Design, synthesis, and DNA sequence selectivity of formaldehyde-mediated DNA-adducts of the novel N-(4-aminobutyl) acridine-4-carboxamide

Article abstract of DOI:10.1016/j.bmcl.2014.10.062

A novel derivative of the anti-tumor agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) was prepared by reduction of 9-oxoacridan-4-carboxylic acid to acridine-4-carboxylic acid with subsequent conversion to N-(4-aminobutyl)acridine-4-carboxamide (C4-DACA). Molecular modeling studies suggested that a DACA analogue comprising a side chain length of four carbons was optimal to form formaldehyde-mediated drug-DNA adducts via the minor groove. An in vitro transcription assay revealed that formaldehyde-mediated C4-DACA-DNA adducts selectively formed at CpG and CpA dinucleotide sequences, which is strikingly similar to that of formaldehyde-activated anthracenediones such as pixantrone.

Full text of DOI:10.1016/j.bmcl.2014.10.062

Accepted Manuscript
Design, synthesis, and DNA sequence selectivity of formaldehyde-mediated
DNA-adducts of the novel N-(4-aminobutyl) acridine-4-carboxamide
Elizabeth A. Ankers, Benny J. Evison, Don R. Phillips, Robert T.C. Brownlee,
Suzanne M. Cutts
PII:
S0960-894X(14)01122-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.10.062
BMCL 22117
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
8 September 2014
13 October 2014
17 October 2014
Please cite this article as: Ankers, E.A., Evison, B.J., Phillips, D.R., Brownlee, R.T.C., Cutts, S.M., Design, synthesis,
and DNA sequence selectivity of formaldehyde-mediated DNA-adducts of the novel N-(4-aminobutyl) acridine-4-
carboxamide, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.10.062
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis, and DNA sequence selectivity of formaldehyde-mediated
DNA-adducts of the novel N-(4-aminobutyl) acridine-4-carboxamide
a
b,c
b
a
b,
Elizabeth A. Ankers , Benny J. Evison , Don R. Phillips , Robert T.C. Brownlee and Suzanne M. Cutts
a
Department of Chemistry, La Trobe Institute for Molecular Sciences, La Trobe University, Bundoora 3086, Australia
Department of Biochemistry, La Trobe Institute for Molecular Sciences, La Trobe University, Bundoora 3086, Australia
b
c
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A novel derivative of the anti-tumor agent N-[2-(dimethylamino)ethyl] acridine-4-carboxamide
(DACA) was prepared by reduction of 9-oxoacridan-4-carboxylic acid to acridine-4-carboxylic
acid with subsequent conversion to N-(4-aminobutyl) acridine-4-carboxamide (C4-DACA).
Molecular modeling studies suggested that a DACA analogue comprising a side chain length of
four carbons was optimal to form formaldehyde-mediated drug-DNA adducts via the minor
groove. An in vitro transcription assay revealed that formaldehyde-mediated C4-DACA-DNA
adducts selectively formed at CpG and CpA dinucleotide sequences, which is strikingly similar
to that of formaldehyde-activated anthracenediones such as pixantrone.
Keywords:
Acridine-4-carboxamide
DNA adducts
Transcription assay
Formaldehyde activation
Energy minimized 3D model
2
014 Elsevier Ltd. All rights reserved.
Daunorubicin and doxorubicin are well characterized
topoisomerase II poisons and are among the most widely used
anticancer agents. Although these drugs have been extremely
useful in the treatment of a wide range of cancers, there have
been problems associated with unwanted side-effects including
2
mitoxantrone (see Figure 1) that showed no signs of acute or
delayed cardiotoxicity in mice. Phase I and II studies indicated
7
that pixantrone is well tolerated with the main toxicity being
8, 9
leucopenia.
Pixantrone is currently in phase III trials, and has
achieved significantly superior results relative to the comparator
group in the treatment of relapsed aggressive non-Hodgkin
1
,
dose-limiting cardiotoxicity and drug resistance.
1
0
Anthracyclines like doxorubicin and daunorubicin are still
obtained from culture extractions and their synthesis is costly in
lymphoma.
