Synthesis of Small Molecules Targeting Multiple DNA Structures using Click Chemistry

Article abstract of DOI:10.1002/cmdc.201200060

The ability of small molecules to target DNA forms the basis of many clinically used antitumour agents. This study examines the effects of novel 9-aminoacridine carboxamides, synthesised by click chemistry based upon the reactions of either 9-(2-azidoethy

Full text of DOI:10.1002/cmdc.201200060

MED
DOI: 10.1002/cmdc.201200060
Synthesis of Small Molecules Targeting Multiple DNA
Structures using Click Chemistry
Lesley A. Howell,[a] Richard A. Bowater,[b] Maria A. O’Connell,[a] Anthony P. Reszka,[c]
Stephen Neidle,[c] and Mark Searcey*[a]
The ability of small molecules to target DNA forms the basis of
many clinically used antitumour agents. This study examines
the effects of novel 9-aminoacridine carboxamides, synthesised
by click chemistry based upon the reactions of either 9-(2-azi-
doethyl)amino or 9-propargylaminoacridine compounds, on
various types of DNA tertiary structures. This gave either mon-
omeric or dimeric compounds, the dimeric derivatives being
the first unsymmetrical acridine dimers to be described. The
compounds were assayed for duplex DNA, quadruplex DNA
and four-way junction DNA binding. Their antiproliferative ac-
tivity in the Human promyelocytic leukaemia cell line, HL60,
was also assessed. Although for some of the compounds, nota-
bly the acridine 4-carboxamides, activity correlated with DNA
binding affinity, for others it did not, with the rigidly linked
dimers in particular showing a complicated relationship be-
tween 3- and 4-carboxamide structure and biological activity.
The monomeric 3-carboxamides were more effective at stabilis-
ing G-quadruplex structures and also gave more hits in the
four-way junction stabilisation assay. There is clear evidence
from the binding of the 3-carboxamides that these compounds
destabilise the open X form of the junction at lower concentra-
tions and stabilise the X-stacked at higher concentrations. This
might have implications for the biological activity of these
compounds against proteins that bind to the Holliday junction
(HJ).
Introduction
Since its first description in 2001,[1] click chemistry has devel-
oped as a useful tool in the design of chemical probes and po-
tential small-molecule therapeutics.[2] In its original form, the
idea of click methodology was to use simple and efficient reac-
tions to generate libraries of products that need little if any pu-
rification. While this concept is still under exploration, the reac-
tion that has garnered most focus is the Huisgen triazole[3] syn-
thesis as adapted by Meldal[4] and Sharpless[1] to run under
mild conditions in the presence of a copper(I) catalyst. Numer-
ous examples of the application of this methodology to ex-
plore biological targets have been disclosed, and herein we
describe the use of this approach to generate compounds to
target multiple forms of DNA.
been the focus of binding studies. Both three-way[7] and four-
way junctions[8,9] can bind small molecules, and the antitumour
activity of the inorganic complexes that target three-way junc-
tions has been established.[10] We have used 9-aminoacridine
carboxamides to target Holliday junctions (HJs), four-way junc-
tions that are involved in bacterial integration processes and in
double-strand break DNA repair mechanisms.[11,12] The original
dimeric 9-aminoacridine 4-carboxamide 1 was shown by high-
resolution crystallography to bind to a HJ via a new binding
mode in which the acridine forms a pseudo-base pair with thy-
mine, displacing an adenine at the junction crossover.[11] This
process is driven by hydrophobic stacking and facilitated by
the longer hydrogen bonds between the bases at the cross-
over site.
Clinically used antitumour agents that bind to or disrupt the
functions of duplex DNA still form the mainstay of current
therapy. Most of these compounds act through general cyto-
toxic mechanisms, and it is clear that the small therapeutic
window of such agents will no longer be tolerated and that
more focussed, targeted therapeutics need to be developed.
While proteins form the main target of such approaches, there
is scope for DNA targeting through the design of agents that
are selective for higher-order DNA structures. The paradigm for
this area has been the G-quadruplex, which was demonstrated
to have potential as a target through inhibition of telomerase,
although this has yet to transfer into a clinically useful thera-
peutic agent.[5] Extension of this work to target promoter and
other sequences within genes suggests that quadruplex target-
ing, should selective agents be found, remains an important
goal.[6] More recently, other higher-order DNA structures have
In order to screen for other HJ binding compounds, we de-
veloped a click chemistry approach to the derivatisation of 9-
aminoacridines at the 9-position and disclosed the use of
Larock and Moses benzyne reactions to generate new com-
[a] Dr. L. A. Howell, Dr. M. A. O’Connell, Prof. M. Searcey
School of Pharmacy, University of East Anglia
Norwich Research Park, Norwich NR4 7TJ (UK)
E-mail: m.searcey@uea.ac.uk
[b] Dr. R. A. Bowater
School of Biological Sciences, University of East Anglia
Norwich Research Park, Norwich NR4 7TJ (UK)
[c] A. P. Reszka, Prof. S. Neidle
Cancer Research UK Biomolecular Structure Group
UCL School of Pharmacy, University College London
29–39 Brunswick Square, London WC1N 1AX (UK)
792
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Small Molecules Targeting Multiple DNA Structures
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in fact, the stability of these compounds was also compro-
mised and, in our hands, the 1,4-substitution pattern was not
isolated. The synthesis of these novel compounds followed the
same general route as the other 9-substituted compounds and
gave the anilinoacridines in reasonable yields (see section
below).
Synthesis of the monomer acridine click library
9-Aminoacridine 4-carboxamides are extremely polar com-
pounds, as exemplified by the requirement for high polarity
mobile phases for chromatographic purification. As such, we
were concerned that this would affect the click reaction using
the classical tert-butanol/water conditions as described by
Sharpless.[1] Initially, we explored other reaction solvents, par-
ticularly N,N-dimethylformamide (DMF), but gratifyingly the re-
actions worked smoothly under the normal conditions to give
reasonable to good yields of the target compounds
(Scheme 1). The main drawback here was that, unlike less
pounds.[13] We have also recently shown that compound 2a
from the Huisgen/Sharpless/Meldal click library has the previ-
ously unknown ability to generate a HJ structure at room tem-
perature without an annealing step.[12] Herein, we describe the
chemistry of the click reactions used to generate a small library
of acridine compounds, including the first—to our knowl-
edge—unsymmetrical acridine dimers. We also disclose bind-
ing studies of these compounds to duplex, quadruplex and
four-way junction DNA, and the antiproliferative activity of the
compounds, demonstrating once again the power of the Huis-
gen/Meldal/Sharpless chemistry in generating new compounds
with interesting activities.
Results and Discussion
Synthesis of the acridine monomers
The synthesis of the 9-(azidoethyl)amino and 9-propargylami-
noacridine carboxamides has been previously described.[12,13]
Briefly, this involves the generation of the 9-chloroacridine car-
boxamides with the appropriate side chain, treatment with
phenol at 1108C, cooling to 558C and addition of the 9-sub-
stituent as the free amine or hydrochloride salt. In the majority
of these studies, we used the N,N-dimethylaminoethyl side
chain. Extensive studies have focussed on this side chain, and
it was present in compound 1, the parent compound that
binds a HJ. We previously explored other side chains, varying
the bulk properties in order to change the kinetics of binding
with duplex DNA,[14] and others have also proposed differing
side chains. We briefly explored the piperidine side chain as
described by Ferguson and co-workers,[15] but the structure–ac-
tivity relationships (SARs) of the side chain will be the focus of
future studies with regard to quadruplex and HJ binding.
Scheme 1. General click chemistry routes for compounds 2–13 (for full struc-
tures and yields, see Table 1). Reagents and conditions: a) CuSO4 (1mol%), Na
ascorbate (10mol%), t-BuOH/H2O (1:1, 0.2m), RT, 24 h; b) CuSO4 (1mol%),
Na ascorbate (10mol%), t-BuOH/H2O (1:1, 0.2m), RT, 24 h.
polar compounds, the products did not precipitate from solu-
tion and a chromatographic step was required to generate
pure material. The 9-propargylaminoacridine carboxamides
were unstable in our hands, and only a limited number of
compounds were generated from this approach. We are cur-
rently exploring protection of the alkyne, for example with a tri-
methylsilyl (TMS) group, and in situ deprotection as an ap-
proach to generating more compounds in this series. Follow-
ing purification by column chromatography, all of the com-
pounds were converted to their hydrochloride salts through
treatment with hydrochloric acid and precipitation or lyophili-
sation.
In the synthesis of the dimers (see section below), it soon
became clear that the 9-propargyl was too unstable to react
with 9-azidoethyl, and we also reasoned that the inter-chromo-
phore distance would be very small for these compounds.
Therefore, we designed a new monomer based upon the am-
sacrine-4-carboxamide nucleus in which an anilinoacridine car-
rying an alkyne on the appended aromatic ring was generated.
These compounds could also allow the generation of a small li-
brary containing meta-, ortho- and para-substituents, although
Synthesis of acridine dimers via click chemistry
Dimeric 9-aminoacridine 4-carboxamides have previously been
studied as DNA bisintercalators and potential topoisomerase
inhibitors.[16] Although these compounds have contained link-
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ers differing in rigidity or length, they have tended to have
symmetrical chromophores. The nature of the click reaction
offers the potential to have a rigid, nonsymmetrical linker as
well as chromophores with different substitution patterns. As
1 was the parent compound, it seemed reasonable from
a design perspective to pursue a more rigid linker to potential-
ly extend across the Holliday junction and also to further inves-
tigate these compounds as bisintercalators in duplex DNA. Ini-
tial studies of a reaction between the 9-propargylamino com-
pounds and the 9-azidoethyl-compounds were unsuccessful,
presumably due to the lack of stability of the alkyne as noted
above. The compounds were redesigned to incorporate an ani-
line group; however, the para-substituted compounds and the
ortho-substituted 3-carboxamides displayed a similar lack of
stability to the aliphatic alkyne and were not isolated. A small
series of unsymmetrical dimers carrying varying substituents
on the acridine (either 3- or 4- carboxamide) as well as either
ortho- or meta-substitution on the ring was generated under
the usual conditions (Scheme 2), with good yields following
column chromatography. These compounds were also convert-
ed to the hydrochloride salts.
Table 1. Monoacridines: yield and DNA binding affinity.
[b]
Compd Structure[a] R group
Yield
[%]
Kapp
[m(bp)ꢀ1 ꢁ106]
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11
A-3
A-4
A-3
A-4
A-3
A-4
A-3
A-4
A-3
A-4
A-3
A-4
A-3
A-4
A-3
A-4
A-4
Phenyl
Phenyl
50
77
50
68
52
58
73
61
47
63
80
88
87
65
96
37
100
2.40
2.18
2.43
2.70
3.03
3.43
1.77
3.83
1.46
1.71
1.49
1.59
41.6
1.61
70.6
3.32
1.18
2-Bromophenyl
2-Bromophenyl
3-Chlorophenyl
3-Chlorophenyl
4-Bromophenyl-
4-Bromophenyl-
4-Chlorophenyl
4-Chlorophenyl
4-Methylphenyl
4-Methylphenyl
4-Trifluoromethylphenyl
4-Trifluoromethylphenyl
Hydroxymethyl
Hydroxymethyl
Carboxylate
12a
A-3
54
2.08
12b
2a
2b
13a
13b
A-4
B-3
B-4
B-3
B-4
As for 20
Benzyl
Benzyl
4-Bromobenzyl
4-Bromobenzyl
73
55
62
13
61
1.14
0.13
5.85
6.50
0.17
[a] A-3: structure A with a 3-carboxamide side chain; A-4: structure A with
a 4-carboxamide side chain; etc. [b] Apparent binding constant (Kapp); see
Experimental Section and Ref. [17].
carboxamide more favourably oriented along the groove but
this does not seem to affect the binding affinity in most cases.
The dimer series is of interest as the DNA binding affinity of
the meta-substituted compounds is of a similar order of mag-
nitude (Kapp = ~26–59ꢁ106 m(bp)ꢀ1; Table 2) as parent bisinter-
Scheme 2. Synthetic route to unsymmetrical diacridine carboxamides using
click chemistry. Reagents and conditions: a) 1. Phenol, 1108C, 15 min; 2. 55
8C, 6-24 h, appropriate ethynylaniline (shown); b) CuSO4 (1mol%), Na ascor-
bate (10mol%), t-BuOH/H2O (1:1, 0.2m), RT, 72 h.
calator 1, which in our hands has a binding affinity of Kapp
=
69ꢁ106 m(bp)ꢀ1 for calf thymus (CT) DNA. It appears that
these compounds are able to bisintercalate or duplex cross-
link[18] double-stranded DNA and that the regiochemistry of
either of the chromophores or the 9-anilino-substituent is suffi-
cient to allow for this. When the aniline is ortho-substituted,
there appears to be a detrimental effect on DNA binding, with
both these compounds having binding affinities lower than
the regioisomers, and the 3-carboxamide in the A-ring having
a strong negative effect on binding that is not seen in the
other compounds. The binding affinity of compound 16 is
around fivefold lower than that of compound 14, and the af-
finity of compound 17 is around threefold lower than that of
15.
