Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents: Part I. Alkylation ortho to the amino function

Article abstract of DOI:10.1016/S1383-5718(01)00259-5

Alkyl-substituted derivatives of 2-aminonaphthalene (2-AN) 1, 2-aminofluorene (2-AF) 6 and 4-aminobiphenyl (4-ABP) 11 were synthesized and the mutagenic activity of these compounds determined in Salmonella typhimurium strains TA98 and TA100 with and without S9 mix. In the case of the ortho-substituted 4-aminobiphenyls 12-15 (3-alkyl = ethyl, iso-propyl, n-butyl, tert-butyl) the substituent with the strongest steric demand (3-tert-butyl) shows the strongest influence on the decrease of mutagenicity if compared with the parent compound. In the series of the bis-ortho-disubstituted compounds 16-18 (3,5-dimethyl-, 3,5-diethyl- and 3,5-diisopropyl-4-aminobiphenyl) generation of non-mutagenic species occurs already with the introduction of two ethyl groups. For the 4-aminobiphenyl derivatives 12-15 and 16-18, as well as for the 1-alkylated 2-aminofluorenes 7-10 and the 1-alkylated 2-aminonaphthalenes 2-5 a smaller mutagenicity was observed if compared with predicted mutagenicities as calculated by the QSAR equations of Debnath et al. (Environ. Mol. Mutagen. 19 (1992) 37). The largest differences resulted in the cases of the tert-butyl substituted compounds. Only with smaller alkyl groups like ethyl the QSAR predictions and the experimentally determined mutagenicities come close to each other. Thus, these results show that appropriate alkyl substitution reduces (eliminates) mutagenicity, secondly, it is necessary to introduce steric parameters to predict the mutagenicity of such compounds correctly.

Full text of DOI:10.1016/S1383-5718(01)00259-5

Mutation Research 498 (2001) 19–37
Transformation of mutagenic aromatic amines into
non-mutagenic species by alkyl substituents
Part I. Alkylation ortho to the amino function
Carsten Glendea, Heimo Schmitta, Lothar Erdingerb,
Günter Engelhardtc, Gernot Bochea,∗
Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein Strasse, 35032 Marburg, Germany
a
b
Hygieneinstitut Heidelberg, Im Neuenheimer Feld, 69120 Heidelberg, Germany
BASF AG, Experimental Toxicology and Ecology, 67056 Ludwigshafen/Rhein, Germany
c
Received 20 March 2001; received in revised form 12 June 2001; accepted 26 June 2001
Abstract
Alkyl-substituted derivatives of 2-aminonaphthalene (2-AN) 1, 2-aminofluorene (2-AF) 6 and 4-aminobiphenyl (4-ABP)
11 were synthesized and the mutagenic activity of these compounds determined in Salmonella typhimurium strains TA98 and
TA100 with and without S9 mix. In the case of the ortho-substituted 4-aminobiphenyls 12–15 (3-alkyl = ethyl, iso-propyl,
n-butyl, tert-butyl) the substituent with the strongest steric demand (3-tert-butyl) shows the strongest influence on the de-
crease of mutagenicity if compared with the parent compound. In the series of the bis-ortho-disubstituted compounds 16–18
(3,5-dimethyl-, 3,5-diethyl- and 3,5-diisopropyl-4-aminobiphenyl) generation of non-mutagenic species occurs already with
the introduction of two ethyl groups. For the 4-aminobiphenyl derivatives 12–15 and 16–18, as well as for the 1-alkylated
2-aminofluorenes 7–10 and the 1-alkylated 2-aminonaphthalenes 2–5 a smaller mutagenicity was observed if compared with
predicted mutagenicities as calculated by the QSAR equations of Debnath et al. (Environ. Mol. Mutagen. 19 (1992) 37). The
largest differences resulted in the cases of the tert-butyl substituted compounds. Only with smaller alkyl groups like ethyl
the QSAR predictions and the experimentally determined mutagenicities come close to each other. Thus, these results show
that appropriate alkyl substitution reduces (eliminates) mutagenicity, secondly, it is necessary to introduce steric parameters
to predict the mutagenicity of such compounds correctly. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Aromatic amines; 2-Aminonaphthalene; 2-Aminofluorene; 4-Aminobiphenyl; Salmonella; Mutagenicity; Alkyl substituents; QSAR
1. Introduction
cides, synthetic materials or pharmaceutical products
[1]. To exert their mutagenic and carcinogenic effects
aromatic amines require metabolic activation. They
are first oxidized by cytochrome P450 to hydroxy-
lamines, and then further activated, e.g. by O-acylation
[2,3]. From these compounds a highly electrophilic
species, the corresponding nitrenium ion, may be gen-
erated and react with bionucleophiles. For mutagenic-
ity and carcinogenicity the main target molecule is the
deoxyribonucleic acid (DNA) [4]. To investigate the
Aromatic amines are a class of ubiquitous envi-
ronmental pollutants. They are found, e.g. in tobacco
smoke, diesel exhaust and tar, and they are used for
preparation of industrial products like azo dyes, pesti-
∗ Corresponding author. Tel.: +49-6421-282-2030;
fax: +49-6421-282-8917.
E-mail address: boche@chemie.uni-marburg.de (G. Boche).
1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(01)00259-5

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C. Glende et al. / Mutation Research 498 (2001) 19–37
mutagenicity of such compounds the most widely used
bacterial short-time mutagenicity assay is the Ames
test with Salmonella typhimurium TA98 and TA100,
with and without S9 mix. The metabolic activation is
simulated by rat liver S9 mix [5].
activity relationship (QSAR) studies that try to predict
the mutagenicity of chemical compounds. In the case
of aromatic amines, Debnath et al. developed QSAR
equations for the tester strains TA98 and TA100 with
S9 mix activation [7]. The most important factor in
these equations is the log P-value (octanol–water
distribution factor), followed by the electronic param-
eters EHOMO and ELUMO. We calculated the values
of our test compounds and compared them with the
experimental data. The aim was to investigate the
effect of alkylation, and whether these equations are
able to predict the mutagenicity of aromatic amines
with sterically demanding alkyl substituents.
In many mutagenicity studies of aromatic amines
the parent compounds 2-AN 1, 2-AF 6 and 4-ABP
11 were tested in the above mentioned Salmonella
strains [6]. In our investigations, the main fo-
cus was on the influence of alkyl groups attached
ortho to the amino function in 1, 6 and 11. There-
fore, we synthesized homologous series of aromatic
alkyl amines with growing steric demand of the alkyl
groups. Here, we concentrate on the ortho-alkylated
1-alkyl-2-aminonaphthalenes 2–5, 1-alkyl-2-amino-
fluorenes 7–10, 3-alkyl-4-aminobiphenyls 12–15 and
3,5-dialkyl-4-aminobiphenyls 16–18 (Table 1). All
samples were tested in the Ames test with S. ty-
phimurium tester strains TA98 and TA100, both with
and without metabolic activation using rat liver S9
mix.
2. Experimental section
2.1. General comments
All solvents were reagent grade and used without
further purification with the following exceptions.
Tetrahydrofuran (THF) and diethyl ether (Et2O) were
dried by distillation from Na/K alloy under argon.
Chemicals used were reagent grade and used without
To understand the parameters which influence
mutagenicity many investigations have been under-
taken [7–10]. One of them are quantitative structure
Table 1
Structures of the tested compounds 1–18
Structure
R
No.
Compound
Abbreviation
H
Et
1
2
2-Aminonaphthalene
1-Ethyl-2-aminonaphthalene
2-AN
1-Et-2AN
iPr
nBu
tBu
3
4
5
1-iPropyl-2-aminonaphthalene
1-nButyl-2-aminonaphthalene
1-tButyl-2-aminonaphthalene
1-iPr-2AN
1-nBu-2AN
1-tBu-2AN
H
Et
6
7
2-Aminofluorene
1-Ethyl-2-aminofluorene
2-AF
1-Et-2AF
iPr
8
1-iPropyl-2-aminofluorene
1-iPr-2AF
nBu
tBu
9
10
1-nButyl-2-aminofluorene
1-tButyl-2-aminofluorene
1-nBu-2AF
1-tBu-2AF
H
Et
iPr
11
12
13
4-Aminobiphenyl
3-Ethyl-4-aminobiphenyl
3-iPropyl-4-aminobiphenyl
4-ABP
3-Et-4ABP
3-iPr-4ABP
nBu
tBu
14
15
3-nButyl-4-aminobiphenyl
3-tButyl-4-aminobiphenyl
3-nBu-4ABP
3-tBu-4ABP
Me
Et
16
17
18
3,5-Dimethyl-4-aminobiphenyl
3,5-Diethyl-4-aminobiphenyl
3,5-Diipropyl-4-aminobiphenyl
3,5-diMe-4ABP
3,5-diEt-4ABP
3,5-diiPr-4ABP
iPr

