Structure-based drug discovery of novel penta- or hexa-bicyclo-pyrazolone derivatives as potent and selective AXL inhibitors

Abstract

AXL is a promising antitumor target due to its important role in tumor growth, poor survival, metastasis, immunosuppression, and drug resistance. Herein, we employed molecular modeling-assisted structural optimization strategies to design and synthesize a series of penta- or hexa-bicyclo-pyrazolone derivatives as novel AXL inhibitors. Compounds with high enzymatic and cellular potencies against AXL are described. Compound w11 showed desirable selectivity for AXL kinase, preferable pharmacokinetic properties, and excellent antitumor efficiency in the MV-4-11 xenograft model. These favorable results demonstrated that compound w11 may serve as a promising therapeutic candidate for hematological malignancy.

AXL is a promising antitumor target due to its important role in tumor growth, poor survival, metastasis, immunosuppression, and drug resistance.

Introduction

Cancer is a leading cause of death worldwide, accounting for about 10 million deaths in 2022, or nearly one in six deaths.¹ Lung cancer and breast cancer are the two most frequently diagnosed cancers.² The choices for cancer treatment are increasingly diverse, which include targeted small-molecule drugs, anti-PD-1 antibodies, antibody–drug conjugates (ADCs), etc.^3–5 However, tumor cells can develop different mechanisms to evade monotherapy.⁶ Combination therapy, especially to improve immune therapy through remolding of the tumor microenvironment, is a focus of current cancer drug development.

The TAM receptor tyrosine kinase (RTK) family, which comprises TYRO3, AXL, and MER, plays intricate roles in innate homeostasis.⁷ These receptors have several ligands, including GAS6, galectin-3, Tubby, and Tubby-like protein 1.⁸ Among these ligands, GAS6 showed the highest binding affinity to AXL. GAS6-dependent dimerization is currently considered the most common activation mechanism of AXL.⁹ Abnormal activation of GAS6/AXL signaling is involved in various biological processes of tumor initiation and progression, such as tumor cell survival, proliferation, invasion, and cancer therapy resistance.¹⁰ Studies also suggest that the GAS6/AXL signaling axis shapes the tumor immune microenvironment through diverse mechanisms, such as modulating angiogenesis, controlling secretions of certain cytokines, and regulating immune-related cells and markers.¹¹ Accordingly, therapeutic AXL inhibition would promote tumor cell apoptosis, inhibit tumor growth, and enhance the sensitivity of cancer to immunotherapy.¹²

Several small-molecule AXL inhibitors are approved for marketing or in clinical trials. However, most of the reported AXL inhibitors are non-selective towards AXL RTK. As listed in Fig. 1, cabozantinib and gilteritinib, which have been approved for marketing, are multi-kinase inhibitors with AXL inhibitor activity.^13,14 Bemcentinib is claimed to be a selective AXL inhibitor, demonstrating potent AXL activity and selectivity based on kinase profiling and cell-based data.¹⁵ Bemcentinib has been studied in combination with immune checkpoint inhibitors in a phase II study for recurrent NSCLC and a phase II study for advanced/metastatic NSCLC with STK11 mutations, both showing positive clinical benefits.¹⁶ Gilteritinib, which targets both FLT3 and AXL, received its first global approval in 2019 for the treatment of acute myelocytic leukemia (AML) harboring FLT3 mutations.¹⁷

Fig. 1. Representative type I or type II small-molecule inhibitors against AXL kinase.

Based on the characteristics of AXL inhibitor structures, we can classify the drugs into type I and type II inhibitors. Gilteritinib, bemcentinib, and our previously reported compound m16 are three representatives of U-shaped type I ATP-competitive inhibitors.¹⁸ Type II AXL inhibitors generally occupy the additional allosteric pocket of the kinase and cause the aspartate-phenylalanine-glycine motif to move away from the active site (DFG-out conformation).¹⁹ The inhibitory potency of cabozantinib against VEGFR2 is 200-fold higher than against AXL.²⁰ Several other reported type II AXL inhibitors, such as ONO-7475, XL-092, RXDX-106, merestinib, foretinib, and sitravatinib, have been in different stages of clinical trials.^21,22 Compounds 10 and 11 were reported to have strong inhibitory activities against AXL with nanomolar-level IC₅₀ values.^23,24 In general, these compounds also inhibited kinase targets such as c-MET, FLT3, and VEGFR-2. The development of selective AXL inhibitors remains a huge challenge.

Type II AXL inhibitors share similar structural features. Numerous preclinical type II AXL inhibitors are mainly optimized at the “tail” segment, which contains a dual hydrogen bond acceptor (DHBA group).²⁵ Most of these inhibitors have an aminophenoxyl linker that connects the DHBA group and the head region. According to the similar features on the head region, type II AXL inhibitors formed one or bidentate H-bonding interactions with the hinge region of AXL kinase by nitrogen of pyridine or quinoline. The benzene ring substituted with fluorine improves the AXL binding potency and pharmacokinetic profile.²⁶ Guided by molecular modeling, we modified three structural domains, including the DHBA group, linker, and head region, to design a series of pyrazolone bicyclic derivatives as selective AXL inhibitors. The synthesis, structure–activity relationship (SAR), and antitumor efficacy of the exemplified compounds as selective type II AXL inhibitors were reported.

Results and discussion

Rational design exploration

The crystal structure of the AXL kinase domain in a complex with a macrocyclic compound was characterized in 2017 (PDB 5U6B).²⁷ However, the crystal structure for the AXL kinase domain in complex with a type II inhibitor is unavailable to date. As a type II multiple-target inhibitor, NPS-1034 exhibited potent inhibitory activities for AXL and MER kinase. The X-ray cocrystal structure of MER and NPS-1034 was already determined (PDB 7AVX).²⁸ As there are 68.94% sequence identities between AXL and MER kinase domains, the MER DFG-out homology model can serve as a binding model to provide structural information for the design of AXL inhibitors.²⁹ Our potent AXL inhibitor discovery initially focused on the head region, starting with compounds 12 and 13, containing a similar DHAB group of NPS-1034 and compound X26.³⁰ The head region, containing an indazole motif, which was derived from merestinib, exhibited remarkable inhibitory activities against AXL kinase. The IC₅₀ values of compound 13 and the benchmark (BGB324) were 8 nM and 15 nM, respectively.

A binding mode study of compound 13 with the MER kinase domain was carried out. As depicted in Fig. 3A, the N-phenylated pyrazolone moiety of this compound is located in the allosteric pocket of the MER kinase domain, while forming two hydrogen bonds with Asp741 and Lys619. The docking study also revealed that the nitrogen of indazole formed a hydrogen bond with the backbone peptide residue Met674. To our success, in the first-round screening, compared to BGB324, compound 13 displayed potent enzymatic AXL inhibition. However, this compound exhibited an unsatisfactory half-life of 5 min in rat liver microsomes (ESI† Table S4). Considering the metabolic instability of diphenyl ether between the head region and the linker, most type II kinase inhibitors change the benzene ring to pyridine or quinoline to improve the stability of the diaryl ether bond (Fig. 1, type II AXL inhibitors). We performed structure-based modifications of compound 13 by (a) the elimination of the C–O bond and (b) a scaffold hopping strategy that replaced benzopyrazole with the pyridine-amine group. Binding mode analysis indicated that the nitrogen atom of the pyrimidine group on compound 14 was far from the residue Met674 (Fig. 3B). The simplest way to shorten the distance is to alter the direction of pyrimidine-4-amine. Compound w0 showed acceptable inhibitory potency with an IC₅₀ value of 17 nM, which was comparable to that of BGB324. Binding mode analysis indicated that the sp² nitrogen of compound w0 formed favorable hydrogen bond interactions with the residues Met674 and Pro672. The phenyl ring of the DHBA moiety faced the residue Phe719, generating a π–π interaction, which is consistent with the characteristics of type II kinase inhibitors (Fig. 3C). The inhibitory activity of compound w0 was slightly reduced, but its stability in rat liver microsomes was improved (cpd. 13vs.w0, T_1/2 = 5 min vs. 43 min, ESI† Table S4). The amino acids of the kinase domain surrounding compound w0 were surfaced in PyMol (expanded by 4 Å) and revealed a possible hydrophobic region (Fig. 3D). The methyl-substituted pyrazolone group is relatively small in volume and cannot extend into the possible hydrophobic cavity, which was speculated to be the other key for AXL inhibition. We retained the scaffold of the head region of compound w0 and next rationally carried out structural modifications on the DHBA group (Fig. 2).

Fig. 3. Rational modification of the head region to provide novel type II AXL inhibitors. (A) Molecular docking of compound 13 with the MER kinase domain (PDB 7AVX). (B) Binding mode analysis of the head region of compound 14 to the MER kinase domain (PDB 7AVX). (C) Molecular docking of compound w0 with the MER kinase domain (PDB 7AVX). (D) Schematic illustration of the proposed hydrophobic region (PDB 7AVX, around compound w0 and expanded by 4 Å).

