Isaindigotone derivatives: A new class of highly selective ligands for telomeric G-quadruplex DNA

Article abstract of DOI:10.1021/jm801600m

Four isaindigotone derivatives (5a,b and 6a,b) designed as telomeric G-quadruplex ligands have been synthesized and characterized. The unfused aromatic rings in these compounds allow a flexible and adaptive conformation in G-quadruplex recognition. The in

Full text of DOI:10.1021/jm801600m

J. Med. Chem. 2009, 52, 2825–2835
2825
Isaindigotone Derivatives: A New Class of Highly Selective Ligands for Telomeric
G-Quadruplex DNA
Jia-Heng Tan,†,‡ Tian-Miao Ou,†,‡ Jin-Qiang Hou,‡ Yu-Jing Lu,‡ Shi-Liang Huang,‡ Hai-Bin Luo,‡ Jian-Yong Wu,§
Zhi-Shu Huang,‡,* Kwok-Yin Wong,§ and Lian-Quan Gu‡,*
School of Pharmaceutical Sciences, Sun Yat-sen UniVersity, Guangzhou 510080, China, Department of Applied Biology and Chemical
Technology and the Central Laboratory of the Institute of Molecular Technology for Drug DiscoVery and Synthesis, The Hong Kong
Polytechnic UniVersity, Hung Hom, Kowloon, Hong Kong, China
ReceiVed December 18, 2008
Four isaindigotone derivatives (5a,b and 6a,b) designed as telomeric G-quadruplex ligands have been
synthesized and characterized. The unfused aromatic rings in these compounds allow a flexible and adaptive
conformation in G-quadruplex recognition. The interaction of human telomeric G-quadruplex DNA with
these designed ligands was explored by means of FRET melting, fluorescence titration, CD spectroscopy,
continuous variation, and molecular modeling studies. Our results showed that the adaptive scaffold might
not only allow the ligands to well occupy the G-quartet but also perfectly bind to the grooves of the
G-quadruplex. The synergetic effect of the multiple binding modes might be responsible for the improved
binding ability and high selectivity of these ligands toward G-quadruplex over duplex DNA. Long-term
exposure of HL60 and CA46 cancer cells to compound 5a showed a remarkable decrease in population
growth, cellular senescence phenotype, and shortening of the telomere length, which is consistent with the
behavior of an effective telomeric G-quadruplex ligand and telomerase inhibitor.
1. Introduction
Human telomeric DNA, located at the very end of chromo-
somes, consists of tandem repeats of sequence d[(TTAGGG)n],
which ends as the single strand.1 This guanine-rich single strand
can adopt higher-order and functionally useful structures called
the G-quadruplexes.2,3 The building blocks of G-quadruplexes
are the G-quartets (Figure 1a), which stack up with one on top
of another to form such secondary DNA structures.4
Induction and stabilization of the telomeric G-quadruplexes
by small molecules have been shown to interfere telomere
Figure 1. Structures of G-quartet (a) and isaindigotone (b).
biological functions, inhibit telomerase activity, and eventually
alter telomere maintenance.5-7 Telomere maintenance is crucial
for the unlimited proliferative potential of cancer cells,8,9 thus
the design of drugs targeting at the telomeric G-quadruplex is
a rational and promising approach for cancer chemotherapy.10-12
However, further efforts in appropriate drug design are needed
to control the G-quadruplex selectivity over duplex DNA
because ligand interaction with duplex DNA leads to acute toxic
and intolerable side effects on normal tissues.13 Several small
molecules, such as trisubstituted anilinoacridines and telom-
estatin, have been identified to selectively bind and stabilize
the telomeric G-quadruplex DNA in vitro.14,15
Isaindigotone (Figure 1b) is a naturally occurring alkaloid.
It is isolated from the root of Isatis indigotica Fort (Ban-Lan-
Gen in Chinese), which is commonly used in traditional Chinese
medicine for treatment of influenza, epidemic hepatitis, and
epidemic encephalitis.16 The structure of isaindigotone com-
prises a pyrrolo[2,1-b]quinazoline moiety conjugated with a
benzylidene group. Such a compound is neither polycyclic nor
macrocyclic and has free rotation around the double bond that
enables the adoption of possible twisted and coplanar conforma-
tions of the aryl groups. Hence, this adaptive structural feature
might allow the unfused aromatic scaffold not only to occupy
the G-quartet but also bind to the grooves of G-quadruplex.17,18
Both the G-quartet and groove regions are binding sites for a
ligand selectively targeting the G-quadruplex.13 In view of this
feature, and enlightened by the tempting G-quadruplex selectiv-
ity exhibited by several unfused aromatic compounds such as
1,4-triazole derivatives,19 biarylpyrimidines,20 triarylpyridines,17
and diarylethynyl amides,18 we perceived that isaindigotone
might offer an attractive template for the design of selective
G-quadruplex ligands. Furthermore, isaindigotone possesses a
unique asymmetric chromophore with an aliphatic five-member
ring in the middle core. This scaffold would be a new type of
unfused aromatic G-quadruplex ligands. The scaffold itself
alone, however, is probably not sufficient as a selective and
effective G-quadruplex ligand, as previous experience indicated
that the presence of at least two cationic side chains to the
chromophore is usually required to gain high G-quadruplex
DNA binding potency and selectivity as well as the solubility
in aqueous medium.21,22
* To whom correspondence should be addressed. Phone: 8620-39943056
(Z.-S.H.); 8620-39943055 (L.-Q.G.). Fax: 8620-39943056 (Z.-S.H. and L.-
Q.G.). E-mail: ceshzs@mail.sysu.edu.cn (Z.-S.H.); cesglq@mail.sysu.edu.cn
(L.-Q.G.).
†
These authors contributed equally to this paper.
School of Pharmaceutical Sciences, Sun Yat-sen University.
Department of Applied Biology and Chemical Technology and the
‡
§
To explore novel and selective G-quadruplex ligands for
cancer chemotherapy, we have therefore synthesized a series
Central Laboratory of the Institute of Molecular Technology for Drug
Discovery and Synthesis, The Hong Kong Polytechnic University.
10.1021/jm801600m CCC: $40.75
2009 American Chemical Society
Published on Web 04/08/2009

2826 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Scheme 1. Synthesis of Isaindigotone Derivativesa
Tan et al.
a Reagents: (a) pyrrolidin-2-one, POCl3, toluene (reflux); (b) 4-nitrobenzaldehyde, Ac2O (reflux); (c) Na2S·9H2O, NaOH, EtOH (reflux); (d) Cl(CH2)2COCl
(reflux); (e) R2NH, KI, ethanol (reflux).
