Discovery of Dual MER/AXL Kinase Inhibitors as Bifunctional Small Molecules for Inhibiting Tumor Growth and Enhancing Tumor Immune Microenvironment

Abstract

A series of bifunctional compounds have been discovered for their dual functionality as MER/AXL inhibitors and immune modulators. The furanopyrimidine scaffold, renowned for its suitability in kinase inhibitor discovery, offers at least three distinct pharmacophore access points. Insights from molecular modeling studies guided hit-to-lead optimization, which revealed that the 1,3-diketone side chain hybridized with furanopyrimidine scaffold that respectively combined amino-type substituent and 1H-pyrazol-4-yl substituent on the top and bottom of the aryl regions to produce 22 and 33, exhibiting potent antitumor activities in various syngeneic and xenograft models. More importantly, 33 demonstrated remarkable immune-modulating activity by upregulating the expression of total T-cells, cytotoxic CD8⁺ T-cells, and helper CD4⁺ T-cells in the spleen. These findings underscored the bifunctional capabilities of 33 (BPR5K230) with excellent oral bioavailability (F = 54.6%), inhibiting both MER and AXL while modulating the tumor microenvironment and highlighting its diverse applicability for further studies to advance its therapeutic potential.

Introduction

The TAM (TYRO3, AXL, and MER) family of receptor tyrosine kinases (RTKs) are characterized by a combination of two immunoglobulin-like (IgL) domains, dual fibronectin type III (FNIII) repeats in the extracellular region and a cytoplasmic kinase domain.¹ TAM RTKs play important roles in innate immunity and homeostasis,^2,3 and dysregulation of TAM RTKs has been implicated in the pathogenesis and progression of cancer.^4,5 Of note, MER and AXL are known to be overexpressed in various types of hematological and solid tumor cancers and have been reported as poor prognostic factors. Their signaling pathways in primary tumors are associated with cancer progression, mesenchymal phenotype, metastasis, and drug resistance in both solid tumors and hematologic cancers.⁶ AXL and MER have also been recognized as potential negative immune regulators that suppress host tumor immune responses. AXL signaling suppresses proinflammatory Toll-like receptor (TLR) responses in antigen-presenting cells (APCs).⁷ During apoptotic cell ingestion, MER suppresses the M1 macrophage proinflammatory cytokine response and enhances M2 macrophage anti-inflammatory cytokine production.⁸ Recent preclinical studies demonstrated that MER mediates intrinsic and adaptive resistance to AXL-targeting agents in human head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC). The combined MER and AXL therapies led to a more potent blockade of downstream signaling and resulted in synergistic growth inhibition in human TNBC and HNSCC xenograft models.⁹ Using mouse models of murine breast tumors, it is demonstrated that both MER and AXL receptors cooperate to promote breast cancer progression and immune escape. AXL drives the aggressiveness of tumors by affecting stemness, migration, invasion, and epithelial-to-mesenchymal transition (EMT), while MER affects immunogenic signals in the tumor microenvironment through efferocytosis and production of cytokines that impinge on the tumor milieu.¹⁰ Thus, therapeutic MER and AXL inhibition would reverse treatment resistance, reduce tumor cell survival and metastatic capacity, and, at the same time, create a more robust antitumor immune response. Successfully developing MER/AXL dual kinase inhibitors would have significant impacts on cancer patients’ treatment outcomes and overall survival. Furthermore, modulating the immune system through small molecule approaches could potentially synergize with biological modalities when used in combination.

As shown in Figure 1, several type I (1–4) and type II (5–8) ATP-competitive MER or AXL inhibitors are currently under various investigation stages (Figure 1). UNC2025 (1) is a well-known MER-selective inhibitor with an extraordinary IC₅₀ value of 0.74 nM and also potently suppressed FLT3 activity with an IC₅₀ value of 0.80 nM.^11,12 UNC5293 (2), another potent and orally bioavailable MER selective inhibitor (IC₅₀ = 0.9 nM), exhibits a highly selective profile.¹³ Bemcentinib (BGB324 or R428, 3) is a potent AXL selective inhibitor (IC₅₀ = 14 nM) with more than 10-fold selectivity against MER and TYRO3 and also showed good inhibitory activities against TIE-2/TEK (4-fold), RET (9-fold), and FLT1/4 (8-fold and 5.5-fold).¹⁴ Currently, bemcentinib (3) is in a phase II clinical trial with pembrolizumab for treating advanced NSCLC patients.¹⁵ Dubermatinib (TP-0903, 4) is another promising AXL selective inhibitor (IC₅₀ = 27 nM), which is under investigation in several clinical trials in patients with advanced solid tumors or leukemia (e.g., AML, CLL, SLL).¹⁶ Regarding the similarities of ATP binding sites between TAMs and other RTKs, drugs targeting TAMs may also exhibit inhibitory effects against various oncogenic RTKs, such as MET, RON, FLT3, VEGFR2, and so forth.¹⁷ Several multitargeting MET inhibitors have shown strong inhibitory abilities against MER and AXL, including glesatinib (5), cabozantinib (6), and others with nanomolar-level IC₅₀ values (Figure 1).¹⁸⁻²¹ Notably, merestinib (LY2801653, 7) and tamnorzatinib (ONO-7475, 8), identified as highly potent dual MER/AXL inhibitors, also behaved strong inhibitory abilities against MET protein.^22,23 In addition to the aforementioned clinical candidates, numerous MER-selective, AXL-selective, or dual MER/AXL inhibitors are currently under study at various stages of development.²⁴⁻³¹

Figure 1.

Representative MER-selective, AXL-selective, or dual MER/AXL inhibitors.

Previously, we have reported a series of furanopyrimidine compounds being second-generation or third-generation epidermal growth factor receptor (EGFR) orally active inhibitors for the treatment of NSCLC.^32,33 We continued to take advantage of the drug-like properties and the structural advantage of furanopyrimidine scaffold to develop orally bioavailable drugs. To achieve the discovery of dual MER/AXL inhibitors, we executed the hybridization of our furanopyrimidine scaffold and 1,3-diketone fragments widely utilized by MET inhibitors (applied in 5–8) to identify a series of furanopyrimidine compounds as promising dual MER/AXL inhibitors in this study. Inhibitors 22 and 33 demonstrated strong in vitro with both MER and AXL at nanomolar levels and in vivo potent efficacy in both syngeneic and mouse models as well as human xenograft tumor models.

Results and Discussion

Initial SAR Study Started with the Hybridization with an EGFR Clinical Candidate Followed by Attenuation of the EGFR Inhibition

Our dual MER/AXL inhibitors discovery campaign began with the analogue (10)³⁴ of our clinical candidate DBPR112 (9),³² which was potent to suppress wild-type (WT) EGFR and double mutant (DM; T790M/L858R) EGFR enzymes, but no effect against MER and AXL enzymes (Table 1). In the first-round screening, MER-selective inhibitor 1 and AXL-selective inhibitor 3 were used as reference compounds. To replace the hydrophobic moiety of 10 to achieve MER and AXL activity with attenuating EGFR activity, we obtained 11 by taking the place of the (S)-2-phenylglycinol moiety of 10 with a 1,3-diketone fragment, which was inspired by the pan-TAM inhibitor 6 and our previous studies.³⁴ However, despite that 11 showed potent inhibition against AXL enzyme, 11 exhibited weak activity against MER enzyme. In addition, the Michael acceptor moiety still made 11 a potent anti-EGFR^DM compound with an IC₅₀ value of 42 nM. In order to further abolish EGFR activities by removing the Michael acceptor of 11 to obtain 12, we unexpectedly observed that the MER activity of 12 was increased significantly accompanied by a massive decrease in wild-type and double mutant EGFR activities. Furthermore, bearing 5-phenyl (13) or 5-hydrogen (14) furanopyrimidine analogues attenuated the MER and AXL activities. In summary, the 5-aniline and 1,3-diketone fragments of hit 12 played crucial roles that thoroughly overturned its enzymatically inhibitory ability from EGFR^WT and EGFR^DM to MER and AXL.

Table 1. Initial SAR Exploration from Hybridization^a.

^aThe inhibition values of MER, AXL, EGFR^WT, and EGFR^DM were performed using in-house Kinase-Glo assay; UNC2025 (1) and bemcentinib (3) were used as controls; all data are expressed as the mean of at least two independent experiments and are mostly within 15% error margins.

^bIC50 value.

To examine the role of oxygen at the 4-position of the furanopyrimidine scaffold, 15 was synthesized and displayed a significant loss on MER inhibition, but AXL inhibition remained, as compared to 13. By introducing different side chains, which are favorable to providing MET activity, 16 and 17 were implanted in the side chains of 5 and 7, respectively, which maintained the anti-AXL potency. However, 16 lost the inhibitory activity against MER enzyme while 17 remained identical ability as 12, showing that the 1,3-diketone moiety is pivotal for MER inhibition. To our success of first-round screening, both 12 and 17 displayed potent enzymatic MER and AXL inhibition. More importantly, 17 even showed excellent single-digit antiproliferative potency against Ba/F3-MER cells with an IC₅₀ value of 8.4 nM, which was much better than 12 (9-fold) and the reference compound 1 (11-fold). These findings offered an opportunity for further investigation of the structure–activity relationship study of compound 17.

Binding Mode Analysis of 17

To understand the binding modes of 17 with MER protein, the molecular modeling study was carried out with the X-ray cocrystal structure of MER and merestinib (7) (PDB: 7AAY).³⁵ Merestinib (7) is a known type II MET inhibitor with potent IC₅₀ values of 0.8 and 11 nM against MER and AXL enzymes, respectively.²² The superposition of type II cocrystal MER-merestinib complex and 17 is shown in Figure 2A, and the binding mode analysis is shown in Figure 2B. As depicted in Figure 2A, 17 adopts a type II binding mode with MER, while 17 and 7 overlap extensively in the ATP binding site. In addition, the 1,3-diketone moiety of 17 located in the allosteric back pocket and the two phenyl rings on the furanopyrimidine scaffold faced the solvent-accessible region. Binding mode analysis (Figure 2B) indicated that the 1-nitrogen of the scaffold formed the crucial hinge interaction with the backbone of Met674. The phenyl ring, acting as a linker between the scaffold and the 1,3-diketone group, faced the Phe742 from the DFG motif. This π–π interaction, a typical type II binding mode, achieved the DFG-out conformation. The oxygen and amide NH from the backbone of Asp741 individually formed bidentate hydrogen bonds with 17. The terminal phenyl ring was perpendicular to the Phe719 on the α-helix A forging another π–π interaction. Besides, the amino group on the 5-phenyl ring reacted with the Glu595, which is close to the solvent accessible region. Intriguingly, the phenyl ring on the 6-position of the scaffold made none of the contribution to the interaction between 17 and MER protein, and this finding spurred us to explore potential functional groups to raise inhibitory activity. We would also like to demonstrate the binding modes between 17 and AXL; however, none of the X-ray cocrystal structures of AXL kinase in complex with a type II inhibitor has been published so far. Notably, the kinase domains of MER and AXL are highly similar sharing nearly 70% identity overall (Figure 2C),³⁶ suggesting that a type II inhibitor might bind to MER and AXL enzymes with similar binding modes.

Figure 2.

Binding mode analysis of 17. (A) Superimposition of merestinib (7, pink) and 17 (yellow) (PDB: 7AAY). (B) Molecular docking of 17 (yellow) into MER kinase domain (PDB: 7AAY). Hydrogen bonding is shown as violet. Bond length unit, Å. (C) Sequence alignment of MER and AXL kinase domains, where the identical amino acid residues are colored in red.

SAR Exploration of the Functional Groups Close to the Solvent Accessible Region

To enhance the inhibitory ability against MER enzyme and maintain the inhibitory ability against AXL enzyme, several substituents were introduced to replace the 6-phenyl ring in a series of analogue 17 (Table 2). Removal of the 6-phenyl ring in 18 or replacement with bromide in 19 resulted in a significant loss of potency against MER kinase and Ba/F3-MER cells. The cellular potency was recovered by the introduction of a methyl group in 20 but still 2-fold weaker than 17 against Ba/F3-MER cells. The above results showed that the modification at the 6-position affected the activity in MER rather than AXL, suggesting that the solvent accessible region near the 6-position may not play an important role in determining the activity of AXL. The strong potency was retrieved by the incorporation of a 1H-pyrazol-4-yl group in 21. The inhibitory abilities of 21 against both enzymatic and cellular assays were nearly identical to those of 17, indicating that an aromatic substituent at 6-position is pivotal for the activity of MER protein. More interestingly, the introduction of an additional methyl group in 22, building upon the modifications from 21, has resulted in the best IC₅₀ value observed so far with both single-digit enzyme and cellular activities. Moreover, 22 demonstrated superior antiproliferative activity against Ba/F3-MER cells, further highlighting its potential as a promising candidate for MER/AXL dual inhibitory activities. Intriguingly, the introduction of an additional methyl group of methyl-1H-pyrazol-4-yl 22 to obtain 1,3-dimethyl-1H-pyrazol-4-yl 23 led to a decrease in potency. It is speculated that the additional methyl group may have interfered with the rotation of the pyrazole ring. At the same token, this hypothesis was also observed that the 3,5-dimethylisoxazol-4-yl group in 24 abolished MER activity as well as dramatically decreased AXL activity. The two methyl groups blocked the rotation of the 5-membered aromatic ring and further caused a loss of important interactions or conformational changes necessary for optimal binding to the target proteins. By changing the orientation of the pyrazole ring from 1H-pyrazol-4-yl 22 to 1H-pyrazol-3-yl 25, 25 exhibited 2- to 7-fold weaker activity in all enzymatic and cellular assays. In the presence of a thiophen-3-yl ring at the 6-position in 26, an enhancement of inhibitory activity against MER and AXL enzymes was observed, suggesting that MER activity largely affected different heteroaromatic rings, but AXL activity was slightly influenced by heteroaromatic rings. The most promising dual MER/AXL inhibitor 22 was then examined for its pharmacokinetics (PK) profile and potential for further development (Table 4). The PK study was conducted in mice, and key parameters such as half-life, area under the curve (AUC), and others were monitored following intravenous (iv) and oral (po) administration of 3 mg/kg of 22. To improve the moderate solubility of 22, it was converted into a hydrochloride salt. 22 exhibited a reasonable PK profile, including an acceptable drug exposure represented by the area under the curve (AUC_(0–inf)). However, the oral bioavailability (F) of 22 was determined to be 19.2%, which was considered relatively low.

Table 2. SAR Exploration of 6-Substituted Furanopyrimidines^a.

^aThe IC₅₀ values of MER and AXL were performed using in-house Kinase-Glo assay; UNC2025 (1) and bemcentinib (3) were used as controls; all data are expressed as the mean of at least two independent experiments and are mostly within 15% error margins.

Table 4. Pharmacokinetics Profile of Potential Compounds in Mice^a.

^aT_1/2, half-life; CL, clearance; V_ss, steady state distribution volume; DNAUC_(0–inf), dose-normalized area under curve from zero to time infinity; DNC_max, dose-normalized maximum plasma concentrations; T_max, time to C_max; F, oral bioavailability; ND, not determined; NA, not available.

^bDNAUC_(0–inf) = AUC_(0–inf)/dose.

^cDNC_max = C_max/dose.

^div dosage and po dosage are 3 mg/kg.

^eI.V. dosage is 2 mg/kg, and P.O. dosage is 10 mg/kg.

