Chemical Research in Toxicology

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Article

Figure 6. N-methyl nitroso substituted alkyl compounds considered in the present study. The AI values from TD50s as well as the predicted CPCA
AI values (in red) are shown.

Table 2. Gibbs Free Energy of Activation (in kcal/mol) for the Mechanistic Steps Involved in the N-methylalkyl Series
nitrosamine

ΔG‡AB

ΔG‡AB′

ΔG‡BC

ΔG‡BC′

ΔG‡DE

ΔG‡DG

kα′/kαa

9
10
11
12
13
14
15
16
17
18
19

21.6
21.7
21.2
20.6
18.4
21.6
20.9
20.1
21.6
20.5
19.9

21.6
19.6
19.8
21.2
17.3
19.6
19.0
18.9
18.6
18.4
20.2

23.1
22.3
22.5
24.7
22.3
22.5
22.8
22.1
23.5
22.9
23.0

23.1
18.5
19.4
18.2
20.7
18.7
19.3
18.7
18.7
19.0
12.1

15.3
12.1
13.3
14.5
19.3
12.4
11.7
14.1
11.5
11.6
12.5

13.2
12.2
12.8
13.4
16.3
11.5
11.9
12.2
10.0
11.3
12.9

1.0b
34.6
10.6
0.4
6.4
29.2
24.7
7.6
158.1
34.6
0.6

a

Ratio of rate constants kα′ and kα calculated from ΔG‡AB and ΔG‡AB′, respectively. bNDMA (9) has same alkyl groups on both sides of N-NO
group, so the kα′/kα is 1. When hydroxylation occurs at the alkyl side of the nitrosamines 9−19, ΔG‡DE and ΔG‡DG will remain same as for the
molecule 9.

WOE arguments on potency and nitrosamines supporting AI
determination.
N-methylalkylnitrosamines. Figure 6 shows the Nmethyl nitrosoalkylamines (compounds 9−19). All these
nitrosamines have a CH3 group on one side of the N-nitroso
moiety and a substituted alkyl group with two α-hydrogens on
the other side. As α-hydrogens are present on both sides, αhydroxylation can occur on either side. These molecules cover
β-hydroxy substitution, carbonyl, electron-donating NMe2, and
benzyl and hydroxy-substituted benzyl groups with an AI in the
range of 10−95,200 ng/day.
We performed QM calculations on the carcinogenic
metabolic pathway (Figure 1b) to understand the potency
trends for these compounds. The activation energies for both
sides of the C−H bonds (Table 2) are evaluated, ΔG‡AB and
ΔG‡AB′ represent α-hydroxylation activation energy for the Nmethyl group and CH2 of the more substituted alkyl side,
respectively. ΔG‡AB is in the range of 18.4−21.7 kcal/mol
whereas ΔG‡AB′ 17.3−21.6 kcal/mol and the ratio of calculated
rate constants from the kα′/k is 0.4 to 158 orders of magnitude
suggesting that α-hydroxylation can occur on both α−C-H
bonds but are kinetically preferable on the more substituted
alkyl side for a majority of the nitrosamines shown in Figure 6.
Compound 9 is NDMA, with ΔG‡AB′ being 21.6 kcal/mol,
while for compound 10, it is 19.6 kcal/mol which is lower
because of the alkyl CH3 group substitution which provides
additional stabilization to radical formed during C−H
activation.32 ΔG‡AB′ for compound 11 is 19.8 kcal/mol

which is comparable to the 10 indicating that OH substitution
at β-carbon shows a minor influence on the α-hydroxylation
process.
Interestingly, ΔG‡AB′ for compound 12 is 21.2 kcal/mol
which is an increase of 1.4 kcal/mol from compound 11 and
which indicates that when double substitution is present at βcarbon, stereo electronic effects play a crucial role in the
hydroxylation process. Furthermore, the ΔG‡AB for compound
12 is 20.6 kcal/mol which is slightly lower than ΔG‡AB′ which
would be a kinetically preferable site. However, compound 12
also has a secondary alcoholic group (AI is 46 ng/day) and is a
much more potent carcinogen compared to compound 11 (AI
is 1290 ng/day). Interestingly, compound 11, which has a
primary alcoholic group, can undergo alcohol oxidation to
aldehyde instead of α-hydroxylation at C−H site whereas
compound 12 is likely to undergo α-hydroxylation. Recently,
Snodin et al. reported that compounds with primary as well as
secondary hydroxy groups undergo competitive phase I and/or
phase II metabolic pathways (Sulfation, glucuronidation and
primary oxidation). Further, in the case of β-hydroxy
compounds, the increased potency is expected because of
alternative ways of forming alkyl diazonium ions.36b This could
be a potential reason for a significant difference in potency
between 11 and 12 (the effect of alternative metabolic
pathways is not included in this study).33,34 Compound 13 has
the lowest ΔG‡AB′ of 17.3 kcal/mol and also a very low AI of
17 ng/day which could be because of the extended conjugation
provided by the carbonyl substitution at the α-carbon. Thomas
1016

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https://doi.org/10.1021/acs.chemrestox.4c00087
Chem. Res. Toxicol. 2024, 37, 1011−1022