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Discovery of Potent, Selective Stem Cell Factor Receptor/Platelet Derived Growth Factor Receptor Alpha (c-KIT/PDGFRα) Dual Inhibitor for the Treatment of Imatinib-Resistant Gastrointestinal Stromal Tumors (GISTs) Yanli Lu,†,‡ Fei Mao,†,‡ Xiaokang Li,† Xinyu Zheng,† Manjiong Wang,† Qing Xu,† Jin Zhu,† and Jian Li*,† †

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China S Supporting Information *

ABSTRACT: Stem cell factor receptor (c-KIT) and platelet derived growth factor receptor alpha (PDGFRα) kinases play an important role in gastrointestinal stromal tumors (GISTs). Here, we have discovered an c-KIT/PDGFRα dual inhibitor, compound 31, with single-digit nanomolar potency against c-KIT and PDGFRα. Compared to Imatinib (1), 31 showed better antiproliferative efficacy against various TEL-c-KIT/PDGFRα-BaF3 isogenic cells, including three 1-resistant BaF3 cell lines, as well as against GIST-T1 and GIST-882 cell lines. Furthermore, compound 31 showed a good KinomeScan selectivity (468 kinases) (S score (1) = 0.01 at 1 μM concentration), good metabolic stability in liver microsomes, and no hERG inhibitory activity. It was worth noting that 31 inhibited GIST-T1 tumor growth (TGI = 81.5%) and even the BaF3-TEL-cKIT-T670I tumor progression (TGI = 41.9%, 1-resistant GISTs) at a dosage of 100 mg/kg/day without exhibiting apparent toxicity.

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INTRODUCTION Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms originating from the intestitial cells of Cajal, the pacemaker cells of the gut and pervading the entire area of the gastrointestinal tract.1 Epidemiological data shows that the annual incidence of GISTs is about 1−2/100000, and approximately 30% of GISTs are malignant.2 The treatment of GISTs faces great challenges due to its complex etiology. Prior to 2010,3 surgical resection appeared to be the only available treatment for GISTs, resulting in five-year survival rates of 48−54% for resectable cases,4 while the median survival period was only 19 months, and just 5−10% of irresectable or metastasized GISTs patients have a five-year survival rate.5 In addition, over one-third of patients will suffer tumor metastases after macroscopically complete surgery.6 Unfortunately, GISTs respond poorly to conventional cytotoxic chemotherapy, and radiation therapy has not been shown to be effective.7 The year 1998 witnessed a critical breakthrough for GISTs, which began with the discovery of the proto-oncogene stem cell © 2017 American Chemical Society

factor receptor (c-KIT). Approximately 90% of GISTs are positive for c-KIT expression,8 which has facilitated the diagnosis of GISTs. About 80%−85% of GISTs arise from gain-of-function mutations in c-KIT or the platelet derived growth factor receptor-α (PDGFRα) gene,9 both of which encode type III receptor tyrosine kinases (RTK). The common sites of mutations are usually located in c-KIT exon 11 (∼70%), c-KIT exon 9 (∼10%), or PDGFRα exons 12 or 18 (∼10%),10 which cause sustained activation of the tyrosine kinase resulting in uncontrolled cellular proliferation and resistance to apoptosis.11 Because of the critical role of c-KIT and PDGFRα kinases in cellular transformation and differentiation to the tumorigenesis of GISTs, c-KIT and PDGFRα kinases have been extensively evaluated as important drug discovery targets for anti-GIST therapy. Currently, there are three nonselective c-KIT/PDGFRα kinase inhibitors approved for clinical use (Figure 1). Imatinib (1),12 a Received: March 27, 2017 Published: May 25, 2017 5099

DOI: 10.1021/acs.jmedchem.7b00468 J. Med. Chem. 2017, 60, 5099−5119

Journal of Medicinal Chemistry

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Figure 1. Chemical structures of representative nonselective c-KIT/PDGFRα kinase inhibitors.

Figure 2. Schematic illustration of the discovery of selective potent dual c-KIT/PDGFRα inhibitor 31.

Moreover, a number of multitarget inhibitors (Figure 1) are undergoing clinical investigation such as nilotinib (4),22 masitinib (5),23 and dovitinib (6).24 Although the above agents for the treatment of GISTs have achieved some success stories, the clinical resistance still demand close attention for the heterogeneity of patterns of mutations in the c-KIT and PDGFRα proteins. In addition, nonselective profiles targeting c-KIT and PDGFRα frequently induced different and more side effects. The patients occurring serious adverse events (grade 3 or 4) with 1 treatment were 21.1% and suffering mild or moderate edema, and fatigue was frequently seen with 1 treatment, while 61% patients suffered the drug-related adverse events of grade 3 or higher with 3 treatment.12,21,25 Hypertension, diarrhea, palmar plantar erythrodysesthesia, and fatigue were commonly observed in therapy with 2 or 3.26 Moreover, skin discoloration (up to 30%, yellow color) and hair discoloration were more representative with 2 treatment,27 while weight loss, hypophosphatemia, and hyperlipasemia were more often observed with 3 treatment. In this text, we planned to design and synthesize a series of novel, potent, and selective dual-target c-KIT/PDGFRα inhibitors, with powerful efficacy against c-KIT/PDGFRα kinase and mutants, and anticipated finding a good lead compound for the treatment of GISTs, especially 1-resistant GISTs.

small molecule tyrosine kinase inhibitor (TKI) with activity against ABL/c-KIT/PDGFR kinases, was the first TKI approved by the U.S. Food and Drug Administration (FDA) as frontline therapy for metastatic or unresectable GISTs. Compound 1 binds competitively to the adenosine triphosphate (ATP) binding site of the target kinases, preventing substrate phosphorylation and signaling, thereby inhibiting tumor proliferation.13 However, approximately 14% patients are initially insensitive to 1,14 and one-half of the responding patients can acquire secondary c-KIT mutations within 2 year of 1 treatment, which contains a T670I substitution within the ATP binding pocket.15 The T670 residue is situated at the gatekeeper position of c-KIT kinase and provides one of the key hydrogen bonds for the binding to 1.16 This mutation prevents the full access of 1 to the ATP binding pocket due to steric hindrance in the drug binding site.17 In addition, experimental evidence showed that c-KIT/T670I mutant will contribute to an early, more aggressive metastasis and shorter progression-free survival.18 Additionally, nearly 5%−7% of patients with GISTs who have PDGFRα mutations are generally 1-resistant.19 Sunitinib (2),20 a VEGFR/PDGFR/c-KIT/ FLT3 TKI, is approved as second-line therapy for patients with advanced GISTs who are 1 resistant or intolerant. Regorafenib (3),21 a VEGFR/PDGFR/FGFR/c-KIT/RET/Raf-1 TKI, is a third-line therapy for GISTs that progressed on 1 and 2. 5100



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Table 1. Structures and Kinase Inhibitory Activities of Compounds 7−17

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DESIGN OF C-KIT/PDGFRα DUAL INHIBITORS Linifanib (7) (Figure 2),28 a 3-aminoindazole-based orally active drug containing an N,N′-diaryl urea moiety (Figure 2), is a novel available multitarget inhibitor of the vascular endothelial growth factor receptor (VEGFR) and PDGFR family members with minimal activity against unrelated RTKs. Inhibitor 7 has demonstrated potent antitumor efficacy in a broad range of clinical trials.29 At the beginning of our efforts to discover new antitumor chemotypes targeting VEGFR2 and PDGFRα, 7 was selected as a hit compound (Table 1; PDGFRα, IC50 = 15 nM; VEGFR2, IC50 = 250 nM). With the aid of a well-documented structure−activity relationship (SAR) as well as the synthetic accessibility of 7, we modified three regions (R1−R3, Figure 2) of this molecule to obtain new SAR information and improve TKI activity. To obtain novel analogues and clarify the SARs under the premise of minimizing the structural changes, we designed and synthesized compounds 8−15 (R1, Table 1) to determine if the 3-aminoindazole moiety is necessary for VEGFR2 and PDGFRα inhibitory activities, compounds 16,17 (R3, Table 1) to demonstrate the significance of the oxygen atom in the urea group as a key linker, and compounds 18−22 (R1, Table 3) to examine the influence of different substituents on the 3-position of pyrazolo[3,4-b]pyridine scaffold. Furthermore, compounds 23−65 (R2, Table 4) were prepared to determine if the type of N-substitutions would affect c-KIT/PDGFRα inhibitory activities.

elimination reaction with zinc and hydrazine hydrate then produced the last key intermediates 73a,b, followed by coupling with various commercially available substituted isocyanates and isothiocyanates to achieve target compounds 12−17 and 20−27. The synthetic route for the preparation of target compounds 18,19 and 41−65 is shown in Scheme 3. Boc protection of 73b gave trisubstitued intermediate 74, followed by bromination to provide 75, which was hydrolyzed to produce 76. The nucleophilic addition of substituted isocyanates with 76 generated target compounds 18,19. Different isocyanates were in situ synthesized using N,N′-carbonyl diimidazole (CDI) from various commercially available aliphatic amines, aryl amines, and aralkyl amines. Nucleophilic addition of these isocyanates with 73b gave target compounds 41−65. Intermediates 77a−m were reduced by the catalytic hydrogenation of Pd/C to provide the corresponding amines 78a−m, respectively, then reacted with phenyl chloroformate and 73b to generate target compounds 28−40 (Scheme 4). The details of the synthetic procedures and structural characterizations of target compounds 8−65 are described in the Experimental Section. The purities of all targeted compounds were determined by HPLC (Table S1, Supporting Information). The details of the synthetic procedures and structural characterizations of intermediates 66−78 are shown in the Supporting Information.