3
Doxorubicin is known to form covalent formaldehyde-
mediated adducts with DNA almost exclusively at GpC
both time and money. Their complex substituents (Figure 1)
require multiple protection and deprotection steps, while some
reactions proceed with low stereoselectivity. The development of
a novel bioactive compound that is easily synthesized, yet is
devoid of the cardiotoxicity associated with the anthracyclines,
has been highly sought after.
11
sequences, with an absolute requirement for the exocyclic
12
amine of guanine. High resolution X-ray crystallography (1.6
Å) of the analogous epidoxorubicin-formaldehyde adduct
revealed that the anthracycline chromophore intercalates between
the DNA base pairs, with the amino sugar in the minor groove,
which forms a covalent methylene bridge between the N3’ of
epidoxorubicin and the N2 of guanine (see Figure 2 which
Mitoxantrone is an anthracenedione that has similar biological
properties to the anthracyclines but exhibits a decrease in
unwanted side effects. The major dose-limiting factor for
mitoxantrone is leucopoenia, but side effects still include
1
3
depicts the covalent mode of binding). The adduct derived from
formaldehyde activation of doxorubicin was verified by another
group independently using NMR to be the same as seen in the X-
4, 5
myelosuppression and cardiotoxicity.
Although the clinical
1
4
cardiotoxic properties of mitoxantrone were more tolerable
relative to doxorubicin, a residual cardiotoxic effect was
attributed to the 5,8-para-hydroxyl substituents on the
ray structure. Compounds which can covalently bind to DNA
via a methylene bridge are new, potentially useful, drug leads.
6
Another class of drugs with similar DNA-intercalating
properties to the anthracyclines is the acridines. N-[2-
anthracenedione (see Figure 1). Screening of novel derivatives
of mitoxantrone led to the discovery of BBR 2778. Pixantrone
(
dimethylamino)ethyl]acridine-4-carboxamide (DACA,
see
(BBR 2778) is a topoisomerase II poison, with a mono-aza
15
Figure 3) is a dual topoisomerase I/II poison, also lacking the
chromophore lacking the 5,8-hydroxyl substituents of
—
——

Corresponding author. Tel.: +613-9479-1517; fax: +613-9479-1266; e-mail: s.cutts@latrobe.edu.au

cardiotoxic 5,8-para-hydroxyl substituents of mitoxantrone and is
much more economically favorable to synthesize than
compounds like doxorubicin (Figure 1).
Figure 3 The structure of N-[2-(dimethylamino)ethyl]acridine-4-
carboxamide (DACA) and substituent position numbering.
Specifically, this involved a modified version of DACA with a
terminal primary amine replacing the dimethylamino to allow
reaction with formaldehyde, and the modification of the side
chain length to enable DNA binding. The starting point for this
work was the X-ray crystal structure of epidoxorubicin
covalently bound to a short section of DNA via a formaldehyde-
mediated methylene bridge (depicted in Figure 2) reported by
1
3
Podell et al.
A series of acridine-like DNA intercalators and representative
DNA structures were obtained from www.pdblite.org and
www.rcsb.org
(Research
Collaboratory for
Structural
Bioinformatics). The structures were reviewed and geometric
1
7
information derived using RasMol.
Manipulations and
calculations on the structures were performed using a Silicon
Graphics Iris Computer using the MSI software Insight and
Discover. The structures needed to be “cleaned up” with the
water molecules in the crystal structure deleted, the correct
number of hydrogen atoms added, and the atom potentials and
Figure 1 Structures of daunorubicin, doxorubicin, pixantrone and
mitoxantrone
1
8
charges set according to the CVFF forcefield using Insight II. In
general, the literature X-ray structures positioned the acridine
ring system parallel to the DNA base pair axis, while the side
chains of acridine intercalators occupied both DNA grooves.
1
3
The literature crystal structure from 1qda
epidoxorubicin bound to the hexanucleotide d(CGCGCG)
Figure 2) and was used to generate the initial model. The
contained
(
1
9
intercalated drug from 465d (DACA) was manually overlapped
1
3
onto the epidoxorubicin chromophore. The acridine ring was
placed in the same orientation as the 465d PDB structure, with
the acridine ring parallel to the DNA bases. This allowed
examination of the binding sites in more detail and the
investigation of the base pair interaction with the intercalating
drug. The bottom three base pairs of the DNA structure were
removed to simplify the computational study and minimizations.
