Rational design and synthesis of 2,4-dichloro-6-methyl pyrimidine derivatives as potential selective EGFRT790M/L858R inhibitors for the treatment of non-small cell lung cancer.

Many patients with non-small cell lung cancer (NSCLC) initially benefit from epidermal growth factor receptor (EGFR) targeted therapy. Unfortunately, varying degrees of resistance or side effects eventually develop. Overcoming and preventing the resistance and side effects of EGFR inhibitors has become a hot topic of research today. Based on the previous studies on AZD-9291, we designed and synthesized two series of 2,4-dichloro-6-methylpyrimidine derivatives, 19 compounds in total, as potential inhibitors of the EGFR kinase. The most promising compound, L-18, showed better inhibitory activity (81.9%) and selectivity against EGFRT790M/L858R kinase. In addition, L-18 showed strong antiproliferative activity against H1975 cells with an IC50 value of 0.65 ± 0.06 µM and no toxicity to normal cells (LO-2). L-18 was able to dose-dependently induce the apoptosis of H1975 cells and produced a cell-cycle-blocking effect, and it can also dose-dependently inhibit the migration and invasion of H1975 cells. L-18 also showed in vivo anticancer efficacy in H1975 cells xenograft mice. We also performed a series of in vivo and in vitro toxicological evaluations of compound L-18, which did not cause obvious injury in mice during administration. These results suggest that L-18 may be a promising drug candidate that warrants further investigation.

Discovery of novel 4-arylamino-quinazoline derivatives as EGFRL858R/T790M inhibitors with the potential to inhibit the non-small cell lung cancers.

Three series of quinazoline derivatives (7a-j, 8a-o, 9a-l) were designed and synthesized as EGFRL858R/T790M inhibitors. Series 7a-j and 8a-o are urea and thiourea derivatives while category 9a-l contain the Michael receptor active warhead. Most of the compounds exhibited excellent anti-proliferative activity in vitro against several cancer cell lines, including non-small cell lung cancer (NSCLC) cell lines A549 and H1975, among which 14 compounds had strong antiproliferative activity against A549 and H1975 cancer cells. What's more, they also showed moderate to excellent kinase inhibitory activity against EGFRWT and EGFRL858R/T790M. 8o exhibited the best kinase inhibitory activity with IC50 values of 0.8, 2.7 nM against EGFRWT and EGFRL858R/T790M, respectively. Moreover, AO single staining and Annexin V-FITC/PI staining results also indicated that both 8o and 9b significantly induced apoptosis in A549 cells. 8o arrested the cell cycle at S phase and 9b arrested the cell cycle at G1 phase.

Design, synthesis and biological evaluation of novel 4-anlinoquinazoline derivatives as EGFR inhibitors with the potential to inhibit the gefitinib-resistant nonsmall cell lung cancers.

A series of quinazoline derivatives with benzylidene hydrazine carboxamide were designed and synthesised as EGFR inhibitors. Most compounds exhibited exceptional anti-proliferative activity against A549, HepG2, MCF-7 and H1975 cells. Furthermore, six compounds demonstrated excellent inhibition activity against EGFRWT with the IC50 value both less than 2 nM. Among the six compounds, 44 exhibited the strongest activity (0.4 nM) and potently inhibited EGFRL858R/T790M (0.1 µM). Excitingly, the most potent compound 14 showed excellent enzyme inhibitory activity with 6.3 nM and 8.4 nM for both EGFRWT and EGFRT790M/L858R. The result of AO single staining and Annexin V/PI staining showed that the compound 14 and 44 could induce remarkable apoptosis of A549 cells. The compound 14 arrested the cell cycle at the S phase and compound 44 arrested the cell cycle at the G0 phase in A549 cells. These preliminary results demonstrate that compound 14 and 44 may be promising lead compound-targeting EGFR.

Design, Synthesis, and Biological Evaluation of Novel Thienopyrimidine Derivatives as PI3Kα Inhibitors.

