The effects of 4′-alkyl substituents on the mutagenic activity of 4-amino- and 4-nitrostilbenes in Salmonella typhimurium

Article abstract of DOI:10.1016/S1383-5718(01)00142-5

Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4′-position and six of the corresponding nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and TA100. Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl. More bulky substituents reduced the mutagenic potential in the order iso-propyl < sec-butyl < tert-butyl for the aminostilbenes. The corresponding nitrostilbenes showed a similar trend under these conditions although the mutagenic activity of the tert-butyl-substituted compound was unexpectedly high in TA100. In the series without metabolic activation the nitrostilbenes showed a continuous decrease of mutagenic activity with the size of the substituents (methyl > ethyl > iso-propyl > sec-butyl > tert-butyl). These trends have been compared with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes. The unexpected result for the tert-butyl nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway.

Full text of DOI:10.1016/S1383-5718(01)00142-5

Mutation Research 491 (2001) 195–209
0
The effects of 4 -alkyl substituents on the mutagenic activity of
-amino- and 4-nitrostilbenes in Salmonella typhimurium
4
a
a
b
Björn Ludolph , Markus Klein , Lothar Erdinger ,
a,∗
Gernot Boche
a
Philipps-Universität, Marburg Hans-Meerwein Straße, 35032 Marburg, Germany
Hygieneinstitut Heidelberg, Im Neuenheimer Feld, 69120 Heidelberg, Germany
b
Received 24 October 2000; received in revised form 16 January 2001; accepted 17 January 2001
Abstract
0
Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4 -position and six of the corresponding
nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and
TA100. Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and
trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl. More bulky substituents reduced the mutagenic
potential in the order iso-propyl < sec-butyl < tert-butyl for the aminostilbenes. The corresponding nitrostilbenes showed a
similar trend under these conditions although the mutagenic activity of the tert-butyl-substituted compound was unexpectedly
high in TA100. In the series without metabolic activation the nitrostilbenes showed a continuous decrease of mutagenic activity
with the size of the substituents (methyl > ethyl > iso-propyl > sec-butyl > tert-butyl). These trends have been compared
with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an
important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes. The unexpected result for the tert-butyl
nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway. © 2001 Elsevier Science
B.V. All rights reserved.
Keywords: Salmonella; Nitrostilbenes; Aminostilbenes; Alkyl substituents; Para; Mutagenicity
1
. Introduction
to find general strategies to reduce the mutagenic
potential of these N-substituted aromatics [5]. It is well
established that metabolic transformation into elec-
trophilic intermediates (hydroxylamines, nitrenium
ions), that form DNA-adducts, is necessary for this
class of compounds to develop their genotoxic proper-
ties. Thus, useful ways to reduce mutagenic responses
should be either to inhibit this activation process or to
hinder the binding of the reactive species to the DNA.
Recently we found that bulky alkyl substituents
in the para-position of 4-aminobiphenyl 1a–e [6,7]
Aromatic amines and nitroarenes are used in
industrial processes and in the manufacturing of drugs,
pesticides and plastics [1]. Several azo compounds
that are widely used as dyes can be metabolically
cleaved into the corresponding amines [2–4]. Since
many aromatic amines are known for their mutagenic
and carcinogenic potential attempts have been made
∗
Corresponding author. Tel.: +49-6421-282-2030;
(paper in preparation), 4-nitrobiphenyl 2a–e [8]
fax: +49-6421-282-8917.
E-mail address: boche@chemie.uni-marburg.de (G. Boche).
and 2-nitrofluorene 3a–e [8] (Fig. 1) decrease the
1
383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S1383-5718(01)00142-5

1
96
B. Ludolph et al. / Mutation Research 491 (2001) 195–209
Fig. 1. Structures of recently examined compounds.
mutagenicity in the Ames-test assayed with strains
TA98 and TA100 of Salmonella typhimurium.
Most remarkably the mutagenicity of those
compounds with large substituents like tert-butyl
carcinogenic activity in 1948 [12]. Recent QSAR
studies of the genotoxic potential of various trans-4-
aminostilbenes were carried out by Sinsheimer
[13,14]. In these investigations mutagenicity could be
correlated with the electronic properties of the sub-
(1e–3e) was almost completely eliminated. In contrast
0
small alkyl substituents like methyl (1b–3b) gener-
ally increased the mutagenic response significantly
compared to the parent compounds 1a–3a.
In the early nineties Debnath and Hansch devel-
oped quantitative structure activity relationship
stituents (in the 4 -position) whereas no correlation
was found between hydrophobicity and mutagenicity.
The mutagenic potential of 4-nitrostilbene and some
of its derivatives was demonstrated by Mullin [15] and
Hoobermann [16]. Much effort has also been under-
taken to characterize DNA-adducts of 4-aminostilbene
derivatives (see Neumann [17], a review of earlier
publications is given by Beland and Kadlubar [18]).
4-Aminostilbenes form structurally different adducts
if compared to those of the 4-aminobiphenyls or
2-aminofluorenes mentioned above: 4-aminostilbene
reacts via the double bond with the nitrogen and
oxygen atoms of the DNA bases to form cyclic
adducts. Some typical adducts (4a–c) are shown in
Fig. 2.
(QSAR) models to predict to mutagenicity of amino
aromatics [9] and nitro aromatics, [10,11] which
are based on hydrophobicity (log P) and electronic
energies (EHOMO, ELUMO). A comparison of our
experimentally determined mutagenicities with those
predicted by Debnath and Hansch led to the con-
clusion that the decrease of mutagenicity with the
size of the alkyl substituents is mainly a steric effect
[6–8]. For alkyl-substituted compounds the
observed mutagenic response was correlated with the
modification of the molecular shape. We suggested
the steric demand of bulky alkyl groups to interfere
with the molecule’s ability to intercalate between the
planar DNA bases. Regarding the small alkyl groups
Is the change of the molecular shape by introduc-
tion of bulky alkyl substituents far away from the
functional group also suitable to reduce the mutageni-
city of 4-amino- and 4-nitrostilbene derivatives as it
is for the corresponding amino- and nitrobiphenyles
(1a–e and 2a–e) and aminofluorenes (3a–e)? To ex-
amine this question we investigated the mutagenicity
(methyl, ethyl), steric demand is less important. In
this case mutagenicity is determined by the electronic
and hydrophobic effects of these substituents. The
introduction of bulky para-alkyl groups “far away”
from the functional group (NH2 or NO2) seems to
be a promising new method to detoxify mutagenic
substances of this class. The chemical properties of
the nitro or amino group, or the degree of aromaticity,
remain essentially unaffected.
0
of 4 -alkyl-substituted trans-4-amino- and trans-4-
nitrostilbenes such as 5a–f and 6a–f, respectively
(Fig. 3).
Furthermore we compared our experimental results
with the predictions of the QSAR models of Deb-
nath and Hansch (applicable for aminoarenes in the
presence of S9-mix and nitroarenes in the absence of
S9-mix) [11] and Sinsheimer, respectively (applicable
Another amino–aromatic model compound of great
interest is trans-4-aminostilbene, first tested for its

