Indazoles as potential c-met inhibitors: Design, synthesis and molecular docking studies

Indazoles as potential c-met inhibitors: Design, synthesis and molecular docking studies

European Journal of Medicinal Chemistry 65 (2013) 112e118 Contents lists available at SciVerse ScienceDirect European Journal of Medicinal Chemistry...

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European Journal of Medicinal Chemistry 65 (2013) 112e118

Contents lists available at SciVerse ScienceDirect

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

Original article

Indazoles as potential c-met inhibitors: Design, synthesis and molecular docking studies Lianbao Ye a,1, Xiaomin Ou a, Yuanxin Tian b,1, Bangwei Yu a, Yan Luo a, Binghong Feng a, Hansen Lin a, Jiajie Zhang b, *, Shuguang Wu b, * a b

Medicinal Chemistry Department, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong, China School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, Guangdong, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 December 2012 Received in revised form 31 March 2013 Accepted 2 April 2013 Available online 29 April 2013

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is considered as an attractive target for small molecule drug discovery. In this study, a series of indazoles were designed, synthesized and evaluated as novel c-Met inhibitors. The results showed that the majority of the compounds exhibited significant inhibition on c-Met and compound 4d showed highest activity against c-Met with IC50 value of 0.17 mM in TR-FRET-based assay and IC50 value of 5.45 mM in cellbased assay as compared to other tested compounds. Molecular docking experiments verified the results and explained the molecular mechanism of pretty activities to c-Met. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: c-Met inhibitor Indazole Bioisosterism principles Molecular docking Suzuki coupling reaction Isomer

1. Introduction c-Met has been shown to be deregulated and associated with high tumor grade and poor prognosis in a number of human cancers. c-Met can become activated by a variety of mechanisms, including gene amplification and mutation inducing motility, invasiveness and tumor genicity into the transformed cells. Activation leads to various signaling cascades that induce survival, as well as mitogenic and motogenic responses [1]. Targeting the ATP binding site of c-Met is a popular strategy for inhibition of the kinase, many small molecules selectively targeting the ATP binding site of c-Met kinase have been identified and exerted significant therapeutic effects in treating human cancers clinically [2]. Impressive numbers of c-Met kinase inhibitors have been developed over the past 10 years and a number of novel therapeutic agents that target the c-Met/HGF pathway have been tested in early-phase clinical studies with promising results [3e7]. Currently, the different binding modes of the available c-Met inhibitors have

* Corresponding authors. Tel.: þ86 20 62789415; fax: þ86 20 61648548. E-mail addresses: [email protected] (L. Ye), [email protected] (Y. Tian), [email protected] (J. Zhang), [email protected] (S. Wu). 1 These authors contributed equally to this work. 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.04.004

implications for specificity and activity, but the body of data available remains incomplete [8,9]. Several pharmaceutical companies including Pfizer, SGX, Sugen, Bayer and formerly Pharmacia, have or have had also ongoing programs in this area. SGX’s SGX-523 (2) is a potent c-Met inhibitor which was withdrawn from Phase I clinical trial due to a reversible acute renal failure in one dose group caused by a metabolite compound crystallizing in the kidney [10], and Pfizer’s PF04217903 (1) and Janssen’s JNJ-33377605 (3) are currently in Phase I clinical trials [11,12]. In our previous study, we designed and synthesized spiro[indoline-3,40 -piperidine]-2-ones and 2,3,4,5tetrahydro-1H-pyrido[4,3-b]indoles as potent c-Met inhibitors using compounds 1 and 3 as lead compounds [13,14], but the synthetic construction of these compounds requires a number of synthetically challenging manipulations and the raw material is relatively expensive. In an ongoing effort to discover novel c-Met inhibitors and build diversification of chemical library, we continued to develop ten structurally relevant novel compounds 4ae4e and 5ae5e by replacing 3H-[1,2,3]triazolo[4,5-b]pyrazine of 1, 3H-[1,2,4]triazolo [4,3-b]pyridazine of 2 and 3 with cheap and readily available indazole moiety (Figs. 1 and 2), and investigated their inhibitions on c-Met, verified the results and explained the molecular mechanism of pretty activities to c-Met through molecular docking experiments. The synthetic route for indazoles

L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118

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HO

N N

N

N

R

N

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N N

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N N

1 4a-4e

F

N N

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F N

+

R

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N N

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N

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2 N N

N

S N

N

N

5a-5e

N N

3 Fig. 1. Design of compounds.

was convenient for raw materials availability and industrial manufacture.

gave information about carbon atoms and all the Mþ ion peaks corresponding to molecular weight of confirmed novel compounds.

