DNA threading bis(9-aminoacridine-4-carboxamides): Effects of piperidine sidechains on DNA binding, cytotoxicity and cell cycle arrest

Article abstract of DOI:10.1016/j.bmc.2008.02.063

We describe the synthesis of a series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylpiperidino and N-methylpiperidin-4-yl sidechains, joined via neutral flexible alkyl chains, charged flexible polyamine chains and a semi-rigid charged piperazine linker. Their cytotoxicity towards human leukaemic cells gives IC₅₀ values ranging from 99 to 1100 nM, with the ethylpiperidino series generally being more cytotoxic than the N-methylpiperidin-4-yl series. Measurements with supercoiled DNA indicate that they bisintercalate.

Full text of DOI:10.1016/j.bmc.2008.02.063

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4390–4400
DNA threading bis(9-aminoacridine-4-carboxamides): Effects
of piperidine sidechains on DNA binding,
cytotoxicity and cell cycle arrest
a
a
a
a
a
Zhicong He, Xianyong Bu, Alexandra Eleftheriou, Malik Zihlif, Zhang Qing,
b,c
a,
*
Bernard W. Stewart and Laurence P. G. Wakelin
a
School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia
School of Women’s and Children’s Health, University of New South Wales, Sydney, NSW 2052, Australia
b
c
Public Health Unit, South East Sydney Area Health Service, Sydney, Australia
Received 17 January 2008; revised 17 February 2008; accepted 19 February 2008
Available online 23 February 2008
Abstract—We describe the synthesis of a series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylpiperidino
and N-methylpiperidin-4-yl sidechains, joined via neutral flexible alkyl chains, charged flexible polyamine chains and a semi-rigid
charged piperazine linker. Their cytotoxicity towards human leukaemic cells gives IC50 values ranging from 99 to 1100 nM, with
the ethylpiperidino series generally being more cytotoxic than the N-methylpiperidin-4-yl series. Measurements with supercoiled
DNA indicate that they bisintercalate.
ꢀ
2008 Elsevier Ltd. All rights reserved.
1
. Introduction
tion with various linkers, in which the carboxamide side-
chains comprise N,N-dimethylaminoethyl and
8
Cytotoxic DNA intercalating agents inhibit cell growth
by two well-established mechanisms: poisoning topoi-
somerases and inhibiting transcription. The majority
of intercalating cancer drugs in clinical use are of the
former type, with examples including adriamycin,
ethylmorpholino groups. The compounds were de-
signed to bisintercalate into DNA, sandwiching two
basepairs and to thread their sidechains through the he-
lix to form hydrogen bonding contacts with the O6/N7
atoms of guanine in the major groove: a hydrogen bond-
ing scheme observed in DNA complexes of the 9-amino-
1
–3
4
1
–3
mitoxantrone and amsacrine.
Surprisingly, little
9
attention has been paid to the development of template
transcription inhibitors, despite their impressive experi-
mental antitumour activity, in part, because of the diffi-
culty of endowing chemically accessible compounds
with the capacity to form the bulky long-lived reversible
complexes needed to inhibit the passage of RNA poly-
merase. Intercalating transcription inhibitors are exem-
plified by the antitumour antibiotics actinomycin D,
the bisintercalator echinomycin and the threading agent
acridine-4-carboxamide monomer 9-amino-DACA.
Our initial findings showed that the targeted bis(9-ami-
noacridine-4-carboxamides) bisintercalate via a thread-
8
ing mode as anticipated, and that their complexes
dissociate many orders of magnitude more slowly than
simple intercalators, some members forming complexes
that have lifetimes comparable to those of the natural
8
products. The most active agents are cytotoxic towards
5
–7
nogalamycin.
In previous work, we combined the
human leukaemic CCRF-CEM cells with IC50 values of
35–50 nM in a range extending over 20-fold, with nei-
ther the dimethylaminoethyl nor the ethylmorpholino
bisintercalation motif of echinomycin with the threading
mode of nogalamycin, to synthesize a series of bis(9-
aminoacridine-4-carboxamides) linked via the 9-posi-
8
series being intrinsically more toxic. Cell cycle progres-
sion studies indicated that the ethylmorpholino series
generally has no effect on cycle distribution in CEM cell
populations, whereas the N,N-dimethylaminoethyl ser-
ies, with two exceptions, cause G2/M arrest in the man-
ner of topoisomerase poisons, consistent with possible
Keywords: Bisintercalator; Transcriptional inhibitor; Threading agent;
Diacridine.
*
Corresponding author. Tel.: +61 2 9385 2563; fax: +61 2 9385
059; e-mail: l.wakelin@unsw.edu.au
1
0
968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.063

Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
4391
8
involvement of topoisomerases in their mode of action.
Subsequent studies of drug effects on global gene expres-
sion using DNA microarrays, showed that selected
examples of the bis(9-aminoacridine-4-carboxamides)
have profound effects on transcription, in a similar man-
ner to that of actinomycin, echinomycin and nogalamy-
cin, producing patterns of perturbation that depend
the binding affinity of the N-methylpiperidin-4-yl series
is lower than that of its congeners.
2. Results
2.1. Chemistry
1
0
both on linker and sidechain structure.
The acridine compounds studied (Fig. 1) were synthe-
sized as outlined in Scheme 1. The 9-oxoacridan-4-car-
boxylic acid was prepared as described by Denny and
Here we provide a further attempt to develop bisinterca-
lative transcription template inhibitors utilizing the
bis(9-aminoacridine-4-carboxamide) framework. Previ-
ous crystallographic studies of monomeric 9-aminoacri-
dine-4-carboxamides with dimethylaminoethyl and
1
2
colleagues, the 9-chloroacridine-4-carbonyl chloride
being obtained by treatment with thionyl chloride in
the presence of catalytic amounts of DMF. The 4-car-
boxamide sidechains were coupled using an excess of
N-piperidinylethyleneamine or 1.1 equivalents of
N-methyl-4-aminopiperidine in the presence of sodium
carbonate in anhydrous dichloromethane. N-Methyl-4-
aminopiperidine was prepared via a reductive amination
by treatment of N-methyl-4-piperidone with ammonium
formate and 10% palladium on carbon in methanol-water
9
,11
ethylmorpholino sidechains
established that the com-
pounds bind to DNA with their carboxamide sidechains
lying in the major groove, forming hydrogen bonds with
the O6/N7 atoms of guanine. In these structures, the eth-
ylmorpholino sidechain, being more bulky, covers two
basepairs, in contrast to the N,N-dimethylaminoethyl
9
,11
sidechain, which extends just to the bonding guanine.
1
3
Surprisingly, our previous work revealed that the kinetic
stability of the complex of the C3NC3-linked ethylmor-
pholino compound is no greater than that of its
(Scheme 2).
The 9-aminoacridine-4-carboxamide
monomers were prepared by reacting the 9-chloroacri-
dine-4-carboxamides with ammonia gas in hot phenol
solution. The bis(9-aminoacridine-4-carboxamides) were
8
N,N-dimethylaminoethyl analogue. This was taken to
8
suggest that either the hydrogen bonding capacity of
the cyclic sidechain is weaker than that of N,N-dimethyl-
aminoethyl, or that during dissociation the sidechain
rotates into the intercalation plane, thereby minimizing
the difference in ‘structural drag’’ between the two
threading groups. In the present work, we have replaced
the N,N-dimethylaminoethyl and ethylmorpholino
groups with ethylpiperidino and N-methylpiperidin-4-
yl sidechains in an attempt to ameliorate these effects.
Our intention with the ethylpiperidino sidechain is to en-
hance the strength of the guanine hydrogen bond, since
piperidine, with a pK of ꢀ11, is substantially more basic
than morpholine, whose pK is about 8, and the N-meth-
ylpiperidin-4-yl group is designed to remove a number
of degrees of freedom from the flexible N,N-dimethyl-
aminoethyl sidechain, while maintaining the protonated
amine in a suitable position for hydrogen bond
formation.
coupled in the manner previously described by us, as
shown in Scheme 1. The 9-chloroacridine-4-carboxam-
ides were dissolved in hot phenol and treated with a half
molecular equivalent of the a,x-diamine linkers. After
heating at 125 ꢁC for 2 h, and suitable workup, the diac-
ridine free bases were recovered from chloroform and
purified by flash chromatography or recrystallization.
