The synthesis of 4-arylamido-2-arylaminoprimidines as potent EGFR T790M/L858R inhibitors for NSCLC

A series of 4-arylamido-2-arylaminoprimidines bearing acrylamide pharmacophore were synthesized as potent EGFR^T790M/L858R inhibitors among which 9c (IC₅₀?=?0.5872?nM), 9d (IC₅₀?=?2.213?nM), or 9h (IC₅₀?=?12.57?nM) showed more potent anti-EGFR^T790M/L858R activity compared with AZD–9291 (IC₅₀?=?20.80?nM) and possessed high SI displaying 307.6, 56.5, or 12.5 for EGFR^T790M/L858R over the wild-type respectively. 9h also showed pretty good activity against H 1975 cells with an IC₅₀ of 1.664?μM and exhibited low toxicity against the normal HBE cells (IC₅₀?>?20?μΜ). 9h had moderate selectivity for H 1975 over A 431 (SI?=?7.0) and the other selected cell lines. Morphological staining results further indicated that 9h could promote apoptosis. Hence, 9h was a promising compound for further investigation as a potential EGFR^T790M/L858R inhibitor for the treatment of NSCLC.     

Synthesis and biological evaluation of azole-diphenylpyrimidine derivatives (AzDPPYs) as potent T790M mutant form of epidermal growth factor receptor inhibitors

A series of novel azole-diphenylpyrimidine derivatives (AzDPPYs) were synthesized and biologically evaluated as potent EGFR^T790M inhibitors. Among these analogues, the most active inhibitor 6e not only displayed high activity against EGFR^T790M/L858R kinase (IC₅₀ = 3.3 nM), but also was able to repress the replication of H1975 cells harboring EGFR^T790M mutation at a concentration of 0.118 μmol/L. In contrast to the lead compound rociletinib, 6e slightly reduces the key EGFRT790M-minduced drug resistance. Significantly, inhibitor 6e demonstrates high selectivity (SI = 299.3) for T790M-containing EGFR mutants over wild type EGFR, hinting that it will cause less side effects.     

Molecular dynamics guided development of indole based dual inhibitors of EGFR (T790M) and c-MET

Secondary acquired mutation in EGFR, i.e. EGFR T790M and amplification of c-MET form the two key components of resistant NSCLC. Thus, previously published pharmacophore models of EGFR T790M and c-MET were utilized to screen an in-house database. On the basis of fitness score, indole-pyrimidine scaffold was selected for further evaluation. Derivatives of indole-pyrimidine scaffold with variedly substituted aryl substitutions were sketched and then docked in both the targets. These docked complexes were then subjected to molecular dynamic simulations, to study the stability of the complexes and evaluate orientations of the designed molecules in the catalytic domain of the selected kinases. Afterwards, the complexes were subjected to MM-GBSA calculation, to study the effect of substitutions on binding affinity of double mutant EGFR towards these small molecules. Finally, the designed molecules were synthesized and evaluated for their inhibitory potential against both the kinases using in vitro experiments. Additionally, the compounds were also evaluated against EGFR (L858R) to determine their selectivity towards double mutant, resistant kinase [EGFR (T790M)]. Compound 7a and 7c were found to be possess nanomolar range inhibitory (IC₅₀) potential against EGFR (T790M), 7 h showed good inhibitory potential against c-MET with IC₅₀ value of 0.101 µM. Overall, this work is one of the earliest report of compounds having significant dual inhibitory potential against secondary acquired EGFR and cMET, with IC₅₀ values in nanomolar range.     

Design and synthesis of novel 2,4-disubstituted aminopyrimidines: reversible non-covalent T790M EGFR inhibitors

Abstract

Cardiotoxicity is one amongst the adverse effect of Osimertinib delineate in clinical trials and related to escalating doses. To triumph over the drawbacks of Osimertinib, in this study, we tend to delineate the design, synthesis, in vitro biological analysis of a series of novel reversible selective T790M inhibitors with minimal cardiotoxicity. Amongst the virtually sorted compounds; compound 18 and 74 have been located to be the foremost active compounds of the series with IC₅₀ value of 0.88, 0.92?μM in cellular assay and 0.56, 0.62?μM in enzymatic assay, against double mutant L858R/T790M EGFR. Additionally, they showed much less affinity toward wild-type (WT)-EGFR with minimal cardiotoxicity.     

Discovery of selective irreversible inhibitors for EGFR-T790M

Targeting the epidermal growth factor receptor kinase (EGFR) with ATP-competitive kinase inhibitors results in dramatic but short-lived responses in patients with EGFR mutant non small cell lung cancer. A series of novel covalent EGFR kinase inhibitors with selectivity for the clinically relevant T790M ‘gatekeeper’ resistance mutation relative to wild-type EGFR were discovered by library screening. A representative compound 3i was obtained through a systematic SAR study guided by mutant EGFR-dependent cellular proliferation assays.     