2. Results and discussions

2.2. Evaluation of biological activity

2.1. Chemistry

The results of anti-c-Met enzyme activities in vitro showed that all the title compounds were active against c-Met enzyme to some extent. Among the compounds tested, most of them showed higher activity. Noticeably, the IC50 values of 4ae4e were, lower than those of their isomers 5ae5e against the c-Met enzyme in vitro, in which 4d had the best inhibitory effect with IC50 of 0.17 mM. The IC50 values derived from in vitro screening studies revealed that all the compounds posed moderate cytotoxicity against MKN-45 cell lines (Table 1). In accordance with the results of c-Met enzyme, Compounds 4ae4e, 5b and 5e showed higher inhibition against MKN45 cell lines with IC50 values of 12.86, 14, 6.72, 11, 5.45,12 and 13.42 mM respectively. Compound 4e was found to show highest activity against MKN-45 cell line with an IC50 value of 5.45 mM. These results indicated that indazoles could afford good potency against activity, so we could elementary make sure that designed compounds should be well worth studying based on molecular docking theory and preliminary biological tests. Further research works on activities are currently under investigation and will be reported in due course.

The target compounds 4ae4e and 5ae5e were prepared as shown in Scheme 1. The 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)pyrazole groups (7e9) reacted with 6-bromo-1H-indazole (6) to obtain 6-(1-methyl-pyrazol-3-yl)-1H-indazole (10), 6-[1-(2tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole (11), and 6-{1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]1H-pyrazol-4-yl}-1H-indazole (12) with yields of 68%, 55%, and 65% respectively via Suzuki coupling reaction. This coupling reaction with pinacol borane esters gave good yields under basic conditions using DMF/H2O as a solvent and Pd (PPh3)4 as a catalyst and provided a range of applicability [15]. The indazole compounds 10, 11, and 12 reacted with commercially available 6-chloromethylquinoline to give three pairs of isomers 4a and 5a, 4b and 5b, 4d and 5d as a result of two isomeric forms of indazole. Then 4b and 5b, 4d and 5d transformed to 4c and 5c, 4e and 5e by hydrolysis. Due to the distinct difference in polarity between isomers allowed them to be separated easily by silica gel column chromatography. Using this producer, two series of compounds (4ae4e and 5ae5e) were synthesized in overall yield of 17.2e26%. A systematic study of the indazole scaffold revealed that the 6position would be suitable for further potency optimization studies. It was found that a range of substituents could be accommodated at this position. The derivatives were characterized by 1H NMR, 13C NMR, elemental analyses and ESI-MS. The analytical data for target compounds can be seen from Experimental and showed that the characteristic signals of 1H NMR for indazole were 7.20e8.10 ppm, 7.60e 8.90 ppm for quinoline, and w5.70 ppm for methane between indazole and quinoline, and substituents data of 6-position in indazole was consistent with the reference. 13C NMR and ESI-MS

2.3. Docking studies Docking experiments indicated that our synthesized compounds adopted a U-shape binding mode with the active site of cMet (shown in Fig. 3), which was the typical conformation of type-I c-Met inhibitors [2]. A direct hydrogen bond was formed between the backbone NH of Met1160 and the quinoline-N, other notable interaction was pep stacking interaction between the indazole group core with Tyr1230 and Met1211. Comparing 5ae5e, the methylene of 4ae4e could better match the sub hydrophobic pocket composed of Val1109, Lys1110, Ala1226 and Val1092 and enhanced the pep interaction. Therefore compounds 4ae4e showed more potent activities than 5ae5e. The binding modes of

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H3C N

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Si O

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HO N

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N

5e Fig. 2. Novel indazoles.

4b and 5b were shown in Fig. 4. Both of them formed two hydrogen bonds with Asn1167 and Arg1166 except the hydrogen bonds composed of Met1160 and quinoline group. The interaction extruded 5a to make methylene match the sub hydrophobic pocket, and then promoted the pep interaction from indazole group and Tyr1230, so the 5b also had potent activity. The same phenomena appeared in compounds 4e and 5e. The activities of 4a and 5a were the lowest in their own series. The docking studies suggested that their short adjacent hydrophilic ring pyrazole could not match the active sites exactly to stabilize the conformation. So we deduced that the 1-position substituent of indazole was better for the Ushape conformation and would enhance activity through the pep interaction. Moreover the hydrophilic group interacted with residue of adjacent solvent area outside pocket could increase the activity, such as 4d and 4b. 3. Conclusions In conclusion, our strategy to replace 3H-[1,2,3]triazolo[4,5-b] pyrazine of 1, 3H-[1,2,4]triazolo[4,3-b]pyridazine of 2 and 3 with indazole moiety was successful in leading to compounds, which demonstrated inhibition of c-Met kinase activity. These compounds would be useful as lead compounds of developing c-Met inhibitors.