Their corresponding hydrochloride salts were prepared
by treating the free base in methanol with concentrated
hydrochloric acid at pH 3. Most of the a,x-diamine
linkers used are commercially available. The tetraamine
required for the C6N2 linker was purchased as the tetr-
ahydrochloride salt, and treated as described previously
8
by us before use. 1,4-Bis(aminoethyl)piperazine, re-
quired for the C2pipC2 linker, was prepared as reported
8
,14
previously.
2.2. DNA binding
In this report, we describe the synthesis of a series of
bis(9-aminoacridine-4-carboxamides) with ethylpiperidi-
no and N-methylpiperidin-4-yl sidechains, joined via
neutral flexible alkyl chains, charged flexible polyamine
chains and a semi-rigid charged piperazine linker
To establish whether the dimers bind to DNA by a bis-
intercalative threading mode, we investigated their
capacity to remove and reverse the supercoiling of
closed circular pBR322 DNA as assessed by electropho-
8
,15,16
retic mobility measurements on agarose gels.
(
Fig. 1). Their cytotoxicity towards human leukaemic
CCRF-CEM cells gives IC50 values ranging from 99 to
100 nM, with the ethylpiperidino series generally being
Figure 2 shows typical titration curves for C3NC3 Pip,
C2pipC2 Pip and C3NC4 NMP in 40 mM TEA buffer,
and reveals equivalence binding ratios, where all the
supercoiling have been removed and the closed and
nicked circular species co-migrate, in the range 0.13–
0.21 ligand molecules per base pair. Calibrating the
titrations using the C8-linked DMAE threading dimer
1
more cytotoxic than the N-methylpiperidin-4-yl series.
However, comparison of the structure–activity relation-
ships of the new agents with that of their N,N-dimethyl-
aminoethyl- and ethylmorphorpholino homologues is
8
8
not straightforward. Analysis of their effects on cell
of known unwinding angle 43ꢁ, and equivalence bind-
cycle progression shows that the ethylpiperidino series
fail to invoke G2/M arrest, whereas the N-methylpiperi-
din-4-yl series does, providing some evidence for topoi-
somerase poisoning in their mode of action. Helix
unwinding angle measurements with supercoiled DNA
indicate that the new agents intercalate, but suggest that
ing ratio of 0.075, enables simple calculation of helix
unwinding angles, as recorded in Table 1. The DNA
complexes of both the ethylpiperidino and N-methylpi-
peridin-4-yl monomers dissociate on the gels, so pre-
cluding a determination. The ethylpiperidino dimers
behave uniformly, with unwinding angles of 20ꢁ to

4392
Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
HN
L
NH
N
R =
N
N
O
N
O
NHR
NHR
Linker
name
Linker
charge
Inter-chromophore
Linker (L)
Flexibility
a
distance, Å
(
(
CH
2
2
)
6
8
C6
Flexible
Flexible
Flexible
Flexible
Flexible
Semi-rigid
Neutral
Neutral
+1
8.8
11.3
9.8
CH
)
C8
(
(
CH
CH
2
2
)
3
NH(CH
2
2
)
3
4
C3NC3
C3NC4
C6N2
)
3
NH(CH
)
+1
11.4
11.3
10.1
(
CH
2
)
2
NH(CH
2
)
2
NH(CH
2
)
2
+2
(CH
2
)
2
N(CH CH
2
2
)
2
N(CH
2
)
2
C2pipC2
+2
Figure 1. Structure of the threading diacridines studied. Compounds are described in the text by reference to their linker and sidechain structure, the
abbreviations for the latter being Pip, ethylpiperidino and NMP, N-methylpiperidin-4-yl. Energy-minimised structures of the NMP monomer with
the sidechain in chair (left) and twisted-boat (right) conformations are also shown. These structures were modeled using Discovery Studio 1.7 and
illustrated using PyMOL. The chair conformer would not be able to make hydrogen bonding interactions to guanine, whereas the twisted-boat
conformer would. In our previous report we described compounds with an N,N-dimethylaminoethyl sidechain, designated DMAE, and an
8
a
2 2 2
ethylmorpholino sidechain, designated morpholino . N(CH CH ) N denotes piperazine. Ref. 15.
2
5ꢁ, their complexes giving no evidence of dissociation
on the gel in 40 mM buffer. Whilst this range is low com-
pared to the expected values of 30–35ꢁ for bisintercala-
tion, given that the 9-aminoacridinecarboxamides have
unwinding angles of 15–17ꢁ, it is comparable to the
values found for bisintercalating simple piperazine-
linked diacridines and to the piperazine-linked DMAE
Cl
N
Cl
N
O
a)
b)
N
H
1
7
COOH
Cl
O
Cl
O NHR
NH2
c)
1
5
and morpholino bis(9-aminoacridinecarboxamides).
N
O
N
O
In contrast, the N-methylpiperidin-4-yl dimers give titra-
tion responses which depend on the ionic strength of the
buffer and the nature of the linker, indicating that their
affinity for DNA is somewhat lower than that of their
ethylpiperidino homologues. Accordingly, for the N-
methylpiperidin-4-yl dimers, Table 1 records the find-
ings in 10 mM TAE buffer, pH 7.5, where the cation
concentration is about 8–9 mM. Under these conditions,
the observed unwinding angles range from 13 to 30ꢁ.
The values for the alkyl- and C3NC3-linked dimers
should be viewed as lower limits since these agents show
the greatest salt-dependent variations (Table S1). In
contrast, the C3NC4- and C6N2-linked dimers give
the same result in both 10 and 40 mM buffer, indicating
that these are quantitatively bound in both circum-
stances, and have unwinding angles of 16 and 30ꢁ which
span the traditional range of mono- to bifunctional reac-
tion. In distinction to the other members of the N-meth-
ylpiperidin-4-yl series, the semi-rigidly linked C2pipC2
NMP dimer removes but fails to reverse DNA supercoil-
ing in 10 mM TAE, and there is a clear evidence of com-
plex dissociation. Thus, it would seem that its DNA
affinity is anomalously low for a dimer with a doubly
charged linker.
NHR
NHR
Cl
N
HN
N
L
NH
d)
N
O
NHR
N
O
NHR
L = -(CH ) -;
L = -(CH ) -;
L = -(CH ) NH(CH ) -;
L = -(CH ) NH(CH ) -;
L = -(CH ) NH(CH ) NH(CH ) -;
L = -(CH ) N(CH CH ) N(CH ) -.
O NHR
N
2 6
R =
2
8
2
3
2 3
2
4
2 3
2
2
2
2
2 2
2
2
2
2
2
2 2
Scheme 1. Synthesis of 9-aminoacridine-4-carboxamides and dimers.
Reagents and conditions: (a) SOCl2; (b) RNH /dcm; (c) NH (gas),
phenol, 125 ꢁC; (d) a, x-diamine, phenol, 125 ꢁC.
2
3
O
N
NH2
N
a)
Scheme 2. Synthesis of N-methyl-4-aminopiperidine. Reagent: (a) 10%
Pd/C, HCOONH , CH OH.
4
3

Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
4393
NMP dimers are, respectively, 6-, 3.5- and 2-fold more
cytotoxic than the NMP monomer, whereas the NMP
dimers with doubly charged linkers, C6N2 and C2pipC2
are both less potent than the monomer. Once again, the
octamethylene linker yields the most active compound.
2.4. G2/M arrest
The effects of the threading agents on cell cycle progres-
sion were investigated by using flow cytometry. CEM
cells were fixed, labeled with propidium iodide and ana-
lyzed after incubation for 24 h in the presence of the li-
gands at concentrations ranging from the IC50 (based on
72 h incubation), upward to 2, 5 and 10 times that level.
