Twenty novel pyrimidine derivatives of deuterated and non-deuterated aminobenzenesulfonamide molecules were designed and synthesized, and their potential application as epidermal growth factor receptor (EGFR) inhibitors for non-small cell lung cancer (NSCLC) was evaluated. The anti-tumor cell proliferation assay demonstrated that most of the synthesized compounds had good inhibitory activity against H1975-EGFR L858R/T790M and PC9-EGFR Del19 tumor cells, with IC₅₀ values in the nanomolar range. Among them, the IC₅₀ values of the lead compound 12b against H1975 and PC9 cells were 12 nM and 16 nM respectively. In rat liver microsomal stability assays, the half-life of deuterated compound 12b was significantly longer than that of its non-deuterated analog 12l. Mechanistic studies revealed that 12b inhibits tumor cell proliferation, induces cell apoptosis, and suppresses tumor cell migration by binding to EGFR and inhibiting its phosphorylation as well as downstream signaling pathways. In H1975 xenograft nude mouse models, 12b achieved a tumor growth inhibition (TGI) rate of 94% at an oral dose of 10 mg/kg. Collectively, these results suggest that 12b holds promising research potential as a highly effective anti-cancer agent for NSCLC.

Ten undescribed coumarins, including five pairs of enantiomers (±)-toddacoumalin C (3a/3b), (±)-toddacoumalin D (4a/4b), (±)-toddacoumalin E (5a/5b), (±)-toddacoumalin F (6a/6b), and (±)-toddacoumalin I (9a/9b), along with 13 known compounds (11-23) were isolated from the roots of Toddalia asiatica. Their structures were elucidated by spectroscopic analyses, and absolute configurations were determined via electronic circular dichroism (ECD) calculations and Mo2(OAc)4-induced ECD. Notably, the isolation of toddacoumalin H (8) provided crucial evidence to revise the double bond geometry of the previously reported toddasirin F from E to Z. All compounds were assessed for their anti-inflammatory potential against lipopolysaccharide-induced nitric oxide (NO) production in RAW264.7 cells. Compound 17 emerged as the most potent inhibitor, exhibiting an IC50 value of 1.109 μM, which was approximately 5-fold more potent than the positive control indomethacin (IC50 = 5.625 μM). Considering the interplay between oxidative stress and inflammation, we further evaluated the antioxidant capacity of the isolates using the FRAP assay. Compounds 10 and 17 exhibited promising antioxidant effects, with the values (expressed as Fe2+ equivalent, mM) of 5.786 ± 0.049 and 2.275 ± 0.070, respectively. Cytotoxicity assays further confirmed that compound 17 had an acceptable safety profile in normal cells. Collectively, these findings highlight compound 17 as a promising dual-functional lead candidate with significant anti-inflammatory and antioxidant activities.

c-Met inhibitors have demonstrated encouraging efficacy in the treatment of non-small cell lung cancer. However, some of these drugs are conditionally approved and still face certain limitations and challenges, primarily manifested as poor blood-brain barrier penetration, off-target toxicity, drug resistance, and low oral bioavailability. These factors restrict their clinical efficacy and widespread application. To discover novel c-Met inhibitors with high potency, minimal toxic side effects, and the ability to penetrate the blood-brain barrier, we designed and synthesized 33 new pyrimidine derivatives using Tepotinib as the lead, employing bioisosterism and conformational restriction strategies. Their anti-tumor activities were evaluated in vitro and in vivo. Among these derivatives, the optimal compound 11g exhibited IC50 values of 4.01 nM and 3.50 nM against MHCC97H and EBC-1 cells, respectively. In the EBC-1 xenograft mouse model, at a dose of 4 mg / kg, 11g achieved a tumor growth inhibition (TGI) rate of 64.9%, which was significantly higher than that of Tepotinib (33.5%) at the same dose. Pathological evaluation further confirmed that 11g possessed improved safety and reduced toxic side effects. In addition, 11g displayed superior blood-brain barrier permeability and metabolic stability compared with the lead compound. Mechanistic studies demonstrated that 11g effectively inhibits tumor cell proliferation and migration by binding to the c-Met protein and induces cell apoptosis. In summary, as a novel and highly potent c-Met inhibitor, 11g shows promising potential for the treatment of NSCLC, particularly in the prevention and treatment of tumor brain metastasis.