Table 1. Stabilization Temperatures (∆Tm) Determined by FRET
of new isaindigotone derivatives by attaching cationic amino
Experiment and Equilibrium Binding Constants (Kb) by Fluorescence
Titration
side chains to the unfused aromatic chromophore. Their interac-
tions with telomeric G-quadruplex DNA as well as the inhibitory
∆Tm (°C) at 1 µM ligand concentrationa Kb (× 106 M-1
)
b
effect on telomerase activity were examined by biophysical and
biochemical assays, and the ligand-quadruplex interaction was
further investigated by molecular modeling studies. In addition,
cellular studies were employed to evaluate the cell senescence
and telomere shortening effect induced by the new quadruplex
ligands.
compd
5a
6a
5b
6b
F21T
F10T
HTG21
21.9 ( 1.2
22.0 ( 0.7
17.6 ( 1.6
17.6 ( 0.9
10.4 ( 0.2
0.1 ( 0.1
0.0 ( 0.0
0.4 ( 0.1
0.1 ( 0.1
5.7 ( 0.1
10.1 ( 0.6
9.8 ( 0.4
7.2 ( 0.8
8.1 ( 1.0
nd
SYUIQ-5
a ∆Tm ) Tm (DNA + ligand) - Tm (DNA). The concentrations of F21T
and F10T were both 0.2 µM. In the absence of ligand, Tm values of annealed
F21T and F10T are 60 and 64 °C, respectively. Isaindigotone was not
included due to its poor solubility. b nd: Not determined.
2. Results and Discussion
2.1. Synthesis of Isaindigotone Derivatives. The facile
synthetic pathway for isaindigotone derivatives is shown in
Scheme 1. Compound 2a was prepared by the condensation of
2-amino-4-nitrobenzoic acid (1a) and pyrrolidin-2-one in the
presence of POCl3. Treatment of 2a with 4-nitrobenzaldehyde
yielded the dinitro intermediate product by Claisen-Schmidt
condensation.23 Without further purification, the product was
reduced to the corresponding diamino compound 3a. Compound
3a was assigned as the E configuration on the basis of
NOEa analysis. In the NOE experiment, upon irradiation of
proton 7′-H of 3a, only enhancement of the equivalent protons
2′-H and 6′-H was observed. Besides, upon irradiation of 2′-H
and 6′-H, signals of protons 7′-H and 2-H were simultaneously
enhanced (Supporting Information). Further acylation of 3a with
3-chloropropionyl chloride gave compound 4a. The target
compounds 5a and 6a were then prepared by substitution of 4a
with appropriate secondary amines. Synthesis of position
isomers 5b and 6b was accomplished following the same
procedures from 2-amino-5-nitrobenzoic acid (1b).
The free-base form of compounds 5a,b and 6a,b were
converted into the hydrochloride salts by treatment with
hydrochloride acid in ethanol to enhance the chemical stability
and aqueous solubility for the following assays.
2.2. FRET and Fluorescence Titration Studies. To evaluate
the stabilization and selectivity of isaindigotone derivatives for
telomeric G-quadruplex DNA, FRET melting experiments were
carried out and the quindoline derivative SYUIQ-5 previously
reported by us was used as a reference compound.24-27
Table 1 shows the effect of derivatives on the enhanced
melting temperature (∆Tm) of two labeled oligonucleotides in
K+ solution, and Figure 2A gives the concentration dependent
melting curves for compounds 5a and 5b. F21T (5′-FAM-
d(GGG[TTAGGG]3)-TAMRA-3′) represents the human telom-
eric DNA sequence, while F10T (5′-FAM-dTATAGCTATA-
HEG-TATAGCTATA-TAMRA-3′) is a hairpin duplex DNA.
The FRET-melting data demonstrate that all of the derivatives
effectively stabilize the telomeric G-quadruplex. At 1 µM of
ligands, the ∆Tm values are in the range of 17.6-22.0 °C, which
is much higher than that of the reference compound SYUIQ-5.
The 4′,6-disubstituted isomers 5a and 6a were shown to be better
quadruplex stabilizers as compared with the 4′,7-disubstituted
isomers (5b and 6b). On the other hand, no significant effect
a Abbreviations: FRET, fluorescence resonance energy transfer; CD,
circular dichroism; MD, molecular dynamics; TRAP, telomere repeat
amplification protocol; SA-ꢀ-Gal, senescence-associated ꢀ-galactosidase;
TRF, telomeric restriction fragment; NOE, nuclear overhauser effect; EMSA,
electrophoretic mobility shift assays; IC50, 50% inhibitory concentration;
Tm, the temperature of midtransition; rmsd, root-mean-square deviation.

Isaindigotone DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2827
Figure 2. (A) Concentration dependent melting curves (∆Tm vs ligand concentration) for ligands 5a and 5b upon binding to F21T and F10T. (B)
Competitive FRET results for ligands 5a,b (0.5, 1, and 2 µM), 6a,b, and SYUIQ-5 (2 µM), without (black) and with 15-fold (3 µM; dark-gray) or
50-fold (10 µM; light-gray) excess of duplex DNA competitor (ds26). The concentration of F21T was 0.2 µM.
Figure 3. CD titration spectra of HTG21 (5 µM) at increasing concentrations (arrows: 0-5 mol equiv) of 5a and 5b in 10 mM Tris-HCl buffer,
pH 7.2, without (A,B) and with (C,D) 100 mM KCl. Changes in ellipticity at characteristic wavelengths (288 and 368 nm for A and B, 366 nm for
C and D) as functions of ligand/HTG21 molar ratio were plotted in the inner panels.
on Tm values was observed if a pyrrolidinyl group was placed
at the terminal of side chains instead of a piperidinyl group.
As shown in Table 1, 5a,b and 6a,b exhibit strong preference
for binding to telomeric G-quadruplex than duplex DNA. None
of these compounds were observed to significantly increase the
melting temperature of F10T, suggesting their poor binding to
the duplex DNA.18,19,28 Furthermore, the G-quadruplex selectiv-
ity of 5a,b and 6a,b was assessed by a FRET-based competition
assay where the ability of ligand to retain G-quadruplex
stabilizing affinity was challenged by nonfluorescent duplex
DNA (ds26).19,29 In the presence of various amounts of
competitor ds26, the thermal stabilization of F21T enhanced
by 5a,b and 6a,b was slightly affected (Figure 2A,B), while
the competitor sharply disrupted the binding of SYUIQ-5 to
the G-quadruplex. The combined results of these assays
demonstrate that isaindigotone derivatives can be considered
as a new class of highly selective G-quadruplex binding ligands.
6a showed stronger quadruplex affinity and their binding
constants are around 1.0 × 107 M-1
.
2.3. CD Studies. Circular dichroism (CD) spectroscopy was
used to investigate the binding property of the isaindigotone
derivatives to telomeric G-quadruplex.31
In the absence of salt, the CD spectrum of randomized
HTG21 oligonucleotide was found to have a negative band
centered at 238 nm, a major positive band at 257 nm, a minor
negative band at 280 nm, and a positive band at near 295 nm
(Figure 3A,B, black line).32 Upon titration with 5a, dramatic
changes in the CD spectra were observed (Figure 3A). The
bands at 238 and 257 nm gradually disappeared with addition
of compound and eventually led to the appearance of a positive
band at 240 nm and a major negative band at 258 nm, while
the band centered at 292 nm significantly increased and shifted
toward 286 nm. These changes are consistent with the induction
of the guanine-rich DNA to form the G-quadruplex structure
by compound 5a.32,33 Meanwhile, a strong and negative induced
CD signal was observed between 310 and 410 nm. The induced
CD signal was additional evidence for the interaction between
the G-quadruplex and compound 5a because its appearance in
the CD spectra is indicative of an achiral ligand binding tightly
to a chiral host.34 Also, the stoichiometry of the binding of
Equilibrium binding constants (Kb) were measured by fluo-
rescence titration assay.30 Oligonucleotide d[G3(T2AG3)3]
(HTG21) was used as the telomeric sequence. As shown in
Table 1, all of isaindigotone derivatives exhibit high affinity
for the preformed G-quadruplex DNA in K+ solution, and in
parallel with FRET results, 4′,6-disubstituted isomers 5a and

2828 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Tan et al.
isaindigotone derivative to G-quadruplex could be determined
from CD spectra.28,32,35,36 The changes in ellipticity at 288 and
368 nm as a function of 5a/HTG21 were plotted on an inner
panel, and the inflection point observed in each curve was
indicative of the reliable formation of a 4:1 5a-oligonucleotide
complex.
