Impact of planarity of unfused aromatic molecules on G-quadruplex binding: Learning from isaindigotone derivatives

Article abstract of DOI:10.1039/c1ob05884c

G-quadruplex structures are a new class of attractive targets for DNA-interactive anticancer agents. The primary building block of this structure is the G-quartet, which is composed of four coplanar guanines and serves as the major binding site for small

Full text of DOI:10.1039/c1ob05884c

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PAPER
Impact of planarity of unfused aromatic molecules on G-quadruplex binding:
Learning from isaindigotone derivatives†
Jin-Qiang Hou, Jia-Heng Tan, Xiao-Xiao Wang, Shuo-Bin Chen, Si-Yuan Huang, Jin-Wu Yan, Shu-Han Chen,
Tian-Miao Ou, Hai-Bin Luo, Ding Li, Lian-Quan Gu* and Zhi-Shu Huang*
Received 3rd June 2011, Accepted 20th June 2011
DOI: 10.1039/c1ob05884c
G-quadruplex structures are a new class of attractive targets for DNA-interactive anticancer agents.
The primary building block of this structure is the G-quartet, which is composed of four coplanar
guanines and serves as the major binding site for small molecules. NMR studies and molecular
dynamics simulations have suggested that the planarity of G-quartet surface has been highly dynamic
in solution. To better investigate how the planarity of unfused aromatic ligand impacts on its
quadruplex binding properties, a variety of planarity controllable isaindigotone derivatives were
designed and synthesized. The interaction of G-quadruplex DNA with these designed ligands was
systematically explored using a series of biophysical studies. The FRET-melting, SPR, and CD
spectroscopy results showed that reducing the planarity of their unfused aromatic core resulted in their
decreased binding affinity and stabilization ability for G-quadruplex. NMR studies also suggested that
these compounds could stack on the G-quartet surface. Such results are in parallel with subsequent
molecular modeling studies. A detailed binding energy analysis indicated that van der Waals energy
(DEvdw) and entropy (TDS) are responsible for their decreased quadruplex binding and stabilization
effect. All these results provided insight information about how quadruplex recognition could be
controlled by adjusting the planarity of ligands, which shed light on further development of unfused
aromatic molecules as optimal G-quadruplex binding ligands.
unfused aromatic compounds, such as 1,4-triazole derivatives,22
Introduction
biarylpyrimidines,23 triarylpyridines,24 have been well character-
The guanine-rich single strand can adopt higher-order structures
ized as G-quadruplex binding ligands. The common and the most
called the G-quadruplexes under appropriate cation conditions.1–3
The primary building block of this structure is the p-stacked
G-quartets, each of which is connected by a network of eight
Hoogsteen hydrogen bonds. Possible G-quadruplex sequences are
widely dispersed in eukaryotic genomes, including telomeres,4
rDNA,5 immunoglobulin switch regions,6 and series of gene
promoter regions, such as c-myc,7 c-kit,8 bcl-2,9 and so on. G-
Quadruplexes play important roles in many biological events.
For example, the stable G-quadruplexes in telomeric DNA result
in the inhibition of telomere elongation in cancer cells,10,11 and
the G-quadruplexes in many oncogene promoters function as a
transcriptional repressor element.12–17 Therefore, the binding and
stabilization of the G-quadruplex by small molecule opens an
opportunity for cancer therapeutics.
favorable binding site for these ligands is the G-quartet surface.25,26
Recently, from molecular dynamics (MD) simulation, we have
suggested that fused aromatic ligand stacking on the G-quartet has
a positive impact on G-quartet stabilization, and helps the overall
G-quadruplex stabilization.27 On the other hand, crystallographic
or NMR structures of G-quadruplex have showed that the G-
quartet surface is not completely coplanar.28–31 MD studies have
also suggested that the planarity of G-quartet surface is highly
dynamics in solution.27,32,33 Based on these considerations, unfused
aromatic ligands with adaptive structural feature may be more
suitable to be accommodated on the dynamic G-quartet surface
rather than polycyclic compounds with rigid aromatic rings. Fur-
thermore, this adaptive structural feature can prevent ligand from
intercalating into the duplex DNA, and is consequently important
for its strong quadruplex selectivity. However, in contrast to fused
aromatic compounds, much less is known about how planarity of
an unfused aromatic ligand influence its G-quadruplex binding
properties.
So far, some fused aromatic compounds, such as BRACO-
19,10,18 RHPS4,19,20 MMQ1,21 and SYUIQ-513 (Fig. 1a), and some
School of Pharmaceutical Sciences, Sun Yat-sen University,
Guangzhou, 510006, People’s Republic of China. E-mail: cesglq@mail.
sysu.edu.cn, ceshzs@mail.sysu.edu.cn; Fax: 8620-39943056; Tel: 8620-
39943056
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05884c
Isaindigotone derivatives have been developed in our lab as
selective telomeric G-quadruplex binding ligands.11 The structure
of a representative compound SYUID-5a is shown in Fig.
1b. Isaindigotone derivative has a unique asymmetric unfused
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Fig. 1 Structures of SYUIQ-5 (a) and SYUID-5a (b).
aromatic chromophore with an aliphatic five-member ring in the
center core. Such a compound is neither polycyclic nor macro-
cyclic, and has free rotation around the single bond, which enables
the adoption of possible twisted or coplanar conformations of
the aryl groups. Our previous studies have suggested that these
isaindigotone derivatives could occupy the G-quartet surface, and
may also prevent ligand from intercalating into the duplex DNA.11
Base on above studies, we perceive that isaindigotone derivatives
offer an attractive template to investigate the influence of ligand
planarity on the G-quadruplex binding properties. And a simple
way to change the planarity of these molecules is to vary the
aliphatic ring size which may also adjust the rotation of their
benzylidene group. In the present study, isaindigotone derivatives
with varying aliphatic ring size from five to seven were synthesized.
Using telomeric G-quadruplex as a model, their interactions
with this G-quadruplex were studied with FRET assay, SPR and
CD. Molecular dynamics simulations were further carried out to
elucidate this effect in detail.
The isaindigotone derivatives were prepared following the pro-
cedure reported previously by our group.11 The synthetic pathway
for isaindigotone derivatives is shown in Scheme 1A and 1B.
Compounds 2a–e were synthesized from 2-amino-4-nitrobenzoic
acid and 2-amino-5-nitrobenzoic acid in the presence of POCl3.
The reactions of compounds 2a–e with 4-nitrobenzaldehyde or 4-
dimethylaminobenzaldehyde through Claisen–Schmidt condensa-
tion yielded dinitro-compounds 3a–e, and 5a–d. Without further
purification, these products were reduced to the corresponding
diamino compounds 4a–e, and 6a–d. These products were assigned
as E configuration on the basis of nuclear overhauser effect
(NOE) analysis of NMR spectroscopy. In the NOE experiment,
upon irradiation of proton 6-H and 6¢-H, signals for protons
3-H and 4-H were simultaneously enhanced (as shown in the
ESI†). Acylation of compounds 4a–e, and 6a–d were performed
through their reactions with neat acid chloride. Their further
amination was carried out with the required tertiary amine
base (pyrrolidine/piperidine/dimethylamine) in the presence of
ethanol to give desired target compounds.
Results and discussion
FRET-melting studies
Design and syntheses of isaindigotone derivatives
The stabilization of isaindigotone derivatives on telomeric G-
quadruplex and duplex DNA was studied with FRET (fluores-
cence resonance energy transfer) based melting assay.34–36 The
isaindigotone derivative SYUID-5a and the quindoline derivative
SYUIQ-5 have been previously reported by us and were used as
reference compounds in the present study.11,37 Table 1 shows the
effect of the ligand library on the enhanced melting temperature
(DTm) of two labeled oligonucleotides in solution containing K+.
F21T represents the human telomeric DNA, while F10T is a hair-
pin duplex DNA. Generally, the FRET-melting data showed that
all of the derivatives could stabilize the telomeric G-quadruplex.
Compounds 7a–7p with double side chains were better quadruplex
stabilizing ligands than compounds 7q–7x with single side chain
To determine how the aliphatic ring affect the planar state of a
molecule, the structures of compounds SYUID-5a, 7e and 7h,
which have varied aliphatic ring size from five to seven, were
constructed and optimized with GAUSSIAN using the HF/6-
31G* basis set. As shown in Fig. 2, the nonplanarity of these
compounds can be measured with the heights of their core region,
which are best indicated from their side view. The heights for
˚
compounds SYUID-5a, 7e and 7h are 1.87, 2.41, and 3.65 A,
respectively. We can conclude that increasing the aliphatic ring
size results in decreased planarity of their unfused aromatic core.
To produce ligand diversity, a library of isaindigotone derivatives
including 24 compounds was synthesized.
Fig. 2 Top view and side view of compounds SYUID-5a, 7e and 7h. The structure were calculated at the HF/6-31G* level.
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Scheme 1 Syntheses of isaindigotone derivatives. reagents: (i) pyrrolidin-2-one/piperidin-2-one/2- oxohexamethyleneimine, POCl3, toluene (reflux);
(ii) 4-nitrobenzaldehyde, Ac2O (reflux); (iii) Na2S·9H2O, NaOH, EtOH (reflux); (iv) Cl(CH2)n = 1,2COCl (reflux); (v) R2NH, KI, ethanol (reflux); (vi)
4-dimethylaminobenzaldehyde, glacial acetic acid (reflux).
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Table 1 Stabilization temperatures (DTm) determined with FRET experimenta
Compound
F21T (DTm/◦C)
F10T (DTm/◦C)
Compound
F21T (DTm/◦C)
F10T (DTm/◦C)
7a
7b
7c
7d
7e
7f
7g
7h
7i
19.5 1.7
7.2 0.8
7.3 0.7
8.2 0.9
9.5 1.2
9.4 0.9
10.0 1.5
6.3 0.7
7.1 0.6
17.2 1.8
6.1 0.6
6.6 0.8
6.5 0.4
1.1 0.1
0.3 0.1
0.3 0.3
0.4 0.1
0.6 0.3
0.4 0.1
0.2 0.3
0.3 0.1
0.2 0.2
0.6 0.1
0.3 0.2
0.2 0.1
0.2 0.0
7n
9.0 1.2
8.5 0.8
8.2 1.1
4.1 0.8
4.4 0.5
2.0 0.4
2.0 0.2
2.6 0.2
3.2 0.4
1.3 0.2
1.2 0.3
21.0 1.1
10.2 0.2
0.3 0.1
0.2 0.2
0.2 0.2
0.1 0.2
0.1 0.2
0.2 0.0
0.1 0.2
0.1 0.1
0.1 0.0
0.1 0.0
0.1 0.0
0.7 0.2
5.1 0.3
7o
7p
7q
7r
7s
7t
7u
7v
7j
7k
7l
7w
7x
SYUID-5a
SYUIQ-5
7m
a DTm = Tm (DNA + ligand) - Tm (DNA). The concentrations of F21T and F10T were both 0.2 mM. The concentrations of the compounds were 1mM.