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RESULTS AND DISCUSSION Kinase Enzymatic Assay. On the basis of our initial investigations of 7, the novel designed analogues were first optimized according to the potencies (IC50) against PDGFRα, VEGFR2, and fibroblast growth factor receptor 1 (FGFR1). From Table 1, we could see that compounds 8−15 caused a distinct loss of activity against VEGFR2 (IC50 > 1000 nM) compared to 7 (IC50 = 250 nM) and weak inhibitory activity against FGFR1 (IC50 > 1000 nM), similar to 7 (IC50 = 5479 nM). This finding indicated that the potencies against VEGFR2 and FGFR1 may not tolerate the change of the core scaffold. Furthermore, in the studied set of three new core scaffolds (R1), the potency (especially PDGFRα) was increased in the order 3-methyl-pyrazolo[3,4-b]pyridine (12,13) > 3-methyl6-hydroxy- pyrazolo[3,4-b]pyridine (10,11) > 3-methyl-6hydroxy-4,5-dihydro-pyrazolo[3,4-b] pyridine (8,9). A small set of analogues with various N-aryl substituents (12−15, Table 1, R2) showed that R2 substituent enabled good variability for potent PDGFRα inhibitory activity. With the novel good core scaffold retained, we turned our attention to the “urea linker” (R3) and prepared thiourea compounds (16,17). Unfortunately, these two compounds both obviously lost activity against all three kinases (IC50 > 1000 nM). Considering the multitargeted profiles of 7, we decided to select compound 13, the most potent PDGFRα inhibitor among compounds 8−17, as the lead compound (Figure 2) to preliminarily explore its spectrum of activity against other RTKs (Table 2). Surprisingly, compound 13 showed very potent inhibitory activity against c-KIT at the single-digit nanomolar concentration (IC50 = 2.1 nM). It is interesting that compound 13 seemed to be a selective c-KIT/PDGFRα dual inhibitor. On the basis of this result, the subsequent work was aimed to developing a dualtargeted compound against c-KIT and PDGFRα, and VEGFR2 was used as a typical kinase to monitor the selective profile. To examine the influence of different substituents on the 3-position of pyrazolo[3,4-b]pyridine scoffold, we replaced

CHEMISTRY As outlined in Scheme 1, ethyl trifluoroacetate was selected as the starting material and was reacted with acetonitrile by Scheme 1. Synthesis of Intermediate 67aa

Reagents and conditions: (a) NaH, THF, reflux, 15 h; (b) N2H4· H2O, CH3HSO3, 80 °C, overnight. a

like-nucleophilic substitution to produce 4,4,4-trifluoro-3-oxobutanenitrile (66), which was not further purified because of its difficult detection by TLC and low boiling point. Then, 66 was cyclized in the presence of hydrazine hydrate to yield the intermediate 67a. Scheme 2 depicts the synthetic route for the preparation of target compounds 8−17 and 20−27. Intermediate 68a,b was synthesized by coupling 67a and 67b (commercially available) with 4-nitro benzaldehyde and 2,2-dimethyl-1,3-dioxane-4,6dione through an effective one-container three-component approach. The reduction of 68b by catalytic hydrogenation of Pd/C yielded the key intermediate 69, followed by treatment with two substituted phenyl isocyanates to produce target compounds 8,9. 68a,b was treated with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) and N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) to provide aromatic intermediates 70a,b via oxidative dehydrogenation. The nitro group of 70b was reduced using SnCl2 to form another key intermediate 71, followed by treatment with isocyanates to produce target compounds 10,11. The chlorination of 70a,b with phenylphosphonic dichloride (BPOD) gave intermediates 72a,b, and a reductive 5102



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Scheme 2. Synthesis of Compounds 8−17 and 20−27a

a Reagents and conditions: (a) for 68a, EtOH, reflux, 4 h; (b) for 68b, DMF, reflux, 4 h; (c) Pd/C, H2, MeOH, rt, 5 h; (d) R2NCO or R2NSO, Et3N, DMSO, rt, overnight; (e) for 70a, DDQ, BSTFA, chlorobenzene, reflux, overnight; (f) for 70b, DDQ, BSTFA, 1,4-dioxane, reflux, overnight; (g) SnCl2, EtOH, reflux, overnight; (h) BPOD, 110 °C, overnight; (i) N2, Zn, N2H4·H2O, THF, reflux, 5 h.

Scheme 3. Synthesis of Compounds 18,19 and 41−65a

a

Reagents and conditions: (a) (Boc)2O, DMPA, THF, rt, 1 h; (b) N2, NBS, BPO, CCl4, reflux, 20 h; (c) H2O, 1,4-dioxane, reflux, overnight; (d) R2NCO, Et3N, DMSO, rt, overnight; (e) R2NH2, CDI, Et3N, DMSO, rt, overnight.

the 3-methyl substituent with hydroxymethyl (18,19) and trifluoromethyl (20,21). The results in Table 3 showed that replacement of the 3-methyl group with a hydroxymethyl group (18 vs 13) maintained fairly potent c-KIT inhibition

(IC50 = 2.4 vs 2.1 nM) but led to a 2-fold loss of PDGFRα activity (IC50 = 87 vs 40 nM). Changing the 3-methyl group to the trifluoromethyl group (20 vs 13) could not be tolerated, leading to a complete loss of all kinase activities (IC50 > 50,000 nM). 5103



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Scheme 4. Synthesis of Compounds 28−40a

a

Reagents and conditions: (a) Pd/C, H2, MeOH, rt, overnight; (b) (1) pyridine, DMF, phenyl chloroformate, rt, 3 h, (2) 1,4-dioxane, reflux, overnight.

Table 2. Kinase Spectrum of 13 against RTK IC50 (nM) compd

PDGFRα

VEGFR2

FGFR1

c-KIT

PDGFRβ

FLT3

VEGFR1

VEGFR3

13

40

1225

>50000

2.1

197

1880

3493

376

Therefore, the 3-methyl-pyrazolo[3,4-b] pyridine moiety represented seemingly the best pharmacophore for inhibiting both c-KIT and PDGFRα. Considering the 3-methyl-1H-pyrazolo[3,4-b]pyridine scaffold and urea linker as a promising novel template for a dual-targeted c-KIT/PDGFRα inhibitor, we next focused SAR exploration on substituent (R2, Figure 2) at the terminal nitrogen of urea. Shifting the methyl group from 3-position (13) to 2-position (23) or 4-position (24) both led to significant loss of activity against c-KIT/PDGFRα (Table 4). Among various 3-substituents (13−15 and 25−28), 3-methyl group was the best one for c-KIT/PDGFRα inhibition. Surprisingly, a N-methyl piperazinyl group at 3-position (28) only moderately decreased the c-KIT/PDGFRα inhibitory activities compared to 13 (Table 4). Inspired by this result, a combination of 3-methyl and N-methyl piperazinyl or morpholinyl group on the terminal phenyl obtained compounds 29−31, of which 31 displayed more potent inhibitory activities than lead compound 13 against c-KIT/PDGFRα kinases (IC50 = 2.4 and 7.2 nM, respectively) and also retained the selectivity over VEGFR2. However, when we changed the combined type or position of methyl, N-methyl piperazinyl, or morpholinyl group on the terminal phenyl (32−35), it caused a significant loss of

activities against all three kinases. Furthermore, introducing diethylamino group (36,37) or dimethylamino group (38,39) on the terminal phenyl started to gain back the c-KIT/PDGFRα activities but inferior to 31. Replacement of 3-dimethylamino group with 3-dimethylaminomethyl group (40) also led to complete loss of activities to all three kinases. To explore more effective compounds, further screening of R2 group (41−65) was implemented (Table 4). For the linear alkyl groups (41−45), the c-KIT/PDGFRα inhibitory activity was substantially improved with an increasing length of alkyl groups. Changing linear alkyl (43) into branched alkyl (46,47) or cycloalky (48−51) groups could not enhance the c-KIT/ PDGFRα inhibitory activity compared to 13. For the aralkyl groups (52−59), the heteroarylmethyl groups (52−54) were favorable for c-KIT/PDGFRα inhibition compared to the phenylmethyl group (55) but inferior to 13. With the different length set of phenylalkyl groups (55−59), the potency against c-KIT/PDGFRα was increased in the order phenylpropyl (57) > phenylethyl (56) > 2,3-dihydro-indene (59) > phenylbutyl (58). Although the c-KIT inhibitory activity of 57 was slightly better than that of 13 and 31, the PDGFRα inhibitory activity of 57 was weaker than that of 31 (IC50 = 24 nM vs 7.2 nM). Additionally, replacement of the aryl group (R2) with heteroaryl 5104



