(trifluoromethyl)benzamide (CHMFL-KIT-110) as a ... - ACS Publications

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Discovery of N‑(3-((1-Isonicotinoylpiperidin-4-yl)oxy)-4methylphenyl)-3-(trifluoromethyl)benzamide (CHMFL-KIT-110) as a Selective, Potent, and Orally Available Type II c‑KIT Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs) Qiang Wang,†,‡,§ Feiyang Liu,†,∥ Beilei Wang,†,‡,§ Fengming Zou,†,‡,§ Cheng Chen,†,‡,§ Xiaochuan Liu,†,⊥ Aoli Wang,†,∥ Shuang Qi,†,‡,§ Wenchao Wang,†,‡,§ Ziping Qi,†,‡,§ Zheng Zhao,†,‡,§ Zhenquan Hu,†,‡,§ Wei Wang,†,‡,§ Li Wang,†,‡,§ Shanchun Zhang,‡,# Yuexiang Wang,○,∇,◆ Jing Liu,*,†,‡,§ and Qingsong Liu*,†,‡,§,∥ †

High Magnetic Field Laboratory, Chinese Academy of Sciences, Mailbox 1110, 350 Shushanhu Road, Hefei, Anhui 230031, P. R. China ‡ CHMFL-HCMTC Target Therapy Joint Laboratory, 350 Shushanhu Road, Hefei, Anhui 230031, P. R. China § Center for Precision Medicine, CAS (Hefei) Institute of Technology Innovation, Hefei Institute of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R. China ∥ University of Science and Technology of China, Hefei, Anhui 230036, P. R. China ⊥ Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230036, P. R. China # Hefei Cosource Medicine Technology Co., LTD, 358 Ganquan Road, Hefei, Anhui 230031, P. R. China ○ SIBS (Institute of Health Sciences)-Changzheng Hospital Joint Center for Translational Medicine, Institute of Health Sciences, Shanghai Changzheng Hospital, Institutes for Translational Medicine (CAS-SMMU), Shanghai 200031, China ∇ Key Laboratory of Stem Cell Biology, Institute of Health Sciences, SIBS, Chinese Academy of Sciences/Shanghai Jiao Tong University School of Medicine, Shanghai 200031, China ◆ Collaborative Innovation Center of Systems Biomedicine, Shanghai 200025, China S Supporting Information *

ABSTRACT: c-KIT kinase is a validated drug discovery target for gastrointestinal stromal tumors (GISTs). Clinically used c-KIT kinase inhibitors, i.e., Imatinib and Sunitinib, bear other important targets such as ABL or FLT3 kinases. Here we report our discovery of a more selective c-KIT inhibitor, compound 13 (CHMFL-KIT-110), which completely abolished ABL and FLT3 kinase activity. KinomeScan selectivity profiling (468 kinases) of 13 exhibited a high selectivity (S score (1) = 0.01). 13 displayed great antiproliferative efficacy against GISTs cell lines GIST-T1 and GIST-882 (GI50: 0.021 and 0.043 μM, respectively). In the cellular context, it effectively affected c-KIT-mediated signaling pathways and induced apoptosis as well as cell cycle arrest. In addition, 13 possessed acceptable bioavailability (36%) and effectively suppressed the tumor growth in GIST-T1 cell inoculated xenograft model without apparent toxicity. 13 currently is undergoing extensive preclinical evaluation and might be a potential drug candidate for GISTs.

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INTRODUCTION Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract, and they are generally resistant to chemotherapy and radiation therapy.1 Most GISTs express constitutively activated forms of the c-KIT kinase whose © XXXX American Chemical Society

activation is an essential driving force in most of the early development stages of GISTs.2 c-KIT kinase belongs to the type Received: February 6, 2016

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DOI: 10.1021/acs.jmedchem.6b00200 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Chemical structures of the representative c-KIT kinase inhibitors: (A) FDA approved c-KIT inhibitors for GISTs and (B) multitarget inhibitors possess c-KIT inhibitory activity.

medicinal chemistry effort starting with the compound 1 core scaffold which led to the discovery of a selective, potent, and orally available type II c-KIT inhibitor compound 13 (CHMFLKIT-110) that abolished both the ABL and FLT3 kinase activities (Figure 2).

III receptor tyrosine kinase (RTK) family, which also includes platelet-derived growth factor receptor alpha/beta (PDGFRα/β), FMS-related tyrosine kinase 3 (FLT3), and colony stimulating factor-1 receptor (CSF1R).3 Structurally, the type III RTKs are characterized by five extracellular immunoglobulin-like repeats and a kinase insert separating the ATP-binding and phosphotransferase regions of the kinase domain. The physiologic activation of wild-type KIT is accomplished by binding to its ligand, stem cell factor (SCF), which promotes KIT dimerization, kinase activation, and tyrosine cross phosphorylation. However, these normal mechanisms of KIT activation can be subverted by oncogenic mutations, which contribute to constitutive activation.4 All c-KIT mutations in GISTs appear to be associated with constitutive c-KIT tyrosine phosphorylation,5 and this makes c-KIT a validated target for the anti-GIST therapy. Currently two of the kinase inhibitors that bear c-KIT kinase inhibitory activity have been approved for the clinical use of GISTs. Compound 1 (Imatinib, Figure 1A),2 as the first clinically approved type II c-KIT kinase inhibitor for GIST treatment, binds to the inactive conformation (DFG-out) of c-KIT and achieved excellent overall success. However, the potent ABL kinase inhibitory activity of compound 1 did not contribute to the GISTs treatment and has been arguably reported to link to potential cardiotoxicity.6 Compound 2 (Sunitinib, Figure 1A),7 the second FDA-approved (type I inhibitor, which binds to the active conformation (DFG-In) of kinases) second line drug for GISTs, was reported to possess FLT3/c-KIT dual inhibition which might be related to potential myelosuppression toxicity.8 Several other multitarget inhibitors, such as 3 (Amuvatinib),9 4 (Masitinib),10 5 (Nilotinib),11 6 (Pexidartinib),12 7 (OSI930),13 8 (Dovitinib),14 etc. were also reported to bear c-KIT kinase inhibitory activity (Figure 1B). However, for most of them, c-KIT is not their primary target. In order to trim the off-target profile and increase the therapeutic safety window, herein we report our

Figure 2. Schematic illustration of discovery of compound 13.

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RESULTS AND DISCUSSION Structure−Activity Relation (SAR) Exploration. We started with examination of the c-KIT/compound 1 X-ray crystal complex (PDB ID: 1T46) and envisioned that this is a typical type II binding mode (Figure 3A). The key binding elements include the hinge binding moiety, linker moiety, DFG-out hydrophobic pocket binding moiety, and the wellknown “Flag” methyl fragment.15 The two key hydrogen bonds formed between Glu640 located in the α-helix and Asp810 located in the DFG motif with the amide group in the linker moiety stabilize the DFG-out conformation of the c-KIT kinase. The hydrogen bond formed between Cys673 and the terminal pyridine in the hinge binding area and the hydrogen bond formed between the gatekeeper residue Thr670 with the −NH of the aminopyrimidine help to improve the binding affinity. Comparison of the three available type II c-KIT kinase inhibitors, compounds 1, 4, and 5, revealed that they all share the same linker moiety and only slightly differ from the hinge binding B



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Figure 3. Schematic illustration of SAR exploration rationale. (A) Analysis of c-KIT/compound 1 binding mode (PDB ID: 1T46). (B) Comparison of compounds 1, 4, and 5 type II key binding moieties.

For the SAR exploration of the R1 and R3 moieties, we choose to start with fixing the R2 as the ether linked piperidine ring (Table 2). Introduction of the methyl group at the 4-position of the benzene ring (22) to compound 13 gained 6-fold c-KIT activity improvement (GI50: 0.031 versus 0.19 μM). However, its selectivity window against parental BaF3 (GI50: 6.39 μM) and K562 (GI50: 2.32 μM) has remarkably narrowed down. Further variation of the R1 fragment (23, 24, and 25) all led to gaining back of BCR-ABL inhibitory activity, although the c-KIT inhibitory activities were better than 13. Introducing the −Cl (26, 27) or −F (28) atom at the 5-position of the benzene ring in the R3 fragment started to gain back the BCR-ABL activity. Interestingly, changing the benzene ring to pyridine ring in R3 (29 and 30) led to complete loss of activity to both the c-KIT and BCR-ABL. Replacement of −CF3 group with a larger −OMe group in R3 (31) brought about 15-fold activity loss against c-KIT compared to 12 (GI50: 1.48 versus 0.11 μM). A tert-butyl isoxazole moiety at R3 position (32) only moderately decreased the c-KIT activity (GI50: 0.34 versus 0.11 μM) compared to 12. Benzodioxole (33) and quinolone (34) moieties at the R3 position both led to around 50-fold activity loss against c-KIT kinase. Changing the amide linkage between the linker moiety and R3 fragment to the urea linkage (35) increased the BCR-ABL activity meanwhile lowering the c-KIT activity. Biochemical and Cellular Property Evaluation. Among all of the compounds tested, 13 displayed the best potency against c-KIT kinase and selectivity over ABL kinase, therefore we chose it for further characterization. We first examined its kinome wide selectivity profile with DiscoveRx’s KinomeScan technology.17 The results demonstrated that 13 bore a high selectivity (S score (1) = 0.01 at 1 μM) among 468 kinases and mutants tested. Besides high binding affinity to c-KIT, it also displayed strong binding against CSF1R, DDR1, and PDGFRβ kinases (percent activity remaining 10 μM), it also exhibited moderate activity against K562 cell (GI50: 2.94 μM), indicating the moderate BCR-ABL inhibitory activity. Shifting the nitrogen atom position in the R1 fragment, i.e., the terminal pyridine (12 and 13), not only retained the c-KIT activity (GI50: 0.33 and 0.19 μM, respectively) but also abolished the BCR-ABL activity completely (K562 cell, GI50: >10 μM), meanwhile retaining the selectivity over the parental BaF3 cells (GI50: >10 μM). However, when the pyridine group was replaced by the isoxazole group (14) in R1, it started to gain back the BCR-ABL activity (GI50: 0.16 μM). Changing the R1 fragment from the aromatic rings to the aliphatic chains such as ethyl (15) and ethylene (16) both led to the c-KIT activity loss. Switching the ether linkage from the 4-position of the piperidine to the 3-position in R2 (17 and 18) replaced the “flag” methyl to hydrogen atom completely lost the c-KIT activity. In addition, replacing the “flag” methyl group with hydrogen atom (19) led to the slight c-KIT activity loss compared to the most active lead compound 11 (GI50: 0.49 versus 0.11 μM). Substitution of the “flag” methyl with −Cl (20) further decreased the c-KIT activity (GI50: 0.96 μM) and −OMe group (21) led to the complete c-KIT activity loss (GI50: > 10 μM). These results implied that the piperidine ring in the R2 fraction is well tolerated and that the “flag” methyl in the R4 fraction is required for the high inhibitory activity. C



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Table 1. SAR Exploration Focused on the R1/R2/R4 Positionsa

a

All GI50 values were obtained by triplet testing.