In the absence of ligand, Tm values of annealed F21T and F10T were 60 and 64 ◦C, respectively.
(Fig. 3A). For ligands 7a–7p with double side chains, it was
apparent that analogs 7a and 7j with five-member-ring were more
effective quadruplex stabilizing ligands, with their DTm values of
isomers showed that the compounds with one methylene group
(n = 1) on their side chain (7b–d) were slightly less effective in
quadruplex stabilization than the compounds with two methylene
groups (n = 2) on their side chain (7e–g). This trend was
consistent with that for M-substituted isomers (7k–m and 7n–
p). Furthermore, no significant effect on DTm values was observed
if a pyrrolidinyl group was placed at the terminal of side chains
instead of a piperidinyl or diethylaminyl group. The concentration
dependent melting curves for ligands 7a,7e,7h,7j and 7n upon
binding to F21T were showed in Fig. 3B.
The selectivity of these compounds for G-quadruplex over du-
plex DNA was also assessed by using FRET-based method. None
of these compounds were found to significantly increase the melt-
ing temperature of FRET-tagged duplex DNA F10T (Table 1),
suggesting their poor binding to the duplex DNA.22,38 Further-
more, the G-quadruplex selectivity of some selected compounds
◦
19.5 and 17.2 C, respectively, while analogs 7e–g and 7n–p with
six-member-ring were less effective quadruplex stabilizing ligands
with their DTm values in the range of 8.2–10.0 ◦C. The analogs 7h
and 7i with seven-member-ring were the least effective quadruplex
stabilizing ligands with their DTm values of 6.3 and 7.1 ◦C,
respectively. These results suggested that increasing aliphatic ring
size resulted in a weaker quadruplex stabilizing effect. Such trend
was consistent with their decreasing planarity.
It was also apparent that all compounds with P (para position)-
substitution pattern, including compounds 7a–7g and 7q–7t, had
generally an enhanced quadruplex stabilization compared to those
with M (meta position)-substitution pattern including compounds
7j–7p and 7u–7x. In addition, the DTm values for P-substituted
Fig. 3 (A) Stabilization temperatures of the isaindigotone derivatives. m represents the size of aliphatic ring, n represents the length of side chains, P and
M represent para and meta position substitution, respectively. (B) Concentration dependent melting curves (DTm vs. ligand concentration) for ligands
7a,7e,7h,7j and 7n upon binding to F21T.
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Table 2 Kinetic parameters determined with SPR spectroscopya
SYUID-5a
7e
7h
Htelob
Duplexc
Htelob
Duplexc
Htelob
Duplexc
d
d
d
ka (M-1 s-1)
kd (s-1)
1.53( 0.04) ¥105
2.70( 0.03)¥10-2
1.76 ¥ 10-7
—
—
—
3.87( 0.11)¥103
2.05( 0.02)¥10-3
5.31 ¥ 10-7
—
—
—
3.08( 0.12) ¥102
1.67( 0.02) ¥10-3
5.43 ¥ 10-6
—
—
—
d
d
d
d
d
d
KD (M)
Chi2
7.99
10.7
10.4
a ka is association constant, while kd is dissociation constant. KD denotes the equilibrium dissociation constant, given by kd/ka. The chi2 value is a
standard statistical measure of the closeness of fit. b Htelo (Quadruplex): 5-biotin-[(GTTAGG)5]-3. c Duplex: 5-biotin-T9CGAATTCGT5CGAATTCG-3.
d No significant binding was found for addition of up to 20 mM ligand.
was examined by using a FRET-based competition assay, and their
activity in stabilizing G-quadruplex was challenged with nonfluo-
rescent duplex DNA (ds26). In the presence of varying amount of
competitor ds26, the thermal stabilization of F21T by the selected
compounds was slightly affected (Fig. 4), while the stabilization of
quadruplex by SYUIQ-5 was significantly decreased. The overall
results of these assays demonstrated that isaindigotone derivatives
could selectively bind to the quadruplex over duplex DNA, and
an optimal G-quadruplex ligand should have a five-member-ring,
P-substitution pattern, and double side chain.
SPR assays
The kinetic constants of the compounds SYUID-5a, 7e, and
7h were assessed by surface plasmon resonance (SPR) against
the human telomere and duplex DNA (Table 2, Fig S3). KD
was calculated by global fitting of the kinetic data from various
concentrations of isaindigotone derivatives using 1 : 1 Langmuir
binding. As showed in the Table 2, the binding affinity were in the
order of SYUID-5a > 7e > 7h. This result suggested that increasing
aliphatic ring size decreases the planarity of the compounds, and
consequently resulted in a decrease of their quadruplex binding
affinity. This result is consistent with that from the above FRET
studies. These compounds exhibited good G-quadruplex/duplex
DNA selectivity, with no duplex DNA interaction detected under
the experimental conditions used.
CD studies
Circular dichroism (CD) spectroscopy was used to study the
binding property of the isaindigotone derivatives to telomeric
G-quadruplex.39 In the absence of salt, the CD spectrum of the
human telomeric sequence d[G3(T2AG3)3] (HTG21) was found
to exhibit a negative band at 238 nm, a major positive band at
255 nm, a minor negative band at 278 nm, and a positive band at
295 nm (Fig. 5A).40 Upon addition of excess compound SYUID-5a
Fig. 4 Competitive FRET results for ligands 7a,7e–f, 7j, 7n–o, 7h–i, and
SYUIQ-5 (1 mM), without (black) and with 15-fold (3 mM; dark-gray) or
50-fold (10 mM; light-gray) excess of duplex DNA competitor (ds26). The
concentration of F21T was 0.2 mM.
Fig. 5 (A) CD spectra of 5 mM HTG21 in 10 mM Tris–HCl buffer, pH 7.2, without cations and drug (ꢀ), with 20 mM compound SYUID-5a ( ), 20 mM
᭹
compound 7e ( ), 20 mM compound 7n (ꢁ), and 20 mM compound 7h (᭜). (B) CD spectra of 5 mM HTG21 in 10 mM Tris–HCl buffer, pH 7.2, with
᭺
150 mM K+ (ꢀ), 20 mM compound SYUID-5a ( ), 20 mM compound 7e ( ), 20 mM compound 7n (ꢁ), and 20 mM compound 7h (᭜).
᭹
᭺
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(4 mol equiv) to the HTG21, a dramatic change in the CD spectra
was observed. The bands at 238, 255, and 278 nm disappeared
and a major negative band at 258 nm appeared, while the band
centered at 292 nm significantly increased and shifted toward
286 nm. These changes suggested that the G-rich DNA HTG21
was induced by compound SYUID-5a to form G-quadruplex
structure. In addition, the strong and negative induced CD signal
around 360 nm was additional evidence for the interaction between
the G-quadruplex and SYUID-5a because its appearance in the
CD spectra is indicative of an achiral ligand binding tightly to
a chiral host.41 The CD spectra of the HTG21 oligonucleotide
for the addition of compound 7e and 7h in the absence of salt
were similar to that for the addition of compound SYUID-5a.
However, the band intensity for compound 7h was weaker than
that of compound SYUID-5a and 7e. This result indicated that
compound 7h was less effective in induction of G-quadruplex
formation than compound SYUID-5a and 7e. In addition, upon
addition of compound 7n to the HTG21 oligonucleotide, slight
changes were observed in the CD spectra, where a band centered at
295 nm increased and a band at 255 nm decreased without obvious
band shift. These results indicated that compound 7n could also
induce the telomeric G-rich DNA to form the G-quadruplex
structure, but the ligand was much less effective in initiating the
topological change of G-rich DNA compared to compound 7e
and 7h. Overall, these results suggested that increasing the size of
aliphatic ring had a negative impact on the ability of ligands in
inducing quadruplex formation. The ligands with P-substitution
were more effective in inducing G-quadruplex formation than
the ligands with M-substitution. These results are consistent with
those from FRET and SPR studies.
for SYUID-5a-quadruplex complex rather than 7e-quadruplex
complex and 7h-quadruplex complex. This result is consistent
with those from the above studies. Decreasing ligands’ planarity
resulted in their decreased interactions with quadruplex.
Molecular docking and molecular dynamics simulations were
carried out in order to understand the nature of the ligand–
DNA interactions, and help rationalize the experimental result.44
Molecular docking studies were used to predict the possible inter-
actions between the ligands and G-quadruplex DNA. The NMR-
determined mixed hybrid-type G-quadruplex structure (PDB
2HY9)45 was used as the template for the modeling studies because
it might be a more relevant form in the presence of K+ ion in
dilute solution.46 Our previous studies have shown that compound
SYUID-5a bound to the telomeric quadruplex via multiple binding
modes. Besides the end-stacking mode, groove binding mode
has also been possible. However, the end-stacking mode has
shown higher binding energy than that of groove binding mode
from MM-PBSA calculations, and G-quartet might be the major
binding site for the increased stability of G-quadruplex.11,27 Hence,
the modeling of end-stacking interaction can help rationalize the
FRET and SPR results. In the present study, we only focused on
the end-stacking binding mode. One hundred docking conformers
of ligand-quadruplex complexes from molecular docking studies
showed that ligands predominantly stacked on the 5¢ end of the G-
quadruplex (Fig. S5). Therefore, the most probable conformation
of each ligand on 5¢ end was chosen, which showed a good
stacking arrangement with the G-tetrads. On the basis of the
docking results, MD simulations (10 ns) were performed on two
complexes formed by hybrid-type G-quadruplexes with ligand 7e
and 7h (MD simulation of SYUID-5a-quadruplex complex has
been reported by our group11). Both models were quite stable
during the dynamics runs (Fig. S6). The complexes formed by
G-quadruplex with 7e and 7h are shown in Fig. 6. These two
ligands bound to the G-quartet in a very similar pattern. The
unfused aromatic rings that form the central pharmacophore
stacked over the guanine tetrads with maximized p–p stacking
interactions. The presence of the tertiary amine at the ends of
the side chains contributed to stabilization of the G-quadruplex
by forming hydrogen bonds to phosphate groups. The terminal
tertiary amine is usually protonated at physiological pH, and
the presence of positive charge would enhance its electrostatic
interactions with the negatively charged atoms of the loops.
