Synthesis of bis-indole carboxamides as G-quadruplex stabilizing and inducing ligands

Article abstract of DOI:10.1002/chem.201102556

The design and synthesis of a series of bis-indole carboxamides with varying amine containing side chains as G-quadruplex DNA stabilising small molecules are reported. Their interactions with quadruplexes have been evaluated by means of Foerster resonance

Full text of DOI:10.1002/chem.201102556

DOI: 10.1002/chem.201102556
Synthesis of Bis-indole Carboxamides as G-Quadruplex Stabilizing and
Inducing Ligands
Jyotirmayee Dash,*[a, b] Rabindra Nath Das,[b] Nagaratna Hegde,[a] G. Dan Pantos¸,[a, e]
Pravin S. Shirude,[a] and Shankar Balasubramanian*[a, c, d]
Abstract: The design and synthesis of a
series of bis-indole carboxamides with
varying amine containing side chains as
G-quadruplex DNA stabilising small
molecules are reported. Their interac-
tions with quadruplexes have been
evaluated by means of Fçrster reso-
nance energy transfer (FRET) melting
analysis, UV/Vis spectroscopy, circular
dichroism spectroscopy and molecular
modelling studies. FRET analysis indi-
cates that these ligands exhibit signifi-
cant selectivity for quadruplex over
duplex DNA, and the position of the
carboxamide side chains is of para-
mount importance in G-quadruplex
stabilisation. UV/Vis titration studies
reveal that bis-indole ligands bind
tightly to quadruplexes and show a
three- to fivefold preference for c-kit2
over h-telo quadruplex DNA. CD stud-
ies revealed that bis-indole carboxa-
mide with a central pyridine ring indu-
ces the formation of a single, antiparal-
lel, conformation of the h-telo quadru-
plex in the presence and absence of
added salt. The chirality of h-telo quad-
ruplex was transferred to the achiral
ligand (induced CD) and the formation
of a preferred atropisomer was ob-
served.
Keywords: atrop chirality
·
G-
quadruplexes · indoles · molecular
recognition · Sonogashira coupling
Introduction
the expression of the genes.[5] The biological functions asso-
ciated with these structures make them attractive targets for
potential drug design and development.[1,6] Recently we
have reported G-quadruplex recognition by two bis-indole
carboxamides containing N,N-dimethylpropylamine side
chains (1a and 4a).[7] We have further investigated this
ligand family by varying the substitution position, length
and stereochemistry of different amine containing side
chains. Herein, we describe the synthesis, thermal melting
profiles, molecular modelling and UV/Vis and CD spectra
of these novel bis-indole carboxamides complexed with h-
telo and c-kit2 quadruplex sequences.
Guanine-rich four-stranded DNA G-quadruplex sequences
have received considerable interest due to their key func-
tional roles in biological processes.[1] In the human genome,
G-quadruplex motifs are found in the telomere[2] as well as
in promoter regions of certain oncogenes, including c-myc,
k-ras and c-kit.[3] Small molecules are known to stabilise
human telomeric G-quadruplex DNA and can disrupt the
telomere function.[4] We have recently reported that small
molecules can bind the c-kit promoter quadruplex and alter
[a] Dr. J. Dash, N. Hegde, Dr. G. D. Pantos¸, P. S. Shirude,
Prof.Dr. S. Balasubramanian
Department of Chemistry, University of Cambridge
Lensfield Road, Cambridge CB2 1EW (UK)
Fax : (+44)1223-336-913
Results and Discussion
Synthesis: The indole ring system represents one of the
most versatile frameworks found in numerous natural prod-
ucts and pharmaceuticals with a wide range of biological ac-
tivities.[8] We reasoned that designing G-quadruplex ligands
based on indole ring systems might lead to novel nontoxic
compounds suitable for cellular studies. There are several
methodologies reported for the preparation of indole ring
systems.[8] However, few approaches have been reported for
the 2,6-bis(2’-indolyl)pyridine core of ligand 2, which has
been used as a tridentate ligand and as a synthetic recep-
tor.[9–11] Methods utilising the Fischer indole synthesis, Stille
coupling and nitrene insertion starting from o-nitrostyryl de-
rivatives have been reported.[9–11] We have developed a gen-
eral approach to prepare diverse water soluble bis-indole
derivatives 1–4; this involves the application of Sonogashira
coupling,[12] and the 5-endo-dig cyclisation[13] as the key steps
E-mail: sb10031@cam.ac.uk
[b] Dr. J. Dash, R. Nath Das
Department of Chemical Sciences
Indian Institute of Science Education and Research-Kolkata
Mohanpur Campus, Mohanpur 7412 52, Nadia, West Bengal (India)
Fax : (+91)33-25873020
E-mail: jyotidash@iiserkol.ac.in
[c] Prof.Dr. S. Balasubramanian
Cancer Research UK, Cambridge Research Institute
Li Ka Shing Center, Cambridge, CB2 0RE (UK)
[d] Prof.Dr. S. Balasubramanian
School of Clinical Medicine, University of Cambridge
Cambridge, CB2 0SP (UK)
[e] Dr. G. D. Pantos¸
Department of Chemistry, University of Bath
Bath BA2 7AY (UK)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102556.
554
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 554 – 564

FULL PAPER
boxylic building block 10 was prepared in near quantitative
yield by base mediated hydrolysis (2N NaOH in THF/
MeOH 1:1) of the diester intermediate 9 (Scheme 2). The
diacid 10 was coupled with six different amines 7a–f by
using EDC, HOBT, and NMM in DMF to give the corre-
sponding water soluble carboxamides 1a–f in 71–87% yields
(Table 1).
Table 1. Amide coupling of bis-indole carboxylic acid 10.
R4-NH2
7
Reaction
time [h]
Yield 1
[%]
Scheme 1. A general approach for the synthesis of bis-indole carboxa-
mides.
12
1a, 83
(Scheme 1). The bis-indole core can be synthesised from
easily accessible starting materials, such as iodoaniline deriv-
atives 5 and 1,3-dialkynes 6. Amide coupling with a variety
of amines 7 was used to incorporate the amine containing
side chains.
16
10
1b, 81
1c, 87
Sonogashira coupling of iodoaniline derivative 5a and 1,3-
diethynylbenzene (6a) was carried out by using PdCl2-
ACHTUNGTRENNUNG
18
15
1d, 75
1e, 71
ACHTUNGTRENNUNG
NaAuCl4·2H2O (6 mol%) in EtOH at 708C afforded the bi-
sindole derivative 9 in excellent yield. The bis-indole dicar-
15
1 f, 73
To examine the effect of carboxamide side chains at dif-
ferent positions of the bis-indole pharmacophore, ligands
2a–b were prepared (Scheme 3). Using similar methodology
(Scheme 1 and Table 1), treatment of methyl 3-amino-4-io-
dobenzoate (5b) with 6a afforded the diester 11, which was
subsequently cyclised to give the bis-indole derivative 12.
Ester hydrolysis of 12 and subsequent coupling of the result-
ing diacid 13 with amines 7a and 7b furnished ligands 2a
and 2b, respectively, in good overall yields (Scheme 3).
The same sequence of reactions was followed with iodoa-
niline 5a and 1,3-diethynyl-5-fluorobenzene (15) to synthe-
sise
fluoro-substituted
bis-indole
carboxamide
3a
(Scheme 4). The dialkyne derivative 15 was prepared in two
steps through Sonogashira coupling of 1,3-dibromo-5-fluoro-
benzene (14) with trimethylsilylacetylene and base mediated
desilylation. Ligand 3a was obtained in 43% yield over
three steps from diester 16 via the intermediates 17 and 18.
