Design and Evaluation of Potent EGFR Inhibitors through the

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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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Journal of Medicinal Chemistry

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*,†,‡ †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

1 ACS Paragon Plus Environment

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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 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 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. 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.

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% 5 ACS Paragon Plus Environment


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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.

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

R

Compound

R

Compound

a

f

k

b

g

l

c

h

m

d

i

n

e

j

o

R

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 6 ACS Paragon Plus Environment

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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 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 High Expressing EGFR.a Compound

Anti-proliferation IC50 (μM)b A549

1a

1.01 ± 0.04

20.21 ± 3.24

1b

1.11 ±0.22

1c

Anti-proliferation IC50 (μM) A431

A549

2a

1.314 ± 0.356

＞50

28.55 ± 4.91

2b

2.231 ± 0.544

＞50

1.047 ± 0.014

14.68 ± 2.26

2c

2.322 ± 0.812

34.27 ± 3.61

1d

1.839 ± 0.233

15.195 ± 1.036

2d

0.561 ± 0.051

16.08 ± 2.34

1e

1.578 ± 0.411

11.84 ± 2.31

2e

1.064 ± 0.341

34.27 ± 1.26

1f

0.958 ± 0.034

3.4 ± 0.4

2f

0.442 ± 0.068

7.25 ± 0.89

1g

1.707 ± 0.129

5.35 ± 1.21

2g

2.98 ± 0.26

6.23 ± 0.91

1h

1.952 ± 0.387

15.73 ± 3.66

2h

1.562 ± 0.183

37.3 ± 3.3

1i

1.424 ± 0.481

27.1 ± 3.49

2i

1.125 ± 0.312

＞50

1j

3.21 ± 0.53

＞50

2j

8.526 ± 0.569

＞50

1k

1.86 ± 0.44

＞50

2k

7.914 ± 0.063

＞50

1l

2.539 ± 0.512

14.53 ± 2.31

2l

2.215 ± 0.991

26.16 ± 2.71

1m

4.385 ± 0.302

23.79 ± 6.14

2m

4.077 ± 0.517

44.24 ± 2.82

1n

6.369 ± 0.981

＞50

2n

12.69 ± 0.71

＞50

1o

5.653 ± 0.737

＞50

2o

18.77 ± 1.26

＞50

Gefitinib

2.47 ± 0.07

aThe

A431

Compound

11.08 ± 2.29

Lapatinib

2.66 ± 0.27

data are the mean ± SD of at least three independent experiments.

bThe

3.64 ± 0.77

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 8 ACS Paragon Plus Environment

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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.

Table 3. In Vitro Enzymatic Inhibitory activity of Selected Compounds against Different Statues of EGFR and HER2.a Compound

aThe

Kinase inhibition IC50 (nM)

1a

EGFRwt 6.6 ± 0.4

HER2 NTb

EGFRT790M NT

2a

8.3 ± 0.6

NT

NT

2d

28.2 ± 5.3

NT

NT

2e

5.6 ± 0.8

NT

NT

1f

1.4 ± 0.2

2.1 ± 0.6

16.5 ± 2.3

2f

0.3 ± 0.1

9.6 ± 0.4

40.6 ± 4.1

Gefitinib

5.8 ± 0.2

NT

148.7 ± 6.3

Lapatinib

10.85 ± 0.11

32.7 ± 1.4

> 2500

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).



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% 11 ACS Paragon Plus Environment


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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).

Table 4. IC50 and inhibition of compounds and positive drugs against L929 at the concentration of 1 μM.a

aThe

1f

Gefitinib

Lapatinib

Inhibition at the concentration of 1 μM

37.63%

25.95%

35.03%

IC50 (μM)

2.74 ± 0.42

1.98 ± 0.13

2.05 ± 0.78

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 the tumor site with compound 1f treatment, as shown in Figure 4E.

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. 13 ACS Paragon Plus Environment


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.

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 14 ACS Paragon Plus Environment

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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.

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 visualization. 1H 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



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%). 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 solid (yield 68.4%). 1H 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 (m, 8H), 2.93-2.85 (m, 10H), 2.02-2.05 (m, 2H).

13C

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 olin-4-amine (1b). Compound 1b was obtained as a light yellow solid (yield 70.2%). 1H 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 (m, 8H), 2.93-2.86 (m, 10H), 2.05-2.03 (m, 2H).