This resulted in the d(GCG) sequence with the drug in the
appropriate position (not shown).
Figure 2 The X-ray structure of the formaldehyde-mediated
1
3
As the calculations are generally performed without solvent,
distance constraints were applied to simulate hydrogen bonding
between base pairs and between the DNA and the terminal
nitrogen of the DACA side chain (see supplementary Table S1
and Figure S1). The structures were energy minimized using
steepest descent, then conjugate gradient methods (100,000
iterations). The application of distance constraints created a
stable platform for drug optimization, as the drug itself was not
constrained. This elucidated how long the side arm should be to
reach the guanine without too much strain or puckering of the
DNA.
epidoxorubicin-DNA adduct. The formaldehyde bridges are highlighted in
green.
DACA intercalates in DNA with its side chain in the minor
groove and the dimethylamino group associated with the base
1
6
pair directly beneath the acridine ring. In order to develop a
compound capable of forming formaldehyde-activated covalent
DNA adducts, molecular modeling of DACA intercalated in
DNA was undertaken. The compound resulting from this was the
novel N-(4-aminobutyl) acridine-4-carboxamide 4 in Scheme 1,
referred to as C4-DACA, with results from drug-DNA interaction
studies reported here.
The robustness of this procedure was tested with DACA
manually intercalated in d(GCG), which was energy minimized
to verify that the constraints used would be sufficient to stabilize
the DNA duplex. After minimizations were finished, the terminus
of the DACA side chain was altered from N(Me) to NH and the
It was clear from a comparison of the X-ray structure of the
formaldehyde-mediated epidoxorubicin-DNA adduct (Figure 2),
with the DACA structure shown in Figure 3, that changes to the
side chain length and structure were required.
2
2
complete minimization using the correct DNA restraints was
performed. The resulting structure showed a reasonable drug-

DNA interaction with minimal steric issues or distortion and
planar DNA bases around the intercalating section. The terminal
base pair showed minor distortion, as expected.
A series of simulations were then performed where the NH2
side chain of DACA (C2-DACA) was artificially activated with
formaldehyde, and bound to the same free amine on the guanine
as the epidoxorubicin molecule from 1qda to generate a covalent
bond with the amino group of guanine. The side chain length was
also optimized to determine the structure with least distortion of
the DNA bases. The side chain of C2-DACA was extended by
one to three atoms and bound to the DNA d(GCG) via the minor
groove. This was performed for the Schiff base of each DACA
analogue-formaldehyde complex where a covalent bond was
formed from the terminal drug-imine to the guanine amino group
of DNA. Constraints were again applied only to the DNA, and
the drug-DNA adduct was geometry optimized. It was found that
Scheme 1 Reagents and conditions (a) HgCl /Al/KOH, 1 h,
2
reflux; (b) 50% aq KOH, HCl/FeCl , 0.5 h, reflux, 32%; (c)
3
CDI/dioxane,
4 h, reflux; (d) 1,4-diaminobutane/CH Cl ,
2 2
overnight, RT, 68%.
extending the side chain by two CH groups (Figure 4) resulted
2
in the stable and relatively strain free structure of the drug bound
to d(GCG) represented in Figure 5.
The first step (1 to 2) was scaled down by a factor of 6 from
Atwell et al . 9-oxoacridan-4-carboxylic acid 1 was reduced by
2
1
mercury amalgam by adopting the literature precedent to afford
2
. Acridine-4-carboxamide (2) was activated by CDI in dioxane
and used without further purification. Subsequent treatment of 3
with 1,4-diaminobutane in CH Cl afforded 4. The oxalate salt of
2
2
4
was prepared and recrystallized in i-PrOH to afford the pale
yellow solid.
