Kinetic study of the nitrosation of 1,3-dialkylureas in aqueous-perchloric acid medium

Article abstract of DOI:10.1002/kin.10192

The kinetics of the nitrosation of 1,3-dimethyl (DMU), 1,3-diethyl (DEU), 1,3-dipropylurea (DPU), 1,3-dibuthyl (DBU), and 1,3-diallylurea (DAU) were studied in a conventional UV/vis spectrophotometer in aqueous-perchloric acid media. The kinetic study was carried out using the initial rate method. The reaction rate observed was r = k[H⁺]²/[H⁺] + K_a [Diurea][Nitrite] where K_a is the acidity constant of nitrous acid. The diureas exhibited the reactivity order DMU ? DEU > DPU > DAU, which can be interpreted as a function of the steric impediment generated by the R alkyl group in the rate controlling step. A probable relationship between both the chemical reactivity and structure of the nitrosable substrate with the biological activity of the N-nitroso compounds generated is proposed.

Full text of DOI:10.1002/kin.10192

Kinetic Study
of the Nitrosation
of 1,3-Dialkylureas
in Aqueous-Perchloric
Acid Medium
1
2
´
´
GUILLERMO GONZALEZ-ALATORRE, SALVADOR H. GUZMAN-MALDONADO, ELEAZAR
1
3
1
˜
´
´
M. ESCAMILLA-SILVA, GUADALUPE LOARCA-PINA, CARLOS HERNANDEZ BENITEZ
1Departamento de Ingenierı´a Quı´mica, Instituto Tecnolo´gico de Celaya, C. P. 38010, Celaya, Guanajuato, Me´xico
2Instituto Nacional de Investigaciones Forestales, Agrı´colas y Pecuarias, Campo Experimental Bajı´o, Celaya Guanajuato,
Me´xico
3Universidad Auto´noma de Quere´taro, Divisio´n Estudios de Posgrado, Quere´taro, Quere´taro, Me´xico
Received 18 April 2003; accepted 12 November 2003
DOI 10.1002/kin.10192
ABSTRACT: The kinetics of the nitrosation of 1,3-dimethyl (DMU), 1,3-diethyl (DEU), 1,3-
dipropylurea (DPU), 1,3-dibuthyl (DBU), and 1,3-diallylurea (DAU) were studied in a conven-
tional UV/vis spectrophotometer in aqueous-perchloric acid media. The kinetic study was car-
ried out using the initial rate method. The reaction rate observed was
k[H+]2
[H+] + Ka
r =
[Diurea][Nitrite]
where Ka is the acidity constant of nitrous acid. The diureas exhibited the reactivity order DMU
ꢀ DEU > DPU > DAU, which can be interpreted as a function of the steric impediment gener-
ated by the R alkyl group in the rate controlling step. A probable relationship between both the
chemical reactivity and structure of the nitrosable substrate with the biological activity of the
N-nitroso compounds generated is proposed. ꢁ 2004 Wiley Periodicals, Inc. Int J Chem Kinet
C
36: 273–279, 2004
INTRODUCTION
For many years, nitrosoalkylureas have served as an
example of direct carcinogenic agents, those that do
not require enzymatic activation (as do nitrosamines)
to reveal their mutagenic and carcinogenic capacity
[1–4].
Correspondence to: Guillermo Gonza´lez-Alatorre; e-mail:
alatorre@iqcelaya.itc.mx.
Contract grant sponsor: Consejo Nacional de Ciencia y Tec-
nolog´ıa de Guanajuato (CONCYTEG).
Contract grant number: Project 02-09-201-016.
Contract grant sponsor: Consejo del Sistema de Educacio´n Tec-
nolo´gica (COSNET).
In the case of the biological activity of the ni-
trosamines, statistically significant correlations have
Contract grant number: Project 900.03-P.
2004 Wiley Periodicals, Inc.
c
ꢁ