DNA binding affinity
Both monomeric and dimeric 9-aminoacridines were assayed
for DNA binding affinity using the ethidium bromide displace-
ment methodology.[17] Both the 3- and 4-carboxamides in the
monomeric series displayed high affinity for duplex DNA with
little apparent change due to the repositioning of the carboxa-
mide side chain (Table 1). The exceptions to this were com-
pounds 9a,b and 10a,b in which the change in duplex DNA
binding affinity was more pronounced (25-fold and 21-fold, re-
spectively). Simple modelling might suggest that the 3-carbox-
amide is more likely to have the side chain lying closer to the
sugar phosphate backbone following intercalation, with the 4-
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Table 2. Dimeric acridines: yield and DNA binding affinity.
[b]
Compd
Structure[a]
B
o or m
Yield
[%]
Kapp
A
[m(bp)ꢀ1 ꢁ106]
1
–
–
–
m
m
o
o
m
m
–
64
73
65
54
81
100
69
59
26
12
8
14
15
16
17
18
19
4-
3-
4-
3-
4-
3-
4-
4-
4-
4-
3-
3-
32
43
[a] As in Table 1. [b] Apparent binding constant (Kapp); see Experimental
Section and Ref. [17].
Holliday junction binding
In order to assess the ability of the compounds to bind to
a HJ, we developed a screen based upon the annealing of four
strands that could form a junction in the presence of divalent
metal ions (Scheme 3). When the four strands are annealed in
the absence of metal ions, the DNA is present as a mixture of
Figure 1. HJ-screening assay of a) 3-carboxamides and b) 4-carboxamides.
Single strands b, h, r and x (5 mm) were heated to 908C in Tris-borate
(pH 8.0) and then allowed to cool to room temperature in the presence or
absence of either MgCl2 or the ligands. All of the compounds synthesised
were assessed for their ability to promote the formation of the HJ at a con-
centration of 50 mm in the absence of Mg2+. For full details and sequences,
see the Experimental Section. Higher concentrations of Mg2+ promote the
formation of the Holliday junction (HJ) and cause the disappearance of
single-stranded DNA. Only strand x is labelled (5’-FAM; 3’-TAMRA). Polyacryl-
amide gel electrophoresis was used to distinguish single-stranded (SS) oligo-
nucleotides and HJ. With no Mg2+, the majority of the oligonucleotides
remain SS (Lane 0). At 2 mm Mg2+, the majority of the DNA is in the form of
the HJ (Lane C).
Scheme 3. Schematic of the HJ binding assay. In solution, in the absence of
metal ions or the ligands, the oligos exist either in the single-stranded or
open X form. Heating to 908C to denature and then cooling in the presence
of either Mg2+ (Lanes C in gels shown in Figure 1) or ligands can promote
the formation of the X-stacked form.
single strands and the open X form, which can be separated
on a polyacrylamide gel and visualised via fluorescence when
one of the strands is labelled at the 3’ and 5’ ends, as in
strand x which is labelled with 6-carboxyfluorescein (FAM) at
the 5’ end and 6-carboxytetramethylrhodamine (TAMRA) at the
3’ end (Lane 0 in gels shown in Figure 1). In the presence of in-
creasing concentrations of Mg2+, the concentration of single
strands is decreased and the equilibrium shifts to generate
only X-stacked HJ (Lane C in gels shown in Figure 1).
whereas two compounds (8b and 10a) appeared to destabi-
lise the duplex. A concentration profile for the compounds
demonstrated that the monomers 2, 11 and 13 all stabilised
the HJ at a concentration of 20–50 mm (data not shown). At
lower concentrations, these compounds all seemed to pro-
mote single-stranded DNA over HJ DNA, and we reasoned that
this is because the small molecules bind with high affinity to
the X-stacked form of DNA, rather than with the open X that
occurs in the absence of ligand/metal ion (Scheme 3) and
which seems to be destabilised by the presence of low con-
centrations of ligands.
All of the acridine 3- and 4-carboxamides were screened at
a concentration of 50 mm (Figure 1). Compound 1, used as
a control for comparison, affects the mobility of a HJ in the
gel. We believe this is due to the base-displacement mode of
binding of this compound,[11] and it is notable that none of the
other acridines have this effect. Surprisingly, only three of the
monomers (2a, 7a, and 8a) and two of the dimers (18 and 19,
data not shown) promoted annealing at this concentration,
This high affinity for the X-stacked form of the junction was
confirmed by circular dichroism studies of compound 2a that
have been previously reported.[12] The inability of any of the 4-
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carboxamides and most of the 3-carboxamides to stabilise
a HJ structure demonstrates that this is not just a consequence
of intercalation. The SAR data derived from this screen are lim-
ited but do generate some information. For example, a 4-car-
boxamide structure is clearly unable to stabilise the junction. A
4-bromo-substituent in the 9-position of a 3-carboxamide is
less efficient than the equivalent chloro-substituent, and move-
ment of the halide to a meta-position abolishes activity. The
methyl substituent in the 4-position of the aromatic ring is fav-
oured, although there is some evidence for stabilisation by the
4-trifluoromethyl-substituent. In the B series, a bromo-substitu-
ent in the para-position abolished activity.
differences in binding of the 3- and 4-carboxamides were not
clear-cut, and both compounds 4b and 6b showed good sta-
bilisation of the quadruplex structures, particularly the c-Kit2
quadruplex. Compounds 4a and 5a demonstrated little selec-
tivity for the different quadruplex structures, which was of in-
terest as compounds 4a and 4b carry the same 9-position
substitutent, suggesting that the selectivity exhibited by com-
pound 4b stems from the carboxamide side chain. Unfortu-
nately, compound 6a could not be screened against c-Kit2 as
the intrinsic fluorescence of the compound interfered with the
assay so a direct comparison with 6b was not possible. 3-Car-
boxamides 3a and 10a both showed some selectivity for sta-
bilisation of c-Kit2, whereas 4-position-substituted compound
5b had the largest selectivity of all the compounds. Effects in
the stabilisation of the duplex T-loop were small for all the
compounds, which might reflect the known GC selectivity of
the acridine carboxamides and the relatively high AT content
in this duplex sequence.
Further investigation of one of the compounds (10a) that
destabilised the junction at 50 mm showed that it, in fact, stabi-
lised the X-stacked junction at higher concentrations and that
at the original test concentration the compound destabilises
the open X form of the junction. Concentration-dependence
studies of the dimers revealed that these agents generated
multiple DNA structures on the gel, suggesting duplex cross-
linking[18] or other higher-order structures (data not shown),
but these observations were not pursued further.
Antiproliferative activity
Initial studies of the 9-aminoacridine carboxamide nucleus car-
ried out by Denny et al. demonstrated that the 3-carboxamides
tended to have little antiproliferative activity compared with
the 4-carboxamides in spite of their ability to bind to duplex
DNA.[20] More recently, Ferguson and co-workers have demon-
strated modest (low micromolar) antitproliferative activity in
pancreatic tumour cells for a series of 3-carboxamides carrying
a piperidine side chain and an indole linked to the 9-posi-
tion.[15] Although we failed to replicate biological activity in an
analogous benzimidazole based series,[13] the results for the
click library demonstrate that several of the acridine 3-carboxa-
mides studied here do maintain modest activity, notably com-
pounds 6a, 7a and 9a with a halogen substituent in the 4-po-
sition. It is noteworthy that 4-methyl derivative 8a is inactive,
whereas trifluoromethyl compound 9a is active. Compound
2a also displays low micromolar antiproliferative activity
(Table 4).
G-Quadruplex binding
With the disclosure of the co-crystal structure of the BRACO19
complex with a human telomeric quadruplex,[19] it was clear
that 9-aminoacridine 3-carboxamides are suitably substituted
to place the 9- and 3-position substituents in the grooves of
the G-quadruplex, and compounds such as the click chemistry
products described here have the potential to build in en-
hanced recognition due to the ability to “read” these grooves.
A small subset of the click products were assayed in a fluores-
cence melting assay to ascertain their ability to stabilise three
differing G-quadruplex structures: c-Kit1, c-Kit2 and the F21T
from the human telomere. They were compared with a control
duplex DNA loop sequence. Stabilisation of the quadruplex
structures was relatively modest for all compounds that
showed an effect (Table 3).
The 9-aminoacridine 4-carboxamide monomers maintain
their potency across the series with the 9-alkynyl-substituted
click precursors leading to particularly potent products. Al-
However, there were some interesting differences in affinity
for different quadruplex structures. Somewhat surprisingly, the
Table 3. Change in melting temperature of G-quadruplex or double-
stranded (duplex) DNA for a subset of the click library.[a]
Table 4. Antiproliferative (IC50) activities against the HL60 human leukae-
mic cell line.
Compd
DTmelt (G-quadruplex) [8C]
DTmelt (duplex) [8C]
F21T
CKit1
CKit2
T-loop
Compd
IC50[a] [mm]
Compd
IC50[a] [mm]
Compd
IC50[a] [mm]
3a
4a
4b
5a
5b
6a
6b
10a
10b
11.9
15.9
10.1
14
ꢀ1.7
14.1
10.2
12.2
6.7
5.9
13
1.7
12.6
1.3
13.9
1.9
18.6
10.6
18.9
11.5
20.3
–
18.6
15.4
–
1.7
2.4
1.1
2.3
0.8
3.9
1.3
3.2
2.1
1
0.33
9.1
7a
7b
8a
8b
9a
9b
10a
10b
11
17.3
12.1
n.a.
12b
2b
13a
13b
14
15
16
17
18
32.4
1.2
n.a.
1.5
16.7
12.4
2.4
28.7
26.2
n.a.
2a
3a
3b
4a
4b
5a
5b
6a
6b
n.a.
12.9
n.a.
39.4
n.a.
29.4
4.9
3.7
59.8
20.1
n.a.
53.3
25.9
n.a.
8.4
2.1
3.6
12a
19
[a] Change in melting temperature (DTmelt) at a concentration of 10 mm;
values are ꢁ0.18C.
[a] Not active (n.a.).
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water or phosphate-buffered saline (PBS) (200 mL) to prevent evap-
oration of cell media. The remaining wells were seeded with HL-60
cells (3ꢁ104/100 mL) and left untreated or treated with water (vehi-
cle control), doxorubicin (positive control, 100 mm–10pm) or acri-
dines (100 mm–1 nm) in triplicate for 72 h at 378C with 5% CO2.
Following this, MTS reagent was added to each well, and the
plates were incubated for 2.5 h at 378C with 5% CO2. The absorb-
ance was measured at 492 nm using a POLARstar OPTIMA micro-
plate reader (BMG Labtech). For statistical analysis, the control of
cells alone was taken as 100% cell proliferation. IC50 values were
calculated using GraphPad Prism version 5.0.
though none of the compounds were as potent as the parent
C6-linked diacridine (1), the alternative click structures of 2b
and 13b represent monointercalators with activity less than
tenfold lower than the parent. All of the unsymmetrical dimers
were 10–100-fold less active than parent compound 1 with the
exception of compound 19, which has both chromophores as
the 3-carboxamide and was inactive. As all the other com-
pounds contain a 4-carboxamide structure, it would suggest
that this configuration is necessary in at least one of the dimer
chromophores for antiproliferative activity.
UV Absorbance screen: To ensure the fluorescence of the acridine
moiety did not interfere with the ethidium bromide displacement
assay, 10 mm solutions of each compound in water were analysed
using UV absorbance spectroscopy between 200 and 800 nm using
a Lamda 25 UV/Vis spectrometer (PerkinElmer).
Conclusions
Click methodology is a powerful tool for the generation of li-
braries of compounds and, as demonstrated here, can be ap-
plied to synthesise relatively complex molecules that can inter-
act with more than one biological target—in this case, duplex
DNA and higher-order DNA structures. The acridine 3-carboxa-
mides are especially interesting as they have been shown to
induce the formation of a Holliday junction and can also be
used to target G-quadruplex structures with some selectivity,
perhaps generated by the variations in structure on the 3- and
9-positions being able to interact with the grooves of the
higher-order DNA.
