Mutagenicity and DNA damage studies of N-acyloxy-N-alkoxyamides--the role of electrophilic nitrogen.

Article abstract of DOI:10.1039/b301618h

N-Acyloxy-N-alkoxyamides are anomeric amides that are direct-acting mutagens. They have been shown to damage DNA in the major and the minor grooves in a pH and sequence-selective manner. In acidic media, they damage adenines at N3 in the minor groove but above neutral pH, only guanine is damaged at N7 in the major groove. Both the acyloxy leaving group and the alkoxy group at the amide nitrogen are responsible for their electrophilicity and Salmonella mutagenicities in TA 100 and DNA damage data confirm that the mutagens react with DNA in an intact form, rather than by solvolysis to electrophilic nitrenium ions in the cytosol, or in vitro, prior to reacting with DNA. Hydrophobicity plays a role in both mutagenicity and DNA damage.

Full text of DOI:10.1039/b301618h

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Mutagenicity and DNA damage studies of N-acyloxy-
N-alkoxyamides — the role of electrophilic nitrogen†
Tony M. Banks,a Antonio M. Bonin,b Stephen A. Glovera and Arungundrum S. Prakash
a Division of Chemistry, School of Biological, Biomedical and Molecular Sciences,
University of New England, Armidale 2351, N.S.W., Australia
b School of Chemistry, University of Sydney, NSW 2006, Australia
c National Research Centre for Environmental Toxicology, University of Queensland,
39 Kessels Rd., Coopers Plains, Qld 4108, Australia
Received 10th February 2003, Accepted 16th May 2003
First published as an Advance Article on the web 2nd June 2003
N-Acyloxy-N-alkoxyamides are anomeric amides that are direct-acting mutagens. They have been shown to damage
DNA in the major and the minor grooves in a pH and sequence-selective manner. In acidic media, they damage
adenines at N3 in the minor groove but above neutral pH, only guanine is damaged at N7 in the major groove. Both
the acyloxy leaving group and the alkoxy group at the amide nitrogen are responsible for their electrophilicity and
Salmonella mutagenicities in TA100 and DNA damage data confirm that the mutagens react with DNA in an intact
form, rather than by solvolysis to electrophilic nitrenium ions in the cytosol, or in vitro, prior to reacting with DNA.
Hydrophobicity plays a role in both mutagenicity and DNA damage.
Introduction
N-Acyloxy-N-alkoxybenzamides 1 constitute a general class of
electrophilic amides first synthesised by our group in 1989 and
which we have shown to be direct-acting mutagens.1–5 We have
synthesised a wide range of these substances with structural
variation on all three side chains3,4,6 and, to date, almost all that
we have tested have exhibited linear dose-response relationships
in the TA100 strain of Salmonella typhimurium in the absence
of metabolic activation. In a recent study, mutagenic activity of
a diverse range of these compounds has been shown to corre-
late linearly with hydrophobicity but with a modest dependence
(log TA100 = 0.23 log P ϩ1.97) which we have interpreted to
reflect the ease of non-covalent binding to bacterial DNA;7
indirect mutagens show a much larger log P dependence of
between 0.65 and 1.15 and both Debnath et al.8 and more
recently Tuppurainen9 have suggested that the absence of a
hydrophobic dependence may even be characteristic of direct-
acting mutagens. Our results indicate that a weaker hydro-
phobic dependence is operative. In addition, for at least one
series that have similar log P values, 2, mutagenic activity is
inversely correlated with chemical reactivity.7 Compounds of
type 1 and 2 undergo both SN1 and SN2 reactions at the amide
nitrogens.3,4,6 Acid-catalysed solvolysis leads to alkoxy-
nitrenium ion formation and such reactions are facilitated by
electron-withdrawing groups on the benzoate leaving group of
2 leading to a Hammett σ correlation with ρ = ϩ0.32.6 The
amides are also susceptible to direct SN2 displacement of
carboxylate by a variety of nucleophiles including inorganic
hydroxide6 and azide,10,11 organic amines,12,13 glutathione and
thiols.14 With hydroxide6 and N-methylaniline,13 2 afforded
Hammett σ-correlations with reaction constants ρ = 1.69 and
0.55, respectively. Mutagenicity of Series 2, on the other hand,
correlated with Hammett σ-substituent constants with a
negative slope (LogTA100 = Ϫ0.53σ).7
a naphthyl substituent present on the side chains exhibit
enhanced mutagenic activity in TA100 largely independent of
other factors.16 Both situations reflect different binding
characteristics; bulky mutagens would be less capable of close
association with DNA while naphthyl groups could intercalate
between base pairs, thereby increasing residence times on DNA.
As such, the mutagenicities of N-acyloxy-N-alkoxyamides are
proving to be useful as probes of the influence of non-reactive
organic substituents upon DNA binding.
N-Acyloxy-N-alkoxybenzamides can be expected to behave
as molecular electrophiles towards DNA or generate electro-
philic N-acyl-N-alkoxynitrenium ions which would be expected
to attack DNA or other biological substrates. Hard and soft
Lewis acids are known to damage DNA through covalent bind-
ing to nucleophilic centres on DNA, the most reactive of which
is widely acknowledged as N7 on guanine residues (N7-G).