Gastric and colorectal cancers are common and deadly across the globe. Preclinical findings propose that the pairing of MET inhibitors with anti-inflammatory drugs could synergistically impede tumor growth and reshape the tumor microenvironment. This study introduces AspMet, a novel dual-targeting inhibitor of c-Met and COX-2. In vitro experiments demonstrated that AspMet significantly inhibited the proliferation of MKN45 (IC50 = 1.05 ± 0.02 nM) and SW480 (IC50 = 1.32 ± 0.01 μM) cell lines. The experimental data indicate that AspMet effectively blocks several cancer-promoting signaling pathways, including c-Met、TRKB、COX-2 and HIF-1α, significantly inhibits epithelial-mesenchymal transition, thus decreasing tumor cell migration and invasion, and causes DNA damage, resulting in G0/G1 cell cycle arrest and the initiation of apoptosis. Furthermore, AspMet has strong anti-angiogenic properties. In animal models, AspMet significantly reduced the growth of subcutaneous tumors in both gastric and colorectal cancers，and it has an extremely high bioavailability. Therefore, the dual inhibition strategy targeting c-Met and COX-2 offers a promising novel approach for the treatment of cancers, particularly inflammatory cancers.

The development of novel anti-cancer compounds that overcome acquired resistance to third-generation EGFR inhibitors such as osimertinib is of great significance. This study designed and synthesized eighteen methoxyquinazoline sulfonamide derivatives, and evaluated their anti-tumor activity using three EGFR triple-mutant tumor cell lines. Among them, the optimal compound 9f exhibited IC₅₀ values of 33.3-95.3 nM against Baf3-L858R/C797S/T790M, Baf3-Del19/C797S/T790M, and H1975-L858R/C797S/T790M cancer cell lines, which were consistent with the results of colony formation assays and 3D spheroid suspension culture assays. In anti-tumor experiments in mice bearing H1975-L858R/C797S/T790M tumors, compound 9f achieved a tumor growth inhibition rate of 67.3%. Mechanistic studies showed that 9f exerts excellent anti-inflammatory effects, including downregulating the expression of inflammation-related proteins iNOS, COX-2, and NF-κB (p65) in Raw264.7 cells (Western blot assay), increasing NO secretion in LPS-stimulated Raw264.7 cells (fluorescence intensity assay), and reducing IL-6 secretion in Raw264.7 and THP-1 cells (ELISA). Mechanistic studies also indicated that 9f promotes apoptosis of tumor cells and inhibits phosphorylation of the key tumor growth protein EGFR.

Twenty new 5-(1H-1,2,4-triazol-3-yl)-1,2,4-oxadiazole derivatives were synthesized and evaluated as OXPHOS inhibitors against cancers. The anti-proliferation assay exhibited that most of the compounds possessed good to excellent inhibitory activity on PC9 non-small cell lung cancer cells and Bxpc-3 pancreatic cancer cells. Among them, the optimal compound 28c, provided 0.0123 μM of IC50 value on PC9 cells, 0.25 μM and 0.0173 μM of the IC50 values against Bxpc-3 cells in glucose and galactose medium, respectively. In PC9 xenograft nude mice, TGI of 28c is 74.4 % compared with 44.16 % of the reference IACS-010759 when oral administration at dosage of 7.5 mg/kg. Mechanism research showed that 28c can bind with respiratory chain complex I, modulate the levels of NADH, NAD+ et al. in PC9 cells, activate cellular ROS and down regulate NRF2 and cause DNA damage in tumor cells.

To discover novel active compounds against non-small cell lung cancer, thirty one amino pyrazine derivatives were synthesized and evaluated as inhibitors against EGFR-mutation cancers. The optimal compound 14a, exhibited 15.4 nM and 18.5 nM of the IC50 values against PC9 and H1975 cancer cell lines, respectively. In H1975 xenograft nude mice, 14a exhibited 90.0 % of the TGI when oral administration at dosage of 10 mg/kg. Kinase activity assay showed that 14a not only has excellent inhibitory activity against EGFR kinase, but also has good activity against JAK2 and JAK3 kinases, exhibiting a dual target characteristics. Mechanism study indicated that 14a could inhibit the phosphorylation of EGFR protein and decrease the active form p-JAK2 for JAK2, induce an increase in intracellular ROS, which may disrupt the balance of the redox system in cancer cells and cause the death of tumor cells. In addition, 14a could increase cellular lipid oxide MDA, meanwhile decrease GSH content, which suggests that 14a have caused ferroptosis in cancer cells, Finally, 14a exhibited high selectivity towards EGFRwt cells with a selectivity ratio of 583.76, which is significant to avoid toxic side effects of drugs.