The CD spectra of compound 5b titrated into the HTG21
oligonucleotide in the absence of salt were similar to that of 5a
in the wavelength region below 300 nm (Figure 3B). In the
region of 300-450 nm, a strong and biphasic induced CD signal
of 5b with a triple negative peak centered at 387 nm and a minor
positive band at near 340 nm was observed. Besides, 5b exhibits
a 3:1 stoichiometry binding to the HTG21. These results
indicated that compound 5b could also induce the guanine-rich
DNA to form the G-quadruplex structure. In addition, ligand-
induced formation of telomeric G-quadruplex in the absence
of salt was further proved by EMSA and thermodynamic
stability experiments (Supporting Information).
In the presence of K+, HTG21 oligonucleotide forms the
hybrid-type G-quadruplex structure.37-39 The CD spectrum of
such preformed structure shows a strong positive band at 290
nm, a minor positive band at 265 nm, and a negative band at
235 nm (Figure 3C,D, black line).40 Upon titration of 5a into
this DNA solution, the CD spectra changed with enhancement
of the maximum at 290 nm and suppression of the band at 265
nm. A strong negative induced CD signal also emerged between
310 and 410 nm. The negative band at 366 nm gradually
increased until the ratio of 5a to HTG21 reached 4:1 (Figure
3C). Compound 5b induced similar CD changes and also
exhibited a 4:1 stoichiometry under the same experimental
conditions (Figure 3D).
Figure 4. Job plots resulting from the method of continuous variation
analysis for 5a and 5b with telomeric G-quadruplex DNA.
MD simulations, and evaluation of binding affinity was per-
formed. The parallel propeller-type X-ray G-quadruplex struc-
ture (PDB 1KF1)42 and a mixed hybrid-type NMR G-quadruplex
structure (PDB 2HY9)43 were used as the templates for the
modeling studies because they might be the more biologically
relevant forms in the presence of K+ ion.44
Molecular docking studies were first carried out to predict
the plausible interactions between the ligands and G-quadruplex
DNA. It has been previously shown that G-quadruplex binders
can stack on the surface of both terminal G-quartet planes.45 In
this study, besides such end-stacking interactions, docking results
also showed that such flexible ligands could be readily accom-
modated in the groove regions of both propeller-type and hybrid-
type G-quadruplex DNA (Supporting Information).
On the basis of the docking results, MD simulations (8 ns)
were performed on eight complexes formed by G-quadruplexes
(propeller-type and hybrid-type) with ligands (5a and 5b) in
two binding modes (end-stacking and groove-binding). All of
the models were quite stable during the dynamics runs (rmsd
values, see Supporting Information). The complexes formed by
G-quadruplex with 5a are shown in Figure 5, and the estimated
free energies of binding of 5a and 5b in MM-PBSA calculations
for each model are shown in Table 2. It was found the ligand
5a exhibited superior binding energies than that of 5b in most
of the models and that was in agreement with the change trend
of FRET and fluorescence titration data. It was also noteworthy
that both of the ligands possessed much more favorable groove
binding interactions (lower binding free energy) with hybrid-
type G-quadruplex than propeller-type G-quadruplex, and
whatever binding of the ligand to the hybrid-type G-quadruplex
showed much more comparable binding free energy.
In view of the binding free energies and the obtained CD
and continuous variation analysis results in K+ solution, hybrid-
type G-quadruplex was chosen as the template for further studies
of the multiple ligand-quadruplex interactions with 4:1 stoi-
chiometry. Both models were quite stable during the dynamics
runs (rmsd values, see Supporting Information). The complex
of hybrid-type G-quadruplex with 5a (1:4) was shown in Figure
5E,F. In the complex, two molecules stacked on both 5′ and 3′
G-quartet planes and the other two were deeply bound in two
grooves of the G-quadruplex. The binding fashion of central
pharmacophore, tertiary amine side chains, and the amide groups
was similar with that in 1:1 ligand-quadruplex interactions.
Meanwhile, the average binding free energies of four 5a
molecules exhibited much superior binding energies than that
of 5b (Table 2). The finding was additional evidence for the
parallel correlations between FRET, fluorescence titration data,
and calculated binding free energy. We recognize exact binding
modes could only be identified by NMR or crystal studies.
Results from all CD experiments showed that the strong
induced CD signal and the high stoichiometry ratio appeared
to be characteristic of the interaction of isaindigotone derivatives
with G-quadruplex structures. Actually, the CD signal of DNA
is usually localized below 300 nm, while induced CD signal
above 300 nm corresponds to the absorbance of the bound
ligand. Thus, the induced CD signal can be used in the
investigation of binding stoichiometry and binding mode
between the ligand and G-quadruplex.18 It is interesting to note
that several quadruplex ligands (e.g., telomestatin, macrocyclic
oligoamide, and SYUIQ-5) have been reported to bind to
telomeric G-quadruplexes in the end-stacking mode with a 2:1
ligand-oligonucleotide stoichiometry.28,32,36 The higher stoichi-
ometry determined here might suggest more complicated and
multiple binding modes for isaindigotone derivatives. Besides
the familiar end-stacking mode, groove binding and loop binding
modes of the ligands are also possible. In addition, the presence
of large induced CD signal in the titration spectra has been
treated as evident of groove binding mode for the ligands,18,34
which may further support our hypothesis that isaindigotone
derivative binds to the quadruplex via multiple binding modes.
2.4. Continuous Variation Studies. To further validate the
meaningful binding stoichiometries obtained in CD studies,
continuous variation analysis (Job plot) was performed (Figure
4).41 The point of intersection of the two best fit lines for the
Job plot for 5a with telomeric G-quadruplex (preformed HTG21
in K+ solution) is 0.799 and for 5b is 0.805. Both of the results
gave the same binding stoichiometry of 4 mol of ligand/mol of
telomeric G-quadruplex DNA, and that is consistent with the
CD studies.
2.5. Molecular Modeling Studies. To gain more details on
the interactions of isaindigotone derivatives with telomeric
G-quadruplexes, an approach that combined molecular docking,

Isaindigotone DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2829
Figure 5. Models of 5a-quadruplex complex. (A,B) 5a stacking on the surface of G-quartet or binding to the groove of propeller-type G-quadruplex
with 1:1 stoichiometry. (C,D) 5a stacking on the surface of G-quartet or binding to the groove of hybrid-type G-quadruplex with 1:1 stoichiometry.
