Discovery of 2-(4-Chloro-3-(trifluoromethyl)phenyl ... - ACS Publications

Article Cite This: J. Med. Chem. 2019, 62, 6083−6101

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Discovery of 2‑(4-Chloro-3-(trifluoromethyl)phenyl)‑N‑(4-((6,7dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (CHMFL-KIT-64) as a Novel Orally Available Potent Inhibitor against Broad-Spectrum Mutants of c‑KIT Kinase for Gastrointestinal Stromal Tumors Yun Wu,† Beilei Wang,†,‡ Junjie Wang,†,‡ Shuang Qi,† Fengming Zou,†,§,∥ Ziping Qi,†,§,∥ Feiyang Liu,†,§,∥ Qingwang Liu,§,∥ Cheng Chen,†,‡ Chen Hu,† Zhenquan Hu,† Aoli Wang,†,§,∥ Li Wang,†,‡ Wenchao Wang,†,§,∥ Tao Ren,§,∥,⊥ Yujiao Cai,# Mingfeng Bai,¶ Qingsong Liu,*,†,‡,§,∥,∇ and Jing Liu*,†,§,∥ †

High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031, China ‡ University of Science and Technology of China, Hefei, Anhui 230026, P. R. China § Precision Medicine Research Laboratory of Anhui Province, Hefei, Anhui 230088, P. R. China ∥ Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R. China ⊥ Precedo Pharmaceuticals Inc, Hefei, Anhui 230088, P. R. China # Department of General Surgery, Second Hospital Affiliated to Army Medical University, Xinqiao Road, Chongqing 400037, P. R. China ¶ Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, United States ∇ Institutes of Physical Science and Information Technology, Anhui University, Hefei, Anhui 230601, P. R. China S Supporting Information *

ABSTRACT: Starting from our previously developed c-KIT kinase inhibitor CHMFL-KIT-8140, through a type II kinase inhibitor binding element hybrid design approach, we discovered a novel c-KIT kinase inhibitor compound 18 (CHMFL-KIT-64), which is potent against c-KIT wt and a broad spectrum of drug-resistant mutants with improved bioavailability. 18 exhibits single-digit nM potency against cKIT kinase and c-KIT T670I mutants in the biochemical assay and displays great potencies against most of the gain-offunction mutations in the juxtamembrane domain, drugresistant mutations in the ATP binding pocket (except V654A), and activation loops (except D816V). In addition, 18 exhibits a good in vivo pharmacokinetic (PK) profile in different species including mice, rats, and dogs. It also displays good in vivo antitumor efficacy in the c-KIT T670I, D820G, and Y823D mutant-mediated mice models as well as in the c-KIT wt patient primary cells which are known to be imatinib-resistant. The potent activity against a broad spectrum of clinically important c-KIT mutants combining the good in vivo PK/pharmacodynamic properties of 18 indicates that it might be a new potential therapeutic candidate for gastrointestinal stromal tumors.

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INTRODUCTION

80−85% GISTs carry activation mutations such as V559A/D/ G, V560D, L576P, and so forth, which are mainly located in the juxtamembrane region of c-KIT encoded by exon 11.4 cKIT kinase belongs to type III receptor tyrosine kinase family which also includes FLT3, CSF1R, and PDGFRα/β.It plays

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract.1,2 Approximately 20 individuals in a million are diagnosed with this disease each year in the United States.3 Identification of the c-KIT mutant was a major breakthrough in the pathology of GISTs, which provided a therapeutic target for the treatment of GISTs. 95% of GISTs express c-KIT, and © 2019 American Chemical Society

Received: February 12, 2019 Published: June 17, 2019 6083

DOI: 10.1021/acs.jmedchem.9b00280 J. Med. Chem. 2019, 62, 6083−6101

Journal of Medicinal Chemistry

Article

Figure 1. Chemical structures of representative inhibitors that bear c-KIT kinase activity.

Figure 2. Schematic illustration of discovery of compound 18 (CHMFL-KIT-64) from 9.

effects.10 In addition, ponatinib (4), which was clinically approved for CML, could also potently inhibit KIT exon 11 primary mutants and a range of secondary mutants, including those within the activation loop.11 However, it was suspended by FDA due to serious side effects in 2013 and later was listed in black box warning status for clinical usage. Cabozantinib (5) (a TKI targeting c-KIT, MET, VEGFR2, and AXL) also displays potency against different c-KIT mutations in the patient-derived xenograft (PDX) models of GISTs.12 In addition, BLU285 (6) which is under clinical investigation is a potent and highly selective inhibitor of mutated c-KIT and PDGFRα. It exhibits a good antitumor effect in the PDX models of GISTs,13 but it could not overcome the c-KIT T670I mutant. 14 Furthermore, DCC-2618 (7) 15 and AZD3229 (8), which are under clinical and preclinical development, respectively, exhibited different broad mutant inhibition profiles.14 However, given the fact that there are over a dozen of different c-KIT mutants emerged in the clinic, there is still a great demand to develop new therapeutics which bears different potency profiles against the broad spectrum of the c-KIT mutants. Our previously developed quinolone-based type II c-KIT kinase inhibitor CHMFL-KIT-8140 (9)16 could also overcome a variety of imatinib-resistant secondary mutants including T670I but with poor pharmacokinetic (PK) properties. Through the type II kinase inhibitor binding element hybrid

critical roles in cell proliferation, migration, and survival upon activation in different physiological and pathological contexts. Drug discovery targeting c-KIT has attracted extensive attention in the past decades. Imatinib (1), an inhibitor of c-KIT, PDGFRs, and ABL kinases, was approved as the first-line treatment for advanced GISTs (Figure 1).5 However, with the exception of 20% of the patients with GISTs exhibiting primary resistance to 1, most patients will eventually relapse due to secondary mutations upon chronic treatment.6 Most of the secondary mutations include those that occurred in the ATP binding pocket such as T670I/E, V654A, outside the ATP binding pocket S709F, and activation loops such as D816E/H/V, D820E/G/Y, D822K, Y823D, A829P, and so forth. Multitargeted kinase inhibitor sunitinib (2) was then approved as the second line treatment for patients with GISTs who are refractory to or intolerant of imatinib therapy. Although 2 could overcome some of imatinib-resistant mutations such as V654A and T670I,7 certain imatinib-resistant mutants located in the activation loop remain resistant to it.8 Later, multitargeted kinase inhibitor regorafenib (3) was approved as the third-line therapy for patients with GISTs who are not amenable to surgery and refractory to both 1 and 2.9 Similarly, 3 also could not overcome some of the secondary resistance mutations in the c-KIT activation loops. Furthermore, both 2 and 3 have limited overall clinical response and exhibit severe side 6084



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Table 1. SAR Exploration Focused on the L2/R1 Moietiesa

a

All GI50 values were obtained by triplet testing.

and “tail” (R1) moieties. The growth inhibition of IL-3independent BaF3 cells (GI50) expressing the c-KIT wt (BaF3tel-c-KIT) and c-KIT T670I (BaF3-tel-c-KIT-T670I), which has been used for the development of 9 and shows a correlation with biochemical IC50,16 was used as the primary readout to evaluate the potency and selectivity of the new compounds (Table 1). Replacement of the urea of L2 in 9 with acetamide and the “tail” moiety R1 with the simple phenyl ring (10) or pyridine (11) resulted in great activity loss. Switching L2 in 10 to cyclopropanecarboxamide (12) did not achieve apparent potency. Interestingly, introduction of the CF3 group in the para-position of R1 (13) significantly increased the activity both to the c-KIT wt (GI50 = 0.022 μM) and the c-KIT T670I (GI50 = 0.011 μM); meanwhile, it showed good selectivity against parental BaF3 cells (GI50 = 2.16 μM). The analogous compound with the m-CF3 substituent (14) also achieved good potency against c-KIT wt and c-KIT T670I (GI50 = 0.020 and 0.001 μM, respectively). However, changing the substituent of R1 from m-CF3 to m-F (15) or m-OMe (16) all resulted in significant activity loss. Addition of the p-F substituent to the R1 moiety of 14 (17) lost about 13-fold activity against c-KIT T670I in comparison with 14, although

drug design approach,17 we designed a series of compounds and screened them with transformed BaF3 cells which led to the discovery of a novel highly potent c-KIT kinase inhibitor CHMFL-KIT-64 (18) (Figure 2). It was potent against a broad spectrum of c-KIT primary gain-of-function mutants, secondary mutants, and A-loop mutants. In addition, 18 also displayed good in vivo PK properties and antitumor efficacy in different preclinical mice models of GISTs.

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RESULTS AND DISCUSSION

Structure−Activity Relationship Exploration. Compound 9 is a highly potent type II c-KIT kinase inhibitor which is capable of overcoming the T670I gatekeeper mutant; however, the poor PK profiles limit its further development. In order to address this deficiency, we initiated a medicinal chemistry study that resulted in the identification of a highly potent inhibitor which could overcome a broad spectrum of cKIT mutants. Using the type II kinase inhibitor binding element hybrid design approach,17 we started to explore the structure−activity relationships (SAR) by varying the “head”, “linker”, and “tail” moieties. We first fixed the hinge-binding part as 6,7-dimethoxyquinolin and explored the “linker2” (L2) 6085



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it still displayed a similar potency toward c-KIT wt. Interestingly, changing the F atom to a larger Cl atom (18) not only remarkably increased the potency against c-KIT wt (GI50 = 0.001 μM) and c-KIT T670I (GI50 = 0.004 μM) but also improved the selectivity window toward the parental BaF3 cells (GI50 = 5.97 μM). However, further increasing the size of the substituent with a methyl group (19) slightly decreased the activity toward c-KIT wt and c-KIT T670I, even though it still kept good selectivity against the parental BaF3 cells. Replacement of the CF3 group in 19 with a methyl group (20) displayed similar trends to 19. Further investigating the effects of other substituents at the meta- and para-positions of the benzene ring, such as 3,4-dimethoxy (21), 3,4-difluoro (22), and 3,4-dichloro (23) all resulted in significant activity loss against c-KIT wt. Installing a F or CF3 group at the orthoposition of the benzene ring (24−26) also led to activity loss. Relatively larger groups at R1, for example, benzodioxole (27) and naphthyl (28), both resulted in activity loss. Additionally, aliphatic rings such as cyclohexyl (29) and N-methyl piperazine (30) at R1 both significantly decreased the potency. Furthermore, reversed amide counterpart of L2 in 13 (31) and 14 (32) lost the activity to both c-KIT wt and c-KIT T670I as well. Because 18 exhibited potent antiproliferative efficacy in the BaF3-tel-c-KIT and BaF3-tel-c-KIT-T670I cells, we then fixed the L2 moiety as acetamide and R1 moiety as 4-chloro-3(trifluoromethyl)phenyl and further explored the “linker 1” (L1) moiety (Table 2). Replacement of the oxygen atom linkage in 18 with −NH (33), −NMe (34), or S atom (35) all resulted in significant activity loss against c-KIT wt and c-KIT T670I. Therefore, we kept the phenoxy group and explored a series of substituents of the benzene ring in the L1 moiety. Addition of one F atom at the ortho-position of the acetamide (36) increased the activity against c-KIT wt (GI50 = 0.049 μM) and c-KIT T670I (GI50 = 0.018 μM). Changing the F atom to Cl atom (37) or the methyl group (38) retained similar activities compared with 36. Switching the F atom from the ortho- to meta-position (39) led to decrease of the activity against c-KIT wt (GI50 = 0.116 μM). Variation of different substituents at this position such as Cl (40), methyl (41), methoxyl (42), trifluoromethyl (43), and nitrile (44) groups all resulted in activity loss in comparison with 18. Selectivity Profiling of Compound 18. Because 18 displayed the best potency against both c-KIT wt and T670I and meanwhile exhibited the best selectivity window to parental BaF3 cells, we then further screened it in a panel of broad-spectrum mutants with BaF3 cell lines expressing a variety of clinically relevant secondary mutants of c-KIT kinase, including those in the juxtamembrane domain (V559D/A/G, V560D, and L576P), ATP binding pocket (V654A, V654A/ V559D, T670E/I, T670I/V559D, and S709F) and the activation loop (D816E/H/V, D820G/Y, N822K, Y823D, and A829P) (Table 3). The results showed that 18 displayed the best potency against c-KIT wt and selectivity window to parental BaF3 cells compared with 1 and 2. In addition, 18 exhibited similar or better activities compared with 1 and 2 against all of the primary gain-of-function mutations of c-KIT in exon 11 including V559A/D/G, V560D, and L576P. Furthermore, 18 showed better potencies than 2 against the compound 1-resistant c-KIT gatekeeper residue T670I/E mutation. 18 and 2 could also overcome compound 1-resistant secondary mixed mutation T670I/V559D and display better potencies against S709F mutation which is located in exon 14