To study the heat stability of the G-quartet after ligand binding,
we elongated the simulation time for another 2 ns at 350 K. The
hydrogen bond (N1–H ◊ ◊ ◊ N7 and N2–H ◊ ◊ ◊ O6) occupancies of
the G-quartet were calculated as shown in Fig. 7. An interaction
was considered to be a hydrogen bond if the distance between the
It has been reported that HTG21 oligonucleotide has formed the
hybrid-type G-quadruplex structure in the presence of K+, with a
large positive band at 290 nm, a small positive band at 265 nm, and
a negative band at 235 nm (Fig. 5B).29,42 Upon our titration of this
DNA solution with compound SYUID-5a, the band at 265 nm
disappeared, while the band at 257 nm appeared. An additional
induced CD signal around 360 nm was also observed. The CD
spectrum of G-quadruplex for the titration with compound 7e
was similar to that with compound SYUID-5a. However, upon
the addition of compound 7h and 7n into the solution of hybrid
G-quadruplex with K+, no significant change was observed in the
resulting CD spectra.
NMR studies and molecular modeling
To further investigate the binding modes between the isaindigo-
tone derivatives and G-quadruplex structures, nuclear magnetic
resonance (NMR) studies and molecular modeling studies were
performed. The intermolecular four-stranded parallel quadruplex
[d(T2AG3)] derived from human telomeric DNA sequences was
used in this study.43 NMR titration experiments showed most 1H
NMR signals exhibited progressive shifts and line broadening
at increasing ligand concentration (Fig S4). This effect was
slightly more apparent for imino proton signals of G4 in all
complexes. Furthermore, the A3H2, A3H8, and G4H8 imino
proton signals exhibited significant line broadening at increasing
ligand concentration. These results suggested that the studied
ligands preferred to stack between the A3 and G4 residues. In
˚
hydrogen donor and acceptor was less than 3.5 A, and the binding
angle was greater than 120.0◦. The Hoogsteen hydrogen bonds in a
G-quadruplex are the main forces for keeping quartets stable. The
SYUID-5a-quadruplex complex has the highest average hydrogen
bond occupancy (98.56%), the next is 7e-quadruplex complex
(98.45%), and the remaining is 7h-quadruplex complex (97.83%).
These results are consistent with those from FRET studies, which
suggested that a larger aliphatic ring size resulted in a weaker
G-quadruplex stabilization effect.
Besides, the binding free energy was further calculated with
MM-PBSA method. Table 3 shows all of the energy terms given
for each compound. According to Table 3, the calculated binding
1
addition, the changes of H NMR signals were more significant
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Fig. 6 Averaged structures obtained by averaging 50 MD simulation snapshots from the last 2 ns on the MD trajectory with an interval of 20 ps. (A)
7e-quadruplex complex; (B) 7h-quadruplex complex. Pictures were generated by PYMOL.47
Fig. 7 Hydrogen bond occupancies within every G-quartet during MD simulations at 350 K. Occupancy is provided as a percentage of the investigated
time period.
Table 3 Energetic analysis of ligand-quadruplex complex formations (kcal mol-1)a
Compound
DEelec
DEvdw
DGnp
DGPB
DGtot
-TDS
DGbind
SYUID-5a
7e
7h
-913.84
-924.59
-849.18
-54.94
-54.16
-49.75
-6.60
-6.01
-5.64
926.98
936.15
861.69
-48.41
-48.60
-42.88
27.14
29.88
28.46
-21.27
-18.12
-14.42
a DEelec is the electrostatic molecular-mechanical energy. DEvdw is the van der Waals molecular-mechanical energy. DGnp is the nonpolar contribution to
the solvation energy. DGPB is the electrostatic contribution to the solvation energy calculated by the PB approach. DGtot is the total energy without solute
entropic contribution (DEelec + DEvdw + DGnp + DGPB). -TDS is the solute entropic contribution, where T = temperature and S = sum of translational,
rotational, and vibrational entropy. DGbind is the total energy with solute entropic contribution (DGtot - TDS).
free energy (DGbind) for ligands was in agreement with the results
from SPR studies. A detailed analysis suggested that, van der
Waals (DEvdw) and electrostatic energy (DEelec) had major favorable
contribution to the binding, while polar solvation energy (DGPB)
and entropic effect (-TDS) were unfavorable for the binding. On
the other hand, nonpolar solvation energy (DGnp) had slightly
favorable contribution. However, the favorable electrostatic energy
was counteracted by the unfavorable polar solvation energy, and
as a result, the van der Waals energy and the entropic effect became
the major factors. The van der Waals energy for compounds
SYUID-5a and 7e was very similar, while that for compound
7h was ~5 kcal mol-1 higher. This result suggested that both
compound 7e and SYUID-5a stacked on the G-quartet surface
well. However, the seven member aliphatic ring in the central
pharmacophore of compound 7h would interfere with its stacking
interaction with G-quartet surface, thus leading to decreased
binding affinity.
To further investigate the structural feature of compounds that
impact on the G-quadruplex binding properties in detail, the
distance deviations between the quinazoline motifs and G-quartet
surface were calculated (Fig. 8). As shown in Fig. 8B, the distance
deviation was in the order of SYUID-5a-quadruplex complex >
7e-quadruplex complex > 7h-quadruplex complex. The average
distances between the quinazoline motifs and G-quartet surface
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Fig. 8 (A) Distance between the quinazoline motifs and G-quartet surface. (B) Distance deviations between the quinazoline motifs and G-quartet
surface calculated from the last 2 ns trajectories.
˚
˚
˚
were 3.59 A, 3.63 A, and 3.89 A for SYUID-5a-quadruplex,
7e-quadruplex, and 7h-quadruplex complex, respectively. This
result indicated that SYUID-5a stabilized G-quadruplex better
than 7e, and 7e did better than 7h. Due to the steric clashes
between their unfused aromatic core and G-quartet surface,
increasing the aliphatic ring size resulted in increased distance
between the quinazoline motifs and G-quartet surface. This result
again supported the experimental observation that increasing the
aliphatic ring size resulted in decreased stacking interaction with
G-quartet.
400.132, 100.614 and 400.132 MHz, respectively. Mass spectra
(MS) were recorded on a Shimadzu LCMS-2010A instrument with
an ESI or ACPI mass selective detector, and high resolution mass
spectra (HRMS) on Shimadzu LCMS-IT-TOF. Melting points
(mp) were determined using a SRS-OptiMelt automated melting
point instrument without correction.
3-Nitro-8,9-dihydro-6H -pyrido[2,1-b]quinazolin-11(7H)-one
(2b). To a stirred suspension of piperidin-2-one (0.50 g, 5 mmol)
and 2-amino-4-nitrobenzoic acid (1.8 g, 10 mmol) in 250 mL
anhydrous toluene, POCl3 (3 mL) was added dropwise at room
temperature, and the mixture was heated under reflux for 8 h.
The reaction product was poured onto ice, and then concentrated
ammonium hydroxide was added to make the solution basic.
The precipitate was separated from water through filtration, and
the filtrate was extracted with 3 ¥ 100 mL of ethyl acetate. The
combined organic phase was washed with 100 mL of water con-
taining 10 mL of concentrated ammonium hydroxide, dried over
magnesium sulfate, and concentrated to dryness. The resulting
residue combined with previous precipitate was purified using
flash chromatography with cyclohexane/ethyl acetate elution to
afford compound 2b as a pale-yellow solid with a yield of 37%.
Conclusion
A series of isaindigotone derivatives as telomeric quadruplex
ligands were designed, synthesized and characterized by using
CD spectroscopy, FRET-melting assay, and SPR. NMR studies
suggested that these compounds could stack over the G-quartet
surface. Through changing the aliphatic ring size in the middle
core of these ligands, the planarity of main core was adjusted in
three levels. The decreased planarity resulted in their decreased
binding affinity and stabilization ability. Molecular modeling
studies confirmed our hypothesis that the aliphatic ring in a
ligand molecule was the key structural basis responsible for this
effect. Comparing to the fused aromatic ligands with rigid core,
the unfused aromatic ligands are flexible and more suitable to
be accommodated on the dynamic G-quartet surface. However,
there are flexibility limitations for the ligands to achieve both
optimal binding affinity and optimal binding selectivity for G-
quadruplex. In fact, for isaindigotone derivatives, more planar
compounds have generally stronger G-quadruplex binding affinity
and stabilization ability. This study provided insight information
about how quadruplex binding affinity and selectivity could be
adjusted by controlling the planarity of a ligand, which shed light
on further development of isaindigotone derivatives for selective
and potent G-quadruplex binding ligands.
◦
1
Mp: 180–182 C. H NMR (400 MHz, CDCl3) d 8.44 (d, J =
2.1 Hz, 1H), 8.41 (d, J = 8.8 Hz, 1H), 8.17 (dd, J = 8.8, 2.1 Hz,
1H), 4.10 (t, J = 6.2 Hz, 2H), 3.05 (t, J = 6.6 Hz, 2H), 2.10 – 1.95
(m, 4H). ESI-MS m/z calcd C12H11N3O3 [M + H]+ 246.1, found
246.1.
3-Nitro-7,8,9,10-tetrahydroazepino[2,1-b]quinazolin-12(6H)-one
(2c). The method for the preparation of compound 2b was used
except replacing piperidin-2-one with 2-oxohexamethyleneimine.
Compound 2c was synthesized as a pale-yellow solid with a yield
of 25%. Mp: 147–150 ◦C. 1H NMR (400 MHz, CDCl3) d 8.41 (d,
J = 2.1 Hz, 1H), 8.38 (d, J = 8.8 Hz, 1H), 8.16 (dd, J = 8.8, 2.1 Hz,
1H), 4.46 – 4.36 (m, 2H), 3.17 – 3.07 (m, 2H), 1.97 – 1.80 (m, 6H).
ESI-MS m/z calcd C13H13N3O3 [M + H]+ 260.1, found 260.1.
Experimental section
2-Nitro-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-one (2e).
The method for the preparation of compound 2b was used
except replacing 2-amino-4-nitrobenzoic acid with 2-amino-5-
nitrobenzoic acid. Compound 2e was synthesized as a pale-yellow
solid with a yield of 50%. Mp: 182–183 ◦C. 1H NMR (400 MHz,
CDCl3) d 9.13 (d, J = 2.6 Hz, 1H), 8.50 (dd, J = 9.0, 2.6 Hz,
1H), 7.71 (d, J = 9.0 Hz, 1H), 4.11 (t, J = 6.2 Hz, 2H), 3.06 (t,
Synthesis and characterization
All chemicals were obtained from commercial sources unless
1
otherwise specified. H NMR, 13C NMR and NOESY spectra
were recorded using TMS as the internal standard in DMSO-
d6 or CDCl3 with a Bruker BioSpin GmbH spectrometer at
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J = 6.6 Hz, 2H), 2.09–1.96 (m, 4H). ESI-MS m/z calcd C12H11N3O3
4-dimethylaminobenzaldehyde to afford compound 5c as a red
solid with a yield of 81%. Mp: 287–290 ◦C. 1H NMR (400 MHz,
CDCl3) d 9.11 (d, J = 2.0 Hz, 1H), 8.48 (d, J = 9.0 Hz, 1H), 7.87
(s, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.50 (d, J = 8.8 Hz, 2H), 6.75
(d, J = 8.8 Hz, 2H), 4.38–4.30 (m, 2H), 3.35–3.27 (m, 2H), 3.08 (s,
6H). ESI-MS m/z calcd C20H18N4O3 [M + H]+ 363.1, found 363.2.
