Design,synthesis and biological evaluation of novel 4-phenoxy-6,7-disubstituted quinolines possessing (thio)semicarbazones as c-Met kinase inhibitors

In continuing our efforts to identify small molecules able to inhibit c-Met kinase, three series of novel 6,7-disubstituted-4-phenoxyquinoline derivatives (23a–w, 26a–d and 30a–d) bearing (thio)semicarbazone scaffold were designed, synthesized and evaluated for their cytotoxicity. The biological data revealed that most compounds exhibited moderate-to-excellent activity against HT-29, MKN-45, A549 cancer cell lines and relative poor potency toward MDA-MB-231 cell as well as hardly any cytotoxicity in normal PBL cell. Eleven compounds were further examined for their inhibitory activity against c-Met kinase and three compounds (23h, 23n and 26a) demonstrated good inhibitory activity. This work resulted in the discovery of a potent c-Met inhibitor 23n, bearing 2-hydroxy-3-allylphenyl group at R² moiety, as a valuable lead molecule, which possessed remarkable cytotoxicity and high selectivity against A549 and HT-29 cell lines with IC₅₀ values of 11 nM and 27 nM. Besides, it displayed excellent c-Met kinase inhibition on a single-digital nanomolar level (IC₅₀ = 1.54 nM). Meanwhile, the results from preliminarily in vivo study reflected that compound 23n showed promising overall PK profiles, consistent with the efficacy in both MKN-45 and HT-29 tumor xenograft mice model. These results clearly indicated that compound 23n is a potent and highly selective c-Met inhibitor and its favorable in vitro and in vivo profiles warrant further investigation.     

Discovery of novel 4-(2-fluorophenoxy)quinoline derivatives bearing 4-oxo-1,4-dihydrocinnoline-3-carboxamide moiety as c-Met kinase inhibitors

A series of novel 4-(2-fluorophenoxy)quinoline derivatives containing 4-oxo-1,4-dihydrocinnoline-3-carboxamide moiety were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase and six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG and SMMC-7721). All the prepared compounds showed moderate to excellent antiproliferative activity, and the analysis of their structure–activity relationships indicated that 2-chloro or 2-trifluoromethyl substituted phenyl group on the 1-position of cinnoline ring was more favorable for antitumor activity. In this study, a promising compound 33, with a c-Met IC₅₀ value of 0.59 nM, was identified as a multitargeted receptor tyrosine kinase inhibitor.     

Discovery of novel 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 5-(aminomethylene)pyrimidine-2,4,6-trione moiety as c-Met kinase inhibitors

A series of novel quinoline derivatives bearing 5-(aminomethylene)pyrimidine-2,4,6-trione moiety were designed, synthesized, and evaluated for their c-Met kinase inhibitory activities and antiproliferative activities against 5 cancer cell lines (HT-29, H460, MKN-45, A549, and U87MG) in vitro. Most compounds showed moderate to excellent potency, with the most promising analogue 45 (c-Met half-maximal inhibitory concentration [IC₅₀] = 1.15 nM) showing high selectivity versus 5 other tyrosine kinases, VEGFR-2, Flt-3, PDGFR-β, c-Kit, and EGFR. Structure–activity relationship studies indicated that electron-donating groups on the phenyl ring at the 3-position of pyrimidine-2,4,6-trione were required to increase the electron density on the 5-(aminomethylene)pyrimidine-2,4,6-trione moiety.     

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

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 c-Met kinase inhibition and cytotoxicity against H460, MKN-45, HT-29 and MDA-MB-231 cancer cell lines in vitro. Most compounds displayed good to excellent potency against four tested cancer cell lines as compared with foretinib. The SAR analyses indicated that compounds with halogen groups, especially fluoro groups at 4-position on the phenyl ring (moiety B) were more effective than those with nitro groups or methoxy groups. In this study, a promising compound 33 (c-Met IC₅₀ = 1.63 nM) was identified, which showed the most potent antitumor activities with IC₅₀ values of 0.055 μM, 0.071 μM, 0.13 μM, and 0.43 μM against H460, MKN-45, HT-29 and MDA-MB-231 cell lines, respectively.     