Competitive ethidium bromide displacement assay: CT DNA stock
was prepared as a 1 mgmLꢀ1 solution in water and stored between
2–88C. This is equal to 3067 mm (bp) calculated from its UV absorb-
ance at 260 nm. Prior to use, CT DNA was diluted with autoclaved
water to 88 mm (bp) and stored between 2–88C. Each well of a 96-
well, black plate was loaded with Tris buffer containing ethidium
bromide (88 mL; 0.1m Tris, 0.1m NaCl, pH 7, 4.4 mm ethidium bro-
mide final concentration). CT DNA (10 mL; 8.8 mm (bp) final concen-
tration) was added to each well followed by test compound (2 mL)
in increasing concentrations of 0.1 mm–10 mm (2 nm–200 mm final
concentration). After incubation in the dark for 30 min at RT, the
plate was read (average of 25 readings per well) on a POLARstar
OPTIMA fluorescent microplate reader (lex =560 nm; lem =590 nm)
in triplicate experiments with six control wells (3ꢁno test com-
pound=100% fluorescence; 3ꢁno DNA=0% fluorescence). Fluo-
rescence readings are reported as % fluorescence relative to con-
trols. IC50 values were calculated using GraphPad Prism version 5.0.
Experimental Section
Biological evaluation
Biochemicals: Calf thymus (CT) DNA was purchased from Aldrich,
diluted to 1 mgmLꢀ1 in water and stored at 48C. Ethidium bromide
was purchased from Fisher Scientific as a 10 mgmLꢀ1 solution in
water. RPMI-1640 media and l-glutamine were purchased from In-
vitrogen. Foetal calf serum (FCS) was purchased from Biosera. 3-
(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)- 2H-tetrazolium (TMS) was purchased from Promega. Acryl-
amide/Bis solution (30%) and tetramethylethylenediamine (TEMED)
were purchased from BioRad. Tris base was purchased from Mel-
ford Laboratories Ltd (Ipswich). Boric acid and ethylenediaminete-
traacetic acid (EDTA) were both purchased from Fisher Scientific as
was ammonium persulfate. All oligonucleotides were synthesised
and purified by Eurogentec S.A. (Seraing, Belgium) and stocks were
diluted with water and stored at ꢀ208C. All water was autoclaved
on site before use.
PAGE Holliday junction binding assay: Oligonucleotides b, h and r
(b=5’-GCCTAGCATGATACTGCTACCG-3’; h=5’-CGGTAGCAGTACCG-
TTGGTGGC-3’; r=5’-GCCACCAACGGCGTCAACTGCC-3’) were dilut-
ed to 5 mm in 1xTris borate (pH 8.0) buffer and oligonucleotide x
(x=5’-FAM-GGCAGTTGACGTCATGCTAGGC-TAMRA-3’, where FAM is
6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhoda-
mine) was diluted to 5 mm in water. The Holliday junction was pre-
pared at a concentration of 1.25 mm by annealing stoichiometric
amounts of oligonucleotides b, h, r and x in the presence of test
compound (2 mL) at the desired concentration (50-fold dilution).
This was carried out by heating the solution at 808C for 5 min fol-
lowed by slow cooling to 108C over a period of several hours. Two
control solutions were prepared and annealed: one in which the
1ꢁTris borate was substituted for 450 mm NaCl, 24 mm sodium cit-
rate, pH 7.0 and 2 mm MgCl2; and one in which test compound
was omitted. Electrophoresis was then carried out using a Mini-
PROTEAN 3 cell system (BioRad) with a 12% native polyacrylamide
gel for 2 h at 50 V using 0.5ꢁTris/borate/EDTA (TBE) as the running
buffer. Gels were visualised using a Molecular Imager FX Pro Plus
Cell culture: HL-60 is a promyelocytic cell line derived from periph-
eral blood leukocytes from a 36 year old female with acute pro-
myelocytic leukaemia and was purchased from the European Col-
lection of Cell Cultures (ECACC, Porton Down, UK). HL-60 cells were
cultured in RPMI-1640 media containing 2 mm l-glutamine and
10% FCS. Cells were maintained at 378C and 5% CO2. HL-60 cells
were maintained between 1ꢁ105 and 9ꢁ105 cellsmLꢀ1 in 75 cm2
flasks, split with fresh media every 3.5 days and used for experi-
mentation until passage 30. For cell number and viability, HL-60
cells were diluted to one in five using trypan blue and counted
using a Neubaeur haemocytometer (Fisher) with light microscopy.
set (BioRad) to detect the FAM fluorophore (lex =488 nm; lem
=
530 nm) and processed using Quantity One version 4.5.1.
DNA Melting experiments using fluorescence resonance energy trans-
fer (FRET): Dual-labelled oligonucleotides (Eurogentec Ltd., UK)
with the following sequences were used (where FAM is the donor
fluorophore and TAMRA is the acceptor): G4 DNA 5’-FAM-
GGGTTAGGGTTAGGGTTAGGGTTAGGG-TAMRA-3’; ds DNA 5’-FAM-
TATAGCTATATTTTTTTATAGCTATA-TAMRA-3’; c-kit1 5’-FAM-AGAGG-
MTS Cytotoxicity assay: The cytotoxicity of the compounds studied
was assessed using a CellTiter 96 AQueous One Solution Cell Prolif-
eration (MTS) assay (Promega) following the manufacturer’s in-
structions. Briefly, the outer wells of a 96-well plate were filled with
ChemMedChem 2012, 7, 792 – 804
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M. Searcey et al.
MED
GAGGGCGCTGGGAGGAGGGGCT-TAMRA-3’;
CCCGGGCGGGCGCGAGGGAGGGGAGG-TAMRA-3’.
c-kit2
5’-FAM-
propargylamine (19.9 mL, 0.31 mmol) was added. The reaction mix-
ture was then stirred at 558C for 1 h. On cooling, aq NaOH (2m,
10 mL) was added. A small amount of CH2Cl2 (5 mL) was added to
a separating funnel, and the reaction mixture was poured on top.
After separation of the organic phase, the aqueous phase was ex-
tracted successively with CH2Cl2 (3ꢁ10 mL). The combined organic
extracts were dried (MgSO4), filtered and concentrated in vacuo to
yield a yellow solid. Column chromatography (10% MeOH in
EtOAc+1% Et3N) yielded the desired compound as an orange oil,
which was unstable (84.2 mg, 80%): 1H NMR (400 MHz, CD3OD):
dH =8.63 (dd, J1 =0.8 Hz, J2 =7.2 Hz, 1H), 8.55 (dd, J1 =1.2 Hz, J2 =
8.8 Hz, 1H), 8.39 (dd, J1 =0.4 Hz, J2 =8.8 Hz, 1H), 8.00 (d, J=8.4 Hz,
1H), 7.77–7.73 (m, 1H), 7.48–7.42 (m, 2H), 4.54 (d, J=2.4 Hz, 2H),
3.76 (t, J=6.4 Hz, 2H), 2.82 (t, J=6.4 Hz, 2H), 2.48 (s, 6H),
Each oligonucleotide was initially diluted to 100 mm in nuclease-
free water (not DEPC-treated), purchased from Ambion Applied
Biosystems UK. Stock solution of 20 mm and subsequent dilutions
were obtained in FRET buffer (60 mm KCl, K-cacodylate, pH 7.4).
Oligonucleotides (400 mm) were annealed by heating at 858C for
10 min and then cooling to RT. Test compounds were prepared as
10 mm DMSO stock solutions and diluted to 1 mm using 1 mm HCl
in HPLC-grade water. The rest of the dilutions were performed
using FRET buffer. Annealed DNA (50 mL) and test compound solu-
tion (50 mL) were distributed across 96-well RT-PCR plates (BioRad;
MJ Research, Waltham, MA, USA) and processed in a DNA Opticon
Engine (MJ Research). Experiments were performed in triplicate.
Fluorescence readings were taken at intervals of 0.58C over the
range 30–1008C, with a constant temperature being maintained
for 30 s prior to each reading. The incident radiation was 450–
495 nm and the detection was conducted at 515–545 nm. Raw
data were imported into the Origin program (version 7.0, Origin
Lab Corp., Northampton, MA, USA), and the graphs were smooth-
ed using a ten-point running average and subsequently normal-
ised. All data were analysed with the Origin Pro 7.0 software pack-
age (Origin Lab Corp.). The change in melting temperature at
a 10 mm ligand concentration (DTm) was calculated from three ex-
periments by subtraction of the blank from the averaged 10 mm
ligand melting temperature.
2.47 ppm (s, 1H); IR (neat): n˜max =3292, 2936, 2863, 1636 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C21H23ON4: 347.1866, found:
347.1863.
N-(2-(Dimethylamino)ethyl)-9-(prop-2-ynylamino)acridine-3-car-
boxamide: 9-Chloro-N-(2-(dimethylamino)ethyl)acridine-3-carboxa-
mide[13] (100 mg, 0.31 mmol) was stirred in excess phenol (500 mg)
at 1108C for 15 min. The temperature was reduced to 558C and
propargylamine (19.9 mL, 0.31 mmol) was added. The reaction mix-
ture was then stirred at 558C for 2 h. On cooling, aq NaOH (2m,
10 mL) was added. A small amount of CH2Cl2 (5 mL) was added to
a separating funnel, and the reaction mixture poured on top. After
separation of the organic phase, the aqueous phase was extracted
successively with CH2Cl2 (3ꢁ10 mL). The combined organic extracts
were dried (MgSO4), filtered and concentrated in vacuo to yield
a yellow solid. Column chromatography (10% MeOH in EtOAc+
1% Et3N) yielded the desired compound as an orange oil, which
Synthesis
General: 1H and 13C NMR spectra were recorded in fourier trans-
form mode either on a Varian Unity Plus 400 or a Gemini 300 spec-
trometer operating at a nominal 1H NMR frequency of 400 MHz
and 300 MHz, respectively, using the specified deuterated solvent.
All spectra were processed using MestRe-C 4.9.9.6 software. Chemi-
1
was unstable (87.5 mg, 83%): H NMR (400 MHz, CD3OD): dH =8.48
(d, J=8.8 Hz, 1H), 8.42 (d, J=8.8 Hz, 1H), 8.29 (d, J=1.2 Hz, 1H),
7.89 (d, J=8.0 Hz, 1H), 7.78–7.74 (m, 2H), 7.46–7.42 (m, 1H), 4.61
(d, J=2.8 Hz, 2H), 3.62 (t, J=6.8 Hz, 2H), 2.69 (t, J=6.8 Hz, 2H),
2.39 (s, 6H), 2.38 ppm (s, 1H); IR (neat): n˜max =3275, 2940, 2827,
1
cal shifts (d) for both H and 13C spectra were recorded in parts per
1635 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C21H23ON4:
million (ppm) and were referenced to the residual solvent peak.
Multiplicities in the NMR spectra are described as: s=singlet, d=
doublet, t=triplet, q=quartet, m=multiplet, br=broad; coupling
constants (J) are reported in hertz (Hz). High-resolution mass spec-
tra (HRMS) were recorded at the EPSRC National Mass Spectrome-
try Service Centre (Swansea, UK). Melting points (mp) were record-
ed using open capillary tubes on a Mel-Temp electrothermal melt-
ing point apparatus and are uncorrected. Infrared (IR) spectra were
recorded as neat samples using a PerkinElmer Spectrum BX FT-IR
and manipulated using Spectrum v5.0.1 software. Ultraviolet (UV)
spectra were obtained using a PerkinElmer Lamda 25 UV/Vis spec-
trometer at the wavelengths specified.
347.1866, found: 347.1866.
9-((1-Benzyl-1H-1,2,3-triazole-4-yl)methylamino)-N-(2-dimethyla-
mino)ethyl)acridine-3-carboxamide (2): N-(2-(Dimethylamino)eth-
yl)-9-(prop-2-ynylamino)acridine-3-carboxamide
0.30 mmol) and benzyl azide (39.9 mg, 0.30 mmol) were dissolved
in tBuOH:H2O (1:1, 1.5 mL). Sodium ascorbate (10 mol%, 30.0 mL,
1m stock) was added followed by aq CuSO4 (1 mol%, 3.0 mL, 1m
stock). The reaction mixture was stirred at RT for 72 h. On comple-
tion, water (50 mL) was added, and the resultant precipitate was
isolated by filtration. The solid was washed well with water and
dried in vacuo to yield the free base as a bright yellow solid
(103.8 mg,
1
(79.1 mg, 55%): mp: 117.1–118.48C; H NMR (400 MHz, CDCl3): dH =
Thin layer chromatography (TLC) was performed on Merck alumini-
um plates coated with 0.2 mm silica gel-60 F254. After elution, TLC
plates were visualised either under UV light or by staining with
phosphomolybdic acid (10% in EtOH) or vanillin followed by heat-
ing. Chromatographic separations were performed on silica gel for
column chromatography (particle size 60 mm). All samples purified
using more than 20% MeOH were filtered after purification.
