From mutagenic to non-mutagenic nitroarenes: Effect of bulky alkyl substituents on the mutagenic activity of nitroaromatics in Salmonella typhimurium. Part II. Substituents far away from the nitro group

Article abstract of DOI:10.1016/S1383-5718(00)00013-9

Derivatives of 4-nitrobiphenyl, 4-nitrosobiphenyl, 2-phenyl-5-nitropyridine (2-aza-4-nitrobiphenyl) and 2-nitrofluorene, bearing various alkyl substituents far away from the nitro group (4'-position in nitrobiphenyls, 7-position in 2-nitrofluorenes) were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. In the absence of S9 in both strains, mutagenicity of all compounds decreased with increasing steric demand of the attached alkyl groups as, e.g., for the 4-nitrobiphenyl series in the order 4'-H>4'-Me>4'-Et>4'-nBu>4'-iPr>4'-tBu>4'-Ad (Ad=adamantyl). Changes of the molecular shape from 'planar' to non-planar caused by the bulk of the introduced substituents (without influencing the twisting of the nitro substituent or the phenyl rings in the biphenyl compounds) may be responsible for this effect by interfering with an efficient intercalation into DNA.A comparison between experimental and theoretical values as calculated from recently developed equations (QSAR) confirmed our previous results (see the preceding paper) that mutagenicity of alkyl-substituted nitroaromatics cannot be predicted by hydrophobicity and LUMO-energies alone without including steric parameters. Copyright (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S1383-5718(00)00013-9

Mutation Research 467 Ž2000. 69–82
www.elsevier.comrlocatergentox
Community address: www.elsevier.comrlocatermutres
From mutagenic to non-mutagenic nitroarenes: effect of bulky
alkyl substituents on the mutagenic activity of nitroaromatics in
Salmonella typhimurium
Part II. Substituents far away from the nitro group
a
b
a,)
Markus Klein , Lothar Erdinger , Gernot Boche
a
Fachbereich Chemie, Philipps-UniÕersit a¨ t Marburg, Hans-Meerwein Straße, 35032 Marburg, Germany
b
Hygieneinstitut Heidelberg, In Neuenheimer Feld, 69120 Heidelberg, Germany
Received 27 October 1999; received in revised form 18 January 2000; accepted 25 January 2000
Abstract
Derivatives of 4-nitrobiphenyl, 4-nitrosobiphenyl, 2-phenyl-5-nitropyridine Ž2-aza-4-nitrobiphenyl. and 2-nitrofluorene,
X
bearing various alkyl substituents far away from the nitro group Ž4 -position in nitrobiphenyls, 7-position in 2-nitrofluorenes.
were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. In the absence
of S9 in both strains, mutagenicity of all compounds decreased with increasing steric demand of the attached alkyl groups
X
X
X
X
X
X
X
as, e.g., for the 4-nitrobiphenyl series in the order 4 -H)4 -Me)4 -Et)4 -nBu)4 -iPr)4 -tBu)4 -Ad ŽAdsadamantyl..
Changes of the molecular shape from ‘planar’ to non-planar caused by the bulk of the introduced substituents Žwithout
influencing the twisting of the nitro substituent or the phenyl rings in the biphenyl compounds. may be responsible for this
effect by interfering with an efficient intercalation into DNA.
A comparison between experimental and theoretical values as calculated from recently developed equations ŽQSAR.
confirmed our previous results Žsee the preceding paper. that mutagenicity of alkyl-substituted nitroaromatics cannot be
predicted by hydrophobicity and LUMO-energies alone without including steric parameters. q 2000 Elsevier Science B.V.
All rights reserved.
Keywords: Salmonella; Nitroaromatics; 4-Nitrobiphenyl; 2-Nitrofluorene; Nitroso; Alkyl substituents; Para; Mutagenicity; QSAR
1
. Introduction
in industry and research, nowadays their use is re-
stricted because of their well known mutagenic and
carcinogenic properties w1,2x. The same is true for
several azo compounds that are widely used as dyes,
since these compounds can be metabolically cleaved
into the corresponding amines w3,4x. Some attempts
have been undertaken to find general strategies to
reduce the mutagenic potential of these N-substituted
aromatics w5x.
Although nitroaromatics and related compounds
as aromatic amines, hydroxylamines and nitroso
compounds are important products and intermediates
)
Corresponding author. Tel.: q49-6421-282-2030; fax: q49-
6
1
421-282-8917.
E-mail address: boche@chemie.uni-marburg.de ŽG. Boche..
383-5718r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S1383-5718Ž00.00013-9

7
0
M. Klein et al.rMutation Research 467 (2000) 69–82
It is well established that metabolic transforma-
oped empirical equations to predict mutagenicity, it
is known that in the Ames test mutagenicity of
aromatic amines ŽTA98 and TA100 w6x. and nitro
compounds ŽTA98 w7x, TA100 w8x. is generally corre-
lated with electronic energies and especially hy-
drophobicity. Introduction of very hydrophilic sul-
fonic acid substituents, for example, was shown to
suppress the carcinogenic and mutagenic properties
of benzidine, aminobiphenyl and N,N-dimethyl-
tion into electrophilic intermediates Žhydroxyl-
amines, nitrenium ions., that form DNA-adducts, is
necessary for these class of compounds to develop
their genotoxic properties. Thus, useful ways to re-
duce mutagenic responses should be to inhibit this
activation process or to hinder the approach of the
reactive species to the DNA. From recent QSAR
investigations by Debnath and Hansch, who devel-
Fig. 1. Structures of synthesised compounds.

M. Klein et al.rMutation Research 467 (2000) 69–82
71
aminoazobenzene w5,9x. In this case, the decreased
Fig. 1, these substances include derivatives of 4-
nitrobiphenyl Žcompounds 1–11., two aza-analogues
of 4-nitrobiphenyl Žcompounds 12, 13. and deriva-
tives of 2-nitrofluorene Žcompounds 14–17.. The
mutagenicities of all compounds were examined in
the Ames test using TA98 and TA100. To investi-
gate whether the effects of para alkyl substituents
on the mutagenic activity can be modelled by hy-
drophobicity and ELUMO alone, we compared pre-
dicted and experimental data as introduced in the
preceding paper.
hydrophobicity may slow down the penetration of
the reactive intermediate through the cell wall or
may weaken the binding of the N-substituted muta-
gen to the activating enzymes.
Inspired by the observation that some methylated
amines w10x and nitroaromatics w11x were less muta-
genic than their unsubstituted parent compounds, we
investigated the influence of steric effects by alkyl
groups on the mutagenicity of nitroaromatics in the
preceding paper. By comparing the mutagenicities of
various alkyl-substituted derivatives of 4-nitro-
biphenyl, we found that introduction of more bulky
alkyl substituents than methyl in the ortho position
2
. Materials and methods
X
of the nitro group or in the 2 -position reduced
2
.1. Instrumentation
mutagenicity efficiently, or even eliminated it. In
contrast, the empirical QSAR equations mentioned
above, which do not explicitly include steric parame-
ters, predicted the mutagenicity of such derivatives
to be much higher. With regard to the ortho-sub-
stituted derivatives, the bulky alkyl groups forced the
nitro substituent to adopt a perpendicular orientation
and therefor inhibited the activation pathway. On the
NMR spectra were obtained with Bruker spec-
trometers ARX-200 and AC-300 and are referenced
to tetramethylsilane. Mass spectra were recorded with
Variant MAT CH-7-A ŽEI, 70 eV., Elementary anal-
ysis was done with Heraeus Rapid Elemen-
taranalysator.
X
other hand, alkyl substituents in the 2 -position desta-
2.2. Chemicals
bilised the reactive intermediates by interfering with
the resonance of the p-electrons in the biphenyl
system. However, since regioselective introduction
of bulky alkyl substituents ortho to a nitro group
often is difficult, and chemical properties of the nitro
functionality are changed by bulky neighbouring
substituents significantly Želectronically, sterically.,
4
-Nitrobiphenyl 1, 2-nitrofluorene 14 and starting
materials were purchased from Aldrich Chemical.
,5-Ditertbutylbromobenzene w13x and 4-adaman-
tylbromobenzene w20x were prepared according to
literature procedures. The alkyl-substituted nitro-
biphenyls 3, 6, 9 and 10 were synthesised in good
yields Ž80–95%. by palladium catalysed coupling of
3
this strategy to reduce mutagenicity may be of lim-
substituted benzene boronic acids with 4-bromo-
nitrobenzene using the Suzuki protocol w14x as de-
scribed in the preceding paper w15x. The boronic
acids were available by standard methods w16x. For
the preparation of compounds 2, 4, 5, 7, 8 and 11
instead of the acids benzene boronic esters were used
that were generated in situ from the corresponding
bromobenzenes by halide–metal exchange with BuLi
followed by treatment with triisopropyl borate w17x.
ited applicability.
In addition to the QSAR approach by Debnath
and Hansch, a more recent Comparative Molecular
Field Analysis ŽCoMFA. of mutagenicity of ni-
troaromatics by Fan et al. w12x indicated that the
mutagenicity should decrease by increasing the bulk
perpendicular to the aromatic plane. What happens if
alkyl substituents are introduced in positions that
neither affect the nitro group nor the p-system di-
rectly but change the molecular shape from flat to
three-dimensional? Is there any steric effect or does
mutagenicity increase with growing hydrophobicity?
We have synthesised alkyl-substituted nitroaro-
matics in which the additional alkyl group Žand in
one example a phenyl group. is located far away
Overall, yields were slightly lower than for the two
step Suzuki procedure indicated above Ž58–86%.
ŽScheme 1..
from, or opposite to the nitro group. As shown in
Scheme 1.