Target identification,lead optimization and antitumor evaluation of some new 1,2,4-triazines as c-Met kinase inhibitors

In silico target fishing approach using PharmMapper server identified c-Met kinase as the selective target for our previously synthesized compound NCI 748494/1. This approach was validated by in vitro kinase assay which showed that NCI 748494/1 possessed promising inhibitory activity against c-Met kinase (IC₅₀ = 31.70 μM). Assessment of ADMET profiling, drug-likeness, drug score as well as docking simulation for the binding pose of that compound in the active site of c-Met kinase domain revealed that NCI 748494/1 could be considered as a promising drug lead. Based on target identification and validation, it was observed that there is structure similarity between NCI 748494/1 and the reported type II c-Met kinase inhibitor BMS-777607. Optimization of our lead NCI 748494/1 furnished newly synthesized 1,2,4-triazine derivatives based on well-established structure-activity relationships, whereas three compounds namely; 4d, 7a and 8c displayed excellent in vitro cytotoxicity against three c-Met addicted cancer cell lines; A549 (lung adenocarcinoma), HT-29 (colon cancer) and MKN-45 (gastric carcinoma); with IC₅₀ values in the range 0.01–1.86 µM. In vitro c-Met kinase assay showed 8c to possess the highest c-Met kinase inhibition profile (IC₅₀ = 4.31 µM). Docking of the active compounds in c-Met kinase active site revealed strong binding interactions comparable to the lead NCI 748494/1 and BMS-777607, suggesting that c-Met inhibition is very likely to be the mechanism of the antitumor effect of these derivatives.     

Celecoxib analogs possessing a N-(4-nitrooxybutyl)piperidin-4-yl or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridin-4-yl nitric oxide donor moiety: Synthesis,biological evaluation and nitric oxide release studies

A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs) wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl 15a–b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a–b, NO-donor moiety was synthesized. All compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH 7.4 (2.4–5.8% range). In comparison, the percentage NO released was higher (3.1–8.4% range) when these nitrate prodrugs were incubated in the presence of l-cysteine. In vitro COX-1/COX-2 isozyme inhibition studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of the COX-2 relative to the COX-1 enzyme. AI structure–activity relationship data acquired showed that compounds having a MeSO₂ COX-2 pharmacophore exhibited superior AI activity compared to analogs having a H₂NSO₂ substituent. Compounds having a MeSO₂ COX-2 pharmacophore in conjunction with a N-(4-nitrooxybutyl)piperidyl (ED₅₀ = 132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl (ED₅₀ = 118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin (ED₅₀ = 128.7 mg/kg po) but lower than ibuprofen (ED₅₀ = 67.4 mg/kg po).     

Design,synthesis, and docking studies of phenylpicolinamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety as c-Met inhibitors

Four series of phenylpicolinamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (12a–e, 13a–f, 14a–f and 15a–i) were designed, synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, PC-3 and MCF-7) and c-Met kinase. Five selected compounds (13b, 15b, 15d, 15e and 15f) were further evaluated for the activity against HepG2 and Hela cell lines. Eighteen of the compounds showed excellent cytotoxicity activity and selectivity with the IC₅₀ valuables in single-digit μM to nanomole range. Seven of them are equal to more active than positive control Foretinib against one or more cell lines. The most promising compound 15f showed superior activity to Foretinib, with the IC₅₀ values of 1.04 ± 0.11 μM, 0.02 ± 0.01 μM and 9.11 ± 0.55 μM against A549, PC-3 and MCF-7 cell lines, which were 0.62 to 19.5 times more active than Foretinib (IC₅₀ values: 0.64 ± 0.26 μM, 0.39 ± 0.11 μM, 9.47 ± 0.22 μM), respectively. Structure–activity relationships (SARs) and docking studies indicated that replacement of quinoline nucleus of the previous active compounds with 1H-pyrrolo[2,3-b]pyridine moiety maintained even improved the potent cytotoxic activity. The results suggested that the introduction of fluoro atoms to the aminophenoxy part of target compounds or the phenyl group of pyrimidine substituted on C-4 position was benefit for the activity.     