Improvement of Pharmacokinetics Profile

The AUC_(0–inf) value per dose of 22 by i.v. administration is much higher than that of 22 by p.o. administration, which indicates that 22 may have an oral permeability problem. Having established the low water solubility of 22, our subsequent efforts focused on implementing polar or solubilizing substituents at the 5-phenyl ring for the PK improvement campaign (Table 3). We initially removed the m-amino group from 22, 27 attenuated its MER inhibitory ability, and AXL inhibitory ability was decreased by 2-fold. The findings suggested that the amino group of aryl substituents at the 5-position of the furanopyrimidine scaffold is critical for enzymatic binding affinity, particularly in relation to MER activity. These observations aligned with the results obtained for 12 and 13, as highlighted in the initial stage of the SAR study. Adding an acyl group on the m-amino in 28 showed similar potency against MER enzyme compared to 22 but 8-fold weaker potency against AXL enzyme. Substituting the m-amino group of 22 with one or two methyl groups led to different outcomes; monomethyl 29 showed identical potency to 22, but dimethyl 30 showed poor inhibition against MER and AXL kinases. Furthermore, when the m-amino of 22 was replaced by an m-aminomethyl in 31 or a m-(methylamino)methyl in 32, the inhibitory ability against MER protein decreased by 3- to 6-fold but maintained its ability to AXL protein. Most interestingly, 33 with the N,N-(dimethylamino)methyl substituent at the meta-position on the 5-phenyl ring exhibited single-digit nanomolar potency against both MER and AXL enzymatically activities as well as an IC₅₀ value of 5 nM suppressing Ba/F3-MER cells.

Table 3. SAR Exploration of Substitution on 5-Phenyl Ring^a.

^aThe IC₅₀ values of MER and AXL were performed using in-house Kinase-Glo assay; UNC2025 (1) and bemcentinib (3) were used as controls; all data are expressed as the mean of at least two independent experiments and are mostly within 15% error margins.

We also introduced cyclic solubilizing groups at the meta-position, a piperazinyl in 34 and a 4-methylpiperazinyl in 35, and both compounds showed strong inhibitory effects against MER and AXL enzymes. Next, we altered the position of substituents to explore how different structural modifications affect inhibitory abilities. 36 and 37, the former with an ortho-amino group and the latter with a para-amino group, exhibited equal-level inhibitory potency against AXL kinase compared to 22, but only 36 exhibited one-third of the inhibitory potency against MER kinase. By imitating the strategy of introducing solubilizing groups at the meta-position and observing their effect, we attempted to introduce similar solubilizing groups at the para-position. Among 38–41, only 38 with an acyl substituent showed a 3.5-fold decrease in potency against MER protein compared to its counterpart 28. The others (39–41) all displayed increased inhibitory abilities against MER enzyme and maintained identical potency in inhibiting AXL enzyme, compared to their counterparts (33–35).

Having several promising dual MER/AXL inhibitors in hand, we selected 28, 29, 31, 33, 34, 35, and 39 to evaluate their pharmacokinetics parameters due to their excellent antiproliferative ability against Ba/F3-MER cells and compared them to 22. Apart from 28, the analogues were also converted into hydrochloride salts as 22. Among the tested compounds, 28, 29, and 31 demonstrated unfavorable PK profiles, possibly attributable to their poor solubility characteristics. Both 34 and 35, which feature piperazine substituents, exhibited high clearance rates after administration with 42.9 mL/min/kg for 34 and 87.1 mL/min/kg for 35. In our system, the presence of N,N-(dimethylamino)methyl group has a significant impact on the PK data. 33 and 39 displayed improved PK profiles compared to 22, and the oral bioavailability of both compounds exceeded 20%. According to the parameters of DNC_max and DNAUC_(0–inf) of 33, we speculated that 33 would demonstrate satisfactory efficacy in further development.

Effect of 33 on Inhibition of MER and AXL Phosphorylation In Vitro

The ability of 33 to inhibit MER and AXL activity was studied in a human melanoma cell line G361 with a high level of MER expression and a human NSCLC cell line H1299 with a high level of AXL expression by Western blotting. As shown in Figure 3, 33 treatment effectively blocked MER phosphorylation induced by anti-human MER monoclonal antibody MAB8912 in G361 cells and Gas6-mediated AXL phosphorylation in H1299 cells. These results validated the capability of 33 to effectively inhibit MER and AXL within the cellular environment.

Figure 3.

Inhibition of MER and AXL phosphorylation by 33 in tumor cell lines. (A) G361 cells were treated with 33 for 48 h followed by 30 min of incubation with a MER agonist antibody, MAB8912. (B) NCI-H1299 cells were treated with 33 for 1 h and then stimulated with recombinant human GAS6 for 30 min. At the end of drug treatment, cells were lysed, and the soluble protein was separated using electrophoresis on a sodium dodecyl sulfate and polyacrylamide gel and analyzed through Western blotting. Proteins were detected by exposure to the X-ray film.

Antitumor Efficacy and Immunomodulatory Activities of 22 and 33In Vivo

The antitumor efficacy and the immunomodulatory activity of 22 were examined in a murine colon tumor MC38 syngeneic model. MC38 tumor does not express MER and AXL kinases on the tumor and is utilized to evaluate the effect of 22 on the tumor immune microenvironment and its impact on tumor growth.²¹ Six to seven-week-old immunocompetent female C57BL/6 tumor-bearing mice at an average tumor volume of 50–60 mm³ were treated with 22 orally at 25 or 50 mg/kg twice a day (BID) with a five-days-on and two-days-off (FOTO) treatment schedule for 3 weeks. As shown in Figure 4A, the treatment with 22 delayed the tumor growth with tumor growth inhibition (TGI) values of 65.9 ± 6.0 and 82.4 ± 2.6% at 25 and 50 mg/kg, respectively. No significant body weight loss was observed during the treatment period (Figures 4A and S1).

Figure 4.

Antitumor efficacy and immunomodulatory activities of 22in vivo. (A) Dose-dependent antitumor effect of 22. MC38 murine colon tumor-bearing mice were dosed orally BID at 25 or 50 mg/kg and FOTO regimen for 3 weeks. (B) Immunomodulatory activities of 22. MC38 tumor-bearing mice were treated with 50 mg/kg 22 orally BID and FOTO regimen for 2 weeks. Thereafter, tumor and spleen tissues were harvested and isolated into single cells. The immune cells were analyzed by flow cytometry analyses. (C) MER and AXL dual inhibition within the tumor by 22in vivo. MDA-MB-231 human triple-negative breast xenograft tumors were treated with 22, MER-selective agent 1, and AXL-selective agent 3, either alone or in combination. All compounds were dosed at 25 mg/kg orally BID and FOTO regimen for 4 weeks. Data were expressed as mean ± SEM, n = 8 mice per group for panels (A) and (C) and n = 5–7 per group for panel (B). *p < 0.05 vs vehicle control, **p < 0.05 vs 25 mg/kg. 22 measured using one-way ANOVA and Bonferroni post-test comparison. #: p < 0.05 vs 1 and 3 single agents. Error bars in some data points are smaller than the symbols.

To evaluate the immunomodulatory activity of 22, tumor-bearing animals (average tumor volume around 150 mm³) were treated with 50 mg/kg of 22 orally BID and FOTO for 2 weeks. Thereafter, tumor and spleen tissues from the control and treated animals were harvested and the immune cells were analyzed by flow-cytometry analysis. As seen in Figure 4B (top panel), 22 produced a 50% decrease in the percentage of pro-tumor M2-like macrophages (Cd206⁺ Cd11b⁺ F4/80⁺) without altering the percentage of antitumor M1-like macrophages (Cd86⁺ Cd11b⁺ F4/80⁺), resulting in an overall reduction in intratumoral Mo macrophage populations (CD45⁺ Cd11b⁺ F4/80⁺).³⁷ The modulation of the tumor microenvironment by 22 was further confirmed by a 3-fold increase in the ratio of CD8⁺ cytotoxic T cells to M2-like macrophages within the tumor (Figure 4B, middle panel). An increase in the intratumoral M1-to-M2 ratio was also observed although it was not statistically significant (Figure 4B, middle panel). In the spleen, 22 produced an average of 1.5-fold increase in the percentage of Cd3⁺ pan-T cell and Cd4⁺ helper T cell populations and a 2-fold increase in Cd8⁺ cytotoxic T cells (Figure 4B, bottom panel). Notably, the increase in Cd4⁺ and Cd8⁺ cell populations by 22 treatment was associated with a reduction in the expression of MER and AXL enzymes on these cells in the spleen (Figure 4B, bottom panel). Collectively, these data indicate that 22 is an effective immunomodulatory agent and its antitumor efficacy is mediated by remodeling the tumor immune microenvironment.

The effect 22 on MER and AXL dual inhibition within the tumor in vivo was evaluated in a human MER and AXL overexpression triple-negative breast tumor MDA-MB-231 xenograft model. Female immunodeficient NOD/SCID mice-bearing subcutaneous tumors at an average tumor volume of 150 mm³ were treated with vehicle control, 22 at 25 mg/kg orally BID, FOTO treatment schedule for 4 weeks. MER-selective agent 1 and AXL-selective agent 3, either alone or in combination, were included for comparison. As seen in Figure 4C (body weight change in Figure S2), 4-week treatment of either 1 and 3 alone had a limited antitumor effect, with TGI values of 15.9 ± 8.6 and 7.7 ± 10.7%, respectively. The combination of 1 and 3 delayed tumor growth with a TGI value of 52.4 ± 4.3%. Notably, 22 decreased MDA-MB-231 tumor volume by 43% vs vehicle control (TGI value is 43.0 ± 5.1% of control) and was equally efficacious as the combination of 1 and 3, verifying 22’s dual MER and AXL inhibitory activities.

To determine whether the improved PK profiles of 33 could translate into enhanced antitumor effect and immunomodulatory activities in vivo, we compared the antitumor efficacy of 22 BID and 33 at 50 mg/kg, either orally once a day (QD) or BID and FOTO regimen, for 3 weeks in the MC38 tumor model. As seen in Figure 5A, 22 QD and BID inhibited tumor growth by 66.5 ± 5.3 and 81.8 ± 3.9%, respectively. Importantly, treatment with 33 QD was equally efficacious as treatment with 22 BID, producing a TGI value of 84.8 ± 2.0%. No further increase in tumor growth inhibition by 33 with the BID treatment schedule was observed (TGI = 90.4 ± 1.6%). Both agents were well-tolerated without significant body weight loss during the course of the study (Figure S3).

Figure 5.

Antitumor efficacy and immunomodulatory activities of 33in vivo. (A) Comparison of antitumor effect of 22 and 33 in the MC38 murine colon tumor model. Tumor-bearing mice were dosed orally with either QD or BID at 50 mg/kg and FOTO regimen for 3 weeks. (B) Immunophenotyping of MC38 tumor-bearing mice treated with 50 mg/kg 33 QD and FOTO regimen for 2 weeks. Thereafter, tumor and spleen tissues were harvested and isolated into single cells. The immune cells were analyzed by flow cytometry analyses. Data were expressed as mean ± SEM, n = 8 mice per group for antitumor efficacy studies and n = 4 mice per group for immunophenotyping experiment. *p < 0.05 vs vehicle control, **p < 0.05 vs 33 measured using one-way ANOVA and Bonferroni post-test comparison. Error bars in some data points are smaller than the symbols.

As an immunomodulatory agent, 33 produced more pronounced effects than 22 on the immune cells in the tumor microenvironment and in the spleen. As seen in Figure 5B upper left panel, treatment with 33 reduced 50% intratumoral Mo macrophages and 70% M2-like macrophages and increased 2-fold in M1-like macrophages. The immunomodulatory activities of 33 were further demonstrated by a 4-fold increase in the ratio of M1/M2 within the tumor and a 3-fold increase in the ratio of Cd8⁺ T cells to M2 within the tumor (Figure 5B, lower left panel). In the spleen, 33 produced a 2-fold increase in the percentage of Cd3⁺ pan-T cell and Cd4⁺ helper T cell populations and a 6-fold increase in the percentage of Cd8⁺ cytotoxic T cells (Figure 5B, right panel). Compared to 22, the expression of MER and AXL on the Cd4⁺ and Cd8⁺ cell surface was reduced to a greater extent by the treatment of 33 in the spleen (Figure 5B, right panel). The above findings demonstrated that the modification of the solubilizing group of 22 improved the PK profiles and enhanced the antitumor efficacy and immunomodulatory effects of 33.

To extend the utility of 33 as a dual MER and AXL targeted agent and immunotherapeutic agent, we further evaluated the antitumor efficacy of 33 in four different tumor models and compared the treatment effect of 33 with that of the clinical stage dual MER and AXL inhibitor tamnorzatinib (8). 8 showed potent dual inhibitory ability against MER and AXL with IC₅₀ values of 9.8 and 2.2 nM, respectively. Both MC38 and Hepa1–6 do not express MER and AXL kinases on the tumors and were used to evaluate the antitumor immunomodulatory activities of 33. 4T1 expresses AXL enzyme on the tumor and both MER and AXL enzymes on the immune cells and was used to examine the effect of 33 on tumor growth and host antitumor immunity. MDA-MB-231 coexpresses MER and AXL proteins on the tumor and was used to evaluate the effect of 33 on dual MER and AXL inhibition within the tumor. Both compounds were used at 30 mg/kg QD and FOTO regimen in Hepa1–6 tumor model and 50 mg/kg QD and FOTO regimen in MC38, 4T1, and MDA-MB-231 tumor models. Animals were treated at various lengths based on the tumor growth rate of the given tumor model. Figure 6 summarizes the results of these experiments. Treatment with 33 produced TGI values of 49, 44, 86, and 81% in 4T1, MDA-MB-231, MC38, and Hepa1–6, respectively. On the other hand, treatment with 8 produced TGI values of 12, 9, 45, and 17% in these tumor models. During the treatment, none of the mice showed severe adverse effects or massive body weight loss (Figures S4–S7).

Figure 6.

Antitumor effect of 8 and 33in vivo. (A) 4T1 murine triple-negative breast tumors, (B) MDA-MB-231 human triple-negative breast xenograft tumors, (C) MC38 murine colon tumors, (D) Hepa1–6 murine liver tumors. 4T1, MDA-MB-231, and MC38 tumor-bearing mice were dosed with QD at 50 mg/kg and FOTO regimen; Hepa1–6 tumor-bearing mice were dosed QD orally at 30 mg/kg and FOTO regimen. Treatment length depended on the individual tumor growth rate of each model. Data were expressed as mean ± SEM, n = 8–9 mice per group. *p < 0.05 vs vehicle control, **p < 0.05 vs 8 measured using one-way ANOVA and Bonferroni post-test comparison. Error bars in some data points are smaller than the symbols.

To understand the specificity and potential off-target effects of 33, it was profiled across a panel of 658 human protein kinases (including 288 mutant kinases) at a screening concentration of 1 μM by the radioactive HotSpot kinase assay.³⁸ The results revealed that 33 possessed a superior target selectivity with a kinome selectivity S(10) score of 0.032 (12/370 nonmutant kinases) (Table S1). In addition to desired target kinases, 33 also strongly inhibits TYRO3, the homology kinase of MER and AXL, and other kinases that are known to be targeted by type II kinase inhibitors, including FLT3, KIT, TRKA/B/C, and so forth. To further understand to safety profile of 33, we also have carried out a 14-day toxicity examination with repeated dose in ICR mice, and the clinical candidate tamnorzatinib (8) was utilized as the positive control (Figures S8–S11). 33 showed no significant toxicity at any dose level, only a mild decline in kidney serum biomarker CRE at 30 and 100 mpk. These results were similar to those observed for 8 at a 100 mpk dosage. To sum up, the in vivo data suggested that 33 could function as an excellent immune modulator and an effective anticancer agent simultaneously, which could be a promising candidate for further investigation as a potential therapeutic agent for cancer treatment.

