Synthesis and evaluation of osimertinib derivatives as potent EGFR inhibitors

Bioorganic & Medicinal Chemistry 25 (2017) 4553–4559

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Synthesis and evaluation of osimertinib derivatives as potent EGFR inhibitors Hongying Gao a,c,d, Zimo Yang b,d, Xinglin Yang b, Yu Rao b,⇑ a

Tsinghua University-Peking University Joint Center for Life Sciences, Beijing 100084, PR China MOE Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, PR China c MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, PR China b

a r t i c l e

i n f o

Article history: Received 25 February 2017 Revised 29 May 2017 Accepted 4 June 2017 Available online 8 July 2017 Keywords: Osimertinib N-Oxide metabolites Fluoride Non-small cell lung cancer Epidermal growth factor receptor-tyrosine kinase inhibitors

a b s t r a c t Osimertinib has been identified as a promising therapeutic drug targeting for EGFR T790M mutant nonsmall cell lung cancer (NSCLC). A new series of N-oxidized and fluorinated osimertinib derivatives were designed and synthesized. The cellular anti-proliferative activity, kinase inhibitory activity and the activation of EGFR signaling pathways of 1–6 in vitro were determined against L858R/T790M and wild-type EGFR, the antitumor efficacy in NCI-H1975 xenografts in vivo were further studied. Compound 2, the newly synthesized N-oxide metabolite in N,N,N0 -trimethylethylenediamine side chain of osimertinib, showed a comparable kinase selectivity in vitro and a slightly better antitumor efficacy in vivo to osimertinib, making it valuable and suitable for the potential lung cancer therapy. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction The epidermal growth factor receptor (EGFR) is regarded as a rational target for cancer therapy since it is commonly expressed at a high level in the pathogenesis and progression of a variety of carcinoma types.1,2 EGFR tyrosine kinases has been implicated at the head of a complex signaling cascade that modulates cell proliferation, maturation, survival, metastasis, migration, adhesion, differentiation and angiogenesis.3,4 Mutations of the EGFR tyrosine kinase domain have been recently discovered in clinical carcinomas. In non-small cell lung cancer (NSCLC), EGFR mutations are identified in 10–30% of patients,5,6 and common EGFR alterations include exon 19 deletions and the L858R point mutation in exon 21.7 Recently, selective small-molecule EGFR tyrosine kinase inhibitors (EGFR TKI) are becoming the evidence-based first-line therapy for advanced NSCLC that harbors sensitizing EGFR mutations (exon 19 deletion, L858R substitutions in exon 21). Although patients with sensitizing EGFR mutations could achieve good responses to therapy with the first-generation EGFR TKIs, such as gefitinib and erlotinib,8–10 most patients acquired drug resistance to EGFR TKI within 1 year treatment, which is mostly driven in about 60% of cases by a second-site EGFR T790M point mutation ⇑ Corresponding author. d

E-mail address: [email protected] (Y. Rao). These authors contributed equally.

http://dx.doi.org/10.1016/j.bmc.2017.06.004 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.

occurring within exon 20.11,12 Although the second-generation EGFR TKIs, such as afatinib, neratinib and dacomitinib, demonstrated a promising activity against T790M in preclinical models, they have failed to overcome T790M-mediated resistance in patients due to dose-limiting toxicity resulting from nonselective inhibition of wild-type (WT) EGFR.13,14 Osimertinib (AZD9291), an oral, novel mono-anilino-pyrimidine third-generation EGFR TKI, has been developed to selectively target the T790M mutation EGFR in a covalent irreversible manner.14–17 The tablet formulation of osimertinib (TagrissoTM) has been granted accelerated approval by United States Food and Drug Administration (FDA) in November 2015, for the therapy of patients with metastatic EGFR T790M mutation-positive NSCLC who have progressed on or after the first and second generation of EGFR TKI therapy. Currently reports have indicated that osimertinib undergoes extensive metabolism in the body, leading to low levels of the parent compound in feces and urine,18 and it is metabolized to yield at least two circulating metabolites, AZ5104 and AZ7550, with major metabolic reactions N-demethylation (Fig. 1). For these two metabolites, AZ5104 displayed a comparable or greater potency to osimertinib in vitro, but with a lower kinase selectivity for WT and mutant EGFR. In contrast, AZ7550 showed a decreased potency toward the mutant and WT EGFR in vitro, but offered a similar selectivity profile to osimertinib.18,19 Drug metabolism research is one of the most important steps in new drug developments. It plays a critical role in drug discovery

