Discovery and structure-activity relationship of novel diphenylthiazole derivatives as BTK inhibitor with potent activity against B cell lymphoma cell lines

European Journal of Medicinal Chemistry 178 (2019) 767e781

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Discovery and structure-activity relationship of novel diphenylthiazole derivatives as BTK inhibitor with potent activity against B cell lymphoma cell lines Xiaofeng Guo a, Dongyan Yang a, **, Zhijin Fan a, *, Nailou Zhang a, Bin Zhao a, Chun Huang b, Fangjie Wang b, Rongji Ma b, Meng Meng b, ***, Youcai Deng b, **** a b

State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin, 300071, PR China Institute of Materia Medica, College of Pharmacy, Army Medical University (Third Military Medical University), Chongqing, 400038, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2018 Received in revised form 9 June 2019 Accepted 10 June 2019 Available online 15 June 2019

By the analysis of different binding modes with Bruton's tyrosine kinase (BTK), series of novel diphenylthiazole derivatives were rationally designed, synthesized and characterized. Biologically evaluation in biochemistry and cellular assay indicated that, compounds 5m, 5o, 6b, 6c, 6g, 6i, 7h, 7i, 7k, 7m, 7n, 7o and 7s exhibited improved potency against Ramos cell (IC50 ¼ 1.36e8.60 mM) and Raji cell (IC50 ¼ 1.20 e14.04 mM) as compared with ibrutinib (IC50 ¼ 14.69 and 15.99 mM, respectively). Especially, compounds 7m and 7n showed 10-time improved potency against Ramos cell viability over ibrutinib. Compound 6b improved 13-fold activity against Raji cell viability than ibrutinib. In addition, active compound 7o potently inhibited C481S mutant BTK with IC50 value of 0.061 mM. Apoptosis analysis of both Ramos and Raji cells indicated that 7o was remarkably more potent than CGI-1746 and ibrutinib. Compound 7o potently inhibited BTK Y223 phosphorylation in Raji cells, and arrested cell cycle progression in the G0/ G1 phase in Raji and Ramos cells. This study expanded the structural diversity of BTK inhibitors and compound 7o was discovered as an active lead inhibitor with great potential for further studies. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: BTK inhibitor Diphenylthiazole B cell lymphoma Docking mode Activity

1. Introduction BTK (Bruton's tyrosine kinase) gene was originally identified in 1993 by several groups, which was involved in XLA (X-link gglobulinemia) [1,2]. It was named for Ogden Bruton, who firstly diagnosed XLA in an 8-year-old boy with complete absence of gglobulin in 1952 [3]. The BTK protein was later categorized as the non-receptor tyrosine kinase TEC family (i.e., Itk, Rlk, Tec and Bmx) as significant structural and sequence homology [1]. BTK is expressed in hematopoietic cells, including myeloid cells, mast cells and B cells, but not in T cells and plasma cells [4]. It is an important

* Corresponding author. State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, No. 94, Weijin Road, Nankai District, Tianjin, 300071, PR China. ** Corresponding author. 30# Gaotanyan Rd, Shapingba District, Chongqing, 400038, PR China. *** Corresponding author. **** Corresponding author. E-mail addresses: [email protected] (D. Yang), [email protected] (Z. Fan), [email protected] (M. Meng), [email protected] (Y. Deng). https://doi.org/10.1016/j.ejmech.2019.06.035 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

signaling molecule in B cell receptor (BCR) or Fcg receptor (FcgR) signaling pathway, which plays a key role in cell development and survival [5]. Given its crucial role in B-cell development and function, BTK becomes a potential drug target of B cell malignancies and autoimmune disorders, including mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL) and Waldenstrom's macroglobulinemia (WM), which especially is validated by the success of potent BTK inhibitor ibrutinib [6]. Many BTK inhibitors have been advanced to preclinical or clinical studies, which is mainly divided into two different categories, covalent and noncovalent inhibitors. Covalent BTK inhibitors share a similar electrophilic acrylamide moiety, which react irreversibly with Cys481 of BTK, such as ibrutinib, acalabrutinib, spebrutinib and HM-71224 (Fig. 1) [6e10]. Ibrutinib is the first-in-class BTK inhibitor with excellent potency and efficacy and approved by FDA for treatment of MCL, CLL and WM [11]. However, acquired resistance to ibrutinib was recently reported mainly due to the mutation of Cys481 or Thr474 in BTK kinase domain [12e14]. The mutation of Cys481 precluded reaction between BTK with ibrutinib or other covalent inhibitor, which made them losing potency. Another class

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Fig. 1. Structures of the representative BTK inhibitors.

of BTK inhibitors is noncovalent, including CGI-1746, GDC-0834, GDC-0853 and RN-486 [15e18] (Fig. 1), which displays excellent potency and selectivity by occupying a specific H3 pocket. By structural analysis of BTK in complex with noncovalent inhibitors and covalent inhibitors, we superimposed two different binding modes of ibrutinib (modeled) and CGI-1746 with BTK (PDB: 4YHF and 3OCS) [16,19], As shown in Fig. 2, the acrylamide group of ibrutinib was essential to react with Cys481 of BTK and 2aminopyrimidine core formed contact with the residues Thr474 and Glu475 in hinge region of BTK. As for CGI-1746, the pyrazinone

core contacted with Met477 in hinge region, while t-butylphenyl ring occupied the H3 pocket. To discover novel BTK inhibitors with higher efficacy and activity not relying on Cys481, the ring-opening strategy was used in pyrrolo[2,3-d]pyrimidine core of ibrutinib by intramolecular hydrogen bond and then thiazole core was introduced by scaffold hopping strategy, at the same time, phenyl-linked amide moiety of CGI-1746 was introduced at thiazole ring to occupy the H3 pocket, novel diphenylthiazole derivatives were designed as target compounds.

Fig. 2. Diphenylthiazole derivatives rationally designed.

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2. Results and discussion 2.1. Chemistry The synthetic procedures for the target compounds were shown in Scheme 1. 4-Hydroxythiazole 1 was obtained by N-Boc protection, thionation and condensation of commercially available 3aminobenzamide. Compound 1 was transformed to the triflate 2 in good yield and then underwent a subsequent Buchwald-Hartwig amination [20e23] to give compounds 3a-3d. Compounds 3a-3d were deprotected under acidic condition to afford amines 4a-4d and then reacted with corresponding acyl chlorides or acids to give amides 5a-5t. Hydrolysis of ester group at the 5-position of the thiazole afforded corresponding acids 6a-6i or crude acids and then were coupled to amides 7a-7u. The molecular structure of the target compound 5e was further determined by single crystal X-ray analysis (CCDC: 1877408) for structure identification as shown in Fig. 3.

2.2. Biological evaluation The newly synthesized target compounds were evaluated for BTK enzyme inhibitory activity by using promega ADP-Glo™ Kinase Assay [24,25]. Their antiproliferative activities against B lymphoma cell lines, Ramos and Raji, were tested according to the Cell Counting Kit-8 (CCK-8) protocol [26]. Two potent BTK inhibitors, ibrutinib and CGI-1746, were selected as positive reference controls. The results were shown in Table 1 and Table 2. We initially explored the effect of substituted anilines at 4position of thiazole and substituents at 5-position of thiazole. These synthesized compounds were evaluated in biochemistry assay and the results were shown in Table 1, as for para-substituents of anilines at 4-position of thiazole, compound 7h (IC50 ¼ 0.206 mM) with N-methyl-piperazinyl at this position, showed improved potency than compound 7e (4morpholinylcarbonyl with IC50 ¼ 0.617 mM) and 7a (morpholinyl with IC50 ¼ 6.754 mM). However, when changing the substituents at 2-position of thiazole, a different trend was observed. BTK inhibitory activity of derivatives with different substituents at 5-position of thiazole decreased as: primary carboxamide (7c) > carboxylic

Fig. 3. X-ray crystal structure of compound 5e (CCDC: 1877408).

acid (6c) > substituted carboxamides (7t and 7u) > ester (5c). In most cases, compounds with primary carboxamide at 5-position of thiazole showed improved potency. In cell-based assay, most of the compounds displayed good inhibitory activity against proliferation of Ramos and Raji cells. Among them, compounds 6b, 6c, 7h, 7i and 7j exhibited remarkably stronger inhibitory activity against Ramos cell (IC50 ¼ 1.42e8.60 mM) than the positive control ibrutinib (IC50 ¼ 14.69 mM). Compound 7i showed 10-time improved potency against Ramos cell over ibrutinib. Compounds 6b, 6c, 7h, 7i and 7t showed stronger inhibitory activity against Raji cell (IC50 ¼ 1.20e8.96 mM) than ibrutinib (IC50 ¼ 15.99 mM), compound 6b improved over 13-time than that of ibrutinib. This indicated that these compounds with excellent potency against B lymphoma cell lines mostly contained the N-methyl-piperazinyl aniline at 4position and the primary carboxamide at 5-position of thiazole, such as compounds 7h, 7i and 7j. Considering the above biochemistry and cellular activity, our attention turned into the modifications of the amide moiety at 30 position with N-methyl-piperazinyl aniline at 4-position of thiazole, and the bioassay results were shown in Table 2. In biochemistry assay, the substituents at the para-position (7i) of the ring gave better activity than those substituted at the meta- or ortho-positions (7l and 7m). Fluoro (7o) and chloro (6h) group at the paraposition of the phenyl group were favorable for BTK inhibitory activity. Replacement of the phenyl group by heterocyclic or cyclohexanyl rings, including furan, thiadiazole, pyridine and cyclohexanyl rings (compounds 5q-5t and 7p-7s), were also tested. Among them, compounds 7q (furan, IC50 ¼ 0.073 mM) and 7s

Scheme 1. General synthetic route of target compounds. Reagents and conditions: (i) Tf2O, DIPEA, CH2Cl2, r.t., 6 h, 75%; (ii) ArNH2, Pd(OAc)2, BINAP, Cs2CO3, dry THF, 66  C, 8 h, 77e93%; (iii) CF3COOH, dry CH2Cl2, r.t., 2 h, 84e99%; (iv) R4COCl, Et3N, CH2Cl2, r.t., 10 h, 62e81%; (v) DIPEA, PyAOP, R2COOH, CH2Cl2, r.t., 10 h, 63e95%; (vi) LiOH, ethanol, THF, H2O, 66  C, 4 h, then 2 mol/L HCl, 50e96%; (vii) DIPEA, PyAOP, NH4Cl or R3NH2, DMF, r.t., 10 h, 42e95%.

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Table 1 Biological activity of target compounds (5a-5j, 6a-6d, 7a-7j, 7t and 7u) in biochemistry and cellular assay.a.

Compd.

R1

R2

R3

Enzymatic activity

Antiproliferative activity

BTK

Ramos

ibrutinib CGI1746 a b c d

morpholinyl morpholinyl morpholinyl morpholinyl 4-morpholinylcarbonyl 4-morpholinylcarbonyl 4-morpholinylcarbonyl N-methyl-piperazinyl N-methyl-piperazinyl N-methyl-piperazinyl morpholinyl morpholinyl morpholinyl morpholinyl morpholinyl morpholinyl morpholinyl 4-morpholinylcarbonyl 4-morpholinylcarbonyl 4-morpholinylcarbonyl N-methyl-piperazinyl N-methyl-piperazinyl N-methyl-piperazinyl morpholinyl morpholinyl

4-CF3 4-F 4-C(CH3)3 4-N(CH3)2 4-CF3 4-F 4-C(CH3)3 4-CF3 4-F 4-C(CH3)3 4-CF3 4-F 4-C(CH3)3 4-N(CH3)2 4-CF3 4-F 4-C(CH3)3 4-CF3 4-F 4-C(CH3)3 4-CF3 4-F 4-C(CH3)3 4-C(CH3)3 4-C(CH3)3

COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOEt COOH COOH COOH COOH CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONHCH3

Raji IC50 (mM)

Inhibitory rate (%)c

IC50 (mM)

21.76 ± 9.35 3.96 ± 2.34 13.96 ± 7.53 12.34 ± 6.26 49.85 ± 8.99 31.13 ± 2.79 82.56 ± 3.09 18.39 ± 7.15 3.53 ± 4.45 21.18 ± 9.92 77.44 ± 0.58 90.52 ± 2.50 94.27 ± 4.86 74.42 ± 6.72 25.21 ± 6.51 3.53 ± 6.43 5.57 ± 8.30 83.83 ± 0.62 91.55 ± 0.49 85.22 ± 0.70 97.79 ± 1.55 95.92 ± 6.69 80.11 ± 0.99 65.84 ± 8.34 27.90 ± 8.31

e e e e e e 25.16 e e e 29.48 3.51 4.37 24.53 e e e 29.79 13.00 28.43 1.58 1.42 8.60 34.09 e

5.44 ± 6.54 25.83 ± 6.35 6.21 ± 5.96 7.96 ± 2.23 6.06 ± 5.75 37.26 ± 5.79 13.25 ± 8.10 33.00 ± 1.98 16.81 ± 6.98 4.22 ± 6.15 18.83 ± 4.94 96.39 ± 2.91 93.81 ± 5.24 24.41 ± 1.81 7.48 ± 7.58 9.92 ± 3.16 4.42 ± 5.99 43.81 ± 2.44 82.51 ± 4.11 33.35 ± 6.00 91.85 ± 0.23 95.31 ± 1.38 12.09 ± 4.42 94.05 ± 5.03 6.39 ± 5.13

e e e e e e e e e e e 1.20 5.74 e e e e 75.31 35.36 e 3.21 2.82 e 8.96 e

81.20 ± 7.88 25.67 ± 4.83

14.69 90.76

76.23 ± 3.96 1.17 ± 2.85

15.99 e

IC50 (mM)

Inhibitory rate (%)

84.03 ± 4.54 29.05 ± 10.87 16.60 ± 4.60 71.74 ± 1.27 17.53 ± 9.41 47.72 ± 5.77 21.90 ± 6.69 22.04 ± 5.20 69.02 ± 6.47 38.18 ± 0.57 47.92 ± 8.59 80.30 ± 2.57 82.47 ± 8.87 54.46 ± 4.75 75.74 ± 6.17 94.80 ± 4.48 85.04 ± 5.13 82.18 ± 5.53 87.28 ± 1.08 88.37 ± 4.60 93.87 ± 1.49 99.78 ± 1.04 76.66 ± 1.43 79.35 ± 8.38 28.24 ± 8.71

2.422 –d e 4.195 e e e e e e 38.109 1.120 5.323 21.045 6.754 0.258 2.229 0.617 0.733 0.333 0.206 0.862 5.254 8.529 62.845

99.76 ± 1.08 95.21 ± 1.20

0.0018 0.0089

Inhibitory rate (%) 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 6a 6b 6c 6d 7a 7b 7c 7e 7f 7g 7h 7i 7j 7t 7u

b

c

Data represent the mean of at least three separate experiments. Inhibitory activity against BTK enzyme at 10 mM, dates are expressed as the mean ± standard deviation. Inhibitory activity against B lymphoma cell lines at 40 mM, dates are expressed as the mean ± standard deviation. Not determined.