BEBT-109, a pan-mutant-selective EGFR inhibitor with potent antitumor activity in EGFR-mutant non-small cell lung cancer

EGFR mutation-positive NSCLC tumors are highly heterogeneous, therefore, exploring an agent simultaneously targeting multiple EGFR mutations may be valuable for clinical practice. Compared with osimertinib, BEBT-109 shows more sensitive and extensive antitumor activity in EGFR mutant NSCLC, while sparing wild-type EGFR cell lines. Meanwhile, unlike the metabolite of osimertinib AZ5104, the main metabolites of BEBT-109 are found lacking in activity against wild-type EGFR cell lines. Preclinical and clinical studies demonstrate a unique pharmacokinetic profiles of BEBT-109 with rapid absorption and quick in vivo clearance without accumulation, which are conducive to minimizing the off-target toxicity of the covalent irreversible EGFR inhibitor. Oral administration of BEBT-109 induces tumor regression in EGFR exon 20 insertion xenografts, and even tumor disappearance in PC-9, HCC827 and H1975 xenograft models. Furthermore, in clinical trials, the objective responses were observed in NSCLC patients with EGFR T790M mutation in the first and second dosing cohorts. These findings demonstrate that BEBT-109, a potent pan-mutant-selective EGFR inhibitor with improved pharmacokinetic properties, might offer a promising new option for the treatment of multiple mutant-EGFR-driven NSCLC.     

Discovery of new [1,4]dioxino[2,3-f]quinazoline-based inhibitors of EGFR including the T790M/L858R mutant

A novel series of 2,3-dihydro-[1,4]dioxino[2,3-f]quinazoline derivatives were designed, synthesized and evaluated as reversible and noncovalent epidermal growth factor receptor (EGFR) inhibitors. Most of the compounds exhibited good potency against EGFR^wt and some showed moderate to excellent potency against EGFR^T790M/L858R mutant. The half-maximal inhibitory concentration (IC₅₀) values of twenty-one compounds against EGFR^wt were less than 50 nM, and those of six compounds were less than 10 nM. The IC₅₀ values of eleven compounds against EGFR^T790M/L858R were less than 100 nM. Among these, compound b1 displayed the most potent inhibitory activity against EGFR^wt (IC₅₀ = 2.0 nM) and EGFR^T790M/L858R (IC₅₀ = 6.9 nM). Compounds with excellent inhibitory activities against EGFR^wt and EGFR^T790M/L858R kinase inhibitory activities showed good antiproliferative activities against H358 and A549 cells. Docking study was performed to position compound b1 into the EGFR active pocket to determine the probable binding conformation.     

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of β-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-β-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-β-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib–simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/β-catenin signaling-dependent down-regulation of survivin and apoptosis induction.     

Design and synthesis of quinazolinones as EGFR inhibitors to overcome EGFR resistance obstacle

The epidermal growth factor receptor (EGFR) T790M mutant is found in about 50% of clinically acquired resistance to gefitinib among patients with non-small cell lung cancer (NSCLC). New derivatives of 4(3H)-quinazolinones were synthesized and evaluated for their inhibitory activity against NSCLC. The results of the study demonstrated that compound 79, 7-chloro-3-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)-2-phenylquinazolin-4(3H)-one was found to be the most potent compounds of the series with IC₅₀ value of 0.031 μM against mutant T790M/L858R EGFR. Compounds 15, 51, 73, 75, 78, 79 and 96 were less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index. The obtained results showed that compounds 15, 51, 73, 75, 78, 79 and 96 could be the promising template to overcome drug resistance mediated by the EGFR T790 Mutant.     

In silico evaluation of the resistance of the T790M variant of epidermal growth factor receptor kinase to cancer drug Erlotinib

Epidermal growth factor receptor kinase is implicated in cancer development due to either overexpression or activation variants in its functional intracellular kinase domain. Threonine to methionine (Thr 790 Met) is one such variant observed commonly in patients showing resistance to kinase inhibitor drug Erlotinib. Two mechanisms for resistance have been proposed (1) steric hindrance and (2) enhanced binding to ATP. In this study, we employed molecular dynamics simulations and studied both the mechanisms. Extensive simulations and free energy of binding analyses has shown that steric hindrance does not explain appropriately the mechanism for resistance against Erlotinib therapy for this variant. It has been observed that conformational switching from an intermediate intrinsically disordered C-helix conformation is required for completion of the kinase’s catalytic cycle. Our study substantiates that T790M variant has greater tendency for early transition to this intrinsically disordered C-helix intermediate state. We propose that enhanced catalytic efficiency in addition to enhanced ATP binding explains mechanism of T790M resistance to drug Erlotinib.     

