7412
J. Am. Chem. Soc. 1996, 118, 7412-7413
Scheme 1
Effects of Structure on the Reactivity of
r-Hydroxydialkylnitrosamines in Aqueous
Solutions1
Milan Mesic´, Cynthia Revis, and James C. Fishbein*
Department of Chemistry, Wake Forest UniVersity
Winston-Salem, North Carolina 27109
ReceiVed April 10, 1996
The carcinogenic activity of certain dialkylnitrosamines (1,
Scheme 1) is thought to be a consequence of DNA alkylation
by diazonium ions and carbocations that are ultimately derived
from decomposition of R-hydroxydialkylnitrosamines (2, Scheme
1).2,3 The R-hydroxydialkylnitrosamines are the products of
enzymatic hydroxylation of dialkylnitrosamines, and it has been
speculated that biological derivatives may act as transport forms
(3, Scheme 1, X ) glucuronyl, -PO3H-, or -SO3-) for the
diffusion of the R-hydroxydialkylnitrosamine moiety to distant
tissues where it, and the alkylating equivalents therein, might
be liberated.3 Thus, R-hydroxydialkylnitrosamines are central
intermediates in the deleterious biological activities of a large
number of nitrosodialkylamines. It was once believed that
R-hydroxydialkylnitrosamines were too unstable to be isolated,
but through the ingenious efforts of the Okada group, the
syntheses and stabilities of a few such compounds were reported
in the early 1980s.4-6 Synthesis of only R-hydroxymethyl-
alkylnitrosamines (2, R ) H) was achieved. These compounds
(2, R′ ) Me, Et, n-Pr, n-Bu, sec-Bu, and tert-Bu) were
reportedly “stable”5 in acidic media with reactivities otherwise
varying with substitution by less than a factor of 10. Since the
first reports, there has been no other report of the effects of
structure on the chemistry and reactivity of these important
reactive intermediates in nitrosamine carcinogenesis. It was
presciently suggested7 that substitution on the hydroxymethyl
group (2, R * H) might significantly enhance the reactivity of
R-hydroxydialkylnitrosamines. We report here a kinetic study
of the decay of some R-hydroxydialkylnitrosamines, all deriva-
tives of potent carcinogenic dialkylnitrosamines, that indicates
that their stability is indeed dramatically affected by substitution
on the hydroxymethyl group.
chloride to survive separation from the triphenylphosphine oxide
by chromatography, as previously reported,4-6 while 2b-d were
not. For kinetic studies, compounds 2b-d were generated in
freshly dried (from CaH) argon-purged acetonitrile by treatment,
in an argon tent for 2d, of the R-hydroperoxy precursor with
1-10 equiv of tributylphosphine. Kinetics were monitored upon
mixing volumes of acetonitrile solutions, after solvent exchange4
in the case of 2a, with a 25-fold (or 10-fold for 2d) excess
volume of aqueous buffer using an Applied Photophysics DX17
MV stopped-flow spectrophotometer.9
The reaction kinetics, monitored by disappearence of 2a-c
at 230 nm and by appearence of benzaldehyde from 2d at 255
or 265 nm, exhibited excellent first-order behavior. Typically
five determinations of kobsd under a single set of conditions
yielded a mean value of kobsd with a standard deviation (σn-1
)
of (2%. In the case of 2d, the kinetic constants were
independent of the concentrations of the reducing phosphine
concentration between 1 and 10 molar equiv, and, at 10-3
M
HCl, UV spectra of the reaction progress as a function of time
indicated a clean isosbestic point at 238 nm.10 Plots of kobsd
against buffer concentration containing 4-5 buffer concentra-
tions were linear with increases of less than 15% above the
value, k0, of kobsd extrapolated to the zero intercept of the buffer
concentration axis. Most values of k0 were thus obtained from
such plots that generally contained two or three points.