8.38 (s, 1H), 8.17 (d, J=9.2 Hz, 1H), 8.13 (d, J=8.8 Hz, 1H), 8.03 (br
d, J=7.2 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.71–7.67 (m, 1H), 7.42–
7.38 (m, 1H), 7.33–7.32 (m, 3H), 7.21 (s, 1H), 7.15–7.13 (m, 2H),
5.46 (s, 2H), 5.02 (, s, 2H), 3.58 (q, J=5.6 Hz, 2H), 2.56 (t, J=5.6 Hz,
2H), 2.29 ppm (s, 6H); IR (neat): n˜max =3338, 2938, 1650 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C28H30N7O: 480.2506, found:
480.2510.
All chemicals were reagent grade and purchased from Aldrich or
Fisher Scientific. All glassware was oven dried prior to use. Distilled
water was used in all cases.
The free base was converted into the dihydrochloride salt by stir-
ring in 1.25m HCl in MeOH (1 mL) for 10 min followed by precipita-
1
tion with EtOAc (quant yield): H NMR (400 MHz, CD3OD): dH =8.63
N-(2-(Dimethylamino)ethyl)-9-(prop-2-ynylamino)acridine-4-car-
boxamide: 9-Chloro-N-(2-(dimethylamino)ethyl)acridine-4-carboxa-
mide[13] (100 mg, 0.31 mmol) was stirred in excess phenol (500 mg)
at 1108C for 15 min. The temperature was reduced to 558C and
(d, J=9.2 Hz, 1H), 8.54 (d, J=8.8 Hz, 1H), 8.34 (d, J=1.6 Hz, 1H),
8.22 (s, 1H), 7.98–7.92 (m, 2H), 7.84 (d, J=8.0 Hz, 1H), 7.52 (t, J=
8.0 Hz, 1H), 7.37–7.30 (m, 5H), 5.63 (s, 2H), 5.43 (s, 2H), 3.87 (t, J=
5.6 Hz, 2H), 3.49 (t, J=5.6 Hz, 2H), 3.03 ppm (s, 6H).
798
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Small Molecules Targeting Multiple DNA Structures
MED
9-(((1-Benzyl-1H-1,2,3-triazole-4-yl)methylamino)-N-(2-dimethyla-
7.92 (m, 2H), 7.62–7.55 (m, 2H), 7.52 (d, J=8.4 Hz, 2H), 7.26 (d, J=
8.4 Hz, 2H), 5.61 (s, 2H), 5.42 (s, 2H), 3.92 (t, J=5.6 Hz, 2H), 3.52 (t,
J=5.6 Hz, 2H), 3.05 ppm (s, 6H); IR (neat): n˜max =3380, 2973,
1636 cmꢀ1; HRMS (ES+): m/z [M+H]+ calcd for C28H29BrN7O:
558.1611, found: 558.1613.
mino)ethyl)acridine-4-carboxamide
no)ethyl)-9-(prop-2-ynylamino)acridine-4-carboxamide
(2b):
N-(2-(Dimethylami-
(40.9 mg,
0.12 mmol) and benzyl azide (15.7 mg, 0.12 mmol) were dissolved
in tBuOH:H2O (1:1, 590 mL). Sodium ascorbate (10 mol%, 11.8 mL,
1m stock) was added followed by aq CuSO4 (1 mol%, 1.18 mL, 1m
stock), and the reaction mixture was stirred at RT for 24 h. The sol-
vent was removed in vacuo, and the crude was purified by column
chromatography (5% MeOH in EtOAc+1% Et3N) to afford the free
base as an orange oil (35.3 mg, 62%).
9-(2-Azidoethylamino)-N-(2-(dimethylamino)ethyl)acridine-4-car-
boxamide: 9-Chloro-N-(2-(dimethylamino)ethyl)acridine-4-carboxa-
mide[13] (500 mg, 1.5 mmol) was stirred in excess phenol (1.5 g) at
1008C for 15 min. On cooling to 558C, 2-azidoethanamine hydro-
chloride[13] (205 mg, 1.7 mmol) was added, and the reaction was
stirred at 558C overnight. On cooling, aq NaOH (2m, 30 mL) was
added, and the reaction mixture extracted with CH2Cl2 (3ꢁ20 mL).
The combined organic extracts were dried (MgSO4), filtered and
concentrated in vacuo. Column chromatography (5% MeOH in
EtOAc+1% Et3N) yielded the desired product as a yellow oil
(555.9 mg, 97%): 1H NMR (300 MHz, CD3OD): dH =8.54 (d, J=
6.6 Hz, 1H), 8.36 (dd, J1 =1.4 Hz, J2 =8.7 Hz, 1H), 8.21 (d, J=8.7 Hz,
1H), 7.87 (d, J=8.1 Hz, 1H), 7.69–7.64 (m, 1H), 7.37–7.32 (m, 2H),
3.92 (t, J=6.3 Hz, 2H), 3.69 (t, J=6.3 Hz, 2H), 3.60 (t, J=5.7 Hz,
2H), 2.70 (t, J=6.5 Hz, 2H), 2.39 ppm (s, 6H); IR (neat): n˜max =3313,
2935, 2856, 2095, 1638 cmꢀ1; HRMS (ES+): m/z [M+H]+ calcd for
C20H24N7O: 378.2037, found: 378.2039.
The free base was further purified through conversion to the dihy-
drochloride salt by stirring in 1.25m HCl in MeOH (0.5 mL) for
10 min followed by precipitation with EtOAc. The resulting yellow
solid was freeze-dried from water to give the dihydrochloride salt
(37.7 mg, 93%): mp: 124.3–125.88C; 1H NMR (400 MHz, CD3OD):
dH =8.78 (d, J=8.8 Hz, 1H), 8.58–8.55 (m, 2H), 8.20 (s, 1H), 8.02–
7.93 (m, 2H), 7.63–7.54 (m, 2H), 7.38–7.32 (m, 5H), 5.63 (s, 2H),
5.42 (s, 2H), 3.92 (t, J=5.6 Hz, 2H), 3.52 (t, J=5.6 Hz, 2H),
3.05 ppm (s, 6H); IR (neat): n˜max =3354, 2945, 1636 cmꢀ1; HRMS
(ES+): m/z [M+H]+ calcd for C28H30N7O: 480.2506, found:
480.2507.
9-(((1-(4-Bromobenzyl)-1H-1,2,3-triazole-4-yl)methylamino)-N-(2-
dimethylamino)ethyl)acridine-3-carboxamide (13a): N-(2-(Dime-
thylamino)ethyl)-9-(prop-2-ynylamino)acridine-3-carboxamide
(63.8 mg, 0.18 mmol) and 4-bromobenzyl azide (39.1 mg,
0.18 mmol) were dissolved in tBuOH:H2O (1:1, 921 mL). Sodium as-
corbate (10 mol%, 18.4 mL, 1m stock) was added followed by aq
CuSO4 (1 mol%, 1.84 mL, 1m stock), and the reaction mixture was
stirred at RT for 24 h. The solvent was removed in vacuo, and the
crude was purified by column chromatography (10% MeOH in
EtOAc+1% Et3N) to afford the free base as an orange oil (12.6 mg,
13%).
9-(2-Azidoethylamino)-N-(2-(dimethylamino)ethyl)acridine-3-car-
boxamide: 9-Chloro-N-(2-(dimethylamino)ethyl)acridine-3-carboxa-
mide[13] (500 mg, 1.5 mmol) was stirred in excess phenol (1.5 g) at
1008C for 15 min. On cooling to 558C, 2-azidoethanamine hydro-
chloride[13] (205 mg, 1.7 mmol) was added, and the reaction was
stirred at 558C overnight. On cooling, aq NaOH (2m, 30 mL) was
added, and the reaction mixture was extracted with CH2Cl2 (3ꢁ
20 mL). The combined organic extracts were dried (MgSO4), filtered
and concentrated in vacuo. Column chromatography (10%!20%
MeOH in EtOAc+1% Et3N) yielded the desired product as a yellow
oil (502.6 mg, 87%): 1H NMR (300 MHz, CDCl3): dH =8.32 (d, J=
1.5 Hz, 1H), 8.16–8.10 (m, 2H), 7.93 (d, J=8.7 Hz, 1H), 7.78 (dd, J1 =
1.8 Hz, J2 =9.0 Hz, 1H), 7.69–7.63 (m, 1H), 7.42–7.36 (m, 2H), 3.95
(t, J=5.4 Hz, 2H), 3.65–3.55 (m, 4H), 2.56 (t, J=5.9 Hz, 2H),
2.29 ppm (s, 6H); IR (neat): n˜max =3275, 2936, 2819, 2094,
The free base was further purified through conversion to the dihy-
drochloride salt by stirring in 1.25m HCl in MeOH (0.5 mL) for
10 min followed by precipitation with EtOAc. The resulting yellow
solid was freeze-dried from water to give the dihydrochloride salt
(12.1 mg, 85%): mp: 165.6–168.28C; 1H NMR (400 MHz, CD3OD):
dH =8.67 (d, J=8.8 Hz, 1H), 8.56 (d, J=8.8 Hz, 1H), 8.37 (d, J=
1.6 Hz, 1H), 8.20 (s, 1H), 8.03–7.98 (m, 1H), 7.94 (d, J=8.8 Hz, 1H),
7.88 (dd, J1 =8.8 Hz, J2 =1.2 Hz, 1H), 7.58–7.52 (m, 3H), 7.37 (d, J=
8.4 Hz, 2H), 5.61 (s, 2H), 5.45 (s, 2H), 3.87 (t, J=5.6 Hz, 2H), 3.47 (t,
J=5.6 Hz, 2H), 3.03 ppm (s, 6H); IR (neat): n˜max =3369, 2971,
1636 cmꢀ1; HRMS (ES+): m/z [M+H]+ calcd for C28H29BrN7O:
558.1611, found: 558.1613.
1643 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C20H24N7O:
378.2037, found: 378.2035.
Click chemistry general method A: The appropriate acridine azide
was dissolved in tBuOH:H2O (1:1, 0.2m), and the solution was treat-
ed with the desired alkyne (1 equiv). The reaction mixture was
stirred at RT for 2 min followed by the addition of sodium ascor-
bate (10 mol%) and CuSO4 (1 mol%). This mixture was stirred at RT
overnight. Water (5 mL) was added, and the mixture was extracted
with CH2Cl2 or EtOAc (3ꢁ10 mL). The combined organic extracts
were dried (MgSO4), filtered and concentrated in vacuo. The prod-
uct was purified by column chromatography.
9-((1-(4-Bromobenzyl)-1H-1,2,3-triazole-4-yl)methylamino)-N-(2-
dimethylamino)ethyl)acridine-4-carboxamide (13b): N-(2-(Dime-
thylamino)ethyl)-9-(prop-2-ynylamino)acridine-4-carboxamide
(34.2 mg, 0.10 mmol) and 4-bromobenzyl azide (21.0 mg,
0.10 mmol) were dissolved in tBuOH:H2O (1:1, 495 mL). Sodium as-
corbate (10 mol%, 10.0 mL, 1m stock) was added followed by
CuSO4 (1 mol%, 1.0 mL, 1m stock), and the reaction mixture was
stirred at RT for 24 h. The solvent was removed in vacuo, and the
crude was purified by column chromatography (5% MeOH in
EtOAc+1% Et3N) to afford the free base as an orange oil (33.7 mg,
61%).
Click chemistry general method B: The appropriate acridine azide
was dissolved in tBuOH:H2O (1:1, 0.2m), and the solution was treat-
ed with the desired alkyne (1 equiv). The reaction mixture was
stirred at RT for 2 min followed by the addition of sodium ascor-
bate (10 mol%) and CuSO4 (1 mol%). This mixture was stirred at RT
overnight. The solvents were removed in vacuo, and the product
was purified by column chromatography.