4. Experimental part All chemicals were obtained from Aladdin or J&K. Solvents were purified and dried by standard procedures, and stored over 3- A molecular sieves. Reactions were followed by TLC using SILG/UV 254 silica-gel plates. Flash chromatography (FC): silica gel (SiO2; 40 mm, 230e400 mesh). 1H NMR and 13C NMR spectra: Bruker Digital NMR Spectrometer, rep. d in parts per million (ppm), J in hertz (Hz). EI-MS: Waters ZQ4000. 4.1. Synthesis of compounds 4.1.1. Synthesis of 10 In a three-necked round bottom flask (50 ml) equipped with a condenser and magnetic stirring bar, Pd(PPh3)4 (11.6 mg, 0.01 mmol) was added portion wise to a solution of the 6-bromo1H-indazole (6; 1.97 g, 10 mmol), 1-methyl-4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)pyrazole (7; 2.50 g, 12 mmol), K2CO3 (4.145 mg, 30 mmol) in DMF/H2O (4/1, 15 ml), and nitrogen was bubbled through the mixture for 5 min. Then, the mixture was stirred for 18 h at 80  C (LCeMS control), then cooled to r.t., H2O (10 ml) was added, and the mixture was extracted with CH2Cl2 (3  30 ml). The org. layer was dried (1 g of Na2SO4), concentrated

L. Ye et al. / European Journal of Medicinal Chemistry 65 (2013) 112e118

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O Si O

R=

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O

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O

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Si O

4c 4e

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O

O

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Si O

c c

5c 5e

Scheme 1. Synthesis of 4ae4e and 5ae5e. Reagents and conditions: (a) Pd(PPh3)4, DMF/H2O, N2,K2CO3, 80  C, 18 h; (b) 6-chloromethylquinoline, KOH, THF, r.t., 2 h; (c) 4 M HCl, 1,4dioxane, THF, r.t., 1 h.

to obtain crude product, which was purified by FC with MeOH/ CH2Cl2. 4.1.2. Synthesis of 11 and 12 Compounds 11 and 12 were prepared in analogy to 10. 4.1.3. Synthesis of 4a and 5a In a double-necked round bottom flask (100 ml) equipped with magnetic stirring bar, a mixture of 10 (39.6 mg, 0.2 mmol), 6chloromethylquinoline (53.3 mg, 0.3 mmol), and THF (5 ml) was stirred for 2 h in nitrogen at r.t. (TLC control). The solvent was removed under reduced pressure, and the crude product was purified by FC with MeOH/CH2Cl2 to obtain the target products 4a and 5a.

4.1.6. Synthesis of 4c In a three-necked round bottom flask (50 ml) equipped with a condenser and magnetic stirring bar, a solution of 4 M HCl in 1,4dioxane was added portion wise to a solution of 4b (45.4 mg, 0.1 mmol) in THF (3 ml), the mixture was stirred for 1 h at r.t., and the solvent was removed under reduced pressure, 10% NaOH (5 ml) was added, the mixture was extracted with CH2Cl2 (3  5 ml). The organic layer was dried (1 g of Na2SO4) and concentrated to afford the crude product, which was purified by FC with MeOH/CH2Cl2. 4.1.7. Compounds 5c, 4e and 5e were prepared in analogy to 4c. 4.1.7.1. 6-(1-Methyl-pyrazol-3-yl)-1H-indazole (10). Yield: 68%: mp: 213.1e214.7  C; 1H NMR (400 MHz, CDCl3): d ¼ 3.97 (s, 3H),

4.1.4. Synthesis of 4b and 5b Compounds 4b and 5b were prepared in analogy to 4a and 5a. 4.1.5. Synthesis of 4d and 5d Compounds 4d and 5d were prepared in analogy to 4a and 5a.

Table 1 Inhibition of 4ae4e and 5ae5e on c-Met and MKN-45. Compound

c-Met IC50 (mM)

MKN-45 IC50 (mM)

4a 4b 4c 4d 4e 5a 5b 5c 5d 5e

0.42 0.29 0.38 0.17 0.64 37.00 0.45 0.83 19.00 0.58

12.86 14.00 6.72 11.00 5.45 105.60 12.00 110.00 1300.00 13.42

Fig. 3. Compact U-shaped binding modes of all compounds (PDB code: 3dkf).