(cyclohexanyl, IC50 ¼ 0.105 mM) showed improved BTK inhibitory activity than compound 7k (phenyl, IC50 ¼ 0.159 mM). As for cellbased assay, compounds 5m, 5o, 6g, 6i, 7k, 7m, 7n, 7o and 7s exhibited remarkably improved potency against Ramos cell (IC50 ¼ 1.36e6.30 mM) and Raji cell(IC50 ¼ 2.62e14.04 mM)as compared with ibrutinib (IC50 ¼ 14.69 and 15.99 mM, respectively). Unfortunately, the most active compounds 7o and 7q in biochemistry assay against BTK kinase (IC50 ¼ 0.090 and 0.073 mM, respectively) were poorer than positive control ibrutinib and CGI1746 (IC50 ¼ 0.0018 and 0.0089 mM, respectively). In biochemical assay against the C481S mutant BTK, as showed in Table 3, ibrutinib was 8 fold less potent (IC50 ¼ 0.017 mM) than WT BTK, however, 7o was more potent (IC50 ¼ 0.061 mM) than WT BTK, which indicated 7o was promising BTK inhibitor lead. In addition, many synthesized compounds remarkably showed improved potency against B lymphoma cell lines than ibrutinib. Compounds 5m, 5o, 6b, 6c, 6g, 6i, 7h, 7i, 7k, 7m, 7n, 7o and 7s exhibited improved potency against Ramos cell (IC50 ¼ 1.36e8.60 mM) and Raji cell (IC50 ¼ 1.20e14.04 mM) as compared with ibrutinib (IC50 ¼ 14.69 and 15.99 mM, respectively). In addition, compounds 5l, 7f and 7j with improved activity against Ramos cell and compounds 5r and 7t with stronger activity against Raji cell also were observed.

2.3. Flow cytometry analysis To further study the effects of these inhibitors on B-cell lymphoma cells, the effects of the active compound 7o was determined on apoptosis and the cell cycle in Ramos and Raji cell line using flow cytometry analysis [27,28], with ibrutinib and CGI-1746 as the positive reference compounds. As shown in Fig. 4, compound 7o significantly induced the Ramos and Raji cells apoptosis with the rates of 72.68% and 67.79% at a concentration of 5 mM for Ramos and Raji cells, respectively. As compared to ibrutinib (6.01% and 1.15%) and CGI-1746 (4.63% and 1.15%) at the same concentration, the novel inhibitor 7o displayed remarkably stronger activity to induce apoptosis than the positive control ibrutinib and CGI-1746. The results of cell cycle analysis were shown in Fig. 5 and Fig. S1. After incubation with 0.1 mM, 1 mM and 5 mM of inhibitor 7o for 48 h, the DNA contents of the Ramos cells (Fig. 5) were 40.13%, 55.48% and 83.68% in G0/G1 phase and 51.54%, 33.13% and 14.14% in S phase, respectively. It indicated that the newly synthesized compound 7o blocked Ramos cells at the G0/G1 phase, as ibrutinib and CGI-1746 arrested the cell cycle. The similar results were observed in Raji cells (Fig. S1).

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Table 2 Biological activity of target compounds (5k-5t, 6e-6i and 7h-7s) in biochemistry and cellular assay.a.

Compd.

R3

R

Enzymatic activity

Antiproliferative activity

BTK

Ramos

Raji

Inhibitory rate (%)b

IC50 (mM)

Inhibitory rate (%)

IC50 (mM)

Inhibitory rate (%)c

IC50 (mM)

COOEt COOEt COOEt COOEt COOEt COOEt COOH COOH COOH COOH COOH CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 CONH2 COOEt CONH2

46.35 ± 7.51 47.70 ± 2.24 85.25 ± 4.49 74.71 ± 11.15 73.05 ± 7.77 22.08 ± 2.37 60.97 ± 3.83 43.39 ± 4.49 46.35 ± 8.71 94.31 ± 0.84 42.85 ± 8.27 93.87 ± 1.49 99.78 ± 1.04 76.66 ± 1.43 93.54 ± 1.59 75.19 ± 6.59 94.21 ± 3.73 92.12 ± 3.10 97.56 ± 0.81 28.01 ± 9.69 85.80 ± 3.38

37.114 19.155 2.096 –d 5.169 e e 27.822 21.888 0.134 e 0.206 0.862 5.254 0.159 e 2.671 0.614 0.090 e 0.637

77.13 ± 1.71 85.17 ± 4.15 96.35 ± 1.16 46.85 ± 0.51 97.54 ± 1.25 72.51 ± 8.49 26.27 ± 2.69 82.65 ± 3.24 93.35 ± 2.19 71.64 ± 3.78 98.60 ± 0.53 97.79 ± 1.55 95.92 ± 6.69 80.11 ± 0.99 98.06 ± 2.09 16.20 ± 1.91 95.50 ± 0.37 98.99 ± 2.46 97.49 ± 0.46 19.04 ± 8.86 96.63 ± 0.09

24.81 12.77 2.00 e 4.28 29.24 e 14.02 6.30 23.28 5.60 1.58 1.42 8.60 1.76 e 1.36 1.36 4.83 e 19.75

42.02 ± 6.41 82.83 ± 0.36 89.82 ± 1.39 24.33 ± 9.85 79.12 ± 0.97 20.72 ± 7.90 11.94 ± 5.82 76.29 ± 2.56 86.85 ± 0.28 90.91 ± 0.09 87.93 ± 1.44 91.85 ± 0.23 95.31 ± 1.38 12.09 ± 4.42 91.26 ± 1.10 2.78 ± 2.14 97.33 ± 0.26 92.58 ± 0.39 94.41 ± 0.60 20.83 ± 1.54 84.63 ± 1.08

e 21.24 9.27 e 14.04 e e 35.14 13.86 7.02 8.74 3.21 2.82 e 6.36 e 3.27 5.70 6.23 e 24.46

5r 7q

COOEt CONH2

93.57 ± 4.87 90.33 ± 5.05

0.161 0.073

50.22 ± 1.27 96.73 ± 0.41

e 19.72

87.62 ± 3.04 84.27 ± 0.67

15.08 37.39

5s 7r

COOEt CONH2

6.05 ± 3.61 94.87 ± 0.26

e 0.213

78.83 ± 2.79 97.55 ± 0.35

24.29 4.73

43.45 ± 7.42 96.06 ± 0.49

e 2.62

5t 7s

COOEt CONH2

38.02 ± 4.47 93.57 ± 2.94

e 0.105

19.87 ± 5.55 55.05 ± 4.43

e e

26.14 ± 0.93 22.38 ± 1.12

e e

99.76 ± 1.08 95.21 ± 1.20

0.0018 0.0089

81.20 ± 7.88 25.67 ± 4.83

14.69 90.76

76.23 ± 3.96 1.17 ± 2.85

15.99 e

5k 5l 5m 5n 5o 5p 6e 6f 6g 6h 6i 7h 7i 7j 7k 7l 7m 7n 7o 5q 7p

phenyl 3-F-phenyl 2-F-phenyl 4-Cl-phenyl 4-CN-phenyl 3-Cl, 4-F-phenyl phenyl 3-F-phenyl 2-F-phenyl 4-Cl-phenyl 3-Cl, 4-F-phenyl 4-CF3-phenyl 4-F-phenyl 4-C(CH3)3-phenyl phenyl 3-F-phenyl 2-F-phenyl 4-Cl-phenyl 3-Cl, 4-F-phenyl

ibrutinib CGI1746 a b c d

Data represent the mean of at least three separate experiments. Inhibitory activity against BTK enzyme at 10 mM, dates are expressed as the mean ± standard deviation. Inhibitory activity against B lymphoma cell lines at 40 mM, dates are expressed as the mean ± standard deviation. Not determined.

2.4. Signaling pathway analysis

2.5. Binding model analysis

To evaluate the effects of compound 7o on the BTK mediated signaling pathway, the western blotting study was performed with BTK inhibitor ibrutinib and CGI-1746 as positive control (Fig. 6). After anti-IgM stimulation, compound 7o potently inhibited BTK Tys223 auto-phosphorylation in Raji cells at the low concentrations of 5 mM and in a dose-dependent manner, while less potently in Ramos cells. The inhibition of BTK downstream mediator PLCg2 Tys1217 phosphorylation was observed in the both cell lines. The results exhibited the same trends as ibrutinib.

To understand the binding mode of the newly synthesized diphenylthiazole compounds with the ATP-binding pocket of the BTK kinase, two representative compounds 7g and 7q, were docked into BTK enzyme (PDB code: 3OCS) by Autodock 4.2 [29]. As shown in Fig. 7, the NeH and carbonyl group of carboxamide at 5-position of thiazole formed hydrogen bonds with Thr474, Glu475 and Met477 in hinge region of BTK, this could explain decrease of BTK inhibitory activity by replacement the carboxamide with ester, carboxylic acid or other N-substituted carboxamide. Another two carbonyl groups of compound 7g (Fig. 7a) interacted with Asp539 and Lys430, respectively; While for compound 7o (Fig. 7b), the NeH and carbonyl group of carboxamide formed hydrogen bonds with Asp539 and Gln412, respectively. This indicated that as compared with 7g, compound 7o exhibited stronger interaction with BTK, which was consistent with the results of experimental activity. It should be noted that, 7g and 7o both were far away from the

Table 3 Biochemical potency of 7o against the C481S mutant BTK.a Compd.

7

ibrutinb

CGI-1746

IC50 (mM)

0.061

0.017

0.016

a

Data represent the mean of at least three separate experiments.

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Fig. 4. Effects of ibrutinib, CGI-1746 and 7o on cell apoptosis in Ramos (A) and Raji cells (B). One representative of three separate experiments is shown. C. Dates are expressed as the mean ± standard deviation.

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Fig. 5. Effects of ibrutinib, CGI-1746 and 7o on cell cycle in Ramos cells. One representative of three separate experiments is shown. Dates are expressed as the mean ± standard deviation.

residue Cys481 and exhibited different binding mode as compared with ibrutinib. 3. Conclusion In summary, 49 novel diphenylthiazole derivatives were synthesized and their activities in biochemistry and cellular assay were evaluated. The most active compounds 7o and 7q against BTK kinase (IC50 ¼ 0.090 and 0.073 mM, respectively) were poorer than the positive control ibrutinib and CGI-1746 (IC50 ¼ 0.0018 and 0.0089 mM, respectively). Biochemical assay against the C481S mutant BTK indicated that, ibrutinib was 8 fold less potent (IC50 ¼ 0.017 mM) than WT BTK, however, the activity of 7o against the C481S mutant BTK was more potent (IC50 ¼ 0.061 mM) than that

of WT BTK, the results indicated 7o was active in the biochemical assay against WT and C481S BTK. In addition, as compared with ibrutinib, many compounds synthesized remarkably showed improved potency against B lymphoma cell lines, Ramos and Raji cell lines. Among these, compounds 7m and 7n (IC50 ¼ 1.36 mM) showed 10-time improved potency against Ramos cell over ibrutinib (IC50 ¼ 14.69 mM). As for Raji cell, compound 6b (IC50 ¼ 1.20 mM) improved 13-time activity than ibrutinib (IC50 ¼ 15.99 mM). Compound 7o potently inhibited the two cell lines with IC50 of 4.83 and 6.23 mM, respectively. A docking model study showed that 7o formed strong interaction with BTK kinase. Apoptosis analysis in the Ramos and Raji cell lines also indicated that 7o was remarkably more potent than CGI-1746 and ibrutinib. Compound 7o potently inhibited BTK Y223 phosphorylation in Raji

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27.0 mmol) and DIPEA (7.11 g, 55.0 mmol) in CH2Cl2 (100 mL) at 0  C under a nitrogen atmosphere and the reaction mixture was warmed to room temperature and stirred for 6 h. The reaction was quenched with sat. aqueous NaHCO3 solution (150 mL). The organic phase was separated, washed with sat. brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether (60e90  C fraction):EtOAc (30:1, v/v) as eluent to afford compound 8 (10.0 g, 75%) as white solid; 1H NMR (400 MHz, CDCl3) d 7.99 (s, 1H), 7.59 (d, J ¼ 7.6 Hz, 2H), 7.38 (t, J ¼ 7.9 Hz, 1H), 6.82 (s, 1H), 4.43 (q, J ¼ 7.1 Hz, 2H), 1.55 (s, 9H), 1.43 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 168.7, 159.0, 152.6, 151.1, 139.6, 132.2, 129.9, 122.1, 120.8, 118.6 (d, J ¼ 320.9 Hz), 115.9, 81.2, 62.5, 28.3, 14.1; HRMS (ESI) m/z calcd for C18H21F3N2O7S2 [M þ H]þ: 497.0664; found: 497.0658.