Synthesis and anticancer activity of novel 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives bearing chromone moiety

Herein, we designed and synthesized of a novel series of 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives bearing chromone moiety (10a–j, 13a–j). All the compounds were evaluated for the IC₅₀ values against five cancer cell lines (A549, PC-3, MCF-7, Hela and HepG2). Seven of the target compounds exhibited moderate to excellent cytotoxicity. For these compounds, we tested their inhibitory activities against mTOR kinase, and four of them were tested their inhibitory activities against PI3Kα kinase in further. The results indicated that the optimized compound 10j showed excellent inhibitory activity and cytotoxicity against mTOR kinase, PI3Kα kinase and five cancer cell lines with IC₅₀ values of 1.1 μM, 0.92 μM and 8.77–14.3 μM. Structure–activity relationships (SARs) and docking studies indicated that the thiopyrano[4,3-d]pyrimidine scaffolds exerted little effect on antitumor activities of target compounds. Substitutions of chromone moiety at C-6 position with carboxyl were benefit to the antitumor activities.     

Design,synthesis and SAR studies of 4-allyoxyaniline amides as potent 15-lipoxygensae inhibitors

A group of 4-allyloxyaniline amides 5a–o were designed, synthesized and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compound 5e showed the best IC₅₀ in SLO inhibition (IC₅₀ = 0.67 ± 0.06 μM). All compounds were docked in SLO active site retrieved from RCSB Protein Data Bank (PDB entry: 1IK3) and showed that allyloxy group of compounds is oriented towards the Fe³⁺-OH moiety in the active site of enzyme and fixed by hydrogen bonding with two conserved His⁵¹³ and Gln⁷¹⁶. It is resulted that molecular volume of the amide moiety would be a major factor in inhibitory potency variation of the synthetic amides, where the hydrogen bonding of the amide group could also involve in the activity of the inhibitors.     

Discovery of quinazolin-4-amines bearing benzimidazole fragments as dual inhibitors of c-Met and VEGFR-2

Both c-Met and VEGFR-2 are important targets for the treatment of cancers. In this study, a series of N-(2-phenyl-1H-benzo[d]imidazol-5-yl)quinazolin-4-amine derivatives were designed and identified as dual c-Met and VEGFR-2 inhibitors. Among these compounds bearing quinazoline and benzimidazole fragments, compound 7j exhibited the most potent inhibitory activity against c-Met and VEGFR-2 with IC₅₀ of 0.05 μM and 0.02 μM, respectively. It also showed the highest anticancer activity against the tested cancer cell lines with IC₅₀ of 1.5 μM against MCF-7 and 8.7 μM against Hep-G2. Docking simulation supported the initial pharmacophoric hypothesis and suggested a common mode of interaction at the ATP-binding site of c-Met and VEGFR-2, which demonstrates that compound 7j is a potential agent for cancer therapy deserving further researching.     

Synthesis,and docking studies of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety as c-Met inhibitors

Four series of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (14a–e, 15a–g, 16a–e and 17a–g) were designed, synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, PC-3 and MCF-7). Four selected compounds (15e, 16a–b and 17a) were further evaluated for the activity against c-Met kinase, HepG2 and Hela cell lines. Most of the compounds showed excellent cytotoxicity activity and selectivity with the IC₅₀ valuables in single-digit μM to nanomole range. Eleven of them are equal to more active than positive control Foretinib against one or more cell lines. The most promising compound 15e showed superior activity to Foretinib against A549, PC-3 and MCF-7 cell lines, with the IC₅₀ values of 0.14 ± 0.08 μM, 0.24 ± 0.07 μM and 0.02 ± 0.01 μM, which were 4.6, 1.6 and 473.5 times more active than Foretinib (0.64 ± 0.26 μM, 0.39 ± 0.11 μM, 9.47 ± 0.22 μM), respectively. Structure–activity relationships (SARs) and docking studies indicated that the replacement of phenylpicolinamide scaffold with phenylpyrimidine fragment of the target compounds was benefit for the activity. What’s more, the introduction of fluoro atom to the aminophenoxy part played no significant impact on the activity and any substituent group on aryl group is unfavourable for the activity.     