C. Glende et al. / Mutation Research 498 (2001) 19–37
21
further purification unless noted. Flash chromatog-
raphy was carried out using Merck silica gel 60
(230–400 mesh) purchased from Merck & Co. Analyt-
ical thin layer chromatography (TLC) was performed
using Merck precoated silica gel 60 F-254 sheets. All
reactions were carried out under argon atmosphere
unless otherwise specified.
Scheme 1.
2.2. Instrumentation
1H NMR and 13C NMR spectra were recorded
on Bruker ARX-200 and AC-300 spectrometers in
CDCl3 solution using tetramethylsilane as internal
standard. Mass spectra were recorded with a Variant
MAT CH-7-A (EI, 70 eV). Elementary analyses were
performed with a Heraeus Rapid Elementalanalyser.
Scheme 2.
lation agent for the aromatic nucleus and a reducing
agent for the nitro group). Yields vary in the range of
10–25%.
3,5-Dialkyl-4-aminobiphenyls 16–18 were synthe-
sized by a modified procedure of Miller and Dugar
[14]. 4-Bromo-2,6-dialkylanilines 19–20 were first
synthesized by selective bromination methods as de-
scribed in the literature [15,16]. Yields vary between
80 and 85%. In a second step the ortho-disubstituted
4-aminobiphenyls were synthesized by coupling of
4-bromo-2,6-dialkylanilines with phenylboronic acid.
Yields were in the range of 35–50%, see Schemes 3
and 4.
2.3. Chemicals
4-Aminobiphenyl 11 [92-67-1], 4-nitrobiphenyl
[92-93-3], 2-aminofluorene 6 [153-78-6], 2-nitro-
fluorene [607-57-8], 2-nitronaphthalene [581-89-5],
2,6-dimethylaniline [87-62-7], 2,6-diethylaniline
[579-66-8], 2,6-diisopropylaniline [24544-04-5] and
phenylboronic acid [98-80-6] were purchased from
Aldrich Chemical Co.
2.4. Synthesis of the test compounds
2.4.1. 2-Aminonaphthalene 1
To a solution of 4.33 g (2.5 mmol) 2-nitronaphthalene
in 100 ml ethanol were added 0.72 g Pd/C (10 wt.%)
and 18 ml hydrazine hydrate (80 wt.%). After re-
fluxing for 10 min the solution was cooled to room
temperature, filtered and the solvent removed. The
solid 1 was purified by dissolving in ligroin. Removal
of the solvent in vacuo yielded 2.7 g (76%) of the
crystalline solid.
2-Aminonaphthalene 1 was prepared according to
a procedure described by Dewar and Mole [11], see
Scheme 1.
Grignard reagents were synthesized by standard
methods [12]. 1-Alkyl-2-aminonaphthalenes 2–5,
1-alkyl-2-aminofluorenes 7–10 and 3-alkyl-4-amino-
biphenyls 12–15 were synthesized according to a
method introduced by Bartoli et al. [13], see Scheme 2
(the Grignard reagent acts simultaneously as an alky-
Spectroscopic and physical data were in agreement
with literature data [17,18].
Scheme 3.

22
C. Glende et al. / Mutation Research 498 (2001) 19–37
Scheme 4.
3
2.4.2. 1-Ethyl-2-aminonaphthalene 2;
typical procedure
1H, CH), 1.54 (d, 6H, J = 7.0 Hz, CH3). 13C NMR
(CDCl3, 75 MHz): δ = 141.2, 133.0, 129.4, 129.0,
127.7, 125.9, 122.9, 122.5, 121.9, 120.11, 26.5 (CH),
20.7 (CH3). MS (70 eV): m/z (%) = 185 (M+, 63),
170 (M+ − CH3, 100). Calc. C 84.28%, H 8.16%, N
7.56%; found C 83.94%, H 8.24%, N 7.80%.
A Grignard solution (ethylmagnesiumchloride)
was cooled to −15◦C and 0.45 g (2.36 mmol)
copper(I)-iodide was added. Then, a solution of
20 mmol of the appropriate nitro compound in 90 ml
THF was added slowly. The reaction mixture was
stirred at room temperature for 18 h. After careful
addition of 10 ml of a 2 N HCl solution at 0◦C, the so-
lution was stirred for 20 min and brought to pH 10 by
treatment with concentrated aqueous ammonia. The
solid residue was separated by filtration and washed
with dichloromethane. The combined organic layers
were extracted with water (4×75 ml), dried (MgSO4),
and evaporated to dryness in vacuo. The solid was
purified by flash chromatography with CHCl3 as the
eluting solvent. 642 mg (19%) dark red crystalline
solid.
2.4.4. 1-nButyl-2-aminonaphthalene 4
1051 mg (27%) reddish-brown viscous liquid.
1H NMR (CDCl3, 300 MHz): δ = 7.84 (d, 1H,
3J = 8.6 Hz, H5), 7.69 (d, 1H, 3J = 8.1 Hz, H4), 7.54
3
3
(d, 1H, J = 8.7 Hz, H8), 7.41 (t, 1H, J = 7.7 Hz,
H7), 7.23 (t, 1H, 3J = 7.5 Hz, H6), 6.93 (d, 1H, 3J =
3
8.7 Hz, H3), 3.82 (s, 2H, NH2), 2.88 (t, 2H, J =
7.9 Hz, CH2), 1.66–1.44 (m, 4H, CH2), 0.98 (t, 3H,
3J = 7.1 Hz, CH3). 13C NMR (CDCl3, 75 MHz): δ =
141.0, 133.3, 128.8, 128.7, 127.3, 126.3, 122.4, 122.0,
118.7, 118.1, 31.2 (CH2), 26.1 (CH2), 23.3 (CH2),
14.2 (CH3). MS (70 eV): m/z (%) = 199 (M+, 26),
157 (21%), 156 (M+ − C3H7, 100). Calc. C 84.37%,
H 8.60%, N 7.03%; found C 83.95%, H 8.32%, N
7.46%.
1H NMR (CDCl3, 300 MHz): δ = 7.95 (d, 1H,
3J = 8.6 Hz, H5), 7.80 (d, 1H, J = 8.1 Hz, H4),
3
3
3
7.63 (d, 1H, J = 8.6 Hz, H8), 7.53 (t, 1H, J =
3
6.7 Hz, H7), 7.34 (t, 1H, J = 6.4 Hz, H6), 6.98 (d,
1H, 3J = 8.6 Hz, H3), 3.88 (s, 2H, NH2), 2.98 (q, 2H,
3J = 7.6 Hz, CH2), 1.32 (t, 3H, J = 7.6 Hz, CH3).
2.4.5. 1-tButyl-2-aminonaphthalene 5
436 mg (12%) dark-red oily liquid.
3
13C NMR (CDCl3, 75 MHz): δ = 140.6, 132.9, 128.8,
127.3, 126.3, 122.2, 122.0, 119.2, 118.8, 19.3 (CH2),
13.0 (CH3). MS (70 eV): m/z (%) = 171 (M+, 57),
156 (M+ − CH3, 100). Calc. C 84.17%, H 7.65%, N
8.18%; found C 84.33%, H 7.66%, N 7.95%.
Analogously compounds 3–5, 7–10 and 12–15 were
prepared.
1H NMR (CDCl3, 300 MHz): δ = 8.35 (d, 1H,
3J = 8.9 Hz, H5), 7.69 (d, 1H, J = 8.0 Hz, H4),
3
3
3
7.48 (d, 1H, J = 8.7 Hz, H8), 7.39 (t, 1H, J =
3
8.0 Hz, H7), 7.25 (t, 1H, J = 7.9 Hz, H6), 6.74 (d,
1H, 3J = 8.7 Hz, H3), 4.11 (s, 2H, NH2), 1.81 (s, 9H,
CH3). 13C NMR (CDCl3, 75 MHz): δ = 141.9, 134.0,
130.4, 129.1, 128.4, 126.7, 124.5, 122.9, 122.3, 121.4,
37.3, 33.6 (CH3). MS (70 eV): m/z (%) = 199 (M+,
50), 184 (M+ − CH3, 100), 144 (24), 143 (33). Calc.
C 84.15%, H 8.60%, N 7.03%; found C 84.15%, H
8.67%, N 7.10%.
2.4.3. 1-iPropyl-2-aminonaphthalene 3
468 mg (17%) dark-red oily liquid.
1H NMR (CDCl3, 300 MHz): δ = 8.08 (d, 1H,
3
3J = 8.6 Hz, H5), 7.73 (d, 1H, J = 8.0 Hz, H4),
3
3
7.55 (d, 1H, J = 8.6 Hz, H8), 7.41 (t, 1H, J =
3
7.7 Hz, H7), 7.26 (t, 1H, J = 7.4 Hz, H6), 6.90 (d,
2.4.6. 1-Ethyl-2-aminofluorene 7
495 mg (12%) dark-red oily liquid.
3
1H, J = 8.6 Hz, H3), 3.97 (s, 2H, NH2), 3.85 (s,

