formate (636.7 mg, 77%)
as a colorless oil. TLC (pentane:Et2O, 10:1) Rf ) 0.48; 1H NMR (400
3
MHz, CDCl3) δ 5.21 (d, JC-H ) 3 Hz, 2H), 7.37 (m, 5H), 8.14 (d,
1JC-H ) 226 Hz, 1H); EIMS: [M]+ ) 137. This intermediate was
converted to N-methyl-[13C]-formanilide in 84% isolated yield (485
mg). TLC (pentane:Et2O, 2:1) Rf ) 0.21; 1H NMR (400 MHz, CDCl3)
3
δ 3.33 (d, JC-H ) 3 Hz, 3H), 7.18 (d, J ) 7.6 Hz, 2H), 7.28 (t, J )
1
7.2 Hz, 1H), 7.42 (t, J ) 7.9 Hz, 2H), 8.48 (d, JC-H ) 197 Hz, 1H);
EIMS: [M]+ ) 136. Conversion to [1′-13C]-3 was effected using the
same procedure as described for 3, i.e., N-methyl-[13C]-formanilide (415
mg, 3.1 mmol), titanium isopropoxide (1.3 mL, 4.4 mmol), ethylmag-
nesium bromide (3.0 M in Et2O, 3.9 mL, 11.7 mmol) and THF (15
mL) afforded [1′-13C]-3 (165.9 mg, 37%) as a colorless oil. TLC
N-Cyclopropyl-N-methylaniline (3)41 and Its Isotopically Labeled
Forms. Ethylmagnesium bromide (3.0 M in Et2O, 4 mL, 12 mmol)
was added dropwise to a vigorously stirred solution of N-methyl-
formanilide (500 µL, 4.1 mmol) and titanium isopropoxide (1.6 mL,
5.4 mmol) in THF (20 mL). The solution was heated at 65 °C for 30
min and then stirred at room temperature for 24 h. After quenching
the reaction with saturated NH4Cl (15 mL) and removing the THF under
reduced pressure, the crude solution was diluted with 1 N HCl (15
mL), extracted with Et2O (5 × 5 mL), dried (MgSO4), and concentrated
in vacuo. The crude oil was purified by flash chromatography (2.4%
Et2O in pentane) to afford 3 (187 mg, 31%) as a colorless oil. TLC
1
(pentane:Et2O, 40:1) Rf ) 0.71; H NMR (400 MHz, CDCl3) δ 0.62
1
3
(m, 2H), 0.80 (m, 2H), 2.37 (m, JC-H ) 173 Hz, 1H), 2.98 (d, JC-H
) 3 Hz, 3H), 6.76 (t, J ) 7.3 Hz, 1H), 6.99 (d, J ) 8.7 Hz, 2H), 7.24
(t, J ) 5.4 Hz, 2H); EIMS: [M]+ ) 148.
[2′,3′-13C2]-3 was prepared using [1,2-13C2]-ethyl iodide as the source
of the Grignard reagent. Thus, [1,2-13C2]-iodoethane (500 mg, 3.2 mmol)
and Et2O (1 mL) were drawn into a syringe and added dropwise to a
flask containing freshly crushed magnesium turnings (84.3 mg, 3.5
mmol), a tiny crystal of elemental iodine, and Et2O (3 drops). The
solution was stirred until refluxing ceased and nearly all the Mg was
consumed. The [1,2-13C2]-EtMgI ethereal solution (∼3.2 M) was
immediately added via syringe to a mixture of N-methylformanilide
(190 µL, 1.5 mmol), titanium isopropoxide (600 µL, 2.0 mmol), and
THF (7.5 mL). Standard workup afforded [2′,3′-13C2]-3 (31.5 mg, 7%)
as a colorless oil. TLC (pentane:Et2O, 40:1) Rf ) 0.67; 1H NMR (400
1
(pentane:Et2O, 40:1) Rf ) 0.70; H NMR (400 MHz, CDCl3) δ 0.62
(m, 2H), 0.81 (m, 2H), 2.37 (m, J ) 3.4 Hz, 1H), 2.97 (s, 3H), 6.76 (t,
J ) 7.2 Hz, 1H), 6.99 (d, J ) 7.9 Hz, 2H), 7.25 (t, J ) 8.0 Hz, 2H);
13C NMR (100.6 MHz, CDCl3) δ 9.26, 33.48, 39.30, 113.97, 117.57,
129.01, 151.05; GC tR ) 12.1 min; EIMS: [M]+ ) 147.
[1′-14C]-3 was synthesized as described above from [formyl-14C]-
N-methylformanilide, which was obtained in two steps as follows.