Pullman and Pullman illustrated that the electrostatic potential
in the region of N7-G is not only the most negative, but that the
magnitude of the negative potential increases significantly in
single-stranded and duplex DNA.17 It is this site in the major
groove that is most often modified by alkylating agents such as
mustards, alkyl sulfonates or dialkyl sulfates.18,19 These form
primary alkyl cations, or, as in the case of aziridinium ions from
nitrogen mustards, masked alkyl cations which are hard Lewis
acids and would be expected to react at N7-G according to
Pearson’s hard-soft acid/base theory. Arylnitrenium ions, the
ultimate carcinogenic metabolites formed from aromatic
amines, are also nitrogen-centred electrophiles but these are
highly delocalised20 and one of their principal points of
attachment to DNA is C8 on guanine (C8-G).21,22 This is in
accord with recent findings by McClelland which illustrated
There are several notable exceptions to these activity trends.
The presence of sterically demanding substituents such as
p-tert-butyl groups reduce mutagenicity15 while mutagens with
† Electronic supplementary information (ESI) available: a partial
sequence of the pBR322 DNA, solvolysis rate constants and primary
bimolecular rate constants. See http://www.rsc.org/suppdata/ob/b3/
b301618h/
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 3 8 – 2 2 4 6
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Table 1 Rate constants, log P values, Ames mutagenicities and
predicted mutagenicities for N-benzoyloxy-N-benzyloxybenzamide 3,
N-benzyloxy-N-(4-methylbenzoyloxy)benzamide 4, N-acetoxy-N-(4-
phenylbenzyloxy)benzamide
amide 6
5 and N-acetoxy-N-butoxy-2-naphth-
3
4
5
6
102K3H08/dm3 molϪ1 sϪ1 a
104K3208/dm3 molϪ1 sϪ1 b
Log Pc
0.37 0.41 0.876 4.69f
261
110
n.d.g
654f
4.33
3.60
2.96
5.03 5.49 5.04
3.22 3.29 3.09
Log TA100/1 µmol plateϪ1 d
Calculated LogTA100/1 µmol plateϪ1 e 3.02 3.13 3.02
a Rate of AAl1 acid-catalysed solvolysis in acetonitrile–water at
308 K.3,4,6 b Rate of SN2 reaction with N-methylaniline in methanol at
308 K.13 c Computed value using ACD Laboratories software.36 d Scaled
relative to 9 (see experimental section); data from previous studies.7
e Computed relative to 9 from the relationship in equation (1). f This
study. g Not determined.
that the intermediate formed from direct attack of various
arylnitrenium ions on C8-G is the initial species observed by
laser flash photolysis experiments.20,23,24 Alkoxyacylnitrenium
ions are strongly delocalised onto electronegative oxygen and,
as such, must be regarded as being harder than arylnitrenium
ions.25 If they are intermediates in the genotoxicity of N-acyl-
oxy-N-alkoxyamides, they would be expected to attack DNA at
N7-G. On the other hand, nucleophilic sites on the DNA bases
could also react bimolecularly with 1 in an SN2 reaction.
Other relatively nucleophilic centres on DNA include N3 on
guanine (N3-G), the exocyclic 2-amino group on guanine, (N2-
G)26–29 and N3 on adenine (N3-A) and reactivity at all these
centres is dependent upon base sequence and accessibility;29
purine N3 in the minor groove is regarded as less accessible to
electrophiles than N7 of purines. It is well known that N7-G at
the 5Ј-end of poly-G sequences is the most nucleophilic centre
in DNA and will react more readily with electrophiles.30–32
Base stacking of this kind is also responsible for preferential
oxidative damage at 5Ј-guanines in poly-G sequences.33–35
To date, we have assumed that mutation of Salmonella
typhimurium must involve modification of prokaryotic DNA in
the cytosol by amides of type 1. In this paper we describe DNA
damage studies that point to site and groove preferences in the
chemical modification by 1. In addition, we provide unequi-
vocal evidence for the requirement for activation of the nitrogen
by both an alkoxy and an acyloxy functionality and evidence
that alkoxynitrenium ions are unlikely to be formed in vitro or
in the cytosol prior to reaction with DNA.
mutagenic activity is inversely correlated with reactivity, the
mutagenicity of 6 would be expected to be lower than predicted
and its enhanced activity cannot be ascribed to its greater
survival under the assay conditions. We have interpreted this
to indicate that the naphthyl group promotes a stronger
association with DNA leading to a greater than expected
mutagenicity.7
Fig. 1 illustrates the strand cleavage profile at pH 5.8 and 7.8
for the three polyaromatic mutagens 3–5 determined with
untreated DNA at both pH’s as reference. All three showed
significant cleavage due to reaction at N7-G in the major
groove. Comparison of the pH 5.8 and 7.8 lanes for each sub-
stance indicated that guanine damage appeared to be pH
dependent with greater reaction at basic rather than acidic pH.
The low pH lane for the naphthalene-containing substrate 6 did
not load on this gel. However, the high pH reaction was similar
to that of 3–5. In addition, while all guanines were reactive, a
sequence dependence was evident in a greater preference for the
5Ј-end of GG (G80-81) and GGG (G129-131) sequences.
