Discovery of Novel 2-Substituted Aniline Pyrimidine Based Derivatives as Potent Mer/c-Met Dual Inhibitors with Improvement Bioavailability

Abstract

This study reports the rational design and systematic evaluation of a novel series of 2-substituted aniline pyrimidine derivatives as dual Mer/c-Met inhibitors. Among the synthesized compounds, 17c demonstrated potent dual kinase inhibition, with IC₅₀ values of 6.4 ± 1.8 nM (Mer) and 26.1 ± 7.7 nM (c-Met). The compound exhibited significant antiproliferative activity across multiple cancer cell lines (HepG2, MDA-MB-231, and HCT116), while showing minimal hERG channel inhibition (IC₅₀ > 40 μM), indicating favorable cardiac safety. Pharmacokinetic profiling revealed high metabolic stability in human liver microsomes (t_1/2 = 53.1 min) and moderate oral bioavailability (F: 45.3%), with strong plasma protein-binding affinity (>95%). Mechanistic studies further demonstrated that 17c dose-dependently suppressed HCT116 cell migration and induced apoptosis. These integrated pharmacological properties position 17c as a promising therapeutic candidate for dual Mer/c-Met drive malignancies.

1. Introduction

Mer kinase (monocytes epithelial tissue reproductive tissue), a key member of the TAM (Tyro3-Axl-Mer) receptor tyrosine kinase family [1,2,3], orchestrates multiple cellular processes critical to immune regulation, apoptosis resistance, and inflammatory signaling. Structurally, Mer is characterized by a conserved intracellular tyrosine kinase domain and extracellular ligand-binding region. Its expression is predominantly localized to immune cells (e.g., macrophages and dendritic cells), where it mediates efferocytosis (apoptotic cell clearance) and suppresses pro-inflammatory cytokine production, thereby maintaining immune tolerance [4]. Mechanistically, Mer activation occurs through Gas6 ligand binding, initiating downstream signaling cascades, e.g., PI3K/AKT (phosphatidylinositol 3-kinase/protein kinase B) and MAPK (mitogen-activated protein kinase), that promote cell survival, chemotaxis, and tissue remodeling [4]. Importantly, Mer dysregulation has been pathologically linked to oncogenesis: overexpression in tumor cells enhances survival under chemotherapy (via BCL-2 upregulation), facilitates metastatic dissemination (through EMT activation), and fosters immunosuppressive microenvironments (by polarizing tumor-associated macrophages) [5,6,7,8]. These multifaceted roles have established Mer as a high-value therapeutic target for precision oncology strategies targeting both tumor-intrinsic and immune evasion mechanisms.

Current Mer kinase inhibitors are structurally categorized into two major chemotypes: aminopyrimidine-pyrazole (pyrrole) and aminopyrimidine derivatives (Figure 1). The first-generation inhibitor UNC569 [9,10], a pyrazole-based compound, selectively inhibits Mer kinase (IC₅₀ = 1.2 nM) and suppresses downstream ERK (extracellular signal-regulated kinase/AKT signaling. However, its clinical translation is hindered by its rapid metabolism (human microsomal t_1/2< 15 min) and low oral bioavailability (F < 10%) [11]. The structural optimization of UNC569 yielded second-generation analogs. UNC2025 enhanced metabolic stability (t_1/2 = 68 min in human hepatocytes) and maintained Mer potency (IC₅₀ = 1.8 nM) [12,13]. MRX2843, a dual Mer/Flt3 (fms-like tyrosine kinase 3) inhibitor (Mer IC₅₀ = 1.2 nM), showed improved CNS penetration (brain/plasma ration = 0.4) [11] and is currently in phase II trails for relapsed/refractory acute lymphoblastic leukemia (NCT04872478) [14]. Notably, the aminopyrimidine-class inhibitor UNC2250 demonstrates exceptional Mer targeting (IC₅₀ = 1.7 nM) and unique activity against Mer-EGFR (epidermal growth factor receptor) fusion protein [15,16]. In xenograft models, UNC2250 induced tumor regression (~60% volume reduction vs. control) by the dual blockade of Mer-mediated survival signaling and EGFR-driven proliferation [16].

The c-Met kinase (cellular mesenchymal–epithelial transition factor), also known as the hepatocyte growth factor receptor (HGFR) [17,18], is a transmembrane receptor tyrosine kinase that plays a pivotal role in regulating critical cellular processes, including proliferation, survival, migration, and angiogenesis [19,20]. The activation of c-Met occurs through binding to its specific ligand HGF, which triggers receptor dimerization and autophosphorylation. This activation initiates downstream signaling cascades, such as the MAPK, PI3K/AKT, and STAT (signal transducer and activator of transcription) pathways. Aberrant c-Met signaling, caused by overexpression, mutations, or gene amplification, is strongly associated with tumorigenesis, promoting cancer progression, metastasis, and therapy resistance. Given its oncogenic significance, c-Met has become a promising therapeutic target. Current clinical investigations are actively exploring both small-molecule inhibitors and monoclonal antibodies targeting c-Met for the treatment of diverse malignancies.

Multiple c-Met inhibitors have entered clinical practice or are under clinical investigation (Figure 2); these could be classified into Type I and Type II inhibitors based on their binding modes to the target kinase. Notably, crizotinib (Type I), approved by the FDA in 2011, is primarily used for ALK (anaplastic lymphoma kinase)-positive metastatic non-small cell lung cancer (NSCLC) but also exhibits c-Met inhibitory activity as part of its multi-target mechanism [21,22]. Cabozantinib (Type II), another tyrosine inhibitor targeting c-Met, VEGFR (vascular endothelial growth factor receptor), Mer, and Kit, has been approved for multiple indications: metastatic medullary thyroid cancer, as a second-line treatment of advanced renal cell carcinoma post-antiangiogenic therapy, and as a first-line therapy for advanced renal cell carcinoma [23,24]. Additionally, savolitinib (Type I), a highly selective c-Met inhibitor, is currently in Phase III clinical trials for papillary renal cell carcinoma and other c-Met-driven malignancies [25]. Capmatinib (Type I), a selective c-Met inhibitor, received FDA approval specifically for metastatic NSCLC with MET exon 14 skipping mutations [26,27].

Dual-target inhibition strategies have gained significant traction in anti-tumor drug development, as exemplified by the FDA-approved agent cabozantinib-a potent c-Met/VEGFR-2 dual kinase inhibitor. Structurally, cabozantinib’s quinoline core competitively binds to the hinge region of c-Met kinase, while its cyclopropylamide side chain enables optimal hydrophobic interaction within the VEGFR-2 active site. The bifunction design confers robust inhibitory activity against both targets, underpinning its clinical approval for advanced thyroid carcinoma, hepatocellular carcinoma (HCC), and renal cell carcinoma (RCC). Additionally, the dual-target strategy has also been effectively applied in other disease areas, such as in the treatment of neglected diseases and neurological disorders [28,29]. Consequently, the development of dual inhibitors represents a paradigm shift in oncology drug discovery, balancing enhanced therapeutic outcomes with improved safety profiles through rationally engineered polypharmacology.

Both Mer and c-Met, members of the receptor tyrosine kinase family, possess structurally homologous extracellular domains and share overlapping downstream signaling pathways. Notably, their functional convergence in regulating tumor proliferation, metastasis, and immune evasion establishes a compelling rationale for the dual targeting of Mer and c-Met kinases. To date, cabozantinib and its derivatives remain the only clinical available agent with demonstrated inhibitory activity against both targets [30]. However, cabozantinib’s clinical utility is predominantly attributed to its potent inhibition of c-Met and VEGFR-2, while the structural determinants governing its Mer kinase inhibitory activity remains poorly characterized. Furthermore, the emergence of cabozantinib resistance underscores the critical need to develop novel Mer/c-Met dual inhibitors with distinct chemical scaffolds. Importantly, the structure–activity relationship (SAR) of Mer/c-Met dual inhibitors remains underexplored, and the systematic exploration of these aspects through rational drug design is therefore essential to advance the development of next-generation dual-targeting agents.

