Chemical Research in Toxicology

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Article

Figure 9. Optimized transition state geometries of compounds 20-24. All the bond lengths are shown in Å.

exothermic. As discussed before, the diazonium cation will
have competing reactions with DNA bases or reaction water,
which is also one of the factors that could influence the
mutagenic potential of nitrosamines. For this series, the
hydrolysis and DNA base adenine reaction are found to be
competitive as the difference between activation energies is in
the range of 0.9−1.2 kcal/mol. It should be noted that
compound 23 is noncarcinogenic as it may undergo phase II
metabolic pathway related to the carboxylic acid group and, in
this study, we show how the electron withdrawing group can
influence the overall nitrosamine metabolic pathway.
In the previous section, we showed that CPCA underestimates the AI for compound 15, and herein, we showed an
example that AI is overestimated for compound 24. Within
compound series 20−24, the α-hydroxylation process is not
the only parameter that can define the trend in the TD50. In
the CPCA, the nitroso group which is part of the fivemembered ring gets a higher deactivating score than the
corresponding six-membered ring. The predicted CPCA-AI for
N-nitroso pyrrolidine is 400 ng/day, which is quite reasonable
when compared with the actual 679 ng/day so that it
conservatively defines the limit for NDSRIs that would have
nitroso pyrrolidines. However, N-nitroso pyrrolidine series,
shows examples wherein predicted CPCA-AI overestimates
and/or underestimates than the actual AI. Notably, molecule
24 predicted AI is 1500 ng/day but the actual AI is 95.2 ng/
day.
For compound 24, Ma et al.28 recently studied the probable
metabolic pathways which are summarized in Figure 11
showing the potential toxifying and detoxifying pathways.
Among all of the possible reactivity sites, α-hydroxylation is the
most preferred pathway for NNN (24). Though CPCA
predicts 1500 ng/day AI for this compound because of the
preferred carcinogenic metabolic pathway actual AI is only
95.7 ng/day. This is one more example wherein QM
calculations emphasize the importance of understanding the
nitrosamine reactivity and help in addressing AI limits for
NDSRIs. Additionally, Thomas and coworkers19 suggested
that molecules with multiple sets of structural features can
undergo various metabolic pathways that require additional
expertise and analysis to understand the potency trend, and
QM calculations could be useful to understand and justify the
AI limits.
Heteroatom Effect on Six-Membered Saturated Rings
wherein N-NO Is Part of the Ring. In this section, we
explored the reactivity of molecules wherein the nitrosamine is
part of the six-membered saturated ring with a heteroatom part
of the ring system. We selected four molecules, namely, Nnitroso piperidine (25), N-nitroso piperazine (26), N-nitroso
morpholine (27), and N-nitroso thiomorpholine (28) shown

Table 3. Gibbs Activation Free Energies (kcal/mol) for
Various Mechanistic Steps for Compounds 20−24
ΔG‡AB
ΔG‡BC
ΔG‡DE
ΔG‡DG

20

21

22

23

24

19.4
22.1
13.5
14.4

18.4
21.8
15.7
16.6

20.3
18.8
13.5
13.3

20.3
23.6
12.1
13.3

20.0
22.6
6.9
4.0

The second step in the metabolic pathway is the aldehyde
formation (Figure 1a) due to hydrogen transfer from the αhydroxyl group to the nitrosamine group as shown in Figure
10. The ΔG‡BC values are higher than the ΔG‡AB except for

Figure 10. Aldehyde formation step transition state structures of
compounds 20 and 22. All of the bond lengths are shown in Å.

compound 22 (N-nitroso oxazolidine) which suggests that the
rate-determining step is the ring opening of five-membered
pyrrolidine affording to the aldehyde. This compound formed
after the ring opening, can further undergo deactivating
metabolism or oxidation to acids which can be glucuronidated,
and in general, phase 2 metabolism should be considered. This
compound formed after the ring opening, can be further
undergo deactivating metabolism or oxidation to acids which
can be glucuronidated, and in general, phase 2 metabolism
should be considered.36b In case of compound 22, the ratedetermining step is α-hydroxylation. To explain the reactivity
differences between pyrrolidine and oxazolidine nitrosamines,
the transition state geometries are observed. The transition
state geometries of the ring-opening reaction showed a clear
difference. The C−N bond distance in TSBC of 22 is 1.750 Å
while it is 1.845 Å in 20 which could be potentially due to the
reduced ring strain in 22.36 The calculated reaction-free
energies for 22 are also thermodynamically more favorable
than other N-nitroso pyrrolidines (Table S1). This is most
likely due to the formation of formate in 22 after ring opening
whereas in other molecules ring opening leads to the formation
of aldehydes.
The next step in the metabolic pathway is the diazonium
cation formation via the elimination of the hydroxyl group
from the N�NOH group, and this process is highly
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https://doi.org/10.1021/acs.chemrestox.4c00087
Chem. Res. Toxicol. 2024, 37, 1011−1022