Chemistry

Syntheses of compounds 11–14 are outlined in Scheme 1. Similar to our prior studies,^32,33 the 4-chloride in building blocks 42a–c was initially substituted by dicarboxamide 43 through nucleophilic aromatic substitution reaction (S_NAr), resulting in the formation of intermediate 44, as well as inhibitors 13 and 14. The nitro group of 44 was subsequently reduced to an amino group with iron powder under acidic conditions to generate inhibitor 12. Finally, amide bond formation was accomplished by treating 12 with acrylic acid, resulting in the synthesis of inhibitor 11 in a moderate yield.

Scheme 1. Synthetic Route for MER/AXL Inhibitors 11–14.

Reagents and conditions: (a) DMF, rt, 16–24 h, 58–99%; (b) iron powder, sat. NH₄Cl_(aq), ethanol, CH₂Cl₂, H₂O, 80 °C, 1.5 h, 65%; (c) acrylic acid, EDCI, CH₂Cl₂, rt, 3.5 h, 62%.

In Scheme 2, we detailed the syntheses of inhibitors 15–21 and 23–26. To synthesize 15, building block 42a was coupled with benzene-1,4-diamine initially to form intermediate 45a. The amino group of 45a was then substituted with a fluorophenyl-containing carboxylic acid to generate intermediate 47. Subsequently, the nitro-reduction of 47 using stannic chloride generated 15 in a 42% yield. By a similar token, the 4-chloride of scaffolds 42a, 42d, and 42e were first substituted by 4-aminophenol under basic conditions to form corresponding anilines 46a, 46d, and 46e, respectively. Inhibitor 16 was obtained by coupling the amino group of 46a with (4-fluorophenyl)acetyl isothiocyanate, followed by nitro group reduction on the 5-phenyl ring of 48. Additionally, 46a reacted with 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid under basic conditions to yield 49a, which was then transformed into inhibitor 17 through nitro-reduction with iron powder and saturated ammonium chloride. Following the same campaign, 46d and 46e were converted into 49d and 49e, and then, both compounds were subsequently reduced to yield inhibitors 18 and 19, respectively. Notably, 19 and 49e, both featuring a 6-bromo functional group, served as valuable intermediates for further synthesis. Inhibitor 20 was generated from 19via Suzuki coupling reaction using methylboronic acid in a moderate yield. In a similarity, 49e was coupled with different boronic esters to produce 49f–h or coupled with thiophen-3-ylboronic acid to receive 49j. The same as the abovementioned reductions, 49f–j were converted into inhibitors 21, 23, 24, and 26 with stannic chloride or iron powder under acid conditions. For 25, the 6-bromo group of 42e was first coupled with SEM-protected 1H-pyrazol-3-yl boronic ester, forming building block 42i. Through the S_NAr reaction, 42i was treated with the presynthesized carboxamide to generate 49i. Subsequently, an iron-catalyzed reduction was performed, and the protecting group of 50i was removed using trifluoroacetic acid to receive the desired inhibitor 25.

Scheme 2. Synthetic Route for MER/AXL Inhibitors 15–21 and 23–26.

Reagents and conditions: (a) benzene-1,4-diamine, ethanol, reflux, 16 h, 89%; (b) 4-aminophenol, NaH or K₂CO₃, DMF, rt, 8–16 h, 75–99%; (c) 1-[(4-fluorophenyl)carbamoyl]cyclopropane-1-carboxylic acid, EDCI, DMA, rt, 3 h, 20%; (d) (4-fluorophenyl)acetyl isothiocyanate, toluene, ethanol, rt, 45 min, 68%; (e) 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid, DIPEA, CH₂Cl₂, 80 °C, 1 h, sealed tube, 28% for 49a; TBTU, DIPEA, CH₂Cl₂, rt, 12 h, 60% for 49d; EDCI, DMAP, DMF, rt, 16 h, 55% for 49e; (f) appropriate boronic acids or boronic esters, Pd(dppf)Cl₂, Na₂CO₃, 1,4-dioxane or DMF/THF, 75–110 °C, 5–16 h, 64–88%; (g) 1-(4-fluorophenyl)-N-(4-hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide, NaH, DMF, rt, 2 h, 77%; (h) SnCl₂·2H₂O, ethanol, reflux, 4 h, 42%; (i) iron powder, sat. NH₄Cl_(aq), ethanol, CH₂Cl₂, H₂O, 70 °C, 16 h, 67%; (j) iron powder, sat. NH₄Cl_(aq), ethanol, CH₂Cl₂, H₂O, 70–80 °C, 4–16 h, 5–93% for 17, 19, 26, 50i; (k) SnCl₂·2H₂O, ethanol, CH₂Cl₂/H₂O or DMF, rt–75 °C, 1.5–20 h, 20–85% for 18, 21, 23, 24; (l) from 19, methylboronic acid, Pd₂dba₃, SPhos, K₃PO₄, 90 °C, 24 h, 54%; (m) from 50i, trifluoroacetic acid, CH₂Cl₂, rt, 16 h, 39%.

Inhibitors 22, and 27–41 were obtained following the procedure presented in Scheme 3. The process commenced with the iodination of commercially available 6-chloropyrimidin-4-ol 51 using N-iodosuccinimide under acidic conditions, generating 52 in an 86% yield. The 5-iodide of 52 was then subjected to Sonogashira coupling reaction with 4-ethynyl-1-methyl-1H-pyrazole, facilitated by a tetrakis(triphenylphosphine)palladium catalyst. This was subsequently followed by a ring closure reaction to obtain the pyrazolyl-containing furanopyrimidine scaffold 53. To further functionalize the scaffold, the bromination of 53 was taken place using N-bromosuccinimide to receive scaffold 54 in a good yield. Building upon the approach outlined in Scheme 2, the 4-chloride of 54 was reacted with 4-aminophenol under basic conditions to offer aniline 55. This aniline was subsequently coupled with 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid to provide 56 in excellent yields. Suzuki coupling of 56 with various boronic acids or boronic esters employing Pd(dppf)Cl₂ as the catalyst. This approach produced inhibitors 22, 27, 29–33, 36–39, and 41, as well as intermediates 57k and 57l. Additionally, the amino group on the 5-phenyl ring of 22 was reacted with acetyl chloride under basic conditions to obtain inhibitor 28 in a moderate yield. The intermediates 57k and 57l, both containing Boc protecting groups, were transformed into inhibitors 34 and 40, respectively, by treatment with trifluoroacetic acid. For inhibitor 35, reductive amination of 34 was performed using formaldehyde with NaBH(OAc)₃ as the reducing agent, resulting in desired inhibitor 35 with a moderate yield.

Scheme 3. Synthetic Route for MER/AXL Inhibitors 22 and 27–41.

Reagents and conditions: (a) N-iodosuccinimide, trifluoroacetic acid, CH₂Cl₂, rt, 12 h, 86%; (b) 4-ethynyl-1-methyl-1H-pyrazole, Pd(PPh₃)₄, CuI, Et₃N, 70 °C, 12 h, 30%; (c) N-bromosuccinimide, DMF, rt, 2 h, 72%; (d) 4-aminophenol, NaH, DMF, THF, rt, 12 h, 92%; (e) 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid, TBTU, DIPEA, DMF, rt, 10 h, quant.; (f) appropriate boronic acids or boronic esters, Pd(dppf)Cl₂, Na₂CO₃, DMF or DMF/THF, 80–120 °C, 5–16 h, 17–82%; (g) from 22, acetyl chloride, Et₃N, CH₂Cl₂, rt, 16 h, 64%; (h) from 57k or 57l, trifluoroacetic acid, CH₂Cl₂, rt, 16 h, 71–94%; (i) from 34, formaldehyde, NaBH(OAc)₃, methanol, CH₂Cl₂, rt, 1.5 h, 38%.

Conclusions

Kinase-targeted cancer therapies have been extensively studied for decades, while cancer immunotherapy has witnessed remarkable advancements in recent years. Both approaches have shown potential to significantly enhance patient outcomes, and ongoing research continues to explore combining them for even more effective and personalized cancer treatments. In this study, we discovered a series of promising MER/AXL inhibitors possessed a furanopyrimidine scaffold that exhibited excellent immunomodulatory effects and effective antitumor efficacy in murine syngeneic and xenograft tumor models.

Initially, leveraging the advantages of our EGFR clinical candidate and known privileged pharmacophores, we designed and synthesized 34 MER/AXL kinase inhibitors in order to increase enzymatic activities against both MER and AXL as well as improve drug-like properties. Through SAR studies, we revealed that the pharmacophore side chain (17 > 15 ≫ 16) hybridized with a furanopyrimidine scaffold, leading to 17 as an initial lead. Optimal combinations, including an amino and N-(dimethylamino)methyl substituent at the meta-position on top aryl region and 1H-pyrazol-4-yl substituent on the bottom of the aryl region, produced dual inhibitors 22 and 33, exhibiting excellent biological inhibitory activities with acceptable PK profiles.

More importantly, 22 and 33 not only demonstrated remarkable in vitro inhibitory potency at the single-digit nanomolar level but also exhibited excellent immune-modulating activity by enhancing the expression of total T-cells, cytotoxic CD8⁺ T-cells, and helper CD4⁺ T-cells in the spleen. Additionally, 33 also remarkably exhibited anticancer activity, achieving a TGI value of nearly 90% at 50 mg/kg BID orally in the MC38 syngeneic murine colorectal model. By the same token, 33 suppressed tumor growth significantly in both the 4T1 syngeneic and MDA-MB-231 xenograft triple-negative breast cancer models, with TGI values exceeding 50%. Moreover, 33 significantly delayed tumor growth in the Hepa1–6 liver cancer model, showing an extraordinary TGI value of 81% with a 30 mg/kg dose regimen. In summary, the immunomodulatory property and antitumor capability of 33 (BPR5K230) highlight its potential as a promising targeted therapy for cancer. Further preclinical studies are ongoing and will be revealed in due course.

Experimental Section

General Methods for Chemistry

All commercial chemicals and solvents are of reagent grade and were used without further purification unless otherwise stated. All reactions were carried out under a dry nitrogen or argon atmosphere and were monitored for completion by TLC using Merck 60 F254 silica gel glass-backed plates or aluminum plates; zones were detected visually under UV irradiation (254 nm) or by spraying with potassium permanganate reagent (Aldrich) followed by heating at 80 °C. Flash column chromatography was carried out using silica gel (Silicycle SiliaFlash P60, R12030B, 230–400 mesh or Merck grade 9385, 230–400 mesh). ¹H and ¹³C NMR spectra were recorded with Bruker 400 or 600 MHz AVANCE III spectrometers. Data analysis was done using Mnova software (Mestrelab Research). Chemical shift (δ) was reported in ppm and referenced to solvent residual signals as follows: DMSO-d₆ at 2.50 ppm, CDCl₃ at 7.26 ppm for ¹H NMR; DMSO-d₆ at 39.5 ppm, CDCl₃ at 77.0 ppm for ¹³C NMR. Splitting patterns are indicated as follows: s = singlet; d = doublet; q = quartet; dd = doublet of doublets; ddd = doublet of doublets of doublets; m = multiplet. Coupling constants (J) were given in Hertz (Hz). Low-resolution mass spectra (LRMS) data were measured with an Agilent MSD-1100 ESI-MS/MS system or Agilent Infinity II 1290 LC/MS (ESI) systems. High-resolution mass spectra (HRMS) data were measured with a Varian 901-MS FT-ICR HPLC/MS-MS system. The purity of the final compounds was determined using high-performance liquid chromatography (HPLC) system (Hitachi 2000 series) equipped with a C18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm. 4.6 mm × 150 mm) and operating at 25 °C, or ultra-performance liquid chromatography (UPLC) system (Waters Acquity UPLC/BSM) equipped with a C18 column (Waters Acquity BEH-C18 1.7 μm. 2.1 mm × 50 mm) and operating at 25 °C. For the HPLC system, elution was carried out using acetonitrile as mobile phase A, and water containing 0.1% formic acid and 2 mmol NH₄OAc as mobile phase B. Elution conditions: at 0.0 min, phase A 10% + phase B 90% with 0.5 mL/min flow rate of the mobile phase; at 25.0 min, phase A 90% + phase B 10% with 0.5 mL/min flow rate of the mobile phase; at 30.5 min, phase A 10% + phase B 90% with 1.0 mL/min flow rate of the mobile phase; at 34.5 min, phase A 10% + phase B 90% with 0.5 mL/min flow rate of the mobile phase; at 37.0 min, phase A 10% + phase B 90% with 0.5 mL/min flow rate of the mobile phase. The injection volume of the sample was 20 μL. For the UPLC system, elution was carried out using acetonitrile as mobile phase A and water containing 0.1% formic acid and 2 mmol NH₄OAc as mobile phase B. Elution conditions: at 0.00 min, phase A 10% + phase B 90%; at 4.15 min, phase A 90% + phase B 10%; at 5.00 min, phase A 10% + phase B 90%; at 6.50 min, phase A 10% + phase B 90%. The flow rate of the mobile phase was 0.6 mL/min, and the injection volume of the sample was 5 μL. Peaks were detected at 254 nm. The purity of all tested compounds was determined and confirmed to be greater than 95% by HPLC or UPLC analysis except for compounds 13 (94.1%), 16 (80.2%), 17 (86.9%), 21 (91.4%), 22 (94.1%), 25 (87.7%), 26 (91.8%), 29 (93.5%), 30 (94.2%), 31 (92.5%), 32 (93.3%), 40 (94.6%), and 42 (91.9%). IUPAC nomenclature of compounds was obtained with Mnova software (Mestrelab Research).

N¹-[4-({5-[3-(Acryloylamino)phenyl]-6-phenylfuro[2,3-d]pyrimidin-4-yl}oxy)phenyl]-N¹-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (11)

To a solution of 12 (89 mg, 0.15 mmol, 1.0 equiv) in dichloromethane (1 mL) were added acrylic acid (15 μL, 0.22 mmol, 1.5 equiv) and EDCI (43 mg, 0.22 mmol, 1.5 equiv), and then, the reaction mixture was stirred at room temperature. After stirring for 3.5 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (50% ethyl acetate in hexane) to yield the title compound 11 (60 mg, 0.09 mmol, 62%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.25 (s, 1H), 10.11 (s, 1H), 10.06 (s, 1H), 8.54 (s, 1H), 7.96 (dd, J = 2.1, 1.8 Hz, 1H), 7.74 (ddd, J = 8.4, 2.1, 1.2 Hz, 1H), 7.65–7.61 (m, 4H), 7.60–7.56 (m, 2H), 7.46–7.40 (m, 4H), 7.33 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 7.17–7.11 (m, 4H), 6.41 (dd, J = 16.8, 10.2 Hz, 1H), 6.24 (dd, J = 16.8, 2.1 Hz, 1H), 5.74 (dd, J = 10.2, 2.1 Hz, 1H), 1.45 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.13, 168.09, 166.82, 163.29, 158.25 (d, J_C–F = 240.2 Hz), 152.93, 148.67, 147.69, 139.31, 136.38, 135.20, 131.73, 131.36, 129.61, 129.17, 128.97, 128.50, 127.13, 126.91, 125.30, 122.36 (d, J_C–F = 8.0 Hz), 121.77, 121.49, 120.62, 119.15, 115.03 (d, J_C–F = 22.2 Hz), 114.92, 106.41, 31.45, 15.43. LRMS (ESI) m/z 654.2 [M + H]⁺. HRMS (ESI) m/z for C₃₈H₂₈FN₅NaO₅ [M + Na]⁺, calcd 676.1972, found 676.1972. HPLC purity 98.02% (t_R = 27.83 min).