Cleavage at adenines was also evident but only in the low pH
lanes (A56, 57, 61, 62, 65, 73, 83, 85, 87, 92, 94, 100, 132, 136
and 139). According to Pullman and Pullman,17 the recognised
order of reactivity of nucleophilic centres on DNA purines with
electrophilic species is N7-G >> N3-G∼N3-A > O6-G > N7-A
and presumably adenine damage arises out of reaction at N3 in
the minor groove. Adenine damage also appears to be substrate
dependent. Relative to 3, the biphenyl-containing substrate 5 is
less reactive at most adenines but appears to react significantly
at A83. The naphthamide 6 damages some adenines (e.g. A61,
A62 and A92) even at basic pH and, relative to the corre-
sponding lanes of benzamides 3–5, guanine damage would
appear to be more extensive.
Results
The mutagenicity of amides 1–6 has been described previously.
Relative activities of 3–5 are adequately described by their
hydrophobicity. The most recent QSAR for all mutagens of
this class is given in equation (1)7 and predicted, together with
log TA100 = 1.78 ϩ 0.25 log P.
(r = 0.889, s = 0.161, n = 35)‡
(1)
experimental values for all three are presented in Table 1. Table
1 also gives rate constants for both AAl1 acid-catalysed ester
solvolysis and SN2 reactivity with N-methylaniline.
Based upon its log P value, the naphthamide 6 is predicted to
be much less mutagenic than is observed. All four substrates
reacted by both AAl1 acid-catalysed solvolysis (SN1 at nitrogen)
and SN2 reactions with N-methylaniline, and the naphthamide 6
is, if anything, predicted to be more reactive than the other
three substrates. Since we have shown in the case of 2 that
The influence of pH upon base selectivity is illustrated
for mutagen 3 in Fig. 2. All reactions were incubated for 16 h
‡ r = correlation coefficient; s = standard error; n = number of
observations.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 3 8 – 2 2 4 6
2239

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Fig. 1 Strand cleavage patterns obtained by the chemical treatment of
the 3Ј-end-labelled EcoR1 to BamH1 fragment of pBR322 DNA with 4
(lanes 3 and 4), 3 (lanes 5 and 6), 5 (lanes 7 and 8) and 6 (lanes 9 and
10). All lanes incubated for 16 hours followed by heating in neutral
buffer then hot piperidine. Lane 1 and 2 are the DMSO control lanes.
Lanes 1, 3, 5, 7, 9 = pH 5.8. Lanes 2, 4, 6, 8, 10 = pH 7.8. Lanes 11, 12
and 13 are guanine, purines and thymine, respectively.
followed by a heat and piperidine treatment. At pH 6.6 and
above, only guanine is damaged. Analysis of densitometry pro-
files for lanes 4 to 9 (Fig. 3) indicates that all guanines are
damaged and that damage increases with pH. However, at pH
5.8 (lane 2), the DNA is severely damaged as evidenced by the
lack of DNA at the origin and increasing intensity towards the
lower end of the gel. Densitometry on lane 2 clearly shows
excessive damage at adenines 33–35, 43, 57, 65, 94, 117 and 132
and, in at least three clear cut cases, the adenine is 5Ј- to a
guanine (G32, A33–35; G64, A65 and G93, A94). A far higher
degree of minor groove damage at pH 5.8 was observed in this
experiment when compared to the reaction of 3 at pH 5.8 in the
previous study. Significant variability of the degree of minor
groove damage was noted in this and in subsequent in vitro
DNA damage studies using these mutagens and this will be the
subject of a future publication.37
Fig. 2 Strand cleavage patterns obtained by the chemical treatment of
the 3Ј-end-labelled EcoR1 to BamH1 fragment of pBR322 DNA with
3. All lanes incubated for 16 hours followed by heating in neutral buffer
then hot piperidine. Lane 1 is the DMSO control. Lanes 2–9 are pH 5.8,
6.2, 6.6, 7.0, 7.4, 7.8, 8.2 and 8.6, respectively.
The requirement for biological activity of both alkoxy as
well as acyloxy functionality at the amide nitrogen is evident
from a comparison of the mutagenicities of 3 and the par-
tially functionalised analogues 7 and 8. 7 lacks an activating
N-alkoxy group while in 8, the N-acyloxy group is replaced by
benzyl. The mutagenicities in TA100 for 3, 7 and 8 are given in
Table 2.
In line with previous studies on this class of compounds,
N-benzoyloxy-N-benzyloxybenzamide 3 afforded an excellent
dose response over the dose range 0–0.5 µmol plateϪ1 but both 7
and 8 were inactive over this range (Fig. 4). While this would be
expected for 8 which lacks a suitable leaving group at nitrogen,
the lack of activity for 7 must be attributed to the absence of an
Fig. 3 Combined densitometry for lanes 2–9 (pH 5.8–8.6) from Fig. 2.
alkoxy group. Clearly, both a donor N-alkoxy group and an
N-acyloxy leaving group are required for mutagenic activity.
