Design, synthesis and biological evaluation of 4-anilinoquinazoline derivatives as new c-myc G-quadruplex ligands

Article abstract of DOI:10.1016/j.ejmech.2016.06.040

A series of 4-anilinoquinazoline derivatives were designed and synthesized as novel c-myc promoter G-quadruplex binding ligands. Subsequent biophysical and biochemical evaluation demonstrated that the introduction of aniline group at 4-position of quinazoline ring and two side chains with terminal amino group improved their binding affinity and stabilizing ability to G-quadruplex DNA. RT-PCR assay and Western blot showed that compound 7a could down-regulate transcription and expression of c-myc gene in Hela cells, which was consistent with the behavior of an effective G-quadruplex ligand targeting c-myc oncogene. More importantly, RTCA and colony formation assays indicated that 7a obviously inhibited Hela cells proliferation, without influence on normal primary cultured mouse mesangial cells. Flow cytometric assays suggested that 7a induced Hela cells to arrest in G0/G1 phase both in a time-dependent and dose-dependent manner.

Full text of DOI:10.1016/j.ejmech.2016.06.040

Accepted Manuscript
Design, synthesis and biological evaluation of 4-anilinoquinazoline derivatives as new
c-myc G-quadruplex ligands
Yin Jiang, Ai-Chun Chen, Guo-Tao Kuang, Shi-Ke Wang, Tian-Miao Ou, Jia-Heng
Tan, Ding Li, Zhi-Shu Huang
PII:
S0223-5234(16)30530-X
10.1016/j.ejmech.2016.06.040
EJMECH 8701
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 April 2016
Revised Date: 17 June 2016
Accepted Date: 20 June 2016
Please cite this article as: Y. Jiang, A.-C. Chen, G.-T. Kuang, S.-K. Wang, T.-M. Ou, J.-H. Tan, D.
Li, Z.-S. Huang, Design, synthesis and biological evaluation of 4-anilinoquinazoline derivatives as
new c-myc G-quadruplex ligands, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.06.040.
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ACCEPTED MANUSCRIPT
Graphic Abstract
Design, synthesis and biological evaluation of 4-anilinoquinazoline derivatives as new c-myc
G-quadruplex ligands
Yin Jiang†, Ai-Chun Chen†, Guo-Tao Kuang, Shi-Ke Wang, Tian-Miao Ou, Jia-Heng Tan, Ding Li, Zhi-Shu
Huang*
A series of compounds with an introduction of aniline group into 4-position to previous reported quinazoline derivatives
were synthesized and evaluated. These compounds showed good c-myc promoter G-quadruplex binding activity and
selectivity.