Table 2. SAR Exploration Focused on the Substitution (L1) Moietya

a


but outside the ATP binding pocket. Only 2 but not 1 or 18 was resistant to the V654A mutant which is located in exon 13 and inside the ATP binding pocket. Interestingly, 18 exhibited much better activities than 1 and 2 against activation loop mutants including D816E/H, D820E/G/Y, D822K, Y823D, and A829P except D816V which was resistant to all of these three compounds. We then moved forward to investigate the selectivity profile of 18 against other protein kinases with DiscoverX’s KINOMEscan assay.18 The data demonstrated that 18 bears a good selectivity (S score (1) = 0.02) in a panel of 468 kinases and mutants at 1 μM concentration (Figure 3A and Table S1). Besides high binding affinity to c-KIT and c-KIT mutant kinases, 18 also displayed strong binding to CSF1R, DDR1, FLT4, PDGFRα, PDGFRβ, LOK, and RET kinases (percent 6086



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Table 3. Antiproliferative Effects of Compounds 1, 2, and 18 against a Panel of c-KIT wt/Mutant-Transformed BaF3 Cells cells

c-KIT status

compd. 1 (GI50: μM)



BaF3 BaF3-tel-c-KIT BaF3-tel-c-KIT-V559A BaF3-tel-c-KIT-V559D BaF3-tel-c-KIT-V559G BaF3-tel-c-KIT-V560D BaF3-tel-c-KIT-L576P BaF3-tel-c-KIT-V654A/V559D BaF3-tel-c-KIT-V654A BaF3-tel-c-KIT-T670E BaF3-tel-c-KIT-T670I BaF3-tel-c-KIT-T670I/V559D BaF3-tel-c-KIT-S709F BaF3-tel-c-KIT-D816E BaF3-tel-c-KIT-D816H BaF3-tel-c-KIT-D816V BaF3-tel-c-KIT-D820E BaF3-tel-c-KIT-D820G BaF3-tel-c-KIT-D820Y BaF3-tel-c-KIT-N822K BaF3-tel-c-KIT-Y823D BaF3-tel-c-KIT-A829P

parental WT exon 11/juxtamembrane domain exon 11/juxtamembrane domain exon 11/juxtamembrane domain exon 11/juxtamembrane domain exon 11/juxtamembrane domain exon 13/11/ATP binding pocket exon 13/ATP binding pocket exon 14/ATP binding pocket exon 14/ATP binding pocket exon 14/11/ATP binding pocket exon 14/outside the ATP binding pocket exon 17/activation loop exon 17/activation loop exon 17/activation loop exon 17/activation loop exon 17/activation loop exon 17/activation loop exon 17/activation loop exon 17/activation loop exon 18/activation loop

>10 0.35 0.905 0.01 0.008 0.003 0.085 0.608 1.41 4.08 >10 >10 0.115 0.174 0.651 >10 0.035 0.337 0.435 1.47 5.87 0.58

1.01 0.003 0.02 0.001 0.004 0.035 0.006 0.019 0.005 0.089 0.01 0.012 0.028 0.059 0.315 1.72 0.093 0.389 0.172 0.384 0.704 0.018

5.97 0.001 0.021 0.006 10 >10 >10

0.011 0.014 0.004 0.035 0.74

0.006 0.013 0.011 0.073 1.37

a


the hinge-binding area. The amide in the linker formed two hydrogen bonds with Asp810 in the DFG motif and Glu640 in the c-Helix. The “tail” part occupied the hydrophobic pocket generated by the DFG-out shifting. In the homology model of the c-KIT T670I mutant, 18 adopted a similar type II binding mode (Figure 4H). The V654A mutant might weaken the van der Waals interactions of 18 or the conformational change resulted in a similar effect to 1 which made it insensitive to 18. The O-bridged phenyl moiety in 18 oriented to an angle that provided enough space for the bulky isoleucine residue, which could explain its potency against the T670I mutant (Figure 4I). The D816V mutant was also resistant to 18, but it was located outside of the ATP binding pocket and hence very hard to model. The modeling study also helps to understand the SAR observed in Table 1. The residues of Val643, Leu644, His790, and Cys809 formed the hydrophobic pocket generated by DFG-out shifting. The trifluoromethyl in the “tail” (R1) pointed to Val643 and Leu644, which helped to gain better potency due to the hydrophobic interaction. In addition, the substituent p-Cl of phenyl in the “tail” moiety could achieve higher binding affinity due to the halogen bond interaction with His790.20 Loss of hydrophobicity (29, 30) or the halogen bond resulted in the activity loss. The amide moiety (L2)

These data indicated that c-KIT, PDGFRα, PDGFRβ, and CSF1R kinases are the main targets of 18. In order to better understand the sensitivity of 18 among different c-KIT mutants and the SAR, we then compared the binding modes of 18 with 1 and 2. As shown in the X-ray crystal structure of 1 with c-KIT wt, 1 fitted well with c-KIT wt via four hydrogen bonds, including the N atom of the pyridine ring with Cys673 in the hinge-binding area, the NH of aminopyrimidine with Thr670, and two canonical type II inhibitor hydrogen bonds between the amide and Glu640/ Asp810 (Figure 4A). Compound 1 was resistant to c-KIT V654A because the conformational change affected the binding of 1 to c-KIT19 (Figure 4B). The hydrogen bond of the NH of aminopyrimidine with Thr670 was lost when Thr670 mutated to Ile670, which resulted in the resistance to 1 (Figure 4C). As shown in the X-ray crystal structure of 2 with c-KIT wt, 2 bounds to inactivated c-KIT wt through two hydrogen bonds with Cys673 and Glu671 in the hinge-binding area with the N and O atom of hydroxy indole (Figure 4D). The mutants of Val654 and Thr670 did not affect the binding of 2; therefore, they were still sensitive to 2 (Figure 4E,F). Docking of 18 into c-KIT wt (PDB ID: 6GQK) showed that 18 adopted a canonical type II binding mode (Figure 4G). As expected, the quinoline nitrogen formed a hydrogen bond with Cys673 in 6088



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Figure 5. Cellular effects of compound 18 in GIST cell lines GIST-T1, GIST-T1-T670I, and GIST-5R. (A) Effects of 18 on c-KIT mediated signaling pathways. (B) Effects of 18 on apoptosis. (C) Effects of 18 on cell cycle progression.

cells through the on-target inhibition of c-KIT but not general toxicity. We also investigated the effects of 18 on the c-KIT-mediated signaling pathways in the c-KIT wt and c-KIT T670I-driven GISTs cell lines GIST-T1, GIST-T1-T670I, and GIST-5R, respectively (Figure 5A). 18 blocked the autophosphorylation of c-KIT pY703, pY719, and pY823 in GIST-T1, GIST-T1T670I, and GIST-5R, respectively, cells at a concentration of 30 nM and also remarkably inhibited the downstream signaling mediators pAKT (T308/S473), pS6 (S235/236), and pERK (T202/204) (EC50 less than 100 nM). 1 and 2 displayed similar effects on the signaling pathways. In compound 1resistant cell lines GIST-T1-T670I and GIST-5R (c-KIT T670I), 18 also significantly affected the phosphorylation of cKIT pY703, pY719, and pY823 and downstream mediators (EC50 less than 100 nM). Similar effects were observed from 2 but not 1. It is worth noting that both 18 and 2 strongly inhibited the phosphorylation of STAT3 in GIST-T1-T670I and GIST-5R cells but moderately in GIST-T1 cells. Furthermore, 18 and 2 affected the autophosphorylation of AKT308 in GIST-T1 and GIST-5R cells but not GIST-T1T670I cells. These discrepancies reflected the different genetic

which could form two typical hydrogen bonds with Glu640 and Asp810 were critical for the binding. Elongation of L2 (31, 32) would result in the loss of these bonds. In addition, π−π stack between the phenyl in the L1 moiety and Phe811 might help to stabilize the binding conformation. Smaller substituents in the L1 moiety (36−40) would not affect the rotation of phenyl. However, larger substituents in the L1 moiety (43− 44) might distort the conformation and lead to decreased activity to c-KIT wt and the T670I mutant. Cellular Evaluation of Compound 18. Next, we examined the antiproliferative effects of 18 against a panel of established GISTs cancer cell lines. Not surprisingly, it did potently inhibit the growth of c-KIT-dependent GIST cancer cells such as GIST-T1 (GI50: 0.006 μM) and GIST-882 (GI50: 0.013 μM) which are also sensitive to 1 and 2 (Table 4). In the compound 1-resistant cell line GIST-T1-T670I (generated by CRISPR knock-in method) and GIST-5R (generated by continuous treatment of 1) cell lines, both 18 and 2 exhibited strong growth inhibition effects. In addition, 18 also displayed a good selectivity window over GIST-48B (GI50: 1.37 μM), whose growth is c-KIT independent. This further confirmed that 18 exerted its antiproliferative effect against GIST cancer 6089



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potent and effective than 1 in the genetically more complicated GIST cells and therefore indicated potential clinical benefits. In Vivo PK/PD and Efficacy Evaluation of Compound 18. The in vivo PK properties of 18 were evaluated in different species including mice, Sprague Dawley rats, and beagle dogs following intravenous (iv) injection and oral administration (Table 5 and Figure S3). 18 exhibited good drug exposure (AUC0−t) in all of these three species, that is, 25 054 ng/mL.h at 10 mg/kg in rats, 12 608 ng/mL.h at 10 mg/kg in mice, and 19 065 ng/mL.h at 5 mg/kg in dogs. This compound displayed different half-lives (T1/2) in these three different species after oral administration, that is, 4.5 h in mice and relative longer time in rats (6.4 h) and in dogs (19.4 h). These results indicated that 18 was metabolized very slowly in dogs than in mice and rats. 18 possessed acceptable bioavailability in mice (F = 43%), rats (F = 50%), and dogs (F = 81%). These PK properties indicated that 18 would be suitable for oral administration. We also evaluated the pharmacodynamic (PD) profile of 18 in the GIST-T1-T670I mouse model at the 80 mg/kg/day dosage (p.o.) for the impact on phosphorylation levels of cKIT and downstream signaling mediators such as ERK and AKT and so forth (Figures 7 and S4). Meanwhile, we also

backgrounds of these cells. In addition, 18 could induce dosedependent cell apoptotic death (by examining the cleaved PARP and cleaved caspase 3) and arrest the cell cycle into the G0/G1 phase in all of these three cell lines (Figure 5B,C). In order to exclude the off-target effects (such as PDGFRs and CSF1R) on the signaling pathway, we also detected the expression and phosphorylation of PDGFRα/β and CSF1R kinases in GIST-T1 cells (Figure S1). The results showed that only PDGFRα expressed and phosphorylated in GIST-T1 cells, and 18 could inhibit its autophosphorylation at the Y849 site. CSF1R did not express in GIST-T1 cells, and phosphorylated PDGFRβ was not detected either. In order to further exclude the effects of downstream mediators caused by the inhibition of PDGFRα, we also used highly selective PDGFRα inhibitor CHMFL-PDGFR-15921 to test the PDGFR signaling pathway and the antiproliferative effect in GIST-T1 cells (Figure S2). The data demonstrated that although pPDGFR was potently inhibited, the downstream mediators such as AKT, STAT3, and ERK were less potently inhibited. Furthermore, the antiproliferative effect of the selective PDGFRα inhibitor was much less than 1, which further proved that the PDGFRα inhibition did not play essential roles in the inhibition of 18 to the c-KIT mediated signaling pathway. All these data suggested that 18 exerted its inhibitory effect to the GIST cancer cells through the on-target inhibition of c-KIT. Inhibition of the Proliferation of Primary GIST Patient Cells. We then investigated the effects of 18 in a more pathologically relevant setting of ex vivo culture of primary cells derived from GIST patients (PDC) (Figure 6). In cells

Figure 7. PD effect of 18 on tumor p-c-KIT, p-ERK, and p-AKT in the GIST-T1-T670I mouse xenograft model with a single dose of 80 mg/kg/day (p.o.).