(GI50: 0.021 μM) and GIST-882 (GI50: 0.043 μM) but not the c-KIT-independent GIST cell line GIST48B (GI50: > 10 μM) (Table 4). In addition, unlike 1, 13 did not exhibit potent antiproliferative activity against BCR-ABL driven CML cell lines such as K562 (GI50: > 10 μM), MEG-01 (GI50: 7.43 μM), and

mutants, but it also affected the parental BaF3 cells growth (GI50: 2.78 μM) which indicated the multiple target features. Compound 13 was next tested against a variety of intact cancer cell lines. Not surprisingly, it did potently inhibit the growth of c-KIT-dependent GIST cancer cells such as GIST-T1 D



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Table 2. SAR Exploration Focused on the R1/R3 Positionsa

a

All GI50 values were obtained by triplet testing. E



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Figure 4. Kinome wide selectivity profiling of 13. (A) KinomeScan profiling of 13 at a concentration of 1 μM against 468 kinases. (B) Biochemical assay characterization of 13’s inhibitory activity against c-KIT, PDGFRβ (Promega ADP-Glo), DDR1, and CSF1R (Invitrogen Z’lyte).

Table 3. Antiproliferative Effects of 1, 2, and 13 against a Variety of TEL-c-KIT-BaF3 Isogenic Cellsa cell line

1 GI50 (μM)

2 GI50 (μM)

13 GI50 (μM)

parental BaF3 Tel-c-KIT-BaF3 Tel-c-KIT/V559D-BaF3 Tel-c-KIT/V559D/V654A-BaF3 Tel-c-KIT/N822K-BaF3 Tel-c-KITt/T670I-BaF3 Tel-c-KIT/V654A-BaF3 Tel-c-KITt/L567P-BaF3 Tel-c-KIT/T670I/V559D-BaF3 Tel-c-KIT/D816V-BaF3

>10 0.40 0.039 3.0 1.29 >10 2.49 0.10 6.67 >10

2.78 0.078 0.009 0.006 0.51 0.002 0.02 0.023 0.053 1.38

>10 0.19 0.04 1.05 1.06 8.24 1.03 0.15 5.31 >10

a

Table 4. Antiproliferation Effects of 1, 2, and 13 against a Variety of Intact Cancer Cell Linesa


KU812 (GI50: 6.71 μM). Furthermore, 13 did not show any apparent inhibitory activity against MV4-11 and MOLM13 cells that are FLT3-ITD growth dependent, which further confirmed that it had no FLT3 kinase activity. 13 did not inhibit the growth of other leukemic cell lines such as U937, HL-60, REC-1, and the normal Chinese hamster ovary cells CHO and CHL cells either, indicating a good safety profile. Comparably, 2 exhibited weaker antiproliferative effect against c-KIT-dependent cell lines and moderate inhibitory activities against BCR-ABL-dependent CML cell lines. However, it did show potent efficacies against several AML cell lines such as MV4-11, MOLM14, and HL-60, which reflected its multiple target features such as FLT3-ITD and others (Table 4). In order to understand the structural binding mechanism of 13 to c-KIT kinase and the selectivity mechanism between the c-KIT and ABL kinase, 13 was docked into c-KIT kinase and ABL kinase, respectively. The docking results demonstrated that 13 could adopt the DFG-out conformation of c-KIT kinase

a

cell line

1 GI50 (μM)

2 GI50 (μM)

13 GI50 (μM)

GIST-T1 GIST-882 GIST48B K562 MEG-01 KU812 U937 MV-4−11 MOLM-14 HL-60 REC-1 CHL CHO

0.008 0.014 >10 0.12 0.074 0.16 >10 >10 >10 >10 >10 >10 >10

0.041 0.11 2.01 1.00 1.20 1.10 1.70 0.001 0.005 0.001 0.87 2.48 2.03

0.021 0.043 >10 >10 7.43 6.71 >10 >10 >10 >10 >10 >10 >10


(PDB ID: 1T46) with a typical type II binding mode (Figure 5A). Surprisingly, instead of using the terminal pyridine as the hinge binding like 1 does, 13 forms an O−H−N hydrogen bond by the amide carbonyl connecting the terminal pyridine moiety and piperidine moiety with the Cys673 in the hinge binding area at a distance about 2.9 Å. There are also two typical hydrogen bonds formed between the Glu640 and Asp810 with the amide moiety in the tail part of 13. In the ABL kinase (PDB ID: 2HYY), although the docking results showed that 13 could also adopt the similar type II binding mode, the hydrogen bond formed in the hinge binding area is a little longer than in the c-KIT kinase (3.6 versus 2.9 Å) (Figure 5B). In addition, the spatially adjacent Tyr253 (3.1 Å to the pyridine moiety) in the P-loop introduces a possible steric hindrance which prevents 13 from stably binding to the ABL kinase. This could be more clearly illustrated when superimposing 13 and 1 in the 1-ABL kinase X-ray crystal structure F



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Figure 5. Structural basis of the binding and selectivity mechanism for 13 against c-KIT and ABL kinase. Hydrogen bonds are indicated by red-hatched lines to key amino acid residues (highlighted). (A) 13 (in color by atoms) was docked into c-KIT kinase (PDB ID: 1T46) (in white). (B) 13 (in color by atoms) was docked into ABL kinase (PDB ID: 2HYY) (in white). (C) Superimposition of 1-ABL kinase X-ray crystal structure (in purple) and docking model of 13 (in color by atoms) with ABL kinase (PDB ID: 2HYY) (in white). (D) Superimposition of docked model of 13 with ABL kinase (PDB ID: 2HYY) (in purple) and 13 with c-KIT kinase (PDB ID: 1T46) (in white).

Compound 13’s antitumor efficacy was tested in GIST-T1 cell inoculated xenograft mouse model. With oral gavage, none of the 25, 50, and 100 mg/kg/day administrations affected the mice body weight (Figure 8A). However, a 50 and 100 mg/kg dosage significantly suppressed tumor progression (Figure 8B−D). Immunohistochemistry stain showed that 13 could strongly suppress the antiproliferation (Ki67 stain) and induce the apoptosis effect (TUNEL stain) in the tumor tissues (Figure 8E). It is worth mentioning that the PK profile obtained from rats may not be directly transformed into mice due to the species difference.

(PDB ID: 2HYY) (Figure 5C). The pyridine moiety in 1 forms a hydrogen bond in the hinge binding area with a distance of 2.9 Å, and this aromatic moiety moves slightly further from the Tyr253 compared to the pyridine moiety in 13, which presumably could avoid the potential steric hindrance. While in the c-KIT kinase, the Tyr253 was replaced by the Gly596, which would provide enough space for this terminal pyridine moiety to be accommodated (Figure 5D). Compound 13’s effect on the signaling pathways was evaluated in the intact GIST cancer cell lines (Figure 6). As expected, it significantly affected c-KIT pY719, pY703, and pY823 autophosphorylation sites and downstream signaling mediators such as pAKT(S473), pERK(T202/204), pS6K(T389), and pSTAT5(Y694) in the c-KIT growth-dependent GIST cancer cell lines GIST-T1 and GIST-882. However, in the c-KITindependent GIST cancer cell line GIST-48B, despite the c-KIT (Y719, Y703) autophosphorylation site inhibition, it did not or only weakly affected pc-KIT(Y823) and downstream mediators pAKT, pERK, and pS6K, though it still strongly affected pSTAT5(Y694). In addition, in all of these three cell lines, 13 greatly inhibited pPDGFR which matched the in vitro biochemical characterizations. It is noteworthy that in all of the signaling pathways tested, 13 exhibited similar trends to the well-established c-KIT inhibitor 1, which further proved its c-KIT kinase inhibitory activity. In accordance with 13’s effect on the c-KIT-mediated signaling pathways, it could also induce cell cycle arrest and apoptosis in GIST-T1 and GIST-882 cells but not GIST-48B cells (Figure 7). In Vivo PK/PD Evaluation. Compound 13’s PK properties were tested in rats following intravenous and oral administration (Table 5). The results demonstrated that 13 bore an acceptable bioavailability (F = 36%) and a suitable half-life (T1/2 = 4.11 h) for the oral administration. It is noteworthy that in the iv injection, 13 bore a short half-life (T1/2, 0.45h) and quick clearance (CLz, 4.988 L/h/kg), which indicated that it might not be very metabolically stable, which required further detailed full spectrum of PK characterization.