7
2
M. Klein et al.rMutation Research 467 (2000) 69–82
the mixture was poured onto water and extracted
with ether Ž3=150 ml.. The organic layer was
separated, washed with brine, dried ŽMgSO . and
4
Scheme 2.
evaporated to dryness in vacuo. Column chromatog-
raphy Žpetrol etherrethyl acetate 7:1. afforded 2
Ž2.27 g, 71%. which was recrystallised from hexane.
Colourless crystals, mp 142 8C ŽRef. w21x 140..
Since Suzuki coupling of benzeneboronic acids
1
3
with 2-bromo-5-nitropyridin was not successful due
to the instability of the bromide under basic aqueous
conditions, the heterocyclic compounds 12 and 13
were prepared by palladium catalysed coupling of
zinc aromatics with 2-bromo-5-nitropyridin in THF
at room temperature w18x. In spite of sustained efforts
using modified catalyst systems yields remained
moderate Ž45–60%. ŽScheme 2..
The nitroso compounds 1a and 5a were prepared
as described in the preceding paper from the corre-
sponding nitro compounds by reduction to the amines
and reoxidation using the peroxy molybdenum com-
H NMR ŽCDCl , 200 MHz. ds8.26 Žd, 2H, Js
3
3
8.9 Hz, H3., 7.71 Žd, 2H, Js8.9 Hz, H2., 7.51 Žd,
3
X
3
2H, Js8.2 Hz, H2 ., 7.29 Žd, 2H, Js8.2 Hz,
X
13
H3 ., 2.42 Žs, 3H, –CH ,.. C NMR ŽCDCl , 50
3
3
MHz. ds147.5, 146.5, 139.0, 135.0, 129.8, 127.4,
127.1, 124.0, 21.1. MS Ž70 eV.: mrz Ž%.s213
q
q
ŽM , 100., 165 Ž42., 152 ŽM –NO –CH , 65..
2 3
Calc. C 73.23%, H 5.20%, N 6.57% found C 73.35%,
H 5.14%, N 6.52%.
X
2
.2.2. 4 -Ethyl-4-nitrobiphenyl (3)
1
Pale yellow crystals, mp 848C. H NMR ŽCDCl ,
3
plex MoŽO.ŽO . ŽH O.ŽHMPA. w19x. The fluorene
3
2
2
2
. Ž .
2
00 MHz ds8.26 d, 2H, Js8.9 Hz, H3 , 7.72
derivatives 15–17 were prepared by a regiospecific
reaction sequence of coupling, reduction and ring
closure as previously shown w20x.
3
3
Žd, 2H, Js8.9 Hz, H2., 7.54 Žd, 2H, Js8.4 Hz,
X
3
X
H2 ., 7.32 Žd, 2H, Js8.4 Hz, H3 ., 2.72 Žq, 2H,
3
3
Js7.5 Hz, –CH ., 1.28 Žt, 3H, Js7.5 Hz,
2
All synthesised compounds were purified by re-
crystallisation andror repeated column chromatogra-
phy to yield high purities. Purities were confirmed
by elemental analysis and gas chromatography. Syn-
thetic procedures and analytical data are given be-
low.
13
–
1
1
CH ,.. C NMR ŽCDCl , 50 MHz. ds147.5,
3
3
46.7, 145.3, 136.0, 128.6, 127.4, 127.2, 124.0, 28.5,
q
5.4. MS Ž70 eV.: mrz Ž%.s227 ŽM , 90., 212
q
q
ŽM –CH , 100., 165 Ž43., 152 ŽM –NO –CH ,
3
2
3
2
5.. Calc. C 73.99%, H 5.77%, N 6.16% found C
7
3.97%, H 5.61%, N 6.18%.
X
2
.2.1. 4 Methyl-4-nitrobiphenyl (2); typical proce-
X
2
.2.3. 4 -isoPropyl-4-nitrobiphenyl (4)
dure
Halide–metal exchange was carried out by addi-
1
Pale yellow crystals, mp 83–848C. H NMR
3
ŽCDCl , 300 MHz. ds8.26 Žd, 2H, Js8.9 Hz,
tion of t-BuLi Ž1.5 M in pentane, 22.7 ml, 34 mmol.
3
3
H3., 7.71 Žd, 2H, Js8.9 Hz, H2., 7.55 Žd, 2H,
with a syringe to a solution of 4-bromotoluene Ž2.89
3
X
3
X
Js8.3 Hz, H2 ., 7.34 Žd, 2H, Js8.3 Hz, H3 .,
g, 17 mmol. in Et O Ž20 ml. under argon at y788C.
2
3
2
.97 Žsept, 1H, Js6.9 Hz, –CH., 1.29 Žd, 6H,
After stirring for 1 h 25 ml THF were added, then
the mixture was cooled to y1008C, charged with
triisopropylborate Ž3.39 g, 18 mmol., stirred for 30
3
13
Js6.9 Hz, –CH .. C NMR ŽCDCl , 75 MHz.
3
3
ds150.0, 147.6, 146.9, 136.2, 127.5, 127.3, 127.2,
q
1
3
1
5
24.0, 33.9, 23.8. MS Ž70 eV.: mrz Ž%.s241 ŽM ,
min, warmed to room temperature while stirring for
q
q
6., 226 ŽM –CH , 100., 180 ŽM –NO –CH , 39.,
1
.5 additional hours, and finally quenched by addi-
3
2
3
65 Ž180-CH , 24.. Calc. C 74.67%, H 6.27%, N
tion of 10 ml degased water. Under reduced pressure
pentane and ether were partly removed. 3.03 g Ž15
mmol. 4-nitrobromobenzene, 7.09 g BaŽOH. P8H O
3
.81% found C 74.44%, H 5.85%, N 5.99%.
2
2
X
Ž22 mmol., 184 mg PdŽPPh . Ž0.34 mmol. and 30
2.2.4. 4 -tertButyl-4-nitrobiphenyl (5)
3
4
1
ml dimethoxyethane were added and the mixture was
refluxed until 4-nitrobromobenzene had disappeared
completely according to TLC Ž10 h.. After cooling
Colourless crystals, mp 1078C. H NMR ŽCDCl ,
3
3
300 MHz. ds8.27 Žd, 2H, Js8.8 Hz, H3., 7.72
3
3
Žd, 2H, Js8.8 Hz, H2., 7.55 Žd, 2H, Js8.6 Hz,

