New quinazoline derivatives for telomeric G-quadruplex DNA: Effects of an added phenyl group on quadruplex binding ability

Article abstract of DOI:10.1016/j.ejmech.2013.01.051

To improve the selectivity of indoloquinoline or benzofuroquinoline derivatives, we previously reported several quinazoline derivatives [17]. These compounds could mimic a tetracyclic aromatic system through intramolecular hydrogen bond. Studies showed that these quinazoline derivatives were effective and selective telomeric G-quadruplex ligands. With this encouragement, here we synthesized a series of N-(2-(quinazolin-2-yl)phenyl)benzamide (QPB) compounds as modified quinazoline derivatives. In this modification, a phenyl group was introduced to the aromatic core. The evaluation results showed that part of QPB derivatives had stronger binding ability and better selectivity for telomeric G-quadruplex DNA than LZ-11, the most potential compound of reported quinazoline derivatives. Furthermore, telomerase inhibition of QPB derivatives and their cellular effects were studied.

Full text of DOI:10.1016/j.ejmech.2013.01.051

European Journal of Medicinal Chemistry 63 (2013) 1e13
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New quinazoline derivatives for telomeric G-quadruplex DNA: Effects of an added
phenyl group on quadruplex binding ability
Jin-Hui He, Hui-Yun Liu, Zeng Li, Jia-Heng Tan, Tian-Miao Ou, Shi-Liang Huang, Lin-Kun An, Ding Li,
*
Lian-Quan Gu, Zhi-Shu Huang
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 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:
To improve the selectivity of indoloquinoline or benzofuroquinoline derivatives, we previously reported
several quinazoline derivatives [17]. These compounds could mimic a tetracyclic aromatic system
through intramolecular hydrogen bond. Studies showed that these quinazoline derivatives were effective
and selective telomeric G-quadruplex ligands. With this encouragement, here we synthesized a series of
N-(2-(quinazolin-2-yl)phenyl)benzamide (QPB) compounds as modified quinazoline derivatives. In this
modification, a phenyl group was introduced to the aromatic core. The evaluation results showed that
part of QPB derivatives had stronger binding ability and better selectivity for telomeric G-quadruplex
DNA than LZ-11, the most potential compound of reported quinazoline derivatives. Furthermore, telo-
merase inhibition of QPB derivatives and their cellular effects were studied.
Received 18 December 2012
Received in revised form
19 January 2013
Accepted 24 January 2013
Available online 9 February 2013
Keywords:
Quinazoline derivatives
Telomeric G-quadruplex DNA
Inducing ability
Ó 2013 Elsevier Masson SAS. All rights reserved.
Telomerase inhibitor
Telomere shortening
1. Introduction
proteins from telomeres, e.g. POT1 [11e13]. Such changes lead to
a fast induction of tumor cell senescence and apoptosis. Thus, the
Telomeres are essential chromosomal components protecting
the end of eukaryotic chromosomes and maintaining proper rep-
lication [1]. These special elements consist of approximately 2e
20 kb of TTAGGG duplex repeats and 50ꢀ500 bases of single-
stranded DNA overhang running 50e30 toward the end of chro-
mosome. During the replication in normal cell proliferation, telo-
meres erode by about 100 bp with each cell division which finally
triggering cellular senescence [2,3]. However, in most cancer cells,
telomere is protected from shortening by telomerase enzyme [4].
Studies showed that the extreme 30- ends of telomeres could adopt
G-quadruplex structures from stacked tetrads of hydrogen-bonded
guanine bases [5e7]. The formation of stable G-quadruplex struc-
tures through the interaction with various ligands alters the over-
hang structure, inhibits catalytic functions of the telomerase
enzyme [8e10] and induces the degradation of telomere through
a DNA-damage repair pathway and release of one of shelterin
design of telomeric G-quadruplex binding ligands is a rational and
promising strategy for cancer therapy.
Indoloquinoline or benzofuroquinoline derivative SYUIQ-05
(Fig. 1A) modified from cryptolepine was an effective quadruplex
binding ligand developed by our group [14,15]. This polycyclic aro-
matic compound interacted with telomeric G-quadruplex DNA and
showed inhibition of telomerase. However, it also intercalated into
duplex DNA to a certain extent [16]. In our recent study, a new
G-quadruplex ligand LZ-11 (Fig. 1B) which shares the quinazoline
scaffold was synthesized and its binding properties towards telo-
meric G-quadruplex DNA were reported [17]. This molecule has an
“imitative” tetracyclic aromatic system (like indoloquinoline)
formed through intramolecular hydrogen bond, which enables
adoption of moderate twisted and co-planar conformations of the
aryl groups, and allows the ligand to well stack on the G-quartet. Our
experimental results showed that LZ-11 was a promising selective
telomeric G-quadruplex binding ligand with strong discrimination
against the duplex DNA. Aiming to develop this kind of G-quad-
ruplex ligands, we tried to modify the main scaffold of the molecule.
Recent research suggested that the decreased binding ability of
flexible ligands might result from the lessened aromaticity or pla-
narity [18]. Therefore, increasing aromaticity or expanding planarity
may be one of feasible modification strategies. Bis-substituted
Abbreviations: CD, circular dichroism; SPR, surface plasmon resonance; TRAP,
telomere repeat amplification protocol; MTT, methyl thiazolyl tetrazolium; SA-
Gal, senescence-associated-galactosidase; TRF, telomeric restriction fragment.
* Corresponding author. Tel.: þ86 20 39949056; fax: þ86 20 39943056.
E-mail address: ceshzs@mail.sysu.edu.cn (Z.-S. Huang).
b-
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.051

2
J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
anthraquinone (AQ) derivatives were proved to be effective G-
quadruplex stabilizers and powerful telomerase inhibitors [19e22].
Studies showed that the amide bond direction on side chains could
modulate their G-quadruplex recognition and telomerase inhibition
[23]. The AQeNHeCOe arrangement performed invariantly better
activity than the AQeCOeNHe arrangement. Theoretical calcula-
tions suggested that the former amide arrangement was co-planar
with the aromatic system. These results indicated that amide bond
not only served as a space linker, but also expanded the aromatic
plane.
configuration of the acylamide between the two phenyl groups, we
performed 2D NOESY (Fig. S1) using an important intermediate 8a.
Results indicated that the two phenyl groups were in anti position
of double bond.
3. Results and discussion
3.1. G-quadruplexeligand interaction studies with circular
dichroism (CD)
On this basis, we modified the original quinazoline derivative by
linking a new substituted benzene ring to the 20 position of phenyl
group of original core through amide bond (Fig. 1C). The partial
double-bond feature of the amide bond might place the newly
added benzene on the same quinazoline plane, which was expected
To investigate the interaction between the synthesized com-
pounds and telomeric G-quadruplex DNA, CD experiments were
carried out. CD spectroscopy is a conventional method for deter-
mining the conformation of G-quadruplex structures and the effect
of ligand binding on quadruplex structure [29]. Here, we used
quindoline derivative SYUIQ-05 and quinazoline derivative LZ-11 as
reference compounds [15,17]. As shown in Fig. 2, in the presence of
150 mM Kþ, the CD spectrum of HTG21 in the absence of any ligand
showed a major positive band at 290 nm, a shoulder at around
270 nm, a small positive band at 250 nm, and a minor negative band
near 234 nm. This indicated the formation of a mixture of anti-
parallel and parallel G-quadruplexes, possibly including hybrid-
types as well. Upon the addition of different compounds to the
above solution, the CD spectra changed differently. Compounds
without the second phenyl group (11a, 12a and LZ-11) had little
conformational change ability to HTG21, while QPB derivatives
changed the spectra significantly. The positive band at 290 nm
dropped, while a new major positive band showed up at 265 nm,
together with a major negative band at around 245 nm. The changes
revealed that a conformational conversion from antiparallel types to
parallel ones was induced in the presence of QPB derivatives. The
ability to induce conformational conversion varied among different
compounds (Table 1). The N-methyl piperazino analogues (13d, 14d
and 15d) and 3-dimethylaminopropylamino analogues (14e and
15e) had the most powerful conformational conversion ability. The
results above demonstrated that QPB derivatives had better
inducing ability than the original quinazoline compounds.
to facilitate the p-p interaction. Meanwhile, the substituted amino
side chain at C-10 position was reserved and a second side chain
was allocated to para-position of the new benzene ring. Therefore,
we obtained a series of N-(2-(quinazolin-2-yl)phenyl)benzamide
(QPB) derivatives (Fig. 1C). The interactions of synthesized com-
pounds with telomeric G-quadruplex DNA were examined using
circular dichroism spectroscopy (CD) and surface plasmon reso-
nance (SPR). Further studies on their inhibitory effects on telo-
merase activity were performed using TRAP-LIG, and their cellular
effects were evaluated through the measurements of cell sen-
escence and telomere shortening.
2. Chemistry
The synthetic pathway for new quinazoline derivatives is shown
in Scheme 1. Compounds 11ae11d and 12ae12e were prepared
mainly according to the previously reported procedure [17]. And we
used stannous chloride dihydrate as reductant [24,25] instead of
hydrazine hydrate to obtain 6a and 6b. Compounds 8a and 8b were
prepared by mixing 6a and 6b with 4-nitrobenzoyl chloride in
dichloromethane under ice bath condition. The resulting solution
was stirred over night at ambient temperature. A second reduction
reaction was then performed using stannous chloride dihydrate to
give compounds 9a and 9b. Reaction of 9a and 9b with acylchloride
in dichloromethane gave compounds 10a, 10b and 10c. Finally,
compounds 13ae13d, 14ae14e, and 15ae15e were obtained
through reaction of the compounds 10a, 10b, and 10c with different
primary and secondary amines under reflux condition, respectively.
In agreement with our previous report [17], the 1H NMR
chemical shifts of acylamide protons, which locate between two
We next performed CD melting assay to evaluate the stabiliza-
tion to G-quadruplex by all synthesized compounds. CD melting
assays is a useful method to measure the melting temperature of G-
quadruplex [30,31]. In this experiment, CD spectra are recorded
every 3 ꢁC when heating. Then CD profiles at a specific wavelength
are used for obtaining melting curves. Here, we used CD profiles at
265 nm and 290 nm to calculate the
DTm values. A comparison of
the CD melting results indicated that the introduction of phenyl
group had a significant effect on the stabilizing ability of these
compounds. With an addition of a phenyl group on the main
structure, compounds 13aꢀ13d, 14aꢀ14e and 15aꢀ15e had
phenyl groups, are in the range of
d 13.11e14.62 ppm in CDCl3 or
DMSO-d6. These data indicated the formation of a relatively strong
intramolecular H-bonding [26e28]. In order to determine the
Fig. 1. Structures of (A) SYUIQ-05, (B) LZ-11 and (C) QPB derivatives.