C. Glende et al. / Mutation Research 498 (2001) 19–37
23
1H NMR (CDCl3, 300 MHz): δ = 7.64 (d, 1H,
7.46 (d, 1H, J = 7.4 Hz, H8), 7.31 (t, 1H, J =
3
3
3
3
3J = 7.6 Hz, H5), 7.49 (d, 1H, J = 7.1 Hz, H4),
7.4 Hz, H6), 7.20 (t, 1H, J = 7.4 Hz, H7), 6.72 (d,
3
3
3
7.46 (d, 1H, J = 8.0 Hz, H8), 7.31 (t, 1H, J =
1H, J = 8.0 Hz, H3), 4.16 (s, 2H, H9), 3.95 (s, 2H,
3
7.3 Hz, H6), 7.20 (t, 1H, J = 7.4 Hz, H7), 6.74 (d,
NH2), 1.69 (s, 9H, CH3). 13C NMR (CDCl3, 75 MHz):
δ = 144.8, 142.5, 141.7, 141.6, 134.7, 131.0, 126.6,
125.2, 124.1, 118.8, 118.6, 118.2, 40.8, 37.13, 31.68
(CH3). MS (70 eV): m/z (%) = 237 (M+, 100), 222
(M+ −CH3, 70). Calc. C 86.03%, H 8.07%, N 5.90%;
found C 86.04%, H 8.27%, N 5.35%.
3
1H, J = 8.0 Hz, H3), 3.80 (s, 2H, H9), 3.71 (s, 2H,
3
NH2), 2.69 (q, 2H, J = 7.6 Hz, CH2), 1.24 (t, 3H,
CH3). 13C NMR (CDCl3, 75 MHz): δ = 143.0, 142.7,
142.1, 132.9, 126.4, 124.8, 124.5, 118.5, 118.2, 114.8,
35.4, 21.4, 12.3 (CH3). MS (70 eV): m/z (%) = 209
(M+, 100), 194 (M+ − CH3, 35), 180 (M+ − C2H5,
49). Calc. C 86.08%, H 7.22%, N 6.69%; found C
85.61%, H 7.51%, N 6.67%.
2.4.10. 3-Ethyl-4-aminobiphenyl 12
844 mg (22%) dark red-brownish viscous liquid.
1H NMR (CDCl3, 300 MHz): δ = 7.76–7.41 (m,
7H), 6.87 (d, 1H, 3J = 8.1 Hz, H5), 3.77 (s, 2H,
2.4.7. 1-iPropyl-2-aminofluorene 8
3
NH2), 2.71 (q, 2H, J = 7.5 Hz, CH2), 1.46 (t, 3H,
365 mg (9%) dark-red oily liquid.
3J = 7.5 Hz, CH3). 13C NMR (CDCl3, 75 MHz): δ =
143.8, 141.7, 131.9, 128.8, 128.4, 127.4, 126.6, 125.6,
115.9, 24.4, 13.3 (CH3). MS (70 eV): m/z (%) = 197
(M+, 78), 182 (M+ − CH3, 100). Calc. C 85.24%, H
7.66%, N 7.10%; found C 85.13%, H 7.79%, N 7.01%.
1H NMR (CDCl3, 300 MHz): δ = 7.63 (d, 1H,
3J = 7.5 Hz, H5), 7.48 (d, 1H, 3J = 7.3 Hz, H4), 7.45
3
3
(d, 1H, J = 8.0 Hz, H8), 7.31 (t, 1H, J = 7.2 Hz,
H6), 7.18 (t, 1H, 3J = 7.4 Hz, H7), 6.73 (d, 1H, 3J =
8.0 Hz, H3), 3.89 (s, 2H, H9), 3.78 (s, 2H, NH2), 3.30
(m, 1H, 3J = 7.2 Hz, CH), 1.43 (d, 6H, 3J = 7.2 Hz,
CH3). 13C NMR (CDCl3, 75 MHz): δ = 143.1, 142.4,
142.2, 133.8, 126.5, 125.0, 124.4, 118.5, 118.3, 116.4,
36.9, 29.1, 20.5 (CH3). MS (70 eV): m/z (%) = 223
(M+, 100), 208 (M+ − CH3, 80), 180 (M+ − C3H7,
40). Calc. C 86.05%, H 7.67%, N 6.27%; found C
85.73%, H 7.50%, N 6.19%.
2.4.11. 3-iPropyl-4-aminobiphenyl 13
382 mg (9%) dark reddish-brown viscous liquid.
1H NMR (CDCl3, 300 MHz): δ = 7.65–7.33 (m,
7H), 6.79 (d, 1H, 3J = 8.2 Hz, H5), 3.77 (s, 2H,
3
NH2), 2.99 (m, 1H, J = 6.2 Hz, CH), 1.38 (d, 6H,
3J = 6.8 Hz, CH3). 13C NMR (CDCl3, 75 MHz):
δ = 142.9, 141.9, 133.0, 132.2, 128.8, 126.7, 126.3,
125.4, 124.5, 116.4, 28.0, 22.5 (CH3). MS (70 eV):
m/z (%) = 211 (M+, 88), 196 (M+ −CH3, 100). Calc.
C 85.26%, H 8.11%, N 6.63%; found C 84.84%, H
8.34%, N 6.93%.
2.4.8. 1-nButyl-2-aminofluorene 9
653 mg (14%) orange-brown solid.
1H NMR (CDCl3, 300 MHz): δ = 7.58 (d, 1H,
3J = 7.6 Hz, H5), 7.43 (d, 1H, 3J = 6.3 Hz, H4), 7.40
3
3
(d, 1H, J = 7.9 Hz, H8), 7.25 (t, 1H, J = 7.3 Hz,
H6), 7.12 (t, 1H, 3J = 7.4 Hz, H7), 6.69 (d, 1H, 3J =
8.0 Hz, H3), 3.84 (s, 2H, H9), 3.74 (s, 2H, NH2), 2.60
2.4.12. 3-nButyl-4-aminobiphenyl 14
254 mg (6%) light brown oily liquid.
1H NMR (CDCl3, 300 MHz): δ = 7.68–7.15 (m,
3
(t, 2H, J = 7.8 Hz, CH2), 1.55 (m, 2H, CH2), 1.42
3
3
(m, 2H, CH2), 0.92 (t, 3H, J = 7.2 Hz, CH3). 13C
6H), 7.07 (s, 1H, H2), 6.63 (d, 1H, J = 8.1 Hz, H5),
3
3.53 (s, 2H, NH2), 2.44 (t, 2H, J = 7.9 Hz, CH2),
NMR (CDCl3, 75 MHz): δ = 144.8, 142.5, 141.7,
141.5, 134.7, 131.0, 126.6, 125.0, 124.7, 118.6, 118.4,
115.0, 35.9, 30.4 (CH2, A), 28.4 (CH2, B), 23.2 (CH2,
C), 14.1 (CH3). MS (70 eV): m/z (%) = 237 (M+, 88),
194 (M+ − C3H7, 100). Calc. C 86.03%, H 8.07%, N
5.90%; found C 85.93%, H 8.48%, N 5.42%.
3
1.58–1.31 (m, 4H, CH2), 0.87 (t, 3H, J = 7.2 Hz,
CH3). 13C NMR (CDCl3, 75 MHz): δ = 142.5, 140.4,
130.6, 127.6, 127.2, 126.1, 125.4, 125.1, 124.5, 114.8,
30.1, 29.9, 21.7, 13.0 (CH3). MS (70 eV): m/z (%) =
225 (M+, 62), 182 (M+−C3H7, 100). Calc. C 85.29%,
H 8.50%, N 6.22%; found C 84.65%, H 8.49%, N
6.10%.
2.4.9. 1-tButyl-2-aminofluorene 10
271 mg (8%) dark reddish-brown viscous liquid.
1H NMR (CDCl3, 300 MHz): δ = 7.63 (d, 1H,
2.4.13. 3-tButyl-4-aminobiphenyl 15
408 mg (10%) dark red-brownish solid.
3J = 7.6 Hz, H5), 7.50 (d, 1H, J = 8.1 Hz, H4),
3