M. Klein et al.rMutation Research 467 (2000) 69–82
73
X
3
X
X
H2 ., 7.50 Žd, 2H, Js8.6 Hz, H3 ., 1.36 Žs, 9 H,
2.2.8. 3 -Methyl-4-nitrobiphenyl (9)
1
3
–
1
3
CŽCH . .. C NMR ŽCDCl , 75 MHz. ds152.2,
47.4, 146.8, 135.7, 127.4, 127.0, 126.1, 124.0, 34.6,
Pale yellow needles, mp 608C ŽRef. w22x 59–608C..
3
3
3
1
3
H NMR ŽCDCl , 300 MHz. ds8.27 Žd, 2H, Js
3
q
3
1.2. MS Ž70 eV.: mrz Ž%.s255 ŽM , 46., 240
8.9 Hz, H3., 7.71 Žd, 2H, Js8.9 Hz, H2., 7.43–7.34
q
q
ŽM –CH , 100., 212 ŽM –C H , 21., 165 Ž17..
Žm, 3H, H
., 7.27–7.22 Žm, 1H, H
., 2.43 Žs,
3
3
7
arom
arom
1
3
Calc. C 75.27%, H 6.71%, N 5.49% found C 75.09%,
H 6.52%, N 5.36%.
3H, –CH .. C NMR ŽCDCl , 75 MHz. ds147.9,
3
3
147.1, 138.9, 138.8, 129.7, 129.1, 128.2, 127.8,
1
24.6, 124.1, 21.6. MS Ž70 eV.: mrz Ž%.s213
q
q
q
X
ŽM , 95., 183 ŽM –NO, 46., 165 Ž71., 152 ŽM –
2
.2.5. 4 -nButyl-4-nitrobiphenyl (6)
1
NO –CH , 100.. Calc. C 73.22%, H 5.20%, N
Yellow solid, mp n.d. H NMR ŽCDCl , 300
2
3
3
3
6.57% found C 73.31%, H 5.14%, N 6.48%.
MHz. ds8.27 Žd, 2H, Js9.0 Hz, H3., 7.71 Žd,
3
3
2
H, Js9.0 Hz, H2., 7.53 Žd, 2H, Js8.2 Hz,
X
3
X
X
X
H2 ., 7.30 Žd, 2H, Js8.2 Hz, H3 ., 2.66 Žt, 2H,
2.2.9. 3 ,5 -Dimethyl-4-nitrobiphenyl (10)
3
3
1
Js7.6 Hz, 4NBp–CH ., 1.63 Žpen, Js7.6 Hz,
Pale yellow crystals, mp 1258C. H NMR ŽCDCl ,
2
3
3
3
2
H, 4NBp–CH –C H –., 1.38 Žsex, 2H, Js7.6
300 MHz. ds8.26 Žd, 2H, Js8.8 Hz, H3., 7.70
2
2
3
3
X
Hz, –C H –CH ., 0.94 Žt, 3H, Js7.6 Hz, –CH ..
Žd, 1H, Js8.8 Hz, H2., 7.21 Žs, 2H, H4 ., 7.08 Žs,
2
3
3
1
3
X
13
C NMR ŽCDCl , 75 MHz. ds147.6, 146.8, 144.1,
2H, H2 ., 2.39 Žs, 6 H,–CH .. C NMR ŽCDCl , 75
3 3
3
1
1
2
36.0, 129.2, 127.4, 127.2, 124.1, 35.3, 33.5, 22.3,
MHz. ds147.9, 146.9, 138.8, 130.6, 127.8, 125.3,
q
q
3.9. MS Ž70 eV.: mrz Ž%.s241 ŽM –CH , 35.,
124.0, 21.4. MS Ž70 eV.: mrz Ž%.s227 ŽM ,
2
q
q
q
26 Ž241–CH , 100., 180 Ž226–NO ., 152 ŽM –
100., 197 ŽM –NO, 4., 166 ŽM –NO ,–CH , 49.,
3
2
2 3
NO –C H , 45.. Calc. C 75.27%, H 6.71%, N
165 Ž36., Calc. C 73.99%, H 5.77%, N 6.16% found
2
4
9
5
.49% found C 75.06%, H 6.54%, N 5.52%.
C 73.79%, H 6.01%, N 6.00%.
X
X
X
2
.2.6. 4 -Adamantyl-4-nitrobiphenyl (7)
2.2.10. 3 ,5 -Ditertbutyl-4-nitrobiphenyl (11)
1
1
Yellow crystals, mp 194–1968C. H NMR
Colourless crystals, mp 106–1088C. H NMR
3
3
ŽCDCl , 300 MHz. ds8.27 Žd, 2H, Js8.9 Hz,
ŽCDCl , 200 MHz. ds8.29 Žd, 2H, Js8.7 Hz,
3
3
3
3
H3., 7.72 Žd, 1H, Js8.9 Hz, H2., 7.59 Žd, 2H,
H3., 7.73 Žd, 2H, Js8.7 Hz, H2., 7.53 Žt, 1H,
3
X
3
X
4
X
4
X
Js8.5 Hz, H2 ., 7.49 Žd, 1H, Js8.5 Hz, H3 .,
Js1.7 Hz, H4 ., 7.43 Žd, 2H, Js1.7 Hz, H2 .,
13
2
1
.17–2.08 Žm, 3H, –Ad., 1.98–1.92 Žm, 6 H, –Ad.,
1.39 Žs, 9 H, –CŽCH . .. C NMR ŽCDCl , 50
3 3 3
1
3
.86–1.72 Žm, 6 H, –Ad.. C NMR ŽCDCl , 75
MHz. ds151.7, 148.9, 146.9, 138.3, 128.1, 124.0,
3
MHz. ds152.5, 147.5, 146.9, 135.8, 127.5, 127.1,
123.1, 121.8, 35.5, 31.5. MS Ž70 eV.: mrz Ž%.s
q
q
1
25.7,124.0, 43.1, 36.7, 36.3, 28.9. MS Ž70 eV.:
311 ŽM ; 37. 296 ŽM –CH ; 100.. Calc. C 77.14%,
3
q
q
mrz Ž%.s333 ŽM , 100., 276 ŽM –C H , 39.,
H 8.09%, N 4.50% found C 76.99%, H 8.38%, N
4
9
q
2
6
3
30 ŽM –C H –NO , 14.. Calc. C 79.25%, H
4.40%.
4 9 2
.95%, N 4.20% found C 79.31%, H 6.84%, N
.98%.
2
.2.11. 2-Phenyl-5-nitropyridine (12); typical proce-
dure
X
2
.2.7. 4 -Nitroterphenylene (8)
To a solution of bromobenzene Ž1.77 g, 11.3
1
Yellow crystals, mp 196–1988C. H NMR
mmol. in Et O Ž40 ml. was added under argon
2
3
ŽCDCl , 300 MHz. ds8.30 Žd, 2H, Js8.9 Hz,
slowly t-BuLi Ž1.5 M in pentane, 16.7 ml. with a
3
3
H3., 7.73 Žd, 2H, Js8.9 Hz, H2., 7.72–7.57 Žm,
syringe at y788C to give the lithiated species. After
stirring for 1 h the cooling bath was removed and the
mixture was charged with THF Ž20 ml. and concen-
trated under diminished pressure. Transmetallation
was achieved by adding a solution of anhydrous
ZnCl Ž1.53 g, 11.3 mmol. in THF Ž30 ml. and
stirring for 1 h. The resulting liquid was treated
1
3
6
H, H
., 7.47–7.31 Žm, 3H, H
.. C NMR
arom
arom
ŽCDCl , 75 MHz. ds147.1, 141.8, 140.1, 137.5,
3
1
28.9, 127.8 Ž2C., 127.7, 127.5, 127.0, 124.1. MS
q
q
Ž70 eV.: mrz Ž%.s275 ŽM , 100., 245 ŽM –NO,
6., 228 Ž68., Calc. C 78.53%, H 4.76%, N 5.09%
found C 78.32%, H 4.81%, N 4.91%.
2
2