J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
3
Scheme 1. Synthesis of QPB 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-
(dimethylamino)-1-propylamine, THF, reflux, 5 h; (v) SnCl2$2H2O, HCl, EtOH, reflux, 1 h; (vi) Cl(CH2)nCOCl, CH2Cl2, rt, 24 h; (vii) 4-nitrobenzoyl chloride, CH2Cl2, rt, 24 h; (viii)
amine, methanol, reflux, 6 h.
a noticeable enhancement of G-quadruplex stabilizing ability. Most
weakest effect on the stability of telomeric G-quadruplex. Mean-
while, compounds with a more basic amino terminal (N-methyl-
piperazino analogues, 11d, 12d, 13d, 14d and 15d) or a longer amino
terminal (3-dimethylaminopropylamino analogues, 12e, 14e and
15e) showed more activity for G-quadruplex DNA. These results
demonstrated the importance of length and basicity of amide side
chain for strong G-quadruplex interactions. Besides, it should be
noted that the chlorine substituent had a strong influence on the
stabilizing ability of quinazoline derivatives. Compounds with
a chlorine substituent (LZ-11 and 15aꢀ15e) showed enhanced ac-
tivity for G-quadruplex over their counterparts (12d and 14aꢀ14e).
This might be the result of changes of electric density or in-
teractions of chlorine atom with G-quadruplex backbone. More-
over, a correlation between inducing ability and stabilizing ability
of the compounds was observed. Compounds with the most
inducing ability (13d, 14d, 14e, 15d and15e) showed the best
QPB derivatives showed strong stabilizing ability for telomeric G-
quadruplex (D
Tm values above 20 ꢁC at 265 nm and 8 ꢁC at 290 nm).
In some QPB compound cases, such as 14e and 15e, the melting
temperatures were above 94 ꢁC at 265 nm. However, the original
quinazoline derivatives 11aꢀ11d, 12aꢀ12e and LZ-11 showed weak
effect on G-quadruplex stability with D
Tm values lower than 15 ꢁC
at 265 nm and 7 ꢁC at 290 nm. The lack of additional aromatic ring
significantly decreases the stabilizing ability of quinazoline de-
rivatives for G-quadruplex DNA. On the other hand, the increase of
Tm values of QPB compounds at 290 nm (
D
Tm less than 15 ꢁC) was
not as much as those at 265 nm (
D
Tm up to 28 ꢁC). These results
indicated that QPB derivatives might mainly strengthen the sta-
bility of parallel G-quadruplex in buffer solution.
Among the compounds owning the same core structures, the
less basic morpholino analogues (11c, 12c, 13c, 14c and 15c) had the

4
J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
Table 2
Kinetic parameters determined with SPR spectroscopy.
Compound KD (M)
Compound KD (M)
HTG21
Duplex
HTG21
Duplex
11d
12d
12e
13a
13d
14a
14d
ea
ea
ea
ea
14e
15a
1.11 ꢂ 10ꢀ6 ea
3.02 ꢂ 10ꢀ7 ea
3.36 ꢂ 10ꢀ6 7.90 ꢂ 10ꢀ6 15b
8.34 ꢂ 10ꢀ6 1.19 ꢂ 10ꢀ5 15c
2.75 ꢂ 10ꢀ6 3.97 ꢂ 10ꢀ5 15d
1.61 ꢂ 10ꢀ6 2.20 ꢂ 10ꢀ5 15e
2.27 ꢂ 10ꢀ7 ea
3.06 ꢂ 10ꢀ6 ea
2.76 ꢂ 10ꢀ7 ea
3.79 ꢂ 10ꢀ7 ea
1.48 ꢂ 10ꢀ6 1.32 ꢂ 10ꢀ5 SYUIQ-05 2.80 ꢂ 10ꢀ5 5.02 ꢂ 10ꢀ6
a
No significant binding was found for addition of up to 20 mM ligand.
DNA and duplex DNA, while 12e bound to both telomeric G-
quadruplex and duplex DNA with similar affinity. The data dem-
onstrated that the introduction of a new phenyl group onto the
scaffold of quinazoline derivatives gave an improvement of both
binding ability and selectivity for G-quadruplex. Consistent with CD
melting results, compounds 12e and 14e were of the best binding
affinity and selectivity for G-quadruplex when compared to their
counterparts, and 15c showed the least binding affinity among the
chlorine substituted QPB derivatives. Both CD and SPR exper-
imental results indicated that the introduction of phenyl group on
the quinazoline derivatives had beneficial effects on their binding
ability for telomeric G-quadruplex DNA.
Fig. 2. CD spectra of HTG21 in 10 mM TriseHCl buffer, pH 7.4, 150 mM KCl. CD spectra
of HTG21 (black line), and HTG21 in the presence of 5 equiv of 11a, 12a, 13a, 13c, 13d,
14a, 14c, 14d, 14e, LZ-11, 15a, 15c, 15d and 15e, respectively.
stabilizing ability for G-quadruplex. Together, CD experiment re-
sults suggested that QPB derivatives had an improved inducing and
stabilizing ability for telomeric G-quadruplex DNA.
3.2. Telomeric G-quadruplex binding affinity and selectivity studies
with surface plasmon resonance (SPR)
3.3. Telomerase inhibition
To investigate the binding affinity and selectivity of the syn-
thesized compounds for telomeric G-quadruplex, we performed
SPR with biotinylated telomeric G-quadruplex DNA HTG21 and
biotinylated duplex DNA attached to a streptavidin-coated sensor
chip [32,33]. By simultaneously injecting a range of concentrations
of ligands to the immobilized telomeric G-quadruplex DNA, the
duplex DNA and the blank reference, SPR sensorgrams were
recorded, which were used to building binding plots for calculating
equilibrium constants later.
The ability of the synthesized compounds to inhibit human
telomerase activity was examined by TRAP-LIG assay [34]. Con-
sidering that the compounds might interfere the PCR step through
interacting with the extension products, the ligands were removed
after incubation with the enzyme and prior to the amplification
step [35]. In the experiments, solutions of derivatives were added to
the telomerase reaction mixture containing extract from cracked
leukemia cell HL60, and the IC50 values of the three chosen
As shown in Table 2, the KD values of QPB compounds binding to
telomeric G-quadruplex showed strong binding affinity (KD from
0.227 to 8.34
the KD values for G-quadruplex were lower than 1
obvious binding was observed for these compounds to duplex DNA
even at a at a concentration of 20 M. Quinazoline compounds 11d
m
M). Especially for compounds 15a, 15b, 15d and 15e,
Table 3
Telomerase inhibition by compounds 12e, 14e and 15e in cell-free assay.
mM, while no
12e
14e
15e
m
IC50
(
m
M)
>50
30.9
2.3
and 12d had little binding affinity to both telomeric G-quadruplex
Table 1
Inducing G-quadruplex conformational change potential and G-quadruplex stabilization ability obtained from CD experiments in KCl buffer (10 mM TriseHCl, 60 mM KCl, pH
7.4).
Compound
Inducing abilitya
D
Tm/ꢁCb
Compound
Inducing abilitya
D
Tm/ꢁCb
265 nm
290 nm
265 nm
290 nm
11a
11b
11c
11d
12a
12b
12c
12d
12e
13a
13b
13c
13d
*
*
2.1
7.3
0.5
3.1
8.2
8.7
4.3
8
12.4
21.6
21.4
11.9
21.8
0.7
2.6
0
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
LZ-11
SYUIQ-05
****
***
****
*****
*****
**
19.6
18.7
15.3
21.7
>28
21.9
>28
21.6
>28
>28
14.4
11.2
5.4
4.6
8.2
8.2
ndd
2.8
5.7
4.7
3.4
14.8
4.4
8
ec
ec
*
0.6
2.5
3.6
0.9
2.1
6.2
11.6
11.7
4.7
7.4
*
ec
****
***
**
*
****
****
***
*****
*****
*****
**
ec
a
Inducing ability depends on the ratio of the signal values from 265 nm to 290 nm on the CD spectrum.
b
c
DTm values were determined using the CD profiles at 265 nm and 290 nm.
No observed conformational change.
Not detected.
d

J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
5
Fig. 3. Senescence induced by compound 15e on HL60 cells. (A) Long-term incubation of HL60 with compound 15e at subcytotoxic concentrations. Expression of SA-
b
-Gal in HL60
cells after treatment with (B) 0.1% DMSO or (C) 1 mM compound 15e continuously for 12 days.
compounds were obtained as shown in Table 3. Based on CD and
SPR results, we chose quinazoline compound 12e and QPB de-
rivatives 14e, 15e for telomerase inhibition studies. It was clearly
found that the inhibitory effect of chlorine substituted QPB ligand
15e was significantly stronger than none chlorine substituted
compound 14e, while quinazoline derivative 12e showed little
inhibitory ability. Obviously, the telomerase inhibition results had
a structureeactivity relationship, which was in agreement with CD
and SPR results.
indicated that dysfunctional telomere could activate p53 to initiate
cellular senescence or apoptosis to suppress tumorigenesis [14].
Therefore, the induction of senescence by compound 15e might be
due to the shortening of telomere length. This result is consistent
with the biological activity expected for efficient telomeric G-
quadruplex ligand and telomerase inhibitor [40].
4. Conclusions
To enhance the telomeric G-quadruplex binding ability of qui-
nazoline derivatives, we designed and synthesized a series of novel
N-(2-(quinazolin-2-yl)phenyl)benzamide (QPB) derivatives. Based
on previous reports of the use of amide bonds on quadruplex ligand
design, a new benzene ring was attached to the scaffold of quina-
zoline derivatives through amide bond. The present studies showed
that QPB derivatives were more effective then the parental quina-
zoline compounds. An inducing ability from hybrid type to parallel
type G-quadruplex was observed for QPB derivatives on CD spectra.
And CD melting results indicated that the existence of an additional
phenyl group and a chlorine substituent could greatly increase the
telomeric G-quadruplex stabilization ability of quinazoline com-
pounds. The same SAR was also found in SPR and TRAP-LIG results.
QPB derivatives, especially chlorine substituted ones, showed
strong binding ability and selectivity for telomeric G-qudruplex
DNA. Compound 15e was found to be a strong telomerase inhibitor.
Cellular experiments demonstrated that 15e could remarkable
decrease the population of HL60 cells with a shortening of the
telomere length, which might due to its telomerase inhibitory
ability. These results proved that the introduction of a phenyl group
through amide bond is an effective and efficient approach for
obtaining more biologically active telomeric G-quadruplex ligands.
3.4. Cell senescence induced by compound 15e
Based on the study results above, compound 15e was proved to be
one of the most promising ligand as a telomerase inhibitor and
telomeric G-quadruplex binding ligand. Thus, the following studies
were carried out using this compound. To examine the effectof ligand
15e on leukemia cell HL60, short-term cell viability was determined
by MTTassay first. The results showed that15e has a potent inhibitory
effect, with an IC50 value of 1.7
mM. Thus, subcytotoxic concentrations
of 15e were set at 0.01, 0.1, 0.5 and 1
mM for long-term HL60 cell
culture experiments. The inhibitory effect of 15e to HL60 cells was
obviously dose-dependent (Fig. 3A). Treatment of HL60 cells with
1
mM compound 15e resulted in a significant inhibitory effect. In the
presence of even 0.5 M compound 15e, a discernible difference was
m
observed between the control and treated cells.
Morphological examination of the cells in long-term studies
showed an increased proportion of enlarged and flattened cells
with phenotypic characteristics of senescence [36,37]. These flat-
tened cells also stained positively for the senescence-associated
b-
galactosidase (SA- -Gal) activity after continuous treatment by
b
compound 15e (Fig. 3B and C). Results indicated that compound 15e
induced accelerated senescence of HL60 cancer cells.
5. Experimental section
3.5. Telomere shortening by compound 15e
5.1. Synthesis and characterization
It had been reported that treatment of cancer cells with telo-
meric G-quadruplex ligands could disrupt telomere length main-
tenance and cause telomeres to erode [38,39]. To investigate
whether 15e could shorten telomeres, the telomere length was
evaluated using the telomeric restriction fragment (TRF) length
assay. The results showed that the length of telomere shortened
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; 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. Elemental analysis was carried
out on an Elementar Vario EL CHNS Elemental Analyzer. Melting
points (m.p.) were determined using an SRS-OptiMelt automated
about 1.1 kb for HL60 cells after long-term exposure to 1
compound 15e (Fig. 4). Telomere shortening was also observed
after 0.01, 0.1, and 0.5 M of 15e treatment. Previous studies
mM of
m

