Disubstituted quinazoline derivatives as a new type of highly selective ligands for telomeric G-quadruplex DNA

Article abstract of DOI:10.1016/j.ejmech.2011.10.057

A series of 2,4-disubstituted quinazoline derivatives found to be a new type of highly selective ligand to bind with telomeric G-quadruplex DNA, and their biological properties were reported for the first time.Their interactions with telomeric G-quadruplex DNA were evaluated by using fluorescence resonance energy transfer (FRET) melting assay, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and molecular modeling. Our results showed that these derivatives could well recognize G-quadruplex and have high selectivity toward G-quadruplex over duplex DNA. The structure-activity relationships (SARs) study revealed that the disubstitution of quinazoline and the length of the amide side chain were important for its interaction with the G-quadruplex. Furthermore, telomerase inhibition of the quinazoline derivatives and their cellular effects were studied.

Full text of DOI:10.1016/j.ejmech.2011.10.057

European Journal of Medicinal Chemistry 47 (2012) 299e311
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Disubstituted quinazoline derivatives as a new type of highly selective ligands
for telomeric G-quadruplex DNA
*
*
Zeng Li, Jia-Heng Tan, Jin-Hui He, Yi Long, Tian-Miao Ou, Ding Li , Lian-Quan Gu, Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,4-disubstituted quinazoline derivatives found to be a new type of highly selective ligand to
bind with telomeric G-quadruplex DNA, and their biological properties were reported for the first
time.Their interactions with telomeric G-quadruplex DNA were evaluated by using fluorescence reso-
nance energy transfer (FRET) melting assay, circular dichroism (CD) spectroscopy, surface plasmon
resonance (SPR), nuclear magnetic resonance (NMR), and molecular modeling. Our results showed that
these derivatives could well recognize G-quadruplex and have high selectivity toward G-quadruplex over
duplex DNA. The structureeactivity relationships (SARs) study revealed that the disubstitution of qui-
nazoline and the length of the amide side chain were important for its interaction with the G-quad-
ruplex. Furthermore, telomerase inhibition of the quinazoline derivatives and their cellular effects were
studied.
Received 23 September 2011
Received in revised form
25 October 2011
Accepted 28 October 2011
Available online 7 November 2011
Keywords:
Quinazoline derivatives
G-quadruplex DNA
Interaction
Ó 2011 Elsevier Masson SAS. All rights reserved.
Selectivity
Telomerase inhibition
1. Introduction
activity and interfere with telomere biology, or alter G-quadruplex
related gene expression, or inhibit rRNA biogenesis in most cancer
Human nucleic acid sequences containing multiple close
segments of three or more consecutive guanosine residues can
form higher-order and functionally useful structures called the G-
quadruplexes [1e3]. They are a type of particular secondary
structure that originates in the assembly of four G-rich DNA strands
held together via the formation of a G-quartet [3,4]. Such motifs
have been identified in biologically relevant regions of the
eukaryotic genome, particularly enriched in telomeric regions [5,6],
insulin-linked polymorphic region [7], regulatory sequences of
muscle-specific genes [8], and upstream of transcription initiation
sites of proto-oncogenes [9e12]. This finding was the initial para-
digm to search for small molecules that have the ability to stabilize
G-quadruplex in order to control gene expression and reverse
tumor cell immortalization [13,14]. Many groups have reported
their results targeting G-quadruplex nucleic acids with small
molecules as antitumor agents, since they could inhibit telomerase
cells but not in normal cells [15e18]. Numerous G-quadruplex
binders have been reported, and most of these molecules comprise
a planar, aromatic core presumed to stack on the terminal tetrads of
G-quadruplex. Examples include polyaromatic hydrocarbons and
macrocyclic frameworks, which have exhibited a strong G-quad-
ruplex stability [19e26]. Besides these compounds, some flexible
ligands with unfused aromatic scaffold have aroused wide interest
recently because of their adaptive structural feature arising from
the rotatable bonds, which prevent the ligands from intercalating
into duplex DNA but can still allow the ligands to stack on the G-
quartet [14,27e33]. On the basis of above results, the introduction
of a moiety as a switch to control the transformation of the ligand
between the rigidity and flexibility is a new strategy in G-quad-
ruplex ligands design.
Previous studies have shown that the moiety of intramolecular
hydrogen bond is one of the most important chemical interactions
that form active structures/foldings of molecules and provide crit-
ical functions in medicinal chemistry [34e36]. The beneficial effect
of intramolecular hydrogen bond on ligand-receptor binding can be
rationalized with conformational restriction in which the small
molecular substituents are favorably aligned with the protein
pockets. The improved potency of caspase-1 inhibitors have been
reported through rigidifying the molecules with an intramolecular
hydrogen bond [37]. The increased binding affinity and
Abbreviations: FRET, fluorescence resonance energy transfer; CD, circular
dichroism; SPR, surface plasmon resonance; NMR, nuclear magnetic resonance;
TRAP, telomere repeat amplification protocol; MTT, methyl thiazolyl tetrazolium;
SA-b-Gal, senescence-associated-galactosidase; TRF, telomeric restriction fragment.
* Corresponding authors. Tel./fax: þ86 20 39943056.
E-mail addresses: liding@mail.sysu.edu.cn (D. Li), ceshzs@mail.sysu.edu.cn
(Z.-S. Huang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.057

300
Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
pharmacological activity have also been observed for aldose
reductase inhibitors by utilizing an intramolecular hydrogen bond
to position the key structural elements of the pharmacophore in
a certain conformation [38]. In addition, rational design of internal
hydrogen bonds for conformational preorganization has been
pursued in scaffold replacements for a diverse set of kinase inhib-
itors, such as 4-aminoquinazoline scytalone dehydratase inhibitors
[39], anthranilamide kinase inhibitors [40], pyrimidin-4-ylurea
kinase inhibitors [41], and alkoxybenzamide poly(ADP-ribose)
polymerase inhibitors [42].
ligands with telomeric G-quadruplex DNA were examined using
the fluorescence resonance energy transfer-melting (FRET-melting)
method, surface plasmon resonance (SPR), circular dichroism
spectroscopy (CD), and the ligandequadruplex interactions and
binding modes were investigated using NMR and molecular
modeling. In addition, their inhibitory effects on telomerase activity
were studied using TRAP-LIG, and their cellular effects were eval-
uated through the measurement of cell senescence and telomere
shortening.
On the other hand, isaindigotone derivatives have been devel-
oped in our lab as selective telomeric G-quadruplex binding ligands
[33]. Recent studies suggest that the decreased planarity of the
flexible ligands would result in their decreased binding affinity
and stabilization ability [43]. So a planar central core may be
essential for the binding of ligand with the G-quadruplex. And the
intramolecular hydrogen bond as one important moiety could
rigidify the molecules and maintain a planar structure. Based
on these discoveries, we designed and synthesized a series of
2,4-disubstituted quinazoline derivatives conformationally con-
strained with intramolecular hydrogen bonds as a new type of G-
quadruplex binding ligands (Scheme 1). The internally organized
hydrogen bond is positioned between the NH group at the ortho-
position of the benzene ring and the lone pair electrons of nitrogen
in pyrimidine ring. This could give a flexible scaffold, which can
prevent ligand from intercalating into the duplex DNA. It could also
provide a coplanar conformation of the main core, which allows
ligand to effectively stack on the G-quartet. Therefore, the ligand’s
quadruplex binding affinity and selectivity may be all improved.
However, based on previous reports [44], we hypothesized that the
scaffold of quinazoline itself alone is probably not a sufficiently to
be an effective G-quadruplex ligand. The addition of two cationic
side chains to the quinazoline parent core, which could interact
with the grooves and loops of the G-quadruplex, may be necessary
to enhance its G-quadruplex binding potency and selectivity, as
well as its aqueous solubility. The interactions of above-mentioned
2. Chemistry
The facile synthetic pathway for quinazoline derivatives is
shown in Scheme 1. The synthetic methodology commenced with
the synthesis of acylchloride through the chlorination of 4-chloro-
2-nitrobenzoic acid, followed with its amidation using anthranila-
mide and triethylamine to give the uncyclized amide intermediates
2 [45]. Subsequently, compound 2-(4-chloro-2-nitrophenyl)-3H-
quinazolin-4-one 3 was prepared through the oxidative ring
closure of compound 2 under basic conditions, using potassium
hydroxide and ethanol, with excellent yield and purity [46]. The
chlorination of 3 with excess phosphorus oxychloride was carried
out to give compound 4 [47]. Coupling of 3-aminopropyl-dime-
thylamine with 4-chloroquinazoline intermediate 4 in THF at 66 ꢀC,
afforded compound 5. In order to obtain the amine functionality,
attempts were made using the conventional procedure for the
reduction of the nitro group with 80% hydrazine hydrate in the
presence of 10% Pd/C and isopropanol as a solvent, which furnished
amino-substituted compound 6 at 96% yield [48]. A significant
feature of this reaction was that product isolation could be facili-
tated through the development of workup procedures without
purification using chromatography. Reaction of amine 6 with
acylchloride and potassium carbonate in dichloromethane gave
compounds 7e9 at 69e85% yield. Finally, the target compounds
10ae10i, 11ae11e, 12a, 12d, and 12e were obtained through
Scheme 1. Synthesis of quinazoline derivatives. Reagent: (i) 2 equiv of TEA, CHCl3, rt, 5 h; (ii) 10% aqueous KOH, EtOH, reflux, 2 h; (iii) N,N-diethylaniline, POCl3, toluene, reflux, 6 h;
(iv) 3-aminopropyldimethyl- amine, THF, reflux, 5 h; (v) 10% Pd/C, 80% N2H4. H2O, isopropanol, reflux, 2 h; (vi) Cl(CH2)nCOCl, K2CO3, CH2Cl2, rt, 24 h; (vii) R2NH, KI, ethanol, reflux.

Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
301
Table 1
aminolysis of the compounds 7, 8, and 9 under reflux through the
treatment with the appropriate primary and secondary amines.
The 1H NMR chemical shifts of acylamide protons of all the
Stabilization temperatures (D
Tm) determined with FRET experiment.a
Compound
F21T
Tm/ꢀC)
F10T
Tm/ꢀC)
Compound
F21T
Tm/ꢀC)
F10T
(D
Tm/ꢀC)
(D
(
D
(
D
ligands in CDCl3 are in the range of
d 13.51e14.12 ppm, which
10a
10b
10c
10d
10e
10f
10g
10h
10i
5.4 ꢁ 0.6
7.4 ꢁ 0.5
4.5 ꢁ 0.6
0.2 ꢁ 0.0
0.1 ꢁ 0.1
0.1 ꢁ 0.0
0.3 ꢁ 0.1
0.0 ꢁ 0.0
0.3 ꢁ 0.1
0.2 ꢁ 0.1
0.3 ꢁ 0.1
0.2 ꢁ 0.1
0.2 ꢁ 0.1
11b
11c
12.8 ꢁ 0.5
14.4 ꢁ 0.4
15.5 ꢁ 0.6
8.4 ꢁ 0.6
11.6 ꢁ 0.3
14.7 ꢁ 0.8
9.8 ꢁ 0.6
0.7 ꢁ 0.3
9.9 ꢁ 0.5
0.2 ꢁ 0.1
0.1 ꢁ 0.1
0.3 ꢁ 0.0
0.1 ꢁ 0.1
0.2 ꢁ 0.0
0.4 ꢁ 0.1
0.1 ꢁ 0.1
0.3 ꢁ 0.1
5.5 ꢁ 0.1
indicate a relatively strong intramolecular H-bonding [49e51]. In
order to determine how the intramolecular hydrogen bond affect
the folded state of the molecule, we performed molecular modeling
experiment for compound 10h using the semi-empirical method
including a solvent model, followed with 1D selective NOESY and
2D NOESY (Figure S1). In the 1D selective NOESY experiment, upon
the irradiation of proton 17-H of 10h, only the enhancement of the
equivalent protons 3-H and 13-H was observed. The cross-peak,
between 17-H and 3-H, was observed on both 2D NOESY and 1D
selective NOESY spectra. All these results suggested the formation
of intramolecular hydrogen bond between the acylamide-NH and
the pyrimidine-N7 atoms. Based on molecular modeling experi-
ment, compound 10h adopts a Z-shaped coplanar conformation as
shown in Fig. 1.
11d
11e
12a
12d
12e
6
10.1 ꢁ 0.3
3.9 ꢁ 0.7
10.4 ꢁ 0.8
9.6 ꢁ 0.4
13.0 ꢁ 0.9
12.3 ꢁ 0.7
11.8 ꢁ 0.7
SYUIQ-5
11a
a
The melting point of native DNA quadruplex was 60 ꢀC ΔTm, change in melting
temperature at 1
mM compound concentration and 200 nM DNA concentration.
The different derivatives were found to have varying effect in
stabilizing the telomeric G-quadruplex. A comparison of the FRET
assay results indicated that existence and the length of the amide
side chain at the ortho- position of the aromatic group had
a significant effect. The intermediate 6 without the amide side
chain didn’t show any effect on G-quadruplex stability. With the
same basic terminus, compounds 11ae11e with longer side chains
(n ¼ 2, with three bonds between basic N terminus and carbonyl
group) showed stronger affinity to the telomeric G-quadruplex
than compounds 10ae10e with shorter side chains (n ¼ 1, with two
bonds between basic N terminus and carbonyl group), while no
distinct effect on ΔTm values was observed with the further
extension of amide side chain, as shown for compounds 12a, 12d,
12e, and 10fe10i, with four or five bonds between basic N terminus
and carbonyl group. On the other hand, alterations of the basic
terminus of the amide side chains showed that the N-methyl
piperazino analogues (10d, 11d and 12d) had strongly enhanced
affinity for G-quadruplex DNA over the piperidino, pyrrolidino, and
diethylamino analogues, with the less basic morpholino analogues
(10e, 11e and 12e) proving detrimental to G-quadruplex affinity.
These results demonstrated the importance of length and basicity
of amide side chain for strong G-quadruplex interactions.
3. Results and discussion
3.1. Studies of the stabilization and selectivity of the synthetic
ligands to telomeric G-quadruplex with FRET assays
The stabilization and selectivity of the quinazoline derivatives to
G-quadruplex DNA were evaluated using an FRET melting assay
[52], with our previously reported quindoline derivative SYUIQ-5
as a reference compound [53]. In order to study the selectivity of
these compounds for G-quadruplex DNA over duplex DNA, a duplex
oligomer ds26 was used in the present study [28,54].
Table 1 shows the effect of derivatives on the enhanced melting
temperature (ΔTm) of two labeled oligonucleotides in Kþ-contain-
ing solution. F21T (50-FAM-d(GGG[TTAGGG]3)-TAMRA-30) repre-
sents the human telomeric DNA sequence, while F10T (50-FAM-
dTATAGCTATA-HEG-TATAGCTATA- TAMRA-30) is a hairpin duplex
DNA. A comparison of the FRET assay results for the reference
compound SYUIQ-5 with fused polycyclic system and synthesized
quinazoline derivatives revealed that all these ligands had high
selectivity towards G-quadruplex DNA, and most of these
compounds had much stronger stabilizing ability to the telomeric
G-quadruplex than SYUIQ-5. Moreover, these derivatives had weak
effect on the thermal stability of the duplex DNA F10T (Table 1,
ΔTm < 1 ꢀC), implied that these derivatives were not duplex DNA-
binding ligands [29].
Furthermore, the selectivity of these derivatives to G-quad-
ruplex was assessed with an FRET-based competition assay, and the
ability of the ligand to stabilize G-quadruplex was challenged with
nonfluorescent duplex DNA (ds26, 50-CAATCGGATCGAATT-
CGATCCGATTG-30) [28,55]. In the presence of varying amount of
competitor ds26, the thermal stabilization of F21T enhanced by
Fig. 1. Structure of 10h (A); energy minimized structures (Chem 3D Ultra 10.0, CambridgeSoft Corp, MA) of 10h, at side view (B) and bottom view (C). The NOE cross peaks are
indicated with arrow. A intramolecular hydrogen bond was marked as dash line in A and green line in B. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