[M + H]+ 246.1, found 246.1.
(E)-3-Nitro-6-(4-nitrobenzylidene)-8,9-dihydro-6H-pyrido[2,1-
b]quinazolin-11(7H)-one (3b). A mixture of compound 2b
(1.2 g, 5.0 mmol), 4-nitrobenzaldehyde or 4-dimethyl-
aminobenzaldehyde (10 mmol), and NaOAc (0.082 g, 1.0 mmol)
in glacial acetic acid (10 mL) was heated under reflux for 8 h. After
cooling to room temperature, the precipitate was separated from
solvent through filtration and rinsed with chloroform (100 mL)
and acetone (200 mL) to afford compound 3b as a pale-yellow
solid with a yield of 93%. Mp: 276–278 ◦C. 1H NMR (400 MHz,
DMSO) d 8.45 (d, J = 2.1 Hz, 1H), 8.41–8.26 (m, 4H), 8.22 (dd, J =
8.8, 2.1 Hz, 1H), 7.85 (d, J = 8.7 Hz, 2H), 4.15–4.04 (m, 2H), 3.00–
2.88 (m, 2H), 2.07–1.94 (m, 2H). ESI-MS m/z calcd C19H14N4O5
[M + H]+ 379.1, found 379.1.
(E)-6-(4-(Dimethylamino)benzylidene)-2-nitro-8,9-dihydro-6H-
pyrido[2,1-b]quinazolin-11(7H)-one (5d). The method for the
preparation of compound 3b was used. Compound 2e was reacted
with 4-dimethylaminobenzaldehyde to afford compound 5d as
a red solid with a yield of 82%. Mp: 206–208 ◦C. 1H NMR
(400 MHz, CDCl3) d 9.05 (d, J = 2.6 Hz, 1H), 8.40 (dd, J =
9.0, 2.6 Hz, 1H), 8.25 (s, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.42 (d, J =
8.8 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 4.14–4.08 (m, 2H), 2.99 (s,
6H), 2.97–2.91 (m, J = 5.7 Hz, 2H), 2.04–1.95 (m, 2H). ESI-MS
m/z calcd C21H20N4O3 [M + H]+ 377.2, found 377.1.
(E)-3-Nitro-6-(4-nitrobenzylidene)-7,8,9,10-tetrahydroazepino-
[2,1-b]quinazolin-12(6H)-one (3c). The method for the prepara-
tion of compound 3b was used, with compound 2c as starting
material. Compound 3c was synthesized as a pale-yellow solid
with a yield of 42%. Mp: 250–253 ◦C. 1H NMR (400 MHz, CDCl3)
d 8.60 (d, J = 2.1 Hz, 1H), 8.47 (d, J = 8.7 Hz, 1H), 8.30 (d, J =
8.7 Hz, 2H), 8.26 (dd, J = 8.7, 2.1 Hz, 1H), 7.67 (d, J = 8.7 Hz,
2H), 7.23 (s, 1H), 4.38–4.30 (m, 2H), 2.87–2.79 (m, 2H), 2.03–1.90
(m, 4H). ESI-MS m/z calcd C20H16N4O5 [M + H]+ 393.1, found
393.1.
(E)-3-Amino-6-(4-aminobenzylidene)-8,9-dihydro-6H-pyrido[2,
1-b]quinazolin-11(7H)-one (4b). To a stirred suspension of com-
pound 3b (1.9 g, 5.0 mmol) 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 under reflux for 6 h. The
ethanol was removed in vacuo, and the residue was cooled to 0–
5
◦C. The resulting precipitate was collected through filtration,
repeatedly washed with water, and dried to afford compound 4b
as an orange–red solid with a yield of 91%. Mp: 230–232 ◦C. 1H
NMR (400 MHz, DMSO) d 7.91 (s, 1H), 7.73 (d, J = 8.6 Hz, 1H),
7.27 (d, J = 8.5 Hz, 2H), 6.65 (dd, J = 8.6, 2.1 Hz, 1H), 6.61 (d,
J = 8.5 Hz, 2H), 6.60–6.57 (m, 1H), 5.96 (s, 2H), 5.57 (s, 2H),
4.00–3.90 (m, 2H), 2.87–2.78 (m, 2H), 1.93–1.83 (m, 2H). ESI-MS
m/z calcd C19H18N4O [M + H]+ 319.2, found 319.2.
(E)-2-Nitro-6-(4-nitrobenzylidene)-8,9-dihydro-6H-pyrido[2,1-
b]quinazolin-11(7H)-one (3e). The method for the preparation of
compound 3b was used, with compound 2e as starting material.
Compound 3e was synthesized as a pale-yellow solid with a yield
◦
1
of 93%. Mp: 244–247 C. H NMR (400 MHz, DMSO) d 8.84
(d, J = 2.4 Hz, 1H), 8.56 (dd, J = 9.0, 2.4 Hz, 1H), 8.37 (s, 1H),
8.31 (d, J = 8.7 Hz, 2H), 7.90 (d, J = 9.0 Hz, 1H), 7.85 (d, J =
8.7 Hz, 2H), 4.13–4.05 (m, 2H), 2.98–2.91 (m, 2H), 2.04–1.96 (m,
2H). ESI-MS m/z calcd C19H14N4O5 [M + H]+ 379.1, found 379.1.
(E)-3-Amino-6-(4-aminobenzylidene)-7,8,9,10-tetrahydroazepi-
no[2,1-b]quinazolin-12(6H)-one (4c). The method for the prepa-
ration of compound 4b was used, with compound 3c as starting
material. Compound 4c was synthesized as an orange–red solid
with a yield of 61%. Mp: 240–241 ◦C. 1H NMR (400 MHz, DMSO)
d 7.76 (d, J = 8.6 Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H), 6.79 (s, 1H),
6.70 (dd, J = 8.6, 2.2 Hz, 1H), 6.64–6.59 (m, 3H), 6.03 (s, 2H), 5.44
(s, 2H), 4.09–4.03 (m, 2H), 2.70–2.64 (m, 2H), 1.80–1.67 (m, 4H).
ESI-MS m/z calcd C20H20N4O [M + H]+ 333.2, found 333.2.
(E)-3-(4-(Dimethylamino)benzylidene)-6-nitro-2,3-dihydropy-
rrolo[2,1-b]quinazolin-9(1H)-one (5a). The method for the prepa-
ration of compound 3b was used. Compound 2a was reacted with
4-dimethylaminobenzaldehyde to afford compound 5a as a red
solid with a yield of 82%. Mp: 271–272 ◦C. 1H NMR (400 MHz,
CDCl3) d 8.52 (d, J = 2.1 Hz, 1H), 8.38 (d, J = 8.7 Hz, 1H), 8.10
(dd, J = 8.7, 2.1 Hz, 1H), 7.81 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 6.73
(d, J = 8.7 Hz, 2H), 4.34–4.23 (m, 2H), 3.34–3.23 (m, 2H), 3.05 (s,
6H). ESI-MS m/z calcd C20H18N4O5 [M + H]+ 363.1, found 363.1.
(E)-2-Amino-6-(4-aminobenzylidene)-8,9-dihydro-6H-pyrido[2,
1-b]quinazolin-11(7H)-one (4e). The method for the preparation
of compound 4b was used, with compound 3e as starting material.
Compound 4e was synthesized as an orange–red solid with a yield
of 90%. Mp: 207–209 ◦C. 1H NMR (400 MHz, DMSO) d 7.83 (d,
J = 2.0 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.24 (d, J = 8.5 Hz, 2H),
7.18 (s, 1H), 7.07 (dd, J = 8.7, 2.0 Hz, 1H), 6.61 (d, J = 8.5 Hz,
2H), 5.55 (s, 2H), 5.51 (s, 2H), 4.05–3.95 (m, 2H), 2.87–2.79 (m,
2H), 1.94–1.84 (m, 2H). ESI-MS m/z calcd C19H18N4O [M + H]+
319.2, found 319.2.
(E)-6-(4-(Dimethylamino)benzylidene)-3-nitro-8,9-dihydro-6H-
pyrido[2,1-b]quinazolin-11(7H)-one (5b). The method for the
preparation of compound 3b was used except replacing 4-
nitrobenzaldehyde with 4-dimethylaminobenzaldehyde. Com-
pound 5b was◦ synthesized as a red solid with a yield of 78%.
1
Mp: 206–209 C. H NMR (400 MHz, CDCl3) d 8.43 (d, J =
2.1 Hz, 1H), 8.29 (d, J = 8.7 Hz, 1H), 8.17 (s, 1H), 8.01 (dd, J =
8.7, 2.1 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 6.66 (d, J = 8.8 Hz, 2H),
4.14–4.02 (m, 2H), 2.97 (s, 6H), 2.95–2.89 (m, 2H), 2.02–1.93 (m,
2H). ESI-MS m/z calcd C21H20N4O3 [M + H]+ 377.2, found 377.2.
(E)-6-Amino-3-(4-(dimethylamino)benzylidene)-2,3-dihydropy-
rrolo[2,1-b]quinazolin-9(1H)-one (6a). The method for the prepa-
ration of compound 4b was used, with compound 5a as starting
material. Compound 6a was synthesized as an orange–red solid
with a yield of 92%. Mp: 256–258 ◦C. 1H NMR (400 MHz, DMSO)
d 7.77 (d, J = 8.5 Hz, 1H), 7.54 (s, 1H), 7.47 (d, J = 8.8 Hz,
2H), 6.77 (d, J = 8.7 Hz, 2H), 6.69–6.63 (m, 2H), 6.01 (s, 2H),
(E)-3-(4-(Dimethylamino)benzylidene)-7-nitro-2,3-dihydropy-
rrolo[2,1-b]quinazolin-9(1H)-one (5c). The method for the prepa-
ration of compound 3b was used. Compound 2d was reacted with
6430 | Org. Biomol. Chem., 2011, 9, 6422–6436
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4.08–4.03 (m, 2H), 3.17–3.10 (m, 2H), 2.97 (s, 6H). ESI-MS m/z
(E)-N-(4-((11-Oxo-3-(2-(pyrrolidin-1-yl)acetamido)-7,8,9,11-
tetrahydro-6H -pyrido[2,1-b]quinazolin-6-ylidene)methyl)phenyl)-
2-(pyrrolidin-1-yl)acetamide (7b). The method for the prepara-
tion of compound 7a was used, replacing 4a with 4b, and replacing
3-chloropropanoyl chloride with chloracetyl chloride. Compound
7b was synthesized as a pale-yellow solid with a yield of 60%. Mp:
96–98 ◦C. 1H NMR (400 MHz, CDCl3) d 9.45 (s, 1H), 9.25 (s, 1H),
8.18–8.26 (m, 2H), 7.96 (d, J = 1.8 Hz, 1H), 7.62–7.71 (m, 3H), 7.48
(d, J = 8.5 Hz, 2H), 4.19–4.11 (m, 2H), 3.33 (d, J = 9.2 Hz, 4H),
2.90–3.00 (m, 2H), 2.63–2.80 (m, 8H), 1.98–2.08 (m, 2H), 1.82–
1.94 (m, 8H). 13C NMR (101 MHz, CDCl3) d 169.50, 161.67,
152.36, 148.70, 142.92, 137.73, 135.06, 132.02, 131.03, 128.91,
127.81, 119.21, 118.04, 116.06, 115.49, 59.78, 54.62, 42.02, 25.90,
24.09, 22.00. HRMS m/z calcd C31H36N6O3 [M + Na]+ 563.2747,
found 563.2727.
calcd C20H20N4O [M + H]+ 333.2, found 333.1.