DNA damage studies mirror these results. Fig. 5 illustrates
the DNA cleavage patterns for untreated DNA, and DNA
treated with 3, 7 and 8 at pH 6.6, 7.4 and 8.6. DNA treated
with 7 or 8 shows only faint damage which is little more than
background. Lanes 2, 6 and 10, corresponding to DNA treated
with mutagen 3, show increasing damage with pH once again
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 3 8 – 2 2 4 6
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Table 2 Salmonella mutagenicities of N-benzoyloxy-N-benzyloxybenzamide 3, N-benzoyloxy-N-benzylbenzamide 7, N-benzyl-N-benzyloxy-
benzamide 8 and N-benzoyloxy-N-butoxybenzamide 11 in TA100a, b
µmol plateϪ1
9d, e f
µmol plateϪ1
3d
7d
8d
µmol plateϪ1
11d, f
0.000
211(12)
[165(20)]
441(6)
[312(19)]
634(30)
[395(21)]
939(69)
[712(46)]
—
0.000
0.060
0.130
0.250
0.500
211(12)
471(27)
645(68)
947(31)
1544(49)
2572.3
211.3(12)
208.3(5)
213.3(17)
221.7(26)
219.0(14)
—
211(12)
214(8)
213(3)
199(4)
202(6)
—
0.000
211(12)
[165(24)]
250(17)
[512(13)]
550(60)
[662(11)]
730(37)
[1073(46)]
1062(63)
[1382(103)]
1117.5
0.250
[0.120]
0.500
[0.250]
1.000
[0.500]
1.500
0.120
0.250
0.500
1.000
[1.000]
[1269(92)]
717.71
[1106.1]
Dose responsec
Dose responsec
[1733.4]
a Without S9 homogenate; mutagens in this class are direct-acting and do not require metabolic activation. b Solvent DMSO. c Revertants at 1 µmol
plateϪ1 calculated from gradient of revertants plateϪ1 versus dosage. d 1 Standard deviation in parentheses. e Reference mutagen. f Mutagenicity of 11
was repeated; duplicate values for reference mutagen 9 and 11 in square brackets.
Fig. 4 Comparison of dose-response for 3,7 and 8 in TA100 without
metabolic activation.
accentuated at the 5Ј-G of stacked GG sequences (Fig. 6). In
addition, there is evidence for weak adenine damage.
While mutagenicity and DNA damage studies confirm the
requirement for an amide nitrogen activated towards displace-
ment of carboxylate through the geminal alkoxy group, from
these studies no distinction can be drawn between SN1 or SN2
displacement reactions with DNA. The former process would
invoke the intermediacy of highly electrophilic N-acyl-N-
alkoxynitrenium ions that, like one ultimate carcinogenic form
of arylamine metabolites, N-acetyl-N-arylnitrenium ions,
would be expected to react with DNA. Arylnitrenium ions react
at near diffusion rates with purine nucleosides yielding mostly
the C8 adduct and these adducts have been formed from in vitro
and in vivo studies with DNA. However, this depends upon
their lifetimes in water. Most arylnitrenium ions react rapidly
Fig. 5 Strand cleavage patterns obtained by the chemical treatment of
with water (kw = 1 × 105–1 × 107) even though they are highly
the 3Ј-end-labelled EcoR1 to BamH1 fragment of pBR322 DNA.
delocalised.20,38–40 Precursors such as 2-aminofluorene or 4-
Lanes 1–4 = pH 6.6, 5–8 = pH 7.4 and 9–12 = pH 8.6. Lanes 1, 5 and 9
aminobiphenyl, which form nitrenium ions that are configur-
ationally more resistant to attack by water, react more select-
ively with bionucleophiles and are more mutagenic.38,41–43 In
contrast, N-acyl-N-alkoxynitrenium ions are far less delocalised
and are likely to be very reactive with water. This has been
illustrated in the analysis of the products from the acid-cata-
lysed solvolysis of N-acyloxy-N-alkoxyamides where N-acyl-N-
alkoxynitrenium ions, formed by AAl1 solvolysis, are rapidly
quenched by water.3,4,6 They are unlikely to survive long enough
to reach target DNA during in vitro DNA studies or in the
Ames mutagenicity assay.
are DMSO controls. Lanes 2, 6 and 10 were incubated with 3. Lanes 3, 7
and 11 were incubated with 7. Lanes 4, 8 and 12 were incubated with 8.
All lanes incubated for 16 hours followed by heating in neutral buffer
then hot piperidine. Lanes 13, 14 and 15 are guanine (faint), purines
and thymine, respectively.
A comparative study of the reactivities, mutagenicities and
DNA damage profiles of four mutagens 3, 9–11 confirms that
N-acyloxy-N-alkoxyamides bind intact with DNA.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 3 8 – 2 2 4 6
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Table 3 Rate constants, log P values, Ames mutagenicities and predicted mutagenicities for N- benzoyloxy-N- benzyloxybenzamide 3, N-acetoxy-N-
butoxybenzamide 9, N-acetoxy-N-benzyloxybenzamide 10 and N-benzoyloxy-N-butoxybenzamide 11
9
10
11
3
102K3H08/l molϪ1 sϪ1 a
2.6
581
3.1
2.50
2.54
0.68
174f
3.28
2.63
2.59
1.57f
2553f
4.85
0.41
261
5.03
3.05f
3.02
104K3208/l molϪ1 sϪ1 b
Log Pc
Log TA100/1 µmol plateϪ1 d
Calculated Log TA100/1 µmol plateϪ1 e
2.70f
2.98
a Rate of AAl1 acid-catalysed solvolysis in acetonitrile–water at 308 K.3,4,6 b Rate of SN2 reaction with N-methylaniline in methanol at 308 K.13
c Computed value using ACD Laboratories software.36 d Scaled relative to 9 (see Experimental section); data for 10 and 11 from previous studies.7
e Computed relative to 9 from the relationship in equation (1). f This study.