Fig. 6. Effects of ibrutinib, CGI-1746 and 7o on the BTK mediated signaling pathways in Raji (A) and Ramos (B) cells. One representative of three separate experiments was shown.

cells, and arrested cell cycle progression in the G0/G1 phase and induced apoptosis in Raji and Ramos cells. Our studies expanded the structural diversity of lead structure for BTK inhibitors and these results indicated that compound 7o was an active candidate inhibitor with great potential for further studies. 4. Experimental section 4.1. Chemistry All commercial chemicals were used as received from the supplier. Melting points of new compounds were determined in an X-4 melting point apparatus. 1H NMR (400 MHz) and 13C NMR (101 MHz) spectra were obtained on a Bruker Avance 400 MHz spectrometer in CDCl3 and DMSO‑d6 solution. High resolution mass spectra (HRMS) were given by using a 7.0 T FTICR-MS instrument. Crystal structure was recorded on a Bruker SMART 1000CCD diffraction meter. Reactions were monitored by Thin-layer chromatography (TLC), using silica gel F254 plates and UV light (254 nm) visualization. Flash chromatography was carried out on Silica Gel (200e300 mesh). 4.1.1. Synthetic procedure for compound 2 Trifluoromethanesulfonic anhydride (Tf2O, 15.48 g, 55.0 mmol) was added dropwise to a solution of compound 1 (10.0 g,

4.1.2. General procedures for compounds 3a-3d Dry tetrahydrofuran (THF, 60 mL) was added to a round bottom flask containing compound 2 (3.00 g, 6.04 mmol), cesium carbonate (2.76 g, 8.46 mmol), palladium (II) acetate (0.06 g, 0.30 mmol) and BINAP (0.30 g, 0.48 mmol). A solution of aromatic amine (6.04 mmol) in THF (20 mL) was added under a nitrogen atmosphere and heated at 66  C for 8 h. The mixture was filtered through Celite and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether (60e90  C fraction):EtOAc (3:1e1:5, v/v) as eluent to afford compounds 3a-3d (77e93%). Ethyl 2-(3-((tert-butoxycarbonyl)amino)phenyl)-4-((4morpholinophenyl)amino) thiazole-5-carboxylate (3a). Yellow solid; yield, 86%; m.p. ¼ 213e215  C; 1H NMR (400 MHz, CDCl3) d 8.99 (s, 1H, NH), 7.94 (s, 1H, Ph-H), 7.66 (t, J ¼ 11.0 Hz, 1H, Ph-H), 7.53 (dd, J ¼ 15.9, 6.1 Hz, 3H), 7.35 (t, J ¼ 7.9 Hz, 1H), 6.92 (d, J ¼ 9.0 Hz, 2H), 6.77 (s, 1H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.92e3.82 (m, 4H), 3.18e3.06 (m, 4H), 1.53 (s, 9H), 1.38 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.0, 164.7, 160.2, 152.6, 146.9, 139.2, 133.7, 133.6, 129.7, 121.2, 120.3, 116.8, 116.4, 94.7, 80.9, 67.0, 60.7, 50.2, 28.4, 14.6. HRMS (ESI) m/z calcd for C27H34N4O5S [M þ H]þ: 525.2172; found: 525.2169. Ethyl 2-(3-((tert-butoxycarbonyl)amino)phenyl)-4-((4-(4methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxylate (3b). Yellow crystal; yield, 93%; m.p. ¼ 188e190  C; 1H NMR (400 MHz, CDCl3) d 9.00 (s, 1H), 7.95 (s, 1H), 7.68 (d, J ¼ 7.7 Hz, 1H), 7.58 (t, J ¼ 9.2 Hz, 3H), 7.38 (t, J ¼ 7.9 Hz, 1H), 6.97 (d, J ¼ 8.3 Hz, 2H), 6.74 (s, 1H), 4.36 (q, J ¼ 7.0 Hz, 2H), 3.20 (s, 4H), 2.62 (s, 4H), 2.38 (s, 3H), 1.56 (s, 9H), 1.40 (t, J ¼ 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 167.0, 164.7, 160.3, 152.6, 146.9, 139.2, 133.7, 133.3, 129.6, 121.2, 120.3, 117.1, 116.4, 94.5, 80.9, 60.6, 55.2, 49.9, 46.1, 28.4, 14.5; HRMS (ESI) m/z calcd for C28H37N5O4S [M þ H]þ: 538.2488; found:

Fig. 7. (A) Putative binding model of compound 7g within BTK (PDB code: 3OCS). (B) Putative binding model of compound 7o within BTK (PDB code: 3OCS). Hydrogen bonds are indicated by yellow lines to the key amino acid residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

X. Guo et al. / European Journal of Medicinal Chemistry 178 (2019) 767e781

538.2484. Ethyl 2-(3-((tert-butoxycarbonyl)amino)phenyl)-4-((4-(morpholine-4-carbonyl) phenyl)amino)thiazole-5-carboxylate (3c). Yellow crystal; yield, 77%; m.p. ¼ 196e198  C; 1H NMR (400 MHz, CDCl3) d 9.17 (s, 1H), 7.92 (s, 1H), 7.57 (d, J ¼ 8.5 Hz, 2H), 7.49 (dd, J ¼ 12.4, 8.1 Hz, 2H), 7.40 (s, 1H), 7.31 (d, J ¼ 8.5 Hz, 2H), 7.22 (d, J ¼ 7.8 Hz, 1H), 4.22 (q, J ¼ 7.1 Hz, 2H), 3.61 (s, 8H), 1.44 (s, 9H), 1.27 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.5, 170.1, 164.3, 158.7, 152.8, 141.9, 139.5, 133.1, 129.4, 128.7, 127.7, 121.4, 120.8, 117.6, 116.4, 96.9, 80.5, 66.8, 60.9, 28.3, 14.3; HRMS (ESI) m/z calcd for C28H34N4O6S [M þ H]þ: 553.2121; found: 553.2111. Ethyl 2-(3-((tert-butoxycarbonyl)amino)phenyl)-4-((5-(4methylpiperazin-1-yl) pyridin-2-yl)amino)thiazole-5-carboxylate (3d). Yellow crystal; yield, 78%; m.p. ¼ 132e134  C; 1H NMR (400 MHz, CDCl3) d 9.35 (s, 1H), 8.12 (s, 1H), 7.92 (s, 2H), 7.53 (s, 2H), 7.42e7.11 (m, 3H), 4.25 (s, 2H), 3.12 (s, 4H), 2.67 (s, 4H), 2.34 (s, 3H), 1.45 (s, 9H), 1.28 (s, 3H); 13C NMR (101 MHz, CDCl3) d 169.8, 163.7, 157.6, 152.8, 146.9, 142.3, 139.5, 136.8, 133.4, 129.6, 126.9, 121.3, 120.9, 116.4, 112.5, 97.0, 80.8, 60.9, 54.2, 49.0, 45.1, 28.4, 14.5; HRMS (ESI) m/z calcd for C27H36N6O4S [M þ H]þ: 539.2440; found: 539.2441. 4.1.3. General procedures for compounds 4a-4d Trifluoroacetic acid (10 mL) was added to a solution of compounds 4 (6.0 mmol) in dry CH2Cl2 (50 mL) at 0  C and the reaction mixture was warmed to room temperature and stirred for 2 h. The reaction was quenched with sat. aqueous NaHCO3 solution (40 mL) and the mixture was extracted with CH2Cl2 (40 mL  2). The organic layer was combined, washed with sat. brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with CH2Cl2:methanol (20:1, v/v) as eluent to afford compounds 4a-4d (84e99%). Ethyl 2-(3-aminophenyl)-4-((4-morpholinophenyl)amino)thiazole-5-carboxylate (4a). Yellow crystal; yield, 84%; m.p. ¼ 115e117  C; 1H NMR (400 MHz, CDCl3) d 9.03 (s, 1H), 7.62e7.57 (m, 2H), 7.36 (ddd, J ¼ 8.6, 5.0, 1.3 Hz, 2H), 7.23 (t, J ¼ 7.8 Hz, 1H), 6.98e6.92 (m, 2H), 6.79 (ddd, J ¼ 7.9, 2.2, 0.7 Hz, 1H), 4.36 (q, J ¼ 7.1 Hz, 2H), 3.92e3.87 (m, 4H), 3.18e3.11 (m, 4H), 1.40 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.7, 164.7, 160.2, 147.0, 146.8, 134.0, 133.7, 129.9, 120.1, 118.0, 117.1, 116.8, 112.7, 94.4, 67.0, 60.7, 50.2, 14.6; HRMS (ESI) m/z calcd for C22H26N4O3S [M þ H]þ: 425.1647; found: 425.1645. Ethyl 2-(3-aminophenyl)-4-((4-(4-methylpiperazin-1-yl)phenyl) amino)thiazole-5 -carboxylate (4b). Yellow solid; yield, 91%; m.p. ¼ 156e158  C; 1H NMR (400 MHz, CDCl3) d 8.99 (s, 1H), 7.56 (d, J ¼ 7.5 Hz, 2H), 7.36 (d, J ¼ 10.5 Hz, 2H), 7.22 (t, J ¼ 7.4 Hz, 1H), 6.96 (d, J ¼ 7.6 Hz, 2H), 6.79 (d, J ¼ 7.6 Hz, 1H), 4.34 (q, J ¼ 6.4 Hz, 2H), 3.82 (s, 2H), 3.19 (s, 4H), 2.63 (s, 4H), 2.37 (s, 3H), 1.38 (t, J ¼ 6.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.7, 164.7, 160.2, 146.9, 146.8, 134.0, 133.5, 129.9, 120.1, 118.0, 117.2, 117.1, 112.7, 94.3, 60.6, 55.1, 49.9, 46.0, 14.6; HRMS (ESI) m/z calcd for C23H29N5O2S [M þ H]þ: 438.1964; found: 438.1961. Ethyl 2-(3-aminophenyl)-4-((4-(morpholine-4-carbonyl)phenyl) amino)thiazole-5 -carboxylate (4c). Yellow solid; yield, 99%; m.p. ¼ 217e219  C; 1H NMR (400 MHz, CDCl3) d 9.34 (s, 1H), 7.74 (d, J ¼ 8.2 Hz, 2H), 7.45 (d, J ¼ 8.3 Hz, 2H), 7.41e7.33 (m, 2H), 7.30e7.22 (m, 1H), 6.83 (d, J ¼ 7.9 Hz, 1H), 4.38 (q, J ¼ 7.1 Hz, 2H), 3.73 (s, 8H), 1.41 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.9, 170.6, 164.5, 159.0, 147., 142.0, 133.7, 130.0, 128.8, 128.0, 118.2, 117.8, 117.0, 112.6, 96.8, 67.0, 61.0, 14.5; HRMS (ESI) m/z calcd for C23H26N4O4S [M þ H]þ: 453.1597; found: 453.1601. Ethyl 2-(3-aminophenyl)-4-((5-(4-methylpiperazin-1-yl)pyridin2-yl)amino) thiazole-5-carboxylate (4d). Yellow solid; yield, 90%; m.p. ¼ 177e179  C; 1H NMR (400 MHz, DMSO‑d6) d 9.40 (s, 1H), 8.21

775

(d, J ¼ 8.1 Hz, 1H), 8.07 (s, 1H), 7.55 (s, 1H), 7.24 (d, J ¼ 32.5 Hz, 3H), 6.73 (d, J ¼ 56.8 Hz, 1H), 5.56 (s, 1H), 4.31 (s, 2H), 3.39 (s, 4H), 3.26 (s, 4H), 2.76 (d, J ¼ 20.7 Hz, 3H), 1.31 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 171.6, 163.8, 157.7, 145.0, 146.4, 141.9, 136.9, 133.0, 130.4, 127.0, 118.0, 114.4, 112.1, 111.6, 95.7, 61.3, 52.9, 46.8, 43.0, 14.7; HRMS (ESI) m/z calcd for C22H28N6O2S [M þ H]þ: 439.1916; found: 439.1912. 4.1.4. General procedures for compounds 5a-5t Methold A, Et3N (4.4 mmol) and corresponding acyl chlorides (4.0 mmol) were added to a solution of compounds 4a-4d (2.0 mmol) in dry CH2Cl2 (10 mL) cooled to 0  C and the reaction mixture was stirred at room temperature for 3e10 h. The reaction mixture was partitioned between CH2Cl2 (20 mL  2) and sat. aqueous NaHCO3 solution (25 mL). The organic layer was combined, washed with sat. brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether (60e90  C fraction):EtOAc (4:1e1:10, v/v) as eluent to afford compounds 5a-5q (63e95%). Method B, N,N-diisopropylethylamine (DIPEA, 0.52 g, 4.0 mmol) and (3-hydroxy-3H-1,2,3-thiazolo[4,5-b]pyridinato-O)tri-1pyrrolidinylphosphonium hexafluorophosphate (PyAOP, 1.15 g, 2.2 mmol) were added to a solution of corresponding acids (2.0 mmol) in CH2Cl2 (10 mL) at 0  C. The mixture was stirred at 0  C for 15 min and amines 4a-4d (1.80 mmol) was added and stirred at room temperature for 10 h. The reaction mixture was partitioned between CH2Cl2 (20 mL  2) and sat. aqueous NaHCO3 solution (25 mL). The organic layer was combined, washed with sat. brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether (60e90  C fraction):EtOAc (5:1e1:3, v/v) as eluent to afford compounds 5r, 5s and 5t (62e81%). Ethyl 4-((4-morpholinophenyl)amino)-2-(3-(4-(trifluoromethyl) benzamido) phenyl)thiazole-5-carboxylate (5a). Yellow solid; yield, 89%; m.p. > 220  C; 1H NMR (400 MHz, CDCl3) d 8.98 (s, 1H), 8.17 (s, 1H), 8.04 (s, 1H), 8.00 (d, J ¼ 8.0 Hz, 2H), 7.92 (d, J ¼ 8.3 Hz, 1H), 7.77 (d, J ¼ 7.8 Hz, 2H), 7.66 (d, J ¼ 8.1 Hz, 1H), 7.55 (d, J ¼ 8.6 Hz, 2H), 7.47 (t, J ¼ 7.9 Hz, 1H), 6.92 (d, J ¼ 8.5 Hz, 2H), 4.35 (q, J ¼ 7.1 Hz, 2H), 3.90e3.82 (m, 4H), 3.10 (s, 4H), 1.39 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, DMSO‑d6) d 169.9, 165.2, 163.9, 159.9, 147.3, 140.2, 138.9, 133.0, 132.9, 130.3, 129.2, 127.4, 125.9, 123.9, 122.4, 120.7, 118.4, 116.4, 94.4, 66.6, 61.1, 49.5, 14.8; HRMS (ESI) m/z calcd for C30H29F3N4O4S [M þ H]þ: 597.1783; found: 597.1781. Ethyl 2-(3-(4-fluorobenzamido)phenyl)-4-((4-morpholinophenyl) amino)thiazole -5-carboxylate (5b). Yellow crystal; yield, 83%; m.p. > 220  C; 1H NMR (400 MHz, CDCl3) d 8.99 (s, 1H), 8.15 (s, 1H), 7.96e7.85 (m, 4H), 7.77 (d, J ¼ 7.3 Hz, 1H), 7.56 (d, J ¼ 8.7 Hz, 2H), 7.46 (t, J ¼ 7.9 Hz, 1H), 7.19 (t, J ¼ 8.4 Hz, 2H), 6.93 (d, J ¼ 8.8 Hz, 2H), 4.35 (q, J ¼ 7.0 Hz, 2H), 3.91e3.81 (m, 4H), 3.15e3.06 (m, 4H), 1.39 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 169.7, 165.1 (d, J ¼ 253.3 Hz), 164.9, 164.6, 160.2, 146.9, 138.5, 133.8, 133.5, 130.8, 129.8, 129.5 (d, J ¼ 9.1 Hz), 123.0, 122.8, 120.3, 118.1, 116.8, 116.0 (d, J ¼ 21.9 Hz), 100.0, 67.0, 60.7, 50.1, 14.5; HRMS (ESI) m/z calcd for C29H29FN4O4S [M þ H]þ: 547.1815; found: 547.1805. Ethyl 2-(3-(4-(tert-butyl)benzamido)phenyl)-4-((4morpholinophenyl)amino) thiazole-5-carboxylate (5c). Yellow solid; yield, 81%; m.p. >220  C; 1H NMR (400 MHz, CDCl3) d 9.00 (s, 1H), 8.17 (s, 1H), 7.99 (d, J ¼ 4.6 Hz, 1H), 7.91 (d, J ¼ 7.8 Hz, 1H), 7.84 (d, J ¼ 8.2 Hz, 2H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.51 (d, J ¼ 8.3 Hz, 2H), 7.44 (t, J ¼ 7.9 Hz, 1H), 6.93 (d, J ¼ 8.8 Hz, 2H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.90e3.82 (m, 4H), 3.15e3.06 (m, 4H), 1.43e1.33 (m, 12H); 13C NMR (101 MHz, CDCl3) d 169.8, 165.9, 164.7, 160.2, 155.7, 146.9, 138.8, 133.7, 133.6, 131.8, 129.7, 127.0, 125.8, 123.0,