The bis-indole ligands 4a and 4c separated by a central
pyridine ring were synthesised from 5a and 2,6-diethynyl-
pyridine (6b). The pyridyl bis-acetylene derivative 19 under-
Scheme 2. Synthesis of bis-indole carboxylic acid building block 10.
a) PdCl2ACHTUNGTRENNUNG(PPh3)2, CuI, Et3N, DMF, room temperature, 9 h, 79%;
b) NaAuCl4·2H2O, EtOH, 708C, 10 h, 91%; c) NaOH (2N), THF/MeOH
(1:1), room temperature, 18 h, 96%.
Chem. Eur. J. 2012, 18, 554 – 564
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
555

J. Dash, S. Balasubramanian et al.
Scheme 3. Synthesis of bis-indole carboxamide 2. a) 6a, PdCl2ACTHNUTRGEN(UNG PPh3)2,
Scheme 4. Synthesis of fluoro-substituted bis-indole amide 3a. a) PdCl2-
AHCTUNGTRENNUNG
CuI, Et3N, DMF, room temperature, 9 h, 77%; b) NaAuCl4·2H2O, EtOH,
708C, 18 h, 87%; c) NaOH (2N), THF/MeOH (1:1), room temperature,
18 h; 83%; d) 7a,b, EDC, HOBT, NMM, DMF, room temperature,
2 days.
CTHUNGTRENNUNG
d) NaAuCl4·2H2O, EtOH, 708C, 24 h; e) NaOH (2N), THF/MeOH (1:1),
room temperature, 18 h; f) 7a, EDC, HOBT, NMM, DMF, room temper-
ature, 10 h, 43% over three steps.
went microwave promoted cyclisation[15] by using CuI as cat-
alyst in N-methylpyrrolidinone (NMP; Scheme 5). The pyri-
dine derivative 19 did not undergo gold
(III) catalysed heter-
length of the central core of ligands 1–3 varies between 14.9
and 15.4 ꢁ, which is comparable with the distance (13.6 ꢁ)
between the N9 of the opposite guanine bases in the G-
tetrad and the distance (10.6–12.1 ꢁ) between the two hy-
drogen atoms of the non-hydrogen-bonding G-NH2 func-
tional group of the opposite guanine bases in the G-tetrad
(Figure S1 in the Supporting Information).[16]
oannulation, probably due to the coordination of AuIII with
the pyridine nitrogen. Subsequent saponification of the re-
sulting crude bis-indole diester 20 afforded the diacid build-
ing block 21 in 81% yield based on 19. The diacid 21 was
treated with amines 7a and 7c to give the corresponding li-
gands 4a and 4c in high yield.
Compounds 1–4 also possess the potential for multiple
H bonding sites on the bis-indole core. Molecular modelling
shows that these ligands are not planar as evident from the
energy minimised structures of the central cores (Figure 1
and Figure S2 in the Supporting Information). Ligand 4 has
a smaller deviation from planarity due to the presence of an
intramolecular bifurcated H bond between the indole NH
and the pyridine lone-pair electrons as seen in the lowest
energy conformation (4A) in which the angle a between the
median planes containing the two indole rings is approxi-
mately 368. Ligands 1–3 adopt butterfly-type conformations
Design: Bis-indole carboxamides 1–4 were designed as po-
tential G-quadruplex binding ligands based on their compa-
rable molecular size to the G-quartet and their ability to
adopt v-shaped and twisted conformations depending on
substituents on the central ring (Scheme 4, Figure 1, Fig-
ure S1 in the Supporting Information).[16] Selected dimen-
sions from energy minimised structures with B3LYP-
631G(d) (Gamess.oct12010R1) of the bis-indole core are
shown in Figure 1 (Figure S2 in the Supporting Informa-
tion). The end-to-end (H atom-to-H atom) total calculated
556
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 554 – 564

G-Quadruplex Stabilizing Bis-indole Carboxamides
FULL PAPER
sis.[17] The FRET melting data, obtained by using dual-la-
belled sequences, are summarised in Table 2 (Figure 2 and
Figure S3 in the Supporting Information). As a part of the
Table 2. G-quadruplex stabilisation (DTm) potential measured by FRET-
melting.[a]
Ligand
DTm [K] at 1 mm ligand
Ligand [mm] at DTm =15 K
h-telo
c-kit2
dup
h-telo
c-kit2
dup (28C)
1a
1b
1c
1d
1e
1 f
2a
2b
3
21.5
16.6
18.3
10.1
14.6
14.2
9.8
21.7
16.6
19.5
10.1
17.3
19.6
9.8
4.2
3.2
3.4
2.7
2.9
1.5
1.0
0
0.58
0.87
0.80
1.45
1.03
1.41
3.20
2.11
0.35
1.46
0.52
0.87
0.76
1.43
0.89
1.41
2.67
1.52
0.42
0.68
0.50
0.70
0.60
0.65
0.67
2.10
1.47
2.09
0.64
2.0
4.1
4.1
27.8
9.8
21.7
16.0
20.4
12.3
3.1
0.5
4a
20.4[b]
22.6
4c
0.8
0.55
1.34
1.65
[a] Tm for h-telo=(59Æ1), c-kit2=(71Æ1), dup (duplex)=(58Æ1) in po-
tassium cacodylate (60 mm) buffer, pH 7.4, without ligand; [b] DTm at
2 mm ligand.
study we examined the influence of stereochemical varia-
tions as well as the importance of the amide side chains of
the bis-indole derivatives on quadruplex stabilisation. We
first evaluated the stabilisation potential of the ligand series
1a–f, in which the bis-indoles are separated by a benzene
ring. Ligand 1a with the N,N-dimethylaminopropyl side
chain appears as the best quadruplex stabiliser. It shows
maximum stabilisation potential for c-kit2 quadruplex
(DTm =21.7 K at 1 mm, that is a Tm of 938C) comparable to
the data obtained for the natural product telomestatin
(DTm =20.4 K at 1 mm).[18] By altering the side chain to a
N,N-dimethylaminoethyl group (1b), a diminished stabilisa-
tion for quadruplex sequences was observed (Figure 2A,
Table 2). Ligand 1b shows a DTm of 16.6 K at 1 mm for both
c-kit2 and h-telo quadruplex sequences. Ligand 1d prepared
from 2-(1-methylpyrrolidin-2-yl)ethanamine (7d) shows
weak stabilisation potential in the series (Table 2). Ligands
1e and 1 f prepared from (R)- and (S)-quinuclidin-3-amines,
respectively (7e and 7 f), show comparable stabilisation of
h-telo quadruplex (DTm = ~14 K at 1 mm). However, ligand
1 f (Figure 2C) shows better stabilisation of c-kit2 quadru-
plex (DTm =19.6 K) and a substantially reduced stabilisation
of duplex (DTm =1.5 K) compared to ligand 1e (Figure 2B),
which shows a DTm of 17.3 K for c-kit2 and 2.9 K for duplex
(Table 2). This suggests that in this case changing the stereo-
chemistry of the end side chains does not significantly affect
homochiral quadruplex stabilisation.
Scheme 5. Synthesis of pyridyl bis-carboxamide 4. a) PdCl2ACTHNUTRGEN(UGN PPh3)2, CuI,
Et3N, DMF, room temperature, 9 h, 83%; b) CuI, NMP, 1908C, micro-
wave, 1 h; c) NaOH (2N), THF/MeOH (1:1), room temperature, 18 h,
81% over two steps; d) 7a,c, EDC, HOBT, NMM, DMF, room tempera-
ture, 12 h.
due to the steric repulsion between the indole NH and the
aromatic CH; this is evidenced by the angle a between the
indole planes, which varies from 628 to 698 (Figure 1).
The ligands containing the bis-indole unit linked to cen-
tral benzene rings show no preference for syn and anti con-
formations whereas ligand 4 shows a preference for the syn
conformation (4A, Figure 1) as its energy of formation is
lower than the other two conformers. We propose that the
indole NH of ligands 1–4 and the lone pair of pyridine nitro-
gen in the case of ligand 4 might form H bond interactions
with the grooves or loops of the G-quadruplex DNA as well
as electrostatic interaction of the amine side chain with the
negatively charged sugar phosphate backbone. Thus, 1–4
have the potential to target G-quadruplex loops and grooves
in addition to stacking interactions with the top of a G-
tetrad.