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Title page
Design, synthesis and biological evaluation of 4-anilinoquinazoline derivatives as new c-myc
G-quadruplex ligands
Yin Jiang†, Ai-Chun Chen†, Guo-Tao Kuang, Shi-Ke Wang, Tian-Miao Ou, Jia-Heng Tan, Ding Li, Zhi-Shu
Huang*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, People’s Republic of China
*
To whom correspondence should be addressed. Tel. and Fax: 8620-39943056, E-mail: ceshzs@mail.sysu.edu.cn.
†
These authors contributed equally.
Abstract
A series of 4-anilinoquinazoline derivatives were designed and synthesized as novel c-myc promoter
G-quadruplex binding ligands. Subsequent biophysical and biochemical evaluation demonstrated that the
introduction of aniline group at 4-position of quinazoline ring and two side chains with terminal amino group
improved their binding affinity and stabilizing ability to G-quadruplex DNA. RT-PCR assay and Western blot
showed that compound 7a could down-regulate transcription and expression of c-myc gene in Hela cells,
which was consistent with the behavior of an effective G-quadruplex ligand targeting c-myc oncogene. More
importantly, RTCA and colony formation assays indicated that 7a obviously inhibited Hela cells proliferation,
without influence on normal primary cultured mouse mesangial cells. Flow cytometric assays suggested that
7
a induced Hela cells to arrest in G0/G1 phase both in a time-dependent and dose-dependent manner.
Keywords
-Anilinoquinazoline derivatives; G-quadruplex; c-myc; transcriptional regulation; antitumor.
4
Abbreviations
FRET, fluorescence resonance energy transfer;
Tm, melting temperature;
SPR, surface plasmon resonance;
CD, circular dichroism;
MTT, methyl thiazolyl tetrazolium;
RT-PCR, reverse transcription-polymerase chain reaction;
RTCA, real time cell analysis.
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1. Introduction
G-quadruplexes are nucleic acid secondary structures formed from guanine-rich sequences, and comprise
a planar arrangement of four guanines (G-quartet, Fig. 1A) stabilized by Hoogsteen hydrogen bonding and
monovalent cations [1]. Since G-quadruplex structures are widely located in plenty of important regulatory
regions including telomeric DNA, oncogene promoters (such as c-myc, VEGF, bcl-2, k-RAS, h-RAS, c-kit, HIF
and HSP90) and 5′-UTR, it is considered playing a significant role in regulating biological processes such as
replication, translation and splicing [2, 3]. The presence of G-quadruplex structures in human cells is now
firmly established with antibody [4], which provides the basis for the elucidation of their function in normal
and disease states.
c-MYC is a transcription factor whose expression is associated with cell proliferation. Increased levels of
c-myc expression are observed in 80% of human cancer cells, and its increase promotes tumorigenesis [5].
The nuclear hypersensitivity element III (NHE III ), a guanine-rich strand of the DNA containing a 27 base
1
1
pair sequence, which is upstream of c-myc promoter, controls 80–90% of the c-myc transcription [6]. The
NHE III can form intramolecular G-quadruplex structures and functions as a transcriptional repressor [7].
1
The transcription of c-myc can be down-regulated through stabilization of the G-quadruplexes by using
specific G-quadruplex binders [8]. A number of c-myc G-quadruplex ligands have been reported, including
phenanthroimidazole derivatives [9], acridine derivatives [10], prolinamide derivatives [11], platinum(II)
complex [12], and showed pronounced antitumor activity.
Inspecting the structures of these G-quadruplex ligands, it is easy to find that most of them are based on a
planar aromatic system that interacts with the G-quartet through π–π stacking interaction, with cationic side
chains that interact with the negatively charged phosphate backbones in G-quadruplex [13,14]. For these
molecules, optimal activity has been achieved through substitution of side chains with terminal amino group,
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especially with an amide bond conjugated with the aromatic ring system to maintain planarity on the edge of
the G-quartet [15]. Besides, intensive research has suggested that extended aromatic surface always favor
ligand-quadruplex binding interactions [16-18]. Neidle and co-workers have increased G-quadruplex binding
affinity and biological activity of 3,6-disubstituted acridines by rationally adding an anilino subsituent at the
9
-position to give 3,6,9-trisubstituted acridine molecules [19-21], one of which was BRACO-19 (Fig. 1B).
Quinazoline derivatives, which have a planar chromophore, may interact with G-quartet of G-quadruplex
DNA. In our recent study, a few 2,4-disubstituted quinazoline derivatives have been synthesized and found to
be telomeric G-quadruplex binders, one of which is compound LZ-11c (Fig. 1C) [22]. Subsequently, a new
substituted benzene ring has been linked to the ortho-position of 2-phenyl group of original quinazoline
derivatives through amide bond, like compound QPB-15e (Fig. 1C), which have shown stronger binding
ability and better selectivity for telomeric G-quadruplex [23]. In order to further develop new c-myc
G-quadruplex binding ligands, we designed a series of new 4-anilinoquinazoline derivatives, in which
aromatic system of the quinazoline is expanded by introducing an anilino group into the 4-position of
quinazoline moiety for improving its G-quadruplex binding activity and stability. Unlike rigid aromatic
compounds, such unfused aromatic molecules with adaptive structural feature could prevent themselves from
intercalating into the duplex DNAs [6]. Based on above consideration, two series of derivatives were designed,
including series I and II (Fig. 1D). For series I, we introduced an amide side chain with basic amino terminal
at the para-position of aniline group (1a, 1b, 2a, and 2b), which could be protonated at physiological pH, to
establish additional electrostatic interaction with the target. Besides, we also replaced the amide bond with
ether bond for the derivatives to uncover their influence on the G-quadruplex recognition (4a and 4b). For
Series II, a phenyl group was added at the 2-position of 4-anilinoquinazoline ring (3a~3b, 5a~5f, 6a~6d,
7
a~7i, 8a, 8b, and 9a) to further extend the planarity of unfused aromatic system. In order to investigate the
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importance of two side chains on ligand-quadruplex interaction, the second amide side chain was appended to
ortho-position of 2-phenyl group (6a~6d, 7a~7i, 8a, 8b, and 9a). Also it should be noted that the
intramolecular hydrogen bond is formed between the NH group at the ortho-position of 2-phenyl group and
the lone pair electrons of nitrogen on pyrimidine ring. Therefore, thirty one 4-anilinoquinazoline derivatives
were synthesized. The interactions of synthesized compounds with c-myc G-quadruplex DNA were examined
through fluorescence resonance energy transfer (FRET), circular dichroism (CD) spectroscopy, surface
plasmon resonance (SPR), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot. In
addition, effects of these compounds on cell proliferation and cell cycle were also evaluated.
2. Chemistry
The synthetic routes for compounds 1a~1b, 2a~2b, 3a~3c and 4a~4b were shown in Scheme 1. The
chlorination of 1H, 3H-quinazoline-2,4-dione with excess phosphorus oxychloride was carried out to give
dichlorinated intermediate 10 [24]. Aniline substitution occurred selectively at C-4 position of 10, yielding
4
-aniline-2-chloroquinazoline 11 [25]. Reaction of 11 with acylchloride gave intermediates 12a and 12b [21],
followed by treatment of 12a or 12b with excess diethylamine, potassium iodide, and potassium carbonate to
give target compounds 1a and 1b, respectively. In comparison, 12a or 12b was treated with diethylamine and
potassium carbonate to give target compound 2a or 2b as major product. Compounds 3a~3c were prepared via
Suzuki coupling reaction of 2a or 2b with appropriate phenylboronic acid [26]. Intermediate 10 was reacted
with 4-methoxyaniline to form intermediate 13, followed by demethylation reaction with BBr to give
3
intermediate 14, and subsequently by substituting with dibromoalkane to give intermediate 15a or 15b.
Compounds 4a and 4b were obtained through reaction of 15a or 15b with diethylamine, respectively.
The synthetic routes for compounds 5a~5f, 6a~6d, 7a~7i, 8a, 8b, and 9a were shown in Scheme 2. The
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synthetic method started with the synthesis of acylchloride through the chlorination of 2-methoxybenzoic acid
or 2-nitrobenzoic acid, followed with its amidation using anthranilamide to give amide intermediate 16 or 22,
and subsequently, intermediate 17 or 23 was respectively prepared through the oxidative ring closure of 16 or
2
2 under basic conditions [21]. The chlorination of 17 with excess thionyl chloride was carried out to give
intermediate 18 [24]. The chlorination of 23 with excess phosphorus oxychloride and phosphorus
pentachloride was carried out to give intermediate 24. Coupling of 4-methoxyaniline with 18 afforded
intermediate 19, followed with demethylation to give intermediate 20. Compound 20 was reacted with
1
,2-dibromoethane to afford intermediate 21 [27]. Coupling of 24 with 4-nitroaniline afforded intermediate 25.
The reduction of the nitro group with 80% hydrazine hydrate in the presence of 10% Rany-Ni gave
amino-substituted intermediate 26. Reaction of 26 with acylchloride gave intermediates 27a~27d.
Intermediates 21 or 27a~27d were treated with different alkylamines to give target compounds 5a~5f, 6a~6d,
7
a~7i, 8a, 8b, and 9a, respectively.
3. Results and Discussion
3.1 Studies on the stabilization and selectivity of derivatives on c-myc G-quadruplex DNA through
FRET
To investigate the stabilizing ability of 4-anilinoquinazoline derivatives on c-myc gene promoter
G-quadruplex DNA, FRET-melting experiment was employed with promoter G-quadruplex sequence of
c-myc containing fluorophores at both 5′-end and 3′-end (FPu22T) [28, 29]. An oligonucleotide that formed
hairpin duplex DNA structure (F10T) was used as a non-quadruplex control [30]. Quinazoline derivatives
LZ-11c and QPB-15e (Fig. 1C) previously reported by us were used as reference compounds [21, 22]. The
results of the FRET-melting experiment were shown in Table 1 and Figure S1, which suggested that these
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compounds had a wide range of G-quadruplex stabilization ability. A comparison of the FRET-melting results
indicated that some of new synthetic compounds, such as 7a, 7e~7i, showed better stabilizing ability than
reference compounds LZ-11c and QPB-15e, which indicated that the introduction of anilino group had a
significant effect on stabilizing G-quadruplex.
Among Series I compounds, it was found that amino (1a, 1b, 4a, 4b) substituent at 2-position of
quinazoline had obviously higher stabilizing ability for G-quadruplex than chlorine substituent (2a and 2b). In
addition, the ꢀT values of 1a and 1b for G-quadruplex stabilization were higher than that of 4a and 4b,
m
respectively, which revealed that amide bond at the para-position of anilino is better than ether bond to
enhance the interaction of ligand with G-quadruplex DNA. This result was consistent with that for 3a~3c over
5
a~5f for Series II compounds, indicating the importance of amide bond conjugated with aromatic ring. For
Series II compounds, most of the compounds with the second amide side chain in the ortho-position of
-phenyl group, such as 6a~6c, 7a~7c, 7e~7i, 8a, 8b, and 9a, showed stronger stabilizing ability for
2
G-quadruplex, compared to compounds 3a~3c. These results indicated that the introduction of the second
alkylamino side chain had a significant effect on the stabilizing ability of these compounds. Analysis of the
ꢀ
Tm values of compounds 6a~6d, 7a~7i, 8a, 8b, and 9a, gave the following conclusions: (1) Compounds with
polyamine side chains (7f~7i) could raise the melting temperature of G-quadruplex greatly by about
8.0-26.6 °C. Meanwhile, 7g and 7i had higher activity than 7f and 7h. Both results indicated that the number
1
and distances apart of positive charges in polyamine side chains could significantly influence their ability of
stabilizing G-quadruplex, just as that reported by Savino et al. [31, 32]. (2) With the same terminal bases,
compounds 7a~7d with longer side chains (n = 2, with two bonds between carbonyl group and basic N
terminus) showed stronger stabilizing ability to the G-quadruplex than compounds 6a~6d with shorter side
chains (n = 1), while no distinct effect on ꢀT values was observed with further extension of amide side chain,
m
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as shown for compounds 8a, 8b, and 9a (n = 3 or 4). These results demonstrated the importance of the length
of amide side chain for their strong interactions with G-quadruplex. (3) Among the compounds with the same
length of side chain and different basic terminal, the least basic compounds (morpholino analogues 6d and 7d)
had the weakest effect on stabilization of G-quadruplex, which indicated the importance of the basicity of
terminal. Besides, the flexibility of amino terminal of compounds might be another influencing factor, for
example, diethylamino analogue 7a with flexible amino terminal showed stronger stabilizing activity for
G-quadruplex over pyrrolidino analogue 7b, N-methyl piperazino analogue 7c, and piperidino analogue 7e.
In comparison, it was quite evident that most studied ligands could barely stabilize the hairpin structure
formed by F10T, suggesting their poor binding to the duplex DNA. Only 7g and 7i showed weak effect on
hairpin structure with ꢀT values of 3.6 and 3.7 °C. The selectivity of these derivatives to G-quadruplex was
m
characterized by using a competitive FRET-melting experiment, and the ability of the ligands to stabilize
G-quadruplex was challenged with nonfluorescent duplex DNA ds26 [30]. As shown in Figure 2, in the
presence of excess competitor ds26, the thermal stabilization of FPu22T enhanced by some selected
compounds was slightly affected, which demonstrated that 4-anilinoquinazoline derivatives could specifically
stabilize G-quadruplex without significant effect for duplex DNA.
3.2 Studies on the binding and selectivity of derivatives on c-myc G-quadruplex DNA by using SPR
To investigate the binding affinity and selectivity of the synthesized compounds for c-myc G-quadruplex,
SPR experiments were carried out quantitatively by using biotinylated c-myc G-quadruplex DNA Pu22 and
biotinylated duplex DNA attached to a streptavidin-coated sensor chip [33]. The binding constants were
determined through equilibrium analysis. As shown in Table 2, the K values for most of the derivatives
D
binding to c-myc G-quadruplex showed strong binding affinity, ranged from 0.14 to 8.08 µM except 2a, 3c,
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and 5a~5f. Most of the compounds with two alkylamino side chains (7a~7c, 7e, 7f, 7h, 7i, 8a, 8b, and 9a)
exhibited strong binding affinity for c-myc G-quadruplex among 4-anilinoquinazoline derivatives, with their
K values lower than 1 ꢁM, while weaker or no obvious binding was found for these compounds to duplex
D
DNA. These results demonstrated that the introduction of two alkylamino side chains onto the scaffold of
4
-anilinoquinazoline derivatives had improved binding ability and selectivity for G-quadruplex. In addition, it
was found that amide bond at the para-position of anilino is better than ether bond to improve the interaction
of ligand with G-quadruplex structure, which was in parallel with FRET-melting data. Moreover, SPR
experimental data also indicated that the amide side chains of the tested compounds with two bonds between
carbonyl group and basic N terminus were optimal for their interactions with G-quadruplex. A comparison of
Pu22
duplex
the SPR assay results for the reference compound LZ-11c (KD
= 0.49 µM, KD
= 9.18 µM) with some
Pu22
duplex
of 4-anilinoquinazoline derivatives, such as 7a, KD
= 0.21 µM, KD
> 10 µM, revealed that the
introduction of substituted anilino at the 4-position of quinazoline core had beneficial effect on their binding
ability and selectivity for c-myc G-quadruplex. The results from above FRET and SPR experiments all
supported that these 4-anilinoquinazoline derivatives could selectively bind and stabilize G-quadruplexs DNA
over duplex DNA, which could act as a new class of highly selective G-quadruplex binding ligands.
3.3 Studies on the binding property of derivatives on G-quadruplex by using CD
CD spectroscopy is an extremely sensitive method for determining the conformation of G-quadruplex
structures and the interaction between ligands and G-quadruplex [33]. Oligonucleotide Pu22
5'-TGAGGGTGGGTAGGGTGGGTAA-3') was used as the c-myc sequence. The binding property of some
-anilinoquinazoline derivatives (6b, 7a, 7b, 7c, 7e, 7f, and 7h) to c-myc G-quadruplex was further studied
(
4
with this method. As show in Figure 3A, in the presence of 100 mM KCl, Pu22 showed a positive signal at
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about 262 nm and a negative signal at 242 nm, which suggested a typical parallel G-quadruplex structure.
Upon addition of compounds to Pu22, no significant change was observed while the peak height was slightly
decreased, which indicated that 4-anilinoquinazoline derivatives could still maintain parallel c-myc
G-quadruplex conformation in the presence of potassium ion.
The CD spectra of Pu22 without any metal cations at room temperature exhibited a negative peak at 240
nm and a positive peak at 260 nm. After the treatment with 4-anilinoquinazoline derivatives, the positive peak
at 260 nm and the negative peak at 240 nm were greatly increased (Fig. 3B), which was similar with the CD
spectrum of Pu22 in the presence of KCl. The result indicated that 4-anilinoquinazoline derivatives could
effectively induce the formation of parallel c-myc G-quadruplex conformation in the absence of metal cations
and further confirmed a strong interaction between G-quadruplex DAN and our derivatives.
3.4 Studies of the derivatives on antitumor activity by using MTT
To investigation the antitumor activity of the derivatives, MTT assay was employed to evaluate the
cytotoxicity of 4-anilinoquinazoline derivatives against cervical cancer cell line Hela, lung adenocarcinoma
cell line A549, lymphoma cell lines Raji and CA46, as well as primary cultured mouse mesangial cell line (in
which the proliferation does not depend on c-myc expression [34]). In the assay, cell viability was determined
through reduction of tetrazolium salt to blue formazan based on mitochondrial enzyme activity of succinate
dehydrogenase in living cells. As shown in Table 3, although compounds 6b, 7a, 7b, 7c, 7e, 7f, 7h exhibited
strong stabilizing ability and affinity to c-myc G-quadruplex, different cytotoxicity occurred among these
compounds. Subsequently, we performed cell uptake assay for the compounds, and the result (Fig. S2)
showed that cellular uptake of compounds 6b, 7a, and 7b were much higher than that of compounds 7f and 7h,
which might explain the results that compounds had obviously different cytotoxicity. Based on the above
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results, we found that 7a stabilized c-myc G-quadruplex with high ꢀT value, bound the G-quadruplex with a
m
submicromolar binding constant, and exhibited stronger inhibitory effects on the tumor cells while weaker
inhibitory effect on primary cultured mouse mesangial cells. Therefore, compound 7a may be a good
candidate for further cellular studies.
3.5 Down-regulation of c-myc transcription and expression by 7a in Hela cells
RT-PCR was performed to determine the effect of compound 7a on the transcription of c-myc gene. Hela
cells were incubated with compound 7a at 0, 1.25, 2.5, 5, 7.5, 10 ꢁM for 3 h. The total RNA was extracted
and reversely transcribed to cDNA. The cDNA was then used as a template for quantitative PCR amplification
of the c-myc sequence. As shown in Figure 4A, derivative 7a showed inhibitory activity on the transcription of
c-myc in a concentration-dependent manner in Hela cells. Next, Western blot was performed to test the effect
of compound 7a on expression of c-myc. As shown in Figure 4B, expression level of c-myc also decreased
upon treatment with compound 7a. The above results suggested that 7a might target the G-quadruplex
structure in the promoter region of c-myc gene and hence down-regulated its expression.
3.6 Inhibition of cell proliferation by derivative 7a
Since stabilization of c-myc G-quadruplex DNA could influence c-myc transcription and inhibit cancer
cell growth, to further evaluate such inhibitory effect of compound 7a, RTCA assays [35] and colony
formation assays were carried out. Hela cells (with overexpression of c-myc) and primary cultured mouse
mesangial cells (the proliferation does not depend on c-myc expression) were treated with various
concentrations of compound 7a (0, 1.25, 2.5, 5, 7.5, 10 ꢁM) in RTCA for 80 h, and the cell proliferation
results were shown in Figure 5. The compound 7a showed significant growth arrest on Hela cells, while no
1
0