6
J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
yield. 1H NMR (400 MHz, DMSO-d6):
d 12.56 (s, 1H), 8.44 (d,
J ¼ 8.2 Hz, 1H), 8.39 (s, 1H), 8.26 (d, J ¼ 1.6 Hz, 1H), 8.04e7.96 (m,
1H), 7.93e7.85 (m, 2H), 7.81 (s, 1H), 7.59 ¼ 5.6 Hz, 2H) ¼ 7.8 Hz, 1H),
7.24 ¼ 5.6 Hz, 2H) ¼ 7.6 Hz, 1H); LCeMS m/z: 320 [M þ H]þ.
5.1.2. General procedure B: preparation of 3a and 3b
A mixture of benzamide (88 mmol) in 10% aqueous KOH
(100 mL) and EtOH (100 mL) was heated under reflux for 2 h.
Ethanol was removed in vacuo, and the aqueous layer was acidified
with hydrochloric acid to pH 3 to give a solid residue. The solid was
filtered and washed with water, which was purified by using col-
umn chromatography, eluting with CH2Cl2/MeOH (20:1) to give
products 3a and 3b.
5.1.2.1. 2-(2-Nitrophenyl)quinazolin-4(3H)-one (3a). The com-
pound 2a was treated with KOH according to general procedure B
to afford 3a as a white solid in 98% yield. 1H NMR (400 MHz, DMSO-
d6):
d 12.86 (s, 1H), 8.27e8.16 (m, 2H), 7.95e7.81 (m, 4H), 7.67 (d,
J ¼ 7.8 Hz, 1H), 7.62e7.56 (m, 1H). LCeMS m/z: 268 [M þ H]þ.
5.1.2.2. 2-(4-Chloro-2-nitrophenyl)quinazolin-4(3H)-one
(3b).
The compound 2b was treated with KOH according to general pro-
cedure B to afford 3b as a white solid in 91% yield. 1H NMR
(400 MHz, DMSO-d6):
d
12.87 (s, 1H), 8.19 (d, J ¼ 2.0 Hz, 1H), 8.05 (d,
J ¼ 8.0 Hz, 1H), 7.89 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.78 (d, J ¼ 8.4 Hz, 1H),
7.71 ¼5.6 Hz, 2H) ¼ 7.8 Hz,1H), 7.51 (d, J ¼ 8.2 Hz,1H), 7.44 ¼ 5.6 Hz,
2H) ¼ 7.7 Hz, 1H); LCeMS m/z: 302 [M þ H]þ.
5.1.3. General procedure C: preparation of 4a and 4b
To a mechanically stirred suspension of 3a or 3b (80 mmol) in
100 mL toluene was added N,N-diethylaniline (133 mmol), followed
with phosphorus oxychloride (96 mmol). The reaction was heated
to 80 ꢁC for 6 h, allowed to cool to room temperature, and then
washed with ice water (200 mL). The organic layer was then
washed sequentially with 20% NaOH (2 ꢂ 100 mL), iced water
(200 mL), and brine (200 mL), and dried over anhydrous sodium
sulfate. The organic solvent was removed in vacuo, and the residue
was purified by using column chromatography with CH2Cl2/MeOH
(20:1) to give 4a or 4b as a colorless powder. The aqueous layers
were combined and extracted with dichloromethane. The organic
layer was reduced in vacuo to yield a yellow solid, which was also
purified by using column chromatography as above.
Fig. 4. Effect of quinazoline derivative 15e on telomere length. TRF analysis of HL60
cells treated with or without compound 15e for 12 days. Lane 1, 0.1% DMSO; lane 2,
0.01
mM compound 15e; lane 3, 0.1
mM compound 15e; lane 4, 0.5 mM compound 15e;
and lane 5, 1
m
M compound 15e.
melting point instrument without correction. Flash column chro-
matography was performed with silica gel (200ꢀ300 mesh) pur-
chased from Qingdao Haiyang Chemical Co. Ltd. The purities of
synthesized compounds were confirmed to be higher than 95% by
analytical HPLC performed with a dual pump Shimadzu LC-20AB
system equipped with an Ultimate XB-C18 column and eluted
with methanol-water (32 : 68 to 60 : 40) containing 0.1% TFA at
a flow rate of 0.6 mL minꢀ1
.
5.1.1. General procedure A: preparation of 2a and 2b
5.1.3.1. 4-Chloro-2-(2-nitrophenyl)quinazoline (4a). The compound
3a was treated with phosphorus oxychloride according to general
procedure C to afford 4a as a pale solid in 70% yield. 1H NMR
The mixture of appropriate nitrobenzoic acid (100 mmol) and
thionyl chloride (50 mL) was heated under reflux for 1.5 h at 80 ꢁC.
The solution was allowed to cool at room temperature followed
with the evaporation of thionyl chloride in vacuo. The resulting
wine red solution was added dropwise to a solution of anthrani-
lamide (1; 125 mmol) and triethylamine (250 mmol) in chloroform
(250 mL), and stirred at room temperature for 5 h. The precipitated
solid was collected through filtration, washed with ethanol, and
dried. Its recrystallization from ethanol afforded the products 2a
and 2b.
(400 MHz, DMSO-d6):
d 8.26e8.10 (m, 2H), 7.97e7.82 (m, 4H), 7.69
(d, J ¼ 7.7 Hz, 1H), 7.63e7.58 (m, 1H); LCeMS m/z: 286 [M þ H]þ.
5.1.3.2. 4-Chloro-2-(4-chloro-2-nitrophenyl)quinazoline
(4b).
The compound 3b was treated with phosphorus oxychloride ac-
cording to general procedure C to afford 4b as a pale solid in 61%
yield. 1H NMR (400 MHz, DMSO- d6):
d 8.36e8.31 (m, 1H), 8.26 (d,
J ¼ 2.1 Hz, 1H), 8.23e8.17 (m, 2H), 8.10 (d, J ¼ 8.1 Hz, 1H), 8.01e7.92
(m, 2H); LCeMS m/z: 321 [M þ H]þ.
5.1.1.1. N-(2-carbamoylphenyl)-2-nitrobenzamide (2a). The com-
pound 1 was treated with 2-nitrobenzoic acid according to general
procedure A to afford 2a as a white solid in 88% yield. 1H NMR
5.1.4. General procedure D: preparation of 5a and 5b
To a suspension of 4a or 4b (56 mmol) in 100 mL THF was
slowly added 3-aminopropyldimethylamine (168 mmol). The
mixture was heated under reflux for 5 h, and TLC analysis indi-
cated reaction completion. After concentration in vacuo, the
resulting residue was dissolved in CHCl3, washed with water and
brine, dried over anhydrous MgSO4, and evaporated. The residue
was chromatographed on silica gel eluting with CH2Cl2/MeOH
(20:1) to give 5a or 5b.
(400 MHz, DMSO-d6):
d
12.57 (s, 1H), 8.53 (d, J ¼ 8.2 Hz, 1H), 8.43 (s,
1H), 8.13 (d, J ¼ 8.3 Hz, 1H), 7.95e7.78 (m, 5H), 7.62 (dd, J ¼ 11.4,
4.0 Hz, 1H), 7.29e7.22 (m, 1H); LCeMS m/z: 286 [M þ H]þ.
5.1.1.2. N-(2-carbamoylphenyl)-4-chloro-2-nitrobenzamide
The compound 1 was treated with 4-chloro-2-nitrobenzoic acid
according to general procedure A to afford 2b as a white solid in 80%
(2b).