(E,F) Top and side view of 5a binding to the hybrid-type G-quadruplex via multiple binding modes with 4:1 stoichiometry.
Table 2. Estimated Free Energy of Binding (∆G, in kcal ·mol-1) in MM-PBSA Calculationsa
∆G/kcal·mol-1
propeller-type G-quadruplex
hybrid-type G-quadruplex
groove-binding (1:1)
compd
end-stacking (1:1)
groove-binding (1:1)
end-stacking (1:1)
multiple-binding (4:1)
5a
5b
-42.23
-37.19
-31.73
-32.99
-48.41
-46.24
-46.16
-45.60
-43.76a
-40.19a
a Average free binding energy per molecule.
However, these MD results in combination with the data
obtained from CD and continuous variation analysis might
reinforce the hypothesis that isaindigotone derivatives bound
to telomeric G-quadruplex via multiple binding modes.
After an array of biophysical studies, it was necessary to
clarify the molecular basis for the high selectivity of isaindigo-
tone derivatives. Because of the geometrical freedom arising
from the rotatable bond, the central pharmacophore of these
ligands could adopt different conformations (i.e., coplanar and
noncoplanar) for G-quadruplex recognition. The high selectivity
of isaindigotone derivatives might first be attributed to their long
dimension of the putatively coplanar chromophore, which
matches the G-quartet better than that of the base pair of duplex
DNA (Supporting Information). On the other hand, the selectiv-
ity might also be a consequence of geometrical freedom and
bulkiness of the aliphatic five-member ring of these compounds,
which do not allow intercalative binding into duplex DNA to
take place.19 Such reliable end-stacking of compounds onto the

2830 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Tan et al.
Table 3. Telomerase Inhibition by Isaindigotone Derivatives in
Cell-Free Assay
difference was observed between the control and treated cells
(Figure 6A). Equally comparable results were also observed in
CA46 cells (Figure 6B).
Morphologic examination of the cells during long-term cell
studies displayed an increased proportion of enlarged and
flattened cells with phenotypic characteristics of senescence.50,51
These flattened cells also stained positively for the senescence-
associated ꢀ-galactosidase (SA-ꢀ-Gal) activity after continuous
5a treatment (Figure 7).14 As a result, compound 5a induced
accelerated senescence in both cancer cell lines.
5a
6a
5b
6b
TelIC50 (µM)
7.8
9.3
9.0
15.2
G-quartet are in agreement with previous unfused aromatic
quadruplex ligands.46,47 Second, besides the end-stacking bind-
ing mode, isaindigotone derivatives might bind to the grooves
of G-quadruplex DNA. Because the grooves of quadruplex DNA
have different groove geometries from duplex DNA as well as
different patterns of donor-acceptor sites, it has been reported
that compounds bound to the quadruplex grooves should be able
to achieve excellent structure-specific recognition affinity and
specificity.18,34 Thanks to the rotatable bond, the unfused
aromatic chromophore in the quadruplex groove can be twisted
to form the suitable noncoplanar conformation for well fitting
groove geometry and maximizing H-bond intermolecular in-
teractions. Such specific ligand-quadruplex interactions might
also contribute to the high selectivity of isaindigotone deriva-
tives. Meanwhile, different positional attachment of the side
chains did not influence selectivity and the multiple binding
modes of the compounds, and that was additional evidence for
the key role of their scaffold. Thus, the synergetic effect of
multiple binding modes arising from isaindigotone derivatives
might be the major reason for enhancing their G-quadruplex
selectivity over duplex DNA.
In addition, modeling studies with duplex DNA were also
performed to reinforce these arguments concerning the selectiv-
ity of ligands (Supporting Information).47 Assessments of the
binding free energies of interaction models demonstrated that
binding to the G-quadruplex was significantly favored relative
to duplex binding. This is consistent with the proposed G-
quadruplex/duplex DNA selectivity demonstrated by these
ligands in biophysical studies. These results with potential
quadruplex multiple-binding compounds provide a new choice
for design of highly selective G-quadruplex ligands.
2.6. Telomerase Inhibition. The ability of isaindigotone
derivatives to inhibit human telomerase activity were evaluated
by a modified TRAP assay, the TRAP-LIG assay,48 because
the original TRAP assay was reported to suffer from interference
by ligands associated with the PCR step.49 In the experiments,
solutions of derivatives were added to the telomerase reaction
mixture containing extract from cracked MCF-7 breast carci-
noma cell lines, and the inhibitory concentrations by half (TelIC50)
values of these compounds are listed in Table 3. It was found
that all of the compounds were effective inhibitors of human
telomerase (TelIC50 values ranging from 7.8 to 15.2 µM). In
parallel with above biophysical studies, compound 5a exhibited
the best inhibitory effect to the enzyme.
2.7. Senescence Induction. To examine the effects of
representative compound 5a on leukemia cell HL60 and
lymphoma cell CA46, short-term cell viability was first deter-
mined in a 2-day cytotoxic assay (MTT assay). The results show
that 5a has a potent inhibitory effect, with an IC50 value of 33
µM in HL60 cells and 29 µM in CA46 cells.
2.8. Telomere Shortening. Treatment of cancer cells with
telomeric G-quadruplex ligands have been previously reported
to disrupt telomere length maintenance and caused telo-
meres to erode.52-54 To investigate whether representative
isaindigotone derivative 5a could cause telomeres to shorten,
the telomere length was evaluated using the telomeric restriction
fragment (TRF) length assay (Figure 8) on leukemia cell HL60
and lymphoma cell CA46. The results showed that 1.8 µM of
5a triggered telomere shortening about 1.1 kb against HL60
cells, and telomere shortening was also observed after 0.9 µM
of 5a treatment. Similar reduction in telomere length was
observed in CA46 cells: 1.8 µM of 5a caused telomere
shortening of about 2.3 kb against CA46 cells.
For a dysfunctional telomere that could activate p53 to initiate
cellular senescence or apoptosis to suppress tumorigenesis, the
induction of senescence by 5a might result from this shortening
of telomere length,36 and this is consistent with the behavior
expected for efficient telomeric G-quadruplex ligand and
telomerase inhibitor.54,55 It was reported that the cellular changes
expected following exposure to telomere-targeting compounds
were varied and complex, reflecting the interactions of telomeric
proteins, DNA, and RNA involved in telomere maintenance.
Thus, the cellular effects of 5a could not be simply explained
by telomeric G-quadruplex interactions or telomerase inhibition.
Other possible telomere-independent genomic targets (promoter
G-quadruplex) could be involved in drug action. Nevertheless,
these compounds represented a novel group of selective
G-quadruplex ligands that could be considered as useful leads
for anticancer approach.
3. Conclusion
The design of drugs targeting at the telomeric G-quadruplex
DNA is a rational and promising approach. On this basis, several
unfused aromatic compounds have been designed, synthesized,
and evaluated as effective and selective telomeric G-quadruplex
ligands. The present study extended these observations to a new
group of isaindigotone derivatives with an asymmetric drug-
like chromophore. Our results show that isaindigotone deriva-
tives are a promising class of telomeric G-quadruplex binding
ligands with strong discrimination against the duplex DNA.