776

X. Guo et al. / European Journal of Medicinal Chemistry 178 (2019) 767e781

122.6, 120.3, 118.0, 116.8, 94.8, 70.0, 60.7, 50.2, 35.0, 31.2, 14.5; HRMS (ESI) m/z calcd for C33H38N4O4S [M þ H]þ: 585.2536; found: 585.2523. Ethyl 2-(3-(4-(dimethylamino)benzamido)phenyl)-4-((4morpholinophenyl) amino)thiazole-5-carboxylate (5d). Yellow crystal; yield, 65%; m.p. > 220  C; 1H NMR (400 MHz, CDCl3) d 9.00 (s, 1H), 8.16 (s, 1H), 7.90 (d, J ¼ 10.0 Hz, 2H), 7.81 (d, J ¼ 8.7 Hz, 2H), 7.73 (d, J ¼ 7.7 Hz, 1H), 7.57 (d, J ¼ 8.8 Hz, 2H), 7.42 (t, J ¼ 7.9 Hz, 1H), 6.94 (d, J ¼ 8.8 Hz, 2H), 6.71 (d, J ¼ 8.7 Hz, 2H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.89e3.84 (m, 4H), 3.14e3.09 (m, 4H), 3.06 (s, 6H), 1.39 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.1, 165.7, 160.2, 157.9, 152.8, 146.8, 139.3, 133.7, 133.7, 129.7, 128.7, 122.9, 122.1, 120.9, 120.2, 117.8, 116.9, 111.2, 67.0, 60.7, 50.2, 40.1, 14.5; HRMS (ESI) m/z calcd for C31H35N5O4S [M þ H]þ: 572.2332; found: 572.2322. Ethyl 4-((4-(morpholine-4-carbonyl)phenyl)amino)-2-(3-(4-(trifluoromethyl) benzamido)phenyl)thiazole-5-carboxylate (5e). Yellow solid; yield, 83%; m.p. ¼ 156e158  C; 1H NMR (400 MHz, CDCl3) d 9.52 (s, 1H), 9.19 (s, 1H), 8.21 (s, 1H), 8.02 (d, J ¼ 7.6 Hz, 2H), 7.89 (d, J ¼ 7.7 Hz, 1H), 7.78e7.49 (m, 5H), 7.38 (dd, J ¼ 16.0, 8.0 Hz, 1H), 7.24 (d, J ¼ 8.5 Hz, 1H), 4.33 (dd, J ¼ 14.1, 7.0 Hz, 2H), 3.64 (s, 8H), 1.39 (t, J ¼ 7.0 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.7, 169.8, 165.3, 164.4, 158.7, 141.9, 138.9, 137.9, 133.3, 129.6, 128.5, 128.1, 127.5, 125.5, 123.8, 118.9, 117.8, 100.0, 66.7, 61.0, 14.5; HRMS (ESI) m/z calcd for C31H29F3N4O5S [M þ H]þ: 625.1733; found: 625.1732. Ethyl 2-(3-(4-fluorobenzamido)phenyl)-4-((4-(morpholine-4carbonyl)phenyl) amino)thiazole-5-carboxylate (5f). Yellow solid; yield, 88%; m.p. > 220  C; 1H NMR (400 MHz, CDCl3) d 9.45 (s, 1H), 9.09 (s, 1H), 8.05 (s, 1H), 7.92e7.79 (m, 2H), 7.70 (s, 1H), 7.52 (d, J ¼ 6.6 Hz, 1H), 7.45 (d, J ¼ 8.1 Hz, 2H), 7.28e7.08 (m, 3H), 6.91 (dd, J ¼ 10.9, 5.2 Hz, 2H), 4.19 (q, J ¼ 6.8 Hz, 2H), 3.53 (s, 8H), 1.26 (t, J ¼ 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 170.7, 169.9, 165.5, 164.8 (d, J ¼ 252.7 Hz), 164.3, 158.6, 142.0, 139.1, 133.0, 130.7 (d, J ¼ 2.9 Hz), 130.1 (d, J ¼ 8.9 Hz), 129.4, 128.5, 127.4, 123.9, 122.3, 119.0, 117.6, 115.4 (d, J ¼ 21.8 Hz), 97.1, 66.8, 61.0, 14.4; HRMS (ESI) m/z calcd for C30H27FN4NaO5S [M þ Na]þ: 597.1584; found: 597.1583. Ethyl 2-(3-(4-(tert-butyl)benzamido)phenyl)-4-((4-(morpholine4-carbonyl) phenyl)amino)thiazole-5-carboxylate (5g). Yellow crystal; yield, 99%; m.p. ¼ 181e183  C; 1H NMR (400 MHz, CDCl3) d 9.25 (s, 1H), 8.78 (s, 1H), 8.25 (s, 1H), 7.88 (d, J ¼ 8.1 Hz, 3H), 7.67 (dd, J ¼ 17.7, 8.1 Hz, 3H), 7.44 (d, J ¼ 8.3 Hz, 2H), 7.37 (dd, J ¼ 15.6, 8.1 Hz, 3H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.66 (s, 8H), 1.41e1.31 (m, 12H); 13C NMR (101 MHz, CDCl3) d 170.64, 170.07, 166.20, 164.47, 158.84, 155.52, 141.97, 139.24, 133.28, 131.69, 130.95, 129.60, 128.67, 127.28, 125.60, 123.47, 122.26, 118.44, 117.80, 99.98, 66.91, 61.01, 35.00, 31.17, 14.46; HRMS (ESI) m/z calcd for C34H38N4O5S [M þ H]þ: 613.2485; found: 613.2486. Ethyl 4-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-(3-(4-(trifluoromethyl) benzamido)phenyl)thiazole-5-carboxylate (5h). Yellow solid; yield, 91%; m.p. ¼ 188e190  C; 1H NMR (400 MHz, DMSO‑d6) d 10.83 (d, J ¼ 12.5 Hz, 2H), 9.00 (s, 1H), 8.54 (d, J ¼ 11.9 Hz, 1H), 8.20 (d, J ¼ 7.9 Hz, 1H), 7.98 (dd, J ¼ 36.8, 8.2 Hz, 2H), 7.78 (s, 1H), 7.59 (d, J ¼ 8.2 Hz, 3H), 7.02 (d, J ¼ 8.9 Hz, 2H), 4.41e4.26 (m, 2H), 3.49 (s, 4H), 3.13 (s, 4H), 2.80 (s, 3H), 1.40e1.27 (m, 3H); 13C NMR (101 MHz, DMSO‑d6) d 169.5, 164.7, 163.3, 159.2, 145.0, 139.8, 138.4, 133.3, 132.5, 129.8, 128.7, 126.9, 125.4, 123.5, 121.9, 120.1, 118.0, 117.0, 94.3, 60.6, 52.1, 46.1, 41.9, 14.3; HRMS (ESI) m/z calcd for C31H32F3N5O [M þ H]þ: 610.2100; found: 610.2092. Ethyl 2-(3-(4-fluorobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxylate (5i). Yellow solid; yield, 73%; m.p. ¼ 155e157  C; 1H NMR (400 MHz, CDCl3) d 9.00 (s, 1H), 8.15 (s, 1H), 7.98e7.92 (m, 3H), 7.90 (s, 1H), 7.80 (d, J ¼ 7.8 Hz, 1H), 7.56 (d, J ¼ 8.9 Hz, 2H), 7.49 (t, J ¼ 7.9 Hz, 1H), 7.22 (t, J ¼ 8.5 Hz, 2H), 6.98 (d, J ¼ 8.9 Hz, 2H), 4.37 (q, J ¼ 7.1 Hz, 2H), 3.23e3.17 (m, 4H), 2.65e2.59 (m, 4H), 2.39 (s, 3H), 1.41 (t, J ¼ 7.1 Hz,

3H); 13C NMR (101 MHz, CDCl3) d 169.6, 166.4, 164.8, 164.6, 160.3, 147.0, 138.5, 133.9, 133.2, 130.8, 129.8, 129.5 (d, J ¼ 9.1 Hz), 123.0, 122.9, 120.4, 118.0, 117.1, 116.1, 115.9, 116.0 (d, J ¼ 22.0 Hz), 60.7, 55.2, 50.0, 46.2, 14.5; HRMS (ESI) m/z calcd for C30H32FN5O3 [M þ H]þ: 560.2132; found: 560.2123. Ethyl 2-(3-(4-(tert-butyl)benzamido)phenyl)-4-((4-(4methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxylate (5j). Yellow solid; yield, 72%; m.p. ¼ 178e180  C; 1H NMR (400 MHz, CDCl3) d 8.98 (s, 1H), 8.14 (s, 1H), 7.94 (d, J ¼ 8.9 Hz, 2H), 7.84 (d, J ¼ 8.4 Hz, 2H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.53 (dd, J ¼ 12.2, 5.3 Hz, 4H), 7.48e7.42 (m, 1H), 6.95 (d, J ¼ 9.0 Hz, 2H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.23e3.13 (m, 4H), 2.64e2.55 (m, 4H), 2.36 (s, 3H), 1.42e1.34 (m, 12H); 13C NMR (101 MHz, CDCl3) d 169.8, 165.9, 164.7, 160.3, 155.7, 146.9, 138.8, 133.8, 133.3, 131.7, 129.8, 127.0, 125.8, 123.0, 122.6, 120.3, 118.0, 117.2, 100.0, 60.7, 55.2, 49.9, 46.2, 35.1, 31.2, 14.5; HRMS (ESI) m/z calcd for C34H41N5O3S [M þ H]þ: 598.2852; found: 598.2837. Ethyl 2-(3-benzamidophenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino) thiazole-5-carboxylate (5k). Yellow solid; yield, 87%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.57 (s, 1H), 9.00 (s, 1H), 8.54 (s, 1H), 8.09e7.93 (m, 3H), 7.75 (d, J ¼ 7.7 Hz, 1H), 7.67e7.50 (m, 6H), 7.02 (d, J ¼ 9.0 Hz, 2H), 4.31 (q, J ¼ 7.1 Hz, 2H), 3.36 (s, 4H), 3.18 (s, 4H), 2.73 (s, 3H), 1.32 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, DMSO‑d6) d 170.1, 166.3, 163.9, 159.6, 145.8, 140.5, 133.4, 132.9, 132.3, 130.2, 129.7, 128.9, 128.2, 122.0, 120.6, 120.5, 118.3, 117.3, 94.6, 61.1, 53.1, 47.0, 43.0, 14.8; HRMS (ESI) m/z calcd for C30H33N5O3S [M þ H]þ: 542.2226; found: 542.2231. Ethyl 2-(3-(3-fluorobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxylate (5l). Yellow solid; yield, 63%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.63 (s, 1H), 9.01 (s, 1H), 8.52 (s, 1H), 8.03 (d, J ¼ 7.9 Hz, 1H), 7.92e7.82 (m, 2H), 7.77 (d, J ¼ 7.7 Hz, 1H), 7.68e7.43 (m, 5H), 7.02 (d, J ¼ 8.7 Hz, 2H), 4.32 (q, J ¼ 7.0 Hz, 2H), 3.34 (s, 4H), 3.25 (s, 4H), 2.76 (s, 3H), 1.32 (t, J ¼ 7.0 Hz, 3H); 13C NMR (101 MHz, DMSO‑d6) d 170.0, 164.9, 163.8, d 162.4 (d, J ¼ 244.3 Hz), 159.7, 145.7, 140.4, 137.3 (d, J ¼ 7.1 Hz), 133.6, 133.0, 131.1 (d, J ¼ 8.2 Hz), 130.3, 124.5 (d, J ¼ 2.6 Hz), 124.0, 122.2, 120.7, 119.2 (d, J ¼ 21.0 Hz), 118.5, 117.3, 115.1 (d, J ¼ 22.7 Hz), 94.7, 61.1, 52.8, 46.7, 42.6, 14.8; HRMS (ESI) m/z calcd for C30H32FN5O3S [M þ H]þ: 560.2132; found: 560.2126. Ethyl 2-(3-(2-fluorobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxylate (5m). Yellow solid; yield, 70%; m.p. ¼ 190e192  C; 1H NMR (400 MHz, DMSO‑d6) d 10.74 (s, 1H), 9.00 (s, 1H), 8.48 (s, 1H), 7.94 (d, J ¼ 8.1 Hz, 1H), 7.78e7.73 (m, 1H), 7.71 (d, J ¼ 7.2 Hz, 1H), 7.57 (d, J ¼ 9.1 Hz, 2H), 7.53 (d, J ¼ 8.0 Hz, 1H), 7.40 (d, J ¼ 11.2 Hz, 1H), 7.37e7.34 (m, 1H), 7.33e7.24 (m, 1H), 7.00 (d, J ¼ 8.9 Hz, 2H), 4.31 (q, J ¼ 7.0 Hz, 2H), 3.34 (s, 4H), 3.12 (d, J ¼ 7.1 Hz, 4H), 2.68 (s, 3H), 1.32 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, DMSO‑d6) d 169.9, 165.3, 163.8, 163.6, 159.8, 159.4 (d, J ¼ 249.0 Hz), 158.1, 146.0, 140.3, 133.4, 133.2 (d, J ¼ 8.0 Hz), 133.1, 131.4 (d, J ¼ 7.8 Hz), 130.4, 128.6 (d, J ¼ 4.0 Hz), 125.3 (d, J ¼ 3.3 Hz), 125.1 (d, J ¼ 3.3 Hz), 123.3, 122.3, 120.6, 117.6, 117.2, 116.7 (d, J ¼ 21.6 Hz), 116.2 (d, J ¼ 21.1 Hz), 94.6, 61.1, 53.2, 47.1, 43.0, 14.8; HRMS (ESI) m/z calcd for C30H32FN5O3S [M þ H]þ: 560.2132; found: 560.2127. Ethyl 2-(3-(4-chlorobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxylate (5n). Yellow solid; yield, 91%; m.p. ¼ 180e182  C; 1H NMR (400 MHz, CDCl3) d 8.96 (s, 1H), 8.44 (s, 1H), 8.16 (s, 1H), 7.94 (d, J ¼ 8.3 Hz, 1H), 7.84 (s, 1H), 7.82 (s, 1H), 7.70 (d, J ¼ 7.8 Hz, 1H), 7.53 (s, 1H), 7.44e7.37 (m, 3H), 7.27 (s, 1H), 6.90 (s, 1H), 6.88 (s, 1H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.19e3.10 (m, 4H), 2.69e2.60 (m, 4H), 2.39 (s, 3H), 1.38 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 169.7, 165.1, 164.6, 160.1, 146.6, 138.6, 138.3, 133.7, 133.4, 133.0, 129.7, 129.0, 128.7, 123.2, 122.8, 120.2, 118.2, 117.3, 60.7, 54.9, 49.5, 45.8, 14.5;