N¹-(4-{[5-(3-Aminophenyl)-6-phenylfuro[2,3-d]pyrimidin-4-yl]oxy}phenyl)-N¹-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (12)

To a solution of 44 (209 mg, 0.33 mmol, 1.0 equiv) in ethanol (6.6 mL), dichloromethane (6.6 mL) and water (1.3 mL) were added iron powder (56 mg, 1.00 mmol, 3.0 equiv) and sat. NH₄Cl_(aq) (0.7 mL), and then, the reaction mixture was stirred at 80 °C. After stirring for 4 h, the reaction mixture was cooled down to room temperature, filtered through Celite, washed with methanol (10 mL) and dichloromethane (10 mL), concentrated in vacuo, and purified by flash chromatography (50% ethyl acetate in hexane) to yield the title compound 12 (130 mg, 0.22 mmol, 65%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.12 (s, 1H), 10.08 (s, 1H), 8.51 (s, 1H), 7.67–7.57 (m, 6H), 7.45–7.38 (m, 3H), 7.17–7.07 (m, 5H), 6.76 (dd, J = 1.2, 1.2 Hz, 1H), 6.71 (dd, J = 9.0, 2.7 Hz, 1H), 6.60 (ddd, J = 8.4, 2.7, 1.2 Hz, 1H), 5.19 (s, 2H), 1.46 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.14, 168.10, 166.74, 163.32, 158.25 (d, J_C–F = 240.1 Hz), 152.75, 148.84, 148.25, 147.82, 136.34, 135.20, 131.28, 129.34, 129.14, 128.82, 126.77, 122.37 (d, J_C–F = 7.9 Hz), 121.83, 121.51, 117.27, 115.87, 115.16, 115.03 (d, J_C–F = 22.2 Hz), 113.93, 106.54, 31.46, 15.44. LRMS (ESI) m/z 600.2 [M + H]⁺. HRMS (ESI) m/z for C₃₅H₂₇FN₅O₄ [M + H]⁺, calcd 600.2047, found 600.2051. HPLC purity 94.06% (t_R = 28.21 min).

N¹-{4-[(5,6-Diphenylfuro[2,3-d]pyrimidin-4-yl)oxy]phenyl}-N¹-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (13)

To a solution of sodium hydride (15 mg, 0.38 mmol, 1.5 equiv) in DMF (1 mL) at 0 °C was added a solution of N¹-(4-fluorophenyl)-N¹-(4-hydroxyphenyl)cyclopropane-1,1-dicarboxamide (43) (79 mg, 0.25 mmol, 1.0 equiv) in DMF (1 mL), and then, the reaction mixture was stirred at room temperature. After stirring for 20 min, the reaction mixture was cooled down to 0 °C, and 4-chloro-5,6-diphenylfuro[2,3-d]pyrimidine (42b) (77 mg, 0.25 mmol, 1.0 equiv) was added and then stirred at room temperature. After stirring for 16 h, the reaction mixture was filtered through Celite, concentrated in vacuo, and purified by thin-plate chromatography (2% methanol in dichloromethane) to yield the title compound 13 (85 mg, 0.15 mmol, 58%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.12 (s, 1H), 10.08 (s, 1H), 8.51 (s, 1H), 7.67–7.57 (m, 6H), 7.45–7.38 (m, 3H), 7.17–7.07 (m, 5H), 6.76 (dd, J = 1.2, 1.2 Hz, 1H), 6.71 (dd, J = 9.0, 2.7 Hz, 1H), 6.60 (ddd, J = 8.4, 2.7, 1.2 Hz, 1H), 5.19 (s, 2H), 1.46 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.13, 168.08, 166.83, 163.4, 158.24 (d, J_C–F = 240.2 Hz), 152.88, 148.71, 147.75 136.38, 135.20, 130.77, 130.16, 129.55, 128.90, 128.59, 128.42, 126.97, 122.35 (d, J_C–F = 7.7 Hz), 121.74, 121.55, 115.02 (d, J_C–F = 22.8 Hz), 106.43, 31.44, 15.45. LRMS (ESI) m/z 585.2 [M + H]⁺. HRMS (ESI) m/z for C₃₅H₂₅FN₄NaO₄ [M + Na]⁺, calcd 607.1758, found 607.1758. HPLC purity 98.24% (t_R = 31.08 min).

N¹-(4-Fluorophenyl)-N¹-{4-[(6-phenylfuro[2,3-d]pyrimidin-4-yl)oxy]phenyl}cyclopropane-1,1-dicarboxamide (14)

To a solution of sodium hydride (15 mg, 0.38 mmol, 1.5 equiv) in DMF (1 mL) at 0 °C was added a solution of N¹-(4-fluorophenyl)-N¹-(4-hydroxyphenyl)cyclopropane-1,1-dicarboxamide (43) (79 mg, 0.25 mmol, 1.0 equiv) in DMF (1 mL), and then, the reaction mixture was stirred at room temperature. After stirring for 20 min, the reaction mixture was cooled down to 0 °C, 4-chloro-6-phenylfuro[2,3-d]pyrimidine (42c) (58 mg, 0.25 mmol, 1.0 equiv) and then stirred at room temperature. After stirring for 16 h, the reaction mixture was filtered through Celite, concentrated in vacuo, and purified by thin-plate chromatography (2% methanol in dichloromethane) to yield the title compound 14 (76 mg, 0.15 mmol, 59%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.19 (s, 1H), 10.09 (s, 1H), 8.50 (s, 1H), 7.98 (d, J = 7.2 Hz, 2H), 7.73 (d, J = 9.0 Hz, 2H), 7.67–7.63 (m, 3H), 7.54 (dd, J = 7.8, 7.2 Hz, 2H), 7.49–7.45 (m, 1H), 7.27 (d, J = 9.0 Hz, 2H), 7.15 (dd, J = 9.0, 9.0 Hz, 2H), 1.48 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.13, 167.98, 162.88, 158.27 (d, J_C–F = 240.2 Hz), 153.92, 152.58, 147.78, 136.60, 135.21 (d, J_C–F = 2.6 Hz), 129.77, 129.21, 128.41, 124.97, 122.39 (d, J_C–F = 8.0 Hz), 121.83, 121.71, 115.03 (d, J_C–F = 22.2 Hz), 106.44, 98.06, 31.50, 15.46. LRMS (ESI) m/z 509.1 [M + H]⁺. HRMS (ESI) m/z for C₂₉H₂₁FN₄NaO₄ [M + Na]⁺, calcd 531.1445, found 531.1443. HPLC purity 98.25% (t_R = 29.04 min).

N¹-(4-{[5-(3-Aminophenyl)-6-phenylfuro[2,3-d]pyrimidin-4-yl]amino}phenyl)-N¹-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (15)

To a solution of 47 (83 mg, 0.13 mmol, 1.0 equiv) in ethanol (5.0 mL) was added SnCl₂·2H₂O (118 mg, 0.52 mmol, 4.0 equiv), and then, the reaction mixture was stirred at reflux. After stirring for 4 h, the reaction mixture was cooled down to room temperature and concentrated in vacuo. Then, the mixture was dissolved in ethyl acetate (10 mL) and washed with sat. NaHCO_3(aq) (10 mL × 3) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo and purified by thin-plate chromatography (50% ethyl acetate in hexane) to yield the title compound 15 (33 mg, 0.06 mmol, 42%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.06 (s, 1H), 10.04 (s, 1H), 8.50 (s, 1H), 7.64–7.60 (m, 4H), 7.56 (d, J = 9.0 Hz, 2H), 7.41 (dd, J = 8.4, 7.2 Hz, 2H), 7.37–7.31 (m, 4H), 7.14 (dd, J = 9.0, 8.4 Hz, 2H), 7.04 (s, 1H), 6.81 (ddd, J = 8.4, 2.7, 0.9 Hz, 1H), 6.79 (ddd, J = 2.7, 2.4 Hz, 1H), 6.76 (ddd, J = 7.2, 2.4, 0.9 Hz, 1H), 5.54 (s, 2H), 1.45 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.30, 167.97, 164.49, 158.26 (d, J_C–F = 238.5 Hz), 154.38, 153.67, 150.21, 146.01, 135.15, 134.38, 134.01, 131.93, 130.80, 128.96, 128.91, 125.91, 122.38 (d, J_C–F = 7.8 Hz), 121.12, 119.63, 115.98, 115.54, 115.04 (d, J_C–F = 21.9 Hz), 114.68, 113.88, 103.68, 31.28, 15.48. LRMS (ESI) m/z 599.2 [M + H]⁺. HRMS (ESI) m/z calcd for C₃₅H₂₆FN₆O₃ [M – H]⁺, calcd 597.2050, found 597.2050. HPLC purity 80.18% (t_R = 28.72 min).

(4-{[5-(3-Aminophenyl)-6-phenylfuro[5,4-d]pyrimidin-4-yl]oxy}phenyl)-N-(phenylacetyl)carbamothioic amide (16)

To a solution of 48 (166 mg, 0.27 mmol, 1.0 equiv) in ethanol (6.5 mL), dichloromethane (6.5 mL), and water (1.3 mL) were added iron powder (94 mg, 1.68 mmol, 6.3 equiv) and sat. NH₄Cl_(aq) (0.7 mL), and then, the reaction mixture was stirred at 80 °C. After stirring for 4 h, the reaction mixture was cooled down to room temperature, filtered through Celite, washed with methanol (10 mL) and dichloromethane (10 mL), and concentrated in vacuo to yield the title compound 16 (142 mg, 0.25 mmol, 93%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 12.35 (s, 1H), 11.73 (s, 1H), 8.54 (s, 1H), 7.65–7.59 (m, 4H), 7.45–7.39 (m, 3H), 7.37–7.33 (m, 4H), 7.31–7.26 (m, 1H), 7.24 (d, J = 9.0 Hz, 2H), 7.10 (dd, J = 8.1, 7.5 Hz, 1H), 6.76 (dd, J = 2.1, 1.5 Hz, 1H), 6.71 (ddd, J = 7.5, 1.5, 1.2 Hz, 1H), 6.59 (ddd, J = 8.1, 2.1, 1.2 Hz, 1H), 5.18 (s, 2H), 3.82 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 179.17, 173.14, 166.79, 162.98, 152.74, 149.94, 148.82, 148.33, 135.09, 134.26, 131.19, 129.53, 129.36, 129.14, 128.82, 128.77, 128.43, 126.99, 126.78, 125.76, 121.94, 117.26, 115.85, 115.16, 113.96, 106.67, 42.36. LRMS (ESI) m/z 572.3 [M + H]⁺. HRMS (ESI) m/z for C₃₃H₂₅N₅NaO₃S [M + Na]⁺, calcd 594.1576, found 594.1576. HPLC purity 86.91% (t_R = 28.92 min).

N-(4-{[5-(3-Aminophenyl)-6-phenylfuro[5,4-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (17)

To a solution of 49a (310 mg, 0.48 mmol, 1.0 equiv) in ethanol (17.8 mL), dichloromethane (17.8 mL), and water (3.3 mL) were added iron powder (165 mg, 2.95 mmol, 6.1 equiv) and sat. NH₄Cl_(aq) (1.2 mL), and then, the reaction mixture was stirred at 80 °C. After stirring for 4 h, the reaction mixture was cooled down to room temperature, filtered through Celite, washed with methanol (10 mL) and dichloromethane (10 mL), concentrated in vacuo, and purified by flash chromatography (3% methanol in dichloromethane) to yield the title compound 17 (17 mg, 0.03 mmol, 5%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.58 (dd, J = 7.2, 2.4 Hz, 1H), 8.52 (s, 1H), 8.11 (dd, J = 6.6, 1.8 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 7.64–7.58 (m, 4H), 7.45–7.39 (m, 5H), 7.20 (d, J = 9.0 Hz, 2H), 7.11 (dd, J = 7.8, 7.8 Hz, 1H), 6.76 (dd, J = 2.4, 1.8 Hz, 1H), 6.74–6.69 (m, 2H), 6.60 (ddd, J = 8.4, 2.4, 1.2 Hz, 1H), 5.19 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.75, 163.22, 161.85, 161.20, 152.78, 148.84, 148.26,147.96, 144.78, 143.94, 136.33, 135.76, 131.263, 129.35 (d, J_C–F = 8.6 Hz), 129.33, 129.15, 128.83, 126.77, 122.35, 120.74, 120.43, 117.26, 116.16, 116.00, 115.89, 115.16, 113.93, 106.97, 106.58. LRMS (ESI) m/z 610.5 [M + H]⁺. HRMS (ESI) m/z for C₃₆H₂₄FN₅NaO₄ [M + Na]⁺, calcd 632.1710, found 632.1717. HPLC purity 97.46% (t_R = 28.27 min).

N-(4-{[5-(3-Aminophenyl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (18)

To a solution of 49d (150 mg, 0.27 mmol, 1.0 equiv) in ethanol (5.3 mL) was added SnCl₂·2H₂O (480 mg, 2.13 mmol, 8.0 equiv), and then, the reaction mixture was stirred at 70 °C. After stirring for 1.5 h, the reaction mixture was cooled down to room temperature and concentrated in vacuo. Then, the mixture was dissolved in ethyl acetate (10 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash chromatography (1–2% methanol in dichloromethane) to yield the title compound 18 (121 mg, 0.23 mmol, 85%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.99 (s, 1H), 8.59 (dd, J = 7.8, 2.4 Hz, 1H), 8.52 (s, 1H), 8.31 (s, 1H), 8.10 (dd, J = 6.9, 2.4 Hz, 1H), 7.77 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 9.0, 8.4 Hz, 2H), 7.31 (d, J = 9.0 Hz, 2H), 7.09 (dd, J = 7.8, 7.8 Hz, 1H), 6.97 (dd, J = 2.1, 1.8 Hz, 1H), 7.93 (ddd, J = 7.8, 2.1, 1.8 Hz, 1H), 6.72 (dd, J = 7.8, 6.9 Hz, 1H), 6.58 (ddd, J = 7.8, 2.1, 2.1 Hz, 1H), 5.19 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.53, 163.48, 161.88 (d, J_C–F = 244.4 Hz), 161.85, 161.23, 152.78, 148.78, 147.90, 144.79, 143.93, 140.90, 136.32, 135.85, 130.33, 129.35 (d, J_C–F = 9.0 Hz), 129.02, 122.50, 121.24, 120.82, 120.44, 116.19, 116.09 (d, J_C–F = 22.8 Hz), 113.94, 113.65, 106.97, 103.28. LRMS (ESI) m/z 534.2 [M + H]⁺. HRMS (ESI) m/z for C₃₀H₂₀FN₅NaO₄ [M + Na]⁺, calcd 556.1397, found 556.1393. HPLC purity 95.03% (t_R = 23.03 min).