Fig. 2. Initial SAR exploration from the head region.

Structure–activity relationship (SAR) exploration of the functional groups

Based on this information, we designed compounds w1–w4 and further supported the exploration of the effect of steric size and hydrophobicity of the pyrazole moiety (Table 1). The benzene amine of the linker with the mono F atom at the meta-position displayed similar potency against AXL kinase (cpd. w1vs.w0). Methylpropan-2-ol-substituted monocyclic pyrazolone w2 exhibited slightly lower inhibitory activity comparable to that of methyl-substituted monocyclic pyrazolone w1. The introduction of tetrahydrofuran led to a significant decrease in potency (cpd. w3). Hexa-penta-bicyclo-pyrazolone derivative w4 displayed a slight increase comparable to that of methyl-substituted pyrazolone derivative w3. The inhibitory activities of compounds w1–w4 suggested that the steric size of the DHBA group may be a crucial influencing factor for the potency. Otherwise, the penta-bicyclo-pyrazolone group is conformationally restricted and relatively bulky, which was speculated to be an important favorable factor for better AXL inhibition activity.

Table 1. SAR exploration of the DHBA group.

Cpd.	R1	Linker	AXL IC50 (nM)
w0			17
w1			26
w2			34
w3			>1000
w4			14

Guided by the above-mentioned information, we further explored different bicyclo-pyrazolone derivatives as novel AXL inhibitors (Table 2). The benzene amine of the linker with a mono-fluorine atom at the meta-position displayed higher potency compared to that at the ortho-position (cpd. w5vs.w4). The relatively higher hydrophilic substituent for this position displayed the lowest inhibitory activity with an IC₅₀ value of 30 nM (cpd. w6vs.w9). The isopropyl-substituted derivative w8 showed higher potency compared to the cyclopropyl-substituted compound w7. Increasing the hydrophobicity and steric size of the substituents could improve the AXL inhibitory activity to an IC₅₀ value of 4 nM (cpd. w10). Therefore, the balance of hydrophobicity and hydrophilicity of the molecule may increase the AXL inhibitory activity, especially for w5–w10. The molecular docking analysis of compounds w6 and w9 possibly indicated the absence of hydrogen bond interaction between the hydroxy group and the kinase domain (ESI† Fig. S1).

Table 2. SAR exploration of the linker and substituents in the head group.

Cpd.	R1	Linker	R2	AXL IC50 (nM)
w5			Methyl-	20
w6				30
w7				8
w8				5
w9				17
w10				4
w11			Methyl-	5
w12			Methyl-	5
w13			Methyl-	24
w14				6
w15				8
w16				11
w17				10
w18			Methyl-	5
w19			Methyl-	16
w20			Methyl-	51
w21			Methyl-	35
w22			Methyl-	18
w23			Methyl-	12
w24				6
w25				15

Compound w12 displayed similar AXL enzymatic inhibitory activities with w11, indicating that different hexa-penta-bicyclo-pyrazolone rings could fit well into the DFG-out domain of AXL kinase. Oxygen-containing or carbon replacement on the same position of the hexa-bicyclic ring did not reduce the kinase inhibitory activity (cpd. w12vs.w18). Small changes in the hydrophobicity of the hexa-bicyclic-pyrazolone ring did not result in decreased potency (cpd. w12 and w18). Replacing the benzene ring of the linker with pyridine resulted in a significant loss of potency (cpd. w13vs.w12, w18vs.w19, and w20vs.w21). The introduction of mono-fluorine in the benzene ring exhibited pharmacokinetic profile improvement.²³ In addition, the position of the fluorine atom resulted in different inhibitory activities for receptor tyrosine kinases. For these compounds, the introduction of a single fluorine atom with a benzene ring in the DHBA motif resulted in a slight loss in enzymatic potency (cpd. w20 and w21vs.w18, and w4 and w5).

We next explored the effect of various substituents on the 7-position of the head group. For hexa-bicyclo-pyrazolones w12–w17, hydroxy-containing compounds showed higher potency in enzymatic inhibition with an IC₅₀ value of 6–10 nM; isopropyl-substituted w16 showed the lowest potency against AXL kinase with an IC₅₀ value of 11 nM. Similarly, for the oxygen-containing hexa-bicyclo-pyrazolones, the introduction of an extra carbon atom and the change in the direction of the hydroxyl group resulted in a significant decrease in activity (IC₅₀: 6 nM for w24vs. 15 nM for w25). On account of their enzymatic inhibition potencies, substituents on NH of the pyrrole ring possibly stretched to the solvent-accessible region. The inhibitory activity results of different substituents on the 7-position of the pyrrolo[5,4-d]pyrimidin-4-amine group suggested that we could explore more potential functional substituents to improve the enzymatic inhibitory activity. Compound w11 was docked to the MER DFG-out model and, as depicted in Fig. 4, exhibited similar allosteric interactions to compound w0 and formed a hydrogen bond interaction with the backbone peptide residue Met674 in the hinge region of the kinase domain.

Fig. 4. Schematic diagram of the proposed binding mode of compound w11 with the MER kinase domain (PDB 7AVX). (A) The hydrophobic pocket binding model and hydrogen bond interaction of compound w11 with the MER kinase domain. (B) Molecular docking of compound w11 into the MER kinase domain.

Molecular modeling

Docking studies were carried out on Schrodinger Suite 2018.1 using the MER DFG-out homology model, while the protein preparation, optimization, and minimization were performed in the preparation process wizard following the procedure guidelines. After rationalizing the structural conformation via LigPrep, the docking site was defined using a grid box size with the centroid of the workspace ligand (compound NPS-1034). The docking site was defined using a grid box size with the centroid of the workspace ligand. The dock ligand is similar in size to the workspace ligand. Next, compound w11 was then docked into the ATP site using Glide release with standard settings, and poses were retained except 5. The binding model analysis of w11 is shown in Fig. 4, which indicated diketone of the DHBA group and a benzene ring located in the allosteric back pocket (Fig. 4B), and the penta-bicyclo-pyrazolone ring possibly embedded a plausible hydrophobic cavity composed of Phe742, Leu744, and Met641 (Fig. 4A). Penta- or hexa-bicyclo-pyrazolone ring derivatives demonstrated similar enzymatic inhibitory activity. The π–π interactions, which are consistent with the typical characteristics of type II kinase inhibitors, originated from the phenyl ring facing the Phe634 from the DFG motif. The linker, the p-phenylamino group, occupied the hydrophobic channel (Fig. 4B). The nitrogen from the pyrrolo[5,4-d]pyrimidin-4-amine fragment formed a hydrogen bond with the residue Met674 on the hinge region. All of these results further demonstrated the rationality of structure-based drug design (SBDD) for AXL inhibitors. The simulated result also provided useful information for further rational design of the substituents on the pyrrole ring, which possibly stretched to the solvent-accessible region.

Kinase selectivity of compound w11

The kinase selectivity profile of compound w11 was further evaluated over a panel of 54 kinases. As shown in Fig. 5, in contrast to its highest potencies against MER and AXL, it exhibited 4-fold and 6-fold weaker inhibition against the additional Aurora B and the highly homologous kinase MET, respectively. No obvious inhibitory effect was observed in the other 50 tested kinases. These results indicated that compound w11 was a selective type II inhibitor, which possessed high selectivity for AXL and MER.

Fig. 5. Kinase selectivity profile of compound w11 at 1 μM.

Cellular potencies of five compounds

Based on comprehensive consideration of the synthesis difficulty and their inhibitory activity against AXL kinase, six compounds were picked to test their cellular potency in BaF3/TEL-AXL in vitro. As shown in Table 3, hexa-bicyclo-pyrazolone compound w12 displayed satisfactory potencies with IC₅₀ values less than 5 nM. Oxygen-containing hexa-bicyclo-pyrazolone compound w18 exhibited a slight decrease in cellular inhibitory potency. The penta-bicyclo-pyrazolone belonging to the DHBA group resulted in a 5-fold loss in cellular potency (cpd. w11vs.w12). The kinase-inhibitory activities of the hydroxyethyl-substituted compound w13 and the tert-butyl hydroxyl-substituted compound w24 are comparable. However, the activity of the cell is distinct, maybe due to the higher hydrophobicity, which makes the membrane permeability stronger.

Table 3. The inhibitory activities against BaF3/TEL-AXL cell viability of six compounds.

