Design, synthesis and biological evaluation of pyrazolo[3,4-b]pyridine derivatives as TRK inhibitors

Abstract

Tropomyosin receptor kinases (TRKs) are associated with the proliferation and differentiation of cells, and thus their continuous activation and overexpression cause cancer. Herein, based on scaffold hopping and computer-aid drug design, 38 pyrazolo[3,4-b]pyridine derivatives were synthesised. Further, we evaluated their activities to inhibit TRKA. Among them, compound C03 showed acceptable activity with an IC₅₀ value of 56 nM and it inhibited the proliferation of the Km-12 cell line with an IC₅₀ value of 0.304 μM together with obvious selectivity for the MCF-7 cell line and HUVEC cell line. Furthermore, compound C03 possessed good plasma stability and low inhibitory activity to a panel of cytochrome P450 isoforms except CYP2C9. Overall, C03 has potential for further exploration.

---

1 Introduction

TRKs, tropomyosin receptor kinases, have three subtypes, namely TRKA, TRKB and TRKC, which belong to the receptor tyrosine kinase (RTK) family.¹ As a member of the transmembrane protein kinase family, TRKs are composed of an extracellular domain, which combines with endogenous ligands, an intercellular domain and a transmembrane domain.^2,3 TRKA mainly combines with nerve growth factor (NGF), while TRKB is mainly activated by brain-derived neurotrophic factor (BDNF) and TRKC is activated by neurotrophin-3 (NT-3).^4–9 Once activated, the intramembrane kinase domain is phosphorylated and the downstream signal transduction pathways (including Ras/Erk, PLC-γ, and PI3K/Akt) are triggered, which are associated with the proliferation, differentiation and survival of cells.^10–13 TRKs are encoded by neurotrophic receptor tyrosine kinase 1 (NTRK1), NTRK2 and NTRK3 genes, respectively.¹ Although NTRK fusion is the primary mechanism of TRK mutations, NTRK fusion means that the 3′ sequences of the NTRK gene fuse with the 5′ sequences of other genes that encode irrelevant proteins. Structurally, mutant TRK proteins lack an extracellular domain. Functionally, TRK proteins are manifested as continuous activation of the intracellular kinase domain, further leading to cancers,¹⁴ including colorectal cancer,¹⁵ non-small cell lung cancer (NSCLC),¹⁶ glioblastoma¹⁷ and head and neck squamous cell carcinoma.¹⁸ Thus, small-molecule TRK inhibitors are important for the treatment of these cancers.

Recently, many potent TRK inhibitors have been reported (Fig. 1). In 2018, larotrectinib (1) was the first TRK inhibitor to be approved by the Food and Drug Administration (FDA) for the treatment of patients with tumours harbouring NTRK fusions. It is a highly selective TRK inhibitor with low nanomole potency (IC₅₀ < 20 nM).^19–21 In 2019, the multiple target inhibitor entrectinib (2) discovered by Roche was approved by the FDA. It exhibited strong inhibitory activities towards ALK (IC₅₀ = 12 nM), ROS1 (IC₅₀ = 7 nM) and all the subtypes of TRKs (IC₅₀ values for TRKA/B/C were 1 nM, 3 nM, and 5 nM, respectively).^22,23 Compound 3, which was reported by Duan et al., exhibited inhibitory activities on TRKA/B/C with IC₅₀ values of 1.6 nM, 2.9 nM, and 2.0 nM, respectively. Moreover, 3 exhibited strong activity for the suppression of the growth of BaF3 cells harboring xDFG motif mutations.²⁴ Compounds 4 and 5 were reported by our research group recently, which are both pan-TRK inhibitors with low nanomole enzyme activities (4, IC₅₀ values for TRKA/B/C were 17 nM, 28 nM and 11 nM and 5, IC₅₀ values for TRKA/B/C were 12 nM, 22 nM and 15 nM, respectively). Meanwhile, 5 showed a potent anticancer effect in vitro and in vivo.^25,26 To suppress on the resistant TRK mutants, LOXO-195 (6) was developed as a next-generation TRK inhibitor, which has advanced to a phase II clinical trial for patients with TRK fusion-positive cancers that acquired resistant mutations to larotrectinib.²⁷

Fig. 1 Chemical structure of potent TRK inhibitors.

Herein, based on scaffold hopping, rational drug design and computer-aid drug design (CADD), 38 pyrazolo[3,4-b]pyridine derivatives were synthesised. In addition, most of them exhibited nanomole inhibitory activities against TRKA.

2 Chemistry

As shown in Scheme 1, the commercially available 5-bromo-1H-pyrazolo [3,4-b]pyridine (7) was iodized by NIS to obtain intermediate 8, and then the NH of intermediate 8 was protected by PMB-Cl to produce the key intermediate 9. Simultaneously, meta-aminobenzoic acid was reacted with morpholine to produce intermediate 12. Intermediate 12 and intermediate 9 were coupled through Buchwald–Hartwig reaction to get intermediate 13. The Miyaura borylation reaction and Suzuki reaction were performed in one pot in sequence with intermediate 13 to produce intermediates 15a–15o, and the protecting group was removed by trifluoroacetic acid to obtain the target compounds A01–A15.

Scheme 1 Synthesis of compounds A01–A15. Reagents and conditions: (a) HOBt, EDCI, DIPEA, DMF, r.t., 82.3%; (b) NIS, DMF, 60 °C, 12 h, 82.4%; (c) NaH, DMF, PMBCl, 0–25 °C, 50.2%; (d) Pd₂(dba)₃, xantphos, Cs₂CO₃, dry dioxane, Ar, 90 °C, 8 h, 48.5%; (e) bis(pinacolato)diboron, Pd(dppf)Cl₂, KOAc, dry dioxane, Ar, 100 °C, 4 h; (f) substituted benzyl bromide, Pd(PPh₃)₄, Cs₂CO₃, dioxane : H₂O = 5 : 1, Ar, 80 °C, 2 h, 30.3–34.7%; and (g) CF₃COOH, 60 °C, 8 h, 48.5–52.3%.

Compounds B01–B13 were synthesized, as shown in Scheme 2. Intermediate 9 was coupled with ethyl 3-aminobenzoate or ethyl 4-aminobenzoate through Buchwald–Hartwig reaction to produce intermediate 16 or 22, respectively. The palladium-catalyzed Miyaura borylation reaction and Suzuki reaction were performed in one pot by sequence to produce intermediate 18 or 24. Trifluoroacetic acid was used to deprotect the para-methoxy benzyl and NaOH was used to deprotect the ethyl, and then crucial intermediate 20 or 26 was obtained, respectively. Condensation reactions occurred between 20(26) and different amino segments, together with deprotection reactions, if necessary, to obtain the target compounds B01–B13.

Scheme 2 Synthesis of compounds B01–B13. Reagents and conditions: (a) Pd₂(dba)₃, xantphos, Cs₂CO₃, dry DMF, Ar, 120 °C, 8 h, 65.9%; (b) bis(pinacolato)diboron, Pd(dppf)Cl₂, Cs₂CO₃, dry dioxane, Ar, 100 °C, 4 h; (c) 2,5-difluorobenzyl bromide, Pd(PPh₃)₄, Cs₂CO₃, dioxane : H₂O = 5 : 1, Ar, 80 °C, 4 h, 48.6%; (d) CF₃COOH, 60 °C, 5 h; (e) NaOH, methanol : H₂O = 3 : 1, 60 °C, 4 h, 65.7%; and (f) I) appropriate amine, PyBop, DIPEA, THF, 0 °C, 2 h and II) 4 N HCl/ethyl acetate, r.t., 4 h, 28.8–55.5%.

Scheme 3 shows the synthetic route for compounds C01–C10. Starting with intermediate 9, it was coupled with 3-ethoxycarbonylphenyl-boronic acid or 4-ethoxycarbonylphenyl-boronic acid through Suzuki reaction to produce intermediate 28 or 34, respectively. Intermediate 28 or 34 was coupled with 2,5-difluorobenzyl through Miyaura borylation reaction and Suzuki reaction in sequence in one pot to generate intermediate 30 or 36, then deprotection was performed to generate the key intermediate 32 or 38, respectively. Condensation reactions or condensation reactions followed by deprotection reactions occurred to yield the target compounds C01–C10.

Scheme 3 Synthesis of compounds C01–C10. Reagents and conditions: (a) Pd(pph₃)₄, S-Phos, KF, dioxane : H₂O = 5 : 1, Ar, 100 °C, 24 h, 53.7%; (b) bis(pinacolato)diboron, Pd(OAc)₂, X-Phos, KOAc, dry dioxane, Ar, 100 °C, 14 h; (c) 2,5-difluorobenzyl bromide, Pd(PPh₃)₄, Cs₂CO₃, dioxane : H₂O = 5 : 1, Ar, 100 °C, 7 h, 52.3%; (d) CF₃COOH, 60 °C, 5 h; (e) NaOH, methanol : H₂O = 3 : 1, 60 °C, 4 h, 54.2%; and (f) I) appropriate amine, PyBop, DIPEA, THF, 0 °C, 2 h and II) 4 N HCl/ethyl acetate, r.t., 4 h, 31.6–59.0%.

3 Results and discussion

3.1 Design and in vitro activities of target compounds against TRKA

All the novel synthesized compounds were evaluated for their in vitro inhibitory activities against TRKs using homogeneous time-resolved fluorescence (HTRF) assays and larotrectinib (1) was employed as a positive comparison. Under the experimental conditions, larotrectinib exhibited suppressive activity on TRKA, TRKB and TRKC with IC₅₀ values of 3.0, 13 and 0.2 nM, respectively, which were similar to previously reported data.

The analysis of the co-crystal structure between entrectinib and TRKA, as shown in Fig. 2A, indicated that the aminopyrazole fragment is anchored to the hinge region through three hydrogen bonds with Glu590 and Met592 residues; the fluorine of the 3,5-difluorophenyl moiety creates favorable fluorine bonds with Asn655, Cys656 and Gly667 which comes from the DFG motif; the benzene ring of the indazole scaffold has a π–π stacking interaction with gatekeeper Phe589; and N-methylpiperazine is oriented toward the solvent zone. Guided molecular docking showed that larotrectinib has a similar binding mode, while the hydroxy orients toward Asp596 and formed a hydrogen bond (Fig. 2B). Observing the structure of entrectinib and larotrectinib, it can be found that they both have a hydrophilic head extending into the solvent zone, a fluorine substituted aromatic ring as hydrophobic tail, which extends to the DFG motif, and a linker that binds to the hinge area as a hydrogen bond center (Fig. 3).^28,29

Fig. 2 (A) Binding mode of entrectinib (blue) and TRKA. (B) Predicted binding mode of larotrectinib (blue) and TRKA. (C) Predicted binding mode of A01 (blue) and TRKA. (D) Overlap of B09 (yellow) and B10 (blue) with entrectinib (green). (PDB code: 5KVT. Residues are depicted in green; the interactions are depicted in dash lines with different colours, hydrogen bond is depicted in green, fluorine bond is depicted in red and π–π stacking interaction or δ–π interaction is depicted in orange).

Fig. 3 Structural analysis of larotrectinib and entrectinib, and the design strategy of target compounds.