Fig. 6 Densitometry of lanes 2, 6 and 10 (for 3) from Fig. 5.
Table 3 illustrates comparative AAl1 (SN1) and SN2 reaction
rate constants at 308 K for mutagens 3, 9–11, together with
their mutagenicities at 1 µmol plateϪ1, computed log P values
and predicted mutagenicities. Replicate Ames mutagenicity
data for the new mutagen 11 was highly reproducible (after
scaling to 9) and is presented together with that for 3, 7 and 8
in Table 2. While relative reactivities are similar for all four
substances with the exception of 11, which undergoes SN2 reac-
tions with N-methylaniline much more rapidly than 3, 9 or 10,
mutagenicities vary significantly and correlate with their hydro-
phobicity in line with our previously reported dependence.
Clearly, overall mutagenicity levels are dependent upon the
hydrophobicity of the full structure. If nitrenium ion inter-
mediates were to be formed prior to association with DNA, 9
and 11 would form the same nitrenium ion, as would 3 and 10
and mutagenicities would have been expected to be comparable
for each pair of substrates.
In vitro DNA damage studies support this assertion. Fig. 7
illustrates damage for 3, 9–11 and untreated DNA at pH 6.6,
7.4 and 8.6. 3 and 10 showed normal guanine (all guanines) and
adenine damage (e.g. A56, A57, A61, A62, A65, A83, A85 and
A87), but guanine damage in the case of 3 strongly increases
with pH whereas the corresponding damage with 10 does not.
(lanes 4–6), 9 (lanes 7–9), 10 (lanes 10–12) and 11 (lanes 13–15). All
Fig. 7 Strand cleavage patterns obtained by the chemical treatment of
the 3Ј-end-labelled EcoR1 to BamH1 fragment of pBR322 DNA with 3
This is best illustrated in densitometry on the well-resolved
lower regions of the gel (Fig. 8 (b) and (d)). Similarly, 9 and 11
both damage guanines without discernable adenine damage but
their guanine damage profiles are different. Guanine damage
increases from low to high pH for 11 but not significantly for 9
(Fig. 8 (a) and (c)). Solvolysis of N-benzoyloxy-N-benzyloxy-
benzamide 3 and N-acetoxy-N-benzyloxybenzamide 10 would
yield the same N-benzoyl-N-benzyloxynitrenium ion which
would have led to identical pH and selectivity profiles from
both reactions. Similarly, N-acetoxy-N-butoxybenzamide 9 and
N-benzoyloxy-N-butoxybenzamide 11 would have formed a
common N-benzoyl-N-butoxynitrenium ion. Thus, it is appar-
ent that reactivity profiles are determined in each case by the
intact mutagen leading to four distinct DNA damage patterns.
lanes incubated for 16 hours followed by heating in neutral buffer then
hot piperidine. Lane 1–3 are the DMSO control. Lanes 1, 4, 7, 10, 13 =
pH 6.6. Lanes 2, 5, 8, 11, 14 = pH 7.4. Lanes 3, 6, 9, 12, 15 = pH 8.6
partition coefficients, log P (Table 1).7 The naphthamide
analogue 6 has a lower calculated log P (4.38) but its significant
DNA reactivity is in line with the mutagenicity of this substrate,
which is nearly double that of 3 and has been attributed to an
intercalative association with DNA. In a future paper, we will
show that a series of naphthalene-containing substrates show
similarly increased reactivities relative to other members of this
class of mutagens, in support of an alternative intercalative
binding mode with DNA.37
The mutagenic behaviour of 3–6, as well as that of other
mutagens,7 while clearly dependent upon hydrophobicity or
possibly intercalation in the case of 6, has, to date, not been
unequivocally ascribed to reactivity at nitrogen or, more speci-
fically, to either an SN1 or an SN2 process. These results point to
the fact that the process whereby these mutagens damage DNA
is clearly electrophilic and requires the presence of, not only a
leaving group, but an activating alkoxy group at the amide
Discussion
Mutagens 3, 4 and 5 show similar reactivity towards DNA in
line with their mutagenicities that correlate linearly with their
hydrophobicities. This is demonstrated by the similarities in
their calculated log TA100 values based upon octanol–water
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 3 8 – 2 2 4 6
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Fig. 8 Densitometry of (a) lanes 7, 8 and 9 (for 9), (b) lanes 10, 11 and 12 (for 10), (c) 13, 14 and 15 (for 11), and (d) 4, 5 and 6 (for 3) from Fig. 7.
nitrogen. The limiting reagents, N-benzoyloxy-N-benzyl-
benzamide 7 (log P = 4.58) and N-benzyl-N-benzyloxybenz-
amide 8 (log P = 5.02), which are computed to have similar
hydrophobicities to 3, are inactive towards DNA in both the
DNA damage studies as well as in Salmonella mutagenicity
tests.