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groups (60−62) all led to a reduction of kinase potency greater than 6-fold, but introduction of large fused aryl groups (63−65) seemingly was beneficial, leading to a visible increase of c-KIT/PDGFRα inhibition compared to 13 but still not improving PDGFRα inhibition at the single-digit nanomolar concentration. Collectively, the structural optimization and SAR studies led to the discovery of a number of novel compounds, such as 31, 45, 57, and 63 that exhibited a higher dual-target potency against c-KIT (IC50 < 3 nM) and PDGFRα (IC50 < 30 nM) as well as maintained the appropriate selective window against VEGFR2 (IC50 > 1800 nM). Especially, 31 had the most surprising single-digit nanomolar potency against PDGFRα, similar to the launched compound 1 (IC50= 7.2 vs 4.6 nM, Table 4), and had powerful c-KIT activity (IC50 = 2.4 nM), providing a 17-fold improvement compared to 1 (Table 4). Cellular Property and Biochemical Evaluation. In light of the previous research of kinase inhibitory activities, compound 31 was the best c-KIT/PDGFRα dual inhibitor. To further evaluate whether 31 was the optimal compound, we selected 11 compounds to examine the inhibitory activities against cellular c-KIT and PDGFRα, including four BaF3 cells,30 whose viability were KIT-dependent or PDGFRαdependent. As shown in Table 5, the 3-methyl-1H-pyrazolo[3,4-b]pyridine scaffold was favorable for the antiproliferation against c-KIT/PDGFRα cell lines. Moreover, the inhibitory activities of cellular level were consistent with the kinase

inhibitory activities in vitro. Excitingly, compound 31 showed the best antiproliferation activities against the four BaF3 cell lines, which was superior to the hit compound 7, with a 5-fold increase of Tel-c-KIT-BaF3 inhibitory potency (GI50 = 0.0181 vs 0.0887 μM, Table 5). Therefore, 31 was chosen for further in-depth studies of antitumor potencies. First, the inhibitory activities of optimal compound 31 against c-KIT/PDGFRα-BaF3 cell lines were comprehensively evaluated. The results are exhibited in Table 6 and Figure 3, including the four cell lines examined above. According to the data, 31 was more potent against all of the mutations except Tel-c-KIT/V559D-BaF3 (GI50 = 0.0965 vs 0.0310 μM) mutation compared to 1. On the basis of exhibiting a similar trend to 1 in the parental BaF3, 31 improved the antiproliferative activity more than 21-fold toward the native c-KIT (GI50 = 0.0181 vs 0.3819 μM). The inhibitory activity of Tel-c-KIT/ C674S-BaF3 mutation (GI50 = 0.0105 μM) was consistent with a point mutation31 Tel-c-KIT/L576P-BaF3 (GI50 = 0.0106 μM) in exon 11, which showed poor sensitivity for 1 (Figure 3). In addition, 31 was an effective inhibitor of all four ATP pocket mutations (Figure 3)32 and brought approximately more than 250-fold activity improvement in the 1-resistance mutations of Tel-c-KIT/T670I-BaF3 (GI50 = 0.0392 vs >10 μM) and Tel-cKIT/V559D/T670I-BaF3 (GI50 = 0.0364 vs >10 μM) compared to 1. With four A-loop mutations (Figure 3), 31 exhibited a highly inhibitory activity of Tel-c-KIT/N822 K-BaF3 mutation (GI50 = 0.0111 μM) and Tel-c-KIT/N829 K-BaF3 5105



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Table 4. continued

Table 5. Antiproliferative Effect of 11 Compounds against Four TEL-c-KIT/PDGFRα-BaF3 Isogenic Cells kinases in vitro: IC50 (nM)

BaF3 isogenic cells: GI50 (μM)

compd

c-KIT

PDGFRα

c-KIT

c-KIT/T670I

PDGFRα

PDGFRα/T674I

7 8 10 13 18 20 31 45 50 57 63

35 >50000 367 2.1 2.4 >50000 2.4 1.4 144 1.7 1.9

15 >50000 1179 40 87 >50000 7.2 27 576 24 22

0.0887 >10 >10 0.2480 1.068 >10 0.0181 3.028 >10 3.025 0.0833

0.0371 >10 >10 0.3176 1.841 >10 0.0392 1.779 >10 1.669 0.2173

0.0104 4.254 0.2045 0.0104 0.0104 0.4652 0.0104 0.0104 0.0133 0.0104 0.0104

0.1041 >10 >10 1.171 4.996 >10 0.1348 3.178 >10 2.054 1.513

mutation (GI50 = 0.0116 μM) in contrast to Tel-c-KIT/D816 V-BaF3 mutation (GI50 > 10 μM) but showed a moderate activity (GI50 = 0.1341 μM) against Tel-c-KIT/D816H-BaF3 mutation. More excitingly (Table 5), 31 improved the antiproliferation activity of Tel-PDGFRα/T674I-BaF3 mutation (GI50 = 0.1348 μM), which was insensitive to 1 (GI50 > 10 μM), while retaining activity against the native PDGFRα (GI50 = 0.0104 μM). Combining the results from all of these different BaF3 cell lines, we concluded that 31 is promising as a powerful dual-targeted c-KIT/PDGFRα kinase inhibitor that is even superior to 1. Next, the inhibitory effects of compound 31 against a variety of intact cancer cell lines were investigated (Table 7). The results showed that the inhibitory effect of 31 against proliferation was more potent than 1 against the GIST-T133 cell line; these cells expressed the characteristics of GISTs that were strongly positive for CD34 and c-KIT but not for desmin, S-100 protein, or α-smooth muscle actin. Most interestingly, 31 exhibited powerful antiproliferative activity against the GIST-88234 cell line with single c-KIT mutation and gained significant improvement of antiproliferation activity compared to 1 (GI50 < 0.003 μM vs 0.020 μM). For the GIST 48B35 cell

line harboring the activating c-KIT mutation but expressing no detectable c-KIT transcript or c-KIT protein, 31 showed the same drug resistance as 1. In addition, 31 completely abolished the inhibitory activity against the BCR-ABL driven CML cell lines, such as K562 (GI50 > 10 μM), KU812 (GI50 > 10 μM), and MEG-01 (GI50 = 9.092 μM) cells in contrast with 1. Moreover, 31 had no antiproliferative activity against normal Chinese hamster ovary CHL and CHO cells, implying a good safety profile. To understand kinase spectrum selective of 31 comprehensively, we then examined its kinome wide selectivity profile with DiscoveRx’s KinomeScan technology.36 The results are shown in Figure 4 and Table S2 (Supporting Information). We were delighted to find that 31 possessed a good selectivity (S score (1) = 0.01) in a panel of 468 kinases and mutants at 1 μM concentration. Besides high binding against c-KIT and PDGFRα kinases, it also exhibited strong binding affinity to PDGFRβ, CSF1R kinases and mutant kinases including c-KIT/V559D/ T670I, c-KIT/L576P, c-KIT/V559D, and FLT3/N841I (percent activity remaining 10 0.8030 >10 0.1482 1.2450 >10 1.4660 0.0107 >10

4.0310 0.0181 0.0105 0.0106 0.0965 0.1365 0.0392 0.3303 0.0364 0.0116 0.1341 >10 0.0111 0.0104 0.1348

CHL CHO GIST-T1 GIST-882 GIST-48B K562 KU812 MEG-01

>10 >10 0.003 0.020 6.986 0.147 0.110 0.045

7.036 >10 10 >10 9.092

tyrosine kinase family. In addition, compound 31 showed high percent of control values (POC > 35%) against all kinds of ABL kinases and mutants (Supporting Information, Table S4), indicating weak binding and consistent with the results that compound 31 showed almost no inhibitory activity against the BCR-ABL driven CML cell lines which had been evaluated in Table 7. Pharmacokinetic and in Vitro Safety Properties. Because of its potency and attractive selectivity profile, compound 31 was evaluated in an in vivo rat pharmacokinetic (PK) model following intravenous (IV), oral, and intraperitoneal (IP) administration. As shown in Table 9, 31 demonstrated a short half-life (T1/2 = 0.69 h), a moderate plasma clearance (CLz = 0.95 L/h/kg), small volume distribution (Vss = 0.97 L/kg), and a good plasma exposure in Sprague−Dawley rats (AUC(0−t) = 2243.82 ng/mL·h) with intravenous (IV) injection at a dose of 2 mg/kg. However, there was no absorption in the oral administration with a dose of 10 mg/kg, which prevented 31 from the oral application in the animal model. And then, pharmacokinetic characterization of 31 with IP injection was tested with two dosages of 25 and 100 mg/kg (Table 10). At the dosage of 100 mg/kg, 31 showed a good half-life (T1/2 = 10.97 h) and a better plasma exposure in Sprague−Dawley rats (AUC(0−t) = 4245.24 ng/mL·h), which was much better

at 1 μM) illustrated that 31 exhibited a good selectivity at 1 μM concentration (Supporting Information, Table S3). Furthermore, considering the fact that KinomeScan is a competitive binding assay and may not fully reflect the inhibitory activity, 31’s inhibitory activities against six kinds of available kinases (CSF1R, PDGFRβ, FLT1, FLT3, FLT4, and RET kinases) with POC < 10 at 1 μM were further tested by using Invitrogen’s Z′-LYTE assay and enzyme-linked immunosorbent assay (ELISA),37 and the results were shown in Table 8. The Z′-LYTE assay demonstrated that 31 inhibited CSF1R kinase with an IC50 of 160 nM. The ELISA showed that 31 inhibited PDGFRβ kinase with an IC50 below 1 nM, FLT1 with an IC50 of 159 nM, FLT3 with an IC50 of 2.0 nM, FLT4 with an IC50 of 72 nM, and RET with an IC50 above 1000 nM (Table 8). The good inhibitory activity against PDGFRβ and FLT3 kinases was understandable because c-KIT, CSF1R, PDGFRα, PDGFRβ, and FLT3 kinases are members of the type III receptor

Figure 3. Antiproliferative effect of 31 against a variety of TEL-c-KIT/PDGFRα -BaF3 isogenic cells. (Note: no comparison for GI50 > 10 μM). 5108



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Figure 4. Kinome wide selectivity profiling of 31. (A) KinomeScan profiling of compound 31 at a concentration of 1 μM against 468 kinases and mutants. (B) Kinases mutants that remained activity less than 1% of control in the presence of 1 μM 31.

that the PK profile obtained from rats cannot be transformed into mice due to the differences of species. Figure 5A showed that no animal lethality or obvious weight loss was observed by the administration of 25 or 100 mg/kg of 31 during 28 consecutive treatment days. Furthermore, 31 showed a high inhibition of tumor growth (TGI = 81.5%) which was slightly weaker than 1 (TGI = 87.3%) at the dosage of 100 mg/kg. When the dosage of 31 was decreased to 25 mg/kg, the treatment also resulted in a modest suppression against tumor progression (TGI = 20.2%) (Figure 5B−D). Immunohistochemistry staining demonstrated that 31 could strongly suppress the cellular proliferation marker (Ki-67 stain) and induce the apoptosis effect (TUNEL stain) in the tumor tissues (Figure 5E). In Vivo Evaluation of 31 in Inoculated Xenograft Mouse Model with BaF3-TEL-cKIT-T670I Cell. Considering 31 with a strong suppression against GIST-T1 progression, we next wished to determine the antiproliferation efficacy in BaF3-TEL-cKIT-T670I cells inoculated xenograft mouse model. We also chose the IP injection administration with the dosage of 25 and 100 mg/kg/day for 11 days of continuous treatment, and the results were shown in Figure 6. The changes