The 2-substituted aniline scaffold has been widely applied in anti-tumor drug development. For instance, the Mer inhibitor UNC2250 (Figure 1) exhibits potent Mer inhibitory activity [15,16]. Additionally, our previous study identified compound 18c (Figure 5) as a dual Mer/c-Met dual inhibitor with potent dual-target inhibitory activity [31]. The IC₅₀ values were 18.5 ± 2.3 nM and 33.6 ± 4.3 nM, respectively. Furthermore, compound 18c also demonstrated potent antiproliferation activities against HepG2, MDA-MB-231, and HCT116 cancer cell lines and showed a low risk in hERG assays. However, pharmacokinetic analysis revealed low oral bioavailability (F: 2.84%), promoting structural optimization. To elucidate the key interactions in dual-target inhibition, we analyzed the binding modes of cabozantinib with Mer (PDB: 4M3Q) [32,33] and c-Met (PDB: 3LQ8) [34,35]. Molecular docking showed that in Mer kinase, cabozantinib’s amide group forms hydrogen bonds with the hinge region (Pro672 and Met674), while the quinoline moiety occupies a hydrophobic cavity without direct hydrogen bonding (Figure 3). In c-Met kinase, both the quinoline core and the amide group participate in hydrogen bonding with residues in the ATP-binding pocket (Figure 4). Based on these observations and the binding mode of 18c [31], we propose modifying the 2-position of the pyrimidine ring to introduce hydrophilic groups. This strategy aims to enhance solubility and bioavailability while preserving dual-target activity.

Building upon our previous work (Figure 5), we designed and synthesized a series of 2-substituted aniline pyrimidine derivatives, with a subsequent evaluation of their biological activities. Among these, compound 17c emerged as a lead candidate due to its superior metabolic stability in human liver microsomes and potent antiproliferative activities against the HepG2 (liver), MDA-MB-231 (breast), and HCT116 (colon) cancer cell lines. 17c also revealed favorable pharmacokinetic properties, plasma protein binding, and a favorable safety profile in hERG assays. Mechanistic studies further showed that 17c induced dose-dependent apoptosis in HCT116 cells and effectively inhibited HCT116 cell migration. These results position 17c as a promising dual inhibitor of Mer and c-Met with significant development potential.

2. Materials and Methods

2.1. Chemical Part

All reagents and solvents were sourced from Titan Chemical Co., Ltd. (Shanghai, China), and Shijiazhuang Kehai Huabo Instruments Co., Ltd. (Shijiazhuang, China), respectively, and used without further purification unless specified. The reaction process was monitored by TLC analysis on silica GF254 Plates. High-resolution mass spectra (HRMS) data were acquired in ESI mode using a Water Q-Tof mass spectrometer (Waters Xevo G2-XS QTof). ¹H- and ¹³C-NMR spectra were recorded on Bruker AM-400/500 spectrometers (Bruker Bioscience, USA) with DMSO-d₆ used as solvent and tetramethylsilane (TMS) used as the internal reference, and chemical shift values were reported in ppm. The multiplicity of the signals is denoted as follows: s (singlet); d (doublet), t (triplet), q (quadruplet), qui (quintuplet), m (multiplet), dd (doublet of doublets), and dt (doublet of triplets). Coupling constants (J) are given in hertz (Hz). Flash chromatography was performed using silica gel (200–300 mesh) as the stationary phase, with a mobile phase consisting of a mixture of methanol (MeOH), ethyl acetate (EA), and petroleum ether (PE). The purity of all synthesized compounds was confirmed to be >95% by high-performance liquid chromatography (HPLC) using a Waters HPLC system equipped with a UV/visible detector (Waters Corporation, MA, USA), monitored at 254 nm. HPLC analysis was carried out on a 5 μm, 4.6 × 250 mm Hypersil ODS2 column, with a mobile phase consisting of a 3:7 (v/v) mixture of potassium dihydrogen phosphate solution and methanol, at a flow rate of 1.0 mL/min and an injection volume of 10 μL. All chemicals were of analytical grade and used without further purification.

2.1.1. Preparation of N-(4-Fluorophenyl)-N-(4-hydroxyphenyl)cyclopropane-1,1-dicarboxamide (Intermediate 11)

Intermediate 11 was prepared as a white solid in an 80.6% yield according to our previous work [31]. Consistent with prior methodology, the crude product required no purification and was employed directly in subsequent steps.

2.1.2. Preparation of N-(4-((2-Chloropyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (Intermediate 12)

Intermediate 12 was prepared as a white solid in a 75.7% yield according to our previous work [31].

2.1.3. General Procedure for Preparation of Target Compounds 13a–13n

Target compounds 13a–13n were prepared according to our previous work [31].

N-(4-fluorophenyl)-N-(4-((2-((4-((1-methylpiperidin-4-yl)oxy)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (compound 13a): White solid, yield: 23.27%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.18 (s, 1H), 10.11 (s, 1H), 9.41 (s, 1H), 8.30 (d, J = 5.6 Hz, 1H), 7.70 (d, J = 8.9 Hz, 2H), 7.66 (dd, J = 9.1, 5.1 Hz, 2H), 7.43 (d, J = 7.5 Hz, 2H), 7.19–7.14 (m, 4H), 6.80 (d, J = 8.7 Hz, 2H), 6.35 (d, J = 5.6 Hz, 1H), 4.44 (s, 1H), 3.16–2.87 (m, 4H), 2.63 (s, 3H), 2.10–2.01 (m, 2H), 1.85 (s, 2H), 1.51 (d, J = 4.6 Hz, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 170.06, 168.73 (d, J = 4.3 Hz, 1C), 160.19, 159.70, 157.79, 151.65, 148.59, 136.63, 135.66, 134.42, 122.86 (d, J = 7.7 Hz, 1C), 122.43 (d, J = 10.0 Hz, 1C), 120.91,116.61, 115.62, 115.44, 98.07, 78.48, 50.54, 43.37, 32.01, 28.45, 15.91. HRMS: m/z C₃₃H₃₃FN₆O₄ [M + H]⁺ 597.2547, Found 597.2628. Purity: >95% (HPLC).

N-(4-fluorophenyl)-N-(4-((2-((3-((2-methoxyethyl)amino)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (compound 13b): White solid, yield: 20.08%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.15 (s, 1H), 10.08 (s, 1H), 9.27 (s, 1H), 8.31 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.66 (dd, J = 9.0, 5.1 Hz, 2H), 7.21–7.14 (m, 4H), 6.86–6.79 (m, 3H), 6.34 (d, J = 5.6 Hz, 1H), 6.16 (d, J = 7.3 Hz, 1H), 5.21 (s, 1H), 3.44 (t, J = 5.9 Hz, 2H), 3.26 (s, 3H), 3.06 (t, J = 5.8 Hz, 2H), 1.49 (s, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.90, 168.67, 160.37, 160.22, 159.70, 157.79, 148.56, 141.27, 136.65, 135.67 (d, J = 2.0 Hz, 1C), 129.15, 122.82 (d, J = 7.7 Hz, 1C), 122.27, 115.61, 115.43, 107.93, 106.71, 103.23, 98.27, 70.93, 58.45, 42.93, 31.95, 15.86. HRMS: m/z C₃₀H₂₉FN₆O₄ [M + H]⁺ 557.2234, Found 557.2312. Purity: >95% (HPLC).

N-(4-((2-((1H-pyrazol-4-yl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 13c): White solid, yield: 31.52%. ¹H NMR (500 MHz, DMSO-d₆) δ 12.31 (s, 1H), 10.17 (s, 1H), 10.08 (s, 1H), 9.65–9.18 (m, 1H), 8.29 (s, 1H), 7.72 (s, 2H), 7.65 (dd, J = 8.7, 5.0 Hz, 3H), 7.30–7.11 (m, 5H), 6.30 (d, J = 5.5 Hz, 1H), 1.50 (s, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ 170.46, 168.68 (d, J = 11.7 Hz, 1C), 160.42, 159.84, 159.71, 157.80, 148.56, 136.67, 135.63, 130.12, 122.87 (d, J = 6.0 Hz, 1C), 122.40, 122.13, 115.62, 115.44, 98.04, 31.89, 15.97. HRMS: m/z C₂₄H₂₀FN₇O₃ [M + H]⁺ 474.1612, Found 474.1686. Purity: >95% (HPLC).