Our studies showed that the N,N-dimethylaminopropyl
side chain 1a had superior binding stabilisation over others,
and amine 7a was chosen as a suitable side chain precursor
for G-quadruplex recognition. In order to examine the
effect of carboxamide substitution at different positions, we
prepared ligands 2a and 2b incorporating the carboxamide
side chains at the meta position relative to the indole NH
Fçrster resonance energy transfer (FRET) melting analysis:
The ability of small molecules 1–4 to stabilise the G-quadru-
plex DNA was evaluated by thermal FRET melting analy-
Chem. Eur. J. 2012, 18, 554 – 564
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
557

J. Dash, S. Balasubramanian et al.
Figure 1. Energy minimised structure of bis-indole core (Gamess, B3LYP-631G(d)). The width of the molecule (d) and the relative energies compared to
the lowest energy conformation are given under each structure; colour codes: H=white, C=black, N=blue, F=yellow.
(Scheme 3). These ligands were found to show very weak
stabilisation of G-quadruplexes compared to the para substi-
tuted analogues from series 1 (Figure 2D, Table 2). The re-
sults obtained with ligands 1 and 2 suggest that the amino-
propyl-substituted ligands demonstrate improved stabilisa-
tion over the corresponding aminoethyl derivatives. These
studies also suggest that there is an optimal orientation
(para substituted) for the amide containing side chains po-
tentially to enable interactions with the G-quadruplex
grooves and loops.
Introducing a fluorine atom on the central benzene ring
of the bis-indole scaffold was found to enhance the stabilisa-
tion potential for h-telo and c-kit2 quadruplex forming se-
quences as compared to ligand 1a (Table 2). Ligand 3a
showed the highest DTm that can be measured by this
method, DTm value of 21.7 K for c-kit2 at 1 mm ligand con-
centration (Figure 2E). Also, it showed better stabilisation
of c-kit2 (DTm =17.3 K) as compared to ligand 1a (DTm =
14.6 K) at 0.5 mm. Similarly, the DTm value for h-telo quadru-
plex at 0.5 and 1 mm ligand concentration is higher (20.2 and
27.8 K) for 3a than for 1a (14.0 and 21.5 K, respectively).
The highest DTm value recorded in this study was 32.9 K
and was obtained for the interaction of 3a (2 mm) with h-
telo. The concentrations of ligand 3a to reach saturation,
which was visualised by the flattening of the curve in
Figure 2, were in the low micromolar range; approximately
0.9 mm for c-kit2 and about 2.8 mm for h-telo quadruplex.
Ligand 3a also exhibits a low DTm (3.1 K) for duplex DNA
compared to 1a (4.2 K). Both ligands 4a (Figure S3 in the
Supporting Information) and 4c (Figure 2F) in the pyridine
series show very low stabilisation of duplex DNA compared
to ligands 1 and 3 as evident from their melting profiles and
DTm value. Ligands 4a and 4b show good DTm for h-telo
and c-kit2 quadruplexes (Table 1). The concentrations of
ligand 4c required to achieve a DTm value of 15 K are 0.55
and 1.34 mm for h-telo and for c-kit2 quadruplexes, respec-
tively. The pyridyl-based ligands were found to be better at
discriminating quadruplex and duplex DNA compared to
phenylene bis-indoles; this could be due to the near-planar
structure of pyridyl ligands as seen from the molecular mod-
elling studies described above.
UV/Vis binding titrations: UV/Vis binding titration was
used to investigate the DNA binding behaviour of ligands
1a, 2a, 3a and 4a with h-telo and c-kit2 quadruplex. The
equilibrium binding constants (Kd) were obtained[19]
(Figure 3 and Figures S4–S6 in the Supporting Information)
by sequential addition of aliquots of h-telo and c-kit2 quad-
ruplex sample into ligand solutions, with absorbance spectra
recorded after each addition. The results are summarised in
Table 3. Ligands 1a–4a showed strong absorbance at 246–
265 nm and shoulders between 321–342 nm. The peaks at
558
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 554 – 564

G-Quadruplex Stabilizing Bis-indole Carboxamides
FULL PAPER
10.0 mm for c-kit2 quadruplex.
The pyridine containing indole
derivative 4a was found to
bind h-telo and c-kit2 with dis-
sociation constants (Kd) of 8.3
and
3.5 mm,
respectively
(Figure 3). The hypochromicity
and red shift varies with the
substitution pattern on the li-
gands and the quadruplex se-
quences. All the ligands show
moderate red shift (6–7 nm)
upon binding to c-kit2 quadru-
plex. In the series, ligands 2a
and 4a showed maximal hypo-
chromicities (58–60%) with c-
kit2 quadruplex (Figure 3D
and Figure S5D in the Sup-
porting Information). Interac-
tion of ligands 1a and 3a with
c-kit2 produced up to 41–49%
hypochromicities (Figures S4D
and S6D in the Supporting In-
formation). Carboxamides with
a central benzene ring, 1a and
2a, show a red shift of up to
11–12 nm with h-telo, which is
approximately double that ob-
tained with c-kit2 quadruplex
(Figures S4A and S5A in the
Supporting
Information).
There was only 35% hypo-
chromicity along with 6 nm
shift of the band at 321 nm of
ligand 3a with h-telo quadru-
plex (Table 3, Figure S6A in
the Supporting Information).
Ligand 4a produced a maximal
red shift (19 nm) upon binding
&
*
Figure 2. FRET stabilisation curves for a selection of ligands 1–4 upon binding to h-telo ( ), c-kit2 ( ), and
~
duplex DNA ( ); buffer: potassium cacodylate (60 mm, pH 7.4).
246–265 nm overlap with DNA and therefore dissociation
constants (Kd) were calculated, by using the peaks at 321–
342 nm for the ligands (Figure 3 and Figures S4–S6 in the
Supporting Information). UV/Vis absorption titration ex-
periments show that the intensity of the band at 321–342 nm
decreased with addition of G-quadruplex DNA.
In agreement with FRET results the ligands exhibited a
preference for c-kit2 quadruplex (three- to fivefold) over h-
telo quadruplex. The ligands show diversity in binding affini-
ties and the Kd varies from 0.5 to 27.1 mm. The fluoro-substi-
tuted ligand 3a, which showed higher stabilisation tempera-
tures, was found to exhibit correspondingly high binding af-
finities to quadruplex sequences (0.5 mm for c-kit2 and
2.3 mm for h-telo). Ligand 1a shows fivefold selectivity for c-
kit2 with a Kd of 1.2 mm over h-telo quadruplex (Kd =
6.0 mm). Ligand 2a, which shows relatively low stabilisation
potential (DTm) in the series, was found to exhibit lower
binding affinities with Kd value 27.1 mm for h-telo and
to h-telo quadruplex in the series (Figure 3A). For determin-
ing duplex–quadruplex selectivity, similar UV/Vis titration
experiments with ligands 1a and 4a with calf thymus DNA
(CT DNA) were carried out (Figures S7 and S9 in the Sup-
porting Information). No detectable change in UV absorp-
tion of ligands 1a and 4a were observed upon addition of
CT DNA (100 mm). The binding constants, Kd, were deter-
mined upon titration of excess CT DNA and found to be 1.8
and 7.2 mm for ligands 1a and 4a, respectively (Figures S7–
S10 in the Supporting Information). Ligand 1a (1 mm),
which shows the highest DTm value of 4.2 K for duplex
DNA, exhibits 300- and 1500-fold selectivity for h-telo and
c-kit2 quadruplex sequences, respectively, over duplex
DNA. Similarly ligand 4a shows 870- and 2060-fold prefer-
ence for h-telo and c-kit2 quadruplexes, respectively, over
CT DNA.