Three series of novel thienopyrimidine derivatives 9a-l, 15a-l, and 18a-h were designed and synthesized, and their IC50 values against four cancer cell lines HepG-2, A549, PC-3, and MCF-7 were evaluated. Most compounds show moderate cytotoxicity against the tested cancer cell lines. The most promising compound 9a showed moderate activity with IC50 values of 12.32 ± 0.96, 11.30 ± 1.19, 14.69 ± 1.32, and 9.80 ± 0.93 µM, respectively. The inhibitory activities of compounds 9a and 15a against PI3Kα and mTOR kinase were further evaluated. Compound 9a exhibited PI3Kα kinase inhibitory activity with IC50 of 9.47 ± 0.63 µM. In addition, docking studies of compounds 9a and 15a were also investigated.

Discovery of novel 2-substituted-4-phenoxypyridine derivatives as potential antitumor agents.

A series of 2-substituted-4-phenoxypyridine derivatives were designed, synthesized, and evaluated for their antiproliferative activity against 4 cancer cell lines (A549, HT-29, H460, and U87MG) in vitro. Most compounds showed moderate to excellent potency. Nine tyrosine kinases (c-Met, Flt-3, ALK, VEGFR-2, VEGFR-3, PDGFR-α, PDGFR-ß, c-Kit, and EGFR) were used to evaluate the inhibitory activities with the most promising analogue 39, which showed the Flt-3/c-Met IC50 values of 2.18/2.61 nM. Structure-activity relationship studies indicated that n-Pr served as R1 group showed a higher preference, and stronger mono-EWGs on the phenyl ring (such as R2 = 4-F) was benefited to the potency.

Design, Synthesis and Biological Evaluation of Novel Phenylsulfonylurea Derivatives as PI3K/mTOR Dual Inhibitors.

Five series of novel phenylsulfonylurea derivatives, 19a⁻d, 20a⁻d, 21a⁻d, 22a⁻d and 23a⁻d, bearing 4-phenylaminoquinoline scaffold were designed, synthesized and their IC50 values against four cancer cell lines (HepG-2, A549, PC-3 and MCF-7) were evaluated. Most compounds showed moderate cytotoxicity activity against the cancer cell lines. Structure⁻activity relationships (SARs) and pharmacological results indicated that introduction of 4-aminoquinoline scaffold and phenylsulfonylurea scaffold were beneficial for anti-tumor activity. Moreover, para-methoxyl substitution of 4-anilino moiety and para-halogen substitution of phenylsulfonylurea have different impacts on different series of compounds. Furthermore, the micromolecule group substitution in the 6-position of the quinoline ring have a slight impact on the cellular activity of the target compounds.

Design, synthesis, and docking studies of quinazoline analogues bearing aryl semicarbazone scaffolds as potent EGFR inhibitors.

Two series of quinazoline derivatives bearing aryl semicarbazone scaffolds (9a-o and 10a-o) were designed, synthesized and evaluated for the IC50 values against four cancer cell lines (A549, HepG2, MCF-7 and PC-3). The selected compound 9o was further evaluated for the inhibitory activity against EGFR kinases. Four of the compounds showed excellent cytotoxicity activity and selectivity with the IC50 values in single-digit µM to nanomole range. Two of them are equal to more active than positive control afatinib against one or more cell lines. The most promising compound 9o showed the best activity against A549, HepG2, MCF-7 and PC-3 cancer cell lines and EGFR kinase, with the IC50 values of 1.32±0.38µM, 0.07±0.01µM, 0.91±0.29µM and 4.89±0.69µM, which were equal to more active than afatinib (1.40±0.83µM, 1.33±1.28µM, 2.63±1.06µM and 3.96±0.59µM), respectively. Activity of the most promising compound 9o (IC50 56nM) against EGFR kinase was slightly lower to the positive compound afatinib (IC50 1.6nM) but more active than reference staurosporine (IC50 238nM). The result of flow cytometry, with the dose of compound 9o increasing, which indicated the compound 9o could induce remarkable apoptosis of A549 and cells in a dose dependent manner. Structure-activity relationships (SARs) and docking studies indicated that replacement of the cinnamamide group by aryl semicarbazone scaffolds slightly decreased the anti-tumor activity. The results suggested that hydroxy substitution at C-4 had a significant impact on the activity and replacement of the tetrahydrofuran group by methyl moiety was not beneficial for the activity.