Table 8. Compound 31’s Inhibitory Activities against CSF1R, PDGFRβ, FLT1, FLT3, FLT4, and RET Kinases targets % activity remaining IC50 (nM)

CSF1R

PDGFRβ

0 160

0 1000

3.5 2

than the plasma level at the dosage of 25 mg/kg (AUC(0−t) = 691.08 ng/mL·h). Moreover, the metabolic stability of compound 31 in rat liver microsomes was further tested in vitro and the result showed that its half-life was 46.76 min, indicating 31 was metabolically stable (Supporting Information). What is more, the safety evaluation experiment (Supporting Information) displayed that 31 possessed almost no hERG inhibitory activity (IC50 > 40 μM). In Vivo Evaluation of 31 in Inoculated Xenograft Mouse Model with GIST-T1 Cell. To assess the potential of 31 for further medicinal chemistry development, we investigated its antitumor efficacy using the GIST-T1 cell inoculated xenograft mouse model. Intraperitoneal (IP) injection was selected based upon the PK data (Table 10). It was noteworthy

Table 9. Pharmacokinetic Characterization of 31 with IV Injection in Intact Rats

a

31

AUC(0−t) (ng·h/mL)

AUC(0−∞) (ng·h/mL)

MRT(0−t) (h)

T1/2 (h)

Tmax (h)

Vss (L/kg)

CLz (L/h/kg)

Cmax (ng/mL)

IV (2 mg/kg)a SD (n = 3)

2243.82 773.33

2253.60 786.16

1.13 0.56

0.69 0.32

0.083 0.000

0.97 0.13

0.95 0.28

1889.02 118.37

Formulation: 5% DMSO, 40% PEG400, and 55% (30%-HP-β-CD).

Table 10. Pharmacokinetic Characterization of 31 with IP Injection in Intact Rats 31 IP (25 mg/kg)a SD (n = 3) IP (100 mg/kg)a SD (n = 3) a

AUC(0−t) (ng·h/mL)

AUC(0−∞) (ng·h/mL)

MRT(0−t) (h)

MRT(0−∞) (h)

T1/2 (h)

Tmax (h)

Cmax (ng/mL)

691.08 205.32 4245.24 1288.30

713.00 210.66 5388.11 1621.92

5.67 0.40 7.99 0.89

6.48 0.32 14.80 3.65

5.09 0.68 10.97 1.94

4.00 0.00 4.00 0.00

116.96 23.78 656.01 338.05

Formulation: HKI solution (0.5% methocellulose and 0.4% Tween 80 in ddH2O) 5109



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Figure 5. Antitumor efficacy of compound 31 in GIST-T1 xenograft model. Female nu/nu mice harboring established GIST-T1 tumor xenografts were treated with 31 at 25 and 100 mg/kg/d or vehicle or with 1 at 100 mg/kg/d. Daily IP injection was initiated when GIST-T1 tumors had reached a volume of 100−200 mm3. Each group contained five animals. The data are expressed as the mean ± SEM (A) body weight and (B) tumor size measurements from GIST-T1 xenograft mice after 31 or 1 administration. Initial body weight and tumor size were set as 100%. (C) Comparison of the final tumor volume in each group after 28-day treatment period with 31 or 1; ns, p > 0.05, *p < 0.05, ***p < 0.001. (D) Representative photographs of tumors in each group after 31 or 1 treatment. (E) Immunohistochemical staining assay.

of all mice body weights were in a reasonable range as shown in Figure 6A. As expected, 1 could not suppress the c-KIT T670I tumor growth while compound 31 demonstrated moderate tumor growth inhibition (TGI = 41.9%) at 100 mg/kg/day dosage (Figure 6B,C) without obvious toxicity. In addition,

The Figure 6D showed that 31 affected c-KIT pY719, pY703, and pY823 autophosphorylation sites of c-KIT at the dosage of 100 mg/kg/day. The results of immunohistochemistry staining also indicated the proliferation of tumor cells was greatly inhibited, and apoptosis was well induced (Figure 6E). 5110



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Figure 6. Antitumor efficacy of compound 31 in BaF3-TEL-cKIT-T670I xenograft model. Female nu/nu mice harboring established BaF3-TELcKIT-T670I tumor xenografts were treated with 31 at 25 and 100 mg/kg/d or vehicle or with 1 at 100 mg/kg/d. Daily IP injection was initiated when BaF3-TEL-cKIT-T670I tumors had reached a volume of 100−200 mm3. Each group contained five animals. The data are expressed as the mean ± SEM (A) body weight and (B) tumor size measurements from BaF3-TEL-cKIT-T670I xenograft mice after 31 or 1 administration. Initial body weight and tumor size were set as 100%. (C) Comparison of the final tumor volume in each group after 11-day treatment period with 31 or 1; ns, p > 0.05, *p < 0.05. (D) Western blot analysis with antibodies specific to the indicated proteins from tumor lysates prepared from the BaF3-TELcKIT-T670I tumor xenografts upon the completion of the indicated treatments. (E) Immunohistochemical staining assay.

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CONCLUSIONS In conclusion, the application of lead optimization based on multitargeted RTK inhibitor 7 contributed to the design and synthesis of 58 new compounds (8−65), and the novel 3-substituted-pyrazolo[3,4-b]pyridine scaffold shifted the kinase inhibitory spectrum from VEGFR/PDGFR to c-KIT/PDGFRα. Using kinase inhibitory assays provided an integrated assessment of kinase selectivity and potency of all compounds. Preliminary SARs were obtained, which showed that the 3-methyl-pyrazolo[3,4-b] pyridine core scaffold of new compounds was an important pharmacophore of the c-KIT/ PDGFRα dual inhibitor; the urea linker and terminal N-substituents, e.g., 3-methyl-4-(morpholin-4-yl)phenyl, long linear alkyl, aralkyl, and large fused heteroaryl, substantially improved the potency.

The preferred compound 31, harboring single-digit nanomolar potency against c-KIT (IC50 = 2.4 nM) and PDGFRα (IC50 = 7.2 nM), also exhibited better inhibition activity against four TEL-c-KIT/PDGFRα cell lines compared to the tested compounds. So its further in-depth anti-GISTs investigations in vitro and in vivo were performed. In vitro, broad antiproliferative efficacy of 31 was found across a range of TEL-c-KIT/ PDGFRα -BaF3 isogenic cells, which were almost suppressed except for the TEL-c-KIT/D816 V-BaF3 cells that were also insensitive to 1. In particular, 31 showed more potent inhibitory activities against the c-KIT/T670I-BaF3, c-KIT/V559D/ T670I-BaF3, and PDGFRα/T674I-BaF3 cell lines, which were all 1-resistant. In parallel, the potency of 31 against a variety of intact cancer cell lines was observed and improved almost 10 times or more against GIST-882 cells and completely 5111