Initially, a crosslinking assay was employed to mimic results
Figure 4 Structure of the drug C4-DACA (4).
involving doxorubicin-DNA adducts reported by Cullinane et
1
2
al. Conditions are noted in Supplementary CA1. The DNA
2
2
source used was linearized pCC1 plasmid end labelled with
3
2
[
α P]dATP. There was a distinct retardation or electromobility
shift of the DNA, indicating double-stranded DNA evident with
increasing drug concentration in the presence of formaldehyde as
depicted in Figure 6a and 6b, implying that C4-DACA had
formed a covalent link to the DNA in a drug dependent manner.
It was noted that the total radioactive DNA present after the
phenol/chloroform wash decreased with an increase in C4-
DACA concentration. This was attributed to partitioning of the
DNA into the phenol layer at high adduct levels, which was
detected by radioactivity in the phenol layer. When 4 was
incubated with DNA in the absence of formaldehyde and
subjected to phenol extractions, >96% of drug was extracted,
consistent with a non-covalent intercalative mode of interaction.
Furthermore, 4 alone (in the absence of formaldehyde) was
insufficient to stabilize double-stranded DNA under denaturing
conditions, even prior to phenol extraction (Supplementary CA2
Figure 5 Resultant structure from a geometry optimization of C4-DACA
bound to the 3’ guanine of d(GCG) via a formaldehyde bridge. The
formaldehyde bridge is highlighted in green.
2
3, 24
Figure S4).
It was therefore concluded that 4 was efficient in
forming drug-DNA adducts only in the presence of
formaldehyde, consistent with a formaldehyde-mediated mode of
covalent binding. C2-DACA, a modified derivative of 4 with a
two carbon side chain, was also tested for its ability to stabilize
double-stranded DNA in the presence of formaldehyde. Adduct
formation was not observed in the presence of this compound
The structure represented in Figure 5 shows minimal
disruptions of the base pairs to yield the drug-DNA adduct,
including reasonable angles and torsion angles in the side chain.
This structure was the formaldehyde-mediated C4-DACA-DNA
adduct, which was the basis for further experimentation.
(
Supplementary CA3 Figure S6) supporting the requirement of a
N-[4-aminobutyl]acridine-4-carboxamide lacks the cardiotoxic
,8-para-hydroxyl substituents of mitoxantrone (Figure 1) and is
four carbon side chain linker for adduct formation.
5
likely to be a dual topoisomerase I/II poison by analogy to the
2
0
parent compound. The lack of chiral centers, readily available
precursors and minimal step synthesis makes 4 much simpler and
more economically favorable to synthesize than compounds like
doxorubicin (Figure 1).The full synthesis and characterization of
4
is given in Supplementary Synthesis and Characterization.

2
0
longer side chain derivatives. The in vitro transcription assay
was therefore appropriate and the method of choice for the
detection of formaldehyde-activated drug-DNA lesions. See
Supplementary Methods for the full in vitro transcription assay
method.
In vitro transcription results in an accumulation of truncated
transcripts that arise from the impairment of RNA polymerase
movement along double stranded DNA by the presence of drug-
DNA lesions. The length of each drug-induced truncated
transcript provides a direct measure of the location of the lesion
on the DNA template. In the absence of drug-induced blockages
RNA polymerase can progress through DNA templates to yield
3
1
full length transcripts (FLT) 379 nucleotides in length.
ds
ss
5
10
15
20
30
40
A simple drug concentration-dependence assay was used to
test whether C4-DACA could form sequence selective DNA
adducts. Prior to transcription, the 512 bp fragment containing
the lac UV5 promoter was reacted with 10 mM formaldehyde
and concentrations of 4 of 0, 20 or 80 µM overnight, and was
then partially purified by ethanol precipitation. In the absence of
4 (lane 0, Figure 7), RNA polymerase transcribed through the
DNA template efficiently to yield the 379 FLT, indicating that
formaldehyde alone was insufficient to induce any transcriptional
blockages (Figure 7). The progression of RNA polymerase
through each drug-reacted DNA template was increasingly
impaired at discrete sites with increasing concentrations of 4
Figure 6a Crosslinking assay of various concentrations of 4 (5 to 40 µM
as indicated) in the presence of formaldehyde (79 mM) at 37C overnight. All
lanes contain 42 M bp DNA, 53 mM phosphate buffer (pH 8.0). Lanes one
and two are the ds and ss controls (no drug present) where ds denotes the
double stranded DNA control not subjected to denaturation conditions, and ss
denotes the single stranded DNA control subjected to denaturation of 65C
for 5 min. The ds DNA in the ss lane represents residual background levels of
ds DNA after the denaturation step.