Figure 6. Antiproliferative effects of compounds 18 and 1 against primary cells derived from GIST patients upon 6 days of drug treatment with the CellTiter-Glo assay.

monitored the drug concentration in the blood plasma. Following a single-dose treatment, in 4, 8, and 12 h time courses, the phosphorylation levels of c-KIT/ERK/AKT were decreased time dependently and then started to get back at 24 h. The drug concentration in the plasma reached the highest level in 4 h and then gradually decreased; even up to 24 h, it did not reduce to the basal level. These data indicated that the drug concentration in the plasma was well correlated to the phosphorylation levels of the signaling mediators. In addition,

from patient A which harbor the c-KIT V559D gain-offunction mutation, 18 displayed a better growth inhibitory effect than 1 although both of them exhibited dose-dependent antiproliferative effects. Interestingly, 18 also exhibited potent and dose-dependent inhibitory effects against cells from patient B, harboring c-KIT wt which are known to be compound 1resistant.22−24 These data suggested that 18 might be more

Table 5. PK Study of Compound 18 in Mice, Sprague Dawley Rats, and Beagle Dogsa rats

18 parameter AUC(0−t) (ng/mL·h) Tmax (h) T1/2 (h) Cmax (ng/mL) F (%)

mice

beagle dogs

iv (1 mg/kg)

p.o. (10 mg/kg)

iv (1 mg/kg)

p.o. (10 mg/kg)

± ± ± ±

25054 ± 7060 9±0 6.41 ± 0.34 1643 ± 462 50

2989 0.03 5.4 520

12 608 4 4.5 1390 43

2553 0 15.85 660

155.1 0 3.72 17

iv (1 mg/kg)

p.o. (5 mg/kg)

± ± ± ±

19065 ± 5696 5.33 ± 1.15 19.4 ± 4.02 1163 ± 288 81

5323 0.033 20.61 1107

569 0 4.82 117

a

All testing data were obtained from three independent mice (±SD). 6090



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Figure 8. (A) Antitumor efficacy of 18 in the BaF3-tel-c-KIT-T670I xenograft mouse model. Female BALB/C-nu mice bearing established BaF3tel-c-KIT-T670I tumor xenografts were treated with 18 at 40 and 100 mg/kg/day dosage or vehicle. (B) Antitumor efficacy of 18 in the BaF3-tel-cKIT-D820G xenograft mouse model. Female BALB/C-nu mice bearing established BaF3-tel-c-KIT-D820G tumor xenografts were treated with 18 at 40 and 80 mg/kg/day dosage or vehicle. Daily oral administration was initiated when BaF3-tel-c-KIT-D820G tumors had reached a size of 200− 400 mm3. (C) Antitumor efficacy of 18 in the BaF3-tel-c-KIT-Y823D xenograft mouse model. Female BALB/C-nu mice bearing established BaF3tel-c-KIT-Y823D tumor xenografts were treated with 18 at 40 and 80 mg/kg/day dosage or vehicle.

it is worth noting that the plasma protein binding rate of 18 was over 90%. We finally evaluated the in vivo antitumor efficacy of 18 in three transformed BaF3 cells, that is, BaF3-tel-c-KIT-T670I (1 resistant, 2 and 18 sensitive), BaF3-tel-c-KIT-D820G (18 sensitive, 1 and 2 resistant), and BaF3-tel-c-KIT-Y823D (18 sensitive, 1 and 2 resistant) and three GISTs intact cells, that is, GIST-T1 (1, 2 and 18 sensitive)-, GIST-T1-T670I (1 resistant, 2 and 18 sensitive)-, and GIST-5R (1 resistant, 2 and 18 sensitive)-inoculated xenograft mouse models by oral administration (Figures 8 and 9). All dosages of 18 (20, 40, 80, and 100 mg/kg/day) did not affect the animal weights which indicated that there was no general cytotoxicity at those dosages (Figures 8A−C and 9A−C left lane). During 11 days of continuous treatment, 18 dose dependently inhibited the BaF3-tel-c-KIT-T670I tumor progression and exhibited almost 100% TGI (tumor growth inhibition) at a dosage of 100 mg/ kg/day (Figure 8A). After 9 days of continuous treatment, 18 dose dependently inhibited the BaF3-tel-c-KIT-D820G and BaF3-tel-c-KIT-Y823D tumor progression and exhibited 92.9 and 90.2% TGI, respectively, at a dosage of 80 mg/kg/day (Figure 8B,C). Comparably, 2 did not show apparent efficacy in the BaF3-tel-c-KIT-D820G or BaF3-tel-c-KIT-Y823D xenograft mouse model. In the GIST-T1 cell-inoculated xenograft mouse models, 18 dose dependently inhibited the tumor progression, and a dosage of 20 mg/kg/day exhibited 95.6% TGI (Figure 9A) which was much better than the

efficacy of 1 at 100 mg/kg/day. In addition, for the 1-resistant cell lines GIST-T1-T670I and GIST-5R, 18 dose dependently inhibited the tumor progression and a dosage of 40 mg/kg/day exhibited 76.4% and 96.9% TGI, respectively (Figure 9B,C). It is worth noting that the mice treated with 2 started to die at the third day in the GIST-T1-T670I xenograft models, and there was only one mouse alive at the 28th day. However, the mice treated with 18 were in good condition. All these in vivo data suggested that 18 bore suitable anti-GISTs efficacy in different c-KIT mutant spectra from 1 and 2 and it might have a potential usage in the clinic.

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CHEMISTRY Scheme 1 depicts the synthetic route for the preparation of target compounds 10−30, 36−37, and 39. 4-Chloro-6,7dimethoxyquinoline was reacted with aminophenols using NaH as the base in DMSO at 110 °C to give the quinoline intermediates 46a-b. 46c-d were prepared with 4-chloro-6,7dimethoxyquinoline and nitrophenols in Ph2O at 140 °C followed by reduction with Fe powder in EtOH under reflux. The desired quinoline derivatives were obtained from direct amide coupling of 46a−d and various substituted phenylacetic acids or acyl chloride. As shown in Scheme 2, intermediate 47 was prepared by nucleophilic substitution of 4-chloro-6,7-dimethoxyquinoline with methyl 4-hydroxybenzoate at 145 °C. Then, hydrolysis of the ester under basic conditions resulted in carboxylic acid 48, 6091



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Figure 9. (A) Antitumor efficacy of 18 in the GIST-T1 xenograft mouse model. Female BALB/C-nu mice bearing established GIST-T1 tumor xenografts were treated with 18 at 20 and 40 mg/kg/day dosage or vehicle. (B) Antitumor efficacy of 18 in the GIST-T1-T670I xenograft mouse model. Female BALB/C-nu mice bearing established GIST-T1-T670I tumor xenografts were treated with 18 at 20 and 40 mg/kg/day dosage or vehicle. (C) Antitumor efficacy of 18 in the GIST-5R xenograft mouse model. Female BALB/C-nu mice bearing established GIST-5R tumor xenografts were treated with 18 at 20 and 40 mg/kg/day dosage or vehicle.

only exhibited better potency than 1 against the c-KIT wt kinase but also potently inhibited a broad spectrum of mutants including the primary gain-of-function mutations and secondary drug-acquired mutations in the ATP binding pocket and activation loops, some of which are resistant to 1 (first-line treatment in clinic) or 2 (second-line treatment in clinic). For example, 18 was potent to 2-insensitive c-KIT A-loop mutants such as D816H, D820G, Y823G, N822K, and so forth, which are important mutants observed in the clinic. In addition, from the results of KINOMEscan, in-vitro biochemical assay and antiproliferative assay of transformed BaF3 cell lines, 18 exhibited a good selectivity profile in the kinome and only showed inhibitory activities to the type III kinase family including CSF1R and PDGFRα/β kinases but not other protein kinases. Importantly, 18 also displayed a potent growth inhibitory effect against the patients’ primary cells harboring cKIT wt which is known to be compound 1-resistant. This might benefit from the different target profiles of 18 from 1. Combining the in vivo PK/PD and efficacy properties, the current data suggest that 18 might be a potential first-line/ second-line drug candidate as a supplementation for the current anti-GIST therapies.

which underwent an amidation reaction with substituted phenethyl amine to provide the final products 31 and 32. Quinoline analogues 33−34 were prepared as illustrated in Scheme 3. 4-Chloro-6,7-dimethoxyquinoline was reacted with 4-nitroaniline in NMP at 120 °C followed by treating with TsOH to give phenylamine derivative 50, and then intermediate 52 was obtained by further methylation. Reduction of the nitro group in 50 and 52 with Pd/C under a hydrogen atmosphere provided amines 51 and 53, which were then coupled with 2-(4-chloro-3-(trifluoromethyl)phenyl)acetic acid to furnish the title compounds. 35 was obtained from an amidation reaction of 2-(4-chloro3-(trifluoromethyl)phenyl)acetic acid and amine 54, which was synthesized by nucleophilic substitution of 4-chloro-6,7dimethoxyquinoline and 4-aminobenzenethiol (Scheme 4). As outlined in Scheme 5, intermediates 45c−h were synthesized by nucleophilic substitution of 6,7-dimethoxyquinolin-4-ol with a variety of substituted 4-fluoronitrobenzene 49a−f using tBuOK or Cs2CO3 as the base in DMSO at 100 °C. The nitro group was then reduced with Fe powder in EtOH under reflux to afford aniline intermediates 46e−j. Final quinoline products 38 and 40−44 were obtained from amidation reaction of 46e−j and 2-(4-chloro-3(trifluoromethyl)phenyl)acetic acid.

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EXPERIMENTAL SECTION

Chemistry. All reagents and solvents were purchased from commercial sources and used without further purification. All the reference compounds were purchased from MedChemExpress (China). 1H NMR and 13C NMR spectra were recorded on a Bruker 400 (1H: 400 MHz, 13C: 100 MHz) or Bruker 500 (1H: 500 MHz, 13 C: 125 MHz) spectrometer and referenced to deuterium DMSO (DMSO-d6). Chemical shifts are expressed in ppm. In the NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet; m,

CONCLUSIONS Starting from the previously developed type II c-KIT kinase inhibitor 9, which has good c-KIT potency but poor PK and in-vivo efficacy properties, through the type II kinase inhibitor binding element hybrid drug design approach, we have discovered novel potent c-KIT kinase inhibitor 18. It not 6092



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Scheme 1. Synthesis of Compounds 10−30, 36−37, and 39a

Reagents and conditions: (a) 4-aminophenol or 4-amino-3-chlorophenol, NaH, DMSO, 110 °C, overnight; (b) R1CH2COOH or 1-phenyl-1cyclopropanecarboxylic acid, HATU, DIPEA, DMF, rt, overnight; (c) cyclohexylacetyl chloride, DIPEA, DCM, 0 °C, 30 min; (d) 2-fluoro-4nitrophenol or 3-fluoro-4-nitrophenol, Ph2O, 140 °C, overnight; (e) Fe, NH4Cl, EtOH, reflux, 5 h; (f) 2-(4-chloro-3-(trifluoromethyl)phenyl)acetic acid, HATU, DIPEA, DMF, rt, overnight. a

Scheme 2. Synthesis of Compounds 31−32a

a Reagents and conditions: (a) methyl 4-hydroxybenzoate, 145 °C, overnight; (b) LiOH, CH3OH/THF, rt, overnight; (c) R1CH2NH2, EDCI, HOBt, Et3N, DMF, rt, overnight.

pressure, and the crude product was purified by silica gel flash chromatography [eluting with MeOH in dimethyl chloride (DCM) 5%] to give title compound 10 as a white solid (53 mg, yield 80%). 1 H NMR (400 MHz, DMSO-d6): δ 10.34 (s, 1H), 8.47 (s, 1H), 7.75 (s, 2H), 7.52 (s, 1H), 7.36−7.23 (m, 8H), 6.44 (s, 1H), 3.94 (s, 6H), 3.67 (s, 2H). LC/MS (ESI, m/z): 415.1 [M + H]+. Compounds 11−28, 30, and 33−44 were prepared following the synthetic procedure of 10. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(pyridin4-yl)acetamide (11). It was obtained with a yield of 65%. 1H NMR (500 MHz, DMSO-d6): δ 10.42 (s, 1H), 8.54−8.52 (m, 2H), 8.47 (d, J = 5.3 Hz, 1H), 7.75−7.72 (m, 2H), 7.51 (s, 1H), 7.39 (s, 1H), 7.38−7.37 (m, 2H), 7.25−7.22 (m, 2H), 6.45 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.74 (s, 2H). LC/MS (ESI, m/z): 416.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-1-phenylcyclopropane-1-carboxamide (12). It was obtained with a yield of 77%. 1H NMR (400 MHz, DMSO-d6): δ 9.29 (s, 1H), 8.45 (s, 1H),

multiplet; and br, broad peak. LC/MS was performed on Agilent 6224 TOF using an ESI source coupled to an Agilent 1260 Infinity highperformance liquid chromatography system operating in the reverse mode with an Agilent Eclipse Plus C18 1.8 μm, 3.0 mm × 50 mm column. Flash column chromatography was conducted using silica gel (SiliCycle 40−64 μm). All of the compounds were determined to be >95% pure. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-phenylacetamide (10). To a solution of 45a (50 mg, 0.16 mmol) in anhydrous N,N-dimethylformamide (DMF) (1 mL) were added 2phenylacetic acid (34 mg, 0.25 mmol), hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) (95 mg, 0.25 mmol) and N,N-diisopropylethylamine (DIPEA) (62 mg, 0.48 mmol), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with EtOAc (3 × 10 mL). The EtOAc layer was washed with a saturated aqueous NaCl solution and was dried over anhydrous Na2SO4. The solvent was removed by distillation under reduced 6093



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Scheme 3. Synthesis of Compounds 33−34a

Reagents and conditions: (a) 4-nitroaniline, TsOH, NMP, 120 °C, overnight; (b) Pd/C, MeOH, rt, overnight; (c) 2-(4-chloro-3(trifluoromethyl)phenyl)acetic acid, HATU, DIPEA, DMF, rt, overnight; (d) MeI, NaH, anhydrous THF, 0 °C-rt, 1 h. a

Scheme 4. Synthesis of Compound 35a

a

Reagents and conditions: (a) 4-aminobenzenethiol, rt, overnight; (b) 2-(4-chloro-3-(trifluoromethyl)phenyl)acetic acid, HATU, DIPEA, DMF, rt, overnight.

