[Frontiers in Bioscience S5, 600-610, January 1, 2013]

Quantum-chemical studies on mutagenicity of aromatic and heteroaromatic amines

Gabriela L. Borosky1

1Departamento de Matematica y Fisica, INFIQC, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Ciudad Universitaria, Cordoba 5000, Argentina


1. Abstract
2. Introduction
3. Structure-activity relationships
4. The role of nitrenium ions
4.1. Formation of nitrenium ions
4.2. Stability of nitrenium ions
5. Aromatic amides
6. The influence of hydrophobicity
7. Summary and perspective
8. Acknowledgements
9. References


Arylamines are well-known as widespread industrial and environmental mutagens and carcinogens. Their bioactivity stems from enzymatic metabolic activation to reactive and highly electrophilic intermediates. In this work, computational investigations related to the biological activity of these compounds have been reviewed, especially focusing on studies reporting results from quantum-mechanical calculations. Correlations between relative mutagenicities and structural and electronic features of the parent amines and of their derived nitrenium ion intermediates were examined, with the aim of achieving a clearer comprehension of the main factors determining the genotoxic potential of this type of compounds.


Numerous aromatic amines (AAs) present in different industrial activities (dyes used in textile, paper, plastic, pharmaceutical, cosmetic, and food industries), as well as in tobacco smoke, are widely distributed environmental carcinogens (1-3). Similarly, heteroaromatic amines (HAs), found in well-done or over cooked meats and protein rich foods, have also been confirmed as mutagens and carcinogens (4-9). The genotoxic potential of these compounds is developed by enzymatic metabolic activation (figure 1) (10-12). The initial step involves oxidation of the exocyclic amine nitrogen by cytochrome P450 enzymes to form aryl N-hydroxylamines (10), which can undergo N-O bond cleavage to arylnitrenium ions under mildly acidic conditions (12). Additionally, further bioactivation of N-hydroxylamines to sulfuric or acetic acid esters facilitates the heterolysis of the N-O bond (10-12). The very electrophilic and highly reactive nitrenium ions generated are the ultimate metabolites that covalently bind and modify DNA (10-12). Replication of covalent DNA adducts may lead to mutations and cancer induction (13-15). Besides this major mutagenic pathway, it has been suggested that interaction of a number of AAs and HAs with P450 enzymes and peroxidases causes formation of reactive oxygen species (ROS), which may also provoke DNA mutations and cancer (figure 1) (16-20).


During the last decades, many efforts have been made to correlate mutagenicity and carcinogenicity data for AAs and HAs with observed and/or calculated properties of the amines or of their derived nitrenium ions. As an indication of the marked interest in this topic, several structure-activity relationship (SAR) and quantitative structure-activity relationship (QSAR) studies have been reported (21-43). Some of them, based on the same set of 95 aromatic and HAs, have lately been compared (44). It was found that the QSAR model developed by Maran et al. (33) (equivalent to the one of Karelson et. al. (38)) was the most adequate to describe the mutagenicity of the 95 amines studied (44). This linear model combined one constitutional descriptor, related to the hydrophobically active surface area of the compounds, and five descriptors derived from quantum-chemical calculations. QSAR studies relative to in vitro mutagenicity and animal carcinogenicity of aromatic and HAs have been recently reviewed (45).

According to QSAR analyses having a mechanistic orientation, the toxic activity of amines was shown to correlate with the ease of formation of the N-hydroxylamine, with the stability of the nitrenium ion, and with the ease of formation of epoxides on the aromatic ring (46). On the other hand, various studies pointed to the central role of hydrophobicity in the mutagenic and carcinogenic potency of aromatic and HAs (45). Electronic descriptors related to reactivity have also been implied, as the energies of the highest occupied molecular orbital (HOMO) and of the lowest unoccupied molecular orbital (LUMO) (45). The HOMO energy describes the tendency of a molecule to be oxidized, and in this case can account for the propensity to form the hydroxylamine metabolite, whereas the mechanistic reason for the correlation of the mutagenic potential with the LUMO energy is not clear. Regarding steric effects, bulky substituents at the nitrogen amino group, and ortho to the amino function, were observed to decrease the activity of arylamines (45). Several authors have found correlations between potency and topological parameters, as the number of fused aromatic rings and the type of ring system (45). In a very recent publication, mutagenicity of arylamines has been ascribed to three factors: (i) high binding affinity in the productive binding mode within the catalytic cavity of P450 enzymes, primarily CYP1A2, (ii) resonance stabilization of the anionic form generated by proton abstraction from the amino group, and (iii) exothermicity of the heterolytic cleavage of N-O bonds of hydroxylamines and their bioconjugates (47).