B. Ludolph et al. / Mutation Research 491 (2001) 195–209
197
Fig. 2. Structures of typical DNA-adducts of N-acetylaminostilbene.
for trans-4-aminostilbenes in the presence of S9-mix)
13].
to tetramethylsilane. Mass spectra were recorded
with Varian MAT CH-7-A (EI, 70 eV). Elemen-
tary analyses were performed by a Heraeus Rapid
Elementaranalysator.
[
2. Materials and methods
2
.2. Chemicals
2.1. Instrumentation
The alkyl-substituted trans-4-nitrostilbenes 6a–f
NMR spectra were obtained with Bruker spectro-
meters ARX-200 and AC-300 and are referenced
(Fig. 3) were prepared either by Wittig reactions as
described by Sinsheimer [12] from (4-nitrobenzyl)-
Fig. 3. Structures of synthesized compounds.

1
98
B. Ludolph et al. / Mutation Research 491 (2001) 195–209
0
triphenylphosphoniumbromide and an appropriate
substituted benzaldehyde (route A, 72–41% yield), or
by Horner-Wadsworth-Emmons reactions as descri-
bed by Hanna [19] from 4-nitrobenzylphosphonic
acid diethylester (prepared according to Deussen
2.2.3. trans-4 -Methyl-4-nitrostilbene (route A)
1
Yellow needles, H NMR (300 MHz, CDCl3): δ
3
3
(ppm) = 8.20 (d, J = 8.9 Hz, 2H), 7.60 (d, J =
3
8.6 Hz, 2H), 7.44 (d, J = 8.14 Hz, 2H), 7.16 (m, 3H),
1
3
2.38 (s, 3H). C NMR (75 MHz, CDCl3): δ (ppm) =
[20], 80% yield) and an appropriate benzalde-
146.6, 144.1, 139.0, 133.4, 129.6, 126.9, 126.6, 125.2,
+
hyde (route B, 71–90% yield). The correspond-
124.1, 21.2. MS (70 eV): m/z (%) = 239 (M , 100),
ing benzaldehydes were purchased from Aldrich;
178 (52).
0
4
-sec-butylbenzaldehyde was prepared in two steps
0
from 4 -sec-butylaniline by a Sandmeyer transfor-
2.2.4. 4-Nitrobenzylphosphonic acid diethylester
1
mation (85% yield) and reaction of the obtained
Red oil, H NMR (CDCl3, 200 MHz): δ (ppm) =
0
3
3
4
-sec-butyliodobenzene with BuLi and N-formylpipe-
8.13 (d, J = 8.8 Hz, 2H), 7.43 (dd, J = 8.8 Hz,
4
ridine as described by Schlüter [21] (84% yield). The
-nitrostilbenes 6c–6e contained up to 5% of the cor-
JP–H = 2.5 Hz, 2H), 4.06 − 3.92 (m, 4H), 3.25
3
3
4
(d, J = 17.3 Hz, 2H), 1.27 (t, J = 6.8 Hz, 6H).
13
responding cis-isomers, which could not be separated
by recrystallization and column chromatography. As
cis-isomers of 4-nitrostilbene derivatives turn out to
be less active than the corresponding trans-isomers
C NMR (75 MHz, CDCl3) δ = 146.8 (d, JC–P =
4 Hz), 139.6 (d, JC–P = 9 Hz), 130.5 (d, JC–P =
6.5 Hz), 123.5 (d, JC–P = 3 Hz), 62.3 (d, JC–P =
7 Hz), 33.9 (d, JC–P = 137 Hz), 16.2 (d, JC–P =
+
[16], the cis-content should not affect the Ames-test
6 Hz). MS (70 eV): m/z (%) = 273 (M , 25), 227
+
+
+
results seriously. However, to investigate the effect
of the cis-isomer on the mutagenicity a second
charge of pure trans-6e, prepared by iodine-catalyzed
(M − NO2, 11), 137 (M − 4-nitrobenzyl, 36) 136
+
(M − PO(OEt)2, 25), 109 (M , 100).
0
isomerization, was also included in the test series
2.2.5. trans-4 -Ethyl-4-nitrostilbene (route B)
0
◦
1
(
6e ).
Yellow needles, mp 98 C, H NMR (CDCl3,
3
Reduction of the nitro compounds 6a–f to the
trans-4-aminostilbenes 5a–f (Fig. 3) was achieved
using zinc and ammonium chloride [22] (route C,
300 MHz): δ (ppm) = 8.20 (d, J = 8.8 Hz, 2H),
3
3
7.60 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.1 Hz, 2H),
3 3
7.25 (d, J = 16.2 Hz, 1H), 7.25 (d, J = 8.1 Hz,
3 3
71–31% yield) or SnCl2 (route D, 65% yield) [23].
2H), 7.09 (d, J = 16.3 Hz, 1H), 2.70 (q, J =
3 13
All compounds were structurally characterized by
NMR and by MS. Purities were checked by TLC and
gas chromatography.
7.7 Hz, 2H), 1.29 (t, J = 7.7 Hz, 3H). C NMR
(75 MHz, CDCl3) δ (ppm) = 146.5, 145.3, 144.0,
133.6, 133.2, 128.3, 127.2, 126.6, 125.3, 124.0, 28.6,
+
1
5.3. MS (70 eV): m/z (%) = 253 (M , 100), 238
+
+
+
2
.2.1. (4-Nitrobenzyl)-triphenylphosphonium-
bromide
White precipitate, 1H NMR (200 MHz, CDCl3):
δ (ppm)
(M −CH3, 47), 192 (M −NO2, 16), 191 (21), 178
(M − CH3 − NO2, 42). HRMS (70 eV): m/z calcu-
lated for C H NO2 253.1103, found 253.1102. The
1
6
15
=
7.85 − 7.31 (m, 19H), 6.01 (d,
substance contained 5% cis-product (NMR), which
could not be separated by recrystallization or column
chromatography.
3
13
JH–P = 15.8, 2H). C NMR (50 MHz, CDCl3): δ
ppm) = 147.2, 135.7, 135.0, 134.4, 132.9, 130.1,
(
1
1
1
4
2
23.1, 117.2 (d,
2 Hz). P NMR (81 MHz, CDCl3): δ (ppm) =
5.5.
J
= 86 Hz), 29.6 (d,
J
=
3
1
0
2.2.6. trans-4 -iso-Propyl-4-nitrostilbene (route B)
◦
1
Yellow needles, mp 134 C, H NMR (CDCl3,
3
4
00 MHz): δ (ppm) = 8.20 (d, J = 8.8 Hz, 2H),
3
3
2
.2.2. trans-4-Nitrostilbene (route A)
7.61 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H),
3 3
1
Yellow needles, H NMR (300 MHz, CDCl3): δ
7.25 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 16.3 Hz,
3 3
3
(
ppm) = 8.20 (d, J = 8.9 Hz, 2H), 7.63 − 7.10 (m,
1H), 7.09 (d, J = 16.3 Hz, 1H), 2.93 (p, J =
3 13
1
3
8
1
H). C NMR (75 MHz, CDCl3): δ (ppm) = 146.8,
7.0 Hz, 1H), 1.27 (d, J = 7.0 Hz, 6H). C NMR
(CDCl3, 100 MHz) δ (ppm) = 150.0, 146.6, 144.1,
133.8, 133.3, 127.1, 127.0, 126.7, 125.3, 124.1, 34.0,
43.9, 136.2, 133.3, 128.9, 127.0, 126.9, 126.3, 124.1.
+
MS (EI): 225.1 (M , 72), 178.1 (100).