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1H), 2.76 (dd, J = 15.6, 6.8 Hz, 1H), 2.54 (dd, J = 15.6, 6.4 Hz, 1H), 2.27 (s, 3H), 1.85 (s, 3H). HRMS (ESI) m/z calcd for C21H21N5O2 [M + H]+ 376.1772, found 376.1773. N-(2-Fluoro-5-methylphenyl)-N′-(4-(3-methyl-6-hydroxy-2Hpyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (10). Light-yellow solid; yield = 11%; mp 131−134 °C. 1H NMR (400 MHz, DMSO-d6): δ 12.89 (s, 1H), 11.56 (s, 1H), 9.38 (s, 1H), 8.60 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.12 (dd, J = 11.2, 8.4 Hz, 1H), 6.82 (m, 1H), 5.85 (s, 1H), 2.28 (s, 3H), 2.09 (s, 3H). HRMS (ESI) m/z calcd for C21H18FN5O2 [M + H]+ 392.1524, found 392.1523. N-(3-Methylphenyl)-N′-(4-(3-methyl-6-hydroxy-2H-pyrazolo[3,4b]pyridin-4-yl)phenyl)urea (11). Yellow solid; yield = 18%; mp 173− 175 °C. 1H NMR (400 MHz, DMSO-d6): δ 12.87 (s, 1H), 11.57 (s, 1H), 8.91 (s, 1H), 8.71 (s, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.31 (s, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 5.87 (s, 1H), 2.29 (s, 3H), 2.10 (s, 3H). HRMS (ESI) m/z calcd for C21H19N5O2 [M + H]+ 374.1618, found 374.1617. N-(2-Fluoro-5-methylphenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4b]pyridin-4-yl)phenyl)urea (12). White solid; yield = 62%; mp 215− 222 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.41 (s, 1H), 8.64 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 8.09−7.94 (m, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.12 (dd, J = 11.3, 8.4 Hz, 1H), 7.03 (d, J = 4.7 Hz, 1H), 6.82 (s, 1H), 2.27 (d, J = 13.8 Hz, 6H). HRMS (EI) m/z calcd for C21H18FN5O (M+) 375.1495, found 375.1499. N-(3-Methylphenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin4-yl)phenyl)urea (13). White solid; yield = 52%; mp 225−229 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.16 (s, 1H), 8.92 (s, 1H), 8.47 (d, J = 4.6 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.03 (d, J = 4.6 Hz, 1H), 6.81 (d, J = 7.2 Hz, 1H), 2.28 (d, J = 13.5 Hz, 6H). HRMS (EI) m/z calcd for C21H19N5O (M+) 357.1590, found 357.1593. N-(3-Bromophenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)urea (14). White solid; yield = 86%; mp 232−235 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.19 (s, 2H), 8.47 (d, J = 4.3 Hz, 1H), 7.89 (s, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 7.9 Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H), 7.26 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.03 (d, J = 4.2 Hz, 1H), 2.25 (s, 3H). HRMS (EI) m/z calcd for C20H16BrN5O (M+) 423.0518, found 423.0523. N-(3-(Trifluoromethyl)phenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4b]pyridine-4-yl)phenyl)urea (15). White solid; yield = 82%; mp 234− 237 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.35 (s, 1H), 9.41 (d, J = 50.8 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 8.06 (s, 1H), 7.65 (dd, J = 16.5, 8.5 Hz, 3H), 7.53 (dd, J = 13.4, 8.3 Hz, 3H), 7.33 (d, J = 7.6 Hz, 1H), 7.03 (d, J = 4.7 Hz, 1H), 2.25 (s, 3H). HRMS (EI) m/z calcd for C21H16F3N5O (M+) 411.1307, found 411.1306. N-(3-Bromophenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)thiourea (16). Light-yellow solid; yield = 60%; mp 93−101 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.36 (s, 1H), 10.17 (s, 1H), 10.06 (s, 1H), 8.49 (d, J = 4.7 Hz, 1H), 7.83 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 6.7 Hz, 1H), 7.32 (s, 2H), 7.05 (d, J = 4.7 Hz, 1H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C20H17BrN5S [M + H]+ 438.0368, found 438.0383. N-(3-(Trifluoromethyl)phenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4b]pyridine-4-yl)phenyl)thiourea (17). Light-yellow solid; yield = 64%; mp 86−95 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.37 (s, 1H), 10.25 (s, 1H), 10.18 (s, 1H), 8.50 (dd, J = 9.1, 4.7 Hz, 1H), 7.97 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.63−7.52 (m, 3H), 7.48 (d, J = 7.8 Hz, 1H), 7.05 (d, J = 4.7 Hz, 1H), 2.24 (s, 3H). HRMS (ESI) m/z calcd for C21H17F3N5S [M + H]+ 428.1151, found 428.1151. N-(3-Methylphenyl)-N′-(4-(3-hydroxymethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (18). White solid; yield = 59%; mp 211− 214 °C. 1H NMR (400 MHz, MeOD): δ 8.52 (d, J = 4.7 Hz, 1H), 8.45 (d, J = 4.9 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.31 (s, 1H), 7.27 (d, J = 8.2 Hz, 1H), 7.19 (dd, J = 16.4, 6.2 Hz, 2H), 7.10 (d, J = 4.9 Hz, 1H), 6.88 (t, J = 9.5 Hz, 3H), 4.70 (d, J = 8.4 Hz, 2H), 2.36 (s, 3H).

abolished the inhibitory activity against BCR-ABL driven CML cell lines compared to 1. Moreover, 31 showed a good selectivity (S score (1) = 0.01 at 1 μM) among 468 kinases and mutants tested with DiscoveRx’s KinomeScan technology and exhibited almost no effects on the growth of two normal cells (CHL, GI50 = 7.036 μM; CHO, GI50 > 10 μM) as well as possessing almost no hERG inhibitory activity (IC50 > 40 μM), indicating a good therapeutic window. 31 also displayed a high metabolic stability in rat liver microsomes with good half-life (T1/2 = 46.76 min). Furthermore, 31 was assessed in a GISTT1 xenograft mouse model and showed significant in vivo antitumor activities, suppressing tumor growth (TGI = 81.5%) and inducing apoptosis. More excitingly, 31 demonstrated 41.9% tumor growth inhibition in the BaF3-TEL-cKIT-T670I isogenic cell inoculated xenograft mouse model (insensitive to 1) and blocked cKIT pY703, pY719, and pY823 autophosphorylation sites at the dosage of 100 mg/kg/day. In general, the favorable in vitro and in vivo potency of 31 makes it possible for serving as a dual-targeted compound against c-KIT and PDGFRα for the treatment of GISTs, especially 1-resistant GISTs, as well as a useful prototype for medicinal chemistry programs to further study the GISTs.

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EXPERIMENTAL SECTION

General Chemistry. Synthetic starting materials, reagents, and solvents were purchased from Energy Chemical, J&K, Adamas-beta, TCI, Shanghai Shaoyuan Co. Ltd., and Alfa Aesar and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on an HSGF 254 (150−200 μm thickness; Yantai Huiyou Co., China), and the peak purity was verified with UV spectroscopy (254 and 365 nm). Melting points were measured in capillary tubes on an SGW X-4 melting point apparatus without correction. Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker AMX-400 NMR (IS as TMS). Chemical shifts were reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). High-resolution mass spectra (LRMS and HRMS) were obtained with electric ionization (EI) and electrospray ionization (ESI) produced by a Waters GCT Premie and Waters LCT. HPLC data analysis of compounds 8−65 were performed on an Agilent 1100 with a quaternary pump and diode array detector (DAD). All compounds were confirmed to have a purity ≥95%. General Procedure for the Preparation of Compounds 8−27. To a solution of 69/71/73/76 (0.5 mmol) in DMSO (5 mL) was added Et3N (50 μL) and corresponding isocyanates or isothiocyanates (0.6 mmol). The reaction mixture was stirred at room temperature for 12 h. Water (15 mL) was added, and the mixture was extracted with EtOAc (3 × 50 mL). The organic layer was separated and dried over Na2SO4. Removal of the solvents produced a residue, which was purified by column chromatography eluted with a mixture of MeOH/DCM to yield corresponding target compounds 8−27. N-(2-Fluoro-5-methylphenyl)-N′-(4-(3-methyl-6-hydroxy-4,5-dihydro-2H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (8). Yellow solid; yield = 21%; mp 163−166 °C. 1H NMR (400 MHz, DMSO-d6): δ 11.81 (s, 1H), 10.29 (s, 1H), 9.04 (s, 1H), 8.45 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.0, 2.0 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.13−7.05 (m, 3H), 6.81−6.76 (m, 1H), 4.49−4.43 (m, 1H), 3.39 (d, J = 5.2 Hz, 1H), 3.17 (d, J = 5.2 Hz, 1H), 2.26 (s, 3H), 1.84 (s, 3H). HRMS (ESI) m/z calcd for C21H20FN5O2 [M + H]+ 394.1679, found 394.1679. N-(3-Methylphenyl)-N′-(4-(3-methyl-6-hydroxy-4,5-dihydro-2Hpyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (9). Yellow solid; yield = 43%; mp 231−233 °C. 1H NMR (400 MHz, DMSO-d6): δ 11.81 (s, 1H), 10.29 (s, 1H), 8.62 (s, 1H), 8.56 (s, 1H), 7.37 (d, J = 8.4 Hz, 2H), 7.29 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 7.2 Hz, 1H), 4.08 (t, J = 6.4 Hz, 5112



Article

HRMS (ESI) m/z calcd for C21H19N5NaO2 [M + Na]+ 396.1434, found 396.1431. N-(3-Ethylphenyl)-N′-(4-(3-hydroxymethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (19). White solid; yield = 34%; mp 209− 213 °C. 1H NMR (400 MHz, MeOD): δ 8.40 (d, J = 4.8 Hz, 1H), 8.33 (d, J = 4.8 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.22 (s, 1H), 7.19−7.08 (m, 2H), 7.05 (d, J = 4.8 Hz, 1H), 6.98 (d, J = 4.8 Hz, 1H), 6.80 (d, J = 7.3 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 4.58 (d, J = 8.0 Hz, 2H), 2.54 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H). HRMS (ESI) m/z calcd for C22H21N5NaO2 [M + Na]+ 410.1588, found 410.1588. N-(3-Methylphenyl)-N′-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (20). White solid; yield = 46%; mp 294− 298 °C. 1H NMR (400 MHz, DMSO-d6): δ 14.71 (s, 1H), 8.88 (s, 1H), 8.68 (d, J = 4.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.33 (s, 1H), 7.28−7.23 (m, 2H), 7.18 (t, J = 8.0 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 2.29 (s, 3H). HRMS (EI) m/z calcd for C21H16F3N5O (M+) 411.1307, found 411.1305. N-(3-Ethylphenyl)-N′-(4-(3-trifluoromethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (21). White solid; yield = 65%; mp 297− 299 °C. 1H NMR (400 MHz, DMSO-d6): δ 14.71 (s, 1H), 8.87 (s, 1H), 8.71−8.67 (m, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.35 (s, 1H), 7.30−7.24 (m, 2H), 7.20 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H), 2.59 (q, J = 7.6 Hz, 1H), 1.19 (t, J = 7.6 Hz, 1H). HRMS (EI) m/z calcd for C22H18F3N5O (M+) 425.1463, found 425.1464. N-(3-Ethylphenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)urea (22). White solid; yield = 48%; mp 165−170 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.07 (s, 1H), 8.86 (s, 1H), 8.47 (d, J = 4.6 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 7.36 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 7.03 (d, J = 4.6 Hz, 1H), 6.84 (d, J = 7.4 Hz, 1H), 2.59 (q, J = 7.5 Hz, 2H), 2.26 (s, 3H), 1.19 (s, 3H). HRMS (EI) m/z calcd for C22H21N5O (M+) 371.1746, found 371.1744. N-(2-Methylphenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin4-yl)phenyl)urea (23). White solid; yield = 45%; mp 222−226 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.54 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 8.20 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H), 7.18 (dd, J = 15.0, 7.5 Hz, 2H), 7.03 (d, J = 4.7 Hz, 1H), 6.97 (dd, J = 10.6, 4.1 Hz, 1H), 2.27 (d, J = 11.0 Hz, 6H). HRMS (EI) m/z calcd for C21H19N5O (M+) 357.1590, found 357.1595. N-(4-Methylphenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin4-yl)phenyl)urea (24). White solid; yield = 25%; mp 234−238 °C, 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 9.30 (s, 1H), 9.05 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 7.03 (d, J = 4.7 Hz, 1H), 2.26 (s, 6H). HRMS (EI) m/z calcd for C21H19N5O (M+) 357.1590, found 357.1595. N-(3-Fluorophenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)urea (25). White solid; yield = 65%; mp 263−266 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.26 (d, J = 11.4 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.60−7.46 (m, 3H), 7.32 (dd, J = 15.2, 8.1 Hz, 1H), 7.22−7.11 (m, 1H), 7.03 (d, J = 4.7 Hz, 1H), 6.80 (td, J = 8.4, 2.0 Hz, 1H), 2.26 (s, 3H). HRMS (EI) m/z calcd for C20H16FN5O (M+) 361.1339, found 361.1344. N-(3-Chlorphenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)urea (26). White solid; yield = 84%; mp 238−245 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.56 (d, J = 7.9 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 7.75 (d, J = 0.9 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 1.0 Hz, 2H), 7.17− 6.89 (m, 2H), 2.25 (s, 3H). HRMS (EI) m/z calcd for C20H16ClN5O (M+) 377.1043, found 377.1042. N-(3-Cyanophenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)urea (27). White solid; yield = 53%; mp 173−177 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.35 (s, 1H), 9.54 (d, J = 37.3 Hz, 2H), 8.47 (d, J = 4.7 Hz, 1H), 8.02 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.58−7.48 (m, 3H), 7.44 (d, J = 7.6 Hz, 1H), 7.04 (d, J = 4.7 Hz, 1H), 2.25 (s, 3H). HRMS (EI) m/z calcd for C21H16N6O (M+) 368.1386, found 368.1395. General Procedure for the Preparation of Compounds 28− 40. Under ice cooling, after a solution of phenyl chloroformate