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O

H N N

N N O O

H N

N

NH

HN

AZ 5104

N

N

N

Fluorine, the most electronegative element, will decrease electron density of indole, which may protect the indole from N-demethylation. In addition, the binding mode of osimertinib with EGFRT790M (Fig. S1A) shows that indole motif was buried in protein pocket. Introduction of fluorine in this motif may add new polar contacts which may increase binding affinity towards target protein. 3. Results and discussion 3.1. Synthesis and characterization

N

NH

O

N

O

1 (AZD 9291)

O

N N

N NH

H N

NH

N

AZ 7550 Fig. 1. Chemical structures of the known N-demethylated metabolites of osimertinib.

The N-oxide metabolites of osimertinib were readily synthesized via oxidation of AZD9291 with m-CPBA, as shown in Scheme 1. Fluorinated N-Methylindoles were used for synthesis of fluorinated derivatives of osimertinib according to literature reported procedures (Scheme 1).29 3.2. In vitro anti-proliferative activity assay on tumor cell line and non-tumor cell line

process, including modification drug structures, identifying candidates with good characteristics, designing hard drugs and soft drugs, etc.20–22 It can accelerate the process of drug development and also help directly discover the new chemical entity.23 Fluorinated compounds comprise a substantial proportion in the therapeutic drugs.24,25 To introduce fluorine or fluorinated substitutes in the small molecule is an important strategy for structure-based medicinal chemistry,26 which can modulate the physicochemical and pharmacokinetic properties to improve bioavailability, or alter the conformation of a molecule to enhance the selectivity and binding affinity to target proteins, or block metabolically labile sites to increase the metabolic stability of drugs.27,28 Dealkylation and oxidation (mainly by cytochrome P450, family 3, subfamily A, also known as CYP3A) are the main metabolic pathways of osimertinib, the biologic activity of the N-demethylation metabolites of osimertinib have been reported by Finlay et al.18 Although they also mentioned that a small amount of the side chain N-oxide was evident at low levels in plasma samples of rat and mouse, there is no any structure identification and pharmacological studies in details. Thus, in this study, we designed and synthesized a new series of osimertinib derivatives, including the N-oxide metabolite-based derivatives and fluorinated derivatives. To the best of our knowledge, this is the first time to chemically synthesis and evaluation of the N-oxide metabolite-based and fluorinated derivatives of osimertinib

To evaluate the pharmacologic activity of the novel synthesized derivatives 1–6 against L858R/T790M EGFR and WT EGFR, we investigated their anti-proliferation activities on human lung cancer NCI-H460 (WT EGFR), PC9 (Exon 19 deletion EGFR) and NCIH1975 (L858R/T790M EGFR) cell lines in vitro by MTS (Promega, G5421) method (Table 1). As shown in Table 1, the fluoride compound 3 (IC50 = 15.3 nM; IC50 = 8.1 nM; IC50 = 429.5 nM) displayed higher levels of phenotype potency than other derivatives in mutant and WT EGFR cell lines. While the N-oxide compound 2 (IC50 = 17.4 nM; IC50 = 16.1 nM; IC50 = 611.2 nM) showed higher anti-proliferation activities than other two fluorinated N-oxide derivatives in mutant and WT EGFR cell lines, but lower than compound 3, In Table S1, it was showed that compound 2 with a lower QP log Po/w (4.6) than compound 3 (5.3), this will decrease its cell permeability in vitro, maybe that is the main reason for the decreased anti-proliferation activity in vitro. Furthermore, the activity of the fluorinated N-oxide molecules (5 and 6) almost decreased 2–3-fold in WT EGFR cell lines, and 4–9-fold in mutant EGFR cell lines to osimertinib, which may be due to the changes of the integrated physicochemical properties (Table S1). In addition, in order to test the toxicity of the compound 1–6, the non-tumor Beas-2B cell line was used. The result showed that although the fluoride compound 3 displayed a higher anti-tumor activity than other derivatives on human lung cancer cell lines, it also possesses a higher toxicity on human non-tumor cell line (IC50 = 7.9 mM for Beas-2B, Table 1). The N-oxide compound 2 showed a similar toxicity levels to osimertinib on human nontumor Beas-2B cell line (15.1 mM vs. 14.9 mM).