7
4
M. Klein et al.rMutation Research 467 (2000) 69–82
slowly with 2-bromo-5-nitropyridine Ž2.09 g, 10.3
mmol. and a catalyst solution prepared of PdŽdba.2
Ž118 mg, 2 mol%. and trifurylphosphine Ž191 mg, 8
mol%. in 2 ml THF. After stirring at room tempera-
ture for 18 h under argon, the reaction mixture was
concentrated under diminished pressure, poured into
water and extracted with ethyl acetate. The organic
152 Ž17.. Calc. C 80.30% H 7.16% N 5.85% found
C 79.95%, H 7.13%, N 5.73%.
2
.3. Computational methods
Structural parameters and LUMO energies were
calculated from fully optimised geometries as de-
scribed w15x. Log P coeffcients were calculated by
means of the program KOWWIN 1.54.
layer was washed with brine, dried ŽMgSO . and
4
evaporated to dryness in vacuo. Chromatography
Žchloroformrethyl acetate 4:1. afforded 2PhNP 12
Ž0.93 g, 45%..
1
Colourless crystals, mp 1208C. H NMR ŽCDCl ,
3
2.4. Mutagenicity tests
3
3
00 MHz. ds9.47 Žd, 1H, Js2.6 Hz, H ., 8.50
py
3 3
Ždd, 1H, Js8.8 Hz, Js2.6 Hz, H ., 8.03–8.10
py
w x
All compounds were tested as described 15 in
the Hygiene-Institut, Universit ¨a t Heidelberg, Ger-
many. The compounds were divided into five inde-
pendent test series. Within one series, all compounds
were tested simultaneously under identical condi-
tions. Compounds that induced revertant numbers
below twice the number of the solvent control at all
concentrations, were classified as non-mutagenic.
X
3
Žm, 2H, H2 ., 7.89 Žd, 1H, Js8.8 Hz, H ., 7.48–
py
1
3
7
.54 Žm, 3H, H
.. C NMR ŽCDCl , 75 MHz.
arom 3
ds162.5, 145.2, 143.3, 137.0, 131.9, 130.8, 129.1,
q
1
1
27.7, 120.0. MS Ž70 eV.: mrz Ž%.s200 ŽM ,
q
00., 154 ŽM –NO , 49., 127 Ž71.. Calc. C 65.99%,
2
H 4.03%, N 13.99% found C 65.88%, H 4.02%, N
1
4.07%.
2
.2.12. 2-(4-tertButylphenyl)-5-nitropyridine (13)
1
Colourless crystals, mp 1528C. H NMR ŽCDCl ,
3
3. Results
3
3
00 MHz. ds9.46 Žd, 1H, Js2.6 Hz, H ., 8.48
py
3
3
Ždd, 1H, Js8.8 Hz, Js2.6 Hz, H ., 8.01 Žd,
py
3
X
3
The revertant numbers of compounds 1–17 are
shown in Tables 1–5. Whereas the number of spon-
taneous revertants of TA98 remained stable over the
whole test period Ž;1 year. there were some changes
2
H, Js8.6 Hz, H2 ., 7.86 Žd, 1H, Js8.8 Hz,
3
X
H ., 7.53 Žd, 2H, Js8.6 Hz, H3 ., 1.35 Žs,.9 H,
py
1
3
tBu.. C NMR ŽCDCl , 75 MHz. ds162.4, 154.5,
3
1
3
45.3, 142.5, 134.3, 131.8, 127.5, 126.1, 119.6, 34.9,
in TA100 as indicated by the solvent controls. Due
to the limited solubility of compounds 7, 8, 12–17 in
DMSO the last two series were carried out with THF
as solvent.
q
1.2. MS Ž70 eV.: mrz Ž%.s256 ŽM , 100.. Calc.
C 70.29%, H 6.29%, N 10.93% found C 70.14%, H
6
.31%, N 11.02%.
Regarding 4NBp and the para-substituted nitro-
biphenyls 2–6 ŽTable 1. in TA98 with and without
metabolic activation, 4NBp 1, the 4 -methyl-and 4 -
ethyl-substituted compounds 2 and 3 showed compa-
rably high activity even at low concentrations Ž20,
100 mg.. At higher concentrations, cell toxicity re-
sulted in a flattening of the corresponding dose
response curves. In the presence of S9 mix the
activity of the methyl compound 2 was further en-
hanced. In contrast to 1–3 mutagenicity and cell
toxicity of compounds 4 and 5 bearing the more
bulky isopropyl and tertbutyl groups, respectively,
was much lower in TA98. Moderate activity was
found only at the highest concentrations, while up to
X
2
.2.13. 4 -tertButyl-4-nitrosobiphenyl (5a)
X
X
This compound was synthesized as described us-
X
ing 4 -tertbutyl-4-aminobiphenyl Ž0.53 g, 2.35 mmol.
and MoŽO.ŽO . ŽHMPT.ŽH O. solution Ž1.5 ml, 2
2
2
2
h.. The reaction furnished 5a Ž0.37 g, 65%..
1
Dark green crystals, mp 1098C. H NMR ŽCDCl ,
3
3
2
00 MHz. ds7.95 Žd, 2H Js8.6 Hz, H2., 7.81
3
3
Žd, 2H, Js8.7 Hz, H3., 7.62 Žd, 2H, Js8.6 Hz,
X
3
X
H2 ., 7.51 Žd, 2H, Js8.7 Hz, H3 ., 1.37 Žs, 9H,
1
3
tBu.. C NMR ŽCDCl , 50 MHz. ds165.0, 152.3,
3
1
47.9, 136.1, 127.5, 127.1, 126.1, 121.7, 34.7, 31.2.
q
q
MS Ž70 eV. mrz Ž%.s239 ŽM , 93., 224 ŽM –
CH 100., 210 Ž97., 194 Ž88., 178 Ž25., 165 Ž25.,
3