N-(4-{[5-(3-Aminophenyl)-6-bromofuro[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (19)

To a solution of 49e (200 mg, 0.31 mmol, 1.0 equiv) in ethanol (6.2 mL), dichloromethane (6.2 mL) and water (1.2 mL) were added iron powder (104 mg, 1.86 mmol, 6.0 equiv) and sat. NH₄Cl_(aq) (0.6 mL), and then, the reaction mixture was stirred at 80 °C. After stirring for 6 h, the reaction mixture was cooled down to room temperature and concentrated in vacuo. Then, the mixture was dissolved in dichloromethane (10 mL) and washed with water (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash chromatography (1% methanol in dichloromethane) to yield the title compound 19 (178 mg, 0.29 mmol, 93%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.58 (dd, J = 7.8, 2,4 Hz, 1H), 8.52 (s, 1H), 8.10 (dd, J = 7.2, 2.4 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 9.0, 4.8 Hz, 2H), 7.41 (dd, J = 9.0, 8.4 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H), 7.11 (dd, J = 7.8, 7.2 Hz, 1H), 6.88 (d, J = 2.4, 1.5 Hz, 1H), 6.82 (d, J = 7.5 Hz, 1H), 6.71 (dd, J = 7.5, 7.5 Hz, 1H), 6.60 (ddd, J = 7.5, 1.5, 0.6 Hz, 1H), 5.26 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.67, 162.15, 161.87 (d, J_C–F = 246.1 Hz), 161.84, 161.21, 152.80, 148.56, 147.74, 144.77, 143.92, 136.30, 135.90, 129.34 (d, J_C–F = 9.1 Hz), 129.28, 128.72, 125.80, 122.38, 120.77, 120.41, 119.74, 117.16, 116.07 (d, J_C–F = 23.0 Hz), 115.07, 113.94, 106.96, 105.10. LRMS (ESI) m/z 612.1 [M + H]⁺. HRMS (ESI) m/z for C₃₀H₂₀BrFN₅O₄ [M + H]⁺, calcd 612.0683, found 612.0683. HPLC purity 95.64% (t_R = 25.70 min).

N-(4-{[5-(3-Aminophenyl)-6-methylfuro[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (20)

To a solution of 19 (89 mg, 0.15 mmol, 1.0 equiv) in toluene (7.0 mL) were added methylboronic acid (35 mg, 0.58 mmol, 4.0 equiv), Pd₂dba₃ (5.32 mg, 0.01 mmol, 4 mol %), SPhos (9.54 mg, 0.02 mmol, 16 mol %), and K₃PO₄ (93 mg, 0.44 mmol, 3.0 equiv), and then, the reaction mixture was stirred at 90 °C. After stirring for 24 h, the reaction mixture was cooled down to room temperature, added with water, and extracted into dichloromethane (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (0.5–1% methanol in dichloromethane) to yield the title compound 20 (43 mg, 0.08 mmol, 54%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.58 (dd, J = 7.2, 2.1 Hz, 1H), 8.44 (s, 1H), 8.10 (dd, J = 6.9, 2.1 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 9.0, 4.8 Hz, 2H), 7.42 (dd, J = 9.0, 8.4 Hz, 2H), 7.24 (d, J = 9.0 Hz, 2H), 7.09 (dd, J = 7.8, 7.8 Hz, 1H), 6.81 (dd, J = 2.4, 1.8 Hz, 1H), 6.74 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.71 (dd, J = 7.2, 6.9 Hz, 1H), 6.56 (ddd, J = 7.8, 2.4, 1.2 Hz, 1H), 5.18 (s, 2H), 2.49 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.99 162.35, 161.87 (d, J_C–F = 246.1 Hz), 161.84, 161.18, 151.77, 150.19, 148.51, 148.00, 144.75, 143.89, 136.31 (d, J_C–F = 3.0 Hz), 135.70, 130.85, 129.33 (d, J_C–F = 9.1 Hz), 128.66, 122.42, 120.75, 120.43, 117.39, 116.07 (d, J_C–F = 22.8 Hz), 115.36, 115.20, 113.19, 106.95, 104.83, 12.48. LRMS (ESI) m/z 548.2 [M + H]⁺. HRMS (ESI) m/z for C₃₁H₂₂FN₅NaO₄ [M + Na]⁺, calcd 570.1554, found 570.1547. HPLC purity 91.44% (t_R = 24.11 min).

N-(4-{[5-(3-Aminophenyl)-6-(1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (21)

To a solution of 49f (86 mg, 0.14 mmol, 1.0 equiv) in ethanol (8.0 mL) and N,N-dimethylformamide (0.75 mL) was added SnCl₂·2H₂O (93 mg, 0.41 mmol, 3.0 equiv), and then, the reaction mixture was stirred at room temperature. After stirring for 20 h, the reaction mixture was concentrated in vacuo. Then, the mixture was dissolved in dichloromethane (10 mL) and quenched with 6N NaOH until the pH value was over 9.0. The combined organic layers were washed with brine, dried over MgSO₄, concentrated in vacuo, and purified by flash chromatography (3% methanol in dichloromethane with 1% NH₄OH) to yield the title compound 21 (16 mg, 0.03 mmol, 20%) as a beige solid. ¹H NMR (400 MHz, CDCl₃) δ 11.85 (s, 1H), 8.75 (dd, J = 7.0, 2.0 Hz, 1H), 8.48 (s, 1H), 7.84 (s, 2H), 7.76 (d, J = 9.0 Hz, 2H), 7. 7.61 (dd, J = 6.8, 2.0 Hz, 1H), 7.41 (dd, J = 9.2, 4.8 Hz, 2H), 7.30–7.23 (m, 3H), 7.11 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 7.6 Hz, 1H), 6.89 (dd, J = 2.4, 1.6 Hz, 1H), 6.74 (d, J = 8.8 Hz, 1H), 6.61 (dd, J = 7.0, 6.8 Hz, 1H), 3.72 (s, 2H). ¹³C NMR (151 MHz, 5% CD₃OD in CDCl₃) δ 166.89, 162.90, 162.59 (d, J_C–F = 250.4 Hz), 162.31, 161.52, 161.42, 151.58, 148.50, 146.31, 145.09, 144.99, 141.77, 135.62, 135.40, 135.29, 131.51, 129.31, 128.26 (d, J_C–F = 8.9 Hz), 121.72, 121.42, 121.32, 120.40, 116.61 (d, J_C–F = 20.5 Hz), 115.25, 112.80, 111.17, 107.27, 107.12. LRMS (ESI) m/z 600.5 [M + H]⁺. HRMS (ESI) m/z for C₃₃H₂₂FN₇NaO₄ [M + Na]⁺, calcd 622.1615, found 622.1614. UPLC purity 94.12% (t_R = 2.259 min).

N-(4-{[5-(3-Aminophenyl)-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (22)

To a solution of 56 (3.11 g, 5.17 mmol, 1.0 equiv) in N,N-dimethylformamide (51.7 mL) and tetrahydrofuran (51.7 mL) were added (3-aminophenyl)boronic acid (1.06 g, 7.75 mmol, 1.5 equiv), Pd(dppf)Cl₂ (1.14 g, 1.56 mmol, 30 mol %), and 2 M Na₂CO_3(aq) (10.2 mL, 4.0 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 110 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over MgSO₄, concentrated in vacuo, and purified by flash chromatography (2–3% methanol in dichloromethane) to yield the title compound 22 (2.74 g, 4.47 mmol, 86%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.58 (dd, J = 7.6, 2.4 Hz, 1H), 8.45 (s, 1H), 8.11 (dd, J = 7.0, 2.4 Hz, 1H), 8.05 (d, J = 0.8 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 8.8, 4.8 Hz, 2H), 7.52 (d, J = 0.8 Hz, 1H), 7.42 (dd, J = 8.8, 8.4 Hz, 2H), 7.20 (d, J = 9.0 Hz, 2H), 7.13 (dd, J = 7.8, 7.6 Hz, 1H), 6.81 (dd, J = 2.2, 1.6 Hz, 1H), 6.74 (ddd, J = 7.6, 1.6, 1.0 Hz, 1H), 6.72 (dd, J = 7.6, 7.0 Hz, 1H), 6.61 (ddd, J = 7.8, 2.2, 1.0 Hz, 1H), 5.21 (s, 2H), 3.87 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.71, 162.53, 161.85 (d, J_C–F = 242.6 Hz), 161.84, 161.19, 151.87, 148.69, 148.03, 144.77, 144.34, 143.93, 136.65, 136.32, 135.70, 130.89, 129.44, 129.35 (d, J_C–F = 9.0 Hz), 128.96, 122.37, 120.72, 120.43, 117.41, 116.08 (d, J_C–F = 23.1 Hz), 115.38, 113.80, 112.69, 110.95, 106.96, 106.24, 38.78. LRMS (ESI) m/z 614.3 [M + H]⁺. HRMS (ESI) m/z for C₃₄H₂₄FN₇NaO₄ [M + Na]⁺, calcd 636.1772, found 636.1771. UPLC purity 99.67% (t_R = 2.479 min).

N-(4-{[5-(3-Aminophenyl)-6-(1,3-dimethyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (23)

To a solution of 49g (100 mg, 0.15 mmol, 1.0 equiv) in ethanol (9.0 mL) and N,N-dimethylformamide (2.0 mL) was added SnCl₂·2H₂O (103 mg, 2.13 mmol, 8.0 equiv), and then, the reaction mixture was stirred at 75 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature and concentrated in vacuo. Then, the mixture was dissolved in dichloromethane (10 mL) and quenched with 6 N NaOH until the pH value was over 9.0. The combined organic layers were washed with brine, dried over MgSO₄, concentrated in vacuo, and purified by Combiflash automated flash chromatography (3% methanol in dichloromethane) to yield the title compound 23 (50 mg, 0.08 mmol, 52%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 11.85 (s, 1H), 8.75 (dd, J = 7.2, 2.1 Hz, 1H), 8.45 (s, 1H), 7.76 (d, J = 9.0 Hz, 2H), 7.60 (dd, J = 6.9, 2.1 Hz, 1H), 7.41 (dd, J = 9.0, 4.8 Hz, 2H), 7.33 (s, 1H), 7.27 (dd, J = 9.0, 7.8 Hz, 2H), 7.19 (dd, J = 7.8, 7.8 Hz, 1H), 7.12 (d, J = 9.0 Hz, 2H), 6.91 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.85 (dd, J = 2.4, 1.8 Hz, 1H), 6.69 (ddd, J = 7.8, 2.4, 1.2 Hz, 1H), 6.60 (dd, J = 7.2, 6.9 Hz, 1H), 3.80 (s, 3H), 3.68 (s, 2H), 2.31 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.86, 162.59, 161.87 (d, J_C–F = 246.0 Hz), 161.84, 161.19, 151.86, 148.55, 148.05, 146.08, 145.05, 144.76, 143.92, 136.32, 135.69, 131.63, 131.30, 129.34 (d, J_C–F = 8.9 Hz), 128.78, 122.38, 120.74, 120.44, 117.51, 116.07 (d, J_C–F = 23.0 Hz), 115.39, 114.06, 113.46, 108.45, 106.96, 105.79, 38.49, 12.94. LRMS (ESI) m/z 628.2 [M + H]⁺. HRMS (ESI) m/z for C₃₅H₂₆FN₇NaO₄ [M+ Na]⁺, calcd 650.1928, found 650.1925. HPLC purity 99.82% (t_R = 22.61 min).

N-(4-{[5-(3-Aminophenyl)-6-(3,5-dimethyl-1,2-oxazol-4-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (24)

To a solution of 49h (87 mg, 0.13 mmol, 1.0 equiv) in ethanol (18.0 mL) and N,N-dimethylformamide (0.5 mL) was added SnCl₂·2H₂O (75 mg, 0.33 mmol, 2.5 equiv), and then, the reaction mixture was stirred at 75 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature and concentrated in vacuo. Then, the mixture was dissolved in dichloromethane (10 mL) and quenched with 6 N NaOH until the pH value was over 9.0. The combined organic layers were washed with brine, dried over MgSO₄, concentrated in vacuo, and purified by Combiflash automated flash chromatography (25–50% ethyl acetate in dichloromethane) to yield the title compound 24 (38 mg, 0.06 mmol, 46%) as a white solid. ¹H NMR (600 MHz, CDCl₃) δ 11.88 (s, 1H), 8.75 (dd, J = 7.5, 2.4 Hz, 1H), 8.53 (s, 1H), 7.80 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 7.2, 2.4 Hz, 1H), 7.41 (dd, J = 9.0, 4.8 Hz, 2H), 7.30–7.25 (m, 2H), 7.18–7.14 (m, 3H), 6.86 (ddd, J = 7.8, 1.8, 0.9 Hz, 1H), 6.80 (dd, J = 2.4, 1.8 Hz, 1H), 6.66 (ddd, J = 8.4, 2.4, 0.0 Hz, 1H), 6.61 (dd, J = 7.5, 7.2 Hz, 1H), 3.67 (s, 2H), 2.16 (s, 3H), 2.14 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 169.42, 168.04, 163.60, 162.75 (d, J_C–F = 250.5 Hz), 162.48, 161.28, 159.45, 153.13, 148.29, 146.54, 145.08, 141.69, 141.47, 136.09, 135.90, 131.26, 129.56, 128.44 (d, J_C–F = 9.1 Hz), 122.39, 121.96, 121.53, 120.13, 118.54, 116.86 (d, J_C–F = 23.1 Hz), 116.15, 114.91, 107.24, 106.73, 105.69, 12.12, 10.96. LRMS (ESI) m/z 629.2 [M + H]⁺. HRMS (ESI) m/z for C₃₅H₂₅FN₆NaO₅ [M + Na]⁺, calcd 651.1768, found 651.1762. HPLC purity 87.74% (t_R = 25.17 min).

N-(4-{[5-(3-Aminophenyl)-6-(1H-pyrazol-3-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (25)

To a solution of 50i (113 mg, 0.15 mmol, 1.0 equiv) in dichloromethane (6.0 mL) at 0 °C was added trifluoroacetic acid (534 μL, 6.97 mmol, 45.0 equiv), and then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and dissolved in dichloromethane (4.0 mL), methanol (1.0 mL), and NH₄OH (1.0 mL) at 0 °C and then stirred at room temperature. After stirring for a further 2 h, the reaction mixture was concentrated in vacuo and purified by Combiflash automated flash chromatography (2% methanol in dichloromethane) to yield the title compound 25 (36 mg, 0.06 mmol, 39%) as a beige solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.96 (s, 1H), 8.57 (dd, J = 7.5, 2.4 Hz, 1H), 8.50 (s, 1H), 8.09 (dd, J = 6.6, 2.4 Hz, 1H), 7.79 (s, 1H), 7.74 (d, J = 9.0 Hz, 2H), 7.60 (dd, J = 9.0, 4.8 Hz, 2H), 7.41 (dd, J = 9.0, 8.4 Hz, 2H), 7.21 (d, J = 9.0 Hz, 2H), 7.09 (dd, J = 8.1, 7.5 Hz, 1H), 6.83 (dd, J = 2.4, 1.8 Hz, 1H), 6.77 (d, J = 7.5 Hz, 1H), 6.71 (dd, J = 7.5, 6.6 Hz, 1H), 6.60 (d, J = 8.1 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H), 5.14 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.81, 163.09, 161.88 (d, J_C–F = 245.8 Hz), 161.84, 161.20, 152.52, 148.35, 148.00, 144.77, 143.88, 140.70, 136.31, 135.74, 130.87, 129.64, 129.33 (d, J_C–F = 8.9 Hz), 128.55, 122.38, 120.76, 120.45, 117.84, 116.08 (d, J_C–F = 23.3 Hz), 115.86, 115.44, 113.75, 106.98, 106.09, 104.56, 69.79. LRMS (ESI) m/z 600.2 [M + H]⁺. HRMS (ESI) m/z for C₃₃H₂₂FN₇NaO₄ [M + Na]⁺, calcd 622.1615, found 622.1768. UPLC purity 91.84% (t_R = 2.298 min).

