Table 1 Product distributions of rat liver microsomal and
CYP2B1-catalyzed N-dealkylation of N-cyclopropyl-N-(2-methyl)
cyclopropyl-p-chloroanilines (2 & 3): (5), N-cyclopropyl-p-chloro-
aniline; (6), N-(2-methyl)cyclopropyl-p-chloroaniline; and (7), 2-Me-
hydroxylated product
Table 2 Product distributions of CYP2B1 and HRP catalyzed
N-dealkylation of 4a–c: (10), N-ethyl-p-chloroaniline; (11), N-b-fluoro-
ethyl-p-chloroaniline
CYP2B1a
HRPa
10
a
CYP2B1a
10
11a–c
11a–c
Microsomal P450
5
6
7
5
6
7
4a
4b
4c
a
68.2 (3)
30.0 (3)
7.9 (2)
31.8 (3)
70.0 (3)
92.1 (2)
77 (2)
70 (2)
94 (1)
23 (2)
30 (2)
6 (1)
2
3
a
4 (1)
8 (2)
18 (1)
92 (2)
77 (1)
NDb
6 (1)
7 (2)
16 (1)
93 (2)
78 (1)
ND
Each entry is an average of at least three independent
determinations. The standard deviations for the last significant
figures are given in parentheses.
Each entry is an average of at least three independent
determinations. The standard deviation for the last significant figures
are given in parentheses. ND, not detected.
b
which was increased up to 94% for the trifluoro derivative (4c).
These results unequivocally demonstrate that the chemistries of the
two systems are quite contrasting and distinct with respect to
substrates 4a–c.
under similar incubation conditions demonstrating that, while
HRP reactions proceed exclusively through a cyclopropyl ring
opening pathway, P450 reactions proceed through a pathway that
does not involve the opening of the cyclopropyl rings. On the other
hand, Ca–Hs of the cyclopropyl rings in P450 catalyzed
N-dealkylation of 2 & 3 are removed at isotopically sensitive steps
along the catalytic pathway.
The incremental fluorine substitution in 4a–c increases the
production of the N-fluoroethyl group cleaved product (10) in
HRP catalyzed reactions, most likely due to the increased acidity
of the Ca–Hs of the N-b-fluoroethyl group relative to the N-ethyl
group of the SET intermediate, nitrogen cation radical. In
contrast, the incremental fluorine substitution disfavours the
N-b-fluoroethyl group cleavage in P450 reactions, suggesting that
the acidities of the Ca–Hs are not an important determinant for
P450-catalyzed N-dealkylations. On the other hand, the decrease of
the formation of 10 with the incremental fluorine substitution in
P450/4a–c reactions correlates well with the increase in Ca–H bond
dissociation energies rather than with the acidities.11
P450 catalyzed N-dealkylations of
2 & 3, should have
preferentially produced the 2-methylcyclopropyl ring opened/
cleaved products, if a nitrogen cation radical is a transient
intermediate, based on the product distributions of HRP catalyzed
oxidation of 2 & 3. In contrast, as shown in Table 1, P450 catalyzed
reactions preferentially produces the N-cyclopropyl cleaved
product 6. Although, the steric constraints of the active site
residues may be responsible for these differences, the high steric
tolerance of the P450 active site10 toward structurally diverse
substrates rules out this possibility. Similarly, since the acidity of
the Ca–H should not be significantly affected by the presence of
the methyl group on the cyclopropyl ring, the difference in product
distribution could not be due to the difference in acidities of the
Ca–H. Therefore, these findings strongly suggest that the nitrogen
cation radical could not be an intermediate in P450-catalyzed
N-dealkylation of 2 & 3, which is in excellent agreement with our
previous results.6
The apparent similarity in the product distributions of
monofluoro derivative 4a in P450 and HRP reactions may be a
consequence of the relative magnitudes of bond dissociation and
Ca-radical stabilization energies of this particular derivative. For
example, theoretical studies have shown that the Ca–H abstraction
from the N-b-monofluoroethyl is favoured over N-ethyl due to the
high relative stability of the b-fluoromethyl-Ca-radical in compar-
ison to the b-methyl-Ca-radical.11 Therefore, both Ca–H atom
abstraction from the neutral substrate and Ca–H deprotonation of
the cation radical intermediate could occur favourably from the
N-b-monofluoroethyl group giving rise to the favourable cleavage
of N-b-fluoroethyl group regardless of whether the SET/H+ or
HAT mechanisms are operative. Taken together, the above results
suggest that while the partition ratios of HRP/4a–c reactions were
determined by the relative acidities of the Ca–H of the
N-substituents, partition ratios of P450/4a–c reactions may be
determined by the bond dissociation energies.
In order to further test whether the acidities of Ca–Hs of
N-substituents determine the product distributions of HRP or P450
catalyzed N-dealkylations under competitive conditions, we have
synthesized and characterized a series of b-fluoro analogs of N,
N-diethyl-p-chloroaniline (4a–c). Incubations of 4a–c with P450
produced both N-ethyl-p-chloroaniline (10) and N-b-fluoroethyl-
p-chloroaniline (11a–c) as the primary products (Scheme 3). The
partition ratios were highly dependent on the number of fluorine
atoms on the substrate and preferential formation of 11a–c
observed as the number of fluorine atoms on the N-ethyl
substituent is increased as shown in Table 2. On the other hand,
incubations of 4a–c with HRP produced 10 as the major product,
In conclusion, the lack of cyclopropyl ring opened or ring
opened radical cyclized products from P450/2 & 3 reactions along
with the product profiles of P450/4a–c reactions suggest against a
SET/H+ transfer mechanism for P450 catalyzed N-dealkylations
and provide strong support for the hydrogen atom abstraction
mechanism.
We thank the National Institutes of Health, GM 45026 (K.W.)
and CA16954 (P. F. H.) for their financial support.
Mehul Bhakta,a Paul F. Hollenbergb and Kandatege Wimalasena*a
aDepartment of Chemistry, Wichita State University, Wichita, Kansas,
67260, USA. E-mail: kandatege.wimalasena@wichita.edu;
Fax: 316-978-3431; Tel: 316-978-7386
Scheme 3 Product distribution of CYP2B1 and HRP catalyzed
N-dealkylations of N-ethyl-N-(b-fluoro)ethyl-p-chloroaniline derivatives
(4a–c).
bDepartment of Pharmacology, The University of Michigan, Ann Arbor,
266 | Chem. Commun., 2005, 265–267
This journal is ß The Royal Society of Chemistry 2005