Design, synthesis and biological evaluation of EGFR kinase inhibitors that spans the orthosteric and allosteric sites.

Acquired drug resistance occurred in the treatment of non-small-cell lung cancer is a persistent challenge, especially in EGFR mutant type. In this study, we present design, synthesis and biological evaluation of novel quinazoline and pyrrolopyrimidine derivatives that simultaneously occupy the orthosteric and allosteric sites of EGFR. Among them, compound A-7 was confirmed as a potential EGFRL858R/T790M/C797S and EGFRDel19/T790M/C797S inhibitor. Docking study indicated that compound A-7 could simultaneously occupy two binding sites of EGFR and form three key H-bonds with the residues Met793, Lys745 and Met766 in two regions.

Discovery of novel conjugates of quinoline and thiazolidinone urea as potential anti-colorectal cancer agent.

Based on the obtained SARs, further structural optimisation of compound BC2021-104511-15i was conducted in this investigation, and totally ten novel quinoline derivates were designed, synthesised and optimised for biological activity. Among them, compound 10a displayed significant in vitro anticancer activity against COLO 205 cells with an IC50 value of 0.11 µM which was over 90-fold more potent than that of Regorafenib (IC50>10.0 µM) and Fruquintinib (IC50>10.0 µM). Furthermore, compound 10a exhibited over 90-fold selectivity towards COLO 205 relative to human normal colorectal mucosa epithelial cell FHC cells. Flow cytometry study demonstrated that compound 10a could induce apoptosis in COLO 205 cells, however, it could not induce cell cycle arrest in COLO 205 cells. The results of preliminary kinase profile study showed that compound 10a was a potential HGFR and MST1R dual inhibitor, with IC50 values of 0.11 µM and 0.045 µM, respectively.

Discovery of 4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)-N,N-diethylpiperidine-1-carboxamide as kinase inhibitor for the treatment of colorectal cancer.

In this study, a novel series of 4,6,7-trisubstituted quinoline analogues bearing thiazolidinones were designed and synthesized based on our previous study. Among them, the most potent compound 15i, 4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)-2-fluorophenoxy)-6-methoxyquinolin-7-yl)oxy)-N,N-diethylpiperidine-1-carboxamide was identified as a multi-kinase inhibitor. The results of MTT assay revealed in vitro antitumor activities against HT-29 cells of compound 15i with an IC50 value of 0.19 µM which was 14.5-fold more potent than that of Regorafenib. In the cellular context, significant antiproliferation, cytotoxicity and induction of apoptosis on HT-29 cells in a dose- and time-dependent manner were confirmed by IncuCyte live-cell imaging assays. Moreover, compound 15i strongly induced apoptosis by arresting cell cycle into the G2/M phase. No antiproliferation and cytotoxicity against human normal colorectal mucosa epithelial cell FHC was observed at 10.0 µg/mL or lower concentrations which indicated that the toxicity to normal cells of compound 15i was much lower than that of Regorafenib. Based on the above findings, further structural modification will be conducted for the development of more potent kinase inhibitors as anticancer agents.

Optimization and biological evaluation of nicotinamide derivatives as Aurora kinase inhibitors.

Aurora kinases are known to be overexpressed in various solid tumors and implicated in oncogenesis and tumor progression. A series of nicotinamide derivatives were synthesized and their biological activities were evaluated, including kinase inhibitory activity against Aur A and Aur B and in vitro antitumor activity against SW620, HT-29, NCI-H1975 and Hela cancer cell lines. In addition, the study of antiproliferation, cytotoxicity and apoptosis was performed meanwhile. As the most potent inhibitor of Aur A, 4-((3-bromo-4-fluorophenyl)amino)-6-chloro-N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)nicotinamide (10l) showed excellent antitumor activity against SW620 and NCI-H1975 with IC50 values were 0.61 and 1.06 µM, while the IC50 values of reference compound were 3.37 and 6.67 µM, respectively. Furthermore, binding mode studies indicated that compound 10l forms better interaction with Aur A.

Discovery of thiazolidin-4-one urea analogues as novel multikinase inhibitors that potently inhibit FLT3 and VEGFR2.