Pyrazolo[3,4-b]pyridine is a common fragment used in the synthesis of kinase inhibitors, whose pyrazolo portion is suitable as a hydrogen bond center. Simultaneously, the pyridine is thought to have a π–π stacking interaction with Phe589. Thus, the scaffold hopping strategy was used to investigate a structurally novel TRK inhibitor. Firstly, we designed and synthesized compound A01 (Table 1), which had weak inhibitory activity on TRKA (IC₅₀ = 0.293 μM). Also, the molecular docking study preliminarily revealed the possible binding mode (Fig. 2C), where the aminopyrazole fragment is bound to Glu590 and Met592 with three hydrogen bonds; the pyridine forms a π–π stacking interaction with Phe589; the 3-fluorophenyl is oriented toward the hydrophobic pocket near to the DFG motif; and simultaneously the morpholine extends to the solvent zone. Next, we investigated the hydrophobic tail and the structures are shown in Table 1. When we removed the fluorine atom, compound A02 showed weaker potency (IC₅₀ = 0.479 μM), which indicates that a suitable substituent group is necessary to achieve the desired activity. Subsequently, chlorine, bromine, and stronger electrophilic substituents such as nitro and trifluoromethyl were introduced in the meta-position of the benzene ring to generate compounds A03–A06, respectively; unfortunately they all showed reduced potency. This indicated that the fluorine is important for the hydrophobic tail. Then, we investigated the effect of the number and position of the fluorine on the benzene ring, obtaining compounds A07–A15. All the compounds had various degrees of activity decline except compound A11 with 2,5-difluorophenyl, which showed a moderate IC₅₀ value of 0.178 μM. This can be ascribed to its suitable orientation and electrical distribution of the meta-fluorine on the benzene ring.

Table 1 In vitro TRKA kinase inhibitory activities of A01 to A15

Entry	R 1	TRKA	Entry	R 1	TRKA
		IC50 (μM)			IC50 (μM)
a Used as a positive comparison.
A01		0.294	A09		1.966
A02		0.479	A10		0.788
A03		4.430	A11		0.178
A04		0.802	A12		0.838
A05		1.400	A13		>100
A06		16.690	A14		0.700
A07		0.978	A15		0.660
A08		2.780	1		0.003

---

Then, we fixed the hydrophobic group as 2,5-difluorophenyl and explored the types and positions of the hydrophilic fragments to get compounds B01–B13. All the structures are shown in Table 2. When the hydrophilic fragment was sited at the meta-position of the benzene ring, it was B01 with the methylpiperazinyl and B04 with piperazine, which showed a slight improvement in potency (IC₅₀ = 0.118 and 0.117 μM, respectively). Moreover, we moved the fragments to the para-position and compounds B07–B13 were obtained, but none of them had better inhibitory activity. B09 with 4-hydroxypiperidinyl and B10 with 3-hydroxypiperidinyl showed a weak potency promotion (IC₅₀ = 0.129 and 0.156 μM, respectively) compared with A11. Molecule docking was used to explain the possible reasons for this (Fig. 2D), where both of them showed a good binding mode at the hydrophobic tail and the hydrogen bond center; simultaneously, the hydroxy formed a hydrogen bond with Asp596, which is similar to larotrectinib. Regrettably, the benzene ring had an uncomfortable conformation, which may be due to the rotatable single bond, leading to the production of additional entropy and preventing the binding between the compound and the protein.

Table 2 In vitro TRKA kinase inhibitory activities of B01 to B13

Entry	R 2	TRKA	Entry	R 2	TRKA
		IC50 (μM)			IC50 (μM)
a Used as a positive comparison.
B01		0.118	B08		0.389
B02		0.613	B09		0.129
B03		0.205	B10		0.156
B04		0.117	B11		0.409
B05		0.437	B12		0.263
B06		0.630	B13		0.321
B07		0.980	1		0.003

---

Referring to the binding mode of larotrectinib, conformational restriction was used and we removed the NH group. Compounds C01–C10 were obtained, as shown in Table 3. When the substitution was located at the para-position, compounds C05 and C08 had a slight potency improvement compared with B08 and B11, but still worse than B04, while at the meta-position, C02 and C03 had better potency. Excitedly, compound C03 with 3-hydroxypiperidinyl at the meta-position of the benzene ring showed acceptable potency with an IC₅₀ value of 0.056 μM. Then, the chirality of C03 was considered, and C09–C10 were synthesized and evaluated for their inhibitory activities on TRKA. Alternatively, compound C10 with (S)-3-hydroxypiperidinyl showed slight superiority with an IC₅₀ value of 0.026 μM.

Table 3 In vitro TRKA kinase inhibitory activity of C01 to C10

Entry	R 3	TRKA	Entry	R 3	TRKA
		IC50 (μM)			IC50 (μM)
a Used as a positive comparison.
C01		0.106	C06		0.383
C02		0.254	C07		0.571
C03		0.056	C08		0.251
C04		0.391	C09		0.057
C05		0.296	C10		0.026
1		0.003

---

3.2 Kinase selectivity

We chose compounds C03, C09 and C10 for further kinase selectivity assay. As shown in Table 4, they were all pan-TRK inhibitors with similar suppression on each subtype of TRK, while C03 had the most similar potency on each subtype of TRKs. Then, the inhibitory activities of C03, C09 and C10 on focal adhesion kinase (FAK), p21-activated kinase 4 (PAK4) and polo-like kinase 4 (PLK4) were tested. As shown in Fig. S1 (ESI†), C03, C09 and C10 exhibited low suppression on PLK4 (IC₅₀ = 1.65, 0.69 and 1.32 μM, respectively), together with significant selectivity for FAK and PAK4 (IC₅₀ for both was over 100 μM). Among them, considering the selectivity and synthetic cost, C03 may more suitable for further exploration.

Table 4 In vitro TRK kinase inhibitory activities of some compounds

Entry	TRKA	TRKB	TRKC
	IC50 (μM)	IC50 (μM)	IC50 (μM)
C03	0.056	0.090	0.058
C09	0.057	0.090	0.083
C10	0.026	0.118	0.076
LOXO-101	0.003	0.013	0.0002

---

3.3 Molecular docking study

The molecular docking study was used to evaluate the binding mode between C03, C09 and C10 and TRKA. Given that compounds C09 and C10 are isomers of C03, both of them were separately used for the molecular docking study. As shown in Fig. 4, compounds C09 and C10 had good overlap with entrectinib and suitably occupied the ATP pocket. Simultaneously, the chiral center of 3-hydroxypiperidinyl did not have an obvious influence on the binding mode. Similar to entrectinib and larotrectinib, the pyrazolyl is bound to the hinge region with Glu590 and Met592 residues through two hydrogen bonds; the 2,5-difluorophenyl is oriented toward the DFG motif and bound to Asn655 and Cys656 with fluorine bonds; pyridine forms a π–π stacking interaction with Phe589; and the 3-hydroxy on the piperidine is bound to Asp596 with a hydrogen bond. Consequently, compound C03 was chosen for further assessments.

Fig. 4 Molecular docking between C09 and C10 with TRKA (PDB code: 5KVT). (A) Overlap of C09 (blue) and C10 (yellow) with entrectinib (white). (B) Predicted binding mode between C09 and TRKA. (C) Predicted binding mode between C10 and TRKA. The residues are depicted in green; the interactions are depicted as dash lines with different colours, hydrogen bond is depicted in green, fluorine bond is depicted in red and π–π stacking interaction is depicted in orange.

3.4 Antiproliferative activities

Compound C03 was further evaluated for its antiproliferative activities against three different cell lines, including Km-12, MCF-7 and HUVEC. Km-12 is a natural colorectal cancer line with TPM3-NTRK1 gene fusion. The breast cancer cell line MCF-7 does not harbour any rearrangement of TRKs. Also, HUVEC are human umbilical vein endothelial cells, a normal cell line without cancer. As shown in Table 5, compound C03 inhibited the proliferation of Km-12 cells with the IC₅₀ value of 0.304 μM. Furthermore, the inhibitory activity against the TRKA-independent cell lines MCF-7 and HUVEC was weak (IC₅₀ > 40 μM and IC₅₀ > 36.69 μM, respectively). This indicated that C03 may have a low risk of off-target effects.

Table 5 Antiproliferation activities of C03 against Km-12, MCF-7 and HUVEC cells

Entry	IC50^a	MCF-7 (μM)	HUVECs (μM)
	Km-12 (μM)
a IC50: concentration of the compound (μM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the CCK-8 assay.
C03	0.304	>40	36.69
LOXO-101	0.012	>40	N.D.

---

3.5 The plasma stability and CYPs inhibition

The plasma stability and CYPs inhibition of C03 were evaluated. As shown in Table 6, compound C03 possessed excellent plasma stability with t_1/2 > 289.1 min. Then, compound C03 was evaluated for its inhibition rates on a panel of cytochrome P450 isoforms (1A2, 2C9, 2C19, 2D6 and 3A4). As shown in Table 7, C03 exhibited relatively strong suppression on only CYP2C9 at the concentration of 10 μM, which means that C03 may have a low risk of drug–drug interactions (DDIs).

Table 6 Plasma stability of compound C03

Time (min)	0	10	30	60	120
Remaining (%)	100.0	103.7	88.0	93.6	89.1
T 1/2 (min)	>289.1

---

Table 7 Cytochrome P450 inhibition by compound C03

Isozyme	CYP1A2	CYP2C9	CYP2C19	CYP2D6	CYP3A4
Inhibition at 10 μM (%)	16.6	66.6	48.6	25.8	0.0

---

4 Conclusion

Herein, based on scaffold hopping, rational drug design and CADD, we designed and synthesized 38 novel TRKA inhibitors with a pyrazolo[3,4-b]pyridine core. The in vitro enzyme activity assays suggested that most of them had nanomole inhibitory activity against TRKA, while compounds C03, C09 and C10 showed acceptable potency with IC₅₀ values of 56/57/26 nM, respectively. The kinase selectivity assays suggested that C03, C09 and C10 are pan-TRK inhibitors combined with significant selectivity for FAK, PAK4 and PLK4. The proliferation assay revealed that C03 also had acceptable antiproliferative activity in the Km-12 cell line with an IC₅₀ value of 0.304 μM, together with selectivity for the MCF-7 cell line and HUVEC cell line. Furthermore, compound C03 exhibited good plasma stability and low inhibitory activities to CYPs except CYP2C9. Thus, this compound has potential for further exploration and related works are undergoing.

5 Experiment

The starting materials, reagents and solvents were obtained from a commercial supplier and used without further purification unless otherwise indicated. Anhydrous solvents were dried and stored according to standard procedures. All reactions were monitored by thin-layer chromatography (TLC) on silica gel plates with fluorescence F-254 and visualized with UV light (254 nm or 365 nm). Column chromatography was performed on silica gel (200–300 mesh). ¹H NMR and ¹³C NMR spectra were recorded in DMSO-d₆ on Bruker ARX-600 NMR and Bruker ARX-400 NMR spectrometers with TMS as the internal standard. Coupling constants (J) are expressed in hertz (Hz). The chemical shifts (δ) of NMR are reported in parts per million (ppm). Liquid chromatography-mass spectrometry (LC-MS) (Agilent, Palo Alto, CA, USA) or high-resolution accurate mass spectrometry (HRMS) (Agilent, Q-TOF6550) was used in the determination of the structure of all the final compounds. Reverse-phase high-performance liquid chromatography (HPLC, SHIMADAZU SPD-20A) was performed to measure the purities of the target compounds (the flow rate was 1.0 mL min⁻¹ and the eluents were 100% methanol and purified water).

5.1 Chemistry

5.1.1 General procedure for the synthesis of intermediate 8. Yield: 82.4%, 32.5 g, faint yellow solid. 5-Bromo-1H-pyrazolo [3,4-b]pyridine (7, 24 g, 121.20 mmol) and N-iodosuccinimide (133.00 mmol) were dissolved by DMF, and then heated at 60 °C and stirred for 12 h. The mixture was cooled to room temperature and poured into water, resulting in the formation of a faint yellow precipitate, which was filtered under reduced pressure to obtain 8 as a faint yellow solid and used in next step without additional purification.

5.1.2 General procedure for the synthesis of intermediate 9. Yield: 50.2%, 20.65 g, white solid. A solution of 8 (30 g, 92.90 mmol) in dry DMF was cooled to 0 °C and sodium hydride (111.48 mmol) was added to the mixture slowly, and after stirring for 2 h, 4-methoxy-benzyl chloride (102.19 mmol) was added, the flask moved to room temperature and stirred for 4 h. Then the mixture was poured in ice water and a faint yellow precipitate was formed. Ammonia chloride was used to adjust the pH to 7, then filtered under reduced pressure to obtain a faint yellow solid, stirred and washed with MeOH : EA = 1 : 1, and then filtered under reduced pressure. The crude solid was recrystallized using 200 mL DMF at 120 °C to obtain 9.