(E)-3-Amino-6-(4-(dimethylamino)benzylidene)-8,9-dihydro-
6H-pyrido[2,1-b]quinazolin-11(7H)-one (6b). The method for the
preparation of compound 4b was used, with compound 5b as
starting material. Compound 6b was synthesized as an orange–red
solid with a yield of 86%. Mp: 196–197 ◦C. 1H NMR (400 MHz,
DMSO) d 7.96 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.41 (d, J = 8.6 Hz,
2H), 6.74 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.8 Hz, 1H), 6.62 (s,
1H), 6.02 (s, 2H), 4.02–3.89 (m, 2H), 2.94 (s, 6H), 2.90–2.80 (m,
2H), 1.96–1.82 (m, 2H). ESI-MS m/z calcd C21H22N4O [M + H]+
347.2, found 347.2.
(E)-7-Amino-3-(4-(dimethylamino)benzylidene)-2,3-dihydropy-
rrolo[2,1-b]quinazolin-9(1H)-one (6c). The method for the prepa-
ration of compound 4b was used, with compound 5c as starting
material. Compound 6c was synthesized as an orange–red solid
with a yield of 81%. Mp: 249–253 ◦C. 1H NMR (400 MHz, DMSO)
d 7.48–7.43 (m, 3H), 7.41 (d, J = 8.7 Hz, 1H), 7.22 (d, J = 2.5 Hz,
1H), 7.07 (dd, J = 8.7, 2.5 Hz, 1H), 6.76 (d, J = 8.7 Hz, 2H), 5.59
(s, 2H), 4.10 (t, J = 7.2 Hz, 2H), 3.15 (t, J = 6.3 Hz, 2H), 2.96 (s,
6H). ESI-MS m/z calcd C20H20N4O [M + H]+ 333.2, found 333.1.
(E)-N -(4-((11-Oxo-3-(2-(piperidin-1-yl)acetamido)-7,8,9,11-
tetrahydro-6H -pyrido[2,1-b]quinazolin-6-ylidene)methyl)phenyl)-
2-(piperidin-1-yl)acetamide (7c). The method for the preparation
of compound 7b was used, replacing pyrrolidine with piperidine.
Compound 7c was synthesized as a pale-yellow solid with a yield
of 62%. Mp: 124–125 ◦C. 1H NMR (400 MHz, CDCl3) d 9.64 (s,
1H), 9.43 (s, 1H), 8.26–8.18 (m, 2H), 7.93 (d, J = 2.0 Hz, 1H),
7.71–7.62 (m, 3H), 7.48 (d, J = 8.6 Hz, 2H), 4.20–4.11 (m, 2H),
3.12 (d, J = 7.9 Hz, 4H), 2.96 (t, J = 5.5 Hz, 2H), 2.68–2.48 (m,
8H), 2.09–1.99 (m, 2H), 1.71–1.65 (m, 8H), 1.56–1.46 (m, 4H).
13C NMR (101 MHz, CDCl3) d 169.30, 161.67, 152.45, 148.74,
142.93, 137.74, 135.10, 132.03, 131.08, 128.98, 127.92, 119.11,
117.99, 116.10, 115.38, 62.81, 54.95, 42.03, 26.35, 25.92, 23.61,
22.04. HRMS m/z calcd C33H40N6O3 [M + Na]+ 591.3060, found
591.3053.
(E)-2-Amino-6-(4-(dimethylamino)benzylidene)-8,9-dihydro-
6H-pyrido[2,1-b]quinazolin-11(7H)-one (6d). The method for the
preparation of compound 4b was used, with compound 5d as
starting material. Compound 6d was synthesized as an orange–red
solid with a yield of 79%. Mp: 244–246 ◦C. 1H NMR (400 MHz,
CDCl3) d 8.01 (s, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.47–7.40 (m, 3H),
7.10 (dd, J = 8.7, 2.8 Hz, 1H), 6.73 (d, J = 8.9 Hz, 2H), 5.55(s,
2H),4.18–4.11 (m, 2H), 3.02 (s, 6H), 3.00–2.94 (m, 2H), 2.05–1.96
(m, 2H). ESI-MS m/z calcd C21H22N4O [M + H]+ 347.2, found
347.2.
(E)-2-(Diethylamino)-N-(4-((3-(2-(diethylamino)acetamido)-11-
oxo-7,8,9,11-tetrahydro-6H-pyrido[2,1-b]quinazolin-6-ylidene)me-
thyl)phenyl)acetamide (7d). The method for the preparation of
compound 7b was used, replacing pyrrolidine with diethylamine.
Compound 7d was synthesized as a pale-yellow solid with a yield
of 58%. Mp: 110–112 ◦C. 1H NMR (400 MHz, CDCl3) d 9.74 (s,
1H), 9.54 (s, 1H), 8.26–8.18 (m, 2H), 7.93 (s, 1H), 7.71–7.63 (m,
3H), 7.48 (d, J = 8.2 Hz, 2H), 4.18–4.11 (m, 2H), 3.21 (s, 4H), 2.96
(t, J = 6.1 Hz, 2H), 2.77–2.60 (m, 8H), 2.07–1.99 (m, 2H), 1.12
ppm (t, J = 6.9 Hz, 12H). 13C NMR (101 MHz, CDCl3) d 170.42,
161.67, 152.40, 148.74, 142.87, 137.69, 135.08, 132.03, 131.07,
128.96, 127.93, 119.03, 117.92, 116.10, 115.32, 58.16, 48.91, 42.02,
25.91, 22.03, 12.46. HRMS m/z calcd C31H40N6O3 [M + Na]+
567.3060, found 567.3051.
(E)-3-(Diethylamino)-N-(4-((6-(3-(diethylamino)propanamido)-
9-oxo-1,2-dihydropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)-
phenyl)propanamide (7a). A suspension of compound 4a (0.15
g, 0.50 mmol) in 3-chloropropanoyl chloride (10 mL) was heated
under reflux for 4 h, until TLC indicated completion of reaction.
◦
After cooling to 0–5 C, the mixture was filtered, and the crude
solid was washed with three 15 mL portions of ether to give an
intermediate. To a stirred refluxing suspension of this intermediate
and KI (0.05 g) in EtOH (5 mL) was added dropwise pyrrolidine
(0.35 mL, 5.0 mmol) in EtOH (5 mL). The mixture was stirred
under reflux for 3 h, and 20 mL cool water was added. The solution
was filtered, and washed with ether (10 mL). After recrystallization
from CHCl3-EtOH (2 : 1 v/v), compound 7a was obtained as a
pale-yellow solid with a yield of 69%. Mp: 179–182 ◦C.1H NMR
(400 MHz, CDCl3) d 11.74 (s, 1H), 11.57 (s, 1H), 8.22 (d, J =
8.6 Hz, 1H), 7.83–7.78 (m, 2H), 7.73 (dd, J = 8.6, 1.8 Hz, 1H),
7.63 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 4.28 (d, J = 6.9 Hz,
2H), 3.33–3.23 (m, J = 6.1 Hz, 2H), 2.85–2.77 (m, J = 10.4, 4.8 Hz,
4H), 2.77–2.65 (m, J = 7.2 Hz, 8H), 2.59–2.51 (m, 4H), 1.23–1.12
(m, J = 7.0 Hz, 12H). 13C NMR (101 MHz, CDCl3) d 171.30,
171.06, 160.85, 156.23, 151.02, 144.26, 139.51, 130.81, 130.39,
129.95, 127.45, 119.48, 118.23, 116.37, 115.41, 48.83, 46.00, 43.96,
33.20, 25.54, 11.47. HRMS m/z calcd C32H42N6O3 [M + Na]+
581.3216, found 581.3227.
(E)-N -(4-((11-Oxo-3-(3-(pyrrolidin-1-yl)propanamido)-7,8,9,
11-tetrahydro-6H-pyrido[2,1-b]quinazolin-6-ylidene)methyl)phen-
yl)-3-(pyrrolidin-1-yl)propanamide (7e). The method for the
preparation of compound 7b was used, replacing chloroacetyl
chloride with 3-chloropropionyl chloride. Compound 7e was
synthesized as a pale-yellow solid with a yield of 73%. Mp: 146–
◦
1
148 C. H NMR (400 MHz, CDCl3) d 11.68 (s, 1H), 11.45 (s,
1H), 8.23–8.13 (m, 2H), 7.83 (d, J = 1.8 Hz, 1H), 7.62–7.51 (m,
3H), 7.45 (d, J = 8.5 Hz, 2H), 4.18–4.10 (m, 2H), 2.95 (t, J =
5.6 Hz, 2H), 2.92–2.84 (m, 4H), 2.78–2.65 (m, 8H), 2.61–2.53 (m,
4H), 2.06–2.00 (m, 2H), 1.98–1.88 (m, 8H). 13C NMR (101 MHz,
CDCl3) d 171.31, 171.02, 161.69, 152.50, 148.76, 144.24, 138.98,
135.24, 131.54, 131.07, 128.80, 127.73, 119.33, 118.52, 115.70,
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115.57, 53.16, 51.33, 41.94, 34.73, 25.95, 23.76, 22.11. HRMS
1.70 (m, 8H), 1.62–1.53 (m, 4H). 13C NMR (101 MHz, CDCl3)
d 171.23, 170.88, 161.17, 159.35, 149.10, 144.3, 138.76, 135.81,
133.63, 131.21, 130.50, 127.99, 119.20, 118.82, 116.30, 115.73,
54.24, 53.65, 42.39, 32.61, 28.98, 26.19, 26.10,24.21, 24.12. HRMS
m/z calcd C36H46N6O3 [M + Na]+ 633.3529, found 633.3544.
m/z calcd C33H40N6O3 [M + Na]+ 591.3060, found 591.3065.