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such effect was found on primary cultured mouse mesangial cells.
Colony formation assays showed a more visualized result about cell proliferation inhibition of compound
7
a. Hela cells and primary cultured mouse mesangial cells were treated with various concentrations of 7a (2, 1,
0
.5, 0.25 ꢁM and DMSO control) for 8 days. As shown in Figure 6, 2 ꢁM of 7a almost completely inhibited
Hela cell proliferation, without effect on primary cultured mouse mesangial cells.
3.7 G0/G1 phase arrest of Hela cells by derivative 7a
As shown in RTCA and colony formation assays, compound 7a could obviously inhibit Hela cell
proliferation. To explore the mechanism of this inhibition, we performed cell cycle analysis to detect Hela
cells treated with 7a at various concentrations and on different time intervals. Compound 7a treatment gave an
increase in G0/G1 phase from 30.8% to 61.1% in a time-dependent manner (Figure 7A), and an increase from
3
5.9% to 54.8% in a dose-dependent manner (Fig. 7B) for 48 h measured by using EPICS XL flow cytometer.
These results indicated that 7a could arrest Hela cells in G0/G1 phases.
4. Conclusions
Some unfused aromatic ligands have been known as effective and selective G-quadruplex binding
ligands. Based on previous reports for the use of anilino on G-quadruplex ligand design, in the present study,
an aniline group was attached to the scaffold of our previously reported quinazoline derivatives to give two
series of novel unfused aromatic quinazoline derivatives, including 4-anilinoquinazoline derivatives (Series I)
and 4-anilino-2-phenylquinazoline derivatives (Series II). Positively charged side chain has been shown to
play an important role in recognizing G-quadruplex, therefore, various positively charged side chains were
1
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incoporated into the molecules. Our FRET and SPR experiments suggested that two positively charged amide
side chains showed strong affinity and selectivity to c-myc G-quadruplex against the duplex DNA. The
introduced two positively charged side chains could participate in electrostatic and H-bonding interactions
with the grooves and loops of the G-quadruplex DNA. Further cellular studies showed that compound 7a
significantly down-regulated c-myc gene transcription and expression in Hela cells, presumably through the
stabilization of c-myc G-quadruplex structure. In addition, RTCA and colony formation assays indicated that
7
a significantly inhibited Hela cell proliferation, without influence on normal primary cultured mouse
mesangial cells. From flow cytometric assays, we found that 7a arrested Hela cell cycle in G0/G1 phase in
both a time-dependent and a dose-dependent manner. These results showed that the introduction of an anilino
group to 2-position of quinazoline core is an efficient approach for obtaining promising c-myc promoter
G-quadruplex binding ligands. Further studies are needed to investigate the properties of compounds 7a, e.g.
basic experimental ADME test and the inhibitory effect to tumor xenografts growth in vivo, and based on
these data, more powerful analogs could be designed.
5. Experimental Section
5
.1. Synthesis and characterization
All commercial chemicals used as starting materials were analytical grade and utilized without further
1
13
purification. H and C NMR spectra were recorded using TMS as the internal standard in DMSO-d or
6
CDCl with a Bruker BioSpin GmbH spectrometer at 400 MHz and 100 MHz, respectively; Mass spectra (MS)
3
were recorded on a Shimadzu LCMS-2010A instrument with an ESI or ACPI mass selective detector, and
high resolution mass spectra (HRMS) were recorded on Shimadzu LCMS-IT-TOF. Melting points (m.p.) were
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determined by using capillary tubes with a MSRS-OptiMelt automated melting point instrument without
correction. The purities of synthesized compounds were confirmed to be higher than 95% by using analytical
HPLC equipped with a dual pump Shimadzu LC-20AB system with an Ultimate XB-C18 column.
5
.1.1 Synthesis of intermediates
The intermediates 10 ~ 27 were prepared following the process shown in Scheme 1 and 2, and the detail of
synthesis was described in Supporting Information.
5
.1.2. General procedure A: preparation of 1a and 1b
To a stirred suspension of 12a or 12b (1 mmol), K CO (2 mmol), KI (1 mmol) in DMF (10 mL) was
2
3
added diethylamine (20 mmol). The solution was stirred under 130 °C for 12 h, cooled to room temperature,
and diluted with 25 mL water. The reaction mixture was extracted with ethyl acetate (50 mL×2), the combined
the organic layers washed six times with water and dried over Na SO , and then evaporated under vacuum.
2
4
The crude solid was purified by using chromatography with CH Cl /MeOH/NH ·H O elution to afford 1a and
2
2
3
2
1
b.
5
.1.2.1. 2-(diethylamino)-N-(4-((2-(diethylamino)quinazolin-4-yl)amino)phenyl)acetamide (1a). Compound
2a was treated with excess diethylamine according to general procedure A to afford 1a. After column
1
chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale
2
2
3
2
1
yellow solid in 71% yield. m.p. 131.1-133.1 °C; H NMR (400 MHz, CDCl ): δ 9.41 (s, 1H), 7.74 (d, J = 8.9
3
Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.59 (d, J = 8.9 Hz, 2H), 7.56 – 7.47 (m, 2H), 7.34 (s, 1H), 7.08 (t, J = 8.0
Hz, 1H), 3.71 (q, J = 7.0 Hz, 4H), 3.16 (s, 2H), 2.66 (q, J = 7.1 Hz, 4H), 1.23 (t, J = 7.0 Hz, 6H), 1.11 (t, J =
1
3