Once again, the topoisomerase poisons amsacrine and 9-
amino-DACA, and the transcription inhibitor actino-
mycin D, were used as comparators. Figure 3a and b
show typical profiles for amsacrine and 9-amino-
8
DACA, where, as found previously, the cells
accumulate in G2/M in a dose-dependent manner as a
consequence of causing topoisomerase II-induced
2
DNA strand breaks. The proportion of cells in G2/M
not exposed to drugs was routinely found to be about
8
2
0%. By contrast, also as previously reported, actino-
mycin has no effect on the cell cycle (data not shown),
an outcome it shares with other transcription template
8
inhibitors like echinomycin and nogalamycin. With
Figure 2. Effects of the C3NC3 Pip, C2pipC2 Pip, and C3NC4 NMP
threading diacridines on the supercoiling of pBR 322 DNA. Measure-
ments were made in 40 mM tris acetate buffer. Drug-to-DNA ratios in
lanes 1–15 for C3NC3 Pip are 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14,
respect to the threading dimers, none of the ethylpiperi-
dino series, including the monomer, caused perturbation
of the cycle (data not shown), from which we infer that
the mechanism of action of these agents does not involve
topoisomerase poisoning. However, all members of the
NMP series studied, including the monomer, arrest cells
in G2/M as shown in Table 1, examples of the profiles
for the monomer and C8 NMP being illustrated in
Figure 3c and d. We take this finding to infer that com-
pounds bearing an N-methylpiperdin-4-yl sidechain, do
engage topoisomerase II in their mode of action, at least
in part.
0
1
0
.16, 0.18, 0.20, 0.25, 0.30, 0.35, and 0. Drug-to-DNA ratios in lanes 1–
5 for C2pipC2 Pip are 0, 0.04, 0.05, 0.06, 0.07, 0.08, 0.10, 0.10, 0.12,
.14, 0.16, 0.18, 0.20, 0.25, and 0. Drug-to-DNA ratios in lanes 1–12
for C3NC4 NMP are 0, 0.05, 0.075, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35,
.40, 0.50 and 0.
0
2
.3. Cytotoxicity
The cytotoxicity of the target bis(9-aminoacridine-4-car-
boxamides) and related monomers against human leu-
kaemia CEM cells is shown in Table 1. Data for the
topoisomerase II poisons amsacrine and 9-amino-
DACA, and the transcription inhibitor actinomycin D,
3. Discussion
The bis(9-aminoacridine-4-carboxamides) were designed
to bisintercalate into DNA in a ‘‘staple-like’’ conforma-
tion, sandwiching 2 basepairs, with the carboxamide
sidechains threading through the helix to form hydrogen
bonding contacts with the O6/N7 atoms of guanine in
8
are presented for the purposes of comparison. The
monofunctional 9-aminoacridinecarboxamides have
IC50 values of 600 and 800 nM, the more potent being
the NMP analogue, and are thus about 5- to 7-fold less
toxic than 9-amino-DACA with the N,N-dimethylami-
noethyl sidechain. In the ethylpiperidino dimer series,
all compounds are more potent than the parental mono-
mer, around 3- to 4-fold more so in the case of the C6-,
the C3NC4-, the C3NC3- and C6N2-linked compounds,
and about 7- to 8-fold more so in the case of the C8- and
C2pipC2-linked dimers. Thus, it is apparent that for the
ethylpiperidino series dimerization consistently en-
hances cytotoxicity, with the uncharged octamethylene
linker promoting the greatest activity. In contrast, for
the N-methylpiperidin-4-yl series, dimerization can en-
hance or weaken the biological potency with IC50 values
varying up to ꢁ10-fold, depending on the nature of the
linker. Here, the C8-, the C6- and the C3NC4-linked
8
the major groove. Our previous work illustrated that
bis(9-aminoacridine-4-carboxamides) with N,N-dimeth-
ylaminoethyl and ethylmorpholino sidechains do indeed
bisintercalate by a threading mode, that their DNA
complexes dissociate slowly, and that they are potently
cytotoxic in a manner that depends both on linker and
sidechain structure. The intention was to develop inhib-
itors of transcription that mimic the RNA polymerase-
blocking activity of antitumour antibiotics like actino-
mycin, echinomycin and nogalamycin,
ments with DNA microarrays illustrated that they
perturb transcription profiles in a similar way to these
natural products. In the work presented here we
sought to enhance the putative sidechain-guanine
hydrogen bonding interaction in two ways, and to deter-
8
5
–7
and experi-
1
0

4394
Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
Table 1. Cytotoxicity, cell cycle effects and helix unwinding angles for the compounds studied
Compound
Cytotoxicity IC50 (nM)
Equivalence binding ratio
Helix unwinding angle (deg)
G2/M arrest
Controls
a
b
a
Amsacrine
9-Amino-DACA
Actinomycin D
130 ± 39
120 ± 25
1.6 ± 1.0
—
—
—
21
17
Yes: 1 · IC50 (68%)
a
c
a
Yes: 10 · IC50 (61%)
No
a
d
18
a
Pip series
Monomer
C6 Pip
800 ± 50
200 ± 10
99 ± 24
No effect on supercoiling
0.140 ± 0.005
0.160 ± 0.010
—
No
No
No
No
—
23 ± 2
20 ± 3
23 ± 2
24 ± 2
20 ± 3
25 ± 1
C8 Pip
C3NC3 Pip
C3NC4 Pip
C6N2 Pip
C2pipC2 Pip
260 ± 100
230 ± 50
270 ± 30
130 ± 5
0.140 ± 0.010
0.135 ± 0.010
0.160 ± 0.015
0.130 ± 0.005
No
No
NMP series
Monomer
C6 NMP
600 ± 130
210 ± 20
130 ± 25
680 ± 90
340 ± 5
No effect on supercoiling
0.250 ± 0.050
0.175 ± 0.025
—
Yes: 5 · IC50 (60%)
—
13 ± 2
19 ± 3
14 ± 2
16 ± 2
30 ± 4
—
C8 NMP
Yes: 10 · IC50 (43%)
Yes: 10 · IC50 (50%)
—
C3NC3 NMP
C3NC4 NMP
C6N2 NMP
C2pipC2 NMP
0.240 ± 0.030
0.210 ± 0.025
1100 ± 50
800 ± 150
0.110 ± 0.020
No reversal
Yes: 10 · IC50 (40%)
Yes: 5 · IC50 (52%)
Cytotoxicity was measured after 72 h incubation with CCRF-CEM human lukaemia cells. Cell cycle effects were studied on the basis of proportion
%) of CCRF-CEM cells arrested at G2/M phase determined by flow cytometry after treatment with the compounds at concentrations equal to the
(
IC50 or at 2·, 5· or 10· that concentration, control preparations being left untreated. Helix unwinding angles were measured by agarose gel
electrophoresis using pBR322 covalently closed circular DNA at pH 7.5. Pip dimers measured in 40 mM tris acetate buffer, and NMP dimers
measured in 10 mM tris acetate buffer. Equivalence binding ratios are expressed in basepair units.
a
Data taken from Ref. 8.
Data taken from Ref. 18.
b
c
Data taken from Ref. 17.
Data taken from Ref. 19.
d
mine the effects of the modifications on cytotoxicity.
Replacing the morpholino group, pK ꢀ8, with the more
basic piperidine moiety, pK ꢀ11, was intended to
strengthen the hydrogen bond energy directly, whereas
the N-methylpiperidin-4-yl group was designed as a ri-
gid analogue of the N,N-dimethylaminoethyl group:
one that would restrict rotation of the sidechain about
the carboxamide bond and reduce its degrees of freedom
from 3 to 1, so making it more difficult to withdraw the
bulky sidechain back through the helix.
