Design and Evaluation of Potent EGFR Inhibitors through the Incorporation of Macrocyclic Polyamine Moieties into the 4-Anilinoquinazoline Scaffold

Article abstract of DOI:10.1021/acs.jmedchem.8b01612

Adenosine triphosphate (ATP)-competitive inhibitors of the epidermal growth factor receptor (EGFR) have provided a significant improvement in the disease outcome of nonsmall cell lung cancer (NSCLC). Unfortunately, some marketed drugs affect a transient beneficial response in EGFR mutant NSCLC patients. We reported a series of potential EGFR inhibitors through incorporation of macrocyclic polyamine into 4-anilinoquinazoline scaffold. It is expected that anilinoquinazoline part effectively bind to EGFR domain, while ATP molecules are captured by a macrocyclic polyamine moiety. In vitro experiments exhibited that most of tested compounds suppressed tumor cell proliferation more strongly than Gefitinib and Lapatinib (dual inhibitor of EGFR/HER2) as controls. In kinase assays, the compound 1f showed excellent dual inhibition activity toward EGFR^WT (IC₅₀ = 1.4 nM) and HER2 (IC₅₀ = 2.1 nM). In vivo pharmacology evaluation of 1f showed significant antitumor activity (TGI = 44.2%) in A549 xenografts mice. The current work provided a feasible solution to optimize anilinoquinazoline-based inhibitors.

Full text of DOI:10.1021/acs.jmedchem.8b01612

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Article
Design and Evaluation of Potent EGFR Inhibitors through the Incorporation
of Macrocyclic Polyamine Moieties into the 4-Anilinoquinazoline Scaffold
Yilan Ju, Jintao Wu, Xi Yuan, Luqing Zhao, Ganlin Zhang, Chao Li, and Renzhong Qiao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01612 • Publication Date (Web): 03 Dec 2018
Downloaded from http://pubs.acs.org on December 3, 2018
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Design and Evaluation of Potent EGFR Inhibitors
through the Incorporation of Macrocyclic Polyamine
Moieties into the 4-Anilinoquinazoline Scaffold
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Yilan Ju,†,‖ Jintao Wu,†,‖ Xi Yuan,† Luqing Zhao,¶ Ganlin Zhang,§ Chao Li,*,† and Renzhong
Qiao*,†,‡
†State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical
Technology, Beijing 100029, P. R. China
‡State Key Laboratory of Natural and Biomimetic Drugs, Peking University, 100871, P. R. China
¶Digestive Disease Center and §Oncology Department of Beijing Hospital of Traditional Chinese
Medicine Affiliated to Capital Medical University, 100010, P. R. China
Keywords: protein kinase inhibitor, non-small cell lung cancer, macrocyclic polyamine,
quinazoline derivatives, epidermal growth factor receptor
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Abstract: ATP-competitive inhibitors of the epidermal growth factor receptor (EGFR) have
provided a significant improvement in the disease outcome of non-small cell lung cancer
(NSCLC). Unfortunately, some marketed drugs affect a transient beneficial response in EGFR
mutant NSCLC patients. We reported a series of potential EGFR inhibitors through
incorporation of macrocyclic polyamine into 4-anilinoquinazoline scaffold. It is expected that
anilinoquinazoline part effectively bind to EGFR domain while ATP molecules are captured by
macrocyclic polyamine moiety. In vitro experiments exhibited that most of tested compounds
suppressed tumor cell proliferation more strongly than Gefitinib and Lapatinib (dual inhibitor of
EGFR/HER2) as controls. In kinase assays, the compound 1f showed excellent dual inhibition
activity toward EGFRWT (IC50 = 1.4 nM) and HER2 (IC50 = 2.1 nM). In vivo pharmacology
evaluation of 1f showed significant antitumor activity (TGI = 44.2%) in A549 xenografts mice.
The current work provided a feasible solution to optimize anilinoquinazoline-based inhibitors.
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Introduction
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The human epidermal growth factor receptor (HER) family kinases play a crucial regulatory
role associated with a variety of malignancies, making this protein family an attractive drug
target.1-4 Among these kinases, EGFR and HER2 are both expressed at low levels in normal
human tissues, but overexpressed in 20-40% of solid tumors such as breast, ovarian, prostate and
colon cancer.5 They are often associated with increased metastasis and poor prognosis.6,7
Overexpression of HER2 increases glucose uptake, lactate production and oxygen consumption,
resulting in the reprogramming of tumor metabolism.8
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Several drugs have been approved by the Food and Drug Administration (FDA) for clinical
use as EGFR inhibitors. Gefitinib is the first orally targeted therapy drug to be approved for the
treatment of NSCLC (non-small cell lung cancer).9 Its good tolerability, response rates and
symptomatic improvements were demonstrated in early trials,10-14 accelerated approval for
Gefitinib treatment in patients with advanced NSCLC who had advanced on chemotherapy.
However, the emergence of acquired point mutations, particular T790M mutation has weakened
their therapeutic efficacy, leading to drug resistance.15-17 Previous studied showed that the
interactions of gefitinib in the active site of the wild-type EGFR consists of hydrogen bonding
between quinazoline ring with the main chain amide of Met793.4 However, the morpholine
moiety in Gefitinib is not involved in any interaction with EGFR and is randomly ordered due to
its lower electron density. Thus, the suitable 6-substituted group in anilinoquinazoline instead of
morpholine probably provided the inhibitors with improved potency.18-21
It is known that several macrocyclic polyamines can catalyze the chemical hydrolysis of
ATP,22 and even bind in the ATP pocket as potent Mer tyrosine kinase (MerTK)-specific
inhibitor.23-25 Some reports indicated depletion of cellular ATP related to growth inhibition of
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tumor cell lines and to drug resistance in MDR (multidrug resistance) cells.26,27 Frydman et al.
investigated five macrocyclic polyamines, related to the budmunchamine family of alkaloids, to
inhibit human prostate tumor cell proliferation through catalyzing the hydrolysis of ATP.28 Only
2 μM of the most efficient macrocycle reduced cellular ATP levels by 3 orders of magnitude
after a 24 h of incubation.29 A rough correlation was established between the cytotoxic effect of
the macrocyclic polyamines and their ATP-ase like activity in cancer cell line, which may
depend on the ring size of the macrocycle.
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Herein, we reported a group of novel quinazoline derivatives with macrocyclic polyamine side
chain at the C-6 position as potent protein kinase inhibitors (Figure 1).
1,4,7,10-tetraazacyclododecane (cyclen) and 1,4,7-triazonane (tacn) were used to replace the
morpholine moiety in clinical drug Gefitinib, mainly considering its affinity to cellular ATP.30,31
If ATP molecules are captured and subsequent hydrolyzed as expected, it would effectively
prevent the binding of ATP to hydrophobic cavity in EGFR, blocking the signals pathway and
inducing cancer cell apoptosis. Meanwhile, protonated polyamine in acidic environment is more
likely to combine and hydrolyze ATP molecules,32-34 which facilitate the passive targeting of
drugs in acidic tumor microenvironment. On the other side, Lapatinib as a reversible dual TKI
with proven effectiveness in clinical trials was approved by FDA in 2007.35 Considering its
selectively inhibition of HER2 and EGFR as well as the corresponding SAR, the featured
substituent in 4-position of quinazoline, 3-chloro-4-((3-fluorobenzyl)oxy)phenyl, was used in
current design as one of the candidates. Thirty compounds with a series of 4-substituted anilines
were investigated to optimize the inhibitor activity and further explore the structure-activity
relationship. Most candidates showed much lower IC50 compared to positive controls against
A431 and A549 cancer cell lines, and further kinase testing on 1f and 2f revealed that both of
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them possessed high inhibitory activity against EGFRwt and HER2. Moreover, in vivo antitumor
efficacy were evaluated using A549 NSCLC xenografts mice model, and results showed the
favorable tumor growth inhibition (TGI). The current work is attempted to explore the
macrocyclic polyamine potential application in antitumor activity by its ATP-binding ability, and
improve the physicochemical properties of clinical quinazoline compounds.
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Figure 1. The structure of Gefitinib, Lapatinib and designed new quinazoline derivatives as
potential EGFR inhibitors.