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CHEMISTRY Starting from commercially available 2-substituted nitrophenol derivatives 36a−d, etherification with various Boc-protected R2 groups followed by hydrogenation afforded aniline compound 38. Amide formation with carboxylic acid format of R3 group followed by Boc-deprotection furnished intermediate 40. Finally installment of R1 group with amide formation conditions generated compounds 9−30 (Scheme 1). Compounds 31−34 were obtained with a slightly modified procedure (Scheme 2). Starting from compound 37a, Boc-deprotection was followed by amide formation to install the R1 group first. Then hydrogenation reduction of nitro group and subsequent introduction of various R3 groups were by amide formation conditions affording compounds 31−34. The synthesis of urea compound 35 started from 38a, which upon urea formation was first to install the R3 group, and then R1 group was introduced via 42a and 42b intermediates (Scheme 3).

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CONCLUSIONS Starting with the well-known type II BCR-ABL/c-KIT dual inhibitor Imatinib’s core scaffold, with the hybrid type II inhibitor design approach, we have discovered a highly selective c-KIT inhibitor 13 which almost completely abolished the ABL kinase activity. In addition, 13 has no FLT3 kinase activity which might avoid the potential myeloid suppression problem of G



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Figure 6. Compound 13’s effects on the signaling transduction pathways in the c-KIT-dependent and -independent intact GIST cancer cell lines. spectra were recorded with a Bruker 400 NMR spectrometer and referenced to deuterium dimethyl sulfoxide (DMSO-d6) or deuterium chloroform (CDCl3). Chemical shifts are expressed in ppm. In the NMR tabulation, s indicates singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak. LC/MS were performed on an Agilent 6224 TOF using an ESI source coupled to an Agilent 1260 Infinity HPLC system operating in reverse mode with an Agilent Eclipse Plus C18 1.8 μm 3.0 × 50 mm column. Flash column chromatography was conducted using silica gel (Silicycle 40−64 μm). The purities of all compounds were determined to be above 95% by HPLC. Compounds 9−30 and 35 were prepared following the synthetic procedure of 13. N-(2-(2-Methyl-5-(3-(trifluoromethyl)benzamido)phenoxy)ethyl)nicotinamide (9). Yield 68%. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.05 (s, 1H), 8.95 (s, 1H), 8.72 (s, 1H), 8.26 (m, 3H), 7.98 (d, J = 7.0 Hz, 1H), 7.80 (t, J = 6.8 Hz, 1H), 7.51 (m, 2H), 7.32 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 7.1 Hz, 1H), 4.16 (s, 2H), 3.74 (s, 2H), 2.15 (s, 3H). LC/MS (ESI, m/z) = 444.1466 [M + H+]. N-(2-(2-Methyl-5-(3-(trifluoromethyl)benzamido)phenoxy)ethyl)isoxazole-5-carboxamide (10). Yield 71%. 1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.19 (s, 1H), 8.76 (s, 1H), 8.30 (s, 2H), 7.98 (s, 1H), 7.81 (s, 1H), 7.47 (s, 1H), 7.31 (s, 1H), 7.11 (s, 2H), 4.13 (s, 2H), 3.71 (s, 2H), 2.13 (s, 3H). LC/MS (ESI, m/z) = 434.1933 [M + H+]. N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-3(trifluoromethyl)benzamide (11). Yield 58%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.65 (s, 2H), 8.27 (s, 2H), 7.87−7.78 (m, 3H), 7.48 (s, 2H), 7.30 (s, 1H), 7.14 (s, 1H), 4.61 (s, 1H), 3.86−3.36

c-KIT/FLT3 dual kinase inhibitors. The extensive biological activity study also revealed that 13 potently inhibited PDGFRβ, DDR1, and CSF1-R kinase, which might strengthen its antitumor activity since PDGFR plays critical roles in the angiogenesis,18 DDR1 kinase plays a role in the tumor proliferation, migration, and invasion,19 and CSF1-R is essential for the cell survival, proliferation, and differentiation.20 13 strongly inhibited the cell proliferation and also induced the cell cycle arrest and apoptosis in the c-KIT-dependent GISTs cancer cells (GIST-T1 and GIST-882) but not in the c-KIT-independent GIST-48B cells. It is worth noting that 13 is not sensitive to compound 1-resistant c-KIT mutants such as V654A, D816V, etc., which are important mutants observed in the clinic. Further detailed SAR study based on this new pharmacophore or combinatorial with other related signaling mediator inhibitors was required for achievement of sensitivity against those drug-resistant mutants. The acceptable selectivity profile, PK profile, and antitumor efficacy suggest that 13 might be a useful pharmacological tool to further study the pathology of GISTs, and currently it is under extensive preclinical PK/PD evaluation to determine whether it might serve as a good potential drug candidate for further clinical development.

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EXPERIMENTAL SECTION

Chemistry. All reagents and solvents were purchased from commercial sources and used as obtained. 1H NMR and 13C NMR H



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Figure 7. Compound 13’s induction of cell cycle arrest and apoptosis in GIST-T1 and GIST-882 cell lines but not in GIST-48B cell line.

Table 5. Pharmacokinetic Characterization of 13 13

AUC(0‑t) (ng/mL·h)

AUC(0‑∞) (ng/mL·h)

MRT(0‑t) (h)

T1/2 (h)

Tmax (h)

Vz (L/kg)

CLz (L/h/kg)

Cmax (ng/mL)

F (%)

iv (1 mg/kg) mean SD po (10 mg/kg) mean SD

198.069 22.942 714.588 153.466

202.038 21.202 779.793 65.913

0.484 0.103 4.799 0.941

0.451 0.056 4.114 0.252

0.017 0.000 0.5 0

3.219 0.142 76.407 6.419

4.988 0.545 12.888 1.145

510.610 82.438 125.167 35.483

NA NA 36.08 NA

N-(4-Methyl-3-((1-propionylpiperidin-4-yl)oxy)phenyl)-3(trifluoromethyl)benzamide (15). Yield 72%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.29 (s, 2H), 7.99 (d, J = 7.4 Hz, 1H), 7.81 (t, J = 7.0 Hz, 1H), 7.50 (s, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 7.9 Hz, 1H), 4.57 (s, 1H), 3.68 (s, 2H), 3.44 (s, 2H), 2.37 (m, 2H), 2.16 (s, 3H), 1.95 (s, 2H), 1.68 (s, 2H), 1.02 (m, 3H). LC/MS (ESI, m/z) = 435.1816 [M + H+]. N-(3-((1-Acryloylpiperidin-4-yl)oxy)-4-methylphenyl)-3(trifluoromethyl)benzamid (16). Yield 76%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.27 (s, 2H), 7.97 (s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.48 (s, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.84 (dd, J = 16.5, 10.2 Hz, 1H), 6.11 (d, J = 16.1 Hz, 1H), 5.68 (d, J = 10.2 Hz, 1H), 4.58 (s, 1H), 3.77 (s, 2H), 3.56 (s, 2H), 2.14 (s, 3H), 1.97 (s, 2H), 1.70 (s, 2H). LC/MS (ESI, m/z) = 433.1668 [M + H+]. N-(3-((1-Nicotinoylpiperidin-3-yl)oxy)phenyl)-3-(trifluoromethyl)benzamide (17). Yield 63%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.66−8.56(m, 3H), 8.27 (s, 2H), 7.97 (s, 1H), 7.83−7.19 (m, 5H), 6.84 (s, 0.4H), 6.64 (s, 0.6H), 4.51(s, 1H), 4.05−3.30 (m, 4H), 2.07−1.58 (m, 4H). LC/MS (ESI, m/z) = 470.1620 [M + H+]. N-(3-((1-(Isoxazole-5-carbonyl)piperidin-3-yl)oxy)phenyl)-3(trifluoromethyl)benzamide (18). Yield 56%. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 0.43H), 10.40 (s, 0.57H), 8.78 (s, 0.43H), 8.63 (s, 0.57H), 8.27 (s, 2H), 7.99 (d, J = 7.4 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H), 7.35 (m, 3H), 6.96 (s, 0.43H), 6.78 (s, 1H), 6.68 (d, J = 7.9 Hz, 0.57H), 4.55 (s, 1H), 3.99−3.30 (m,4H), 2.06−1.85 (m, 3H), 1.61 (s, 1H). LC/MS (ESI, m/z) = 460.1412 [M + H+]. N-(3-((1-Nicotinoylpiperidin-4-yl)oxy)phenyl)-3-(trifluoromethyl)benzamide (19). Yield 53%. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.64 (s, 2H), 8.27 (s, 2H), 8.04−7.69 (m, 3H), 7.50 (s, 1H), 7.48(s, 1H), 7.34(s, 1H), 7.27 (s, 1H), 6.78 (s, 1H), 4.64 (s, 1H), 3.95 (s, 1H), 3.52 (s, 2H), 1.98 (s, 2H), 1.70 (s, 2H). LC/MS (ESI, m/z) = 470.1619 [M + H+].