5.1.3 General procedure for the synthesis of intermediate 12. Yield: 82.3%, 6.19 g, brown oil. Morphine (26.69 mmol), 1-hydroxybenzotriazole (29.11 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (29.11 mmol) and N,N-diisopropylethylamine (48.51 mmol) were dissolved in DMF and stirred at room temperature. Then 3-aminobenzoic acid (5.0 g, 25.00 mmol) in DMF was added dropwise and stirred at room temperature for 2 h, and the resulting mixture was poured into water and extracted with ethyl acetate (250 mL × 3). The organic layer was washed with saturated ammonium chloride solution (100 mL × 2) and saturated brine (100 mL × 2) and dried using anhydrous sodium sulfate. Then the organic layer was concentrated in vacuo to give 12.

5.1.4 General procedure for the synthesis of intermediate 13. Yield: 48.5%, 3.99 g, yellow solid. A mixture of 9 (7 g, 15.76 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethyl-xanthene (1.57 mmol), 12 (18.91 mmol) and Cs₂CO₃ (31.53 mmol) in dry 1,4-dioxane was degassed with argon for 10 min, Pd₂(dba)₃ (1.57 mmol) was added, and the mixture was degassed again for 10 min. The resulting mixture was heated at 90 °C under an argon atmosphere for 8 h. Then the mixture was concentrated in vacuo and purified by silica chromatography (DCM : MeOH = 150 : 1) to obtain 13.

5.1.5 General procedure for the synthesis of intermediate 14. A solution of 13 (200 mg, 0.38 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (0.96 mmol) and KOAc (1.15 mmol) in dry 1,4-dioxane (10 mL) was degassed with argon for 10 min, then the Pd(dppf)Cl₂ (0.019 mmol) was added under an argon atmosphere, degassed again for 10 min and heated at 100 °C under an argon atmosphere for 4 h. Then it was cooled to room temperature and used for next step without additional purification.

5.1.6 General procedure for the synthesis of intermediate 15a–15o. Yield: 30.3–34.7%, yellow solid. 2 mL water, Cs₂CO₃ (1.15 mmol), substituted bromobenzyl (1.15 mmol) and Pd(PPh₃)₄ (0.019 mmol) were added to the reaction mixture of 14, degassed with argon for 10 min, and heated at 80 °C under an argon atmosphere for 2 h. Then the mixture was concentrated in vacuo and the resulting suspension was redissolved in DCM and MeOH, filtered under reduced pressure and the filtrate was concentrated to get the crude product, which was purified by silica chromatography (DCM : MeOH = 150 : 1) to give 15a–15o.

5.1.7 General procedure for the synthesis of target compounds A01–A15. 15a–15o was dissolved in 5 mL CF₃COOH, heated at 60 °C for 8 h. The mixture was concentrated in vacuo, 1 mL MeOH was added, and the suspension was adjusted to pH = 8 using saturated sodium bicarbonate, filtered under reduced pressure and the filter cake was washed with water. Then the residue was stirred and washed with MeOH to obtain A01–A15.
5.1.7.1 (3-((5-(3-Fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A01). Yield: 49.9%, 30 mg, faint yellow solid. m.p. 208.5–209.6 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.55 (s, 1H), 9.19 (s, 1H), 8.45 (d, J = 2.0 Hz, 1H), 8.18 (d, J = 1.8 Hz, 1H), 7.80(t, J = 3.2, J = 1.6, 1H), 7.61 (dd, J = 8, J = 1.6, 1H), 7.30–7.39 (m, 2H), 7.11 (dd, J = 8.8, J = 1.2, 2H), 7.06–7.01 (m, 1H), 6.83 (dt, J = 7.4, 1.3 Hz, 1H), 4.11 (s, 2H), 3.60–3.57 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.94, 163.61, 161.99, 151.25, 151.00, 144.79 (d, J = 7.6 Hz), 143.81, 143.06, 136.57, 130.98 (d, J = 8.8 Hz), 129.29 (d, J = 3.3 Hz), 127.68, 125.26 (d, J = 3.2 Hz), 117.93, 117.31, 115.87 (d, J = 20.5 Hz), 114.69, 113.44 (d, J = 21.3 Hz), 106.94, 66.68 (2C), 38.06. HRMS (ESI, m/z) calcd for C₂₄H₂₃FN₅O₂ [M + H]⁺, 432.1836; found 432.1836. HPLC purity (MeOH : H₂O = 80 : 20): 99.6%, t_R = 12 min.
5.1.7.2 (3-((5-Benzyl-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A02). Yield: 51.4%, 18 mg, faint yellow solid. m.p.: 190.3–191.2 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.70 (s, 0.2H), 12.54 (s, 0.8H), 9.44 (s, 0.2H), 9.25 (s, 0.8H), 8.80 (s, 0.2H), 8.75 (s, 0.2H), 8.43 (s, 0.8H), 8.21 (s, 0.8H), 7.89 (s, 0.2H), 7.81 (s, 0.8H), 7.75–7.68 (m, 0.8H), 7.62 (d, J = 8.3 Hz, 0.8H), 7.54–7.51 (m, 0.4H),7.41–7.39 (m, 0.2H), 7.36 (d, J = 7.8 Hz, 0.2H), 7.33 (s, 0.4H), 7.31 (s, 0.8H), 7.30 (s, 0.8H), 7.28 (s, 0.8H), 7.26 (s, 0.8H), 7.20 (t, J = 7.3 Hz, 1H), 6.87 (d, J = 7.8 Hz, 0.2H), 6.83 (d, J = 7.5 Hz, 0.8H), 4.23–4.08 (m, 2H), 3.60 (s, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.95, 151.21, 151.01, 143.80, 143.09, 141.87, 136.54, 129.64, 129.11 (2C), 129.08 (2C), 128.26, 127.19, 126.62, 117.89, 117.30, 114.67, 106.94, 66.68 (2C), 38.54. Ms (ESI) m/z 413.2, [M + H^]+ 414.2.
5.1.7.3 (3-((5-(3-Chlorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A03). Yield: 52.1%, 22 mg, faint yellow solid. m.p.: 195.9–197.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.58 (s, 1H), 9.23 (s, 1H), 8.46 (d, J = 1.8, 1H), 8.18 (s, 1H), 7.81 (s, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.36–7.31 (m, 3H), 7.28 (d, J = 7.7 Hz, 1H), 7.25 (d, J = 7.2, 1H), 6.84 (d, J = 7.2 Hz, 1H), 4.11 (s, 2H), 3.62–3.39 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.93, 151.25, 151.01, 144.47, 143.81, 143.05, 136.57, 133.65, 130.95, 129.30, 128.95, 127.90, 127.64, 126.66, 117.93, 117.31, 114.70, 106.94, 66.68 (2C), 37.94. Ms (ESI) m/z 447.1, [M + H]⁺ 448.1.
5.1.7.4 (3-((5-(3-Bromobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A04). Yield: 49.1%, 25 mg, faint yellow solid. m.p.: 187.8–189.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.57 (s, 1H), 9.23 (s, 1H), 8.45 (d, J = 1.9 Hz, 1H), 8.17 (d, J = 1.7 Hz, 1H), 7.81 (t, J = 1.8, 1H), 7.61 (dd, J = 7.8, J = 1.2, 1H), 7.50 (s, 1H), 7.41 (dq, J = 5.6, 3.6, 2.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.30–7.23 (m, 2H), 6.83 (d, J = 7.8 Hz, 1H), 4.10 (s, 2H), 3.62 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.94, 151.25, 151.00, 144.78, 143.81, 143.05, 136.56, 131.81, 131.26, 129.55, 129.31 (2C), 128.29, 127.64, 122.36, 117.93, 117.32, 114.70, 106.94, 66.68 (2C), 65.39 (2C), 37.91. Ms (ESI) m/z 492.0, [M + H]⁺ 493.0.
5.1.7.5 Morpholino(3-((5-(3-nitrobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)methanone (A05). Yield: 51.2%,31 mg, faint yellow solid. m.p.: 221.3–222.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.60 (s, 1H), 9.25 (s, 1H), 8.50 (d, J = 1.8 Hz, 1H), 8.22 (d, J = 2.2 Hz, 1H), 8.16 (t, J = 2.0 Hz, 1H), 8.09 (dd, J = 8.4, J = 1.8, 1H), 7.81 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.64–7.60 (m, 2H), 7.32 (t, J = 7.8 Hz, 1H), 6.83 (m, 1H), 4.26 (s, 2H), 3.62–3.38 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.93, 151.27, 151.06, 148.44, 144.23, 143.84, 143.04, 136.56, 136.08, 130.62, 129.52, 129.29, 127.23, 123.66, 121.78, 117.94, 117.32, 114.70, 106.97, 66.68 (2C), 37.74. Ms (ESI) m/z 458.2, [M + H]⁺ 459.1.
5.1.7.6 Morpholino(3-((5-(3-(trifluoromethyl)benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)methanone (A06). Yield: 50.1%, 25 mg, faint yellow solid. m.p.: 218.7–219.9 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.59 (s, 1H), 9.25 (s, 1H), 8.48 (d, J = 2.0 Hz, 1H), 8.20 (s, 1H), 7.81 (t, J = 1.9 Hz, 1H), 7.66 (s, 1H), 7.62–7.55 (m, 4H), 7.32 (t, J = 7.8 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1H), 4.21 (s, 2H), 3.66–3.40 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.93, 151.25, 151.06, 143.82, 143.40, 143.04, 136.56, 133.38, 130.16 (2C), 129.86 (d, J = 31.2 Hz), 129.40 (d, J = 16.2 Hz), 127.51, 125.56 (m), 123.60 (m), 117.93, 117.31, 114.70, 106.95, 66.68 (2C), 37.98. Ms (ESI) m/z 481.2, [M + H]⁺ 482.1.
5.1.7.7 (3-((5-(2-Fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A07). Yield: 49.6%, 35 mg, faint yellow solid. m.p.: 287.7–288.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.56 (s, 1H), 9.24 (s, 1H), 8.44 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.81 (t, J = 1.9 Hz, 1H), 7.62 (dd, J = 8.2, 2.3 Hz, 1H), 7.37 (td, J = 7.7, 1.9 Hz, 1H), 7.33–7.28 (m, 2H), 7.20–7.16 (m, 2H), 6.83 (d, J = 7.8 Hz, 1H), 4.11 (s, 2H), 3.66–3.61 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.94, 161.64, 160.02, 151.23, 150.85, 143.77, 143.06, 136.55, 131.64 (d, J = 4.4 Hz), 129.28, 129.04 (d, J = 10.0 Hz), 128.45 (d, J = 15.6 Hz), 126.89, 125.15, 117.91, 117.32, 115.87 (d, J = 21.8 Hz), 114.71, 106.85, 66.68 (2C), 31.82. Ms (ESI) m/z 431.2, [M + H]⁺ 432.1.
5.1.7.8 (3-((5-(4-Fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A08). Yield: 50.5%, 37 mg, faint yellow solid. m.p.: 194.5–195.9 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.56 (s, 1H), 9.21 (s, 1H), 8.44 (d, J = 1.8, 1H), 8.15 (s, 1H), 7.81 (s, 1H), 7.61 (dd, J = 8.1, 2.3 Hz, 1H), 7.33–7.29 (m, 3H), 7.16–7.13 (m, 2H), 6.83 (d, J = 7.2 Hz, 1H), 4.08 (s, 2H), 3.61 (s, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.93, 162.08, 160.48, 151.22, 150.99, 143.78, 143.07, 138.00 (d, J = 2.8 Hz), 136.56, 130.95 (d, J = 7.9 Hz, 2C), 129.30, 129.16, 128.19, 117.91, 117.30, 115.84 (d, J = 21.3 Hz, 2C), 114.67, 66.68 (2C), 37.56. Ms (ESI) m/z 431.2, [M + H]⁺ 432.2.
5.1.7.9 (3-((5-(2,3-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A09). Yield: 51.3%, 40 mg, faint yellow solid. m.p.: 151.3–152.3 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.59 (s, 1H), 9.24 (s, 1H), 8.47 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.82 (t, J = 1.9 Hz, 1H), 7.62 (ddd, J = 8.3, 2.4, 1.0 Hz, 1H), 7.34–7.30 (m, 2H), 7.20–7.18 (m, 2H), 6.84 (dt, J = 7.8, 1.8 Hz 1H), 4.17 (s, 2H), 3.51 (m, 8H). ¹³C NMR (151 MHz, DMSO) δ 169.93, 151.23, 151.16 (dd, J = 245.3, 12.4 Hz) 150.83, 149.36, (dd, J = 245.3, 12.4 Hz), 143.81, 143.00, 136.56, 131.15 (d, J = 12.4 Hz), 129.29, 129.14, 126.76 (t, J = 3.3 Hz), 126.32, 125.48 (dd, J = 6.7, 4.6 Hz), 117.96, 117.34, 116.19 (d, J = 16.8 Hz), 114.73, 106.85, 66.68 (2C), 31.53. Ms (ESI) m/z 449.2, [M + H]⁺ 450.1.
5.1.7.10 (3-((5-(2,4-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A10). Yield: 52.3%, 30 mg, faint yellow solid. m.p.: 212.4–213.2 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.57 (s, 1H), 9.23 (s, 1H), 8.44 (s, 1H), 8.15 (s, 1H), 7.81 (s, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.43 (q, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.26–7.22 (m, 1H), 7.09–7.06 (m, 1H), 6.84 (m, 1H), 4.09 (s, 2H), 3.66–3.62 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.94, 162.45 (dd, J = 128.9, 12.4 Hz), 160.83 (dd, J = 131.0, 12.3 Hz), 151.22, 150.83, 143.79, 143.03, 136.56, 132.63 (dd, J = 9.8, 6.1 Hz), 129.29, 128.99, 126.73, 124.81 (dd, J = 15.7, 3.9 Hz), 117.94, 117.33, 114.73, 112.17 (dd, J = 21.0, 3.7 Hz), 106.83, 104.60 (t, J = 26.0 Hz), 66.68 (2C), 31.26. Ms (ESI) m/z 449.2, [M + H]⁺ 450.1.
5.1.7.11 (3-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A11). Yield: 50.1%, 27 mg, faint yellow solid. m.p.: 207.0–208.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.58 (s, 1H), 9.24 (s, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.82 (t, J = 1.9 Hz, 1H), 7.62 (dd, J = 8.3, 2.4 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.29–7.23 (m, 2H), 7.16–7.12 (m, 1H), 6.83 (d, J = 7.2 Hz, 1H), 4.10 (s, 2H), 3.66–3.39 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.93, 159.43, 157.84 (d, J = 5.2 Hz), 156.22, 151.23, 150.84, 143.81, 143.01, 136.56, 130.59 (dd, J = 18.3, 7.9 Hz), 129.29, 129.10, 126.32, 118.01 (m), 117.85 (dd, J = 58.9, 6.8 Hz), 117.48 (m), 115.41 (dd, J = 23.8, 8.9 Hz), 114.73, 106.84, 66.68 (2C), 31.82. HRMS (ESI, m/z) calcd for C₂₄H₂₂F₂N₅O₂ [M + H]⁺, 450.1742; found 450.1737. HPLC purity (MeOH : H₂O = 80 : 20): 99.5%, t_R = 11 min.
5.1.7.12 (3-((5-(2,6-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A12). Yield: 49.9%, 35 mg, yellow solid. m.p.: 206.2–207.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.87 (m, 0.23H), 12.59 (s, 0.77H), 9.43 (m, 0.15H), 9.29 (s, 0.77H), 9.26 (m, 0.08H), 8.82 (m, 0.08H), 8.74 (m, 0.15H), 8.54 (d, J = 2.4 Hz, 0.08H), 8.44 (d, J = 2.1 Hz, 0.77H), 8.30 (d, J = 2.7 Hz, 0.08H), 8.19 (d, J = 2.1 Hz, 1H), 8.14 (m, 0.08H), 7.90 (m, 0.08H), 7.85(m, 1H), 7.76 (m, 0.17H), 7.70 (m, 0.08H), 7.65 (m, 1H), 7.56 (m, 0.23H), 7.38 (tt, J = 8.1, 6.5 Hz, 1H), 7.32 (t, J = 7.8 Hz, 0.77H), 7.22 (m, 0.23H), 7.15 (t, J = 7.8 Hz, 1.54H), 6.88 (d, J = 6.96 Hz, 0.23H), 6.84 (dt, J = 7.5, 1.3 Hz, 0.77H), 5.33 (m, 0.08H), 5.19 (s, 0.15H), 4.12 (s, 1.54H), 3.51 (m, 8H). Ms (ESI) m/z 449.2, [M + H]⁺ 450.1.
5.1.7.13 (3-((5-(3,4-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A13). Yield: 49.5%, 32 mg, faint yellow solid. m.p.: 216.9–217.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.58 (s, 1H), 9.20 (s, 1H), 8.45 (d, J = 2.1 Hz, 1H), 8.15 (d, J = 2.1 Hz, 1H), 7.81 (t, J = 1.9 Hz, 1H), 7.61 (dd, J = 8.2, 2.4 Hz, 1H), 7.40–7.35 (m, 2H), 7.32 (t, J = 7.9 Hz, 1H), 7.12–7.10 (m, 1H), 6.83 (d, J = 7.8 Hz, 1H), 4.09 (s, 2H), 3.74–3.61 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.94, 151.26, 150.97, 150.67 (d, J = 12.6 Hz), 149.39 (dd, J = 53.0, 12.4 Hz), 147.78 (d, J = 12.4 Hz), 143.81, 143.06, 139.64 (t, J = 4.7 Hz), 136.57, 129.30, 129.23, 127.60, 125.86, 118.12 (d, J = 10.5 Hz), 118.01(d, J = 11.7 Hz), 117.32, 114.70, 106.94, 66.68 (2C), 37.41. Ms (ESI) m/z 449.2, [M + H]⁺ 450.1.
5.1.7.14 (3-((5-(3,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (A14). Yield: 48.5%, 22 mg, faint yellow solid. m.p.: 218.1–219.2 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.57 (s, 1H), 9.21 (s, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.19 (d, J = 2.1 Hz, 1H), 7.81 (t, J = 1.9 Hz, 1H), 7.62 (dd, J = 8.2, 2.3 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 7.10–7.05 (m, 1H), 7.05–7.00 (m, 2H), 6.83 (dt, J = 7.5, 1.2 Hz, 1H), 4.12 (s, 2H), 3.60–3.44 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.94, 163.82 (d, J = 13.5 Hz), 162.18 (d, J = 12.7 Hz), 151.24, 150.94, 146.55, 146.49 (t, J = 9.0 Hz), 143.03, 136.56, 129.42 (d, J = 17.8 Hz), 127.08, 117.95, 117.33, 114.70, 112.39 (dd, J = 19.7, 4.9 Hz), 106.97, 102.37 (t, J = 25.9 Hz), 66.68 (2C), 55.38 (2C), 37.89. Ms (ESI) m/z 449.2, [M + H]⁺ 450.1. HPLC purity (MeOH : H₂O = 80 : 20): 99.0%, t_R = 11 min.
5.1.7.15 Morpholino(3-((5-(3,4,5-trifluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)methanone (A15). Yield: 49.7%, 20 mg, faint yellow solid. m.p.: 229.2–231.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.60 (s, 1H), 9.20 (s, 1H), 8.46 (s, 1H), 8.15 (s, 1H), 7.81 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.27 (t, J = 7.8 Hz, 2H), 6.84 (d, J = 7.8 Hz, 1H), 4.10 (s, 2H), 3.66–3.61 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.93, 151.53 (dd, J = 9.9, 4.0 Hz), 151.28, 150.95, 149.89 (dd, J = 10.1, 3.2 Hz), 143.83, 143.04, 139.26 (m), 138.74 (dt, J = 247.0, 15.6 Hz), 138.64, 138.54, 137.11, 137.00, 136.90, 136.58, 129.31 (2C), 127.00, 117.95, 117.32, 114.70, 113.80 (d, J = 4.0 Hz), 113.69 (d, J = 4.0 Hz), 106.94, 66.68 (2C), 55.77 (2C), 37.43. Ms (ESI) m/z 467.2, [M + H]⁺ 468.1. HPLC purity (MeOH : H₂O = 80 : 20): 95.4%, t_R = 10 min.