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
(Figure 7, lanes 20 and 80).
The data from Figure
7
allows the individual
binding/blockage sites for the drug to be elucidated. Sequencing
lanes A and C use the chain terminators 3´-O-methoxy-ATP or
3
´-O-methoxy-CTP respectively, and indicate termination of the
transcript by adenine or cytosine respectively. The bold bands in
lanes 20 and 80 show the blockage sites resulting from
formaldehyde-activated 4. Due to the 4-fold increase in drug
concentration, the intensity of blockages (lane 80) is noticeably
greater than in lane 20, as expected. A negligible amount of
background transcriptional blockages can be seen in lane X and
is comparable to that seen from 10 mM formaldehyde treatment
in lane 0, indicating that essentially no blockages result without
the presence of 4.
0
5
10 15 20 25 30 35 40 45
C4-DACA concentration (µM)
Figure 6b Percentage of ds DNA (with respect to total DNA per lane) vs.
increasing C4-DACA concentration (5 to 40 µM) in the presence of
formaldehyde.
The increase in transcriptional blockages with increasing
concentrations of 4 was also associated with a decrease in the
fraction of full length transcript. This is a direct reflection of
transcriptional inhibition by formaldehyde-mediated C4-DACA-
DNA adducts. The presence of discrete transcriptional blockages
induced by 4 indicates that the drug-DNA adduct demonstrates a
well-defined degree of sequence specificity. The DNA damage
caused by formaldehyde-activated C4-DACA-DNA adducts may
therefore ultimately contribute towards the compound’s
cytotoxicity.
2
5
An in vitro transcription assay by Phillips et al. showed that
doxorubicin blocked transcription at specific sites of DNA in the
presence of a reducing environment containing Fe /Fe and
DTT. Doxorubicin-DNA adducts were found to have a high
sequence selectivity, forming almost exclusively at GpC sites. It
was later found that this adduct formation was mediated by
2
+
3+
2
5
2
6
accumulation of formaldehyde in solution.
Furthermore,
doxorubicin was found to be covalently linked to only one strand
of DNA with the ability to stabilize double stranded DNA
through non-covalent interactions with the opposite DNA
The sites of highest transcriptional blockages (shown in bold)
are summarized in Table 1, with likely adduct sites indicated by
the rectangular boxes. This table highlights the sequence
selectivity of formaldehyde-mediated C4-DACA-DNA adduct
formation. Although the activated drug may intercalate reversibly
between any base pair combination, the drug-DNA adducts
exhibited a distinctively sequence-dependent occurrence, with the
sequence-selective blockage of RNA polymerase at and
immediately prior to CpG and CpA dinucleotide sequences
1
4
strand.
The in vitro transcription assay was subsequently employed to
confirm if 4 formed covalent drug-DNA adducts, and to elucidate
any potential sequence selectivity. This technique does not
recognize drugs that merely intercalate into DNA with rapid
dissociation kinetics as lesions must have a half-life of at least
00 seconds on the DNA. DNA-intercalation interactions
typically have a much shorter half-life than that of a covalently
bound drug-DNA adduct (e.g. intercalated daunomycin t  1
s, formaldehyde mediated doxorubicin-DNA adducts t1/2  7.5
2
7
3
(
Figure 7 and Table 1). The formation of C4-DACA-DNA
adducts at these sequence specific sites indicates that there is a
favorable three-dimensional access point to receptive
1
/2
2
8
a
2
9
h, formaldehyde mediated mitoxantrone-DNA adducts t1/2  3
h ). Furthermore, DACA exhibits extremely fast dissociation
kinetics (in the order of milliseconds) and this is accentuated for
nucleophilic site on the DNA, suggesting the formaldehyde-
activated 4 reacts reversibly with guanine and/or cytosine. The
3
0

N7 and N2 positions of guanine have been established as the
most reactive sites in nucleic acids for numerous alkylating
3
2, 33
agents in neutral aqueous solutions,
thus by analogy with
pixantrone, N2 is most likely the site of adduct formation. It is
evident from Figure 7 that 4 has a remarkably similar CpG
sequence selectivity to that reported for formaldehyde-activated
3
0, 34
35, 36
pixantrone
and formaldehyde-activated mitoxantrone.