(m, 3H), 2.16 (s, 3H), 2.02 (s, 2H), 1.79 (s, 2H). LC/MS (ESI, m/z) = 484.1775 [M + H+]. N-(4-Methyl-3-((1-picolinoylpiperidin-4-yl)oxy)phenyl)-3(trifluoromethyl)benzamide (12). Yield 62%. 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.61 (s, 1H), 8.28 (s, 2H), 7.96 (m, 2H), 7.80 (t, J = 7.9 Hz, 1H), 7.61 (d, J = 7.2 Hz, 1H), 7.50 (s, 2H), 7.31 (d, J = 7.7 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 3.96−3.33 (m, 4H), 2.18 (s, 3H), 2.03 (s, 2H), 1.76 (s, 2H). LC/MS (ESI, m/z) = 484.1773 [M + H+]. N-(3-(1-Isonicotinoylpiperidin-4-yloxy)-4-methylphenyl)-3(trifluoromethyl)benzamide (13). HATU (0.06 mmol, 23 mg), 40a (0.05 mmol, 19 mg), and DIPEA (0.075 mmol, 10 mg) were dissolved in 0.5 mL of DMF and cooled to 0 °C. Isonicotinic acid (0.06 mmol, 7.4 mg) was added to the system, and the mixture was stirred at room temperature for 2 h. The system was extracted with EtOAc and dried with anhydrous Na2SO4. The solvent was removed under vacuum, and the residue was purified by silica gel flash chromatography (DCM/ MeOH = 10/1) to offer the product 13 (16.2 mg, 67%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.67 (s, 2H), 8.27 (s, 2H), 7.96 (d, J = 6.8 Hz, 1H), 7.79−7.76 (m, 1H), 7.48 (s, 1H), 7.43 (s, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 4.60 (s, 1H), 3.83−3.28 (m, 4H), 2.16 (s, 3H), 1.95 (brs, 2H), 1.77 (brs, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 165.02, 164.34, 155.06, 152.22, 143.67, 138.37, 136.22, 132.38, 130.92, 130.14, 128.52, 124.94, 122.94, 113.44, 106.97, 71.90, 44.21, 39.38, 38.94, 30.94, 30.19, 16.15. LC/MS (ESI, m/z) = 484.1781 [M + H+]. N-(3-((1-(Isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4-methylphenyl)-3-(trifluoromethyl)benzamide (14). Yield 57%, 1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.76 (s, 1H), 8.27 (s, 2H), 7.96 (s, 1H), 7.79 (s, 1H), 7.49 (s, 1H), 7.29 (d, J = 7.2 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 6.96 (s, 1H), 4.64 (s, 1H), 3.79−3.50 (m, 4H), 2.17 (s, 3H), 2.03 (s, 2H), 1.81 (s, 2H). LC/MS (ESI, m/z) = 474.1568 [M + H+]. I



Article

Figure 8. Compound 13’s antitumor efficacy in GIST-T1 xenograft mouse model. Female nu/nu mice bearing established GIST-T1 tumor xenografts were treated with 13 at 25.0, 50.0, and 100 mg/kg/d or vehicle. Daily oral administration was initiated when GIST-T1 tumors had reached a size of 100 to 200 mm3. Each group contained six animals. Data, mean ± SEM (A) body weight and (B) tumor size measurements from GIST-T1 xenograft mice after 13 administration. Initial body weight and tumor size were set as 100%. (C) Representative photographs of tumors in each group after 25.0, 50.0, or 100 mg/kg/d 13 or vehicle treatment. (D) Comparison of the final tumor weight in each group after 21-day treatment period of 13; ns, p > 0.05, *p < 0.05, **p < 0.01. N-(4-Chloro-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-3(trifluoromethyl)benzamide (20). Yield 55%. 1H NMR (400 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.67 (s, 2H), 8.27 (s, 2H), 8.00 (d, J = 7.4 Hz, 1H), 7.90 (d, J = 7.1 Hz, 1H), 7.82 (t, J = 7.5 Hz, 1H), 7.72 (s, 1H), 7.58−7.38 (m, 3H), 4.71 (s, 1H), 3.91−3.34 (m, 4H), 2.07 (s, 2H), 1.81 (s, 2H). LC/MS (ESI, m/z) = 504.1229 [M + H+]. N-(4-Methoxy-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-3(trifluoromethyl)benzamide (21). Yield 66%. 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.65 (s, 2H), 8.27 (s, 2H), 8.04−7.72 (m, 3H), 7.59−7.44 (m, 2H), 7.38 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 4.52 (s, 1H), 3.97 (s, 1H), 3.78 (s, 3H), 3.5−3.32 (m, 3H), 2.02 (s, 2H), 1.74 (s, 2H). LC/MS (ESI, m/z) = 500.1726 [M + H+]. N-(3-((1-Isonicotinoylpiperidin-4-yl)oxy)-4-methylphenyl)-4methyl-3-(trifluoromethyl)benzamide (22). Yield 66%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.68 (d, J = 4.1 Hz, 2H), 8.23 (s, 1H), 8.15 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.55−7.36 (m, 3H), 7.29 (d, J = 8.1 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.61 (s, 1H), 3.84 (s, 1H), 3.68 (s, 1H), 3.48 (s, 1H), 3.30 (s, 1H), 2.17 (s, 3H), 2.00 (s, 2H), 1.78 (s, 2H). LC/MS (ESI, m/z) = 498.1933 [M + H+]. 4-Methyl-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)3-(trifluoromethyl)benzamide (23). Yield 63%. 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 8.65 (s, 2H), 8.22 (s, 2H), 7.88 (s, 1H), 7.62 (s, 1H), 7.49 (s, 2H), 7.28 (s, 1H), 7.12 (s, 1H), 4.60 (s, 1H), 3.98−3.43 (m, 4H), 2.16 (s, 3H), 2.00 (s, 2H), 1.77 (s, 2H). LC/MS (ESI, m/z) = 498.1923 [M + H+]. 4-Methyl-N-(4-methyl-3-((1-(thiazole-5-carbonyl)piperidin-4-yl)oxy)phenyl)-3-(trifluoromethyl)benzamide (24). Yield 65%. 1H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.24 (s, 1H), 8.22 (s, 2H), 8.14 (s, 1H), 7.62 (s, 1H), 7.49 (s, 1H), 7.27 (s, 1H), 7.12 (s, 1H),

4.62 (s, 1H), 3.79 (s, 2H), 3.66 (s, 2H), 2.16 (s, 3H), 2.02 (s, 2H), 1.79 (s, 2H). LC/MS (ESI, m/z) = 504.1496 [M + H+]. N-(3-((1-(Isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4-methylphenyl)-4-methyl-3-(trifluoromethyl)benzamide (25). Yield 75%. 1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.76 (s, 1H), 8.22 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.49 (s, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.13 (d, J = 7.9 Hz, 1H), 6.96 (s, 1H), 4.63 (s, 1H), 3.67 (t, J = 47.8 Hz, 4H), 2.16 (s, 3H), 2.02 (s, 2H), 1.79 (s, 2H). LC/MS (ESI, m/z) = 488.1722 [M + H+]. 3-Chloro-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)5-(trifluoromethyl)benzamide (26). Yield 72%. 1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.65 (s, 2H), 8.31 (s, 1H), 8.23 (s, 1H), 8.13 (s, 1H), 7.87 (s, 1H), 7.46 (s, 2H), 7.27 (s, 1H), 7.15 (d, J = 6.8 Hz, 1H), 4.60 (s, 1H), 3.92−3.34 (m, 4H), 2.17 (s, 3H), 2.01 (s, 2H), 1.77 (s, 2H). LC/MS (ESI, m/z) = 518.1385 [M + H+]. 3-Chloro-N-(3-((1-(isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4methylphenyl)-5-(trifluoromethyl)benzamide (27). Yield 56%. 1 H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.76 (s, 1H), 8.32 (s, 1H), 8.24 (s, 1H), 8.13 (s, 1H), 7.47 (s, 1H), 7.28 (s, 1H), 7.15 (d, J = 7.4 Hz, 1H), 6.96 (s, 1H), 4.63 (s, 1H), 3.79−3.54 (m, 4H), 2.17 (s, 3H), 2.03 (s, 3H), 1.81 (s, 2H). LC/MS (ESI, m/z) = 508.1169 [M + H+]. 3-Fluoro-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)5-(trifluoromethyl)benzamide (28). Yield 66%. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.65 (s, 2H), 8.15−8.09 (m, 2H), 7.96 (s, 1H), 7.87 (s, 1H), 7.47 (s, 2H), 7.27 (s, 1H), 7.15 (d, J = 7.4 Hz, 1H), 4.60 (s, 1H), 3.82−3.35 (m, 3H), 2.17 (s, 3H), 1.99 (s, 2H), 1.77 (s, 2H). LC/MS (ESI, m/z) = 502.1669 [M + H+]. N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)-6(trifluoromethyl)picolinamide (29). Yield 58%. 1H NMR (400 MHz, J



Article

Scheme 1. Synthesis of Compounds 9−30a

a

Reagents and conditions: (a) for 37a−d: tert-butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate; for 37e: tert-butyl 3-hydroxypiperidine-1carboxylate, DIAD, PPh3, THF, 0 °C to rt; for 37f: tert-butyl (2-bromoethyl)carbamate, K2CO3, DMF, 90 °C, overnight; (b) Pd/C, H2, EtOAc, rt, 6 h; (c) R3CO2H, HATU, DIPEA, DMF, rt, 2 h; (d) 4 N HCl in EtOAc, rt, overnight; (e) R1CO2H, HATU, DIPEA, DMF, rt, 2 h.

Scheme 2. Synthesis of Compounds 31−34a

a Reagents and conditions: (a) 4 N HCl in EtOAc, rt, overnight; (b) nicotinic acid, HATU, DIPEA, DMF, rt, 2 h; (c) Pd/C, H2, EtOAc, rt, 6 h; (d) R3CO2H, HATU, DIPEA, DMF, rt, 2 h.

