Kinetic study of the nitrosation of N-alkylureas in dioxane-acetic acid mixtures

Article abstract of DOI:10.1002/(SICI)1097-4601(1998)30:2<145::AID-KIN6>3.0.CO;2-S

The rate constants were determined for the nitrosation reactions of the following substrates: Methyl (MU), Ethyl (EU), Propyl (PU)Butyl (BU), and Allylurea (AU). The rate equation found at a constant pH was: v = k[HNO₂] [Urea]. The reactions were carried out in predominantly organic media(dioxane-acetic acid-water) with differing polarities. The proposed reaction mechanism involves the proton transfer from the protonated N-alkyl-N-nitrosourea to the acetate anion. As the polarity of the medium decreased, an approximation of the rate constants of the nitrosation of the different substrates was observed. This approximation can be interpreted as a function of the impediment generated by the R alkyl radical in the rate controlling step. Accordingly, the substrate reactivity will be associated with the ease in which the protonated N-alkyl-N nitrosurea can transfer the proton to the acetate anion. The results achieved in this study are in accordance with there activities observed in the nitrosation of these substrates in aqueous media MU ? (EU ≈ PU ≈ BU) > AU.

Full text of DOI:10.1002/(SICI)1097-4601(1998)30:2<145::AID-KIN6>3.0.CO;2-S

Kinetic Study of the
Nitrosation of N-Alkylureas
in Dioxane-Acetic Acid
Mixtures
´
GUILLERMO GONZALEZ ALATORRE, JAVIER G. ZAPIAIN S., PEDRO A. QUINTANA H.,
´
GLORIA M. MARTINEZ G.
Departamento de Ingenierı´a Qu´ımica, Instituto Tecnolo´gico deCelaya, E-38010, Celaya, Guanajuato, Me´xico
Received 23 January 1997; accepted 26 May 1997
ABSTRACT: The rate constants were determined for the nitrosation reactions of the following
substrates: Methyl (MU), Ethyl (EU),Propyl (PU)Butyl (BU), and Allylurea (AU). The rate equa-
tion found at a constant pH was: v ϭ k[HNO2] [Urea]. The reactions were carried out in pre-
dominantly organic media(dioxane–acetic acid–water) with differing polarities. The proposed
reaction mechanism involves the proton transfer from the protonated N-alkyl-N-nitrosourea
to the acetate anion. As the polarity of the medium decreased, an approximation of the rate
constants of the nitrosation of the different substrates was observed. This approximation can
be interpreted as a function of the impediment generated by the R alkyl radical in the rate
controlling step. Accordingly, the substrate reactivity will be associated with the ease in which
the protonated N-alkyl-N nitrosurea can transfer the proton to the acetate anion. The results
achieved in this study are in accordance with there activities observed in the nitrosation of
these substrates in aqueous media MUϾϾ(EU Ϸ PU Ϸ BU) Ͼ AU. ᭧ 1998 John Wiley & Sons, Inc.
Int J Chem Kinet 30: 145–150, 1998.
INTRODUCTION
trosamides, in general, and alkylnitrosoureas, in
particular, are concerned, studies carried out are few
and far between [11,12], even though the biological
[13] and kinetic [14–16] differences between both
types of nitrosocompounds are well known. From the
biological point of view, it is a well known fact that
where as nitrosamides require an enzymatic activation
prior to acting as carcenogenic agents [17], nitrosou-
reas do not need this activation [13]. The above can
be explained in terms of the formation of DNA alkyl-
izing diazone ions. Therefore, whereas nitrosamides
are stable compounds, needing to be attacked by en-
zymes such as microsomal mono-oxygenase [17], ni-
trosamides are unstable in aqueous solutions generat-
ing the above mentioned ions as they decompose [13].
In a recent study [12] of the nitrosation of ureas
Nitrosation reactions have attracted wide interest in
recent years due to the fact that most nitrosocom-
pounds are toxic [1], carcinogenic [2,3], teratogenic
and mutagenic [4–8]. In the case of nitrosamines, a
large number of studies have been carried out to de-
termine both their kinetic characteristics and their re-
action mechanisms [9,10]. Nevertheless, as far as ni-
Correspondence to: G. Gonza´lez Alatorre
Contract grant Sponsor: Consejo del Sistema Nacional de Ed-
ucacio´n Tecnolo´gica, COSNET
Contract grant Sponsor: Mexican Consejo Nacional de Ciencia
y Tecnolog´ıa, CONACYT
᭧ 1998 John Wiley & Sons, Inc. CCC 0538-8066/98/020145-06