Fig. 3). This is most notable for the uncharged C6 and
C8 alkyl-linked dimers, and for the C2PipC2-linked di-
mer. These results strongly suggest that the DNA affin-
ity of the N-methylpiperidin-4-yl series is diminished,
and implies that the rigid sidechain is unable to make
the desired favourable interactions with the guanine
O6/N7 atoms. The most likely explanation for this is
that the cyclic N-methylpiperidin-4-yl moiety more-
readily adopts a chair-like conformation in the complex,
and directs the protonated nitrogen away from the
guanine base (Fig. 1). Notwithstanding this, the unwind-
ing angles of the C8-linked and all the linear polyamine-
linked dimers have values approaching, or are typical of,
bisintercalation (Table 1), implying that the N-methylpi-
peridin-4-yl moiety does not intrinsically inhibit bifunc-
tional reaction in this structural framework. However,
the failure of the C2PipC2 NMP dimer to reverse
DNA supercoiling at any ionic strength implies that this
compound is unable to bisintercalate, despite the posi-
tive changes on its linker and chromophore, suggesting
a structural impediment to bifunctional reaction in this
case.
The helix unwinding angle measurements (Table 1 and
Fig. 3) show that the ethylpiperidino series form stable
complexes with circular DNA on agarose gels, and that
they intercalate with unwinding angles in the range
2
0–25ꢁ, in a manner independent of linker structure.
Although these values are low for typical bisintercalat-
ing diacridines, they are within the range found for
simple piperazine-linked diacridines and the pipera-
zine-linked DMAE and morpholino bis(9-aminoacridin-
8
,15
ecarboxamides).
The findings suggest that the
ethylpiperidino group binds as anticipated, that DNA
affinity remains high, but that the helicoidal parameters
of the complexes are distorted compared to those of the
N,N-dimethylaminoethyl complexes. In contrast, the
complexes of several members of the N-methylpiperi-
din-4-yl series show clear evidence of dissociation on
agarose gels at the standard TEA concentration of
The cytotoxicity of the ethylpiperidino series appears
essentially independent of linker structure (Table 1),
the IC50 values varying over a range of about 3-fold
implying that all bisintercalated ethylpiperidino com-
plexes are equally biologically active, caveats about cel-
lular uptake notwithstanding. Taken as whole, this is a
different response to the N,N-dimethylaminoethyl and
40 mM, and stable complexes are only observed at the
lower buffer concentration of 10 mM (Tables 1 and S1,

Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
4395
Figure 3. Flow cytometric profiles for selected agents, based on staining of CCRF-CEM cells with propidium iodide, illustrating accumulation of
cells having G2+M DNA content >40% following exposure to amsacrine at 1· IC50 (a), 9-amino DACA at 10· IC50 (b), C8 NMP at 10 · IC50 (c)
and the NMP monomer at 5 · IC50 (d) for 24 h.
1
0
the ethylmorpholino series, whose toxicities spread over
8
a greater range and are more linker-specific. Despite
these different profiles, the C8- and C2pipC2-linked di-
mers are amongst the most potent in both the DMAE
and ethylpiperidino series (IC50 in the range 35–
nomycin, nogalamycin and actinomycin, fails to elicit
the typical G2/M block (Table 1) associated with
DNA strand breaks caused by trapping of the topoiso-
merase II cleavable complex, which mitigates against
this possibility. In contrast, the N-methylpiperidin-4-yl
series, as a whole, including the monomer (Table 1),
causes clear evidence of G2/M arrest, implying that
the rigid cyclic analogue of the N,N-dimethylaminoethyl
sidechain may interact favourably with topoisomerase
II.
1
30 nM), comparable activity being found for the
8
C3NC3- and C3NC4-linked ethylmorpholino dimers.
The cytotoxicity of the N-methylpiperidin-4-yl series
presents yet a different structure activity profile, the C8
NMP dimer, with an IC50 of 130 nM, being the only
example that might be considered amongst the ‘active’
bisthreading agents (Table 1). Curiously, although the
linear amine-linked NMP dimers have greater DNA
affinity, they are less cytotoxic, a tendency being most
pronounced with the clearly bisintercalating C6N2 di-
mer which is the least toxic of the series. More re-assur-
ingly, the C2pipC2 NMP dimer, which appears not to be
a bisthreader, is also amongst the least active (Table 1).
Whilst DNA microarray studies confirm the threading
bis(9-aminoacridine-4-carboxamides) as transcription
4. Conclusion
In this work we have attempted to enhance the strength
of the putative hydrogen bonding interaction between
the carboxamide sidechain of bisintercalated threading
bis(9-aminoacridine-4-carboxamides) and the O6/N7
atoms of guanine in their DNA complexes, and to assess
the effects of these modifications on cytotoxicity. We
find that an ethylpiperidino sidechain, employed be-
cause of its greater basicity, yields compounds with high
DNA affinity and cytotoxic potency, whereas an N-
methylpiperidin-4-yl moiety, designed as a rigid side-
chain with reduced degrees of rotational freedom to
1
0
inhibitors, the possibility exists that these agents, being
dimers of known topoisomerase II poisons, may also in-
volve topoisomerase biology in their mode of action.
However, the cell cycle measurements show that the eth-
ylpiperidino series, like the transcription inhibitors acti-

4396
Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
provide greater kinetic stability, destabilizes DNA
binding and diminishes biological activity. We ascribe
the latter effect to the N-methylpiperidin-4-yl moiety
adopting an unfavourable chair-like conformation that
directs hydrogen bonding away from the guanine base
5.3. N-[2-(N-Piperidinyl)ethyl]-9-chloroacridine-4-
carboxamide
An ice-cold solution of 1-(2-aminoethyl)piperidine
(0.96 g, 7.5 mmol) in dichloromethane (10 mL), contain-
ing sodium carbonate (1 g), was added over 5 min to a
stirred suspension of the above carbonyl chloride
(7.5 mmol) in dichloromethane (10 mL) cooled on ice.
The mixture was stirred at room temperature for 4 h,
the sodium carbonate removed by filtration, and the or-
ganic filtrate washed and condensed to give the product
as a pale yellow solid (2.69 g). It was recrystallized from
benzene and petroleum ether as a yellow powder (1.98 g,
(
Fig. 1). Evidently, the design of bulky threading side-
chains remains a challenging task in the development
of bisthreading agents with enhanced biological
activity.
5. Experimental
1
Melting points were measured on a Reichert ‘ther-
mopan’ microscope hot-stage apparatus up to
71%), mp 116–117 ꢁC. H NMR: 1.48–1.54 (m, 2H,
CH ), 1.65–1.73 (m, 4H, 2CH ), 2.56–2.60 (m, 4H,
2CH ), 2.75 (t, 2H, J = 6.2 Hz, CH ), 3.81 (q, 2 H,
2 2
2
2
3
00 ꢁC and are recorded uncorrected. Microanalyses
were performed by either the Microanalytical Unit,
Australian National University, Canberra, Australia,
or the Campbell Laboratory, University of Otago,
New Zealand. NMR spectra were recorded on a
Bruker AM-300 spectrometer operating at 300.17
J = 6.2 Hz, CH ), 7.65–7.76 (m, 2H, 2ArH), 7.83–7.88
2
(m, 1H, ArH), 8.35 (d, 1H, J = 8.6 Hz, ArH), 8.43 (d,
1H, J = 8.6 Hz, ArH), 8.59 (dd, 1H, J = 8.6, 1.5 Hz,
ArH), 8.99 (dd, 1H, J = 7.2, 1.5 Hz, ArH), 11.74 (br s,
1
3
1H, CONH). C NMR: 24.4 (CH ), 25.9 (CH ), 37.3
2
2
1
13
and 75.48 MHz for H and C, respectively. Chem-
ical shifts are reported as d values in ppm relative
to internal tetramethylsilane. The solvent was deuter-
ated chloroform, unless otherwise specified. DEPT-
(CH ), 54.6 (CH ), 58.0 (CH ), 123.8 (C), 124.4 (C),
2 2 2
124.6 (CH), 126.5 (CH), 127.4 (CH), 128.3 (CH), 129.0
(C), 129.6 (CH), 131.1 (CH), 135.4 (CH), 142.7 (C),
146.3 (C), 147.3 (C), 165.3 (C).