Results and discussion
Chemistry. A series of novel quinazoline derivatives linked with macrocyclic polyamine at
C-6 position and different aniline at C-4 position were synthesized. The synthetic route of the
titled compounds is depicted in Table 1, starting from compound 3, which is commercially
available. After protection of 6-OH by acetic anhydride (step i), carbonyl in compound 3 was
chlorinated by thionyl chloride to afford 4 in 74% yield (step ii). Subsequently, various
substituted anilines were coupled to 4-Cl in quinazoline (step iii), and hydrolysis of the acetyl
group using ammonium hydroxide gave corresponding phenol intermediates in average 75%
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yield (5a-o, step iv). The alkylation of the 6-OH on compounds 5a-o were performed with two
kinds of macrocyclic polyamine moieties containing bromopropyl (step v). Finally, the boc
groups were removed with 12 M hydrochloride acid (HCl) in anhydrous methanol solution, to
give the titled compounds 1a-o and 2a-o in average 70% yield (step vi), respectively.
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Table 1. General Procedure for the Synthesis of 4-anilinoquinazoline Derivatives 1a-o and 2a-o.
Reagents and Conditions: (i) (CH3CO)2O, pyridine, reflux; (ii) SOCl2, DMF, reflux; (iii)
substituted aniline, CH3CN, reflux; (iv) NH3·H2O, CH3OH, reflux; (v) cyclen (or tacn) moiety,
DMF, reflux; (vi) HCl (l), CH3OH.
Compound
a
R
Compound
f
R
Compound
k
R
b
c
g
h
i
l
m
n
o
d
e
j
Anti-Proliferative Activity Assay. The anti-proliferation activity of the titled compounds
against A549 and A431 cell lines with abnormally high expressing EGFR were evaluated by
MTT assay, as shown in Table 2. Gefitinib, the first selective inhibitor of EGFR, was used as
control compound. All synthesized compounds showed more effective anti-proliferative
activities in A431 cell lines (IC50 from 0.442 to 18.77 μM) than that in A549 cell lines. We were
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encouraged that anti-proliferative activity of 1a and 2a with the same 4-substituted aniline
(3-chloro-4-fluorophenyl) as Gefitinib was well accepted with IC50 values of 1.01 μM and 1.31
μM against A431 cell lines, respectively. Macrocyclic polyamine substituted for morpholine
could contribute to increased inhibitory effects. For A549 cell lines, the compounds attached
cyclen moiety (1a-o) showed better anti-proliferative activity than those with tacn moiety (2a-o),
while no such difference in A431 cell lines. The best results were from compound 1f with IC50
values of 0.958 μM and 3.4 μM against A431 and A549 cell lines, respectively, which were
much better than the inhibitory effects of Gefitinib (IC50 2.47 μM against A431 and 11.08 μM
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against
A549).
It
should
be
noted
that
the
4-substituted
aniline,
3-chloro-4-((3-fluorobenzyl)oxy)phenyl, is the same as the featured substituent of Lapatinib,
which may have a positive effect on the activity of 1f or 2f. Thus, Lapatinib was also used as
control to evaluate the anti-proliferative activity in both cell lines. Similarly, more than half of
the tested compounds showed better anti-proliferative activity than that of Lapatinib against
A431 cell lines, but only 1f gave acceptable IC50 value of 3.4 μM in A549 cell lines. Compound j,
k, n, and o hardly exhibited any inhibitory effects against A549 (IC50 > 50 μM), which may
result from electronic or steric effect of substituent. Despite the benefits from the featured side
chain of Lapatinib, the positive effect of macrocyclic polyamine on anti-proliferative activity is
also not negligible.
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Table 2. Anti-Proliferative Activities of 1a-o and 2a-o against A549 and A 431 Cells Harboring
5
6
High Expressing EGFR.a
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Anti-proliferation IC50 (μM)b
Anti-proliferation IC50 (μM)
Compound
Compound
A431
A549
A431
A549
＞50
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1a
1b
1.01 ± 0.04
1.11 ±0.22
1.047 ± 0.014
20.21 ± 3.24
28.55 ± 4.91
14.68 ± 2.26
2a
1.314 ± 0.356
2.231 ± 0.544
2.322 ± 0.812
0.561 ± 0.051
1.064 ± 0.341
0.442 ± 0.068
2.98 ± 0.26
2b
＞50
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2c
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16.08 ± 2.34
34.27 ± 1.26
7.25 ± 0.89
6.23 ± 0.91
37.3 ± 3.3
＞50
1d
1.839 ± 0.233 15.195 ± 1.036
2d
1e
1.578 ± 0.411
0.958 ± 0.034
1.707 ± 0.129
1.952 ± 0.387
1.424 ± 0.481
3.21 ± 0.53
11.84 ± 2.31
3.4 ± 0.4
5.35 ± 1.21
15.73 ± 3.66
27.1 ± 3.49
＞50
2e
1f
2f
1g
2g
1h
2h
1.562 ± 0.183
1.125 ± 0.312
8.526 ± 0.569
7.914 ± 0.063
2.215 ± 0.991
4.077 ± 0.517
12.69 ± 0.71
18.77 ± 1.26
2.66 ± 0.27
1i
2i
1j
2j
＞50
1k
1.86 ± 0.44
＞50
2k
＞50
1l
2.539 ± 0.512
4.385 ± 0.302
6.369 ± 0.981
5.653 ± 0.737
2.47 ± 0.07
14.53 ± 2.31
23.79 ± 6.14
＞50
2l
2m
26.16 ± 2.71
44.24 ± 2.82
＞50
1m
1n
2n
1o
＞50
2o
＞50
Gefitinib
11.08 ± 2.29
Lapatinib
3.64 ± 0.77
aThe data are the mean ± SD of at least three independent experiments. bThe anti-proliferative
effects of the synthesized compounds against human cancer cells gathered with MTT assay.
Kinases Inhibitory Activity. The inhibitory activity of the selected compounds against two
EGFRs (WT and T790M) and HER2 were evaluated using a well-established LANCE Ultra
kinase assay, with Gefitinib and Lapatinib employed as positive controls. As shown in Table 3,
most of the tested compounds exhibited inhibitory activities against EGFRWT, in which 2f
suppressed EGFRWT (IC50 0.3 nM) more potently relative to Gefitinib (IC50 5.8 nM) and
Lapatinib (IC50 10.85 nM). Also, 1f (IC50 2.1 nM) and 2f (IC50 9.6 nM) showed better inhibitory
activities against HER2 than Lapatinib (IC50 32.7 nM). For mutant EGFR, 1f and 2f showed
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moderate inhibitory effect on T790M (with IC50 of 16.5 nM and 40.6 nM, respectively), which
was still much better than Gefitinib (IC50 148.7 nM). These results suggested that 1f and 2f can
serve as dual inhibitors against both EGFRWT and HER2, and were more active than Lapatinib.
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Table 3. In Vitro Enzymatic Inhibitory activity of Selected Compounds against Different Statues
of EGFR and HER2.a
Kinase inhibition IC50 (nM)
Compound
EGFRwt
6.6 ± 0.4
8.3 ± 0.6
28.2 ± 5.3
5.6 ± 0.8
1.4 ± 0.2
0.3 ± 0.1
5.8 ± 0.2
10.85 ± 0.11
HER2
NTb
EGFRT790M
NT
1a
2a
NT
NT
2d
2e
NT
NT
NT
NT
1f
2.1 ± 0.6
9.6 ± 0.4
NT
16.5 ± 2.3
40.6 ± 4.1
148.7 ± 6.3
> 2500
2f
Gefitinib
Lapatinib
32.7 ± 1.4
aThe data are the mean ± SD of at least three independent experiments. bNT means not test.
Cellular ATP Depletion. Cellular ATP levels in A549 cells were measured at 24 and 72 h
after incubation with 1f, 2f, and control drugs. Cellular ATP concentrations were measured using
the luciferaseluciferin system. After a 24 h treatment with 0-2 μM of all samples, no obvious
change in ATP levels was obtained but at 8 μM, 1f was found to effectively deplete intracellular
ATP, which was better than 2f and Gefitinib under the same incubation conditions (Figure 2A).
After Lapatinib treatment with the maximal dosage of 6 μM, ATP remained at moderate levels.
For a 72 h treatment, 1f only required 2 μM concentration to deplete intracellular ATP to low
level (0.1 μM), while 2f required 8 μM concentration to achieve comparable inhibition. Gefitinib
and Lapatinib exhibited moderate inhibition of intracellular ATP at maximal dosage (Figure 2B).