J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
7
5.1.4.1. N1,N1-dimethyl-N3-(2-(2-nitrophenyl)quinazolin-4-yl)pro-
pane-1,3-diamine (5a). The compound 4a was treated with 3-
aminopropyldimethylamine according to general procedure D to
afford 5a as a yellow solid in 87% yield. 1H NMR (400 MHz, CDCl3):
(400 MHz, CDCl3):
d
14.42 (s, 1H), 9.07 (d, J ¼ 1.9 Hz, 1H), 8.96 (s,
1H), 8.73 (d, J ¼ 8.6 Hz, 1H), 8.03 (d, J ¼ 8.4 Hz, 2H), 7.81 (d,
J ¼ 8.3 Hz, 1H), 7.72 (t, J ¼ 7.6 Hz, 1H), 7.64 (d, J ¼ 8.2 Hz, 1H), 7.48e
7.42 (m, 1H), 7.12 (dd, J ¼ 8.7, 2.0 Hz, 1H), 6.80 (d, J ¼ 8.5 Hz, 2H),
4.05 (s, 2H), 3.88 (dd, J ¼ 10.2, 5.4 Hz, 2H), 2.65 (t, J ¼ 5.6 Hz, 2H),
2.42 (s, 6H), 1.96e1.90 (m, 2H); LCeMS m/z: 476 [M þ H]þ.
d
8.78 (s, 1H), 8.11 (dd, J ¼ 7.7, 1.3 Hz, 1H), 7.77 (d, J ¼ 8.0 Hz, 1H),
7.67e7.60 (m, 2H), 7.57e7.50 (m, 2H), 7.43 (td, J ¼ 7.8, 1.4 Hz, 1H),
7.38e7.33 (m, 1H), 3.60 (dd, J ¼ 10.4, 5.8 Hz, 2H), 2.50 ¼ 5.6 Hz,
2H) ¼ 5.6 Hz, 2H), 2.29 (s, 6H), 1.76 (dt, J ¼ 11.5, 5.9 Hz, 2H); LCeMS
m/z: 352 [M þ H]þ.
5.1.6. General procedure F: preparation of 7a, 7b, 10a, 10b and 10c
A solution of the appropriate acid halide (9.6 mmol) in CH2Cl2
(10 mL) was added to a well-stirred mixture of the amino-
substituted compound (8 mmol) in CH2Cl2 (50 mL) at 0 ꢁC. The
reaction was then allowed to warm up to room temperature, and
then stirred overnight. After cooling to 0 ꢁC, the precipitate formed
was filtered off, and was further purified by using flash column
chromatography with CH2Cl2/MeOH (15:1 to 10:1) elution to give
the compounds 7a, 7b, 10a, 10b and 10c.
5.1.4.2. N1-(2-(4-chloro-2-nitrophenyl)quinazolin-4-yl)-N3,N3-dime-
thylpropane-1,3-diamine (5b). The compound 4b was treated with
3-aminopropyldimethylamine according to general procedure D to
afford 5b as a yellow solid in 84% yield. 1H NMR (400 MHz, CDCl3):
d
8.95 (s, 1H), 8.21 (d, J ¼ 8.4 Hz, 1H), 7.84 (d, J ¼ 8.3 Hz, 1H), 7.75e
7.66 (m, 2H), 7.62e7.56 (m, 2H), 7.45 ¼ 5.6 Hz, 2H) ¼ 7.3 Hz, 1H),
3.66 (dd, J ¼ 10.4, 5.6 Hz, 2H), 2.63e2.55 (m, 2H), 2.38 (s, 6H), 1.88e
1.82 (m, 2H); LCeMS m/z: 386 [M þ H]þ.
5.1.6.1. 2-Chloro-N-(2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)acetamide (7a). The compound 6a was treated
with chloroacetyl chloride according to general procedure F to
afford 7a as a white solid in 85% yield. 1H NMR (400 MHz, CDCl3):
5.1.5. General procedure E: preparation of 6a, 6b, 9a and 9b
To a suspension of appropriate nitrobenzene (48 mmol) and
stannous chloride dihydrate (144 mmol) in 80 mL ethanol was slowly
added 10 M hydrochloric acid (14.4 mL). The mixture was heated
under reflux for 5 h, and TLC analysis indicated reaction completion.
After concentration in vacuo, the resulting residue was slowly dis-
solved in sodium hydroxide solution (pH 13ꢀ14) with icewater. Then
the mixture was quickly extracted with dichloromethane. The
organic layer was then dried over anhydrous MgSO4 and evaporated.
The residue was chromatographed on silica gel eluting with CH2Cl2/
MeOH (15:1) to give corresponding aminobenzene product.
d
14.03 (s, 1H), 8.80 (s, 1H), 8.62e8.57 (m, 2H), 7.87 (dd, J ¼ 8.4,
0.6 Hz,1H), 7.64 (ddd, J ¼ 8.4, 7.0, 1.3 Hz,1H), 7.57 (dd, J ¼ 8.1, 0.7 Hz,
1H), 7.39e7.34 (m, 2H), 7.16e7.12 (m, 1H), 4.21 (s, 2H), 3.80 (dd,
J ¼ 10.3, 5.8 Hz, 2H), 2.57 (t, J ¼ 5.6 Hz, 2H), 2.33 (s, 6H), 1.88e1.81
(m, 2H); LCeMS m/z: 398 [M þ H]þ.
5.1.6.2. 3-Chloro-N-(2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)propanamide (7b). The compound 6a was treated
with 3-chloropropanoyl chloride according to general procedure F
to afford 7b as a white solid in 84% yield. 1H NMR (400 MHz, CDCl3):
5.1.5.1. N1-(2-(2-aminophenyl)quinazolin-4-yl)-N3,N3-dimethylpro-
pane-1,3-diamine (6a). The compound 5a was treated with stan-
nous chloride dihydrate according to general procedure E to afford
d
14.20 (s,1H), 8.95 (s,1H), 8.74 (d, J ¼ 8.1 Hz, 2H), 7.73 (d, J ¼ 3.9 Hz,
2H), 7.62 (d, J ¼ 8.1 Hz,1H), 7.46e7.40 (m, 2H), 7.17 (t, J ¼ 7.6 Hz,1H),
3.97 (t, J ¼ 6.9 Hz, 2H), 3.86 (dd, J ¼ 10.3, 5.5 Hz, 2H), 3.01 (t,
J ¼ 6.9 Hz, 2H), 2.63 (t, J ¼ 5.6 Hz, 2H), 2.40 (s, 6H), 1.95e1.88 (m,
2H).; LCeMS m/z: 412 [M þ H]þ.
6a as a white solid in 88% yield. 1H NMR (400 MHz, CDCl3):
d 8.49
(dd, J ¼ 8.0, 1.6 Hz, 1H), 8.44 (s, 1H), 7.69 (d, J ¼ 8.3 Hz, 1H), 7.56
(ddd, J ¼ 8.3, 7.0, 1.3 Hz, 1H), 7.48 (d, J ¼ 8.1 Hz, 1H), 7.26 (ddd,
J ¼ 8.1, 7.0, 1.1 Hz, 1H), 7.11 (ddd, J ¼ 8.6, 7.2, 1.7 Hz, 1H), 6.81e6.21
(m, 4H), 3.74 (dd, J ¼ 10.4, 5.9 Hz, 2H), 2.49 ¼ 5.6 Hz, 2H) ¼ 5.6 Hz,
2H), 2.28 (s, 6H), 1.79 (dt, J ¼ 11.6, 5.9 Hz, 2H); LCeMS m/z: 322
[M þ H]þ.
5.1.6.3. 4-(2-Chloroacetamido)-N-(2-(4-((3-(dimethylamino)propyl)
amino)quinazolin-2-yl)phenyl)benzamide (10a). The compound 9a
was treated with chloroacetyl chloride according to general pro-
cedure F to afford 10a as a pale solid in 85% yield. 1H NMR
(400 MHz, CDCl3):
d
14.47 (s, 1H), 9.50 (s, 1H), 8.94 (d, J ¼ 8.2 Hz,
5.1.5.2. N1-(2-(2-amino-4-chlorophenyl)quinazolin-4-yl)-N3,N3-
dimethylpropane-1,3-diamine (6b). The compound 5b was treated
with stannous chloride dihydrate according to general procedure E
to afford 6b as a white solid in 85% yield. 1H NMR (400 MHz, CDCl3):
1H), 8.84 (s, 1H), 8.77 (d, J ¼ 7.9 Hz, 1H), 8.17 (d, J ¼ 8.5 Hz, 2H),
7.81e7.72 (m, 4H), 7.65 (d, J ¼ 8.0 Hz, 1H), 7.56e7.40 (m, 2H), 7.19 (t,
J ¼ 7.3 Hz, 1H), 3.87 (dd, J ¼ 10.1, 5.3 Hz, 2H), 3.13 (s, 2H), 2.67e2.62
(m, 2H), 2.41 (s, 6H), 1.95e1.89 (m, 2H); LCeMS m/z: 518 [M þ H]þ.
d
8.57 (s, 1H), 8.51 (d, J ¼ 8.7 Hz, 1H), 7.78e7.73 (m, 1H), 7.70e7.61
(m, 2H), 7.41e7.35 (m, 1H), 6.77 (s, 2H), 6.71 (dt, J ¼ 8.3, 1.9 Hz, 2H),
3.84 (dd, J ¼ 10.5, 5.8 Hz, 2H), 2.69e2.62 (m, 2H), 2.42 (s, 6H), 1.99e
1.91 (m, 2H); LCeMS m/z: 356 [M þ H]þ.
5.1.6.4. 4-(3-Chloropropanamido)-N-(2-(4-((3-(dimethylamino)pro-
pyl)amino)quinazolin-2-yl)phenyl)benzamide (10b). The compound
9a was treated with 3-chloropropanoyl chloride according to gen-
eral procedure F to afford 10b as a pale solid in 85% yield. 1H NMR
5.1.5.3. 4-Amino-N-(2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)benzamide (9a). The compound 8a was treated
with stannous chloride dihydrate according to general procedure E
to afford 9a as a white solid in 77% yield. 1H NMR (400 MHz, CDCl3):
(400 MHz, CDCl3):
d
14.45 (s, 1H), 11.15 (s, 1H), 8.94 (d, J ¼ 8.3 Hz,
1H), 8.89 (s, 1H), 8.77 (d, J ¼ 8.0 Hz, 1H), 8.14 (d, J ¼ 8.6 Hz, 2H), 7.81
(d, J ¼ 8.2 Hz, 1H), 7.73 (dt, J ¼ 7.0, 2.6 Hz, 3H), 7.60 (d, J ¼ 7.8 Hz,
1H), 7.49 (t, J ¼ 7.0 Hz, 1H), 7.43 (t, J ¼ 7.1 Hz, 1H), 7.19 (t, J ¼ 7.6 Hz,
1H), 3.94 (t, J ¼ 5.6 Hz, 2H), 3.88 (dd, J ¼ 10.1, 5.4 Hz, 2H), 3.03 (t,
J ¼ 5.6 Hz, 2H), 2.63 (t, J ¼ 5.6 Hz, 2H), 2.40 (s, 6H), 1.93e1.88 (m,
2H); LCeMS m/z: 532 [M þ H]þ.
d
14.26 (s,1H), 8.94 (d, J ¼ 8.4 Hz,1H), 8.81 (s,1H), 8.76 (d, J ¼ 7.9 Hz,
1H), 8.02 (d, J ¼ 7.9 Hz, 2H), 7.81 (d, J ¼ 8.3 Hz,1H), 7.74e7.67 (m, 2H),
7.49e7.42 (m, 2H), 7.16 ¼ 5.6 Hz, 2H) ¼ 7.6 Hz,1H), 6.78 (d, J ¼ 8.1 Hz,
2H), 4.00 (s, 2H), 3.90 (dd, J ¼ 10.3, 5.0 Hz, 2H), 2.69 (t, J ¼ 5.6 Hz, 2H),
2.44 (s, 6H), 1.99e1.93 (m, 2H); LCeMS m/z: 441 [M þ H]þ.
5.1.6.5. N-(5-chloro-2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)-4-(3-chloropropanamido)benzamide
(10c).
5.1.5.4. 4-Amino-N-(5-chloro-2-(4-((3-(dimethylamino)propyl)
amino)quinazolin-2-yl)phenyl)benzamide (9b). The compound 8b
was treated with stannous chloride dihydrate according to general
procedure E to afford 9b as a white solid in 74% yield. 1H NMR
The compound 9b was treated with 3-chloropropanoyl chloride
according to general procedure F to afford 10c as a pale solid in 84%
yield. 1H NMR (400 MHz, DMSO-d6):
d
14.49 (s, 1H), 10.75 (s, 1H),
8.98 (t, J ¼ 5.3 Hz, 1H), 8.89 (d, J ¼ 2.2 Hz, 1H), 8.75 (d, J ¼ 8.7 Hz,