Design, synthesis and antitumor activity of Novel Sorafenib derivatives bearing pyrazole scaffold.

Four series of Sorafenib derivatives bearing pyrazole scaffold (8a-m, 9a-c, 10a-e and 11a) were synthesized and characterized by NMR and MS. All of the target compounds were evaluated for the cytotoxicity against A549, HepG2, MCF-7, and PC-3 cancer cell lines and some selected compounds were further evaluated for the activity against VEGFR-2/KDR, BRAF, CRAF, c-Met, EGFR and Flt-3 kinases. Compounds 8b and 8i were more active than that of compounds 8h, 9a, especially the IC50 value of compounds 8b on VEGFR-2 kinase was 0.56µM. And compound 8b exhibited moderate to good activity toward c-Met and showed moderate to no activity against CRAF, c-Met, EGFR, Flt-3 kinases. Eleven of the target compounds exhibited moderate to good antitumor activities. The most promising compound 8b showed strong antitumor activities against A549, HepG2 and MCF-7 cell lines with IC50 values of 2.84±0.78µM, 1.85±0.03µM and 1.96±0.28µM, which were equivalent to Sorafenib (2.92±0.68µM, 3.44±0.50µM and 3.18±0.18µM). Structure-activity relationships (SARs) and docking studies indicated that the pyrazole scaffolds exerted key effect on antitumor activities of target compounds. Substitutions of aryl group at C-3 positions had a significant impact on the antitumor activities, and 3-Br substitution produced the best potency.

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±0.43µM. Meanwhile, compound 17n revealed more active than Sorafenib against A549 cell line, with the IC50 values of 3.39±0.37µM. 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.

Synthesis and Biological Evaluation of Novel 4-Morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine Derivatives Bearing Phenylpyridine/ Phenylpyrimidine-Carboxamides.

Four series of novel 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives 11a-j, 12a-j, 13a-g and 14a-g bearing phenylpyridine/phenylpyrimidine- carboxamide scaffolds were designed, synthesized and their IC50 values against three cancer cell lines (A549, PC-3 and MCF-7) were evaluated. Eleven of the compounds showed moderate cytotoxicity activity against the cancer cell lines. Structure-activity relationships (SARs) and pharmacological results indicated that the introduction of phenylpyridine-carboxamide scaffold was beneficial for the activity. What's more, the oxidation of the sulfur atom in thiopyran and various types of substituents on the aryl group have different impacts on different series of compounds. Furthermore, the positions of aryl group substituents have a slight impact on the activity of the phenylpyridine-carboxamide series compounds.

Design, synthesis, and biological evaluation of diphenyl ether substituted quinazolin-4-amine derivatives as potent EGFRL858R/T790M/C797S inhibitors.

Epidermal growth factor receptor (EGFR) is a validated target for non-small-cell lung cancer (NSCLC). However, the treatment for EGFR-C797S mutation induced by third-generation EGFR inhibitors remains a concern. Therefore, the development of the fourth-generation EGFR inhibitors to overcome the EGFR-C797S mutation has great potential for clinical treatment. In this article, we designed and synthesized a series of diphenyl ether substituted quinazolin-4-amine derivatives that simultaneously occupy the ATP binding pocket and the allosteric site of EGFR. Among the newly synthesized compounds, 9d displayed excellent kinase activity against EGFRL858R/T790M/C797S with an IC50 value of 0.005 µM, and exhibited anti-proliferation activity in BaF3-EGFRL858R/T790M/C797S cells with the IC50 value of 0.865 µM. Furthermore, 9d could suppress phosphorylation of EGFR and induce cell apoptosis and cycle arrest at G2 phase in a dose-dependent manner in BaF3-EGFRL858R/T790M/C797S cells. More importantly, 9d displayed significant antitumor effects in BaF3-EGFRL858R/T790M/C797S xenograft mouse model (30 mg/kg, TGI = 71.14 %). All the results indicated compound 9d might be a novel fourth-generation EGFR inhibitor for further development in overcoming the EGFR-C797S resistance mutation.