302
Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
Both FRET and SPR experimental results indicated that the
amide side chains of the tested compounds with at least three
bonds between basic N terminus and C]O bond of amide group
(10h, 11d, 12d) were optimal for their interactions with telomeric
G-quadruplex DNA. For the study of their binding selectivity for
quadruplex over duplex by SPR, incubation of these compounds
with duplex DNA immobilized on the chips showed neither
significant nor specific binding interactions, which prevented the
determination of their binding affinity (Figure S5). This might
indicate that these disubstituted quinazoline derivatives had good
selectivity for G-quadruplex DNA, which was in agreement with the
FRET data.
3.3. Studies of the binding property of the synthetic ligands to
telomeric G-quadruplex with circular dichroism (CD)
CD spectroscopy is a highly sensitive method for determining
the conformation of G-quadruplex structures and the effect of
ligand binding on quadruplex structure [57]. The binding property
of the disubstituted quinazoline derivatives to telomeric G-quad-
ruplex was further studied with this method. It has been reported
that HTG21 has formed a hybrid-type of quadruplex DNA con-
taining parallel and anti-parallel structure in the presence of Kþ,
with a large positive band at 290 nm, a shoulder at around 270 nm,
a small positive band at 255 nm, and a minor negative band near
234 nm on CD spectra [56,57]. In the present study, upon the
addition of compound 11d to the above solution, the CD spectrum
significantly changed, with a maximum band at 290 nm increased
and shifted toward 286 nm, and the shoulder at 270 nm gradually
enhanced and merged into the band at 286 nm. Meanwhile, the
small positive band at 255 nm gradually disappeared and led to the
appearance of a positive band at 240 nm and a major negative band
at 260 nm (Fig. 3A). Compound 11h and 12d induced similar CD
changes (Figure S6). However, dramatically different changes in the
CD spectra were observed when the compound 10d was titrated
into the HTG21 oligonucleotide in the presence of Kþ. Addition of
10d resulted in stronger intensities of both the positive band at
290 nm and the shoulder at around 270 nm, and only a remarkable
attenuation of the band at 255 nm (Fig. 3B). These results are
consistent with those obtained from other experiments in the
present study, and may further support the results of the thermo-
dynamic stability studies indicating the importance of amide side
chain length for strong G-quadruplex interactions. Also, all of above
changes were obviously concentration dependent, but we were not
able to figure out particular conformational changes occurring for
HTG21 based on the present information.
Fig. 2. Competitive FRET results for SYUIQ-5 and ligands 10d, 10e, 10h, 11d, 11e, 12d,
and 12e (1
mM), without and with excess of duplex DNA competitor (ds26). The
concentrations of ds26 were 0, 3, 10
mM.
some selected compounds was slightly affected (Fig. 2), while the
competitor significantly disrupted the binding of SYUIQ-5 to the G-
quadruplex. Our above experimental results demonstrated that
unfused quinazoline derivatives were found to be a new type of
highly selective human telomeric G-quadruplex binding ligands.
3.2. Studies of binding and selectivity of the synthetic ligands to
telomeric G-quadruplex with surface plasmon resonance (SPR)
SPR is a useful technique to monitor molecular reactions in real
time, which has been applied to investigate the interactions between
small molecular ligands and human G-quadruplex DNA [55,56].
Here, SPR experiments were carried out in order to quantitatively
determine the kinetic constants of the quinazoline derivatives
binding to either G-quadruplex or duplex DNA. Figure S4 show the
SPR sensorgrams of 10d, 10h, 11d, and 12d binding to the immobi-
lized telomeric G-quadruplex (HTG21, 50-d(GGG[TTAGGG]3)-30) at
different concentrations. Then KD was calculated by global fitting of
the kinetic data from various concentrations of quinazoline deriva-
tives using Langmuir binding model (Table 2). As the data shown,
The binding constants of the disubstituted compounds to telomeric
G-quadruplex showed strong binding affinity (KD, 0.337e2.11
while no obvious binding was observed using SPR for mono
substituted compound 6 even at a concentration of 20 M, which
mM),
m
was consistent with very low ΔTm values obtained from FRET
melting. These results further confirmed the importance of the dis-
ubstitution for the binding of quinazoline derivatives to the telo-
meric G-quadruplex.
3.4. Studies of the binding property of the synthetic ligands to
telomeric G-quadruplex with 1H NMR
In order to further investigate the binding affinity and binding
models between quinazoline derivatives and G-quadruplex at
a molecular level, we carried out ligand titration experiments with
an intermolecular four-stranded parallel G-quadruplex DNA
formed from [d(TTAGGG)]4 (HT-6) [58,59], a single repeat sequence
of the human telomere, followed with analysis using 1H NMR.
Based on the 1D selective NOESY and 2D NOESY studies
mentioned above, the intramolecular hydrogen bond between the
acylamide-NH and the pyrimidine-N7 atoms of the quinazoline
derivatives was formed in CDCl3. To explore the structural state of
the compound 11d in the experimental solution, 1H NMR studies in
Kþ-containing buffer using a pH Gradient Method was carried out.
As shown in Figure S7, the 1H NMR spectrum of 11d in Kþ-con-
taining buffer revealed that the signal of an acylamide proton at
Table 2
Kinetic parameters determined with SPR spectroscopy.a
ka (Mꢂ1 sꢂ1
)
kd (sꢂ1
)
KD (M)
Htelob
Duplexc
Htelob
Duplexc
Htelob
Duplexc
d
d
d
10d
10h
11d
12d
4.26 ꢃ 104
2.76 ꢃ 105
2.83 ꢃ 105
2.52 ꢃ 105
_
_
_
_
0.09
0.05
0.09
0.07
_
2.11 ꢃ 10ꢂ6
3.37 ꢃ 10ꢂ7
3.14 ꢃ 10ꢂ7
2.78 ꢃ 10ꢂ7
_
d
d
d
_
_
d
d
d
_
_
d
d
d
_
_
a
ka is association constant, while kd is dissociation constant. KD denotes the
equilibrium dissociation constant, given by kd/ka.
b
Htelo (Quadruplex): 50-biotin-[(GTTAGG)5]-30.
c
Duplex: 50-biotin-T9CGAATTCGT5CGAATTCG-30.
d
No significant binding was found for addition of up to 10 or 20
m
M ligand. IC50
.
d 15.65 ppm still exist at pH 4.0, which indicated intramolecular

Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
303
Fig. 3. CD titration spectra of HTG21 (5 mM) at increasing concentrations of 11d (A) and 10d (B) (arrows: 0e5 Mol equiv; dashed line indicated HTG21 in the absence of ligands) in
10 mM TriseHCl buffer, pH 7.2, with 100 mM KCl.
hydrogen bond would keep a steady state during the NMR
experiments.
Compound 11d was selected for this study as one of the best
binding ligands. The crystal structure of the mixed hybrid-type
telomeric G-quadruplex (d[AG3(T2AG3)3], PDB code: 2HY9
[61,62]) with potassium ion was used as the starting point for the
modeling because it might be a more biologically relevant form.
The hybrid-type G-quadruplex conformation, with its 50- and 30-
ends pointing to opposite directions, can provide an efficient scaf-
fold for the formation of compact-stacking multimer structures in
the human telomeric DNA. Our results showed that quinazoline
derivatives could stack on both external G-quartet planes. In this
experiment,100 random starting structures were generated, and 28
structures with lowest energy (average intermolecular binding
In the titration experiments, both downfield- and upfield-
shifted portions of 400 MHz 1H NMR spectrum of [d(TTAGGG)]4
at 25 ꢀC were observed in the absence of compound 11d as shown
in Fig. 4. The imino proton signals attribute to G4-G6 were resolved
in the downfield-shifted region (10e12 ppm). The binding of 11d to
the [d(TTAGGG)]4 was confirmed by a general line-broadening of
the [d(TTAGGG)]4 resonance signals upon addition of the ligand
[60]. There were marked upfield shifts in the imino proton region of
the [d(TTAGGG)]4, indicative of
pep interactions between the
aromatic surface of the ligand and the terminal G-tetrad(s) of the
[d(TTAGGG)]4 [60]. Interestingly, at a ratio of 0.5:1 for ligand:qua-
druplex stoichiometry, another set of new proton signal was
energy,
DG ¼ ꢂ11.72 kcal/mol) were obtained after the docking
protocol were analyzed for 11d (Fig. 5A). As shown in Fig. 5B,
preferable binding site of 11d was found to be 50-terminal G-quartet
plane (with an edgewise loop) for 2HY9. The aromatic core with
a planer “imitative” tetracyclic structure formed through intra-
molecular hydrogen bond (2.6 Å) stacks on at least two guanines
with its side chains in adjacent grooves. The ligand had three to
four hydrogen bonds with either guanine nitrogen or backbone
oxygen atoms, contributing to the stabilization of the complex. This
is consistent with thermal stability of G-quadruplex in the presence
of these quinazoline derivatives, which is attributed to the stable
observed in the downfield (
d
¼ 14.51 ppm), which became domi-
nant at increasing ligand concentration. These results demon-
strated that the intramolecular hydrogen bond of the ligand still
remained in the process of the interaction between quinazoline
derivatives and G-quadruplex DNA. Besides, for a number of proton
resonance signals from the aromatic nucleobases (A3: H8, H2; G4:
H8), the line-broadening changes started to level with an
increasing 11d concentration, suggesting that 11d binds to
[d(TTAGGG)]4 near the A3 and G4 residues, and demonstrating that
the time scale for the formation of the complex of 11d and
[d(TTAGGG)]4 is faster than the 1H NMR time scale. In summary,
these data support that the ligands bind to and stabilize the G-
quadruplex formed by the [d(TTAGGG)]4.
hydrogen bondings,
interactions.
pep stacking interactions, and electrostatic
The combinations of NMR experimental results and molecular
modeling studies indicated that both intramolecular hydrogen
bond and
pep stacking interaction with G-quartet may maintain
the planarity of the aromatic region in 11d during its interaction
with G-quadruplex DNA. G-Quartets are derived from the associa-
tion of four guanines into a cyclic Hoogsteen hydrogen-bonding
arrangement with two hydrogen bonds between two adjacent
guanine bases, which supplied a lager square aromatic surface than
3.5. Molecular modeling studies
Molecular docking experiments were performed on some of
ligand-G-quadruplex complexes to investigate the best binding
mode between the G-quadruplex and the quinazoline derivatives.
Fig. 4. 1H NMR spectra (400 MHz) of [d(TTAGGG)]4 for which R11d{[11d]/[[d(TTAGGG)]4]} ¼ 0, 0.25, 0.5, 1 and 2 in 0.5 mM [d(TTAGGG)]4, 25 mM potassium phosphate buffer (pH
7.0), and 150 mM KCl at 25 ꢀC. Most DNA signals exhibited upfield shifts and were line-broadened with an increase in the R11d value. The assignments of some signals are given in
the spectra.

304
Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
Fig. 5. Results of the docking calculation showing 11d complexed to the 2HY9 human intramolecular quadruplex. (A) Overlay of 28 structures with the lowest energy, illustrating
the unique position of the aromatic ring over the 50-G-quartet (in Green). (B) representative structure showing 11d and the solvent-excluded molecular surface area of the
quadruplex. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
products would possibly interfere with the PCR step, the ligands
were removed prior to the amplification step [64]. In the experi-
ments, solutions of derivatives were added to the telomerase
reaction mixture containing extract from cracked MCF-7 breast
carcinoma cell lines, and the inhibitory concentrations by half
values (TelIC50) of these compounds are listed in Table 3. It was
found that the inhibitory effects of disubstituted quinazoline
derivatives with longer amide side chain (with at least three bonds
between basic N terminus and C]O bond of amide group) on
telomerase activity were significantly enhanced when compared to
that of the shorter one 10d. In general, a good correlation was found
between G-quadruplex stabilization potency and telomerase inhi-
bition among quinazoline derivatives synthesized.
Table 3
Telomerase inhibition by quinazoline derivatives in cell-free assay.
10d
11d
12d
IC50
(m
M)
29.3
7.4
7.7
the Watson-Crick base pairs of duplex DNA [1]. The formation of
intramolecular hydrogen bond in 11d could increase the area of
pep stacking of the compound and finally result in an increase of
its binding affinity to G-quadruplex DNA.
3.6. Telomerase inhibition
The ability of quinazoline derivatives to inhibit human telome-
rase activity were evaluated by a modified TRAP assay, the TRAP-
LIG assay [63]. This method provided qualitative and quantitative
estimates of telomerase inhibition by the small molecules.
Considering that the presence of the compounds in the extended
3.7. Senescence induced by quinazoline derivative 11d
Since compound 11d was found to be one most promising
compound as a telomerase inhibitor and telomeric G-quadruplex
binding ligand in the above studies, the following further in-depth
Fig. 6. Senescence induced by compound 11d on HL60 cells. (A) Long-term incubation of HL60 with compound 11d at subcytotoxic concentrations. Expression of SA-
b-Gal in HL60
cells after treatment with (B) 0.1% DMSO and (C) 1.25 M compound 11d continuously for 16 days.
m

Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
305
experimental studies were carried out only for this particular
compound. To examine the effect of ligand 11d on leukemia cell
HL60, short-term cell viability was determined in a two-day cyto-
toxic assay (MTT assay) first. The results show that 11d has a potent
inhibitory effect, with an IC50 value of 9.6 mM.
To avoid acute cytotoxicity and other nonspecific events that
could lead to difficulty in result interpretation, subcytotoxic
compound 11d might be due to the shortening of telomere length,
which is consistent with those reported previously for efficient
telomeric G-quadruplex ligands and telomerase inhibitors [68]. But
we must be noted that the cellular effect of compound 11d may not
be simply explained through telomeric G-quadruplex interactions
or telomerase inhibition. This molecule could also be targeted for
other possible telomere-independent genomic DNAs, such as
promoter G-quadruplex. Nevertheless, compound 11d still could be
considered as useful lead for anticancer approach for its optimal
selectivity towards G-quadruplex DNA.
concentrations (0.625, 1.25 and 2.5
HL60 for long-term exposure. Upon treatment of HL60 cells with
2.5 M 11d, a significantly inhibitory effect was found after 8 days
and the growth was halted after 12 days. At 1.25 M, a delayed
effect was also observed, with the number of population doublings
significantly decreased after 12 days, and even with 0.625 M 11d,
mM) of 11d were evaluated on
m
m
4. Conclusion
m
a discernible difference was observed between the control and
treated cells (Fig. 6A).
Morphological examination of the cells in long-term studies
showed an increased proportion of enlarged and flattened cells
with phenotypic characteristics of senescence [65,66]. These flat-
tened cells were also stained positively for the senescence-
Rational design of molecules targeting the telomeric G-quad-
ruplex DNA is a promising approach. On this basis, several unfused
aromatic compounds were designed, synthesized, and evaluated as
effective and selective telomeric G-quadruplex binding ligands.
Compared with many G-quadruplex ligands with rigid or flexible
structures, the compounds synthesized in the present study are
novel “imitative” tetracyclic aromatic system formed through
intramolecular hydrogen bond, which enable adoption of moderate
twisted and coplanar conformations of the aryl groups, and allow
the ligands to well stack on the G-quartet. Our experimental results
show that these derivatives are a promising type of selective telo-
meric G-quadruplex binding ligands with strong discrimination
against the duplex DNA. The introduced two positively charged side
chains could participate in electrostatic and H-bonding interactions
with the grooves and loops of the G-quadruplex DNA. The amide
side chain attaching the terminal N-methyl piperazino group with
at least three bonds between basic terminus and carboxyl group
was proved to be optimal for G-quadruplex binding. Moreover, the
disubstituted quinazoline derivatives were found to be strong
telomerase inhibitors, and long-term incubation of HL60 cancer
cells with compound 11d showed a remarkable decrease of cell
population accompanied with a shortening of the telomere length,
which are consistent with those previously reported for effective
telomerase inhibitors and telomeric G-quadruplex ligands. Such
interesting results provide us with another new entry into G-
quadruplex ligand design and conformation studies.
associated-galactosidase (SA-b-Gal) activity after continuous
treatment with compound 11d (Fig. 6B and C), and our result
showed that compound 11d induced accelerated senescence of
HL60 cancer cells.
3.8. Telomere shortening by quinazoline derivative 11d
Long term treatment of tumor cells at subapoptotic dosage with
telomeric G-quadruplex ligands have been previously reported to
disrupt telomere length maintenance and caused telomeres to
erode [67]. To investigate whether representative quinazoline
derivative 11d could cause telomeres to shorten, the telomere
length was evaluated using the telomeric restriction fragment (TRF)
length assay (Fig. 7) on leukemia cell HL60. The results showed that
1.25
HL60 cells, and telomere shortening was also observed after
0.625 M of 11d treatment. Telomere dysfunction could activate
mM of 11d triggered telomere shortening about 1.2 kb against
m
p53 to initiate cellular senescence or apoptosis to suppress
tumorigenesis [25], and here the induction of senescence by
5. Experimental section
5.1. Synthesis and characterization
1H and 13C NMR spectra were recorded using TMS as the internal
standard in DMSO-d6 or CDCl3 with a Bruker BioSpin GmbH spec-
trometer at 400 MHz and 100 MHz, respectively. Melting points
(mp) were determined using an SRS-OptiMelt automated melting
point instrument without correction. MS spectra were recorded on
a Shimadzu LCMS-2010A instrument with an ESI or ACPI mass
selective detector. Elemental analysis was carried out on an Ele-
mentar Vario EL CHNS Elemental Analyzer.
5.1.1. General aminolysis procedure: preparation of 10ae10i,
11ae11e and 12a, 12d, 12e
To a stirred refluxing suspension of the chloride compounds 7, 8
or 9 (0.5 mmol) and KI (0.08 g) in EtOH (15 mL) was added drop-
wise appropriate secondary amine (2.0 mL) in EtOH (5 mL). The
mixture was stirred under reflux for 5 h, cooled to 0 ꢀC, and diluted
with distilled water. The resulting solution was filtered, washed
with ether and water, and then evaporated under vacuum. The
crude solid was purified by using chromatography with CHCl3/
MeOH/NH3$H2O elution to afford 10ae10i, 11ae11e and 12a, 12d,
12e.
Fig. 7. Effect of quinazoline derivative 11d on telomere length. TRF analysis of HL60
cells treated with or without compound 11d for 16 days. Lane 1, 0.1% DMSO; lane 2,
0.625 mM compound 11d; lane 3, 1.25 mM compound 11d.