EGFR and ALK are common driver genes in NSCLC, and more patients with these mutations are being identified due to medical advances. Thus, developing dual-target EGFR/ALK inhibitors is crucial. In this study, 10 novel small molecules were designed and synthesized. CCK8 experiments revealed that compound (-)-9a exhibited the best anti-tumor activity, with IC50 values of 1.08 +/- 0.07 nM for EGFR and 2.395 +/- 0.023 nM for ALK mutant tumor cells. Studies show that compound (-)-9a can inhibit phosphorylated proteins in EGFR, ALK, and BRK signaling pathways and halt the cell cycle, leading to reduced mitochondrial membrane potential and apoptosis in tumor cells. Additionally, (-)-9a not only directly targets tumor cells but also exhibits potential immuneenhancing effects. Furthermore, evaluations conducted in animal models have demonstrated that this drug effectively reduces tumor growth in vivo. In summary, (-)-9a boasts dual-targeting, potent antitumor activity, and immune-enhancing potential, presenting vast potential as a next-gen anticancer drug.

Twenty pyrimidine derivatives with aminophenylsulfonamide moiety were synthesized and evaluated as inhibitors against EGFR-mutation cancers. The anti-proliferation assay showed that most of the synthesized compounds had excellent inhibitory activity against H1975-EGFRL858R/T790M and PC9-EGFRDel19 tumor cells. Among them, the optimal compound 12e, exhibited 0.6 nM and 4 nM of the IC50 values against H1975 cells and PC9 cells, respectively. In PC9 and H1975 xenograft nude mice, TGI of 12e is 98.5 %and 97.7 % when oral administration at dosage of 20 mg/kg. Molecular docking study showed 12e gave preferable affinity upon EGFR then Osimertinib. As for the anti-tumor mechanism, 12e inhibits phosphorylation and downstream signaling by binding to EGFR, then inhibits the proliferation of tumor cell lines, promotes apoptosis, and prohibits the migration and invasion of the tumor cell lines.

Abstract Third-generation cutaneous growth factor receptor tyrosine kinase inhibitors have led to impressive advances in the treatment of non-small cell lung cancer. However, acquired resistance significantly limits the clinical application of these agents, with the epidermal growth factor receptor (EGFR)C797S mutation being the primary resistance mechanism. In this study, a series of novel EGFR kinase inhibitors was designed and synthesized for the treatment of the EGFRC797S mutation in non-small cell lung cancer. Structure activity relationship screening revealed that compound 5d was the most effective inhibitor of the C797S mutation. It strongly inhibited Baf3-EGFRL858R/T790M/C797S and Baf3-EGFR 19del/T790M/C797S cell lines, with IC50 values of 0.01836±0.0065 and 0.025±0.005 μM, respectively. Molecular docking studies showed that 5d binds to the EGFRL858R/T790M/C797S and EGFR19del/T790M/C797S proteins effectively. A mechanistic study showed that 5d induced cell cycle arrest in the G2/M phase. Compound 5d caused cell apoptosis in a dose-dependent manner, accompanied by a significant increase in the level of the apoptotic protein cleaved caspase-3. An in vivo xenograft assay of mice with Baf3-EGFR19del/T790M/C797S cells showed that 5d effectively inhibited tumor growth, with inhibition rates of 45.2% (70 mg/kg) and 55.7% (100 mg/kg) being observed. Therefore, 5d is worthy of further investigation as a fourth-generation EGFR inhibitor.

In this study, we discovered and identified a novel AXL/triple angiokinase inhibitor 11b by rational structural modification based on the structure of triple angiokinase inhibitor Nintedanib. We found that 11b potently inhibited AXL expression with the IC50 value of 3.75 nM and possessed similar inhibitory activity on KDR as Nintedanib. In the assay of antiproliferative activity on NIH/3T3, HUVEC, Bxpc-3, and MDA-MB-231, 11b showed better inhibitory ability than Nintedanib. In pancreatic cancer xenograft mouse models from Bxpc-3 cells, even when the dosage was halved, 11b exhibited better or comparable effects to Nintedanib (tumor growth inhibition (TGI) based on tumor volume change during the trial or tumor weight). Notably, we also found that 11b prohibited Bxpc-3 resulted lung metastasis by inhibiting its epithelial-mesenchymal transition (EMT) process. Another mechanism assay also proved that 11b inhibited the function of blood vessels and fibroblasts, promoted apoptosis of cancer and fibroblast cells, and exhibited low toxicity and good metabolic stability.