The relative stability of the nitrenium intermediates has been highlighted to be essential in determining the bioactivity of AAs (10). In line with this, earlier theoretical works have proposed that mutagenicity increases with the rate of nitrenium ion formation, that is, with a greater stability of the nitrenium intermediate, as computed with the semiempirical method AM1 (23-25). Multiple variable models, including variables related to nitrenium ion stability calculated at higher level ab initio methods, have later suggested that these variables were of only limited use in regression models (21,22,27-29). The significance of nitrenium ion stability in ruling the mutagenic potency of amines was thus questioned (29).

Considering the observed discrepancies between semiempirical and Hartree-Fock ab initio results, this issue was then examined by using density functional theory (DFT) methods (41,42). Thus, the importance of nitrenium ions on the mutagenicity of this type of compounds was evaluated at a higher level of theory including electron correlation. Formation of these electrophilic intermediates from their precursors was analyzed, and correspondences were explored between experimental mutagenic potencies reported in the literature and calculated reaction energies and electronic properties for a series of AAs and HAs of diverse structure. In this way, clear correlations were observed for compounds of related structure, indicating that mutagenic activity increased with nitrenium ion stability (41-42). Subsequently, DFT calculations were applied in several studies relating the stability of nitrenium ions with mutagenicity (47-55).

4.1. Formation of nitrenium ions

Aniline was selected as the model amine to analyze the reactivity of different precursors towards nitrenium ion generation at the B3LYP/6-31+G* level (41). Calculations on the N-O bond dissociation process were performed for the N-hydroxyl derivative (1a), as well as for its acetic (1b) and sulfuric esters (1c) (the deprotonated (negatively charged) analogue 1d was also considered) (figure 2) (41). The nitrenium ion derived from aniline presented a geometry more consistent with an imino carbenium ion, the dominant canonical form being the one in which the positive charge is localized at the para-carbon (figure 2). This fact, evidenced by natural population analysis (NPA) derived charges, the C-N bond distance, and the C-C bond length alternation in the ring, was in agreement with previous reports (56,57). Spectroscopic 13C NMR studies on protonated aromatic imines had shown charge delocalization into the phenyl ring, establishing the ambident carbocationic nature of the iminium ion via the aminocarbenium ion form (58).

The most feasible N-O bond cleavage reaction corresponded to the sulfuric acid ester 1c, whereas the process from the deprotonated derivative 1d was much more endothermic. Acetic acid ester 1b was the second one in reactivity, and the heterolytic dissociation of 1a resulted the most endothermic reaction (41). These observations were in agreement with the lower reactivity of hydroxyarylamines as compared with their esters (10). In aqueous solution, the charged products were remarkably stabilized, as it would be expected, diminishing the endothermicity of the reaction. Nevertheless, the relative reactivity order for the bond breaking process was identical to the gas-phase trend (41).

Several reactions involved in the mutagenicity of aromatic and HAs were evaluated at the B3LYP/6-31G* level for a set of 312 amines (48). The reactions considered were those corresponding to the generation of a reactive species (hydroxylamines and their conjugates, nitrenium ions, nitrosoarenes), as well as those involving the reaction of these species with DNA. Results showed that formation of nitrenium ions (from hydroxylamines activated either by protonation or conjugation) presented the higher levels of discrimination between active and inactive compounds (48). In this way, chemical reactivity, computed by quantum mechanical methods as energy changes to form reactive intermediates, closely correlated with activity in the Ames test. Solvation corrections by IEFPCM single point energy calculations did not enhance the agreement between computations and experiments (48).

For a large collection of 846 arylamines, the reaction energies of metabolically relevant pathways were calculated at the B3LYP/6-31G* level (50). The nitrenium-forming reaction was determined as the most relevant energetic step for discriminating among mutagenic and nonmutagenic compounds. As shown in previous studies (41,48), solvation energies had an observable effect but did not improve the splitting between both groups. A threshold of 283 kcal/mol for the nitrenium formation energy was suggested for differentiating active (Ames+, lower reaction energies) from inactive (Ames-, higher reaction energies) amines (50). Nitrenium formation energy computations were thus performed for over 14,000 arylamines, with the aim of building a database of potential mutagenicity hazards of use in the pharmaceutical industry (50).