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Figure 2. Effect of macrocyclic polyamines on the intracellular ATP levels of A549 cells after
incubating for (A) 24 h and (B) 72 h. The treated cells for each concentration of macrocycle were
run in quadruplicate. ATP concentrations were determined using Enlitten reagent and
luminescence was read after a 1 min mixture time. Finally, relative light units were converted to
ATP concentrations using a reference plot.
Induction of Apoptosis in Vitro. To further investigate the mechanism of inhibiting tumor
cells, a flow cytometry-based apoptosis assay was performed, and cells under initiation of
apoptosis were stained by Annexin V-FITC while late apoptotic cells labeled by PI (Figure 3).
A549 cells harboring high expressing EGFR were treated with a variable level of 1f (1 μM, 4 μM,
and 8 μM) for 72 h. It was found that treatment with 1f induced apoptosis in a dose-dependent
manner, and total cellular apoptosis increased from 17.41%-89.47%. At concentration of 8 μM,
1f (89.47%) induced more cellular apoptosis of A549 cell than Gefitinib (27.77%) and Lapatinib
(57.77%), in which early apoptosis (81.53%) accounted for a large proportion. The ability to
significantly induce early apoptosis enabled compound 1f to be a potential candidate for
anti-tumor drugs.
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Figure 3. Cell apoptosis analysis on A549 cells treated with compound 1f, Gefitinib and
Lapatinib at indicated concentrations detected by flow cytometer. The inserted table showed
quantitative early/late cellular apoptosis for tested compounds.
Cytotoxicity. The inhibitory effect on non-tumor cell line was also tested to verify the
tumor-targeting potency of the best compound 1f. The mouse fibroblast cells L929 without
overexpressed EGFR or HER2 were used as non-tumor cell model in cytotoxicity assay. As it
can be seen from Table 4, Gefitinib, Lapatinib and 1f exhibited 25.95%, 35.03%, and 37.63%
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growth inhibition of L929 cells, respectively, implying low cell inhibition at 1 μM. Although
Gefitinib showed the lowest inhibition rate, a surge of growth inhibition against L929 was
observed with increasing drug concentration. Subsequently, the IC50 of 1f was tested with the
value of 2.74 μM, indicating its comparable cytotoxicity to the clinical drugs Gefitinib (1.98 μM)
and Lapatinib (2.05 μM).
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Table 4. IC50 and inhibition of compounds and positive drugs against L929 at the concentration
of 1 μM.a
1f
Gefitinib
Lapatinib
Inhibition at the concentration of 1 μM
IC50 (μM)
37.63%
25.95%
35.03%
2.74 ± 0.42
1.98 ± 0.13
2.05 ± 0.78
aThe data are the mean ± SD of at least three independent experiments.
In Vivo Antitumor Activity. The antitumor activity of compound 1f and Gefitinib was
compared by recording the tumor volumes and weights of A549 tumor-bearing nude mice at a
dose of 30 mg/kg, and saline-treated mice were used as controls. After 19 days treatment, all
animals in different treatment group were kept a stable body weights (Figure 4A). The tumors
size were measured averagely 2433 mm3 for the control and 1598 mm3 for Gefitinib, respectively,
while those of 1f-treated animals were measured 1357 mm3 (Figure 4B-C). The tumor weights
treated by 1f significantly reduced, and showed statistical difference compared to those of
control and Gefitinib (Figure 4D). Tumor growth inhibition (TGI) values of Gefitinib and 1f
were also calculated by empirical equation (see Experimental Section), with the values of
34.31% and 44.23% respectively at dosages of 30mg/kg. These results indicate effective ability
of 1f to inhibit tumor growth in nude mice bearing A549 xenografts via proliferation suppression.
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The histologic images using H&E staining showed a massive cancer cell underwent necrosis in
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the tumor site with compound 1f treatment, as shown in Figure 4E.
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Figure 4 Antitumor efficacy of compound 1f in the A549 xenografts mouse model. (A) Body
weight and (B) Tumor volume measurements from A549 xenografts mice after 19 days of 1f
administration. (C) Representative photographs of tumors in each group (D) Comparison of the
final tumor weights in each group after the 19 days treatment period of 1f. *p < 0.05 compared
with the control group. (E) H&E staining images of tumor tissue from each treatment group. The
scale bar is 50 μm.
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Molecular Docking Studies. In order to predict the possible binding mode of this series of
compounds with EGFR, the docking study of compound 1f with EGFR kinase was performed
using Gefitinib and Lapatinib as positive controls (Figure 5A-B). In the binding model, the
nitrogen atoms in quinazoline of 1f can bind to MET793 residue by hydrogen bonds, which is
consistent with the binding site of Gefitinib and Lapatinib in EGFR domain.
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Figure 5. Docking modes of compounds 1f (green) with EGFR comparing with (A) Gefitinib
(grey, PDB code 2ITY) and (B) Lapatinib (grey, PDB code 1XKK), respectively. Selected
residues MET793 in EGFR are shown. Green dashed lines indicate hydrogen bonds.
Conclusions
In summary, a series of anilinoquinazoline derivatives containing macrocyclic polyamine were
designed, synthesized, and evaluated as potential EGFR-competitive inhibitors. These
compounds exhibited strong anti-proliferative activity against A431 cells bearing overexpressed
EGFR. Among them, both 1f and 2f showed stronger dual inhibition against EGFRWT and HER2
compared with Gefitinib and Lapatinib in kinase assays. Moreover, compound 1f exhibited
significant tumor inhibition in the human NSCLC xenografts mouse model of A431. The use of
macrocyclic polyamine moiety in inhibitor is intended to selectively capture the native substrate
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ATP, thus assisting to block EGFR downstream signal pathway. This design offered an option
for the optimization and development of anilinoquinazoline-based NSCLC agents and drugs.
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Experimental section
General Methods for Chemistry. All reagents and solvents were used as purchased from
commercial sources. Flash chromatography was performed using 400 mesh silica gel. All
reactions were monitored by TLC, using silica gel plates with fluorescence F254 and UV light
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visualization. H NMR spectra and 13C NMR spectra were recorded on a Bruker AV-400
spectrometer at 400 MHz. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ)
of NMR are reported in parts per million (ppm) units relative to an internal standard (TMS).
High-resolution mass spectra (HRMS) were recorded using an Agilent 6540 UHD Q-TOF MS
equipped with a gas nebulizer probe, capable of analyzing ions up to m/z 6000. Purification of
the final compounds were performed with reverse-phase high-performance liquid
chromatography (HPLC). All final compounds were purified to ≥95% chemical purity as
determined by HPLC with UV detection at 254 nm. Further details on the analytical conditions
used for individual compounds may be found in the Supporting Information. We further used the
latest version of ZINC15 database to virtually test all final compounds for structural elements
representing pan-assay interference compounds (PAINS). We did not get any PAIN hit from this
database search.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-chloro-4-fluorophenyl)-7-methox
yquinazolin-4-amine (1a). General procedure for syntheses of 1a-o. A mixture of compound
(5a, 0.67 g, 2.1 mmol),
10-(3-chloropropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
tricarboxylate (1.27g, 2.3 mmol, 1.1 eq.) and K2CO3 (0.9 g, 6.5 mmol, 3 eq.) was dissolved in
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DMF (30 mL). And the solution was stirred at 130 °C for 6 h, filtered to discard inorganic salt
and concentrated in vacuo. The resulting solid was purified by chromatography on silica
(petroleum ether/EtOAc, 10:1) to afford tri-tert-butyl 10-(3-((4-((3-chloro-4-fluorophenyl)
amino)-7-methoxyquinazolin-6-yl)oxy)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxyl
ate as a yellow solid (yield 92.8%).
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A mixture of product above (0.86 g, 1.03 mmol) was dissolved in CH3OH (45 mL) and
hydrochloric acid (12 mol/L, 15 mL) was added at room temperature with continues stirring.
After stirring for an additional 6 h, the resulted solution was concentrated in vacuo. The acquired
solid was re-dissolved in methanol and subsequently solution was filtered, the filter cake was
washed with methanol. The resulted solid was dried under vacuum for 1 day to afford the yellow
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solid (yield 68.4%). H NMR (400MHz, D2O) δ 8.52(s,1H), 7.60-7.58 (m, 2H), 7.39-7.37 (m,
1H), 7.10-7.08 (t, J = 8.96, 7.03 Hz, 2H), 4.17-4.15 (t, J = 6.21 Hz, 2H), 3.98 (s, 3H), 3.16-3.10
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(m, 8H), 2.93-2.85 (m, 10H), 2.02-2.05 (m, 2H). C NMR (101 MHz, D2O) δ 157.27, 156.58,
154.09, 149.55, 147.83, 134.51, 132.60, 125.05, 123.42, 119.99, 116.44, 106.91, 103.46, 99.52,
68.30, 56.88, 49.61, 48.03, 44.04, 42.04, 23.19. HRMS (ESI) m/z calcd. for C26H35ClFN7O2
[M+H]+: 532.2603. Found: 532.2597.
