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Theoretical Studies of Chemical Reactivity of Metabolically Activated Forms of Aromatic Amines toward DNA Igor Shamovsky,*,† Lena Ripa,† Niklas Blomberg,† Leif A. Eriksson,∥ Peter Hansen,† Christine Mee,‡ Christian Tyrchan,§ Mike O'Donovan,‡ and Peter Sjö† †

Department of Medicinal Chemistry, R&I iMed, AstraZeneca R&D, Pepparedsleden 1, S-431 83 Mölndal, Sweden Genetic Toxicology, AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom § Department of Medicinal Chemistry, CVGI iMed, AstraZeneca R&D, Pepparedsleden 1, S-431 83 Mölndal, Sweden ∥ Department of Chemistry and Molecular Biology, University of Gothenburg, S-412 96 Göteborg, Sweden ‡

S Supporting Information *

ABSTRACT: The metabolism of aromatic and heteroaromatic amines (ArNH2) results in nitrenium ions (ArNH+) that modify nucleobases of DNA, primarily deoxyguanosine (dG), by forming dG-C8 adducts. The activated amine nitrogen in ArNH+ reacts with the C8 of dG, which gives rise to mutations in DNA. For the most mutagenic ArNH2, including the majority of known genotoxic carcinogens, the stability of ArNH+ is of intermediate magnitude. To understand the origin of this observation as well as the specificity of reactions of ArNH+ with guanines in DNA, we investigated the chemical reactivity of the metabolically activated forms of ArNH2, that is, ArNHOH and ArNHOAc, toward 9-methylguanine by DFT calculations. The chemical reactivity of these forms is determined by the rate constants of two consecutive reactions leading to cationic guanine intermediates. The formation of ArNH+ accelerates with resonance stabilization of ArNH+, whereas the formed ArNH+ reacts with guanine derivatives with the constant diffusion-limited rate until the reaction slows down when ArNH+ is about 20 kcal/mol more stable than PhNH+. At this point, ArNHOH and ArNHOAc show maximum reactivity. The lowest activation energy of the reaction of ArNH+ with 9-methylguanine corresponds to the charge-transfer π-stacked transition state (πTS) that leads to the direct formation of the C8 intermediate. The predicted activation barriers of this reaction match the observed absolute rate constants for a number of ArNH+. We demonstrate that the mutagenic potency of ArNH2 correlates with the rate of formation and the chemical reactivity of the metabolically activated forms toward the C8 atom of dG. On the basis of geometric consideration of the π-TS complex made of genotoxic compounds with long aromatic systems, we propose that precovalent intercalation in DNA is not an essential step in the genotoxicity pathway of ArNH2. The mechanism-based reasoning suggests rational design strategies to avoid genotoxicity of ArNH2 primarily by preventing N-hydroxylation of ArNH2.

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are able to diffuse to genomic DNA and cause mutations.2,19 These metabolically activated forms cause the formation of covalent DNA adducts.2,3,9,20 The DNA adduct formation is recognized as a common property of most potent chemical mutagens and carcinogens,9,21 even though the relationship between DNA adducts and carcinogenesis is not fully understood.22 Metabolically activated forms of ArNH2 covalently modify nucleobases of DNA, primarily deoxyguanosine (dG), by forming dG-C8 adducts, where the C8 atom of dG reacts with the activated amine nitrogen of ArNH2.1,21,23 It has been established that formation of covalent dG adducts is mediated by short-lived cationic electrophilic species, nitrenium ions (ArNH+), formed by heterolytic dissociation of N−O bonds of the metabolically activated forms of ArNH2 under slightly acidic conditions.9,20,24−36 The lifetime of ArNH+ in water is in the

INTRODUCTION Aromatic and heteroaromatic amines (ArNH2) represent a class of organic compounds that can cause mutations in DNA and thereby result in a variety of toxic effects including cancer.1−3 Mutagenic and carcinogenic ArNH2 are present in our environment;2−6 they have been identified in food,2,7−9 in cigarette smoke,3−10 in petrol and diesel exhaust fumes,11 and in various industrial settings.12 Like most other classes of chemical mutagens and carcinogens, ArNH2 have to be metabolically activated to be able to change the genetic code in DNA.1,2,13,14 The mutagenic activation pathway of ArNH2 in most cases starts with N-hydroxylation by P450 enzymes, predominantly by the 1A2 and 1A1 isoforms, and may get further activated by arylamine N-acetyltransferases (NAT) and sulfotransferases (SULT).2,14−18 The resulting metabolically activated species, hydroxylamines ArNHOH and especially their bioconjugates ArNHOAc and ArNHOSO3H, are well documented as the ultimate mutagenic and carcinogenic forms of ArNH2, which © 2012 American Chemical Society

Received: July 9, 2012 Published: September 4, 2012 2236

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without these fragments70−72 or were found to be overall less significant than the link to the electron affinity of the parent ArNH2.56,73−75 Additionally, it has been shown that for various classes of ArNH2, including analogues of aniline, 2-aminofluorene, 4-aminobiphenyl, and aminoimidazoazaarenes (AIA), the mutagenic potency significantly increases with the addition of electron-withdrawing functions that destabilize ArNH+.51,56,73,75,76 Promutagenic effects of electron-withdrawing groups are captured in many quantinative structure−activity relationship (QSAR) studies as the importance of lowering the lowest unoccupied molecular orbital (LUMO) of ArNH2 for increasing mutagenic potency.5,56,65,75,77,78 On the other hand, when the mutagenic potency was not taken into consideration but the Ames mutagenicity of ArNH2 was binary flagged as Ames positive (Ames+) or Ames negative (Ames−), stabilization of ArNH+ again turned out to be the most important single descriptor of Ames mutagenicity.68 Thus, both stabilization and destabilization of ArNH+ can be promutagenic, with the promutagenic effects of electron-withdrawing groups being detected only when the value of mutagenic potency is taken into consideration.51,56,65,72,74,75 The apparently contradictory observations on the role of ArNH+ can be explained if one hypothesizes that apart from the established involvement of cationic forms,2,24,25,33,66 a number of chemical events along the metabolic activation pathway, such as N-hydroxylation by P450 enzymes and bioconjugation by Phase II enzymes like NAT or SULT, involve anionic forms, ArNH−. In that case, pyridine-like nitrogens or other electronwithdrawing groups would accelerate these events by facilitating proton abstraction from ArNH2.17,79 In this report, we study the kinetics of formation of guanine adducts by the metabolically activated forms of ArNH2 by exploring the rate constants of the two consecutive steps, formation and quenching of ArNH+ with 9-methylguanine (9MG) through quantum mechanical calculations. Our purpose was to understand the observed structure−genotoxicity relationships within the entire class of ArNH2, rather than to build predictive QSAR equations. Specifically, we provide mechanistic insights to the roles that the rate of metabolic activation and the chemical reactivity of the metabolically activated forms play in the overall mutagenic potency of ArNH2. Results suggest that the mutagenic potency of ArNH2 correlates with the rate of formation of dG-C8 DNA adducts, which is proportional to the rate of metabolic activation and the chemical reactivity of the metabolically activated forms toward the C8 atom of guanine. Mechanistic considerations of the fundamental chemical events of the mutagenic pathway allowed us to explain the apparent contradictory effects of resonance stabilizations of ArNH+ and ArNH− on the mutagenic potency of ArNH2. The stability of the anionic form plays a significant role in the rate of metabolic activation of ArNH2. Chemical reactivities of ArNHOH and ArNHOAc with almost planar aromatic systems are at a maximum when the corresponding ArNH+ are more stable than PhNH+ by 20 ± 10 kcal/mol in gas phase. This “area −20” is the common region of the most mutagenic aromatic and heteroaromatic amines of diverse structures because their metabolically activated forms, such as ArNHOH, ArNHOAc, and ArNHOSO3H, are most reactive with DNA. More stable ArNH+ are inherent in genotoxic ArNH2 that causes oxidative DNA damage, rather than forms DNA adducts. On the basis of our calculations, comparison with experimental kinetics of quenching of ArNH+, and orbital considerations, we propose that reaction of ArNH+ with guanine derivatives undergoes π-stacked charge-transfer transition states (TSs) that lead to the direct formation of C8 intermediates.