N-(4-((2-((3,5-difluorophenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 13d): White solid, yield: 19.40%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.23 (s, 1H), 10.07 (s, 1H), 10.01 (s, 1H), 8.47 (d, J = 5.6 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.69 (dd, J = 8.7, 5.1 Hz, 2H), 7.34 (d, J = 9.4 Hz, 2H), 7.28–7.17 (m, 4H), 6.67 (t, J = 9.1 Hz, 1H), 6.59 (d, J = 5.6 Hz, 1H), 1.53 (d, J = 4.1 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.09, 168.56, 160.40, 159.61, 157.59, 148.25, 143.31, 137.04, 135.59 (d, J = 2.6 Hz, 1C), 122.93 (d, J = 7.8 Hz, 1C), 122.29, 122.05, 115.62, 115.40, 101.74, 101.44, 99.97, 31.88, 15.93. HRMS: m/z C₂₇H₂₀F₃N₅O₃ [M + H]⁺ 520.1518, Found 520.1594. Purity: >95% (HPLC).

N-(4-((2-((3-(4-acetylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 13e): White solid, yield: 23.07%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.15 (s, 1H), 10.07 (s, 1H), 9.38 (s, 1H), 8.34 (d, J = 5.6 Hz, 1H), 7.70 (d, J = 9.0 Hz, 2H), 7.64 (dd, J = 9.1, 5.1 Hz, 2H), 7.20–7.13 (m, 5H), 7.08 (d, J = 8.1 Hz, 1H), 6.97 (t, J = 8.1 Hz, 1H), 6.47 (dd, J = 8.1, 1.7 Hz, 1H), 6.39 (d, J = 5.6 Hz, 1H), 3.56–3.47 (dd, J = 10.5, 7.0 Hz, 4H), 2.98 (t, J = 4.7 Hz, 2H), 2.93 (t, J = 4.9 Hz, 2H), 2.03 (s, 3H), 1.47 (s, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.82, 168.71 (d, J = 4.3 Hz, 1C), 168.60, 160.30 (d, J = 7.60 Hz, 1C), 159.95, 157.56, 151.52, 148.56, 141.30, 136.59, 135.66 (d, J = 2.5 Hz, 1C), 129.26, 122.85 (d, J = 7.9 Hz, 1C), 122.10, 115.62, 115.40, 111.05, 110.13, 107.05, 98.72, 49.23, 45.87, 31.97, 21.64, 15.85. HRMS: m/z C₃₃H₃₂FN₇O₄ [M + H]⁺ 610.2500, Found 610.2576. Purity: >95% (HPLC).

N-(4-fluorophenyl)-N-(4-((2-((3-(piperidin-3-ylcarbamoyl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (compound 13f): White solid, yield: 31.00%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.11 (s, 1H), 10.07 (s, 1H), 8.23 (d, J = 5.6 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.9 Hz, 2H), 7.64 (dd, J = 8.9, 5.1 Hz, 2H), 7.19–7.12 (m, 4H), 7.07 (t, J = 7.8 Hz, 1H), 7.02 (s, 1H), 6.94 (d, J = 7.7 Hz, 1H), 6.69 (dd, J = 7.9, 1.5 Hz, 1H), 6.07 (d, J = 5.4 Hz, 1H), 5.27 (s, 1H), 4.59–4.20 (m, 2H), 3.83–3.74 (m, 1H), 2.88–2.76 (m, 2H), 1.93–1.85 (m, 1H), 1.75–1.68 (m, 1H), 1.64–1.54 (m, 1H), 1.46 (s, 4H), 1.44–1.34 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.81, 168.62 (d, J = 11.21 Hz, 1C), 167.20, 161.64, 160.43, 159.70, 157.79, 148.95, 148.31, 136.40, 136.11, 135.63 (d, J = 1.61 Hz, 1C), 129.02, 122.88 (d, J = 7.70 Hz, 1C), 121.96, 116.86, 115.58, 115.41, 115.05, 113.40, 95.84, 48.53, 46.15, 43.81, 31.91, 30.76, 23.97, 15.91. HRMS: m/z C₂₄H₂₀FN₇O₃ [M + H]⁺ 610.2500, Found 610.2576. Purity: >95% (HPLC).

N-(4-fluorophenyl)-N-(4-((2-((4-(piperidin-3-ylcarbamoyl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (compound 13g): White solid, yield: 28.15%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.10 (s, 1H), 10.08 (s, 1H), 8.22 (d, J = 5.5 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.69–7.61 (m, 4H), 7.58 (d, J = 8.4 Hz, 2H), 7.15 (t, J = 8.2 Hz, 4H), 6.54 (d, J = 8.4 Hz, 2H), 6.06 (d, J = 4.9 Hz, 1H), 5.64 (s, 1H), 4.62–4.15 (m, 2H), 3.83–3.72 (m, 1H), 2.82 (t, J = 11.5 Hz, 2H), 1.88 (d, J = 9.6 Hz, 1H), 1.71 (d, J = 11.6 Hz, 1H), 1.61–1.51 (m, 1H), 1.46 (s, 4H), 1.44–1.33 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 169.81, 168.62 (d, J = 9.89 Hz, 1C), 166.27, 161.62, 160.39, 159.70, 157.79, 152.00, 148.32, 136.37, 135.62 (d, J = 1.39 Hz, 1C), 129.33, 122.88 (d, J = 7.70 Hz, 1C), 122.04, 121.94, 115.59, 115.41, 112.95, 95.78, 48.76, 45.99, 43.83, 31.88, 30.93, 24.04, 15.92. HRMS: m/z C₂₄H₂₀FN₇O₃ [M + H]⁺ 610.2500, Found 610.2567. Purity: >95% (HPLC).

N-(4-fluorophenyl)-N-(4-((2-((4-(4-methylpiperazine-1-carbonyl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (compound 13h): White solid, yield: 18.28%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.18 (s, 1H), 10.09 (s, 1H), 9.80 (s, 1H), 8.38 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.65 (dd, J = 9.1, 5.1 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 9.0 Hz, 2H), 7.19–7.13 (m, 4H), 6.48 (d, J = 5.6 Hz, 1H), 3.46 (s, 4H), 2.32 (s, 4H), 2.21 (s, 3H), 1.50 (s, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.11, 169.51, 168.71, 160.41, 159.90, 157.56, 148.52, 141.96, 136.78, 135.62 (d, J = 2.5 Hz, 1C), 128.19, 122.89 (d, J = 7.9 Hz, 1C), 122.51, 122.33, 118.39, 115.62, 115.40, 99.15, 54.92, 45.97, 31.81, 31.62, 16.07. HRMS: m/z C₃₃H₃₂FN₇O₄ [M + H]⁺ 610.2500, Found 610.2568. Purity: >95% (HPLC).

N-(4-((2-((4-(2-((2-(diethylamino)ethyl)amino)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 13i): White solid, yield: 16.50%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.15 (s, 1H), 10.09 (s, 1H), 9.86 (s, 1H), 9.48 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.65 (dd, J = 9.0, 5.1 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.20–7.13 (m, 4H), 6.36 (d, J = 5.6 Hz, 1H), 3.36 (s, 3H), 2.90 (s, 2H), 2.75 (s, 6H), 1.51 (d, J = 5.2 Hz, 4H), 1.06 (t, J = 7.1 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.02, 169.51, 168.74 (d, J = 2.1 Hz, 1C), 160.17, 159.94, 157.55, 148.51, 136.72, 136.35, 135.64 (d, J = 2.6 Hz, 1C), 133.07, 122.86 (d, J = 7.8 Hz, 1C), 122.25 (d, J = 12.7 Hz, 1C), 120.10, 119.64, 115.62, 115.40, 98.40, 52.32, 51.46, 46.90, 45.95, 31.92, 15.99, 10.73. HRMS: m/z C₃₅H₃₉FN₈O₄ [M + H]⁺ 655.3078, Found 655.3160. Purity: >95% (HPLC).

N-(4-((2-((4-(2-((2-(dimethylamino)ethyl)amino)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (compound 13j): White solid, yield: 30.11%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.16 (s, 1H), 10.12 (s, 1H), 9.91 (s, 1H), 9.48 (s, 1H), 8.32 (s, 1H), 7.71 (s, 2H), 7.65 (s, 2H), 7.49 (s, 2H), 7.42 (s, 2H), 7.18 (s, 4H), 6.36 (s, 1H), 3.32 (s, 3H), 2.71 (s, 2H), 2.55 (s, 2H), 2.32 (s, 6H), 1.51 (s, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.03, 169.76, 168.76 (d, J = 3.0 Hz, 1C), 160.18, 157.57, 148.54, 136.70, 136.30, 135.61 (d, J = 2.6 Hz, 1C) 133.12, 122.89 (d, J = 7.9 Hz, 1C), 122.32, 122.19, 120.01, 119.65, 115.62, 115.40, 98.39, 58.06, 52.53, 46.03, 44.87, 31.88, 16.02. HRMS: m/z C₃₃H₃₅FN₈O₄ [M + H]⁺ 627.2765, Found 627.2841. Purity: >95% (HPLC).