1
in
35 was used to identify proton-bound carbon atoms
C NMR spectra. MALDI-TOF mass spectra
1
3
5.4. N-(N-Methylpiperidin-4-yl)-9-chloroacridine-4-
carboxamide
were obtained using a hydroxycinnamic acid matrix
on a PerSeptive Biosystems Voyager mass spectrom-
eter. All solvents were laboratory grade and used
without further purification unless otherwise stated.
Phenol was redistilled and kept under nitrogen.
Dichloromethane was stored over anhydrous calcium
chloride, refluxed for 4 h, and then distilled and
This compound was prepared as above, using N-methyl-
4-aminopiperidine (0.45 g, 4.0 mmol) and 9-chloroacri-
dine-4-carbonyl chloride (2.0 mmol), to give N-(N-
methyl-4-piperidinyl)-9-chloroacridine-4-carboxamide
1
as a pale yellow solid (0.4 g, 57%). H NMR: 1.99–2.06
(m, 4H, 2CH ), 2.25–2.30 (m, 2H, CH ), 2.46 (s+m,5H,
˚
stored over 4 A molecular sieves. All chemical re-
2
2
agents were used as obtained from commercial sup-
pliers without further purification unless otherwise
indicated.
CH +2CH ), 3.0–3.05 (m, 2H, CH ), 4.20–4.30 (m, H,
3 2 2
CH), 7.67–7.78 (m, 3H, 3ArH), 7.85–7.88 (m, 1H,
ArH), 8.17 (d, 1H, J = 9.0 Hz, ArH), 8.43 (d, 1H,
J = 9.0 Hz, ArH), 8.61 (dd, 1H, J = 9.0, 1.6 Hz, ArH),
8.99 (dd, 1H, J = 7.1, 1.6 Hz, ArH), 11.80 (br s, 1H,
1
3
5
.1. N-Methyl-4-aminopiperidine
1
3
CONH). C NMR: 34.0 (CH ), 47.7 (CH ), 48.0
2
2
A solution of 4-methylpiperidone (4.9 g, 43 mmol) in
MeOH (112 mL) was vigorously mixed with ammo-
nium formate (25 g, 400 mmol) in water (12.5 mL).
After complete dissolution, 10% Pd/C (5.1 g, 4.8 mmol)
was added and the reaction mixture stirred overnight
at room temperature. On completion of the reaction,
the catalyst was filtered off on Celite and the solution
concentrated under reduced pressure. N-methyl-4-
aminopiperidine was distilled off under reduced pres-
(CH ), 56.1 (CH ), 125.6 (C), 126.3 (C), 126.5 (CH),
2 2
128.3 (CH), 129.2 (CH), 130.2 (CH), 130.5 (C), 130.8
(CH), 133.4 (CH), 137.4 (CH), 144.8 (C), 148.1 (C),
148.9 (C), 166.3 (C).
5.5. N-[2-(N-Piperidinyl)ethyl]-9-aminoacridine-4-
carboxamide
N-[2-(N-Piperidinyl)ethyl]-9-chloroacridine-4-carbox-
amide (0.37 g, 1.0 mmol) was dissolved in anhydrous
phenol (3 g) in a flask immersed in a 100 ꢁC oil bath.
Dry ammonia gas was bubbled through the mixture
for 15 min, and the temperature raised to 115 ꢁC for
30 min, whilst the addition of the ammonia continued.
After cooling, the reaction mixture was stirred with
sure as a colourless oil (1.8 g, 37%), bp 120 ꢁC
1
28 mm Hg). H NMR: 1.22–1.35 (m, 2 H, CH ),
(
2
1
2
.73–1.78 (m, 2H, CH ), 2.02–2.10 (m, 2H, CH ),
2 2
.16 (s, 3H, CH ), 2.56–2.65 (m, 1H, CH), 2.74–2.78
3
1
3
(
m, 2H, CH2); C NMR: 35.7 (CH ), 46.0 (CH ),
3 2
4
7.9 (CH ), 54.49 (CH).
2
2
0% sodium hydroxide (20 mL), and the resulting sus-
5
.2. 9-Chloroacridine-4-carbonyl chloride
-Oxoacridan-4-carboxylic acid was prepared as de-
pension extracted with chloroform. The organic extract
was washed with saturated sodium carbonate and dried
over sodium sulfate. Evaporation of the solvent gave the
title compound, which was recrystallized from benzene/
petroleum ether as a yellow solid (0.21 g, 60%), mp
9
scribed by Rewcastle et al. and the acid chloride, pre-
pared according to Atwell et al., used in the next step
without further purification.
1
2
1
7
1
178.5–180 ꢁC. H NMR: 1.49–1.53 (m, 2H, CH2),

Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
4397
1
.65–1.72 (m, 4H, 2CH ), 2.56–2.59 (m, 4H, 2CH ), 2.77
2
258 ꢁC (Found: C, 52.89; H, 7.57; N, 10.09;
C H N O Æ4HClÆ9H O requires: C, 53.04; H, 7.42;
N, 10.31).
2
(
t, 2H, J = 6.2 Hz, CH ), 3.81 (q, 2H, J = 6.2 Hz, CH ),
2 2
48 58
8
4
2
6
.01 (br s, 2H, NH ), 7. 23 (t, 1H, J = 7.2 Hz, ArH), 7.36
2
(
t, 1H, J = 7.5 Hz, ArH), 7.66 (t, 1H, J = 7.2 Hz, ArH),
7
1
.86–7.99 (m, 3H, 3ArH), 8.73 (d, 1H, J = 6.8 Hz, ArH),
2.49 (br s, 1H, CONH). The di-hydrochloride salt of
5.8. 1,6-Bis{4-[N-(N-methylpiperidin-4-yl)carbam-
oyl]acridin-9-yl}-diaminohexane (C6 NMP)
this compound was obtained as a yellow solid, mp
20.5–222 ꢁC, by treating a solution of the free base in
methanol with hydrogen chloride gas (Found: C,
4.89; H, 6.55; N, 12.05; C H N OÆ2HClÆ2H O re-
2
This was prepared in the manner of the C8 NMP dimer,
from
N-(N-methylpiperidin-4-yl)-9-chloroacridine-4-
5
carboxamide (0.22 g, 0.62 mmol) and hexamethylenedi-
amine (0.036 g, 0.31 mmol), to give the product as a yel-
low solid (0.18 g, 77%). H NMR: 1.30–1.50 (m, 4H,
2
1
24
4
2
quires: C, 55.14; H, 6.61; N, 12.25).
1
5.6. N-(N-Methylpiperidin-4-yl)-9-aminoacridine-4-
carboxamide
2CH ), 1.65–1.70 (m, 8H, 4CH ), 1.80–1.90 (m, 4H,
2
2CH ), 2.10–2.20 (m, 4H, 2CH ), 2.31 (s, 6H, 2CH ),
2 2 3
2
2.81–2.84 (m, 4H, 2CH ), 3.72–3.8 (m, 4H, 2CH ),
2 2
This compound was prepared as above, using N-(N-
Methyl-4-piperidinyl)-9-chloroacridine-4-carboxamide
4.14 (br s, 2H, 2CH), 5.11 (br s, 2H, 2NH), 7.29–7.39
(m, 4H, 4ArH), 7.60–7.65 (m, 2H, 2ArH), 7.90–7.98
(m, 4H, 4ArH), 8.09–8.12 (d, 2H, 2ArH), 8.81 (d, 2H,
2ArH), 12.45 (br s, 2H, 2CONH). MALDI-MS:
(
9
(
0.2 g, 0.56 mmol), to give N-(N-Methyl-4-piperidinyl)-
-aminoacridine-4-carboxamide as brown solid
ꢁ0.2 g) which was further recrystallized from acetoni-
a
+
748.45 (M ). The tetrahydrochloride salt was obtained
as a yellow solid, mp 225–226.5 ꢁC (Found: C, 50.80;
1
trile as a yellow solid (0.13 g, 68%). H NMR: 1.65–
.72 (m, 2H, CH ), 1.78–1.90 (m, 2H, CH ), 2.02–2.15
1
H, 7.12; N, 10.11; C H N O Æ4HClÆ10H O requires:
2
2
48 54
8
2
2
(
m, 2H, CH ), 2.27 (s, 3H, CH ), 4.14 (br s, 1H,
2
C, 51.30; H, 7.30; N, 10.40).