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13
7
.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ 170.1, 158.1, 157.3, 153.2, 135.5, 133.2, 132.6, 126.1, 121.4,
3
-
1
20.6, 120.5, 119.8, 110.1, 58.1, 48.9, 41.7, 29.7, 13.6, 12.5. HRMS (ESI): Calcd for [M-H] (C H N O)
2
4
32
6
requires m/z 419.2565, found 419.2550.
5
.1.2.2. 3-(diethylamino)-N-(4-((2-(diethylamino)quinazolin-4-yl)amino)phenyl)propanamide (1b). Compound
2b was treated with excess diethylamine according to general procedure A to afford 1b. After column
1
chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale
2
2
3
2
1
yellow solid in 62% yield. m.p. 135.3-137.4°C; H NMR (400 MHz, CDCl ): δ 11.27 (s, 1H), 7.69 (d, J = 8.9
3
Hz, 2H), 7.65 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 9.0 Hz, 3H), 7.51 – 7.49 (m, 1H), 7.33 (s, 1H), 7.09 – 7.05 (m,
1
7
1
H), 3.70 (q, J = 7.0 Hz, 4H), 2.81 – 2.77 (m, 2H), 2.69 (q, J = 7.1 Hz, 4H), 2.55 – 2.49 (m, 2H), 1.22 (t, J =
1
3
.0 Hz, 6H), 1.14 (t, J = 7.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ 171.0, 158.2, 157.4, 153.1, 135.0, 134.4,
3
32.5, 126.0, 121.6, 120.7, 120.5, 119.9, 110.2, 48.9, 46.0, 41.7, 33.1, 13.5, 11.5. HRMS (ESI): Calcd for
-
[
M-H] (C H N O) requires m/z 433.2721, found 433.2704.
25
34
6
5
.1.3. General procedure B: preparation of 2a and 2b
The mixture of 12a or 12b (2 mmol), and K CO (4 mmol) in 10 mL DMF was added diethylamine (10
2
3
mmol). The solution was stirred at 80 °C for 12 h, cooled to room temperature, and diluted with 25 mL water.
The resulting solution was extracted with CH Cl (50 mL×2), the combined organic phase washed six times
2
2
with water and dried over Na SO , and then evaporated under vacuum. The crude solid was purified by using
2
4
chromatography with CH Cl /MeOH/NH ·H O elution to afford 2a and 2b.
2
2
3
2
5
.1.3.1. N-(4-((2-chloroquinazolin-4-yl)amino)phenyl)-2-(diethylamino)acetamide (2a). Compound 12a was
1
4

ACCEPTED MANUSCRIPT
treated with excess diethylamine according to general procedure B to afford 2a. After column chromatography
with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale yellow solid in
2
2
3
2
1
5
8% yield. m.p. 213.0-215.4°C; H NMR (400 MHz, CDCl ): δ 9.56 (s, 1H), 8.04 (d, J = 7.9 Hz, 2H), 7.85 –
3
7
.77 (m, 2H), 7.72 (d, J = 8.9 Hz, 2H), 7.60 (d, J = 8.9 Hz, 2H), 7.56 – 7.52 (m, 1H), 3.22 (s, 2H), 2.70 (d, J =
1
3
6
.8 Hz, 4H), 1.13 (t, J = 7.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ 158.8, 157.3, 151.4, 134.4, 133.8, 133.7,
3
+
1
28.0, 126.5, 122.9, 121.3, 120.4, 113.6, 58.0, 48.9, 12.4. HRMS (ESI): Calcd for [M+H] (C H N O)
2
6
35
7
requires m/z 384.1586, found 384.1588.
5
.1.3.2. N-(4-((2-chloroquinazolin-4-yl)amino)phenyl)-3-(diethylamino)propanamide (2b). Compound 12b
was treated with excess diethylamine according to general procedure B to afford 2b. After column
chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale
2
2
3
2
1
yellow solid in 40% yield. m.p. 171.0-172.3°C; H NMR (400 MHz, DMSO-d ): δ 10.21 (s, 1H), 10.17 (s,
6
1
2
H), 8.55 (d, J = 8.1 Hz, 1H), 7.91 – 7.84 (m, 1H), 7.73 – 7.67 (m, 1H), 7.65 (d, J = 5.9 Hz, 4H), 7.62 (s, 1H),
1
3
.75 (t, J = 6.9 Hz, 2H), 2.55 – 2.50 (m, 4H), 2.43 (t, J = 7.0 Hz, 2H), 0.99 (t, J = 7.1 Hz, 6H); C NMR (101
MHz, DMSO-d ): δ 170.4, 159.4, 156.4, 150.8, 136.2, 133.9, 133.0, 126.8, 126.5, 123.6, 123.4, 119.1, 113.7,
6
+
4
3
8.4, 46.1, 34.1, 11.8. HRMS (ESI): Calcd for [M+H] (C H N OCl) requires m/z 398.1742, found
2
1
24
5
98.1741.
5
.1.4. General procedure C: preparation of 3a ~ 3c
A mixture of 2a or 2b (0.3 mmol), appropriate phenylboronic acid (0.45 mmol), Na CO (0.9 mmol), and
2
3
Pd(PPh ) (0.03 mmol) in 10 mL DMF and 2 mL H O was purged with nitrogen, then was heated at 100 °C
3
4
2
for 12 h under nitrogen protection. The solution was cooled to room temperature, and diluted with 25 mL
1
5

ACCEPTED MANUSCRIPT
water. The resulting solution was extracted with CH Cl (50 mL×2), the combined organic phase washed three
2
2
times with water and dried over Na SO , and then evaporated under vacuum. The crude solid was purified by
2
4
using chromatography with CH Cl /MeOH/NH ·H O elution to afford 3a ~ 3c.
2
2
3
2
5
.1.4.1.
2-(diethylamino)-N-(4-((2-(4-hydroxyphenyl)quinazolin-4-yl)amino)phenyl)acetamide
(3a).
Compound 2a was treated with 4-hydroxyphenylboronic acid according to general procedure C to afford 3a.
After column chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was
2
2
3
2
1
obtained as a pale yellow solid in 45% yield. m.p. 205.8-207.4 °C; H NMR (400 MHz, DMSO-d ): δ 9.88 (s,
6
1
7
2
1
1
H), 9.76 (s, 1H), 9.70 (s, 1H), 8.52 (d, J = 8.3 Hz, 1H), 8.28 (d, J = 8.6 Hz, 2H), 7.90 (d, J = 8.9 Hz, 2H),
.84 – 7.76 (m, 2H), 7.73 (d, J = 8.9 Hz, 2H), 7.54 (t, J = 7.3 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 3.19 (s, 2H),
1
3
.64 (d, J = 6.2 Hz, 4H), 1.06 (t, J = 7.1 Hz, 6H); C NMR (101 MHz, DMSO-d ): δ 170.1, 160.1, 159.7,
6
58.0, 151.1, 135.4, 134.6, 133.4, 130.1, 129.8, 128.3, 125.6, 123.4, 122.9, 119.9, 115.6, 114.1, 57.9, 48.4,
+
2.5. HRMS (ESI): Calcd for [M+H] (C H N O ) requires m/z 442.2238, found 442.2221.
26
27
5
2
5
.1.4.2.
N-(4-((2-(2-aminophenyl)quinazolin-4-yl)amino)phenyl)-3-(diethylamino)propanamide
(3b).
Compound 2b was treated with 2-aminophenylboronic acid according to general procedure C to afford 3b.
After column chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was
2
2
3
2
1
obtained as a pale yellow solid in 40% yield. m.p. 120.9-123.4 °C; H NMR (400 MHz, CDCl ): δ 11.30 (s,
3
1
7
2
H), 8.50 (d, J = 7.9 Hz, 1H), 7.94 – 7.86 (m, 2H), 7.77 (m, 3H), 7.64 (d, J = 8.3 Hz, 2H), 7.54 – 7.47 (m, 2H),
.22 (t, J = 7.5 Hz, 1H), 6.79 (t, J = 7.5 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.54 (s, 2H), 2.85 (d, J = 5.3 Hz,
1
3
H), 2.75 (dd, J = 13.8, 6.8 Hz, 4H), 2.59 (t, J = 5.3 Hz, 2H), 1.20 (t, J = 6.9 Hz, 6H); C NMR (101 MHz,
DMSO-d ): δ 170.8, 161.8, 157.5, 150.5, 150.0, 136.0, 134.6, 133.6, 131.4, 131.0, 127.9, 125.9, 123.7, 123.4,
6
1
6