´
GONZALEZ-ALATORRE ET AL.
274
been found between carcinogenic activity and water–
hexane partition coefficients [5]. As far as the authors
are aware, there is no published data regarding a cor-
relation between partition coefficients and the biologi-
cal activity of nitrosoalkylureas. This lack of data is
possibly due to the difference between the carcino-
genesis mechanism of nitrosamides and that of ni-
trosamines. In nitrosamines, which are more stable in
aqueous solutions than nitrosoalkylureas [6], the re-
quirements for carcinogenic potency appear dominated
by the structure of the unmetabolized precarcinogen
[5]. Therefore, the transport of the original molecule
to its active site has an important effect on its potency.
Nitrosoalkylureas, unstable in an aqueous medium [3],
and therefore direct carcinogenic agents, are unlikely
to have the transport of molecules to their active sites as
the rate limiting step in the carcinogenesis mechanism.
The mechanistic model for the biological activity of
nitrosoureas is based on the relative ease with which
these compounds decompose, giving way to the diazo-
nium ions responsible for the alkylation of the DNA
molecules [4–6]. Diverse studies have been conducted
to relate the chemical structure of the nitrosoureas (ni-
trosomonoalkylureas and nitrosodialkylureas) to their
biological activity to form a more solid base for this
model [2,3]. The experiments were carried out using
different experimental animals and nitrosoureas with
different stabilities and chemical structures [7–9]. It
has not been possible to establish a simple pattern that
indicates that the observed mutagenic power is the sole
consequence of the stability of the associated nitroso-
compound; rather, it depends on sex and type of ex-
perimental animal, application form, dose and func-
tional group linked to the N-nitroso compound, among
others.
EXPERIMENTAL
Solutions of 1,3-dimethylurea (DMU), 1,3-diethylurea
(DEU), 1,3-dipropylurea (DPU), 1,3-dibuthylurea
(DBU), 1,3-diallylurea (DAU), and sodium nitrite
(NaNO2) were prepared from analytical reagents
(Aldrich Co. Mexico). Ionic strength (I) was main-
tained with NaClO4 monohydrate (Aldrich Co.
Mexico).
It was found that N-nitrosodimethylurea, N-
nitrosodimethylurea, N-nitrosodipropylurea, and N-
nitrosodibuthylurea products showed a maximum ab-
sorbance of 249 nm whereas the N-nitrosodiallylurea
had 238 nm. At these wavelengths, the nitrite mo-
lar absorption coefficient is negligible with respect
to the nitrosoalkylureas (e.g., for nitrosomethylurea
ε240 = 3500 M−1 cm−1 and ε240 = 8 M−1 cm−1 for
nitrite).
Nitrosation kinetics were recorded using a Perkin-
Elmer UV/vis spectrophotometer lambda 25 equipped
with a thermoelectric cell holder thermostat Haake
DC1 maintaining the temperature within 0.1◦C.
Acidity was measured with an Ultra Basic Bench top
Denver pH meter.
The initial rate method was applied to the nitrosation
of diureas.
RNHCONHR + NaNO2
(DRU)
(nitrite)
H3+
O
−→ RN(NO)CONHR + Na+ + 2H2O
(NDRU)
where R = Methyl, Ethyl, Propyl, Buthyl, and Allyl
groups.
Studies have been undertaken on the reactions of
the nitrosation of ureas [10–15] and as a result, a reac-
tion mechanism for the nitrosation of monoureas with
the general formula RNHCONH2 (R = Methyl, ethyl,
propyl, buthyl, and allylurea) has been proposed [11].
The results of the kinetic study on a series of diureas
with the general formula RNHCONHR (R = Methyl,
ethyl, propyl, buthyl, and allylurea), in an aqueous-
perchloric acid medium, using the initial rate method,
are presented in this work. By using this method it was
possible to carry out the kinetic study at slow reaction
rates where it would have been impossible to use the
integral method [10,11]. This study produced an ex-
perimental rate equation consistent with the reaction
mechanism proposed in previous works [10–15].
The kinetic study yielded the reactivity order associ-
ated with the five nitrosation reactions and was related
to the structure of the substrate to be nitrosated and the
N-nitroso compound produced.
The rate expression is
ro = k[DRU]αo [nitrite]βo
(1)
In order to determine the reaction orders, Eq. (1)
became
ꢀ
ꢁꢀ
ꢁ
1
εNDRU
dA
dt
ro =
= k[DRU]αo [nitrite]βo (2)
t→0
where εNDRU is the molar absorption coefficient of the
corresponding nitrosodiurea and A is the absorbance of
the NDRU at the wavelength of maximum absorption.
In the determination of the influence of acidity on
k, all the variables involved in the nitrosation, such
as nitrite concentrations and DRU, ionic strength, and
temperature, were kept constant whereas the proton
concentration was varied in the interval 1 × 10−4
<
[H+] < 1.7 × 10−3 M. The following expression,