(0.5 mL, 4 mmol) in THF (0.5 mL) was added dropwise to a solution of the intermediate 78/80 (3.2 mmol) and pyridine (1.2 mL) in DMF (5 mL), the mixture was stirred at room temperature for 3 h. Ethyl acetate (40 mL) was added thereto, and the whole mixture was washed with water (40 mL) twice and brine (40 mL) twice. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The resulting solid was collected by filtration with diethyl ether and dried under reduced pressure without further purification. This product was dissolved in 1,4-dioxane (10 mL) with 73b (0.5 mmol) and 1-methyl pyrrolidine (20 μL). The mixture was stirred at 80 °C for 12 h and then cooled to room temperature and concentrated. The residue was purified by column chromatography eluting with a mixture of MeOH/DCM to afford corresponding target compounds 28−40. N-(3-(4-Methylpiperazin-1-yl)phenyl)-N′-(4-(3-methyl-1Hpyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (28). Yellow solid; yield = 27%; mp 108−109 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 8.91 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.20 (s, 1H), 7.12 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 4.8 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 3.13 (brs, 4H), 2.50 (brs, 4H), 2.25 (d, 6H). HRMS (ESI) m/z calcd for C25H28N7O [M + H]+ 442.2350, found 442.2348. N-(5-Methyl-2-(4-methylpiperazin-1-yl)phenyl)-N′-(4-(3-methyl1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (29). White solid; yield = 19%; mp 247−250 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 9.81 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 8.08 (s, 1H), 7.89 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 4.8 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 2.79 (t, J = 4.0 Hz, 4H), 2.64 (brs, 4H), 2.31 (s, 3H), 2.25 (s, 6H). HRMS (ESI) m/z calcd for C26H30N7O [M + H]+ 456.2506, found 456.2504. N-(3-Methyl-4-(4-methylpiperazin-1-yl)phenyl)-N′-(4-(3-methyl1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (30). White solid; yield = 20%; mp 236- 238 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 8.87 (s, 1H), 8.58 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.27 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 4.8 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 2.79 (t, J = 4.4 Hz, 4H), 2.45 (brs, 4H), 2.24 (t, 9H). HRMS (ESI) m/z calcd for C26H30N7O [M + H]+ 456.2506, found 456.2506. N-(3-Methyl-4-(morpholin-4-yl)phenyl)-N′-(4-(3-methyl-1Hpyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (31). White solid; yield = 18%; mp 253−255 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.32 (s, 1H), 8.85 (s, 1H), 8.57 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.28 (s, 1H), 7.26 (d, J = 8.4 Hz,1H), 7.03 (d, J = 4.8 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 3.73 (t, J = 4.4 Hz, 4H), 2.79 (t, J = 4.4 Hz, 4H), 2.25 (s, 6H). HRMS (ESI) m/z calcd for C25H27N6O2 [M + H]+ 443.2190, found 443.2188. N-(2-Methyl-4-(4-methylpiperazin-1-yl)phenyl)-N′-(4-(3-methyl1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (32). Pink solid; yield = 24%; mp 263−264 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 9.09 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.86 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 8.4 Hz, 3H), 7.02 (d, J = 4.8 Hz, 1H), 6.81 (s, 1H), 6.75 (d, J = 8.8 Hz, 1H), 3.07 (t, J = 4.4 Hz, 4H), 2.45 (t, J = 4.4 Hz, 4H), 2.25 (s, 3H), 2.21 (s, 6H). HRMS (ESI) m/z calcd for C26H30N7O [M + H]+ 456.2506, found 456.2504. N-(2-(4-Methylpiperazin-1-yl)phenyl)-N′-(4-(3-methyl-1Hpyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (33). White solid; yield = 89%; mp 253−253 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 8.91 (s, 1H), 8.68 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 3H), 7.49 (d, J = 8.4 Hz, 2H), 7.20 (s, 1H), 7.12 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 4.8 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.59 (d, J = 8.0 Hz, 1H), 3.13 (brs, 4H), 2.50 (brs, 4H), 2.25 (d, 6H). HRMS (ESI) m/z calcd for C25H28N7O [M + H]+ 442.2350, found 442.2346. N-(5-Methyl-2-(morpholin-4-yl)phenyl)-N′-(4-(3-methyl-1Hpyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (34). Yellow solid; yield = 48%; mp 268−270 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 9.76 (s, 1H), 8.47 (d, J = 4.4 Hz, 1H), 8.19 (s, 1H), 7.94 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 4.4 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 3.86 (t, J = 3.6 Hz, 4H), 2.79 (t, J = 3.6 Hz, 4H), 2.26 (d, 6H). HRMS (ESI) m/z calcd for C25H27N6O2 [M + H]+ 443.2190, found 443.2188. 5113



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N-Butyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (43). Yellow solid; yield = 72%; mp 177−180 °C. 1H NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 8.49 (d, J = 4.8 Hz, 1H), 7.27 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 4.8 Hz, 1H), 6.81 (d, J = 7.6 Hz, 2H), 3.58 (dd, J = 13.2, 6.4 Hz, 2H), 2.38 (s, 3H), 1.81−1.64 (m, 2H), 1.48 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z calcd for C18H22N5O [M + H]+ 324.1824, found 324.1819. N-n-Pentyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (44). Yellow solid; yield = 68%; mp 233−236 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.31 (s, 1H), 8.64 (s, 1H), 8.45 (d, J = 4.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.22 (t, J = 5.6 Hz, 1H), 3.10 (dd, J = 12.4 Hz, 6.4 Hz, 2H), 2.24 (s, 3H), 1.45 (m, 2H), 1.30 (m, 4H), 0.89 (t, J = 6.4 Hz, 3H). HRMS (ESI) m/z calcd for C19H24N5O [M + H]+ 338.1981, found 338.1975. N-n-Hexyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (45). Light-yellow solid; yield = 68%; mp 233−236 °C. 1 H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.63 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.21 (t, J = 5.6 Hz, 1H), 3.10 (dd, J = 12.8, 6.4 Hz, 2H), 2.23 (s, 3H), 1.43 (m, 2H), 1.29 (m, 6H), 0.88 (t, J = 6.4 Hz, 3H). HRMS (ESI) m/z calcd for C20H25N5NaO [M + Na]+ 374.1957, found 374.1951. N-Isobutyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (46). White solid; yield = 69%; mp 262−265 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.64 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.27 (t, J = 6.0 Hz, 1H), 2.95 (t, J = 6.4 Hz, 2H), 2.23 (s, 3H), 1.71 (dt, J = 13.6, 6.8 Hz, 1H), 0.89 (d, J = 6.8 Hz, 6H). HRMS (ESI) m/z calcd for C18H22N5O [M + H]+ 324.1825, found 324.1819. N-tert-Butyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (47). Yellow solid; yield = 56%; mp 244−251 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.47 (s, 1H), 8.44 (d, J = 4.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 4.4 Hz, 1H), 6.08 (s, 1H), 2.23 (s, 3H), 1.31 (s, 9H). HRMS (ESI) m/z calcd for C18H21N5NaO [M + Na]+ 346.1644, found 346.1638. N-Cyclopropyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (48). White solid; yield = 63%; mp 247−250 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.53 (s, 1H), 8.45 (d, J = 4.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.4 Hz, 1H), 6.49 (d, J = 2.0 Hz, 1H), 2.60−2.55 (m, 1H), 2.23 (s, 3H), 0.65 (q, J = 6.8 Hz, 2H), 0.46−0.39 (m, 2H). HRMS (ESI) m/z calcd for C17H18N5O [M + H]+ 308.1511, found 308.1506. N-Cyclobutyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (49). White solid; yield = 67%; mp 240−243 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.56 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 4.15 (dd, J = 16.4, 8.4 Hz, 1H), 2.23 (s, 3H), 2.19 (dd, 2H), 1.86 (dd, J = 19.6, 10.4 Hz, 2H), 1.70−1.55 (m, 2H). HRMS (ESI) m/z calcd for C18H20N5O [M + H]+ 322.1668, found 322.1662. N-Cyclopentyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (50). White solid; yield = 89%; mp 258−262 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.49 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.25 (d, J = 7.2 Hz, 1H), 3.96 (dq, J = 13.2, 6.8 Hz, 1H), 2.23 (s, 3H), 1.85 (dt, J = 12.2, 6.0 Hz, 2H), 1.62 (dd, J = 16.4, 6.0 Hz, 2H), 1.56 (dd, J = 14.2, 7.2 Hz, 2H), 1.39 (dt, J = 12.2, 6.4 Hz, 2H). HRMS (ESI) m/z calcd for C19H22N5O [M + H]+ 336.1824, found 336.1819. N-Cyclohexyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (51). Light-yellow solid; yield = 52%; mp 233−237 °C. 1 H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.53 (s, 1H), 8.44 (d, J = 4.4 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.17 (d, J = 7.6 Hz, 1H), 3.54−3.41 (m, 1H), 2.23 (s, 3H), 1.82 (d, J = 8.8 Hz, 2H), 1.67 (d, J = 12.8 Hz, 2H), 1.55 (d, J = 12.0 Hz, 1H), 1.32 (dd, J = 23.6, 11.6 Hz, 2H), 1.18 (dd, J = 21.8, 12.0 Hz, 3H). HRMS (ESI) m/z calcd for C20H24N5O [M + H]+ 350.1981, found 350.1975.

