Alkylating potential of N-phenyl-N-nitrosourea

Article abstract of DOI:10.1002/poc.1456

Alkylating agents are considered to be archetypical carcinogens. Since the decomposition of alkylnitrosoureas gives rise to alkyldiazonium ions without any need for metabolic activation, they offer an ideal series of compounds with which to examine the ef

Full text of DOI:10.1002/poc.1456

Research Article
Received: 29 July 2008,
Revised: 5 September 2008,
Accepted: 5 September 2008,
Published online in Wiley InterScience: 27 October 2008
(www.interscience.wiley.com) DOI 10.1002/poc.1456
Alkylating potential of N-phenyl-N-nitrosourea
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a
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J. A. Manso , M. T. Perez-Prior , R. Gomez-Bombarelli , M. Gonzalez-Perez ,
a
a
a
a
*
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I. F. Cespedes , M. P. Garcı´a-Santos , E. Calle and J. Casado
Alkylating agents are considered to be archetypical carcinogens. Since the decomposition of alkylnitrosoureas gives
rise to alkyldiazonium ions without any need for metabolic activation, they offer an ideal series of compounds with
which to examine the effect of variations in chemical structure on alkylating potential. Due to its instability
N-phenyl-N-nitrosourea is easily hydrolyzed to a benzenediazonium ion. Since N-phenyl-N-nitrosourea shows a
particular behavior when compared with other nitrosoureas, here its alkylating potential was studied. The nucleo-
phile 4-(p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents, was used as an alkylation substrate. Conclusions
were drawn as follows: (i) the mechanism of the alkylation reaction of NBP by phenylnitrosourea is different from that
caused by other nitrosoureas; (ii) the formation of the NBP–N-phenyl-N-nitrosourea adduct occurs about 20,000-fold
slower than with N-methyl-N-nitrosourea, one of the most effective carcinogenic nitrosoureas; and, (iii) a correlation
was found between the alkylating potential of nitrosoureas and their tumorigenicity. Copyright ß 2008 John Wiley &
Sons, Ltd.
Keywords: reactivity of N-phenyl-N-nitrosourea; alkylation reactions
previously used this method to investigate the alkylating
INTRODUCTION
potential of strong alkylating reagents such as lactones[12–16]
as well as to determine the reactivity of much weaker alkylating
molecules, such as sorbates.[17,18]
Alkylating agents are considered to be archetypical carcino-
gens.[1,2] Since the decomposition of alkylnitrosoureas (ANU)
gives rise to alkyldiazonium ions (RNþ2 ) without any need for
metabolic activation, they offer an ideal series of compounds with
which to examine the effect of variations in chemical structure on
alkylating potential.
The NBP alkylation reaction by N-phenyl-N-nitrosourea was
investigated. The effective alkylating agent resulting from the
decomposition of PhNU is the benzenediazonium ion, giving rise
to NBP-PhNU adduct (Scheme 1).
Previous studies (Chapter 3[3]) have shown that some ANUs
induce tumors in different organs of rats, but does not have a
favorable partition coefficient into organic solvents from water. If
physical properties such as solubility and partition coefficient
play any role, it must be minor and perhaps limited to effects on
potency. Some results[4] have indicated the structure of the alkyl
group adjacent to the nitroso group as the most important
determinant of the carcinogenic effect.
In order to render the NBP soluble, the PhNUþ NBP alkylation
mixtures were prepared in a 7:3 (vol) water/dioxane medium.
Reactions were carried out in the 5.0–6.0 pH range. Acetic/acetate
buffer was used to maintain pH constant. To monitor the alkylation
reactions, 2.4 mL aliquots of the alkylation mixture were removed
at different times and added to a cuvette containing 0.6 mL of 99%
triethylamine reagent (Et3N), which stopped the alkylation process
and generated a yellow color, the absorbance of which was
measured at the wavelength of maximum absorption (see below).
The observation of N2 bubbles along the reaction shows that this
occurs through a dediazoniation mechanism.