NITROSATION OF 1,3-DIALKYLUREAS IN AQUEOUS-PERCHLORIC ACID MEDIUM
275
Table I Reaction Orders for the Nitrosation of Diureas
(Figs. 1 and 2)
obtained from Eq. (2), was used to find the functionality
between k and [H+]
Substrate
α
β
(1/εNDRU)(dA/dt)t→0
k =
DMU
DEU
DPU
DBU
DAU
1.06 0.05
1.03 0.02
1.03 0.00
1.01 0.03
1.03 0.05
1.01 0.030
0.98 0.010
1.03 0.010
1.00 0.028
1.00 0.048
[DRU]ꢀo [nitrite]βo
ꢀ
ꢁ
dA
dt
= M
= f ([H+])
(3)
t→0
All the terms that remained constant throughout the
experimentation were grouped in parameter M.
where b and c are constants resulting from the lineal
correlation. Equations (3) and (4) yield
RESULTS AND DISCUSSION
M[H+]2
b + c[H+]
k =
(5)
The reaction orders for the nitrosation of the five di-
ureas are shown in Table I (Figs. 1 and 2).
The functionality between k and [H+], Eq. (3), was
consistent with that observed in monoureas, Eq. (4),
using the integral method [10,11]. See Fig. 3.
Substituting orders α = 1 and β = 1 in Eq. (1), the
experimental rate for the nitrosation of diureas is
reached.
M[H+]2
b + c[H+]
[H+]2
r =
[nitrite][DRU]
(6)
= b + c[H+]
(4)
(dA/dt)t→0
Figure 2 Order with respect to the diureas under study
Figure 1 Order reaction with respect to the nitrite [Eq. (3)]
in the nitrosation of the five diureas under study, T = 298 K
[Eq. (4)], T = 298 K and I = 0.1 M; : 1.7 × 10−4
<
◦
[DMU]o < 1 × 10−3 M, [nitrite]o = 3 × 10−4 M, [H+] =
and I = 0.1 M; : [DMU]o = 5 × 10−4 M, 1 × 10−4
<
◦
5 × 10−4 M; : 2.5 × 10−3 < [DEU]o < 1.2 × 10−2 M,
[nitrite]o < 5 × 10−4 M, [H+] = 5 × 10−4 M; : [DEU]
ꢀ
ꢀ
o
[nitrite]o = 3 × 10−4 M, [H+] = 1 × 10−2 M; ꢁ: 4.1 × 10−4
= 5 × 10−4 M, 7.4 × 10−4 < [nitrite]o < 3.6 × 10−2 M,
< [DPU]o < 1.53 × 10−3 M, [nitrite]o = 5 × 10−5 M, [H+]
[H+] = 1 × 10−2 M; ꢁ: [DPU] = 5 × 10−4 M, 2 × 10−4
<
= 1.5 × 10−2 M; : 6.1 × 10−4 < [DBU]o < 1 × 10−3 M,
[nitrite]o < 1.5 × 10−3 M, [H+] = 1 × 10−2 M; : [DBU]o
ꢀ
ꢀ
[nitrite]o = 2.5 × 10−4 M, [H+] = 2.5 × 10−2 M; +: 3 ×
10−4 < [DAU]o < 7.1 × 10−4 M, [nitrite]o = 2.5 × 10−4
M, [H+] = 2.5 × 10−2 M.
= 2.5 × 10−4 M, 3 × 10−4 < [nitrite]o < 1 × 10−3 M, [H+]
= 2.5 × 10−2 M; +: [DAU]o = 2.5 × 10−4 M, 2.4 × 10−4
< [nitrite]o < 1.7 × 10−3M, [H+] = 2.5 × 10−2 M.