N—O acyl bond, together with the sp3 hybridisation of nitro-
gen, facilitates Љcarbon-typeЉ substitution reactions at the
amide nitrogen. These mutagens can be expected to react with
nucleophilic centres on DNA by either an SN1 or an SN2
process.
Mutagenicities in TA100 (Table 3) and the DNA damage
profiles of 3, 9–11 (Fig. 7) suggest that nitrenium ions are not
the reactive species that interact with DNA as the mutagens
clearly associate with DNA intact. These results indicate that
the reaction with N7-G and N3-A is most probably best
described as an SN2, rather than an SN1 process. The difference
between the mutagenicity and DNA damage reactivity of 3
and 7, where the anomerically destabilising oxygen atom is
absent, can be accounted for on the basis of either SN1 or SN2
reactivity. However, recent ab initio calculations on model SN2
reactions of ammonia with N-formyloxy-N-methoxyform-
amide 12 and N-formyloxy-N-methylformamide 13 predict an
activation barrier twice as large where N-methyl (EA = 140 kJ
molϪ1) as opposed to N-methoxy (EA = 72 kJ molϪ1) is present.47
A similar outcome was computed for the SN2 reaction with
azide.10 Furthermore, the computed transition state geometry
for the reaction of ammonia with N-formyloxy-N-methoxy-
formamide (Fig. 9(c)), allows maximal overlap between the
p-type lone pair on the methoxy oxygen and the departing
N—OCHO bond pointing to the importance of this inter-
action in promoting such reactions by nucleophiles at the amide
nitrogen.
The role of an alkoxy or an amino group in anomeric weak-
ening of an N—O acyl, N—O alkyl or N—halogen bond has
been described previously.5,10,44–48 In these anomeric amides, the
nitrogen is highly pyramidalised, a consequence of the com-
bined electron demand of the two electronegative oxygen atoms
which is better met with longer sp3 hybrid orbitals at nitrogen
(Fig. 9(a)). The lone pair is consequently also sp3 and hence
most, if not all, resonance delocalisation onto the carbonyl is
lost. Infrared carbonyl stretch frequencies of all these mutagens
reflect this; they absorb in the region of 1720–1740 cmϪ1 which
is completely atypical of simple amides.5 In addition, we have
evidence from theoretical studies,5,44,47 and more recently from
X-ray analysis,49 that the conformation about the N—O alkyl
bond facilitates an anomeric interaction between the p-type
lone pair on oxygen and the N—O acyl σ* orbital in a classical
anomeric interaction (Fig. 9(b)). This destabilisation of the
Analysis of reactivity patterns of 3 at low pH (Figs. 1–3)
indicates that the mutagen damages DNA in pH-dependent
fashion. Minor groove damage at N3-A, prevalent from reac-
tions at acidic pH (Figs. 1–3), is disfavoured in neutral to basic
buffers. By analogy with alkylation reactions, the ammonium
ion adduct from N3-A attack, leads to direct fragmentation of
DNA upon application of heat (90 ЊC).50,51 At 37 ЊC and under
basic reaction conditions, this adduct might be unstable under-
going hydrolysis to hydroxamic acid resulting in regeneration of
Fig. 9 (a) Pyramidal nitrogen and (b) anomeric interaction in N-
acyloxy-N-alkoxyamides; (c) HF/6-31G* transition state for reaction of
ammonia with N-formyloxy-N-methoxyformamide.
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Scheme 1
adenine (Scheme 1). Evidence will be presented elsewhere which
supports this proposal.37
Guanine damage is ascribed to the reaction of mutagens at
N7-G in the major groove which leads to lability of the purine–
deoxyribose bond resulting in DNA cleavage upon treatment
with hot piperidine.52 While the trend is not as evident with all
mutagens, the pH dependence of guanine damage, particularly
evident with mutagens 3–5 and 11 is interesting. For instance,
Scheme 2
with N-benzoyloxy-N-benzyloxybenzamide 3,
a
marked
increase in damage is evident with increasing pH in Figs. 1, 2, 5
and 7. While the N7-G adduct, like the N3-A adduct in the
minor groove, may be labile to base, hydrolysis is more likely to
involve attack on C8 of the purine which becomes highly sus-
ceptible to nucleophilic attack upon functionalisation of N7
(Scheme 2). Thus, in neutral to basic buffer, hydrolysis and ring-
opening of the imidazole ring, which corresponds to the first
step in the DNA cleavage sequence, would occur.
The difference between the N-benzoyloxy and N-acetoxy
mutagens is not clear although the pH dependence may well be
more evident with more strongly hydrophobic mutagens. While,
in Figs. 7 and 8, 9 and 10 show relatively similar damage across
the pH profile, 5, which also bears an N-acetoxy leaving group,
but which has a greater overall hydrophobicity (log P =5.04 as
opposed to 3.1 and 3.28), also exhibits greater guanine damage
at higher pH (Fig. 1).