Chem. Eur. J. 2012, 18, 554 – 564
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
559

J. Dash, S. Balasubramanian et al.
Figure 3. Hypochromicity changes observed for ligand 4a on progressive addition of A) h-telo, and D) c-kit2 quadruplexes in buffer containing KCl
(100 mm) and Tris-HCl (10 mm) at pH 7.4. Binding isotherms of ligand 4a upon binding to: B) h-telo, and E) c-kit2 quadruplexes. Scatchard plots of bind-
ing data of ligand 4a with: C) h-telo, and F) c-kit2 quadruplexes.
Table 3. Dissociation constants (Kd) obtained by UV/Vis titration studie-
nantly as the parallel structure with a positive signal at
260 nm and no further change in ellipticity was observed
upon titration with ligand 4a (Figure S11 in the Supporting
Information).
s.[a]
Ligand lmax [nm]
h-telo
c-kit2
Kd
red shift hc
Kd
red shift hc
[mm]
[nm]
[%] [mm]
[nm]
[%]
The human telomeric quadruplex is highly polymorphic
and has been shown to exist as parallel, antiparallel, and
mixed-type parallel/antiparallel structures.[20] We have stud-
ied the recognition of ligand 4a by h-telo structures in the
absence and presence of added salt. The CD spectrum of
1a
2a
3a
4a
260, 321
6.0Æ0.6 12
53
45
35
39
1.2Æ0.2
6
7
6
6
49
60
41
58
246, 324 27.1Æ2.8 11
10.0Æ1.0
0.5Æ0.1
3.5Æ0.9
265, 321
256, 342
2.3Æ0.6
6
8.3Æ0.9 19
[a] Kd and %hyprochromicity (hc) were measured at lmax between 321–
342 nm.
the human telomeric d[AACHTNUTRGNE(UNG G3T2A)3G3T] oligonucleotide ex-
hibits a negative band centred at 235 nm and a major posi-
tive band at 256 nm (Figure 4A). Upon addition of the
ligand 4a (0.5–3 mol equiv), the peak at 256 nm decreased
and a new peak appeared at 293 nm indicating induction of
the antiparallel conformation.- No further changes in signal
were observed even after incubation for longer time periods
(Figure 4A), and at higher ligand concentrations signals
were observed to decrease, consistent with the precipitation
of the ligand–DNA complex. This indicates that ligand 4a
templates the folding of the h-telo sequence to an antiparal-
lel structure.
In potassium containing buffer the h-telo sequence shows
peaks at 263 and 293 nm corresponding to a mixture of par-
allel and antiparallel structures. Upon addition of ligand 4a
at room temperature, the peak at 263 nm (parallel confor-
mation) disappeared and a new, negative signal developed
at this wavelength (Figure 4B), indicating induction of the
antiparallel conformation of h-telo. The peak at 293 nm (an-
tiparallel) did not change significantly throughout the titra-
Circular dichroism (CD) spectroscopic studies: CD spectros-
copy can be used to distinguish between parallel and anti-
parallel G-quaduplex structures,[20,-21] and reveal the prefer-
ence of ligands to induce or recognise a particular quadru-
plex structure and to provide insights into the binding mode
of ligands.[20,-21] We have reported that a small molecule 4a
templates the folding of the c-kit2 quadruplex into the paral-
lel conformation in the absence of any added salt (Fig-
ure S11 in the Supporting Information).[7] Upon titration of
ligand 4a to the c-kit2 quadruplex, a significant increase in
the ellipticity was observed at 260 nm indicating an increase
in the parallel conformation in equilibrium.[7] We cannot
rule out the possibility that induced CD (ICD) bands be-
longing to 4a at 264 nm might overlap in this region since
4a absorbs at 264, 341 nm. We have not observed, however,
any induced signal at other wavelengths around 340 nm. In
the presence of K+, the c-kit2 quadruplex exists predomi-
560
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 554 – 564

G-Quadruplex Stabilizing Bis-indole Carboxamides
FULL PAPER
In the presence of NaCl (100 mm) the h-telo quadruplex
exhibits a positive band at 293 nm and a negative band
around 264 nm, and a smaller positive band at 244 nm char-
acteristic of an antiparallel G-quadruplex structure (Fig-
ure 4C).[20] Upon titration of 4a (1–10 equiv) an increase in
ellipticity of the band at 293 nm (antiparallel) was observed.
This result suggests that ligand 4a induces the antiparallel
conformation of human telomeric DNA quadruplex in the
presence of sodium containing buffer.
The CD spectra of h-telo in the presence of ligand 4a, in
the absence or presence of 100 mm K+ or Na+, are consis-
tent with an antiparallel G-quadruplex being the major con-
formation in the 4a–quadruplex complex. Furthermore,
upon titration of ligand 4a into h-telo quadruplex, an in-
duced CD signal at 372 nm was observed in all the titrations
(Figure 4A and C). This is indicative of a change in the chir-
ality of the proximal chemical environment of ligand 4a.
However, no induced signal was observed with the addition
of 4a to c-kit2 quadruplex (Figure 4). This suggests that the
antiparallel conformation of h-telo quadruplex stabilises a
preferred atropisomer in ligand 4a. The induced CD signal
is an example of structure-specific recognition[-21] of quadru-
plex DNA by ligand 4a and further suggests different bind-
ing modes of ligand 4a to h-telo and c-kit2 quadruplexes.
Molecular modelling: To understand the structural basis for
the differential behaviours of this class of ligands molecular
modelling of ligand 4a with c-kit2 (PDB ID: 2KQG) and h-
telo (PDB ID: 1KF1) quadruplexes was performed by using
two possible end-stacking binding modes (Figure 5A–D).
Docking results indicate that for c-kit2 ligand 4a shows pref-
erable end-stacking mode in which the pyridyl bis-indole
core covers a larger surface area of the G-tetrad (Figure 5A
and B). Between the possible end-stacking binding modes,
Figure 4. CD spectra of a solution of h-telo (12.5 mm) in Tris-HCl buffer
(pH 7.4): A) without added salt, B) with KCl (100 mm), and C) with NaCl
(100 mm), each titrated with ligand 4a (0–10 equiv).
tion. Thus, ligand 4a appears to induce the formation of a
single, antiparallel, conformation of the h-telo quadruplex.
Figure 5. Amber99 forcefield minimisation of: A), B) c-kit2 quadruplex,
and C), D) h-telo with ligand 4a, top view.[22]
Chem. Eur. J. 2012, 18, 554 – 564
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
561

J. Dash, S. Balasubramanian et al.
(10 mol%), and CuI (20 mol%) was evacuated and filled with argon.
DMF (5 mL, 1 mmol) and triethylamine (10 equiv) were added to the
mixture. After being stirred at room temperature under an argon atmos-
phere for the specified time, the mixture was dried, in vacuo. The residue
was purified by chromatography on silica gel to afford the corresponding
cross coupled products 8, 11, 16 or 19.
the model (Figure 5A) is found to be lower in energy mode
(200 kcal more stable) than the other alternate possible ar-
rangement (Figure 5B) with c-kit2 quadruplex. In the case
of h-telo there is no significant preference for one stacking
over the other (DEꢀ2 kcal; Figure 5C and D). Modelling
of the binding of ligand 4a with the h-telo quadruplex is in-
dicative of hydrogen bonding between the two species; this
restricts the rotation of indole cores of 4a to give one atrop
isomer preferentially (Figure S12 in the Supporting Informa-
tion). This is in agreement with the observed induced CD
signature on the absorbance of 4a (vide supra).
Cyclisation reaction: NaAuCl4·2H2O (0.04 mmol) was added to a stirred
solution of 8, 11 or 16 (1.0 mmol) in EtOH (20 mL) and stirred at 708C
for the specified time (Schemes 2–4). The solvent was evaporated, in
vacuo, and the mixture was washed with Et2O. The solid product was
dried under vacuum to give the corresponding bis-indole derivatives 9, 12
or 17.