B. Ludolph et al. / Mutation Research 491 (2001) 195–209
199
+
+
266 (C17H NO2 , 100). HRMS (70 eV): m/z cal-
16
2
(
3.8. MS (70 eV): m/z (%) = 267 (M , 100), 252
+
+
M −CH3, 75), 235 (14). 178 (M −C3H7−NO2, 7).
culated for C18H19NO2 281.1416, found 281.141.
The compound prepared via route B contained up
to 5% cis-product, which could not be separated by
recrystallization or column chromatography.
CHN: calculated C 76.38%, H 6.41%, N 5.24%, found
C 76.51%, H 6.32%, N 5.31%. The substance con-
tained 5% cis-product (NMR), which could not be sep-
arated by recrystallization or column chromatography.
2
.2.11. trans-4-Aminostilbene
1
2
.2.7. 4-sec-Butyliodobenzene
3
Off-white precipitate, H NMR (300 MHz, CDCl ):
1
3
Colourless liquid, H NMR (200 MHz, CDCl3): δ
δ (ppm) = 7.46 (d, J = 7.7 Hz, 2H), 7.34 − 7.30
3 5
3
3
(
ppm) = 7.60 (d, J = 12.9 Hz, 2H), 6.93 (d, J =
(m, 4H), 7.20 (tt, J = 7.3 Hz, J = 1.3 Hz, 1H),
3 3
3
1
6
2.9, 2H), 2.54 (m, 1H), 1.56 (m, 2H), 1.20 (d, J =
7.02 (d, J = 16.3 Hz, 1H), 6.91 (d, J = 16.3 Hz,
3
13
3
.9 Hz, 3H), 0.80 (t, J = 7.3 Hz, 3H). C NMR
1H), 6.66 (d, J = 8.6 Hz, 2H), 3.72 (s, 2H, NH ).
2
1
3
(
9
2
50 MHz, CDCl3): δ (ppm) = 147.3, 137.4, 129.3,
C NMR (75 MHz, CDCl ): δ (ppm) = 146, 136.9,
3
0.7, 41.3, 31.0, 21.7, 12.2. MS (70 eV): m/z (%) =
127.8, 127.5, 126.5, 125.6, 125.3, 124.8, 122.8, 113.7.
+
+
+
60 (M , 55), 231 (C8H8I , 100).
MS (70 eV): m/z (%) = 195.2 (M , 100), 167.1 (7).
CHN: calculated C 86.12%, H 6.71%, N 7.17%, found
C 86.19%, H 6.70%, N 7.31%.
2
.2.8. 4-sec-Butylbenzaldehyde
Colourless liquid, H NMR (200 MHz, CDCl3): δ
1
3
0
(
ppm) = 9.97 (s, 1H), 7.81 (d, J = 8.0 Hz, 2H),
2.2.12. trans-4 -Methyl-4-aminostilbene
3
1
7
2
3
1
.34 (d, J = 8.0 Hz, 2H), 2.67 (m, 1H), 1.63 (m,
Off-white precipitate, H NMR (300 MHz, CDCl3):
3
3
3
3
H), 1.26 (d, J = 7.0 Hz, 3H), 0.82 (t, J = 7.4 Hz,
δ (ppm) = 7.37 (d, J = 8.1 Hz, 2H), 7.32 (d, J =
1
3
3 3
H). C NMR (50 MHz, CDCl3): δ (ppm) = 191.8,
8.4 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 6.98 (d, J =
3
55.0, 134.5, 129.8, 127.6, 41.9, 35.4, 21.4, 12.0. MS
16.3 Hz, 1H), 6.88 (d, J = 16.3 Hz, 1H), 6.66 (d,
+
+
3
(
1
70 eV): m/z (%) = 162 (M , 32), 133 (C9H9O ,
J = 8.6 Hz, 2H), 3.71 (s, 2H, NH2), 2.34 (s, 3H).
3
1
00). HRMS (70 eV): m/z calculated for C11H14O
C NMR (75 MHz, CDCl3): δ (ppm) = 145, 136.6,
162.1045, found 162.105.
135.2, 129.3, 128.3, 127.7, 127.6, 126.0, 125.1, 115.2,
+
2
1.1. MS (70 eV): m/z (%) = 209.2 (M , 100), 194.2
0
2
.2.9. trans-4 -sec-Butyl-4-nitrostilbene
(16). CHN: calculated C 86.08%, H 7.22%, N 6.69%,
found C 85.92%, H 7.09%, N 6.83%.
1
Yellow needles, H NMR (300 MHz, CDCl3): δ
3
3
(
ppm) = 8.21 (d, J = 8.9 Hz, 2H), 7.61 (d, J =
.8 Hz, 2H), 7.48 (d, 3J = 8.18 Hz, 2H), 7.28 −
2.2.13. trans-4 -Ethyl-4-aminostilbene
0
8
1
7
.15 (m, 4H), 2.63 (m, 1H), 1.62 (m, 2H), 1.25 (d,
Off-white precipitate, H NMR (300 MHz, CDCl3):
3
3
13
3
J = 6.9 Hz), 0.84 (t, J = 7.3 Hz, 3H). C NMR
75 MHz, CDCl3): δ (ppm) = 148.8, 146.6, 144.1,
δ (ppm) = 7.40 (d, J = 8.0 Hz, 2H), 7.32 (d,
3
3
3
(
J = 8.3 Hz, 2H), 7.17 (d, J = 7.9 Hz, 2H), 6.98 (d,
3
1
3
4
33.8, 133.3, 127.6, 127.0, 126.7, 125.3, 124.1, 41.5,
J = 16.3 Hz, 1H), 6.90 (d, J = 16.3 Hz, 1H), 6.66
+
3
1.0, 21.6, 12.2. MS (70 eV): m/z (%) = 281 (M ,
(d, J = 8.3 Hz, 2H), 3.71 (s, 2H, NH2), 2.64 (q,
+
3
13
5), 252 (C H14NO2 , 100). HRMS (70 eV): m/z cal-
2H), 1.23 (t, J = 7.6 Hz, 3H). C NMR (75 MHz,
CDCl3): δ (ppm) = 145.9, 143.1, 135.4, 128.2, 128.0,
1
6
culated for C18H19NO2 281.1416, found 281.1419.
1
27.7, 127.5, 126.0, 125.0, 115.1, 28.5, 15.5. MS
0
+
+
2
.2.10. trans-4 -tert-Butyl-4-nitrostilbene (route A)
(70 eV): m/z (%) = 223 (M , 100), 208 (C H N ,
1
5
15
1
Yellow needles, H NMR (200 MHz, CDCl3):
47). CHN: calculated C 86.05%, H 7.67%, N 6.24%,
found C 85.85%, H 7.61%, N 6.27%.
3
δ (ppm) = 8.18 (d, J = 8.7 Hz, 2H), 7.59 (d,
3
3
J = 8.8 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H) 7.41
3
3
0
(
7
(
1
3
d, J = 8.7 Hz, 2H), 7.25 (d, J = 16.2 Hz, 1H),
2.2.14. trans-4 -iso-Propyl-4-aminostilbene
3
13
1
.08 (d, J = 16.2 Hz, 1H), 1.34 (s, 9H). C NMR
Off-white precipitate, H NMR (300 MHz, CDCl3):
3
50 MHz, CDCl3): δ (ppm) = 152.2, 146.5, 144.0,
δ (ppm) = 7.41 (d, J = 8.2 Hz, 2H), 7.32 (d,
3
3
33.3, 133.1, 126.8, 126.6, 125.8, 125.4, 124.0,
J = 8.5 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 6.99
+
3
3
4.7, 31.2. MS (70 eV): m/z (%) = 281 (M , 42),
(d, J = 16.3 Hz, 1H), 6.90 (d, J = 16.3 Hz, 1H),

