Design, synthesis and biological evaluation of novel 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 4-oxo-3,4-dihydrophthalazine-1-carboxamide moieties as c-Met kinase inhibitors
Graphical abstract
A series of 6,7-disubstituted-4-phenoxyquinoline derivatives was designed, synthesized and evaluated for their cytotoxicity, enzymatic assays and docking analysis.
Introduction
The receptor tyrosine kinase, c-Met, and its natural ligand, hepatocyte growth factor (HGF), are involved in cell proliferation, migration and invasion, and are essential for normal embryonic development.1 Binding of active HGF ligand to the c-Met extracellular domain causes receptor polymerization and phosphorylation of tyrosine residues in the intracellular c-Met domains.2, 3 Aberrant HGF/c-Met signaling has been identified in a wide range of human malignancies, including bladder, breast, cervical, colorectal, endometrial, gastric, kidney, liver, lung, pancreatic, prostate, and thyroid cancers.4, 5, 6, 7, 8, 9, 10 Therefore, c-Met has gained considerable attention as a potential target for cancer treatment.
In recent years, research has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt HGF/c-Met signaling. Both small-molecule c-Met kinase inhibitors and antibodies targeting c-Met or HGF have exhibited antitumor activities in preclinical models.11, 12, 13, 14, 15, 16, 17, 18, 19 Recently, significant progress has been made on the development of c-Met inhibitors resulting in more than ten candidates have been in clinical trials, including foretinib (1, Phase II), cabozantinib (2, Phase III), as well as several newly developed compounds bearing 6,7-disubstituted-4-phenoxyquinoline frameworks, such as AM7 (3, Preclinical), MG10 (4, Preclinical) and Amgen (5, Preclinical) et al., as the primary pharmacophoric scaffolds (Fig. 1).20, 21, 22, 23, 24
The structure–activity relationships (SARs) of 6,7-disubstituted-4-phenoxyquinoline based inhibitors suggested that quinoline pharmacophores were responsible for forming hydrogen bonds with the backbone of c-Met kinase, and an aryl fragment (moiety B) probably extended into the hydrophobic pocket.25, 26, 27 Apparently, there are two structural characteristics in the linkers between moiety A and moiety B. One is ‘5 atoms regulation’, which means the distance of six chemical bonds exists between moiety A and moiety B; the other is the linker containing both hydrogen-bond donor or acceptor and at least one amide group. In view of above-mentioned results, our research group have introduced different biologically active fragments, such as pyridine, pyrimidine, semicarbazone, quinoline, 1,4-dihydrocinnoline and pyrimidine-2,4,6-trione into the 5-atom linker and the resulting derivatives 6–10 (Fig. 2) showed excellent potency.28, 29, 30, 31, 32, 33, 34
As an extension of our work on the development of novel potent c-Met inhibitors, we noticed that compounds 11–13 (Fig. 3), which contain a 2-phenylphthalazin-1(2H)-one (PHTZ) framework, displayed a multitude of biological activities, including antitumor, anti-autoimmune and anti-inflammatory activities.35, 36, 37 Recently, some PHTZ derivatives have been reported as the core skeleton for the design of potent and selective human A3 adenosine receptor (hA3 AR) antagonists, especially the 2,5-dimethoxyphenylphthalazin-1(2H)-one 12 being the most potent and selective hA3 AR antagonist (Ki = 0.776 nM; hA1/hA3 and hA2A/hA3 >12,000).38 Considering its strong potency and basing on the ‘5 atoms regulation’, we introduced PHTZ as a part of the 5-atom linker to the 6,7-disubstituted-4-phenoxyquinoline moiety via an amide bond, which was called 4-oxo-3,4-dihydrophthalazine-1-carboxamide moiety (Fig. 3).
According to our previous studies, the 3-carbon tether at the 7-position of quinoline was reserved, while the morpholinyl group was replaced by four other water-soluble substituents, including piperidinyl, 4-methyl piperidinyl, pyrrolidinyl, and 4-methyl piperazinyl groups, to observe the effects of the different cyclic tertiary amino groups on activity. Furthermore, various substituents were introduced at the phenyl ring (moiety B) with the aim to explore the effect of substituents on biological activity. Accordingly, we designed a novel series of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 4-oxo-3,4-dihydrophthalazine-1-carboxamide moieties as c-Met kinase inhibitors (Fig. 4).