Scheme 5. Synthesis of Compounds 38 and 40−44a

a Reagents and conditions: (a) tBuOK or Cs2CO3, anhydrous DMSO, rt-100 °C, 3 h; (b) Fe, NH4Cl, EtOH, reflux, 5 h; (c) 2-(4-chloro-3(trifluoromethyl)phenyl)acetic acid, HATU, DIPEA, DMF, rt, overnight.

(d, J = 5.2 Hz, 1H), 7.75−7.72 (m, 3H), 7.67−7.63 (m, 2H), 7.61− 7.58 (m, 1H), 7.51 (s, 1H), 7.40 (s, 1H), 7.25−7.23 (m, 2H), 6.45 (d, J = 5.3 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.82 (s, 2H). LC/MS (ESI, m/z): 483.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(3fluorophenyl)acetamide (15). It was obtained with a yield of 83%. 1 H NMR (500 MHz, DMSO-d6): δ 10.34 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.75−7.72 (m, 2H), 7.50 (s, 1H), 7.41−7.37 (m, 2H), 7.24− 7.18 (m, 4H), 7.12−7.08 (m, 1H), 6.43 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.71 (s, 2H). LC/MS (ESI, m/z): 433.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(3methoxyphenyl)acetamide (16). It was obtained with a yield of 81%. 1H NMR (500 MHz, DMSO-d6): δ 10.33 (s, 1H), 8.53 (d, J = 5.6 Hz, 1H), 7.76 (d, J = 9.0 Hz, 2H), 7.56 (s, 1H), 7.42 (s, 1H), 7.27−7.24 (m, 3H), 6.94−6.92 (m, 2H), 6.85−6.83 (m, 1H), 6.53 (d,

7.71 (s, 2H), 7.50 (s, 1H), 7.44−7.39 (m, 5H), 7.32 (s, 1H), 7.20− 7.18 (m, 2H), 6.42 (s, 1H), 3.94 (s, 6H), 1.47 (s, 2H), 1.15 (s, 2H). LC/MS (ESI, m/z): 441.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(4(trifluoromethyl)phenyl)acetamide (13). It was obtained with a yield of 78%. 1H NMR (500 MHz, DMSO-d6): δ 10.40 (s, 1H), 8.47 (d, J = 5.3 Hz, 1H), 7.75−7.71 (m, 4H), 7.58 (d, J = 8.0 Hz, 2H), 7.51 (s, 1H), 7.39 (s, 1H), 7.25−7.22 (m, 2H), 6.44 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.80 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 168.86, 160.52, 153.07, 149.81, 149.71, 149.23, 146.79, 141.20, 137.11, 130.54, 127.84 (q, J = 32.0 Hz), 125.61 (q, J = 4.0 Hz), 124.86 (q, J = 272.0 Hz), 121.93, 121.33, 115.61, 108.21, 103.50, 99.57, 56.18, 56.15, 43.35. LC/MS (ESI, m/z): 483.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(3(trifluoromethyl)phenyl)acetamide (14). It was obtained with a yield of 80%. 1H NMR (500 MHz, DMSO-d6): δ 10.40 (s, 1H), 8.47 6094



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(d, J = 5.3 Hz, 1H), 7.74−7.71 (m, 3H), 7.67 (t, J = 7.5 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.52 (s, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.40 (s, 1H), 7.24 (d, J = 9.0 Hz, 2H), 6.47 (d, J = 5.3 Hz, 1H), 3.96−3.95 (m, 5H), 3.94 (s, 3H). LC/MS (ESI, m/z): 483.1 [M + H]+. 2-(2,6-Difluorophenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (25). It was obtained with a yield of 67%. 1 H NMR (500 MHz, DMSO-d6): δ 10.45 (s, 1H), 8.50 (d, J = 5.4 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.53 (s, 1H), 7.44−7.37 (m, 2H), 7.25 (d, J = 9.0 Hz, 2H), 7.12 (t, J = 7.8 Hz, 2H), 6.49 (d, J = 5.4 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.81 (s, 2H). LC/MS (ESI, m/z): 451.1 [M + H]+. 2-(2,4-Difluorophenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (26). It was obtained with a yield of 77%. 1 H NMR (500 MHz, DMSO-d6): δ 10.36 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.51 (s, 1H), 7.49−7.44 (m, 1H), 7.39 (s, 1H), 7.26−7.20 (m, 3H), 7.08 (tt, J = 8.6, 1.7 Hz, 1H), 6.44 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.75 (s, 2H). LC/MS (ESI, m/z): 451.1 [M + H]+. 2-(Benzo[d][1,3]dioxol-5-yl)-N-(4-((6,7-dimethoxyquinolin4-yl)oxy)phenyl)acetamide (27). It was obtained with a yield of 75%. 1H NMR (500 MHz, DMSO-d6): δ 10.25 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.50 (s, 1H), 7.39 (s, 1H), 7.22 (d, J = 9.0 Hz, 2H), 6.92 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.81 (dd, J = 7.9, 1.7 Hz, 1H), 6.43 (d, J = 5.2 Hz, 1H), 5.99 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 3.57 (s, 2H). LC/MS (ESI, m/z): 459.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(naphthalen-2-yl)acetamide (28). It was obtained with a yield of 84%. 1H NMR (500 MHz, DMSO-d6): δ 10.40 (s, 1H), 8.47 (d, J = 5.3 Hz, 1H), 7.91−7.89 (m, 3H), 7.86 (s, 1H), 7.76 (d, J = 9.0 Hz, 2H), 7.54−7.47 (m, 4H), 7.39 (s, 1H), 7.23 (d, J = 9.0 Hz, 2H), 6.45 (d, J = 5.3 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.85 (s, 2H). LC/MS (ESI, m/z): 465.1 [M + H]+. 2-Cyclohexyl-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (29). To a solution of 45a (50 mg, 0.16 mmol) in anhydrous DCM (1 mL) was added DIPEA (62 mg, 0.48 mmol). Next, commercially available 2-cyclohexylacetyl chloride (38 mg, 0.24 mmol) was added thereto, and the mixture was stirred at 0 °C for 30 min. The mixture was removed by distillation under reduced pressure, and the crude product was purified by silica gel flash chromatography (eluting with MeOH in DCM 5%) to give title compound 29 as a white solid (53 mg, yield 79%). 1H NMR (500 MHz, DMSO-d6): δ 10.00 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.51 (s, 1H), 7.39 (s, 1H), 7.21 (d, J = 8.9 Hz, 2H), 6.43 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 2.21 (d, J = 7.1 Hz, 2H), 1.81−1.61 (m, 6H), 1.28−1.13 (m, 3H), 1.06−0.95 (m, 2H). LC/MS (ESI, m/ z): 421.2 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(4-methylpiperazin-1-yl)acetamide (30). It was obtained with a yield of 82%. 1H NMR (500 MHz, DMSO-d6): δ 9.84 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.78 (d, J = 9.0 Hz, 2H), 7.51 (s, 1H), 7.39 (s, 1H), 7.23 (d, J = 8.9 Hz, 2H), 6.44 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.14 (s, 2H), 2.53 (s, 4H), 2.40 (s, 4H), 2.19 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 168.78, 160.46, 153.02, 149.78, 149.77, 149.29, 146.90, 136.64, 121.85, 121.64, 115.60, 108.30, 103.49, 99.55, 62.25, 56.18, 56.15, 54.96, 53.11, 46.15. LC/MS (ESI, m/z): 437.2 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-N-(4-(trifluoromethyl)benzyl)benzamide (31). To a solution of 48 (20 mg, 0.06 mmol) in anhydrous DMF (1 mL) were added (4-(trifluoromethyl)phenyl)methanamine (12.6 mg, 0.072 mmol), EDCI (17.2 mg, 0.09 mmol), HOBt (12.1 mg, 0.09 mmol), and Et3N (12.1 mg, 0.12 mmol) and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with EtOAc (3 × 5 mL). The EtOAc layer was washed with a saturated aqueous NaCl solution and was dried over anhydrous Na2SO4. The solvent was removed by distillation under reduced pressure, and the crude product was purified by silica gel flash chromatography (eluting with EtOAc in PE 50%) to give title compound 31 as a white solid (14 mg, yield 48%). 1H NMR (500 MHz, DMSO-d6): δ 9.22 (t, J =

J = 5.5 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.76 (s, 3H), 3.63 (s, 2H). LC/MS (ESI, m/z): 445.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(4-fluoro3-(trifluoromethyl)phenyl)acetamide (17). It was obtained with a yield of 68%. 1H NMR (500 MHz, DMSO-d6): δ 10.38 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.77 (dd, J = 7.1, 2.2 Hz, 1H), 7.74−7.70 (m, 3H), 7.51−7.48 (m, 2H), 7.39 (s, 1H), 7.23 (d, J = 9.0 Hz, 2H), 6.43 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.81 (s, 2H). LC/MS (ESI, m/z): 501.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (18). It was obtained with a yield of 72%. 1H NMR (500 MHz, DMSO-d6): δ 10.40 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.86 (s, 1H), 7.75−7.66 (m, 4H), 7.51 (s, 1H), 7.40 (s, 1H), 7.24 (d, J = 8.8 Hz, 2H), 6.44 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.83 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 168.71, 160.45, 153.05, 149.80, 149.75, 149.24, 146.85, 137.03, 136.44, 135.59, 131.95, 129.42, 129.20 (q, J = 5.1 Hz), 126.80 (q, J = 30.6 Hz), 123.40 (q, J = 273.0 Hz), 121.93, 121.35, 115.60, 108.26, 103.51, 99.56, 56.18, 56.15, 42.31. LC/MS (ESI, m/z): 517.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(4-methyl-3-(trifluoromethyl)phenyl)acetamide (19). It was obtained with a yield of 74%. 1H NMR (400 MHz, DMSO-d6): δ 1.37 (s, 1H), 8.47 (s, 1H), 7.75−7.67 (m, 3H), 7.54−7.51 (m, 2H), 7.44−7.40 (m, 2H), 7.24−7.22 (m, 2H), 6.43 (s, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.76 (s, 2H), 2.44 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 169.18, 160.46, 153.03, 149.79, 149.69, 149.26, 146.86, 137.12, 134.71, 134.61, 133.63, 132.67, 127.69 (q, J = 29.2 Hz), 126.81 (q, J = 5.5 Hz), 125.09 (q, J = 273.8 Hz), 121.93, 121.32, 115.60, 108.28, 103.50, 99.56, 56.18, 56.16, 42.75, 18.86. LC/MS (ESI, m/z): 497.1 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(3,4dimethylphenyl)acetamide (20). It was obtained with a yield of 72%. 1H NMR (500 MHz, DMSO-d6): δ 10.25 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.50 (s, 1H), 7.39 (s, 1H), 7.21 (d, J = 9.0 Hz, 2H), 7.13−7.04 (m, 3H), 6.42 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.57 (s, 2H), 2.21 (s, 3H), 2.19 (s, 3H). LC/MS (ESI, m/z): 443.2 [M + H]+. 2-(3,4-Dimethoxyphenyl)-N-(4-((6,7-dimethoxyquinolin-4yl)oxy)phenyl)acetamide (21). It was obtained with a yield of 77%. 1 H NMR (500 MHz, DMSO-d6): δ 10.24 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.50 (s, 1H), 7.39 (s, 1H), 7.22 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 2.1 Hz, 1H), 6.93−6.84 (m, 2H), 6.43 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 3.58 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 169.89, 160.52, 153.04, 149.79, 149.57, 149.22, 149.04, 148.14, 146.81, 137.33, 128.76, 121.89, 121.56, 121.29, 115.61, 113.53, 112.32, 108.22, 103.46, 99.57, 56.15, 56.13, 56.00, 55.93, 43.40. LC/MS (ESI, m/z): 475.1 [M + H]+. 2-(3,4-Difluorophenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (22). It was obtained with a yield of 85%. 1 H NMR (500 MHz, DMSO-d6): δ 10.33 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.50 (s, 1H), 7.44−7.37 (m, 3H), 7.23 (d, J = 9.0 Hz, 2H), 7.20−7.17 (m, 1H), 6.43 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.70 (s, 2H). LC/MS (ESI, m/z): 451.1 [M + H]+. 2-(3,4-Dichlorophenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (23). It was obtained with a yield of 84%. 1 H NMR (500 MHz, DMSO-d6): δ 10.35 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.63 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.50 (s, 1H), 7.39 (s, 1H), 7.35 (dd, J = 8.3, 2.1 Hz, 1H), 7.23 (d, J = 9.0 Hz, 2H), 6.43 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.72 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 168.74, 160.44, 153.02, 149.79, 149.74, 149.25, 146.89, 137.41, 137.05, 131.77, 131.23, 130.84, 130.17, 129.82, 121.92, 121.35, 115.61, 108.28, 103.49, 99.56, 56.16, 56.13, 42.39. LC/MS (ESI, m/ z): 483.0 [M + H]+. N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-2-(2(trifluoromethyl)phenyl)acetamide (24). It was obtained with a yield of 66%. 1H NMR (500 MHz, DMSO-d6): δ 10.37 (s, 1H), 8.48 6095