N-(4-fluorophenyl)-N-(4-((2-((4-(2-(4-(2-methoxyethyl)piperazin-1-yl)acetamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (compound 13k): White solid, yield: 33.25%. ¹H NMR (400 MHz, DMSO-d₆) δ 10.14 (s, 1H), 10.09 (s, 1H), 9.48 (s, 1H), 9.46 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.64 (dd, J = 8.9, 5.1 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.16 (q, J = 8.6 Hz, 4H), 6.36 (d, J = 5.6 Hz, 1H), 3.42 (t, J = 5.8 Hz, 2H), 3.22 (s, 3H), 3.04 (s, 2H), 2.50 (s, 2H), 2.47 (s, 8H), 1.51 (d, J = 8.2 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.05, 168.72, 168.18, 160.15, 157.56, 148.54, 136.71, 136.39, 135.63 (d, J = 2.5 Hz, 1C), 132.92, 122.87 (d, J = 7.9 Hz, 1C), 122.35, 122.17, 120.33, 119.54, 115.62, 115.40, 98.36, 70.34, 62.22, 58.46, 57.46, 53.42, 53.27, 31.89, 16.01. HRMS: m/z C₃₆H₃₉FN₈O₅ [M + H]⁺ 683.3027, Found 683.3107. Purity: >95% (HPLC).

2.1.4. Preparation of N-(4-Hydroxyphenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (Intermediate 15)

A mixture of 4-antipyrine carboxylic acid (14, 2.0 g, 8.61 mmol) and 4-aminophenol (10, 1.13 g, 10.33 mmol) in DMF (20 mL) was treated with HBTU (3.92 g, 10.33 mmol) and TEA (2.61 g, 25.84 mmol). After stirring at ambient temperature for 8 h (reaction monitored by TLC), the mixture was quenched by pouring into ice water (200 mL). The resulting precipitate was collected by filtration, washed thoroughly, and vacuum-dried to afford intermediate 15 as a white solid (1.75 g, 63.0%). This product was used directly in subsequent without purification.

2.1.5. Preparation of N-(4-((2-Chloropyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (Intermediate 16)

A mixture of intermediate 15 (1.75 g, 5.41 mmol) and 2,4-dichloropyrimidine (0.81 g, 5.41 mmol) in DMF (15 mL) was treated with K₂CO₃ (0.82 g, 5.95 mmol). The mixture was stirred at 80 °C for 4.5 h. The reaction solution was poured into ice water (100 mL), and the precipitate was filtered off, washed, and dried in a vacuum to yield intermediate 16 as a white solid (1.85 g, 78.0%), which can be used directly without any further purification.

2.1.6. General Procedure for Preparation of Target Compounds 17a–17h

Intermediate 16 (1.2 mmol) and substituted aniline (1.0 mmol) were dissolved in DMF (8 mL), followed by the addition of PTSA (4.00 mmol). The reaction mixture was stirred at 90 °C for 4 h under N₂ atmosphere. After cooling to ambient temperature, the solution was quenched with ice water (100 mL). The precipitate solid was isolated by filtration, washed, and vacuum-dried. Crude product purification via silica gel chromatography (gradient elution: DCM/MeOH 100:1–30:1) afforded compounds 17a–17h.

1,5-dimethyl-N-(4-((2-((4-((1-methylpiperidin-4-yl)oxy)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17a): White solid, yield: 15.05%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.82 (s, 1H), 9.41 (s, 1H), 8.29 (d, J = 5.6 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.60 (t, J = 7.7 Hz, 2H), 7.53 (d, J = 7.3 Hz, 1H), 7.45 (d, J = 7.5 Hz, 2H), 7.40–7.34 (m, 2H), 7.18 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 7.7 Hz, 2H), 6.38 (d, J = 5.6 Hz, 1H), 4.25 (s, 1H), 3.38 (s, 3H), 2.77 (s, 2H), 2.74 (s, 3H), 2.45 (s, 2H), 2.31 (d, J = 13.7 Hz, 3H), 1.90 (s, 2H), 1.68 (s, 2H).¹³C NMR (126 MHz, DMSO-d₆) δ 170.12, 163.52, 161.67, 160.36, 160.15, 154.26, 151.89, 148.17, 136.67, 134.09, 133.49, 129.97, 129.33, 127.59, 123.09, 120.95, 120.76, 116.33, 97.94, 97.60, 44.97, 33.83, 21.25, 11.98. HRMS: m/z C₃₄H₃₆N₇O₄ [M + H]⁺ 606.2751, Found 606.2832. Purity: >95% (HPLC).

N-(4-((2-((3-((2-methoxyethyl)amino)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17b): White solid, yield: 5.80%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.82 (s, 1H), 9.28 (s, 1H), 8.31 (d, J = 5.4 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.60 (t, J = 7.4 Hz, 3H), 7.52 (t, J = 7.2 Hz, 1H), 7.45 (d, J = 7.4 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 6.87–6.77 (m, 3H), 6.36 (d, J = 5.4 Hz, 1H), 6.16 (d, J = 6.8 Hz, 1H), 3.42 (t, J = 5.5 Hz, 2H), 3.37 (s, 3H), 3.24 (s, 3H), 3.05 (s, 2H), 2.72 (s, 3H).¹³C NMR (126 MHz, DMSO-d₆) δ 169.92, 163.53, 161.65, 160.35, 160.23, 154.29, 149.19, 148.09, 141.27, 136.65, 133.52, 129.95, 129.32, 129.04, 127.58, 122.77, 120.61, 107.94, 106.65, 103.26, 98.29, 97.57, 70.93, 58.42, 42.94, 33.79, 11.93. HRMS: m/z C₃₁H₂₁N₇O₄Na [M + Na]⁺ 588.2438, Found 588.2337. Purity: >95% (HPLC).

1,5-dimethyl-N-(4-((2-((3-((1-methylpiperidin-4-yl)oxy)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17c): White solid, yield: 7.01%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.81 (s, 1H), 9.51 (s, 1H), 8.35 (d, J = 5.5 Hz, 1H), 7.68 (d, J = 8.7 Hz, 2H), 7.60 (t, J = 7.5 Hz, 2H), 7.52 (t, J = 7.4 Hz, 1H), 7.45 (d, J = 7.5 Hz, 2H), 7.27 (s, 1H), 7.20 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 7.8 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.51 (d, J = 7.7 Hz, 1H), 6.42 (d, J = 5.5 Hz, 1H), 4.30 (s, 1H), 3.34 (s, 2H), 2.93 (s, 2H), 2.72 (s, 3H), 2.45 (s, 3H), 2.00 (s, 2H), 1.76 (s, 2H).¹³C NMR (126 MHz, DMSO-d₆) δ 169.95, 163.51, 161.64, 160.33, 160.13, 157.35, 154.28, 147.98, 141.90, 136.69, 133.50, 129.96, 129.49, 129.33, 127.59, 122.70, 120.68, 112.14, 108.92, 107.25, 98.85, 97.57, 51.75, 44.37, 33.80, 31.62, 30.30, 11.94. HRMS: m/z C₃₄H₃₆N₇O₄ [M + H]⁺ 606.2751, Found 606.2823. Purity: >95% (HPLC).

1,5-dimethyl-N-(4-((2-((4-(4-methylpiperazine-1-carbonyl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17d): White solid, yield: 63.0%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.85 (s, 1H), 9.82 (s, 1H), 8.38 (d, J = 5.6 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.60 (t, J = 7.6 Hz, 2H), 7.49–7.56 (m, 3H), 7.45 (d, J = 7.3 Hz, 2H), 7.21 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H), 6.51 (d, J = 5.6 Hz, 1H), 3.37 (s, 4H), 3.33 (s, 3H), 2.73 (s, 3H), 2.27 (s, 4H), 2.15 (s, 3H).¹³C NMR (126 MHz, DMSO-d₆) δ 170.17, 169.40, 163.51, 161.67, 160.46, 159.84, 154.35, 148.05, 141.99, 136.85, 133.49, 129.95, 129.30, 128.45, 128.17, 127.57, 123.12, 120.68, 118.37, 99.11, 97.65, 54.90, 33.80, 11.98. HRMS: m/z C₃₄H₃₄N₈O₄Na [M + Na]⁺ 641.2703, Found 641.2603. Purity: >95% (HPLC).