´
GONZALEZ-ALATORRE ET AL.
146
with the general formula RNHCONH2 in aqueous me-
dia, the following order in the rate constants was
found: MUϾϾ(EU Ͼ PU Ͼ BU) Ͼ AU. There action
mechanism involved considers the proton transfer
from the N-alkyl-N-nitrosourea to the solvent (water)
as the rate controlling step. On the basis of the above
mechanism, the observed reactivity was explained as
a function of the impediment generated by the alkyl
chains, which when found in apolar medium (water),
double back on themselves, partially obstructing exit
of the proton to the solvent.
This article describes the results of a kinetic study
of the same series of ureas in predominantly organic
media (dioxane–acetic acid–water). The polarity of
the medium was gradually decreased so as to observe
the effect on the rate constants, revealing an approxi-
mation of them as the polarity was reduced. This was
interpreted as being due to partial unfolding of the
aliphatic chains.
Lastly, a relationship is proposed between the struc-
ture and the reactivity of the nitrosable substrate with
the biological activity of the product (nitrosocom-
pound).
Figure 1 Determination of the order with respect to the
nitrite for the series of reactions studied: (ࠗ) MU; (ٗ) EU;
(᭝) PU; (᭛) BU; and (ϩ) AU. [urea] ϭ 0.223 M, 25% ace-
tic acid, 70% dioxane, 5% water, and T ϭ 298 K.
Given the insolubility of nitrites in organic
mixtures (dioxane–acetic acid) it was found to be nec-
essary to add a small percentage (5%) of water to the
solutions. Water was omitted in the proposed reaction
mechanism, given both its low nucleophic power in
relation to the acetate anion and its low concentration
in the reactive solutions.
EXPERIMENTAL
Solutions of MU, EU, BU, and AU were made up by
weight from p.a. products from Aldrich (St. Louis,
Missouri, USA.) with nominal purities of 97–99%.
Solutions of PU were made up by weight from a prod-
uct from Alfa (Karlsruhe, Germany) after prior re-
crystalization from ethanol/water. NaNO2, 99% from
Sigma (St. Louis, Missouri USA), 1–4 dioxane, 99%
from Merck (Tlanepantla, Mexico), and CH3COOH,
99ϩ% from Sigma, were all used without previous
purification.
Nitrosation kinetics were recorded by monitoring
absorbance by nitrosocompounds at 260 nm in a
PerkinElmer UV/VIS spectrophotometer equipped
with a thermoelectric cell holder thermostat maintain-
ing temperature within Ϯ0.1ЊC. Under these condi-
tions, the molar absorptivity of the nitrosoureas
RESULTS AND DISCUSSION
The kinetic studies were begun with the determination
of the orders with respect to the nitrite and urea in a
predominantly organic medium. The first-order with
respect to the nitrite became established on analyzing
the adjustment of the experimental data to eq. (1) (See
Fig. 1)
ln[Aϱ Ϫ A] ϭ ϪkЈt ϩ ln[Aϱ Ϫ Ao]
(1)
where
studied is much greater (e.g., for MNU ⑀260
ϭ
ϭ
1
1
3405 MϪ cmϪ ) than that of nitrite (⑀260
kЈ ϭ k[urea]␣
(2)
6MϪ cmϪ ), and absorbance by both alkylureas and
carboxylic acids is constant. Absorbance time data
were analyzed by the integration method following the
reactions until 90%. The Aϱ was estimated using the
Greg [19] programme for the nonlineal data adjust-
ment. All kinetic experiments were carried out with a
large excess of alkylurea (henceforth called urea) over
nitrite.
1
1
[urea] ϾϾ [nitrite]
When the values of kЈ vs. [urea] were plotted, the ex-
perimental data formed straight lines, indicative of a
first-order with respect to the urea (see Fig. 2).
On the basis of the above results, the experimental
rate equation was established as follows:

NITROSATION OF N-ALKYLUREAS
147
Figure 2 Determination of the order with respect to the
urea for the series of reactions studied: (ࠗ) MU; (ٗ) EU;
(᭝) PU; (᭛) BU; and (ϩ) AU. [nitrite] ϭ 1 ϫ 10 M, 25%
acetic acid, 70% dioxane, 5% water, and T ϭ 298 K.
Figure 4 Determination of the rate constant k for the ni-
trosation of the substrates: (ࠗ) MU; (ٗ) EU; (᭝) PU; (᭛)
BU; and (ϩ) AU. [nitrite] ϭ 1 ϫ 10 M, 10% acetic acid,
85% dioxane, 5% water, and T ϭ 298 K.
Ϫ4
Ϫ4
d[nit]
The values of k (Table 1) rise in proportion to the
increase in the percentage of acetic acid. The above
effect on k cannot be attributed exclusively to the
change in the [Hϩ] in the medium, given the known
catalytic effect [11,23] of the carboxylate anions on
the reaction rate both in aqueous [12,14] and in or-
ganic media [20].
To discern between the influence of [CH3COOϪ]
and [Hϩ] on k, and therefore to be able to interpret the
results of Table I, the reaction mechanism shown in
Scheme I is proposed. This mechanism for the nitro-
sation reactions in an organic medium, is founded as
much on the results of the kinetic study (first-order in
nitrite and urea) as on previous studies that consider
the nitrosation of the same kind of substrates both in
aqueous and an organic media [12,20].
v ϭ Ϫ
ϭ k[urea][nitrite]
(3)
(4)
dt
kЈ ϭ k[urea]
where the constant k ϭ f(pH, [CH3COOH], I, and T)
and I ϭ ionic strength
In addition to the values for k determined with 25%
acetic acid Figure 2, values were also determined with
35% and 10%, Figures 3 and 4, respectively.
Figure 3 Determination of the rate constant k for the ni-
trosation of the substrates: (ࠗ) MU; (ٗ) EU; (᭝) PU; (᭛)
Ϫ4
BU; and (ϩ) AU. [nitrite] ϭ 1 ϫ 10 M, 35% acetic acid,
60% dioxane, 5% water, and T ϭ 298 K.
Scheme I Proposed mechanism for the nitrosation of ureas
in an organic medium (dioxane–acetic acid–water).