DMSO-d6) δ 10.28 (s, 1H), 8.65 (s, 2H), 8.38 (s, 2H), 8.17 (s, 1H), 7.87 (s, 1H), 7.68−7.27 (m, 3H), 7.15 (s, 1H), 4.66 (s, 1H), 3.98−3.34 (m, 4H), 2.17 (s, 3H), 1.97 (s, 2H), 1.77 (s, 2H). HRMS (ESI, m/z) = 485.1729 [M + H+]. N-(3-((1-(Isoxazole-5-carbonyl)piperidin-4-yl)oxy)-4-methylphenyl)-6-(trifluoromethyl)picolinamide (30). Yield 61%. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 8.75 (s, 1H), 8.37 (d, J = 8.4 Hz, 2H), 8.18 (d, J = 7.6 Hz, 1H), 7.56 (s, 1H), 7.41 (d, J = 7.3 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.02−6.86 (m, 1H), 4.69 (s, 1H), 3.79−3.54

(m, 4H), 2.17 (s, 3H), 2.03 (s, 2H), 1.80 (s, 2H). LC/MS (ESI, m/z) = 475.1522 [M + H+]. Compounds 31−34 were prepared following the synthetic procedure of 33. 3-Methoxy-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)benzamide (31). Yield 63%. 1H NMR (400 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.64 (s, 2H), 7.87 (s, 1H), 7.50−7.46(m, 5H), 7.27 (s, 1H), 7.12 (s, 2H), 4.59 (s, 1H), 3.83−3.55 (m, 7H), 2.15 (s, 3H), 2.00 (s, 2H), 1.76 (s, 2H). LC/MS (ESI, m/z) = 446.2010 [M + H+]. K



Article

Scheme 3. Synthesis of Compound 35a

a

Reagents and conditions: (a) 1-chloro-4-isocyanato-2-(trifluoromethyl)benzene, CH2Cl2, rt, overnight; (b) 4 N HCl in EtOAc, rt; (c) nicotinic acid, HATU, DIPEA, DMF, rt, 2 h.

5-(tert-Butyl)-N-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)isoxazole-3-carboxamide (32). Yield 59%. 1 H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.67 (s, 2H), 7.90 (d, J = 7.8 Hz, 1H), 7.49 (m, 2H), 7.36 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 6.68 (s, 1H), 4.58 (s, 1H), 3.96−3.32 (m, 4H), 2.17 (s, 3H), 2.01 (s, 2H), 1.78 (s, 2H), 1.37 (s, 9H). LC/MS (ESI, m/z) = 463.2273 [M + H+]. N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)benzo[d][1,3]dioxole-5-carboxamide (33). To a solution of benzo[d][1,3]dioxole-5-carboxylic acid (0.05 mmol, 8.3 mg) and 41c (0.05 mmol, 16 mg) in 0.5 mL DMF were added HATU (0.06 mmol, 22.8 mg) and DIPEA (0.1 mmol, 13 mg). The mixture was stirred at room temperature for 2 h, and the system was quenched with water, extracted with EtOAc, and dried with anhydrous Na2SO4. The solvents were removed under vacuum, and the crude product was purified by silica gel flash chromatography (DCM: MeOH = 10:1) to give the product 33 (15.3 mg, 67%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.67 (s, 2H), 7.89 (s, 1H), 7.61−7.40 (m, 4H), 7.28 (s, 1H), 7.09 (m, 2H), 6.15 (s, 2H), 4.60 (s, 1H), 3.95−3.37 (m, 4H), 2.17 (s, 3H), 2.04 (s, 2H), 1.78 (s, 2H). LC/MS (ESI, m/z) = 460.1803 [M + H+]. N-(4-Methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)quinoline3-carboxamide (34). Yield 57%. 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.38 (s, 1H), 8.97 (s, 1H), 8.68 (s, 2H), 8.18−8.13 (m, 2H), 7.90 (s, 2H), 7.76 (s, 1H), 7.57 (s, 1H), 7.51(s, 1H), 7.35 (s, 1H), 7.18 (d, J = 7.1 Hz, 1H), 4.64 (s, 1H), 3.87−3.55 (m, 4H), 2.20 (s, 3H), 2.03 (s, 2H), 1.81 (s, 2H). LC/MS (ESI, m/z) = 467.2001 [M + H+]. 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-((1-nicotinoylpiperidin-4-yl)oxy)phenyl)urea (35). Yield 53%. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.82 (s, 1H), 8.65 (s, 2H), 8.08 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.70−7.56 (m, 2H), 7.50 (d, J = 4.7 Hz, 1H), 7.25 (s, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.87 (d, J = 6.9 Hz, 1H), 4.62 (s, 1H), 3.83−3.52 (m, 4H), 2.14 (s, 3H), 2.00 (s, 2H), 1.78 (s, 2H). LC/MS (ESI, m/z) = 533.1486 [M + H+]. tert-Butyl 4-(5-Amino-2-methylphenoxy) piperidine-1-carboxylate (38a). 2-Methyl-5-nitrophenol (36a) (5 mmol, 0.77g) and tertbutyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate (10 mmol, 2.79g) were dissolved in 15 mL DMF. Then K2CO3 (10 mmol, 1.38g) was added to the system and heated at 90 °C overnight. The system was extracted with EtOAc and dried with anhydrous Na2SO4. The solvent was removed under vacuum, and the residue was purified by silica gel flash column chromatography (petroleum ether:EtOAc = 6:1) to give 37a as a yellow solid. LC/MS (ESI, m/z) [M + Na+] = 359.1693. The obtained 37a was directly dissolved in 20 mL EtOAc, and Pd/C (5%) was added. The mixture was stirred under hydrogen balloon at room temperature for 6 h. The system was filtered by diatomaceous earth, and the filtered organic system was concentrated under vacuum. The residue was purified by silica gel flash chromatography (petroleum ether:EtOAc = 8:1) to give 38a (1.12g) as a yellow oil with a two-step yield of 73%. 1H NMR (400 MHz, DMSO-d6) δ 6.74(d, J = 6.0 Hz, 1H), 6.23 (s, 1H), 6.07 (d, J = 6.0 Hz, 1H), 4.78 (s, 2H), 4.37−4.35 (m, 1H), 3.55 (m, 2H), 3.26 (m, 2H), 1.97 (s, 3H), 1.83 (s, 2H), 1.55 (s, 2H), 1.40 (s, 9H). LC/MS (ESI, m/z) = 329.1949 [M + Na+]. Compounds 38b−d were prepared following the synthetic procedure of 38a. tert-Butyl 4-(5-Amino-2-chlorophenoxy)piperidine-1-carboxylate (38b). Yield (two-step) 71%. 1H NMR (400 MHz, CDCl3) δ 7.09

(d, J = 7.6 Hz, 1H), 6.29−6.23 (m, 2H), 4.32−4.28 (m, 1H), 3.64−3.62 (m, 2H), 3.46−3.42 (m, 2H), 1.82 (s, 4H), 1.45 (s, 9H). LC/MS (ESI, m/z) = 349.1403 [M + Na+]. tert-Butyl 4-(5-Amino-2-methoxyphenoxy)piperidine-1-carboxylate (38c). Yield (two-step) 69%. 1H NMR(400 MHz, DMSO-d6) δ 6.67 (d, J = 7.6 Hz, 1H), 6.30 (s, 1H), 6.13 (d, J = 7.6 Hz, 1H), 4.61 (s, 2H), 4.32−4.28 (m, 1H), 3.63−3.60 (m, 5H), 3.16 (s, 2H), 1.97 (s, 3H), 1.81 (s, 2H), 1.51 (s, 2H), 1.40 (s, 9H). LC/MS (ESI, m/z) = 345.1898 [M + Na+]. tert-Butyl 4-(3-Aminophenoxy)piperidine-1-carboxylate (38d). Yield (two-step) 58%. 1H NMR (400 MHz, DMSO-d6) δ 6.87 (d, J = 4.4 Hz, 1H), 6.15−6.08 (m, 3H), 4.98 (s, 2H), 4.38−4.36 (m, 1H), 3.61 (s, 2H), 3.16 (s, 2H), 1.97 (s, 3H), 1.84 (s, 2H), 1.50 (s, 2H), 1.40 (s, 9H). LC/MS (ESI, m/z) = 315.1783 [M + Na+]. tert-Butyl 3-(3-Aminophenoxy)piperidine-1-carboxylate (38e). Diisopropyl azodicarboxylate (DIAD) (6 mmol, 1.21g) and triphenylphosphine (6 mmol, 1.57g) in 20 mL dry THF were stirred at 0 °C for 0.5 h. tert-Butyl 3-hydroxypiperidine-1-carboxylate (6 mmol, 1g) and 3-nitrophenol (5 mmol, 595 mg) were added to the system, which was warmed to room temperature and stirred overnight. The solvent was removed under vacuum, and the crude product was purified by silica gel flash chromatography (petroleum ether:EtOAc = 6:1) to give the intermediate 37e as a yellow solid. The obtained 37e was dissolved in 20 mL EtOAc, and Pd/C (5%) was added. The mixture was stirred under hydrogen balloon at room temperature for 6 h. Then the system was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum to give 38e (1.17g) as yellow oil with a two-step yield 63%. 1H NMR (400 MHz, DMSO-d6) δ 6.90 (t, J = 7.7 Hz, 1H), 6.17−6.10 (m, 3H), 5.04 (s, 2H), 4.20 (s, 1H), 3.79−3.11 (m, 4H), 1.90−1.71 (m, 3H), 1.40−1.31 (m, 10H). LC/MS (ESI, m/z) = 315.1793 [M + Na+]. tert-Butyl (2-(5-amino-2-methylphenoxy)ethyl)carbamate (38f). 2-Methyl-5-nitrophenol (5 mmol, 0.77g) and tert-butyl (2-bromoethyl)carbamate (8 mmol, 1.79g) were dissolved in 15 mL DMF. K2CO3 (10 mmol, 1.38g) was added to the system and heated at 90 °C overnight. The system was extracted with EtOAc and dried with anhydrous Na2SO4. The solvent was removed under vacuum, and the residue was purified by silica gel flash chromatography (petroleum ether:EtOAc = 6:1) to give the intermediate 37f as a yellow solid. 37f (4 mmol, 1.13 g) was dissolved in 20 mL EtOAc, and Pd/C (5%) was added. The mixture was stirred under hydrogen balloon at room temperature for 6 h. The system was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum to give compound 38f (780 mg) as a yellow oil in two-step yield 59%. 1H NMR (400 MHz, DMSO-d6) δ 6.95 (s, 1H), 6.74 (d, J = 7.7 Hz, 1H), 6.17 (s, 1H), 6.07 (d, J = 7.6 Hz, 1H), 4.82 (s, 2H), 3.82 (s, 2H), 3.30 (d, J = 5.5 Hz, 2H), 1.98 (s, 3H), 1.40 (s, 9H). LC/MS (ESI, m/z) = 267.1626 [M + H+]. N-(4-Methyl-3-(piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)benzamide (40a). To a solution of 3-(trifluoromethyl)benzoic acid (3 mmol 570 mg) and 38a (3 mmol, 918 mg) in 15 mL DMF were added HATU (3.6 mmol, 1.37g) and DIPEA (4.5 mmol, 585 mg). The mixture was stirred at room temperature for 2 h, and the system was quenched with water, extracted with EtOAc, and dried with anhydrous Na2SO4. The solvents were removed under vacuum and provided the crude product 39a. LC/MS (ESI, m/z) [M + H+] = 479.2069. Then 39a L