N-(2-(Morpholin-4-yl)phenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4b]pyridin-4-yl)phenyl)urea (35). White solid; yield = 39%; mp 225− 228 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 9.76 (s, 1H), 8.47 (d, J = 4.4 Hz, 1H), 8.20 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 7.2 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 4.4 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 3.87 (brs, 4H), 2.83 (brs, 4H), 2.25 (s, 3H). HRMS (ESI) m/z calcd for C24H25N6O2 [M + H]+ 429.2034, found 429.2033. N-((3-Diethylamino)phenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (36). Green-brown solid; yield = 26%; mp 125−128 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 8.84 (s, 1H), 8.59 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.03 (t, J = 4.8 Hz,8.4 Hz, 2H), 6.92 (s, 1H), 6.63 (d, J = 8.4 Hz, 1H), 6.31 (d, J = 8.4 Hz, 1H), 3.31 (q, J = 7.2 Hz, 4H), 2.25 (s, 3H), 1.10 (t, J = 7.2 Hz, 6H). HRMS (ESI) m/z calcd for C24H27N6O [M + H]+ 415.2241, found 415.2241. N-((4-Diethylamino)phenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (37). Brown solid; yield = 73%; mp 242− 246 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.32 (s, 1H), 8.76 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 8.33 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.64 (d, J = 8.8 Hz, 2H), 3.27 (q, J = 6.8 Hz, 4H), 2.26 (s, 3H), 1.07 (t, J = 6.8 Hz, 6H). HRMS (ESI) m/z calcd for C24H27N6O [M + H]+ 415.2241, found 415.2238. N-((3-Dimethylamino)phenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4b]pyridin-4-yl)phenyl)urea (38). Light-yellow solid; yield = 19%; mp 226−228 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 8.84 (s, 1H), 8.61 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.08 (t, J = 8.0 Hz, 1H), 7.03 (d, J = 4.8 Hz, 1H), 6.95 (s, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.38 (d, J = 8.0 Hz, 1H), 2.89 (s, 7H), 2.25 (s, 3H). HRMS (ESI) m/z calcd for C22H23N6O [M + H]+ 387.1928, found 387.1925. N-((4-Dimethylamino)phenyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4b]pyridin-4-yl)phenyl)urea (39). Brown solid; yield = 10%; mp 218− 221 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 8.78 (s, 1H), 8.46 (d, J = 4.4 Hz, 1H), 8.41 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 4.4 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 2.82 (d, 6H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C22H23N6O [M + H]+ 387.1928, found 387.1928. N-((3-((Dimethylamino)methyl)phenyl)-N′-(4-(3-methyl-1Hpyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (40). Yellow solid; yield = 23%; mp 185−186 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.39 (s, 1H), 9.64 (s, 1H), 9.25 (s, 1H), 8.52 (d, J = 2.8 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.2 Hz, 2H), 7.55 (d, J = 7.2 Hz, 2H), 7.31 (t, J = 6.4 Hz, 1H), 7.23 (d, J = 6.4 Hz, 1H), 7.08 (d, J = 2.8 Hz, 1H), 7.04 (t, J = 6.4 Hz, 1H), 3.50 (s, 2H), 2.30 (s, 3H), 2.24 (s, 6H). HRMS (ESI) m/z calcd for C23H25N6O [M + H]+ 401.2084, found 401.2082. General Procedure for the Preparation of Compounds 41− 65. A solution of corresponding substituted amine (37.4 mmol) in DMSO was added: Et3N (5.73 mL, 41.1 mmol), CDI (3.64 g, 22.4 mmol), and 73b. After stirring overnight at room temperature, the mixture was partitioned between EtOAc and water. Upon separation of the layers, the organic layer was washed with brine and then dried over Na2SO4. The desiccant was filtered and the solvent removed under vacuum to provide raw products, which were purified by column chromatography eluted with a mixture of MeOH/ DCM to obtain the corresponding target compounds. N-Ethyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (41). Yellow solid; yield = 69%; mp 211−213 °C. 1H NMR (400 MHz, CDCl3): δ 9.17 (s, 1H), 8.49 (d, J = 4.8 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 4.8 Hz, 1H), 6.83 (d, J = 8.0 Hz, 2H), 3.62 (q, 2H), 2.36 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z calcd for C16H18N5O [M + H]+ 296.1511, found 296.1506. N-Propyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (42). Yellow solid; yield = 64%; mp 208−210 °C. 1H NMR (400 MHz, CDCl3): δ 9.15 (t, J = 5.6 Hz, 1H), 8.56 (d, J = 4.8 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 4.8 Hz, 1H), 6.72 (d, J = 8.4 Hz, 2H), 3.42−3.35 (t, 2H), 2.30 (s, 3H), 1.68−1.55 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H). HRMS (ESI) m/z calcd for C17H20N5O [M + H]+ 310.1668, found 310.1662. 5114



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N-(Pyridin-2-yl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (61). Yellow solid; yield = 30%; mp 244−248 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.32 (s, 1H), 10.74 (s, 1H), 9.53 (s, 1H), 8.46 (d, J = 4.7 Hz, 1H), 8.30 (d, J = 4.9 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.51 (t, J = 8.0 Hz, 3H), 7.02 (dd, J = 8.1, 4.0 Hz, 2H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C19H16N6NaO [M + Na]+ 367.1283, found 367.1278. N-(Pyridin-3-yl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (62). White solid; yield = 52%; mp 245−247 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 9.06 (s, 1H), 8.64 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 8.21 (d, J = 4.0 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.34 (dd, J = 8.4, 4.8 Hz, 1H), 7.03 (d, J = 4.4 Hz, 1H), 6.66 (s, 1H), 2.25 (s, 3H). HRMS (ESI) m/z calcd for C19H16N6NaO [M + Na]+ 367.1283, found 367.1278. N-(Quinolin-3-yl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)urea (63). Yellow solid; yield = 30%; mp 226−229 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 9.23 (s, 1H), 9.15 (s, 1H), 8.87 (d, J = 2.4 Hz, 1H), 8.56 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 4.7 Hz, 1H), 7.93 (dd, J = 13.8, 8.3 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.64−7.55 (m, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 4.8 Hz, 1H), 2.26 (s, 3H). HRMS (ESI) m/z calcd for C23H19N6O [M + H]+ 395.1620, found 395.1615. N-(Naphthalen-2-yl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin4-yl)phenyl)urea (64). White solid; yield = 34%; mp 232−235 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.32 (s, 1H), 8.99 (d, J = 5.6 Hz, 2H), 8.47 (d, J = 4.8 Hz, 1H), 8.14 (s, 1H), 7.89−7.78 (m, 3H), 7.68 (d, J = 8.8 Hz, 2H), 7.55−7.49 (m, 3H), 7.49−7.43 (m, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 4.8 Hz, 1H), 2.25 (s, 3H). HRMS (ESI) m/z calcd for C24H20N5O [M + H]+ 394.1668, found 394.1662. N-(Naphthalen-1-yl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin4-yl)phenyl)urea (65). Yellow solid; yield = 69%; mp 225−228 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.34 (s, 1H), 8.87 (s, 1H), 8.48 (d, J = 4.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.75−7.47 (m, 9H), 7.04 (d, J = 4.4 Hz, 1H), 2.26 (s, 3H). HRMS (ESI) m/z calcd for C24H20N5O [M + H]+ 394.1668, found 394.1662. Kinase Enzyme Assays. Enzyme inhibition assays were carried by mobility shift assay under the condition of ATP at km concentration. c-KIT, PDGFRα−β, VEGFR1−3, FLT3, and FGFR-1 kinase activities were tested in vitro, with staurosporine and 1 serving as a reference and positive control compound, respectively. An excess (2.5×) of corresponding enzyme was added into the plate except to control wells (no enzyme), where the buffer (1×) (50 mM HEPES pH 7.5, 0.01% Brij-35, 10 mM MgCl2, 2 mM DTT) was added instead. FAM-labeled peptide and ATP were simultaneously added to the 1× kinase base buffer to prepare 2.5× peptide solution. The initial concentration of each compound was prepared at 50 μM in 100% DMSO solution and then diluted 5-fold to eight different concentrations; a 5 μL volume was added for dose response in 384-well reaction plates. A 10 μL volume of the above diluted enzyme solution was sequentially added, and the assay plates were incubated at room temperature for 10 min. A 10 μL volume of the above 2.5× peptide solution was added and incubated at 28 °C for the specified period of time. The reaction was stopped with the addition of 50 mM EDTA containing 25 mL of 100 mM HEPES, pH 7.5, 0.015% Brij-35, and 0.2% Coating Reagent no. 3. After detection by a single span, the conversion values were read with a caliper and converted into inhibition values according to the following formula: percent inhibition = (max-conversion)/(max − min) × 100 (“max” stands for DMSO control; “min” stands for low control with no kinase control). Finally, the data were fitted in XLfit excel add-in version 4.3.1 to obtain IC50 values. The equation used is Y = Bottom + (Top − Bottom)/(1 + (LogIC50/X) × HillSlope)). Cellular Antiproliferation Assays. TEL-Isogenic Cell Generation. On the basis of the pMSCVpuro (Clontech) backbone, retroviral constructs for BaF3-c-KIT and BaF3-PDGFRα mutants were prepared according to published procedures. For the TEL-c-KIT/ PDGFRα vector, the first 1 kb of human TEL gene with an artificial myristoylation site sequence (MGCGCSSHPEDD) was cloned into the pMSCVpuro retroviral vector, followed by a 3xFLAG tag sequence