Hydrolysis of esters: NaOH (10 equiv, 2N solution in water) was added
to a solution of the methyl ester 9, 12, 17 or 20 (1 equiv) in methanol and
THF (1:1) at 08C. After being stirred for 12 h at room temperature, the
reaction mixture was acidified with formic acid. The precipitate formed
was collected by filtration and washed with water and diethyl ether to
give the corresponding crude diacid derivatives 10, 13, 18 or 21. The
products were used without further purification.
Conclusion
We have synthesised a series of bis-indole derivatives de-
signed by using information from G-quadruplex ligands with
the carboxamide side chains at different positions and exam-
ined their binding potential with h-telo and c-kit2 promoter
quadruplexes. Our results have shown that introduction of
the carboxamide side chain onto the para position of indole
NH produces compounds demonstrating high stabilisation
potential of the G-quadruplexes. These ligands show signifi-
cant selectivity for G-quadruplex over duplex DNA, bind
tightly with G-quadruplexes and show a three- to fivefold
preference for c-kit2 over h-telo quadruplex DNA. The pyri-
dine containing bis-indole carboxamide induces the forma-
tion of a single, antiparallel, conformation of the h-telo
quadruplex in the presence and absence of added salt. The
chirality of h-telo quadruplex was transferred to the achiral
ligand (induced CD) and CD studies further suggest differ-
ential recognition of c-kit2 and h-telo quadruplex sequences.
Amide coupling:
(0.25 mmol), EDC (1.06 mmol, 4.24 equiv), HOBt (1.06 mmol,
4.24 equiv), NMM (2.10 mmol, 8.40 equiv), amine (0.78 mmol,
A mixture of dicarboxylic acid 10, 13, 18 or 21
7
3.12 equiv) in DMF (5 mL) was stirred at room temperature for the
specified time (Table 1, Scheme 3–5). The solvent was removed, in vacuo,
and purified by HPLC (gradient: starting from H2O/MeCN (1% TFA)
20:80 to pure MeCN (1% TFA) over 35 min, 12 mLminÀ1), to provide
the target small molecule 1–4.
Diester 11: 1H NMR (CDCl3): d=7.71 (d, J=1.5 Hz, 1H), 7.51 (dd, J=
8.0, 1.6 Hz, 2H), 7.42–7.40 (m, 4H), 7.39–7.37 (m, 3H), 4.39 (s, 4H),
3.90 ppm (s, 6H); 13C NMR (CDCl3): d=166.5 (s), 147.7 (s), 134.4 (d),
132.1 (d), 131.5 (d), 131.0 (s), 130.8 (s), 129.0 (d), 123.2 (s), 118.6 (d),
115.3 (d), 111.6 (s), 96.3 (s), 86.1 (s), 52.2 ppm (q); HRMS (ESI); calcd
for C26H21N2O4 [M+H]+ 425.1500, found 425.1495.
Bis-indole derivative 9: 1H NMR ([D6]DMSO): d=12.03 (d, J=1.3 Hz,
2H), 8.42 (t, J=1.6 Hz, 1H), 8.28–8.27 (m, 2H), 7.85 (dd, J=7.8, 1.7 Hz,
2H), 7.76 (dd, J=8.6, 1.7 Hz, 2H), 7.59 (t, J=7.8 Hz, 1H), 7.51 (d, J=
8.6 Hz, 2H), 7.19 (d, J=1.5 Hz, 2H), 3.85 ppm (s, 6H); 13C NMR
([D6]DMSO): d=167.3 (s), 139.8 (s), 139.3 (s), 132.4 (s), 129.8 (d), 128.3
(s), 124.6 (d), 122.8 (d), 122.7 (d), 122.3 (d), 121.1 (s), 111.4 (d), 100.5 (d),
51.8 ppm (q); calcd for C26H21N2O4 [M+H]+ 425.1500, found 425.1495.
Bis-indole derivative 12: 1H NMR ([D6]DMSO): d=12.08 (s, 2H), 8.49
(s, 1H), 8.18 (s, 2H), 8.08 (s, 2H), 7.91 (dd, J=6.0, 1.5 Hz, 2H), 7.65–7.64
(m, 1H), 7.57 (d, J=7.5 Hz, 2H), 7.18 (s, 2H), 3.85 ppm (s, 6H);
13C NMR (CDCl3): d=167.2 (s), 141.0 (s), 136.4 (s), 132.3 (s), 129.8 (d),
125.1 (d), 122.8 (d), 122.6 (s), 120.3 (d), 120.0 (d), 113.2 (d), 99.8 (d),
51.9 ppm (q); HRMS (ESI): calcd for C26H21N2O4 [M+H]+: 425.1505,
found 425.1495.
Experimental Section
General: Experiments were carried out under an inert atmosphere unless
otherwise stated. Solvents were purified by standard procedures. All
starting materials were obtained from commercial suppliers and used as
received. H and 13C NMR spectra were recorded at 500 and 125 MHz by
1
1
using a Bruker DRX 500 instrument. H spectra were recorded in deuter-
1
Diacid 10: H NMR ([D6]DMSO): d=12.03 (brs, 2H), 12.05 (s, 2H), 8.46
ated solvents as detailed and at ambient probe temperature (300 K).
Chemical shifts are reported in parts per million (ppm) and are refer-
enced to the residual solvent peak. The following notations are used: sin-
glet (s); doublet (d); triplet (t); quartet (q); multiplet (m); broad (br).
Data are reported in the following manner: chemical shift (multiplicity,
coupling constant if appropriate, integration). Signals are quoted as d
values in ppm and coupling constants (J) are reported in Hertz. Using re-
(s, 2H), 8.28 (s, 1H), 8.18–8.17 (m, 2H), 7.86 (dd, J=8.0, 1.0 Hz, 2H),
7.51 (d, J=8.0 Hz, 2H), 7.21 ppm (s, 2H); 13C NMR ([D6]DMSO): d=
168.4 (s), 139.7 (s), 137.8 (s), 132.4 (s), 129.7 (d), 128.1 (d), 124.4 (d),
123.1 (d), 122.4 (d), 121.2 (s), 111.2 (d), 101.0 ppm (d); HRMS (ESI):
calcd for C24H17N2O4 [M+H]+: 397.1188, found 397.1182.
1
Diacid 12: H NMR ([D6]DMSO): d=12.56 (brs, 2H), 11.99 (s, 2H), 8.46
sidual protonated solvent signals as internal standard (1H: d
7.26 ppm, ((CH3)2SO)=2.50 ppm, (CH3OH)=3.31 ppm,
4.67 ppm; and 13C: d
(CHCl3)=77.16 ppm, d((CH3)2SO)=39.52 ppm, d-
ACHTUNGTRENNUNG(CH3OH)=49.00 ppm). Mass spectra were recorded on Micromass Q-
ACHTUNGTRENNUNG
(s, 1H), 8.07 (m, 2H), 7.89 (dd, J=6.0, 1.5 Hz, 2H), 7.64–7.59 (m, 5H),
7.17 ppm (s, 2H); 13C NMR ([D6]DMSO): d=168.3 (s), 140.7 (s), 136.5
(s), 132.4 (s), 129.8 (d), 124.9 (d), 123.8 (d), 122.6 (s), 120.6 (d), 119.8 (d),
113.3 (d), 99.7 ppm (d); HRMS (ESI): calcd for C24H17N2O4 [M+H]+:
397.1188, found 397.1182.
dG
d
A
dACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
ToF (ESI) spectrometer. Thin layer chromatography (TLC) was per-
formed on Merck Kieselgel 60 F254 glass plates and visualised under UV
light. Flash column chromatography (FC) was performed by using Merck
Kieselgel 60 silica gel with distilled solvents. HPLC was performed by
using a Varian Pursuit C18, 5m column (250ꢂ21.2 mm) and a gradient
elution with 0.1% TFA/MeCN and 0.1% TFA/H2O at a flow rate of
1
Diacid 18: H NMR ([D6]DMSO): d=12.63 (brs, 2H), 12.10 (s, 2H), 8.34
(s, 1H), 8.28 (d, J=1.2 Hz, 2H), 7.79 (dd, J=8.5, 1.6 Hz, 2H), 7.71 (dd,
J=10.0, 1.1 Hz, 2H), 7.52 (d, J=8.5 Hz, 2H), 7.30 ppm (d, J=1.4 Hz,
2H); 13C NMR ([D6]DMSO): d=168.1 (s), 163.1 (d, J1C,F =242.2 Hz),
139.6 (s), 137.7 (d, JC,F =3.0 Hz), 134.5 (d, JC,F =9.2 Hz), 127.8, 123.4 (d),
122.9 (d), 122.3, 118.0 (d), 111.2 (d), 110.6 (d, JC,F =23.7 Hz), 101.4 ppm
(d); HRMS (ESI): calcd for C24H16N2O4F [M+H]+: 415.1094, found
415.1068.