Article

7.91−7.65 (m, 2H), 7.47 (d, J = 8.1 Hz, 1H), 4.92 (s, 1H), 3.16 (s, 4H), 2.29 (s, 3H), 2.17 (s, 2H), 1.95 (s, 2H). LC/MS (ESI, m/z) = 237.1159 [M + H+]. (4-(5-Amino-2-methylphenoxy)piperidin-1-yl) (pyridin-3-yl)methanone (41c). To a solution of nicotinic acid (3 mmol, 369 mg) and 41a (3 mmol, 816 mg) in DMF (15 mL) were added HATU (3.6 mmol, 1.37g) and DIPEA (4.5 mmol, 585 mg). The resulting mixture was stirred at room temperature for 2 h, and the system was quenched with water, extracted with EtOAc, and dried with anhydrous Na2SO4. The solvents were removed under vacuum to yield the crude product 41b, which was dissolved in EtOAc (20 mL), and Pd/C (5%) was added. The mixture was stirred under hydrogen balloon at room temperature for 6 h. The system was filtered through diatomaceous earth, and the filtrate was concentrated under vacuum to give the product 41c (727 mg) as yellow oil. Yield (two-step) 78%. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 2H), 7.88 (d, J = 7.5 Hz, 1H), 7.49 (dd, J = 7.5, 4.7 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.28 (s, 1H), 6.09 (d, J = 7.6 Hz, 1H), 4.87 (s, 2H), 4.49 (s, 1H), 3.96−3.38 (m, 4H), 2.02 (s, 5H), 1.72 (s, 3H). LC/MS (ESI, m/z) = 312.1640 [M + H+]. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-methyl-3-(piperidin4-yloxy)phenyl)urea Hydrochloride (42b). Compound 38a (5 mmol, 1.53 g) in CH2Cl2 (20 mL) was added to 1-chloro-4-isocyanato-2(trifluoromethyl)benzene (5 mmol, 1.1 g), and the mixture was stirred overnight at room temperature. The system was extracted with EtOAc and dried with anhydrous Na2SO4. The solvents were removed under vacuum to give the crude product 42a, which was added directly into 4 N HCl in EtOAc (30 mL). The mixture was stirred overnight at room temperature. The solid was collected and washed with EtOAc to give the product 42b (1.6 g) as a white solid. Yield (two step) 69%. 1H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.33 (s, 1H), 8.90 (s, 2H), 8.10 (s, 1H), 7.72−7.52 (m, 2H), 7.30 (s, 1H), 7.06 (d, J = 7.9 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H), 4.59 (s, 1H), 3.20 (s, 2H), 3.14 (s, 2H), 2.13 (s, 5H), 1.92 (s, 2H). LC/MS (ESI, m/z) = 428.1281 [M + H+]. Cell Lines and Cell Culture. The human GIST-T1 cell line was purchased from Cosmo Bio Co., Ltd. Tokyo, Japan. 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, USA. K562 (CML), KU812 (CML), MEG-01 (CML), MV4-11 (AML), MOLM14 (AML), U937 (AML), REC-1 (human B-cell lymphoma cell), HL-60 (human promyelocytic leukemia cells), MEC-1(CLL), Kasumi-1 (AML), CHL (hamster lung cell), and CHO (hamster ovary cell) were obtained from American Type Culture Collection (Manassas, VA). All the cells were grown in a humidified incubator (Thermo, USA) at 37 °C under 5% CO2. GIST-T1, CHO cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. MV4-11 (AML), GIST-882, and GIST-48B were grown in IMDM supplemented with 10% FBS and 1% penicillin/streptomycin. All other cell lines and all the isogenic BaF3 cells were grown in RPMI 1640 medium supported with 10% FBS and 1% penicillin/streptomycin. Kasumi-1 (AML) was grown in RPMI 1640 medium supported with 20% FBS and 1% penicillin/streptomycin. Adherent cells were grown in tissue culture flasks until they were 85−95% confluent prior to use. For suspension cells, cells were collected by spinning down at 700 rpm/min for 4 min before use. General Procedure for Antiproliferation Assays. A density of 1 to 3 × 104 cells/mL cells were mixed with various concentrations of compounds, then 100 μL suspension was added to each well and incubated for 72 h. Cell viability was determined using the CellTiter-Glo (Promega, USA) or CCK-8 (Beboy, China). Both assays were performed according to the manufacturer instructions. For CellTiter-Glo assay, luminescence was determined in a multilabel reader (Envision, PerkinElmer, USA). For CCK-8 assay, absorbance was measured in a microplate reader (iMARK, Bio-Rad, USA) at 450 and 655 nm. Data were normalized to control group (DMSO). GI50 values were calculated using Prism 5.0 (GraphPad Software, San Diego, CA). TEL-Isogenic Cell Generation. Retroviral constructs for BaF3-KIT mutants were made based on the pMSCVpuro (Clontech) backbone. For TEL-KIT vector, the first 1 kb of human TEL gene with an artificial myristoylation sequence (MGCGCSSHPEDD) was cloned into the pMSCVpuro retroviral vector, followed by a 3xFLAG tag sequence and

was dissolved in 10 mL 4 N HCl in EtOAc. The mixture was stirred at room temperature overnight and neutralized by NaHCO3 (aq), then extracted by EtOAc and dried with anhydrous Na2SO4. The concentrated residue was purified by silica gel flash chromatography (DCM:MeOH = 10:1) to give the product 40a (782 mg) as a white solid with two-step yield 69%. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.27 (s, 2H), 7.94 (s, 1H), 7.77 (s, 1H), 7.48 (s, 1H), 7.28 (s, 1H), 7.11 (s, 1H), 4.44−4.42 (m, 1H), 3.07 (s, 2H), 2.82 (s, 2H), 2.13 (s, 1H), 1.97 (s, 3H), 2.01 (s 2H), 1.70 (s, 2H). LC/MS (ESI, m/z) = 379.1564 [M + H+]. Compounds 40b−i were prepared following the synthetic procedure of 40a. 4-Methyl-N-(4-methyl-3-(piperidin-4-yloxy)phenyl)-3(trifluoromethyl)benzamide Hydrochloride (40b). Yield (two-step) 63%. 1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.31 (s, 2H), 8.24 (s, 2H), 7.59−7.49 (m, 2H), 7.31 (s, 1H), 7.11 (s, 1H), 4.57−4.55 (m, 1H), 3.17 (brs, 2H), 3.08 (s, 2H), 2.5 (s, 1H), 2.14 (s, 5H), 1.96 (s, 3H). LC/MS (ESI, m/z) = 393.1719 [M + H+]. 3- Fluoro-N-(4-methyl-3-(piperidin-4-yloxy)phenyl)-5(trifluoromethyl)benzamide (40c). Yield (two-step) 58%. 1H NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.27−8.05 (m, 2H), 7.97 (d, J = 7.1 Hz, 1H), 7.43 (s, 1H), 7.28 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.40 (s, 1H), 3.03 (s, 2H), 2.72 (s, 2H), 2.13 (d, s, 3H), 1.93 (s, 2H), 1.64 (s, 2H). LC/MS (ESI, m/z) = 397.1466 [M + H+]. 3-Chloro-N-(4-methyl-3-(piperidin-4-yloxy)phenyl)-5(trifluoromethyl)benzamide (40d). Yield (two-step) 61%. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.34 (s, 1H), 8.26 (s, 1H), 8.13 (s, 1H), 7.47 (s, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 4.43 (s, 1H), 3.07 (s, 2H), 2.77 (s, 2H), 2.16 (s, 3H), 1.95 (s, 2H), 1.70 (s, 2H). LC/MS (ESI, m/z) = 413.1172 [M + H+]. N-(4-Methyl-3-(piperidin-4-yloxy)phenyl)-6-(trifluoromethyl)picolinamide Hydrochloride (40e). Yield (two-step) 53%. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.26 (s, 2H), 8.96 (s, 1H), 8.45− 8.27 (m, 2H), 8.19 (d, J = 6.8 Hz, 1H), 7.53 (s, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 4.65 (s, 1H), 3.21 (s, 2H), 3.12 (s, 2H), 2.18 (s, 5H), 1.97 (s, 2H). LC/MS (ESI, m/z) = 416.1280 [M + H+]. N-(4-Chloro-3-(piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)benzamide (40f). Yield (two-step) 56%. 1H NMR (400 MHz, DMSOd6) δ 10.69 (s, 1H), 8.32 (s, 2H), 8.00 (d, J = 6.6 Hz, 1H), 7.81 (d, J = 9.3 Hz, 2H), 7.46 (s, 2H), 4.64 (s, 1H), 3.19 (s, 2H), 3.04 (s, 2H), 2.13 (s, 2H), 1.91 (s, 2H). LC/MS (ESI, m/z) = 399.1006 [M + H+]. N-(4-Methoxy-3-(piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)benzamide (40g). Yield (two-step) 59%. 1H NMR (400 MHz, DMSOd6) δ 10.46 (s, 1H), 9.05 (s, 2H), 8.32 (s, 2H), 7.98 (d, J = 7.6 Hz, 1H), 7.80 (s, 1H), 7.58 (s, 1H), 7.40 (d, J = 9.1 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 4.50 (s, 1H), 3.80 (s, 3H), 3.25 (s, 2H), 3.08 (s, 2H), 2.11 (s, 2H), 1.92 (s, 2H). LC/MS (ESI, m/z) [M + H+] = 395.1513. N-(3-(Piperidin-4-yloxy)phenyl)-3-(trifluoromethyl)benzamide Hydrochloride (40h). Yield (two-step) 47%. 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 9.18 (s, 2H), 8.28 (s, 2H), 7.98 (d, J = 7.2 Hz, 1H), 7.80 (s, 1H), 7.57 (s, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 4.65 (s, 1H), 3.20 (s, 3H), 3.10 (s, 2H), 2.15 (s, 2H), 1.91 (s, 2H). LC/MS (ESI, m/z) = 365.1396 [M + H+]. N-(3-(piperidin-3-yloxy)phenyl)-3-(trifluoromethyl)benzamide (40i). Yield (two-step) 52%. 1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.29 (s, 2H), 7.98 (d, J = 6.6 Hz, 1H), 7.81 (d, J = 7.2 Hz, 1H), 7.59−7.12 (m, 3H), 6.74 (d, J = 7.4 Hz, 1H), 4.31 (s, 1H), 3.19−2.58 (m, 4H), 2.04 (s, 1H), 1.73 (s, 1H), 1.57−1.50 (m, 2H), 1.23−1.20 (m, 1H). LC/MS (ESI, m/z) = 365.1407 [M + H+]. N-(3-(2-aminoethoxy)-4-methylphenyl)-3-(trifluoromethyl)benzamide Hydrochloride (40j). Yield (two-step) 56%. 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 8.46 (s, 3H), 8.35 (d, J = 7.4 Hz, 2H), 7.97 (d, J = 7.4 Hz, 1H), 7.79 (t, J = 7.4 Hz, 1H), 7.54 (s, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.20 (s, 2H), 3.26 (s, 2H), 2.19 (s, 3H). LC/MS (ESI, m/z) = 339.1249 [M + H+]. 4-(2-Methyl-5-nitrophenoxy)piperidine Hydrochloride (41a). Compound 37a (5 mmol, 1.68 g) was added into 20 mL EtOAc (4 N HCl), and the system was stirred at room temperature for 6 h. The solid was collected and dried to give the product 41a HCl salt as a yellow solid (1.22g, 90%). 1H NMR (400 MHz, DMSO-d6) δ 9.35 (s, 2H), M