Their cellular effects are also consistent with the behavior
expected for efficient telomeric G-quadruplex ligand and
telomerase inhibitor. Encouraged by the highly quadruplex
selectivity, telomerase inhibition, and cellular effects of these
ligands, more detailed investigations on the chemical biology
and pharmacology of these compounds are now underway.
To evaluate the long-term effects of 5a on these cancer cells,
subcytotoxic concentrations (0.9, 1.8, and 4.5 µM) of 5a were
employed to avoid acute cytotoxicity and other nonspecific
events that could lead to difficulty in result interpretation. Upon
treatment of HL60 cells with 4.5 µM 5a, a significantly
inhibitory effect was found after 8 days and the growth was
halted after 12 days. At 1.8 µM, a delayed effect was also
observed, with the number of population doublings significantly
decreased after 12 days, and even with 0.9 µM 5a, a discernible
4. Experimental Section
Synthesis and Characterization. All chemicals were obtained
from commercial sources unless otherwise specified. Melting points
were determined using an X-6 microscope melting point instrument
and are uncorrected. 1H NMR spectra and NOESY experiment were
performed on a Varian Mercury-Plus 300 NMR and INOVA 500NB
spectrometry, respectively, with tetramethylsilane (TMS) as an

Isaindigotone DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2831
Figure 6. Long-term exposure of HL60 (A) and CA46 cells (B) with 5a at subcytotoxic concentrations. Cells were exposed to indicated concentrations
of 5a or 0.1% DMSO, respectively. Every 4 days, the cells in control and drug-exposed flasks were counted and flasks reseeded with cells. Each
experiment was performed three times at each point. This experiment was a representative of three experiments.
Figure 7. Expression of SA-ꢀ-Gal in HL60 and CA46 cells after continuous treatment with 5a. HL60 cells were treated with 0.9 µM 5a (A) or
0.1% DMSO (B) continuously for 16 days. CA46 cells were also treated with 0.9 µM 5a (C) or 0.1% DMSO (D) continuously for 16 days. Then
cells were fixed, stained with SA-ꢀ-Gal staining kit, and photographed (×400). The experiment was repeated twice.
hydroxide was added to make the solution basic. The precipitate
was separated from water by filtration, and the filtrate was extracted
with 3 × 100 mL portions of ethyl acetate. The combined organic
phase was washed with 100 mL of water containing 10 mL of
concentrated ammonium hydroxide, dried over magnesium sulfate,
and concentrated to dryness. The resulting residue combined with
previous precipitate was purified by flash chromatography with
cyclohexane/ethyl acetate (20:80) elution to afford a pale-yellow
solid 2a (0.69 g, 60%); mp 195-196 °C (lit.56 mp 198-199 °C).
1H NMR (300 MHz, CDCl3) δ (ppm): 8.45 (d, J ) 2.1 Hz, 1H),
8.40 (d, J ) 8.7 Hz, 1H), 8.17 (dd, J ) 8.7, 2.1 Hz, 1H), 4.24 (t,
J ) 7.2 Hz, 2H), 3.23 (t, J ) 7.8 Hz, 2H), 2.30-2.40 (m, 2H).
ESI-MS: m/z 232 [M + H]+.
(E)-6-Amino-3-(4-aminobenzylidene)-2,3-dihydropyrrolo[2,1-
b]quinazolin-9(1H)-one (3a). A mixture of 2a (1.16 g, 5 mmol),
4-nitrobenzaldehyde (6.04 g, 40 mmol), and acetic anhydride (50
mL) was heated at reflux temperature for 28 h. After cooling, the
precipitate was separated from solvent by filtration and rinsed with
chloroform (100 mL) and acetone (200 mL) to afford a dinitro
intermediate. To a stirred suspension of dinitro intermediate (1.64
g) in ethanol (50 mL) was added a solution of Na2S·9H2O (4.8 g,
20 mmol) and NaOH (2 g, 50 mmol) in water (80 mL). The mixture
was heated at reflux for 6 h and left to stand overnight. The ethanol
was removed in vacuo and the residue cooled to 0-5 °C. The
resulting precipitate was collected by filtration, repeatedly washed
with water, and dried. Recrystallization from ethanol/acetone
afforded the product 3a (0.91 g, 60%) as an orange-red solid; mp
Figure 8. Effect of isaindigotone derivative 5a on telomere length.
TRF of cancer cells treated or untreated with 5a was analyzed using
the Telo TAGGG telomere length assay. (A) TRF analysis of HL60
cells treated or untreated with 5a for 16 days. Lane 1, 0.1% DMSO;
lane 2, 0.9 µM 5a; lane 3, 1.8 µM 5a. (B) TRF analysis of CA46 cells
treated or untreated with 5a for 16 days. Lane 1, 0.1% DMSO; lane 2,
0.9 µM 5a; lane 3, 1.8 µM 5a.
internal standard. ESI-MS spectra were obtained using a Thermo
LCQ DECA XP LC-MS spectrometry. Elemental analysis was
carried out on an Elementar Vario EL CHNS elemental analyzer,
and purity of all target compounds used in the biophysical and
biological studies was g95%. All compounds were routinely
checked by TLC with Merck Silica Gel 60F-254 glass plates.
6-Nitro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (2a).
To a stirred suspension of pyrrolidin-2-one (0.43 g, 5 mmol) and
2-amino-4-nitrobenzoic acid (1.82 g, 10 mmol) in 250 mL
anhydrous toluene, POCl3 (2 mL) was added dropwise at room
temperature and the mixture was refluxed for 8 h. The reaction
product was poured onto ice, and then concentrated ammonium
1
>280 °C. H NMR (500 MHz, DMSO-d6) δ (ppm): 7.75 (d, J )
8.5 Hz, 1H), 7.48 (br s, 1H), 7.34 (d, J ) 8.5 Hz, 2H), 6.62-6.66
(m, 4H), 5.95 (br s, 2H), 5.64 (br s, 2H), 4.05 (t, J ) 7.5 Hz, 2H),
3.11-3.14 (m, 2H). ESI-MS: m/z 305 [M + H]+.
(E)-3-Chloro-N-(4-((6-(3-chloropropanamido)-9-oxo-1,2-dihy-
dropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)phenyl)pro-
panamide (4a). A suspension of 3a (1.52 g, 5 mmol) in 3-chlo-
ropropanoyl chloride (10 mL) was heated at reflux for 4 h, until

2832 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Tan et al.
TLC indicated completion of reaction. After cooling to 0-5 °C,
the mixture was filtered and the crude solid washed with three 15
mL portions of ether. Recrystallization from DMF-EtOH (4:1 v/v)
afforded 4a (1.82 g, 75%) as a yellow solid; mp >280 °C. 1H NMR
(300 MHz, DMSO-d6) δ (ppm): 10.55 (br s, 1H), 10.34 (br s, 1H),
8.14 (d, J ) 2.1 Hz, 1H), 8.05 (d, J ) 8.7 Hz, 1H), 7.73 (d, J )
8.7 Hz, 2H), 7.70 (br s, 1H), 7.63 (d, J ) 8.7 Hz, 2H), 7.53 (dd,
J ) 8.7, 2.1 Hz, 1H), 4.16 (t, J ) 6.6 Hz, 2H), 3.87-3.94 (m,
4H), 3.23-3.27 (m, 2H), 2.84-2.93 (m, 4H). ESI-MS: m/z 485
[M - H]-.