Cpds.	IC50 (nM)
	Axl kinase	BaF3/TEL-AXL proliferation
w11	5	15.5
w12	5	2.8
w13	8	16.0
w18	5	7.4
w24	6	97.9

Pharmacokinetic parameters of three compounds w11, w12, and w18

Given their excellent inhibitory activity against AXL kinase, three compounds, w11, w12, and w18, were selected for pharmacokinetic studies in vivo. Preliminary pharmacokinetic tests were conducted in rats via oral administration and intravenous injection. Specific parameters are summarized in Table 4. Three compounds displayed comparable moderate oral exposures and satisfactory bioavailability in rats at an oral dose of 5 mg kg⁻¹. Although the pharmacokinetic parameters were not significantly different, the penta-cyclo-pyrazolone compound w11 was the best one (cpd. w11vs.w18). In contrast, compound w12 exhibited a relative loss in oral exposures and bioavailability in rats. Additionally, this compound displayed a short half-life of 0.55 h, which may be a factor in its low oral exposure. Consequently, compound w11 was selected for further profiling.

Table 4. Pharmacokinetic data of three compounds in rats.

Parameter	w11		w12		w18
	PO	IV	PO	IV	PO	IV
	5 mg kg−1	1 mg kg−1	5 mg kg−1	1 mg kg−1	5 mg kg−1	1 mg kg−1
AUClast (ng h mL−1)	20 700	3530	14 300	2770	20 300	4640
C max (ng mL−1)	4800	5180	5230	5570	5700	6200
CL (mL h−1 kg−1)	NA	0.281	NA	0.377	NA	0.214
T 1/2 (h)	3.1	1.0	0.55	0.7	3.0	1.2
F (%)	117.5		103.2		103.2

Antiproliferative activities of compound w11

Based on the differences between the cellular activity of six compounds in BaF3-AXL cells and the pharmacokinetic parameters in vivo, one of the top three was selected to evaluate the anti-proliferative activities against 13 cancer cell lines. Penta-cyclo-pyrazolone compound w11 had high proliferation inhibitory activity in two hematological cancer cell lines, MV-4-11 and MOLM13, with CC₅₀ values of 24.8 nM and 5.9 nM, respectively. Table 5 also shows that the compound exhibited high potency against RAJI cell proliferation with a CC₅₀ value of 17.8 nM. On the other hand, the selected compound displayed moderate to extremely low anti-proliferative activities in the ten other cancer cell lines.

Table 5. Cell line proliferation inhibitory profile of compound w11.

Cpd.	Cell line	CC50 (nM)	Max inhibition%
w11	A549	1871.785	92.75
	MGC803	2063.036	85.14
	BGC823	3737.924	70.99
	BXPC-3	3463.780	79.28
	MV-4-11	24.862	93.57
	PC-3	3821.662	76.81
	RAJI	17.850	61.07
	SNU5	1761.978	87.58
	SW620	1641.024	95.64
	NALM-6	482.325	61.13
	MDA-MB-231	>10 μM	54.34
	MOLM13	5.881	73.00
	U87MG	7255.813	60.41

Antitumor efficacy of compounds w11 and w12in vivo

We further assessed the antitumor efficacy of two compounds, w11 and w12, against the MV-4-11 xenograft model at a dose of 50 mg kg⁻¹ once daily for 38 consecutive days (Fig. 6). Immunodeficient NOD/SCID mice bearing subcutaneous tumors at an average tumor volume of 160 mm³ were treated with vehicle control, compound w11, and w12 for 5 weeks. Fig. 5 shows that two compounds significantly suppressed tumor growth. This may be related to the high potency against MV-4-11 cell proliferation, although there are no experimental cellular data for compound w12.

Fig. 6. In vivo effect of two compounds in MV-4-11 xenograft tumor model. (A) Efficacy of AXL inhibitors w11 and w12 in the MV-4-11 xenograft tumor model. (B) Effect of compounds w11 and w12 on body weight of MV-4-11 xenograft tumor model.

Conclusion

In summary, through SAR exploration and structure-based drug design (SBDD) efforts, we designed and synthesized a series of penta- or hexa-bicyclo-pyrazolone derivatives as new AXL inhibitors. Several compounds exhibited excellent enzymatic and cellular potency against AXL. The candidate compound w11 remarkably suppressed the proliferation of 3 cancer cell lines (MV-4-11, RAJI, and MOLM13) and displayed great selectivity for AXL and MER kinases. Two compounds, w11 and w12, demonstrated acceptable (pharmacokinetic) PK profiles in rats and exhibited significant antitumor activity in vivo in the MV-4-11 xenograft tumor model. Compound w11 may serve as a promising targeted therapy agent for hematological malignancies. Further biological studies, including immunomodulatory effects, are ongoing and will be revealed in due course.

Experimental section

Chemistry

All starting materials, reagents, and solvents are of reagent grade and commercially available. Unless otherwise noted, all purchased chemicals were directly used without further purification. All chemical reactions were monitored for completion by thin-layer chromatography (TLC) or LC-MS. Purification was performed by automated flash chromatography on a Biotage instrument using silica gel (200–300 mesh). An Agilent HPLC 1260-MS6120 system and a chromatographic separation column (Agilent-SB-C18, 2.5 mm × 30 mm, 3.5 μm) were used for LC-MS. ¹H NMR and ¹³C NMR spectra were obtained by using CDCl₃, CD₃OD, or DMSO-d₆ as a solvent on a Bruker Avance III 400 or 600 M frequency spectrometer. NMR data were recorded as follows: coupling constants (J: hertz), chemical shift (TMS as an internal standard), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br. = broad, m = multiplet, dt = double triplet), chemical shift for solvent residual signals (DMSO-d₆ at 2.50 ppm, CD₃OD at 3.31 ppm, CDCl₃ at 7.26 ppm for ¹H NMR; DMSO-d₆ at 39.5 ppm, CD₃OD at 49.0 ppm, CDCl₃ at 77.0 ppm for ¹³C NMR). The purity of all final compounds was confirmed to be greater than 95% by HPLC analysis. High-resolution ESI-MS was performed on an Agilent G6500 series Q-TOF spectrometer.

The syntheses of key intermediates 24–27 and 37–40 are outlined in Schemes 1 and 2. Specifically, phenyl hydrazine block 15 were initially acylated with 4-chlorobutanoyl chloride or 5-chloropentanoyl chloride, resulting in the formation of compounds 16 and 17, then followed by another acylation with ethyl 3-chloro-3-oxopropanoate to afford intermediates 18 and 19. Next, intramolecular nucleophilic substitution and aldol condensation reactions were sequentially carried out in the presence of NaH or DBU with intermediates 20 and 21, resulting in the corresponding ester derivatives 22 and 23. Finally, they were hydrolyzed to afford compounds 24 and 25. As needed, acyl chlorination derivatives 26 and 27 were obtained by treating compounds 24 and 25 with oxalyl chloride.

Scheme 1. Syntheses of intermediates 16–27. Reagents and conditions: (a) 4-chlorobutanoyl chloride or 5-chloropentanoyl chloride, Na₂CO₃, DCM/H₂O, 0 °C–rt. (b) Ethyl 3-chloro-3-oxopropanoate, Na₂CO₃, DCM, rt. (c) NaH, DMF, 0 °C–rt. (d) DBU, 50 °C. (e) NaOH, ethanol, THF/H₂O, rt. (f) Oxalyl chloride, DMF, THF, 0 °C–rt.

Scheme 2. Syntheses of intermediates 37–40. Reagents and conditions: (a) ethyl acetate, lithium diisopropylamide (2.0 M solution in THF), THF, −78 °C–rt. (b) I₂, PPh₃, imidazole, DCM, -5 °C–rt. (c) Phenylhydrazine or 4-fluorophenylhydrazine, AcOH, AcONa, ethanol, reflux. (d) (i) (Chloromethylene)dimethyliminium chloride, DCM, rt.; (ii) H₂O, rt. (e) NaClO₂, DMSO, H₃PO₄, DCM, H₂O, rt. (f) Oxalyl chloride, DMF, THF, 0 °C–rt.

Commercially available anhydrous ethyl acetate was initially dehydrogenated with lithium diisopropylamide and reacted with compound 28 to open the hexa-ring and form the carbon–carbon bond to afford intermediate 29. Subsequently, the conversion of primary alcohols to iodine-substituted compound 30 was carried out under appropriate conditions of the Appel reaction. Next, this intermediate was treated with phenylhydrazine or 4-fluorophenylhydrazine to generate compounds 33 and 34. The Vilsmeier–Haack reaction was utilized to form aldehyde intermediates 35 and 36, followed by oxidation to obtain compounds 37 and 38. Finally, acyl chlorination derivatives 39 and 40 were prepared from compounds 37 and 38 with oxalyl chloride.