ACCEPTED MANUSCRIPT
-
1
19.5, 118.2, 116.9, 115.1, 113.6, 48.9, 46.6, 34.6, 12.3. HRMS (ESI): Calcd for [M-H] (C H N O) requires
2
7
30
6
m/z 453.2408, found 453.2391.
5
.1.4.3. 3-(diethylamino)-N-(4-((2-phenylquinazolin-4-yl)amino)phenyl)propanamide (3c). Compound 2b was
treated with phenylboronic acid according to general procedure C to afford 3c. After column chromatography
with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale yellow solid in
2
2
3
2
1
5
0% yield. m.p. 152.5-154.7 °C; H NMR (400 MHz, CDCl ): δ 11.28 (s, 1H), 8.53 (d, J = 7.0 Hz, 2H), 7.98
3
(
d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.78 (t, J = 7.7 Hz, 1H), 7.63 (d, J = 8.1
Hz, 2H), 7.57 (s, 1H), 7.53 – 7.43 (m, 4H), 2.86 – 2.79 (m, 2H), 2.72 (q, J = 7.0 Hz, 4H), 2.60 – 2.51 (m, 2H),
1
3
1
1
.17 (t, J = 7.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ 171.1, 160.4, 157.5, 151.0, 138.8, 134.8, 134.5,
3
32.8, 130.2, 129.1, 128.5, 128.4, 125.9, 122.2, 120.8, 120.2, 114.0, 77.4, 77.1, 76.8, 48.9, 46.0, 33.1, 11.6.
+
HRMS (ESI): Calcd for [M+H] (C H N O) requires m/z 440.2445, found 440.2428.
2
7
29
5
5
.1.5. General procedure D: preparation of 4a and 4b
To a stirred suspension of 15a or 15b (0.55 mmol) and K CO (1.1 mmol) in DMF (10 mL) was added
2
3
dropwise diethylamine (11 mmol). The mixture was stirred under 110 °C for 12 h, cooled to room temperature,
and diluted with 25 mL water. The resulting solution was extracted with CH Cl (50 mL×2), the combined
2
2
organic phase washed six times with water and dried over Na SO , and then evaporated under vacuum. The
2
4
crude solid was purified by using chromatography with CH Cl /MeOH/NH ·H O elution to afford 4a and 4b.
2
2
3
2
4
2
2
5
.1.5.1. N -(4-(2-(diethylamino)ethoxy)phenyl)-N ,N -diethylquinazoline-2,4-diamine (4a). Compound 15a
was treated with excess diethylamine according to general procedure D to afford 4a. After column
17

ACCEPTED MANUSCRIPT
chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale
2
2
3
2
1
yellow solid in 73% yield. m.p. 149.5-152.5 °C; H NMR (400 MHz, CDCl ): δ 7.64 – 7.57 (m, 3H), 7.52 (m,
3
2
H), 7.13 (s, 1H), 7.07 (dd, J = 11.2, 5.0 Hz, 1H), 6.92 (d, J = 9.0 Hz, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.69 (q, J
7.0 Hz, 4H), 2.91 (t, J = 6.2 Hz, 2H), 2.68 (q, J = 7.1 Hz, 4H), 1.20 (t, J = 7.0 Hz, 6H), 1.10 (t, J = 7.1 Hz,
=
13
6
H); C NMR (101 MHz, CDCl ): δ 158.2, 157.6, 155.2, 153.1, 132.5, 132.2, 126.1, 122.9, 120.5, 120.4,
3
-
1
14.6, 110.0, 77.4, 77.0, 76.7, 66.9, 51.8, 47.9, 41.6, 13.5, 11.8. HRMS (ESI): Calcd for [M-H] (C H N O)
2
4
33
5
requires m/z 406.2612, found 406.2597.
4
2
2
5
.1.5.2. N -(4-(3-(diethylamino)propoxy)phenyl)-N ,N -diethylquinazoline-2,4-diamine (4b). Compound 15b
was treated with excess diethylamine according to general procedure D to afford 4b. After column
chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale
2
2
3
2
1
yellow solid in 70% yield. m.p. 133.0-134.9 °C; H NMR (400 MHz, CDCl ): δ 7.64 – 7.57 (m, 3H), 7.55 –
3
7
.50 (m, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.10 (s, 1H), 7.09 – 7.04 (m, 1H), 6.91 (d, J = 8.9 Hz, 2H), 4.03 (t, J =
.3 Hz, 2H), 3.68 (q, J = 7.0 Hz, 4H), 2.67 – 2.61 (m, 2H), 2.57 (q, J = 7.1 Hz, 4H), 2.00 – 1.90 (m, 2H), 1.20
6
1
3
(
t, J = 7.0 Hz, 6H), 1.05 (t, J = 7.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ 158.3, 157.5, 155.4, 153.2, 132.5,
3
1
32.1, 126.2, 122.8, 120.4, 120.3, 114.5, 110.0, 66.7, 49.4, 47.0, 41.6, 27.1, 13.6, 11.8. HRMS (ESI): Calcd
-
for [M-H] (C H N O) requires m/z 420.2769, found 420.2752.
2
5
35
5
5
.1.6. General procedure E: preparation of 5a ~ 5f
To a stirred suspension of 21 (0.3 mmol) and K CO (0.6 mmol) in DMF (10 mL) was added dropwise
2
3
appropriate amine (3 mmol). The mixture was stirred under 100 °C for 12 h, cooled to room temperature, and
diluted with 25 mL water. The resulting solution was extracted with CH Cl (50 mL×2), the combined organic
2
2
1
8

ACCEPTED MANUSCRIPT
phase washed six times with water and dried over Na SO , and then evaporated under vacuum. The crude
2
4
solid was purified by using chromatography with CH Cl /MeOH/NH ·H O elution to afford 5a ~ 5f.
2
2
3
2
5
.1.6.1. 2-(4-((4-(2-(diethylamino)ethoxy)phenyl)amino)quinazolin-2-yl)phenol (5a). Compound 21 was
treated with excess diethylamine according to general procedure E to afford 5a. After column chromatography
with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale yellow solid in
2
2
3
2
1
4
7
7
2
1
0% yield. m.p. 139.7-141.1 °C; H NMR (400 MHz, CDCl ): δ 14.53 (s, 1H), 8.39 (dd, J = 7.9, 1.5 Hz, 1H),
3
.87 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.80 – 7.75 (m, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.54 (s, 1H),
.52 – 7.47 (m, 1H), 7.38 – 7.31 (m, 1H), 7.02 – 6.97 (m, 3H), 6.94 – 6.88 (m, 1H), 4.13 (t, J = 6.2 Hz, 2H),
1
3
.94 (t, J = 6.1 Hz, 2H), 2.71 (q, J = 7.1 Hz, 4H), 1.12 (t, J = 7.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ
3
61.5, 161.3, 157.2, 156.3, 148.0, 133.4, 132.6, 130.7, 129.5, 127.5, 126.1, 124.2, 120.6, 119.6, 118.6, 117.6,
-
1
14.9, 113.3, 66.8, 51.8, 47.9, 11.8. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 427.2139,
2
6
28
4
2
found 427.2127.
5
.1.6.2. 2-(4-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)amino)quinazolin-2-yl)phenol (5b). Compound 21 was
treated with excess pyrrolidine according to general procedure E to afford 5b. After column chromatography
with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale yellow solid in
2
2
3
2
1
6
7
7
0% yield. m.p. 183.4-186.6 °C; H NMR (400 MHz, CDCl ): δ 14.52 (s, 1H), 8.39 (dd, J = 7.9, 1.6 Hz, 1H),
3
.89 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 7.81 – 7.75 (m, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.56 (s, 1H),
.51–7.49 (m, 1H), 7.35–7.32 (m, 1H), 7.06 – 6.98 (m, 3H), 6.94 – 6.88 (m, 1H), 4.20 (t, J = 5.8 Hz, 2H), 2.99
1
3
(
t, J = 5.8 Hz, 2H), 2.73 (s, 4H), 1.88–1.84 (m, 4H); C NMR (101 MHz, CDCl ): δ 161.5, 161.3, 157.2,
3
1
56.3, 148.0, 133.4, 132.6, 130.8, 129.5, 127.5, 126.1, 124.2, 120.6, 119.6, 118.6, 117.6, 115.0, 113.3, 67.4,
1
9