We tested the alkylating potential of various N-alkyl-
N-nitrosoureas. We found that these alkylation reactions occur
mainly through steric control.[5]
Due to its instability, N-phenyl-N-nitrosourea[6] (PhNU) is easily
hydrolyzed to a benzenediazonium ion (PhNþ2 )[7] and induces
local tumors in rats.[8] However, painting PhNU on mouse skin
failed or induced few skin tumors.[9] The reason for its failure to
induce skin tumors is, therefore, not clear.
Despite its chemical relevance, to our knowledge, the
chemical reactivity of N-phenyl-N-nitrosourea as an alkylating
agent has not been investigated in a comparative quantitative
way, and accordingly, we were prompted to address this issue.
The NBP-PhNU adduct showed maximum absorption at
l ¼ 425 nm. As an example, Fig. 1 shows the increase in
absorption caused by the formation of the NBP-PhNU adduct
over time until no change in absorbance, A, was observed.
Because the NBP was in large excess, it can be assumed that all
the nitrosourea was consumed.
*
Correspondence to: J. Casado, Departamento de Qu´ımica f´ısica, Universidad
de Salamanca, E-37008 Salamanca, Spain.
E-mail: jucali@usal.es
RESULTS AND DISCUSSION
´ ´ ´ ´
J. A. Manso, M. T. Perez-Prior, R. Gomez-Bombarelli, M. Gonzalez-Perez, I. F.
a
The nucleophile 4-(p-nitrobenzyl)pyridine (NBP), a trap for
alkylating agents[10] with nucleophilic characteristics similar to
DNA bases,[11] was used as the alkylation substrate. We had
´
Cespedes, M. P. Garc´ıa-Santos, E. Calle, J. Casado
Departamento de Qu´ımica f´ısica, Universidad de Salamanca, E-37008
Salamanca, Spain
J. Phys. Org. Chem. 2009, 22 386–389
Copyright ß 2008 John Wiley & Sons, Ltd.

REACTIVITY OF N-PHENYL-N-NITROSOUREA
Scheme 1.
Figure 3. Formation of the NBP-Ph adduct in a 7:3 water/dioxane med-
ium; [PhNU]o ¼ 2 10À4 M; [NBP] ¼ 0.02 M; pH ¼ 5.72; T ¼ 37.5 8C. Ao and
At, designate the absorbance values of the adducts at times, respectively,
of 0 and t
Figure 1. Spectrograms showing the formation of the NBP-Ph adduct
over time in a 7:3 water/dioxane medium. Variation in absorbance in the
10–90 min interval. [PhNU]o ¼ 2 10À4 M; [NBP] ¼ 0.01 M; T ¼ 35 8C; and
pH ¼ 5.72
Table 1. Alkylation rate constants as a function of
temperature for benzenediazonium ion in a 7:3 water/dioxane
medium
T (8C)
kalk (MÀ1 minÀ1 a
)
25.0
27.5
30.0
32.5
35.0
37.5
1.0 Æ 0.2
1.4 Æ 0.2
1.7 Æ 0.2
2.2 Æ 0.4
3.0 Æ 0.4
3.5 Æ 0.4
a Values are given with their standard deviations
medium was observed, unlike other nitrosoureas.[5] Therefore,
the mechanism of the NBP alkylation by PhNU, through
the formation of the benzenediazonium ion, which is more
stable than aliphatic alkyldiazonium ions, must be different from
that of other nitrosoureas. Since the intercept of the k1/[NBP] plot
is not significantly different from 0 (Fig. 2), the possibility of some
competitive reactions, such as solvolytic dediazoniations,[19] was
discarded in the current conditions.