(E)-N-(4-((11-Oxo-3-(3-(piperidin-1-yl)propanamido)-7,8,9,11-
tetrahydro-6H -pyrido[2,1-b]quinazolin-6-ylidene)methyl)phenyl)-
3-(piperidin-1-yl)propanamide (7f). The method for the prepa-
ration of compound 7e was used, replacing pyrrolidine with
piperidine. Compound 7f was sy◦nthesized as a pale-yellow solid
with a yield of 76%. Mp: 142–143 C. 1H NMR (400 MHz, CDCl3)
d 11.77 (s, 1H), 11.60 (s, 1H), 8.23–8.14 (m, 2H), 7.97–7.90 (m, 1H),
7.66–7.55 (m, J = 8.5 Hz, 3H), 7.47 (d, J = 8.5 Hz, 2H), 4.18–4.11
(m, 2H), 2.96 (t, J = 5.7 Hz, 2H), 2.80–2.46 (m, 16H), 2.07–2.01 (m,
2H), 1.81–1.70 (m, 8H), 1.67–1.50 (m, 4H). 13C NMR (101 MHz,
CDCl3) d 171.25, 170.96, 161.68, 152.45, 148.81, 144.20, 139.01,
135.25, 131.50, 131.10, 128.77, 127.71, 119.15, 118.36, 115.60,
54.24, 53.64, 41.93, 32.61, 26.24, 25.94, 24.17, 22.09. HRMS m/z
calcd C35H44N6O3 [M + H]+ 597.3567, found 597.3553.
(E)-3-(Diethylamino)-N-(4-((7-(3-(diethylamino)propanamido)-
9-oxo-1,2-dihydropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)methyl)-
phenyl)propanamide (7j). The method for the preparation of
compound 7a was used, replacing compound 4a with compound
4d. Compound 7j was synthesized as a pale-yellow solid with a
◦
1
yield of 67%. Mp: 220–222 C. H NMR (400 MHz, CDCl3) d
11.63 (s, 1H), 11.57 (s, 1H), 8.29 (dd, J = 8.9, 2.1 Hz, 1H), 8.09 (s,
J = 2.1 Hz, 1H), 7.77 (s, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.63 (d, J =
8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 4.29 (t, J = 7.1 Hz, 2H), 3.29
(d, J = 7.1 Hz, 2H), 2.85–2.77 (m, 4H), 2.77–2.66 (m, 8H), 2.58–
2.51 (m, 4H), 1.21–1.13 (m, 12H). 13C NMR (101 MHz, CDCl3)
d 171.12, 160.99, 154.63, 145.92, 139.47, 137.21, 130.86, 130.75,
129.86, 129.81, 128.11, 126.91, 121.17, 119.50, 114.98, 48.86,
46.00, 44.02, 33.13, 25.58, 11.52. HRMS m/z calcd C32H42N6O3
[M + H]+ 559.3397, found 559.3390.
(E)-3-(Diethylamino)-N-(4-((3-(3-(diethylamino)propanamido)-
11-oxo-7,8,9,11-tetrahydro-6H-pyrido[2,1-b]quinazolin-6-ylidene)-
methyl)phenyl)propanamide (7g). The method for the prepa-
ration of compound 7e was used, replacing pyrrolidine with
diethylamine. Compound 7g was synthesized as a pale-yellow solid
with a yield of 70%. Mp: 101–104 ◦C. 1H NMR (400 MHz, CDCl3)
d 11.69 (s, 1H), 11.45 (s, 1H), 8.19 (d, J = 8.7 Hz, 1H), 8.16 (s,
1H), 7.85 (d, J = 1.8 Hz, 1H), 7.63 (dd, J = 8.7, 1.8 Hz, 1H),
7.60 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 4.18–4.10 (m,
2H), 2.95 (t, J = 5.5 Hz, 2H), 2.86–2.78 (m, 4H), 2.78–2.66 (m,
8H), 2.61–2.51 (m, 4H), 2.05–1.98 (m, 2H), 1.24–1.10 (m, 12H).
13C NMR (101 MHz, CDCl3) d 171.20, 170.87, 161.65, 152.44,
148.78, 144.08, 138.85, 135.20, 131.54, 131.06, 128.79, 127.74,
119.15, 118.35, 115.71, 115.41, 48.86, 46.00, 41.90, 33.20, 25.92,
22.10, 11.34. HRMS m/z calcd C33H44N6O3 [M + Na]+ 595.3373,
found 595.3386.
(E)-N-(4-((11-Oxo-2-(2-(pyrrolidin-1-yl)acetamido)-7,8,9,11-
tetrahydro-6H -pyrido[2,1-b]quinazolin-6-ylidene)methyl)phenyl)-
2-(pyrrolidin-1-yl)acetamide (7k). The method for the prepara-
tion of compound 7b was used, replacing 4b with 4e. Compound
7k was sy◦nthesized as a pale-yellow solid with a yield of 52%. Mp:
1
113–115 C. H NMR (400 MHz, CDCl3) d 9.46 (s, 1H), 9.26
(s, 1H), 8.41 (dd, J = 8.9, 2.3 Hz, 1H), 8.15 (s, 1H), 8.07 (d, J =
2.3 Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.47
(d, J = 8.5 Hz, 2H), 4.23–4.12 (m, 2H), 3.38–3.27 (m, 4H), 2.97
(t, J = 5.6 Hz, 2H), 2.81–2.67 (m, 8H), 2.11–1.98 (m, 2H), 1.94–
1.86 (m, 8H). 13C NMR (101 MHz, CDCl3) d 168.32, 160.88,
149.88, 143.14, 136.70, 135.19, 133.57, 131.07, 129.98, 128.04,
127.38, 125.60, 119.32, 118.19, 114.21, 58.73, 53.65, 41.24, 24.90,
23.11, 21.06. HRMS m/z calcd C31H36N6O3 [M + Na]+ 563.2750,
found 563.2750.
(E)-N -(4-((12-Oxo-3-(3-(pyrrolidin-1-yl)propanamido)-7,8,9,
10-tetrahydroazepino[2,1-b]quinazolin-6(12H)-ylidene)methyl)ph-
enyl)-3-(pyrrolidin-1-yl)propanamide (7h). The method for the
preparation of compound 7e was used, replacing compound 4b
with compound 4c. Compound 7h was synthesized as apale-yellow
solid with a yield of 57%. Mp: 138–140 ◦C. 1H NMR (400 MHz,
CDCl3) d 11.61 (s, 1H), 11.32 (s, 1H), 8.14 (d, J = 8.7 Hz, 1H), 7.70
(d, J = 8.7 Hz, 1H), 7.66 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.38 (d,
J = 8.4 Hz, 2H), 6.98 (s, 1H), 4.27–4.16 (m, 2H), 2.85–2.77 (m, 4H),
2.76–2.70 (m, 2H), 2.68–2.59 (m, 8H), 2.53–2.47 (m, 4H), 1.89–
1.77 (m, 12H). 13C NMR (101 MHz, CDCl3) d 170.30, 169.94,
160.14, 158.38, 147.98, 143.34, 137.69, 134.82, 132.55, 130.20,
129.44, 127.00, 118.36, 117.91, 115.25, 114.73, 52.14, 50.31, 41.39,
33.69, 27.94, 25.08, 23.21, 22.72. HRMS m/z calcd C32H42N6O3
[M + Na]+ 605.3216, found 605.3242.
(E)-N -(4-((11-Oxo-2-(2-(piperidin-1-yl)acetamido)-7,8,9,11-
tetrahydro-6H -pyrido[2,1-b]quinazolin-6-ylidene)methyl)phenyl)-
2-(piperidin-1-yl)acetamide (7l). The method for the preparation
of compound 7c was used, replacing compound 4b with compound
4e. Compound 7l was synth◦esized as a pale-yellow solid with a
1
yield of 53%. Mp: 169–173 C. H NMR (400 MHz, CDCl3) d
9.61 (s, 1H), 9.46 (s, 1H), 8.39 (dd, J = 8.9, 2.3 Hz, 1H), 8.16
(s, 1H), 8.09 (s, J = 2.3 Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.66
(d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 4.21–4.14 (m,
2H), 3.23–3.07 (m, 4H), 2.97 (t, J = 5.6 Hz, 2H), 2.69–2.52 (m,
8H), 2.08–2.00 (m, 2H), 1.77–1.64 (m, 8H), 1.57–1.48 (m, 4H).
13C NMR (101 MHz, CDCl3) d 168.87, 161.86, 150.98, 144.17,
137.68, 136.15, 134.57, 132.17, 130.98, 129.07, 128.38, 126.58,
120.36, 119.12, 115.19, 62.71, 54.94, 42.25, 26.19, 25.89, 23.54,
22.06. HRMS m/z calcd C33H40N6O3 [M + Na]+ 591.3060, found
591.3060.
(E)-N-(4-((12-Oxo-3-(3-(piperidin-1-yl)propanamido)-7,8,9,10-
tetrahydroazepino[2,1-b]quinazolin-6(12H)-ylidene)methyl)phen-
yl)-3-(piperidin-1-yl)propanamide (7i). The method for the
preparation of compound 7h was used, replacing pyrrolidine with
piperidine. Compound 7i was sy◦nthesized as a pale-yellow solid
with a yield of 55%. Mp: 145–147 C. 1H NMR (400 MHz, CDCl3)
d 11.72 (s, 1H), 11.53 (s, 1H), 8.23 (d, J = 8.7 Hz, 1H), 7.84 (s,
1H), 7.77 (s, J = 8.7 Hz, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.47 (d, J =
8.3 Hz, 2H), 7.07 (s, 1H), 4.33–4.24 (m, 2H), 2.86–2.79 (m, 2H),
2.72–2.66 (m, 4H), 2.65–2.48 (m, 12H), 1.95–1.85 (m, 4H), 1.77–
(E)-2-(Diethylamino)-N-(4-((2-(2-(diethylamino)acetamido)-11-
oxo-7,8,9,11-tetrahydro-6H-pyrido[2,1-b]quinazolin-6-ylidene)me-
thyl)phenyl)acetamide (7m). The method for the preparation of
compound 7d was used, replacing 4b with 4e. Compound 7m was
synt◦hesized as a pale-yellow solid with a yield of 53%. Mp: 107–
108 C. 1H NMR (400 MHz, CDCl3) d 9.72 (s, 1H), 9.54 (s, 1H),
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8.41 (dd, J = 8.9, 2.3 Hz, 1H), 8.16 (s, 1H), 8.06 (s, J = 2.3 Hz,
1H), 7.73 (d, J = 8.9 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.48 (d,
J = 8.6 Hz, 2H), 4.20–4.13 (m, 2H), 3.25–3.12 (m, J = 4.5 Hz, 4H),
3.00–2.93 (m, 2H), 2.74–2.61 (m, 8H), 2.08–1.99 (m, 2H), 1.15–
1.08 (m, 12H). 13C NMR (101 MHz, CDCl3) d 170.31, 161.90,
150.87, 144.16, 137.67, 136.16, 134.59, 132.08, 131.03, 129.07,
128.44, 126.48, 120.38, 119.04, 115.06, 58.11, 48.96, 42.28, 25.92,
22.09, 12.45. HRMS m/z calcd C31H40N6O3 [M + Na]+ 567.3060,
found 567.3060.