´
GONZALEZ-ALATORRE ET AL.
148
Table I Values of k Eq. (4)
Acetic Acid Percentage
MU
EU
PU
BU
AU
Ϫ
Ϫ1
k, M 1s
0a
10.1 Ϯ 0.01
3.03 Ϯ 0.05
2.77 Ϯ 0.07
3.02 Ϯ 0.15
1.94 Ϯ 0.04
Ϫ
Ϫ1
k ϫ 104, M 1s
35
25
10
80.1 Ϯ 0.32
32.9 Ϯ 0.18
4.90 Ϯ 0.32
41.7 Ϯ 0.28
18.8 Ϯ 0.29
3.28 Ϯ 0.01
36.6 Ϯ 0.10
16.5 Ϯ 0.10
2.59 Ϯ 0.13
35.1 Ϯ 0.29
18.7 Ϯ 0.08
2.61 Ϯ 0.10
23.6 Ϯ 0.23
9.64 Ϯ 0.03
2.20 Ϯ 0.08
a Aqueous media [28].
In order to determine the theoretical equation cor-
responding to this mechanism the following supposi-
tions were made: (a) Both the concentration of water
and of the nitrite ion are insignificant compared to
those of acetic acid and dioxane and (b) The nu-
cleophic power of both water and the nitrite ion are
equally insignificant in relation to that of the acetate
anion acetate anion. On the basis of the above, it is
possible to eliminate the final two elemental reactions
in Scheme I.
sation of the dimethylurea in dioxane–water media.
This study found that for percentages of dioxane
greater than 75%, the nitrosation mechanism is no
longer that proposed in Scheme I but corresponds to
that presented in Scheme II. This effect had already
been observed by Crookes and Williams [21,22] when
the reaction medium was changed from aqueous to
organic during the nitrosation reactions.
d[nit]
v ϭ Ϫ
ϭ kCH COOϪK1K2KRKЈa
3
dt
Scheme II Proposed mechanism for the nitrosation of
ureas in a dioxane–water medium, for a percentage of di-
oxane greater than 75% [20].
[H2O][CH3COOH][Hϩ3 O][urea][sodium nitrite]
ϫ
(KЈa[H2O]2)([H3ϩO] ϩ KЈa[H2O])
(5)
The equation related to this mechanism is:
Where KЈa is the dissociation constant of H2NO2 in wa-
ter. If we compare eqs. (3) and (5), we end up with:
d[nitrite]
v ϭ Ϫ
dt
k ϭ k
ϪK1K2KRKЈa
CH3COO
K1k2[DMU]
1 ϩ K1[Hϩ]
ϭ
ϩ kϪ [nitrite] ϩ kϪ [nitrite]o
2 2
ͩ
ͪ
[H2O][CH3COOH][Hϩ3 O]
(KЈa[H2O]2)([H3ϩO] ϩ KЈa[H2O])
ϫ
(6)
(8)
When the pH, [H2O], [CH3COOH], T, and I remain
constant throughout the kinetic study, the k in eq. (6)
Given the impossibility of equivalence between the
theoretical, eq. (8) and the experimental, eq. (3), it can
be concluded that the reaction mechanism involved for
the three percentages of acetic acid shown in Table II,
is that described in Scheme I.
The fact that the mechanism described in Scheme
II has still not occurred with a 90% concentration of
dioxane can be attributed to the nucleophile nature of
the CH3COOϪ anion which enables it to extract pro-
tons (in the rate controlling step) even at low concen-
trations. In the case of the dioxane–water system[20],
this situation does not occur because a greater per-
centage of water (25%) is needed to extract protons at
the rate controlling step.
Ϫ
will be the exclusive function of kCH COO given that
3
K1, K2, KR, and KЈa are constants common to the five
substrates (See Scheme I). Given the above, it is pos-
sible to express eq. (5) as ␯ ϭ k[urea][nitrite] eq. (3).
Ϫ
Given that kCH COO corresponds to the limiting step
3
of the mechanism, it will be a measurement of the
reaction rate, function of the substrate (see Scheme I)
eq. (7).
Ϫ
k ϭ f(kCH COO
)
3
ϭ f(substrate characteristics) ϭ f(R)
(7)
Before applying eq. (7) in the interpretation of the re-
sults, it is vital to take into consideration the kinetic
studies carried out by Bravo et al. [20] on the nitro-
Given that the rate constants (Table II) were deter-
mined under different conditions of acidity and ionic
intensity, it was necessary to refer the value of the rate