The syntheses of inhibitors w6–w10, w14–w17, w24 and w25 are illustrated in Scheme 3. The starting compound 50 was initially substituted by different alkyl halides (such as 2-iodopropane, (chloromethyl)cyclopropane, bromoacetone, and 2-bromoethanol) through biomolecular nucleophilic substitution (S_N2 reaction) under various alkaline conditions, resulting in the formation of compounds 43, 44, 46 and 47. The same raw material was treated with 2,2-dimethyl oxirane in the presence of NaH to obtain intermediate 42. However, cyclopropylboronic acid was used to generate compound 45 under the Chan–Lam coupling condition.³¹ Building blocks 42–47 were coupled with tert-butyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)carbamate to form intermediates 48–54. The chloride of these compounds was further transformed into the corresponding amine blocks 55–59 with ammonium hydroxide. Reduction of the acetonyl group generated 60 in the presence of NaBH₄. Then, the end products w6–w10, w14–w17, w24 and w25 were separately obtained through condensation between amines 55–59 and carboxylic acids 24, 25, and 37.

Scheme 3. Syntheses of AXL inhibitors w6–w10, w14–w27, w24 and w25. Reagents and conditions: (a) 2,2-dimethyloxirane, NaH (60% dispersion in mineral oil), DMF, rt. (b) 2-Iodopropane, potassium tert-butoxide, THF, 70 °C. (c) (Chloromethyl)cyclopropane, KOH, DMSO, 50 °C. (d) Cyclopropylboronic acid, Cu(OAc)₂, Na₂CO₃, 2,2′-bipyridine, 1,2-DCE, 80 °C. (e) Bromoacetone, Cs₂CO₃, DMF, rt. (f) 2-Bromoethanol, Cs₂CO₃, DMF, rt. (g) Pd(PPh₃)₄ or Pd(dppf₂)Cl₂, Na₂CO₃, 1,4-dioxane, 100 °C. (h) (i) Ammonium hydroxide, 1,4-dioxane, 130 °C. (ii) HCl (4 M solution in 1,4-dioxane), 1,4-dioxane. (i), NaBH₄, MeOH, rt. (j) EDCI, HOAT, DIPEA, DCM/THF, 45 °C. ^a The Boc group was unexpectedly cleaved.

The other twelve inhibitors were synthesized according to the procedures outlined in Scheme 4. The starting material organic anilines 61, 63, and 64 were separately condensed with different carboxylic acids 24, 25, and 37 to afford intermediates 65–71. The amide bond formation was accomplished by treating organic anilines 62–64 with acid chlorides 26, 27, 39, and 40, resulting in the formation of intermediates 72–74. Then, the corresponding boronic acid pinacol ester was prepared. Finally, inhibitors w4, w5, w11–13, and w18–23 were obtained by coupling the bromo group of commercially available 85 with boronic acid pinacol ester derivatives 75–84.

Scheme 4. Syntheses of AXL inhibitors w4, w5, w11–w13, and w18–w23. Reagents and conditions: (a) HATU, DCM, rt. (b) EDCI, HOAT, DCM, 40 °C. (c) Et₃N, DCM, rt. (d) Pd(PPh₃)₄ or Pd(dppf₂)Cl₂, KOAc, 1,4-dioxane, 100 °C. (e) Pd(PPh₃)₄, K₂CO₃ or Cs₂CO₃, 1,4-dioxane, 100 °C.

Experimental

Chemistry

The synthetic routes of six compounds 12 and 13 and characterization data of intermediates are described in ESI† Schemes S1–S15.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-3-fluorophenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (w0)

To a solution of N-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (410 mg, 0.91 mmol) in dioxane (8 mL) and H₂O (1.5 mL) were added 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (217 mg, 0.91 mmol), Pd(dppf)₂Cl₂·CH₂Cl₂ (75 mg, 0.091 mmol) and Cs₂CO₃ (604 mg, 1.8 mmol). The reaction was degassed with nitrogen. After being stirred at reflux for 16 h, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: DCM/MeOH (v/v) = 500/1) to give a yellow solid (78 mg, 0.16 mmol, 18%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.05 (s, 1H), 8.54–8.50 (t, J = 8.4 Hz, 1H), 8.16 (s, 1H), 7.62–7.59 (t, J = 7.5 Hz, 2H), 7.55–7.51 (m, 1H), 7.46–7.44 (d, J = 7.6 Hz, 2H), 7.35–7.30 (m, 2H), 7.26–7.24 (d, J = 8.3 Hz, 1H), 6.17 (s, 2H), 3.74 (s, 3H), 3.38 (s, 3H), 2.73 (s, 3H). MS (ESI⁺) m/z: 472.1 [M + H]⁺.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (w1)

To a solution of 1,5-dimethyl-3-oxo-2-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2,3-dihydro-1H-pyrazole-4-carboxamide (280 mg, 0.65 mmol) in dioxane (6.0 mL) and H₂O (1.0 mL) were added intermediate 92 (154 mg, 0.66 mmol), Pd(dppf)₂Cl₂·CH₂Cl₂ (53 mg, 0.064 mmol) and Cs₂CO₃ (430 mg, 1.3 mmol). The reaction was degassed with nitrogen, followed by stirring and reflux for 16 h. The mixture was cooled and concentrated in vacuo. The residue was purified via flash column chromatography (eluent: DCM/MeOH (v/v) = 500/1) to give a yellow solid (68 mg, 0.15 mmol, 23%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.82 (s, 1H), 8.15 (s, 1H), 7.71–7.69 (d, J = 8.5 Hz, 2H), 7.62–7.58 (t, J = 7.6 Hz, 2H), 7.54–7.50 (t, J = 7.4 Hz, 1H), 7.46–7.44 (d, J = 7.4 Hz, 2H), 7.41–7.39 (d, J = 8.4 Hz, 2H), 7.28 (s, 1H), 6.06 (s, 2H), 3.74 (s, 3H), 3.37 (s, 3H), 2.72 (s, 3H). MS (ESI⁺) m/z: 454.3 [M + H]⁺.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-1-(2-hydroxy-2-methyl propyl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (w2)

To a solution of 5-(4-aminophenyl)-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine dihydrochloride (100 mg, 0.36 mmol) in CH₂Cl₂ (2 mL) were added 1-(2-hydroxy-2-methyl-propyl)-5-methyl-3-oxo-2-phenyl-pyrazole-4-carboxylic acid (115 mg, 0.38 mmol), EDCI (82 mg, 0.42 mmol), and HOAT (9 mg, 0.065 mmol). After being stirred at reflux for 6 h, the reaction mixture was concentrated in vacuo. The residue was purified via flash column chromatography (eluent: ethyl acetate/MeOH (v/v) = 100/1) to give the title compound as a yellow solid (124 mg, 0.24 mmol, 66.8%).¹H NMR (400 MHz, DMSO-d₆) δ 10.81 (s, 1H), 8.15 (s, 1H), 7.71–7.68 (d, J = 8.5 Hz, 2H), 7.59–7.55 (t, J = 7.7 Hz, 2H), 7.48–7.44 (t, J = 7.4 Hz, 1H), 7.41–7.39 (d, J = 8.4 Hz, 2H), 7.36–7.35 (d, J = 7.6 Hz, 2H), 7.28 (s, 1H), 6.07 (s, 2H), 4.84 (s, 1H), 3.86 (s, 2H), 3.74 (s, 3H), 2.81 (s, 3H), 0.97 (s, 6H). MS (ESI⁺) m/z: 512.3 [M + H]⁺.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-5-methyl-3-oxo-2-phenyl-1-((tetrahydrofuran-2-yl)methyl)-2,3-dihydro-1H-pyrazole-4-carboxamide (w3)

To a solution of 5-(4-aminophenyl)-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine dihydrochloride (50 mg, 0.18 mmol) in CH₂Cl₂ (2 mL) were added 5-methyl-3-oxo-2-phenyl-1-((tetrahydrofuran-2-yl)methyl)-2,3-dihydro-1H-pyrazole-4-carboxylic acid (64 mg, 0.2 mmol) and HATU (150 mg, 0.4 mmol). After being stirred at room temperature for 10 h, the reaction mixture was concentrated in vacuo. The residue was purified via flash column chromatography (eluent: DCM/MeOH (v/v) = 100/1) to give a yellow solid (10.3 mg, 19.7 μmol, 9.4%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.78 (s, 1H), 8.16 (s, 1H), 7.81–7.79 (d, J = 8.4 Hz, 2H), 7.71–7.69 (d, J = 7.8 Hz, 2H), 7.56–7.52 (t, J = 7.8 Hz, 2H), 7.44–7.39 (m, 3H), 7.29 (s, 1H), 6.07 (s, 2H), 4.14–4.02 (m, 3H), 3.75 (s, 3H), 3.71–3.61 (m, 2H), 2.38 (s, 3H), 1.89–1.81 (m, 1H), 1.78–1.71 (m, 2H), 1.55–1.47 (m, 1H). MS (ESI⁺) m/z: 525.2 [M + H]⁺.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-3-fluorophenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w4)