2. Design

3.3. In vitro kinase activity assay

As shown in Fig. 1, the N-demethylation metabolic reactions mostly happened in N,N,N0 -trimethylethylenediamine side chain and the N-Methylindole motif of osimertinib. Therefore, in our study, we mainly designed and synthesized two kinds of novel osimertinib derivatives (Fig. 2): 1. Side chain N-oxide metabolites of osimertinib. On the one hand, introduction of N-oxide in the N,N,N0 -trimethylethylenediamine side chain of osimertinib would change the polarity of the compound, increase the solubility and further improve its bioavailobility. On the other hand, it was reported that the metabolite with N-demethylation in N,N,N0 trimethylethylenediamine side chain showed a decreased activity toward the mutant and WT EGFR cell lines,18 therefore, to some extent, the side chain N-oxide modification would decrease the N-demethylation in vivo. 2. Fluorinated derivatives in indole motif.

In terms of the anti-proliferation potency of compound 1–6 in mutant and WT EGFR cell lines, the kinase inhibitory activity of compound 1–6 on L858 R/T790M and WT EGFR were simultaneously detected in vitro (Table 2) using Z0 -LYTETM kinase assay kit (Invitrogen, PV3193). Compound 2 potently inhibited the kinase activity of L858R/T790M and WT EGFR with IC50 values of 6.4 and 317.6 nM respectively, which is similar to those of osimertinib and showed an equal kinase selectivity for L858R/T790M EGFR over WT EGFR (49.9 versus 49.3). Compound 3 has a good kinase inhibitory activity for L858R/T790M EGFR (IC50 = 4.9 nM), more nearly to osimertinib (IC50 = 4.6 nM), but it processes a poor kinase selectivity between L858R/T790M and WT EGFR (34.3), The decreased selectivity may be due to the introduction of fluorine in indole, which decreased the electron density of indole, and further

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O

O

N

+

O

H N N

N

N NH

O

N

O

+ N

N

F

NH

O

N

N

H N

N O F

NH

N+

N N

N NH

O

N

F

4

O

H N N

O

N N

N N

3

2 O

H N

N N

N

NH

O

O

H N

N

5

N F

6

Fig. 2. Chemical structures of newly designed derivatives of osimertinib.

O

O

H N

m-CPBA

N

N

O

N O

H N

N

NH

N

N N O

N

1 (AZD9291)

N

NH

N

2 (I.Y. 85%) O

N

N

O

F

N N

N

reference 29

O

H N

NH

m-CPBA

O

F

3 5-F (I.Y. 70%) 4 7-F (I.Y. 75%)

N N

N N O

N

H N

NH

N

5 5-F (I.Y. 75%) 6 7-F (I.Y. 80%)

F

Scheme 1. Preparation of osimertinib derivatives. Reagents and conditions: 1.0 equiv. m-CPBA, DCM, 0 °C, 30 min.

Table 1 Anti-proliferation activity of 1–6 against NCI-H460 (WT EGFR), PC9 (Exon 19 deletion EGFR), NCI-H1975 (L858R/T790M EGFR) and non-tumor Beas-2B cell lines. IC50 (mean ± SD)a

Compound

1 (Osimertinib) 2 3 4 5 6 a b c

NCI-H460b

PC9b

NCI-H1975b

Beas-2Bc

415.9 ± 32.9 611.2 ± 37.7 429.5 ± 38.4 874.9 ± 40.0 892.5 ± 41.3 1138.7 ± 45.1

6.5 ± 0.5 16.1 ± 0.8 8.1 ± 0.4 20.5 ± 0.9 26.2 ± 1.1 46.7 ± 1.8

10.5 ± 0.8 17.4 ± 1.6 15.3 ± 1.0 32.9 ± 1.0 45.6 ± 1.3 89.9 ± 2.6

14.9 ± 0.6 15.1 ± 0.4 7.9 ± 1.8 15.6 ± 0.3 15.7 ± 1.4 28.4 ± 2.4

IC50 values were determined in triplicate with MTS assay. The unit of the IC50 values is nM. The unit of the IC50 values is mM.