N-(4-{[5-(3-Aminophenyl)-6-(thiophen-3-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (26)

To a solution of 49j (375 mg, 0.58 mmol, 1.0 equiv) in ethanol (11.6 mL), dichloromethane (11.6 mL), and water (2.3 mL) were added iron powder (195 mg, 3.49 mmol, 6.0 equiv) and sat. NH₄Cl_(aq) (1.2 mL), and then, the reaction mixture was stirred at 80 °C. After stirring for 6 h, the reaction mixture was cooled down to room temperature and concentrated in vacuo. Then, the mixture was dissolved in dichloromethane (10 mL) and washed with water (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash chromatography (0.5–1% methanol in dichloromethane) to yield the title compound 26 (83 mg, 0.13 mmol, 23%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.58 (dd, J = 7.2, 2.4 Hz, 1H), 8.50 (s, 1H), 8.10 (dd, J = 6.6, 2.4 Hz, 1H), 7.91 (dd, J = 3.0, 1.2 Hz, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.62 (dd, J = 5.4, 3.0 Hz, 1H), 7.61 (dd, J = 9.0, 4.8 Hz, 2H), 7.42 (dd, J = 9.0, 8.4 Hz, 2H), 7.19 (d, J = 9.0 Hz, 2H), 7.13 (dd, J = 8.1, 7.5 Hz, 1H),7.08 (dd, J = 5.4, 1.2 Hz, 1H), 6.79 (dd, J = 2.1, 1.8 Hz, 1H), 6.73 (ddd, J = 7.5, 1.8, 1.2 Hz, 1H), 6.71 (dd, J = 7.2, 6.6 Hz, 1H), 6.63 (ddd, J = 8.1, 2.1, 1.2 Hz, 1H), 5.22 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.63, 163.09, 161.87 (d, J_C–F = 246.1 Hz), 161.84, 161.19, 152.56, 148.74, 147.97, 145.76, 144.76, 143.92, 136.32, 135.74, 131.60, 131.06, 129.69, 129.34 (d, J_C–F = 8.9 Hz), 129.03, 127.61, 125.35, 125.04, 122.35, 120.73, 120.43, 117.43, 116.07 (d, J_C–F = 23.1 Hz), 115.39, 114.48, 113.98, 106.96, 106.43. LRMS (ESI) m/z 616.2 [M + H]⁺. HRMS (ESI) m/z for C₃₄H₂₂FN₅NaO₄S [M + Na]⁺, calcd 638.1274, found 638.1272. HPLC purity 99.59% (t_R = 27.95 min).

1-(4-Fluorophenyl)-N-(4-{[6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (27) and 1-(4-Fluorophenyl)-N-[4-({5-[3-(methylamino)phenyl]-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-2-oxo-1,2-dihydropyridine-3-carboxamide (29)

To a solution of 56 (76 mg, 0.13 mmol, 1.0 equiv) in N,N-dimethylformamide (2.5 mL) and tetrahydrofuran (2.5 mL) were added N-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (44 mg, 0.19 mmol, 1.5 equiv), Pd(dppf)Cl₂ (19 mg, 0.03 mmol, 21 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.0 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 110 °C. After stirring for 12 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3), The combined organic layers were washed with brine, dried over MgSO₄, concentrated in vacuo, and purified by flash chromatography (2% methanol in dichloromethane) to yield the title compound 27 (19 mg, 0.04 mmol, 29%) as a white solid and 29 (41 mg, 0.07 mmol, 52%) as a light-yellow solid. For 27: ¹H NMR (600 MHz, DMSO-d₆) δ 12.00 (s, 1H), 8.60 (dd, J = 7.5, 1.8 Hz, 1H), 8.45 (s, 1H), 8.35 (s, 2H), 8.12 (dd, J = 6.6, 1.8 Hz, 1H), 8.02 (s, 1H), 7.79 (d, J = 9.0 Hz, 2H), 7.62 (dd, J = 9.0, 4.8 Hz, 2H), 7.43 (dd, J = 9.0, 8.4 Hz, 2H), 7.30 (d, J = 9.0 Hz, 2H), 7.13 (s, 1H), 6.73 (dd, J = 7.5, 6.6 Hz, 1H), 3.92 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.58, 162.13, 161.88 (d, J_C–F = 245.8 Hz), 161.85, 161.26, 151.63, 149.38, 148.08, 144.81, 143.97, 136.72, 136.33, 135.91, 129.35 (d, J_C–F = 8.9 Hz), 129.21, 122.29, 120.95, 120.44, 116.09 (d, J_C–F = 23.08 Hz), 111.50, 106.97, 106.39, 95.19, 38.84. LRMS (ESI) m/z 523.2 [M + H]⁺. HRMS (ESI) m/z for C₂₈H₁₉FN₆NaO₄ [M + Na]⁺, calcd 545.1350, found 545.1361. HPLC purity 99.68% (t_R = 22.82 min). For 29: ¹H NMR (600 MHz, DMSO-d₆) δ 11.96 (s, 1H), 8.58 (dd, J = 7.2, 2.4 Hz, 1H), 8.45 (s, 1H), 8.11 (dd, J = 6.6, 2.4 Hz, 1H), 8.06 (s, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 9.0, 4.8 Hz, 2H), 7.54 (s, 1H), 7.42 (dd, J = 9.0, 8.4 Hz, 2H), 7.21–7.16 (m, 3H), 6.80 (dd, J = 2.4, 1.5 Hz, 1H), 6.78 (ddd, J = 7.8, 1.5, 1.2 Hz, 1H), 6.71 (dd, J = 7.2, 6.6 Hz, 1H), 6.59 (ddd, J = 8.4, 2.4, 1.2 Hz, 1H), 5.78 (q, J = 4.8 Hz, 1H), 3.86 (s, 3H), 2.65 (d, J = 4.8 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.74, 162.65, 161.88 (d, J_C–F = 246.1 Hz), 161.84, 161.20, 151.90, 149.83, 148.10, 144.77, 144.36, 143.93, 136.67, 136.32, 135.71, 130.93, 129.49, 129.35 (d, J_C–F = 8.9 Hz), 128.86, 122.35, 120.79, 120.44, 117.13, 116.08 (d, J_C–F = 23.1 Hz), 113.03, 112.83, 111.87, 110.96, 106.97, 106.31, 29.66. LRMS (ESI) m/z 628.3 [M + H]⁺. HRMS (ESI) m/z for C₃₅H₂₆FN₇NaO₄ [M + Na]⁺, calcd 650.1928, found 650.1934. HPLC purity 94.22% (t_R = 24.66 min).

N-[4-({5-[3-(Acetylamino)phenyl]-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (28)

To a solution of 22 (32 mg, 0.05 mmol, 1.0 equiv) in dichloromethane (1.0 mL) were added triethylamine (30 μL, 0.22 mmol, 4.1 equiv) and acetyl chloride (12 μL, 0.17 mmol, 3.2 equiv), and then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was quenched with iced water (10 mL) and washed with sat. NaHCO_3(aq) (10 mL × 3) and brine. The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash chromatography (5% methanol in dichloromethane) to yield the title compound 28 (22 mg, 0.03 mmol, 64%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.96 (s, 1H), 10.08 (s, 1H), 8.57 (dd, J = 7.4, 2.2 Hz, 1H), 8.47 (s, 1H), 8.12 (s, 1H), 8.10 (dd, J = 6.4, 2.2 Hz, 1H), 7.93 (dd, J = 2.8, 1.8 Hz, 1H), 7.72 (d, J = 10.2 Hz, 2H), 7.65–7.53 (m, 4H), 7.45–7.36 (m, 3H), 7.33 (dd, J = 7.6, 1.8 Hz, 1H), 7.19 (d, J = 10.2 Hz, 2H), 6.71 (dd, J = 7.4, 6.4 Hz, 1H), 3.86 (s, 3H), 2.05 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.59, 166.75, 162.53, 161.88 (d, J_C–F = 246.8 Hz), 161.85, 161.19, 152.04, 147.96, 144.77, 144.64, 143.92, 139.31, 136.71, 136.32 (d, J_C–F = 3.0 Hz), 135.74, 130.84, 129.72, 129.35 (d, J_C–F = 9.1 Hz), 128.88, 125.02, 122.32, 120.76, 120.68, 120.43, 118.89, 116.08 (d, J_C–F = 23.1 Hz), 111.85, 110.65, 106.97, 106.15, 38.80, 24.03. LRMS (ESI) m/z 656.2 [M + H]⁺. HRMS (ESI) m/z for C₃₆H₂₆FN₇NaO₅ [M + Na]⁺, calcd 678.1877, found 678.1872. HPLC purity 93.47% (t_R = 22.07 min).

N-[4-({5-[3-(Dimethylamino)phenyl]-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (30)

To a solution of 56 (80 mg, 0.13 mmol, 1.0 equiv) in N,N-dimethylformamide (4.0 mL) were added (3-(dimethylamino)phenyl)boronic acid (44 mg, 0.27 mmol, 2.0 equiv), Pd(dppf)Cl₂ (29 mg, 0.04 mmol, 30 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 100 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3), The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (1% methanol in dichloromethane) to yield the title compound 30 (51 mg, 0.08 mmol, 60%) as a brown solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.95 (s, 1H), 8.58 (dd, J = 7.5, 2.4 Hz, 1H), 8.46 (s, 1H), 8.11 (dd, J = 6.6, 2.4 Hz, 1H), 8.07 (s, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.61 (dd, J = 9.0, 4.8 Hz, 2H), 7.54 (s, 1H), 7.42 (dd, J = 9.0, 8.4 Hz, 2H), 7.28 (dd, J = 8.1, 7.5 Hz, 1H), 7.19 (d, J = 8.7 Hz, 2H), 7.04 (dd, J = 2.1, 1.8 Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 6.77 (dd, J = 8.1, 2.1 Hz, 1H), 6.72 (dd, J = 7.5, 6.6 Hz, 1H), 3.87 (s, 3H), 2.88 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.63, 162.35, 161.90 (d, J_C–F = 248.4 Hz), 161.78, 160.72, 151.14, 149.91, 147.85, 144.41, 144.32, 136.80, 135.35, 130.79, 128.49, 128.37, 128.17 (d, J_C–F = 9.1 Hz), 121.43, 121.11, 120.60, 117.40, 115.99 (d, J_C–F = 23.1 Hz), 113.66, 112.64, 111.80, 111.35, 106.74, 106.48, 28.97, 28.94. LRMS (ESI) m/z 642.2 [M + H]⁺. HRMS (ESI) m/z for C₃₆H₂₈FN₇NaO₄ [M + Na]⁺, calcd 664.2085, found 664.2092. UPLC purity 92.53% (t_R = 2.944 min).

N-[4-({5-[3-(Aminomethyl)phenyl]-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (31)

To a solution of 56 (80 mg, 0.13 mmol, 1.0 equiv) in N,N-dimethylformamide (2.2 mL) and tetrahydrofuran (2.2 mL) were added (3-(aminomethyl)phenyl)boronic acid (30 mg, 0.20 mmol, 1.5 equiv), Pd(dppf)Cl₂ (20 mg, 0.03 mmol, 21 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 100 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (7–8% methanol in dichloromethane) to yield the title compound 31 (63 mg, 0.10 mmol, 75%) as a beige solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.96 (s, 1H), 8.57 (dd, J = 6.9, 1.8 Hz, 1H), 8.48 (s, 1H), 8.11 (dd, J = 6.9, 1.8 Hz, 1H), 8.07 (s, 1H), 7.72 (d, J = 9.0 Hz, 2H), 7.60 (dd, J = 9.0, 5.4 Hz, 2H), 7.55 (s, 1H), 7.42 (dd, J = 9.0, 8.4 Hz, 2H), 7.20 (d, J = 9.0 Hz, 2H), 7.09 (dd, J = 10.8, 3.0 Hz, 1H), 6.71 (dd, J = 7.2, 6.6 Hz, 1H), 3.88–3.83 (m, 5H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.77, 162.60, 161.88 (d, J_C–F = 246.0 Hz), 161.84, 161.20, 152.05, 147.99, 144.77, 144.61, 143.94, 136.63, 136.31, 135.73, 130.35, 129.56, 129.34 (d, J_C–F = 8.6 Hz), 129.23, 128.66, 128.36, 127.45, 122.28, 120.79, 120.42, 116.08 (d, J_C–F = 23.0 Hz), 111.93, 110.70, 106.97, 106.21, 44.56, 39.09. LRMS (ESI) m/z 628.2 [M + H]⁺. HRMS (ESI) m/z for C₃₅H₂₇FN₇O₄ [M + H]⁺, calcd 628.2109, found 628.2110. HPLC purity 93.29% (t_R = 15.57 min).

1-(4-Fluorophenyl)-N-{4-[(5-{3-[(methylamino)methyl]phenyl}-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl)oxy]phenyl}-2-oxo-1,2-dihydropyridine-3-carboxamide (32)

To a solution of 56 (80 mg, 0.13 mmol, 1.0 equiv) in N,N-dimethylformamide (2.6 mL) and tetrahydrofuran (2.6 mL) were added N-methyl[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanamine (66 mg, 0.27 mmol, 2.0 equiv), Pd(dppf)Cl₂ (29 mg, 0.04 mmol, 30 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 100 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (2–3% methanol in dichloromethane) to yield the title compound 32 (58 mg, 0.09 mmol, 68%) as a beige solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.95 (s, 1H), 8.57 (dd, J = 7.2, 2.1 Hz, 1H), 8.47 (s, 1H), 8.11 (dd, J = 6.9, 2.1 Hz, 1H), 8.05 (s, 1H), 7.72 (d, J = 9.0 Hz, 2H), 7.63–7.57 (m, 3H), 7.54–7.50 (m, 2H), 7.45–7.39 (m, 3H), 7.36 (ddd, J = 7.2, 1.8, 1.2 Hz, 1H), 7.19 (d, J = 9.0 Hz, 2H), 6.71 (dd, J = 7.2, 6.9 Hz, 1H), 3.86 (s, 3H), 3.67 (s, 2H), 2.18 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.79, 162.63, 161.88 (d, J_C–F = 246.1 Hz), 161.84, 161.20, 152.03, 148.03, 144.77, 144.56, 143.93, 141.05, 136.58, 136.32, 135.70, 130.15, 129.80, 129.54, 129.35 (d, J_C–F = 8.9 Hz), 128.29, 128.24, 128.01, 122.28, 120.74, 120.43, 116.08 (d, J_C–F = 23.3 Hz), 112.11, 110.76, 106.97, 106.25, 54.82, 38.77, 35.39. LRMS (ESI) m/z 642.2 [M + H]⁺. HRMS (ESI) m/z for C₃₆H₂₉FN₇O₄ [M + H]⁺, calcd 642.2265, found 642.2258. HPLC purity 97.39% (t_R = 16.35 min).