2
00
B. Ludolph et al. / Mutation Research 491 (2001) 195–209
3
6
(
.65 (d, J = 8.6 Hz, 2H), 3.70 (s, 2H, NH2), 2.90
S9-mix (+S9) and without metabolic activation
3
3
13
m, J = 6.9 Hz, 1H), 1.26 (t, J = 6.9 Hz, 3H).
C
(−S9). Arochlor-induced S9 from rat liver was pur-
chased from CCR, Germany. The concentration of
the S9 fraction in the mix was 10%. For the aminos-
tilbenes a five-point and for the nitrostilbenes a
six-point dose-response curve was established using
appropriate dilutions of the compound to be tested.
Each revertant number presented in Tables 1 and 2
is the average of three values. In order to guarantee
optimal comparability all compounds within one se-
ries were tested under identical conditions within 1
week. At higher concentrations for most substances
cell toxicity resulted in a flattening of the correspond-
ing dose-response curves. Experimental mutation
rates (revertant/nmol) are calculated as the slope of
a regression analysis over the linear portion of the
dose-response curve.
NMR (75 MHz, CDCl3): δ (ppm) = 147.7, 145.9,
1
3
2
8
35.5, 128.2, 127.8, 127.5, 126.6, 126.0, 125.0, 115.1,
+
2.8, 23.9. MS (70 eV): m/z (%) = 237 (M , 100),
+
22 (C H N , 47). CHN: calculated C 86.03%, H
1
6
16
.07%, N 5.90%, found C 85.81%, H 8.08%, N 5.61%.
0
2
.2.15. trans-4 -sec-Butyl-4-aminostilbene
Off-white precipitate, H NMR (300 MHz, CDCl3):
1
3
3
δ (ppm) = 7.39 (d, J = 8.2 Hz, 2H), 7.32 (d, J =
3
3
8
.4 Hz, 2H), 7.14 (d, J = 8.2 Hz, 2H), 6.99 (d, J =
3
1
6.3 Hz, 1H), 6.89 (d, J = 16.3 Hz, 1H), 6.67 (d,
3
J = 8.4 Hz, 2H) 3.52 (s, 2H, NH2), 2.58 (m, 1H),
3
3
1
7
1
1
.59 (m, 2H), 1.23 (d, J = 6.9 Hz, 3H), 0.83 (t, J =
13
.4 Hz, 3H). C NMR (75 MHz, CDCl3): δ (ppm) =
46.6, 145.6, 135.5, 128.5, 127.7, 127.5, 127.3, 126.0,
25.3, 115.3, 41.4, 31.1, 21.7, 12.2. MS (70 eV): m/z
+
+
(
%) = 251 (M , 93), 222 (C H N , 100). CHN:
16
16
calculated C 86.01%, H 8.42%, N 5.57%, found C
3
. Results
8
5.81%, H 8.31%, N 5.52%.
3
.1. Aminostilbenes 5a–f
0
2
.2.16. trans-4 -tert-Butyl-4-aminostilbene
1
Off-white precipitate, H NMR (300 MHz, CDCl3):
The Ames-test results of the compounds 5a–f are
3
δ (ppm) = 7.39 − 7.28 (m, 6H), 6.96 (d, J =
listed in Table 1. As typically found for amines, with-
out metabolic activation the numbers of revertants do
not differ from the solvent controls. A graphical com-
parison of experimental mutagenicities obtained in
the presence of S9-mix is given in Fig. 4. The com-
pounds in Fig. 4 are ordered by growing steric demand
of the substituents: methyl < ethyl < iso-propyl <
sec-butyl < tert-butyl.
3
1
6.3 Hz, 1H), 6.87 (d, J = 16.3 Hz, 1H), 6.64 (d,
3
J = 8.5 Hz, 2H), 3.32 (s, 2H, NH2), 1.29 (s, 9H).
1
3
C NMR (75 MHz, CDCl3): δ (ppm) = 150.0, 145.8,
1
3
2
35.2, 128.4, 127.9, 127.6, 125.8, 125.5, 125.1, 115.3,
+
4.6, 31.3. MS (70 eV): m/z (%) = 251 (M , 100),
+
36 (C17H18N , 66). HRMS (70 eV): m/z calculated
for C18H21N 251.1674, found 251.1678.
2.3. Mutagenicity tests
The mutagenicity of all compounds was determined
using the Ames-test with the Salmonella typhimurium
strains TA98(ꢀS9) and TA100(ꢀS9). All compounds
were tested in the Hygiene-Institut, Universität Hei-
delberg, Germany. The Ames-test protocol employed
in these investigations followed the standard method-
ology described by Baron and Ames [24]. Both tester
strains (TA98 and TA100) were a gift from Bruce
Ames.
In all cases DMSO was used as the diluting sol-
vent. Positive and negative controls (DMSO) were
included in each experiment. The test results shown
here were obtained using metabolic activation with
Fig. 4. Experimental mutagenicity of aminostilbenes 5a–f with S9.