Table 2 Kinase inhibitory activity of 1–6 on WT EGFR and L858 R/T790M EGFR in vitro. Compound

1 (Osimertinib) 2 3 4 5 6 a

IC50 (mean ± SD, nM)a

Kinase Selectivity

WT EGFR

L858R/T790M EGFR

WT: L858R/T790M

225.2 ± 21.3 317.6 ± 17.3 166.3 ± 20.2 174.6 ± 26.7 357.3 ± 36.5 595.2 ± 38.5

4.6 ± 0.4 6.4 ± 0.5 4.9 ± 0.3 6.0 ± 0.3 10.2 ± 0.3 17.8 ± 0.4

49.3 49.9 34.3 29.2 35.0 33.4

IC50 values were determined in triplicate.

altered the interaction model of the compound and enzyme binding site of the target proteins. Since in the molecular docking result of compound 3, we did not get the similar binding mode with osimertinib (Fig. S1), which is also beyond our expectations in the design of the compound. 3.4. Inhibitory effects on EGFR phosphorylation and the downstream signaling transduction Considering the remarkable kinase and cellular inhibitory activities in vitro, we continue analyzed the inhibitory effects of

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Fig. 3. Inhibitory effects of compound 2 on EGFR phosphorylation (p) and its downstream signaling pathways in mutant (H1975 and PC9) and WT (LoVo) EGFR cell lines. Cell lysates were harvested for western blot analysis after a 6 h treatment. The representative data are at least three separate experiments.

Fig. 4. In vivo antitumor efficacy of compound 2 (5 mg/kg) in subcutaneous NCI-H1975 (L858R/T790M) xenografts following 30 days of daily treatment through oral administration (n = 10 per group). (A) The average tumor weights in the end of the experiment. (B) The average tumor volume curves during the period of the drug treatment. (C) The average body weights during the period of the drug treatment. (D) Tumor morphology of all the experimental animal. Data represents mean ± SD.

compound 2–6 on the phosphorylation of EGFR and the downstream signaling transduction by Western blot in mutant (H1975 and PC9) and WT (LoVo) EGFR cell lines (Figs. 3 and S2). The result showed that compound 2 selectively inhibited the phosphorylation of mutant EGFR, ERK and AKT on H1975 and PC9 cell lines, especially on H197, which showed a similar inhibitory activity to osimertinib.16 Meanwhile, compound 2 exhibited less inhibitory activity on WT EGFR phosphorylation on LoVo cell line, even at 1000 nM (Fig. 3). The result confirmed that compound 2 was a potent and selective L858 R/T790M mutant EGFR inhibitor. In addition, the inhibitory effects of compound 3–6 on EGFR phosphorylation and the downstream signaling pathways showed in details in the supplementary information (Fig. S2), and the inhibitory activity results were consistent with the in vitro kinase activity.

3.5. In vivo xenograft experiments To further confirm the antitumor efficacy of the derivatives in vivo, we administered compound 1–6 as monotherapy against NCI-H1975 (L858R/T790M) xenografts at 5mg/kg/day in the preexperimental study (Fig. S3). As the previous study described, the tumor shrinkage was observed after oral administration of 5 mg/ kg/day osimertinib.19 Interestingly, once-daily dosing of compound 2 induced significant tumor regression after 30 days, which is slightly better than osimertinib (Figs. S3 and 4, the repeated experimental study). To our surprisingly, the in vivo antitumor efficacy is not consistent with the in vitro kinase and cellular activity, which may be due to an increased solubility (>10 mg/mL) and an increased human oral absorption value of 3.0, which was predicted and calculated using Qikprop in Maestro software package