To a solution of N-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (210 mg, 0.45 mmol) in dioxane (6 mL) and H₂O (1 mL) were added 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (108 mg, 0.45 mmol), Pd(dppf)₂Cl₂·CH₂Cl₂ (37 mg, 0.045 mmol) and Cs₂CO₃ (301 mg, 0.91 mmol). The reaction was degassed with nitrogen. After being stirred at reflux for 16 h, the reaction mixture was concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 500 : 1) to give a yellow solid (46 mg, 0.095 mmol, 21%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.38 (s, 1H), 8.15 (s, 1H), 7.88–7.85 (d, J = 12.8 Hz, 1H), 7.60–7.52 (m, 4H), 7.45–7.41 (t, J = 7.3 Hz, 1H), 7.36–7.35 (m, 2H), 7.30 (s, 1H), 6.03 (s, 2H), 3.85–2.81 (t, J = 6.9 Hz, 2H), 3.75 (s, 3H), 3.20–3.17 (t, J = 6.8 Hz, 2H), 2.46–2.43 (m, 2H). MS (ESI⁺) m/z: 484.2 [M + H]⁺.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-fluorophenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w5)

To a solution of N-(2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (135 mg, 0.29 mmol) in dioxane (6 mL) and H₂O (1 mL) were added 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (70 mg, 0.29 mmol), Pd(dppf)₂Cl₂·CH₂Cl₂ (24 mg, 0.029 mmol) and Cs₂CO₃ (194 mg, 0.58 mmol). The reaction was degassed with nitrogen. After being stirred at reflux for 16 h, the reaction mixture was concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 500 : 1) to give a yellow solid (46 mg, 0.095 mmol, 33%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.51–10.50 (d, J = 1.8 Hz, 1H), 8.53–8.49 (t, J = 8.4 Hz, 1H), 8.16 (s, 1H), 7.59–7.52 (m, 4H), 7.45–7.41 (t, J = 7.0 Hz, 1H), 7.35–7.32 (m, 2H), 7.27–7.25 (d, J = 8.5 Hz, 1H), 6.18 (s, 2H), 3.84–3.81 (t, J = 6.8 Hz, 2H), 3.74 (s, 3H), 3.20–3.17 (t, J = 7.2 Hz, 2H), 2.48–2.41 (m, 2H). MS (ESI⁺) m/z: 484.2 [M + H]⁺.

N-(4-(4-Amino-7-(2-hydroxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w6)

EDCI (900.0 mg, 4.60 mmol) and HOAT (86 mg, 0.619 mmol) were added to a mixture of 2-[4-amino-5-(4-aminophenyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethanol (760 mg, 2.8 mmol) and 2-oxo-1-phenyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-carboxylic acid (831 mg, 3.4 mmol) in DCM (15 mL). The mixture was stirred and refluxed for 16 h. H₂O (20 mL) was added into the mixture and the resulting mixture was extracted with DCM (100 mL). The organic phase was combined and concentrated in vacuo to afford a yellow solid. The solid was purified by flash column chromatography (eluent: DCM : MeOH (v/v) = 50 : 1) to give a yellow solid (366.4 mg, 0.67 mmol, 23.8%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.21 (s, 1H), 8.09 (s, 1H), 7.68 (d, J = 8.1 Hz, 2H), 7.57–7.44 (m, 4H), 7.37 (dd, J = 7.5, 4.6 Hz, 3H), 7.26 (s, 1H), 6.03 (s, 2H), 4.93 (t, J = 5.4 Hz, 1H), 4.17 (t, J = 5.7 Hz, 2H), 3.73 (dt, J = 17.1, 6.3 Hz, 4H), 3.13 (t, J = 7.3 Hz, 2H), 2.37 (q, J = 7.2 Hz, 2H). MS (ESI⁺) m/z: 496.3 [M + H]⁺.

N-(4-(4-Amino-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w7)

To a solution of 5-(4-aminophenyl)-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (0.25 g, 0.94 mmol) in DCM (30 ml) were added 2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxylic acid (0.23 g, 0.94 mmol), EDCI (0.36 g, 1.89 mmol), and HOAt (0.26 g, 1.89 mmol). The reaction mixture was refluxed for 16 h. The reaction mixture was quenched with water (50 ml) and extracted with DCM (600 mL). The combined organic layer was washed with saturated NaHCO₃ aqueous solution (50 mL) and brine (100 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Then, the mixture was purified by flash column chromatography (eluent: MeOH/DCM (v/v) = 20 : 1) to give a yellow solid (300 mg, 0.6 mmol, 65%).¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.26 (s, 1H), 8.18 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.54 (dd, J = 14.5, 7.2 Hz, 4H), 7.42 (d, J = 8.3 Hz, 3H), 7.25 (s, 1H), 6.17 (s, 2H), 3.81 (t, J = 6.9 Hz, 2H), 3.59–3.48 (m, 1H), 3.18 (t, J = 7.3 Hz, 2H), 2.47–2.38 (m, 2H), 1.04 (dd, J = 7.9, 6.1 Hz, 4H). MS (ESI⁺) m/z: 492.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₈H₂₆N₇O₂ [M + H]⁺: 492.2148, found 492.2140.

N-(4-(4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,5, 6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w8)

To a solution of 5-(4-aminophenyl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (100 mg, 0.37 mmol) in CH₂Cl₂ (2 mL) were added 2-oxo-1-phenyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-carboxylic acid (110 mg, 0.45 mmol), EDCI (110 mg, 0.56 mmol) and HOAT (10 mg, 0.072 mmol). After being stirred at reflux for 9 h, the reaction mixture was concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH = 100 : 1) to give a yellow solid (94 mg, 0.19 mmol, 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.13 (s, 1H), 7.74–7.72 (d, J = 8.3 Hz, 2H), 7.59–7.51 (m, 4H), 7.44–7.41 (m, 4H), 6.06 (s, 2H), 5.00–4.92 (td, J = 13.3, 6.6 Hz, 1H), 3.82–3.79 (t, J = 6.8 Hz, 2H), 3.20–3.17 (t, J = 5.8 Hz, 3H), 2.46–2.40 (m, 2H), 1.47–1.46 (d, J = 6.7 Hz, 6H). MS (ESI⁺) m/z: 494.2 [M + H]⁺.

N-(4-(4-Amino-7-(2-hydroxypropyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w9)

EDCI (380.0 mg, 1.94 mmol) and HOAT (33.0 mg, 0.238 mmol) were added to a mixture of 2-oxo-1-phenyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-carboxylic acid (308 mg, 1.26 mmol), 1-[4-amino-5-(4-aminophenyl)pyrrolo[2,3-d]pyrimidin-7-yl]propan-2-ol (300 mg, 1.06 mmol) in DCM (10 mL). The mixture was stirred and refluxed for 16 h. H₂O (20 mL) was added into the mixture and the resulting mixture was extracted with DCM (200 mL). The organic phase was combined and concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 20 : 1) to give a yellow solid (79.6 mg, 0.14 mmol, 13.7% yield). 1H NMR (400 MHz, CDCl₃) δ 10.28 (s, 1H), 8.31 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.55 (t, J = 7.8 Hz, 2H), 7.42 (dd, J = 18.0, 9.3 Hz, 5H), 7.02 (s, 1H), 5.81 (s, 2H), 4.35–4.29 (m, 1H), 4.29–4.21 (m, 1H), 4.20–4.13 (m, 1H), 3.75 (t, J = 6.9 Hz, 2H), 3.35 (t, J = 7.4 Hz, 2H), 3.22 (s, 1H), 2.61–2.50 (m, 2H), 1.28 (d, J = 6.1 Hz, 3H). MS (ESI⁺) m/z: 510.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₈H₂₈N₇O₂ [M + H]⁺: 510.2175, found 510.2246.