5.1.8 General procedure for the synthesis of intermediate 16. Yield: 65.9%, 1.79 g, yellow solid. 9 (2.5 g, 5.19 mmol) and ethyl 3-aminobenzoate (5.71 mmol) were dissolved in dry DMF, and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.51 mmol) and Cs₂CO₃ (10.38 mmol) were added, degassed with argon for 10 min. Then Pd₂(dba)₃ (0.26 mmol) was added under an argon atmosphere and degassed again for 10 min and heated at 120 °C under argon atmosphere for 8 h. The mixture was cooled to room temperature and poured into water. Then it was filtered under reduced pressure and the crude solid was purified by silica chromatography (dichloromethane) and stirred and washed using MeOH to give 16.

5.1.9 General procedure for the synthesis of intermediate 17. 4,4,4′,4′,5,5,5′,5′-Octamethyl-2,2′-bi(1,3,2-dioxaborolane) (6.23 mmol), Cs₂CO₃ (6.23 mmol) and 16 (1.5 g, 3.12 mmol) were dissolved in dry 1,4-dioxane (50 mL) and degassed with argon for 10 min, and Pd(dppf)Cl₂ (0.16 mmol) was added under an argon atmosphere and degassed again for 10 min. Then it was heated at 100 °C under an argon atmosphere for 4 h to produce 17 as a crude product, and then used in the next step without additional purification.

5.1.10 General procedure for the synthesis of intermediate 18. Yield: 48.6%, 800 mg, yellow oil. 10 mL water, Cs₂CO₃ (3.12 mmol), 2,5-difluorobenzyl bromide (9.35 mmol) and Pd(PPh₃)₄ (0.16 mmol) were added to a mixture of 17 and degassed for 10 min and heated at 80 °C under an argon atmosphere for 4 h. Then the mixture was cooled to room temperature and concentrated in vacuo and purified through silica chromatography (PE : EA = 5 : 1).

5.1.11 General procedure for the synthesis of intermediate 19. 18 (600 mg, 1.14 mmol) was dissolved in 10 mL CF₃COOH and heated at 60 °C for 5 h. Then the mixture was concentrated in vacuo and used for the next step without additional purification.

5.1.12 General procedure for the synthesis of intermediate 20. Yield: 65.7%, 284 mg, brown solid. 19 was dissolved in 9 mL MeOH and 3 mL H₂O and NaOH (2.0 g) were added to the suspension and heated at 60 °C for 4 h. Then the mixture was cooled to room temperature and poured into water. The solution was washed with EA and the aqueous phase was adjusted to pH = 4 with hydrochloric acid and a brown precipitate was formed, which was filtered under reduced pressure and the filter cake was washed with water, and the residue was dried in a blast dryer.

5.1.13 General procedure for the synthesis of intermediate 26. The synthesis of intermediate 26 was similar to the synthesis of intermediate 20.