13C

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. 6-(3-(1,4,7,10-tetraazacyclododecan-1-yl)propoxy)-N-(3-bromophenyl)-7-methoxyquinazo lin-4-amine (1c). Compound 1c was obtained as a light yellow solid (yield 66.8%). 1H 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 Hz, 2H), 4.00(s, 3H), 3.16-3.09 (m, 8H), 2.91-2.83 (m, 10H) 2.04-2.01 (m, 2H). 13C 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 (m, 2H). 13C 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. 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 Hz, 1H), 4.18 (s, 2H), 4.04 (s, 2H), 3.41 (s, 3H), 3.22-2.74 (m, 18H), 2.01 (s, 2H).

13C

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, 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). 13C 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. 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, 10.2 Hz, 8H), 3.01-2.67 (m, 10H), 2.01 (dt, J = 8.1, 5.6 Hz, 2H). 13C 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) quinazolin-4-amine (1j). Compound 1j was obtained as a light yellow solid (yield 74.5%). 1H 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 lin-4-amine (1k). Compound 1k was obtained as a light yellow solid (yield 67.5%). 1H NMR



(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. 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, 5.2 Hz, 2H).

13C

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 nazolin-4-amine (1m). Compound 1m was obtained as a light yellow solid (yield 67.8%). 1H 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 quinazolin-4-amine (1o). Compound 1o was obtained as a white solid (yield 69.8%). 1H 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 Hz, 3H). 13C 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-4amine (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



Page 22 of 39

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%). 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. 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 (2c). Compound 2c was obtained as a white solid (yield 67.5%). 1H 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. 6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(4-bromo-2-fluorophenyl)-7-methoxyquinazolin-4amine (2d). Compound 2d was obtained as a light yellow solid (yield 69.8%). 1H 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-4amin (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 = 7.82 Hz, 2H), 2.02-1.98 (m, 2H). 13C 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.



Page 24 of 39

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), 3.01 (t, J = 5.6 Hz, 4H), 2.93 – 2.83 (m, 2H), 2.04 (d, J = 4.5 Hz, 2H).

13C

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 = 5.6 Hz, 4H), 3.06 – 2.88 (m, 6H), 2.11 – 2.01 (m, 2H).

13C

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)quinazolin4-amine (2h). Compound 2h was obtained as a white solid (yield 74.9%). 1H 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, 4H), 2.88-2.77 (m, 2H), 1.93 (d, J = 9.2 Hz, 2H). 13C 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. 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 (2j). Compound 2j was obtained as a yellow solid (yield 69.6%). 1H 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), 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).

13C

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 (2k). Compound 2k was obtained as a yellow solid (yield 75.1%). 1H 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), 2.94-2.83 (m, 2H), 2.12-1.92 (m, 2H).

13C

NMR (101 MHz, D2O) δ 163.20, 160.78, 157.17,



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. 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 itrile (2n). Compound 2n was obtained as a yellow solid (yield 69.6%). 1H 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. 6-(3-(1,4,7-triazonan-1-yl)propoxy)-N-(4-bromo-2-ethylphenyl)-7-methoxyquinazolin-4-a mine (2o). Compound 2o was obtained as a yellow solid (yield 71.8%). 1H 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), 1.00 (t, J = 7.5 Hz, 3H). 13C 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



Page 28 of 39

extracted with CHCl2 (2×), and the combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo to afford a yellow solid (yield 98.4%). 1H NMR (400MHz, DMSO) δ 8.51 (s, 1H), 7.82 (s, 1H), 7.36 (s, 1H), 3.94 (s, 3H), 2.31 (s, 3H).

13C

NMR (101

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. 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



