Discovery of Novel c-Met Inhibitors Bearing a 3-Carboxyl Piperidin-2-one Scaffold

A series of compounds containing a novel 3-carboxypiperidin-2-one scaffold based on the lead structure BMS-777607 were designed, synthesized and evaluated for their c-Met kinase inhibition and cytotoxicity against MKN45 cancer cell lines. The results indicated that five compounds exhibited significant inhibitory effect on c-Met with IC50 values of 8.6−81 nM and four compounds showed potent inhibitory activity against MKN45 cell proliferation, with IC50s ranging from 0.57−16 μM.


Introduction
c-Met kinase is a transmembrane receptor tyrosine kinase (RTK). Upon binding of its endogenous ligand hepatocyte growth factor (HGF, also known as scatter factor, SF), c-Met receptor undergoes dimerization and in turn triggers signal transducers to mediate a variety of cellular responses such as cell growth, invasion, migration and survival [1,2]. The normal c-Met/HGF pathway plays an important role in embryogenesis and wound healing, but aberrant forms of this pathway (for example, as a result of overexpression of c-Met and HGF) have frequently been observed in a variety of human solid tumors and hematologic malignancies. Importantly, both increased levels of c-Met and HGF have OPEN ACCESS been associated with poor clinical outcomes [3][4][5]. Therefore, c-Met has been pursued as an attractive anticancer drug target for the past two decades [6,7]. Several approaches to inhibition of the HGF/c-Met pathway in cancer cells have been reported, such as antagonistic ligands to c-Met, antibodies against HGF or c-Met, and small molecule c-Met inhibitors [8][9][10].
During the development of small molecular c-Met kinase inhibitors, a compound disclosed by Kirin Brewery Company in 2003 [11] could be regarded as a milestone ( Figure 1). Structurally, this compound (1) is composed of four moieties: a phenyl group (moiety A), a bridge moiety B, an ortho-fluoro phenol and a 6,7-dimethoxyquinoline. Initiated by this discovery, numerous c-Met kinase inhibitors bearing diverse chemical scaffolds have been reported. Generally, structural optimization based on compound 1 mainly focused on moiety D and B. Replacement of the 6,7-dimethoxyquinoline moiety by various N-containing heterocycles, such as substituted quinoline [12], thienopyridine [13][14][15], pyrrolopyridine [16], aminopyridine [17], thienopyrimidine [18], furopyrimidine [18], imidazopyridine [19] or imidazopyridazine [19], has been investigated. The bridge moiety B connecting moiety A and C was designed as linear [20][21][22] or cyclic [14,15,[23][24][25][26], bearing at least one amide bond with 5-atoms in the main chain [22,24] (i.e., six chemical bonds distance between moiety A and C, Figure 1). However, there are little changes to moiety A and C, except for phenyl ring or substituted phenyl ring modifications to the former. A good example for these inhibitors is BMS-777067, which is now in phase 2 trial because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles [17]. Taking BMS-777607 as leading compound, the design and synthesis of new derivatives with novel structures are under study in our laboratory. Preliminary investigation indicated that 3-carboxypiperidin-2-one is a promising scaffold for the design of new c-Met inhibitors. Herein we would like to report our efforts in this respect ( Figure 2).

Chemistry
As shown in Scheme 1, saponification of isobutyl ester 2 with lithium hydroxide gave the piperidinone 3-carboxylic acid 3, which could be further brominated giving compound 4 in 92% yield. On the other hand, deprotonation of compound 2 with sodium hydride, followed by treatment with an alkyl halide (MeI, EtBr, or n-BuBr) led to the corresponding α-substituted piperidinones. Saponification of these esters 5a-c gave the corresponding carboxylic acids smoothly. In this way, we had five carboxylic acids (compounds 3, 4, 6a-c) in hand, which would be used in next coupling step. Scheme 1. Synthesis of the piperidinone 3-carboxylic acids 3, 4 and 6a-c.

Evaluation of Biological Activity
As illustrated in Table 1, all of the compounds bearing a 3-carboxypiperidin-2-one scaffold exhibit potent c-Met kinase inhibition activity. However, compounds lacking an α-substituent group (15a, 17a, 18a, 19a, 20a) only showed much less potent anti-c-Met kinase activity. When the α-proton was substituted by chlorine, the activity generally increased (cf. 15b vs. 15a, 16b vs. 16a, 20b vs. 20a). When alkyl groups (Me, Et, or n-Bu) were introduced to this position, the inhibitory effects were greatly enhanced (20c, 20d and 20e vs. 20a). Among these three derivatives, the smallest methyl group was the most favorable among the compounds exerting inhibitory activity against c-Met kinase activity and c-Met-driven cell proliferation. Generally, 6,7-dimethoxyquinoline -containing analogues showed more potency than the pyrropyridine, pyrimidine, or aminopyrimidine counterparts (20b vs. 15b, 16b, 17b, 18b, 19b) according to the biological activity results. The most potent analogue 20b exhibited significant potency against c-Met kinase and c-Met-driven MKN45 cell proliferation, with IC 50 values of 8.6 nM and 0.57 μM, respectively. Other three analogues 20c-e with alkyl substitution in the piperidone moiety are also promising, showing inhibitory activity against c-Met enzymatic activities with the IC 50 s of 11.2~64.0 nM and inhibiting MKN45 cell proliferation with IC 50 s of 0.65~16.0 μM, individually.