5.1.14 General procedure for the synthesis of B01–B13. A mixture of 20(26) (40 mg, 0.11 mmol) and PyBop (0.21 mmol) in dry THF was cooled to 0 °C, and N,N-diisopropylethylamine (0.32 mmol) was added and stirred for 1 h. A solution of N-methylpiperazine (0.21 mmol) in dry THF was added dropwise and stirred for 1 h. Then the mixture was poured into water and extracted with EA (10 mL × 3) and the organic layer was washed with saturated ammonium chloride solution (10 mL) and purified by silica chromatography (DCM : MeOH = 50 : 1). If the compound was protected by Boc group, then the solid was treated with 4 N HCl/EA at room temperature for 1 h. The mixture was concentrated in vacuo, and then the oil was dissolved in water and saturated sodium bicarbonate solution was added to adjust the pH = 8. Subsequently, it was extracted with EA (10 mL × 3), dried with anhydrous sodium sulfate, and finally the solvent was removed to get the product.
5.1.14.1 (3-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(4-methylpiperazin-1-yl)methanone (B01). Yield: 51.4%, 25 mg, white solid. m.p.: 239.1–241.4 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.58 (s, 1H), 9.22 (s, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.77 (t, J = 2.0 Hz, 1H), 7.63 (dd, J = 8.2, 2.3 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.29–7.23 (m, 2H), 7.13 (ddd, J = 8.7, 6.4, 3.6 Hz, 1H), 6.80 (dt, J = 7.4, 1.3 Hz, 1H), 4.10 (s, 2H), 3.61 (s, 2H), 3.33 (s, 2H), 2.35–2.28 (m, 4H), 2.19 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.81, 159.43 (d, J = 240.6 Hz), 157.80 (d, J = 240.0 Hz), 151.24, 150.83, 143.82, 142.99, 136.99, 130.59 (dd, J = 18.3, 7.9 Hz), 129.28, 129.11, 126.32, 118.00 (dd, J = 24.5, 5.1 Hz) 117.81, 117.46 (dd, J = 25.2, 8.8 Hz), 117.18, 115.39, 115.28 (dd, J = 24.2, 8.5 Hz), 114.58, 106.86, 55.38–54.83 (m, 2C), 49.08 (2C), 46.07, 31.83. HRMS (ESI, m/z) calcd for C₂₅H₂₅F₂N₆O [M + H]⁺, 463.2058; found 463.2069. HPLC purity (MeOH : H₂O = 80 : 20): 98.1%, t_R = 11 min.
5.1.14.2 (3-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(4-hydroxypiperidin-1-yl)methanone (B02). Yield: 55.4%, 27 mg, white solid. m.p.: 169.9–171.1 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.57 (s, 1H), 9.23 (s, 1H), 8.46 (d, J = 1.8, 1H), 8.19 (s, 1H), 7.76 (s, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.32–7.23 (m, 3H), 7.14 (tt, J = 8.3, 3.6 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 4.80 (d, J = 3.7 Hz, 1H), 4.10 (s, 2H), 4.02 (s, 1H), 3.75–3.73 (m, 1H), 3.55 (s, 1H), 3.20–3.16 (m, 2H), 2.02–1.70 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.77, 159.42(d, J = 240.2 Hz), 157.80 (d, J = 240.7 Hz), 151.22, 150.82, 143.84, 142.96, 137.44, 130.60 (dd, J = 18.3, 7.9 Hz), 129.26, 129.14, 126.29, 118.01 (dd, J = 24.2, 4.8 Hz), 117.54, 117.47 (dd, J = 24.9, 8.9 Hz), 117.01, 115.40 (dd, J = 23.8, 8.9 Hz), 114.31, 106.85, 66.04, 45.12 (2C), 31.82. Ms (ESI) m/z 463.2, [M + H]⁺ 464.1.
5.1.14.3 (3-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(3-hydroxypiperidin-1-yl)methanone (B03). Yield: 47.2%, 23 mg, white solid. m.p.: 197.5–199.2 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.57 (s, 1H), 9.25 (s, 1H), 8.45 (s, 1H), 8.21 (s, 1H), 8.16 (d, J = 8.5 Hz, 1H), 7.99 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 7.27–7.24 (m, 2H), 7.15–7.12 (m, 1H), 4.10 (s, 2H), 3.82–3.78 (m, 1H), 3.00 (m, 4H), 2.96–2.94 (m, 1H), 1.73–1.71 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 170.16, 159.43 (d, J = 240.0 Hz), 157.82 (d, J = 243.6 Hz), 151.23, 150.81, 143.85, 142.96, 137.52, 130.60 (dd, J = 18.4, 7.7 Hz), 129.15 (2C), 126.30, 118.00 (dd, J = 24.3, 4.9 Hz), 117.59, 117.46 (dd, J = 24.9, 9.0 Hz), 116.97, 115.39 (dd, J = 24.1, 8.6 Hz), 114.51 (d, J = 45.9 Hz), 106.87, 65.74–65.58 (m), 49.13, 47.64, 31.82, 27.29, 23.94. Ms (ESI) m/z 463.2, [M + H]⁺ 464.1.
5.1.14.4 (3-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(piperazin-1-yl)methanone (B04). Yield: 48.7%, 23 mg, faint yellow solid. m.p.: 234.9–236.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.58 (s, 1H), 9.24 (s, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.19 (d, J = 2.1 Hz, 1H), 7.77 (t, J = 1.9 Hz, 1H), 7.62 (dd, J = 8.2, 2.3 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.28–7.23 (m 2H), 7.13 (tt, J = 8.2, 3.6 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 4.10 (s, 2H), 3.54 (s, 2H), 3.28–3.26 (m, 2H), 2.72–2.64 (m, 4H), 2.43 (s, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.79, 159.42 (d, J = 240.7 Hz), 157.80 (d, J = 240.8 Hz), 151.23, 150.82, 143.84, 142.97, 137.25, 130.60 (dd, J = 18.9, 7.9 Hz), 129.25, 129.15, 126.30, 118.00 (dd, J = 24.3, 5.1 Hz), 117.77, 117.46 (dd, J = 24.9, 9.0 Hz), 117.05, 115.39 (dd, J = 23.9, 8.2 Hz), 114.54, 106.86, 46.51, 46.34, 46.31, 46.03, 31.82. HRMS (ESI, m/z) calcd for C₂₄H₂₃F₂N₆O [M + H]⁺, 449.1901; found 449.1900. HPLC purity (MeOH : H₂O = 85 : 15): 94.5%, t_R = 15 min.
5.1.14.5 (4-Aminopiperidin-1-yl)(3-((5-(2,5-difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)methanone (B05). Yield: 41.1%, 20 mg, faint yellow solid. m.p.: 163.8–164.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.58 (s, 1H), 9.22 (s, 1H), 8.46 (s, 1H), 8.18 (s, 1H), 7.76 (s, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.31–7.25 (m, 3H), 7.14 (s, 1H), 6.78 (d, J = 7.5 Hz, 1H), 4.28 (s, 1H), 4.10 (s, 2H), 3.61–3.33 (m, 4H), 1.99–1.66 (m, 4H). Ms (ESI) m/z 462.2, [M + H]⁺ 463.2.
5.1.14.6 3-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)-N-(piperidin-4-yl)benzamide (B06). Yield: 51.4%, 25 mg, faint yellow solid. m.p.: 173.4–174.5 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.57 (s, 1H), 9.25 (s, 1H), 8.45 (s, 1H), 8.21 (s, 1H), 8.16 (d, J = 8.5 Hz, 1H), 7.99 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.27–7.23 (m, 3H), 7.15–7.12 (m, 1H), 4.10 (s, 2H), 3.81 (s, 1H), 3.01 (s, 4H), 1.72 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 166.47, 159.43 (d, J = 239.4 Hz), 157.81 (d, J = 240.5 Hz), 151.27, 150.74, 143.95, 142.96, 136.26, 130.60 (dd, J = 18.8, 7.7 Hz), 129.20, 128.96, 126.24, 118.41, 118.11, 118.00 (dd, J = 24.4, 5.0 Hz), 117.46 (dd, J = 24.9, 9.0 Hz), 115.91, 115.39 (dd, J = 23.9, 8.3 Hz), 106.91, 46.34, 46.31, 45.82, 31.8, 26.41, 26.36. Ms (ESI) m/z 462.2, [M + H]⁺ 463.1.
5.1.14.7 (4-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(morpholino)methanone (B07). Yield: 46.6%, 22 mg, faint yellow solid. m.p.: 227.5–229.4 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.65 (s, 1H), 9.36 (s, 0.87 H),8.99 (s, 0.13 H), 8.70 (s, 0.13 H), 8.47 (s, 0.87 H), 8.20 (s, 0.87 H), 8.12 (s, 0.13 H), 7.83 (d, J = 8.4 Hz, 0.26H), 7.69 (d, J = 8.3 Hz, 1.74H), 7.44 (d, J = 7.8 Hz, 0.26 H), 7.37 (d, J = 8.3 Hz, 1.74 H), 7.26 (m, 2H), 7.13 (tt, J = 8.2, 3.5 Hz, 1H), 4.11 (s, 2H), 3.60–3.51 (m, 8H). Ms (ESI) m/z 449.2, [M + H]⁺ 450.1.
5.1.14.8 (4-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(4-methylpiperazin-1-yl)methanone (B08). Yield: 55.5%, 27 mg, faint yellow solid. m.p.: 168.7–170.4 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.64 (s, 1H), 9.33 (s, 1H), 8.47 (d, J = 2.1 Hz, 1H), 8.19 (d, J = 2.1 Hz, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.29–7.23 (m, 2H), 7.16–7.12 (m, 1H), 4.11 (s, 2H), 3.51 (s, 4H), 2.33–2.31 (m, 4H), 2.20 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.84, 159.43 (d, J = 240.0 Hz), 157.80 (d, J = 240.1 Hz), 151.23, 150.86, 144.30, 143.56, 130.59 (dd, J = 18.8, 7.9 Hz), 129.15, 129.04 (2C), 126.41, 126.29, 118.01 (dd, J = 24.3, 4.9 Hz), 117.47 (dd, J = 24.8, 9.0 Hz), 115.48 (2C), 115.40 (dd, J = 23.9, 8.9 Hz), 115.34, 115.24, 115.18, 106.92, 55.07 (2C), 49.07 (2C), 46.08, 31.82. Ms (ESI) m/z 462.2, [M + H]⁺ 463.2.
5.1.14.9 (4-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(4-hydroxypiperidin-1-yl)methanone (B09). Yield: 43.1%, 21 mg, faint yellow. m.p.: 171.4–172.3 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.63 (s, 1H), 9.31 (s, 1H), 8.47 (d, J = 2.2 Hz, 1H), 8.19 (d, J = 2.0 Hz, 1H), 7.67 (m, 2H), 7.33 (m, 2H), 7.26 (m, 2H), 7.14 (m, 1H), 4.77 (d, J = 4.0 Hz, 1H), 4.11 (s, 2H), 3.82–3.70 (m, 2H), 3.19–3.15 (m, 2H), 1.74 (s, 2H), 1.38–1.23 (m, 3H). HRMS (ESI, m/z) calcd for C₂₅H₂₄F₂N₅O₂ [M + H]⁺, 464.1898; found 464.1894. HPLC purity (MeOH : H₂O = 80 : 20): 98.6%, t_R = 12 min.
5.1.14.10 (4-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(3-hydroxypiperidin-1-yl)methanone (B10). Yield: 36.9%, 18 mg, faint yellow solid. m.p.: 178.7–179.9 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.75 (s, 0.25H), 12.62 (s, 0.75H), 9.41 (s, 0.25H), 9.30 (s, 0.75H), 8.81 (s, 0.25H), 8.71 (s, 0.25H), 8.47 (s, 0.75H), 8.19 (s, 0.75H), 7,74 (m, 0.5H), 7.67 (d, J = 8.4 Hz, 1.5H), 7.53 (t, J = 7.5 Hz, 0.5H), 7.42 (d, J = 7.2 Hz, 0.25H), 7.38 (d, J = 8.2 Hz, 0.5H), 7.33 (d, J = 8.1 Hz, 1.5H), 7.29–7.24 (m, 1.5H), 7.13 (dq, J = 8.2, 3.9, 3.5 Hz, 0.75H), 4.90 (s, 1H), 4.11 (s, 2H), 3.81–3.48 (m, 3H), 3.09–2.88 (m, 2H), 1.87 (s, 1 H), 1.70 (S, 1H), 1.41–1.38 (m, 2H). HRMS (ESI, m/z) calcd for C₂₅H₂₄F₂N₅O₂ [M + H]⁺, 464.1898; found 464.1893. HPLC purity (MeOH : H₂O = 90 : 10): 96.4%, t_R = 10 min.
5.1.14.11 (4-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)(piperazin-1-yl)methanone (B11). Yield: 48.8%, 23 mg, faint yellow solid. m.p.: 218.8–220.1 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.63 (s, 1H), 9.31 (s, 1H), 8.47 (s, 1H), 8.19 (s, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.26 (m, 2H), 7.14 (m, 1H), 4.11 (s, 2H), 3.43 (s, 4H), 2.68 (s, 4H), 1.23 (s, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.82, 159.42 (d, J = 240.7 Hz), 157.81 (d, J = 242.1 Hz), 151.23, 150.84, 144.16, 143.59, 130.59 (dd, J = 18.6, 7.9 Hz), 129.15, 128.98, 126.59, 126.39, 118.00 (dd, J = 23.9, 3.6 Hz), 117.46 (dd, J = 24.9, 9.1 Hz), 115.48, 115.40 (dd, J = 24.5, 9.4 Hz), 106.92, 46.27 (2C), 31.82. Ms (ESI) m/z 448.2, [M + H]⁺ 449.4.
5.1.14.12 (4-Aminopiperidin-1-yl)(4-((5-(2,5-difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)phenyl)methanone (B12). Yield: 32.9%, 16 mg, faint yellow solid. m.p.: 234.4–235.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.63 (s, 1H), 9.33 (s, 0.8H), 8.99 (s, 0.2H), 8.69 (s, 0.2H), 8.46 (s, 0.8H), 8.20 (s, 0.8H), 8.12 (s, 0.2H), 7.80 (d, J = 8.4 Hz, 0.4H), 7.67 (d, J = 8.2 Hz, 1.6H), 7.37 (d, J = 8.4 Hz, 0.4H), 7.31 (d, J = 8.3 Hz, 1.6H), 7.25 (m, 2H), 7.13 (tt, J = 8.2, 3.5 Hz, 1H), 4.11 (s, 2H), 3.33 (s, 2H), 2.97 (s, 2H), 2.82–2.77 (m, 1H), 1.71 (s, 2H), 1.19–1.14 (m, 2H). Ms (ESI) m/z 462.2, [M + H]⁺ 463.2. HPLC purity (MeOH : H₂O = 85 : 15): 98.9%, t_R = 28 min.
5.1.14.13 4-((5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)amino)-N-(piperidin-4-yl)benzamide (B13). Yield: 28.8%, 14 mg, faint yellow solid. m.p.: 245.6–247.6 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 12.78 (s, 0.2H), 12.66 (s, 0.8H), 9.51 (s, 0.2H), 9.40 (s, 0.8H), 8.82 (s, 0.2H), 8.73 (s, 0.2H), 8.47 (s, 0.8H), 8.21 (s, 0.8H), 7.97 (d, J = 7.8 Hz, 0.4H), 7.85 (d, J = 8.4 Hz, 0.4H), 7.80 (d, J = 8.3 Hz, 2H), 7.75–7.72 (m, 1H), 7.67 (d, J = 8.3 Hz, 1.6H), 7.54–7.52 (m, 0.4H), 7.42–7.39 (m, 0.2H), 7.29–7.24 (m, 1.6H), 7.15–7.12 (m, 0.8H), 4.11 (s, 2H), 3.81 (s, 1H), 3.33 (s, 2H), 2.95 (d, J = 11.9 Hz, 2H), 1.99 (m, 0.5H), 1.72–1.70 (m, 2H), 1.43–1.38 (m, 2H). Ms (ESI) m/z 462.2, [M + H]⁺ 463.1. HPLC purity (MeOH : H₂O = 90 : 10): 97.5%, t_R = 25 min.