Experimental
Chemicals
General syntheses of mutagens 3–6, 9 and 10 have been
described elsewhere3,4,6,7 as has the synthesis of N-benzoyloxy-
N-benzylbenzamide 7.10 N-Benzoyloxy-N-butoxybenzamide 11
and 8 were synthesised for the first time in this study. Pet. spirit
was the fraction boiling between 60 and 80 ЊC. DCM is
dichloromethane. 40% (19
: 1) acrylamide–bisacrylamide
was purchased from Bio-Rad Laboratories Pty Ltd. pBr322,
Klenow fragment, BamH1, EcoR1, BSA (bovine serum
albumin) and NE buffer 2, were purchased from (New England
Biolabs) Genesearch Pty Ltd. α-dATP([α-32P]-deoxyadenosine-
5Ј-triphosphate (10 µCi µlϪ1), isoblue stabilized) was purchased
from ICN Biomedicals Inc.
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Synthesis of N-benzoyloxy-N-butoxybenzamide 11
Depurination and subsequent strand breaks at all purines
modified at N7-G, N3-A and N7-A50,52,53 were effected by heat-
ing at 90Њ (15 minutes) in water and then at 90Њ in 1.1 M
piperidine.
N-Butoxy-N-chlorobenzamide. N-Butoxybenzamide3 (1.5 g,
7.76 mmol) and neat tert-butyl hypochlorite (2.53 g, 23.3 mmol)
was stirred for 10 minutes in the dark. Removal of excess hypo-
chlorite in vacuo provided the title compound which was used
immediately without further purification. νmax(CHCl3)cmϪ1
Where appropriate, sequencing lanes were produced by the
Maxam–Gilbert sequencing reactions.50,51 Samples were
denatured at 95 ЊC and loaded onto a 0.4 mm 6% denaturing
acrylamide–bisacrylamide (19 : 1) polyacrylamide gel which
was run at a constant 75 W until the xylene cyanol had
migrated 26 cm from the bottom of the loading wells (ca. 2 h).
At the completion of the run, an autoradiogram was generated
with Kodak XAR-5 film (Ϫ80 ЊC for 15–20 hours) using a
Kodak Biomax MS intensifying screen.
1718s (C᎐O), 1239m; δ (300 MHz, CDCl ) 0.90 (3H, t,
᎐
H
3
CH3), 1.32 (2H, m, CH2CH3), 1.58 (2H, m, OCH2CH2),
4.14 (2H, t, OCH2), 7.46 (2H, t, m-Ar–H), 7.57 (1H, t, p-Ar–H),
7.79 (2H, d, o-Ar–H); δC(75 MHz CDCl3) 13.6 (q), 19.0 (t),
29.4 (t), 74.5 (t), 128.3 (d), 129.3 (d), 131.2 (s), 132.8 (d), 174.2
(s).
N-Benzoyloxy-N-butoxybenzamide. Sodium benzoate (0.89 g,
6.15 mmol) was stirred at room temperature with N-butoxy-N-
chlorobenzamide (1.0 g, 4.4 mmol) in dry acetone for 24 hours.6
The solvent was removed in vacuo and purification was
achieved by centrifugal chromatography with ethyl acetate–pet
spirit (0.91 g, 66%). The oil was characterised spectroscopically.
νmax(CHCl3)cmϪl 1758s (C᎐O), 1728s (C᎐O), 1238s; δ (300
MHz, CDCl3) 0.91 (3H, t, CH3), 1.39 (2H, sextet, CH2CH3),
1.69 (2H, quintet, OCH2CH2), 4.31 (2H, t, OCH2), 7.43 (2H, t,
m-Ar–H), 7.46 (2H, t, mЈ-Ar–H), 7.54 (lH, t, p-Ar–H), 7.62
(1H, t, pЈ-Ar–H), 7.87 (2H, d, o-Ar–H), 8.03 (2H, d, oЈ-Ar–H);
δC(75 MHz, CDCl3) 13.7 (q), 19.0 (t), 30.1 (t), 75.6 (t), 127.4 (d),
128.3 (d), 128.6 (d), 129.1 (d), 130.0 (d), 131.9 (s), 132.7 (d),
134.0 (s), 164.3 (s), 174.5 (s).
Mutagenicity assays
Salmonella typhimurium strain TA100 was obtained from Pro-
fessor B. N. Ames, University of California, Berkeley, U.S.A.
and cultured as described54 with the exception that fresh broth
cultures were incubated at 37 ЊC in a shaking water bath for 10
h prior to use in each assay. Top agar, supplemented with a trace
of histidine and biotin, was dispensed in 2 ml volumes into 5 ml
plastic vials and maintained at 45 ЊC in a water bath. Before
pouring on the surface of minimal agar plates, 0.1 ml of
the broth culture, 0.1 ml of the test chemical dissolved in dry,
analytically pure DMSO were added to each vial. Tripli-
cate plates at each dose level were incubated at 37 ЊC for 72 h
before counting revertant colonies with an Artek model 880
counter.
Assays at different dose levels were carried out in triplicate
together with negative controls. Responses for each compound
at 1 µmol plateϪ1 were obtained from least squares analysis of
linear regions of the plots of mean revertants/plate versus dose.
Comparative data were derived by scaling mutagenicities at
1 µmol plateϪ1 to that of a single standard, N-acetoxy-N-
butoxybenzamide 9 (Log TA100 = 2.5),7 which was always
analysed in parallel with new mutagens.