H. Gao et al. / Bioorganic & Medicinal Chemistry 25 (2017) 4553–4559

(Table S1). What’s more, from the statistic data of the body weight, it seems that the mice in compound 2 treated group have a better appetite than osimertinib (Fig. 4C), which implies that the synthesized N-oxide metabolite compound 2 may had lower side effects in mice in vivo. Furthermore, the docking result of compound 2 with EGFRT790M (PDB ID code: 3IKA) exhibited that except the covalent bond with CYS797 and other two hydrogen bond with MET793 (same to osimertinib, Fig. S1A), compound 2 could form a new salt bridge with LYS716 (Fig. S1B), which may be another reason for its good antitumor efficacy in vivo. 4. Conclusion A novel series of osimertinib derivatives were synthesized and evaluated for their in vitro anti-proliferation activities, kinase inhibitory activities, EGFR signaling pathway activation and in vivo antitumor efficacy in NCI-H1975 (L858R/T790M) xenografts. In the current results, although compound 3 showed the best in vitro anti-proliferation activity and the kinase inhibitory activity against mutant and WT EGFR, the N-oxide metabolite compound 2 displayed a better kinase selectivity and a comparable antitumor efficacy in vivo to osimertinib. As we know, most of the metabolic enzyme used to binding the lipophilic groups,30 while in our study, on the one hand, the enhanced solubility of the compound 2 (Table S1) may weaken its binding activity to metabolic enzyme, delay the compounds metabolism in vivo, and further improve its bioavailability. On the other hand, in the molecular docking result (Fig. S1), compound 2 formed a new salt bridge with LYS716 residue of the target protein, which will help to increase its binding affinity to the target protein, and then improve its activity in vivo. The above synergistic actions may be one of the reasons for compound 2 for its maintaining antitumor effects in vivo. In addition, introduction of fluorine in N-methylindoles motif of the N-oxide metabolite, the antitumor activities in vitro and in vivo were significantly decreased, and also decreased the kinase selectivity. These may be due to the changes of the physicochemical parameters (Table S1) and the changes of the interaction model between compound and the target proteins (Fig. S1). In conclusion, the N-oxide metabolite-based derivatives of osimertinib was reported for the first time with the relevant pharmacological activity evaluation. These findings provided valuable clues for further discovery of more active L858R/T790M EGFR inhibitors, the newly N-oxide metabolite compound 2 may serve as one of the potential therapeutic drugs in future. 5. Experimental 5.1. General All commercial materials (Alfa Aesar, Aladdin, J&K Chemical Ltd.) were used without further purification. All solvents were analytical grade. The 1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz spectrometer in DMSO-d6 or CDCl3 using TMS or solvent peak as a standard. All 13C NMR spectra were recorded with complete proton decoupling. Low-resolution mass spectral analysis were performed with a Waters AQUITY UPLCTM/MS. Analytical TLC was performed on Yantai Chemical Industry Research Institute silica gel 60 F254 plates and flash column chromatography was performed on Qingdao Haiyang Chemical Co. Ltd silica gel 60 (200–300 mesh). The rotavapor was BUCHI’s Rotavapor R-3. 5.2. General procedure for the preparations of compound 2–6 N-oxide derivatives (compound 2, 5, 6) was prepared as, 1.0 equiv. of m-CPBA was added into a solution of AZD9291 or its