X. Guo et al. / European Journal of Medicinal Chemistry 178 (2019) 767e781

HRMS (ESI) m/z calcd for C30H32ClN5O3S [M þ H]þ: 576.1836; found: 576.1816. Ethyl 2-(3-(4-cyanobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxylate (5o). Yellow solid; yield, 92%; m.p. ¼ 205e207  C; 1H NMR (400 MHz, CDCl3) d 9.06 (s, 1H), 8.95 (s, 1H), 8.24 (s, 1H), 7.97 (t, J ¼ 7.9 Hz, 3H), 7.71 (d, J ¼ 7.9 Hz, 1H), 7.66 (d, J ¼ 8.4 Hz, 2H), 7.50 (d, J ¼ 8.9 Hz, 2H), 7.41 (t, J ¼ 8.0 Hz, 1H), 6.84 (d, J ¼ 9.0 Hz, 2H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.16e3.06 (m, 4H), 2.70 (d, J ¼ 4.3 Hz, 4H), 2.41 (s, 3H), 1.39 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 169.6, 164.6, 164.6, 160.1, 146.3, 138.6, 133.6, 133.5, 132.3, 129.8, 128.1, 123.4, 123.0, 120.1, 118.5, 118.0, 117.3, 115.2, 94.9, 77.4, 77.1, 76.8, 60.8, 54.5, 49.0, 45.2, 14.5; HRMS (ESI) m/z calcd for C31H32N6O3S [M þ H]þ: 567.2178; found: 567.2170. Ethyl 2-(3-(3-chloro-4-fluorobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxylate (5p). Yellow crystal; yield, 82%; m.p. ¼ 210e212  C; 1H NMR (400 MHz, CDCl3) d 8.93 (s, 1H), 8.21 (s, 1H), 8.09 (s, 1H), 7.94 (d, J ¼ 6.0 Hz, 1H), 7.85 (d, J ¼ 7.3 Hz, 1H), 7.74 (s, 1H), 7.69 (d, J ¼ 7.4 Hz, 1H), 7.49 (d, J ¼ 8.3 Hz, 2H), 7.38 (t, J ¼ 7.8 Hz, 1H), 7.20 (t, J ¼ 8.4 Hz, 1H), 6.89 (d, J ¼ 8.3 Hz, 2H), 4.33 (q, J ¼ 7.1 Hz, 2H), 3.11 (s, 4H), 2.56 (s, 4H), 2.35 (s, 3H), 1.38 (t, J ¼ 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 160.3 (d, J ¼ 255.3 Hz), 164.6, 163.8, 160.2, 159.0, 146.9, 138.3, 133.8, 133.1, 131.8 (d, J ¼ 3.4 Hz), 130.2, 129.8, 127.4 (d, J ¼ 7.7 Hz), 123.2, 123.0, 121.9 (d, J ¼ 18.0 Hz), 120.3, 118.3, 117.0, 116.8, 100.0, 60.7, 55.2, 49.8, 46.1, 14.5; HRMS (ESI) m/z calcd for C30H31ClFN5O3S [M þ H]þ: 594.1742; found: 594.1726. Ethyl 4-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-(3-(5-(trifluoromethyl) picolinamido)phenyl)thiazole-5-carboxylate (5q). Yellow crystal; yield, 95%; m.p. ¼ 210e212  C; 1H NMR (400 MHz, CDCl3) d 10.06 (s, 1H), 9.02 (s, 1H), 8.93 (s, 1H), 8.48 (d, J ¼ 8.1 Hz, 1H), 8.35 (s, 1H), 8.21 (d, J ¼ 7.7 Hz, 1H), 8.01 (d, J ¼ 8.1 Hz, 1H), 7.84 (d, J ¼ 7.8 Hz, 1H), 7.59 (d, J ¼ 8.7 Hz, 2H), 7.51 (t, J ¼ 7.9 Hz, 1H), 7.00 (d, J ¼ 8.7 Hz, 2H), 4.37 (q, J ¼ 7.1 Hz, 2H), 3.21 (t, J ¼ 8.0 Hz, 4H), 2.64 (s, 4H), 2.40 (s, 3H), 1.41 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 169.7, 164.7, 160.8, 160.2, 152.4, 146.9, 145.2, 138.0, 135.3, 134.0, 133.3, 129.9, 124.4, 122.9, 122.4, 121.7, 120.2, 117.7, 117.2, 94.8, 60.7, 55.2, 49.9, 46.2, 14.5; HRMS (ESI) m/z calcd for C30H31F3N6O3S [M þ H]þ: 611.2052; found: 611.2051. Ethyl 2-(3-(furan-2-carboxamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxylate (5r). Yellow solid; yield, 75%; m.p. ¼ 177e179  C; 1H NMR (400 MHz, CDCl3) d 8.99 (s, 1H), 8.40 (s, 1H), 8.22 (s, 1H), 7.91 (dd, J ¼ 8.1, 1.3 Hz, 1H), 7.74 (d, J ¼ 7.8 Hz, 1H), 7.53 (dd, J ¼ 14.4, 4.9 Hz, 3H), 7.41 (t, J ¼ 8.0 Hz, 1H), 7.27 (d, J ¼ 2.1 Hz, 1H), 6.93 (d, J ¼ 9.0 Hz, 2H), 6.55 (dd, J ¼ 3.5, 1.7 Hz, 1H), 4.32 (q, J ¼ 7.1 Hz, 2H), 3.27e3.13 (m, 4H), 2.79e2.64 (m, 4H), 2.43 (s, 3H), 1.37 (t, J ¼ 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 169.7, 164.6, 160.2, 156.3, 147.6, 146.4, 144.5, 138.2, 133.8, 133.7, 129.8, 122.7, 122.6, 120.1, 117.8, 117.4, 115.6, 112.7, 94.9, 60.7, 54.6, 49.3, 45.3, 14.5; HRMS (ESI) m/z calcd for C28H31N5O4S [M þ H]þ: 532.2019; found: 532.2014. Ethyl 2-(3-(1,2,3-thiadiazole-5-carboxamido)phenyl)-4-((4-(4methylpiperazin- 1-yl)phenyl)amino)thiazole-5-carboxylate (5s). Yellow solid; yield, 62%; m.p. ¼ 218e220  C; 1H NMR (400 MHz, CDCl3) d 9.47 (s, 1H), 9.36 (s, 1H), 9.00 (s, 1H), 8.28 (s, 1H), 7.95 (d, J ¼ 7.9 Hz, 1H), 7.81 (d, J ¼ 7.6 Hz, 1H), 7.55 (d, J ¼ 7.7 Hz, 2H), 7.48 (t, J ¼ 8.0 Hz, 1H), 6.96 (d, J ¼ 7.7 Hz, 2H), 4.33 (q, J ¼ 6.8 Hz, 2H), 3.18 (s, 4H), 2.59 (s, 4H), 2.35 (s, 3H), 1.37 (t, J ¼ 6.9 Hz, 3H); 3C NMR (101 MHz, CDCl3) d 169.5, 164.6, 160.3, 157.9, 156.7, 146.9, 140.8, 137.9, 134.0, 133.2, 129.9, 123.3, 122.7, 120.1, 117.9, 117.2, 94.8, 60.7, 55.2, 49.9, 46.2, 14.5; HRMS (ESI) m/z calcd for C26H29N7O3S [M þ H]þ: 550.1695; found: 550.1683. Ethyl 2-(3-((1R,4R)-4-methylcyclohexane-1-carboxamido) phenyl)-4-((4(4-methylpiperazin-1-yl)phenyl)amino)thiazole-5carboxylate (5t). Yellow crystal; yield, 81%; m.p. ¼ 101e103  C; 1H

777

NMR (400 MHz, CDCl3) d 8.85 (s, 1H), 7.95 (s, 1H), 7.73 (d, J ¼ 7.6 Hz, 2H), 7.58 (d, J ¼ 7.5 Hz, 1H), 7.41 (d, J ¼ 8.7 Hz, 2H), 7.24 (t, J ¼ 7.9 Hz, 1H), 6.80 (d, J ¼ 8.6 Hz, 2H), 4.22 (q, J ¼ 7.0 Hz, 2H), 3.04 (s, 4H), 2.49 (s, 4H), 2.25 (s, 3H), 2.04 (t, J ¼ 11.9 Hz, 1H), 1.81 (d, J ¼ 12.2 Hz, 2H), 1.68 (d, J ¼ 16.9 Hz, 4H), 1.47 (dd, J ¼ 24.0, 11.7 Hz, 2H), 1.27 (t, J ¼ 7.0 Hz, 3H), 0.78 (d, J ¼ 6.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 175.0, 170.1, 164.6, 160.3, 146.9, 139.1, 133.5, 133.2, 129.6, 122.6, 122.1, 120.4, 117.6, 117.1, 100.0, 60.7, 55.1, 49.8, 46.2, 46.0, 34.3, 31.9, 29.6, 22.5, 14.5; HRMS (ESI) m/z calcd for C31H41N5O3S [M þ H]þ: 562.2852; found: 562.2851. 4.1.5. General procedures for compounds 6a-6i Aqueous LiOH solution (4 mol/L, 15.0 mmol) was added to a solution of compounds 5a-5t (1.50 mmol) in ethanol (4 mL) and THF (4 mL) and the reaction mixture was heated at 66  C for 2e4 h. The organic solvent was removed under reduced pressure and the residual aqueous solution was acidified to about pH 2 with 2 mol/L HCl. The suspension was filtered and filter cake was dried to obtain the acids 6a-6i (50e96%) or the crude acids. 4-((4-morpholinophenyl)amino)-2-(3-(4-(trifluoromethyl)benzamido)phenyl)thiazole-5-carboxylic acid (6a). Yellow solid; yield, 55%; m.p. ¼ 190e192  C; 1H NMR (400 MHz, DMSO‑d6) d 10.75 (d, J ¼ 10.9 Hz, 1H), 9.05 (d, J ¼ 9.7 Hz, 1H), 8.45 (d, J ¼ 9.6 Hz, 1H), 8.17 (dd, J ¼ 20.5, 11.5 Hz, 3H), 8.04 (s, 1H), 7.92 (dd, J ¼ 24.7, 8.7 Hz, 3H), 7.78 (d, J ¼ 7.8 Hz, 1H), 7.55 (s, 3H), 6.97 (s, 2H), 3.74 (s, 4H), 3.06 (s, 4H); 13C NMR (101 MHz, DMSO‑d6) d 168.8, 165.0, 164.7, 159.2, 139.7, 139.6, 138.4, 132.7, 130.1, 129.8, 128.7, 125.6, 125.4, 123.3, 122.5, 121.9, 119.9, 117.9, 116.0, 66.1, 49.1; HRMS (ESI) m/z calcd for C27H23FN4O4S [M]þ: 568.1387; found: 568.1381. 2-(3-(4-fluorobenzamido)phenyl)-4-((4-morpholinophenyl) amino)thiazole-5-carboxylic acid (6b). Yellow solid; yield, 80%; m.p. ¼ 156e158  C; 1H NMR (400 MHz, DMSO‑d6) d 10.58 (s, 1H), 9.21 (s, 1H), 8.48 (s, 1H), 8.10 (dd, J ¼ 8.4, 5.5 Hz, 2H), 8.04 (d, J ¼ 8.0 Hz, 1H), 7.77 (d, J ¼ 7.7 Hz, 1H), 7.70 (d, J ¼ 7.7 Hz, 2H), 7.55 (t, J ¼ 8.0 Hz, 2H), 7.41 (t, J ¼ 8.8 Hz, 4H), 3.89 (s, 4H), 3.30 (s, 4H); 13C NMR (101 MHz, DMSO‑d6) d 165.9, 165.1, 164.8, 164.0, 163.4, 153.9, 140.4, 133.9, 131.5, 130.1, d 131.0 (d, J ¼ 9.0 Hz), 122.3, 121.3, 117.7, 116.8, 115.8 (d, J ¼ 21.8 Hz), 100.0, 66.3, 53.5; HRMS (ESI) m/z calcd for C27H24FN4O4S [M þ H]þ: 519.0512; found: 519.0517. 2-(3-(4-(tert-butyl)benzamido)phenyl)-4-((4-morpholinophenyl) amino)thiazole-5-carboxylic acid (6c). Yellow solid; yield, 95%; m.p. ¼ 197e199  C; 1H NMR (400 MHz, DMSO‑d6) d 10.41 (d, J ¼ 12.1 Hz, 1H), 10.33 (d, J ¼ 12.2 Hz, 1H), 8.32 (d, J ¼ 11.6 Hz, 1H), 7.94 (d, J ¼ 8.3 Hz, 3H), 7.66 (s, 1H), 7.56 (d, J ¼ 11.9 Hz, 2H), 7.47 (s, 3H), 6.90 (d, J ¼ 9.1 Hz, 2H), 3.72 (s, 4H), 2.98 (s, 4H), 1.32 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) d 167.5, 166.6, 163.8, 155.2, 155.1, 145.0, 139.3, 135.0, 133.7, 131.3, 129.7, 127.4, 125.4, 117.9, 117.5, 116.9, 96.8, 66.0, 49.7, 34.5, 30.7; HRMS (ESI) m/z calcd for C31H34N4O4S [M þ H]þ: 557.2223; found: 557.2214. 2-(3-(4-(dimethylamino)benzamido)phenyl)-4-((4morpholinophenyl)amino)thiazole-5-carboxylic acid (6d). Yellow solid; yield, 50%; m.p. ¼ 160e162  C; 1H NMR (400 MHz, DMSO‑d6) d 10.16 (d, J ¼ 27.2 Hz, 1H), 9.26 (s, 1H), 8.49 (s, 1H), 8.05 (d, J ¼ 8.1 Hz, 1H), 7.91 (dd, J ¼ 16.2, 8.6 Hz, 3H), 7.74 (t, J ¼ 6.9 Hz, 2H), 7.63 (d, J ¼ 7.5 Hz, 1H), 7.47 (ddd, J ¼ 26.2, 17.2, 8.2 Hz, 3H), 6.81 (d, J ¼ 7.9 Hz, 2H), 4.04e3.94 (m, 4H), 3.37 (s, 4H), 3.02 (s, 6H); 13C NMR (101 MHz, DMSO‑d6) d 165.4, 164.6, 152.8, 152.2, 143.4, 140.5, 134.3, 133.3, 129.4, 129.3, 121.8, 121.2, 120.2, 117.1, 115.8, 111.2, 94.9, 63.7, 54.1; HRMS (ESI) m/z calcd for C29H30N5O4S [M þ H]þ: 544.2019; found: 544.2023. 2-(3-benzamidophenyl)-4-((4-(4-methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxylic acid (6e). Yellow solid; yield, 90%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.55 (s, 1H), 10.20 (d, J ¼ 14.6 Hz, 1H), 8.38 (s, 1H), 8.01 (dd, J ¼ 15.4, 7.6 Hz, 3H), 7.70 (d, J ¼ 7.7 Hz, 1H), 7.65e7.59 (m, 1H), 7.56 (t, J ¼ 7.3 Hz, 2H), 7.53e7.30