24
C. Glende et al. / Mutation Research 498 (2001) 19–37
1H NMR (CDCl3, 300 MHz): δ = 7.62–7.32 (m,
1H NMR (CDCl3, 200 MHz): δ = 7.78 (d, 2H,
3J = 7.78 Hz, H6), 7.68–7.53 (m, 3H, H7/8), 7.50
(s, 2H, H3), 4.02 (s, 2H,NH2), 3.13 (p, 2H, 3J =
6.91 Hz, H9), 1.51 (d, 12H, 3J = 6.91 Hz, H10).
13C NMR (CDCl3, 75 MHz): δ = 144.78, 138.64,
134.98, 129.21, 127.65, 126.94, 126.56. MS (70 eV):
m/z (%) = 253 (M+, 100%), 237 (76%). Calc. C
85.32%, H 9.15%, N 5.53%; found C 84.98%, H
8.94%, N 5.89%
7H), 6.74 (d, 1H, 3J = 8.1 Hz, H5), 3.88 (s, 2H, NH2),
1.53 (s, 9H, CH3). 13C NMR (CDCl3, 75 MHz): δ =
144.1, 141.9, 133.9, 131.6, 128.7, 126.6, 126.2, 125.6,
118.2, 34.4, 29.7 (CH3). MS (70 eV): m/z (%) = 225
(M+, 100), 210 (M+ − CH3, 70). Calc. C 85.29%, H
8.50%, N 6.22%; found C 84.85%, H 8.37%, N 6.25%.
2.4.14. 3,5-Dimethyl-4-aminobiphenyl 16;
typical procedure
A solution of 10 ml toluene, 5 ml Na2CO3 (2 M)
and 0.17 g (150 ␮mol) tetra-kis(triphenylphosphine)-
palladium was treated with a solution of 1.00 g
(5.00 mmol) 4-bromo-2,6-dimethylaniline in ethanol
and a solution of 0.67 g (5.50 mmol) phenylboronic
acid in ethanol, and refluxed for 48 h. The reaction
mixture was separated by filtration and the solid 16
extracted with Et2O (3 × 20 ml). The organic layer
was extracted with brine (3 × 20 ml), dried with
MgSO4 and evaporated in vacuo. Repeated column
chromatography (petrol ether:ethyl acetate, 8:1) af-
forded 16.
2.4.17. 4-Bromo-2,6-dimethylaniline 19;
typical procedure
2,6-Dimethylaniline (2.42 g, 0.02 mmol) was dis-
solved in 50 ml dichloromethane and cooled to
−10◦C. The bromination agents 2,4,4,6-tetrabromo-
cyclohexane (8.20 g, 0.02 mmol), dissolved in 130 ml
dichloromethane, was added dropwise and the reac-
tion mixture was stirred for 2.5 h at 20◦C. After ex-
traction with 3×30 ml 2N NaOH and 3×30 ml H2O,
the solvent was evaporated in vacuo. Purification by
flash chromatography (petrol ether:ethyl acetate, 8:1)
afforded 19.
0.76 g (39%) violet solid.
3.41 g (85%) violet solid.
1H NMR (CDCl3, 200 MHz): δ = 7.62 (d, 2H,
3J = 7.80, H6), 7.46(m, 3H, H7/8), 7.29 (s, 2H, H3),
3.91 (s, 2H, NH2), 2.32 (s, 6H, H9). 13C NMR (CDCl3,
75 MHz): δ = 142.50, 141.94, 131.63, 129.04, 127.50,
126.97, 122.60, 18.27 (CH3). MS (70 eV): m/z (%) =
197 (M+, 100%); 182 (18.2%; M+ − 15). Calc. C
85.24%, H 7.66%, N 7.10%; found C 85.06%, H
7.64%, N 7.13%
1H NMR (CDCl3, 200 MHz): δ = 6.98 (s, 2H, H3),
3.47 (s, 2H, NH2), 2.07 (s, 6H, H5). 13C NMR (CDCl3,
75 MHz): δ = 143.39, 131.14, 125.04, 117.58, 18.65
(CH3). MS (70 eV): m/z (%) = 201 (M+, 88%), 199
(87%), 186 (5%), 184 (6%), 120 (100%). Calc. C
47.84%, H 5.02%, N 6.97%; found C 47.62%, H
5.31%, N 6.83%.
Analogously compounds 20 and 21 were prepared
Analogously compounds 17–18 were prepared.
2.4.18. 2,6-Diethyl-4-bromoaniline 20
3.77 g (85%) deep green oil.
2.4.15. 3,5-Diethyl-4-aminobiphenyl 17
1H NMR (CDCl3, 200 MHz): δ = 6.94 (s, 2H, H3),
630 mg (32%) reddish-brown oil.
1H NMR (CDCl3, 200 MHz): δ = 7.47 (d, 2H,
3
3.47 (s, 2H, NH2), 2.32 (q, 4H, J = 7.59 Hz, H5),
3J = 7.76 Hz, H6), 7.33–7.25 (m, 3H, H7/8), 7.15
3
1.14 (t, 6H, J = 7.59 Hz, H6). 13C NMR (CDCl3,
3
(s, 2H, H3), 3.71 (s, 2H, NH2), 2.45 (q, 4H, J =
75 MHz,) δ = 142.21, 131.09, 130.23, 111.67, 25.78
(CH2), 14.25 (CH3). MS (70 eV): m/z (%) = 229 (M+,
62%), 227 (61%), 214 (82%), 212 (72%), 149 (63%),
134 (100%). Calc. C 52.43%, H 6.16%, N 6.11%;
found C 52.14%, H 6.32%, N 5.95%.
3
7.41 Hz, H9), 1.21 (t, 6H, J = 7.41 Hz, H10). 13C
NMR (CDCl3, 75 MHz): δ = 141.20, 139.61, 138.41,
127.57, 127.10, 124.60, 124.48, 24.28 (CH2), 13.26
(CH3). MS (70 eV): m/z (%) = 225 (M+, 100%), 210
(M+ − 15, 54.9%), 195 (M+ − 30, 15.5%). Calc.
C 85.28%, H 8.50%, N 6.22%; found C 84.30%, H
8.56%, N 6.44%
2.4.19. 2,6-Diisopropyl-4-bromoaniline 21
4.17 g (81%) deep red oil.
1H NMR (CDCl3, 200 MHz): δ = 7.02 (s, 2H,
3
2.4.16. 3,5-Diisopropyl-4-aminobiphenyl 18
980 mg (52%) red-coloured oil.
H3), 3.59 (s, 2H, NH2), 2.84 (p, 2H, J = 6.92 Hz,
3
H5), 1.15 (d, 6H, J = 6.92 Hz, H6-H7). 13C NMR

C. Glende et al. / Mutation Research 498 (2001) 19–37
25
(CDCl3, 75 MHz) δ
=
139.10, 134.35, 125.51,
the dose-response curve (linear regression analysis).
Cytotoxicity is indicated by thinning or absence of
background lawn.
110.87, 27.69 (CH), 22.01 (CH3). MS (70 eV): m/z
(%) = 257 (M+, 66%), 255 (61%), 242 (97%), 240
(100%), 177 (20%), 162 (56%), 146 (23%). Calc.
C 56.01%, H 7.05%, N 5.44%; found C 56.42%, H
7.19%, N 4.82%
3. Results
3.1. Salmonella assays
2.5. Computational methods
The mutagenicities (log rev/nmol) of the arylamines
(1, 6, 11) and the alkylated arylamines (2–5, 7–10,
12–18) as determined in the Ames tests are summa-
rized in Table 2.
Details of the mutagenicity tests of 2-AN 1 and
its alkylated derivatives 2–5, 2-AF 6 and the alky-
lated 2-aminofluorenes 7–10, 4-ABP 11 and the
alkylated 4-aminobiphenyls 12–15 as well as the
ortho- dialkylated 4-aminobiphenyls 16–18, are
shown in Tables 3–6.
EHOMO and ELUMO of 1–18 were calculated by the
semi-empirical molecular orbital method AM1, after
optimizing the starting geometries by MM2 force
field calculations [19]. The initial geometry was built
from standard bond lengths and angles. All calcu-
lations were performed using the program MOPAC
Version 6.0. Hydrophobicity is expressed in terms
of log P as calculated with the program KOWWIN
Version 1.54.
2.6. Mutagenicity tests
3.1.1. 2-Aminonaphthalene 1 and its ortho-alkyl
derivatives 2–5
The mutagenic activity of the aromatic amines
1–18 was evaluated in S. typhimurium TA98 and
TA100, with and without metabolic activation (S9
mix), using the procedure of Maron and Ames [5].
Tester strains S. typhimurium TA98 and TA100 were
gifts from Bruce Ames, University of California,
Berkeley, CA. Aroclor induced S9 rat liver mix was
purchased from CCR, Germany. In all cases, DMSO
was used as the diluting solvent, and dosage solutions
were prepared immediately prior to testing. Incuba-
tions were carried out in triplicate; solvent controls
were run with each experiment. Compounds were
mostly tested at five concentrations both with TA98
and TA100. Each data point represents the average
value of three parallel test experiments. Appropriate
vehicle controls (DMSO) were conducted both with
and without metabolic activation. The positive con-
trol for TA98 + S9 was 2-aminoanthracene (2-AA),
for TA98-S9 4-nitro-o-phenylenediamine (NOPD) or
nitropyrene (NP), for TA100 + S9 2-AA, and for
TA100 − S9 N-methyl-Nꢀ-nitro-N-nitrosoguanidine
(MNNG) or sodium azide (NaN3). Compounds were
considered to be mutagenic if the average number
of histidine revertants obtained at any dose level as-
sayed was at least twice that of the solvent control.
Revertants per nanomoles (rev/nmol) were calcu-
lated from the linear slope of the increasing part of
The number of revertants induced by the test com-
pounds 1–5 in TA98 and TA100 with and without
metabolic activation are shown in Table 3.
The parent compound 2-AN 1 shows in the tester
strain TA98 with (+S9) and without (−S9) metabolic
Table 2
Experimental mutagenicities (log rev/nmol) of compounds 1–18
Number
Compound
log rev/nmol
TA98 + S9
TA100 + S9
1
2
2-AN
1-Et-2AN
−1.43
0.29
1.22
0.36
3
4
5
6
1-iPr-2AN
1-nBu-2AN
1-tBu-2AN
2-AF
−0.62
−0.29
<−2
1.54
0.68
1.04
−0.82
1.03
7
1-Et-2AF
1.85
1.08
8
9
1-iPr-2AF
1-nBu-2AF
1-tBu-2AF
4-ABP
3-Et-4ABP
3-iPr-4ABP
3-nBu-4ABP
3-tBu-4ABP
3,5-DiMe-4ABP
3,5-DiEt-4ABP
3,5-DiiPr-4ABP
2.23
2.86
0.07
0.65
1.29
0.82
10
11
12
13
14
15
16
17
18
−0.26
0.92
0.17
0.89
0.97
0.27
0.40
−0.37
−0.30
−1.40
−0.01
−0.17
−0.39
−1.34
−1.71
−1.95