N-(Furan-2-ylmethyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin4-yl)phenyl)urea (52). White solid; yield = 70%; mp 232−234 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.31 (s, 1H), 8.77 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.60 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.65 (t, J = 5.6 Hz, 1H), 6.41 (s, 1H), 6.28 (s, 1H), 4.32 (d, J = 5.6 Hz, 2H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C19H18N5O2 [M + H]+ 348.1460, found 348.1455. N-(Thiophen-2-ylmethyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (53). White solid; yield = 67%; mp 233− 235 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.31 (s, 1H), 8.81 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 4.8 Hz, 1H), 7.04−6.94 (m, 3H), 6.77 (t, J = 5.6 Hz, 1H), 4.49 (d, J = 5.6 Hz, 2H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C19H17N5NaOS [M + Na]+ 386.1052, found 386.1046. N-(Pyridin-2-ylmethyl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (54). Light-yellow solid; yield = 68%; mp 218−222 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.31 (s, 1H), 8.97 (s, 1H), 8.52 (d, J = 4.4 Hz, 2H), 8.45 (d, J = 4.4 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 4.8 Hz, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.85 (t, J = 6.0 Hz, 1H), 4.36 (d, J = 6.0 Hz, 2H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C20H19N6O [M + H]+ 359.1620, found 359.1615. N-Benzyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (55). Light-yellow solid; yield = 50%; mp 91−97 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.56 (t, J = 6.0 Hz, 1H), 8.55 (d, J = 4.8 Hz, 1H), 7.37 (m,4H), 7.27 (t, J = 7.8 Hz, 3H), 7.23 (d, J = 5.2 Hz, 1H), 6.71 (d, J = 8.4 Hz, 2H), 4.63 (d, J = 6.0 Hz, 2H), 2.32 (s, 3H). HRMS (ESI) m/z calcd for C21H20N5O [M + H]+ 358.1668, found 358.1662. N-Phenylethyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (56). White solid; yield = 82%; mp 221−224 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.31 (s, 1H), 8.73 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 7.6 Hz, 2H), 7.24 (dd, J = 18.6, 7.2 Hz, 3H), 7.00 (d, J = 4.8 Hz, 1H), 6.22 (t, J = 5.6 Hz, 1H), 3.37 (t, 2H), 2.78 (t, J = 7.2 Hz, 2H), 2.23 (s, 3H). HRMS (ESI) m/z calcd for C22H22N5O [M + H]+ 372.1824, found 372.1819. N-Phenylpropyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (57). Yellow solid; yield = 70%; mp 223−225 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.31 (s, 1H), 8.68 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.23 (d, J = 7.2 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 7.00 (d, J = 4.8 Hz, 1H), 6.31 (t, J = 5.6 Hz, 1H), 3.13 (dd, J = 12.8, 6.4 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.24 (s, 3H), 1.83−1.67 (m, 2H). HRMS (ESI) m/z calcd for C23H23N5NaO [M + Na]+ 408.1800, found 408.1795. N-Phenylbutyl-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenyl)urea (58). White solid; yield = 92%; mp 224−227 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.63 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.28 (t, J = 7.2 Hz, 2H), 7.24−7.13 (m, 3H), 6.99 (d, J = 4.4 Hz, 1H), 6.24 (t, J = 5.6 Hz, 1H), 3.13 (dd, J = 12.6, 6.4 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.60 (dd, J = 14.8, 7.2 Hz, 2H), 1.47 (dd, J = 14.5, 7.0 Hz, 2H). HRMS (ESI) m/z calcd for C24H25N5NaO [M + Na]+ 422.1957, found 422.1951. N-(2,3-Dihydro-1H-inden-2-yl)-N′-(4-(3-methyl-1H-pyrazolo[3,4b]pyridin-4-yl)phenyl)urea (59). White solid; yield = 20%; mp 249− 251 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.30 (s, 1H), 8.55 (s, 1H), 8.44 (d, J = 4.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.30−7.22 (m, 2H), 7.21−7.14 (m, 2H), 7.00 (d, J = 4.8 Hz, 1H), 6.55 (d, J = 7.2 Hz, 1H), 4.45 (m, 1H), 3.23 (d, J = 7.2 Hz, 1H), 3.19 (d, J = 6.8 Hz, 1H), 2.82 (d, J = 5.2 Hz, 1H), 2.78 (d, J = 4.8 Hz, 1H), 2.22 (s, 3H). HRMS (ESI) m/z calcd for C23H22N5O [M + H]+ 384.1824, found 384.1819. N-(1H-Pyrazol-5-yl)-N′-(4-(3-methyl-1H-pyrazolo[3,4-b]pyridin-4yl)phenyl)urea (60). White solid; yield = 30%; mp 260−263 °C. 1H NMR (400 MHz, DMSO-d6): δ 13.33 (s, 1H), 12.28 (s, 1H), 9.26 (s, 1H), 9.06 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 3H), 7.49 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 4.8 Hz, 1H), 6.29 (s, 1H), 2.24 (s, 3H). HRMS (ESI) m/z calcd for C17H15N7NaO [M + Na]+ 356.1236, found 356.1230. 5115



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and tumor growth were measured daily after treatment with 31, and the growth tumor curves were determined by measuring the tumor volume using the following equation: V(mm3) = (L × W2)/2, where length (L) is defined as the larger of the two measurements, and width (W) is defined as the smaller of the two measurements. HE Staining. HE staining was performed as previously reported.38 The tissue sections on a slide were hydrated and dipped into a Coplin jar containing Mayer’s hematoxylin and agitated for 30 s. The slide was stained with 1% eosin Y solution for 10−30 s with agitation after rinsing it in H2O for 1 min. The sections were dehydrated with two changes of 95% alcohol and two changes of 100% alcohol for 30 s each, followed by removal of alcohol with two changes of xylene. Eventually, one or two drops of mounting medium were added, and the slides were covered with coverslips. Ki-67 Staining. For IHC demonstration of Ki-67, the 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, the slides were incubated in mouse mAb anti-Ki-67 (ZSGBBIO, China) solution at a 1:50 dilution overnight at 4 °C, followed by the secondary detection system to visualize antibody binding. Staining was developed with 3,3′-diaminobenzidine (DAB), and slides were counterstained with hematoxylin, dehydrated, and mounted. TUNEL Staining. TUNEL staining was carried out using the POD In Situ Cell Death Detection Kit (Roche, USA). Briefly, the sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Subsequently, the sections were treated by nuclease-free Proteinase K at room temperature for 15 min before the endogenous peroxidase was blocked with 3% H2O2 in methanol. Terminal deoxynucleotidyl transferase in a reaction buffer was applied to sections at 37 °C for 1 h. Following washes, the slides were covered with converter−POD solution for 30 min at 37 °C. Finally, apoptotic cells were tested after incubation in 3,3′-diaminobenzidine (DAB) chromogen (Beyotime Biotechnology, China) for nearly 8 min, and slides were counterstained with hematoxylin. BaF3-TEL-cKIT-T670I Xenograft Model. The in vivo anti-1resistant GIST activity of compound 31 was tested in BaF3-TELcKIT-T670I xenograft model. The experimental conditions and methods were similar as the GIST-T1 xenograft model. Differently, to obtain orthotopic xenograft of human mammary tumor in the mice, one million BaF3-TEL-cKIT-T670I cells which were harvested during exponential growth in PBS were suspended in a 1:1 mixture with Matrigel (BD Biosciences).

and 3976a stop codon. Thereafter, the kinase domain coding sequence of c-KIT/PDGFRα was inserted in-frame between TEL and 3xFLAG sequences. For full-length expression vectors, the coding sequences of c-KIT/PDGFRα 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 c-KIT/PDGFRαcontaining MSCV vectors together with two helper plasmids. Virus supernatants were harvested 48 h after transfection and filtered before infection. BaF3 cells were then infected with harvested virus supernatants using an inoculation protocol, and stable cell lines were obtained after 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. Cell Lines and Cell Culture. All of the cells were obtained and maintained as described. All the isogenic BaF3 cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/ streptomycin. 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, Boston (USA). K562 (CML), KU812 (CML), MEG-01 (CML), CHL (hamster lung cell), and CHO (hamster ovary cell) were obtained from American Type Culture Collection (Manassas, VA). GIST-T1 and CHO cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/ streptomycin. GIST-882 and GIST-48B cells were grown in IMDM supplemented with 10% FBS and 1% penicillin/streptomycin. General Procedure for Antiproliferation Study. A series of concentrations of compound 31 were added into the 96-well culture plates (3000/well) seeded with cells and treated for 72 h to observe the cell proliferation. According to the manufacturer’s instructions, cell viability was detected using the CellTiter-Glo assay (Promega, USA), and luminescence was measured using a multilabel reader (Envision, PerkinElmer, USA). The data were normalized to control groups (DMSO) and represented as the mean of three independent measurements with standard error of

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