order of 100 ns to 100 ms depending on the structure of the aromatic system, long enough to predominantly attack arenetrapping agents if present in solution.37,38 The nitrogens of ArNH+ have been demonstrated to react with the C8 atom of guanine and guanine derivatives much faster than with other aromatic nucleophiles including other nucleobases34,37,39−41 to form cationic C8 intermediates that after proton loss result in C8 guanine adducts.38,42−44 The reaction mechanism for ArNH+ with the C8 of guanine derivatives is not fully understood, nor is it clear what feature of guanine that makes it such an effective target for ArNH+.37 Although the formation of cationic C8 intermediates have been observed by laser flash photolysis and time-resolved resonance Raman studies,38,45,46 it is still unclear why this reaction is so fast.37 Besides, apart from the direct formation of C8 intermediates, a stepwise mechanism of the reaction that undergoes initial formation of N7 intermediates has been hypothesized; it should be noted, however, that this concept is currently regarded as controversial since it did not receive sufficient experimental support.2,32,34,37,40−42,47 It has been established that the mechanism of reactions of ArNH+ with C8 atoms of dG in DNA is similar to the mechanism with monomeric guanine derivatives, such as guanosine, dG, guanosine-5′-phosphate, 2′-dexyguanosine-5′-phosphate, and DNA oligomers.34,37 The intrinsic reactivity of ArNH+ to the C8 position of guanine derivatives is linked to the observed predominant formation of the dG-C8 adducts in DNA in vitro and in vivo.42,48,49 However, it has been noted that single-stranded DNA hexamers react with ArNH+ to form dG-C8 adducts, whereas double-stranded hexamers do not.50 On the other hand, in double-stranded DNA, a minor adduct (dG-N2) has been observed.2,9,21,34,37,48,50 The mutagenic potency of ArNH2 is determined by many factors because of the involvement of several enzymes and chemical steps.2,5,14,51,52 High reactivity and specificity of ArNH+ toward dG is only one of them; other important factors that are known to increase the mutagenic potency include higher rates of N-hydroxylation and bioconjugation,53−56 a higher rate of heterolysis of the N−O bonds in the ultimate mutagenic forms,57 and slower rates of detoxification.2,5,16 The maximum rate of formation of DNA adducts will be made by compounds, which exhibit the highest rates in all promutagenic steps and minimal rates of detoxification. However, the large number of DNA adducts caused by mutagenic metabolites of ArNH2 do not necessarily result in the large number of DNA mutations.58 Most of the C8-dG adducts get repaired by several cellular DNA repair pathways59 but not evenly in all places. The site of DNA damage, miscoding potential, and persistence in DNA seem to be as important features of DNA adducts as the rate of their formation in determining mutagenic potency.58,60−64 Thus, particular DNA adducts can block DNA replication by inhibiting DNA polymerases, which dramatically decreases the mutagenic potency of the corresponding ArNH2.58 The mutagenicity of ArNH2 is conventionally associated with a high resonance stabilization of ArNH+.65−69 Thus, classical works of Ford et al. and Borosky70−72 showed correlations between Ames mutagenic potency of different classes of ArNH2 and the stability of ArNH+. However, data on ArNH2 with pyridine-like nitrogens have been difficult to reconcile with the nitrenium stabilization concept as they destabilize ArNH+ and simultaneously dramatically increase the mutagenic potency of ArNH2.56,73,74 Thus, when pyridine-like aromatic fragments are included, the overall correlation of mutagenic potency with stabilization of ArNH+ either had to be split into separate subsets with and 2237

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Article

MATERIALS AND METHODS

Fully optimized density functional theory (DFT) calculations at the M06-2X/6-31+G(d) level of theory80 were utilized to study free energy profiles of key reactions of metabolically activated forms of a variety of ArNH2 to study the physical origin of their reactivity and selectivity toward dG in DNA. The stability of the TSs of reactions of dG with ArNH+ is determined by delocalization of the excess positive charge over the frontier orbitals, the highest occupied molecular orbital (HOMO) of dG, and the LUMO of ArNH+. Both of these orbitals are of π-character, which are located in the conjugated π-systems of the reactants. The influence of the adjacent saturated moieties on the structure and stability of such molecular systems is minimal. For this reason, in our studies, 9MG was used as a model of dG, in line with prior theoretical efforts,42,81 and because different guanine derivatives, both neutral and acidic, show similar kinetics of reactions with ArNH+.37 Because thermochemistry, kinetics, noncovalent interactions, and internuclear distances at TSs were all important for this study, the M06-2X density functional was chosen, consistent with published recommendations.82,83 The medium-sized basis set 631+G(d) with polarization and sp type diffuse functions in nonhydrogen atoms was used to properly describe molecular systems with unusually long interatomic distances that occur in TSs, to reduce basis set superposition errors (BSSE), and to enable calculations of large molecular systems. Optimizations of equilibrium geometries and TSs were performed at the M06-2X/6-31+G(d) level of theory using redundant internal coordinates of molecular systems84 and the maximum grid density in the program Jaguar (version 9.2.109; Schrödinger Inc.). Subsequent single-point vibrational analyses were carried out to confirm the nature of all identified stationary points and to calculate standard Gibbs energies. The polarizable continuum model (PCM) that uses the integral equation formalism variant (IEF-PCM)85 was utilized to take water solvation effects into account through single point calculations using the program Gaussian 09.86 Single-point calculations using the M06-2X and ωB97X-D functionals87 and the extended basis set 6-311++G(3df,3p) were performed to evaluate the magnitude of the BSSE and to compare predicted and experimentally determined rate constants of the quenching of ArNH+ by guanine derivatives.37,88 The structure of the intercalated complexes of a 20 base pair long double-stranded oligonucleotide (dG)20/(dC)20 with ArNHOAc placed in the middle of the DNA motif was obtained by molecular mechanics calculations. The sequence of the utilized DNA motif, repeated guanines, was chosen because it represents a mutation-susceptible sequence motif for mutagenic ArNH2.89−91 The energy minimizations were carried out using OPLS 2005 force field with GB/SA implicit water solvation92 as incorporated in MacroModel (version 9.9.2; Schrödinger Inc.). All energy minimizations started with B-DNA geometry, and nitrogens of intercalated ArNHOAc were juxtaposed with C8 atoms of dG.

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Figure 1. Content of AstraZeneca corporate database of ArNH2 with molecular weights less than 400 g/mol as a histogram against relative formation energy of ArNH+ in the gas phase obtained by DFT calculations at the B3LYP/6-31G(d) level of theory. A compound is considered Ames positive if it is mutagenic in at least one of two bacterial strains, TA98 or TA100, in the presence or absence of metabolic activation (S9). TA98 detects frameshift mutations and TA100 detects base-pair substitutions. Both strains are UV repair deficient (ΔuvrB), and both contain the plasmid pKM101, which gives error-prone repair. The formation energy of PhNH+ was used as a reference. The number of Ames positive and Ames negative compounds at each bin is shown by red and green bars, respectively. The approximate location of the maximum local fraction of Ames positive compounds is framed.

not depend on the size limit of the included ArNH2,68 suggesting that the nature of the maximum does not depend on the structure of ArNH2 but is probably linked to an enhanced promutagenic propensity of ArNH+ around this particular stabilization energy. To study the origin of the enhanced promutagenic propensity of ArNH+ in this area, we focus on 12 representative ArNH2 that cover a range of stabilities for the corresponding ArNH+ from −46 to 16 kcal/mol with respect to PhNH+ in gas phase and calculate kinetics of formation and quenching of ArNH+ with 9MG. Figure 2 gives the structures of the focused molecule set (1−12), and in addition, 15 mutagenic ArNH2 selected from different classes (13−27) are exemplified to illustrate and verify our conclusions.56,73,74,76,78 The kinetics of formation of guanine adducts is determined by the rate constants of two fundamental consecutive reactions, formation and quenching of ArNH+. Formations of ArNH+ from ArNHOH and ArNHOAc under slightly acidic conditions are described by reactions 1a and 1b, respectively. The quenching of ArNH+ with 9MG undergoes reaction 2, but one has to know the rate-determining TS of this reaction to obtain the relevant reaction rate constant. Although quenching of ArNH+ by guanine derivatives in water has been studied experimentally37,45,88 and is known to result only in C8 intermediates,21,45,33,93 the actual mechanism of this reaction remains disputed.32,34,42,45,47,94,95 It should be noted that the reaction rate constants predicted by DFT and high level ab initio calculations of the direct covalent binding of the nitrenium ion of 2-aminofluorene 11 to the C8 atom of 9MG were at least 2 orders of magnitude too slow with respect to the observed kinetics.40 To determine the structure of the rate-limiting TS, three possible sites of the initial attack of 2-fluorenylnitrenium ions in 9MG, specifically C8, N7, and N2, were investigated.