N-(4-((2-((4-(4-acetylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17e): White solid, yield: 15.79%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.85 (s, 1H), 9.36 (s, 1H), 8.28 (d, J = 5.5 Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.60 (t, J = 7.6 Hz, 2H), 7.52 (t, J = 7.4 Hz, 1H), 7.47 (d, J = 7.4 Hz, 2H), 7.26 (s, 2H), 7.16 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 6.9 Hz, 2H), 6.38 (d, J = 5.5 Hz, 1H), 3.48 (s, 4H), 3.40 (s, 3H), 2.99 (s, 2H), 2.87 (s, 2H), 2.74 (s, 3H), 2.00 (s, 3H).¹³C NMR (126 MHz, DMSO-d₆) δ 170.26, 168.66, 163.48, 161.69, 160.29, 160.03, 154.04, 148.29, 145.98, 136.72, 133.47, 133.25, 129.97, 129.38, 127.69, 123.28, 120.82, 120.48, 116.55, 97.44, 50.18, 49.24, 45.98, 33.75, 21.60, 11.88. HRMS: m/z C₃₄H₃₄N₈O₄Na [M + Na]⁺ 641.2703, Found 641.2607. Purity: >95% (HPLC).

N-(4-((2-((3-(4-acetylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17f): White solid, yield: 20.0%. ¹H NMR (500 MHz, DMSO-d₆) δ 10.82 (s, 1H), 9.40 (s, 1H), 8.34 (d, J = 5.5 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.60 (t, J = 7.6 Hz, 2H), 7.52 (t, J = 7.4 Hz, 1H), 7.45 (d, J = 7.4 Hz, 2H), 7.19 (d, J = 8.5 Hz, 3H), 7.09 (d, J = 7.3 Hz, 1H), 6.96 (t, J = 8.0 Hz, 1H), 6.51 (d, J = 7.5 Hz, 1H), 6.41 (d, J = 5.5 Hz, 1H), 3.51 (t, J = 5.7 Hz, 4H), 3.37 (s, 4H), 2.96 (s, 4H), 2.72 (s, 3H), 2.01 (s, 3H).¹³C NMR (126 MHz, DMSO-d₆) δ 169.84, 168.70, 163.53, 161.64, 160.33, 160.26, 154.27, 151.57, 148.05, 141.31, 136.61, 133.51, 129.95, 129.32, 129.14, 127.60, 122.59, 120.57, 111.11, 110.15, 107.18, 98.73, 97.56, 49.29, 48.88, 45.86, 33.78, 21.63, 11.92. HRMS: m/z C₃₄H₃₄N₈O₄Na [M + Na]⁺ 641.2703, Found 641.2599. Purity: >95% (HPLC).

1,5-dimethyl-N-(4-((2-((6-(2-(4-methylpiperazin-1-yl)acetamido)pyridin-3-yl)amino)pyrimidin-4-yl)oxy)phenyl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17g): White solid, yield: 18.6%. ¹H NMR (600 MHz, DMSO-d₆) δ 10.81 (s, 1H), 9.72 (s, 1H), 9.67 (s, 1H), 8.49 (s, 1H), 8.35 (d, J = 5.6 Hz, 1H), 7.98 (s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.69 (d, J = 8.9 Hz, 2H), 7.60 (t, J = 7.7 Hz, 2H), 7.52 (t, J = 7.5 Hz, 1H), 7.48–7.43 (m, 2H), 7.20 (d, J = 8.9 Hz, 2H), 6.43 (d, J = 5.6 Hz, 1H), 3.37 (s, 5H), 3.11 (s, 2H), 2.72 (s, 3H), 2.52–2.50 (m, 2H), 2.38 (s, 4H), 2.18 (s, 3H).¹³C NMR (126 MHz, DMSO-d₆) δ 170.10, 168.61, 163.52, 161.66, 161.66, 160.40, 160.08, 154.31, 147.90, 145.69, 139.23, 136.78, 133.75, 133.50, 129.95, 129.32, 128.77, 127.57, 122.68, 120.68, 113.01, 99.15, 97.62, 61.50, 55.13, 52.97, 46.02, 33.78, 11.94. HRMS: m/z C₃₄H₃₆N₁₀O₄Na [M + Na]⁺ 671.2921, Found 671.2823. Purity: >95% (HPLC).

1,5-dimethyl-N-(4-((2-((4-(2-(4-methylpiperazin-1-yl)propanamido)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (compound 17h): White solid, yield: 33.8%. ¹H NMR (600 MHz, DMSO-d₆) δ 10.81 (s, 1H), 9.55 (s, 1H), 9.49 (s, 1H), 8.32 (d, J = 5.6 Hz, 1H), 7.71–7.67 (m, 2H), 7.60 (t, J = 7.7 Hz, 2H), 7.52 (dt, J = 8.3, 1.5 Hz, 1H), 7.51–7.47 (m, 2H), 7.45–7.42 (m, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.22–7.18 (m, 2H), 6.37 (d, J = 5.6 Hz, 1H), 3.37 (s, 3H), 3.15 (q, J = 6.8 Hz, 1H), 2.72 (s, 3H), 2.52–2.50 (m, 2H), 2.40 (d, J = 71.7 Hz, 6H), 2.13 (s, 3H), 1.13 (d, J = 6.9 Hz, 3H).¹³C NMR (126 MHz, DMSO-d₆) δ 171.20, 170.01, 163.52, 161.65, 160.31, 160.17, 154.32, 148.03, 136.71, 133.49, 133.13, 129.95, 129.31, 127.53, 122.77, 120.64, 120.16, 119.56, 98.41, 97.65, 63.77, 55.38, 49.39, 46.16, 33.78, 13.29, 11.94. HRMS: m/z C₃₆H₄₀N₉O₄ [M + H]⁺ 662.3125, Found 662.3207. Purity: >95% (HPLC).

2.2. Inhibitory Activity Assessment Against Mer and c-Met

In vitro Mer/c-Met kinase inhibition profiling was outsourced to Shanghai Bioduro Biological Technology Co., Ltd. (Shanghai, China) using the following optimized protocol: Assay buffer (1X, MgCl₂ 5 mM, DTT 1 mM) served as the base matrix. Test compounds were diluted in DMSO to 100× final concentration and dispensed (100 nL/well) into a 384-well plate via an automated liquid handler, maintaining 1% DMSO% throughout. Enzymes were diluted to a 2X assay working concentration (1.2 nM) in buffer. Then, 5 μL was added to the assay plate manually (final 1 nM) with a multichannel pipette, span down at 1000 rpm and centrifuged for 30 s, and incubated for 15 min at 25 °C temperatures. The substrate solutions were diluted with 1X assay buffer. Then, 5 μL of mix or buffer was added to the assay plate manually (ATP final 3.5 μM and TK-Sub-Biotin final 0.5 μM) with a multichannel pipette, span down at 1000 rpm and centrifuged for 30 s. After 30 °C 60 min, 10 μL of detection solution (TK-antidody-cryptate final 0.25X and streptavidine-XL 665 final 0.3125 μM) was added to each well of the assay. This was mixed briefly with centrifuge and equilibrated for 60 min. The luminescence was recorded on Envision.

2.3. Antiproliferation Assay

The HepG2, MDA-MB-231, and HCT116 cell lines were derived from our own laboratory (the laboratory of our collaborator, Dr. Xiaolei Zhou, at College of Food Science and Biology in Hebei University of Science and Technology) [31]. The antiproliferative activities were assessed using the CCK-8 assay. Cell viability was measured according to the manufacturer’s instructions for the Cell Counting Kit-8 (Meilunbio, Dalian, China). In this assay, 2.5 × 10³ cells per well were seeded into 96-well plates.

After an initial 12 h incubation, cancer cells were exposed to varying concentrations of compound 17c for 72 h. Following CCK-8 solution addition and 2 h incubation, the absorbance at 450 nm was measured using a BioTek Synergy HTX microplate reader. Experiments included triplicate wells with three independent replicates. The IC₅₀ values for compound 17c were derived from an analysis of the dose–response curves in GraphPad PRISM 9.5.