The free base was further purified through conversion to the dihy-
drochloride salt by stirring in 1.25m HCl in MeOH (0.5 mL) for
10 min followed by precipitation with EtOAc. The resulting yellow
solid was freeze-dried from water to give the dihydrochloride salt
(30.5 mg, 80%): mp: 161.3–162.28C; 1H NMR (400 MHz, CD3OD):
dH =8.78 (d, J=8.4 Hz, 1H), 8.58–8.55 (m, 2H), 8.23 (s, 1H), 8.01–
N-(2-(Dimethylamino)ethyl)-9-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)e-
thylamino)acridine-3-carboxamide (3a): Prepared according to
general method A. Column chromatography (10%!20% MeOH in
EtOAc+1% Et3N) afforded the free base as an orange oil (46.4 mg,
50%). The free base was converted into the dihydrochloride salt by
stirring the compound in 1.25m HCl in MeOH (2 mL) for 10 min fol-
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M. Searcey et al.
MED
lowed by precipitation with EtOAc. The resultant solid was freeze-
dried from water to give the salt as a yellow oil (32.5 mg, 61%):
1H NMR (400 MHz, CD3OD): dH =8.56 (d, J=9.2 Hz, 1H), 8.47 (d, J=
8.4 Hz, 1H), 8.28 (s, 2H), 8.00–7.94 (m, 2H), 7.79 (d, J=8.4 Hz, 1H),
7.62–7.56 (m, 3H), 7.40–7.32 (m, 3H), 5.01 (t, J=5.2 Hz, 2H), 4.81 (t,
J=5.4 Hz, 2H), 3.85 (t, J=5.8 Hz, 2H), 3.48 (t, J=5.8 Hz, 2H),
3.03 ppm (s, 6H); IR (neat): n˜max =3256, 3028, 2950, 2811,
compound in 1.25m HCl in MeOH (2 mL) for 10 min followed by
precipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a yellow oil (57.8 mg, 88%): 1H NMR
(400 MHz, CD3OD): dH =8.52 (d, J=8.8 Hz, 1H), 8.46 (d, J=8.8 Hz,
1H), 8.35 (s, 1H), 8.26 (s, 1H), 7.98–7.92 (m, 2H), 7.76 (d, J=8.4 Hz,
1H), 7.60–7.52 (m, 2H), 7.52 (dd, J1 =1.4 Hz, J2 =7.4 Hz, 1H), 7.38–
7.30 (m, 2H), 5.01 (br s, 2H), 4.80 (br s, 2H), 3.86 (t, J=5.6 Hz, 2H),
3.49 (t, J=5.6 Hz, 2H), 3.03 ppm (s, 6H); IR (neat): n˜max =3241,
3070, 2944, 2821, 1637 cmꢀ1; HRMS (ES+): m/z [M+H]+ calcd for
C28H29ClN7O: 514.2117 (35Cl), found: 514.2114.
1636 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C28H30N7O:
480.2506, found: 480.2510.
N-(2-(dimethylamino)ethyl)-9-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)e-
thylamino)acridine-4-carboxamide (3b): Prepared according to
general method A. Column chromatography (10% MeOH in
EtOAc+1% Et3N) afforded a yellow oil (48.8 mg, 77%), which was
converted into the dihydrochloride salt by stirring the compound
in 1.25m HCl in MeOH (2 mL) for 10 min followed by precipitation
with EtOAc. The resultant solid was freeze-dried from water to give
9-(2-(4-(3-Chlorophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-4-carboxamide (5b): Prepared ac-
cording to general method A. Column chromatography (10%
MeOH in EtOAc+1% Et3N) afforded a yellow oil (39.2 mg, 58%),
which was converted into the dihydrochloride salt by stirring the
compound in 1.25m HCl in MeOH (2 mL) for 10 min followed by
precipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a yellow oil (49.6 mg, quant): 1H NMR
(400 MHz, CD3OD): dH =8.67 (dd, J1 =0.8 Hz, J2 =8.8 Hz, 1H), 8.54
(dd, J1 =1.2 Hz, J2 =7.6 Hz, 1H), 8.48 (d, J=8.4 Hz, 1H), 8.27 (s, 1H),
8.00–7.96 (m, 1H), 7.86 (d, J=8.4 Hz, 1H), 7.68–7.60 (m, 3H), 7.52–
7.49 (m, 1H), 7.39–7.32 (m, 2H), 4.99 (t, J=5.4 Hz, 2H), 4.80 (t, J=
5.2 Hz, 2H), 3.89 (t, J=5.8 Hz, 2H), 3.50 (t, J=5.8 Hz, 2H), 3.04 ppm
(s, 6H).); IR (neat): n˜max =3231, 3057, 2972, 1623 cmꢀ1; HRMS (ES+):
m/z [M+H]+ calcd for C28H29ClN7O: 514.2117 (35Cl), found:
514.2123.
1
the salt as a yellow oil (55.8 mg, 99%): H NMR (400 MHz, CD3OD):
dH =8.68 (d, J=8.8 Hz, 1H), 8.54 (d, J=7.6 Hz, 1H), 8.49 (d, J=
8.4 Hz, 1H), 8.30 (s, 1H), 7.99–7.95 (m, 1H), 7.85 (d, J=8.4 Hz, 1H),
7.67–7.58 (m, 4H), 7.41–7.32 (m, 3H), 5.02 (t, J=5.4 Hz, 2H), 4.80 (t,
J=5.4 Hz, 2H), 3.88 (t, J=5.6 Hz, 2H), 3.50 (t, J=5.6 Hz, 2H),
3.04 ppm (s, 6H); IR (neat): n˜max =3228, 3031, 2966, 1623 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C28H30N7O: 480.2506, found:
480.2506.
9-(2-(4-(2-Bromophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-3-carboxamide (4a): Prepared ac-
cording to general method B. Column chromatography (10%!
20% MeOH in EtOAc+1% Et3N) afforded an orange oil (49.9 mg,
50%), which was converted into the dihydrochloride salt by stirring
the compound in 1.25m HCl in MeOH (2 mL) for 10 min followed
by precipitation with EtOAc. The resultant solid was freeze-dried
from water to give the salt as a yellow oil (57.8 mg, quant):
1H NMR (400 MHz, CD3OD): dH =8.58 (d, J=8.8 Hz, 1H), 8.49 (d, J=
8.4 Hz, 1H), 8.42 (s, 1H), 8.33 (d, J=1.2 Hz, 1H), 8.02–7.98 (m, 2H),
7.84 (dd, J1 =0.8 Hz, J2 =8.8 Hz, 2H), 7.65–7.61 (m, 3H), 7.42–7.37
(m, 1H), 7.27–7.22 (m, 1H), 5.05 (t, J=5.4 Hz, 2H), 4.83 (s, 2H, un-
derneath water signal), 3.86 (t, J=6.0 Hz, 2H), 3.47 (t, J=6.0 Hz,
2H), 3.03 ppm (s, 6H); IR (neat): n˜max =3240, 3075, 2943, 2811,
1636 cmꢀ1; HRMS (ES+): m/z [M+H]+ calcd for C28H29BrN7O:
558.1611 (79Br), found: 558.1610.
9-(2-(4-(4-Bromophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-3-carboxamide (6a): Prepared ac-
cording to general method B. Column chromatography (10%!
15% MeOH in EtOAc+1% Et3N) afforded an orange oil (66.8 mg,
73%). The orange oil was converted into the dihydrochloride salt
by stirring the compound in 1.25m HCl in MeOH (2 mL) for 10 min
followed by precipitation with EtOAc. The resultant solid was
freeze-dried from water to give the salt as a bright yellow solid
(58.0 mg, quant): 1H NMR (400 MHz, CD3OD): dH =8.54 (d, J=
8.8 Hz, 1H), 8.46 (d, J=8.4 Hz, 1H), 8.27 (d, J=1.6 Hz, 1H), 8.26 (s,
1H), 7.99–7.95 (m, 2H), 7.79 (dd, J1 =0.8 Hz, J2 =8.8 Hz, 1H), 7.63–
7.59 (m, 1H), 7.55–7.49 (m, 4H), 4.98 (t, J=5.2 Hz, 2H), 4.80 (t, J=
5.6 Hz, 2H), 3.86 (t, J=5.8 Hz, 2H), 3.48 (t, J=5.6 Hz, 2H), 3.03 ppm
(s, 6H); IR (neat): n˜max =3251, 3033, 2970, 2811, 1638 cmꢀ1; HRMS
(ES+): m/z [M+H]+ calcd for C28H29BrN7O: 558.1611 (79Br), found:
558.1610.
9-(2-(4-(2-Bromophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-4-carboxamide (4b): Prepared ac-
cording to general method A. Column chromatography (10%
MeOH in EtOAc+1% Et3N) afforded a yellow oil (50.5 mg, 68%),
which was converted into the dihydrochloride salt by stirring the
compound in 1.25m HCl in MeOH (2 mL) for 10 min followed by
precipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a yellow oil (58.5 mg, quant): 1H NMR
(400 MHz, CD3OD): dH =8.68 (dd, J1 =0.8 Hz, J2 =8.8 Hz, 1H), 8.56
(dd, J1 =1.2 Hz, J2 =7.6 Hz, 1H), 8.49 (d, J=8.4 Hz, 1H), 8.43 (s, 1H),
8.03–7.97 (m, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.70–7.61 (m, 4H), 7.42–
7.38 (m, 1H), 7.27–7.23 (m, 1H), 5.05 (t, J=5.2 Hz, 2H), 4.82 (t, J=
5.6 Hz, 2H), 3.89 (t, J=5.8 Hz, 2H), 3.50 (t, J=5.6 Hz, 2H), 3.04 ppm
(s, 6H); IR (neat): n˜max =3231, 3059, 2951, 2816, 1623 cmꢀ1; HRMS
(ES+): m/z [M+H]+ calcd for C28H29BrN7O: 558.1611 (79Br), found:
558.1601.
9-(2-(4-(4-Bromophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-4-carboxamide (6b): Prepared ac-
cording to general method A. Column chromatography (10%
MeOH in EtOAc+1% Et3N) afforded a yellow oil (44.8 mg, 61%),
which was converted into the dihydrochloride salt by stirring the
compound in 1.25m HCl in MeOH (2 mL) for 10 min followed by
precipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a bright yellow solid (48.3 mg, 94%):
1H NMR (400 MHz, CD3OD): dH =8.66 (dd, J1 =1.2 Hz, J2 =8.8 Hz,
1H), 8.53 (dd, J1 =1.2 Hz, J2 =7.6 Hz, 1H), 8.48 (d, J=8.8 Hz, 1H),
8.23 (s, 1H), 8.00–7.96 (m, 1H), 7.87 (d, J=8.4 Hz, 1H), 7.68–7.60
(m, 2H), 7.56–7.49 (m, 4H), 4.98 (t, J=5.4 Hz, 2H), 4.80 (t, J=
5.4 Hz, 2H), 3.89 (t, J=5.8 Hz, 2H), 3.50 (t, J=5.8 Hz, 2H), 3.04 ppm
(s, 6H); IR (neat): n˜max =3236, 3054, 1623 cmꢀ1; HRMS (ES+): m/z
[M+H]+ calcd for C28H29BrN7O: 558.1611 (79Br), found: 558.1605.
9-(2-(4-(3-Chlorophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-3-carboxamide (5a): Prepared ac-
cording to general method B. Column chromatography (10%
MeOH in EtOAc+1% Et3N) afforded an orange oil (52.3 mg, 52%),
which was converted into the dihydrochloride salt by stirring the
9-(2-(4-(4-Chlorophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-3-carboxamide (7a): Prepared ac-
cording to general method B. Column chromatography (15%!
17.5% MeOH in EtOAc+1% Et3N) afforded an orange/yellow oil
800
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 792 – 804

Small Molecules Targeting Multiple DNA Structures
MED
(74.2 mg, 86%), which was converted into the dihydrochloride salt
by stirring the compound in 1.25m HCl in MeOH (2 mL) for 10 min
followed by precipitation with EtOAc. The resultant solid was
freeze-dried from water to give the salt as a bright yellow solid
(12% MeOH in EtOAc+1% Et3N) afforded an orange/yellow oil
(62.1 mg, 79%), which was converted into the dihydrochloride salt
by stirring the compound in 1.25m HCl in MeOH (2 mL) for 10 min
followed by precipitation with EtOAc. The resultant solid was
freeze-dried from water to give the salt as a bright yellow solid
1
(39.6 mg, 47%): H NMR (400 MHz, CD3OD): dH =8.54 (d, J=9.2 Hz,
1
1H), 8.46 (d, J=8.8 Hz, 1H), 8.26 (s, 2H), 7.98–7.94 (m, 2H), 7.78
(dd, J1 =0.8 Hz, J2 =8.8 Hz, 1H), 7.62–7.55 (m, 3H), 7.38 (d, J=
8.8 Hz, 2H), 4.98 (t, J=5.4 Hz, 2H), 4.80 (t, J=5.6 Hz, 2H), 3.85 (t,
J=6.0 Hz, 2H), 3.48 (t, J=6.0 Hz, 2H), 3.03 ppm (s, 6H); IR (neat):
n˜max =3244, 3065, 2956, 1637 cmꢀ1; HRMS (ES+): m/z [M+H]+
calcd for C28H29ClN7O: 514.2117, found: 514.2120.
