Discovery of 4,6-pyrimidinediamine derivatives as novel dual EGFR/FGFR inhibitors aimed EGFR/FGFR1-positive NSCLC
Abstract
Activation of the FGF2-FGFR1 autocrine signaling pathway diminishes the effectiveness of EGFR inhibitors, such as Gefitinib, in treating non-small cell lung cancer (NSCLC). As a result, the development of dual-specific inhibitors that selectively target both EGFR and FGFR is crucial for overcoming this resistance. By analyzing the structural features of highly effective EGFR and FGFR inhibitors, a series of 33 4,6-pyrimidinediamine derivatives were designed and synthesized, aiming to achieve dual inhibition of EGFR and FGFR. Following preliminary cell screening, BZF 2 emerged as a promising candidate for further investigation.
Subsequent kinase assays and western blot analyses confirmed that BZF 2 functions as a selective and potent dual EGFR/FGFR inhibitor. Further biological evaluations conducted on NSCLC cell lines exhibiting the FGF2-FGFR1 autocrine loop demonstrated that BZF 2 significantly reduced cell proliferation, with IC50 values of 2.11 µM for H226 cells and 0.93 µM for HCC827 GR cells. Additionally, BZF 2 inhibited cell migration, induced apoptosis, and caused cell cycle arrest, thereby disrupting the progression of cancerous growth.
In vivo anti-tumor activity studies provided further validation of its therapeutic potential, as treatment with BZF 2 led to a marked reduction in tumor size. Given its high selectivity and potency, BZF 2 represents a promising therapeutic candidate for treating NSCLC cases characterized by EGFR and FGFR1 positivity. Its dual-targeting mechanism could offer an effective approach for overcoming drug resistance and improving treatment outcomes.
Introduction
A significant proportion of non-small cell lung cancer (NSCLC) patients with gain-of-function mutations in the epidermal growth factor receptor (EGFR) experience favorable responses to first-generation EGFR tyrosine kinase inhibitors (TKIs). However, the emergence of drug resistance substantially limits the long-term efficacy of these treatments. Multiple mechanisms contribute to EGFR-TKI resistance, including mutations within EGFR itself, alterations in genes encoding downstream effectors, epithelial-mesenchymal transition, and activation of alternative signaling pathways. Despite extensive studies, these resistance mechanisms remain incompletely understood, posing a persistent challenge in treating NSCLC patients who initially respond to EGFR-TKIs.
Recent research has identified the activation of the FGF2-FGFR1 autocrine loop in EGFR-TKI-insensitive NSCLC cell lines, demonstrating its role in regulating key cellular processes such as proliferation, differentiation, and migration. Co-expression of FGF2 and FGFR1 has been observed in both primary drug-resistant NSCLC cell lines, including H226, H1703, and H520, as well as in acquired resistant cell models such as HCC827GR and PC-9GR, which exhibit resistance to Gefitinib, and PC-9AR, which is resistant to Afatinib. Studies suggest that EGFR and FGFR1 both signal through the PI3K/AKT pathway, contributing to their synergistic effects in cancer progression.
Given these findings, simultaneous inhibition of EGFR and FGFR1 pathways is considered a necessary approach for effectively eliminating NSCLC tumor cells harboring the activated FGF2-FGFR1 autocrine loop. Combined treatment with EGFR and FGFR inhibitors, such as Gefitinib and PD173074, has demonstrated substantial therapeutic efficacy. However, administering selective inhibitors targeting individual proteins may lead to unintended toxic side effects due to drug interactions. Consequently, the concept of developing a single dual EGFR/FGFR inhibitor as a treatment strategy for NSCLC cases characterized by EGFR and FGFR1 positivity has gained traction.
Over the past several years, research efforts have focused on the design and evaluation of 4,6-disubstituted pyrimidine derivatives, with many exhibiting strong inhibitory effects against gain-of-function EGFR mutations. Among these compounds, Yfq07 displayed the highest potency. Structural studies indicate that the substitution of 6-aniline enhances selectivity and affinity for EGFR, with methoxy groups contributing to hydrophobic interactions, alkyl substituents facilitating binding within hydrophobic pockets, and halogen substitutions increasing liposolubility. Investigations further demonstrate that 4,6-disubstituted pyrimidines establish crucial hydrogen bonding interactions with mutant EGFR, while pendant aniline rings improve kinase selectivity. For example, Compound G has been identified as a potent inhibitor of EGFR mutations, with an IC50 of 63 nm.