B. Ludolph et al. / Mutation Research 491 (2001) 195–209
201
Table 1
Experimental mutagenicity data of aminostilbenes 5a–f
Code
Dose (␮g/plate)
Revertants, induction factors
TA98−S9
TA98+S9
TA100−S9
TA100+S9
Positive controla
Negative control
3017
35
35
34
30
39
37
49
35
33
49
43
38
39
35
34
36
34
33
32
35
40
33
36
27
32
35
38
41
28
37
29
35
33
36
30
33
27
1311
60
60
1032
102
102
101
117
129
134
182
102
100
102
98
113
121
102
95
106
108
117
99
102
87
102
113
101
89
102
105
94
1740
102
102
189
265
435
848
469
102
265
355
496
775
597
102
154
265
476
651
594
102
227
332
641
610
458
102
118
112
114
119
107
102
162
213
295
319
211
DMSO
0
5a
5b
5c
5d
5e
5f
1.0
1.0
0.9
1.1
1.1
1.4
1.0
0.9
1.4
1.3
1.1
1.1
1.0
1.0
1.0
1.0
0.9
0.9
1.0
1.1
0.9
1.0
0.8
0.9
1.0
1.1
1.2
0.8
1.1
0.8
1.0
1.0
1.0
0.9
1.0
0.8
1.0
1.5
1.7
3.3
5.4
7.8
1.0
2.7
4.7
6.3
9.0
10.6
1.0
2.8
5.3
7.7
9.1
9.2
1.0
2.5
4.2
7.4
6.4
6.6
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.7
1.9
2.6
2.5
2.1
1.0
1.0
1.1
1.3
1.3
1.8
1.0
1.0
1.0
1.0
1.1
1.2
1.0
0.9
1.0
1.1
1.1
1.0
1.0
0.9
1.0
1.1
1.0
0.9
1.0
1.0
0.9
0.9
0.9
0.9
1.0
1.0
0.9
0.9
0.9
0.8
1.0
6
.25
92
1.8
2.6
4.3
8.3
4.6
1.0
2.6
3.5
4.9
7.6
5.9
1.0
1.5
2.6
4.7
6.4
5.8
1.0
2.2
3.3
6.3
6.0
4.5
1.0
1.2
1.1
1.1
1.2
1.0
1.0
1.6
2.1
2.9
3.1
2.1
1
2
5
2.5
5
0
104
200
328
469
60
160
283
383
541
642
60
172
322
465
546
557
60
149
255
445
387
396
60
51
58
59
61
57
60
104
114
159
151
130
b
b
b
b
1
1
1
1
1
1
00
0
6
.25
.25
.25
.25
.25
1
2
5
2.5
5
0
00
0
6
b
1
2
5
2.5
5
0
b
00
0
6
1
2
5
2.5
5
0
b
b
00
0
6
1
2
5
2.5
5
0
96
92
94
102
99
96
93
93
77
00
0
6
b
1
2
5
2.5
5
0
b
00
a
TA98−S9 1-nitropyren 2.5 ␮g; TA100−S9 sodium azide 5 ␮g; TA98+S9 and TA100+S9 2-aminoanthracene 2.5 ␮g.
b
Indicates the highest dose used in regression analysis.
In TA98+S9 the mutagenic potential increases from
the sterically even more demanding tert-butyl com-
pound 5e. The compound 5e is non-mutagenic at all
concentrations. As shown in Fig. 4 the mutagenicity
in TA100+S9 is within the same range as in strain
TA98+S9. In TA100+S9 also a similar trend in the
mutagenicity is observed. Again the activity of the
sec-butyl compound 5f and the tert-butyl compound
5e is lower than those of all other 4-aminostilbenes.
the parent compound 5a to the ethyl-substituted com-
pound 5c, which is strongly mutagen. In contrast ac-
tivity drops continuously from 5c to 5e with increasing
steric demand of the substituents. While the number
of revertants exceeds the solvent control up to 1.5-fold
for the sec-butyl compound 5f, no revertants above the
solvent controls and no cell toxicity are observed for