A series of novel thiazolidine-4-one urea analogues were designed, synthesized and biologically evaluated. The structure-activity relationship (SAR) at several positions of the scaffolds was investigated and its binding mode was analyzed by molecular modeling studies. Compound 17b proved to be the most potent one, and IC50 values against A549 and HT-29 cancer cell lines were 0.65 µM and 0.11 µM, respectively. The results of kinase profile demonstrated that compound 17b is a multikinase inhibitor that potently inhibits FLT3 (IC50 = 8.6 nM) and VEGFR2 (IC50 = 18.7 nM). The results of real-time live-cell imaging indicated that compound 17b showed excellent cytotoxicity and anti-proliferative activity against HT-29 cancer cells in a time- and dose-dependent manner, which was significantly potent than that of Cabozantinib. In addition, in vitro antitumor activity was associated with inducing cancer cell apoptosis and suppression of cancer cell migration.

Danggui Buxue Tang, an ancient Chinese herbal decoction, protects ß-amyloid-induced cell death in cultured cortical neurons.

BACKGROUND: Danggui Buxue Tang (DBT) is a historical Chinese herbal decoction, and which has more than 800 years of applications. This herbal decoction solely contains two materials: Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) at a weight ratio of 5:1. Clinically, DBT aims to improve anemia syndrome. In complementary and alternative medicine theory, the cause of neurodegenerative disease is proposed to be related with anemia. In line to this notion, low levels of hemoglobin and red blood cell have been reported in patients suffering from Alzheimer's disease (AD), a chronic neurodegenerative disease caused by ß-amyloid peptide (Aß) accumulation. Therefore, we would like to probe the neuroprotective functions of this ancient herbal formula in vitro. METHOD: The neuroprotective effects of DBT in the Aß-induced cell death were detected in cultured cortical neurons by multiple techniques, i.e. confocal and western blot. RESULTS: In the cultures, application of DBT reduced Aß-induced apoptosis rate in a dose-dependent manner. In Aß-treated cortical neurons, the expression ratio of Bcl2 to Bax was altered by DBT. In parallel, application of DBT markedly suppressed the Aß-induced expressions of apoptotic markers, i.e. cleaved-caspase 3/9 and PARP. CONCLUSION: Taken these results, DBT shows promising protective effects against Aß-induced stress or insult in cultured neurons.

Separation and purification of five alkaloids from Aconitum duclouxii by counter-current chromatography.

C19 -diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter-current chromatography. In the process of separation, the critical conditions of counter-current chromatography were optimized. The two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1, 24.1 mg of 2, 250.6 mg of 3, 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra-high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation.

Identification of novel aminopyrimidine derivatives for the treatment of mutant NSCLC.

Starting from the binding mode of allosteric EGFR inhibitor JBJ-04-125-02 and the key pharmacophore of the third-generation EGFR inhibitors, we designed and synthesized a novel series of EGFR inhibitors, represented by (R)-N-(4-((2-aminopyrimidin-4-yl)amino)phenyl)-2-(5-(4-(4-methylpiperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenylacetamide (6q). Docking study demonstrated that top compound 6q spanned orthosteric and allosteric sites of EGFR, and formed three key H-bonds with the residues Asp855, Lys745, and Met793 located in two sites. Biological evaluation indicated that compound 6q showed potential inhibitory activity against Ba/F3-EGFRL858R/T790M/C797S and Ba/F3-EGFRDel19/T790M/C797S cells, with IC50 values of 0.42 µM and 0.41 µM, respectively. Furthermore, compound 6q showed excellent activity against mutant NSCLC cell line NCI-H1975-EGFRL858R/T790M/C797S cells, with IC50 value of 0.82 µM which was superior to that of osimertinib (IC50 = 2.94 µM), JBJ-04-125-02 (IC50 = 3.66 µM), and coadministration of JBJ-04-125-02 and osimertinib (IC50 = 1.25 µM). Cell cycle arrest and cell apoptosis assay indicated that compound 6q could promote apoptosis of NCI-H1975-EGFRL858R/T790M/C797S cells at the concentration of 0.8 µM and no obvious cell cycle arrest was found.

Discovery and optimization of novel 4-phenoxy-6,7-disubstituted quinolines possessing semicarbazones as c-Met kinase inhibitors.