306
Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
5.1.1.1. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-(piperidin-1-yl)acetamide (10a). The compound
7 was treated with excess piperidine according to general ami-
nolysis procedure to afford 10a. After column chromatography with
CHCl3/CH3OH/NH3$H2O (50:1:0.1) elution, the desired product was
obtained as a white solid in 78% yield. mp 180e182 ꢀC; 1H NMR
2.72e2.67 (m, 2H), 2.67e2.51 (m, 4H), 2.46 (s, 6H), 2.40e2.24 (m,
4H), 2.12 (s, 3H), 2.01e1.91 (m, 2H); 13C NMR (101 MHz, CDCl3):
d
169.36, 160.52, 159.45, 148.55, 139.93, 136.37, 132.42, 131.75,
128.46, 126.01, 123.79, 122.85, 121.29, 120.67, 113.86, 64.20, 59.29,
54.15, 53.35, 45.80, 45.20, 42.04, 24.31; HRMS (ESI): (M ꢂ H)-
(C26H34ClN7O) cacld 494.2435, found 494.2421; elemental analysis
calcd (%) for C26H34ClN7O.H2O: C 60.75, H 7.06, N 19.07; found: C
60.66, H, 6.97, N 18.89.
(400 MHz, CDCl3):
d
13.51 (s, 1H), 9.00 (s, 1H), 8.89 (d, J ¼ 2.2 Hz,
1H), 8.51 (d, J ¼ 8.6 Hz,1H), 7.99 (d, J ¼ 8.2 Hz,1H), 7.72 (t, J ¼ 7.6 Hz,
1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.45 (t, J ¼ 7.4 Hz, 1H), 7.12 (dd, J ¼ 8.6,
2.2 Hz,1H), 3.86 (dd, J ¼ 10.2, 5.6 Hz, 2H), 3.26 (s, 2H), 2.67e2.61 (m,
2H), 2.57e2.44 (m, 4H), 2.41 (s, 6H), 1.94e1.87 (m, 2H), 1.46 (dt,
J ¼ 10.8, 5.6 Hz, 4H), 1.37e1.29 (m, 2H); 13C NMR (101 MHz, CDCl3):
5.1.1.5. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-morpholinoacetamide (10e). The compound
7
was treated with excess morpholine according to general ami-
nolysis procedure to afford 10e. After column chromatography with
CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the desired product was
obtained as a white solid in 78% yield. m.p. 179e181 ꢀC; 1H NMR
d
170.12, 160.56, 159.41, 148.66, 140.14, 136.35, 132.17, 131.77, 128.41,
125.85, 123.76, 122.69, 121.02, 120.66, 113.84, 65.22, 59.93, 54.81,
45.50, 42.73, 25.28, 24.45, 23.84; HRMS (ESI): (M
ꢂ
H)-
(C26H33ClN6O) cacld 479.2326, found 479.2320; elemental analysis
calcd (%) for C26H33ClN6O.H2O: C 62.57, H 7.07, N 16.84; found: C
62.46, H 7.03, N, 16.84.
(400 MHz, CDCl3)
d
13.67 (s,1H), 9.09 (s, 1H), 8.88 (d, J ¼ 2.2 Hz, 1H),
8.53 (d, J ¼ 8.6 Hz, 1H), 7.92 (d, J ¼ 8.2 Hz, 1H), 7.77e7.69 (m, 1H),
7.62 (d, J ¼ 8.0 Hz,1H), 7.46 (t, J ¼ 7.6 Hz,1H), 7.13 (dd, J ¼ 8.6, 2.2 Hz,
1H), 3.86 (dd, J ¼ 10.2, 5.6 Hz, 2H), 3.64e3.53 (m, 4H), 3.32 (s, 2H),
2.68e2.62 (m, 2H), 2.63e2.53 (m, 4H), 2.42 (s, 6H), 1.94e1.85 (m,
5.1.1.2. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-(pyrrolidin-1-yl)acetamide (10b). The compound
7 was treated with excess pyrrolidine according to general ami-
nolysis procedure to afford 10b. After column chromatography with
CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the desired product was
obtained as a white solid in 74% yield. mp 174e175 ꢀC; 1H NMR
2H); 13C NMR (101 MHz, CDCl3)
d 168.97, 160.56, 159.40, 148.43,
140.04, 136.45, 132.35, 131.83, 128.02, 126.02, 123.51, 122.86, 121.22,
120.55, 113.89, 66.34, 64.81, 59.95, 53.78, 45.51, 42.80, 24.41; HRMS
(ESI): (M ꢂ H)- (C25H31ClN6O2) cacld 481.2119, found 481.2107;
elemental analysis calcd (%) for C25H31ClN6O2: C 62.17, H 6.47, N
17.40; Found: C 62.24, H 6.36, N 17.55.
(400 MHz, CDCl3):
d
13.84 (s, 1H), 8.93 (s, 2H), 8.57 (d, J ¼ 8.2 Hz,
1H), 7.92 (d, J ¼ 7.8 Hz,1H), 7.69 (d, J ¼ 6.6 Hz,1H), 7.60 (d, J ¼ 7.6 Hz,
1H), 7.43 (t, J ¼ 7.0 Hz, 1H), 7.12 (d, J ¼ 8.2 Hz, 1H), 3.86 (dd, J ¼ 10.0,
5.8 Hz, 2H), 3.43 (s, 2H), 2.68 (t, J ¼ 7.0 Hz, 4H), 2.65e2.52 (m, 2H),
2.40 (s, 6H), 1.97e1.84 (m, 2H), 1.71 (t, J ¼ 7.2 Hz, 4H); 13C NMR
5.1.1.6. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-(2-(pyrrolidin-1-yl)ethylamino)acetamide
(10f). The compound 7 was treated with excess 2-(pyrrolidin-1-yl)
ethanamine according to general aminolysis procedure to afford
10f. After column chromatography with CHCl3/MeOH/NH3$H2O
(50:1:0.1) elution, the desired product was obtained as a white
solid in 44% yield. mp 146e148 ꢀC; 1H NMR (400 MHz, CDCl3):
(101 MHz, CDCl3):
d 170.55, 160.50, 159.38, 148.72, 140.36, 136.42,
131.99, 131.76, 128.01, 125.79, 123.54, 122.66, 121.01, 120.69, 113.80,
62.22, 59.78, 54.52, 45.44, 42.53, 24.51, 23.93; HRMS (ESI): Cacld for
(M ꢂ H)- (C25H31ClN6O) requires m/z 465.2170, found 465.2162;
elemental analysis calcd (%) for C25H31ClN6O.H2O: C 61.91, H 6.86, N
17.33; found: C 61.88, H 6.53, N 17.38.
d
14.04 (s, 1H), 8.94 (s, 1H), 8.90 (s, 1H), 8.60 (d, J ¼ 8.6 Hz, 1H), 7.95
(d, J ¼ 8.2 Hz, 1H), 7.68 (t, J ¼ 7.6 Hz, 1H), 7.58 (d, J ¼ 8.2 Hz, 1H), 7.41
(t, J ¼ 7.6 Hz, 1H), 7.10 (d, J ¼ 8.4 Hz, 1H), 3.84 (dd, J ¼ 10.0, 5.2 Hz,
2H), 3.58 (s, 2H), 2.76 (t, J ¼ 6.0 Hz, 2H), 2.63e2.57 (m, 2H), 2.51 (t,
J ¼ 6.0 Hz, 2H), 2.42 (t, J ¼ 6.0 Hz, 4H), 2.37 (s, 6H), 2.11 (s, 1H),
1.96e1.88 (m, 2H), 1.80e1.67 (m, 4H); 13C NMR (101 MHz, CDCl3):
5.1.1.3. N-(5-chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-(diethylamino)acetamide (10c). The compound 7
was treated with excess diethylamine according to general ami-
nolysis procedure to afford 10c. After column chromatography with
CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the desired product was
obtained as a white solid in 81% yield. mp 170e172 ꢀC; 1H NMR
d
171.48, 160.57, 159.36, 148.62, 140.53, 136.60, 132.36, 131.85,
128.03, 125.83, 123.16, 122.67, 121.06, 120.49, 113.91, 59.93, 55.63,
54.79, 54.11, 48.42, 45.51, 42.71, 24.54, 23.49; HRMS (ESI): (M ꢂ H)-
(C27H36ClN7O) Cacld 508.2592, found 508.2585; elemental analysis
calcd (%) for C27H36ClN7O. H2O: C 61.41, H 7.25, N, 18.57; found: C
61.22, H 7.21, N 18.50.
(400 MHz, CDCl3):
d
13.60 (s, 1H), 8.92 (d, J ¼ 2.2 Hz, 2H), 8.53 (d,
J ¼ 8.6 Hz,1H), 7.90 (d, J ¼ 8.4 Hz, 1H), 7.71 (t, J ¼ 7.2 Hz, 1H), 7.60 (d,
J ¼ 8.0 Hz, 1H), 7.43 (t, J ¼ 7.6 Hz, 1H), 7.13 (dd, J ¼ 8.6, 2.2 Hz, 1H),
3.86 (dd, J ¼ 10.4, 5.6 Hz, 2H), 3.30 (s, 2H), 2.68 (q, J ¼ 7.2 Hz, 4H),
2.65e2.55 (m, 2H), 2.40 (s, 6H), 1.90 (dd, J ¼ 16.8, 5.8 Hz, 2H), 0.99
5.1.1.7. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-(2-(piperidin-1-yl)ethylamino)acetamide
(t, J ¼ 7.2 Hz, 6H); 13C NMR (101 MHz, CDCl3):
d 172.03, 160.53,
159.44, 148.88, 140.01, 136.26, 132.04, 131.93, 128.02, 125.71, 124.22,
122.73, 121.02, 120.86, 113.85, 59.92, 58.81, 49.09, 45.51, 42.65,
24.53, 11.64; HRMS (ESI): (M ꢂ H)ꢂ (C25H33ClN6O) cacld 467.2326,
found 467.2319; elemental analysis calcd (%) for C25H33ClN6O.H2O:
C 61.65, H 7.24, N 17.26, found: C 61.58, H 7.21, N 17.04.
(10g). The compound 7 was treated with excess 2-(piperidin-1-yl)
ethanamine according to general aminolysis procedure to afford
10g. After column chromatography with CHCl3/MeOH/NH3$H2O
(50:1:0.1) elution, the desired product was obtained as a white
solid in 47% yield. mp 140-142 ꢀC; 1H NMR (400 MHz, CDCl3):
d
14.04 (s,1H), 8.95 (s,1H), 8.91 (d, J ¼ 2.0 Hz,1H), 8.61 (d, J ¼ 8.6 Hz,
5.1.1.4. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
1H), 7.96 (d, J ¼ 8.4 Hz,1H), 7.69 (t, J ¼ 7.6 Hz,1H), 7.59 (d, J ¼ 8.0 Hz,
1H), 7.43 (t, J ¼ 7.4 Hz, 1H), 7.11 (dd, J ¼ 8.6, 1.9 Hz, 1H), 3.86 (dd,
J ¼ 10.0, 5.4 Hz, 2H), 3.59 (s, 2H), 2.75 (t, J ¼ 6.2 Hz, 2H), 2.66e2.60
(m, 2H), 2.40 (s, 6H), 2.38e2.34 (m, 2H), 2.34e2.13 (m, 4H), 2.07 (s,
1H), 1.90 (dt, J ¼ 10.8, 5.6 Hz, 2H), 1.55 (dt, J ¼ 10.6, 5.2 Hz, 4H),
lin-2-yl)phenyl)-2-(4-methylpiperazin-1-yl)acetamide
(10d). The
compound was treated with excess N-methyl piperazine
7
according to general aminolysis procedure to afford 10d. After
column chromatography with CHCl3/MeOH/NH3$H2O (50:1:0.1)
elution, the desired product was obtained as a white solid in 66%
1.44e1.35 (m, 2H); 13C NMR (101 MHz, CDCl3):
d 171.55, 160.49,
yield. mp 174e175 ꢀC; 1H NMR (400 MHz, CDCl3):
d
13.51 (s, 1H),
159.30, 148.58, 140.48, 136.50, 132.25, 131.83, 127.97, 125.78, 123.17,
122.61, 121.04, 120.43, 113.87, 59.90, 58.24, 54.84, 54.59, 46.56,
8.98 (s, 1H), 8.86 (d, J ¼ 2.2 Hz, 1H), 8.49 (d, J ¼ 8.6 Hz, 1H), 7.95 (d,
J ¼ 8.2 Hz, 1H), 7.77e7.67 (m, 2H), 7.47 (t, J ¼ 7.6 Hz, 1H), 7.13 (dd,
J ¼ 8.6, 2.2 Hz, 1H), 3.87 (dd, J ¼ 10.4, 5.6 Hz, 2H), 3.31 (s, 2H),
45.50, 42.68, 26.01, 24.53, 24.46; HRMS (ESI): (M
ꢂ
H)-
(C28H38ClN7O) calcd 522.2748, found 522.2745. elemental analysis

Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
307
calcd (%) for C28H38ClN7O.H2O: C 62.03, H 7.44, N, 18.09; found: C
62.14, H 7.45, N 18.17.
compound 8 was treated with excess pyrrolidine according to
general aminolysis procedure to afford 11b. After column chro-
matography with CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the
desired product was obtained as a white solid in 71% yield. mp
5.1.1.8. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-(3-(dimethylamino)propylamino)a-cetamide
(10h). The compound 7 was treated with excess N1,N1-dimethyl-
propane-1,3-diamine according to general aminolysis procedure to
afford 10h. After column chromatography with CHCl3/MeOH/
NH3$H2O (50:1:0.1) elution, the desired product was obtained as
a white solid in 55% yield. mp 153e154 ꢀC; 1H NMR (400 MHz,
163e165 ꢀC; 1H NMR (400 MHz, CDCl3):
d 14.12 (s, 1H), 9.07 (s, 1H),
8.86 (d, J ¼ 2.0 Hz, 1H), 8.67 (d, J ¼ 8.6 Hz, 1H), 7.74 (dt, J ¼ 15.0,
7.6 Hz, 2H), 7.60 (d, J ¼ 8.2 Hz,1H), 7.45 (dd, J ¼ 11.0, 4.0 Hz, 1H), 7.10
(dd, J ¼ 8.6, 2.2 Hz, 1H), 3.87 (dd, J ¼ 10.2, 5.4 Hz, 2H), 3.01 (t,
J ¼ 7.6 Hz, 2H), 2.82 (t, J ¼ 7.6 Hz, 2H), 2.74e2.55 (m, 6H), 2.41 (s,
6H), 1.97e1.88 (m, 2H), 1.86e1.74 (m, 4H); 13C NMR (101 MHz,
CDCl3):
d
14.05 (s, 1H), 8.99 (s, 1H), 8.89 (d, J ¼ 2.2 Hz, 1H), 8.62 (d,
CDCl3): d 170.62, 160.68, 159.16, 148.07, 141.15, 136.84, 132.64,
J ¼ 8.6 Hz, 1H), 7.88 (d, J ¼ 8.2 Hz, 1H), 7.71 (t, J ¼ 7.6 Hz, 1H), 7.60 (d,
J ¼ 8.0 Hz, 1H), 7.44 (t, J ¼ 7.4 Hz, 1H), 7.12 (dd, J ¼ 8.6, 2.2 Hz, 1H),
3.87 (dd, J ¼ 10.2, 5.6 Hz, 2H), 3.60 (s, 2H), 2.74 (t, J ¼ 7.2 Hz, 2H),
2.67e2.61 (m, 2H), 2.41 (s, 6H), 2.29 (t, J ¼ 7.2 Hz, 2H), 2.15 (s, 6H),
1.96 (s, 1H), 1.93e1.90 (m, 2H), 1.70e1.62 (m, 2H); 13C NMR
131.73, 127.50, 125.94, 122.39, 121.81, 121.19, 120.09, 113.88, 59.96,
54.18, 52.08, 45.51, 42.84, 38.41, 24.40, 23.52; HRMS (ESI): Cacld for
(M ꢂ H)- (C26H33ClN6O) requires m/z 479.2326, found 479.2318;
elemental analysis calcd (%) for C26H33ClN6O: C 64.92, H 6.91, N
17.47; found: C 65.02, H 6.68, N 17.55.
(101 MHz, CDCl3):
d 171.21, 160.47, 159.25, 148.37, 140.47, 136.55,
132.41, 131.83, 127.74, 125.87, 122.90, 122.66, 121.12, 120.34, 113.86,
59.89, 57.80, 54.62, 48.45, 45.51, 45.43, 42.72, 27.88, 24.44,; HRMS
(ESI): (M ꢂ H)- (C26H36ClN7O) cacld 496.2592, found 496.2588;
elemental analysis calcd (%) for C26H36ClN7O.H2O: C 60.51, H 7.42, N
19.00; found: C 60.47, H 7.45, N 19.03.
5.1.1.12. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)-
quinazolin-2-yl)phenyl)-3-(diethylamino)propanamide
(11c). The
compound 8 was treated with excess diethylamine according to
general aminolysis procedure to afford 11c. After column chroma-
tography with CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the
desired product was obtained as a white solid in 60% yield. mp
5.1.1.9. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-2-(3-(diethylamino)propylamino)ac-etamide
158e160 ꢀC; 1H NMR (400 MHz, CDCl3):
d 14.08 (s, 1H), 9.07 (s, 1H),
8.86 (d, J ¼ 2.1 Hz, 1H), 8.67 (d, J ¼ 8.6 Hz, 1H), 7.77e7.68 (m, 2H),
7.59 (d, J ¼ 8.1 Hz, 1H), 7.44 (ddd, J ¼ 8.1, 6.1, 2.0 Hz, 1H), 7.09 (dd,
J ¼ 8.6, 2.2 Hz,1H), 3.86 (dd, J ¼ 10.2, 5.6 Hz, 2H), 3.07e2.95 (m, 2H),
2.76e2.68 (m, 2H), 2.64 (t, J ¼ 4.2 Hz, 2H), 2.63e2.56 (m, 4H), 2.41
(s, 6H), 1.97e1.85 (m, 2H), 1.05 (t, J ¼ 7.2 Hz, 6H); 13C NMR
(10i). The compound 7 was treated with excess N1,N1-dieth-
ylpropane-1,3-diamine according to general aminolysis procedure
to afford 10i. After column chromatography with CHCl3/MeOH/
NH3$H2O (50:1:0.1) elution, the desired product was obtained as
a white solid in 57% yield. mp 149e151 ꢀC; 1H NMR (400 MHz,
(101 MHz, CDCl3):
d 171.05, 160.60, 159.10, 147.99, 141.19, 136.74,
CDCl3):
d
14.05 (s, 1H), 9.00 (s, 1H), 8.89 (d, J ¼ 2.2 Hz, 1H), 8.62 (d,
132.56, 131.74, 127.35, 125.93, 122.31, 121.74, 121.24, 119.95, 113.87,
59.90, 48.93, 47.01, 45.49, 42.73, 36.64, 24.41, 11.90. HRMS (ESI):
(M ꢂ H)- (C26H35ClN6O) cacld 481.2483, found 481.2472; elemental
analysis calcd (%) for C26H35ClN6O.H2O: C 62.32, H 7.44, N 16.77;
found: C 62.15, H 7.57, N 16.63.
J ¼ 8.6 Hz, 1H), 7.89 (d, J ¼ 8.2 Hz, 1H), 7.75e7.68 (m, 1H), 7.61 (d,
J ¼ 8.2 Hz, 1H), 7.48e7.41 (m, 1H), 7.13 (dd, J ¼ 8.6, 2.2 Hz, 1H), 3.87
(dd, J ¼ 10.2, 5.6 Hz, 2H), 3.60 (s, 2H), 2.74 (t, J ¼ 7.0 Hz, 2H),
2.68e2.62 (m, 2H), 2.51 (dd, J ¼ 13.2, 6.8 Hz, 6H), 2.42 (d, J ¼ 5.6 Hz,
6H), 2.08 (s, 1H), 1.91 (dd, J ¼ 11.2, 5.6 Hz, 2H), 1.73e1.63 (m, 2H),
0.99 (t, J ¼ 7.2 Hz, 6H); 13C NMR (CDCl3, 101 MHz):
d
171.33, 160.50,
5.1.1.13. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)-
quinazolin-2-yl)phenyl)-3-(4-methylpiperazin-1-yl)propane-mide
(11d). The compound 8 was treated with excess N-methyl pipera-
zine according to general aminolysis procedure to afford 11d. After
column chromatography with CHCl3/MeOH/NH3$H2O (50:1:0.1)
elution, the desired product was obtained as a white solid in 44%
159.28, 148.46, 140.48, 136.53, 132.34, 131.83, 127.82, 125.82, 123.01,
122.63, 121.06, 120.38, 113.87, 59.94, 54.64, 51.13, 48.84, 46.79,
45.51, 42.75, 27.13, 24.49, 11.68; HRMS (ESI): (M
ꢂ
H)-
(C28H40ClN7O) calcd 524.2905, found 524.2900; elemental analysis
calcd (%) for C28H40ClN7O.H2O: C 61.80, H 7.78, N 18.02; found: C
61.62, H 7.61, N 17.93.
yield. mp 167e168 ꢀC; 1H NMR (400 MHz, CDCl3):
d 14.07 (s, 1H),
9.07 (s, 1H), 8.85 (d, J ¼ 1.6 Hz, 1H), 8.66 (d, J ¼ 8.6 Hz, 1H), 7.72 (d,
J ¼ 4.0 Hz, 2H), 7.60 (d, J ¼ 8.2 Hz, 1H), 7.45 (dt, J ¼ 8.2, 4.0 Hz, 1H),
7.09 (dd, J ¼ 8.6, 1.8 Hz, 1H), 3.86 (dd, J ¼ 10.2, 5.4 Hz, 2H), 2.92 (t,
J ¼ 7.4 Hz, 2H), 2.75 (t, J ¼ 7.4 Hz, 2H), 2.67e2.63 (m, 2H), 2.59 (dd,
J ¼ 19.2, 11.0 Hz, 4H), 2.49e2.41 (m, 4H), 2.41 (s, 6H), 2.26 (s, 3H),
5.1.1.10. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)-
quinazolin-2-yl)phenyl)-3-(piperidin-1-yl)propanamide (11a). The
compound 8 was treated with excess piperidine according to
general aminolysis procedure to afford 11a. After column chroma-
tography with CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the
desired product was obtained as a white solid in 69% yield. mp
1.95e1.87 (m, 2H); 13C NMR (101 MHz, CDCl3):
d 170.62, 160.71,
159.19, 148.08, 141.12, 136.87, 132.69, 131.77, 127.40, 125.98, 122.46,
121.85, 121.28, 120.12, 113.92, 59.92, 55.08, 54.20, 52.99, 46.01,
45.51, 42.80, 36.39, 24.42; HRMS (ESI): (M ꢂ H)- (C27H36ClN7O)
cacld 508.2592, found 508.2588; elemental analysis calcd (%) for
C27H36ClN7O: C 63.58, H 7.11, N 19.22; found: C 63.49, H 7.26, N
19.36.
178e179 ꢀC; 1H NMR (400 MHz, CDCl3):
d 14.06 (s, 1H), 9.06 (s, 1H),
8.85 (d, J ¼ 2.0 Hz, 1H), 8.66 (d, J ¼ 8.6 Hz, 1H), 7.79e7.68 (m, 2H),
7.59 (d, J ¼ 8.2 Hz, 1H), 7.48e7.40 (m, 1H), 7.08 (dd, J ¼ 8.6, 2.2 Hz,
1H), 3.84 (dd, J ¼ 10.2, 5.6 Hz, 2H), 2.92e2.82 (m, 2H), 2.80e2.71
(m, 2H), 2.67e2.58 (m, 2H), 2.54e2.42 (m, 4H), 2.40 (s, 6H),
1.96e1.85 (m, 2H), 1.61e1.53 (m, 4H), 1.46e1.37 (m, 2H); 13C NMR
(101 MHz, CDCl3):
d
170.93, 160.60, 159.11, 148.02, 141.16, 136.75,
5.1.1.14. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-3-morpholinopropanamide (11e). The compound 8
was treated with excess morpholine according to general ami-
nolysis procedure to afford 11e. After column chromatography with
CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the desired product was
obtained as a white solid in 67% yield. mp 165e167 ꢀC; 1H NMR
132.58, 131.74, 127.45, 125.93, 122.33, 121.79, 121.21, 120.01, 113.86,
59.94, 54.99, 54.42, 45.51, 42.83, 36.52, 25.97, 24.39, 24.30; HRMS
(ESI): (M ꢂ H)- (C27H35ClN6O) cacld 493.2483, found 493.2472;
elemental analysis calcd (%) for C27H35ClN6O: C 65.51, H 7.13, N
16.98; found: C 65.70, H 7.24, N 16.86.
(400 MHz, CDCl3):
d
14.10 (s, 1H), 9.08 (s, 1H), 8.84 (d, J ¼ 2.0 Hz,
5.1.1.11. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)-
quinazolin-2-yl)phenyl)-3-(pyrrolidin-1-yl)propanamide (11b). The
1H), 8.67 (d, J ¼ 8.6 Hz, 1H), 7.75e7.69 (m, 2H), 7.62 (d, J ¼ 8.2 Hz,
1H), 7.45 (ddd, J ¼ 8.2, 5.2, 3.0 Hz, 1H), 7.10 (dd, J ¼ 8.6, 2.2 Hz, 1H),