Toward discovery of mutant EGFR inhibitors; Design,synthesis and in vitro biological evaluation of potent 4-arylamino-6-ureido and thioureido-quinazoline derivatives

A new series of 4-anilinoquinazolines with C-6 ureido and thioureido side chains and various substituents at the C-4 anilino moiety was designed, synthesized and evaluated as wild type (WT) and mutant EGFR inhibitors. Most of the compounds inhibited EGFR kinase wild type (EGFR WT) with IC₅₀ values in the low nanomolar range (<0.495–9.05 nM) and displayed more potent cytotoxic effect in BaF/3 expressing EGFR WT than reference compound gefitinib. The anti-proliferative effect of all synthesized compounds against gefitinib insensitive double mutant cell lines Ba/F3 expressing Del19/T790M and Ba/F3 expressing L858R/T790M were assayed. Compounds 4d, 6f, 7e showed significant inhibition (IC₅₀ = 1.76–2.38 μM) in these mutant lines and significant Her2 enzyme inhibition (IC₅₀ = 19.2–40.6 nM) compared to lapatinib (60.1 nM). The Binding mode of compounds 6d, 6f, 7a, 7b and 8b were demonstrated. Furthermore, growth inhibition against gefitinib insensitive cell lines PC9-GR4 (Del19/T790M) were tested, compounds 6f and 7e showed about eight and three folds respectively greater potency than gefitinib. Our structure–activity relationships (SAR) studies suggested that presence of ethyl piperidino urea/thiourea at 6-position and bulky group of (3-chloro-4-(3-fluorobenzyloxy)phenyl)amino at 4-position of quinazoline may serve as promising scaffold for developing inhibitors against wild type and mutant EGFR.     

Design and synthesis of 4-methoxyphenylacetic acid esters as 15-lipoxygenase inhibitors and SAR comparative studies of them

A group of 4-methoxyphenylacetic acid esters were designed, synthesized and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compounds 7d–e showed the best IC₅₀ in SLO inhibition (IC₅₀ = 3.8 and 1.9 μM, respectively). All compounds were docked in SLO active site and showed that carbonyl group of compounds is oriented toward the Fe^III–OH moiety in the active site of enzyme and fixed by hydrogen bonding with hydroxyl group. It is assumed that lipophilic interaction of ligand–enzyme would be in charge of inhibiting the enzyme activity. The selectivity of the synthetic esters in inhibiting of 15-HLOb was also compared with 15-HLOa by molecular modeling and multiple alignment techniques.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

Design,synthesis and biological evaluation of 1H-pyrrolo[2,3-b]pyridine and 1H-pyrazolo[3,4-b]pyridine derivatives as c-Met inhibitors

Five novel 1H-pyrrolo[2,3-b]pyridine or 1H-pyrazolo[3,4-b]pyridine derivatives, with a methylene, sulfur, sulfoxide or cyclopropyl group as a linker, were designed, synthesized and biologically evaluated against c-Met and ALK. The development of these methods of compound synthesis may provide an important reference for the construction of novel 7-azaindole and 7-azaindazole derivatives with a single atom linker. The enzyme assay and cell assay in vitro showed that compound 9 displayed strong c-Met kinase inhibition with IC₅₀ of 22.8 nM, moderate ALK kinase inhibition, and strong cell inhibition with MKN-45 IC₅₀ of 329 nM and EBC-1 IC₅₀ of 479 nM. In order to find the better candidate compounds, compounds 8, 9 and 10 have been selected as tool compounds for further optimization.     

A novel and potent VLA-4 antagonist based on trans-4-substituted cyclohexanecarboxylic acid

During the course of our study, it was revealed that the poor pharmacokinetic properties of a series of benzoic acid derivatives such as 1 should be attributed to the diphenylurea moiety. Thus, we replaced the diphenylurea moiety in 1 with a 2-(2-methylphenylamino)benzoxazole moiety which mimics the diphenylurea structure. However, this modification resulted in a significant decrease (3, IC₅₀ = 19 nM) in VLA-4 inhibitory activity compared to 1 (IC₅₀ = 1.6 nM). To address this discrepancy, we worked on optimization of the carboxylic acid moiety in compound 3. As a result, our efforts have led to the discovery of trans-4-substituted cyclohexanecarboxylic acid derivative 11b (IC₅₀ = 2.8 nM) as a novel and potent VLA-4 antagonist. In addition, compound 11b exhibited favorable pharmacokinetic properties (CL = 3.3 ml/min/kg, F = 51%) in rats.     