N-{4-[(5-{3-[(Dimethylamino)methyl]phenyl}-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl)oxy]phenyl}-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (33)

To a solution of 56 (80 mg, 0.13 mmol, 1.0 equiv) in N,N-dimethylformamide (2.6 mL) and tetrahydrofuran (2.6 mL) was added N,N-dimethyl[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanamine (52 mg, 0.20 mmol, 1.5 equiv), Pd(dppf)Cl₂ (29 mg, 0.04 mmol, 30 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 100 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (4–5% methanol in dichloromethane) to yield the title compound 33 (51 mg, 0.08 mmol, 58%) as a beige solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.95 (s, 1H), 8.58 (dd, J = 7.2, 2.4 Hz, 1H), 8.44 (s, 1H), 8.10 (dd, J = 6.9, 2.4 Hz, 1H), 7.91 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.60 (dd, J = 9.0, 4.8 Hz, 2H), 7.42 (dd, J = 9.0, 9.0 Hz, 2H), 7.39 (d, J = 1.2 Hz, 1H), 7.17 (dd, J = 7.5, 1.8 Hz, 1H), 7.15–7.10 (m, 3H), 6.77 (dd, J = 8.1, 1.2 Hz, 1H), 6.71 (dd, J = 7.2, 6.9 Hz, 1H), 6.61 (ddd, J = 8.1, 7.5, 1.2 Hz, 1H), 5.03 (s, 2H), 3.85 (s, 3H). ¹³C NMR (151 MHz, 10% DMSO-d₆ in CDCl₃) δ 166.76, 162.50, 162.13 (d, J_C–F = 249.9 Hz), 161.93, 160.77, 151.48, 147.85, 144.51, 144.42, 141.38, 138.87, 137.00, 135.44, 135.40, 130.52, 130.27, 128.56, 128.26, 128.22, 128.04 (d, J_C–F = 8.8 Hz), 127.91, 121.57, 121.43, 120.71, 116.22 (d, J_C–F = 23.1 Hz), 112.08, 111.53, 106.80, 106.50, 63.66, 44.86, 38.66. LRMS (ESI) m/z 656.3 [M + H]⁺. HRMS (ESI) m/z for C₃₇H₃₁FN₇O₄ [M + H]⁺, calcd 656.2422, found 656.2430. HPLC purity 98.64% (t_R = 16.15 min).

1-(4-Fluorophenyl)-N-[4-({6-(1-methyl-1H-pyrazol-4-yl)-5-[3-(piperazin-1-yl)phenyl]furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-2-oxo-1,2-dihydropyridine-3-carboxamide (34)

To a solution of 57k (107 mg, 0.14 mmol, 1.0 equiv) in dichloromethane (1.4 mL) at 0 °C was added trifluoroacetic acid (260 μL, 3.40 mmol, 24.8 equiv), and then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo, and purified by flash chromatography (8% methanol in dichloromethane) to yield the title compound 34 (88 mg, 0.13 mmol, 94%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.96 (s, 1H), 8.58 (dd, J = 7.2, 2.4 Hz, 1H), 8.47 (s, 1H), 8.11 (dd, J = 6.6, 2.4 Hz, 1H), 8.08 (s, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.60 (dd, J = 9.0, 4.8 Hz, 2H), 7.54 (s, 1H), 7.42 (dd, J = 9.0, 9.0 Hz, 2H), 7.32 (dd, J = 8.4, 8.1 Hz, 1H), 7.26 (dd, J = 2.7, 1.8 Hz, 1H), 7.19 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.98 (dd, J = 8.1, 2.7 Hz, 1H), 6.72 (dd, J = 7.2, 6.6 Hz, 1H), 3.87 (s, 3H), 3.11 (s, 4H), 2.85 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.76, 161.89 (d, J_C–F = 247.0 Hz), 161.84, 161.20, 151.97, 151.16, 148.15, 144.77, 144.45, 143.94, 136.62, 136.31, 135.70, 130.95, 129.60, 129.35 (d, J_C–F = 9.1 Hz), 128.98, 122.30, 120.78, 120.44, 120.20, 117.43, 116.09 (d, J_C–F = 23.0 Hz), 115.10, 112.60, 110.85, 106.98, 106.35, 48.45, 45.00. LRMS (ESI) m/z 683.3 [M + H]⁺. HRMS (ESI) m/z for C₃₈H₃₂FN₈O₄ [M + H]⁺, calcd 683.2531, found 683.2531. HPLC purity 98.01% (t_R = 16.43 min).

1-(4-Fluorophenyl)-N-[4-({5-[3-(4-methylpiperazin-1-yl)phenyl]-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-2-oxo-1,2-dihydropyridine-3-carboxamide (35)

To a solution of 34 (70 mg, 0.10 mmol, 1.0 equiv) in methanol (3.5 mL) and dichloromethane (3.5 mL) were added formaldehyde (61 μL, 0.21 mmol, 2.0 equiv) and NaBH(OAc)₃ (108 mg, 0.51 mmol, 5.0 equiv), and then, the reaction mixture was stirred at room temperature. After stirring for 1.5 h, the reaction mixture was concentrated in vacuo, dissolved in dichloromethane (20 mL), and washed with water (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (5–6% methanol in dichloromethane) to yield the title compound 35 (27 mg, 0.04 mmol, 38%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.96 (s, 1H), 8.58 (dd, J = 7.5, 2.1 Hz, 1H), 8.47 (s, 1H), 8.11 (dd, J = 6.9, 2.1 Hz, 1H), 8.08 (s, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 9.0, 8.4 Hz, 2H), 7.31 (dd, J = 8.4, 7.8 Hz, 1H), 7.27 (dd, J = 3.0, 2.1 Hz, 1H), 7.19 (d, J = 9.0 Hz, 2H), 7.03 (ddd, J = 7.8, 2.1, 1.2 Hz, 1H), 6.98 (ddd, J = 8.4, 3.0, 1.2 Hz, 1H), 6.72 (dd, J = 7.5, 6.9 Hz, 1H), 3.87 (s, 3H), 3.14 (s, 4H), 2.40 (s, 4H), 2.18 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.75, 161.89 (d, J_C–F = 250.2 Hz), 161.84, 161.19, 151.96, 150.77, 148.13, 144.76, 144.43, 143.94, 136.63, 136.32, 135.71, 130.93, 129.58, 129.34 (d, J_C–F = 8.9 Hz), 128.95, 122.30, 120.75, 120.44, 120.09, 117.36, 116.08 (d, J_C–F = 23.1 Hz), 115.08, 112.60, 110.84, 106.96, 106.33, 69.78, 54.44, 47.78, 45.65. LRMS (ESI) m/z 697.3 [M + H]⁺. HRMS (ESI) m/z for C₃₉H₃₄FN₈O₄ [M + H]⁺, calcd 697.2687, found 697.2684. UPLC purity 100.00% (t_R = 2.091 min).

N-(4-{[5-(2-Aminophenyl)-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (36)

To a solution of 56 (100 mg, 0.17 mmol, 1.0 equiv) in N,N-dimethylformamide (1.6 mL) and tetrahydrofuran (1.6 mL) were added 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (47 mg, 0.21 mmol, 1.3 equiv), Pd(dppf)Cl₂ (38 mg, 0.05 mmol, 31 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.0 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 80 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), washed with NaHCO_3(aq) (20 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over MgSO₄, concentrated in vacuo, and purified by Combiflash automated flash chromatography (5% methanol in dichloromethane) to yield the title compound 36 (20 mg, 0.03 mmol, 20%) as a brown solid. ¹H NMR (600 MHz, CDCl₃) δ 11.82 (s, 1H), 8.73 (dd, J = 7.2, 2.4 Hz, 1H), 8.47 (s, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 0.6 Hz, 1H), 7.59 (dd, J = 6.9, 2.4 Hz, 1H), 7.51 (s, 1H), 7.40 (dd, J = 9.0, 4.8 Hz, 2H), 7.29–7.21 (m, 4H), 7.08 (d, J = 9.0 Hz, 2H), 6.87–6.80 (m, 2H), 6.59 (dd, J = 7.2, 6.9 Hz, 1H), 3.88 (s, 3H), 3.73 (s, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 167.64, 163.14, 162.73 (d, J_C–F = 250.1 Hz), 162.45, 161.17, 152.34, 148.60, 145.73, 144.99, 144.86, 141.38, 137.67, 135.92, 135.80, 131.44, 129.88, 128.61, 128.43 (d, J_C–F = 8.6 Hz), 122.45, 121.87, 121.35, 118.58, 116.81 (d, J_C–F = 23.1 Hz), 115.84, 115.65, 111.97, 108.22, 107.30, 107.17, 39.20. LRMS (ESI) m/z 614.2 [M + H]⁺. HRMS (ESI) m/z for C₃₄H₂₄FN₇NaO₄ [M + Na]⁺, calcd 636.1772, found 636.1772. UPLC purity 96.65% (t_R = 2.669 min).

N-(4-{[5-(4-Aminophenyl)-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl]oxy}phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (37)

To a solution of 56 (80 mg, 0.13 mmol, 1.0 equiv) in N,N-dimethylformamide (2.6 mL) and tetrahydrofuran (2.6 mL) were added 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (44 mg, 0.20 mmol, 1.5 equiv), Pd(dppf)Cl₂ (29 mg, 0.04 mmol, 30 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 110 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (2% methanol in dichloromethane) to yield the title compound 37 (45 mg, 0.07 mmol, 55%) as beige solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.97 (s, 1H), 8.58 (dd, J = 7.2, 2.1 Hz, 1H), 8.43 (s, 1H), 8.11 (dd, J = 6.6, 2.1 Hz, 1H), 8.02 (s, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 9.0, 4.8 Hz, 2H), 7.54 (d, J = 0.6 Hz, 1H), 7.42 (dd, J = 9.0, 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 9.0 Hz, 2H), 6.72 (dd, J = 7.2, 6.6 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 5.30 (s, 2H), 3.87 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.73, 161.87 (d, J_C–F = 244.0 Hz), 161.84, 161.17, 151.73, 148.74, 148.09, 144.76, 143.92, 143.81, 136.46, 136.32, 135.67, 130.78, 129.34 (d, J_C–F = 8.8 Hz), 129.15, 122.32, 120.75, 120.44, 116.71, 116.07 (d, J_C–F = 23.0 Hz), 113.59, 112.75, 111.21, 106.96, 106.49, 73.50, 24.95. LRMS (ESI) m/z 614.2 [M + H]⁺. HRMS (ESI) m/z for C₃₄H₂₄FN₇NaO₄ [M + Na]⁺, calcd 636.1772, found 636.1773. HPLC purity 96.96% (t_R = 22.14 min).

N-[4-({5-[4-(Acetylamino)phenyl]-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (38)

To a solution of 56 (90 mg, 0.15 mmol, 1.0 equiv) in N,N-dimethylformamide (2.5 mL) and tetrahydrofuran (2.5 mL) were added [4-(acetylamino)phenyl]boronic acid (40 mg, 0.22 mmol, 1.5 equiv), Pd(dppf)Cl₂ (16 mg, 0.02 mmol, 15 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 100 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (3% methanol in dichloromethane) to yield the title compound 38 (81 mg, 0.12 mmol, 83%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.95 (s, 1H), 10.97 (s, 1H), 8.57 (dd, J = 7.5, 2.4 Hz, 1H), 8.45 (s, 1H), 8.09 (dd, J = 6.9, 2.4 Hz, 1H), 8.03 (s, 1H), 7.72 (d, J = 9.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.60 (dd, J = 9.0, 4.8 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.52 (s, 1H), 7.41 (dd, J = 9.0, 8.4 Hz, 2H), 7.19 (d, J = 9.0 Hz, 2H), 6.71 (dd, J = 7.5, 6.9 Hz, 1H), 3.86 (s, 3H), 2.06 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 168.45, 166.73, 162.61, 161.86 (d, J_C−F = 246.1 Hz), 161.83, 161.16, 151.95, 147.97, 144.74, 144.43, 143.87, 139.30, 136.55, 136.30, 135.72, 130.55, 129.44, 129.32 (d, J_C−F = 8.9 Hz), 124.70, 122.28, 120.75, 120.43, 118.66, 116.07 (d, J_C−F = 23.1 Hz), 111.78, 110.78, 106.95, 106.29, 38.76, 24.04. LRMS (ESI) m/z 656.2 [M + H]⁺. HRMS (ESI) m/z for C₃₆H₂₆FN₇NaO₅ [M + Na]⁺, calcd 678.1877, found 678.1877. HPLC purity 97.91% (t_R = 21.33 min).

N-{4-[(5-{4-[(Dimethylamino)methyl]phenyl}-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl)oxy]phenyl}-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (39)

To a solution of 56 (100 mg, 0.17 mmol, 1.0 equiv) in N,N-dimethylformamide (1.7 mL) and tetrahydrofuran (1.7 mL) were added N,N-dimethyl-1-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methanamine hydrochloride (74 mg, 0.25 mmol, 1.5 equiv), Pd(dppf)Cl₂ (33 mg, 0.05 mmol, 27 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 100 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (3% methanol in dichloromethane) to yield the title compound 39 (70 mg, 0.11 mmol, 64%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.94 (s, 1H), 8.57 (dd, J = 7.2, 1.8 Hz, 1H), 8.47 (s, 1H), 8.10 (dd, J = 6.6, 1.8 Hz, 1H), 8.06 (s, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.63–7.57 (m, 4H), 7.45 (s, 1H), 7.44–7.38 (m, 4H), 7.18 (d, J = 9.0 Hz, 2H), 6.71 (dd, J = 7.2, 6.6 Hz, 1H), 3.86 (s, 3H), 3.47 (s, 2H), 2.18 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.78, 162.60, 161.87 (d, J_C–F = 246.0 Hz), 161.83, 161.18, 151.99, 148.02, 144.76, 144.58, 143.89, 138.76, 136.50, 136.30, 135.70, 129.96, 129.58, 129.32 (d, J_C–F = 8.9 Hz), 129.04, 128.82, 122.23, 120.79, 120.43, 116.06 (d, J_C–F = 23.0 Hz), 111.91, 110.75, 106.96, 106.25, 62.97, 44.92, 38.78. LRMS (ESI) m/z 656.2 [M + H]⁺. HRMS (ESI) m/z for C₃₇H₃₁FN₇O₄ [M + H]⁺, calcd 656.2422, found 656.2427. UPLC purity 94.62% (t_R = 1.962 min).

1-(4-Fluorophenyl)-N-[4-({6-(1-methyl-1H-pyrazol-4-yl)-5-[4-(piperazin-1-yl)phenyl]furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-2-oxo-1,2-dihydropyridine-3-carboxamide (40)

To a solution of 57l (99 mg, 0.13 mmol, 1.0 equiv) in dichloromethane (1.3 mL) at 0 °C was added trifluoroacetic acid (240 μL, 3.13 mmol, 24.8 equiv), and then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by flash chromatography (8% methanol in dichloromethane) to yield the title compound 40 (61 mg, 0.09 mmol, 71%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.96 (s, 1H), 8.58 (dd, J = 7.2, 2.4 Hz, 1H), 8.44 (s, 1H), 8.10 (dd, J = 6.6, 2.4 Hz, 1H), 8.05 (s, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.60 (dd, J = 9.0, 4.8 Hz, 2H), 7.51 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.42 (dd, J = 9.0, 8.4 Hz, 2H), 7.19 (d, J = 9.0 Hz, 2H), 7.01 (d, J = 8.7 Hz, 2H), 6.71 (dd, J = 7.2, 6.6 Hz, 1H), 3.86 (s, 3H), 3.16 (s, 4H), 2.88 (s, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.76, 162.72, 161.88 (d, J_C–F = 246.9 Hz), 161.85, 161.19, 151.83, 151.00, 148.09, 144.77, 144.11, 143.90, 136.54, 136.31 (d, J_C–F = 3.1 Hz), 135.73, 130.80, 129.346, 129.342 (d, J_C–F = 9.0 Hz), 122.41, 120.81, 120.43, 119.67, 116.09 (d, J_C–F = 23.1 Hz), 114.43, 112.15, 111.03, 106.98, 106.32, 48.30, 45.38, 38.78. LRMS (ESI) m/z 683.3 [M + H]⁺. HRMS (ESI) m/z for C₃₈H₃₂FN₈O₄ [M + H]⁺, calcd 683.2531, found 683.2543. HPLC purity 98.08% (t_R = 16.07 min).