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2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-2-methylphenyl)acetamide (38). It was obtained with a yield of 60%. 1H NMR (500 MHz, DMSOd6): δ 9.67 (s, 1H), 8.48 (d, J = 5.3 Hz, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.68 (dd, J = 8.3, 2.0 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.49 (s, 1H), 7.40 (s, 1H), 7.16 (d, J = 2.8 Hz, 1H), 7.07 (dd, J = 8.7, 2.8 Hz, 1H), 6.48 (d, J = 5.3 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.85 (s, 2H), 2.22 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 168.89, 160.24, 153.09, 151.51, 149.84, 149.22, 146.82, 136.79, 135.48, 134.86, 133.93, 131.99, 129.35, 129.04 (q, J = 5.3 Hz), 127.31, 126.82 (q, J = 30.9 Hz), 123.41 (q, J = 273.0 Hz), 122.94, 118.81, 115.71, 108.21, 103.88, 99.53, 56.20, 56.15, 41.83, 18.26.LC/ MS (ESI, m/z): 531.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)acetamide (39). It was obtained with a yield of 68%. 1H NMR (500 MHz, DMSO-d6): δ 10.58 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.87−7.84 (m, 2H), 7.72 (d, J = 8.2 Hz, 1H), 7.66 (dd, J = 8.4, 2.0 Hz, 1H), 7.52 (s, 1H), 7.44−7.42 (m, 2H), 7.41 (s, 1H), 6.44 (dd, J = 5.2, 1.0 Hz, 1H), 3.95 (s, 3H), 3.95 (s, 3H), 3.85 (s, 2H). LC/MS (ESI, m/z): 535.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(3-chloro-4-((6,7dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (40). It was obtained with a yield of 66%. 1H NMR (500 MHz, DMSO-d6): δ 10.57 (s, 1H), 8.48 (d, J = 5.3 Hz, 1H), 8.04 (d, J = 2.5 Hz, 1H), 7.86 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.66 (dd, J = 8.3, 2.1 Hz, 1H), 7.60 (dd, J = 8.9, 2.5 Hz, 1H), 7.54 (s, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.41 (s, 1H), 6.36 (d, J = 5.3 Hz, 1H), 3.96 (s, 3H), 3.95 (s, 3H), 3.85 (s, 2H). LC/MS (ESI, m/z): 551.0 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-methylphenyl)acetamide (41). It was obtained with a yield of 62%. 1H NMR (500 MHz, DMSOd6): δ 10.32 (s, 1H), 8.43 (d, J = 5.2 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.66 (dd, J = 7.4, 2.2 Hz, 2H), 7.56 (s, 1H), 7.54 (dd, J = 8.7, 2.6 Hz, 1H), 7.39 (s, 1H), 7.15 (d, J = 8.7 Hz, 1H), 6.26 (d, J = 5.2 Hz, 1H), 3.95 (s, 6H), 3.81 (s, 2H), 2.08 (s, 3H). LC/MS (ESI, m/z): 531.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-methoxyphenyl)acetamide (42). It was obtained with a yield of 82%. 1H NMR (500 MHz, DMSO-d6): δ 10.41 (s, 1H), 8.42 (d, J = 5.3 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.67 (dd, J = 8.3, 2.0 Hz, 1H), 7.63 (d, J = 2.2 Hz, 1H), 7.52 (s, 1H), 7.38 (s, 1H), 7.24−7.20 (m, 2H), 6.28 (d, J = 5.3 Hz, 1H), 3.94 (s, 3H), 3.94 (s, 3H), 3.83 (s, 2H), 3.68 (s, 3H). LC/ MS (ESI, m/z): 547.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-(trifluoromethyl)phenyl)acetamide (43). It was obtained with a yield of 72%. 1H NMR (500 MHz, DMSO-d6): δ 10.67 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 8.24 (d, J = 2.6 Hz, 1H), 7.89 (dd, J = 9.0, 2.6 Hz, 1H), 7.86 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.67 (dd, J = 8.3, 2.1 Hz, 1H), 7.45−7.43 (m, 2H), 7.42 (s, 1H), 6.55 (d, J = 5.2 Hz, 1H), 3.96 (s, 3H), 3.91 (s, 3H), 3.86 (s, 2H). LC/MS (ESI, m/z): 585.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(3-cyano-4-((6,7dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (44). It was obtained with a yield of 77%. 1H NMR (500 MHz, DMSO-d6): δ 10.72 (s, 1H), 8.55 (d, J = 5.3 Hz, 1H), 8.24 (d, J = 2.6 Hz, 1H), 7.90 (dd, J = 9.1, 2.6 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.66 (dd, J = 8.3, 2.1 Hz, 1H), 7.51 (s, 1H), 7.47−7.45 (m, 2H), 6.61 (d, J = 5.3 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.87 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 169.37, 159.51, 153.51, 151.22, 150.31, 148.96, 146.52, 137.46, 136.01, 135.70, 131.97, 129.53, 129.32 (q, J = 5.3 Hz), 126.82 (q, J = 30.5 Hz), 126.49, 123.93, 123.38 (q, J = 273.0 Hz), 123.23, 115.58, 115.50, 107.91, 105.91, 104.28, 99.33, 56.34, 56.31, 42.17. LC/MS (ESI, m/z): 542.1 [M + H]+. 4-(3-Fluoro-4-nitrophenoxy)-6,7-dimethoxyquinoline (45a). A mixture of 4-chloro-6,7-dimethoxyquinoline (0.71 g, 3.18 mmol) and 3-fluoro-4-nitrophenol (1.0 g, 6.37 mmol) in Ph2O (12 mL) was heated at 140 °C for overnight. The reaction mixture was allowed to cool to room temperature; then, the solid was collected by filtration and washed with Et2O to give title compound 45a as a yellow solid

6.0 Hz, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 7.9 Hz, 2H), 7.47 (s, 1H), 7.43 (s, 1H), 7.36 (d, J = 8.7 Hz, 2H), 6.61 (d, J = 5.2 Hz, 1H), 4.58 (d, J = 5.8 Hz, 2H), 3.96 (s, 3H), 3.92 (s, 3H). LC/MS (ESI, m/z): 483.1 [M + H]+. Compound 32 was prepared following the synthetic procedure of 31. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-N-(3-(trifluoromethyl)benzyl)benzamide (32). It was obtained with a yield of 40%. 1H NMR (500 MHz, DMSO-d6): δ 9.21 (t, J = 6.0 Hz, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H), 7.69 7.58 (m, 4H), 7.46 (s, 1H), 7.42 (s, 1H), 7.36 (d, J = 8.7 Hz, 2H), 6.62 (d, J = 5.2 Hz, 1H), 4.58 (d, J = 5.9 Hz, 2H), 3.95 (s, 3H), 3.91 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 165.95, 159.43, 157.37, 153.25, 150.05, 149.17, 146.90, 141.65, 131.95, 131.38, 130.23, 129.89, 129.50 (q, J = 31.3 Hz), 124.76 (q, J = 272.2 Hz), 124.27 (q, J = 3.8 Hz), 124.04 (q, J = 4.1 Hz), 120.74, 116.00, 108.17, 105.05, 99.50, 56.26, 56.19, 42.81. LC/MS (ESI, m/z): 483.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)amino)phenyl)acetamide (33). It was obtained with a yield of 66%. 1H NMR (500 MHz, DMSO-d6): δ 10.29 (s, 1H), 8.76 (s, 1H), 8.24 (d, J = 5.4 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.72−7.64 (m, 5H), 7.28 (d, J = 8.8 Hz, 2H), 7.24 (s, 1H), 6.68 (d, J = 5.5 Hz, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.81 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 167.43, 151.14, 147.59, 147.12, 146.68, 144.56, 135.55, 135.22, 134.60, 134.51, 130.89, 128.33, 128.11 (q, J = 5.3 Hz), 125.74 (q, J = 31.1 Hz), 122.96, 122.36 (d, J = 273.0 Hz), 119.63, 112.93, 107.13, 100.47, 99.57, 55.35, 54.91, 41.28. LC/MS (ESI, m/z): 516.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)(methyl)amino)phenyl)acetamide (34). It was obtained with a yield of 62%. 1HNMR (500 MHz, DMSO-d6): δ 10.17 (s, 1H), 8.59 (d, J = 5.3 Hz, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.62 (dd, J = 8.3, 2.0 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.30 (s, 1H), 7.10 (d, J = 5.3 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H), 6.77 (s, 1H), 3.89 (s, 3H), 3.76 (s, 2H), 3.43 (s, 3H), 3.41 (s, 3H). 13 C NMR (125 MHz, DMSO-d6): δ 167.34, 151.54, 151.18, 147.34, 146.58, 144.53, 144.10, 135.49, 134.49, 133.60, 130.86, 128.29, 128.06 (q, J = 5.3 Hz), 125.71 (q, J = 30.7 Hz), 122.33 (q, J = 273.1 Hz), 121.19, 119.90, 116.42, 109.71, 106.26, 102.77, 55.07, 54.32, 41.66, 41.16. LC/MS (ESI, m/z): 530.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)thio)phenyl)acetamide (35). It was obtained with a yield of 63%. 1H NMR (500 MHz, DMSO-d6): δ 10.52 (s, 1H), 8.41 (d, J = 4.9 Hz, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.77 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 8.3 Hz, 1H), 7.66 (dd, J = 8.4, 2.0 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.39 (s, 1H), 7.31 (s, 1H), 6.63 (d, J = 4.8 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.85 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ 169.18, 152.73, 150.07, 147.73, 145.48, 144.75, 141.01, 136.38, 136.24, 135.64, 131.96, 129.46, 129.26 (q, J = 5.2 Hz), 126.81 (q, J = 30.9 Hz), 123.39 (q, J = 273.0 Hz), 122.77, 121.01, 120.76, 116.56, 108.84, 101.52, 56.22, 56.14, 42.36. LC/MS (ESI, m/z): 533.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-2-fluorophenyl)acetamide (36). It was obtained with a yield of 73%. 1H NMR (500 MHz, DMSO-d6): δ 10.14 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 7.96 (t, J = 8.9 Hz, 1H), 7.87 (d, J = 2.0 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.66 (dd, J = 8.3, 2.0 Hz, 1H), 7.47 (s, 1H), 7.41 (s, 1H), 7.35 (dd, J = 11.4, 2.7 Hz, 1H), 7.10−7.08 (m, 1H), 6.57 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.92 (s, 3H), 3.89 (s, 2H). LC/MS (ESI, m/z): 535.1 [M + H]+. 2-(4-Chloro-3-(trifluoromethyl)phenyl)-N-(2-chloro-4-((6,7dimethoxyquinolin-4-yl)oxy)phenyl)acetamide (37). It was obtained with a yield of 70%. 1H NMR (500 MHz, DMSO-d6): δ 9.93 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H), 7.89 (d, J = 1.9 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 2.7 Hz, 1H), 7.47 (s, 1H), 7.41 (s, 1H), 7.25 (dd, J = 8.9, 2.7 Hz, 1H), 6.58 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.92 (s, 3H), 3.90 (s, 2H). LC/MS (ESI, m/z): 551.0 [M + H]+. 6096