5.1.15 General procedure for the synthesis of intermediate 28. Yield: 53.7%, 1.69 g, white solid. 9 (3.0 g, 6.76 mmol), (3-(ethoxycarbonyl)phenyl)boronic acid (7.43 mmol), KF (13.5 mmol), and dicyclohexyl(2′,6′-dimethoxy-[1,1′-biphenyl]-2-yl)phosphine (1.35 mmol) were dissolved in 50 mL 1,4-dioxane and 10 mL H₂O and degassed with argon for 10 min, and then Pd(PPh₃)₄ (0.34 mmol) was added under an argon atmosphere, degassed again for 10 min and heated at 100 °C for 24 h. The mixture was concentrated in vacuo, then purified by silica chromatography (dichloromethane) and stirred and washed with MeOH to get 28.

5.1.16 General procedure for the synthesis of intermediate 29. 4,4,4′,4′,5,5,5′,5′-Octamethyl-2,2′-bi(1,3,2-dioxaborolane) (6.43 mmol), KOAc (6.43 mmol), 28 (1.5 g, 3.22 mmol) and 2-(dicyclohexylphosphino)-2′,4′,6′-tri-i-propyl-1,1′-biphenyl (0.64 mmol) were dissolved in dry 1,4-dioxane (40 mL) and degassed with argon for 10 min. Then Pd(OAc)₂ (0.16 mmol) was added under an argon atmosphere, degassed again for 10 min and heated at 100 °C under an argon atmosphere for 14 h to produce 29 as a crude product, which was used in the next step without additional purification.

5.1.17 General procedure for the synthesis of intermediate 30. Yield: 52.3%, 864 mg, white oil. 8 mL water, Cs₂CO₃ (6.43 mmol), 2,5-difluorobenzyl bromide (9.65 mmol) and Pd(PPh₃)₄ (0.32 mmol) were added to a mixture of 29, degassed for 10 min and heated at 100 °C under an argon atmosphere for 7 h. Then the mixture was cooled to room temperature and concentrated in vacuo, and then purified through silica chromatography (PE : EA = 5 : 1).

5.1.18 General procedure for the synthesis of intermediate 31. 30 (864 mg, 1.68 mmol) was dissolved in 10 mL CF₃COOH and heated at 60 °C for 5 h. Then the mixture was concentrated in vacuo and used in next step without additional purification.

5.1.19 General procedure for the synthesis of intermediate 32. Yield: 54.2%, 333 mg, white solid. 31 was dissolved in 9 mL MeOH and 3 mL H₂O, NaOH (2.0 g) was added into the suspension and heated at 60 °C for 4 h. Then the mixture was cooled to room temperature and poured into water. The solution was washed with EA and the aqueous phase was adjusted to pH = 4 with hydrochloric acid, resulting in the formation of a white precipitate, which was filtered under reduced pressure, the filter cake was washed with water and the residue was dried in a blast dryer to obtain 32.

5.1.20 General procedure for the synthesis of intermediate 38. The synthesis of intermediate 38 was similar to the synthesis of intermediate 32.