Additional studies on FGFR inhibitors have revealed that the presence of an N-pyrimidin-4-yl-urea motif allows for the formation of a pseudo six-membered ring, providing a novel strategy in FGFR-targeted drug design. This discovery has contributed to the development of promising FGFR inhibitors, including BGJ398 and FIIN-3. The strong inhibitory activity of these compounds is attributed to the structural features of 4-aniline, where methoxy groups positioned at C(3) and C(5) occupy hydrophobic pockets, and chlorine atoms introduced at ortho positions reduce deconjugation energy penalties.
Guided by these structural insights, researchers have defined the 4,6-di-substituted pyrimidine scaffold, incorporating both 4-urea aniline and 6-aniline as essential components. Through careful molecular analysis, two series of novel compounds have been designed and synthesized, each featuring 1-phenyl-3-(6-(phenylamino)pyrimidin-4-yl)urea with methoxy, alkyl, or halogen substitutions. Subsequent biological experiments have been conducted to assess the anti-tumor efficacy of selected compounds, further advancing their potential as viable therapeutic candidates for drug-resistant NSCLC treatment.
Materials and methods
Chemistry
General
All materials and reagents were purchased from commercial suppliers and used without further purification unless otherwise stated. The progress of the reaction was monitored by analytical thin layer chromatography (TLC) using a silica gel GBZF 254 plate (Qingdao Haiyang Chemical Plant, Qingdao, China), and the spots were observed under UV light of 254 nm or 365 nm. Column chromatography was carried out on silica gel (90-150 µM; Qingdao Ocean Chemical Co. Ltd.). The melting point measured on the XT-4 micro melting point apparatus without corrected.
1 H NMR and 13 C NMR spectra were measured on a Bruker Avanve NMR spectrometer using CDCl3 or DMSO-d6 as a solvent. Mass spectra were obtained on an MS Agilent 1200 Series LC / MSD Trap Mass Spectrometer (ESI-MS). Elemental analysis for C, H and N were performed using elemental analyser (Flash EA 1112) and were found within ± 0.5% of the theoretical values.
Biological Activities
Cell Lines and Reagents
NSCLC (HCC827, PC-9, H520, H1581, H226) were purchased from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). HCC827GR cells (HCC827 Gefitinib resistance cells) are cultured in our laboratory (For confirmation of FGF2-FGFR1 bypass activation after gefitinib resistance in HCC827 cells, please refer to the supplementary data.). H520, H1581, H226 and HCC827GR were cultured in RPMI 1640 (GIBCO) medium supplemented with 10% FBS (GIBCO), 50 µg/mL penicillin and 50 µg/mL streptomycin; HCC827, PC-9 were cultured in DMEM (GIBCO) medium supplemented with 10% FBS, 50 µg/mL penicillin and 50 µg/mL streptomycin; All cells were cultured at 37 °C under a 5% CO2 and 90% humidified atmosphere.
Kinase Inhibition Assay
The experimental procedure involved multiple preparation steps for enzyme and peptide solutions followed by kinase activity assessment. A 2.5× enzyme solution was prepared by dissolving the kinase in a 1× kinase base buffer. Simultaneously, a 2.5× peptide solution was prepared, incorporating FAM-labeled peptide along with ATP in a 1× kinase base buffer. The assay plate had been preloaded with 5 µL of the test compound dissolved in 10% dimethyl sulfoxide (DMSO).
The enzyme solution was then transferred to the assay plate, with 10 µL being added to each well of a 384-well plate. Following this, incubation was carried out at room temperature for 10 minutes. Subsequently, the prepared peptide solution was introduced into the assay plate, with 10 µL added to each well, followed by an incubation step at 28°C for a specified duration. The reaction was terminated by adding 25 µL of buffer to quench the enzymatic activity.