In a very recent study, B3LYP/6-31G* calculations were applied to the rational design of nonmutagenic 4-aminobiphenyls for a drug discovery project (55). For this compound set, 4-aminobiphenyls with a computed energy change for nitrenium ion formation above 145 kcal/mol were inactive in the Ames test (55). These resulted to be the first nonmutagenic derivatives, as no aminobiphenyls that were not positive in an Ames test were previously known.

4.2. Stability of nitrenium ions

Relative nitrenium ion stabilities were calculated for a set of 17 N-acetoxy esters of diverse aromatic and HAs by applying DFT methods (41). The parent amines are displayed in figure 3. Related structures were selected in order to consider the influence of the number of rings, the effect of the nitrogen atom (HAs vs. carbocyclic amines), and the presence of a methyl substituent. Relative stabilities were assessed by the computed changes in energy for Reaction 1.

Because of extensive resonance delocalization of the cationic charge through the aromatic system, the resulting structures corresponded to imino carbenium ions (in accordance to references 56 and 57). For every cation, the exocyclic nitrogen presented a negative NPA charge density. With nonsymmetrical aryl substituents, alternative orientations of the NH bond generates two distinct configurational isomers, designated syn if the hydrogen of the NH group is oriented toward the β-ring carbon of higher priority (in the Kahn-Ingold-Prelog sense), and anti otherwise (59), both isomers being separated by substantial activation barriers (57,60). The most stable isomer for each cation was considered to determine the relative stability order (41). It should be noticed that the traditional nitrenium ion designation will be conserved along this review, in spite of the actual imino carbenium nature of all the species.

No correlation was found between the experimental mutagenic potencies and the calculated changes in energy for nitrenium ion formation (Reaction 1) for the complete set of 17 N-acetoxy esters derived from the aromatic and HAs considered (41). In contrast, clear correlations were noticed for groups of compounds of related structure, denoted as aromatic, imidazocarbocyclic, and imidazoheterocyclic. According to this, mutagenic activity was observed to increase with nitrenium ion stability (lower ΔEr in Reaction 1), and with the development of a more negative charge density at the exocyclic nitrogen of the nitrenium ion (qN), although each group of structures followed a different functional relationship (41). It was found that ΔEr and qN were strongly correlated, and whilst both heteroaromatic sets fitted almost the same function, the aromatic compounds followed another line. Hence, qN was pointed out as an important factor in determining nitrenium ion stability, which was also favored by the increment in the number of fused rings (resonance effect), and by methyl substitution (hyperconjugation and inductive effects) (41). In aqueous phase, the correlations for each group of amines matched the gas-phase results. Solvation significantly stabilized the charged products, decreasing the endothermicity in Reaction 1.

Subsequently, the previous series of 17 amines in reference 41 was extended to 43 compounds, and the relative stabilities of their corresponding nitrenium ions were evaluated by B3LYP/6-31G* calculations (42). In order to properly represent this group of chemicals, a diverse set of AAs and HAs was selected, covering a wide range of experimental values for mutagenic potencies reported in the literature (5.790 - -3.390) (29). These amines are displayed in figure 4. Relative nitrenium ion stabilities were gauged by comparing the computed change in energy (ΔEr) for the formation of the ion from the corresponding parent amine in gas phase (Reaction 2).

Correlations were analyzed between mutagenic potencies expressed as LogMP (logarithm of the number of histidine revertants per nanomole of chemical in the Ames assay for Salmonella typhimurium strain TA98 + S9 microsomal preparation) and calculated properties. The theoretical parameters evaluated were ΔEr (nitrenium ion stability, as defined by Reaction 2), and the NPA charge at the exocyclic nitrogen atom of the nitrenium ion (qN). As previously noticed (41), better correlations were found for compounds of related structure. Distinct correlation functions were observed for the amines grouped according to their classification as aromatic (Ar), heteroaromatic (HAr), imidazocarbocyclic (ImiAr, amines presenting an imidazole ring fused with a carbocyclic aromatic moiety), imidazoheterocyclic (ImiH, imidazole fused to a heterocyclic system), dipyridoimidazoles (PI), and quinoxalines (Qx) (42).