M. Klein et al.rMutation Research 467 (2000) 69–82
75
Table 1
Mutagenicity of nitrobiphenyls with para alkyl groups
Table 2
Mutagenicity of nitroso compounds 1a and 5a
Code Compound
Dose
Žmgrplate. TA 98
yS9 qS9 yS9 qS9
37 67 207 206
Revertants
Code Compound
Dose
Žmgrplate. TA 98
Revertants
TA100
TA100
yS9 qS9 yS9 qS9
32 43 158 146
3136 1628 892 2351
DMSO
50 ml
DMSO
Positive control
a
Positive control
4NBp
2149 1151 902 795
96 131 347 488
233 406 795 1285
290 508 885 1294
382 660 904 1377
721 830 1177 1567
a
1
2
20
b
c
1
5
a
a
4NOBp
10
249
516
340 557
778 311 1112
769
b
b
1
5
2
5
00
00
500
000
c
2
5
5
0
513 1111 272 1005
347 1104 167
1
5
00
00
10
531
51
c
0
58
68
98
101
135
614
0
X
4 Me-4NBp
10
108
–
–
–
X
4 tBu-4NOBp
55 264
54 323
51 451
47 455
71 797
250
330
438
498
678
2
1
5
2
5
0
134 286 313 794
c
c
2
5
5
0
b
b
00
00
500
000
174 677 348 1682
188 682 368 1518
222 700 389 1213 tox
286 838 477 1516
84 295 281 556
1
5
00
00
c
X
3
4
5
6
4 Et-4NBp
20
a
TA98 yS9 and TA98qS9 1-nitropyrene 2.5 mg; TA100
b
1
5
2
5
00
00
500
000
165 297 330 860
yS9 sodium azide 5 mg; TA100 qS9 2-aminoantracene 2.5 mg.
b
297 304 343 883
b
Data from preceding paper.
Indicates highest dose used in regression analysis.
c
c
338 430 436 998
566 650 632 1105
c
X
4 iPr-4NBp
20
50
50
66
87 200 449
86 213 481
1
5
2
5
00
00
500
000
substituted nitrobiphenyls was rather weak and only
the unsubstituted 4NBp 1 exhibited significant muta-
92 213 412
c
c
b
b
158 198 293 400 tox
271 279 327 119 tox
X
4 tBu-4NBp
20
45
53
50
62 206 209
63 203 209
Table 3
1
5
2
5
00
00
500
000
X
Mutagenicity of 3 -alkyl-substituted nitrobiphenyls
68 210 227
c
c
b
b
Code Compound
Dose
Žmgrplate. TA 98
yS9 qS9 yS9 qS9
Revertants
140 196 409 382
151 219 510 591
52 107 248 330
74 107 269 350
TA100
X
4 nBu-4NBp
20
1
5
2
5
00
00
500
000
b
b
DMSO
50 ml
36
44
168
177
185 141 290 453
200 248 373 824
255 456 414 906
a
c
c
Positive control
2767 1890 916
1759
1027
1073
1158
X
9
3 Me-4NBp
10
39
42
77
234 228
244 237
310 438
2
0
a
TA98 yS9 1-nitropyrene 2.5 mg; TA100 yS9 sodium azide
100
500
2500
b
b
5
mg; TA98 and TA100 qS9 2-aminoantracene 2 mg.
390 819 1663 1442
0 tox 0 tox 0 tox 0 tox
b
Indicates highest dose used in regression analysis.
Precipitation: effective dose is lower than specified.
X
X
c
10
3 ,5 DiMe-4NBp 10
37
35
42
40
36
35
35
39
36
39
97
189
621
699
781
1028
673
216
242
224
214
222
2
1
5
2
0
103 217
138 255
149 256
100 247
49
46
45
47
42
b
b
00
00
500
c
c
X
X
1
1
3 ,5 DitBu-4NBp 10
205
229
5
00 mg revertant numbers remained below twice the
2
1
0
00
solvent control. Notably, the nbutyl-substituted com-
pound 6 was considerably more mutagenic in TA98
than the tertbutyl-substituted compound 5. When
assayed in TA100 a similar trend as in TA98 was
observed, especially in the presence of S9 mix.
Compounds 4 and 5 were the weakest mutagens in
this series. In the absence of S9 mix, activity of all
b
b
231
232
209
500
2500
a
TA98 yS9 and TA98 qS9 1-nitropyrene 2.5 mg; TA100
yS9 sodium azide 5 mg; TA100 qS9 2-aminoantracene 2.5 mg.
b
Indicates highest dose used in regression analysis.
Precipitation: effective dose is lower than specified.
c

7
6
M. Klein et al.rMutation Research 467 (2000) 69–82
Table 4
Mutagenicity of alkyl-substituted nitrobiphenyls and azanitrobiphenyls
Code
Compound
Dose
Revertants
TA 98
Žmgrplate.
TA100
yS9
qS9
yS9
qS9
THF
50 ml
39
2757
32
31
28
61
1579
56
50
50
123
1019
146
145
139
148
136
2339
150
148
142
a
Positive control
X
7
8
4Ad-4NBp
20
5
1
5
1
0
00
00
000
30
28
55
56
184
187
b
c
182
4NTp
20
41
35
36
34
43
47
85
219
764
614 tox
37
123
109
102
99
108
71
143
341
730
301 tox
56
149
156
154
208
216
213
203
187
330
651
1158
747 tox
68 tox
199
297
317
5
1
5
1
8
3
1
5
1
8
3
1
5
1
0
00
00
000
c
160
b
157
185
321
1
1
2
3
2PhNP
1
c
25
00
000
905
c
1110
759 tox
157
208
219
4tBu-2PhNP
1
34
34
37
122
52
52
52
52
c
25
00
000
250
301
326
413
c
a
b
c
TA98 yS9 1-nitropyrene 2.5 mg; TA100 yS9 sodium azide 5 mg; TA98 and TA100 qS9 2-aminoantracene 2.5 mg.
No positive slope found.
Indicates highest dose used in regression analysis.
genicity. Under these conditions, the isopropyl com-
pound 4 was non-mutagenic at all concentrations.
Compared to the unsubstituted 4-nitrosobiphenyl
tions. In analogy to the first series Žcompound 2., the
presence of S9 mix increased the mutagenicity of
both methyl-substituted compounds Ž9, 10. in TA98
X
X
1
a ŽTable 2. which was highly mutagenic as ex-
and TA100. Under these conditions 3 ,5 -dimethyl-
4-nitrobiphenyl 10 became moderately mutagenic as
well. Following the same trend as in the first series,
compound 11 with its two bulky tertbutyl groups
remained inactive in both strains in the absence and
the presence of S9.
pected even at very low concentrations the activity
X
of its 4 -tertbutyl-substituted derivative 5a was
strongly reduced in TA98 without S9 mix. More-
over, in the presence of S9 mix, the revertant num-
bers of 5a did not reach twice the solvent control at
any test concentration. In TA100 introduction of the
tertbutyl group resulted in a similar decrease of
mutagenicity compared to compound 1a, although
the effect was less pronounced in this strain.
To check the mutagenicity-reducing effect of
bulky substituents in the para-position of 4NBp, in
the fourth series ŽTable 4. compounds 7 and 8
bearing a sterically very demanding adamantyl and a
phenyl substituent, respectively, were tested. Con-
firming our previous results, compound 7 was non-
mutagenic at all concentrations in both test strains,
but exhibited some cell toxicity in TA98. Interest-
ingly, also compound 8 turned out to be non-muta-
genic in the absence of S9 mix, although a phenyl
With respect to compounds 9–11 in the third
X
series ŽTable 3., only the 3 -methyl-substituted com-
pound 9 was highly mutagenic in both test strains,
whereas in the absence of metabolic activation the
X
3
X
,5 -dimethyl-substituted compound 10 showed al-
most no activity, not even in the highest concentra-

M. Klein et al.rMutation Research 467 (2000) 69–82
77
Table 5
Cell toxicity was observed in concentrations above
00 mg. In excellent agreement with 5, the
Mutagenicity of 2-nitrofluorenes with alkyl groups in the 7-posi-
tion
5
tertbutyl-substituted compound 13 was much less
mutagenic and showed no ŽyS9. or weak ŽqS9.
activity in TA98. In TA100, the reduction of muta-
genicty was less pronounced but still noticeable.
Finally, in the last series, the 7-alkyl-2-
Code Compound
Dose
Žmgrplate. TA 98
yS9 qS9 yS9 qS9
33 38 134 114
1794 1428 836 1774
Revertants
TA100
THF
100 ml
a
Positive control
2NF
Ž
.
nitrofluorenes 14–17 were investigated Table 5 .
The parent fluorene 14 and the methyl-substituted
derivative 15 were potent mutagens in both test
strains. The extremely high revertant numbers in
TA98 indicate that these flat molecules act as
frameshift mutagens. In the presence of S9 mix the
methyl compound 15 turned out to be highly cyto-
toxic at concentrations above 10 mg. Similar to the
previous series the tertbutyl substituent rendered the
fluorene 16 much less active in TA98 independent of
whether S9 mix was added or not. Moreover, com-
pound 17 with its very bulky adamantyl group was
non-mutagenic in TA98 at all concentrations. The
same trend was found in TA100. In the absence of
S9 mix both compounds 16 and 17 were inactive. In
the presence of S9 mix 16 exhibited moderate and 17
weak mutagenicity.
14
15
16
17
2,5
5
285
551
615
260 208 188
600 261 351
1
2
5
5
1
2
5
1
5
1
2
5
1
5
1
2
5
1
0
0
0
809 365 520
b
b
1512 1244 834 867
1605 2196
0
1840
7Me-2NF
7tBu-2NF
7Ad-2NF
671
761
834
753 379 257
800 422 0 tox
858 475 0 tox
b
b
0
0
0
00
906 1810 475 0 tox
1960 2394 702 0 tox
43
57
61
76
67
32
33
38
45
47
62 130 319
68 149 437
88 155 511
0
0
0
00
b
b
98 189 466
119 214 550
46 133 150
46 141 160
47 118 194
52 134 229
0
0
0
00
b
c
58 126 214
Summarising these results, it turns out that alkyl
groups that are located far away from the nitro
group, exhibit a size-dependent effect on the muta-
genicity of nitroaromatics like 4NBp 1, 2PhNBp 12
and 2NF 14 and nitroso aromatics like 4NOBp 1a.
Whereas small methyl substituents do not change the
activity of the parent compound significantly in the
absence of S9 and even enhance it in the presence of
S9, mutagenic responses are reduced or even inhib-
ited completely by introduction of more bulky groups
like isopropyl, tertbutyl, or adamantyl.
a
TA98 yS9 1-nitropyrene 2.5 mg; TA100 yS9 sodium azide
5
mg; TA98 and TA100 qS9 2-aminoantracene 2.5 mg.
b
Indicates highest dose used in regression analysis.
No positive slope found.
c
substituent is less bulky than a tertbutyl group ac-
cording to Taft’s model w23x. However, in TA98 in
the presence of S9 mix, revertant numbers ap-
proached twice the solvent control already at low test
concentrations, but no linear dose–response curve
was observed. This may be due to cell toxicity. At
the moment it remains open whether 8 is mutagenic
or not under these conditions.
4
. Discussion
In order to investigate, whether similar substituent
effects on the mutagenicity are apparent in other
nitroaromatics as well, we included compounds 12
Žunsubstituted. and 13 Žbearing a tertbutyl sub-
stituent. in these investigations. 12 and 13 differ
4
.1. Comparison of predicted and experimental mu-
tagenicities
In Table 6, log P values are summarised, LUMO
from their structural analogues 1 and 5 by containing
a nitrogen atom in the biphenyl skeleton. Similar to
energies, experimental and predicted mutagenicities
of all compounds investigated in this work.
4
NBp 1 the aza analogue 12 was mutagenic in both
Since hydrophobicity for a given aromatic system
increases with the number and size of the attached
test strains in the presence and absence of S9 mix.