Dried, finely ground sodium formate (67.6 mg, 0.994 mmol) and sodium
[14C]-formate (9.09 × 10-3 mmol in 5 mL of aqueous EtOH, 500 µCi)
were transferred to a 16 × 100 mm glass culture tube and concentrated
to dryness in vacuo. Methanol (6 mL) and 18-crown-6 (568 mg, 2.2
mmol) were added to the culture tube, and the homogeneous solution
was concentrated slowly under reduced pressure to afford an amorphous
solid. THF (5 mL) was added, and the tube was sealed with a Teflon-
lined screw-cap and heated to 70 °C. After being heated for 24 h, the
tube was cooled to room temperature, benzyl bromide (150 µL, 1.3
mmol) was added, and the solution was again heated at 70 °C for
another 60 h. Upon cooling, the THF was removed by careful Vigreux
distillation, and the residue was diluted with H2O (8 mL), extracted
with Et2O (3 × 4 mL), dried (MgSO4), and concentrated by Vigreux
distillation. The crude oil was purified by flash chromatography (9%
Et2O in pentane) to afford benzyl [14C]-formate (282 µCi, 56%) as a
colorless oil. TLC (pentane:Et2O, 10:1) Rf ) 0.47. The latter was
combined with Kugelrohr-distilled N-methylaniline (500 µL, 4.6 mmol)
and heated at 95 °C for 48 h in a screw-cap culture tube sealed under
N2. After being cooled to room temperature, the reaction solution was
diluted with 1 N HCl (5 mL) and extracted with Et2O (5 × 2 mL). The
combined extracts were dried (MgSO4) and concentrated by Vigreux
distillation, and the crude oil was purified by flash chromatography
1
1
MHz, CDCl3) δ 0.62 (dm, JC-H ) 162 Hz, 2H), 0.82 (dm, JC-H
)
158 Hz, 2H), 2.37 (m, J ) 3.2 Hz, 1H), 2.98 (s, 3H), 6.76 (t, J ) 6.9
Hz, 1H), 6.99 (d, J ) 7.8 Hz, 2H), 7.25 (t, J ) 8.0 Hz, 2H); 13C NMR
(100.6 MHz, CDCl3) δ 9.24, 33.13 (1JC-C ) 37.7 Hz), 39.31, 114.01,
117.61, 129.02, 151.06; EIMS: [M]+ ) 149.
General Procedure for the Synthesis of 9, 10, and 18. Sodium
cyanoborohydride (15 mmol) was added to a stirred solution of the
respective Kugelrohr-distilled aniline analogue (5 mmol) and the
requisite carbonyl compound (15 mmol) in MeCN (10 mL).42 Glacial
acetic acid (500 µL, 9.3 mmol) was added to the solution over a 10
min period, and the reaction was stirred at room temperature. After 2
h, glacial acetic acid (500 µL, 9.3 mmol) was again added to the
solution, which was stirred at room temperature for an additional 15
h. The reaction was quenched with 2 N KOH (20 mL) and extracted
with Et2O (5 × 5 mL), and the extracts were washed with brine (10
mL), dried (Na2SO4), and concentrated in vacuo. The residue was
purified by flash chromatography (9% Et2O in pentane) to afford the
desired compound. N-Isopropyl-N-methylaniline (9, from 4 and
acetone) was obtained as a colorless oil (29% yield). TLC (pentane:
Et2O, 10:1) Rf ) 0.65; 1H NMR (400 MHz, CDCl3) δ 1.16 (d, J ) 6.6
Hz, 6H), 2.72 (s, 3H), 4.09 (sp, J ) 6.6 Hz, 1H), 6.69 (d, J ) 7.3 Hz,
1H), 6.79 (d, J ) 8.0 Hz, 2H), 7.23 (t, J ) 8.0 Hz, 2H); 13C NMR
(100.6 MHz, CDCl3) δ 19.52, 29.97, 49.09, 113.50, 116.58, 129.31,
150.41; GC tR ) 11.3 min; EIMS: [M]+ ) 149. N-Isopropylaniline
(41) Chaplinski, V.; de Meijere, A. Angew. Chem., Int. Ed. Engl. 1996,
35, 413-414.
(42) Borch, R. F.; Hassid, A. I. J. Org. Chem. 1972, 37, 1673-1674.

8508 J. Am. Chem. Soc., Vol. 123, No. 35, 2001
Shaffer et al.