(E)-N-(9-oxo-3-(4-(3-(Pyrrolidin-1-yl)propanamido)ben-
zylidene)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-6-yl)-3-
(pyrrolidin-1-yl)propanamide (5a). To a stirred refluxing sus-
pension of 4a (0.25 g, 0.5 mmol) and KI (0.05 g) in EtOH (10
mL) was added dropwise pyrrolidine (0.35 mL, 5 mmol) in EtOH
(1.6 mL). The mixture was stirred at reflux for 3 h, cooled to 0 °C,
filtered, and washed with ether (10 mL). Recrystallization from
CHCl3-EtOH (2:1 v/v) gave 5a (0.19 g, 70%) as a pale-yellow solid;
mp 183-185 °C. 1H NMR (300 MHz, CDCl3) δ (ppm): 11.64 (br
s, 1H), 11.45 (br s, 1H), 8.22 (d, J ) 8.7 Hz, 1H), 7.80 (t, J ) 2.4
Hz, 1H), 7.78 (d, J ) 2.1 Hz, 1H), 7.68 (dd, J ) 8.7, 2.1 Hz, 1H),
7.59 (d, J ) 8.7 Hz, 2H), 7.52 (d, J ) 8.7 Hz, 2H), 4.29 (t, J ) 6.9
Hz, 2H), 3.27-3.32 (m, 2H), 2.90-2.94 (m, 4H), 2.64-2.80 (m,
8H), 2.59-2.64 (m, 4H), 1.95-1.98 (m, 8H). ESI-MS: m/z 555
[M + H]+. Trihydrochloride salt, anal. (C32H38N6O3 ·3HCl·3.5H2O)
C, H, N.
(E)-N-(9-oxo-3-(4-(3-(Piperidin-1-yl)propanamido)benzylidene)-
1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-6-yl)-3-(piperidin-1-
yl)propanamide (6a). Following the method for production of 5a,
a yellow solid 6a (0.20 g, 68%) was obtained; mp 179-182 °C.
1H NMR (300 MHz, CDCl3) δ (ppm): 11.70 (br s, 1H), 11.58 (br
s, 1H), 8.20 (d, J ) 8.7 Hz, 1H), 7.86 (d, J ) 1.8 Hz, 1H), 7.79 (br
s, 1H), 7.64 (dd, J ) 8.7, 1.8 Hz, 1H), 7.62 (d, J ) 8.7 Hz, 2H),
7.51 (d, J ) 8.4 Hz, 2H), 4.26 (t, J ) 7.2 Hz, 2H), 3.25-3.30 (m,
2H), 2.67-2.71 (m, 4H), 2.52-2.58 (m, 12H), 1.71-1.76 (m, 8H),
1.50-1.63 (m, 4H). ESI-MS: m/z 583 [M + H]+. Trihydrochloride
salt, anal. (C34H42N6O3 ·3HCl·5H2O) C, H, N.
MS: m/z 555 [M + H]+. Trihydrochloride salt, anal. (C32H38N6O3 ·
3HCl·1.5H2O) C, H, N.
(E)-N-(9-oxo-3-(4-(3-(Piperidin-1-yl)propanamido)benzylidene)-
1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-(piperidin-1-
yl)propanamide (6b). A yellow solid 6b (0.19 g, 65%); mp
1
240-241 °C. H NMR (300 MHz, CDCl3) δ (ppm): 11.69 (br s,
1H), 11.59 (br s, 1H), 8.26 (dd, J ) 9, 2.4 Hz, 1H), 8.13 (d, J )
2.4 Hz, 1H), 7.77 (br s, 1H), 7.72 (d, J ) 9 Hz 1H), 7.64 (d, J )
8.7 Hz, 2H), 7.53 (d, J ) 8.7 Hz, 2H), 4.30 (t, J ) 7.2 Hz, 2H),
3.28-3.33 (m, 2H), 2.69-2.74 (m, 4H), 2.54-2.60 (m, 12H),
1.74-1.79 (m, 8H), 1.60-1.61 (m, 4H). ESI-MS: m/z 583 [M +
H]+. Trihydrochloride salt, anal. (C34H42N6O3 ·3HCl·4H2O) C, H,
N.
Materials. All oligomers/primers used in this study were
purchased from Invitrogen (China). Acrylamide/bisacrylamide
solution and N,N,N′,N′-tetramethyl-ethylenediamine were purchased
from Sigma. Taq DNA polymerase was purchased from Sangon
(China). Stock solutions of all the isaindigotone derivatives (2 mM)
were made using double-distilled deionized water containing 50%
DMSO and stored at -80 °C. Further dilutions to working
concentrations were made with relevant buffer immediately prior
to use. All tumor cell lines were obtained from the American Type
Culture Collection (ATCC, Rockville, MD). The cell culture was
maintained in a RPMI-1640 medium supplemented with 10% fetal
bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin
in 25 cm2 culture flasks at 37 °C humidified atmosphere with 5%
CO2.
FRET Assay. FRET assay was performed as a high-throughput
screen following previously published procedures.29,36 The labeled
oligonucleotides F21T:5′-FAM-d(GGG[TTAGGG]3)-TAMRA-3′ and
F10T:5′-FAM-dTATAGCTATA-HEG-TATAGCTATA-TAMRA-
3′ (donor fluorophore FAM is 6-carboxyfluorescein; acceptor
fluorophore TAMRA is 6-carboxytetramethylrhodamine; HEG linker
is [(-CH2-CH2-O-)6]) were used as the FRET probes. Fluores-
cence melting curves were determined with a Roche LightCycler
2 real-time PCR machine, using a total reaction volume of 20 µL,
with 0.2 µM of labeled oligonucleotide in Tris-HCl buffer (10 mM,
pH 7.2) containing 60 mM KCl. Fluorescence readings with
excitation at 470 nm and detection at 530 nm were taken at intervals
of 1 °C over the range 37-99 °C, with a constant temperature being
maintained for 30 s prior to each reading to ensure a stable value.
The melting of the G-quadruplex was monitored alone or in the
presence of various concentrations of compounds and/or of double-
stranded competitor ds26 (5′-CAATCGGATCGAATTCGATC-
CGATTG-3′). Final analysis of the data was carried out using Origin
7.5 (OriginLab Corp.).
Using the 2-amino-5-nitrobenzoic acid as the starting material
and following the method for production of 2a-6a, compounds
2b-6b were synthesized.
7-Nitro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one (2b).
A pale-yellow solid 2b (0.75 g, 65%); mp 191-192 °C (lit.57 mp
187-188 °C). 1H NMR (300 MHz, CDCl3) δ (ppm): 9.11 (d, J )
2.7 Hz, 1H), 8.48 (dd, J ) 8.7, 2.7 Hz, 1H), 7.72 (d, J ) 8.7 Hz,
1H), 4.24 (t, J ) 7.2 Hz, 2H), 3.23 (t, J ) 7.8 Hz, 2H), 2.29-2.40
(m, 2H). ESI-MS: m/z 232 [M + H]+.