Data collection was performed using Caliper, and conversion values were transformed into inhibition percentages using the formula: inhibition percentage = (max – conversion) / (max – min) × 100, where “max” corresponds to the DMSO control, and “min” represents the low control. Further analysis was conducted using the XL Fit Excel add-in (version 4.3.1) to derive IC50 values, applying the equation: Y = Bottom + (Top – Bottom) / (1 + (LogIC50 / X) × Hill Slope).
The study tested various kinase targets, including EGFR, FGFR1, EGFR L858R, and EGFR (d746-750). Additionally, kinase selectivity testing was performed across a panel of 32 tyrosine kinases, which encompassed several EGFR variants (WT, d746-750, T790M, and L858R), members of the FGFR family (FGFR1-4), as well as other signaling kinases such as JAK2, JAK3, AKT1, AKT2, Erk1, Erk2, MET, CDK7, IKKβ, CDK4/cyclin D3, CDK6/cyclin D3, WEE1, CDK9/CycT1, PLK1, PDGFRα, PDGFRβ, SRC, FLT1, and FLT4. Kinases such as JNK1 and JNK2 were obtained from Invitrogen, whereas CDC2/CycB1, CDK2/CycA2, and CDK5/P35 were sourced from Eurofins.
These steps ensured a systematic and rigorous approach to evaluating kinase inhibition profiles and selectivity, contributing to the characterization of the test compound’s therapeutic potential.
MTT Assay
In this experiment, MTT assay was used to detect cell viability in HCC827, PC-9, H520, H1581, H226 in the logarithmic growth phase and was cultured in a 96-well plate at a concentration of 6 × 103 cells per well, incubated for 24 h at 37 °C. Then, 1 µL of the positive compound G, BGJ398 or G:BGJ398 and 48 target compound solutions dissolved in DMSO at a final concentration of 10 µM or different concentration gradients (0.1-2 µM) were administered; After 48 h of administration, 20 µL of MTT solution dissolved in PBS to 5 mg/mL was added to each well, and continued to culture for 4 h. Discard the solution in each well, add 150 µL of DMSO to each well to dissolve the formazan crystals and shake it on a shaker for 10 min. Finally, the absorbance of each well at the ultraviolet absorption wavelength of 490 nm was detected by a microplate reader, and the corresponding cell survival rate, inhibition rate or IC50 value were calculated. This experiment requires at least three repeated experiments to reduce the experimental error.
Western Blot Analysis
PC-9, HCC827, H520, and HCC827GR cells were seeded into six-well plates and allowed to adhere overnight under standard culture conditions. Following incubation, varying concentrations of the active compound BZF 2 (0 µM, 1 µM, 5 µM, 10 µM) were introduced, alongside treatments with 10 µM G, BGJ398, or a combination of G and BGJ398 for a duration of two hours. The treated cells were subsequently harvested, washed thoroughly with phosphate-buffered saline (PBS), and lysed using RIPA buffer containing 1% Triton X-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) to facilitate total protein extraction.
The extracted proteins were loaded onto polyacrylamide gels and subjected to SDS-PAGE electrophoresis for separation. Following electrophoresis, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated overnight with primary antibodies specific to FGFR1, phosphorylated FGFR1 (p-FGFR1), EGFR, phosphorylated EGFR (p-EGFR), AKT, phosphorylated AKT (p-AKT), phosphorylated FRS2 (p-FRS2), FRS2, and GAPDH. After overnight incubation, the membranes were washed to remove unbound antibodies, and secondary antibody incubation was performed to enable signal detection.
The final detection of bound immune complexes was carried out using the ChemiDoc™ XRS+ system from Bio-Rad Laboratories. This procedure allowed for the evaluation of the effects of BZF 2 and other treatment conditions on key signaling proteins within these NSCLC cell lines, providing insights into the molecular interactions and potential therapeutic responses.
Flow Cytometry Analysis of Apoptotic Cells
H226 and HCC827GR cells at a density of 5×105 per well were cultured in regular growth medium in 6-well plates for 24 h and treated with different concentrations of BZF 2 or G:BGJ398 for 24 h. The cells were harvested, washed and stained with 3 µL Annexin V and 3 µL Propidium Iodide (PI) at room temperature for 15 min and 1 × binding buffer was used for dilution to 500 µL. The cells were then stained with PI, Annexin-V alone as positive control. The samples were measured with a BD Accuri™ C6 flow cytometer (Becton Dickinson) and the data were processed using Flow Jo 7.6.1.