8
J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
1H), 8.44 (d, J ¼ 8.0 Hz, 1H), 8.04 (d, J ¼ 8.7 Hz, 2H), 7.92 (d,
J ¼ 8.7 Hz, 2H), 7.84 (t, J ¼ 7.7 Hz, 1H), 7.69 (d, J ¼ 8.1 Hz, 1H), 7.58 (t,
J ¼ 7.4 Hz, 1H), 7.29 (dd, J ¼ 8.7, 2.2 Hz, 1H), 3.93 (d, J ¼ 6.1 Hz, 2H),
3.74 (d, J ¼ 5.7 Hz, 2H), 3.06 (t, J ¼ 7.0 Hz, 2H), 2.95 (t, J ¼ 6.2 Hz, 2H),
2.66 (s, 6H), 2.15e2.08 (m, 2H); LCeMS m/z: 566 [M þ H]þ.
5.1.8.2. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-2-(pyrrolidin-1-yl)acetamide (11b). The compound 7a was
treated with excess pyrrolidine according to general procedure H to
afford 11b. After column chromatography with CHCl3/MeOH/
NH3$H2O (25:1:0.1) elution, the desired product was obtained as
a white solid in 74% yield. m.p. 147ꢀ149 ꢁC; 1H NMR (400 MHz,
5.1.7. General procedure G: preparation of 8a and 8b
CDCl3):
d
13.58 (s, 1H), 8.78 (d, J ¼ 8.3 Hz, 2H), 8.56 (dd, J ¼ 7.9,
The mixture of 4-nitrobenzoic acid (52.5 mmol) and thionyl
chloride (50 mL) was heated under reflux for 1.5 h at 80 ꢁC. The
solution was allowed to cool at room temperature followed with
the evaporation of thionyl chloride in vacuo. The resulting wine red
solution was added dropwise to a solution of 6a or 6b (1;
125 mmol) in dichloromethane (150 mL) at 0 ꢁC, then stirred at
room temperature for 5 h. The precipitated solid was collected
through filtration, washed with ethanol, and dried. Its recrystalli-
zation from ethanol afforded the products 8a or 8b.
1.5 Hz, 1H), 7.93 (d, J ¼ 8.2 Hz, 1H), 7.76e7.66 (m, 2H), 7.48e7.39 (m,
2H), 7.16 (t, J ¼ 7.1 Hz,1H), 3.90 (dd, J ¼ 10.6, 5.5 Hz, 2H), 3.42 (s, 2H),
2.79e2.70 (m, 2H), 2.67 (t, J ¼ 5.8 Hz, 4H), 2.48 (s, 6H), 2.03e1.97
(m, 2H), 1.74e1.65 (m, 4H); 13C NMR (101 MHz, CDCl3):
d 170.30,
161.25, 159.46, 148.89, 139.22, 131.93, 130.70, 130.56, 128.04, 125.70,
125.66, 122.69, 121.06, 121.02, 113.73, 62.21, 59.68, 54.52, 45.47,
42.39, 24.70, 23.92; HRMS (ESI): Calcd for [M ꢀ H]ꢀ (C25H32N6O)
requires m/z 431.2559, found 431.2550.
5.1.8.3. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-2-morpholinoacetamide (11c). The compound 7a was
treated with excess morpholine according to general procedure H
to afford 11c. After column chromatography with CHCl3/MeOH/
NH3$H2O (25:1:0.1) elution, the desired product was obtained as
a white solid in 78% yield. m.p. 144ꢀ145 ꢁC; 1H NMR (400 MHz,
5.1.7.1. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-4-nitrobenzamide (8a). The compound 6a was treated with
4-nitrobenzoic acid according to general procedure G to afford 8a
as a pale solid in 85% yield. 1H NMR (400 MHz, CDCl3):
d 14.89 (s,
1H), 9.05 (s, 1H), 8.92 (d, J ¼ 7.9 Hz, 1H), 8.81 (d, J ¼ 7.5 Hz, 1H), 8.40
(d, J ¼ 7.9 Hz, 2H), 8.32 (d, J ¼ 7.7 Hz, 2H), 7.74 (t, J ¼ 8.0 Hz,1H), 7.67
(d, J ¼ 5.9 Hz, 1H), 7.62 (d, J ¼ 8.1 Hz, 1H), 7.56e7.42 (m, 3H), 3.90
(dd, J ¼ 8.9, 5.3 Hz, 2H), 2.68 (t, J ¼ 5.6 Hz, 2H), 2.44 (s, 6H), 1.99e
1.90 (m, 2H); LCeMS m/z: 471 [M þ H]þ.
CDCl3):
d
13.31 (s, 1H), 8.84 (s, 1H), 8.66 (d, J ¼ 8.2 Hz, 1H), 8.46
(dd, J ¼ 7.9, 1.5 Hz, 1H), 7.86 (d, J ¼ 8.2 Hz, 1H), 7.66 (t, J ¼ 7.6 Hz,
1H), 7.59 (d, J ¼ 8.1 Hz, 1H), 7.42e7.33 (m, 2H), 7.11 (t, J ¼ 7.1 Hz,
1H), 3.80 (dd, J ¼ 10.4, 5.6 Hz, 2H), 3.54e3.47 (m, 4H), 3.23 (s, 2H),
2.59e2.55 (m, 2H), 2.53e2.46 (m, 4H), 2.34 (s, 6H), 1.87e1.80 (m,
5.1.7.2. N-(5-chloro-2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)-4-nitrobenzamide (8b). The compound 6b was
treated with 4-nitrobenzoic acid according to general procedure G
to afford 8b as a pale solid in 80% yield. 1H NMR (400 MHz, CDCl3):
2H); 13C NMR (101 MHz, CDCl3):
d 168.73, 161.34, 159.51, 148.69,
138.87, 132.30, 130.74, 130.69, 128.08, 125.87, 125.69, 122.92,
121.21, 120.95, 113.80, 66.34, 64.79, 59.67, 53.78, 45.47, 42.47,
24.62; HRMS (ESI): Calcd for [M ꢀ H]ꢀ (C25H32N6O2) requires m/z
447.2508, found 447.2500.
d
14.89 (s,1H), 9.06 (t, J ¼ 4.4 Hz,1H), 8.92 (d, J ¼ 8.2 Hz,1H), 8.81 (dd,
J ¼ 8.0,1.4 Hz,1H), 8.40 (d, J ¼ 8.7 Hz, 2H), 8.32 (d, J ¼ 8.8 Hz, 2H), 7.73
(d, J ¼ 8.0 Hz, 1H), 7.63 (t, J ¼ 6.7 Hz, 2H), 7.54e7.49 (m, 1H), 7.46 (t,
J ¼ 7.6 Hz, 1H), 3.89 (dd, J ¼ 10.2, 5.6 Hz, 2H), 2.65 (t, J ¼ 5.6 Hz, 2H),
2.42 (s, 6H), 1.95e1.89 (m, 2H); LCeMS m/z: 505 [M þ H]þ.
5.1.8.4. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-2-(4-methylpiperazin-1-yl)acetamide (11d). The compound
7a was treated with excess N-methyl piperazine according to gen-
eral procedure H to afford 11d. After column chromatography with
CHCl3/MeOH/NH3$H2O (25:1:0.1) elution, the desired product was
obtained as a white solid in 76% yield. m.p. 152ꢀ154 ꢁC; 1H NMR
5.1.8. General procedure H: preparation of 11ae11d, 12ae12e,
13ae13d, 14ae14e and 15ae15e
To a stirred refluxing suspension of the chloride compounds 7a,
7b, 10a, 10b and 10c (0.5 mmol) in MeOH (15 mL) was added
dropwise appropriate secondary amine (2.0 mL) in EtOH (5 mL).
The mixture was stirred under reflux for 6 h, cooled to room
temperature, and diluted with distilled water. The resulting solu-
tion was filtered, washed with ether and water, and then evapo-
rated under vacuum. The crude solid was purified by using
chromatography with CHCl3/MeOH/NH3$H2O elution to afford
11ae11d, 12ae12e, 13ae13d, 14ae14e and 15ae15e.
(400 MHz, CDCl3):
d
13.11 (s,1H), 8.83 (s,1H), 8.65 (d, J ¼ 8.3 Hz,1H),
8.41 (d, J ¼ 9.1 Hz, 1H), 7.89 (d, J ¼ 8.3 Hz, 1H), 7.66 (t, J ¼ 7.6 Hz, 1H),
7.58 (d, J ¼ 8.1 Hz,1H), 7.43e7.31 (m, 2H), 7.10 (t, J ¼ 7.6 Hz,1H), 3.80
(dd, J ¼ 10.4, 5.5 Hz, 2H), 3.22 (s, 2H), 2.72e2.40 (m, 6H), 2.34 (s, 6H),
2.28e2.12 (m, 4H), 2.02 (s, 3H), 1.86 (dt, J ¼ 7.1, 3.4 Hz, 2H); 13C NMR
(101 MHz, CDCl3):
d 168.11,160.39,158.51, 147.83, 137.69, 131.31,
129.58, 127.60, 124.78, 121.90, 120.08, 120.04, 63.18, 58.68, 53.17,
52.36, 44.79, 44.42, 41.46, 23.46; HRMS (ESI): Calcd for [M ꢀ H]ꢀ
(C26H35N7O) requires m/z 460.2825, found 460.2814.
5.1.8.1. 2-(Diethylamino)-N-(2-(4-((3-(dimethylamino)propyl)
amino)quinazolin-2-yl)phenyl)acetamide (11a). The compound 7a
was treated with excess diethylamine according to general pro-
cedure H to afford 11a. After column chromatography with CHCl3/
MeOH/NH3$H2O (25:1:0.1) elution, the desired product was
obtained as a white solid in 76% yield. m.p. 128ꢀ129 ꢁC; 1H NMR
5.1.8.5. 3-(Diethylamino)-N-(2-(4-((3-(dimethylamino)propyl)
amino)quinazolin-2-yl)phenyl)propanamide (12a). The compound
7b was treated with excess diethylamine according to general
procedure H to afford 12a. After column chromatography with
CHCl3/MeOH/NH3$H2O (25:1:0.1) elution, the desired product was
obtained as a white solid in 79% yield. m.p. 112ꢀ113 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
13.35 (s, 1H), 8.75 (t, J ¼ 6.0 Hz, 2H), 8.51 (dd,
(400 MHz, CDCl3): d 13.94 (s, 1H), 8.93 (s, 1H), 8.77e8.69 (m, 2H),
J ¼ 7.9, 1.6 Hz, 1H), 7.95e7.86 (m, 1H), 7.77e7.65 (m, 2H), 7.43
(ddd, J ¼ 8.6, 6.5, 2.1 Hz, 2H), 7.17 (t, J ¼ 8.1 Hz, 1H), 3.89 (dd,
J ¼ 10.7, 5.6 Hz, 2H), 3.28 (s, 2H), 2.76e2.58 (m, 6H), 2.45 (s, 6H),
1.97 (dt, J ¼ 11.4, 5.9 Hz, 2H), 0.97 (t, J ¼ 7.2 Hz, 6H); 13C NMR
7.80 (d, J ¼ 7.9 Hz, 1H), 7.73 (t, J ¼ 7.6 Hz, 1H), 7.64 (d, J ¼ 8.0 Hz,
1H), 7.47e7.40 (m, 2H), 7.15 (t, J ¼ 8.2 Hz, 1H), 3.89 (dd, J ¼ 10.4,
5.7 Hz, 2H), 3.10e3.01 (m, 2H), 2.81e2.73 (m, 2H), 2.71e2.62 (m,
6H), 2.42 (s, 6H), 1.97e1.91 (m, 2H), 1.09 (t, J ¼ 7.2 Hz, 6H); 13C
(101 MHz, CDCl3):
d
171.77, 161.31, 159.56, 149.05, 138.82, 131.99,
NMR (101 MHz, CDCl3) : d 170.87, 161.40, 159.22, 148.27, 140.25,
130.87, 130.38, 128.05, 126.43, 125.58, 122.78, 121.26, 121.06,
113.77, 59.63, 58.77, 49.04, 45.47, 42.31, 24.78, 11.63; HRMS (ESI):
Calcd for [M ꢀ H]ꢀ (C25H34N6O) requires m/z 433.2716, found
433.2709.
132.50, 131.01, 130.67, 127.47, 125.78, 123.74, 122.36, 121.21, 120.35,
113.80, 59.68, 49.02, 46.98, 45.46, 42.50, 36.63, 24.62, 11.85; HRMS
(ESI): Calcd for [M ꢀ H]ꢀ (C26H36N6O) requires m/z 447.2872,
found 447.2864.