ACCEPTED MANUSCRIPT
-
5
5.2, 54.8, 23.5. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 425.1983, found 425.1969.
2
6
26
4
2
5
.1.6.3. 2-(4-((4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)amino)quinazolin-2-yl)phenol (5c). Compound 21
was treated with excess N-methyl piperazine according to general procedure E to afford 5c. After column
chromatography with CH Cl /MeOH/NH ·H O (25:1:0.1) elution, the desired product was obtained as a pale
2
2
3
2
1
yellow solid in 64% yield. m.p. 142.3-143.3 °C; H NMR (400 MHz, CDCl ): δ 14.53 (s, 1H), 8.39 (dd, J =
3
7
2
4
.9, 1.5 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.79– 7.75 (m, 1H), 7.65 (d, J = 8.9 Hz,
H), 7.57 (s, 1H), 7.53 – 7.47 (m, 1H), 7.38 – 7.32 (m, 1H), 7.00 (d, J = 8.9 Hz, 3H), 6.94 – 6.88 (m, 1H),
1
3
.16 (t, J = 5.8 Hz, 2H), 2.86 (t, J = 5.8 Hz, 2H), 2.69 (s, 4H), 2.55 (s, 4H), 2.34 (s, 3H); C NMR (101 MHz,
CDCl ): δ 161.4, 161.2, 157.2, 156.1, 147.9, 133.3, 132.5, 131.0, 129.5, 127.3, 126.1, 124.2, 120.8, 119.6,
3
+
1
18.6, 117.6, 114.9, 113.4, 66.2, 57.2, 55.0, 53.5, 46.0. HRMS (ESI): Calcd for [M+H] (C H N O ) requires
2
7
29
5
2
m/z 456.2394, found 456.2398.
5
.1.6.4. 2-(4-((4-(2-morpholinoethoxy)phenyl)amino)quinazolin-2-yl)phenol (5d). Compound 21 was treated
with excess morpholine according to general procedure E to afford 5d. After column chromatography with
CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale yellow solid in 70%
2
2
3
2
1
yield. m.p. 162.5-164.1 °C; H NMR (400 MHz, CDCl ): δ 14.55 (s, 1H), 8.39 (d, J = 6.9 Hz, 1H), 7.85 (d, J
3
=
8.3 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.77 (t, J = 7.5 Hz, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.54 (s, 1H), 7.49 (t,
J = 7.4 Hz, 1H), 7.38 – 7.32 (m, 1H), 7.00 (d, J = 8.8 Hz, 3H), 6.91 (t, J = 7.5 Hz, 1H), 4.17 (t, J = 5.6 Hz,
1
3
2
1
H), 3.82 – 3.73 (m, 4H), 2.85 (t, J = 5.6 Hz, 2H), 2.63 (d, J = 4.2 Hz, 4H); C NMR (101 MHz, CDCl ): δ
3
61.4, 161.2, 157.1, 156.0, 147.9, 133.4, 132.6, 130.9, 129.5, 127.4, 126.1, 124.1, 120.6, 119.5, 118.6, 117.6,
+
1
14.9, 113.3, 66.9, 66.0, 57.7, 54.1. HRMS (ESI): Calcd for [M+H] (C H N O ) requires m/z 443.2078,
2
6
26
4
3
2
0

ACCEPTED MANUSCRIPT
found 443.2080.
5
.1.6.5. 2-(4-((4-(2-(piperidin-1-yl)ethoxy)phenyl)amino)quinazolin-2-yl)phenol (5e). Compound 21 was
treated with excess piperidine according to general procedure E to afford 5e. After column chromatography
with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as a pale yellow solid in
2
2
3
2
1
5
5% yield. m.p. 131.2-134.0 °C; H NMR (400 MHz, CDCl ): δ 14.56 (s, 1H), 8.40 (d, J = 7.9 Hz, 1H), 7.89
3
(
d, J = 8.1 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.57 (s, 1H),
7
5
.51 (t, J = 7.5 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.01 (d, J = 8.2 Hz, 3H), 6.92 (t, J = 7.5 Hz, 1H), 4.20 (t, J =
1
3
.7 Hz, 2H), 2.87 (t, J = 6.1 Hz, 2H), 2.61 (s, 4H), 1.72 – 1.62 (m, 4H), 1.49 (s, 2H); C NMR (101 MHz,
CDCl ): δ 161.3, 161.2, 157.2, 156.0, 147.9, 133.3, 132.5, 130.8, 129.5, 127.3, 126.1, 124.2, 120.8, 119.6,
3
+
1
18.6, 117.5, 114.8, 113.4, 66.0, 57.9, 55.1, 25.7, 24.0. HRMS (ESI): Calcd for [M+H] (C H N O ) requires
2
7
28
4
2
m/z 441.2285, found 441.2275.
5
.1.6.6.
Compound 21 was treated with excess N,N-diethylethylenediamine according to general procedure E to afford
f. After column chromatography with CH Cl /MeOH/NH ·H O (25:1:0.1) elution, the desired product was
2-(4-((4-(2-((2-(diethylamino)ethyl)amino)ethoxy)phenyl)amino)quinazolin-2-yl)phenol
(5f).
5
2
2
3
2
1
obtained as a pale yellow solid in 40% yield. m.p. 122.9-124.8 °C; H NMR (400 MHz, CDCl ): δ 14.60 (s,
3
1
H), 8.39 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.85 – 7.73 (m, 3H), 7.64 (d, J = 8.4 Hz, 2H), 7.49 (t, J
7.4 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 9.0 Hz, 3H), 6.91 (t, J = 7.5 Hz, 1H), 4.12 (t, J = 4.8 Hz,
H), 3.06 (t, J = 4.9 Hz, 2H), 2.81 (t, J = 6.1 Hz, 2H), 2.64 (t, J = 6.1 Hz, 2H), 2.57 (m, 4H), 2.42 (s, 1H), 1.05
=
2
1
3
(
t, J = 7.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ 161.4, 161.2, 157.3, 156.2, 148.0, 133.3, 132.5, 130.9,
3
1
29.5, 127.3, 126.1, 124.3, 121.0, 119.6, 118.6, 117.6, 114.7, 113.5, 67.5, 52.6, 49.0, 47.5, 47.0, 11.7. HRMS
2
1

ACCEPTED MANUSCRIPT
+
(
ESI): Calcd for [M+H] (C H N O ) requires m/z 472.2707, found 472.2690.
28 33 5 2
5
.1.7. General procedure F: preparation of 6a~6d, 7a~7i
To a stirred suspension of the chloride compound 27a or 27b (1 mmol) and K CO (2 mmol) in DMF (10
2
3
mL) was added dropwise appropriate amine (20 mmol). The mixture was stirred under 80-100 °C for 12 h,
cooled to room temperature, and diluted with 25 mL water. The resulting solution was extracted with CH Cl2
2
(
50 mL×2), the combined organic phase washed six times with water and dried over Na SO , and then
2 4
evaporated under vacuum. The crude solid was purified by using chromatography with
CH Cl /MeOH/NH ·H O elution to afford 6a ~ 6d, 7a ~ 7i.
2
2
3
2
5
.1.7.1.
2-(diethylamino)-N-(2-(4-((4-(2-(diethylamino)acetamido)phenyl)amino)quinazolin-2-yl)phenyl)-
acetamide (6a). Compound 27a was treated with excess diethylamine according to general procedure F to
afford 6a. After column chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product
2
2
3
2
1
was obtained as a pale yellow solid in 60% yield. m.p. 202.7-204.4 °C; H NMR (400 MHz, CDCl ): δ 13.29
3
(
s, 1H), 9.40 (s, 1H), 8.71 (d, J = 8.2 Hz, 1H), 8.37 (dd, J = 7.9, 1.3 Hz, 1H), 8.13 (s, 1H), 8.03 (d, J = 8.2 Hz,
H), 7.90 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 8.8 Hz, 2H), 7.68 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.40
t, J = 7.1 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 3.18 (s, 2H), 3.11 (s, 2H), 2.58 (m, 8H),
1
(
1
3
1
1
5
5
.03 (t, J = 7.0 Hz, 6H), 0.88 (t, J = 7.0 Hz, 6H); C NMR (101 MHz, CDCl ): δ 172.0, 170.4, 160.7, 157.5,
3
49.6, 138.8, 135.2, 133.7, 132.6, 131.0, 130.6, 128.3, 126.2, 126.0, 123.1, 122.6, 121.6, 121.4, 120.1, 113.7,
-
8.7, 58.1, 49.1, 48.9, 12.5, 11.7. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 552.3092, found
3
2
39
7
2
52.3079.
2
2

ACCEPTED MANUSCRIPT
5
.1.7.2. 2-(pyrrolidin-1-yl)-N-(2-(4-((4-(2-(pyrrolidin-1-yl)acetamido)phenyl)amino)quinazolin-2-yl)phenyl)-
acetamide (6b). Compound 27a was treated with excess pyrrolidine according to general procedure F to afford
b. After column chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was
6
2
2
3
2
1
obtained as a pale yellow solid in 70% yield. m.p. 248.4-251.1 °C; H NMR (400 MHz, CDCl ): δ 13.43 (s,
3
1
H), 9.19 (s, 1H), 8.71 (d, J = 8.0 Hz, 1H), 8.42 (dd, J = 8.0, 1.5 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.90 (d, J
8.2 Hz, 1H), 7.73 (dd, J = 12.3, 5.3 Hz, 3H), 7.65 (s, 1H), 7.61 (d, J = 8.9 Hz, 2H), 7.48 (t, J = 7.6 Hz, 1H),
.40 – 7.35 (m, 1H), 7.11 (t, J = 7.1 Hz, 1H), 3.32 (s, 2H), 3.27 (s, 2H), 2.73 – 2.59 (m, 8H), 1.83 (s, 4H), 1.66
=
7
1
3
(
s, 4H); C NMR (101 MHz, DMSO-d ): δ 169.6, 168.6, 159.9, 157.6, 148.8, 139.0, 134.9, 134.2, 133.0,
6
1
30.6, 130.4, 127.4, 126.4, 124.6, 122.9, 122.6, 120.3, 119.5, 113.3, 61.7, 59.5, 54.0, 53.7, 23.5, 23.4. HRMS
-
(
ESI): Calcd for [M-H] (C H N O ) requires m/z 548.2779, found 548.2782.
3
2
35
7
2
5
.1.7.3.
2-(4-methylpiperazin-1-yl)-N-(2-(4-((4-(2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)-
quinazolin-2-yl)phenyl)acetamide (6c). Compound 27a was treated with excess N-methyl piperazine
according to general procedure F to afford 6c. After column chromatography with CH Cl /MeOH/NH ·H O
2
2
3
2
(
25:1:0.1) elution, the desired product was obtained as a pale yellow solid in 68% yield. m.p. 237.5-240.0 °C;
1
H NMR (400 MHz, CDCl ): δ 13.09 (s, 1H), 9.23 (s, 1H), 8.77 (d, J = 8.3 Hz, 1H), 8.43 (dd, J = 7.9, 1.5 Hz,
3
1
H), 8.09 (d, J = 8.3 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.90 (s, 1H), 7.85 (t, J = 8.3 Hz, 3H), 7.67 (d, J = 8.8
Hz, 2H), 7.59 (t, J = 7.6 Hz, 1H), 7.50 – 7.43 (m, 1H), 7.24 – 7.17 (m, 1H), 3.24 (s, 2H), 3.20 (s, 2H), 2.71 (s,
1
3
4
H), 2.58 (s, 8H), 2.37 (s, 3H), 2.32 (s, 4H), 2.13 (s, 3H); C NMR (101 MHz, CDCl ): δ 169.2, 168.5, 160.8,
3
1
57.2, 149.5, 138.8, 134.8, 133.9, 133.0, 130.9, 130.8, 129.1, 126.5, 125.4, 123.1, 122.4, 121.1, 121.0, 120.2,
-
1
13.5, 64.0, 61.9, 55.2, 54.2, 53.5, 53.3, 46.0, 45.8. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z
3
4
41
9
2
6
06.3310, found 606.3290.
2
3