NITROSATION OF N-ALKYLUREAS
149
Table II Relative Values of k with Respect to k(MU)
k (MU)/k(urea)
Acetic Acid Percentage
MU
EU
PU
BU
AU
0a
35
25
10
1.00 Ϯ 0.00
1.00 Ϯ 0.00
1.00 Ϯ 0.00
1.00 Ϯ 0.00
3.32 Ϯ 0.12
1.92 Ϯ 0.20
1.75 Ϯ 0.40
1.49 Ϯ 0.10
3.63 Ϯ 0.16
2.18 Ϯ 0.15
1.99 Ϯ 0.20
1.88 Ϯ 0.23
3.33 Ϯ 0.23
2.27 Ϯ 0.30
1.76 Ϯ 0.18
1.87 Ϯ 0.20
5.18 Ϯ 0.21
3.39 Ϯ 0.42
3.41 Ϯ 0.31
2.27 Ϯ 0.23
a Aqueous media [28].
constants pertaining to all the substrates k (ureas) to
the value corresponding to that of the methylurea
k(MU). The above was carried out using the relation-
ship k(MU)/k(urea). The above values are shown in
Table II.
probably as a result of the combination of experimen-
tal error and the low gradient of the concentration of
acetic acid involved.
In order to be certain that the tendency observed in
the data obeys a correlation between polarity and the
unfolding of the R groups, it would be necessary to
carry out experiments at lower polarities until the
total unfolding of the alkyl chain was achieved,
k(MU)/k(urea) ϭ 1. However, this would be impos-
sible as it would require the elimination of both the
5% of water used in the solution of the sodium nitrite
and the acetic acid used in the extraction of protons in
the rate controlling step of the reaction mechanism.
The effect the R alkyl group has on the reactions
of the nitrosation of ureas could also have biomedical
implications. It is a well known fact that the carcen-
ogenic activity of the nitrosocompounds depends on
the facility with which diazone ions are formed, them-
selves responsible for the alkylation of the DNA mol-
ecules [5,24]. Whereas for nitrosamines, a previous
enzimatic activation is required for the formation of
such ions, in the case of nitrosoureas, only the hy-
drolysis of such compounds is needed(probably be-
cause of their instability) [13]. In function of what has
been presented in this study, it is to be expected that
the most stable nitrosourea is methylnitrosourea,
which, because of the low steric impediment of the R
alkylic radical, would be more difficult to hydrolize
and therefore hindering the formation of the diazone
ions in relation to the other substrates. If this were to
occur, the methylnitrosourea would be the least bio-
logically active. In fact there is experimental evidence
from various studies that show the low biological ac-
tivity (in laboratory animals) of the methylnitrosourea,
compared to the activity of other alkyl nitrosoureas
[25–27].
Table II shows how a reduction in the polarity in
the medium is accompanied by an approximation of
the k(MU)/k(urea) values for the different substrates.
The highest separation between the values of k(MU)/
k(urea) for different substrates occurs in the case of an
aqueous medium (0% of acetic acid). This can be in-
terpreted in terms of the high polarity of the water.
Such polarity will cause the folding of the alkylic rad-
ical R (see Fig. 5) which partially impedes the depar-
ture of the proton from the protonated N-alkyl-N-ni-
trosourea to the proton extracter (CH3COOϪ), in the
rate controlling step of the reaction mechanism. As the
polarity is reduced in the medium, it is expected that
the alkylic chains R tend to linealize, allowing for the
approximation of the rate constants for the different
substrates. In Table II we can observe a certain tend-
ency towards unity in the relationship k(MU)/k(urea)
as the polarity of the medium decreases. At first,
sharply as they pass from the aqueous to the organic
medium with 35% acetic acid, then gradually as the
polarity declines in proportion to the reduction in the
concentration of acetic acid. Note how neither the BU
nor the AU follow the expected sequence when the
acetic acid reduces from a 35 to 25% concentration,
CONCLUSION
The kinetic studies of the nitrosation of the five ureas
in a predominantly organic medium have shown the
following results: (a) First-order with respect to both
Figure 5 Impediment of the R alkyl radical on the carbox-
ilic anion CH3COO during the rate controlling step of the
reaction mechanism.
Ϫ

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GONZALEZ-ALATORRE ET AL.
150
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The authors wish to thank the Consejo del Sistema Nacional
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out and the Mexican Consejo Nacional de Ciencia Tecnol-
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