A novel series of N(1)-(3-fluoro-4-(6,7-disubstituted-quinolin-4-yloxy)phenyl)-N(4)-arylidenesemicarbazide derivatives were synthesized and evaluated for their c-Met kinase inhibition and cytotoxicity against A549, HT-29, MKN-45 and MDA-MB-231 cancer cell lines in vitro. Several potent compounds were further evaluated against three other cancer cell lines (U87MG, NCI-H460 and SMMC7721). Most of compounds tested exhibited moderate to excellent activity. The studies of SARs identified the most promising compound 28 (c-Met IC50=1.4nM) as a c-Met kinase inhibitor. In this study, a promising compound 28 was identified, which displayed 2.1-, 3.3-, 48.4- and 3.6-fold increase against A549, HT-29, U87MG and NCI-H460 cell lines, respectively, compared with that of Foretinib.

Synthesis and biological evaluation of 4-phenoxy-6,7-disubstituted quinolines possessing semicarbazone scaffolds as selective c-Met inhibitors.

Novel quinoline derivatives bearing acyclic semicarbazones were prepared and their chemical structures as well as the relative stereochemistry were confirmed. All the synthesized compounds were evaluated for their c-Met kinase inhibitory activity and their cytotoxicity against the cell lines HT-29, MKN-45, and MDA-MB-231 in vitro. Several potent compounds were further evaluated against A549 cells. Most compounds displayed moderate to excellent activity, and the structure-activity relationship studies identified the most promising compound 35 as a selective c-Met kinase inhibitor (IC50 = 4.3 nM). Compound 35 showed a 3.5- and 18.8-fold increase in cytotoxicity in vitro against HT-29 and A549 cells, respectively, compared to that of foretinib. Poor off-target effects of compound 35 were further confirmed by the antiproliferative activity against the c-Met inhibition less sensitive MDA-MB-231 cell line (IC50 = 0.77 µM).

Dual target inhibitors based on EGFR: Promising anticancer agents for the treatment of cancers (2017-).

The EGFR family play a significant role in cell signal transduction and their overexpression is implicated in the pathogenesis of numerous human solid cancers. Inhibition of the EGFR-mediated signaling pathways by EGFR inhibitors is a widely used strategy for the treatment of cancers. In most cases, the EGFR inhibitors used in clinic were only effective when the cancer cells harbored specific activating EGFR mutations which appeared to preserve the ligand-dependency of receptor activation but altered the pattern of downstream signaling pathways. Moreover, cancer is a kind of multifactorial disease, and therefore manipulating a single target may result in treatment failure. Although drug combinations for the treatment of cancers proved to be successful, the use of two or more drugs concurrently still was a challenge in clinical therapy owing to various dose-limiting toxicities and drug-drug interactions caused by pharmacokinetic profiles changed. Therefore, a single drug targeting two or multiple targets could serve as an effective strategy for the treatment of cancers. In recent, drugs with diverse pharmacological effects have been shown to be more advantageous than combination therapies due to their lower incidences of side effects and more resilient therapies. Accordingly, dual target-single-agent strategy has become a popular field for cancer treatment, and researchers became more and more interest in the development of novel dual-target drugs in recent years. In this review, we briefly introduce the EGFR family proteins and synergisms between EGFR and other anticancer targets, and summarizes the development of potential dual target inhibitors based on wild-type and/or mutant EGFR for the treatment of solid cancers in the past five years. Additionally, the rational design and SARs of these dual target agents are also presented in detailed, which will lay a significant foundation for the further development of novel EGFR-based dual inhibitors with excellent druggability.

Identification of (S)-1-(2-(2,4-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinolin-4-yl)oxy)-3,5-difluorophenyl)urea as a potential anti-colorectal cancer agent.

In our previous study, 1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinolin-4-yl)oxy)-3,5-difluorophenyl)urea (1) was obtained as a potent tyrosine kinase inhibitor. Further structural optimization was performed in this investigation, and a series of novel quinoline derivates were designed, synthesized and evaluated for their biological activity. Among them, compound 8m possessed nanomolar c-Met and Ron inhibitory activity, with IC50 values of 4.32 nM and 2.39 nM, respectively. Kinase profile study demonstrated that it could also inhibit ABL, PDGFRß, AXL, RET, and FLT3 with submicromolar potency. It also exhibited moderate to excellent cytotoxic activity against different types of human cancer cell lines, especially against COLO 205 cells (IC50 = 0.035 µM) which was remarkably superior to that of Cabozantinib (IC50 = 6.6 µM) and Fruquintinib (IC50 > 10.0 µM). Compared to ( ± )-8m, isomer (S)-8m and (R)-8m showed similar kinase inhibitory activity against c-Met/RON and in vitro anticancer activity against COLO 205 cells. Differently, compound (S)-8m showed an over 238-fold selectivity toward COLO 205 (IC50 = 0.042 µM) cells to FHC cells (IC50 > 10.0 µM), which indicated its low cytotoxicity against human normal tissue cells. Flow cytometry study demonstrated that compound (S)-8m could significantly induce apoptosis in COLO 205 cells in a dose-dependent manner. Cell cycle arrest assays showed that compound (S)-8m could not arrest the cell-cycle progression due to the massive dead cells.