RESULTS AND DISCUSSION

Figure 1 presents the content of AstraZeneca corporate Ames mutagenicity database of ArNH2 with molecular weights below 400 g/mol. This data set mostly contains chemical building blocks and fragments rather than entire druglike molecules. The average fraction of Ames+ ArNH2 in this compound set is about 30%. Consistent with previous studies of this and other databases of ArNH2,66−69 the local fraction of mutagenic ArNH2 increases as soon as ArNH+ becomes more stable. We further note that the local fraction of mutagenic ArNH2 in our compound collection is maximal in the region where the ArNH+ is more stable than PhNH+ by approximately 20 kcal/mol in the gas phase; in this region, the proportion of Ames+ compounds reaches 50%. Identical locations of the maximum density of Ames+ ArNH2 in the axis of relative formation energy of ArNH+ have been observed in three databases collected by other organizations.67,68 Remarkably, the location of the maximum does

k1a

ArNHOH + H3O+ → ArNH+ + 2H 2O 2238

(1a)

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Figure 2. Structures of the investigated ArNH2. Molecules 1−12 represent the focused set. The structures of mutagenic compounds 13−27 are included to verify conclusions. Names and logarithmic values of mutagenic potencies (LogMP) are as follows:56,73,74,76,78 1, aniline, −3.39; 2, oanisidine, −1.672; 3, p-anisidine, −2.305; 4, dimethyl-4-phenylenediamine (DMPD), generates free radicals;111 5, 4-cyano-aniline, Ames positive; 6, 2-naphthylamine, −0.67; 7, biphenyl-4-amine, −0.14; 8, 2,6-dimethylbiphenyl-4-amine, likely mutagenic; 9, 4′-methoxybiphenyl-4-amine, likely mutagenic; 10, benzidine, −0.39; 11, 2-aminofluorene, 1.93; 12, 3-amino-4,6-dimethylpyrido(1,2-a:3′,2′-d)imidazole, 4.83; 13, 3-aminodipyrido(1,2a:3′,2′-d)imidazole, 2.26; 14, 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2), 4.09; 15, 2-amino-1-methylimidazolo[4,5-f ]quinoline, 5,80; 16, 2-amino-1-methylnaphtho[2,1-d]imidazole, 2.29; 17, 2-amino-3,8-dimethylimidazolo[4,5-f ]quinoxaline (MeIQx), 4.33; 18, 2-amino-3methylimidazolo[4,5-f ]quinoline (IQ), 4.50; 19, 2-amino-3-methylnaphtho[2,1-d]imidazole, 0.59; 20, 2-amino-1-methyl-6-phenylimidazole[4,5b]pyridine (PhIP), 2.75; 21, 2-amino-3-methyl-6-phenylimidazole[4,5-b]pyridine, 0.65; 22, 2-amino-3-methylimidazolo[4,5-b]6-methylfuro[2,3e]pyridine, 3.31; 23, 3-aminophenanthrene, 3.77; 24, 2-aminoanthracene, 2.62; 25, 2,7-diaminophenazine, 3.97; 26, 4-amino-6-methyl-1H2,5,10,10b-tetraazafluoranthene (Orn-P-1), 4.17; and 27, 8-aminofluoranthene, 3.80. k1b

ArNHOAc + H3O+ → ArNH+ + H 2O + HOAc k2

ArNH+ + dG → ArNH‐dG+

Table 1. Standard Gibbs Activation Energies of Different TSs of Reaction 2 of ArNH+ of 11 with 9MG with Respect to Separate Reactants in the Gas Phase (ΔGg#) and in Water (ΔGw#) Calculated at Different Levels of theorya

(1b) (2)

Table 1 presents standard Gibbs energies of activation barriers hindering covalent binding of 2-fluorenylnitrenium ion to C8, N7, and N2 of 9MG in the gas phase and water with respect to the separate reactants. This ArNH+ was chosen for detailed DFT studies and comparison with experimental data because it is known to react with guanine derivatives in the kinetic region.37 As can be seen, the results obtained with the M06-2X functional are not very sensitive to the basis set, which suggests that the M06-2X/6-31+G(d) level of theory is good enough to remove most of the superposition error. Consistent with prior theoretical considerations, water considerably increased the activation barriers of the reactions.40,95 The other functional, ωB97X-D, predicts systematically higher activation barriers by 1.5−2.5 kcal/mol. The obtained absolute values of Gibbs energies of TS in water appear too high to explain the observed rate constant (7.6 × 108 M−1 s−1)37 for all considered reactions except for one, in which the direct C8 covalent binding undergoes a TS of π-stacked charge-transfer type. The involvement of π-stacking intermediates in the reactions of ArNH+ with arene

kcal/mol ΔGg#

ΔGw#

9MG atom

TS type

F1/B1

F1/B2// F1/B1

F1/ B1

F1/B2// F1/B1

F2/B2// F1/B1

N2 N7 C8 C8

σ σ σ π

16.9 −4.4 2.2 −9.5

19.7 −1.3 3.2 −7.7

19.4 15.1 17.3 2.6

24.2 17.0 17.3 3.0

25.4 17.3 19.5 4.6

a F1 and F2 signify density functionals M06-2X and ωB97X-D, respectively. B1 and B2 denote basis sets 6-31+G(d) and 6-311+ +G(3df,3p), respectively. The observed effective Gibbs activation energy of the reaction in water is 5.23 kcal/mol.37

traps has previously been hypothesized based on results of laser flash photolysis experiments96,97 but has not yet been supported theoretically. The data presented in Table 1 indicate that the Gibbs energy, ΔGw#, of the π-stacked TS predicted by the DFT calculations 2239

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with the ωB97X-D functional (4.6 kcal/mol) agrees well with the effective activation barrier of 5.23 kcal/mol obtained in kinetic experiments in water,37 whereas the previously described σ-TS of the direct C8 binding in water40,42,43 is found to be about 14 kcal/mol too high. It should be noted that the effective activation barrier calculated from the observed reaction rate constants has to be somewhat higher than ΔGw# of this reaction because the actual reaction kinetics slows down by a closely located diffusion limit.37 Figure 3 presents the optimized structure of the π-TS and the frontier orbitals of 2-fluorenylnitrenium ion and 9MG and

DFT calculations at the M06-2X/6-311++G(3df,3p)//M062X/6-31+G(d) level of theory in water as functions of relative Gibbs energies of formation of ArNH+ in water calculated at the same level. Table 2 presents detailed information including results obtained by the single-point ωB97X-D/6-311++G(3df,3p)//M06-2X/6-31+G(d) calculations in water. As can be seen, the M06-2X/6-31+G(d) and ωB97X-D/6-311++G(3df,3p)//M06-2X/6-31+G(d) calculations give a very similar picture of the activation energies of reactions 1a and 1b for the formation of ArNH+, whereas the barrier heights predicted at the M06-2X/6-311++G(3df,3p)//M06-2X/6-31+G(d) level are about 4 kcal/mol higher. All three DFT levels predict similar activation barriers of reaction 2, the quenching of ArNH+ with 9MG, for the entire focused set, with the ωB97XD/6-311++G(3df,3p)//M06-2X/6-31+G(d) calculations giving 1.5−2.5 kcal/mol higher values of ΔGw#. It should be noted that at all theoretical levels employed, we see the same trend in that as soon as ArNH+ becomes more stabilized, the activation barriers of the formation and quenching linearly change in the opposite directions but with similar slopes, such that the former gets faster and the latter slows down. There are some disturbances from linearity of Gibbs activation energy for all four trends, but really significant deviation is observed only for compound 8 with nonplanar ArNH+ and only in π-stacked TS. According to the M06-2X/6-311++G(3df,3p)//M06-2X/ 6-31+G(d) level of theory, the Gibbs energy of the π-TS of 8 is about 5.4 kcal/mol higher than anticipated from a linear regression built on the remaining quasi-planar compounds (Figure 5). Naturally, the energy of the π-stacked TS with unusually short interatomic distances between two aromatic molecules is especially sensitive to the planarity of the reacting molecules. If reaction 2 is indeed mediated by the π-stacked TS, one should expect higher activation barriers for reactions of ArNH+ that are essentially nonplanar. The experimental fact that quenching of nonplanar ArNH+ of 8 with dG is much slower than the anticipated rate based on its high solvent reactivity37 confirms that reaction 2 undergoes the π-TS. Figure 6 presents the lengths of the covalent bonds in the TS structures that are broken in reactions 1a and 1b and formed in 2 as functions of relative energy of ArNH+ obtained at the M06-2X/6-31+G(d) level of theory in the gas phase. As can be seen, the bond lengths of the reaction coordinates are linearly linked to the stabilization energy of ArNH+ in all TSs, suggesting that it is the stabilization energy of ArNH+ that primarily determines the structure of the TS within each reaction. As expected, the bond lengths in all TSs correlate with the chemical reactivity of ArNH+. Unsurprisingly, the N−C8 bond in the π-TS of the reaction of nonplanar ArNH+ of 8 with 9MG significantly deviates from the linear regression line (0.12 Å shorter). The optimized π-stacked TS structures of reaction 2 for 7 and 8 are illustrated in Figure 7. As is seen, the angular character of the mutual orientation of the reactants in TS increases because of the essential nonplanarity of ArNH+ of 8. Rate constants of reactions 1a, 1b, and 2 were obtained from results of DFT calculations of the focused compound set at the M06-2X/6-311++G(3df,3p)//M06-2X/6-31+G(d) and ωB97X-D/6-311++G(3df,3p)//M06-2X/6-31+G(d) levels of theory in water (Table 2), by the following expression derived from the TS theory:

Figure 3. Origin of stabilization of the π-stacked charge-transfer TSs, in which N of ArNH+ eclipses the C8 atom of 9MG. (a) The mutual orientation of the reactants in the optimized π-TS structure, which gives favorable electronic coupling between HOMO of 9MG (b) and LUMO of 2-fluorenylnitrenium ion (c). The optimized mutual orientation of reactants inherent in the TS (a) is kept in parts b and c. Nitrogens, oxygens, and hydrogens are shown in dark blue, red, and gray, respectively. Carbon atoms of 9MG and 2-fluorenylnitrenium ion are shown in dark green and cyan, respectively. Different colors in the frontier molecular orbitals (red and dark blue) signify different signs of the wave functions.

suggests a possible explanation of the additional stabilization of the π-stacked TS with respect to σ-TS. As can be seen, in the π-stacked TS, where the nitrogen of ArNH+ eclipses the C8 of guanine, the node structures of the LUMO of 2-fluorenylnitrenium ion and the HOMO of 9MG exhibit the maximum overlap, which implies maximum electron coupling and maximum charge transfer.98 This suggests that direct C8 binding of ArNH+ to guanine is mediated by the π-stacked TS, with the stability of this structure explained by the maximum orbital coupling of the frontier π-orbitals of the reactants when N of ArNH+ is preorganized for direct covalent binding to C8 of guanine. Figure 4 illustrates TS structures that mediate reactions 1a, 1b, and 2 of derivatives of 2-aminofluorene, with both π-TS and σ-TS being shown for reaction 2. It should be noted that in both TSs that mediate formation of ArNH+ in reactions 1a and 1b, the proton is already transferred from the hydronium ion to oxygens of ArNHOH and ArNHOAc, respectively, as seen in Figure 4a,b. Figure 5 is a plot of Gibbs activation barriers hindering reactions 1a, 1b, and 2 obtained for the focused set of molecules by

k = (kBT /h) ·e−ΔGw 2240

#

/ RT

(3)

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Figure 4. Geometries of TSs of formation (a and b) and quenching (c and d) of ArNH+ of 2-aminofluorene 11 obtained by DFT calculations at the M06-2X/6-31+G(d) level of theory. (a) TS of reaction 1a. (b) TS of reaction 1b. (c) The σ-TS of reaction 2. (d) The π-TS of reaction 2. The optimized lengths of breaking and forming covalent bonds are given in Å.

is the Gibbs free energy of activation in water. The predicted average rate constants that interpolate the obtained DFT results for the quasi-planar compounds of the focused molecular set (Table 2) are illustrated in Figure 8 as functions of relative Gibbs energy of formation of ArNH+ in water. Experimentally determined rate constants of reaction 2 of four quasi-planar compounds, 3, 7, 9, and 11, and one nonplanar compound 837,88 are shown in Figure 8. As can be seen, the reaction rate constants of the π-stacked TS of reaction 2 obtained from DFT calculations fit to the experimental kinetics of ArNH+ observed in laser flash photolysis studies for 3, 9, and 11. Because of the planarity and relative stability of their ArNH+, they react with 9MG in the kinetic region, according to eq 3. On the other hand, the ArNH+ of 7 is less stable, such that the rate constant of reaction 2 hits the encounter limit. The ArNH+ of 8 is even less stable, but because of its nonplanarity, the activation barrier of π-TS is too high, which slows down its kinetics of quenching and keeps it significantly below the encounter level. Thus, experimental data are consistent with absolute rate constants predicted for the π-stacked TS of all five exemplified compounds, provided the encounter limit is taken into account. The reaction rate constants predicted for the σ-TS of reaction 2 by both density functionals are far away from experimental data (Figure 8). This together suggests that reaction 2 undergoes the π-stacked charge-transfer TS. It should be noted that both M06-2X/6-311++G(3df,3p)//M06-2X/6-31+G(d) and ωB97X-D/6-311++G(3df,3p)//M06-2X/6-31+G(d) levels of theory augmented with the PCM, which models solvation effects in water, exhibit a remarkable agreement with experimental kinetics data. Reactions 1a and 2, and 1b and 2 represent two pairs of consecutive reactions of the metabolically activated forms of ArNH2, undergoing intermediate formation of ArNH+. Strictly speaking, all of these reactions are of the second order. However, it can be readily assumed that they are of the pseudofirst order because the concentrations of H3O+ and dG at any one instance are much higher than those of the other reactants under slightly acidic conditions and therefore represent constants during the course of events. Thus, concentrations of the common product, the cationic C8 intermediate of dG, resulting from

Figure 5. Standard Gibbs energies of TSs in kcal/mol obtained for the focused set of ArNH2 at the M06-2X/6-311++G(3df,3p)//M06-2X/631+G(d) level of theory with respect to the separate reactants in water as functions of relative Gibbs energy of formation of ArNH+ in water. The Gibbs energy of formation of PhNH+ is used as a reference. Circles in A and triangles in B indicate activation barriers of formation and quenching of ArNH+ by 9MG, respectively. TSs of reactions 1a, 1b, π-TS of 2 and σ-TS of 2 for each compound are shown in red, blue, green, and purple, respectively. The activation energy of π-TS for nonplanar ArNH+ of 8 is additionally highlighted with a green square. Structures of the focused compound set are given in Figure 2.

where kB is Boltzmann's constant, h is Planck's constant, T is temperature (298 K), R is the universal gas constant, and ΔGw# 2241

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0.0 −15.1 −21.2 −40.7 15.4 −6.8 −7.9 −6.5 −15.8 −24.1 −15.5 −9.5 c1 c2

F1/B2//F1/ B1

7.2 0.92 −1.5 −6.6 16.4 5.3 5.3 5.5 3.8 0.79 2.5 3.6 0.39 ± 0.03 8.4 ± 0.5

F1/B1

4.9 −1.5 −3.1 −8.2 13.6 2.9 3.1 3.4 0.75 −0.94 −0.90 1.1 0.37 ± 0.03 5.8 ± 0.5

ΔGw#(1a)

5.2 −0.91 −3.9 −10.9 14.6 3.6 3.4 3.5 0.85 −0.85 −0.16 −1.3 0.42 ± 0.04 6.3 ± 0.7

0.25 −2.7 −4.9 −14.6 10.3 −0.09 −1.0 −0.56 −1.9 −7.1 −5.1 −2.1 0.41 ± 0.03 2.6 ± 0.5

3.3 −1.4 −2.7 −7.1 12.1 2.8 2.7 2.3 1.9 0.24 0.72 1.6 0.31 ± 0.03 5.2 ± 0.6

0.90 −5.7 −6.7 −11.6 10.2 0.37 0.68 −0.27 −1.65 −3.7 −3.3 −4.6 0.36 ± 0.04 2.4 ± 0.8

F2/B2//F1/ B1 9.2 12.9 18.3 27.6 6.0 12.4 13.0 11.7 17.9 22.9 17.3 10.2 −0.43 ± 0.05 9.5 ± 0.9

F1/B1 10.1 15.8 18.7 28.1 7.0 13.0 14.0 13.1 19.2 23.5 17.3 13.2 −0.41 ± 0.03 10.9 ± 0.6

F2/B2//Fl/Bl −4.6 4.8 6.2 17.8 −11.3 −4.4 −0.83 4.7 4.9 11.8 3.0 3.3 −0.55 ± 0.04 −4.2 ± 1.2

Fl/Bl −4.7 4.8 6.1 17.5 −10.7 −5.0 −1.5 4.9 4.0 11.0 2.58 0.97 −0.54 ± 0.04 −4.6 ± 0.8