N-(4-(4-Amino-7-(cyclopropylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w10)

To a mixture of 2-oxo-1-phenyl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole-3-carboxylic acid (139.6 mg, 0.57 mmol) and 5-(4-aminophenyl)-7-(cyclopropylmethyl)pyrrolo[2,3-d]pyrimidin-4-amine (159.9 mg, 0.5725 mmol) in DCM (10 mL) were added HOAT (24.3 mg, 0.18 mmol) and EDCI (198.5 mg, 1.03 mmol). The mixture was stirred at 40 °C for 12 h. The mixture was concentrated in vacuo to give a yellow solid. The solid was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 50 : 1) to give a yellow solid (103.5 mg, 0.2 mmol, 35%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.14 (s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.63–7.49 (m, 4H), 7.46–7.33 (m, 4H), 6.09 (s, 2H), 4.02 (d, J = 7.1 Hz, 2H), 3.80 (t, J = 6.8 Hz, 2H), 3.18 (dd, J = 9.5, 4.9 Hz, 2H), 2.47–2.38 (m, 2H), 0.92–0.77 (m, 1H), 0.57–0.46 (m, 2H), 0.43 (d, J = 4.0 Hz, 2H). MS (ESI⁺) m/z: 503.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₉H₂₇N₇O₂ [M + H]⁺: 506.2226, found 506.2306.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (w11)

To a solution of 2-oxo-1-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2,4,5,6-tetrahydro-1H-pyrrolo[1,2-b]pyrazole-3-carboxamide (600 mg, 1.34 mmol) in 1,4-dioxane (20 mL) were added Pd(dppf)Cl₂ (200 mg, 0.269 mmol), Cs₂CO₃ (900 mg, 2.69 mmol), and H₂O (2 mL). Then, the mixture was stirred at reflux for 7 h under a nitrogen atmosphere and concentrated in vacuo. The residue was purified by flash column chromatography (eluent: MeOH/DCM (v/v) = 1 : 20) to give a white solid (120 mg, 0.25 mmol, 19%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 8.15 (s, 1H), 7.73 (q, J = 15.3, 11.7 Hz, 2H), 7.61–7.52 (m, 2H), 7.50–7.25 (m, 4H), 7.03 (d, J = 57.4 Hz, 1H), 6.08 (s, 2H), 3.75 (dq, J = 41.8, 22.3, 14.5 Hz, 5H), 3.19 (d, J = 21.8 Hz, 2H), 2.48–2.26 (m, 2H). MS (ESI⁺) m/z: 466.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₆H₂₃N₇O₂ [M + H]⁺: 466.1913, found 466.1980.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-1,2,4,5,6,7-hexahydropyrazolo[1,5-a]pyridine-3-carboxamide (w12)

To a solution of 2-oxo-1-phenyl-N-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxamide (368 mg, 0.8 mmol) and 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (182 mg, 0.8 mmol) in 1,4-dioxane (8 mL) were added Pd(dppf)Cl₂·CH₂Cl₂ (134 mg, 0.16 mmol) and Cs₂CO₃ (522 mg, 1.6 mmol). The mixture was stirred at 110 °C for 15 h under a N₂ atmosphere and concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 20 : 1) to give a yellow solid (14.7 mg, 0.0277 mmol, 3.5%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.66 (s, 1H), 8.33 (s, 1H), 7.75 (d, J = 8.5 Hz, 2H), 7.55 (t, J = 7.6 Hz, 2H), 7.45 (t, J = 7.5 Hz, 1H), 7.39 (dd, J = 14.3, 7.9 Hz, 4H), 5.11 (s, 2H), 3.83 (s, 3H), 3.57 (t, J = 5.9 Hz, 2H), 3.42 (t, J = 6.4 Hz, 3H), 2.13–2.05 (m, 3H), 1.97–1.89 (m, 3H). MS (ESI⁺) m/z: 480.4 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₇H₂₆N₇O₂ [M + H]⁺: 480.2070, found 482.2155.

N-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyridine-2-yl)-2-oxo-1-phenyl-1,2,4,5,6,7-hexahydropyrazolo[1,5-a]pyridine-3-carboxamide (w13)

To a solution of 2-oxo-1-phenyl-N-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxamide (1.5 g, 3.3 mmol) and 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (1.0 g, 4.4 mmol) in H₂O (5 mL) and 1,4-dioxane(10 mL) were added Pd(dppf)Cl₂·CH₂Cl₂ (0.66 g, 0.79 mmol) and Cs₂CO₃ (2.6 g, 8.0 mmol). After stirring at 105 °C for 16 h under a nitrogen atmosphere, the mixture was concentrated in vacuo. The residue was multiply purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 50 : 1–10 : 1) to give a yellow solid (6.9 mg, 0.014 mmol, 0.5% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 11.84 (s, 1H), 8.62 (d, J = 8.3 Hz, 1H), 8.48 (s, 1H), 8.15 (dd, J = 7.9, 1.4 Hz, 1H), 7.51–7.48 (m, 1H), 7.26 (d, J = 8.7 Hz, 2H), 7.25–7.21 (m, 1H), 6.92 (d, J = 8.7 Hz, 2H), 5.41 (s, 2H), 3.72 (s, 4H), 2.55 (s, 3H). MS (ESI⁺) m/z: 481.4 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₆H₂₄N₈O₂ [M + H]⁺: 481.2022, found 481.2077.

N-(4-(4-Amino-7-(2-hydroxy-2-methylpropyl)-7H-pyrrolo[2,3-d]pyrimidin-5-l)phenyl)-2-oxo-1-phenyl-1,2,4,5,6,7-hexahydropyrazolo[1,5-a]pyridine-3-carboxamide (w14)

To a solution of 2-oxo-1-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxylic acid (305 mg, 1.181 mmol) in DCM (20 ml) were added EDCI (350 mg, 1.177 mmol) and HOAT (33 mg, 0.238 mmol). The reaction mixture was refluxed for 16 h. The reaction mixture was quenched with water (50 ml) and extracted with DCM (200 mL). The organic phase was washed with H₂O (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The mixture was purified by flash gel column chromatography (eluent: DCM : MeOH (v/v) = 100 : 1–10 : 1) to give a yellow solid (231 mg, 0.407 mmol, 34.6%). ¹H NMR (600 MHz, CDCl₃) δ 10.66 (s, 1H), 8.25 (s, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.53 (t, J = 7.7 Hz, 2H), 7.45 (t, J = 7.4 Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 7.8 Hz, 2H), 6.94 (s, 1H), 5.25 (s, 2H), 4.72 (s, 1H), 4.19 (s, 2H), 3.56 (t, J = 5.9 Hz, 2H), 3.40 (t, J = 6.4 Hz, 2H), 2.07 (dt, J = 11.5, 5.7 Hz, 2H), 1.94–1.89 (m, 2H), 1.24 (s, 6H). MS (ESI⁺) m/z: 538.3 [M + H]⁺. HRMS (ESI⁺) calcd for C₃₀H₃₂N₇O₃ [M + H]⁺: 538.2488, found 538.2569.

N-(4-(4-Amino-7-(2-hydroxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-1,2,4,5,6,7-hexahydropyrazolo[1,5-a]pyridine-3-carboxamide (w15)

To a solution of 2-(4-amino-5-(4-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethanol (0.12 g, 0.45 mmol) in DCM (50 ml) were added 2-oxo-1-phenyl-1,2,4,5,6,7-hexahydropyrazolo[1,5-a]pyridine-3-carboxylic acid (0.12 g, 0.45 mmol), EDCI (0.17 g, 0.89 mmol), and HOAt (0.12 g, 0.89 mmol). The reaction mixture was refluxed for 16 h. The reaction mixture was quenched with water (50 ml) and extracted with DCM (200 mL). The organic phase was washed with saturated NaHCO₃ aqueous solution (50 mL) and brine (100 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by flash gel column chromatography (eluent: MeOH/DCM (v/v) = 1/30) to give a white solid (100 mg, 0.19 mmol, 45.1%). ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.67 (s, 1H), 8.13 (s, 1H), 7.70 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 6.7 Hz, 2H), 7.51 (d, J = 6.6 Hz, 1H), 7.43 (dd, J = 24.5, 7.2 Hz, 4H), 7.30 (s, 1H), 6.07 (s, 2H), 4.98 (s, 1H), 4.22 (s, 2H), 3.75 (s, 2H), 3.56 (s, 2H), 3.22 (s, 2H), 1.98 (s, 2H), 1.82 (s, 2H). MS (ESI⁺) m/z: 510.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₈H₂₈N₇O₃ [M + H]⁺: 510.2253, found 510.2248.

N-(4-(4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-1,2,4,5,6,7-hexahydropyrazolo[1,5-a]pyridine-3-carboxamide (w16)

To a solution of 5-(4-aminophenyl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (100 mg, 0.37 mmol) in CH₂Cl₂ (2 mL) were added 2-oxo-1-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxylic acid (116 mg, 0.45 mmol), EDCI (110 mg, 0.56 mmol) and HOAT (10 mg, 0.072 mmol). After being stirred at 40 °C for 8 h, the reaction mixture was concentrated in vacuo. The residue was purified by flash gel column chromatography (eluent: DCM/MeOH (v/v) = 100 : 1) to give a yellow solid (97 mg, 0.19 mmol, 51%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.68 (s, 1H), 8.13 (s, 1H), 7.71–7.69 (d, J = 8.4 Hz, 2H), 7.61–7.57 (t, J = 7.5 Hz, 2H), 7.53–7.51 (d, J = 7.2 Hz, 1H), 7.48–7.46 (d, J = 7.4 Hz, 2H), 7.43–7.41 (m, 3H), 6.05 (s, 2H), 5.02–4.92 (m, 1H), 3.59–3.56 (t, J = 5.7 Hz, 2H), 3.24–3.21 (t, J = 6.2 Hz, 2H), 1.99–1.98 (m, 2H), 1.84–1.81 (m, 2H), 1.47–1.45 (d, J = 6.7 Hz, 6H). MS (ESI⁺) m/z: 508.4 [M + H]⁺.