3
1
CH), 5.67 (s, 2H, NH ), 7.38 (m, 2H, 2ArH), 7.64
2
(
m, 1H, ArH), 7.83–7.97 (m, 3H, 3ArH), 8.81 (m, 1H,
ArH), 12.51 (br s, 1H, CONH); MALDI-MS: 335.18
5.9. 1,7-Bis{4-[N-(N-methylpiperidin-4-yl)carbam-
oyl]acridin-9-yl}-1,4,7-triaza-heptane (C3NC3 NMP)
+
M+1) . The di-hydrochloride salt of this compound
(
was prepared by treating a methanolic solution with
hydrogen chloride gas followed by precipitation with
ethyl acetate. It was obtained as a yellow solid, mp
This was prepared as for the C8 NMP dimer, from
N-(N-methylpiperidin-4-yl)-9-chloroacridine-4-carboxam-
ide (0.212 g, 0.6 mmol) and N-(3-aminopropyl)-1,3-pro-
pane-diamine (0.0393 g, 0.3 mmol). The product so
obtained was impure, purification being achieved by
converting the free base to its pentahydrochloride salt
followed by re-basification with 3% sodium hydroxide.
The free base was then extracted with chloroform and
2
35–236 ꢁC (Found: C, 51.72; H, 6.73; N, 11.67;
C H N OÆ2HClÆ3H O requires: C, 52.06; H, 6.55; N,
2
0
22
4
2
1
2.14).
5
.7. 1,8-Bis{4-[N-(N-methylpiperidin-4-yl)carbam-
oyl]acridin-9-yl}-diaminootane (C8 NMP), and general
procedure for synthesis of bis(9-aminoacridine-4-
carboxamides)
recrystallized from chloroform/hexane as a yellow pow-
der (0.09 g, 39%). H NMR: 1.48–1.51 (m, 4H, 2CH ),
1
2
1.67–1.72 (m, 8H, 4CH ), 2.08–2.12 (m, 4H, 2CH ),
2
2
2.29 (s, 6H, 2CH ), 2.79–2.87 (m, 8H, 4CH ), 3.95–
3.97 (m, 4H, 2CH ), 4.15 (br m, 2H, 2CH), 7.16–7.30
3
2
A mixture of N-(N-methylpiperidin-4-yl)-9-chloroacri-
dine-4-carboxamide (0.177 g, 0.5 mmol), 1,8-diaminooc-
tane (0.036 g, 0.25 mmol) and phenol (2.0 g) was heated
at 125 ꢁC with stirring for 2 h. On cooling, the reaction
mixture was added to a mixture of diethyl ether/ethyl
acetate (90 mL/45 mL) and the solution decanted. The
solid residue was dissolved in methanol (3–5 mL), added
to a 3% sodium hydroxide solution (100 mL) and ex-
tracted twice with chloroform (2· 50 mL). The com-
bined organic extract was washed with 10% sodium
carbonate, then with saturated brine and dried over so-
dium sulfate. Evaporation of the solvent gave the prod-
uct as a brownish oil (0.13 g), which was recrystallized
2
(m, 4H, 4ArH), 7.56 (d, 2H, 2ArH), 7.86–8.14 (m, 6H,
6ArH), 8.76 (d, 2H, 2ArH), 12.53 (br s, 2H, 2CONH).
MALDI-MS: 763.46 (M ). The pentahydrochloride salt
+
was obtained as a yellow solid, mp 284.5–286 ꢁC
(Found: C, 55.40; H, 6.81; N, 12.40; C H N O Æ5H-
4
8
55
9
2
ClÆ3H O requires: C, 55.12; H, 6.64; N, 12.58).
2
5.10. 1,10-Bis{4-[N-(N-methylpiperidin-4-yl)carbam-
oyl]acridin-9-yl}-1,5,10-triaza-decane (C3NC4 NMP)
This was prepared as for the C8 NMP dimer, using N-
(N-methylpiperidin-4-yl)-9-chloroacridine-4-carboxamide
(0.203 g, 0.57 mmol) and spermidine (0.041 g,
0.28 mmol), followed by flash chromatography on alu-
mina (ethyl acetate/di(isopropyl)amine) to give a yellow
solid (0.05 g, 22%) (Found: C, 72.13; H, 7.24; N, 16.46;
from hot acetonitrile as a yellow solid (0.09 g, 46%).
H NMR: 1.10–1.35 (m, 12H, 6CH ), 1.66–1.73 (m,
1
2
4
2
3
H, 2CH ), 1.80–2.0 (m, 4H, 2CH ), 2.1–2.2 (m, 4H,
2 2
CH ), 2.34 (s, 6H, 2CH ), 2.86–2.89 (m, 4H, 2CH ),
2
3
2
1
.65–3.80 (m, 4H, 2CH ), 4.16 (br m, 2H, 2CH), 5.16
2
C H N O requires: C, 72.37; H, 7.37; N, 16.16). H
46 55
9
2
(
br s, 2H, 2NH), 7.34–7.42 (m, 4H, 4ArH), 7.66 (t,
NMR: 1.55–1.95 (m, 12H, 6CH ), 2.23 (m, 2H, CH ),
2 2
2
8
2
2
H, J = 7.5 Hz, 2ArH), 7.93 (d, 2H,J = 8.3 Hz, 2ArH),
.01 (d, 2H,J = 8.3 Hz, 2ArH), 8.15 (d, 2H,J = 7.9 Hz,
ArH), 8.81 (d, 2H, J = 6.8 Hz, 2ArH), 12.49 (s, 2H,
2.36 (s, 6H, 2CH ), 2.67 (m, 2H, CH ), 2.84 (m, 4H,
3 2
2CH ), 3.36 (m, 2H, CH ), 3.79 (m, 2H, CH ), 3.95
2
2
2
(m, 2H, CH ), 3.98 (t, 2H, CH ), 4.14 (br, 2H, 2CH),
2
2
+
CONH). MALDI-MS: 776.48 (M ). The tetrahydro-
7.25–7.31 (m, 4H, 4ArH), 7.60 (m, 2H, 2ArH), 7.89–
8.17 (br m, 6H, 6ArH), 8.77 (br m, 2H, 2ArH), 12.45
chloride salt was obtained as a yellow solid, 255–

4398
Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
+
br s, 2H, 2CONH). MALDI-MS: 777.47 (M ). The
(
pentahydrochloride salt was obtained as a yellow solid,
4CH2), 1.50–1.53 (m, 4H, 2CH2), 2.69 (t, 8H,
J = 5.6 Hz, 4CH ), 2.85 (t, 4H, J = 6.2 Hz, 2CH ), 3.78
2
2
mp 235–236 ꢁC.
(t, 8H, J = 6.2 Hz, 4CH ), 5.22 (br s, 2H, 2NH), 7.35
2
(t, 4H, J = 7.5 Hz, 4ArH), 7.67 (t, 2H, J = 7.5 Hz,
5.11. 1,10-Bis{4-[N-(N-methylpiperidin-4-yl)carbam-
oyl]acridin-9-yl}-1,4,7,10-tetra-azadecane (C6N2 NMP)
ArH), 7.99 (d, 2H, J = 8.6 Hz, 2ArH), 8.11 (d, 2H,
J = 8.6 Hz, 2ArH), 8.27 (d, 2H, J = 8.3 Hz, 2ArH),
8
.69 (d, 2H, J = 7.2 Hz, 2ArH), 11.81 (br s, 2H,
+
This was prepared as for the C8 NMP dimer, using N-
(N-methylpiperidin-4-yl)-9-chloroacridine-4-carboxamide
2CONH). MALDI-MS: 807.62 (M ). The tetrahydro-
chloride salt was obtained as a yellow solid, mp 199–
200 ꢁC.