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X. Guo et al. / European Journal of Medicinal Chemistry 178 (2019) 767e781

(m, 4H), 6.89 (d, J ¼ 8.8 Hz, 2H), 3.03 (s, 4H), 2.45 (s, 4H), 2.21 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.5, 166.3, 162.9, 155.1, 145.3, 140.4, 135.6, 135.3, 134.6, 132.2, 129.9, 128.9, 128.3, 126.5, 122.2, 121.3, 117.8, 117.7, 117.3, 109.5, 55.3, 49.8, 46.3; HRMS (ESI) m/z calcd for C28H29N5O3S [M þ H]þ: 514.1913; found: 514.1904. 2-(3-(3-fluorobenzamido)phenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxylic acid (6f). Yellow solid; yield, 84%; m.p. > 220  C; 1H NMR (400 MHz, CD3OD) d 8.37 (s, 1H), 7.90 (d, J ¼ 7.9 Hz, 1H), 7.77 (t, J ¼ 7.2 Hz, 2H), 7.69 (d, J ¼ 9.6 Hz, 1H), 7.58 (d, J ¼ 8.8 Hz, 2H), 7.51 (dd, J ¼ 13.7, 8.0 Hz, 1H), 7.45 (t, J ¼ 7.9 Hz, 1H), 7.36e7.26 (m, 1H), 6.96 (d, J ¼ 8.8 Hz, 2H), 3.08 (s, 4H), 2.58 (s, 4H), 2.30 (s, 3H); 13C NMR (101 MHz, CD3OD) d 167.0, 166.1, 165.3, 162.7 (d, J ¼ 245.9 Hz), 156.4, 145.2, 139.1, 137.1 (d, J ¼ 7.3 Hz), 135.8, 134.5, 130.2 (d, J ¼ 7.9 Hz), 129.1, 123.2 (d, J ¼ 2.9 Hz), 122.5, 121.9, 118.3 (d, J ¼ 21.4 Hz), 118.1, 118.0, 117.7, 114.3 (d, J ¼ 23.4 Hz), 100.0, 54.6, 49.9, 44.7; HRMS (ESI) m/z calcd for C28H28FN5O3S [M þ H]þ: 532.1819; found: 532.1809. 2-(3-(2-fluorobenzamido)phenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxylic acid (6g). Yellow solid; yield, 85%; m.p. ¼ 194e196  C; 1H NMR (400 MHz, CD3OD) d 8.39 (s, 1H), 7.87 (d, J ¼ 7.9 Hz, 1H), 7.82e7.65 (m, 2H), 7.59 (d, J ¼ 8.9 Hz, 2H), 7.53 (dd, J ¼ 11.0, 4.3 Hz, 1H), 7.46 (t, J ¼ 7.9 Hz, 1H), 7.23 (ddd, J ¼ 32.5, 19.3, 7.6 Hz, 2H), 6.99 (t, J ¼ 20.8 Hz, 2H), 3.07 (s, 4H), 2.57 (s, 4H), 2.29 (s, 3H); 13C NMR (101 MHz, CD3OD) d 170.0, 165.2, 164.4, 159.7 (d, J ¼ 249.8 Hz), 156.4, 145.2, 139.1, 135.8, 134.5, 132.7 (d, J ¼ 8.6 Hz), 129.9 (d, J ¼ 1.8 Hz), 124.3 (d, J ¼ 3.3 Hz), 124.0 (d, J ¼ 14.3 Hz), 121.9, 121.9, 118.0, 117.7, 117.6, 115.9 (d, J ¼ 22.5 Hz), 105.4, 54.6, 49.9, 44.7; HRMS (ESI) m/z calcd for C28H28FN5O3S [M þ H]þ: 532.1819; found: 532.1816. 2-(3-(4-chlorobenzamido)phenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxylic acid (6h). Yellow solid; yield, 46%; m.p. > 220  C; 1H NMR (400 MHz, CD3OD) d 8.37 (s, 1H), 7.90 (t, J ¼ 11.9 Hz, 3H), 7.74 (d, J ¼ 7.2 Hz, 1H), 7.57 (d, J ¼ 8.8 Hz, 2H), 7.45 (t, J ¼ 7.8 Hz, 3H), 6.94 (d, J ¼ 8.8 Hz, 2H), 3.05 (s, 4H), 2.55 (s, 4H), 2.29 (s, 3H); 13C NMR (101 MHz, CD3OD) d 171.4, 167.8, 166.7, 157.8, 146.6, 140.6, 139.0, 137.2, 135.8, 134.8, 132.0, 130.5, 129.8, 123.9, 123.3, 119.5, 119.4, 119.0, 56.0, 51.3, 46.1; HRMS (ESI) m/z calcd for C28H28ClN5O3S [M þ H]þ: 548.1523; found: 548.1514. 2-(3-(3-chloro-4-fluorobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl)amino)thiazole-5-carboxylic acid (6i). Yellow solid; yield, 95%; m.p. ¼ 185e187  C; 1H NMR (400 MHz, CD3OD) d 8.36 (t, J ¼ 1.8 Hz, 1H), 8.09 (dd, J ¼ 7.0, 2.2 Hz, 1H), 7.93 (ddd, J ¼ 8.6, 4.5, 2.3 Hz, 1H), 7.90e7.85 (m, 1H), 7.77e7.73 (m, 1H), 7.60e7.57 (m, 1H), 7.57e7.54 (m, 1H), 7.44 (dd, J ¼ 10.3, 5.6 Hz, 1H), 7.34 (t, J ¼ 8.8 Hz, 1H), 6.98e6.95 (m, 1H), 6.95e6.93 (m, 1H), 3.15e3.00 (m, 4H), 2.66e2.52 (m, 4H), 2.31 (s, 3H); 13C NMR (101 MHz, CD3OD) d 167.0, 165.2, 165.0, 160.0 (d, J ¼ 253.0 Hz), 156.4, 145.1, 139.1, 135.8, 134.5, 132.2 (d, J ¼ 3.5 Hz), 130.3, 129.1, 128.2 (d, J ¼ 8.1 Hz), 122.4, 121.9, 120.8 (d, J ¼ 18.4 Hz), 118.1, 118.0, 117.6, 116.4 (d, J ¼ 21.9 Hz), 100.0, 54.6, 49.9, 44.7; HRMS (ESI) m/z calcd for C28H27ClFN5O3S [M þ H]þ: 566.1429; found: 566.1409. 4.1.6. General procedures for compounds 7a-7u DIPEA (0.28 g, 2.2 mmol) and PyAOP (0.57 g, 1.1 mmol) were added to a solution of acids 6a-6i or the crude acids (1.0 mmol) in CH2Cl2 (10 mL), at 0  C. The mixture was stirred at 0  C for 15 min and NH4Cl or amines (2.0 mmol) were added. The reaction was stirred at room temperature for 10 h. The reaction mixture was partitioned between CH2Cl2 (20 mL  2) and H2O (25 mL). The organic layer was combined, washed with sat. brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether (60e90  C fraction):EtOAc (1:5e1:10, v/v) as eluent to afford compounds 7a-7u (42e95%). 4-((4-morpholinophenyl)amino)-2-(3-(4-(trifluoromethyl)