The following correlation coefficients were obtained: for the Ar group, r2 = 0.914 (13 compounds); for HAr, r2 = 0.943 (8 compounds); for ImiAr, r2 = 0.991 (4 compounds); for Qx, r2 = 0.956 (4 compounds); for PI, r2 = 0.994 (4 compounds); and for the ImiH group, r2 = 0.854, (9 compounds) (42). Mutagenic activity increased with the stability of the nitrenium ions. Correlations for each set are shown in figure 5. It should be remarked that combination of the different heteroaromatic groups into larger sets afforded very good correlations, some of them even better than those for the separate groups. Thus, for HAr + Qx (12 compounds), r2 = 0.953; for HAr + Qx + PI (16 compounds), r2 = 0.933; for HAr + Qx + PI + ImiH (25 compounds), r2 = 0.914. Accordingly, almost all the HAs fitted one curve, indicating that mutagenic activity is significantly influenced by nitrenium ion stability (42).

Mutagenicity was also found to increase with the development of negative charge at the exocyclic nitrogen of the nitrenium ion (qN) (42). As qN and ΔEr were strongly correlated between each other, the negative charge density at the exocyclic nitrogen was signaled as an important factor in determining nitrenium ion stability, and considered as an estimation of the extent of delocalization of the net positive charge within the π system of the cation. Nitrenium ions derived from AAs were more stable than those from the HAs of related structure. Among related heteroaromatic compounds, nitrenium ion stability decreased with the increment in the number of nitrogen atoms. In this way, ImiAr derived ions were more stable than the related ImiH and Qx intermediates. In all cases, qN was more negative for the most stable structures (42).

Nitrenium stability was also favored by the increase in the number of aromatic rings (resonance effect), and by methyl substitution (hyperconjugation and inductive effects) (42). However, for some pairs of amines differing in the number or position of the methyl groups, the stability of the cations did not match the mutagenicity order. According to this, bioactivity should probably be also affected by some other factors, as steric interactions within the active site of the enzymes involved in the activation pathway, or intercalation into the DNA.

More recently, quantum mechanical calculations at different levels of theory (semiempirical, ab initio, DFT) were applied to compute the stability of nitrenium ions derived from a set of 257 primary aromatic and HAs (49). These calculations could correctly differentiate between Ames active and inactive compounds, as well as rationalize and predict SAR trends within structurally related chemical series. Therefore, the authors proposed the use of nitrenium ion stability calculations as a valuable tool for medicinal chemists to select nonmutagenic aromatic and heteroaromatic primary amines in preclinical drug discovery programs (49).


Aromatic amides follow the same metabolic pathway as aromatic amines (10-12), even though esterification of the N-hydroxyarylamides intermediates (hydroxamic acids) is required for subsequent reactivity with DNA (10). Although amides generally appear to be no less carcinogenic than amines (1), the acetamido derivatives were always less mutagenic when compared to their parent amines (61,62). In agreement with this, heterolysis of the N-O bond to give a nitrenium ion was calculated by DFT to be less favorable in the arylamides because of the loss of amide resonance in their precursors (41); but not owing to inductive destabilization of the arylnitrenium ion by the N-acetyl group, as proposed earlier according to semiempirical computations (63,64).

Reactions of type 1 were calculated for the N-acetoxy esters of acetanilide and 2-acetylaminofluorene at the B3LYP/6-31+G* level in order to compare their results with those from the esters of the respective amines (41). The N-O bond-breaking reaction was more favorable (less endothermic) for the amines (41). The approximately orthogonal conformation adopted by the carbonyl group in the N-acetyl nitrenium ions precluded resonance with the aromatic system. According to the NPA charges, nitrenium ions derived from the amides presented less negative charge density at the nitrogen atom than those derived from the amines. However, the change in charge density for the exocyclic nitrogen (ΔqN) was more favorable (more negative) for the nitrenium ion derived from acetanilide than that from aniline. In contrast, electron donation brought about by the acetyl group resulted in an important decrease of the negative charge at the oxygen atom of the carbonyl. Thus, the acetyl substituent, despite of being generally regarded as a powerful electron withdrawing group, in the nitrenium ion derived from acetanilide was found to act as a σ-electron donor (41,63,64). On this basis, the formation of N-acetyl nitrenium ions seems to be hindered by an unfavorable polarization of the carbonyl group.