(
0
0.253 g, 0.7 mmol) and triethylenetetramine (0.052 g,
.35 mmol). The product was dissolved in hot acetoni-
trile and cooled at 4 ꢁC to give an orange semi-solid
which was solidified by tritriation with cold acetonitrile
5.14. 1,6-Bis{4-[N-(2-piperidinylethyl)carbamoyl]acridin-
9-yl}-diaminohexane (C6 Pip)
1
0.09 g, 32%). H NMR: 1.60–1.90 (m, 8H, 4CH ), 2.22
(
2
(
m, 4H, 2CH ), 2.32 (m, 4H, 2CH ), 2.36 (s, 6H, 2CH ),
2
.84 (m, 8H, 4CH ), 3.87 (m, 4H, 2CH ), 4.20 (br m, 2H,
2 2
CH), 7.25–7.38 (m, 4H, 4ArH), 7.64 (m, 2H, 2ArH),
.94 (br, 2H, 2ArH), 8.11 (d, 2H, J = 8.6, 2ArH), 8.26
This was prepared in the manner of the C8 Pip dimer
using the same chloro-precursor and hexamethylenedi-
amine, followed by flash chromatography on alumina
(ethyl acetate then methanol/chloroform) to give the
product as a yellow solid (60% yield), mp 171–172 ꢁC
(Found: C, 71.72; H, 7.52; N, 13.73; C H N OÆ1.5H O
2
3
2
2
7
(d, 2H, J = 8.6, 2ArH), 8.84 (br s, 2H, 2ArH), 12.54
(br s, 2H, 2CONH). MALDI-MS: 778.47 (M ). The
+
4
8
1
58
8
2
hexahydrochloride salt was obtained as a yellow solid,
mp 232.5–234 ꢁC (Found: C, 50.70; H, 6.96; N, 12.93;
C H N O Æ6HClÆ5H O requires: C, 50.69; H, 6.73;
requires: C, 71.52; H, 7.63; N, 13.90). H NMR: 1.46–
1.50 (m, 8H, 4CH ), 1.65–1.70 (m, 12H, 6CH ), 2.58–
2
2
2.61 (m, 8H, 4CH ), 2.69–2.73 (m, 4H, 2CH ), 3.77–
2
4
6
56 10
2
2
2
N, 12.64).
3.84 (m, 8H, 4CH ), 5.20 (br s, 2H, 2NH), 7.37–7.43
2
(
m, 4H, 4ArH), 7.62–7.68 (m, 2H, 2ArH), 8.02–8.22
(m, 6H, 6ArH), 8.85 (br s, 2H, 2ArH), 12.39 (br s, 2H,
5
.12. N,N-Bis{4-[N-(N-methylpiperidin-4-yl)carbam-
oyl]acridin-9-yl}-[1,4-bis(2-aminoethyl)-piperazine]
C2pipC2 NMP)
+
2CONH). MALDI-MS: 779.53 (M ). The tetrahydro-
(
chloride salt was obtained as a yellow solid, mp 234.5–
2
36 ꢁC.
This was prepared in the manner of the C8 NMP dimer,
from N-(N-methylpiperidin-4-yl)-9-chloroacridine-4-
5.15. 1,7-Bis{4-[N-(2-piperidinylethyl)carbamoyl]acridin-
9-yl}-1,4,7-triazaheptane (C3NC3 Pip)
carboxamide (0.177 g, 0.5 mmol) and 1,4-bis(2-amino-
ethyl)piperazine (0.043 g, 0.25 mmol), and purified by
column chromatography (alumina, ethyl acetate/di(iso-
propyl)amine (v/v = 10/1) then methanol/chloroform).
The product obtained contains trace amounts of impu-
rities which were removed by repeated washing with
hot ethyl acetate, and recrystallization from hot acetoni-
This was prepared in the manner of the C8 Pip dimer
using the same chloro-precursor and N-(3-aminopro-
pyl)-1,3-propane-diamine, followed by flash chromatog-
raphy on alumina (ethyl acetate/di(isopropyl)amine, v/
v = 10/1, then methanol/di(isopropyl)amine, v/v = 5/1).
The product so obtained was impure, purification being
achieved by converting the free base to its pentahydro-
chloride salt followed by re-basification with 3% sodium
hydroxide. The free base was then recrystallized from
chloroform/hexane as a yellow powder, mp 102–103 ꢁC
(Found: C, 71.21; H, 7.52; N, 15.39; C H N O ÆH O
1
trile to give a yellow powder (0.07 g, 35%). H NMR:
1
.55–1.70 (m, 4H, 2CH ), 1.82 (m, 4H, 2CH ), 2.11
2 2
(
m, 4H, 2CH ), 2.28 (m, 4H, 2CH ), 2.30 (s, 6H,
2 2
2
CH ), 2.42 (m, 8H, 4CH ), 2.49 (m, 4H, 2CH ), 2.62
3 2 2
(
m, 4H, 2CH ), 2.80 (m, 4H, 2CH ), 4.14 (br s, 2H,
2 2
2
7
8
1
CH), 5.69 (br s, 1H, 1NH), 6.68 (br s, 1H, 1NH),
.34–7.39 (m, 4H, 4ArH), 7.63 (m, 2H, 2ArH), 7.80–
.15 (m, 6H, 6ArH), 8.81 (d, 2H, J = 8.6 Hz, 2ArH),
4
8
59
9
2
2
1
requires: C, 70.99; H, 7.57; N, 15.52). H NMR: 1.48–
1.51 (m, 4H, 2CH ), 1.67–1.72 (m, 8H, 4CH ), 2.08–
2
2
+
2.53 (br s, 2H, 2CONH). MALDI-MS: 804.48 (M ).
2.12 (m, 4H, 2CH ), 2.68–2.71 (m, 8H, 4CH ), 2.85 (t,
2 2
The hexahydrochloride salt was obtained as a yellow so-
lid, mp 233.5–235 ꢁC (Found: C, 50.71; H, 6.72; N,
4H, J = 6.2 Hz, 2CH ), 2.96–3.01 (m, 4H, 2CH ),
2 2
3.76–3.79 (m, 4H, 2CH ), 3.95–3.97 (m, 4H, 2CH ),
2 2
6.63 (br s, 2H, 2NH), 7.16–7.23 (m, 4H, 4ArH), 7.56
(t, 2H, J = 7.7 Hz, 2ArH), 7.74 (d, 2H, J = 8.2 Hz,
1
2.35; C H N O Æ5.5H O requires: C, 51.25; H,
4
8
58 10
2
2
6
.72; N, 12.45).
2ArH), 8.12 (d, 2H, J = 8.2 Hz, 2ArH), 8.27 (d, 2H,
J = 7.9 Hz, 2ArH), 8.45 (d, 2H, J = 7.2 Hz, 2ArH),
5
9
.13. 1,8-Bis{4-[N-(2-piperidinylethyl)carbamoyl]acridin-
-yl}-diaminooctane (C8 Pip)
+
11.27 (br s, 2H, 2CONH). MALDI-MS: 794.23 (M ).
The pentahydrochloride salt was obtained as a yellow
solid, mp 199.5–201 ꢁC (some sublimation at >165 ꢁC).