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4.1.7.5. 1-(Quinolin-6-ylmethyl)-6-[1-(2-tetrahydro-2H-pyran-2yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole (4b). Yield: 38%: mp: 284.3e285.8  C; 1H NMR (400 MHz, CDCl3): d ¼ 1.45e1.79 (m, 6H), 3.45 (m, 1H), 3.67 (m, 1H), 3.81 (m, 1H), 4.11 (m, 1H), 4.36e4.39 (m, 2H), 4.56 (t, J ¼ 3.6 Hz, 1H), 5.78 (s, 2H), 7.27 (dd, J ¼ 1.6, 8.8 Hz, 1H), 7.45 (dd, J ¼ 4.0, 8.0 Hz, 1H), 7.64 (dd, J ¼ 1.2, 8.8 Hz, 1H), 7.66 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.71 (s, 1H), 7.80 (dd, J ¼ 0.8, 2.0 Hz, 1H), 7.83 (dd, J ¼ 0.8, 11.6 Hz, 1H), 7.95 (d, J ¼ 0.8 Hz, 1H), 8.16e8.18 (m, 2H), 8.92 ppm (dd, J ¼ 2.0, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 21.1, 26.2, 30.7, 56.4, 61.9, 63.4, 64.1, 106.5, 108.7 (C-7), 119.1, 121.2, 121.7, 123.9 (C-3a), 126.1, 127.2, 128.3, 128.5, 129.1, 130.9, 131.8, 132.9 (C-3), 134.1, 135.4, 136.1, 141.1 (C-7a), 145.2, 149.3 ppm; EI-MS: 454.04 [M þ Hþ]. Anal. Calcd for C27H27N5O2 (453.22): C 71.50, H 6.00, N 15.44. Found: C 71.52, H 5.97, N 15.44.

Fig. 4. Conformations of 4b (gray) and 5b (magenta) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

7.31 (dd, J ¼ 1.2, 8.4, 1H), 7.56 (t, J ¼ 1.2, 1H), 7.67 (s, 1H), 7.73 (d, J ¼ 8.0, 1H), 7.83 (d, J ¼ 0.4, 1H), 8.03 (s, 1H), 10.27ppm (br s, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 44.3, 109.1, 119.3, 121.4, 124.7, 126.3, 127.5, 128.5, 132.1, 134.3, 142.8 ppm; EI-MS: 199.00 [M þ Hþ]. Anal. Calcd for C11H10N4 (198.09): C 66.65, H 5.08, N 28.27. Found: C 66.71, H 5.03, N 28.26. 4.1.7.2. 6-[1-(2-Tetrahydro-2H-pyran-2-yloxyethyl)-1H-pyrazol-4yl]-1H-indazole (11). Yield: 55%: mp: 244.3e246.1  C; 1H NMR (400 MHz, CDCl3): d ¼ 1.47e1.59 (m, 6H), 3.47e1.59 (m, 1H), 3.68 (m, 1H), 3.83 (m, 1H), 4.11 (m, 1H), 4.36e4.39 (m, 2H), 4.57 (t, J ¼ 4.0, 1H), 7.31 (dd, J ¼ 1.2, 8.4, 1H), 7.56 (s, 1H), 7.73 (d, J ¼ 8.4, 1H), 7.81 (d, J ¼ 0.8, 1H), 7.85 (d, J ¼ 0.8, 1H), 8.03 (s, 1H), 10.27 ppm (br s, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 19.1, 25.3, 30.2, 57.1, 62.8, 64.1, 106.6, 108.3, 119.0, 121.2, 123.7, 125.1, 128.3, 128.7, 131.4, 133.6, 142.7 ppm; EI-MS: 313.06 [M þ Hþ]. Anal. Calcd for C17H20N4O2 (312.16): C 65.37, H 6.45, N 17.94. Found: C 65.35, H 6.47, N 17.94. 4.1.7.3. 6-{1-[(1R,4R)-4-(tert-Butyldimethylsilyloxy)cyclohexyl]-1Hpyrazol-4-yl}-1H-indazole (12). Yield: 65%: mp: 237.1e238.5  C; 1H NMR (400 MHz, CDCl3): d ¼ 0.08 (s, 6H), 0.91 (s, 9H), 1.48e1.58 (m, 2H), 1.84e1.94 (m, 2H), 2.00e2.04 (m, 2H), 2.21 (m, 2H), 3.72 (m, 1H), 4.14 (m, 1H), 7.29 (d, J ¼ 8.4, 1H), 7.55 (s, 1H), 7.70 (s, 1H), 7.71 (d, J ¼ 8.4, 1H), 7.82 (s, 1H), 8.04 ppm (s, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 4.6, 19.1, 25.3, 25.4, 32.8, 43.7, 65.1, 107.9, 117.8, 119.6, 123.5, 126.1, 128.2, 128.4, 132.81, 133.2, 143.3 ppm; EI-MS: 397.09 [M þ Hþ]. Anal. Calcd for C22H32N4OSi (396.23): C 66.62, H 8.13, N 14.13. Found: C 66.57, H 8.12, N 14.18. 4.1.7.4. 1-(Quinolin-6-ylmethyl)-6-(1-methyl-pyrazol-3-yl)-1Hindazole (4a). Yield: 23.7 mg (35%): mp: 276.5e277.7  C; 1H NMR (400 MHz, CDCl3): d ¼ 3.93 (s, 3H), 5.79 (s, 2H), 7.29 (dd, J ¼ 0.8, 8.0 Hz, 1H), 7.38 (dd, J ¼ 4.4, 8.4 Hz, 1H), 7.41 (dd, J ¼ 0.8, 2.0 Hz, 1H), 7.57 (d, J ¼ 0.8 Hz, 1H), 7.60 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.61 (s, 1H), 7.74 (dd, J ¼ 1.2, 8.8 Hz, 1H), 7.76 (d, J ¼ 0.8 Hz, 1H), 8.05 (d, J ¼ 0.8 Hz, 1H), 8.07e8.10 (m, 2H), 8.88 ppm (dd, J ¼ 2.0, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 43.1, 61.9 (eCH2e), 108.7 (C-7), 118.6, 121.3, 121.7, 125.4 (C-3a), 126.1, 126.9, 127.6, 128.2, 128.9, 131.3, 132.1, 132.9, 133.8 (C-3), 135.6, 136.1, 140.4 (C-7a), 145.3, 148.7 ppm; EIMS: 340.07 [M þ Hþ]. Anal. Calcd for C21H17N5 (339.15): C 74.32, H 5.05, N 20.63. Found: C 74.26, H 5.10, N 20.64.