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fluorinated derivatives in DCM (25 mL). The mixture was allowed to stir at 0 °C for 30 min. Then water was added to quench the reaction. The reaction mixture was extracted with DCM for 3 times. The combined organic layer was dried over anhydrous Na2SO4 and concentrated on rotavapor under reduced pressure. The residue was purified by HPLC (MeOH:H2O = 10:1) to give desired products as yellow solid. Fluorinated N-Methylindoles were used for synthesis of fluorinated derivatives of osimertinib (compound 3, 4) according to literature reported procedures.29 5.2.1. 2-((2-Acrylamido-5-methoxy-4-((4-(1-methyl-1H-indol-3-yl) pyrimidin-2-yl)amino)phenyl) (methyl)amino)-N,N-dimethylethan-1amine oxide (2) Yellow solid. Yield 85%. 1H NMR (400 MHz, DMSO-d6, d (ppm)), 12.50 (s, 1H), 9.00 (s, 1H), 8.57 (s, 1H), 8.32–8.27 (m, 2H), 7.90 (s, 1H), 7.51 (d, J = 8.16 Hz, 1H), 7.26–7.14 (m, 4H), 6.20–6.15 (m, 1H), 5.62–5.59 (m, 1H), 3.89 (s, 3H), 3.86 (s, 3H), 3.46–3.44 (m, 2H), 3.33 (s, 3H), 3.30 (s, 3H), 3.25–3.24 (m, 2H), 2.74 (s, 3H). 13C NMR (100 MHz, DMSO-d6, d (ppm)), 163.5, 161.68, 160.1, 157.5, 146.2, 139.5, 137.6, 133.7, 133.6, 126.8, 125.4, 124.8, 124.6, 122.0, 121.6, 120.9, 116.8, 112.5, 110.4, 107.0, 104.4, 64.3, 59.8, 55.9, 53.8, 33.0; ESI-LRMS calcd. for C16H18Cl2N2O [M+H]+: 516.27, found 516.35. 5.2.2. N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-((4-(5fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-4methoxyphenyl)acrylamide (3) Yellow solid. Yield 70%. 1H NMR (400 MHz, CDCl3, d (ppm)), 10.17 (s, 1H), 9.80 (s, 1H), 9.03 (s, 1H), 8.36 (d, J = 5.2 Hz, 1H), 7.73–7.71 (m, 2H), 7.28–7.25 (m, 1H), 7.05 (d, J = 5.2 Hz, 1H), 7.00 (t, J = 8.8 Hz, 1H), 6.79 (s, 1H), 6.42–6.38 (m, 2H), 5.69 (dd, J = 3.5 Hz, J = 9.1 Hz, 1H), 3.94 (d, 3H), 3.87 (s, 3H), 2.88 (t, J = 5.2 Hz, 1H), 2.69 (s, 3H), 2.28 (t, J = 5.2 Hz, 1H), 2.26 (s, 6H); 13 C NMR (100 MHz, CDCl3, d (ppm)), 162.92, 161.78, 160.06, 159.66, 158.01, 157.72, 144.43, 136.45, 134.89, 132.91, 129.59, 127.53, 126.32, 125.43, 113.66, 110.70, 110.08, 107.58, 105.95, 105.71, 104.66, 57.35, 56.38, 56.12, 45.42, 43.85, 33.39; ESI-LRMS calcd. for C28H32FN7O2 [M+H]+: 518.26, found 518.34. 5.2.3. N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-((4-(7fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-4methoxyphenyl)acrylamide (4) Yellow solid. Yield 75%. 1H NMR (400 MHz, CDCl3, d (ppm)), 10.14 (s, 1H), 9.82 (s, 1H), 9.00 (s, 1H), 8.37 (d, J = 5.2 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.14 (d, J = 5.2 Hz, 1H), 7.09 (td, 1H), 6.90 (dd, J = 12.4 Hz, J = 8.0 Hz, 1H), 6.78 (s, 1H), 6.43– 6.41 (m, 2H), 5.69 (dd, J = 3.5 Hz, J = 8.4 Hz, 1H), 4.19 (d, 3H), 3.87 (s, 3H), 2.89 (t, J = 5.2 Hz, 1H), 2.69 (s, 3H), 2.30 (t, J = 5.2 Hz, 1H), 2.27 (s, 6H); 13C NMR (100 MHz, CDCl3, d (ppm)), 163.08, 161.75, 159.48, 157.96, 149.46, 144.36, 136.43, 134.61, 132.72, 129.20, 127.57, 125.78, 121.21, 121,14, 115.98, 114.34, 110.09, 107.98, 107.53, 107.35, 104.35, 56.49, 56.09, 54.98, 44.60, 43.78, 36.19; ESI-LRMS calcd. for C28H32FN7O2 [M + H]+: 518.26, found 518.34. 5.2.4. 2-((2-Acrylamido-4-((4-(5-fluoro-1-methyl-1H-indol-3-yl) pyrimidin-2-yl)amino)-5-methoxyphenyl)(methyl)amino)-N,Ndimethylethan-1-amine oxide (5) Yellow solid. Yield 75%. 1H NMR (400 MHz, CDCl3, d (ppm)), 9.96 (s, 1H), 9.68 (s, 1H), 8.85 (s, 1H), 8.35 (d, J = 5.2 Hz, 1H), 7.74 (dd, J = 10.2 Hz, J = 1.9 Hz, 1H), 7.65 (s, 1H), 7.28–7.25 (m, 1H), 7.05 (d, J = 5.2 Hz, 1H), 7.00 (td, 1H), 6.76 (s, 1H), 6.69 (dd, J = 16.8 Hz, J = 10.2 Hz, 1H), 6.39 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 5.70 (dd, J = 10.2 Hz, J = 1.6 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.45 (t, J = 5.7 Hz, 1H), 3.32 (t, J = 5.7 Hz, 1H), 3.28 (s, 6H), 2.71 (s, 3H); 13 C NMR (100 MHz, CDCl3, d (ppm)), 165.84, 163.97, 160.87,