10.6 16.6 19.8 30.6 8.3 14.6 15.3 13.6 21.2 26.2 19.5 15.0 −0.44 ± 0.04 12.2 ± 0.8

−2.3 7.2 9.1 19.9 −9.0 −2.2 0.87 3.3 7.0 14.5 4.6 6.1 −0.55 ± 0.05 −2.0 ± 0.9

F2/B2//Fl/ Bl

ΔGw#(2) π-TS 9MG-C8

F2/B2//Fl/ Bl

F1/B2//F1/ B1

F1/B2//F1/ B1

F1/B1

F2/B2//F1/ B1

ΔGw#(2) σ-TS 9MG-C8

ΔGw#(1b)

a

Stuctures of the focused set are given in Figure 2. All data are in kcal/mol. Fl and F2 are density functionals M06-2X and ωB97X-D, respectively. Bl and B2 are basis sets 6-31+G(d) and 6-311+ +G(3df,3p). The very left column gives compound numbers illustrated in Figure 2. c1 and c2 are the coefficients of linear regression ΔG# = c1 × ΔΔG + c2. The underlined values of nonplanar nitrenium ion of 8 were not taken into account when calculating regression coefficients. bRelative Gibbs energy of formation of ArNH+ obtained by DFT calculations in water with respect to PhNH+.

1 2 3 4 5 6 7 8 9 10 11 12

no.

F1/ B2// F1/B1

ΔΔGwb

Table 2. Standard Gibbs Activation Energies (ΔGW#) of Reactions 1a, 1b, and 2 in Water for the Focused Set of ArNH2 Obtained at the Specified Level of Theorya

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Figure 6. Lengths of breaking (N−O) or forming (N−C8) bonds in the TS structures obtained at the M06-2X/6-31+G(d) level of theory. Compound structures are shown in Figure 2. Symbols, colors, and numbers as defined in Figure 5. Bond lengths in the TS structures with the essentially nonplanar compound 8 are indicated as squares.

Figure 7. Comparison of optimized geometries of π-stacked TS structures of ArNH+ of quasi-planar 7 (a) and nonplanar 8 (b) with 9MG. Carbons of ArNH+ and 9MG are shown in cyan and dark green, respectively. Methyl groups in 8 decrease planarity of the ArNH+ moiety in the π-TS with 9MG, thereby increasing the activation barrier of reaction 2.

these two pairs of consecutive reactions, are determined by the following equations:

[ArNH‐dG+](t )

= [ArNHOH](0) ·{1 + (k 2·e−k1a·t − k1a·e−k 2·t )

/(k1a − k 2)}

/(k1b − k 2)}

2242

(4a)

[ArNH‐dG+](t )

= [ArNHOAc](0) ·{1 + (k 2·e−k1b·t − k1b·e−k 2·t )

(4b)

where k1a, k1b, and k2 are rate constants of reactions 1a, 1b, and 2, respectively, and t is the time of the course of the reaction. It is assumed that concentrations of the product ([ArNH-dG+]) and the reaction intermediate ([ArNH+]) are equal to zero at

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product in 4a or 4b, and, consequently, the higher the chemical reactivity of the metabolically activated form. This constant reference time (tref) should be long enough to enable deviations of values of R from 0 and short enough to ensure that they did not become 1 for any compound as yet. Analogous to IC50 determination, we suggest choosing the reference time by limiting the value of R for the most reactive compound in the fastest reaction pair, R1b‑2(t), to be equal to 50%. In other words, tref is defined as a specific time point in the reaction course for the faster pair of the consecutive reactions such that 50% of the final concentration of the product in 4b formed by the most reactive imaginary reactant is obtained. Because the slopes of the rate constants of the consecutive reactions 1b and 2 shown in Figure 8 are opposite, the maximum R at each given time in 5b is achieved when k1b = k2. In this particular case, to avoid singularity, one has to use the eq 6, which describes the most reactive compound: R max(t ) = 1 − e−k·t − k·t ·e−k·t

where k = k1b = k2. When one defines tref from eq 6, values of R(tref) for any compound in 5a and 5b at this particular time are below Rmax(tref). The value of the reference time given in eq 7 corresponds to the concentration of the product of the most reactive imaginary compound formed in the course of the fastest reaction pair 1b-2 to be Rmax(tref) = 50%:

Figure 8. Predicted rate constants of reactions 1a, 1b, and 2 at standard conditions in water obtained at the M06-2X/6-311+ +G(3df,3p)//M06-2X/6-31+G(d) (A) and the ωB97X-D/6-311+ +G(3df,3p)//M06-2X/6-31+G(d) (B) levels of theory vs relative Gibbs energy of formation of ArNH+ with respect to PhNH+ calculated at the M06-2X/6-311++G(3df,3p)//M06-2X/6-31+G(d) level in water. The diffusion limit of reactivity of ArNH+ in water (2.2 × 109 M−1 s−1) is shown by the dashed horizontal line.37 Experimental data on reactivity of ArNH+ of quasi-planar compounds 3, 7, 9, and 11 with 2′-deoxyguanoside in water are illustrated by green circles; the reactivity of the nonplanar compound 8 is shown by a green square.37 The upper limit of reactivity of 4-alkoxyphenylnitrenium ion 3 is indicated.88 Colors of the lines are defined in Figure 5. The red arrows in A and B point to the values of the relative stability of ArNH+, at which the rate constant functions of reaction 2 calculated for π-TS with the M06-2X and ωB97X-D density functionals intersect the experimentally determined diffusion limit (−16 and −12 kcal/mol, respectively). The intersection points of k1b and k2 at both levels of theory are encircled in red. These points define the reference times, tref, which are 5.2 × 10−12 and 8.2 × 10−13 s for the M06-2X and ωB97X-D functionals, respectively. Intersection points that define the maximum reactivity of the metabolically activated forms in water are shown by the black arrows in A and B.

tref = 1.6783/k

(5a)

R1b‐2(t ) = 1 + (k 2·e−k1b·t − k1b·e−k 2·t )/(k1b − k 2)

(5b)

(7)

Thus, the chemical reactivities of different metabolically activated forms of various ArNH2 can be compared using eqs 5a and 5b at the fixed reference time, tref. The reference time is defined by eq 7 at the intersection point of the straight lines of k1b and k2 (Figure 8), where k = k1b = k2. The following expressions give the concentrations of the C8 intermediates of dG at the fixed reference time in the course of reactions 1a, 1b, and 2 as products of the initial concentration of the metabolically activated form and its chemical reactivity toward the C8 atom of dG: [ArNH‐dG+](tref ) = [ArNHOH](0) ·{1 + (k 2·e−k1a·tref − k1a·e−k 2·tref ) /(k1a − k 2)}

(8a)

[ArNH‐dG+](tref ) = [ArNHOAc](0) ·{1 + (k 2·e−k1b·tref − k1b·e−k 2·tref )

t = 0. If the initial concentrations of the metabolically activated forms of different ArNH2 are considered to be equal, concentrations of the products in 4a and 4b at time (t) are determined by the chemical reactivities of these forms in two consecutive reactions, 1a-2 or 1b-2, which are given by the following expressions: R1a‐2(t ) = 1 + (k 2·e−k1a·t − k1a·e−k 2·t )/(k1a − k 2)

(6)

/(k1b − k 2)}

(8b)

where the expressions within the curly brackets are dimensionless constants. Figure 9 presents the chemical reactivities of ArNHOH and ArNHOAc in logarithmic scale given by expressions within the curly brackets of eqs 8a and 8b based on linear regressions of actual DFT data presented in Figure 8 and Table 2 and the calculated reference times tref of 5.2 × 10−12 and 8.2 × 10−13 s obtained for M06-2X and ωB97X-D functionals, respectively. As can be seen, the maximum chemical reactivity of ArNHOH and ArNHOAc is located in the vicinity of −10 kcal/mol on the horizontal axis for results obtained with the M06-2X density functional (Figure 9A) and at −4 kcal/mol for the ωB97X-D functional (Figure 9B). The identical left slopes of the chemical reactivity curves for ArNHOH and ArNHOAc in Figures 9 represent situations, where the overall rate of the consecutive

where R1a‑2(t) and R1b‑2(t) are dimensionless monotonous functions of time. These functions vary from 0 to 1 as time runs from zero to infinity. More reactive metabolically activated forms reach the maximum R faster. To compare values of R for different ArNH2 using eqs 5a and 5b, one has to define a certain intermediate time at which these values differ. The higher magnitude of R at this time, the higher concentration of the 2243