The plots of log k0 against pH for reactions of compounds
2a-d at 25 °C, ionic strength 1 M (NaClO4), 4% or 9%
acetonitrile, are presented in Figure 1. Where comparable, the
data for 2a are quantitatively similar to the data previously
reported for 2a under similar reaction conditions.4 The data in
Figure 1 are consistent with rate law of eq 1, which contains
three terms; a pH independent term, k1; a term whose contribu-
tion is inversely proportional to hydrogen ion concentration,
k2; a term whose contribution is directly proportional to
hydrogen ion concentration, k3. The latter term has not been
previously reported. The fit of the data to the rate law of eq 1
is indicated by the solid lines in Figure 1 for the values of the
Synthesis of the R-hydroxydialkylnitrosamines 2a-d was
effected in a manner that was strategically analogous to one of
the two original4 methods, entailing triphenylphosphine reduc-
tion of the R-hydroperoxy precursors (Scheme 1, 3, with X )
OH).8 The compound 2a was sufficiently stable in methylene
(1) Warning! The R-hydroxydialkylnitrosamines and their R-hydro-
peroxy precursors are presumed carcinogens and must be handled accord-
ingly.
(2) Lawley, P. D. In Chemical Carcinogens; Searle, C. D., Ed.; ACS
Monograph 182; American Chemical Society: Washington, DC, 1984.
(3) Lijinsky. Chemistry and Biology of N-nitroso Compounds; Cambridge
University Press: Cambridge, 1992.
(4) Mochizuki, M.; Anjo, T.; Okada, M. Tetrahedron Lett. 1980, 21,
3693.
(5) Okada, M.; Mochizuki, M.; Anjo, T.; Sone, T.; Wakabayashi, Y.;
Suzuki, E. IARC Sci. Publ. 1980, 31, 71.
(6) Mochizuki, M.; Anjo, T.; Takeda, K.; Suzuki, E.; Sekiguchi, N.;
Huang, G. F.; Okada, M. IARC Sci. Publ. 1980, 31, 71.
(9) In the case of 2d, the traditional arrangement of the two-syringe
mixing aparatus was replaced by introducing the substrate solution in
acetonitrile, initially protected from the reaction cell by a 10-20 µL bolus
of dry acetonitrile, into the reaction cell supply line by means of a T-junction
and an additional filling syringe that contained the substrate solution and
that could be isolated from the flow system by a third valve. The collinear
ends of the T-junction were attached to the reaction cell by means of a 120
µL loop and to one of the two mixing syringes containing freshly dried
(from CaH) acetonitrile.
(10) The λmax ()252 nm) of the product was identical with that of
benzaldehyde. HPLC analysis (C-18 column, CH3CN/H2O eluant) indicated
a 90((1)% yield of benzaldehyde for reactions in 10-3 M HCl. In the
case of 2d, a few runs with a 25-fold excess of aqueous buffer gave values
of k0 that were within 7% of those obtained in experiments in which a
10-fold excess of aqueous buffer was used.
(7) Loeppky, R. N. IARC Sci. Publ. 1980, 31, 82.
(8) R-Hydroperoxydialkylnitrosamines (3, Scheme 1, X ) OH). 3b: 1H
NMR (CDCl3) δ 1.14, (t, J ) 7.2 Hz, 3 H); 1.61, (d, J ) 6.5 Hz, 3 H);
3.41-3.73, (m, 1 H); 6.33, (q, J ) 6.4 Hz, 1 H); 8.9, (s, broad, 1 H). 3c:
1H NMR (CDCl3) δ 0.87, (d, J ) 6.8 Hz, 3 H); 1.20, (d, J ) 6.6 Hz, 3 H);
2.00-2.19, (m, 1 H); 2.99, (s, 3 H); 5.85, (d, J ) 9.8 Hz, 1 H); 8.72, (s, 1
H). 3d: 1H NMR (CDCl3) δ 2.83, (s, 3 H); 7.26, (s, 1 H); 7.35-7.52, (m,
5 H); 10.39, (s, 1 H). R-Hydroxydialkylnitrosamines (2, Scheme 1). 2b:
1H NMR (CDCl3) δ 1.10, (t, J ) 7.2 Hz, 3 H); 1.64, (d, J ) 6.4 Hz, 3 H);
3.5-3.75, (m, 2 H); 4.2 (s, broad, 1 H); 6.25, (o, J ) 6.2 Hz, 1 H). 2c:
1H NMR (CDCl3) δ 0.78, (d, J ) 6.7 Hz, 3 H); 1.16, (d, J ) 6.6, 3 H);
2.00-2.2, (m, 1 H); 2.97 (s, 3 H); 5.59, (q, J ) 4.88 Hz, 1 H); 6.16, (d, J
) 4.85 Hz, 1 H). 2d: 1H NMR (CDCl3) δ 2.83, (s, 3 H); 7.23, (s, 1 H);
7.34-7.50, (m, 5 H); 11.3 (br, 1 H).
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