12.0 mLminÀ1
Sonogashira reaction: A flask containing a mixture of iodoaniline deriva-
tive 5 (2 equiv), diethynyl compounds 6 or 15 (1 equiv), PdCl2A(PPh3)2
.
CTHUNGTRENNUNG
562
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 554 – 564

G-Quadruplex Stabilizing Bis-indole Carboxamides
FULL PAPER
1
Diacid 21: H NMR ([D6]DMSO): d=12.50 (brs, 2H), 12.01 (s, 2H), 8.32
Bis-indole amide 3a: 1H NMR (CD3CN): d=10.79 (s, 2H), 9.58 (brs,
2H), 8.18 (s, 1H), 8.16 (s, 2H), 7.67 (dd, J=8.5, 1.0 Hz, 2H), 7.56 (d, J=
8.5 Hz, 2H), 7.51 (dd, J=9.9, 1.0 Hz, 2H), 7.09 (d, J=1.2 Hz, 2H), 3.50
(q, J=6.1 Hz, 4H), 3.09 (q, J=6.1 Hz, 4H), 2.85 (s, 6H), 2.84 (s, 6H),
2.03–1.98 ppm (m, 4H); 13C NMR (CD3CN): d=171.3 (s), 165.2 (d,
(s, 2H), 7.95 (brs, 3H), 7.84 (d, J=8.3 Hz, 2H), 7.64 (d, J=8.3 Hz, 2H),
7.45 ppm (s, 2H); 13C NMR ([D6]DMSO): d=168.0 (s), 148.9 (s), 139.0
(s), 138.4 (s), 138.0 (d), 128.1 (s), 123.8 (d), 123.5 (d), 122.1 (s), 118.4 (d),
111.3 (d), 102.0 ppm (d); HRMS (ESI): calcd for C23H16N3O4 [M+H]+:
398.1143, found 398.1135.
J1C,F =242.9 Hz), 140.3 (s), 139.1 (d, J4C,F =3.0 Hz), 135.7 (d, J3
=
C,F
Bis-indole amide 1a: 1H NMR (CD3CN): d=10.91 (s, 2H), 9.92 (brs,
2H), 8.42 (s, 1H), 8.18 (s, 2H), 7.81 (d, J=7.7 Hz, 2H), 7.70 (d, J=
8.4 Hz, 2H), 7.59 (d, J=8.4 Hz, 1H), 7.56 (t, J=7.7 Hz, 2H), 7.07 (s,
2H), 3.52 (q, J=5.9 Hz, 4H), 3.11 (q, J=5.9 Hz, 4H), 2.86 (s, 12H),
2.04 ppm (q, J=5.9 Hz, 4H); 13C NMR (CD3CN): d=171.2 (s), 140.3 (s),
133.5 (s), 130.6 (d), 129.4 (s), 126.1 (s), 125.7 (d), 122.8 (d), 122.2 (d),
121.3 (d), 112.2 (d), 101.2 (d), 55.6 (t), 43.4 (q), 36.5 (t), 25.8 ppm (t);
HRMS (ESI): calcd for C34H41N6O2 [M+H]+: 565.3306, found 565.3286.
Bis-indole amide 1b: 1H NMR (CD3CN): d=10.68 (s, 2H), 9.63 (brs,
2H), 8.32 (s, 1H), 8.15 (s, 2H), 7.76 (d, J=7.4 Hz, 2H), 7.67 (d, J=
8.4 Hz, 2H), 7.54–7.53 (m, 3H), 7.03 (s, 2H), 3.72 (q, J=4.9 Hz, 4H),
3.30–3.31 (m, 4H), 2.91 ppm (s, 12H); 13C NMR (CD3CN): d=171.2 (s),
140.3 (s), 140.2 (s), 133.5 (s), 130.6 (d), 129.5 (s), 125.9 (s), 125.7 (d),
122.8 (d), 122.3 (d), 121.4 (d), 112.1 (d), 101.2 (d), 60.0 (t), 44.0 (q),
36.5 ppm (t); HRMS (ESI): calcd for C32H37N6O2 [M+H]+: 536.2899,
found 536.2889.
Bis-indole amide 1c: 1H NMR (CD3CN): d=11.02 (s, 2H), 10.24 (brs,
2H), 8.42 (s, 1H), 8.15 (s, 2H), 7.78 (dd, J=7.8, 1.2 Hz, 2H), 7.66 (dd,
J=8.4, 1.4 Hz, 2H), 7.56–7.51 (m, 3H), 7.04 (s, 2H), 3.63–3.60 (m, 4H),
3.50 (q, J=6.2 Hz, 4H), 3.13 (q, J=6.2 Hz, 4H), 2.95–2.92 (m, 4H), 2.09–
2.03 (m, 4H), 2.02–1.98 ppm (m, 8H); 13C NMR (CD3CN): d=170.8 (s),
140.29 (s), 140.27 (s), 133.6 (s), 130.6 (d), 129.3 (s), 126.3 (s), 125.7 (d),
122.6 (d), 122.2 (d), 121.2 (d), 112.1 (d), 101.1 (d), 54.5 (t), 52.9 (t), 44.0
(t), 36.8 (t), 26.9 (t), 23.7 ppm (t); HRMS (ESI): calcd for C38H45N6O2
[M+H]+: 617.3629, found 617.3599.
9.1 Hz), 129.2 (s), 126.1 (s), 122.6 (d), 121.6 (d), 121.3 (s), 118.1 (d,
merged with CD3CN), 112.3 (d), 112.1 (d, J2C,F =23.7 Hz), 102.9 (d), 55.6
(t), 43.5 (q), 36.3 (t, 2C), 25.9 ppm (t, 2C); HRMS (ESI): calcd for
C34H40N6O2F [M+H]+: 583.3197, found 518.3187.
Bis-indole amide 4a: 1H NMR (CD3CN): d=10.84 (s, 2H), 9.19 (brs,
2H), 8.22 (s, 2H), 7.93–7.86 (m, 3H), 7.74 (dd, J=8.6, 1.4 Hz, 2H), 7.67–
7.63 (m, 2H), 7.28 (d, J=1.2 Hz, 2H), 3.52 (q, J=6.1 Hz, 4H), 3.11 (q,
J=6.1 Hz, 4H), 2.88 (s, 6H), 2.87 (s, 6H), 2.04 ppm (q, J=6.2 Hz, 4H);
13C NMR (CD3CN): d=171.6 (s), 150.2 (s), 139.7 (s), 139.4 (s), 139.0 (d),
129.6 (s), 126.0 (s), 123.1 (d), 122.3 (d), 119.7 (d), 112.4 (d), 102.6 (d),
55.6 (t), 43.6 (q), 36.2 (t), 25.9 ppm (t); HRMS (ESI): calcd for
C33H40N7O2 [M+H]+ 566.3238, found 566.3265.