(60.4 mg, 86%): H NMR (400 MHz, CD3OD): dH =8.57 (d, J=8.8 Hz,
1H), 8.48 (d, J=8.0 Hz, 1H), 8.39 (s, 1H), 8.28 (d, J=1.2 Hz, 1H),
8.00–7.95 (m, 2H), 7.81–7.79 (m, 3H), 7.69 (d, J=8.0 Hz, 2H), 7.64–
7.60 (m, 1H), 5.02 (t, J=5.2 Hz, 2H), 4.82 (t, J=5.4 Hz, 2H), 3.85 (t,
J=5.8 Hz, 2H), 3.47 (t, J=5.8 Hz, 2H), 3.03 ppm (s, 6H); IR (neat):
n˜max =3273, 3074, 1636 cmꢀ1; HRMS (ES+): m/z [M+H]+ calcd for
C29H29F3N7O: 548.2380, found: 548.2376.
9-(2-(4-(4-Chlorophenyl)-1H-1,2,3-triazol-1-yl)ethylamino)-N-(2-
(dimethylamino)ethyl)acridine-4-carboxamide (7b): Prepared ac-
cording to general method B. Column chromatography (5%!10%
MeOH in EtOAc+1% Et3N) afforded an orange/yellow oil (96.0 mg,
79%), which was converted into the dihydrochloride salt by stirring
the compound in 1.25m HCl in MeOH (2 mL) for 10 min followed
by precipitation with EtOAc. The resultant solid was freeze-dried
from water to give the salt as a bright yellow solid (69.2 mg, 63%):
1H NMR (400 MHz, CD3OD): dH =8.66 (d, J=8.8 Hz, 1H), 8.53 (dd,
J1 =0.8 Hz, J2 =7.6 Hz, 1H), 8.47 (d, J=8.8 Hz, 1H), 8.23 (s, 1H),
8.00–7.95 (m, 1H), 7.87–7.85 (m, 1H), 7.67–7.60 (m, 2H), 7.56 (d,
J=8.8 Hz, 2H), 7.39 (d, J=8.8 Hz, 2H), 4.98 (t, J=5.2, 2H), 4.79 (t,
J=5.4 Hz, 2H), 3.88 (t, J=5.8 Hz, 2H), 3.50 (t, J=5.8 Hz, 2H),
N-(2-(Dimethylamino)ethyl)-9-(2-(4-(4-(trifluoromethyl)phenyl)-
1H-1,2,3-triazol-1-yl)ethylamino)acridine-4-carboxamide
(9b):
Prepared according to general method B. Column chromatography
(10% MeOH in EtOAc+1% Et3N) afforded an orange/yellow oil
(64.0 mg, 61%), which was converted into the dihydrochloride salt
by stirring the compound in 1.25m HCl in MeOH (2 mL) for 10 min
followed by precipitation with EtOAc. The resultant solid was
freeze-dried from water to give the salt as a bright yellow solid
1
(47.0 mg, 65%): H NMR (400 MHz, CD3OD): dH =8.68 (d, J=8.8 Hz,
1H), 8.53 (d, J=7.2 Hz, 1H), 8.49 (d, J=8.8 Hz, 1H), 8.38 (s, 1H),
8.00–7.96 (m, 1H), 7.88 (d, J=8.4 Hz, 1H), 7.80 (d, J=8.0 Hz, 2H),
7.70 (d, J=8.0 Hz, 2H), 7.67–7.61 (m, 2H), 5.01 (t, J=5.2, 2H), 4.81
(t, J=5.2 Hz, 2H), 3.87 (t, J=5.6 Hz, 2H), 3.49 (t, J=5.8 Hz, 2H),
3.04 ppm (s, 6H); IR (neat): n˜max =3230, 3048, 1623 cmꢀ1; HRMS
(ES+): m/z [M+H]+ calcd for C29H29F3N7O: 548.2380, found:
548.2375.
3.04 ppm (s, 6H); IR (neat): n˜max =3212, 3036, 2972, 1623 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C28H29ClN7O: 514.2117, found:
514.2112.
N-(2-(Dimethylamino)ethyl)-9-(2-(4-p-tolyl-1H-1,2,3-triazol-1-yl)e-
thylamino)acridine-3-carboxamide (8a): Prepared according to
general method B. Column chromatography (10% MeOH in
EtOAc+1% Et3N) afforded an orange/yellow oil (92.4 mg, 80%),
which was converted into the dihydrochloride salt by stirring the
compound in 1.25m HCl in MeOH (2 mL) for 10 min followed by
precipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a bright yellow solid (85.2 mg, 80%):
1H NMR (400 MHz, CD3OD): dH =8.54 (d, J=8.8 Hz, 1H), 8.46 (d, J=
8.8 Hz, 1H), 8.34 (s, 1H), 8.26 (d, J=1.6 Hz, 1H), 7.98–7.92 (m, 2H),
7.77 (d, J=8.4 Hz, 1H), 7.60–7.56 (m, 1H), 7.47 (d, J=8.0 Hz, 2H),
7.20 (d, J=8.0 Hz, 2H), 5.04 (t, J=5.2 Hz, 2H), 4.80 (t, J=5.0 Hz,
2H), 3.86 (t, J=5.8 Hz, 2H), 3.49 (t, J=5.8 Hz, 2H), 3.03 (s, 6H),
N-(2-(dimethylamino)ethyl)-9-(2-(4-(hydroxymethyl)-1H-1,2,3-tri-
azol-1-yl)ethylamino)acridine-3-carboxamide (10a): Prepared ac-
cording to general method B. Column chromatography (20%
MeOH in EtOAc+1% Et3N) afforded an orange oil (69.4 mg, 96%),
which was converted into the dihydrochloride salt by stirring the
compound in 1.25m HCl in MeOH (2 mL) for 10 min followed by
precipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a yellow solid (43.1 mg, 53%): 1H NMR
(400 MHz, CD3OD): dH =8.58 (d, J=8.8 Hz, 1H), 8.49 (d, J=8.8 Hz,
1H), 8.38 (d, J=1.2 Hz, 1H), 8.05 (s, 1H), 8.03–8.01 (m, 2H), 7.89 (d,
J=8.4 Hz, 1H), 7.65–7.62 (m, 1H), 5.05 (t, J=5.4 Hz, 2H), 4.78 (t,
J=5.2 Hz, 2H), 4.57 (s, 2H), 3.87 (t, J=5.8 Hz, 2H), 3.84 (t, J=
5.8 Hz, 2H), 3.03 pm (s, 6H); IR (neat): n˜max =3262, 3064, 2868,
2.34 ppm (s, 3H); IR (neat): n˜max =3240, 3032, 2956, 1636 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C29H32N7O: 494.2663, found:
1636 cmꢀ1 HRMS (ES+): m/z [M+H]+ calcd for C23H28N7O2:
;
494.2663.
434.2299, found: 443.2295.
N-(2-(Dimethylamino)ethyl)-9-(2-(4-p-tolyl-1H-1,2,3-triazol-1-yl)e-
thylamino)acridine-4-carboxamide (8b): Prepared according to
general method B. Column chromatography (10% MeOH in
EtOAc+1% Et3N) afforded an orange/yellow oil (61.7 mg, 75%),
which was converted into the dihydrochloride salt by stirring the
compound in 1.25m HCl in MeOH (2 mL) for 10 min followed by
precipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a yellow solid (62.2 mg, 88%): 1H NMR
(400 MHz, CD3OD): dH =8.66 (d, J=8.8 Hz, 1H), 8.53 (d, J=7.6 Hz,
1H), 8.48 (d, J=8.8 Hz, 1H), 8.22 (s, 1H), 7.98–7.94 (m, 1H), 7.84 (d,
J=8.4 Hz, 1H), 7.66–7.59 (m, 2H), 7.45 (d, J=8.0 Hz, 2H), 7.19 (d,
J=8.0 Hz, 2H), 4.99 (t, J=5.2 Hz, 2H), 4.79 (t, J=5.2 Hz, 2H), 3.88
(t, J=5.6 Hz, 2H), 3.50 (t, J=5.6 Hz, 2H), 3.04 (s, 6H), 2.35 ppm (s,
3H); IR (neat): n˜max =3228, 3040, 2966, 1623 cmꢀ1; HRMS (ES+): m/
z [M+H]+ calcd for C29H32N7O: 494.2663, found: 494.2656.
N-(2-(Dimethylamino)ethyl)-9-(2-(4-(hydroxymethyl)-1H-1,2,3-tri-
azol-1-yl)ethylamino)acridine-4-carboxamide (10b): Prepared ac-
cording to general method A. Column chromatography (10%!
20% MeOH in EtOAc+1% Et3N) afforded a yellow oil (27.1 mg,
38%), which was converted into the dihydrochloride salt by stirring
the compound in 1.25m HCl in MeOH (2 mL) for 10 min followed
by precipitation with EtOAc. The resultant solid was freeze-dried
1
from water to give the salt as a yellow oil (29.8 mg, 94%): H NMR
(400 MHz, CD3OD): dH =8.67 (dd, J1 =1.2 Hz, J2 =8.8 Hz, 1H), 8.58
(dd, J1 =0.8 Hz, J2 =7.6 Hz, 1H), 8.48 (d, J=8.8 Hz, 1H), 8.06–8.02
(m, 1H), 7.98–7.94 (m, 2H), 7.70–7.63 (m, 2H), 5.01 (br s, 2H), 4.76
(t, J=5.6 Hz, 2H), 4.53 (s, 2H), 3.92 (t, J=5.6 Hz, 2H), 3.52 (t, J=
5.6 Hz, 2H), 3.05 ppm (s, 6H); IR (neat): n˜max =3318, 3041,
1622 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C23H28N7O2:
434.2299, found: 443.2295.
N-(2-(Dimethylamino)ethyl)-9-(2-(4-(4-(trifluoromethyl)phenyl)-
1-(2-(4-(2-(Dimethylamino)ethylcarbamoyl)acridin-9-ylamino)eth-
yl)-1H-1,2,3-triazole-4-carboxylic acid (11): Prepared according to
general method A, affording a yellow oil (61.5 mg, quant), which
1H-1,2,3-triazol-1-yl)ethylamino)acridine-3-carboxamide
(9a):
Prepared according to general method B. Column chromatography
ChemMedChem 2012, 7, 792 – 804
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801

M. Searcey et al.
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was converted into the dihydrochloride salt by stirring the com-
pound iin 1.25m HCl in MeOH (2 mL) for 10 min followed by pre-
cipitation with EtOAc. The resultant solid was freeze-dried from
water to give the salt as a yellow oil (66.1 mg, 92%): 1H NMR
(400 MHz, CD3OD): dH =8.64 (d, J=8.4 Hz, 1H), 8.58 (d, J=7.2 Hz,
1H), 8.47 (d, J=8.4 Hz, 1H), 8.41 (br s, 1H), 8.05–8.00 (m, 1H), 7.94
(d, J=8.4 Hz, 1H), 7.68–7.63 (m, 2H), 4.98 (t, J=4.8 Hz, 2H), 4.79 (t,
J=5.0 Hz, 2H), 3.92 (t, J=5.4 Hz, 2H), 3.54 (t, J=5.6 Hz, 2H),
J=5.4 Hz, 2H), 4.52 (s, 2H), 3.88 (t, J=6.0 Hz, 2H) 3.49 (t, J=
6.0 Hz, 2H), 3.03 ppm (s, 6H); IR (neat): n˜max =3232, 1623 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C30H34O2N9: 552.2830, found:
552.2830.
9-(2-(4-((2-Aminobenzamido)methyl)-1H-1,2,3-triazol-1-yl)ethyla-
mino-N-(2-dimethylamino)ethyl)acridine-4-carboxamide (12b): 9-
(2-Azidoethylamino)-N-(2-(dimethylamino)ethyl)acridine-4-carboxa-
mide (50 mg, 0.13 mmol) and 2-amino-N-(prop-2-ynyl)benzamide
(23.1 mg, 0.13 mmol) were dissolved in tBuOH:H2O (1:1, 650 mL).