2.4. hERG Potassium Channel Activity Assessment

hERG channel activity was evaluated in CHO cells (Cell Bank of Chinese Academy of Sciences, SCSP-507) stably expressing hERG transcripts using Sophion’s QPatch automated patch clamp system (Shanghai Institute of Materia Medica, Shanghai, China). Cells cultured in serum-supplemented F12 medium (37 °C/5% CO₂) to 70–80% confluency were PBS-washed, detached with Detachin (37 °C/2 min), and resuspended in growth medium (2–5 × 10⁶ cells/mL) before QPatch loading. Following centrifugation and washing, recording was performed using extracellular solution (140 NaCl, 5 KCl, 1 CaCl₂, 1.25 MgCl₂, 10 HEPES and 10 Glucose, pH 7.4 with NaOH) and intracellular solution (140 KCl, 1 MgCl₂, 1 CaCl₂, 10 EGTA and 10 HEPES, pH 7.2 KOH). A voltage protocol measured tail current peaks at −50 mV. After a 5 min baseline recording, compounds were applied for 2.5 min/concentration across six escalating doses (n ≥ 3 cells/dose), with terminal cisapride reference. The IC₅₀ values were derived from concentration–inhibition curves (four-parameter logistic fit, GraphPad Prism 8) using Assay Software v5.6.4 for acquisition and Excel for supplementary analysis.

2.5. Liver Microsome Stability Assay

Each test compound was initially dissolved in DMSO to prepare a 10 mM stock solution. Test compound and positive controls were pre-diluted in acetonitrile to 200 μM. Incubation mixtures (200 μL total volume) comprised 0.1 M PBS (pH 7.4), 2 mM of NADPH, 0.2 mg/mL liver microsomes, and the diluted compound/control. Following a 5 min pre-incubation of all components (excluding NADPH) at 37 °C, reactions were initiated withNADPH addition. The mixture was pipette-mixed thoroughly, and 20 μL was immediately transferred to a “Quenching” plate as a 0 min sample, followed by pipette-mixing. At subsequent time points (5, 15, 30, and 60 min), 20 μL aliquots were transferred from the incubate to the “Quenching” plate after pipette-mixing. To each well of the plate, 200 μL of acetonitrile containing the internal standard was added. After centrifugation (4000 rpm, 10 min), 50 μL of supernatant was diluted 1:1 with deionized water and subjected to LC-MS/MS analysis.

2.6. Docking Study

Molecular docking simulations were performed in our lab. The binding interactions of the compounds were investigated with the aid of Discovery Studio 2019. For this analysis, the X-ray crystallographic structures of MerTK (PDB: 4M3Q) and c-Met (PDB: 3LQ8), both resolved at 1.84 Å resolution, were utilized. The protein–ligand interactions were visualized and analyzed through the interaction mode diagram generated by Discovery Studio 2019.

2.7. Western Blot Assay

Western blot assay was performed according to established protocols. HCT colon cancer cells were exposed to the specified concentration of compound 17c for 24 h, after which whole cell lysates were prepared using RAPI cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Primary antibodies, purchased from Beyotime Institute of Biotechnology, were diluted at a ratio of 1:2000. The protein bands on polyvinylidene fluoride membranes were visualized and analyzed using Glyco Band-Scan Software Version 4.50. The antibody details are as follows:

Name: Phospho-MER/TYRO3 (Tyr753/Tyr685) antibody, Species: Rabit, Sequence: synthesized peptide derived from human MER/TYRO3 around the Phosphorylation site of Tyr753/Tyr685, Dilution: 1:2000, RRID number: AB_2840500, Catalogue number: AF8443, Supplier: Affinity Biosciences (Beijing, China).

Name: Phospho-c-Met (Tyr1234) antibody, Species: Rabit, Sequence: synthesized peptide derived from human c-Met around the Phosphorylation site of Tyr1234, Dilution: 1:2000, RRID number: AB_2834564, Catalogue number: AF3129, Supplier: Affinity Biosciences (Beijing, China).

2.8. Apotosis Study

Apoptosis was analyzed in a dose-dependent manner using the One Step TUNEL Apoptosis Assay Kit (Meilunbio, Dalian, China). HCT116 cells (1.5 × 10⁴ cells/well) were seeded onto TC-treated glass coverslips in 24-well plates and incubated for 24 h. Following this, the cells were exposed to varying concentrations of compound 17c for 48 h. After treatment, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton-X-100. TUNEL staining was performed as per the manufacturer’s guidelines, with DAPI used for nuclear counterstaining. Fluorescent images were obtained using an Olympus microscope. The ED₅₀ value for compound 17c was calculated using GraphPad PRISM 9.5. Each assay was performed in triplicate and repeated three times for consistency.

2.9. Transwell Assay

Migration assays were conducted using Transwell^® inserts (Costar, Cambridge, MA, USA) with an 8 μm pore size polycarbonate membrane. HCT116 cells (1.5 × 10⁵) were seeded into the upper chamber with serum-free medium containing 0.2% BSA, while the lower chamber contained medium supplemented with 15% FBS. Compound 17c was applied at the specified concentrations to both sides of the membrane. After 24 h of incubation at 37 °C, the cells were fixed in methanol and stained with 0.1% crystal violet. Non-migrated cells on the upper surface of the membrane were removed using a cotton swab. Five random fields per well were imaged under brightfield microscopy, and the number of migrated cells was quantified using ImageJ software (1.53c). Each experiment was performed in triplicate.

2.10. Plasma Protein Binding in Plasma

A stock solution (20 mM) of compound 17c was prepared in DMSO. The stock solution was then diluted into 100 μM with acetonitrile. The HT Dialysis apparatus was assembled according to the manufacturer’s instructions. Then, 150 μL of the plasma spiked with 1 μM final compound concentrations was added to one side of the well and 150 μL of PBS was added to the opposite side of the well (n = 3). The plate was equilibrated for 6 h at 37 °C in a 5% CO₂ incubator. For T0 samples, 200 μL of spiked plasma was stored at –20 °C. For T6 samples, 200 μL of spiked plasma was incubated at 37 °C in a 5% CO₂ incubator for 6 h. After incubation, 50 μL of the samples (plasma or PBS) was collected into a vessel containing 50 μL of the opposite matrix. Then, 300 μL of acetonitrile with IS was added into the vessels. The 96-well plate was centrifuged at 4000 rpm for 10 min. Then, 50 μL of supernatant was mixed with 50 μL of ddH₂O and then injected onto the LC-MS/MS system for analysis.

2.11. Pharmacokinetic Properties

Male and female SD rats (SLRC Laboratory Animal Inc., Shanghai, China) were used for the study. In the oral (PO) administration, 3 rats per group were given a single 10 mg/kg dose. Blood samples (100 μL per time point) were collected via jugular vein cannula and taken at 5, 15, and 30 min, and 1, 2, 4, 8, and 24 h post-dose. For the intravenous (IV) administration, 3 rats per group received 2.0 mg/kg, and blood samples were collected at 5, 15, and 30 min, and 1, 2, and 4 h after dosing. Rats were fasted overnight prior to dosing and remained fasted for 6 h post-dose. Plasma was separated by centrifugation and stored at –40 °C until analysis. Compound concentrations were determined by LC/MS/MS (Shimadzu LC-30AD), and pharmacokinetic parameters, including C_max, T_max, T_1/2, and AUC_0-inf, were calculated.

3. Results and Discussion

3.1. Chemistry

The synthesis of target compounds and the key intermediates are described in Scheme 1 and Scheme 2, respectively. The structures of compounds were confirmed by ¹H-NMR, ¹³C-NMR and HRMS spectroscopy.

As depicted In Scheme 1, a condensation reaction where 1-((4-fluorophenyl)carbamoyl)cyclopropane-1-carboxylic acid (9) was combined with readily accessible 4-aminophenol (10) at ambient temperature was employed, yielding the intermediate N-(4-fluorophenyl)-N-(4-hydroxyphenyl)cyclopropane-1,1-dicarboxamide (11) [36]. Subsequently, the intermediate N-(4-((2-chloropyrimidin-4-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (12) was produced via an S_N2 reaction between intermediate 11 with 2,4-dichloropyrimidine [37]. Finally, the desired compounds 13a–13k were obtained by reacting intermediate 12 with various substituted anilines, which were either commercially available or prepared in our prior research [38].