benzamido)phenyl)thiazole-5-carboxamide (7a). Yellow solid; yield, 82%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.78 (s, 1H), 9.97 (s, 1H), 8.51 (s, 1H), 8.20 (d, J ¼ 8.1 Hz, 2H), 7.96 (t, J ¼ 7.1 Hz, 3H), 7.75 (d, J ¼ 7.6 Hz, 1H), 7.57 (t, J ¼ 8.0 Hz, 1H), 7.51 (d, J ¼ 8.9 Hz, 2H), 6.95 (d, J ¼ 9.0 Hz, 2H), 3.80e3.70 (m, 4H), 3.10e3.02 (m, 4H); 13 C NMR (101 MHz, DMSO‑d6) d 166.5, 165.8, 159.1, 146.6, 140.3, 133.8, 133.4, 129.2, 125.9, 123.5, 119.6, 118.1, 116.7, 66.7, 49.7; HRMS (ESI) m/z calcd for C28H26F3N5O3S [M þ H]þ: 568.1630; found: 568.1609. 2-(3-(4-fluorobenzamido)phenyl)-4-((4-morpholinophenyl) amino)thiazole-5-carboxamide (7b). Yellow solid; yield, 77%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.56 (d, J ¼ 12.4 Hz, 1H), 9.96 (d, J ¼ 13.4 Hz, 1H), 8.49 (d, J ¼ 13.1 Hz, 1H), 8.08 (d, J ¼ 5.4 Hz, 2H), 7.94 (s, 1H), 7.72 (dd, J ¼ 13.2, 7.7 Hz, 1H), 7.61e7.33 (m, 7H), 6.99e6.87 (m, 2H), 3.72 (d, J ¼ 4.5 Hz, 4H), 3.03 (d, J ¼ 4.5 Hz, 4H); 13 C NMR (101 MHz, DMSO‑d6) d 166.6, 165.8, 165.3, 164.7 (d, J ¼ 245.9 Hz), 159.1, 146.5, 140.5, 133.8, 133.4, 131.5, 131.0 (d, J ¼ 8.9 Hz), 130.3, 123.4, 121.8, 119.6, 118.1, 116.7, 115.9 (d, J ¼ 21.1 Hz), 97.2, 66.6, 49.7; HRMS (ESI) m/z calcd for C27H26FN5O3S [M þ H]þ: 518.1662; found: 518.1647. 2-(3-(4-(tert-butyl)benzamido)phenyl)-4-((4-morpholinophenyl) amino)thiazole-5-carboxamide (7c). Yellow solid; yield, 83%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.47 (s, 1H), 9.99 (s, 1H), 8.54 (s, 1H), 7.95 (t, J ¼ 8.8 Hz, 3H), 7.73 (d, J ¼ 7.8 Hz, 1H), 7.59e7.49 (m, 6H), 6.95 (d, J ¼ 8.7 Hz, 2H), 3.74 (s, 4H), 3.04 (s, 4H), 1.33 (s, 9H); 13 C NMR (101 MHz, DMSO‑d6) d 166.7, 166.4, 165.9, 159.2, 155.2, 146.5, 140.6, 137.3, 133.8, 133.3, 130.2, 128.1, 125.7, 123.4, 121.7, 119.6, 118.0, 116.7, 97.2, 66.6, 49.7, 35.2, 31.4; HRMS (ESI) m/z calcd for C31H35N5O3S [M þ H]þ: 556.2382; found: 556.2366. 4-((4-(morpholine-4-carbonyl)phenyl)amino)-2-(3-(4-(trifluoromethyl)benzamido)phenyl)thiazole-5-carboxamide (7e). Yellow solid; yield, 74%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.78 (s, 1H), 10.40 (s, 1H), 8.59 (s, 1H), 8.46e7.04 (m, 11H), 4.05e3.14 (m, 8H); 13C NMR (101 MHz, DMSO‑d6) d 169.6, 166.6, 165.5, 165.2, 157.8, 142.5, 140.3, 138.9, 133.2, 130.5, 129.3, 129.2, 128.0, 126.0, 123.5, 122.1, 118.1, 117.4, 100.2, 66.6; HRMS (ESI) m/z calcd for C29H24F3N5NaO4S [M þ Na]þ: 618.1399; found: 618.1398. 2-(3-(4-fluorobenzamido)phenyl)-4-((4-(morpholine-4-carbonyl) phenyl)amino)thiazole-5-carboxamide (7f). Yellow crystal; yield, 42%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.58 (s, 1H), 10.39 (s, 1H), 8.58 (s, 1H), 8.16e8.04 (m, 2H), 7.98 (d, J ¼ 7.8 Hz, 1H), 7.78 (d, J ¼ 7.4 Hz, 1H), 7.72 (d, J ¼ 6.8 Hz, 2H), 7.63e7.53 (m, 2H), 7.42 (t, J ¼ 8.0 Hz, 5H), 3.61 (s, 4H), 3.54 (s, 4H); 13C NMR (101 MHz, DMSO‑d6) d 169.6, 166.7, 165.9, 165.5, 165.3, 164.7 (d, J ¼ 246.3 Hz), 163.4, 157.8, 142.5, 140.6, 133.2, 131.6, 131.0 (d, J ¼ 9.3 Hz), 130.4, 129.3, 128.0, 123.4, 121.8, 118.0, 117.4, 115.9 (d, J ¼ 21.8 Hz), 100.1, 66.6; HRMS (ESI) m/z calcd for C28H24FN5NaO4S [M þ Na]þ: 568.1431; found: 568.1430. 2-(3-(4-(tert-butyl)benzamido)phenyl)-4-((4-(morpholine-4carbonyl)phenyl)amino)thiazole-5-carboxamide (7g). Yellow solid; yield, 64%; m.p. > 220  C; 1H NMR (400 MHz, THF-d8) d 10.42 (s, 1H), 9.55 (s, 1H), 8.56 (s, 1H), 7.81 (t, J ¼ 9.0 Hz, 3H), 7.67 (d, J ¼ 7.8 Hz, 1H), 7.63 (s, 1H), 7.61 (s, 1H), 7.41 (d, J ¼ 8.4 Hz, 2H), 7.34 (d, J ¼ 8.0 Hz, 1H), 7.30 (d, J ¼ 8.5 Hz, 2H), 6.81 (s, 2H), 2.49 (s, 4H), 1.62 (s, 4H), 1.25 (s, 9H); 13C NMR (101 MHz, THF-d8) d 169.7, 166.9, 165.8, 158.6, 155.0, 143.0, 141.1, 133.8, 133.1, 129.6, 129.2, 128.2, 127.6, 125.4, 122.5, 121.3, 117.8, 117.3, 66.6, 34.9, 30.9; HRMS (ESI) m/ z calcd for C32H33N5NaO4S [M þ Na]þ: 606.2151; found: 606.2150. 4-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-(3-(4-(trifluoromethyl)benzamido)phenyl)thiazole-5-carboxamide (7h). Yellow solid; yield, 91%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.82 (d, J ¼ 15.4 Hz, 1H), 10.01 (d, J ¼ 15.4 Hz, 1H), 8.56 (d, J ¼ 14.9 Hz, 1H), 8.36e8.14 (m, 2H), 7.97 (d, J ¼ 7.1 Hz, 3H), 7.87e7.70 (m, 1H), 7.68e7.38 (m, 5H), 6.97 (d, J ¼ 6.1 Hz, 2H), 3.12 (s, 4H), 2.60 (s, 4H), 2.33 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.5, 165.9,

X. Guo et al. / European Journal of Medicinal Chemistry 178 (2019) 767e781

165.3, 159.1, 146.3, 140.2, 138.9, 133.7, 133.4, 132.2, 131.8, 130.3, 129.2, 125.9, 125.9, 123.5, 123.0, 122.1, 119.6, 118.1, 117.1, 97.2, 54.7, 48.9, 45.5; HRMS (ESI) m/z calcd for C29H29F3N6O2S [M þ H]þ: 581.1947; found: 581.1941. 2-(3-(4-fluorobenzamido)phenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxamide (7i). Yellow crystal; yield, 45%; m.p. ¼ 176e178  C; 1H NMR (400 MHz, DMSO‑d6) d 10.55 (s, 1H), 9.95 (s, 1H), 8.50 (s, 1H), 8.09 (dd, J ¼ 8.8, 5.5 Hz, 2H), 7.95 (d, J ¼ 8.2 Hz, 1H), 7.73 (d, J ¼ 7.8 Hz, 1H), 7.55 (t, J ¼ 8.0 Hz, 2H), 7.48 (d, J ¼ 9.0 Hz, 3H), 7.41 (t, J ¼ 8.8 Hz, 2H), 6.94 (d, J ¼ 9.0 Hz, 2H), 3.11e3.05 (m, 4H), 2.46 (s, 4H), 2.23 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.6, 165.8, 165.3, 164.7 (d, J ¼ 249.3 Hz),163.4, 159.2, 146.6, 140.5, 133.5, 133.4, 131.6, 131.0 (d, J ¼ 9.1 Hz), 131.0, 130.3, 123.4, 121.8, 119.6, 118.1, 116.9, 115.9 (d, J ¼ 21.9 Hz), 97.2, 55.2, 49.3, 46.2; HRMS (ESI) m/z calcd for C28H29FN6O2 [M þ H]þ: 531.1978; found: 531.1966. 2-(3-(4-(tert-butyl)benzamido)phenyl)-4-((4-(4-methylpiperazin1-yl)phenyl)amino)thiazole-5-carboxamide (7j). Yellow solid; yield, 45%; m.p. ¼ 165e167  C; 1H NMR (400 MHz, DMSO‑d6) d 10.42 (d, J ¼ 17.9 Hz, 1H), 8.76 (s, 1H), 8.49 (s, 1H), 7.94 (d, J ¼ 8.4 Hz, 2H), 7.87 (d, J ¼ 8.2 Hz, 1H), 7.64 (d, J ¼ 7.8 Hz, 1H), 7.57 (d, J ¼ 8.4 Hz, 2H), 7.47 (t, J ¼ 7.9 Hz, 1H), 7.19 (d, J ¼ 8.9 Hz, 2H), 6.89 (d, J ¼ 8.9 Hz, 2H), 6.51 (s, 1H), 3.11e2.98 (m, 4H), 2.57e2.47 (m, 4H), 2.26 (s, 3H), 1.33 (s, 9H); 13C NMR (101 MHz, DMSO‑d6) d 172.6, 166.2, 164.5, 155.5, 155.1, 145.3, 140.5, 136.2, 134.1, 132.4, 130.0, 128.1, 125.7, 122.1, 121.2, 118.0, 117.6, 117.4, 90.7, 55.0, 49.4, 45.8, 35.2, 31.4; HRMS (ESI) m/z calcd for C32H38N6O2S [M þ H]þ: 569.2699; found: 569.2683. 2-(3-benzamidophenyl)-4-((4-(4-methylpiperazin-1-yl)phenyl) amino)thiazole-5-carboxamide (7k). Yellow solid; yield, 93%; m.p. ¼ 125e127  C; 1H NMR (400 MHz, DMSO‑d6) d 10.54 (s, 1H), 9.97 (s, 1H), 8.55 (s, 1H), 8.03e7.94 (m, 3H), 7.73 (d, J ¼ 7.5 Hz, 1H), 7.61 (d, J ¼ 7.1 Hz, 1H), 7.59e7.54 (m, 4H), 7.49 (d, J ¼ 7.5 Hz, 3H), 6.93 (d, J ¼ 8.6 Hz, 2H), 3.09 (s, 4H), 2.56 (s, 4H), 2.29 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.1, 165.9, 165.3, 158.7, 145.8, 140.1, 134.7, 133.2, 132.9, 131.7, 129.7, 128.4, 127.7, 122.9, 121.2, 119.1, 117.6, 116.5, 96.7, 54.4, 48.5, 45.2; HRMS (ESI) m/z calcd for C28H30N6O2S [M þ H]þ: 513.2073; found: 513.2067. 2-(3-(3-fluorobenzamido)phenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxamide (7l). Yellow solid; yield, 87%; m.p. > 220  C; 1H NMR (400 MHz, DMSO‑d6) d 10.62 (s, 1H), 9.97 (s, 1H), 8.52 (s, 1H), 8.00e7.93 (m, 1H), 7.87 (d, J ¼ 7.8 Hz, 1H), 7.83 (dd, J ¼ 9.8, 2.3 Hz, 1H), 7.75 (d, J ¼ 8.2 Hz, 1H), 7.63 (td, J ¼ 8.0, 6.0 Hz, 1H), 7.56 (t, J ¼ 8.0 Hz, 2H), 7.51 (d, J ¼ 3.4 Hz, 2H), 7.48 (d, J ¼ 4.1 Hz, 2H), 6.94 (d, J ¼ 9.0 Hz, 2H), 3.13e3.07 (m, 4H), 2.52 (d, J ¼ 4.3 Hz, 4H), 2.27 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.5, 165.8, 165.0, 162.4 (d, J ¼ 244.4 Hz), 159.2, 146.4, 140.3, 137.4 (d, J ¼ 7.2 Hz), 133.6, 133.4, 131.1 (d, J ¼ 8.0 Hz), 130.3, 124.5, 123.4, 122.0, 119.1 (d, J ¼ 27.6 Hz), 119.3, 118.1, 117.0, 115.1 (d, J ¼ 22.9 Hz), 97.3, 55.0, 49.1, 46.0; HRMS (ESI) m/z calcd for C28H29FN6O2S [M þ H]þ: 531.1978; found: 531.1969. 2-(3-(2-fluorobenzamido)phenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxamide (7m). Yellow solid; yield, 78%; m.p. ¼ 169e171  C; 1H NMR (400 MHz, DMSO‑d6) d 10.73 (s, 1H), 10.02 (s, 1H), 8.54 (s, 1H), 7.85 (d, J ¼ 8.1 Hz, 1H), 7.80e7.68 (m, 2H), 7.68e7.44 (m, 6H), 7.44e7.32 (m, 2H), 7.00 (d, J ¼ 9.0 Hz, 2H), 3.28 (s, 4H), 3.18 (d, J ¼ 4.2 Hz, 4H), 2.74 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.5, 165.8, 163.7, 159.4 (d, J ¼ 249.4 Hz), 159.0, 145.1, 140.3, 134.5, 133.5, 133.2 (d, J ¼ 8.5 Hz), 130.4, 125.3, 125.2, 125.1 (d, J ¼ 3.2 Hz), 122.8, 121.9, 119.5, 117.6, 117.4, 116.7 (d, J ¼ 21.6 Hz), 97.6, 53.4, 47.5, 43.3; HRMS (ESI) m/z calcd for C28H29FN6O2S [M þ H]þ: 531.1978; found: 531.1975. 2-(3-(4-chlorobenzamido)phenyl)-4-((4-(4-methylpiperazin-1-yl) phenyl)amino)thiazole-5-carboxamide (7n). Yellow solid; yield, 64%; m.p. ¼ 160-160  C; 1H NMR (400 MHz, DMSO‑d6) d 10.60 (s, 1H), 10.06e9.92 (m, 1H), 8.52 (s, 1H), 8.03 (d, J ¼ 8.6 Hz, 2H), 7.95 (d,