J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
9
5.1.8.6. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-3-(pyrrolidin-1-yl)propanamide (12b). The compound 7b
was treated with excess pyrrolidine according to general procedure
H to afford 12b. After column chromatography with CHCl3/MeOH/
NH3$H2O (25:1:0.1) elution, the desired product was obtained as
a white solid in 74% yield. m.p. 156ꢀ157 ꢁC; 1H NMR (400 MHz,
5.1.8.10. 4-(2-(Diethylamino)acetamido)-N-(2-(4-((3-(dimethyla-
mino)propyl)amino)quinazolin-2-yl)phenyl)benzamide (13a).
The compound 10a was treated with excess diethylamine according
to general procedure H to afford 13a. After column chromatography
with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired product
was obtained as a white solid in 78% yield. m.p. 160ꢀ162 ꢁC; 1H
CDCl3):
d
13.99 (s, 1H), 8.95 (s, 1H), 8.72 (d, J ¼ 8.1 Hz, 2H), 7.84 (d,
NMR (400 MHz, CDCl3): d 14.36 (s, 1H), 9.53 (s, 1H), 8.86 (d,
J ¼ 8.3 Hz,1H), 7.74 (t, J ¼ 7.3 Hz,1H), 7.64 (d, J ¼ 8.1 Hz,1H), 7.43 (dt,
J ¼ 8.3, 4.3 Hz, 2H), 7.15 (t, J ¼ 7.6 Hz, 1H), 3.90 (dd, J ¼ 10.3, 5.6 Hz,
2H), 3.09 (t, J ¼ 7.5 Hz, 2H), 2.89 (t, J ¼ 7.5 Hz, 2H), 2.73 (t, J ¼ 6.4 Hz,
4H), 2.68e2.63 (m, 2H), 2.43 (s, 6H), 1.94 (dt, J ¼ 11.3, 5.8 Hz, 2H),
J ¼ 8.3 Hz, 1H), 8.69 (d, J ¼ 8.0 Hz, 2H), 8.09 (d, J ¼ 8.5 Hz, 2H), 7.73e
7.67 (m, 3H), 7.65 (d, J ¼ 7.6 Hz, 1H), 7.59 (d, J ¼ 8.1 Hz, 1H), 7.41 (t,
J ¼ 7.3 Hz,1H), 7.35 (t, J ¼ 7.4 Hz,1H), 7.11 (t, J ¼ 7.5 Hz,1H), 3.80 (dd,
J ¼ 10.3, 5.2 Hz, 2H), 3.12 (s, 2H), 2.60 (m, 6H), 2.34 (s, 6H), 1.88e
1.83 (m, 2H), 1.05 (t, J ¼ 7.1 Hz, 6H); 13C NMR (101 MHz, CDCl3):
1.84 (dt, J ¼ 6.7, 3.4 Hz, 4H); 13C NMR (101 MHz, CDCl3):
d 170.44,
161.38, 159.20, 148.25, 140.30, 132.50, 130.97, 130.68, 127.51, 125.75,
123.72, 122.31, 121.18, 120.36, 113.80, 59.85, 54.15, 52.25, 45.50,
42.67, 38.54, 24.59, 23.53; HRMS (ESI): Calcd for [M ꢀ H]ꢀ
(C26H34N6O) requires m/z 445.2716, found 445.2705.
d 169.47, 164.74,160.41, 158.23, 147.22,139.57, 139.52, 131.71, 130.99,
130.02, 129.87, 128.00, 125.98, 124.80, 123.30, 121.50, 120.33, 119.41,
117.69, 112.74, 58.42, 57.17, 47.87, 44.27, 41.17, 23.47, 11.41; HRMS
(ESI): Calcd for [M ꢀ H]ꢀ (C32H39N7O2) requires m/z 552.3092,
found 552.3115.
5.1.8.7. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-3-morpholinopropanamide (12c). The compound 7b was
treated with excess morpholine according to general procedure H to
afford 12c. After column chromatography with CHCl3/MeOH/
NH3$H2O (25:1:0.1) elution, the desired product was obtained as
awhite solidin75% yield. m.p.151ꢀ153 ꢁC; 1H NMR(400 MHz, CDCl3):
5.1.8.11. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-4-(2-(pyrrolidin-1-yl)acetamido)benzamide
(13b). The
compound 10a was treated with excess pyrrolidine according to
general procedure H to afford 13b. After column chromatography
with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired product
was obtained as a white solid in 72% yield. m.p.174ꢀ176 ꢁC; 1H NMR
d
13.91 (s, 1H), 8.92 (s, 1H), 8.71 (t, J ¼ 7.0 Hz, 2H), 7.79e7.68 (m, 3H),
7.49e7.41 (m, 2H), 7.15 (t, J ¼ 7.6 Hz,1H), 3.91 (dd, J ¼ 10.5, 5.3 Hz, 2H),
3.69e3.61 (m, 4H), 2.90 (t, J ¼ 7.2 Hz, 2H), 2.79e2.69 (m, 4H), 2.57e
2.50 (m, 4H), 2.47 (s, 6H), 2.02e1.96 (m, 2H); 13C NMR (101 MHz,
(400 MHz, CDCl3):
d
14.48 (s,1H), 9.34 (s,1H), 8.96 (d, J ¼ 8.4 Hz,1H),
8.88 (s,1H), 8.78 (d, J ¼ 7.9 Hz,1H), 8.18 (d, J ¼ 8.5 Hz, 2H), 7.83e7.75
(m, 4H), 7.66 (d, J ¼ 8.0 Hz, 1H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.45 (t,
J ¼ 7.4 Hz, 1H), 7.20 (t, J ¼ 7.6 Hz, 1H), 3.89 (dd, J ¼ 10.1, 5.3 Hz, 2H),
3.35 (s, 2H), 2.75 (t, J ¼ 6.3 Hz, 4H), 2.66 (t, J ¼ 6 Hz, 2H), 2.43 (s, 6H),
CDCl3): d 170.29, 161.40, 159.21, 148.21, 140.20, 132.52, 131.02, 130.72,
127.32, 125.79, 123.75, 122.42, 121.29, 120.37, 113.82, 66.92, 59.83,
54.78, 53.54, 45.50, 42.66, 36.42, 24.58; HRMS (ESI): Calcd for
[M ꢀ H]ꢀ (C26H34N6O2) requires m/z 461.2665, found 461.2655.
1.96e1.90 (m, 6H); 13C NMR (101 MHz, CDCl3):
d 168.52, 164.71,
160.34, 158.15, 147.14, 139.67, 139.51, 131.65, 130.93, 129.95, 129.90,
127.91, 125.87, 124.74, 123.27, 121.47, 120.28, 119.34, 117.88, 112.71,
58.87, 58.47, 53.62, 44.33, 41.25, 23.55, 23.08; HRMS (ESI): Calcd for
[M þ H]þ (C32H37N7O2) requires m/z 552.3082, found 552.3082.
5.1.8.8. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-3-(4-methylpiperazin-1-yl)propanamide (12d). The com-
pound 7b was treated with excess N-methyl piperazine according to
general procedure H to afford 12d. After column chromatography
with CHCl3/MeOH/NH3$H2O (25:1:0.1) elution, the desired product
was obtained as a white solid in 74% yield. m.p.153ꢀ155 ꢁC; 1H NMR
5.1.8.12. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-4-(2-morpholinoacetamido)benzamide (13c). The com-
pound 10a was treated with excess morpholine according to gen-
eral procedure H to afford 13c. After column chromatography with
CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired product was
obtained as a white solid in 79% yield. m.p. 188ꢀ189 ꢁC; 1H NMR
(400 MHz, CDCl3):
d
13.87 (s,1H), 8.91 (s,1H), 8.71 (t, J ¼ 8.7 Hz, 2H),
7.82e7.65 (m, 3H), 7.49e7.40 (m, 2H), 7.15 (t, J ¼ 7.3 Hz,1H), 3.90 (dd,
J ¼ 10.5, 5.5 Hz, 2H), 2.93 (t, J ¼ 7.4 Hz, 2H), 2.76 (t, J ¼ 7.4 Hz, 2H),
2.72e2.68 (m, 2H), 2.67e2.51 (m, 4H), 2.50e2.37 (m, 10H), 2.27 (s,
(400 MHz, CDCl3):
d
14.41 (s,1H), 9.19 (s,1H), 8.87 (d, J ¼ 8.3 Hz,1H),
3H), 2.01e1.94 (m, 2H); 13C NMR (101 MHz, CDCl3):
d
170.39, 161.43,
8.80 (s,1H), 8.70 (d, J ¼ 7.9 Hz,1H), 8.10 (d, J ¼ 8.3 Hz, 2H), 7.73e7.65
(m, 4H), 7.57 (d, J ¼ 8.0 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 7.36 (t,
J ¼ 8 Hz, 1H), 7.12 (t, J ¼ 7.5 Hz, 1H), 3.81 (dd, J ¼ 10.4, 5.9 Hz, 2H),
3.74 (t, J ¼ 6 Hz, 4H), 3.13 (s, 2H), 2.62e2.55 (m, 6H), 2.34 (s, 6H),
159.22, 148.28, 140.22, 132.53, 131.01, 130.67, 127.47, 125.77, 123.77,
122.36, 121.19, 120.39, 113.82, 59.92, 55.09, 54.34, 52.99, 46.00,
45.51, 42.74, 36.47, 24.55; HRMS (ESI): Calcd for [M ꢀ H]ꢀ
(C27H37N7O) requires m/z 474.2981, found 474.2970.
1.87e1.83 (m, 2H); 13C NMR (101 MHz, CDCl3):
d 167.24, 164.65,
160.50, 158.27, 147.26, 139.50, 139.32, 131.73, 131.31, 130.09, 129.89,
128.03, 126.01, 124.81, 123.32, 121.57, 120.30, 119.45, 117.86, 112.79,
66.03, 61.57, 58.64, 52.83, 44.39, 41.41, 23.49; HRMS (ESI): Calcd for
[M ꢀ H]ꢀ (C32H37N7O3) requires m/z 566.2885, found 566.2871.
5.1.8.9. 3-((3-(Dimethylamino)propyl)amino)-N-(2-(4-((3-(dimethy-
lamino)propyl)amino)quinazolin-2-yl)phenyl)propanamide
(12e).
The compound 7b was treated with excess 3-(dimethylamino)-1-
propylamine according to general procedure H to afford 12e. Af-
ter column chromatography with CHCl3/MeOH/NH3$H2O (25:1:0.1)
elution, the desired product was obtained as a white solid in 73%
5.1.8.13. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-4-(2-(4-methylpiperazin-1-yl)acetamido)benzamide (13d).
The compound 10a was treated with excess N-methylpiperazine
according to general procedure H to afford 13d. After column
chromatography with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the
desired product was obtained as a white solid in 71% yield. m.p.
yield. m.p. 124ꢀ126 ꢁC; 1H NMR (400 MHz, CDCl3):
d 14.01 (s, 1H),
8.91 (s, 1H), 8.72 (t, J ¼ 6.8 Hz, 2H), 7.80e7.70 (m, 2H), 7.62 (d,
J ¼ 8.1 Hz, 1H), 7.47e7.40 (m, 2H), 7.16 (t, J ¼ 7.6 Hz, 1H), 3.88 (dd,
J ¼ 9.9, 4.8 Hz, 2H), 3.10 (t, J ¼ 6.0 Hz, 2H), 2.84 (t, J ¼ 6.2 Hz, 2H),
2.77 (t, J ¼ 6.8 Hz, 2H), 2.64 (t, J ¼ 4.8 Hz, 2H), 2.40 (s, 6H), 2.34 (t,
J ¼ 7.2 Hz, 2H), 2.20 (s, 6H), 1.95e1.90 (m, 2H), 1.73e1.67 (m, 2H);
183ꢀ184 ꢁC; 1H NMR (400 MHz, CDCl3):
d 14.41 (s, 1H), 9.28 (s, 1H),
8.87 (d, J ¼ 8.3 Hz, 1H), 8.75 (s, 1H), 8.70 (d, J ¼ 7.9 Hz, 1H), 8.10 (d,
J ¼ 8.4 Hz, 2H), 7.73e7.67 (m, 4H), 7.62 (d, J ¼ 8.2 Hz, 1H), 7.42 (t,
J ¼ 7.8 Hz,1H), 7.36 (t, J ¼ 7.4 Hz,1H), 7.12 (t, J ¼ 7.6 Hz,1H), 3.80 (dd,
J ¼ 9.4, 5.3 Hz,2H), 3.12 (s, 2H), 2.65e2.57 (m, 6H), 2.51e2.44 (m,
4H), 2.35 (s, 6H), 2.09 (s, 3H),1.90e1.84 (m, 2H); 13C NMR (101 MHz,
13C NMR (101 MHz, CDCl3):
d 170.35, 161.28, 159.22, 148.22, 140.09,
132.60, 131.02, 130.69, 127.62, 127.59, 125.83, 122.50, 121.18, 120.28,
113.78, 59.79, 58.09, 48.55, 45.56, 45.46, 45.35, 42.63, 37.74, 27.03,
24.52; HRMS (ESI): Calcd for [M þ H]þ (C27H39N7O) requires m/z
478.3289, found 478.3288.
CDCl3):
d 167.67, 164.70, 160.42, 158.26, 147.22, 139.50, 139.47,