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Cell Cycle Analysis. GIST-T1, GIST-882, and GIST-48B cells were treated with DMSO, serially diluted compound 13 (0.3, 1, and 3 μM), and 3 μM Imatinib for the indicated periods. The cells were fixed in 70% cold ethanol and incubated at −20 °C overnight then stained with PI/RNase staining buffer (BD Pharmingen). Flow cytometry was performed using a FACS Calibur (BD), and results were analyzed by ModFit software. In Vivo Pharmacokinetics Study. Compound 13 was dissolved in 55% saline containing 5% DMSO and 40% PEG400 by vortex. The final concentration of the stock solution was 1 mg/mL for administration. Six 8 week old male Sprague−Dawely rats were fasted overnight before starting drug treatment via intravenous and oral administration. Animal blood collection time points were as follows: for groups 1, 3, 5 (intravenous): 1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h before and after administration was selected; for groups 2, 4, 6 (oral): 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h before and after dosing. Each time about 0.2 mL of blood was collected through the jugular vein adding heparin for anticoagulation and kept on ice. Then plasma was separated by centrifugation at 8000 rpm for 6 min at 2−8 °C. The obtained plasma was stored at −80 °C before analysis. After finishing the test, all surviving animals were transferred to the repository or euthanasia (CO2 asphyxiation). GIST-T1 Xenograft Model. Six week old female nu/nu mice were purchased from the Shanghai Experimental Center, Chinese Academy of Sciences (Shanghai, China). All animals were maintained 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), and all efforts were made to minimize animal suffering. To obtain orthotopic xenograft of human mammary tumor in the mice, cells were harvested during exponential growth. Five million GIST-T1 cells in PBS were suspended in 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 GIST-T1 tumors had reached a size of 100−200 mm3. Animals were then randomized into treatment groups of six mice each for efficacy studies. Compound 13 was delivered daily in a HKI solution (0.5% methocellulose/0.4% Tween80 in ddH2O) by orally gavages. A range of doses of 13 or its vehicle was administered, as indicated in Figure 8 legends. Body weight and tumor growth were measured daily after 13 treatment. Tumor volumes were calculated as follows: tumor volume (mm3) = [(W2 × L)/2], where width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements. HE Staining. HE staining was carried out according to the previous report.20 First the sections were hydrated, and then the slide was dipped into a Coplin jar containing Mayer’s hematoxylin and agitated for 30 s. After rinsing the slide in H2O for 1 min, it was stained with 1% eosin Y solution for 10−30 s with agitation. Subsequently, the sections were dehydrated with two changes of 95% alcohol and two changes of 100% alcohol for 30 s each. The alcohol was extracted with two changes of xylene. Finally, one or two drops of mounting medium were added and covered with a coverslip. Ki-67 Staining. For IHC demonstration of Ki-67, tissue sections were quenched for endogenous peroxides and placed in an antigen retrieval solution (0.01 M citrate buffer, pH 6.0) for 15 min in a microwave oven at 100 °C at 600 W. After incubation in the casein block, mouse mAb anti-Ki-67 (ZSGB-BIO, China) was applied to the sections at dilutions of 1:50. Incubations with primary antibodies lasted overnight at 4 °C. The secondary detection system was used to visualize antibody binding. Staining was developed with 3,3′-diaminobenzidine (DAB), and the slides were counterstained with hematoxylin, dehydrated, and mounted. TUNEL Staining. TUNEL staining was performed using the POD in Situ Cell Death Detection kit (Roche, USA). Briefly, sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and then treated by nuclease-free Proteinase K for 15 min at room temperature before the endogenous peroxidase was blocked in 3% H2O2 in methanol. Terminal deoxynucleotidyl transferase in reaction buffer was applied to sections for 1 h at 37 °C. Following washes, the slides were covered by converter-POD solution for 30 min at 37 °C.

a stop codon. Then, the kinase domain coding sequence of KIT was inserted in-frame between TEL and 3xFLAG sequences. For full-length expression vectors, the coding sequences of KIT variants were directly cloned in pMSCVpuro vector with a 3xFLAG tag at the C-terminal end. All mutagenesis was performed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer’s instructions. Retrovirus was packaged in HEK293T cells by transfecting KIT-containing MSCV vectors together with two helper plasmids. Virus supernatants were harvested 48 h after transfection and filtered before infection. Then BaF3 cells were infected with harvested virus supernatants using spinoculation protocol, and stable cell lines were obtained by puromycin selection for 48 h. The IL-3 concentrations in the culture medium were gradually withdrawn until cells were able to grow in the absence of IL-3. Kinase Biochemical Assay. The fluorescence resonance energytransfer-based Z′-LYTE kinase assay kit−Tyr 06 peptide (Invitrogen, Carlsbad, CA) was used to evaluate the IC50 value of compound 13 for inhibition of c-KIT kinase. The reaction was performed on a 384-well plate with a 10 μL reaction volume per well containing 2 μM Tyr 06 peptide substrate in reaction buffer and 2.5 μL c-KIT kinase with a serial 3-fold dilution of compound 13 (2.5 μL, 10 μM to 1.5 nM). The final reaction concentration of ATP was 300 μM. After 1 h incubation, a reaction was developed and terminated, and the fluorescence measured with an automated plate reader (SpectraMax I3, MD, USA). A dose− response curve was fitted using Prism 5.0 (GraphPad Software Inc., San Diego, CA). The biochemical tests of CSF1R and DDR1 were provided by Invitrogen (Carlsbad, CA, USA). The ADP-Glo kinase assay (Promega, Madison, WI) was used to screen compound 13 for its PDGFRβ inhibition effects. The kinase reaction system contains 9 μL PDGFRβ (10 ng/μL), 1 μL of serially diluted compound 13, and 10 μL substrate Poly(4:1 Glu, Tyr) peptide (0.4 μg/μL) (Promega, Madison, WI) with 100 μM ATP (Promega, Madison, WI). The reaction in each tube was started immediately by adding ATP and kept going for 1 h under 37 °C. After the tube was cooled for 5 min at room temperature, 5 μL solvent reactions were carried out in a 384-well plate. Then 5 μL of ADP-Glo reagent was added into each well to stop the reaction and consume the remaining ATP within 40 min. At the end, 10 μL of kinase detection reagent was added into the well and incubated for 30 min to produce a luminescence signal. The luminescence signal was measured with an automated plate reader (Envision, PE, USA), and the dose−response curve was fitted using Prism 5.0 (GraphPad Software Inc., San Diego, CA). Signaling Pathway Study. GIST-T1, GIST-882, and GIST-48B cells were treated with DMSO, serially diluted compound 13, and 1 μM Imatinib for 2 h. Cells were then washed with cold PBS and lysed in RIPA buffer (Beyotime, China). Western blotting was performed by standard methods, as previously described.21 The following antibodies were used at a range of antibody concentrations as indicated by the manufacturers to probe for specific proteins: rabbit polyclonal antibodies to total c-KIT (cat. no. 3308) and phospho-KIT Y719 (cat. no. 3308) and Y703 (cat. no. 3308) were from Cell Signaling Technology. Rabbit polyclonal antibodies to phospho-KIT Y823 was from Invitrogen (cat. no. 44−498G). Polyclonal rabbit antibodies to total p42/44 mitogen activated protein kinase (MAPK) (cat. no. 4695), phospho-p44/42 MAPK T202/Y204 (cat. no. 4370), phospho-AKT S473 (cat. no. 4060), total AKT (cat. no. 4691), phospho-Stat5 (cat. no. 9314), total Stat5 (cat. no. 9363), phospho-Stat3 (cat. no. 9145), total Stat3 (cat. no. 12640), phospho-S6K T389 (cat. no. 9206), total S6K (cat. no. 9202), phospho-S6 S235/236 (cat. no. 2211), total S6 (cat. no. 2217), phospho-Src Y416 (cat. no. 6943), total Src (cat. no. 2123), phospho-PDGFRα Y849/PDGFRβ Y857 (cat. no. 3308), and total PDGFRα (cat. no. 3308) were from Cell Signaling Technology. Beta actin antibodies were purchased from Sigma (cat. no. A5316). Apoptosis Effect Examination. GIST-T1, GIST-882, and GIST48B cells were treated with DMSO, serially diluted compound 13, 1 μM Imatinib for the indicated periods. Cells were collected and analyzed by Western blotting using the following antibodies: PARP (cat. no. 9532) and Caspase-3 (cat. no. 9665) from Cell Signaling Technology. Beta actin antibodies were purchased from Sigma (cat. no. A5316). N