Compound 1b-o were synthesized using similar route according to 1a.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-ethynylphenyl)-7-methoxyquinaz
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olin-4-amine (1b). Compound 1b was obtained as a light yellow solid (yield 70.2%). H NMR
(400 MHz, D2O) δ 8.53 (s, 1H), 7.75-7.73 (dd, J = 5.93, 2.54 Hz, 1H), 7.61 (s, 1H), 7.46-7.42
(m, 1H), 7.11-7.07 (t, J = 8.75 Hz, 2H), 4.20-4.17 (t, J = 5.65 Hz, 2H), 4.01 (s, 3H), 3.17-3.09
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(m, 8H), 2.93-2.86 (m, 10H), 2.05-2.03 (m, 2H). C NMR (101 MHz, D2O) δ 157.42, 156.50,
149.53, 147.72, 135.85, 134.51, 130.04, 129.08, 126.60, 124.19, 121.98, 106.99, 103.50, 99.55,
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82.63, 79.24, 68.31, 56.85, 49.41, 47.91, 44.07, 41.98, 23.17. HRMS (ESI) m/z calcd. for
C28H37N7O2 [M+H]+: 504.3087. Found: 504.3097.
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6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-bromophenyl)-7-methoxyquinazo
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lin-4-amine (1c). Compound 1c was obtained as a light yellow solid (yield 66.8%). H NMR
(400 MHz, D2O) δ 8.52 (s, 1H), 7.71 (s, 1H), 7.65 (s, 1H), 7.46-7.44 (d, J = 8.30 Hz, 1H),
7.37-7.35 (d, J = 7.52 Hz, 1H), 7.26-7.22 (t, J = 8.04 Hz, 1H), 7.10 (s, 1H), 4.20-4.18 (t, J = 5.71
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Hz, 2H), 4.00(s, 3H), 3.16-3.09 (m, 8H), 2.91-2.83 (m, 10H) 2.04-2.01 (m, 2H). C NMR (101
MHz, D2O) δ 157.02, 156.38, 149.55, 147.56, 137.04, 134.38, 130.10, 129.05, 125.37, 121.53,
106.86, 103.32, 99.43, 68.33, 56.88, 49.60, 48.05, 44.02, 42.07, 23.15. HRMS (ESI) m/z calcd.
for C26H36BrN7O2 [M+H]+: 558.2192. Found: 558.2185.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(4-bromo-2-fluorophenyl)-7-methox
yquinazolin-4-amine (1d). Compound 1d was obtained as a light yellow solid (yield 72.4%). 1H
NMR (400 MHz, D2O) δ 8.48 (s, 1H), 7.60-7.57 (d, J = 11.41 Hz, 2H), 7.50-7.48 (d, J = 7.06
Hz, 1H), 7.33-7.32 (m, 2H), 7.05 (s 1H), 4.15-4.12 (t, J = 5.98 Hz, 2H), 3.98 (s, 3H), 3.55 (s,
5H), 3.30-3.27 (t, J = 5.98 Hz, 4H), 2.99-2.96 (t, J = 5.98 Hz, 4H), 2.90-2.86 (m, 2H), 2.04-2.00
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(m, 2H). C NMR (101 MHz, D2O) δ 158.85, 157.28, 154.90, 149.68, 148.22, 135.02, 128.85,
128.01, 122.48, 121.09, 119.79, 107.26, 103.71, 99.75, 68.12, 56.93, 49.59, 48.00, 44.05, 42.00,
23.27. HRMS (ESI) m/z calcd. for C26H35BrFN7O2 [M+H]+: 576.2098. Found: 576.2085.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-bromo-4-fluorophenyl)-7-methox
yquinazolin-4-amine (1e). Compound 1e was obtained as a light yellow solid (yield 74.3%). 1H
NMR (400 MHz, D2O) δ 8.50 (s, 1H), 7.59 (s, 1H), 7.34-7.26 (m, 3H), 7.17 (s 1H), 4.14-4.11 (t,
J = 5.92 Hz, 2H), 4.02 (s, 3H), 3.56 (s, 4H), 3.32-3.29 (t, J = 5.92 Hz, 4H), 3.04-3.01 (t, J = 5.92
Hz, 4H), 2.96-2.92 (t, J = 7.82 Hz, 2H), 2.11-2.07 (m, 2H). 13C NMR (101 MHz, D2O) δ 156.99,
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156.59, 156.51, 149.53, 147.76, 134.41, 132.89, 127.45, 123.84, 116.07, 107.91, 106.83, 103.40,
99.51, 68.31, 56.91, 49.68, 48.08, 44.02, 42.07, 23.21. HRMS (ESI) m/z calcd. for
C26H35BrFN7O2 [M+H]+: 576.2098. Found: 576.2096.
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6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-chloro-4-((3-fluorobenzyl)oxy)ph
enyl)-7-methoxyquinazolin-4-amine (1f). Compound 1f was obtained as a yellow solid (yield
64.3%). 1H NMR (400 MHz, D2O) δ 8.45 (s, 1H), 7.43 (s, 1H), 7.31 (s, 1H), 7.19 (t, J = 7.6 Hz,
2H), 6.90 (t, J = 8.2 Hz, 1H), 6.78 (d, J = 7.4 Hz, 1H), 6.62 (d, J = 13.7 Hz, 2H), 6.41 (d, J = 8.8
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Hz, 1H), 4.18 (s, 2H), 4.04 (s, 2H), 3.41 (s, 3H), 3.22-2.74 (m, 18H), 2.01 (s, 2H). C NMR
(101 MHz, D2O) δ 163.40, 160.95, 158.71, 155.22, 149.07, 147.13, 138.05, 133.79, 129.83,
123.04, 121.38, 120.32, 114.01, 111.37-111.17, 106.29, 102.73, 98.60, 67.78, 55.96, 49.68,
48.85, 48.21, 44.03, 42.03, 23.64. HRMS (ESI) m/z calcd. for C33H41ClFN7O3 [M+H]+:
638.3022. Found: 638.3009
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(4-chloro-3-(trifluoromethyl)phenyl
)-7-methoxyquinazolin-4-amine (1g). Compound 1g was obtained as a light yellow solid (yield
72.9%). 1H NMR (400 MHz, D2O) δ 8.63 (s, 1H), 7.90 (d, J = 2.5 Hz, 1H), 7.73 (dd, J = 8.7, 2.5
Hz, 1H), 7.59 (s, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.04 (s, 1H), 4.17 (t, J = 5.8 Hz, 2H), 3.99 (s,
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3H), 3.13 (dd, J = 17.4, 12.3 Hz, 8H), 3.02-2.80 (m, 10H), 2.04 (dd, J = 10.1, 5.3 Hz, 2H). C
NMR (101 MHz, D2O) δ 156.68, 149.63, 147.62, 135.11, 134.38, 131.32, 127.12, 126.58,
126.07, 123.39, 120.75, 106.65, 103.10, 99.45, 68.46, 68.09, 67.85, 56.90, 49.45, 47.96, 44.08,
42.02, 23.14. HRMS (ESI) m/z calcd. for C27H35ClF3N7O2 [M+H]+: 582.2571. Found: 582.2559
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-7-methoxy-N-(3-(trifluoromethyl)phen
yl) quinazolin-4-amine (1h). Compound 1h was obtained as a light yellow solid (yield 69.7%).
1H NMR (400 MHz, D2O) δ 8.54 (s, 1H), 7.90 (s, 1H), 7.72 (dd, J = 5.4, 3.5 Hz, 1H), 7.66 (s,
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1H), 7.52 (d, J = 0.9 Hz, 2H), 7.02 (s, 1H), 4.17 (s, 2H), 3.96 (s, 3H), 3.12 (dd, J = 13.3, 8.1 Hz,
8H), 3.04-2.75 (m, 10H), 2.05-1.89 (m, 2H). 13C NMR (101 MHz, D2O) δ 157.72, 156.61,
149.61, 147.86, 136.32, 134.55, 130.31, 129.99, 127.21, 124.97, 123.34, 122.27, 120.68, 106.93,
103.50, 99.34, 68.35, 56.79, 49.31, 47.83, 44.07, 41.97, 23.03. HRMS (ESI) m/z calcd. for
C27H36F3N7O2 [M+H]+: 548.2961. Found: 548.2947.