Astragaloside IV, a saponin from Astragalus membranaceus var. mongholicus, induces expressions of heme recycle proteins via signaling of Nrf2/ARE in cultured macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM) theory, "Qi" is classified as energetic essence supporting the life activities in human. "Blood" is categorized as nourishing essence and circulating in the body. "Blood" and "Qi" have an intimate relationship. Astragali Radix (AR; root of Astragalus membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao) has a broad spectrum of application for "Qi-Blood" enrichment. Astragaloside IV, a major saponin in AR, has therapeutic functions in erythropoietic, cardiovascular and immune systems. However, the efficacy of astragaloside IV in erythrophagocytosis has not been elucidated. AIM OF THE STUDY: The possible functions of astragaloside IV in heme iron recycling during erythrophagocytosis in cultured macrophage were elucidated. METHODS: The translational and transcriptional expressions of heme recycling enzymes were determined after incubating of astragaloside IV for 24 h in cultured macrophage. RESULTS: In astragaloside IV-treated macrophage, the expressions, both RNA and protein levels, of regulators of heme recycling, e.g. heme oxygenase-1 (HO-1), ferroportin (FPN), biliverdin reductase A and B (BVRA, BVRB), were markedly induced in dose-dependent manners. In parallel, the transcriptional activity of antioxidant response element, cloned within an expression vector as pARE-Luc and transfected in cultured macrophages, was markedly induced after a challenge with astragaloside IV in a dose-dependent manner. Moreover, the translocation of Nrf2, a transcriptional factor in regulating expression of heme recycling protein, was induced by astragaloside IV, leading to an enrichment at nucleus fraction. CONCLUSION: Astragaloside IV shed lights in enhancing the expression of heme recycle proteins via Nrf2/ARE signaling pathway.

Identification of novel quinoline analogues bearing thiazolidinones as potent kinase inhibitors for the treatment of colorectal cancer.

In this investigation, a novel series of quinoline analogues bearing thiazolidinones were designed and synthesized based on our previous study. Among them, the most potent compound 11k, 4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide, possessed submicromolar c-Met and Ron inhibitory activities. In addition, enzymatic assays against a mini-panel of kinases (c-Kit, B-Raf, c-Src, IGF1R, PDGFRα and AXL) were performed, the results showed that compound 11k exhibited moderate inhibitory activity against PDGFRα, c-Src and AXL. MTT assay revealed in vitro antitumor activities against HT-29 cells of compound 11k with an IC50 value of 0.31 µM which was 9.3- and 34.2-fold more potent than that of Regorafenib (IC50 = 2.87 µM) and Cabozantinib (IC50 = 10.6 µM). Preliminary antitumor mechanisms were also investigated by cellular assays. Considerable cytotoxicity, antiproliferation and induction of apoptosis of compound 11k in a dose- and time-dependent manner were confirmed by IncuCyte live-cell imaging assays. Treatment with compound 11k caused slight G2-or M-phase arrest in HT-29 cells. Further cell selectivity of compound 11k showed that it was not active against human normal colorectal mucosa epithelial cell FHC at 10.0 µg/mL. The above results support further structural modification of compound 11k to improve its inhibitory activity, which will lead to more potent anticancer agents.

The effect of methanol extract from Saussurea involucrata in the lipopolysaccharide-stimulated inflammation in cultured RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussureae Involucratae Herba (SIH), known as "snow lotus" in Uyghur and/or Chinese medicines, is generated from the dried aerial part of Saussurea involucrata (Kar. et Kir.) Sch.-Bip. (Asteraceae). The major pharmaceutical value of SIH has been recorded in China Pharmacopoeia, i.e. to balance the immune system, and thus SIH is commonly used for rheumatoid arthritis treatment. Nevertheless, the detailed mechanism of SIH in immune function is still unresolved. AIM OF THE STUDY: Here, we employed macrophage RAW 264.7 cell as a model to demonstrate the signaling pathways, triggered by SIH, in regulating the LPS-induced inflammation. METHODS: The application of SIH methanolic extract suppressed the expression of cytokines, a hallmark of chronic inflammation, in lipopolysaccharide (LPS)-stimulated cultures. RESULTS: The anti-inflammatory functions of SIH were shown to be triggered via NF-κB/PI3K/MAPK signaling pathways by revealing the specific biomarkers, i.e. translocation activities of NF-κB and phosphorylations of Erk1/2, JNK and Akt. CONCLUSION: The aforementioned results showed the underlying action mechanism of SIH in chronic inflammation mitigation, and which might shed light on clinical applications of SIH in traditional Chinese and/or Uyghur medicines.