Bis-indole amide 4b: 1H NMR (CD3CN): d=11.28 (s, 2H), 10.38 (brs,
2H), 8.19 (s, 2H), 7.87–7.81 (m, 3H), 7.71–7.62 (m, 4H), 7.20 (s, 2H),
3.64–3.61 (m, 4H), 3.52–3.51 (m, 4H), 3.16–3.12 (m, 4H), 2.96–2.94 (m,
4H), 2.09–2.05 (m, 4H), 2.02–2.00 ppm (m, 8H); 13C NMR (CD3CN): d=
170.8 (s), 150.2 (s), 139.7 (s), 139.5 (s), 138.8 (d), 126.4 (s), 123.0 (d),
121.9 (d), 119.5 (d), 112.5 (d), 102.3 (d), 54.5 (t), 52.9 (t), 36.8 (t), 26.9 (t),
23.8 ppm (t); HRMS (ESI): calcd for C37H44N7O2 [M+H]+: 618.3577,
found 618.3551.
Acknowledgements
Bis-indole amide 1d: 1H NMR (CD3CN): d=10.92 (s, 2H), 10.22 (brs,
2H), 8.39 (brs, 1H), 8.19 (s, 2H), 7.79 (dd, J=7.6, 1.6 Hz, 2H), 7.70 (dd,
J=8.5, 1.6 Hz, 2H), 7.57 (d, J=8.5 Hz, 2H), 7.57–7.54 (m, 1H), 7.06 (d,
J=1.6 Hz, 2H), 3.88–3.84 (m, 4H), 3.60–3.52 (m, 8H), 3.42–3.38 (m,
4H), 3.12–3.04 (m, 4H), 2.22–2.16 (m, 4H), 2.08–2.00 ppm (m, 4H);
13C NMR (CD3CN): d=172.5 (s), 140.5 (s), 140.4 (s), 133.5 (s), 130.7 (d),
129.5 (s), 125.7 (s), 125.3 (d), 122.8 (d), 122.3 (d), 121.6 (d), 112.3 (d),
101.2 (d), 69.6 (d), 54.5 (t), 51.6 (t), 41.7 (q), 27.6 (t), 23.9 (t), 11.3 ppm
(q); HRMS (ESI): calcd for C38H45N6O2 [M+H]+: 617.3604, found
617.3591.
We thank the BBSRC for project funding, Cancer Research UK for pro-
gramme funding and the European Commission (Marie Curie Incoming
Postdoctoral Fellowship to J.D.) for funding. J.D. thanks CSIR (scheme
no: 01ACTHNUGRTENUNG(2371)10/EMR-II) and DBT India. G.D.P. thanks Pembroke Col-
lege, Cambridge and University of Bath, UK for funding, and R.N.D.
thanks CSIR India for a research fellowship. We thank Abhiket Gaurav
for his help with UV titration experiments, Dr. Zoꢃ A. E. Waller and Dr.
Chris Lowe for critical reading of the manuscript.
Bis-indole amides 1e and 1 f: 1H NMR (CD3CN): d=10.45 (s, 2H), 9.03
(brs, 2H), 8.26 (brs, 1H), 8.11 (s, 2H), 7.71 (d, J=7.7 Hz, 2H), 7.64 (dd,
J=8.5, 1.3 Hz, 2H), 7.48 (d, J=8.5 Hz, 2H), 7.37–7.36 (m, 1H), 6.98
(brs, 2H), 4.38–4.37 (m, 2H), 3.73–3.69 (m, 2H), 3.39–3.33 (m, 2H),
3.29–3.18 (m, 8H), 2.32–2.30 (m, 2H), 2.25–2.20 (m, 2H), 2.00–1.97 (m,
4H), 1.86–1.80 ppm (m, 2H); 13C NMR (CD3CN): d=170.2 (s), 140.1 (s),
140.0 (s), 130.6 (d), 129.3 (s), 126.7 (s), 125.6 (d), 122.9 (d), 122.4 (d),
121.3 (d), 112.0 (d), 101.1 (d), 53.7 (t), 47.4 (t), 46.9 (t), 46.1 (d), 25.4 (d),
22.5 (t), 18.2 ppm (t); HRMS (ESI) of 1e: calcd for C38H41N6O2 [M+H]+
: 613.3304, found 613.3286.
Bis-indole amide 2a: 1H NMR (CD3CN): d=10.50 (s, 2H), 9.12 (s, 2H),
8.30 (s, 1H), 8.01 (s, 2H), 7.78 (d, J=7.5 Hz, 2H), 7.77 (d, J=7.5 Hz,
2H), 7.64 (d, J=8.5 Hz, 2H), 7.54 (t, J=7.5 Hz, 1H), 6.99 (s, 2H), 3.49
(q, J=6.0 Hz, 4H), 3.08 (q, J=6.5 Hz, 4H), 2.82 (s, 12H), 2.0 ppm (q, J=
6.5 Hz, 4H); 13C NMR (CD3CN): d=171.5 (s), 141.7 (s), 137.6 (s), 133.4
(d), 130.9 (s), 127.7 (s), 126.5 (d), 123.0 (d), 119.6 (d), 115.7 (d), 112.4 (d),
100.6 (d), 55.8 (t), 43.5 (q), 36.7 (t), 25.8 ppm (t); HRMS (ESI): calcd for
C34H41N6O2 [M+H]+: 565.3257, found 565.3285.
[1] a) A. M. Burger, F. Dai, C. M. Schultes, A. P. Reszka, M. J. B.
Moore, J. A. Double, S. Neidle, Cancer Res. 2005, 65, 1489–1496;
b) G. Pennarun, C. Granotier, L. R. Gauthier, D. Gomez, F. Hoff-
schir, E. Mandine, J.-F. Riou, J.-L. Mergny, P. Mailliet, F. D. Boussin,
Oncogene 2005, 24, 2917–2928; c) D. Monchaud, M.-P. Teulade-
Fichou, Org. Biomol. Chem. 2008, 6, 627–636; d) T.-M. Ou, Y.-J.
Lu, J.-H. Tan, Z.-S. Huang, K.-Y. Wong, L.-Q. Gu, ChemMedChem
2008, 3, 690–713; e) Y. Qin, L. H. Hurley, Biochimie 2008, 90, 1149–
1171; f) S. Balasubramanian, S. Neidle, Curr. Opin. Chem. Biol.
2009, 13, 345–353; g) S. N. Georgiades, N. H. Abd Karim, K. Sun-
tharalingam, R. Vilar, Angew. Chem. 2010, 122, 4114–4128; Angew.
Chem. Int. Ed. 2010, 49, 4020–4034; h) S. Balasubramanian, L. H.
Hurley, S Neidle, Nat. Rev. Drug Discovery 2011, 10, 261–275.
[2] a) E. H. Blackburn, J. G. Gall, J. Mol. Biol. 1978, 120, 33–53; b) J.
Shampay, J. W. Szostak, E. H. Blackburn, Nature 1984, 310, 154–
157; c) C. W. Greider, E. H. Blackburn, Cell 1987, 51, 887–898.
[3] a) J. L. Huppert, S. Balasubramanian, Nucleic Acids Res. 2007, 35,
406–413; b) A. K. Todd, M. Johnston, S. Neidle, Nucleic Acids Res.
2005, 33, 2901–2907.
[4] a) D. Sun, B. Thompson, B. E. Cathers, M. Salazar, S. M. Kerwin,
J. O. Trent, T. C. Jenkins, S. Neidle, L. H. Hurley, J Med. Chem.
1997, 40, 2113–2116; b) W. Duan, A. Rangan, H. Vankayalapati,
M.-Y. Kim, Q. Zeng, D. Sun, H. Han, O. Y. Fedoroff, D. Nishioka,
S. Y. Rha, E. Izbicka, D. D. Von Hoff, L. H. Hurley, Mol. Cancer
Ther. 2001, 1, 103–120; c) R. Rodriguez, S. Muller, J. A. Yeoman, C.