N-(4-(4-Amino-7-(2-hydroxypropyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-1,2,4,5,6,7-hexahydropyrazolo[1,5-a]pyridine-3-carboxamide (w17)

EDCI (360.0 mg, 1.84 mmol) and HOAT (32.6 mg, 0.235 mmol) were added to a mixture of 2-oxo-1-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-3-carboxylic acid (300 mg, 1.161 mmol) and 1-[4-amino-5-(4-aminophenyl)pyrrolo[2,3-d]pyrimidin-7-yl]propan-2-ol (300.0 mg, 1.059 mmol) in DCM (10 mL). The mixture was refluxed for 16 h. H₂O (20 mL) was added into the mixture and the resulting mixture was extracted with DCM (100 mL). After separation, the organic phase was concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 20 : 1) to give a yellow solid (207 mg, 0.348 mmol, 32.8%). ¹H NMR (400 MHz, CDCl₃) δ 10.68 (s, 1H), 8.29 (s, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.55 (t, J = 7.6 Hz, 2H), 7.46 (t, J = 7.4 Hz, 1H), 7.43–7.33 (m, 4H), 7.00 (s, 1H), 5.88 (s, 2H), 4.39–4.05 (m, 4H), 3.58 (t, J = 5.9 Hz, 2H), 3.41 (t, J = 6.4 Hz, 2H), 2.09 (tq, J = 6.0, 3.9, 2.4 Hz, 2H), 1.99–1.88 (m, 2H), 1.26 (d, J = 6.0 Hz, 3H).MS (ESI⁺) m/z: 524.3 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₉H₃₀N₇O₃ [M + H]⁺: 524.2332, found 524.2403.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w18)

To a solution of 2-oxo-1-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (306 mg, 0.66 mmol) in dioxane (7.0 mL) and H₂O (1.0 mL) were added intermediate 92 (151 mg, 0.66 mmol), Pd(dppf)₂Cl₂·CH₂Cl₂ (58 mg, 0.066 mmol) and Cs₂CO₃ (446.5 mg, 1.35 mmol). The reaction was degassed with nitrogen, followed by stirring and reflux for 8 h. The mixture was cooled and concentrated in vacuo. The residue was purified via flash column chromatography (eluent: DCM/MeOH (v/v) = 500/1) to give a yellow solid (65.6 mg, 0.13 mmol, 20.5%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.48 (s, 1H), 8.12 (s, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.64–7.57 (m, 2H), 7.56–7.48 (m, 6H), 7.15 (s, 2H), 6.64 (s, 1H), 5.13 (s, 2H), 4.10 (t, J = 4.8 Hz, 2H), 3.73–3.68 (m, 5H). MS (ESI⁺) m/z: 482.1 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₆H₂₄N₇O₃ [M + H]⁺: 482.1862, found 482.1928.

N-(5-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyridin-2-yl)-2-oxo-1-phenyl-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w19)

To a solution of 2-oxo-1-phenyl-N-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (425 mg, 0.92 mmol) and 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (235 mg, 1.0 mmol) in H₂O (5 mL) and 1,4-dioxane (10 mL) were added Pd(PPh₃)₄ (216 mg, 0.19 mmol) and K₂CO₃ (390 mg, 2.8 mmol). The mixture was stirred at 105 °C for 20 h under a nitrogen atmosphere and concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 50 : 1) to give a yellow solid (73.0 mg, 0.15 mmol, 16.5%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 8.37 (d, J = 1.7 Hz, 1H), 8.27 (d, J = 8.5 Hz, 1H), 8.16 (s, 1H), 7.84 (dd, J = 8.5, 2.1 Hz, 1H), 7.64–7.58 (m, 2H), 7.56–7.50 (m, 3H), 7.37 (s, 1H), 6.18 (s, 2H), 5.13 (s, 2H), 4.12–4.08 (m, 2H), 3.74 (s, 3H), 3.70 (t, J = 4.5 Hz, 2H). MS (ESI⁺) m/z: 483.1 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₅H₂₂N₈O₃ [M + H]⁺: 483.1815, found 483.1886.

N-(5-(4-amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyridin-2-yl)-1-(4-fluorophenyl)-2-oxo-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w20)

To a solution of 1-(4-fluorophenyl)-2-oxo-N-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (1.1 g, 2.3 mmol) and 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (550 mg, 2.4 mmol) in H₂O (2.5 mL) and 1,4-dioxane (30 mL) were added Pd(PPh₃)₄ (530 mg, 0.46 mmol) and K₂CO₃ (0.96 g, 6.9 mmol). The mixture was stirred at 105 °C for 16 h under a nitrogen atmosphere. Then, the mixture was cooled and concentrated in vacuo. The residue was multiply purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 50 : 1) to give a yellow solid (36.3 mg, 0.07 mmol, 2.9%). ¹H NMR (400 MHz, chloroform-d) δ 10.77 (s, 1H), 8.57–8.25 (m, 3H), 7.80 (d, J = 8.5 Hz, 1H), 7.41 (s, 2H), 6.98 (s, 1H), 5.28 (s, 2H), 5.10 (s, 2H), 4.17 (t, J = 4.8 Hz, 2H), 3.87 (s, 3H), 3.66 (t, J = 4.7 Hz, 2H). ¹⁹F NMR (376 MHz, CDCl3) δ −110.64 (s). MS (ESI⁺) m/z: 501.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₅H₂₁FN₈O₃ [M + H]⁺: 501.1721, found 501.1842.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-1-(4-fluorophenyl)-2-oxo-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w21)

To a solution of 1-(4-fluorophenyl)-2-oxo-N-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (1.16 g, 2.42 mmol) and 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (580 mg, 2.55 mmol) in H₂O (2.5 mL) and 1,4-dioxane (30 mL) were added Pd(PPh₃)₄ (566 mg, 0.49 mmol) and K₂CO₃ (1.1 g, 7.9 mmol). The mixture was stirred at 105 °C for 16 h under a nitrogen atmosphere and concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH = 50 : 1) to give a yellow solid (26 mg, 0.04 mmol, 1.7%). ¹H NMR (600 MHz, DMSO-d₆) δ 10.38 (s, 1H), 8.15 (s, 1H), 7.74–7.68 (m, 2H), 7.63–7.59 (m, 2H), 7.50–7.39 (m, 4H), 7.28 (s, 1H), 6.07 (s, 2H), 5.12 (s, 2H), 4.10 (t, J = 5.1 Hz, 2H), 3.74 (s, 3H), 3.71–3.66 (m, 2H). MS (ESI⁺) m/z: 500.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₅H₂₂FN₇O₃ [M + H]⁺: 500.1768, found 500.1872.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-fluorophenyl)-2-oxo-1-phenyl-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w22)

To a solution of N-[2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]-2-oxo-1-phenyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (555 mg, 1.16 mmol) and 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (279 mg, 1.23 mmol) in H₂O (1.5 mL) and 1,4-dioxane (20 mL) were added Pd(PPh₃)₄ (270 mg, 0.23 mmol) and K₂CO₃ (488 mg, 3.5 mmol). The mixture was stirred at 105 °C for 16 h under a nitrogen atmosphere and concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 50 : 1) to give a yellow solid (26 mg, 0.04 mmol, 3.8%). ¹H NMR (400 MHz, CDCl₃) δ 10.62 (s, 1H), 8.51 (t, J = 8.3 Hz, 1H), 8.34 (s, 1H), 7.56 (t, J = 7.6 Hz, 2H), 7.47 (t, J = 7.4 Hz, 1H), 7.41 (d, J = 7.5 Hz, 2H), 7.24 (s, 1H), 7.21 (d, J = 3.4 Hz, 1H), 6.93 (s, 1H), 5.27 (s, 2H), 5.13 (s, 2H), 4.20–4.10 (m, 2H), 3.83 (s, 3H), 3.70–3.63 (m, 2H). MS (ESI⁺) m/z: 500.1 [M + H]⁺.

N-(4-(4-Amino-7-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-3-fluorophenyl)-2-oxo-1-phenyl-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w23)

To a solution of N-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-oxo-1-phenyl-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (555 mg, 1.16 mmol) and 5-bromo-7-methyl-pyrrolo[2,3-d]pyrimidin-4-amine (278 mg, 1.22 mmol) in H₂O (1.5 mL) and 1,4-dioxane (20 mL) were added Pd(PPh₃)₄ (270 mg, 0.23 mmol) and K₂CO₃ (490 mg, 3.51 mmol). The mixture was stirred at 105 °C overnight under a nitrogen atmosphere and concentrated in vacuo. The residue was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 50 : 1) to give a yellow solid (64 mg, 0.12 mmol, 10%). ¹H NMR (400 MHz, CDCl₃) δ 10.46 (s, 1H), 8.33 (s, 1H), 7.77 (d, J = 11.4 Hz, 1H), 7.57 (t, J = 7.5 Hz, 2H), 7.49 (t, J = 7.4 Hz, 1H), 7.39 (d, J = 7.5 Hz, 2H), 7.36–7.30 (m, 2H), 7.00 (s, 1H), 5.27 (s, 2H), 5.03 (s, 2H), 4.15 (t, J = 4.9 Hz, 2H), 3.84 (s, 3H), 3.67 (t, J = 4.9 Hz, 2H). MS (ESI⁺) m/z: 500.1 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₆H₂₂FN₇O₃ [M + H]⁺: 500.1768, found 500.1838.