Colony Survival Assay
H226 and HCC827GR cells were cultured 1000 per well in 6-well plate with regular growth medium. Cells were treated by BZF 2 and G:BGJ398 on the following day for 24 h. Cells were allowed to grow for 10-14 d until the colonies were visible. Crystalviolet solution (Sigma, St. Louis, MO, USA) was used to stain the colonies for 4 h and imaged.
Wound Healing Assay
HCC827GR cells were cultured in 6-well culture plates until its concentration reached 90-100%. A vertical “-” scratch mark was created on adherent cells using a 10 µL micropipette tip and non-adherent cells were washed out by applying PBS. The adherent cells were incubated with different concentrations of the test compound or the reference standard. At the time points 0 h, 24 h, 48 h, 72 h, the cells were observed and photographed under microscope.
Pharmacokinetics Study in Rats in Vivo
Four female and four male Sprague-Dawley rats (weight: 200-220 g) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd (ID Number: wxdw2018-1022). All rats were housed at the Wenzhou Medical University Laboratory Animal Research Center. All animals were housed under controlled conditions (22 °C) with a natural light-dark cycle. All experimental procedures were conducted according to the Institutional Animal Care guidelines and approved by the Administration Committee of Experimental Animals, Laboratory Animal Center of Wenzhou Medical University. The rats were fasted for 12 h before the beginning of all drug treatments via oral administration (0.5% CMC-Na suspension at 80 mg/kg dose). At time points 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, after administration, blood samples were collected from each animal and separated by centrifugation (4000 r/min for 10 min). Then, the samples were analyzed by UPLC−MS/MS and the acquired data were analyzed using Masslynx 4.0.
Tumor xenograft experiment
In vivo studies were conducted following approval from the Animal Policy and Ethics Committee of Wenzhou Medical University (ID Number: wxdw2018-1206). Female BALB/c (NU/NU) mice, six weeks of age, were used for tumor implantation. Each mouse was subcutaneously injected with 0.2 mL of HCC827GR tumor cells, totaling approximately 1 × 10⁶ cells per injection. Once tumor growth was established, the mice were randomly divided into three experimental groups, with five mice per group.
The first group served as the blank control (solvent group), the second group received treatment with the active compound BZF 2 at a dosage of 15 mg/kg, and the third group was administered a combination treatment of G and BGJ398 in a 1:1 ratio at 15 mg/kg. Intraperitoneal injections were performed every two days, continuing until the animals were sacrificed. Throughout the study, body weight and tumor volume measurements were recorded at two-day intervals to monitor changes over time. On the day of sacrifice, the tumor weight of each mouse was carefully documented.
Following euthanasia, excised solid tumors were rapidly frozen in liquid nitrogen to preserve their molecular integrity for subsequent immunoblotting analyses. This study design enabled a comprehensive evaluation of the therapeutic impact of BZF 2 and the combination regimen on tumor progression and growth dynamics in this NSCLC xenograft model.
Extraction of total protein from tissues
A small portion of tumor tissue is placed into a 15 mL centrifuge tube, and the tissue is finely cut using clean scissors while ensuring that all operations are conducted on ice to preserve sample integrity. Following this, 400 µL of RIPA buffer containing PMSF is added, and the mixture undergoes homogenization at a speed of 5000 r/min.
After a few minutes, further grinding is performed, and the sample is intermittently placed on ice to maintain stability. The grinding process is repeated multiple times to achieve thorough tissue disruption. Subsequently, the lysate is incubated for 30 minutes to allow for complete cellular breakdown.
After lysis, the mixture is carefully transferred to a 1.5 mL centrifuge tube using a pipette, followed by centrifugation at 14,000 rpm for 5 minutes at 4°C. The supernatant is then collected in a separate centrifuge tube and stored at -80°C for subsequent analysis.
The final step involves performing protein quantification and electrophoresis following the outlined protocol to ensure accurate characterization of the extracted proteins.