In Scheme 2, the preparation of the crucial intermediate N-(4-hydroxyphenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (15) commenced with a condensation process involving 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxylic acid (14) and 4-aminophenol (10) [32]. Intermediate 15 was then reacted with 2,4-dichloropyrimidine, yielding the pivotal intermediate N-(4-((2-chloropyrimidin-4-yl)oxy)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (16). The target compounds 17a–17h were subsequently obtained by carrying out a nucleophilic substituted reaction between intermediate 16 and various substituted anilines.

3.2. Kinase Inhibitory Activities

All the target compounds (Supplementary Material, S1) were initially screened for their inhibitory activities on Mer and c-Met kinases compared to the lead compound (18c), and the results are depicted in

Table 1. Inhibitory activities of compound 13a–13k against Mer and c-Met kinase.

Compd.	R1	IC50 (nM) 1
		Mer	c-Met
13a		21.7 ± 5.8	63.3 ± 9.1
13b		24.3 ± 5.7	25.9 ± 7.7
13c		110.1 ± 15.7	48.9 ± 6.4
13d		640.2 ± 82.2	728.1 ± 131.4
13e		7.5 ± 1.6	7.5 ± 1.1
13f		>1000	>1000
13g		>1000	>1000
13h		72.0 ± 8.9	41.6 ± 8.5
13i		95.3 ± 20.1	88.6 ± 13.9
13j		13.1 ± 2.1	48.6 ± 11.2
13k		37.3 ± 10.6	35.4 ± 10.0
18c		18.5 ± 2.3	33.6 ± 4.3
cabozantinib		0.6 ± 0.1	1.4 ± 0.2

¹ Data are means from three independent experiments.

and

Table 2. Inhibitory activities of compound 17a–17h against Mer and c-Met kinase.

Compd.	R2	IC50 (nM) 1
		Mer	c-Met
17a		26.2 ± 7.1	233.4 ± 25.4
17b		12.1 ± 1.3	100.7 ± 18.5
17c		6.4 ± 1.8	26.1 ± 7.7
17d		84.1 ± 13.9	137.1 ± 34.3
17e		69.8 ± 12.5	235.6 ± 25.6
17f		5.5 ± 0.8	25 ± 3.5
17g		>1000	139.7 ± 18.9
17h		11.9 ± 1.7	45.0 ± 6.8
18c		18.5 ± 2.3	33.6 ± 4.3
cabozantinib		0.6 ± 0.1	1.4 ± 0.2

¹ Data are means from three independent experiments.

. The corresponding curves are shown in Supplementary S3 and S4.

As summarized in Table 1, the substituents (R¹) at the terminal pyrimidine amine group significantly modulate inhibitory potency against Mer and c-Met kinases. Compounds 13b, 13j, and 13k exhibited moderate to potent dual inhibitory, with Mer IC₅₀ values ranging from 13.1 ± 2.1~37.3 ± 10.6 nM and c-Met IC₅₀ values of 25.9 ± 7.7~48.6 ± 11.2 nM, compared to the lead compound 18c (Mer IC₅₀: 18.5 ± 2.3 nM, c-Met IC₅₀: 33.6 ± 4.3 nM). Strikingly, compound 13e demonstrated the highest dual potency, achieving IC₅₀ values of 7.5 ± 1.6 nM (Mer) and 7.5 ± 1.1 nM (c-Met), representing a 2.5-fold and 4.5-fold improvement over 18c, respectively. In contrast, compounds 13c and 13h showed reduced Mer inhibitory activity while maintaining c-Met inhibitory activity comparable to 18c. Conversely, compounds 13d and 13i exhibited significantly reduced inhibitory activities against both Mer and c-Met kinase.

A novel series of pyrimidine derivatives were designed to optimize dual Mer/c-Met inhibition. As shown in Table 2, the Mer kinase inhibitory activities varied significantly across the series. Compounds 17d, 17e, and 17g exhibited reduced inhibitory potency, with Mer IC₅₀ values of 84.1 ± 13.9, 69.8 ± 12.5, and >1000 nM, respectively. In contrast, compounds 17a, 17b, and 17h retained activity comparable to the lead compound, featuring IC₅₀ values of 26.2 ± 7.1, 12.1 ± 1.3, and 11.9 ± 1.7 nM. Notably, compounds 17c and 17f showcased superior inhibition, achieving Mer IC₅₀ values of 6.4 ± 1.8 nM and 5.5 ± 0.8 nM.

For c-Met kinase inhibition, compounds 17c, 17f, and 17h, exhibited comparable IC₅₀ values ranging from 25.0 ± 3.5 to 45.0 ± 6.8 nM. However, the potency of 17a, 17b, 17d, 17e, and 17g decreased (IC₅₀ values from 100.7 ± 18.5 nM to 235.6 ± 25.6 nM), indicating that specific structural optimization at the pyrimidine scaffold detrimentally impacts c-Met binding.

The findings converge to demonstrate that the designed compounds exhibit robust inhibitory effects on both Mer and c-Met targets. The steric bulk, electronic properties, and hydrophobicity of the R¹ substituents have a decisive impact on dual activity. The R¹ structure of 13e likely optimizes interactions with the kinase ATP pocket (e.g., hydrogen bonding, hydrophobic packing), whereas the R¹ groups in 13d and 13i may introduce steric hindrance or disrupt critical binding interactions. Modification of the pyridine ammine scaffold can selectively enhance Mer inhibition (e.g., 17c/f), while most modifications (e.g., 17a/b/d/e/g) weaken c-Met binding, suggesting that c-Met is more sensitive to structural alterations. There may be local differences in the ATP-binding pockets of Mer and c-Met (e.g., flexibility or key amino acids), which could result in differing structural optimization on the two targets. A general structure–activity relationship can be visualized in Figure 6 and Figure 7. While the designed compounds demonstrate potent Mer kinase inhibition, their inhibitory activity still falls short of the positive control, indicating the need for further optimization to improve potency.

3.3. In Vitro Stability in Liver Microsomes

Metabolic stability is a critical determinant of drug-like properties. To prioritize compounds for further development, we evaluate the human liver microsomal stability of selected candidates demonstrating potent dual Mer/c-Met dual inhibition in vitro. As shown in

Table 3. Microsome stability study of compound 13b, 13e, 13j, 13k, 13m, 13n, 17c, and 17f.

Cpd.	HUMAN
	T1/2 (min)	CL (mL/min/mg)
13b	38.3	0.0905
13e	30.4	0.1142
13j	27.9	0.1521
13k	38.4	0.1243
17c	147.0	0.0236
17f	51.0	0.0680
Testosterone	32.1	0.11

, compounds 13b, 13e, 13j, 13k, 17c, and 17f were subjected to microsome assays to determine their half-life and intrinsic clearance. Compound 17c demonstrated superior metabolic stability, with an extended t_1/2 of 147.0 min and low CLint of 0.026 mL/min/mg, whereas 17f exhibited a shorter t_1/2 of 51.0 min and higher CLint of 0.068 mL/min/mg. Notably, all other compounds showed rapid hepatic clearance (t_1/2 < 40 min, CL_int > 0.068 mL/min/mg), suggesting limited metabolic resistance. The results highlight 17c as the most metabolically stable candidate within this series.

3.4. Antiproliferation Assay In Vitro

Compound 17c was further evaluated for its antiproliferative activity against three cancer cell lines with high Mer and c-Met expression (HCT116 colon cancer, CAKI-1 renal cancer, and PC-3 prostate cancer) using the CCK-8 assay [5,39,40,41,42]. As summarized in

Table 4. Cytotoxic activities of compound 17c on cancer cell lines in vitro.

Cpds	IC50 (μM) 1 of 3 Cell Lines
	HepG2	MDA-MB-231	HCT116
17c	0.46 ± 0.06	2.31 ± 0.67	3.79 ± 1.09
Cabozantinib	7.87 ± 2.11	4.62 ± 0.54	15.57 ± 4.39

¹ Data are means from three independent experiments.