Trentesaux, J.-F. Riou, S. Balasubramanian, J. Am. Chem. Soc. 2008,
130, 15758–15759.
1
Bis-indole amide 2b: H NMR (CD3CN): d=11.10 (s, 2H), 10.61 (s, 2H),
8.47 (s, 1H), 8.09 (s, 2H), 8.03 (t, J=7.5 Hz, 2H), 7.81 (d, J=5.0 Hz,
2H), 7.62 (d, J=8.5 Hz, 2H), 7.55 (m, 1H), 7.01 (s, 2H), 3.75 (q, J=
5.5 Hz, 4H), 3.30 (t, J=5.5 Hz, 4H), 2.88 ppm (s, 12H); 13C NMR
(CD3CN): d=170.2, 141.5, 137.8, 133.5 (d), 130.6, 128.0, 126.0 (d), 122.9
(d), 119.7 (d), 118.5 (d), 112.5 (d), 100.3 (d), 59.2 (t), 43.9 (q), 36.2 ppm
(q); HRMS (ESI): calcd for C32H37N6O2 [M+H]+: 536.2899, found
536.2889.
Chem. Eur. J. 2012, 18, 554 – 564
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
563

J. Dash, S. Balasubramanian et al.
[5] a) M. Bejugam, S. Sewitz, P. S. Shirude, R. Rodriguez, R. Shahid, S.
Balasubramanian, J. Am. Chem. Soc. 2007, 129, 12926–12927;
b) Z. A. E. Waller, S. A. Sewitz, S.-T. D. Hsu, S. Balasubramanian, J.
Am. Chem. Soc. 2009, 131, 12628–12633; c) M. Bejugam, M. Gunar-
atnam, S. Mueller, D. A. Sanders, S. Sewitz, J. A. Fletcher, S. Neidle,
S. Balasubramanian, ACS Med. Chem. Lett. 2010, 1, 306–310;
d) K. I. E. McLuckie, Z. A. E. Waller, D. A. Sanders, D. Alves, R.
Rodriguez, J. Dash, G. J. McKenzie, A. R. Venkitaraman, S. Balasu-
bramanian, J. Am. Chem. Soc. 2011, 133, 2658–2663.
[6] For specific recent examples, see: a) J. Dash, P. S. Shirude, S.-T. D.
Hsu, S. Balasubramanian, J. Am. Chem. Soc. 2008, 130, 15950–
15956; b) K. Shinohara, Y. Sannohe, S. Kaieda, K. Tanaka, H.
Osuga, H. Tahara, Y. Xu, T. Kawase, T. Bando, H. Sugiyama, J. Am.
Chem. Soc. 2010, 132, 3778–3782; c) S. G. Rzuczek, D. S. Pilch, A.
Liu, L. Liu, E. J. LaVoie, J. E. Rice, J. Med. Chem. 2010, 53, 3632–
3644; d) J. Dash, Z. A. E. Waller, G. D. Pantos, S. Balasubramanian,
Chem. Eur. J. 2011, 17, 4571–4581; e) F. Hamon, E. Largy, A.
Guꢄdin-Beaurepaire, M. Rouchon-Dagois, A. Sidibe, D. Monchaud,
J.-L. Mergny, J.-F. Riou, C.-H. Nguyen, M.-P. Teulade-Fichou,
Angew. Chem. 2011, 123, 8904–8908; Angew. Chem. Int. Ed. 2011,
50, 8745–8749.
[12] For reviews, see: a) K. Sonogashira, in Metal-Catalyzed Cross-Cou-
pling Reactions (Eds: F. Diederich, P. J. Stang), Wiley-VCH, Wein-
heim, 1998, Chapter 5; b) E.-I. Negishi, L. Anastasia, Chem. Rev.
2003, 103, 1979–2017.
[13] For a review, see: T. E. Mꢅller, M. Beller, Chem. Rev. 1998, 98, 675–
703.
[14] A. Arcadi, G. Bianchi, F. Marinelli, Synthesis 2004, 610–618.
[15] S. E. Pearson, S. Nandan, Synthesis 2005, 2503–2506.
[16] Molecular dimensions of indole derivative 1 are determined from an
energy minimised chem 3D diagram and compared with the X-ray
crystal structure of the G-tetrad; a) A. De Cian, E. DeLemos, J.-L.
Mergny, M.-P. Teulade-Fichou, D. Monchaud, J. Am. Chem. Soc.
2007, 129, 1856–1857; b) G. N. Parkinson, M. P. H. Lee, S. Neidle,
Nature 2002, 417, 876–880.
[17] R. A. J. Darby, M. Sollogoub, C. McKeen, L. Brown, A. Risitano, N.
Brown, C. Barton, T. Brown, K. R. Fox, Nucleic Acids Res. 2002, 30,
39e; b) A. De Cian, L. Guittat, M. Kaiser, B. Sacca, S. Amrane, A.
Bourdoncle, P. Alberti, M.-P. Teulade-Fichou, L. Lacroix, J.-L.
Mergny, Methods 2007, 42, 183–195.
[18] P. S. Shirude, E. R. Gilles, S. Ladame, F. Godde, K. Shin-ya, I. Huc,
S. Balasubramanian, J. Am. Chem. Soc. 2007, 129, 11890–11891.
[19] a) J. B. Chaires, Methods Enzymol. 2001, 340, 3–22; b) B. A. Brown,
Y. Li, J. C. Brown, C. C. Hardin, J. F. Roberts, S. C. Pelsue, L. D.
Shultz, Biochemistry 1998, 37, 16325–16337, and references therein.
[20] a) P. Balagurumoorthy, S. K. Brahmachari, J. Biol. Chem. 1994, 269,
21858–21869; b) R. Jin, B. L. Gaffney, C. Wang, R. A. Jones, K. J.
Breslauer, Proc. Natl. Acad. Sci. USA 1992, 89, 8832–8866; c) S.
Paramasivan, I. Rujan, P. H. Bolton, Methods 2007, 43, 324–331.
[21] E. W. White, F. Tanious, M. A. Ismail, A. P. Reszka, S. Neidle, D. W.
Boykin, W. D. Wilson, Biophys. Chem. 2007, 126, 140–153.
[22] E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M.
Greenblatt, E. C. Meng, T. E. Ferrin, J. Comput. Chem. 2004, 25,
1605–1612.
[7] J. Dash, P. S. Shirude, S. Balasubramanian, Chem. Commun. 2008,
3055–3057.
[8] For reviews, see: a) G. R. Humphrey, J. T. Kuethe, Chem. Rev. 2006,
106, 2875–2920; b) D. A. Horton, G. T. Bourne, M. L. Smythe,
Chem. Rev. 2003, 103, 893–930; c) H.-J. Knçlker, K. R. Reddy,
Chem. Rev. 2002, 102, 4303–4427.
[9] W.-L. Jia, Q.-D. Liu, R. Wang, S. Wang, Organometallics 2003, 22,
4070–4078.
[10] a) Q. Liu, L. Thorne, I. Kozin, D. Song, C. Seward, M. D’Iorio, Y.
Tao, S. Wang, J. Chem. Soc. Dalton Trans. 2002, 3234–3240; b) R. P.
Thummel, V. Hegde, J. Org. Chem. 1989, 54, 1720–1725; c) V.
Hegde, C. Y. Hung, P. Madhukar, R. Cunningham, T. Hopfner, R. P.
Thummel, J. Am. Chem. Soc. 1993, 115, 872–878.
[11] a) S. Tollari, S. Cenini, A. Rossi, G. Palmisano, J. Mol. Catal. A
1998, 135, 241–248; b) G. Palmisano, M. Santagostino, Helv. Chim.
Acta 1993, 76, 2356–2366.
Received: August 17, 2011
Published online: December 7, 2011
564
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 554 – 564