Design,synthesis, and docking studies of afatinib analogs bearing cinnamamide moiety as potent EGFR inhibitors

Two series of afatinib derivatives bearing cinnamamide moiety (10a–n and 11a–h) were designed, synthesized and evaluated for the IC₅₀ values against four cancer cell lines (A549, PC-3, MCF-7 and Hela). Two selected compounds (10e, 10k) were further evaluated for the inhibitory activity against EGFR and VEGFR2/KDR kinases. Seven of the compounds showed excellent cytotoxicity activity and selectivity with the IC₅₀ values in single-digit μM to nanomole range. Three of them are equal to more active than positive control afatinib against one or more cell lines. The most promising compound 10k showed the best activity against A549, PC-3, MCF-7 and Hela cancer cell lines and EGFR kinase, with the IC₅₀ values of 0.07 ± 0.02 μM, 7.67 ± 0.97 μM, 4.65 ± 0.90 μM and 4.83 ± 1.28 μM, which were equal to more active than afatinib (0.05 ± 0.01 μM, 4.1 ± 2.47 μM, 5.83 ± 1.89 μM and 6.81 ± 1.77 μM), respectively. Activity of compounds 10e (IC₅₀ 9.1 nM) and 10k (IC₅₀ 3.6 nM) against EGFR kinase were equal to the reference compound afatinib (IC₅₀ 1.6 nM). Structure–activity relationships (SARs) and docking studies indicated that replacement of the aqueous solubility 4-(dimethylamino)but-2-enamide group by cinnamamide moiety didn’t decrease the antitumor activity. The results suggested that methoxy substitution had a significant impact on the activity and methoxy substituted on C-4 or C-2,3,4 position was benefit for the activity.     

Synthesis,activity and docking studies of phenylpyrimidine–carboxamide Sorafenib derivatives

Two series of Sorafenib derivatives bearing phenylpyrimidine–carboxamide moiety (16a–g and 17a–p) were designed, synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, MCF-7 and PC-3). Two selected compounds (17f and 17n) were further evaluated for the activity against VEGFR2/KDR kinase. More than half of the synthesized compounds showed moderate to excellent activity against three cancer cell lines. Compound 17f showed equal activity to Sorafenib against MCF-7 cell line, with the IC₅₀ values of 6.35 ± 0.43 μM. Meanwhile, compound 17n revealed more active than Sorafenib against A549 cell line, with the IC₅₀ values of 3.39 ± 0.37 μM. Structure–activity relationships (SARs) and docking studies indicated that the second series (17a–p) showed more active than the first series (16a–g). What’s more, the introduction of fluoro atom to the phenoxy part played no significant impact on activity. In addition, the presence of electron-donating on aryl group was benefit for the activity.     

Discovery of imidazopyridine derivatives as novel c-Met kinase inhibitors: Synthesis,SAR study,and biological activity

Receptor tyrosine kinase c-Met acts as an alternative angiogenic pathway in the process and contents of cancers. A series of imidazopyridine derivatives were designed and synthesized according to the established docking studies as possible c-Met inhibitors. Most of these imidazopyridine derivatives displayed nanomolar potency against c-Met in both biochemical enzymatic screens and cellular pharmacology studies. Especially, compound 7 g exhibited the most inhibitory activity against c-Met with IC₅₀ of 53.4 nM and 253 nM in enzymatic and cellular level, respectively. Following that, the compound 7 g was docked into the protein of c-Met and the structure-activity relationship was analyzed in detail. These findings indicated that the novel imidazopyridine derivative compound 7 g was a potential c-Met inhibitor deserving further investigation for cancer treatment.     

Discovery of novel 2-substituted-4-(2-fluorophenoxy) pyridine derivatives possessing pyrazolone and triazole moieties as dual c-Met/VEGFR-2 receptor tyrosine kinase inhibitors

In our efforts to develop novel dual c-Met/VEGFR-2 inhibitors as potential anticancer agents, a series of 2-substituted-4-(2-fluorophenoxy) pyridine derivatives bearing pyrazolone scaffold were designed and synthesized. The cell proliferation assay in vitro demonstrated that most target compounds had inhibition potency on both c-Met and VEGFR-2, especially compound 9h, 12b and 12d. Based on the further enzyme assay in vitro, compound 12d was considered as the most promising one, the IC₅₀ values of which were 0.11 μM and 0.19 μM for c-Met and VEGFR-2, respectively. Further molecular docking studies suggested a common mode of interaction at the ATP-binding site of c-Met and VEGFR-2, indicating that 12d was a potential compound for cancer therapy deserving further study.