The C797S mutation of EGFR leads to Osimertinib resistance by blocking the covalent binding of Cys797. To develop new agents that can overcome EGFR mutation resistance, thirty seven new cyclopropane sulfonamide derivatives were synthesized and evaluated as EGFRL858R/T790M/C797S or EGFRDel19/T790M/C797S inhibitors by structure-based screening. Most of the synthesized compounds exhibit good to excellent anti proliferation activity against to BaF3-EGFR L858R/T790M/C797S and BaF3-C797S/Del19/T790M cancer cell lines. Representative compounds 8l showed inhibitory activity against the two cancer cell lines with the IC50 values of 0.0012 and 0.0013 mu M, respectively. Another compound 8h, exhibited slightly lower activity (0.0042 and 0.0034 mu M of the IC50 values) to both of the two tri-mutation cell lines, but excellent activities against H1975 and PC9 cells with IC50 values of 13 and 19 nM, respectively. Considering the acquired drug resistance of tumors is a gradual process, we chose 8h for further in vivo and mechanism study. 8h was demonstrated significantly inhibited tumor growth with 72.1 % of the TGI in the BaF3/EGFR-TM xenograft tumor model and 83.5 % in the H1975-DM xenograft tumor model. Compound 8h was confirmed to be safe with no significant side effects as showed by the results of in vitro assay of human normal cells and the sections of animals major organs. Mechanism studies showed that in addition to inhibiting EGFR mutations, 8h can also target the tumor microenvironment and induce tumor cell apoptosis. All these results indicate that 8h deserves further investigation as an EGFR inhibitor to overcome C797S-mediated resistance.

A series of new deuterated and non-deuterated N 2 , N 4 -diphenylpyridine - 2,4-diamine derivatives were synthesized and evaluated as EGFR C797S-mediated resistance inhibitors. Most of these compounds exhibited potent antiproliferative activity against Baf3-EGFR L858R/T790M/C797S and Baf3-EGFR Del19/T790M/C797S cancel cell lines, with IC 50 values in the nanomolar concentration range. Among them, compound 14l represented the most active compound with IC 50 values of 8 -11 nM. Interestingly, metabolic stability assay with rat liver microsomes indicated that the half-life of the deuterated derivative 14o was significantly increased compared to that of 14l . In xenograft mice models, 1 4o inhibited tumor growth with excellent inhibitory rate of 75.1 % at the dosage of 40 mg/kg, comparing 73.2 % of the TGI with its non-deuterated compound 14l , at a dosage of 80 mg/kg. Mechanism studies revealed that 14o was a potent EGFR L858R/T790M/C797S and EGFR Del19/T790M/C797S kinase inhibitor, which could downregulate the protein phosphorylation of EGFR and m -TOR signaling pathways, arrest cell cycle at G2/M phase by affecting the expression of CDC25C, and promote cell apoptosis by regulating the expression of cleaved caspase-3. In summary, 14o could serve as a promising deuterated compound for the development of highly efficient anticancer agents.

With ceritinib as the lead, a series of novel compounds containing the sulfoxide structure were synthesized and evaluated as anaplastic lymphoma kinase inhibitors.

Multiple tumors are synergistically promoted by c-Met and TRK, and blocking their cross-signalling pathway may give better effects. In this study, we developed a tyrosine kinase inhibitor 1D228, which exhibited excellent anti-tumor activity by targeting c-Met and TRK. Models in vitro, 1D228 showed a significant better inhibition on cancer cell proliferation and migration than the positive drug Tepotinib. Models in vivo, 1D228 showed robust anti-tumor effect on gastric and liver tumor growth with 94.8% and 93.4% of the TGI, respectively, comparing 67.61% and 63.9% of Tepotinib. Importantly, compared with the combination of Larotrectinib and Tepotinib, 1D228 monotherapy in MKN45 xenograft tumor models showed stronger antitumor activity and lower toxicity. Mechanistic studies showed that 1D228 can largely inhibit the phosphorylation of TRKB and c-Met. Interestingly, both kinases, TRKs and c-Met, have been found to be co-expressed at high levels in patients with gastric cancer through IHC. Furthermore, bioinformatics analysis has revealed that both genes are abnormally co-expressed in multiple types of cancer. Cell cycle analysis found that 1D228 induced G0/G1 arrest by inhibiting cyclin D1. Additionally, vascular endothelial cells also showed a pronounced response to 1D228 due to its expression of TRKB and c-Met. 1D228 suppressed the migration and tube formation of endothelial cells, which are the key functions of tumor angiogenesis. Taken together, compound 1D228 may be a promising candidate for the next generation of c-Met and TRK inhibitors for cancer treatment, and offers a novel potential treatment strategy for cancer patients with abnormal expressions of c-Met or NTRK, or simultaneous of them.