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Figure 9. Chemical reactivity of the metabolically activated forms of quasi-planar ArNH2 toward the C8 atom of 9MG in water predicted from first principles, expressed in logarithmic scale as functions of relative Gibbs energy of formation of ArNH+ in kcal/mol with respect to PhNH+. A and B give results obtained with the M06-2X and ωB97X-D density functionals, respectively. The reactivities of ArNHOH and ArNHOAc are shown in red and blue, respectively. Dashed lines illustrate theoretical reactivities based on the linear regressions obtained from the Gibbs activation barriers of the focused set (Table 2) by expressions within the curly brackets in eqs 9a and 9b. Solid lines show the chemical reactivities that take the observed diffusion limit of reactivity of ArNH+ for guanine derivatives in water into account.37 The vertical red arrows point to the predicted locations of the maximum of chemical reactivity of the metabolically activated forms toward guanine derivatives in water. The locations of the maximum reactivity points are determined by the intersections of the rate constant k2 lines with the diffusion limit (Figure 8). The ratios of the reactivities of ArNHOAc and ArNHOH are defined in the area, where they are determined by the rate of the hydrolytic dissociation. (C) Rescale of the relative Gibbs energy of formation of ArNH+ in water calculated with the extended basis set to their relative formation energy in gas phase using M06-2X/6-31+G(d) calculations. Energies of PhNH+ are used as references. The blue bracket in the vertical axis outlines the “area −20”, the region of the maximum chemical reactivity of metabolically activated forms of ArNH2. Structures of the focused compound set are given in Figure 2.

energy of ArNH+ in the gas phase (vertical axis), which represents a conventional scale of resonance stabilities of ArNH+. As a result, the maximum chemical reactivity of both metabolically activated forms is located in “area −20” in the gas phase. This explains the mentioned similar location of the maximal mutagenic fraction in different databases of ArNH2 in the ArNH+ stabilization axis in the gas phase. It is important to add that the bioconjugated form, ArNHOAc, is roughly 60 times more reactive than hydroxylamine, ArNHOH (Figure 9A,B), which is in line with experimental data. However, both metabolically activated forms of compounds that produce less reactive ArNH+, which react with dG in the kinetic region, have identical reactivities, which is, for example, the case for DMPD 4 or benzidine 10 (Figure 9). Differentiation of eqs 8a and 8b leads to eqs 9a and 9b that give the rates of formation of dG-C8 adducts. The chemical reactivities of the metabolically activated forms of ArNH2 are represented by the expressions within the curly brackets. The other important factor of each equation is the rate of metabolic formation of these activated forms:

reactions 1 and 2 is determined by the rates of quenching of ArNH+ with 9MG, which is inherent in especially stable ArNH+. The right slopes reflect situations where the overall rate of the consecutive reactions is determined by the rates of dissociation of the metabolically activated species under acidic conditions, characteristic of more unstable ArNH+. The intersection of the lines of the reaction rate constants of formation and quenching of ArNH+ (Figure 8) defines the position of the maximum chemical reactivity of the metabolically activated forms. The existence of the diffusion limit of reaction 2 in water does not allow the corresponding rate constant, k2, to be higher that the constant value of 2.2 × 109 M−1 s−1.37 As is seen in Figure 9A,B, when this condition is applied to eqs 5a and 5b at t = tref, the chemical reactivities of both metabolically activated forms in the right part of the curves decrease, and the maximum reactivities shift to the left toward the area around −15 kcal/mol in the horizontal axis. Positions of the new maxima are determined by the intersection points of k2 with the diffusion limit (Figures 8 and 9). This implies that the maximum chemical reactivity is inherent in metabolically activated forms of such ArNH2, which undergo ArNH+ of intermediate stability, such that they are sufficiently stable to be readily formed and sufficiently reactive to exhibit the diffusion-controlled reactivity toward the C8 atoms of guanine derivatives in water. As is seen, the existence of the diffusion limit decreases the maximum chemical reactivity of ArNHOAc toward dG by 2 orders of magnitude. Figure 9C rescales the relative Gibbs energy of formation of ArNH+ in water (horizontal axis) to the relative formation

d[ArNH‐dG+](t ) d[ArNHOH](t ) ∼ dt dt ·{1 + (k 2· e−k1a·tref − k1a· e−k 2·tref )/(k1a − k 2)}

(9a)

d[ArNH‐dG+](t ) d[ArNHOAc](t ) ∼ dt dt ·{1 + (k 2· e−k1b·tref − k1b· e−k 2·tref )/(k1b − k 2)} 2244

(9b)

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where k2 is equal to the experimentally determined encounter limit if the intrinsic reactivity of ArNH+ in reaction 2 exceeds that limit. The rate of formation of guanine adducts is considered to be a measure of mutagenic potency of ArNH2.21,27,99 Recent experimental data obtained for few ArNH2 suggest that there is no direct connection between the number of DNA adducts and the number of DNA mutations or between the number of DNA mutations and the mutagenic potency of ArNH2.58−64 Nevertheless, the rate of formation of DNA adducts given by eqs 9a and 9b may still be linked to the mutagenic potency within the entire class of ArNH2 at least as a correlation. To verify this assumption, we formed a structurally diverse set of mutagenic ArNH2 to check whether behavior inherent in each of the two factors of eq 9b can be detected in experimental mutagenicity data. Figure 10 presents two plots of Ames mutagenic potency of 91 ArNH2 taken from different classes56,73,74,76,78 versus relative

that have been shown to be mutagenic and in some cases also carcinogenic.9,70,71,78,100−102 Structures, mutagenic potencies, absolute energies, and relative stabilities of the cationic and anionic forms obtained at the M06-2X/6-31+G(d) level of theory for all of these compounds are presented in Table 1S in the Supporting Information. Structures of distinct representatives of this set are shown in Figure 2. The chemical reactivity function of ArNHOAc as it is seen within the curly brackets of eq 9b is taken from Figure 9A and after rescaling to the gas phase by the linear correlation presented in Figure 9C overlaid with data in Figure 10A. Figure 10A reflects the promutagenic effects of the chemical reactivity of the metabolically activated forms: the maximum mutagenic potency of ArNH2 observed at a given level of stability of ArNH+ tends to follow the chemical reactivity function. Consistent with data presented in Figure 10, mutagenic potencies of ArNH2 have been noticed to span 10 orders of magnitude.74 Notably, the chemical reactivity of ArNHOAc also covers 10 orders of magnitude (Figure 10A). As can be seen, all of the most mutagenic compounds are located in the “area −20” in Figure 10A, more precisely between −30 and −10 kcal/mol. Thus, the chemical reactivity of metabolically activated forms toward the C8 atom of 9MG seems to represent the primary pro-mutagenic factor of planar polycyclic ArNH2. Figure 10B reflects the relative ease of proton abstraction from ArNH2, which has been hypothesized to be the ratelimiting step in the metabolic activation of planar ArNH2 by the 1A2 isoform of cytochrome P450 (P450 1A2) and NAT.17,79 Consistent with this concept, the rates of N-hydroxylation by P450 subfamily 1 enzymes tend to increase with resonance stabilization of the anionic form. A classical example of the importance of the rate of metabolic activation in the overall mutagenic potency of ArNH2 is aniline 1. Although it is not mutagenic by itself,103 its bioactivated form, PhNHOAc, has been demonstrated to be mutagenic and forms the dG-C8 adducts with dG and DNA.104 The electron-withdrawing pcyano group that stabilizes the anionic form makes 4-cyanoaniline 5 mutagenic by facilitating the proton abstraction event. Furthermore, PhIP 20, which has the most stable anionic form of the compounds in the data set, was well documented to be N-hydroxylated by P450 enzymes faster than Trp-P-2 14, MeIQx 17, IQ 18, and 2-amino-3-methyl-6-phenylimidazole[4,5-b]pyridine 21.56,58,60,105 Naturally, there are other factors that affect the rate of N-hydroxylation of a particular ArNH2 apart from the anionic stabilization, such as the geometric fit to the binding sites of P450 enzymes, residence time of the productive binding mode in the binding site, and existence of competing routes of detoxification. Nevertheless, if the mechanism of metabolic activation of all ArNH2 by P450 family 1 enzymes includes the proton abstraction event, the general trend of accelerating the metabolic activation by the resonance stabilization of the anionic form has to be seen in the entire class of ArNH2. Actual data presented in Figure 10A,B are used for statistical evaluation of the relevance of the rate of formation of DNA adducts given by eqs 9a and 9b to the mutagenic potency of ArNH2. A χ-squared test (χ2 = 19.3, p value = 4.0 × 10−6) shows the significance of the link between the high chemical reactivity of the metabolically activated forms (relative stability of ArNH+ being in the “area −20”) and the high mutagenic potency of ArNH2 (Log MP > 2 in Figure 10A). This implies that high chemical reactivity of ArNHOH and ArNHOAc is required for the high mutagenic potency of ArNH2. The second