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(700 mg, yield 63%), 1H NMR (500 MHz, DMSO-d6): δ 8.92 (d, J = 6.4 Hz, 1H), 8.42 (t, J = 8.8 Hz, 1H), 7.89−7.85 (m, 2H), 7.68 (s, 1H), 7.54 (dd, J = 9.1, 2.5 Hz, 1H), 7.23 (d, J = 6.5 Hz, 1H), 4.04 (s, 3H), 4.02 (s, 3H). LC/MS (ESI, m/z): 345.0 [M + H]+. Compound 45b was prepared following the synthetic procedure of 45a. 4-(2-Fluoro-4-nitrophenoxy)-6,7-dimethoxyquinoline (45b). Yield 55%. 1H NMR (500 MHz, DMSO-d6): δ 8.90 (d, J = 6.3 Hz, 1H), 8.57 (dd, J = 10.2, 2.7 Hz, 1H), 8.33−8.31 (m, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.74 (s, 1H), 7.73 (s, 1H), 7.17 (d, J = 6.4 Hz, 1H), 4.05 (s, 3H), 4.03 (s, 3H). LC/MS (ESI, m/z): 345.0 [M + H]+. 6,7-Dimethoxy-4-(3-methyl-4-nitrophenoxy)quinoline (45c). To a solution of 6,7-dimethoxyquinolin-4-ol (3.0 g, 14.6 mmol) in anhydrous DMSO (30 mL) was added tBuOK (1.63 g, 14.6 mmol) in batches, and the mixture was stirred at room temperature for 20 min. Next, commercially available 4-fluoro-2-methyl-1nitrobenzene 49a (2.71 g, 17.5 mmol) was added thereto, and the mixture was stirred at 100 °C for 3 h. Water was added to the reaction solution, and the mixture was extracted with EtOAc (3 × 300 mL). The EtOAc layer was washed with water and a saturated aqueous NaCl solution and was dried over anhydrous Na2SO4. EtOAc was concentrated to give the crude product which was then purified by silica gel flash chromatography (eluting with EtOAc in petroleum ether 50%) to give product 45c as a yellow solid (1.2 g, yield 24%). 1 H NMR (500 MHz, DMSO-d6): δ 8.59 (d, J = 5.1 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.45 (s, 1H), 7.40 (d, J = 2.5 Hz, 2H), 7.26 (dd, J = 9.0, 2.7 Hz, 1H), 6.80 (d, J = 5.1 Hz, 1H), 3.96 (s, 3H), 3.90 (s, 3H), 2.56 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 157.69, 157.13, 152.20, 149.15, 148.32, 146.27, 144.53, 135.98, 126.84, 122.70, 117.49, 115.14, 107.39, 105.34, 98.21, 55.21, 55.14, 19.37. LC/MS (ESI, m/z): 341.1 [M + H]+ Compounds 45d−g were prepared following the synthetic procedure of 45c. Compound 45h was prepared following the same procedure; only the base tBuOK was replaced with Cs2CO3. 4-(2-Chloro-4-nitrophenoxy)-6,7-dimethoxyquinoline (45d). It was obtained with a yield of 64%, 1H NMR (500 MHz, DMSO-d6): δ 8.59−8.58 (m, 2H), 8.29 (dd, J = 9.0, 2.8 Hz, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.46 (s, 1H), 7.42 (s, 1H), 6.73 (d, J = 5.1 Hz, 1H), 3.97 (s, 3H), 3.91 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 157.73, 155.99, 153.37, 150.37, 149.33, 147.33, 145.10, 126.95, 126.44, 125.23, 122.78, 115.59, 108.49, 105.64, 99.11, 56.32, 56.26. LC/MS (ESI, m/z): 361.0 [M + H]+ 6,7-Dimethoxy-4-(2-methyl-4-nitrophenoxy)quinoline (45e). It was obtained with a yield of 65%, 1H NMR (500 MHz, DMSO-d6): δ 8.55 (d, J = 5.1 Hz, 1H), 8.36 (d, J = 2.4 Hz, 1H), 8.16 (dd, J = 8.9, 2.9 Hz, 1H), 7.46 (s, 1H), 7.45 (s, 1H), 7.31 (d, J = 8.9 Hz, 1H), 6.61 (d, J = 5.1 Hz, 1H), 3.97 (s, 3H), 3.92 (s, 3H), 2.35 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ 158.47, 158.19, 153.27, 150.20, 149.37, 147.24, 144.62, 132.00, 127.44, 124.02, 121.17, 115.75, 108.46, 105.15, 99.34, 56.23, 16.09. LC/MS (ESI, m/z): 341.1 [M + H]+ 6,7-Dimethoxy-4-(2-methoxy-4-nitrophenoxy)quinoline (45f). It was obtained with a yield of 37%, 1H NMR (500 MHz, DMSO-d6): δ 8.48 (d, J = 5.2 Hz, 1H), 8.04 (d, J = 2.6 Hz, 1H), 7.97 (dd, J = 8.8, 2.6 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.47 (s, 1H), 7.42 (s, 1H), 6.49 (d, J = 5.2 Hz, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 3.89 (s, 3H). LC/MS (ESI, m/z): 357.1 [M + H]+. 6,7-Dimethoxy-4-(4-nitro-2-(trifluoromethyl)phenoxy)quinoline (45g). It was obtained with a yield of 64%, 1H NMR (500 MHz, DMSO-d6): δ 8.68 (d, J = 5.1 Hz, 1H), 8.62−8.61 (m, 1H), 8.53−8.50 (m, 1H), 7.49 (d, J = 1.4 Hz, 1H), 7.44 (d, J = 9.1 Hz, 1H), 7.25 (s, 1H), 7.06 (dd, J = 5.0, 1.0 Hz, 1H), 3.97 (s, 3H), 3.85 (s, 3H). LC/MS (ESI, m/z): 395.1 [M + H]+. 2-((6,7-Dimethoxyquinolin-4-yl)oxy)-5-nitrobenzonitrile (45h). It was obtained with a yield of 44%, 1H NMR (500 MHz, DMSO-d6): δ 8.98 (d, J = 2.8 Hz, 1H), 8.70 (d, J = 5.1 Hz, 1H), 8.48 (dd, J = 9.3, 2.8 Hz, 1H), 7.50 (s, 1H), 7.34−7.32 (m, 2H), 7.12 (d, J = 5.1 Hz, 1H), 3.97 (s, 3H), 3.89 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 162.31, 156.62, 153.57, 150.75, 149.47, 147.66, 143.77,

131.22, 130.91, 119.67, 116.30, 114.47, 108.62, 108.48, 105.14, 98.97, 56.39, 56.35. LC/MS (ESI, m/z): 352.1 [M + H]+. 4-((6,7-Dimethoxy-4-quinolyl)oxy)aniline (46a). To a solution of 4-aminophenol (10 g, 91.7 mmol) in anhydrous DMSO (60 mL) was added NaH (60 wt %, 4.0 g, 100.9 mol) in batches, and the mixture was stirred at room temperature for 20 min. Next, commercially available 4-chloro-6,7-dimethoxyquinoline (20 g, 91.7 mmol) was added thereto, and the mixture was stirred at 110 °C for overnight. Water was added to the reaction solution, and the mixture was extracted with EtOAc (3 × 50 mL). The EtOAc layer was washed with a saturated aqueous NaHCO3 solution and was dried over anhydrous Na2SO4. The solvent was removed by distillation under reduced pressure. Methanol was added to the residue, and the precipitated crystal was collected by suction filtration to give 46a (21.6 g, yield 80%). 1H NMR (500 MHz, DMSO-d6): δ 8.44 (d, J = 5.3 Hz, 1H), 7.52 (s, 1H), 7.38 (s, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.70 (d, J = 8.7 Hz, 2H), 6.39 (d, J = 5.2 Hz, 1H), 5.18 (s, 2H), 3.95 (s, 3H), 3.94 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 160.37, 151.82, 148.52, 148.26, 146.08, 145.70, 142.88, 121.20, 114.44, 114.31, 107.22, 101.71, 98.62, 55.08, 55.05. LC/MS (ESI, m/z): 297.1 [M + H]+. Compound 46b was prepared following the synthetic procedure of 46a. 2-Chloro-4-((6,7-dimethoxyquinolin-4-yl)oxy)aniline (46b). It was obtained with a yield of 85%. 1H NMR (500 MHz, DMSOd6): δ 8.45 (d, J = 5.2 Hz, 1H), 7.50 (s, 1H), 7.38 (s, 1H), 7.21 (d, J = 2.7 Hz, 1H), 7.00 (dd, J = 8.7, 2.6 Hz, 1H), 6.92 (d, J = 8.7 Hz, 1H), 6.43 (d, J = 5.2 Hz, 1H), 5.44 (s, 2H), 3.95 (s, 3H), 3.94 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 159.92, 151.89, 148.61, 148.27, 145.74, 142.84, 142.27, 121.39, 120.50, 116.51, 115.43, 114.34, 107.22, 101.90, 98.55, 55.09, 55.07. LC/MS (ESI, m/z): 331.0 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-2-fluoroaniline (46c). A mixture of 45a (1.0 g, 2.9 mmol), Fe (0.81 g, 14.5 mmol), and NH4Cl (5 mL) in EtOH (15 mL) was stirred reflux for 5 h. The reaction mixture was removed by distillation under reduced pressure, and the crude product was purified by silica gel flash chromatography (eluting with EtOAc in PE 50%) to give product 46c as a yellow solid (0.8 g, yield 88%), 1H NMR (500 MHz, DMSO-d6): δ 8.45 (d, J = 5.3 Hz, 1H), 7.49 (s, 1H), 7.38 (s, 1H), 7.06 (dd, J = 11.8, 2.5 Hz, 1H), 6.92−6.82 (m, 2H), 6.43 (d, J = 5.3 Hz, 1H), 5.21 (s, 2H), 3.94 (s, 3H), 3.94 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 160.99, 152.93, 150.66 (d, J = 239.9 Hz), 149.66, 149.33, 146.79, 143.33 (d, J = 9.2 Hz), 134.92 (d, J = 12.8 Hz), 117.95 (d, J = 3.4 Hz), 116.91 (d, J = 5.6 Hz), 115.39, 109.62 (d, J = 21.0 Hz), 108.28, 102.96, 99.58, 56.15, 56.14. LC/MS (ESI, m/z): 315.1 [M + H]+. Compounds 46d−j were prepared following the synthetic procedure of 46c. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-fluoroaniline (46d). It was obtained with a yield of 85%. 1H NMR (500 MHz, DMSOd6): δ 8.46 (d, J = 5.3 Hz, 1H), 7.52 (s, 1H), 7.39 (s, 1H), 7.08 (t, J = 9.0 Hz, 1H), 6.57 (dd, J = 13.1, 2.6 Hz, 1H), 6.48 (dd, J = 8.7, 2.5 Hz, 1H), 6.41 (dd, J = 5.2, 1.1 Hz, 1H), 5.50 (s, 2H), 3.95 (s, 6H). 13C NMR (125 MHz, DMSO-d6): δ 159.58, 153.79 (d, J = 243.5 Hz), 151.91, 148.67, 148.29, 147.96 (d, J = 10.2 Hz), 145.67, 128.71 (d, J = 12.8 Hz), 123.55, 113.94, 109.55, 107.21, 100.98, 100.87 (d, J = 21.1 Hz), 98.47, 55.11. LC/MS (ESI, m/z): 315.1 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-2-methylaniline (46e). It was obtained with a yield of 88%. 1H NMR (500 MHz, DMSO-d6): δ 8.42 (d, J = 5.2 Hz, 1H), 7.51 (s, 1H), 7.37 (s, 1H), 6.86 (d, J = 2.7 Hz, 1H), 6.82 (dd, J = 8.5, 2.8 Hz, 1H), 6.72 (d, J = 8.5 Hz, 1H), 6.38 (d, J = 5.3 Hz, 1H), 4.93 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H), 2.09 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 160.40, 151.80, 148.50, 148.25, 145.65, 143.94, 142.97, 122.27, 121.94, 118.52, 114.42, 114.13, 107.19, 101.77, 98.58, 55.07, 55.03, 16.85. LC/MS (ESI, m/ z): 311.1 [M + H]+. 3-Chloro-4-((6,7-dimethoxyquinolin-4-yl)oxy)aniline (46f). It was obtained with a yield of 83%. 1H NMR (500 MHz, DMSOd6): δ 8.46 (d, J = 5.2 Hz, 1H), 7.53 (s, 1H), 7.40 (s, 1H), 7.10 (d, J = 8.7 Hz, 1H), 6.80 (d, J = 2.6 Hz, 1H), 6.65 (dd, J = 8.7, 2.6 Hz, 1H), 6097