7
8
M. Klein et al.rMutation Research 467 (2000) 69–82
Table 6
Structural parameters, predicted and experimental mutagenicity
Code
Compound
Log Pa
ELUMO
Log revertants rnmol
TA98
TA100
Predicted
Experimental
Predicted
Experimental
1
2
3
4
5
6
4NBp
3.57
4.12
4.61
5.03
5.48
5.59
y1.229
y1.208
y1.206
y1.201
y1.193
y1.198
y1.189
y1.270
y1.207
y1.184
y1.151
y0.979
y0.907
y1.515
y1.475
y1.302
y1.279
y1.257
y1.257
y0.15
0.14
0.35
0.39
0.19
0.09
y0.42
y0.66
y0.98
y1.96
-y2
y1.14
-y2
-y2
y0.81
y1.84
y1.93
0.55
y0.53
y0.54
y1.70
1.16
0.40
0.99
1.50
1.82
1.94
1.93
0.06
y0.62
y0.62
-y2
X
b
4 Me-4NBp
X
4 Et-4NBp
X
4 iPr-4NBp
X
4 tBu-4NBp
y1.67
y1.51
X
4 nBu-4NBp
X
7
8
9
4 A` d-4NBp
7.19
5.34
4.12
4.67
7.39
3.63
5.54
2.39
4.30
-y2
0.39
0.14
0.34
-y2
y
y0.43
2.09
0.99
1.51
y0.94
–
y1.83
y1.97
y0.20
y0.76
y0.88
0.86
0.18
0.09
y0.80
0.85
0.81
4NTp
X
3 Me-4NBp
X
X
1
1
1
5
1
1
1
1
1
1
0
1
a
a
2
3
4
5
6
7
3 ,5 DiMe-4NBp
X
X
3 ,5 DitBu-4NBp
4NOBp
X
4 tBu-4NOBp
–
–
2PhNP
y0.51
0.60
1.71
2.25
1.89
y1.40
y0.42
1.74
0.32
1.42
1.98
y0.83
X
4 tBu-2PhNP
c
b
2NF
3.37
7Me-2NF
7tBu-2NF
7Ad-2NF
4.38
5.74
7.44
1.19
y0.43
y1.03
y0.51
-y2
a
Log P values calculated with KOWWIN slightly differ from those calculated with CLOGP which was used by Debnath and Hansch.
Since LUMO energies were calculated from optimised structures, there are slight deviations to the energies used by Debnath and
b
Hansch.
c
Experimental log P.
alkyl substituents, compounds 7 Žadamantyl., 11 Ž2
tertbutyl. and 17 Žadamantyl. with log P values
In Fig. 2, data of all derivatives of 4NBp Žcom-
pounds 1–11, three test series. are shown. In TA98,
the activity — mainly determined by hydrophobicity
— is predicted to increase from the unsubstitued
exceeding 7 belong to the most hydrophobic ni-
troaromatics ever tested. Compared to the hydropho-
bicity the impact of alkyl groups on the LUMO
energies is rather weak and can be attributed to their
qI effect. As expected exchange of a carbon to a
nitrogen atom Ž1 12, 5 13. lowers the LUMO
energies rather significantly. The equations by Deb-
nath and Hansch used here to predict mutagenicities
are briefly discussed in the preceding paper w15x.
Experimental mutation rates Žrevrnmol. were ob-
tained as the slope of a regression analysis over the
linear portion of the dose–response curve. Graphical
comparisons between predicted and experimental
mutagenicities for both test strains are shown in Figs.
2
–4. The compounds are ordered by increasing hy-
drophobicity which approximately parallels the steric
demand of the substituents.
Fig. 2. Experimental and predicted mutagenicity for compounds
1–11.

M. Klein et al.rMutation Research 467 (2000) 69–82
79
4
NBp 1 in the sequence 2–9-3–10-4–8 and then
to drop slowly for the more hydrophobic compounds
)5)6. The highly hydrophobic compounds 7
8
X
X
X
Ž4 tBu-4NBp. and 11 Ž3 ,5 DitBu-4NBp. are pre-
dicted to be inactive. In contrast to this prediction the
overall experimental trend is to decrease continu-
ously with number and size of the substituents. Only
X
compound 6 Ž4 nBu-4NBp. with its more flexible
and less sterically demanding nbutyl substituent ex-
hibits comparatively high activity. Very similar
curves are obtained in TA100. Consequently, there is
good agreement between prediction and experiment
for 4NBp 1, but deviations are growing with the
steric demand of the substituents. The inactivity of
the most hydrophobic compounds Ž7, 11. is well
Fig. 4. Experimental and predicted mutagenicity for compounds
1
4–17.
predicted.
the general results observed in the absence of S9 also
hold in the presence of S9. Nevertheless, the muta-
genicity of some compounds — especially those
containing methyl or ethyl groups like 2, 3, 9 and 10
— was markedly enhanced in the presence of S9 in
both strains. Aromatic methyl groups in nitrotoluenes
are known to be oxidised metabolically to benzyl
alcohols, followed by further transformation to the
corresponding sulfates which are strong electrophiles
w24x and therefor principally capable to attack DNA
Regarding the aza-analogues ŽFig. 3. the same
general outcome is observed. Predictions are rela-
tively accurate for the unsubstituted compound 12 in
both strains, but far too high for the inactive tert-
butyl-substituted species 13. This is also true for the
alkyl-substituted fluorenes 14–17 ŽFig. 4.. Indeed
the experimental curve and the deviations from the
predicted curve in Fig. 4 are very similar to those in
Fig. 2. Again the highly hydrophobic adamantyl
compound 17 is predicted to be inactive, as experi-
mentally found. Interestingly, the difference between
predicted and experimental behaviour of the com-
pounds tested in this work is almost identical to the
results we obtained for the ortho-substituted deriva-
tives of 4NBp investigated in the previous paper.
So far no QSAR equations exist to predict muta-
genicities of nitroaromatics in the presence of S9
mix. Regarding the experimental data in Tables 1–5
bases. A similar activation process should account
for the observed increase of mutagenicity of com-
pounds 2, 3, 9 and 10.
4.2. Reduction of mutagenicity — possible explana-
tions
As predicted by Hansch and Debnath, the highly
hydrophobic compounds 7, 11 and 17 Žlog P)7.
exhibited weak activity confirming a trend which we
already observed in the preceding publication. High
log P values generally render nitroaromatics non-
mutagenic. Low solubility may play some role, too,
and it remains unclear whether these molecules are
really able to penetrate the cell wall and to approach
the DNA. However, the strong steric effect of bulky
para alkyl groups in the more soluble compounds
was rather surprising at first glance. In contrast to the
X
alkyl-substituted Žortho, 2 -position. derivatives of
4
NBp, investigated in the preceding paper, the para
alkyl substituents of the compounds studied here are
far away from the nitro groups and do neither affect
the charge delocalisation nor the nitro group orienta-
Fig. 3. Experimental and predicted mutagenicity for compounds
1
2 and 13.