´
GONZALEZ-ALATORRE ET AL.
276
as the rate limiting step in the formation mechanism
of nitrosoamides has been confirmed by the following
observations:
i. absence of nucleophilic catalysis on the nitrosa-
tion of amides [12,14]
ii. presence of primary solvent isotope effects on
the nitrosation of mono and dialkylureas [11,15]
iii. general base catalysis in the mono and dialky-
lureas nitrosation [11,15]
The initial attack of the nitrosating agent (Scheme 1)
occurs on the oxygen atom given that it is the most
nucleophilic site of the molecule [12,14].
T−heoretical rate Eq. (7) is obtained considering that
[NO2 ] = 1 × 10−4 M ꢂ [H2O], and that [NaNO2] =
[HNO2] + [NO−2 ]:
Figure 3 Fitting of experimental data to Eq. (14) [DMU]
= [DEU] = [DPU] = [DBU] = [DAU] = 8.2 × 10−2 M,
[nitrite] = 1 × 10−4 M, 1 × 10−4 < [H+] < 9.8 × 10−4 M,
k
H O K1 K2[H+]2
2
r =
[DRU][NaNO2]
[H+] + Ka
T = 298 K, I = 0.03 M. : DMU, : DEU, ꢁ: DPU,
:
ꢀ
[H+]2
◦
ꢀ
DBU, +: DAU.
=
[DRU][NaNO2] (7)
[H+]/kw exp + Ka/kw exp
The mechanism for the nitrosation of the series of di-
ureas under study, Scheme 1, is consistent with the ex-
perimental rate equation [compare Eqs. (6) and (7)] and
is similar to that proposed for the nitrosation of mono
and dialkylureas [10–12,14,15]. The proton transfer
from the protonated N-nitroso compound to the water
where kw exp = kH O K1 K2.
2
Parameter Ka = 7 × 10−4 [16], [see Eq. (7)], is ob-
tained from parameters b and c, Eq. (4), for the five
diureas under study. The differences between the value
of Ka determined by direct methods and the values
obtained from the kinetic study (see Table II) can be
Scheme 1 Proposed mechanism for the nitrosation of diureas in an aqueous-perchloric acid medium.

NITROSATION OF 1,3-DIALKYLUREAS IN AQUEOUS-PERCHLORIC ACID MEDIUM
277
Table II Values of Ka [Eq. (4)]
Table III Values of kw exp [Eq. (9)]
kw exp (s−1 M−2
)
Substrate
Ka (M)
Substrate
Dimethylurea
Diethylurea
Dipropylurea
Dibuthylurea
Diallylurea
6.7 × 10−4
3.6 × 10−4
2.0 × 10−4
1.9 × 10−4
3.9 × 10−4
Dimethylurea
Diethylurea
Dipropylurea
Diallylurea
258.4 9.1
32.5 0.7
19.8 0.9
10.5 0.4
Note: The substrate dibuthylurea was discontinued when this
experiment was carried out.
attributed to experimental error and/or the presence of
other chemical species in the reagent solution.
obtained that fit Eq. (9).
The reactivity of the substrates was related to the
kw exp parameter (see Scheme 1), determined by the
integral method, considering (α = 1, β = 1) and using
the concentrations of the diureas in excess with respect
to the nitrite concentration. To enable the use of the
integral method, it was necessary to take [H+] > 3.5 ×
k = m[H+]
(9)
Therefore, under these conditions, the experimental
rate equation is
10−3 M in such a way that the value of A could be
∞
r = m[H+][nitrite][DRU]
(10)
obtained in a reaction time of less than 6 h. Under
these conditions, the rate equation for the nitrosation
of diureas is r = kꢃ [nitrite], where kꢃ = k [DRU]. The
corresponding integrated equation in function of the
absorbance of the product is
Equations (10) and (7) are consistent when [H+] ꢀ Ka,
where m = kw exp (see Table III).
According to the proposed mechanism, the longi-
tude of the R groups will affect the rate controlling
step. This can be justified by bearing in mind that
the R alkyl groups are hydrophobic and will there-
fore tend to fold back on themselves in an aque-
ln(A∞ − A) = −kt + ln(A∞ − Ao)
(8)
Applying this equation to a study of the influence of
acidity on k values, Fig. 4, experimental data were
ous medium, Fig. 5(A) [10]. This affects the kw exp
,
and by extension, the chemical reactivity of the sub-
strates giving way to the sequence DMU > DEU >
DPU > DAU (Table III). This sequence can also be
related to the stability of the resulting nitrosoureas
and their biological activity. The instability of the N-
nitrosoamides in an aqueous medium is attributed to the
closeness of the two highly electropositive functional
groups (NNO and CO), which in turn gives way to the
breaking up of the molecules as shown in Fig. 5(B)
[17,18].
The volume of the R (hydrophobic) alkyl group
could be another factor that facilitates the break-
ing up of the nitrosourea by increasing the steric
Table IV Stability of Nitrosoalkylureas Half-Life at pH
7 and pH 8 (22◦C), [3,7]
t1/2 (h)
Figure 4 Determination of kw exp for the four substrates un-
der study. I = 1.0 M, T = 298 K. : [DMU] = 1.5 × 10−2
Compound
pH 7
pH 8
◦
M, [nitrite] = 1 × 10−4 M, 3.3 × 10−3 < [H+] < 1.35 ×
1-Nitroso-
1,3-dimethylurea
1,3-diethylurea
1-methyl-3-ethylurea
1-ethyl-3-methylurea
10−2 M; : [DEU] = 1.5 × 10−2 M, [nitrite] = 1 × 10−4
ꢀ
300
87
230
84
43
18
38
18
M, 4.2 × 10−3 < [H+] < 1.6 × 10−2 M; ꢁ: [DPU] =
6.25 × 10−3 M, [nitrite] = 1 × 10−4 M, 1 × 10−3 < [H+] <
3.6 × 10−2 M; +: [DAU] = 3 × 10−2 M, [nitrite] = 1 × 10−4
M, 4.9 × 10−3 < [H+] < 1.6 × 10−2 M.