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6.31 (d, J = 5.2 Hz, 1H), 5.49 (s, 2H), 3.95 (s, 6H). 13C NMR (125 MHz, DMSO-d6): δ 160.33, 152.97, 149.71, 149.31, 148.61, 146.76, 138.79, 126.40, 124.59, 115.09, 114.83, 114.26, 108.29, 102.35, 99.55, 56.18. LC/MS (ESI, m/z): 331.0 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-methylaniline (46g). It was obtained with a yield of 77%. 1H NMR (500 MHz, DMSO-d6): δ 8.42 (d, J = 5.2 Hz, 1H), 7.56 (s, 1H), 7.38 (s, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 2.6 Hz, 1H), 6.51 (dd, J = 8.5, 2.7 Hz, 1H), 6.26 (d, J = 5.2 Hz, 1H), 5.09 (s, 2H), 3.95−3.94 (m, 6H), 1.96 (s, 3H). 13 C NMR (125 MHz, DMSO-d6): δ 160.80, 152.89, 149.58, 149.39, 147.17, 146.72, 142.15, 130.52, 122.56, 116.72, 115.24, 113.19, 108.31, 102.16, 99.67, 56.15, 16.10. LC/MS (ESI, m/z): 311.1 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-methoxyaniline (46h). It was obtained with a yield of 78%. 1H NMR (500 MHz, DMSO-d6): δ 8.40 (d, J = 5.3 Hz, 1H), 7.50 (s, 1H), 7.35 (s, 1H), 6.88 (d, J = 8.5 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 6.27 (d, J = 5.2 Hz, 1H), 6.21 (dd, J = 8.5, 2.4 Hz, 1H), 5.20 (s, 2H), 3.93 (s, 6H), 3.62 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 160.05, 151.68, 151.05, 148.36, 148.23, 147.45, 145.55, 130.81, 122.36, 114.11, 107.15, 105.14, 101.01, 98.68, 98.49, 55.05, 54.58. LC/MS (ESI, m/z): 327.1 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)-3-(trifluoromethyl)aniline (46i). It was obtained with a yield of 82%. 1H NMR (500 MHz, DMSO-d6): δ 8.48 (d, J = 5.3 Hz, 1H), 7.48 (s, 1H), 7.40 (s, 1H), 7.15 (d, J = 8.7 Hz, 1H), 7.02 (d, J = 2.7 Hz, 1H), 6.92 (dd, J = 8.7, 2.7 Hz, 1H), 6.43 (d, J = 5.3 Hz, 1H), 5.66 (s, 2H), 3.96 (s, 3H), 3.92 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 160.76, 153.02, 149.79, 149.31, 147.53, 146.78, 140.19, 124.94, 123.81 (q, J = 272.9 Hz), 122.46 (q, J = 30.4 Hz), 118.83, 115.35, 111.31 (q, J = 4.8 Hz), 108.29, 102.81, 99.32, 56.20, 56.07. LC/MS (ESI, m/z): 365.1 [M + H]+. 5-Amino-2-((6,7-dimethoxyquinolin-4-yl)oxy)benzonitrile (46j). It was obtained with a yield of 88%. 1H NMR (500 MHz, DMSO-d6): δ 8.50 (d, J = 5.2 Hz, 1H), 7.53 (s, 1H), 7.43 (s, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.00−6.97 (m, 2H), 6.42 (d, J = 5.2 Hz, 1H), 5.70 (s, 2H), 3.96 (s, 3H), 3.95 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 159.30, 152.09, 148.88, 148.28, 146.76, 145.76, 143.81, 123.03, 119.53, 115.91, 115.19, 114.17, 107.23, 105.19, 101.83, 98.36, 55.17. LC/MS (ESI, m/z): 322.1 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)oxy)benzoic Acid (48). A mixture of 4-chloro-6,7-dimethoxyquinoline (1.0 g, 4.48 mmol) and 4-hydroxybenzoate (817 mg, 5.38 mmol) was heated at 145 °C for overnight. The reaction mixture was cooled and treated with saturated NaHCO3. The solution was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated. The mixture was purified by silica gel flash chromatography (eluting with MeOH in DCM 5%) to give 47 as a white solid (700 mg, yield 46%), LC/MS (ESI, m/z): 340.1 [M + H]+. To a solution of 47 (600 mg, 1.77 mmol) in MeOH/THF (6 mL/6 mL) was added LiOH (212 mg, 8.85 mmol). The mixture was stirred at room temperature overnight. The mixture was removed by distillation under reduced pressure, and the crude product was acidized with conc. HCl until pH = 5−6. The white solid was filtered and was washed with water and diethyl ether. Then, the solid was dried to give title compound 48 as a white solid (310 mg, yield 54%). 1 H NMR (500 MHz, DMSO-d6): δ 8.82 (d, J = 6.7 Hz, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.81 (s, 1H), 7.71 (s, 1H), 7.58 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 6.6 Hz, 1H), 4.04 (s, 3H), 4.04 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 166.83, 164.81, 156.44, 156.39, 151.58, 143.02, 137.72, 132.61, 129.87, 121.90, 116.05, 104.30, 100.63, 100.20, 57.04, 56.91. LC/MS (ESI, m/z): 326.1 [M + H]+. 6,7-Dimethoxy-N-(4-nitrophenyl)quinolin-4-amine (50). A mixture of 4-chloro-6,7-dimethoxyquinoline (1.0 g, 4.48 mmol), 4nitroaniline (742 mg, 5.38 mmol), and TsOH (925 mg, 5.38 mmol) in NMP (10 mL) was heated at 120 °C for overnight. The reaction mixture was allowed to cool to room temperature; then, the solid was collected by filtration and washed with saturated NaHCO3 to give title compound 50 as a yellow solid (1.1 g, yield 75%), 1H NMR (500 MHz, DMSO-d6): δ 9.70 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.21 (d, J

= 9.2 Hz, 2H), 7.62 (s, 1H), 7.44 (d, J = 9.2 Hz, 2H), 7.36 (s, 1H), 7.26 (d, J = 5.2 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 151.68, 148.48, 148.34, 147.09, 145.16, 142.96, 139.56, 125.11, 116.82, 115.42, 107.15, 106.09, 100.62, 55.31, 55.08. LC/MS (ESI, m/z): 326.1 [M + H]+. N1-(6,7-Dimethoxyquinolin-4-yl)benzene-1,4-diamine (51). A mixture of 50 (1.0 g, 4.48 mmol) and Pd/C (250 mg) in MeOH (30 mL) was stirred at room temperature for 5 h under H2 atmosphere. The reaction mixture was filtered, and the filtrate was removed by distillation under reduced pressure to afford title compound 51 as a yellow solid (1.2 g, yield 91%), 1H NMR (500 MHz, DMSO-d6): δ 8.60 (s, 1H), 8.15 (d, J = 5.5 Hz, 1H), 7.74 (s, 1H), 7.21 (s, 1H), 7.00 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 6.38 (d, J = 5.5 Hz, 1H), 5.07 (br, 2H), 3.92 (s, 3H), 3.89 (s, 3H). 13 C NMR (125 MHz, DMSO-d6): δ 152.00, 149.58, 148.34, 148.00, 146.83, 145.22, 128.61, 126.70, 115.02, 113.34, 107.99, 101.71, 99.53, 56.44, 55.90. LC/MS (ESI, m/z): 296.1 [M + H]+. 6,7-Dimethoxy-N-methyl-N-(4-nitrophenyl)quinolin-4amine (52). To a solution of 50 (1.0 g, 3.07 mmol) in anhydrous THF (20 mL) was added NaH (60 wt %, 147 mg, 3.69 mmol) in batches, and the mixture was stirred at 0 °C for 20 min. Next, CH3I (0.23 mL, 3.69 mmol) was added thereto, and the mixture was stirred at 0 °C for 1 h. The solvent was removed by distillation under reduced pressure, and the crude product was purified by silica gel flash chromatography (eluting with EtOAc) to give product 52 as a yellow solid (0.8 g, yield 80%). 1H NMR (500 MHz, DMSO-d6): δ 8.78 (d, J = 4.8 Hz, 1H), 8.07 (d, J = 9.3 Hz, 2H), 7.50 (s, 1H), 7.36 (d, J = 4.8 Hz, 1H), 6.89 (s, 1H), 6.71 (d, J = 9.0 Hz, 2H), 3.96 (s, 3H), 3.71 (s, 3H), 3.52 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 152.76, 152.00, 149.57, 148.68, 148.16, 146.51, 137.20, 125.06, 119.33, 117.11, 112.51, 108.03, 99.93, 55.26, 55.06. LC/MS (ESI, m/z): 340.1 [M + H]+. Compound 53 was prepared following the synthetic procedure of 51. N1-(6,7-Dimethoxyquinolin-4-yl)-N1-methylbenzene-1,4-diamine (53). It was obtained with a yield of 85%. 1H NMR (500 MHz, DMSO-d6): δ 8.49 (s, 1H), 7.29 (s, 1H), 6.96 (d, J = 5.7 Hz, 1H), 6.87 (d, J = 8.2 Hz, 2H), 6.76 (s, 1H), 6.58 (d, J = 8.2 Hz, 2H), 3.87 (s, 3H), 3.40 (s, 3H), 3.37 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 152.96, 151.32, 146.33, 146.24, 143.39, 140.76, 137.43, 125.19, 114.02, 113.95, 104.99, 104.33, 103.52, 55.11, 54.12, 43.37. LC/MS (ESI, m/z): 310.1 [M + H]+. 4-((6,7-Dimethoxyquinolin-4-yl)thio)aniline (54). A mixture of 4-chloro-6,7-dimethoxyquinoline (1.0 g, 4.48 mmol) and 4aminobenzenethiol (672 mg, 5.38 mmol) was stirred at room temperature overnight. The solid was collected by filtration and washed with Et2O to give title compound 54 as a yellow solid (0.8 g, yield 57%).1H NMR (500 MHz, DMSO-d6): δ 8.59 (d, J = 6.2 Hz, 1H), 7.71 (s, 1H), 7.42 (s, 1H), 7.36−7.29 (m, 2H), 6.83−6.80 (m, 3H), 4.05 (s, 3H), 4.01 (s, 3H). LC/MS (ESI, m/z): 313.1 [M + H]+. Cell Lines, Cell Culture Antibodies, and Chemicals. The human GIST-T1 cell line was purchased from Cosmo Bio Co., Ltd. Tokyo, Japan. GIST-T1-T670I cell line was constructed using CRISPR-Cas 9 by our group. GIST-882 and GIST-48B cell lines were kindly provided by the group of Professor Jonathan A. Fletcher, Brigham, and Women’s Hospital in Boston. The GIST-5R cell line was provided by Prof. Brian Rubin, Department of Molecular Genetics, Lerner Research Institute, and Department of Anatomic Pathology and Taussing Cancer Center, Clevel and Clinic, Cleveland, OH 44195. All cells were grown in a humidified incubator (Thermo, USA) at 37 °C under 5% CO2. GIST-T1 and GIST-T1-T670I cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. GIST-882 and GIST-48B were grown in Iscove’s modified Dulbecco’s media (Gibco, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. All other cell lines and all isogenic BaF3 cells were grown in RPMI 1640 (Gibco, USA) medium supported with 10% FBS and 1% penicillin/streptomycin. Adherent cells were grown in tissue culture flasks until they were 85−95% 6098



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Apoptosis Effect Examination. GIST-T1, GIST-T1-T670I, and GIST-5R cells were cultured in 10% FBS-containing DMEM medium. Serially diluted compound 18, 1 μM imatinib, and 0.1 μM sunitinib were added to cells for 24 h. Then, apoptosis of GIST-T1, GIST-T1T670I, and GIST-5R cells was detected by the western blot using PARP, caspase 3, and α-tubulin antibodies (Cell Signaling Technology). Cell Cycle Analysis. GIST-T1, GIST-T1-T670I, and GIST-5R cells were treated with DMSO, serially diluted compound 18, 1 μM imatinib, and 0.1 μM sunitinib for indicated periods. The cells were fixed in 70% cold ethanol and incubated at −20 °C overnight and then stained with PI/RNase staining buffer (BD Pharmingen). Flow cytometry was performed using a FACS Calibur (BD), and the results were analyzed by ModFit software. PK Study. This study protocol was approved by the Animal Ethics Committee of Hefei Institutes of Physical Science, Chinese Academy of Sciences (Hefei, China). The male Sprague−Dawley rats (190− 210 g) were provided by Laboratory Animal Center of Anhui Medical University (Hefei, China). The animals were housed in an airconditioned animal room at a temperature of 23 ± 2 °C and a relative humidity of 50 ± 10% and allowed free access to tap water and lab. The mice, rats, and dogs were acclimatized to the facilities for one week and then fasted for 12 h with free access to water prior to the experiment. The six mice, rats, or dogs were randomly and equally divided into two groups for one compound’s PK study. One group was injected with iv formulation at a dose of 1 mg/kg compound 18, and the other group was treated by oral administration of p.o. formulation at doses of 10 mg/kg compound 18. The p.o. formulation (2 or 3 mg/mL) consisted of 10 mg compound dissolved in 0.5 mL of DMSO and 4.5 mL of 5% glucose water. The iv formulation is made with 0.5 mL of the p.o. formulation and 4.5 mL of 5% glucose water. About 300 μL of blood samples were collected into heparinized tubes at 2, 5, 15, 30, 60, 120, 240, 360, 540, and 720 min after iv injection and at 5, 15, 30, 60, 90, 120, 240, 360, 540, and 720 min after oral administration. Plasma (100 μL) was harvested by centrifuging the blood sample at 4 °C and 5000 rpm for 3 min and then stored at −80 °C until analysis. An aliquot of 100 μL of each plasma sample was mixed with 20 μL of internal standard working solution (200 ng/mL of caffeine). Methanol (400 μL) was then added for precipitation. After vortexing for 5 min and centrifuging at 14 000 rpm for 10 min, 5 μL of the supernatant was injected for LC−MS/MS analysis. The PK parameters were analyzed through the noncompartment model using WinNonlin 6.1 software (Pharsight Corporation, Mountain View, USA), including half-life (T1/2), plasma concentration at 0 min (C0), the peak of the plasma concentration (Cmax), the time to peak of the plasma concentration (Tmax), the area under the plasma concentration−time curve during the period of observation (AUC0−t), the area under the plasma concentration−time curve from zero to infinity (AUC0−∞), clearance (CL), apparent volume of distribution (Vd), and the mean residence time. The oral bioavailability (F) is calculated according to the following equation: F = AUC0−∞ (oral)/AUC0−∞ (iv) × dose (iv)/dose (oral) × 100%. Xenograft Tumor Model. Five-week old female BALB/C-nu mice were purchased from the Shanghai Experimental Center, Chinese Academy of Sciences (Shanghai, China). All animals were housed in a specific pathogen-free facility and used according to the animal care regulations of Hefei Institutes of Physical Science, Chinese Academy of Sciences (Hefei, China). Prior to implantation, cells were harvested during exponential growth. One to five million BaF3-tel-c-KIT-T670I, BaF3-tel-c-KIT-D820G, BaF3-tel-c-KITY823D, GIST-T1, GIST-T1-T670I, and GIST-5R cells in phosphate-buffered saline were formulated as a 1:1 mixture with Matrigel (BD Biosciences) and injected into the subcutaneous space on the right flank of nu/nu mice. Daily oral administration was initiated when tumors had reached a size of 200−400 mm3. Animals were then randomized into treatment groups of 5 mice each for efficacy studies. Compound 18 was delivered daily in a HKI solution (0.5% methocellulose/0.4% Tween80 in ddH2O) by oral gavage. A range of doses of compound 18 or its vehicle as the control were administered. Body weight was measured daily, and tumor growth was