Figure 10. Ames mutagenic potency (MP) in logarithmic scale observed for 91 ArNH2 of different classes vs relative formation energy of cationic (A) and anionic (B) forms in gas phase with respect to PhNH+ and PhNH−, respectively, obtained by fully optimized DFT calculations at the M06-2X/6-31+G(d) level. The most mutagenic compounds that are above the red horizontal line are located within “area −20”, which is indicative of maximal chemical reactivity of metabolically activated forms of ArNH2. The chemical reactivity function of ArNHOAc taken from Figure 9A and rescaled for the gas phase nitrenium stability is shown as a blue line in A; the values in logarithmic scale are illustrated by the blue axis to the left from the plot A. The thick red arrow in B illustrates the general promutagenic trend of increasing resonance stabilization of the anionic form. Shapes and colors of the data points signify the chemical class of ArNH2: yellow rhombs, monocyclic; green rhombs, fused aromatics; black squares, aminoimidazoazaarenes; purple circles, analogues of biphenyl4-amine; red circles, analogues of 2-aminofluorene; and yellow triangles, analogues of amino-carbolines. Structures of selected compounds are given in Figure 2, and the whole set is detailed in Table 1S in the Supporting Information.

stabilities of their cationic (Figure 10A) and anionic (Figure 10B) forms in the gas phase. Included are monocyclic, bicyclic, tricyclic, and tetracyclic aromatic and heteroaromatic amines 2245

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a well-known mutagen and human carcinogen,106 and four included derivatives are located to the left of the “area −20”, which is indicative of too stable ArNH+ for forming DNA adducts. This result is in line with experimental results that demonstrated that the mechanism of mutagenicity and carcinogenicity of these compounds is different and primarily caused by their ability to generate free radicals and oxidative stress, which cause the oxidative damage of DNA.79,107−114 The standard bacterial Ames mutagenicity tests in strains TA98 and TA100 are just not sensitive to oxidative genotoxicity.107 As is seen in Figure 10A, in addition to the most mutagenic ArNH2 known to man, “area −20” includes many slightly genotoxic compounds, for example, monocyclic and a number of bicyclic ArNH2. The chemical reactivity of the metabolically activated forms of these compounds toward DNA is as high as for the most mutagenic ones (see Figure 5). It is the rate of metabolic activation, the first factor in eqs 9a and 9b, which is the primary factor that keeps their mutagenicity low. Figure 10B illustrates that monocyclic ArNH2 have relatively unstable anionic states, which slows down the proton abstraction events in P450 1A2 and NAT.79 Extension of the aromatic system in ArNH2, which has previously been established as a major promutagenic factor,5,65,74,77,78,100,115,116 stabilizes the anionic form (ArNH−) and thereby accelerates the metabolic activation in these enzymes. The other important factor, which is known to be able to dramatically reduce mutagenic potency of polycyclic ArNH2, is reduction of the miscoding propensity of the dG-C8 adduct because of strong inhibition of DNA polymerases.58 Thus, the maximum chemical reactivity of the metabolically activated forms inherent in ArNH2 of “area −20” caused by particular stabilization energy of ArNH+ is required but not sufficient for high mutagenic potency. The decrease of the chemical reactivity of the metabolically activated forms by 10 orders of magnitude from the maximum levels is still not enough to completely remove compound mutagenicity. How could the mutagenicity of compounds be possibly eliminated? The decrease of compound lipophilicity does not bring mutagenic potency to zero.5 As demonstrated with compound 8, essential nonplanarity of ArNH+ reduces its chemical reactivity toward dG but does not completely abolishes it.37 The addition of a number of electron-withdrawing groups, which destabilize ArNH+, moves compounds away to the right from “area −20” in Figure 10A, but if they remain planar, the rate of formation of DNA adducts and mutagenic potency will roughly follow the chemical reactivity of the metabolically activated forms, which in this range of the nitrenium stabilization energy may not be enough to make compounds Ames−. For example, we note that monocyclic 4-cyano-aniline 5 (that would be located to the very right in Figures 10A) is still mutagenic in TA98 and TA100 strains. Removal of mutagenicity of ArNH2 with an extended aromatic system by adding electron-withdrawing functions is even more difficult because one has to start with a higher mutagenic potency. On the other hand, by making ArNH2 sufficiently nonplanar or by using structural elements that are incompatible with the planar substrate binding site of P450 1A2, their geometric fit to P450 1A2 could be disrupted, such that the rate of metabolic activation will approach zero and the chemical reactivity of the metabolically activated forms will not make the difference.79 Thus, we recommend disrupting the complementarity of ArNH2 to the substrate sites of the N-hydroxylating P450 enzymes as the most reliable method to design nongenotoxic ArNH2, even if they appear in “area −20”. This is in line with prior publications that showed that steric

statistical test that we make is significance of the Pearson correlation between the mutagenic potency of ArNH2 and the relative anionic stability in Figure 10B using the Fischer statistic. The value of r2 = 0.3 (Df = 89, FStat = 39.44, and p value = 1.2 × 10−8) indicates that there is a significant link between the anionic stability and the mutagenic potency of ArNH2. This means that there is a statistically significant trend of increasing the overall mutagenic potency of ArNH2 by facilitating the proton abstraction event. The third statistical test is whether the combination of two factors of eqs 9a and 9b improves the latter correlation. The chemical reactivity of the metabolically activated forms was simplified for this test as closeness to the value of −20 kcal/mol in the relative stability of ArNH+. According to the Fischer statistic, a combination of the two factors, the simplified chemical reactivity of the metabolically activated forms and the rate of their formation (represented by the anionic stability) as required by eqs 9a and 9b, makes the correlation with the mutagenic potency stronger: r2 = 0.4 (Df = 89, FStat = 59.22, and p value = 1.8 × 10−11). This means that both the high stability of the anionic form and the high reactivity of the metabolically activated forms are statistically required for the high mutagenic potency of the diverse set of ArNH2. Thus, the rate of formation of DNA adducts is likely to be statistically valid as a measure of mutagenic potency within the entire class of ArNH2. The rate of formation of DNA adducts given by eqs 9a and 9b incorporates many contradictory factors that have been demonstrated to be critical for the mutagenic potency of ArNH+, such as (i) the conventional nitrenium stabilization concept, which reflects the importance of the ease of dissociation of ArNHOAc in the right slopes of the curves in Figure 9A,B;67−71 (ii) the intrinsic DNA reactivity of ArNH+ that is critical in the left slopes of the curves and works against the nitrenium stabilization concept; (iii) the geometric fit of ArNH2 to the enzymes involved in the metabolic activation of ArNH2, like P450 1A2 and NAT;79 and (iv) the ease of proton abstraction by P450 1A2 and NAT.17,79 The nitrenium stabilization concept can be illustrated for example by comparison of 2-naphthylamine 6 and biphenyl-4-amine 7. The promutagenic propensity of chemical reactivity of ArNH+ is clearly seen in the analogues of 2-aminofluorene (shown in Figure 10A by red circles), in which removal of a π-electron-donating group in the resonance position as well as its replacement by electronwithdrawing functions increases mutagenic potency. Insufficient shape complementarity of ArNH2 with the substrate binding sites of the metabolizing enzymes is likely the reason of low mutagenic potency three amino-carbolines illustrated in Figure 10A; in these compounds, the amino group is about perpendicular to the long axis of the molecule.79 Another example of this kind is 1-naphthylamine, which is much less mutagenic than its isomer, 2-naphthylamine. The former is N-hydroxylated much slower than the latter, even though the metabolically activated form of the former is more reactive.54−56 The promutagenic effects of the ease of proton abstraction from ArNH2 are seen in Figure 10B. The drop of mutagenic potency to the right of the “area −20” in Figure 10A is linked to the destabilization of ArNH+ caused by the decrease of the size of the aromatic system in ArNH2 and additional electron-withdrawing functions. This area consists of monocyclic and bicyclic ArNH2, which are much less mutagenic than polycyclic ArNH2, consistent with previous studies.56,66−72,78 The drop of mutagenic potency for ArNH2 with very stable ArNH+ in the left part of Figure 10A does not make them less genotoxic. Notably, benzidine 10, 2246