26
C. Glende et al. / Mutation Research 498 (2001) 19–37
Table 3
Number of revertants of 2-aminonaphthalene 1 and the ortho-alkylated 2-aminonaphthalenes 2–5 in TA98 and TA100a
Number
Compound
Dose (␮g per plate)
Revertants
TA98
+S9
TA100
+S9
−S9
−S9
1
DMSO
Positive control
2-AN
42
691
53
70
37
15
7
37
1129
31
39
37
106
992
108
1002
126
143
214
32
␣
␤
␣
␤
20
100
500
2500
5000
381
429
315
50
34
toxT
toxT
toxT
2
DMSO
Positive control
1-Et-2-AN
42
691
356
684
558
toxT
toxA
37
1129
30
29
35
toxT
toxA
106
992
108
1002
112
114
130
toxT
toxA
␣
␥
␣
␤
20
100
500
2500
5000
1139
1554
1317
toxT
toxA
3
4
DMSO
Positive control
1-iPr-2-AN
47
516
34
44
56
32
1021
32
29
28
29
124
793
152
205
635
117
707
132
140
166
159
␣
␥
␣
␥
0.8
4
20
100
168
2700
DMSO
Positive control
1-nBu-2AN
33
1005
80
289
584
toxT
toxA,P
27
1011
27
113
1431
114
1025
␣
␥
␣
␤
5
20
100
500
2500
306
1198
1644
toxT
128
141
127
toxT
28
26
toxT
toxA,P
toxA,P
toxA,P
5
DMSO
Positive control
1-tBu-2AN
42
691
42
39
23
toxT
toxT
37
1129
35
31
13
toxT
toxT
106
992
196
204
43R
toxA
toxA
108
1002
115
␣
␥
␣
␤
20
100
500
2500
5000
126
29R
toxA
toxA
a toxT: thinning of background lawn; toxA: absence of background lawn; P: precipitation of test compound; R: reduced number of
revertants by thinning of background lawn; ␣: 2-AA, (2.5 ␮g); ␤: MNNG, (5 ␮g); ␥: NOPD, (10 ␮g).
activation no mutagenic activity. The regression anal-
ysis of the linear portion of the dose-response curve
leads to a value of −1.43 log rev/nmol (TA98 + S9).
In the case of TA98 − S9 cytotoxic effects are ob-
served at a concentration of 5000 ␮g per plate. In
TA100 + S9, a different situation is seen: the num-
ber of revertants shown in Table 3 lead to 0.29 log
rev/nmol (Table 2) which clearly indicates muta-
genic activity. Cytotoxicity is seen at a concentration
of 5000 ␮g per plate. Without S9 mix no activity
could be found in TA98 and TA100. In the case
of 1-Et-2AN 2, in both tester strains with S9 mix,
an increase of mutagenicity is observed as com-
pared to 1, see Tables 2 and 3: in TA98 + S9 the

C. Glende et al. / Mutation Research 498 (2001) 19–37
27
Table 4
Number of revertants of 2-aminonaphthalene 6 and the ortho-alkylated 2-aminofluorenes 7–10 in TA98 and TA100a
Number
Compound
Dose (␮g per plate)
Revertants
TA98
+S9
TA100
+S9
−S9
−S9
6
DMSO
Positive control
2-AF
16
527
17
49
29
16
17
352
9
6
8
102
1448
453
1289
869
toxA
110
920
125
␣
␥
␣
␤
5
20
100
500
31R
12R
3
toxA
7
8
1-Et-2AF
5
20
100
500
20
75
82
toxA
10
10
24
386
1305
1483
816
153
293
608
toxA
toxA
DMSO
Positive control
1-iPr-2AF
31
1555
81
166
737
toxA
37
965
33
53
172
toxA
102
1448
419
1825
1933
toxA
110
920b
149
264
452
toxA
␣
␥
␣
5
20
100
500
9
DMSO
Positive control
1-nBu-2AF
33
1005
192
601
1198
1153P
396P
27
1011
31
28
113
1431
170
650
1061
363P
38P
114
1025
114
124
110
75P
44P
␣
␥
␣
␤
5
20
100
500
2500
27
28P
22P,R
10
DMSO
Positive control
1-tBu-2AF
16
527
9
18
18
toxA
17
352
10
8
6
toxA
102
1448
135
154
184
43R
110
920
115
110
112
3R
␣
␥
␣
␤
5
20
100
500
a toxA: absence of background lawn; P: precipitation of test compound; R: reduced number of revertants by thinning of background
lawn; ␣: 2-AA, (2.5 ␮g); ␤: MNNG, (5 ␮g); ␥: NOPD, (10 ␮g).
mutagenicity of 2 (0.36 log rev/nmol) is nearly 60
times higher than that of 1 (−1.43 log rev/nmol).
Similarly, in TA100+S9 a strong mutagenic potential
is revealed (1.22 log rev/nmol). In both strains, with-
out metabolic activation, no mutagenicity is detected.
Thinning of background lawn was observed for both
tester strains, TA98 and TA100, with and without
metabolic activation at concentrations of 2500 ␮g
per plate. Absence of background lawn is found at
5000 ␮g per plate. 1-Isopropyl-2-aminonaphthalene
3 shows no mutagenic activity in TA98 − S9 and in
TA100 − S9. In TA98 + S9, for 3 a linear slope up
to a concentration of 100 ␮g per plate is observed,
leading to −0.62 log rev/nmol, which indicates weak
mutagenicity. In TA100 + S9, up to a concentra-
tion of 100 ␮g per plate, the mutagenic properties of
1-isopropyl-2-aminonaphthalene 3 led to a value of
0.68 log rev/nmol clearly indicating mutagenic behav-
ior of 3. Cytotoxicity is neither observed in TA98 nor
in TA100. For the n-butyl derivative 4 in TA98 with
S9 mix, a value of −0.29 log rev/nmol, and in TA100
with S9 mix, 1.04 log rev/nmol are observed. With-
out S9 mix also compound 4 shows no mutagenicity.
Above 500 ␮g per plate cytotoxicity is detected in