Hydrophobicity plays an important role in the absorption and transport of chemicals to their sites of activation and chemical reaction, as well as in the interaction of the compounds with the bioreceptors. The potential importance of hydrophobic interactions on the genotoxic activity of molecules led to the use of hydrophatic factors in a variety of mutagenicity QSARs. Particularly, earlier QSARs for the mutagenicity of amines had established that activity was primarily determined by hydrophobicity (30). However, different QSAR studies for AAs and HAs where hydrophatic factors were considered generated contradictory results. Whereas earlier reports indicated that bioactivity was predominantly determined by the hydrophobicity of the amines (30,32), further studies observed that hydropatic factors made only a small contribution to mutagenic potency (22,29).

Afterwards, the influence of the logarithm of the n-octanol/water partition coefficient (LogP) on the mutagenic activity of aromatic and HAs was analyzed (figure 6) (42). Positive correlations were observed for each series of compounds in reference 42, although they were not as good as those achieved between calculated nitrenium ion stabilities (ΔEr for Reaction 2) and LogMP. In general, within each group of amines, a higher bioactivity was associated with relatively large LogP values (figure 6A). Similarly, the more stable nitrenium ions were those derived from the amines with higher LogP values (figure 6B) (42). On the other hand, when the whole set of compounds was considered, the more active HAs, presenting lower LogP values, formed relatively less stable nitrenium ions than the correspondingly related AAs, which are more hydrophobic and less active (42).

Hydrophobicity can differently affect the variety of processes involved in chemical genotoxicity, and this could lead to confusing conclusions if sets of amines of very diverse structure are considered. The observed discrepancies between QSAR studies using hydrophatic factors could arise from this fact (22,29,30,32). Therefore, the influence of hydrophobicity on mutagenic potency seems to be better interpreted when analyzing congeneric amines. Considering related compounds, mutagenicity increases with LogP, in correspondence with greater nitrenium ion stability (42). In contrast, when comparing amines that differ in the replacement in an aromatic ring of a carbon atom by nitrogen, an inverse correlation of the mutagenic activity with LogP was observed (42).


Aromatic and HAs are compounds of particular importance in the pharmaceutical industry and occur frequently as substructures in drug candidates (65). Because of their synthetic feasibility, valuable physicochemical properties, and potential interaction with binding sites of target proteins, they are convenient pharmaceutical building blocks. However, their mutagenic and carcinogenic potential restrict their use in drug discovery programs. Bioactivity of this class of compounds is brought about by complex processes involving a number of metabolic and chemical steps. Nevertheless, the studies presented in this review indicate that current computational methods can provide proper estimations of relative mutagenic potencies, denoting its potential value as predictive tools. A calculated probability of activity in the Ames test can help the rational design of safer drugs. The use of DFT calculations appears particularly encouraging on the basis of their accuracy at moderate computational costs.

Nitrenium ion stability is considered as a key factor in determining mutagenic potency. Since better correlations were obtained for groups of related compounds (41,42), it is fairly probable that the influence on activity of additional aspects, such as solubility, transport, specific interactions with the biological environment, etc., needs to be also considered. In this regard, activation pathways of a series of para-substituted anilines and 2-aminopyridines in the CYP1A2 enzyme have been lately investigated via SAR analysis and DFT computations (47). Furthermore, a recent study has shown that the interactions of nitroaromatic compounds and their reductive enzymes play an important role in the formation of nitrenium ions, and correspond to differences in the occurrence of DNA adducts (66).

Additional comparisons of calculated activity trends with data obtained by mutagenicity and tumorigenicity assays, and by DNA binding studies, would be desirable in order to achieve a more comprehensive understanding of SARs between arylamines and their toxicological potential.


The author acknowledges financial support from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (Secyt-UNC).


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Abbreviations: AA: aromatic amine; HA: heteroaromatic amine; ROS: reactive oxygen species; SAR: structure-activity relationship; QSAR: quantitative structure-activity relationship; HOMO: highest occupied molecular orbital; LUMO: lowest unoccupied molecular orbital; DFT: density functional theory; NPA: natural population analysis; Ar: aromatic; HAr: heteroaromatic; ImiAr: imidazocarbocyclic; ImiH: imidazoheterocyclic; PI: dipyridoimidazoles; Qx: quinoxalines

Key Words: Aromatic and heteroaromatic amines, Mutagenic activity, Quantum-mechanical calculations, Nitrenium ions, Review

Send correspondence to: Gabriela L. Borosky, Departamento de Matematica y Fisica, INFIQC, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Ciudad Universitaria, Cordoba 5000, Argentina, Tel: 54-351-4344972, Fax: 54-351-4344970, E-mail: gborosky@fcq.unc.edu.ar