(10, from aniline and acetone) was obtained as a colorless oil (66%
yield). TLC (hexanes:EtOAc, 4:1) Rf ) 0.77; 1H NMR (400 MHz,
CDCl3) δ 1.25 (d, J ) 6.3 Hz, 6H), 3.67 (sp, J ) 6.2 Hz, 1H), 6.62 (d,
J ) 8.6 Hz, 2H), 6.71 (t, J ) 7.3 Hz, 1H), 7.20 (t, J ) 7.5 Hz, 2H);
13C NMR (100.6 MHz, CDCl3) δ 23.18, 44.33, 113.38, 117.09, 129.44,
147.68; GC tR ) 10.0 min; EIMS: [M]+ ) 135. N-Methyl-1,2,3,4-
tetrahydroquinoline (18, from 1,2,3,4-tetrahydroquinoline and form-
aldehyde) was obtained as a colorless oil (64% yield). TLC (pentane:
Et2O, 10:1) Rf ) 0.56; 1H NMR (400 MHz, CDCl3) δ 2.04 (m, J ) 6.3
Hz, 2H), 2.82 (t, J ) 6.4 Hz, 2H), 2.94 (s, 3H), 3.27 (t, J ) 5.7 Hz,
2H), 6.67 (t, J ) 7.5 Hz, 2H), 7.00 (d, J ) 7.1 Hz, 1H), 7.13 (t, J )
7.5 Hz, 1H); 13C NMR (100.6 MHz, CDCl3) δ 22.90, 28.23, 39.56,
51.72, 111.40, 116.64, 123.30, 127.48, 129.25, 147.19; GC tR ) 13.6
min; EIMS: [M]+ ) 147.
to 12 was observable by using 1H NMR to monitor the disappearance
of the two doublets from the methylene protons of 1-ethoxycyclopro-
panol and the concomitant appearance of a singlet for the methylene
1
protons of 12. H NMR (400 MHz, D2O) δ 0.86 (s, 4H); 13C NMR
(100.6 MHz, D2O) δ 14.20, 79.87.
3-Hydroxypropanal (6).45 Acrolein (5, 14 µL, 210 µmol), H2O (493
µL), and D2O (50 µL) were combined in a 5 mm NMR tube and left
at room temperature for 14 days. Direct NMR analysis of the solution
1
detected 6 in 50% yield. H NMR (400 MHz, D2O) δ 2.74 (td, J )
5.8, 1.7 Hz, 2H), 3.92 (t, J ) 5.8 Hz, 2H), 9.69 (t, J ) 1.7 Hz, 1H);
13C NMR (100.6 MHz, D2O) δ 39.78, 58.32, 207.12.
Acknowledgment. Fellowship support for C.L.S. was pro-
vided by the American Foundation for Pharmaceutical Education
and an NIH Predoctoral Training Grant (GM-07775).
1-Ethoxycyclopropanol.43 [(1-Ethoxycyclopropyl)oxy]trimethyl-
silane (3 mL, 14.9 mmol) was added all at once to MeOH (10 mL),
and the solution was stirred at room temperature for 21 h. The title
compound (965.2 mg, 64%) was isolated from the reaction solution
1
Supporting Information Available: 1H, H-1H COSY,
1
by rotary evaporation as a colorless oil. H NMR (400 MHz, CDCl3)
heteronuclear multiple quantum correlation (HMQC), and
heteronuclear multiple bond correlation (HMBC) spectra for
incubation solutions containing 16 and 17 upon the aerobic
oxidation of 3 by HRP/H2O2; complete 1H and 13C NMR
chemical shift assignments for 16 and 7. This material is
available free of charge via the Internet at http://pubs.acs.org.
δ 0.84 (d, J ) 10.2 Hz, 4H), 1.13 (t, J ) 7.1 Hz, 3H), 3.68 (q, J ) 7.1
1
Hz, 2H), 4.04 (br s, 1H); H NMR (400 MHz, D2O) δ ppm 0.96 (dd,
J ) 5.3, 5.2 Hz, 4H), 1.19 (t, J ) 7.2 Hz, 3H), 3.77 (q, J ) 7.2 Hz,
2H); 13C NMR (100.6 MHz, CDCl3) δ 14.08, 15.34, 61.90, 85.23.
Cyclopropanone Hydrate (12).44 A solution of 1-ethoxycyclopro-
panol (7.8 mg, 76 µmol) in 500 µL of KH2PO4 buffer (0.4 M, pH 5.5)
and 50 µL of D2O was heated at 100 °C for 7 min in a 5 mm NMR
tube to afford 12 in quantitative yield. The conversion of the hemiketal
JA0111479
(43) Salau¨n, J.; Marguerite, J. Org. Synth. 1989, 63, 147-153.
(44) Wiseman, J. S.; Abeles, R. H. Biochemistry 1979, 18, 427-435.
(45) Mao, J.; Doane, R.; Kovacs, M. F. J. J. Liq. Chrom. 1994, 17 (8),
1811-1819.