This sequence selectivity differs significantly from the 5’-GpC
sequence preferred for formaldehyde-activated doxorubicin
1
4
37, 38
adducts and formaldehyde-activated daunorubicin.
Despite
the differences in sequence selectivity, the four aforementioned
formaldehyde-activated drugs have an absolute requirement for
2
3, 30, 39, 40
the exocyclic N2 amino group of guanine.
The structural
similarities between 4 and these drugs indicate that 4 most likely
forms formaldehyde mediated adducts with DNA via the
exocyclic N2 amino group of guanine.
The similarity of the C4-DACA binding site to that of
pixantrone is quite promising, implying that 4 may have similar
success. A natural progression from this result would be to
investigate 4 in a biological setting to ascertain cellular
responses. The potential to combine formaldehyde-releasing
prodrugs with 4 to explore its viability as a therapeutic alternative
to doxorubicin and mitoxantrone could be valuable. Alternatively
there is potential for such covalent adduct formation in the
absence of exogenous formaldehyde. Endogenous formaldehyde
is ubiquitously present in human tissues with the concentration of
formaldehyde in human blood and tissues being approximately
4
1
0
.1 mM. Moreover, cancer patients have been shown to have
42
higher levels of formaldehyde in their urine and expired air
4
3
samples compared to healthy individuals. Indeed doxorubicin-
DNA adducts have been detected in breast cancer cells in culture
at levels of 1300 adducts per cell at 25 nM drug concentration in
4
4
the absence of formaldehyde supplementation.
Table 1 C4-DACA-DNA adduct blockage sites. Nucleotides
shown in bold font indicate the most intense blockage sites.
Numbers represent the length of each truncated transcript
beginning from the transcription start site at ˗1. The likely
adduct attachment sites are shown within the rectangular
boxes.
Blockage site
Blockage Sequence
4
5
4
2
4
5
7
7
4
2
1
9
A
T
C
A
C
C
C
A
C
G
G
G
A
G
T
A
C
G
C
T
G
G
G
C
A
A
7
1
T
7
9
G
A
C
A
A1
C
C
09
12
16
1
1
1
09
12
16
1
1
C
In summary, results from the molecular modeling based on
derivatives of DACA showed that an increase in chain length of
approximately two to three atoms would produce a more
favorable binding interaction. There was a requirement of a side
chain of approximately four carbons to facilitate favorable
interactions between the formaldehyde-activated drug and DNA.
There is potential for these compounds to be further developed to
be efficiently activated by formaldehyde in vivo.
Figure 7 In vitro transcription assay of formaldehyde-activated 4. Lane X
is the control (no formaldehyde or drug) and denotes the elongation of this
complex to FLT. Lanes designated 0, 20, and 80 contain 10 mM
formaldehyde with 0, 20, and 80 µM 4 respectively. Lanes A and C are
sequencing lanes using the chain terminators 3´-O-methoxy-ATP or 3´-O-
methoxy-CTP respectively. Numbering at the left hand side of the
phosphorimage indicates the length of select truncated transcripts.
Acknowledgements
We thank Dr Les Deady, La Trobe University, for very
considerable help in the synthesis of the C4-DACA.

3
4
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Taatjes, D. J.; Gaudiano, G.; Koch, T. H.; Fenick, D. J. Chem.
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Design, synthesis, and DNA sequence
selectivity of formaldehyde-mediated DNA-
adducts of the novel N-(4-aminobutyl)
acridine-4-carboxamide
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Elizabeth A. Ankers, Benny J. Evison, Don R. Phillips, Robert T.C. Brownlee and Suzanne M. Cutts