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Apoptotic cells were detected after incubation in DAB chromogen (Beyotime Biotechnology, China) for approximately 8 min, and the slides were counterstained with hematoxylin. Molecular Modeling. Molecular docking of compound 13 to the ABL1 kinase and the c-KIT kinase was performed with software YetiX 8.3.22 The kinase domain of chain A in the PDB was used for docking (PDB ID: 2HYY and 1T46 for ABL1 and c-KIT, respectively). Alternative conformations of the side chains were manually confirmed, missing side chains were automatically added using AmberTools,23 the protonation and tautomeric state at physiological pH were confirmed by software Reduce,24 and the receptor side-chain structure was further optimized using YetiX 8.3. Compound 13 was constructed using BioX 4.6,25 and the atomic partial charges were generated by AmberTools. Template-based induced-fit docking of small molecules to the two kinases: ABL1 and c-Kit were performed using YetiX 8.3. The docked models were optimized by the directional Yeti force field.26

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REFERENCES

(1) Rubin, B. P.; Singer, S.; Tsao, C.; Duensing, A.; Lux, M. L.; Ruiz, R.; Hibbard, M. K.; Chen, C. J.; Xiao, S.; Tuveson, D. A.; Demetri, G. D.; Fletcher, C. D.; Fletcher, J. A. KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res. 2001, 61, 8118−8121. (2) (a) Joensuu, H.; Roberts, P. J.; Sarlomo-Rikala, M.; Anderson, L. C.; Tervahartiala, P.; Tuveson, D.; Siberman, S. L.; Capdeville, R.; Dimitrijevic, S.; Druker, B.; Demetri, G. D. Effect of the tyrosine inhibitor STI571 in a patient with a metastatis gastrointestinal stromal tumor. N. Engl. J. Med. 2001, 344, 1052−1056. (b) Demetri, G. D.; von Mehren, M.; Blanke, C. D.; Van den Abbeele, A. D.; Eisenberg, B.; Roberts, P. J.; Heinrich, M. C.; Tuveson, D. A.; Singer, S.; Janicek, M.; Fletcher, J. A.; Silverman, S. G.; Silberman, S. L.; Capdeville, R.; Kiese, B.; Peng, B.; Dimitrijevic, S.; Druker, B. J.; Corless, C.; Fletcher, C. D.; Joensuu, H. Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N. Engl. J. Med. 2002, 347, 472−480. (3) Yarden, Y.; Kuang, W. J.; Yang-Feng, T.; Coussens, L.; Munemitsu, S.; Dull, T. J.; Chen, E.; Schlessinger, J.; Francke, U.; Ullrich, A. Human proto-oncogene kit: a new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987, 6, 3341−3351. (4) (a) Hirota, S.; Isozaki, K.; Moriyama, Y.; Hashimoto, K.; Nishida, T.; Ishiguro, S.; Kawano, K.; Hanada, M.; Kurata, A.; Takeda, M.; Tunio, G. M.; Matsuzawa, Y.; Kanakura, Y.; Shinomura, Y.; Kitamura, Y. Gainof-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998, 279, 577−580. (b) Heinrich, M. C.; Corless, C. L.; Demetri, G. D.; Blanke, C. D.; von Mehren, M.; Joensuu, H.; McGreevey, L. S.; Chen, C. J.; Van den Abbeele, A. D.; Druker, B. J.; Kiese, B.; Eisenberg, B.; Roberts, P. J.; Singer, S.; Fletcher, C. D.; 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. (c) Sommer, G.; Agosti, V.; Ehlers, I.; Rossi, F.; Corbacioglu, S.; Farkas, J.; Moore, M.; Manova, K.; Antonescu, C. R.; Besmer, P. Gastrointestinal stromal tumors in a mouse model by target mutation of the kit receptor tyrosine kinase. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 6706−6711. (5) (a) Ernst, S. I.; Hubbs, A. E.; Przygodzki, R. M.; Emory, T. S.; Sobin, L. H.; O’Leary, T. J. Kit mutation portends poor prognosis in gastrointestinal stromal/smooth muscle tumors. Lab Invest. 1998, 78, 1633−1636. (b) Lasota, J.; Jasinski, M.; Sarlomo-Rikala, M.; Miettinen, M. Mutations in exon 11 of c-kit occur preferentially in malignant versus benign gastrointestinal stromal tumors and do not occur in leiomyomas or leiomyosarcomas. Am. J. Pathol. 1999, 154, 53−60. (6) (a) Kerkelä, R.; Grazette, L.; Yacobi, R.; Iliescu, C.; Patten, R.; Beahm, C.; Walters, B.; Shevtsov, S.; Pesant, S.; Clubb, F. J.; Rosenzweig, A.; Salomon, R. N.; Van Etten, R. A.; Alroy, J.; Durand, J. B.; Force, T. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate. Nat. Med. 2006, 12, 908−916. (b) Perik, P. J.; Rikhof, B.; de Jong, F. A.; Verweij, J.; Gietema, J. A.; van der Graaf, W. T. Results of plasma n-terminal pro b-type natriuretic peptide and cardiac troponin monitoring in gist patients do not support the existence of imatinibinduced cardiotoxicity. Ann. Oncol. 2008, 19, 359−361. (c) Rosti, G.; Martinelli, G.; Baccarani, M. In reply to ’cardiotoxicity of the cancer therapeutic agent imatinib mesylate’. Nat. Med. 2007, 13, 13−14. (7) Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui, J.; Fukuda, J. Y.; Chu, J. Y.; Nematalla, A.; Wang, X.; Chen, H.; Sistla, A.; Luu, T. C.; Tang, F.; Wei, J.; Tang, C. Discovery of 5-[5-Fluoro-2-oxo1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-Diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase. J. Med. Chem. 2003, 46, 1116−1119. (8) Warkentin, A. A.; Lopez, M. S.; Lasater, E. A.; Lin, K.; He, B. L.; Leung, A. Y. H.; Smith, C. C.; Shah, N. P.; Shokat, K. M. Overcoming myelosuppression due to synthetic lethal toxicity in FLT3-targeted acute myeloid leukemia therapy. eLife 2014, 3, e03445. (9) Bearss, D. J.; Joshi-hangal, R.; Liu, X.-H., Phiasivongsa, P.; Redkar, S. G.; Vankayalapati, H. Pharmaceutical formulations comprising salts of a protein kinase inhibitor and methods of using same. U.S. Patent US 20080226747, September 18, 2008.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00200.

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Article

Table S1 listing the DiscoveRx’s KinomeScan selectivity profiling data of 13 (PDF) Compound data (CSV)

AUTHOR INFORMATION

Corresponding Authors

*Phone: 86-551-65593186. E-mail: jingliu@hmfl.ac.cn. *Phone: 86-551-65595161. E-mail: qsliu97@hmfl.ac.cn. Author Contributions

Q.W., F.L., B.W., F.Z., and C.C. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): Dr. Shanchun Zhang is a shareholder of Hefei Cosource Medicine Technology Co., LTD.

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ACKNOWLEDGMENTS Q.L. is supported by the Joint Funds of Research on Major Science Facilities, Key Program of the National NSFC (no. U1432250). W.W., J.L., and Q.L. are supported by the grant of “Cross-disciplinary Collaborative Teams Program for Science, Technology, and Innovation (2014-2016)” from Chinese Academy of Sciences. We are grateful for the China “Thousand Talents Program” support for Q.L. and “Hundred Talents Program” of the Chinese Academy of Sciences support for J.L. and W.W. J.L. is also supported by the National Program for Support of Top-notch Young Professionals. Q.L. is also supported by the CAS/SAFEA international partnership program for creative research teams.

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ABBREVIATIONS USED CML, chronic myeloid leukemia; GISTs, gastrointestinal stromal tumors; ABL kinase, Abelson kinase; KIT kinase, v-kit Hardy− Zuckerman 4 feline sarcoma viral oncogene homologue; FLT3, FMS-related tyrosine kinase 3; RTK, receptor tyrosine kinase; PDGFR, platelet derived growth factor receptor; CSF1R, stimulating factor-1 receptor; SAR, structure−activity relationship O



Article

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