5.1.21 General procedure for the synthesis of C01–C10. The synthesis of C01–C10 was the same as the synthesis of B01–B13.
5.1.21.1 (3-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(4-methylpiperazin-1-yl)methanone (C01). Yield: 51.0%, 25 mg, white solid. m.p.: 122.3–123.9 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.89 (s, 1H), 8.48 (m, 2H), 8.08 (d, J = 7.8 Hz, 1H), 7.93 (s, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.27–7.21 (m, 2H), 7.12–7.09 (m, 1H), 4.18 (s, 2H), 3.66 (s, 4H), 2.42–2.32 (m, 4H), 2.22 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.11, 159.43 (d, J = 240.0 Hz), 157.67 (d, J = 239.9 Hz), 152.48, 150.56, 142.02, 137.19, 133.79, 130.59 (dd, J = 18.2, 7.9 Hz), 130.01, 129.75, 128.89, 127.93, 127.03, 125.00, 117.85 (dd, J = 23.9, 4.6 Hz), 117.38 (dd, J = 24.9, 9.0 Hz), 115.36 (dd, J = 23.9, 8.9 Hz), 112.35, 46.17 (2C), 45.97 (3C), 31.79. Ms (ESI) m/z 447.2, [M + H]⁺ 448.2.
5.1.21.2 (3-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(4-hydroxypiperidin-1-yl)methanone (C02). Yield: 59.0%, 29 mg, white solid. m.p.: 120.6–121.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.88 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.44 (d, J = 2.0 Hz, 1H), 8.07 (dt, J = 7.8, 1.4 Hz, 1H), 7.92 (t, J = 1.7 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.42 (dt, J = 7.6, 1.4 Hz, 1H), 7.28 (ddd, J = 9.1, 5.9, 3.2 Hz, 1H), 7.23 (td, J = 9.2, 4.6 Hz, 1H), 7.13–7.09 (m, 1H), 4.80 (d, J = 4.0 Hz, 1H), 4.18 (s, 2H), 4.05 (s, 1H), 3.78–3.74 (m, 1H), 3.56 (s, 1H), 3.26–3.16 (m, 2H), 1.83–1.71 (m, 2H), 1.44–1.35 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.05, 159.43 (d, J = 240.6 Hz), 157.69 (d, J = 240.2 Hz), 152.46, 150.54, 142.08, 137.63, 133.77, 130.58 (dd, J = 18.2, 7.9 Hz), 129.99, 129.73, 128.91, 127.78, 126.78, 124.75, 117.89 (dd, J = 24.4, 4.9 Hz), 117.40 (dd, J = 25.2, 8.7 Hz), 115.37 (dd, J = 24.2, 8.5 Hz), 112.35, 65.9, 46.22 (2C), 31.80. Ms (ESI) m/z 448.2, [M + H]⁺ 449.1.
5.1.21.3 (3-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(3-hydroxypiperidin-1-yl)methanone (C03). Yield: 57.0%, 28 mg, white solid. m.p.: 185.0–186.1 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.87 (s, 1H), 8.51 (s, 1H), 8.45 (s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 13.5 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.28 (ddd, J = 9.0, 5.8, 3.3 Hz, 1H), 7.23 (td, J = 9.2, 4.5 Hz, 1H), 7.13–7.09 (m, 1H), 5.01–4.85 (m, 1H), 4.18 (s, 2H), 3.82 (s, 1H), 3.57–3.42 (m, 2H), 3.31–2.87 (m, 2H), 1.90–1.40 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.52 (m), 159.43 (d, J = 240.1 Hz), 157.68 (d, J = 240.2 Hz), 152.46, 150.52, 142.12, 137.71, 133.70, 130.59 (dd, J = 18.3, 7.9 Hz), 130.00, 129.65, 128.91, 127.70, 127.13 (m), 125.18 (m), 117.89 (dd, J = 24.3, 4.9 Hz), 117.38 (dd, J = 24.9, 9.0 Hz), 115.36 (dd, J = 24.2, 8.5 Hz), 112.36, 65.50, 54.41 (0.5C), 49.20 (0.5C), 47.73 (0.5C), 46.20 (0.5C), 33.43 (0.5C), 32.96 (0.5C), 31.81, 23.87 (0.5C), 22.29 (0.5C). HRMS (ESI, m/z) calcd for C₂₅H₂₃F₂N₄O₂ [M + H]⁺, 449.1789; found 449.1785. HPLC purity (MeOH : H₂O = 80 : 20): 98.7%, t_R = 10 min.
5.1.21.4 (3-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(piperazin-1-yl)methanone (C04). Yield: 31.6%, 15 mg, white solid. m.p.: 81.0–82.2 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.89 (s, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.44 (d, J = 2.0 Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 1.9 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.28 (ddd, J = 9.1, 5.9, 3.2 Hz, 1H), 7.24 (dt, J = 9.2, 4.6 Hz, 1H), 7.12 (m, 1H), 4.19 (s, 2H), 3.59 (s, 4H), 2.71 (m, 4H), 2.00 (m, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.08, 159.43 (d, J = 239.5 Hz), 157.68 (d, J = 240.0 Hz), 152.47, 150.54, 142.03, 137.45, 133.78, 130.58 (dd, J = 18.8, 8.1 Hz),129.97, 129.71, 128.92, 127.78, 126.99, 124.97, 117.89 (dd, J = 24.7, 4.6 Hz), 117.39 (dd, J = 24.9, 9.0 Hz), 115.38 (dd, J = 23.9, 8.8 Hz), 112.34, 65.50 (2C), 46.34 (2C), 31.80. Ms (ESI) m/z 433.2, [M + H]⁺ 434.1. HPLC purity (MeOH : H₂O = 90 : 10): 93.7%, t_R = 12 min.
5.1.21.5 (4-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(4-methylpiperazin-1-yl)methanone (C05). Yield: 55.1%, 27 mg, white solid. m.p.: 209.7–210.5 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.90 (s, 1H), 8.54 (d, J = 2.0 Hz, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.28 (ddd, J = 9.2, 5.9, 3.2 Hz, 1H), 7.24 (td, J = 9.3, 4.6 Hz, 1H), 7.13–7.09 (m, 1H), 4.18 (s, 2H), 3.64–3.42 (m, 4H), 2.37 (m, 4H), 2.23 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.14, 159.43 (d, J = 240.1 Hz), 157.66 (d, J = 240.3 Hz), 152.49, 150.54, 142.00, 135.82, 134.73, 130.62 (dd, J = 18.2, 7.8 Hz), 130.20, 128.93, 128.21 (2C), 126.80 (2C), 117.85 (dd, J = 24.3, 5.0 Hz), 117.38 (dd, J = 24.9, 9.0 Hz), 115.35 (dd, J = 24.1, 8.6 Hz), 112.44, 49.20 (2C), 46.00 (2C), 31.82, 27.30 (3C). Ms (ESI) m/z 447.2, [M + H]⁺ 448.1.
5.1.21.6 (4-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(4-hydroxypiperidin-1-yl)methanone (C06). Yield: 40.8%, 20 mg, white solid. m.p.: 155.1–156.8 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.89 (s, 1H), 8.53 (d, J = 2.0 Hz, 1H), 8.50 (d, J = 1.9 Hz, 1H), 8.07 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 7.9 Hz, 2H), 7.27 (ddd, J = 9.4, 5.9, 3.4 Hz, 1H), 7.22 (dd, J = 9.2, 4.5 Hz, 1H), 7.12–7.08 (m, 1H), 4.81 (d, J = 3.6 Hz, 1H), 4.17 (s, 2H), 4.06–4.04 (m, 1H), 3.78–3.73 (m, 2H), 3.22–3.16 (s, 2H), 1.81–1.73 (m, 2H), 1.39–1.34 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.11, 159.43 (d, J = 240.0 Hz), 157.66 (d, J = 239.9 Hz), 152.47, 150.51, 142.08, 136.29, 134.58, 130.63 (dd, J = 18.9, 7.9 Hz), 130.23, 128.91, 127.95 (2C), 126.79 (2C), 117.85 (dd, J = 24.3, 4.9 Hz), 117.37 (dd, J = 24.9, 9.0 Hz), 115.34 (dd, J = 24.2, 8.6 Hz), 112.45, 65.98 (2C), 31.82. Ms (ESI) m/z 448.2, [M + H]⁺ 449.1. HPLC purity (MeOH : H₂O = 80 : 20): 97.8%, t_R = 11 min.
5.1.21.7 (4-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(3-hydroxypiperidin-1-yl)methanone (C07). Yield: 44.8%, 22 mg, white solid. m.p.: 230.9–232.7 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.88 (s, 1H), 8.52 (m, 2H), 8.07 (d, J = 7.9 Hz, 2H), 7.54 (s, 2H), 7.28 (m, 1H), 7.22 (dd, J = 9.2, 4.5 Hz, 1H), 7.10 (m, 1H), 5.01–4.86 (m, 1H), 4.18 (s, 2H), 3.83 (s, 1H), 3.54 (s, 2H), 3.16–2.86 (m, 2H), 1.86–1.78 (m, 2H), 1.45 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.50, 159.43 (d, J = 240.3 Hz), 157.66 (d, J = 240.0 Hz), 152.47, 150.51, 142.09, 136.38, 134.52, 130.64 (dd, J = 18.2, 7.9 Hz), 130.23, 128.92, 128.28, 127.96, 126.73, 117.86 (dd, J = 24.3, 4.9 Hz), 117.38 (dd, J = 24.9, 9.0 Hz), 115.34 (dd, J = 23.9, 8.8 Hz), 112.45, 65.52 (2C), 31.82. Ms (ESI) m/z 448.2, [M + H]⁺ 449.1.
5.1.21.8 (4-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(piperazin-1-yl)methanone (C08). Yield: 31.6%, 15 mg, white solid. m.p.: 107.8–109.2 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.89 (s, 1H), 8.52 (m, 2H), 8.07 (d, J = 7.9 Hz, 2H), 7.53 (d, J = 7.9 Hz, 2H), 7.28–7.25 (m, 1H), 7.23 (td, J = 9.3, 4.5 Hz, 1H), 7.12–7.08 (m, 1H), 4.23–4.20 (m, 1H), 4.17 (s, 2H), 3.57 (s, 4H), 2.74–2.68 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.11, 159.42 (d, J = 240.2 Hz), 157.66 (d, J = 239.9 Hz), 152.49, 150.52, 142.03, 136.10, 134.58, 132.00, 130.63 (dd, J = 18.8, 8.0 Hz), 130.21, 129.14, 128.91, 128.17, 126.78, 117.84 (dd, J = 24.1, 4.7 Hz), 117.37 (dd, J = 24.9, 9.0 Hz), 115.34 (dd, J = 24.4, 8.7 Hz), 112.44, 65.50 (2C), 31.82. HRMS (ESI, m/z) calcd for C₂₄H₂₂F₂N₅O [M + H]⁺, 434.1792; found 434.1788.
5.1.21.9 (R)-(3-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(3-hydroxypiperidin-1-yl)methanone (C09). Yield: 46.9%, 23 mg, white solid. m.p.: 86.5–87.5 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.88 (s, 1H), 8.51 (d, J = 1.9 Hz, 1H), 8.45 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.96–7.34 (m, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.43 (t, J = 7.3 Hz, 1H), 7.27 (ddd, J = 9.1, 5.9, 3.2 Hz, 1H), 7.22 (td, J = 9.2, 4.5 Hz, 1H), 7.10 (tt, J = 8.2, 3.5 Hz, 1H), 5.03–4.874.95 (m, 1H), 4.18 (s, 2H), 3.83–3.82 (m, 1H), 3.58–3.50 (m, 2H), 3.28–2.85 (m, 2H), 1.93–1.42 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.55–169.31 (m), 158.63 (d, J = 240.2 Hz), 156.89 (d, J = 239.6 Hz), 152.47, 150.52, 142.13, 137.69, 133.70, 130.48 (dd, J = 18.2, 7.9 Hz), 129.99, 129.66, 128.92 (2C), 127.72, 117.78 (dd, J = 24.4, 4.9 Hz), 117.25 (dd, J = 24.9, 9.0 Hz), 115.23 (dd, J = 24.2, 8.5 Hz), 112.37 (2C), 65.50, 51.80 (d, J = 784.9 Hz), 44.97 (d, J = 831.7 Hz), 33.18 (d, J = 71.0 Hz), 31.82, 23.05 (d, J = 237.4 Hz). HRMS (ESI, m/z) calcd for C₂₅H₂₃F₂N₄O₂ [M + H]⁺, 449.1789; found 449.1785. HPLC purity (MeOH : H₂O = 80 : 20): 94.0%, t_R = 10 min.
5.1.21.10 (S)-(3-(5-(2,5-Difluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)(3-hydroxypiperidin-1-yl)methanone (C10). Yield: 57.0%, 28 mg, white solid. m.p.: 110.1–111.0 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 13.88 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.45 (s, 1H), 8.07 (dt, J = 7.8, 1.5 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.44–7.41 (m, 1H), 7.27 (ddd, J = 9.1, 5.9, 3.2 Hz, 1H), 7.22 (td, J = 9.2, 4.6 Hz, 1H), 0.10 (ddd, J = 12.1, 8.3, 3.5 Hz, 1H), 5.03–4.87 (m, 1H), 4.18 (s, 2H), 3.82 (s, 1H), 3.59–3.50 (m, 2H), 3.20–2.85 (m, 2H), 1.92–1.41 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.54–169.31 (m), 158.63 (d, J = 240.5 Hz), 156.89 (d, J = 240.0 Hz), 152.46, 150.51, 137.70, 133.71, 130.49 (dd, J = 18.9, 7.9 Hz), 129.99, 129.64, 128.91, 128.29, 127.72, 119.58, 117.78 (dd, J = 23.9, 4.6 Hz), 117.25 (dd, J = 24.9, 9.0 Hz), 115.23 (dd, J = 24.2, 8.5 Hz), 112.37, 110.11, 65.50, 51.81 (d, J = 785.0 Hz), 44.97 (d, J = 831.3 Hz), 33.18 (d, J = 71.1 Hz), 31.81, 23.06 (d, J = 238.6 Hz). HRMS (ESI, m/z) calcd for C₂₅H₂₃F₂N₄O₂ [M + H]⁺, 449.1789; found 449.1786. HPLC purity (MeOH : H₂O = 80 : 20): 97.6%, t_R = 15 min.

5.2 Pharmacological assay

5.2.1 TRKs HTRF assay. The purified kinases were purchased from Carna Biosciences (Japan) and the commercial TK kinEASE kit was purchased from Cisbio Bioassays (France). The HTRF assays were performed according to the manufacturer's instructions. Some optimization experiments were implemented to determine the optimal concentration of kinases. The concentration of TRKA, TRKB and TRKC used was 0.111 ng μL⁻¹, 0.037 ng μL⁻¹ and 0.037 ng μL⁻¹. The concentration of ATP was determined by similar experiments, which was 14.68 μM (TRKA), 4.77 μM (TRKB) and 25.64 μM (TRKC).

Firstly, the compounds were diluted from a concentrated stock of 8 mM in 100% DMSO with serial kinase reaction buffer dilutions. Secondly, a 5 μM TK solution, 0.5 μM XL-665 solution, kinase solution and ATP solution whose concentrations were displayed previously were prepared. For each assay, 4 μL of dispensed compound, 2 μL of ATP, 2 μL of substrate TK and 2 μL of kinase were added to the assay wells. The assay wells were incubated at 25 °C for 30–40 min. The enzymatic reaction time was related to the type of kinase (the reaction time of TRKA was 30 min, while that for TRKB and TRKC was 40 min). Finally, a mixture of 5 μL of XL-665 and 5 μL of TK-antibody-cryptate was added to terminate the reactions. The assay wells were incubated at 25 °C for another 60 min, and then HTRF signals were obtained by reading plates using an Infinite® F500 microplate reader (Tecan, Switzerland). The ratio of fluorescence at 665 nm to fluorescence at 620 nm was calculated for each well. The data were analyzed using GraphPad Prism 8. Every experiment was repeated at least two times.

5.2.2 PLK4, FAK, and PAK4 enzymatic assay. The inhibitory activities of the representative compounds on PLK4, FAK and PAK4 kinases were investigated according to the methods reported by our research group.^30–32

5.2.3 Cell proliferation assay. The antiproliferative activities of the compounds were evaluated against the Km-12, MCF-7, and HUVEC cell lines using the standard Cell Counting Kit-8 (CCK-8) assay in vitro. The cells were seeded in 96 well plates. After incubation for 24 h, the cells were exposed to various concentrations of compounds for an additional 72 h. The IC₅₀ values were calculated by concentration-response curve fitting using GraphPad Prism 8.

5.2.4 Plasma stability assay. The pooled frozen human plasma (BioreclamationIVT, batch no. BRH1569252) was thawed in a water bath at 37 °C prior to the experiment. The plasma was centrifuged at 4000 rpm for 5 min and if any clots were present, they were removed. The pH was adjusted to 7.4 ± 0.1 if required. 100 mM propantheline bromide solution was prepared by diluting 5 μL of 10 mM stock solution with 495 μL H₂O as a positive control. The other detected compound solution was prepared in the same way. 98 μL of blank plasma was spiked with 2 μL of dosing solution (100 μM) to achieve 2 μM of the final concentration in duplicate and the samples were incubated at 37 °C in a water bath. At each time point (0, 10, 30, 60 and 120 min), 400 μL of stop solution (200 ng mL⁻¹ tolbutamide and 200 ng mL⁻¹ labetalol in ACN) was added to precipitate the protein and mixed thoroughly. The sample plates were centrifuged at 4000 rpm for 15 min. An aliquot of supernatant (50 μL) was transferred from each well and mixed with 100 μL ultra-pure water. The samples were shaken at 800 rpm for about 10 min before they were analyzed by LC-MS/MS.