In the current study, all target compounds were evaluated for their antiproliferative activity in vitro against four cancer cell lines: H460 (human lung cancer), MKN-45 (human gastric cancer), HT-29 (human colon cancer) and MDA-MB-231 (human breast cancer). Additionally, to determine c-Met kinase inhibition and selectivity, the enzymatic assays of several potent compounds were evaluated, and most of them showed promising inhibition. Furthermore, a docking analysis was also performed to elucidate the binding mode of the target compound 33 with c-Met kinase.
Section snippets
Chemistry
The synthesis of the key intermediates of 4-(2-fluorophenoxy)quinolines 21a–e was achieved in 8 steps from commercially available 1-(4-hydroxy-3-methoxyphenyl)ethanone as shown in Scheme 1, which has been illustrated in detail in our previous study28, 29, 30, 31, 32, 33, 34 (see also Supplementary information).
Target compounds 25–59 were prepared as outlined in Scheme 2. The side chains 24a–h were synthesized from naphthalene, which was dissolved in 0.5 N NaOH and refluxed with KMnO4 aq. for 2 h
Cytotoxic activities against tumor cells assay
The antiproliferative activities of compounds 25–59 were evaluated against H460, MKN-45, HT-29 and MDA-MB-231 cell lines by the standard MTT assay in vitro, with foretinib as the positive control.41, 42 The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS). Approximate 4 × 103 cells, suspended in MEM medium, were plated into each well of a 96-well plate and incubated in 5% CO2 at 37 °C for 24 h. The tested compounds at the indicated final
In vitro cytotoxic activities and structure activity relationships
The cytotoxic activities of the target compounds 25–59 were evaluated in H460 (human lung cancer), MKN-45 (human gastric cancer), HT-29 (human colon cancer), and MDA-MB-231 (human breast cancer) cell lines using the MTT assay. Foretinib was used as the positive control. The results were expressed as half-maximal inhibitory concentration (IC50) values and were presented in Table 1, as mean values of experiments performed in triplicate.
As illustrated in Table 1, all the target compounds showed
Binding model analysis
To further elucidate the binding mode of compounds, a docking analysis was performed. In our study, the co-crystal structure of foretinib (GSK1363089) with c-Met kinase was selected as the docking model (PDB ID: 3LQ8). The docking simulation was conducted with Glide XP (Schrödinger 2014), since Glide uses a hierarchical series of filters to search for possible locations of the ligand in the active-site region of the receptor. The shape and properties of the receptor were represented on a grid
Conclusions
In summary, a series of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 4-oxo-3,4-dihydrophthalazine-1-carboxamide moieties were designed, synthesized and evaluated for their biological activity. The preliminary investigation showed that most compounds displayed good to excellent potency against four tested cancer cell lines as compared with foretinib. In particular, the most promising compound 33 (c-Met IC50 = 1.63 nM, a multi-target tyrosine kinase inhibitor) showed the most potent
Experimental
Unless otherwise noted, all materials were obtained from commercial suppliers and were used without further purification. Reactions’ time and purity of the products were monitored by TLC on FLUKA silica gel aluminum cards (0.2 mm thickness) with fluorescent indicator 254 nm. Column chromatography was run on silica gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil,
Acknowledgments
The work was supported by Program for Innovative Research Team of the Ministry of Education of the People’s Republic of China and Program for Liaoning Innovative Research Team in University (IRT1073).
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2019, Bioorganic and Medicinal ChemistryCitation Excerpt :Studies demonstrated that type II inhibitors may be more active than type I inhibitors against the mutations that disrupt the binding sites due to their binding interactions extend to beyond the entrance of the active site of Met.34–38 As shown in Fig. 1, on the whole, the structures of type II c-Met inhibitors can be disconnected into four units (block A–D) according to their interaction modes with target protein.39–41 Among them, the main modification of these type II c-Met inhibitors was focused on the moiety C (a 5-atom linker), which is between the moiety B and the moiety D.