10
J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
131.73, 131.14, 130.04, 129.89, 128.00, 125.96, 124.81, 123.31, 121.55,
120.37, 119.42, 117.82, 112.77, 60.95, 58.23, 54.20, 52.44, 44.94,
44.21, 41.06, 23.52; HRMS (ESI): Calcd for [M ꢀ H]ꢀ (C33H40N8O2)
requires m/z 579.3201, found 579.3181.
elution, the desired product was obtained as a white solid in 71%
yield. m.p. 164ꢀ166 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d 14.32 (s,
1H),10.49 (s, 1H), 8.80 (d, J ¼ 8.1 Hz,1H), 8.75e8.66 (m, 2H), 8.28 (d,
J ¼ 7.8 Hz, 1H), 8.05 (d, J ¼ 8.0 Hz, 2H), 7.86e7.79 (m, 3H), 7.73 (d,
J ¼ 8.1 Hz, 1H), 7.57 (t, J ¼ 7.4 Hz, 1H), 7.52 (t, J ¼ 7.6 Hz, 1H), 7.24 (t,
J ¼ 7.2 Hz, 1H), 3.70 (dd, J ¼ 11.4, 6.4 Hz, 2H), 2.67 (t, J ¼ 6.4 Hz, 2H),
2.56e2.50 (m, 4H), 2.40e2.31 (m, 6H), 2.19 (s, 6H), 2.16 (s, 3H),
5.1.8.14. 4-(3-(Diethylamino)propanamido)-N-(2-(4-((3-(dimethyla-
mino)propyl)amino)quinazolin-2-yl)phenyl)benzamide
(14a).
The compound 10b was treated with excess diethylamine accord-
ing to general procedure H to afford 14a. After column chroma-
tography with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired
1.93e1.83 (m, 4H); 13C NMR (101 MHz, CDCl3):
d 169.78, 164.88,
160.39, 158.44, 147.32, 140.71, 139.57, 131.76, 130.62, 130.12, 129.80,
128.01, 126.03, 124.91, 123.29, 121.50, 120.76, 119.50, 117.94, 112.82,
56.81, 54.23, 52.54, 51.16, 44.89, 43.43, 39.77, 31.64, 23.31; HRMS
(ESI): Calcd for [M ꢀ H]ꢀ (C34H42N8O3) requires m/z 593.3358,
found 593.3359.
product was obtained as
a white solid in 79% yield. m.p.
155ꢀ157 ꢁC; 1H NMR (400 MHz, CDCl3):
d 14.37 (s, 1H), 11.55 (s, 1H),
8.88 (d, J ¼ 8.3 Hz, 1H), 8.70 (d, J ¼ 7.2 Hz, 2H), 8.06 (d, J ¼ 8.2 Hz,
2H), 7.74 (d, J ¼ 8.6 Hz, 1H), 7.64 (d, J ¼ 7.9 Hz, 4H), 7.43e7.36 (m,
2H), 7.11 (t, J ¼ 7.7 Hz, 1H), 3.82 (dd, J ¼ 9.3, 5.8 Hz, 2H), 2.75 (t,
J ¼ 5.5 Hz, 2H), 2.68e2.61 (m, 6H), 2.49 (t, J ¼ 5.5 Hz, 2H), 2.38 (s,
6H), 1.92e1.87 (m, 2H), 1.11 (t, J ¼ 7.0 Hz, 6H); 13C NMR (101 MHz,
5.1.8.18. 4-(3-((3-(Dimethylamino)propyl)amino)propanamido)-N-
(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)phenyl)
benzamide (14e). The compound 10b was treated with excess 3-
(dimethylamino)-1-propylamine according to general procedure H
to afford 14e. After column chromatography with CHCl3/MeOH/
NH3$H2O (5:1:0.1) elution, the desired product was obtained as
a white solid in 73% yield. m.p. 108ꢀ109 ꢁC; 1H NMR (400 MHz,
CDCl3):
d 170.20, 164.95, 160.47, 158.30, 147.31, 140.79, 139.63,
131.66, 130.51, 130.07, 129.84, 127.98, 126.12, 124.81, 123.26, 121.44,
120.36, 119.43,117.83,112.78, 58.17, 47.90, 44.98, 44.14, 40.99, 32.24,
23.49, 10.51; HRMS (ESI): Calcd for [M ꢀ H]ꢀ (C33H41N7O2) requires
m/z 566.3249, found 566.3225.
CDCl3):
d
14.38 (s, 1H), 11.08 (s, 1H), 8.87 (d, J ¼ 8.3 Hz, 1H), 8.82 (t,
J ¼ 3.9 Hz, 1H), 8.71 (dd, J ¼ 8.0, 1.5 Hz, 1H), 8.07 (d, J ¼ 8.6 Hz, 2H),
7.74 (d, J ¼ 8.3 Hz, 1H), 7.68e7.65 (m, 3H), 7.53 (d, J ¼ 7.8 Hz, 1H),
7.44e7.39 (m, 1H), 7.38e7.33 (m, 1H), 7.14e7.10 (m, 1H), 3.81 (dd,
J ¼ 10.1, 5.4 Hz, 2H), 2.96 (t, J ¼ 5.6 Hz, 2H), 2.74 (t, J ¼ 6.7 Hz, 2H),
2.56 (t, J ¼ 5.5 Hz, 2H), 2.49 (t, J ¼ 5.6 Hz, 2H), 2.37e2.31 (m, 8H),
2.17 (s, 6H), 1.86e1.81 (m, 2H), 1.73e1.69 (m, 3H); 13C NMR
5.1.8.15. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-4-(3-(pyrrolidin-1-yl)propanamido)benzamide
(14b).
The compound 10b was treated with excess pyrrolidine according
to general procedure H to afford 14b. After column chromatography
with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired product
was obtained as a white solid in 70% yield. m.p. 175ꢀ176 ꢁC; 1H
(101 MHz, CDCl3):
d 171.40, 165.94, 161.53, 159.27, 148.32, 141.73,
NMR (400 MHz, CDCl3):
d
14.46 (s, 1H), 11.61 (s, 1H), 8.96 (dd,
140.59, 132.74, 131.54, 131.09, 130.89, 128.93, 127.14, 125.79, 124.34,
122.49, 121.19, 120.46, 119.06, 113.80, 59.82, 58.19, 47.87, 45.57,
45.52, 45.34, 42.60, 36.05, 27.71, 24.59; HRMS (ESI): Calcd for
[M þ 2H]2þ (C34H44N8O2) requires m/z 299.1866, found 299.1864.
J ¼ 8.3, 0.7 Hz, 1H), 8.89 (t, J ¼ 4.4 Hz, 1H), 8.79 (dd, J ¼ 8.0, 1.5 Hz,
1H), 8.15 (d, J ¼ 8.6 Hz, 2H), 7.82 (d, J ¼ 8.2 Hz, 1H), 7.76e7.71 (m,
1H), 7.68 (d, J ¼ 8.6 Hz, 2H), 7.62 (d, J ¼ 7.9 Hz, 1H), 7.52e7.47 (m,
1H), 7.46e7.42 (m, 1H), 7.22e7.17 (m, 1H), 3.89 (dd, J ¼ 10.2, 5.7 Hz,
2H), 2.91 (t, J ¼ 5.8 Hz, 2H), 2.73 (t, J ¼ 6.4 Hz, 4H), 2.64 (t, J ¼ 5.5 Hz,
2H), 2.59 (t, J ¼ 5.8 Hz, 2H), 2.41 (s, 6H), 1.98e1.88 (m, 6H); 13C NMR
5.1.8.19. N-(5-chloro-2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)-4-(3-(diethylamino)propanamido)benzamide
(15a). The compound 10c was treated with excess diethylamine
according to general procedure H to afford 15a. After column
chromatography with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the
desired product was obtained as a white solid in 54% yield. m.p.
(101 MHz, CDCl3):
d 170.15, 164.90, 160.56, 158.28, 147.35, 140.87,
139.63, 131.66, 130.52, 130.09, 129.86, 127.96, 126.20, 124.77, 123.30,
121.44, 120.17, 119.44, 117.99, 112.80, 58.86, 52.11, 50.24, 44.49,
41.64, 33.75, 23.56, 22.76; HRMS (ESI): Calcd for [M ꢀ H]ꢀ
(C33H39N7O2) requires m/z 564.3092, found 564.3067.
173ꢀ174 ꢁC; 1H NMR (400 MHz, CDCl3):
d 14.59 (s, 1H), 11.67 (s, 1H),
9.07 (d, J ¼ 1.9 Hz, 1H), 8.91 (s, 1H), 8.71 (d, J ¼ 8.6 Hz, 1H), 8.13 (d,
J ¼ 8.6 Hz, 2H), 7.80 (d, J ¼ 8.0 Hz, 1H), 7.75e7.70 (m, 4H), 7.45 (t,
J ¼ 7.5 Hz, 1H), 7.13 (dd, J ¼ 8.6, 2.0 Hz, 1H), 3.89 (dd, J ¼ 10.1, 5.0 Hz,
2H), 2.83 (t, J ¼ 5.8 Hz, 2H), 2.75e2.70 (m, 6H), 2.57 (t, J ¼ 5.8 Hz,
2H), 2.48 (s, 6H), 2.01e1.97 (m, 2H), 1.18 (t, J ¼ 7.1 Hz, 6H); 13C NMR
5.1.8.16. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-4-(3-morpholinopropanamido)benzamide (14c). The com-
pound 10b was treated with excess morpholine according to gen-
eral procedure H to afford 14c. After column chromatography with
CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired product was
obtained as a white solid in 73% yield. m.p. 199ꢀ201 ꢁC; 1H NMR
(101 MHz, CDCl3):
d 170.16, 165.06, 159.76, 158.28, 147.18, 140.96,
140.56, 135.95, 131.89, 130.85, 130.10, 128.03, 126.06, 125.09, 121.48,
121.41, 120.56, 119.20, 117.88, 112.83, 47.94, 45.03, 43.99, 38.18,
32.24, 28.68, 23.16, 10.49; HRMS (ESI): Calcd for [M þ 2H]2þ
(C33H40ClN7O2) requires m/z 301.6539, found 301.6547.
(400 MHz, CDCl3):
d
14.41 (s, 1H), 11.01 (s, 1H), 8.87 (d, J ¼ 8.3 Hz,
1H), 8.80 (s, 1H), 8.70 (d, J ¼ 7.8 Hz, 1H), 8.08 (d, J ¼ 8.5 Hz, 2H), 7.73
(d, J ¼ 8.2 Hz, 1H), 7.67e7.62 (m, 3H), 7.58 (d, J ¼ 8.1 Hz, 1H), 7.42 (t,
J ¼ 7.8 Hz, 1H), 7.36 (t, J ¼ 7.4 Hz, 1H), 7.12 (t, J ¼ 7.3 Hz, 1H), 3.84e
3.77 (m, 6H), 2.71 (t, J ¼ 5.6 Hz, 2H), 2.64e2.56 (m, 6H), 2.53 (t,
J ¼ 5.8 Hz, 2H), 2.35 (s, 6H), 1.89e1.84 (m, 2H); 13C NMR (101 MHz,
5.1.8.20. N-(5-Chloro-2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)-4-(3-(pyrrolidin-1-yl)propanamido)benzamide
(15b). The compound 10c was treated with excess pyrrolidine ac-
cording to general procedure H to afford 15b. After column chro-
matography with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the
desired product was obtained as a white solid in 53% yield. m.p.
CDCl3):
d 169.54, 164.79, 160.44, 158.21, 147.20, 140.59, 139.54,
131.58, 130.67, 130.05, 129.89, 128.01, 125.97, 124.79, 123.26, 121.51,
120.26, 119.35,117.87,112.77, 66.05, 58.67, 53.06, 51.80, 44.42, 41.46,
31.44, 23.54; HRMS (ESI): Calcd for [M ꢀ H]ꢀ (C33H39N7O3) requires
m/z 580.3042, found 580.3027.
195ꢀ196 ꢁC; 1H NMR (400 MHz, CDCl3):
d 14.60 (s,1H), 11.66 (s, 1H),
9.06 (s, 1H), 8.97 (s, 1H), 8.72 (d, J ¼ 8.7 Hz, 1H), 8.13 (d, J ¼ 8.1 Hz,
2H), 7.79 (d, J ¼ 8.1 Hz, 1H), 7.75e7.66 (m, 3H), 7.60 (d, J ¼ 7.5 Hz,
1H), 7.43 (t, J ¼ 7.2 Hz, 1H), 7.13 (d, J ¼ 8.0 Hz, 1H), 3.86 (dd, J ¼ 9.3,
4.9 Hz, 2H), 2.90 (t, J ¼ 5.0 Hz, 2H), 2.73 (t, J ¼ 5.6 Hz, 4H), 2.65e2.57
(m, 4H), 2.40 (s, 6H), 1.97e1.89 (m, 6H); 13C NMR (101 MHz, CDCl3):
5.1.8.17. N-(2-(4-((3-(dimethylamino)propyl)amino)quinazolin-2-yl)
phenyl)-4-(3-(4-methylpiperazin-1-yl)propanamido)benzamide
(14d). The compound 10b was treated with excess N-methyl
piperazine according to general procedure H to afford 14d. After
column chromatography with CHCl3/MeOH/NH3$H2O (5:1:0.1)
d
170.20, 165.03,159.85, 158.22, 147.17,141.09, 140.52, 135.91, 131.80,