1-(4-Fluorophenyl)-N-[4-({5-[4-(4-methylpiperazin-1-yl)phenyl]-6-(1-methyl-1H-pyrazol-4-yl)furo[2,3-d]pyrimidin-4-yl}oxy)phenyl]-2-oxo-1,2-dihydropyridine-3-carboxamide (41)

To a solution of 56 (100 mg, 0.17 mmol, 1.0 equiv) in N,N-dimethylformamide (2.8 mL) and tetrahydrofuran (2.8 mL) were added 1-methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]piperazine (75 mg, 0.25 mmol, 1.5 equiv), Pd(dppf)Cl₂ (36 mg, 0.05 mmol, 30 mol %), and 2 M Na₂CO_3(aq) (0.3 mL, 4.1 equiv). The reaction mixture was degassed for 30 min, refilled with Argon_(g), and stirred at 100 °C. After stirring for 16 h, the reaction mixture was cooled down to room temperature, filtered through Celite, added with water (10 mL), and extracted into CH₂Cl₂ (10 mL × 3). The combined organic layers were washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by flash chromatography (6–8% methanol in dichloromethane) to yield the title compound 41 (20 mg, 0.03 mmol, 17%) as a brown solid. ¹H NMR (600 MHz, DMSO-d₆) δ 11.95 (s, 1H), 8.58 (dd, J = 7.2, 2.4 Hz, 1H), 8.44 (s, 1H), 8.10 (dd, J = 6.6, 2.4 Hz, 1H), 8.05 (s, 1H), 7.73 (d, J = 9.0 Hz, 2H), 7.61 (dd, J = 8.7, 4.8 Hz, 2H), 7.52 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.42 (dd, J = 9.0, 8.7 Hz, 2H), 7.20 (d, J = 9.0 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.72 (dd, J = 7.2, 6.6 Hz, 1H), 3.87 (s, 3H), 3.21 (s, 4H), 2.46 (s, 4H). 2.23 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.73, 162.69, 161.84 (d, J_C–F = 246.0 Hz), 161.80, 161.15, 151.80, 150.44, 148.08, 144.72, 144.11, 143.86, 136.52, 136.27 (d, J_C–F = 2.4 Hz), 135.67, 130.75, 129.32, 129.29 (d, J_C–F = 8.8 Hz), 122.34, 120.78, 120.43, 119.73, 116.02 (d, J_C–F = 23.1 Hz), 114.46, 112.12, 110.97, 106.91, 106.31, 54.53, 47.25, 40.04, 38.73. LRMS (ESI) m/z 697.3 [M + H]⁺. HRMS (ESI) m/z for C₃₉H₃₄FN₈O₄ [M + H]⁺, calcd 697.2687, found 697.2688. HPLC purity 91.88% (t_R = 16.59 min).

Computational Studies

The protein structure of MER (PDB: 7AAY) was utilized in this study which was downloaded from RCSB Protein Data Bank (PDB).³⁵ All docking analyses were conducted using the Discovery Studio 2021//LigandFit program (BIOVIA Inc., San Diego, CA, USA) with the CHARMm force field through Align and Superimpose Proteins, CDOCKER, and Flexible Docking protocols with default parameters.³⁹ The number of docking poses was set as 10 with default parameters. The decision of the best pose was according to the lowest binding energy of the compound as well as the nitrogen atom of the compound forming a hydrogen bond with the backbone amide nitrogen of Met274 in MER. This was imposed based on the observation of hydrogen bonds in the cocrystal structures of the MET kinase complexed to merestinib (PDB: 7AAY). The docking results were shown as the cartoon model processed by PyMoL and Discovery Studio 2021.^40,41

MER and AXL Enzyme Inhibition Assays

A purified kinase (MER or AXL) was incubated with a compound or DMSO (control) for 15 min in an assay buffer (25 mM Tris pH 7.4, 10 mM MgCl₂, 4 mM MnCl₂, 2 mM DTT, 0.01% BSA, 0.02% TritonX-100, 0.01% Brij35 and 0.5 mM Na₃VO₄ for MER; and 40 mM Tris pH 7.4, 20 mM MgCl₂, 2 mM DTT, 0.01% BSA and 0.5 mM Na₃VO₄ for AXL). The above-prepared substrate and ATP (12 μM for MER; 50 μM for AXL) were added. The mixture was incubated for 3 h at 30 °C. The luminescence was calculated to determine the kinase activity using a Kinase Glo assay kit for MER and an ADP-Glo Kinase Assay kit for AXL following the manufacturer’s instructions (Promega Corp., Madison, Wisconsin).

Cell Culture

The Ba/F3-TMEM-MerTK cells were maintained in RPMI 1640 (Gibco, New York, NY, USA). The culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin and streptomycin (HyClone, Logan, UT, USA). The cells were maintained at 37 °C in an incubator (Thermo Fisher Scientific, New York, NY, USA) with an atmosphere of 5% CO₂.

Cell Viability Assay

Ba/F3-TMEM-MerTK cells, which overexpressed MER, were seeded in 96-well clear plates at a density of 8 × 10³ cells per well overnight. Then, cells were treated with indicated concentrations of test compounds for 72 h. At the end of incubation, for a 96-well microtiter plate, MTS Mix reagent containing culture medium, MTS (tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]; Promega, Madison, WI, USA), and PMS (phenazine methosulfate; Sigma, St. Louis, MO, USA) in a ratio of 8:2:0.1, respectively. The medium in the well was removed, and the MTS Mix reagent was then added to cells (100 μL/well). The plates were incubated for 1.5 h at 37 °C in a humidified 5% CO₂ atmosphere, and the absorbance was then recorded at 490 nm by a Victor2 plate reader (PerkinElmer, Ramsey, MN, USA).

Western Blotting Analysis

To measure the effect of 33 on AXL phosphorylation, NCI-H1299 nonsmall cell lung cancer cells were plated at 4 × 10⁵ cells/well in 6 well plates and incubated at 37 °C with 5% CO₂ for 3 to 4 h and then switched to serum-free culture medium overnight. The next day, diluted 33 was added to the cells and incubated for 1 h. Cells were then stimulated with recombinant human Gas6 (400 ng/mL final concentration) for 30 min. For phosphorylated MER measurement, human melanoma G361 cells were seeded 2 × 10⁶ cell/well in 6 well plates for 2 days followed by serum-free culture medium at 37 °C with 5% CO₂ overnight. Diluted 33 was then added and incubated for another 48 h followed by stimulation with MER-agonist antibody MAB8912 (500 ng/mL final concentration) for 30 min. The treated and untreated cells were incubated in PBS with 2 mM Na₃VO₄ for 5 min on ice and washed with PBS. Cells were lysed in RIPA Lysis and Extraction (Thermo Scientific, Waltham, MA, USA) with 2 mM sodium orthovanadate, 1xHalt Phosphatase Inhibitor Cocktail, and 1xHalt Protease Inhibitor Cocktail. Protein lysates were resolved in SDS-PAGE and then transferred onto apolyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were immunoblotted with appropriate antibodies and reacted with the SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA), followed by exposure to X-ray film. The sources of primary antibodies were as follows: antiphospho-AXL (Tyr702) and anti-AXL (C89E7) were procured from Cell Signaling Technology; antiphospho-MER proto-oncogene tyrosine kinase (MER) (Y749 + Y753 + Y754) and anti-MERTK (Y323) were from Abcam. β-actin antibody was purchased from Thermo Fisher, MA5-15739. The secondary antibody horseradish peroxidase (HRP)-linked goat antirabbit IgG (111-035-003) was purchased from Cell Signaling Technology, human Gas6 from Abcam and MER agonist antibody MAB8912 from R&D System. Proteins were detected using the SuperSignal reagent (Pierce, Rockford, IL) followed by exposure to the X-ray film.

In Vivo Pharmacokinetics Study

The animal studies were performed according to NHRI institutional animal care and committee-approved procedures. Male ICR mice (25–35 g) were obtained from BioLASCO (Taiwan Co., Ltd., Ilan, Taiwan). A single 2.0, 3.0, or 10 mg/kg dose of the compounds, as a PEG400/DMA (80/20, v/v) for both iv and oral dosing or DMSO/CrEL/D5W (5%/5%/90%,v/v/v)(iv) and 1%CMC+0.5%Tween80 (oral) solution, was separately administered to mice. At 0 (before dosing), 0.5, 1, 2, 4, 6, 8, 16, and 24 h after dosing, a blood sample was collected from groups of three mice at each time point by cardiac puncture and plasma was separated from the blood by centrifugation and stored in a freezer (−70 °C) before analysis. All samples were analyzed for the parent drug by LC-MS/MS. Plasma concentration data were analyzed with a noncompartmental method.

In Vitro Microsomal Stability Assay

The potent compounds (1 μM) were incubated with mouse liver microsomes to perform the metabolic stability study. All incubations were initiated with the addition of NADPH-generating system at 37 °C for 30 min. At 0 and the end of incubation, 100 μL of aliquots were taken from the incubation mixture and placed into centrifuge tubes containing 100 μL of ice-cold acetonitrile to terminate the metabolic reaction. The samples were vortexed and centrifuged, and then, the supernatant injected onto LC/MS. Percent of remaining of each compound was calculated by comparing peak areas at 30 min to that at the initiation of incubation.

Animal Studies

Female C7BL/6 mice were used for MC38 murine colon cancer cells and Hepa1–6 murine liver cancer cells, Balb/c mice for 4T1 murine triple-negative breast cancer cells, and NOD/SCID mice for MDA-MB-231 human triple-negative breast cancer cells. All mice were used between 6 and 7 weeks of age. MC38 cells and Hepa1–6 cells were prepared at 10⁵ and 10⁶ cells, respectively, per mouse in 100 μL of culture medium and implanted subcutaneously into the left flank region of mice with a 25–5/8 gauge needle. 4T1 was prepared at 1 million cells and MDA-MB-231 was prepared at 5 million cells in 1:1 matrigel and culture medium. For the 4T1 tumor model, cells were injected into the fourth left mammary fat pad in 50 μL per mouse. For the MDA-MB-231 xenograft tumor model, tumor cells were either implanted subcutaneously or into mammary fat pad as described in the text. Tumor cells were detected as free of Mycoplasma spp prior to injection into animals. Treatment was initiated after randomization when the average tumor size reached approximately 50–60 mm³ for MC38 and 4T1 and 150 to 200 mm³ for Hepa1–6 and MDA-MB-231. Animals received vehicle control, 50 mg/kg twice a day (BID) of 22 or 33, or once a day (QD). The reference compound 8 (tamnorzatinib) was given at 50 mg/kg QD. MER-selective compound 1 (UNC2025) and AXL-selective compound 3 (bemcentinib) were dosed at 25 mg/kg BID, alone or in combination. A five-days-on and two-days-off (FOTO) treatment schedule was used; the treatment length for each model was indicated in figure legends. All compounds were freshly prepared daily in 10%DMA/40%PEG400/50% (1%CMC). Tumor growth was measured with an electronic caliper, and volumes were calculated as L × W × W/2. Tumor size and animal body weight were measured once a week after tumor cell inoculation. Tumor growth inhibition (% TGI) = [1 – ΔT/ΔC] × 100, where ΔT is the difference of average tumor volume on the measured day and day 0 of treated groups and ΔC is the difference of average tumor volume on the measured day and day 0 of control groups. The uses and experimental procedures in animals were approved by the Institutional Animal Care and Use Committees (IACUCs) of the National Health Research Institutes. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals“.

Flow-Cytometry Analysis

MC38 tumor-bearing mice were treated with 50 mg/kg of 22 BID and FOTO for 2 weeks. Tumor and spleen tissues from the control and treated animals were harvested 2 h after the last dose (day 11). Non-necrotic tissues were carefully removed from the tumors followed by digestion with 0.1% collagenase III in DMEM medium for 30 min at 37 °C with agitation every 10 min. The digestion was terminated by adding 10% FBS containing PBS. The single-cell suspension was filtered through a 40 μm filter after red blood cell lysis. Aliquots of the cell suspension were preincubated with the mouse Fc receptor blocker for 10 min before staining with appropriate antimouse antibody conjugate for Cd45, Cd3, Cd4, Cd8, Cd11b, Cd86, F4/80, AXL, and MER (all antibody conjugates from BioLegend) at 4 °C for 30 min followed by two washes in 1% BSA containing buffer and analyzed by flow cytometry to quantify the accumulation of infiltrating immune cells into the tumor. For analysis of Cd206, the cells were fixed using the Cyto X/Cytoperm kit (BD Biosciences) after surface marker staining and then stained with antimouse Cd206 antibody.

Statistical Analysis

Data are expressed as mean ± standard error of the mean (SEM). Differences in mean values between groups were analyzed through a nonparametric t test. One-way analysis of variance (ANOVA) test, followed by Bonferroni post-test comparison, was employed for multiple comparison analysis. A p value of <0.05 indicated significant differences. GraphPad Prism 9 was used for conducting the statistical analysis.

Glossary

Abbreviations Used

AML

acute myeloid leukemia

APCs

antigen-presenting cells

CLL

chronic lymphocytic leukemia

CMC

carboxymethyl cellulose

CrEL

cremophor EL

D5W

5% dextrose in water

DIPEA

N,N-diisopropylethylamine

DM

double mutant

DMEM

Dulbecco’s modified eagle medium

EDCI

1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide

EMT

epithelial-to-mesenchymal transition

FBS

fetal bovine serum

FLT1

FMS-like tyrosine kinase 1

FLT3

FMS-like tyrosine kinase 3

FLT4

FMS-like tyrosine kinase 4

FNIII

fibronectin type III

FOTO

five-days-on and two-days-off

HNSCC

head and neck squamous cell carcinoma

IgL

immunoglobulin-like

LRMS

low-resolution mass spectra

NA

not available

ND

not detected/determined

ng

nanogram

NHRI

National Health Research Institutes

nM

nanomolar

PMS

phenazine methosulfate

RIPA

radio-immunoprecipitation assay

RTKs

receptor tyrosine kinases

SDS-PAGE

sodium dodecyl-sulfate polyacrylamide gel electrophoresis

SEM

2-(trimethylsilyl)ethoxymethyl/standard error of the mean

SLL

small lymphocytic lymphoma

Sphos

2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl

TAM

TYRO3, AXL, and MER

TBTU

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate

TGI

tumor growth inhibition

TNBC

triple-negative breast cancer

UPLC

ultraperformance liquid chromatography

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00400.

• SMILES string for compounds 11–41 (CSV)

• 7AAY_17 (docking pose of 17 in MER) (PDB)

• ¹H NMR, ¹³C NMR, HPLC purity, and UPLC purity spectra of final compounds 11–41, synthesis procedure and compound characterization data for intermediate compounds 60–64, 66, 67, and 69–72, mice body weight change during drug treatment, and kinase profiling data of 33 (PDF)

The authors declare no competing financial interest.