28
C. Glende et al. / Mutation Research 498 (2001) 19–37
Table 5
Number of revertants of 4-aminobiphenyl 11 and the ortho-alkylated 4-aminobiphenyls 12–15 in TA98 and TA100a
Number
Compound
Doses (␮g per plate)
Revertants
TA98
+S9
TA100
+S9
−S9
−S9
11
DMSO
Positive control
4-ABP
39
950
188
576
572
375R
toxA
28
842
28
28
27
113
1431
336
1093
1116
1281
toxA
114
1025
115
146
148
␣
␥
␣
␤
5
20
100
500
2500
21
toxA
141
toxA
12
DMSO
Positive control
3-Et-4ABP
36
721
–
88
toxA,C
304
449
603
671
691
32
1176
134
479
12
1051
␣
␥
␣
␤
2.40
4.75
9.5
19
37.5
75
–
–
–
184
290
395
876
1573
–
–
–
–
–
–
30
33
38
41
145
142
152
69
150
300
–
–
13
14
DMSO
Positive control
3-iPr-4-ABP
31
1555
37
115
487
toxA
37
965
28
23
25
toxA
102
1448
176
945
1539
toxA
110
920
103
99
113
toxA
␣
␥
␣
␤
5
20
100
500
DMSO
Positive control
3-nBu-4-ABP
33
1005
53
96
334
293
82P
27
1011
34
31
24
29
16P,R
113
1431
132
269
114
1025
␣
␥
␣
␤
5
20
100
500
2500
115
112
79
921
65R
26R
toxA,P
toxA,P
15
DMSO
Positive control
3-tBu-4-ABP
41
1379
116
177
304
325
32
1176
27
17
17
21
125
1837
643
964
1053
1493
123
1051
103
104
101
92
␣
␥
␣
␤
37.5
75
150
300
a toxA: absence of background lawn; P: precipitation of test compound; R: reduced number of revertants by thinning of background
lawn; C: contamination; (–): no measurement at this concentration; ␣: 2-AA, (2.5 ␮g); ␤: MNNG, (5 ␮g); ␥: NOPD, (10 ␮g).
both tester strains, and at a concentration of 2500 ␮g
per plate precipitation of the test compound together
with the absence of background lawn is observed. In
contrast to compounds 2–4, the tert-butyl derivative
5 shows in TA98, with metabolic activation, no mu-
tagenic potential (≤2 log rev/nmol for TA98 + S9).
Also in TA100 + S9 no mutagenicity is found (−0.82
log rev/nmol). In TA98 with and without S9 mix cyto-
toxicity sets in at a concentration of 2500 ␮g per plate
indicated by thinning of the background lawn. At the
same concentration absence of background lawn is
observed for TA100 + S9 and TA100 − S9. Thus, all

C. Glende et al. / Mutation Research 498 (2001) 19–37
29
Table 6
Number of revertants of the ortho-dialkylated 4-aminobiphenyls 16–18 in TA98 and TA100a
Number
Compound
Dose (␮g per plate)
Revertants
TA98
+S9
TA100
+S9
−S9
−S9
16
DMSO
Positive control
3,5-DiMe-4ABP
48
1167
44
57
70
31
1487
30
34
49
106
1612
118
150
184
219
24
118
898
␣
␦
␣
⑀
10
20
100
500
2500
108
111
123
38
143
toxT
33
toxT
toxT
17
18
3,5-DiEt-4ABP
10
20
100
500
2500
46
57
57
57
57
30
34
38
39
42
117
150
152
150
55
107
122
129
123
36
3,5-DiiPr-4ABP
10
20
100
500
2500
49
48
53
54
60
30
30
30
30
40
136
117
136
137
131
100
103
116
90
107
a toxT: thinning of background lawn; ␣: 2-AA, 2.5 ␮g; ␦: NP 2.5 ␮g; ⑀: NaN3 (5 ␮g).
alkyl-substituted derivatives of 2-AN 1, except the
tert-butyl derivative 5, are in TA98 + S9 of slightly
higher mutagenicity than 2-AN 1. The same trend is
observed for the tester strain TA100 + S9. Interest-
ingly 1-tbutyl-2-aminonaphthalene 5 shows in TA98
and TA100, with and without metabolic activation,
no mutagenicity.
2-AF 6, as reported in the literature [20], is a muta-
genic compound. This is clearly illustrated by the log
rev/nmol values in TA98+S9 (1.54 log rev/nmol) and
TA100 + S9 (1.03 log rev/nmol), see Table 2. With-
out S9 mix, in both tester strains, a positive response
was not seen. In TA100 with and without S9 mix, ab-
sence of background lawn was observed at a concen-
tration of 500 ␮g per plate. In TA98 − S9, the number
of revertants was at all concentrations (5–500 ␮g per
plate) lower than the solvent control. The ethyl deriva-
tive 1-ethyl-2-aminofluorene 7 is in TA98 + S9 more
mutagenic (1.85 log rev/nmol) than the non-alkylated
6, see Table 2. In TA100 + S9, the mutagenicity 1.08
log rev/nmol is in the range of 6. At concentrations
of 500 ␮g per plate in strain TA98 with and with-
out S9 mix and in TA100 with S9 mix, cytotoxic-
ity was indicated by the absence of background lawn.
In TA98 without S9 mix no mutagenicity is found.
In TA100 − S9, mutagenicity is clearly seen as indi-
cated by the number of revertants, see Table 4. The
iso-propyl-substituted 1-iPr-2AF 8 is even more mu-
tagenic than 6 and 7 in both tester strains with S9 mix:
2.23 log rev/nmol (TA98 + S9) and 1.29 log rev/nmol
3.1.2. 2-Aminofluorene 6 and its ortho-alkyl
derivatives 7–10
The number of revertants induced by the test com-
pounds 6–10 in TA98 and TA100 with and without
metabolic activation are shown in Table 4. 1
1 In the test series of the 2-aminofluorenes 6–10 in TA98, due to
the quality of the tester strain, the number of revertants in the case
of the parent compound 2-AF 6 was much smaller than reported in
the literature [20]. Since, the results of the alkyl-substituted 7–10
should be similarly affected, the revertants of 2-AF 6 were related
to the literature value of 34.6 rev/nmol [20], and the same relation
as found for 6 was used for the determination of the values of
7–10. As one can see from Fig. 2, the determined values of 6–10
in TA98 + S9 show exactly the same tendency as those correctly
determined in TA100+S9. Lack of compounds 7–10, unfortunately,
did not allow a second mutagenicity study in TA98 + S9.

30
C. Glende et al. / Mutation Research 498 (2001) 19–37
(TA100 + S9), see Table 2. Cytotoxicity is found at
500 ␮g per plate. Both, for TA98 and TA100 without
S9 mix mutagenicity is observed (Table 4). 1-nBu-2AF
9, the n-butyl-substituted derivative, shows the high-
est mutagenicity in this series: in TA98 + S9 it is
20 times higher (2.86 log rev/nmol) than that of the
parent compound 6 (Table 2). In TA100 + S9, 9 has
a lower mutagenic activity (0.82 log rev/nmol) com-
pared to 8 (1.29 log rev/nmol). Precipitation of the test
compound was observed at concentrations of 500 and
2500 ␮g per plate. The only alkyl-substituted deriva-
tive having lower mutagenicity than the unsubstituted
6 (1.54 log rev/nmol in TA98 + S9, 1.03 log rev/nmol
in TA100 + S9) in both tester strains, is 1-tBu-2AF
10: 0.07 log rev/nmol in TA98 + S9 and −0.26 log
rev/nmol in TA100 + S9 (Table 2). The latter value
together with the number of revertants in Table 4 indi-
cates weak mutagenicity. Cytotoxicity is observed at
concentrations of 500 ␮g per plate for TA98 with and
without S9 mix. In TA100, a reduced number of re-
vertants caused by a reduction of the background lawn
is observed at the same concentration. As expected,
mutagenicity is not observed without metabolic acti-
vation. In conclusion, with increasing log P-values of
compounds 6–9, mutagenicity in general increases. In
the case of the tert-butyl-substituted 10, however, the
sterically crowded substituent leads to a reduction al-
most an elimination of mutagenicity.
S9 mix. In the absence of S9 mix no mutagenicity
was observed in TA98, but in TA100 mutagenic-
ity is seen as indicated by the number of revertants
(Table 5). 3-iPr-4ABP 13 is non-mutagenic in both
tester strains without activation. The number of rever-
tants per plate are in the range of the negative control.
The compound shows mutagenicity in TA98 + S9
(−0.01 log rev/nmol) and TA100 + S9 (0.97 log
rev/nmol), see Tables 5 and 2. At concentrations of
500 ␮g per plate 13 is cytotoxic in both tester strains
with and without metabolic activation. 3-nBu-4ABP
14 is non-mutagenic without S9 mix (Table 5). With
S9 mix, 14 showed in TA98 (−0.17 log rev/nmol) and
in TA100 (0.27 log rev/nmol) a lower mutagenicity
than 11. In TA100 with and without S9 mix cytotox-
icity was observed at concentrations of 2500 ␮g per
plate. The compound with the bulkiest alkyl group
within this series, 3-tBu-4-ABP 15, reveals in both
tester strains with S9 mix (−0.39 log rev/nmol for
TA98, and 0.40 log rev/nmol for TA100) reduced
mutagenicity as compared to 11, especially in TA98.
When 3-tBu-4ABP 15 was tested without S9 mix,
the compound was non-mutagenic in both strains. In
contrast to 1-tBu-4AN 5, the mutagenic activity of
1-tBu-4ABP 15 in TA98 + S9 was not completely
extinguishable. A similar observation is made in
TA100 + S9.
In conclusion, also in the alkylated 4-aminobiphenyl
test series 12–15, the mutagenicity decreases with
increasing steric demand of the ortho-alkyl sub-
stituents.
3.1.3. 4-Aminobiphenyl 11 and its ortho-alkylated
derivatives 12–15
The number of revertants induced by the test com-
pounds 11–15 in TA98 and TA100 with and without
metabolic activation are shown in Table 5.
3.1.4. 3,5-Dialkyl-4-aminobiphenyl 16–18
The number of revertants induced by the test com-
pounds 16–18 in TA98 and TA100 with and without
metabolic activation is given in Table 6.
As expected, the parent compound 4-aminobiphenyl
11 requires metabolic activation to become a muta-
gen. In both tester strains with S9 mix, 11 shows a
positive mutagenic response, being stronger in TA100
(0.92 log rev/nmol) than in TA98 (0.65 log rev/nmol),
see Tables 5 and 2. Up to a concentration of 20 ␮g
per plate, in TA98 + S9, the mutagenicity of 4-ABP
11 rises linearly, while at concentrations of 2500 ␮g
per plate absence of background lawn is observed
in both tester strains with and without S9 mix. In
both strains, without S9 mix, no mutagenicity was
recorded (Table 5). 3-Et-4ABP 12 shows clearly mu-
tagenic properties in TA98 (0.17 log rev/nmol) and
in TA100 (0.89 log rev/nmol), in the presence of
It was shown above that due to alkylation ortho to
the amino function mutagenicity of arylamines may
decrease, especially in the case of bulky substituents.
Double-alkylation ortho to the amino function should
enhance this effect. Indeed, 3,5-DiMe-4ABP 16 con-
firms this suggestion: 16 shows very weak mutagenic
potential in TA98 + S9 (−1.34 log rev/nmol), see
Table 2. In comparison to the parent compound
4-ABP 11 (0.65 log rev/nmol), mutagenicity is nearly
nine times lower. In TA100 + S9 weak mutagenicity
is also observed (−0.37 log rev/nmol). In both tester
strains, without metabolic activation, a significant