4.1.7.6. 1-(Quinolin-6-ylmethyl)-6-[1-(2-hydroxyethyl)-1H-pyrazol4-yl]-1H-indazole (4c). Yield: 36.0 mg (97%): mp: 301.3e303.1  C. 1 H NMR (400 MHz, CDCl3): d ¼ 4.03 (t, J ¼ 4.8 Hz, 2H), 4.28 (t, J ¼ 4.4 Hz, 2H), 5.82 (s, 2H), 7.32 (dd, J ¼ 1.2, 8.0 Hz, 1H), 7.40 (dd, J ¼ 1.2, 2.0 Hz, 1H), 7.52 (m, 1H), 7.61 (s, 1H), 7.70e7.72 (m, 2H), 7.76 (dd, J ¼ 1.2, 8.8 Hz, 1H), 7.81 (d, J ¼ 0.8 Hz, 1H), 8.08 (d, J ¼ 0.8 Hz, 1H), 8.26 (m, 1H), 8.33 (m, 1H), 8.91 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 58.3, 59.4, 63.1, 108.6 (C-7), 118.7, 121.2, 121.5, 123.9 (C-3a), 126.5, 127.2, 128.6, 128.8, 129.1, 130.9, 132.4, 133.1 (C-3), 134.1, 135.4, 136.1, 140.5 (C-7a), 145.3, 149.2 ppm; EI-MS: 370.08 [M þ Hþ]. Anal. Calcd for C22H19N5O (369.16): C 71.53, H 5.18, N 18.96. Found: C 71.53, H 5.21, N 18.94. 4.1.7.7. 1-(Quinolin-6-ylmethyl)-6-{1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1H-pyrazol-4-yl}-1H-indazole (4d). Yield: 40%: mp: 313.2e314.3  C. 1H NMR (400 MHz, CDCl3): d ¼ 0.07 (s, 6H), 0.91 (s, 9H), 1.47e1.56 (m, 2H), 1.81e1.92 (m, 2H), 1.98e2.04 (m, 2H), 2.16e 2.21 (m, 2H), 3.70 (m, 1H), 4.12 (m, 1H), 5.30 (s, 2H), 7.30 (dd, J ¼ 1.2, 8.4 Hz, 1H), 7.39 (dd, J ¼ 4.4, 8.0 Hz, 1H), 7.41 (d, J ¼ 1.2 Hz, 1H), 7.56 (d, J ¼ 1.2 Hz, 1H), 7.61 (dd, J ¼ 2.0, 8.8 Hz, 1H), 7.66 (d, J ¼ 0.8 Hz, 1H), 7.74 (dd, J ¼ 0.8, 8.0 Hz, 1H), 7.77 (d, J ¼ 1.2 Hz, 1H), 8.05 (d, J ¼ 1.2 Hz, 1H), 8.08e8.12 (m, 2H), 8.88 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 5.2, 18.7, 25.8, 26.1, 33.1, 62.3, 65.1, 73.9, 108.5 (C-7), 118.6, 121.3, 121.7, 124.1 (C-3a), 126.7, 127.5, 129.1, 129.5, 129.7, 131.2, 131.9, 132.8 (C-3), 133.9, 135.4, 136.2, 141.0 (C-7a), 144.9, 148.8 ppm; EI-MS: 538.16 [M þ Hþ]. Anal. Calcd for C32H39N5OSi (537.29): C 71.47, H 7.31, N 13.02. Found: C 71.49, H 7.30, N 13.03. 4.1.7.8. 1-(Quinolin-6-ylmethyl)-6-[1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazol-4-yl]-1H-indazole (4e). Yield: 99%: mp: 325.4e 327.1  C; 1H NMR (400 MHz, CDCl3): d ¼ 1.57e1.66 (m, 2H), 1.88e 1.94 (m, 2H), 2.14e2.17 (m, 2H), 2.23e2.26 (m, 2H), 3.77 (m, 1H), 4.23 (m, 1H), 5.89 (s, 2H), 7.34 (dd, J ¼ 1.6, 8.4 Hz, 1H), 7.42 (s, 1H), 7.70e7.74 (m, 2H), 7.79 (d, J ¼ 8.4 Hz, 1H), 7.85e7.88 (m, 2H), 7.99 (d, J ¼ 8.8 Hz, 1H), 8.11 (s, 1H), 8.71 (d, J ¼ 8.0 Hz, 1H), 8.93 (d, J ¼ 8.4 Hz, 1H), 8.98 ppm (d, J ¼ 5.6 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 25.3, 31.7, 62.3, 64.7, 74.1, 108.7 (C-7), 119.1, 121.3, 121.6, 124.1 (C3a), 126.9, 127.4, 128.7, 128.8, 129.3, 131.1, 132.5, 133.4 (C-3), 133.9, 135.7, 135.9, 139.8 (C-7a), 145.3, 149.0 ppm; EI-MS: 424.05 [M þ Hþ]. Anal. Calcd for C26H25N5O (423.21): C 73.74, H 5.95, N 16.54. Found: C 73.75, H 5.94, N 16.53. 4.1.7.9. 2-(Quinolin-6-ylmethyl)-6-(1-methyl-pyrazol-3-yl)-1Hindazole (5a). Yield: 30%: mp: 278.5e280.1  C; 1H NMR (400 MHz, CDCl3): d ¼ 3.96 (s, 3H), 5.78 (s, 2H), 7.26 (dd, J ¼ 1.6, 8.8 Hz, 1H), 7.43 (dd, J ¼ 4.4, 8.4 Hz, 1H), 7.63e7.65 (m, 2H), 7.66 (s, 1H), 7.71 (d, J ¼ 1.2 Hz, 1H), 7.80 (dd, J ¼ 1.6, 2.0 Hz, 1H), 7.82 (d, J ¼ 0.8 Hz, 1H), 7.95 (d, J ¼ 0.8 Hz, 1H), 8.13e8.15 (m, 2H), 8.92 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 42.7, 60.9, 108.5 (C7), 118.6, 121.1 (C-3a), 121.7, 122.8 (C-3), 124.6, 126.1, 126.9, 127.6,