2
02
B. Ludolph et al. / Mutation Research 491 (2001) 195–209
Table 2
Experimental mutagenicity data of nitrostilbenes 6a–f
Code
Dose (␮g/plate)
Revertants, induction factors
TA98−S9
TA98+S9
TA100−S9
TA100+S9
Positive controla
Negative control
2450
36
36
55
76
118
162
183
209
36
54
59
86
101
105
118
36
–
41
59
58
76
78
142
36
–
41
40
48
46
41
61
36
–
36
41
36
40
44
52
36
–
1661
47
47
90
92
1206
176
176
308
460
774
625
0
1830
130
130
125
124
148
195
411
773
130
493
631
945
1062
0
DMSO
0
6a
6b
6c
1.0
1.6
2.1
3.3
4.5
4.9
5.0
1.0
1.5
1.7
2.4
2.8
2.9
3.3
1.0
–
1.1
1.6
1.6
2.1
2.2
4.0
1.0
–
1.1
1.1
1.3
1.3
1.1
1.7
1.0
–
1.0
1.1
1.0
1.1
1.2
1.5
1.0
–
1.0
1.9
2.0
2.0
2.2
2.9
4.8
1.0
5.0
5.7
7.3
12.5
16.5
5.4
1.0
9.3
11.5
14.5
20.9
22.7
24.4
–
1.0
2.0
2.0
2.4
3.6
5.5
11.4
–
1.0
2.9
3.0
3.4
4.5
5.2
6.3
–
1.0
1.8
2.6
4.4
3.5
0.0
0.0
1.0
1.7
2.0
2.7
2.8
3.0
3.0
1.0
–
1.4
1.7
1.7
1.8
1.8
2.3
1.0
–
1.3
1.4
1.4
1.4
1.4
1.5
1.0
–
1.0
1.2
1.5
1.5
1.5
1.5
1.0
–
1.0
1.0
1.0
1.1
1.5
3.2
6.0
1.0
3.8
4.9
7.3
8.2
0.0
0.0
1.0
3.3
5.1
8.9
4.8
0.0
0.0
–
1
3
6
.6
.1
.25
b
b
b
95
b
1
2
5
2.5
5
0
105
136
225
47
235
267
344
585
774
254
47
435
541
681
981
1069
1145
–
47
94
94
111
171
259
536
–
b
b
0
0
176
301
359
471
491
522
520
176
–
238
298
299
312
315
412
176
–
234
245
248
249
249
265
176
–
183
213
273
270
260
259
176
–
1
3
6
.6
.1
.25
b
b
1
2
5
2.5
5
0
0
0
130
433
668
1150
619
0
1
3
6
.6
.1
.25
b
b
1
2
5
2.5
5
0
b
0
–
1
1
1
1
00
0
1
3
6
6
6
d
130
191
226
358
788
1156
375
–
130
365
589
795
909
923
973
–
130
394
511
857
1022
1125
873
–
1.0
1.5
1.7
2.8
6.1
8.9
2.9
–
.6
.1
.25
b
6
b
1
2
5
2.5
5
0
b
00
0
1
3
6
e
47
1.0
2.8
4.5
6.1
7.0
7.1
7.2
–
.6
.1
.25
135
140
160
212
246
298
–
b
b
b
1
2
5
2.5
5
0
00
0
0
e
47
1.0
2.6
3.1
4.0
4.4
5.2
6.2
–
1.0
3.0
3.9
6.6
7.9
8.7
6.7
–
1
.6
124
145
188
205
243
291
–
3
.1
37
41
43
42
43
48
1.0
1.1
1.2
1.2
1.2
1.3
237
267
285
240
236
236
1.3
1.5
1.6
1.4
1.3
1.3
b
6
.25
b
b
1
2
5
2.5
5
0
00

B. Ludolph et al. / Mutation Research 491 (2001) 195–209
203
Table 2 (Continued)
Code Dose (␮g/plate)
Revertants, induction factors
TA98−S9
TA98+S9
TA100−S9
TA100+S9
6
f
0
1
3
6
36
–
1.0
–
47
74
74
119
126
196
246
–
1.0
1.6
1.6
2.5
2.7
4.2
5.2
–
176
–
1.0
–
130
170
208
264
389
686
897
–
1.0
.6
.1
.25
1.3
1.6
2.0
3.0
5.3
6.9
37
37
42
53
76
90
1.0
1.0
1.2
1.5
2.1
2.5
226
230
233
254
275
253
1.3
1.3
1.3
1.4
1.6
1.4
b
1
2
5
2.5
5
0
b
b
1
00
a
TA98−S9 1-nitropyren 2.5 ␮g; TA100−S9 sodium azide 5 ␮g; TA98+S9 and TA100+S9 2-aminoanthracene 2.5 ␮g.
b
Indicates the highest dose used in regression analysis.
3
.2. Nitrostilbenes 6a–f
the tert-butyl-nitrostilbene containing 5% cis-product
0
6
e and the pure tert-butyl-nitrostilbene 6e . In TA98
0
The revertant numbers of the nitrostilbene series
a–f in the presence and in the absence of S9-mix are
the number of revertants of 6e and 6e remain below
twice the solvent control at all concentrations (see Ta-
ble 2) but exceed the solvent control up to 2.5-fold in
case of the sec-butyl-substituted compound 6f at a dose
6
summarized in Table 2.
0
3
.2.1. Absence of S9-mix
of 100 ␮g. In TA100 the revertants of 6d, 6e, 6e and 6f
A graphical comparison of the mutagenicities of
do not reach twice the solvent control at any concen-
tration tested. In the absence of S9-mix all these com-
pounds must be classified as weakly or non-mutagenic
according to the definition of Ames [24].
compounds 6a–f without S9-mix is shown in Fig. 5. In
both Salmonella strains the parent compound 6a and
the methyl-substituted 6b derivative are highly muta-
genic. The activity of 6a and 6b is in TA100 markedly
higher than in TA98, in good agreement with former
results of Hoobermann [16]. Concerning the influence
of the alkyl substituents in both strains a continuous
decrease of mutagenicity is observed from the parent
compound 6a to the tert-butyl-substituted nitrostilbene
3.2.2. Presence of S9-mix
In the presence of S9-mix a strong increase of muta-
genicity in both strains is observed for all compounds
if compared with the same test series in the absence
of S9-mix (see Table 2). In Fig. 6 the mutagenic-
ity of the compounds 6a–f with S9-mix is shown.
In strain TA98 the mutagenicity increases from the
6
e. Notably there is no significant difference between
Fig. 5. Experimental mutagenicity of nitrostilbenes 6a–f without
S9.
Fig. 6. Experimental mutagenicity of nitrostilbenes 6a–f with S9.