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6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-chlorophenyl)-7-methoxyquinazo
lin-4-amine (1i). Compound 1i was obtained as a white solid (yield 77.3%). 1H NMR (400 MHz,
D2O) δ 8.52 (s, 1H), 7.61 (s, 1H), 7.54 (t, J = 2.0 Hz, 1H), 7.44-7.36 (m, 1H), 7.26 (t, J = 8.1 Hz,
1H), 7.19-7.09 (m, 1H), 7.04 (s, 1H), 4.16 (t, J = 5.9 Hz, 2H), 3.98 (s, 3H), 3.12 (dd, J = 15.4,
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10.2 Hz, 8H), 3.01-2.67 (m, 10H), 2.01 (dt, J = 8.1, 5.6 Hz, 2H). C NMR (101 MHz, D2O) δ
157.28, 156.48, 149.57, 147.66, 136.93, 134.50, 133.57, 130.00, 126.36, 122.94, 121.46, 106.95,
103.40, 99.45, 68.35, 56.84, 49.44, 47.93, 44.07, 42.00, 23.15. HRMS (ESI) m/z calcd. for
C26H36ClN7O2 [M+H]+: 514.2697. Found: 514.2682.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-7-methoxy-N-(3-methoxyphenyl)
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quinazolin-4-amine (1j). Compound 1j was obtained as a light yellow solid (yield 74.5%). H
NMR (400 MHz, D2O) δ 8.47 (s, 1H), 7.59 (s, 1H), 7.24 (t, J = 8.1 Hz, 1H), 7.07 (t, J = 4.3 Hz,
2H), 7.02 (s, 1H), 6.79-6.74 (m, 1H), 4.14 (t, J = 5.9 Hz, 2H), 3.96 (s, 3H), 3.71 (s, 3H), 3.12
(dd, J = 14.6, 9.4 Hz, 8H), 3.00-2.69 (m, 10H), 2.05-1.93 (m, 2H). 13C NMR (101 MHz, D2O) δ
158.86, 157.37, 156.33, 149.37, 147.65, 136.84, 134.38, 129.84, 116.14, 111.92, 109.46, 106.95,
103.56, 99.40, 68.29, 56.79, 55.34, 49.43, 47.94, 44.06, 42.01, 23.09. HRMS (ESI) m/z calcd. for
C27H39N7O3 [M+H]+: 510.3193. Found: 510.3176.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-fluorophenyl)-7-methoxyquinazo
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lin-4-amine (1k). Compound 1k was obtained as a light yellow solid (yield 67.5%). H NMR
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(400 MHz, D2O) δ 8.51 (s, 1H), 7.66 (s, 1H), 7.43-7.25 (m, 3H), 7.07 (s, 1H), 7.02 (td, J = 8.4,
1.8 Hz, 1H), 4.18 (t, J = 5.9 Hz, 2H), 3.96 (s, 3H), 3.12 (dd, J = 15.7, 10.5 Hz, 8H), 2.93 (d, J =
4.9 Hz, 8H), 2.87 (dd, J = 9.4, 6.4 Hz, 2H), 2.04 (dd, J = 10.6, 5.5 Hz, 2H). 13C NMR (101 MHz,
D2O) δ 163.19, 160.78, 157.27, 156.48, 149.51, 147.65, 137.13, 134.46, 130.32, 119.17, 113.38,
110.59, 106.83, 103.34, 99.43, 68.21, 56.80, 49.89, 48.29, 43.91, 42.22, 23.01. HRMS (ESI) m/z
calcd. for C26H36FN7O2 [M+H]+: 498.2993. Found: 498.2980.
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6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-iodophenyl)-7-methoxyquinazoli
n-4-amine (1l). Compound 1l was obtained as a white solid (yield 68.6%). 1H NMR (400 MHz,
D2O) δ 8.43 (s, 1H), 7.73 (t, J = 1.8 Hz, 1H), 7.44 (s, 1H), 7.40 – 7.32 (m, 1H), 7.29 (d, J = 8.2
Hz, 1H), 6.91 (s, 1H), 6.88 (t, J = 8.0 Hz, 1H), 4.05 (t, J = 5.8 Hz, 2H), 3.92 (s, 3H), 3.05 (dd, J
= 14.2, 9.1 Hz, 8H), 2.85 (d, J = 22.5 Hz, 8H), 2.74 (dd, J = 9.5, 6.4 Hz, 2H), 1.90 (dd, J = 10.3,
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5.2 Hz, 2H). C NMR (101 MHz, D2O) δ 156.70, 156.27, 149.53, 147.46, 136.77, 134.82,
134.21, 130.84, 129.98, 121.70, 106.76, 103.28, 99.43, 93.42, 68.37, 56.95, 49.57, 48.03, 44.03,
42.06, 23.21. HRMS (ESI) m/z calcd. for C26H36IN7O2 [M+H]+: 606.2053. Found: 606.2031.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(2,4-dichlorophenyl)-7-methoxyqui
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nazolin-4-amine (1m). Compound 1m was obtained as a light yellow solid (yield 67.8%). H
NMR (400 MHz, D2O) δ 8.48 (s, 1H), 7.73 (s, 1H), 7.58 (d, J = 2.3 Hz, 1H), 7.43 (d, J = 8.6 Hz,
1H), 7.35 (dd, J = 8.6, 2.3 Hz, 1H), 7.22 (s, 1H), 4.20 (t, J = 5.9 Hz, 2H), 4.04 (s, 3H), 3.13 (dd,
J = 17.9, 12.6 Hz, 8H), 3.02-2.76 (m, 10H), 2.09 (td, J = 11.2, 5.9 Hz, 2H). 13C NMR (101 MHz,
D2O) δ 158.99, 157.08, 149.68, 148.11, 134.79, 133.97, 131.53, 129.78, 128.02, 107.04, 103.61,
99.61, 68.30, 56.94, 51.51, 47.22, 43.40, 41.87, 23.75. HRMS (ESI) m/z calcd. for
C26H35Cl2N7O2 [M+H]+: 548.2308. Found: 548.2293.
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5-((6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-7-methoxyquinazolin-4-yl)amino)-
2-fluorobenzonitrile (1n). Compound 1n was obtained as a light yellow solid (yield 73.2%). 1H
NMR (400 MHz, D2O) δ 8.57 (s, 1H), 7.91 (dd, J = 5.3, 2.6 Hz, 1H), 7.85 – 7.75 (m, 1H), 7.64
(s, 1H), 7.22 (t, J = 8.9 Hz, 1H), 7.09 (s, 1H), 4.19 (t, J = 5.4 Hz, 2H), 3.99 (s, 3H), 3.18 (s, 4H),
3.12 (d, J = 4.7 Hz, 4H), 2.96 (d, J = 12.4 Hz, 8H), 2.94 – 2.87 (m, 2H), 2.08 (s, 2H). 13C NMR
(101 MHz, D2O) δ 161.54, 158.98, 157.42, 156.90, 149.71, 148.10, 134.76, 132.80, 130.72,
127.70, 116.86, 113.64, 106.96, 103.47, 100.25-99.56 (m), 68.31, 56.97, 49.90, 48.26, 43.94,
42.17, 23.14. HRMS (ESI) m/z calcd. for C27H35FN8O2 [M+H]+: 523.2945. Found: 523.2932.