308
Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
3.86 (dd, J ¼ 10.2, 5.4 Hz, 2H), 3.69e3.61 (m, 4H), 2.89 (t, J ¼ 7.2 Hz,
2H), 2.75 (t, J ¼ 7.2 Hz, 2H), 2.68e2.62 (m, 2H), 2.58e2.48 (m, 4H),
(ESI): (M ꢂ H)- (C27H35ClN6O2) cacld 509.2432, found 509.2429;
elemental analysis calcd (%) for C27H35ClN6O2 : C 63.45, H 6.90, N
16.44; found: C 63.31, H 7.05, N 16.51.
2.42 (s, 6H),1.95e1.89 (m, 2H); 13C NMR (101 MHz, CDCl3):
d 170.52,
160.59, 159.12, 147.94, 141.04, 136.77, 132.63, 131.77, 127.20, 125.99,
122.46, 121.80, 121.34, 120.02, 113.88, 66.88, 59.72, 54.61, 53.53,
45.46, 42.61, 36.28, 24.44; HRMS (ESI): (M ꢂ H)- (C26H33ClN6O2)
cacld 495.2275, found 495.2271; elemental analysis calcd (%) for
C26H33Cl N6O.2H2O: C 60.63, H 6.85, N 16.32; found: C 60.51, H 6.92,
N 16.36.
5.2. Materials
All oligomers/primers used in this study were purchased from
Invitrogen (China). Stock solutions of all the derivatives (10 mM)
were made using DMSO (10%) or double-distilled deionized water.
Further dilutions to working concentrations were made with
double-distilled deionized water.
5.1.1.15. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)-
quinazolin-2-yl)phenyl)-4-(piperidin-1-yl)butanamide
(12a). The
compound 9 was treated with excess piperidine according to
general aminolysis procedure to afford 12a. After column chro-
matography with CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the
desired product was obtained as a white solid in 73% yield. mp
5.3. FRET assay
FRET assay was carried out on a real-time PCR apparatus
following previously published procedures [52]. The fluorescently
labeled oligonucleotides F21T: 50-FAM-d(GGG[TTAGGG]3)-TAMRA-
30 and F10T: 50-FAM-dTATAGCTATA-HEG-TATAGCTATA-TAMRA-30
(donor fluorophore FAM is 6-carboxyfluorescein; acceptor fluo-
rophore TAMRA is 6-carboxytetramethylrhodamine; HEG linker is
[(CH2eCH2eO)6]) were used as the FRET probes. Fluorescence
melting curves were determined with a Roche LightCycler 2 real-
159e160 ꢀC; 1H NMR (400 MHz, CDCl3):
d 14.04 (s, 1H), 9.03 (s, 1H),
8.86 (d, J ¼ 2.2 Hz, 1H), 8.68 (d, J ¼ 8.6 Hz, 1H), 7.80e7.70 (m, 2H),
7.61 (d, J ¼ 8.0 Hz, 1H), 7.45 (ddd, J ¼ 8.2, 6.2, 2.0 Hz, 1H), 7.10 (dd,
J ¼ 8.6, 2.2 Hz,1H), 3.87 (dd, J ¼ 10.2, 5.6 Hz, 2H), 2.69e2.62 (m, 2H),
2.59 (t, J ¼ 7.4 Hz, 2H), 2.53e2.46 (m, 2H), 2.46e2.41 (m, 4H), 2.41
(s, 6H), 2.04 (dt, J ¼ 14.8, 7.4 Hz, 2H), 1.92 (dt, J ¼ 11.2, 5.8 Hz, 2H),
1.56 (dt, J ¼ 11.0, 5.4 Hz, 4H), 1.45e1.36 (m, 2H); 13C NMR (101 MHz,
time PCR machine, using a total reaction volume of 20
mL, with
CDCl3):
d
171.94, 160.74, 159.20, 148.17, 141.29, 136.90, 132.64,
0.2 M of labeled oligonucleotide in TriseHCl buffer (10 mM, pH
m
131.77, 127.54, 125.94, 122.33, 121.81, 121.23, 120.05, 113.92, 60.00,
58.70, 54.56, 45.53, 42.86, 36.90, 25.95, 24.45, 23.42, 22.94; HRMS
(ESI): (M ꢂ H)- (C28H37ClN6O) calcd 507.2639, found 507.2629;
elemental analysis calcd (%) for C28H37ClN6O: C 66.06, H 7.33, N
16.51; found: C 66.10, H 7.49, N 16.32.
7.4) 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 37e99 ꢀ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. Final analysis of the data was carried out using
Origin7.5 (OriginLab Corp.).
5.1.1.16. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-4-(4-methylpiperazin-1-yl)butanami-de (12d). The
compound
9 was treated with excess N-methyl piperazine
according to general aminolysis procedure to afford 12d. After
column chromatography with CHCl3/MeOH/NH3$H2O (50:1:0.1)
elution, the desired product was obtained as a white solid in 58%
5.4. Surface plasmon resonance
SPR measurements were performed on a ProteOn XPR36 Protein
Interaction Array system (Bio-Rad Laboratories, Hercules, CA) using
a Neutravidin-coated GLH sensor chip. In a typical experiment,
biotinylated duplex DNA and biotinylated HTG21 (50-d(GGG
[TTAGGG]3)-30) were folded in filtered and degassed running buffer
(TriseHCl 50 mM pH 7.2, 100 mM KCl). The DNA samples were then
captured (w1000 RU) in flow cells 1 and 2, leaving the third flow
cell as a blank. Ligand solutions (at 0.15625, 0.3125, 0.625, 1.25, 2.5,
yield. mp 155e157 ꢀC; 1H NMR (400 MHz, CDCl3):
d 14.03 (s, 1H),
9.02 (s, 1H), 8.85 (d, J ¼ 2.2 Hz, 1H), 8.66 (d, J ¼ 8.6 Hz, 1H), 7.72 (dd,
J ¼ 4.0, 1.6 Hz, 2H), 7.60 (d, J ¼ 8.2 Hz, 1H), 7.43 (ddd, J ¼ 8.2, 5.4,
2.8 Hz, 1H), 7.08 (dd, J ¼ 8.6, 2.2 Hz, 1H), 3.89 (dd, J ¼ 10.2, 5.6 Hz,
2H), 2.75e2.69 (m, 2H), 2.66 (t, J ¼ 7.4 Hz, 2H), 2.59 (t, J ¼ 7.4 Hz,
2H), 2.55e2.46 (m, 4H), 2.43 (s, 6H), 2.36e2.28 (m, 4H), 2.23 (s, 3H),
2.02 (dt, J ¼ 14.6, 7.4 Hz, 2H), 1.97e1.90 (m, 2H); 13C NMR (101 MHz,
CDCl3):
d
171.86, 160.64, 159.12, 148.05, 141.26, 136.80, 132.60,
5, 10, and 20 mM) were prepared with running buffer by serial
131.75, 127.42, 125.91, 122.26, 121.71, 121.26, 119.95, 113.89, 59.89,
57.81, 55.10, 53.11, 45.99, 45.49, 42.76, 36.75, 24.41, 22.93; HRMS
(ESI): (M ꢂ H)- (C28H38ClN7O) cacld 522.2748, found 522.2747;
elemental analysis calcd (%) for C28H38ClN7O: C 64.17, H 7.31, N
18.71; found: C 64.03, H 7.26, N 18.59.
dilutions from stock solutions. Six concentrations were injected
simultaneously at a flow rate of 100 mL minꢂ1 for 150 s of associ-
ation phase, followed with 300 s of dissociation phase at 25 ꢀC. The
NLC sensor chip was regenerated with short injection of 1 M NaCl
between consecutive measurements. The final graphs were ob-
tained by subtracting blank sensorgrams from the duplex or
quadruplex sensorgrams. Data are analyzed with ProteOn manager
software, using the Langmuir model for fitting kinetic data.
5.1.1.17. N-(5-Chloro-2-(4-(3-(dimethylamino)propylamino)quinazo-
lin-2-yl)phenyl)-4-morpholinobutanamide (12e). The compound 9
was treated with excess morpholine according to general ami-
nolysis procedure to afford 12e. After column chromatography with
CHCl3/MeOH/NH3$H2O (50:1:0.1) elution, the desired product was
obtained as a white solid in 65% yield. mp 152e154 ꢀC; 1H NMR
5.5. CD measurements
The oligomer HTG21 (50-d(GGG[TTAGGG]3)-30) at
concentration of 5 M was resuspended in TriseHCl buffer (10 mM,
a final
(400 MHz, CDCl3):
d
14.07 (s, 1H), 9.03 (s, 1H), 8.87 (d, J ¼ 2.2 Hz,
m
1H), 8.68 (d, J ¼ 8.6 Hz, 1H), 7.78e7.69 (m, 2H), 7.65 (d, J ¼ 8.2 Hz,
1H), 7.46 (ddd, J ¼ 8.2, 5.0, 3.2 Hz, 1H), 7.10 (dd, J ¼ 8.6, 2.2 Hz, 1H),
3.88 (dd, J ¼ 10.4, 5.6 Hz, 2H), 3.70e3.56 (m, 4H), 2.73e2.64 (m,
2H), 2.61 (t, J ¼ 7.2 Hz, 2H), 2.47 (t, J ¼ 6.4 Hz, 2H), 2.44 (s, 6H),
2.08e1.99 (m, 2H), 1.98e1.91 (m, 2H), 1.86e1.72 (m, 4H); 13C NMR
pH 7.2) containing the derivatives to be tested. The samples were
heated to 95 ꢀC, then gradually cooled to room temperature, and
incubated at 4 ꢀC for at least 6 h. The CD spectra were recorded on
Chirascan (AppliedPhotophysics) spectrophotometer.
A quartz
cuvette with 4 mm path length was used for the spectra recorded
over a wavelength range of 230e450 at 1 nm bandwidth, 1 nm step
size, and 0.5 s time per point. The CD spectra were obtained by
taking the average of two scans made from 230 to 450 nm. Final
(101 MHz, CDCl3):
d 171.82, 160.65, 159.12, 148.02, 141.24, 136.83,
132.60, 131.77, 127.30, 125.96, 122.32, 121.68, 121.32, 119.93, 113.89,
66.97, 59.81, 58.27, 53.67, 45.48, 42.68, 36.62, 24.43, 22.52; HRMS

Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
309
analysis of the data was carried out using Origin 7.5 (OriginLab
Corp.).
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,
5.6. NMR spectroscopy
NMR experiments were performed on a Bruker AVANCE AV
400 MHz spectrometer. The 1H NMR spectrum of 11d was obtained
in a 90% H2O/10% D2O solution containing 150 mM KCl and 25 mM
potassium phosphate buffer with varying pH values of 3.0, 3.5, 4.0,
5.0, 6.0, and 7.0 respectively. All of the titration experiments were
carried out at 25 ꢀC in a 90% H2O/10% D2O solution containing
150 mM KCl and 25 mM potassium phosphate buffer (pH ¼ 7.0).
Water suppression was achieved by the Watergate method. The
oligonucleotide d(TTAGGG) was purified by using HPLC, and the
concentration was 0.5 mM for the NMR measurements.
a second PCR master mix was prepared consisting of 1
m
M ACX
reverse primer (50-GCGCGG[CTTACC]3CTAACC-30), 0.1
mg
TS
forward primer (50-AATCCGTCGAGCAGAGTT-30), TRAP buffer, 5
mg
BSA, 0.5 mM dNTPs, and 2 units of Taq polymerase. A 10 mL 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. Tel IC50 values were then
calculated from the optical density quantitated from the AlphaEa-
seFC software.
5.7. Molecular modelling
5.9. Short-term cell viability
The crystal structure of the mixed hybrid-type 26-mer telomeric
G-quadruplex (PDB ID 2HY9) [61,62] was used as an initial model to
study the interaction between the quindoline derivatives and
telomeric DNA. The terminal 50 adenine residue was removed to
generate a 21-mer structure, for comparison with the d(GGG
[TTAGGG]3) DNA we used in the FRET and CD experiments. Water
molecules were removed from the PDB file, and the missing
hydrogen atoms were added to the system using the Biopolymer
module implemented in the SYBYL7.3.5 molecular modeling soft-
ware from Tripos Inc. (St. Louis, MO). Ligand structures were con-
structed by adopting the empirical Gasteiger Huckel (GH) partial
atomic charges and then were optimized (Tripos force field) with
a nonbond cutoff of 12 Å and a convergence of 0.01 kcal molꢂ1/Å
over 10,000 steps using the Powell conjugate-gradient algorithm.
Docking studies were carried out using the AUTODOCK 4.0
program [69]. By using ADT [70], 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
resulting G-quadruplex structure was used as an input for the
HL60 leukemia cell line was seeded on 96-well plates (1.0 ꢃ 103
wellꢂ1) and exposed to various concentrations of derivatives. After
48 h of treatment at 37 ꢀC in a humidified atmosphere of 5% CO2,
10 m
L of 5 mg/mLꢂ1 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 mL 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.
5.10. Long-term cell culture experiments
Long-term proliferation experiments were carried out using the
HL60 leukemia cell line. Cells (1.0 ꢃ 105) were grown in 10 cm Petri
dishes and exposed to a subcytotoxic concentration of a ligand or
an equivalent volume of 0.1% DMSO every 4 days. The cells in
control and drug-exposed flasks were counted and flasks reseeded
with 1.0 ꢃ 105 cells. The remaining cells were collected and used for
the measurements described below. This process was continued for
16 days.
AUTOGRID program. AUTOGRID gave
a precalculated atomic
affinity grid maps for each atom type in the ligand, plus an elec-
trostatics map and a separate desolvation map present in the
substrate molecule. The dimensions of the active site box, which
was placed at the center of the G-quadruplex, were set to
54 Å ꢃ 54 Å ꢃ 54 Å with the grid points 0.375 Å apart. 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 25,000,000 energy
evaluations, a maximum number of generations of 27,000, and
a mutation rate of 0.02. One hundred independent docking runs
were carried out for each ligand. The resulting positions were
clustered according to a root-mean-square criterion of 0.5 Å.
5.11. SA-b-Gal assay
After the long-term incubation, the growth medium was aspi-
rated and the cells were fixed in 2% formaldehyde/0.2% glutaral-
dehyde 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
b-Gal stain solution containing 1 mg/mL of
5-bromo-4-chloro-3-indolyl-
b-D
-galactoside, followed by incuba-
tion 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.
5.8. TRAP-LIG assay
The ability of quinazoline derivatives to inhibit telomerase in
a cell-free system was assessed with the TRAP-LIG assay following
previously published procedures [63]. Protein extracts from expo-
nentially growing MCF-7 breast carcinoma cells were used. Briefly,
5.12. Telomere length assay
Cells were incubated with the ligand for 16 days. To measure the
telomere length, genomic DNA was digested with Hinf1/Rsa1
restriction enzymes. The digested DNA fragments were separated
on 0.8% agarose gel, transferred to a nylon membrane, and the
transferred DNA fixed on the wet blotting membrane by baking the
membrane at 120 ꢀC for 20 min. The membrane was hybridized
with a DIG-labeled hybridization probe for telomeric repeats and
incubated with anti-DIG-alkaline phosphatase. TRF was performed
with chemiluminescence detection.
0.1
m
g of TS forward primer (50-AATCCGTC-GAGCAGAGTT-30) was
elongated by telomerase (500 ng protein extract) in TRAP buffer
(20 mM TriseHCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA,
and 0.05% Tween 20) containing 125 mM dNTPs and 0.05 mg 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

310
Z. Li et al. / European Journal of Medicinal Chemistry 47 (2012) 299e311
and reduces tumourigenicity of human melanoma cells, Eur. J. Cancer 42
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We thank the Natural Science Foundation of China (Grants
U0832005, 90813011), the International S&T Cooperation Program
of China (2010DFA34630), the Specialized Research Fund for the
Doctoral Program of Higher Education (20090171110050), and the
Science Foundation of Guangzhou (2009A1-E011-6) for financial
support of this study.
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Appendix. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.10.057.
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