Molecular Modeling
To further elucidate the binding mode of compounds, docking analysis was performed. In our study, the co-crystal structure of BMS-777607 with c-Met kinase (PDB ID:3F82) was selected as the docking model. The inhibitor was docked using the GLIDE docking algorithm [27] in the XP (extra precision) mode. A binding model for (R)-20b in the ATP binding site is presented in Figure 3a. The resulting model successfully identifies key hydrogen bond interaction and hydrophobic interactions between the ligands and residues of the protein's ATP binding pocket. The carbonyl oxygen of the 3-carboxypiperidin-2-one and the nitrogen atom of the quinoline ring in 20b formed hydrogen bonding interactions with Asp1222 and Met1160, respectively. π-π Interactions were formed between the phenyl ring (moiety C) and Phe1223. In addition, hydrophobic interactions were formed between the phenyl ring (moiety A) in 20b and Phe1134, Phe1200. A binding model for (S)-20b in the ATP binding site is presented in Figure 3b. However, this compound failed to dock into the binding pocket, as the orientation of the ligand in the binding model was opposite to that of BMS-777607. Therefore, we postulate that the requisite chirality for these compound may be the R-configuration. We are now seeking an efficient route to access the enantiomers, and the optical pure compounds will be synthesized and evaluated in the due course.

General Information
All chemical reagents were used as supplied unless indicated. Solvents used in organic reactions were distilled under an inert atmosphere. Unless otherwise noted, all reactions were carried out at room temperature and performed under a positive pressure of argon. Flash column chromatography was performed on silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd, Qingdao, China). Analytical thin layer chromatography (TLC) was performed on glass plates pre-coated with a 0.25 mm thickness of silica gel. 1 H-NMR and 13 C-NMR spectra were taken on a Jeol JNM-ECP 600 spectrometer (Jeol Ltd., Tokyo, Japan) at room temperature. Chemical shifts of the 1 H-NMR spectra are expressed in ppm relative to the solvent residual signal 7.26 in CDCl 3 or to tetramethylsilane (δ = 0.00). Chemical shifts of the 13 C-NMR spectra are expressed in ppm relative to the solvent signal 77.00 in CDCl 3 or to tetramethylsilane (δ = 0.00) unless otherwise noted. Electrospray (ESI) mass spectra were recorded on a Global Q-TOF mass spectrometer (Waters, Wilford, MA, USA).

Preparation of 16a and 16b
To amide 15a or 15b (0.2 mmol) in ethyl acetate (2 mL), acetonitrile (2 mL), and water (1 mL) at 0 °C was added iodobenzene diacetate (82 mg, 0.26 mmol). After stirring at room temperature for 2 h, saturated NaHCO 3 (3 mL) was added, followed by 30 mL of ethyl acetate. The mixture was filtered, and the filtrate was washed with brine (3 × 5 mL), dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel to give compounds 16a-b.

c-Met Kinase Assay
The effects of indicated compound on the activities of c-Met kinases were determined using enzyme-linked immunosorbent assays (ELISAs) with purified recombinant proteins. Briefly, 20 μg/mL poly (Glu,Tyr) 4:1 (Sigma, St. Louis, MO, USA) was pre-coated in 96-well plates as a substrate. A 50-μL aliquot of 10 μmol/L ATP solution diluted in kinase reaction buffer (50 mmol/L HEPES [pH 7.4], 50 mmol/L MgCl 2 , 0.5 mmol/L MnCl 2 , 0.2 mmol/L Na 3 VO 4 , and 1 mmol/L DTT) was added to each well; 1 μL of various concentrations of indicated compound diluted in 1% DMSO (v/v) (Sigma) were then added to each reaction well. DMSO (1%, v/v) was used as the negative control. The kinase reaction was initiated by the addition of purified c-Met tyrosine kinase proteins diluted in 49 μL of kinase reaction buffer. After incubation for 60 min at 37 °C, the plate was washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T-PBS). Anti-phosphotyrosine (PY99) antibody (100 μL; 1:500, diluted in 5 mg/mL BSA T-PBS) was then added. After a 30-min incubation at 37 °C, the plate was washed three times, and 100 μL horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000, diluted in 5 mg/mL BSA T-PBS) was added. The plate was then incubated at 37 °C for 30 min and washed 3 times. A 100-μL aliquot of a solution containing 0.03% H 2 O 2 and 2 mg/ml o-phenylenediamine in 0.1 mol/L citrate buffer (pH 5.5) was added. The reaction was terminated by the addition of 50 μL of 2 mol/L H 2 SO 4 as the color changed, and the plate was analyzed using a multi-well spectrophotometer (SpectraMAX 190, Molecular Devices, Sunnyvale, CA, USA) at 490 nm. The inhibition rate (%) was calculated using the following equation: [1 − (A490/A490 control)] × 100%. The IC 50 values were calculated from the inhibition curves in two separate experiments.

Cell Proliferation Assay
Cells were seeded in 96-well tissue culture plates. On the next day, the cells were exposed to various concentrations of compounds and further cultured for 72 h. Cell proliferation was then determined using sulforhodamine B (SRB, Sigma, St. Louis, MO, USA). The IC 50 values were calculated by concentration-response curve fitting using the four-parameter method.

Conclusions
In summary, a series of compounds based upon the 3-carboxylpiperidin-2-one scaffold were designed, synthesized and evaluated for their c-Met kinase inhibition and cytotoxicity against MKN45 cancer cell lines. Five compounds (16b, 20b-e) exhibited moderate to excellent activity against c-Met kinase, with IC 50 values ranging from 8.6-81 nM. Moreover, four compounds (20b-e) showed potent inhibitory activity against MKN45 cell proliferation, with IC 50 s ranging from 0.57-16 μM. Further structure-activity relationship studies are under way in our laboratory and will be reported in due course.