8
0
M. Klein et al.rMutation Research 467 (2000) 69–82
tion. Although it cannot be excluded that bulky para
alkyl groups interfere with the binding to the nitrore-
ductase which may slow down the metabolic activa-
tion process somehow, it is unlikely that this effect
alone is responsible for the strong reduction of muta-
genicity in compounds 4, 5, 13 and 16. Further
evidence that some other effect may be involved,
arises from the related nitroso compounds. Despite
of the fact that the more difficult first reduction step
of the activation process has already taken place in
this class of compounds, the tertbutyl substituent
exhibited a strong effect on the mutagenicity, too
induction rates of all compounds, excluding the very
hydrophobic ones, to be similar in TA100. Neverthe-
less, experimental data showed the steric effect of
alkyl groups to be present even in TA100. Thus,
does planarity play some role as well? It is known
that metabolic intermediates of 4-aminobiphenyl and
2-aminofluorene, or of the corresponding nitro com-
pounds in vivo w26,27x and in vitro w28,29x mainly
form C8-deoxyguanosine adducts w30x. In recent years
it has been indicated that apart from the number and
kinds of adducts the conformation of these adducts
within the DNA plays an important role in determin-
ing mutation rates. NMR studies and molecular dy-
namics of isolated alkyl-aniline C8-substituted de-
oxyguanosine adducts showed that the location of
the alkyl substituents can change the ratio of syn and
anti conformers w31x. Furthermore, also in DNA-
X
Žsee Table 6.. In TA98 4 -tertbutyl-4-nitrosobiphenyl
5
a, the reduction intermediate of 5, is a rather weak
mutagen ŽTA98 yS9 log revrnmol s y0.53.
whereas the unsubstituted 4-nitrosobiphenyl 1a is
highly active ŽTA98 yS9 log revrnmolsq0.55..
In TA100 the effects are similar: 5a ŽTA100 yS9
log revrnmolsq0.18. is significantly less muta-
genic than 1a ŽTA100 yS9 log revrnmols0.86..
oligonucleotides containing adducts of 4-amino-
biphenyl, 2-aminofluorene or structurally similar
compounds two major conformations were detected
Žconformational heterogenicity. by various NMR
It has been demonstrated that a flat aromatic
system, capable to intercalate in DNA, is an impor-
tant structural feature for a molecule to react as
frameshift mutagen w25x. In TA98, the nitrated planar
fluorene derivatives 14 and 15 are much more active
than their corresponding less planar biphenyl deriva-
tives 1 and 2 Žthe dihedral angles between the phenyl
rings are about 408.. By introduction of bulky sub-
1
13
19
studies Ž H, C, F. w32–35x. Whereas in the so-
called ‘‘B-type’’ conformer, the aromatic moiety of
the mutagen is located within the major or minor
groove of the DNA helix and the overall structure is
relatively undisturbed, in the ‘‘stacked’’ conformer
the adduct moiety is inserted within the helix. It has
been suggested that this stacked conformer is associ-
ated with an increased mutation frequency per adduct
w35x. While para alkyl substituents have no effect on
the conformation of a single adduct Ž4-amino-
biphenyl, 2-aminofluorene. modified nucleotide, this
stituents this planarity is lost. Consequently, com-
pounds like 4, 5, 7, 11, 13, 16 and 17 lose their
ability to intercalate well into DNA which should
reduce their mutagenic properties in TA98. Espe-
X
cially by comparing compounds 2 Ž4 Me-4NBp. and
may be different in double-stranded DNA containing
adducts. Sterically demanding alkyl substituents at
the aromatic adduct moiety may probably destabilise
the stacked conformer by interfering with the interca-
lation into the helix, and therefore change the ratio of
the ‘‘B-type’’ and the ‘‘stacked conformer’’. To
examine the extent of conformational heterogenicity,
X
X
5
2
Ž4 tBu-4NBp. or 15 Ž7 Me-2NF. and 16 Ž7tBu-
NF., it is impressive to see how small changes —
replacement of a methyl group by a tertbutyl group
—
render a molecule from highly mutagenic to weak
or non-mutagenic. Most remarkably, this reduction
of activity is due only to a modification of the
molecular shape while the chemical properties of the
nitro group and the degree of aromaticity remain
essentially unaffected.
In TA100, base substitution mutations instead of
frameshift mutations are detected and planarity
should be less important than in TA98. Conse-
quently, assuming that bulky para alkyl groups do
neither inhibit the nitro reduction process nor the
adduct formation significantly, one might expect the
X
the incorporation guanosine adducts of 4 -tertbutyl-
4-aminobiphenyl into DNA oligonucleotides is
planned. Conformational changes may be also re-
sponsible for the reduced mutagenicity of other
X
non-planar nitroaromatics, e.g., in the series of 2 -
substituted 4-nitrobiphenyls investigated in the pre-
ceding paper.
In conclusion, it is shown that otherwise strongly
mutagenic nitroaromatics like 4NBp 1, 2PhNP 12 or