This was prepared as for the C8 NMP dimer, using
N-[2-(N-piperidine)ethyl]-9-chloroacridine-4-carboxamide
and 1,8-diaminooctane. The pure product was obtained
as a yellow solid following flash chromatography on alu-
mina (ethyl acetate then methanol/chloroform, v/v = 1/
5.16. 1,10-Bis{4-[N-(2-piperidinylethyl)carbamoyl]acri-
din-9-yl}-1,5,10-triazadecane (C3NC4 Pip)
1
) in 57% yield, mp 112–113 ꢁC (Found: C, 70.99; H,
This was prepared as for the C8 Pip dimer, from N-[2-
(N-piperidine)ethyl]-9-chloroacridine-4-carboxamide
(0.50 mmol) and spermidine (0.25 mmol). The product
7.81; N, 13.24; C H N OÆ2H O requires: C, 71.23;
H, 7.89; N, 13.29). H NMR: 1.25–1.37 (m, 8H,
5
0
62
1
8
2

Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
4399
was purified by chromatography on an alumina col-
5.19. DNA helix unwinding assay
umn (ethyl acetate then methanol) and recrystallized
from chloroform/hexane. The dimer was obtained as
a yellow solid (0.145 g, 35%) (Found: C, 72.83; H,
Helix unwinding angles were measured using agarose
8
gels as previously described. Briefly, supercoiled
7
7
4
2
2
3
7
8
.61; N, 15.60; C H N O Æ0.5H O requires: C,
pBR322 DNA was purchased from Progen Industries
Ltd as a 0.5 mg per ml solution in 10 mM Tris–HCl
buffer, pH 7.5. Solutions of both series of dimers were
prepared in 40 mM TAE buffer [40 mM tris(hydroxy-
methyl)aminomethane, 30 mM glacial acetic acid,
1 mM EDTA, pH 7.5] at the required concentrations,
and 5 ll of solution was mixed with an equal volume
of a 30 lg per ml solution of DNA (150 ng of DNA,
23.6 lM in base pairs) in TAE buffer pH 7.5. NMP di-
mer–DNA complexes were also prepared in 10 mM
TAE buffer [10 mM tris(hydroxymethyl)aminomethane,
4
9
61 19
2
2
1
0.47; H, 7.52; N, 17.12). H NMR: 1.47–1.49 (m,
H, 2CH ), 1.63–1.66 (m, 8H, 4CH ), 2.55 (m, 4H,
2
2
CH ), 2.72 (m, 8H, 4CH ), 2.88 (m, 8H, 4CH ),
2
2
2
.98 (m, 2H, CH ), 3.76–3.78 (m, 4H, 2CH ), 3.83–
2
2
.87 (m, 2H, CH ), 3.99–4.09 (m, 2H, CH ), 7.21–
2
2
.30 (m, 4H, 4ArH), 7.57–7.65 (m, 2H, 2ArH), 8.01–
.24 (m, 6H, 6ArH), 8.79 (br s, 2H, 2ArH), 12.41
+
(br s, 2H, 2CONH). MALDI-MS: 805.50 (M ). The
hexahydrochloride salt was obtained as a yellow solid,
mp 201–202.5 ꢁC.
7.5 mM glacial acetic acid, 1 mM EDTA, pH 7.5]. Com-
5.17. 1,10-Bis{4-[N-(2-piperidinylethyl)carbamoyl]acri-
din-9-yl}-1,4,7,10-tetraaza-decane (C6N2 Pip)
plexes were incubated in the dark for 1 h at room tem-
perature prior to application to a 0.8% agarose gel.
Each titration was repeated at least 3 times. Following
incubation, 3 ll of loading buffer [0.25%(w/v) bromo-
phenol blue and 30%(w/v) glycerol] was mixed with
the complex samples and the samples electrophoresed
for 1 h and 15 min at 74 V (5 V/cm) in either 10 or
40 mM tris buffer at room temperature. After electro-
phoresis, the gel was stained with ethidium bromide
and visualized by fluorescence using a CCD camera cou-
This was prepared as for the C8 Pip dimer, using N-[2-
(
(
N-piperidine)ethyl]-9-chloroacridine-4-carboxamide
0.50 mmol) and triethylenetetramine. Following alu-
mina column chromatography (ethyl acetate then meth-
anol/chloroform) the product was still impure, the clean
free base finally being obtained by re-basifying its
hydrochloride salt, followed by recrystallization from
acetonitrile. The dimer was obtained as a yellow solid
in 39% yield, mp 119–121 ꢁC (Found: C, 70.78; H,
TM
pled to a Bio-Rad Gel Doc 2000 apparatus.
7
7
2
6
2
2
2
2
.40; N, 17.27; C H N O Æ0.5H O requires: C,
5.20. Growth inhibition assay and flow cytometry
4
8
60 10
2
2
1
0.47; H, 7.52; N, 17.12). H NMR: 1.47–1.49 (m, 4H,
CH ), 1.63–1.66 (m, 8H, 4CH ), 2.52–2.55 (m, 12H,
2
Growth inhibition and flow cytometry were performed
8
as previously described. Briefly, for growth inhibition
2
CH ), 2.67–2.70 (m, 4H, 2CH ), 2.93–2.95 (m, 4H,
2
2
5
CH ), 3.74–3.77 (m, 4H, 2CH ), 3.83–3.86 (m, 4H,
2
measurements, 1 · 10 CCFR-CEM cells/ml were incu-
2
CH ), 7.26–7.34 (m, 4H, 4ArH), 7.59–7.64 (m, 2H,
2
ArH), 8.03-8.23 (m, 6H, 6ArH), 8.80 (br s, 2H,
ArH), 12.42 (br s, 2H, 2CONH). The hexahydrochlo-
bated in 24-well plates with test agents for 72 h at
37 ꢁC in RPMI medium 1640 (Gibco BRL) supple-
mented with 10% foetal bovine serum (Trace). Cells
numbers were determined using a Coulter Counter, each
drug concentration being measured in duplicate, and the
experiment repeated at least 3 times. IC50 values were
obtained using GraphPad Prism software. For flow
ride salt was obtained as a yellow solid, mp 239.5–
2
41 ꢁC.
5
.18. N,N-Bis{4-[N-(2-piperidinylethyl)carbamoyl]acri-
5
din-9-yl}-[1,4-bis(2-aminoethyl)-piperazine] (C2pipC2
Pip)
cytometry, cells were seeded at a density of 1 · 10 per
ml in 25 ml flasks and incubated for 24 h before addi-
tion of drugs. Drugs were added at 1·, 2·, 5·, and
10· the measured IC concentrations, and the cells
This was prepared as for the C8 Pip dimer, using N-[2-
5
0
(N-piperidine)ethyl]-9-chloroacridine-4-carboxamide
(0.50 mmol) and 1,4-bis(2-aminoethyl)piperazine, and
incubated for a further 24 h. The cells were then washed
in phosphate-buffered saline and fixed in ice-cold etha-
nol, stained with propidium iodide, and analysed on a
Becton–Dickson FACSort using CellQuest software.
Suitable control cells were prepared in a similar manner.
purified by column chromatography (alumina, ethyl ace-
tate/di(isopropyl)amine, v/v = 10/1, then methanol/chlo-
roform). The product obtained this way still contains
trace amounts of impurities which were removed by re-
peated washing with hot ethyl acetate. Recrystallization
from chloroform/hexane gave the pure dimer as a yellow
powder, mp 119.5–121 ꢁC (Found: C, 69.64; H, 7.51; N,
Acknowledgment
1
6.48; C H N O Æ1.5H O requires: C, 69.66; H, 7.60;
This work was supported by the Australian National
Health and Medical Research Council (L.P.G.W.,
B.W.S.).
5
0
62 10
2
2
1
N, 16.25). H NMR: 1.46–1.51 (m, 4H, 2CH ), 1.65–
2
1
.72 (m, 8H, 4CH ), 2.55–2.78 (m, 24H, 12CH ), 3.79
2 2
(
q, 4H, J = 5.7 Hz, 2CH ), 3.94 (t-like, 4H, 2CH ),
2 2
6
.68 (br s, 2H, 2NH), 7.37–7.46 (m, 4H, 4ArH), 7.71
(
8
t, 2H, J = 7.2 Hz, 2ArH), 8.16–8.20 (m, 4H, 4ArH),
.30 (d, 2H, J = 7.9 Hz, 2ArH), 8.83 (d, 2H,
J = 7.2 Hz, 2ArH), 12.41 (br s, 2H, 2CONH). MAL-
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.02.063.
+
DI-MS: 835.31 (M ). The hexahydrochloride salt was
obtained as a yellow solid, mp 217.5–219 ꢁC.

4400
Z. He et al. / Bioorg. Med. Chem. 16 (2008) 4390–4400
10. Zihif, M. Ph.D thesis Thesis, University of New South
Wales, 2005.
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9