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157.95, 148.14, 138.57, 136.36, 136.21, 133.05, 127.64, 127.51, 127.29, 115.99, 114.41, 112.00, 111.45, 111.18, 108.40, 107.74, 107.48, 105.51, 67.93, 58.63, 56.69, 51.31, 44.06, 33.72; ESI-LRMS calcd. for C28H32FN7O3 [M+H]+: 534.26, found 534.35. 5.2.5. 2-((2-Acrylamido-4-((4-(7-fluoro-1-methyl-1H-indol-3-yl) pyrimidin-2-yl)amino)-5-methoxyphenyl)(methyl)amino)-N,Ndimethylethan-1-amine oxide (6) Yellow solid. Yield 80%. 1H NMR (400 MHz, CDCl3, d (ppm)), 9.89 (s, 1H), 9.73 (s, 1H), 8.84 (s, 1H), 8.37 (d, J = 5.2 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.70 (s, 1H), 7.14 (d, J = 5.2 Hz, 1H), 7.10 (td, 1H), 6.90 (dd, J = 12.4 Hz, J = 8.0 Hz, 1H), 6.77 (s, 1H), 6.67 (dd, J = 16.8 Hz, J = 10.2 Hz, 1H), 6.43 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 5.72 (dd, J = 10.2 Hz, J = 1.6 Hz, 1H), 4.15 (d, 3H), 3.88 (s, 3H), 3.47 (t, J = 6.0 Hz, 1H), 3.30 (t, J = 6.0 Hz, 1H), 3.27 (s, 6H), 2.71 (s, 3H); 13C NMR (100 MHz, CDCl3, d (ppm)), 163.55, 159.60, 158.15, 144.66, 136.18, 134.38, 132.83, 129.89, 128.38, 127.71, 126.27, 121.33, 116.15, 114.44, 111.46, 108.21, 107.72, 107.55, 103.76, 66.90, 60.11, 56.26, 52.31, 43.19, 36.36, 36.32, 29.80; ESI-LRMS calcd. for C28H32FN7O3 [M+H]+: 534.26, found 534.35. 5.3. Bioassay 5.3.1. Cell Culture The human lung cancer cell lines NCI-H460, PC9 and NCI-H1975 were derived from American Type Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS according to the supplier’s instructions. All the cells were Mycoplasma free and used within passages 3–5 from thawing. Cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C. 5.3.2. In vitro anti-proliferative activity assay The in vitro anti-proliferation activity of the osimertinib and its derivatives 1–5 was evaluated using the CellTiter 96 AQueous One Solution Cell Proliferation Assay from Promega. Exponentially growing NCI-H460, PC9 and NCI-H1975 cells were seeded into a 96-well flat bottom plates at a density of 7000 cells per well, and incubated at 37 °C. 24 h later, replaced the medium with fresh medium containing test compounds (0.5 mL of a 3-fold serial dilution in DMSO to 99.5 mL of media), and incubated at 37 °C. 72 h later, 20 lL of MTS solution was added to each well and incubated for 2 h. The plates were then measured using a microplate reader at a wavelength of 490 nm. Each sample was performed in triplicates and repeated at least three times. 5.3.3. L858R/T790M EGFR and WT EGFR inhibition assay In vitro kinase assays were detected with Z0 -LYTETM kinase assay kit (Invitrogen, PV3193), and the detection procedures were conducted by the protocols provided by the kit. The detected compounds were diluted to 3-fold serial dilutions in 4% DMSO/H2O solutions. The fluorescence signals were monitored at 400 nm (excitation) and 445/520 nm (emission) using an EnVision multilabel plate reader (PerkinElmer Life Sciences, Boston, MA, USA). IC50 values were calculated from the inhibitory curves according to the kit protocol. 5.4. Western blot analysis Cells were washed with cold PBS on ice and collected in RIPA buffer (150 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% NP40, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche), 40 mmol/L sodium fluoride and 1 mmol/L sodium orthovanadate. Protein concentrations were measured with BCA kit (Pierce). Equal protein amounts were loaded and separated by 10% SDS-PAGE followed by transfer to polyvinylidene difluoride (PVDF) membranes.