Discovery of hybrid Hsp90 inhibitors and their anti-neoplastic effects against gefitinib-resistant non-small cell lung cancer (NSCLC)

Heat shock protein 90 (Hsp90) represents an attractive cancer therapeutic target due to its role in the stabilization and maturation of many oncogenic proteins. We have designed a series of hybrid Hsp90 inhibitors by connecting the resorcinol ring of VER-49009 (2) and the trimethoxyphenyl ring of PU3 (3) using structure-based approach. Subsequent testing established that compound 1f inhibited gefitinib-resistant H1975 cell proliferation, brought about the degradation of Hsp90 client proteins including EGFR, Met, Her2 and Akt and induced the expression of Hsp70. The design, synthesis, and evaluation of 1f are described herein.     

Design,synthesis and biological evaluation of potent EGFR kinase inhibitors against 19D/T790M/C797S mutation

The efficacy of EGFR inhibitors is frequently affected by acquired resistance. EGFR^{19D/T790M/C797S} mutation is one of the primary reasons for the emergence of resistance after treatment with the third-generation EGFR inhibitors such as AZD9291, CO1686 and Olmutinib. To overcome the resistance mutation 19D/T790M/C797S, we designed and prepared a series of indole derivatives with the terminal hydroxyl of alkyl chain to increase extra interaction with the Asp855 in the conservative DFG site. Activity evaluation, structure-activity relationship and docking analysis were also carried out. Among them, compound 12e displayed significant inhibitory activity against EGFR^{19D/T790M/C797S} (IC₅₀ = 15.3 nM) and good selectivity over EGFR WT (IC₅₀ > 1000 nM), L858R/T790M (IC₅₀, 156.6 nM) and L858R/T790M/C797S (IC₅₀, 218.3 nM) respectively. Furthermore, 12e exhibited good growth inhibition activity, induced G1 phase cell cycle arrest and apoptosis in BaF3/EGFR^{19D/T790M/C797S} cells by suppressing EGFR phosphorylation signaling pathway. In all, our study might provide a novel structural design method and lay the solid foundation for the development of the 4th generation EGFR^{19D/T790M/C797S} inhibitors.     

Structure based designing of triazolopyrimidone-based reversible inhibitors for kinases involved in NSCLC

Secondary acquired mutant EGFR (L858R-T790M) overexpressed NSCLC forms one of the prevalent form of resistant NSCLC. Another subset of resistant NSCLC includes amplified cMET in mutant EGFR derived tumours. Thus, in continuation to our previous work on these two major targets of resistant NSCLC, i.e., EGFR (L858R-T790M) and cMET, we are hereby reporting reversible inhibitors of these kinases. Out of 11 lead molecules reported in our previous study, we selected triazolo-pyrimidone (BAS 09867482) scaffold for further development of small molecule dual and reversible inhibitors. Analogues of lead with different substituents on the side ring were sketched and docked in both the target kinases, followed by molecular dynamic simulations. Analogues maintaining hydrophobic interaction with M790 in secondary acquired mutant EGFR (L858R-T790M) were selected and duly synthesized. In vitro biochemical evaluation of these molecules against EGFR (L858R-T790M) and cMET kinase, along with EGFR (L858R) kinase disclosed that three molecules were having significant dual kinase inhibitory potential with IC₅₀ values well below 100 nM. Further, in vitro anti-proliferative assay against three cell lines (A549, A431 and H460) was performed. Out of all, two compounds were having significant potency against these cell lines.     

C-2 (E)-4-(Styryl)aniline substituted diphenylpyrimidine derivatives (Sty-DPPYs) as specific kinase inhibitors targeting clinical resistance related EGFRT790M mutant

With the aim to overcome the drug resistance induced by the EGFR T790M mutation (EGFR^T790M), herein, a family of diphenylpyrimidine derivatives (Sty-DPPYs) bearing a C-2 (E)-4-(styryl)aniline functionality were designed and synthesized as potential EGFR^T790M inhibitors. Among them, the compound 10e displayed strong potency against the EGFR^T790M enzyme, with the IC₅₀ of 11.0 nM. Compound 10e also showed a higher SI value (SI = 49.0) than rociletinib (SI = 21.4), indicating its less side effect. In addition, compound 10e could effectively inhibit the proliferation of H1975 cells harboring the EGFR^T790M mutation, within the concentration of 2.91 μM. Significantly, compound 10e has low toxicity against the normal HBE cell (IC₅₀ = 22.48 μM). This work provided new insights into the discovery of potent and selective inhibitor against EGFR^T790M over wild-type (EGFR^WT).     