Efficacy of WWQ-131, a highly selective JAK2 inhibitor, in mouse models of myeloproliferative neoplasms.

Hyperactivation of the Janus kinase 2 (JAK2) signaling pathway leads to myeloproliferative neoplasms (MPNs) and targeting JAK2 can be used as an effective strategy for the treatment of MPNs. Here, our study indicated that WWQ-131 was a highly selective JAK2 inhibitor (IC50 =2.36 nM), with 182-fold and 171-fold more selective to JAK1 and JAK3, respectively. In JAK2V617F-dependent cell lines, WWQ-131 efficaciously inhibited cell proliferation, induced cell cycle arrest at the G2/M phase and apoptosis, and blocked the aberrant activation of JAK2 signaling pathway. In a mouse Ba/F3_JAK2V617F driven disease model, WWQ-131 effectively suppressed STAT5 phosphorylation in spleen and liver, and inhibited Ba/F3_JAK2V617F cells spreading and proliferation in vivo. In addition, WWQ-131 suppressed rhEPO-induced extramedullary erythropoiesis and polycythemia in mice, as well as hematocrits and spleen sizes, especially had no effect on white blood cell count. Furthermore, WWQ-131 (75 mg/kg) exhibited stronger therapeutic effects than fedratinib (120 mg/kg) in these two MPN models. Taken together, this study suggests that WWQ-131 will be a promising candidate for the treatment of MPNs.

Discovery and optimization of 4-anilinoquinazoline derivatives spanning ATP binding site and allosteric site as effective EGFR-C797S inhibitors.

Epidermal growth factor receptor (EGFR) is an effective drug target for the treatment of non-small cell lung cancer (NSCLC). However, a tertiary point mutation (C797S) at the ATP binding pocket of the EGFR induces resistance to the third-generation EGFR inhibitors, due to the loss of covalent interaction with Cys797. Here, we designed a series of 4-anilinoquinazoline derivatives that simultaneously occupied the ATP binding pocket and the allosteric site. The newly-synthesized compounds displayed high potency against EGFR-C797S resistance mutation. Among them, compound 14d presented high anti-proliferative effect against BaF3-EGFRL858R/T790M/C797S (IC50 = 0.75 µM) and BaF3-EGFR19del/T790M/C797S (IC50 = 0.09 µM) cells. Moreover, 14d resulted in obvious inhibition activities against EGFR and its downstream signaling pathways in a dose-dependent manner in BaF3-EGFR19del/T790M/C797S cells. Finally, 14d significantly inhibited tumor growth in BaF3-EGFR19del/T790M/C797S xenograft model (30 mg/kg, TGI = 67.95%). These results demonstrated that 14d is a novel and effective EGFR-C797S inhibitor which spanning the ATP binding pocket and the allosteric site and effective both in vitro and in vivo.

Discovery of novel quinazoline derivatives bearing semicarbazone moiety as potent EGFR kinase inhibitors.

Aimed at discovering effective EGFR inhibitors, six series of quinazoline derivatives bearing a semicarbazone moiety were designed, synthesized and evaluated in different cancer cell lines (A549, HepG2, MCF-7 and PC-3). Most of the selected compounds showed remarkable cytotoxicity with IC50 values reaching the nanomole range. Further, the inhibition efficacy of 11 compounds against EGFR kinases was tested, which demonstrated excellent IC50 values in nanomolar level. Importantly, 2 compounds exhibited IC50 values of 0.05 nM and 0.1 nM against wild type EGFR respectively, suggesting more potent activities than that of the positive control, Afatinib (4.0 nM). Excitingly, 2 compounds showed excellent enzyme inhibitory activity with 8.6 nM and 5.6 nM for double T790 M/L858R mutant EGFRs, which is almost the same as Afatinib (3.8 nM). Structure-activity relationships (SARs) analysis indicated that the type of small molecule amine in pyrrole moiety or the chain length of pyrrolamine moiety had no obvious impact on the inhibition efficacy of our synthesized compounds against cancer cells. In addition, results of cell cycle analysis indicated that the G2/M phase of A549 cells was efficiently arrested by the selected compounds. These preliminary results demonstrate that 2 compounds may be promising lead compound-targeting EGFR.