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128.2, 128.9, 131.5, 132.1, 133.8, 135.5, 136.0, 145.3, 148.3 (C-7a), 149.1 ppm; EI-MS: 340.07 [M þ Hþ]. Anal. Calcd for C21H17N5 (339.15): C 74.32, H 5.05, N 20.63. Found: C 74.31, H 5.07, N 20.62. 4.1.7.10. 2-(Quinolin-6-ylmethyl)-6-[1-(2-tetrahydro-2H-pyran-2yloxyethyl)-1H-pyrazol-4-yl]-1H-indazole (5b). Yield: 32%: mp: 287.1e288.6  C; 1H NMR (400 MHz, CDCl3): d ¼ 1.42e1.72 (m, 6H), 3.42 (m, 1H), 3.63 (m, 1H), 3.79 (m, 1H), 4.08 (m, 1H), 4.35 (dt, J ¼ 1.2, 4.8 Hz, 2H), 4.54 (t, J ¼ 3.6 Hz, 1H), 5.80 (s, 2H), 7.31 (dd, J ¼ 1.6, 8.4 Hz, 1H), 7.41 (dd, J ¼ 4.0, 8.4 Hz, 1H), 7.42 (d, J ¼ 1.2 Hz, 1H), 7.61 (d, J ¼ 1.6 Hz, 1H), 7.64 (dd, J ¼ 2.0, 6.0 Hz, 1H), 7.75 (dd, J ¼ 0.8, 8.4 Hz, 1H), 7.78 (dd, J ¼ 0.8, 5.2 Hz, 1H), 8.06 (d, J ¼ 0.8 Hz, 1H), 8.11e 8.14 (m, 2H), 8.89 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 19.8, 25.3, 30.4, 56.6, 61.9, 62.8, 64.1, 107.3, 107.9 (C7), 119.1, 120.9, 121.3, 122.9 (C-3), 124.5 (C-3a), 126.1, 126.8, 128.3, 128.7, 129.1, 131.7, 132.2, 133.8, 134.9, 136.1, 145.1, 148.4 (C-7a), 149.1 ppm; EI-MS: 454.04 [M þ Hþ]. Anal. Calcd for C27H27N5O2 (453.22): C 71.50, H 6.00, N 15.44. Found: C 71.52, H 5.97, N 15.44. 4.1.7.11. 2-(Quinolin-6-ylmethyl)-6-[1-(2-hydroxyethyl)-1H-pyrazol4-yl]-1H-indazole (5c). Yield: 98%: mp: 304.7e306.3  C; 1H NMR (400 MHz, CDCl3): d ¼ 4.06 (t, J ¼ 4.8 Hz, 2H), 4.30 (t, J ¼ 4.8 Hz, 2H), 5.80 (s, 2H), 7.26 (dd, J ¼ 1.6, 8.0 Hz, 1H), 7.50 (m, 1H), 7.66 (dd, J ¼ 1.2, 8.8 Hz, 1H), 7.69 (d, J ¼ 8.4 Hz, 1H), 7.73e7.74 (m, 2H), 7.81 (dd, J ¼ 1.2, 2.8 Hz, 1H), 7.87 (d, J ¼ 0.8 Hz, 1H), 7.97 (d, J ¼ 0.8 Hz, 1H), 8.23 (m, 2H), 8.94 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 58.1, 59.3, 61.2, 109.1 (C-7), 119.2, 121.1, 121.5, 123.1 (C-3), 124.5 (C-3a), 126.2, 126.9, 128.4, 128.9, 129.2, 131.7, 132.2, 134.1, 135.0, 135.9, 145.3, 148.5 (C-7a), 149.3 ppm; EIMS: 370.01 [M þ Hþ]. Anal. Calcd for C22H19N5O (369.16): C 71.53, H 5.18, N 18.96. Found: C 71.49, H 5.19, N 18.97. 