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aromatic moieties fit to the space between base pairs in doublestranded DNA, such that the long axis of the intercalator almost aligns with the long axis of the intercalation site.126,134,135 The free energy of precovalent intercalated complexes of neutral compounds is rather small (around −5 kcal/mol) because of the substantial free energy cost of forming the intercalation cavity;127 therefore, deviations from this structural feature make the intercalation impossible. To test whether metabolically activated forms of ArNH2 could also undergo precovalent intercalation before the formation of ArNH+, we tested two ArNH2 that are too long to fit to the intercalation site. Figure 11 shows important features of the optimized intercalated complexes with three mutagenic compounds, ethidium bromide 28136 and the bioconjugated forms of amino-carbazoles, 29 and 30.137 As is seen, long axes of three intercalators are approximately directed along the intercalation site. An additional noncoplanar dimethyl-oxazole ring was added to the positions of 29 and 30 to form 31 and 32, respectively, to cause maximum sterical clashes with the cytosine DNA backbone. If precovalent intercalation was a critical step of the mutagenic pathway of ArNH2, 31 and 32 would not be mutagenic. On the contrary, Ames mutagenicity tests in bacterial strains TA98 and TA100 showed that these compounds are mutagenic, which suggests that the mutagenicity pathway of ArNH2 does not necessarily involve DNA intercalation. This result is in line with publications that demonstrated that the formation of dG-C8 adducts in DNA occurs during replication or transcription, when DNA is in the single-stranded form.50 Correspondingly, geometric incompatibility with the intercalation site in double-stranded DNA cannot be used to design nongenotoxic ArNH2.

hindrance caused by bulky ortho-substituents disrupted the metabolic oxidation of the NH2 group in ArNH2 by the enzymes, thereby decreasing or removing their mutagenicity.117−119 We demonstrated that reactions of ArNH+ with guanine derivatives are mediated by π-stacked TS structures, in which the planar aromatic reactants are approximately parallel to each other, and the nitrogen of ArNH+ eclipses C8 of 9MG. When it comes to reactions of ArNH+ with dG in double-stranded DNA, the required initial orientation of the reactants prior to reaction could result from intercalation of metabolically activated forms of ArNH2. Precovalent intercalation is proposed to be a distinct step in the mutagenic pathways of several classes of mutagens and carcinogens, including ArNH2.52,120−133 It has been noted that the increase of the size of the aromatic system in ArNO2 beyond a certain limit leads to a drop of compound mutagenicity, and this effect was ascribed to exceeding the size limit imposed by the intercalation site in DNA.51 If the DNA intercalation is an essential step in the mutagenic pathway of ArNH2, disruption of the geometric fit to the intercalation site of DNA could be used in drug discovery programs to remove genotoxocity of aromatic amines to be used as building block in drugs. In the intercalated binding mode, quasi-planar hydrophobic

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CONCLUSIONS We have developed a mechanistic model for the genotoxicity of aromatic amines supported by calculations on the kinetics of formation and quenching of nitrenium ions with 9MG in water. Thus, in line with experimental rate constants, we propose that binding of nitrenium ions to guanine derivatives is mediated by the π-stacked TS prior to a direct reaction with the C8 atom. This TS corresponds to the lowest Gibbs activation energy with respect to the previously described σ TSs leading to reactions with C8, N7, or N2 of guanine. It follows that the efficiency of formation of guanine adducts by the metabolically activated forms of aromatic amines is determined by a balance of rate constants of formation and quenching of nitrenium ions, which are determined by their stability and planarity. The most mutagenic and carcinogenic aromatic amines are shown to be quasi-planar and have similar nitrenium ion stabilities, being 20 ± 10 kcal/mol more stable than PhNH+ in the gas phase. The origin of this effect is that the metabolically activated forms of such compounds, ArNHOH and ArNHOAc, exhibit the maximum chemical reactivity toward the C8 atoms of guanine, the primary target of nitrenium ions in DNA. Nitrenium ions of intermediate stability react with guanine derivatives with diffusion-controlled rate constants and are readily formed under slightly acidic conditions. Nitrenium ions with a higher stability exhibit lower DNA reactivity, whereas less stable nitrenium ions display the same DNA reactivity but lower rates of formation. It is the maximum DNA reactivity of the metabolically activated forms that makes “area −20” the region with the highest density of mutagenic quasi-planar aromatic and heteroaromatic amines of diverse structures in various databases. This result can be used in predictive QSAR models of Ames mutagenic

Figure 11. Test of the precovalent intercalation concept as a necessary step in the mutagenic pathway of ArNH2: (A) mutagenic compounds and (B) overlaid intercalated binding modes of ethidium bromide 28 (shown in the ball-and-stick fashion) and metabolically activated forms of amino-carbazoles 29 and 30 in a 20 base pair guanine repeat predicted by molecular modeling. Atoms of the oligonucleotide and nonpolar hydrogens of intercalated molecules are not shown. The molecular surface of the oligonucleotide backbone around the intercalation site is shown in red. This surface defines the boundaries of the intercalation site. The long axis of the intercalation site is oriented horizontally. Carbon atoms of 28 and ArNHOAc forms of 29 and 30 are shown in gray, cyan, and dark red, respectively. Arrows point to positions in 29 and 30, which are located by the boundaries of the intercalations site; therefore, DNA intercalation should be disrupted by bulky substitutions added to this place like in 31 and 32. 2247

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Chemical Research in Toxicology potency of ArNH2. We recommend splitting the whole range of the relative formation energy of nitrenium ions with respect to PhNH+ into two regions, lower and higher than the borderline of −20 kcal/mol in the gas phase, such that the nitrenium ion stability and the relevant descriptors will be antimutagenic and promutagenic in these regions, respectively. We propose eqs 9a and 9b that link the rate of formation of DNA adducts to the rate of formation and the chemical reactivity of the metabolically activated forms of ArNH2. These equations are derived from first principles and provide mechanistic understanding of the factors that affect mutagenic potency of aromatic amines. In addition to the chemical reactivity of the metabolically activated species of planar ArNH2, which is primarily determined by the structure of the aromatic system, the mutagenic potency is influenced by the rate of metabolic formation of these forms by several enzymes, which is dependent on the intrinsic propensity of the aromatic system to stabilize the anionic forms and on the structure of the entire molecule that has to fit to the substrate binding sites of the metabolizing enzymes. To completely remove the mutagenicity of aromatic and heteroaromatic amines, we recommend primarily focusing on chemical alterations that lead to the prevention of the N-hydroxylation by P450 enzymes, mostly by the P450 subfamily 1, by engineering steric incompatibility of the productive binding mode with the substrate binding site, for example, by making them essentially nonplanar.79 It is also possible to prevent hydrolytic dissociation of the metabolically activated forms of monocyclic aromatic amines by significant destabilization of nitrenium ions using one or more electron-withdrawing groups.79 Thus, we provide a mechanism-based reasoning that supports rational design strategies for avoidance of Ames mutagenicity in drug discovery programs.

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ABBREVIATIONS

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REFERENCES

P450 1A2, the 1A2 isoform of cytochrome P450; P450 1A1, the 1A1 isoform of cytochrome P450; NAT, arylamine Nacetyltransferase; SULT, sulfotransferase; dG, 2′-deoxyguanosine; 9MG, 9-methylguanine; QSAR, quantinative structure− activity relationship; DFT, density functional theory; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital; BSSE, basis set superposition error; PCM, polarizable continuum model; r2, the squared correlation coefficient; Df, degree of freedom; FStat, Fischer statistics; p value, probability value

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ASSOCIATED CONTENT

S Supporting Information *

(i) Absolute energies and energy-optimized atomic Cartesian coordinates of the key compounds and TSs of the focused set and (ii) the M062X/6-31+G(d) optimized absolute energies and mutagenic potencies of a set of aromatic amines taken from different structural classes as well as absolute and relative energies of their cationic and anionic forms discussed in this article. This material is available free of charge via the Internet at http://pubs.acs.org.

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Corresponding Author

*Tel: +4631-7064347. Fax: +4631-7763818. E-mail: igor. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We express our gratitude to the High-Performance Computing Team of AstraZeneca R&D Mölndal and in particular Roger Andersson, Martin Budsjö, Andreas Loong, Mikael Bengtsson, Joakim Castenström, and Lisa Hamberg for their terrific job of keeping our corporate supercomputing facilities in a fantastic shape. We thank Ulrika Ekstrand for her help in preparation of the manuscript. 2248

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