CD Measurements. CD experiments were performed on a
Chirascan circular dichroism spectrophotometer (Applied Photo-
physics). A quartz cuvette with 4 mm path length was used for the
spectra recorded over a wavelength range of 230-450 at 1 nm
bandwidth, 1 nm step size, and 0.5 s time per point. The oligomer
HTG21 (5′-d(GGG[TTAGGG]3)-3′) was diluted from stock to the
correct concentration (5 µM) in Tris-HCl buffer (10 mM, pH 7.2)
with or without 100 mM KCl and then annealed by heating to 90
°C for 5 min, gradually cooled to room temperature, and incubated
at 4 °C overnight. Then, CD titration was performed at a fixed
HTG21 concentration (5 µM) with various concentrations (0-5
mol equiv) of the ligands in buffer with or without 100 mM KCl
at 25 °C. After each addition of ligand, the reaction was stirred
and allowed to equilibrate for at least 13 min (until no elliptic
changes were observed) and a CD spectrum was collected. A buffer
baseline was collected in the same cuvette and subtracted from the
sample spectra. Final analysis of the data was carried out using
Origin 7.5 (OriginLab Corp.).
(E)-7-Amino-3-(4-aminobenzylidene)-2,3-dihydropyrrolo[2,1-
b]quinazolin-9(1H)-one (3b). An orange-red solid 3b (0.94 g,
62%); mp >280 °C. 1H NMR (500 MHz, DMSO-d6) δ (ppm): 7.42
(br s, 1H), 7.40 (d, J ) 8.5 Hz, 1H), 7.31 (d, J ) 8.5 Hz, 2H), 7.22
(d, J ) 2.5 Hz, 1H), 7.07 (dd, J ) 8.5, 2.5 Hz, 1H), 6.64 (d, J )
8.5 Hz, 2H), 5.56 (br s, 4H), 4.11 (t, J ) 7.5 Hz, 2H), 3.13-3.16
(m, 2H). ESI-MS: m/z 305 [M + H]+.
(E)-3-Chloro-N-(4-((7-(3-chloropropanamido)-9-oxo-1,2-dihy-
dropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)phenyl)pro-
panamide (4b). A yellow solid 4b (1.60 g, 66%); mp >280 °C.
1H NMR (300 MHz, DMSO-d6) δ (ppm): 10.49 (br s, 1H), 10.36
(br s, 1H), 8.50 (d, J ) 2.4 Hz, 1H), 7.96 (dd, J ) 8.7, 2.4 Hz 1H),
7.72-7.76 (m, 4H), 7.61 (d, J ) 9 Hz, 2H), 4.20 (t, J ) 7.1 Hz,
2H), 3.87-3.93 (m, 4H), 3.24-3.29 (m, 2H), 2.84-2.90 (m, 4H).
ESI-MS: m/z 519 [M + Cl]-.
(E)-N-(9-oxo-3-(4-(3-(pyrrolidin-1-yl)propanamido)ben-
zylidene)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-7-yl)-3-
(pyrrolidin-1-yl)propanamide (5b). A yellow solid 5b (0.18 g,
Fluorescence Titration. Fluorescence titrations were performed
on a Perkin-Elmer LS-55 luminescence spectrophotometer following
previously published procedures at 25 °C.58 A quartz cuvette with
1 cm × 1 cm path length was used for the spectra recorded at 5
nm excitation and emission slit widths. Small aliquots of a stock
solution of HTG21 (preformed G-quadruplex) were added into the
solution containing ligand at fixed concentration (2 µM) in Tris-
1
66%); mp 247-248 °C. H NMR (300 MHz, CDCl3) δ (ppm):
11.53 (br s, 1H), 11.49 (br s, 1H), 8.20 (dd, J ) 9, 2.4 Hz, 1H),
8.02 (d, J ) 2.4 Hz, 1H), 7.74 (t, J ) 2.7 Hz, 1H), 7.68 (d, J ) 9
Hz 1H), 7.55 (d, J ) 9 Hz, 2H), 7.49 (d, J ) 9 Hz, 2H), 4.27 (t,
J ) 6.9 Hz, 2H), 3.24-3.30 (m, 2H), 2.84-2.89 (m, 4H),
2.65-2.75 (m, 8H), 2.53-2.59 (m, 4H), 1.91-1.92 (m, 8H). ESI-

Isaindigotone DeriVatiVes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2833
HCl buffer (10 mM, pH 7.2) with 100 mM KCl. The final
concentration of DNA was varied from 0 to 2 µM. After each
addition of DNA, the reaction was stirred and allowed to equilibrate
for at least 10 min and fluorescence measurement was taken at Ex
343 nm for 5a and 6a or 353 nm for 5b and 6b. Fluorescence
emission (448 nm for 5a and 6a or 450 nm for 5b and 6b) data
points at each step were then obtained, and the equilibrium binding
constants were derived from the Scatchard analysis.
The hydrogen bonds were constrained using SHAKE.68 For the
nonbonded interactions, a residue-based cutoff of 10 Å was used.
Temperature regulation was achieved by Langevin coupling with
a collision frequency of 1.0. The solvated structures were subjected
to initial minimization to equilibrate the solvent and counter cations.
The G-quadruplex and inner K+ ions were initially fixed with force
constants of 100 kcal·mol-1. The system was then heated from 0
to 300 K in a 100 ps simulation and followed by a 100 ps simulation
to the equilibrate the density of the system. Afterward, constant
pressure MD simulation of 8 ns was then performed in an NPT
ensemble at 1 atm and 300 K. The output and trajectory files were
saved every 0.1 and 1 ps for the subsequent analysis, respectively.
All trajectory analysis was done with the Ptraj module in the Amber
10.0 suite and examined visually using the VMD software pack-
age.69
Continuous Variation Analysis. Continuous variation analysis
was performed on a Shimadzu UV-2450 spectrophotometer fol-
lowing previously published procedures.41 Stock solutions of 10
µM ligand and 10 µM HTG21 were prepared in Tris-HCl buffer
(10 mM, pH 7.2) containing 100 mM KCl. Two series of solutions
were used for the experiments: one with varying mole fractions of
ligand and HTG21, and another one with varying concentrations
of ligand. The sum of the ligand and HTG21 concentrations was
always 10 µM. Their absorbance spectra were collected from 300
to 500 nm using a 1 cm path length quartz cuvette at 25 °C.
Absorbance of ligand solution without oligonucleotide minus that
of corresponding ligand solution with oligonucleotide gave the
difference spectra. Such difference in the absorbance values at λmax
wavelength (343 nm for 5a and 353 nm for 5b) was plotted versus
the ligand mole fraction to generate a Job plot. Final analysis of
the data was carried out using Origin 7.5 (OriginLab Corp.).
Molecular Modeling. The crystal structure of the parallel
propeller-type 22-mer telomeric G-quadruplex (PDB 1KF1)42 and
the NMR structure of the mixed hybrid-type 26-mer telomeric
G-quadruplex (PDB 2HY9)43 were used as the initial templates to
study the interaction between the isaindigotone derivatives and
telomeric DNA. For comparison with the d(GGG[TTAGGG]3)
DNA we used in the FRET and CD experiments, we removed the
terminal 5′ adenine residue from the propeller-type structure and
five adenines from each end of the hybrid-type structure to generate
the 21-mer structures. Water molecules were removed from the
PDB file, whereas the missing hydrogen atoms were added to the
systemusingtheBiopolymermoduleimplementedintheSYBYL7.3.5
molecular modeling software from Tripos Inc. (St. Louis, MO).