After blocking in 5% non-fat milk-TBST, membranes were blotted with phospho-EGFR (Y1068; Cell Signaling Technology; #3777S), total EGFR (Cell Signaling Technology; #4267S), phospho-AKT (Ser473; Cell Signaling Technology; #9271L), total AKT (Cell Signaling Technology; #9272S), phospho-ERK (T202/Y204; Cell Signaling Technology; #9101L), total ERK (Cell Signaling Technology; #9102L) and GAPDH (Santa Cruz; sc-32233) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology; anti-rabbit #7074 or anti-mouse #7076). Signals were detected with ECL detection reagents (Pierce; 34080) by Bio-Rad Molecular imager (ChemiDocTM XRS+ With image LabTM Software). 5.5. In vivo xenografts The in vivo animal experiments were performed at the Laboratory Animal Research Center, Tsinghua University (Beijing, China). The animal experimental protocol was evaluated and approved by the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University (Protocol number: 16-RY2). Female Nude mice (6–8 weeks, Laboratory Animal Research Center, Tsinghua University) were inoculated subcutaneously into the right flank of mice with NCI-H1975 cells (1  106/mouse), respectively. After the xenograft solid tumors were established and allowed to reach about 150–200 mm3, the mice were randomized and treated intragastrically (ig) with compound 1 and 2 (5 mg/kg/day) or the same volume of vehicle consisting of DMSO/Cremophor-EL/PBS (1:1:8) daily for 30 consecutive days. The tumor size was recorded every two days from the measurement of length and width using a vernier caliper and calculated as tumor volumes with the following formula: Tumor volume (mm3) = width  width  length/2. Acknowledgments We thank the Laboratory Animal Research Center, Tsinghua University for the animal care support. This work was supported by the National Natural Science Foundation of China – China (No. 81502567), the China Postdoctoral Science Foundation – China (No. 2015M571027) and the Tsinghua-Peking University Life Science Center Postdoctoral Fellowship. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2017.06.004. References 1. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res. 2001;7:2958–2970. 2. Ciardiello F, Tortora G. Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur J Cancer. 2003;39:1348–1354. 3. Yarden Y. The EGFR family and its ligands in human cancer. Signalling mechanisms and therapeutic opportunities. Eur J Cancer. 2001;37:S3–8. 4. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2:127–137. 5. Maione P, Sacco PC, Sgambato A, Casaluce F, Rossi A, Gridelli C. Overcoming resistance to targeted therapies in NSCLC: current approaches and clinical application. Ther Adv Med Oncol. 2015;7:263–273. 6. Hirano T, Yasuda H, Tani T, et al. In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer. Oncotarget. 2015;6:38789–38803. 7. Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007;7:169–181. 8. Tiseo M, Bartolotti M, Gelsomino F, Bordi P. Emerging role of gefitinib in the treatment of non-small-cell lung cancer (NSCLC). Drug Design, Development and Therapy. 2010;4:81–98.

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