2
04
B. Ludolph et al. / Mutation Research 491 (2001) 195–209
parent compound 6a to the ethyl-substituted deriva-
tive 6c. Similar to the aminostilbene series larger alkyl
groups generally reduce the mutagenicity with the
size of the attached substituent. Compounds 6d–f are
markedly less active than the corresponding deriva-
tives bearing smaller alkyl groups. Compared to the
sec-butyl-nitrostilbene 6f a slightly enhanced activity
properties like hydrophobicity (log P) and energies
of the frontier orbitals (EHOMO, ELUMO) [25,26].
Based on these parameters Debnath and Hansch de-
veloped empirical equations that allow to predict the
mutagenicity of aminoarenes [9] (in the presence of
S9-mix) and nitroarenes (in the absence of S9-mix)
in TA98 [10] and TA100 [11]. The influence of log P
and orbital energies on the processes underlying
mutagenesis has been discussed by several authors
[9–11,25,26]. In general it is assumed that electronic
factors determine whether the mutagen is active or
not whereas the log P of a certain mutagen correlates
with the magnitude of its mutagenic potency [25].
Applying the Debnath/Hansch model to predict the
mutagenicity of N-substituted stilbenes in quantitative
terms one has to be careful due to the special reactivity
of stilbenes with the DNA via the double bond (see
Fig. 2). It has to be noted further that some of the
alkyl-substituted 4-aminostilbenes investigated in this
work have a higher hydrophobicity than those that
were used in their QSAR study.
0
for the tert-butyl-nitrostilbenes 6e and 6e is found. In
strain TA100 the same overall trend is followed but
the differences in activity between the compounds are
much larger. Maximum activity again is reached for
the ethyl nitrostilbene 6c, followed by a decrease from
6
c to 6f. Remarkably the tert-butyl-substituted com-
0
pounds 6e and 6e exhibit an exceptional behavior un-
der these conditions. Instead of being non-mutagenic,
0
6
e and 6e are highly active. Apart from this exper-
iment we have never observed a similar effect for
any other tert-butyl compound. As in the test series
without S9-mix one finds only a minor difference
0
between the cis-containing 4 -tert-butyl-4-nitrostilbene
6
0
e and the pure trans-compound 6e in both
strains TA98 and TA100. This finding suggests that
0
cis-4 -tert-butyl-4-nitrostilbene is just as, or less,
4.1. Aminostilbenes 5a–f
0
mutagenic than trans-4 -tert-butyl-4-nitrostilbene.
Further investigations using pure cis-4-nitrostilbene
derivatives have to clarify this point.
The mutagenicities of the aminostilbenes 5a–f
according to the Debnath/Hansch model are listed
in Table 3. They are compared graphically with our
experimental results in Fig. 7.
4
. Discussion
The log P values and the electronic energies were
calculated as previously described [27]. In both strains
the mutagenicity is predicted to increase continuously
with the size of the alkyl group. Orbital energy cal-
culations for the aminostilbenes 5a–f revealed that
In the last two decades QSAR studies have revealed
a number of chemical and biological factors that af-
fect mutagenicity, including structural and chemical
Table 3
Experimental and predicted mutagenicity of aminostilbenes 5a–f
Code log Pa
EHOMO
ELUMO
σ
+
Log revertants/nmol
TA98+S9
TA100+S9
Experimental Sinsheimer’s Debnath/Hansch’s Experimental Debnath/Hansch’s
prediction
prediction
prediction
5a
5b
5c
5d
5e
5f
3.61
4.15
4.65
5.06
5.52
5.55
−8.017 −0.351
0.00
0.02
0.57
0.67
0.75
0.47
0.48
0.50
0.50
0.51
1.09
1.74
2.27
2.69
3.31
3.24
0.47
0.63
0.58
1.71
2.26
2.71
3.05
3.50
3.53
−7.963 −0.344 −0.31
−7.967 −0.341 −0.30
−7.977 −0.323 −0.28
7.958 −0.325 −0.27
7.967 −0.338 −0.26
0.57
0.71
b
−1.47
−1.09
−0.03
0.35
a
Log P values calculated with KOWWIN.
Value extrapolated.
b

B. Ludolph et al. / Mutation Research 491 (2001) 195–209
205
Fig. 7. Experimental and predicted mutagenicity of aminostilbenes 5a–f.
alkyl substituents exert only a minor influence on the
HOMO and LUMO energies (see Table 3), ruling out
that electronic factors cause the predicted increase
of mutagenic activity. An analysis of the parame-
ters used to calculate the mutagenicity indicated that
hydrophobicity almost exclusively accounts for the
predicted increase of mutagenicity in the aminostil-
bene series investigated in this work. For the first
compounds of the test series (5a–c) the experimen-
tal data agree decently well with the prediction (see
Fig. 7). However, regarding the derivatives with
larger alkyl groups (5d–f), the deviations between
experimental and predicted mutagenicities become
larger, growing with the size of the substituent. Hy-
drophobicity alone, clearly does not correlate with the
experimental results as shown in Table 3. Although
the tert-butyl-substituted 5e is approximately as hy-
drophobic as the sec-butyl-substituted 5f, 5e is less
active than 5f. Likewise 5f is more hydrophobic than
the higher mutagenic iso-propyl-substituted 5d.
values are very similar for different alkyl groups,
Sinsheimer’s model essentially predicts no significant
change in mutagenicity for the differently alkylated
compounds. For 5b–d with small alkyl groups these
predictions agree very well with the experimental
results in absolute numbers, whereas neither the ac-
tivity increase from 5a to 5c nor the activity drop for
5e–f is predicted correctly. Summarizing these com-
parisons, both models are unsuitable to predict the
low mutagenicity of compounds bearing large alkyl
substituents. Thus, in addition to orbital energies,
+
hydrophobicity and Hammet coefficients σ , other
parameters should be considered in order to explain
the experimental activities of 5e and 5f.
Apart from the parameters discussed above com-
pounds 5a–f strongly differ in the steric demand of
their substituents. Since the mutagenicity decreases
with the size of the attached alkyl groups we conclude
that the deviations between prediction and experiment
must be attributed mainly to steric factors. This con-
clusion is in accordance with our previous findings
for the alkyl-substituted amino- and nitrobiphenyls
(1a–f, 2a–f) and nitrofluorenes (3a, b, e). As dis-
cussed for these compounds [8] most probably the
reduced ability of the sterically demanding aminos-
tilbenes to intercalate well into DNA accounts for the
reduced mutagenicity.
In 1994 Sinsheimer developed a QSAR equation
0
describing the mutagenic activity of 4 -substituted
4
-aminostilbenes in TA98+S9 based on the Hammet
+
coefficients σ of the attached substituents [14]. We
also applied this rather simple QSAR model to the
aminostilbenes 5a–f as shown in Table 3 and Fig. 7.
+
+
The σ values were taken from [28]. Since the σ