Sodium ascorbate (10 mol%, 13.3 mL, 1m stock) was added fol-
lowed by aq CuSO4 (1 mol%, 1.33 mL, 1m stock), and the reaction
mixture was stirred overnight at RT. The solvents were removed in
vacuo to yield a dark orange oil. Column chromatography (10%
MeOH in EtOAc+1% Et3N) gave the free base as a yellow oil
(41.7 mg, 57%).
3.07 ppm (s, 6H); IR (neat): n˜max =3237, 3069, 2970, 1622 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C23H27N7O3: 448.2092, found:
448.2093.
2-Nitro-N-(prop-2-ynyl)benzamide:
2-Nitrobenzoyl
chloride
(355 mL, 2.7 mmol), propargylamine (173 mL, 2.7 mmol) and Et3N
(375 mL, 2.7 mmol) were stirred in CH2Cl2 (5 mL) at RT for 2 h. On
completion, the solvents were removed in vacuo, and the resulting
solid was purified by column chromatography (5% MeOH in
CH2Cl2) to yield the title compound as a light brown solid
(398.5 mg, 72%): mp: 107.3–109.18C; 1H NMR (400 MHz, CD3OD):
dH =8.11 (dd, J1 =1.2 Hz, J2 =8.0 Hz, 1H), 7.80–7.76 (m, 1H), 7.71–
7.66 (m, 1H), 7.57 (dd, J1 =1.6 Hz, J2 =7.6 Hz, 1H), 4.15 (d, J=
2.4 Hz, 2H), 2.67 ppm (t, J=2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3):
dH =166.1, 146.3, 133.8, 132.1, 130.7, 128.7, 124.6, 78.5, 72.4,
The free base was further purified through conversion to the trihy-
drochloride salt by stirring the compound in 1.25m HCl in MeOH
(2 mL) for 10 min followed by precipitation with EtOAc to give
a
bright yellow hygroscopic solid (36.4 mg, 73%): 1H NMR
(400 MHz, CD3OD): dH =8.69 (d, J=8.4 Hz, 1H), 8.59 (dd, J1 =1.2 Hz,
J2 =7.6 Hz, 1H), 8.48 (d, J=8.8 Hz, 1H), 8.08 (s, 1H), 8.02–7.94 (m,
2H), 7.84 (dd, J1 =1.2 Hz, J2 =8.0 Hz, 1H), 7.72–7.51 (m, 5H), 5.02 (t,
J=5.4 Hz, 2H), 4.75 (t, J=5.6 Hz, 2H), 4.52 (s, 2H), 3.92 (t, J=
5.8 Hz, 2H), 3.52 (t, J=5.8 Hz, 2H), 3.05 ppm (s, 6H); IR (neat):
30.0 ppm; IR (neat): n˜max =3287, 3261, 1647, 1525, 1348 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C10H8N2O3: 204.0529, found:
204.0525.
n˜max =3245, 1654 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for
2-Amino-N-(prop-2-ynyl)benzamide: 2-Nitro-N-(prop-2-ynyl)benza-
mide (100 mg, 0.49 mmol) was dissolved in Et2O/EtOH/H2O (22:7:1,
10 mL). Aluminium foil (132 mg, 4.9 mmol) was cut into strips and
rolled into coils. Each coil was dipped in Et2O and then subse-
quently immersed in a stirred solution of 2% aq HgCl2 for 30 s.
The amalgated foil was then dipped briefly into Et2O to remove
excess H2O and then added to the reaction mixture. After addition
of the Al/Hg amalgam, the reaction was stirred at RT for 2 h until
completion (TLC). The resulting solution was filtered through celite
and concentrated in vacuo to yield the title compounds as a light
brown solid (85 mg, 99%): mp: 90.4–92.68C; 1H NMR (400 MHz,
CDCl3): dH =7.33 (dd, J1 =1.6 Hz, J2 =8.0 Hz, 1H), 7.24–7.20 (m, 1H),
6.70–6.63 (m, 2H), 6.24 (br s, 1H), 4.20 (dd, J1 =2.4 Hz, J2 =5.2 Hz,
2H), 2.27 ppm (t, J=2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): dC =
168.9, 148.8, 132.7, 127.2, 117.4, 116.6, 114.9, 79.6, 71.7, 29.4 ppm;
IR (neat): n˜max =3426, 3288, 1636 cmꢀ1; HRMS (ES+): m/z [M+H]+
calcd for C10H12N2O: 175.0866, found: 175.0866.
C30H34O2N9: 552.2830, found: 552.2835.
N-(2-(Dimethylamino)ethyl)-9-(3-ethynylphenylamino)acridine-4-
carboxamide: 9-Chloro-N-(2-(dimethylamino)ethyl)acridine-4-car-
boxamide (300 mg, 0.92 mmol) was stirred in excess phenol (1.5 g)
at 1108C for 15 min. The temperature was reduced to 558C and 3-
ethynylaniline (96 mL, 0.92 mmol) was added. The reaction mixture
was then stirred at 558C for 6 h. On cooling, aq NaOH (2m, 20 mL)
was added. A small amount of CH2Cl2 (5 mL) was added to a sepa-
rating funnel, and the reaction mixture was poured on top. After
separation of the organic phase, the aqueous phase was extracted
with CH2Cl2 (3ꢁ15 mL). The combined organic extracts were dried
(MgSO4), filtered and concentrated in vacuo to yield an orange oil.
Column chromatography (2.5% MeOH in EtOAc+1% Et3N) yielded
1
the desired compound as an orange oil (327.9 mg, 88%): H NMR
(400 MHz, CD3OD): dH =8.60 (d, J=7.2 Hz, 1H), 8.46 (dd, J1 =0.8 Hz,
J2 =8.8 Hz, 1H), 8.20 (d, J=8.8 Hz, 1H), 8.11–8.04 (m, 2H), 7.60–
7.45 (m, 6H), 3.94 (t, J=5.8 Hz, 2H), 3.68 (s, 1H), 3.26 (t, J=5.8 Hz,
9-(2-(4-((2-Aminobenzamido)methyl)-1H-1,2,3-triazol-1-yl)ethyla-
mino-N-(2-dimethylamino)ethyl)acridine-3-carboxamide (12a): 9-
(2-Azidoethylamino)-N-(2-(dimethylamino)ethyl)acridine-3-carboxa-
mide (50 mg, 0.13 mmol) and 2-amino-N-(prop-2-ynyl)benzamide
(23.1 mg, 0.13 mmol) were dissolved in tBuOH:H2O (1:1, 650 mL).
Sodium ascorbate (10 mol%, 13.3 mL, 1m stock) was added fol-
lowed by aq CuSO4 (1 mol%, 1.33 mL, 1m stock), and the reaction
mixture was stirred overnight at RT. The solvents were removed in
vacuo to yield a dark orange oil. Column chromatography (10%!
15% MeOH in EtOAc+1% Et3N) gave the free base as a yellow oil
(32 mg, 44%).
2H), 3.06 ppm (s, 6H); IR (neat): n˜max =3288, 2943, 2820, 1639 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C26H25N4O: 409.2023, found:
409.2023.
N-(2-(Dimethylamino)ethyl)-9-(2-ethynylphenylamino)acridine-4-
carboxamide: 9-Chloro-N-(2-(dimethylamino)ethyl)acridine-4-car-
boxamide (300 mg, 0.92 mmol) was stirred in excess phenol (1.5 g)
at 1108C for 15 min. The temperature was reduced to 558C and 2-
ethynylaniline (106 mL, 0.92 mmol) was added. The reaction mix-
ture was then stirred at 558C for 6 h. On cooling, aq NaOH (2m,
20 mL) was added. A small amount of CH2Cl2 (5 mL) was added to
a separating funnel, and the reaction mixture was poured on top.
After separation of the organic phase, the aqueous phase was ex-
tracted with CH2Cl2 (3ꢁ15 mL). The combined organic extracts
were dried (MgSO4), filtered and concentrated in vacuo to yield an
orange oil. Column chromatography (2.5% MeOH in EtOAc+1%
Et3N) yielded the desired compound as an orange oil (256.2 mg,
69%): 1H NMR (400 MHz, CD3OD): dH =8.59 (dd, J1 =1.2 Hz, J2 =
7.4 Hz, 1H), 8.40 (dd, J1 =1.2 Hz, J2 =8.8 Hz, 1H), 8.13 (d, J=8.8 Hz,
1H), 8.10–8.04 (m, 2H), 7.70–7.66 (m, 3H), 7.59–7.48 (m, 3H), 3.93
The free base was further purified through conversion to the trihy-
drochloride salt by stirring the compound in 1.25m HCl in MeOH
(2 mL) for 10 min followed by precipitation with EtOAc to give
a
bright yellow hygroscopic solid (17.9 mg, 54%): H NMR
(400 MHz, CD3OD): dH =8.55 (d, J=8.8 Hz, 1H), 8.46 (d, J=8.8 Hz,
1H), 8.36 (d, J=1.6 Hz, 1H), 8.07 (s, 1H), 8.00–7.96 (m, 2H), 7.87 (d,
J=8.4 Hz, 1H), 7.80 (dd, J1 =1.2 Hz, J2 =7.6 Hz, 1H), 7.67 (m, 1H),
7.61–7.57 (m, 1H), 7.52–7.46 (m, 2H), 5.02 (t, J=5.6 Hz, 2H), 4.75 (t,
802
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 792 – 804

Small Molecules Targeting Multiple DNA Structures
MED
(t, J=5.8 Hz, 2H), 3.52 (t, J=5.6 Hz, 2H), 3.49 (s, 1H), 3.06 ppm (s,
6H); IR (neat): n˜max =3278, 2932, 2820, 1641 cmꢀ1; HRMS (ES+): m/
z [M+H]+ calcd for C26H25N4O: 409.2023, found: 409.2023.
The free base was further purified through conversion to the tetra-
hydrochloride salt by stirring the compound in 1.25m HCl in MeOH
(2 mL) for 10 min followed by precipitation with EtOAc to give an
1
orange hygroscopic glassy solid (67.8 mg, 98%): H NMR (400 MHz,
N-(2-(Dimethylamino)ethyl)-9-(3-ethynylphenylamino)acridine-3-
carboxamide: 9-Chloro-N-(2-(dimethylamino)ethyl)acridine-3-car-
boxamide (150 mg, 0.46 mmol) was stirred in excess phenol (1.5 g)
at 1108C for 15 min. The temperature was reduced to 558C and 3-
ethynylaniline (48 mL, 0.46 mmol) was added. The reaction mixture
was then stirred at 558C overnight. On cooling, aq NaOH (2m,
20 mL) was added. A small amount of CH2Cl2 (5 mL) was added to
a separating funnel, and the reaction mixture was poured on top.
After separation of the organic phase, the aqueous phase was ex-
tracted with CH2Cl2 (3ꢁ15 mL). The combined organic extracts
were dried (MgSO4), filtered and concentrated in vacuo to yield an
orange oil. Column chromatography (10% MeOH in EtOAc+1%
Et3N) yielded the desired compound as an orange oil (50.8 mg,
CD3OD): dH =8.62–8.58 (m, 2H), 8.53 (d, J=8.8 Hz, 1H), 8.49 (d, J=
8.4 Hz, 1H), 8.40 (s, 1H), 8.35 (d, J=1.2 Hz, 1H), 8.27 (d, J=8.4 Hz,
1H), 8.11–8.05 (m, 2H), 8.03–7.96 (m, 2H), 7.86–7.82 (m, 2H), 7.74
(d, J=7.6 Hz, 1H), 7.63–7.48 (m, 4H), 7.41 (d, J=7.6 Hz, 1H), 5.02
(br s, 2H), 4.82–4.81 (m, 2H), 3.94 (t, J=5.6 Hz, 2H), 3.84 (t, J=
6.0 Hz, 2H), 3.54 (t, J=5.6 Hz, 2H), 3.46 (t, J=6.0 Hz, 2H), 3.06 (s,
6H), 3.02 ppm (s, 6H); IR (neat): n˜max =3233, 2979, 2946, 1624 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C46H48N11O2: 786.3987, found:
786.3982.
N-(2-(Dimethylamino)ethyl)-9-(2-(1-(2-(4-(2-(dimethylamino)e-
thylcarbamoyl)acridin-9-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)phe-
nylamino)acridine-4-carboxamide (16): 9-(2-Azidoethylamino)-N-
1
27%): H NMR (400 MHz, CD3OD): dH =8.50 (d, J=1.2 Hz, 1H), 8.26–
(2-(dimethylamino)ethyl)acridine-4-carboxamide
(50 mg,
8.23 (m, 2H), 8.08–8.00 (m, 2H), 7.86 (dd, J1 =1.6 Hz, J2 =9.2 Hz,
1H), 7.59–7.52 (m, 4H), 7.49–7.46 (m, 1H), 3.86 (t, J=5.8 Hz, 2H),
3.68 (s, 1H), 3.47 (t, J=5.8 Hz, 2H), 3.02 ppm (s, 6H); IR (neat):
n˜max =3278, 3066, 2932, 2820, 1641 cmꢀ1; HRMS (ES+): m/z [M+
H]+ calcd for C26H25N4O: 409.2023, found: 409.2025.