ACCEPTED MANUSCRIPT
5
.1.7.4. 2-morpholino-N-(2-(4-((4-(2-morpholinoacetamido)phenyl)amino)quinazolin-2-yl)phenyl)acetamide
(6d). Compound 27a was treated with excess morpholine according to general procedure F to afford 6d. After
column chromatography with CH Cl /MeOH/NH ·H O (50:1:0.1) elution, the desired product was obtained as
2
2
3
2
1
a pale yellow solid in 70% yield. m.p. 273.5-275.7 °C; H NMR (400 MHz, DMSO-d ): δ 13.15 (s, 1H), 10.03
6
(
s, 1H), 9.79 (s, 1H), 8.66 (d, J = 8.2 Hz, 1H), 8.62 (d, J = 8.2 Hz, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.03 (d, J =
.2 Hz, 1H), 7.98 – 7.92 (m, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.71 – 7.66 (m, 1H), 7.46
t, J = 7.8 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 3.71 – 3.62 (m, 4H), 3.41 (s, 4H), 3.18 (s, 2H), 3.16 (s, 2H), 2.54
8
(
(
1
3
s, 4H), 2.41 (s, 4H); C NMR (101 MHz, DMSO-d ): δ 168.5, 167.9, 159.9, 157.6, 148.8, 138.6, 134.8,
6
1
34.3, 133.3, 130.6, 130.4, 127.9, 126.5, 124.9, 123.0, 122.9, 122.7, 120.2, 119.6, 113.4, 66.1, 65.5, 64.0, 62.0,
-
5
3.2. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 580.2678, found 580.2656.
3
2
35
7
4
5
.1.7.5. 3-(diethylamino)-N-(2-(4-((4-(3-(diethylamino)propanamido)phenyl)amino)quinazolin-2-yl)phenyl)-
propanamide (7a). Compound 27b was treated with excess diethylamine according to general procedure F to
afford 7a. After column chromatography with CH Cl /MeOH/NH ·H O (15:1:0.1) elution, the desired product
2
2
3
2
1
was obtained as a pale yellow solid in 58% yield. m.p. 150.3-152.7 °C; H NMR (400 MHz, CDCl ): δ 13.60
3
(
s, 1H), 11.39 (s, 1H), 8.70 (d, J = 7.7 Hz, 1H), 8.56 (dd, J = 8.0, 1.6 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.96 (s,
1
7
7
H), 7.86 (d, J = 7.6 Hz, 1H), 7.82 – 7.76 (m, 1H), 7.74 (d, J = 8.9 Hz, 2H), 7.59 (d, J = 8.9 Hz, 2H), 7.55 –
.49 (m, 1H), 7.45 – 7.38 (m, 1H), 7.17 – 7.09 (m, 1H), 3.02 – 2.95 (m, 2H), 2.83 – 2.77 (m, 2H), 2.70 (q, J =
1
3
.1 Hz, 4H), 2.64 – 2.57 (m, 6H), 2.56 – 2.52 (m, 2H), 1.16 (t, J = 7.1 Hz, 6H), 1.05 (t, J = 7.2 Hz, 6H); C
NMR (101 MHz, CDCl ): δ 171.3, 171.0, 160.9, 157.2, 148.9, 140.0, 135.0, 134.3, 133.1, 131.1, 130.1, 127.7,
3
1
26.3, 123.6, 122.9, 122.7, 121.8, 120.5, 120.3, 113.6, 48.9, 48.9, 46.9, 46.0, 36.3, 33.1, 11.7, 11.5. HRMS
2
4

ACCEPTED MANUSCRIPT
-
(
ESI): Calcd for [M-H] (C H N O ) requires m/z 580.3405, found 580.3386.
34 43 7 2
5
.1.7.6.
3-(pyrrolidin-1-yl)-N-(2-(4-((4-(3-(pyrrolidin-1-yl)propanamido)phenyl)amino)quinazolin-2-yl)-
phenyl)propanamide (7b). Compound 27b was treated with excess pyrrolidine according to general procedure
F to afford 7b. After column chromatography with CH Cl /MeOH/NH ·H O (15:1:0.1) elution, the desired
2
2
3
2
1
product was obtained as a pale yellow solid in 64% yield. m.p. 198.6-202.3 °C; H NMR (400 MHz, CDCl ):
3
δ 13.60 (s, 1H), 11.30 (s, 1H), 8.74 – 8.68 (m, 1H), 8.54 (dd, J = 8.0, 1.6 Hz, 1H), 8.09 (s, 1H), 8.07 (d, J =
8
–
2
.1 Hz, 1H), 7.86 – 7.81 (m, 1H), 7.79 – 7.74 (m, 1H), 7.73 (d, J = 8.9 Hz, 2H), 7.53 (d, J = 8.9 Hz, 2H), 7.51
7.47 (m, 1H), 7.43 – 7.38 (m, 1H), 7.15 – 7.10 (m, 1H), 2.94 (dd, J = 9.4, 5.7 Hz, 2H), 2.90 – 2.84 (m, 2H),
13
.68 (dd, J = 10.4, 4.6 Hz, 6H), 2.57 (t, J = 5.9 Hz, 6H), 1.91 (s, 4H), 1.79 – 1.71 (m, 4H); C NMR (101
MHz, CDCl ): δ 171.2, 170.6, 160.8, 157.2, 148.9, 140.0, 135.1, 134.4, 133.1, 131.1, 130.8, 127.7, 126.3,
3
1
23.6, 122.9, 122.7, 121.73, 120.5, 120.4, 113.6, 54.1, 53.2, 52.1, 51.4, 38.2, 34.7, 23.7, 23.5. HRMS (ESI):
+
Calcd for [M+H] (C H N O ) requires m/z 578.3238, found 578.3265.
3
4
39
7
2
5
.1.7.7.
3-(4-methylpiperazin-1-yl)-N-(2-(4-((4-(3-(4-methylpiperazin-1-yl)propanamido)phenyl)amino)-
quinazolin-2-yl)phenyl)propanamide (7c). Compound 27b was treated with excess N-methyl piperazine
according to general procedure F to afford 7c. After column chromatography with CH Cl /MeOH/NH ·H O
2
2
3
2
(
15:1:0.1) elution, the desired product was obtained as a pale yellow solid in 63% yield. m.p. 210.6-210.8 °C;
1
H NMR (400 MHz, CDCl ): δ 13.47 (s, 1H), 11.08 (s, 1H), 8.70 (d, J = 8.3 Hz, 1H), 8.56 (dd, J = 8.0, 1.5 Hz,
3
1
H), 8.02 (d, J = 8.2 Hz, 1H), 7.89 – 7.84 (m, 1H), 7.83 (t, J = 7.0 Hz, 2H), 7.75 (d, J = 8.9 Hz, 2H), 7.63 (d, J
8.9 Hz, 2H), 7.59 – 7.54 (m, 1H), 7.46 – 7.41 (m, 1H), 7.18 – 7.12 (m, 1H), 2.88 (t, J = 7.4 Hz, 2H), 2.79 –
=
13
2
.75 (m, 2H), 2.72 – 2.51 (m, 16H), 2.45 (s, 4H), 2.37 (s, 3H), 2.27 (s, 3H); C NMR (101 MHz, CDCl ): δ
3
2
5