4.1.7.12. 2-(Quinolin-6-ylmethyl)-6-{1-[(1R,4R)-4-(tert-butyldimethylsilyloxy)cyclohexyl]-1H-pyrazol-4-yl}-1H-indazole (5d). Yield: 31%: mp: 311.2e312.5  C; 1H NMR (400 MHz, CDCl3): d ¼ 0.08 (s, 6H), 0.90 (s, 9H), 1.45e1.54 (m, 2H), 1.79e1.91 (m, 2H), 1.97e2.03 (m, 2H), 2.14e2.20 (m, 2H), 3.68 (m, 1H), 4.03 (m, 1H), 5.28 (s, 2H), 7.24 (dd, J ¼ 1.6, 8.8 Hz, 1H), 7.41 (dd, J ¼ 4.4, 8.4 Hz, 1H), 7.62e7.65 (m, 2H), 7.63 (s, 1H), 7.70 (d, J ¼ 1.2 Hz, 1H), 7.78 (dd, J ¼ 1.6, 2.0 Hz, 1H), 7.80 (d, J ¼ 0.8 Hz, 1H), 7.91 (d, J ¼ 0.8 Hz, 1H), 8.11e8.14 (m, 2H), 8.91 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13C NMR (75 MHz, (D6) DMSO): d ¼ 4.9, 17.8, 24.8, 25.7, 32.5, 60.1, 63.9, 73.9, 108.1 (C-7), 118.2, 120.9, 121.3, 123.0 (C-3), 124.2 (C-3a), 126.1, 126.7, 128.5, 128.9, 129.2, 131.6, 132.1, 133.8, 135.2, 135.9, 144.7, 148.1 (C-7a), 149.2 ppm; EI-MS: 538.09 [M þ Hþ]. Anal. Calcd for C32H39N5OSi (537.29): C 71.47, H 7.31, N 13.02. Found: C 71.46, H 7.30, N 13.04. 4.1.7.13. 2-(Quinolin-6-ylmethyl)-6-[1-((1R,4R)-4-hydroxy-cyclohexyl)-1H-pyrazol-4-yl]-1H-indazole (5e). Yield: 99%: mp: 327.7e 329.3  C; 1H NMR (400 MHz, CDCl3): d ¼ 1.59e1.67 (m, 2H), 1.90e 1.95 (m, 2H), 2.16e2.19 (m, 2H), 2.25e2.28 (m, 2H), 4.12 (m, 1H), 4.27 (m, 1H), 6.01 (s, 2H), 7.28 (dd, J ¼ 1.6, 8.8 Hz, 1H), 7.47 (dd, J ¼ 4.4, 8.4 Hz, 1H), 7.64e7.69 (m, 2H), 7.67 (s, 1H), 7.78 (d, J ¼ 1.2 Hz, 1H), 7.81 (dd, J ¼ 1.6, 2.0 Hz, 1H), 7.87 (d, J ¼ 0.8 Hz, 1H), 8.03 (d, J ¼ 0.8 Hz, 1H), 8.13e8.16 (m, 2H), 8.94 ppm (dd, J ¼ 1.6, 4.4 Hz, 1H); 13 C NMR (75 MHz, (D6) DMSO): d ¼ 25.3, 31.3, 61.1, 63.7, 72.1, 108.2 (C-7), 118.9, 120.9, 121.4, 123.3 (C-3), 124.1 (C-3a), 125.9, 126.8, 128.4, 128.9, 129.2, 131.6, 132.4, 134.3, 135.1, 136.2, 145.4, 148.6 (C7a), 149.4 ppm; EI-MS: 424.05 [M þ Hþ]. Anal. Calcd for C26H25N5O (423.21): C 73.74, H 5.95, N 16.54. Found: C 73.74, H 5.93, N 16.55. 4.2. c-Met kinase assay The c-Met kinase activities were evaluated according to a published literature [3]. IC50 values for inhibitors of c-Met were