Figure 2. Influence of [NBP] in the alkylation reaction of NBP by PhNU at
35 8C in a 7:3 water/dioxane medium. [PhNU]o ¼ 2 10À4 M and pH ¼ 5.72
In agreement with the mechanism in Scheme 1, and
½PhNþ2 ꢀo ffi ½PhNUꢀo, the alkylation reaction rate of NBP by
phenylnitrosourea is
d½ADꢀ
rate ¼
¼ kalk½PhNþ2 ꢀ½NBPꢀ ¼ k1½PhNþ2 ꢀ;
(1)
dt
By designating as Ao, At, and A , the absorbance values of the
1
NBP-PhNU adduct at times, respectively, of 0, t, and infinity (i.e.,
where, [AD] represents the concentration of the NBP-PhNU
adduct, and k1 ¼ kalk [NBP] is the pseudo-first-order rate constant.
In the 5.0–6.0 pH range, no dependence on the acidity of the
when the plateau is reached; see Fig. 3), "PhNþ , "NBP, and "AD
2
being the molar absorption coefficients of PhNþ2 , NBP, and
J. Phys. Org. Chem. 2009, 22 386–389
Copyright ß 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

J. A. MANSO ET AL.
Table 2. Alkylating potential of N-phenyl-N-nitrosourea and N-methyl-N-nitrosourea and their tumorigenicity
N-alkyl-N-nitrosourea
Alkylating potential (Table 1) (35 8C)
kalk (MÀ1 minÀ1 a
Tumorigenicity[9]
)
Number of Swiss mice with skin tumors
N-methyl-N-nitrosourea
N-phenyl-N-nitrosourea
62,000 Æ 2,000
3.0 Æ 0.4
19
1
a Values are given with their standard deviations.
adducts, respectively, and x the adduct concentration, Eqns
(2)–(4) can be written.
þ
þ
Ao ¼ "
½PhN ꢀ þ "NBP½NBPꢀ
(2)
(3)
(4)
PhN2
2
o
þ
þ
At ¼ "
ð½PhN ꢀ À xÞ þ "NBP½NBPꢀ þ x"AD
PhN2
2
o
A
¼ "AD½PhNþꢀ þ "NBP½NBPꢀ
1
2
o
Integration of Eqn (1) gives
At À Ao ¼ ðA1 À AoÞð1 À eÀk t
Þ
(5)
1
Figure 3 shows the fitting of the experimental data to Eqn (5).
In order to calculate the alkylation rate constant,
alk(Scheme 1), experiments at different [NBP] were carried out
k
(Fig. 2). The intercept, not significantly different from 0, revealed
first order with respect to the concentration of [NBP] according to
Eqn (1).
Table 1 gives the values of kalk for the alkylation reaction as a
function of temperature (T ).
Figure 4. Absorption of NBP-PhNU adduct in a 7:3 water/dioxane med-
ium
The value of the rate constants for NBP alkylation by
phenylnitrosourea is consistent with its biological activity in
comparison with other nitrosoureas.[9] The NBP alkylation rate
constant by the methyldiazonium ion (previously calculated with
data from[5] and[20]) is about 20,000-fold greater than that of the
phenyldiazonium ion. This result is consistent with their relative
biological activities, can be observed in Table 2.
The structures of the NBP-PhNU adduct obtained by geometry
optimization (refer to Experimental Section) revealed (Fig. 5) a
planar adduct, with an angle between the phenyl rings, a,
approximately equal to 08. Thus, the e value should be greater
The structural characteristics of alkylnitrosoureas, such as
molecular size, are important as regard to their biological activity,
hindered alkylnitrosoureas being relatively inactive biologi-
cally.[3,21] This is consistent with the sequence of the alkylating
rate constants found here for the respective methyldiazonium
and benzenediazonium ions.
The calculated activation energy is Ea ¼ 75 Æ 2 kJ molÀ1
.
The kinetic results here observed are consistent with the fact
that the biological activity of alkylnitrosoureas decreases when
their molecular size increases hindered alkylnitrosoureas being
biologically inactive.[3]
Molar absorption coefficient of the NBP-PhNU adduct
We were also interested in knowing the molar absorption
coefficient (e) of NBP-PhNU adduct. Knowledge of these values
should permit easy determination of the concentration of adduct
by simply measuring the absorbance.