Docking Simulations
The 3D coordinate of X-ray crystal structure of EGFR and FGFR1 were downloaded from the RCSB protein data bank (PDB ID: 2ITZ, 3TT0). Before docking, the protein was prepared using PyMol software, which removed water molecules and added hydrogen atoms. Prepare ligand 4.py and prepare recptor 4.py scripts from AutoDockTools 1.5.6 were used to calculate Gasteiger partial charge and check hydrogens. Then this docking job was carried out by the Autodock 4.2 program with Lamarckian genetic algorithm (LGA) adopted to search the best binding poses. The grid spacing was set to 0.375 Å with a box size of 60 × 60 × 60 enclosed the whole binding site. The ligand was docked 100 times and the top docking pose associated with the lowest docking score was extracted for visual analysis.
Results and discussion
Chemistry
As shown in Scheme 1, 20 C6 aniline-substituted 4-amino-6-chloropyrimidine compounds were initially synthesized by the nucleophilic aromatic substitution (SNAr). Two anilines were used to synthesize isocyanate intermediates by the phosgene method, which were then used to synthesize urea by nucleophilic aromatic substitution at the C4 of the 20 aniline-substituent pyrimidines. The two series of compounds were structurally validated by mass spectrometry (MS), 13C, 1H-nuclear magnetic resonance (NMR) and elemental analysis (EA).
Structure-Activity Relationship
To investigate the structure-activity relationship (SAR) of 4,6-disubstituted pyrimidine diamines, derivatives were examined for their kinase inhibitory effects and half-maximal inhibitory concentrations (IC50) against EGFR, EGFR/d746-750, EGFR L858R, and FGFR1 using a mobility shift assay. Additionally, all synthesized compounds were tested for their antiproliferative effects on non-small cell lung cancer (NSCLC) models that overexpress EGFR or FGFR1. Comparative evaluations included individual treatments with G, BGJ398, and their combination in a 1:1 ratio as controls. Further assessment of select compounds was conducted using a proliferation assay to determine their IC50 values.
Analysis revealed that compounds from the BZY series exhibited strong kinase inhibition against EGFR, but their effectiveness against FGFR1 was relatively weak. In contrast, BZF series compounds demonstrated robust inhibitory activity against both EGFR and FGFR1. Consistent with these findings, BZF series compounds showed superior efficacy against NSCLC compared to the BZY series. The data indicated that the presence of chlorine atoms at the ortho positions of the 4-aniline moiety played a crucial role, particularly in enhancing inhibition of FGFR1. It was hypothesized that the introduction of chlorine atoms stabilized the perpendicular conformation, improving binding affinity with both EGFR and FGFR1. Overall, kinase inhibition and antiproliferative effects were significantly enhanced when chlorine atoms were incorporated into the ortho position of 4-aniline.
Within the BZY series, compounds such as BZY1, BZY2, BZY3, BZY6, and BZY13, which contained methoxy substitutions at the C(5) position of 6-aniline, exhibited stronger inhibitory effects against EGFR kinase. Similarly, in the BZF series, compounds including BZF1, BZF2, BZF8, and BZF11 displayed high inhibition against both EGFR and FGFR1, with methoxy substitutions at the C(5) position of 6-aniline contributing to increased potency. At the cellular level, BZY1, BZY2, BZY6, BZY13, BZF1, BZF2, and BZF8 effectively suppressed NSCLC proliferation, reinforcing the hypothesis that the C(5) methoxy substitution enhances activity.
Selected compounds were further evaluated against a panel of five NSCLC cell lines. Results demonstrated that six compounds exhibited general inhibitory activity against EGFR-overexpressing cells, while a subset (BZF1, BZF2, BZF8) also suppressed FGFR-overexpressing cells. Specifically, BZF1, which contained 3,5-dimethoxy substitutions on the benzene ring at the 6-position, showed effects on PC-9 (IC50 = 3.18 µM) and H1581 (IC50 = 5.32 µM). BZF2, with 2,5-dimethoxy substitutions on the benzene ring at the 6-position, exhibited broad inhibitory effects across all tested cell lines, with IC50 values ranging from 1.25 to 3.03 µM. BZF8, featuring 3-fluoro-5-methyl substitutions at the 6-position, effectively inhibited cell growth in all tested lines except HCC827, with IC50 values between 2.79 µM and 4.78 µM.