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determined using an IMAP Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay. 50 nM 6 His-tagged recombinant human c-Met residues 974-end (Millipore) was incubated in 20 mM Tris, 10 mM MgCl2, 2.5 mM MnCl2, 0.01% Tween 20 and 2 mM DTT with 5 mM ATP and 200 nM 5FAM-KKKSPGEYVNIGFG-NH2 in a total volume of 25 ml for 60 min at ambient temperature. Inhibitors were tested at 10 concentrations starting from 20 mM at a final concentration of 1% DMSO. The reaction was stopped by addition of 50 ml of IMAP stop solution containing 60% buffer A:40% buffer B and a 1 in 400 dilution of beads and Terbium reagent. Plates were read after an overnight incubation at 4  C on an Analyst HT reader. Reported IC50s are from a minimum of 2 experiments (n ¼ 2). Data analysis was carried out using a four parameter curve fit. The standard errors of the mean were calculated and expressed as a percentage of the mean IC50. The average for this value was 12%. 4.3. Cell assay MKN-45 cells (purchased from Riken Cell Bank of Japan) were seeded in RPMI 1640 medium containing 10% FCS and pretreated for 1 h at 37  C with increasing concentrations of compound or DMSO. Cells were then lysed in RIPA buffer containing appropriate inhibitors. Western blotting was carried out following standard procedures and probed with the following antibodies: antiphosphor c-Met (pYpYpY1230/1234/1235), anti-c-Met (Cell Signaling Technologies Inc.), following the manufacturer’s protocol. Total cellular protein loadings were semi-quantified by probing with anti-GAPDH. 4.4. Molecular docking Molecular docking was performed with Surflex-Dock program that is interfaced with Sybyl 7.3. The programs adapted an empirical scoring function and a patented searching engine [16,17]. Ligand was docked into the corresponding protein’s binding site guided by protomol, which is an idealized representation of a ligand that makes every potential interaction with binding site. In this work, the protomol was established by ligand from the crystal structure of c-Met (PDB entry: 3dkf). Beginning of docking, all the water and ligands were removed and the random hydrogen atoms were added. Then the receptor structure was minimized in 10,000 cycles with Powell method in Sybyl 7.3. All the compounds were constructed using Sketch Molecular module. Hydrogen and GasteigereHückel charges were added to every molecule. Then their geometries are optimized by conjugate gradient method in TRIPOS force field. The energy convergence criterion is 0.001 kcal/mol. Default values are chosen to finish this work except that the threshold was 1 when the protomol was generated. Acknowledgments This study was supported by grants from Guangdong Strategic Emerging Project of Guangdong Provincial Department of Science and Technology (no. 2012A080800012), International Science and Technology Cooperation Base of Guangdong Provincial Department of Science and Technology (no. 2009B050900006) and Science and Technology Bureau of Guangzhou (no. 2009A1-E011-8 and no. 2010V1-E00531-3). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.ejmech.2013. 04.004. These data include MOL files and InChiKeys of the most important compounds described in this article.

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