was added to each well. Luminescence were determined by a microplate reader (Flexstation 3) within 1 min after adding reconstitution reagent. Flow Cytometry Analysis. A549 Cells (5×105 cells/mL) were seeded in six-well plates and treated with compounds at different concentrations for 72 h. The cells were then harvested by trypsinization and washed twice with cold PBS. After centrifugation and removal of the supernatants, cells were resuspended in 300 μL of binding buffer which was then added to 5 μL of annexin V-FITC and 10 μL of PI, then the cells were incubated at room temperature for another 15 min in the dark. The stained cells were analyzed by a flow cytometer (FACS-MolFlo XDP Beckman Coulter, USA). The result was analyzed using summit 5.2. Mouse Tumor Xenograft Efficacy Study. The A549 is considered as a highly metastatic cancer cell line.36 Six-week-old female BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals. Therefore, an exponential rapid increase is observed in the tumor volume for the control group. A549 NSCLC xenografts were established by 2.0 × 106 cells subcutaneously inoculated in nude mice. Treatments were initiated when tumors reached a mean group size of 200-400 mm3. All of 21 mice were randomized to control (30 mL/kg, water, ig administration), Gefitinib (30 mg/kg, ig administration), 1f (30 mg/kg, ig administration), every 2 days for 19 days. The size of tumors were measured individually twice a week with microcalipers. Tumor volume (V) was calculated as V = length × width2 /2. Tumor growth inhibition (TGI) was calculated using the following formula: TGI = [1-(T-T0)/(C-C0)] × 100%, where T and T0 are the mean tumor weight on a specific experimental day and on the first day of treatment, respectively, for the


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experimental groups, and likewise where C and C0 are the mean tumor weight for the control group. HE Staining. HE staining was carried out according to a previous publication.37 First, the sections were hydrated and then the slide was dipped into a Coplin jar containing Mayer’s hematoxylin and agitated for 30 s. After rinsing the slide in H2O for 1 min, it was stained with 1% eosin Y solution for 10-30 s with agitation. Subsequently, the sections were dehydrated by using 95% alcohol and 100% alcohol twice for 30 s each, and then the alcohol was extracted using xylene twice. Finally, one or two drops of mounting medium was added and covered with a coverslip. Molecular Docking. All the calculations were carried out on a Dell PC with Windows 7.0 system using OMEGA (version 2.4.6; OpenEye Scientific Software, Inc.: Santa Fe, USA, 2016) and Hybrid (version 3.0.1; OpenEye Scientific Software, Inc.: Santa Fe, USA), 2016. The crystal structural data of EGFR kinase domain complexed with Gefitinib (PDB code: 2ITY) Lapatinib (PDB code: 1XKK) was obtained from RCSB Protein Data Bank. Molecular docking was carried out as previous reported.38,39

Associated content Supporting Information. The supporting information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.xxxxxxx. 1H

NMR, 13C NMR, HRMS spectra, and HPLC analysis of all compounds (PDF)

Compound characterization checklist (XLS) Molecular formula strings and some data (CSV)



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Author information Corresponding Author *86 10 64413899. E-mail: [email protected], [email protected] ORCID Renzhong Qiao: 0000-0002-6672-9609 Chao Li: 0000-0002-0320-5509 Author Contributions Yilan Ju and Jintao Wu contributed equally to this work. Notes The authors declare no competing financial interests.

Acknowledgment This work was supported by the National Natural Science Foundation of China

[Nos

21672021, 21572018, 21372024 and 21232005]; the State Key Laboratory of Natural and Biomimetic Drugs; the Fundamental Research Funds for the Central Universities (PYBZ1824); Beijing Municipal Administration of Hospitals' Youth Programme [No 20161002]; Xu-Xia Fund for Young Scholars [No XX-201703].

Abbreviations Used HER2, human epidermal growth factor receptor 2; MerTK, mer tyrosine kinase; TGI, tumor growth inhibition; SD, standard deviation; H&E, hematoxylin and eosin; DMEM, Dulbecco's modified eagle medium.


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(15) Sequist, L. V.; Martins, R. G.; Spigel, D.; Grunberg, S. M.; Spira, A.; Jänne, P. A.; Joshi, V. A.; McCollum, D.; Evans, T. L.; Muzikansky, A.; Kuhlmann, G. L.; Han, M.; Goldberg, J. S.; Settleman, J.; Iafrate, A. J.; Engelman, J. A.; Haber, D. A.; Johnson, B. E.; Lynch, T. J. First-line gefitinib in patients with advanced non–small-cell lung cancer harboring somatic egfr mutations. J. Clin. Oncol. 2008, 26, 2442-2449. (16) Chen, L.; Fu, W.; Zheng, L.; Liu, Z.; Liang, G. Recent progress of small-molecule epidermal growth factor receptor (EGFR) inhibitors against C797S resistance in non-small-cell lung cancer. J. Med. Chem. 2017, 61, 4290-4300. (17) Song, Z.; Ge, Y.; Wang, C.; Huang, S.; Shu, X.; Liu, K.; Zhou, Y.; Ma, X. Challenges and

perspectives

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Design and Evaluation of Potent EGFR Inhibitors through the

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