5.2.5 Cytochrome P450 inhibition assay. Cytochrome P450 inhibition was evaluated in human liver microsomes (0.253 mg mL⁻¹) using five probe substrates (CYP1A2, phenacetin; CYP2C9, diclofenac; CYP2C19, S-mephenytoin; CYP2D6, dextromethorphan; and CYP3A4, midazolam) in the presence of compound C03 (10 μM). After preincubation for 10 min at 37 °C, an NADPH-regenerating system was added. After the mixed system was incubated for 15 min at 37 °C, the reaction was stopped by adding 400 μL of cold stop solution (200 ng mL⁻¹ tolbutamide and 200 ng mL⁻¹ labetalol in acetonitrile). Then the incubation mixtures were centrifuged and the supernatants analyzed by LC-MS/MS.

5.3 Molecular docking study

The crystal structure of TRKA (PDB code: 5KVT) downloaded from the Protein Data Bank was processed with the Protein Preparation Wizard in Schrodinger suite. The protein structure was adjusted and modified, followed by the addition of hydrogen atoms, deleting solvent water molecules, and defining right bonds orders using Prime. The protonation and tautomeric states of Asp, Lys, and His were assigned at pH 7.4 state. Afterward, all the hydrogen atoms of the TRKA complexes were optimized with the OPLS_2005 force field, which minimized and converged heavy atoms to an RMSD of 0.3. The selected inhibitors were prepared by using LigPrep from the Schrodinger suite with the OPLS_2005 force field. The structure of the inhibitors was also adjusted and modified, followed by the addition of all hydrogen atoms and checking the bond order and atom types. Receptor grids were generated before docking with the allosteric site determined according to the literature. The prepared protein ligand complex was imported into Glide 9.7, which defined it as the receptor structure with a size box of (15 Å × 15 Å × 15 Å). Based on the OPLS_2005 force field, the grid of the TRKA crystal structure was generated. The extra precision (XP) mode was set for docking studies with the two crucial residues Glu-590 and Met-592 in constrained binding to get accurate results. The ligand entrectinib in co-crystallization was extracted for re-docking to evaluate the accuracy of the docking method. The results showed that the conformation obtained by docking was consistent with that in co-crystallization.

Abbreviations used

DCM	Dichloromethane
DIPEA	N,N-Diisopropylethylamine
DMF	N,N-Dimethylformamide
EA	Ethyl acetate
EDCI	N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
HOBt	1-Hydroxybenzotriazole
MeOH	Methanol
NaH	Sodium hydride
PE	Petroleum ether
THF	Tetrahydrofuran
PMB-Cl	4-Methoxybenzene-1-sulfonyl chloride
PyBop	((1H-Benzo[d][1,2,3]triazol-1-yl)oxy)tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate
NIS	N-Iodosuccinimide

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We gratefully acknowledge the Program for Innovative Research Team of the Ministry of Education and the Program for Liaoning Innovative Research Team in University.

Notes and references

1. E. J. Huang and L. F. Reichardt, Neurotrophins: roles in neuronal development and function, Annu. Rev. Neurosci., 2001, 24, 677–736.
2. M. H. Ultsch, C. Wiesmann, L. C. Simmons, J. Henrich, M. Yang and D. Reilly, et al., Crystal structures of the neurotrophin-binding domain of TrkA, TrkB and TrkC 1 1Edited by I. A. Wilson, J. Mol. Biol., 1999, 290(1), 149–159.
3. C. Wiesmann, M. H. Ultsch, S. H. Bass and A. M. de Vos, Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor, Nature, 1999, 401(6749), 184–188.
4. D. R. Kaplan, B. L. Hempstead, D. Martin-Zanca, M. V. Chao and L. F. Parada, The trk Proto-Oncogene Product: a Signal Transducing Receptor for Nerve Growth Factor, Science, 1991, 252(5005), 554–558.
5. D. R. Kaplan, D. Martin-Zanca and L. F. Parada, Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF, Nature, 1991, 350(6314), 158–160.
6. R. Klein, S. Jing, V. Nanduri, E. O'Rourke and M. Barbacid, The trk proto-oncogene encodes a receptor for nerve growth factor, Cell, 1991, 65(1), 189–197.
7. F. Lamballe, R. Klein and M. Barbacid, trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3, Cell, 1991, 66(5), 967–979.
8. S. P. Squinto, T. N. Stitt, T. H. Aldrich, S. Davis, S. M. Blanco and C. RadzieJewski, et al., trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor, Cell, 1991, 65(5), 885–893.
9. E. C. Yuen and W. C. Mobley, Early BDNF, NT-3, and NT-4 Signaling Events, Exp. Neurol., 1999, 159(1), 297–308.
10. C. Miranda, A. Greco, C. Miele, M. A. Pierotti and E. Van Obberghen, IRS-1 and IRS-2 are recruited by TrkA receptor and oncogenic TRK-T1, J. Cell. Physiol., 2000, 186(1), 35–46.
11. V. Ranzi, S. O. Meakin, C. Miranda, P. Mondellini, M. A. Pierotti and A. Greco, The Signaling Adapters Fibroblast Growth Factor Receptor Substrate 2 and 3 Are Activated by the Thyroid TRK Oncoproteins, Endocrinology, 2003, 144(3), 922–928.
12. E. Roccato, C. Miranda, V. Ranzi, M. Gishizki, M. A. Pierotti and A. Greco, Biological activity of the thyroid TRK-T3 oncogene requires signalling through Shc, Br. J. Cancer, 2002, 87(6), 645–653.
13. K. Bartkowska, A. Paquin, G. AeS, D. R. Kaplan and F. D. Miller, Trk signaling regulates neural precursor cell proliferation and differentiation during cortical development, Development, 2007, 134(24), 4369–4380.
14. W. Yan, N. R. Lakkaniga, F. Carlomagno, M. Santoro, N. Q. McDonald and F. Lv, et al., Insights into Current Tropomyosin Receptor Kinase (TRK) Inhibitors: Development and Clinical Application, J. Med. Chem., 2019, 62(4), 1731–1760.
15. E. Ardini, R. Bosotti, A. L. Borgia, C. De Ponti, A. Somaschini and R. Cammarota, et al., The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition, Mol. Oncol., 2014, 8(8), 1495–1507.
16. R. Wang, H. Hu, Y. Pan, Y. Li, T. Ye and C. Li, et al., RET Fusions Define a Unique Molecular and Clinicopathologic Subtype of Non–Small-Cell Lung Cancer, J. Clin. Oncol., 2012, 30(35), 4352–4359.
17. J. Kim, Y. Lee, H. J. Cho, Y.-E. Lee, J. An and G.-H. Cho, et al., NTRK1 fusion in glioblastoma multiforme, PLoS One, 2014, 9(3), e91940.
18. M. Mourad, T. Jetmore, A. A. Jategaonkar, S. Moubayed, E. Moshier and M. L. Urken, Epidemiological Trends of Head and Neck Cancer in the United States: A SEER Population Study, J. Oral Maxillofac. Surg., 2017, 75(12), 2562–2572.
19. R. C. Doebele, L. E. Davis, A. Vaishnavi, A. T. Le, A. Estrada-Bernal and S. Keysar, et al., An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101, Cancer Discovery, 2015, 5(10), 1049–1057.
20. A. Drilon, T. W. Laetsch, S. Kummar, S. G. DuBois, U. N. Lassen and G. D. Demetri, et al., Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children, N. Engl. J. Med., 2018, 378(8), 731–739.
21. T. W. Laetsch, S. G. DuBois, L. Mascarenhas, B. Turpin, N. Federman and C. M. Albert, et al., Larotrectinib for paediatric solid tumours harbouring NTRK gene fusions: phase 1 results from a multicentre, open-label, phase 1/2 study, Lancet Oncol., 2018, 19(5), 705–714.
22. E. Ardini, M. Menichincheri, P. Banfi, R. Bosotti, C. De Ponti and R. Pulci, et al., Entrectinib, a Pan–TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications, Mol. Cancer Ther., 2016, 15(4), 628–639.
23. M. Menichincheri, E. Ardini, P. Magnaghi, N. Avanzi, P. Banfi and R. Bossi, et al., Discovery of Entrectinib: A New 3-Aminoindazole As a Potent Anaplastic Lymphoma Kinase (ALK), c-ros Oncogene 1 Kinase (ROS1), and Pan-Tropomyosin Receptor Kinases (Pan-TRKs) inhibitor, J. Med. Chem., 2016, 59(7), 3392–3408.
24. Y. Duan, J. Wang, S. Zhu, Z.-C. Tu, Z. Zhang and S. Chan, et al., Design, synthesis, and Structure–Activity Relationships (SAR) of 3-vinylindazole derivatives as new selective tropomyosin receptor kinases (Trk) inhibitors, Eur. J. Med. Chem., 2020, 203, 112552.
25. T. Wu, C. Zhang, R. Lv, Q. Qin, N. Liu and W. Yin, et al., Design, synthesis, biological evaluation and pharmacophore model analysis of novel tetrahydropyrrolo[3,4-c]pyrazol derivatives as potential TRKs inhibitors, Eur. J. Med. Chem., 2021, 223, 113627.
26. T. Wu, Q. Qin, R. Lv, N. Liu, W. Yin and C. Hao, et al., Discovery of quinazoline derivatives CZw-124 as a pan-TRK inhibitor with potent anticancer effects in vitro and in vivo, Eur. J. Med. Chem., 2022, 238, 114451.
27. A. Drilon, R. Nagasubramanian, J. F. Blake, N. Ku, B. B. Tuch and K. Ebata, et al., A Next-Generation TRK Kinase Inhibitor Overcomes Acquired Resistance to Prior TRK Kinase Inhibition in Patients with TRK Fusion-Positive Solid Tumors, Cancer Discovery, 2017, 7(9), 963–972.
28. L. S. Zhuo, M. S. Wang, F. X. Wu, H. C. Xu, Y. Gong and Z. C. Yu, et al., Discovery of Next-Generation Tropomyosin Receptor Kinase Inhibitors for Combating Multiple Resistance Associated with Protein Mutation, J. Med. Chem., 2021, 64(20), 15503–15514.
29. T. Wu, Q. Qin, N. Liu, C. Zhang, R. Lv and W. Yin, et al., Rational drug design to explore the structure-activity relationship (SAR) of TRK inhibitors with 2,4-diaminopyrimidine scaffold, Eur. J. Med. Chem., 2022, 230, 114096.
30. C. Hao, F. Zhao, H. Song, J. Guo, X. Li and X. Jiang, et al., Structure-Based Design of 6-Chloro-4-aminoquinazoline-2-carboxamide Derivatives as Potent and Selective p21-Activated Kinase 4 (PAK4) Inhibitors, J. Med. Chem., 2018, 61(1), 265–285.
31. Y. Sun, L. Wang, Y. Sun, J. Wang, Y. Xue and T. Wu, et al., Structure-based discovery of 1-(3-fluoro-5-(5-(3-(methylsulfonyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)phenyl)-3-(pyrimidin-5-yl)urea as a potent and selective nanomolar type-II PLK4 inhibitor, Eur. J. Med. Chem., 2022, 243, 114714.
32. R. Wang, S. Yu, X. Zhao, Y. Chen, B. Yang and T. Wu, et al., Design, synthesis, biological evaluation and molecular docking study of novel thieno[3,2-d]pyrimidine derivatives as potent FAK inhibitors, Eur. J. Med. Chem., 2020, 188, 112024.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2md00334a

---