6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(4-bromo-2-ethylphenyl)-7-methoxy
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quinazolin-4-amine (1o). Compound 1o was obtained as a white solid (yield 69.8%). H NMR
(400 MHz, D2O) δ 8.32 (s, 1H), 7.68 (s, 1H), 7.48 (d, J = 1.9 Hz, 1H), 7.22 (dd, J = 8.4, 1.9 Hz,
1H), 7.12 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 4.10 (s, 2H), 3.97 (d, J = 14.4 Hz, 3H), 3.08 (dd, J =
16.1, 11.1 Hz, 8H), 2.98 – 2.74 (m, 10H), 2.44 (q, J = 7.5 Hz, 2H), 2.01 (s, 2H), 0.92 (t, J = 7.5
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Hz, 3H). C NMR (101 MHz, D2O) δ 159.65, 156.90, 149.59, 148.27, 143.91, 134.92, 132.65,
130.05, 129.60, 122.23, 107.14, 104.19, 99.99, 68.15, 56.83, 49.75, 48.10, 44.01, 42.05, 24.03,
23.25, 13.45. HRMS (ESI) m/z calcd. for C28H40BrN7O2 [M+H]+: 586.2505. Found: 586.2493.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-chloro-4-fluorophenyl)-7-methoxyquinazolin-4-
amine (2a). General procedure for syntheses of 2a-o. A mixture of compound (5) (0.67 g, 1.86
mmol), di-tert-butyl 7-(3-chloropropyl)-1,4,7-triazonane-1,4-dicarboxylate (1.27g, 2.046 mmol,
1.1 eq.) and K2CO3 (0.9 g, 6.5 mmol, 3 eq.) was dissolved in DMF (30 mL). And the solution
was stirred at 130 °C for 6 h. Filtered to discard inorganic salt and concentrated in vacuo. The
resulting solid was purified by chromatography on silica (petroleum ether/EtOAc, 15:1) to afford
di-tert-butyl
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7-(3-((4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)propyl)-1,4,7-triazona
ne-1,4-dicarboxylate as a yellow solid(1.13 g, yield 89.8%).
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A mixture of above mentioned product (0.96 g, 1.39 mmol) was dissolved in CH3OH (45 mL)
and hydrochloric acid (12 mol/L, 15 mL) was added at room temperature with stirring. After
stirring for an additional 6 h, the resulted solution was concentrated in vacuo. And the solid was
re-dissolved in methanol. After that solution was filtered, the filter cake was washed with
methanol. The solid was dried under vacuum for 1 days to afford the pure yellow solid (yield,
66.3%). 1H NMR (400 MHz, D2O) δ 8.49 (s, 1H), 7.61-7.58 (d, J = 12.22 Hz, 2H), 7.50-7.48 (d,
J = 7.06 Hz, 1H), 7.34-7.28 (m, 2H), 7.08 (s 1H), 4.17-4.15 (t, J = 5.98 Hz, 2H), 3.99 (s, 3H),
3.53 (s, 1H), 3.15-3.08 (m, 8H), 2.89-2.81 (m, 10H), 2.02-1.98 (m, 2H). 13C NMR (101 MHz,
D2O) δ 157.25, 156.56, 154.13, 149.61, 147.79, 134.46, 132.63, 125.11, 123.46, 120.02, 116.47,
106.89, 103.27, 99.44, 68.42, 56.85, 51.48, 47.21, 43.46, 41.89, 23.76. HRMS (ESI) m/z calcd.
for C24H30ClFN6O2 [M+H]+: 489.2181. Found: 489.2176.
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Compound 2b-o were synthesized using similar route according to 2a.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-ethynylphenyl)-7-methoxyquinazolin-4-amine
(2b). Compound 2b was obtained as a yellow solid (yield 74.6%). 1H NMR (400 MHz, D2O) δ
8.51 (s, 1H), 7.67 (s, 1H), 7.39-7.35 (m, 3H), 7.21 (s, 1H), 4.20-4.17 (t, J = 5.71 Hz, 2H), 4.03 (s,
3H), 3.17-3.09 (m, 8H), 2.94-2.87 (m, 10H), 2.08 (s, 2H). 13C NMR (101 MHz, D2O) δ 157.36,
156.45, 149.57, 147.66, 135.86, 134.43, 130.05, 129.11, 126.62, 124.19, 122.00, 106.95, 103.28,
99.45, 82.64, 79.25, 68.36, 56.81, 51.43, 47.18, 43.44, 41.87, 23.73. HRMS (ESI) m/z calcd. for
C26H32N6O2 [M+H]+: 461.2665. Found: 461.2665.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-bromophenyl)-7-methoxyquinazolin-4-amine
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(2c). Compound 2c was obtained as a white solid (yield 67.5%). H NMR (400 MHz, D2O) δ
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8.52 (s, 1H), 7.56-7.54 (dd, J = 6.79, 2.2 Hz, 1H), 7.48 (s, 1H), 7.38-7.35 (m, 1H), 7.02-6.98 (t J
= 8.96, 6.48 Hz 2H), 4.10-4.08 (t, J = 5.98 Hz, 2H), 3.95 (s, 3H), 3.57 (s, 4H), 3.32-3.29 (t, J =
5.45 Hz, 4H), 3.01-2.99 (t, J = 5.71 Hz, 4H) 2.91-2.87 (t, J = 8.3 Hz, 2H) 2.04-2.01 (m, 2H). 13C
NMR (101 MHz, D2O) δ 157.12, 156.40, 149.62, 147.57, 137.04, 134.36, 130.20, 129.19,
125.65, 121.66, 106.89, 103.21, 99.35, 68.46, 56.84, 51.45, 47.18, 43.44, 41.87, 23.76. HRMS
(ESI) m/z calcd. for C24H31BrN6O2 [M+H]+: 515.1770. Found: 515.1763.
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6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(4-bromo-2-fluorophenyl)-7-methoxyquinazolin-4-
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amine (2d). Compound 2d was obtained as a light yellow solid (yield 69.8%). H NMR (400
MHz, D2O) δ 8.53 (s, 1H), 7.76-7.74 (dd, J = 6.51, 2.48 Hz, 1H), 7.59 (s, 1H), 7.47-7.43 (m,
1H), 7.12-7.08 (t, J = 6.86 Hz, 2H), 4.19-4.16 (t, J = 5.92 Hz, 2H), 4.00 (s, 3H), 3.56 (s, 4H),
3.32-3.29 (t, J = 4.97 Hz, 4H), 3.02-3.00 (t, J = 5.68 Hz, 4H), 2.95-2.91 (t, J = 7.34 Hz, 2H),
2.09-2,05 (m, 2H). 13C NMR (101 MHz, D2O) δ 158.65, 157.19, 154.77, 149.70, 148.13, 134.88,
128.73, 127.93, 122.44, 121.01, 119.72, 107.16, 103.42, 99.61, 68.21, 56.93, 51.53, 47.24, 43.44,
41.89, 23.75. HRMS (ESI) m/z calcd. for C24H30BrFN6O2 [M+H]+: 533.1676. Found: 533.1665.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-bromo-4-fluorophenyl)-7-methoxyquinazolin-4-
amin (2e). Compound 2e was obtained as a white solid (yield 76.7%). 1H NMR (400 MHz,
D2O) δ 8.51 (s, 1H), 7.68 (s, 1H), 7.54 (s, 1H), 7.44-7.42 (d, J = 8.19 Hz, 1H), 7.31-7.26 (d, J =
8.19 Hz, 1H), 7.19-7.15 (t, J = 8.53 Hz, 1H), 7.00 (s 1H), 4.13-4.10 (t, J = 5.92 Hz, 2H), 3.97 (s,
3H), 3.55 (s, 4H), 3.29-3.28 (d, J = 6.16 Hz, 4H), 2.98-2.96 (t, J = 5.68 Hz, 4H), 2.89-2.86 (t, J =
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7.82 Hz, 2H), 2.02-1.98 (m, 2H). C NMR (101 MHz, D2O) δ 157.03, 156.50, 154.98, 149.60,
147.72, 134.36, 132.86, 127.57, 123.88, 116.12, 107.94, 106.82, 103.23, 99.43, 68.45, 56.87,
51.51, 47.21, 43.47, 41.89, 23.78. HRMS (ESI) m/z calcd. for C24H30BrFN6O2 [M+H]+:
533.1676. Found: 533.1675.
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6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-methox
yquinazolin-4-amine (2f). Compound 2f was obtained as a yellow solid (yield 67.3%). 1H NMR
(400 MHz, D2O) δ 8.43 (s, 1H), 7.43 (d, J = 2.6 Hz, 1H), 7.30 (s, 1H), 7.26-7.14 (m, 2H), 6.93
(td, J = 8.7, 2.2 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.68 (d, J = 9.8 Hz, 1H), 6.63 (s, 1H), 6.44 (d,
J = 9.1 Hz, 1H), 4.24 (s, 2H), 4.05 (s, 2H), 3.58 (s, 4H), 3.44 (s, 3H), 3.31 (t, J = 5.4 Hz, 4H),
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3.01 (t, J = 5.6 Hz, 4H), 2.93 – 2.83 (m, 2H), 2.04 (d, J = 4.5 Hz, 2H). C NMR (101 MHz,
D2O) δ 163.46, 161.04, 155.40, 149.49, 149.01, 147.28, 138.08, 133.74, 129.98, 123.17, 121.65,
120.50, 113.90, 111.50, 106.38, 102.71, 98.74, 68.21, 67.68, 56.00, 51.63, 47.25, 43.44, 41.88,
24.01. HRMS (ESI) m/z calcd. for C31H36ClFN6O3 [M+H]+: 595.2600. Found: 595.2585.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(4-chloro-3-(trifluoromethyl)phenyl)-7-methoxy
quinazolin-4-amine (2g). Compound 2g was obtained as a yellow solid (yield 71.1%). 1H NMR
(400 MHz, D2O) δ 8.62 (s, 1H), 7.91 (d, J = 2.5 Hz, 1H), 7.77 – 7.68 (m, 1H), 7.57 (s, 1H), 7.28
(d, J = 8.8 Hz, 1H), 7.02 (s, 1H), 4.15 (t, J = 5.9 Hz, 2H), 3.98 (s, 3H), 3.58 (s, 4H), 3.32 (t, J =
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5.6 Hz, 4H), 3.06 – 2.88 (m, 6H), 2.11 – 2.01 (m, 2H). C NMR (101 MHz, D2O) δ 156.64,
149.69, 147.58, 135.11, 134.34, 131.35, 127.38, 126.94, 126.63, 126.09, 123.40, 120.79, 106.63,
102.96, 99.38, 68.61, 56.86, 51.43, 47.18, 43.46, 41.89, 23.69. HRMS (ESI) m/z calcd for
C25H30ClF3N6O2 [M+H]+: 539.2149. Found: 539.2142.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-7-methoxy-N-(3-(trifluoromethyl)phenyl)quinazolin-
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4-amine (2h). Compound 2h was obtained as a white solid (yield 74.9%). H NMR (400 MHz,
D2O) δ 8.51 (d, J = 1.1 Hz, 1H), 7.91 (s, 1H), 7.72 (d, J = 4.5 Hz, 1H), 7.56 (s, 1H), 7.52 (s, 2H),
6.94 (s, 1H), 4.11 (t, J = 5.3 Hz, 2H), 3.92 (s, 3H), 3.55 (s, 4H), 3.28 (s, 4H), 2.93 (d, J = 5.0 Hz,
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4H), 2.88-2.77 (m, 2H), 1.93 (d, J = 9.2 Hz, 2H). C NMR (101 MHz, D2O) δ 157.48, 156.42,
149.59, 147.87, 136.37, 134.69, 130.28, 129.82, 127.10, 125.00, 123.23, 122.30, 120.58, 106.84,
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103.16, 99.32, 68.42, 56.71, 51.28, 47.09, 43.41, 41.86, 23.64. HRMS (ESI) m/z calcd. for
C25H31F3N6O2 [M+H]+: 505.2539. Found: 505.2531.
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6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-chlorophenyl)-7-methoxyquinazolin-4-amine
(2i). Compound 2i was obtained as a white solid (yield 71.8%). 1H NMR (400 MHz, D2O) δ 8.50
(s, 1H), 7.51 (dd, J = 4.0, 1.8 Hz, 2H), 7.38 (dd, J = 8.1, 1.1 Hz, 1H), 7.22 (t, J = 8.1 Hz, 1H),
7.11 (dd, J = 8.0, 1.0 Hz, 1H), 6.96 (s, 1H), 4.09 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H), 3.56 (s, 4H),
3.30 (t, J = 5.6 Hz, 4H), 2.97 (t, J = 5.8 Hz, 4H), 2.92 – 2.81 (m, 2H), 1.99 (td, J = 11.2, 6.0 Hz,
2H). 13C NMR (101 MHz, D2O) δ 157.03, 156.29, 149.55, 147.63, 136.99, 134.58, 133.53,
129.98, 126.23, 122.77, 121.30, 106.86, 103.08, 99.44, 68.44, 56.77, 51.40, 47.17, 43.45, 41.87,
23.73. HRMS (ESI) m/z calcd. for C24H31ClN6O2 [M+H]+: 471.2275. Found: 471.2260.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-7-methoxy-N-(3-methoxyphenyl)quinazolin-4-amine
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(2j). Compound 2j was obtained as a yellow solid (yield 69.6%). H NMR (400 MHz, D2O) δ
8.46 (s, 1H), 7.53 (s, 1H), 7.24 (t, J = 8.5 Hz, 1H), 7.08 (dd, J = 4.4, 2.2 Hz, 2H), 6.97 (s, 1H),
6.77 (dd, J = 8.3, 1.7 Hz, 1H), 4.10 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H), 3.72 (s, 3H), 3.56 (s, 4H),
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3.29 (t, J = 5.7 Hz, 4H), 2.97 (t, J = 5.8 Hz, 4H), 2.92-2.79 (m, 2H), 2.11-1.88 (m, 2H). C
NMR (101 MHz, D2O) δ 158.82, 157.16, 156.18, 149.36, 147.61, 136.91, 134.42, 129.80,
115.98, 111.84, 109.27, 106.89, 103.25, 99.36, 68.37, 56.73, 55.33, 51.35, 47.14, 43.43, 41.87,
23.68. HRMS (ESI) m/z calcd. for C25H34N6O3 [M+H]+: 467.2771. Found: 467.2760.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-fluorophenyl)-7-methoxyquinazolin-4-amine
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(2k). Compound 2k was obtained as a yellow solid (yield 75.1%). H NMR (400 MHz, D2O) δ
8.50 (s, 1H), 7.59 (s, 1H), 7.38-7.32 (m, 2H), 7.32-7.27 (m, 1H), 7.05-6.94 (m, 2H), 4.12 (t, J =
6.0 Hz, 2H), 3.94 (s, 3H), 3.56 (s, 4H), 3.29 (dd, J = 10.8, 4.8 Hz, 4H), 2.99 (t, J = 5.8 Hz, 4H),
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2.94-2.83 (m, 2H), 2.12-1.92 (m, 2H). C NMR (101 MHz, D2O) δ 163.20, 160.78, 157.17,
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156.41, 149.57, 147.57, 137.13, 134.34, 130.33, 119.14, 113.38, 110.56, 106.78, 103.12, 99.31,
68.47, 56.76, 51.33, 47.12, 43.39, 41.84, 23.67. HRMS (ESI) m/z calcd. for C24H31FN6O2
[M+H]+: 455.2571. Found: 455.2558.
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6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(3-iodophenyl)-7-methoxyquinazolin-4-amine (2l).
Compound 2l was obtained as a yellow solid (yield 72.7%). 1H NMR (400 MHz, D2O) δ 8.49 (s,
1H), 7.84 (t, J = 1.6 Hz, 1H), 7.49 (s, 1H), 7.43 (dd, J = 12.8, 4.9 Hz, 2H), 7.00 (d, J = 8.0 Hz,
1H), 6.97 (s, 1H), 4.10 (t, J = 5.9 Hz, 2H), 3.98 (s, 3H), 3.55 (s, 4H), 3.28 (dd, J = 9.9, 4.1 Hz,
4H), 2.96 (t, J = 5.7 Hz, 4H), 2.89-2.80 (m, 2H), 1.98 (td, J = 11.2, 5.9 Hz, 2H). 13C NMR (101
MHz, D2O) δ 156.81, 156.24, 149.56, 147.54, 136.81, 134.67, 131.23, 130.12, 122.03, 106.80,
103.13, 99.42, 93.40, 68.47, 57.41, 56.89, 51.50, 47.20, 43.47, 41.86, 23.81, 16.78. HRMS (ESI)
m/z calcd. for C24H31IN6O2 [M+H]+: 563.1631. Found: 563.1620.
6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(2,4-dichlorophenyl)-7-methoxyquinazolin-4-amine
(2m). Compound 2m was obtained as a yellow solid (yield 70.4%). 1H NMR (400 MHz, D2O) δ
8.47 (s, 1H), 7.70 (s, 1H), 7.56 (d, J = 2.3 Hz, 1H), 7.42 (d, J = 8.6 Hz, 1H), 7.33 (dd, J = 8.6,
2.3 Hz, 1H), 7.20 (s, 1H), 4.17 (t, J = 5.9 Hz, 2H), 4.03 (s, 3H), 3.56 (s, 4H), 3.31 (t, J = 5.6 Hz,
4H), 3.03 (t, J = 5.8 Hz, 4H), 2.98-2.87 (m, 2H), 2.11 (td, J = 11.1, 6.0 Hz, 2H). 13C NMR (101
MHz, D2O) δ 159.16, 157.14, 149.67, 148.20, 135.08, 134.06, 131.61, 129.84, 128.09, 107.14,
103.85, 99.77, 68.16, 56.95, 49.71, 48.11, 44.02, 42.06, 23.26. HRMS (ESI) m/z calcd. for
C24H30Cl2N6O2 [M+H]+: 505.1886. Found: 505.1876.