M. Klein et al.rMutation Research 467 (2000) 69–82
81
2
NF 14 can be transformed even in non-mutagenic
Shusterman, C. Hansch, Structure–activity relationship of
mutagenic aromatic and hetreoaromatic nitro compounds.
Correlation with molecular orbital energies and hydrophobic-
ity, J. Med. Chem. 34 Ž1991. 786–797.
ones simply by introduction of bulky para alkyl
groups without changing the chemical properties sig-
nificantly. This reduction which might be attributed
to the incapability to intercalate into the DNA, is not
predicted by the empirical equations of Debnath et
al. w7,8x. Hopefully, the data presented here and in
the preceding paper will help to improve these equa-
tions by adding steric parameters. It has to be inves-
tigated further whether this strategy of reducing the
mutagenicity by changing the molecular shape is of
general applicability also for other N-substituted aro-
matics. So far, we obtained similar results for the
corresponding aromatic amines that will be pub-
lished soon.
w8x A.K. Debnath, R.L. Lopez de Compadre, A.J. Shusterman,
C. Hansch, Quantitative structure–activity relationship inves-
tigation of the role of hydrophobicity in regulating muta-
genicity in the Ames test: 2. Mutagenicity of aromatic and
heteroaromatic nitro compounds in Salmonella typhimurium
TA100, Environ. Mol. Mutagen. 19 Ž1992. 53–70.
w9x H.A. Nieper, Failure of methyl orange to show a carcino-
genic effect on rats, Naturwissenschaften 43 Ž1956. 500.
w10x J. Ashby, D. Paton, P.A. Lefevre, J.A. Styles, F.L. Rose,
Evaluation of two suggested methods of deactivating organic
carcinogens by molecular modification, Carcinogenesis 3
Ž1982. 1283–1287.
w11x K. El-Bayoumy, E.J. Vavoie, S.S. Hecht, E.A. Fow, D.
Hoffmann, The influence of methyl substitution on the muta-
genicity of nitronaphthalenes and nitrobiphenyls, Mutat. Res.
8
1 Ž1981. 143–153.
w12x M. Fan, C. Byrd, C.M. Compadre, R.L. Compadre, Compari-
son of CoMFA models for Salmonella typhimurium TA98,
TA100, TA98qS9 and TA100qS9 mutagenicity of ni-
troaromatics, SAR QSAR Environ. Res. 9 Ž1998. 187–215.
w13x S. Murphy, X. Yang, G.B. Schuster, Cyanine borate salts that
Acknowledgements
We are very grateful to the BASF, Ludwigshafen,
Germany for the continuous financial support of this
work.
form penetrated ion pairs in benzene solution: synthesis,
properties, and structure, J. Org. Chem. 60 Ž1995. 2411–
2
422.
w14x A. Suzuki, T. Watanabe, N. Miyaura, Synthesis of sterically
hindered biaryls via the palladium-catalysed cross-coupling
reaction of arylboronic acids or their esters with haloarenes,
Synlett Ž1992. 207–210.
References
w15x M. Klein, U. Voigtmann, T. Haack, L. Erdinger, G. Boche,
From mutagenic to non-mutagenic nitroarenes: effect of bulky
alkyl substituents on the mutagenic activity of 4-nitro-
biphenyl in Salmonella typhimurium. Part I. Substituents
w1x D.R. Hartter, The use and importance of nitroaromatic chem-
icals in the chemical industry, in: D.E. Rickert ŽEd.., Toxic-
ity of Nitroaromatic Compounds, Hemisphere, New York,
X
ortho to the nitro group and in 2 -position, Mutat. Res. 467
1
985, pp. 1–13.
Ž2000. 55–68, this issue.
w16x F.A. Carey, R.J. Sundberg, Advanced Organic Chemistry,
Part B, 3rd edn., Plenum, New York, pp. 461–462.
w2x Lebensmittel- und Bedarfsgegenst ¨a ndegesetz Bundesrepublik
Deutschland 15.7.1994
w3x R.J. Allan, J.J. Roxon, Metabolism by intestinal bacteria: the
w17x M. Klein, Mutagenitatsuntersuchungen an Substituierten 4-
¨
effect of bile salts on tatrazine azo reduction, Xenobiotica 4
Ž1974. 637–643.
Nitrobiphenylen, Diplomarbeit, Philipps-Universit ¨a t, Mar-
burg, 1997.
w4x R.P. Mason, F.J. Peterson, J.H. Holtzman, Inhibition of
w18x E. Negishi, T. Takahashi, A.O. King, Synthesis of biaryls via
palladium-catalysed cross coupling, Org. Synth. 66 Ž1987.
azoreductase by oxygen, the role of the azo anion free radical
metabolite in the reduction of oxygen to superoxide, Mol.
Pharmacol. 14 Ž1978. 665–671.
67–74.
w19x S. Tollari, M. Cuscela, F. Porta, Catalytic oxidation of
w5x J. Ashby, D. Paton, P.A. Lefevre, J.A. Styles, F.L. Rose,
primary aromatic amines to the corresponding nitroso com-
Evaluation of two suggested methods of deactivating organic
carcinigens by molecular modification, Carcinogenesis 3
Ž1982. 1277–1282.
pounds by H O and MoŽO.ŽO . ŽH O.ŽHMPA., Chem.
2
2
2
2
2
Commun. Ž1993. 1510–1511.
w20x M. Klein, G. Boche, Regiospecific synthesis of substituted
nitrofluorenes and aminofluorenes with the Negishi coupling
reaction as key step, Synthesis 7 Ž1999. 1246–1250.
w21x W.S.N. Grieve, D.H. Hey, Substitution in compounds con-
taining two or more phenyl groups: Part I. The nitration of
4-methyldiphenyl, J. Chem. Soc. Ž1932. 1888–1894.
w22x K. Shudo, T. Ohta, T. Okamoto, Acid-catalyzed reactions of
w6x A.K. Debnath, G. Debnath, A.J. Shusterman, C. Hansch, A
QSAR investigation of the role of hydrophobicity in regulat-
ing mutagenicity in the Ames test: mutagenicity of aromatic
and heteroaromatic amines in Salmonella typhimurium TA98
and TA100, Environ. Mol. Mutagen. 19 Ž1992. 37–52.
w7x A.K. Debnath, R.L. Lopez de Compadre, G. Debnath, A.J.

8
2
M. Klein et al.rMutation Research 467 (2000) 69–82
N-arylhydroxylamines and related compounds with benzene.
Iminium-benzenium ions, J. Am. Chem. Soc. 103 Ž1981.
45–653.
w29x F. Bosold, G. Boche, The ultimate carcinogen, O-acetyl-N-
Ž2-fluorenyl.hydroxylamine Ž‘‘N-acetoxy-2-aminofluorene’’.,
and its reaction in vitro to form 2-w N-Ždeoxyguanosin-8-
yl.aminoxfluorene, Angew. Chem. Int. Ed. Engl. 29 Ž1990.
6
w23x C. Hansch, A. Leo, Exploring QSAR fundamentals and
applications in chemistry and biology, in: ACS Professional
Reference Book, American Chemical Society, Washington,
DC, 1995, pp. 69–96.
63–64.
w30x F.A. Beland, F.F. Kadlubar, in: C.S. Cooper, P.L. Grover
ŽEds.., Handbook of Experimental Pharmacology Vol 94rI
w24x D.E. Rickert, Metabolism of nitroaromatic compounds, Drug
Springer, Heidelberg, 1990, pp. 267–325.
Metab. Rev. 18 Ž1987. 23–53.
w31x F.A. Beland, W.B. Melchior Jr., L.L.G. Mourato, M.A.
w25x T. Hirayama, T. Watanabe, M. Akita, S. Shimomura, Y.
Santos, M.M. Marques, Arylamine–DNA adduct conforma-
tion in relation to mutagenesis, Mutat. Res. 376 Ž1997.
13–19.
Fujioka, S. Ozasa, S. Fukui, Relationships between structure
of nitrated arenes and their mutagenicity in Salmonella ty-
phimurium; 2- and 2,7-nitro substituted fluorene, phenan-
threne and pyrene, Mutat. Res. 209 Ž1988. 67–74.
w32x B.P. Cho, F.A. Beland, M.M. Marques, NMR structural
studies of a 15-mer DNA sequence from a ras protoonco-
gene, modified at the first base of codon 61 with the carcino-
gen 4-aminobiphenyl, Biochemistry 31 Ž1992. 9587–9602.
w33x B.P. Cho, F.A. Beland, M.M. Marques, NMR structural
studies pf a 15-mer DNA duplex from a ras protooncogene
modified with the carcinogen 2-aminofluorene: conforma-
tional heterogeneity, Biochemistry 33 Ž1994. 1373–1384.
w34x L. Zhou, M. Rajabzadeh, D. Traficante, B.P. Cho, Conforma-
w26x W.T. Allaben, C.C. Weis, N.F. Fullerton, F.A. Beland, For-
mation and persistence of DNA adducts from the carcinogen
N-hydroxy-2-acetylaminofluorene in rat mammary gland in
vivo, Carcinogenesis 4 Ž1983. 1067–1070.
w27x R.C. Gupta, N.R. Dighe, Formation and removal of DNA
adducts in rat liver treated with N-hydroxy derivatives of
2
-acetylaminofluorene, 4-acetylaminobiphenyl and 2-
acetylaminophenanthrene, Carcinogenesis 5 Ž1984. 343–349.
w28x M. Famulok, F. Bosold, G. Boche, Synthese von O-acetyl-N-
Ž4-biphenylyl.hydroxyl Ž‘‘N-acetoxy-4-aminobiphenyl’’.,
tional heterogeneity of arylamine-modified DNA: F evi-
dence, J. Am. Chem. Soc. 119 Ž1997. 5384–5389.
w35x L. Eckel, T.R. Krugh, Structural characterisation of two
einem entscheidenden Metaboliten des carcinogenen 4-
Aminobiphenyls, und seine Reaktion mit Desoxyguanosin,
Angew. Chem. 101 Ž1989. 349–350.
interchangeable conformations of a 2-aminofluorene-mod-
ified DNA oligomer by NMR and energy minimisation,
Biochemistry 33 Ž1994. 13611–13624.