᎐
᎐
H
Compound 8 was synthesised by benzylation of N-benzyl-
oxybenzamide the synthesis of which was described previ-
ously.3
Synthesis of N-benzyl-N-benzyloxybenzamide 8. N-Benzyl-
oxybenzamide (1.00 g, 0.0044 mol) and benzyl bromide (0.75 g,
0.0044 mol) were dissolved in 10% aqueous methanol (20 ml).
Potassium hydroxide (0.35 g, 0.0062 mol) was added and the
solution was stirred for 24 h. The methanol was removed in
vacuo and water was added (25 ml). The solution was extracted
with DCM (3 × 20 ml) which was washed with dilute HCl
(20 ml), H2O (20 ml), 10% Na2CO3 (20 ml) and dried over
MgSO4. The pure product was obtained by centrifugal chrom-
atography using ethyl acetate–pet spirit (0.47 g, 34%) as eluant
(Found: C, 79.88; H, 6.15; N, 4.29%. C21H19NO2 requires C,
Kinetic studies
AAl1 acid-catalysed solvolysis. The procedure for determin-
ing rates of acid-catalysed solvolysis has been described pre-
viously3,4,6 and involves dissolving a small quantity of the
N-acyloxy-N-alkoxyamide (typically between 10–40 mg) in 26%
(D2O)–(CD3CN) in an NMR tube. The mixture was preheated
and shimmed in the probe of the NMR spectrometer before an
appropriate volume of sulfuric acid–D2O solution was injected
79.47; H, 6.03; N, 4.41%); νmax(CHCl3)cmϪ1 1636s (C᎐O);
᎐
δH(300 MHz, CDCl3) 4.56 (2H, s, NCH2), 4.94 (2H, s, OCH2),
6.97 (2H, d, Ar–H), 7.28 (3H, m, Ar–H), 7.36–7.46 (8H, m, Ar–
H), 7.70 (2H, d, Ar–H); δC(75 MHz, CDCl3) 51.5 (t), 77.1 (t),
127.8 (d), 128.0 (d), 128.3 (d), 128.4 (d), 128.6 (d), 128.7 (d),
128.8 (d), 129.5 (d), 130.6 (d), 134.1 (s), 134.5 (s), 136.4 (s).
Carbonyl carbon absent.
1
to initiate solvolysis. H NMR spectra were then acquired at
intervals using an automated program. Rates were obtained by
integration of appropriate resonances. Solvolysis rate constants
were obtained at four different temperatures in the range of
293 K to 323 K with correlation coefficients close to unity and
are presented in Table S4 of supplementary material to this
paper.†
Labelling and isolation of a plasmid DNA fragment
A 375 base pair EcoRI to BamHI fragment of plasmid pBR322
DNA was 3Ј end labelled at the EcoRI site using Klenow
fragment and [α-32P]dATP(3000 Ci mmolϪ1) according to a
published procedure.51 The fragment was isolated on a 4% non-
denaturing polyacrylamide gel. A partial sequence of the
pBR322 DNA used in this work is presented as supplementary
material to this paper.
SN2 reactions with N-methylaniline. Rate constants for the
bimolecular reaction between N-acyloxy-N-alkoxybenzamides
6, 10 and 11 and N-methylaniline were determined using H
1
NMR spectroscopy.12,13 5–20 mg of N-acyloxy-N-alkoxy-
benzamide in 400 µl of methanol-d4 was equilibrated in a 5 mm
NMR tube at the required temperature prior to addition of
N-methylaniline (5–30 µl). The exact time of mixing was noted.
1H NMR spectra were collected using an automated program
after which progress of the reaction was monitored by inte-
gration of representative NMR resonances of the substrate and
the N-methyl resonance of N-methylaniline. Initial substrate
concentrations were obtained by back extrapolation of concen-
tration plots for both reagents to the initial time of mixing,
to. Primary bimolecular rate constants were obtained at four
temperatures between 290 K and 325 K and are presented in
Table S5 of supplementary material to this paper.†
DNA reaction studies32
The compound to be tested was prepared as a 50 mmol stock
solution in dry DMSO. The labelled DNA was prepared in a
solution of TE buffer such that the radiation level was 15000
cpm µlϪ1. A typical alkylation reaction mixture consisted of 1 µl
of labelled DNA in 97 µl of 10 mM phosphate buffer and 2 µl
of compound stock solution (final volume 100 µl). Samples
were incubated for 16 h at 37 ЊC whereupon they were immedi-
ately precipitated with ethanol and lyophilised.
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25 S. A. Glover and A. P. Scott, Tetrahedron, 1989, 45, 1763.
Acknowledgements
26 D. E. Thurston and D. S. Bose, Chem. Rev., 1994, 94, 433.
27 N. Cooper, D. R. Hagan, A. Tiberghien, T. Ademefun, C. S.
Matthews, P. W. Howard and D. E. Thurston, Chem. Commun.,
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The authors are grateful to the Australian Research Council for
a Large Grant and to the University of New England for a
Ph.D. scholarship to Tony Banks.
28 A. Kamal, G. Ramesh, N. Laxman, P. Ramulu, O. Srinivas,
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