C. Glende et al. / Mutation Research 498 (2001) 19–37
31
number of mutated revertants was not observed. 16
exhibits cytotoxicity in TA98 + S9, TA98 − S9 and
TA100 − S9 at concentrations of 2500 ␮g per plate.
3,5-DiEt-4ABP 17, in TA98 with S9 mix, shows no
mutagenicity (−1.71 log rev/nmol), see Table 2. In
the strain TA100 + S9 the mutagenicity value (−0.30
log rev/nmol) is in the range of 3,5-DiMe-4ABP
16. 3,5-DiiPr-4ABP 18, in TA98 and TA100 with
and without metabolic activation, is non-mutagenic
(−1.95 log rev/nmol for TA98 + S9, and −1.40 log
rev/nmol for TA100 + S9).
According to their results mutagenic activity of an
aromatic amine is mainly determined by its hydropho-
bicity (log P) and HOMO and LUMO energies. The
correlations for aromatic and heteroaromatic amines,
assayed in TA98 and TA100 with rat liver S9 mix, are
shown in Eqs. (1) and (2).
log TA98 = 1.08 log P + 1.28 EHOMO
−0.73 ELUMO + 1.46 IL + 7.20
(1)
log TA100 = 0.92 log P + 1.17 EHOMO
−1.18 ELUMO + 7.35
In conclusion, double-alkylation ortho to the amino
function with (bulky) alkyl groups causes an enhanced
decrease of mutagenicity in the 4-aminobiphenyl
derivatives 16–18.
(2)
The strongest influence results from hydrophobicity
(log P). Increasing hydrophobicity increases mu-
tagenicity linearly. In the case of TA98 mutagenic
activity is furthermore influenced by the indicator
variable IL. It is 1 for compounds containing three
or more fused rings, and 0 for all other species. The
electronic properties (EHOMO and ELUMO) have only
smaller influence on the mutagenicity. They account
for 17% in TA100 and 4% in TA98. Nevertheless,
there exists a positive correlation between mutagenic
activity and EHOMO. Compounds with higher EHOMO
3.2. Comparison of predicted and
experimental mutagenicities
The aim of a QSAR is to predict the mutagenic po-
tential of chemical compounds as a function of their
structure. Debnath et al. developed empirical corre-
lations for aromatic amines tested in the Salmonella
strains TA98 and TA100 with metabolic activation [7].
Table 7
The log P, ELUMO, EHOMO values, and calculated mutagenicities of 1–18 in comparison with the experimental mutagenicities
Number
Compound
log P
ELUMO (eV)
EHOMO (eV)
log rev/nmol
TA98 + S9
Calculated
TA100 + S9
Expected
Calculated
Expected
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
2-AN
1-Et-2AN
1-iPr-2AN
1-nBu-2AN
1-tBu-2AN
2-AF
2.28a
3.29
3.71
4.27
4.16
3.14a
4.14
4.55
5.12
5.01
2.86a
3.88
4.30
4.86
4.75
3.93
4.92
5.75
−0.175
−0.169
−0.172
−0.171
−0.179
−0.098
−0.073
−0.051
−0.065
−0.041
0.045
0.073
0.076
0.072
0.108
0.088
0.096
0.114
−8.227
−8.127
−8.102
−8.124
−8.052
−8.107
−8.060
−8.023
−8.045
−7.997
−8.274
−8.214
−8.210
−8.212
−8.170
−8.166
−8.150
−8.144
−0.33
0.82
1.28
1.88
1.79
1.75
2.87
3.34
3.94
3.86
−0.33
0.82
1.28
1.88
1.79
0.92
2.01
2.90
−1.43
0.36
−0.62
−0.29
<−2
1.54
1.85
2.23
2.86
0.07
0.00
1.07
1.49
1.98
1.97
0.87
1.82
2.21
2.72
2.65
0.25
1.22
1.61
2.13
2.03
1.31
2.23
2.98
0.29
1.22
0.68
1.04
−0.82
1.03
1-Et-2AF
1.08
1.29
0.82
1-iPr-2AF
1-nBu-2AF
1-tBu-2AF
4-ABP
3-Et-4ABP
3-iPr-4ABP
3-nBu-4ABP
3-tBu-4ABP
3,5-DiMe-4ABP
3,5-DiEt-4ABP
3,5-DiiPr-4ABP
−0.26
0.65
0.17
0.92
0.89
0.97
0.27
0.40
−0.37
−0.30
−1.40
−0.01
−0.17
−0.39
−1.34
−1.71
−1.95
a Experimental log P.

32
C. Glende et al. / Mutation Research 498 (2001) 19–37
energies are more easily oxidized, which leads to
faster transformation into the corresponding hydrox-
ylamines and, thus, e.g. O-acylhydroxylamines, the
ultimate mutagens. The QSAR correlations as devel-
oped by Debnath et al. are based on a large number of
aromatic amines with different aromatic ring systems.
Different (alkyl) substituents, however, have not been
considered. It is noticed that “steric effects and other
structural factors, which have not been built into the
QSARs, must be responsible for some of the ob-
served variation in activity” [7]. In our investigations,
we exactly studied these effects of alkyl substituents
ortho to the amino function, and it is obvious from
the results shown above that steric effects dramati-
cally influence the mutagenicity of the compounds
tested.
4. Discussion
As shown in Table 7 hydrophobicity of a certain
compound increases with the number and the size of
the alkyl groups attached ortho to the amino function.
The highest log P-values are calculated for com-
pounds containing the n-butyl group (4: 4.27, 9: 5.12
and 14: 4.86), and for the disubstituted species 16–18
(16: 3.93, 17: 4.92 and 18: 5.75). The log P-values of
compounds with linear alkyl side chains are higher
than those of the isomeric branched ones (1-nBu-2AN
4: 4.27 versus 1-tBu-2AN 5: 4.16). The LUMO and
the HOMO energies are slightly raised by alkyl groups
because of their +I-effects (4-ABP 11: ELUMO
=
0.045 eV, EHOMO = −8.274 eV versus 3-Et-4ABP
12: ELUMO = 0.073 eV, EHOMO = −8.214 eV). The
only exception is 1-tBu-2AN 5 showing a slightly
lower LUMO (ELUMO = −0.179 eV) than the par-
ent compound 2-AN 1 (ELUMO = −0.175 eV).
Ortho-disubstituted aromatic amines show a stronger
In Table 7, log P-values and electronic properties
(EHOMO and ELUMO) of compounds 1–18 are listed
along with their calculated and experimental muta-
genicities (log rev/nmol).
Fig. 1. Experimental and calculated mutagenicities of compounds 1–5.