, 17c demonstrated superior antiproliferative activity compared to cabozantinib across all tested cell lines. Notably, 17c exhibited the greatest potency with HCT116 cells, with an IC₅₀ value of 0.46 ± 0.06 μmol/L—a 17.1-fold improvement over cabozantinib. In CAKI-1 cells, 17c showed an IC₅₀ of 2.31 ± 0.67 μmol/L, representing a 2.0-fold enhancement compared to cabozantinib. Similarly, against PC-3 cells, 17c achieved an IC₅₀ of 3.79 ± 1.09 μmol/L, which is 4.1 times more effective than cabozantinib. These results collectively establish compound 17c as a potent dual Mer/c-Met inhibitor with significant therapeutic potential. The dose–response curves are provided in Supplementary S5.

3.5. Molecular Docking Study of Compound 17c

Molecular docking was conducted to validate the binding mode of compound 17c with Mer kinase (PDB: 4M3Q) and c-Met (PDB: 3LQ8) kinases. As illustrated in Figure 8, the docking pose of 17c in Mer kinase revealed three critical interactions: (1) the aminopyrimidine moiety acts as hydrogen bond acceptor with Leu593; (2) the amide side chain forms a hydrogen bond with Lys619; and (3) the benzene ring of the side chain engages in a π-π stacking interaction. The docking interaction energy was −68.7952 KJ/mol. For c-Met kinase (Figure 9), 17c exhibited a similar binding mode: (1) the aminopyrimidine core forms a hydrogen bond with Met1160; (2) the amide group forms hydrogen bonds with Lys1110 and Phe1223; and (3) the pyrimidine group participates in π-π interaction. The docking interaction energy was −91.7674 KJ/mol. These results confirm that the 2-substitutedaniline pyrimidine group effectively occupies the ATP-binding pockets of both kinases through conserved hydrogen bonding and hydrophobic interactions. The structural coherence between the predicted binding modes and enzymes inhibitory data strongly supports the rational design strategy, validating the scaffold’s potential for dual Mer/c-Met inhibition.

We also systematically compared our compound’s docking results with that of cabozantinib and foretinib (XL880). For c-Met kinase, the docking results of cabozantinib with c-Met kinase showed that the quinoline core could form a hydrogen bond with Met1160, and that the amide group participates in hydrogen bonding with Lys1110 and Asp1222. The docking results of foretinib with c-Met showed that the quinoline core could form a hydrogen bond with Met1160, and that the amide group participates in hydrogen bonding with Lys1110 and Asp1222. The docking results confirm that compound 17c shares a conserved binding mode with both the reference drug cabozantinib and the co-crystallized c-Met inhibitor foretinib (PDB: 3LQ8).

For Mer kinase, the binding mode of cabozantinib with Mer kinase remains unreported, and co-crystallized Mer ligands possess distinct chemotypes. We conducted the docking of cabozantinib and 17c with Mer kinase, and the results showed that cabozantinib’s amide group forms hydrogen bonds with the hinge region (Pro672 and Met674), while the quinoline moiety occupies a hydrophobic cavity without direct hydrogen bonding. Compound 17c’s aminopyrimidine moiety acts as a hydrogen bond acceptor with Leu593 and the amide side chain forms a hydrogen bond with Lys619. The docking results showed that compound 17c possesses better affinity than that of the reference drug cabozantinib.

3.6. Western Blot Assay

The inhibitory effects of compound 17c on Mer and c-Met kinase phosphorylation were assessed using Western blot analysis. As shown in Figure 10, increasing concentrations of 17c resulted in a reduction in the phosphorylation levels of both Mer and c-Met kinases. At a concentration of 2.5 μM, 17c significantly inhibited the phosphorylation of these kinases compared to the positive control, demonstrating its potential to effectively block Mer and c-Met kinase activation.

3.7. The hERG Test

To evaluate the potential cardiotoxicity risk associated with compound 17c, we assessed its inhibitory activity against the hERG potassium channel, a key determinant of cardiac safety [33]. As summarized in

Table 5. Activity on hERG potassium currents of compound 17c.

Cpd.	IC50 (μM)
17c	>40
Cisapride	0.04

, 17c demonstrated negligible hERG inhibition (IC₅₀ > 40 μM), indicating that a low risk of hERG activity was observed despite its potent dual inhibition of Mer and c-Met kinases.

3.8. Apoptosis Assay

To evaluate the apoptosis-inducing capability of compound 17c, the HCT116 colon cancer cell line was treated with varying concentrations of the compound. Apoptosis was evaluated using the TUNEL assay. DAPI staining (blue) served as the positive control, while apoptotic cells were identified by TUNEL staining (green) after 48 h of incubation with compound 17c, as shown in Figure 11. The quantity of apoptotic cells was measured at different concentrations of 17c, resulting in an ED₅₀ value of 2.036 μM. These results demonstrate that compound 17c efficiently triggers apoptosis in HCT116 cells, thereby emphasizing its potential as a highly promising candidate for additional research and development.

3.9. Transwell Assay

The Transwell migration assay shown in Figure 12 was performed to evaluate the effect of compound 17c on the migration of HCT116 cells. A decrease in the number of cells migrating through the membrane was observed with an increasing concentration of 17c. These results suggest that compound 17c effectively inhibits the migration capacity of HCT116 colon cancer cells in a concentration-dependent manner.

3.10. Plasma Protein-Binding Affinity

The plasma protein-binding (PPB) affinity significantly influences the compound’s free fraction, bioavailability, tissue distribution, and elimination kinetics, thereby impacting the pharmacokinetic properties. The PPB profile of compound 17c was assessed via equilibrium dialysis, with warfarin serving as a positive control. As shown in

Table 6. Stability recovery of compound 17c and positive control in plasma.

Cpd.	Con. (μM)	Species	Mean Fu	Mean Fb	Stability (%)
17c	1	Human	0.0158	98.4%	99.3%
17c	1	Rat	0.0178	98.2%	102.2%
Warfarin	1	Human	0.00877	99.1%	99.8%
Warfarin	1	Rat	0.0102	99.0%	98.2%

Fu (Free drug fraction): refers to the portion of the drug in the plasma that is not bound to proteins. Fu = Area ratio of compound in buffer/Area ratio of compound in plasma. Fb (Bound drug fraction): Refers to the portion of the drug that is bound to plasma proteins. Fb = 100 × (1 − Fu).

, 17c exhibited high species-independent protein binding, with unbound fractions of 1.58% in human plasma and 1.78% in mouse plasma, corresponding to PPB values of 98.4% and 98.2%, respectively. Combined with its extended half-life and low clearance, the high PPB of 17c may contribute to sustained target engagement.

3.11. Pharmacokinetic Properties

The Pharmacokinetic (PK) profiles of compound 17c were evaluated in Sprague Dawley (SD) rats following single-dose administration via the oral (PO) and intravenous (iv) route, and the Animal Care and Use Committee (ACUC) approval documentation was IACUC-ALM-02. As summarized in

Table 7. Pharmacokinetic parameters of compound 17c.

Adm.	Dose (mg/kg)	AUClast (h*ng/mL)	T1/2 (h)	Tmax (h)	Cmax (ng/mL)	CL_obs (mL/min/kg)	MRTINF_obs (h)	F (%)
p.o.	10	4626	3.46	4.00	740	–	6.52	45.3
i.v.	2	2154	3.33	–	–	16.4	3.23

, the oral administration of 17c achieved a peak plasma concentration (Cmax) of 740 ng/mL, at Tmax = 4.0 h, with a half-time (T_1/2) of 3.46 h and a mean residence time (MRT_inf) of 6.52 h. Compared to lead compound 18c, compound 17c demonstrated significantly improved PK properties, including a prolonged oral half-time, reduced clearance, an extended mean residence time, and enhanced oral bioavailability (45.3%). These data indicate that 17c exhibits favorable PK properties, with sustained drug exposure and optimized absorption characteristics.

4. Conclusions

We report a novel series of dual Mer/c-Met inhibitors, with 17c emerging as the lead candidate. Molecular docking revealed that 17c forms hydrogen bonds with Mer (Leu593/Lys619) and c-Met (Met1160/Lys1110/Phe1223), complemented by π-π interaction. 17c exhibited potent enzymatic inhibition and superior antiproliferative activity over cabozantinib against three cancer cell lines. Pharmacokinetically, 17c showed 45.3% oral bioavailability (16-fold higher than 18c), an extended half-life (3.46 h), and low hepatic clearance (0.026 mL/min/mg). Safety profiling indicated minimal hERG inhibition. Mechanistically, 17c induced apoptosis and suppressed HCT116 migration. These data position 17c as a promising dual Mer/c-Met inhibitor warranting further development.