confluent prior to use. For suspension cells, cells were collected by spinning down at 800 rpm for 4 min before use. The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): AKT (pan) (C67E7) rabbit mAb (no. 4691), Phospho-AKT (Thr308) (244F9) rabbit mAb (no. 4056), Phospho-AKT (Ser473) (D9E) XP rabbit mAb (no. 4060), PARP (46D11) rabbit mAb (no. 9532), caspase-3 (8G10) rabbit mAb (no. 9665), Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (197G2) rabbit mAb (no. 4377), p44/42 MAPK (ERK1/2) (137F5) rabbit mAb (no. 4695), c-Kit (Ab81) mouse mAb (no. 3308), Phospho-c-Kit (Tyr703) (D12E12) rabbit mAb (no. 3073), Phospho-c-Kit (Tyr719) rabbit mAb (no. 3391), Phospho-anti-c-Kit (pY823) rabbit mAb (no. 77522), Phospho-STAT3 (Tyr705) (D3A7) XP rabbit mAb (Biotinylated) (no. 4093), STAT3 (D3Z2G) rabbit mAb no. 12640, phospho-S6 ribosomal protein (Ser235/236) rabbit mAb (no. 2211), and S6 ribosomal protein (5G10) rabbit mAb no. 2217. α-Tubulin (TU-02) mouse mAb (no. sc-8035) was purchased from Santa Cruz Biotechnology. Imatinib and sunitinib were purchased from MedChemExpress (Shanghai, China). Generation of the GIST-T1-T670I Cell Line Using the CRISPR/Cas9 System. SgRNA targeting the genomic region surrounding the T670 site of the KIT gene was designed and synthesized; annealed sgRNAoligos were inserted into pSpCas9(BB)2A-Puro (PX459) V2.0 vector (Addgene, Cambridge, MA, USA) and transfected into GIST-T1 cells together with a 90 nt oligo containing T670I mutation as the HDR template by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Antibiotic selection with puromycin was performed two days after transfection and then cells were diluted and cultured in 96-well plates with only one cell per well for 3 weeks. Correctly edited T670I knock-in cell clones were then confirmed by Sanger sequencing at the targeted genomic region. General Procedure for Antiproliferation Assays. For the nonadherent BaF3 cells, a density of 1.5 × 104 cells/mL cells was mixed with various concentrations of compounds; then, 100 μL suspension was added to each well and then incubated at 37 °C with 5% CO2 for 72 h. For the adherent GIST cells, a density of 2−5 × 104 cells/mL was added to the 96-well plate, incubated at 37 °C with 5% CO2 overnight. The next day, the supernatant was changed with fresh medium containing different various concentrations of compounds and then incubated for 72 h. Cell viability was determined using CCK-8 assay (MedChemExpress, China) according to the instruction manual. The absorbance was measured in a microplate reader (iMARK, Bio-Rad, USA) at 450 nm. Data were normalized to the control group (DMSO). GI50 values were calculated using Prism 7.0 (GraphPad Software, San Diego, CA). Culture of Primary Human GIST Cells. Human GIST samples were obtained with informed consent from the patients of the People’s Liberation Army Joint Service Support Force no. 901 Hospital. All procedures were performed according to the hospital guidelines of the Research Ethics Committees and the World Medical Association Declaration of Helsinki. Fresh tumor tissue fragments were dissociated using collagenase/hyaluronidase and dispase (StemCell Technologies, Canada) at 37 °C for 2 h with shaking. Primary cells were placed in flasks coated with collagen I (Corning, USA) in the culture medium. The culture medium included DMEM/ F12 medium freshly supplemented with 5% FBS (Gibco, USA), GlutaMAX-I (Gibco, USA), primocin (Invivogen, USA), 25 μg/mL hydrocortisone (Sigma, USA), 5 μg/mL insulin (Gibco, USA), 125 ng/mL EGF (Sigma, USA), and 10 μM Rho kinase inhibitor Y27632 (MedChemExpress). Cells were grown in humidified 37 °C incubators in an atmosphere of 5% CO2 and 2% O2. Medium was replaced every 3−4 days, and the primary cells were cultured for a period of 3−4 weeks. Signaling Pathway Study. GIST-T1, GIST-T1-T670I, and GIST-5R cells were cultured in 10% FBS-containing DMEM medium. Serially diluted compound 18, 1 μM imatinib, and 0.1 μM sunitinib were added to cells for 2 h. The cells were collected and lysed. The cell lysates were analyzed by western blotting. Western blotting was performed by standard methods, as previously described.16 6099



Article

ZR201711290017), the Key Program of 13th five-year plan of CASHIPS (grant KP-2017-26), the Presidential Foundation of CASHIPS (grant YZJJ2018QN17), and the Frontier Science Key Research Program of CAS (grant QYZDB-SSW-SLH037). We are also grateful for the National Program for Support of Top-Notch Young Professionals for J.L. and the support of Hefei leading talent in 2017 for F.Z.

measured every day after compound 18 treatment. Tumor volume was calculated as follows: tumor volume (mm3) = [(W2 × L)/2] in which width (W) is defined as the smaller of the two measurements, and length (L) is defined as the larger of the two measurements. Molecular Modeling. All calculations were performed using the Schrödinger Suite. The DFG-out c-KIT complex (PDB ID: 6GQK) was used for docking studies. The crystal structure was prepared using the Protein Preparation Wizard, and the T670I mutant was modeled in situ within Maestro. The ligand structures were built in Maestro and prepared for docking using LigPrep (LigPrep 3.4, Schrödinger, LLC, New York, NY) and further docked into the receptor by the IFD protocol (Induced Fit Docking protocol, Schrödinger, LLC, New York, NY).

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ABBREVIATIONS GISTs, gastrointestinal stromal tumors; PDGFR, plateletderived growth factor receptor; CSF1R, colony stimulating factor 1 receptor; SAR, structure−activity relationship; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; DCM, dimethyl chloride; THF, tetrahydrofuran; DIPEA, N,Ndiisopropylethylamine; HATU, hexafluorophosphate azabenzotriazole tetramethyl uronium; HOBT, hydroxybenzotriazole; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00280. DiscoverX’s KINOMEScan selectivity profiling data of 18, expression and autophosphorylation inhibition of PDGFRα, PDGFRβ, and CSF1R in GIST-T1 cells, effects of selective PDGFRα inhibitor CHMFL-PDGFR159 on PDGFR-mediated signaling pathways in GISTT1 cells and antiproliferation against GIST-T1 cells, PK profiles of 18 in mice, rats, and beagle dogs, and PD effect of 18 on tumor p-c-KIT, p-ERK, and p-AKT in the GIST-T1-T670I mouse xenograft model from a single dose of 80 mg/kg/day (p.o.) (PDF) Molecular formula strings (CSV) Docking poses for 1, 2, and 18 with c-KIT wt, c-KIT V654A, and c-KIT T670I (PDB) (ZIP)

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REFERENCES

(1) Rubin, B. P.; Heinrich, M. C.; Corless, C. L. Gastrointestinal stromal tumour. Lancet 2007, 369, 1731−1741. (2) Steigen, S. E.; Eide, T. J. Gastrointestinal stromal tumors (GISTs): a review. APMIS 2010, 117, 73−86. (3) Lennartsson, J.; Rönnstrand, L. Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol. Rev. 2012, 92, 1619−1649. (4) Hirota, S.; Isozaki, K.; Moriyama, Y.; Hashimoto, K.; Nishida, T.; Ishiguro, S.; Kawano, K.; Hanada, M.; Kurata, A.; Takeda, M.; Muhammad Tunio, G.; Matsuzawa, Y.; Kanakura, Y.; Shinomura, Y.; Kitamura, Y. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998, 279, 577−580. (5) Heinrich, M. C.; Corless, C. L.; Demetri, G. D.; Blanke, C. D.; Mehren, M. v.; Joensuu, H.; McGreevey, L. S.; Chen, C.-J.; Abbeele, A. D. V. d.; Druker, B. J.; Kiese, B.; Eisenberg, B.; Roberts, P. J.; Singer, S.; Fletcher, C. D. M.; Silberman, S.; Dimitrijevic, S.; Fletcher, J. A. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J. Clin. Oncol. 2003, 21, 4342−4349. (6) Zalcberg, J. R.; Verweij, J.; Casali, P. G.; Le Cesne, A.; Reichardt, P.; Blay, J.-Y.; Schlemmer, M.; Van Glabbeke, M.; Brown, M.; Judson, I. R. Outcome of patients with advanced gastro-intestinal stromal tumours crossing over to a daily imatinib dose of 800mg after progression on 400mg. Eur. J. Cancer 2005, 41, 1751−1757. (7) Gajiwala, K. S.; Wu, J. C.; Christensen, J.; Deshmukh, G. D.; Diehl, W.; DiNitto, J. P.; English, J. M.; Greig, M. J.; He, Y.-A.; Jacques, S. L.; Lunney, E. A.; McTigue, M.; Molina, D.; Quenzer, T.; Wells, P. A.; Yu, X.; Zhang, Y.; Zou, A.; Emmett, M. R.; Marshall, A. G.; Zhang, H.-M.; Demetri, G. D. KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1542−1547. (8) Heinrich, M. C.; Maki, R. G.; Corless, C. L.; Antonescu, C. R.; Harlow, A.; Griffith, D.; Town, A.; Mckinley, A.; Ou, W.-B.; Fletcher, J. A.; Fletcher, C. D. M.; Huang, X.; Cohen, D. P.; Baum, C. M.; Demetri, G. D. Primary and secondary kinase genotypes correlate with the biological and clinical activity of sunitinib in imatinibresistant gastrointestinal stromal tumor. J. Clin. Oncol. 2008, 26, 5352−5359. (9) George, S.; Wang, Q.; Heinrich, M. C.; Corless, C. L.; Zhu, M.; Butrynski, J. E.; Morgan, J. A.; Wagner, A. J.; Choy, E.; Tap, W. D.; Yap, J. T.; Van den Abbeele, A. D.; Manola, J. B.; Solomon, S. M.; Fletcher, J. A.; von Mehren, M.; Demetri, G. D. Efficacy and safety of regorafenib in patients with metastatic and/or unresectable GI stromal tumor after failure of imatinib and sunitinib: A multicenter phase II trial. J. Clin. Oncol. 2012, 30, 2401−2407. (10) Serrano, C.; George, S.; Valverde, C.; Olivares, D.; GarcíaValverde, A.; Suárez, C.; Morales-Barrera, R.; Carles, J. Novel insights

AUTHOR INFORMATION

Corresponding Authors

*E-mail: qsliu97@hmfl.ac.cn. Phone: 86-551-65595161 (Q.L.). *E-mail: jingliu@hmfl.ac.cn. Phone: 86-551-65593186 (J.L.). ORCID

Qingsong Liu: 0000-0002-7829-2547 Jing Liu: 0000-0001-9513-3591 Author Contributions

The manuscript was written through contributions of all the authors. All the authors have given approval to the final version of the manuscript. Y.W., B.W., J.W., and S.Q. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grants 81872748, 81603123, 81673469, 81773777, 81803366, 81703007, and 81370054), the “Personalized Medicines-Molecular Signature-Based Drug Discovery and Development”, Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDA12020224), the Natural Science Foundation of Anhui Province (grants 1808085MH268, 1808085QH263), the Major Science and Technology Program of Anhui Province (grant 18030801116), the Key Research and Development Program of Anhui Province (grant 1704a0802140), the Key Technology Research and Development Program of Hefei (grant 6100



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