Experiments were performed with [NBP] ¼ 0.02 M and PhNU
concentrations in the 4 10À5–3.5 10À4 M range.
The plot in Fig. 4 shows the absorption coefficients to be
eNBP-PhNU ¼ 7,674 Æ 343 MÀ1 cmÀ1 (l ¼ 425 nm).
Figure 5. Lack of coplanarity in the NBP-ANU adducts
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Copyright ß 2008 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2009, 22 386–389

REACTIVITY OF N-PHENYL-N-NITROSOUREA
Procedures: synthesis of N-phenyl-N-nitrosourea
Table 3. Correlation between the structure of NBP-ANU
adducts and their molar absorption coefficients, eAD
.
PhNU were prepared from the respective N-phenylurea (this urea
was obtained from Fluka). PhNU was prepared as by Werner.[22]
N-phenylurea (approx. 1 g) was dissolved in water and 10 g of
sodium nitrite was added. The mixture was cooled to below 0 8C
and ice-cold sulphuric acid (10%) was added dropwise during
continuous stirring. The nitrosourea precipitated as yellow
crystals. After vacuum filtration, the crystals collected were
repeatedly washed with cool water and then desiccated.
N-alkyl-N-nitrosourea
a (8)
eAD (MÀ1 cmÀ1
)
N-phenyl-N-nitrosourea
N-methyl-N-nitrosoureaa
N-allyl-N-nitrosoureaa
N-ethyl-N-nitrosoureaa
N-propyl-N-nitrosoureaa
N-butyl-N-nitrosoureaa
ꢂ 0
29.0
31.0
31.2
31.6
31.6
7,674 Æ 343
521 Æ 30
282 Æ 20
204 Æ 8
93 Æ 11
65 Æ 8
Acknowledgements
a Values obtained from Reference [5].
´
We thank the Spanish Ministerio de Ciencia e Innovacion
´
(CTQ2007-63263), as well as the Spanish Junta de Castilla y Leon
(Grant SA040 A08) for supporting the research reported in this
´
paper. R.G.B. thanks the Ministerio de Ciencia e Innovacion, I. F. C.
thanks the Spanish Ministerio de Asuntos Exteriores y de Co-
than that of the other nitrosoureas studied.[5] The greater the
non-planarity in the NBP-ANU adducts, disrupting the p-electron
cloud to interlink the two phenyl rings, the smaller the values of e,
as can be seen in Table 3.
´
operacion as well as Agencia Espanola de Cooperacion Inter-
nacional and J.A.M., M.T.P.P., and M. G. P. thank the Junta de
˜
´
A similar argument can be made to rationalize the fact that the
biological activity of ANUs decreases when their molecular size
increases:[3] the smaller the alkyl group of the alkyldiazonium ion,
the stronger the linkage between DNA nucleophile sites and the
electrophilic alkyldiazonium ions.
´
Castilla y Leon, for PhD grants. Thanks are also given for the
valuable comments made by the referees.
REFERENCES
[1] P. D. Lawley, in: Chemical Carcinogens, 2nd ed. (Ed.: C. E. Searle), ACS
Monograph 182, American Chemical Society, Washington DC, 1984.
325.
[2] S. J. Lu, M. C. Archer, Mol. Carcinogen. 1992, 6, 260–265.
[3] W. Lijinsky, Chemistry and Biology of N-Nitroso Compounds. Cam-
bridge University Press, Cambridge, UK, 1992.
[4] W. Lijinsky, R. K. Elespuru, A. W. Andrews, Mutat. Res. 1987, 178,
157–165.
CONCLUSIONS
ꢁ The mechanism of the alkylation reaction of NBP by
N-phenyl-N-nitrosourea is different from that caused by other
nitrosoureas.
ꢁ The formation of the NBP–N-phenyl-N-nitrosourea adduct
occurs about 20,000-fold slower than with N-methyl-
N-nitrosourea, one of the most effective carcinogenic nitro-
soureas.