Synthesis and bioevaluation and doking study of 1H-pyrrolo[2,3-b]pyridine derivatives bearing aromatic hydrazone moiety as c-Met inhibitors.

Two series of aromatic hydrazone derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (7a-r, 8a-i, 12a-b, 13a-c, 16a-d and 17a-e) were designed, synthesized and evaluated for the IC50 values against four cancer cell lines (A549, HepG2, MCF-7and PC-3). Two selected compounds (7c and 17e) were further evaluated for the activity against c-Met, Flt-3, VEGFR-2 and EGFR kinases. The data indicated that targets compounds were selective for c-Met kinase. And the most promising compound 7c was further studied in terms of dose-dependent, time-dependent and cell apoptosis. Most of the compounds showed excellent cytotoxicity activity, especially the most promising compound 7c with the IC50 values of 0.82 ±â€¯0.08 µM, 1.00 ±â€¯0.11 µM, 0.93 ±â€¯0.28 µM and 0.92 ±â€¯0.17 µM against A549, HepG2, MCF-7 and PC-3 cell lines and 0.506 µM against c-Met kinase. Structure-activity relationships (SARs) and docking studies indicated that the activities of the phenyl hydrazone derivatives (7a-r and 8a-i) were superior to that of the heterocyclic hydrazone series (12a-b, 13a-c, 16a-d and 17a-e). What's more, the further studies indicated that the target compounds can induce apoptosis of A549 cells and arrest efficiently the cell cycle progression in G2/M phase of A549 cells.

Discovery of novel pyrrolopyrimidine/pyrazolopyrimidine derivatives bearing 1,2,3-triazole moiety as c-Met kinase inhibitors.

Six series of pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine derivatives bearing 1,2,3-triazole moiety were designed and synthesized, and some bio-evaluation was also carried out. As a result, four points can be summarized: Firstly, some of compounds exhibited excellent cytotoxicity activity and selectivity with the IC50 values in single-digit µm level. In particular, the most promising compound 16d showed equal activity to lead compound foretinib against A549, HepG2, and MCF-7 cell lines, with the IC50 values of 4.79 ± 0.82, 2.03 ± 0.39, and 2.90 ± 0.43 µm, respectively. Secondly, the SARs and docking studies indicated that the in vitro antitumor activity of pyrrolo[2,3-d]pyrimidine derivatives bearing 1,2,3-triazole moiety was superior to the pyrazolo[3,4-d]pyrimidine derivatives bearing 1,2,3-triazole moiety. Thirdly, three selected compounds (16d, 18d, and 20d) were further evaluated for inhibitory activity against the c-Met kinase, and the 16d could inhibit the c-Met kinase selectively by experiments of enzyme-based selectivity. What is more, 16d could induce apoptosis of HepG2 cells and inhibitor the cell cycle of HepG2 on G2/M phase by acridine orange staining and cell cycle experiments, respectively.

Synthesis and antiproliferative activity of pyrrolo[2,3-b]pyridine derivatives bearing the 1,8-naphthyridin-2-one moiety.

A series of pyrrolo[2,3-b]pyridine derivatives bearing the 1,8-naphthyridin-2-one moiety were synthesized, and evaluated for their antiproliferative activity against four cancer cell lines (HT-29, A549, H460, and U87MG) and six tyrosine kinases (c-Met, Flt-3, PDGFR-ß, VEGFR-2, EGFR, and c-Kit) inhibitory activities in vitro. Most compounds showed moderate to excellent potency, with the most promising analogue 32 showing Flt-3/c-Met IC50 value of 1.16/1.92 nM. Structure-activity relationship studies indicated that the hydrogen atom served as R1 group was benefited to the potency, and mono-electron-withdrawing groups (mono-EWGs) on the phenyl ring (such as R3 = 4-F) showed a higher preference for antiproliferative activity.