0.13 mmol) and N-(2-(dimethylamino)ethyl)-9-(2-ethynylphenylami-
no)acridine-4-carboxamide (54 mg, 0.13 mmol) were stirred in
tBuOH:H2O (1:1, 663 mL) for 2 min. Sodium ascorbate (10 mol%,
13.3 mL, 1m stock) and aq CuSO4 (1 mol%, 1.33 mL, 1m stock) were
added, and the reaction mixture was stirred at RT for 48 h. On
completion (as seen by TLC), the solvents were removed in vacuo,
and the crude was purified by column chromatography (20%!
30% MeOH in EtOAc+1% Et3N) to yield the free base as an
orange oil (67.7 mg, 65%).
N-(2-(Dimethylamino)ethyl)-9-(3-(1-(2-(4-(2-(dimethylamino)e-
thylcarbamoyl)acridin-9-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)phe-
nylamino)acridine-4-carboxamide (14): 9-(2-Azidoethylamino)-N-
(2-(dimethylamino)ethyl)acridine-4-carboxamide
(100 mg,
0.27 mmol) and N-(2-(dimethylamino)ethyl)-9-(3-ethynylphenylami-
no)acridine-4-carboxamide (108 mg, 0.27 mmol) were stirred in
tBuOH:H2O (1:1, 1.3 mL) for 2 min. Sodium ascorbate (10 mol%,
26.5 mL, 1m stock) and aq CuSO4 (1 mol%, 2.65 mL, 1m stock) were
added, and the reaction mixture was stirred at RT for 72 h. On
completion (as seen by TLC), the solvents were removed in vacuo,
and the crude was purified by column chromatography (10%!
40% MeOH in EtOAc+1% Et3N) to yield the free base as an
orange oil (134 mg, 64%).
The free base was further purified through conversion to the tetra-
hydrochloride salt by stirring the compound in 1.25m HCl in MeOH
(2 mL) for 10 min followed by precipitation with EtOAc to give
1
a dark brown hygroscopic solid (48.9 mg, 61%): H NMR (400 MHz,
CD3OD): dH =8.54 (dd, J1 =7.4 Hz, J2 =1.2 Hz, 1H), 8.49 (s, 1H),
8.45–8.43 (m, 1H), 8.39 (br s, 1H), 8.23 (d, J=8.4 Hz, 1H), 8.18 (br s,
1H), 7.95–7.86 (m, 4H), 7.82–7.79 (m, 2H), 7.65–7.61 (m, 1H), 7.52–
7.48 (m, 1H), 7.45–7.36 (m, 4H), 7.28–7.24 (m, 1H), 5.06–5.03 (m,
2H), 4.62 (t, J=5.6 Hz, 2H), 3.94–3.92 (m, 4H), 3.56–3.51 (m, 4H),
3.06 (s, 6H), 3.05 ppm (s, 6H); IR (neat): n˜max =3189, 3022, 2967,
1623 cmꢀ1 HRMS (ES+): m/z [M+H]+ calcd for C46H48N11O2:
;
786.3987, found: 786.3981.
The free base was further purified through conversion to the tetra-
hydrochloride salt by stirring the compound in 1.25m HCl in MeOH
(2 mL) for 10 min followed by precipitation with EtOAc to give
1
a red glassy hygroscopic solid (156.2 mg, 99%): H NMR (400 MHz,
N-(2-(Dimethylamino)ethyl)-9-(2-(4-(2-(4-(2-(dimethylamino)e-
thylcarbamoyl)acridin-9-ylamino)phenyl)-1H-1,2,3-triazol-1-yl)e-
thylamino)acridine-3-carboxamide (17): 9-(2-Azidoethylamino)-N-
CD3OD): dH =8.69 (d, J=8.8 Hz, 1H), 8.63 (d, J=7.6 Hz, 1H), 8.59
(d, J=7.2 Hz, 1H), 8.53–8.49 (m, 2H), 8.40 (s, 1H), 8.26 (d, J=
9.2 Hz, 1H), 8.10–8.04 (m, 2H), 7.98–7.96 (m, 1H), 7.93–7.90 (m,
1H), 7.78–7.75 (m, 2H), 7.67–7.42 (m, 6H), 5.04–5.02 (m, 2H), 4.80
(t, J=5.6 Hz, 2H), 3.94 (t, J=5.6 Hz, 2H), 3.87 (t, J=5.6 Hz, 2H),
3.54 (t, J=5.6 Hz, 2H), 3.50 (t, J=5.6 Hz, 2H), 3.06 (s, 6H),
(2-(dimethylamino)ethyl)acridine-3-carboxamide
(54 mg,
0.14 mmol) and N-(2-(dimethylamino)ethyl)-9-(2-ethynylphenylami-
no)acridine-4-carboxamide (58 mg, 0.14 mmol) were stirred in
tBuOH:H2O (1:1, 714 mL) for 2 min. Sodium ascorbate (10 mol%,
14.3 mL, 1m stock) and aq CuSO4 (1 mol%, 1.43 mL, 1m stock) were
added, and the reaction mixture was stirred at RT for 24 h. On
completion (as seen by TLC), the solvents were removed in vacuo,
and the crude was purified by column chromatography (10%!
40% MeOH in EtOAc+1% Et3N) to yield the free base as an
orange oil (60.6 mg, 54%).
3.03 ppm (s, 6H); IR (neat): n˜max =3213, 3030, 2978, 1623 cmꢀ1
;
HRMS (ES+): m/z [M+H]+ calcd for C46H48N11O2: 786.3987, found:
786.3987.
N-(2-(Dimethylamino)ethyl)-9-(2-(4-(3-(4-(2-(dimethylamino)e-
thylcarbamoyl)acridin-9-ylamino)phenyl)-1H-1,2,3-triazol-1-yl)e-
thylamino)acridine-3-carboxamide (15): 9-(2-Azidoethylamino)-N-
(2-(dimethylamino)ethyl)acridine-3-carboxamide
(38.5 mg,
The free base was further purified through conversion to the tetra-
hydrochloride salt by stirring the compound in 1.25m HCl in MeOH
(2 mL) for 10 min followed by precipitation with EtOAc to give
a dark brown hygroscopic glassy solid (61.9 mg, 86%): 1H NMR
(400 MHz, CD3OD): dH =8.56 (s, 1H), 8.47 (dd, J1 =7.6 Hz, J2 =
1.2 Hz, 1H), 8.22 (d, J=1.6 Hz, 1H), 8.03–7.90 (m, 5H), 7.81–7.62
(m, 6H), 7.50–7.46 (m, 1H), 7.31–7.19 (m, 4H), 5.10 (t, J=5.2 Hz,
2H), 4.60 (t, J=5.2 Hz, 2H), 3.98–3.94 (m, 2H), 3.89 (t, J=6.2 Hz,
2H), 3.59 (t, J=5.8 Hz, 2H), 3.50 (t, J=6.0 Hz, 2H), 3.10 (s, 6H),
0.10 mmol) and N-(2-(dimethylamino)ethyl)-9-(3-ethynylphenylami-
no)acridine-4-carboxamide (41.7 mg, 0.10 mmol) were stirred in
tBuOH:H2O (1:1, 511 mL) for 2 min. Sodium ascorbate (10 mol%,
10.2 mL, 1m stock) and aq CuSO4 (1 mol%, 1.02 mL, 1m stock) were
added, and the reaction mixture was stirred at RT for 24 h. On
completion (as seen by TLC), the solvents were removed in vacuo,
and the crude was purified by column chromatography (10%!
40% MeOH in EtOAc+1% Et3N) to yield the free base as an
orange oil (58.4 mg, 73%).
3.04 ppm (s, 6H); IR (neat): n˜max =3205, 3030, 2961, 1624 cmꢀ1
;
ChemMedChem 2012, 7, 792 – 804
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
803

M. Searcey et al.
MED
HRMS (ES+): m/z [M+H]+ calcd for C46H48N11O2: 786.3987, found:
786.3992.
Acknowledgements
We thank the University of East Anglia (Norwich, UK) for funding
(L.H.) and the UK Engineering and Physical Sciences Research
Council (EPSRC) mass spectrometry service at Swansea for per-
forming HRMS. Cancer Research UK is thanked for programme
grant support (S.N.). We also thank Dr. Zoꢀ Waller (University of
East Anglia) for drawing and allowing us to use the top image in
the graphical abstract.
N-(2-(Dimethylamino)ethyl)-9-(3-(1-(2-(4-(2-(dimethylamino)e-
thylcarbamoyl)acridin-9-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)phe-
nylamino)acridine-3-carboxamide (18): 9-(2-Azidoethylamino)-N-
(2-(dimethylamino)ethyl)acridine-4-carboxamide
(18.5 mg,
0.05 mmol) and N-(2-(dimethylamino)ethyl)-9-(3-ethynylphenylami-
no)acridine-3-carboxamide (20.1 mg, 0.05 mmol) were stirred in
tBuOH:H2O (1:1, 246 mL) for 2 min. Sodium ascorbate (10 mol%,
4.91 mL, 1m stock) and aq CuSO4 (1 mol%, 0.49 mL, 1m stock) were
added, and the reaction mixture was stirred at RT for 24 h. On
completion (as seen by TLC), the solvents were removed in vacuo,
and the crude was purified by column chromatography (20%
MeOH in EtOAc+1% Et3N!40% MeOH in EtOAc+2% Et3N) to
yield the free base as an orange oil (31.5 mg, 81%).
Keywords: acridines
· click chemistry · DNA · Holliday
junction · quadruplexes
The free base was further purified through conversion to the tetra-
hydrochloride salt by stirring the compound in 1.25m HCl in MeOH
(2 mL) for 10 min followed by precipitation with EtOAc to give an
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orange hygroscopic glassy solid (25.8 mg, 69%): H NMR (400 MHz,
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8.43 (m, 3H), 8.29–8.25 (m, 2H), 8.01–7.92 (m, 3H), 7.88–7.82 (m,
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1636 cmꢀ1 HRMS (ES+): m/z [M+H]+ calcd for C46H48N11O2:
;
786.3987, found: 786.3993.
N-(2-(Dimethylamino)ethyl)-9-(3-(1-(2-(3-(2-(dimethylamino)e-
thylcarbamoyl)acridin-9-ylamino)ethyl)-1H-1,2,3-triazol-4-yl)phe-
nylamino)acridine-3-carboxamide (19): 9-(2-Azidoethylamino)-N-
(2-(dimethylamino)ethyl)acridine-3-carboxamide
(21.0 mg,
0.06 mmol) and N-(2-(dimethylamino)ethyl)-9-(3-ethynylphenylami-
no)acridine-3-carboxamide (22.7 mg, 0.06 mmol) were stirred in
tBuOH:H2O (1:1, 279 mL) for 2 min. Sodium ascorbate (10 mol%,
5.57 mL, 1m stock) and aq CuSO4 (1 mol%, 0.56 mL, 1m stock) were
added, and the reaction mixture was stirred at RT for 24 h. On
completion (as seen by TLC), the solvents were removed in vacuo,
and the crude was purified by column chromatography (20%!
100% MeOH in EtOAc+1% Et3N) to yield the free base as an
orange oil (44.8 mg, quant).
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1
orange hygroscopic glassy solid (24.6 mg, 46%): H NMR (400 MHz,
CD3OD): dH =8.57 (d, J=8.8 Hz, 1H), 8.49–8.46 (m, 2H), 8.41 (s,
1H), 8.32–8.27 (m, 3H), 8.03–7.93 (m, 4H), 7.86–7.80 (m, 3H), 7.72
(d, J=7.6 Hz, 1H), 7.61–7.54 (m, 2H), 7.50–7.46 (m, 1H), 7.40–7.38
(m, 1H), 5.04–5.01 (m, 2H), 4.82–4.79 (m, 2H), 3.87–3.81 (m, 4H),
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3.49–3.45 (m, 4H), 3.02 (s, 6H), 3.02 ppm (s, 6H); IR (neat): n˜max
=
3240, 3038, 2966, 1636 cmꢀ1; HRMS (ES+): m/z [M+H]+ calcd for
Received: January 30, 2012
C46H48N11O2: 786.3987, found: 786.3989.
Published online on February 29, 2012
804
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