´
[5] J. A. Manso, M. T. Perez Prior, M. P. Garc´ıa Santos, E. Calle, J. Casado,
J. Phys. Org. Chem. 2008, DOI: 10.1002/poc.1402, 21, 932–938.
[6] R. Uehara, Bull. Chem. Soc. Jpn. 1960, 33, 12–14.
[7] J. Casado, A. Castro, F. M. Lorenzo, F. Meijide, M. Mosquera, Bull. Soc.
Chim. Fr. 1985, 4, 597–601.
[8] R. Preussmann, H. Druckrey, J. Bucheler, Z. Krebsforsch. 1968, 71,
63–65.
ꢁ A correlation was found between the alkylating potential of
nitrosoureas and their tumorigenicity.
[9] W. Lijinsky, C. Winter, J. Cancer Res. Clin. Oncol. 1981, 102, 13–20.
[10] J. H. Kim, J. J. Thomas, Bull. Environ. Contam. Toxicol. 1992, 49,
879–885.
EXPERIMENTAL
[11] S. E. Shephard, W. K. Lutz, Cancer Surv. 1989, 8, 401–421.
´
´
[12] M. P. Garc´ıa Santos, S. Gonzalez Mancebo, J. Hernandez Benito, E.
Calle, J. Casado, J. Am. Chem. Soc. 2002, 124, 2177–2182.
General remarks
´
[13] J. A. Manso, M. T. Perez Prior, M. P. Garc´ıa Santos, E. Calle, J. Casado,
Chem. Res. Toxicol. 2005, 18, 1161–1166.
[14] M. P. Garc´ıa Santos, E. Calle, J. Casado, J. Am. Chem. Soc. 2001, 123,
7506–7510.
A Shimadzu UV-2401-PC spectrophotometer with a thermoelec-
tric six-cell holder temperature control system (Æ0.1 8C) was used.
The reaction temperature was kept constant (Æ0.05 8C) with a
Lauda Ecoline RE120 thermostat.
´
[15] J. A. Manso, M. T. Perez Prior, M. P. Garc´ıa Santos, E. Calle, J. Casado,
J. Solution Chem. 2008, 37, 451–457.
´
´
´
´
A Crison Micro pH 2000 pH-meter was used to perform pH
measurements (Æ0.01).
[16] R. Gomez Bombarelli, M. Gonzalez Perez, M. T. Perez Prior, J. A. Manso,
E. Calle, J. Casado, Chem. Res. Toxicol. 2008, in press, DOI: 10.1021/
tx800153j
Water was deionized with a MilliQ-Gradient (Millipore).
All kinetic runs were performed in triplicate.
´
[17] M. T. Perez Prior, J. A. Manso, M. P. Garc´ıa Santos, E. Calle, J. Casado,
J. Solution Chem. 2008, 37, 459–466.
Numerical treatment of data was performed using the
7.1.44 Data Fit software. Geometry optimization of the NBP-
alkyldiazonium adducts was carried out with the Chem3D Ultra
Molecular Modeling and Analysis software, version 9.0, and with
Gaussian 03W Client Pro 9.0. The PM3 semiempirical method was
used.
´
[18] M. T. Perez Prior, J. A. Manso, M. P. Garc´ıa Santos, E. Calle, J. Casado,
J. Agric. Food Chem. 2005, 53, 10244–10247.
´
[19] R. Pazo Llorente, M. J. Sarabia Rodr´ıguez, C. Bravo D´ıaz, E. Gonzalez
Romero, Int. J. Chem. Kinet. 1999, 31, 73–82.
[20] F. McGarrity, T. Smyth, J. Am. Chem. Soc. 1980, 102, 7303–7308.
[21] W. Lijinsky, Qual. Assur. 1992, 1, 115–119.
[22] E. A. Werner, J. Chem. Soc. 1919, 115, 1093–1102.
J. Phys. Org. Chem. 2009, 22 386–389
Copyright ß 2008 John Wiley & Sons, Ltd.
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