Design, synthesis and biological evaluation of N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide derivatives as novel P-glycoprotein inhibitors reversing multidrug resistance
2019, Bioorganic ChemistryCitation Excerpt :Intermediate 8 was prepared from Naphthalene oxidized by KMnO4. The synthesized 8 was stirred in EtOH with substituted phenyl hydrazine at R.T. for 15 h to afford intermediate 9a–s [15]. Under reflux condition, 9a–s reacted with SOCl2 to produce intermediate 10a–s. 3,4-dimethoxyphenylethylamine and paraformaldehyde were mixed in EtOH and stirred at R.T. for 3 h and then refluxed for 4 h after acidification with concentrated hydrochloric acid to afford intermediate 12.
Recent advances in the discovery of small molecule c-Met Kinase inhibitors
2018, European Journal of Medicinal ChemistryCitation Excerpt :In their research work for development of c-Met kinase inhibitors, the modification was focused on the “5-atom linker”. They had incorporated various biologically active scaffolds like 4-oxo-1,4-dihydroquinoline (102, c-Met IC50 = 1.35 nM, MKN-45 IC50 = 0.010 μM) [167], quinoline (103, c-Met IC50 = 1.32 nM, MKN-45 IC50 = 0.011 μM) [168], semicarbazone (104, c-Met IC50 = 4.3 nM, MKN-45 IC50 = 0.016 μM) [169] (105, c-Met IC50 = 1.4 nM, MKN-45 IC50 = 0.51 μM) [170], pyridine (106, c-Met IC50 = 1.39 nM, MKN-45 IC50 = 0.073 μM) [171], pyrimidine-2,4,6-trione (107, c-Met IC50 = 1.15 nM, MKN-45 IC50 = 0.057 μM) [172], pyridazinone (108, c-Met IC50 = 2.15 nM, MKN-45 IC50 = 0.03 μM) [173], 4-oxo-3,4-dihydrophthalazine (109, c-Met IC50 = 1.63 nM, MKN-45 IC50 = 0.071 μM) [174], pyrazolone (110, c-Met IC50 = 2.20 nM, MKN-45 IC50 = 0.03 μM) [175], 1,2,3-triazole (111, c-Met IC50 = 1.04 nM, MKN-45 IC50 = 0.021 μM) [176] [177], acylhydrazone (112, c-Met IC50 = 1.86 nM, MKN-45 IC50 = 0.37 μM) [178], 4-oxo-1,4-dihydrocinnoline (113, c-Met IC50 = 0.59 nM, MKN-45 IC50 = 0.022 μM) [179], imidazolone (114, c-Met IC50 = 1.42 nM, MKN-45 IC50 = 0.014 μM) [180], 2-oxo-4-chloro-1,2-dihydroquinoline (115, c-Met IC50 = 1.21 nM, MKN-45 IC50 = 0.16 μM) [181], 1,2,4-triazolone (116, c-Met IC50 = 1.57 nM, MKN-45 IC50 = 0.03 μM) [182], 1H-imidazole (117, c-Met IC50 = 1.1 nM, MKN-45 IC50 = 0.22 μM), (E)-3-hydrosulfonylacrylamido motifs (118, c-Met IC50 = 1.52 nM, MKN-45 IC50 = 0.45 μM) [183] [184], benzo[d]thiazole -2-yl urea (119, c-Met IC50 = 17.6 nM, MKN-45 IC50 = 0.06 μM) [185], thiosemicarbazone (120, c-Met IC50 = 8.92 nM, MKN-45 IC50 = 0.71 μM) [186], 3-amino-2-cyano-acrylamide (121, HT-29 IC50 = 0.04 μM, MKN-45 IC50 = 0.39 μM) [187] and 3-oxo-3,4-dihydroquinoxaline (122, c-Met IC50 = 0.90 nM, MKN-45 IC50 = 0.023 μM) [188] in the 5 atom linker to restrict the flexibility. Different human cancer cell lines (MKN-45, A549, H460, U87MG and/or HT-29) were also used for in vitro cytotoxic activity of the synthesized compounds.
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2017, Journal of Molecular Graphics and ModellingCitation Excerpt :And the structure-activity relationships of the c-Met type II inhibitors to c-Met were systematically investigated based on the computationally determined binding modes. The large number of c-Met inhibitors with reported activities [9,15–31] as well as the six c-Met crystal structures (pdb code: 2WGJ, 2WKM, 3ZXZ, 3ZZE, 3LQ8 and 3U6I) [11,12,14,32] in complex with some of these inhibitors gave rise to the possibility to test our methods. Molecular docking was performed using MOE [33].