Ligand structures were constructed and optimized with GAUSSIAN
0359 using the HF/6-31G* basis set.
Docking studies were performed using the AUTODOCK 4.0
program.60 Using ADT,61 nonpolar hydrogens of telomeric G-
quadruplex were merged to their corresponding carbons and partial
atomic charges were assigned. The nonpolar hydrogens of the
ligands were merged, and rotatable bonds were assigned. The
propeller-type or hybrid-type G-quadruplex structure was used as
an input for the AUTOGRID program. AUTOGRID performed a
precalculated atomic affinity grid maps for each atom type in the
ligand plus an electrostatics map and a separate desolvation map
present in the substrate molecule. The dimensions of the active site
box that was placed at the center of the G-quadruplex were set to
110 Å × 110 Å × 110 Å with a grid spacing of 0.375 Å. Docking
calculations were carried out using the Lamarckian genetic algo-
rithm (LGA). Initially, we used a population of random individuals
(population size: 150), a maximum number of 2500000 energy
evaluations, a maximum number of generations of 27000, and a
mutation rate of 0.02. Two hundred independent docking runs were
done for each ligand. The resulting positions were clustered
according to a root-mean-square criterion of 0.5 Å.
The MM/PBSA method70 implemented in the AMBER 10 suite
was used to calculate the binding free energy between the
G-quadruplex and the isaindigotone derivatives. All the waters and
counterions were stripped off but included the K+ present within
the negative charged central channel. The set of 1000 snapshots
from MD trajectories were collected to calculate the binding free
energies.
Molecular modeling studies of the interactions between isain-
digotone derivatives with duplex DNA were performed following
analogous procedures and parameters shown above. A self-
complementary duplex DNA was built in SYBYL from the
sequence d[(TA)2GC(TA)2] for these studies.47
TRAP-LIG Assay. The ability of isaindigotone derivatives to
inhibit telomerase in a cell-free system was assessed with the TRAP-
LIG assay following previously published procedures.48 Protein
extracts from exponentially growing MCF-7 breast carcinoma cells
were used. Briefly, 0.1 µg of TS forward primer (5′-AATCCGTC-
GAGCAGAGTT-3′) was elongated by telomerase (500 ng protein
extract) in TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl,
1.5 mM MgCl2, 1 mM EGTA, and 0.05% Tween 20) containing
125 µM dNTPs and 0.05 µg BSA. The mix was added to tubes
containing freshly prepared ligand at various concentrations and
to a negative control containing no ligand. The initial elongation
step was carried out for 20 min at 30 °C, followed by 94 °C for 5
min and a final maintenance of the mixture at 20 °C. To purify the
elongated product and to remove the bound ligands, the QIA quick
nucleotide purification kit (Qiagen) was used according to the
manufacturer’s instructions. The purified extended samples were
then subject to PCR amplification. For this, a second PCR master
mix was prepared consisting of 1 µM ACX reverse primer (5′-
GCGCGG[CTTACC]3CTAACC-3′), 0.1 µg TS forward primer (5′-
AATCCGTCGAGCAGAGTT-3′), TRAP buffer, 5 µg BSA, 0.5
mM dNTPs, and 2 units of Taq polymerase. A 10 µL aliquot of
the master mix was added to the purified telomerase extended
samples and amplified for 35 cycles of 94 °C for 30 s, of 61 °C for
1 min, and of 72 °C for 1 min. Samples were separated on a 16%
PAGE and visualized with silver-stained. TelIC50 values were then
calculated from the optical density quantitated from the AlphaE-
aseFC software.
Short-Term Cell Viability. HL60 leukemia cell line and CA46
lymphoma cell line were seeded on 96-well plates (1.0 × 103/well)
and exposed to various concentrations of ligand. After 48 h of
treatment at 37 °C in a humidified atmosphere of 5% CO2, 10 µL
of 5 mg/mL methyl thiazolyl tetrazolium (MTT) solution was added
to each well and further incubated for 4 h. The cells in each well
were then treated with dimethyl sulfoxide (DMSO) (200 µL for
each well), and the optical density (OD) was recorded at 570 nm.
All drug doses were parallel tested in triplicate, and the IC50 values
were derived from the mean OD values of the triplicate tests versus
drug concentration curves.
Molecular dynamics simulations were performed using the sander
module of the AMBER 10.0 program suite. The nucleic acids
studied were as treated using the parm99 parameters.62 Partial-atomic
charges for the ligand molecules were derived using the HF/6-31G*
basis set followed by RESP calculation, while force-field parameters
were taken from the generalized Amber force field (GAFF)63 using
ANTECHAMBER module. The K+ radius was kept at 2.025 Å.64,65
Periodic boundary conditions were applied with the particle-mesh
Ewald (PME) method66 used to treat long-range electrostatic
interactions. The quadruplex and ligand complexes were solvated
in a rectangular box of TIP3P67 water molecules with solvent layers
10 Å. And the potassium counterions were added to neutralize the
complexes.
Long-Term Cell Culture Experiments. Long-term proliferation
experiments were carried out using the HL60 leukemia cell line
and CA46 lymphoma cell line. Cells were grown in T80 tissue
culture flasks at 1.0 × 105 per flask and exposed to a subcytotoxic
concentration of ligand or an equivalent volume of 0.1% DMSO
every 4 days. The cells in control and drug-exposed flasks were

2834 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Tan et al.
counted and flasks reseeded with 1.0 × 105 cells. The remaining
cells were collected and used for measurements described below.
This process was continued for 16 days or stopped until the cell
number was not enough to reseed.
SA-ꢀ-Gal Assay. Cells treated with the ligand were incubated
for 16 days. After the incubation, the growth medium was aspirated
and the cells were fixed in 2% formaldehyde/0.2% glutaraldehyde
for 15 min at room temperature. The fixing solution was removed,
and the cells were gently washed twice with PBS and then stained
using the ꢀ-Gal stain solution containing 1 mg/mL of 5-bromo-4-
chloro-3-indolyl-ꢀ-D-galactoside, followed by incubation overnight
at 37 °C. The staining solution was removed, and the cells were
washed three times with PBS. The cells were viewed under an
optical microscope and photographed.
(12) Ou, T.-M.; Lu, Y.-J.; Tan, J.-H.; Huang, Z.-S.; Wong, K.-Y.; Gu, L.-
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Acknowledgment. We thank the National Nature Science
Foundation of China (20772159, 90813011, U0832005), the
NSFC/RGC joint Research Scheme (30731160006 and N_PolyU
508/06), the Science Foundation of Guangzhou (2006Z2-E402),
the NCET, the Shenzhen Key Laboratory Fund, and the
University Grants Committee Areas of Excellence Scheme in
Hong Kong (AoE P/10-01) for financial support of this study.
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Supporting Information Available: NOE analysis of 3a and
3b, elemental analysis used to determine the degree of purity for
compounds, UV analysis, FRET-melting studies, fluorescence
titration assay, EMSA and thermodynamic stability experiments,
molecular modeling studies, and telomerase inhibition results. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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