ACCEPTED MANUSCRIPT
1
1
70.8, 170.6, 160.8, 157.2, 149.0, 139.9, 135.0, 134.4, 133.2, 131.1, 130.8, 127.7, 126.3, 123.7, 123.0, 122.7,
21.7, 120.6, 120.3, 113.6, 55.3, 54.9, 54.2, 53.6, 52.8, 52.3, 46.0, 45.9, 36.2, 32.6. HRMS (ESI): Calcd for
-
[
M-H] (C H N O ) requires m/z 634.3623, found 634.3594.
36
45
9
2
5
.1.7.8.
3-morpholino-N-(2-(4-((4-(3-morpholinopropanamido)phenyl)amino)quinazolin-2-yl)phenyl)-
propanamide (7d). Compound 27b was treated with excess morpholine according to general procedure F to
afford 7d. After column chromatography with CH Cl /MeOH/NH ·H O (15:1:0.1) elution, the desired product
2
2
3
2
1
was obtained as a pale yellow solid in 62% yield. m.p. 239.0-242.0 °C; H NMR (400 MHz, DMSO-d ): δ
6
1
3.22 (s, 1H), 10.09 (s, 1H), 10.06 (s, 1H), 8.58 (d, J = 8.1 Hz, 1H), 8.54 (dd, J = 8.3, 0.9 Hz, 1H), 8.44 (dd, J
8.0, 1.6 Hz, 1H), 7.96 – 7.86 (m, 2H), 7.76 (d, J = 9.0 Hz, 2H), 7.72 – 7.64 (m, 3H), 7.48 – 7.41 (m, 1H),
.20 – 7.14 (m, 1H), 3.63 – 3.57 (m, 4H), 3.46 – 3.40 (m, 4H), 2.68 – 2.59 (m, 4H), 2.53 – 2.50 (m, 2H), 2.43
=
7
1
3
(
s, 6H), 2.36 – 2.30 (m, 4H); C NMR (101 MHz, DMSO-d ): δ 169.9, 169.9, 160.0, 157.4, 148.5, 139.5,
6
1
5
6
35.8, 133.6, 133.6, 130.8, 130.4, 127.2, 126.5, 123.5, 123.4, 123.1, 122.4, 120.1, 119.2, 113.4, 66.2, 66.0,
-
4.2, 53.1, 53.0, 35.4, 33.9. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 608.2991, found
3
4
39
7
4
08.2967.
5
.1.7.9. 3-(piperidin-1-yl)-N-(2-(4-((4-(3-(piperidin-1-yl)propanamido)phenyl)amino)quinazolin-2-yl)phenyl)-
propanamide (7e). Compound 27b was treated with excess piperidine according to general procedure F to
afford 7e. After column chromatography with CH Cl /MeOH/NH ·H O (15:1:0.1) elution, the desired product
2
2
3
2
1
was obtained as a pale yellow solid in 60% yield. m.p. 211.9-214.3 °C; H NMR (400 MHz, CDCl ): δ 13.53
3
(
s, 1H), 11.48 (s, 1H), 8.71 (d, J = 8.0 Hz, 1H), 8.56 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.88 (d, J =
.4 Hz, 1H), 7.85 (s, 1H), 7.84 – 7.78 (m, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 8.1 Hz, 2H), 7.54 (t, J =
8
2
6

ACCEPTED MANUSCRIPT
7
.3 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.4 Hz, 1H), 2.83 (t, J = 7.2 Hz, 2H), 2.73 – 2.67 (m, 2H),
.67 – 2.62 (m, 2H), 2.56 (d, J = 5.2 Hz, 6H), 2.44 (s, 4H), 1.72 (d, J = 4.3 Hz, 4H), 1.60 – 1.50 (m, 6H), 1.40
2
1
3
(
s, 2H); C NMR (101 MHz, CDCl ): δ 171.0, 170.8, 161.0, 157.1, 149.0, 140.1, 135.5, 133.9, 133.2, 131.3,
3
1
2
30.9, 128.1, 126.4, 123.6, 122.9, 122.7, 121.2, 120.6, 120.2, 113.5, 55.0, 54.4, 54.3, 53.7, 36.3, 32.5, 26.3,
-
5.9, 24.3, 24.2. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 604.3405, found 604.3388.
3
6
43
7
2
5
3
.1.7.10.
-((2-(diethylamino)ethyl)amino)-N-(2-(4-((4-(3-((2-(diethylamino)ethyl)amino)propanamido)-phenyl)-
amino)quinazolin-2-yl)phenyl)propanamide (7f). Compound 27b was treated with excess
N,N-diethylethylenediamine according to general procedure F to afford 7f. After column chromatography with
CH Cl /MeOH/NH ·H O (5:1:0.1) elution, the desired product was obtained as a yellow solid in 30% yield.
2
2
3
2
1
m.p. 140.0-143.0 °C; H NMR (400 MHz, CDCl ): δ 13.52 (s, 1H), 11.01 (s, 1H), 8.66 (d, J = 8.1 Hz, 1H),
3
8.55 (d, J = 7.9 Hz, 1H), 8.07 (s, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.79 – 7.73 (m, 1H),
7.70 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.49 (t, J = 7.3 Hz, 1H), 7.41 (t, J = 7.1 Hz, 1H), 7.13 (t, J =
7.2 Hz, 1H), 3.02 (t, J = 6.0 Hz, 4H), 2.82 – 2.71 (m, 4H), 2.66 – 2.47 (m, 16H), 1.04 (t, J = 7.1 Hz, 6H), 0.98
1
3
(
t, J = 7.1 Hz, 6H); C NMR (101 MHz, CDCl ): δ 171.4, 170.6, 160.6, 157.1, 149.0, 139.9, 135.2, 134.2,
3
1
4
6
33.1, 131.1, 130.8, 127.8, 126.3, 123.6, 123.0, 122.7, 121.6, 120.7, 120.5, 113.5, 52.3, 47.3, 47.1, 47.0, 46.7,
-
5.7, 45.3, 37.9, 36.0, 11.7, 11.5. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 666.4249, found
3
8
53
9
2
66.4273.
5
.1.7.11. 3-((3-(diethylamino)propyl)amino)-N-(2-(4-((4-(3-((3-(diethylamino)propyl)amino)propanamido)-
phenyl)amino)quinazolin-2-yl)phenyl)propanamide (7g). Compound 27b was treated with excess
27

ACCEPTED MANUSCRIPT
N,N-diethyl-1,3-propanediamine according to general procedure F to afford 7g. After column chromatography
with CH Cl /MeOH/NH ·H O (5:1:0.1) elution, the desired product was obtained as a yellow solid in 35%
2
2
3
2
1
yield. m.p. 143.5-146.2 °C; H NMR (400 MHz, CDCl ): δ 13.53 (s, 1H), 10.96 (s, 1H), 8.67 (d, J = 8.3 Hz,
3
1
H), 8.56 (dd, J = 8.0, 1.5 Hz, 1H), 8.04 (d, J = 8.2 Hz, 1H), 8.00 (s, 1H), 7.86 – 7.82 (m, 1H), 7.77 (t, J = 7.6
Hz, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.62 (d, J = 8.9 Hz, 2H), 7.54 – 7.49 (m, 1H), 7.45 – 7.39 (m, 1H), 7.16 –
7
1
.12 (m, 1H), 2.99 (dd, J = 8.0, 4.7 Hz, 4H), 2.78 (t, J = 6.7 Hz, 2H), 2.69 (t, J = 6.9 Hz, 2H), 2.61 – 2.42 (m,
1
3
6H), 1.79 – 1.71 (m, 2H), 1.65 (m, 2H), 1.07 – 1.00 (t, J = 7.1 Hz, 6H), 0.98 (t, J = 7.1 Hz, 6H); C NMR
(
101 MHz, CDCl ): δ 171.4, 170.7, 160.6, 157.2, 148.9, 139.8, 135.1, 134.3, 133.1, 131.0, 130.8, 127.7, 126.3,
3
1
23.6, 123.0, 122.7, 121.8, 120.5, 120.5, 113.5, 51.5, 51.0, 48.6, 48.3, 46.8, 46.7, 45.7, 45.4, 37.8, 36.0, 27.2,
-
2
6.8, 11.5, 11.4. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z 694.4562, found 694.4581.
4
0
57
9
2
5
.1.7.12. 3-((2-(dimethylamino)ethyl)amino)-N-(2-(4-((4-(3-((2-(dimethylamino)ethyl)amino)propanamido)-
phenyl)amino)quinazolin-2-yl)phenyl)propanamide (7h). Compound 27b was treated with excess
N,N-dimethylethylenediamine according to general procedure F to afford 7h. After column chromatography
with CH Cl /MeOH/NH ·H O (5:1:0.1) elution, the desired product was obtained as a yellow solid in 32%
2
2
3
2
1
yield. m.p. 140.1-143.2 °C; H NMR (400 MHz, CDCl ): δ 13.40 (s, 1H), 10.76 (s, 1H), 8.55 (d, J = 7.8 Hz,
3
1
H), 8.45 (dd, J = 8.0, 1.5 Hz, 1H), 8.24 (s, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 7.4 Hz, 1H), 7.67 – 7.62
m, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 7.41 – 7.36 (m, 1H), 7.34 – 7.29 (m, 1H), 7.07 –
.01 (m, 1H), 2.94 (t, J = 6.2 Hz, 4H), 2.70 (m, 4H), 2.52 – 2.47 (m, 2H), 2.46 – 2.40 (m, 4H), 2.35 (t, J = 6.2
(
7
1
3
Hz, 2H), 2.19 (s, 6H), 2.13 (s, 6H); C NMR (101 MHz, CDCl ): δ 171.3, 170.5, 160.4, 157.2, 148.9, 139.7,
3
1
4
35.0, 134.4, 133.0, 131.0, 130.8, 127.6, 126.2, 123.7, 123.1, 122.7, 122.0, 120.7, 120.4, 113.5, 58.6, 58.5,
-
7.0, 46.4, 45.6, 45.4, 45.4, 45.3, 37.5, 36.0. HRMS (ESI): Calcd for [M-H] (C H N O ) requires m/z
3
4
45
9
2
2
8