(E)-N-(3-(4-(Dimethylamino)benzylidene)-9-oxo-1,2,3,9-tetra-
hydropyrrolo[2,1-b]quinazolin-6-yl)-3-(pyrrolidin-1-yl)propanami-
de (7q). The method for the preparation of compound 7a
was used, replacing diethylamide with pyrrolidine, and replacing
compound 4a with compound 6a. Compound 7q was synthesized
as a yellow solid with a yield of 65%. Mp: 194–196 ◦C. 1H NMR
(400 MHz, CDCl3) d 11.49 (s, 1H), 8.20 (d, J = 8.7 Hz, 1H),
7.76 (s, 1H), 7.73 (d, J = 1.9 Hz, 1H), 7.68 (dd, J = 8.7, 1.9 Hz,
1H), 7.49 (d, J = 8.9 Hz, 2H), 6.75 (d, J = 8.9 Hz, 2H), 4.26 (t,
J = 6.9 Hz, 2H), 3.29–3.23 (m, 2H), 3.04 (s, 6H), 2.96–2.91 (m,
2H), 2.82–2.73 (m, 4H), 2.67–2.59 (m, 2H), 2.00–1.94 (m, 4H).
13C NMR (101 MHz, CDCl3) d 171.34, 161.02, 156.94, 151.21,
150.59, 144.30, 131.62, 131.33, 127.39, 125.81, 123.64, 117.94,
116.21, 115.36, 111.94, 53.18, 51.34, 44.00, 40.12, 34.80, 25.54,
23.78. HRMS m/z calcd C27H31N5O2 [M + H]+ 458.2556, found
458.2552.
(E)-N -(4-((11-Oxo-2-(3-(pyrrolidin-1-yl)propanamido)-7,8,9,
11-tetrahydro-6H-pyrido[2,1-b]quinazolin-6-ylidene)methyl)phen-
yl)-3-(pyrrolidin-1-yl)propanamide (7n). The method for the
preparation of compound 7e was used, replacing compound 4b
with compound 4e. Compound 7n was synthesized as apale-yellow
solid with a yield of 65%. Mp: 192–194 ◦C. 1H NMR (400 MHz,
CDCl3) d 11.56 (s, 1 H), 11.45 (s, 1H), 8.24 (dd, J = 8.8, 1.9 Hz,
1H), 8.13 (s, 1H), 8.01 (d, J = 1.9 Hz, 1H), 7.69 (d, J = 8.8 Hz,
1H), 7.55 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 4.22–
4.09 (m, 2H), 3.01–2.93 (m, 2H), 2.93–2.83 (m, 4H), 2.78–2.65 (m,
8H), 2.63–2.52 (m, 4H), 2.06–1.99 (m, 2H), 1.96–1.89 (m, 8H).
13C NMR (101 MHz, CDCl3) d 170.89, 161.86, 150.72, 143.86,
138.88, 137.24, 134.57, 131.57, 130.97, 128.83, 128.18, 127.10,
120.33, 119.31, 115.35, 53.18, 51.36, 42.17, 34.61, 25.92, 23.75,
22.10. HRMS m/z calcd C33H40N6O3 [M + Na]+ 591.3060, found
591.3057.
(E)-N-(3-(4-(Dimethylamino)benzylidene)-9-oxo-1,2,3,9-tetra-
hydropyrrolo[2,1-b]quinazolin-6-yl)-3-(piperidin-1-yl)propanamide
(7r). The method for the preparation of compound 7q was
used, replacing pyrrolidine with piperidine. Compound 7r was
synthesized as a yellow solid with a yield of 61%. Mp: 202–204 ◦C.
1H NMR (400 MHz, CDCl3) d 11.58 (s, 1H), 8.13 (d, J = 8.7 Hz,
1H), 7.77 (d, J = 1.9 Hz, 1H), 7.70 (s, 1H), 7.59 (dd, J = 8.7,
1.9 Hz, 1H), 7.41 (d, J = 8.9 Hz, 2H), 6.67 (d, J = 8.9 Hz, 2H),
4.22–4.14 (m, 2H), 3.22–3.14 (m, 2H), 2.96 (s, 6H), 2.67–2.45 (m,
8H), 1.74–1.64 (m, 4H), 1.57–1.47 (m, 2H). 13C NMR (101 MHz,
CDCl3) d 170.14, 159.99, 155.88, 150.30, 149.56, 143.22, 130.60,
130.31, 126.35, 124.82, 122.65, 116.77, 115.23, 114.36, 110.91,
53.22, 52.69, 42.96, 39.10, 31.68, 25.18, 24.53, 23.14. HRMS m/z
calcd C28H33N5O2 [M + H]+ 472.2713, found 472.2711.
(E)-N-(4-((11-Oxo-2-(3-(piperidin-1-yl)propanamido)-7,8,9,11-
tetrahydro-6H -pyrido[2,1-b]quinazolin-6-ylidene)methyl)phenyl)-
3-(piperidin-1-yl)propanamide (7o). The method for the prepa-
ration of compound 7f was used, replacing compound 4b with
compound 4e. Compound 7o was synt◦hesized as a pale-yellow
solid with a yield of 67%. Mp: 198–199 C. 1H NMR (400 MHz,
CDCl3) d 11.71 (s, 1H), 11.55 (s, 1H), 8.28 (dd, J = 8.9, 2.1 Hz,
1H), 8.13 (s, 1H), 8.09 (s, J = 2.1 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H),
7.61 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 4.22–4.10 (m,
2H), 2.96 (t, J = 6.0 Hz, 2H), 2.78–2.43 (m, 16H), 2.07–1.99 (m,
2H), 1.80–1.70 (m, 8H), 1.64–1.53 (m, 4H). 13C NMR (101 MHz,
CDCl3) d 170.89, 161.88, 150.75, 143.85, 138.96, 137.34, 134.60,
131.57, 131.04, 128.85, 128.26, 126.92, 120.40, 119.18, 115.15,
54.30, 53.68, 42.18, 32.56, 26.22, 25.96, 24.17, 22.14. HRMS m/z
calcd C35H44N6O3 [M + Na]+ 619.3373, found 619.3391.
(E)-N-(6-(4-(Dimethylamino)benzylidene)-11-oxo-7,8,9,11-tet-
rahydro-6H-pyrido[2,1-b]quinazolin-3-yl)-3-(pyrrolidin-1-yl)prop-
anamide (7s). The method for the preparation of compound 7q
was used, replacing compound 6a with compound 6b. Compound
7s was synthesized as a yellow solid with a yield of 73%. Mp:
181–183 ◦C. 1H NMR (400 MHz, CDCl3) d 11.53 (s, 1H), 8.17 (d,
J = 8.7 Hz, 1H), 8.13 (s, 1H), 7.82 (d, J = 1.4 Hz, 1H), 7.56 (dd,
J = 8.7, 1.4 Hz, 1H), 7.45 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.7 Hz,
2H), 4.18–4.08 (m, 2H), 3.02 (s, 6H), 3.00–2.91 (m, 4H), 2.80–
2.72 (m, 4H), 2.68–2.60 (m, 2H), 2.08–1.99 (m, 2H), 1.98–1.91
(m, 4H). 13C NMR (101 MHz, CDCl3) d 171.07, 161.76, 153.22,
150.21, 148.99, 144.06, 136.28, 132.00, 127.70, 125.19, 124.37,
118.10, 115.49, 111.68, 53.24, 51.34, 41.72, 40.18, 34.72, 26.25,
23.73, 22.19. HRMS m/z calcd C28H33N5O2 [M + H]+ 472.2713,
found 472.2714.
(E)-3-(Diethylamino)-N-(4-((2-(3-(diethylamino)propanamido)-
11-oxo-7,8,9,11-tetrahydro-6H-pyrido[2,1-b]quinazolin-6-ylidene)-
methyl)phenyl)propanamide (7p). The method for the prepara-
tion of compound 7g was used, replacing compound 4b with
compound 4e. Compound 7p was synt◦hesized as a pale-yellow
solid with a yield of 64%. Mp: 173–174 C. 1H NMR (400 MHz,
CDCl3) d 11.61 (s, 1H), 11.47 (s, 1H), 8.29 (dd, J = 8.9, 2.3 Hz,
1H), 8.13 (s, 1H), 8.04 (d, J = 2.3 Hz, 1H), 7.69 (d, J = 8.9 Hz,
1H), 7.59 (d, J = 8.5 Hz, 2H), 7.44 (d, J = 8.5 Hz, 2H), 4.19–
4.11 (m, 2H), 2.95 (t, J = 5.8 Hz, 2H), 2.84–2.76 (m, 4H), 2.76–
2.65 (m, 8H), 2.59–2.49 (m, 4H), 2.07–1.98 (m, 2H), 1.22–1.11
(m, 12H).13C NMR (101 MHz, CDCl3) d 171.02, 161.87, 150.71,
143.85, 138.80, 137.18, 134.57, 131.55, 130.99, 128.80, 128.23,
126.95, 120.37, 119.14, 115.05, 48.87, 46.01, 42.16, 33.12, 25.92,
22.11, 11.44. HRMS m/z calcd C33H44N6O3 [M + H]+ 573.3508,
found 573.3571.
(E)-N-(6-(4-(Dimethylamino)benzylidene)-11-oxo-7,8,9,11-tet-
rahydro-6H-pyrido[2,1-b]quinazolin-3-yl)-3-(piperidin-1-yl)prop-
anamide (7t). The method for the preparation of compound 7s
was used, replacing pyrrolidine with piperidine. Compound 7t
was ◦synthesized as a yellow solid with a yield of 74%. Mp: 196–
197 C. 1H NMR (400 MHz, CDCl3) d 11.66 (s, 1H), 8.18 (d, J =
8.7 Hz, 1H), 8.15 (s, 1H), 7.91 (d, J = 1.8 Hz, 1H), 7.56 (dd, J =
8.7, 1.8 Hz, 1H), 7.46 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz,
2H), 4.17–4.10 (m, 2H), 3.03 (s, 6H), 2.97 (t, J = 5.8 Hz, 2H),
2.74–2.69 (m, 2H), 2.67–2.51 (m, 6H), 2.04–1.98 (m, 2H), 1.80–
1.73 (m, 4H), 1.64–1.52 (m, 2H). 13C NMR (101 MHz, CDCl3)
d 171.20, 161.77, 153.19, 150.21, 149.07, 144.09, 136.30, 132.00,
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127.71, 125.24, 124.39, 117.97, 115.46, 111.68, 54.25, 53.69, 41.71,
40.18, 32.69, 26.24, 24.16, 22.20. HRMS m/z calcd C29H35N5O2
[M + H]+ 486.2869, found 486.2864.