5-((6-(3-(1,4,7-triazonan-1-yl)propoxy)-7-methoxyquinazolin-4-yl)amino)-2-fluorobenzon
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itrile (2n). Compound 2n was obtained as a yellow solid (yield 69.6%). H NMR (400 MHz,
D2O) δ 8.56 (s, 1H), 7.94 (dd, J = 5.1, 2.4 Hz, 1H), 7.83 (dd, J = 8.6, 4.2 Hz, 1H), 7.64 (s, 1H),
7.29 (t, J = 8.9 Hz, 1H), 7.12 (s, 1H), 4.20 (t, J = 5.6 Hz, 2H), 4.00 (s, 3H), 3.57 (s, 4H), 3.31 (d,
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J = 5.2 Hz, 4H), 3.04 (t, J = 5.5 Hz, 4H), 2.99-2.92 (m, 2H), 2.11 (s, 2H). 13C NMR (101 MHz,
D2O) δ 161.57, 159.01, 157.38, 156.78, 149.74, 148.06, 135.04, 132.86, 130.79, 127.76, 116.90,
113.70, 107.00, 103.21, 100.31-99.43, 68.48, 56.89, 51.46, 47.22, 43.46, 41.90, 23.78. HRMS
(ESI) m/z calcd. for C25H30FN7O2 [M+H]+: 480.2523. Found: 480.2511.
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6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(4-bromo-2-ethylphenyl)-7-methoxyquinazolin-4-a
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mine (2o). Compound 2o was obtained as a yellow solid (yield 71.8%). H NMR (400 MHz,
D2O) δ 8.40 (s, 1H), 7.74 (s, 1H), 7.55 (s, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.19 (s, 1H), 7.10 (d, J =
8.4 Hz, 1H), 4.15 (s, 2H), 4.02 (s, 3H), 3.57 (s, 4H), 3.30 (dd, J = 12.7, 7.0 Hz, 4H), 3.03 (t, J =
5.6 Hz, 4H), 2.95 (dd, J = 9.4, 6.4 Hz, 2H), 2.52 (q, J = 7.5 Hz, 2H), 2.09 (d, J = 5.7 Hz, 2H),
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1.00 (t, J = 7.5 Hz, 3H). C NMR (101 MHz, D2O) δ 159.62, 156.87, 149.64, 148.21, 143.90,
134.84, 132.78, 132.53, 129.81, 122.22, 107.13, 103.93, 99.88, 68.18, 56.79, 51.53, 47.25, 43.42,
41.89, 24.04, 23.75, 13.42. HRMS (ESI) m/z calcd. for C26H35BrN6O2 [M+H]+: 543.2083. Found:
543.2072.
4-chloro-7-methoxyquinazolin-6-yl acetate (4). A mixture of commercially available
6-hydroxy-7-methoxyquinazolin-4(3H)-one (3, 1 g, 5.2 mmol) and pyridine (4 mL) was
dissolved in acetic anhydride (20 mL). The solution was stirred at 100 °C for 6 h. Cold water
(200 mL) was added to the solution to precipitate the product and stirring was continued for 20
min after the solution was cooled down to room temperature. The solid was filtered under
vacuum and washed with water. The solid was dried under vacuum for 2 days to afford the white
solid (yield 74%).
A mixture of product above (1 g, 4.27 mmol) and DMF (75 μL) was dissolved in SOCl2 (15
mL). The solution was stirred at 90 °C for 2 h after, it was cooled down to room temperature.
The reaction mixture was extracted between water and CHCl2. The aqueous phase was further
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extracted with CHCl2 (2×), and the combined organic phases were dried over anhydrous sodium
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sulfate and concentrated in vacuo to afford a yellow solid (yield 98.4%). H NMR (400MHz,
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DMSO) δ 8.51 (s, 1H), 7.82 (s, 1H), 7.36 (s, 1H), 3.94 (s, 3H), 2.31 (s, 3H). C NMR (101
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MHz, D2O) δ 169.07, 164.29, 156.34, 159.15, 149.17, 144.13, 118.26, 115.83, 107.9, 55.85,
20.33. HRMS (ESI) m/z calcd. for C11H9ClN2O3 [M+H]+: 397.0396. Found: 397.0391.
4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-ol (5a-o). General procedure
for syntheses of 5a-o. A mixture of 4-chloro-7-methoxyquinazolin-6-yl acetate (4) (1.1g,
4.36mmol) and 3-chloro-4-fluoroaniline (0.96g, 6.62 mmol, 1.2 eq.) was dissolved in CH3CN
(150mL). The solution was stirred at 90 °C for 1 h. The sediment was filtered under vacuum and
washed with acetonitrile. The solid was dried under vacuum for 1 day to afford the yellow solid.
A mixture of product above (1 g, 2.77 mmol) and NH3·H2O (2 mL) was dissolved in CH3OH
(40 mL). The solution was stirred at 75 °C for 2 h. The sediment was filtered under vacuum and
washed with methanol. The solid was dried under vacuum for 1 day to give 5a as yellow solid
(yield 72%).1H NMR (400 MHz, DMSO) δ 9.69 (s, 1H), 9.47 (s, 1H), 8.47 (s, 1H), 8.22-8.20
(dd, J = 6.87, 2.75 Hz, 1H), 7.84-7.82 (m, 1H), 7.77 (s, 1H), 7.41-7.38 (t, J = 9.02 Hz, 1H), 7.21
(s, 1H), 3.97 (s, 3H).
Cell Lines and Reagents. The human epidermal carcinoma cell line A431 and human
non-small cell lung cancer cell line A549 Cell lines were obtained from China Infrastructure of
Cell Line Resources. The cells were maintained at 37 °C in 5% CO2 incubator in DMEM
(Corning) containing 10% fetal bovine serum (Corning). The EGFR gene of every cell line was
sequenced before use. Gefitinib and lapatinib were purchased from Damas-beta.
Cell Proliferation Assay. The human epidermal carcinoma cell line A431 and human
non-small cell lung cancer cell line A549 were used to evaluate the potency of synthesized
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analogues in cell-based level. Assays were performed using the MTT assay. A431 and A549
cells were seeded in density of 8000 cells/well and 6000 cells/well, respectively, in 96-well
plates (Corning) for 12 h. Duplicate wells were treated with test or reference compounds for 72 h
at various concentrations or DMSO (Sigma) as control. Plates were incubated at 37 °C in 5%
CO2 atmosphere. Cell proliferation was measured according to the manufacturer’s protocol. The
IC50 was calculated using GraphPad Prism 5.0.
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Kinase Inhibition Assay. The assays used an ULight™-labeled peptide substrate and an
appropriate Europium-labeled anti-phospho antibody. EGFR, HER2 and EGFRT790M were
purchased from Carna, and ATP was purchased from Promega. Compounds with concentration
ranging from 2.5 µM to 0.04 nM and kinases were added into the assay plate and centrifuged for
1 min to mix them. Then ATP-substrate mixture were added to start the enzyme reaction. The
assays were conducted at room temperature for 1 hour and stopped by EDTA buffer. Finally
detected solution was added and incubated for 1 hour. For HPE (hundred percent effect) wells no
kinase or compound but ATP, substrate and 1% DMSO were contained. ZPE (zero percent
effect) wells contained no compound but kinase, ATP, substrate and 1% DMSO were contained.
The IC50 was calculated using GraphPad Prism 5.0. Compound inhibition rate= (ZPE
-“compound” reading) / (ZPE-HPE) *100%.
Cellular ATP Measurement. Cellular ATP was detected with the ATP assay kit (BioAssay)
according to the manufacturer’s instructions. Briefly, 4000 cells (A549) in per well were plated
in white opaque 96-well plates (Corning). After incubated for 12 h, the cells were treated with
compound or DMSO (as negative control) for 24 h or 72 h. The culture medium was removed
immediately before adding the reconstitution reagent. For each 96-well plates, 95 μL Assay
Buffer with 1 μL Substrate and 1 μL ATP Enzyme were mixed. 90 μL Reconstituted Reagent
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