779

J ¼ 8.2 Hz, 1H), 7.73 (t, J ¼ 10.7 Hz, 1H), 7.64 (d, J ¼ 8.5 Hz, 2H), 7.59e7.53 (m, 2H), 7.52 (s, 1H), 7.50 (s, 1H), 6.95 (d, J ¼ 9.0 Hz, 2H), 3.15 (s, 4H), 2.73 (s, 4H), 2.42 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.6, 165.9, 165.4, 159.1, 146.0, 140.3, 137.1, 133.9, 133.8, 133.3, 130.3, 130.2, 129.0, 123.5, 122.0, 119.6, 118.1, 117.2, 97.2, 54.4, 48.5, 45.0; HRMS (ESI) m/z calcd for C28H29ClN6O2S [M þ H]þ: 547.1683; found: 547.1669. 2-(3-(3-chloro-4-fluorobenzamido)phenyl)-4-((4-(4methylpiperazin-1-yl)phenyl)amino)thiazole-5-carboxamide (7o). Yellow solid; yield, 68%; m.p. ¼ 148e150  C; 1H NMR (400 MHz, DMSO‑d6) d 10.65 (s, 1H), 9.96 (s, 1H), 8.46 (d, J ¼ 26.9 Hz, 1H), 8.26 (d, J ¼ 7.0 Hz, 1H), 8.05 (s, 1H), 7.95 (d, J ¼ 7.9 Hz, 1H), 7.74 (d, J ¼ 7.7 Hz, 1H), 7.67e7.52 (m, 3H), 7.49 (d, J ¼ 8.3 Hz, 3H), 6.93 (d, J ¼ 8.4 Hz, 2H), 3.10 (s, 4H), 2.55 (s, 4H), 2.28 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 166.0, 165.3, 163.5, 158.7, 159.2 (d, J ¼ 251.8 Hz), 145.9, 139.8, 133.2, 132.9, 132.2 (d, J ¼ 3.3 Hz), 130.3, 129.8, 129.2 (d, J ¼ 8.4 Hz), 122.9, 121.5, 119.7 (d, J ¼ 18.4 Hz), 119.2, 117.7, 117.1, 116.9, 116.5, 96.8, 54.4, 48.5, 45.3; HRMS (ESI) m/z calcd for C28H27ClFN6O2S [M þ H]þ: 565.1589; found: 565.1588. 4-((4-(4-methylpiperazin-1-yl)phenyl)amino)-2-(3-(5-(trifluoromethyl)picolinamido)phenyl)thiazole-5-carboxamide (7p). Yellow crystal; yield, 45%; m.p. ¼ 145e147  C; 1H NMR (400 MHz, THFd8) d 10.41 (s, 1H), 10.03 (s, 1H), 8.89 (s, 1H), 8.63 (s, 1H), 8.35 (d, J ¼ 8.2 Hz, 1H), 8.27 (dd, J ¼ 8.2, 1.7 Hz, 1H), 7.85 (d, J ¼ 8.0 Hz, 1H), 7.71 (d, J ¼ 7.8 Hz, 1H), 7.51 (d, J ¼ 8.7 Hz, 2H), 7.36 (t, J ¼ 7.9 Hz, 1H), 6.85 (d, J ¼ 8.7 Hz, 2H), 6.73 (s, 2H), 3.23 (s, 4H), 3.17 (s, 4H), 2.74 (s, 3H); 13C NMR (101 MHz, THF-d8) d 166.1, 161.1, 160.6, 159.3, 153.4, 145.1, 144.8, 139.2, 135.4, 135.2, 133.8, 130.0, 122.6, 122.4, 121.9, 119.1, 117.7, 117.5, 100.0, 54.2, 48.1, 43.4; HRMS (ESI) m/z calcd for C28H27F3N7O2S [M þ H]þ: 582.1899; found: 582.1897. 2-(3-(furan-2-carboxamido)phenyl)-4-((4-(4-methylpiperazin-1yl)phenyl)amino)thiazole-5-carboxamide (7q). Yellow solid; yield, 63%; m.p. ¼ 155e157  C; 1H NMR (400 MHz, THF-d8) d 9.98 (s, 1H), 9.54 (s, 1H), 8.41 (s, 1H), 7.84 (d, J ¼ 8.2 Hz, 1H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.58 (s, 1H), 7.43 (d, J ¼ 8.8 Hz, 2H), 7.29 (t, J ¼ 8.0 Hz, 1H), 7.10 (d, J ¼ 3.1 Hz, 1H), 6.79 (d, J ¼ 8.1 Hz, 2H), 6.65 (s, 1H), 6.58 (d, J ¼ 7.9 Hz, 1H), 6.48 (dd, J ¼ 3.3, 1.7 Hz, 1H), 3.08 (s, 4H), 2.79e2.66 (m, 4H), 2.40 (s, 3H); 13C NMR (101 MHz, THF-d8) d 166.3, 165.8, 159.5, 156.2, 148.4, 145.8, 144.7, 139.8, 134.5, 133.7, 129.2, 122.4, 121.4, 119.2, 117.7, 117.0, 114.7, 112.0, 96.3, 54.8, 48.9, 44.5; HRMS (ESI) m/z calcd for C26H27N6O3S [M þ H]þ: 503.1865; found: 503.1864. N-(3-(5-carbamoyl-4-((4-(4-methylpiperazin-1-yl)phenyl)amino) thiazol-2-yl)phenyl)-1,2,3-thiadiazole-5-carboxamide (7r). Yellow crystal; yield, 60%; m.p. ¼ 166e168  C; 1H NMR (400 MHz, DMSO‑d6) d 9.97 (s, 1H), 9.54 (s, 1H), 9.20 (s, 1H), 8.57 (s, 1H), 7.95 (d, J ¼ 7.9 Hz, 1H), 7.81 (d, J ¼ 7.6 Hz, 1H), 7.55 (d, J ¼ 7.7 Hz, 2H), 7.48 (t, J ¼ 8.0 Hz, 1H), 6.96 (d, J ¼ 7.7 Hz, 2H), 3.23 (s, 4H), 2.60 (s, 4H), 2.34 (s, 3H); 13C NMR (101 MHz, DMSO‑d6) d 169.5, 167.6, 160.3, 159.5, 156.7, 146.9, 140.8, 137.9, 134.0, 133.2, 129.9, 123.3, 122.7, 120.1, 117.8, 117.1, 95.8, 55.2, 49.9, 45.3. 2-(3-((1R,4R)-4-methylcyclohexane-1-carboxamido)phenyl)-4((4-(4-methylpiperazin-1-yl)phenyl)amino)thiazole-5-carboxamide (7s). Yellow solid; yield, 62%; m.p. ¼ 136e138  C; 1H NMR (400 MHz, DMSO‑d6) d 10.13 (s, 1H), 9.93 (d, J ¼ 9.5 Hz, 1H), 8.34 (d, J ¼ 7.9 Hz, 1H), 7.69 (d, J ¼ 44.9 Hz, 2H), 7.46 (s, 4H), 6.92 (s, 1H), 3.03 (d, J ¼ 29.3 Hz, 4H), 2.48 (s, 4H), 2.24 (s, 3H), 1.77 (d, J ¼ 43.4 Hz, 5H), 1.61e1.06 (m, 4H), 0.91 (d, J ¼ 24.3 Hz, 4H); 13C NMR (101 MHz, DMSO‑d6) d 175.2, 166.6, 165.8, 159.1, 146.5, 140.9, 133.6, 133.3, 130.2, 122.0, 120.9, 119.6, 116.9, 116.8, 97.1, 55.1, 49.2, 46.3, 46.1, 45.2, 34.4, 32.1, 29.5, 26.4, 26.3, 23.0; HRMS (ESI) m/z calcd for C29H37N6O2S [M þ H]þ: 533.2698; found: 533.2695. 2-(3-(4-(tert-butyl)benzamido)phenyl)-N-methyl-4-((4morpholinophenyl)amino)thiazole-5-carboxamide (7t). Yellow solid; yield, 92%; m.p. ¼ 216e218  C; 1H NMR (400 MHz, CDCl3) d 9.73 (s,

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X. Guo et al. / European Journal of Medicinal Chemistry 178 (2019) 767e781

1H), 8.23 (s, 1H), 8.01 (s, 1H), 7.86 (d, J ¼ 8.2 Hz, 2H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.74 (d, J ¼ 7.6 Hz, 1H), 7.54 (t, J ¼ 8.4 Hz, 3H), 7.44 (t, J ¼ 7.8 Hz, 1H), 6.93 (d, J ¼ 8.5 Hz, 2H), 5.44 (s, 1H), 3.86 (s, 4H), 3.11 (s, 4H), 2.99 (d, J ¼ 4.6 Hz, 3H), 1.37 (s, 9H); 13C NMR (101 MHz, CDCl3) d 167.8, 166.1, 165.8, 165.0, 155.8, 147.1, 133.6, 132.3, 131.7, 130.9, 128.9, 127.1, 125.8, 122.8, 122.5, 119.8, 118.1, 117.0, 67.0, 50.4, 31.4, 29.7, 22.7; HRMS (ESI) m/z calcd for C32H37N5O3S [M þ H]þ: 570.2539; found: 570.2536. 2-(3-(4-(tert-butyl)benzamido)phenyl)-N-isopropyl-4-((4morpholinophenyl)amino)thiazole-5-carboxamide (7u). Yellow crystal; yield, 95%; m.p. > 220  C; 1H NMR (400 MHz, CDCl3) d 9.72 (s, 1H), 8.32 (s, 1H), 8.14 (s, 1H), 7.86 (d, J ¼ 8.2 Hz, 2H), 7.73 (t, J ¼ 7.7 Hz, 2H), 7.52 (t, J ¼ 9.3 Hz, 4H), 7.41 (t, J ¼ 7.9 Hz, 1H), 6.91 (d, J ¼ 8.8 Hz, 2H), 5.34 (d, J ¼ 7.6 Hz, 1H), 4.25 (dq, J ¼ 13.2, 6.6 Hz, 1H), 3.95e3.79 (m, 4H), 3.15e3.02 (m, 4H), 1.36 (s, 9H), 1.28 (d, J ¼ 6.5 Hz, 6H); 13C NMR (101 MHz, CDCl3) d 165.9, 165.5, 163.6, 159.1, 155.8, 146.4, 138.8, 134.4, 133.7, 131.7, 129.7, 127.0, 125.8, 122.5, 122.4, 119.8, 118.0, 117.0, 96.6, 67.0, 50.4, 42.0, 35.1, 31.2, 23.1; HRMS (ESI) m/z calcd for C34H41N5O3S [M þ H]þ: 598.2852; found: 598.2839. 4.2. Single crystal X-ray diffraction data collection Single crystal X-ray diffraction data of compound 5e were collected on a Rigaku Saturn 724 CCD system using Mo Ka radiation (l ¼ 0.71073 Å). A total of 17761 reflections were collected in the range of 1.578  q  27.897 (12  h  13, 14  k  15, 18  l  18) with 6991 unique reflections (Rint ¼ 0.0925), of which 3709 had I > 2s(I) for refinements. The structure was directly solved by SHELXS-97 program and hydrogens were introduced in idealized positions according to theoretical models. The derived atomic parameters were refined through full matrix least-squares with R ¼ 0.1308; wR ¼ 0.1392 (w ¼ 1/[s2(Fo2)þ(0.0420P)2], where P¼(Fo2þ2Fc2)/3; (D/s)max ¼ 0.002 and S ¼ 1.051. 4.3. Biological evaluation 4.3.1. Cell culture and reagents The BTK enzyme and the ADP-Glo™ Kinase Assay system were purchased from Promega Corporation (V9071). The BTK (C481S) enzyme was purchased from SignalChem Corporation (B10e12CH05). The Cell Counting Kit-8 (CCK-8) reagent was purchased from MedChemExpress (HYeK0301). The Annexin V-FITC Apoptosis Detection Kit and Cell Cycle Assay were purchased from eBioscience (BMS500FI-20). All cell lines were cultured in RPMI-1640 (Gibco™) supplemented with 10% FBS (Gibco™), 1% penicillin streptomycin. The cells were maintained in media at 37  C in humidified 5% CO2. 4.3.2. In vitro BTK kinase assay The BTK enzymatic assay was performed using the ADP-Glo™ Kinase Assay (Promega, V9071). Firstly, in 384-well plate, a kinase reaction of 5 mL in the presence or absence of inhibitor was incubated for 60 min at room temperature. Secondly, 5 mL of ADP-Glo™ Reagent was added and incubated for 40 min at room temperature to deplete remaining ATP. Thirdly, 10 mL of Kinase Detection Reagent was added and incubated for 30 min at room temperature to convert ADP to ATP and introduce luciferase/luciferin reaction to quantity the newly synthesized ATP. Finally, luminescence of plate was recorded. The inhibitory rates were calculated against positive control. Inhibitory rates ¼ (RLUcontrol-RLUtest)/(RLUcontrol-RLUblank)  100. The inhibitory activity of synthesized compounds against BTK was initially tested at 10 mM. Active compounds were tested at eight concentrations (105-102 mM.) with each concentration in triplicates and the IC50 values were calculated using GraphPad Prim version 6.02.

4.3.3. Cellular antiproliferative assay The antiproliferative activities of compounds against Ramos and Raji cell lines were tested according to CCK-8 protocol. The cells were seeded in 96-well culture plates at a density of 2000e3000 cells/well for one day before compounds of various concentrations were added. Cell proliferation was determined after treatment with compounds for 72 h and the absorbance at 450 nm was obtained by a microplate reader. Compounds were tested at appropriate concentrations (1.25e80 mM) with each concentration in triplicates. The IC50 values were calculated using GraphPad Prim version 6.02. 4.3.4. Flow cytometry assay The apoptosis of cell was detected with a kit purchased from Biolegend (# 640930), as described previously [30]. Briefly, the cells were washed twice with cold cell staining buffer and then suspended with 100 mL Annexin V binding buffer (containing antiAnnexin V antibodies and 7-AAD) and incubated for 15 min at room temperature in dark. After centrifugation, cells was added 400 ml Annixin V binding buffer and analyzed by flow cytometry. The method for cell cycle analysis was referenced from a published reference study [31]. Briefly, the cells were collected and washed twice with PBS. Then they were stained with nuclei staining buffer (0.1% Triton X-100 in PBS, 200 g/ml RNase, and 20 g/ml PI) for 2 h at 4  C. Then the cells were analyzed by flow cytometry and the data were processed by Flowjo (Version 10.5). 4.3.5. Signaling pathway assay Ramos and Raji cells were treated with serially diluted compound 7o, ibrutinib and CGI-1746 for 48 h. Cells were harvested and lysed in 2  Laemmli buffer (Bio-Rad) supplemented with 2.5% bmercaptoethanol at 4  C for 15 min and then boiled for 5 min. Proteins were fractionated on a 10% SDS polyacrylamide gel and transferred onto nitrocellulose membranes. Incubation of primary antibodies was performed at 4  C for about 12e18 h using 1:1000 Phospho-BTK (Tyr223), Phospho-PLCg2 (Tyr1217) and b-actin. Anti-rabbit horseradish peroxidase (Cell Signaling Technology, 7074 S) was used as secondary antibody, followed by detection with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher, 34579). 4.4. Molecular docking study The crystal structure (PDB code: 3OCS) of BTK in complex with CGI-1746 and the crystal structure (PDB code: 4YHF) of BTK bound to ibrutinib (modeled) were superimposed using Pymol to analyze the two different binding modes, after removing water molecules and nonpolar hydrogen atoms. Compound 7g and 7o were docked into BTK enzyme (PDB code: 3OCS) by the Autodock 4.2 program. 100 docking poses were generated for each compound and the best-scoring ligand protein complexes were selected for analyses. AutoDockTools and Pymol were used to analyze the docking results. Acknowledgments This work was supported in part by the National Natural Science Foundation of China (No. 31571991, No. 31872007), the Tianjin Natural Science Foundation (No. 18JCZDJC33500), the International Science & Technology Cooperation Program of China (No. 2014DFR41030), and The Fundamental Research Funds for the Central Universities, Nankai University (No 63191323, No 63191743).

X. Guo et al. / European Journal of Medicinal Chemistry 178 (2019) 767e781

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.06.035. [15]

Abbreviations BTK XLA BCR FcgR MCL CLL WM CCK-8 SAR DIPEA PyAOP

Bruton's tyrosine kinase X-link agammaglobulinemia B cell receptor Fcg receptor mantle cell lymphoma chronic lymphocytic leukemia Waldenstrom's macroglobulinemia Cell Counting Kit-8 structure-activity relationship N,N-diisopropylethylamine 3-hydroxy-3H-1,2,3-thiazolo[4,5-b]pyridinato- O)tri-1pyrrolidinylphosphonium hexafluorophosphate

[16]

[17]

[18]

[19]

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