Wif1 hypermethylation as unfavorable prognosis of non-small cell lung cancers with EGFR mutation

Lung cancer is a leading cause of cancer-related mortality across the world and tobacco smoking is the major risk factor. The Wnt signaling pathway is known to be involved in smoke-induced tumorigenesis in the lung. Promoter hypermethylation of Wnt inhibitory factor 1 (Wif1) has become a common event in a number of human tumors. Using a methylation-specific PCR, hypermethylation of the Wif1 gene promoter was evaluated in 139 primary nonsmall cell lung cancers (NSCLCs) and its correlation with clinicopathological and prognostic parameters was evaluated. Methylation of Wif1 was observed in 47.5% and 20.9% of neoplastic and adjacent normal lung tissues, respectively. Its methylation rate tended to be higher in stage I than stages II-IIIA. Results of Kaplan-Meier analysis showed no significant difference in overall survival according to Wif1 methylation status. However, Wif1 methylation showed an association with unfavorable prognosis of adenocarcinoma (AC) patients with EGFR mutation. According to our current findings, Wif1 promoter methylation is an early, frequent event as an epigenetic field manner and could be considered as a useful prognostic marker for AC patients with EGFR mutation. Further investigation into the therapeutic potential of this finding is warranted.     

Design,synthesis and SAR study of 2-aminopyrimidines with diverse Michael addition acceptors for chemically tuning the potency against EGFRL858R/T790M

The covalent binding nature of irreversible kinase inhibitors potentially increases the severity of “off-target” toxicity. Based on our continual strategy of chemically tuning the Michael addition acceptors, herein, we further explore the relationship among the electronic nature of Michael addition acceptors and EGFR^T790M mutation selectivity as well as “off-target” toxicity balance. By perturbing the electronic nature of acrylamide moiety, compound 8a with a chloro-group at the α-position of the Michael addition acceptor was identified. It was found that 8a retained the excellent EGFR ^L858R/T790M potency (IC₅₀ = 3.9 nM) and exhibited good anti-proliferative activities against the gefitinib-resistant NCI-H1975 cells (IC₅₀ = 0.75 μM). Moreover, 8a displayed a significant EGFR^WT selectivity and much weaker inhibitory activity against non-EGFR dependent SW620 cell and COS7. Preliminary study showed that 8a could arrest NCI-H1975 cells in G0/G1 phase. This work provides a promising chemical tuned strategy for balancing the mutant-EGFR potency and selectivity as well as “off-target” toxicity.     

Discovery of selective EGFR modulator to inhibit L858R/T790M double mutants bearing a N-9-Diphenyl-9H-purin-2-amine scaffold

Based upon the modeling binding mode of marketed AZD9291 with T790M, a series of N-9-Diphenyl-9H-purin-2-amine derivatives were designed and synthesized with the purpose to overcome the drug resistance resulted from T790M/L858R double mutations. The most potent compound 23a showed excellent enzyme inhibitory activities and selectivity with nanomolar IC₅₀ values for both the single T790M and double T790M/L858R mutant EGFRs, and was more than 8-fold selective for wild type EGFR. Compound 23a displayed strong antiproliferative activity against the H1975 non-small cell lung cancer (NSCLC) cells bearing T790M/L858R. And it was less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index.     

Novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one derivatives as potential inhibitors of chorismate mutase

A series of novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one (DHPM) derivatives were designed, synthesized and evaluated in vitro as potential inhibitors of chorismate mutase (CM). All these compounds were prepared via a multi-component reaction (MCR) involving sequential I₂-mediated Biginelli reaction followed by Cu-free Sonogashira coupling. Some of them showed promising inhibitory activities when tested at 30 μM. One compound showed dose dependent inhibition of CM with IC₅₀ value of 14.76 ± 0.54 μM indicating o-alkynylphenyl substituted DHPM as a new scaffold for the discovery of promising inhibitors of CM.     

Staurosporine scaffold–based rational discovery of the wild‐type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog‐sensitive kinase technology

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first‐line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small‐molecule kinase inhibitors in EGFR‐targeted chemotherapy. Previously, the reversible pan‐kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR^T790M) over wild‐type kinase (EGFR^WT), suggesting that the staurosporine scaffold is potentially to develop the wild‐type sparing reversible inhibitors of EGFR^T790M. Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold–based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico–in vitro analog‐sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild‐type sparing inhibitors UCN‐01, UCN‐02, AFN941, and SB‐218078 with high or moderate selectivity of 30‐, 45‐, 5‐, and 8‐fold for EGFR^T790M over EGFR^WT, respectively, which are comparable with or even better than that of the parent compound staurosporine (24‐fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN‐02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase‐inhibitor binding. A hydroxyl group of UCN‐02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN‐02 than staurosporine for mutant over wild‐type kinase.     

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ET_A receptor (R) and ET_BR on cancer cells. High levels of tumor ET-1 and ET_AR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ET_AR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ET_AR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca²⁺ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.