C. Glende et al. / Mutation Research 498 (2001) 19–37
33
Fig. 2. Experimental and calculated mutagenicities of compounds 6–10.
influence on the orbital energies than monosubsti-
tuted ones, see, e.g. 3,5-DiMe-4ABP 16 (ELUMO
1-tBu-2-AN 5, in TA98 + S9, the experimental value
deviates from the straight line by nearly 3.8 units
(log rev/nmol; this corresponds to a factor of ∼6000).
These results nicely illustrate that the decrease of mu-
tagenicity in compounds 2–5 is strongly related to the
steric influence of the alkyl groups. The steric demand
of the n-butyl group is in the range between that of
the ethyl and the iso-propyl groups [21]. Therefore,
and because of its high hydrophobicity, 1-nBu-2AN
4 shows higher mutagenic activity than 1-iPr-2AN 3
and 1-tBu-2AN 5. The only species showing smaller
mutagenicity than the parent compound 2-AN 1, is
1-tBu-2AN 5 with its sterically strongly demanding
tert-butyl substituent [21]. Related results were also
observed in the case of the tester strain TA100 + S9,
see also Fig. 1.
=
0.088 eV, EHOMO = −8.166 eV). In Figs. 1–4 the
calculated and the measured mutagenicity values (log
rev/nmol) are graphically compared with each other.
In Fig. 1, the parent compound 2-aminonaphthalene
1 and the alkylated derivatives 2–5 are shown. For
both Salmonella strains TA98 and TA100, with
metabolic activation, the Debnath–Hansch correla-
tions predict a linear increase of mutagenicity with
increasing hydrophobicity resulting from the alkyl
groups (Et < iPr < tBu < nBu). In the case of
the parent compound 2-AN 1 and of 1-Et-2AN 2
mutagenicity is decently predicted. However, with
growing steric demand of the alkyl groups, the pre-
dicted and the experimental values differ increasingly.
The graphs show strong deviations in the case of the
iso-propyl-substituted 3, the n-butyl-substituted 4, and
especially the tert-butyl-substituted 5. In the case of
The same tendency as for 1–5 was similarly ob-
served in the case of the alkylated 2-aminofluorenes
7–10 if compared with the parent compound 6, see

34
C. Glende et al. / Mutation Research 498 (2001) 19–37
Fig. 3. Experimental and calculated mutagenicities of compounds 11–15.
Fig. 2. The diagrams for both tester strains (Fig. 2) re-
veal that the compounds bearing bulkier alkyl groups
have less mutagenic activity than predicted by the
Debnath–Hansch equations (Eqs. (1) and (2)). In con-
trast to the series of 1–5 only weak increase of muta-
genicity was observed due to the ethyl group in 7. As in
the case of 1-nBu-2AN 4, see Fig. 1, also 1-nBu-2AF
9 in TA98+S9 shows stronger mutagenic activity than
the other alkylated species 7, 8 and 10, see Fig. 2.
Apparently, the strong hydrophobicity of 9 dominates
here over the steric demand of the n-butyl group. The
tert-butyl substituted 10 has again the lowest muta-
genicity value within the 2-AF series. It was, however,
not possible to suppress completely the mutagenic ac-
tivity in the case of 10.
mental and the predicted values deviate strongly in
these cases: for 3-tBu-4ABP 15 the Debnath–Hansch
correlation predicts a mutagenicity in TA98 + S9 of
1.79 log rev/nmol, while the experimental data lead
to −0.39 log rev/nmol. The largest difference is ob-
served for the dialkyl compounds 16–18, and here es-
pecially for 3,5-DiiPr-4ABP 18 (calculated: 2.90 log
rev/nmol; experimental: −1.95 log rev/nmol). These
results strongly support the necessity to include steric
parameters into the QSAR correlations if reasonable
mutagenicity values are to be excepted for compounds
bearing sterically demanding substituents.
Similar observations were made by Klein et al. in
the case of ortho-alkylated nitrobiphenyls [22]. In
their work the synthesis of non-mutagenic derivatives
of 4-nitrobiphenyls is reported by introducing bulky
alkyl groups, as similarly shown here. The predicted
mutagenicities of alkyl-substituted nitro compounds
are also too high, because the existing equations for
the calculation of mutagenicities likewise do also
The results of the ortho-alkylated 4-aminobiphenyls
12–15 (Fig. 3) and the dialkylated 16–18 (Fig. 4) show
similarly the importance of steric factors on muta-
genicity. Again growing steric demand of the alkyl
groups leads to decreased mutagenicity. The experi-

C. Glende et al. / Mutation Research 498 (2001) 19–37
35
Fig. 4. Experimental and calculated mutagenicities of compounds 11, 16–18.
not take into account steric effects of aliphatic sub-
stituents.
genic. In contrast, in the case of the respective hy-
droxylamines, it is 1-hydroxylaminonaphthalene that
shows a 10-fold higher induction rate of tumors than
2-hydroxylaminonaphthalene [23]. Related results are
observed for the 2-amino and 3-amino positional iso-
mers of 4-aminobiphenyl 11 and their hydroxylamino
derivatives [24], and for 3-tBu-4ABP 15 [25].
What is the reason for the comparatively high muta-
genicity of the ethyl-substituted arylamines 1-Et-2AN
2, 1-Et-2AF 7 and 3-Et-4ABP 12, as observed in this
work, and for 3-methyl-4-aminobiphenyl as reported
by Bayoumy et al. [26], and for ortho-alkylated ani-
lines as described by Beland et al. [27]? First, the
steric hindrance caused by the methyl and the ethyl
group is small if compared to the increase of the log
P-value resulting from these substituents. One should
also consider that ortho-alkyl groups, due to their
+I-effect, stabilize the intermediate nitrenium ions. In-
deed, one has observed a linear relationship between
4.1. Reduced and increased mutagenicity of
ortho-alkyl substituted aromatic amines
One reason for the decrease of the mutagenicity
of arylamines with sterically demanding alkyl groups
as the iso-propyl and tert-butyl substituents could be
the steric hindrance of the metabolic oxidation of the
amino group by enzymes, which is a pre-requisite
for the transformation of the non-mutagenic aromatic
amines into mutagens. If the oxidation step to the re-
spective hydroxylamine is blocked, or aggravated, the
amine is not transformed into its ultimate mutagen. In
the literature similar cases are mentioned, e.g. the com-
pletely different mutagenicity of 1-aminonaphthalene
and 2-aminonaphthalene: 1-aminonaphthalene is
non-mutagenic, while 2-aminonaphthalene is muta-

36
C. Glende et al. / Mutation Research 498 (2001) 19–37
mutagenicity and the stability of the respective ni-
trenium ion [28]. Secondly, substituents like methyl,
and probably ethyl, are oxidized, leading to the cor-
responding benzylic alcohol which are transformed
by sulfation or phosphation into ultimate mutagens
which react with DNA [29]. A prominent example
for such a metabolic pathway was observed in the
case of 1-methylpyrene [30,31]. Thus, methyl- and
ethyl-substituted arylamines possibly are not only ac-
tivated via oxidation of the amino group, but also via
oxidation of the small alkyl groups bonded to the aro-
matic rings.
In summary, it is shown in this work that the mu-
tagenicity of aromatic amines decreases with growing
steric demand of the alkyl groups in the ortho-position
of the amine functionality. Generally, steric demand
leads to lower mutagenicities than predicted by the
Debnath–Hansch equations (Eqs. (1) and (2)). Thus,
these QSAR correlations are not appropriate to eval-
uate the mutagenicity of aromatic amines substituted
with such alkyl groups.
[7] A.K. Debnath, G. Debnath, A.J. Shusterman, C. Hansch,
A QSAR investigation of the role of hydrophobicity in
regulating mutagenicity in the Ames test: Part 1. Mutagenicity
of aromatic and heteroaromatic amines in Salmonella
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mutagenicity in the Ames test. Part 2. Mutagenicity of
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[10] Z. You, M.-D. Brezell, S.K. Das, B.H. Hoobermann, J.E.
Sinsheimer, Substituent effects on the in vitro and in
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derivatives, Mutat. Res. 320 (1/2) (1994) 45–58.
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[12] Organikum, VEB Deutscher Verlag der Wissenschaften, 16.
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Due to these findings, it was possible in the
cases of 1-tBu-2AN 5, 3,5-DiEt-4ABP 17 and
3,5-DiiPr-4-ABP 18 to synthesize non-mutagenic
derivatives of rather mutagenic parent aromatic
amines.
The investigations of aromatic amines bearing alkyl
groups “far away” from the amino functionality will
be discussed in a following paper.
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