N-(4-(4-Amino-7-(2-hydroxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w24)

EDCI (116 mg, 0.59 mmol) and HOAT (11 mg, 0.08 mmol) were added to a mixture of 2-[4-amino-5-(4-aminophenyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethanol (105 mg, 0.39 mmol) and 2-oxo-1-phenyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine-3-carboxylic acid (112 mg, 0.43 mmol) in DCM (6 mL). The mixture was stirred and refluxed for 16 h. Then, H₂O (20 mL) was added into the mixture, and the resulting mixture was extracted with DCM (100 mL). After separation, the organic phase was concentrated in vacuo. The solid was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 100 : 1) to give a yellow solid (33.1 mg, 0.06 mmol, 15.7%). ¹H NMR (400 MHz, CDCl₃) δ 10.30 (s, 1H), 8.18 (d, J = 3.7 Hz, 1H), 7.64 (dd, J = 8.5, 3.3 Hz, 2H), 7.49 (dd, J = 9.5, 5.9 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 7.37–7.29 (m, 4H), 6.95 (d, J = 3.6 Hz, 1H), 5.20 (d, J = 12.8 Hz, 4H), 4.87 (s, 1H), 4.25 (q, J = 4.8, 4.3 Hz, 2H), 4.07 (t, J = 5.0 Hz, 2H), 3.94–3.85 (m, 2H), 3.59 (t, J = 5.0 Hz, 2H). MS (ESI⁺) m/z: 512.3 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₇H₂₆N₇O₄ [M + H]⁺: 512.2068, found 512.2037.

N-(4-(4-Amino-7-(2-hydroxypropyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)-2-oxo-1-phenyl-2,4,6,7-tetrahydro-1H-pyrazolo[5,1-c][1,4]oxazine-3-carboxamide (w25)

EDCI (119.6 mg, 0.61 mmol) and HOAT (11.1 mg, 0.08 mmol) were added to a mixture of 2-oxo-1-phenyl-6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazine-3-carboxylic acid (101.7 mg, 0.39 mmol) and 1-[4-amino-5-(4-aminophenyl)pyrrolo[2,3-d]pyrimidin-7-yl]propan-2-ol (100.1 mg, 0.35 mmol) in DCM (8 mL). The mixture was stirred and refluxed for 16 h. H₂O (20 mL) was added into the mixture and the resulting mixture was extracted with DCM (100 mL). After separation, the organic phase was concentrated in vacuo. The solid was purified by flash column chromatography (eluent: DCM/MeOH (v/v) = 50 : 1) to give a yellow solid (23.2 mg, 0.04 mmol, 12%). ¹H NMR (400 MHz, CDCl₃) δ 10.41 (s, 1H), 8.30 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.59 (t, J = 7.7 Hz, 3H), 7.47 (dt, J = 29.1, 7.5 Hz, 6H), 6.97 (s, 1H), 5.32 (d, J = 11.1 Hz, 3H), 4.35–4.24 (m, 2H), 4.22–4.13 (m, 3H), 3.69 (t, J = 5.1 Hz, 2H), 1.30 (s, 3H). MS (ESI⁺) m/z: 526.2 [M + H]⁺. HRMS (ESI⁺) calcd for C₂₈H₂₈N₇O₄ [M + H]⁺: 526.2125, found 526.2200.

Enzyme inhibition assay

The kinases selected for the test were incubated with a candidate compound for 1 h in different buffers. Specific and general methods: kinase inhibitory reactions were performed in a total volume of 34 μL of kinase buffer (example: 5 mM HEPES pH 7.6, 15 mM NaCl, 0.01% BSA (Sigma #I-5506), 10 mM MgCl₂, 1 mM DTT, 0.02% Triton X-100). Compound w11 (1 μM) was dissolved in DMSO and added to each well to a final concentration of 1% in DMSO (each test was repeated twice). The above-mentioned substrate and ATP (unlabeled ATP or (γ-33P)-labeled ATP) were added and incubated for 1 h. After washing with PBS, the radioactivity was counted using a Wallac TriLux counter. The kinase selectivity assay of compound w11 was screened against other kinases performed by Eurofins standard KinaseProfiler™.

Cell viability assay

BaF3/TEL-AXL cell lines with cell viability higher than 90% were seeded in 96-well plates and incubated overnight. RXDX-106 or selected compounds were dissolved in DMSO and added into each well. The cell and compounds were further co-incubated for 72 h. Then, the cell viability of BaF3/TEL-AXL cells was determined by CellTiter-Glo assay. IC₅₀ values were calculated by concentration–response curve fitting using a curve integration method (GraphPad Prism, version 5.0).

Cell proliferation assay

The cell proliferation assay was carried out using a cell counting kit (CCK-8) as previously reported. Various cells were independently seeded in 96-well plates with growth medium at a low density and incubated overnight. Designated different concentrations of compounds or staurosporine were added into each well and incubated for 72 h. The CC₅₀ values were calculated by concentration–response curve fitting using a curve integration method (GraphPad Prism, version 8.3).

Pharmacokinetic study

The animal studies were performed according to the institutional ethical guidelines on animal care which were approved by the animal care and use committee at Dongguan Institute of Sunshine Lake Pharma Co. Ltd. (Study no. 16020-5015). The pharmacokinetic parameters were calculated based on the mean plasma concentration following drug administration. A single 5 mg kg⁻¹ oral dose or 1 mg kg⁻¹ intravenous dose of the compounds was separately administered to male rats. The formulation for our compounds was 5% DMSO/60% PEG400/35% saline. The blood samples were collected at 0.25, 0.5, 1, 2, 5, 7, and 24 h in the oral group. All samples were analyzed utilizing the LC-MS/MS system.

Molecular modeling

Docking studies were carried out on Schrodinger Suites 2018.1 using the protein structure of MER kinase (PDB 7AVX). The protein preparation, optimization, and minimization were performed in the preparation process wizard (Schrödinger, LLC, New York, NY, 2019). All docking compounds were optimized by LigPrep. The best conformation was selected according to the lowest binding energy of the compound. The docking site was determined using a grid box size that docks ligands similar in size to the workspace ligand. The docking results are shown as a cartoon model from PyMol.

Animal studies

The antitumor activity assay was performed according to the institutional ethical guidelines on animal care which were approved by the animal care and use committee at Dongguan Institute of Sunshine Lake Pharma Co. Ltd. (Study no. 16020-5015). NOD/SCID mice were used for MV-4-11 cells. All mice were used between 5 and 6 weeks of age. MV-4-11 cells were cultured in IMDM and placed in a 5% carbon dioxide incubator at 37 °C. The original medium was removed after the cells grew to the logarithmic growth phase. The cells were collected and centrifuged. Subsequently, a cell count was performed. The cell density was adjusted to 1.6 × 10⁸ cells per mL using a mixture of serum-free RPMI 1640 medium and Matrigel agent (1 : 1). For the MV-4-11 tumor model, cells were injected into the subcutaneous tissue at 0.1 mL per mice. Treatment was initiated after randomization when the average tumor size reached approximately 160 mm³. The animals were randomly divided into vehicle groups (n = 10) and treatment groups (n = 10) according to the tumor volume. Compounds w11 and w12 were formulated in 15% HP-β-CD (pH = 2.2–2.5), respectively. Treatment groups received two compounds (50 mg kg⁻¹, QD) at the indicated doses via PO administration once daily for the indicated days. The tumor sizes were measured once per week. The tumor volume (TV) was calculated as TV = (length × width²)/2. Tumor growth inhibition (TGI) was measured as TGI (%) = {1 − [(TV_{treated final day} − TV_{treated day 0})/(TV_{vehicle final day} − TV_{vehicle day 0})]} × 100.

Author contributions

All authors have approval for the final version of the manuscript. Mingming Sun: data collection and analysis, the design and synthesis of compounds, pharmacokinetic assay, manuscript preparation. Shuang Wu: synthesis, liver microsomal stability assay, model docking, manuscript preparation. Ning Xi: research design, supervision of the whole project, manuscript preparation. Qianyong Cao: research design, supervision of the whole project, manuscript preparation.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All data generated or analyzed during this study are included in this published article and its ESI† files.