´
GONZALEZ-ALATORRE ET AL.
278
Figure 5 (A) Proposed hydrogen bonding between water and protonated N-alkyl-N-nitrosodiureas; (B) breaking up of ni-
trosodiureas molecules to form diazone ions [4].
impediment and weakening the N-C link. This hypoth-
esis is founded on the following arguments:
form alkylating agents. It is to be expected, therefore,
that the sequence in the biological activity of the ni-
trosodialkylureas be the opposite of that observed in
the reactivity DNMU ꢂ DNEU < DNPU < DNAU.
Lijinsky et al. [3] have carried out studies on the bio-
logical activities of the first two members of the series,
NDMU and NDEU, where the greatest differences in
chemical reactivity are observed. In the studies done
on rats, Table V, the biological activity of the NDEU
is markedly greater than that observed for the DMEU.
These results agree with our hypothesis regarding the
relationship that exists between the chemical reactiv-
ity/reaction mechanism/structure of the substrate to be
nitrosated and the biological activity of the N-nitroso
compound generated.
As mentioned in the introduction, the differences
in the qualitative and quantitative carcinogenic effect
between these two nitrosodiureas can only be partly
explained by their alkylating properties. Other fac-
tors exist, such as receptors in the cells of certain
experimental animals that have a specific affinity to
the structure of one N-nitroso compound in particular
[4].
a. One reaction path to the generation of potent alky-
lating agents involves the attack of a nucleophile
on the C O group to generate a tetrahedral in-
termediate which decomposes with the genera-
tion of diazotate ion (R N=N O ) [4,14,15].
The length of the R alkyl group could favor this
path.
b. The reported instability of the nitrosoureas in an
aqueous medium (see Table IV) [3,7]. At pH val-
ues of 7 and 8, the N-nitroso compounds with
methyl groups are markedly more stable than
those with ethyl groups. In this same table, the
greater stability of the nitrosoalkylureas at low
pH values can be observed which agrees with the
studies carried out for the decontamination and
disposal of nitrosoureas [19].
The results shown in Table IV indicate that the biolog-
ical activity of the N-nitroso compounds will depend
on the ease with which their molecules decompose to
Table V Carcinogenesis by Nitrosodialkylureas in Rats [4]
Rats (Gavage)
Rats (Drinking Water)
Dose 1.2 mmol
Dose 0.6 mmol
Dose 1.2 mmol
Dose 0.6 mmol
RN(NO)CONHR
R
t1/2
M
t1/2
F
t1/2
M
t1/2
F
t1/2
M
t1/2
F
t1/2
M
t1/2
F
CH3
C2H5
81
33
40
–
83
36
80
29
86
49
71
32
95
58
100
47
t1/2 M = median week of death in a male rat, t1/2 F = median week of death in a female rat.

NITROSATION OF 1,3-DIALKYLUREAS IN AQUEOUS-PERCHLORIC ACID MEDIUM
279
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CONCLUSION
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The kinetic study of the nitrosation of diureas in an
aqueous medium showed the following characteris-
tics: (1) the application of the initial rate method to
the study of the effect of the [H+] on the reaction rate
constant and consequently on the establishment of the
reaction mechanism, showed several significant advan-
tages over the integral method. (2) The reactivity ob-
served can be justified in function of the rate-limiting
step of the mechanism and of the hypothesis of the
folding of the hydrophobic alkyl group. (3) A probable
relationship between the chemical reactivity and struc-
ture of the substrate to be nitrosated and the activity of
the nitrosourea generated has been established.
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The authors thank Dr. Joel Mart´ınez Me´ndez (Director of
Health Services for the State of Guanajuato) and Dr. Samuel
Villalpando Rodriguez for the interest shown in this project.
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