2
06
B. Ludolph et al. / Mutation Research 491 (2001) 195–209
Table 4
Experimental and predicted mutagenicity of nitrostilbenes 6a–f
Code
log Pa
ELUMO
Log revertants/nmol
TA98−S9
TA100−S9
Experimental
Debnath/Hansch’s prediction
Experimental
Debnath/Hansch’s prediction
6
6
6
6
6
6
6
a
b
c
4.2
−1.356
−1.339
−1.333
−1.332
−1.329
−1.329
−1.333
0.47
0.26
0.39
0.57
0.52
1.34
1.03
0.38
0.00
0.36
0.36
0.00
1.38
1.90
2.19
2.18
1.79
1.79
1.74
4.75
5.24
5.66
6.11
6.11
6.15
−0.42
−1.00
−1.52
−1.40
−0.64
d
e
0.21
−0.44
−0.44
−0.50
0
e
f
a
Log P values calculated with KOWWIN.
4
.2. Nitrostilbenes 6a–f
For TA98 and TA100 the QSAR model predicts
first an increase of the mutagenic activity with in-
creasing size of the substituents, a maximum around
those substances with a log P value of 4.9 (TA98)
or 5.4 (TA100), followed by a decrease for the
more hydrophobic sec-butyl and tert-butyl-substituted
nitrostilbenes 6d–f. Since electronic energies are not
influenced significantly by different alkyl substituents,
the predicted trend is, as for the aminostilbenes,
mainly determined by the increasing hydrophobicity
of the molecules. In Debnath/Hansch’s QSAR model
The nitrostilbenes 6a–f tested without S9-mix
showed a continuous decrease of the activity with
the size of the alkyl substituents in both Salmonella
strains. Only in TA100−S9 the tert-butyl compounds
0
6e and 6e are found to be slightly more active than
the preceding compounds. In Table 4 the experimental
and predicted values according to Debnath/Hansch’s
QSAR model are compared. A graphical comparison
is given in Fig. 8 for both strains.
Fig. 8. Experimental and predicted mutagenicity of nitrostilbenes 6a–f.

B. Ludolph et al. / Mutation Research 491 (2001) 195–209
207
mutagenic activity of nitroarenes is bilinearly depen-
dent on the hydrophobicity and causes a reduction of
mutagenicity for compounds with log P values exceed-
ing the optimum. In fact this decrease of mutagenic
activity for very hydrophobic compounds as 6d–f is
corroborated by our experimental results. Neverthe-
less there are still large gaps between the curves in
Fig. 8 (more so in TA100 than in TA98) confirming
additional effects to be important here as well. Our
investigations suggest that mainly steric effects cause
these deviations. As can be seen from the striking dif-
ferences between predicted and experimental results
especially in TA100, even for sterically less demand-
ing compounds like 6b and 6c the Debnath/Hansch
model seems to be generally less suitable to predict
mutagenicity of nitrostilbene derivatives in TA100 cor-
rectly. As mentioned before, this may be due to the
special reactivity of nitrostilbenes via the double bond.
Apart from a recent CoMFA study, which consid-
ers mutagenicities of nitroarenes in TA98(ꢀS9) and
TA100(ꢀS9) [29], there exists no simple 2D-QSAR
model for 4-nitrostilbenes tested with metabolic acti-
vation. The overall experimental trends (see Fig. 6) in
the presence of S9-mix are comparable to the results
obtained for the aminostilbenes in both Salmonella
strains (see Fig. 4). Again the trend for the nitrostil-
benes turns out to be determined by hydrophobicity
and steric demand of the substituents as this is the
case for the aminostilbenes. However, striking excep-
benzylic activation is also supported by the findings of
Hanna who described that P450 mediated metaboliza-
0
tion of N-acetyl-4-aminostilbenes bearing a 4 -methyl
0
or 4 -tert-butyl substituent takes place at the alkyl
0
groups predominately. In the case of 4 -tert-butyl-N-
acetyl-4-aminostilbene oxidation occurred almost
exclusively at the aliphatic alkyl group producing
a primary alcohol, and not at the N-acetylamino
group. Thus, we suggest a different metabolic path-
way to be operative in the presence of S9-mix,
which does not involve the hydroxylamine as the
important intermediate. Since no metabolic studies
0
on 4 -alkyl-substituted 4-nitrostilbenes have been
performed so far we cannot explain doubtlessly the
0
unexpected high mutagenicity of 4 -tert-butyl-4-
nitrostilbene 6e at the moment. This area has to be
the subject of further research.
A definite reason for the reduced mutagenic poten-
tial of the stilbenes with sterically demanding alkyl
groups (like iso-propyl or tert-butyl) can neither be
given. Concerning the molecular shape it has been
demonstrated that a flat aromatic system, capable to
intercalate in DNA, is an important structural feature
for a molecule to react as frameshift mutagen [30].
We discussed this topic recently to explain the steric
effect of the alkyl-substituted nitrobiphenyls 2a–f and
nitrofluorenes 3a–f [8]. Since adducts of N-substituted
stilbenes are quite different (see Fig. 2) from adducts
of, e.g. amino- or nitrobiphenyls, it is not clear
whether a similar explanation holds here, too. Apart
from intercalation into DNA the stilbenes’ bulky
alkyl groups may also interfere with its binding to the
N-esterification enzymes, which is essential to exhibit
the mutagenic potential. Experimentally, however,
it is obvious that sterically demanding alkyl groups
reduce the mutagenicity of amino- and nitrostilbenes.
0
0
tions are the 4 -tert-butyl-4-nitrostilbenes 6e and 6e
in TA100+S9, which are much more mutagenic than
the sec-butyl- and iso-propyl-substituted compounds
6f and 6d. It is therefore obvious that the mutagenic
activity of the tert-butyl compound 6e in TA100+S9
cannot be determined exclusively by the factors al-
ready discussed (hydrophobicity, electronic energies
and steric demand). This exceptional behavior points
to another factor. The strong increase of mutagenicity
of all nitrostilbenes tested in the presence of S9 may be
caused by an additional oxidative activation process.
In principal this activation can occur at the benzylic
position of the alkyl groups or at the double bond
instead of the nitrogen atom. Indeed, Hoobermann
5. Conclusion
Summarizing the results, it turns out that alkyl
groups that are located far away from the func-
tional groups –NH2 and –NO2, respectively, exhibit a
size-dependent effect on the mutagenicity of amino-
and nitrostilbenes (5a–f, 6a–f). Therefore, these
results confirm the effects we found recently for the
amino- 1a–f and nitrobiphenyls 2a–f [8] and nitroflu-
orenes 3a–f [8]. Based on the results of this and our
0
showed that mutagenicity of 4 -methyl-4-nitrostilbene
6
b in the nitroreductase deficient strains TA98NR,
with S9-mix added, is much stronger than for
-nitrostilbene 6a [16]. This activation is not believed
to proceed via the corresponding hydroxylamine. A
4

2
08
B. Ludolph et al. / Mutation Research 491 (2001) 195–209
recent work, the strategy to reduce the mutagenicity by
changing the molecular shape seems to be of promis-
ing general applicability for N-substituted aromatic
compounds. However, there are limitations. Apart
from steric considerations, the mutagenic potential is
also influenced by several other parameters like
hydrophobicity, and experimentally observed muta-
genicity is the result of various factors. A striking
example for an “exception from the rule” is the
unexpected high mutagenicity of the tert-butyl ni-
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[
0
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Acknowledgements
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