These findings highlighted the significance of the 5-methoxy substitution on the benzene ring at the 6-position, suggesting that the presence of additional electron-withdrawing groups may further enhance inhibitory effects. In summary, the incorporation of C(5)-methoxy substituents on 6-aniline and 2,6-dichlorophenyl substitutions on 4-urea aniline was found to significantly improve inhibition potency, leading to the selection of BZF2 as a promising therapeutic candidate.
The identification of BZF 2 as a dual EGFR and FGFR inhibitor
To establish whether BZF 2 functions as a dual EGFR and FGFR inhibitor, its kinase activity and selectivity were assessed through kinase assay and Western blot analysis. The first stage of evaluation involved determining its kinase selectivity across a panel of 32 kinases using an in vitro ATP-site competition binding assay conducted at a concentration of 10 µM. To confirm specificity, BZF 2 was screened against a range of kinase receptors, including JAK, AKT, and MET, with inhibition percentages reflecting the compound’s binding affinity. Higher inhibition percentages corresponded to stronger binding interactions.
The results demonstrated that BZF 2 exhibited potent inhibition against EGFR (WT) at 92.81 ± 1.13%, EGFR L858R at 98.45 ± 1.15%, EGFR (d746-750) at 95.47 ± 1.21%, EGFR T790M at 86.82 ± 1.02%, and FGFR1 at 75.17 ± 0.93%. In contrast, its inhibitory effect on JAK, AKT, and MET was relatively weak, with the highest inhibition percentage reaching only 28.01%, confirming its selectivity for EGFR and FGFR1.
To further validate its inhibitory potential at the cellular level, PC-9, HCC827, and H520 cell lines were exposed to different concentrations of BZF 2, followed by Western blot analysis. Serum-starved PC-9 and HCC827 cells, which stably express EGFR, were treated with BZF 2 or G as a control for one hour. The data revealed that BZF 2 effectively blocked EGFR activation and inhibited the phosphorylation of downstream effectors, particularly AKT. Notably, its EGFR inhibition was slightly stronger than that of G.
Similarly, serum-starved H520 cells, which stably express FGFR1, were treated with either BZF 2 or BGJ398 as a control for one hour. The results showed that BZF 2 significantly inhibited FGFR1 activation and suppressed phosphorylation of downstream effectors, including AKT. At comparable concentrations, BGJ398 also displayed strong inhibitory effects on FGFR1 and AKT phosphorylation.
Based on findings from kinase assays and Western blot experiments, it was concluded that BZF 2 is a selective and highly potent dual EGFR/FGFR inhibitor, demonstrating its potential as an effective therapeutic candidate in targeted cancer treatment.
Conclusion
Multi-target inhibitors can be developed through molecular integration of single-target inhibitors. By screening various kinases and NSCLC cell lines that overexpress EGFR and FGFR1, BZF 2 emerged as a potent dual inhibitor with remarkable antiproliferative activity across five NSCLC cell lines. In studies involving H226 and HCC827GR, which exhibit resistance to Gefitinib, BZF 2 effectively suppressed cell proliferation, with IC50 values of 2.11 µM and 0.93 µM, respectively. Additionally, it significantly induced cell cycle arrest at the G1 phase, promoted apoptosis, and strongly inhibited cell migration, demonstrating an efficacy comparable to the combination treatment of G and BGJ398.
Furthermore, BZF 2 exhibited powerful inhibition of the cellular phosphorylation of EGFR L858R and FGFR1, as well as key downstream signaling molecules such as AKT. In vivo studies reinforced these findings, revealing that BZF 2 effectively impeded tumor progression.
These results indicate that BZF 2 incorporates 2,5-dimethoxy substitutions on the 6-aniline moiety and 2,6-dichlorophenyl substitutions on the 4-urea aniline, Irpagratinib along with a 4,6-substituted pyrimidine scaffold, establishing it as a promising dual EGFR and FGFR inhibitor with potential therapeutic applications.