Saussureae Involucratae Herba (Snow Lotus): Review of Chemical Compositions and Pharmacological Properties.

Saussureae Involucratae Herba is the dried ground part of Saussurea involucrata (Kar. et Kir.) Sch.-Bip, which is also named as "Snow lotus" and being used in traditional Uyghur and/or Chinese medicine. This rare herb can be found at 4,000 m elevation in western part of Tianshan Mountain, Xinjiang China. According to China Pharmacopoeia (2015), the major pharmaceutical values of "Snow lotus" (Xue liánhua in Chinese) are alleviating rheumatoid arthritis, accelerating blood circulation and mitigating other "cold" syndromes. Traditionally, the clinical application of "Snow lotus" includes the treatments in inflammation-associated disorder, blood circulation acceleration and heat and dampness elimination. Recent studies suggested that "Snow lotus" possessed therapeutic effects associating with anti-cancer, anti-oxidation, adipogenesis suppression and neuroprotection activities, which were proposed to be related with its bioactive constitutes, i.e. acacetin, hispidulin, and rutin. In the present review, we aim to summarize pharmacological effects and underlying cell signaling pathways of "Snow lotus" in treating various medical problems.

Discovery of N1-(4-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinolin-4-yl)oxy)-3,5-difluorophenyl)-N3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)urea as a multi-tyrosine kinase inhibitor for drug-sensitive and drug-resistant cancers treatment.

A series of 21 novel N1-(2-aryl-1,3-thiazolidin-4-one)-N3-aryl urea derivatives based on the previously identified lead compound I were synthesized and biologically characterized. The most promising compound 19a was identified as a multi-tyrosine kinase inhibitor, including c-Met, Ron, c-Kit, AXL and IGF-1R, etc. The results of real-time live-cell imaging indicated that compound 19a showed improved cytotoxicity and anti-proliferative activity against HT-29 cancer cells in a time- and dose-dependent manner, with an efficacy that was significantly greater than Cabozantinib. Flow cytometry and western blot analysis demonstrated the fact that anticancer activity was closely related with cancer cell apoptosis and the blockade of the phosphorylation of c-Met and its downstream signaling ERK and Akt. Furthermore, compound 19a also displayed slightly stronger effects on HT-29 cancer cells migration than that of Cabozantinib.

Identification of novel N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl ureas showing potent multi-tyrosine kinase inhibitory activities.

A total of 29 novel compounds bearing N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl ureas were designed, synthesized and evaluated for their biological activities. The structure-activity relationships (SARs) and binding modes of this series of compounds were clarified together. Compound 29b was identified possessing high potency against multi-tyrosine kinases including Ron, c-Met, c-Kit, KDR, Src and IGF-1R, etc. In vitro antiproliferation and cytotoxicity of compound 29b against A549 cancer cell line were confirmed by IncuCyte live-cell imaging.

Synthesis and biological evaluation of novel 2-(2-arylmethylene)hydrazinyl-4-aminoquinazoline derivatives as potent antitumor agents.

Two series of novel 2-(2-arylmethylene)hydrazinyl-4-aminoquinazoline derivatives were synthesized and evaluated for their cytotoxicity against H-460, HT-29, HepG2 and SGC-7901 cancer cell lines in vitro. Most compounds displayed moderate to excellent activity, with IC(50) values ranging from 0.015 to 4.09 µM against all tested cell lines, respectively. The most promising compound 9p (E)-2-(2-((1-(2,3-dichlorobenzyl)-1H-imidazol-2-yl)methylene)hydrazinyl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine with IC(50) values of 0.031 µM, 0.015 µM, 0.53 µM and 0.58 µM, which was 4- to 224 times more active than references 10 and Iressa, had emerged as a lead for further structural modifications.