J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
11
130.92, 130.07, 128.01, 126.11, 124.94, 121.49, 121.44, 120.22, 119.16,
118.02, 112.86, 58.88, 52.10, 50.23, 44.47, 41.70, 33.71, 23.40, 22.75;
HRMS (ESI): Calcd for [M þ 2H]2þ (C33H38ClN7O2) requires m/z
300.6461, found 300.6455.
Further dilutions to working concentrations were made with
double-distilled deionized water.
5.3. CD measurements
5.1.8.21. N-(5-Chloro-2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)-4-(3-morpholinopropanamido)benzamide (15c).
The compound 10c was treated with excess morpholine according
to general procedure H to afford 15c. After column chromatography
with CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired product
was obtained as a white solid in 54% yield. m.p. 212ꢀ214 ꢁC; 1H
The CD spectra were recorded on Chirascan (Applied Photo-
physics) spectrophotometer. A quartz cuvette with 1 mm path
length was used for the spectra recorded over a wavelength range
of 230e320 at 1 nm bandwidth, 1 nm step size, and 0.5 s per point.
The oligomer HTG21 (50-d(GGG[TTAGGG]3)-30) was diluted from
stock to the required concentration (5
buffer, pH 7.4, 150 mM KCl, and then annealed by heating at
95 ꢁC in the presence of 25
M compound for 5 min, gradually
mM) in 10 mM TriseHCl
NMR (400 MHz, CDCl3):
d 14.62 (s, 1H), 11.13 (s, 1H), 9.07 (d,
J ¼ 2.1 Hz,1H), 8.99 (t, J ¼ 3.6 Hz,1H), 8.73 (d, J ¼ 8.6 Hz,1H), 8.15 (d,
J ¼ 8.6 Hz, 2H), 7.79 (d, J ¼ 7.8 Hz, 1H), 7.74e7.70 (m, 3H), 7.64 (d,
J ¼ 8.2 Hz,1H), 7.47e7.41 (m, 1H), 7.14 (dd, J ¼ 8.6, 2.2 Hz,1H), 3.89e
3.85 (m, 6H), 2.82e2.78 (m, 2H), 2.71e2.64 (m, 6H), 2.61 (t,
J ¼ 5.8 Hz, 2H), 2.43 (s, 6H), 1.97e1.90 (m, 2H); 13C NMR (101 MHz,
m
cooled to room temperature, and incubated at 4 ꢁC overnight. CD
melting experiments were carried out by recording the CD values at
265 or 290 nm every 3 ꢁC in a temperature range of 25e94 ꢁC at
a heating rate of 1 ꢁC/min in buffer containing 60 mM KCl. Final
analysis of the data was carried out using Origin 8.0 (OriginLab
Corp.).
CDCl3):
d 169.53, 164.91, 159.85, 158.24, 147.15, 140.75, 140.49,
136.63, 135.95, 131.78, 130.92, 130.40, 128.11, 126.02, 125.01, 121.55,
121.43, 120.35, 119.17, 117.91, 66.09, 58.67, 53.11, 51.82, 44.36, 41.46,
31.34, 23.33; HRMS (ESI): Calcd for [M þ 2H]2þ (C33H38ClN7O3) re-
quires m/z 308.6435, found 308.6431.
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
(50 mM TriseHCl, 100 mM KCl, pH 7.4, 0.005% Tween20). 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 0.015625,
0.025, 0.03125, 0.05, 0.0625, 0.1, 0.125, 0.15625, 0.2, 0.25, 0.3125,
5.1.8.22. N-(5-Chloro-2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)-4-(3-(4-methylpiperazin-1-yl)propanamido)ben-
zamide (15d). The compound 10c was treated with excess N-
methyl piperazine according to general procedure H to afford 15d.
After column chromatography with CHCl3/MeOH/NH3$H2O
(5:1:0.1) elution, the desired product was obtained as a white solid
in 60% yield. m.p.182ꢀ183 ꢁC; 1H NMR (400 MHz, CDCl3):
d 14.60 (s,
1H), 11.35 (s, 1H), 9.07 (d, J ¼ 2.2 Hz, 1H), 8.92 (t, J ¼ 4.0 Hz, 1H), 8.71
(d, J ¼ 8.6 Hz, 1H), 8.15 (d, J ¼ 8.6 Hz, 2H), 7.80 (d, J ¼ 7.8 Hz, 1H),
7.76e7.71 (m, 4H), 7.48e7.43 (m, 1H), 7.14 (dd, J ¼ 8.6, 2.2 Hz, 1H),
3.90 (dd, J ¼ 10.6, 5.4 Hz, 2H), 2.88e2.52 (m, 14H), 2.49 (s, 6H), 2.39
0.4, 0.5, 0.625, 0.8, 1, 1.25, 2, 2.5, 5, 10, and 20 mM) were prepared
with running buffer by serial dilutions from stock solutions. Six
concentrations were injected simultaneously at a flow rate of
(s, 3H), 2.02e1.98 (m, 2H); 13C NMR (101 MHz, CDCl3):
d
169.83,
50 m m m
L minꢀ1 for 400 s (or 30 L minꢀ1 for 666 s, 30 L minꢀ1 for
164.96, 159.81, 158.20, 147.13, 140.93, 140.48, 135.90, 131.75, 130.93,
130.20, 128.06, 126.03, 124.93, 121.51, 121.42, 120.29, 119.13, 117.93,
112.87, 58.72, 54.38, 52.55, 51.30, 45.04, 44.42, 41.54, 31.59, 23.39;
HRMS (ESI): Calcd for [M þ 2H]2þ (C34H41ClN8O2) requires m/z
315.1593, found 315.1583.
800 s) of association phase, followed with 400 s (or 666 s, 800 s) of
dissociation phase at 25 ꢁC. The GLH sensor chip was regenerated
with short injection of 50 mM NaOH between consecutive mea-
surements. The final graphs were obtained 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.
5.1.8.23. N-(5-chloro-2-(4-((3-(dimethylamino)propyl)amino)quina-
zolin-2-yl)phenyl)-4-(3-((3-(dimethylamino)propyl)amino)prop-
anamido)benzamide (15e). The compound 10c was treated with
excess 3-(dimethylamino)-1-propylamine according to general
procedure H to afford 15e. After column chromatography with
CHCl3/MeOH/NH3$H2O (5:1:0.1) elution, the desired product was
obtained as a white solid in 56% yield. m.p. 127ꢀ128 ꢁC; 1H NMR
5.5. TRAP-LIG assay
The ability of synthesized compounds to inhibit telomerase in
a cell-free system was assessed with the TRAP-LIG assay following
previously published procedures [36]. Protein extracts from expo-
(400 MHz, CDCl3):
d
14.58 (s,1H),11.16 (s,1H), 9.04 (d, J ¼ 1.9 Hz,1H),
nentially growing HL60 leukemia cells were used. Briefly, 0.1 mg of
8.91 (s,1H), 8.70 (d, J ¼ 8.6 Hz,1H), 8.11 (d, J ¼ 8.5 Hz, 2H), 7.75e7.68
(m, 4H), 7.58 (d, J ¼ 8.1 Hz,1H), 7.40 (t, J ¼ 7.2 Hz,1H), 7.11 (dd, J ¼ 8.6,
1.9 Hz, 1H), 3.82 (dd, J ¼ 9.6, 5.2 Hz, 2H), 3.03 (t, J ¼ 5.6 Hz, 2H), 2.80
(t, J ¼ 6.6 Hz, 2H), 2.61 (t, J ¼ 5.2, 2H), 2.56 (t, J ¼ 5.6 Hz, 2H), 2.43e
TS forward primer (50-AATCCGTC-GAGCAGAGTT-30) was elongated
by telomerase (500 ng protein extract) in TRAP buffer (20 mM Trise
HCl [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
2.37 (m, 8H), 2.23 (s, 6H), 1.91e1.86 (m, 2H), 1.81e1.74 (m, 2H); 13
C
added to tubes containing freshly prepared ligand at various con-
centrations and to a negative control containing no ligand. The
initial elongation step was carried out for 20 min at 30 ꢁC, followed
by 94 ꢁC for 5 min and a final maintenance of the mixture at 20 ꢁC.
To purify the elongated product and to remove the bound ligands,
the QIA quick nucleotide purification kit (Qiagen) was used ac-
cording to the manufacturer’s instructions. The purified extended
samples were then subject to PCR amplification. For this, a second
NMR (101 MHz, CDCl3):
d 171.39, 166.00, 160.73, 159.15, 148.08,
141.93, 141.49, 136.79, 132.79, 131.94, 131.08, 128.96, 126.97, 125.91,
122.45, 122.42, 121.20, 120.09, 119.07, 113.83, 59.83, 58.20, 47.90,
45.54, 45.49, 45.32, 42.66, 36.01, 27.63, 24.46; HRMS (ESI): Calcd for
[M þ 2H]2þ (C34H43ClN8O2) requires m/z 316.1672, found 316.1683.
5.2. Materials
PCR master mix was prepared consisting of 1
primer (50-GCGCGG[CTTACC]3CTAACC-30), 0.1
g TS forward primer
(50-AATCCGTCGAGCAGAGTT-30), TRAP buffer, 5
g BSA, 0.5 mM
dNTPs, and 2 units of Taq polymerase. A 10 L aliquot of the master
mM ACX reverse
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.
m
m
m

12
J.-H. He et al. / European Journal of Medicinal Chemistry 63 (2013) 1e13
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 AlphaEaseFC
software.
Appendix A. Supplementary data
The supplementary data associated with this article can be
found in the online version at doi:10.1016/j.ejmech.2013.01.051.
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We thank the Natural Science Foundation of China (Grants
91213302, 21172272, 81001400), the International S&T Cooperation
Program of China (2010DFA34630), and the Science Foundation of
Guangzhou (2009A1-E011-6) for financial support of this study.

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