For non-small cell lung cancer patients with dual mutations in EGFR and ALK, there are currently no effective therapies. Consequently, novel EGFR/ALK dual-target inhibitors are urgently needed for the treatment of NSCLC. Here, we designed a series of highly effective small molecule dual inhibitors of ALK and EGFR. The biological evaluation highlighted that most of these new compounds could effectively inhibit both ALK and EGFR in enzymatic and cellular assays. Compound (+)-8l was investigated for its antitumor properties, and it was found that (+)-8l blocked the phosphorylation of EGFR and ALK induced by ligands and inhibited phosphorylation-ERK and phosphorylation-AKT induced by ligands. Furthermore, (+)-8l also induces apoptosis and G0/G1 cell cycle arrest in cancer cells and inhibits proliferation, migration, and invasion. Notably, (+)-8l significantly suppressed tumor growth in the H1975 cell-inoculated xenograft model (20 mg/kg/d, TGI: 96.11%), PC9 cell-inoculated xenograft model (20 mg/kg/d, TGI: 96.61%) and EML4 ALK-Baf3 cell-inoculated xenograft model (30 mg/kg/d, TGI: 80.86%). These results highlight the differentiated potential of (+)-8l to inhibit ALK rearrangement and EGFR mutation in NSCLC.

Fourteen new compounds bearing sulfonamide groups that target EGFRT790M/L858R mutations and ALK rearrangement were synthesized and evaluated as dual-target tumor inhibitors. The study on the anti-proliferation activity on cancer cells showed that the sulfonamide derivative with pyrimidine nucleus had much better activities compared with those with quinazoline nucleus. Among them, compound 19e exhibited excellent activity against H1975 cancer cell lines (EGFRT790M/L858R high express) and H2228 cells (ALK rearrangement) with the IC50 values of 0.0215 μM and 0.011 μM, respectively. The ALK and EGFR kinase inhibition assays also provided similar results. Genotype selectivity of EGFR on kinase and cell level, cytotoxicity towards human normal cell lines and cell morphology assay implied that 19e had acceptable selectivity and low toxicity. In addition, the inhibitory activity of 19e on H1975 and H2228 cells cloning and its apoptosis-inducing effect on the two cell lines were studied, and its inhibitory effect on the invasion and migration of tumor cells were also investigated. All the results show that 19e is worthy of further study.

Third-generation cutaneous growth factor receptor tyrosine kinase inhibitors have made impressive advances against non-small cell lung cancer. Even so, acquired resistance has significantly limited their clinical application, EGFRC797S mutation as the primary resistance mechanism. In this study, a series of novel EGFR kinase inhibitors were designed and synthesized as the inhibitors for the treatment of EGFRC797S mutation of non-small cell lung cancer. SAR screenings revealed that compound 5d was the most effective inhibitor targeting the C797S mutation. It exhibited strong inhibition against Baf3-EGFRL858R/T790M/C797S and Baf3-EGFR 19del/T790M/C797S cell liness, with IC50 values of 0.01836±0.0065 and 0.025±0.005 μM, respectively. Molecular docking studies showed that 5d binds to EGFRL858R/T790M/C797S and EGFR19del/T790M/C797S proteins effectively. Mechanism study showed that 5d induced cell cycle arrest in the G2/M phase by upregulating Cdc25C protein expression in cells with the C797S mutation. 5d caused cell apoptosis in a dose-dependent increase, accompanied by a significant elevation of the apoptotic protein cleaved caspase-3. The in vivo xenografts assay of mice with Baf3-EGFR19del/T790M/C797S cells showed 5d could effectively inhibit tumor growth with 45.2% (70 mg/kg) and 55.7% (100 mg/kg) of the tumor growth inhibition rates (TGI), respectively. Based above, 5d is worth of further study as the fourth EGFR inhibitor.

Hepatocellular carcinoma (HCC) is a type of liver cancer that is known for its poor prognosis and high mortality rate. Researchers have been exploring potential treatments for HCC, and in this study a series of novel tepotinib derivatives were synthesized and evaluated as anti-cancer agents for liver cancer. Among the synthesized compounds, compound (R)-10 which possess a chiral center and methyl pyrazole as the side chain showed promising results in both in vitro and in vivo studies. (R)-10 exhibited good in vitro activity (0.010 μM of the IC50 value) against MHCC-97H, a human liver cancer cell line with high c-Met expression, and favorable in vivo activity in tumor transplantation model. In addition, we also attempted to use (R)-10 in a novel oral nanodrug delivery system and achieved encouraging result with 87.67% of the TGI at the dosage of 8 mg/kg of the (R)-10. Taken together, these findings indicate that (R)-10 shows potential as a potent anti-HCC agent. Furthermore, the successful incorporation of (R)-10 into a novel oral nanodrug delivery system suggests that alternative administration methods for this compound are promising and deserving of further investigation.