151.53, 150.16, 144.22, 136.72, 135.62, 131.89, 128.03, 126.86,
125.27, 124.41, 120.04, 115.30, 111.68, 54.15, 53.72, 41.94, 40.16,
32.44, 26.22, 25.67, 23.83, 22.19. HRMS m/z calcd C29H35N5O2
[M + H]+ 486.2869, found 486.2874.
(E)-N-(3-(4-(Dimethylamino)benzylidene)-9-oxo-1,2,3,9-tetra-
hydropyrrolo[2,1-b]quinazolin-7-yl)-3-(pyrrolidin-1-yl)propanami-
de (7u). The method for the preparation of compound 7q was
used, replacing compound 6a with compound 6c. Compound 7u
was◦synthesized as a yellow solid with a yield of 58%. Mp: 240–
242 C. 1H NMR (400 MHz, CDCl3) d 11.39 (s, 1H), 8.21 (dd, J =
8.9, 2.1 Hz, 1H), 8.05 (d, J = 2.1 Hz, 1H), 7.74 (s, 1H), 7.69 (d,
J = 8.9 Hz, 1H), 7.48 (d, J = 8.9 Hz, 2H), 6.75 (d, J = 8.9 Hz, 2H),
4.30–4.24 (m, 2H), 3.31–3.24 (m, 2H), 3.04 (s, 6H), 2.95–2.89 (m,
2H), 2.81–2.70 (m, 4H), 2.65–2.58 (m, 2H), 1.98–1.92 (m, 4H).
13C NMR (101 MHz, CDCl3) d 171.02, 161.12, 155.36, 150.54,
146.27, 136.77, 131.52, 130.74, 127.81, 127.01, 125.79, 123.73,
120.87, 115.32, 111.97, 53.24, 51.42, 44.04, 40.14, 34.60, 25.57,
23.75. HRMS m/z calcd C27H31N5O2 [M + H]+ 458.2556, found
458.2543.
Biological activity and molecular modeling
FRET-melting assay. FRET assay was carried out on
a real-time PCR apparatus following previously published
procedures. The fluorescently labeled oligonucleotide F21T
[5¢-FAM-d(GGG[TTAGGG]3)-TAMRA-3¢] and F10T [5¢-FAM-
dTATAGCTATA-HEG-TATA-GCTATA-TAMRA-3¢]
(HEG
linker: [(-CH2-CH2-O-)6]) were used as G-quadruplex and duplex
model respectively. Fluorescence melting curves were determined
with a Roche LightCycler 2 real-time PCR machine, using a total
reaction volume of 20 mL, with 0.2 mM 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¢-GTTAGCCTAGCTTAAGCTAGGCTAAC-
3¢]. Final analysis of the data was carried out using Origin 7.5
(OriginLab Corp.).
(E)-N-(3-(4-(Dimethylamino)benzylidene)-9-oxo-1,2,3,9-tetra-
hydropyrrolo[2,1-b]quinazolin-7-yl)-3-(piperidin-1-yl)propanamide
(7v). The method for the preparation of compound 7u was
used, replacing pyrrolidine with piperidine. Compound 7v was
synthesized as a yellow solid with a yield of 56%. Mp: 243–245 ◦C.
1H NMR (400 MHz, CDCl3) d 11.60 (s, 1H), 8.18 (dd, J = 8.9,
2.4 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.66 (s, 1H), 7.62 (d, J =
8.9 Hz, 1H), 7.41 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H),
4.24–4.14 (m, 2H), 3.24–3.15 (m, 2H), 2.96 (s, 6H), 2.69–2.39 (m,
8H), 1.74–1.62 (m, 4H), 1.55–1.45 (m, 2H). 13C NMR (101 MHz,
CDCl3) d 169.88, 160.07, 154.29, 149.50, 145.18, 135.84, 130.48,
129.67, 126.83, 125.73, 124.78, 122.71, 119.88, 114.04, 110.94,
53.28, 52.6, 42.98, 39.10, 31.48, 25.22, 24.55, 23.19. HRMS m/z
calcd C28H33N5O2 [M + H]+ 472.2713, found 472.2705.
CD measurements
The oligomer HTG21 [5¢-d(GGG[TTAGGG]3)-3¢] was diluted
from stock to the required concentration (5 mM) in Tris-HCl
buffer (10 mM, pH 7.2) with or without 150 mM KCl, and
then annealed by heating to 90 ◦C for 5 min, gradually cooled
to room temperature, and incubated at 4 ◦C overnight. CD
experiments were performed on a Chirascan circular dichroism
spectrophotometer (Applied Photophysics). A quartz cuvette with
4 mm path length was used for the spectra recorded over a
wavelength range of 230–450 nm at 1 nm bandwidth, 1 nm step
size, and 0.5 s time per point. 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)-N-(6-(4-(Dimethylamino)benzylidene)-11-oxo-7,8,9,11-tet-
rahydro-6H-pyrido[2,1-b]quinazolin-2-yl)-3-(pyrrolidin-1-yl)prop-
anamide (7w). The method for the preparation of compound 7u
was used, replacing compound 6c with compound 6d. Compound
7w was synthesized as a yellow solid with a yield of 60%. Mp: 218–
220 ◦C. 1H NMR (400 MHz, CDCl3) d 11.26 (s, 1H), 8.20 (dd, J =
8.9, 2.3 Hz, 1H), 8.10 (s, 1H), 8.04 (d, J = 2.3 Hz, 1H), 7.67 (d,
J = 8.9 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H),
4.21–4.11 (m, 2H), 3.03 (s, 6H), 3.01–2.92 (m, 4H), 2.85–2.73 (m,
4H), 2.70–2.60 (m, 2H), 2.07–1.99 (m, 2H), 1.99–1.91 (m, 4H).
13C NMR (101 MHz, CDCl3) d 168.99, 161.92, 151.60, 150.15,
144.43, 136.27, 135.63, 131.92, 127.93, 126.99, 125.23, 124.41,
119.90, 115.77, 111.68, 53.83, 51.37, 41.98, 40.16, 33.89, 26.21,
23.52, 22.13. HRMS m/z calcd C28H33N5O2 [M + H]+ 472.2713,
found 472.2718.
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. Oligonucleotides
used were Biotin-Htelo (Quadruplex): 5-biotin-[(GTTAGG)5]-
3, Biotin-Atelo (single-stranded control): 5-biotin-[(AGTTAG)5]-
3, and Biotin-duplex: 5-biotin-T9CGAATTCGT5CGAATTCG-3.
In a typical experiment, biotinylated DNA was folded in filtered
and degassed running buffer (50 mM Tris-HCl, pH 7.2, 100 mM
KCl). The DNA samples were then captured (~1000 RU) in flow
cell 1, leaving the fourth flow cell as a blank. Ligand solutions were
prepared with running buffer through serial dilutions of stock
solution. Five concentrations were injected simultaneously at a
flow rate of 100 mL min-1 for 300 s (or 400 s) of association
phase, followed with 300 s (or 400 s) of dissociation phase
at 25 ◦C. The GLH sensor chip was regenerated with short
(E)-N-(6-(4-(Dimethylamino)benzylidene)-11-oxo-7,8,9,11-tet-
rahydro-6H-pyrido[2,1-b]quinazolin-2-yl)-3-(piperidin-1-yl)prop-
anamide (7x). The method for the preparation of compound 7w
was used, replacing pyrrolidine with piperidine. Compound 7x was
synthesized as a yellow solid with a yield of 62%. Mp: 215–217 ◦C.
1H NMR (400 MHz, CDCl3) d 11.48 (s, 1H), 8.27–8.08 (m, 3H),
7.70–7.64 (m, 1H), 7.45 (d, J = 8.9 Hz, 2H), 6.74 (d, J = 8.9 Hz,
2H), 4.19–4.12 (m, 2H), 3.03 (s, 6H), 3.01–2.95 (m, 2H), 2.87–2.76
(m, 2H), 2.73–2.58 (m, 4H), 2.06–1.98 (m, 2H), 1.86–1.72 (m, 4H),
1.65–1.51 (m, 4H). 13C NMR (101 MHz, CDCl3) d 170.34, 161.89,
6434 | Org. Biomol. Chem., 2011, 9, 6422–6436
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57
˚
injection of 50 mM NaOH between consecutive measurements.
The final graphs were obtained by subtracting black and single-
stranded control sensorgrams from quadruplex sensorgrams. Data
were analyzed with ProteOn manager software, using the kinetic
method for kinetic data fitting.
2.025 A. A total of 100 snapshots were taken from the last
2 ns trajectory with an interval of 20 ps. Each snapshot was
minimized using the conjugate gradient method for 10,000 steps
with a distance-dependent dielectric constant of 4 inter-atomic
distances. The terminating criterion for minimization was set to
0.01 kJ mol-1 A gradient RMS. To investigate the heat stability of
˚
the ligand-quadruplex complex, we elongated the simulation time
for another 2 ns at 350 K.
NMR spectroscopy
NMR experiments were performed on a Bruker 600 MHz spec-
trometer. 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 7.2). The oligonucleotide
d(T2AG3) was purified by HPLC, and the concentration was
0.5 mM for the NMR measurements.
Acknowledgements
We thank the Natural Science Foundation of China (Grants
U0832005, 90813011, 81001400), the International S&T Co-
operation 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.
Molecular modeling
The NMR structure of the mixed hybrid-type 26-mer telomeric
G-quadruplex (PDB 2HY9))45 was used as the initial template
to study the interactions between isaindigotone derivatives and
telomeric DNA. For comparison with the d(GGG[TTAGGG]3)
DNA used in the FRET and CD experiments, we removed five
adenines from 5¢ end of the hybrid-type quadruplex to generate
the 21-mer structure. Ligand structures were constructed with
SYBYL7.3 (Tripos Inc., St Louis, MO, USA) and optimized with
GAUSSIAN 03 using the HF/6-31G* basis set.48 Docking studies
were performed using the AUTODOCK 4.0 program.49 The
dimensions of the active site box were chosen to be large enough to
encompass the entire DNA molecule. Docking calculations were
carried out using the Lamarckian genetic algorithm (LGA). A
maximum number of 2,500,000 energy evaluation was used. Each
docking experiment was consisted of 100 independent runs. All the
MD simulations were performed using the AMBER 10.0 program
suite.50 Based on the electrostatic potential (ESP) calculations at
the ab initio HF/6-31G* level,48 the partial atomic charges for
ligands were refined by using RESP calculation in antechamber
module.51 Other parameters for ligands were taken from the
Generalized Amber Force field (GAFF).52 Two K+ ions were
placed manually in the central core of the complexes. Every ligand-
receptor system was then solvated in a truncated octahedron box of
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